ZA200210133B - Methods and compositions utilizing quinazolinones. - Google Patents

Description

o 5

METHODS AND COMPOSITIONS UTILIZING QUINAZOLINONES

FIELD OF THE INVENTION .

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

BACKGROUND OF THE INVENTION :

Interest in the medicinal chemistry of quinazoline derivatives was stimulated in the, carly 1950's with the elucidation of the structure of a quinazoline alkaloid, 3-{8-keto- gamma-(3-hydroxy-2-piperidyl)-propyl]-4-quinazolone, from an Asian plant knowr! for its antimalarial properties. In a quest to find additional antimalarial ageats, various substituted quinazolines have been synthesized. Of particular import was the synthesis of the derivative 2-methyi-3-0-tolyl-4-(3H)-quinazolinone. This compound, known by the name methaqualone, though ineffective against protozoa, was found to bea potent hypnotic.

Since the introduction of methaqualone and its discovery as a hypnotic, the : pharmacological 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 of 1 -substituted-4-aryl-2(1 H)-quinazolinone derivatives useful as anti-inflammatory agents. European patent publication EP 0 056 637 Bl claims a class of 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 treat nourodegencrative, psychotropic, and drug and alcohol induced central and peripheral nervous system disorders.

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 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 thymidylaté 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 derivatives used to treat cancers characterized by over-activity or inappropriate activity of tyrosine receptor kinases. U.S. Patent No. 5,037,829 claims (1H-azol-1-- 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 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 spindle. The mitotic spindle is responsible for distribution of replicate copies of the 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.

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, dramatic improvements in the treatment of cancer are associated with identification of therapeutic agents acting through novel mechanisms, Examples of this include not 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 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 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, 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 0 one another, thus forcing the two spindle poles spart. KSP may also mediate in anaphase B spindle elongation and focussing of microtubules at the spindle pole.

Human KSP (also termed HsEgS5) has been described [Blangy, et al., Cell, 83:1159-69 (1995); Whitehead, et al, Arthritis Rheum., 39:1635-42 (1996); Galgio etal, J. Cell

Biol., 135:339-414 (1996); Blangy, et al., J Biol. Chem., 272:19418-24 (1997);

Blangy, ct al, Cell Motil Cytoskeleton, 40:174-82 (1998); Whitehead and Rattner, J.

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

38 ers forts k gene (TRIPS) bas been described [Lee, et al., Mol Endocrinol., 9:243-54 (1995);

GenBank accession number 140372]. 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 novel

S mitotic 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 : compositions and methods that can be used to treat diseases of proliferating cells. The 10 compositions are KSP inhibitors, particularly human KSP inhibitors.

In one aspect, the invention relates to methods for treating cellular proliferative for treating disord iated with KSP kinesin activity, and for inhibit

KSP kinesin, The methods employ compounds chosen from the group consisting of: 0 Re 0 Re

AN Re Ph Re “| “

SNP CN(PZN

N Ry N Ry 0, N Re N

Y Nr, d Ng,

Re Ry 0 Re

Ry Re o Re

Nn

Re Ry Re

Ry" Nn Sy

N Ry Ra

Ry xn

N Re N Ry so” aS

N Re

L and nw’ Sr wherein:

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

Rand Ry’ are independently chosen from hydrogen, alkyl, oxaalkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; or Ra and Ry’ taken together form a 3- to 7-membered ring;

Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, : substituted alkylheteroaryl, oxaalkyl, oxaalkylaryl, substituted oxaalkylaryl, oxaalkyl heteroaryl, substituted oxaalkylheteroaryl, R;5O- and R;s-NH-;

Ry: is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyiheteroaryl and Rs-NH-;

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

Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, gubstituted alkylheteroaryl, and Ryg-alkylene-;

Rs, Rg R7 and Ry are independently chosen from hydrogen, alkyl, alkoxy, halogen, fluoroalkyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkyisulfonamido, sulfonamidoalkyl, sulfonamidoaryl, alkylthio, carboxyalkyl, carboxamido, aminocarborryl, aryl and heretoaryl;

Ris is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted ~~ alkylheteroaryl;

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

Diseases and disorders that respond to therapy with compounds of the invention include cancer, hyperplasia, restenosis, cardiac hypertrophy, immune disorders and inflammation; especially cancer, hyperplasia, restenosis, and cardiac hypertrophy; particularly cancer.

In another aspect, the invention relates to compounds useful in inhibiting KSP kinesin.

The compounds have the structures shown above.

In an additional aspect, the present invention provides methods of screening for compounds that will bind 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 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.

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.

Figure 4 depicts a synthetic route to substantially pure single enantiomers.

Figure 5 depicts synthetic routes to sulfonamides (5a), carbamates (5b), ureas (5c) and amines (5d).

DETAILED DESCRIPTION OF THE INVENTION

The 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 modulating mitotic kinesins, but not other kinesins (e.g., transport kinesins), specific inhibition of cellular proliferation is accomplished. Thus, the present invention capitalizes on the finding that perturbation of mitotic kinesin function causes malformation or dysfunction of mitotic spindles, frequently resulting in cell cycle arrest and cell death. The methods of inhibiting a human KSP kinesin comprise contacting an inhibitor of the invention with a KSP kinesin, particularly human KSP Kinesins, including fragments and variants of KSP. The inhibition can be of the ATP ’ hydrolysis activity of the KSP kinesin and/or the 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 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 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 1a: 0 Re

R Re “NN ]

Re' x

N Ry 0 N hi NR,

Re 1a quinazolinone sulfonamides of formula 1b 0 Re

R Re

BN wl]

Ry" Nn . N Ry

N Re il

Ry 1b and quinazolinone amines of formulae 1c and 1d 0 Re

R Re

Ny

Re

Re! xX

N Ry

N Re vd Nr lc 0 Rs

Ry Re

Ny no]

Re’ "Nn.

ON Re

N Re [

Re 1d wherein:

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

Ry and Ry’ are independently chosen from hydrogen, alkyl, oxaalkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; or Ra and Ry’ taken together form a 3- to 7-membered ring;

= ®

Rj is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, oxaalkyl, oxaalkylaryl, substituted oxaalkylaryl, oxaalkylheteroaryl, substituted oxaalkylheteroaryl, R1sO- and Ris-NH-;

Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, and Rys-NH-

Ry is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substitated alkyiheteroaryl;

Ry is chosen from hydrogen, alkyl, ary], alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkytheteroaryl, and R;¢-alkylene-;

Rs, Re, Ry and Ry are independently chosen from hydrogen, alkyl, alkoxy, halogen, fluoroalkyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkyisulfonamido, sulfonamidoalkyl, sulfonamidoaryl, alkylthio, carboxyalkyl, carboxamido,

Rys is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, alkylheteroaryl;

Ry 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 kinesin inhibitors, but not all the compounds are novel. In particular, certain ureas (i.e. compounds in which R; is RisNH) are disclosed in US patent 5,756,502 as agents which modify cholecystokinin 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.

Definitions

The term "optional" or "optionally" 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, “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 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, ethyl, propyl, isopropyl, butyl, s-and t-butyl and the like. Preferred alkyl groups are those of Cz or below. More preferred alkyl groups are those of Cys 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, norbormyl, adamantyl and the like. In this application, alkyl refers to alkanyl, as well as the unsaturated alkenyl and alkynyl 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 (-CH2CH;-), ethenyiene (-CHz~CHy-), propylene (-CH;CH,CHy-), dimethyipropylene (-CHzC(CH) 2CHz) and cyclohexyipropylene (-CH;CH;CH(C¢His)-). When an alkyl residue having a 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”.

Alkoxy or alkoxyl refers to groups of from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy,

oO 0 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 to the parent structure through a carbonyl functionality. One or more carbons in the acyl residue may be replaced by nitrogen, oxygen or sulfur as long as the point of attachment to the 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.

Aryl and heteroaryl mean a S- or 6-membered aromatic or heteroaromatic ring containing 0-3 heteroatoms selected from O, N, or S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O,

N, or S; or a tricyclic 13- or 14-membered aromatic or heteroaromatic ring system containing 0-3 heteroatoms selected from O, N, or S. The aromatic 6- to 14- membered carbocyclic rings include, e.g., benzene, naphthalene, indane, tetralin, and fluorene and the 5- to 10-membered aromatic heterocyclic rings includs, 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 via an alkylene residue. Examples arc benzyl, phenethyl, phenylvinyl, phenylallyl 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), benzyloxymethy! and pheaoxymethyl; in general, glycol ethers, such as polyethyleneglycol, are intended to 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

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 heterocycles that fall within the scope of the invention include imidazoline,

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, isoxazole, dioxane, tetrahydrofuran and the like. “N-heterocyclyl” 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-1-piperazinyl and 4-benzyl-1-

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., methrylenedioxy), flucroalkyl, carboxy (-COOH), carboalkoxy (i.., acyloxy -O(O)CR), carboxyalkyl (i.c., esters -C(O)OR), carboxamido, sulfonamidoalkyl, sulfonamidoaryl, aminocarbonyl, benzyloxycarbonylamino (CBZ-amino), cyano, carbonyl, nitro, primary-, secondary- and tertiary-amino (e.g., alkylamino and dialkylamino) and aminoektylene, alkylthio, alkyisulfinyl, alkyisulfonyl, alkylsul fonamido, arylthio, arylsulfinyl, arylsulfonyl, amidino, aryl (e.g., phenyl and benzyl), heteroaryl, heterocyclyl, phenoxy, benzyloxy, or hetercaryloxy. For the purpoees 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 axsalkylaryl refer to residues where either or both of the alkylene and aryl moieties are substituted. Substituted alkylheteroaryl and 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 evea three substitution groups, R, R’ and R” (e.g., diethylamino and trifluoromethyl).

Halogen refers to fluorine, chlorine, bromine or iodine. Fluorine, chlorine and bromine are prefered. Dihaloaryl, dihaloalkyl, trihaloaryl, etc. refer to aryl and alkyl o 0 substituted with a plurality of halogens, but not necessarily a plurality of the same halogen; thus 4-chloro-3-fluorophemnyl is within the scope of dihaloaryl.

Most of the compounds described herein contain one or more asymmetric centers (€.g. the carbon to which R; and Ry’ are attached) and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. 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 skilled in the art, for example by formation of diastereoisomeric salts or complexes which may be separated, for example, by crystallisation; via formation of diastersoisomeric derivatives which may be separated, for example, by crystallisation, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic oxidation or reduction, followed 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 bound chiral ligand or in the presence of a chiral solvent. It will be 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 required to liberate the desired enantiomeric form. Alternatively, specific enantiomer may be 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.

In one embodiment, as will be appreciated by those in the art, the two adjacent Ry groups may be fused together to form a ring structure. Again, the fused ring structure may contain heteroatoms and may be substituted with one or more substitution groups "R*. It should additionally be noted that for cycloalkyl (i.e., saturated ring structures), certain positions may contain two substitution groups, R and R'.

Preferred Embodiments

Considering formulae 1a, 1b, 1c and 1d, but focusing on 1a, in a preferred embodiment R, is selected from hydrogen, alkyl, aryl, substituted alkyl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl and substituted alkylaryl.

