105807.001076 – PCT Application BRM AND BRG1 TARGETING COMPOUNDS AND ASSOCIATED METHODS OF USE CROSS REFERENCE TO RELATED APPLICATIONS [1] This application claims the benefit of U.S. Provisional Application No.63/560,057, filed March 1, 2024, the entirety of which is incorporated by reference herein. TECHNICAL FIELD [2] The description provides bifunctional compounds comprising a target protein binding moiety and a E3 ubiquitin ligase binding moiety, and associated methods of use. The bifunctional compounds are useful as modulators of targeted ubiquitination, especially with respect to Switch/Sucrose Non-Fermentable (SWI/SNF)-Related, Matrix-Associated, Actin- Dependent Regulator of Chromatin, Subfamily A, Member 2 (SMARCA2, i.e., BRAHMA or BRM) and Member 4 (SMARCA4, i.e., BRG1), both of which are degraded and/or otherwise inhibited by bifunctional compounds according to the present disclosure. BACKGROUND [3] The human SWItch/Sucrose Non-Fermentable (SWI/SNF) complexes are ATP-dependent chromatin remodelers. These large complexes play important roles in essential cellular processes, such as transcription, DNA repair and replication by regulating DNA accessibility. [4] Mutations in the genes encoding up to 20 canonical SWI/SNF subunits are observed in nearly 20% of all human cancers with the highest frequency of mutations observed in rhabdoid tumors, female cancers (including ovarian, uterine, cervical and endometrial), lung adenocarcinoma, gastric adenocarcinoma, melanoma, esophageal, and renal clear cell carcinoma. [5] SMARCA2 (BRM) and SMARCA4 (BRG1) are the subunits containing catalytic ATPase domains and they are essential for the function of SWI/SNF in perturbation of histone-DNA contacts, thereby providing access points to transcription factors and cognate DNA elements that facilitate gene activation and repression. [6] Previous studies have demonstrated the strong synthetic lethality using gene expression manipulation such as RNAi; downregulating SMARCA2 gene expression in SMARCA4 mutated cancer cells results in suppression of cancer cell proliferation. However, SMARCA2/4 bromodomain inhibitors (e.g., PFI-3) exhibit little to no effect on cell proliferation inhibition [Vangamudi et al. Cancer Res 2015]. Thus, a phenotypic discrepancy exists between gene expression downregulation and small molecule-based inhibition approaches. [7] Therapeutic compounds that inhibit SMARCA2 and SMARCA4 are needed.
105807.001076 – PCT Application SUMMARY [8] The present disclosure is directed to compounds of Formula (I):
or a pharmaceutically acceptable salt or solvate thereof; wherein R1a is H, C1-3 alkyl, or halo; Rh is C1-3 alkyl; Z1 and Z2 are each independently CH or N; and A is CH2 or CO. [9] Stereoisomers of the compounds of Formula (I), and the pharmaceutical salts and stereoisomers thereof, are also contemplated, described, and encompassed herein. Methods of using compounds of Formula (I) are described, as well as pharmaceutical compositions including the compounds of Formula (I). DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS [10] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the disclosure. [11] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise (such as in the case of a group containing a number of carbon atoms in which case each carbon atom number falling within the range is provided), between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. - 2 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [12] The following terms are used to describe the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure. [13] The articles “a” and “an” as used herein and in the appended claims are used herein to refer to one or to more than one (e.g., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, “an element” means one element or more than one element. [14] The terms “co-administration” and “co-administering” or “combination therapy” refer to both concurrent administration (administration of two or more therapeutic agents at the same time) and time varied administration (administration of one or more therapeutic agents at a time different from that of the administration of an additional therapeutic agent or agents), as long as the therapeutic agents are present in the patient to some extent, preferably at effective amounts, at the same time. [15] The term “compound”, as used herein, unless otherwise indicated, refers to any specific chemical compound disclosed herein and includes tautomers, regioisomers, geometric isomers, and where applicable, stereoisomers, including optical isomers (enantiomers) and other stereoisomers (diastereomers) thereof, as well as pharmaceutically acceptable salts and/or deuterated forms thereof where applicable, in context. Deuterated small molecules contemplated are those in which one or more of the hydrogen atoms contained in the drug molecule have been replaced by deuterium. [16] Within its use in context, the term compound generally refers to a single compound, but also may include other compounds such as stereoisomers, regioisomers and/or optical isomers (including racemic mixtures) as well as specific enantiomers or enantiomerically enriched mixtures of disclosed compounds. It is noted that in describing the present compounds, numerous substituents and variables associated with same, among others, are described. It is understood by those of ordinary skill that molecules which are described herein are stable compounds as generally described hereunder. [17] The term “ubiquitin ligase” refers to a family of proteins that facilitate the transfer of ubiquitin to a specific substrate protein, targeting the substrate protein for degradation. For example, an E3 ubiquitin ligase protein that alone or in combination with an E2 ubiquitin- conjugating enzyme causes the attachment of ubiquitin to a lysine on a target protein, and subsequently targets the specific protein substrates for degradation by the proteasome. Thus, E3 ubiquitin ligase alone or in complex with an E2 ubiquitin conjugating enzyme is responsible for the transfer of ubiquitin to targeted proteins. In general, the ubiquitin ligase is involved in polyubiquitination such that a second ubiquitin is attached to the first; a third is attached to the - 3 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application second, and so forth. Polyubiquitination marks proteins for degradation by the proteasome. However, there are some ubiquitination events that are limited to mono-ubiquitination, in which only a single ubiquitin is added by the ubiquitin ligase to a substrate molecule. Mono- ubiquitinated proteins are not targeted to the proteasome for degradation but may instead be altered in their cellular location or function, for example, via binding other proteins that have domains capable of binding ubiquitin. Further complicating matters, different lysines on ubiquitin can be targeted by an E3 to make chains. The most common lysine is Lys48 on the ubiquitin chain. This is the lysine used to make polyubiquitin, which is recognized by the proteasome. [18] As used herein, the term “alkyl”, by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical having up to twelve carbon atoms. In some embodiments, the number of carbon atoms is designated (i.e., C1-C3 means one to three carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t- butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. In some embodiments, the alkyl group is a C1-C3 alkyl; in some embodiments, it is a C1-C4 alkyl. [19] When a range of carbon atoms is used herein, for example, C1-C6, all ranges, as well as individual numbers of carbon atoms are encompassed. For example, “C1-C3” includes C1-C3, C1- C2, C2-C3, C1, C2, and C3. [20] The terms “halo” or “halogen”, by itself or as part of another substituent, means a fluorine, chlorine, bromine, or iodine atom. As used herein, the term “stereoisomers” refers to compounds which have identical chemical constitution but differ with regard to the arrangement of the atoms or groups in space, e.g., enantiomers, diastereomers, tautomers. [21] The term “patient” or “subject” is used throughout the specification to describe an animal, preferably a human or a domesticated animal, to whom treatment, including prophylactic treatment, with the compositions according to the present disclosure is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal, including a domesticated animal such as a dog or cat or a farm animal such as a horse, cow, sheep, etc. In general, in the present disclosure, the term patient refers to a human patient unless otherwise stated or implied from the context of the use of the term. [22] The term “effective” is used to describe an amount of a compound, composition or component which, when used within the context of its intended use, effects an intended result. The term effective subsumes all other effective amount or effective concentration terms, which are otherwise described or used in the present application. - 4 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [23] “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, e.g., in humans. [24] “Pharmaceutically acceptable salt” refers to a salt of a compound of the disclosure that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic may be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4- hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N- methylglucamine and the like. Salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like. [25] A “pharmaceutically acceptable excipient” refers to a substance that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or otherwise used as a vehicle, carrier, or diluent to facilitate administration of an agent and that is compatible therewith. Examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. [26] A “solvate” refers to a physical association of a compound of Formula (I) with one or more solvent molecules. [27] “Treating” or “treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (e.g., arresting or reducing the development of the disease or - 5 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application at least one of the clinical symptoms thereof). In another embodiment “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to delaying the onset of the disease or disorder. [28] In one aspect, the disclosure is directed to a compound of Formula (I):
or a pharmaceutically acceptable salt or solvate thereof; wherein R1a is H, C1-3 alkyl, or halo; Rh is C1-3 alkyl; Z1 and Z2 are each independently CH or N; and A is CH2 or CO. [29] In some embodiments, R1a in Formula (I) is H, C1-3 alkyl, or halo. [30] In some embodiments, R1a in Formula (I) is H. [31] In some embodiments, R1a in Formula (I) is halo, such as, for example, F, Cl, Br, or I. In other embodiments, R1a in Formula (I) is F. [32] In other embodiments, R1a in Formula (I) is C1-3 alkyl, such as, for example, C1 alkyl, C2, alkyl, C3 alkyl, methyl, ethyl, propyl, isopropyl, and the like. In yet other embodiments, R1a in Formula (I) is methyl. [33] In some embodiments, Rh in Formula (I) is C1-3 alkyl, such as, for example, C1 alkyl, C2, alkyl, C3 alkyl, methyl, ethyl, propyl, isopropyl, and the like. In some embodiments, Rh in Formula (I) is methyl. In other embodiments, Rh in Formula (I) is ethyl. [34] In some embodiments, Z1 in Formula (I) is CH or N. In some embodiments, Z1 in Formula (I) is CH. In other embodiments, Z1 in Formula (I) is N. [35] In some embodiments, Z2 in Formula (I) is CH or N. In some embodiments, Z2 in Formula (I) is CH. In other embodiments, Z2 in Formula (I) is N. - 6 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [36] In some embodiments, A in Formula (I) is CH2 or CO (i.e., C=O). In some embodiments, A in Formula (I) is CH2. In other embodiments, A in Formula (I) is CO. [37] In one aspect, the disclosure is directed to compounds of Formula (I):
or a pharmaceutically acceptable salts or solvates thereof; wherein R1a is H, methyl, or F; Rh is methyl or ethyl; Z1 and Z2 are each independently CH or N; and A is CH2 or CO. [38] In some embodiments, R1a in Formula (I) is H, methyl, or F. In some embodiments, R1a in Formula (I) is H. In other embodiments, R1a in Formula (I) is F. In yet other embodiments, R1a in Formula (I) is methyl. [39] In some embodiments, Rh in Formula (I) is methyl or ethyl. In some embodiments, Rh in Formula (I) is methyl. In other embodiments, Rh in Formula (I) is ethyl. [40] In some embodiments, Z1 in Formula (I) is CH or N. In some embodiments, Z1 in Formula (I) is CH. In other embodiments, Z1 in Formula (I) is N. [41] In some embodiments, Z2 in Formula (I) is CH or N. In some embodiments, Z2 in Formula (I) is CH. In other embodiments, Z2 in Formula (I) is N. [42] In some embodiments, A in Formula (I) is CH2 or CO. In some embodiments, A in Formula (I) is CH2. In other embodiments, A in Formula (I) is CO. [43] In another aspect, the disclosure is directed to a compound of Formula (I) that is: (S)-3-(6-(4-((6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3-yl)methyl)piperazin-1-yl)-1- oxoisoindolin-2-yl)piperidine-2,6-dione; (S)-3-(6-(4-((5-Fluoro-6-(((6aS,8R)-2-(2-hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3-yl)methyl)piperazin-1- yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione; - 7 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application (S)-3-(6-(1-((6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylpyridin-3-yl)methyl)piperidin-4- yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione; (S)-3-(6-(4-((6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylpyridin-3-yl)methyl)piperazin-1- yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione; (S)-3-(6-(4-(6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinoyl)piperazin-1-yl)-1- oxoisoindolin-2-yl)piperidine-2,6-dione; (3S)-3-[5-[4-[[6-[[(4R,6S)-6-Ethyl-12-(2-hydroxyphenyl)-2,8,10,11-tetrazatricyclo- [7.4.0.02,6]trideca-1(9),10,12-trien-4-yl]oxy]-5-methyl-3-pyridinyl]methyl]piperazin-1-yl]-3- oxo-1H-isoindol-2-yl]piperidine-2,6-dione; (S)-3-(6-(4-(6-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinoyl)piperazin-1-yl)-1- oxoisoindolin-2-yl)piperidine-2,6-dione; (S)-3-(6-(4-((5-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-6-methylpyrazin-2-yl)methyl)piperazin-1- yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione; (S)-3-(6-(4-((6-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3-yl)methyl)piperazin-1-yl)-1- oxoisoindolin-2-yl)piperidine-2,6-dione; (S)-3-(6-(1-((6-(((6aS,8R)-6a-ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3-yl)methyl)piperidin-4-yl)-1- oxoisoindolin-2-yl)piperidine-2,6-dione; or a pharmaceutically acceptable salt thereof. [44] In another aspect, the disclosure is directed to a compound of Formula (I) that is:
or a pharmaceutically acceptable salt or solvate thereof. [45] In another aspect, the disclosure is directed to a compound of Formula (I) that is: - 8 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
or a pharmaceutically acceptable salt or solvate thereof. [46] In yet another aspect, the disclosure is directed to a compound of Formula (I) that is in the form of a pharmaceutically acceptable salt. [47] In yet another aspect, the disclosure is directed to a compound of Formula (I) that is in the form of a pharmaceutically acceptable solvate. [48] In yet another aspect, the disclosure is directed to a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient. [49] It will be apparent that the compounds of Formula (I), including all subgenera described herein, may have multiple stereogenic centers. As a result, there exist multiple stereoisomers (enantiomers and diastereomers) of the compounds of Formula (I) (and subgenera described herein). The present disclosure contemplates and encompasses each stereoisomer of any compound of Formula (I) (and subgenera described herein), as well as mixtures of said stereoisomers. [50] Pharmaceutically acceptable salts and solvates of the compounds of Formula (I) (including all subgenera described herein) are also within the scope of the disclosure. [51] Isotopic variants of the compounds of Formula (I) (including all subgenera described herein) are also contemplated by the present disclosure. Pharmaceutical Compositions and Methods of Administration [52] The subject pharmaceutical compositions are typically formulated to provide a therapeutically effective amount of a compound of the present disclosure as the active ingredient, or a pharmaceutically acceptable salt, or solvate (including hydrate) thereof. Where desired, the pharmaceutical compositions contain pharmaceutically acceptable salt and/or coordination complex thereof, and one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants. - 9 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [53] The subject pharmaceutical compositions can be administered alone or in combination with one or more other agents, which are also typically administered in the form of pharmaceutical compositions. Where desired, the one or more compounds of the invention and other agent(s) may be mixed into a preparation or both components may be formulated into separate preparations to use them in combination separately or at the same time. [54] In some embodiments, the concentration of one or more compounds provided in the pharmaceutical compositions of the present invention is less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% (or a number in the range defined by and including any two numbers above) w/w, w/v or v/v. [55] In some embodiments, the concentration of one or more compounds of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25%, 13%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25%, 7%, 6.75%, 6.50%, 6.25%, 6%, 5.75%, 5.50%, 5.25%, 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 1.25% , 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% (or a number in the range defined by and including any two numbers above) w/w, w/v, or v/v. [56] In some embodiments, the concentration of one or more compounds of the invention is in the range from approximately 0.0001% to approximately 50%, approximately 0.001% to approximately 40%, approximately 0.01% to approximately 30%, approximately 0.02% to approximately 29%, approximately 0.03% to approximately 28%, approximately 0.04% to approximately 27%, approximately 0.05% to approximately 26%, approximately 0.06% to approximately 25%, approximately 0.07% to approximately 24%, approximately 0.08% to approximately 23%, approximately 0.09% to approximately 22%, approximately 0.1% to approximately 21%, approximately 0.2% to approximately 20%, approximately 0.3% to approximately 19%, approximately 0.4% to approximately 18%, approximately 0.5% to approximately 17%, approximately 0.6% to approximately 16%, approximately 0.7% to - 10 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application approximately 15%, approximately 0.8% to approximately 14%, approximately 0.9% to approximately 12%, approximately 1% to approximately 10% w/w, w/v or v/v. [57] In some embodiments, the concentration of one or more compounds of the invention is in the range from approximately 0.001% to approximately 10%, approximately 0.01% to approximately 5%, approximately 0.02% to approximately 4.5%, approximately 0.03% to approximately 4%, approximately 0.04% to approximately 3.5%, approximately 0.05% to approximately 3%, approximately 0.06% to approximately 2.5%, approximately 0.07% to approximately 2%, approximately 0.08% to approximately 1.5%, approximately 0.09% to approximately 1%, approximately 0.1% to approximately 0.9% w/w, w/v or v/v. [58] In some embodiments, the amount of one or more compounds of the invention is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g (or a number in the range defined by and including any two numbers above). [59] In some embodiments, the amount of one or more compounds of the invention is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, , 0.15 g, 0.2 g, , 0.25 g, 0.3 g, , 0.35 g, 0.4 g, , 0.45 g, 0.5 g, 0.55 g, 0.6 g, , 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5g, 7 g, 7.5g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g (or a number in the range defined by and including any two numbers above). [60] In some embodiments, the amount of one or more compounds of the invention is in the range of 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1- 3 g. [61] The compounds according to the invention are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. An exemplary dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician. - 11 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [62] A pharmaceutical composition of the invention typically contains an active ingredient (e.g., a compound of the disclosure) of the present invention or a pharmaceutically acceptable salt and/or coordination complex thereof, and one or more pharmaceutically acceptable excipients, carriers, including but not limited to inert solid diluents and fillers, diluents, sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants. [63] Described below are non- limiting exemplary pharmaceutical compositions and methods for preparing the same. Pharmaceutical Compositions for Oral Administration [64] In some embodiments, the invention provides a pharmaceutical composition for oral administration containing a compound of the invention, and a pharmaceutical excipient suitable for oral administration. [65] In some embodiments, the invention provides a solid pharmaceutical composition for oral administration containing: (i) an effective amount of a compound of the invention; optionally (ii) an effective amount of a second agent; and (iii) a pharmaceutical excipient suitable for oral administration. In some embodiments, the composition further contains: (iv) an effective amount of a third agent. [66] In some embodiments, the pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption. Pharmaceutical compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non- aqueous liquid, an oil-in- water emulsion, or a water-in-oil liquid emulsion. Such dosage forms can be prepared by any of the methods of pharmacy, but all methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free- flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. - 12 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [67] This invention further encompasses anhydrous pharmaceutical compositions and dosage forms comprising an active ingredient, since water can facilitate the degradation of some compounds. For example, water may be added (e.g., 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf- life or the stability of formulations over time. Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms of the invention which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs. [68] An active ingredient can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro- crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. [69] Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre- gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof. [70] Examples of suitable fillers for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or - 13 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. [71] Disintegrants may be used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which may disintegrate in the bottle. Too little may be insufficient for disintegration to occur and may thus alter the rate and extent of release of the active ingredient(s) from the dosage form. Thus, a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the compounds disclosed herein. The amount of disintegrant used may vary based upon the type of formulation and mode of administration, and may be readily discernible to those of ordinary skill in the art. About 0.5 to about 15 weight percent of disintegrant, or about 1 to about 5 weight percent of disintegrant, may be used in the pharmaceutical composition. Disintegrants that can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums or mixtures thereof. [72] Lubricants which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, or mixtures thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, or mixtures thereof. A lubricant can optionally be added, in an amount of less than about 1 weight percent of the pharmaceutical composition. [73] When aqueous suspensions and/or elixirs are desired for oral administration, the active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof. [74] The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium - 14 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil. [75] Surfactant which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed. [76] A suitable hydrophilic surfactant may generally have an HLB value of at least 10, while suitable lipophilic surfactants may generally have an HLB value of or less than about 10. An empirical parameter used to characterize the relative hydrophilicity and hydrophobicity of non- ionic amphiphilic compounds is the hydrophilic-lipophilic balance (" HLB" value). Surfactants with lower HLB values are more lipophilic or hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have greater solubility in aqueous solutions. [77] Hydrophilic surfactants are generally considered to be those compounds having an HLB value greater than about 10, as well as anionic, cationic, or zwitterionic compounds for which the HLB scale is not generally applicable. Similarly, lipophilic (e.g., hydrophobic) surfactants are compounds having an HLB value equal to or less than about 10. However, HLB value of a surfactant is merely a rough guide generally used to enable formulation of industrial, pharmaceutical and cosmetic emulsions. [78] Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acyl lactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof. [79] Within the aforementioned group, ionic surfactants include, by way of example: lecithins, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di- acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof. - 15 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [80] Ionic surfactants may be the ionized forms of lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG-phosphatidylethanolamine, PVP - phosphatidylethanolamine, lactylic esters of fatty acids, stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides, mono/diacetylated tartaric acid esters of mono/diglycerides, citric acid esters of mono/diglycerides, cholylsarcosine, caproate, caprylate, caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate, linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate, lauroyl carnitines, palmitoyl carnitines, myristoyl carnitines, and salts and mixtures thereof. [81] Hydrophilic non-ionic surfactants may include, but are not limited to, alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, derivatives, and analogues thereof; polyoxyethylated vitamins and derivatives thereof; polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof; polyethylene glycol sorbitan fatty acid esters and hydrophilic transesterification products of a polyol with at least one member of the group consisting of triglycerides, vegetable oils, and hydrogenated vegetable oils. The polyol may be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, or a saccharide. [82] Other hydrophilic-non-ionic surfactants include, without limitation, PEG- 10 laurate, PEG- 12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG- 12 oleate, PEG- 15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG- 15 stearate, PEG-32 distearate, PEG-40 stearate, PEG- 100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40 castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6 caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides, polyglyceryl-10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, - 16 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application PEG-30 soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, tocopheryl PEG- 100 succinate, PEG-24 cholesterol, polyglyceryl-lOoleate, Tween 40, Tween 60, sucrose monostearate, sucrose mono laurate, sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG 15-100 octyl phenol series, and poloxamers. [83] Suitable lipophilic surfactants include, by way of example only: fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acids esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and di-glycerides; hydrophobic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols; oil- soluble vitamins/vitamin derivatives; and mixtures thereof. Within this group, preferred lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters, and mixtures thereof, or are hydrophobic transesterification products of a polyol with at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils, and triglycerides. [84] In one embodiment, the composition may include a solubilizer to ensure good solubilization and/or dissolution of the compound of the present invention and to minimize precipitation of the compound of the present invention. This can be especially important for compositions for non-oral use, e.g., compositions for injection. A solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion. [85] Examples of suitable solubilizers include, but are not limited to, the following: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediols and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycols having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (glycofurol) or methoxy PEG ; amides and other nitrogen-containing compounds such as 2-pyrrolidone, 2-piperidone, ε-caprolactam, N- alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone; esters such as ethyl propionate, tributylcitrate, acetyl triethylcitrate, acetyl tributyl citrate, triethylcitrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, ε-caprolactone and - 17 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application isomers thereof, δ-valerolactone and isomers thereof, β-butyrolactone and isomers thereof; and other solubilizers known in the art, such as dimethyl acetamide, dimethyl isosorbide, N-methyl pyrrolidones, monooctanoin, diethylene glycol monoethyl ether, and water. [86] Mixtures of solubilizers may also be used. Examples include, but not limited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N- hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide. Particularly preferred solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol and propylene glycol. [87] The amount of solubilizer that can be included is not particularly limited. The amount of a given solubilizer may be limited to a bioacceptable amount, which may be readily determined by one of skill in the art. In some circumstances, it may be advantageous to include amounts of solubilizers far in excess of bioacceptable amounts, for example to maximize the concentration of the drug, with excess solubilizer removed prior to providing the composition to a subject using conventional techniques, such as distillation or evaporation. Thus, if present, the solubilizer can be in a weight ratio of 10%, 25%o, 50%), 100%o, or up to about 200%> by weight, based on the combined weight of the drug, and other excipients. If desired, very small amounts of solubilizer may also be used, such as 5%>, 2%>, 1%) or even less. Typically, the solubilizer may be present in an amount of about 1%> to about 100%, more typically about 5%> to about 25%> by weight. [88] The composition can further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, anti- foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof. [89] In addition, an acid or a base may be incorporated into the composition to facilitate processing, to enhance stability, or for other reasons. Examples of pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine, tris(hydroxymethyl)aminomethane (TRIS) and the like. Also suitable are bases that are salts of a pharmaceutically acceptable acid, such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, - 18 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application isoascorbic acid, lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid, and the like. Salts of polyprotic acids, such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate can also be used. When the base is a salt, the cation can be any convenient and pharmaceutically acceptable cation, such as ammonium, alkali metals, alkaline earth metals, and the like. Example may include, but not limited to, sodium, potassium, lithium, magnesium, calcium and ammonium. [90] Suitable acids are pharmaceutically acceptable organic or inorganic acids. Examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, and the like. Examples of suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p- toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid and the like. Pharmaceutical compositions for injection [91] In some embodiments, the invention provides a pharmaceutical composition for injection containing a compound of the present invention and a pharmaceutical excipient suitable for injection. Components and amounts of agents in the compositions are as described herein. [92] The forms in which the novel compositions of the present invention may be incorporated for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. [93] Aqueous solutions in saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, for the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. [94] Sterile injectable solutions are prepared by incorporating the compound of the present invention in the required amount in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are - 19 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, certain desirable methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Pharmaceutical Compositions for Topical (e.g., Transdermal) Delivery [95] In some embodiments, the invention provides a pharmaceutical composition for transdermal delivery containing a compound of the present invention and a pharmaceutical excipient suitable for transdermal delivery. [96] Compositions of the present invention can be formulated into preparations in solid, semisolid, or liquid forms suitable for local or topical administration, such as gels, water soluble jellies, creams, lotions, suspensions, foams, powders, slurries, ointments, solutions, oils, pastes, suppositories, sprays, emulsions, saline solutions, dimethylsulfoxide (DMSO)-based solutions. In general, carriers with higher densities are capable of providing an area with a prolonged exposure to the active ingredients. In contrast, a solution formulation may provide more immediate exposure of the active ingredient to the chosen area. [97] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients, which are compounds that allow increased penetration of, or assist in the delivery of, therapeutic molecules across the stratum corneum permeability barrier of the skin. There are many of these penetration- enhancing molecules known to those trained in the art of topical formulation. [98] Examples of such carriers and excipients include, but are not limited to, humectants (e.g., urea), glycols (e.g., propylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleic acid), surfactants (e.g., isopropyl myristate and sodium lauryl sulfate), pyrrolidones, glycerol monolaurate, sulfoxides, terpenes (e.g., menthol), amines, amides, alkanes, alkanols, water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. [99] Another exemplary formulation for use in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of a compound of the present invention in controlled amounts, either with or without another agent. [100] The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Pat. Nos.5,023,252, 4,992,445 and 5,001,139. Such - 20 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. Pharmaceutical Compositions for Inhalation [101] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner. Other Pharmaceutical Compositions [102] Pharmaceutical compositions may also be prepared from compositions described herein and one or more pharmaceutically acceptable excipients suitable for sublingual, buccal, rectal, intraosseous, intraocular, intranasal, epidural, or intraspinal administration. Preparations for such pharmaceutical compositions are well-known in the art. See, e.g., Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw- Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 20037ybg; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001 ; Remingtons Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all of which are incorporated by reference herein in their entirety. [103] Administration of the compounds or pharmaceutical composition of the present invention can be affected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, intraarterial, subcutaneous, intramuscular, intravascular, intraperitoneal or infusion), topical (e.g., transdermal application), rectal administration, via local delivery by catheter or stent or through inhalation. Compounds can also be administered intraadiposally or intrathecally. - 21 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [104] In some embodiments, the compounds or pharmaceutical composition of the present invention are administered by intravenous injection. [105] The amount of the compound administered will be dependent on the subject being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compound and the discretion of the prescribing physician. However, an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, preferably about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, preferably about 0.05 to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, e.g., by dividing such larger doses into several small doses for administration throughout the day. [106] In some embodiments, a compound of the invention is administered in a single dose. [107] Typically, such administration will be by injection, e.g., intravenous injection, in order to introduce the agent quickly. However, other routes may be used as appropriate. A single dose of a compound of the invention may also be used for treatment of an acute condition. [108] In some embodiments, a compound of the invention is administered in multiple doses. Dosing may be about once, twice, three times, four times, five times, six times, or more than six times per day. Dosing may be about once a month, once every two weeks, once a week, or once every other day. In another embodiment a compound of the invention and another agent are administered together about once per day to about 6 times per day. In another embodiment the administration of a compound of the invention and an agent continues for less than about 7 days. In yet another embodiment the administration continues for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous dosing is achieved and maintained as long as necessary. [109] Administration of the compounds of the invention may continue as long as necessary. In some embodiments, a compound of the invention is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In some embodiments, a compound of the invention is administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, a compound of the invention is administered chronically on an ongoing basis, e.g., for the treatment of chronic effects. [110] An effective amount of a compound of the invention may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, or as an inhalant. - 22 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [111] The compositions of the invention may also be delivered via an impregnated or coated device such as a stent, for example, or an artery-inserted cylindrical polymer. Such a method of administration may, for example, aid in the prevention or amelioration of restenosis following procedures such as balloon angioplasty. Without being bound by theory, compounds of the invention may slow or inhibit the migration and proliferation of smooth muscle cells in the arterial wall which contribute to restenosis. A compound of the invention may be administered, for example, by local delivery from the struts of a stent, from a stent graft, from grafts, or from the cover or sheath of a stent. In some embodiments, a compound of the invention is admixed with a matrix. Such a matrix may be a polymeric matrix and may serve to bond the compound to the stent. Polymeric matrices suitable for such use, include, for example, lactone-based polyesters or copolyesters such as polylactide, polycaprolactonglycolide, polyorthoesters, polyanhydrides, polyaminoacids, polysaccharides, polyphosphazenes, poly (ether-ester) copolymers (e.g. PEO- PLLA); polydimethylsiloxane, poly(ethylene-vinylacetate), acrylate-based polymers or copolymers (e.g. polyhydroxyethyl methylmethacrylate, polyvinyl pyrrolidinone), fluorinated polymers such as polytetrafluoroethylene and cellulose esters. Suitable matrices may be non- degrading or may degrade with time, releasing the compound or compounds. Compounds of the invention may be applied to the surface of the stent by various methods such as dip/spin coating, spray coating, dip-coating, and/or brush-coating. The compounds may be applied in a solvent and the solvent may be allowed to evaporate, thus forming a layer of compound onto the stent. Alternatively, the compound may be located in the body of the stent or graft, for example in microchannels or micropores. When implanted, the compound diffuses out of the body of the stent to contact the arterial wall. Such stents may be prepared by dipping a stent manufactured to contain such micropores or microchannels into a solution of the compound of the invention in a suitable