WO1999052365A1 - Anticancer agents - Google Patents

Abstract

The invention relates to the use of quinoxaline-5,8-dione derivatives in treating cancer. The present invention also provides novel quinoxaline-5,8-dione compounds.

Description

- 1- A TICA CER AGENTS

The present invention relates to the use of quinoxaline-5 , 8-dione derivatives as inhibitors of GTP binding to mutant Ras . The present invention further relates to the use of these compounds in inhibiting cell proliferation and also in treating cancer. The present invention even further relates to novel compounds.

Cancer is a major health problem in the Western world, ranking second only to cardiovascular disease as a cause of mortality. The overarching theory in cancer is that tumor cells represent fundamentally normal cells that have escaped their regulatory processes, resulting in uncontrolled cell proliferation. It is now understood that this uncontrolled cell proliferation occurs as a result of certain cancer causing gene (oncogene) mutations. Ras proteins are an important class of oncogene products implicated in a wide variety of human tumors, including tumors of virtually every tumor group e.g., lung, colorectal, pancreatic, breast, urinary tract, stomach, liver, cervix, ovary, pancreas, gall bladder, leukemias and myelodisplatic syndrome (MDS) (See :

- 2 - Bos.J., Ras Oncogenes in Human Cancer: A Review, Cancer Res. , 9_:4682 (1989) and Mutation Research, 195: 255-271 (1988) ) .

There are three highly related Ras genes in mammalian cells: Ha-Ras, Ki-Ras and N-Ras . These genes are involved in Ras signaling pathways that regulate cellular proliferation and differentiation inside the nucleus. Ras encoded by these genes is a small (21kD) guanine nucleotide-binding and hydrolyzing protein containing intrinsic GTPase activity. Ras proteins cycle between guanosine diphosphate (GDP) -bound (inactive) and guanosine triphosphate (GTP) -bound (active) forms. The switch between GDP-bound and GTP-bound forms of Ras is regulated by its interaction with a Ras GTPase activating protein (GAP) , neurofibromin and GDP/GTP exchange modulators. In its activated form, Ras binds with Raf, resulting in the activation of Raf kinase and the mitogen-activated protein kinase (MAPK) /extracellular signal-regulated kinase (ERK) pathway. The subsequent phosphorylation and activation of MAPK leads to a cascade of events that regulate cell proliferation and differentiation. Krontiris, The New England Journal of Medicine, 333 : 303-306 (1995) .

Non-mutant, GTP-bound Ras is returned to its inactive state upon hydrolysis of GTP by the intrinsic GTPase activity of Ras. Point mutations in Ras, in particular the subtype ki-Ras, are highly prevalent in human cancers. Mutations at codons 12, 13 and 61 are most common, and generally lead to a defective intrinsic GTPase activity of Ras, which in turn results in a perpetually activated and uncontrolled signal to make cells divide. Due to the pivotal role played by the GTP-bound form of Ras in many cancer cells, compounds that inhibit or reduce the interactions between GTP and mutant Ras may be efficacious for treating cancer resulting from mutant Ras. - 3 - The treatment of cancer often requires a multi- disciplinary approach. For patients with localized disease, therapy often consists of an integrated effort utilizing surgery and radiation therapy to gain local control of a primary tumor followed by (or sometimes preceded by) chemotherapy delivered systemically to delay or prevent the emergence of metastatic disease. The use of chemotherapy in this setting corresponds to secondary prevention in medicine. For patients who are diagnosed with metastatic cancer or who are not able to undergo surgery for medical reasons, chemotherapy is often the sole therapeutic modality. In this setting, radiation therapy may be used as an adjunct to palliate localized pain.

The use of standard cytotoxic chemotherapeutic agents is associated with significant toxicity to normal tissues. These drugs possess very narrow therapeutic indices, and when administered at prescribed doses, may also cause bone marrow suppression (which may result in life-threatening neutropenic fever) , diarrhea, ucositis, alopecia, nausea, vomiting, and end organ damage to the liver, kidneys, heart and nervous system. Although considerable progress has been made in the discovery and development of curative chemotherapy regimens to treat several cancers, improved preventative and curative therapies are still greatly needed. Accordingly, it would be advantageous to find additional means of arresting the growth of cancer cells and tumors that can be used alone or in conjunction with other treatments .

The compounds of the present invention have been shown to inhibit the binding of GTP to mutant Ras in the binding assay described below. This inhibition of GTP and mutant Ras binding was confirmed in a whole cell-based luciferase assay designed to measure activation of the Ras pathway downstream of the Ras-GTP interaction. Importantly, the - 4- series of compounds is also active in an _in vitro whole cell proliferation assay utilizing the Ki-Ras dependent human cancer cell line HCT116.

Summary of the Invention The present invention is directed to a method for inhibiting the binding of GTP to mutant Ras by administering a compound of the formula I :

where R]_ and R2 are independently hydrogen, halogen, cyano, trifluoromethyl, C^-Cg alkyl, C^-Cg alkoxy, carboxy, C1-C4 alkoxycarbonyl , amino, mono (C1-C4₎ alkylamino, di(C_]_-C4) alkylamino, amido, C1-C4 alkylamido, aryl, or a heterocycle, where the aryl or heterocycle is optionally substituted with C1-C4 alkyl, C1-C4 alkoxy, halo, cyano or trifluoromethyl ;

R3 is hydrogen, halogen or cyano;

R is SR₅, N(Rg)(R₇), -NH(-L-)R₈, -NHCHR₉R₁₀ or a heterocycle where the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C1-C4 alkyl, C_.-C4 alkoxy, C1-C4 alkoxy (C1-C4 alkyl) or C1-C4 alkoxycarbonyl; - 5 - where R5 is hydrogen, Cχ-C4 alkyl or aryl, where the aryl is optionally substituted with 1, 2 or 3 groups selected from ^cl^_c4 alkyl, C1-C4 alkoxy, halo, cyano or trifluoromethyl;

Rg is hydrogen or methyl;

R7 is C_]_-Cg alkyl, C2-C_]_ alkenyl, benzyl, unsubstituted or substituted C3-C7 cycloalkyl, triphenylmethyl, aryl or a heterocycle, where the C]_-Cg alkyl is optionally substituted with 1, 2, 3, 4 or 5 hydroxy groups and where the aromatic ring of benzyl, the aryl or the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C -C4 alkyl, C1-C4 alkoxy, hydroxy, carboxy, halo, cyano, nitro, trifluoromethyl, C1-C4 alkoxycarbonyl, phenyl, benzyl, morpholino, or piperidinyl ;

L is C2-C3 alkanediyl;

R3 is aryl, C3-C7 cycloalkyl, heterocycle, amino, mono(C^-Cg alkylamino), di(C;j_-Cg alkyl)amino, mono(C -Cg alkoxy)amino or di(C]_-Cg alkoxy) amino, hydroxy (C -C4 alkyl), where the aryl or the heterocycle is optionally substituted with 1, 2 or 3 groups selected from Cχ-C4 alkyl, C1-C4 alkoxy, hydroxy, carboxy, halo, cyano or trifluoromethyl;

R9 and R]_Q are independently hydrogen, C1-C4 alkyl, hydroxy (C1-C4 alkyl), benzyl, C3-C7 cycloalkyl, - (CH₂ ) _ZS (CH₂ ) _z • CH₃ or heterocycle, where the heterocycle or aromatic ring of the benzyl is optionally substituted with from 1 to 3 groups selected from C1-C4 alkyl, Cχ-C4 alkoxy, hydroxy, oxo or halo; and

Z and Z¹ are independently 0, 1, 2 or 3 ; - 6 - or a pharmaceutically acceptable salt or solvate thereof;

provided that :

(1) when R]_ , R₂ and R3 are hydrogen, R4 is not 4-substituted piperazinyl and R5 is not hydrogen or C -C4 alkyl ; and

(2) when R]_ and R₂ are hydrogen or C -C4 alkyl and R3 is hydrogen, R7 is not phenyl, substituted phenyl, morpholinyl or pyrrolidinyl .

In addition, the present invention provides for a method for inhibiting cell proliferation by administering a mammal in need of such treatment a compound of the formula I.

The present invention also provides a method for treating cancer by administering to a mammal in need of such treatment a compound of the formula I. Another aspect of the invention provides for compounds of the formula I and pharmaceutically acceptable salts and solvates thereof as well as pharmaceutical formulations comprising a compound of formula I, or pharmaceutically acceptable salts and solvates thereof, in association with one or more pharmaceutically acceptable carriers, diluents, or excipients.

Detailed Description of the Invention

The terms and abbreviations used in the instant specification have their normal meanings unless otherwise designated. For example, "°C" refers to degrees Celsius; "N" refers to normal or normality; "mmol" refers to millimole or millimoles; "g" refers to gram or grams; "ml" means milliliter or milliliters; "M" refers to molar or molarity; "MS" refers to mass spectrometry ; "IR" refers to infrared spectroscopy ; "NMR" refers to nuclear magnetic resonance spectroscopy and "m/e" refers to the mass to - 7 - charge ratio of ions which appear in the mass spectra of the products (In general, the values correspond to molecular weights of the major peaks, and are so designated "M⁺") . The term "aryl", as used herein, represents an unsaturated aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (such as phenyl) or a multiple fused ring system (such as naphthyl and anthracyl) . When substituted, the substituents may be located at any available position on the aryl ring(s) that are sterically feasible and afford a stable structure.

The term "substituted C3-C7 cycloalkyl", as used herein, represents a cycloalkyl group of 3 to 7 carbon atoms substituted with one or two moieties chosen from the group consisting of halo and Cχ-Cg alkyl. Examples of substituted cycloalkyls include 2-isopropylcyclohexyl,

5-methylcyclohexyl, 2 -isopropyl-5-methylcyclohexyl, 4-chlorocyclohexyl, 3-chlorocyclohexyl, 4-iodocyclohexyl , 3-iodocyclohexyl, 3-chlorocyclopentyl , and 3 -chlorocycloheptyl . The term "heterocycle", as used herein, represents a monovalent saturated or unsaturated group having a single 5 or 6 membered ring and up to 4 nitrogen atoms and/or up to 2 oxygen atoms and/or up to 2 sulfur atoms, arranged to afford a stable structure that is sterically feasible. Exemplary heterocycles include: 2-thienyl or 3-thienyl, 2-furyl or 3-furyl, pyrrolyl, pyridyl, pyrimidyl, imidazolyl, pyrrolidinyl, orpholinyl and piperidinyl .

The term "C]_-Cg alkyl", as used herein, represents a branched or linear, monovalent alkyl group having from one to six carbon atoms. Typical C_]_-Cg alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t.-butyl, n-pentyl, isopentyl, n-hexyl, 2-methylpentyl, and the like. Also encompassed within the term C]_-Cg alkyl is the more narrow range C1-C4 alkyl. The term "C₂-C;]_o alkenyl", as used herein, represents a straight or branched, monovalent, unsaturated aliphatic chain having from two to ten carbon atoms with one or two double bonds. Typical C₂-C o alkenyl groups include ethenyl (also known as vinyl) , 1-methylethenyl , 1-methyl-1-propenyl, 1-hexenyl, 2-methyl-2-propenyl , 1-propenyl, 1-butenyl, 2-pentenyl, and the like.

The term "C]_-Cg alkoxy", as used herein, represents a straight or branched -0-(C]_-Cg alkyl) chain. The oxygen atom bonds at the point of attachment to the parent molecule. Typical C -Cg alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, t.-butoxy, pentoxy and the like. Also encompassed within the term Cχ-Cg alkoxy is the more narrow range C1-C4 alkoxy. The term "halo" or "halogen", as used herein, means fluorine, chlorine, bromine, or iodine.

The term "C1-C4 alkoxycarbonyl, as used herein, represents a C -C4 alkoxy group attached to a carbonyl group [-C (0) - (C1-C4 alkoxy)]. The alkoxycarbonyl is bonded to the parent molecule via the carbonyl group.

The term "hydroxymethylene" , as used herein, represents the radical -CH₂OH.

The term "amido" means an aminocarbonyl (-C(0)NH ) group . The term "C1-C4 alkylamino" or "mono (Cχ-C4 alkylamino)" means a group - H(Cχ-C4 alkyl) .

The term "C]_-Cg alkylamino" or "mono(C -Cg alkylamino)" means a group -NH(C_]_-Cg alkyl) .

The term "di (C -Cg₎ alkylamino" or " (C_]_-Cg) dialkylamino) " means a group -N(Cχ-Cg) .

The term "di (C1-C4 ₎ alkylamino" or " (C1-C4 ) dialkylamino" means a group -N(C]_-C4 alkyl) ₂.

The term "C1-C4 alkylamido" means a group -C (O)NH (C1-C4 alkyl) . - 9 -

The term "Cχ-Cg alkylamido" means a group -C (0) NH (Ci-C alkyl) .

The term "C]_-Cg alkoxyamino" means -NH(C]_-Cg alkoxy).

The term "C^-Cg dialkoxyamino" or "di (C^-Cg alkoxyamino)" means (-N(Cχ-Cg alkoxy)₂).

The term "oxo" means a double bond to an oxygen atom.

The term "C1-C4 alkoxy (C1-C4 alkyl)" means a C1-C4 alkoxy group bonded to a C -C4 alkyl group where the C1-C4 alkyl group is bonded to the parent molecule. The term "hydroxy (C1-C4 alkyl)" means a C1-C4 alkyl group substituted with an -OH group.

The term "treating" as used herein includes prophylaxis of the named physical condition or delay in the onset of the named physical condition or amelioration or elimination of the disease or condition once it has been established.

