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WO1993020813A1 - Derives de 1,3,-propanediamine ayant une activite d'inhibition de la proteine kinase c - Google Patents

Derives de 1,3,-propanediamine ayant une activite d'inhibition de la proteine kinase c Download PDF

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Publication number
WO1993020813A1
WO1993020813A1 PCT/US1993/003177 US9303177W WO9320813A1 WO 1993020813 A1 WO1993020813 A1 WO 1993020813A1 US 9303177 W US9303177 W US 9303177W WO 9320813 A1 WO9320813 A1 WO 9320813A1
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alkyl
independently
compound
alkynyl
alkenyl
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PCT/US1993/003177
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English (en)
Inventor
Jeffrey B. Nichols
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Sphnix Pharmaceuticals Corporation
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Priority to AU40464/93A priority Critical patent/AU4046493A/en
Publication of WO1993020813A1 publication Critical patent/WO1993020813A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole

Definitions

  • the present invention relates to compositions for diagnosis and treatment of inflammatory, cardiovascular and neoplastic diseases. More particularly, the present invention relates to compositions containing
  • 1,3-propanediamine derivatives for inhibition of the activity of the enzyme protein kinase C.
  • Protein kinase C is a family of calcium- and phospholipid-dependent serine/threonine-specific protein kinases which play an important role in cellular growth control, regulation, and differentiation. Protein kinase C is also fundamental to the processes involved in tumorigenicity, since it is the major high-affinity receptor for several classes of tumor promoters as well as for endogenous cellular diacylglycerols. These tumor promoters also stimulate protein kinase C catalysis. Castagna, et al . , J. Biol . Chem . , 1982, 257 , 7847, reported direct activation of protein kinase C by tumor promoting phorbol esters.
  • Protein kinase C is activated by diacylglycerol (DAG) , a neutral lipid, and when activated will transfer the 7-phosphate of MgATP to a serine or threonine residue on a substrate protein.
  • DAG diacylglycerol
  • protein kinase C Since the activation of protein kinase C has been implicated in several human disease processes, including 5 cancer tumors, inflammation, and reperfusion injury, inhibition of protein kinase C should be of great therapeutic value in treating these conditions.
  • protein kinase C inhibitors have been reported to potentiate the antitumor activity of cis-platin 0 both in vitro and in vivo. See, Grunicke, et al . , Adv. Enzyme Regul . , 1989, 28 , 201, and German Offenlegungsschrift DE 3827974.
  • protein kinase C would be a potential target for therapeutic design because of its central role in cell 5 growth. See, Tritton, et al.. Cancer Cells, 1990, 2, 95-102.
  • inflammation and reperfusion injury particularly pertaining to cardiac injury, are common conditions for which there exists no definitive treatment despite extensive research, and appropriate treatments for 0 these conditions are needed.
  • Certain protein kinase C inhibitors have been demonstrated to block platelet aggregation and release of neutrophil activating agents such as platelet activating factor, PAF. See, Schachtele, et al . , Biochem, Biophy. Res .
  • Protein kinase C inhibitors have also been shown to inhibit neutrophil activation, and chemotactic migration. See, Mclntyre, et al . , J. Biol . Chem. , 1987,
  • inhibitors of protein kinase C have the potential for blocking all three of the most significant mechanisms of pathogenesis associated with myocardial reperfusion injury, and should thus have a 0 decided therapeutic advantage. Additionally, the inhibitory effect of protein kinase C inhibitors on keratinocytes, and on the oxidative burst in neutrophils will lead to an anti-inflammatory effect.
  • German Offenlegungsschrift DE 3827974 Al 5 discloses therapeutic preparations comprising a protein kinase C inhibitor in combination with a lipid, a lipid analog, a cytostatic agent or phospholipase inhibitor useful for cancer therapy.
  • N,N,N , -trimethyl-N'-alkyl-l,3-propanediamines have been 0 reported for antifungal and antimicrobial uses, and as cosmetic components and fabric softeners.
  • compositions comprising a pharmaceutically acceptable carrier or diluent and: (a) a compound having the formula (I) 1
  • R 1f R 2 and R 3 are, independently, C 1 to about C 20 alkyl, C 1 to about C 20 alkenyl, or C, to about C 20 alkynyl; and R 4 is C 8 to about C 20 alkyl; or
  • compositions of the invention may also comprise at least one compound having formula I and at least one compound having formula II.
