Methods and compositions utilizing quinazolinones

153554/2 -2 mini iniN D,l7Onn ninpn n'tiQm |in-4-

2-(1(N-3-AMINOPROPYL)BENZAMIDO)ISOBUTYL)QUINAZOLIN-4-ONE DERIVATIVES AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

Applicants: Cyclokinetics, Inc. CYTO 90232/1.2 153554/2

FIELD OF THE INVENTION

This invention relates to quinazolinone derivatives, which are inhibitors ofthe mitotic kinesin KSP and are useful in the treatment of cellular proliferative diseases, for example cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders and inflammation.

BACKGROUND OF THE INVENTION

Interest in the medicinal chemistry of quinazoline derivatives was stimulated in the early 1950's with the elucidation ofthe structure ofa quinazoline alkaloid,3- [B-kQto-gamma- (3-hydroxy-2-piperidyl)-propyl]-4-quinazolone, from an Asian plant known / for its antimalarial properties. In a quest to find additional antimalarial agents, various substituted quinazolines have been synthesized. Of particular import was the synthesis ofthe derivative2-methyl-3-o-tolyl-4-(3H)-quinazolinone. This compound, known by the name methaqualone, though ineffective against protozoa, was found to be a potent hypnotic.

Since the introduction of methaqualone and its discovery as a hypnotic, thepharmacological activity of quinazolinones and related compounds has been investigated. Quinazolinones and derivatives thereof are now known to have a wide variety of biological properties including hypnotic, sedative, analgesic, anticonvulsant, antitussive and anti-inflammatory activities.

Quinazolinone derivatives for which specific biological uses have been described include U. S. Patent No. 5,147,875 describing 2- (substituted phenyl)-4-oxo quinazolines with bronchodilator activity. U. S. Patent Nos. 3,723,432,3,740,442, and 3,925,548 describe a class of1-substituted-4-aryl-2 (1 H)-quinazolinone derivatives useful as anti-inflammatory agents. European patent publication EP 0 056 637 B 1 claims a classof 4 (3H)-quinazolinone derivatives for the treatment of hypertension. European patent publication EP 0 884 319 Al describes pharmaceutical compositions of quinazolin-4-one derivatives used to treatneurodegenerative, psychotropic, and drug and alcohol induced central and peripheral nervous system disorders. - 1 - WO 01/98278 PCT/USO1/13901

Quinazolinones are among a growing number of therapeutic agents used to treat cell proliferative disorders, including cancer. For example, PCT WO 96/06616 describes a pharmaceutical composition containing a quinazolinone derivative to inhibit vascular smooth cell proliferation. PCT WO 96/19224 uses this same quinazolinone 5 derivative to inhibit mesengial cell proliferation. U.S. Patent Nos. 4,981,856, 5,081,124 and 5,280,027 describe the use of quinazolinone derivatives to inhibit thymidylate synthase, the enzyme that catalyzes the methylation of deoxyuridine monophosphate to produce thymidine monophosphate which is required for DNA synthesis. U.S. Patent Nos. 5,747,498 and 5,773,476 describe quinazolinone 10 derivatives used to treat cancers characterized by over-activity or inappropriate activity of tyrosine receptor kinases. U.S. Patent No. 5,037,829 claims (lH-azol-l~ ylmethyl) substituted quinazoline compositions to treat carcinomas which occur in epithelial cells. PCT WO 98/34613 describes a composition containing a quinazolinone derivative useful for attenuating neovascularization and for treating 15 malignancies. U.S. Patent 5,187,167 describes pharmaceutical compositions comprising quinazolin-4-one derivatives which possess anti-tumor activity.

Other therapeutic agents used to treat cancer include the taxanes and vinca alkaloids. Taxanes and vinca alkaloids act on microtubules, which are present in a variety of cellular structures. Microtubules are the primary structural element of the mitotic 20 spindle. The mitotic spindle is responsible for distribution of replicate copies ofthe genome to each of the two daughter cells that result from cell division. It is presumed that disruption of the mitotic spindle by these drugs results in inhibition of cancer cell division, and induction of cancer cell death. However, microtubules form other types of cellular structures, including tracks for intracellular transport in nerve processes. 25 Because these agents do not specifically target mitotic spindles, they have side effects that limit their usefulness.

Improvements in the specificity of agents used to treat cancer is of considerable interest because of the therapeutic benefits which would be realized if the side effects associated with the administration of these agents could be reduced. Traditionally, 30 dramatic improvements in the treatment of cancer are associated with identification of therapeutic agents acting through novel mechanisms. Examples of this include not -2- WO 01/98278 PCT/USO1/13901 only the taxanes, but also the camptothecin class of topoisomerase I inhibitors. From both of these perspectives, mitotic kinesins are attractive targets for new anti-cancer agents.

Mitotic kinesins are enzymes essential for assembly and function of the mitotic 5 spindle, but are not generally part of other microtubule structures, such as in nerve processes. Mitotic kinesins play essential roles during all phases of mitosis. These enzymes are "molecular motors" that transform energy released by hydrolysis of ATP into mechanical force which drives the directional movement of cellular cargoes along microtubules. The catalytic domain sufficient for this task is a compact structure of 10 approximately 340 amino acids. During mitosis, kinesins organize microtubules into the bipolar structure that is the mitotic spindle. Kinesins mediate movement of chromosomes along spindle microtubules, as well as structural changes in the mitotic spindle associated with specific phases of mitosis. Experimental perturbation of mitotic kinesin function causes malformation or dysfunction of the mitotic spindle, 15 frequently resulting in cell cycle arrest and cell death.

Among the mitotic kinesins that have been identified is KSP. KSP belongs to an evolutionarily conserved kinesin subfamily of plus end-directed microtubule motors that assemble into bipolar homotetramers consisting of antiparallel homodimers. During mitosis KSP associates with microtubules of the mitotic spindle. • 20 Microinjection of antibodies directed against KSP into human cells prevents spindle pole separation during prometaphase, giving rise to monopolar spindles and causing mitotic arrest and induction of programmed cell death. KSP and related kinesins in other, non-human, organisms, bundle antiparallel microtubules and slide them relative to one another, thus forcing the two spindle poles apart. KSP may also mediate in 25 anaphase B spindle elongation and focussing of microtubules at the spindle pole.

Human KSP (also termed HsEg5) has been described [Blangy, et al., Cell, 83:1159-69 (1995); Whitehead, et al., Arthritis Rheum., 39:1635-42 (1996); Galgio et al., J. Cell Biol., 135:339-414 (1996); Blangy, et al., J Biol. Chem., 272:19418-24 (1997); Blangy, et al., Cell Motil Cytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J.

30 Cell Sci., 111:2551-61 (1998); Kaiser, et al., JBC 274:18925-31 (1999); GenBank accession numbers: X85137, NM004523 and U37426], and a fragment of the KSP -3- * 153554/2 gene (TR1P5) has been described [Lee, et al., Mol Endocrinol., 9: 243-54 (1995);

GenBank accession number L40372], Xenopus KSP homologs (Eg5), as well as

Drosophila KLP61 F/KRP1 30 have been reported.

Mitotic kinesins are attractive targets for the discovery and development of noveimitotic chemotherapeutics. Accordingly, it is an object of the present invention to provide methods and compositions useful in the inhibition of KSP, a mitotic kinesin.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present invention provides compounds that are KSP inhibitors, particularly human KSP inhibitors. These, compounds and compositions containing them can be used to treat diseases of proliferating cells, disorders associated with KSP kinesin activity, and to inhibit KSP kinesin.

The compounds provided according to one aspect of the present invention have the formula: 0

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R3 including pharmaceutically acceptable salts thereof; wherein the isopropyl group is attached in the R-configuration; Rt is a benzyl, chlorobenzyl, methyibenzyl, methoxybenzyl, cyanobenzyl or hydroxy benzyl group; R2 is halo or cyano; and R3 is a methyl- and/or halo- substituted phenyl group.

In a particular embodiment, in the compounds and salts of the invention, the compound is in the form of a substantially pure optical R-isomer. -4- -¼ 153554/2

In another particular embodiment, R! is a benzyl group. In yet another particular embodiment, R2 is chloro. In still another particular embodiment, R, is a benzyl group; R2 is chloro; and R3 is a 4-methylphenyl group, and the compound and its salts are preferably in the form of a substantially pure optical R-isomer.

In accordance with a further aspect of the invention, an in-vitro method of inhibiting KSP kinesin comprises contacting KSP kinesin with a compound or salt as defined above.

In accordance with a further aspect of the invention, there is provided a compound or salt as defined, preferably in the form of a substantially pure optical R-isomer, for use as a medicament.

According to yet a further aspect of the invention, there is provided the use, in the manufacture of a medicament for treating cellular proliferative disease, of a compound or salt as defined above, substantially as described herein. The disease just referred to may be e.g. cancer, hyperplasia, restenosis, cardiac hypertrophy, immune disorder or inflammation.

According to still a further aspect of the invention, there is provided a pharmaceutical composition which comprises a compound or salt of as defined above and a carrier thereof.

Statement according to the Registrar’s Circular No. 23(P) of April 5,1992

The passages in the present specification which do not fall within the scope of the present claims do not relate directly to the claimed invention.

Diseases and disorders that respond to therapy with compounds of the invention include cancer, hyperplasia, restenosis, cardiac hypertrophy, immune disorders and - 5 - WO 01/98278 PCT/USO1/13901 inflammation; especially cancer, hyperplasia, restenosis, and cardiac hypertrophy, particularly cancer.

In another aspecb the invention relates to compounds useful in inhibiting KSP kinesin. The compounds have die structures shown above, 5 In an additional aspect, the present invention provides methods of screening for compounds that will hind to a KSP kinesin, for example compounds that will displace or compete with the binding of the compositions of the invention. The methods comprise combining a labeled compound of the invention, a KSP kinesin, and at least one candidate agent and determining the binding of the candidate bioactive agent to 10 the KSP kinesin.

In a further aspect, the invention provides methods of screening for modulators of KSP kinesin activity. The methods comprise combining a composition of the invention, a KSP kinesin, and at least one candidate agent and determining the effect of the candidate bioactive agent on the KSP kinesin activity.

15 BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a generic synthetic scheme to make compositions of the invention.

Figure 2 depicts a synthetic route for the synthesis of quinazolinone KSP inhibitors.

Figure 3 depicts representative chemical structures of quinazolinone KSP inhibitors.

Figured depicts a synthetic route to substantially pure single enantiomers. 20 Figure 5 depicts synthetic routes to sulfonamides (5a), carbamates (5b), ureas (5c) and amines (5d).

DETAILED DESCRIPTION OF THE INVENTION I'he present invention is directed to a class of novel compounds, based on a core quinazolinone structure, that are modulators of mitotic kinesins. By inhibiting or 25 modulating mitotic kinesins, but not other kinesins (e.g., transport kinesins), specific inhibition of cellular proliferation is accomplished. Thus, the present invention -6- WO 01/98278 PCT/USO1/13901 capitalizes on the finding that perturbation of mitotic kmesin function causes ' malformation or dysfunction of mitotic spindles, frequently resulting in cell cycle arrest and cell death. The methods of inhihiting a human KSP kinesin comprise

contacting an inhibitor of the invention with a KSP kinesin, particularly human KSP 5 kinesins, including fragments and variants of KSP. The inhibition can be of tkv ATP hydrolysis activity of the KSP kinesin and/or die mitotic spindle formation activity, such that the mitotic spindles are disrupted. Meiotic spindles may also be disrupted.