The compounds of the present invention are known to form solvates with appropriate solvents. Preferred solvents for the preparation of solvate forms include water, alcohols, tetrahydrofuran (THF), DMF, and DMSO. Preferred alcohols are methanol and ethanol. Other appropriate solvents may be selected based on the size of the solvent molecule. Small solvent molecules are preferred to facilitate the corresponding solvate formation. The solvate is typically formed in the course of recrystallization or in the course of salt formation. One useful reference concerning solvates is Sykes, Peter, A Guidebook to Mechanism in Organic Chemistry, _6, 56 (1986), John Wiley & Sons, New York. The term "solvate" as used herein includes hydrate forms such as monohydrate and dihydrates . The compounds claimed herein can also form acid addition salts with a wide variety of inorganic and organic acids. Typical acids which can be used include sulfuric, hydrochloric, hydrobromic, phosphoric, hypophosphoric, hydroiodic, sulfamic, citric, acetic, maleic, malic, - 10 - succinic, tartaric, cinnamic, benzoic, ascorbic, mandelic, p-toluenesulfonic, benzenesulfonic, methanesulfonic, trifluoroacetic, hippuric and the like. The preferred pharmaceutically acceptable salts are those formed with hydrochloric acid or acetic acid.

If a compound of formula I contains a chiral element, it may exist in, and be isolated in, optically active and racemic forms. If a compound of formula I contains an additional chiral element, such compound of formula I may exist in, and be isolated in, the form of a diastereomeric mixture or as a single diastereomer . The present invention encompasses a compound of formula I as an individual diastereomer, mixtures of diastereomers, racemic mixtures and individual enantiomers. One skilled in the art will appreciate that individual enantiomers can be isolated using well-known resolution techniques. One useful reference describing various techniques is Jacques et al . , Enantiomers, Racemates, and Resolutions (John Wiley and Sons 1981) . Appropriate resolution methods include direct crystallization, entrainment, and crystallization by optically active solvents. Chrisey, L.A. Heterocycles, 267 , 30 (1990) .

While the compounds of formula I are important in the concept of the present invention, certain groups of compounds constitute preferred aspects of the invention. The following list sets out a number of such preferred groups, each of which constitutes a preferred aspect of the invention, and the formulations, methods of use and the like associated with each such group are also preferred aspects. It will be understood that the reader can combine groups of preferred aspects from the list below to produce additional more limited or more comprehensive aspects:

R]_ and R are independently hydrogen, halogen, C_.-C4 alkyl, C -C4 alkoxy, aryl, or a 5 or 6 membered heterocycle, where the aryl and/or 5 or 6 membered heterocycle is - 11 - optionally substituted with Cχ-C4 alkyl, C1-C4 alkoxy, halo, or cyano ;

Ri and R₂ is independently hydrogen, methyl, phenyl,

4-methoxyphenyl, 2-furyl, or 2-pyridyl; R3 is hydrogen or halogen;

R3 is bromo;

R4 is SR5 where R5 is hydrogen, C -C4 alkyl or aryl, where the aryl is optionally substituted with 1, 2 or 3 groups selected from Cχ-C4 alkyl, C -C4 alkoxy, halo, cyano or trifluoromethyl;

R5 is phenyl;

R is N(Rg) (R₇) ;

Rg is hydrogen or methyl;

R7 is hydrogen, methyl, benzyl, phenyl, 2-methylphenyl, 3- methylphenyl , 4-methylphenyl , 2-fluorophenyl , 3- fluorophenyl, 4-fluorophenyl, 2 , 4-difluorophenyl, 4- chlorophenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4- methoxyphenyl , 4-ethoxycarbonylphenyl, 4-cyanophenyl, 2- hydroxyphenyl , 4-hydroxyphenyl, 4-fluorobenzyl, triphenylmethyl, cyclohexylmethyl, 1-benzyl-4-piperidyl,

2, 3-dihydroxypropyl, 2 , 3 , 4, 5 , 6-pentahydroxyhexyl , 3,4- dimethoxyphenyl , 2 , 4-dimethoxyphenyl , 3,4,5- trimethoxyphenyl, 4-morpholinophenyl , cyclohexyl, triphenylmethyl, 3 , 7-dimethyl-2 , 6-dioctenyl , 3, 3-dimethylpropyl, 2 -methoxyethyl , l-benzyl-4-piperazinyl , 4-methylpiperazin-l-yl, l-ethylpiperidin-3-yl , 4-piperidinylpiperidin-l-yl, l-benzylpyrrolidin-3-yl, or 4-nitrophenyl ; R is -NH(-L-)R₈; L is C₂-C₈ alkanediyl,

R3 is N,N-dimethylamino, N, N-diethylamino, N,N- diisopropylamino, N,N-di-2-ethoxyamino, N,N-diethoxyamino, N,N-2-hydroxyethylamino, 1-imidazol, 1-pyrrolidinyl, - 12 - pyrrolidin-2-on-l-yl , 1-piperidinyl, 1-morpholinyl , 4-methylpiperazinyl or 1-piperazinyl ; R₄ is -NHCHRgRxo;

Rg and Rχo are independently hydrogen, l-ethylpyrrolidin-2- yl, hydroxymethylene, phenyl, benzyl, 4-hydroxybenzyl , -CH₂CH₂SCH3, isopropyl or l-ethyl-3-piperidin-3-yl ; or R4 is 1-pyrrolidinyl, 2-methoxymethylpyrrolidin-l-yl or 2-t- butoxycarbonylpyrrolidin-1-yl .

The following group is illustrative of some preferred compounds contemplated within the scope of this invention: 6- (phenylamino) quinoxaline-5 , 8-dione 5Aa; 6-[(2- methylphenyl) amino] quinoxaline-5 , 8-dione 5Ab; 6-[(3- methylphenyl) amino] quinoxaline-5 , 8-dione 5Ac; 6-[(4- methylphenyl) amino] quinoxaline-5, 8-dione 5Ad; 7-bromo-6- [ (4- methylphenyl) amino] quinoxaline-5, 8-dione 5Ae; 6-[(3- fluorophenyl) amino] quinoxaline-5, 8-dione 5Af; 6-[(4- fluorophenyl) amino] quinoxaline-5 , 8-dione 5Ag; 6-[(4- methoxyphenyl) amino] quinoxaline-5, 8-dione 5Ah; 6- [ [4- (ethyloxycarbonyl ) phenyl] amino] quinoxaline-5 , 8-dione 5Ai ; 6- [ (4-cyanophenyl) amino] quinoxaline-5, 8-dione 5Aj ; 6- (benzylamino) uinoxaline-5 , 8-dione 5Ak; 6-[(4- fluorobenzyl) amino] quinoxaline-5 , 8-dione 5A1 ; 6- (triphenylmethyl) amino] quinoxaline-5 , 8-dione 5Am; 6- (cyclohexylmethylamino) quinoxaline-5, 8-dione 5Ao; 6- [ [ 1- (benzyl)piperidin-4-yl] amino] quinoxaline-5, 8-dione 5Ap;

6- [ [3- (dimethylamino)propyl] amino] quinoxaline-5 , 8-dione 5Aq; 6- [ [3- (imidazol-l-yl)propyl] amino] quinoxaline-5 , 8-dione 5Ar ; 6- [ [ (1- ( S) -hydroxymethyl-2 -phenyl) ethyl] amino] quinoxaline- 5,8-dione 5As ; 6- [ [ [1- ( S) -hydroxymethyl-2- (4- hydroxyphenyl) ] ethyl] amino] uinoxaline-5, 8-dione 5At ; 6- [ [ (1- ( S) -hydroxymethyl-3 -methylthio)propyl] amino] - quinoxaline-5, 8-dione 5Au; 6- [ [ ( 1- ( S) -hydroxymethyl-2- methyl)propyl] amino] quinoxaline-5, 8-dione 5Av; 6-[2-((5)- methoxymethyl)pyrrolidin-l-yl] quinoxaline-5 , 8-dione 5Aw; - 13 - 6- [2- ( ( S) -tert-butyloxycarbony) pyrrolidin-1-yl] quinoxaline- 5,8-dione 5Ax; 6- (pyrrolidin-1-yl ) quinoxaline-5 , 8-dione 5Ay; 6- [ (2 ( S) , 3-dihydroxypropyl) amino] quinoxaline-5 , 8-dione 5Az ; 6- [ [ ( 2 -hydroxyl-1-hydroxymethyl) ethyl] amino] quinoxaline-5 , 8- dione 5Aa; 6- [ [2 ( S) , 3 ( S) , 4 (R) , 5 ( R) , 6- (pentahydroxyl) hexyl] - amino] quinoxaline-5, 8-dione 5Ab; 6- (phenylthio) quinoxaline- 5,8-dione 5Ac; 2 , 3-dimethyl-6- (phenylamino) quinoxaline-5 , 8- dione 5Ba; 2 , 3-dimethyl-6- [ (4-methylphenyl ) amino] - quinoxaline-5, 8-dione 5Bb; 2 , 3-dimethyl-6- [ (4-fluorophenyl ) - amino] quinoxaline-5, 8-dione 5Bc; 2 , 3-dimethyl-6- [ (4- chlorophenyl) amino] quinoxaline-5, 8-dione 5Bd; 2 , 3-dimethyl- 6- [ (4-methoxyphenyl ) amino] quinoxaline-5 , 8-dione 5Be; 2,3- dimethyl-6- [ (2 -hydroxyphenyl) amino] quinoxaline-5 , 8-dione 5Bf ; 2 , 3 -dimethyl-6- [ ( 2 -hydroxyphenyl ) amino] quinoxaline-5 , 8- dione 5Bg; 2 , 3-dimethyl-6- [ (3 , 4-dimethoxyphenyl ) amino] - quinoxaline-5 , 8-dione 5Bh; 2 , 3-dimethyl-6- [ (2 , 4- dimethoxyphenyl) amino] quinoxaline-5, 8-dione 5Bi ; 2,3- dimethyl-6-[ (4-cyanophenyl ) amino] uinoxaline-5 , 8-dione 5B ; 2 , 3-dimethyl-6- [ (phenyl ethyl ) amino] quinoxaline-5 , 8-dione 5Bk; 2, 3-dimethyl-6- [ [2- (phenyl) ethyl] amino] quinoxaline-5 , 8- dione 5B1 ; 2 , 3-dimethyl-6- [ [3- (dimethylamino) - propyl] amino] quinoxaline-5 , 8-dione 5Bm; 2 , 3-diphenyl-6- (phenylamino) quinoxaline-5, 8-dione 5Ca; 2 , 3-diphenyl-6- [ (4- methoxyphenyl) amino] uinoxaline-5, 8-dione 5Cb; 2 , 3-diphenyl- 6- [ [3- (dimethylamino) propyl] amino] quinoxaline-5 , 8-dione 5Cc; 2 , 3-di (pyrid-2-yl) -6- (phenylamino) quinoxaline-5 , 8-dione 5Da; 2, 3-di (pyrid-2-yl) -6- [ (4-methylphenyl) amino] quinoxaline-5 , 8- dione 5Db; 2 , 3-di (pyrid-2-yl) -6- [ (3-fluorophenyl ) amino] - quinoxaline-5, 8-dione 5Dc; 2 , 3-di (pyrid-2-yl) -6- [ (4- fluorophenyl) amino] quinoxaline-5, 8-dione 5Dd; 2 , 3-di (pyrid- 2-yl) -6- [ (2 , 4-difluorophenyl) amino] quinoxaline-5 , 8-dione 5De; 2 , 3-di (pyrid-2-yl) -6- [ (4-methoxyphenyl) amino] - quinoxaline-5, 8-dione 5Df; 2 , 3-di (pyrid-2-yl) -6- [ (4- hydroxyphenyl) amino] quinoxaline-5, 8-dione 5Dg; 2 , 3-di (pyrid- - 14- 2-yl) -6- [ (3,4, 5-trimethoxyphenyl ) amino] quinoxaline-5 , 8-dione 5Dh; 2,3-di(pyrid-2-yl) -6- [ (4- morpholinophenyl ) amino] uinoxaline-5 , 8-dione 5Di ; 2 , 3-di (pyrid-2-yl) -6- [ [3- (1-pyrrolidino) propyl] amino] - quinoxaline-5, 8-dione 5Dj ; 2 , 3-di (fur-2-yl) -6- [ (4- fluorophenyl ) amino] uinoxaline-5 , 8-dione 5Ea; 2 , 3-di ( fur-2- yl) -6- [ (4-methoxyphenyl ) amino] quinoxaline-5 , 8-dione 5Eb; 6- ( cyclohexylamino) -2 , 3-di (fur-2-yl) quinoxaline-5 , 8-dione 5Ec; and 2, 3-di (fur-2-yl) -6- [ [3- (pyrrolidin-2-on-l-yl) propyl] - amino] quinoxaline-5 , 8-dione 5Ed.

The compounds of the present invention, or their precursors, are prepared using procedures known to those of ordinary skill in the art. Preparation of the compounds of this invention may be performed by the following reaction Scheme 1 :

Scheme 1

OH OH

..

0 ♦ ^HzN S ] F r^N

FV KJ-I -^ A R ¹ - V

III IV v o

V r^N ι Λ / ^R3 V

V V ^ V rY

0 0

VII VI

where R^, R₂ , R3 and R4 are as previously defined for formula I .

