WO2009067697A1 - Composés thérapeutiques - Google Patents

Composés thérapeutiques Download PDF

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Publication number
WO2009067697A1
WO2009067697A1 PCT/US2008/084409 US2008084409W WO2009067697A1 WO 2009067697 A1 WO2009067697 A1 WO 2009067697A1 US 2008084409 W US2008084409 W US 2008084409W WO 2009067697 A1 WO2009067697 A1 WO 2009067697A1
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Prior art keywords
compound
alkyl
crc
cycloalkyl
aryl
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PCT/US2008/084409
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English (en)
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Chengguo Xing
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Regents Of The University Of Minnesota
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Publication of WO2009067697A1 publication Critical patent/WO2009067697A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/36—Sulfur atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/10—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing aromatic rings

Definitions

  • Prostate cancer is the most common malignancy and the second leading cause of cancer- related deaths among males in the United States of America with about 218,890 new cases and 27,050 prostate cancer-related deaths expected for 2007 (Jemal A, et al, CA Cancer J. Clin. 2007;57:43-66). This rate translates to one in six men being diagnosed with prostate cancer in their lifetime. While treatment for localized therapy can be highly effective, current treatment strategies for metastatic prostate cancer are temporizing as the disease following initial remission in response to androgen ablation relapses to a hormone-refractory phenotype (HRPC), a generally fatal form of prostate cancer. It was estimated that a median expected survival for patients with HRPC is only 10 months ( Sciarra A, Salciccia S., Eur. Urol. 2007;52:964-72). Therefore, the search for novel agents and approaches for the treatment of HRPC must be considered a high priority.
  • HRPC hormone-refractory phenotype
  • docetaxel-based chemotherapy has recently been demonstrated to modestly improve the overall survival of HRPC patients by approximately 6 months ( Petrylak DP., Rev. Urol. 2006;8:S48-55). These results have now made docetaxel the standard of care for the treatment of HPRC.
  • docetaxel is a microtubule-stabilizing agent - one subfamily of anti-microtubule agents which halts the cell cycle at the metaphase-anaphase transition, eventually resulting in apoptosis.
  • docetaxel has been demonstrated to have moderate success in the treatment of
  • HRPC patients most of these patients develop resistance to docetaxel and die from the drug- resistant phenotype ( Mancuso A, et al., Crit. Rev. Oncol. Hematol. 2007;61: 176-85).
  • Drug- resistant HRPC is often genetically associated with the over-expression of Bcl-2 protein, a key regulator in the prohibition of apoptosis and strongly implicated in the development of the drug- resistant phenotype of multiple cancers (Mancuso A, et al., Crit. Rev. Oncol. Hematol.
  • Applicant has identified compounds with activity as dual antagonists against anti- apoptotic Bcl-2 proteins and microtubule. These compounds have been shown to have activity against HRPC. Considering the pivotal function of anti-apoptotic Bcl-2 proteins in the development and metastasis of HRPC and the promising future of antimicrotubule agents against HRPC, a compound possessing dual antagonism provides a new opportunity for the effective treatment of HRPC. The ability to obtain dual activity within a single molecule eliminates the complicated dosing requirements associated with the administration of two or more agents.
  • the invention provides a compound of the invention which is a compound of formula I:
  • R 4 Is OH OrNHSO 2 R 1 ;
  • R 1 is aryl, heteroaryl, (C 1 -C 6 )alkyl, (C 3 -C 6 )cycloalkyl, aryl(C r C 6 )alkyl, heteroaryKOr C 6 )alkyl, or (C 3 -C 6 )cycloalkyl(C 1 -C 6 )alkyl, wherein any aryl or heteroaryl is optionally substituted with one or more (e.g.
  • each R 3 is independently selected from halo, cyano, hydroxy, (Ci-C 6 )alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C r C 6 )alkyl, (C r C 6 )alkoxy, (d-C 6 )alkanoyl, (C 1 - C 6 )alkoxycarbonyl, (Ci-
  • the invention also provides a composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the invention also provides a therapeutic method for treating cancer (e.g. lymphoma, prostate cancer, hormone-refractory phenotype prostate cancer and leukemia) in an animal (e.g. a mammal such as a human) comprising, administering to the animal an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.
  • cancer e.g. lymphoma, prostate cancer, hormone-refractory phenotype prostate cancer and leukemia
  • the invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof as described herein for use in medical therapy.
  • the invention also provides the use of a compound of formula I or a pharmaceutically acceptable salt thereof as described herein for the manufacture of a medicament useful for the treatment of cancer (e.g. lymphoma, prostate cancer, hormone- refractory phenotype prostate cancer and leukemia) in an animal (e.g. a mammal such as a human).
  • cancer e.g. lymphoma, prostate cancer, hormone- refractory phenotype prostate cancer and leukemia
  • an animal e.g. a mammal such as a human
  • the invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in the prophylactic or therapeutic treatment of cancer (e.g. lymphoma, prostate cancer, hormone-refractory phenotype prostate cancer and leukemia) in an animal (e.g. a mammal such as a human).
  • the invention also provides a method to antagonize anti-apoptotic Bcl-2 proteins in an animal (e.g. a mammal such as a human) comprising administering to the animal an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.
  • an animal e.g. a mammal such as a human
  • the invention also provides a method to antagonize microtubule formation in an animal (e.g. a mammal such as a human) comprising administering to the animal an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.
  • an animal e.g. a mammal such as a human
  • the invention also provides a method to antagonize anti-apoptotic Bcl-2 proteins and to antagonize microtubule formation in an animal (e.g. a mammal such as a human) comprising administering to the animal an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.
  • an animal e.g. a mammal such as a human
  • the invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful for antagonizing anti-apoptotic Bcl-2 proteins in an animal (e.g. a mammal such as a human).
  • the invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful for antagonizing microtubule formation in an animal (e.g. a mammal such as a human).
  • the invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful for antagonizing anti-apoptotic Bcl-2 proteins and for antagonizing microtubule formation in an animal (e.g. a mammal such as a human).
  • the invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in the prophylactic or therapeutic treatment of a pathological condition or symptom in an animal (e.g. a mammal such as a human) wherein the antagonism of anti-apoptotic Bcl-2 proteins is desired.
  • an animal e.g. a mammal such as a human
  • the invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in the prophylactic or therapeutic treatment of a pathological condition or symptom in an animal (e.g. a mammal such as a human)wherein the antagonism of microtubule formation is desired.
  • an animal e.g. a mammal such as a human
  • the invention also provides a compound of formula I or a pharmaceutically acceptable salt thereof for use in the prophylactic or therapeutic treatment of a pathological condition or symptom in an animal (e.g. a mammal such as a human) wherein the antagonism of anti-apoptotic Bcl-2 proteins and the antagonism of microtubule formation is desired.
  • the invention also provides processes and intermediates disclosed herein that are useful for preparing compounds of formula (I) or salts thereof.
  • Figure 1 The chemical structures of the representative Bcl-2 antagonists screened for microtubule interactions.
  • FIG. 1 Competition of Compound 1 against Bak peptides to Bcl-2 and BCI-X L proteins. Analysis was carried out as described in Materials and Methods. In brief, Bcl-2 or BCI-XL was first mixed with a fluorescein-labeled Bak peptide to form a protein-peptide complex, which resulted in fluorescence polarization (FP) increase. Upon the addition of Compound 1, FP was measured, which reflects the amount of remaining protein-peptide complex.
  • FP fluorescence polarization
  • FIG. 3 The effect of Compound 1 on microtubule and cell cycle.
  • A. Dose-dependent Compound 1 induction of microtubule depolymerization.
  • B. Compound 1 (80 ⁇ M) induction of cell cycle arrest.
  • A-E Merged images of microtubules (red), centrosomes (green), and D API- stained nuclei (blue) are shown in A-E, and distribution of filamin-containing microfilaments is also shown in the same cells (A' and E').
  • Compound 1 caused partial depolymerization of cytoplasmic microtubules (B-E) originated from the centrosome in interphase cells (A). Microtubules tended to bundle around the nucleus (A, C) and at the cell periphery (D, E), and stress fibers became less prominent in drug treated cells (A' and E'). Bar, 10 ⁇ m.
  • Figure 4 in vitro cytotoxicity and apoptotic induction by Compound 1.
  • B and C Evidence of apoptotic death in drug-treated PC-3 cells.
  • PC-3 cells were treated with Compound 1 at the indicated concentration for 24 hours and caspase-3/-7 activity was evaluated by Apo-ONE® Caspase-3/-7 reagent.
  • C DNA fragmentation in PC-3 cells upon Compound 1 treatment.
  • PC-3 cells were treated with Compound 1 at the indicated concentration for 6 hours and DNA fragmentation was assessed by using Apoptotic DNA Ladder Extraction Kit.
  • FIG. 5 Overcoming drug resistance by Compound 1.
  • A. Sensitivity of various anticancer agents and Compound 1 to Jurkat cells with varied level of Bcl-2 and Bcl-X L .
  • A. Tumor volumes. Points, mean; bars, SE (n 8; * ⁇ 0.05 compared with the control group).
  • B. Serum level of Compound 1. Points, mean; bars, SE (n 4; * ⁇ 0.05 between the two treated groups).
  • halo is fluoro, chloro, bromo, or iodo.
  • Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as propyl embraces only the straight chain radical, a branched chain isomer such as isopropyl being specifically referred to.
  • Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic.
  • Heteroaryl encompasses a radical of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C 1 - C 4 )alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
  • (C 1 -C 6 )alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec- butyl, pentyl, 3-pentyl, or hexyl;
  • (C 3 -C 6 )cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl;
  • (C 1 -C 6 )EIkOXy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;
  • (Ci-C 6 )alkanoyl can be acetyl, propanoyl or butanoyl;
  • (CrC 6 )alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbony
  • a specific value for R 4 is OH.
  • groups independently selected from halo, cyano, hydroxy, (CrC 6 )alkyl, (CrC 6 )alkoxy, (C 1 - C 6 )alkanoyl, (CrC 6 )alkoxycarbonyl, (CrC 6 )alkanoyloxy, (C 3 -C 6 )cycloalkyl
  • a specific value for R 1 is phenyl, optionally substituted with one or more (e.g. 1, 2, 3, or
  • R 1 is aryl optionally substituted with one or more (e.g. 1, 2, 3, or 4) groups independently selected from (d-C 6 )alkyl, NR 3 R b and nitro.
  • R 1 is phenyl optionally substituted with one or more (e.g. 1, 2, 3, or 4) groups independently selected from nitro and NR 3 R b .
  • R 1 is phenyl optionally substituted with one or more (e.g. 1, 2, 3, or 4) groups independently selected from nitro and NR 3 R b wherein each R 3 and Rb is independently hydrogen or (C 1 -C 6 )alkyl wherein any (d-C 6 )alkyl is optionally substituted with S Aryl or OAryl.
  • a specific value for Rj is 3-nitro-4-(2-phenylthioethylamino)phenyl.
  • a specific value for R 1 is phenyl, optionally substituted with one or more (e.g. 1, 2, 3, or
  • R 1 is aryl optionally substituted with one or more (e.g. 1, 2, 3, or 4) (C r C 6 )alkyl.
  • Ri is phenyl optionally substituted with one or more (e.g. 1, 2, 3, or 4) (Ci-C 6 )alkyl.
  • Ri 4-methylphenyl
  • a specific value for R 5 is A specific value for m is 0, 1 or 2. A specific value for m is 0. A specific value for R 5 benzyl. A specific value for R 5 is (Ci-C 6 )alkyl.
  • R 5 is isobutyl.
  • a specific value for n is 1 or 2.
  • a specific value for n is 1, 2, or 3.
  • groups independently selected from halo, cyano, hydroxy, (CrC6)alkoxy, (Ci-C6)alkanoyl, (Ci-C 6 )alkoxycarbonyl, (Ci-C 6 )alkanoyloxy, (C 3 -C 6 )
  • R 3 is hydroxy, phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl wherein any phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl of R 3 is optionally susbtituted with one or more (e.g. 1, 2, 3, or 4) groups independently selected from halo, cyano, hydroxy, (CpC 6 )alkyl, (Cp C 6 )alkoxy, (C]-C 6 )alkanoyl, (C]-C 6 )alkoxycarbonyl, (C !
  • a specific value for R 3 is hydroxy, phenyl, 2-pyridyl, 3-pyridyl, or 4-pyridyl, wherein any phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl of R 3 is optionally susbtituted with one or more (e.g. 1, 2, 3, or 4) groups independently selected from hydroxy and (Ci-C 6 )alkoxy.
  • R 3 is hydroxy, phenyl, 2-pyridyl, 3-pyridyl, or 4-pyridyl, wherein any phenyl, 2-pyridyl, 3-pyridyl or 4-pyridyl of R 3 is optionally substituted with one or two groups independently selected from hydroxy and (Q-C ⁇ alkoxy.
  • R 6 A specific value for R 6 is 4-biphenylyl, 4'-hydroxy-biphenyl-4-yl, 3'-hydroxy-4'- methoxybiphenyl-4-yl, 3'-hydroxybiphenyl-4-yl, 3', 5'-dihydroxybiphenyl-4-yl, T- hydroxybiphenyl-4-yl, 3-hydroxybiphenyl-4-yl, 2-hydroxybiphenyl-4-yl, 4-(4-pyridyl)phenyl, 4- (2-pyridyl)phenyl or 4-(3-pyridyl)phenyl.
  • R 3 A specific value for R 3 is -A-B-C-D.
  • a specific value for R 3 is -A-B-C-D wherein C is aryl and D is aryl.
  • a specific value for R 3 is -A-B-C-D wherein A is piperazino, B is methylene, C is aryl and D is aryl.
  • R 3 A specific value for R 3 is -A-B-C-D wherein A is piperazino, B is methylene, C is phenyl and D is phenyl.
  • a specific group of compounds are compounds wherein n is 1 and R 3 is phenyl or phenyl(CrC 6 )alkyl, wherein any phenyl of R 3 is optionally susbtituted with one or more (e.g.
  • one or more (e.g. 1, 2, 3, or 4) groups independently selected from halo, cyano, hydroxy, (C r C 6 )alkyl, (CrC 6 )alkoxy, (CrC 6 )alkanoyl, (CrC 6 )alk
  • a specific group of compounds are compounds wherein n is 1 and R 3 is phenyl.
  • the invention provides compounds of formula I wherein the group R 5 -CH-C(O)-R 4 has the absolute stereochemistry corresponding to a natural L amino acid.
  • the invention also provides compounds of formula I wherein the group Rs-CH-C(O)-R 4 has the absolute stereochemistry corresponding to a D amino acid.
  • n is 1 and R 3 is on the 4-position of the phenyl ring in Formula I.
  • R 1 is aryl, heteroaryl, (C r C 6 )alkyl, (C 3 -C 6 )cycloalkyl, aiyl(Ci -C 6 )alkyl, heteroaryl(C r C 6 )alkyl, or (C 3 -C 6 )cycloalkyl(Ci-C 6 )alkyl, wherein any aryl or heteroaryl is optionally substituted with one or more (e.g.
  • each R 3 is independently selected from halo, cyano, hydroxy, (d-C 6 )alkyl, (C 3 - C 6 )cycloalkyl, (C 3 -C 6 )cycloalkyl(C r C 6 )alkyl, (C r C 6 )alkoxy, (C r C 6 )alkanoyl, (d-
  • each R a and R b is independently hydrogen, (CrC 6 )alkyl, or (C 1 -C 6 )alkanoyl; or R a and R b taken together with the nitrogen to which they are attached form a pyrrolidino, piperidino, piperazino, or morpholino ring; each R 0 and R d is independently hydrogen, (d-C 6 )alkyl, or (C 1 -C 6 )alkan
  • a specific compound of formula I is a compound of the following formula:
  • a specific compound of formula I is a compound of the following formula:
  • a specific compound of formula I is a compound of the following formula:
  • a compound of formula I wherein R 4 is NHSO 2 R 1 can be prepared by converting a corresponding acid of formula 3:
  • an intermediate acid of formula 3 is useful for preparing a compound of formula I.
  • the invention also provides a method for preparing a salt of a compound of formula I comprising converting a corresponding compound of formula I to the salt.
  • a salt of a compound of formula I can be useful as an intermediate for isolating or purifying a compound of formula I.
  • administration of a compound of formula I as a pharmaceutically acceptable acid or base salt may be appropriate.
  • pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, ⁇ - ketoglutarate, and ⁇ -glycerophosphate.
  • Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
  • salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
  • a sufficiently basic compound such as an amine
  • a suitable acid affording a physiologically acceptable anion.
  • Alkali metal for example, sodium, potassium or lithium
  • alkaline earth metal for example calcium
  • the compounds of formula I can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
  • a mammalian host such as a human patient
  • the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations will typically contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.
  • the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
  • the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and devices.
  • the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or 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.
  • isotonic agents for example, sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • useful dermatological compositions which can be used to deliver the compounds of formula I to the skin are known to the art; for example, see Jacquet et al. (U.S.
  • Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat.
  • the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
  • the compound can be conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
  • the active ingredient should typically be administered to achieve peak plasma concentrations of the active compound of from about 0.25 to about 75 ⁇ M, preferably, about 1 to
  • 50 ⁇ M most preferably, about 2 to about 30 ⁇ M.
  • This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
  • the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
  • Compounds of the invention can also be administered in combination with other therapeutic agents, for example, other agents that are useful for the treatment of cancer. Examples of such agents include anti-microtubule agents such as docetaxel, paclitaxel, vincrystine and vinblastine.
  • the invention also provides a composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, and a pharmaceutically acceptable diluent or carrier.
  • the invention also provides a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, packaging material, and instructions for administering the compound of formula I or the pharmaceutically acceptable salt thereof and the other therapeutic agent or agents to an animal to treat cancer (e.g. HRPC).
  • a kit comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, at least one other therapeutic agent, packaging material, and instructions for administering the compound of formula I or the pharmaceutically acceptable salt thereof and the other therapeutic agent or agents to an animal to treat cancer (e.g. HRPC).
  • Test A Binding of Compound 1 with recombinant Bcl-2 or Bcl-X L protein.
  • the ability of a compound of the invention to bind with recombinant Bcl-2 or BCI-X L protein may be determined using pharmacological models which are well known to the art, or using Test A described below.
  • the binding interaction of Compound 1 with recombinant Bcl-2 or BCI-X L protein was evaluated by following an established procedure (Doshi JM, et al, J. Med. Chem. 2006;49:7731- 9).
  • Controls included dose-response measurements in the absence of proteins to assess for any interactions between Compound 1 and the Flu-Bak peptide with such effects taken into account by subtraction.
  • Inhibitory constant (Ki) was determined by fitting the FP values to the concentrations of the small molecule using a single-site competition model in GraphPad (Doshi JM, et al., J. Med. Chem. 2006;49:7731-9).
  • test B Induction of microtubule depolymerization.
  • Microtubule Stabilization Destabilization Assay Kit was purchased from Cytoskeleton (Denver, CO). Cell Lysis Buffer, 2 x Reaction Buffer, Dithiothreitol (DTT) and Apoptotic DNA Ladder Extraction Kit were purchased from Biovision (Mountain View, CA). CellTilter-Blue® Cell Viability Assay kit and Apo-ONE® Homogeneous Caspase-3/7 Assay kit were purchased from Promega (Madison, WI).
  • the influence of the Bcl-2 antagonists on microtubules was evaluated using the Microtubule Stabilization/Destabilization Assay Kit from Cytoskeleton (Denver, CO) by following the manufacture procedures. Briefly, the buffers and tubulin were prepared and stored according to manufacture's protocol. The compounds to be tested were dissolved in DMSO to 3 mM and further diluted to varying concentrations in Buffer 3 with 10 % DMSO in the final solution. The solutions of 2 x 0.5 ml of Buffer 1, 12 ⁇ l of GTP stock, 10 ⁇ l of paclitaxel were defrosted.
  • MT BUFFER The following components was mixed in a tube placed on ice: Buffer 1 (1 ml), Buffer 2 (88 ⁇ l), GTP stock solution (12 ⁇ l), and Paclitaxel (2.8 ⁇ l, 200 ⁇ M) and the solution was labeled as MT BUFFER.
  • Tubulin (170 ⁇ l) was suspended with 800 ⁇ l of MT BUFFER by pipetting up and down for one minute. The solution was then incubated for exactly 30 min at 37 °C in a water bath and labeled as microtubule stock (MT STOCK). MT STOCK was incubated at room temperature for two hours.
  • Buffer 3 (5 ⁇ l) was pipetted into the control wells and the small molecules of varying concentrations (5 ⁇ l) were pipetted into the other wells as appropriate.
  • 2 mM CaCl 2 (5 ⁇ l) was used as a destabilization control and 200 ⁇ M paclitaxel (5 ⁇ l) was used as a stabilization control.
  • MT STOCK (45 ⁇ l) was then slowly pipetted into each well and the solution was mixed through gentle shaking.
  • the fluorescence intensity in each well was measured by using the GENios Pro multi-well plate reader from Tecan with excitation at 360 nm and emission at 420 nm for 15 min with recording every 30 sec.
  • the small-molecule controls included dose-response measurements in the absence of tubulin to assess for any fluorescence background from the small molecules. Eventual effects were taken into account by subtraction.
  • Compound 1 was found to induce microtubule depolymerization at a single concentration of 30 ⁇ M. The dose-dependent effect of Compound 1 towards microtubule depolymerization was then assessed (Fig. 3 A). Over the concentrations ranging from 5 - 60 ⁇ M, Compound 1 enhanced the depolymerization rate of microtubule in a dose-dependent manner. Compound 1 at 20 ⁇ M was of equal potency as Ca 2+ (the standard depolymerizer) at 300 ⁇ M in inducing the microtubule depolymerization, suggesting that Compound 1 is a potent destabilizing agent against microtubule.
  • Compound 1 changed the distribution of cells among the different phases of the cell cycle. With 80 ⁇ M Compound 1 treatment, more cells were accumulated at G2/M phase (34.4% vis 13.3%) and less cells were at the S phase (11.9% vis 38.7%) compared to the control cell samples, further supporting that Compound 1 may interfere with microtubule.
  • the in vitro effect of Compound 1 towards microtubule was further explored by using microscopy analysis of cytoskeleton of CHO (Chinese hamster ovary) cells upon Compound 1 treatment (Fig. 3C).
  • Control cells included a well-developed network of interphase microtubules emanating from the centrosome at the juxanuclear position (arrows in Fig. 3CA). Those cells also revealed the presence of prominent actin-containing stress fibers as shown in Figure 3CA'. Compound 1 treatment caused disappearance of microtubules in a dose-dependent manner. Although little difference in microtubule organization between control cells and cells treated with the drug below 20-25 ⁇ M was noted, administration of 75 ⁇ M Compound 1 for 12- 18 hr resulted in partial depolymerization of cytoplasmic microtubules (Fig. 3 CB-CE).
  • Microtubules tended to be bundled around the nuclei (Fig. 3CC) or at the periphery of cells (Fig. 3CD and CE). Concomitantly, stress fibers became disorganized in drug-treated cells (Fig. 3CE'). Besides CHO cells, similar effects of Compound 1 were noted in HeLa cells. We were unable to detect complete depolymerization of microtubule in cells treated with Compound 1 at higher concentrations. This is mainly due to the change of cell morphology caused by drug treatment: as a result of quick entrance into apoptotic pathways, the cells became round-up which made it difficult to detect microtubule architectures. Thus, Compound 1 caused partial depolymerization of interphase microtubules in vitro. Together, these ex vitro and in vitro data suggest that Compound 1 interferes with cell cycle likely through its ability to induce microtubule depolymerization.
  • Bcl-2 over-expressing and BCI-X L over-expressing Jurkat cells were provided by Dr. Claus Belka at University of Tuebingen and Dr. Daniel Johnson at the University of Pittsburgh respectively and characterized as described (22).
  • Jurkat cells and various prostate cancer cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (VfV), 100 units/ml penicillin G, 100 ⁇ g/ml streptomycin, and 5 % CO 2 at 37 °C.
  • Jurkat cells 1 xlO 4 cells / well were plated in a 96-well plate.
  • prostate cancer cells 3000 cells / well were plated in a 96-well plate.
  • the cells were treated with either a vehicle control or various concentrations of Compound 1 for 24 or 48 hours.
  • cell viability was measured by using CellTilter-Blue® Cell Viability Assay kit from Promega. Briefly, 20 ⁇ l of the dye was added to each well with 100 ⁇ l of culture media and incubated for 1 hour at 37 0 C. The fluorescence intensity was recorded with excitation at 560 nm and emission at 590 run, and normalized to the vehicle-treated control.
  • the in vitro cytotoxicity of Compound 1 was evaluated against several prostate cancer cell line, along with its ability to induce apoptosis.
  • the IC 50 S of Compound 1 against these four malignancies were all in the low micromolar concentration (10 - 20 ⁇ M, Fig. 4 A), suggesting that Compound 1 was effective against prostate cancer of different stages of progression.
  • Compound 1 was studied to determine if it could overcome drug resistance induced through prolonged drug exposure, which mimics the natural development of drug resistance.
  • three drug-resistant PC-3 cells were developed by culturing parent PC-3 cells in the presence of sublethal dosage of cisplatin, doxorubicin, or taxol respectively for at least 6 months.
  • the PC-3 cells all acquired resistance to the corresponding agents though the extent of resistance varied, with relative high drug resistance for cisplatin and taxol (Fig. 5 B).
  • AU three drug-resistant PC-3 cells were more sensitive to Compound 1 compared to the parent PC-3 cells (Fig. 5 C), indicating that Compound 1 would be potentially an effective anticancer agent against drug-resistant prostate malignancies.
  • DNA fragmentation was assessed by Apoptotic DNA Ladder Extraction Kit from Biovision. Briefly, PC-3 cells were treated by Compound 1 for 6 hours. 2.0 x 10 6 cells were harvested and washed with PBS. The cells were suspended in 50 ⁇ l DNA Ladder Extraction Buffer. After incubation at 23 0 C for 10 seconds with gentle pipetting, the mixture was centrifuged for 5 min at 1600 x g. The supernatant was transferred to a fresh tube and the cell pellet was extracted again with DNA Ladder Extraction Buffer (50 ⁇ l). The supernatants were combined and 5 ⁇ l Enzyme A solution was added into the supernatant. The solution was mixed by gentle vortex and incubated at 37 0 C for 10 min.
  • Enzyme B solution (5 ⁇ l) was then added into the mixture and further incubated overnight at 50 °C.
  • Ammonium acetate solution from Biovision (5 ⁇ l) was added to the sample and mixed well.
  • Isopropanol 100 ⁇ l was added and the solution was mixed well and kept at - 20 °C for 20 min.
  • DNA pellet was obtained by centrifugation at 13,000 x g for 10 min. The pellet was washed twice with cold 75 % ethanol, dried, and re-suspended in 20 ⁇ l DNA Suspension Buffer. Samples were loaded onto a 1.2 % agarose gel containing 0.5 ⁇ g/ml Ethidium bromide in both gel and running buffer. Electrophoresis was run at 50 V for 1 hour. DNA was visualized with UV light and photographed.
  • the effects of a compound of the invention on caspase activity may be determined using pharmacological models which are well known to the art, or using Test E described below.
  • Apo-ONE® Homogeneous Caspase-3/-7 Assay kit from Promega was used to measure the caspase-3/-7 activity according to the manufacturer's instructions. Briefly, after Compound 1 treatment, the cell culture medium was removed and fresh RPMI cell culture medium (50 ⁇ l) with Apo-ONE® Caspase-3/-7 reagent (50 ⁇ l) was added to each well. The solution was mixed gently and incubated at 37 0 C for 45 min. The fluorescence intensity of each well was measured with excitation at 485 nm and emission at 530 nm. Caspase-3/-7 activity was normalized to the vehicle-treated control. Results are shown in Figures 4B and 4C.
  • the effect of Compound 1 to cell cycle was evaluated by following a reported procedure from Nicoletti et al (Nicoletti I, et al., J. Immunol. Methods. 1991;l39:271-9). Briefly, Jurkat cells were treated with varying concentrations of Compound 1 for 24 hours. The cells were pelleted and suspended in hypotonic fluorochrome solution to stain cellular DNA with propidium iodide. Upon overnight incubation, the nuclei of the cells were analyzed by flow cytometry and the distribution of cells along the cell cycle was determined by the relative amount of cellular DNA.
  • Test G Induction of microtubule depolymerization by Compound 1 in vitro. Effects of Compound 1 on the cytoskeleton were analyzed by fluorescence staining of cultured mammalian cells with antibodies specific to a microtubule subunit protein ( ⁇ -tubulin) and an actin-binding protein (filamin).
  • CHO Choinese hamster ovary cells constitutively expressing GFP-tagged centrin2 (24) were prepared as reported previously (25) and grown as monolayers in Ham's F-IO medium containing 10% fetal bovine serum. Cells on coverslips were treated with 75 ⁇ M Compound 1 for 10-12 h and fixed with methanol for 5 min at - 2O 0 C.
  • mice Male athymic BALB/c nude mice (obtained from the Frederick Cancer Research Facility, National Cancer Institute, Frederick, MD) were maintained in a laminar airflow cabinet under pathogen-free conditions and used at 8 to 12 weeks of age. All facilities were approved by the American Association for Accreditation of Laboratory Animal Care in accordance with the current regulations and standards of the U.S. Department of Agriculture, U.S. Department of Health and Human Services, and NIH.
  • PC3-LN4 cells (60-70% confluent) were prepared for injection as described previously (26). Mice were anesthetized with isoflurane. Viable tumor cells (2 x 10 6 per 0.2 mL) in PBS were implanted subcutaneously into the flank. Formation of a bulla indicated a satisfactory injection. Beginning on day three after injection, groups of mice were then treated with intraperitoneal saline, 50 mg/kg Compound 1, or 100 mg/kg Compound 1 for 12 days. Tumor size and volume were assessed every 2 days. Mice were subjected to necropsy 24 hours after the last Compound 1 treatment. The tumors were removed and weighed. The tumors were quickly frozen in liquid nitrogen or fixed in 10% buffered formalin for additional analysis.
  • serum of each mouse was collected and stored at -80 °C till usage.
  • the serum level of Compound 1 was determined by recovering Compound 1 from the serum and quantifying the recovered Compound 1 through HPLC at 395 nm with 4-phenylphenol as the internal standard. Briefly, serum (200 ⁇ L) was mixed a mixture of methanol and acetonitrile (600 ⁇ L, v: v 1 : 1 ) and the mixture was mixed through vortex for 10 sec. The suspension was centrifuged at 14,000 x g for 10 min.
  • the supernatant was recovered, 500 ⁇ L of which was mixed with a solution of 4-phenylphenol (100 ⁇ M, 500 ⁇ L of H 2 O : MeOH : acetonitrile 2 : 1 : 1).
  • the solution was then analyzed by HPLC.
  • HPLC analysis was performed on a Beckman Coulter System Gold 126 solvent module and 168 detector.
  • a Phenomenex Polar RP column (5 ⁇ m, 250 x 4.6 mm) was used for the analyses. The flow rate used was 0.6 mL/min.
  • the mobile phase A was water with
  • the serum stability of a compound of the invention may be determined using pharmacological models which are well known to the art, or using Test I described below. Test I. Serum Stability
  • Example 2 Compounds 4, 5 and 6 of formula I were prepared utilizing the procedure outlined in

Abstract

L'invention concerne un composé de formule I: dans laquelle R4, R5 et R6 ont l'une quelconque des valeurs décrites ici ou un sel de celui-ci, ainsi que des procédés de synthèse et des intermédiaires utiles pour préparer de tels composés. Les composés ont une action anticancéreuse.
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Citations (3)

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