An object of the present invention is to develop inhibitors and modulators of mitotic kinesins, in particular KSP, for the treatment of disorders associated with cell 10 proliferation. Traditionally, dramatic improvements in the treatment of cancer, one type of cell proliferative disorder, have been associated with identification of therapeutic agents acting through novel mechanisms. Examples of this include not only the taxane class of agents that appear to act on microtubule formation, but also the camptothecin class of topoisomerase I inhibitors. The compositions and methods 15 described herein can differ in their selectivity and are preferably used to treat diseases of proliferating cells, including, but not limited to cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders and inflammation.

Accordingly, the present invention relates to methods employing quinazolinone amides of formula la:

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la WO 01/98278 PCT/USO1/13901

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lb 5 and quinazolinone amines of foimulae lc and Id

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Id 10 wherein:

Ri is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl;

Rz and R2’ are independently chosen from hydrogen, alkyl, oxaalkyl, aryl, alkylaryl, 15 heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; or R2 and R2’ taken together form a 3- to 7-membered ring; -8- WO 01/98278 PCT/US01/13901 R3 is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, oxaalkyl, oxaalkylaiyl, substituted oxaalkylaiyl, oxaalkylheteroaiyl, substituted oxaalkylheteroaryl, RuO- and R15-NH-; 5 R3· is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkyiheteroaryi, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaiyl, substituted alkylheteroaryl, and R15-NH-; R3.. is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted 10 alkylheteroaryl; R4 is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaiyl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaiyl, substituted alkylheteroaryl, and Ru-alkylene-; R5, Ri, R7 and Rg are independently chosen from hydrogen, alkyl, alkoxy, halogen, 15 fluoroalkyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, sulfonamidoalkyl, sulfonamidoaiyl, alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, aryl and heteroaiyl;

Ru is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaiyl, and substituted 20 alkylheteroaryl;

Ru is chosen from alkoxy, amino, alkylamino, dialkylamino, N-heterocyclyl and substituted N-heterocyclyl.

All of the compounds falling within the foregoing parent genus and its subgenera are useful as kinesm inhibitors, but not all the compounds are novel. In particular, certain 25 ureas (i.e. compounds in which R3 is R15NH) are disclosed in US patent 5,756,502 as agents which modify cholecystokimn action. The specific exceptions in the claims reflect applicants' intent to avoid claiming subject matter that, while functionally part of the inventive concept, is not patentable to them for reasons having nothing to do with the scope of the invention. -9- WO 01/98278 PCT/USO1/13901

Definitions

Tbe term "optional" or "optionally11 means that the subsequently described event or circumstance 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, 5 “optionally substituted alkyl” means either “alkyl” or “substituted alkyl,” as defined below. It will be understood by those skilled in the art with respect to any group containing one or more substituents that such groups are not intended to introduce any substitution or substitution patterns (e.g., substituted alkyl includes optionally substituted cycloalkyl groups, which in tum are defined as including optionally 10 substituted alkyl groups, potentially ad infinitum) that are sterically impractical and/or synthetically non-feasible.

Alkyl is intended to include linear, branched, or cyclic hydrocarbon structures and combinations thereof. Lower alkyl refers to alkyl groups of from 1 to 5 carbon atoms. Examples of lower alkyl groups include methyl, etbyl, propyl, isopropyl, butyl, s-and 15 t-butyl and the like. Preferred alkyl groups are those of Ca or below. More preferred alkyl groups are those of Cu or below. Cycloalkyl is a subset of alkyl and includes cyclic hydrocarbon groups of from 3 to 13 carbon atoms. Examples of cycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbomyl, adamantyl and tbe like. In this application, alkyl refers to alkanyl, as well as the unsaturated alkenyl and alkynyl 20 residues; it is intended to include cyclohexylmethyl, vinyl, allyl, isoprenyl and the like. Alkylene refers to the same residues as alkyl, but having two points of attachment. Examples of alkylene include ethylene (-CH2CH2-), ethenylene (-CH2=CH2-), propylene (-CH2CH2CH2-), dimethylpropylene (-CH2C(CH3) 2CHr) and cyclohexylpropylene (-CH2CH2CH(C6Hi3)-). When an alkyl residue having a 25 specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed; thus, for example, “butyl” is meant to include n-butyl, sec-butyl, isobutyl and t-butyl; “propyl” includes n-propyl and isopropyl (or “i-propyl”). 1

Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of a straight, branched, 30 cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, -10- WO 01/98278 PCT/USO1/13901 cyclopropyloxy, cyclohexyloxy and the like. Lower-alkoxy refers to groups containing one to four carbons.

Acyl refers to groups of from 1 to 8 carbon atoms of a straight, branched, cyclic configuration, saturated, unsaturated and aromatic and combinations thereof, attached 5 to the parent structure through a carbonyl functionality. One or more carbons in the acyl residue may be replaced hy nitrogen, oxygen or sulfur as long as the point of attachment to die parent remains at the carbonyl. Examples include acetyl, benzoyl, propionyl, isobutyryl, t-butoxycarbonyl, benzyloxycarbonyl and the like. Lower-acyl refers to groups containing one to four carbons. 10 Aryl and heteroaryl mean a 5- or 6-membered aromatic or heteroaromatic ring containing 0-3 heteroatoms selected from Ο, N, or S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from 0, N, or S; or a tricyclic 13- or 14-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from Ο, N, or S. The aromatic 6- to 14- 15 membered carbocyclic rings include, e.g., benzene, naphthalene, indane, tetralin, and fluorene and the 5- to 10-membered aromatic heterocyclic rings include, e.g., imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole and pyrazole.

Alkylaryl refers to a residue in which an aryl moiety is attached to the parent structure 20 via an alkylene residue. Examples are benzyl, phenethyl, phenylvinyl, phenylaflyl and the like. Oxaalkyl and oxaalkylaryl refer to alkyl and alkylaryl residues in which one or more methylenes have been replaced by oxygen. Examples of oxaalkyl and oxaalkylaryl residues are ethoxyethoxyethyl (3,6-dioxaoctyl), benzyloxymethyl and phenoxymethyl; in general, glycol ethers, such as polyethyleneglycol, are intended to 25 be encompassed by this group. Alkylheteroaryl refers to a residue in which a heteroaryl moiety is attached to the parent structure via an alkylene residue. Examples include furanylmethyl, pyridinylmethyl, pyrimidinylethyl and the like.

Heterocycle means a cycloalkyl or aryl residue in which one to four of the carbons is replaced by a heteroatom such as oxygen, nitrogen or sulfur. Examples of 30 heterocycles that fall within the scope of the invention include imidazoline, -11 - WO 01/98278 PCT/USO1/13901 pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazole, morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, 5 isoxazole, dioxane, tetrahydrofuran and the like. “N-heterocycly!” refers to a nitrogen-containing heterocycle as a substituent residue. The term heterocyclyl encompasses heteroaryl, which is a subset of heterocyclyl. Examples of N-heterocyclyl residues include 4-morpholinyl, 4-thiomorpholinyl, 1-piperidinyl, 1-pyrrolidinyl, 3-thiazolidinyl, piperazinyl and 4-(3,4-dihydrobenzoxazinyl). 10 Examples of substituted heterocyclyl include 4-methyl-l-piperazinyl and 4-benzyl-l-piperidinyl.

Substituted alkyl, aryl and heteroaryl refer to alkyl, aryl or heteroaryl wherein one or more H atoms are replaced with alkyl, halogen, hydroxy, alkoxy, alkylenedioxy (e.g., methylenedioxy), fluoroalkyl, carboxy (-COOH), carboalkoxy (i.e., acyloxy 15 -O(O)CR), carboxyalkyl (i.e., esters -C(O)OR), carboxamido, sulfonamidoalkyl, sulfonamidoaiyl, aminocarbonyl, benzyloxycarbonylamino (CBZ-amino), cyano, carbonyl, nitro, primary-, secondary- and tertiary-amino (e.g., alkylamino and dialkylamino) and aminoaklylene, alkyltiiio, alkylsulfinyl, alkylsulfonyl, alkylsulfonamido, arylthio, arylsulfinyl, arylsulfonyl, amidino, aryl (e.g., phenyl and 20 benzyl), heteroaiyl, heterocyclyl, phenoxy, benzyloxy, or heteroaryloxy. For the purposes of the present invention, substituted alkyl also includes oxaalkyl residues, i.e. alkyl residues in which one or more carbons has been replaced by oxygen. Substituted alkylaryl and substituted oxaalkylaryl refer to residues where either or . both of the alkylene and aryl moieties are substituted. Substituted alkylheteroaryl and 25 substituted oxaalkylheteroaryl residues where either or both of the alkylene and heteroaryl moieties are substituted. It should additionally be noted that certain positions may contain two or even three substitution groups, R, R’ and R” (e.g., diethylamino and trifluoromethyl).

Halogen refers to fluorine, chlorine, bromine or iodine. Fluorine, chlorine and 30 bromine are preferred. Dihaloaryl, dihaloalkyl, trihaloaryl, etc. refer to aryl and alkyl -12- WO 01/98278 PCT/US01/13901 substituted with a plurality of halogens, but not necessarily a plurality of the same halogen; thus 4-chloro-3-fluorophenyl is within the scope of dihaloaryl.

Most of the compounds described herein contain one or more asymmetric centers (e.g. the cafbon to which Ri and Ro’ are attached) and may thus give rise to enantiomers, 5 diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. The present invention is meant to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures. Optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When 10 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.

When desired, the R- and S-isomers may be resolved by methods known to those 15 skilled in the art, for example by formation of diastereoisomeric salts or complexes which may be separated, for example, by crystallisation; via formation of diastereoisomeric derivatives which maybe separated, for example, by crystallisation, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagenζ for example enzymatic oxidation or reduction, followed 20 by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support, such as silica with a hound chiral ligand or in the presence of a chiral solvent It will he appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step may be 25 required to liberate the desired enantiomeric form. Alternatively, specific enantiomer may he synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting on enantiomer to the other by asymmetric transformation. An example of a synthesis from optically active starting materials is shown in Figure 4. 30 In one embodiment, as will be appreciated by those in the art, the two adjacent Rj groups may be fused together to form a ring structure. Again, the fused ring structure -13- WO 01798278 PCT/USO1/13901 may contain heteroatoms and may be substituted with one or more substitution groups "R". It should additionally be noted that for cycloaikyl (i.e., saturated ring structures), certain positions may contain two substitution groups, R and R'.

Preferred Embodiments 5 Considering formulae la, lb, lc and Id, but focusing on la, in a preferred embodiment Ri is selected from hydrogen, alkyl, aryl, substituted alkyl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl and substituted alkylaryl.

In a more preferred embodiment Ri is selected from hydrogen, lower alkyl,substituted lower alkyl, benzyl, substituted benzyl, phenyl, naphtbyl and substituted phenyl. 10 In a most preferred embodiment Ri is chosen from hydrogen, ethyl, propyl, methoxyethyl, naphtbyl, phenyl, bromophenyl, chlorophenyl, methoxyphenyl, ethoxyphenyl, tolyl, dimethylphenyl, chorofluorophenyl, methylchlorophenyl, ethylphenyl, phenethyl, benzyl, chlorobenzyl, methylhenzyl, methoxybenzyl, cyanobenzyl, hydroxybenzyl, tetrahydrofuranylmethyl and (ethoxycarbonyl)ethyl. 15 In a preferred embodiment R2 is hydrogen, alkyl,cycloaikyl or substituted alkyl. As will be appreciated by those in the art, Formulae la, lb, lc and Id possess a potentially chiral center at the carbon to which R2 is attached. Thus, the R2 position may comprise two substitution groups, R2 and Rj*. The R2 and Ra' groups may be the same or different; if different, the composition is chiral. When the R2 and R2 are 20 different, preferred embodiments utilize only a single non-hydrogen R2. The invention contemplates the use of pure enantiomers and mixtures of enantiomers, including racemic mixtures, although the use of tiie substantially optically pure eutomer will generally be preferred, particularly tiie R enantiomer.

