WO2003101373A2 - Thiazolidinedione derivatives in therapeutic and prophylactic methods - Google Patents

Thiazolidinedione derivatives in therapeutic and prophylactic methods Download PDF

Info

Publication number
WO2003101373A2
WO2003101373A2 PCT/CA2003/000815 CA0300815W WO03101373A2 WO 2003101373 A2 WO2003101373 A2 WO 2003101373A2 CA 0300815 W CA0300815 W CA 0300815W WO 03101373 A2 WO03101373 A2 WO 03101373A2
Authority
WO
WIPO (PCT)
Prior art keywords
alkyl
group
hydrogen
aralkyl
aryl
Prior art date
Application number
PCT/CA2003/000815
Other languages
French (fr)
Other versions
WO2003101373A3 (en
Inventor
Timothy S. Daynard
Zaihui Zhang
Donna L. Morrison
Original Assignee
Qlt Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Qlt Inc. filed Critical Qlt Inc.
Priority to AU2003233716A priority Critical patent/AU2003233716A1/en
Publication of WO2003101373A2 publication Critical patent/WO2003101373A2/en
Publication of WO2003101373A3 publication Critical patent/WO2003101373A3/en

Links

Classifications

    • 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
    • C07D417/04—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 directly linked by a ring-member-to-ring-member bond
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings

Definitions

  • This invention is directed to thiazolidinedione derivatives, pharmaceutical compositions containing thiazolidinedione derivatives, and methods of using thiazolidinedione derivatives for therapeutic and/or prophylactic effect in mammals.
  • Protein phosphorylation is a regulatory mechanism used by cells to selectively modify proteins carrying regulatory signals to the nucleus from outside the cell.
  • the proteins that mediate protein phosphorylation are a group of enzymes known as protein kinases and protein phosphatases, and as critical members of essential biological pathways, are sometimes associated with disease. While a plethora of extracellular molecules exist that modulate cellular functions via binding to membrane receptors inside the cell, their actions are mediated by relatively few signaling mechanisms within the cell.
  • PI-3K phosphatidylinositol 3-kinase
  • PKA protein kinase B
  • PKB Activated PKB mediates a number of metabolic effects of insulin; and protects cells from apoptosis (Kane et al. (1999), Curr Biol 9(11): 601-4).
  • survival factors can suppress apoptosis in a transcription- independent manner by activating the serine/threonine kinase PKB, which then phosphorylates and inactivates components of the apoptotic machinery. Survival factor withdrawal triggers apoptosis by inducing the expression of genes that are critical for cell death.
  • Signal transduction also plays a key regulatory role in the growth and metastatic potential of tumor cells. These signaling pathways form an interconnecting grid that serves to regulate the homeostatic, survival and invasive functions of the cell.
  • the key regulatory molecules in these pathways are the serine/threonine-protein kinases cyclic AMP-dependent protein kinase (PKA), Akt (PKB) and protein kinase C (PKC). These protein kinases modulate pathways associated with tumor proliferation, cell survival and multidrug resistance, and at a molecule level are likely to serve as effective targets for drug design.
  • the protein kinase B sequence is described by Coffer and Woodgett (1991) Eur. J. Biochem. 201(2): 475-481, and may be found in Genbank, indexed as accession number X61037.
  • PKB is a widely expressed cytoplasmic serine-threonine kinase, and its aberrant expression has been implicated in tumorigenesis (Testa and Bellacosa (2001) PNAS 98(20): 10983-10985; Nicholson and Anderson (2002) Cellular Signalling 14(5):381-395).
  • PKB contains at its NH 2 -terminus a domain termed the pleckstrin homology (PH) domain, which may regulate the activation of PKB by binding D3- phosphorylated phosphoinositides that are the products of PI3-K (Franke et al., (1997) Science 275(5300): 665-668). Phosphorylation of PKB also influences its activation, and the PH domain may influence the activation of PKB by promoting its translocation to the plasma membrane (Andjelkovic, M. et al., (1997) J. Biol. Chem. 272(50):31515-31524).
  • PH pleckstrin homology
  • This invention is directed to certain thiazolidinedione compounds, pharmaceutical compositions containing certain thiazolidinedione compounds, and the use of certain thiazolidinedione compounds in treating hyperproliferative disorders, e.g., cancer, inflammation, etc. in a mammal.
  • hyperproliferative disorders e.g., cancer, inflammation, etc. in a mammal.
  • hyperproliferative disorders associated with cellular modulation of protein phosphorylation states, e.g. altered activity of protein tyrosine kinases.
  • compounds and pharmaceutical compositions of the invention are used to treat inflammation and cell migration in a mammal.
  • compounds and pharmaceutical compositions of the invention are used to treat cancer, angiogenesis, and neurological disorders in a mammal.
  • the compounds and pharmaceutical compositions of the invention are used to prevent inflammation, cell migration, cancer, angiogenesis, and neurological disorders in a mammal.
  • the compounds and pharmaceutical compositions of the invention are used to promote apoptosis in proliferating cells in a mammal.
  • compounds and pharmaceutical compositions of the invention are used to inhibit the activity of protein kinase B (PKB).
  • PPKB protein kinase B
  • This enzyme has been associated with alterations in the phosphorylation state of cellular proteins.
  • R 0 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoiso ers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
  • R 8 is hydrogen, alkyl or aralkyl; each R 9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
  • R 10 is hydrogen, alkyl or aralkyl
  • R 11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to twenty five carbon atoms, and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (/so-propyl), n-butyl, ⁇ -pentyl, 1 ,1-dimethylethyl (/-butyl), and the like.
  • the alkyl radical optionally has: 1 to 25 carbons, i.e., is a C 1 -C 25 alkyl radical, or is a C ⁇ -C 20 alkyl radical, or is a C C 15 alkyl radical, or is a C ⁇ -C 10 alkyl radical, or is a lower alkyl radical, i.e., a C ⁇ -C 6 alkyl radical, or is a C 2 -C 25 alkyl radical, or is a C 2 - C 20 alkyl radical, or is a C 2 -C 15 alkyl radical, or is a C 2 -C 10 alkyl radical, or is a C 2 -C 6 alkyl radical.
  • the alkyl radical is unsubstituted. In another aspect, the alkyl radical has a single substituent. In another aspect, the alkyl radical has two substituents. Unless stated otherwise specifically in the specification, it is understood that for radicals, as defined below, that contain a substituted alkyl group that the substitution can occur on any carbon of the alkyl group.
  • Alkenyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to eight carbon atoms, and which is attached to the rest of the molecule by a single bond or a double bond, e.g., ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1 ,4-dienyl, and the like.
  • the alkenyl radical optionally has: 1 to 25 carbons, i.e., is a C C 25 alkenyl radical, or is a C C 20 alkenyl radical, or is a CrC 15 alkenyl radical, or is a C ⁇ -C 10 alkenyl radical, or is a lower alkenyl radical, i.e., a C C 6 alkenyl radical, or is a C 2 -C 5 alkenyl radical, or is a C 2 -C 20 alkenyl radical, or is a C 2 -C 15 alkenyl radical, or is a C 2 -C 10 alkenyl radical, or is a C 2 -C 6 alkebyl radical.
  • the alkenyl radical is unsubstituted. In another aspect, the alkenyl radical has a single substituent. In another aspect, the alkenyl radical has two substituents. Unless stated otherwise specifically in the specification, it is understood that for radicals, as defined below, that contain a substituted alkenyl group that the substitution can occur on any carbon of the alkenyl group.
  • the aryl radical is unsubstituted, i.e., is substituted only with hydrogens.
  • the aryl radical has a single substituent.
  • the aryl radical has two substituents.
  • “Aralkylene” refers to a radical of the formula -R a R b where R a is an alkylene radical as defined above and R b is one or more aryl radicals as defined above, e.g., benzyl, diphenylmethyl and the like.
  • the aryl radical(s) may be optionally substituted as described above.
  • the aralkylene radical is unsubstituted.
  • aralkylene radical has a single substituent.
  • “Aralkenylene” refers to a radical of the formula -R 0 R b where R c is an alkenylene radical as defined above and R b is one or more aryl radicals as defined above, e.g., 3-phenylprop-1-enyl, and the like.
  • the aryl radical(s) and the alkenyl radical may be optionally substituted as described above.
  • Alkylene and “alkylene chain” refer to a straight or branched divalent hydrocarbon chain consisting solely of carbon and hydrogen, containing no unsaturation and having from one to eight carbon atoms, e.g., methylene, ethylene, propylene, n-butylene, and the like.
  • Alkenylene chain refers to a straight or branched divalent hydrocarbon chain consisting solely of carbon and hydrogen, containing at least one double bond and having from two to eight carbon atoms, e.g., ethenylene, prop-1-enylene, but-1-enylene, pent-1-enylene, hexa-1,4-dienylene, and the like.
  • R d is independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl, and R e is hydrogen, alkyl or aralkyl.
  • the alkenylene chain may be attached to the rest of the molecule through any two carbons within the chain.
  • Cycloalkyl refers to a stable monovalent monocyclic or bicyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, having from three to ten carbon atoms, and which is saturated and attached to the rest of the molecule by a single bond, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, decalinyl and the like.
  • the cycloalkyl radical has 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 carbons. Independent of the number of carbons in the cycloalkyl radical, in various aspects the cycloalkyl radical has 0, or 1, or 2 or 3 substituents.
  • Cycloalkylalkylene refers to a radical of the formula -R a R d where R a is an alkylene radical as defined above and R d is a cycloalkyl radical as defined above.
  • the alkyl radical and the cycloalkyl radical may be optionally substituted as defined above.
  • Halo refers to bromo, chloro, fluoro or iodo.
  • Haloalkyl refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, 1-bromomethyl-2-bromoethyl, and the like.
  • Haloalkoxy refers to a radical of the formula -OR c where R c is an haloalkyl radical as defined above, e.g., trifluoromethoxy, difluoromethoxy, trichloromethoxy, 2,2,2-trif luoroethoxy, 1 -fluoromethyl-2-fluoroethoxy, 3-bromo-2-fluoropropoxy, 1-bromomethyl-2-bromoethoxy, and the like.
  • Heterocyclyl refers to a stable 3- to 15-membered ring radical which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur.
  • the heterocyclyl radical may be a monocyclic, bicyclic or tricyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be aromatic or partially or fully saturated.
  • the heterocyclyl radical may not be attached to the rest of the molecule at any heteroatom atom.
  • heterocyclyl radicals include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzothiadiazolyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, carbazolyl, cinnolinyl, decahydroisoquinolyl, dioxolanyl, furanyl, furanonyl, isothiazolyl, imidazolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, indolizinyl, isoxazolyl,
  • Heterocyclylalkyl refers to a radical of the formula -R a R e where R a is an alkyl radical as defined above and R e is a heterocyclyl radical as defined above, and if the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl may be attached to the alkyl radical at the nitrogen atom.
  • the heterocyclyl radical may be optionally substituted as defined above.
  • compounds which are "commercially available” may be obtained from standard commercial sources including Acros Organics (Pittsburgh, PA), Aldrich Chemical (Milwaukee, Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park, UK), Avocado Research (Lancashire, U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester, PA), Crescent Chemical Co. (Hauppauge, NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, PA), Fisons Chemicals (Leicestershire, UK), Frontier Scientific (Logan, UT), ICN Biomedicals, Inc.
  • suitable conditions for carrying out a synthetic step are explicitly provided herein or may be discerned by reference to publications directed to methods used in synthetic organic chemistry.
  • the reference books and treatise set forth above that detail the synthesis of reactants useful in the preparation of compounds of the present invention, will also provide suitable conditions for carrying out a synthetic step according to the present invention.
  • "methods known to one of ordinary skill in the art” may be identified though various reference books and databases. Suitable reference books and treatise that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R.
  • Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C., www.acs.org may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.
  • Prodrugs is meant to indicate a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound of the invention.
  • prodrug refers to a metabolic precursor of a compound of the invention that is pharmaceutically acceptable.
  • a prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the invention.
  • Prodrugs are typically rapidly transformed in vivo to yield the parent compound of the invention, for example, by hydrolysis in blood.
  • the prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam).
  • prodrugs are provided in Higuchi, T., et al., "Pro-drugs as Novel Delivery Systems," A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated in full by reference herein.
  • prodrug is also meant to include any covalently bonded carriers which release the active compound of the invention in vivo when such prodrug is administered to a mammalian subject.
  • Prodrugs of a compound of the invention may be prepared by modifying functional groups present in the compound of the invention in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound of the invention.
  • Prodrugs include compounds of the invention wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the compound of the invention is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively.
  • Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of the invention and the like.
  • Solid compound and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
  • “Mammal” includes humans and domestic animals, such as cats, dogs, swine, cattle, sheep, goats, horses, rabbits, and the like.
  • Optional or “optionally” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.
  • optionally substituted aryl means that the aryl radical may or may not be substituted and that the description includes both substituted aryl radicals and aryl radicals having no substitution.
  • “Pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.
  • “Pharmaceutically acceptable salt” includes both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, triflu
  • “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
  • “Therapeutically effective amount” refers to that amount of a compound of formulae (1) or (2) which, when administered to a mammal, preferably a human, is sufficient to effect treatment, as defined below, for inhibiting cancer, inflammation, neurological disease, angiogenesis and cell migration in the mammal.
  • the amount of a compound of formulae (1) or (2) which constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.
  • Treating covers the treatment of a hyperproliferative disease as disclosed herein, in a mammal, preferably a human, and includes: preventing cancer, inflammation, neurological disease angiogenesis and cell migration from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; inhibiting cancer, inflammation, neurological disease angiogenesis and cell migration, i.e., arresting its development; and/or relieving cancer, inflammation, neurological disease angiogenesis and cell migration, i.e., causing regression of the condition.
  • the compounds of formulae (1 ) and (2), or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids.
  • the present invention is meant to include all such possible isomers, as well as, their racemic and optically pure forms.
  • Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC.
  • the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.
  • the present invention provides a compound of formula (1):
  • R 7 is hydrogen, alkyl or aralkyl
  • each R 8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl
  • R 9 is hydrogen, alkyl or aralkyl
  • R 10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
  • the compounds of the present invention described by formula (1) do not include homologs of the "excluded compounds" as identified above, where a homolog of an excluded compound differs by having, or not having, a methylene group relative to the excluded compound.
  • the compounds of the present invention described by formula (1) do not include compounds wherein R 3 is methyl when R 4 is methyl and R s is phenyl.
  • the present invention provides a compound of formula (2):
  • each of R 4 , R 5 and R 6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R 7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
  • R 8 is hydrogen, alkyl or aralkyl; each R 9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
  • R 10 is hydrogen, alkyl or aralkyl
  • R 11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
  • compounds of formula (1) can be prepared according to schemes known to those of ordinary skill in the art. See, for example, Lesyk, R., Bollettino Chimico Farmaceutico (1998), Vol. 137(6), pp. 210-217.
  • a compound of formula (C) may be prepared in situ by reacting carbon disulfide (i.e., a compound of formula (A)) with an amine compound of formula (B) in aqueous potassium hydroxide at room temperature.
  • Compounds of formula (D) may be obtained from a source such as Aldrich, or prepared according to schemes known to those of ordinary skill in the art.
  • Compounds of formula (1 ) may then be prepared by adding an aqueous solution containing a compound of formula (D), most preferably as its alkali metal salt if R is H, to a solution containing the compound of formula (C) prepared as described above (the mole ratio of (C) to (D) being about 1 to 1).
  • the admixture is stirred for two days at which point it may be acidified with hydrochloric acid and heated in a water bath for two hours.
  • the solution may then be allowed to cool to room temperature and the compound of formula (1) may be isolated as a solid by filtration.
  • the compound of formula (1) may be further purified by a technique know to those of ordinary skill in the art such as crystallization from a suitable solvent.
  • starting components (A') and (B') may be obtained from a source such as Aldrich, or prepared according to schemes known to those of ordinary skill in the art.
  • compound (1a) can be prepared by reacting (B') (0.01 mole) with 0.01 mole of reagent (A') in 10-40 mL of glacial acetic acid, the admixture then being boiled for about 15 minutes. Upon cooling, the resulting precipitate may be isolated by filtration, washed with water and allowed to dry, affording a compound of formula (1a).
  • compounds of formula (E) can be prepared following the procedure described in Scheme 1.
  • compounds of formula (1b) can be prepared by reacting a compound of formula (E) (5 mmoles) with a compound of formula (F) (5 mmoles) in an aprotic solvent (100 mL), such as toluene, benzene, etc., containing a catalytic amount of piperidinium acetate under boiling conditions. The admixture is boiled in a Dean-Stark apparatus for about 2 hours. The compound of formula (1b) is obtained upon cooling.
  • compounds of formula (C) may be prepared by reacting an isothiocyanate (i.e., a compound of formula (A)) with an amine compound of formula (B) (the mole ratio of (A) to (B) being about 1 to 1), in the presence of hydrochloric acid.
  • the admixture is dried and the residue is heated to ambient temperature for up to about 6 hours.
  • the resulting substance is a compound of formula (C).
  • Compounds of formula (D) may be obtained from a source such as Aldrich, or prepared according to schemes known to those of ordinary skill in the art. For instance, a hydroxycarbonylmethylenehalide compound may be reacted with about an equimolar amount of diphosphorus pentasulfide to afford a hydroxythiocarbonylmethylenehalide compound of formula (D), which can then be used in the reaction scheme as set forth herein.
  • Compounds of formula (2a) can be prepared under cyclization conditions according to schemes known to those of ordinary skill in the art. See, for example, Minoru, M., Yakugaku Zasshi (1962), Vol. 82, pp. 36-45.
  • a compound of formula (2a) may be formed by combining the foregoing quantity of a compound of formula (C) at a reduced temperature with a compound of formula (D) or a basic salt thereof, in an aqueous solution alkalized with diluted base solution.
  • the reaction mixture is warmed to ambient temperature, admixed with hydrochloric acid, and heated to about 100°C for up to 1 hour. After cooling, the resulting precipitate may be isolated by filtration, washed with water and allowed to dry, affording a compound of formula (2a).
  • starting components (A') and (B') may be obtained from a source such as Aldrich, or prepared according to schemes known to those of ordinary skill in the art.
  • compound (2b) can be prepared by reacting (B') (0.01 mole) with 0.01 mole of reagent (A') in 10-40 mL of glacial acetic acid, the admixture then being boiled for about 15 minutes. Upon cooling, the resulting precipitate may be isolated by filtration, washed with water and allowed to dry, affording a compound of formula (2b).
  • compounds of formula (E) can be prepared following the procedure described in Scheme 3.
  • Compounds of formula (2c) can be prepared by reacting a compound of formula (E) (5 mmoles) with a compound of formula (F) (5 mmoles) in an aprotic solvent (100 mL), such as toluene, benzene, etc., containing a catalytic amount of piperidinium acetate under boiling conditions. The admixture is boiled in a Dean-Stark apparatus for about 2 hours. The compound of formula (2c) is obtained upon cooling.
  • the present invention provides a pharmaceutical composition that comprises a compound of formula (1) and a pharmaceutically acceptable carrier, diluent or excipient:
  • R 9 is hydrogen, alkyl or aralkyl
  • R 10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
  • compositions of the present invention include a compound of formula (1) as defined above, including in separate embodiments, compositions including compounds of the specific embodiments defined above, e.g., compounds that do not include "excluded compounds” referred to above in connection with compounds of formula (1).
  • the present invention provides a pharmaceutical composition that comprises a compound of formula (2) and a pharmaceutically acceptable carrier, diluent or excipient:
  • R 8 is hydrogen, alkyl or aralkyl; each R 9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
  • compositions of the present invention include a compound of formula (2) as defined above, with the proviso that compositions of the present invention exclude compounds having the Registry Numbers of 93293-15-3 and 93312-04-0 as the exclusive compound of formula (2) within the composition.
  • compositions further exclude homologs of the compounds of Registry Numbers 93293-15-3 and 93312-04-0 as the exclusive compound of formula (2) within the composition, where two compounds that are homologs differ structurally by a single methylene (-CH 2 -) group.
  • present invention provides a composition as defined above with the proviso that when R 3 is methyl and R 4 is methyl and R 5 is phenyl, then R 1 and R 2 are not selected from hydrogen, methyl or ethyl.
  • This invention is directed to methods of using compounds of formulae (I) and (2), as set forth above in the Summary of the Invention, and pharmaceutical compositions containing compounds of formulae (I) and (2) in treating various conditions, which are associated with activation of PKB. These conditions include cancer, inflammation, angiogenesis, cell migration, and neurological disorders.
  • PKB has been associated with an inappropriate lack of apoptosis in proliferating cells, e.g. in tumor cells, activated lymphocytes, and the like.
  • the methods disclosed herein are useful in treating disorders and physiological conditions associated with hyperproliferation when administered to a subject in need of such treatment.
  • compounds and pharmaceutical compositions of the invention are used to inhibit the activity of PKB.
  • the compounds and pharmaceutical compositions of the invention are administered to a subject having a cancer or a pathological inflammation in order to inhibit tumor growth by inducing apoptosis, angiogenesis and/or cell migration.
  • the methods of the invention can be used prophylactically (i.e., to prevent the disorder of interest from occurring) or therapeutically (i.e., to inhibit or relieve the disorder).
  • the term "treating" is used to refer to both prevention of disease, and treatment of pre-existing conditions.
  • the prevention of symptoms is accomplished by administration of the compounds and pharmaceutical compositions of the invention prior to development of overt disease, e.g., to prevent the regrowth of tumors, prevent metastatic growth, diminish restenosis associated with cardiovascular surgery, to prevent or reduce cell migration leading to inflammation and associated tissue damage.
  • the compounds and pharmaceutical compositions of the invention may be administered to a subject in need thereof to treat an ongoing disease, by stabilizing or improving the clinical symptoms of the patient.
  • the subject, or patient may be from any mammalian species, e.g. primates, particularly humans; rodents, including mice, rats and hamsters; rabbits; equines; bovines; canines; felines; etc. Animal models are of interest for experimental investigations, providing a model for treatment of human disease.
  • Sphingosine 1-phosphate (S ⁇ P)-induced endothelial cell migration requires the
  • PKB-mediated phosphorylation of a G protein-coupled receptor This activation is indispensable for RAC activation, cortical actin assembly, angiogenesis and chemotaxis.
  • Transactivation of GPCRs by PKB may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration. Inhibition of PKB may prevent this activation and induction of angiogenesis and cell migration.
  • Neurodethelial neuronal survival is important for the survival of cerebellar neurons, through a signaling pathway by which insulin-like growth factor-1 activation of PIK3 triggers the activation of PKB.
  • PKB is a critical mediator of growth factor- induced neuronal survival.
  • Neuroologic disorder is defined here and in the claims as a disorder in which dysfunction of neurons occurs either in the peripheral nervous system or in the central nervous system.
  • neurologic disorders include: chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, diabetic peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis; aging; and acute neurodegenerative disorders including: stroke, traumatic brain injury, peripheral nerve damage, hypoglycemia, spinal cord injury, epilepsy, and anoxia and hypoxia.
  • PKB directly phosphorylates eNOS and activates the enzyme leading to nitric oxide (NO) production.
  • Activated PKB increases basal nitric oxide release from endothelial cells, and activation-deficient PKB attenuated NO production stimulated by vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • eNOS is an PKB substrate linking signal transduction by PKB to the release of the gaseous second messenger nitric oxide.
  • Phosphorylation of eNOS by PKB represents a calcium-independent regulatory mechanism for activation
  • Hyperproliferative disorders refers to excess cell proliferation, relative to that occurring with the same type of cell in the general population and/or the same type of cell obtained from a patient at an earlier time.
  • the term denotes malignant as well as non-malignant cell populations.
  • Such disorders have an excess cell proliferation of one or more subsets of cells, which often appear to differ from the surrounding tissue both morphologically and genotypically.
  • the excess cell proliferation can be determined by reference to the general population and/or by reference to a particular patient, e.g. at an earlier point in the patient's life.
  • Hyperproliferative cell disorders can occur in different types of animals and in humans, and produce different physical manifestations depending upon the affected cells.
  • Hyperproliferative cell disorders include cancers; blood vessel proliferative disorders such as restenosis, atherosclerosis, in-stent stenosis, vascular graft restenosis, etc.; fibrotic disorders; psoriasis; inflammatory disorders, e.g. arthritis, etc.; glomerular nephritis; endometriosis; macular degenerative disorders; benign growth disorders such as prostate enlargement and lipomas; and autoimmune disorders.
  • Cancers of particular interest include carcinomas, e.g.
  • hyperproliferative disorders that may be associated with altered activity of PKB include a variety of conditions where there is proliferation and/or migration of smooth muscle cells, and/or inflammatory cells into the intimal layer of a vessel, resulting in restricted blood flow through that vessel, i.e. neointimal occlusive lesions.
  • Occlusive vascular conditions of interest include atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement, and the like.
  • disorders and conditions where there is hyperproliferation and/or tissue remodeling or repair of reproductive tissue e.g.
  • uterine, testicular and ovarian carcinomas, endometriosis, squamous and glandular epithelial carcinomas of the cervix, etc. are reduced in cell number by administration of the compounds and pharmaceutical compositions of the invention.
  • Other disorders and conditions of interest relate to epidermal hyperproliferation, tissue remodelling and repair.
  • the chronic skin inflammation of psoriasis is associated with hyperplastic epidermal keratinocytes.
  • disorders of interest include inflammatory disorders and autoimmune conditions including, but not limited to, psoriasis, rheumatoid arthritis, multiple sclerosis, scleroderma, systemic lupus erythematosus, Sjogren's syndrome, atopic dermatitis, asthma, and allergy.
  • Target cells susceptible to the treatment include cells involved in instigating autoimmune reactions as well as those suffering or responding from the effects of autoimmune attack or inflammatory events, and include lymphocytes and fibroblasts.
  • the susceptibility of a particular cell to treatment according to the invention may be determined by in vitro testing.
  • a culture of the cell is combined with a subject compound at varying concentrations for a period of time sufficient to allow the active agents to induce cell death or inhibit migration, usually between about one hour and one week.
  • cultured cells from a biopsy sample may be used.
  • the dose will vary depending on mode of administration, specific disorder, patient status, etc. Typically a therapeutic dose will be sufficient to substantially decrease the undesirable cell population in the targeted tissue, while maintaining patient viability. Treatment will generally be continued until there is a substantial reduction, e.g. at least about 50%, decrease in the clinical manifestation of disease, and may be continued until there are essentially none of the undesirable cellular activity detected in the relevant tissue.
  • the compounds of formulae (1) and (2) may also find use in the specific inhibition of signaling pathways mediated by PKB, and as a "positive" control in high throughput screening for other modulating compounds. In particular, this invention directed to methods of using compounds of formulae (1) and (2) and pharmaceutical compositions containing such compounds in treating cancer or inflammation associated with PKB activity.
  • methods are provided for using compounds of formulae (1) and (2) and pharmaceutical compositions containing such compounds in treating hyperproliferative disorders.
  • the methods disclosed herein are useful in treating disorders and physiological conditions associated with hyperproliferation and tissue remodeling or repair when administered to a subject in need of such treatment.
  • the compounds and pharmaceutical compositions of the invention are administered to a subject having a cancer or a pathological inflammation in order to inhibit tumor growth by impeding cell division, and to decrease inflammation by inhibiting cell adhesion and cell migration.
  • the compounds of formulae (1) and (2) may also find use as affinity reagents for the isolation and/or purification of PKB using the biochemical affinity of the enzyme for inhibitors that act on it.
  • the compounds are coupled to a matrix or gel.
  • the coupled support is then used to separate the enzyme, which binds to the compound, from a sample mixture, e.g., a cell lysate, which may be optionally partially purified.
  • the sample mixture is contacted with the compound-coupled support under conditions that minimize non-specific binding.
  • Methods known in the art include columns, gels, capillaries, etc.
  • the unbound proteins are washed free of the resin and the bound proteins are then eluted in a suitable buffer.
  • the compounds of formulae (1 ) and (2) may also be useful as reagents for studying signal transduction or any of the clinical disorders listed throughout this application, and for use as a positive control in high throughput screening.