  • compositions of the invention inhibit protein kinase C and exert anti-inflammatory, anti-cancer, and reperfusion injury protection effects through their anti-proliferative and anti-inflammatory activities in human neutrophils and tumor cells. Accordingly, the present invention also provides novel methods useful for treating conditions related to or affected by inhibition of protein kinase C activity, particularly cancer tumors, inflammatory disease, reperfusion injury, and cardiac dysfunctions related to reperfusion injury.
  • the present invention provides 1,3-propanediamines and pharmaceutically acceptable salts thereof that have protein kinase C inhibiting activity and that exert anti-inflammatory, anti-cancer, and reperfusion injury protection effects through their anti-proliferative and anti-inflammatory activities in human neutrophils and tumor cells.
  • the compounds and pharmaceutical compositions of the invention are useful for treating conditions related to or affected by inhibition of protein kinase C activity, particularly cancer tumors, inflammatory disease, reperfusion injury, and cardiac dysfunctions related to reperfusion injury.
  • the compounds useful in the methods of the invention are selective for protein kinase C and have no effect on cyclic AMP (cAMP) dependent protein kinase activity.
  • cAMP cyclic AMP
  • the compounds useful in the invention should thus have no effect on the metabolic pathways associate with stimulation of protein kinase by cAMP.
  • Compounds useful in the invention inhibit tumor cell proliferation and are not cross-resistant to the multi-drug-resistant family of agents such as adriamycin.
  • compositions according to the invention comprise a pharmaceutically acceptable carrier or diluent and a compound of formula I:
  • R 3 are preferably, i independently, C, to about C 20 alkyl, C, to about C 20 alkenyl, or C, to about C 20 alkynyl.
  • R 1f R 2 and R 3 more preferably are, independently, C, to about C 8 alkyl, C, to about C 8 alkenyl, or C, to about C 8 alkynyl, even more preferably methyl or ethyl.
  • R 4 preferably is C 8 to about C 20 alkyl. More preferably R 4 is C 12 to about C 16 alkyl.
  • compositions of the invention comprise a pharmaceutically acceptable carrier or diluent and a compound having the formula (II) :
  • R s and R 6 preferably are, independently, C 1 to about C 5 alkyl, C, to about C 5 alkenyl, or c t to about C 5 alkynyl.
  • R j and R 6 are more preferably, independently, C, to about C 5 alkyl, even more preferably C, to C j alkyl.
  • R 7 preferably is C 8 to about C 20 alkyl.
  • R 7 more preferably is C 12 to C 20 alkyl, even more preferably C 12 to C 18 alkyl.
  • compositions of the invention comprise a compound of formula I and a compound of formula II, along with a pharmaceutically acceptable carrier or diluent. Accordingly, the present invention provides methods for inhibiting protein kinase c which comprise contacting protein kinase C with an inhibitory amount of a compound of formula I or Formula II, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention.
  • Another aspect of the invention provides methods of inhibiting an oxidative burst in neutrophils, comprising contacting a neutrophil with an amount of a compound of formula I or formula II, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention, said amount being effective either to inhibit such oxidative burst or to inhibit protein kinase C.
  • protein kinase C inhibitory amount refers to an amount of a compound that will inhibit protein kinase C activity, reduce the amount of histone 1 that is phosphorylated by protein kinase C, or inhibit the activity of protein kinase C by any other measure of protein kinase C activity.
  • the invention also provides methods for treating inflammation which comprise administering to a mammal suffering from inflammation an amount of a compound of formula I or formula II, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention, said amount being effective to inhibit inflammation or to inhibit protein kinase C.
  • Another aspect of the invention provides methods for inhibiting growth of mammalian tumor cells which comprise contacting a mammalian tumor cell with an amount of a compound of formula I or formula II, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention, said amount being effective to inhibit the growth of the tumor cell or to inhibit protein kinase C.
  • Yet another aspect of the invention provides methods for treating mammalian tumors which comprise administering to a mammal having a tumor an amount of a compound of formula I or formula II, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention, said amount being effective to inhibit growth of the tumor or to inhibit protein kinase C.
  • Still another aspect of the invention provides methods of inhibiting keratinocyte proliferation comprising administering to a keratinocyte an amount of a compound of formula I or formula II, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the invention, said amount being effective to inhibit proliferation of the keratinocyte or to inhibit protein kinase C.
  • the pharmaceutical compositions and compounds of the invention may be administered by any method that produces contact of the active ingredient with the agent's site of action in the body of a mammal, or in the body fluid or tissue including but not limited to oral, topical, hypodermal, intramuscular, intravenous, and intraparenteral.
  • the compounds and pharmaceutical compositions may be administered singly or in combination with other compounds of the invention, other pharmaceutical compounds, such as chemotherapeutic compounds, or in conjunction with other therapies, such as radiation treatment.
  • 1,3-Propanediamine derivatives useful in the methods of the invention are preferably administered with a pharmaceutically acceptable carrier or diluent selected on the basis of the selected route of administration and standard pharmaceutical practice.
  • the compounds and pharmaceutical compositions are administered to mammals, preferably humans, in therapeutically effective amounts to inhibit protein kinase c, to inhibit tumor cell growth, to inhibit inflammation of tissue, to inhibit keratinocyte proliferation, to inhibit oxidative burst from neutrophils or to inhibit platelet aggregation.
  • the dosage administered in any particular instance will depend upon factors such as the pharmacodynamic characteristics of the particular compound, its mode and route of administration, the age, health, and weight of the recipient, the nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • Pharmaceutically acceptable salts of the compounds and pharmaceutical compositions are also within the scope of the invention.
  • Pharmaceutically acceptable salts include hydrochlorides, hydrobromides, succinates, fumarates, oxalates, methanesulfonates, sulfates, maleates, malonates, acetates or lactates.
  • the daily dosage of the compounds or active ingredient of the pharmaceutical compositions will be in the range of from about 0.1 to about 40 g per kg of body weight, preferably from about 1 to about 20 mg per I;g body weight.
  • the pharmaceutical compositions of the invention may be administered in any dosage form, including a single dosage, divided dosages, or in sustained release form. Persons of ordinary skill will be able to determine dosage forms and amounts with only routine experimentation based upon the considerations of the invention. Isomers of the compounds and pharmaceutical compositions, particular optically active stereoisomers, are also within the scope of the present invention.
  • the compounds and pharmaceutical compositions of the invention may also be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. They may also be administered parenterally in sterile liquid dosage forms or topically in a carrier.
  • the pharmaceutical compositions of the invention may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. See, Remington's Pharmaceutical Sciences, A. Osol, Mack Publishing Company, Easton, Pennsylvania.
  • the pharmaceutical compositions of the invention can be prepared by mixing a compound of formula I or formula II or both with pharmaceutically acceptable powdered carriers, such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, and stearic acid for insertion into gelatin capsules, or for forming into tablets.
  • pharmaceutically acceptable powdered carriers such as lactose, sucrose, mannitol, starch, cellulose derivatives, magnesium stearate, and stearic acid for insertion into gelatin capsules, or for forming into tablets.
  • Both tablets and capsules may be manufactured as sustained release products for continuous release of medication over a period of hours.
  • Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere or enteric coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration may contain coloring and flavoring to increase patient acceptance, in addition to a pharmaceutically acceptable diluent such as water, buffer or saline solution.
  • a pharmaceutically acceptable diluent such as water, buffer or saline solution.
  • compounds useful in the invention may be mixed with a suitable carrier or diluent such as water, an oil, saline solution, aqueous dextrose (glucose) , and related sugar solutions, and glycols such as propylene glycol or polyethylene glycols.
  • Solutions for parenteral administration contain preferably a water soluble salt of a compound useful in the invention.
  • Stabilizing agents, antioxidizing agents and preservatives may also be added.
  • Suitable antioxidizing agents include sodium bisulfite, sodium sulfite, and ascorbic acid, citric acid and its salts, and sodium EDTA.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol.
  • Cancer is a disease characterized in part by uncontrolled cell growth. Protein kinase C is directly involved in cellular growth control and is believed to be involved in tumor formation. Protein kinase C is the major, if not exclusive, intracellular receptor of phorbol esters, which are very potent tumor promoters. Phorbol esters and other tumor promoters bind to and activate protein kinase C. Since diacylglyerol (DAG) and phorbol esters interact at the same site, DAG's have been suggested to be the "endogenous phorbol esters" by analogy with the opiate receptor, where the conservation of a high affinity receptor implied the existence of an endogenous analogue.
  • DAG diacylglyerol
  • DAG has been shown to increase the affinity of protein kinase C for Ca +2 and phospholipid and thus activates protein kinase C at cellular levels of these essential cofactors.
  • Extracellular signals including hormones, growth factors, and neurotransmitters are known to stimulate phosphatidylinositol turnover resulting in the generation of IP3 and DAG.
  • Structures of 40 distinct oncogenes of viral and cellular origin have revealed that oncogenes encode altered forms of normal cellular proteins.