For compounds of Scheme I where R^ and R is hydrogen, glyoxal is reacted with 2 , 3-diaminophenol III to afford the - 15 - 8-hydroxyquinoxaline IV. For compounds of Scheme I where R_]_ and R₂ is other than hydrogen, the appropriately substituted dione is reacted with 2 , 3-diaminophenol III to afford the 8- hydroxyquinoxaline IV. 8-Hydroxyquinoxaline IV, in tetrahydrofuran (THF) , can be further reacted with

[bis ( trifluoroacetoxy) iodo] benzene to afford the 5,8- quinoxalinedione V. Treatment of the 5, 8-quinoxalinedione V with an appropriately substituted amine in the presence of cerium chloride affords the 5 , 8-quinoxalinediones VI bearing the appropriate amino substituted R4 group.

Treatment of 5, 8-quinoxalinedione V with an appropriate thiol, for example, thiophenol in tetrahydrofuran, followed by addition of DDQ affords the R4 thio-substituted diones . The compounds VI may be further reacted with an appropriate halogenating agent to afford a desired dione where R3 is halogen VII . The dione bearing a cyano group at R3 may be prepared by methods known to those skilled in the art, for example, by reacting VI with N-bromosuccinimide , followed by reaction with potassium cyanide in DMSO . The following Preparations and Examples further illustrate the compounds of the present invention and the methods of their synthesis . The Examples are not intended to limit the scope of the invention in any respect, and should not be construed as such. The starting materials used to prepare the compounds of the present invention are commercially available and/or are prepared by methods familiar to those skilled in the art.

Preparation 1 5-hydroxyquinoxaline 3A

Glyoxal (40% aqueous solution, 7.37 mL, 64.5 mmol) was added to a stirred solution of 2 , 3-diaminophenol (5.00 g, 40.3 mmol) in THF (50 mL) at ambient temperature. The resultant solution was stirred for 2 h. After concentration - 16- at 35 °C, the gummy residue was chromatographed on silica (gradient 0-50% ethyl acetate (EtOAc) in methylene chloride (CH₂C1₂)) to give the compound of Preparation 1 (5.03 g, 85%) as a yellow solid, which was recrystallized from CH₂Cl /hexane: mp 98.0-99.5 °C ; MS m/e 146 (M+) ; Anal.

Calcd. for C₈HgN2θ: C, 65.74; H, 4.14; N, 19.17. Found: C, 65.82; H, 4.15; N, 19.10.

Preparation 2 2,3-dimethyl-8-hydroxyquinoxaline 3B

Butane-2 , 3-dione (11.4 mL, 129 mmol) was added to a stirred solution of 2 , 3-diaminophenol ( 10.0 g, 80.6 mmol) in THF (100 mL) at ambient temperature. The resultant solution was stirred for 4-5 h. After concentration at 35 °C, the residue was suspended in ethanol (EtOH, 50 mL) and sonicated prior to being filtered. The compound of Preparation 2 (13.5 g, 96%) was obtained as a tan solid, mp 147.0-149.0 °C ; MS m/e 174 (M+); Anal. Calcd. for C!oH_{10 2}0: C, 68.95; H, 5.79; N, 16.08. Found: C, 68.81; H, 5.91; N, 16.10.

Following the procedure described in Example 2, the following compounds were prepared:

Preparation 3 2,3-diphenyl-8-hydroxyquinoxaline, 3C

(90%)as a yellow solid. mp 94 °C; MS m/e 298 (M+) ; Anal. Calcd. for C₂₀H₁ N₂O: C, 80.52; H, 4.73; N, 9.39. Found: C, 80.42; H, 4.72; N, 9.45.

Preparation 4

2,3-di-(2-furyl) -8-hydroxyquinoxaline 3D, (99%) as a brown solid, mp 89-90 °C; MS m/e 279 (M++1); Anal. Calcd. for C₁gH₁₀N₂O₃: C, 69.06; H, 3.60; N, 10.07. Found: C, 68.76; H, 3.51; N, 9.82. - 17

Preparation 5 2, 3-di- (2-pyridyl) -8-hydroxyquinoxaline 3E, (95%) as a brownish-yellow solid, mp 178-180 °C; MS m/e 299 (M^_-l); 1H NMR (CDCI3) d 7.26 (br s, 3H) , 7.74 (s, 2H) , 7.81 - 7.86 (m, 2H) , 7.92 ( , IH) , 8.08 (br s, IH) , 8.36 (br s, 2H) .

Preparation 6 <ϊuinoxaline-5,8-dione 4A A solution of 3A (3.00 g, 20.5 mmol) in THF (120 mL) was added in a small stream to a stirred solution of [bis ( trifluoroacetoxy) iodo] benzene (18.1 g, 42.1 mmol) in THF (90 mL) /H₂0 (60 mL) at 0 °C . The resultant solution was stirred for 1-2 h. After concentration at 35 °C, the residue was chromatographed on silica (gradient 0-50% EtOAc in CH2CI2 ) to give 4A (1.50 g, 46%) as a yellow solid, which was recrystallized from CH Cl₂/hexane: mp 175 °C (dec); MS m/e 160 (M⁺); Anal. Calcd. for C₈H N₂θ2 : C, 60.01; H, 2.52; N, 17.49. Found: C, 59.92; H, 2.66; N, 17.22.

The following compounds were prepared according to the procedure as described in Example 6.

Preparation 7 2,3-dimethylquinoxaline-5,8-dione 4B

(57%) as a yellow solid. mp 185-187 °C; MS m/e 188 (M⁺) ; Anal. Calcd. for C₁₀H₈N₂O2 : C, 63.83; H, 4.29; N, 14.89. Found: C, 63.71; H, 4.12; N, 14.69.

Preparation 8

2, 3-diphenylquinoxaline-5, 8-dione 4C

(68%) as a yellow solid. mp 228 °C (dec); MS m/e 312 (M⁺); Anal. Calcd. for C₂₀H₁₂N₂O2: C, 76.91; H, 3.87; N, 8.97. Found: C, 76.85; H, 4.00; N, 9.01. Preparation 9 2, 3-di (fur-2-yl)quinoxaline-5, 8-dione 4D

(58%) as a red crystalline solid. mp 165 °C (sub) ; MS m/e 292 (M⁺) ; Anal. Calcd. for C₁gH₈N 0 : C, 65.76; H, 2.76; N, 9.59. Found: C, 65.89; H, 2.84; N, 9.36.

Preparation 10 2,3-di(pyrid-2-yl)quinoxaline-5, 8-dione 4E To a stirred solution of 3E (6.00 g, 20.0 mmol) in 5:1 THF/H2O (360 mL) was added [bis ( trifluoroacetoxy) iodo] - benzene (18.04 g, 42.0 mmol) in THF (120 mL)at 0 °C . The resultant solution was stirred at 0 °C for 1 h. After concentration at 42 °C, the residue was taken up in CH2CI2, then the flask was washed with methanol (MeOH, 3mL) before loading onto a dry silica gel column. About 0.5 L CH2CI2 was initially used to elute with, then gradient elution using 2-4% MeOH/AcOH (1:1) in CH₂C1₂. The fractions were neutralized with a saturated solution of sodium bicarbonate (NaHC03), washed with brine, dried over magnesium sulfate (MgSθ4), then concentrated to give 4E (2.66 g, 42%) as a darkish-yellow solid. mp 186-189 °C (dec) ; MS m/e 314 (M⁺) ; ^XH NMR (acetone-dg) d 7.28 (s, 2H) , 7.32-7.36 ( , 2H) , 7.96 (td, J = 8 and 1.5 Hz, 2H) , 8.09 (d, J = 8.0 Hz, 2H) , 8.24 (br d, J = 4.4 Hz, 2H) .

Example 11 6- (phenylamino)quinoxaline-5, 8-dione 5Aa

Aniline (105 mg, 1.12 mmol) was added to a stirred suspension of compound 4A (180 mg, 1.12 mmol) and

CeCl3.7(H₂0) (42 mg, 0.11 mmol) in EtOH (10 mL) at room temperature, the resultant mixture was allowed to stir exposed to air for 2.5 h. After concentration, the residue - 19 - was chromatographed on silica [gradient 0-10% MeOH in EtOAc/CH₂Cl₂ (1/1)] to give compound 5Aa (226 mg, 80%) as a purple/red solid, which was recrystallized from CH2Cl2/Et2θ. mp 178 °C(sub); MS m/e 251 (M⁺) ; Anal. Calcd. for C₁ H₉N3θ2 : C, 66.93; H, 3.61; N, 16.72. Found: C, 67.08; H, 3.64; N, 16.55.

The following compounds were prepared according to the procedure described in Example 11:

Example 12 6- t (2-methylphenyl)amino]quinoxaline-5, 8-dione 5Ab was obtained as a purple red solid in a 82% yield, which was recrystallized from CH₂Cl2/Et 0. mp 190-192 °C; MS m/e 265 (M⁺); Anal. Calcd. for C15HH 3O2 : C, 67.92; H, 4.18; N, 15.84. Found: C, 67.76; H, 4.11; N, 15.78.

Example 13 6- [ (3-methylphenyl) amino]quinoxaline-5, 8-dione 5Ac was obtained as a purple red solid in a 86% yield, which was recrystallized from CH2Cl₂/Et2θ. mp 170 °C(sub); MS m/e 265 (M⁺) ; Anal. Calcd. for C₁₅H_1:IN3θ2 : C, 67.92; H, 4.18; N, 15.84. Found: C, 67.98; H, 4.19; N, 15.80.

Example 14

6- [ (4-methylphenyl) amino] quinoxaline-5, 8-dione 5Ad was obtained as a purple red solid in a 86% yield, which was recrystallized from CH2Cl₂/Et2θ. mp 205 °C(sub); MS m/e 265 (M⁺); Anal. Calcd. for C15H11N3O2 : C, 67.92; H, 4.18; N, 15.84. Found: C, 68.15; H, 4.26; N, 15.77. - 20 - Example 15 6- [ (3-fluorophenyl)amino] quinoxaline-5, 8-dione 5Af was obtained as a purple red solid in a 50% yield, which was recrystallized from CH₂C1₂/Et₂0. mp 220.0-222.0 °C; MS m/e 269 (M⁺); Anal. Calcd. for C₁₄H₈FN θ2 : C, 62.45; H, 2.97; N, 15.61. Found: C, 62.19; H, 3.10; N, 15.49.

Example 16 6- [ (4-fluorophenyl) amino] quinoxaline-5, 8-dione 5Ag was obtained as a purple red solid in a 68% yield, which was recrystallized from CH2Cl2/Et2θ. mp 275 °C (dec); MS /e 269 (M⁺) ; Anal. Calcd. for C₁₄H₈FN30₂ : C, 62.46; H, 3.00; N, 15.61. Found: C, 62.75; H, 2.82; N, 15.34.

Example 17

6- [ (4-methoxyphenyl) amino] φiinoxaline-5, 8-dione 5Ah was obtained as a black solid in a 81% yield, which was recrystallized from CH Cl2/Et₂0. mp 207-208 °C ; MS m/e 281 (M⁺) ; Anal. Calcd. for Ci5H11N3O3 : C, 64.05; H, 3.94; N, 14.94. Found: C, 64.35; H, 4.04; N, 15.02.

Example 18 6- [ [4- (ethyloxycarbonyl)phenyl] amino]quinoxaline-5, 8-dione 5Ai was obtained as a orange red solid in a 47% yield, which was recrystallized from CH₂Cl2/Et2θ. mp 265-268 °C ; MS m/e 323 (M⁺); !H NMR (DMSO-dg) d 1.29 (t, J = 7.1 Hz, 3H) , 4.28 (q, J = 7.1 Hz, 2H) , 6.47 (s, IH) , 7.54 (d, J = 8.6 Hz, 2H) , 7.98 (d, J = 8.6 Hz, 2H) , 8.98 (d, J = 2.1 Hz, IH) , 9.01 (d, J = 2.1 Hz, IH) , 9.64 (s, IH) . - 21 - Example 19 6- [ (4-cyanophenyl)amino]quinoxaline-5, 8-dione 5Aj was obtained as a dark purple solid in a 95% yield, which was recrystallized from CH₂Cl2/Et₂0. mp >280 °C ; MS m/e 276 (M⁺); NMR (DMSO-dg) d 6.50 (s, IH) , 7.58 (d, J = 8.4 Hz, 2H) , 7.84 (d, J = 8.4 Hz, 2H) , 8.99 (s, IH) , 9.02 (s, IH) , 9.69 (s, IH) .

Example 20 6- (benzylamino)quinoxaline-5, 8-dione 5Ak was obtained as an orange red solid in a 72% yield, which was recrystallized from CH₂C1₂/Et₂0. mp 235 °C (dec) ; MS m/e 265 (M⁺) ; Anal. Calcd. for C15HHN3O2 : C, 67.92; H, 4.18; N, 15.84. Found: C, 67.87; H, 4.06; N, 15.77.

Example 21 6- [ (4-fluorobenzyl)amino]quinoxaline-5, 8-dione 5A1 was obtained as an orange red solid in a 78% yield, which was recrystallized from CH2C1₂/Et₂0. mp 243-245 °C ; MS m/e 283 (M⁺) ; Anal. Calcd. for Cχ₅H₁₀FN3θ₂ : C, 63.60; H, 3.56; N, 14.83. Found: C, 63.68; H, 3.59; N, 15.04.

Example 22 6- (triphenylmethyl) amino] quinoxaline-5, 8-dione 5Am was obtained as a red solid in a 2% yield. mp 220 °C (dec); MS m/e 417 (M+); E NMR (CDCI3) d 1.62 (s, IH) , 5.53 (s, IH) , 7.20-7.40 (m, 15H) , 8.90 (d, J = 2.1 Hz, IH) , 8.94 (d, J = 2.1 Hz, IH) .