  • Administration of the Compounds and Pharmaceutical Compositions of the Invention may also be useful as reagents for studying signal transduction or any of the clinical disorders listed throughout this application, and for use as a positive control in high throughput screening.
  • compositions of the invention can be prepared by combining a compound of the invention with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
  • Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units.
  • composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disorder or condition associated with hyperproliferation and tissue remodelling or repair in accordance with the teachings of this invention.
  • a pharmaceutical composition of the invention may be in the form of a solid or liquid.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, e.g., inhalatory administration.
  • the pharmaceutical composition When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • a solid composition will typically contain one or more inert diluents or edible carriers.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, PrimogelTM, corn starch and the like; lubricants such as magnesium stearate or SterotexTM; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, PrimogelTM, corn starch and the like
  • lubricants such as magnesium stearate or SterotexTM
  • glidants such as colloidal silicon dioxide
  • sweetening agents such as sucrose or saccharin
  • a flavoring agent such as peppermint
  • the pharmaceutical composition when in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil.
  • a liquid carrier such as polyethylene glycol or a fatty oil.
  • the pharmaceutical composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.
  • the liquid pharmaceutical compositions of the invention may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride
  • fixed oils such as synthetic mono or diglycerides which may
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Physiological saline is a preferred adjuvant.
  • An injectable pharmaceutical composition is preferably sterile.
  • a liquid pharmaceutical composition of the invention intended for either parenteral or oral administration should contain an amount of a compound of the invention such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of a compound of the invention in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition.
  • Preferred oral pharmaceutical compositions contain between about 4% and about 80% of the compound of the invention.
  • Preferred pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 1% by weight of the compound of the invention.
  • the pharmaceutical composition of the invention may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.
  • the base for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.
  • Thickening agents may be present in a pharmaceutical composition for topical administration.
  • the composition may include a transdermal patch or iontophoresis device.
  • Topical formulations may contain a concentration of the compound of the invention from about 0.1 to about 10% w/v (weight per unit volume).
  • the pharmaceutical composition of the invention may be intended for rectal administration, in the form, e.g., of a suppository, which will melt in the rectum and release the drug.
  • the composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient.
  • bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
  • the pharmaceutical composition of the invention may include various materials that modify the physical form of a solid or liquid dosage unit.
  • the composition may include materials that form a coating shell around the active ingredients.
  • the materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents.
  • the active ingredients may be encased in a gelatin capsule.
  • the pharmaceutical composition of the invention in solid or liquid form may include an agent that binds to the compound of the invention and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include a monoclonal or polyclonal antibody, a protein or a liposome.
  • the pharmaceutical composition of the invention may consist of dosage units that can be administered as an aerosol.
  • aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One skilled in the art, without undue experimentation may determine preferred aerosols.
  • the pharmaceutical composition of the present invention may contain one or more known pharmacological agents used in the treatment of cancer or inflammation in a mammal, particularly, cancer or inflammation associated with hyperproliferation and tissue remodelling or repair.
  • compositions of the invention may be prepared by methodology well known in the pharmaceutical art.
  • a pharmaceutical composition intended to be administered by injection can be prepared by combining a compound of the invention with water so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non-covalently interact with the compound of the invention so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
  • the compounds of the invention are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • a therapeutically effective daily dose is from about 0.1 mg to about 20 mg/kg of body weight per day of a compound of the invention, or a pharmaceutically acceptable salt thereof; preferably, from about 0.1 mg to about 10 mg/kg of body weight per day; and most preferably, from about 0.1 mg to about 7.5 mg/kg of body weight per day.
  • the present invention provides a method of treating selected medical conditions in a mammal, using a thiazolidinedione derivative compound as described herein.
  • the medical condition is cancer and/or angiogenesis, and/or a neurological disorder.
  • the medical condition is inflammation and/or cell migration. In various other aspects, any two or more of these medical conditions can be combined in describing a method of the present invention.
  • the present invention provides a method of treating a medical condition in a mammal comprising administering to the mammal in need thereof a therapeutically effective amount of a compound of formula (1):
  • R 7 is hydrogen, alkyl or aralkyl
  • each R 8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl
  • R 9 is hydrogen, alkyl or aralkyl
  • R 10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
  • the present invention provides a method of treating a condition selected from cancer, inflammation, angiogenesis, cell migration and neurological disorders in a mammal, which method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound or composition as described herein containing a compound of formula (2):
  • R 8 is hydrogen, alkyl or aralkyl; each R 9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
  • R 10 is hydrogen, alkyl or aralkyl
  • R 11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
  • the present invention additionally provides a method of treating cancer, a method of treating inflammation, a method of treating angiogenesis, a method of treating cell migration and a method of treating neurological disorders with compounds and compositions as described herein.
  • the present invention provides a method for preventing a selected medical condition in a mammal.
  • the medical condition being prevented in a mammal is cancer and/or angiogenesis and/or a neurological disorder.
  • the medical condition being prevented in a mammal is inflammation and/or cell migration.
  • the inventive method of preventing a medical condition in a mammal comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formulae (1) or (2) as identified herein. In various other aspects, any two or more of these medical conditions can be combined in describing a method of the present invention.
  • the present invention provides a method of promoting apoptosis in proliferating cells in a mammal.
  • the inventive method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formulae (1) or (2) as identified herein.
  • the present invention provides a method of inhibiting PKB activity in a mammal.
  • the inventive method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formulae (1) or (2) as identified herein.
  • the present invention provides a method of restoring the normal foot process architecture of podocytes in glomerular diseases associated with proteinuria in a mammal.
  • the inventive method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formulae (1) or (2):
  • any of the following provisos may be applied: (1) The compounds do not include any one or more of the "excluded compounds” as defined above in connection with compounds of formula (1), i.e., 4- thiazolidinone, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)-2-thioxo (RN 34931-00-5), etc.
  • the compounds do not include homologs of any of the above-identified excluded compounds, where an excluded compound and its homolog differ structurally by the presence or absence of a methylene (-CH 2 -) group.
  • R 5 is not phenyl when both of R 3 and R 4 are methyl.
  • the present invention provides pharmaceutical composition
  • a compound of formula (1) and a pharmaceutically acceptable carrier, diluent or excipient:
  • R 9 is hydrogen, alkyl or aralkyl
  • R 10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
  • compositions of the present invention contain a compound of formula (1) with the proviso that R 5 is not phenyl when both of R 3 and R 4 are methyl.
  • the present invention provides compounds useful in the methods.
  • the present invention provides a compound of formula (1):
  • each of R 4 and R 5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R 6 is independently a direct bond or an optionally substituted alkylene of one to six carbons; R 7 is hydrogen, alkyl or aralkyl; each R 8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or
  • R 9 is hydrogen, alkyl or aralkyl
  • R 10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
  • the compounds of the present invention having formula (1) do not include any two or more of the excluded compounds set forth in the Table above.
  • R 1 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl;
  • R 1 is selected from the group consisting of aryl, aralkylene and aralkenylene;
  • R 1 is selected from the group consisting of heterocyclyl and heterocyclyalkylene;
  • the compound may be utilized as a mixture of steroisomers, and/or the compound is a pharmaceutically acceptable salt, and/or the compound has a molecular weight between 100 and 1,000 Daltons; and/or the compound has a molecular weight between 250 and 750 Daltons.
  • any of the following provisos may be applied: (1) The compounds do not include any of the "excluded compounds" as defined above in connection with compounds of formula (2), i.e., 5-thiazolidineacetic acid, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)-2,4-dioxo- (RN 405924-24-5) . . . 2,4-thiazolidinedione, 3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)- (RN 93293-15-3).
  • the compound may be utilized as a mixture of steroisomers, and/or the compound is a pharmaceutically acceptable salt, and/or the compound has a molecular weight between 100 and 1 ,000 Daltons; and/or the compound has a molecular weight between 250 and 750 Daltons.
  • aryl e.g., unsubstituted phenyl.
  • the present invention provides compounds useful in the methods, and pharmaceutical compositions containing the compounds, which are also useful in the inventive methods.
  • the original bovine PKB ⁇ cDNA was obtained from Dr. Jim Woodgett (Coffer and
  • the PKB ⁇ enzyme was expressed using the baculovirus expression system in a two-step process.
  • the infectious baculovirus particles were amplified prior to the recombinant overexpression of the protein.
  • infectious baculovirus was generated by co-transfecting recombinant pAcG2T-PKB ⁇ plasmid with linear AcNPV (Autographa californica nuclear polyhedrosis virus (BD PharMingen) DNA into adherent Spodoptera frugiperda Sf9 insect cells (Invitragen) following the manufacturer's instructions.
  • AcNPV Autographa californica nuclear polyhedrosis virus (BD PharMingen) DNA into adherent Spodoptera frugiperda Sf9 insect cells (Invitragen) following the manufacturer's instructions.
  • the expression cells were lysed by mild sonication in the lysis buffer (50 mM Tris-HCI, PH7.5, 2.5 mM EDTA, 150 mM NaCI, 1% NP-40, 0.1% ⁇ -mercaptoethanol, 0.5 mM sodium orthovanadate, 50 mM ⁇ -glycerophosphate, 1 mM benzamidine and 0.5 %(V ⁇ ) protease inhibitor cocktail set III (CalBiochem)).
  • the lysate was cleared of cellular debris by centrifugation.
  • the active recombinant protein was purified from the supernatant using a GST-glutathione affinity system according the manufacturer's instructions (Pharmacia).
  • the matrix was transferred to a 1X10 cm Flex-column (Kontes Glass) chromatography system.
  • the column was washed with high-salt buffer (50 mM Tris-HCI, PH7.5, 1 mM EDTA, 500 mM NaCI, 0.1% NP-40, 0.1% ⁇ -mercaptoethanol, 0.5 mM sodium orthovanadate, 50 mM ⁇ -glycerophosphate, 1 mM benzamidine and 0.1 mM PMSF), then low-salt buffer (50 mM Tris-HCI, PH7.5, 1 mM EDTA, 50 mM NaCI, 0.1% ⁇ - mercaptoethanol, 0.5 mM sodium orthovanadate, 50 mM ⁇ -glycerophosphate, 1 mM benzamidine and 0.1 mM PMSF).
  • high-salt buffer 50 mM Tris-HCI, PH7.5, 1 mM EDTA, 500 mM NaCI, 0.
  • the GST-PKB ⁇ fusion protein was released from the matrix using a glutathione buffer (50 mM Tris-HCI, PH7.5, 50 mM NaCI, 10 mM glutathione, 0.1% ⁇ -mercaptoethanol and 0.1 mM PMSF).
  • a complete quantitative and qualitative analysis of the protein was monitored using Coomassie blue staining and Western blot analysis using anti-GST and anti-PKB-PH specific antibodies (Kinetek).
  • PKB ⁇ protein comprised approximately 5% to 7% of the total insect cellular protein concentration.
  • Biochemical analysis of the activated enzyme was performed on recombinant bovine GST-PKB ⁇ fusion protein using the experimental protocol outlined in the section entitled "IN VITRO ACTIVITY PROFILE FOR KINASES".
  • the GST-PKB ⁇ preparations were found to exhibit protein phosphotransferase activity in the order of about 200 pmol/min/ug in the presence of 50 uM [ ⁇ - 32 P]-ATP and 162 uM PKB substrate peptide (amino acid sequence: CKRPRAASFAE) during a 15 min reaction at ambiant temperature.
  • Inhibition of the PKB in vitro was measured by scintillation counting, which involves the incorporation of radioactive phosphate onto a specific substrate that is immobilized onto a filter paper at the end of the assay.
  • the assay was performed in the absence and presence of specific and known inhibitors of PKB (as controls), and the amount of incorporated radioactivity was compared.
  • Test compounds were lyophilized and stored at -20°C. Stock solutions were made by weighing out the compounds and dissolving them in dimethyl sulfoxide (DMSO) to a standard concentration, usually 20 mM, and stored at -20°C.
  • DMSO dimethyl sulfoxide
  • the compounds were diluted to a starting intermediate concentration of 250 ⁇ M in 1 % DMSO, then serially diluted across a row of a 96 well plate using serial 2 fold dilution steps. Diluted 100% DMSO was used as a negative control. 5 ⁇ L of each compound dilution were robotically pipetted to Costar brand serocluster plates maintaining the same plate layout. All assay mixtures consisted of the following volumes: 5 ⁇ L diluted compound 10 ⁇ L target enzyme preparation 1 ⁇ L substrate
  • the “baseline activity” is the amount of radioactivity incorporated in the absence of a target inhibitor.
  • the amount of radioactivity incorporated in the presence of a target inhibitor is called the “sample activity”, and the % inhibition is expressed by the following formula:
  • the IC 50 of an inhibitor is estimated (i.e. the concentration at which enzymatic activity is reduced by 50%).
  • the IC 50 of various inhibitors against a particular target can be compared, where a lower IC 50 indicates a more potent inhibitor.
  • tumour cells are harvested cells as per normal procedures: i.e. trypsinize, centrifuge and count cells. A volume of 90 ⁇ L is used to seed 5,000 cells/well in a 96 well plate. Cells are incubated for 24 hours at 37°C under 5% CO 2 . After incubation, cells should be 80-90% confluent.
  • 3 H-thymidine (Amersham) is diluted in cell culture media to a concentration of 100 ⁇ Ci/mL.
  • the test compound is diluted in the thymidine broth to 10X the final desired concentration.
  • the principle of this assay is based on the presence of ubiquitous intracellular esterase activity found in live cells.
  • esterase By enzymatic reaction of esterase, non-fluorescent cell-permeant calcein AM is converted to the intensely fluorescent calcein.
  • the polyanionic dye calcein is retained within live cells, producing a green fluorescence in live cells. It is a faster, safer, and better-correlated indicator of cytotoxicity than alternative methods (e.g. 3H-Thymidine incorporation).
  • Calcein AM is susceptible to hydrolysis when exposed to moisture. Therefore, prepare aqueous working solutions containing calcein AM immediately prior to use, and used within about one day.
  • a kit available to do this assay is "LIVE/DEAD® Viability/Cytotoxicity Kit (L-3224)" by Molecular Probes.
  • Cells were collected from tissue culture flasks and trypsinized, centrifuged, resuspended and counted. Cells were seeded to obtain 80-90% confluence (for normal cells, 10,000 cells/well (8000 cells/well for HUVEC cells)). A cell concentration of 110,000 cells/mL (88,000 cells/well for HUVEC cells) is prepared as 90 ⁇ L volume is used per well.
  • cell culture media e.g., RPMI + 10%FBS
  • 10X compound solution of final desired concentration from 20 mM stock compounds was prepared.
  • 10 ⁇ l of this 10X compound solution is added to the 90 ⁇ L of cells already present in the 96 well plates and a known cytotoxic compound from previous testing is used as a positive control.
  • the negative control is 100% DMSO diluted to the same factor as the compounds.
  • the plates are incubated at 37°C for approximately 24 hours, and media is aspirated after plates are spun at 2400 rpm for 10 min at ambient temperature. 100 ⁇ L of 1X DPBS (without calcium chloride, without magnesium chloride (GibcoBRL, cat#14190-144)) is added to each well.
  • 1X DPBS without calcium chloride, without magnesium chloride (GibcoBRL, cat#14190-144)
  • Annexin V assay is used as a measure of apoptosis in cells treated with cytotoxic compounds. Cells undergoing apoptosis have many characteristic changes, and one of these is the externalization of cell membrane phosphatidylserine. Annexin V is a protein that has a very high affinity for phosphatidylserine, and as an assay reagent conjugated to fluorescence such as FITC or PE, it can be used to label phosphatidylserine on the membrane surface.
  • Cultured tumour cells are harvested cells as per normal procedures: i.e. trypsinize (if adherent), centrifuge and count cells. Cells are adjusted to 9 x 10 4 cells/ml for adherent cells (ie. A549, B16-F1, or H460), or 1 x 10 6 cells/ml for non-adherent cells (ie. Jurkat). For adherent cells, 1.8ml of cells are seeded into 6-well tissue culture plates and incubated overnight at 37°C and 5% CO 2 . For non-adherent cells, pre-seeding is not required, and on the day of treatment, 450ul of cells are seeded into 24-well tissue culture plates.
  • Compounds are prepared to 10X final concentration, along with DMSO controls and a positive control (ie. Staurosporin).
  • a positive control ie. Staurosporin
  • To the 1.8ml of adherent cells in the 6-well dishes is added 200 ⁇ l of 0X stock, while 50 ⁇ is added to the 450 ⁇ l of suspension cells in the 24-well dishes. Cells are treated overnight at 37°C and 5% CO 2 , although this time may be adjusted as required for the compound/cell line to obtain an optimal percentage of stained cells.
  • cells are harvested by collecting all cells, centrifuging, and washing in ice-cold PBS before resuspending in 1X binding buffer (BD Pharmingen, Cat# 66121 E) at a concentration of 1 x 10 6 cells/ml.
  • 1X binding buffer BD Pharmingen, Cat# 66121 E
  • 5ml standard FACS tubes ie. Falcon, Cat# 352008
  • 5 ⁇ l of cell suspension (1 x 10 5 cells
  • 5 ⁇ l of Annexin V-PE BD Pharmingen, Cat # 65875H or 65875X
  • 7-AAD BD Pharmingen, Cat# 68981 E
  • Controls are set up for use in setting up compensation and quadrants, including unstained cells, cells stained with Annexin V-PE only, and cells stained with 7-AAD only. Samples are incubated in the dark at room temperature for 15 minutes. To each tube is then added 400 ⁇ l of 1X binding buffer and flow cytometry acquisition using laser excitation at 488nm is carried out immediately (within 1-2 hours for all samples). Cells undergoing early stage apoptosis are quantitated as staining Annexin V positive and 7-AAD negative. Necrotic cells and/or late stage apoptotic cells are Annexin V and 7-AAD double positive.
  • the percentage of Annexin V positive cells (without 7-AAD staining) is calculated to confirm that the mechanism of action of cytotoxicity was apoptosis.
  • the total percentage of Annexin V positive cells (with or without 7-AAD staining) is calculated to determine the degree of apoptosis occurring as a result of compound treatment.
  • Cultured tumour cells are harvested cells as per normal procedures: i.e. trypsinize (if adherent), centrifuge and count cells. Cells are adjusted to 5.5 x 10 4 cells/ml and seeded at 90ul/well into 96-well plates. Cells are incubated overnight at 37°C, 5% CO 2 . Compounds are prepared to 10X final concentration in cell culture media (serum free), along with DMSO controls and a positive control. To each well is added 10ul of this 10X stock solution and the cells incubated for 24-48 hours at 37°C, 5% CO 2 .
  • MTS solution is prepared by first preparing PMS solution, dissolving 0.92g of PMS (phenazine methosulfate) powder (Sigma, Cat# P-9625) in 1.0L of 1X PBS (GibcoBRL, Cat# 14190-144). The solution is filter sterilized with a 0.2um filter. The final MTS reagent is prepared by dissolving 4.0g of MTS powder (Fisher Sci, Cat# G1111) in 1.8L of 1X PBS (GibcoBRL, Cat# 14190-144). The pH is adjusted to pH 6.2 and the solution made up to a volume of 2L. To the 2L is added 100ml of PMS solution and the mixture filtered.
  • PMS phenazine methosulfate
  • MTS reagent Before addition of MTS reagent to the 96-well plates, plates are inspected under the microscope and observations made on the health of the cells. To each well is slowly added 20ul of MTS reagent and the plates are further incubated for 2-4 hours (depending on the cell type and number) at 37 C C, 5% CO 2 . The plates are read using a microplate reader (ie. BIO-TEK Instruments Inc., EL312e) at 490 nm wavelength. The amount of cytotoxicity is determined by the following formula:
  • % inhibition 100 - [(AVG treatment -AVG positive control)/100(AVG negative control - AVG positive control) ]
  • EXAMPLE 6 IN vivo TUMOUR EFFICACY STUDY. To test the efficacy of test compounds on H460 subcutaneous xenograft alone and in combination with doxorubicin.
  • Athymic nude female mice are used for this experiment.
  • a group of 60 mice are inoculated with five million H460 cells in 100 ⁇ L MatrigelTM(VWR Canada) excipient.
  • Tumours are measured three times a week with digital calipers and the tumour volumes calculated. When tumours have reached an average size of 100 mm 3 , about two weeks after tumour implantation. At that time any nongrowing Outliers' are removed so that animals can be distributed into groupings that are equal and statistically the same tumour mass, i.e. divided into six groups with about 10 mice per group.
  • Treatments with test compounds continue for about 20 days, and will be oral (gavage), intravenous, subcutaneous, or intraperitoneal depending on the known solubility of the test compound.
  • a dose of 25mg/kg is typical for such testing, but the dose selected will reflect the potency of the compound and the route of administration.
  • Positive controls may alternately be doxorubicin or cisplatin, or cyclophosphamide.
  • mice are anesthetized 3 hours after the last dose of test compound, and plasma and tissues are harvested and frozen. Tumours are divided into the desired number of aliquots and fast frozen for later analysis.
  • the cell lines used are HT 1080 (ATCC, Cat# CCL - 121), DU-145 (ATCC, Cat# HTB-81), PC3 (ATCC, Cat# CRL-1435) or B16F1 (ATCC, Cat# CRL-6323).
  • the invasion test system is removed from the package from -20°C storage and allowed to warm to ambient temperature. PBS is added to the interior of the inserts and they are allowed to rehydrate for 2 hours at 37 C C. Then the medium is removed and 450 ⁇ L cell suspensions of tumour cells (grown to 50-70% confluence, trypsinized, and resuspended in medium without serum at 1 x 10 6 /mL) is added to the top chamber. Test compounds are added to the top chamber at 10X the desired final concentration in 50 ⁇ L volumes. DMSO acts as control.
  • the insert plate is transferred into a second 24-well plate containing 0.5 mL of 5 ⁇ g/mL calcein AM (Molecular Probes) in Hanks buffered salt solution (HBSS), and plates are incubated for 1 hour at 37°C, 5% CO 2 .
  • calcein AM Molecular Probes
  • HBSS Hanks buffered salt solution
  • Fluorescence data indicating cell invasion is read in a Fluoroskan Ascent FLTM (LabSystems) with bottom reading at excitation/emission wavelength of 485/538 nm.
  • FU fluorescence unit
  • EXAMPLE 8 PERITONEAL MACROPHAGE STIMULATION AND ANALYSIS A. Establishment of inflammation assay panel.
  • Macrophages are important elements of innate immunity to infection and are among the first cell type in the immune response to be exposed to and activated by infectious agents.
  • IFN- ⁇ and LPS are potent activators of macrophages, priming them for a variety of biological effects.
  • IFN- ⁇ initially secreted by NK and T cells in response to infection, converts macrophages from a resting to an activated state (inflammatory macrophages), priming them for antimicrobial activity manifested by increased killing of intracellular pathogens, and antigen processing and presentation to lymphocytes.
  • IFN- ⁇ is synergized with the LPS second messenger, enhancing the stimulation of macrophages through the activation of NF- ⁇ B, that results in the transcriptional up-regulation of a number of genes involved in the cell-mediated immune response, including inducible iNOS (nitric oxide synthase).
  • Activated macrophages are qualitatively different from quiescent macrophages. These differences are typically observed by an increased proliferation index, up-regulated expression of MHC-II, and production of various bioactive molecules. The latter biological effects are mediated by NO (nitric oxide) release and increased production of pro-inflammatory cytokines (IL-6, TNF- ⁇ , IL-1).
  • Primary macrophages derived from Balb/c mice and RAW 264.7 cells (Balb/c background) were used to establish in vitro inflammatory models with fast and reliable readouts.
  • iNOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and murine rlFN- ⁇ are purchased from Calbiochem, (San Diego, CA). Protein-free, phenol/water-extracted LPS (from E. coli serotype 0111 :B4 0127:B8), Zymosan ATM, dexamethasone and hydrocortisone, sulfanilar ⁇ ide and N-(1-naphthyl)-ethylenediamine, arare purchased from Sigma (St. Louis, MO). Human recombinant vascular endothelial growth factor (VEGF) is purchased from R&D Systems (Minneapolis, MN).
  • VEGF vascular endothelial growth factor
  • ELISA dual-set kit for detection of IL-6 is purchased from PharMingen (San Diego, CA).
  • Anti-murine iNOS/NOS type II and cyclooxygenase-2 (COX-2) antibodies are obtained from Transduction Laboratories (Lexington, KY).
  • mice Female BALB/c inbred mice, 6-12 weeks of age, are purchased from Harlan Inc. (Indianapolis, IN) and housed under fluorescent light for 12 h per day. Mice are housed in cages, and maintained in compliance with the Canadian Council on Animal Care standards.
  • Peritoneal exudate macrophages are isolated by peritoneal lavage with ice-cold sterile physiological saline 24 hours after intraperitoneal injection of BALB/c mice with 0.5 mL of sterile Zymosan ATM (1 mg/0.5 mL 0.9% saline). Cells are ished, resuspended in RPM1 1640 supplemented with 1 mM D-glucose, 1mM sodium pyruvate, 100 units/mL penicillin, 100 ⁇ g/mL streptomycin, and 5% FBS.
  • Primary macrophages (1.5 * 105 cells/well) are grown in 96-well plates (nitrite assay), or 6-well plates (2 * 106 cells/well) for measurement of iNOS and COX-2 expression. Following 3 hours incubation, at 37°C, 5% CO 2 (allowing macrophages to attach) cells are stimulated with LPS (5 ⁇ g/mL) and IFN- ⁇ (100 U/mL) in the absence or presence of various concentrations of test compounds (all treatments are replicated six times). Cells are incubated for an additional 24 hours, and cell free culture supernatants from each well are collected for NO and cytokine determination. The remaining cells are stained with crystal violet or MTS to determine effect of the test compounds on cell survival.
  • NO production Following stimulation, the production of NO is determined by assaying culture supernatants for NO 2 , a stable reaction product of NO with molecular oxygen. Briefly, 100 ⁇ L of culture supernatant is reacted with an equal volume of Griess reagent at ambient temperature for 10 minutes. The absorbance at 550 nm is determined. All measurements are performed six times. The concentration of NO 2 is calculated by comparison with a standard curve prepared using NaNO 2 . 5. Western blot analysis.
  • cells (duplicate samples, 2x10 6 cell/6-wells plate) are washed in PBS and lysed on ice in 60 ⁇ L of lysis buffer.
  • the protein content of each sample is determined using the Bradford protein assay kit (Bio-Rad, Richmond, CA). Absorbance is measured at 750 nm with a Beckman DU530 spectrophotometer (Palo Alto, CA). Proteins are mixed with 45xSDS sample buffer. Following separation of proteins by SDS-PAGE, using 8% bis-acrylamide in the separation gel, the proteins are transferred from the gels onto PVDF membranes using a MiniProteanTM III Cell (Bio-Rad), at 100 V for 1.5 hours.
  • Equal amounts of protein (5 ⁇ g) are loaded onto SDS-PAGE gels and examined by Western blot analysis with anti-actin, anti-iNOS, anti-COX-2 murine monoclonal antibodies, according to the manufacturer's specifications (Transduction Laboratories).
  • Primary antibodies in 5% blocking buffer (5% NFM TTBS), are incubated with blots 2 hours or overnight at 4°C, followed by incubation with peroxidase-conjugated secondary antibody.
  • Chemiluminescence substrates are used to reveal positive bands. The bands are exposed on X-ray films.
  • the films are used to analyze the impact of inhibitors on expression of iNOS and COX-2 compared to various controls and "house-keeping" protein (Actin) concentration to control the protein loading and detect any non-specific effects on protein production.
  • the Multi-AnalystTM/PC system from Biorad is used to quantitate the bands of the expressed protein on the film.
  • This version of Multi-Analyst is used with the Bio-Rad Gel Doc 1000TM imaging system.
  • White light is chosen as the selected light source, thus the signal strength is measured in OD (optic density) units. The OD of each band is being subtracted to a global background area of the gel.
  • HUVEC cells cultured for 24 hours in M199 with 0.5% FCS are plated at 6 x 105 cells/well in 12-well plates pre-coated with 300 ⁇ L of Matrigel (10.7 mg/mL; Becton Dickinson) in M199 with 0.5% FCS in the presence of VEGF (1ng/mL), and in the absence or presence of positive control (Z)-3-[2,4-dimethyl-5-(2-oxo-1 ,2-dihydroindol- 3-ylidenemethyl)-1/V-pyrrol-3-yl]propionic acid or various inhibitors.
  • IL-6 levels are determined with PharMingen's OptEIATM ELISA set developed using an anti-mouse IL-6 Ab pair and mouse rlL-6 standard (PharMingen). Maxisorp F16 multiwell strips (Nunc, Roskilde, Denmark) are coated with anti-mouse IL-6 capture Ab (at recommended concentration) in 0.1 M NaHCO 3 , pH 9.5, 100 ⁇ L ⁇ /vell, overnight at 4°C. Plates are washed three times with 0.05% Tween 20 in PBS (PBST) and blocked for 1 hour at ambient temperature with 200 ⁇ lJwell of 10% FCS in PBS (blocking and dilution buffer).
  • Plates are washed three times with PBST and duplicate samples (100 ⁇ lJwell) or standards (100 ⁇ lJwell) in diluent buffer are incubated for 2 hours at ambient temperature. Plates are washed five times with PBST and incubated with biotinylated anti-mouse IL-6 and avidin-horseradish peroxidase conjugate (at concentrations recommended by the manufacturer) for 1 hour at ambient temperature. Plates are washed seven times with PBST and 100 ⁇ L of 3,3'5,5' tetramethylbenzidine substrate solution (TMB substrate reagent set, BD PharMingen) is added to each well.
  • TMB substrate reagent set 3,3'5,5' tetramethylbenzidine substrate solution

