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WO2018187294A1 - Pyrimido-pyridazinone compound combinations, methods, kits and formulations thereof - Google Patents

Pyrimido-pyridazinone compound combinations, methods, kits and formulations thereof Download PDF

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Publication number
WO2018187294A1
WO2018187294A1 PCT/US2018/025841 US2018025841W WO2018187294A1 WO 2018187294 A1 WO2018187294 A1 WO 2018187294A1 US 2018025841 W US2018025841 W US 2018025841W WO 2018187294 A1 WO2018187294 A1 WO 2018187294A1
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WIPO (PCT)
Prior art keywords
pyridazin
amino
phenyl
oxo
piperidin
Prior art date
Application number
PCT/US2018/025841
Other languages
French (fr)
Inventor
Sanjeeva Reddy
Niranjan Rao
Louis Denis
Sandeep Gupta
Original Assignee
Asana Biosciences, Llc
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Publication date
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Publication of WO2018187294A1 publication Critical patent/WO2018187294A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within cells. Almost all kinases contain a similar 250 to 300 amino acid catalytic domain. The kinases can be categorized into families by the substrates they phosphorylate.
  • JAK (Janus kinase) is a family of intracellular non-receptor tyrosine kinases, which includes JAK1, JAK2, JAK3 and TYK2. JAK is expressed in hematopoietic cells and abundantly in primary leukemic cells from children with acute lymphoblastic leukemia.
  • the downstream substrates of JAK include the signal tranducer activator of transcription (STAT) proteins. STAT proteins function both as signaling molecules and transcription factors and ultimately bind to specific DNA sequences present in the promoters of cytokine-responsive genes.
  • JAK/STAT signaling has been implicated in the mediation of many abnormal immune responses such as allergies, asthma, autoimmune diseases such as transplant (allograft) rejection, rheumatoid arthritis, amyotrophic lateral sclerosis and multiple sclerosis, as well as in solid and hematologic malignancies such as leukemia and lymphomas.
  • Spleen tyrosine kinase is a member of the syk family of protein tyrosine kinases and plays a crucial role in inflammatory and allergic responses. Syk triggers IgE and IgG receptor mediated signaling in mast cells, basophils, and macrophages leading to
  • ITAM-mediated signaling has emerged as a primary event in signaling pathways responsible for human pathologies.
  • ITAM-mediated signaling is responsible for relaying activation signals initiated at classical immune receptors such as T-cell receptors, B-cell receptors, and Fc receptors in immune cells and at GPVI and FcyRIIa in platelets to downstream intracellular molecules such as Syk.
  • the binding of a ligand to an ITAM-containing receptor triggers signaling events which allows for the recruitment of proteins from a family of nonreceptor tyrosine kinases called the Src family. These kinases phosphorylate tyrosine residues within the ITAM sequence, a region with which the tandem SH2 domains on either Syk or ZAP-70 interact.
  • Syk with diphosphorylated ITAM sequences induces a conformation change in the kinases that allows for tyrosine phosphorylation of the kinase itself.
  • these kinases contribute to normal host defense, they also play roles in the pathogenesis of diseases. Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events. These diseases include autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and
  • Immunotherapy has been used as a treatment for various diseases by inducing, enhancing or modulating an immune response.
  • cancer immunotherapy attempts to stimulate the immune system to destroy tumors, or inhibit immune system checkpoint molecules so that they no longer block proteins on cancer cells or immune cells (such as T cells) that respond to them.
  • immune system checkpoint molecules are designed to overcome the ability of cancer cells to mask themselves from the patient's immune system.
  • oncologic therapies e.g., which act by inhibition of specific biochemical pathways
  • immunotherapeutic agents for example immune checkpoint inhibitors or therapies with immunostimulatory potential
  • combination treatments may result in increases in toxicity and adverse side effects, for example by increasing cytokine production and other inflammatory reactions related to the non-tumor suppressive activities of the immunotherapeutic agents.
  • the present disclosure provides pharmaceutical combinations of a compound of Formula (I), wherein R 1 and R 2 are defined herein
  • methods of treating cancer include administering a compound of Formula (I) and at least one immunotherapeutic agent to a patient in need thereof.
  • methods of inhibiting tumor growth or metastasis include administering a compound of Formula (I) and at least one immunotherapeutic agent to a patient in need thereof.
  • dosing regimens include administering to a patient in need thereof a compound of Formula (I) and at least one immunotherapeutic agent to a patient in need thereof.
  • kits include a dosage form comprising a compound of Formula (I) and a dosage form comprising at least one immunotherapeutic agent and therapeutic instructions for administering the dosage forms.
  • uses of a medicament in treating cancer include administering to a patient in need thereof a multi-part pharmaceutical combination including a compound of Formula (I) and at least one immunotherapeutic agent to a patient in need thereof.
  • Figs. 1 and 2 provide comparative data illustrating the anti-inflammatory effects of methotrexate, a known anti-inflammatory, and a compound described herein which is encompassed by the compound of Formula (I), using the Collagen Induced Arthritis (CIA) model of human rheumatoid arthritis (RA) in female lewis rats.
  • CIA Collagen Induced Arthritis
  • RA rheumatoid arthritis
  • Figure 1 A illustrates anti- inflammatory effects as a function of the amount of edema (mL) vs. time (days).
  • the black diamonds ( ⁇ ) represent results for the control.
  • Figure 1 B illustrates anti-inflammatory effects as a function of the amount of edema (mL) vs. time (days).
  • the circles ( ⁇ ) represent results for the control.
  • the inverted triangles represent results for the compound of Example 62.
  • the astericks (*) represent results for the compound of Example 108.
  • the squares ( ⁇ ) represent results for the compound of Example 189.
  • the diamonds ( ⁇ ) represent results for the compound of Example 191.
  • Figure 2A illustrates anti-inflammatory effects as a function of arhtritic score (per rat) vs. time (days).
  • the black diamonds ( ⁇ ) represent results for the control.
  • the triangles represent results for the compound of Example 19.
  • the crosses (x) represent results for methotrexate.
  • Figure 2B illustrates anti-inflammatory effects as a function of arhtritic score (per rat) vs. time (days).
  • the circles ( ⁇ ) represent results for the control.
  • the triangles represent
  • Figure 3 illustrates percent decrease in inflammation biomarkers on Day 15 after initiation of Compoud A treatment with the compound of Example 189 ("COMPOUND A") (BID dosing).
  • the percent change shown in Figure 3 are average values of all patients in each cohort.
  • B2M p2-Microglobulin
  • CRP C-Reactive Protein
  • IL-18 Interleukin-18
  • ⁇ -1 ⁇ Macrophage Inflammatory Protein- 1 ⁇
  • TNFR2 Tumor necrosis factor receptor 2.
  • Figure 4 illustrates average tumor volume (mm 3 ) in mice treated with COMPOUND A alone or in combination with mouse anti-PD 1 antibody after different days of treatment initiation. Table shown in Figure 4 shows % TGI for various treatments on days 5 to 15 and statistical comparisons between groups on day 15.
  • Figure 5 illustrates average tumor volume (mm 3 ) in mice treated with COMPOUND A alone or in combination with epacadostat (IDO-1 inhibitor) after different days of treatment initiation. Table shown in Figure 5 shows % TGI for various treatments on days 5 to 15 and statistical comparisons between groups on day 15.
  • FIG. 6 illustrates changes in average body weight of mice treated with
  • COMPOUND A alone or in combination with mouse anti-PD 1 antibody after treatment on day 15.
  • FIG. 7 illustrates changes in average body weight of mice treated with
  • COMPOUND A alone or in combination with epacadostat after treatment on day 15.
  • Figure 8 illustrates percent change in body weight of mice treated with COMPOUND A alone or in combination with mouse anti-PD 1 antibody measured on different days.
  • Figure 9 illustrates percent change in body weight of mice treated with COMPOUND A alone or in combination with epacadostat measured on different days.
  • Figure 10 presents raw data for tumor volume and body weight measurement on day
  • Figure 11 presents raw data for tumor volume and body weight measurement on day
  • Figure 12 presents raw data for tumor volume and body weight measurement on day
  • Figure 13 presents raw data for tumor volume and body weight measurement on day 11.
  • Figure 14 presents raw data for tumor volume and body weight measurement on day
  • Figure 15 presents raw data for COMPOUND A concentrations in plasma analyzed 2 h after last dose on day 15.
  • Figure 16 presents raw data for COMPOUND A concentrations in tumor analyzed 2 h after last dose on day 15.
  • Figure 17 illustrates average tumor volume (mm 3 ) in mice treated with COMPOUND
  • Table shown in Figure 17 shows % TGI for various treatments on days 4 to 15 and statistical comparisons between groups on day 15.
  • Figure 18 illustrates average tumor volume (mm 3 ) in mice treated with COMPOUND
  • Table shown in Figure 18 shows % TGI for various treatments on days 4 to 15 and statistical comparisons between groups on day 15.
  • FIG 19 illustrates changes in average body weight of mice treated with
  • COMPOUND A alone or in combination with mouse anti-PDl antibody after treatment on day 15.
  • FIG. 20 illustrates changes in average body weight of mice treated with
  • COMPOUND A alone or in combination with epacadostat after treatment on day 15.
  • Figure 21 illustrates percent change in body weight of mice treated with
  • COMPOUND A alone or in combination with epacadostat measured on different days.
  • Figure 23 presents raw data for tumor volume and body weight measurement on day
  • Figure 24 presents raw data for tumor volume and body weight measurement on day
  • Figure 25 presents raw data for tumor volume and body weight measurement on day
  • Figure 26 presents raw data for tumor volume and body weight measurement on day 11. The shaded rows in Figure 26 indicate animal mortality.
  • Figure 27 presents raw data for tumor volume and body weight measurement on day 15. The shaded rows in Figure 27 indicate animal mortality.
  • Figure 28 presents raw data for COMPOUND A concentrations in plasma on day 15 two hours of last dose.
  • Figure 29 presents raw data for COMPOUND A concentration in tumor on day 15 two hours of last dose.
  • Figure 30 illustrates average tumor volume (mm 3 ) in mice treated with COMPOUND A alone or in combination with mouse anti-PD 1 antibody after different days of treatment initiation. Table shown in Figure 30 shows % TGI for various treatments on days 4 to 15 and statistical comparisons between groups on day 15 .
  • Figure 31 illustrates average tumor volume (mm 3 ) in mice treated with COMPOUND A alone or in combination with epacadostat after different days of treatment initiation. Table shown in Figure 31 shows % TGI for various treatments on days 4 to 15 and statistical comparisons between groups on day 15.
  • Figure 32 illustrates changes in average body weight of mice treated with
  • COMPOUND A alone or in combination with mouse anti-PDl antibody after treatment on day 15.
  • Figure 33 illustrates changes in average body weight of mice treated with
  • COMPOUND A alone or in combination with epacadostat after treatment on day 15.
  • Figure 34 illustrates percent change in body weight of mice treated with
  • COMPOUND A alone or in combination with epacadostat measured on different days.
  • Figure 36 presents raw data for tumor volume and body weight measurement on day
  • Figure 37 presents raw data for tumor volume and body weight measurement on day
  • Figure 38 presents raw data for tumor volume and body weight measurement on day
  • Figure 39 presents raw data for tumor volume and body weight measurement on day
  • Figure 40 presents raw data for tumor volume and body weight measurement on day
  • Figure 41 presents raw data for COMPOUND A concentrations in plasma analyzed 2 h after last dose on day 15.
  • Figure 42 presents raw data for COMPOUND A concentrations in tumor analyzed 2 h after last dose on day 15.
  • the disclosure provides pharmaceutical combinations comprising compounds and immunotherapeutic agents, which are capable of reducing or eliminating inflammation caused by tissue insult, injury, or pathology, and treating cancer and other diseases.
  • the compounds disclosed herein can function through a protein kinase inhibitory mechanism.
  • the present disclosure provides a pharmaceutical combination comprising a compound of Formula (I):
  • R 1 is NR 4 R 5 , optionally substituted C 1 to C 6 alkoxy, optionally substituted C 6 to C 14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic orbicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic or bicyclic heterocyclyl.
  • the 3-4 membered cycloalkyl and heterocyclyl are saturated.
  • the hydrogen atoms on the same carbon atom of the cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl.
  • the hydrogen atoms on the same atom of the cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent.
  • R 1 is N(C 1 to C 6 alkyl)(C 1 to C 6 alkyl) or C 1 to C 6 alkoxy. ii. In still a further embodiment, R 1 is N(CH(CH 3 ) 2 )2, N(CH 3 ) 2 , OCH 2 CH 3 , or OCH 3 . iii. In another embodiment, R 1 is optionally substituted phenyl.
  • R 1 is of the structure:
  • R 22 , R 23 , R 24 , R 25 , and R 26 are, independently, H, C(O)(C 1 to C 6 alkoxy), C(O)OH, 0(C 1 to C 3 perfluoroalkyl), 0(C 1 to C 6 perfluoroalkoxy), Ci to Ce alkoxy, halogen, (C 1 to C 6 alkyl)heterocyclyl, or (C 1 to C 6 alkyl)CN.
  • R 1 is:
  • R 1 is optionally substituted 5-9 membered saturated heterocyclyl.
  • R 1 is of the structure:
  • R 34 , R 35 , R 36 , and R 37 are, independently, H, C 1 to C 6 alkyl, or CN;
  • R 7 and R 8 are, independently, H, C 1 to C 6 alkyl, C(O)OH, (C 1 to C 6 alkyl)CN, (C 1 to C 6 alkyl)C(O)OH, C(O)(C 1 to C 6 alkyl)CN, or CN; and
  • x is 0 to 2.
  • R is:
  • R 1 is of the structure:
  • R 1 is:
  • R 1 is a heteroaryl
  • R 1 is thiophene, benzooxole, or pyridine.
  • R 1 is a monocyclic C 3 to C 8 cycloalkyl.
  • R 1 is cycloheptyl or cyclohexyl, both optionally substituted with -N(C 1 to C 6 alkyl)(C 1 to C 6 alkyl).
  • R 1 is piperidine substituted with C(O)(C 1 to C 6 alkyl)CN.
  • xvi. In still a further embodiment, R is:
  • R 2 is phenyl substituted with C(O)NR 4 R 5 .
  • R 2 is phenyl substituted with
  • R 2 is phenyl substituted with NR 4 R 5 .
  • R 2 is phenyl substituted with (C 1 to C 6 alkyl)NR 4 R 5 .
  • R 2 is phenyl substituted with NR 4 R 5 or (C 1 to C 6 alkyl)NR 4 R 5 and R 4 and R 5 are taken together with the nitrogen atom to which they are attached to form a 6-membered ring.
  • R 2 is phenyl substituted with NR 4 R 5 or (C 1 to C 6
  • R 10 , R 11 , R 12 , and R 13 are, independently, H or C 1 to C 6 alkyl;
  • R 14 is halogen, OH, C(O)OH, C 1 to C 6 alkoxy, (C 1 to C 6 alkyl)halogen, (C 1 to C 6 alkyl)C(O)OH, C 1 to C 6 hydroxyalkyl, C 3 to C 8 cycloalkyl, (C 1 to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)C(O)NH(C 1 to C 6 hydroxyalkyl), (C 1 to C 6 alkyl)C(O)N(C 1 to C 6 hydroxyalkyl) 2 , (C 1 to C 6 alkyl)CN, (C 1 to C 6 alkyl)heteroaryl, or heteroaryl; and R 18 is C 1 to C 6 hydroxyalkyl or (C 1 to C 6 alkyl)C(O)OH.
  • R 2 is phenyl substituted with NR 4 R 5 or (C 1 to C 6
  • R 2 is phenyl substituted with NR 4 R 5 or (C 1 to C 6 alkyl)NR 4 R 5 and R 4 and R 5 are joined to form:
  • R 2 is phenyl substituted with NR 4 R 5 or (C 1 to C 6
  • R 2 is phenyl substituted with NR 4 R 5 or (C 1 to C 6
  • R 2 is phenyl substituted with NR 4 R 5 or (C 1 to C 6
  • R 10 , R 11 , R 12 , and R 13 are, independently, H or C 1 to C 6 alkyl; Y is O or NR 9 ; and R 9 is H, C 1 to C 6 alkyl, OH, C(O)OH, C 1 to C 6 hydroxyalkyl, (Ci to C 6 alkyl)NH 2 , (C 1 to C 6 alkyl)N(C 1 to C 6 alkyl)(C 1 to C 6 alkyl), (C 1 to C 6 alkyl)(C 1 to C 6 alkoxy), C(O)(C 1 to C 6 alkyl)NH 2 , (C 1 to C 6 alkyl)C(O)OH, C(O)(C 1 to C 6 hydroxyalkyl), C(O)(C 1 to C 6 alkyl)CN, (C 1 to C 6 alkyl)CN, (C 1 to C 6 alkyl)halogen, or (C 1 to C 6 alkyl)0(C 1 to C 6 alkyl;
  • R 2 is phenyl substituted with NR 4 R 5 or (C 1 to C 6
  • R 2 is phenyl substituted with NR 4 R 5 or (C 1 to C 6
  • alk 4 R 5 and R 4 and R 5 are taken together to form:
  • R 2 is phenyl substituted with (C 1 to C 6 alkyl)NR 4 R 5 .
  • R 2 is phenyl substituted with (C 1 to C 6 alkyl)NR 4 R 5 and
  • R 4 and R 5 are (C 1 to C 6 hydroxyalkyl).
  • R 2 is phenyl substituted with (C 1 to C 6 alkyl)NR 4 R 5 and NR 4 R 5 is:
  • R 2 is phenyl substituted with (C 1 to C 6 alkyl)NR 4 R 5 and R 4 and R 5 are joined to form an optionally substituted 6-membered ring.
  • R 2 is phenyl substituted with (C 1 to C 6 alkyl)NR 4 R 5 and NR 4 R 5 are joined to form the 6-membered ring:
  • R 14 is H, OH, C(O)OH, C 1 to C 6 alkyl, or (C 1 to C 6 alkyl)CN.
  • R 2 is phenyl substituted with (C 1 to C 6 alkyl)NR 4 R 5 and NR 4 R 5 are joined to form the 6-membered ring: wherein, Y is O or NR 9 ; and R 9 is H, C 1 to C 6 alkyl, OH, C 1 to C 6 hydroxyalkyl, C(O)(C 1 to C 6 hydroxyalkyl), C(O)(C 1 to C 6 alkyl)CN, (Ci to C 6 alkyl)CN, (C 1 to C 6 alkyl)NH 2 , (C 1 to C 6 alkyl)halogen, C(O)(C 1 to C 6 alkyl)CN or (C 1 to C 6 alkyl)0(C 1 to C 6 alkyl)C(O)(C 1 to C 6 alkyl)NH 2 .
  • R 2 is phenyl substituted with (C 1 to C 6 alkyl)NR 4 R 5 and
  • NR 4 R 5 are joined to form the 6-membered ring
  • a, b, c, d, and e are, independently, absent, (CH 2 ), CH(R 3 ), or O; and R 3 is H or C(O)OH.
  • R 2 is a heteroaryl substituted with (C 1 to C 6 alkyl)NR 4 R 5 . v. In still another embodiment, R 2 is:
  • R 2 is a heteroaryl substituted with NR 4 R 5 .
  • R 2 is a heteroaryl substituted with NR 4 R 5 and the heteroaryl is pyridine.
  • R 2 is of the structure:
  • R 2 is of the structure:
  • R 80 is OH, -(C 1 to C 6 alkyl)CN, C 1 to C 6 hydroxyalkyl, (C 1 to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)heterocycle or -(C 1 to C 6 alkyl) C(O)OH.
  • R 80 is OH, -(C 1 to C 6 alkyl)CN, C 1 to C 6 hydroxyalkyl, (C 1 to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)heterocycle or -(C 1 to C 6 alkyl) C(O)OH.
  • p is 1 to 6; and R is H or C(O)OH.
  • R is of the structure:
  • R 90 is H, C 1 to C 6 alkyl, C(O)(C 1 to C 6 alkyl)CN, (C 1 to C 6 alkyl)C(O)OH, or C(O)C 1 to C 6 hydroxyalkyl.
  • heterocyclyl optionally substituted heteroaryl, -O-(C 1 to C 6 alkyl)C(O)OH, -O-(Ci to C 6 alkyl)NR 4 R 5 , -0(optionally substituted heterocycle), -0(C 1 to C 6 alkyl)N(C 1 to C 6 alkyl)(C 1 to C 6 alkyl), -0(C 1 to C 6 alkyl)NH 2 , C 1 to C 6 hydroxyalkyl, -0(C 1 to C 6 hydroxyalkyl), -0(C 1 to C 6 alkyl)C(O)OH, -C 1 to C 6 alkoxy-C 1 to C 6 alkoxy, - 0(heterocycle)(C 1 to C 6 hydroxyalkyl), -S0 2 (C 1 to C 6 alkyl), or -(C 1 to C 6 alkyl)(Ci to Ce alkoxy)halogen.
  • R 2 is of the structure:
  • R 6 is H, (C 1 to C 6 alkyl)C(O)OH, or (C 1 to C 6 alkyl)CN. ee.
  • R 2 is of the structure:
  • z is 1 , 2, 3, 4, 5, or 6.
  • R 2 is of the structure:
  • R 6 is H or (C 1 to C 6 alkyl)C(O)OH.
  • R 2 is -0(C 1 to C 6 alkyl)NR 4 R 5 .
  • R 2 is of the structure:
  • y is 2 to 6; and R is H, OH, C 1 to C 6 alkyl, C 1 to C 6 hydroxyalkyl, or -(C 1 to C 6 alkyl)C(O)OH.
  • R 2 is of the structure:
  • R 2 is of the structure: wherein, r is 2 to 6; and R is H, C(O)OH or C 1 to C 6 hydroxyalkyl.
  • R 2 is:
  • R 2 is aryl substituted with -O-(C 1 to C 6 alkyl)-heterocycle. mm. In a further embodiment, R 2 is:
  • pharmaceutical combination comprising a compound of Formula (I) is provided, wherein R 1 is NR 4 R 5 , optionally substituted C 1 to C 6 alkoxy, optionally substituted C 6 to C 14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic or bicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic or bicyclic heterocyclyl.
  • R 1 is NR 4 R 5 , optionally substituted C 1 to C 6 alkoxy, optionally substituted C 6 to C 14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic or bicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic or bicyclic heterocyclyl.
  • R 1 is NR 4 R 5 , optionally substituted C 1 to C 6 alkoxy, optionally substituted C 6 to C 14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocycl
  • Hydrogen atoms on the same carbon atom of the cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl.
  • hydrogen atoms on the same atom of the cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent.
  • R 2 is phenyl or 5-6 membered heteroaryl containing at least one N or NH in the backbone, wherein R 2 is optionally substituted with one or more R 19 and when R 2 is 4- pyridyl, the 4-pyridyl lacks a carbonyl substituent at the 2 nd position.
  • R 19 is NR 4 R 5 , (C 1 to C 6 alkyl)NR 4 R 5 , C 1 to C 6 alkyl, C(O)NR 4 R 5 , C 3 to C 8 cycloalkyl substituted with one or more R 21 , or heterocyclyl substituted with one or more R 21 .
  • R 21 is (C 1 to C 6 alkyl)CN, (C 1 to C 6 alkyl)C(O)OH, (C 1 to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)C(O)NHCH 2 CH 2 OH, or (C 1 to C 6 alkyl)C(O)N(CH 2 CH 2 OH) 2 .
  • R 4 and R 5 are independently selected from among H, C 1 to C 6 alkyl, C 1 to C 6 hydroxyalkyl, and (C 1 to C 6 alkyl)N(C 1 to C 6 alkyl)(C 1 to C 6 alkyl).
  • R 4 and R 5 are joined to form an optionally substituted 3-8 membered heterocyclyl optionally further containing one or more O, S(O) n , or NR 9 .
  • R 9 is H, OH, (Ci to C 6 alkyl)C(O)OH, (C 1 to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)C(O)NHCH 2 CH 2 OH, (C 1 to C 6 alkyl)C(O)N(CH 2 CH 2 OH) 2 , C(O)(C 1 to C 6 alkyl)NH 2 , C(O)(C 1 to C 6 alkyl)OH, C 1 to C 6 hydroxyalkyl, or C 1 to C 6 alkyl and n is 0 to 2.
  • R 9 is CH 2 CH 2 OH.
  • Hydrogen atoms on the same carbon atom of the heterocyclyl are optionally replaced with a 3-6 membered cycloalkyl or heterocyclyl optionally substituted with one or more R to form a spirocycloalkyl or spiroheterocyclyl.
  • R 20 is C(O)0(C 1 to C 6 alkyl), C(O)OH, (C 1 to C 6 alkyl)C(O)OH, (C 1 to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)C(O)NHCH 2 CH 2 OH, or (C 1 to C 6 alkyl)C(O)N(CH 2 CH 2 OH) 2 .
  • hydrogen atoms on the same atom of any of the heterocyclyls or cycloalkyls of R 9 are optionally replaced with O to form an oxo substituent; or a pharmaceutically acceptable salt or ester thereof.
  • R 1 is NR 4 R 5 , C 1 to C 6 alkoxy, optionally substituted phenyl, heteroaryl, optionally substituted 3-10 membered cycloalkyl, or optionally substituted 3-10 membered monocyclic or bicyclic heterocyclyl.
  • Hydrogen atoms on the same carbon atom of the cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl.
  • R 2 is phenyl or pyrazole, wherein R 2 is optionally substituted with one or more R 19 .
  • R 19 is NR 4 R 5 , (C 1 to C 6 alkyl) R 4 R 5 , Q to Ce alkyl, C(O)NR 4 R 5 , C 3 to C 8 cycloalkyl substituted with one or more R 21 , or heterocyclyl substituted with one or more R 21 .
  • R 21 is (C 1 to C 6 alkyl)CN, (C 1 to C 6 alkyl)C(O)OH, (Ci to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)C(O) HCH 2 CH 2 OH, (C 1 to C 6
  • R 4 and R 5 are (a) independently selected from among H, Ci to C 6 alkyl, C 1 to C 6 hydroxyalkyl, and (C 1 to C 6 alkyl)N(C 1 to C 6 alkyl)(C 1 to C 6 alkyl) or (b) joined to form an optionally substituted 3-8 membered heterocyclyl optionally further containing one or more O, S(O) impart, or NR 9 .
  • R 9 is H, OH, C 1 to C 6 hydroxyalkyl, (C 1 to C 6 alkyl)C(O)OH, (C 1 to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)C(O)NHCH 2 CH 2 OH, (C 1 to C 6 alkyl)C(O)N(CH 2 CH 2 OH) 2 , C(O)(C 1 to C 6 alkyl)NH 2 , C(O)(C 1 to C 6 alkyl)OH, or C 1 to C 6 alkyl and n is 0 to 2.
  • R 9 is CH 2 CH 2 OH.
  • R 20 is C(O)0(C 1 to C 6 alkyl), C(O)OH, (C 1 to C 6 alkyl)C(O)OH, (C 1 to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)C(O)NHCH 2 CH 2 OH, or (C 1 to C 6
  • R 1 is NR 4 R 5 , C 1 to C 6 alkoxy, phenyl optionally substituted with C(O)0(C 1 to C 6 alkyl), C(O)OH, 0(C 1 to C 3 perfluoroalkyl), C 1 to C 6 alkoxy, halogen, CH 2 - heterocyclyl, or CH 2 CN, 5-8 membered cycloalkyl, heteroaryl, or 3-10 membered monocyclic or bicyclic heterocyclyl optionally substituted with (C 1 to C 6 alkyl)C(O)OH, Ci to C 6 alkyl, CN, C(O)OH, or (C 1 to C 6 alkyl)CN.
  • Hydrogen atoms on the same carbon atom of the cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl.
  • hydrogen atoms on the same atom of the cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent.
  • R 2 is phenyl or pyrazole, wherein
  • R 2 is optionally substituted with one R 19 .
  • R 19 is NR 4 R 5 , (C 1 to C 6 alkyl)NR 4 R 5 , C 1 to C 6 alkyl, C(O)NR 4 R 5 , C 3 to C 8 cycloalkyl substituted with one or more R 21 , or heterocyclyl substituted with one or more R 21 .
  • R 21 is (C 1 to C 6 alkyl)CN, (C 1 to C 6 alkyl)C(O)OH, (Ci to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)C(O) HCH 2 CH 2 OH, or (C 1 to C 6
  • R 4 and R 5 are (a) independently selected from among H, Q to
  • R 4 and R 5 may also be (b) joined to form a 5-8 membered heterocyclyl optionally further containing one or two O, S(O)n, or NR 9 .
  • R 9 is H, OH, C 1 to C 6 hydroxyalkyl (C 1 to C 6 alkyl)C(O)OH, C(O)(C 1 to C 6 alkyl)NH 2 , C(O)(C 1 to C 6 alkyl)OH, (C 1 to C 6 alkyl)C(O)NH 2 , (C 1 to C 6 alkyl)C(O)NHCH 2 CH 2 OH, (C 1 to C 6 alkyl)C(O)N(CH 2 CH 2 OH) 2 , or C 1 to C 6 alkyl and n is 0 to 2.
  • R 9 is CH 2 CH 2 OH.
  • R 20 is C(O)0(C 1 to C 6 alkyl), C(O)OH, (C 1 to C 6 alkyl)C(O)OH, (C 1 to C 6 alkyl)C(O)NH 2 , (C 1 to C 6
  • the disclosure includes each possible combination of chiral centers within a compound, as well as all possible enantiomeric and diastereomeric mixtures thereof. All chiral, diastereomeric, and racemic forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials.
  • arylalkyloxycabonyl refers to the group (C6-C14 aryl)-(C 1 -C 6 alkyl)-O-C(O)-.
  • Terms not defined herein have the meaning commonly attributed to them by those skilled in the art.
  • Alkyl refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms, for example, a C1-Q2 alkyl group may have from 1 to 12 (inclusive) carbon atoms in it.
  • Examples of C 1 -C 6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert- butyl, isopentyl, neopentyl, and isohexyl.
  • C 1 -C 8 alkyl groups include, but are not limited to, methyl, propyl, pentyl, hexyl, heptyl, 3-methylhex-l-yl, 2,3-dimethylpent-2-yl, 3- ethylpent-l-yl, octyl, 2-methylhept-2-yl, 2,3-dimethylhex-l-yl, and 2,3,3-trimethylpent-l-yl.
  • An alkyl group can be unsubstituted or substituted with one or more of halogen, NH 2 ,
  • alkyl NH, (alkyl)(alkyl)N-, -N(alkyl)C(O)(alkyl), -NHC(O)(alkyl), -NHC(O)H, -C(O)NH 2 , - C(O)NH(alkyl), -C(O)N(alkyl)(alkyl), CN, OH, alkoxy, alkyl, C(O)OH, -C(O)0(alkyl), - C(O)(alkyl), aryl, heteroaryl, heterocyclyl, cycloalkyl, haloalkyl, aminoalkyl-, -OC(O)(alkyl), carboxyamidoalkyl-, and N0 2 .
  • Alkoxy refers to the group R-O- where R is an alkyl group, as defined above.
  • Exemplary C 1 -C 6 alkoxy groups include but are not limited to methoxy, ethoxy, n-propoxy, 1-propoxy, n-butoxy and t-butoxy.
  • An alkoxy group can be unsubstituted or substituted with one or more of halogen, OH, alkoxy, NH 2 , (alkyl)amino-, di(alkyl)amino-, (alkyl)C(O)N(Ci- C 3 alkyl)-, (alkyl)carboxyamido-, HC(O)NH-, H 2 NC(O)-, (alkyl)NHC(O)-, di(alkyl)NC(O)-, CN, C(O)OH, (alkoxy)carbonyl-, (alkyl)C(O)-, aryl, heteroaryl, cycloalkyl, haloalkyl, amino(C 1 -C 6 alkyl)-, (alkyl
  • Aryl refers to an aromatic 6 to 14 membered hydrocarbon group.
  • Ce- Ci4 aryl group include, but are not limited to, phenyl, a-naphthyl, ⁇ -naphthyl, biphenyl, anthryl, tetrahydronaphthyl, fluorenyl, indanyl, biphenylenyl, and acenanaphthyl.
  • Examples of a C6-Cioaryl group include, but are not limited to, phenyl, a-naphthyl, ⁇ -naphthyl, biphenyl, and tetrahydronaphthyl.
  • An aryl group can be unsubstituted or substituted with one or more of alkyl, halogen, haloalkyl, alkoxy, haloalkoxy, OH, hydroxyalkyl, -O- (hydroxyalkyl), -O-(alkyl)-C(O)OH, -(alkyl)-(alkoxy)-halogen, NH 2 , aminoalkyl-, dialkylamino-, C(O)OH, -C(O)0-(alkyl), -OC(O)(alkyl), -O-(alkyl)-N(alkyl)(alkyl) ,N- alkylamido-, -C(O)NH 2 , (alkyl)amido-, N0 2 , (aryl)alkyl, alkoxy, aryloxy, heteroaryloxy, (aryl)amino, (alkoxy)carbonyl-, (alkyl)amido-, (
  • bicycle or "bicyclic” as used herein refers to a molecule that features two fused rings, which rings are a cycloalkyl, heterocyclyl, or heteroaryl.
  • the rings are fused across a bond between two atoms.
  • the bicyclic moiety formed therefrom shares a bond between the rings.
  • the bicyclic moiety is formed by the fusion of two rings across a sequence of atoms of the rings to form a bridgehead.
  • a "bridge” is an unbranched chain of one or more atoms connecting two bridgeheads in a polycyclic compound.
  • the bicyclic molecule is a "spiro" or “spirocyclic” moiety.
  • the spirocyclic group is a carbocyclic or heterocyclic ring which bound through a single carbon atom of the spirocyclic moiety to a single carbon atom of a carbocyclic or heterocyclic moiety.
  • the spirocyclic group is a cycloalkyl and is bound to another cycloalkyl.
  • the spirocyclic group is a cycloalkyl and is bound to a heterocyclyl.
  • the spirocyclic group is a heterocyclyl and is bound to another heterocyclyl. In still another embodiment, the spirocyclic group is a heterocyclyl and is bound to a cycloalkyl.
  • (Aryl)alkyl refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with an aryl group as defined above.
  • (C6-C14 aryl)alkyl- moieties include benzyl, benzhydryl, 1-phenylethyl, 2-phenylethyl, 3- phenylpropyl, 2-phenylpropyl, 1-naphthylmethyl, 2-naphthylmethyl and the like.
  • An (aryl)alkyl group can be unsubstituted or substituted with one or more of of halogen, CN, NH 2 , OH, (alkyl)amino-, di(alkyl)amino-, (alkyl)C(O)N(alkyl)-, (alkyl)carboxyamido-, HC(O)NH-, H 2 NC(O)-, (alkyl)NHC(O)-, di(alkyl)NC(O)-, CN, OH, alkoxy, alkyl, C(O)OH, (alkoxy)carbonyl-, (alkyl)C(O)-, aryl, heteroaryl, cycloalkyl, haloalkyl, amino(alkyl)-, (alkyl)carboxyl-, carboxyamidoalkyl-, or N0 2 .
  • (Alkoxy)carbonyl- refers to the group alkyl-O-C(O)-.
  • Exemplary (C 1 -C 6 alkoxy)carbonyl- groups include but are not limited to methoxy, ethoxy, n-propoxy, 1- propoxy, n-butoxy and t-butoxy.
  • An (alkoxy)carbonyl group can be unsubstituted or substituted with one or more of halogen, OH, NH 2 , (alkyl)amino-, di(alkyl)amino-,
  • alkyl)C(O)N(alkyl)- (alkyl)carboxyamido-, HC(O)NH-, H 2 NC(O)-, (alkyl)NHC(O)-, di(alkyl)NC(O)-, CN, alkoxy, C(O)OH, (alkoxy)carbonyl-, (alkyl)C(O)-, aryl, heteroaryl, cycloalkyl, haloalkyl, amino(alkyl)-, (alkyl)carboxyl-, carboxyamidoalkyl-, orN0 2 .
  • (Alkyl)amido- refers to a -C(O)NH- group in which the nitrogen atom of said group is attached to a C 1 -C 6 alkyl group, as defined above.
  • Representative examples of a (C 1 -C 6 alkyl)amido- group include, but are not limited to, -C(O)NHCH 3 , -C(O)NHCH 2 CH 3 , -C(O)NHCH 2 CH 2 CH 3 , -C(O)NHCH 2 CH 2 CH 2 CH 3 , -C(O)NHCH 2 CH 2 CH 2 CH 2 CH 3 , -C(O)NHCH(CH 3 ) 2 , -C(O)NHCH 2 CH(CH 3 ) 2 , -C(O)NHCH(CH 3 )CH 2 CH 3 , -C(O)NH- C(CH 3 ) 3 and -C(O)NHCH 2 C(CH 3 ) 3 .
  • (Alkyl)amino- refers to an -NH group, the nitrogen atom of said group being attached to a alkyl group, as defined above.
  • Representative examples of an (C 1 -C 6 alkyl)amino- group include, but are not limited to CH 3 NH-, CH 3 CH 2 NH-, CH 3 CH 2 CH 2 NH-, CH 3 CH 2 CH 2 CH 2 NH-, (CH 3 ) 2 CHNH-, (CH 3 ) 2 CHCH 2 NH-, CH 3 CH 2 CH(CH 3 )NH- and (CH 3 ) 3 CNH-.
  • An (alkyl)amino group can be unsubstituted or substituted on the alkyl moiety with one or more of halogen, NH 2 , (alkyl)amino-, di(alkyl)amino-, (alkyl)C(O)N(alkyl)-, (alkyl)carboxyamido-, HC(O)NH-, H 2 NC(O)-, (alkyl)NHC(O)-, di(alkyl)NC(O)-, CN, OH, alkoxy, alkyl, C(O)OH, (alkoxy)carbonyl-, (alkyl)C(O)-, aryl, heteroaryl, cycloalkyl, haloalkyl, amino(alkyl)-, (alkyl)carboxyl-, carboxyamidoalkyl-, or N0 2 .
  • halogen NH 2 , (alkyl)amino-, di(al
  • Aminoalkyl- refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with -NH 2 ; one or both H of the NH 2 may be replaced by a substituent.
  • Alkylcarboxyl- refers to an alkyl group, defined above that is attached to the parent structure through the oxygen atom of a carboxyl (C(O)-O-) functionality. Examples of (C 1 -C 6 alkyl)carboxyl- include acetoxy, propionoxy, propylcarboxyl, and isopentylcarboxyl.
  • (Alkyl)carboxyamido- refers to a -NHC(O)- group in which the carbonyl carbon atom of said group is attached to a C 1 -C 6 alkyl group, as defined above.
  • Representative examples of a (C 1 -C 6 alkyl)carboxyamido- group include, but are not limited to,
  • (Aryl)amino refers to a radical of formula (aryl)-NH-, wherein aryl is as defined above.
  • (Aryl)oxy refers to the group Ar-O- where Ar is an aryl group, as defined above.
  • Cycloalkyl refers to a non-aromatic, saturated, partially saturated, monocyclic, bicyclic or polycyclic hydrocarbon 3 to 12 membered ring system.
  • Representative examples of a C 3 -Ci 2 cycloalkyl include, but are not limited to, cyclopropyl, cyclopentyl, cycloheptyl, cyclooctyl, decahydronaphthalen-l-yl, octahydro-lH-inden-2-yl, decahydro-lH- benzo[7]annulen-2-yl, and dodecahydros-indacen-4-yl.
  • C 3 -Cio cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, decahydronaphthalen-l-yl, and octahydro-lH-inden-2-yl.
  • C 3 -C 8 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and octahydropentalen-2-yl.
  • a cycloalkyl can be unsubstituted or substituted with one or more of halogen, NH 2 , (alkyl)NH, (alkyl)(alkyl)N-, -N(alkyl)C(O)(alkyl), -NHC(O)(alkyl), -NHC(O)H, -C(O)NH 2 ,
  • Halo or "halogen” refers to -F, -CI, -Br and -I.
  • C 1 -C 6 haloalkyl refers to a C 1 -C 6 alkyl group, as defined above, wherein one or more of the C 1 -C 6 alkyl group's hydrogen atoms has been replaced with F, CI, Br, or I. Each substitution can be independently selected from F, CI, Br, or I.
  • an C 1 -C 6 haloalkyl- group include, but are not limited to, -CH 2 F, -CC1 3 , -CF 3 , CH 2 CF 3 , -CH 2 C1, -CH 2 CH 2 Br, -CH 2 CH 2 I, -CH 2 CH 2 CH 2 F, -CH 2 CH 2 CH 2 C1, -CH 2 CH 2 CH 2 CH 2 Br, -CH2CH2CH2I, -CH 2 CH2CH2CH 2 CH2Br, -CH2CH2CH2CH2I, -CH 2 CH(Br)CH 3 , -CH 2 CH(C1)CH 2 CH3, -CH(F)CH 2 CH 3 and -C(CH 3 )2(CH 2 C1).
  • Heteroaryl refers to a monocyclic, bicyclic, or polycyclic aromatic ring system containing at least one ring atom selected from the heteroatoms oxygen, sulfur and nitrogen.
  • C1-C9 heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4- triazole, l-methyl-l,2,4-triazole, lH-tetrazole, 1 -methyltetrazole, benzoxazole,
  • Bicyclic C1-C9 hetroaryl groups include those where a phenyl, pyridine, pyrimidine or pyridazine ring is fused to a 5 or 6-membered monocyclic heteroaryl ring having one or two nitrogen atoms in the ring, one nitrogen atom together with either one oxygen or one sulfur atom in the ring, or one O or S ring atom.
  • Examples of monocyclic C1-C4 heteroaryl groups include 2H-tetrazole, 3H-l,2,4-triazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, imidazole, and pyrrole.
  • a heteroaryl group can be unsubstituted or substituted with one or more of C 1 -C 6 alkyl, halogen, haloalkyl, OH, CN, hydroxyalkyl, N3 ⁇ 4, aminoalkyl-, dialkylamino-, C(O)OH, -C(O)0-(alkyl), -OC(O)(alkyl), N-alkylamido-, -C(O)NH 2 , (alkyl)amido-, -N0 2 , (aryl)alkyl, alkoxy, aryloxy, heteroaryloxy, (aryl)amino, (alkoxy)carbonyl-, (alkyl)amido-, (alkyl)amino, aminoalkyl-, alkylcarboxyl-, (alkyl)carboxyamido-, (aryl)alkyl-, (aryl)amino-, cycloalkenyl, di(alkyl
  • Heterocycle refers to monocyclic, bicyclic, polycyclic, or bridged head molecules in which at least one ring atom is a heteroatom.
  • a heterocycle maybe saturated or partially saturated.
  • Exemplary C1-C9 heterocyclyl groups include but are not limited to aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydro furan, dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine, 1,2,3,6- tetrahydropyridine-l-yl, tetrahydropyran, pyran, thiane, thiine, piperazine, azepane, diazepane, oxazine, 5,6-dihydro-4H-l,3-oxazin-2-yl, 2,5-diazabicyclo[2.2.1]heptane,
  • Ci heterocyclyl radicals would include but are not limited to oxaziranyl, diaziridinyl, and diazirinyl
  • C 2 heterocyclyl radicals include but are not limited to aziridinyl, oxiranyl, and diazetidinyl
  • C heterocyclyl radicals include but are not limited to azecanyl, tetrahydroquinolinyl, and perhydroisoquinolinyl.
  • a heterocyclyl group can be unsubstituted or substituted with one or more of alkyl, halogen, alkoxy, haloalkyl, OH, hydroxyalkyl, -C(O)-(hydroxyalkyl), NH 2 , aminoalkyl-, dialkylamino-, C(O)OH, -C(O)0- (alkyl), -OC(O)(alkyl), N-alkylamido-, -C(O)NH 2 , (alkyl)amido-, -C(O)-(alkyl)-CN, (alkyl)- CN, or N0 2 .
  • Heterocyclyl(alkyl)- refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with a heterocycle group as defined above.
  • Heterocyclyl( C 1 -C 6 alkyl)- moieties include 1 -piperazinylethyl, 4-morpholinylpropyl, 6-piperazinylhexyl, and the like.
  • a heterocyclyl(alkyl) group can be unsubstituted or substituted with one or more of halogen, NH 2 , (alkyl)amino-, di(alkyl)amino-,
  • alkyl)C(O)N(alkyl)- (alkyl)carboxyamido-, HC(O)NH-, H 2 NC(O)-, (alkyl)NHC(O)-, di(alkyl)NC(O)-, CN, OH, alkoxy, alkyl, C(O)OH, (alkoxy)carbonyl-, (alkyl)C(O)-, 4- to 7- membered monocyclic heterocycle, aryl, heteroaryl, or cycloalkyl.
  • Heteroaryl(alkyl) refers to a heteroaryl which is attached to an alkyl group and the heteroaryl is defined above.
  • Hydroalkyl refers to a alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with OH groups.
  • C 1 -C 6 hydroxyalkyl moieties include, for example, -CH 2 OH, -CH 2 CH 2 OH, -CH 2 CH 2 CH 2 OH, -CH 2 CH(OH)CH 2 OH, -CH 2 CH(OH)CH 3 , -CH(CH 3 )CH 2 OH and higher homologs.
  • Perfluoroalkyl- refers to alkyl group, defined above, having two or more fluorine atoms. Examples of a C 1 -C 6 perfluoroalkyl- group include CF3, CH 2 CF3, CF 2 CF3 and CH(CF 3 ) 2 .
  • a "subject” is a mammal, e.g. , a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or gorilla.
  • salts include but are not limited to, e.g., water-soluble and water-insoluble salts, such as the acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, bromide, butyrate, calcium, chloride, choline, citrate, edisylate (camphorsulfonate), fumarate, gluconate, glucuronate, glutamate, hydrobromide, hydrochloride, lauryl sulfate, malate, maleate, mandelate, mesylate, palmitate, pantothenate, phosphate, potassium, propionate, p-toluenesulfonate, salicylate, sodium, stearate, succinate, and sulfate salts.
  • water-soluble and water-insoluble salts such as the acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, bromide,
  • ACN is acetonitrile
  • DMSO dimethylsulfoxide
  • DMF is N,N-dimethylformamide
  • DMF.DMA dimethylformamide dimethylacetal
  • TFA trifluroroacetic acid
  • mCPBA meta- chloroperbenzoic acid
  • RT room temperature
  • THF is tetrahydrofuran
  • NMP N- methyl pyrrolidinone
  • Scheme 1 provides the synthesis of compounds of Formula (I).