Example 23

6- (cyclohexylmethylamino)quinoxaline-5, 8-dione 5Ao was obtained as a red solid in a 72% yield, which was recrystallized from CH C1₂/Et₂0. mp 243-244 °C; MS m/e 271 - 22 - (M⁺) ; Anal. Calcd. for C₁₅H₁₇N3θ2 : C, 66.40; H, 6.32; N, 15.49. Found: C, 66.70; H, 6.26; N, 15.55.

Example 24 6- [ [l-(benzyl)piperidin-4-yl] amino] quinoxaline-5,8-dione 5Ap was obtained as an orange solid in a 68% yield, which was recrystallized from CH₂Cl2/Et₂0. mp 178-180 °C ; IR (KBr) 3360, 1698, 1640, 1600 cm^"1; MS m/e 348 (M+); NMR (CDCI3) d 1.50-1.80 (m, 2H) , 2.00-2.30 (m, 4H) , 2.85-3.00 (m, 2H) , 3.35-3.50 ( , 1 H) , 3.55 (s, 2H) , 6.01 (br s, IH) , 6.04 (s,

IH) , 7.27-7.36 (m, 5H) , 8.91 (d, J = 2.0 Hz, IH) , 8.99 (d, J = 2.0 Hz, IH) .

Example 25 6- [ [3-(dimethylamino)propyl]amino]cιuinoxaline-5,8-dione 5Aq was obtained as a red solid in a 61% yield, which was recrystallized from CH₂C1₂/Et 0. mp 193 °C (dec); IR (KBr) 3295 (br) , 1693, 1631, 1601 cm^"1; MS m/e 260 (M+) ; ^-H NMR (CDCI3) d 1.83-1.92 (m, 2H) , 2.32 (s, 6H) , 2.53 (t, J = 5.8 Hz, 2H) , 3.31-3.38 (m, 2H) , 5.96 (s, IH) , 8.60 (br s, IH) , 8.88 (d, J = 2.1 Hz, IH), 8.97 (d, J^" = 2.1 Hz, IH). Anal. Calcd. for C₁3H₁gN 0₂ : C, 60.00; H, 6.15; N, 21.54. Found: C, 59.93; H, 6.03; N, 21.25.

Example 26

6- [ [3- (imidazol-l-yl)propyl]amino]qιinoxaline-5, 8-dione 5Ar was obtained as a red solid in a 71% yield, which was recrystallized from CH₂C1₂/Et₂0. mp 179-181 °C; IR (KBr) 3200 (br) , 1697, 1634, 1615 cm^"1; MS m/e 283 (M+)and 284 (M⁺+1); MR (DMSO-dg) d 1.95-2.05 (m, 2H) , 3.10-3.18 (m, 2H) , 4.00 (t, J = 6.9 Hz, 2H) , 5.79 (s, IH) , 6.85 (s, IH) , 7.16 (s, IH) , 7.60 (s, IH) , 7.85-7.95 (m, IH) , 8.90 (d, J = 2.1 Hz, IH) , 8.96 (d, J = 2.1 Hz, IH) . 23 -

Example 27 6- [ [ (l-(5) -hydroxymethyl-2-phenyl) ethyl] amino]quinoxaline- 5, 8-dione 5As was obtained as a red solid in a 50% yield, which was recrystallized from CH₂Cl₂/hexane . mp 96-98 °C ; MS m/e 309 (M⁺) ; Anal. Calcd. for C₁₇H₁ N3θ3 -0.17CH₂C1₂: C, 63.70; H, 4.78; N, 12.98. Found: C, 63.62; H, 4.59; N, 13.11.

Example 28

6- [ t [1- (S) -hydroxymethyl-2-(4-hydroxyphenyl) ] ethyl] amino] - guinoxaline-5, 8-dione 5At was obtained as a red solid in a 32% yield, which was recrystallized from CH₂Cl₂/hexane . mp 137-140 °C ; MS /e 326 (M++1); H NMR (DMSO-dg) d 2.74 - 2.78 (m, 2H) , 3.44 -

3.48 (m, 2H) , 3.62 - 3.66 ( , IH) , 4.89 (t, J = 5.6 Hz, IH) , 5.81 (s, IH) , 6.59 (d, J^" = 8.2 Hz, 2H) , 7.01 (d, J = 8.2 Hz, 2H) , 7.25 (d, J = 8.9 Hz, IH) , 8.88 (d, J = 1.9 Hz, IH) , 9.94 (d, J = 1.9 Hz, IH) , 9.12 (s, IH) .

Example 29 6- [ [ (1- ( S) -hydroxymethyl-3-methylthio)propyl] amino] - quinoxaline-5, 8-dione 5Au was obtained as a red foam in a 49% yield. MS m/e 293 (M⁺) ; Anal. Calcd. for C₁₃H₁₅N3θ3 -0.6CH₂Cl2: C, 47.44; H, 4.74; N, 12.20. Found: C, 47.31; H, 4.55; N, 12.46.

Example 30 6- [ [ (1- (S) -hydroxymethyl-2-methyl)propyl]amino]quinoxaline- 5, 8-dione 5Av was obtained as a red foam in a 54% yield. MS m/e 261 (M⁺) ; Anal. Calcd. for C₁₃H₁₅N3θ3 -O.lC^Cl^ C, 58.32; H, 5.68; N, 15.58. Found: C, 58.58; H, 5.85; N, 15.33. 24 -

Example 31 6- [2- ( ( S) -methoxymethyl)pyrrolidin-l-yl]quinoxaline-5,8- dione 5A was obtained as a red solid in a 50% yield, which was recrystallized from CH2C1₂/Et₂0. mp 142-143 °C ; MS m/e 273 (M⁺) ; Anal. Calcd. for C₁ H₁₅N₃θ3 ^: _> 61-53; H, 5.53; N, 15.38. Found: C, 61.32; H, 5.53; N, 15.26.

Example 32

6- [2- ( (5) -tert-butyloxycarbony)pyrrolidin-l-yl]quinoxaline- 5, 8-dione 5Ax was obtained as an orange solid in a 53% yield, which was recrystallized from CH2Cl2/Et2θ. mp 146-147 °C; MS m/e 329 (M⁺) ; Anal. Calcd. for CX HX9N304 -0.5CH2C1₂: C, 61.39; H, 5.77; N, 12.60. Found: C, 61.45; H, 5.78; N, 12.43.

Example 33 6- (pyrrolidin-l-yl)quinoxaline-5, 8-dione 5Ay was obtained as a red solid in a 65% yield, which was recrystallized from CH2Cl /Et2θ. mp 180 °C (dec) ; MS m/e

229 (M⁺) ; Anal. Calcd. for C12H11N3O2 O.IICH2CI2: C, 60.97; H, 4.74; N, 17.61. Found: C, 61.08; H, 4.76; N, 17.46.

Example 34

6- [(2(_?) ,3-dihydroxypropyl)amino]quinoxaline-5,8-dione 5Az was obtained as a violet solid in a 62% yield, which was recrystallized from CH Cl₂/Et2θ. mp 106-108 °C (dec) ;

MS m/e 249 (M⁺); Anal. Calcd. for C11HHN3O4 -0.11H₂O: C, 52.26; H, 4.54; N, 16.62. Found: C, 52.34; H, 4.57; N, 16.45. - 25- Example 35 6- [ [ (2-hydroxyl-1-hydroxymethyl) ethyl] amino]quinoxaline-5, 8- dione 5Aa was obtained as an orange red solid in a 55% yield, which was recrystallized from CH₂C1₂/Et₂0. mp 220 °C (dec) ; MS m/e 249 (M⁺); E NMR (DMSO-dg) d 3.45-3.58 (m, 5H) , 4.86 (br s, OH, 2H) , 5.98 (s, IH) , 7.01 (d, J = 7.0 Hz IH) , 8.90 (d, J^" = 2.0 Hz, IH), 8.96 (d, J = 2.0 Hz, IH). Anal. Calcd. for C H11N3O4 : C, 53.01; H, 4.45; N, 16.86. Found: C, 52.73; H, 4.43; N, 16.63.

Example 36 6- [ [2(5) ,3(5) ,4(1?) ,5 (J?) , 6- (pentahydroxyl)hexyl] amino] - quinoxaline-5,8-dione 5Ab was obtained as an orange red solid in a 25% yield, which was recrystallized from CH₂C1₂/Et₂0. mp 170 °C (dec) ; IR (KBr) 3401 (br) , 3309 (br) , 1694, 1629, 1604 cm^"1; MS m/e 322 (M⁺-OH) , 308 (M⁺-CH₂OH) ; NMR (DMSO-dg) d 3.15-3.65 (m, 7H) , 3.80-3.90 (m, IH) , 4.30-4.58 (m, 4H) , 5.04 (d, J = 4.6 Hz, IH) , 5.89 (s, IH) , 7.48 (br s, IH) , 8.90 (d, J = 1.9 Hz, IH) , 8.96 (d, J = 1.9 Hz, IH) .

Example 37 2, 3-dimethyl-6- (phenylamino)quinoxaline-5, 8-dione 5Ba was obtained as a purple red solid in a 32% yield, which was recrystallized from CH₂Cl₂/Et2θ. mp 223-225 °C ; MS m/e 279 (M⁺); Anal. Calcd. for C₁₆H₁₃N3θ2 -0.25CH₂Cl2: C, 64.94; H, 4.53; N, 13.98. Found: C, 64.73; H, 4.31; N, 13.77. - 26 - Example 38 2, 3-dimethyl-6- [ (4-methylphenyl)amino]quinoxaline-5, 8-dione 5Bb was obtained as a purple red solid in a 37% yield, which was recrystallized from CH2Cl2/Et2θ. mp 224-226 °C; MS m/e 293 (M⁺) ; Anal. Calcd. for C₁₇H₁ N3θ2 : C, 69.61; H, 5.15; N, 14.33. Found: C, 69.89; H, 5.13; N, 14.20.

Example 39 2, 3-dimethyl-6- [ (4-fluorophenyl) amino] guinoxaline-5, 8-dione 5Be was obtained as a purple red solid in a 46% yield, which was recrystallized from CH₂Cl2/Et2θ. mp 255-257 °C; IR (KBr) 3191 (br) , 1695, 1638, 1622, 1610 cm^"1; MS m/e 297 (M⁺); !H NMR (CDC1₃) d 2.78 (s, 3H) , 2.80 (s, 3H) , 6.41 (s, IH) , 7.10-7.19 (m, 2H) , 7.26-7.30 (m, 2H) , 7.53(s, IH) .

Example 40 2, 3-dimethyl-6- [ (4-chlorophenyl)amino]quinoxaline-5, 8-dione 5Bd was obtained as a purple red solid in a 38% yield, which was recrystallized from CH2Cl2/Et₂0. mp 269-270 °C; MS m/e 313 (M⁺) ; Anal. Calcd. for C₁₆H₁ C1N₃0 : C, 61.25; H, 3.86; N, 13.39. Found: C, 61.47; H, 3.83; N, 13.19.

Example 41 2,3-dimethyl-6- [ (4-methoxyphenyl)amino]quinoxaline-5, 8-dione 5Be was obtained as a purple red solid in a 35% yield, which was recrystallized from CH₂Cl2/Et₂0. mp 213-215 °C; MS m/e 309 (M⁺) ; Anal. Calcd. for C₁₇H₁₅N3θ3 : C, 66.01; H, 4.89; N, 13.58. Found: C, 65.92; H, 5.00; N, 13.48. - 27 - Example 42 2, 3-dimethyl-6- [ (2-hydroxyphenyl ) amino] quinoxaline-5, 8-dione 5Bf was obtained as a dark purple solid in a 17% yield, which was recrystallized from CH2Cl2/Et2θ. mp >280 °C; MS m/e 295 (M+) ; Anal. Calcd. for C₁gH₁₃N θ3 : C, 65.08; H, 4.43; N, 14.23. Found: C, 64.82; H, 4.43; N, 14.06.

Example 43 2, 3-dimethyl-6- [ (2-hydroxyphenyl) amino]quinoxaline-5, 8-dione 5Bg was obtained as a purple solid in a 13% yield. mp 265 °C (dec); MS m/e 295 (M⁺) ; Anal. Calcd. for C₁₆H₁₃N3θ3 : C, 65.08; H, 4.44; N, 14.23. Found: C, 65.35; H, 4.40; N, 14.08.

Example 44 2 , 3-dimethyl-6- [ ( 3 , 4-dimethoxypheny1 ) amino] uinoxaline-5,8- dione 5Bh was obtained as a dark purple solid in a 44% yield, which was recrystallized from CH₂C1₂/Et₂0. mp 207.0-208.0 °C; MS m/e 339 (M⁺); Anal. Calcd. for C₁₈H₁₇N₃θ4 : C, 63.71; H, 5.04; N, 12.38. Found: C, 63.63; H, 4.93; N, 12.25.

Example 45

2, 3-dimethyl-6- [ (2,4-dimethoxyphenyl)amino]guinoxaline-5, 8- dione 5Bi was obtained as a purple red solid in a 44% yield, which was recrystallized from CH2Cl2/Et2θ. mp 219.5-220.0 °C; MS m/e 339 (M⁺) ; Anal. Calcd. for C₁₈H₁₇N₃θ4 : C, 63.70; H, 5.04; N, 12.38. Found: C, 63.99; H, 4.85; N, 12.25. - 28 - Example 46 2, 3-dimethyl-6- [ (4-cyanophenyl)amino]quinoxaline-5, 8-dione 5Bj was obtained as a red solid in a 35% yield, which was recrystallized from CH₂Cl2/Et2θ. mp >280 °C; MS m/e 304 (M⁺) ; Anal. Calcd. for C₁₇H₁₂N4θ2 : C, 67.10; H, 3.97; N, 18.41. Found: C, 67.24; H, 4.07; N, 18.40.