In a more prefened embodiment, R2 is chosen from hydrogen, lower alkyl and 25 substituted Ipwer alkyl, and R2 ’ is hydrogen. In a most preferred embodiment R2 is chosen from hydrogen, methyl, ethyl, propyl (particularly z-propyl), butyl (particularly ί-butyl), methylthioethyl, aminobutyl, (CBZ)aminobutyl, cyclohexylmethyl, benzyloxymethyl, methylsulfinylethyl, methylsulfinylmethyl, hydroxymethyl, benzyl and indolylmethyl. Especially preferred is the R enantiomer where R2 is z-propyl. -14- WO 01/98278 PCT/USO1/13901

In a preferred embodiment R3 is selected from chosen from alkyl, substituted alkyl, alkylaryl, heteroaryl, aryl, substituted aryl, substituted oxaalkylaryl, -O-R15 and -NH-R15, and R15 is chosen from alkyl, aryl and substituted aryl.

In a mors preferred embodiment, when R3 is not -NHRis, R3 is chosen from C1.-C13 5 alkyl; substituted lower alkyl;aryl, including phenyl, biphenyl and naphthyl; substituted aryl, including phenyl substituted with one or more halo, lower alkyl, loweralkoxy, nitro, carboxy, methylenedioxy or trifluoromethyl; benzyl; phenoxymethyl; halophenoxymethyl; phenylvinyl; heteroaryl; heteroaryl substituted with lower alkyl; and benzyloxymethyl. 10 In a most preferred embodiment, when R3 is not -NHR15, R3 is chosen from ethyl, propyl, chloropropyl, butoxy, heptyl, butyl, octyl, tridecanyl, (ethoxycarbonyl)ethyl, dimethylaminoethyl, dimethylaminomethyl, phenyl, naphthyl, halophenyl, dihalophenyl, cyanophenyl, halo(trifluoromethyl)phenyl, chlorophenoxymethyl, methoxyphenyl, carboxyphenyl, ethylphenyl, tolyl, biphenyl, methylenedioxyphenyl, 15 methylsulfonylphenyl, methoxychlorophenyl, chloronaphthyl, methylhalophenyl, trifluoromethylphenyl, butylphenyl, pentylphenyl, methylnitrophenyl, phenoxymethyl, dimethoxyphenyl, phenylvinyl, nitrochlorophenyl, nitrophenyl, dinitrophenyl, bis(trifluoromethyl)phenyl, benzyloxymethyl, benzyl, furanyl, benzofuranyl, pyridinyl, indolyl, methylpyridinyl, quinolinyl, picolinyl, pyrazolyl, and imidazolyl. 20 In a more preferred embodiment, when R3 is -NHR15, R15 is chosen from lower alkyl; cyclohexyl; phenyl; and phenyl substituted with halo, lower alkyl, loweralkoxy, or lower alkylthio.

In a most preferred embodiment, when R3 is -NHR15, Ru is isopropyl, butyl, cyclohexyl, phenyl, bromophenyl, dichlorophenyi, methoxyphenyl, ethylphenyl, tolyl, 25 trifluoromethylphenyl or methylthiophenyl.

In a preferred embodiment R4 is chosen from alkyl, aryl, alkylaryl, alkylheteroaryl, substituted alkyl, substituted aryl, and -alkylene-Ru, and Ru is chosen from alkoxy, amino, alkylamino, dialkylamino and N-heterocyclyl. -15- WO 01/98278 PCT/USO1/13901 hi a more preferred embodiment, R4 is selected from lower alkyl, substituted lower alkyl, cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl; heteroarylmethyl; heteroarylethyl; heteroarylpropyl and -alkylene-Ru, wherein Ru is amino, lower alkylamino, di(lower alkyl)amino, lower alkoxy, or N- 5 heteroeyclyl.

In a most preferred embodiment, R4 is chosen from methyl, ethyl, propyl, butyl, cyclohexyl, carboxyethyl, caiboxymethyl, methoxyethyl, hydroxyethyl, hydroxypropyi, dimethylaminoethyl, dimethylaminopropyl, diethylaminoethyl, diethylaminopropyl, aminopropyl, methylaminopropyl, 2,2-dimethyl-3- 10 (dimethylamino)propyl, l-cyclohexyl-4-(diethylamino)butyl, aminoethyl, aminobutyl, aminopentyl, aminohexyl, axninoethoxyethyl, isopropylaminopropyl, diisopropylaminoethyl, l-methyl-4-(diethylamino)butyl, (t-Boc)ammopropyl, hydroxyphenyl, benzyl, methoxyphenyl, methylmethoxyphenyl, dimethylphenyl, tolyl, ethylphenyl, (oxopyrrolidinyl)propyl, (methoxycaibonyljethyl, benzylpiperidinyl, 15 pyridinylethyl, pyridinylmethyl, morpholinylethyl, morpholinylpropyl, piperidinyl, azetidinylmethyl, azetidinylpropyl, pyrrolidinylethyl, pynolidinylpropyl, piperidinylmethyl, piperidinylethyl, imidazolylpropyl, imidazolylethyl, (ethylpyrrolidinyl)methyl, (methylpyrrolidinyl)ethyl, (methylpiperidinyl)propyl, (methylpiperazinyl)propyl, fhranylmethyl and indolylethyl. 20 In other preferred embodiments, R5, Rg, R7 and Rg are chosen from hydrogen, halo (particularly chloro and fluoro), lower alkyl (particularly methyl), substituted lower alkyl (particularly trifluoromethyl), lower alkoxy (particularly methoxy), and cyano; more preferably from hydrogen and halo. Further preferred for each of the specific substituents: Rs is hydrogen or halo; Rg is hydrogen, methyl or halo; R7 is hydrogen, 25 halo, alkyl (particularly methyl), alkoxy (particularly methoxy) or cyano; and Rg is hydrogen or halo. Still further preferred are the compounds where only one of R5, Re, R7 and Rg is not hydrogen, especially R7.

In a particularly preferred subgenus, Ri is benzyl or halobenzyl; R2 is chosen from ethyl and propyl; R2’ is hydrogen; R3 (or R3' or R3-) is substituted phenyl; R4 is - 30 (CH2)mOH where m is two or three, or -(CH2)pRi6 where p is one to three and Rig is -16- WO 01/98278 PCT/USO1/13901 amino, propylamino or azetidinyl; Rs is hydrogen; Re is hydrogen; R7 is halo; and Rg is hydrogen.

When considering primarily the sulfonamides of formula lb, Rj is preferably chosen from hydrogen, lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, phenyl 5 and substituted phenyl; R2 is chosen from hydrogen and lower alkyl and R2’ is hydrogen; R3· is chosen from C1-C13 alkyl; phenyl; naphthyl; phenyl substituted with halo, lower alkyl, lower alkoxy, nitro, methylenedioxy, or trifluoromethyl; biphenylyl and heteroaryl; and R4 is chosen from lower alkyl, cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl; heteroarylmethyl; heteroarylethyl; 10 heteroarylpropyl; heteroarylethyl; heteroarylpropyl and -alkylene-Ru, wherein Rie is di(lower alkyl)amino, (lower alkyl)amino, amino, lower alkoxy, or N-heterocyclyl, particularly pyrrolidino, piperidino or imidazolyl.

When considering primarily the sulfonamides of formula lb, Ri is most preferably chosen from lower alkyl, benzyl, substituted benzyl and substituted phenyl; R2 is 15 hydrogen or lower alkyl; R2’ is hydrogen; R3 is chosen from substituted phenyl and naphthyl; R4 is -alkylene-Rie; R; is hydrogen, fluoro, methyl or chloro; Rs, Rs and Rg are hydrogen; and Rie is chosen from di(lower alkylamino), (lower alkyl)amino, amino, pyrrolidino, piperidino, imidazolyl and morpholino.

When considering primarily the amines of formulae lc and Id, Ri is preferably chosen 20 from hydrogen, lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, phenyl, naphthyl and substituted phenyl; R2 is chosen from hydrogen, lower alkyl and substituted lower alkyl and R21 is hydrogen; R3·· is chosen from C1-C13 alkyl; substituted lower alkyl; phenyl; naphthyl; phenyl substituted with halo, lower alkyl, lower alkoxy, nitro, methylenedioxy, or trifluoromethyl; biphenylyl, benzyl and 25 heterocyclyl; and R4 is chosen from lower alkyl; cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl; substituted benzyl; heterocyclyl; heteroarylmethyl; heteroarylethyl; heteroarylpropyl and -alkylene-Ru, wherein Rie is di(lower alkyl)amino, (lower alkyl)amino, amino, lower alkoxy, or N-heterocyclyl.

When considering primarily the amines of formulae lc and ld, Ri is most preferably 30 chosen from lower alkyl, benzyl, substituted benzyl and substituted phenyl; R2 is -17- WO 01/98278 PCT/USO1/13901 hydrogen or lower alkyl; R2’ is hydrogen; R3" is chosen from substituted phenyl, - heterocyclyl and naphthyl; R4 is chosen from suhtituted benzyl, heterocyclyl and -alkylene-Rj6; R« and R7 are chosen from hydrogen and halo; R5 and Rg are hydrogen; and Rie is chosen from di(lower alkylamino), (lower alkyl)amino, amino, pyrrolidinyl, 5 piperidinyl, imidazolyl and moipholinyl. When R3·· is present (as in Id) it is most preferably chosen from halophenyl, polyhalophenyl, tolyl, dimethylphenyl, methoxyphenyl, dimethoxyphenyl, cyanophenyl, trifluoromethylphenyl, trifluoromethoxyphenyl, bis(trifluoromethyl)phenyl, carboxyphenyl, t-butylphenyl, methoxycarbonylphenyl, piperidinyl and naphthyl. 10 In view of the foregoing, particularly when taken in consideration of the test data presented below, it will be appreciated that preferred for the compounds, pharmaceutical formulations, methods of manufacture and use of the present invention are the following combinations and permutations of substituent groups (subgrouped, respectively, in increasing order of preference): 15 1. Any of formulae la, lb, lc or Id (preferably formulae la or Id) where Rj is hydrogen, lower alkyl, substituted lower alkyl, alkylaryl or substituted alkylaryl (preferably benzyl or substituted benzyl): a. Especially where the stereogenic center to which R2 and R2’ are attached is of die R configuration, where R2· is hydrogen. 20 i. Particularly where Ri is lower alkyl (preferably ethyl, i-proyl, c-propyl or t-butyl). 1. Most preferably where R2 is i-propyl. · b. Especially those whdre R4 is substituted alkyl, (preferably a primary-, secondary- or tertiary-amino-substituted lower alkyl). 25 i. Most preferably where R4 is primary-amino lower alkyl. c. Especially those where Rs, Re, R7 and Rg are chosen from hydrogen, halo, lower alkyl (preferably methyl), substituted lower alkyl, lower alkoxy (preferably methoxy), and cyano. i. Preferably Rs, Rg, and Rg are hydrogen. 30 1. More preferably R7 is halo or cyano, especially fluoro or chloro, and most preferably chloro. -18- WO 01/98278 PCT/USO1/13901 d. hi the case of formulae la and Id, especially those where R3 or R3·· is aryl (preferably phenyl), substituted aryl (preferably lower alkyl- or lower alkoxy-substituted phenyl), alkylaryl (preferably benzyl and phenylviny), alkylheteroaiyl, oxaalkylaiyl (preferably phenoxy lower alkyl), 5 oxaalkylheteroaryl, substituted alkylaryl (preferably substituted benzyl and substituted phenylviny), substituted alkylheteroaiyl, substituted oxaalkylaryl (preferably substituted phenoxy lower alkyl), or substituted oxaalkylheteroaryl. i. Most preferably those where R3 or R3" is aryl, substituted aryl, 10 lower alkylaryl or substituted lower alkylaryl. 2. Any of formulae 1 a, lb, 1 c or 1 d, where the stereogenic center to which R2 and R2’ are attached is of the R configuration, particularly where R2’ is hydrogen: a. Especially where R2 is lower alkyl (preferably ethyl, i-proyl, c-propyl or t-butyl). 15 i. Most preferably where R2 is i-propyl. b. Especially where R4 is substituted alkyl (preferably a primary-, secondary-or tertiaiy-amino-substituted lower alkyl). i. Most preferably where R4 is primary amino-lower alkyl. c. Especially where R5, Re, and Rg are hydrogen. 20 i. Most preferably where R7 is hydrogen, halo (particularly chloro or fluoro), lower alkyl (particularly methyl), substituted lower alkyl, lower alkoxy (particularly methoxy), or cyano 1. Especialy where R7 is chloro. 3. Any of formulae la, lb, lc or Id where R7 is hydrogen, halo (preferably chloro or 25 fluoro), lower alkyl (preferably methyl), substituted lower alkyl, lower alkoxy (preferably methoxy), or cyano. a. Especially those where R7 is halo or cyano. b. Especially those where R5, Re, and Rg are hydrogen. i. Most preferably those where R7 is chloro. -19- WO 01/98278 PCT/USO1/13901 4. Any of formulae lb or lc, where Rf is substituted alkyl (preferably a primary-, secondary- or tertiary-amino-substituted lower alkyl, especially primary-amino lower alkyl). a. Especially where the stereogenic center to which Ra and R2’ are attached is 5 of the R configuration, particularly where Ry is hydrogen: i. Especially where Rais lower alkyl (preferably ethyl, i-proyl, c-propyl or t-butyl). 1. Most preferably where Rais i-propyl. b. Especially where R5, Re, and R« are hydrogen. 10 i. Most preferably where R7 is hydrogen, halo (particularly chloro or fluoro), lower alkyl (particularly methyl), substituted lower alkyl, lower alkoxy (particularly methoxy), or cyano 1. Especialy where R7 is chloro.

Most preferred for the compounds, pharmaceutical formulations, methods of 15 manufacture and use of the present invention is formula la incorporating the following combinations and permutations of substituent groups (sub-grouped, respectively, in increasing order of preference): 1. Ri is alkylaryl or substituted alkylaryl (preferably benzyl or substituted benzyl; most preferably benzyl). 20 a. Especially where the stereogenic center to which R2 and R2’ are attached is of the R configuration, where R2’ is hydrogen. i. Particularly those where R2 is lower alkyl .(preferably ethyl, i-propyl, c-propyl or t-butyl). 1. Most preferably those where R2 is i-propyl. 25 b. Especially those where R3 is aryl (preferably phenyl), substituted aryl (preferably lower alkyl-, lower alkoxy- and/or halo-substituted phenyl), alkylaryl (preferably benzyl and phenylviny), alkylheteroaryl, oxaalkylaiyl (preferably phenoxy lower alkyl), oxaalkylheteroaiyl, substituted alkylaryl (preferably substituted benzyl and substituted phenylviny), substituted 30 alkylheteroaryl, substituted oxaalkylaryl (preferably substituted phenoxy lower alkyl), or substituted oxaalkylheteroaryl. - 20 - WO 01/98278 PCT/USO1/13901 i. Particularly those where R3 is aryl, substituted aryl, lower alkylaryl, substituted lower alkylaryl, oxa(lower)alkylaryl. 1. Most preferably those where R3 is methyl-and/or halo-substituted phenyl. 5 c. Especially those where R4 is substituted alkyl (preferably a primary-, secondary- or tertiary-amino-substituted lower alkyl). i. Particularly those where R» is a primary-amino-substituted lower alkyl. 1. Most preferably those where R4 is 3-amino-n-propyl. 10 d. Especially those whereRj, R, R? and R« are chosen fiom hydrogen, halo (preferably chloro and fluoro), lower alkyl (preferably methyl), substituted lower alkyl, lower alkoxy (preferably methoxy), and cyano. i. Particularly those where Rs, Re, R7 and Re are hydrogen, halo, lower alkyl or cyano. 15 1. Most preferably those where R5, Re and Rg are hydrogen. a. Especially those where R7 is halo or cyano (most preferably chloro). 2. Especially those where R7 is halo or cyano (most preferably chloro).

20 2. Where the stereogenic center to which R2 and R2’ are attached is of the R configuration (preferably where Rj’ is hydrogen). a. Especially those where R2 is lower alkyl (preferably ethyl, i-propyl, c-propyl or t-butyl). i. Most preferably those where Rais i-propyl. 25 b. Especially those where R3 is aryl (preferably phenyl), substituted aryl (preferably lower alkyl-, lower alkoxy-, and/or halo-substituted phenyl), alkylaryl (preferably benzyl andphenylviny), alkylheteroaryl, oxaalkylaryl (preferably phenoxy lower alkyl), oxaalkylheteroaryl, substituted alkylaryl (preferably substituted benzyl and substituted phenylviny), substituted 30 alkylheteroaryl, substituted oxaalkylaryl (preferably substituted phenoxy lower alkyl), or substituted oxaalkylheteroaryl. -21- &lt;· WO 01/98278 PCT/US01/13901 i. Particularly those where R3 is aryl, substituted aryl, lower alkylaryl, substituted lower alkylaryl, oxa(lower)alkylaiyl. 1. Most preferably those where R3 is methyl- and/or halo-substituted phenyl. 5 c. Especially those where R4 is substituted alkyl (preferably a primary-, secondary- or terhary-amino-substituted lower alkyl). i. Particularly those where R4 is a primary-amino-substituted lower allyl. 1. Most preferably those where R4 is 3-amino-n-propyl. 10 d. Especially those where R5, R&amp;amp; R7 and Rj are chosen from hydrogen, halo (preferably chloro and fluoro), lower alkyl (preferably methyl), substituted lower alkyl, lower alkoxy (preferably methoxy), and cyano. i. Particularly those where Rs, Ri, R7 and Rg are hydrogen, halo or lower alkyl. 15 1. Most preferably those where Rj, Ri and Rg are hydrogen. a. Especially those where R7 is halo or cyano (most preferably chloro). 2. Especially those where R7 is halo or cyano (most preferably chloro). 20 3. R3 is aryl (preferably phenyl), substituted aryl (preferably lower alkyl-, lower alkoxy-, and/or halo-substituted phenyl), alkylaryl (preferably benzyl and phenylviny), alkylheteroaiyl, oxaalkylaiyl (preferably phenoxy lower alkyl), oxaalkylheteroaryl, substituted alkylaryl (preferably substituted benzyl and substituted phenylviny), substituted alkylheteroaiyl, substituted oxaalkylaryl y 25 (preferably substituted phenoxy lower alkyl), or substituted oxaalkylheteroaryl. a. Especially those where R3 is aryl, substituted aiyl, lower alkylaryl, substituted lower alkylaryl, oxa(lower)alkylaiyl. i. Most preferably those where R3 is methyl- and/or halo-substituted

phenyL 30 b. Especially those where R4 is substituted alkyl (preferably a primary-, secondary- or tertiaiy-amino-substituted lower alkyl). -22- WO 01/98278 PCT/USO1/13901 i. Particularly those where R4 is a primary-amino-substituted lower alkyl. 1. Most preferably those where Rg is 3-amino-n-propyl. c. Especially those where R5, Rg, R7 and Rg are chosen from hydrogen, halo r 5 (preferably chloro and fluoro), lower alkyl (preferably methyl), substituted lower alkyl, lower alkoxy (preferably methoxy), and cyano. i. Particularly those where Rj, Rg, R7 and Rs are hydrogen, halo, lower alkyl or cyano. 1. Most preferably those where Rs, Rg and Rg are hydrogen. 10 a. Especially those where R7 is halo or cyano (most preferably chloro). 2. Especially those where R7 is halo or cyano (most preferably chloro). 4. R4 is substituted alkyl (preferably a primary-, secondary- or tertiary-amino-15 substituted lower alkyl). a. Particularly those where R4 is a primary-amino-substituted lower alkyl. i. Most preferably those where R4 is 3-amino-n-piopyl. b. Especially those where R5, Rg, R7 and Eg are chosen from hydrogen, halo (preferably chloro and fluoro), lower alkyl (preferably methyl), substituted 20 lower alkyl, lower alkoxy (preferably methoxy), and cyano. i. Particularly those where R5, Rg, R7 and Rg are hydrogen, halo, lower alkyl or cyano. 1. Most preferably those where R5,Rg and Rg are hydrogen. a. Especially those where R7 is halo or cyano (most 25 preferably chloro). 2. Especially those where R7 is halo or cyano (most preferably chloro). 5. Rs, Rg, R7 and Rg are chosen from hydrogen, halo (preferably chloro and fluoro), lower alkyl (preferably methyl), substituted lower alkyl lower alkoxy (preferably 30 methoxy), and cyano. -23- WO 01/98278 PCT/US0V13901 a. Especially those where R5, R&amp;amp; R7 and R« are hydrogen, halo, lower alkyl or cyano. i. Most preferably those where R5, R$ and Rs are hydrogen. 1. Especially those where R7 rs halo or cyano (most preferably* 5 chloro). ii. Especially those where R7 is halo or cyano (most preferably chloro).

Especially preferred for the compounds, pharmaceutical formulations, methods of manufacture and use of the present invention is formula la where Ri is alkylaryl or 10 substituted alkylaryl (particularly benzyl or substituted benzyl), R2 is lower alkyl (particularly i-propyl), R2’ is hydrogen, R3 is substituted aryl (particularly methyl-and/or halo-substituted phenyl), R4 is substituted alkyl (particularly

3-amino-n-propyl), R5, R« and R$ are hydrogen, and R7 is halo or cyano (particularly chloro), where the stereogenic center to which R2 and R2’ are attached is of the R 15 configuration.

Synthesis, Testing and Use

The compositions of the invention are synthesized as outlined below, utilizing techniques well known in the art. For example, as described in Ager et al., J. of Med. 20 Chem., 20:379-386 (1977), hereby incorporated by reference, quinazolinones can be obtained by acid-catalyzed condensation of N-acylanthranilic acids with aromatic primary amines. Other processes for preparing quinazolinones are described in U.S. Patent applications 5,783,577, 5,922,866 and 5,187,167, all of which are incorporated by reference. 25 The compositions of the invention may be made as shown in Figures 1,2,4 and 5. Compounds of formulae Id are made in analogous fashion to Figure 1, except that the acyl halide in the final step is replaced by an alkyl halide.

Once made, the compositions of the invention find use in a variety of applications. As will be appreciated by those in the art, mitosis maybe altered in a variety of ways; that 30 is, one can affect mitosis either by increasing or decreasing the activity of a component in the mitotic pathway. Stated differently, mitosis may be affected (e.g., -24- WO 01/98278 PCT/USO1/13901 disrupted) by disturbing equilibrium, either by inhibiting or activating certain components. Similar approaches may be used to alter meiosis.

In a preferred embodiment, the compositions of the invention are used to modulate mitotic spindle formation, thus causing prolonged cell cycle arrest in mitosis. By 5 "modulate" herein is meant altering mitotic spindle formation, including increasing and decreasing spindle formation. By "mitotic spindle formation" herein is meant organization of microtubules into bipolar structures by mitotic kinesins. By “mitotic spindle dysfunction" herein is meant mitotic arrest and monopolar spindle formation.

The compositions of the invention are useful to bind to and/or modulate the activity of 10 a mitotic kinesin, KSP. In a preferred embodiment, the KSP is human KSP, although KSP kinesins from other organisms may also be used. In this context, modulate means either increasing or decreasing spindle pole separation, causing malformation, i.e., splaying, of mitotic spindle poles, or otherwise causing morphological perturbation of the mitotic spindle. Also included within the definition of KSP for 15 these purposes are variants and/or fragments of KSP. See U.S. Patent Application "Methods of Screening for Modulators of Cell Proliferation and Methods of Diagnosing Cell Proliferation States", filed Oct 27,1999 (U.S. Serial Number 09/428,156), hereby incorporated by reference in its entirety. In addition, other mitotic kinesins may be used in the present invention. However, the compositions of 20 the invention have been shown to have specificity for KSP.

For assay of activity, generally either KSP or a compound according to the invention is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g., a microtiter plate, an array, etc.). The insoluble support may be made of any composition to which the compositions can be bound, is readily separated from 25 soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports maybe solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, Teflon™, etc. Microtiter plates and arrays 30 are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner -25- WO 01/98278 PCT/USO1/13901 of binding of the composition is not crucial so long as it is compatible with die reagents and overall methods of the invention, maintains the activity of the composition and is nondifiusable. Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation 5 sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of die protein or agent, excess unbound material is removed by washing. The sample receiving areas may then he blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety. 10 The antimitotic agents of the invention may be used on their own to modulate the activity of a mitotic kinesin, particularly KSP. In this embodiment, die mitotic agents of the invention are combined with KSP and the activity of KSP is assayed. Kinesin activity is known in die art and includes one or more kinesin activities. Kinesin activities include the ability to affect ATP hydrolysis; microtubule binding; gliding 15 and polymerization/depolymerization (effects on microtubule dynamics); binding to other proteins of the spindle; binding to proteins involved in cell-cycle control; serving as a substrate to other enzymes; such as kinases or proteases; and specific ldnesin cellular activities such as spindle pole separation.

Methods of performing motility assays are well known to those of skill in the art 20 [See e.g., Hall, et al. (1996), Biophys. J., 71:3467-3476, Turner et al., 1996, AnaL Biochem. 242 (l):20-5; Gittes et al., 1996, Biophys. J. 70(1): 418-29; Shirakawa et al., 1995, J. Exp. BioL 198:1809-15; Winkelmann et al., 1995, Biophys. J. 68:2444-53; Winkelmann et al., 1995, Biophys. J. 68:72S.]

Methods known in die art for determining ATPase hydrolysis activity also can be 25 used. Preferably, solution based assays are utilized. U.S. application 09/314,464, filed May 18,1999, hereby incorporated by reference in its entirety, describes such assays. Alternatively, conventional methods are used. For example, Pj release from kinesin can be quantified. In one preferred embodiment the ATPase hydrolysis activity assay utilizes 0.3 M PCA (perchloric acid) and malachite green reagent (8.27 30 mM sodium molybdate H, 0.33 mM malachite green oxalate, and 0.8 mM Triton X-l 00). To perform the assay, 10 pL of reaction is quenched in 90 pL of cold 0.3 M -26- WO 01/98278 PCT/US01/13901 PCA. Phosphate standards are used so data can be converted to mM inorganic phosphate released. When all reactions and standards have been quenched in PCA, 100 pL of malachite green reagent is added to the relevant wells in e.g., a microtiter plate. The mixture is developed for 10-15 minutes and die plate is read at an 5 absorbance of 650 nm. If phosphate standards were used, absorbance readings can be converted to mM Pi and plotted over time. Additionally, ATPase assays known in the art include the luciferase assay. ATPase activity of kinesin motor domains also can be used to monitor the effects of modulating agents. In one embodiment ATPase assays of kinesin are perfonned in the 10 absence of microtubules. In another embodime^ the ATPase assays are perfonned in the presence of microtubules. Different types of modulating agents can be detected in the above assays. In a preferred embodiment, the effect of a modulating agent is independent of the concentration of microtubules and ATP. In another embodiment, the effect of the agents on kinesin ATPase can be decreased by increasing the 15 concentrations of ATP, microtubules or both. In yet another embodiment, the effect of the modulating agent is increased by increasing concentrations of ATP, microtubules or both.

Agents that modulate the biochemical activity of KSP in vitro may then be screened in vivo. Methods for such agents in vivo include assays of cell cycle distribution, cell 20 viability, or the presence, morphology, activity, distribution, or amount of mitotic spindles. Methods for monitoring cell cycle distribution of a cell population, for example, by flow cytometry, are well known to those skilled in the art, as are methods for determining cell viability. See for example, U.S. Patent Application "Methods of Screening for Modulators of Cell Proliferation and Methods of Diagnosing Cell 25 Proliferation States,” filed Oct 22,1999, serial number 09/428,156, hereby incorporated by reference in its entirety.

In addition to the assays described above, microscopic methods for monitoring spindle formation and malformation are well known to those of skill in the art (see, e.g., Whitehead and Rattner (1998), J. Cell Sci. 111:2551-61; Galgio et al, (1996) J. Cell 30 bioL, 135:399-414). -27- WO 01798278 PCT/USO1/13901

The compositions of the invention inhibit the KSP kinesin. One measure of inhibition is IC50, defined as fee concentration of fee composition at which fee activity of KSP is decreased by fifty percent Preferred compositions have ICso's of less than about 1 mM, wife preferred embodiments having ICso's of less than about 100 μΜ, wife more 5 preferred embodiments having ICso's of less than about 10 μΜ, wife particularly preferred embodiments having ICso's of less than about 1 μΜ, and especially preferred embodiments having ICso's of less than about 100 nM, and wife fee most preferred embodiments having ICso's of less than about 10 nM. Measurement ofICso is done using an ATPase assay. 10 Another measure of inhibition is Kj. For compounds wife ICso's less than 1 μΜ, fee Ki or Kd is defined as fee dissociation rate constant for fee interaction of fee quinazolinone with KSP. Preferred compounds have Kj's of less than about 100 μΜ, wife preferred embodiments having K,'s of less than about 10 μΜ, and particularly preferred embodiments having Kj's of less than about 1 μΜ and especially preferred 15 embodiments having Kj's of less than about 100 nM, and wife fee most preferred embodiments having K/s of less than about 10 nM. The Kj for a compound is determined from fee IC50 based on three assumptions. First, only one compound molecule hinds to the enzyme and there is no cooperativity. Second, fee concentrations of active enzyme and fee compound tested are known (i.e., there are no 20 significant amounts of impurities or inactive forms in fee preparations). Third, fee enzymatic rate of fee enzyme-inhibitor complex is zero. The rate (i.e., compound concentration) data are fitted to fee equation: V = VnffiXE0 I- (Ep + Ip + Kd)- V(Eo + Ip + Κά/-4ΕρΙρ 2En

Where V is fee observed rate, V,^ is the rate of the free enzyme, Io is the inhibitor 25 concentration, Eo is fee enzyme concentration, and Kj is fee dissociation constant of fee enzyme-inhibitor complex. -28- WO 01/98278 PCT/US01/13901

Another measure of inhibition is GI50, defined as the concentration of the compound that results in a decrease in the rate of cell growth by fifty percent Preferred compounds have GIso's of less than about 1 mM. The level of preferability of

embodiments is a function of their GI50: those having GIso's of less than about 20 pM 5 are more preferred; those having GIso's of 10 μΜ more so; those having GI50 of less than about 1 μΜ more so; those having GIso's of 100 nM more so; those having GI50 of less than about 10 nM even more so. Measurement of GI50 is done using a cell proliferation assay.

The compositions of the invention are used to treat cellular proliferation diseases. 10 Disease states which can be treated by the methods and compositions provided herein include, hut are not limited to, cancer (further discussed below), autoimmune disease, arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures, including, but not limited to, surgery, angioplasty, and the like. It is appreciated that in some cases the cells may not be in a hyper or hypo proliferation 15 state (abnormal state) and still require treatment. For example, during wound healing, the cells may be proliferating "normally", hut proliferation enhancement may be desired. Similarly, as discussed above, in the agriculture arena, cells may be in a "normal" state, but proliferation modulation may be desired to enhance a crop by directly enhancing growth of a crop, or by inhibiting the growth of a plant or organism 20 which adversely affects the crop. Thus, in one embodiment, the invention herein includes application to cells or individuals afflicted or impending affliction with any one of these disorders or states.

The compositions and methods provided herein are particularly deemed useful for the treatment of cancer including solid tumors such as skin, breast, brain, cervical 25 carcinomas, testicular carcinomas, etc. More particularly, cancers that may he treated by the compositions and methods of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, 30 adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal.· esophagus -29- WO 01/98278 PCT/USO1/13901 (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Kaiposi's sarcoma, leiomyoma, 5 hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal 10 carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bope: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, 15 malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain 20 (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical i dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous 25 cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma], fallopian tubes (carcinoma); Hematologic: blood (myeloid 30 leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant -30- WO 01/98278 PCT/US01/13901 melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma. Thus, the term “cancerous cell" as provided herein, includes a cell afflicted by any one of the above-identified conditions. 5 Accordingly, fee compositions of fee invention are administered to cells. By "administered" herein is meant administration of a therapeutically effective dose of fee mitotic agents of fee invention to a cell either in cell culture or in a patient. By "therapeutically effective dose" herein is meant a dose that produces fee effects for which it is administered. The exact dose will depend on fee purpose of the treatment, 10 and will he ascertainable by one skilled in fee art using known techniques. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of fee condition may be necessary, and will be ascertainable wife routine experimentation by those skilled in fee art. By "cells" herein is meant almost any cell 15 in which mitosis or meiosis can he altered. A "patient" for fee purposes of the present invention includes both humans and other animals, particularly mammals, and other organisms. Thus the methods are applicable to both human therapy and veterinary applications. In fee preferred embodiment fee patient is a mammal, and in fee most preferred embodiment the patient is human. 20 Mitotic agents having fee desired pharmacological activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon fee manner of introduction, fee compounds may be formulated in a variety of ways as discussed below. The concentration of therapeutically active compound in fee formulation may vary from about 0.1-100 wt.%. The agents may be administered 25 alone or in combination wife other treatments, i.e., radiation, or other chemotherapeutic agents.

In a preferred embodiment, fee pharmaceutical compositions are in a water soluble form, such as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to 30 those salts that retain the biological effectiveness of the free bases and that are not -31- WO 01/98278 PCT/US01/13901 biologically or otherwise undesirable, 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, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric 5 acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are 10 the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primaiy, 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, and 15 ethanolamine.

The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically- 20 active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents. The pharmaceutical compositions may also include one or more of the following: carrier proteins such as 25 serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, com and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.

The administration of the mitotic agents of the present invention can be done in a 30 variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, -32- WO 01/98278 PCT/USO1/13901 intramuscularly, intrapulmonaiy, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the anti-mitotic agents may be directly applied as a solution or spray.

To employ the compounds of the invention in a method of screening for compounds 5 that bind to KSP kinesin, the KSP is bound to a support, and a compound of the invention (which is a mitotic agent) is added to the assay. Alternatively, the compound of the invention is bound to the support and KSP is added. Classes of compounds among which novel binding agents may be sought include specific antibodies, non-natural binding agents identified in screens of chemical libraries, 10 peptide analogs, etc. Of particular interest are screening assays for candidate agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like. 15 The determination of the binding of the mitotic agent to KSP may be done in a number of ways. In a preferred embodiment, the mitotic agent (the compound of the invention) is labeled, for example, with a fluorescent or radioactive moiety and binding determined directly. For example, this may be done by attaching all or a portion of KSP to a solid support, adding a labeled mitotic agent (for example a 20 compound of the invention in which at least one atom has been replaced by a detectable isotope), washing off excess reagent, and determining whether the amount of the label is that present on the solid support Various blocking and washing steps may be utilized as is known in the art

By "labeled" herein is meant that the compound is either directly or indirectly labeled 25 with a label which provides a detectable signal, e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles such as magnetic particles, chemiluminescent tag, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule 30 which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal. -33- WO 01/98278 PCT/US01/13901

In some embodiments, only one of the components is labeled. For example, the kinesin proteins may be labeled at tyrosine positions using t25I, or with fluorophores.