solvent, followed by evaporation of the solvent. Excess drug on the surface of the stent may be removed via an additional brief solvent wash. In yet other embodiments, compounds of the invention may be covalently linked to a stent or graft. To link compounds of the invention to the stent or graft, a covalent linker may be used which degrades in vivo, leading to the release of the compound from the stent. Compounds of the invention may additionally be administered intravascularly from a balloon used during angioplasty. Extravascular administration of the compounds via the pericard or via adventitial application of formulations of the invention may also be performed to decrease restenosis. [112] A variety of stent devices which may be used as described are disclosed, for example, in the following references, all of which are hereby incorporated by reference: U.S. Pat. No. 5451233; U.S. Pat. No.5040548; U.S. Pat. No.5061273; U.S. Pat. No.5496346; U.S. Pat. No. 5292331; U.S. Pat. No.5674278; U.S. Pat. No.3657744; U.S. Pat. No.4739762; U.S. Pat. No. - 23 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application 5195984; U.S. Pat. No.5292331 ; U.S. Pat. No.5674278; U.S. Pat. No.5879382; U.S. Pat. No. 6344053. [113] The compounds of the invention may be administered in dosages. It is known in the art that due to intersubject variability in compound pharmacokinetics, individualization of dosing regimen is necessary for optimal therapy. Dosing for a compound of the invention may be found by routine experimentation in light of the instant disclosure. [114] When a compound of the invention is administered in a composition that comprises one or more agents, and the agent has a shorter half- life than the compound of the invention unit dose forms of the agent and the compound of the invention may be adjusted accordingly. [115] The subject pharmaceutical composition may, for example, be in a form suitable for oral administration as a tablet, capsule, pill, powder, sustained release formulations, solution, suspension, for parenteral injection as a sterile solution, suspension or emulsion, for topical administration as an ointment or cream or for rectal administration as a suppository. The pharmaceutical composition may be in unit dosage forms suitable for single administration of precise dosages. The pharmaceutical composition will include a conventional pharmaceutical carrier or excipient and a compound according to the invention as an active ingredient. In addition, it may include other medicinal or pharmaceutical agents, carriers, adjuvants, etc. Exemplary parenteral administration forms include solutions or suspensions of active compound in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Methods of Use [116] The method typically comprises administering to a subject a therapeutically effective amount of a compound of the invention. The therapeutically effective amount of the subject combination of compounds may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., reduction of proliferation or downregulation of activity of a target protein. The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried. [117] In certain embodiment, the present invention provides a pharmaceutical composition comprising a compound of bispecific formula, or pharmaceutically acceptable salt thereof. - 24 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [118] In certain embodiment, the present invention provides a pharmaceutical composition comprising a compound of bispecific formula for use in degrading a target protein in a cell. [119] In certain embodiment, a method of degrading a target protein comprising administering to a cell therapeutically effective amount of a bispecific compound, or pharmaceutically acceptable salt, wherein the compound is effective for degrading the target protein. [120] In some embodiments, the disclosure is directed to methods of degrading a SMARCA2 protein comprising contacting the SMARCA2 protein with a compound or pharmaceutical composition of the disclosure. [121] In other embodiments, the disclosure is directed to methods of degrading a SMARCA4 protein comprising contacting the SMARCA4 protein with a compound or pharmaceutical composition of the disclosure. [122] In certain embodiment, the present invention provides a pharmaceutical composition comprising a compound of bispecific formula, for use in treating or preventing of a disease or disorder in which SMARCA2 and/or SMARCA4 plays a role. [123] In certain embodiment, the present invention provides a pharmaceutical composition comprising a compound of bispecific formula, for use in treating or preventing of a disease or disorder in which SWI/SNF mutations plays a role. [124] In certain embodiment, target proteins are SMARCA2, SMARCA4 and/or PB1. [125] In certain embodiment, target protein complex is SWI/SNF in a cell. [126] In certain embodiment, diseases or disorders dependent on SMARCA2 or SMARCA4 include cancers. [127] In certain embodiment, diseases or disorders dependent on SWI/SNF complex include cancers. [128] Exemplary cancers which may be treated by the present compounds either alone or in combination with at least one additional anti-cancer agent include squamous-cell carcinoma, basal cell carcinoma, adenocarcinoma, hepatocellular carcinomas, and renal cell carcinomas, cancer of the bladder, bowel, breast, cervix, colon, esophagus, head, kidney, liver, lung, neck, ovary, pancreas, prostate, and stomach; leukemias; benign and malignant lymphomas, particularly Burkitt's lymphoma and Non-Hodgkin's lymphoma; benign and malignant melanomas; myeloproliferative diseases; sarcomas, including Ewing's sarcoma, hemangio- sarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, synovial sarcoma, gliomas, astrocytomas, oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas; bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular - 25 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma; carcinosarcoma, Hodgkin's disease, Wilms' tumor and teratocarcinomas. [129] In some embodiments, the cancer which may be treated using compounds according to the present disclosure is non-small cell lung cancer, colorectal cancer, bladder cancer, cancer of unknown primary, glioma, breast cancer, melanoma, non-melanoma skin cancer, endometrial cancer, soft tissue sarcoma, or penile cancer. [130] In certain embodiments, the cancers which may be treated using compounds according to the present disclosure include, for example, acute myeloid leukemia (AML), T-lineage Acute lymphoblastic Leukemia (T-ALL), T-lineage lymphoblastic Lymphoma (T-LL), Peripheral T- cell lymphoma, Adult T-cell Leukemia, Pre-B ALL, Pre-B Lymphomas, Large B-cell Lymphoma, Burkitts Lymphoma, B-cell ALL, Philadelphia chromosome positive ALL and Philadelphia chromosome positive CML.In certain further embodiment, the cancer is a SMARCA2 and/or SMARAC4-dependent cancer. [131] In certain embodiment, the present invention provides a pharmaceutical composition comprising a compound of bispecific formula for use in the diseases or disorders dependent upon SMARCA2 and/or SMARCA4 is cancer. [132] Compounds of the disclosure, as well as pharmaceutical compositions comprising them, can be administered to treat any of the described diseases, alone or in combination with a medical therapy. Medical therapies include, for example, surgery and radiotherapy (e.g., gamma- radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, systemic radioactive isotopes). [133] In other aspects, compounds of the disclosure, as well as pharmaceutical compositions comprising them, can be administered to treat any of the described diseases, alone or in combination with one or more other agents. [134] In other methods, the compounds of the disclosure, as well as pharmaceutical compositions comprising them, can be administered in combination with agonists of nuclear receptors agents. [135] In other methods, the compounds of the disclosure, as well as pharmaceutical compositions comprising them, can be administered in combination with antagonists of nuclear receptors agents. [136] In other methods, the compounds of the disclosure, as well as pharmaceutical compositions comprising them, can be administered in combination with an anti-proliferative agent. - 26 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application Combination Therapies [137] In certain aspects, one or more of the compounds described herein, are co-administered in combination with at least one additional bioactive agent, especially including an anticancer agent. In particularly preferred aspects, the co-administration of compounds results in synergistic activity and/or therapy, including anticancer activity. [138] For treating cancer and other proliferative diseases, the compounds of the invention can be used in combination with chemotherapeutic agents, agonists or antagonists of nuclear receptors, or other anti-proliferative agents. The compounds of the invention can also be used in combination with a medical therapy such as surgery or radiotherapy, e.g., gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, and systemic radioactive isotopes. Examples of suitable chemotherapeutic agents include any of: abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, all-trans retinoic acid, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bendamustine, bevacizumab, bexarotene, bleomycin, bortezombi, bortezomib, busulfan intravenous, busulfan oral, calusterone, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, nofetumomab, oxaliplatin, paclitaxel, pamidronate, panobinostat, panitumumab, pegaspargase, pegfilgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, procarbazine, quinacrine, rasburicase, rituximab, ruxolitinib, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinstat and zoledronate. [139] In some embodiments, the compounds of the invention can be used in combination with a therapeutic agent that targets an epigenetic regulator. Examples of epigenetic regulators include bromodomain inhibitors, the histone lysine methyltransferase inhibitors, histone arginine methyl transferase inhibitors, histone demethylase inhibitors, histone deacetylase inhibitors, histone acetylase inhibitors, and DNA methyltransferase inhibitors. Histone deacetylase inhibitors - 27 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application include, e.g., vorinostat. Histone arginine methyl transferase inhibitors include inhibitors of protein arginine methyltransferases (PRMTs) such as PRMT5, PRMT1 and PRMT4. DNA methyltransferase inhibitors include inhibitors of DNMT1 and DNMT3. [140] For treating cancer and other proliferative diseases, the compounds of the invention can be used in combination with targeted therapies, including JAK kinase inhibitors (e.g. Ruxolitinib), PI3 kinase inhibitors including PI3K-delta selective and broad spectrum PI3K inhibitors, MEK inhibitors, Cyclin Dependent kinase inhibitors, including CDK4/6 inhibitors and CDK9 inhibitors, BRAF inhibitors, mTOR inhibitors, proteasome inhibitors (e.g. Bortezomib, Carfilzomib), HDAC inhibitors (e.g. panobinostat, vorinostat), DNA methyl transferase inhibitors, dexamethasone, bromo and extra terminal family member (BET) inhibitors, BTK inhibitors (e.g. ibrutinib, acalabrutinib), BCL2 inhibitors (e.g. venetoclax), dual BCL2 family inhibitors (e.g. BCL2/BCLxL), PARP inhibitors, FLT3 inhibitors, or LSD1 inhibitors. [141] In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-1, e.g., an anti-PD-1 monoclonal antibody. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab, pembrolizumab (also known as MK-3475), or PDR001. In some embodiments, the anti-PD-1 monoclonal antibody is nivolumab or pembrolizumab. In some embodiments, the anti-PD1 antibody is pembrolizumab. In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of PD-L1, e.g., an anti-PD-L1 monoclonal antibody. In some embodiments, the anti-PD-L1 monoclonal antibody is atezolizumab, durvalumab, or BMS-935559. In some embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CTLA-4, e.g., an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is ipilimumab. [142] In some embodiments, the agent is an alkylating agent, a proteasome inhibitor, a corticosteroid, or an immunomodulatory agent. Examples of an alkylating agent include cyclophosphamide (CY), melphalan (MEL), and bendamustine. In some embodiments, the proteasome inhibitor is carfilzomib. In some embodiments, the corticosteroid is dexamethasone (DEX). In some embodiments, the immunomodulatory agent is lenalidomide (LEN) or pomalidomide (POM). [143] The following Examples are provided to illustrate some of the concepts described within this disclosure. While the Examples are considered to provide an embodiment, it should not be considered to limit the more general embodiments described herein. EXAMPLES - 28 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application Example 1. (S)-3-(6-(4-((6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3-yl)methyl)- piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione
[144] Step 1: 2-Benzyl 1-(tert-butyl) (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate [145] To a mixture of (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (25.0 g, 108 mmol) and K2CO3 (44.8 g, 324 mmol) in DMF (250 mL) was added benzyl bromide (26.7 mL, 216 mmol). The reaction was stirred at room temperature for 2 h then diluted with MTBE (500 mL) and filtered through a pad of Celite®. The filtrate was dried over Na2SO4, filtered then concentrated to give crude give 2-benzyl 1-(tert-butyl) (2S,4R)-4-hydroxy- pyrrolidine-1,2-dicarboxylate (assumed quantitative yield) which was used without further purification. LCMS calcd for C17H24NO5 [M+H]+: m/z = 322.2; Found: 322.5. [146] Step 2: 2-Benzyl 1-(tert-butyl) (2S,4R)-4-(benzyloxy)pyrrolidine-1,2-dicarboxylate [147] To crude 2-benzyl 1-(tert-butyl) (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (34.7 g, 108 mmol) in DMF (300 mL) was added benzyl bromide (19.2 mL, 162 mmol) then KOH (18.2 g, 324 mmol). The reaction was stirred at room temperature for 3 h then diluted with MTBE (500 mL) and filtered through a pad of Celite®. The filtrate was washed with 1 N HCl (aq; 500 mL), sat. NaHCO3 (aq; 500 mL), then brine (500 mL). The organic layer was dried over Na2SO4, - 29 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application filtered and concentrated. The residue was purified via SiO2 FCC (0-40% MTBE in heptanes) to give 2-benzyl 1-(tert-butyl) (2S,4R)-4-(benzyloxy)pyrrolidine-1,2-dicarboxylate (26.0 g, 59%) as a colorless oil. LCMS calcd for C24H30NO5 [M+H]+: m/z = 412.2; Found: 412.5. [148] Step 3: 2-Benzyl 1-(tert-butyl) (4R)-4-(benzyloxy)-2-methylpyrrolidine-1,2-dicarboxylate [149] To 2-benzyl 1-(tert-butyl) (2S,4R)-4-(benzyloxy)pyrrolidine-1,2-dicarboxylate (34.7 g, 84.3 mmol) in THF (8 mL) at 0 °C was added methyl iodide (6.3 mL, 101 mmol) portion-wise. The reaction was stirred for 30 min, after which LiHMDS (16.9 g, 101 mmol) was added. The reaction was allowed to warm to room temperature, stirred for 12 h, then quenched with sat. NH4Cl (aq; 200 mL) and extracted with MTBE (3 x 200 mL). The combined organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified via SiO2 FCC (0-20% MTBE in heptanes) to give 2-benzyl 1-(tert-butyl) (4R)-4-(benzyloxy)-2-methylpyrrolidine-1,2- dicarboxylate (33.2 g, 93%) as a colorless, viscous oil. LCMS calcd for C25H32NO5 [M+H]+: m/z = 426.2; Found: 426.1. [150] Step 4: (4R)-4-(Benzyloxy)-1-(tert-butoxycarbonyl)-2-methylpyrrolidine-2-carboxylic acid
[151] To 2-benzyl 1-(tert-butyl) (4R)-4-(benzyloxy)-2-methylpyrrolidine-1,2-dicarboxylate (33.2 g, 78.0 mmol) in MeOH (150 mL) at 0 °C was added NaOH (12.5 g, 312 mmol) in water (150 mL) dropwise. The reaction was stirred at 75 °C for 12 h, after which MeOH was removed via rotary evaporation. The resultant aqueous phase was diluted with water (150 mL), washed with 2:1 heptanes/MTBE (3 x 150 mL), then acidified to pH ~2 with 4 N HCl (aq) and extracted with MTBE (3 x 150 mL). The combined organic phase from the latter three MTBE extractions was dried over Na2SO4, filtered and concentrated to afford (4R)-4-(benzyloxy)-1-(tert-butoxy- carbonyl)-2-methylpyrrolidine-2-carboxylic acid (27.0 g, quant) which was used without further purification. LCMS calcd for C18H26NO5 [M+H]+: m/z = 336.2; Found: 336.3. [152] Step 5: (6R,7aS)-6-(benzyloxy)-7a-methyltetrahydro-1H,3H-pyrrolo[1,2-c]oxazole-1,3- dione - 30 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[153] To a solution of DMF (4.5 mL, 58 mmol) in DCM (20 mL) at 0 °C was added oxalyl chloride (4.32 mL, 50.4 mmol) dropwise. The reaction was stirred at 0 °C for 30 min, after which a solution of (4R)-4-(benzyloxy)-1-(tert-butoxycarbonyl)-2-methylpyrrolidine-2-carboxylic acid (13.0 g, 38.8 mmol) and pyridine (3.12 mL, 38.8 mmol) in DCM (100 mL) was added, dropwise. After an additional 30 min of stirring at 0 °C, the reaction was washed with cold water (100 mL), then dried over Na2SO4, filtered and concentrated. The residue was purified via SiO2 FCC (0- 100% EtOAc in heptanes) to give “diastereomer A” and the desired “diastereomer B” (6R,7aS)- 6-(benzyloxy)-7a-methyltetrahydro-1H,3H-pyrrolo[1,2-c]oxazole-1,3-dione (820 mg, 8%) as colorless, viscous oils. [154] Step 6: (2S,4R)-4-(Benzyloxy)-N-(4-bromo-6-chloropyridazin-3-yl)-2-methylpyrrolidine- 2-carboxamide
[155] To a cold solution of (6R,7aS)-6-(benzyloxy)-7a-methyltetrahydro-1H,3H-pyrrolo[1,2- c]oxazole-1,3-dione (diastereomer B) (820 mg, 3.14 mmol) and 4-bromo-6-chloropyridazin-3- amine (0.59 g, 2.8 mmol) in THF (4 mL), NaH (60% suspension in mineral oil; 0.188 g, 4.71 mmol) was added in portions at 0 °C. When HPLC analysis indicated the reaction had stalled, additional NaH (60% suspension in mineral oil; 0.093 g, 2.4 mmol) was added. HPLC analysis indicated improved conversion. The reaction was quenched with a 10% citric acid (aq.) (20 mL), and the mixture extracted with 3:1 CHCl3/IPA (15 mL × 5). The combined organic phase was dried over Na2SO4, filtered, and concentrated to provide the crude (2S,4R)-4-(benzyloxy)-N-(4- bromo-6-chloropyridazin-3-yl)-2-methylpyrrolidine-2-carboxamide (assumed quantitative yield) which was used for next reaction without further purification. LCMS calculated for C17H19BrClN4O2 [M+H]+: m/z = 425.0; found: 425.2. [156] Step 7: (6aS,8R)-8-(Benzyloxy)-2-chloro-6a-methyl-6a,7,8,9-tetrahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-6(5H)-one
- 31 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [157] To crude (2S,4R)-4-(benzyloxy)-N-(4-bromo-6-chloropyridazin-3-yl)-2-methyl- pyrrolidine-2-carboxamide in THF (20 mL) was added N-methylmorpholine (228 mg, 2.26 mmol) slowly. The reaction mixture was stirred overnight at 120 °C then concentrated. The residue was taken up in water (10 mL) and DCM (10 mL), and the organic layer was separated. The aqueous layer was extracted with DCM (3 x 50 mL), and the combined organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified via SiO2 FCC (30-50% EtOAc in heptanes) to afford (6aS,8R)-8-(Benzyloxy)-2-chloro-6a-methyl-6a,7,8,9-tetrahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-6(5H)-one (255 mg, 23.8% for two steps), as a white solid. LCMS calculated for C17H18ClN4O2 [M+H]+: m/z = 345.1; found: 345.4. [158] Step 8: (6aS,8R)-8-(Benzyloxy)-2-chloro-6a-methyl-5,6,6a,7,8,9-hexahydropyrrolo [1’,2’:4,5]pyrazino[2,3-c]pyridazine
[159] To (6aS,8R)-8-(benzyloxy)-2-chloro-6a-methyl-6a,7,8,9-tetrahydropyrrolo[1’,2’:4,5] pyrazino[2,3-c]- 32 -yridazine-6(5H)-one (260 mg, 0.74 mmol) in THF (3 mL) was added BH3·SMe2 (0.35 mL, 3.7 mmol) at 0 °C. The reaction mixture was warmed to 50 °C and stirred for 90 min. The LCMS analysis indicated full consumption of starting material. The reaction mixture was cooled to 0 °C and quenched slowly with MeOH and concentrated. The crude intermediate was then dissolved in EtOH (3 mL). NaBH3CN (279 mg, 4.44 mmol) and AcOH (0.51 mL, 8.9 mmol) were added at 0 °C. The reaction mixture was slowly heated to 80 °C and refluxed overnight. The LCMS analysis indicated consumption of the starting material and the formation of the desired product. The reaction mixture was allowed to cool to room temperature and poured into cold sat. NaHCO3 (aq.) (25 mL). The organic layer was separated, and the aqueous layer extracted with DCM (20 mL × 3). The combined organic phase was dried over Na2SO4, filtered, and concentrated. The residue was purified via SiO2 FCC (10~60% EtOAc in DCM) to give (6aS,8R)-8-(benzyloxy)-2-chloro-6a-methyl-5,6,6a,7,8,9-hexahydropyrrolo- [1',2':4,5] pyrazino[2,3-c]pyridazine (210 mg, 0.635 mmol, 85.8% yield) as colorless viscous oil. LCMS calculated for C17H20ClN4O [M+H]+: m/z = 331.1; found: 331.5. [160] Step 9: (6aS,8R)-2-Chloro-6a-methyl-5,6,6a,7,8,9-hexahydropyrrolo[1’,2’:4,5] pyrazino[2,3-c]pyridazine-8-ol - 32 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[161] Trichloroborane (1M in DCM, 425 mg, 3.63 mmol) was added dropwise to a solution of (6aS,8R)-8-(benzyloxy)-2-chloro-6a-methyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]pyrazino [2,3-c]pyridazine (240 mg, 0.73 mmol) in DCM (3 mL) at -78 °C . The resulting mixture was stirred at -78 °C for 30 min, and the reaction quenched with MeOH (10 mL). The volatiles were evaporated, the residue was basified by addition of 10% Na2CO3 (aq.), and the mixture was then extracted with CHCl3/IPA (3:1, 10 mL × 6). The combined organic phase was dried over Na2SO4, filtered, and concentrated. The residue was triturated in MTBE (5 mL) for 30 min. The solid material was filtered, washed with MTBE, and dried under high vacuum to give the title compound (150 mg, 0.62 mmol, 86% yield) as a white solid. LCMS calculated for C10H14ClN4O [M+H]+: m/z = 241.1; found: 241.3. [162] Step 10. Methyl 6-(((6aS,8R)-2-chloro-6a-methyl-5,6,6a,7,8,9-hexahydropyrrolo- [1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)nicotinate
[163] To a solution of (6aS,8R)-2-chloro-6a-methyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5] pyrazino[2,3-c]pyridazin-8-ol (250 mg, 1.04 mmol) and methyl 6-fluoronicotinate (209 mg, 1.35 mmol) in THF (4 mL) and DMF (1 mL) was added sodium hydride (83 mg, 2.1 mmol, 60% dispersion in mineral oil) at 0 °C. The reaction mixture was stirred for 1 h. The reaction was quenched with ice-cold 1 N HCl (aq.) and extracted with DCM. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel chromatography (EtOAc/DCM, then MeOH/DCM) to afford the title compound. LC-MS calc. for C17H19ClN5O3 [M+H]+: m/z = 376.1; Found: 376.0. [164] Step 11. Methyl 6-(((6aS,8R)-2-(2-hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)nicotinate - 33 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[165] A solution of methyl 6-(((6aS,8R)-2-chloro-6a-methyl-5,6,6a,7,8,9-hexahydropyrrolo- [1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)nicotinate (175 mg, 0.466 mmol), 2-hydroxyphenyl- boronic acid (96 mg, 0.70 mmol), potassium carbonate (257 mg, 1.86 mmol), and SPhos Pd G2 (17 mg, 0.023 mmol, CAS 1375325-64-6) in water (1 mL) and 1,4-dioxane (3 mL) was stirred overnight at 90 °C. The reaction was cooled to room temperature and diluted with water and DCM. The layers were separated, and the aqueous layer was extracted with DCM. The combined organic layers were dried over Na2SO4 and concentrated. The crude material was purified by silica gel chromatography to afford the title compound (150 mg, 74%). LC-MS calc. for C23H24N5O4 [M+H]+: m/z = 434.2; Found: 434.1. [166] Step 12.2-((6aS,8R)-8-((5-(Hydroxymethyl)pyridin-2-yl)oxy)-6a-methyl-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-2-yl)phenol
[167] To a solution of methyl 6-(((6aS,8R)-2-(2-hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)nicotinate (77 mg, 0.18 mmol) in THF (4 mL) was added lithium aluminum hydride (68 mg, 1.8 mmol) at 0 °C. The reaction was stirred at room temperature overnight. The reaction was quenched with ice-cold water with the reaction vessel in an ice bath and extracted with CHCl3/IPA (3:1). The organic layers were washed with brine, dried over Na2SO4, and concentrated. The crude material was purified by silica gel chromatography (0–20% MeOH/DCM) to afford the title compound (50 mg, 69% yield). LC-MS calc. for C22H24N5O3 [M+H]+: m/z = 406.2; Found: 406.1. [168] Step 13. tert-Butyl 4-(4-cyano-3-methoxycarbonylphenyl)piperazine-1-carboxylate
[169] To a solution of methyl 2-cyano-5-fluorobenzoate (20.0 g, 112 mmol) and tert-butyl 1- piperazinecarboxylate (31.2 g, 167 mmol) in NMP (20 mL) was added N,N-diisopropyl- - 34 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application ethylamine (0.01 mL, 0.04 mmol) and heated to 120 °C for 4 h. HPLC monitoring after 4 h showed the consumption of starting material. The reaction mixture was cooled to -20°C overnight and product crashed out. The solid was filtered and washed with heptanes to obtain yellow solid as the desired product tert-butyl 4-(4-cyano-3-(methoxycarbonyl)phenyl)piperazine- 1-carboxylate (28.0 g, 72.6 %). LCMS calculated for C13H16N3O2 (M+2H-Boc)+: m/z = 246.17; found: 245.99.1H NMR (300 MHz, DMSO) δ 7.73 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 2.7 Hz, 1H), 7.22 (dd, J = 8.8, 2.7 Hz, 1H), 3.89 (s, 3H), 3.51 – 3.38 (m, 8H), 1.42 (s, 9H). [170] Step 14. tert-Butyl 4-(4-formyl-3-(methoxycarbonyl)phenyl)piperazine-1-carboxylate
[171] To a solution of tert-butyl 4-(4-cyano-3-methoxycarbonylphenyl)piperazine-1- carboxylate (8.10 g, 23.5 mmol) in pyridine (11.4 mL, 141 mmol) and acetic acid (8.05 mL, 141 mmol) was added sodium phosphinate monohydrate (4.97 g, 46.9 mmol) and water (10.0 mL). To the stirring solution was added Raney Nickel (2.75 g, 46.9 mmol) in multiple portions to avoid the emulsion formation. The reaction was heated to 75°C for 7 h and monitored by HPLC to see only ~50% conversion. To the solution was added another 1.0 equiv of Raney Nickel and heated at 75°C overnight. HPLC monitoring showed ~94% conversion. The heating was stopped and the reaction was cooled down, diluted with MeOH and filtered through celite and washed with MeOH to remove Raney Nickel. The filtrate was concentrated to remove pyridine and MeOH. The concentrated solution was then washed with water. The organic phase was dried over Na2SO4, filtered and concentrated to obtain the crude. Purified by FCC (0% to 60% EA/Heptanes) to obtain yellowish solid as desired product tert-butyl 4-(4-formyl-3-methoxy- carbonylphenyl)piperazine-1-carboxylate (5.00 g, 61.1 %). LCMS calculated for C18H25N2O5 (M+H)+: m/z = 349.2; found: 348.9.1H NMR (300 MHz, CDCl3) δ 10.34 (s, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 1.6 Hz, 1H), 7.00 (dd, J = 8.8, 2.6 Hz, 1H), 3.96 (s, 3H), 3.66 – 3.55 (m, 4H), 3.39 (dd, J = 13.4, 8.6 Hz, 4H), 1.48 (s, 9H). [172] Step 15. tert-Butyl (S)-4-(2-(1-amino-5-methoxy-1,5-dioxopentan-2-yl)-3-oxoisoindolin-5- yl)piperazine-1-carboxylate
[173] To a solution of tert-butyl 4-(4-formyl-3-methoxycarbonylphenyl)piperazine-1- carboxylate (4.20 g, 12.0 mmol) in DCM (50.0 mL) was added N,N-diisopropylethylamine (5.25 - 35 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application mL, 30.1 mmol) and methyl (4S)-4,5-diamino-5-oxopentanoate (2.32 g, 14.5 mmol). The reaction was stirred at RT for 1 h and acetic acid (6.89 mL, 121 mmol) was added to this solution. The reaction was further stirred for another hour and sodium triacetoxyborohydride (7.66 g, 36.2 mmol) was added. The reaction was stirred overnight to see full conversion, monitored by HPLC. The reaction mixture was diluted with DCM (30.0 mL) and quenched with water (30.0 mL). Further extraction was done by DCM (30 mL x 2). The organic phase was dried over Na2SO4, filtered, and concentrated to obtain the crude. Crude was further purified by FCC (0% to 7% MeOH/DCM) to obtain white solid as desired compound tert-butyl (S)-4-(2-(1-amino- 5-methoxy-1,5-dioxopentan-2-yl)-3-oxoisoindolin-5-yl)piperazine-1-carboxylate (3.80 g, 68.5 %). LCMS calculated for C23H33N4O6 (M+H)+: m/z = 461.2; found: 461.1.1H NMR (300 MHz, CDCl3) δ 7.33 (dd, J = 10.8, 5.3 Hz, 2H), 7.16 (dd, J = 8.4, 2.4 Hz, 1H), 6.40 (s, 1H), 5.58 (s, 1H), 5.00 – 4.83 (m, 1H), 4.39 (q, J = 16.8 Hz, 2H), 3.63 (s, 3H), 3.62 – 3.55 (m, 4H), 3.22 – 3.13 (m, 4H), 2.49 – 2.28 (m, 3H), 2.20 (tt, J = 11.4, 8.2 Hz, 1H), 1.49 (s, 9H). [174] Step 16. tert-Butyl (S)-4-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)piperazine-1- carboxylate
[175] To a solution of tert-butyl (S)-4-(2-(1-amino-5-methoxy-1,5-dioxopentan-2-yl)-3- oxoisoindolin-5-yl)piperazine-1-carboxylate (3.80 g, 8.25 mmol) in THF (80.0 mL), was added potassium t-butoxide (1.0 M in THF, 9.90 mL, 9.90 mmol) at -78°C. The reaction was stirred at -78°C for 3 h. The reaction temperature was maintained at 0 °C and quenched by addition of 1N HCl to achieve pH 3. To this solution NaHCO3 (sat. aq.) was added dropwise slowly to achieve pH ~6. The reaction mixture was then diluted further by DCM and washed with water. Water phase was further extracted by DCM. Organic phase was collected, dried over Na2SO4, filtered and concentrated to obtain the crude. tert-butyl (S)-4-(2-(2,6-dioxopiperidin-3- yl)-3-oxoisoindolin-5-yl)piperazine-1-carboxylate (2.90 g, 82.0 %). The crude was about 96% pure (HPLC) which was taken to the next step without further purification. LCMS calculated for C18H21N4O5 (M+2H-tBu)+: m/z = 373.2; found: 373.0.1H NMR (300 MHz, CDCl3) δ 8.06 (s, 1H), 7.37 (dd, J = 9.3, 5.3 Hz, 2H), 7.17 (dd, J = 8.4, 2.3 Hz, 1H), 5.22 (dd, J = 13.1, 5.2 Hz, 1H), 4.35 (dd, J = 43.1, 15.6 Hz, 2H), 3.72 – 3.49 (m, 4H), 3.30 – 3.13 (m, 4H), 2.96 – 2.73 (m, 2H), 2.43 – 2.15 (m, 2H), 1.49 (s, 9H). [176] Step 17. (S)-3-(1-Oxo-6-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione - 36 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[177] To a round bottomed flask containing tert-butyl (S)-4-(2-(2,6-dioxopiperidin-3-yl)-3- oxoisoindolin-5-yl)piperazine-1-carboxylate (4.3 g, 10.0 mmol) was added 4N HCl in dioxane (34.0 mL, 136 mmol) at 0 °C. The solution was stirred at 0 °C to RT for 3 h to see full conversion. The precipitate was filtered and dried under air flow to yield (S)-3-(1-oxo-6- (piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione;hydrochloride (3.30 g, 90.1 %). LCMS calculated for C17H21N4O3 (M+H)+: m/z = 329.2; found: 329.0.1H NMR (300 MHz, DMSO) δ 10.98 (s, 1H), 9.27 (s, 2H), 7.49 (d, J = 8.3 Hz, 1H), 7.39 – 7.19 (m, 2H), 5.10 (dd, J = 13.2, 5.1 Hz, 1H), 4.27 (dt, J = 27.7, 13.9 Hz, 2H), 3.49 – 3.40 (m, 4H), 3.22 (s, 4H), 2.98 – 2.83 (m, 1H), 2.59 (d, J = 16.3 Hz, 1H), 2.46 – 2.27 (m, 1H), 2.09 – 1.94 (m, 1H). [178] Step 18. (S)-3-(6-(4-((6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9-hexa- hydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3-yl)methyl)piperazin-1-yl)-1- oxoisoindolin-2-yl)piperidine-2,6-dione [179] A solution of sulfurtrioxide pyridine (59 mg, 0.37 mmol) in DMSO (0.50 mL) was stirred for 20 mins.2-((6aS,8R)-8-((5-(hydroxymethyl)pyridin-2-yl)oxy)-6a-methyl-5,6,6a,7,8,9-hexa- hydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-2-yl)phenol (50.0 mg, 0.12 mmol) and triethyl- amine (155 ^L, 1.11 mmol) were added and the reaction mixture was stirred for 2 h. (S)-3-(1- oxo-6-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione (61 mg, 0.18 mmol) and acetic acid (106 ^L, 1.85 mmol) were added and the reaction mixture was stirred for 1 h. Sodium triacetoxy- borohydride (78 mg, 0.37 mmol) was added and the reaction was stirred overnight. The reaction mixture was diluted with MeCN, filtered, and purified by prep-HPLC on a C18 column (18.0– 23.0% MeCN/0.2% TFA (aq)) to afford the title compound as a TFA salt (4.8 mg, 0.0058 mmol, 4.7% yield), a white solid. LC-MS calc. for C39H42N9O5 [M+H]+: m/z = 716.3; Found: 716.1. Example 2. (S)-3-(6-(4-((5-Fluoro-6-(((6aS,8R)-2-(2-hydroxyphenyl)-6a-methyl- 5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3- yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione - 37 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[180] Step 1.2-((6aS,8R)-8-((3-Fluoro-5-(hydroxymethyl)pyridin-2-yl)oxy)-6a-methyl- 5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-2-yl)phenol
[181] The title compound was synthesized according to procedures analogous to Example 1, Steps 10-12, using methyl 5,6-difluoronicotinate. LC-MS calc. for C22H23FN5O3 [M+H]+: m/z = 424.2; Found: 424.1. [182] Step 2. (S)-3-(6-(4-((5-Fluoro-6-(((6aS,8R)-2-(2-hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3-yl)methyl)piperazin-1- yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione [183] The title compound was synthesized according to procedures analogous to Example 1, Step 18. LC-MS calc. for C39H41FN9O5 [M+H]+: m/z = 734.3; Found: 734.2. Example 3. (S)-3-(6-(1-((6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylpyridin-3- yl)methyl)piperidin-4-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione - 38 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[184] Step 1.2-((6aS,8R)-8-((5-(Hydroxymethyl)-3-methylpyridin-2-yl)oxy)-6a-methyl- 5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-2-yl)phenol
[185] The title compound was synthesized according to procedures analogous to Example 1, Steps 10–12, using methyl 6-fluoro-5-methylnicotinate. LC-MS calc. for C23H26N5O3 [M+H]+: m/z = 420.2; Found: 420.2. [186] Step 2. Methyl 5-bromo-2-(dibromomethyl)benzoate
[187] A suspension of methyl 5-bromo-2-methylbenzoate (6.08 g, 26.6 mmol), NBS (14.0 g, 79.7 mmol), and benzoyl peroxide (0.32 g, 1.33 mmol) in DCE (60.0 mL) was stirred at 80 °C for 18 h. The reaction mixture was cooled to ambient temperature, diluted with DCM (140 mL), washed with 10% Na2S2O3 solution (100 mL), saturated NaHCO3 solution (100 mL), water (100 mL), and brine (100 mL), then dried over Na2SO4, filtered, and concentrated. The residue was dried under high vacuum to give the desired product, methyl 5-bromo-2-(dibromomethyl)- benzoate (9.875 g, 96.1%), as light yellow solid.1H NMR (300 MHz, CDCl3) δ 8.04 (dd, J = 5.4, 3.1 Hz, 2H), 7.97 (s, 1H), 7.74 (dd, J = 8.6, 2.1 Hz, 1H), 3.96 (s, 3H). [188] Step 3. Methyl 5-bromo-2-formylbenzoate - 39 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [189] Silver nitrate (6.57 g, 38.7 mmol) was dissolved in water (60.0 mL). This solution was added dropwise into the stirred solution of methyl 5-bromo-2-(dibromomethyl)benzoate (5.99 g, 15.5 mmol) in IPA (60.0 mL) at 0 °C. The resulting mixture was stirred at ambient temperature in the dark for 4 h, when HPLC indicated the disappearance of the starting material. The precipitated silver salt was filtered off and washed with IPA in small portions. IPA was evaporated under reduced pressure and the remaining aqueous phase was extracted with EtOAc (50.0 mL × 3). The combined organic phase was washed with water (50.0 mL), brine (50.0 mL), then dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was dried under high vacuum to give the desired product, methyl 5-bromo-2-formylbenzoate (3.26 g, 13.486.8 %), as white solid. LCMS calculated for C9H8BrO3 (M+H)+: m/z = 242.9; found: 242.8.1H NMR (300 MHz, CDCl3) δ 10.58 (s, 1H), 8.13 (d, J = 1.5 Hz, 1H), 7.85 – 7.76 (m, 2H), 3.99 (s, 3H). [190] Step 4. tert-Butyl 4-(4-formyl-3-methoxycarbonylphenyl)-3,6-dihydro-2H-pyridine-1- carboxylate
[191] A suspension of N-Boc-1,2,3,6-tetrahydropyridine-4-boronic acid pinacol ester (5.55 g, 17.9 mmol), methyl 5-bromo-2-formylbenzoate (2.91 g, 12.0 mmol), Pd(dppf)Cl2 (0.44 g, 0.6 mmol), and CsOAc (9.18 g, 47.8 mmol) in 1,4-dioxane (40.0 mL) and water (10.0 mL) was heated at 95 °C under N2 atmosphere for 1.5 h, when HPLC indicated the full conversion of the starting material. The reaction was cooled to ambient temperature and partitioned between EtOAc and water (100 mL each). The organic layer was separated, and the aqueous layer extracted with EtOAc (50 mL × 3). The combined organic phase was washed with water and brine (100 mL each), dried over Na2SO4, filtered and concentrated. The residue was purified by flash column chromatography (0~25% EtOAc in heptanes) to give the desired product, tert-butyl 4-(4-formyl-3-methoxycarbonylphenyl)-3,6-dihydro-2H-pyridine-1-carboxylate (3.30 g, 79.9 %), as yellow solid. Rf = 0.4 (25% EtOAc in heptanes).1H NMR (300 MHz, CDCl3) δ 10.57 (s, 1H), 7.93 (t, J = 4.8 Hz, 2H), 7.63 (dd, J = 8.1, 1.5 Hz, 1H), 6.25 (s, 1H), 4.12 (d, J = 2.8 Hz, 2H), 3.98 (s, 3H), 3.66 (t, J = 5.7 Hz, 2H), 2.56 (d, J = 1.4 Hz, 2H), 1.49 (s, 9H). [192] Step 5. tert-Butyl (S)-4-(2-(1-amino-5-methoxy-1,5-dioxopentan-2-yl)-3-oxoisoindolin-5- yl)-3,6-dihydropyridine-1(2H)-carboxylate - 40 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[193] To a suspension tert-butyl 4-(4-formyl-3-methoxycarbonylphenyl)-3,6-dihydro-2H- pyridine-1-carboxylate (Intermediate 5, Step 3) (2.88 g, 8.34 mmol) and H-Glu(OMe)-NH2 hydrochloride (2.05 g, 10.4 mmol) in DCM (40 mL) was slowly added DIPEA (4.36 mL, 25.0 mmol) and the resulting mixture was stirred at ambient temperature for 30 min. AcOH (2.86 mL, 50.0 mol) was then added and the mixture was stirred for 1 h. NaBH(OAc)3 (5.30 g, 25.0 mmol) was added in portions and the reaction was stirred at ambient temperature for 18 h. The reaction was diluted with DCM (150 mL), washed with saturated NaHCO3 solution, dried over Na2SO4, filtered and concentrated. The residue was purified by flash column chromatography (0~5% MeOH in DCM) to give the desired product, tert-butyl (S)-4-(2-(1-amino-5-methoxy-1,5- dioxopentan-2-yl)-3-oxoisoindolin-5-yl)-3,6-dihydropyridine-1(2H)-carboxylate (3.01 g, 78.9 %), as white solid. Rf = 0.3 (5% MeOH in DCM). LCMS calculated for C24H32N3O6 (M+H)+: m/z = 458.2; found: 458.1.1H NMR (300 MHz, DMSO) δ 7.75 – 7.67 (m, 2H), 7.57 (d, J = 8.3 Hz, 2H), 7.20 (s, 1H), 6.26 (s, 1H), 4.75 (dd, J = 10.2, 4.6 Hz, 1H), 4.52 (dd, J = 45.0, 17.7 Hz, 2H), 4.02 (d, J = 7.1 Hz, 2H), 3.56 (t, J = 5.5 Hz, 2H), 3.50 (s, 3H), 2.58 – 2.50 (m, 2H), 2.29 – 1.96 (m, 4H), 1.43 (s, 9H). [194] Step 6. tert-Butyl (S)-4-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)-3,6-dihydro- pyridine-1(2H)-carboxylate
[195] tert-butyl (S)-4-(2-(1-amino-5-methoxy-1,5-dioxopentan-2-yl)-3-oxoisoindolin-5-yl)-3,6- dihydropyridine-1(2H)-carboxylate (1.01 g, 2.20 mmol) in THF (15.0 mL) was added t-BuOK (1.0 M in THF,, 2.75 mmol, 2.75 mL) dropwise at -78 °C. The resulting mixture was stirred at - 78 °C for 2 h. The reaction was quenched by the addition of 1 N HCl solution until pH 6 and extracted with DCM (50 mL × 3). The combined organic phase was washed with brine (50 mL), dried over Na2SO4, filtered and concentrated. The residue was suspended in a minimum amount of DCM (2.00 mL) and MTBE (20.0 mL) was added at 0 °C. The white precipitate was filtered off and washed with cold MTBE. The filter cake was dried under air flow to give tert-butyl (S)-4- (2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)-3,6-dihydropyridine-1(2H)-carboxylate (718 mg, 1.69 mmol, 76.7% yield) as white solid. LCMS calculated for C19H20N3O5 (M+2H-tBu)+: - 41 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application m/z = 370.14; found: 370.09.1H NMR (300 MHz, DMSO) δ 10.99 (s, 1H), 7.75 – 7.71 (m, 2H), 7.58 (d, J = 8.5 Hz, 1H), 6.28 (s, 1H), 5.13 (dd, J = 13.2, 5.1 Hz, 1H), 4.39 (dd, J = 41.6, 17.5 Hz, 2H), 4.02 (s, 2H), 3.56 (t, J = 5.6 Hz, 2H), 3.01 – 2.83 (m, 1H), 2.71 – 2.51 (m, 3H), 2.40 (qd, J = 13.3, 4.5 Hz, 1H), 2.01 (ddd, J = 10.2, 5.1, 3.1 Hz, 1H), 1.43 (s, 9H). [196] Step 7. tert-Butyl (S)-4-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)piperidine-1- carboxylate
[197] tert-butyl (S)-4-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)-3,6-dihydropyridine- 1(2H)-carboxylate (818 mg, 1.92 mmol) in MeOH (5.00 mL) and THF (5.00 mL) was hydrogenated under 30 psi for 2 h with 10% Pd/C. The solid materials were filtered off and washed with MeOH in small portions. The filtrate was concentrated and the residue was purified by flash column chromatography on silica gel (0~4% MeOH in DCM) to give tert-butyl (S)-4-(2- (2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)piperidine-1-carboxylate (809 mg, 98.4 %) as white solid. Rf = 0.25 (5% MeOH in DCM). LCMS calculated for C19H22N3O5 (M+2H-tBu)+: m/z = 372.2; found: 372.0.1H NMR (300 MHz, DMSO) δ 10.99 (s, 1H), 7.55 (d, J = 11.0 Hz, 3H), 5.11 (dd, J = 13.3, 5.1 Hz, 1H), 4.35 (dd, J = 41.3, 17.1 Hz, 2H), 4.08 (d, J = 10.3 Hz, 2H), 2.99 – 2.74 (m, 4H), 2.59 (dd, J = 15.4, 2.2 Hz, 1H), 2.39 (qd, J = 13.1, 4.3 Hz, 1H), 2.10 – 1.92 (m, 1H), 1.79 (d, J = 12.1 Hz, 2H), 1.66 – 1.45 (m, 2H), 1.42 (s, 9H).
[199] tert-butyl (S)-4-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)piperidine-1- carboxylate (809 mg, 1.89 mmol) was suspended in 1,4-dioxane (2.00 mL) and 4 N HCl in 1,4- dioxane (15.0 mL) was added at 0 °C. The reaction was stirred at ambient temperature for 18 h. The white precipitate was filtered off and washed with cold 1,4-dioxane in small portions. The filter cake was dried under air flow to give (S)-3-(1-oxo-6-(piperidin-4-yl)isoindolin-2-yl) piperidine-2,6-dione hydrochloride (688 mg, quant.) as white solid. LCMS calculated for C18H22N3O3 (M+H)+: m/z = 328.2; found: 328.1. - 42 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [200] Step 9. (S)-3-(6-(1-((6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylpyridin-3- yl)methyl)piperidin-4-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione [201] The title compound was synthesized according to procedures analogous to Example 1, Step 18. LC-MS calc. for C41H45N8O5 [M+H]+: m/z = 729.4; Found: 729.2. Example 4. (S)-3-(6-(4-((6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylpyridin-3- yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione
[202] The title compound was synthesized according to procedures analogous to Example 1, Step 18, using 2-((6aS,8R)-8-((5-(hydroxymethyl)-3-methylpyridin-2-yl)oxy)-6a-methyl- 5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-2-yl)phenol (from Example 3, Step 1) and (S)-3-(1-oxo-6-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione (from Example 1, Step 17). LC-MS calc. for C40H44N9O5 [M+H]+: m/z = 730.3; Found: 730.2. Example 5. (S)-3-(6-(4-(6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinoyl)- piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione - 43 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[203] Step 1.6-(((6aS,8R)-2-Chloro-6a-methyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinic acid
[204] To a solution was added (6aS,8R)-2-chloro-6a-methyl-5,6,6a,7,8,9-hexahydropyrrolo- [1',2':4,5]pyrazino[2,3-c]pyridazin-8-ol (50 mg, 0.21 mmol) in DMF (0.50 mL) was added sodium hydride (15 mg, 0.62 mmol, 60% dispersion in mineral oil). The reaction mixture was stirred for 30 min at 40 °C. Methyl 6-fluoro-5-methylnicotinate (42 mg, 0.25 mmol) was added, and the reaction mixture was stirred at 40 °C for 18 h. The reaction was quenched with 10% citric acid (aq.) and extracted with DCM. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude material was purified by prep-HPLC (10.0–50.0% MeCN/0.1% TFA (aq)) to afford the title compound as a TFA salt (32 mg, 0.086 mmol, 41% yield), a yellow solid. LC-MS calc. for C17H19ClN5O3 [M+H]+: m/z = 376.1; Found: 376.2. [205] Step 2.6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9-hexahydropyrrolo- [1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinic acid
[206] The title compound was synthesized according to procedures analogous to Example 1, Step 11. LC-MS calc. for C23H24N5O4 [M+H]+: m/z = 434.2; Found: 434.1. [207] Step 3. (S)-3-(6-(4-(6-(((6aS,8R)-2-(2-Hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinoyl)piperazin-1-yl)-1- oxoisoindolin-2-yl)piperidine-2,6-dione - 44 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [208] To a solution of 6-(((6aS,8R)-2-(2-hydroxyphenyl)-6a-methyl-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinic acid (19 mg, 0.044 mmol) and (S)-3-(1-oxo-6-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione hydrochloride (21 mg, 0.057 mmol) in DCM (1 mL) was added 1-[bis(dimethylamino)-methylene]-1H-1,2,3- triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (20 mg, 0.053 mmol) and triethylamine (24 ^L, 0.18 mmol). The reaction mixture was stirred for 2 h. The reaction mixture was concentrated, dissolved in MeCN and DMSO, and purified by prep-HPLC (21.7–41.7% MeCN/0.2% TFA (aq)) to afford the title compound as a TFA salt (1.5 mg, 0.0017 mmol, 4.1% yield), a white solid. LC-MS calc. for C40H42N9O6 [M+H]+: m/z = 744.3; Found: 744.2. Example 6. (3S)-3-[5-[4-[[6-[[(4R,6S)-6-Ethyl-12-(2-hydroxyphenyl)-2,8,10,11- tetrazatricyclo[7.4.0.02,6]trideca-1(9),10,12-trien-4-yl]oxy]-5-methyl-3-pyridinyl] methyl]piperazin-1-yl]-3-oxo-1H-isoindol-2-yl]piperidine-2,6-dione
[209] Step 1.1-(tert-Butyl) 2-methyl (4R)-4-(benzyloxy)-2-ethylpyrrolidine-1,2-dicarboxylate
[210] To 1-(tert-butyl) 2-methyl (2R,4R)-4-(benzyloxy)pyrrolidine-1,2-dicarboxylate (2.00 g, 5.96 mmol) in THF (55 mL) was added LiHMDS (12 mL, 12 mmol) slowly at −78 °C. The reaction mixture was stirred at −78 °C for 1 h, after which ethyl iodide (1.9 mL, 23.6 mmol) was added. The reaction was stirred at −78 °C for 5 min then allowed to warm to room temperature with stirring. After 3.5 h, the reaction was quenched with MeOH (55 mL). A solution of NaOH (1.91 g, 47.7 mmol) in water (55 mL) was added and the mixture was stirred at room temperature for 20 min then poured into water. The aqueous layer was extracted with EtOAc (3 x). The combined organic layers were washed with brine (1 x) then concentrated and purified via SiO2 FCC (0-30% EtOAc in hexanes) to afford 1-(tert-butyl) 2-methyl (4R)-4-(benzyloxy)-2- - 45 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application ethylpyrrolidine-1,2-dicarboxylate (1.11 g, 51%). LCMS calcd for C15H22NO3 [M−Boc+2H]+: m/z = 264.2; Found: 264.0. [211] Step 2. (4R)-4-(Benzyloxy)-1-(tert-butoxycarbonyl)-2-ethylpyrrolidine-2-carboxylic acid
[212] To 1-(tert-butyl) 2-methyl (4R)-4-(benzyloxy)-2-ethylpyrrolidine-1,2-dicarboxylate (2.72 g, 7.48 mmol) in 1:1 THF/MeOH (40 mL) was added a solution of NaOH (2.99 g, 74.8 mmol) in water (20 mL). The reaction was stirred at 75 °C for 2.5 h then allowed to cool to room temperature. The mixture was poured into water then extracted with MTBE (3 x). The combined organic layers were washed with water (2x). The combined aqueous layers were extracted once more with MTBE then acidified to pH < 2 with 2 N HCl (aq) and subsequently extracted with EtOAc (3x). The combined EtOAc layers were washed with brine, dried with MgSO4, filtered then concentrated to give crude (4R)-4-(benzyloxy)-1-(tert-butoxycarbonyl)-2-ethylpyrrolidine- 2-carboxylic acid (assumed quantitative yield) as a yellow oil. LCMS calcd for C14H20NO3 [M−Boc+2H]+: m/z = 250.1; Found: 250.0. [213] Step 3. tert-Butyl (2R,4R)-4-(benzyloxy)-2-((4-bromo-6-chloropyridazin-3-yl)carbamoyl)- 2-ethylpyrrolidine-1-carboxylate and tert-butyl (2S,4R)-4-(benzyloxy)-2-((4-bromo-6-chloro- pyridazin-3-yl)carbamoyl)-2-ethylpyrrolidine-1-carboxylate
[214] To crude 1-(tert-butyl) 2-methyl (4R)-4-(benzyloxy)-2-ethylpyrrolidine-1,2-dicarboxylate (672 mg, 1.43 mmol) and N,N-diisopropylethylamine (1.21 mL, 6.92 mmol) in MeCN (10 mL) was added HATU (880 mg, 2.31 mmol). The reaction was stirred at room temperature for 20 min then concentrated. The residue was taken up in THF (8 mL) then 4-bromo-6-chloropyridazin-3- amine (802 mg, 3.85 mmol) was added, followed by NaH (60% dispersion in mineral oil; 385 - 46 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application mg, 9.62 mmol). After rinsing the sides of the flask with THF (2 mL), the reaction was stirred at room temperature for 50 min then cooled to 0 °C and quenched with sat. NH4Cl (aq). The mixture was poured into water then extracted with EtOAc (3 x). The combined organic layers were washed with brine (1 x) then concentrated. The residue was purified via SiO2 FCC (0-100% EtOAc in hexanes) to afford “diastereomer A” tert-butyl (2R,4R)-4-(benzyloxy)-2-((4-bromo-6- chloropyridazin-3-yl)carbamoyl)-2-ethylpyrrolidine-1-carboxylate (473 mg, 46%) and “diastereomer B” tert-butyl (2S,4R)-4-(benzyloxy)-2-((4-bromo-6-chloropyridazin-3- yl)carbamoyl)-2-ethylpyrrolidine-1-carboxylate (286 mg, 28%). LCMS calcd for C23H29BrClN4O4 [M+H]+: m/z = 539.1; Found: 539.0 (diastereomer A) and 539.0 (diastereomer B). [215] Step 4. (6aR,8R)-8-(Benzyloxy)-2-chloro-6a-ethyl-6a,7,8,9-tetrahydropyrrolo[1',2':4,5] pyrazino[2,3-c]pyridazin-6(5H)-one and (6aS,8R)-8-(benzyloxy)-2-chloro-6a-ethyl-6a,7,8,9- tetrahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-6(5H)-one
[216] To tert-butyl (2R,4R)-4-(benzyloxy)-2-((4-bromo-6-chloropyridazin-3-yl)carbamoyl)-2- ethylpyrrolidine-1-carboxylate (473 mg, 0.88 mmol) in DCM (17 mL) was added TFA (17 mL, 222 mmol). The reaction was stirred at room temperature for 3 h then concentrated. The residue was dissolved in MeCN (15 mL). N,N-Diisopropylethylamine (0.76 mL, 4.38 mmol) was added and the reaction was stirred at 80 °C for 2 h then allowed to cool to room temperature overnight. The reaction was concentrated to afford crude (6aR,8R)-8-(benzyloxy)-2-chloro-6a-ethyl- 6a,7,8,9-tetrahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-6(5H)-one (Intermediate 8; assumed quantitative yield) which was used directly without further purification. LCMS calcd for C18H20ClN4O2 [M +H]+: m/z = 359.1; Found: 359.1. [217] Step 5. (6aS,8R)-8-(Benzyloxy)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo- [1’,2’:4,5]pyrazino[2,3-c]pyridazine
- 47 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [218] To a solution of (6aS,8R)-8-(benzyloxy)-2-chloro-6a-ethyl-6a,7,8,9-tetrahydropyrrolo- [1’,2’:4,5]pyrazino[2,3-c]- 48 -yridazine-6(5H)-one (1.34 g, 3.73 mmol) in THF (30 mL) was added borane dimethyl sulfide complex (3.54 mL, 37.3 mmol) at 0 °C. The resulting mixture was stirred at 50 °C for 1 h and then was cooled to 0 °C. MeOH (3 mL) was added slowly, and the reaction mixture was stirred at room temperature for 10 min. The mixture was concentrated and diluted with ethanol (30 mL). Sodium cyanoborohydride (1.17 g, 18.7 mmol) and acetic acid (2.14 mL, 37.3 mmol) were added, and the reaction mixture was stirred at 80 °C for 4 h. The reaction was quenched with sat. NaHCO3 (aq.) (30 mL), and the mixture was diluted with EtOAc (30 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (30 mL x 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to afford the title compound (1.29 g, 3.74 mmol, 99% yield) as a yellow solid. LC- MS calc. for C18H22ClN4O [M+H]+ m/z= 345.2/347.2; Found 345.4/347.5. [219] Step 6. (6aS,8R)-2-Chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-ol
[220] To a solution of (6aS,8R)-8-(benzyloxy)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazine (1.45 g, 4.20 mmol) in DCM (30 mL) at –78 °C was added trichloroborane (1 N in DCM, 12.6 mL, 12.6 mmol) dropwise. The resulting mixture was slowly warmed to room temperature and stirred for 30 min. Sat. NaHCO3 (aq.) (50 mL) was added, and the reaction mixture was diluted with DCM (20 mL). The layers were separated, and the aqueous layer was extracted with CHCl3/IPA (3:1) (50 mL x 4). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was slurried in MTBE (100 mL) and collected by filtration to afford the title compound (1.01 g, 3.93 mmol, 93.4% yield) as a white solid.1H NMR (300 MHz, DMSO-d6) δ 7.01 (d, J = 4.3 Hz, 1H), 6.26 (d, J = 0.9 Hz, 1H), 5.25 (d, J = 3.4 Hz, 1H), 4.45 (q, J = 6.0, 5.1 Hz, 1H), 3.52 (ddd, J = 16.1, 11.7, 5.4 Hz, 2H), 3.28 – 3.11 (m, 1H), 2.61 (dd, J = 11.6, 1.2 Hz, 1H), 2.00 (dd, J = 13.3, 1.9 Hz, 1H), 1.86 – 1.68 (m, 1H), 1.68 – 1.57 (m, 1H), 1.55 – 1.41 (m, 1H), 0.82 (t, J = 7.4 Hz, 3H). LC-MS calc. for C11H16ClN4O [M+H]+ m/z= 255.1/257.1; Found 255.3/257.3. [221] Step 7. (6aS,8S)-2-Chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]pyrazin[2,3- c]pyridazin-8-yl 4-nitrobenzoate - 48 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[222] To a solution of (6aS,8R)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-ol (290 mg, 1.14 mmol) in THF (6 mL) was added triphenyl- phosphine (597 mg, 2.28 mmol) and 4-nitrobenzoic acid (381 mg, 2.28 mmol). The resulting mixture was stirred for 5 min, and then diisopropyl azodicarboxylate (0.451 mL, 2.28 mmol) was added dropwise. The resulting mixture was stirred for 1 h. The reaction mixture was concentrated, and the crude material was purified by silica gel chromatography (0–70% EtOAc/heptanes) to afford the title compound (430 mg, 1.06 mmol, 93.5% yield) as a white solid. LC-MS calc. for C18H19ClN5O4 [M+H]+ m/z= 404.1/406.1; Found 404.4/406.4. [223] Step 8. (6aS,8S)-2-Chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]pyrazino- [2,3-c]pyridazin-8-ol
[224] To a solution of (6aS,8S)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-yl 4-nitrobenzoate (430 mg, 1.06 mmol) in methanol (4 mL) and THF (4 mL) was added potassium carbonate (442 mg, 3.19 mmol). The resulting mixture was stirred overnight. The reaction mixture was concentrated, and 2 N HCl (aq.) (20 mL) and EtOAc (20 mL) were added. The layers were separated, and the aqueous layer was extracted with EtOAc (20 mL). The combined organic layers were discarded, and the aqueous layer was adjusted to pH 7–8 with solid sodium bicarbonate. The resulting solution was extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to afford the title compound (220 mg, 0.864 mmol, 81.1% yield) as a white solid. LC-MS calc. for C11H16ClN4O [M+H]+ m/z= 255.1/257.1; Found 255.5/257.3. [225] Step 9.6-Hydroxy-5-methylnicotinic acid
[226] To a solution of 6-fluoro-5-methylnicotinic acid (1.0 g, 6.5 mmol) in water (20 mL) was added sodium hydroxide (1.29 g, 32.2 mmol). The resulting mixture was stirred at 90 °C overnight. The reaction mixture was cooled to room temperature, and the mixture was adjusted to - 49 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application pH 3–4 with 1 N HCl (aq.), producing a white precipitate. The solid was collected by filtration and co-evaporated with toluene (2x) to afford the title compound (930 mg, 6.0 mmol, 93% yield) as a white solid. LC-MS calc. for C7H8NO3 [M+H]+ m/z= 154.0; Found 154.1. [227] Step 10. Methyl 6-hydroxy-5-methylnicotinate
[228] To a solution of 6-hydroxy-5-methylnicotinic acid (930 mg, 6.0 mmol) in methanol (20 mL) was added sulfuric acid (0.32 mL, 6.0 mmol). The reaction mixture was stirred at reflux overnight. The mixture was cooled to room temperature, and the resulting solid was collected by filtration, washed with MeOH, and concentrated to afford the title compound (610 mg, 3.7 mmol, 61% yield) as a white solid. LC-MS calc. for C8H10NO3 [M+H]+ m/z= 168.1; Found 168.2. [229] Step 11. Methyl 6-(((6aS,8R)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinate
[230] To a solution of (6aS,8S)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-ol (45.5 mg, 0.179 mmol) in THF (2 mL) was added triphenyl- phosphine (93.7 mg, 0.357 mmol) and methyl 6-hydroxy-5-methylnicotinate (59.7 mg, 0.357 mmol). The reaction mixture was stirred for 10 min at room temperature. Diisopropyl azodicarboxylate (0.0703 mL, 0.357 mmol) was added at 0 °C, and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated, and the crude material was purified by silica gel chromatography (20–100% EtOAc/heptanes) to afford the title compound (70 mg, 0.17 mmol, 97% yield) as a colorless oil. LC-MS calc. for C19H23ClN5O3 [M+H]+ m/z= 404.1/406.1; Found 404.2/406.4. [231] Step 12: (6-(((6aS,8R)-2-Chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylpyridin-3-yl)methanol