Example 47 2, 3-dimethyl-6- [ (phenylmethyl) amino] uinoxaline-5, 8-dione 5Bk was obtained as a red solid in a 27% yield, which was recrystallized from CH₂Cl2/Et2θ. mp 205.0-207.0 °C; MS m/e 293 (M⁺) ; Anal. Calcd. for C₁₇H₁₅N3θ₂ -0.150^12 : C, 67.30; H, 5.04; N, 13.73. Found: C, 67.29; H, 4.99; N, 13.58.

Example 48 2, 3-dimethyl-6- [ [2- (phenyl)ethyl]amino]quinoxaline-5, 8-dione 5B1 was obtained as a red solid in a 33% yield, which was recrystallized from CH₂Cl2/Et₂0. mp 212.0-214.0 °C ; MS m/e 307 (M⁺) ; Anal. Calcd. for C₁₈H₁₇N₃θ2 : C, 70.34; H, 5.58; N, 13.67. Found: C, 70.10; H, 5.42; N, 13.51.

Example 49

2, 3-dimethyl-6- [ [3- (dimethylamino)propyl] amino] quinoxaline- 5, 8-dione 5Bm was obtained as an orange red solid in a 34% yield, which was recrystallized from CH2Cl2/Et2θ. mp 205 °C (dec) ; IR (KBr) 3423 (br) , 1700, 1627, 1606 cm^"1; MS m/e 288 (M+); !H NMR (CDC1₃) d 1.85-1.95 (m, 2H) , 2.33 (s, 6H) , 2.50-2.95 (m, 2H) , 2.73 (s, 3H) , 2.77 (s, 3H) , 3.28-3.35 (m, 2H) , 5.85 (s, IH) , 8.43 (br s, IH) . 29

Example 50 2,3-diphenyl-6- (phenylamino)quinoxaline-5, 8-dione 5Ca was obtained as a purple red solid in a 78% yield, which was recrystallized from CH2Cl2/Et2θ. mp 136-137 °C ; MS m/e 403 (M⁺); Anal. Calcd. for C2gH₁₇N θ2 : C, 77.42; H, 4.22; N, 10.42. Found: C, 77.71; H, 4.12; N, 10.39.

Example 51 2, 3-diphenyl-6- [ (4-methoxyphenyl)amino]quinoxaline-5, 8-dione 5Cb was obtained as a red solid in a 75% yield, which was recrystallized from CH2Cl /Et2θ. mp 245 °C (dec); MS m/e 433 (M⁺) ; Anal. Calcd. for C27H2_.9N3O3 : C, 74.81; H, 4.42; N, 9.69. Found: C, 75.08; H, 4.44; N, 9.90.

Example 52 2, 3-diphenyl-6- [ [3- (dimethylamino)propyl] amino] qiαinoxaline- 5, 8-dione 5Cc was obtained as a red solid in a 76% yield, which was recrystallized from CH₂Cl₂/Et2θ. mp 200-201 °C; MS m/e 412 (M⁺) ; Anal. Calcd. for C25H24N4O2 : C, 72.82; H, 5.83; N, 13.59. Found: C, 72.94; H, 5.82; N, 13.64.

Example 53

2,3-di(pyrid-2-yl) -6- (phenylamino) quinoxaline-5, 8-dione 5Da was obtained as a red solid in a 77% yield, which was recrystallized from CH₂Cl₂/Et2θ. mp 245-247 °C ; MS m/e 406 (M++1); NMR (CDCI3) d 6.71 (s, IH) , 6.90 - 7.02 (m, IH) , 7.05 - 7.14 ( , 2H) , 7.26 (br s, 3H+CHC1₃), 7.38 - 7.43 (m, IH) , 7.84-7.87 (br m, 3H) , 8.09 (d, J = 7.7 Hz, IH) , 8.19 (d, J = 7.7 Hz, IH) , 8.27 - 8.31 (m, 2H) . - 30 - Example 54 2, 3-di(pyrid-2-yl) -6- [ (4-methylphenyl)amino]quinoxaline-5, 8- dione 5Db was obtained as a purple solid in a 33% yield, which was recrystallized from CH₂Cl2/Et2θ. mp 256-258 °C ; MS m/e 419 (M+) ; Anal. Calcd. for C₂₅H₁₇N₅0₂ : C, 71.59; H, 4.09; N, 16.70. Found: C, 71.43; H, 3.96; N, 16.66.

Example 55 2,3-di(pyrid-2-yl) -6- [ (3-fluorophenyl) amino] quinoxaline-5, 8- dione 5Dc was obtained as a red solid in a 69% yield, which was recrystallized from CH₂C1₂/Et 0. mp 249-251 °C; MS m/e 424 (M++1); NMR (DMSO-dg) d 6.69 (s, IH) , 7.24-7.31 ( , 3H) , 7.34 (d, J = 7.8 Hz, IH) , 7.45 - 7.50 (m, 2H) , 7.72 (s, IH) , 7.84-7.90 (m, 2H) , 8.12 (d, J = 7.8 Hz, IH) , 8.25(d, J = 7.6 Hz, IH) , 8.27 - 8.33 ( , 2H) .

Example 56 2, 3-di(pyrid-2-yl) -6- [ (4-fluorophenyl) amino] quinoxaline-5, 8- dione 5Dd was obtained as a reddish purple solid in a 34% yield, which was recrystallized from CH₂Cl₂/Et2θ. mp 248-250 °C ; MS m/e 423 (M⁺) ; E NMR (DMSO-dg) d 6.18 (s, IH) , 7.26 - 7.45 (m, 6H) , 7.92 - 7.98 (m, 4H) , 8.25 - 8.29 (m, 2H) , 9.55 (s, IH) .

Example 57 2,3-di(pyrid-2-yl) -6- [ (2, 4-difluorophenyl)amino] uinoxaline- 5, 8-dione 5De was obtained as a orange solid in a 67% yield, which was recrystallized from CH₂Cl2/Et2θ. mp 233-234 °C(dec); MS m/e 441 (M⁺) ; ^XH NMR (CDC1₃) d 6.38 (s, IH) , 6.99 - 7.05 (m, - 31- 2H) , 7.26 - 7.30 (m, IH) , 7.42 - 7.48 (m, 3H) , 7.85 - 7.91 (m, 2H) 8.18 (d, J = 7.8 Hz, IH) , 8.22 (d, J = 7.8 Hz, IH) , 8.28 - 8.33 (m, 2H) .

Example 58

2,3-di(pyrid-2-yl) -6- [ (4-methoxyphenyl) amino]quinoxaline- 5, 8-dione 5Df was obtained as a red solid in a 39% yield, which was recrystallized from CH₂Cl2/Et 0. mp 231-233 °C ; MS m/e 435 (M⁺); Anal. Calcd. for C₂5H₁₇N₅θ3: C, 68.95; H, 3.94; N, 16.08. Found: C, 68.84; H, 3.76; N, 15.82.

Example 59 2, 3-di(pyrid-2-yl) -6- [ (4-hydroxyphenyl) amino] quinoxaline- 5, 8-dione 5Dg was obtained as a red solid in a 94% yield, which was recrystallized from CH₂Cl₂/Et2θ. mp >280 °C; MS m/e 421 (M+) ; !H NMR (DMSO-dg) d 6.07 (s, IH) , 6.83 (d, J = 8.6 Hz, 2H) , 7.17 (d, J = 8.6 Hz, 2H) , 7.33 - 7.38 (m, 2H) , 7.91 - 7.97 (m, 4H) , 8.24 - 8.29 (m, 2H) , 9.40 (s, IH) , 9.61 (s, IH) .

Example 60 2,3-di(pyrid-2-yl) -6- [ (3,4, 5-trimethoxyphenyl)amino] - quinoxaline-5,8-dione 5Dh was obtained as a purple solid in a 94% yield, which was recrystallized from CH₂Cl2/Et2θ. mp 190-194 °C ; MS m/e 495 (M⁺) ; NMR (DMSO-dg) d 3.66 (s, 3H) , 3.76 (s, 6H) , 6.31 (s, IH) , 6.71 (s, 2H) , 7.35 - 7.39 (m, 2H) , 7.94 - 7.96 (m, 4H) , 8.25 - 8.29 (m, 2H) , 9.40 (s, IH) . - 32 - Example 61 2, 3-di (pyrid-2-yl) -6- [ (4-morpholinophenyl)amino]quinoxaline- 5, 8-dione 5Di was obtained as a black solid in a 32% yield, which was recrystallized from CH₂C1 /Et₂0. mp 232-236 °C ; MS m/e 490 (M⁺); Anal. Calcd. for C₂₈H₂2Ngθ3 • 0.25 (H₂0) : C, 67.94; H, 4.58; N, 16.98. Found: C, 67.84; H, 4.47; N, 16.67.

Example 62 2,3-di(pyrid-2-yl)-6- [ [3- (1-pyrrolidino)propyl] amino] - quinoxaline-5, 8-dione 5Dj was obtained as a red solid in a 32% yield, which was recrystallized from CH₂Cl₂/Et2θ. mp 197-200 °C ; MS m/e 440 (M⁺) ; Anal. Calcd. for C₂₅H₂₄N θ3 • 0.25H₂0 : C, 67.47; H, 5.54; N, 18.88. Found: C, 67.21; H, 5.58; N, 18.65.

Example 63 2, 3-di (fur-2-yl) -6- [ (4-fluorophenyl) amino] quinoxaline-5, 8- dione 5Ea was obtained as a maroon solid in a 74% yield, which was recrystallized from CH C1₂/Et 0. mp >280 °C; MS m/e 401 (M⁺) ; Anal. Calcd. for C22H12FN3O4: C, 65.84; H, 3.01; N, 10.47. Found: C, 66.06; H, 2.91; N, 10.50.

Example 64

2,3-di(fur-2-yl) -6- [ (4-methoxyphenyl ) amino] quinox line-5, 8- dione 5Eb was obtained as a black solid in a 99% yield, which was recrystallized from CH₂C1₂/Et 0. mp 252-253 °C ; MS m/e 413 (M⁺); Anal. Calcd. for C23H15N3O5: C, 66.83; H, 3.66; N, 10.16. Found: C, 66.73; H, 3.71; N, 10.18. - 33 - Example 65 6- (cyclohexylamino) -2, 3-di (fur-2-yl)quinoxaline-5, 8-dione 5Ec was obtained as an orangeish red solid in a 63% yield, which was recrystallized from CH₂Cl2/Et2θ. mp 235-237 °C ; MS m/e 389 (M⁺) ; Anal. Calcd. for C22H19N3O4: C, 67.86; H, 4.92; N, 10.79. Found: C, 67.66; H, 4.84; N, 10.64.

Example 66 2,3-di(fur-2-yl)-6-[ [3- (pyrrolidin-2-on-l- yl)propyl] amino] quinoxaline-5, 8-dione 5Ed was obtained as a red solid in a 68% yield, which was recrystallized from CH₂Cl2/Et₂0. mp 170-174 °C; MS m/e 432 (M⁺) ; Anal. Calcd. for C23H20N4O5: C, 63.88; H, 4.66; N, 12.96. Found: C, 64.08; H, 4.56; N, 12.93.

Example 67 6- [ (2-fluorophenyl) amino] -5, 8-quinoxalinedione

Red solid. MS m/e 270 (M⁺+1) .

Example 68 6- [ (3-methylbutyl)amino] -5, 8-quinoxalinedione

Red solid; MS m/e 246 (M⁺+1) .

Example 69

6- [ [4- (dimethylamino)butyl] amino] -5, 8-quinoxalinedione

Red solid, mp : 175 °C (dec); FDMS m/e 274 ( +); Anal. Calcd. for C₁ H₁₈N4θ₂: C, 61.31; H, 6.57; N, 20.44. Found: C, 61.26; H, 6.45; N, 20.44. - 34 - Example 70 6- [ [3- (dimethylamino) -2, 2- (dimethyl )propyl] amino] -5, 8- quinoxalinedione

Red solid. MS m/e 289 (M⁺) .

Example 71 6- [ [3- [bis ( -2-hydroxyethyl) amino]propyl] amino] -5,8- quinoxalinedione

Red solid. MS m/e 321 (M++1) .

Example 72 6- [ [3- [4- (methyl)piperazin-l-yl]propyl] amino] -5,8- quinoxalinedione red solid, mp : 188-190 °C; FDMS /e 315 (M+) ; Anal. Calcd. for C₁₆H2iN₅θ2: C, 60.95; H, 6.67; N, 22.22. Found: C, 60.72; H, 6.51; N, 21.94.

Example 73 6- [ [3- (4-morpholinyl)propyl] amino] -5, 8-quinoxalinedione Red solid. FDMS m/e 302 (M+) .

Example 74 6- [ [2- (4-morpholinyl) ethyl] amino] -5, 8-quinoxalinedione

Dark red solid. FDMS m/e 288 (M⁺) .

Example 75 6- [ [2- (1-piperidinyl) ethyl] amino] -5, 8-quinoxalinedione

Red solid. MS m/e 287 (M++1) . - 35 - Example 76 6- [ [2- (1-pyrrolidinyl) ethyl] amino] -5, 8-quinoxalinedione

Red solid. MS m/e 273 (M⁺+1) .

Example 77

6- [ [2-(dibutylamino) ethyl] amino] -5, 8-quinoxalinedione

Red gum. MS m/e 303 (M⁺+1) .

Example 78 6- [ [2- [di- (2-propyl) amino] ethyl] amino] -5, 8-quinoxalinedione

Red solid. MS m/e 331 (M++1) .