Alternatively, more than one component maybe labeled with different labels; using I2SI for the proteins, for example, and a fluorophor for the mitotic agents. 5 The compounds of the invention may also be used as competitors to screen for additional drug candidates. "Candidate bioactive agent" or "drug candidate” or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for bioactivity. They may be capable of directly or indirectly altering the cellular 10 proliferation phenotype or the expression of a cellular proliferation sequence, including both nucleic acid sequences and protein sequences. Ia other cases, alteration of cellular proliferation protein binding and/or activity is screened. Screens of this sort may be performed either in the presence or absence of microtubules. In the case where protein binding or activity is screened, preferred embodiments exclude 15 molecules already known to bind to that particular protein, for example, polymer structures such as microtubules, and energy sources such as ATP. Preferred embodiments of assays herein include candidate agents which do not bind the cellular proliferation protein in its endogenous native state termed herein as "exogenous" agents. In another preferred embodiment, exogenous agents further exclude 20 antibodies to KSP.

Candidate agents can encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly 25 hydrogen bonding and lipophilic binding, and typically include at least an amine, carbonyl, hydroxyl, ether, or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules 30 including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. -34- WO 01/98278 PCT/US01/13901

<img img-format="tif" img-content="drawing" file="IL153554AD00025.tif" id="idf0005" />

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, 5 libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents maybe subjected to directed or random chemical modifications, such as acylation, alkylation, 10 esterification, amidification to produce structural analogs.

Competitive screening assays may be done by combining KSP and a drug candidate in a first sample. A second sample comprises a mitotic agent, KSP and a drug candidate. This may be performed in either the presence or absence of microtubules. The binding of the drug candidate is determined for both samples, and a change, or 15 difference in binding between the two samples indicates the presence of an agent capable of binding to KSP and potentially modulating its activity. That is, if the binding of the drug candidate is different in the second sample relative to the first sample, the drug candidate is capable of binding to KSP.

In a preferred embodiment, the binding of the candidate agent is determined through 20 the use of competitive binding assays. In this embodiment, the competitor is a binding moiety known to bind to KSP, such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the candidate agent and the binding moiety, with the binding moiety displacing the candidate agent 25 In one embodiment, the candidate agent is labeled. Either the candidate agent, or the competitor, or both, is added first to KSP for a time sufficient to allow binding, if present. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40°C.

Incubation periods are selected for optimum activity, but may also be optimized to 30 facilitate rapid high throughput screening. Typically between 0.1 and 1 hour will be -35- WO 01/98278 PCT/US01/13901 sufficient Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.

In a preferred embodiment, fee competitor is added first followed by fee candidate 5 agent Displacement of fee competitor is an indication fee candidate agent is binding to KSP and feus is capable of binding to, and potentially modulating, the activity of KSP. In this embodiment, either component can be labeled. Thus, for example, if fee competitor is labeled, fee presence of label in fee wash solution indicates displacement by fee agent Alternatively, if fee candidate agent is labeled, the 10 presence of fee label on the support indicates displacement

In an alternative embodiment, the candidate agent is added first with incubation and washing, followed by fee competitor. The absence of binding by the competitor may indicate the candidate agent is bound to KSP with a higher affinity. Thus, if fee candidate agent is labeled, the presence of fee label on the support, coupled with a 15 lack of competitor binding, may indicate fee candidate agent is capable of binding to KSP.

It may be of value to identify fee binding site of KSP. This can be done in a variety of ways. In one embodiment once KSP has been identified as binding to fee mitotic agent KSP is fragmented or modified and the assays repeated to identify the necessary 20 components for binding.

Modulation is tested by screening for candidate agents capable of modulating the activity of KSP comprising the steps of combining a candidate agent wife KSP, as above, and determining an alteration in the biological activity of KSP. Thus, in this embodiment fee candidate agent should both bind to KSP (although this may not be 25 necessary), and alter its biological or biochemical activity as defined herein. The methods include both in Yitro screening methods and in vivo screening of cells for alterations in cell cycle distribution, cell viability, or for the presence, moipohology, activity, distribution, or amount of mitotic spindles, as axe generally outlined above.

Alternatively, differential screening may be used to identify drug candidates that bind 30 to fee native KSP, but cannot bind to modified KSP. -36- WO 01/98278 PCT/US01/13901

Positive controls and negative controls may be used in the assays. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non- 5 specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound. A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc which may be used 10 to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. Ihe mixture of components may be added in any order that provides for the requisite binding. 15 The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes. All references cited herein are incorporated by reference in their entirety.

20 EXAMPLES

Abbreviations and Definitions

The following abbreviations and terms have the indicated meanings throughout:

Ac = acetyl ' 25 BNB = 4-bromomethyl-3-nitrobenzoic acid

Boc = t-butyloxy carbonyl

Bu = butyl c- = cyclo CBZ = carbobenzoxy=ben2yloxycafbonyl 30 DBU = diazabicyclo[5.4.0]undec-7-ene DCM = dichloromethane = methylene chloride = CH2CI2 DCE = dichloroethylene DEAD = diethyl azodicarboxylate DIC = diisopropylcarbodiimide -37- WO 01/98278 PCT/USO1/13901

DDBA

DMAP

DMF

DMSO 5 DVB EEDQ Et

Fmoc

GC

10 HATU

HMDS HOAc HOBt 15 Me mesyl MTBE NMO PEG . 20 Ph PhOH PfP PPTS Py 25 PyBroP rt sat=d . 6-t-

30 TBDMS TES TFA THF TMOF 35 TMS tosyl Trt = Ν,Ν-diisopropylethyl amine = 4-N,N~dimethylaininopyridme = N,N-dimethylformamide = dimethyl sulfoxide = 1,4-divinyIbenzene = 2-ethoxy-l -ethoxycarbonyl-1,2-dihydroquinoline = ethyl = 9-fhjorenylmethoxycarbonyl = gas chromatography = O-(7-Azahenzotriazol-l-yl)-l, 1,3,3-tetramethyluronium hexafluorophosphate = hexamethyldisilazane = acetic acid = hydroxybenzotriazole = methyl = methanesulfonyl = methyl t-butyl ether = N-methylmoipholine oxide = polyethylene glycol = phenyl = phenol = pentafluorophenol = pyridinium p-toluenesulfonate = pyridine . = bromo-tris-pyrrolidincnphosphonium hexafluorophosphate = room temperature = saturated = secondary = tertiary = t-butyldimethylsilyl = triethylsilane = trifhioroacetic acid = tetrahydrofuran = trimethyl orthoformate = trimethylsilyl = p-toluenesulfonyl = triphenylmethyl

Example 1 40 Synthesis of Compounds

The general synthesis is shown in Figures 1 and 2.

Step 1: N-butyryl anthranilic acid.

To a three-necked, 500 mL round-bottom flask equipped with a thermometer, -38- WO 01/98278 PCT/US01/13901 dropping funnel, and an efficient magnetic stir bar, was added anthranilic acid (1) (0.5 mole, 68.5 g) and dimethyl formamide (250 mL). To this solution was added butyryl chloride (0.55 mole, 57.1 mL) dropwise at such a rate that the temperature of the mixture did not rise above 40°C. The suspension was stirred vigorously at room 5 temperature for at least an additional 3 h. The mixture was poured into water (2000 mL) and stirred for another 1 h. The precipitated product was collected by filtration, washed with cold water, and dried under reduced pressure over P2O5, yielding compound 2 (67.3 g, 65%).

Step 2: 2-Propyl-3.1-f4H1benzoxazin-4-oiie. 10 Compound 2 (51.8 g, 025 mole) was dissolved in acetic anhydride (180.mL) in a 500 mL round-bottom flask equipped with a magnetic stir bar, a Claisen-distillation bead (with vacuum inlet) and a thermometer. The flask was placed in an oil bath and slowly heated to 170-180°C with vigorous stirring. The acetic acid produced was slowly distilled off under atmospheric pressure. Monitoring the head temperature of 15 the distillation unit was used to follow the progress of the transformation. The reaction mixture was then cooled to 60 °C and the excess of acetic anhydride removed by distillation under reduced pressure (ca. 20 mm Hg). The residue was afterward cooled and the product crystallized. The product was triturated with n-hexane (75 mL) and isolated by filtration to yield 2-propyl-3,l-[4H]benzoxazin-4-one (3) (29.3 g, 20 62%). The above procedure gave compound 3 sufficiently pure to use directly in the next step.

Step 3: 2-Propvl-3-benzvlqumazolin-4-one·

Compound 3 (28.4 g, 0.15 mole) and benzylamine (17.5 mL, 0.16 mole) were refluxed in chloroform (50 ml) in a one-neck 250 mL round-bottom flask for 6 h. 25 After complete consumption of compound 3, the chloroform was evaporated under reduced pressure. Ethylene glycol (100 mL) and NaOH pellets (0.60 g) were added to the residue and the flask equipped with a Claisen-distillation head and a magnetic stir bar. The flask was immersed in an oil bath and reheated to 130-140 °C bath temperature with vigorous stirring and maintained there for 5 h while the water 30 produced was removed by distillation. After completion of the reaction, the clear solution was allowed to cool to room temperature and kept overnight to precipitate the -39- WO 01/98278 PCT/D SO1/13901

<img img-format="tif" img-content="drawing" file="IL153554AD00026.tif" id="idf0006" />

product The pH of the suspension was adjusted to 7-8 by adding 3% aq. HCI, fee crystals were filtered off and washed wife cold water, and then recrystallized from isopropanol (or alternatively from acetone) to provide fee compound, 2-propyl-3-benzylquinazolin-4-one (compound 4) (28.0 g, 67%). 5 Step 4: 2-{I'-bromopropvl)-3-benzvlquinazolin-4-one.

To a three-neck 250 mL round-bottom flask equipped wife a thermometer, dropping funnel, and efficient magnetic stir bar was added compound 4 (27.8 g, 0.10 mole), anhydrous sodium acetate (10.0 g) and glacial acetic acid (130 mL). Bromine (16.0 g, 0.10 mole) dissolved in acetic acid (10 mL) was added dropwise to the above solution 10 at 40 °C for 1-2 E After addition was complete, fee mixture was poured into water (1500 mL) and stirred for 1-2 h at room temperature. The precipitated product, 2-(1-bromopropyl)-3-benzylquinazolin-4-one (5) was isolated by filtration, washed wife warm water to remove traces of acetic acid, and rinsed with a small amount of isopropanoL Drying yielded compound 5 (33.0 g, 92%). 15 Step 5: 2-il'-(N,N-diinethvletovienediammo')piopylT3-beiizylquinazolin-4-oiie·

Compound 5 (10.7 g, 0.03 mole) and Ν,Ν-dimefeylefeylenediamine (6.6 mL, 0.06 mole) were dissolved in abs. ethanol (60 mL) and heated at reflux for 6 h. After completion of fee reaction, fee solvent was evaporated under reduced pressure. The residue was dissolved in dichloromethane (150 mL) and washed wife 3% aq. NaOH 20 solution (ca. 10-20 mL). The organic layer was dried over MgSCb and evaporated to dryness under reduced pressure. The remaining oily product was purified by flash chromatography on a short silica gel pad using an eluent of CHCl3-MeOH-aq.NH3, 90:10:0.1, to give the desired compound (5), 2-[F-{N,N- dimethylefeylenediamino)propyl]-3-benzylquinazolin-4-one (6) (6.0 g, 55%). 25 Step 6: 2-n'-fN-4-fluorobenzovD-&lt;N.N-dimethvlefevlenedianuno,)propvH-3- benzylquinazolin-4-one. A stock solution of compound 5 (1.822 g, 5.0 mmol) was prepared in HPLC grade CHCI3 (0.5 mL). A stock solution of p-flurobenzoyl chloride (160.2 mg, 1 mmol) in HPLC grade 1,2-dichloroethane (2.0 mL) was prepared in a 2.0 mL volumetric flask. 30 A third solution of triethylamine (2.0 mL of 0.5 M) was prepared in HPLC grade 1,2- dichlorethane. A 100 pL aliquot of each solution was pipetted into a glass reaction -40- WO 01/98278 PCT/US01/13901 vessel using a Beckman Biomet 2000 automated liquid dispenser. The reaction mixture was shaken using a mechanical shaker, sonicated in an ultrasonic water bath, and then incubated overnight at room temperature. The mixture was diluted in CHCI3 (300 pL) and washed with 5% aqueous NaHCO3 and water. The solvent was removed 5 in vacuo to provide compound 6 (65%). The purity of the compound was analyzed by TLC eluted with GLCb-ethanol-concentratcd aqueous NH3&gt; 100:10:1.