Abstract

Thiazolidinedione derivatives of the formula (I), methods of using thiazolidinedione derivatives in therapeutic and prophylactic methods for the treatment of hyperproliferative disorders, inflammation, and other disorders associated with overexpression of Akt, as well as pharmaceutical compositions containing such derivatives are disclosed.

Description

THIAZOLIDINEDIONE DERIVATIVES IN THERAPEUTIC AND PROPHYLACTIC
METHODS
BACKGROUND OF THE INVENTION
Field of the Invention
This invention is directed to thiazolidinedione derivatives, pharmaceutical compositions containing thiazolidinedione derivatives, and methods of using thiazolidinedione derivatives for therapeutic and/or prophylactic effect in mammals. Description of the Related Art
Protein phosphorylation is a regulatory mechanism used by cells to selectively modify proteins carrying regulatory signals to the nucleus from outside the cell. The proteins that mediate protein phosphorylation are a group of enzymes known as protein kinases and protein phosphatases, and as critical members of essential biological pathways, are sometimes associated with disease. While a plethora of extracellular molecules exist that modulate cellular functions via binding to membrane receptors inside the cell, their actions are mediated by relatively few signaling mechanisms within the cell. One of these mechanisms is activation of phosphatidylinositol 3-kinase (PI-3K), which results in the generation of membrane-restricted second messenger polyphosphatidyl- inositides containing a 3'-phosphate, and the activation of protein kinase B (PKB).
Activated PKB mediates a number of metabolic effects of insulin; and protects cells from apoptosis (Kane et al. (1999), Curr Biol 9(11): 601-4).
Most proliferating cells are programmed to undergo apoptosis unless specific survival signals are provided. Survival factors can suppress apoptosis in a transcription- independent manner by activating the serine/threonine kinase PKB, which then phosphorylates and inactivates components of the apoptotic machinery. Survival factor withdrawal triggers apoptosis by inducing the expression of genes that are critical for cell death.
Signal transduction also plays a key regulatory role in the growth and metastatic potential of tumor cells. These signaling pathways form an interconnecting grid that serves to regulate the homeostatic, survival and invasive functions of the cell. Among the key regulatory molecules in these pathways are the serine/threonine-protein kinases cyclic AMP-dependent protein kinase (PKA), Akt (PKB) and protein kinase C (PKC). These protein kinases modulate pathways associated with tumor proliferation, cell survival and multidrug resistance, and at a molecule level are likely to serve as effective targets for drug design.
The protein kinase B sequence is described by Coffer and Woodgett (1991) Eur. J. Biochem. 201(2): 475-481, and may be found in Genbank, indexed as accession number X61037. PKB is a widely expressed cytoplasmic serine-threonine kinase, and its aberrant expression has been implicated in tumorigenesis (Testa and Bellacosa (2001) PNAS 98(20): 10983-10985; Nicholson and Anderson (2002) Cellular Signalling 14(5):381-395). PKB contains at its NH2-terminus a domain termed the pleckstrin homology (PH) domain, which may regulate the activation of PKB by binding D3- phosphorylated phosphoinositides that are the products of PI3-K (Franke et al., (1997) Science 275(5300): 665-668). Phosphorylation of PKB also influences its activation, and the PH domain may influence the activation of PKB by promoting its translocation to the plasma membrane (Andjelkovic, M. et al., (1997) J. Biol. Chem. 272(50):31515-31524). Phosphorylation of GSK-3 by PKB is believed to regulate glycogen synthesis (Cross et al., (1995), Nature 378(6559):785-789). Activation of PKB is blocked by wortmannin and by LY294002 (Alessi et al., (1996) EMBO J. 15:6541-6551; King et al., (1997) Mol. Cell Biol. 17(8):4406-4418). BRIEF SUMMARY OF THE INVENTION
This invention is directed to certain thiazolidinedione compounds, pharmaceutical compositions containing certain thiazolidinedione compounds, and the use of certain thiazolidinedione compounds in treating hyperproliferative disorders, e.g., cancer, inflammation, etc. in a mammal. Of particular interest are hyperproliferative disorders associated with cellular modulation of protein phosphorylation states, e.g. altered activity of protein tyrosine kinases.
In one embodiment, compounds and pharmaceutical compositions of the invention are used to treat inflammation and cell migration in a mammal. In another embodiment, compounds and pharmaceutical compositions of the invention are used to treat cancer, angiogenesis, and neurological disorders in a mammal.
In another embodiment of the invention, the compounds and pharmaceutical compositions of the invention are used to prevent inflammation, cell migration, cancer, angiogenesis, and neurological disorders in a mammal.
In another embodiment of the invention, the compounds and pharmaceutical compositions of the invention are used to promote apoptosis in proliferating cells in a mammal.
In one embodiment, compounds and pharmaceutical compositions of the invention are used to inhibit the activity of protein kinase B (PKB). This enzyme has been associated with alterations in the phosphorylation state of cellular proteins.
In another embodiment of the invention, the use of compounds and pharmaceutical compositions of the invention for the treatment or prophylaxis of disease is provided, or for the manufacture of medicaments for the treatment or prophylaxis of disease.
Compounds of formula (1) are defined as follows:
Figure imgf000004_0001
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2> -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form /=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons; R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl; R9 is hydrogen, alkyl or aralkyl; and
R 0 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoiso ers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
Compounds of formula (2) are defined as follows:
Figure imgf000004_0002
wherein, independently at each occurrence: E is selected from the group consisting of O and S; each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O),R9 (where t is 0 to 2), -S(O)tN(R9)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R 0)(R11); each of R4, R5 and R6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R8 is hydrogen, alkyl or aralkyl; each R9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R10 is hydrogen, alkyl or aralkyl; and
R11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof. DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a compound" refers to one or more of such compounds, while "the enzyme" includes a particular enzyme as well as other family members and equivalents thereof as known to those skilled in the art. As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated.
"Alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to twenty five carbon atoms, and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (/so-propyl), n-butyl, π-pentyl, 1 ,1-dimethylethyl (/-butyl), and the like. Unless stated otherwise specifically in the specification or claims, in various aspects the alkyl radical optionally has: 1 to 25 carbons, i.e., is a C1-C25 alkyl radical, or is a Cι-C20 alkyl radical, or is a C C15 alkyl radical, or is a Cι-C10 alkyl radical, or is a lower alkyl radical, i.e., a Cι-C6 alkyl radical, or is a C2-C25 alkyl radical, or is a C2- C20 alkyl radical, or is a C2-C15 alkyl radical, or is a C2-C10 alkyl radical, or is a C2-C6 alkyl radical. Unless stated otherwise specifically in the specification, and independent of the number of carbon atoms in the alkyl group, the alkyl radical may be optionally substituted by hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro, mercapto, alkylthio, -NHOH, -N=N-O-Re, -N(Rd)2, -C(O)ORd, -C(O)N(Rd)2, S(O)tRd (where t is 0 to 2), S(O)tN(Rd)2 (where t is 0 to 2), or -N(Rd)C(O)Rd where each Rd is independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl, and Re is hydrogen, alkyl or aralkyl. In one aspect, the alkyl radical is unsubstituted. In another aspect, the alkyl radical has a single substituent. In another aspect, the alkyl radical has two substituents. Unless stated otherwise specifically in the specification, it is understood that for radicals, as defined below, that contain a substituted alkyl group that the substitution can occur on any carbon of the alkyl group. "Alkenyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing at least one double bond, having from two to eight carbon atoms, and which is attached to the rest of the molecule by a single bond or a double bond, e.g., ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1 ,4-dienyl, and the like. Unless stated otherwise specifically in the specification or claims, in various aspects the alkenyl radical optionally has: 1 to 25 carbons, i.e., is a C C25 alkenyl radical, or is a C C20 alkenyl radical, or is a CrC15 alkenyl radical, or is a Cι-C10 alkenyl radical, or is a lower alkenyl radical, i.e., a C C6 alkenyl radical, or is a C2-C 5 alkenyl radical, or is a C2-C20 alkenyl radical, or is a C2-C15 alkenyl radical, or is a C2-C10 alkenyl radical, or is a C2-C6 alkebyl radical. Unless stated otherwise specifically in the specification, and independent of the number of carbon atoms that forms the alkenyl radical, the alkenyl radical may be optionally substituted by hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto, alkylthio, cycloalkyl, -NHOH, -N=N-O-Re, -N(Rd)2, -C(O)ORd, -C(O)N(Rd)2, S(O)tRd (where t is 0 to 2), S(O)tN(Rd)2 (where t is 0 to 2),or -N(Rd)-C(O)-Rd where each Rd is independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl, and Re is hydrogen, alkyl or aralkyl. In one aspect, the alkenyl radical is unsubstituted. In another aspect, the alkenyl radical has a single substituent. In another aspect, the alkenyl radical has two substituents. Unless stated otherwise specifically in the specification, it is understood that for radicals, as defined below, that contain a substituted alkenyl group that the substitution can occur on any carbon of the alkenyl group.
"Aryl" refers to a phenyl or naphthyl radical. Unless stated otherwise specifically in the specification, the term "aryl" or the prefix "ar-" (such as in "aralkyl") is meant to include aryl radicals optionally substituted by one or more substituents selected from the group consisting of hydroxy, alkoxy, aryloxy, haloalkoxy, cyano, nitro, mercapto, alkylthio, cycloalkyl, -NHOH, -N=N-0-Re, -N(Rd)2, -C(O)ORd, -C(O)N(Rd)2, S(O)tRd (where t is 0 to 2), S(O),N(Rd)2 (where t is 0 to 2),or -N(Rd)C(O)Rd where each Rd is independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl, and Re is hydrogen, alkyl or aralkyl. In one aspect of the invention, the aryl radical is unsubstituted, i.e., is substituted only with hydrogens. In another aspect of the invention, the aryl radical has a single substituent. In a further aspect of the invention, the aryl radical has two substituents. "Aralkylene" refers to a radical of the formula -RaRb where Ra is an alkylene radical as defined above and Rb is one or more aryl radicals as defined above, e.g., benzyl, diphenylmethyl and the like. The aryl radical(s) may be optionally substituted as described above. In one aspect of the invention, the aralkylene radical is unsubstituted. In another aspect the aralkylene radical has a single substituent. "Aralkenylene" refers to a radical of the formula -R0Rb where Rc is an alkenylene radical as defined above and Rb is one or more aryl radicals as defined above, e.g., 3-phenylprop-1-enyl, and the like. The aryl radical(s) and the alkenyl radical may be optionally substituted as described above.
"Alkylene" and "alkylene chain" refer to a straight or branched divalent hydrocarbon chain consisting solely of carbon and hydrogen, containing no unsaturation and having from one to eight carbon atoms, e.g., methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain may be optionally substituted by one or more substituents selected from the group consisting of aryl, halo, hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto, alkylthio, cycloalkyl, -NHOH, -N=N-O-Re, -N(Rd)2, -C(O)ORd, -C(O)N(Rd)2, S(0)tRd (where t is 0 to 2), S(O)tN(Rd)2 (where t is 0 to 2),or -N(Rd)C(O)Rd where each Rd is independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl, and Rβ is hydrogen, alkyl or aralkyl. The alkylene chain may be attached to the rest of the molecule through any two carbons within the chain.
"Alkenylene chain" refers to a straight or branched divalent hydrocarbon chain consisting solely of carbon and hydrogen, containing at least one double bond and having from two to eight carbon atoms, e.g., ethenylene, prop-1-enylene, but-1-enylene, pent-1-enylene, hexa-1,4-dienylene, and the like. The alkenylene chain may be optionally substituted by one or more substituents selected from the group consisting of aryl, halo, hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto, alkylthio, cycloalkyl, - NHOH, -N=N-O-Re, -N(Rd)2> -C(O)ORd, -C(O)N(Rd)2, S(O),Rd (where t is 0 to 2),
S(O)tN(Rd)2 (where t is 0 to 2),or -N(Rd)C(O)Rd where each Rd is independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl, and Re is hydrogen, alkyl or aralkyl. The alkenylene chain may be attached to the rest of the molecule through any two carbons within the chain. "Cycloalkyl" refers to a stable monovalent monocyclic or bicyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, having from three to ten carbon atoms, and which is saturated and attached to the rest of the molecule by a single bond, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, decalinyl and the like. Unless otherwise stated specifically in the specification, the term "cycloalkyl" is meant to include cycloalkyl radicals which are optionally substituted by one or more substituents independently selected from the group consisting of alkyl, aryl, aralkyl, halo, haloalkyl, hydroxy, alkoxy, haloalkoxy, cyano, nitro, mercapto, alkylthio, cycloalkyl, -NHOH, -N=N-O-Re, -N(Rd)2, -C(O)ORd, -C(O)N(Rd)2, S(O)tRd (where t is 0 to 2), S(O)tN(Rd)2 (where t is 0 to 2),or -N(Rd)C(O)Rd where each Rd is independently hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl, and Re is hydrogen, alkyl or aralkyl. In various aspects of the invention, the cycloalkyl radical has 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 carbons. Independent of the number of carbons in the cycloalkyl radical, in various aspects the cycloalkyl radical has 0, or 1, or 2 or 3 substituents.
"Cycloalkylalkylene" refers to a radical of the formula -RaRd where Ra is an alkylene radical as defined above and Rd is a cycloalkyl radical as defined above. The alkyl radical and the cycloalkyl radical may be optionally substituted as defined above. "Halo" refers to bromo, chloro, fluoro or iodo. "Haloalkyl" refers to an alkyl radical, as defined above, that is substituted by one or more halo radicals, as defined above, e.g., trifluoromethyl, difluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, 1-bromomethyl-2-bromoethyl, and the like.
"Haloalkoxy" refers to a radical of the formula -ORc where Rc is an haloalkyl radical as defined above, e.g., trifluoromethoxy, difluoromethoxy, trichloromethoxy, 2,2,2-trif luoroethoxy, 1 -fluoromethyl-2-fluoroethoxy, 3-bromo-2-fluoropropoxy, 1-bromomethyl-2-bromoethoxy, and the like.
"Heterocyclyl" refers to a stable 3- to 15-membered ring radical which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. For purposes of this invention, the heterocyclyl radical may be a monocyclic, bicyclic or tricyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be aromatic or partially or fully saturated. The heterocyclyl radical may not be attached to the rest of the molecule at any heteroatom atom. Examples of such heterocyclyl radicals include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzothiadiazolyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, carbazolyl, cinnolinyl, decahydroisoquinolyl, dioxolanyl, furanyl, furanonyl, isothiazolyl, imidazolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, indolizinyl, isoxazolyl, isoxazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, oxazolyl, oxazolidinyl, oxiranyl, piperidinyl, piperazinyl, 4-piperidonyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, thiazolyl, thiazolidinyl, thiadiazolyl, triazolyl, tetrazolyl, tetrahydrofuryl, triazinyl, tetrahydropyranyl, thienyl, thiamorpholinyl, thiamorpholinyl sulfoxide, and thiamorpholinyl sulfone. Unless stated otherwise specifically in the specification, the term "heterocyclyl" is meant to include heterocyclyl radicals as defined above which are optionally substituted by one or more substituents selected from the group consisting of alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -NHOH, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl, wherein each R6, R7 and R8 are as defined above in the Summary of the Invention.
"Heterocyclylalkyl" refers to a radical of the formula -RaRe where Ra is an alkyl radical as defined above and Re is a heterocyclyl radical as defined above, and if the heterocyclyl is a nitrogen-containing heterocyclyl, the heterocyclyl may be attached to the alkyl radical at the nitrogen atom. The heterocyclyl radical may be optionally substituted as defined above.
As used herein, compounds which are "commercially available" may be obtained from standard commercial sources including Acros Organics (Pittsburgh, PA), Aldrich Chemical (Milwaukee, Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park, UK), Avocado Research (Lancashire, U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester, PA), Crescent Chemical Co. (Hauppauge, NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, PA), Fisons Chemicals (Leicestershire, UK), Frontier Scientific (Logan, UT), ICN Biomedicals, Inc. (Costa Mesa, CA), Key Organics (Cornwall, U.K.), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. Ltd. (Cornwall, U.K.), Parish Chemical Co. (Orem, UT), Pfaltz & Bauer, Inc. (Waterbury, CN), Polyorganix (Houston, TX), Pierce Chemical Co. (Rockford, IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland, OR), Trans World Chemicals, Inc. (Rockville, MD), and Wako Chemicals USA, Inc. (Richmond, VA).
As used herein, "suitable conditions" for carrying out a synthetic step are explicitly provided herein or may be discerned by reference to publications directed to methods used in synthetic organic chemistry. The reference books and treatise set forth above that detail the synthesis of reactants useful in the preparation of compounds of the present invention, will also provide suitable conditions for carrying out a synthetic step according to the present invention. As used herein, "methods known to one of ordinary skill in the art" may be identified though various reference books and databases. Suitable reference books and treatise that detail the synthesis of reactants useful in the preparation of compounds of the present invention, or provide references to articles that describe the preparation, include for example, "Synthetic Organic Chemistry", John Wiley & Sons, Inc., New York; S. R. Sandier et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H. O. House, "Modern Synthetic Reactions", 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, "Heterocyclic Chemistry", 2nd Ed., John Wiley & Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure", 4th Ed., Wiley-lnterscience, New York, 1992. Specific and analogous reactants may also be identified through the indices of known chemicals prepared by the Chemical Abstract Service of the American Chemical Society, which are available in most public and university libraries, as well as through on-line databases (the American Chemical Society, Washington, D.C., www.acs.org may be contacted for more details). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis houses, where many of the standard chemical supply houses (e.g., those listed above) provide custom synthesis services.
"Prodrugs" is meant to indicate a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound of the invention. Thus, the term "prodrug" refers to a metabolic precursor of a compound of the invention that is pharmaceutically acceptable. A prodrug may be inactive when administered to a subject in need thereof, but is converted in vivo to an active compound of the invention. Prodrugs are typically rapidly transformed in vivo to yield the parent compound of the invention, for example, by hydrolysis in blood. The prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam).
A discussion of prodrugs is provided in Higuchi, T., et al., "Pro-drugs as Novel Delivery Systems," A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated in full by reference herein.
The term "prodrug" is also meant to include any covalently bonded carriers which release the active compound of the invention in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound of the invention may be prepared by modifying functional groups present in the compound of the invention in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound of the invention. Prodrugs include compounds of the invention wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the compound of the invention is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of the invention and the like.
"Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
"Mammal" includes humans and domestic animals, such as cats, dogs, swine, cattle, sheep, goats, horses, rabbits, and the like.
"Optional" or "optionally" means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, "optionally substituted aryl" means that the aryl radical may or may not be substituted and that the description includes both substituted aryl radicals and aryl radicals having no substitution. "Pharmaceutically acceptable carrier, diluent or excipient" includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. "Pharmaceutically acceptable salt" includes both acid and base addition salts.
"Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
"Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
"Therapeutically effective amount" refers to that amount of a compound of formulae (1) or (2) which, when administered to a mammal, preferably a human, is sufficient to effect treatment, as defined below, for inhibiting cancer, inflammation, neurological disease, angiogenesis and cell migration in the mammal. The amount of a compound of formulae (1) or (2) which constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure. "Treating" or "treatment" as used herein covers the treatment of a hyperproliferative disease as disclosed herein, in a mammal, preferably a human, and includes: preventing cancer, inflammation, neurological disease angiogenesis and cell migration from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; inhibiting cancer, inflammation, neurological disease angiogenesis and cell migration, i.e., arresting its development; and/or relieving cancer, inflammation, neurological disease angiogenesis and cell migration, i.e., causing regression of the condition.
The compounds of formulae (1 ) and (2), or their pharmaceutically acceptable salts may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present invention is meant to include all such possible isomers, as well as, their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.
The nomenclature used herein for the compounds of formulae (I) and (2) is a modified form of the I.U.P.A.C. nomenclature system wherein the compounds are named herein as thiazolidinedione derivatives. Compounds
In one aspect, the present invention provides a compound of formula (1):
Figure imgf000013_0001
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(0)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form l=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
In one embodiment, the compounds of the present invention described by formula
(1) do not include the following "excluded compounds", where RN stands for Registry
Number as defined by the Chemical Abstract Service of the American Chemical Society
(Washington, D.C.)
Figure imgf000013_0002
Figure imgf000014_0001
Figure imgf000015_0002
In one embodiment, the compounds of the present invention described by formula (1) do not include homologs of the "excluded compounds" as identified above, where a homolog of an excluded compound differs by having, or not having, a methylene group relative to the excluded compound.
In one embodiment, the compounds of the present invention described by formula (1) do not include compounds wherein R3 is methyl when R4 is methyl and Rs is phenyl.
In one aspect, the present invention provides a compound of formula (2):
Figure imgf000015_0001
wherein, independently at each occurrence:
E is selected from the group consisting of O and S; each of R\ R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2,
-N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), -S(O),N(R9)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R and R2 together may form I=(R10)(R11); each of R4, R5 and R6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R8 is hydrogen, alkyl or aralkyl; each R9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R10 is hydrogen, alkyl or aralkyl; and R11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof. In one embodiment, the compounds of the present invention described by formula
(2) do not include the following "excluded compounds", where RN stands for Registry Number as defined by the Chemical Abstract Service of the American Chemical Society (Washington, D.C.).
Figure imgf000016_0001
Figure imgf000017_0001
Compounds of the present invention, and compounds useful in the pharmaceutical compositions and methods described herein may be made in a manner according to, or analogous to, that described in the following references: Minoru, M., Yakugaku Zasshi (1962), Vol. 82, pp. 36-45; Singh, S. R., Bokin Bovai (1984), Vol. 12(6), 273-277; Lesyk, R., Bollettino Chimico Farmaceutico (1998), Vol. 137(6), pp. 210-217; and Lesyk, R. B., Farm. Zh. (Kiev) (1996), Vol. 5-6, pp. 94-98. Alternatively, the compounds may be obtained from commercial sources or made from commercially available starting materials according to methods known to one of ordinary skill in the art, e.g., by a contract laboratory. Many companies that provide commercially available compounds will also provide contract laboratory services.
Compounds of present invention described by formula (1) can also prepared following the following Schemes. In Scheme 1, starting components may be obtained from sources such as Aldrich, or synthesized according to sources known to those of ordinary skill in the art, e.g., Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley Interscience, New York) and other texts identified herein. Groups R1, R2, R3, R4, R5 and E are selected from components as defined in the specification heretofore. R is selected from hydrogen, alkyl or metal ions, such as Na+, K+ etc.
REACTION SCHEME 1
Figure imgf000018_0001
Figure imgf000018_0002
(D) (1)
In general, compounds of formula (1) can be prepared according to schemes known to those of ordinary skill in the art. See, for example, Lesyk, R., Bollettino Chimico Farmaceutico (1998), Vol. 137(6), pp. 210-217. For instance, a compound of formula (C) may be prepared in situ by reacting carbon disulfide (i.e., a compound of formula (A)) with an amine compound of formula (B) in aqueous potassium hydroxide at room temperature.
Compounds of formula (D) may be obtained from a source such as Aldrich, or prepared according to schemes known to those of ordinary skill in the art. Compounds of formula (1 ) may then be prepared by adding an aqueous solution containing a compound of formula (D), most preferably as its alkali metal salt if R is H, to a solution containing the compound of formula (C) prepared as described above (the mole ratio of (C) to (D) being about 1 to 1). The admixture is stirred for two days at which point it may be acidified with hydrochloric acid and heated in a water bath for two hours. The solution may then be allowed to cool to room temperature and the compound of formula (1) may be isolated as a solid by filtration. The compound of formula (1) may be further purified by a technique know to those of ordinary skill in the art such as crystallization from a suitable solvent. REACTION SCHEME 2
Figure imgf000019_0001
(A") (B1) (1a)
In Scheme 2, starting components (A') and (B') may be obtained from a source such as Aldrich, or prepared according to schemes known to those of ordinary skill in the art. In general, compound (1a) can be prepared by reacting (B') (0.01 mole) with 0.01 mole of reagent (A') in 10-40 mL of glacial acetic acid, the admixture then being boiled for about 15 minutes. Upon cooling, the resulting precipitate may be isolated by filtration, washed with water and allowed to dry, affording a compound of formula (1a).
REACTION SCHEME 3
Figure imgf000019_0002
In general, compounds of formula (E) can be prepared following the procedure described in Scheme 1. Thus, compounds of formula (1b) can be prepared by reacting a compound of formula (E) (5 mmoles) with a compound of formula (F) (5 mmoles) in an aprotic solvent (100 mL), such as toluene, benzene, etc., containing a catalytic amount of piperidinium acetate under boiling conditions. The admixture is boiled in a Dean-Stark apparatus for about 2 hours. The compound of formula (1b) is obtained upon cooling.
Compounds of formula (2) may be prepared as described in Scheme 4. In Scheme 4, starting components may be obtained from sources such as Aldrich, or synthesized according to sources known to those of ordinary skill in the art, e.g., Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley Interscience, New York) and other texts identified herein. Groups R1, R2, R3, R4, R5 and E are selected from components as defined in the specification heretofore. R is selected from hydrogen, alkyl or metal ions, such as Na+, K+ etc. X presents halogen atom, such as Cl, Br or I.
REACTION SCHEME 4
Figure imgf000020_0001
(A) (B) (C)
Figure imgf000020_0002
(D) (2a)
In general, compounds of formula (C) may be prepared by reacting an isothiocyanate (i.e., a compound of formula (A)) with an amine compound of formula (B) (the mole ratio of (A) to (B) being about 1 to 1), in the presence of hydrochloric acid. The admixture is dried and the residue is heated to ambient temperature for up to about 6 hours. The resulting substance is a compound of formula (C).
Compounds of formula (D) may be obtained from a source such as Aldrich, or prepared according to schemes known to those of ordinary skill in the art. For instance, a hydroxycarbonylmethylenehalide compound may be reacted with about an equimolar amount of diphosphorus pentasulfide to afford a hydroxythiocarbonylmethylenehalide compound of formula (D), which can then be used in the reaction scheme as set forth herein. Compounds of formula (2a) can be prepared under cyclization conditions according to schemes known to those of ordinary skill in the art. See, for example, Minoru, M., Yakugaku Zasshi (1962), Vol. 82, pp. 36-45. For instance, a compound of formula (2a) may be formed by combining the foregoing quantity of a compound of formula (C) at a reduced temperature with a compound of formula (D) or a basic salt thereof, in an aqueous solution alkalized with diluted base solution. The reaction mixture is warmed to ambient temperature, admixed with hydrochloric acid, and heated to about 100°C for up to 1 hour. After cooling, the resulting precipitate may be isolated by filtration, washed with water and allowed to dry, affording a compound of formula (2a). REACTION SCHEME 5
Figure imgf000021_0001