Example 79 6- [ [2- [di- (2-hydroxyethy1) amino] ethyl] amino] -5,8- quinoxalinedione

Red solid. MS m/e 307 (M++1) .

Example 80 6- [ (2-methoxyethy1) amino] -5, 8-quinoxalinedione red solid; FDMS m/e 233 (M⁺) ; Anal. Calcd. for CnHi1N3O3 : C, 56.65; H, 4.72; N, 18.03. Found: C, 56.25; H, 5.08; N, 17.62.

Example 81 6- [ [ (1-methyl)piperidin-4-yl] (methyl) mino] -5, 8- quinoxalinedione

Red solid. MS m/e 287 (M++1) .

Example 82 6- [4- (l-piperidinyl)piperidin-l-yl] -5, 8-quinoxalinedione

Red solid. MS m/e 327 (M++1) . - 36- Example 83 6- [ (l-ethylpiperidin-3-yl)amino] -5, 8-quinoxalinedione

Red gum. MS m/e 287 (M++1) .

Example 84

6- [ (l-benzylpyrrolidin-3 (R) -yl)amino] -5, 8-quinoxalinedione

Dark red solid. MS m/e 335 (M++1) .

Example 85 6- [ [ (l-ethylpyrrolidin-2 (S) -yl)methyl] amino] -5,8- quinoxalinedione

Red solid. MS m/e 287 (M++1) .

Example 86 2,3-di(fur-2-yl) -6- [ [3- (pyrrolidin-l-yl)propyl] amino] - quinoxaline-5,8-dione (414373, MYI-74-10).

Red solid. MS m/e 419 (M++1) .

Example 87 2,3-di(fur-2-yl)-6-[[3-[di-(2-hydroxyethyl)amino]- propyl] amino]quinoxaline-5, 8-dione

Dark red solid. MS m/e 453 (M++1) .

Example 88 6- [ [2- (dibutylamino) ethyl] amino] -2, 3-di (fur-2- yl)quinoxaline-5, 8-dione

Red solid. MS m/e 462 (M++1) .

Example 89 2,3-di(fur-2-yl)-6-[[2-[di-(l-methylethyl)amino]ethyl]- amino]quinoxaline-5, 8-dione

Dark red solid. MS m/e 435 (M++1) . - 37 - Example 90 2, 3-di (fur-2-yl) -6- [ [2- (dimethylamino) ethyl] amino] - quinoxaline-5, 8-dione

Dark red solid. MS m/e 379 (M++1) .

Example 91 2, 3-di(fur-2-yl) -6- [ [2- (pyrrolidin-l-yl)ethyl]amino] - quinoxaline-5, 8-dione

Dark red solid. MS m/e 405 (M++1) .

Example 92 2,3-di(fur-2-yl)-6- [ [2- (piperidin-1-yl) ethyl] amino] - quinoxaline-5, 8-dione

Dark red solid. MS m/e 419 (M++1) .

Example 93 2, 3-di (fur-2-yl) -6- [ [2- [di- (2-hydroxyethyl)amino] - ethyl] amino] quinoxaline-5, 8-dione

Dark red solid. MS m/e 439 (M++1) .

Example 94 2 , 3-di ( fur-2-yl ) -6- [ [ ( 1-ethylpyrrolidin-2 (S) -yl )methyl] - amino] quinoxaline-5, 8-dione

Dark red solid. MS m/e 418 (M++1) .

Example 95 2,3-di(fur-2-yl)-6- [ [ (1-ethylpyrrolidin-2- yl)methyl] amino] quinoxaline-5, 8-dione

Dark red solid. MS m/e 419 (M++1) . - 38 - Example 96 2,3-di(fur-2-yl)-6-[(l-ethylpiperidin-3- yl) amino]quinoxaline-5, 8-dione

Dark red gum. MS m/e 419 (M++1) .

Example 97 6- [ (l-benzylpyrrolidin-3(R) -yl) amino] -2, 3-di(fur-2- yl)quinoxaline-5, 8-dione

Dark red solid. MS m/e 467 (M⁺+1) .

Example 98 7-bromo-6- [ (4-methylphenyl) amino]quinoxaline-5, 8-dione 5Ae

N-Bromosuccinimide (67.1 mg, 0.377 mmol) was added to a stirred solution of 5Ad (100 mg, 0.377 mmol) in CHC1₃ (4 mL) at room temperature. The mixture was stirred for 1 h. The mixture was directly subject to silica gel chromatography [gradient 0-5% MeOH in EtOAc/CH₂Cl (1/1) ] and the isolated solid product was washed with water (25 mL) to give 5Ae (110 mg, 84%) as a dark brown solid, which was recrystallized from CH₂C1₂/Et₂0. mp 217-218 °C; MS m/e 343

(M⁺, ⁷⁹Br) and 345 (M⁺, ⁸¹Br) ; Anal. Calcd. for C₁₅H₁₀BrN₃O2 : C, 52.35; H, 2.93; N, 12.21. Found: C, 52.07; H, 3.04; N, 12.06.

Example 99

6- (phenylthio)quinoxaline-5, 8-dione 5Ac

Thiophenol (0.108 mL, 1.05 mmol) was added to a stirred suspension of compound 4A (160 mg, 1.00 mmol) and CeCl₃.7 (H₂0) (37 mg, 0.10 mmol) in THF (10 mL) at room temperature. The resultant mixture was allowed to stir uncapped for 3 h. After concentration, the residue was chromatographed on silica [gradient MeOH in EtOAc: 0-1%] to give the dihydro derivative of compound 5Ac (70 mg) as a - 39 - yellow solid. Which was dissolved in THF (5 mL) before it was treated with DDQ (54 mg, 0.24 mmol), and the mixture was stirred for 1 h. After concentration, the residue was chromatographed on silica [gradient 30-100% EtOAc in hexane] to give compound 5Ac (25 mg, 9% yield) as a yellow solid, which was recrystallized from CH₂Cl₂/Et2θ. mp 171-173 °C; MS m/e 268 (M⁺); ¹H NMR (CDCl ) d 6.37 (s, IH) , 7.56 (br s, 5H) , 9.02 (s, 2H) . Anal. Calcd. for C₁ H₈N₂θ2 S-0.9H O: C, 59.10; H, 3.47; N, 9.85. Found: C, 58.84; H, 3.09; N, 9.78.

The biological activity of the compounds of the present invention was evaluated by an initial screening assay which measures the inhibition of Ras binding to GTP by the compound being tested. The scintillation-proximity-based assay described below quantitates the amount of [³H] -GTP bound to a biotinylated form of Ki-Ras that is then captured by scintillation beads coated with streptavidin . The valine-12 form of Ki-Ras (G12V, amino acids 1-166) was fused to glutathione-S-transferase (GST) and produced in E. coli . Neither fusion to GST nor biotinylation affected the [³H] - GTP/GDP exchange activity of Ki-Ras. The assay has been appropriately validated for variability and reproducibility.

MATERIALS AND METHODS Expression and Purification of GST-Ras : cDNA coding for amino acids 1-166 of the oncogenic Ki-JRas/G12V (glycine 12 -> valine 12) was cloned into pGEX-2T, a glutathione-S- transferase (GST) fusion protein expression vector (Pharmacia) , to form pGST-RasK(G12V) . The fusion protein, - 40 - referred to in this report as GST-Ras, was produced in E. coli and purified by affinity chromatography following the procedure recommended by the vendor. The purity of the protein was analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with Pan-Ras antibody

(Oncogene Research Products). Protein concentration was determined by the Bio-Rad Protein Assay (Bio-Rad) . GST was prepared in the same manner and included in the studies for comparison. Biotinylation of GST-Ras: Purified GST-Ras (750 mg) was dialyzed overnight against 2 x 500 ml 0.1 N NaHC0₃ at

4°C. After dialysis, 100 μg of Biotin-X-NHS (Calbiochem) were added to the dialyzed GST-Ras . The mixture was covered with parafilm and incubated in the dark at room temperature for 4.5 hours. The reaction mixture was then dialyzed

overnight at 4°C against 2 x 500 ml phosphate buffered saline (PBS) to remove any unreacted biotin. The biotinylated GST-Ras was quantitated with the Bio-Rad

Protein Assay™ using unbiotinylated GST-Ras as the standard. Approximately 300 μg of the GST-Ras was usually recovered after this procedure.

Biotinylation of GST-Ras was assessed by standard

ELISA. Immulon-4 96-well plates (Dynatech Labs) were coated with 100 μg of bovine serum albumin fused to glutathione (BSA-GSH, 1 mg/ml in PBS) . After incubation (90 minutes at - 41 - 37°C) , the wells were washed 3X with 0.05% Tween-20 in PBS, followed by addition of 200 μl of 5.0% non-fat dry milk in

the same buffer. The plates were incubated at 37°C for 1 hour, then washed 3X with 0.05% Tween-20/PBS . One-hundred microliters of biotinylated GST-Ras or biotinylated GST solutions were diluted in 0.05% Tween-20, 1.0% BSA to final concentrations ranging from 0-700 nM. One-hundred microliters/well of PBS were added to negative control

wells. Plates were incubated at 37°C for 1 hour and washed 3X with 0.05% Tween-20. Following addition of streptavidin- POD (Boehringer Mannheim) , the reaction was developed by standard procedure and measured in an ELISA 96-well plate reader (Titertek Multiskan MCC/340) at 450 nm. An ELISA was performed to determine whether GST-Ras and GST had been successfully biotinylated. Several concentrations (0 - 700 nM) of the biotinylated GST-Ras were added to Immulon-4 microtiter plates which had been precoated with glutathione- BSA. Biotin on the fusion protein bound to the plates through GST-GSH linkage was quantitated by a standard ELISA procedure.

SPA-Based GST-Ras/GTP Binding Assay; Standard binding assay of [³H] -GTP to biotinylated GST-Ras was performed in 96-well plates (Wallac) . The following components were added (in order) : 75 μl binding buffer (25 mM Tris-HCl, pH 7.5; 0.1 M NH₄C1; 0.1 mg/ml BSA; 0.05% NaN₃ ; 10 mM DTT) , 25 - 42 - μl GST-Ras (final concentration, 6.0 nM, except where indicated), and 50 μl guanosine 5 ' -triphosphate, tetrasodium salt, [8,5'-³H] (New England Nuclear; 35 Ci/mM, 59.6 nM final concentration, except where indicated) . The final volume was maintained at 150 ml. After an incubation for 90 minutes at room temperature, 250 μg (unless otherwise noted) of Streptavidin-coated SPA beads were added. (SPA beads were purchased from Amersham and stored at 10 mg/ml in stop buffer containing 25 mM Tris-HCl, pH 7.5; 100 mM NH₄C1; 30 mM MgCl₂.) The reaction was further incubated at room temperature for 30 min. The plates were then loaded onto a Wallac Microbeta 96-well plate counter, and the amount (cpm) of [³H] -GTP bound was determined. In every assay, biotinylated GST was tested in parallel for background determinations.

To test if biotinylated GST-Ras retained the [³H] - GTP-binding function, a direct competition assay with unbiotinylated GST-Ras was performed. In a reaction where the final total concentration of GST-Ras was fixed at 6.0 nM, several concentrations of unbiotinylated GST-Ras (0, 1.2, 2.4, 4.8 and 6 nM) were added with a corresponding decrease in the concentration of biotinylated GST-Ras. Percent reduction in [³H] -GTP bound to biotinylated GST-Ras was calculated. Under the conditions described, biotinylation did not alter the intrinsic guanine nucleotide exchange activity of GST-Ras. - 43 - Competition/Inhibition : Competition with unlabeled GTP

(Sigma) or non-biotinylated GST-Ras was performed in the following manner: Different amounts of competitor were added to the binding buffer prior to the addition of biotinylated GST-Ras in the standard GST-Ras/ [³H] -GTP binding reaction as described above. Background counts were subtracted from each sample before calculation of % inhibition and inference of IC₅α determination. The affects of solvents such as ethanol, methanol or DMSO were also tested in the same manner and did not affect signal outputs in the presence or absence of a competitor up to 6.25%.

To confirm that the Ras/GTP inhibitors were active in human tumor cells, assays were carried out to examine the ability of the compounds to inhibit the proliferation of Ras dependent cell lines (Proliferation Assay). Further confirmation of activity in the Ras pathway was obtained by examining the effect of inhibitors on the downstream Ras pathway component MAPK. This was accomplished using a luciferase reporter assay (Luciferase Assay) .

Proliferation Assay.

The viability of cultured cells can be determined using colormetric methods that depend on the presence of viable cells to convert a non colored compound to a colored one. The CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega) provides a convenient means for these - 44 - evaluations. HCT116 (ATCC# ) human colon carcinoma cells containing mutant K-Ras were grown using standard tissue culture techniques in RPMI 1640 with

L-glutamine (Life Technologies 11875-093) plus 10% dialyzed fetal calf serum and 50 μg/ml gentamycin. Alternatively

K5.2 normal rat kidney cells transfected with mutant K-Ras derived from the human colon carcinoma cell line SW480

(ATCC# ) were grown in Dulbecco's Modified Eagle

Medium (Life Technologies 11965-092). On day 0, cells growing in a T75 flask were removed by trypsinization, washed by trypsinization and suspended in DMEM/F-12 (3 : 1 )

(Life Technologies 93-0152DK) culture medium containing 10% dialyzed fetal calf serum, gentamycin and 0.2 M Hepes (Life

Technologies) . The cells were distributed into 96 well tissue culture plates at a concentration of 2 X 10E4 per well .

On day one, drug dilutions were made with compound reconstituted in DMSO at a final drug concentration of

1 mg/ml. Subsequent dilutions were made in DMEM/F-12 (3 : 1) to yield final drug concentrations of 0, 5, 2.5, 1.25,

0.625, 0.313, 0.156 and 0.078 μg/ml of culture medium added to replicate wells of the 96 well plate.