  • Several of the gene products appear related to growth factors or other elements involved in transmembrane signalling. These oncogene products appear to function by altering the level of critical second messengers.
  • PMA protein kinase C
  • oncogenes such as ras
  • c-fos c-cis
  • c-fms nuclear proto- oncogenes important in cell transformation.
  • ischemic-related myocardial damage can be attributed to polymorphonuclear leukocytes (neutrophils) which accumulate at the site of occlusion. Damage from the accumulated neutrophils may be due to the release of proteolytic enzymes from the activated neutrophils or the release of reactive oxygen intermediated (ROI) .
  • ROI reactive oxygen intermediated
  • Much of the "no reflow" phenomenon associated with myocardial ischemia is attributed to myocardial capillary plugging. The plugging of capillaries has been attributed to both aggregated platelets and aggregated neutrophils. Although both cell types are aggregated during the ischemic event, the relative contribution of each to capillary plugging has not yet been established.
  • SOD Superoxide dismutase
  • SOD Superoxide dismutase
  • Several companies have tackled this problem by creating versions of this enzyme with increased half-lives by techniques such as liposome encapsulation or polyethylene glycol conjugation. Reports on the effectiveness of these new versions are mixed.
  • Catalase, a scavenger of hydrogen peroxide, and hydroxyl radical scavengers have also been tested and found to be effective to varying degrees.
  • 1 ,3-propanediamines having formula I can be synthesized by acylation of N,N,N'-trimethyl- 1,3-propanediamine, such as via reaction with an acyl halide in non-protic solvent, followed by reduction, such as with lithium aluminum hydride in tetrahydrofuran (THF) .
  • THF tetrahydrofuran
  • a RCOC1, CH 2 C1 2 , room temperature
  • b lithium aluminum hydride, tetrahydrofuran, reflux
  • N-[3- (N r ,N r -dimethylamino)propyl]-N-methyldodecanamide with 1 N aqueous sodium hydroxide (18 mL) , methanol (40 mL) , and chloroform (20 mL) .
  • This mixture was transferred to a separatory funnel and 1 N aqueous sodium hydroxide (20 mL) and chloroform (20 mL) were added.
  • the aqueous layer was extracted twice more with chloroform (20 L) and the organic layers were combined and dried with magnesium sulfate.
  • the clear oil was dissolved in dry tetrahydrofuran (10 mL) and added via syringe to a stirring solution of lithium aluminum hydride in tetrahydrofuran (1.0 M, 5.85 mL) to dry tetrahydrofuran (15 mL) . Additional dry tetrahydrofuran (10 mL) was added and the reaction mixture was heated at reflux for 8 hours and then cooled to room temperature. Then the reaction mixture was quenched slowly and sequentially with 6 drops water/6 drops, 15% NaOH then 15 drops water and stirred for 45 minutes to allow a precipitate to form. The precipitate was removed by vacuum filtration and the solvent removed under reduced pressure to yield (1.34 g) a pale clear oil.
  • N'-[3-(N,N-dimethylamino)propyl]-N'-methylnonanamide (1.65 g, 6.43 mmol) was dissolved in dry tetrahydrofuran (7 mL) and added to a solution of lithium aluminum hydride in tetrahydrofuran (1.0 M, 7.0 mL) diluted in tetrahydrofuran (20 mL) .
  • the reaction was refluxed under nitrogen for 8 hrs. After cooling to room temperature the reaction was quenched slowly and sequentially with 7 drops water, 14 drops 15% aqueous sodium hydroxide and 7 drops water. The 5 resulting precipitate was collected after 45 min.
  • N-[3-(N',N # -dimethylaminopropyl]-N-methyltridecanamine dihydrochloride (Compound 6) N-[3-(N',N'-dimethylamino)propyl]-N-methyltridecanamide (2.03 g, 6.50 mmol) was dissolved in anhydrous tetrahydrofuran (10 mL) and added to a solution of lithium aluminum hydride in tetrahydrofuran (1.0M in tetrahydrofuran, 8.50 mL) in dry tetrahydrofuran (30 mL) . The reaction mixture was refluxed for 8 hours under nitrogen.
  • N-[3-(N',N'-dimethylamino)propyl]-N-methyItetradecanamide 5 hydrochloride was dissolved in IN aqueous sodium hydroxide (30 mL) , mixture was transferred to a separatory funnel and IN aqueous sodium hydroxide (30 mL) and chloroform (30 mL) were added. The layers extracted twice with chloroform (30 mL each) . The organic layers were combined and dried with 0 magnesium sulfate. The drying agent was removed by vacuum filtration, and the solvent was removed under reduced pressure to yield the free amine as an oil.
  • Table 1 Exemplary compounds having formula I and formula II as described herein useful in the pharmaceutical compositions and methods of the invention.
  • Table 1 illustrates chemical composition and moieties of 1,3-propanediamine derivatives formula I and II as described herein. The approximate melting point of a compound in degrees centigrade is indicated in the column labeled "MP (°C) .”
  • a protein kinase C (PKC) assay is designed to duplicate the in vivo conditions required for protein kinase C function. Therefore, pH, salt and cofactor concentrations are similar to physiologic levels.
  • a lysine rich histone, HI was used in the assay as the phosphorylation acceptor-protein because it is readily available and serves as a good substrate for protein kinase c.
  • Enzyme was prepared from rat brain and purified to apparent homogeneity as determined by a single band on silver stained SDS-polyacrylamide.
  • phosphatidylserine (PS) and DAG were co-sonicated to form unilamellar and multilamellar vesicles.
  • concentration of lipids in the assay were suboptimal to maximize the detection potential of the assay for inhibitors.
  • Potential inhibitor compounds were added to the assay in dimethylsulfoxide at three concentrations to give final inhibitor concentrations of 4.3, 43 and 218 ⁇ K, respectively.
  • the assay was started with the addition of enzyme and stopped after 10 min by the addition of 25% trichloroacetic acid (TCA) and 1.0 mg/ml bovine serum albumin (BSA) .
  • TCA trichloroacetic acid
  • BSA bovine serum albumin
  • the amount of phosphorylation was determined by the radioactivity measured in a scintillation counter, controls were included in every assay to measure background activity in the absence of enzyme, activity in the absence of lipids, and the maximum enzyme activity with saturating levels of the activator lipids.
  • Table 2 shows the protein kinase C assay components and their concentrations.
  • HEPES is N-[2-hydroxyethyl] piperazine-N'-[ethanesulfonic acid] and EGTA is Ethylene-bis (oxyethylenenitrilo) tetracetic acid.
  • Results of the protein kinase C assay are shown in Table 3 in the column labeled PKC. Results are show as IC 50 , which is the concentration of test compound needed to inhibit 50% of the protein kinase C activity in controls.
  • Compounds of the invention were able to effectively inhibit protein kinase activity.
  • Compounds 8 and 9 had an IC 50 of less than 25 ⁇ M, 24 ⁇ M and 22 ⁇ M, respectively.
  • PKA cAMP dependent protein kinase
  • the assay was started by the addition of 32P-ATP and the reaction was allowed to proceed for 10 min before stopping with 25% trichloroacetic acid (TCA) and 1.0 mg/ml bovine serum albumin (BSA) . Phosphorylated protein was then isolated by filtration and the radioactivity was counted in a beta scintillation counter.
  • TCA trichloroacetic acid
  • BSA bovine serum albumin
  • test compounds 1, 5, 6, 7, and 8 were tested in PKA assays but had no effect on PKA activity.
  • the tested compounds of the invention are selective for protein kinase C, and have no effect on cAMP dependent protein kinase.
  • the compounds of the invention should thus have no effect on the metabolic pathways associated with stimulation of protein kinase by cAMP.
  • MCF-7 a human breast tumor cell line and MCF-7/ADR an adriamycin resistant line of MCF-7 cells were obtained form the National Cancer Institute, Frederick, Maryland.
  • CEM cells ATCC accession number CCL 119 were obtained from the American Type Culture Collection, Rockville, Maryland.
  • Human tumor cells were trypsinized with 0.05% trypsin (GIBCO) , counted with a hemacytometer and seeded at a concentration of 10,000 cells/well in a 96 well microtiter plate. After allowing cells to attach to the surface overnight, the culture medium was aspirated and replaced with 100 ⁇ l of fresh medium. These agents were diluted to determine dose response at 2X final concentration and added in quadruplicate at 100 ⁇ l/well to bring the total volume of each well to 200 ⁇ l. The microtiter plate was then incubated at 37°c 5% C0 2 overnight for 18 to 24 hrs before H-thymidine was added at a concentration of 0.5 ⁇ Ci/well in 50 ⁇ l culture medium. The plate was incubated again for 4 hrs under the same conditions as above. Supernatant was then aspirated and 50 ⁇ l of 0.05% trypsin (GIBCO) was added to each well.
  • GEBCO trypsin
  • IC 50 is the concentration of test compound required to inhibit fifty per cent of the incorporation of H-thymidine into proliferating cells not exposed to test agent. Uptake of H-thymidine is a standard test for measuring the metabolism of cells.
  • Test agents inhibit the uptake of 3H-thymidine thus slow the growth of cells.
  • compounds of the invention were able to inhibit H-thymidine uptake and thus inhibit the proliferation of the tested cell lines.
  • Compound 1 had an IC 50 of greater than 25 ⁇ M for both the MCF-7 cell line and the adriamycin resistant MCF cell line.
  • Compounds 5, 8, and 9 had similar IC gj s for MCF-7 cells, 3.90, 1.50 and 5.10, respectively.
  • Compound 5, 8, and 9 were also able to inhibit thymidine uptake of adriamycin resistant MCF-7 cells, with IC 50 's for the adriamycin-resistant cells of 4.50, 2.60 and 4.60, respectively.
  • compounds useful in the invention not only inhibit tumor cell proliferation but are not cross-resistant to the multi-drug-resistant family of agents such as adriamycin.
  • Neutrophil Superoxide Anion (0 2 ⁇ ) Release Assay Neutrophils were isolated from whole blood collected from human volunteers. All reagent materials are obtained from Sigma Chemical Company with the exception of isotonic saline (Travenol Laboratories, Inc. Deerfield, Illinois) and lymphocyte separation medium (Organon Teknika, Durham, North Carolina) . Neutrophil Isolation
  • the washed pellet was resuspended in 10 ml HBSS and placed on ice before counting on a hemacytometer.
  • Assay Procedure The neutrophil cell concentration was adjusted to 2 x 106 cells/ml with HBSS before adding 0.8 ml cells to 12 X 75 mm polypropylene test tubes (Fisher Scientific) . Test agents were diluted to determine dose response and added at 10X final concentration at a volume of 0.1 ml/tube in duplicate.
  • cytochrome C 15 mg/ml
  • catalase 3000 units/ml
  • PMA phorbol 12-myristate 13-acetate
  • the change in OD of cytochrome c was obtained between PMA-stimulated and non-stimulated tubes, and the dose responses of the test agents were compared to the positive controls which contain HBSS in place of test agents.
  • PMA stimulates 0 2 -production which reduces cytochrome c.
  • Reducing cytochrome c increases its absorbance, and the change in OD of cytochrome c is proportional to the amount of 0 2 -production by PMA stimulation.
  • Inhibition is expressed as IC 50 ⁇ M and is the amount of test compound that will inhibit fifty per cent of the PMA-stimulated respiratory outburst, i.e. 0 2 -production.

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Abstract

Compositions pharmaceutiques comprenant un excipient ou diluant pharmaceutiquement acceptable et (a) un composé de la formule (I) dans lequel m est 2, 3 ou 4; R1, R2 et R3 sont, indépendamment, alkyle C1 à environ C20, alcényle C1 à environ C20 ou alcynyle C1 à environ C20; et R4 est alkyle C8 à environ C20; ou (b) un composé de la formule (II) dans laquelle n est 1, 2, 3 ou 4; R5 et R6 sont, indépendamment, alkyle C1 à environ C5, alcényle C1 à environ C5 ou alcynyle C1 à environ C5; et R7 est alkyle C8 à environ C20; ou (c) un sel pharmaceutiquement acceptable de (a) ou (b). Lesdites compositions pharmaceutiques sont utilisées pour inhiber la protéine kinase C et traiter des pathologies liées à l'inhibition de la protéine kinase C ou influencées par cette inhibition, en particulier les tumeurs cancéreuses, les maladies inflammatoires, les blessures de reperfusion et les dysfonctionnements cardiaques liées aux blessures de reperfusion.
PCT/US1993/003177 1992-04-14 1993-04-05 Derives de 1,3,-propanediamine ayant une activite d'inhibition de la proteine kinase c WO1993020813A1 (fr)

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AU40464/93A AU4046493A (en) 1992-04-14 1993-04-05 1,3,-propanediamine derivatives having protein kinase C inhibitory activity

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US86853792A 1992-04-14 1992-04-14
US07/868,537 1992-04-14

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, Vol. 91: 698862, 1979, EPSTEIN et al., "Carcinogenicity of a Composite Organic Extract of Urban Particulate Atomospheric Pollutants Following Subcutaneous Injection in Infant Mice". *

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