  • Ethyl acetoacetate 1 is converted to the corresponding bis(methylthio)methylene derivative 2 using carbon disulfide, an organic or inorganic base such as K2CO3 and a alkylating agent.
  • the alkylating agent is an alkyl iodide, alkyl triflate, or alkyl sulfonate.
  • the alkylating agent is a methylating agent.
  • the alkylating agent is methyl iodide. Reaction of 2 with an R 1 -optionally substituted amidine hydrochloride in the presence of a base results in pyrimidine 3.
  • the base utilized to form pyrimidine 3 is EtsN or Hiinig's base.
  • the alkyl group on the pyrimidine group of compound 3 is then oxidized using an oxidizing agent. In one embodiment, the oxidation is performed using Se0 2 .
  • the resulting pyrimidine aldehyde 4 is converted to pyrimido-pyridazinone 5 using hydrazine hydrate or hydrazine hydrochloride.
  • the methyl thio group in compound 5 is oxidized to a methane sulfonyl using meta-chloroperoxybenzoic acid (mCPBA) or hydrogen peroxide/acetic acid.
  • mCPBA meta-chloroperoxybenzoic acid
  • the methane sulfonyl group of compound 6 is replaced with an R 2 -substituted aniline to provide compound (I).
  • the R 2 -substituted aniline is an aryl or heteroaryl substituted aniline.
  • Scheme 2 provides the synthesis of compound IB which are encompassed by the structure of Formula (I).
  • ethyl acetoacetate 1 is converted to the corresponding bis(methylthio)methylene derivative 2 using carbon disulfide, K2CO3 and methyl iodide.
  • Reaction of 2 with an R-substituted benzamidine hydrochloride in the presence of Et3N results inpyrimidine 3a.
  • the methyl group bound to the C-atom of pyrimidine 3a is then oxidized using Se0 2 .
  • the resulting pyrimidine aldehyde 4a is converted to pyrimido-pyridazinone 5a using hydrazine hydrate or hydrazine hydrochloride.
  • Scheme 3 provides the synthesis of compounds IC which are encompassed by the structure of Formula (I).
  • Treatment of ethyl chloroformate 7 with ammonium thiocyanate results in the production of ethyl thiocyanato formate 8 which upon treatment with ethyl 3- amino crotanoate results in compound 9.
  • Compound 9 is cyclized to compound 10 by treatment with an organic or inorganic base.
  • the organic or inorganic base is a strong base.
  • the strong base is a tertiary organic base.
  • the strong base is aqueous Et3N.
  • the dichloro pyrimidine 11 is obtained by treating compound 10 with a chlorinating agent.
  • the chlorinating agent is POCI3.
  • this transformation can also be carried out by using other chlorinating agents such as PCI5, SOCl 2 in the presence of an organic base such as TEA, tributyl amine, and ⁇ , ⁇ -dimethylaniline.
  • the 4-position of dichloropyrimidine 11 is then substituted by reaction with an optionally substituted (R 2 ) aniline to afford compound 12.
  • the 2-position of pyrimidine 12 is then R ⁇ -substituted using coupling agents such as boronic acids or boronic ester reagents to provide compound 13.
  • the methyl group at position 4 of pyrimidine 13 is then oxidized using an oxidizing agent such as Se0 2 to provide compound 14.
  • the resulting pyrimidine aldehyde 14 is converted to pyrimido-pyridazinone IC using hydrazine hydrate.
  • Scheme 4 provides the synthesis of compounds ID which are encompassed by the structure of Formula (I).
  • Treatment of ethyl chloroformate 7 with ammonium thiocyanate results in ethyl thiocyanato formate 8 which upon treatment with ethyl 3-amino crotanoate results in compound 9.
  • Compound 9 is cyclized to compound 10 as described in Scheme 3, i.e., by treatment with aqueous Et3N.
  • the dichloro pyrimidine compound 11 is obtained by treating compound 10 with POCI3.
  • the 4-position of dichloropyrimidine 11 is then substituted by reaction with an optionally R-substituted aniline to afford compound 12a.
  • the 2-position of pyrimidine compound 12a is then substituted with an R'-substituted aryl or heteroaryl group using a boronic acid or an boronic ester reagent to provide compound 13a.
  • the boronic acid is (R'-aryl)-B(OH)2 or (R'-heteroaryl)-B(OH)2.
  • the methyl at position-4 on pyrimidine 13a is then oxidized using Se0 2 .
  • the resulting pyrimidine aldehyde 14a is converted to pyrimido-pyridazinone ID using hydrazine hydrate.
  • Scheme 5 provides the synthesis of compound IE which are encompassed by the structure of Formula (I).
  • Compound 12 is reacted with an optionally substituted amine (NHR 4 R 5 ) to provide compound 15.
  • the methyl group at the 4-position of compound 15 is reacted with DMF.DMA to provide compound 16.
  • Compound 17 is obtained by oxidative cleavage of the olefin of compound 16. In one embodiment, oxidative cleavage is performed using NaI0 4 .
  • pyrimido-pyridazinone IE is obtained by cyclizing the aldehyde 17. In one embodiment, the cyclizaton is performed using hydrazine, hydrazine hydrate or hydrazine hydrochloride, as described previously.
  • Scheme 6 provides the synthesis of compounds IF which are encompassed by the structure of Formula (I).
  • Compound 12a is reacted with optionally substituted amines to result in 15a.
  • the methyl group of 15a is reacted with DMF.DMA to give compound 16a.
  • the aldehyde 17a is obtained by the oxidative cleavage of the olefin in 16a. In one embodiment, the oxidative cleavage is performed with NaI0 4 . Finally, the pyrimido- pyridazinone IF is obtained by cyclizing aldehyde 17a. In one embodiment, cyclization is performed using with hydrazine.
  • Scheme 7 provides the synthesis of compound 1G which are encompassed by the structure of Formula (I).
  • Compound 10 is reacted with an alkylating agent to the S-methyl compound 18.
  • the reacted is performed under basic conditions.
  • the alkylating agent is methyl iodide, ethyl iodide, propyl iodide, dimethylsulfate, among others.
  • Compound 19 is obtained by chlorinating compound 18.
  • compound 18 is chlorinated using POCI3.
  • Compound 19 is then NR 4 R 5 substituted with an optionally substituted amine to provide compound 20 [using NHR 4 R 5 ?].
  • the methyl group of compound 20 is reacted with DMF.DMA to give compound 21.
  • the aldehyde 22 is obtained by the oxidative cleavage of the olefin group in compound 21.
  • the oxidative cleavage is performed with NaI0 4 .
  • compound 20 may directly converted to the pyrimidine aldehyde 22 by oxidizing the methyl group using Se0 2 or a combination of C0 2 , t-butyl hydroperoxide, and an alcohol such as Ci to Ce alkyl)H 2 OH.
  • the resulting pyrimidine aldehyde 22 is converted to pyrimido-pyridazinone 23 using hydrazine hydrate or hydrazine hydrochloride.
  • the methyl thio group in compound 23 is oxidized to a methane sulfonyl group.
  • compound 23 is reacted with meta-chloroperoxybenzoic acid (mCPBA) or hydrogen peroxide/acetic acid.
  • mCPBA meta-chloroperoxybenzoic acid
  • the methane sulfonyl group of compound 24 is replaced with suitably substituted aniline to provide compound 1G.
  • Scheme 8 provides the synthesis of compound 1H which is encompassed by the structure of Formula (I).
  • compound 19 is coupled with an optionally substituted boronic acid or boronic ester to give compound 25.
  • the coupling is performed in the presence of a coupling agent such as Pd(PPh3) 4 or PdCl 2 (PPh3) 2 .
  • the methyl group in compound 25 is then oxidized to the corresponding aldehyde.
  • the oxidation is performed using Se0 2 or a combination of C0 2 , t-butyl hydroperoxide, and an alcohol such as C 1 to C 6 alkyl)H 2 OH to give compound 26.
  • Compound 26 is converted to pyrimido-pyridazinone 27 using hydrazine hydrate or hydrazine hydrochloride.
  • the methyl thio group in compound 27 is then oxidized to a methane sulfonyl group.
  • the oxidation is performed using mCPBA or hydrogen peroxide/acetic acid.
  • the methane sulfonyl group of compound 28 is replaced with suitably substituted aniline to provide compound 1H.
  • Pharmaceutical combinations of the disclosure comprise a compound of Formula (I) optionally with other pharmaceutically inert or inactive ingredients.
  • the pharmaceutically inert or inactive ingredient is one or more pharmaceutically acceptable carrier or excipient.
  • the present disclosure also contemplates combining the compound of Formula (I) with one or more therapeutic agents, i.e., active ingredients, as described below.
  • a compound of Formula (I) is combined with one or more inert/inactive ingredients and one or more therapeutic agents.
  • the pharmaceutical combinations of the disclosure contain an amount of a compound of Formula (I) that is effective for treating inflammation in a subject.
  • the dosage of the compound of Formula (I) to achieve a therapeutic effect will depend on factors such as the formulation, pharmacological potency of the drug, age, weight and sex of the patient, condition being treated, severity of the patient's symptoms, specific compound of Formula
  • the treatment and dosage of the compound of Formula (I) maybe administered in unit dosage form and that one skilled in the art would adjust the unit dosage form accordingly to reflect the relative level of activity.
  • the decision as to the particular dosage to be employed (and the number of times to be administered per day) is within the discretion of the ordinarily-skilled physician, and maybe varied by titration of the dosage to the particular circumstances to produce the desired therapeutic effect.
  • the therapeutically effective amount is about 0.0001% to about 25% w/w. In another embodiment, the therapeutically effective amount is less than about 20% w/w, about 15% w/w, about 10% w/w, about 5% w/w, or about 1% w/w. In another embodiment, the therapeutically effective amount is about 0.0001% to about 10% w/w. In a further embodiment, the therapeutically effective amount is about 0.005 to about 5% w/w. In yet another embodiment, the therapeutically effective amount is about 0.01 to about 5% w/w.
  • the therapeutically effective amount is about 0.01% w/w, about 0.05% w/w, about 0.1 % w/w, about 0.2 % w/w, about 0.3% w/w, about 0.4% w/w, about
  • the therapeutically effective amounts maybe provided on regular schedule, i.e., on a less than daily, weekly, monthly, or yearly basis or on an irregular schedule with varying administration days, weeks, months, etc.
  • the therapeutically effective amount to be administered may vary.
  • the therapeutically effective amount for the first dose is higher than the therapeutically effective amount for one or more of the subsequent doses.
  • the therapeutically effective amount for the first dose is lower than the therapeutically effective amount for one or more of the subsequent doses.
  • Equivalent dosages may be administered over various time periods including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every 2 weeks, about every 3 weeks, about every month, about every 2 months, about every 3 months and about every 6 months.
  • the number and frequency of dosages corresponding to a completed course of therapy will be determined according to the judgment of a health-care practitioner.
  • the therapeutically effective amounts described herein refer to total amounts administered for a given time period; that is, if more than one compound of Formula (I) is administered, the therapeutically effective amounts correspond to the total amount administered.
  • the compound of Formula (I) maybe administered by any route, taking into consideration the specific condition for which it has been selected.
  • the compounds of Formula (I) may be delivered orally, by injection (including intravascularly, e.g ,
  • the compound of Formula (I) may be administered by injection, transdermally or topically.
  • the compound of Formula (I) maybe administered topically to the eye, e.g., as solutions, suspensions or ointments.
  • ophthalmically compatible carriers which may be used include, without limitation, an aqueous solution, such as saline solution, oil solution or ointments containing ophthalmically compatible preservatives, surfactants, buffers, and viscosity regulators. These compositions may also contain stabilizing agents, antibacterial agents, and maybe manufactured in different dosage units, suitable for ocular administration. Drug inserts, either soluble or insoluble, may also be used.
  • the compound of Formula (I) maybe administered by injection.
  • Solutions for injection or infusion may be prepared as aqueous solutions.
  • the compound of Formula (I) is present in a concentration of about 0.001 ⁇ g mL to 1 mg/mL, or this amount maybe adjusted higher or lower as needed.
  • These solutions may also contain stabilizing agents, antibacterial agents, buffers and maybe manufactured in different dosage unit ampoules or bottles.
  • the compound of Formula (I) maybe administered rectally.
  • Dosage units for rectal administration may be prepared in the form of ointments or suppositories, which contain the compound of Formula (I) in a mixture with a neutral fat base, or they may be prepared in the form of gelatin-rectal capsules that contain the compound of Formula (I) in a mixture with, e.g., a vegetable oil or paraffin oil.
  • Ointments, suppositories or creams containing at least one compound of Formula (I) are useful for the treatment of hemorrhoids.
  • the compound of Formula (I) maybe administered orally.
  • Dosage units for oral administration include, without limitation, tablets, caplets, capsules, powders, suspensions, microcapsules, dispersible powder, granules, suspensions, syrups,. elixirs, and aerosols, which contain the compound of Formula (I) optionally with one or more excipient.
  • the compositions are compressed into a tablet or caplet.
  • the tablet or caplet maybe administered to the subject.
  • the tablet or caplet may be added to a capsule.
  • the composition containing the compound of Formula (I) is added directly to a capsule.
  • the capsule includes hydroxypropyl methylcellulose, hypromellose capsule, or a hard shell gelatin capsule.
  • the tablets or caplets are optionally film-coated using film-coatings known to those of skill in the art.
  • the film-coating is selected from among polymers such as, without limitation,
  • hydroxypropylmethylcellulose ethyl cellulose
  • polyvinyl alcohols and combinations thereof.
  • the compound of Formula (I) may also be administered in the presence of one or more pharmaceutical carriers that are physiologically compatible.
  • the amount of the pharmaceutical carrier(s) is determined by the solubility and chemical nature of the compound of Formula (I), chosen route of administration, and standard pharmacological practice.
  • the carriers may be in dry (solid) or liquid form and must be pharmaceutically acceptable.
  • Liquid pharmaceutical compositions are typically sterile solutions or suspensions. When liquid carriers are utilized for parenteral administration, they are desirably sterile liquids. Liquid carriers are typically utilized in preparing solutions, suspensions, emulsions, syrups and elixirs. A variety of suitable liquid carriers is known and may be readily selected by one of skill in the art.
  • Such carriers may include, .e.g., dimethylsulfoxide (DMSO), saline, buffered saline, cyclodextrin,
  • the compound of Formula (I) is dissolved a liquid carrier.
  • the compound of Formula (I) is suspended in a liquid carrier.
  • a suitable liquid carrier depending on the route of administration.
  • the compound of Formula (I) may alternatively be formulated in a solid carrier of which a variety of solid carriers and excipients are known to those of skill in the art.
  • the composition maybe compacted into a unit dose form, i.e., tablet or cap let.
  • the composition maybe added to unit dose form, i.e., a capsule.
  • the composition maybe formulated for administration as a powder.
  • the solid carrier may perform a variety of functions, i.e., may perform the functions of two or more of the excipients described below.
  • a solid carrier may also act as a flavoring agent, lubricant, solubilizer, suspending agent, filler, glidant, compression aid, binder, disintegrant, or encapsulating material.
  • compositions may also be sub-divided to contain appropriate quantities of the compound of Formula (I).
  • the unit dosage can be packaged compositions, e.g., packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids.
  • excipients which may be combined with one or more compound of
  • Formula (I) include, without limitation, adjuvants, antioxidants, binders, buffers, coatings, coloring agents, compression aids, diluents, disintegrants, emulsifiers (e g. , polyoxyethylene fatty acid esters), emollients, encapsulating materials, fillers, flavoring agents, glidants, granulating agents, lubricants, metal chelators, osmo-regulators, pH adjustors (e.g., sodium hydroxide), preservatives, solubilizers, sorbents, stabilizing agents, sweeteners (such as saccharin), surfactants, suspending agents, syrups, thickening agents (e.g.,
  • penetration enhancers e.g., hydroxypolyethoxydodecane, DMSO, DMAC, DDM, etc
  • viscosity regulators such as polymers to increase viscosity
  • compositions maybe utilized as inhalants.
  • compositions maybe prepared as fluid unit doses using a compound of Formula (I) and a vehicle for delivery by an atomizing spray pump or by dry powder for insufflation.
  • compositions maybe utilized as aerosols, i.e., oral or intranasal.
  • aerosols i.e., oral or intranasal.
  • the compositions are formulated for use in a pressurized aerosol container together with a gaseous or liquefied propellant, e.g. ,
  • dichlorodifluoromethane carbon dioxide, nitrogen, propane, and the like. Also provided is the delivery of a metered dose in one or more actuations.
  • compositions may be administered by a modified-release delivery device.
  • Modified-release refers to delivery of a compound of Formula (I) which is controlled, for example over a period of at least about 8 hours (e.g., extended delivery) to at least about 12 hours (e.g., sustained delivery). Such devices may also permit immediate release (e.g., therapeutic levels achieved in under about 1 hour, or in less than about 2 hours).
  • suitable modified-release delivery devices For use in such modified-release delivery devices, the compound of Formula (I) is formulated as described herein.
  • the compounds of Formula (I) are combined with other medications or therapeutic agents in a single composition.
  • the present disclosure is not so limited.
  • the compounds of Formula (I) may be administered in one or more separate formulations from other compounds of Formula (I), or other medications or therapeutic agents as described below.
  • agents typically used to treat inflammation may be used in conjunction with a combination of the disclosure in the methods, compositions, and kits described herein.
  • agents include, but are not limited to, non-steroidal anti-inflammatory drugs.
  • the compound of Formula (I) maybe combined with glucose or dextrose when utilized for infusion or as a regional analgesic or anti-pruritic.
  • the compound of Formula (I) maybe combined with thickening agents to form a jelly, or may also contain penetration enhancers, for use in topical or dermal applications such as for urogenital topical procedures.
  • the compound of Formula (I) maybe formulated as an ointment for administration to accessible mucous membranes.
  • kits or packages of pharmaceutical formulations containing the compounds of Formula (I) or compositions described herein are also provided herein.
  • the kits maybe organized to indicate a single formulation or combination of formulations to be taken at each desired time.
  • the kit contains packaging or a container with the compound of Formula (I) formulated for the desired delivery route.
  • the kit contains instructions on dosing and an insert regarding the compound of Formula (I).
  • the kit may further contain instructions for monitoring local or circulating levels of product and materials for performing such assays including, e.g., reagents, well plates, containers, markers or labels, and the like.
  • Such kits are readily packaged in a manner suitable for treatment of a desired indication.
  • the kit may also contain instructions for use of an oral dosage form such as a pill, capsule, patch, spray pump or other delivery device.
  • suitable components to include in such kits will be readily apparent to one of skill in the art, taking into consideration the desired indication and the delivery route.
  • a package or kit can include the compound of Formula (I) in each dosage unit (e.g., solution, lotion, tablet, pill, drug-eluting patch or other unit described above or utilized in drug delivery), and optionally instructions for administering the doses less-than-daily, daily, weekly, or monthly, for a predetermined length of time or as prescribed.
  • a package or kit can include placebos during periods when the compound of Formula (I) is not delivered.
  • a package or kit may contain a sequence of dosage units which provide the desired variability.
  • the package has indicators for each period.
  • the package is a foil or blister package, labeled ampoule, vial or bottle.
  • the packaging means of a kit may itself be geared for administration, such as an inhalant, syringe, pipette, eye dropper, or other such apparatus, from which the formulation may be applied to an affected area of the body, such as the lungs, injected into a subject, or even applied to and mixed with the other components of the kit.
  • kits also may be provided in dried or lyophilized forms.
  • reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another package.
  • kits of the present disclosure also will typically include a means for containing the vials or other suitable packaging means in close confinement for commercial sale such as, e.g. , injection or blow-molded plastic containers into which the desired vials are retained.
  • a means for containing the vials or other suitable packaging means in close confinement for commercial sale such as, e.g. , injection or blow-molded plastic containers into which the desired vials are retained.
  • the kits also may include, or be packaged with a separate instrument for assisting with the injection/administration or placement of the composition within the body of an animal.
  • a separate instrument maybe an inhalant, syringe, pipette, forceps, measuring spoon, eye dropper or any such medically approved delivery means.
  • a kit in one embodiment, contains a compound of Formula (I).
  • the compound of Formula (I) may be in the presence or absence of one or more of the carriers or excipients described above.
  • the kit may optionally contain instructions for administering the compound of Formula (I) to a subject having inflammation.
  • a kit in a further embodiment, contains a compound of Formula (I) in a second dosage unit, and one or more of the carriers or excipients described above in a third dosage unit.
  • the kit may optionally contain instructions for administering the compound of
  • Formula (I) to a subject having inflammation.
  • inflammation as used herein includes all types of inflammation.
  • the inflammation may be acute or chronic.
  • the inflammation may be nociceptive,
  • the inflammation maybe from a migraine, gynecological condition, pre-labor or labor, stroke, surgery, neuralgia, sickle cell, interstitial cystitis, urological condition (such as urethritis), dental work/injury, or headache, among other.
  • Inflammation may also occur in patients with cancer, which may be due to multiple causes, such as nerve compression and mechanical forces resulting from tissue distension as a consequence of invasion by a tumor and tumor metastasis into bone or other tissues.
  • inflammation results from neuropathy, such as post-herpetic neuralgia.
  • the inflammation results from a surgery or procedure.
  • the inflammation results from an infection, cancer, colitis, cystitis, irritable bowel syndrome, or idiopathic neuropathy
  • Somatic inflammation includes inflammation in bone, joint, muscle, skin, or connective tissue.
  • Central inflammation includes inflammation arising as a consequence of brain trauma, stroke, or spinal cord injury.
  • Viceral inflammation includes inflammation in visceral organs, such as the respiratory or gastrointestinal tract and pancreas, the urinary tract and reproductive organs. In one embodiment, visceral inflammation results from tumor involvement of the organ capsule. In another embodiment, visceral inflammation from obstruction of hollow viscus.
  • Idiopathic inflammation refers to inflammation which has no underlying cause or refers to inflammation caused by condition which remains undiagnosed.
  • Disfunctional inflammation refers to inflammation which occurs in the absence of a noxious stimulus, tissue damage or a lesion to the nervous system.
  • dysfunctional inflammation results from rheumatologic conditions such as arthritis and fibromyalgia, tension type headache, irritable bowel disorders and erythermalgia.
  • Nociceptive inflammation includes inflammation caused by noxious stimuli that threaten to or actually injure body tissues.
  • nociceptive inflammation results from a cut, bruise, bone fracture, crush injury, burn, trauma, surgery, labor, sprain, bump, injection, dental procedure, skin biopsy, or obstruction.
  • nociceptive inflammation is located in the skin, musculoskeletal system, or internal organs.
  • Neurotoxic inflammation is inflammation due to abnormal processing of sensory input by the peripheral or central nervous system consequent on a lesion to these systems.
  • neuropathic inflammation is chronic and non-malignant.
  • neuropathic inflammation is due to trauma, surgery, herniation of an intervertebral disk, spinal cord injury, diabetes, infection with herpes zoster (shingles), HIV/AIDS, late-stage cancer, amputation (such as mastectomy), carpal tunnel syndrome, chronic alcohol use, exposure to radiation, and as an unintended side-effect of neurotoxic treatment agents, such as certain anti-HIV and chemotherapeutic drugs.
  • Procedural inflammation includes refers to inflammation arising from a medical procedure.
  • the medical procedure may include any type of medical, dental or surgical procedure.
  • the procedural inflammation is postoperative.
  • the inflammation is associated with an injection, draining an abscess, surgery, dermatological, dental procedure, ophthalmic procedure, arthroscopy and use of other medical instrumentation, and/or cosmetic surgery.
  • a “migraine” is a type of headache, typically defined clinically as being caused by activation of sensory fibers innervating the meninges of the brain.
  • treat means to include therapy utilized to remedy a health problem or condition in a patient or subject.
  • the health problem or condition may be eliminated permanently or for a short period of time.
  • the health problem or condition may be prevented.
  • the severity of the health problem or condition, or of one or more symptoms characteristic of the health problem or condition may be lessened permanently, or for a short period of time.
  • the effectiveness of a treatment of inflammation can be determined using any standard inflammation index, such as those described herein, or can be determined based on the patient's subjective inflammation assessment. A patient is considered “treated” if there is a reported reduction in inflammation, or a reduced reaction to stimuli that should cause inflammation.
  • the treatment methods described herein include administering a compound of Formula (I) to a patient. Additional, optional agents, such as those described above for use in the combination, may be administered to the patient prior to, concurrently with, or subsequent to the compound of Formula (I).
  • Ethyl 2-hydroxy-4-mercapto-6-methylpyrimidine-5-carboxylate (1.4 g, 6.54 mmol) was taken up in POCI3 (13.67 mL) at 0°C, tri-n-butylamine was added and the mixture heated to 90°C for 5 hours. The mixture was cooled, poured slowly on to crushed ice and extracted with dichloromethane. The organic layer was washed with water, dried over Na 2 S0 4 and concentrated under vacuum.
  • Example 340 Inhibition of enzymatic Syk kinase activity
  • the objective of this assay was to examine by radiometric method the ability of compounds to inhibit Syk kinase enzyme.
  • Spleen tyrosine kinase is a cytosolic protein tyrosine kinase that plays a crucial role in inflammatory and allergic responses. Syk triggers IgE and IgG receptor mediated signaling in mast cells, basophils, and macrophages leading to degranulation and cytokine release. Abnormal function of Syk has also been implicated in several instances of hematopoietic malignancies.
  • Syk is capable of phosphorylating substrates such as VAV, LAT, SLP-76, which in turn activate MAPK, PLCy signaling pathways. Crystallization studies of the Syk catalytic domain (360-635) showed more activity compared to the full length Syk enzyme.
  • This in vitro assay tests the ability of syk to phosphorylate a substrate peptide in the presence of ATP. By using a radio-labeled form of ATP, it is possible to measure the amount of phosphorylation of the substrate.
  • the enzyme transfers a radio-labeled phosphate group from ⁇ 32 P labeled ATP to pG4T. Briefly the enzyme was incubated with substrate, radio-labeled
  • the reaction mixture was transferred on to a Multiscreen filter plate and unreacted ⁇ 32 P ATP was washed off.
  • the filter plate was dried and the radioactivity was measured on a scintillation counter to estimate the incorporated radioactivity on the substrate. The percent inhibition of activity of the enzyme was calculated by comparing counts in the presence and absence of compounds.
  • the fragment utilized included a C'-terminal tag of 4 amino acids and a stretch of 15 amino acids N-terminal to the kinase domain, starting at amino acid 356.
  • the assay squares were washed 3 times for 5 minutes each in ortho-phosphoric acid (0.5%) and once in acetone. Assay squares were dried for 15 minutes in a 30°C oven and transferred to 96 well optiplate. Microscint-O® reagent (100 ⁇ L, Perkin Elmer) was added to each well, the plate was sealed with Topseal®-A microplates and incubated for 10 minutes at room temperature at very low speed on rocker and the plate was read in the Topcount® NXL instrument.
  • Fold induction radioactivity counts (uncorrected values) in positive control/substrate control.
  • TR-FRET Time-resolved fluorescence resonance energy transfer
  • JAK (Janus kinase 2) is a family of intracellular non-receptor tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway. These kinases have apparent molecular weight of about 130 Kda. They were initially named “just another kinase” 1 & 2 (since they were just two of a large number of discoveries in a PCR-based screen of kinases), but were ultimately published as "Janus kinase”. JAKs possess two near-identical phosphate-transferring domains. One domain exhibits the kinase activity while the other negatively regulates the kinase activity of the first. They are crucial signal transducers for a variety of cytokines, growth factors and interferons.
  • TR-FRET assays are homogeneous proximity assays where Eu-labeled antiphosphotyrosine antibody binds to the phosphorylated substrates labeled with Ulight fluorescence acceptor. Eu can transfer energy to Ulight accepter in the complex and the interaction of two dye-labeled binding partners is detected by the energy transfer between a donor and an acceptor dye, and the subsequent light emission by the acceptor dye. The intensity of the light emission is proportional to the level of Ulight peptide phosphorylation.
  • the objective of this assay was to examine by Fluorescence method the effect of compounds on ⁇ -hexosaminidase release during immune complex mediated degranulation in RBL2H3 cells.
  • Protocol A 24 well format
  • RBL2H3 cells were maintained in MEM complete media containing 10% FBS at 70% - 80% confluence in a mammalian cell culture C0 2 incubator with 5% C0 2 at 37°C. 2 x 10 5 cells/well were plated in 1 mL of complete media and incubated for 5 hours for cell attachment. Complete media was replaced with 1 mL of serum free MEM media containing 1.2 ⁇ g/mL of anti-DNP rat IgE as sensitizing agent and further incubated overnight with 5% C0 2 at 37°C. The following day, cells were washed with serum free media and further treated with various concentrations of test compounds (in 0.1% DMSO) for 45 minutes at 37°C and 5% C0 2 .
  • test compounds in 0.1% DMSO
  • % release of ⁇ -Hexosaminidase for the test compound was calculated using the following formula:
  • Protocol B 96 well format
  • RBL2H3 cells were maintained in MEM complete media containing 10% FBS at 70-80% confluence in a mammalian cell culture C0 2 incubator with 5% C0 2 at 37°C.
  • 5 x 10 4 cells/well were plated in 200 uL of complete media containing 0.3 ⁇ g/mL of anti-DNP rat IgE as sensitizing agent for 24 hours at 37°C & 5% C0 2 .
  • the following day, cells were washed twice with PIPES buffer for 10 minutes at 37°C and replenished with serum free MEM media. Cells were treated with various concentrations of test compounds (in 0.5% DMSO) for 15 minutes at 37°C and 5% C0 2 .
  • the cells were further stimulated with 0.1 ⁇ g/mL of DNP-BSA for 45 minutes.
  • the plates were spun for 5 minutes at 2000 rpm and 25 ⁇ L of culture supernatant was then transferred from each assay well into a 96 well black coated plate.
  • Fifth ⁇ L ⁇ -NAG substrate was added and incubated at RT for 30 minutes. After incubation with substrate, 150 ⁇ L of stop solution was added and fluorescence was monitored. (Excitation 355 nm; Emission 460 nm). See, Yamamoto, JPET, 306(3):1174- 1181 (2003) and Taylor, MCB, 15(8): 4149-4157 (1995), which are herein incorporated by reference.
  • Example 343 In vivo assay - Chronic study
  • CIA Collagen Induced Arthritis
  • RA rheumatoid arthritis
  • ell type II collagen
  • CIA exhibits several features of human RA such as severe swelling inflammation of joints, synovial hyperplasia, cartilage destruction and bone erosion.
  • Pathophysiology of CIA consists of T cell component, as evidenced by increased infiltration of T-cells in joint synovium and also, by attenuation of CIA in T-cell deficient mice.
  • CIA development involves B cell component too, as is evidenced by circulating ell antibody in disease animals and also, failure to develop the disease in xid mice/B cell deficient mice/CXCR5 null mice. Recently, a significant role of macrophages has also been suggested in the pathogenesis of CIA as well as human RA. See, Pine, "Inflammation and bone erosion are suppressed in models of rheumatoid arthritis following treatment with a novel Syk inhibitor", Clin. Immunol, 2007, 124(3 ):244-57; Xiong cha, "Suppression of the onset and progression of collagen-induced arthritis in rats by QFGJS, a preparation from an anti-arthritic Chinese herbal formula", J.
  • mice Female Lewis rats (8 per group, 6-8 weeks old) were immunized on day 1 with type II collagen (Immunization grade Bovine type II; Chondrex; Cat #20021) emulsified with Complete Freund's Adjuvant (Sigma; Cat# F5881) at a final concentration of 1.2 mg/mL).
  • type II collagen Immunization grade Bovine type II; Chondrex; Cat #20021
  • Complete Freund's Adjuvant Sigma; Cat# F5881
  • a booster injection of the same type II collagen emulsified with Incomplete Freund's Adjuvant (Sigma, Cat #F5506) (0.25 mL/rat) was given to the animals on day 8 at the base of the tail (100 ⁇ g).
  • the final ell concentration in the booster was 0.4 ⁇ g/mL.
  • Animals with an arthritic score of > 1 were grouped and dosing with test compound (30 mg/kg bid) or methotrexate (0.5 mg/kg) started between about day 12 to day 14, with daily dosing of their respective compounds continuing for 10 days.
  • Edema Paw volumes are measured by Plethysmometry for the animals before induction of CIA (Basal readings) and on Day 1, 3, 6 and 9 of dosing period. Both hind paw volumes were measured and edema was calculated by subtracting from the basal mean.
  • Example 344 In vivo assay - Acute study
  • Type III hypersensitivity reactions are immune complex-mediated, and involve the deposition of antigen/antibody complexes mainly in the vascular walls, serosa (pleura, pericardium, synovium), and glomeruli. This involves formation of antigen antibody complexes after the intradermal injection of an antibody. If the animal was previously injected with antigen and dye (has circulating antigen), an Arthus reaction occurs. This manifests as local vasculitis due to deposition of immune complexes in dermal blood vessels. Activation of complement and recruitment of PMNs ensue, resulting in an inflammatory response and extravasation of dye to the skin. Compounds which can inhibit this complex process can have therapeutic implications in wide range of inflammatory and auto-immune disorders. See, Pine,
  • mice Female c57BL/6 mice were given an antigen injection in which the antigen was 0.1% Ovalbumin (OVA) in PBS containing 1% Evans blue (EB) at the concentration of 10 mL/kg intravenously under Isoflurane anesthesia [2.5 mg/mouse with a body weight of 25 g].
  • Ovalbumin Ovalbumin
  • EB Evans blue
  • PBS phosphate buffered saline
  • mice were euthanized by cervical dislocation 4 hours after antigen (Ovalbumin) challenge. Skin tissue was assessed for edema by tracing the edema area on to a transparent plastic sheet. Punch biopsies of the injection sites were collected.
  • Edema area was measured manually by scale. Two diameters were taken and averaged for each animal.
  • This example illustrates that the compounds described herein may be utilized in treating inflammation.
  • combination treatments for treating cancers involving targeted oncologic therapies e.g., inhibition of specific biochemical pathways
  • immunotherapeutic agents would be expected to decrease treatment efficacy and/or increase adverse side effects, because targeted therapies with immuno stimulatory potential can also display immunosuppressive activities.
  • the activity of targeted therapies may therefore cancel out, and even inhibit, therapeutic mechanisms of imunomodulators.
  • the combination of dual inhibitors of Syk/JAK family kinases according to the disclosure and immunotherapeutic agents for the treatment of cancers would be expected to result in a decrease in efficacy.
  • such combination treatments can result in increases in toxicity and adverse side effects, for example by increasing cytokine production and other inflammatory reactions related to the non-tumor suppressive activities of the immunotherapeutic agents.
  • compositions comprising a compound of Formula (I), as described herein, and one or more immunotherapeutic agents can be used to treat cancers, where the combination has a reduced adverse side effect profile and where the activity of the immunotherapeutic agent is not suppressed.
  • the anticancer or antitumor activity of pharmaceutical combinations of the present disclosure may be substantially the same as, or may be increased, as compared to administering a compound of Formula (I) alone.
  • the pharmaceutical combinations of the present disclosure can reduce negative side effects in patients, for example by inhibiting or suppressing chemokine and/or cytokine levels.
  • a pharmaceutical combination comprising the combination comprising a compound of Formula (I), as described herein, and at least one immunotherapeutic agent.
  • the at least one immunotherapeutic agent can comprise one or more immunomodulators.
  • the immunomodulators can target one or more components of the immune system, including (without limitation) PD-1, PD-L1, CTLA-4, 4-1BB, OX40, LAG3, GITR, TIM3, VISTA, KIR, ICOS, BTLA, CD244, CD80, CD86, PD-L2, IDO-1, IDO-2, and B7-H3, and can produce a therapeutic effect.
  • the one or more immunomodulators can comprise (without limitation) inhibitors of immune system components, activators of immune system components, and/or immune checkpoint inhibitors, and can comprise biologic or small molecule therapeutics.
  • TCR agents can include (without limitation) chimeric antigen receptor (CAR) T cells, and TCR agonist or antagonist peptides.
  • CAR T cells can comprise (without limitation) CTL019 and JCAR015 etc.
  • the vaccines can include (without limitation) sipuleucel-T (Provenge ® ) and Talimogene laherparepvec (Imlygic ® ), aslo known as T-Vec.
  • checkpoint inhibitors can include (without limitation) co- inhibitory molecules such as cytotoxic T-lymphocyte-as so dated protein 4 (CTLA-4), PD-1, lymphocyte-activation gene 3, and T-cell immunoglobulin mucin-3, and co-stimulatory molecules such as glucocorticoid-induced tumor necrosis factor receptor and OX40 (CD134, TNFRSF4, tumor necrosis factor receptor superfamily member 4).
  • co- inhibitory molecules such as cytotoxic T-lymphocyte-as so dated protein 4 (CTLA-4), PD-1, lymphocyte-activation gene 3, and T-cell immunoglobulin mucin-3
  • co-stimulatory molecules such as glucocorticoid-induced tumor necrosis factor receptor and OX40 (CD134, TNFRSF4, tumor necrosis factor receptor superfamily member 4).
  • the immune checkpoint inhibitors can include (without limitation), anti-PD-1 antibody, anti-PD- Ll antibody, anti-CTLA-4 antibody, anti-4-lBB antibody, anti-OX40 antibody, anti-LAG3 antibody, anti-GITR antibody, anti-TIM3 antibody, anti- VISTA antibody, anti-KIR antibody, anti-ICOS antibody, anti-BTLA antibody, anti-CD244 antibody, anti-CD80 antibody, anti- CD86 antibody, anti-PD-L2 antibody, anti-IDO-1 antibody, anti-IDO-2 antibody, anti-B7-H3 antibody, and small molecule inhibitors of any of these antibody targets, for example an IDO inhibitor drug.
  • the immune checkpoint inhibitor is anti-PD-1 antibody.
  • the immune checkpoint inhibitor is small molecule IDO-1 inhibitor.
  • the pharmaceutical combination can further comprise at least one pharmaceutically acceptable carrier.
  • the disclosure also provides methods of treating cancer, the methods comprising administering to a patient in need thereof a multi-part pharmaceutical combination comprising a compound of Formula (I), as described herein, and at least one immunotherapeutic agent, as described herein.
  • the compounds of Formula (I) can be administered intravenously or orally, or by any other suitable manner.
  • the immunotherapeutic agent can be administered intravenously, subcutaneously, intramuscularly, or orally, or by any other suitable manner.
  • administration of the compound is intravenous and the administration of the at least one immunotherapeutic agent is intravenous, subcutaneous or intra-muscular.
  • administration of the compound is oral and the administration of the at least one immunotherapeutic agent is oral.
  • administration of the compound is intravenous and the administration of the at least one immunotherapeutic agent is oral. In yet another embodiment, administration of the compound is oral and the administration of the at least one immunotherapeutic agent is intravenous, subcutaneous or intra-muscular.
  • the compound of Formula (I) and the immunotherapeutic agent can be administered by any suitable dosing regimen.
  • the compound of Formula (I) is administered once or twice daily.
  • the compound and the at least one immunotherapeutic agent can be administered simultaneously or sequentially.
  • the compound and the at least one immunotherapeutic agent are administered sequentially.
  • the at least one immunotherapeutic agent is administered intermittently every four to thirty days.
  • the compound of Formula (I) can be administered to a patient at a dosage of about 5 mg/kg to about 70 mg/kg, preferably from about 10 mg/kg to about 60 mg kg. In an embodiment, the compound of Formula (I) is administered at a dosage of about 10 mg/kg, about 30 mg/kg, or about 60 mg/kg. In other embodiments, the compound of Formula (I) can be administered to a patient at a dosage of about 10 mg, 20 mg, 30 mg, 40 mg, 40 m g, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg or 120 mg once or twice per day.
  • cancers can be treated using the pharmaceutical combinations provided here, including (without limitation) the following cancers: prostate, head, neck, eye, mouth, throat, esophagus, bronchus, larynx, pharynx, chest, bone, lung, colon, rectum, stomach, bladder, uterus, cervix, breast, ovaries, vagina, testicles, skin, thyroid, blood, lymph nodes, kidney, liver, intestines, pancreas, brain, central nervous system, adrenal gland, skin or a leukemia and/or lymphoma.
  • methods of the present disclosure include ⁇ identifying an indication or patient population for administering the pharmaceutical combinations of the present disclosure.
  • the disclosure provides methods of inhibiting tumor growth or metastasis in a subject, the methods comprising administering to the subject a multi-part pharmaceutical combination comprising a compound of Formula (I), as described herein, and at least one immunotherapeutic agent, as described herein, wherein the administration of the pharmaceutical combination inhibits tumor growth or metastasis.
  • Inhibition of tumor growth or metastasis means reducing the number of cancer cells or causing the amount of cancer cells to remain substantially the same, reducing tumor size or causing the tumor size to remain substantially the same, inhibiting metastasis (including inhibition of tumor cell migration and/or invasion), inhibiting tumor growth and/or ameliorating one or more of the symptoms of the cancer.
  • TGI tumor growth or metastasis
  • a therapeutically effective amount of a pharmaceutical combination when used for the treatment of cancer is an amount which may inhibit tumor growth or metastasis.
  • administration of the pharmaceutical combinations described herein can achieve TGI in an amount of 0% to 100%, preferably an amount of above about 50%, for example about 50% to 90%, or about 60% to 80%.
  • the disclosure provides dosing regimens comprising administering to a patient in need a multi-part pharmaceutical combination comprising a compound of Formula (I), as described herein, and at least one immunotherapeutic agent, as described herein. Administration of the compound of Formula (I) and the immunotherapeutic agent can be performed as described herein.
  • kits comprising at least one first dosage form comprising a compound of Formula (I), as described herein, and at least one second dosage form comprising at least one immunotherapeutic agent, as described herein, and administering to a patient in need the compound of Formula (I) and the immunotherapeutic agent.
  • Administration of the compound of Formula (I) and the immunotherapeutic agent can be performed as described herein.
  • the disclosure provides uses of a medicament in treating cancer, wherein the medicament is administered by a dosing regimen comprising administering to a patient in need a compound of Formula (I), as described herein, and at least one immunotherapeutic agent, as described herein.
  • Administration and the dosing regimen of the compound of Formula (I) and the immunotherapeutic agent can be performed as described herein.
  • Example 345 demonstrates that the pharmaceutical combinations described herein can suppress cytokines and can prevent cytokine release syndrome. Suppression of cytokines can result in a decrease in adverse side effects and toxicity related to cytokine production in patients.
  • EXAMPLE 345 Evaluation of inflammation biomarkers in cancer patients treated with COMPOUND A
  • Example 189 2-(l-(4-((4-(4-hydroxypiperidin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidm-4-yl)acetonitrile, was tested as described below.
  • the compound of Example 189 is sometimes referred to herein as "COMPOUND A.”
  • Serum samples were collected from patients before the treatment with COMPOUND A on Day 1 and after treatment with COMPOUND A on day 15. The samples were then analyzed for the levels of a panel of 45 inflammation biomarkers. The percent change in the serum levels of each biomarker was calculated by comparing the protein levels before and after treatment. There was a significant inhibition of several inflammation biomarkers on Day 15 at all doses evaluated. A few key inflammation biomarkers that showed a marked inhibition with COMPOUND A treatment were C-Reactive Protein (CRP), p2-microglobulin
  • C-reactive protein is an acute-phase protein, the levels which are elevated in response to inflammation. The levels of this protein are also elevated in patients with various types of cancers and this is considered as a potential predictor of cancer risk and/or survival.
  • p2-microglobulin is a component of MHC class I molecules and is encoded by the gene, B2M. ⁇ 2 -microglobulin is found to be elevated in lymphoma and multiple myeloma.
  • IL-18 is a proinflammatory cytokine involved in several inflammatory disorders.
  • ⁇ - ⁇ also known as CCL4
  • CCR5 chemokine receptor 5
  • the levels of all these inflammation biomarkers have been significantly inhibited in patient serum after treatement with COMPOUND A. Because of its ability to suppress some of the important cytokines such as IL-18, COMPOUND A also has a potential to prevent the cytokine release syndrome when combined with immunotherapies (see also Fig. 3).
  • COMPOUND A was also evaluated for its ability to inhibit signaling pathways (JAK/STAT) in peripheral blood monocular cells stimulated with varuious cytokines such as IL-2, IL-4, IL-6, IL-12, IL-23 etc. In these studies, COMPOUND A potently inhibited the
  • HPC Hydroxypropylcellulose LOQ: Limit of quantification
  • TGI Tumor growth inhibition
  • IDO-1 Indoleamine 2,3-dioxygenase
  • PD-1 Programmed cell Death- 1
  • the inventors have unexpectedly discovered that administration of the pharmaceutical combinations comprising a compound of Formula (I) and an immunotherapeutic agent, as described herein, can produce substantially the same, or an increase in, anti-tumor efficacy compared to administering the compound without an immunotherapeutic agent.
  • Example 346 demonstrates the anti-tumor efficacy of COMPOUND A alone and in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) in a 4T1 - Balb/c mouse syngeneic allograft tumor model.
  • the mice bearing 4T1 allografts in the untreated control group showed a physiological body weight gain of 19 %.
  • COMPOUND A, anti PD-1 antibody, epacadostat, or their combinations were tolerated well with a minimal, yet acceptable, loss of body weight (-2 %).
  • Plasma COMPOUND A concentrations were found to be in the range of 8 to 1813 ng ml; however in tumor samples, it ranged from 72 to 2495 ng/g for mice administered with 10 to 60 mg/kg
  • COMPOUND A There was an 8-fold increase in exposure in tumor vs. plasma samples for single agent COMPOUND A at 10 mg/kg; and a 3-fold increase was obtained when COMPOUND A at 10 mg/kg combined with either anti PD-1 antibody or epacadostat. A lower COMPOUND A concentration in tumor vs. plasma was found at 30 - 60 mg/kg COMPOUND A when combined with anti PD1 antibody. Similarly, COMPOUND A-60 mg kg treatment combined with epacadostat resulted in lower concentration of COMPOUND A in tumor than plasma. No treatment related morbidity or mortality was seen during the study.
  • Epacadostat an inhibitor of IDO-1 has shown efficacy in inhibiting tumor growth in multiple immunocompetant tumor models.
  • PD-1 Programmed cell Death- 1 expression by tumor- infiltrating lymphocytes is shown to be important in anti-inflammatory and tissue injury protection that further mediates tumor-induced immune suppression/immune escape. Since tumor growth inhibitory effect of both anti-PDl antibody and epacadostat have already been established in this model, a single dose of these agents was used in combination with different doses of COMPOUND A to demonstrate the potential benefit to tumor growth inhibition demonstrated by COMPOUND A.