On day 4, cell proliferation was detected using

CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay kit (Promega) exactly according to the manufacturers directions. MTS (Owens reagent) is bioreduced by cells into - 45- a water soluble formazan. The absorbance at 490 nm of formazan in 96 well plates can be directly measured and the amount of formazan is directly proportional to the number of viable cells. The 50% drug inhibitory concentration (IC/50) relative to the 0 μg/ml drug wells was calculated using non linear analysis. Individual values representing the results from separate assays are depicted.

Luciferase Assay An important indicator of activation of Ras and its signal transduction pathway is the activation of the protein MAPK by phosphorylation. Among the targets of phosphorylated MAPK is the protein ELK yielding phosphorylated ELK. A luciferase reporter assay was constructed utilizing standard molecular biology techniques to measure the levels of phosphorylated ELK, which is proportional to the level of activation of the Ras pathway. In this assay a plasmid designated P101 is transfected into target SW480 human colon carcinoma cells. P101 yields a Gal 4 binding domain/ELK activation domain fusion protein.

Simultaneously, a plasmid designated P301 is transfected into the same cells. P301 contains the Gal 4 binding sequence in juxtaposition to the delta 56 FOS promoter, in turn in juxtaposition to a luciferase gene. In the cell, activated MAPK interacts with and phosphorylates the ELK region of the Gal 4/ELK hybrid protein. The hybrid protein - 46 - can then interact with the Gal 4 binding sequence and FOS promoter, resulting in the transcription of the luciferase gene. The amount of luciferase produced is therefore dependent on the level of activation of the MAPK and hence the level of activation of the Ras pathway.

The construction of plasmids P101 and P301 was accomplished using standard molecular biology techniques .

To assay compound activity, 4.5 10E6 mutant Ras containing SW480 cells, grown in Dulbecco's Modified Eagle Medium (Life Technologies 11965-092) plus 10% fetal calf serum were trypsinized and transferred to T75 flasks on day 1. 5-6 hours later cells were transfected with 8.8 μl 301 (0.9 μg/μl) and 4 μl 101 (1.0 μg/μl) in 8 μg carrier DNA using a Calcium Phosphate Transfection System (18306-019 Life Technologies) according to the manufacturers recommendations. After 18 hours of incubation (37 °C in 5% CO2 ) the cells were removed from the flask by trypsinization, and 3.5 X 10E4 cells in 50 μl culture medium were added to each well of a 96 well plate. After an additional 5-6 hr incubation, the test drugs were added in

50 μl of culture medium plus 10% fetal calf serum. After an additional 18 hr incubation, the culture medium containing test compounds were removed, and 80 μl of lysis buffer (100 mM KPO4, 0.2% triton, ImMDTT, pH 7.8) was added to each well and incubated for 15 min. 40 μl were then removed to a luminometer plate. Luciferase assay buffer (40 μl ) (2.688 g - 47 - Tricine, 0.9078 g ATP, 1,848 g MgS0 , 0.7715 g DTT, H₂0 to

500 ml, pH 7.8) was added to each well and the luciferase activity in the well was evaluated using a MC3000

Luminometer (Dynatech) according to manufacturers recommendations. Luciferin Solution for the luminometer consisted of 100 mg luciferin, 9 ml IM glycyl-glycine, 36 ml

40 mM EGTA, 720 μl IM DTT, 5.4 ml 1 M MgS0₄ , and 310 ml H₂0.

The IC/50 concentration of the test compounds was determined graphically . Representative compounds were tested for activity and values representing the GTP-binding assay (GTP) , proliferation assay (Pro) and the luciferase assay (luc) from separate assays are provided below.

Example # GTP(μM) Luc(μM) Pro(μM)

11 0.4, 0.4 >2 6.4

12 0.4 >2

13 0.5 >2

14 0.3, 0.2 2 3.2

15 0.4, 0.4 >2 10

16 0.3, <0.4 1-2 3.5

17 0.3 >2

18 0.8

19 0.7

20 0.4, 0.38 3.2

21 0.3, <0.3 >2 2.3

22 1.2, 0.9 >2 2.1

23 0.22 , 0.37 2 1.1

24 0.3 >2 2.4

25 0.2 >2, >2 >19.2 48

Example # GTP(viM) Luc(μM) Pro(μM)

26 0.2

27 0.4

28 0.5

29 0.4

30 0.4

31 0.9

32 1.1 >2

33 0.5 >2

34 0.3, <0.4 >2 20

35 0.4 >2

36 0.5 >2

37 0.6, 1.2, 1.4, >2, >1 3.1, 1.1, 1.8

38 0.8, 1, l. .5, >2, >1 1.4 <1.0

39 0.9, <1 >1, 1-3 1.3

40 0.6, 1.1, 1.5, >2, 2, >1 2.9 <1.0

41 0.8, 0.7, 0.7, >2, >1 1.5 1.2

42 3.3 >2 >17

43 1, o .9 >2 9.5

44 0.8, <0.9 >1, 3 1.9

45 1.3, 0.6, 1.2, 2, : >1 1.2 0.9

46 1.2, 2.6, 1.3 >2, >1 12.1

47 1.5, 1.7, 1.8, >2, >1 4.1 1.8

48 1.4, 1.8, 1.7 >2, >1 3.6

49 0.9, 0.6, 0.5, >1, >3 4.8, 4. 0.5, 1.1

50 1.6, 3.6 >2 3.9

51 1, 1 .2 >2 2.3

52 0.4, 0.4 0.5 -1 0.9 49

Example # GTP(μM) Luc(μM) Pro(μM)

53 0.5, 0.4, 0.8, 1.1

54 0.5, 0.6, 0.5 1 1.8

55 0.8, 1.3, 0.7

56 0.4, 0.5 1-2 2.3

57 0.6, 1 >2 3.6

58 0.6, <0.7 1, 1 1.0

59 0.5, 0.4 >2 5.9

60 >3.2 2 3.8

61 0.3, 0.4 1 1.5

62 0.5, 0.3 1-2 1.8

63 0.6 >2

64 0.5, 0.5 1 1.2

65 0.6, 0.6 2 1.4

66 0.5 >2

67 0.4, 0.5 >2

68 0.4, 0.5

69 0.1 >4 18.3

70 0.3, 0.3

71 0.2

72 0.1 >2, >4 15.9

73 0.1 >4 >16.6

74 0.1 >4 >17.4

75 0.2, 0.2 >2

76 0.2, 0.2 >2 11.1

77 0.2, 0.2 1-2 3.0

78 0.3, 0.3 4.4

79 0.3, 0.3

80 0.4, 0.4

81 1.3, 0.9 50

Example # GTP(μM) Luc(μM) Pro(μM)

82 1.6, 1. 3

83 0.5, 0. 3 >2

84 0.3, 0. .3 >2 2.2

85 0.2, 0. .3 >3 13.3

86 0.2 <1 0.5

87 0.3 >4 4.2

88 0.3 1-2 1.7

89 0.3 0.5-1, <1 0.5

90 0.3

91 0.1 <1 0.6

92 0.2 1-2 0.7

93 0.3 >4 6.5

94 0.2 0.5-1, <1 0.6

95 0.4

96 0.2 <1 0.4

97 0.4 3, 1-2 1.7

98 0.4 >2

99 2.9

Administration of the compounds of the present invention alone may provide an effective course of treatment for the various Ras related cancers. Still another embodiment of the present invention comprises the administration of compounds of the present invention in combination with other known treatments to provide an effective course of treatment for such cancers.

While it is possible to administer a compound employed in the methods of this invention directly without any formulation, the compounds are usually administered in the form of a pharmaceutically acceptable excipient and at least one active ingredient (a compound of the present invention) . Such compositions contain from about 0.1% by weight to about 90.0% by weight of the present compound. These compositions - 51 - can be administered by a variety of routes including oral, rectal, topical, transdermal, buccally, subcutaneous, intravenous, intramuscular, and intranasal . Many of the compounds employed in the methods of this invention are effective as both injectable, oral and topical compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. See, e.g., Remington's Pharmaceutical Sciences, (16th ed. 1980) . In making the compositions employed in the present invention the active ingredient is usually mixed with an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium) , ointments containing for example up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation.

Some examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, - 52 - alcohol, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates ; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

The compounds of this invention may be delivered transdermally using known transdermal delivery systems and excipients. Most preferably, a compound of this invention may be admixed with permeation enhancers including, but not limited to, propylene glycol, polyethylene glycol monolaurate, and azacycloalkan-2-ones, and incorporated into a patch or similar delivery system. Additional excipients including gelling agents, emulsifiers, and buffers may be added to the transdermal formulation as desired. For topical administration, the compound of this invention ideally can be admixed with any variety of excipients in order to form a viscous liquid or cream-like preparation.

For oral administration, a compound of this invention ideally can be admixed with carriers and diluents and molded into tablets or enclosed in gelatin capsules.

The compositions are preferably formulated in a unit dosage form, each dosage containing from about 0.05 to about 500 mg, usually about 1.0 to about 100 mg, more usually about 1.0 to about 50 mg, of the active ingredient. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. - 53 -

The active compounds are generally effective over a wide dosage range. For examples, dosages per day normally fall within the range of about 0.01 to about 500 mg/kg of body weight, preferably within the range of about 0.01 to about 30 mg/kg, more preferably within the range of about 0.01 to about 10 mg/kg. In the treatment of adult humans, the range of about 0.1 to about 5 mg/kg/day, in single or divided dose, is especially preferred. However, it will be understood that the amount of the compound actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound or compounds administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms, and therefore the above dosage ranges are not intended to limit the scope of the invention in any way. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, provided that such larger doses are first divided into several smaller doses for administration throughout the day.

In order to more fully illustrate the operation of the present invention, the following formulation examples are provided. The examples are illustrative only, and are not intended to limit the scope of the invention in any manner. The formulations may employ as active ingredients (compounds) any of the compounds of the present invention. - 54 -

Formulation Preparation 1 Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule)

Active Ingredient 10.0

Starch 325.0

Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.

Formulation Preparation 2 A tablet formula is prepared using the ingredients below:

Quantity

Ingredient ( g/tablet )

Active Ingredient 100.0

Cellulose, microcrystalline 125.0 Colloidal silicon dioxide 10.0

Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing 240 mg .

Formulation Preparation 3 A dry powder inhaler formulation is prepared containing the following components:

Ingredient Weight % Active Ingredient 5

Lactose 95

The active mixture is mixed with the lactose and the mixture is added to a dry powder inhaling appliance. - 55 -

Formulation Preparation 4 Tablets, each containing 30 mg of active ingredient, are prepared as follows:

Quantity Ingredient (mg/tablet)

Active Ingredient 30.0 mg

Starch 45.0 mg

Microcrystalline cellulose 35.0 mg

Polyvinylpyrrolidone (as 10% solution in water) 4.0 mg

Sodium carboxymethyl starch 4.5 mg

Magnesium stearate 0.5 mg

Talc 1.0 mg

Total 120 mg

The active ingredient, starch and cellulose are passed through a No . 20 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve. The granules so produced are dried at 50-60°C and passed through a 16 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No . 30 mesh U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 120 mg .

Formulation Preparation 5 Capsules, each containing 40 mg of medicament are made as follows: Quantity

Ingredient (mg/capsule)

Active Ingredient 40.0 mg

Starch 109.0 mg

Magnesium stearate 1.0 mg Total 150.0 mg - 56 -

The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No . 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg quantities .

Formulation Preparation 6 Suppositories, each containing 25 mg of active ingredient are made as follows:

Ingredient Amount Active Ingredient 25 mg

Saturated fatty acid glycerides to 2,000 mg

The active ingredient is passed through a No . 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Preparation 7

Suspensions, each containing 50 mg of medicament per 5.0 ml dose are made as follows:

Ingredient Amount

Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%)

Microcrystalline cellulose (89%) 50.0 mg

Sucrose 1.75 g

Sodium benzoate 10.0 mg Flavor and Color q.v.

Purified water to 5.0 ml

The medicament, sucrose and xanthan gum are blended, passed through a No . 10 mesh U.S. sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water. The sodium - 57 - benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.

Formulation Preparation 8

Capsules, each containing 15 mg of medicament, are made as follows :

Quantity

Ingredient (mg/capsule) Active Ingredient 15.0 mg

Starch 407.0 mg

Magnesium stearate 3.0 mg

Total 425.0 mg

The active ingredient, cellulose, starch, and magnesium stearate are blended, passed through a No . 20 mesh U.S. sieve, and filled into hard gelatin capsules in 425 mg quantities .

Formulation Preparation 9 An intravenous formulation may be prepared as follows:

Ingredient Quantity Active Ingredient 250.0 mg

Isotonic saline 1000 ml

Formulation Preparation 10

A topical formulation may be prepared as follows: Ingredient Quantity

Active Ingredient 1-10 g

Emulsifying Wax 30 g

Liquid Paraffin 20 g

White Soft Paraffin to 100 g - 58 -

The white soft paraffin is heated until molten. The liquid paraffin and emulsifying wax are incorporated and stirred until dissolved. The active ingredient is added and stirring is continued until dispersed. The mixture is then cooled until solid.