In Scheme 5, starting components (A') and (B') may be obtained from a source such as Aldrich, or prepared according to schemes known to those of ordinary skill in the art. In general, compound (2b) can be prepared by reacting (B') (0.01 mole) with 0.01 mole of reagent (A') in 10-40 mL of glacial acetic acid, the admixture then being boiled for about 15 minutes. Upon cooling, the resulting precipitate may be isolated by filtration, washed with water and allowed to dry, affording a compound of formula (2b).
REACTION SCHEME 6
Figure imgf000022_0001
In general, compounds of formula (E) can be prepared following the procedure described in Scheme 3. Compounds of formula (2c) can be prepared by reacting a compound of formula (E) (5 mmoles) with a compound of formula (F) (5 mmoles) in an aprotic solvent (100 mL), such as toluene, benzene, etc., containing a catalytic amount of piperidinium acetate under boiling conditions. The admixture is boiled in a Dean-Stark apparatus for about 2 hours. The compound of formula (2c) is obtained upon cooling.
PHARMACEUTICAL COMPOSITIONS
In one aspect, the present invention provides a pharmaceutical composition that comprises a compound of formula (1) and a pharmaceutically acceptable carrier, diluent or excipient:
Figure imgf000022_0002
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O),R8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form I=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons; R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
In various embodiments, the compositions of the present invention include a compound of formula (1) as defined above, including in separate embodiments, compositions including compounds of the specific embodiments defined above, e.g., compounds that do not include "excluded compounds" referred to above in connection with compounds of formula (1).
In one aspect, the present invention provides a pharmaceutical composition that comprises a compound of formula (2) and a pharmaceutically acceptable carrier, diluent or excipient:
wherein, independently at each occurrence: E is selected from the group consisting of O and S; each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), -S(O)tN(R9)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form l=(R10)(R11); each of R4, R5 and R6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R8 is hydrogen, alkyl or aralkyl; each R9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R10 is hydrogen, alkyl or aralkyl; and R11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof. In one embodiment, the compositions of the present invention include a compound of formula (2) as defined above, with the proviso that compositions of the present invention exclude compounds having the Registry Numbers of 93293-15-3 and 93312-04-0 as the exclusive compound of formula (2) within the composition. In another embodiment the compositions further exclude homologs of the compounds of Registry Numbers 93293-15-3 and 93312-04-0 as the exclusive compound of formula (2) within the composition, where two compounds that are homologs differ structurally by a single methylene (-CH2-) group. In another embodiment, the present invention provides a composition as defined above with the proviso that when R3 is methyl and R4 is methyl and R5 is phenyl, then R1 and R2 are not selected from hydrogen, methyl or ethyl.
Methods of Use
This invention is directed to methods of using compounds of formulae (I) and (2), as set forth above in the Summary of the Invention, and pharmaceutical compositions containing compounds of formulae (I) and (2) in treating various conditions, which are associated with activation of PKB. These conditions include cancer, inflammation, angiogenesis, cell migration, and neurological disorders. In particular, PKB has been associated with an inappropriate lack of apoptosis in proliferating cells, e.g. in tumor cells, activated lymphocytes, and the like. Thus, the methods disclosed herein are useful in treating disorders and physiological conditions associated with hyperproliferation when administered to a subject in need of such treatment. In one aspect of the invention, compounds and pharmaceutical compositions of the invention are used to inhibit the activity of PKB. The compounds and pharmaceutical compositions of the invention are administered to a subject having a cancer or a pathological inflammation in order to inhibit tumor growth by inducing apoptosis, angiogenesis and/or cell migration. The methods of the invention can be used prophylactically (i.e., to prevent the disorder of interest from occurring) or therapeutically (i.e., to inhibit or relieve the disorder). As used herein, the term "treating" is used to refer to both prevention of disease, and treatment of pre-existing conditions. The prevention of symptoms is accomplished by administration of the compounds and pharmaceutical compositions of the invention prior to development of overt disease, e.g., to prevent the regrowth of tumors, prevent metastatic growth, diminish restenosis associated with cardiovascular surgery, to prevent or reduce cell migration leading to inflammation and associated tissue damage. Alternatively, the compounds and pharmaceutical compositions of the invention may be administered to a subject in need thereof to treat an ongoing disease, by stabilizing or improving the clinical symptoms of the patient. The subject, or patient, may be from any mammalian species, e.g. primates, particularly humans; rodents, including mice, rats and hamsters; rabbits; equines; bovines; canines; felines; etc. Animal models are of interest for experimental investigations, providing a model for treatment of human disease.
Most proliferating cells are programmed to undergo apoptosis unless specific survival signals are provided. Survival factors can suppress apoptosis in a transcription- independent manner by activating PKB, which then phosphorylates and inactivates components of the apoptotic machinery. Over-expression or inappropriate activation of PKB can lead to an inactivation of apoptosis components, thereby leading to unregulated proliferation of cells. Sphingosine 1-phosphate (SΙP)-induced endothelial cell migration requires the
PKB-mediated phosphorylation of a G protein-coupled receptor. This activation is indispensable for RAC activation, cortical actin assembly, angiogenesis and chemotaxis. Transactivation of GPCRs by PKB may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration. Inhibition of PKB may prevent this activation and induction of angiogenesis and cell migration.
PKB is important for the survival of cerebellar neurons, through a signaling pathway by which insulin-like growth factor-1 activation of PIK3 triggers the activation of PKB. In the developing nervous system PKB is a critical mediator of growth factor- induced neuronal survival. "Neurologic disorder" is defined here and in the claims as a disorder in which dysfunction of neurons occurs either in the peripheral nervous system or in the central nervous system. Examples of neurologic disorders include: chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's chorea, diabetic peripheral neuropathy, multiple sclerosis, amyotrophic lateral sclerosis; aging; and acute neurodegenerative disorders including: stroke, traumatic brain injury, peripheral nerve damage, hypoglycemia, spinal cord injury, epilepsy, and anoxia and hypoxia. PKB directly phosphorylates eNOS and activates the enzyme leading to nitric oxide (NO) production. Activated PKB increases basal nitric oxide release from endothelial cells, and activation-deficient PKB attenuated NO production stimulated by vascular endothelial growth factor (VEGF). eNOS is an PKB substrate linking signal transduction by PKB to the release of the gaseous second messenger nitric oxide. Phosphorylation of eNOS by PKB represents a calcium-independent regulatory mechanism for activation of eNOS.
Hyperproliferative disorders refers to excess cell proliferation, relative to that occurring with the same type of cell in the general population and/or the same type of cell obtained from a patient at an earlier time. The term denotes malignant as well as non-malignant cell populations. Such disorders have an excess cell proliferation of one or more subsets of cells, which often appear to differ from the surrounding tissue both morphologically and genotypically. The excess cell proliferation can be determined by reference to the general population and/or by reference to a particular patient, e.g. at an earlier point in the patient's life. Hyperproliferative cell disorders can occur in different types of animals and in humans, and produce different physical manifestations depending upon the affected cells.
Hyperproliferative cell disorders include cancers; blood vessel proliferative disorders such as restenosis, atherosclerosis, in-stent stenosis, vascular graft restenosis, etc.; fibrotic disorders; psoriasis; inflammatory disorders, e.g. arthritis, etc.; glomerular nephritis; endometriosis; macular degenerative disorders; benign growth disorders such as prostate enlargement and lipomas; and autoimmune disorders. Cancers of particular interest include carcinomas, e.g. colon, prostate, breast, melanoma, ductal, endometrial, stomach, dysplastic oral mucosa, invasive oral cancer, non-small cell lung carcinoma, transitional and squamous cell urinary carcinoma, etc.; neurological malignancies, e.g. neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhood acute leukaemia, non-Hodgkin's lymphomas, chronic lymphocytic leukaemia, malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoid hyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichen planus, etc.; sarcomas, melanomas, adenomas; benign lesions such as papillomas, and the like.
Other hyperproliferative disorders that may be associated with altered activity of PKB include a variety of conditions where there is proliferation and/or migration of smooth muscle cells, and/or inflammatory cells into the intimal layer of a vessel, resulting in restricted blood flow through that vessel, i.e. neointimal occlusive lesions. Occlusive vascular conditions of interest include atherosclerosis, graft coronary vascular disease after transplantation, vein graft stenosis, peri-anastomatic prosthetic graft stenosis, restenosis after angioplasty or stent placement, and the like. Disorders and conditions where there is hyperproliferation and/or tissue remodeling or repair of reproductive tissue, e.g. uterine, testicular and ovarian carcinomas, endometriosis, squamous and glandular epithelial carcinomas of the cervix, etc. are reduced in cell number by administration of the compounds and pharmaceutical compositions of the invention. Other disorders and conditions of interest relate to epidermal hyperproliferation, tissue remodelling and repair. For example, the chronic skin inflammation of psoriasis is associated with hyperplastic epidermal keratinocytes. Other disorders of interest include inflammatory disorders and autoimmune conditions including, but not limited to, psoriasis, rheumatoid arthritis, multiple sclerosis, scleroderma, systemic lupus erythematosus, Sjogren's syndrome, atopic dermatitis, asthma, and allergy. Target cells susceptible to the treatment include cells involved in instigating autoimmune reactions as well as those suffering or responding from the effects of autoimmune attack or inflammatory events, and include lymphocytes and fibroblasts. The susceptibility of a particular cell to treatment according to the invention may be determined by in vitro testing. Typically, a culture of the cell is combined with a subject compound at varying concentrations for a period of time sufficient to allow the active agents to induce cell death or inhibit migration, usually between about one hour and one week. For in vitro testing, cultured cells from a biopsy sample may be used.
The dose will vary depending on mode of administration, specific disorder, patient status, etc. Typically a therapeutic dose will be sufficient to substantially decrease the undesirable cell population in the targeted tissue, while maintaining patient viability. Treatment will generally be continued until there is a substantial reduction, e.g. at least about 50%, decrease in the clinical manifestation of disease, and may be continued until there are essentially none of the undesirable cellular activity detected in the relevant tissue. The compounds of formulae (1) and (2) may also find use in the specific inhibition of signaling pathways mediated by PKB, and as a "positive" control in high throughput screening for other modulating compounds. In particular, this invention directed to methods of using compounds of formulae (1) and (2) and pharmaceutical compositions containing such compounds in treating cancer or inflammation associated with PKB activity.
In one embodiment of the invention, methods are provided for using compounds of formulae (1) and (2) and pharmaceutical compositions containing such compounds in treating hyperproliferative disorders. Thus, the methods disclosed herein are useful in treating disorders and physiological conditions associated with hyperproliferation and tissue remodeling or repair when administered to a subject in need of such treatment. The compounds and pharmaceutical compositions of the invention are administered to a subject having a cancer or a pathological inflammation in order to inhibit tumor growth by impeding cell division, and to decrease inflammation by inhibiting cell adhesion and cell migration.
The compounds of formulae (1) and (2) may also find use as affinity reagents for the isolation and/or purification of PKB using the biochemical affinity of the enzyme for inhibitors that act on it. The compounds are coupled to a matrix or gel. The coupled support is then used to separate the enzyme, which binds to the compound, from a sample mixture, e.g., a cell lysate, which may be optionally partially purified. The sample mixture is contacted with the compound-coupled support under conditions that minimize non-specific binding. Methods known in the art include columns, gels, capillaries, etc. The unbound proteins are washed free of the resin and the bound proteins are then eluted in a suitable buffer.
The compounds of formulae (1 ) and (2) may also be useful as reagents for studying signal transduction or any of the clinical disorders listed throughout this application, and for use as a positive control in high throughput screening. Administration of the Compounds and Pharmaceutical Compositions of the Invention
Administration of the thiazolidinedione derivative compounds as described herein, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The pharmaceutical compositions of the invention can be prepared by combining a compound of the invention with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion techniques. Pharmaceutical compositions of the invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound of the invention in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company, Easton, Pennsylvania, 1990). The composition to be administered will, in any event, contain a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, for treatment of a disorder or condition associated with hyperproliferation and tissue remodelling or repair in accordance with the teachings of this invention.
A pharmaceutical composition of the invention may be in the form of a solid or liquid. In one aspect, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, e.g., inhalatory administration.
When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel™, corn starch and the like; lubricants such as magnesium stearate or Sterotex™; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
When the pharmaceutical composition is in the form of a capsule, e.g., a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil.
The pharmaceutical composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included. The liquid pharmaceutical compositions of the invention, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile. A liquid pharmaceutical composition of the invention intended for either parenteral or oral administration should contain an amount of a compound of the invention such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of a compound of the invention in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Preferred oral pharmaceutical compositions contain between about 4% and about 80% of the compound of the invention. Preferred pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 1% by weight of the compound of the invention.
The pharmaceutical composition of the invention may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a pharmaceutical composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. Topical formulations may contain a concentration of the compound of the invention from about 0.1 to about 10% w/v (weight per unit volume).
The pharmaceutical composition of the invention may be intended for rectal administration, in the form, e.g., of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.
The pharmaceutical composition of the invention may include various materials that modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. The pharmaceutical composition of the invention in solid or liquid form may include an agent that binds to the compound of the invention and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include a monoclonal or polyclonal antibody, a protein or a liposome. The pharmaceutical composition of the invention may consist of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols of compounds of the invention may be delivered in single phase, bi-phasic, or tri-phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One skilled in the art, without undue experimentation may determine preferred aerosols.
Whether in solid, liquid or gaseous form, the pharmaceutical composition of the present invention may contain one or more known pharmacological agents used in the treatment of cancer or inflammation in a mammal, particularly, cancer or inflammation associated with hyperproliferation and tissue remodelling or repair.
The pharmaceutical compositions of the invention may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining a compound of the invention with water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the compound of the invention so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
Methods for the preparation of pharmaceutical compositions as described above are well known in the art.
The compounds of the invention, or their pharmaceutically acceptable salts, are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. Generally, a therapeutically effective daily dose is from about 0.1 mg to about 20 mg/kg of body weight per day of a compound of the invention, or a pharmaceutically acceptable salt thereof; preferably, from about 0.1 mg to about 10 mg/kg of body weight per day; and most preferably, from about 0.1 mg to about 7.5 mg/kg of body weight per day. Preferred Embodiments of the Invention
In one embodiment, the present invention provides a method of treating selected medical conditions in a mammal, using a thiazolidinedione derivative compound as described herein. In various aspects of this embodiment, the medical condition is cancer and/or angiogenesis, and/or a neurological disorder. In various other aspects, the medical condition is inflammation and/or cell migration. In various other aspects, any two or more of these medical conditions can be combined in describing a method of the present invention.
Thus, in one aspect, the present invention provides a method of treating a medical condition in a mammal comprising administering to the mammal in need thereof a therapeutically effective amount of a compound of formula (1):
Figure imgf000032_0001
wherein, independently at each occurrence: each of R\ R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(0)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl; R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
In another aspect, the present invention provides a method of treating a condition selected from cancer, inflammation, angiogenesis, cell migration and neurological disorders in a mammal, which method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound or composition as described herein containing a compound of formula (2):
Figure imgf000033_0001
wherein, independently at each occurrence:
E is selected from the group consisting of O and S; each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(0)tR9 (where t is 0 to 2), -S(O)tN(R9)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R10)(R11); each of R4, R5 and R6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R8 is hydrogen, alkyl or aralkyl; each R9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R10 is hydrogen, alkyl or aralkyl; and
R11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
In separate embodiments, the present invention additionally provides a method of treating cancer, a method of treating inflammation, a method of treating angiogenesis, a method of treating cell migration and a method of treating neurological disorders with compounds and compositions as described herein. Thus, the present invention provides a method for preventing a selected medical condition in a mammal. In various aspects of this embodiment, the medical condition being prevented in a mammal is cancer and/or angiogenesis and/or a neurological disorder. In various other aspects, of this embodiment, the medical condition being prevented in a mammal is inflammation and/or cell migration. The inventive method of preventing a medical condition in a mammal comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formulae (1) or (2) as identified herein. In various other aspects, any two or more of these medical conditions can be combined in describing a method of the present invention.
In another embodiment, the present invention provides a method of promoting apoptosis in proliferating cells in a mammal. The inventive method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formulae (1) or (2) as identified herein. In another embodiment, the present invention provides a method of inhibiting PKB activity in a mammal. The inventive method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formulae (1) or (2) as identified herein.
In another embodiment, the present invention provides a method of restoring the normal foot process architecture of podocytes in glomerular diseases associated with proteinuria in a mammal. The inventive method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formulae (1) or (2):
In each of the inventive methods identified above, in describing the compounds of formula (1), any of the following provisos may be applied: (1) The compounds do not include any one or more of the "excluded compounds" as defined above in connection with compounds of formula (1), i.e., 4- thiazolidinone, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)-2-thioxo (RN 34931-00-5), etc.
(2) The compounds do not include homologs of any of the above-identified excluded compounds, where an excluded compound and its homolog differ structurally by the presence or absence of a methylene (-CH2-) group.
(3) R5 is not phenyl when both of R3 and R4 are methyl.
For example, the present invention provides pharmaceutical composition comprising a compound of formula (1) and a pharmaceutically acceptable carrier, diluent or excipient:
Figure imgf000034_0001
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2l -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O),R8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form I=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons; R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
The three provisos identified above in connection with the inventive methods may also be applied to describing the compounds contained within the scope of the compositions of the present invention. For example, in one embodiment, the pharmaceutical compositions of the present invention contain a compound of formula (1) with the proviso that R5 is not phenyl when both of R3 and R4 are methyl.
As mentioned above, in addition to inventive methods, and inventive pharmaceutical compositions, the present invention provides compounds useful in the methods. In one aspect, the present invention provides a compound of formula (1):
Figure imgf000035_0001
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(0)OR8, -C(O)N(R8)2, -N(R8)2,
-N(R8)C(O)R8, -N(R8)C(O)OR7, -S(0)tR8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons; R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
The three provisos identified above in connection with the inventive methods and inventive compositions may also be applied to describing the compounds of the present invention. For example, in one embodiment, the compounds of the present invention having formula (1) do not include any two or more of the excluded compounds set forth in the Table above.
In further embodiments of the methods, compositions and compounds of the present invention, the compounds utilized in the above methods, or present in the inventive compositions, are compounds having formula (1) wherein R1 and R2 together form J=(R9)(R10), i.e., in this embodiment the compounds of formula (1) are described by formula (4), where compounds of formula (3) are themselves an embodiment of the present invention:
Figure imgf000036_0001
In the compounds, compositions and methods of the present invention that entail compound of formula (1 ) or (3), in separate embodiments, the following criteria may be used according to the present invention to define the group of compounds being utilized in the composition or method, where any two or more of these criteria may be combined, and the present invention provides embodiments where every two or more criteria are combined so long as the combined criteria are not inconsistent with one another: R1 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl; R1 is selected from the group consisting of aryl, aralkylene and aralkenylene; R1 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R1 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(0)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O),R8 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2); R2 is H; R2 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl; R2 is selected from the group consisting of aryl, aralkylene and aralkenylene; R2 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R2 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), and -S(O)tN(R8)2 (where t is 0 to 2); R1 and R2 together form J=(R9)(R10) where optionally R9 is hydrogen, R9 is alkyl, and/or R9 is aralkyl, R10 is hydrogen, R10 is alkyl, R10 is alkenyl, R10 is cycloalkyl, R10 is cycloalkylalkyl, R10 is heterocyclyl, R10 is heterocyclylalkyl, R 0 is aralkyl, and/or R10 is aryl, e.g., R10 may be mono-substituted aryl, e.g., R9 is hydrogen and R10 is aryl; R3 is H; R3 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl; R3 is selected from the group consisting of aryl, aralkylene and aralkenylene; R3 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R3 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(0)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), and -S(O)tN(R8)2 (where t is 0 to 2); R4 is H; R4 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene; R4 is selected from the group consisting of aryl and aralkylene; R4 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R5 is H; R5 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene; R5 is selected from the group consisting of aryl and aralkylene; R5 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R6 is a direct bond; R6 is an optionally substituted alkylene of one to six carbons; R6 is an unsubstituted alkylene of one to six carbons; R7 is hydrogen; R7 is alkyl; R7 is aralkyl; and R8 is hydrogen, R8 is alkyl, R8 is alkenyl, R8 is cycloalkyl, R8 is cycloalkylalkyl, R8 is aralkyl, or R8 is aryl. Additionally, the compound may be utilized as a mixture of steroisomers, and/or the compound is a pharmaceutically acceptable salt, and/or the compound has a molecular weight between 100 and 1,000 Daltons; and/or the compound has a molecular weight between 250 and 750 Daltons.
In each of the inventive methods identified above, in describing the compounds of formula (2), any of the following provisos may be applied: (1) The compounds do not include any of the "excluded compounds" as defined above in connection with compounds of formula (2), i.e., 5-thiazolidineacetic acid, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)-2,4-dioxo- (RN 405924-24-5) . . . 2,4-thiazolidinedione, 3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)- (RN 93293-15-3).
(2) The compounds utilized in the above methods, or present in the inventive compositions, are compounds having formula (2) wherein R and R2 together form 1=(R10)(R11), i.e., in this embodiment the compounds of formula (2) are described by formula (4), where compounds of formula (4) are themselves an embodiment of the present invention:
Figure imgf000038_0001
In the compounds, compositions and methods of the present invention that entail compounds of formulae (2) or (4), in separate embodiments, the following criteria may be used according to the present invention to define the group of compounds being utilized in the composition or method, where any two or more of these criteria may be combined, and the present invention provides embodiments where every two or more criteria are combined so long as the combined criteria are not inconsistent with one another: E is O; E is S; R1 is H; R1 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl; R1 is selected from the group consisting of aryl, aralkylene and aralkenylene; R1 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R1 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(0)tR9 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2); R2 is H; R2 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl; R2 is selected from the group consisting of aryl, aralkylene and aralkenylene; R2 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R2 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2l -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), and -S(O),N(R9)2 (where t is 0 to 2); R1 and R2 together form J=(R10)(R11) where optionally R10 is hydrogen, R10 is alkyl, R10 is aralkyl, R11 is hydrogen, R11 is alkyl, R11 is alkenyl, R11 is cycloalkyl, R11 is cycloalkylalkyl, R11 is heterocyclyl, R11 is heterocyclylalkyl, R is aralkyl, R11 is aryl, R11 is mono-substituted aryl, e.g., R10 is hydrogen and R11 is aryl; R3 is H; R3 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl; R3 is selected from the group consisting of aryl, aralkylene and aralkenylene; R3 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R3 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2) -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2); R4 is H; R4 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene; R4 is selected from the group consisting of aryl and aralkylene; R4 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R5 is H; R5 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene; R5 is selected from the group consisting of aryl and aralkylene; R5 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R6 is H; R6 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene; R6 is selected from the group consisting of aryl and aralkylene; R6 is selected from the group consisting of heterocyclyl and heterocyclyalkylene; R7 is a direct bond; R7 is an unsubstituted alkylene; R7 is a substituted alkylene; R8 is hydrogen; R8 is alkyl; R8 is aralkyl; R9 is H; R9 is selected from the group consisting of alkyl, alkenyl, cycloalkyl and cycloalkylalkylene; and R9 is selected from the group consisting of aryl and aralkylene. Additionally, the compound may be utilized as a mixture of steroisomers, and/or the compound is a pharmaceutically acceptable salt, and/or the compound has a molecular weight between 100 and 1 ,000 Daltons; and/or the compound has a molecular weight between 250 and 750 Daltons. For example, the present invention provides compounds, compositions and methods wherein the compound of formula (2) has R and R2 together forming 1=(R10)(R11) where R10 is hydrogen and R11 is mono-substituted aryl, e.g., para- dialkylamino-phenyl (e.g., para-dimethylamine-phenyl) or para-nitro-phenyl or para- alkoxy-phenyl (e.g., para-methoxy-phenyl), R3 is alkyl, e.g., methyl, R4 is alkyl, e.g., methyl, and R5 is aryl, e.g., unsubstituted phenyl.