  • mice obtained from Envigo, Netherlands.
  • the animals were housed in individually ventilated cages (maximum of 5 animals/cage) with 12 hour dark, 12 hour light conditions.
  • the animals were fed food and water ad libitum.
  • Temperature and relative humidity were maintained at 20 ⁇ 2°C and 65%, respectively.
  • COMPOUND A (Lot # C15K081007A) was formulated in the vehicle containing 20 mg ml hydroxypropylcellulose (HPC, Klucel LF). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout dosing.
  • Epacadostat (Cat # PBLJ9203; CAS # 1204669-58-8, purchased from PharmaBlock): was formulated as a homogenous suspension in the vehicle containing 0.5 % methyl cellulose (MC). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout the dosing.
  • Anti-mouse PD1 antibody 100 ⁇ g in 200 ⁇ antibody was administered (i.p.) per mouse.
  • Antibody was diluted with 1 X sterile PBS (calcium and magnesium-free).
  • COMPOUND A and epacadostat were administered orally by gavage at a dose volume of 10 mL/kg.
  • Mouse breast carcinoma 4T1 cells were sourced from American Type Culture Collection (ATCC), USA. Cells were grown in RPMI-1640 medium (Sigma, Cat # R6504) supplemented with 10% FBS (Invitrogen, Cat # 10438-026), and 1% penicillin streptomycin (Thermo Fisher Scientific, Cat # 15140-122). To establish allografts, 1 million 4T1 cells were suspended in 50 ⁇ of serum-free medium and mixed at 1:1 ratio with matrigel before implanting subcutaneously into the right flank of mice.
  • ATCC American Type Culture Collection
  • Tumor dimensions were measured for all animals on the first treatment day (Day 1) and then two times per week, including the termination day of the study. Tumor volumes were calculated using the formula: tumor length x (tumor width) 2 / 2. Tumor growth inhibition was calculated after normalizing the tumor volume on a given day to that on day 1.
  • Treatment was initiated 9 days after subcutaneous injection of tumor cells when the average tumor volume reached approximately 90 mm 3 .
  • the animals were randomized based on tumor volumes into 12 groups of eight animals each.
  • the dosing schedule of COMPOUND A and epacadostat were twice daily for 15 days by p.o. and anti-PDl antibody was administered once in 4 days (total 5 doses) by i.p.
  • the treatment period was 15 days after which the overall efficacy and tolerability was evaluated based on tumor volume and body weight changes observed during the treatment period.
  • Tumor volumes were analyzed using two-way ANOVA with Bonferonni' s multiple comparisons test for comparison of treatment versus control group.
  • %TGI percent tumor growth inhibition
  • the anti-tumor effect of COMPOUND A was evaluated in tumor allograft bearing Balb/c mice.
  • the anti-tumor efficacy of single agent anti-PDl antibody, epacadostat and COMPOUND A were compared to vehicle control, whereas combination groups, COMPOUND A + anti-PDl antibody, and COMPOUND A + epadacostat were compared with single agent groups and vehicle control.
  • mice treated with COMPOUND A (10, 30, 60 mg kg), anti-PDl antibody with untreated vehicle control indicated a statistically significant decrease ( ⁇ .0001) on day 15 of treatment.
  • the average tumor volume for mice in vehicle control group was 976 ⁇ 86 mm 3 and that for 10, 30, 60 mg/kg COMPOUND A, anti-PDl antibody 0.1 mg/mouse, and epacadostat 30 mg kg treated groups were found to be 549 ⁇ 51 (48 % TGI), 447 ⁇ 38 (60 % TGI), 282 ⁇ 66 (79 % TGI), 530 ⁇ 58 (51 % TGI), and 567 ⁇ 48 mm 3 (46 % TGI), respectively ( Figure 4 and 5; Table 18).
  • mice bearing 4T1 allografts in the untreated control group showed a physiological body weight gain of 19 % (Table 20).
  • Mice treated with COMPOUND A - 10, 30 and 60 mg/kg equally gained 17 % body weight on day 15, not significantly different from controls (p>0.05, Table 19; Figures 6-9).
  • Anti-PDl antibody combinations with COMPOUND A - 10, 30, and 60 mg kg had a similar gain of 18 %, 17 %, and 16 % body weights, respectively ( Figures 6 and 8; Tables 19 and 20).
  • Table 19 Average body weight in mice after different days of treatment initiation
  • COMPOUND A anti PD-1 antibody, and epacadostat were tolerated well with a minimal, yet acceptable, loss of body weight. No treatment related morbidity or mortality was seen during the study. Plasma and tumor concentrations of COMPOUND A
  • Plasma and tumor samples from six mice/group were collected for analyzing COMPOUND A by LC-MS.
  • Plasma COMPOUND A concentrations were found to be in the range of 8 to 1813 ng/ml (Table 21); however in tumor samples, COMPOUND A concentrations ranged from 72 to 2495 ng/g for mice administered with 10 to 60 mg/kg COMPOUND A (Table 22).
  • concentration of COMPOUND A in plasma ranged from 41 to 1889 ng/ml and in tumor it ranged from 126 to 1624 ng/g (Tables 21, 22).
  • concentration of COMPOUND A in plasma ranged from 48 to 4114 ng/ml and in tumor it ranged from 172 to 2517 ng/g in mice administered with 10 to 60 mg/kg COMPOUND A (Tables 21, 22).
  • COMPOUND A-10 mg/kg There was an 8-folds increase in exposure in tumor than plasma samples for single agent COMPOUND A-10 mg/kg; and a 3-fold increase was obtained when COMPOUND A-10 mg/kg combined with either anti PD-1 antibody or epacadostat.
  • a lower COMPOUND A concentration in tumor than plasma was found at 30 - 60 mg/kg COMPOUND A when combined with anti PD 1 antibody.
  • COMPOUND A-60 mg kg treatment combined with epacadostat resulted in lower concentration of COMPOUND A in tumor than plasma.
  • COMPOUND A at 10, 30 and 60 mg/kg doses resulted in a significant and dose- dependent tumor growth inhibition of 48 %, 60 %, and 79 %.
  • a tumor growth inhibition of 51 % and 46 % was obtained for anti-PDl antibody (0.1 mg/mouse) and epacadostat (30 mg kg), respectively. All treatments were tolerated well and no treatment related morbidity or mortality was seen during the study.
  • Combination of COMPOUND A 10 mg kg with anti- PDl antibody showed substantially the same TGI as that of single agent COMPOUND A.
  • There was an enhancement in the tumor growth inhibition when epacadostat combined with COMPOUND A although it was not statistically significant (p>0.05).
  • COMPOUND A- 10 mg/kg A 3 -folds increase was also obtained when COMPOUND A- 10 mg/kg combined with either anti PD-1 antibody or epacadostat.
  • Example 347 demonstrates the anti-tumor efficacy of COMPOUND A alone and in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) in B16-F10 - C57/BL6 syngeneic allograft tumor model.
  • the objective of this study was to test efficacy of COMPOUND A in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) using B16-F10 - C57/BL6 syngeneic allograft tumor model.
  • the average tumor volume for mice in vehicle control group was 2529 ⁇ 110 mm 3 and that for 10, 20, 60 mg/kg COMPOUND A, anti-PDl antibody 0.1 mg/mouse, and epacadostat 30 mg/kg treated groups were found to be 1323 ⁇ 121 (49 % TGI), 1041 ⁇ 98 (60 % TGI), 923 ⁇ 58 (65 % TGI), 1117 ⁇ 82 (57 % TGI), and 1394 ⁇ 90 mm 3 (46 % TGI), respectively.
  • Co-administration of either anti-PDl antibody or epacadostat with COMPOUND A showed substantially the same tumor growth reduction in B16-F10 melanoma cancer syngeneic model.
  • COMPOUND A (10 - 60 mg/kg), anti-PDl antibody and epacadostat were tolerated well without loss of body weight.
  • Combinations of COMPOUND A with anti-PDl antibody or epacadostat were tolerated well without loss of body weight.
  • a widely used B16-F10 tumor was employed in C57/BL6 mouse model to assess the immunomodulatory effect of known immunotherapeutic agents, anti-PDl antibody and IDO- 1 inhibitor (epacadostat) in combination with COMPOUND A. Since tumor growth inhibitory effect of both anti-PDl antibody and epacadostat have already been established in this model, a dose that has resulted in 40-50% inhibition was selected as a single agent and combined with different doses of COMPOUND A to understand any potential benefit to tumor growth inhibition demonstrated by COMPOUND A.
  • the animals were housed in individually ventilated cages (maximum of 5 animals/cage) with 12 hour dark, 12 hour light conditions. The animals were fed food and water ad libitum. Temperature and relative humidity were maintained at 20 ⁇ 2°C and 65%, respectively.
  • COMPOUND A (Lot # C15K081007A) was formulated in the vehicle containing 20 mg/ml hydroxypropylcellulose (HPC, Klucel LF). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout dosing.
  • Epacadostat (Cat # PBLJ9203; CAS # 1204669-58-8, purchased from PharmaBlock): was formulated as a homogenous suspension in the vehicle containing 0.5 % methyl cellulose (MC). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout the dosing.
  • Anti-mouse PD1 antibody 100 ⁇ g in 200 ⁇ antibody was administered (i.p.) per mouse. Antibody was diluted with 1 X sterile PBS (calcium and magnesium-free). COMPOUND A and epacadostat were administered orally by gavage at a dose volume of 10 mL/kg.
  • Cell line and tumor model 100 ⁇ g in 200 ⁇ antibody was administered (i.p.) per mouse. Antibody was diluted with 1 X sterile PBS (calcium and magnesium-free). COMPOUND A and epacadostat were administered orally by gavage at a dose volume of 10 mL/kg.
  • Cell line and tumor model 100 ⁇ g in 200 ⁇ antibody was administered (i.p.) per mouse. Antibody was diluted with 1 X sterile PBS (calcium and magnesium-free). COMPOUND A and epacadostat were administered orally by gavage at a dose volume of 10 mL/kg.
  • Cell line and tumor model 100 ⁇ g in
  • Mouse melanoma B16-F10 cells were sourced from American Type Culture Collection (ATCC), USA. Cells were grown in Dulbecco's Modified Eagel Medium (DMEM) (Gibco, Cat # 31600-034) supplemented with 10% FBS (Invitrogen, Cat # 10438- 026), and 1% penicillin streptomycin (Thermo Fisher Scientific, Cat # 15140-122). To establish allografts, 0.1 million B16-F10 mouse melanoma cells were suspended in 50 ⁇ of serum-free medium and mixed at 1 : 1 ratio with matrigel before implanting subcutaneously into the right flank of mice.
  • DMEM Dulbecco's Modified Eagel Medium
  • FBS Invitrogen, Cat # 10438- 026
  • penicillin streptomycin Thermo Fisher Scientific, Cat # 15140-122
  • Tumor dimensions were measured for all animals on the first treatment day (Day 1) and then two times per week, including the termination day of the study. Tumor volumes were calculated using the formula: tumor length x (tumor width) 2 / 2. Tumor growth inhibition was calculated after normalizing the tumor volume on a given day to that on day 1.
  • Drug Treatments Treatment was initiated 6 days after subcutaneous injection of tumor cells when the average tumor volume had reached approximately 50 mm 3 .
  • the animals were randomized based on tumor volumes into 12 groups of ten animals each.
  • the dosing schedule of COMPOUND A and epacadostat were twice daily for 15 days by p.o. and anti-PDl antibody was administered once in 4 days (total 5 doses) by i.p.
  • the treatment period was 15 days after which the overall efficacy and tolerability was evaluated based on tumor volume and body weight changes observed during the treatment period.
  • Tumor volumes were analyzed using two-way ANOVA with Bonferonni' s multiple comparisons test for comparison of treatment versus control group.
  • %TGI percent tumor growth inhibition
  • % TGI [1 -(Treatment TVpinai - Treatment TV ln i t i_i) / (Control TVpinai - Control
  • the anti-tumor effect of COMPOUND A was evaluated in tumor allograft bearing C57/BL6 mice.
  • the anti-tumor efficacy of single agents, anti-PDl antibody, epacadostat and COMPOUND A were compared to vehicle control, whereas combination groups, COMPOUND A + anti-PDl antibody, and COMPOUND A + epadacostat were compared with single agent groups and vehicle control.
  • mice in vehicle control group was 2529 ⁇ 110 mm 3 and that for 10, 20, 60 mg/kg COMPOUND A, anti-PDl antibody 0.1 mg/mouse, and epacadostat 30 mg/kg treated groups were found to be, respectively 1323 ⁇ 121 (49 % TGI), 1041 ⁇ 98 (60 % TGI), 923 ⁇ 58 (65 % TGI), 1117 ⁇ 82 (57 % TGI), and 1394 ⁇ 90 mm 3 (46 % TGI) ( Figure 17 and 18; Table 26).
  • Co-administration of anti-PDl antibody showed substantially the same tumor growth inhibition obtained for COMPOUND A in B16-F10 tumor syngeneic model (53 %, 62% and 74% TGI for combinations of anti PD-1 antibody and COMPOUND A vs. 49 %, 60% and 65% TGI for single agent COMPOUND A at 10, 20 and 60 mg kg, respectively) ( Figure 17 and 18; Table 26).
  • there was substantially the same benefit of coadministration of epacadostat with COMPOUND A 52 %, 64% and 70% TGI for combinations of epacadostat and COMPOUND A vs. 49 %, 60% and 65% TGI for single agent COMPOUND A at 10, 30 and 60 mg kg, respectively; Figure 17 and 18; Table 26).
  • Table 26 Average tumor volume (mm ) in mice after different days of treatment initiation
  • mice bearing B16-F10 allografts in the untreated control group maintained body weight with negligible change of -0.6 % on day 15 (Table 28).
  • COMPOUND A - 10, 30, and 60 mg/kg, anti-PDl antibody and epacadostat were tolerated well without loss of body weight ( Figures 19 and 21 ; Tables 27 and 28).
  • the highest increase of 17.3 % in body weight was observed in mice treated with mouse anti-PDl antibody (group 5).
  • Table 27 Average body weight of mice after different days of treatment initiation
  • Table 29 Animal mortality was random across all experimental groups and the mortality did not appear to be due to treatment, but could be because of spontaneous metastatic nature of B16F10 cells in the syngeneic C57-BL6 mouse model. Table 29: Animal mortality after different days of treatment initiation
  • Plasma COMPOUND A concentrations were found to be in the range of 32.8 to 935 ng/ml (Table 30), however in tumor samples, COMPOUND A concentrations increased from 883 to 7568 ng/g for mice administered with 10 to 60 mg/kg COMPOUND A (Table 31).
  • concentration of COMPOUND A in plasma ranged from 25 to 1023 ng/ml (Table 30) and in tumor it ranged from 1024 to 9439 ng/g (Table 31).
  • CONCLUSION COMPOUND A at 10, 30 and 60 mg/kg doses resulted in a significant and dose- dependent tumor growth inhibition of 49, 60 and 65%.
  • a tumor growth inhibition of 57 % and 46 % was obtained for anti-PDl antibody (0.1 mg/mouse) and epacadostat (30 mg/kg), respectively.
  • Combination of either anti-PDl antibody or epacadostat showed substantially the same TGI than that obtained from single agent COMPOUND A.
  • Example 348 demonstrates the anti-tumor efficacy of COMPOUND A alone and in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) in CT26 - Balb/c syngeneic allograft tumor model
  • the objective of this study was to demonstrate the anti-tumor acitivity of COMPOUND A in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) using CT26 - Balb/c syngeneic allograft tumor model.
  • the mice bearing CT26 allografts in the untreated control group showed a physiological body weight gain of 18 %.
  • COMPOUND A - 30 and 60 mg/kg treatment has significantly decreased the physiological gain body weight on day 15 compared to controls (p ⁇ 0.05), however there was no loss of body weight compared to their day 1 initial weights (p ⁇ 0.05).
  • Combination of COMPOUND A with anti-PDl antibody or epacadostat had a significant but acceptable loss of body weight compared to controls (p ⁇ 0.0001).
  • Anti PD-1 antibody or epacadostat co-administration decreased the COMPOUND A concentrations both in plasma and tumor. There was a 2-folds increase in exposure in tumor than plasma samples for single agent COMPOUND A-10 mg/kg, a similar increase was also obtained when combined with either anti PD-1 antibody or epacadostat.COMPOUND A, anti PD-1 antibody, and epacadostat were tolerated with acceptable loss of body weight. An increase in the loss of body weight, still within an acceptable limit of 10 % was observed when combined with anti PD-1 antibody or epacadostat.
  • CT26-Balb/c model was used to assess the immunomodulatory effect of known immunomodulatory agents, anti-PDl antibody and IDO-1 inhibitor (epacadostat) in combination with COMPOUND A. Since tumor growth inhibitory effect of both anti-PDl antibody and epacadostat have already been established in this model, a single dose of these agents was used in combination with different doses of COMPOUND A to understand any potential benefit to tumor growth inhibition demonstrated by COMPOUND A.
  • the animals were housed in individually ventilated cages (maximum of 5 animals/cage) with 12 hour dark, 12 hour light conditions. The animals were fed food and water ad libitum. Temperature and relative humidity were maintained at 20 ⁇ 2°C and 65%, respectively.
  • COMPOUND A (Lot # C15K081007A) was formulated in the vehicle containing 20 mg ml hydroxypropylcellulose (HPC, Klucel LF). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout dosing.
  • Epacadostat (Cat # PBLJ9203; CAS # 1204669-58-8, purchased from PharmaBlock): was formulated as a homogenous suspension in the vehicle containing 0.5 % methyl cellulose (MC). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout the dosing.
  • Anti-mouse PD1 antibody 100 ⁇ g in 200 ⁇ antibody was administered (i.p.) per mouse.
  • Antibody was diluted with 1 X sterile PBS (calcium and magnesium-free).
  • COMPOUND A and epacadostat were administered orally by gavage at a dose volume of 10 mL/kg.
  • Mouse colon carcinoma CT26 cells were sourced from American Type Culture Collection (ATCC), USA. Cells were grown in RPMI-1640 medium (Sigma, Cat # R6504) supplemented with 10% FBS (Invitrogen, Cat # 10438-026), and 1% penicillin streptomycin (Thermo Fisher Scientific, Cat # 15140-122). To establish allografts, 1 million CT26 cells were suspended in 50 ⁇ of serum-free medium and mixed at 1:1 ratio with matrigel before implanting subcutaneously into the right flank of mice.
  • ATCC American Type Culture Collection
  • Tumor dimensions were measured for all animals on the first treatment day (Day 1) and then two times per week, including the termination day of the study. Tumor volumes were calculated using the formula: tumor length x (tumor width) 1 12. Tumor growth inhibition was calculated after normalizing the tumor volume on a given day to that on day 1.
  • Treatment was initiated 9 days after subcutaneous injection of tumor cells when the average tumor volume reached approximately 90 mm 3 .
  • the animals were randomized based on tumor volumes into 12 groups of eight animals each.
  • the dosing schedule of COMPOUND A and epacadostat were twice daily for 15 days by p.o. and anti-PDl antibody was administered once in 4 days (total 5 doses) by i.p.
  • the treatment period was 15 days after which the overall efficacy and tolerability was evaluated based on tumor volume and body weight changes observed during the treatment period.
  • Tumor volumes were analyzed using two-way ANOVA with Bonferonni' s multiple comparisons test for comparison of treatment versus control group.
  • %TGI percent tumor growth inhibition
  • % TGI [1 -(Treatment TVFinai - Treatment TV ln iti_i) / (Control TVFinai - Control
  • the average tumor volume for mice in vehicle control group was 2835 ⁇ 500 mm 3 and that for 10, 30, 60 mg/kg COMPOUND A, anti-PDl antibody 0.1 mg/mouse, and epacadostat 30 mg/kg treated groups were found to be 1929 ⁇ 407 (33 % TGI), 965 ⁇ 135 (68 % TGI), 771 ⁇ 90 (76 % TGI), 1264 ⁇ 143 (57 % TGI), and 1901 ⁇ 396 mm 3 (34 % TGI), respectively ( Figure 30 and 31 ; Table 35).
  • Co-administration of COMPOUND A- 10 mg/kg and anti-PDl antibody in CT26 tumor syngeneic model significantly increased tumor growth inhibition (p ⁇ 0.0 ⁇ , 63 % TGI in combination vs.
  • Table 35 Average tumor volume (mm 3 ) in mice after different days of treatment initiation
  • Table 36 Average body weight in mice after different days of treatment initiation
  • COMPOUND A anti PD-1 antibody, and epacadostat were tolerated with acceptable loss of body weight. An increase in the loss of body weight, still within an acceptable limit of 10 % was observed when combined with anti PD-1 antibody or epacadostat.
  • Plasma and tumor samples from six mice/group were collected for analyzing COMPOUND A by LC-MS.
  • Plasma COMPOUND A concentrations were found to be in the range of 208 to 3071 ng/ml (Table 38), however in tumor samples, COMPOUND A concentrations ranged from 382 to 1286 ng/g for mice administered with 10 to 60 mg/kg COMPOUND A (Table 39).
  • concentration of COMPOUND A in plasma ranged from 80 to 711 ng/ml and in tumor it ranged from 147 to 805 ng/g (Tables 38, 39).
  • concentration of COMPOUND A in plasma ranged from 51 to 1774 ng/ml and in tumor it ranged from 151 to 937 ng/g in mice administered with 10 to 60 mg/kg COMPOUND A (Tables 38, 39).
  • COMPOUND A- 10 mg/kg There was a 2-fold increase in exposure in tumor than plasma samples for single agent COMPOUND A- 10 mg/kg, a similar increase was also obtained when combined with either anti PD-1 antibody or epacadostat.
  • COMPOUND A at 10, 30 and 60 mg/kg doses resulted in a significant and dose- dependent tumor growth inhibition of 33, 68 and 79%.
  • a tumor growth inhibition of 57 % and 34 % was obtained for anti-PDl antibody (0.1 mg/mouse) and epacadostat (30 mg/kg), respectively.
  • Combination of COMPOUND A 10 mg/kg with anti-PDl antibody significantly enhanced TGI of single agent COMPOUND A; however, higher concentrations of COMPOUND A did not produce this response.
  • TGI was substantially the same for COMPOUND A and epacadostat combination compared to single agent COMPOUND A.
  • Example 349 demonstrates the clinical activity, safety and tolerabilityof
  • COMPOUND A a dual SYK/JAK inhibitor, in patients with Non-Hodgkins lymphoma (NHL) and solid tumors, including dose-limiting toxicities (DLTs) and determines the maximum tolderated dose (MTD).
  • NDL Non-Hodgkins lymphoma
  • DLTs dose-limiting toxicities
  • MTD maximum tolderated dose
  • COMPOUND A has low nM IC50s against SYK and JAK, decreases proliferation in ibrutinib-resistant cell lines, and suppresses tumor growth in rodent xenograft models of DLBCL.
  • Example 349 demonstrates that COMPOUND A, at dose levels associated with clinical and biomarker activity, is safe and tolerable for development as single agent and in combination with other treatments.
  • COMPOUND A The safety profile of COMPOUND A differentiates favorably from approved JAK inhibitors ruxolitinib and tofacitinib.
  • checkpoint inhibitors PD-1 / PD-1 ligand directed
  • Checkpoint inhibitors can include co-inhibitory molecules such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), PD-1, lymphocyte-activation gene 3, and T-cell immunoglobulin mucin-3, and co-stimulatory molecules such as:
  • glucocorticoid-induced tumor necrosis factor receptor and OX40 CD134, TNFRSF4, tumor necrosis factor receptor superfamily member 4
  • Other immune modulators that can be combined with a compound of Formula (I) include indoleamine (2,3)-dioxygenase (IDO) inhibitors, vaccines and agents that target T-cell receptors (TCR agents).
  • TCR agents can include (without limitation) chimeric antigen receptor (CAR) T cells, and TCR agonist or antagonist peptides.
  • the vaccines can include (without limitation) sipuleucel-T (Provenge®) and Talimogene laherparepvec (Imlygic®), aslo known as T-Vec.
  • COMPOUND A can be thus be clinically developed in combination with checkpoint inhibitors, IDO inhibitors, vaccines (for example, sipuleucel-T (Provenge®) and/or Talimogene laherparepvec
  • T-Vec TCR agents
  • CAR T cells and/or TCR agonist or antagonist peptides TCR agents
  • other immune-modulators to improve outcome of treatment.
  • IDO inhibitors vaccines
  • vaccines for example, sipuleucel-T (Provenge®) and/or Talimogene laherparepvec (Imlygic®), aslo known as T-Vec
  • TCR agents for example CAR T cells and/or TCR agonist or antagonist peptides
  • COMPOUND A could also improve immune mediated toxicities associated with administration of of such treatments.
  • Example 349 demonstrates the safety, tolerability, and preliminary efficacy of COMPOUND A in subjects with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL).
  • Example 349 also demonstrates pharmacokinetic (PK) profile of COMPOUND A after single and multiple doses and the effects of COMPOUND A on Phospho-STAT3, Phospho-S6, Phospho-SYK
  • p2-microglobulin 525/526, p2-microglobulin, ⁇ , VCAM-1, TNFR2, C - reactive protein (CRP), and IL- 18.
  • Rule-based design (3+3) is used in the dose escalation component (Part A of Example 349). This method is commonly used in Phase 1 oncology clinical trials for cytotoxic agents and molecularly targeted agents. In Part A of the study, lymphomas and solid tumor patients were evaluated. The PK properties of COMPOUND A were evaluated after single and multiple dose administrations at different dose levels. Both twice and once-daily administration schedules were evaluated.
  • Expansion cohorts include patients with Diffuse Large B-Cell Lymphoma (DLBCL), Follicular cell Lymphoma FL and Mantle Cell Lymphoma (MCL) treated daily at a dose regimen to identified in Part A. Patients diagnosed with other hematologic malignancies may also be included such as T-cell lymphomas, myelofibrosis, chronic lymphocytic leukemia. Cohort expansion after determination of the MTD is commonly used in Phase 1 oncology study designs.
  • DLBCL Diffuse Large B-Cell Lymphoma
  • MCL Mantle Cell Lymphoma
  • Example 349 Additional studies for Example 349 may be conducted to characterize safety and efficacy of COMPOUND A in additional hematologic malignancies where JAK or SYK signal inhibition could provide clinical benefit. Additional studies to characterize the ADME and metabolism of COMPOUND A maybe conducted.
  • Example 349 demonstrates a Phase 1/2 clinical trial in patients with solid tumors and hematologic malignancies evaluates QD and BID oral COMPOUND A at escalating doses of 10, 20, 30, 40, 50 and 75 mg BID and 80 and 120 mg QD mg (NCT02440685).
  • Phase 1 allows patients with solid tumors or hematologic malignancies;
  • Phase 2 allows only patients with diffuse large B-Cell lymphoma (DLBCL), follicular lymphoma (FL) or mantle cell lymphoma (MCL).
  • Endpoints include safety, tolerability, pharmacokinetics, serum markers of inflammation, and response using RECIST or Lugano Classification System. Table 40 below shows clinical safety data for ongoing Example 349 (up to
  • Stable disease in a patient with primary peritoneal cancer, about 50% reduction in target lesions at 3 months in a FL patient (Lugano, 6 prior lines) and stable disease and reduction of pruritus in a peripheral T-Cell lymphoma patient after 2 months (Lugano, 2 prior lines) of treatment have been observed.
  • COMPOUND A is safe and well tolerated and does not appear to be associated with the significant thrombocytopenia, anemia and neutropenia as reported for the JAK inhibitor ruxolitinib and which should be managed by dose reduction, or interruption, or red blood cell transfusion.
  • Clinical trials report thrombocytopenia, anemia and neutropenia in 70%, 96% and 19% of patients treated with ruxolitinib.
  • COMPOUND A-related diarrhea has been reported, while the BTK signal inhibitor ibrutinib has been associated with diarrhea in over 50% of patients treated in clinical trials and severe/fatal bleeding events in up to 6%.
  • COMPOUND A is also does not appear to be associated with lipid elevation as reported for JAK inhibitor tofacitinib.
  • Clinical trials LDL elevations were reported in 15-19% of patients treated with tofacitinib.
  • Checkpoint inhibitors may serve to increase a baseline T-cell-specific immune response that turns the immune system against the tumor.
  • a disruption in the functioning of immune checkpoint molecules can lead to imbalances in immunologic tolerance that result in an unchecked immune response. This may clinically manifest with autoimmune-like/ inflammatory side-effects, which cause collateral damage to
  • immune-related adverse events are termed immune-related adverse events and are also thought to be principally T-cell mediated.
  • Other immune cells may play a role in the development of immune-related adverse events, including B cells that secrete antibodies that may mediate toxicity, granulocytes that secrete inflammatory mediators, and cytokines.
  • Patients treated with the CTLA-4 PD-1 / PD-L1 directed treatment can experience the following adverse reactions; fatigue, pruritus, diarrhea, decreased appetite, rash, pyrexia, cough, dyspnea, musculoskeletal pain, constipation, and nausea. Warnings and precautions also include immune-mediated pneumonitis, colitis, and other immune mediated adverse reactions.
  • CAR T cells have shown to be associated with similar immune-related adverse reactions and the use of CAR T cells is limited by potentially severe and fatal toxicities. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues.
  • CRS cytokine release syndrome
  • CRS a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells.
  • COMPOUND A does not appear to overlap with checkpoint inhibitors, small molecule inhibitors, TCR agents (for example, CAR T cells and/or TCR agonist or antagonist peptides), vaccines (for example, sipuleucel-T (Provenge®) and/or
  • Example 349 demonstrates that COMPOUND A is safe and well tolerated. Encouraging preliminary evidence of efficacy in NHL patients has been observed. MTD was not reached.

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Abstract

The present application provides novel pyrimido-pyridazinone compound combinations and methods for preparing and using these combinations. These compounds are useful in treating cancer or tumor growth or metastasis in patients by administering one or more of the combinations to a patient. In one embodiment, the novel combination includea a compound of Formula (I) and R1 and R2 are defined herein and an immunotherapeutic agent.

Description

PYRIMIDO-PYRIDAZINONE COMPOUND COMBINATIONS, METHODS, KITS AND
FORMULATIONS THEREOF
BACKGROUND
Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within cells. Almost all kinases contain a similar 250 to 300 amino acid catalytic domain. The kinases can be categorized into families by the substrates they phosphorylate.
JAK (Janus kinase) is a family of intracellular non-receptor tyrosine kinases, which includes JAK1, JAK2, JAK3 and TYK2. JAK is expressed in hematopoietic cells and abundantly in primary leukemic cells from children with acute lymphoblastic leukemia. The downstream substrates of JAK include the signal tranducer activator of transcription (STAT) proteins. STAT proteins function both as signaling molecules and transcription factors and ultimately bind to specific DNA sequences present in the promoters of cytokine-responsive genes. JAK/STAT signaling has been implicated in the mediation of many abnormal immune responses such as allergies, asthma, autoimmune diseases such as transplant (allograft) rejection, rheumatoid arthritis, amyotrophic lateral sclerosis and multiple sclerosis, as well as in solid and hematologic malignancies such as leukemia and lymphomas.
Spleen tyrosine kinase (syk) is a member of the syk family of protein tyrosine kinases and plays a crucial role in inflammatory and allergic responses. Syk triggers IgE and IgG receptor mediated signaling in mast cells, basophils, and macrophages leading to
degranulation and cytokine release.
Immunoreceptor tyrosine activation motif (ITAM)-mediated signaling has emerged as a primary event in signaling pathways responsible for human pathologies. ITAM-mediated signaling is responsible for relaying activation signals initiated at classical immune receptors such as T-cell receptors, B-cell receptors, and Fc receptors in immune cells and at GPVI and FcyRIIa in platelets to downstream intracellular molecules such as Syk.
The binding of a ligand to an ITAM-containing receptor triggers signaling events which allows for the recruitment of proteins from a family of nonreceptor tyrosine kinases called the Src family. These kinases phosphorylate tyrosine residues within the ITAM sequence, a region with which the tandem SH2 domains on either Syk or ZAP-70 interact. The interaction of Syk with diphosphorylated ITAM sequences induces a conformation change in the kinases that allows for tyrosine phosphorylation of the kinase itself. Not only do these kinases contribute to normal host defense, they also play roles in the pathogenesis of diseases. Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events. These diseases include autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and
neurodegenerative diseases, cancer, cardiovascular diseases, allergies, asthma, Alzheimer's disease and hormone-related diseases. As a consequence, there have been substantial efforts in medicinal chemistry to find inhibitors of protein kinases for use as therapeutic agents.
Immunotherapy has been used as a treatment for various diseases by inducing, enhancing or modulating an immune response. For example, cancer immunotherapy attempts to stimulate the immune system to destroy tumors, or inhibit immune system checkpoint molecules so that they no longer block proteins on cancer cells or immune cells (such as T cells) that respond to them. Such checkpoint inhibitors are designed to overcome the ability of cancer cells to mask themselves from the patient's immune system.
Difficulties arise with combination treatments involving targeted oncologic therapies (e.g., which act by inhibition of specific biochemical pathways) and immunotherapeutic agents, for example immune checkpoint inhibitors or therapies with immunostimulatory potential, and thus treatment efficacy with such a combination may decrease. In addition, such combination treatments may result in increases in toxicity and adverse side effects, for example by increasing cytokine production and other inflammatory reactions related to the non-tumor suppressive activities of the immunotherapeutic agents.
There is a need in the art for combinations of compounds that are dual inhibitors of Syk and JAK family kinases and immunotherapeutic agents, as well as for methods for treating conditions that can benefit from such combinations. SUMMARY
In one aspect, the present disclosure provides pharmaceutical combinations of a compound of Formula (I), wherein R1 and R2 are defined herein
Figure imgf000004_0001
at least one immunotherapeutic agent. In another aspect, methods of treating cancer are provided and include administering a compound of Formula (I) and at least one immunotherapeutic agent to a patient in need thereof.
In another aspect, methods of inhibiting tumor growth or metastasis are provided and include administering a compound of Formula (I) and at least one immunotherapeutic agent to a patient in need thereof.
In another aspect, dosing regimens are provided and include administering to a patient in need thereof a compound of Formula (I) and at least one immunotherapeutic agent to a patient in need thereof.
In another aspect, kits are provided and include a dosage form comprising a compound of Formula (I) and a dosage form comprising at least one immunotherapeutic agent and therapeutic instructions for administering the dosage forms.
In another aspect, uses of a medicament in treating cancer are provided and include administering to a patient in need thereof a multi-part pharmaceutical combination including a compound of Formula (I) and at least one immunotherapeutic agent to a patient in need thereof.
Other aspects and advantages of the disclosure will be readily apparent from the following detailed description of the disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
Figs. 1 and 2 provide comparative data illustrating the anti-inflammatory effects of methotrexate, a known anti-inflammatory, and a compound described herein which is encompassed by the compound of Formula (I), using the Collagen Induced Arthritis (CIA) model of human rheumatoid arthritis (RA) in female lewis rats. After type II collagen (cll) induced CIA, the compound of Example 19 (2 x 30 mg/kg) and methotrexate (0.5 mg kg) were administered daily or twice daily, respectively, to separate rats.
Figure 1 A illustrates anti- inflammatory effects as a function of the amount of edema (mL) vs. time (days). The black diamonds (♦) represent results for the control. The triangles
(A) represent results for the compound of Example 19. The crosses (x) represent results for methotrexate.
Figure 1 B illustrates anti-inflammatory effects as a function of the amount of edema (mL) vs. time (days). The circles (·) represent results for the control. The inverted triangles represent results for the compound of Example 62. The astericks (*) represent results for the compound of Example 108. The squares (■) represent results for the compound of Example 189. The diamonds (♦) represent results for the compound of Example 191.
Figure 2A illustrates anti-inflammatory effects as a function of arhtritic score (per rat) vs. time (days). The black diamonds (♦) represent results for the control. The triangles
Figure imgf000006_0002
represent results for the compound of Example 19. The crosses (x) represent results for methotrexate.
Figure 2B illustrates anti-inflammatory effects as a function of arhtritic score (per rat) vs. time (days). The circles (·) represent results for the control. The triangles represent
Figure imgf000006_0001
results for the compound of Example 62. The astericks (*) represent results for the compound of Example 108. The squares (■) represent results for the compound of Example 189. The diamonds (♦) represent results for the compound of Example 191.
Figure 3 illustrates percent decrease in inflammation biomarkers on Day 15 after initiation of Compoud A treatment with the compound of Example 189 ("COMPOUND A") (BID dosing). The percent change shown in Figure 3 are average values of all patients in each cohort. In Figure 3, B2M = p2-Microglobulin; CRP = C-Reactive Protein; IL-18 = Interleukin-18; ΜΙΡ-1β= Macrophage Inflammatory Protein- 1 β; and TNFR2 = Tumor necrosis factor receptor 2.
Figure 4 illustrates average tumor volume (mm3) in mice treated with COMPOUND A alone or in combination with mouse anti-PD 1 antibody after different days of treatment initiation. Table shown in Figure 4 shows % TGI for various treatments on days 5 to 15 and statistical comparisons between groups on day 15.
Figure 5 illustrates average tumor volume (mm3) in mice treated with COMPOUND A alone or in combination with epacadostat (IDO-1 inhibitor) after different days of treatment initiation. Table shown in Figure 5 shows % TGI for various treatments on days 5 to 15 and statistical comparisons between groups on day 15.
Figure 6 illustrates changes in average body weight of mice treated with
COMPOUND A alone or in combination with mouse anti-PD 1 antibody after treatment on day 15.
Figure 7 illustrates changes in average body weight of mice treated with
COMPOUND A alone or in combination with epacadostat after treatment on day 15.
Figure 8 illustrates percent change in body weight of mice treated with COMPOUND A alone or in combination with mouse anti-PD 1 antibody measured on different days. Figure 9 illustrates percent change in body weight of mice treated with COMPOUND A alone or in combination with epacadostat measured on different days.Compound A
Figure 10 presents raw data for tumor volume and body weight measurement on day
1.
Figure 11 presents raw data for tumor volume and body weight measurement on day
5.
Figure 12 presents raw data for tumor volume and body weight measurement on day
8.
Figure 13 presents raw data for tumor volume and body weight measurement on day 11.
Figure 14 presents raw data for tumor volume and body weight measurement on day
15.
Figure 15 presents raw data for COMPOUND A concentrations in plasma analyzed 2 h after last dose on day 15.
Figure 16 presents raw data for COMPOUND A concentrations in tumor analyzed 2 h after last dose on day 15.
Figure 17 illustrates average tumor volume (mm3) in mice treated with COMPOUND
A alone or in combination with mouse anti-PD 1 antibody after different days of treatment initiation. Table shown in Figure 17 shows % TGI for various treatments on days 4 to 15 and statistical comparisons between groups on day 15.
Figure 18 illustrates average tumor volume (mm3) in mice treated with COMPOUND
A alone or in combination with epacadostat after different days of treatment initiation. Table shown in Figure 18 shows % TGI for various treatments on days 4 to 15 and statistical comparisons between groups on day 15.
Figure 19 illustrates changes in average body weight of mice treated with
COMPOUND A alone or in combination with mouse anti-PDl antibody after treatment on day 15.
Figure 20 illustrates changes in average body weight of mice treated with
COMPOUND A alone or in combination with epacadostat after treatment on day 15.
Figure 21 illustrates percent change in body weight of mice treated with
COMPOUND A alone or in combination with mouse anti-PDl antibody measured on different days. Figure 22 illustrates percent change in body weight of mice treated with
COMPOUND A alone or in combination with epacadostat measured on different days.
Figure 23 presents raw data for tumor volume and body weight measurement on day
1.
Figure 24 presents raw data for tumor volume and body weight measurement on day
4.
Figure 25 presents raw data for tumor volume and body weight measurement on day
8.
Figure 26 presents raw data for tumor volume and body weight measurement on day 11. The shaded rows in Figure 26 indicate animal mortality.
Figure 27 presents raw data for tumor volume and body weight measurement on day 15. The shaded rows in Figure 27 indicate animal mortality.
Figure 28 presents raw data for COMPOUND A concentrations in plasma on day 15 two hours of last dose.
Figure 29 presents raw data for COMPOUND A concentration in tumor on day 15 two hours of last dose.
Figure 30 illustrates average tumor volume (mm3) in mice treated with COMPOUND A alone or in combination with mouse anti-PD 1 antibody after different days of treatment initiation. Table shown in Figure 30 shows % TGI for various treatments on days 4 to 15 and statistical comparisons between groups on day 15 .
Figure 31 illustrates average tumor volume (mm3) in mice treated with COMPOUND A alone or in combination with epacadostat after different days of treatment initiation. Table shown in Figure 31 shows % TGI for various treatments on days 4 to 15 and statistical comparisons between groups on day 15.
Figure 32 illustrates changes in average body weight of mice treated with
COMPOUND A alone or in combination with mouse anti-PDl antibody after treatment on day 15.
Figure 33 illustrates changes in average body weight of mice treated with
COMPOUND A alone or in combination with epacadostat after treatment on day 15.
Figure 34 illustrates percent change in body weight of mice treated with
COMPOUND A alone or in combination with mouse anti-PDl antibody measured on different days. Figure 35 illustrates percent change in body weight of mice treated with
COMPOUND A alone or in combination with epacadostat measured on different days.
Figure 36 presents raw data for tumor volume and body weight measurement on day
1 .
Figure 37 presents raw data for tumor volume and body weight measurement on day
4.
Figure 38 presents raw data for tumor volume and body weight measurement on day
8.
Figure 39 presents raw data for tumor volume and body weight measurement on day
1 1 .
Figure 40 presents raw data for tumor volume and body weight measurement on day
15.
Figure 41 presents raw data for COMPOUND A concentrations in plasma analyzed 2 h after last dose on day 15.
Figure 42 presents raw data for COMPOUND A concentrations in tumor analyzed 2 h after last dose on day 15.
DETAILED DESCRIPTION
The disclosure provides pharmaceutical combinations comprising compounds and immunotherapeutic agents, which are capable of reducing or eliminating inflammation caused by tissue insult, injury, or pathology, and treating cancer and other diseases. The compounds disclosed herein can function through a protein kinase inhibitory mechanism.
In one aspect, the present disclosure provides a pharmaceutical combination comprising a compound of Formula (I):
Figure imgf000009_0001
In this formula, R1 is NR4R5, optionally substituted C1 to C6 alkoxy, optionally substituted C6 to C14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic orbicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic or bicyclic heterocyclyl. In one aspect, the 3-4 membered cycloalkyl and heterocyclyl are saturated. In another aspect, the hydrogen atoms on the same carbon atom of the cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl. In a further aspect, the hydrogen atoms on the same atom of the cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent.
i. In another embodiment, R1 is N(C1 to C6 alkyl)(C1 to C6 alkyl) or C1 to C6 alkoxy. ii. In still a further embodiment, R1 is N(CH(CH3)2)2, N(CH3)2, OCH2CH3, or OCH3. iii. In another embodiment, R1 is optionally substituted phenyl.
iv. In still another embodiment, R1 is of the structure:
Figure imgf000010_0001
wherein, R22, R23, R24, R25, and R26 are, independently, H, C(O)(C1 to C6 alkoxy), C(O)OH, 0(C1 to C3 perfluoroalkyl), 0(C1 to C6 perfluoroalkoxy), Ci to Ce alkoxy, halogen, (C1 to C6 alkyl)heterocyclyl, or (C1 to C6 alkyl)CN. v. In a further embodiment, R1 is:
Figure imgf000010_0002
vi. In yet another embodiment, R1 is optionally substituted 5-9 membered saturated heterocyclyl.
vii. In still a further embodiment, R1 is of the structure:
Figure imgf000010_0003
wherein, R34, R35, R36, and R37 are, independently, H, C1 to C6 alkyl, or CN; Y is (C(R8)2)x, NR7(C(R8)2)x, O, (S=0), S02, or NR7; R7 and R8 are, independently, H, C1 to C6 alkyl, C(O)OH, (C1 to C6 alkyl)CN, (C1 to C6 alkyl)C(O)OH, C(O)(C1 to C6 alkyl)CN, or CN; and x is 0 to 2.
viii. In another embodiment, R is:
Figure imgf000011_0001
still a further embodiment, R1 is of the structure:
Figure imgf000011_0002
wherein, f, g, h, j, and m are, independently, absent, (CH2), CH(R3), Z, or C=0; R3 is H, C(O)OH, or C(O)0(C1 to C6 alkyl); R45, R46, R47, and R48 are, independently, H or C1 to C6 alkyl; and Z is O, S, SO, S02, or NH. In yet another embodiment, R1 is:
Figure imgf000011_0003
xi. In a further embodiment, R1 is a heteroaryl.
xii. In yet another embodiment, R1 is thiophene, benzooxole, or pyridine.
xiii. In still another embodiment, R1 is a monocyclic C3 to C8 cycloalkyl.
xiv. In yet a further embodiment, R1 is cycloheptyl or cyclohexyl, both optionally substituted with -N(C1 to C6 alkyl)(C1 to C6 alkyl).
xv. In another embodiment, R1 is piperidine substituted with C(O)(C1 to C6 alkyl)CN. xvi. In still a further embodiment, R is:
Figure imgf000012_0001
a. In one embodiment, R2 is phenyl substituted with C(O)NR4R5.
b. In another embodiment, R2 is phenyl substituted with
Figure imgf000012_0002
c. In a further embodiment, R2 is phenyl substituted with NR4R5.
d. In yet another embodiment, R2 is phenyl substituted with (C1 to C6 alkyl)NR4R5. e. In another embodiment, R2 is phenyl substituted with NR4R5 or (C1 to C6 alkyl)NR4R5 and R4 and R5 are taken together with the nitrogen atom to which they are attached to form a 6-membered ring.
f. In still a further embodiment, R2 is phenyl substituted with NR4R5 or (C1 to C6
alkyl)NR4R5 and R4 and R5 are joined to form a heterocyclyl of the structure:
Figure imgf000012_0003
wherein, R10, R11, R12, and R13 are, independently, H or C1 to C6 alkyl; R14 is halogen, OH, C(O)OH, C1 to C6 alkoxy, (C1 to C6 alkyl)halogen, (C1 to C6 alkyl)C(O)OH, C1 to C6 hydroxyalkyl, C3 to C8 cycloalkyl, (C1 to C6 alkyl)C(O)NH2, (C1 to C6 alkyl)C(O)NH(C1 to C6 hydroxyalkyl), (C1 to C6 alkyl)C(O)N(C1 to C6 hydroxyalkyl)2, (C1 to C6 alkyl)CN, (C1 to C6 alkyl)heteroaryl, or heteroaryl; and R18 is C1 to C6 hydroxyalkyl or (C1 to C6 alkyl)C(O)OH.
g. In yet a further embodiment, R2 is phenyl substituted with NR4R5 or (C1 to C6
alkyl)NR4R5 and wherein R4 and R5 are joined to form an optionally substituted piperidine or diazepane.
h. In another embodiment, R2 is phenyl substituted with NR4R5 or (C1 to C6 alkyl)NR4R5 and R4 and R5 are joined to form:
Figure imgf000013_0001
i. In still a further embodiment, R2 is phenyl substituted with NR4R5 or (C1 to C6
alkyl)NR4R5 and R4 and R5 are joined to form a heterocyclyl of the structure:
Figure imgf000013_0002
wherein, a, b, c, d, and e are, independently, absent, (CH2), CH(R3), Z, or C=0; R3 is H, C(O)OH, C1 to C6 hydroxyalkyl, or C(O)0(C1 to C6 alkyl); R10, R11, R12, and R13 are, independently, H or C1 to C6 alkyl; and Z is O, S, or NH. j. In yet another embodiment, R2 is phenyl substituted with NR4R5 or (C1 to C6
alkyl)NR4R5 and R4 and R5 are joined to form:
Figure imgf000014_0001
k. In a further embodiment, R2 is phenyl substituted with NR4R5 or (C1 to C6
alkyl)NR4R5 and R4 and R5 are taken to ether to form a heterocyclyl of the structure:
Figure imgf000014_0002
wherein, R10, R11, R12, and R13 are, independently, H or C1 to C6 alkyl; Y is O or NR9; and R9 is H, C1 to C6 alkyl, OH, C(O)OH, C1 to C6 hydroxyalkyl, (Ci to C6 alkyl)NH2, (C1 to C6 alkyl)N(C1 to C6 alkyl)(C1 to C6 alkyl), (C1 to C6 alkyl)(C1 to C6 alkoxy), C(O)(C1 to C6 alkyl)NH2, (C1 to C6 alkyl)C(O)OH, C(O)(C1 to C6 hydroxyalkyl), C(O)(C1 to C6 alkyl)CN, (C1 to C6 alkyl)CN, (C1 to C6 alkyl)halogen, or (C1 to C6 alkyl)0(C1 to C6 alkyl)C(O)(C1 to C6 alkyl)NH2; wherein 2 hydrogen atoms attached to the same carbon atom are optionally replaced with =0.