Examples 2 and 3

<img img-format="tif" img-content="drawing" file="IL153554AD00027.tif" id="idf0007" />

10 All anhydrous solvents were purchased from Aldrichchemical company in SureSeal® containers. Most reagents were purchase from Aldrich Chemical Company. Abbreviations: DCM, dichloromethane; DIEA, NJSf-diisopropylethylamine; DMF, Ν,Ν-dimethylformamide; TES, triethylsilane; TFA, trifluoroacetic acid. Array synthesis was conducted in 15 x 75 mm glass round bottom screw-cap vials contained 15 in a 4 x 6 array aluminum synthesis block, sealed with a Teflon-lined rubber membrane. Reagents were added and aqueous extractions performed with single or multichannel pipettors. Filtrations were performed using Whatman/Polyfiltronics 24 well, 10 mL filtration blocks. Evaporation of volatile materials from the array was performed with a Labconco Vortex-Evaporator or by sweeping with a 4 x 6 nitrogen 20 manifold. -41- WO 01/98278 PCT/US01/13901

Example 2 (solid phase synthesis of a single compound) STEP 1: 1,3-Diaminopropane trityl resin (Novabiochem, 1.2 mmol/g) (0.20 g, 0.24 mmol) was weighed into a screw-cap vial and 3 mL of a 1:1 mixture of DMF and chloroform was added. DIEA (0.130 mL, 0.72 mmol) and 2-(1 ’-bromopropyl)-3- 5 benzylquinazolin-4-one (from Example 1) (0.188 g, 0.48 mmol) were added. The vial was sealed, heated to 70 °C and shaken overnight The resin was filtered and washed (3 x DCM, 2 x MeOH, 1 x DCM, 2 x ether) and dried under vacuum. A 27 mg aliquot of resin was treated with 5:5:90 TFA:TES:DCM for 15 min and the mixture was filtered and evaporated, resulting in 8 mg (64% yield) of the quinazolinone- 10 diamine intermediate. LCMS analysis showed &gt;80 % purity.

STEP 2: The resin from Step 1 was swelled in 3 mL of DCM. DIEA (0.130 mL, 0.72 mmol) and 4-bromobenzyl bromide (0.12 g, 0.48 mmol) were added. The vial was sealed and shaken overnight. LCMS analysis of a cleaved aliquot revealed an approximate 1:1 mixture of starting material and product Another 0.130 mL of DIEA 15 and 0.12 g of 4-bromobenzyl bromide were added and the mixture was shaken at 70 °C for 8 h. The resin was filtered, washed (as above), and dried under vacuum. STEP 3: The resin from Step 2 was twice shaken for 30 min with 5:5:90 TFA:TES:DCM and filtered. The filtrates were combined and evaporated, yielding 140 mg of an orange oil. This material was purified by reverse phase preparative 20 HPLC (acetonitrile-water gradient) to provide 27 mg (17% for 3 steps) of file mono-TFA salt.

Example 3 (combinatorial synthesis of multiple compounds) STEP 1: 1,2-Diaminoethane trityl resin (Novabiochem, 0.95 mmol/g) (200 g, 1.9 mmol) and 1,3-Diaminopropane trityl resin (Novabiochem, 1.14 mmol/g) (2.0 g, 2.28 25 mmol) were each placed in different 10 mL polypropylene fritted tubes (Bio-Rad). To each were added 4 mL of DMF, 4 mL of chloroform, 3 eq. of DIEA (1.0 mL and 1.2 mL, respectively) and 2 eq. of 2-(r-bromopropyl)-3-benzylquinazolin-4-one (from Example 1) (1.5 g and 1.8 g, respectively). The mixtures were shaken at 70 °C overnight. Each mixture was washed (3 x DCM, 2 x MeOH, 1 x DCM, 2 x ether) and 3 0 dried under vacuum. Analysis of a cleaved aliquot revealed the presence of the appropriate quinazolinone-diamine for each in &gt;90 % purity. -42- WO 01/98278 PCT/US01/13901 STEP 2: Tlie quinazolinone ethyl-diamine resin (105 mg, 0.10 mmol) was placed into each of tlie vials in the first 2 rows of the array, and the quinazolinone propyl-diamine resin (88 mg, 0.10 mmol) was placed into each vial of the last 2 rows ofthe array. To each vial was added DIEA (0.131 mL, 0.75 mmol). Into each vial ofthe first 2 rows

5 of die array was added a different amine, and the additions were repeated for the last two rows of the array. The reaction block was shaken at 70 °C overnight Liquid was removed from each vial by multichannel pipette using fine-pointed gel-well tips, and the resins were washed (2 x DCM, 1 x MeOH, 1 x DCM) and dried under vacuum. STEP 3: To each vial of the array was added 2 mL of a 10:5:85 TFA:TES:DCM 10 solution. The reaction block was shaken for 45 min and the mixtures were transferred to a filter block, filtered, and washed twice with 0.75 mL DCM. The solutions were evaporated to yield yellow-to-red oils. These thick oils were triturated twice with ether, dissolved in DCM and treated with 4 M HCI in dioxane to provide the HCI salts (unknown number of salts per compound) as tan-to-wliite powdery or amorphous 15 solids. Analysis by LCMS showed all to be &gt;75 % pure.

Examples 4-6

Six racemic quinazolinones were separated into their enantiomers by chiral chromatography. The chiral chromatography of three of these compounds is 20 described below:

<img img-format="tif" img-content="drawing" file="IL153554AD00028.tif" id="idf0008" />

25 Column - Cbiralpak AD, 250 x 4.6 mm (Diacel Inc.). Sample - 0.5 mg/mL in EtOH. -43- WO 01/98278 PCT/US01/13901

Conditions -15 min at 60% EtOH in Hexane, enantiomer 1 elutes at 4.5 min, enantiomer 2 elutes at 4.9 min.

<img img-format="tif" img-content="drawing" file="IL153554AD00029.tif" id="idf0009" />

Column - Chiralcel OJ, 250 x 4.6 mm (Diacel Inc.). Sample - 0.5 mg/mL in EtOH. Conditions -15 min at 10% EtOH in Hexane, (R)-eriantiomer elutes at 8.4 min, (S)- &lt; 10 enantiomer elutes at 9.6 min.

Example 6

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15

Column - Chiralpak AD, 250 x 4.6 min (Diacel Inc.). Sample — 0.5 mg/mL in EtOH. Conditions - 15 min at 70% EtOH in Hexane, enantiomer 1 elutes at 6.5 min, enantiomer 2 elutes at 8.8 min. 20 The table below depicts the IC50 activity of the racemate and the enantiomers of three other compounds separated as above. In all three cases, one enantiomer was significantly more potent than the other. By independent chiral synthesis, it appears that the more active enantiomer is the R enantiomer. -44- WO 01/98278 PCT/US01/13901

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-45- WO 01/98278 PCT/US01/13901

Examples 7 and 8

The following two compounds were synthesized as single enantiomers by the route shown in Figure 4. The data indicate that the more active enantiomer is the R 5 enantiomer. Κ,(μΜ) Κι(μΜ) S enantiomer R enantiomer oO oX 2 &lt;0.1 •9 0 Br &gt;0.5 &lt;0.05

Example 9

Chiral Resolution by Recrystallization with Tartaric Acid 10

Intermediate A, prepared in Example 1, can be converted to an intermediate B, which, upon resolution, provides an alternative to the first five steps shown in Figure 4. The process is shown in the scheme below: -46- WO 01/98278 PCT/US01/13901

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A' B

The R enantiomer of B can he crystallized selectively by heating a mixture of B with 1.1 equivalents of D-tartaric acid in a mixture of isopropanol and methanol and then 5 letting the mixture return to room temperature.

Example 9: X = C1,R=H

Racemic intermediate B (1.5 g), dissolved in 100 mL of boiling isopropanol, was mixed with 0.8 g of Z)-tartaric acid in 100 mL of boiling methanol. The mixture was 10 allowed to slowly reach room temperature. After standing overnight, the solid was removed by filtration and rinsed with ethyl acetate and hexanes, and allowed to air dry. The dried solid (0.8 g) was then dissolved in a boiling mixture of 50 mL of isopropanol and 50 mL of methanol and allowed to slowly cool to room temperature. After standing overnight, the resulting solid was removed by filtration and rinsed with 15 ethyl acetate and hexanes, and allowed to air dry. The dried solid was then stirred . with saturated sodium bicarbonate for 30 min and extracted with ethyl acetate. The organics were dried (MgSCU), filtered and evaporated to dryness. The resulting clear oil weighed 345 mg. Chiral purity of &gt;95% was determined by conversion of a portion to the S-Mosher amide and examination of the product by ’HNMR. 20 The enantiomerically pure compounds below were prepared, according to the remaining steps in Figure 4, from material resulting from the procedure described above using both D- and L-tartaric acid. -47- WO 01/98278 PCT/US01/13901

Racemic 1 C60 (uM) R Isomer ICh, (uM) S Isomer ICk, (uM) οψ ^νη2 &lt;0.05 &lt;0.05 &gt;0.5 ιΊ) τ ch3

Example 10

Induction of Mitotic Arrest in Cell Populations Treated with a Quinazolinone KSP 5 Inhibitor FACS analysis to determine cell cycle stage by measuring DNA content was performed as follows. Skov-3 cells (human ovarian cancer) were split 1:10 for plating in 10cm dishes and grown to subconfluence with RPMI1640 medium containing 5% fetal bovine serum (FBS). The cells were then treated with either lOnM paclitaxel, 10 400nM quinazolinone 1,200nM quinazolinone2, or 0.25% DMSO (vehicle for compounds) for 24 hours. Cells were then rinsed off the plates with PBS containing 5mM EDTA, pelleted, washed once in PBS containing 1% FCS, and then fixed overnight in 85% ethanol at 4°C. Before analysis, the cells were pelleted, washed once, and stained in a solution of 10pg propidium iodide and 250μg of ribonuclease 15 (RNAse) A per milliliter at 37°C for half an hour. Flow cytometry analysis was performed on a Becton-Dickinson FACScan, and data from 10,000 cells per sample was analyzed with Modfit software. -48- WO 01/98278 PCT/USO1/13901

The quinazolinone compounds, as well as the known anti-mitotic agent paclitaxel, caused a shift in the population of cells from a G0/G1 cell cycle stage (2n DNA content) to a G2/M cell cycle stage (4n DNA content). Other compounds of this class were found to have similar effects. 5 Monopolar Spindle Formation following Application of a Quinazolinone KSP Inhibitor

To determine the nature of the G2/M accumulation, human tumor cell lines Skov-3 (ovarian), HeLa (cervical), and A549 (lung) were plated in 96-well plates at densities of 4,000 cells per well (SKOV-3 &amp;amp; HeLa) or 8,000 cells per well (A549), allowed to 10 adhere for 24 hours, and treated with various concentrations of the quinazolinone compounds for 24 hours. Cells were fixed in 4% formaldehyde and stained with antitubulin antibodies (subsequently recognized using fluorescently-labeled secondary antibody) and Hoechst dye (which stains DNA).