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105807.001076 – PCT Application [232] To a solution of methyl 6-(((6aS,8R)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo- [1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinate (70.0 mg, 0.173 mmol) in THF at –78 °C (3 mL) was added lithium diisobutyl-tert-butoxyaluminum hydride (2.5 N in THF/hexanes, 3.47 mL, 0.867 mmol) slowly. The resulting mixture was stirred at –78 °C for 10 min, then slowly warmed to room temperature, and stirred for 30 min. The reaction was quenched with MeOH (2 mL), and the reaction mixture was stirred for 30 min. The reaction mixture was diluted with water (20 mL) and EtOAc (20 mL). The layers were separated, and the aqueous layer was extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to afford the title compound (61.0 mg, 0.162 mmol, 93.6% yield) as a white solid. LC-MS calc. for C18H23ClN5O2 [M+H]+ m/z= 376.2/378.2; Found 376.4/378.5. [233] Step 13: 6-(((6aS,8R)-2-Chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinaldehyde
[234] To a solution of (6-(((6aS,8R)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylpyridin-3-yl)methanol (61.0 mg, 0.160 mmol) in CHCl3 (5 mL) was added manganese(IV) oxide (282 mg, 3.25 mmol). The reaction mixture was stirred at 50 oC for 2 h. The mixture was cooled to room temperature, filtered through Celite, and concentrated. The crude material was further purified by silica gel chromatography (20–100% EtOAc/heptanes) to afford the title compound (37.0 mg, 0.0990 mmol, 61.0% yield) as a white solid. LC-MS calc. for C18H21ClN5O2[M+H]+ m/z= 374.1/376.1; Found 374.2/376.2. [235] Step 14: 6-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9-hexahydropyrrolo- [1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinaldehyde
[236] To a solution of 6-(((6aS,8R)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinaldehyde (37.0 mg, 0.0990 mmol) in 1,4- dioxane (2 mL) and water (0.10 mL) was added 2-hydroxyphenylboronic acid (41.0 mg, 0.297 mmol), XPhos-Pd-G2 (7.8 mg, 0.0099 mmol, CAS 1310584-14-5), and cesium acetate (76.0 mg, - 51 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application 0.396 mmol). The reaction mixture was sparged with N2 for 10 min and stirred at 95 °C for 2 h. The reaction was cooled to room temperature and concentrated. The crude material was diluted with DMSO (3 mL) and centrifuged. The liquid was collected and directly purified by prep- HPLC (10.0–60.0% MeCN/0.1% TFA (aq)) to afford the title compound as a TFA salt (32.0 mg, 0.0742 mmol, 74.9% yield), a brown solid. LC-MS calc. for C24H26N5O3 [M+H]+ m/z= 432.2; Found 432.3. [237] Step 15: (S)-3-(6-(4-((6-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylpyridin-3-yl)methyl)- piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione [238] To a solution of (S)-3-(1-oxo-6-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione (24.4 mg, 0.0742 mmol) in DMSO (2 mL) was added 6-(((6aS,8R)-6a-ethyl-2-(2-hydroxyphenyl)- 5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotin- aldehyde (16.0 mg, 0.0371 mmol) and N,N-diisopropylethylamine (0.0646 mL, 0.371 mmol). After stirring for 20 min, sodium triacetoxyborohydride (31.4 mg, 0.148 mmol) was added. The reaction mixture was stirred at 40 °C overnight and then directly purified by prep-HPLC (10– 60% MeCN/0.1% TFA (aq)) to afford the title compound as a TFA salt (8.7 mg, 0.012 mmol, 32% yield), a white solid.1H NMR (300 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.24 (s, 1H), 8.07 – 7.86 (m, 2H), 7.59 (d, J = 2.2 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.37 – 7.13 (m, 4H), 6.98 (s, 1H), 6.85 - 6.81 (m, 2H), 5.80 (t, J = 6.3 Hz, 1H), 5.11 (dd, J = 13.3, 5.1 Hz, 1H), 4.35 (d, J = 16.8 Hz, 1H), 4.22 (d, J = 16.8 Hz, 1H), 4.03 (dd, J = 13.1, 6.2 Hz, 1H), 3.73 – 3.49 (m, 8H), 3.23 - 3.20 (m, 3H), 2.97 – 2.81 (m, 2H), 2.65 – 2.56 (m, 2H), 2.45 – 2.30 (m, 2H), 2.19 (s, 3H), 2.08 – 1.94 (m, 2H), 1.87 (dd, J = 13.8, 7.3 Hz, 1H), 1.68 (dd, J = 13.8, 7.2 Hz, 1H), 1.49 - 1.44 (m, 1H), 0.91 (q, J = 7.0 Hz, 3H). LC-MS calc. for C41H46N9O5 [M+H]+ m/z= 744.4; Found 746.6. Example 7. (S)-3-(6-(4-(6-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-5-methylnicotinoyl) piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione
- 52 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [239] The title compound was synthesized according to procedures analogous to Example 5, Steps 1–3, using (6aS,8R)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]pyrazino- [2,3-c]pyridazin-8-ol (from Example 6, Step 6). LC-MS calc. for C41H44N9O6 [M+H]+: m/z = 758.3; Found: 758.2. Example 8. (S)-3-(6-(4-((5-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-6-methylpyrazin-2- yl)methyl)piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione
[240] Step 1: 5-(((6aS,8R)-2-Chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-yl)oxy)-6-methylpyrazine-2-carbonitrile
[241] To a solution of (6aS,8S)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-ol (55.0 mg, 0.216 mmol) in THF (2 mL) was added triphenyl- phosphine (113 mg, 0.432 mmol) and 5-hydroxy-6-methylpyrazine-2-carbonitrile (58.4 mg, 0.432 mmol). The reaction mixture was stirred for 10 min, then diisopropyl azodicarboxylate (0.0850 mL, 0.432 mmol) was added at 0 °C. The reaction mixture was stirred at room temperature for 3 h, then concentrated. The crude product was purified by silica gel chromatography (20–100% EtOAc/heptanes) to afford the title compound (80.0 mg, 0.215 mmol, 99.6% yield) as a colorless oil. LC-MS calc. for C17H19ClN7O [M+H]+ m/z= 372.1/374.1; Found 372.4/374.5. [242] Step 2: 5-(((6aS,8R)-2-Chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-yl)oxy)-6-methylpyrazine-2-carbaldehyde - 53 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[243] To a solution of 5-(((6aS,8R)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-yl)oxy)-6-methylpyrazine-2-carbonitrile (60.0 mg, 0.161 mmol) in DCM (2 mL) was added diisobutylaluminum hydride, (1 N in heptane, 0.968 mL, 0.968 mmol). The reaction mixture was stirred at –78 °C for 1 h, and then the reaction was quenched with MeOH (1 mL) at –78 °C. The resulting mixture was warmed to room temperature and stirred overnight. The resulting mixture was filtered through a pad of Celite and concentrated. The crude material was purified by silica gel chromatography (20–100% EtOAc/heptane) to afford the title compound (11.2 mg, 0.0299 mmol, 18.5% yield) as a colorless oil. LC-MS calc. for C17H20ClN6O2 [M+H]+ m/z= 375.1/377.1; found 375.5/377.4. [244] Step 3.5-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9-hexahydropyrrolo- [1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-6-methylpyrazine-2-carbaldehyde
[245] The title compound was synthesized according to procedures analogous to Example 6, Step 14. LC-MS calc. for C23H25N6O3 [M+H]+ m/z= 433.2; Found 433.6. [246] Step 4. (S)-3-(6-(4-((5-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)-6-methylpyrazin-2-yl)methyl)piperazin-1- yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione [247] The title compound was synthesized according to procedures analogous to Example 6, Step 15, using (S)-3-(1-oxo-6-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione (from Example 1, Step 17). LC-MS calc. for C40H45N10O5 [M+H]+ m/z= 745.4; Found 745.8. Example 9. (S)-3-(6-(4-((6-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3-yl)methyl) piperazin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione - 54 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application
[248] Step 1. Methyl 6-(((6aS,8R)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5- ]pyrazino[2,3-c]pyridazin-8-yl)oxy)nicotinate
[249] To a solution of (6aS,8R)-2-chloro-6a-ethyl-5,6,6a,7,8,9-hexahydropyrrolo[1',2':4,5]- pyrazino[2,3-c]pyridazin-8-ol (60.0 mg, 0.236 mmol) in THF (1 mL) was added sodium hydride (37.8 mg, 0.942 mmol, 60% dispersion in mineral oil) at 0 °C. The reaction mixture was stirred at 0 °C for 5 min, then methyl 6-fluoronicotinate (43.8 mg, 0.283 mmol) was added, and the mixture was stirred at room temperature for 2 h. The reaction was quenched with sat. NH4Cl (aq.) (20 mL), and the reaction mixture diluted with EtOAc (20 mL). The aqueous layer was adjusted to pH 3–4 with 1 N HCl (aq.) then extracted with EtOAc (20 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude material was dissolved in MeOH (5 mL), and sulfuric acid (63 µL, 1.2 mmol) was added. The resulting mixture was stirred at reflux overnight. The reaction mixture was cooled to room temperature and sat. NaHCO3 (aq.) was added slowly. The mixture was concentrated, and the resulting aqueous solution was extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The crude material was purified by silica gel chromatography (20–100% EtOAc/heptane) to afford the title compound (59.1 mg, 0.152 mmol, 64.1% yield) as a white solid. LC-MS calc. for C18H21ClN5O3 [M+H]+: m/z = 390.1/392.1; Found 390.4/392.2. [250] Step 2. (S)-3-(6-(4-((6-(((6aS,8R)-6a-Ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9-hexahydro- pyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3-yl)methyl)piperazin-1-yl)-1- oxoisoindolin-2-yl)piperidine-2,6-dione [251] The title compound was synthesized according to procedures analogous to Example 6, Steps 12-15.1H NMR (300 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.22 (br, 1H), 8.16 (d, J = 2.3 Hz, - 55 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application 1H), 7.93 (dd, J = 8.3, 1.6 Hz, 1H), 7.74 (dd, J = 8.4, 2.4 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.34 – 7.12 (m, 4H), 6.99 (s, 1H), 6.89 - 6.81 (m, 3H), 5.81 – 5.64 (m, 1H), 5.11 (dd, J = 13.2, 5.1 Hz, 1H), 4.35 (d, J = 16.8 Hz, 1H), 4.21 (d, J = 16.7 Hz, 1H), 4.05 (dd, J = 12.9, 6.4 Hz, 1H), 3.76 – 3.55 (m, 8H), 3.24 - 3.20 (m, 3H), 2.87 (dd, J = 16.2, 11.9 Hz, 2H), 2.42 – 2.33 (m, 2H), 2.00 (dt, J = 7.7, 4.6 Hz, 3H), 1.81 (dd, J = 13.8, 7.5 Hz, 1H), 1.63 (dd, J = 13.8, 7.2 Hz, 1H), 1.46 (s, 1H), 0.88 (t, J = 7.4 Hz, 3H). LC-MS calc. for C40H44N9O5 [M+H]+: m/z = 730.3; Found 730.4. Example 10. (S)-3-(6-(1-((6-(((6aS,8R)-6a-ethyl-2-(2-hydroxyphenyl)-5,6,6a,7,8,9- hexahydropyrrolo[1',2':4,5]pyrazino[2,3-c]pyridazin-8-yl)oxy)pyridin-3-yl)methyl) piperidin-4-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione
[252] The title compound was synthesized according to procedures analogous to Example 9, Steps 1–2, using (S)-3-(1-oxo-6-(piperidin-4-yl)isoindolin-2-yl)piperidine-2,6-dione (from Example 3, Step 8). LC-MS calc. for C41H45N8O5 [M+H]+: m/z = 729.3; Found 729.3. Example A. SMARCA2 HiBiT and SMARCA4 HiBiT Degradation Assay Preparation of SMARCA2/4-HiBiT knock-in cells [253] HiBiT peptide knock-in of SMARCA2 in LgBiT expressing HEK293T cells was performed by CRISPR-mediated tagging system as described Promega. The homozygous HiBiT knock-in on c-terminus SMARCA2 was confirmed by sanger sequence. SMARCA2-HiBiT knock-in Hela monoclonal cell (CS302366) and SMARCA4-HiBiT knock-in Hela monoclonal cell (CS3023226) were purchased from Promega. The heterozygous HiBiT-knock-in was confirmed by sanger sequence in both SMARCA2-HiBiT and SMARCA4-HiBiT monoclonal cells. SMARCA2 HiBiT and SMARCA4 HiBiT degradation assay in HeLa cells [254] Dispense 10ul aliquot of prepared Hela-SMARCA2-HiBiT or Hela-SMARCA4-HiBiT cells (1:1 ratio of cells:Trypan Blue (#1450013, Bio-Rad)) onto cell counting slide (#145-0011, Bio-Rad) and obtain cell density and cell viability using cell counter (TC20, Bio-Rad). Remove - 56 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application appropriate volume of resuspended cells from culture flask to accommodate 2500 cells/well @ 20 ^L/well. Transfer Hela-HiBiT cells to 50 mL conical (#430290, Corning). Spin down at 1000 rpm for 5 min using tabletop centrifuge (SPINCHRON 15, Beckman). Discard supernatant and resuspend cell pellet in modified EMEM (#30-2003, ATCC) cell culture media containing 10% FBS (F2422-500ML, Sigma), and 1X Penicillin/Streptomycin (200g/L) (30-002-CI, Corning) to a cell density of 125,000 cells/mL. Dispense 20 ^Lof resuspended Hela-HiBit cells per well in 384-well TC treated plate (#12-565-343, Thermo Scientific) using standard cassette (#50950372, Thermo Scientific) on Multidrop Combi (#5840310, Thermo Scientific) inside laminar flow cabinet. [255] Compounds were dissolved in DMSO to make 10 mM stock and 3-fold series dilutions were further conducted keeping the highest concentration 10 µM. Dispense test compounds onto plates using digital liquid dispenser (D300E, Tecan). Incubate plates in humidified tissue culture incubator @37̊ °C for 18 hours. Add 20 ^L of prepared Nano-Glo® HiBiT Lytic detection buffer (N3050, Promega) to each well of 384-well plate using small tube cassette (#24073295, Thermo Scientific) on Multidrop Combi, incubate @ RT for 30-60 min. Read plates on microplate reader (Envision 2105, PerkinElmer) using 384 well Ultra-Sensitive luminescence mode. Raw data files and compound information reports are swept into centralized data lake and deconvoluted using automated scripts designed by TetraScience, Inc. Data analysis, curve-fitting and reporting done in Dotmatics Informatics Suite using Screening Ultra module. Results are summarized below in Table 1. Table 1. SMARCA2 and SMARCA4 Degradation
[256] In Table 1, columns DC50, an “A” denotes DC50 < 0.1 nM; a “B” denotes 0.1 nM ≤ DC50 < 1 nM; a “C” denotes DC50 > 1.0 nM. In Table 1, columns Dmax, an “A” denotes Dmax > 90%; a “B” denotes 50% < Dmax ≤ 90%; a “C” denotes that a Dmax ≤ 50%. - 57 - 105807.001076 4919-6324-9437.1
105807.001076 – PCT Application [257] While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example. - 58 - 105807.001076 4919-6324-9437.1