1. In yet another embodiment, R2 is phenyl substituted with NR4R5 or (C1 to C6
alkyl)NR4R5 and R4 and R5 are taken together to form an optionally substituted morpholine orpiperazine.
m. In still a further embodiment, R2 is phenyl substituted with NR4R5 or (C1 to C6
alk 4R5 and R4 and R5 are taken together to form:
Figure imgf000014_0003
Figure imgf000015_0001
In another embodiment, R2 is phenyl substituted with (C1 to C6 alkyl)NR4R5.
In yet a further embodiment, R2 is phenyl substituted with (C1 to C6 alkyl)NR4R5 and
R4 and R5 are (C1 to C6 hydroxyalkyl).
In still another embodiment, R2 is phenyl substituted with (C1 to C6 alkyl)NR4R5 and NR4R5 is:
Figure imgf000015_0002
a further embodiment, R2 is phenyl substituted with (C1 to C6 alkyl)NR4R5 and R4 and R5 are joined to form an optionally substituted 6-membered ring.
In yet another embodiment, R2 is phenyl substituted with (C1 to C6 alkyl)NR4R5 and NR4R5 are joined to form the 6-membered ring:
Figure imgf000015_0003
wherein, R14 is H, OH, C(O)OH, C1 to C6 alkyl, or (C1 to C6 alkyl)CN. In another embodiment, R2 is phenyl substituted with (C1 to C6 alkyl)NR4R5 and NR4R5 are joined to form the 6-membered ring:
Figure imgf000015_0004
wherein, Y is O or NR9; and R9 is H, C1 to C6 alkyl, OH, C1 to C6 hydroxyalkyl, C(O)(C1 to C6 hydroxyalkyl), C(O)(C1 to C6 alkyl)CN, (Ci to C6 alkyl)CN, (C1 to C6 alkyl)NH2, (C1 to C6 alkyl)halogen, C(O)(C1 to C6 alkyl)CN or (C1 to C6 alkyl)0(C1 to C6 alkyl)C(O)(C1 to C6 alkyl)NH2.
In still a further embodiment, R2 is phenyl substituted with (C1 to C6 alkyl)NR4R5 and
NR4R5 are joined to form the 6-membered ring
Figure imgf000016_0001
wherein, a, b, c, d, and e are, independently, absent, (CH2), CH(R3), or O; and R3 is H or C(O)OH.
u. In yet another embodiment, R2 is a heteroaryl substituted with (C1 to C6 alkyl)NR4R5. v. In still another embodiment, R2 is:
Figure imgf000016_0002
In a further embodiment, R2 is a heteroaryl substituted with NR4R5.
In still another embodiment, R2 is a heteroaryl substituted with NR4R5 and the heteroaryl is pyridine.
In yet a further embodiment, R2 is of the structure:
Figure imgf000016_0003
In another embodiment, R2 is of the structure:
Figure imgf000016_0004
wherein, R80 is OH, -(C1 to C6 alkyl)CN, C1 to C6 hydroxyalkyl, (C1 to C6 alkyl)C(O)NH2, (C1 to C6 alkyl)heterocycle or -(C1 to C6 alkyl) C(O)OH. still a further embodiment 2 is of the structure:
Figure imgf000017_0001
wherein, p is 1 to 6; and R is H or C(O)OH.
bb. In another embodiment, R is of the structure:
Figure imgf000017_0002
wherein, R90 is H, C1 to C6 alkyl, C(O)(C1 to C6 alkyl)CN, (C1 to C6 alkyl)C(O)OH, or C(O)C1 to C6 hydroxyalkyl.
cc. In yet another embodiment, wherein R is phenyl substituted with one or more Q to
Ce alkoxy, (C1 to C6 alkyl)halogen, C1 to C6 trifluoroalkoxy, (C1 to C6 alkyl)C(O)OH, halogen, optionally substituted C3 to C8 cycloalkyl, optionally substituted
heterocyclyl, optionally substituted heteroaryl, -O-(C1 to C6 alkyl)C(O)OH, -O-(Ci to C6 alkyl)NR4R5, -0(optionally substituted heterocycle), -0(C1 to C6 alkyl)N(C1 to C6 alkyl)(C1 to C6 alkyl), -0(C1 to C6 alkyl)NH2, C1 to C6 hydroxyalkyl, -0(C1 to C6 hydroxyalkyl), -0(C1 to C6 alkyl)C(O)OH, -C1 to C6 alkoxy-C1 to C6 alkoxy, - 0(heterocycle)(C1 to C6 hydroxyalkyl), -S02(C1 to C6 alkyl), or -(C1 to C6 alkyl)(Ci to Ce alkoxy)halogen.
dd. In a further embodiment, wherein R2 is of the structure:
Figure imgf000017_0003
wherein, R6 is H, (C1 to C6 alkyl)C(O)OH, or (C1 to C6 alkyl)CN. ee. In still another embodiment, R2 is of the structure:
Figure imgf000018_0001
wherein, z is 1 , 2, 3, 4, 5, or 6.
ff. In another embodiment, R2 is of the structure:
Figure imgf000018_0002
wherein, R6 is H or (C1 to C6 alkyl)C(O)OH.
gg. In yet a further embodiment, R2 is -0(C1 to C6 alkyl)NR4R5.
hh. In still another embodiment, R2 is of the structure:
Figure imgf000018_0003
wherein, y is 2 to 6; and R is H, OH, C1 to C6 alkyl, C1 to C6 hydroxyalkyl, or -(C1 to C6 alkyl)C(O)OH.
ii. In further embodiment, R2 is of the structure:
Figure imgf000018_0004
wherein, m is 2 to 6; y is 0 or 1 ; Z is O or R ; and R is H, C1 to C6 alkyl, C1 to C6 hydroxyalkyl, -(C1 to C6 alkyl)CN, -(C1 to C6 alkyl)C(O)OH, -(Ci to C6 alkyl)CONH2, or -C(O)(C1 to C6 alkyl)OH; wherein 2 hydrogen atoms attached to one carbon atom of the nitrogen-ring are replaced with an oxo or optionally substituted 3-8 membered spirocyclic ring.
jj. In yet another embodiment, R2 is of the structure:
Figure imgf000018_0005
wherein, r is 2 to 6; and R is H, C(O)OH or C1 to C6 hydroxyalkyl.
kk. In still a further embodiment, R2 is:
Figure imgf000019_0001
11. In another embodiment, R2 is aryl substituted with -O-(C1 to C6 alkyl)-heterocycle. mm. In a further embodiment, R2 is:
Figure imgf000019_0002
In one aspect, pharmaceutical combination comprising a compound of Formula (I) is provided, wherein R1 is NR4R5, optionally substituted C1 to C6 alkoxy, optionally substituted C6 to C14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic or bicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic or bicyclic heterocyclyl. The 3-4 membered cycloalkyl and heterocyclyl rings are saturated. Hydrogen atoms on the same carbon atom of the cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl. In addition or alternatively, hydrogen atoms on the same atom of the cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent. R2 is phenyl or 5-6 membered heteroaryl containing at least one N or NH in the backbone, wherein R2 is optionally substituted with one or more R19 and when R2 is 4- pyridyl, the 4-pyridyl lacks a carbonyl substituent at the 2nd position. R19 is NR4R5, (C1 to C6 alkyl)NR4R5, C1 to C6 alkyl, C(O)NR4R5, C3 to C8 cycloalkyl substituted with one or more R21, or heterocyclyl substituted with one or more R21. R21 is (C1 to C6 alkyl)CN, (C1 to C6 alkyl)C(O)OH, (C1 to C6 alkyl)C(O)NH2, (C1 to C6 alkyl)C(O)NHCH2CH2OH, or (C1 to C6 alkyl)C(O)N(CH2CH2OH)2. R4 and R5 are independently selected from among H, C1 to C6 alkyl, C1 to C6 hydroxyalkyl, and (C1 to C6 alkyl)N(C1 to C6 alkyl)(C1 to C6 alkyl).
Alternatively, R4 and R5 are joined to form an optionally substituted 3-8 membered heterocyclyl optionally further containing one or more O, S(O)n, or NR9. R9 is H, OH, (Ci to C6 alkyl)C(O)OH, (C1 to C6 alkyl)C(O)NH2, (C1 to C6 alkyl)C(O)NHCH2CH2OH, (C1 to C6 alkyl)C(O)N(CH2CH2OH)2, C(O)(C1 to C6 alkyl)NH2, C(O)(C1 to C6 alkyl)OH, C1 to C6 hydroxyalkyl, or C1 to C6 alkyl and n is 0 to 2. In one embodiment, R9 is CH2CH2OH. Hydrogen atoms on the same carbon atom of the heterocyclyl are optionally replaced with a 3-6 membered cycloalkyl or heterocyclyl optionally substituted with one or more R to form a spirocycloalkyl or spiroheterocyclyl. R20 is C(O)0(C1 to C6 alkyl), C(O)OH, (C1 to C6 alkyl)C(O)OH, (C1 to C6 alkyl)C(O)NH2, (C1 to C6 alkyl)C(O)NHCH2CH2OH, or (C1 to C6 alkyl)C(O)N(CH2CH2OH)2. Alternatively, or in addition, hydrogen atoms on the same atom of any of the heterocyclyls or cycloalkyls of R9 are optionally replaced with O to form an oxo substituent; or a pharmaceutically acceptable salt or ester thereof.
In another aspect, pharmaceutical combination comprising a compound of Formula (I) is provided, wherein R1 is NR4R5, C1 to C6 alkoxy, optionally substituted phenyl, heteroaryl, optionally substituted 3-10 membered cycloalkyl, or optionally substituted 3-10 membered monocyclic or bicyclic heterocyclyl. Hydrogen atoms on the same carbon atom of the cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl. In addition or alternatively, hydrogen atoms on the same atom of the cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent. R2 is phenyl or pyrazole, wherein R2 is optionally substituted with one or more R19. R19 is NR4R5, (C1 to C6 alkyl) R4R5, Q to Ce alkyl, C(O)NR4R5, C3 to C8 cycloalkyl substituted with one or more R21, or heterocyclyl substituted with one or more R21. R21 is (C1 to C6 alkyl)CN, (C1 to C6 alkyl)C(O)OH, (Ci to C6 alkyl)C(O)NH2, (C1 to C6 alkyl)C(O) HCH2CH2OH, (C1 to C6
alkyl)C(O)N(CH2CH2OH)2. R4 and R5 are (a) independently selected from among H, Ci to C6 alkyl, C1 to C6 hydroxyalkyl, and (C1 to C6 alkyl)N(C1 to C6 alkyl)(C1 to C6 alkyl) or (b) joined to form an optionally substituted 3-8 membered heterocyclyl optionally further containing one or more O, S(O)„, or NR9. R9 is H, OH, C1 to C6 hydroxyalkyl, (C1 to C6 alkyl)C(O)OH, (C1 to C6 alkyl)C(O)NH2, (C1 to C6 alkyl)C(O)NHCH2CH2OH, (C1 to C6 alkyl)C(O)N(CH2CH2OH)2, C(O)(C1 to C6 alkyl)NH2, C(O)(C1 to C6 alkyl)OH, or C1 to C6 alkyl and n is 0 to 2. In one embodiment, R9 is CH2CH2OH. Hydrogen atoms on the same carbon atom of the heterocyclyl are optionally replaced with a 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl optionally substituted with one or more R20. R20 is C(O)0(C1 to C6 alkyl), C(O)OH, (C1 to C6 alkyl)C(O)OH, (C1 to C6 alkyl)C(O)NH2, (C1 to C6 alkyl)C(O)NHCH2CH2OH, or (C1 to C6
alkyl)C(O)N(CH2CH2OH)2. Alternatively, or in addition, hydrogen atoms on the same atom of the heterocyclyl (b) or cycloalkyl are optionally replaced with O to form an oxo substituent; or a pharmaceutically acceptable salt or ester thereof. In a further aspect, pharmaceutical combination comprising a compound of Formula (I) is provided, wherein R1 is NR4R5, C1 to C6 alkoxy, phenyl optionally substituted with C(O)0(C1 to C6 alkyl), C(O)OH, 0(C1 to C3 perfluoroalkyl), C1 to C6 alkoxy, halogen, CH2- heterocyclyl, or CH2CN, 5-8 membered cycloalkyl, heteroaryl, or 3-10 membered monocyclic or bicyclic heterocyclyl optionally substituted with (C1 to C6 alkyl)C(O)OH, Ci to C6 alkyl, CN, C(O)OH, or (C1 to C6 alkyl)CN. Hydrogen atoms on the same carbon atom of the cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl. In addition or alternatively, hydrogen atoms on the same atom of the cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent. R2 is phenyl or pyrazole, wherein
R2 is optionally substituted with one R19. R19 is NR4R5, (C1 to C6 alkyl)NR4R5, C1 to C6 alkyl, C(O)NR4R5, C3 to C8 cycloalkyl substituted with one or more R21, or heterocyclyl substituted with one or more R21. R21 is (C1 to C6 alkyl)CN, (C1 to C6 alkyl)C(O)OH, (Ci to C6 alkyl)C(O)NH2, (C1 to C6 alkyl)C(O) HCH2CH2OH, or (C1 to C6
alkyl)C(O)N(CH2CH2OH)2. R4 and R5 are (a) independently selected from among H, Q to
C6 alkyl, C1 to C6 hydroxyalkyl, and (C1 to C6 alkyl)N(C1 to C6 alkyl)(C1 to C6 alkyl). R4 and R5 may also be (b) joined to form a 5-8 membered heterocyclyl optionally further containing one or two O, S(O)n, or NR9. R9 is H, OH, C1 to C6 hydroxyalkyl (C1 to C6 alkyl)C(O)OH, C(O)(C1 to C6 alkyl)NH2, C(O)(C1 to C6 alkyl)OH, (C1 to C6 alkyl)C(O)NH2, (C1 to C6 alkyl)C(O)NHCH2CH2OH, (C1 to C6 alkyl)C(O)N(CH2CH2OH)2, or C1 to C6 alkyl and n is 0 to 2. In one embodiment, R9 is CH2CH2OH. Hydrogen atoms on the same carbon atom of the heterocyclyl are optionally replaced with a 3-5 membered cycloalkyl optionally substituted with one or more R 20 to form a spirocycloalkyl. R 20 is C(O)0(C1 to C6 alkyl), C(O)OH, (C1 to C6 alkyl)C(O)OH, (C1 to C6 alkyl)C(O)NH2, (C1 to C6
alkyl)C(O)NHCH2CH2OH, or (C1 to C6 alkyl)C(O)N(CH2CH2OH)2. Alternatively, or in addition, hydrogen atoms on the same atom of the heterocyclyl (b) or cycloalkyl (b) are optionally replaced with O to form an oxo substituent; or a pharmaceutically acceptable salt or ester thereof. Some compounds within the present disclosure possess one or more chiral centers, and the present disclosure includes each separate enantiomer of such compounds as well as mixtures of the enantiomers. Where multiple chiral centers exist in compounds of the present disclosure, the disclosure includes each possible combination of chiral centers within a compound, as well as all possible enantiomeric and diastereomeric mixtures thereof. All chiral, diastereomeric, and racemic forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials.
The following definitions are used in connection with the compounds of the present disclosure unless the context indicates otherwise. In general, the number of carbon atoms present in a given group is designated "Cx-Cy", where x and y are the lower and upper limits, respectively. For example, a group designated as " C1-C6" contains from 1 to 6 carbon atoms. The carbon number as used in the definitions herein refers to carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like. Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming from left to right the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkyloxycabonyl" refers to the group (C6-C14 aryl)-(C1-C6 alkyl)-O-C(O)-. Terms not defined herein have the meaning commonly attributed to them by those skilled in the art.
"Alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms, for example, a C1-Q2 alkyl group may have from 1 to 12 (inclusive) carbon atoms in it. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert- butyl, isopentyl, neopentyl, and isohexyl. Examples of C1-C8 alkyl groups include, but are not limited to, methyl, propyl, pentyl, hexyl, heptyl, 3-methylhex-l-yl, 2,3-dimethylpent-2-yl, 3- ethylpent-l-yl, octyl, 2-methylhept-2-yl, 2,3-dimethylhex-l-yl, and 2,3,3-trimethylpent-l-yl. An alkyl group can be unsubstituted or substituted with one or more of halogen, NH2,
(alkyl)NH, (alkyl)(alkyl)N-, -N(alkyl)C(O)(alkyl), -NHC(O)(alkyl), -NHC(O)H, -C(O)NH2, - C(O)NH(alkyl), -C(O)N(alkyl)(alkyl), CN, OH, alkoxy, alkyl, C(O)OH, -C(O)0(alkyl), - C(O)(alkyl), aryl, heteroaryl, heterocyclyl, cycloalkyl, haloalkyl, aminoalkyl-, -OC(O)(alkyl), carboxyamidoalkyl-, and N02.
"Alkoxy" refers to the group R-O- where R is an alkyl group, as defined above.
Exemplary C1-C6 alkoxy groups include but are not limited to methoxy, ethoxy, n-propoxy, 1-propoxy, n-butoxy and t-butoxy. An alkoxy group can be unsubstituted or substituted with one or more of halogen, OH, alkoxy, NH2, (alkyl)amino-, di(alkyl)amino-, (alkyl)C(O)N(Ci- C3 alkyl)-, (alkyl)carboxyamido-, HC(O)NH-, H2NC(O)-, (alkyl)NHC(O)-, di(alkyl)NC(O)-, CN, C(O)OH, (alkoxy)carbonyl-, (alkyl)C(O)-, aryl, heteroaryl, cycloalkyl, haloalkyl, amino(C1-C6 alkyl)-, (alkyl)carboxyl-, carboxyamidoalkyl-, or N02.
Aryl refers to an aromatic 6 to 14 membered hydrocarbon group. Examples of a Ce- Ci4 aryl group include, but are not limited to, phenyl, a-naphthyl, β-naphthyl, biphenyl, anthryl, tetrahydronaphthyl, fluorenyl, indanyl, biphenylenyl, and acenanaphthyl. Examples of a C6-Cioaryl group include, but are not limited to, phenyl, a-naphthyl, β-naphthyl, biphenyl, and tetrahydronaphthyl. An aryl group can be unsubstituted or substituted with one or more of alkyl, halogen, haloalkyl, alkoxy, haloalkoxy, OH, hydroxyalkyl, -O- (hydroxyalkyl), -O-(alkyl)-C(O)OH, -(alkyl)-(alkoxy)-halogen, NH2, aminoalkyl-, dialkylamino-, C(O)OH, -C(O)0-(alkyl), -OC(O)(alkyl), -O-(alkyl)-N(alkyl)(alkyl) ,N- alkylamido-, -C(O)NH2, (alkyl)amido-, N02, (aryl)alkyl, alkoxy, aryloxy, heteroaryloxy, (aryl)amino, (alkoxy)carbonyl-, (alkyl)amido-, (alkyl)amino, aminoalkyl-, alkylcarboxyl-, (alkyl)carboxyamido-, (aryl)alkyl-, (aryl)amino-, cycloalkenyl, di(alkyl)amino-, heteroaryl, (heteroaryl)alkyl-, heterocyclyl, -O-(heterocyclyl), heterocyclyl(alkyl)-, (hydroxyalkyl)NH-, (hydroxyalkyl)2N, -S02(alkyl) or a spiro substituent.
The term "bicycle" or "bicyclic" as used herein refers to a molecule that features two fused rings, which rings are a cycloalkyl, heterocyclyl, or heteroaryl. In one embodiment, the rings are fused across a bond between two atoms. The bicyclic moiety formed therefrom shares a bond between the rings. In another embodiment, the bicyclic moiety is formed by the fusion of two rings across a sequence of atoms of the rings to form a bridgehead.
Similarly, a "bridge" is an unbranched chain of one or more atoms connecting two bridgeheads in a polycyclic compound. In another embodiment, the bicyclic molecule is a "spiro" or "spirocyclic" moiety. The spirocyclic group is a carbocyclic or heterocyclic ring which bound through a single carbon atom of the spirocyclic moiety to a single carbon atom of a carbocyclic or heterocyclic moiety. In one embodiment, the spirocyclic group is a cycloalkyl and is bound to another cycloalkyl. In another embodiment, the spirocyclic group is a cycloalkyl and is bound to a heterocyclyl. In a further embodiment, the spirocyclic group is a heterocyclyl and is bound to another heterocyclyl. In still another embodiment, the spirocyclic group is a heterocyclyl and is bound to a cycloalkyl.
"(Aryl)alkyl" refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with an aryl group as defined above. (C6-C14 aryl)alkyl- moieties include benzyl, benzhydryl, 1-phenylethyl, 2-phenylethyl, 3- phenylpropyl, 2-phenylpropyl, 1-naphthylmethyl, 2-naphthylmethyl and the like. An (aryl)alkyl group can be unsubstituted or substituted with one or more of of halogen, CN, NH2, OH, (alkyl)amino-, di(alkyl)amino-, (alkyl)C(O)N(alkyl)-, (alkyl)carboxyamido-, HC(O)NH-, H2NC(O)-, (alkyl)NHC(O)-, di(alkyl)NC(O)-, CN, OH, alkoxy, alkyl, C(O)OH, (alkoxy)carbonyl-, (alkyl)C(O)-, aryl, heteroaryl, cycloalkyl, haloalkyl, amino(alkyl)-, (alkyl)carboxyl-, carboxyamidoalkyl-, or N02.
"(Alkoxy)carbonyl-" refers to the group alkyl-O-C(O)-. Exemplary (C1-C6 alkoxy)carbonyl- groups include but are not limited to methoxy, ethoxy, n-propoxy, 1- propoxy, n-butoxy and t-butoxy. An (alkoxy)carbonyl group can be unsubstituted or substituted with one or more of halogen, OH, NH2, (alkyl)amino-, di(alkyl)amino-,
(alkyl)C(O)N(alkyl)-, (alkyl)carboxyamido-, HC(O)NH-, H2NC(O)-, (alkyl)NHC(O)-, di(alkyl)NC(O)-, CN, alkoxy, C(O)OH, (alkoxy)carbonyl-, (alkyl)C(O)-, aryl, heteroaryl, cycloalkyl, haloalkyl, amino(alkyl)-, (alkyl)carboxyl-, carboxyamidoalkyl-, orN02.
"(Alkyl)amido-" refers to a -C(O)NH- group in which the nitrogen atom of said group is attached to a C1-C6 alkyl group, as defined above. Representative examples of a (C1-C6 alkyl)amido- group include, but are not limited to, -C(O)NHCH3, -C(O)NHCH2CH3, -C(O)NHCH2CH2CH3, -C(O)NHCH2CH2CH2CH3, -C(O)NHCH2CH2CH2CH2CH3, -C(O)NHCH(CH3)2, -C(O)NHCH2CH(CH3)2, -C(O)NHCH(CH3)CH2CH3, -C(O)NH- C(CH3)3 and -C(O)NHCH2C(CH3)3.
"(Alkyl)amino-" refers to an -NH group, the nitrogen atom of said group being attached to a alkyl group, as defined above. Representative examples of an (C1-C6 alkyl)amino- group include, but are not limited to CH3NH-, CH3CH2NH-, CH3CH2CH2NH-, CH3CH2CH2CH2NH-, (CH3)2CHNH-, (CH3)2CHCH2NH-, CH3CH2CH(CH3)NH- and (CH3)3CNH-. An (alkyl)amino group can be unsubstituted or substituted on the alkyl moiety with one or more of halogen, NH2, (alkyl)amino-, di(alkyl)amino-, (alkyl)C(O)N(alkyl)-, (alkyl)carboxyamido-, HC(O)NH-, H2NC(O)-, (alkyl)NHC(O)-, di(alkyl)NC(O)-, CN, OH, alkoxy, alkyl, C(O)OH, (alkoxy)carbonyl-, (alkyl)C(O)-, aryl, heteroaryl, cycloalkyl, haloalkyl, amino(alkyl)-, (alkyl)carboxyl-, carboxyamidoalkyl-, or N02.
"Aminoalkyl-" refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with -NH2; one or both H of the NH2 may be replaced by a substituent. "Alkylcarboxyl-" refers to an alkyl group, defined above that is attached to the parent structure through the oxygen atom of a carboxyl (C(O)-O-) functionality. Examples of (C1-C6 alkyl)carboxyl- include acetoxy, propionoxy, propylcarboxyl, and isopentylcarboxyl.
"(Alkyl)carboxyamido-" refers to a -NHC(O)- group in which the carbonyl carbon atom of said group is attached to a C1-C6 alkyl group, as defined above. Representative examples of a (C1-C6 alkyl)carboxyamido- group include, but are not limited to,
-NHC(O)CH3, -NHC(O)CH2CH3, -NHC(O)CH2CH2CH3, -NHC(O)CH2CH2CH2CH3, -NHC(O)CH2CH2CH2CH2CH3, -NHC(O)CH(CH3)2, -NHC(O)CH2CH(CH3)2,
-NHC(O)CH(CH3)CH2CH3, -NHC(O)-C(CH3)3 and -NHC(O)CH2C(CH3)3.
"(Aryl)amino" refers to a radical of formula (aryl)-NH-, wherein aryl is as defined above. "(Aryl)oxy" refers to the group Ar-O- where Ar is an aryl group, as defined above.
"Cycloalkyl" refers to a non-aromatic, saturated, partially saturated, monocyclic, bicyclic or polycyclic hydrocarbon 3 to 12 membered ring system. Representative examples of a C3-Ci2 cycloalkyl include, but are not limited to, cyclopropyl, cyclopentyl, cycloheptyl, cyclooctyl, decahydronaphthalen-l-yl, octahydro-lH-inden-2-yl, decahydro-lH- benzo[7]annulen-2-yl, and dodecahydros-indacen-4-yl. Representative examples of a C3-Cio cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, decahydronaphthalen-l-yl, and octahydro-lH-inden-2-yl.
Representative examples of a C3-C8 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and octahydropentalen-2-yl. A cycloalkyl can be unsubstituted or substituted with one or more of halogen, NH2, (alkyl)NH, (alkyl)(alkyl)N-, -N(alkyl)C(O)(alkyl), -NHC(O)(alkyl), -NHC(O)H, -C(O)NH2,
-C(O)NH(alkyl), -C(O)N(alkyl)(alkyl), CN, OH, alkoxy, alkyl, C(O)OH, -C(O)0(alkyl), - C(O) alkyl), aryl, heteroaryl, cycloalkyl, haloalkyl, aminoalkyl-, -OC(O)(alkyl),
carboxyamidoalkyl-, and N02. Additionally, each of any two hydrogen atoms on the same carbon atom of the carbocyclic ring can be replaced by an oxygen atom to form an oxo (=0) substituent.
"Halo" or "halogen" refers to -F, -CI, -Br and -I.
"C1-C6 haloalkyl" refers to a C1-C6 alkyl group, as defined above, wherein one or more of the C1-C6 alkyl group's hydrogen atoms has been replaced with F, CI, Br, or I. Each substitution can be independently selected from F, CI, Br, or I. Representative examples of an C1-C6 haloalkyl- group include, but are not limited to, -CH2F, -CC13, -CF3, CH2CF3, -CH2C1, -CH2CH2Br, -CH2CH2I, -CH2CH2CH2F, -CH2CH2CH2C1, -CH2CH2CH2CH2Br, -CH2CH2CH2CH2I, -CH2CH2CH2CH2CH2Br, -CH2CH2CH2CH2CH2I, -CH2CH(Br)CH3, -CH2CH(C1)CH2CH3, -CH(F)CH2CH3 and -C(CH3)2(CH2C1).
"Heteroaryl" refers to a monocyclic, bicyclic, or polycyclic aromatic ring system containing at least one ring atom selected from the heteroatoms oxygen, sulfur and nitrogen. Examples of C1-C9 heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole, 1,2,4- triazole, l-methyl-l,2,4-triazole, lH-tetrazole, 1 -methyltetrazole, benzoxazole,
benzo thiazole, benzofuran, benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, and isoquinoline. Bicyclic C1-C9 hetroaryl groups include those where a phenyl, pyridine, pyrimidine or pyridazine ring is fused to a 5 or 6-membered monocyclic heteroaryl ring having one or two nitrogen atoms in the ring, one nitrogen atom together with either one oxygen or one sulfur atom in the ring, or one O or S ring atom. Examples of monocyclic C1-C4 heteroaryl groups include 2H-tetrazole, 3H-l,2,4-triazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, imidazole, and pyrrole. A heteroaryl group can be unsubstituted or substituted with one or more of C1-C6 alkyl, halogen, haloalkyl, OH, CN, hydroxyalkyl, N¾, aminoalkyl-, dialkylamino-, C(O)OH, -C(O)0-(alkyl), -OC(O)(alkyl), N-alkylamido-, -C(O)NH2, (alkyl)amido-, -N02, (aryl)alkyl, alkoxy, aryloxy, heteroaryloxy, (aryl)amino, (alkoxy)carbonyl-, (alkyl)amido-, (alkyl)amino, aminoalkyl-, alkylcarboxyl-, (alkyl)carboxyamido-, (aryl)alkyl-, (aryl)amino-, cycloalkenyl, di(alkyl)amino-, heteroaryl, (heteroaryl)alkyl-, heterocyclyl, hetyerocyclyl(alkyl)-,
(hydroxyalkyl)NH-, (hydroxyalkyl)2N or a spiro substituent.
"Heterocycle" or "heterocyclyl" refers to monocyclic, bicyclic, polycyclic, or bridged head molecules in which at least one ring atom is a heteroatom. A heterocycle maybe saturated or partially saturated. Exemplary C1-C9 heterocyclyl groups include but are not limited to aziridine, oxirane, oxirene, thiirane, pyrroline, pyrrolidine, dihydrofuran, tetrahydro furan, dihydrothiophene, tetrahydrothiophene, dithiolane, piperidine, 1,2,3,6- tetrahydropyridine-l-yl, tetrahydropyran, pyran, thiane, thiine, piperazine, azepane, diazepane, oxazine, 5,6-dihydro-4H-l,3-oxazin-2-yl, 2,5-diazabicyclo[2.2.1]heptane, 2,5- diazabicyclo[2.2.2]octane, 3,6-diazabicyclo[3.1.1]heptane, 3,8-diazabicyclo[3.2.1]octane, 6- oxa-3,8-diazabicyclo[3.2.1]octane, 7-oxa-2,5-diazabicyclo[2.2.2]octane, 2,7-dioxa-5- azabicyclo[2.2.2]octane, 2-oxa-5-azabicyclo[2.2.1 ]heptane-5-yl, 2-oxa-5- azabicyclo[2.2.2]octane, 3,6-dioxa-8-azabicyclo[3.2.1]octane, 3-oxa-6- azabicyclo[3.1.1 ]heptane, 3 -oxa-8-azabicyclo[3.2.1 ]octan-8-yl, 5,7-dioxa-2- azabicyclo[2.2.2]octane, 6,8-dioxa-3-azabicyclo[3.2.1]octane, 6-oxa-3- azabicyclo[3.1.1 ]heptane, 8-oxa-3-azabicyclo[3.2.1 ]octan-3-yl, 2-methyl-2,5- diazabicyclo[2.2.1]heptane-5-yl, l,3,3-trimethyl-6-azabicyclo[3.2.1]oct-6-yl, 3-hydroxy-8- azabicyclo[3.2.1]octan-8-yl-, 7-methyl-3-oxa-7,9-diazabicyclo[3.3.1]nonan-9-yl, 9-oxa-3- azab icyclo [3.3.1 ]nonan-3 -yl, 3 -oxa-9-azab icyclo [3.3.1 ]nonan-9 -yl, 3 ,7-dioxa-9- azabicyclo [3.3.1 ]nonan-9-yl, 4-methyl-3 ,4-dihydro-2H- 1 ,4-benzoxazin-7-yl, thiazine, dithiane, and dioxane. The contemplated heterocycle rings or ring systems have a minimum of 3 members. Therefore, for example, Ci heterocyclyl radicals would include but are not limited to oxaziranyl, diaziridinyl, and diazirinyl, C2 heterocyclyl radicals include but are not limited to aziridinyl, oxiranyl, and diazetidinyl, C heterocyclyl radicals include but are not limited to azecanyl, tetrahydroquinolinyl, and perhydroisoquinolinyl. A heterocyclyl group can be unsubstituted or substituted with one or more of alkyl, halogen, alkoxy, haloalkyl, OH, hydroxyalkyl, -C(O)-(hydroxyalkyl), NH2, aminoalkyl-, dialkylamino-, C(O)OH, -C(O)0- (alkyl), -OC(O)(alkyl), N-alkylamido-, -C(O)NH2, (alkyl)amido-, -C(O)-(alkyl)-CN, (alkyl)- CN, or N02.
"Heterocyclyl(alkyl)-" refers to an alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with a heterocycle group as defined above. Heterocyclyl( C1-C6 alkyl)- moieties include 1 -piperazinylethyl, 4-morpholinylpropyl, 6-piperazinylhexyl, and the like. A heterocyclyl(alkyl) group can be unsubstituted or substituted with one or more of halogen, NH2, (alkyl)amino-, di(alkyl)amino-,
(alkyl)C(O)N(alkyl)-, (alkyl)carboxyamido-, HC(O)NH-, H2NC(O)-, (alkyl)NHC(O)-, di(alkyl)NC(O)-, CN, OH, alkoxy, alkyl, C(O)OH, (alkoxy)carbonyl-, (alkyl)C(O)-, 4- to 7- membered monocyclic heterocycle, aryl, heteroaryl, or cycloalkyl.
"Heteroaryl(alkyl)" refers to a heteroaryl which is attached to an alkyl group and the heteroaryl is defined above.
"Hydroxyalkyl" refers to a alkyl group, as defined above, wherein one or more of the alkyl group's hydrogen atoms has been replaced with OH groups. Examples of C1-C6 hydroxyalkyl moieties include, for example, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, -CH2CH(OH)CH2OH, -CH2CH(OH)CH3, -CH(CH3)CH2OH and higher homologs.
"Perfluoroalkyl-" refers to alkyl group, defined above, having two or more fluorine atoms. Examples of a C1-C6 perfluoroalkyl- group include CF3, CH2CF3, CF2CF3 and CH(CF3)2. A "subject" is a mammal, e.g. , a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon or gorilla.
With the proviso that when R2 is 4-pyridyl, then there cannot be a carbonyl substituent at the 2-position of the 4-pyridyl moiety,
Representative "pharmaceutically acceptable salts" include but are not limited to, e.g., water-soluble and water-insoluble salts, such as the acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, bromide, butyrate, calcium, chloride, choline, citrate, edisylate (camphorsulfonate), fumarate, gluconate, glucuronate, glutamate, hydrobromide, hydrochloride, lauryl sulfate, malate, maleate, mandelate, mesylate, palmitate, pantothenate, phosphate, potassium, propionate, p-toluenesulfonate, salicylate, sodium, stearate, succinate, and sulfate salts.
The following abbreviations are used and have the indicated definitions: ACN is acetonitrile; DMSO is dimethylsulfoxide; DMF is N,N-dimethylformamide; DMF.DMA is dimethylformamide dimethylacetal; TFA is trifluroroacetic acid; mCPBA is meta- chloroperbenzoic acid; RT is room temperature; THF is tetrahydrofuran; and NMP is N- methyl pyrrolidinone.
Methods useful for making the compounds of Formula (I) are set forth in the Examples below and generalized in Schemes I-III. One of skill in the art will recognize that Schemes l-III can be adapted to produce the compounds of Formula (I) and pharmaceutically accepted salts of compounds of Formula (I) according to the present disclosure. In the reactions described, reactive functional groups, such as hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, maybe protected to avoid unwanted reactions. Conventional protecting groups maybe used in accordance with standard practice.
Scheme 1
Figure imgf000029_0001
Scheme 1 provides the synthesis of compounds of Formula (I). Ethyl acetoacetate 1 is converted to the corresponding bis(methylthio)methylene derivative 2 using carbon disulfide, an organic or inorganic base such as K2CO3 and a alkylating agent. In one embodiment, the alkylating agent is an alkyl iodide, alkyl triflate, or alkyl sulfonate. In another embodiment, the alkylating agent is a methylating agent. In a further embodiment, the alkylating agent is methyl iodide. Reaction of 2 with an R1 -optionally substituted amidine hydrochloride in the presence of a base results in pyrimidine 3. In one embodiment, the base utilized to form pyrimidine 3 is EtsN or Hiinig's base. The alkyl group on the pyrimidine group of compound 3 is then oxidized using an oxidizing agent. In one embodiment, the oxidation is performed using Se02. The resulting pyrimidine aldehyde 4 is converted to pyrimido-pyridazinone 5 using hydrazine hydrate or hydrazine hydrochloride. The methyl thio group in compound 5 is oxidized to a methane sulfonyl using meta-chloroperoxybenzoic acid (mCPBA) or hydrogen peroxide/acetic acid. Finally, the methane sulfonyl group of compound 6 is replaced with an R2-substituted aniline to provide compound (I). In one embodiment, the R2-substituted aniline is an aryl or heteroaryl substituted aniline.
Figure imgf000030_0001
Scheme 2 provides the synthesis of compound IB which are encompassed by the structure of Formula (I). In this embodiment, ethyl acetoacetate 1 is converted to the corresponding bis(methylthio)methylene derivative 2 using carbon disulfide, K2CO3 and methyl iodide. Reaction of 2 with an R-substituted benzamidine hydrochloride in the presence of Et3N results inpyrimidine 3a. The methyl group bound to the C-atom of pyrimidine 3a is then oxidized using Se02. The resulting pyrimidine aldehyde 4a is converted to pyrimido-pyridazinone 5a using hydrazine hydrate or hydrazine hydrochloride. The methyl thio group in compound 5a is oxidized to a methane sulfonyl group using mCPBA. Finally, the methane sulfonyl group is replaced with an R"-substituted aniline to give compound IB.
Figure imgf000031_0001
Scheme 3 provides the synthesis of compounds IC which are encompassed by the structure of Formula (I). Treatment of ethyl chloroformate 7 with ammonium thiocyanate results in the production of ethyl thiocyanato formate 8 which upon treatment with ethyl 3- amino crotanoate results in compound 9. Compound 9 is cyclized to compound 10 by treatment with an organic or inorganic base. In one embodiment the organic or inorganic base is a strong base. In one embodiment, the strong base is a tertiary organic base. In another embodiment, the strong base is aqueous Et3N. The dichloro pyrimidine 11 is obtained by treating compound 10 with a chlorinating agent. In one embodiment, the chlorinating agent is POCI3. In another embodiment, this transformation can also be carried out by using other chlorinating agents such as PCI5, SOCl2 in the presence of an organic base such as TEA, tributyl amine, and Ν,Ν-dimethylaniline. The 4-position of dichloropyrimidine 11 is then substituted by reaction with an optionally substituted (R2) aniline to afford compound 12. The 2-position of pyrimidine 12 is then R^-substituted using coupling agents such as boronic acids or boronic ester reagents to provide compound 13. The methyl group at position 4 of pyrimidine 13 is then oxidized using an oxidizing agent such as Se02 to provide compound 14. The resulting pyrimidine aldehyde 14 is converted to pyrimido-pyridazinone IC using hydrazine hydrate.
Figure imgf000032_0001
Scheme 4 provides the synthesis of compounds ID which are encompassed by the structure of Formula (I). Treatment of ethyl chloroformate 7 with ammonium thiocyanate results in ethyl thiocyanato formate 8 which upon treatment with ethyl 3-amino crotanoate results in compound 9. Compound 9 is cyclized to compound 10 as described in Scheme 3, i.e., by treatment with aqueous Et3N. The dichloro pyrimidine compound 11 is obtained by treating compound 10 with POCI3. The 4-position of dichloropyrimidine 11 is then substituted by reaction with an optionally R-substituted aniline to afford compound 12a. The 2-position of pyrimidine compound 12a is then substituted with an R'-substituted aryl or heteroaryl group using a boronic acid or an boronic ester reagent to provide compound 13a. In one embodiment, the boronic acid is (R'-aryl)-B(OH)2 or (R'-heteroaryl)-B(OH)2. The methyl at position-4 on pyrimidine 13a is then oxidized using Se02. The resulting pyrimidine aldehyde 14a is converted to pyrimido-pyridazinone ID using hydrazine hydrate.
Figure imgf000033_0001
Scheme 5 provides the synthesis of compound IE which are encompassed by the structure of Formula (I). Compound 12 is reacted with an optionally substituted amine (NHR4R5) to provide compound 15. The methyl group at the 4-position of compound 15 is reacted with DMF.DMA to provide compound 16. Compound 17 is obtained by oxidative cleavage of the olefin of compound 16. In one embodiment, oxidative cleavage is performed using NaI04. Finally, pyrimido-pyridazinone IE is obtained by cyclizing the aldehyde 17. In one embodiment, the cyclizaton is performed using hydrazine, hydrazine hydrate or hydrazine hydrochloride, as described previously.
Figure imgf000034_0001
Scheme 6 provides the synthesis of compounds IF which are encompassed by the structure of Formula (I). Compound 12a is reacted with optionally substituted amines to result in 15a. The methyl group of 15a is reacted with DMF.DMA to give compound 16a.
The aldehyde 17a is obtained by the oxidative cleavage of the olefin in 16a. In one embodiment, the oxidative cleavage is performed with NaI04. Finally, the pyrimido- pyridazinone IF is obtained by cyclizing aldehyde 17a. In one embodiment, cyclization is performed using with hydrazine.
Scheme 7
Figure imgf000035_0001
Scheme 7 provides the synthesis of compound 1G which are encompassed by the structure of Formula (I). Compound 10 is reacted with an alkylating agent to the S-methyl compound 18. In one embodiment, the reacted is performed under basic conditions. In another embodiment, the alkylating agent is methyl iodide, ethyl iodide, propyl iodide, dimethylsulfate, among others. Compound 19 is obtained by chlorinating compound 18. In one embodiment, compound 18 is chlorinated using POCI3. Compound 19 is then NR4R5 substituted with an optionally substituted amine to provide compound 20 [using NHR4R5?]. The methyl group of compound 20 is reacted with DMF.DMA to give compound 21. The aldehyde 22 is obtained by the oxidative cleavage of the olefin group in compound 21. In one embodiment, the oxidative cleavage is performed with NaI04. Alternatively, compound 20 may directly converted to the pyrimidine aldehyde 22 by oxidizing the methyl group using Se02 or a combination of C02, t-butyl hydroperoxide, and an alcohol such as Ci to Ce alkyl)H2OH. The resulting pyrimidine aldehyde 22 is converted to pyrimido-pyridazinone 23 using hydrazine hydrate or hydrazine hydrochloride. The methyl thio group in compound 23 is oxidized to a methane sulfonyl group. In one embodiment compound 23 is reacted with meta-chloroperoxybenzoic acid (mCPBA) or hydrogen peroxide/acetic acid. Finally, the methane sulfonyl group of compound 24 is replaced with suitably substituted aniline to provide compound 1G.
Scheme 8
Figure imgf000036_0001
Scheme 8 provides the synthesis of compound 1H which is encompassed by the structure of Formula (I). In this scheme, compound 19 is coupled with an optionally substituted boronic acid or boronic ester to give compound 25. In one embodiment, the coupling is performed in the presence of a coupling agent such as Pd(PPh3)4 or PdCl2(PPh3)2. The methyl group in compound 25 is then oxidized to the corresponding aldehyde. In one embodiment, the oxidation is performed using Se02 or a combination of C02, t-butyl hydroperoxide, and an alcohol such as C1 to C6 alkyl)H2OH to give compound 26. Compound 26 is converted to pyrimido-pyridazinone 27 using hydrazine hydrate or hydrazine hydrochloride. The methyl thio group in compound 27 is then oxidized to a methane sulfonyl group. In one embodiment, the oxidation is performed using mCPBA or hydrogen peroxide/acetic acid. Finally, the methane sulfonyl group of compound 28 is replaced with suitably substituted aniline to provide compound 1H.