Visual inspection revealed that the quinazolinone compounds caused cell cycle arrest 15 in the prometaphase stage of mitosis. DNA was condensed and spindle formation had initiated, but arrested cells uniformly displayed monopolar spindles, indicating that there was an inhibition of spindle pole body separation. Microinjection of anti-KSP antibodies also causes mitotic arrest with arrested cells displaying monopolar spindles.

Inhibition of Cellular Proliferation in Tumor Cell Lines Treated with Quinazolinone 20 KSP Inhibitors.

Cells were plated in 96-well plates at densities from 1000-2500 cells/well of a 96-well plate (depending on the cell line) and allowed to adhere/grow for 24 hours. They were then treated with various concentrations of drug for 48 hours. The time at which compounds are added is considered To. A tetrazolium-hased assay using the reagent 25 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium (MTS) (I.S&gt; Patent No. 5,185,450) (see Promega product catalog #G3580, CeflTiter 96® AQura» One Solution Cell Proliferation Assay) was used to determine the number of viable cells at To and fee number of cells remaining after 48 hours compound exposure. The number of cells remaining after 48 hours was compared to -49- WO 01/98278 PCT/US01/13901 the number of viable cells at the time of drug addition, allowing for calculation of growth inhibition. 10

The growth over 48 hours of cells in control wells that had been treated with vehicle only (0.25% DMSO) is considered 100% growth and the growth of cells in wells with compounds is compared to this. Quinazolinone KSP inhibitors inhibited cell proliferation in human tumor cell lines of the following tumor types: lung (NCI-H460, A549), breast (MDA-MB-231, MCF-7, MCF-7/ADR-RES), colon (HT29, y HCT15), ovarian (SKOV-3, OVCAR-3), leukemia (HL-60(TB), K-562), central nervous system (SF-268), renal (A498), osteosarcoma (U2-OS), and cervical (HeLa). In addition, a mouse tumor line (B16, melanoma) was also growth-inhibited in the presence of the quinazolinone compounds. A Giso was calculated by plotting the concentration of compound in μΜ vs the percentage of cell growth of cell growth in treated wells. The Giso calculated for tire compounds is the estimated concentration at which growth is inhibited by 50% 15 compared to control, i.e., the concentration at which: 100 x [(Treated^ - To) / (Control^ - To)] = 50.

All concentrations of compounds are tested in duplicate and controls are averaged over 12 wells. A very similar 96-well plate layout and Giso calculation scheme is used by the National Cancer Institute (see Monks, et al., J. NatL Cancer Inst 83:757-766 20 (1991)). However, the method by which the National Cancer Institute quantitates cell number does not use MTS, but instead employs alternative methods.

Calculation Of ICjo:

Measurement of a composition's ICso for KSP activity uses an ATPase assay. The following solutions are used: Solution 1 consists of 3 mM phosphoenolpyruvate 25 potassium salt (Sigma P-7127), 2 mM ATP (Sigma A-3377), 1 mM IDTT (Sigma D-9779), 5 μΜ paclitaxel (Sigma T-7402), 10 ppm antifoam 289 (Sigma A-8436), 25 mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM MgC12 (VWR JT400301), and 1 mM EGTA (Sigma E3889). Solution 2 consists of 1 mM NADH (Sigma N8129), 0.2 mg/ml BSA (Sigma A7906), pyruvate kinase 7U/ml, L-lactate dehydrogenase 10 U/ml -50- WO 01/98278 PCT/US01/13901 (Sigma P0294), 100 nM KSP motor domain, 50 pg/ml microtubules, 1 mM DTT (Sigma D9779), 5 μΜ paclitaxel (Sigma T-7402), 10 ppm antifoam 289 (Sigma A-8436), 25 mM Pipes/KOH pH 6.8 (Sigma P6757), 2 mM MgC12 (VWR JT4003-01), and 1 mM EGTA (Sigma E3889). Serial dilutions (8-12 two-fold dilutions) of the 5 composition are made in a 96-well microtiter plate (Coming Costar 3695) using Solution 1. Following serial dilution each well has 50 μΐ of Solution 1. The reaction is started by adding 50 μΐ of solution 2 to each well. This may be done with a multichannel pipettor either manually or with automated liquid handling devices. The microtiter plate is then transferred to a microplate absorbance reader and multiple 10 absorbance readings at 340 nm are taken for each well in a kinetic mode. The observed rate of change, which is proportional to the ATPase rate, is then plotted as a function of the compound concentration. For a standard IC50 determination the data acquired is fit by the following four parameter equation using a nonlinear fitting program (e.g., Grafit 4): 15 y=

Range

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+ Background

Where y is the observed rate and x the compound concentration.

The quinazolinone compounds inhibit growth in a variety of cell lines, including cell lines (MCF-7/ADR-RES, HCT1 5) that express P-glycoprotein (also known as Multidrug Resistance, or MDR4), which conveys resistance to other chemotherapeutic 20 drugs, such as pacilitaxel. Therefore, the quinazolinones are anti-mitotics that inhibit cell proliferation, and are not subject to resistance by overexpression of MDR+ by drug-resistant tumor lines.

Other compounds of this class were found to inhibit cell proliferation, although GI50 values varied. GI5g values for the quinazolinone compounds tested 25 ranged from 200 nM to greater than the highest concentration tested. By this we mean fliat although most of the compounds that inhibited KSP activity biochemically did inhibit cell proliferation, for some, at the highest concentration tested (generally about 20 μΜ), cell growth was inhibited less than 50%. Many of the compounds have Glso -51- WO 01/98278 PCT/US01/13901 values less than 10 μΜ, and several have GIso values less than 1 μΜ. Antiproliferative compounds that have been successfully applied in the clinic to treatment of cancer (cancer chemotherapeutics) have GIso’s that vary greatly. For example, in A549 cells, paclitaxel GIso is 4 nM, doxorubicin is 63 nM, 5-fluorouracil is 1 μΜ, and 5 hydroxyurea is 500 μΜ (data provided by National Cancer Institute, Developmental

Therapeutic Program, http://dtp.nci.nih.gov/). Therefore, compounds that inhibit cellular proliferation at virtually any concentration may be useful. However, preferably, compounds will have GIso values of less than 1 mM. More preferably, compounds will have GIso values of less than 20 μΜ. Even more preferably, 10 compounds will have GIso values ofless than 10 μΜ. Further reduction in GIso values may also be desirable, including compounds with GI50 values ofless than 1 μΜ.

Some of the quinazolinone compounds of the invention inhibit cell proliferation with GI50 values from below 200 nM to below 10 nM.

Example 11 15 Female nude mice weighing approximately 20 g were implanted s.c. by trocar with fragments of human tumor carcinomas harvested from s.c. growing tumors in nude mice host. When the tumors were approximately 77 mg in size, the animals were pair matched into treatment and control groups. Each group containted 8 tumored mice, each of which was ear-tagged and followed individually throughout the 20 experiment. Initial doses (10 mL/kg of a 66 mM Citrate buffer, pH 5.0 / 0.9% Saline / 10% Tween 80 formulation of each test compound having a maximum concentration of 5 mg/mL) were given on Day 1 following pair matching, dosing at the levels and schedules indicated.

Mice were weighed twice weekly, and tumor measurements were taken by 25 calipers twice weekly, starting on Day 1. These tumor measurements were converted to mg tumor weight by a well-known formula, W2 x L/2. The experiment was terminated when the control group tumor size reached an average of 1 gram. Upon termination, the mice were weighted, sacrificed and their tumors excised. Tumors were weighted and the mean treated tumor weight per group was calculated. In this 30 model, the change in mean treated tumor weight / the change in mean control tumor -52- r 153554/2 weight χ 100% (ΔΤ/ΔΟ) was subtracted from 100% to give the tumor growth inhibition (TGI) for each group.

Compounds 1-5 (below) were tested by the above-described method, giving the results summarized below in Tables A-D. Other compounds of the present invention show comparable activities when tested by this method.

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-53 -

Compound3 Compound 4 153554/1

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Br 53A- WO 01/98278 PCT/US01/13901

Table A SKOV3 tumor xenograft Vehicle COiupOuuu 1 ACUkVl Dose&amp;amp; Schedule Daily x 5 80 mg/kg every 3 daysx4 20 mg/kg dailyx5 Route i.v. i.v. i.p. # Mice at Start .8 8 8 Final Tumor Weight (Mean ± SEM) 904.1 ± 126.5 554.3 ±76.8 90.4 ±36.0 Tumor Growth Inhibition 41.5% 91.7% Mice with Partial Tumor Shrinkage 0 0 4 Mean% Tumor Shrinkage 27.9% Maximum Weight Loss None None 16.5% Mortalities 0 1 0 -54- WO 01/98278 PCT/US01/13901

Table B SK0V3 tumor xenograft Vehicle Compound 2 Compound 3 Taxol Dose&amp;amp; Schedule Daily x 5 50 mg/kg every 3 days x 4 60 mg/kg daily x 5 20 mg/kg daily x 5 Route i.v. i.v. i.v. i-P- # Mice at Start 8 8 8 8 Pinal Tumor Weight (Mean ± SEM) 1506.3 ±227.1 340.8 ±93.0 806.1 ± 163.8 55.9 ±29.4 Tumor Growth Inhibition 81.5% 48.3% 99.7% Mice with Partial Tumor Shrinkage 0 0 0 7 Mean% Tumor Shrinkage 43.5% Maximum Weight Loss None None 2.76% 7.49% Mortalities 0 0 1 0 -55- WO 01/98278 PCT/USO1/13901

Table C SK0V3 tumor xenograft xr.tu. V G1UUK7 CcnipOuiul 4 Taxol Dose&amp;amp; Schedule Daily x 5 4 mg/kg weekly x3 20 mg/kg daily x 5 Route i.v. i.v. i.p. # Mice at Start 8 8 8 Final Tumor Weight (Mean ± SEM) 1191.1 ±239.6 726.9 ±147.2 90.6 ±34.5 Tumor Growth Inhibition 40.1% 87.1% Mice wife Partial Tumor Shrinkage 0 0 4 Mean% Tumor Shrinkage 44.4% Maximum Weight Loss None 0.02% 12.26% Mortalities 1 1 1 -56- WO 01/98278 PCT/US01/13901

Table D SKOV3 tumor xenograft Vehicle Compound 5 Taxol Dose &amp;amp; Schedule Daily x 5 25 mg/kg daily x 5 20 mg/kg daily x 5 Route i.v. i.v. i.p. # Mice at Start 8 8 8 Final Tumor Weight (Mean ± SEM) 1230.4 ± 227.3 405.6 ±124.8 379.0 ±154.0 Tumor Growth Inhibition 71.0% 73.0% Mice with Partial Tumor Shrinkage 0 1 0 Meaa% Tumor Shrinkage 56.3% Maximum Weight Loss None None 8.77% Mortalities 0 0 0 .

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various 5 changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. All 10 patents and publications cited above are hereby incorporated by reference. -57- □nosuzan nnuza , crnxan rwzn ατα inia^n pnow ρηνπ irn πτ -jaoa ,Ρ’ηη ηχζοπ laoana nrrna n^maa πρ^ποζ .□ΌΟΕζΰπ ™·Ώ3 mpuan ρπίι1? oxnnzj □ιηπη pv SB0**3

A'g2)tt 10:2103 4B:(D

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.(mcna navin) crus&amp;amp;'an nwa