In a more preferred embodiment R; is selected from hydrogen, lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, phenyl, naphthyl and substituted phenyl.

Ina most preferred embodiment R, is chosen from hydrogen, ethyl, propyl, methoxyethyl, naphthyl, phenyl, bromophenyl, chlorophenyl, methoxyphenyl, ethoxyphearyl, tolyl, dimethylphenyl, chorofiuorophexyl, methyichlorophenyl, cthryiphenyl, phenethyl, benzyl, chlorobenzyl, methylbenzyl, methoxybenzyl, cyanobenzyl, hydroxybenzyl, tetrahydrofuranyimethyl and (ethoxycarbonyl)ethyl.

Ina preferred embodiment R; is hydrogen, alkyl,cycloalkyl or substituted alkyl. As will be appreciated by those in the art, Formulae 1a, 1b, 1c and 1d possess a ] potentially chiral center at the carbon to which Rj is attached. Thus, the R; position may comprise two substitution groups, Ra and Ra’. The R; and Ry’ groups may be the same or different; if different, the composition is chiral, When the R; and Ry’ are different, preferred embodiments utilize only a single non-hydrogen R;. The invention contemplates the use of pure enantiomers and mixtures of enantiomers, including racemic mixtures, although the use of the substantially optically pure eutomer will generally be preferred, particularly the R enantiomer.

In a more preferred embodiment, R; is chosen from hydrogen, lower alkyl and substituted lower alkyl, and Ra’ is hydrogen. In a most preferred embodiment R; is chosen from hydrogen, methyl, ethyl, propyl (particularly i-propyl), butyl (particularly t-butyl), methylthioethyl, aminobutyl, (CBZ)aminobutyl, cyclohexylmethyl, benzyloxymethyl, methylsulfinrylethyl, methylsulfinylmethyl, hydroxymethyl, benzyl and indolylmethyl. Bspecially preferred is the R enantiomer where R; is i-propyl.

2 ®

In a preferred embodiment Ry; is selected from chosen from alkyl, substituted alkyl, alkylaryl, heteroaryl, aryl, substituted aryl, substituted oxaalkylaryl, -O-R,s and -NH-Rs, and Rys is chosen from alkyl, aryl and substituted aryl.

In a more preferred embodiment, when Ry is not -NHRys, Rj is chosen from Ci-Cy3 alkyl; substituted lower alkyl;ary], 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. Inamost preferred embodiment, when Ry is not -NHR ys, Rs i8 chosen from ethyl, propyl, chioropropyl, butoxy, heptyl, butyl, octyl, tridecanyl, (ethoxycarboryl)ethyl, dimethylaminoethyl, dimethylaminomethyl, phenyl, naphthyl, halophenyl, methoxyphemyl, carboxyphenyl, ethrylphenyl, tolyl, biphenyl, methylenedioxyphenyl, methylsulfonylphenyl, methoxychlorophenyl, chloronaphthyi, methylhalophenyl, trifluoromethyiphenyl, butylphenyl, pentylphenyl, methyinitrophenyl, phenoxymetiryl, dimethoxyphenyl, phenylvinyl, nitrochlorophenryl, nitropheny, dinitropheayl, bis(triffucromethyl)phenyl, benzyloxymethyl, benzyl, furanyl, ‘benzofuranyl, pyridinyl, indolyl, methylpyridinyl, quinolinyl, picolinyl, pyrazolyl, and imidazolyl.

Ina more preferred embodiment, when Ry is -NHRs, Ris is chosen from lower alkyl; cyclohexyl; phenyl; and phenyl substituted with halo, lower alkyl, loweralkoxy, or lower alkylthio.

In a most preferred embodiment, when Rj is -NHRys, Rys is isopropyl, butyl, cyclohexyl, phenyl, bromophenyl, dichlorophenyl, methoxyphenyl, ethylphenyl, tolyl, trifluoromethylphenyl or methylthiophemyl.

In a preferred embodiment Ry is chosen from alkyl, aryl, alkylaryl, alkylheteroaryl, substituted alkyl, substituted aryl, and -alkylene-R1s, and Rig is chosen from alkoxy, amino, alkylamino, dialkylamino and N-heterocyclyl.

2 5

In a more preferred embodiment, Ry is selected from lower alkyl, substituted lower alkyl, cyclohexyl; phenyl substituted with lrydroxy, lower alkoxy or lower alkyl; benzyl; hetsroaryimethyl; heteroarylethyl; heteroarylpropyl and -alkylene-Rig, wherein Ry is amino, lower alkylamino, di(lower alkyl)amino, lower alkoxy, or N- heterocyclyl.

In a most preferred embodiment, Ry is chosen from methyl, ethyl, propyl, butyl, cyclohexyl, carboxyethyl, carboxymethyl, methoxyethyl, kydroxyethyl, hydroxypropyl, dimethylaminoethyl, dimethylaminopropyl, dicthylaminoethyl, diethylaminopropyl, aminopropyl, methylaminopropyl, 2,2-dimethyl-3- (dimethylamino)propyl, 1-cyclohexyl-4-(diethylamino)butyl, aminoethyl, aminobutyl, aminopentyl, aminohexyl, aminoethoxyethyl, isopropylaminopropyl, diisopropylaminoethyl, 1-methyl-4-(diethylamino)butyl, (t-Boc)aminopropyl, hydraxyphenyl, benzyl, methoxyphenyl, methylmethoxyphenyl, dimethytphenyl, tobyl, ethytphenyl, (oxopyrrolidinyl)propyl, (methoxycarbomy)ethiyl, benzylpiperidinyl, pyridinylethyl, pyridinyimetiryl, morpholinylethyl, morpholinylpropyl, piperidinyl, azetidinylmethyl, azetidimylpropyl, pymrolidinylethyl, pyrrotidinylpropyl, piperidinylmethyl, piperidinylethyl, imidazolylpropyl, imidazolyletryl, (cthrylpyrrolidinylmethyl, (methytpyrrolidinyl)ethyl, (methylpiperidinylpropyl, (methylpiperazinylpropyl, furanytmethyl and indolylethryl

In other preferred embodiments, Rs, Rg, R7 and Ry are chosen from hydrogen, halo . (particularly chloro and fluoro), lower alkyl (particularly methyl), substituted lower alkyl (particularly trifluoromethyl), lower alkoxy (particularly methoxy), and cymo; more preferably from hydrogen and halo. Further preferred for each of the specific substituents: Rg is hydrogen or halo; Ry is hydrogen, methyl or halo; R, is hydrogen, halo, alkyl (particularly methyl), alkoxy (particularly methoxy) or cyano; and Ris hydrogen or halo. Still further preferred are the compounds where only one of Rs, Re,

Ry; and Ry is not hydrogen, especially Rs.

In a particularly preferred subgenus, R; is benzyl or halobenzyl; R; is chosen from ethyl and propyl; Ro’ is hydrogen; Rs (or Ry: or Ry) is substituted phenyl; Ry is — (CHa)nOH where m is two or three, or (CHg)p Ris where p is one to three and Ry; is amino, propylamino or azetidinyl; Rs is hydrogen; Re is hydrogen; Ry is halo; and Ry is hydrogen.

When considering primarily the sulfonamides of formula 1b, R is preferably chosen from hydrogen, lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, phenyl and substituted phenyl; R; is chosen from hydrogen and lower alkyl and R;’ is hydrogen; Rj is chosen from C;-C,3 alkyl; phenyl; naphthyl; phenyl substituted with halo, lower alkyl, lower alkoxy, nitro, methylenedioxy, or trifluoromethyl; biphenylyl and heteroaryl; and Ry is chosen from lower alkyl, cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl; heteroarylmethyl; heteroarylethyl; heteroaryipropyl; heteroarylethyl; heteroaryipropyl and -alkylene-Ri¢, wherein Ry is " di(lower alkyl)amino, (lower alkyl)amino, amino, lower alkoxy, or N-beterocyclyl, particularly pyrrolidino, piperidino or imidazolyl.

When considering primarily the sulfonamides of formula 1b, R; is most preferably . chosen from lower alkyl, benzyl, substituted benzyl and substituted phenyl; R; is hydrogen or lower alkyl; Ry’ is hydrogen; R; is chosen from substituted phenyl and naphthyl; Ry is -alkyleno-Rig; Ry is hydrogen, fluoro, methyl or chloro; Rg, Rg and Ry are hydrogen; and Rj is chosen from di(lower alkylamino), (lower alkyl)amino, amino, pyrrolidino, piperidino, imidazolyl and morpholino. :

When considering primarily the amines of formulae 1c and 1d, R; is preferably chosen from hydrogen, lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, phenyl, naphthyl and substituted phenyl; R; is chosen from hydrogen, lower alkyl and substituted lower alkyl and R,’ is hydrogen; Rs is chosen from C,-C); alkyl; : substituted lower alkyl; phenyl; naphthyl; phenyl substituted with halo, lower alkyl, lower alkoxy, nitro, methylenedioxy, or trifluoromethyl; biphenylyl, benzyl and heterocyclyl; and R, is chosen from lower alkyl; cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl; substituted benzyl; heterocyclyl; heteroaryimethyl; heteroarylethyl; heteroarylpropyl and -alkylene-R;¢, wherein Ry¢ is di(lower alkyl)amino, (lower alkyl)amino, amino, lower alkoxy, or N-heterocyclyl.

When considering primarily the amines of formulae 1c and 1d, R; is most preferably chosen from lower alkyl, benzyl, substituted benzyl and substituted phenyl; R; is hydrogen or lower alkyl; Ry’ is hydrogen; Ry is chosen from substituted phenyl, heterocyclyl and naphthyl; Ry is chosen from subtituted benzyl, heterocyclyl and . -alkylene-R;; Rs and R; are chosen from hydrogen and halo; Rs and Ry are hydrogen; and R¢ is chosen from di(lower alkylamino), (lower alkyl)amino, amino, pyrrolidinyl, : 5 piperidinyl, imidazolyl and morpholinyl. When R;- is present (as in 1d) it is most preferably chosen from halophenyl, polyhalophenyl, tolyl, dimethytphenyl, methoxyphenyl, dimethoxyphenyl, cyanophenyl, triflucromethylphenyl, trifluoromethoxyphenyl, bis(trifluoromethyl)phenyl, carboxyphenyl, t-butylphenyl, methoxycarbonylphenyl, piperidinyl and naphthyl.

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 (sub- grouped, respectively, in increasing order of preference): ‘ 1. Any of formulas la, 1b, lc or 1d (preferably formulae 1a or 1d) where R, is hydrogen, lower alkyl, substituted lower alkyl, alkylaryl or substituted alkylaryl (preferably benzyl or substituted benzyl): : a. Especially where the stereogenic center to which R; and R;’ are attached is of the R configuration, where Ry is hydrogen.