Pharmaceutical combinations of the disclosure comprise a compound of Formula (I) optionally with other pharmaceutically inert or inactive ingredients. In one embodiment, the pharmaceutically inert or inactive ingredient is one or more pharmaceutically acceptable carrier or excipient. The present disclosure also contemplates combining the compound of Formula (I) with one or more therapeutic agents, i.e., active ingredients, as described below. In a further embodiment, a compound of Formula (I) is combined with one or more inert/inactive ingredients and one or more therapeutic agents. The pharmaceutical combinations of the disclosure contain an amount of a compound of Formula (I) that is effective for treating inflammation in a subject. Specifically, the dosage of the compound of Formula (I) to achieve a therapeutic effect will depend on factors such as the formulation, pharmacological potency of the drug, age, weight and sex of the patient, condition being treated, severity of the patient's symptoms, specific compound of Formula
(I), route of delivery, and response pattern of the patient. It is also contemplated that the treatment and dosage of the compound of Formula (I) maybe administered in unit dosage form and that one skilled in the art would adjust the unit dosage form accordingly to reflect the relative level of activity. The decision as to the particular dosage to be employed (and the number of times to be administered per day) is within the discretion of the ordinarily-skilled physician, and maybe varied by titration of the dosage to the particular circumstances to produce the desired therapeutic effect.
In one embodiment, the therapeutically effective amount is about 0.0001% to about 25% w/w. In another embodiment, the therapeutically effective amount is less than about 20% w/w, about 15% w/w, about 10% w/w, about 5% w/w, or about 1% w/w. In another embodiment, the therapeutically effective amount is about 0.0001% to about 10% w/w. In a further embodiment, the therapeutically effective amount is about 0.005 to about 5% w/w. In yet another embodiment, the therapeutically effective amount is about 0.01 to about 5% w/w. In still a further embodiment, the therapeutically effective amount is about 0.01% w/w, about 0.05% w/w, about 0.1 % w/w, about 0.2 % w/w, about 0.3% w/w, about 0.4% w/w, about
0.5% w/w, about 0.6% w/w, about 0.7% w/w, about 0.8 % w/w, about 0.8% w/w, about 0.9% w/w, about 1% w/w, about 2% w/w, about 3% w/w, about 4% w/w, or about 5% w/w.
The therapeutically effective amounts maybe provided on regular schedule, i.e., on a less than daily, weekly, monthly, or yearly basis or on an irregular schedule with varying administration days, weeks, months, etc. Alternatively, the therapeutically effective amount to be administered may vary. In one embodiment, the therapeutically effective amount for the first dose is higher than the therapeutically effective amount for one or more of the subsequent doses. In another embodiment, the therapeutically effective amount for the first dose is lower than the therapeutically effective amount for one or more of the subsequent doses. Equivalent dosages may be administered over various time periods including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every 2 weeks, about every 3 weeks, about every month, about every 2 months, about every 3 months and about every 6 months. The number and frequency of dosages corresponding to a completed course of therapy will be determined according to the judgment of a health-care practitioner. The therapeutically effective amounts described herein refer to total amounts administered for a given time period; that is, if more than one compound of Formula (I) is administered, the therapeutically effective amounts correspond to the total amount administered.
The compound of Formula (I) maybe administered by any route, taking into consideration the specific condition for which it has been selected. The compounds of Formula (I) may be delivered orally, by injection (including intravascularly, e.g ,
intravenously or intra-arterially), inhalation (intranasally and intratracheally), ocularly, transdermally (via simple passive diffusion formulations or via facilitated delivery using, for example, iontophoresis, microporation with microneedles, radio-frequency ablation or the like), intravascularly, subcutaneously, intramuscularly, sublingually, intracranially, epidurally, rectally, and vaginally, among others. Desirably, the compound of Formula (I) may be administered by injection, transdermally or topically.
In one embodiment, the compound of Formula (I) maybe administered topically to the eye, e.g., as solutions, suspensions or ointments. Examples of ophthalmically compatible carriers which may be used include, without limitation, an aqueous solution, such as saline solution, oil solution or ointments containing ophthalmically compatible preservatives, surfactants, buffers, and viscosity regulators. These compositions may also contain stabilizing agents, antibacterial agents, and maybe manufactured in different dosage units, suitable for ocular administration. Drug inserts, either soluble or insoluble, may also be used.
In another embodiment, the compound of Formula (I) maybe administered by injection. Solutions for injection or infusion may be prepared as aqueous solutions.
Desirably, the compound of Formula (I) is present in a concentration of about 0.001 μg mL to 1 mg/mL, or this amount maybe adjusted higher or lower as needed. These solutions may also contain stabilizing agents, antibacterial agents, buffers and maybe manufactured in different dosage unit ampoules or bottles.
In a further embodiment, the compound of Formula (I) maybe administered rectally. Dosage units for rectal administration may be prepared in the form of ointments or suppositories, which contain the compound of Formula (I) in a mixture with a neutral fat base, or they may be prepared in the form of gelatin-rectal capsules that contain the compound of Formula (I) in a mixture with, e.g., a vegetable oil or paraffin oil. Ointments, suppositories or creams containing at least one compound of Formula (I) are useful for the treatment of hemorrhoids.
In yet another embodiment, the compound of Formula (I) maybe administered orally. Dosage units for oral administration include, without limitation, tablets, caplets, capsules, powders, suspensions, microcapsules, dispersible powder, granules, suspensions, syrups,. elixirs, and aerosols, which contain the compound of Formula (I) optionally with one or more excipient. In one embodiment, the compositions are compressed into a tablet or caplet. In another embodiment, the tablet or caplet maybe administered to the subject. In another embodiment, the tablet or caplet may be added to a capsule. In a further embodiment, the composition containing the compound of Formula (I) is added directly to a capsule. In one embodiment, the capsule includes hydroxypropyl methylcellulose, hypromellose capsule, or a hard shell gelatin capsule. In yet another embodiment the tablets or caplets are optionally film-coated using film-coatings known to those of skill in the art. In one embodiment, the film-coating is selected from among polymers such as, without limitation,
hydroxypropylmethylcellulose, ethyl cellulose, polyvinyl alcohols, and combinations thereof.
Although the compound of Formula (I) maybe administered alone, i.e., neat, it may also be administered in the presence of one or more pharmaceutical carriers that are physiologically compatible. The amount of the pharmaceutical carrier(s) is determined by the solubility and chemical nature of the compound of Formula (I), chosen route of administration, and standard pharmacological practice. The carriers may be in dry (solid) or liquid form and must be pharmaceutically acceptable. Liquid pharmaceutical compositions are typically sterile solutions or suspensions. When liquid carriers are utilized for parenteral administration, they are desirably sterile liquids. Liquid carriers are typically utilized in preparing solutions, suspensions, emulsions, syrups and elixirs. A variety of suitable liquid carriers is known and may be readily selected by one of skill in the art. Such carriers may include, .e.g., dimethylsulfoxide (DMSO), saline, buffered saline, cyclodextrin,
hydroxypropylcyclodextrin (HPpCD), n-dodecyl-P-D-maltoside (DDM) and mixtures thereof. In one embodiment, the compound of Formula (I) is dissolved a liquid carrier. In another embodiment, the compound of Formula (I) is suspended in a liquid carrier. One of skill in the art of formulations would be able to select a suitable liquid carrier, depending on the route of administration. The compound of Formula (I) may alternatively be formulated in a solid carrier of which a variety of solid carriers and excipients are known to those of skill in the art. In one embodiment, the composition maybe compacted into a unit dose form, i.e., tablet or cap let. In another embodiment, the composition maybe added to unit dose form, i.e., a capsule. In a further embodiment, the composition maybe formulated for administration as a powder. The solid carrier may perform a variety of functions, i.e., may perform the functions of two or more of the excipients described below. For example, a solid carrier may also act as a flavoring agent, lubricant, solubilizer, suspending agent, filler, glidant, compression aid, binder, disintegrant, or encapsulating material.
The composition may also be sub-divided to contain appropriate quantities of the compound of Formula (I). For example, the unit dosage can be packaged compositions, e.g., packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids.
Examples of excipients which may be combined with one or more compound of
Formula (I) include, without limitation, adjuvants, antioxidants, binders, buffers, coatings, coloring agents, compression aids, diluents, disintegrants, emulsifiers (e g. , polyoxyethylene fatty acid esters), emollients, encapsulating materials, fillers, flavoring agents, glidants, granulating agents, lubricants, metal chelators, osmo-regulators, pH adjustors (e.g., sodium hydroxide), preservatives, solubilizers, sorbents, stabilizing agents, sweeteners (such as saccharin), surfactants, suspending agents, syrups, thickening agents (e.g.,
carboxypolymethylene or hydroxypropylmethylcellulose), penetration enhancers (e.g., hydroxypolyethoxydodecane, DMSO, DMAC, DDM, etc) or viscosity regulators (such as polymers to increase viscosity). See, for example, the excipients described in the "Handbook of Pharmaceutical Excipients", 5th Edition, Eds.: Rowe, Sheskey, and Owen, APhA
Publications (Washington, DC), December 14, 2005, which is incorporated herein by reference.
In one embodiment, the compositions maybe utilized as inhalants. For this route of administration, compositions maybe prepared as fluid unit doses using a compound of Formula (I) and a vehicle for delivery by an atomizing spray pump or by dry powder for insufflation.
In another embodiment, the compositions maybe utilized as aerosols, i.e., oral or intranasal. For this route of administration, the compositions are formulated for use in a pressurized aerosol container together with a gaseous or liquefied propellant, e.g. ,
dichlorodifluoromethane, carbon dioxide, nitrogen, propane, and the like. Also provided is the delivery of a metered dose in one or more actuations.
In another embodiment, the compositions may be administered by a modified-release delivery device. "Modified-release" as used herein refers to delivery of a compound of Formula (I) which is controlled, for example over a period of at least about 8 hours (e.g., extended delivery) to at least about 12 hours (e.g., sustained delivery). Such devices may also permit immediate release (e.g., therapeutic levels achieved in under about 1 hour, or in less than about 2 hours). Those of skill in the art know suitable modified-release delivery devices. For use in such modified-release delivery devices, the compound of Formula (I) is formulated as described herein.
Also contemplated is the administration of the compounds of Formula (I) with other medication(s) or therapeutic agent(s). In one embodiment, the compounds of Formula (I) are combined with other medications or therapeutic agents in a single composition. However, the present disclosure is not so limited. In other embodiments, the compounds of Formula (I) may be administered in one or more separate formulations from other compounds of Formula (I), or other medications or therapeutic agents as described below.
Additionally, one or more agents typically used to treat inflammation may be used in conjunction with a combination of the disclosure in the methods, compositions, and kits described herein. Such agents include, but are not limited to, non-steroidal anti-inflammatory drugs.
The compound of Formula (I) maybe combined with glucose or dextrose when utilized for infusion or as a regional analgesic or anti-pruritic.
Further, the compound of Formula (I) maybe combined with thickening agents to form a jelly, or may also contain penetration enhancers, for use in topical or dermal applications such as for urogenital topical procedures.
Finally, the compound of Formula (I) maybe formulated as an ointment for administration to accessible mucous membranes.
Also provided herein are kits or packages of pharmaceutical formulations containing the compounds of Formula (I) or compositions described herein. The kits maybe organized to indicate a single formulation or combination of formulations to be taken at each desired time.
Suitably, the kit contains packaging or a container with the compound of Formula (I) formulated for the desired delivery route. Suitably, the kit contains instructions on dosing and an insert regarding the compound of Formula (I).
Optionally, the kit may further contain instructions for monitoring local or circulating levels of product and materials for performing such assays including, e.g., reagents, well plates, containers, markers or labels, and the like. Such kits are readily packaged in a manner suitable for treatment of a desired indication. For example, the kit may also contain instructions for use of an oral dosage form such as a pill, capsule, patch, spray pump or other delivery device. Other suitable components to include in such kits will be readily apparent to one of skill in the art, taking into consideration the desired indication and the delivery route.
The compounds of Formula (I) or compositions described herein can be a single dose or for continuous or periodic discontinuous administration. For continuous administration, a package or kit can include the compound of Formula (I) in each dosage unit (e.g., solution, lotion, tablet, pill, drug-eluting patch or other unit described above or utilized in drug delivery), and optionally instructions for administering the doses less-than-daily, daily, weekly, or monthly, for a predetermined length of time or as prescribed. When the compound of Formula (I) is to be delivered periodically in a discontinuous fashion, a package or kit can include placebos during periods when the compound of Formula (I) is not delivered. When varying concentrations of a composition, of the components of the composition, or the relative ratios of the compounds of Formula (I) or agents within a composition over time is desired, a package or kit may contain a sequence of dosage units which provide the desired variability.
A number of packages or kits are known in the art for dispensing pharmaceutical agents for periodic oral use. In one embodiment, the package has indicators for each period. In another embodiment, the package is a foil or blister package, labeled ampoule, vial or bottle.
The packaging means of a kit may itself be geared for administration, such as an inhalant, syringe, pipette, eye dropper, or other such apparatus, from which the formulation may be applied to an affected area of the body, such as the lungs, injected into a subject, or even applied to and mixed with the other components of the kit.
One or more components of these kits also may be provided in dried or lyophilized forms. When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another package.
The kits of the present disclosure also will typically include a means for containing the vials or other suitable packaging means in close confinement for commercial sale such as, e.g. , injection or blow-molded plastic containers into which the desired vials are retained. Irrespective of the number or type of packages and as discussed above, the kits also may include, or be packaged with a separate instrument for assisting with the injection/administration or placement of the composition within the body of an animal. Such an instrument maybe an inhalant, syringe, pipette, forceps, measuring spoon, eye dropper or any such medically approved delivery means.
In one embodiment, a kit is provided and contains a compound of Formula (I). The compound of Formula (I) may be in the presence or absence of one or more of the carriers or excipients described above. The kit may optionally contain instructions for administering the compound of Formula (I) to a subject having inflammation.
In a further embodiment, a kit is provided and contains a compound of Formula (I) in a second dosage unit, and one or more of the carriers or excipients described above in a third dosage unit. The kit may optionally contain instructions for administering the compound of
Formula (I) to a subject having inflammation.
As discussed above, the methods, compositions, and kits of the disclosure can be used to treat inflammation resulting from a number of conditions. The term "inflammation" as used herein includes all types of inflammation. In one embodiment, the inflammation may be acute or chronic. In another embodiment, the inflammation may be nociceptive,
dysfunctional, idiopathic, neuropathic, somatic, visceral, and/or procedural. For example, the inflammation maybe from a migraine, gynecological condition, pre-labor or labor, stroke, surgery, neuralgia, sickle cell, interstitial cystitis, urological condition (such as urethritis), dental work/injury, or headache, among other. Inflammation may also occur in patients with cancer, which may be due to multiple causes, such as nerve compression and mechanical forces resulting from tissue distension as a consequence of invasion by a tumor and tumor metastasis into bone or other tissues.
In one embodiment, inflammation results from neuropathy, such as post-herpetic neuralgia. In still another embodiment, the inflammation results from a surgery or procedure. In yet a further embodiment, the inflammation results from an infection, cancer, colitis, cystitis, irritable bowel syndrome, or idiopathic neuropathy
"Somatic inflammation" includes inflammation in bone, joint, muscle, skin, or connective tissue.
"Central inflammation" includes inflammation arising as a consequence of brain trauma, stroke, or spinal cord injury.
"Visceral inflammation" includes inflammation in visceral organs, such as the respiratory or gastrointestinal tract and pancreas, the urinary tract and reproductive organs. In one embodiment, visceral inflammation results from tumor involvement of the organ capsule. In another embodiment, visceral inflammation from obstruction of hollow viscus.
"Idiopathic inflammation" refers to inflammation which has no underlying cause or refers to inflammation caused by condition which remains undiagnosed.
"Dysfunctional inflammation" refers to inflammation which occurs in the absence of a noxious stimulus, tissue damage or a lesion to the nervous system. In one embodiment, dysfunctional inflammation results from rheumatologic conditions such as arthritis and fibromyalgia, tension type headache, irritable bowel disorders and erythermalgia.
"Nociceptive inflammation" includes inflammation caused by noxious stimuli that threaten to or actually injure body tissues. In one embodiment, nociceptive inflammation results from a cut, bruise, bone fracture, crush injury, burn, trauma, surgery, labor, sprain, bump, injection, dental procedure, skin biopsy, or obstruction. In another embodiment, nociceptive inflammation is located in the skin, musculoskeletal system, or internal organs.
"Neuropathic inflammation" is inflammation due to abnormal processing of sensory input by the peripheral or central nervous system consequent on a lesion to these systems. In one embodiment, neuropathic inflammation is chronic and non-malignant. In one embodiment, neuropathic inflammation is due to trauma, surgery, herniation of an intervertebral disk, spinal cord injury, diabetes, infection with herpes zoster (shingles), HIV/AIDS, late-stage cancer, amputation (such as mastectomy), carpal tunnel syndrome, chronic alcohol use, exposure to radiation, and as an unintended side-effect of neurotoxic treatment agents, such as certain anti-HIV and chemotherapeutic drugs.
"Procedural inflammation" includes refers to inflammation arising from a medical procedure. The medical procedure may include any type of medical, dental or surgical procedure. In one embodiment, the procedural inflammation is postoperative. In another embodiment, the inflammation is associated with an injection, draining an abscess, surgery, dermatological, dental procedure, ophthalmic procedure, arthroscopy and use of other medical instrumentation, and/or cosmetic surgery.
A "migraine" is a type of headache, typically defined clinically as being caused by activation of sensory fibers innervating the meninges of the brain.
The term "treat", "treating", or any variation thereof is meant to include therapy utilized to remedy a health problem or condition in a patient or subject. In one embodiment, the health problem or condition may be eliminated permanently or for a short period of time. In a further embodiment, the health problem or condition may be prevented. In another embodiment, the severity of the health problem or condition, or of one or more symptoms characteristic of the health problem or condition, may be lessened permanently, or for a short period of time. The effectiveness of a treatment of inflammation can be determined using any standard inflammation index, such as those described herein, or can be determined based on the patient's subjective inflammation assessment. A patient is considered "treated" if there is a reported reduction in inflammation, or a reduced reaction to stimuli that should cause inflammation.
In one embodiment, the treatment methods described herein include administering a compound of Formula (I) to a patient. Additional, optional agents, such as those described above for use in the combination, may be administered to the patient prior to, concurrently with, or subsequent to the compound of Formula (I).
The present disclosure is further exemplified, but not limited, by the following examples that illustrate the preparation of compounds of Formula (I) according to the disclosure
Examples
Example 1 : 4-(4-morpholinophenylamino)-2-phenylpyrimido [5,4-d]p ridazin-5(6H)-one
Figure imgf000045_0002
a: Ethyl 2-(bis(methylthio)methylene)-3-oxobutanoate
Figure imgf000045_0001
To a solution of ethyl acetoacetate (10 g, 76.9 mmol) in DMF (40 mL) was added K2CO3 (10.6 g, 76.9 mmol) and the reaction mixture was stirred at RT for 2 hours. Carbon disulfide (8.8 g, 230 mmol) was added, stirring was continued. After 2 hours, methyl iodide was added and stirring was continued for additional 10 hours. The reaction mixture was partitioned between ethyl acetate and water, the organic layer washed with water and dried over Na2S04 and the solvent evaporated in vacuum. The crude product was purified by column chromatography over silica gel using ethyl acetate and petroleum ether as eluent to give the desired product (8.8 g). 1H-NMR 400 Hz (CDC13): δ 4.29 (q, J = 7.2 Hz, 2H), 2.44 (s, 6H), 2.34 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H); MS m/z 235 (M+l). b: Ethyl 4-methyl-6-(methylthio)-2-phenylpyrimidine-5-carboxylate
Figure imgf000046_0002
To a solution of ethyl 2-(bis(methylthio)methylene)-3-oxobutanoate (500 mg, 2 mmol) in ethanol was added benzamidine acetate (732 mg, 6 mmol) and triethyl amine (1.41 mL) and the reaction mixture was refluxed overnight. The reaction mixture was concentrated and partitioned between ethyl acetate and water and the organic layer washed with water and dried over Na2S04 and the solvent evaporated in vacuum. The crude product was purified by column chromatography over silica gel using 1 % ethyl acetate in petroleum ether as eluent to give the desired product (350 mg). 1H-NMR 400 Hz (CDC13): δ 8.50 (d, J = 7.6 Hz, 2H), 7.50-7.46 (m, 3H), 4.45 (q, J = 7.2 Hz, 2H), 2.67 (s, 3H), 2.65 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H); MS m/z 288.7 (M+l). c: Ethyl 4-formyl-6-(methylthio)-2-phenylpyrimidine-5-carboxylate
Figure imgf000046_0001
To a solution of ethyl 4-methyl-6-(methylthio)-2-phenylpyrimidine-5-carboxylate (5 g 17.4 mmol) in 1.4-dioxane (50 mL) was added selenium dioxide (9.6 g, 86.5 mmol) and water (1.5 mL) and the reaction mixture was refluxed for 12 hours. Dioxane was removed in a vacuum and the crude solid suspended in ethyl acetate and filtered. The filtrate was concentrated and purified by column chromatography over silica gel using 10% ethyl acetate in petroleum ether as eluent to give the desired product (3.1 g). 1H- MR (400 Hz, CDCI3): δ 10.10 (s, 1H), 8.54 (d, J = 7.2 Hz, 2H), 7.56-7.43 (m, 3H), 4.47 (q, J
3H), 2.64 (s, 3H), 1.42 (t, J = 7.2 Hz, 3H); MS m/z 303.1 (M+l). d: 4-(memylthio)-2-phenylpyrimido[4,5-d]pyridazin-5(6H)-one
Figure imgf000047_0002
To a solution of ethyl 4-formyl-6-(methylthio)-2-phenylpyrimidine-5-carboxylate (600 mg, 1.98 mmol) in ethanol (15 mL) was added hydrazine dihydrochloride (0.2 g, 1.98 mmol) and the mixture refluxed for 1 hour. The precipitated solid was filtered and dried in vacuum. The desired product (520 mg) was obtained by column chromatography over silica gel using methanol/dichloromethane as eluent. 1H-NMR (400 Hz, DMSO-d6): δ 13.19 (s, 1H), 8.54 (d, J = 8.0 Hz, 2H), 8.36 (s, 1H), 7.64-7.58 (m, 3H), 2.67 (s, 3H); MS m/z 271.1 (M+l). e: 4-(4-Morpholinophenylammo)-2-phenylpyrimido[5,4-d]pyridazin-5(6H)-one
4-(Methylthio)-2-phenylpyrimido[4,5-d]pyridazin-5(6H)-one (0.1 g, 0.37 mmol) was dissolved in dichloromethane, m-chloro perbenzoic acid (0.19 g) was added and the reaction mixture was stirred room temperature for 10-12 hours. The solid precipitate was filtered and dried under vacuum. The crude product was dissolved in NMP, 4-(morpholinomethyl)aniline (45 mg, 0.25 mmol) was added and the reaction mixture was heated to 60°C for 30 minutes. The reaction mixture was cooled and ice water was added. The solid precipitate was filtered and dried under vacuum to yield the desired product (38 mg).
Example 2: Methyl 4-(5-oxo-4-(4-(piperazin-l-ylmethyl)phenylamino)-5,6- dihydropyrimido[4,5-d]pyridazin-2-yl)benzoate hydrochloride
Figure imgf000047_0001
a: (Z)-Ethyl 3-amino-2-(ethoxycarbonylcarbamothioyl)but-2-enoate
Figure imgf000048_0001
To a solution of ethyl chloroformate (lg, 9.2 mmol) in 1,4-dioxane cooled to 0°C was added ammonium thiocyanate (0.771 g, 10 mmol) and pyridine (0.726 g, 9.2 mmol) and the mixture was stirred at 0°C for 2 hours. The reaction mixture was extracted with diethyl ether, dried over Na2S04 and concentrated in vacuum to give crude ethyl isothiocyanatoformate. This crude product was added dropwise over a period of 2-3 hours to a solution of ethyl 3- aminocrotanoate (1.19 g, 9.2 mmol) in dioxane at 0-10°C. The reaction mixture was quenched with ice water and the resulting solid filtered and dried to get the desired crude product, which is used without further purification. 1H NMR (400 MHz, CDC13): δ 10.52 (s, 1H), 9.51 (s, 1H), 5.25 (s, 1H), 4.21-4.12 (m, 4H), 2.26 (s, 3H), 1.29-1.21 (m, 6H); MS m/z 261.1 (M+l). b: Ethyl 2-hydroxy-4-mercapto-6-methylpyrimidine-5-carboxylate
Figure imgf000048_0002
(Z)-ethyl 3-amino-2-(ethoxycarbonylcarbamothioyl)but-2-enoate was dissolved in 30% aqueous triethyl amine and the reaction mixture stirred at 70°C. After 2 hours, the reaction mixture was cooled and neutralized with glacial acetic acid and the aqueous mixture extracted with EtOAc. The organic layer was washed with water, dried over Na2S04 and concentrated under vacuum to give the desired product. 1H NMR (400 MHz, DMSO-de): δ 12.54 (s, 1H), 11.85 (s, 1H), 4.21 (q, J = 7.6 Hz, 2H), 2.03 (s, 3H), 1.24 (t. J = 7.6 Hz, 3H); MS m/z 213.1 (M-l). c: Ethyl 2,4-dichloro-6-methylpyrimidine-5-carboxylate
Figure imgf000048_0003
Ethyl 2-hydroxy-4-mercapto-6-methylpyrimidine-5-carboxylate (1.4 g, 6.54 mmol) was taken up in POCI3 (13.67 mL) at 0°C, tri-n-butylamine was added and the mixture heated to 90°C for 5 hours. The mixture was cooled, poured slowly on to crushed ice and extracted with dichloromethane. The organic layer was washed with water, dried over Na2S04 and concentrated under vacuum. The pure product was obtained by column purification over silica gel using 3% EtOAc/hexane as eluent^H NMR (400 MHz, CDC13): δ 4.47 (q, J = 6.8 Hz, 2H), 2.57 (s, 3H), 1.42 (t. J = 6.8 Hz, 3H); MS m/z 235.1 (M+l). d: Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin-l -yl)methyl)phenylamino)-2-chloro-6- methylpyrimidine-5-carboxylate
Figure imgf000049_0001
Ethyl 2,4-dichloro-6-methylpyrimidine-5-carboxylate (0.5 g, 2.1 mmol) and tert-butyl 4-(4-aminobenzyl)piperazine-l-carboxylate (0.6 g, 2.06 mmol) were dissolved in NMP and stirred at 0°C for 2 hour. The reaction mixture was poured onto water and extracted with
EtOAc. The organic layer was washed with water, dried over Na2S04 and evaporated under vacuum to get the desired compound (0.62 g). 1H NMR (400 MHz, CDC13): δ 10.58 (s, 1H), 7.58 (d, J = 6.4 Hz, 2H), 7.31 (d, J = 6.4 Hz, 2H), 4.45 (q, J = 6.8 Hz, 2H), 3.56 (s, 2H), 3.41 (m, 4H), 2.69 (s, 3H), 2.47 (m, 4H), 1.45 (s, 9H), 1.43 (t, J = 6.8 Hz, 3H); MS m/z 490.0
(M+l). e: Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin-l -yl)methyl)phenyli
(methoxycarbonyl)phenyl -6-methylpyrimidine-5-carboxylate
Figure imgf000049_0002
Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin- 1 -yl)methyl)phenylamino)-2-chloro-6- methylpyrimidine-5-carboxylate (0.8 g, 1.63 mmol), K2C03 (0.56g, 4 mmol), Pd(PPh3)4 (0.28 g, 0.24 mmol) were dissolved in dioxane/water (20:1), 4-(methoxycarbonyl)phenylboronic acid (0.38 g, 2.1 mmol) was added the mixture was heated to 100°C for 3 hours. The mixture was diluted with water and extracted with EtOAc. The organic layer was concentrated and purified by column chromatography over silica gel using 30% EtOAc/hexanes as eluent to give the desired product (0.7 g). 1H NMR (400 MHz, DMSO-d6): δ 9.88 (s, 1H), 8.43 (d, J = 8.0 Hz, 2H), 8.10 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 4.39 (q, J = 6.8 Hz, 2H), 3.89 (s, 3H), 3.49 (s, 2H), 2.68 (m, 4H), 2.50 (s, 3H), 2.33 (m, 4H), 1.91 (t, J = 6.8 Hz, 3H), 1.39 (s, 9H); MS m/z 590.2 (M+l). f: Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin- 1 -yl)methyl)phenylamino)-6-formyl-2-(4- (methoxycarbonyl)phenyl)pyrimidine-5-carboxylate
Figure imgf000050_0001
Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin- 1 -yl)methyl)phenylamino)-2-(4- (methoxycarbonyl)phenyl)-6-methylpyrimidine-5-carboxylate (0.63 g, 1.06 mmol) was dissolved in dioxane (10 mL), Se02 (0.6 g, 5.4 mmol) and water (0.096 mL) were added and the reaction mixture heated at 100°C for 3.5 hours. The reaction mixture was concentrated in vacuum and the mixture loaded on to a column of silica gel and eluted with MeOH, dichloromethane as eluent, to give the desired compound (0.52 g). 1H NMR (400 MHz, DMSO-de): δ 10.18 (s, 1H), 9.53 (s, lH), 8.56 (d, J = 8.4 Hz, 2H), 8.08 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 4.39 (q, J = 6.8 Hz, 2H), 3.89 (s, 3H), 3.41 (s, 2H), 3.26 (m, 4H), 2.35 (m, 4H)), 1.39 (s, 9H), 1.36 (t, J = 6.8 Hz, 3H); MS m/z 636.5 (M+l). g: tert-butyl 4-(4-(2-(4-(methoxycarbonyl)phenyl)-5-oxo-5,6-dmydropyrimido[4,5- d]pyridazin-4-ylamino)benzyl)piperazine- 1 -carboxylate
Figure imgf000051_0001
Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin- 1 -yl)methyl)phenylamino)-6-formyl-2- (4-(methoxycarbonyl)phenyl)pyrimidine-5 -carboxylate (0.5 g, 0.82 mmol) was dissolved in ethanol (5 mL), hydrazine hydrate (60 μL, 1.24 mmol) was added and the mixture is refluxed for 2.5 hours. The reaction mixture was cooled to obtain a solid precipitate. The precipitate was filtered and the solid dried under vacuum (0.4 g). 1H NMR (400 MHz, DMSO-d6): δ 13.41 (s, 1H), 11.52 (s, 1H), 8.52 (d, J = 8.4 Hz, 2H), 8.34 (s, 1H), 8.13 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 3.90 (s, 3H), 3.51 (s, 2H), 3.29 (t, J = 4.4 Hz, 4H), 2.34 (t, J = 4.4 Hz, 4H), 1.39 (s, 9H); MS m/z 572.3 (M+l). h: Methyl 4-(5-oxo-4-(4-(piperazin-l-ylmethyl)phenylamino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)benzoate hydrochloride
Tert-butyl 4-(4-(2-(4-(methoxycarbonyl)phenyl)-5-oxo-5,6-dmydropyrimido[4,5- d]pyridazin-4-ylamino)benzyl)piperazine-l -carboxylate (0.1 g, 0.175 mmol) was dissolved in dioxane.HCl (5 mL) and stirred at room temperature for 1.5 hours. The solid obtained was filtered and the residue washed with EtOAc and dried to give the desired product (40 mg).
Example 3: 2-morpholino-4-(4-(piperazin-l-ylmethyl)phenylamino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride
Figure imgf000052_0002
a: Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin-l -yl)methyl)phenylamino)-6-methyl-2- morpholinopyrimidine-5-carboxylate
Figure imgf000052_0001
Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin- 1 -yl)methyl)phenylamino)-2-chloro-6- methylpyrimidine-5-carboxylate (0.6 g, 1.23 mmol) was dissolved in MP (5 mL), morpholine (0.14 mL) was added and the mixture was stirred at room temperature for 45 minutes. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was washed with water, dried over Na2S04 and evaporated to give the desired product (0.53 g). 1H NMR (400 MHz, DMSO-d6): δ 10.48 (s, 1H), 7.56 (d, J = 8.4 Hz, 2H), 7.26 (d, J = 8.4 Hz, 2H), 4.30 (q, J = 6.8 Hz, 2H), 3.75 (s, 2H), 3.64 (m, 4H), 3.46-3.21 (m, 8H), 2.69 (s, 3H), 2.41 (m, 4H), 1.38 (s, 9H), 1.33 (t, J = 6.8 Hz, 3H); MS m/z 541.1 (M+l). b: (E)-ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin- 1 -yl)methyl)phenylamino)-6-(2- (dimethylamino)vinyl)-2-morpholinopyrimidine-5-carboxylate
Figure imgf000053_0001
Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin- 1 -yl)methyl)phenylamino)-6-methyl-2- morpholinopyrimidine-5-carboxylate (350 mg, 0.64 mmol) was dissolved in DMF (5 mL), dimethyl formamide dimethyl acetal (0.26 mL) was added and the mixture was heated tol30°C for 12 hours. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was washed with water, dried over Na2S04 and evaporated to give the desired product (0.23 g). 1H NMR (400 MHz, DMSO-d6): δ 10.44 (s, 1H), 7.94 (d, J = 12.2 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 8.8 Hz, 2H), 5.89 (d, J = 12.2 Hz, 1H), 4.30 (q, J = 6.8 Hz, 2H), 3.80 (s, 2H), 3.75-3.65 (m, 8H), 3.33 (m, 4H), 3.32 (m, 6H), 2.41 (m, 4H), 1.38 (s, 9H), 1.32 (t, J = 6.8 Hz, 3H); MS m/z 596.4 (M+l). c: Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin-l -yl)methyl)phenylamino)-6-formyl-2- morpholinopyrimidine-5-carboxylate
Figure imgf000053_0002
(E)-ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin- 1 -yl)methyl)phenylamino)-6-(2- (dimethylamino)vinyl)-2-morpholinopyrimidine-5-carboxylate (0.2 g, 0.336 mmol) was dissolved in methanol (4 mL) and to this mixture at room temperature was added a solution of sodium periodate (0.21 g, 1 mmol) in methanol (4 mL). The mixture was stirred at room temperature for 3 hours and the precipitate filtered. The filtrate was evaporated, diluted with water and extracted with EtOAc. The organic layer was dried over Na2S04 and evaporated in vacuum. The crude product was purified by column chromatography over silica gel using acetone/dichloromethane as eluent. (0.11 g). 1H NMR (400 MHz, DMSO-d6): δ 10.07 (s, 1H), 10.02 (s, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 4.31 (q, J = 6.8 Hz, 2H), 3.83 (s, 2H), 3.75-3.65 (m, 8H), 3.33 (m, 4H), 2.32 (m, 4H), 1.38 (s, 9H), 1.31 (t, J = 6.8 Hz, 3H); MS m/z 555.5 (M+l). d: 2-morpholino-4-(4-(piperazin- 1 -ylmethyl)phenylamino)pyrimido[4,5-d]pyridazin-5(6H)- one hydrochloride
Ethyl 4-(4-((4-(tert-butoxycarbonyl)piperazin- 1 -yl)methyl)phenylamino)-6-formyl-2- morpholinopyrimidine-5-carboxylate (100 mg, 0.18 mmol) was dissolved in ethanol, hydrazine dihydrochloride (28 mg, 26 mmol) was added and the mixture was refluxed for 3 hours. The solid precipitated was filtered and dried in vacuum to give the desired product (49 mg).
By repeating the procedures described in the above examples, using appropriate starting materials, the following compounds of Formula I, as identified in Table 1, are obtained, together with their spectroscopic data in Table 2.
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
yl)acetic acid
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Figure imgf000114_0001
Figure imgf000115_0001
Figure imgf000116_0001
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
yl)acetic acid
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
Figure imgf000130_0001
Figure imgf000131_0001
Figure imgf000132_0001
Figure imgf000133_0001
Figure imgf000134_0001
Figure imgf000135_0001
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000138_0001
Figure imgf000139_0001
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001
Figure imgf000143_0001
Figure imgf000144_0001
Figure imgf000145_0002
The following compounds in Table A are prepared by one of skill in the art using the above-noted Schemes, descriptions, and Examples 1-339.
Figure imgf000145_0001
Figure imgf000146_0001
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
Figure imgf000155_0001
Figure imgf000156_0001
Figure imgf000157_0001
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
Figure imgf000161_0001
Figure imgf000162_0001
Figure imgf000163_0001
Figure imgf000164_0001
Figure imgf000165_0002
Figure imgf000165_0001
Figure imgf000166_0001
Figure imgf000167_0001
Figure imgf000168_0001
Figure imgf000169_0001
Figure imgf000170_0001
Figure imgf000171_0001
Figure imgf000172_0001
Figure imgf000173_0001
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
Figure imgf000184_0001
Figure imgf000185_0001
Figure imgf000186_0001
Figure imgf000187_0001
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000200_0001
Figure imgf000201_0001
Figure imgf000202_0001
Figure imgf000203_0001
Figure imgf000204_0001
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
Figure imgf000208_0001
Figure imgf000209_0001
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
Figure imgf000219_0001
Figure imgf000220_0001
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001
Figure imgf000225_0001
Figure imgf000226_0001
Figure imgf000227_0001
Figure imgf000228_0001
Figure imgf000229_0001
Figure imgf000230_0001
Figure imgf000231_0001
Figure imgf000232_0001
Figure imgf000233_0001
Figure imgf000234_0001
Figure imgf000235_0001
Figure imgf000236_0001
Figure imgf000237_0001
Figure imgf000238_0001
Figure imgf000239_0001
Figure imgf000240_0001
Figure imgf000241_0001
Figure imgf000242_0001
Figure imgf000243_0001
Figure imgf000244_0001
Figure imgf000245_0001
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
Figure imgf000251_0001
Figure imgf000252_0001
Figure imgf000253_0001
Figure imgf000254_0001
Figure imgf000255_0001
Figure imgf000256_0001
Figure imgf000257_0001
Figure imgf000258_0002
The following compounds are anticipated to result in MS having M values noted in the following Table B.
Figure imgf000258_0001
Figure imgf000259_0001
Figure imgf000260_0001
Figure imgf000261_0001
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Figure imgf000265_0001
Figure imgf000266_0001
Figure imgf000267_0001
Figure imgf000268_0001
Figure imgf000269_0001
Figure imgf000270_0001
Figure imgf000271_0001
Figure imgf000272_0001
Figure imgf000273_0001
Figure imgf000274_0001
Figure imgf000275_0001
Figure imgf000276_0001
Example 340: Inhibition of enzymatic Syk kinase activity
The objective of this assay was to examine by radiometric method the ability of compounds to inhibit Syk kinase enzyme.
A. Background
Spleen tyrosine kinase (Syk) is a cytosolic protein tyrosine kinase that plays a crucial role in inflammatory and allergic responses. Syk triggers IgE and IgG receptor mediated signaling in mast cells, basophils, and macrophages leading to degranulation and cytokine release. Abnormal function of Syk has also been implicated in several instances of hematopoietic malignancies.
Syk is capable of phosphorylating substrates such as VAV, LAT, SLP-76, which in turn activate MAPK, PLCy signaling pathways. Crystallization studies of the Syk catalytic domain (360-635) showed more activity compared to the full length Syk enzyme. This in vitro assay tests the ability of syk to phosphorylate a substrate peptide in the presence of ATP. By using a radio-labeled form of ATP, it is possible to measure the amount of phosphorylation of the substrate. The enzyme transfers a radio-labeled phosphate group from γ32 P labeled ATP to pG4T. Briefly the enzyme was incubated with substrate, radio-labeled
& cold ATP and substrate in buffer with or without compounds. At the end of the reaction, the reaction mixture was transferred on to a Multiscreen filter plate and unreacted γ 32 P ATP was washed off. The filter plate was dried and the radioactivity was measured on a scintillation counter to estimate the incorporated radioactivity on the substrate. The percent inhibition of activity of the enzyme was calculated by comparing counts in the presence and absence of compounds.
B. Reagents and Instruments
Figure imgf000277_0001
* See, Figure 2A of Law, "Molecular Cloning of Human Syk", J. Biol. Chem., 269(16): 12310-12319 (1994) which provides the full-length amino acid sequence for human
Syk. the fragment utilized included a C'-terminal tag of 4 amino acids and a stretch of 15 amino acids N-terminal to the kinase domain, starting at amino acid 356.
See, also, Yagi, "Cloning of the cDNA for the Deleted SYK Kinase Homologous to ZAP-70 from Human Basophilic Leukemia Cell Line (KU812)", Biochem. Biophys. Res. Commun., 200(l):28-34 (1994). Both of these publications are incorporated by reference herein.
Figure imgf000277_0002
Figure imgf000278_0002
C. Protocol
2.5 μL of 10% DMSO or compound in 10% DMSO was added to the wells in a 96 well V-bottom plate. Optimized concentration of in-house Syk enzyme (different batches of Syk (356-635) kinase domain) were used at optimized concentrations) ranging from 0.035 ng to 7.5 ng/reaction diluted in assay buffer was added to a total volume of 12.5 μL).
Compound and protein were incubated for 30 minutes at room temperature on a plate shaker. Ten μL of a substrate mix containing 100 μΜ ATP (0.25 μL), γ-Ρ32-ΑΤΡ (0.1 μL; 10 μCi/μL), pG4T (0.25 uL; 10 mg mL) and IX assay buffer (9.4 μL) was added to all the wells. Samples were incubated at 30°C for 10 minutes after mixing. The reaction was stopped by the addition of 8N HC1 (13 μL) containing 100 mM ATP. Thirty μL of sample was transferred to the center of a 2 x 2 cm Whatman® P81 chromatography paper. After allowing the sample to dry for one minute, the assay squares were washed 3 times for 5 minutes each in ortho-phosphoric acid (0.5%) and once in acetone. Assay squares were dried for 15 minutes in a 30°C oven and transferred to 96 well optiplate. Microscint-O® reagent (100 μL, Perkin Elmer) was added to each well, the plate was sealed with Topseal®-A microplates and incubated for 10 minutes at room temperature at very low speed on rocker and the plate was read in the Topcount® NXL instrument.
The following calculations were made:
Fold induction = radioactivity counts (uncorrected values) in positive control/substrate control.
Percent inhibition was calculated with the corrected values:
Figure imgf000278_0001
The % inhibitions of the compound vs. concentrations of NCE were plotted using Graphpad® Prism software to calculate the IC50 of the active NCE. See, Rossi, J. Allergy Clin. Immunol. (2006), 118(3):749-755 and Eva Papp, "Steady State Kinetics of Spleen Tyrosine Kinase Investigated by a Real Time Fluorescence Assay", Biochemistry (2007) 46:15103-15114, which are hereby incorporated by reference. Example 341: Inhibition of enzymatic JAK kinase activity
The objective of this assay was to screen compounds in a Time-resolved fluorescence resonance energy transfer (TR-FRET) Enzymatic assay method for their potential to inhibit JAK (Janus kinase) activity. Compounds which inhibit Syk and JAK in these studies maybe potentially used in treating inflammation.
A. Background
JAK (Janus kinase 2) is a family of intracellular non-receptor tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway. These kinases have apparent molecular weight of about 130 Kda. They were initially named "just another kinase" 1 & 2 (since they were just two of a large number of discoveries in a PCR-based screen of kinases), but were ultimately published as "Janus kinase". JAKs possess two near-identical phosphate-transferring domains. One domain exhibits the kinase activity while the other negatively regulates the kinase activity of the first. They are crucial signal transducers for a variety of cytokines, growth factors and interferons.
TR-FRET assays are homogeneous proximity assays where Eu-labeled antiphosphotyrosine antibody binds to the phosphorylated substrates labeled with Ulight fluorescence acceptor. Eu can transfer energy to Ulight accepter in the complex and the interaction of two dye-labeled binding partners is detected by the energy transfer between a donor and an acceptor dye, and the subsequent light emission by the acceptor dye. The intensity of the light emission is proportional to the level of Ulight peptide phosphorylation.
See, Rodig, "Disruption of the Jakl gene demonstrates obligatory and nonredundant roles of the JAKs in cytokine-induced biologic responses", Cell, 93(3):373-83 (1998) and Yamaoka, "The Janus kinases (Jaks)", Genome Biology, 5:253 (2004), which are incorporated herein by reference.
B. Reagents and Equipment
Figure imgf000279_0001
Figure imgf000280_0002
Figure imgf000280_0001
C. Protocol
Two μL of 10% DMSO in blank, substrate control and positive control wells and 2 μL of test compound in test wells was added. Thirteen μL of assay buffer in blank and substrate control wells and 13 μL of Enzyme buffer mix in positive and test wells was added. The reaction mixture was incubated for 30 minutes at RT on a plate shaker. Ultra Light-pGT substrate (5 μL) [poly Glu-Tyr (4:1) labeled with U Light TM dye, a tyrosine kinase substrate] and ATP mix was added to all wells. The reaction plate was incubated for 60 minutes at RT on a plate shaker. The reaction was stopped by adding 40 mM EDTA (10 μL) in buffer. Ten μL of antibody was added to all the wells. The plate was read in a Wallac® 1420 Multilabel Counter Victor 3 instrument (Ex: 340 nm Em: 615 & 665 nm) The following calculations were made:
F@ 665 Value-Buffer blank
F@ 615 Value-Buffer blank
Ratio: (F@665 Buffer blank/F@615 Buffer blank)* 10000
Ratio of F@665/F@615- Substrate Blank
% Activity= (Test Sample/Positive control) *100
% Inhibition= (100-% Activity)
Figure imgf000280_0003
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Example 342: Degranulation assay
The objective of this assay was to examine by Fluorescence method the effect of compounds on β-hexosaminidase release during immune complex mediated degranulation in RBL2H3 cells. A. Introduction
Auto-antibodies and their immune complexes (ICs) reacting with self antigens through immunoglobulin receptors have been implicated widely in inflammation and chronic inflammatory disease such as rheumatoid arthritis. Activation of the high affinity receptor for immunoglobulin E (IgE), FCERI, which is expressed on the surface of mast cells and basophils, plays a central role in the initiation of these allergic responses. Following aggregation of the receptor by ICs, the mast cell release a variety of potent biologically active molecules, including cytokines, lipid-derived mediators, amines, protease, and proteoglycans. Anti-DNP (anti-dinitrophenyl) IgE treated RBL2H3 cells on stimulation with DNP-BSA leads to FCERI cross linking which mediates release of various pro-inflammatory molecules including β-hexosaminidase.