In addition to inventive methods, the present invention provides compounds useful in the methods, and pharmaceutical compositions containing the compounds, which are also useful in the inventive methods.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric. EXAMPLES
EXAMPLE 1 PKBα ENZYME PREPARATION:
The original bovine PKBα cDNA was obtained from Dr. Jim Woodgett (Coffer and
Woodgett, (1991) Eur. J. Biochem. 201 (2) -475-481) and subcloned into baculovirus expression vector pAcG2T (BD PharMingen) for recombinant protein expression in insect cells. Expression of bovine PKBα as a fusion protein required that the cDNA be ligated into the polyclonal site situated in frame and downstream of the glutathione-S-transferase gene of the baculovirus transfer vector pAcG2T. The 1443 bp PKBα coding sequence was directionally subcloned from pGEX-PKBα vector into the Bam HI and Eco Rl polyclonal sites of the pAcG2T vector. Sequence analysis of the recombinant clone revealed no discordance with the original wild type sequence. The PKBα enzyme was expressed using the baculovirus expression system in a two-step process. The infectious baculovirus particles were amplified prior to the recombinant overexpression of the protein. In the first step, infectious baculovirus was generated by co-transfecting recombinant pAcG2T-PKBα plasmid with linear AcNPV (Autographa californica nuclear polyhedrosis virus (BD PharMingen) DNA into adherent Spodoptera frugiperda Sf9 insect cells (Invitragen) following the manufacturer's instructions. Recombination between homologous sites allowed the heterologous PKBα gene transfer from the transfer vector pAcG2T-PKBα to the genomic AcNPV DNA and finally the production and amplification of packaged baculovirus particles. Then the recombinant GST-PKBα was overexpressed under the control of the very late polyhedrin promoter activated after virion assembly was complete. The PKBα was activated in vivo using co-infection of Hi5 insect cells (Invitragen) with GST-PKBα recombinant baculovirus and dual-PI3K/ILK recombinant baculovirus. The expression cells were lysed by mild sonication in the lysis buffer (50 mM Tris-HCI, PH7.5, 2.5 mM EDTA, 150 mM NaCI, 1% NP-40, 0.1% β-mercaptoethanol, 0.5 mM sodium orthovanadate, 50 mM β-glycerophosphate, 1 mM benzamidine and 0.5 %(VΛ ) protease inhibitor cocktail set III (CalBiochem)). The lysate was cleared of cellular debris by centrifugation. The active recombinant protein was purified from the supernatant using a GST-glutathione affinity system according the manufacturer's instructions (Pharmacia). Following batch binding of the fusion protein to the glutathione- agarose beads, the matrix was transferred to a 1X10 cm Flex-column (Kontes Glass) chromatography system. The column was washed with high-salt buffer (50 mM Tris-HCI, PH7.5, 1 mM EDTA, 500 mM NaCI, 0.1% NP-40, 0.1% β-mercaptoethanol, 0.5 mM sodium orthovanadate, 50 mM β-glycerophosphate, 1 mM benzamidine and 0.1 mM PMSF), then low-salt buffer (50 mM Tris-HCI, PH7.5, 1 mM EDTA, 50 mM NaCI, 0.1% β- mercaptoethanol, 0.5 mM sodium orthovanadate, 50 mM β-glycerophosphate, 1 mM benzamidine and 0.1 mM PMSF). The GST-PKBα fusion protein was released from the matrix using a glutathione buffer (50 mM Tris-HCI, PH7.5, 50 mM NaCI, 10 mM glutathione, 0.1% β-mercaptoethanol and 0.1 mM PMSF). A complete quantitative and qualitative analysis of the protein was monitored using Coomassie blue staining and Western blot analysis using anti-GST and anti-PKB-PH specific antibodies (Kinetek). PKBα protein comprised approximately 5% to 7% of the total insect cellular protein concentration.
PKBα IN VITRO KINASE ASSAY:
Biochemical analysis of the activated enzyme was performed on recombinant bovine GST-PKBα fusion protein using the experimental protocol outlined in the section entitled "IN VITRO ACTIVITY PROFILE FOR KINASES". Typically, the GST-PKBα preparations were found to exhibit protein phosphotransferase activity in the order of about 200 pmol/min/ug in the presence of 50 uM [γ-32P]-ATP and 162 uM PKB substrate peptide (amino acid sequence: CKRPRAASFAE) during a 15 min reaction at ambiant temperature.
IN VITRO ACTIVITY PROFILE FOR PKBα
Inhibition of the PKB in vitro was measured by scintillation counting, which involves the incorporation of radioactive phosphate onto a specific substrate that is immobilized onto a filter paper at the end of the assay. The assay was performed in the absence and presence of specific and known inhibitors of PKB (as controls), and the amount of incorporated radioactivity was compared. Test compounds were lyophilized and stored at -20°C. Stock solutions were made by weighing out the compounds and dissolving them in dimethyl sulfoxide (DMSO) to a standard concentration, usually 20 mM, and stored at -20°C. The compounds were diluted to a starting intermediate concentration of 250 μM in 1 % DMSO, then serially diluted across a row of a 96 well plate using serial 2 fold dilution steps. Diluted 100% DMSO was used as a negative control. 5 μL of each compound dilution were robotically pipetted to Costar brand serocluster plates maintaining the same plate layout. All assay mixtures consisted of the following volumes: 5 μL diluted compound 10 μL target enzyme preparation 1 μL substrate
5 μL assay ATP The assay mixtures were then incubated 15 minutes at ambient temperature.
From each assay mixture, 10 μL of assay mixture was spotted onto Millipore Multiscreen-PH™ opaque plates and washed twice for 10 minutes in 1% phosphoric acid. The plates were dried at 40°C for 30 minutes, then the substrate phosphate complexes were quantitated by scintillation counting. These Millipore plates are in a 96-well format with immobilized P81 ™ phosphocellulose membranes in the wells. Both the phosphorylated and non-phosphorylated form of the substrate bind to the membrane while ATP (unincorporated phosphate) is removed in the subsequent wash steps.
Calculation oflC50. Inhibition of the targets by the test compounds is measured by scintillation counting of the incorporation of radioactive phosphate onto a specific substrate that is immobilized onto a filter paper at the end of the assay. To provide meaningful measurements of inhibition, the assays are performed both in the absence and presence of specific and known inhibitors, and the amount of incorporated radioactivity is compared to provide a baseline measurement.
The "baseline activity" is the amount of radioactivity incorporated in the absence of a target inhibitor. The amount of radioactivity incorporated in the presence of a target inhibitor is called the "sample activity", and the % inhibition is expressed by the following formula:
% inhibition = 100 -(sample activity/baseline activity*100)
and is usually expressed in conjunction with the compound concentration. By using a range of target inhibitor concentrations, the IC50 of an inhibitor is estimated (i.e. the concentration at which enzymatic activity is reduced by 50%). The IC50 of various inhibitors against a particular target can be compared, where a lower IC50 indicates a more potent inhibitor.
The compound 2,4-thiazolidinedione, 3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl- 1 H-pyrazol-4-yl)-5-[[4-(dimethylamino)phenyl]methylene] had an IC5o of 7.9 μm in this assay, while 4-thiazolidinone, 3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4- yl)-5-[[2-hyrdoxy-3-(2-propenyl)phenyl]methylene]-2-thioxo had one of 3.4 μm. The compound Benzenesulfonamide,
4-[5-[[3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)-4-oxo-2-thioxo-5- thiazolidinylidene]methyl]-2-furanyl]- inhibited enzyme activity 43% at a concentration of
20μm.
EXAMPLE 2 CELL PROLIFERATION This procedure (Jelinkova, R. B. et al., "Antiproliferative effect of a lectin- and anti-Thy-1.2 antibody-targeted HPMA copolymer-bound doxorubicin on primary and metastatic human colorectal carcinoma and on human colorectal carcinoma transfected with the mouse Thy-1.2 gene", Bioconjug. Chem. (2000, Sep-Oct), Vol. 11, No. 5, pp. 664-73) is used to assess the effect compounds have on various cell lines with respect to proliferation. The rate of growth of various tumor cells is quantified by measuring the amount of free isotopic thymidine that has been incorporated into the cells over a period of time. The effect of any compound to inhibit the proliferation of various tumor cells could be used as an indication of its ability to prevent disease progression in cancer. Cultured tumour cells are harvested cells as per normal procedures: i.e. trypsinize, centrifuge and count cells. A volume of 90 μL is used to seed 5,000 cells/well in a 96 well plate. Cells are incubated for 24 hours at 37°C under 5% CO2. After incubation, cells should be 80-90% confluent.
3H-thymidine (Amersham) is diluted in cell culture media to a concentration of 100 μCi/mL. The test compound is diluted in the thymidine broth to 10X the final desired concentration.
Then 10 μL of diluted compound is added to the 90 μL of cells already present in the 96-well plates. Six replicates wells are done per treatment in columns 2 to 11. Plates were mixed by rocking. A known cytotoxic compound such as staurosporine is used in relatively high concentrations as a positive control in column 1. Diluted DMSO is used as a negative control in column 12. The plate is incubated for exactly 24 hours at 37CC.
After incubation, plates are observed under the microscope for obvious cell death, abnormal cell shape, crystal formation of the compound, ete. Then 25 μL volume of cold 50% TCA is added slowly to the 100 μL volume already in each well, and incubated for 1-2 hours at 4°C. The plates are then washed 5X in tap water and allowed to dry completely (usually overnight) at ambient temperature. Finally, 100 μL of scintillation fluid is added to each well and the plates are counted in a Wallac 1450 Microbeta™ counter according to user manual instructions. The amount of inhibition is determined by the following formula:
% inhibition = 100 - [(AVG treatment -AVG positive control)/100(AVG negative control - AVG positive control) ] Where the compound was 2,4-thiazolidinedione, 3-(2,3-dihydro-1 ,5-dimethyl-3- oxo-2-phenyl-1H-pyrazol-4-yl)-5-[[4-(dimethylamino)phenyl]methylene], the results were as follows:
Table 1 : % Inhibition of Proliferation at 50 μM
Figure imgf000043_0001
EXAMPLE 3 CYTOTOXICITY ASSAY
This procedure is used to assess the effects compounds have on various cell lines with respect to cell viability. Cell viability is quantified using calcein AM ((3',6'-Di(O- acetyl)-2',7'-bis[N,N-bis-(carboxymethyl)aminomethyl]-fluorescein, tetraacetoxymethyl ester) and measuring its conversion to a fluorescent product (calcein) with a fluorimeter.
The principle of this assay is based on the presence of ubiquitous intracellular esterase activity found in live cells. By enzymatic reaction of esterase, non-fluorescent cell-permeant calcein AM is converted to the intensely fluorescent calcein. The polyanionic dye calcein is retained within live cells, producing a green fluorescence in live cells. It is a faster, safer, and better-correlated indicator of cytotoxicity than alternative methods (e.g. 3H-Thymidine incorporation). Calcein AM is susceptible to hydrolysis when exposed to moisture. Therefore, prepare aqueous working solutions containing calcein AM immediately prior to use, and used within about one day. A kit available to do this assay is "LIVE/DEAD® Viability/Cytotoxicity Kit (L-3224)" by Molecular Probes.
Cells were collected from tissue culture flasks and trypsinized, centrifuged, resuspended and counted. Cells were seeded to obtain 80-90% confluence (for normal cells, 10,000 cells/well (8000 cells/well for HUVEC cells)). A cell concentration of 110,000 cells/mL (88,000 cells/well for HUVEC cells) is prepared as 90 μL volume is used per well.
Using an 8-channel multi-dispense pipettor, cells were seeded in the central rows of the plate (Nunclon™ 96 well flat-bottom plate), leaving the peripheral top and bottom rows with same volume of media only. The plates were incubated at 37°C, 5% CO2 overnight for approximately 24 hours.
For test compounds, cell culture media (e.g., RPMI + 10%FBS), 10X compound solution of final desired concentration from 20 mM stock compounds was prepared.
10 μl of this 10X compound solution is added to the 90 μL of cells already present in the 96 well plates and a known cytotoxic compound from previous testing is used as a positive control. The negative control is 100% DMSO diluted to the same factor as the compounds.
The plates are incubated at 37°C for approximately 24 hours, and media is aspirated after plates are spun at 2400 rpm for 10 min at ambient temperature. 100 μL of 1X DPBS (without calcium chloride, without magnesium chloride (GibcoBRL, cat#14190-144)) is added to each well.
The calcein AM solution is prepared by added 50 μg of calcein AM crystal (m.w. = 994.87g/mol, Molecular Probes) and anhydrous DMSO (Sigma Aldrich) to make 1 mM stock and diluting stock to 2X the final desired concentration in 1X DPBS just before the assay. 100 μL of this 2X is added to the 100 μL of DPBS in the wells and the plates are incubated at ambient temperature for 30 minutes. Fluorescence data is read and recorded (Fluoroskan Ascent® FL fluorimeter (excitation~485nm, emission~527nm)). The values for replicates (three in this case) are averaged and % inhibition is calculated as follows:
% inhibitions 00 - [(AVG treatment - AVG positive control) / (AVG negative control - AVG positive control)*100] Where the compound was 2,4-thiazolidinedione, 3-(2,3-dihydro-1 ,5-dimethyl-3- oxo-2-phenyl-1H-pyrazol-4-yl)-5-[[4-(dimethylamino)phenyl]methylene], the results were as follows:
Table 2
Figure imgf000045_0001
EXAMPLE 4 CELL APOPTOSIS
The Annexin V assay is used as a measure of apoptosis in cells treated with cytotoxic compounds. Cells undergoing apoptosis have many characteristic changes, and one of these is the externalization of cell membrane phosphatidylserine. Annexin V is a protein that has a very high affinity for phosphatidylserine, and as an assay reagent conjugated to fluorescence such as FITC or PE, it can be used to label phosphatidylserine on the membrane surface.
Cultured tumour cells are harvested cells as per normal procedures: i.e. trypsinize (if adherent), centrifuge and count cells. Cells are adjusted to 9 x 104 cells/ml for adherent cells (ie. A549, B16-F1, or H460), or 1 x 106 cells/ml for non-adherent cells (ie. Jurkat). For adherent cells, 1.8ml of cells are seeded into 6-well tissue culture plates and incubated overnight at 37°C and 5% CO2. For non-adherent cells, pre-seeding is not required, and on the day of treatment, 450ul of cells are seeded into 24-well tissue culture plates.
Compounds are prepared to 10X final concentration, along with DMSO controls and a positive control (ie. Staurosporin). To the 1.8ml of adherent cells in the 6-well dishes is added 200μl of 0X stock, while 50μ is added to the 450μl of suspension cells in the 24-well dishes. Cells are treated overnight at 37°C and 5% CO2, although this time may be adjusted as required for the compound/cell line to obtain an optimal percentage of stained cells. At the selected timepoint after apoptosis induction, cells are harvested by collecting all cells, centrifuging, and washing in ice-cold PBS before resuspending in 1X binding buffer (BD Pharmingen, Cat# 66121 E) at a concentration of 1 x 106 cells/ml. Into 5ml standard FACS tubes (ie. Falcon, Cat# 352008) is transferred 100μl of cell suspension (1 x 105 cells), 5μl of Annexin V-PE (BD Pharmingen, Cat # 65875H or 65875X) and 5μl of 7-AAD (BD Pharmingen, Cat# 68981 E). Controls are set up for use in setting up compensation and quadrants, including unstained cells, cells stained with Annexin V-PE only, and cells stained with 7-AAD only. Samples are incubated in the dark at room temperature for 15 minutes. To each tube is then added 400μl of 1X binding buffer and flow cytometry acquisition using laser excitation at 488nm is carried out immediately (within 1-2 hours for all samples). Cells undergoing early stage apoptosis are quantitated as staining Annexin V positive and 7-AAD negative. Necrotic cells and/or late stage apoptotic cells are Annexin V and 7-AAD double positive. The percentage of Annexin V positive cells (without 7-AAD staining) is calculated to confirm that the mechanism of action of cytotoxicity was apoptosis. The total percentage of Annexin V positive cells (with or without 7-AAD staining) is calculated to determine the degree of apoptosis occurring as a result of compound treatment.
The inhibition of AnnexinV at 50 μM by the compound: 2,4-thiazolidinedione, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)-5-[[4- (dimethylamino)phenyl]methylene] was 59% in Jurkat cells.
EXAMPLE 5 TUMOUR CELL CYTOTOXICITY
This procedure is used to assess the effects compounds have on various tumour cell lines with respect to cell viability. Cell viability is quantified using 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt (MTS). The conversion of MTS into the aqueous soluble formazan product is accomplished by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan product as measured by the amount of 490nm absorbance is directly proportional to the number of living cells in culture.
Cultured tumour cells are harvested cells as per normal procedures: i.e. trypsinize (if adherent), centrifuge and count cells. Cells are adjusted to 5.5 x 104 cells/ml and seeded at 90ul/well into 96-well plates. Cells are incubated overnight at 37°C, 5% CO2. Compounds are prepared to 10X final concentration in cell culture media (serum free), along with DMSO controls and a positive control. To each well is added 10ul of this 10X stock solution and the cells incubated for 24-48 hours at 37°C, 5% CO2. MTS solution is prepared by first preparing PMS solution, dissolving 0.92g of PMS (phenazine methosulfate) powder (Sigma, Cat# P-9625) in 1.0L of 1X PBS (GibcoBRL, Cat# 14190-144). The solution is filter sterilized with a 0.2um filter. The final MTS reagent is prepared by dissolving 4.0g of MTS powder (Fisher Sci, Cat# G1111) in 1.8L of 1X PBS (GibcoBRL, Cat# 14190-144). The pH is adjusted to pH 6.2 and the solution made up to a volume of 2L. To the 2L is added 100ml of PMS solution and the mixture filtered.
Before addition of MTS reagent to the 96-well plates, plates are inspected under the microscope and observations made on the health of the cells. To each well is slowly added 20ul of MTS reagent and the plates are further incubated for 2-4 hours (depending on the cell type and number) at 37CC, 5% CO2. The plates are read using a microplate reader (ie. BIO-TEK Instruments Inc., EL312e) at 490 nm wavelength. The amount of cytotoxicity is determined by the following formula:
% inhibition = 100 - [(AVG treatment -AVG positive control)/100(AVG negative control - AVG positive control) ]
or
% inhibition = 100 - [(AVG treatment -AVG Background)/100(AVG negative control - AVG Background) ]
EXAMPLE 6 IN vivo TUMOUR EFFICACY STUDY. To test the efficacy of test compounds on H460 subcutaneous xenograft alone and in combination with doxorubicin.
Athymic nude female mice are used for this experiment. A group of 60 mice are inoculated with five million H460 cells in 100 μL Matrigel™(VWR Canada) excipient.
Tumours are measured three times a week with digital calipers and the tumour volumes calculated. When tumours have reached an average size of 100 mm3, about two weeks after tumour implantation. At that time any nongrowing Outliers' are removed so that animals can be distributed into groupings that are equal and statistically the same tumour mass, i.e. divided into six groups with about 10 mice per group.
Treatments with test compounds continue for about 20 days, and will be oral (gavage), intravenous, subcutaneous, or intraperitoneal depending on the known solubility of the test compound. A dose of 25mg/kg is typical for such testing, but the dose selected will reflect the potency of the compound and the route of administration.
Up to 200 mg/kg may be selected. Positive controls may alternately be doxorubicin or cisplatin, or cyclophosphamide.
The study breakdown in tabular form:
Figure imgf000048_0001
At study termination, the mice are anesthetized 3 hours after the last dose of test compound, and plasma and tissues are harvested and frozen. Tumours are divided into the desired number of aliquots and fast frozen for later analysis.
EXAMPLE 7
CELL INVASION IN MATRIGEL™
This procedure is used to assess the compound effect on the tumor cell invasion through MatrigeI™-coated Fluoroblok™ inserts. Invasion allows tumor cells to spread to sites other that the primary tumor. BD Bioscience's BioCoat FluoroBlok™ Invasion Systems™ combine the benefits of the BD BioCoat Matrigel™ Invasion Chambers with the fluorescence blocking membrane capabilities of the BD Falcon™ HTS FluoroBlok™ 24-Multiwell Insert System. The following assay uses this system to assess compound effects on the anti-tumor cell invasion through layer of Matrigel™ extracellular matrix. The cell lines used are HT 1080 (ATCC, Cat# CCL - 121), DU-145 (ATCC, Cat# HTB-81), PC3 (ATCC, Cat# CRL-1435) or B16F1 (ATCC, Cat# CRL-6323).
The invasion test system is removed from the package from -20°C storage and allowed to warm to ambient temperature. PBS is added to the interior of the inserts and they are allowed to rehydrate for 2 hours at 37CC. Then the medium is removed and 450 μL cell suspensions of tumour cells (grown to 50-70% confluence, trypsinized, and resuspended in medium without serum at 1 x 106/mL) is added to the top chamber. Test compounds are added to the top chamber at 10X the desired final concentration in 50 μL volumes. DMSO acts as control.
Then 750 μL of medium containing 50% fresh growth medium with 10% FBS and 50% NIH 3T3-conditioned medium is added to each of the bottom wells. The invasion system is then incubated for 24 to 48 hours at 37°C, in a 5% CO2 atmosphere.
Following incubation, the insert plate is transferred into a second 24-well plate containing 0.5 mL of 5 μg/mL calcein AM (Molecular Probes) in Hanks buffered salt solution (HBSS), and plates are incubated for 1 hour at 37°C, 5% CO2.
Fluorescence data indicating cell invasion is read in a Fluoroskan Ascent FL™ (LabSystems) with bottom reading at excitation/emission wavelength of 485/538 nm.
Data is expressed as fluorescence unit (FU) from the sum of middle 25 areas per 24-well or as percentage of invasion inhibition by following formula: % of invasion inhibition = 100 - FU of compound treated cell invasion/ FU of DMSO treated cell invasion times 100. The compounds inhibit invasion in this assay, and thus may be used to prevent metastasis in cancer and tissue remodeling.
EXAMPLE 8 PERITONEAL MACROPHAGE STIMULATION AND ANALYSIS A. Establishment of inflammation assay panel.
Macrophages are important elements of innate immunity to infection and are among the first cell type in the immune response to be exposed to and activated by infectious agents. IFN-γ and LPS are potent activators of macrophages, priming them for a variety of biological effects. IFN-γ, initially secreted by NK and T cells in response to infection, converts macrophages from a resting to an activated state (inflammatory macrophages), priming them for antimicrobial activity manifested by increased killing of intracellular pathogens, and antigen processing and presentation to lymphocytes. The action of IFN-γ is synergized with the LPS second messenger, enhancing the stimulation of macrophages through the activation of NF-κB, that results in the transcriptional up-regulation of a number of genes involved in the cell-mediated immune response, including inducible iNOS (nitric oxide synthase). Activated macrophages are qualitatively different from quiescent macrophages. These differences are typically observed by an increased proliferation index, up-regulated expression of MHC-II, and production of various bioactive molecules. The latter biological effects are mediated by NO (nitric oxide) release and increased production of pro-inflammatory cytokines (IL-6, TNF-γ, IL-1). Primary macrophages derived from Balb/c mice and RAW 264.7 cells (Balb/c background) were used to establish in vitro inflammatory models with fast and reliable readouts. B. Materials and Methods
1. Reagents.
The iNOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and murine rlFN-γ are purchased from Calbiochem, (San Diego, CA). Protein-free, phenol/water-extracted LPS (from E. coli serotype 0111 :B4 0127:B8), Zymosan A™, dexamethasone and hydrocortisone, sulfanilarήide and N-(1-naphthyl)-ethylenediamine, arare purchased from Sigma (St. Louis, MO). Human recombinant vascular endothelial growth factor (VEGF) is purchased from R&D Systems (Minneapolis, MN). Rabbit polyclonal antibody against active (phosphorylated) extracellular signal-regulated kinase (ERK), as well as HRP-conjugated donkey anti-rabbit IgG are obtained from Promega (Madison, Wl). ELISA dual-set kit for detection of IL-6 is purchased from PharMingen (San Diego, CA). Anti-murine iNOS/NOS type II and cyclooxygenase-2 (COX-2) antibodies are obtained from Transduction Laboratories (Lexington, KY).
Female BALB/c inbred mice, 6-12 weeks of age, are purchased from Harlan Inc. (Indianapolis, IN) and housed under fluorescent light for 12 h per day. Mice are housed in cages, and maintained in compliance with the Canadian Council on Animal Care standards.
2. Isolation of primary mouse macrophages.
Peritoneal exudate macrophages are isolated by peritoneal lavage with ice-cold sterile physiological saline 24 hours after intraperitoneal injection of BALB/c mice with 0.5 mL of sterile Zymosan A™ (1 mg/0.5 mL 0.9% saline). Cells are ished, resuspended in RPM1 1640 supplemented with 1 mM D-glucose, 1mM sodium pyruvate, 100 units/mL penicillin, 100 μg/mL streptomycin, and 5% FBS.
3. Treatment of primary macrophages. Primary macrophages (1.5 * 105 cells/well) are grown in 96-well plates (nitrite assay), or 6-well plates (2 * 106 cells/well) for measurement of iNOS and COX-2 expression. Following 3 hours incubation, at 37°C, 5% CO2 (allowing macrophages to attach) cells are stimulated with LPS (5 μg/mL) and IFN-γ (100 U/mL) in the absence or presence of various concentrations of test compounds (all treatments are replicated six times). Cells are incubated for an additional 24 hours, and cell free culture supernatants from each well are collected for NO and cytokine determination. The remaining cells are stained with crystal violet or MTS to determine effect of the test compounds on cell survival.
4. NO production. Following stimulation, the production of NO is determined by assaying culture supernatants for NO2, a stable reaction product of NO with molecular oxygen. Briefly, 100 μL of culture supernatant is reacted with an equal volume of Griess reagent at ambient temperature for 10 minutes. The absorbance at 550 nm is determined. All measurements are performed six times. The concentration of NO2 is calculated by comparison with a standard curve prepared using NaNO2. 5. Western blot analysis.
After incubation with the indicated stimuli in the presence of inhibitors, cells (duplicate samples, 2x106cell/6-wells plate) are washed in PBS and lysed on ice in 60 μL of lysis buffer. The protein content of each sample is determined using the Bradford protein assay kit (Bio-Rad, Richmond, CA). Absorbance is measured at 750 nm with a Beckman DU530 spectrophotometer (Palo Alto, CA). Proteins are mixed with 45xSDS sample buffer. Following separation of proteins by SDS-PAGE, using 8% bis-acrylamide in the separation gel, the proteins are transferred from the gels onto PVDF membranes using a MiniProtean™ III Cell (Bio-Rad), at 100 V for 1.5 hours. Equal amounts of protein (5 μg) are loaded onto SDS-PAGE gels and examined by Western blot analysis with anti-actin, anti-iNOS, anti-COX-2 murine monoclonal antibodies, according to the manufacturer's specifications (Transduction Laboratories). Primary antibodies, in 5% blocking buffer (5% NFM TTBS), are incubated with blots 2 hours or overnight at 4°C, followed by incubation with peroxidase-conjugated secondary antibody. Chemiluminescence substrates are used to reveal positive bands. The bands are exposed on X-ray films. The films are used to analyze the impact of inhibitors on expression of iNOS and COX-2 compared to various controls and "house-keeping" protein (Actin) concentration to control the protein loading and detect any non-specific effects on protein production. The Multi-Analyst™/PC system from Biorad is used to quantitate the bands of the expressed protein on the film. This version of Multi-Analyst is used with the Bio-Rad Gel Doc 1000™ imaging system. White light is chosen as the selected light source, thus the signal strength is measured in OD (optic density) units. The OD of each band is being subtracted to a global background area of the gel. C. In vitro Angiogenesis.
HUVEC cells cultured for 24 hours in M199 with 0.5% FCS are plated at 6 x 105 cells/well in 12-well plates pre-coated with 300 μL of Matrigel (10.7 mg/mL; Becton Dickinson) in M199 with 0.5% FCS in the presence of VEGF (1ng/mL), and in the absence or presence of positive control (Z)-3-[2,4-dimethyl-5-(2-oxo-1 ,2-dihydroindol- 3-ylidenemethyl)-1/V-pyrrol-3-yl]propionic acid or various inhibitors. After 5 hours of incubation in a 5% C02-humidified atmosphere at 37°C, the three-dimensional organization of the cells is examined using an inverted photomicroscope. The cells are fixed with Crystal Violet (0.05% in 20% ethanol) and digitally photographed. D. Enzyme immunoassays for mouse IL-6.
IL-6 levels are determined with PharMingen's OptEIA™ ELISA set developed using an anti-mouse IL-6 Ab pair and mouse rlL-6 standard (PharMingen). Maxisorp F16 multiwell strips (Nunc, Roskilde, Denmark) are coated with anti-mouse IL-6 capture Ab (at recommended concentration) in 0.1 M NaHCO3, pH 9.5, 100 μLΛ/vell, overnight at 4°C. Plates are washed three times with 0.05% Tween 20 in PBS (PBST) and blocked for 1 hour at ambient temperature with 200 μlJwell of 10% FCS in PBS (blocking and dilution buffer). Plates are washed three times with PBST and duplicate samples (100 μlJwell) or standards (100 μlJwell) in diluent buffer are incubated for 2 hours at ambient temperature. Plates are washed five times with PBST and incubated with biotinylated anti-mouse IL-6 and avidin-horseradish peroxidase conjugate (at concentrations recommended by the manufacturer) for 1 hour at ambient temperature. Plates are washed seven times with PBST and 100 μL of 3,3'5,5' tetramethylbenzidine substrate solution (TMB substrate reagent set, BD PharMingen) is added to each well. After 15-30 minute incubation at ambient temperature, color development is terminated by adding 50 μL of 2 N H2SO (Sigma). Absorbance is read at 450 nm with an EL 312e™ microplate reader or the like. The lower limit of detection for IL-6 is 15.6 pg/mL
All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. For example, U.S. Patent Application No. 60/384,437 filed May 31 , 2002; and U.S. Patent Application No. 60/386,330 filed June 4, 2002 are incorporated herein by reference, in their entirety. From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims

1. A pharmaceutical composition comprising a compound of formula (1) and a pharmaceutically acceptable carrier, diluent or excipient:
Figure imgf000053_0001
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(0)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof. with the proviso that R5 is not phenyl when both of R3 and R4 are methyl.
2. A compound of formula (1 ):
Figure imgf000054_0001
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(O),N(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof. with the proviso that R5 is not phenyl when both of R3 and R4 are methyl.
3. A method of treating a condition selected from cancer, angiogenesis, and neurological disorders in a mammal, which method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formula (1):
Figure imgf000054_0002
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O),R8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
4. A method of treating a condition selected from inflammation and cell migration in a mammal, which method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formula (1):
Figure imgf000055_0001
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons; R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof; with the proviso that R5 is not phenyl when both of R3 and R4 are methyl.
5. A method of preventing a condition selected from cancer, angiogenesis, and neurological disorders in a mammal, which method comprises administering to the mammal in need thereof a prophylactically effective amount of a compound of formula (1):
Figure imgf000056_0001
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
6. A method of preventing a condition selected from inflammation and cell migration in a mammal, which method comprises administering to the mammal in need thereof a prophylactically effective amount of a compound of formula (1 ):
Figure imgf000057_0001
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2) -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form I=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof; with the proviso that R5 is not phenyl when both of R3 and R4 are methyl.
7. A method of promoting apoptosis in proliferating cells in a mammal, which method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formula (1):
Figure imgf000058_0001
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form I=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
8. A method of inhibiting PKB activity in a mammal, which method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formula (1 ):
Figure imgf000058_0002
wherein, independently at each occurrence: each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R6-N=N-O-R7, -OR8, -C(O)OR8, -C(O)N(R8)2, -N(R8)2, -N(R8)C(O)R8, -N(R8)C(O)OR7, -S(O)tR8 (where t is 0 to 2), -S(O)tN(R8)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R9)(R10); each of R4 and R5 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R6 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R7 is hydrogen, alkyl or aralkyl; each R8 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R9 is hydrogen, alkyl or aralkyl; and
R10 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof; with the proviso that R5 is not phenyl when both of R3 and R4 are methyl.
9. A method, composition or compound according to any one of claims 1-8 where R1 is H.
10. A method, composition or compound according to any one of claims 1-8 where R1 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl.
11. A method, composition or compound according to any one of claims 1-8 wherein R1 is selected from the group consisting of aryl, aralkylene and aralkenylene.
12. A method, composition or compound according to any one of claims 1-8 wherein R1 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
13. A method, composition or compound according to any one of claims 1-8 wherein R1 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(0)OR9, -C(O)N(R9)2, -N(R9)2, -N(R )C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2).
14. A method, composition or compound according to any one of claims 1-13 where R2 is H.
15. A method, composition or compound according to any one of claims 1-13 where R2 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl.
16. A method, composition or compound according to any one of claims 1-13 wherein R2 is selected from the group consisting of aryl, aralkylene and aralkenylene.
17. A method, composition or compound according to any one of claims 1-13 wherein R2 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
18. A method, composition or compound according to any one of claims 1-13 wherein R2 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, _R7_N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2).
19. A method, composition or compound according to any one of claims 1-18 where R3 is H.
20. A method, composition or compound according to any one of claims 1-18 where R3 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl.
21. A method, composition or compound according to any one of claims 1-18 wherein R3 is selected from the group consisting of aryl, aralkylene and aralkenylene.
22. A method, composition or compound according to any one of claims 1-18 wherein R3 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
23. A method, composition or compound according to any one of claims 1-18 wherein R3 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2).
24. A method, composition or compound according to any one of claims 1-23 wherein R4 is hydrogen.
25. A method, composition or compound according to any one of claims 1-23 wherein R4 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene.
26. A method, composition or compound according to any one of claims 1-23 wherein R4 is selected from the group consisting of aryl and aralkylene.
27. A method, composition or compound according to any one of claims 1-23 wherein R4 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
28. A method, composition or compound according to any one of claims 1-27 wherein R5 is hydrogen.
29. A method, composition or compound according to any one of claims 1-27 wherein R5 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene.
30. A method, composition or compound according to any one of claims 1-27 wherein R5 is selected from the group consisting of aryl and aralkylene.
31. A method, composition or compound according to any one of claims 1 -27 wherein R5 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
32. A method, composition or compound according to any one of claims 1-31 wherein R6 is hydrogen.
33. A method, composition or compound according to any one of claims 1-31 wherein R6 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene.
34. A method, composition or compound according to any one of claims 1-31 wherein R6 is selected from the group consisting of aryl and aralkylene.
35. A method, composition or compound according to any one of claims 1-31 wherein R6 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
36. A method, composition or compound according to any one of claims 1-35 wherein R7 is a direct bond.
37. A method, composition or compound according to any one of claims 1-35 wherein R7 is an unsubstituted alkylene.
38. A method, composition or compound according to any one of claims 1-35 wherein R7 is a substituted alkylene.
39. A method, composition or compound according to any one of claims 1-38 wherein R8 is hydrogen.
40. A method, composition or compound according to any one of claims 1-38 wherein R8 is alkyl.
41. A method, composition or compound according to any one of claims 1-38 wherein R8 is aralkyl.
42. A method, composition or compound according to any one of claims 1-41 wherein the compound is a pharmaceutically acceptable salt.
43. A method, composition or compound according to any one of claims 1-42 wherein the compound has a molecular weight between 100 and 1,000 Daltons.
44. A method, composition or compound according to any one of claims 1-42 wherein the compound has a molecular weight between 250 and 750 Daltons.
45. A method according to any one of claims 1-44 wherein the mammal is a human.
46. A method of treating a condition selected from cancer, inflammation, angiogenesis, cell migration and neurological disorders in a mammal, which method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formula (2):
Figure imgf000062_0001
wherein, independently at each occurrence:
E is selected from the group consisting of O and S; each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2l -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), -S(O),N(R9)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R and R2 together may form J=(R10)(R11); each of R4, R5 and R6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R8 is hydrogen, alkyl or aralkyl; each R9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R10 is hydrogen, alkyl or aralkyl; and
R11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
47. A method of preventing a condition selected from cancer, inflammation, angiogenesis, cell migration and neurological disorders in a mammal, which method comprises administering to the mammal in need thereof a prophylactically effective amount of a compound of formula (2):
Figure imgf000063_0001
wherein, independently at each occurrence:
E is selected from the group consisting of O and S; each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O),R9 (where t is 0 to 2), -S(O),N(R9)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R10)(R11); each of R4, R5 and R6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R8 is hydrogen, alkyl or aralkyl; each R9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R10 is hydrogen, alkyl or aralkyl; and
R 1 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
48. A method of promoting apoptosis in proliferating cells in a mammal, which method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formula (2):
Figure imgf000064_0001
wherein, independently at each occurrence:
E is selected from the group consisting of O and S; each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O),R9 (where t is 0 to 2), -S(O)tN(R9)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form I=(R10)(R11); each of R4, R5 and R6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R8 is hydrogen, alkyl or aralkyl; each R9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R10 is hydrogen, alkyl or aralkyl; and R11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solyate or polymorph; or as a pharmaceutically acceptable salt thereof.
49. A method of inhibiting PKB activity in a mammal, which method comprises administering to the mammal in need thereof a therapeutically effective amount of a compound of formula (2):
Figure imgf000065_0001
wherein, independently at each occurrence:
E is selected from the group consisting of O and S; each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(0)tR9 (where t is 0 to 2), -S(O)tN(R9)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R10)(R11); each of R4, R5 and R6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R8 is hydrogen, alkyl or aralkyl; and each R9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R10 is hydrogen, alkyl or aralkyl; and
R11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof.
50. A pharmaceutical composition comprising a compound of formula (2) and a pharmaceutically acceptable carrier, diluent or excipient:
Figure imgf000066_0001
wherein, independently at each occurrence:
E is selected from the group consisting of O and S; each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O),R9 (where t is 0 to 2), -S(O)tN(R9)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form J=(R10)(R11); each of R4, R5 and R6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R8 is hydrogen, alkyl or aralkyl; and each R9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R10 is hydrogen, alkyl or aralkyl; and
R11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof; with the proviso that when E is O or S and R3 is methyl and R4 is methyl and R5 is phenyl, then R1 and R2 are not selected from hydrogen, methyl or ethyl.
51. A method or composition according to any one of claims 46-50 wherein E is O.
52. A method or composition according to any one of claims 46-50 wherein E is S.
53. A method or composition according to any one of claims 46-52 where R1 is
H.
54. A method or composition according to any one of claims 46-52 where R1 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl.
55. A method or composition according to any one of claims 46-52 wherein R1 is selected from the group consisting of aryl, aralkylene and aralkenylene.
56. A method or composition according to any one of claims 46-52 wherein R1 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
57. A method or composition according to any one of claims 46-52 wherein R1 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2).
58. A method or composition according to any one of claims 46-57 where R2 is H.
59. A method or composition according to any one of claims 46-57 where R2 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl.
60. A method or composition according to any one of claims 46-57 wherein R2 is selected from the group consisting of aryl, aralkylene and aralkenylene.
61. A method or composition according to any one of claims 46-57 wherein R2 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
62. A method or composition according to any one of claims 46-57 wherein R2 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-0-R8, -OR9, -C(O)OR9, -C(0)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), and -S(0)tN(R9)2 (where t is 0 to 2).
63. A method or composition according to any one of claims 46-52 wherein R1 and R2 together form I=(R10)(R11).
64. A method or composition according to any one of claims 46-63 where R3 is H.
65. A method or composition according to any one of claims 46-63 where R3 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl.
66. A method or composition according to any one of claims 46-63 wherein R3 is selected from the group consisting of aryl, aralkylene and aralkenylene.
67. A method or composition according to any one of claims 46-63 wherein R3 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
68. A method or composition according to any one of claims 46-63 wherein R3 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2).
69. A method or composition according to any one of claims 46-68 wherein R4 is hydrogen.
70. A method or composition according to any one of claims 46-68 wherein R4 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene.
71. A method or composition according to any one of claims 46-68 wherein R4 is selected from the group consisting of aryl and aralkylene.
72. A method or composition according to any one of claims 46-68 wherein R4 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
73. A method or composition according to any one of claims 46-72 wherein R5 is hydrogen.
74. A method or composition according to any one of claims 46-72 wherein R5 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene.
75. A method or composition according to any one of claims 46-72 wherein R5 is selected from the group consisting of aryl and aralkylene.
76. A method or composition according to any one of claims 46-72 wherein R5 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
77. A method or composition according to any one of claims 46-76 wherein R6 gen.
78. A method or composition according to any one of claims 46-76 wherein R6 ted from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene.
79. A method or composition according to any one of claims 46-76 wherein R6 ted from the group consisting of aryl and aralkylene.
80. A method or composition according to any one of claims 46-76 wherein R6 ted from the group consisting of heterocyclyl and heterocyclyalkylene.
81. A method or composition according to any one of claims 46-80 wherein R7 ct bond.
82. A method or composition according to any one of claims 46-80 wherein R7 substituted alkylene.
83. A method or composition according to any one of claims 46-80 wherein R7 stituted alkylene.
84. A method or composition according to any one of claims 46-83 wherein R8 gen.
85. A method or composition according to any one of claims 46-83 wherein R8
86. A method or composition according to any one of claims 46-83 wherein R8 yl.
87. A method or composition according to any one of claims 46-86 wherein R9 gen.
88. A method or composition according to any one of claims 46-86 wherein R9 ted from the group consisting of alkyl, alkenyl, cycloalkyl and cycloalkylalkylene.
89. A method or composition according to any one of claims 46-86 wherein R9 ted from the group consisting of aryl and aralkylene.
90. A method or composition according to any one of claims 46-89 as a mixture of steroisomers.
91. A method or composition according to any one of claims 46-90 wherein the compound is a pharmaceutically acceptable salt.
92. A method or composition according to any one of claims 46-91 wherein the compound has a molecular weight between 100 and 1,000 Daltons.
93. A method or composition according to any one of claims 46-91 wherein the compound has a molecular weight between 1 ,000 and 2,000 Daltons.
94. A method according to any one of claims 46-93 wherein the mammal is a human.
95. A method according to any one of claims 46-94 wherein the condition is cancer.
96. A method according to any one of claims 46-95 wherein the condition is inflammation.
97. A compound of formula (1 )
Figure imgf000070_0001
wherein, independently at each occurrence:
E is selected from the group consisting of O and S; each of R1, R2, and R3 is selected from the group consisting of hydrogen, alkoxy, alkyl, alkenyl, aryl, aralkyl, aralkenyl, cycloalkyl, cycloalkylalkyl, halo, haloalkyl, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), -S(O),N(R9)2 (where t is 0 to 2), heterocyclyl and heterocyclylalkyl; or R1 and R2 together may form j=(R10)(R11); each of R4, R5 and R6 is selected from the group consisting of hydrogen, alkyl, aralkyl, aryl, haloalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl or heterocyclylalkyl; each R7 is independently a direct bond or an optionally substituted alkylene of one to six carbons;
R8 is hydrogen, alkyl or aralkyl; and each R9 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl or aryl;
R10 is hydrogen, alkyl or aralkyl; and
R11 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, aryl, heterocyclyl or heterocyclylalkyl; as a single stereoisomer, a mixture of stereoisomers, or as a racemic mixture of stereoisomers; or as a solvate or polymorph; or as a pharmaceutically acceptable salt thereof; with the proviso that compounds of the present invention do not include the following excluded compounds, where RN stands for Registry Number as defined by the Chemical Abstract Service of the American Chemical Society (Washington, D.C.): 5- Thiazolidineacetic acid, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)-2,4- dioxo- (RN 405924-24-5); 2,4-Thiazolidinedione, 5-[(2-chlorophenyl)methylene]-3-(2,3- dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)- (RN 392322-35-9); 2,4- Thiazolidinedione, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)-5-[(3- ethoxy-4-hydroxyphenyl)methylene]- (RN 392322-32-6); 2,4-Thiazolidinedione, 3-(2,3- dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)-5-[(4-hydroxyphenyl)methylene]- (RN 392322-29-1); 2,4-Thiazolidinedione, 5-[(4-chlorophenyl)methylene]-3-(2,3-dihydro- 1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)- (RN 392322-26-8); 2,4-Thiazolidinedione, 3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)-5-[(4- fluorophenyl)methylene]- (RN 392322-23-5); 2,4-Thiazolidinedione, 3-(2,3-dihydro-1 ,5- dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)-5-(3-phenyl-2-propenylidene)- (RN 314275-30- 4); 2,4-Thiazolidinedione, 5-[2-chloro-3-(4-nitrophenyl)-2-propenylidene]-3-(2,3-dihydro- 1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)- (RN 314275-24-6); 2,4-Thiazolidinedione, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)-5-(1 ,2-dihydro-2-oxo-3H- indol-3-ylidene)- (RN 301227-46-3); 2,4-Thiazolidinedione, 3-(2,3-dihydro-1 ,5-dimethyl-3- oxo-2-phenyl-1 H-pyrazol-4-yl)-5-[(4-hydroxy-3-methoxyphenyl)methylene]- (RN 301227- 45-2); 2,4-Thiazolidinedione, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4- yl)-5-[(3,4-dimethoxyphenyl)methylene]- (RN 301227-44-1); 2,4-Thiazolidinedione, 3-(2,3- dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)-5-[[4-
(dimethylamino)phenyl]methylene]- (RN 301227-43-0); 2,4-Thiazolidinedione, 3-(2,3- dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)-5-[(4-methoxyphenyl)methylene]- (RN 301227-42-9); 2,4-Thiazolidinedione, 3-(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H- pyrazol-4-yl)-5-(phenylmethylene)- (RN 301227-41-8); 2,4-Thiazolidinedione, 5-(5-bromo- 1 ,2-dihydro-2-oxo-3H-indol-3-ylidene)-3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H- pyrazol-4-yl)- (RN 299954-23-7); 2,4-Thiazolidinedione, 3-(2,3-dihydro-1 ,5-dimethyl-3- oxo-2-phenyl-1 H-pyrazol-4-yl)-5-(1 ,2-dihydro-1-methyl-2-oxo-3H-indol-3-ylidene)- (RN 299954-22-6); 2,4-Thiazolidinedione, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1H- pyrazol-4-yl)-5-[(3-nitrophenyl)methylene]- (RN 299954-21-5); 2,4-Thiazolidinedione, 3- (2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)-5-[(4-nitrophenyl)methylene]- (RN 299954-20-4); 2,4-Thiazolidinedione, 3-antipyrinyl-5,5-dimethyl- (RN 93312-04-0); 2,4-Thiazolidinedione, 3-(2,3-dihydro-1 ,5-dimethyl-3-oxo-2-phenyl-1 H-pyrazol-4-yl)- (RN 93293-15-3).
98. A compound according to claim 97 wherein E is O.
99. A compound according to claim 97 wherein E is S.
100. A compound according to any one of claims 97-99 where R1 is H.
101. A compound according to any one of claims 97-100 where R1 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl.
102. A compound according to any one of claims 97-100 wherein R1 is selected from the group consisting of aryl, aralkylene and aralkenylene.
103. A compound according to any one of claims 97-100 wherein R1 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
104. A compound according to any one of claims 97-100 wherein R1 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O),R9 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2).
105. A compound according to any one of claims 97-104 where R2 is H.
106. A compound according to any one of claims 97-104 where R2 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl.
107. A compound according to any one of claims 97-104 wherein R2 is selected from the group consisting of aryl, aralkylene and aralkenylene.
108. A compound according to any one of claims 97-104 wherein R2 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
109. A compound according to any one of claims 97-104 wherein R2 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2).
110. A compound according to any one of claims 97-104 wherein R1 and R2 together form J=(R10)(R11).
111. A compound according to any one of claims 97-110 where R3 is H.
112. A compound according to any one of claims 97-110 where R3 is selected from the group consisting of alkyl, alkenyl, cycloalkyl, and cycloalkylalkyl.
113. A compound according to any one of claims 97-110 wherein R3 is selected from the group consisting of aryl, aralkylene and aralkenylene.
114. A compound according to any one of claims 97-110 wherein R3 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
115. A compound according to any one of claims 97-110 wherein R3 is selected from the group consisting of halo, alkoxy, haloalkoxy, nitro, cyano, -R7-N=N-O-R8, -OR9, -C(O)OR9, -C(O)N(R9)2, -N(R9)2, -N(R9)C(O)R9, -N(R9)C(O)OR8, -S(O)tR9 (where t is 0 to 2), and -S(O)tN(R9)2 (where t is 0 to 2).
116. A compound according to any one of claims 97-115 wherein R4 is hydrogen.
117. A compound according to any one of claims 97-115 wherein R4 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene.
118. A compound according to any one of claims 97-115 wherein R4 is selected from the group consisting of aryl and aralkylene.
119. A compound according to any one of claims 97-115 wherein R4 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
120. A compound according to any one of claims 97-115 wherein R5 is hydrogen.
121. A compound according to any one of claims 97-115 wherein R5 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene.
122. A compound according to any one of claims 97-115 wherein R5 is selected from the group consisting of aryl and aralkylene.
123. A compound according to any one of claims 97-115 wherein R5 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
124. A compound according to any one of claims 97-123 wherein R6 is hydrogen.
125. A compound according to any one of claims 97-123 wherein R6 is selected from the group consisting of alkyl, haloalkyl, cycloalkyl, and cycloalkylalkylene.
126. A compound according to any one of claims 97-123 wherein R6 is selected from the group consisting of aryl and aralkylene.
127. A compound according to any one of claims 97-123 wherein R6 is selected from the group consisting of heterocyclyl and heterocyclyalkylene.
128. A compound according to any one of claims 97-123 wherein R7 is a direct bond.
129. A compound according to any one of claims 97-123 wherein R7 is an unsubstituted alkylene.
130. A compound according to any one of claims 97-123 wherein R7 is a substituted alkylene.
131. A compound according to any one of claims 97-130 wherein R8 is hydrogen.
132. A compound according to any one of claims 97-130 wherein R8 is alkyl.
133. A compound according to any one of claims 97-130 wherein R8 is aralkyl.
134. A compound according to any one of claims 97-133 wherein R9 is hydrogen.
135. A compound according to any one of claims 97-133 wherein R9 is selected from the group consisting of alkyl, alkenyl, cycloalkyl and cycloalkylalkylene.
136. A compound according to any one of claims 97-133 wherein R9 is selected from the group consisting of aryl and aralkylene.
137. A compound according to any one of claims 97-133 as a mixture of steroisomers.
138. A compound according to any one of claims 97-137 wherein the compound is a pharmaceutically acceptable salt.
139. A compound according to any one of claims 97-138 wherein the compound has a molecular weight between 100 and 1,000 Daltons.
140. A compound according to any one of claims 97-138 wherein the compound has a molecular weight between 1 ,000 and 2,000 Daltons.
PCT/CA2003/000815 2002-05-31 2003-05-30 Thiazolidinedione derivatives in therapeutic and prophylactic methods WO2003101373A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003233716A AU2003233716A1 (en) 2002-05-31 2003-05-30 Thiazolidinedione derivatives in therapeutic and prophylactic methods