C20 i. Particularly where R; is lower alkyl (preferably ethyl, i-proyl, c-propyl or t-butyl). 1. Most preferably where R; is i-propyl. - b. * Especially those where Ry is substituted alkyl, (preferably a primary-, secondary- or tertiary-smino-substituted lower alkyl). i. Most preferably where Ry is primary-amino lower alkyl. c. Especially those where Rg, Rs, R7 and Rg are chosen from hydrogen, halo, lower alkyl (preferably methyl), substituted lower alkyl, lower alkoxy (preferably methoxy), and cyano. i. Preferably Rs, Re, and Ry are hydrogen. 1. More preferably Ry is halo or cyano, especially fluoro or chloro, and most prefombly chloro.

CB ® d. In the case of formulae 1a and 1d, especially those where R; or R;- is aryl (preferably phenyl), substituted aryl (preferably lower alkyl- or lower alkoxy-substituted phenyl), alkylaryl (preferably benzyl and phenylviny), alkylheteroaryl, oxaalkylaryl (preferably phenoxy lower alkyl), oxaalkylheteroaryl, substituted alkylaryl (preferably substituted benzyl and substituted phenylviny), substituted alkylheteroaryl, substituted oxaalkylaryl (preferably substituted phenoxy lower alkyl), or substituted oxaalkylheteroaryl. i. Most preferably those where R; or Ry- is aryl, substituted aryl, lower alkylaryl or substituted lower alkylaryl. 2. Any of formulae 1a, 1b, 1c or 1d, where the stereogenic center to which R; and

Ry’ are attached is of the R configuration, particularly where Ry: is hydrogen: a. Especially where Ry is lower alkyl (preferably ethyl, i-proyl, c-propyl or t-butyl). i Most preferably where R; is i-propyl. b. Especially where Ry is substituted alkyl (preferably a primary-, secondary- or tertiary-amino-substituted lower alkyl). i. Most preferably where Ry is primary amino-lower alkyl. c. Especially where Rs, Rg, and Ry are hydrogen. i. Most preferably where R; is hydrogen, halo (particularly chloro or fluoro), lower alkyl (particularly methyl), substituted lower alkyl, lower alkoxy (particularly methoxy), or cyano 1. Especialy where Ry is chloro. 3. Any of formulae 1a, 1b, 1c or 1d where Ry is hydrogen, halo (preferably chloro or fluoro), lower alkyl (preferably methyl), substituted lower alkyl, lower alkoxy (preferably methoxy), or cyano. a. Especially those where Ry is halo or cyano. ' b. Especially those where Rs, Re, and Rg are hydrogen. i. Most preferably those where Ry is chloro.

4. Any of formulae 1b or lc, where Ry 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 R; and Ry’ are attached is of the R configuration, particularly where Ry is hydrogen: i Especially where R; is lower alkyl (preferably ethyl, i-proyl, c-propyl or t-butyl). 1. Most preferably where R; is i-propyl. b. Especially where Rs, Re, and Ry are hydrogen. i. Most preferably where R; is hydrogen, halo (particularly chloro or fluoro), lower alkyl (particularly methyl), substituted lower alkyl, : lower alkoxy (particularly methoxy), or cyano 1. Bspecialy where R; is chloro.

Most preferred for the compounds, pharmaceutical formulations, methods of manufacture and use of the present invention is formula 1a incorporating the following combinations and permutations of substituent groups (sub-grouped, respectively, in increasing order of preference): 1. Ry is alkylaryl or substituted alkylaryl (preferably benzyl or substituted benzyl, most preferably benzyl). a. Especially where the stereogenic center to which R; and R;’ are attached is . of the R configuration, where R;’ is hydrogen. i. Particularly those where R; is lower alkyl (preferably ethyl, i-propyl, c-propyl or t-butyl). 1. Most preferably those where R; is i-propyl. b. Especially those where Rj is aryl (preferably phenyl), substituted aryl (preferably lower alkyl-, lower alkoxy- and/or halo-substituted phenyl), alkylaryl (proferably benzyl and pheaylviny), alkylheteroaryl, oxaalkylaryl (preferably phenoxy lower alkyl), oxaalkylheteroaryl, substituted alkylaryl (preferably substituted benzyl and substituted phenylviny), substituted alkylheteroaryl, substituted oxaalkylaryl (preferably substituted phenoxy lower alkyl), or substituted oxaalkylheteroaryl.

i.

Particularly those where Ry is aryl, substituted aryl, lower alkylaryl, substituted lower alkylaryl, oxa(lower)alkylaryl. 1. Most preferably those where R; is methyl- and/or halo-substituted phenyl. c.

Especially those where Ry is substituted alkyl (preferably a primary-, secondary- or tertiary-amino-substituted lower alkyl). i.

Particularly those where Ryis a primary-amino-substituted lower alkyl. 1. Most preferably those where Ry is 3-amino-n-propyl. d.

Especially those where Rs, Rg, Ry and Ry 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, Rg, R7 and Ry are hydrogen, halo, lower alkyl or cyano. 1. Most preferably those where Rs, Re and Ry are hydrogen. a.

Especially those where Ry is halo or cymno (most preferably chloro). 2. Especially those where Ry is halo or cyano (most preferably chloro). : 2. Where the stercogenic center to which Ro and Ry’ are sttached is of the R configuration (preferably where Ry” is hydrogen). a Especially those where Rj is lower alkyl (preferably ethyl, i-propyl, c-propyl or t-butyl). i.

Most preferably those where R; is i-propyl.

b.

Especially those where R; is aryl (preferably phenyl), substituted aryl (preferably lower alkyl-, lower alkoxy-, and/or halo-substituted phenyl), slkylaryl (preferably benzyl and pheaylviny), alkylheteroaryl, oxaalkylaryl (preferably phenoxy lower alkyl), oxaslkyiheteroaryl, substituted alkylaryl (preferably substituted benzyl and substituted phenylviny), substituted alkytheteroaryl, substituted oxaalkylaryl (preferably substituted phenoxy lower alkyl), or substituted esasliyumroasy

= i.

Particularly those where Rj is aryl, substituted aryl, lower alkylaryl, substituted lower alkylaryl, oxa(lower)alkylaryl. 1. Most preferably those where Rj is methyl- and/or halo-gubstituted phenyl. c.

Especially those where Ry is substituted alkyl (preferably a primary-, secondary- or tertiary-amino-substituted lower alkyl). i.

Particularly those where Ry, is a primary-amino-substituted lower alkyl. 1. Most preferably those where Ry is 3-amino-n-propyl.

d.

Especially those where Rg, Rg, Ry and Rg 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, Re, R7 and Rg are hydrogen, halo or lower alkyl.

1. Most preferably those where Rs, Rg and Ry are hydrogen.

a.

Especially those where R7 is halo or cyano (most preferably chloro). 2. Especially those where R; is halo or cyano (most preferably chloro). : 3. Ryis aryl (preferably phenyl), substituted aryl (preferably lower alkyl-, lower alkoxy-, and/or halo-substituted phenyl), alkylaryl (preferably benzyl and phenylviny), alkyiheteroaryl, oxaalkylaryl (preferably phenoxy lower alkyl), oxaalkyTheteroaryl, substituted alkylaryl (preferably substituted benzyl and substituted phenylviny), substituted alkylheteroaryl, substituted oxaalkylaryl (preferably substituted phenoxy lower alkyl), or substituted oxaalkylheteroaryl. a.

Especially those where R; is aryl, substituted aryl, lower alkylaryl, substituted lower alkylaryl, oxa(lower)alkylaryl. i.

Most preferably those where R; is methyl- and/or halo-substituted phenyl. : : b.

Especially those where Ry is substituted alkyl (preferably a primary-, secondary- or tertiary-amino-substituted lower alkyl).

= &

i.

Particularly those where Ry is a primary-amino-substituted lower alkyl. 1. Most preferably those where Ry is 3-amino-n-propyl. c.

Especially those where Rs, Rg, Ry and Rg 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, Rg, R7 and Rg are hydrogen, halo, lower alkyl or cyano. 1. Most preferably those where Rs, Rs and Rg are hydrogen. a.

Especially those where Rs is halo or cyano (most preferably chloro). 2. Especially those where R; is halo or cyano (most preferably chloro). 4. Ry is substitated alkyl (preferably a primary-, secondary- or tertiary-amino- substituted lower alkyl). a.

Particularly those where R4 is a primary-smino-substituted lower alkyl. i.

Most preferably those where Ry is 3-amino-n-propyl. b.

Especially those where Rs, Re, R7 and Rg 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, Rg, R7 and Rg are hydrogen, halo, lower alkyl or cyano. 1. Most preferably those where Rs, Rs and Rg are hydrogen. a.

Especially those where R1 is halo or cyano (most preferably chloro). 2. Especially those where Ry is halo or cyano (most preferably chloro). 5. Rs, Re, Ry and Ry are chosen from hydrogen, halo (preferably chloro and fluoro), lower alkyl (preferably methyl), substituted lower alkyl, lower alkoxy (preferably methoxy), and cyano.

a. Especially those where Rs, Re, R7 and Ry are hydrogen, halo, lower alkyl or : cyano. i. Most preferably those where Rs, Rg and Ry are hydrogen. 1. Especially those where R; is halo or cyano (most preferably chloro). ii. Especially those where Rs 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 1a where R, is alkylaryl or substituted alkylaryl (particularly benzyl or substituted benzyl), R; is lower alkyl (particularly i-propyl), Ro’ is hydrogen, R is substituted aryl (particularly methyl- and/or halo-substituted phenyl), Ry is substituted alkyl (particularly 3-amino-n-propyl), Rs, Rg and Rg are hydrogen, and Ry is halo or cyano (particularly chloro), where the stereogenic center to which R; and R,’ are attached is of the R configuration.

Synthesis, Testing and U

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 Mod.

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. oo

The compositions of the invention may be made as shown in Figures 1,2,4 and 5.

Compounds of formulae 1d 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 may be altered in a variety of ways, that 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.,

® & 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 "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 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 ofherwise cansing morphological perturbation of the mitotic spindle. Also included within the definition of KSP for 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 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 (¢.g., & 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 soluble material, and is otherwise compatible with the overall method of screening.

The surface of such supports may be solid or porous and of any convenicat 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 are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of . antibodies (which do not sterically block cither the ligand binding site or activation 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 the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.

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, the mitotic agents of the invention are combined with KSP and the activity of KSP is assayed. Kinesin activity is known in the art and includes one or more kinesin activities. Kinesin activities include the ability to affect ATP hydrolysis; microtubule binding; gliding 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 kinesin cellular activities such as spindle pole separation. ,

Methods of performing motility assays are well known to those of skill in the art. [See e.g., Hall, et al. (1996), Biophys. J., 71: 3467-3476, Tumer etal., 1996, AnaL

Biochem. 242 (1):20-5; Gittes et al., 1996, Biophys. J. 70(I): 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 the art for determining ATPase hydrolysis activity also can be 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. Altematively, conventional methods are used. For example, P; 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 mM sodium molybdate II, 0.33 mM malachite green oxalate, and 0.8 mM Triton X-1 00). To perform the assay, 10 pL of reaction is quenched in 90 pL of cold 0.3 M

D o

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 the plate i8 read at an absorbance of 650 nm. If phosphate standards were used, absorbance readings can be converted to mM P; 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 performed in the absence of microtubules. In another embodiment, the ATPase assays are performed 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 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 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

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 biol, 135:399-414).