Compounds were tested for their ability to inhibit the ability of this immune complex to mediate β-hexosaminidase release, in an enzyme assay with p-nitrophenyl-P-D- glucosaminide as substrate. The fluorescence of the product 4-methylumbellifernone was monitored (Excitation 355 nm; Emission 460 nm).
B. Reagents and Instruments
Figure imgf000380_0001
Figure imgf000381_0002
Figure imgf000381_0001
C. Protocols
(i) Protocol A: 24 well format
RBL2H3 cells were maintained in MEM complete media containing 10% FBS at 70% - 80% confluence in a mammalian cell culture C02 incubator with 5% C02 at 37°C. 2 x 105 cells/well were plated in 1 mL of complete media and incubated for 5 hours for cell attachment. Complete media was replaced with 1 mL of serum free MEM media containing 1.2 μg/mL of anti-DNP rat IgE as sensitizing agent and further incubated overnight with 5% C02 at 37°C. The following day, cells were washed with serum free media and further treated with various concentrations of test compounds (in 0.1% DMSO) for 45 minutes at 37°C and 5% C02. Cells were further stimulated with 5 μg/mL of D P-BSA for 60 minutes. Plates were centrifuged for 5 minutes at 1000 rpm and 25 μL of culture supernatant was transferred from each assay well into a 96 well black coated plate. 25 μL β-NAG substrate was added to this mixture and incubated at room temperature for 30 minutes. The reaction was terminated with 100 μL of stop solution and fluorescence was monitored. (Excitation 355 nm; Emission 460 nm) See, Sanderson, (2010), Cellular Immunology, 262(1): 28-34 and Silverman, (2006) MCB, 26(5): 1826-1838, which are incorporated herein by reference.
The % release of β-Hexosaminidase for the test compound was calculated using the following formula:
Figure imgf000382_0001
(ii) Protocol B: 96 well format
RBL2H3 cells were maintained in MEM complete media containing 10% FBS at 70-80% confluence in a mammalian cell culture C02 incubator with 5% C02 at 37°C. 5 x 104 cells/well were plated in 200 uL of complete media containing 0.3 μg/mL of anti-DNP rat IgE as sensitizing agent for 24 hours at 37°C & 5% C02. The following day, cells were washed twice with PIPES buffer for 10 minutes at 37°C and replenished with serum free MEM media. Cells were treated with various concentrations of test compounds (in 0.5% DMSO) for 15 minutes at 37°C and 5% C02. The cells were further stimulated with 0.1 μg/mL of DNP-BSA for 45 minutes. The plates were spun for 5 minutes at 2000 rpm and 25 μL of culture supernatant was then transferred from each assay well into a 96 well black coated plate. Fifth μL β-NAG substrate was added and incubated at RT for 30 minutes. After incubation with substrate, 150 μL of stop solution was added and fluorescence was monitored. (Excitation 355 nm; Emission 460 nm). See, Yamamoto, JPET, 306(3):1174- 1181 (2003) and Taylor, MCB, 15(8): 4149-4157 (1995), which are herein incorporated by reference.
Release of β-hexosaminidase during the degranulation process by immune complex mediated FCERI stimulation is through the SYK pathway. The % inhibition of β- hexosaminidase release by Syk inhibitor gives information with regard to its Syk inhibition potency. Thus compounds having lower EC50 values are more potent in inhibiting immune complex mediated Syk signaling during degranulation process.
Figure imgf000383_0001
Figure imgf000384_0001
Figure imgf000385_0001
Figure imgf000386_0001
Example 343: In vivo assay - Chronic study
A. Introduction
Collagen Induced Arthritis (CIA) is a well characterized model of human rheumatoid arthritis (RA) that can be induced in susceptible animals following immunization with type II collagen (ell) in Freund's adjuvant. CIA exhibits several features of human RA such as severe swelling inflammation of joints, synovial hyperplasia, cartilage destruction and bone erosion. Pathophysiology of CIA consists of T cell component, as evidenced by increased infiltration of T-cells in joint synovium and also, by attenuation of CIA in T-cell deficient mice. CIA development involves B cell component too, as is evidenced by circulating ell antibody in disease animals and also, failure to develop the disease in xid mice/B cell deficient mice/CXCR5 null mice. Recently, a significant role of macrophages has also been suggested in the pathogenesis of CIA as well as human RA. See, Pine, "Inflammation and bone erosion are suppressed in models of rheumatoid arthritis following treatment with a novel Syk inhibitor", Clin. Immunol, 2007, 124(3 ):244-57; Xiong cha, "Suppression of the onset and progression of collagen-induced arthritis in rats by QFGJS, a preparation from an anti-arthritic Chinese herbal formula", J. Ethnopharmacology (2007) 110:39-48; and Stolina, "The evolving systemic and local biomarker milieu at different stages of disease progression in rat collagen induced arthritis", Biomarkers (2008) 13(7-8):692-712, which are herein incorporated by reference.
B. Method
(i) Induction of CIA:
Female Lewis rats (8 per group, 6-8 weeks old) were immunized on day 1 with type II collagen (Immunization grade Bovine type II; Chondrex; Cat #20021) emulsified with Complete Freund's Adjuvant (Sigma; Cat# F5881) at a final concentration of 1.2 mg/mL). For the initial immunization, the animals were injected at the base of the tail with 300 μg of the ell (0.25 mL/rat). A booster injection of the same type II collagen emulsified with Incomplete Freund's Adjuvant (Sigma, Cat #F5506) (0.25 mL/rat) was given to the animals on day 8 at the base of the tail (100 μg). The final ell concentration in the booster was 0.4 μg/mL. (ii) Dosage regimen
Animals with an arthritic score of > 1 were grouped and dosing with test compound (30 mg/kg bid) or methotrexate (0.5 mg/kg) started between about day 12 to day 14, with daily dosing of their respective compounds continuing for 10 days.
(iii) Measurements:
Edema: Paw volumes are measured by Plethysmometry for the animals before induction of CIA (Basal readings) and on Day 1, 3, 6 and 9 of dosing period. Both hind paw volumes were measured and edema was calculated by subtracting from the basal mean.
Arthritic score: Animals were scored for the symptoms of arthritis every day starting from Day of onset of disease till the end of the study. Both the hind paws were scored and the total scores were averaged and compared with control. The scoring pattern was as follows:
Figure imgf000388_0003
C. Results
(i) Calculations
The percent inhibition of Edema was calculated with respect to control by the formula:
Figure imgf000388_0002
The percent inhibition of Arthritic score was calculated with respect to control by the formula:
Figure imgf000388_0001
(ii) Statistical analysis
Means of different groups were compared with control using one way ANOVA followed by Dunnett's test. Significance is represented as follows.
This example illustrates that the compounds may be utilized for treating inflammation. See Figures 1-2.
Figure imgf000389_0001
Example 344: In vivo assay - Acute study
A. Introduction
Arthus reaction is a type of local type III hypersensitivity reaction. Type III hypersensitivity reactions are immune complex-mediated, and involve the deposition of antigen/antibody complexes mainly in the vascular walls, serosa (pleura, pericardium, synovium), and glomeruli. This involves formation of antigen antibody complexes after the intradermal injection of an antibody. If the animal was previously injected with antigen and dye (has circulating antigen), an Arthus reaction occurs. This manifests as local vasculitis due to deposition of immune complexes in dermal blood vessels. Activation of complement and recruitment of PMNs ensue, resulting in an inflammatory response and extravasation of dye to the skin. Compounds which can inhibit this complex process can have therapeutic implications in wide range of inflammatory and auto-immune disorders. See, Pine,
"Inflammation and bone erosion are suppressed in models of rheumatoid arthritis following treatment with a novel Syk inhibitor"; Clin. Immunol. (2007) 124 (3): 244-57; and Sylvia, "R-406, an Orally Available Spleen Tyrosine Kinase Inhibitor Blocks Fc Receptor Signaling and Reduces Immune Complex-Mediated Inflammation", JPET 319:998-1008, 2006, which are herein incorporated by reference.
B. Immunization and challenge
Female c57BL/6 mice were given an antigen injection in which the antigen was 0.1% Ovalbumin (OVA) in PBS containing 1% Evans blue (EB) at the concentration of 10 mL/kg intravenously under Isoflurane anesthesia [2.5 mg/mouse with a body weight of 25 g]. Ten minutes after antigen injection; the animals were injected with the rabbit anti-OVA IgG (50 μg in 25 μΐνβϊίε) (Polysciences; Cat #23744) intradermally on the shaved back at two top locations. Animals were also injected with phosphate buffered saline (PBS, 25 μL) intradermally on the back at two bottom and opposite locations to serve as negative control. The mice were euthanized by cervical dislocation 4 hours after antigen (Ovalbumin) challenge. Skin tissue was assessed for edema by tracing the edema area on to a transparent plastic sheet. Punch biopsies of the injection sites were collected.
C. Measurements
(i) Area of extravasation
Edema area was measured manually by scale. Two diameters were taken and averaged for each animal.
(ii) Extent of dye extravasation
Punch biopsies of the injection sites (using 10 mm skin biopsy punches) were incubated in 2 mL of sodium sulfate: acetone mixture (0.6 + 1.4mL) at room temperature for 16-18 hours. The supernatants were removed from digested tissues by centrifuging at 4000 rpm for 10 minutes, filtered and were read spectrophotometrically at 610 nm.
D. Data analysis
The percent inhibition of dye leakage was calculated with respect to control by the formula:
Percent inhibition of OD =
Figure imgf000390_0001
The percent inhibition of edema area was calculated with respect to control by the formula:
Percent inhibition of edema area =
Figure imgf000390_0002
E. Statistical analysis:
Means of different groups were compared with control using One way ANOVA followed by Dunnett's test. Significance was represented as follows.
This example illustrates that the compounds described herein may be utilized in treating inflammation.
Figure imgf000391_0001
As discussed above, combination treatments for treating cancers involving targeted oncologic therapies (e.g., inhibition of specific biochemical pathways) and immunotherapeutic agents would be expected to decrease treatment efficacy and/or increase adverse side effects, because targeted therapies with immuno stimulatory potential can also display immunosuppressive activities. The activity of targeted therapies may therefore cancel out, and even inhibit, therapeutic mechanisms of imunomodulators. For example, the combination of dual inhibitors of Syk/JAK family kinases according to the disclosure and immunotherapeutic agents for the treatment of cancers would be expected to result in a decrease in efficacy. In addition, such combination treatments can result in increases in toxicity and adverse side effects, for example by increasing cytokine production and other inflammatory reactions related to the non-tumor suppressive activities of the immunotherapeutic agents.
The inventors unexpectedly discovered that pharmaceutical combinations comprising a compound of Formula (I), as described herein, and one or more immunotherapeutic agents can be used to treat cancers, where the combination has a reduced adverse side effect profile and where the activity of the immunotherapeutic agent is not suppressed. In an embodiment, the anticancer or antitumor activity of pharmaceutical combinations of the present disclosure may be substantially the same as, or may be increased, as compared to administering a compound of Formula (I) alone. In an embodiment, the pharmaceutical combinations of the present disclosure can reduce negative side effects in patients, for example by inhibiting or suppressing chemokine and/or cytokine levels.
In one embodiment, a pharmaceutical combination is provided, comprising the combination comprising a compound of Formula (I), as described herein, and at least one immunotherapeutic agent.
The at least one immunotherapeutic agent can comprise one or more immunomodulators. The immunomodulators can target one or more components of the immune system, including (without limitation) PD-1, PD-L1, CTLA-4, 4-1BB, OX40, LAG3, GITR, TIM3, VISTA, KIR, ICOS, BTLA, CD244, CD80, CD86, PD-L2, IDO-1, IDO-2, and B7-H3, and can produce a therapeutic effect. The one or more immunomodulators can comprise (without limitation) inhibitors of immune system components, activators of immune system components, and/or immune checkpoint inhibitors, and can comprise biologic or small molecule therapeutics. Other immune modulators that can be combined with a compound of Formula (I) can include, for example (without limitation), indoleamine (2,3)-dioxygenase (IDO) inhibitors, vaccines, and agents that target T-cell receptors (TCR agents). In an embodiment, TCR agents can include (without limitation) chimeric antigen receptor (CAR) T cells, and TCR agonist or antagonist peptides. CAR T cells can comprise (without limitation) CTL019 and JCAR015 etc. In an embodiment, the vaccines can include (without limitation) sipuleucel-T (Provenge®) and Talimogene laherparepvec (Imlygic®), aslo known as T-Vec.
In an embodiment, checkpoint inhibitors can include (without limitation) co- inhibitory molecules such as cytotoxic T-lymphocyte-as so dated protein 4 (CTLA-4), PD-1, lymphocyte-activation gene 3, and T-cell immunoglobulin mucin-3, and co-stimulatory molecules such as glucocorticoid-induced tumor necrosis factor receptor and OX40 (CD134, TNFRSF4, tumor necrosis factor receptor superfamily member 4). In an embodiment, the immune checkpoint inhibitors can include (without limitation), anti-PD-1 antibody, anti-PD- Ll antibody, anti-CTLA-4 antibody, anti-4-lBB antibody, anti-OX40 antibody, anti-LAG3 antibody, anti-GITR antibody, anti-TIM3 antibody, anti- VISTA antibody, anti-KIR antibody, anti-ICOS antibody, anti-BTLA antibody, anti-CD244 antibody, anti-CD80 antibody, anti- CD86 antibody, anti-PD-L2 antibody, anti-IDO-1 antibody, anti-IDO-2 antibody, anti-B7-H3 antibody, and small molecule inhibitors of any of these antibody targets, for example an IDO inhibitor drug. In an embodiment, the immune checkpoint inhibitor is anti-PD-1 antibody. In another embodiment, the immune checkpoint inhibitor is small molecule IDO-1 inhibitor. The pharmaceutical combination can further comprise at least one pharmaceutically acceptable carrier.
The disclosure also provides methods of treating cancer, the methods comprising administering to a patient in need thereof a multi-part pharmaceutical combination comprising a compound of Formula (I), as described herein, and at least one immunotherapeutic agent, as described herein. The compounds of Formula (I) can be administered intravenously or orally, or by any other suitable manner. The immunotherapeutic agent can be administered intravenously, subcutaneously, intramuscularly, or orally, or by any other suitable manner. In one embodiment, administration of the compound is intravenous and the administration of the at least one immunotherapeutic agent is intravenous, subcutaneous or intra-muscular. In another embodiment, administration of the compound is oral and the administration of the at least one immunotherapeutic agent is oral. In yet another embodiment, administration of the compound is intravenous and the administration of the at least one immunotherapeutic agent is oral. In yet another embodiment, administration of the compound is oral and the administration of the at least one immunotherapeutic agent is intravenous, subcutaneous or intra-muscular.
The compound of Formula (I) and the immunotherapeutic agent can be administered by any suitable dosing regimen. In one embodiment, the compound of Formula (I) is administered once or twice daily. The compound and the at least one immunotherapeutic agent can be administered simultaneously or sequentially. In one emdbodiment, the compound and the at least one immunotherapeutic agent are administered sequentially. In one embodiment, the at least one immunotherapeutic agent is administered intermittently every four to thirty days.
In some embodiments, the compound of Formula (I) can be administered to a patient at a dosage of about 5 mg/kg to about 70 mg/kg, preferably from about 10 mg/kg to about 60 mg kg. In an embodiment, the compound of Formula (I) is administered at a dosage of about 10 mg/kg, about 30 mg/kg, or about 60 mg/kg. In other embodiments, the compound of Formula (I) can be administered to a patient at a dosage of about 10 mg, 20 mg, 30 mg, 40 mg, 40 m g, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg or 120 mg once or twice per day.
Any variety of cancers can be treated using the pharmaceutical combinations provided here, including (without limitation) the following cancers: prostate, head, neck, eye, mouth, throat, esophagus, bronchus, larynx, pharynx, chest, bone, lung, colon, rectum, stomach, bladder, uterus, cervix, breast, ovaries, vagina, testicles, skin, thyroid, blood, lymph nodes, kidney, liver, intestines, pancreas, brain, central nervous system, adrenal gland, skin or a leukemia and/or lymphoma.
In one embodiment, methods of the present disclosure include ^identifying an indication or patient population for administering the pharmaceutical combinations of the present disclosure.
The disclosure provides methods of inhibiting tumor growth or metastasis in a subject, the methods comprising administering to the subject a multi-part pharmaceutical combination comprising a compound of Formula (I), as described herein, and at least one immunotherapeutic agent, as described herein, wherein the administration of the pharmaceutical combination inhibits tumor growth or metastasis. Inhibition of tumor growth or metastasis means reducing the number of cancer cells or causing the amount of cancer cells to remain substantially the same, reducing tumor size or causing the tumor size to remain substantially the same, inhibiting metastasis (including inhibition of tumor cell migration and/or invasion), inhibiting tumor growth and/or ameliorating one or more of the symptoms of the cancer. Such inhibition of tumor growth or metastasis is sometimes referred to herein as "TGI." As described herein, a therapeutically effective amount of a pharmaceutical combination when used for the treatment of cancer is an amount which may inhibit tumor growth or metastasis.
In one embodiment, administration of the pharmaceutical combinations described herein can achieve TGI in an amount of 0% to 100%, preferably an amount of above about 50%, for example about 50% to 90%, or about 60% to 80%.
The disclosure provides dosing regimens comprising administering to a patient in need a multi-part pharmaceutical combination comprising a compound of Formula (I), as described herein, and at least one immunotherapeutic agent, as described herein. Administration of the compound of Formula (I) and the immunotherapeutic agent can be performed as described herein.
The disclosure provides kits comprising at least one first dosage form comprising a compound of Formula (I), as described herein, and at least one second dosage form comprising at least one immunotherapeutic agent, as described herein, and administering to a patient in need the compound of Formula (I) and the immunotherapeutic agent. Administration of the compound of Formula (I) and the immunotherapeutic agent can be performed as described herein. The disclosure provides uses of a medicament in treating cancer, wherein the medicament is administered by a dosing regimen comprising administering to a patient in need a compound of Formula (I), as described herein, and at least one immunotherapeutic agent, as described herein. Administration and the dosing regimen of the compound of Formula (I) and the immunotherapeutic agent can be performed as described herein.
As discussed below with respect to Example 345, the inventors unexpectedly discovered that the pharmaceutical combinations of the present disclosure can result in decreased toxicity in patients. Example 345 demonstrates that the pharmaceutical combinations described herein can suppress cytokines and can prevent cytokine release syndrome. Suppression of cytokines can result in a decrease in adverse side effects and toxicity related to cytokine production in patients.
EXAMPLE 345: Evaluation of inflammation biomarkers in cancer patients treated with COMPOUND A
To assess the pharmacodynamic effects of compounds of Formula (I), the compound of Example 189, 2-(l-(4-((4-(4-hydroxypiperidin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidm-4-yl)acetonitrile, was tested as described below. The compound of Example 189 is sometimes referred to herein as "COMPOUND A."
Serum samples were collected from patients before the treatment with COMPOUND A on Day 1 and after treatment with COMPOUND A on day 15. The samples were then analyzed for the levels of a panel of 45 inflammation biomarkers. The percent change in the serum levels of each biomarker was calculated by comparing the protein levels before and after treatment. There was a significant inhibition of several inflammation biomarkers on Day 15 at all doses evaluated. A few key inflammation biomarkers that showed a marked inhibition with COMPOUND A treatment were C-Reactive Protein (CRP), p2-microglobulin
(B2M), IL-18 = Interleukin-18; Macrophage Inflammatory Protein- 1 β (ΜΙΡ-1β) and Tumor necrosis factor receptor 2 (TNFR2) (Table 14). The same data are also illustrated as a waterfall graph in Fig 3. C-reactive protein is an acute-phase protein, the levels which are elevated in response to inflammation. The levels of this protein are also elevated in patients with various types of cancers and this is considered as a potential predictor of cancer risk and/or survival. p2-microglobulin is a component of MHC class I molecules and is encoded by the gene, B2M. β2 -microglobulin is found to be elevated in lymphoma and multiple myeloma. IL-18 is a proinflammatory cytokine involved in several inflammatory disorders. ΜΙΡΙ-β, also known as CCL4, is a chemokine with specificity to chemokine receptor 5 (CCR5), which is a chemo-attractant for natural killer, monocytes and other types of immune cells. The levels of all these inflammation biomarkers have been significantly inhibited in patient serum after treatement with COMPOUND A. Because of its ability to suppress some of the important cytokines such as IL-18, COMPOUND A also has a potential to prevent the cytokine release syndrome when combined with immunotherapies (see also Fig. 3).
Table 14: Percent decrease in inflammation on Day 15 after initiation of COMPOUND A treatment
Figure imgf000396_0001
B2M = p2-Microglobulin; CRP = C-Reactive Protein; IL-18 = Interleukin-18; MIP- 1 β= Macrophage Inflammatory Protein- 1 β; TNFR2 = Tumor necrosis factor receptor 2
Note: The percent change shown in this table are average values of all patients in each cohort.
COMPOUND A was also evaluated for its ability to inhibit signaling pathways (JAK/STAT) in peripheral blood monocular cells stimulated with varuious cytokines such as IL-2, IL-4, IL-6, IL-12, IL-23 etc. In these studies, COMPOUND A potently inhibited the
JAK/STAT pathway involving a range of cytokines. The suppression of multiple signaling pathways in immune cells by COMPOUND A did not affect the efficacy of tumnor growth inhibtion: however, this immunosuppressive effect should minimize the deleterious toxicity associated with immunotherapies.
Examples 346, 347 and 348, discussed below, are presented to demonstrate the tumor efficacy of COMPOUND A alone and in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) in various tumor models. The following abbreviations are used herein:
ATCC: American type culture collection
HBSS: Hanks balanced salt solution
HPC: Hydroxypropylcellulose LOQ: Limit of quantification
BLQ: Below limit of quantification
PD: Pharmacodynamic
PK: Pharmacokinetic
p.o.: Per oral
i.p. : Intraperitoneal
RPM: Rotation per minute
SEM: Standard error mean
TGI: Tumor growth inhibition
IDO-1 : Indoleamine 2,3-dioxygenase
Epa: Epacadostat
Q4D: Once in four days
PD-1 : Programmed cell Death- 1
As discussed below with respect to Examples 346, 347 and 348, the inventors have unexpectedly discovered that administration of the pharmaceutical combinations comprising a compound of Formula (I) and an immunotherapeutic agent, as described herein, can produce substantially the same, or an increase in, anti-tumor efficacy compared to administering the compound without an immunotherapeutic agent.
EXAMPLE 346
Example 346 demonstrates the anti-tumor efficacy of COMPOUND A alone and in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) in a 4T1 - Balb/c mouse syngeneic allograft tumor model. At the end of study, the mice bearing 4T1 allografts in the untreated control group showed a physiological body weight gain of 19 %. COMPOUND A, anti PD-1 antibody, epacadostat, or their combinations were tolerated well with a minimal, yet acceptable, loss of body weight (-2 %). Comparisons of tumor volumes in mice treated with COMPOUND A (10, 30, 60 mg/kg), anti-PDl antibody with untreated vehicle control indicated a statistically significant decrease (p<0.0001) 48 %, 60 %, 79 %, 51 %, and 46 % TGI, respectively on day 15 of treatment. Co-administration of COMPOUND A- 10 mg/kg with anti-PDl antibody in 4T1 tumor syngeneic model showed substantially the same tumor growth inhibition (p>0.05, all three combinations vs. corresponding COMPOUND A alone groups). There was an enhancement in the tumor growth inhibition when epacadostat combined with COMPOUND A, although it was not statistically significant (p>0.05), 63 %, 69 % and 90 % TGI in combinations vs. 48 %, 60 % and 79 % TGI in single agent COMPOUND A - 10, 30 and 60 mg/kg, respectively. Plasma COMPOUND A concentrations were found to be in the range of 8 to 1813 ng ml; however in tumor samples, it ranged from 72 to 2495 ng/g for mice administered with 10 to 60 mg/kg
COMPOUND A. There was an 8-fold increase in exposure in tumor vs. plasma samples for single agent COMPOUND A at 10 mg/kg; and a 3-fold increase was obtained when COMPOUND A at 10 mg/kg combined with either anti PD-1 antibody or epacadostat. A lower COMPOUND A concentration in tumor vs. plasma was found at 30 - 60 mg/kg COMPOUND A when combined with anti PD1 antibody. Similarly, COMPOUND A-60 mg kg treatment combined with epacadostat resulted in lower concentration of COMPOUND A in tumor than plasma. No treatment related morbidity or mortality was seen during the study.
Because they have an intact immune system, syngeneic mouse tumor models have been widely accepted for evaluating the effect of immunomodulating agents on tumor growth and their use when given in combination with other anti-tumor agents in vivo. A 4Tl-Balb/c model was used to assess the immunomodulatory effect of known immunomodulatory agents, anti-PDl antibody and IDO-1 inhibitor (epacadostat) in combination with COMPOUND A. IDO-1 (Indoleamine 2,3-dioxygenase), an enzyme in the tryptophan catabolism pathway that has been shown to be elevated in certain tumors and play a key role in tumor growth.
Epacadostat, an inhibitor of IDO-1 has shown efficacy in inhibiting tumor growth in multiple immunocompetant tumor models. PD-1 (Programmed cell Death- 1) expression by tumor- infiltrating lymphocytes is shown to be important in anti-inflammatory and tissue injury protection that further mediates tumor-induced immune suppression/immune escape. Since tumor growth inhibitory effect of both anti-PDl antibody and epacadostat have already been established in this model, a single dose of these agents was used in combination with different doses of COMPOUND A to demonstrate the potential benefit to tumor growth inhibition demonstrated by COMPOUND A. MATERIALS AND METHODS:
Animals
Experiments were performed in female Balb/c mice obtained from Envigo, Netherlands. The animals were housed in individually ventilated cages (maximum of 5 animals/cage) with 12 hour dark, 12 hour light conditions. The animals were fed food and water ad libitum. Temperature and relative humidity were maintained at 20±2°C and 65%, respectively.
Compound preparation and administration for efficacy study
COMPOUND A (Lot # C15K081007A) was formulated in the vehicle containing 20 mg ml hydroxypropylcellulose (HPC, Klucel LF). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout dosing.
Epacadostat (Cat # PBLJ9203; CAS # 1204669-58-8, purchased from PharmaBlock): was formulated as a homogenous suspension in the vehicle containing 0.5 % methyl cellulose (MC). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout the dosing.
Anti-mouse PD1 antibody 100 μg in 200 μΐ antibody was administered (i.p.) per mouse. Antibody was diluted with 1 X sterile PBS (calcium and magnesium-free).
COMPOUND A and epacadostat were administered orally by gavage at a dose volume of 10 mL/kg.
Cell line and tumor model:
Mouse breast carcinoma 4T1 cells were sourced from American Type Culture Collection (ATCC), USA. Cells were grown in RPMI-1640 medium (Sigma, Cat # R6504) supplemented with 10% FBS (Invitrogen, Cat # 10438-026), and 1% penicillin streptomycin (Thermo Fisher Scientific, Cat # 15140-122). To establish allografts, 1 million 4T1 cells were suspended in 50 μΐ of serum-free medium and mixed at 1:1 ratio with matrigel before implanting subcutaneously into the right flank of mice.
Table 15: Study groups
Figure imgf000400_0001
Tumor volume measurement
Tumor dimensions (length and breadth) were measured for all animals on the first treatment day (Day 1) and then two times per week, including the termination day of the study. Tumor volumes were calculated using the formula: tumor length x (tumor width) 2 / 2. Tumor growth inhibition was calculated after normalizing the tumor volume on a given day to that on day 1.
Drug Treatments
Treatment was initiated 9 days after subcutaneous injection of tumor cells when the average tumor volume reached approximately 90 mm3. The animals were randomized based on tumor volumes into 12 groups of eight animals each. The dosing schedule of COMPOUND A and epacadostat were twice daily for 15 days by p.o. and anti-PDl antibody was administered once in 4 days (total 5 doses) by i.p. The treatment period was 15 days after which the overall efficacy and tolerability was evaluated based on tumor volume and body weight changes observed during the treatment period. Tumor volumes were analyzed using two-way ANOVA with Bonferonni' s multiple comparisons test for comparison of treatment versus control group. Tissue collection and bioanalytical method
Two hours after the last dose of COMPOUND A on day 15 (first dose of b.ld. dosing) blood samples were collected by bleeding retrorbital sinus following which mice were sacrificed. 4T1 allografts were dissected and collected in three aliquots; two-parts were snap-frozen in liquid nitrogen (for tissue concentration analysis and cellular protein/biomarker analysis) and one-part was fixed in 4 % paraformaldehyde (for tissue histology). The whole blood samples were immediately placed on ice after collection following which plasma was separated by centrifuging under 5000 rpm at 2-8 °C for 8 min.
Plasma and tumor samples were stored at -80 °C till bioanalysis. Concentrations of COMPOUND A were estimated by LC-MS/MS.
Bio-Analysis
Quantitative bio-analysis of plasma and tumor homogenate samples was done using
LC-MS/MS. All the study samples were removed from the deep freezer and allowed to thaw to room temperature. Plasma samples and tumor homogenates were analyzed following protein precipitation with acetonitrile containing an internal standard. Calibration curve range was 5.5- 5104 ng/ml for COMPOUND A.
Chromatographic condition
Column: Atlantis CI 8 (50 x 4.6 mm, 3 μm; Waters®)
Mobile phase: A: 0.2% Formic acid in water B: Acetonitrile
Gradient program: Time (min)
Auto sampler temperature: 10°C
Column temperature: 40°C
Injection volume: ΙΟμΙ
Table 16: Chromatographic condition
Figure imgf000401_0001
Table 17: Mass Spectrometer condition
Figure imgf000402_0001
Sample preparation
The following sample extraction procedure was used:
• 45 μL of blank plasma was transferred in to a pre labeled micro centrifuge tube and spiked with 5μl of spiking solution to prepare the calibration standards.
• 50 μL of plasma samples were added to a pre labeled micro centrifuge tubes.
• 50 μL of tumor homogenate samples were added to a pre labeled micro centrifuge tubes.
• The plasma samples and tumor homogenate samples were quenched with 200 μL acetonitrile containing internal standard.
• All the samples were vortexed and centrifuged at 14,000 rpm for 5min at 4° C.
• From all the above samples supernatant was collected and transferred in to vials.
• Vials were then injected to LC-MS/MS system.
Data analysis and statistical evaluation
All statistical calculations were performed using Prism 5.0 (GraphPad Software Inc, USA). Comparisons of tumor size and body weights were made between the treatment groups and respective vehicle control groups by two-way ANOVA, followed by Bonferroni's multiple comparison tests. A p value less than 0.05 were considered significant. As a measure of efficacy the percent tumor growth inhibition (%TGI) value was calculated from recorded tumor volumes (TV) at the end of the experiment using the formula below. % TGI = [1 -(Treatment TVFinai - Treatment TVlniti_i) / (Control TVFinai - Control
TVi„itial)]*100
Changes in percent body weight (BW) were calculated according to the formula below:
% BW change = (BWFinal -BWInitial) / (BWInitial) * 100
Efficacy of COMPOUND A or in combination with anti-PDl antibody or epacadostat in 4T1 -Balb/c mouse model
The anti-tumor effect of COMPOUND A was evaluated in tumor allograft bearing Balb/c mice. The anti-tumor efficacy of single agent anti-PDl antibody, epacadostat and COMPOUND A were compared to vehicle control, whereas combination groups, COMPOUND A + anti-PDl antibody, and COMPOUND A + epadacostat were compared with single agent groups and vehicle control. Dose response/efficacy study
Comparisons of tumor volumes in mice treated with COMPOUND A (10, 30, 60 mg kg), anti-PDl antibody with untreated vehicle control indicated a statistically significant decrease ( θ.0001) on day 15 of treatment. The average tumor volume for mice in vehicle control group was 976 ± 86 mm3 and that for 10, 30, 60 mg/kg COMPOUND A, anti-PDl antibody 0.1 mg/mouse, and epacadostat 30 mg kg treated groups were found to be 549±51 (48 % TGI), 447±38 (60 % TGI), 282±66 (79 % TGI), 530±58 (51 % TGI), and 567±48 mm3 (46 % TGI), respectively (Figure 4 and 5; Table 18). Co-administration of COMPOUND A mg/kg and anti-PDl antibody in 4T1 tumor syngeneic model showed substantially the same tumor growth inhibition (p>0.05, all three combinations vs. corresponding COMPOUND A alone groups) (Figure 4 and 5; Table 18). There was an enhancement in the tumor growth inhibition when epacadostat combined with COMPOUND A, although it was not statistically significant (p>0.05), 63 %, 69 % and 90 % TGI in combinations vs. 48 %, 60 % and 79 % TGI in single agent COMPOUND A - 10, 30 and 60 mg/kg, respectively (Figure 4 and 5; Table 18). Table 18: Average tumor volume (mm3) in mice after different days of treatment initiation
Figure imgf000404_0001
Effect of compounds on body weight in 4T1 allograft efficacy study
At the end of study (Table 15), the mice bearing 4T1 allografts in the untreated control group showed a physiological body weight gain of 19 % (Table 20). Mice treated with COMPOUND A - 10, 30 and 60 mg/kg equally gained 17 % body weight on day 15, not significantly different from controls (p>0.05, Table 19; Figures 6-9). Anti-PDl antibody combinations with COMPOUND A - 10, 30, and 60 mg kg had a similar gain of 18 %, 17 %, and 16 % body weights, respectively (Figures 6 and 8; Tables 19 and 20). Likewise, combinations of epacadostat with COMPOUND A - 10, 30, or 60 mg/kg were also well tolerated with an average gain of 18 %, 17 %, and 17 % in body weights, respectively on day 15 (Figures 7 and 9; Tables 19 and 20).
Table 19: Average body weight in mice after different days of treatment initiation
Figure imgf000405_0001
Table 20: Percent change in body weight on different days after treatment initiation
Figure imgf000406_0001
Tolerability of treatment in vivo
COMPOUND A, anti PD-1 antibody, and epacadostat were tolerated well with a minimal, yet acceptable, loss of body weight. No treatment related morbidity or mortality was seen during the study. Plasma and tumor concentrations of COMPOUND A
Plasma and tumor samples from six mice/group were collected for analyzing COMPOUND A by LC-MS. Plasma COMPOUND A concentrations were found to be in the range of 8 to 1813 ng/ml (Table 21); however in tumor samples, COMPOUND A concentrations ranged from 72 to 2495 ng/g for mice administered with 10 to 60 mg/kg COMPOUND A (Table 22). In the 10 to 60 mg/kg COMPOUND A dosed groups coadministered with anti-PDl antibody, concentration of COMPOUND A in plasma ranged from 41 to 1889 ng/ml and in tumor it ranged from 126 to 1624 ng/g (Tables 21, 22). Similarly, in the epacadostat combination groups, concentration of COMPOUND A in plasma ranged from 48 to 4114 ng/ml and in tumor it ranged from 172 to 2517 ng/g in mice administered with 10 to 60 mg/kg COMPOUND A (Tables 21, 22). There was an 8-folds increase in exposure in tumor than plasma samples for single agent COMPOUND A-10 mg/kg; and a 3-fold increase was obtained when COMPOUND A-10 mg/kg combined with either anti PD-1 antibody or epacadostat. A lower COMPOUND A concentration in tumor than plasma was found at 30 - 60 mg/kg COMPOUND A when combined with anti PD 1 antibody. Similarly, COMPOUND A-60 mg kg treatment combined with epacadostat resulted in lower concentration of COMPOUND A in tumor than plasma.
Table 21: Concentration of COMPOUND A in plasma analyzed in mice after 15 days treatment
Figure imgf000407_0001
Table 22: Concentration of COMPOUND A in tumor analyzed in mice after 15 days treatment
Figure imgf000407_0002
CONCLUSION
COMPOUND A at 10, 30 and 60 mg/kg doses resulted in a significant and dose- dependent tumor growth inhibition of 48 %, 60 %, and 79 %. A tumor growth inhibition of 51 % and 46 % was obtained for anti-PDl antibody (0.1 mg/mouse) and epacadostat (30 mg kg), respectively. All treatments were tolerated well and no treatment related morbidity or mortality was seen during the study. Combination of COMPOUND A 10 mg kg with anti- PDl antibody showed substantially the same TGI as that of single agent COMPOUND A. There was an enhancement in the tumor growth inhibition when epacadostat combined with COMPOUND A, although it was not statistically significant (p>0.05). There was dose dependent increase in COMPOUND A exposure both in plasma and tumor, and the concentrations were 8-folds higher in tumor than plasma in mice administered with
COMPOUND A- 10 mg/kg. A 3 -folds increase was also obtained when COMPOUND A- 10 mg/kg combined with either anti PD-1 antibody or epacadostat. These results suggest substantially the same anti-tumor efficacy for COMPOUND A with mouse anti PD-1 antibody or epacadostat in the 4Tl-Balb/c mouse model of breast cancer.
EXAMPLE 347
Example 347 demonstrates the anti-tumor efficacy of COMPOUND A alone and in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) in B16-F10 - C57/BL6 syngeneic allograft tumor model.
The objective of this study was to test efficacy of COMPOUND A in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) using B16-F10 - C57/BL6 syngeneic allograft tumor model. At the end of the study, the average tumor volume for mice in vehicle control group was 2529 ± 110 mm3 and that for 10, 20, 60 mg/kg COMPOUND A, anti-PDl antibody 0.1 mg/mouse, and epacadostat 30 mg/kg treated groups were found to be 1323±121 (49 % TGI), 1041±98 (60 % TGI), 923±58 (65 % TGI), 1117±82 (57 % TGI), and 1394±90 mm3 (46 % TGI), respectively. Co-administration of either anti-PDl antibody or epacadostat with COMPOUND A showed substantially the same tumor growth reduction in B16-F10 melanoma cancer syngeneic model. COMPOUND A (10 - 60 mg/kg), anti-PDl antibody and epacadostat were tolerated well without loss of body weight. Combinations of COMPOUND A with anti-PDl antibody or epacadostat were tolerated well without loss of body weight. There was 1.6-fold higher exposure in tumor than plasma in COMPOUND A- 30 mg/kg + anti PD-1 antibody combination group compared to COMPOUND A alone. Also similarly, there was 1.3-fold higher exposure in tumor than plasma in COMPOUND A-30 mg kg + epacadostat combination group compared to COMPOUND A alone.
. A widely used B16-F10 tumor was employed in C57/BL6 mouse model to assess the immunomodulatory effect of known immunotherapeutic agents, anti-PDl antibody and IDO- 1 inhibitor (epacadostat) in combination with COMPOUND A. Since tumor growth inhibitory effect of both anti-PDl antibody and epacadostat have already been established in this model, a dose that has resulted in 40-50% inhibition was selected as a single agent and combined with different doses of COMPOUND A to understand any potential benefit to tumor growth inhibition demonstrated by COMPOUND A.
MATERIALS AND METHODS:
Animals
Experiments were performed in female C57/BL6 mice obtained from Charles River
Laboratories, USA (CRL). The animals were housed in individually ventilated cages (maximum of 5 animals/cage) with 12 hour dark, 12 hour light conditions. The animals were fed food and water ad libitum. Temperature and relative humidity were maintained at 20±2°C and 65%, respectively.
Compound preparation and administration for efficacy study:
COMPOUND A (Lot # C15K081007A) was formulated in the vehicle containing 20 mg/ml hydroxypropylcellulose (HPC, Klucel LF). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout dosing.
Epacadostat (Cat # PBLJ9203; CAS # 1204669-58-8, purchased from PharmaBlock): was formulated as a homogenous suspension in the vehicle containing 0.5 % methyl cellulose (MC). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout the dosing.
Anti-mouse PD1 antibody: 100 μg in 200 μΐ antibody was administered (i.p.) per mouse. Antibody was diluted with 1 X sterile PBS (calcium and magnesium-free). COMPOUND A and epacadostat were administered orally by gavage at a dose volume of 10 mL/kg. Cell line and tumor model:
Mouse melanoma B16-F10 cells were sourced from American Type Culture Collection (ATCC), USA. Cells were grown in Dulbecco's Modified Eagel Medium (DMEM) (Gibco, Cat # 31600-034) supplemented with 10% FBS (Invitrogen, Cat # 10438- 026), and 1% penicillin streptomycin (Thermo Fisher Scientific, Cat # 15140-122). To establish allografts, 0.1 million B16-F10 mouse melanoma cells were suspended in 50 μΐ of serum-free medium and mixed at 1 : 1 ratio with matrigel before implanting subcutaneously into the right flank of mice.
Table 23: Study groups:
Figure imgf000410_0001
Tumor volume measurement
Tumor dimensions (length and breadth) were measured for all animals on the first treatment day (Day 1) and then two times per week, including the termination day of the study. Tumor volumes were calculated using the formula: tumor length x (tumor width) 2 / 2. Tumor growth inhibition was calculated after normalizing the tumor volume on a given day to that on day 1.
Drug Treatments Treatment was initiated 6 days after subcutaneous injection of tumor cells when the average tumor volume had reached approximately 50 mm3. The animals were randomized based on tumor volumes into 12 groups of ten animals each. The dosing schedule of COMPOUND A and epacadostat were twice daily for 15 days by p.o. and anti-PDl antibody was administered once in 4 days (total 5 doses) by i.p. The treatment period was 15 days after which the overall efficacy and tolerability was evaluated based on tumor volume and body weight changes observed during the treatment period. Tumor volumes were analyzed using two-way ANOVA with Bonferonni' s multiple comparisons test for comparison of treatment versus control group.
Tissue collection and bioanalytical method
Two hours after the last dose of COMPOUND A on day 15 (first dose of b.ld. dosing) blood samples were collected by bleeding retrorbital sinus following which mice were sacrificed. B16-F10 allografts were dissected and collected in three aliquots; two-parts were snap-frozen in liquid nitrogen (for tissue concentration analysis and cellular protein/biomarker analysis) and one-part was fixed in 4 % paraformaldehyde (for tissue histology). The whole blood samples were immediately placed on ice after collection following which plasma was separated by centrifuging under 5000 rpm at 2-8 °C for 8 min. Plasma and tumor samples were stored at -80 °C till bioanalysis. Concentrations of COMPOUND A were estimated by LC-MS/MS.
Bio-Analysis
Quantitative bio-analysis of plasma and tumor homogenate samples was done using LC-MS/MS. All the study samples were removed from the deep freezer and allowed to thaw to room temperature. Plasma samples and tumor homogenates were analyzed following protein precipitation with acetonitrile containing an internal standard. Calibration curve range was 5.9- 3098 ng/ml for COMPOUND A.
Chromatographic condition
Column: Atlantis CI 8 (50 x 4.6 mm, 3 μm; Waters®)
Mobile phase: A: 0.2% Formic acid in water B: Acetonitrile Gradient program: Time (min)
Auto sampler temperature: 10°C
Column temperature: 40°C
Injection volume: ΙΟμΙ
Table 24: Chromatographic condition
Figure imgf000412_0001
Table 25: Mass Spectrometer condition
Figure imgf000412_0002
Sample preparation
The following sample extraction procedure was used:
• 45 μL of blank plasma was transferred in to a pre labeled micro centrifuge tube and spiked with 5 μl of spiking solution to prepare the calibration standards.
• 50 μL of plasma samples were added to a pre labeled micro centrifuge tubes.
• 50 μL of tumor homogenate samples were added to a pre labeled micro centrifuge tubes.
• The plasma samples and tumor homogenate samples were quenched with 200μΙ_. acetonitrile containing internal standard.
• All the samples were vortexed and centrifuged at 14,000 rpm for 5min at 4° C.
• From all the above samples supernatant was collected and transferred in to vials. Vials were then injected to LC-MS/MS system.
Data analysis and statistical evaluation
All statistical calculations were performed using Prism 5.0 (GraphPad Software Inc, USA). Comparisons of tumor size and weight measurements during and at the termination of the study were made between the treatment groups and respective vehicle control groups by two-way ANOVA, followed by Bonferroni's multiple comparison tests. A p value less than 0.05 were considered significant. As a measure of efficacy the percent tumor growth inhibition (%TGI) value was calculated from recorded tumor volumes (TV) at the end of the experiment using the formula below.
% TGI = [1 -(Treatment TVpinai - Treatment TVlniti_i) / (Control TVpinai - Control
TVi„itial)]*100 Changes in percent body weight (BW) were calculated according to the formula below:
% BW change = (BWFinal -BWInitial) / (BWInitial) * 100
Efficacy of COMPOUND A or in combination with anti-PDl antibody or epacadostat in B16-F10 -C57/BL6 mouse model
The anti-tumor effect of COMPOUND A was evaluated in tumor allograft bearing C57/BL6 mice. The anti-tumor efficacy of single agents, anti-PDl antibody, epacadostat and COMPOUND A were compared to vehicle control, whereas combination groups, COMPOUND A + anti-PDl antibody, and COMPOUND A + epadacostat were compared with single agent groups and vehicle control.
Dose response/efficacy study
Comparisons of tumor volumes of mice after treated with COMPOUND A (10, 30, 60 mg kg), anti-PDl antibody, or epacadostat with untreated vehicle control indicated a statistically significant decrease (p<0.0001) on day 1 1 and 15 (final day) of treatment. The average tumor volume for mice in vehicle control group was 2529 ± 110 mm3 and that for 10, 20, 60 mg/kg COMPOUND A, anti-PDl antibody 0.1 mg/mouse, and epacadostat 30 mg/kg treated groups were found to be, respectively 1323±121 (49 % TGI), 1041±98 (60 % TGI), 923±58 (65 % TGI), 1117±82 (57 % TGI), and 1394±90 mm3 (46 % TGI) (Figure 17 and 18; Table 26). Co-administration of anti-PDl antibody showed substantially the same tumor growth inhibition obtained for COMPOUND A in B16-F10 tumor syngeneic model (53 %, 62% and 74% TGI for combinations of anti PD-1 antibody and COMPOUND A vs. 49 %, 60% and 65% TGI for single agent COMPOUND A at 10, 20 and 60 mg kg, respectively) (Figure 17 and 18; Table 26). Similarly, there was substantially the same benefit of coadministration of epacadostat with COMPOUND A (52 %, 64% and 70% TGI for combinations of epacadostat and COMPOUND A vs. 49 %, 60% and 65% TGI for single agent COMPOUND A at 10, 30 and 60 mg kg, respectively; Figure 17 and 18; Table 26).