Formulation Preparation 11 Sublingual or buccal tablets, each containing 10 mg of active ingredient, may be prepared as follows:

Quantity Ingredient Per Tablet Active Ingredient 10.0 mg Glycerol 210.5 mg Water 143.0 mg Sodium Citrate 4.5 mg Polyvinyl Alcohol 26.5 mg Polyvinylpyrrolidone 15.5 mg

Total 410.0 mg

The glycerol, water, sodium citrate, polyvinyl alcohol, and polyvinylpyrrolidone are admixed together by continuous stirring and maintaining the temperature at about 90°C. When the polymers have gone into solution, the solution is cooled to about 50-55°C and the medicament is slowly admixed. The homogenous mixture is poured into forms made of an inert material to produce a drug-containing diffusion matrix having a thickness of about 2-4 mm. This diffusion matrix is then cut to form individual tablets having the appropriate size. Another preferred formulation employed in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. - 59 -

See, e.g., U.S. Patent 5,023,252, issued June 11, 1991, herein incorporated by reference. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents . Indirect techniques, which are generally preferred, usually involve formulating the compositions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid-soluble drugs or prodrugs. Latentiation is generally achieved through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present on the drug to render the drug more lipid soluble and amenable to transportation across the blood-brain barrier. Alternatively, the delivery of hydrophilic drugs may be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood-brain barrier.

The type of formulation employed for the administration of the compounds employed in the methods of the present invention may be dictated by the particular compounds employed, the type of phar acokinetic profile desired from the route of administration and the compound(s), and the state of the patient.

Claims

- 60We Claim :

1. Use in the manufacture of a medicament for treating cancer of a compound of the formula I :

O

where R^ and R2 are independently hydrogen, halogen, cyano, trifluoromethyl, Cχ-Cg alkyl, Cχ-Cg alkoxy, carboxy, C -C4 alkoxycarbonyl, amino, mono (Cχ-C4 ₎ alkylamino, di(Cχ-C4) alkylamino, amido, C -C4 alkyla ido, aryl, or a heterocycle, where the aryl or heterocycle is optionally substituted with C -C4 alkyl, C -C4 alkoxy, halo, cyano or trifluoromethyl ;

R3 is hydrogen, halogen, cyano or Cχ-C4 alkoxy;

R₄ is SR₅, N(Rg)(R₇), -NH(-L-)R₈, -NHCHR₉R₁₀ or a heterocycle where the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C1-C4 alkyl, C -C4 alkoxy, Cχ-C4 alkoxy (C -C4 alkyl) or C -C4 alkoxycarbonyl;

where R5 is hydrogen, C -C4 alkyl or aryl, where the aryl is optionally substituted with 1, 2 or 3 groups selected from ^cl~^c4 alkyl, C -C4 alkoxy, halo, cyano or trifluoromethyl;

Rg is hydrogen or methyl;

R7 is C -Cg alkyl, C2~C o alkenyl, benzyl, unsubstituted or substituted C3-C7 cycloalkyl, triphenylmethyl, aryl or a heterocycle, where the Cχ-Cg alkyl is optionally substituted - 61- with 1, 2, 3, 4 or 5 hydroxy groups and where the aromatic ring of benzyl, the aryl or the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C -C4 alkyl, ^cl^_c4 alkoxy, hydroxy, carboxy, halo, cyano, nitro, trifluoromethyl, Cχ-C4 alkoxycarbonyl, phenyl, benzyl, morpholino, or piperidinyl;

L is C2-C3 alkanediyl;

Rg is aryl, C3-C7 cycloalkyl, heterocycle, amino mono(C -Cg alkylamino), di(Cχ-Cg alkyl)amino, mono(Cχ-Cg alkoxy)amino or di(Cχ-Cg alkoxy) amino, hydroxy (C1-C4 alkyl), where the aryl or the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C -C4 alkyl, C -C4 alkoxy, hydroxy, carboxy, halo, cyano or trifluoromethyl;

Rg and Rχø ^are independently hydrogen, C -C4 alkyl, hydroxy (C1-C4 alkyl), benzyl, C3-C7 cycloalkyl, - (CH₂ ) zS (CH ) z ' ^CH3 or heterocycle, where the heterocycle or aromatic ring of the benzyl is optionally substituted with from 1 to 3 groups selected from C -C4 alkyl, C -C4 alkoxy, hydroxy, oxo or halo; and

Z and Z' are independently 0, 1, 2 or 3;

or a pharmaceutically acceptable salt or solvate thereof

2. Use in the manufacture of a medicament for treating a cancer selected from lung, colorectal, pancreatic, breast, urinary tract, stomach, liver, cervix, ovary, pancreas, gall bladder, leuke ias and myelodisplatic syndrome (MDS) of a compound of the formula I:

where Rj_ and R2 are independently hydrogen, halogen, cyano, trifluoromethyl, Cχ-Cg alkyl, C -Cg alkoxy, carboxy, Cχ-C alkoxycarbonyl, amino, mono (C1-C4₎ alkylamino, di(C -C4) alkylamino, amido, Cχ-C4 alkylamido, aryl, or a heterocycle, where the aryl or heterocycle is optionally substituted with C -C4 alkyl, C -C4 alkoxy, halo, cyano or trifluoromethyl ;

R3 is hydrogen, halogen, cyano or C -C4 alkoxy;

R4 is SR₅, OR_6? N(R₇)(R₈), -NH(-L-)R₉, -NHCHR₁₀Rn or a heterocycle where the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C -C4 alkyl, Cχ-C4 alkoxy, C -C4 alkoxy (Cχ-C4 alkyl) or C -C4 alkoxycarbonyl;

where R5 is hydrogen, C -C4 alkyl or aryl, where the aryl is optionally substituted with 1, 2 or 3 groups selected from Cχ-C4 alkyl, C -C4 alkoxy, halo, cyano or trifluoromethyl;

Rg is hydrogen or methyl;

R7 is Cχ-Cg alkyl, C2~C o alkenyl, benzyl, unsubstituted or substituted C3-C7 cycloalkyl, triphenylmethyl, aryl or a heterocycle, where the C -Cg alkyl is optionally substituted with 1, 2, 3, 4 or 5 hydroxy groups and where the aromatic ring of benzyl, the aryl or the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C -C4 alkyl, - 63 - ^cl^_c4 alkoxy, hydroxy, carboxy, halo, cyano, nitro, trifluoromethyl, C1-C alkoxycarbonyl, phenyl, benzyl, morpholino, or piperidinyl;

L is C2-Cg alkanediyl;

Rg is aryl, C3-C7 cycloalkyl, heterocycle, amino mono(Cχ-Cg alkylamino), di (Cχ-Cg alkyl)amino, mono(Cχ-Cg alkoxy)amino or di(C -Cg alkoxy) amino, hydroxy (C -C4 alkyl), where the aryl or the heterocycle is optionally substituted with 1, 2 or 3 groups selected from Cχ-C4 alkyl, C -C4 alkoxy, hydroxy, carboxy, halo, cyano or trifluoromethyl;

Rg and R o are independently hydrogen, C -C4 alkyl, hydroxy (C1-C4 alkyl), benzyl, C3-C7 cycloalkyl, - (CH₂ ) _ZS (CH₂ ) _z ' H₃ or heterocycle, where the heterocycle or aromatic ring of the benzyl is optionally substituted with from 1 to 3 groups selected from C -C4 alkyl, C -C4 alkoxy, hydroxy, oxo or halo; and

Z and Z' are independently 0, 1, 2 or 3 ;

or a pharmaceutically acceptable salt or solvate thereof.

3. A pharmaceutical composition containing a compound of the formula I:

O

- 64- where R^ and R2 are independently hydrogen, halogen, cyano, trifluoromethyl, Cχ-Cg alkyl, Cχ-Cg alkoxy, carboxy, C -C4 alkoxycarbonyl, amino, mono (C1-C4₎ alkylamino, di(Cχ-C4) alkylamino, amido, Cχ-C4 alkylamido, aryl, or a heterocycle, where the aryl or heterocycle is optionally substituted with C -C4 alkyl, C -C4 alkoxy, halo, cyano or trifluoromethyl;

R3 is hydrogen, halogen, cyano or C -C4 alkoxy;

R₄ is SR₅, OR₆ N(R₇) (R₈), -NH(-L-)Rg, -NHCHR₁₀Rn or a heterocycle where the heterocycle is optionally substituted with 1, 2 or 3 groups selected from Cχ-C4 alkyl, Cχ-C4 alkoxy, C -C4 alkoxy (C -C4 alkyl) or C -C4 alkoxycarbonyl;

where R5 is hydrogen, C -C alkyl or aryl, where the aryl is optionally substituted with 1, 2 or 3 groups selected from C -C4 alkyl, C1-C4 alkoxy, halo, cyano or trifluoromethyl;

Rg is hydrogen or methyl;

R7 is Cχ-Cg alkyl, C2-Cχo alkenyl, benzyl, unsubstituted or substituted C3-C7 cycloalkyl, triphenylmethyl, aryl or a heterocycle, where the C -Cg alkyl is optionally substituted with 1, 2, 3, 4 or 5 hydroxy groups and where the aromatic ring of benzyl, the aryl or the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C -C4 alkyl, C -C4 alkoxy, hydroxy, carboxy, halo, cyano, nitro, trifluoromethyl, C1-C4 alkoxycarbonyl, phenyl, benzyl, morpholino, or piperidinyl;

L is C2-C3 alkanediyl; - 65- Rg is aryl, C3-C7 cycloalkyl, heterocycle, amino mono(Cχ-Cg alkylamino), di (C -Cg alkyl)amino, mono(C -Cg alkoxy)amino or di(Cχ-Cg alkoxy) amino, hydroxy (C -C4 alkyl), where the aryl or the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C -C4 alkyl, C -C4 alkoxy, hydroxy, carboxy, halo, cyano or trifluoromethyl;

Rg and R o are independently hydrogen, C -C4 alkyl, hydroxy (C1-C4 alkyl), benzyl, C3-C7 cycloalkyl, - (CH₂ ) _ZS (CH₂ ) _z ■ CH₃ or heterocycle, where the heterocycle or aromatic ring of the benzyl is optionally substituted with from 1 to 3 groups selected from C -C4 alkyl, Cχ-C4 alkoxy, hydroxy, oxo or halo; and

Z and Z¹ are independently 0, 1, 2 or 3 ;

or a pharmaceutically acceptable salt or solvate thereof;

provided that : (1) when R^, R2 and R3 are hydrogen, R4 is not

4-substituted piperazinyl and R5 is not hydrogen or C -C4 alkyl ; and

(2) when R]_ and R2 are hydrogen or Cχ-Cg alkyl and R3 is hydrogen, R7 is not phenyl, substituted phenyl, morpholinyl or pyrrolidinyl .

66 -

4. A compound of the formula I

0

where Rj_ and R2 are independently hydrogen, halogen, cyano, trifluoromethyl, Cχ-Cg alkyl, Cχ-Cg alkoxy, carboxy, C -C4 alkoxycarbonyl, amino, mono (C -C4 ₎ alkylamino, di(C -C4) alkylamino, amido, Cχ-C4 alkylamido, aryl, or a heterocycle, where the aryl or heterocycle is optionally substituted with C -C4 alkyl, C -C4 alkoxy, halo, cyano or trifluoromethyl ;

R3 is hydrogen, halogen, cyano or C -C4 alkoxy;

R4 is SR5, OR_6/ N(R₇) (Rg), -NH(-L-)Rg, -NHCHR₁₀ n or a heterocycle where the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C -C4 alkyl, C -C4 alkoxy, C -C4 alkoxy (C -C4 alkyl) or C -C4 alkoxycarbonyl;

where R5 is hydrogen, C -C4 alkyl or aryl, where the aryl is optionally substituted with 1, 2 or 3 groups selected from ^cl^_c4 alkyl, C -C4 alkoxy, halo, cyano or trifluoromethyl;

Rg is hydrogen or methyl;

R7 is Cχ-Cg alkyl, C2-C g alkenyl, benzyl, unsubstituted or substituted C3-C7 cycloalkyl, triphenylmethyl, aryl or a heterocycle, where the Cχ-Cg alkyl is optionally substituted with 1, 2, 3, 4 or 5 hydroxy groups and where the aromatic - 67 - ring of benzyl, the aryl or the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C -C4 alkyl, ^cl~^c4 alkoxy, hydroxy, carboxy, halo, cyano, nitro, trifluoromethyl, C1-C4 alkoxycarbonyl, phenyl, benzyl, morpholino, or piperidinyl ;

L is C2-C3 alkanediyl;

Rg is aryl, C3-C7 cycloalkyl, heterocycle, amino mono(C -Cg alkylamino), di (C -Cg alkyl)amino, mono(C -Cg alkoxy)amino or di(C -Cg alkoxy) amino, hydroxy (C -C4 alkyl), where the aryl or the heterocycle is optionally substituted with 1, 2 or 3 groups selected from C1-C4 alkyl, C -C4 alkoxy, hydroxy, carboxy, halo, cyano or trifluoromethyl;

Rg and R o ^are independently hydrogen, C -C4 alkyl, hydroxy (C1-C4 alkyl), benzyl, C3-C7 cycloalkyl, - (CH₂ ) _ZS (CH₂ ) _z ' CH₃ or heterocycle, where the heterocycle or aromatic ring of the benzyl is optionally substituted with from 1 to 3 groups selected from C -C4 alkyl, C -C4 alkoxy, hydroxy, oxo or halo; and

Z and Z' are independently 0, 1, 2 or 3 ;

or a pharmaceutically acceptable salt or solvate thereof;

provided that :

(1) when R^ , R2 and R3 are hydrogen, R4 is not 4-substituted piperazinyl and R5 is not hydrogen or Cχ-C4 alkyl; and

(2) when R]_ and R2 are hydrogen or C -Cg alkyl and R3 is hydrogen, R7 is not phenyl, substituted phenyl, morpholinyl or pyrrolidinyl .