The compositions of the invention inhibit the KSP kinesin. One measure of inhibition is ICso, defined as the concentration of the composition at which the activity of KSP is decreased by fifty percent. Preferred compositions have ICs's of less than about 1 mM, with preferred embodiments having ICsq's of less than about 100 uM, with more preferred embodiments having ICsq's of less than about 10 uM, with particularly preferred embodiments having ICso's of less than about 1 uM, and especially preferred embodiments having ICsq's of less than about 100 nM, and with the most preferred embodiments having ICso's of less than about 10 nM. Measurement of ICs is done using an ATPase assay.

Another measure of inhibition is K;. For compounds with ICso's less than 1 uM, the

K; or Kq is defined as the dissociation rate constant for the interaction of the quinazolinone with KSP. Preferred compounds have K's of less than about 100 uM, with preferred embodiments having K{'s of less than about 10 uM, and particulady preferred embodiments having K's of less than about 1 pM and especially preferred embodiments having K's of less than about 100 nM, and with the most preferred embodiments having K's of less than about 10 nM. The K| for a compound is determined from the ICs based on three assumptions, First, only one compound molecule binds to the enzyme and there is no cooperativity. Second, the concentrations of active enzyme and the compound tested are known (i.e., there are no significant amounts of impurities or inactive forms in the preparations). Third, the enzymatic rate of the enzyme-inhibitor complex is zero. The rate (i.e., compound concentration) data are fitted to the equation: . - _— 8 (Bo + Ip + Ka). (Bo +l + RAP-4Koly 28,

Where V is the observed rate, Vig is the rate of the free enzyme, I is the inhibitor concentration, By is the enzyme concentration, and Ky is the dissociation constant of the enzyme-inhibitor complex.

Another measure of inhibition is Glsg, defined as the concentration of the compound that results in a decrease in the rate of cell growth by fifty percent. Preferred compounds have Glso's of less than about 1 mM. The level of preferability of embodiments is a function of their Gls : those having Glso's of less than about 20 uM are more preferred; those having Glsy's of 10 uM more so; those having Glso of less than about 1 pM more 50; those having Glsg's of 100 nM more 80; those having Gls of less than about 10 nM even more so. Measurement of Gls is done using a cell proliferation assay.

The compositions of the invention are used to treat cellular proliferation diseases.

Disease states which can be treated by the methods and compositions provided herein include, but are not limited to, cancer (further discussed below), autoimmune discase, 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 state (abnormal state) and still require treatment. For example, during wound healing, the cells may be proliferating "normally", but proliferation enhancement may be desired. Similarly, as discussed above, in the agriculture arena, cells may be ina 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 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 carcinomas, testicular carcinomas, etc. More particularly, cancers that may be treated by the compositions and methods of the invention include, but are not limited to:

Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, thabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus

(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, Karposi's sarcoma, leionryoma, 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 carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma . (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma (osteosarcoma), 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, meningiossrcoma, gliomatosis), brain (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 dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], grannlosa-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 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 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 sbove-identified conditions.

Accordingly, the compositions of the invention are administered to cells. By "administered" herein is meant administration of a therapeutically effective dose of the mitotic agents of the invention to a cell either in cell culture or in a patient. By “therapeutically effective dose" herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, ) 10 and will be ascertainable by one skilled in the 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 the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. By "cells" herein is meant almost any cell in which mitosis or meiosis can be altered.

A "patient" for the 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 the preferred embodiment the patient is 8 mammal, and in the most preferred embodiment the patient is human.

Mitotic agents having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a patient, as described herein. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways as . discussed below. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%. The agents may be administered . 25 alone or in combination with other treatments, i.e., radiation, or other chemotherapeutic agents.

In a preferred embodiment, the pharmaceutical compositions ae 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 those salts that retain the biological effectiveness of the free bases and that are not 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 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 the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, dicthylamine, triethylamine, tripropylamine, and 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- 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 serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Additives are well known in the art, and arc used in a variety of formulations.

The administration of the mitotic agents of the present invention can be done ina variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally,

intramuscularly, intrapulmonary, 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 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, : 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.

The determination of the binding of the mitotic agent to KSP may be dono 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 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 with a label which provides a detectable signal, .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 which provides for detection, in accordance with known procedures, as outlined above, The label can directly or indirectly provide a detectable signal.

o &

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

Alternatively, more than one component may be labeled with different labels; using 13%] for the proteins, for example, and a fluorophor for the mitotic agents.

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 proliferation phenotype or the expression of a cellular proliferation sequence, including both nucleic acid sequences and protein sequences. In 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 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 hezein 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 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 hydrogen bonding and lipophilic binding, and typically include at least an amine, carbonyl, hydroxyl, ether, or carboxyl group, preferably at least two of the finctional 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 including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.

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, 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 may be subjected to directed or random chemical modifications, such as acylation, alkylation, 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 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 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.

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 facilitate rapid high throughput screening. Typically between 0.1 and 1 hour will be 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, the competitor is added first, followed by the candidate agent. Displacement of the competitor is an indication the candidate agent is binding to KSP and thus is capable of binding to, and potentially modulating, the activity of

KSP. In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the candidate agent is labeled, the presence of the label on the support indicates displacement.

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

KSP.

It may be of value to identify the binding site of KSP. This can be done in a variety of ways. In one embodiment, once KSP has been identified as binding to the mitotic agent, KSP is fragmented or modified and the assays repeated to identify the necessary 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 with KSP, as above, and determining an alteration in the biological activity of KSP. Thus, in this embodiment, the candidate agent should both bind to KSP (although this may not be necessary), and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods and in vivo screening of cells for alterations in cell cycle distribution, cell viability, or for the presence, morpohology, activity, distribution, or amount of mitotic spindles, as are generally outlined above.

Alternatively, differential screening may be used to identify drug candidates that bind to the native KSP, but cannot bind to modified KSP.

ES 50%

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- 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 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. The mixture of components may be added in any order that provides for the requisite binding.

The following examples serve to more fully describe the marmer 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.

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 = benzyloxycarbonyl

DBU = diazabicyclo[5.4.0Jundec-7-ene

DCM = dichloromethane = methylene chloride = CHCl,

DCE = dichloroethylene

DEAD = diethyl azodicarboxylate

DIC = diisopropylcarbodiimide . Co .37-

DIEA = N,N-diisopropylethyl amine

DMAP = 4NN-dimethylaminopyridine

DMF = N)N-dimethylformamide

DMSO = dimethyl sulfoxide

DVB = ],4-divinylbenzene

EEDQ = 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

Et = ethyl

Fmoc = 9-flyorenylmethoxycarbonyl

GC = gag chromatography

HATU = O-(7-Azabenzotriazol-1-y)-1,1,3,3-tetramethyluronium hexaftuorophosphate

HMDS = hexamethyldisilazane

HOAc = acetic acid

HOBt = hydroxybenzotriazole

Me = methyl mesyl = methanesulfonyl

MTBE = methyl t-butyl ether

NMO = N-methylmorpholine oxide

PEG = polyethylene glycol

Ph = phenyl

PhOH = phenol

BPfP = pentafluorophenol

PPTS = pyridinium p-toluenesulfonate

Py = pyridine

PyBroP = bromo-tris-pyrrolidino-phosphonium hexafluorophosphate t = room temperature sat=d = saturated s = socondary t- = tottiary

TBDMS = t-butyldimethylsilyl

TES = ftriethylsilane

TFA = trifluoroacetic acid

THF = tetrahydrofuran

TMOF = trimethyl cxthoformate

TMS = trimethylsilyl tosyl = p-toluenesulfonyl

Tt = triphenylmethyl

Example 40 Synthesis of Compounds

The general synthesis is shown in Figures 1 and 2.

To a three-necked, 500 mL round-bottom flask equipped with a thermometer,

BD M dropping fume, 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 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 P20, yielding compound 2 (67.3 g, 65%).

Step 2; 2-Propyi-3,1-[4H]benzoxazin-4-one,

Compound 2 (51.8 g, 0.25 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 the distillation ynit 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-3,1-{4H]benzoxazin-4-one (3) (29.3 g, 62%). The above procedure gave compound 3 sufficiently pure to use directly in the next step.

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.

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

Step 4: 2-(I'bromopropyl)-3-benzylquinazolin-4-one.

To a three-neck 250 mL round-bottom flask equipped with 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 at40 °C for 1-2 h. After addition was complete, the mixture was poured into water (1500 mL) and stirred for 1-2 h at room temperature. The precipitated product, 241- bromopropyl)-3-benzylquinazolin-4-one (5) was isolated by filtration, washed with warm water fo remove traces of acetic acid, and rinsed with a small amount of isopropanol. Drying yielded compound 5 (33.0 g, 92%). :

Compound 5 (10.7 g, 0.03 mole) and N)N-dimethylethylenediamine (6.6 mL, 0.06 mole) were dissolved in abs. ethanol (60 mL) and heated at reflux for 6 h. After completion of the reaction, the solvent was evaporated under reduced pressure. The residue was dissolved in dichloromethane (150 mL) and washed with 3% aq. NaOH solution (ca. 10-20 mL). The organic layer was dried over MgSO, 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 CHC1;-MeOH-aq.NH;, 90:10:0.1, to give the desired compound (5), 2{I'-(N,N- dimethylethylenediamino)propyi}-3-benzylquinazolin-4-one (6) (6.0 g, 55%). benzylquinazo ]lin-4-one o

A stock solution of compound 5 (1.822 g, 5.0 mmol) was prepared in HPLC grade

CHCl; (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.

A third solution of triethylamine (2.0 mL of 0.5 M) was prepared in HPLC grade 1,2- dichlorethane. A 100 uL aliquot of each solution was pipetted into a glass reaction 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 CHCl, (300 pL) and washed with 5% aqueous NaHCOs and water. The solvent was removed in vacuo to provide compound 6 (65%). The purity of the compound was analyzed by

TLC eluted with CH;Cl-ethanol-concentrated aqueous NH;, 100:10:1. . Examples 2 aud 3

Synthesis of compounds of General Formula 1d oo i

NH -Ry

N oS OO pe Br Ry N ?

DIEA, DMF, CHCl HN

NH~~Reosin

I)R3"Br

DIEA, DCM 0 2)5:590 oor TFA:TBS:DCM

Ry a

Ny r

NH,

All anhydrous solvents were purchased from Aldrich chemical company in SureSeal®

Abbreviations: DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DMF,

N,N-dimethylformamide; TES, triethylsilane; TFA, trifluoroacetic acid. Array synthesis was conducted in 15 x 75 mm glass round bottom scrow-cap vials contained ina 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 8 4 x 6 nitrogen manifold

Example 2 (solid phase synthesis of a single compound)

STEP 1: 1,3-Diaminopropane trityl resin (Novabiochem, 1.2 mmol/g) (020g, 024 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- 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- diamine intermediate. LCMS analysis showed >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 scaled and shaken ovemnight. LCMS analysis of a cleaved aliquot revealed an ) approximate 1:1 mixture of starting material and product. Another 0.130 mL of DIEA and 0.12 g of 4-bromobenzyl bromide were added and the mixture was shaken at 70 oC for 8 h. The resin was filtered, washed (as above), and dried under vacuum.

STEP 3: The rosin from Step 2 was twice shaken for 30 min with 5:5:90

TRA:TES:DCM and filtered. The filtrates were combined and evaporated, yielding 140 mg of an orange oil. This material was purified by reverse phase preperative

HPLC (acetonitrile-water gradient) to provide 27 mg (17% for 3 steps) of the mono-

TFA salt.

STEP 1: 1,2-Diaminoethane trityl resin (Novabiochem, 0.95 mmol/g) (200 g, 1.9 mmol) and 1,3-Diaminopropans trityl resin (Novabiochem, 1.14 mmol/g) (2.0 g, 2.28 mmol) were each placed in different 10 mL polypropylene fritted tubes (Bio-Rad). To cach 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-(1’-bromopropyi)-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 dried under vacuum. Analysis of a cleaved aliquot revealed the presence of the appropriate quinazolinone-diamine for each in >90 % purity.

STEP 2: The quinazolinone cthyl-diamine resin (105 mg, 0.10 mmol) was placed into each of the 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 of the array. To cach vial was added DIEA (0.131 mL, 0.75 mmol). Into each vial of the first 2 rows of the 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 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-white powdery or amarphous solids. Analysis by LCMS showed all to be >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 described below:

Example 4 5

Bee cl PN 5

Column - Chiralpak AD, 250 x 4.6 mm (Diacel Inc.). Sample —0.5 mg/mL in EtOH.

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

Example § foe. r

POON

NH;

Column - Chiraleel OJ, 250 x 4.6 mm (Diacel Inc.). Sample ~ 0.5 mg/mL in EtOH.

Conditions - 15 min at 10% EtOH in Hexane, (R)-cnantiomer elutes at 8.4 min, (S)- / enantiomer elutes at 9.6 min.

Example 6 oh p

NH,

Column - Chiralpak AD, 250 x 4.6 mm (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.

The table below depicts the ICs 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.

ICso (uM) ICs (0M) ICs (0M)

Racemate | Enantiomer 1 | Enantiomer2

Bee

N

© 12.7 >>40

Ree

SF

© 2.6 >>40 13 u }

Seo ~~

L,

= 5

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 enantiomer.

I YTV I TT somone mone . oY <0.1 ] 3 h 3 p

Example 9

Chiral Resolution by Recrystallization with Tartaric Acid ’

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:

E ® oO > 1. NaN,, DMF 0 > joe Ro Ter joe R

X NS X NS

Br NH,

A B

The R enantiomer of B can be 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 letting the mixture return to room temperature.

Example 9: X=CL, R=H

Racemic intermediate B (1.5 g), dissolved in 100 mL of boiling isopropanol, was mixed with 0.8 g of D-tartaric acid in 100 mL of boiling methanol. The mixture was 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 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 (MgSO), filtered and evaporated to dryness. The resulting clear oil weighed 345 mg. Chiral purity of >95% was determined by conversion of a portion to the S-Mosher amide and examination of the product by "HNMR.

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.

I = rr rR mw (UM ICgp (UM Cp (UM 2 <0.056 oF

CHy

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 subconfinence with RPMI 1640 medium containing 5% fetal bovine serum (FBS). The cells were then treated with either 10nM paclitaxel, 400nM quinazolinone 1, 200nM quinazolinone2, or 025% DMSO (vehicle for compounds) for 24 hours. Cells were then rinsed off the plates with PBS containing

SmM EDTA, pelleted, washed once in PBS containing 1% FCS, and then fixed overnight in 85% ethanol at 4°C. Before analysis, the cells were polleted, washed once, and stained in a solution of 10pg propidium iodide and 250g of ribonuclease (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.

The quinazolinone compounds, as well as the known anti-mitotic agent paclitaxel, caused a shift in the population of cells from a GO/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.

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 & HeLa) or 8,000 cells per well (A549), allowed to 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 anti- tubulin 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 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 canses mitotic arrest with arrested cells displaying monopolar spindles.

Inhibition of Cellular Proliferation in Tumor Cell Lines Treated with Quinazolinone

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 Ty. A tetrazolium-based assay using the reagent 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium (MTS) (LS> Patent No. 5,185,450) (see Promega product catalog #G3580,

CelITiter 96® AQueos One Solution Cell Proliferation Assay) was used to determine the number of viable cells at To and the number of cells remaining after 48 hours compound exposure. The number of cells remaining after 48 hours was compared to the number of viable cells at the time of drug addition, allowing for calculation of

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,

HCT]15), ovarian (SKOV-3, OVCAR-3), leukemia (HL-60(TB), K-562), central nervous system (SF-268), renal (A498), osteosarcoma (U2-OS), and cervical (HeLa).

Inaddition, a mouse tumor line (B16, melanoma) was also growth-inhibited in the presence of the quinazolinone compounds.

A Gig was calculated by plotting the concentration of compound in pM vs the percentage of cell growth of cell growth in treated wells. The Gisq calculated for the compounds is the estimated concentration at which growth is inhibited by 50% 1S compared to contro, i.c., the concentration at which: 100 x {(Treatedss - To) / (Controle - 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 (1991)). However, the method by which the National Cancer Institute quantitates cell number does not use MTS, but instead employs altemative methods. :

Calculation Of ICs:

Measurement of a composition's ICs for KSP activity uses an ATPase assay. The following solutions are used: Solution 1 consists of 3 mM phosphoenolpyruvate potassium salt (Sigma P-7127), 2 mM ATP (Sigma A-3377), 1 mM IDTT (Sigma D- 9779), 5 uM 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

2 a (Sigma P0294), 100 nM KSP motor domain, 50 pg/ml microtubules, 1 mM DTT (Sigma D9779), 5 uM 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 (Corning Costar 3695) using

Solution 1. Following serial dilution each well has 50 pl of Solution 1. The reaction is started by adding 50 pl 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 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 ICsp determination the data acquired is fit by the following four parameter equation using a nonlinear fitting program (e.g, Grafit 4): y= — Re + Backgromd 1+

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, HCT 5) that express P-glycoprotein (also known as Multi- drug Resistance, or MDR"), which conveys resistance to other chemotherapeutic 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 Glso values varied. Gls; values for the quinazolinone compotmds tested ranged from 200 nM to greater than the highest concentration tested. By this we mean that although most of the compounds that inhibited KSP activity biochemically did inhibit cell proliferation, for some, at the highest concentration tested (generally about 20 pM), cell growth was inhibited less than 50%. Many of the compounds have Gls

8 2 values less than 10 uM, and several have Glsg values loss than 1 pM. Anti- proliferative compounds that have been successfully applied in fhe clinic to treatment of cancer (cancer chemotherapeutics) have Glsy’s that vary greatly. For example, in

A549 cells, paclitaxel Gly is 4 nM, doxorubicin is 63 nM, 5-fluorouracil is 1 uM, and 5S hydroxyurea is 500 pM (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 Gls values of less than 1 mM. More preferably, compounds will have Glsq values of less than 20 uM. Even more preferably, compounds will have Gly values of less than 10 pM. Further reduction in Glso values may also be desirable, including compounds with Glsy values of less than 1 pM.

Some of the quinazolinone compounds of the invention inhibit cell proliferation with

Glsg values from below 200 nM to below 10 nM.

Example 11

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 cantainted 8 tumored mice, each of which was ear-tagged and followed individually throughout the 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 S 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 calipers twice weekly, starting on Day 1. These tumor measurements were converted to mg tumor weight by a well-known formula, W? 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 model, the change in mean treated tumor weight / the change in mean control tumor weight x 100% (AT/AC) was subtracted from 100% to give the tumor growth inhibition (TG) 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.

Compound 1 “

Compound 2 ZL

Compound 3 . Compound 4 : :

Compound 5 2

= a

Table A

SKOV3 tumor xenograft :

Jon

Dose & Deilyx 5 80 mg/kg every 20 mg/kg

Schedule 3 daysx4 daily x 5

NF I I al I NL LI

Final Tumor | 904.1+126.5( 554.3176.8 90.4 + 36.0

Weight (Mean + SEM)

Tumor Growth 91.7%

Inhibition

Mice with

Partial Tumor

Shrinkage

Mean % 27.9%

Tumor }

Shrinkage

Maximum None 16.5%

Weight Loss wim 0 [Ur]

Table B

SKOV?3 tumor xenograft

IE SC cl Ki

Dose & Daily x 5 50 mg/kg every 60 mg/kg 20 mg/kg

Schedule 3daysx4 daily x 5 dailyx 5

Bc HC HN

Waal NN NL FL NL

Final Tumor 15063+227.1 | 34081930 | 806111638 | 55.9+294

Weight (Mean + SEM)

Tumor Growth 81.5% 48.3% 99.7%

Inhibition

Mice with 7

Partial Tumor

Shrinkage

Mean %

Tumor

Shrinkage

Maximum None 2.76% 7.49%

Weight Loss wii | 0 | ©] t ] °

Table C

SKOV3 tumor xenograft

I a

Dose & Daily x 5 4 mg/kg 20 mg/kg

Schedule weekly x 3 daily x § a IL

Fad IL I A

Final Tumor 1191.1 £239.6 | 726911472 | 90.6134.5

Weight (Mean i SEM)

Tumor Growth 40.1% 87.1%

Inhibition

Mice with

Partial Tumor ,

Shrinkage

Mean % 44.4%

Tumor

Shrinkage

Maximum 0.02% 12.26%

Weight Loss a SLI LI RL

Table D

SKOV3 tumor xenograft

I Me cid

Dose & Dailyx 5 25 mg/kg 20 mg/kg

Schedule : dailyx 5 dailyx 5 al EL

J cd I EA NL

Final Tumor 1230.4 £227.3 | 405.61124.8 | 379.0+154.0

Weight (Mean + SEM)

Tumor Growth 71.0% 73.0%

Inhibition

Mice with

Partial Tumor

Shrinkage

Mean %

Tumor

Shrinkage

Maximum 8.77% - | Weight Loss pots | 0 1° 1°]

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 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 patents and publications cited above are hereby incorporated by reference.

Claims (64)

We claim:

1. A compound for use in a method of treating cellular proliferative diseases comprising administering the compound which is chosen from the group consisting of: 0 Rs (o] Rs Ry Re R Re Nn RNY ; ow |] ow Ry’ XN Ry’ A N R; N R; lo} N Rs N Re hd Sg, d “Sr, Rs Ra- 0] Rs Ry Re 0 Rs Sy R, | Ry Rs Ry’ A pe N Ry R, Ry XN N R N R; iil SR, : N Rs Ry and nw Sg, wherein: R; is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; R; and R;’ are independently chosen from hydrogen, alkyl, oxaalkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; or R; and R;’ taken together form a 3- to 7-membered ring; R; 1s chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, oxaalkyl, oxaalkylaryl, substituted oxaalkylaryl, oxaalkylheteroaryl, substituted oxaalkylheteroaryl, R;sO- and R;s-NH-; -58- AMENDED SHEET

Rj is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, and R;s-NH-; Rj; is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl, Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, and R;¢-alkylene-; Rs, Rg, R7 and Rg are independently chosen from hydrogen, alkyl, alkoxy, halogen, fluoroalkyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, sulfonamidoalkyl, sulfonamidoaryl, alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, aryl and heteroaryl; Rs is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; and Rie is chosen from alkoxy, amino, alkylamino, dialkylamino, N-heterocyclyl and substituted N-heterocyclyl, or a pharmaceutically acceptable salt thereof.

2. A compound for use in a method of treating a disorder associated with KSP kinesin activity comprising administering the compound which is chosen from the group consisting of: 0 Rs 0 Rs ow |] ol Ry’ RN Ry AN JA Ry JA Ry NT Rg on Re Ra Rar -59- AMENDED SHEET

0 Re aN Re 0 Re or | AN Re “JA . SE Ry! EY sor” Sr, ~ JA v

L and We ~ wherein:

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

and substituted alkylheteroaryt;

R; and Ry’ are independently chosen from hydrogen, alkyl, oxaalkyl, aryl, alkylaryl, alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; or Ry and Ry’ taken together form a 3- to 7-membered ring;

Rg is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylhsteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkyiheteroaryl, axaalkyl, axsalkylaryl, substituted oxaalkylaryl, oxaalkylheteroaryl, substituted oxaalkylheteroaryl, R1sO-and Rys-NH-;

Ry: is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl,

substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkyiheteroaryl and Rys-NH-;

Rg~ is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkyiheteroaryl;

: 20 Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, and Ris-alkylene-;

Rs, Ré, R7 and R are independently chosen from hydrogen, alkyl, alkoxy, halogen, flucroalkyl, itro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido,

sulfonamidoalicyl, sulforumidoaryl, alkylthio, carboxyalkyl, carboxamido,

aminocarbonyl, aryl and heteroaryl; Ris is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; and Ri is chosen from alkoxy, amino, alkylamino, dialkylamino, N-heterocyclyl and substituted N-heterocyclyl, or a pharmaceutically acceptable salt thereof.

3. A compound for use in a method of inhibiting KSP kinesin comprising contacting KSP kinesin with the compound which is chosen from the group consisting of:

[0] Rs Q Rs Ry Rs R Re NN ENE R, | R, R,' XN Ry’ NY N Ry N Ry lo} N Rg N Re hd Sg, d “Sg, Rs Ra 0 Rs R, Re 0] Rs Sy R; | Ry Re Ry Xn Sn N Ry R; Ry XN N R N R7 i Sg, ° N Rs Ra and vd SR, wherein: Rj is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; R; and R;’ are independently chosen from hydrogen, alkyl, oxaalkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted -61 - AMENDED SHEET alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; or R; and Ry’ taken together form a 3- to 7-membered ring;

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

substituted alkylheteroaryl, oxaalkyl, oxaalkylaryl, substituted oxaalkylaryl, oxaalkylheteroaryl, substituted oxaalkylheteroaryl, RisO-and R;s-NH-;

Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl and R;s-NH-;

Ry~ is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl;

Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl,

substituted alkylheteroaryl, and Ris-alkylene-;

Rs, Re, Ry and Ry are independently chosen from hydrogen, alkyl, alkoxy, halogen, fluoroalkyl, nitro, cyano, dialkylamino, alkylsulforyl, alkylsulfonamido, sulfonamidoalkyl, sulfonamidoaryl, alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, aryl and heteroaryl;

Ry; is chosen from alkyl, aryl, alky'aryl, heteroaryl, alkyiheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; and

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

ora pharmaceutically acceptable salt thereof.

4, A compound according to claim 1, 2 or 3 wherein: R, is chosen from hydrogen, alkyl, aryl, substituted alkyl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl and substituted alkylheteroaryl; Rjis chosen from hydrogen, alkyl and substituted alkyl; R;’ is hydrogen; Ri is chosen from alkyl, substituted alkyl, alkylaryl, heteroaryl, aryl, substituted aryl, substituted hereroaryl, substituted oxaalkylaryl R;sO- and R,s-NH-; R4 is chosen from alkyl, aryl, alkylaryl, alkylheteroaryl, substituted alkyl, substituted aryl, and R,¢-alkylene-; Rs is hydrogen; Re, R7 and Rg are independently chosen from hydrogen, halogen, methyl, cyano and trifluoromethyl; Rs is chosen from alkyl, aryl and substituted aryl; Rye 1s chosen from alkoxy, amino, alkylamino, dialkylamino and N-heterocyclyl.

5. A compound according to claim 1, 2 or 3 wherein: R; is chosen from hydrogen, alkyl and substituted alkyl; R,’ is hydrogen; and the stereogenic center to which R; and R;’ are attached is of the R configuration.

6. A compound according to claim 1, 2 or 3 of the formula: 0} Rs mo] + N R; Ne Re Ra or a pharmaceutically acceptable salt thereof. -63- AMENDED SHEET

7. A compound according to claim 6 wherein R, is chosen from hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, alkylaryl and substituted alkylaryl.

8. A compound according to claim 7 wherein R, is chosen from hydrogen, ethyl, propyl, methoxyethyl, naphthyl, phenyl, bromophenyl, chloropheny!, methoxyphenyl, ethoxyphenyl, tolyl, dimethylphenyl, chorofluorophenyl, methylchlorophenyl, ethylphenyl, phenethyl, benzyl, chlorobenzyl, methylbenzyl, methoxybenzyl, tetrahydrofuranylmethyl and (ethoxycarbonyl)ethyl.

9. A compound according to claim 6 wherein R; is chosen from hydrogen, lower alkyl and substituted lower alkyl; R;’ is hydrogen; and the stereogenic center to which R; and R;’ are attached is of the R configuration.

10. A compound according to claim 9 wherein R; is chosen from hydrogen, methyl, ethyl, propyl, methylthioethyl, aminobutyl, (CBZ)aminobutyl, cyclohexylmethyl, benzyloxymethyl, methylsulfinylethyl, methylsulfinylmethyl, hydroxymethyl, benzyl and indolylmethyl.

11. A compound according to claim 6 wherein Rj is chosen from C,;-C,; alkyl; substituted lower alkyl; aryl, substituted aryl, alkylaryl, alkylheteroaryl, oxaalkylaryl, oxaalkylheteroaryl, substituted alkylaryl, substituted alkylheteroaryl, substituted oxaalkylaryl, and substituted oxaalkylheteroarlyl.

12. A compound according to claim 11 wherein Rj 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, biphenylyl, methylenedioxyphenyl, methylsulfonylphenyl, methoxychlorophenyl, chloronaphthyl, methylhalophenyl, trifluoromethylphenyl, butylphenyl, pentylphenyl, methylnitrophenyl, phenoxymethyi, dimethoxyphenyl, phenylvinyl, nitrochlorophenyl, nitrophenyl, dinitrophenyl, bis(trifluoromethyl)phenyl, benzyloxymethyl, benzyl, furanyl, benzofuranyl, pyridinyl, -64 - AMENDED SHEET indolyl, methylpyridinyl, quinolinyl, picolinyl, pyrazolyl, and imidazolyl.

13. A compound according to claim 6 wherein Rj is Rys-NH- and Rs is chosen from lower alkyl; cyclohexyl; phenyl; and phenyl substituted with halo, lower alkyl, loweralkoxy, or lower alkylthio.

14. A compound according to claim 13 wherein Rs is chosen from isopropyl, butyl, cyclohexyl, phenyl, bromophenyl, dichlorophenyl, methoxyphenyl, ethylphenyl, tolyl, trifluoromethylphenyl and methylthiophenyl.

15. A compound according to claim 6 wherein Ry is chosen from lower alkyl, substituted lower alkyl, cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl; heteroarylmethyl; heteroarylethyl; heteroarylpropyl and Rj¢-alkylene-, wherein R¢ is amino, lower alkylamino, di(lower alkyl)amino, lower alkoxy, or N-heterocyclyl.

16. A compound according to claim 15 wherein Ry is chosen from methyl, ethyl, propyl, butyl, cyclohexyl, carboxyethyl, carboxymethyl, methoxyethyl, hydroxyethyl, hydroxypropyl, dimethylaminoethyl, dimethylaminopropyl, diethylaminoethyl, diethylaminopropyl, aminopropyl, methylaminopropyl, 2,2- dimethyl-3-(dimethylamino)propyl, 1-cyclohexyl-4-(diethylamino)butyl, aminoethyl, aminobutyl, aminopentyl, aminohexyl, aminoethoxyethyl, isopropylaminopropyl, diisopropylaminoethyl, 1-methyl-4-(diethylamino)butyl, (t-Boc)aminopropyl, hydroxyphenyl, benzyl, methoxyphenyl, methylmethoxyphenyl, dimethylphenyl, tolyl, ethylphenyl, (oxopyrrolidinyl)propyl, (methoxycarbonyl)ethyl, benzylpiperidinyl, pyridinylethyl, pyridinylmethyl, morpholinylethyl, morpholinylpropyl, piperidinyl, azetidinylmethyl, azetidinylpropyl, pyrrolidinylethyl, pyrrolidinylpropyl, piperidinylmethyl, piperidinylethyl, imidazolylpropyl, imidazolylethyl, (ethylpyrrolidinyl)methyl, (methylpyrrolidinyl)ethyl, (methylpiperidinyl)propyl, (methylpiperazinyl)propyl, furanylmethyl and indolylethyl. -65 - AMENDED SHEET

17. A compound according to claim 6 wherein R, is chosen from lower alkyl, benzyl, substituted benzyl and substituted phenyl; R; is chosen from hydrogen, alkyl, substituted lower alkyl and benzyl; R;’ is hydrogen; Rjis chosen from substituted phenyl and naphthyl; R4 is chosen from substituted alkyl and R¢-alkylene-; Rs is hydrogen or halo Rs is hydrogen, methyl or halo; R7 is hydrogen, halo, lower alkyl, substituted lower alkyl, lower alkoxy or cyano; Rgis hydrogen or halo; and Ri¢ is chosen from di(lower alkylamino), (lower alkyl)amino, amino, N-heterocyclyl and substituted N-heterocyclyl.

18. A compound according to claim 1, 2 or 3 wherein Ris benzyl or halobenzyl, R; is chosen from ethyl and propyl; R;’ is hydrogen, Rj is substituted phenyl; R3: is substituted phenyl; Rs is substituted phenyl; Rs is -(CH2)mOH or -(CH;)pRi6 wherein mis 2 or 3 and p is 1-3; Rs is hydrogen; Rg is hydrogen; R; is halo; Rgis hydrogen; and Ri¢ 1s chosen from amino, propylamino, and azetidinyl.

19. A compound according to claim 18 wherein the stereogenic center to which R; and R;: are attached is of the R configuration. - 66 - AMENDED SHEET

20. A compound according to claim 1, 2 or 3 of the formula: 0 Rs Ry Re Sy Rr; R,' XN N R; N Rs i Sg, Ry or a pharmaceutically acceptable salt thereof.

21. A compound according to claim 20 wherein: Rj is chosen from hydrogen, lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, phenyl, naphthyl and substituted phenyl; R; is chosen from hydrogen, lower alkyl and substituted lower alkyl and R;’ is hydrogen; R3- 1s chosen from C,-C,; alkyl; phenyl; naphthyl; phenyl substituted with halo, lower alkyl, lower alkoxy, nitro, methylenedioxy, or trifluoromethyl; biphenylyl, benzyl and heteroaryl; and R4 1s chosen from lower alkyl, substituted lower alkyl, cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl; heteroarylmethyl, heteroarylethyl; heteroarylpropyl and R¢-alkylene, wherein Rye is amino, (lower alkyl)amino, di(lower alkyl)amino, lower alkoxy, or N- heterocyclyl.

22. A compound according to claim 20 wherein

R. is chosen from lower alkyl, benzyl, substituted benzyl and substituted phenyl; R; is hydrogen or lower alkyl; Ry’ is hydrogen; Rj: is chosen from substituted phenyl and naphthyl, Rs 1s Ryg-alkylene-, hydroxy lower alkyl or carboxy lower alkyl; Re and R; are chosen from hydrogen and halo; -67 - AMENDED SHEET

Rs and Rg are hydrogen; Ris is chosen from di(lower alkylamino), (lower alkyl)amino, amino, piperidinyl, azetidinyl pyrrolidinyl and morpholinyl.

23. A compound according to claim 1, 2 or 3 of the formula: lo) Rs Ri Re SN ow |] RJ’ NW N R; N Rs rd SR, or a pharmaceutically acceptable salt thereof.

24. A compound according to claim 23 wherein: R; is chosen from hydrogen, lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, phenyl, naphthyl and substituted phenyl; Rj is chosen from hydrogen, lower alkyl and substituted lower alkyl and R;’ is hydrogen; and Ry is chosen from lower alkyl, cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl; heteroarylmethyl; heteroarylethyl; heteroarylpropyl and R¢-alkylene, wherein R)¢ is di(lower alkyl)amino, alkylamino, amino, lower alkoxy, or N-heterocyclyl.

25. A compound according to claim 23 wherein R, is chosen from lower alkyl, benzyl, substituted benzyl and substituted phenyl; R; is hydrogen or lower alkyl, R,’ is hydrogen; Ris Ryg-alkylene-; Re and R; are chosen from hydrogen and halo; - 68 - AMENDED SHEET

Rs and Rg are hydrogen; and Rie is chosen from di(lower alkylamino), (lower alkyl)amino, amino, pyrrolidinyl, piperidinyl, imidazolyl and morpholinyl.

26. A compound according to claim 1, 2 or 3 of the formula: 0 Rs Ry Re Sy R, Ry AN N Ry N Re [ Ry or a pharmaceutically acceptable salt thereof.

27. A compound according to claim 26 wherein: R| is chosen from hydrogen, lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, phenyl, naphthyl and substituted phenyl; Rj is chosen from hydrogen, lower alkyl and substituted lower alkyl and R;’ is hydrogen; R; 1s chosen from C,-C,; alkyl; substituted lower alkyl; phenyl; naphthyl; phenyl substituted with halo, lower alkyl, lower alkoxy, nitro, methylenedioxy, or trifluoromethyl; biphenylyl; benzyl and heterocyclyl; and Rysis chosen from lower alkyl, substituted lower alkyl; cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl; substituted benzyl; heterocyclyl; heteroarylmethyl; heteroarylethyl; heteroarylpropyl and Rie- alkylene, wherein Re is di(lower alkyl)amino, (lower alkyl)amino, amino, lower alkoxy, or N- heterocyclyl. -69 - AMENDED SHEET

28. A compound according to claim 27 wherein R, is chosen from lower alkyl, benzyl, substituted benzyl and substituted phenyl; R; is hydrogen or lower alkyl; R;’ is hydrogen; Rj is chosen from substituted phenyl, heterocyclyl and naphthyl; Rs 1s chosen from subtituted benzyl, heterocyclyl substituted lower alkyl and R¢- alkylene-; Re and Ry are chosen from hydrogen and halo; Rs and Rg are hydrogen; Rye 1s chosen from di(lower alkylamino), (lower alkyl)amino, amino, pyrrolidinyl, azetidinyl piperidinyl, imidazolyl and morpholinyl.

29. A compound according to claim 28 wherein R; is benzyl, R; is ethyl; Rj’ is hydrogen; R;- is chosen from halophenyl, polyhalophenyl, tolyl, dimethylphenyl, methoxyphenyl, dimethoxyphenyl, cyanophenyl, trifluoromethylphenyl, trifluoromethoxyphenyl, bis(trifluoromethyl)phenyl, carboxyphenyl, t-butylphenyl, methoxycarbonylphenyl, piperidinyl and naphthyl, Rais chosen from substituted benzyl, piperidinyl, hydroxy (lower alkyl) and R- alkylene-; R¢ and R; are chosen from hydrogen and halo; Rs and Rg are hydrogen; Ri¢ 1s chosen from dimethylamino, amino, pyrrolidinyl and piperidinyl.

30. A compound according to claim 1 or 2 wherein said disease or disorder is chosen from the group consisting of cancer, hyperplasia, restenosis, and cardiac hypertrophy. -70 - AMENDED SHEET

31. A compound chosen from the group consisting of: fo) Rs 0 Rs Ry 1 Sn h MN . Fe JA | JA Re’ A Ry’ Ax N Ry N Ry 0 N Re N Re J a and Re wherein:

R is chosen from hydrogen, alkyl, alkylaryl, alkylheteroaryl, substituted alkyl, substituted alkylaryl, and substituted alkylhetexoaryl;

Ry and R;’ are independently chosen from hydrogen, alkyl, oxaalkyl, aryl, alkylaryl,

alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; or Ry and Ry’ taken together form a 3- to 7-membered ring;

R; is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, oxaalkyl, oxaalkylaryl, substituted oxsalkylaryl,

oxaalkyliheteroaryl, substituted oxaalkyfheteroaryl, RisO- and R;s-NH-;

Rs~ is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkytheteroaryl, substituted alkyl, alkylheteroaryl;

Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl,

substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, and Ris-alkylene-;

Rs, Rg, Ry and Rg are independently chosen from hydrogen, alkyl, alkoxy, halogen, fluoroalkyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkcylsulfonamido, sulfonamidoalkyl, sulfonamidoaryl, alkylthio, carboxyalkyl, carboxamido,

aminocarbonyl, aryl and heteroaryl; Rus is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkytheteroaryl,

substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkyTheteroaryl; and a. Ris is chosen from alkoxy, amino, alkylamino, dialkylamino, N-heterocyclyl and substituted N-heterocyclyl, or apharmaceutically acceptable salt thereof, with the proviso that when Rj; is R;s-NH-, both of R; and R, must be other than hydrogen.

32. A compound chosen from the group consisting of: © 0 Re Rin Re 0 Re N Ra Ry Re Ll ~ N Ry Ra Ry" Nn il Re N Ry N Re wi HOR wherein: . R, is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkyiheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; R; and R;’ are independently chosen from hydrogen, alkyl, axaalkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; or R; and R,’ taken together form a 3- to 7-membered ring; Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkyiheteroaryl, substituted alkylheteroaryl and R;s-NH-; Ry is chosen from hydrogen, alkyl, aryl, alkylary), heteroaryl, alkylheteroary), substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, and R,¢-alkylene-;

. -T-

= GS Rs, Rg, Ry and Ry are independently chosen from hydrogen, alkyl, alkoxy, halogen, fluoroalkyl, nitro, cyano, dialkylamino, alkyisulfonyl, alkylsuifonamido, sulfonamidoalkyl, sulfonamidoaryl, alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, aryl and heteroaryl; Rysis chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; and Ry¢ is chosen from alkoxy, amino, alkylamino, dialkylamino, N-heterocyclyl and substituted N-heterocyclyl, ora phannaceutically acceptable salt thereof.

33. A compound or salt according to claim 31 or 32 wherein: R; is chosen from hydrogen, alkyl, aryl, substituted alkyl, substituted aryl, heteroaryl, R; is chosen from hydrogen, alkyl and substituted alkyl; R,’ is hydrogen; and the : 15 stereogenic center to which R; and Ry’ are attached is of the R configuration. R; is chosen from alkyl, aryl, alkylaryl, heteroaryl, substituted aryl, substituted alkyl, substituted heteroaryl, oxaalkylaryl, substituted oxaalkylaryl, RisO- and Rys- NH-; . Ry is chosen from alkyl, aryl, alkylaryl, alkylheteroaryl, substituted alkyl, substituted aryl, and Ric-alkylene-; Rs is rydrogen, Re R7 and Ry are independently chosen from hydrogen, halogen, methyl and Rs is chosen from alkyl, aryl and substituted aryl; and Ry is chosen from alkoxy, amino, alkylamino, dialkylamino and N-heterocyclyl.

34. A compound according to claim 31 of formula: 0 Re . R Re "NN JA Ry" x N Ry 0) N J or a pharmaceutically acceptable salt thereof.

35. A compound or salt according to claim 34 wherein R, is chosen from hydrogen, lower alkyl, substituted lower alkyl, alkylaryl or substituted alkylaryl.

36. A compound or salt according to claim 35 wherein R; is chosen from hydrogen, ethyl, propyl, methoxyethyl, phenethyl, benzyl, chiorobenzyl, methylbenzyl, - methoxybenzyl, tetrahydrofuranyimethyl and (ethoxycarbonyl)ethyl.

37. A compound or salt according to claim 34 wherein R; is chosen from hydrogen, lower alkyl and substituted lower alkyl; Ra’ is hydrogen.; and the stereogenic center to which Ry and R,’ are attached is of the R configuration.

38. A compound or salt according to claim 37 wherein R; is chosen from ethyl, i-propyl, c-propyl or t-butyl.

39. A compound or salt according to claim 34 or 37 wherein R; is chosen from aryl, substituted aryl, alkylaryl, alkylheteroaryl, oxaalkylaryl, oxaalkylheteroaryl, substituted alkylaryl, substituted alkylheteroaryl, substituted oxaalkylaryl or substituted oxaalkyiheteroaryl.

40. A compound or salt according to claim 39 wherein Rj is chosen from phenyl, substituted phenyl, benzyl, substituted benzyl, phenylvinyl, substituted phenylvinyl, phenoxy lower alkyl and substituted phenoxy lower alkyl, furanyl, * benzofuranyl, pyridinyl, indolyl, methylpyridinyl, quinolinyl, picolinyl, pyrazolyl, and imidazolyl.

41. A compound or salt according to claim 40 wherein R; is chosen from halophenyl, dihalophenyl, cyanophenyl, halo(trifluoromethyl)phenyl, chlorophenoxymethyl, methoxyphenyl, carboxyphenyl, methyiphenyl, ethylphenyl, tolyl, biphenylyl, methylenedioxyphenyl, methylsulfonylphenyl, methoxychlorophenyl, methylhalophenyl, trifluoromethylphenyl, butylphenyl, pentylphenyl, methylnitrophenyl, dimethoxyphenyl, phenylvinyl, nitrochlorophenyl, : nitrophenyl, dinitrophenyl, bis(trifluoromethyl)phenyl,

42. A compound or salt according to claim 37 wherein R; is R;s-NH- where Rs is chosen from isopropyl, butyl, cyclohexyl, phenyl, bromophenyl, dichlorophenyl, methoxyphenyl, ethylphenyl, tolyl, trifluoromethyiphenyl and. methyithiophenyl.

43. A compound or salt according to claim 34, 37 or 39 wherein R, is chosen from lower alkyl, substituted lower alkyl, and Ri¢-alkylene-, wherein R¢ is amino, lower alkylamino, di(lower alkyl)amino, lower alkoxy, or N-heterocyclyl.

44, A compound or salt according to claim 34, 37 or 39 wherein Rs, Rg, Rs and Ry are chosen from hydrogen, halo, lower alkyl, substituted lower alkyl, lower alkoxy and cyano.

45. A compound or salt according to claim 44 wherein Rs, Rg and Ry are hydrogen.

46. A compound or salt according to claim 45 wherein R is halo.

CB

47. A compound or salt according to claim 34 wherein: R; is chosen from alkylaryl or substituted alkylaryl; Rq is lower alkyl; Ry’ is hydrogen; and the stereogenic center to which Rp and Ry’ are attached is of the R configuration; Ris substituted aryl; R4 is substituted alkyl; Rg is hydrogen; Rg is hydrogen; R; is halo or cyano; and Rgis hydrogen. }

48. The compound or salt according to claim 47 wherein: R; is benzyl or substituted benzyl; Rg is i-propyl; Rj is methyl- and/or halo-substituted phemyl; ' R, is 3-amino-n-propyl, Rs is hydrogen; Rg is hydrogen; Ry is chloro or cyano; and Ryis hydrogen.

49. The compound or salt according to claim 47 wherein: R, is benzyl; R; is i-propyl; Ris p-fluorophenyl; Ry is 3-amino-n-propyl; Rs is hydrogen; Rg is hydrogen, Rj is fluoro; and Rgis hydrogen.

MN ED

50. The compound or salt according to claim 47 wherein: R, is benzyl; R; is i-propyl; R; is p-fluorophenyl; Ry is 3-amino-n-propyl; Rs is hydrogen; Rg is hydrogen, Ry is chloro; and Ry is hydrogen.

51. The compound or salt according to claim 47 wherein: R; is benzyl, R, is i-propyl, Rg is 3-fluoro-4-methylphenyl; Rqis 3-amino-n-propyl; Rs is hydrogen; Rg is hydrogen; Ry is chloro; and Ryis hydrogen.

52. The compound or salt according to claim 47 wherein: R; is benzyl; R; is i-propyl; Ry is p-methylphenyl; Ris 3-amino-n-propyl; Ry is hydrogen; Rg is lrydrogen; Ris chloro; and Ryis hydrogen.

53. The compound or salt according to claim 47 wherein: R, is benzyl; R; is i-propyl; R; is p-methylphenyl; S Ry is 3-amino-n-propyl; Rs is hydrogen; Rg is hydrogen; R9 is cyano; and . . Rgishydrogen.

54. A compound according to claim 32 of formula: : 0 0) Rin Re JO Re! x . N Ry il Re or a pharmaceutically acceptable salt thereof.

55. A compound according to claim 54 wherein: 1S R, is chosen from hydrogen, lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, phenyl, naphthyl and substituted phenyl; R; is chosen from hydrogen, lower alkyl and substituted lower alkyl and R;’ is hydrogen; Ry: is chosen from C;~Cy3 alkyl; phenyl; naphthyl; phenyl substituted with halo, lower alkyl, lower alkoxy, nitro, methylenedioxy, or triftuoromethyl; biphenylyl, benzyl and heteroaryl; and R, is chosen from lower alkyl, substituted lower alkyl, cyclohexyl; phemyl substituted with hydroxy, lower alkoxy or lower alkyl, benzyl; heteroarylmethyl; heteroarylethyl; heteroarylpropyl and Rie-alkylene, wherein Ry is amino, (lower alkyljamino, di(lower alkyDamino, lower alkoxy, or N- heterocyclyl.

56. A compound according to claim 55 wherein: Ry is chosen from lower alkyl, benzyl, substituted benzyl and substituted phenyl, R; is hydrogen or lower alkyl; Ry’ is hydrogen; Ris chosen from substituted phenyl and naphthyl; Ru is Ry-alkylene-, hydroxy(lower alkyl) or carboxy (lower alkyl); Rj is hydrogen, fluoro, chloto or methyl; Rs, Rg and Rg are hydrogen, Ry is chosen from di(lower alkyl)amino, (lower alkyl)amino, amino, pyrrolidinyl and piperidinyl.

57. A compound according to claim 32 of formula: 1% 5] Ry Re Ny JA Re! xX N Ry N Re Ww “Sr, or a pharmaceutically acceptable salt thereof.

58. A compound ar salt according to claim 57 wherein: R, is chosen from hydrogen, lower alkyl, substituted lower alkyl, benzyl, substituted benzyl, phenyl, naphthyl and substituted phenyl; R; is chosen from hydrogen, lower alkyl and substituted lower alkyl and R,’ is hydrogen; and R, is chosen from lower alkyl, cyclohexyl; phenyl substituted with hydroxy, lower alkoxy or lower alkyl; benzyl; hoteroaryimethyl; heteroaryiethyl; heteroarylpropyl and Re-alkylene, wherein Rye is dilower alkyl)amino, (lower alkyl)amino, amino, lower alkaxy, or N-heterocyclyl.

& 0

59. A compound or salt according to claim 58 wherein: R, is chosen from lower alkyl, benzyl, substituted benzyl and substituted phenyl; Rj is hydrogen or lower alkyl; Ry’ is hydrogen; - RsisRjealkylene-; Rj is hydrogen, fluoro, chloro or methyl; Rs, Rs and Rg are hydrogen; Ris is chosen from di(lower alkylamino), (lower alkyl)amino, amino, pyrrolidinyl, piperidinyl, imidazolyl and morpholinyl.

60. A compound according to claim 31 of formula: 0 Re Ris, Re “ N Ry ~ Re or a pharmaceutically acceptable salt thereof.

61. A compound or salt according to claim 60 wherein: R is chosen from hydrogen, lower alkyl, substituted lower alkyl, alkylaryl or substituted alkylaryl; Rg is chosen from hydrogen, lower alkyl and substituted lower alkyl; Ry’ is hydrogen; and the stereogenic center to which Ra and R;’ are attached is of the R configuration; Ry» is chosen from aryl, substituted aryl, alkylaryl, alkylheteroaryl, oxaalkylaryl, oxaalkyiheteroaryl, substituted alkylaryl, substituted alkytheteroaryl, substituted oxaalkylaryl or substituted oxaalkylheteroaryl; and Ry is chosen from lower alkyl, substituted lower alkyl, and R,e-alkylene, wherein Rys is di(lower alkyl)amino, (lower alkyl)amino, amino, lower alkoxy, or N- heterocyclyl.

62. A compound substantially as herein described with reference to Figure

63. A method of screening for KSP kinesin modulators comprising: (a) combining a kinesin, a candidate bioactive agent and a compound chosen from the group consisting of: 0] Rs o] Rs R, Re R Re Ny NY R, | R, R,' AN Ry’ Nn N R; N R; (0) N Rg N Rg hd aS d Sg, Ra Rs 0 Rs R, Re 0 Rs Sn Rs | R; Re Ry’ Xe pa N Ry R, Ry’ NN N R N Ry iil SR, : N Rs Ry and nw Sg, wherein: Rj is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl, R; and R;’ are independently chosen from hydrogen, alkyl, oxaalkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; or R; and R,’ taken together form a 3- to 7-membered ring; Rj; is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, -81 - AMENDED SHEET substituted alkylheteroaryl, oxaalkyl, oxaalkylaryl, substituted oxaalkylaryl, oxaalkylheteroaryl, substituted oxaalkylheteroaryl, RisO- and Ris-NH-;

Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl,

substituted alkylheteroaryl and Rys-NH-;

Ryn is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkcylheteroaryl, substituted alkyl, . substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl;

Ru is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl,

substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, and Rig-alkylene-; .

Rs, Re, Ry and Ry sre independently chosen from hydrogen, alkyl, alkoxy, halogen, fluoroalkyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, sulfonamidoalkyl, sulfonamidoaryl, alkylthio, carboxyalkyl, carboxamido,

aminocarbomnyl, aryl and heteroaryl;

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

Rg is chosen from alkoxy, amino, alkylamino, dialkylamino, N-heterocyclyl and substituted N-heterocyclyl; and (b) determining the effect of said candidate bioactive agent on the activity of said kinesin.

64. A method of screening for compounds that bind to KSP kinesin comprising: (8) combining a kinesin, a candidate bioactive agent and a labeled compound chosen from the group consisting of: 0 Rs 0 Re R Re R Re Ny AN Ra Ra Re" x Re’ x N Ry N Ry 0 NG. . Ry N he NR, d NR, Re 0 Rs ANY Re 0 Re Ry Re Ry Ry! A Sy . wl Re’ x N N Ry iil NR, Re Re ad wo Sr,

wherein:

R, is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkyiheteroaryl,

substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl,

Ra and Ry’ are independently chosen from hydrogen, alkyl, oxaalkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl; orR; and Ry’

taken together form a 3- to 7-membered ring;

R, is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkyiheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl, oxaalkyl, oxaalkylaryl, substituted oxaalkylaryl, oxaalkylheteroaryl, substituted oxaalkyiheteroaryl, RysO- and R;s-NH-;

2 2

Ry is chosen from hydrogen, alkyl, ary}, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, substituted alkylheteroaryl and R;s-NH-;

Rj is chosen from alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryi, substituted alkyl,

S substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroaryl;

Ry is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, } substituted alkylheteroaryl, and Rs-alkylene-;

Rs, Re, R; and Ry are independently chosen from hydrogen, alkyl, alkoxy, halogen, flucroalkyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, . sulfonamidoalkyl, sulfonamidoaryl, alkylthio, carboxyalkyl, carboxamido,

aminocarbonyl, aryl and heteroaryl;

Ris is chosen from hydrogen, alkyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl,

substituted alkyl, substituted aryl, substituted alkylaryl, substituted heteroaryl, and substituted alkylheteroary}; and

Rye is chosen from alkoxy, amino, alkylamino, dialkylamino, N-heterocyclyl and "substituted N-heterocyclyl; and (b) determining the binding of said candidate bioactive agent to : said kinesin.