Table 26: Average tumor volume (mm ) in mice after different days of treatment initiation
[ 1 and 15 after treatment initiation
Figure imgf000414_0001
Figure imgf000415_0002
Effect of compounds on body weight in B16-F10 allograft efficacy study At end of dosing (Table 23), the mice bearing B16-F10 allografts in the untreated control group maintained body weight with negligible change of -0.6 % on day 15 (Table 28). COMPOUND A - 10, 30, and 60 mg/kg, anti-PDl antibody and epacadostat were tolerated well without loss of body weight (Figures 19 and 21 ; Tables 27 and 28). There was an improvement with a gain of animal body weight for all treatment groups on day 15 (not significant, p>0.05; Figures 19 and 21 ; Tables 27 and 28). The highest increase of 17.3 % in body weight was observed in mice treated with mouse anti-PDl antibody (group 5).
Table 27: Average body weight of mice after different days of treatment initiation
Figure imgf000415_0001
Figure imgf000416_0002
Table 28: Percent change in body weight after different days of treatment initiation
Figure imgf000416_0001
Tolerability of treatment in vivo
Mortality events occurred on various days during the study are tabulated below in
Table 29. Animal mortality was random across all experimental groups and the mortality did not appear to be due to treatment, but could be because of spontaneous metastatic nature of B16F10 cells in the syngeneic C57-BL6 mouse model. Table 29: Animal mortality after different days of treatment initiation
Figure imgf000417_0001
Plasma and tumor concentrations of COMPOUND A
Plasma and tumor samples from six mice/group were collected 2 h after last dose for analyzing COMPOUND A by LC-MS. Plasma COMPOUND A concentrations were found to be in the range of 32.8 to 935 ng/ml (Table 30), however in tumor samples, COMPOUND A concentrations increased from 883 to 7568 ng/g for mice administered with 10 to 60 mg/kg COMPOUND A (Table 31). In the 10 to 60 mg/kg COMPOUND A dosed groups coadministered with anti-PDl antibody, concentration of COMPOUND A in plasma ranged from 25 to 1023 ng/ml (Table 30) and in tumor it ranged from 1024 to 9439 ng/g (Table 31). Similarly, in the epacadostat combination groups, concentration of COMPOUND A in plasma ranged from 59.5 to 1054 ng/ml (Table 30) and in tumor it increased from 1002 to 8190 ng/g in mice administered with 10 to 60 mg/kg COMPOUND A (Table 31). There was 1.6-folds higher exposure in tumor than plasma in COMPOUND A-30 mg/kg + anti PD-1 antibody combination group compared to COMPOUND A alone. Also similarly, there was 1.3-folds higher exposure in tumor than plasma in COMPOUND A-30 mg/kg + epacadostat combination group compared to COMPOUND A alone. Table 30: Concentration of COMPOUND A in plasma analyzed in mice after 15 days treatment
Figure imgf000418_0001
Table 31: Concentration of COMPOUND A in tumor analyzed in mice after 15 days treatment
Figure imgf000418_0002
CONCLUSION COMPOUND A at 10, 30 and 60 mg/kg doses resulted in a significant and dose- dependent tumor growth inhibition of 49, 60 and 65%. A tumor growth inhibition of 57 % and 46 % was obtained for anti-PDl antibody (0.1 mg/mouse) and epacadostat (30 mg/kg), respectively. Combination of either anti-PDl antibody or epacadostat showed substantially the same TGI than that obtained from single agent COMPOUND A. There was a dose dependent increase in COMPOUND A exposure with a maximum of 0.93 μg/ml plasma and
7.5 μg/g tumor after 2 h of last dose. Overall, these compounds were well tolerated as single agents or in combinations. These results suggest that combination of COMPOUND A with immunomodulatory drugs may be substantially as efficacious, as single agent administration, although they are safe in the B16-F10-C57/BL6 mouse model of melanoma cancer.
EXAMPLE 348
Example 348 demonstrates the anti-tumor efficacy of COMPOUND A alone and in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) in CT26 - Balb/c syngeneic allograft tumor model
The objective of this study was to demonstrate the anti-tumor acitivity of COMPOUND A in combination with anti-PDl antibody or IDO-1 inhibitor (epacadostat) using CT26 - Balb/c syngeneic allograft tumor model. At the end of study, the mice bearing CT26 allografts in the untreated control group showed a physiological body weight gain of 18 %. COMPOUND A - 30 and 60 mg/kg treatment has significantly decreased the physiological gain body weight on day 15 compared to controls (p<0.05), however there was no loss of body weight compared to their day 1 initial weights (p<0.05). Combination of COMPOUND A with anti-PDl antibody or epacadostat had a significant but acceptable loss of body weight compared to controls (p<0.0001). Treatment of mice with COMPOUND A (10, 30, 60 mg/kg), anti-PDl antibody had significantly lower tumor volume on day 15 (p<0.001 to p<0.0001, depending on treatment), except for epacadostat (30 mg/kg). Coadministration of COMPOUND A-10 mg/kg and anti-PDl antibody in CT26 tumor syngeneic model significantly increased tumor growth inhibition (p<0.01, 63 % TGI in combination vs. 33 % TGI in COMPOUND A-10 mg/kg), except for higher concentrations of COMPOUND A. There was substantially the same tumor volume for the COMPOUND A and epacadostat combination (p>0.05). Plasma COMPOUND A concentrations were in the range of 208 to 3071 ng/ml; however in tumor samples, COMPOUND A concentrations ranged from 382 to 1286 ng/g for mice administered with 10 to 60 mg/kg COMPOUND A.
Anti PD-1 antibody or epacadostat co-administration decreased the COMPOUND A concentrations both in plasma and tumor. There was a 2-folds increase in exposure in tumor than plasma samples for single agent COMPOUND A-10 mg/kg, a similar increase was also obtained when combined with either anti PD-1 antibody or epacadostat.COMPOUND A, anti PD-1 antibody, and epacadostat were tolerated with acceptable loss of body weight. An increase in the loss of body weight, still within an acceptable limit of 10 % was observed when combined with anti PD-1 antibody or epacadostat. CT26-Balb/c model was used to assess the immunomodulatory effect of known immunomodulatory agents, anti-PDl antibody and IDO-1 inhibitor (epacadostat) in combination with COMPOUND A. Since tumor growth inhibitory effect of both anti-PDl antibody and epacadostat have already been established in this model, a single dose of these agents was used in combination with different doses of COMPOUND A to understand any potential benefit to tumor growth inhibition demonstrated by COMPOUND A.
MATERIALS AND METHODS:
Animals
Experiments were performed in female Balb/c mice obtained from Envigo,
Netherlands. The animals were housed in individually ventilated cages (maximum of 5 animals/cage) with 12 hour dark, 12 hour light conditions. The animals were fed food and water ad libitum. Temperature and relative humidity were maintained at 20±2°C and 65%, respectively.
Compound preparation and administration for efficacy study:
COMPOUND A (Lot # C15K081007A) was formulated in the vehicle containing 20 mg ml hydroxypropylcellulose (HPC, Klucel LF). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout dosing.
Epacadostat (Cat # PBLJ9203; CAS # 1204669-58-8, purchased from PharmaBlock): was formulated as a homogenous suspension in the vehicle containing 0.5 % methyl cellulose (MC). The formulation was stirred for 30 min to obtain a homogenous suspension to which polysorbate-80 was added at 1 mg/ml. This formulation was continuously stirred throughout the dosing.
Anti-mouse PD1 antibody 100 μg in 200 μΐ antibody was administered (i.p.) per mouse. Antibody was diluted with 1 X sterile PBS (calcium and magnesium-free).
COMPOUND A and epacadostat were administered orally by gavage at a dose volume of 10 mL/kg.
Cell line and tumor model:
Mouse colon carcinoma CT26 cells were sourced from American Type Culture Collection (ATCC), USA. Cells were grown in RPMI-1640 medium (Sigma, Cat # R6504) supplemented with 10% FBS (Invitrogen, Cat # 10438-026), and 1% penicillin streptomycin (Thermo Fisher Scientific, Cat # 15140-122). To establish allografts, 1 million CT26 cells were suspended in 50 μΐ of serum-free medium and mixed at 1:1 ratio with matrigel before implanting subcutaneously into the right flank of mice.
Table 32: Study groups
Figure imgf000421_0001
Tumor volume measurement
Tumor dimensions (length and breadth) were measured for all animals on the first treatment day (Day 1) and then two times per week, including the termination day of the study. Tumor volumes were calculated using the formula: tumor length x (tumor width) 1 12. Tumor growth inhibition was calculated after normalizing the tumor volume on a given day to that on day 1.
Drug Treatments
Treatment was initiated 9 days after subcutaneous injection of tumor cells when the average tumor volume reached approximately 90 mm3. The animals were randomized based on tumor volumes into 12 groups of eight animals each. The dosing schedule of COMPOUND A and epacadostat were twice daily for 15 days by p.o. and anti-PDl antibody was administered once in 4 days (total 5 doses) by i.p. The treatment period was 15 days after which the overall efficacy and tolerability was evaluated based on tumor volume and body weight changes observed during the treatment period. Tumor volumes were analyzed using two-way ANOVA with Bonferonni' s multiple comparisons test for comparison of treatment versus control group.
Tissue collection and bioanalytical method
Two hours after the last dose of COMPOUND A on day 15 (first dose of b.ld. dosing) blood samples were collected by bleeding retrorbital sinus following which mice were sacrificed. CT26 allografts were dissected and collected in three aliquots; two-parts were snap-frozen in liquid nitrogen (for tissue concentration analysis and cellular protein/biomarker analysis) and one-part was fixed in 4 % paraformaldehyde (for tissue histology). The whole blood samples were immediately placed on ice after collection following which plasma was separated by centrifuging under 5000 rpm at 2-8 °C for 8 min. Plasma and tumor samples were stored at -80 °C till bioanalysis. Concentrations of COMPOUND A were estimated by LC-MS/MS.
Bio-Analysis
Quantitative bio-analysis of plasma and tumor homogenate samples was done using LC-MS/MS. All the study samples were removed from the deep freezer and allowed to thaw to room temperature. Plasma samples and tumor homogenates were analyzed following protein precipitation with acetonitrile containing an internal standard. Calibration curve range was 5.8- 3070 ng ml for COMPOUND A.
Chromatographic condition
Column: Atlantis CI 8 (50 x 4.6 mm, 3 μm; Waters®)
Mobile phase: A: 0.2% Formic acid in water B: Acetonitrile
Gradient program: Time (min)
Auto sampler temperature: 10°C
Column temperature: 40°C
Injection volume: ΙΟμΙ Table 33: Chromatographic condition
Figure imgf000423_0001
Table 34: Mass Spectrometer condition
Figure imgf000423_0002
Sample preparation
The following sample extraction procedure was used:
• 45 μL of blank plasma was transferred in to a pre labeled micro centrifuge tube and spiked with 5 μl of spiking solution to prepare the calibration standards.
• 50 μL of plasma samples were added to a pre labeled micro centrifuge tubes.
• 50 μL of tumor homogenate samples were added to a pre labeled micro centrifuge tubes.
• The plasma samples and tumor homogenate samples were quenched with 200μΙ_. acetonitrile containing internal standard.
• All the samples were vortexed and centrifuged at 14,000 rpm for 5min at 4° C.
• From all the above samples supernatant was collected and transferred in to vials.
• Vials were then injected to LC-MS/MS system.
Data analysis and statistical evaluation
All statistical calculations were performed using Prism 5.0 (GraphPad Software Inc, USA). Comparisons of tumor size and body weights were made between the treatment groups and respective vehicle control groups by two-way ANOVA, followed by Bonferroni's multiple comparison tests. A p value less than 0.05 were considered significant. As a measure of efficacy the percent tumor growth inhibition (%TGI) value was calculated from recorded tumor volumes (TV) at the end of the experiment using the formula below.
% TGI = [1 -(Treatment TVFinai - Treatment TVlniti_i) / (Control TVFinai - Control
TVjnitiaO iOO
Changes in percent body weight (BW) were calculated according to the formula below:
% BW change = (BWFinal -BWInitial) / (BWInitial) * 100
Efficacy of COMPOUND A or in combination with anti-PDl antibody or epacadostat in CT26 -Balb/c mouse model
The anti-tumor effect of COMPOUND A was evaluated in tumor allograft bearing
Balb/c mice. The anti-tumor efficacy of single agent anti-PDl antibody, epacadostat and COMPOUND A were compared to vehicle control, whereas combination groups, COMPOUND A + anti-PDl antibody, and COMPOUND A + Epadacostat were compared with single agent groups and vehicle control.
Dose response/efficacy study
Comparisons of tumor volumes in mice treated with COMPOUND A (10, 30, 60 mg kg), anti-PDl antibody with untreated vehicle control indicated a statistically significant decrease (p<0.001 to / θ.0001) on the final day of treatment, except for epacadostat (30 mg/kg). The average tumor volume for mice in vehicle control group was 2835 ± 500 mm3 and that for 10, 30, 60 mg/kg COMPOUND A, anti-PDl antibody 0.1 mg/mouse, and epacadostat 30 mg/kg treated groups were found to be 1929±407 (33 % TGI), 965±135 (68 % TGI), 771±90 (76 % TGI), 1264±143 (57 % TGI), and 1901±396 mm3 (34 % TGI), respectively (Figure 30 and 31 ; Table 35). Co-administration of COMPOUND A- 10 mg/kg and anti-PDl antibody in CT26 tumor syngeneic model significantly increased tumor growth inhibition (p<0.0\, 63 % TGI in combination vs. 33 % TGI in COMPOUND A-10 mg/kg). However, higher concentrations of COMPOUND A when combined with and anti-PDl antibody showed substantially the same tumor growth inhibition (p>0.05, 72 % and 84 % TGI in combinations vs. 68 % and 76 % TGI in single agent COMPOUND A, 30 and 60 mg kg, respectively) (Figure 30 and 31; Table 35). Similarly, tumor volume stayed substantially the same for the COMPOUND A and epacadostat combination (p>0.05, 51 %, 62 % and 73 % TGI in combinations vs. 33 %, 68 % and 76 % TGI in single agent COMPOUND A - 10, 30 and 60 mg/kg, respectively (Figure 30 and 31 ; Table 35).
Table 35: Average tumor volume (mm3) in mice after different days of treatment initiation
Figure imgf000425_0001
Effect of compounds on body weight in CT26 allograft efficacy study At the end of study (Table 32), the mice bearing CT26 allografts in the untreated control group showed a physiological body weight gain of 18 % (Table 37). COMPOUND A - 30 and 60 mg/kg treatment has significantly decreased the physiological gain body weight on day 15 compared to controls (p<0.05), however there was no loss of body weight compared to their day 1 initial weights (p<0.05, Table 36; Figures 32-35). Anti-PDl antibody combinations with COMPOUND A - 10 mg/kg and 60 mg/kg, except for 30 mg/kg had a significant but acceptable loss of body weight compared to controls (p<0.0001, Figures 32 and 34; Tables 36 and 37). Likewise, combinations of epacadostat with COMPOUND A - 10, 30, or 60 mg/kg have resulted in a significant but acceptable loss of body weight on day 15 (p<0.01, Figures 33 and 35; Tables 36 and 37).
Table 36: Average body weight in mice after different days of treatment initiation
Figure imgf000426_0001
Table 37: Percent change in body weight on different days after treatment initiation
Figure imgf000427_0001
Tolerability of treatment in vivo
COMPOUND A, anti PD-1 antibody, and epacadostat were tolerated with acceptable loss of body weight. An increase in the loss of body weight, still within an acceptable limit of 10 % was observed when combined with anti PD-1 antibody or epacadostat.
Plasma and tumor concentrations of COMPOUND A
Plasma and tumor samples from six mice/group were collected for analyzing COMPOUND A by LC-MS. Plasma COMPOUND A concentrations were found to be in the range of 208 to 3071 ng/ml (Table 38), however in tumor samples, COMPOUND A concentrations ranged from 382 to 1286 ng/g for mice administered with 10 to 60 mg/kg COMPOUND A (Table 39). In the 10 to 60 mg/kg COMPOUND A dosed groups coadministered with anti-PDl antibody, concentration of COMPOUND A in plasma ranged from 80 to 711 ng/ml and in tumor it ranged from 147 to 805 ng/g (Tables 38, 39). Similarly, in the epacadostat combination groups, concentration of COMPOUND A in plasma ranged from 51 to 1774 ng/ml and in tumor it ranged from 151 to 937 ng/g in mice administered with 10 to 60 mg/kg COMPOUND A (Tables 38, 39). There was a 2-fold increase in exposure in tumor than plasma samples for single agent COMPOUND A- 10 mg/kg, a similar increase was also obtained when combined with either anti PD-1 antibody or epacadostat.
Table 38: Concentration of COMPOUND A in plasma analyzed in mice after 15 days treatment
Figure imgf000428_0001
Table 39: Concentration of COMPOUND A in tumor analyzed in mice after 15 days treatment
Figure imgf000428_0002
CONCLUSION
COMPOUND A at 10, 30 and 60 mg/kg doses resulted in a significant and dose- dependent tumor growth inhibition of 33, 68 and 79%. A tumor growth inhibition of 57 % and 34 % was obtained for anti-PDl antibody (0.1 mg/mouse) and epacadostat (30 mg/kg), respectively. Combination of COMPOUND A 10 mg/kg with anti-PDl antibody significantly enhanced TGI of single agent COMPOUND A; however, higher concentrations of COMPOUND A did not produce this response. TGI was substantially the same for COMPOUND A and epacadostat combination compared to single agent COMPOUND A. There was dose dependent increase in COMPOUND A exposure both in plasma and tumor, and the concentrations were at least 2-folds higher in tumor than plasma in mice administered with COMPOUND A- 10 mg/kg. A similar increase was also obtained when COMPOUND A- 10 mg/kg combined with either anti PD-1 antibody or epacadostat. These results suggest an enhancement of efficacy for lower concentration of COMPOUND A (e.g., 10 mg kg) with mouse anti PD-1 antibody in the CT26-Balb/c mouse model of colon cancer.
EXAMPLE 349
Example 349 demonstrates the clinical activity, safety and tolerabilityof
COMPOUND A, a dual SYK/JAK inhibitor, in patients with Non-Hodgkins lymphoma (NHL) and solid tumors, including dose-limiting toxicities (DLTs) and determines the maximum tolderated dose (MTD). Pre-clinical studies indicate that COMPOUND A has low nM IC50s against SYK and JAK, decreases proliferation in ibrutinib-resistant cell lines, and suppresses tumor growth in rodent xenograft models of DLBCL. Example 349 demonstrates that COMPOUND A, at dose levels associated with clinical and biomarker activity, is safe and tolerable for development as single agent and in combination with other treatments. The safety profile of COMPOUND A differentiates favorably from approved JAK inhibitors ruxolitinib and tofacitinib. Despite known immune modulating properties of COMPOUND A, the combination with checkpoint inhibitors (PD-1 / PD-1 ligand directed) or other immune-modulators, does not reduce the activity of such treatments, but rather shows additivity in preclinical models. Checkpoint inhibitors can include co-inhibitory molecules such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), PD-1, lymphocyte-activation gene 3, and T-cell immunoglobulin mucin-3, and co-stimulatory molecules such as:
glucocorticoid-induced tumor necrosis factor receptor and OX40 (CD134, TNFRSF4, tumor necrosis factor receptor superfamily member 4). Other immune modulators that can be combined with a compound of Formula (I) include indoleamine (2,3)-dioxygenase (IDO) inhibitors, vaccines and agents that target T-cell receptors (TCR agents). The TCR agents can include (without limitation) chimeric antigen receptor (CAR) T cells, and TCR agonist or antagonist peptides. The vaccines can include (without limitation) sipuleucel-T (Provenge®) and Talimogene laherparepvec (Imlygic®), aslo known as T-Vec. COMPOUND A can be thus be clinically developed in combination with checkpoint inhibitors, IDO inhibitors, vaccines (for example, sipuleucel-T (Provenge®) and/or Talimogene laherparepvec
(Imlygic®), aslo known as T-Vec), TCR agents (for example CAR T cells and/or TCR agonist or antagonist peptides) or other immune-modulators to improve outcome of treatment. Furthermore, while improving outcome of treatment when combined with checkpoint inhibitors, IDO inhibitors, vaccines (for example, sipuleucel-T (Provenge®) and/or Talimogene laherparepvec (Imlygic®), aslo known as T-Vec), TCR agents (for example CAR T cells and/or TCR agonist or antagonist peptides), or other immune modulators, COMPOUND A could also improve immune mediated toxicities associated with administration of of such treatments.
Example 349 demonstrates the safety, tolerability, and preliminary efficacy of COMPOUND A in subjects with relapsed/refractory diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and mantle cell lymphoma (MCL). Example 349 also demonstrates pharmacokinetic (PK) profile of COMPOUND A after single and multiple doses and the effects of COMPOUND A on Phospho-STAT3, Phospho-S6, Phospho-SYK
525/526, p2-microglobulin, ΜΙΡΙβ, VCAM-1, TNFR2, C - reactive protein (CRP), and IL- 18.
Rule-based design (3+3) is used in the dose escalation component (Part A of Example 349). This method is commonly used in Phase 1 oncology clinical trials for cytotoxic agents and molecularly targeted agents. In Part A of the study, lymphomas and solid tumor patients were evaluated. The PK properties of COMPOUND A were evaluated after single and multiple dose administrations at different dose levels. Both twice and once-daily administration schedules were evaluated.
Once Part A of Example 349, the dose escalation component, was completed and the MTD was determined, Part B of Example 349, the cohort expansion component, was be performed. Expansion cohorts include patients with Diffuse Large B-Cell Lymphoma (DLBCL), Follicular cell Lymphoma FL and Mantle Cell Lymphoma (MCL) treated daily at a dose regimen to identified in Part A. Patients diagnosed with other hematologic malignancies may also be included such as T-cell lymphomas, myelofibrosis, chronic lymphocytic leukemia. Cohort expansion after determination of the MTD is commonly used in Phase 1 oncology study designs.
Additional studies for Example 349 may be conducted to characterize safety and efficacy of COMPOUND A in additional hematologic malignancies where JAK or SYK signal inhibition could provide clinical benefit. Additional studies to characterize the ADME and metabolism of COMPOUND A maybe conducted.
Example 349 demonstrates a Phase 1/2 clinical trial in patients with solid tumors and hematologic malignancies evaluates QD and BID oral COMPOUND A at escalating doses of 10, 20, 30, 40, 50 and 75 mg BID and 80 and 120 mg QD mg (NCT02440685). Phase 1 allows patients with solid tumors or hematologic malignancies; Phase 2 allows only patients with diffuse large B-Cell lymphoma (DLBCL), follicular lymphoma (FL) or mantle cell lymphoma (MCL). Endpoints include safety, tolerability, pharmacokinetics, serum markers of inflammation, and response using RECIST or Lugano Classification System. Table 40 below shows clinical safety data for ongoing Example 349 (up to
COMPOUND A doses of 40mg administered twice daily).
Table 40
Figure imgf000431_0001
Figure imgf000432_0001
Twenty- four patients (19 solid tumor, 5 lymphoma) have completed the 28-day DLT phase at doses of 10 mg - 75 mg BID and at 80 mg QD. All patients had multiple prior lines of treatment (range: 2 - 8). COMPOUND A has been well tolerated. No dose limiting adverse events have been reported at these dose levels. Most drug-related adverse events were Gr 1/2. Steady-state systemic exposure was high (Cmax, AUC (0-12h) and Tm at 40 mg BID was 0.7 μΜ, 6.3 μΜ and 18 h, respectively). High systemic exposure was also observed at 80 mg QD. Robust reduction of CRP, IL-18, MIP1 β, VCAM-1 , TNFR2 have been observed at all doses. Stable disease (RECIST, 9+ months) in a patient with primary peritoneal cancer, about 50% reduction in target lesions at 3 months in a FL patient (Lugano, 6 prior lines) and stable disease and reduction of pruritus in a peripheral T-Cell lymphoma patient after 2 months (Lugano, 2 prior lines) of treatment have been observed.
From the clinical data, it can be concluded that COMPOUND A is safe and well tolerated and does not appear to be associated with the significant thrombocytopenia, anemia and neutropenia as reported for the JAK inhibitor ruxolitinib and which should be managed by dose reduction, or interruption, or red blood cell transfusion. Clinical trials report thrombocytopenia, anemia and neutropenia in 70%, 96% and 19% of patients treated with ruxolitinib.
No COMPOUND A-related diarrhea has been reported, while the BTK signal inhibitor ibrutinib has been associated with diarrhea in over 50% of patients treated in clinical trials and severe/fatal bleeding events in up to 6%. COMPOUND A is also does not appear to be associated with lipid elevation as reported for JAK inhibitor tofacitinib. Clinical trials LDL elevations were reported in 15-19% of patients treated with tofacitinib.
Checkpoint inhibitors may serve to increase a baseline T-cell-specific immune response that turns the immune system against the tumor. However, a disruption in the functioning of immune checkpoint molecules can lead to imbalances in immunologic tolerance that result in an unchecked immune response. This may clinically manifest with autoimmune-like/ inflammatory side-effects, which cause collateral damage to
normal organ systems and tissues, including: the skin, gastrointestinal, hepatic, pulmonary, mucocutaneous, and endocrine systems. Such adverse events are termed immune-related adverse events and are also thought to be principally T-cell mediated. Other immune cells may play a role in the development of immune-related adverse events, including B cells that secrete antibodies that may mediate toxicity, granulocytes that secrete inflammatory mediators, and cytokines. Patients treated with the CTLA-4 PD-1 / PD-L1 directed treatment can experience the following adverse reactions; fatigue, pruritus, diarrhea, decreased appetite, rash, pyrexia, cough, dyspnea, musculoskeletal pain, constipation, and nausea. Warnings and precautions also include immune-mediated pneumonitis, colitis, and other immune mediated adverse reactions.
Indoleamine (2,3)-dioxygenase (IDO) inhibitors have shown to be associated with similar immune-related adverse reactions. Chimeric antigen receptor (CAR) T cells have shown to be associated with similar immune-related adverse reactions and the use of CAR T cells is limited by potentially severe and fatal toxicities. CAR T cells can potentially damage normal tissues by specifically targeting a tumor-associated antigen that is also expressed on those tissues. In addition, cytokine release syndrome (CRS), a systemic inflammatory response caused by cytokines released by infused CAR T cells can lead to widespread reversible organ dysfunction. CRS is the most common type of toxicity caused by CAR T cells.
The safety profile of COMPOUND A does not appear to overlap with checkpoint inhibitors, small molecule inhibitors, TCR agents (for example, CAR T cells and/or TCR agonist or antagonist peptides), vaccines (for example, sipuleucel-T (Provenge®) and/or
Talimogene laherparepvec (Imlygic®), aslo known as T-Vec) or other immune modulators and the addition of COMPOUND A could improve overall tolerability and help manage the reported immune mediated adverse reactions. Example 349 demonstrates that COMPOUND A is safe and well tolerated. Encouraging preliminary evidence of efficacy in NHL patients has been observed. MTD was not reached.
All publications cited in this specification are incorporated herein by reference. While the disclosure has been described with reference to particular embodiments, it will be appreciated that modifications can be made without departing from the spirit of the disclosure. Such modifications are intended to fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A pharmaceutical combination, the combination comprising:
a compound of Formula (I):
Figure imgf000435_0001
wherein:
R1 is R4R5, optionally substituted C1 to C6 alkoxy, optionally substituted C6 to C14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic or bicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic orbicyclic heterocyclyl, wherein:
(i) 3-4 membered cycloalkyl and heterocyclyl are saturated;
(ii) hydrogen atoms on the same carbon atom of said cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and
(iii) hydrogen atoms on the same atom of said cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent;
R is optionally substituted phenyl, -O-(C1 to C6 alkyl)-optionally substituted phenyl, or optionally substituted 5-6 membered heteroaryl, with the proviso that when R2 is 4- pyridyl, the 4-pyridyl lacks a carbonyl substituent at the 2nd position;
R4 and R5 are:
(a) independently selected from the group consisting of H, C1 to C6 alkyl, C1 to C6 hydroxyalkyl, C3 to C8 cycloalkyl, and -(C1 to C6 alkyl)N(Ci to C6 alkyl)(C1 to C6 alkyl);
(b) joined to form an optionally substituted 3-8 membered heterocyclyl, wherein;
(bi) hydrogen atoms on the same carbon atom of said heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and (bii) hydrogen atoms on the same atom of said heterocyclyl (b), cycloalkyl (bi), or heterocyclyl (bi), are optionally replaced with O to form an oxo substituent;
or a pharmaceutically acceptable salt or ester thereof; and
at least one immunotherapeutic agent.
2. The pharmaceutical combination of claim 1 , wherein the at least one
immunotherapeutic agent comprises one or more immunomodulators,
wherein the one or more immunomodulators target one or more of PD-1, PD-L1, CTLA-4, 4-1BB, OX40, LAG3, GITR, TIM3, VISTA, KIR, ICOS, BTLA, CD244, CD80, CD86, PD-L2, IDO-1 , IDO-2, and B7-H3 and
wherein the one or more immunomodulators produce a therapeutic effect.
3. The pharmaceutical combination of claim 1 , wherein the at least one
immunotherapeutic agent comprises an immunomodulator comprising one or more of inhibitors of immune system components, activators of immune system components, immune checkpoint inhibitors, biologic or small molecule therapeutics, indoleamine (2,3)- dioxygenase (IDO) inhibitors, vaccines, and agents that target T-cell receptors (TCR agents), wherein the TCR agents comprise one or more of chimeric antigen receptor (CAR) T cells, TCR agonist peptides and TCR antagonist peptides.
4. The pharmaceutical combination of claim 2, wherein the one or more
immunomodulators comprises one or more of:
an immune checkpoint inhibitor selected from the group consisting of anti-PD-1 antibody, anti-PD-Ll antibody, anti-CTLA-4 antibody, anti-4-lBB antibody, anti-OX40 antibody, anti-LAG3 antibody, anti-GITR antibody, anti-TIM3 antibody, anti- VISTA antibody, anti-KIR antibody, anti-ICOS antibody, anti-BTLA antibody, anti-CD244 antibody, anti-CD80 antibody, anti-CD86 antibody, anti-PD-L2 antibody, anti-IDO-1 antibody, anti- IDO-2 antibody, anti-B7-H3 antibody,
co-inhibitory molecules selected from the group consisting of cytotoxic T- lymphocyte-associated protein 4 (CTLA-4), PD-1, lymphocyte-activation gene 3, and T-cell immunoglobulin mucin-3, co-stimulatory molecules selected from the group consisting of glucocorticoid- induced tumor necrosis factor receptor and OX40 (CD 134, TNFRSF4, tumor necrosis factor receptor superfamily member 4), and
and small molecule inhibitors of any of the foregoing.
5. The pharmaceutical combination of claim 4, wherein the immune checkpoint inhibitors is anti-PD-1 antibody, or a small molecule IDO-1 inihibitor.
6. The pharmaceutical combination of claim 1 , further comprising at least one pharmaceutically acceptable carrier.
7. A method of treating cancer, comprising:
administering to a patient in need thereof a multi-part pharmaceutical combination comprising:
(a) a compound of Formula (I):
Figure imgf000437_0001
wherein:
R1 is R4R5, optionally substituted C1 to C6 alkoxy, optionally substituted C6 to C14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic or bicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic orbicyclic heterocyclyl, wherein:
(i) 3-4 membered cycloalkyl and heterocyclyl are saturated;
(ii) hydrogen atoms on the same carbon atom of said cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and
(iii) hydrogen atoms on the same atom of said cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent;
R2 is optionally substituted phenyl, -O-(C1 to C6 alkyl)-optionally substituted phenyl, or optionally substituted 5-6 membered heteroaryl, with the proviso that when R is 4- pyridyl, the 4-pyridyl lacks a carbonyl substituent at the 2nd position; R4 and R5 are:
(a) independently selected from the group consisting of H, C1 to C6 alkyl, C1 to C6 hydroxyalkyl, C3 to C8 cycloalkyl and -(C1 to C6 alkyl)N(Ci to C6 alkyl)(C1 to C6 alkyl);
(b) joined to form an optionally substituted 3-8 membered heterocyclyl, wherein;
(bi) hydrogen atoms on the same carbon atom of said heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and
(bii) hydrogen atoms on the same atom of said heterocyclyl (b), cycloalkyl (bi), or heterocyclyl (bi), are optionally replaced with O to form an oxo substituent;
or a pharmaceutically acceptable salt or ester thereof; and
(b) at least one immunotherapeutic agent.
8. The method of claim 7, wherein the at least one immunotherapeutic agent comprises one or more immunomodulators,
wherein the one or more immunomodulators target one or more of PD-1, PD-L1, CTLA-4, 4-1BB, OX40, LAG3, GITR, TIM3, VISTA, KIR, ICOS, BTLA, CD244, CD80, CD86, PD-L2, IDO-1 , IDO02, and B7-H3 and
wherein the one or more immunomodulators produce a therapeutic effect.
9. The method of claim 7, wherein the at least one immunotherapeutic agent comprises an immunomodulator comprising one or more of inhibitors of immune system components, activators of immune system components, immune checkpoint inhibitors, biologic or small molecule therapeutics, indoleamine (2,3)-dioxygenase (IDO) inhibitors, vaccines, and agents that target T-cell receptors (TCR agents), wherein the TCR agents comprise one or more of chimeric antigen receptor (CAR) T cells, TCR agonist peptides and TCR antagonist peptides.
10. The method of claim 9, wherein the one or more immunomodulators comprise one or more of: an immune checkpoint inhibitor selected from the group consisting of anti-PD-1 antibody, anti-PD-Ll antibody, anti-CTLA-4 antibody, anti-4-lBB antibody, anti-OX40 antibody, anti-LAG3 antibody, anti-GITR antibody, anti-TIM3 antibody, anti- VISTA antibody, anti-KIR antibody, anti-ICOS antibody, anti-BTLA antibody, anti-CD244 antibody, anti-CD80 antibody, anti-CD86 antibody, anti-PD-L2 antibody, anti-IDO-1 antibody, anti- IDO02 antibody, anti-B7-H3 antibody,
co-inhibitory molecules selected from the group consisting of cytotoxic T- lymphocyte-associated protein 4 (CTLA-4), PD-1, lymphocyte-activation gene 3, and T-cell immunoglobulin mucin-3,
co-stimulatory molecules selected from the group consisting of glucocorticoid- induced tumor necrosis factor receptor and OX40 (CD 134, TNFRSF4, tumor necrosis factor receptor superfamily member 4), and
and small molecule inhibitors of any of the foregoing.
11. The method of claim 10, wherein the one or more immune checkpoint inhibitors is anti-PD-1 antibody, or a small molecule IDO-1 inhibitor.
12. The method of claim 7, further comprising administering the compound and the at least one immunotherapeutic agent with at least one pharmaceutically acceptable carrier.
13. The method of claim 7, wherein administration of the compound is intravenous or oral and the administration of the at least one immunotherapeutic agent is intravenous, subcutaneous, intra-muscular or oral.
14. The method of claim 13, wherein:
administration of the compound is intravenous and the administration of the at least one immunotherapeutic agent is intravenous, subcutaneous or intra-muscular;
administration of the compound is oral and the administration of the at least one immunotherapeutic agent is oral;
administration of the compound is intravenous and the administration of the at least one immunotherapeutic agent is oral; or administration of the compound is oral and the administration of the at least one immunotherapeutic agent is intravenous, subcutaneous or intra-muscular.
15. The method of claim 7, wherein the compound is administered once or twice daily.
16. The method of claim 15, wherein:
the compound and the at least one immunotherapeutic agent are administered simultaneously or sequentially, or
the compound and the at least one immunotherapeutic agent are administered sequentially.
17. The method of claim 13, wherein the at least one immunotherapeutic agent is administered intermittently every four to thirty days.
18 The method of claim 7, wherein the cancer is selected from the group consisting of: prostate, head, neck, eye, mouth, throat, esophagus, bronchus, larynx, pharynx, chest, bone, lung, colon, rectum, stomach, bladder, uterus, cervix, breast, ovaries, vagina, testicles, skin, thyroid, blood, lymph nodes, kidney, liver, intestines, pancreas, brain, central nervous system, adrenal gland, skin or a leukemia and lymphoma.
19. The method of claim 7, further comprising:
identifying an indication or patient population for administering the compound and the at least one immunotherapeutic agent.
20. A method of inhibiting tumor growth or metastasis in a subject, comprising:
administering to the subject a multi-part pharmaceutical combination comprising: (a) a compound of Formula (I):
Figure imgf000440_0001
wherein: R1 is R4R5, optionally substituted C1 to C6 alkoxy, optionally substituted C6 to C14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic or bicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic orbicyclic heterocyclyl, wherein:
(i) 3-4 membered cycloalkyl and heterocyclyl are saturated;
(ii) hydrogen atoms on the same carbon atom of said cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and
(iii) hydrogen atoms on the same atom of said cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent;
R is optionally substituted phenyl, -O-(C1 to C6 alkyl)-optionally substituted phenyl, or optionally substituted 5-6 membered heteroaryl, with the proviso that when R2 is 4- pyridyl, the 4-pyridyl lacks a carbonyl substituent at the 2nd position;
R4 and R5 are:
(a) independently selected from the group consisting of H, C1 to C6 alkyl, C1 to C6 hydroxyalkyl, C3 to C8 cycloalkyl, and -(C1 to C6 alkyl)N(Ci to C6 alkyl)(C1 to C6 alkyl);
(b) joined to form an optionally substituted 3-8 membered heterocyclyl, wherein;
(bi) hydrogen atoms on the same carbon atom of said heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and
(bii) hydrogen atoms on the same atom of said heterocyclyl (b), cycloalkyl (bi), or heterocyclyl (bi), are optionally replaced with O to form an oxo substituent;
or a pharmaceutically acceptable salt or ester thereof; and
(b) at least one immunotherapeutic agent,
wherein the administration of the compound and the at least one immunotherapeutic agent inhibits tumor growth or metastasis.
21. A dosing regimen comprising: administering to a patient in need thereof a multi-part pharmaceutical combination comprising:
(a) a compound of Formula (I):
Figure imgf000442_0001
wherein:
R1 is R4R5, optionally substituted C1 to C6 alkoxy, optionally substituted C6 to C14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic or bicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic orbicyclic heterocyclyl, wherein:
(i) 3-4 membered cycloalkyl and heterocyclyl are saturated;
(ii) hydrogen atoms on the same carbon atom of said cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and
(iii) hydrogen atoms on the same atom of said cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent;
R2 is optionally substituted phenyl, -O-(C1 to C6 alkyl)-optionally substituted phenyl, or optionally substituted 5-6 membered heteroaryl, with the proviso that when R is 4- pyridyl, the 4-pyridyl lacks a carbonyl substituent at the 2nd position;
R4 and R5 are:
(a) independently selected from the group consisting of H, C1 to C6 alkyl, C1 to C6 hydroxyalkyl, C3 to C8 cycloalkyl, and -(C1 to C6 alkyl)N(Ci to C6 alkyl)(C1 to C6 alkyl);
(b) joined to form an optionally substituted 3-8 membered heterocyclyl, wherein;
hydrogen atoms on the same carbon atom of said heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and (bii) hydrogen atoms on the same atom of said heterocyclyl (b), cycloalkyl (bi), or heterocyclyl (bi), are optionally replaced with O to form an oxo substituent;
or a pharmaceutically acceptable salt or ester thereof; and
(b) at least one immunotherapeutic agent.
22. A kit comprising:
at least one first dosage form comprising a compound of Formula (I):
Figure imgf000443_0001
wherein:
R1 is R4R5, optionally substituted C1 to C6 alkoxy, optionally substituted C6 to C14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic or bicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic orbicyclic heterocyclyl, wherein:
(i) 3-4 membered cycloalkyl and heterocyclyl are saturated;
(ii) hydrogen atoms on the same carbon atom of said cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and
(iii) hydrogen atoms on the same atom of said cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent;
R is optionally substituted phenyl, -O-(C1 to C6 alkyl)-optionally substituted phenyl, or optionally substituted 5-6 membered heteroaryl, with the proviso that when R2 is 4- pyridyl, the 4-pyridyl lacks a carbonyl substituent at the 2nd position;
R4 and R5 are:
(a) independently selected from the group consisting of H, C1 to C6 alkyl, C1 to C6 hydroxyalkyl, C3 to C8 cycloalkyl and -(C1 to C6 alkyl)N(Ci to C6 alkyl)(C1 to C6 alkyl);
(b) joined to form an optionally substituted 3-8 membered heterocyclyl, wherein; (bi) hydrogen atoms on the same carbon atom of said heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and
(bii) hydrogen atoms on the same atom of said heterocyclyl (b), cycloalkyl (bi), or heterocyclyl (bi), are optionally replaced with O to form an oxo substituent;
or a pharmaceutically acceptable salt or ester thereof;
at least one second dosage form comprising at least one immuno therapeutic agent; and
therapeutic instructions for administering the at least one first dosage form and the at least one second dosage form, wherein the therapeutic instructions comprise the steps of:
administering to a patient in need thereof the compound and the at least one immunotherapeutic agent.
23. A use of a medicament in treating cancer, wherein the medicament is administered by a dosing regimen comprising:
administering to a patient in need thereof a multi-part pharmaceutical combination comprising:
(a) a compound of Formula (I):
Figure imgf000444_0001
wherein:
R1 is R4R5, optionally substituted C1 to C6 alkoxy, optionally substituted C6 to C14 aryl, optionally substituted heteroaryl, optionally substituted 3-10 membered monocyclic or bicyclic cycloalkyl, or optionally substituted 3-10 membered monocyclic orbicyclic heterocyclyl, wherein:
(i) 3-4 membered cycloalkyl and heterocyclyl are saturated;
(ii) hydrogen atoms on the same carbon atom of said cycloalkyl or heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spirocycloalkyl or spiroheterocyclyl; and (iii) hydrogen atoms on the same atom of said cycloalkyl or heterocyclyl are optionally replaced with O to form an oxo substituent;
R2 is optionally substituted phenyl, -O-(C1 to C6 alkyl)-optionally substituted phenyl, or optionally substituted 5-6 membered heteroaryl, with the proviso that when R2 is 4- pyridyl, the 4-pyridyl lacks a carbonyl substituent at the 2nd position;
R4 and R5 are:
(a) independently selected from the group consisting of H, C1 to C6 alkyl, C1 to C6 hydroxyalkyl, C3 to C8 cycloalkyl and -(C1 to C6 alkyl)N(Ci to C6 alkyl)(C1 to C6 alkyl);
(b) joined to form an optionally substituted 3-8 membered heterocyclyl, wherein;
(bi) hydrogen atoms on the same carbon atom of said heterocyclyl are optionally replaced with an optionally substituted 3-6 membered cycloalkyl or heterocyclyl to form a spiro cycloalkyl or spiroheterocyclyl; and
(bii) hydrogen atoms on the same atom of said heterocyclyl (b), cycloalkyl (bi), or heterocyclyl (bi), are optionally replaced with O to form an oxo substituent;
or a pharmaceutically acceptable salt or ester thereof; and
(b) at least one immunotherapeutic agent.
24. The pharmaceutical combination according to claim 1, wherein R2 is:
phenyl substituted with C(O)NR4R5;
phenyl substituted withNR4R5;
phenyl substituted with (C1 to C6 alkyl)NR4R5;
phenyl substituted with (C1 to C6 alkyl)NR4R5;
a heteroaryl substituted with (C1 to C6 alkyl)NR4R5;
a heteroaryl substituted with NR4R5;
phenyl substituted with one or more C1 to C6 alkoxy, (C1 to C6 alkyl)halogen, C1 to C6 trifluoroalkoxy, (C1 to C6 alkyl)C(0(OH, halogen, optionally substituted C3 to C8 cycloalkyl, optionally substituted heterocyclyl, optionally substituted heteroaryl, -O-(C1 to C6 alkyl)C(O)OH, -O-(C1 to C6 alkyl)-NR4R5, -O-(optionally substituted heterocycle), -0(Ci to Ce alkyl)-N(C1 to C6 alkyl)(C1 to C6 alkyl), -O-(C1 to C6 alkyl)NH2, C1 to C6 hydroxyalkyl, - 0-(Ci to C6 hydroxyalkyl), 0-(C1 to C6 alkyl)-C(O)OH, -C1 to C6 alkoxy-C1 to C6 alkoxy, O- (heterocycle)-(C1 to C6 hydroxyalkyl), S02-(C1 to C6 alkyl), or -(C1 to C6 alkyl)-(C1 to C6 alkoxy)-halogen;
-O-(C1 to C6 alkyl)NR4R5; or
aryl substituted with -O-(C1 to C6 alkyl)-heterocycle.
25. The pharmaceutical combination according to claim 1, wherein R4 and R5 are:
joined to form an optionally substituted piperidine or diazepane;
(C1 to C6 hydroxyalkyl); or
joined to form an optionally substituted 6-membered ring.
26. The pharmaceutical combination according to claim 1, wherein R
N(C1 to C6 alkyl)(C1 to C6 alkyl) or Ci to C6 alkoxy;
optionally substituted phenyl;
optionally substituted 5-9 membered saturated heterocyclyl;
a heterocyclyl of the structure:
Figure imgf000446_0001
wherein f, g, h, j, and m are, independently, absent, (CH2), CH(R ), Z, or C=0, R3 is H, C(O)OH, or C(O)0(C1 to C6 alkyl),
R45, R46, R47, and R48 are, independently, H or C1 to C6 alkyl, and Z is O, S, SO, S02, orNH;
a heteroaryl;
a monocyclic C3 to C8 cycloalkyl; or
piperidine substituted with C(O)(C1 to C6 alkyl)CN;
27. The pharmaceutical combination according to claim 1, wherein the compound is a salt of an acid.
28. The pharmaceutical combination according to claim 27, wherein said acid is selected from the group consisting of acetic, propionic, lactic, citric, tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic,
trifluoroacetic, and camphorsulfonic.
29. The pharmaceutical combination according to claim 1 , wherein the compound is:
4-(4-mo holinophenylamino)-2-phenylpyrimido[4,5-d]pyridazin-5(6H)-one;
methyl 4-(5-oxo-4-(4-(piperazin- 1 -ylmethyl)phenylamino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)benzoate hydrochloride;
2-morpholino-4-(4-(piperazin-l-ylmethyl)phenylamino)pyrimido[4,5-d]pyridazin- 5(6H)-one hydrochloride;
4-(4-(morpholmomethyl)phenylammo)-2-phenylpyrimido[4,5-d]pyridazin-5(6H)-one;
4-(4-(4-ethylpiperazm-l-yl)phenylamino)-2-phenylpyrimido[4,5-d]pyridazin-5(6H)- one;
4-(4-((4-ethylpiperazin-l-yl)methyl)phenylamino)-2-phenylpyrimido[4,5-d]pyridazin- 5(6H)-one;
2-phenyl-4-(4-(piperazm-l-ylmethyl)phenylamino)pyrimido[4,5-d]pyridazin-5(6H)- one hydrochloride;
2-phenyl-4-(4-(piperazm-l-yl)phenylamino)pyrimido[4,5-d]pyridazin-5(6H)-one; 4-(4-(morpholme-4-carbonyl)phenylamino)-2-phenylpyrimido[4,5-d]pyridazin- 5(6H)-one;
4-(4-(bis(2-hydroxyethyl)amino)phenylammo)-2-phenylpyrimido[4,5-d]pyridazin- 5(6H)-one;
4-(4-(4-(2-aminoacetyl)piperazin-l-yl)phenylamino)-2-phenylpyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride;
2-(4-(4-(5-oxo-2-phenyl-5,6-dmydropyrimido[4,5-d]pyridazin-4- ylamino)phenyl)piperazin-l-yl)acetic acid;
l-(4-(5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4- ylamino)phenyl)piperidine-4-carboxylic acid;
4-(4-(4-(2-aminoacetyl)piperazin-l-yl)phenylamino)-2-phenylpyrimido[4,5- d]pyridazin-5(6H)-one;
N-(2-(dimethylammo)ethyl)-N-methyl-4-(5-oxo-2-phenyl-5,6-dihydropyrimido[4,5- d]pyridazin-4-ylamino)benzamide; 4-(4-(2-oxo-l,7-diazaspiro[3.5]nonan-7-yl)phenylamino)-2-phenylpyrimido[4,5- d]pyridazin-5(6H)-one;
4-(4-(2-oxa-7-azaspiro[3.5]nonan-7-yl)phenylamino)-2-phenylpyrimido[4,5- d]pyridazin-5(6H)-one;
4-(4-mo holinophenylamino)-2-(6-azaspiro [2.5 ]octan-6-yl)pyrimido [4,5- d]pyridazin-5(6H)-one;
6-(4-(5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4-ylamino)phenyl)-6- azaspiro[2.5]octane-l -carboxylic acid;
ethyl 6-(4-(5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4-ylamino)phenyl)- 6-azaspiro [2.5 ]octane- 1 -carboxylate;
6-(4-(5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4-ylamino)benzyl)-6- azaspiro[2.5]octane-l -carboxylic acid;
sodium 6-(4-(5-oxo-2-phenyl-5,6-dmydropyrimido[4,5-d]pyridazin-4- ylamino)phenyl)-6-azaspiro[2.5]octane-l-carboxylate;
4-(4-(2-oxa-7-azaspiro[3.5]nonan-7-ylmethyl)phenylammo)-2-phenylpyrimido[4,5- d]pyridazin-5(6H)-one;
4-(4-(piperazin- 1 -ylmethyl)phenylamino)-2-(thiophen-3 -yl)pyrimido[4,5-d]pyridazin- 5(6H)-one hydrochloride;
6-(5-oxo-4-(4-(piperazm-l-ylmethyl)phenylamino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)-6-azaspiro[2.5]octane-l -carboxylic acid hydrochloride;
4-(4-(4-ethylp ip erazin- 1 -yl)phenylamino )-2-morpholinopyrimido [4,5 -d]p yridazin- 5(6H)-one;
4-(5-oxo-4-(4-(piperazm-l-ylmethyl)phenylamino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)benzoic acid hydrochloride;
4-(4-(4-ethylp ip erazin- 1 -yl)phenylamino )-2-(4- (trifluoromemoxy)phenyl)pyrimido[4,5-d]pyridazin-5(6H)-one;
methyl 4-(4-(4-morpholinophenylamino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)benzoate;
4-(4-(piperazin-l-ylmethyl)phenylamino)-2-(piperidm-l-yl)pyrimido[4,5-d]pyrid^ 5(6H)-one hydrochloride;
2-(3-methoxyphenyl)-4-(4-(piperazin-l-ylmethyl)phenylamino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride; 2-(piperazin- 1 -yl)-4-(4-(piperazin- 1 -ylmethyl)phenylamino)pyrimido [4,5 - d]pyridazin-5(6H)-one dihydrochloride;
2-(benzo[d][l,3]dioxol-5-yl)-4-(4-(piperazin-l-ylme1hyl)phenylamino)pyrim d]pyridazin-5(6H)-one hydrochloride;
2-(l-(4-(5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4- ylamino)phenyl)piperidin-4-yl)acetic acid;
1- (4-(5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4- ylamino)benzyl)piperidine-4-carboxylic acid;
2- (2-methoxyphenyl)-4-(4-(piperazin-l-ylmethyl)phenylamino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride;
4-(4-(4-ethylpiperazin-l-yl)phenylamino)-2-(tMophen-3-yl)pyrimido[4,5-d]pyridazin^ 5(6H)-one hydrochloride;
9-(5-oxo-4-(4-(piperazin-l-ylmethyl)phenylamino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)-3,9-diazaspiro[5.5]undecane-2,4-dione hydrochloride;
6-(4-(5-oxo-2-(thiophen-3-yl)-5 ,6-dihydrop yrimido[4,5-d]pyridazin-4- ylamino)phenyl)-6-azaspiro[2.5]octane-l-carboxylic acid;
2-(4-cUorophenyl)-4-(4-(piperazin-l-ylmethyl)phenylamino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride;
2-(4-methoxyphenyl)-4-(4-(piperazin-l-ylmethyl)phenylamino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride;
6-(4-((2-mo holino-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(l -(5-oxo-4-((4-(piperazin- 1 -ylmethyl)phenyl)amino)-5,6-dihydropyrimido [4,5- d]pyridazin-2-yl)piperidin-4-yl)acetic acid hydrochloride;
2-(l-oxidotWomorpholino)-4-((4-(piperazin-l-ylmethyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride;
2-(4-methylpiperazin- 1 -yl)-4-((4-(piperazin- 1 -ylmethyl)phenyl)amino)pyrimido [4,5- d]pyridazin-5(6H)-one hydrochloride;
6-(4-((2-(4-methoxyphenyl)-5 -oxo-5 ,6-dihydrop yrimido [4 ,5 -d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
6-(4-((2-(3 -methoxyphenyl)-5 -oxo-5 ,6-dihydrop yrimido [4 ,5 -d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid; 4-((4-(piperazin- 1 -ylmethyl)phenyl)amino)-2-(pyrrolidin- 1 -yl)p yrimido [4,5- d]pyridazin-5(6H)-one hydrochloride;
2-(dimethylamino)-4-((4-(p^erazm-l-ylmethyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride;
2-emoxy-4-((4-(piperazm-l-ylmethyl)phenyl)amino)pyrimido[4,5-d]pyridazin^ one hydrochloride;
1- (5-oxo-4-((4-(piperazin-l-ylmethyl)phenyl)amino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidine-4-carboxylic acid hydrochloride;
2- (azepan- 1 -yl)-4-((4-(piperazin- 1 -ylmethyl)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one hydrochloride;
6-(4-((2-(2-methoxyphenyl)-5 -oxo-5 ,6-dihydrop yrimido [4 ,5 -d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(diisopropylamino)-4-((4-(piperazin-l-ylmethyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride;
2-(4-(morpholinomethyl)phenyl)-4-((4-(piperazin- 1 - ylmethyl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one hydrochloride;
1- (5-oxo-4-((4-(piperazin-l-ylmethyl)phenyl)amino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidine-4-carbonitrile hydrochloride;
2- (4-ethylpiperazin- 1 -yl)-4-((4-(piperazin- 1 -ylmethyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride;
4-((l-(2-morpholinoethyl)-lH-pyrazol-4-yl)amino)-2-phenylpyrimido[4,5- d]pyridazin-5(6H)-one;
2-( 1 ,4-diazepan- 1 -yl)-4-((4-(piperazin- 1 -ylmethyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one dihydrochloride;
2-(azepan-l-yl)-4-((4-mo holmophenyl)ammo)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-methoxy-4-((4-(piperazin-l-ylmethyl)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one hydrochloride;
6-(4-((2-(azepan-l-yl)-5-oxo-5,6-dmydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-phenyl-4-((l -(2-(piperazin- 1 -yl)ethyl)- lH-pyrazol-4-yl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride;
4-(( 1 -(2-(4-methylp ip erazin- 1 -yl)ethyl)- 1 H-pyrazol-4-yl)amino)-2- phenylpyrimido[4,5-d]pyridazin-5(6H)-one; 4-((l-(2-(4-ethylpiperazin-l-yl)ethyl)-lH-pyrazol-4-yl)amino)-2-phenylpyrimido[4,5- d]pyridazin-5(6H)-one;
6-(4-((2-(4-(cyanomethyl)piperidm-l-yl)-5-oxo-5,6- 4-yl)amino)phenyl)-6-azaspiro[2.5]octane-l -carboxylic acid;
2-(azepan-l-yl)-4-((3,4,5-trime1hoxyphenyl)amino)p^
one;
2-(azepan-l-yl)-4-((4-(morpholine-4-carbonyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
2-(l-(4-((2-(4-(cyanomethyl)piperazin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperidin-4-yl)acetic acid;
2-(l-(4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperidin-4-yl)acetic acid;
6-(4-((2-(l,4-diazq)an-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid hydrochloride;
6-(4-((2-(4-(cyanomethyl)piperazin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)-6-azaspiro[2.5]octane-l -carboxylic acid;
6-(4-((2-(4-cyanopiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(l-(4-((2-(4-cyanopiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
1- (4-((2-(4-cyanopiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)benzyl)piperidine-4-carboxylic acid;
2- (l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)benzyl)piperidin-4-yl)acetic acid;
1- (4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin^ 4-yl)amino)benzyl)piperidine-4-carboxylic acid;
6-(4-((2-cyclohexyl-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4-yl)amino)phenyl)- 6-azaspiro [2.5 ]octane- 1 -carboxylic acid;
2- (l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetonitrile; 6-(4-((2-cycloheptyl-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4-yl)amino)phenyl)- 6-azaspiro [2.5 ]octane- 1 -carboxylic acid;
2-(l-(4-((5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetonitrile;
4-((4-((4-(2-hydroxy-2-methylpropanoyl)piperazin-l-yl)methyl)phenyl)amino)-2- phenylpyrimido[4,5-d]pyridazin-5(6H)-one;
2-(azepan- 1 -yl)-4-((4-((4-(2-hydroxy-2-methylpropanoyl)piperazin- 1 - yl)methyl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(azepan-l-yl)-4-((4-((4-methylpiperazin-l-yl)methyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(4-(4-((5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin- 1 -yl)acetic acid;
2-(4-(4-((5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)cyclohexyl)acetic acid;
2-(l-(4-((2-(azocan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
2-(azepan- 1 -yl)-4-((4-(2-(piperazin- 1 -yl)ethoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one hydrochloride;
2-(l-(4-((2-cycloheptyl-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
2-(4-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)cyclohexyl)acetic acid;
4-(4-((2-(azepan- 1 -yl)-5 -oxo-5 ,6-dihydrop yrimido [4,5 -d]pyridazin-4- yl)amino)phenoxy)butanoic acid;
2-(azepan-l-yl)-4-((4-(2-morpholmoethoxy)phenyl)ammo)pyrimido[4,5-d]pyridazin- 5(6H)-one;
4-((4-(2-mo hoUnoethoxy)phenyl)ammo)-2-phenylpyrimido[4,5-d]pyridazin-5(6H)- one;
2-(azepan-l-yl)-4-((4-(2-(4-memylpiperazin-l-yl)ethoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-( 1 -(2-(4-((2-(azepan- 1 -yl)-5 -oxo-5 ,6-dihydrop yrimido [4 ,5 -d]pyridazin-4- yl)amino)phenoxy)ethyl)piperidin-4-yl)acetic acid; 2-(l-(5-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)pyridin-2-yl)piperidin-4-yl)acetonitrile;
1- (2-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenoxy)ethyl)piperidine-4-carboxylic acid;
2- (l-(5-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)pyridin-2-yl)piperidin-4-yl)acetamide;
2-(l-(4-((2-(4-(2-cyanopropan-2-yl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperidin-4-yl)acetic acid;
2-(l-(5-((5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4-yl)amino)pyridin-2- yl)piperidin-4-yl)acetonitrile;
2-(l-(5-((5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4-yl)amino)pyridin-2- yl)piperidin-4-yl)acetamide;
2-(l-(5-((2-(4-(cyanomethyl)piperidin-l -yl)-5-oxo-5 ,6-dihydrop yrimido[4,5- d]pyridazin-4-yl)amino)pyridin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(piperazine- 1 -carbonyl)phenyl)amino)p yrimido [4,5- d]pyridazin-5(6H)-one hydrochloride;
2-(l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dmydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetamide;
2-(4-(2-(4-((2-(azepan- 1 -yl)-5 -oxo-5 ,6-dihydrop yrimido [4 ,5 -d]pyridazin-4- yl)amino)phenoxy)ethyl)piperazin- 1 -yl)acetic acid;
2-(azepan- 1 -yl)-4-((4-(2-(piperazin- 1 -yl)ethoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(5-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)pyridin-2-yl)piperidin-4-yl)acetic acid;
2-(l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)-N-(2 -hydro xyethyl)acetamide;
2-(azepan- 1 -yl)-4-((4-(((2 S,5 S)-5 -(hydroxymethyl)- 1 ,4-dioxan-2- yl)methoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(4-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperazin- 1 -yl)-2-methylpropanenitrile;
2-(4-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperazin- 1 -yl)-2-methylpropanamide; 2-(l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetonitrile;
2-(l-(5-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)pyridin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(2-(4-(2-hydroxy-2-methylpropanoyl)piperazin-l- yl)ethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(5-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)pyridin-2-yl)piperidin-4-yl)acetic acid;
2-(l-(4-((2-(3,5-dimethylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridaz 4-yl)amino)phenyl)piperidin-4-yl)acetic acid;
2-(l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)-N,N-bis(2-hydroxyethyl)acetamide;
2-methyl-2-( 1 -(4-((5-oxo-2-phenyl-5 ,6-dihydropyrimido [4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)propanenitrile;
2-(4-(2-(4-((2-(azepan- 1 -yl)-5 -oxo-5 ,6-dihydrop yrimido [4 ,5 -d]pyridazin-4- yl)amino)phenoxy)ethyl)piperazin- 1 -yl)-2-methylpropanenitrile;
2-(l-(4-((2-(2,6-dimethylmorphoUno)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
4-((4-(((2S,5S)-5-(hydroxymethyl)-l ,4-dioxan-2-yl)methoxy)phenyl)amino)-2- phenylpyrimido[4,5-d]pyridazin-5(6H)-one;
2-((lS,4S)-4-(4-((2-(azq)an-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)cyclohexyl)acetic acid;
(2R,5S)-5-((4-((2-(azq)an-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenoxy)methyl)-l ,4-dioxane-2-carboxylic acid;
2-(4-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperazin- 1 -yl)acetic acid;
2-(l-(3 -(4-((2-(azepan- 1 -yl)-5 -oxo-5 ,6-dihydrop yrimido [4 ,5 -d]pyridazin-4- yl)amino)phenoxy)propyl)piperidin-4-yl)acetic acid;
6-(4-((2-(cyclohexylammo)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)spiro[2.5]octane-l-carboxylic acid;
6-(4-((5-oxo-2-(piperidin- 1 -yl)-5,6-dihydropyrimido [4,5-d]pyridazin-4- yl)amino)phenyl)spiro[2.5]octane-l-carboxylic acid; 2-((l R,4R)-4-(4-((2-(azepan- 1 -yl)-5-oxo-5 ,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)cyclohexyl)acetic acid;
3 -( 1 -(4-((2-(azepan- 1 -yl)-5-oxo-5,6-dihydropyrimido [4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)propanoic acid;
2- (l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)cyclopropanecarboxylic acid;
3- (l-(4-((5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)propanoic acid;
6-(4-((2-(4-fluorophenyl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
6-(5-((2-(azq)an-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)pyridin-2-yl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
6-(4-((2-(azq)an-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4-yl)amino)-2- fluorophenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(azepan-l-yl)-4-((4-(3-(piperazin-l-yl)propoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
6-(5-((5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4-yl)amino)pyridin-2- yl)-6-azaspiro[2.5]octane-l -carboxylic acid;
6-(4-((5-oxo-2-(pyridin-4-yl)-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(azepan-l-yl)-4-((4-(4-(2-hydroxypropan-2-yl)piperidin-l- yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(4-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperazin- 1 -yl)-2-methylpropanoic acid;
6-(4-((2-(azocan- 1 -yl)-5 -oxo-5 ,6-dihydrop yrimido [4,5 -d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)-2-methylpropanoic acid;
2-methyl-2-(l-(4-((5-oxo-2-phenyl-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)propanoic acid;
2-(2,6-dimethylpiperidin- 1 -yl)-4-((4-(piperazin- 1 - ylmethyl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one; 2-(l-(4-((2-(2,6-dimethylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazm^ 4-yl)amino)phenyl)piperidin-4-yl)acetic acid;
6-(4-((2-(2,6-dimethylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(l-(4-((2-(2,6-dimethylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin- 4-yl)amino)phenyl)piperidin-4-yl)acetonitrile;
2-(2,6-dimethylpiperidin-l-yl)-4-((4-(4-(2-hydroxy-2-methylpropanoyl)piperazin-l- yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l -(5-((2-(2,6-dimethylpiperidm- 1 -yl)-5-oxo-5,6-d
4-yl)amino)pyridin-2-yl)piperidin-4-yl)acetic acid;
2-(3,5-dimethylpiperidin- 1 -yl)-4-((4-(piperazin- 1 - ylmethyl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((2-(3,5-dimethylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridaz 4-yl)amino)phenyl)piperidin-4-yl)acetic acid;
6-(4-((2-(3,5-dimethylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(l-(4-((2-(3,5-dimethylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyr 4-yl)amino)phenyl)piperidin-4-yl)acetonitrile;
2-(3,5-dimethylpiperidin-l-yl)-4-((4-(4-(2-hydroxy-2-methylpropanoyl)piperidin-l- yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(5-((2-(3,5-dimethylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyr 4-yl)amino)pyridin-2-yl)piperidin-4-yl)acetic acid;
2-(2,6-dimethylmorpholino)-4-((4-(piperazin-l-ylmethyl)phenyl)amino)py^ d]pyridazin-5(6H)-one;
2-(l-(4-((2-(2,6-dimethylmorphoUno)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
6-(4-((2-(2,6-dimethylmorpholino)-5-oxo-5,6-dihy^
yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(l-(4-((2-(2,6-dimethylmorphoUno)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetonitrile;
2-(2,6-dimethylmorpholino)-4-((4-(4-(2-hydroxy-2-methylpropanoyl)piperidin-l- yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one; 2-(l-(5-((2-(2,6-dimethylmorphoUno)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)pyridin-2-yl)piperidin-4-yl)acetic acid;
2-(diisopropylamino)-4-((4-(piperazin-l-ylmethyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((2-(diisopropylamino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
6-(4-((2-(diisopropylamino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(l-(4-((2-(diisopropylamino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetonitrile;
2-(diisopropylamino)-4-((4-(4-(2-hydroxy-2-methylpropanoyl)piperidin-l- yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(5-((2-(diisopropylamino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)pyridin-2-yl)piperidin-4-yl)acetic acid;
2-(2-methylpiperidin- 1 -yl)-4-((4-(piperazin- 1 -ylmethyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((2-(2-methylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
6-(4-((2-(2-methylp ip eridin- 1 -yl)-5 -oxo-5 ,6-dihydropyrimido [4,5 -d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(l-(4-((2-(2-methylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetonitrile;
4-((4-(4-(2-hydroxy-2-methylpropanoyl)piperidin-l-yl)phenyl)amino)-2-(2- methylpiperidin-l-yl)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(5-((2-(2-methylpiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)pyridin-2-yl)piperidin-4-yl)acetic acid;
1 -(4-((2-(azepan-l -yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidine-4-carboxylic acid;
1 -(4-((2-(azepan-l -yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)benzyl)piperidine-4-carboxylic acid;
4-((4-((4-(2H-tetrazol-5-yl)piperidin-l-yl)methyl)phenyl)amino)-2-(azepan-l- yl)pyrimido[4,5-d]pyridazin-5(6H)-one; 4-((4-((4-((2H-tetrazol-5-yl)methyl)piperidin-l-yl)methyl)phenyl)amino)-2-(azq)an- l-yl)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((2-cycloheptyl-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
1- (4-((2-cycloheptyl-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidine-4-carboxylic acid;
6-(4-((2-(3-cyanopiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2- (l-(4-((2-(3-cyanopiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
l-(4-((2-(3-cyanopiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidine-4-carboxylic acid;
l-(4-((2-(3-cyanopiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)benzyl)piperidine-4-carboxylic acid;
1- (4-((4-((4-(2H-tetrazol-5-yl)piperidin-l-yl)methyl)phenyl)amino)-5-oxo-5,6- dihydropyrimido [4,5 -d]pyridazin-2 -yl)piperidine-3 -carbonitrile ;
2- (l-(4-((2-(3-cyanopiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)benzyl)piperidin-4-yl)acetic acid;
l-(4-((4-((4-((2H-tetrazol-5-yl)methyl)piperidin-l-yl)methyl)phenyl)amino)-5-oxo- 5,6-dihydropyimido[4,5-d]pyridazin-2-yl)piperidine-3-carbonitrile;
1- (4-((2-cycloheptyl-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)benzyl)piperidine-4-carboxylic acid;
4-((4-((4-(2H-tetrazol-5-yl)piperidin-l-yl)methyl)phenyl)amino)-2- cycloheptylpyrimido[4,5-d]pyridazin-5(6H)-one;
2- (l-(4-((2-cycloheptyl-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)benzyl)piperidin-4-yl)acetic acid;
4-((4-((4-((2H-tetrazol-5-yl)methyl)piperidin-l-yl)methyl)phenyl)amino)-2- cycloheptylpyrimido[4,5-d]pyridazin-5(6H)-one;
l-(4-((2-(4-cyanopiperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidine-4-carboxylic acid;
l-(4-((4-((4-(2H-tetrazol-5-yl)piperidin-l-yl)methyl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidine-4-carbonitrile; l-(4-((4-((4-((2H-tetrazol-5-yl)methyl)piperidin-l-yl)methyl)phenyl)amino)-5-oxo- 5,6-dihydropyimido[4,5-d]pyridazin-2-yl)piperidine-4-carbonitrile;
1 - (4-((2-(4-(cyanomethyl)piperidm-l -yl)-5-oxo-5^
4-yl)amino)benzyl)piperidine-4-carboxylic acid;
2- (l-(4-((4-((4-(2H-tetrazol-5-yl)piperidin-l-yl)methyl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-( 1 -(4-((4-((4-((2H-tetrazol-5-yl)methyl)piperidin- 1 -yl)methyl)phenyl)amino)-5 - oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
l-(4-((2-(4-(cyanomethyl)piperazin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperidine-4-carboxylic acid;
1- (4-((2-(4-(cyanomethyl)piperazin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)benzyl)piperidine-4-carboxylic acid;
2- (4-(4-((4-((4-(2H-tetrazol-5-yl)piperidin-l-yl)methyl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperazin-l-yl)acetonitrile;
2-(l-(4-((2-(4-(cyanomethyl)piperazin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)benzyl)piperidin-4-yl)acetic acid;
2-(4-(4-((4-((4-((2H-tetrazol-5-yl)methyl)piperidin-l-yl)methyl)phenyl)amino)-5- oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperazin-l-yl)acetonitrile;
6-(4-((2-(l ,4-diazepan- 1 -yl)-5-oxo-5 ,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(l-(4-((2-(l,4-diazepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetic acid;
1 -(4-((2-(l ,4-diazepan- 1 -yl)-5-oxo-5 ,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidine-4-carboxylic acid;
1 -(4-((2-(l ,4-diazepan- 1 -yl)-5-oxo-5 ,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)benzyl)piperidine-4-carboxylic acid;
6-(4-((2-(4-(2-cyanoacetyl)piperazin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)-6-azaspiro[2.5]octane-l -carboxylic acid;
2-(l-(4-((2-(4-(2-cyanoacetyl)piperazin-l-yl)-5-oxo-5,6-diliydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperidin-4-yl)acetic acid;
1 -(4-((2-(4-(2-cyanoacetyl)piperazin- 1 -yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperidine-4-carboxylic acid; 1 -(4-((2-(4-(2-cyanoacetyl)piperazin- 1 -yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)benzyl)piperidine-4-carboxylic acid;
3- (4-(4-((4-((4-(2H-tetrazol-5-yl)piperidin-l-yl)methyl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperazin-l-yl)-3-oxopropanenitrile;
2-(l-(4-((2-(4-(2-cyanoacetyl)piperazin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)benzyl)piperidin-4-yl)acetic acid;
4- ((4-((4-((2H-tetrazol-5-yl)methyl)piperidin -yl)methyl)phenyl)amino)-2-(4-(2- isocyanoacetyl)piperazin- 1 -yl)pyrimido[4,5-d]pyridazin-5(6H)-one;
6-(4-((2-(l-(2-cyanoacetyl)piperidin-4-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)-6-azaspiro[2.5]octane-l-carboxylic acid;
2- (l-(4-((2-(l-(2-cyanoacetyl)piperidin-4-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperidin-4-yl)acetic acid;
l-(4-((2-(l-(2-cyanoacetyl)piperidin-4-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperidine-4-carboxylic acid;
1- (4-((2-(l-(2-cyanoacetyl)piperidin-4-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)benzyl)piperidine-4-carboxylic acid;
3- (4-(4-((4-((4-(2H-tetrazol-5-yl)piperidin-l-yl)methyl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-l-yl)-3-oxopropanenitrile;
2- (l-(4-((2-(l-(2-cyanoacetyl)piperidin-4-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)benzyl)piperidin-4-yl)acetic acid;
4- ((4-((4-((2H-tetrazol-5-yl)methyl)piperidin -yl)methyl)phenyl)amino)-2-(l-(2- isocyanoacetyl)piperidin-4-yl)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(4-(5-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)pyridin-2-yl)piperazin- 1 -yl)acetic acid;
2-(4-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)- 1 ,4-diazepan- 1 -yl)acetic acid;
2- (l-(4-((4-(4-(2-hydroxypropan-2-yl)piperidin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
3- (l-(4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperidin-4-yl)propanoic acid;
6-(5-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridaz^ 4-yl)amino)pyridin-2-yl)-6-azaspiro[2.5]octane-l-carboxylic acid; 1- (5-oxo-4-((4-(2-(piperazin-l-yl)ethoxy)phenyl)amino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidine-4-carbonitrile;
2- ( 1 -(5-oxo-4-((4-(2-(piperazin- 1 -yl)ethoxy)phenyl)amino)-5,6-dihydropyrimido [4,5 - d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(2-(4-e1hylpiperazin-l-yl)ethoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-phenyl-4-((4-(2-(piperazin-l-yl)ethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one;
4-((4-(2-(piperazin- 1 -yl)ethoxy)phenyl)amino)-2-(piperidin- 1 -yl)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(4-(4-((5-oxo-2-(piperidin-l-yl)-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)- 1 ,4-diazepan- 1 -yl)acetic acid;
6-(4-((2-(4-methylp ip eridin- 1 -yl)-5 -oxo-5 ,6-dihydropyrimido [4,5 -d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
2-(4-methylpiperidin- 1 -yl)-4-((4-(2-(piperazin- 1 - yl)ethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one hydrochloride;
2-(azepan-l-yl)-4-((4-(2-(3-oxopiperazin-l-yl)ethoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
6-(2-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenoxy)ethyl)-6-azaspiro[2.5]octane-l-carboxylic acid;
2-(azepan-l-yl)-4-((4-(2-(diethylamino)e1hoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(cyclohexyl(methyl)amino)-4-((4-(2-(piperazin- 1 - yl)ethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((2-(cyclohexyl(methyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin- 4-yl)amino)phenyl)piperidin-4-yl)acetic acid;
2-(4-(4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperazin- 1 -yl)acetic acid;
2-(l-(4-((2-(3-methoxyphenyl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-4-yl)acetonitrile;
4-((4-(2-(l,4-diazepan-l-yl)ethoxy)phenyl)amino)-2-(azepan-l-yl)pyrimido[4,5- d]pyridazin-5(6H)-one; 2-(azepan-l-yl)-4-((4-(3-oxopiperazin-l-yl)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one;
2-(3-methoxyphenyl)-4-((4-(2-(piperazin-l-yl)ethoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(azepan-l-yl)-4-((4-(2-(dimethylamino)ethoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(azepan-l-yl)-4-((4-(2-(piperidin-4-yl)ethoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(5-oxo-4-((4-(2-(piperidin-4-yl)ethoxy)phenyl)amino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
6-(4-((2-(cyclohexyl(methyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-6-azaspiro[2.5]octane- 1 -carboxylic acid;
4-((4-(2-( 1 ,4-diazepan- 1 -yl)ethoxy)phenyl)amino)-2-(piperidin- 1 -yl)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(azepan- 1 -yl)-4-((4-(2-(4-hydroxypiperidin- 1 - yl)ethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(azepan-l-yl)-4-((4-(3-(piperazin-l-yl)propyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(5-oxo-4-((4-(3-(piperazin-l-yl)propyl)phenyl)amino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(4-ethyl-3-oxopiperazin- 1 -yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-morpholino-4-((4-(2-(piperazin-l-yl)ethoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(2,6-dimethylmorpholino)-4-((4-(2-(piperazin- 1 - yl)ethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((4-(4-ethyl-3-oxopiperazin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(3-(4-hydroxypiperidin- 1 - yl)propyl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(azepan-l-yl)-4-((4-(4-ethylpiperazin-l-yl)phenyl)amino)pyrimido[4,5-d]pyridazm^ 5(6H)-one; 4-((6-(2-(piperazin- 1 -yl)ethoxy)pyridin-3-yl)amino)-2-(piperidin- 1 -yl)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(azepan-l-yl)-4-((6-(2-(piperazin-l-yl)ethoxy)pyridin-3-yl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
4-((4-(2-(4-hydroxyp iperidin- 1 -yl)ethoxy)phenyl)amino )-2-(p iperidin- 1 - yl)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(azepan- 1 -yl)-4-((4-(2-(4-hydroxy-4-methylpiperidin- 1 - yl)ethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((4-(2-(4-hydroxy-4-methylpiperidin-l-yl)ethoxy)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(4-hydroxypiperidin-l-yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(4-ethylpiperazin-l-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(2-(diisopropylamino)ethoxy)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(4-hydroxypiperidin-l-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(4-(2-hydroxyethyl)piperazin- 1 -yl)phenyl)amino)pyrimido [4,5- d]pyridazin-5(6H)-one;
2-( 1 -(4-((4-(4-(2-hydroxyethyl)piperazin- 1 -yl)phenyl)amino)-5-oxo-5 ,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(piperazin- 1 -yl)phenyl)amino)pyrimido [4,5-d]pyridazin-5(6H)- one;
2-(l -(5-oxo-4-((4-(piperazin- 1 -yl)phenyl)amino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(4-(3-hydroxypropyl)piperidin-l - yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(azepan- 1 -yl)-4-((4-(4-(2-hydroxyethyl)piperidin- 1 -yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(4-(2-hydroxyethyl)piperidin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile; 2-( 1 -(4-((4-(2-(4-ethylp iperazin- 1 -yl)ethyl)phenyl)amino)-5 -oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(2-(piperazin-l-yl)ethyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
4-((4-(2-aminoe1hoxy)phenyl)amino)-2-(azepan-l-yl)pyrimido[4,5-d]pyridazin- 5(6H)-one;
2-(l-(4-((4-(2-aminoe1hoxy)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(2-(4-hydroxypiperidin- 1 -yl)ethyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(azepan-l-yl)-4-((4-(4-(hydroxymethyl)piperidin-l-yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(azepan- 1 -yl)-4-((4-(2-(4-(hydroxymethyl)piperidin- 1 - yl)ethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-( 1 -(2-(4-((2-(azepan- 1 -yl)-5 -oxo-5 ,6-dihydrop yrimido [4 ,5 -d]pyridazin-4- yl)amino)piperidin- 1 -yl)ethyl)piperidin-4-yl)acetonitrile;
2-( 1 -(4-((4-(4-(3-hydroxypropyl)piperidin- 1 -yl)phenyl)amino)-5-oxo-5 ,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(4-(4-((2-(azepan-l-yl)-5-oxo-5,6-dmydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperazin- 1 -yl)acetonitrile;
2- ( 1 -(4-((4-(4-(cyanomethyl)piperazin- 1 -yl)phenyl)amino)-5 -oxo-5 ,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
3- (4-(4-((2-(azepan-l-yl)-5-oxo-5,6-dmydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperazin- 1 -yl)propanoic acid;
2-(azepan-l-yl)-4-((4-(2-(4-emylpiperazin-l-yl)ethyl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(azepan- 1 -yl)-4-((4-(2-(4-hydroxy-4-methylpiperidin- 1 - yl)ethyl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(azepan- 1 -yl)-4-((4-(2-(4-(2-hydroxyethyl)piperazin- 1 - yl)ethyl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-( 1 -(4-((4-(2-(4-(2-hydroxyethyl)piperazin- 1 -yl)ethyl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile; 2-(4-(4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperazin- 1 -yl)-2-methylpropanoic acid;
2- (azepan- 1 -yl)-4-((4-(4-(2-hydroxy-2-methylpropanoyl)piperazin- 1 - yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
3- (4-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperazin- 1 -yl)-3 -oxopropanenitrile;
2-(l-(4-((4-(4-(hydroxymethyl)piperidin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((6-(4-hydroxypiperidin-l-yl)pyridin-3-yl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2- (l-(4-((4-(2-(4-(hydroxymethyl)piperidin-l-yl)ethoxy)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
(phosphonooxy)methyl 6-(4-((2-(azepan- 1 -yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)-6-azaspiro[2.5]octane-l-carboxylate;
3- (4-(4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperazin- 1 -yl)propanoic acid;
2-(azepan- 1 -yl)-4-((6-(piperazin- 1 -yl)pyridin-3-yl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one;
2-(azepan- 1 -yl)-4-((4-(2-(2-hydroxyethyl)-2H-tetrazol-5- yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((4-(4-(2-hydroxy-2-methylpropanoyl)piperazin-l-yl)phenyl)amino)-5-oxo- 5,6-dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((4-(2-(die1hylamino)ethoxy)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(2-ethyl-2H-tetrazol-5-yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(2-ethyl-2H-tetrazol-5-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
4- ((4-(2H-tetrazol-5-yl)phenyl)amino)-2-(az^an-l-yl)pyrimido[4,5-d]pyridazin- 5(6H)-one;
2-(l-(4-((6-(4-ethylpiperazin-l-yl)pyridin-3-yl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile; 2-(azepan- 1 -yl)-4-((6-(4-ethylpiperazin-l -yl)pyridin-3 -yl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(4-(5-oxo-4-((4-(2-(piperazin-l-yl)ethoxy)phenyl)amino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperazin- 1 -yl)acetonitrile;
2-( 1 -(5-oxo-4-((6-(piperazin- 1 -yl)pyridin-3 -yl)amino)-5 ,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-3-yl)acetic acid;
2- (l-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)piperidin-3-yl)acetic acid;
3- (4-(5-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)pyridin-2-yl)piperazin- 1 -yl)-3-oxopropanenitrile;
2-(5-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenyl)-2H-tetrazol-2-yl)acetic acid;
2- (azepan- 1 -yl)-4-((6-(4-(2-hydroxyethyl)piperidin- 1 -yl)pyridin-3 - yl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
3- (4-(5-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)pyridin-2-yl)piperazin-l-yl)-3-oxopropanenitrile;
2-(azepan- 1 -yl)-4-((6-(4-(2-hydroxy-2-methylpropanoyl)piperazin- 1 -yl)pyridin-3- yl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2- (azepan- 1 -yl)-4-((4-(2-(4-(2-hydroxyethyl)piperazin- 1 - yl)ethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
3- (4-(4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperazin- 1 -yl)-3-oxopropanenitrile;
2-(5-(4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)-2H-tetrazol-2-yl)acetic acid;
2-(azepan- 1 -yl)-4-((6-(4-methylpiperazin- 1 -yl)pyridin-3-yl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((6-(4-methylpiperazin-l-yl)pyridin-3-yl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
4- ((4-(4-((2H-tetrazol-5-yl)methyl)piperidin-l-yl)phenyl)amino)-2-(azepan-l- yl)pyrimido[4,5-d]pyridazin-5(6H)-one; 2-(l-(4-((4-(2H-tetrazol-5-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((4-(4-((2H-tetrazol-5-yl)methyl)piperidin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(4-methylpiperazin-l-yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(4-methylpiperazin-l-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(5-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)pyridin-2-yl)piperidin-3 -yl)acetic acid;
2-(l-(4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenyl)piperidin-3-yl)acetic acid;
2-(l-(4-((6-(4-(2-hydroxy-2-methylpropanoyl)piperazin-l-yl)pyridin-3-yl)amino)-5- oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
4-((6-(4-((2H-tetrazol-5-yl)methyl)piperidin-l-yl)pyridin-3-yl)amino)-2-(azepan-l- yl)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((6-(4-((2H-tetrazol-5-yl)methyl)piperidin-l-yl)pyridin-3-yl)amino)-5-oxo- 5,6-dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(4-(4-((2-(azepan-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenoxy)piperidin- 1 -yl)acetic acid;
2-(azepan- 1 -yl)-4-((4-(4-(2-methoxyethyl)piperazin- 1 -yl)phenyl)amino)pyrimido [4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(4-(2-methoxyethyl)piperazin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((4-(4-(2-aminoethyl)piperazin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(l -(hydroxymethyl)-6-azaspiro[2.5]octan-6- yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((4-(l-(hydroxymethyl)-6-azaspiro[2.5]octan-6-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(4-(3-hydroxypropyl)piperazin-l- yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one; 2-(l-(4-((4-(4-(3-hydroxypropyl)piperazin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(4-(4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-4-yl)amino)phenoxy)piperidin-l -yl)acetic acid;
2-(azepan- 1 -yl)-4-((4-(4-(2-hydroxyethyl)- 1 ,4-diazepan- 1 - yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-( 1 -(4-((4-(4-(2-hydroxyethyl)- 1 ,4-diazepan- 1 -yl)phenyl)amino)-5 -oxo-5 ,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(4-(l-hydroxy-2-methylpropan-2-yl)piperazin-l- yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l -(4-((4-(4-(l -hydroxy-2-methylpropan-2-yl)piperazin- 1 -yl)phenyl)amino)-5-oxo- 5,6-dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(4-(2-(dimethylamino)ethyl)piperazin- 1 - yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-( 1 -(4-((4-(4-(2-(dimethylamino )ethyl)piperazin- 1 -yl)phenyl)amino)-5 -oxo-5 ,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(2-(4-ethylpiperazin- 1 -yl)-2- oxoe1hoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-( 1 -(4-((4-(2-(4-ethylp iperazin- 1 -yl)-2-oxoethoxy)phenyl)amino)-5-oxo-5 ,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((4-(2-(4-methylpiperazin-l-yl)-2-oxoethoxy)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(3-(2-hydroxyethyl)piperidin- 1 -yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(azepan- 1 -yl)-4-((4-(4-(2,3-dihydroxypropyl)piperazin- 1 - yl)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((4-(4-(2,3-dihydroxypropyl)piperazin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(4-(2-fluoroe1hyl)piperazin-l-yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-( 1 -(4-((4-(4-(2-fluoroethyl)piperazin- 1 -yl)phenyl)amino)-5-oxo-5 ,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile; 2-(azepan-l-yl)-4-((6-(3-hydroxypiperidin-l-yl)pyridin-3-yl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l -(4-((6-(3-hydroxypiperidin- 1 -yl)pyridin-3 -yl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(4,4-difluoropiperidin- 1 -yl)phenyl)amino)pyrimido [4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(4,4-difluoropiperidin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(3-hydroxypiperidin-l-yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(3-hydroxypiperidin-l-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(2-(4-methylpiperazin-l-yl)-2- oxoe1hoxy)phenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((4-(3-(hydroxymethyl)piperidin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(3-(hydroxymethyl)piperidin-l-yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(3-(2-hydroxyethyl)piperidin-l-yl)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((4-(4-methoxypiperidin-l-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(4-methoxypiperidin-l-yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(azepan- 1 -yl)-4-((4-(4-fluoropiperidin- 1 -yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(4-fluoropiperidin-l-yl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((4-((l-(2-hydroxyethyl)piperidin-4-yl)oxy)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(piperidin-4-yloxy)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one hydrochloride; 2-(l-(4-((4-(2-hydroxyethoxy)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(2-hydroxyethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one;
2-(l-(4-((4-(3-hydroxypropoxy)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(3-hydroxypropoxy)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one;
2-(l-(4-((4-(4-(l-hydroxy-2-methylpropan-2-yl)-l,4-diazepan-l-yl)phenyl)amino)-5- oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(4-(2-fluoroethyl)piperidin- 1 -yl)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l -(4-((4-(4-(2-fluoroethyl)piperidin- 1 -yl)phenyl)amino)-5 -oxo-5 ,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-( 1 -(4-((3 ,5 -dimethoxyphenyl)amino)-5 -oxo-5,6-dihydropyrimido[4,5-d]pyridazin- 2-yl)piperidin-4-yl)acetonitrile;
2-(l-(5-oxo-4-((4-(trifluoromethoxy)phenyl)amino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((3,5-dimethoxyphenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)- one;
2- (azepan-l-yl)-4-((4-(trifluoromethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one;
3 -(4-((2-(azq)an- 1 -yl)-5 -oxo-5 ,6-dihydrop yrimido [4,5 -d]pyridazin-4- yl)amino)phenyl)propanoic acid;
3- (4-((2-(4-(cyanomethyl)piperidin-l-yl)-5-oxo-5,6-dmydropyrimido[4,5-d]pyridazin- 4-yl)amino)phenyl)propanoic acid;
2-(azepan-l-yl)-4-((4-(bis(2-hydroxyethyl)amino)phenyl)amino)pyrimido[4,5- d]pyridazin-5(6H)-one;
2-(l-(4-((4-(bis(2-hydroxyethyl)amino)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(3-hydroxypropyl)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one; 2-(l-(4-((4-(3-hydroxypropyl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((3-(hydroxymethyl)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one;
2-(l-(4-((3-(hydroxymethyl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((4-fluorophenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2- yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-fluorophenyl)amino)pyrimido [4,5 -d]pyridazin-5(6H)-one;
2-(azepan-l-yl)-4-((4-((l-hydroxy-2-methylpropan-2- yl)oxy)phenyl)amino)pyrimido [4,5 -d]pyridazin-5(6H)-one;
2-(l-(4-((4-((l-hydroxy-2-methylpropan-2-yl)oxy)phenyl)amino)-5-oxo-5,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(4-((2-(azq)an-l-yl)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-4- yl)amino)phenoxy)-2-methylpropanoic acid;
2-(4-((2-(4-(cyanomethyl)piperidm-l-yl)-5-oxo-5,^
4-yl)amino)phenoxy)-2-methylpropanoic acid;
2-(azepan-l-yl)-4-((3,4-dimethoxyphenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)- one;
2-(l-(4-((3,4-dimethoxyphenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin- 2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((3-methoxyphenyl)amino)pyrimido[4,5-d]pyridazin-5(6H)-one;
2-(l-(4-((3-methoxyphenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5-d]pyridazin-2- yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(2-hydroxyethyl)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one;
2-(l-(4-((4-(2-hydroxyethyl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(2-methoxyethoxy)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one;
2-(l-(4-((4-(2-methoxyethoxy)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile; 2-(l-(5-oxo-4-((3,4,5-trimemoxyphenyl)amino)-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-(benzo[d][l,3]dioxol-5-ylamino)pyrimido[4,5-d]pyridazin-5(6H)- one;
2-(l-(4-(benzo[d][l,3]dioxol-5-ylamino)-5-oxo-5,6-dmydropyrimido[4,5-d]pyridazin- 2-yl)piperidin-4-yl)acetonitrile;
2-(azepan- 1 -yl)-4-((4-(( 1 -(2-hydroxyethyl)piperidin-4- yl)oxy)phenyl)amino)pyrimido [4,5 -d]pyridazin-5(6H)-one;
sodium (2-(4-(cyanomethyl)piperidin-l-yl)-4-((4-(4-hydroxypiperidin-l- yl)phenyl)amino)-5-oxopyrimido[4,5-d]pyridazin-6(5H)-yl)methyl phosphate;
2-(azepan-l-yl)-4-((4-(hydroxymethyl)phenyl)amino)pyrimido[4,5-d]pyridazin- 5(6H)-one;
2-(l-(4-((4-(hydroxymethyl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((4-(methylsulfonyl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(azepan-l-yl)-4-((4-(methylsulfonyl)phenyl)amino)pyimido[4,5-d]pyridazin- 5(6H)-one;
2-(l-(4-((lH-benzo[d][l,2,3]triazol-5-yl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-( 1 -(5-oxo-4-((2-oxo-2,3 -dihydro- 1 H-benzo [d]imidazol-5-yl)amino)-5 ,6- dihydropyimido[4,5-d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((4-(3-fluoropropyl)phenyl)amino)-5-oxo-5,6-dihydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile;
2-(l-(4-((4-(difiuoromemoxy)phenyl)ammo)-5-oxo-5,6-dmydropyrimido[4,5- d]pyridazin-2-yl)piperidin-4-yl)acetonitrile; or
4-((lH-benzo[d][l,2,3]triazol-5-yl)amino)-2-(azepan-l-yl)pyrimido[4,5-d]pyridazin- 5(6H)-one.
30. The pharmaceutical combination of claim 1, the combination comprising:
(a) a compound of
Figure imgf000473_0001
or a pharmaceutically acceptable salt or ester thereof; and
(b) an anti-PD-1 antibody or a small molecule IDO-1 inhibitor.
31. The pharmaceutical combination of claim 1 , the combination comprising:
(a) a compound of
Figure imgf000473_0002
or a pharmaceutically acceptable salt or ester thereof; and
(b) an anti-PD-1 antibody or a small molecule IDO-1 inhibitor.
PCT/US2018/025841 2017-04-03 2018-04-03 Pyrimido-pyridazinone compound combinations, methods, kits and formulations thereof WO2018187294A1 (en)

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