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US38443702P 2002-05-31 2002-05-31
US60/384,437 2002-05-31
US38633002P 2002-06-04 2002-06-04
US60/386,330 2002-06-04

Publications (2)

Publication Number Publication Date
WO2003101373A2 true WO2003101373A2 (en) 2003-12-11
WO2003101373A3 WO2003101373A3 (en) 2004-04-29

Family

ID=29715329

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2003/000815 WO2003101373A2 (en) 2002-05-31 2003-05-30 Thiazolidinedione derivatives in therapeutic and prophylactic methods

Country Status (2)

Country Link
AU (1) AU2003233716A1 (en)
WO (1) WO2003101373A2 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004086038A2 (en) * 2003-03-28 2004-10-07 Institut Curie A method for screening compounds having the capacity to control tumor cell invasion or metastasis using constitutively active akt
EP1566202A1 (en) * 2004-02-23 2005-08-24 Sahltech I Göteborg AB Use of resistin antagonists in the treatment of rheumatoid arthritis

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; LADNAYA, L. YA. ET AL: "Synthesis and conversion of antipyrine derivatives with a thiazolidine ring" retrieved from STN Database accession no. 76:34166 XP002257283 & KHIM. ISSLED. FARM. (1970) 35-7 FROM: REF. ZH., KHIM. 1970, ABSTR. NO. 23ZH400, *
KUZHELYUK, A. A. ET AL: "Characteristic absorption of the carbonyl group in molecules of thiazolidine and 1,3-thiazane derivatives" IZVESTIYA VYSSHIKH UCHEBNYKH ZAVEDENII, KHIMIYA I KHIMICHESKAYA TEKHNOLOGIYA (1973), 16(10), 1516-21 , XP009018568 *
LESYK, R. B. ET AL: "Synthesis of rhodanine derivatives based on 4-aminoantipyrine and investigation of their antiinflammatory activity" FARMATSEVTICHNII ZHURNAL (KIEV) (1996), (5-6), 94-98 , XP009018576 *
LESYK, R. ET AL: "New thiazolidones-4 with pyrazolone-5 substituent as the potential NSAIDs" BOLLETTINO CHIMICO FARMACEUTICO (1998), 137(6), 210-217 , XP009016670 *
LESYK, R. ET AL: "Synthesis and antiinflammatory activity of novel 3-(2,3-dimethyl-1-phenyl-4-pyrazolon-5-yl) -4-thiazolidones" BOLLETTINO CHIMICO FARMACEUTICO (2002), 141(3), 197-201 , XP009018572 *
MORITA, MINORU ET AL: "Synthesis and antibacterial activity of antipyrine derivatives. I. 4-Thiazoline-2-thiones, dithiocarbamic acid esters, and rhodanines" (1962), 82, 36-45, XP009018570 *
MORITA, MINORU ET AL: "Synthesis and antibacterial activity of antipyrine derivatives. VI. 4-Thiazolidinones" (1962), 82, 69-74, XP009018569 *
SINGH, S.R. ET AL: "Synthesis and antibacterial activity of antipyrine derivatives" BOKIN BOBAI (1984) 12(6) 273-7, XP009018573 *
TURKEVICH, N. M. ET AL: "Infrared spectra of 2-thiono- and 2-oxo-4-thiazolidinone derivatives" IZVESTIYA VYSSHIKH UCHEBNYKH ZAVEDENII, KHIMIYA I KHIMICHESKAYA TEKHNOLOGIYA (1972), 15(6), 859-62 , XP009018571 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004086038A2 (en) * 2003-03-28 2004-10-07 Institut Curie A method for screening compounds having the capacity to control tumor cell invasion or metastasis using constitutively active akt
WO2004086038A3 (en) * 2003-03-28 2005-01-06 Inst Curie A method for screening compounds having the capacity to control tumor cell invasion or metastasis using constitutively active akt
EP1566202A1 (en) * 2004-02-23 2005-08-24 Sahltech I Göteborg AB Use of resistin antagonists in the treatment of rheumatoid arthritis

Also Published As

Publication number Publication date
AU2003233716A8 (en) 2003-12-19
WO2003101373A3 (en) 2004-04-29
AU2003233716A1 (en) 2003-12-19

Similar Documents

Publication Publication Date Title
KR101151647B1 (en) -3 thiadiazolidinones as gsk-3 inhibitors
KR101471999B1 (en) -3 gsk-3 inhibitors
US20220218632A1 (en) Pharmaceutical compositions and combinations comprising inhibitors of the androgen receptor and uses thereof
US11236090B2 (en) Substituted glutarimides as CDK inhibitors
EP2019828A1 (en) N- (2-thiazolyl) -amide derivatives as gsk-3 inhibitors
JP2007509173A (en) Indolinone derivatives and their use in the treatment of disease states such as cancer
HUE030848T2 (en) Benzylideneguanidine derivatives and therapeutic use for the treatment of protein misfolding diseases
US20230183252A1 (en) SUBSTITUTED IMIDAZO[5,1-d][1,2,3,5]TETRAZINES FOR THE TREATMENT OF CANCER
US7393869B2 (en) Methods of using thiazolidine derivatives to treat cancer or inflammation
US20110166191A1 (en) 3-(2-amino-ethyl)-5-(3-cyclohexyl-propylidene)-thiazolidine-2,4-dione and its derivatives as multiple signaling pathway inhibitors and for the treatment of cancer
KR101123071B1 (en) Novel imidazole derivatives or pharmaceutically acceptable salts thereof, preparation method thereof and pharmaceutical composition for the prevention and treatment of melanoma containing the same as an active ingredient
US20060281798A1 (en) Methods of using thiazolidinedithione derivatives
US7919581B2 (en) Bi-dentate compounds as kinase inhibitors
WO2003101373A2 (en) Thiazolidinedione derivatives in therapeutic and prophylactic methods
US11731985B2 (en) Compositions and methods for treating cancer
US8614326B2 (en) Therapeutic quinoline and naphthalene derivatives
JP2005535593A5 (en)
WO2014038894A1 (en) Ethyl(2-methyl-3{(e)-[(naphtha[2,1-b]furan-2-ylcarbonyl)hydrazono]methyl}-1h-indole-1-yl)acetate, and composition comprising analogues thereof as active component, for preventing and treating cancer
US20060148848A1 (en) Compounds and methods for treating cancer and inflammation
WO2009078587A2 (en) Composition for prevention and treatment of cancer containing triazolyl-thio-ethanone derivatives inhibiting activity of protein phosphatases or pharmaceutically acceptable salts thereof as an active ingredient
US20060235061A1 (en) Methods of using benzothiophenone derivatives to treat cancer or inflammation
WO2009078586A1 (en) Composition for prevention and treatment of cancer containing phenyl-amino-thiazolone derivatives inhibiting activity of protein phosphatases or pharmaceutically acceptable salts thereof as an active ingredient
US20070060551A1 (en) Methods of using isothiazole derivatives to treat cancer or inflammation
CZ2002178A3 (en) Therapeutic and prophylactic compounds intended for tumor diseases
TWI452043B (en) Oxo-imidazolyl compounds

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP