Compounds, compositions, and methods

FIELD OF THE INVENTION

0001This invention relates to compounds which are inhibitors of the mitotic kinesin KSP and are useful in the treatment of cellular proliferative diseases, for example cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders, fungal disorders, and inflammation.

BACKGROUND OF THE INVENTION

0002Among the therapeutic agents used to treat cancer are the taxanes and vinca alkaloids, which act on microtubules. 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.

0003Improvements 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 1 inhibitors. From both of these perspectives, mitotic kinesins are attractive targets for new anti-cancer agents.

0004Mitotic 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.

0005Among the mitotic kinesins which 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. Microinjection of antibodies directed against KSP into human cells prevents spindle pole separation during prometaphase, giving rise to monopolar spindles and causing mitotic arrest and induction of programmed cell death. KSP and related kinesins in other, non-human, organisms, bundle antiparallel microtubules and slide them relative to one another, thus forcing the two spindle poles apart. KSP may also mediate in anaphase B spindle elongation and focussing of microtubules at the spindle pole.

0006Human KSP (also termed HsEg5) has been described (<nplcit id="ncit0001" npl-type="s"><text>Blangy, et al., Cell, 83:1159-69 (1995</text></nplcit>); <nplcit id="ncit0002" npl-type="s"><text>Whitehead, et al., Arthritis Rheum., 39:1635-42 (1996</text></nplcit>); <nplcit id="ncit0003" npl-type="s"><text>Galgio et al.,'1. Cell Biol., 135:339-414 (1996</text></nplcit>); <nplcit id="ncit0004" npl-type="s"><text>Blangy, et al., J Biol. Chem., 272:19418-24 (1997</text></nplcit>); <nplcit id="ncit0005" npl-type="s"><text>Blangy, et al., Cell Motil Cytoskeleton, 40:174-82 (1998</text></nplcit>); <nplcit id="ncit0006" npl-type="s"><text>Whitehead and Rattner, J. Cell Sci., 111:2551-61 (1998</text></nplcit>);<nplcit id="ncit0007" npl-type="s"><text> Kaiser, et al., JBC 274:18925-31 (1999</text></nplcit>); GenBank accession numbers: X85137, NM004523 and U37426), and a fragment of the KSP gene (TRIP5) has been described (<nplcit id="ncit0008" npl-type="s"><text>Lee, et al., Mol Endocrinol., 9:243-54 (1995</text></nplcit>); GenBank accession number L40372). Xenopus KSP homologs (Eg5), as well as Drosophila KLP61 F/KRP1 30 have been reported. <patcit id="pcit0001" dnum="WO0198278A"><text>WO 01/98278</text></patcit> describes quinazolinones useful for treating cellular proliferative diseases and disorders associated with KSP kinesin activity

0007Mitotic kinesins, including KSP, are attractive targets for the discovery and development of novel antimitotic chemotherapeutics. Accordingly, it is an object of the present invention to provide compounds, compositions and methods useful in the inhibition

SUMMARY OF THE INVENTION

0008In accordance with the objects outlined above, the present invention provides compounds that can be used to treat cellular proliferative diseases. The compounds are KSP inhibitors, particularly human KSP inhibitors. The present invention also provides compositions comprising such compounds, and the use of such compounds or compositions in the manufacture of a medicament, which can be used to treat cellular proliferative diseases.

0009In one aspect, the invention relates to compounds for treating cellular proliferative diseases, and for treating disorders by inhibiting the activity of KSP. The methods employ compounds represented by Formula I: <chemistry id="chem0001" num="0001"><img file="EP1511734B1_D0001.tif" /></chemistry> wherein: <ul id="ul0001" list-style="none" compact="compact"><li>R₁ is chosen from hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, and optionally substituted heteroaralkyl-;</li><li>R₂ and R₂, are independently chosen from hydrogen, optionally substituted alkyl-, optionally substituted alkoxy, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, and optionally substituted heteroaralkyl-; or R₂ and R₂. taken together form an optionally substituted 3- to 7-membered ring;</li><li>R₁₂ is selected from the group consisting of optionally substituted imidazolyl, optionally substituted imidazolinyl, -NHR₄; -N(R₄)(COR₃); -N(R₄)(SO₂R_3a); and - N(R₄)(CH₂R_3b);</li><li>R₃ is chosen from hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, optionally substituted heteroaralkyl-, R₁₅O- and R₁₇-NH-;</li><li>R_3a is chosen from optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, optionally substituted heteroaralkyl-, and R₁₇-NH-;</li><li>R_3b is chosen from hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, and optionally substituted heteroaralkyl-;</li><li>R₄ is chosen from hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted hetercyclyl-, and optionally substituted heteroaralkyl-;</li><li>R₅, R₆, R₇ and R₈ are independently chosen from hydrogen, acyl, optionally substituted alkyl-, optionally substituted alkoxy, halogen, hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl-, alkylsulfonamido-, alkylthio-, carboxyalkyl-, carboxamido-, aminocarbonyl-, optionally substituted aryl and optionally substituted heteroaryl-;</li><li>R₁₅ is chosen from optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroatyl-, and optionally substituted heteroaralkyl-; and</li><li>R₁₇ is hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, or optionally substituted hetero-aralkyl-, including single stereoisomers, mixtures of stereoisomers; <ul id="ul0002" list-style="none" compact="compact"><li>a pharmaceutically acceptable salt of a compound of Formula I;</li><li>a pharmaceutically acceptable solvate of a compound of Formula I; or</li><li>a pharmaceutically acceptable solvate of a pharmaceutically acceptable salt of a compound of Formula I.</li></ul></li></ul>

0010According to one embodiment, when either R₂ or R₂, is hydrogen, the other is not hydrogen. In another embodiment, R₂ and R₂, are each hydrogen; and R₁ is chosen from optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl, provided however, that R₁ is not substituted phenyl.

0011In one aspect, the invention relates to compounds for treating cellular proliferative diseases and other disorders that can be treated by inhibiting KSP by the administration of a therapeutically effective amount of a compound of Formula I; a pharmaceutically acceptable salt of a compound of Formula I; or a pharmaceutically acceptable solvate of a pharmaceutically acceptable salt of a compound of Formula I. thereof. Such diseases and disorders include cancer, hyperplasia, restenosis, cardiac hypertrophy, immune disorders, fungal disorders and inflammation.

0012In another aspect, the invention relates to compounds useful in inhibiting KSP kinesin. The compounds have the structures shown above in Formula I; a pharmaceutically acceptable salt of a compound of Formula I; or a pharmaceutically acceptable solvate of a pharmaceutically acceptable salt of a compound of Formula I. The invention also relates to pharmaceutical compositions containing a therapeutically effective amount of a compound of Formula I; a pharmaceutically acceptable salt of a compound of Formula I; or a pharmaceutically acceptable solvate of a pharmaceutically acceptable salt of a compound of Formula I, admixed with at least one pharmaceutical excipient. In another aspect, the composition further comprises a chemotherapeutic agent other than a compound of the present invention.

0013In 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 a compound 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 agent to the KSP kinesin.

0014In a further aspect, the invention provides methods of screening for modulators of KSP kinesin activity. The methods comprise combining a compound of the invention, a KSP kinesin, and at least one candidate agent and determining the effect of the candidate agent on the KSP kinesin activity.

BRIEF DESCRIPTION OF THE FIGURES

0015The invention will be better understood by reference to the following description taken in conjunction with the accompanying drawings. <ul id="ul0003" list-style="none" compact="compact"><li><figref idref="f0001">Figures 1</figref> and <figref idref="f0002">2</figref> represent a XRPD and MDSC scan, respectively, of a salt prepared according to Preparation A.</li><li><figref idref="f0003">Figure 3</figref> represents an XRPD scan of a salt prepared according to Preparation B.</li></ul>

DETAILED DESCRIPTION OF THE INVENTION

Definitions

0016As used in the present specification, the following words and phrases are generally intended to have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise. The following abbreviations and terms have the indicated meanings throughout: <dl id="dl0001" compact="compact"><dt>Ac =</dt><dd>acetyl</dd><dt>Boc =</dt><dd>t-butyloxy carbonyl</dd><dt>Bu =</dt><dd>butyl</dd><dt>c- =</dt><dd>cyclo</dd><dt>CBZ =</dt><dd>carbobenzoxy = benzyloxycarbonyl</dd><dt>DCM =</dt><dd>dichloromethane = methylene chloride = CH₂Cl₂</dd><dt>DIEA =</dt><dd>N,N-diisopropylethylamine</dd><dt>DMF =</dt><dd>N,N-dimethylformamide</dd><dt>DMSO =</dt><dd>dimethyl sulfoxide</dd><dt>Et =</dt><dd>ethyl</dd><dt>HBTU =</dt><dd>O-benzotriazol-1-yl-N,N ,N',N'-tetramethyluronium hexafluorophosphate</dd><dt>HMDS =</dt><dd>hexamethyldisilazane</dd><dt>HOAc =</dt><dd>acetic acid</dd><dt>IPA =</dt><dd>isopropyl alcohol</dd><dt>Me =</dt><dd>methyl</dd><dt>Ph =</dt><dd>phenyl</dd><dt>Py =</dt><dd>pyridine</dd><dt>rt =</dt><dd>room temperature</dd><dt>sat'd =</dt><dd>saturated</dd><dt>s- =</dt><dd>secondary</dd><dt>t- =</dt><dd>tertiary</dd><dt>TEA =</dt><dd>triethylamine</dd><dt>TFA =</dt><dd>trifluoroacetic acid</dd><dt>THF =</dt><dd>tetrahydrofuran</dd><dt>Tf =</dt><dd>triflate</dd></dl>

0017<b>Alkyl</b> is intended to include linear, branched, or cyclic aliphatic hydrocarbon structures and combinations thereof, which structures may be saturated or unsaturated. <b>Lower-alkyl</b> refers to alkyl groups of from 1 to 5 carbon atoms, preferably from 1 to 4 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 C₂₀ or below. More preferred alkyl groups are those of C₁₃ or below. <b>Cycloalkyl</b> is a subset of alkyl and includes cyclic aliphatic hydrocarbon groups of from 3 to 13 carbon atoms. Examples of cycloalkyl groups include c-propyl, c-butyl, c-pentyl, norbornyl, adamantyl and the like. <b>Cycloalkyl-alkyl-</b> is another sublet of alkyl and refers to cycloalkyl attached to the parent structure through a non-cyclic alkyl. Examples of cycloalkyl-alkyl- include cyclohexylmethyl, cyclopropylmethyl, cyclohexylpropyl, and the like. In this application, alkyl includes alkanyl, alkenyl and alkynyl residues; it is intended to include vinyl, allyl, isoprenyl and the like. <b>Alkylene-, alkenylene-, and alkynylene-</b> are other subsets of alkyl, including the same residues as alkyl, but having two points of attachment within a chemical structure. Examples of alkylene include ethylene (-CH₂CH₂-), propylene (-CH₂CH₂CH₂-), dimethylpropylene ( -CH₂C(CH₃)₂CH₂-) and cyclohexylpropylene (-CH₂CH₂CH(C₆H₁₃)- ). Likewise, examples of alkenylene include ethenylene (-CH=CH-), propenylene (-CH=CH-CH₂-), and cyclohexylpropenylene (-CH=CHCH(C₆H₁₃)-). Examples of alkynylene include ethynylene (-C≡C-) and propynylene (-CH≡CH-CH₂-). 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, isopropyl, and c-propyl.

0018<b>Alkoxy</b> or <b>alkoxyl</b> refers to an alkyl group, preferably including from 1 to 8 carbon atoms, of a straight, branched, or cyclic configuration, or a combination thereof, attached to the parent structure through an oxygen (i.e., the group alkyl-O-). Examples include methoxy-, ethoxy-, propoxy-, isopropoxy-, cyclopropyloxy-, cyclohexyloxy- and the like. <b>Lower-alkoxy</b> refers to alkoxy groups containing one to four carbons.

0019<b>Acyl</b> refers to groups of from 1 to 8 carbon atoms of a straight, branched, or cyclic configuration or a combination thereof, attached to the parent structure through a carbonyl functionality. Such groups may be saturated or unsaturated, and aliphatic or aromatic. 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. <b>Lower-acyl</b> refers to acyl groups containing one to four carbons.

0020<b>Amino</b> refers to the group -NH₂. The term "substituted amino" refers to the group -NHR or -NRR where each R is independently selected from the group: optionally substituted alkyl, optionally substituted alkoxy, optionally substituted amino carbonyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl and sulfonyl, e.g., diethylamino, methylsulfonylamino, furanyl-oxy-sulfonamino.

0021<b>Aminocarbonyl</b>- refers to the group -NR^cCOR^b, -NR^cCO₂R^b ,or-NR^cCONR^bR^c, where R^b is H or optionally substituted C₁-C₆ alkyl, aryl, heteroaryl, aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl- group; and R^c is hydrogen or C₁-C₄ alkyl; and where each optionally substituted R^b group is independently unsubstituted or substituted with one or more substituents independently selected from C₁-C₄ alkyl, aryl, heteroaryl, aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, -OC₁-C₄ alkyl, -OC₁-C₄ alkylphenyl, -C₁-C₄ alkyl-OH, -OC₁-C₄ haloalkyl, halogen, -OH, -NH₂, -C₁-C₄ alkyl-NH₂, -N(C₁-C₄ alkyl)(C₁-C₄ alkyl), -NH(C₁-C₄ alkyl), -N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), -NH(C₁-C₄ alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), -CO₂H -C(O)OC₁-C₄ alkyl, -CON(C₁-C₄ alkyl)(C₁-C₄ alkyl), -CONH(C₁-C₄ alkyl), -CONH₂, -NHC(O)(C₁-C₄ alkyl), -NHC(O)(phenyl), -N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), -N(C₁-C₄ alkyl)C(O)(phenyl), -C(O)C₁-C₄ alkyl, -C(O)C₁-C₄ phenyl, -C(O)C₁-C₄ haloalkyl, -OC(O)C₁-C₄ alkyl, -SO₂(C₁-C₄ alkyl), - SO₂(phenyl), -SO₂(C₁-C₄ haloalkyl), -SO₂NH₂, -SO₂NH(C₁-C₄ alkyl), -SO₂NH(phenyl), NHSO₂(C₁-C₄ alkyl), -NHSO₂(phenyl and -NHSO₂(C₁-C₄ haloalkyl).

0022<b>Antimitotic</b> refers to a drug for inhibiting or preventing mitosis, for example, by causing metaphase arrest. Some antitumour drugs block proliferation and are considered antimitotics.

0023<b>Aryl</b> and <b>heteroaryl</b> mean a 5- or 6-membered aromatic or heteroaromatic ring containing 0 or 1-4 heteroatoms, respectively, selected from O, N, or S; a bicyclic 9- or 10-membered aromatic or heteroaromatic ring system containing 0 or 1-4 (or more) heteroatoms, respectively, selected from O, N, or S; or a tricyclic 12- to 14-membered aromatic or heteroaromatic ring system containing 0 or 1-4 (or more) heteroatoms, respectively, selected from O, N, or S. The aromatic 6- to 14-membered carbocyclic rings include, e.g., phenyl, naphthyl, indanyl, tetralinyl, and fluorenyl and the 5- to 10-membered aromatic heterocyclic rings include, e.g., imidazolyl, pyridinyl, indolyl, thienyl, benzopyranonyl, thiazolyl, furanyl, benzimidazolyl, quinolinyl, isoquinolinyl, quinoxalinyl, pyrimidinyl, pyrazinyl, tetrazolyl and pyrazolyl.

0024<b>Aralkyl-</b> refers to a residue in which an aryl moiety is attached to the parent structure via an alkyl residue. Examples include benzyl, phenethyl, phenylvinyl, phenylallyl and the like. <b>Heteroaralkyl-</b> refers to a residue in which a heteroaryl moiety is attached to the parent structure via an alkyl residue. Examples include furanylmethyl, pyridinylmethyl, pyrimidinylethyl and the like.

0025<b>Aralkoxy-</b> refers to the group -O-aralkyl. Similarly, <b>heteroaralkoxy-</b> refers to the group -O-heteroaralkyl; <b>aryloxy-</b> refers to the group -O-aryl; <b>acyloxy-</b> refers to the group -O-acyl; <b>heteroaryloxy-</b> refers to the group -O-heteroaryl; and <b>heterocyclyloxy-</b> refers to the group -O-heterocyclyl (i.e., aralkyl, heteroaralkyl, aryl, acyl, heterocyclyl, or heteroaryl is attached to the parent structure through an oxygen).

0026<b>Carboxyalkyl-</b> refers to the group -alkyl-COOH.

0027<b>Carboxamido</b> refers to the group -CONR^bR^c, where R^b is H or optionally substituted C₁-C₆ alkyl, aryl, heteroaryl, aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl- group; and R^c is hydrogen or C₁-C₄ alkyl; and where each optionally substituted R^b group is independently unsubstituted or substituted with one or more substituents independently selected from C₁-C₄ alkyl, aryl, heteroaryl, aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, -OC₁-C₄ alkyl, -OC₁-C₄ alkylphenyl, -C₁-C₄ alkyl-OH, -OC₁-C₄ haloalkyl, halogen, -OH, -NH₂, -C₁-C₄ alkyl-NH₂, -N(C₁-C₄ alkyl)(C₁-C₄ alkyl), -NH(C₁-C₄ alkyl), -N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), -NH(C₁-C₄ alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), -CO₂H -C(O)OC₁-C₄ alkyl, -CON(C₁-C₄ alkyl)(C₁-C₄ alkyl), -CONH(C₁-C₄ alkyl), -CONH₂, -NHC(O)(C₁-C₄ alkyl), -NHC(O)(phenyl), -N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), -N(C₁-C₄ alkyl)C(O)(phenyl), -C(O)C₁-C₄ alkyl, -C(O)C₁-C₄ phenyl, -C(O)C₁-C₄ haloalkyl, -OC(O)C₁-C₄ alkyl, -SO₂(C₁-C₄ alkyl), - SO₂(phenyl), -SO₂(C₁-C₄ haloalkyl), -SO₂NH₂, -SO₂NH(C₁-C₄ alkyl), -SO₂NH(phenyl), - NHSO₂(C₁-C₄ alkyl), -NHSO₂(phenyl and -NHSO₂(C₁-C₄ haloalkyl).

0028<b>Halogen</b> or <b>halo</b> refers to fluorine, chlorine, bromine or iodine. Fluorine, chlorine and bromine are preferred. Dihaloaryl, dihaloalkyl, trihaloaryl etc. refer to aryl and alkyl substituted with the designated plurality of halogens (here, 2, 2 and 3, respectively), but not necessarily a plurality of the same halogen; thus 4-chloro-3-fluorophenyl is within the scope of dihaloaryl.

0029<b>Heterocyclyl</b> 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 azetidinyl, imidazolinyl, pyrrolidinyl, pyrazolyl, pyrrolyl, indolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, benzofuranyl, benzodioxanyl, benzodioxyl (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazolyl, morpholinyl, thiazolyl, pyridinyl, pyridazinyl, piperidinyl, pyrimidinyl, thienyl, furanyl, oxazolyl, oxazolinyl, isoxazolyl, dioxanyl, tetrahydrofuranyl and the like. "N-heterocyclyl" refers to a nitrogen-containing heterocycle. The term heterocyclyl encompasses heteroaryl, which is a subset of heterocyclyl. Examples of N-heterocyclyl residues include azetidinyl, 4-morpholinyl, 4-thiomorpholinyl, 1-piperidinyl, 1-pyrrolidinyl, 3-thiazolidinyl, piperazinyl and 4-(3,4-dihydrobenzoxazinyl). Examples of substituted heterocyclyl include 4-methyl-1-piperazinyl and 4-benzyl-1-piperidinyl.

0030A <b>leaving group</b> or <b>atom</b> is any group or atom that will, under the reaction conditions, cleave from the starting material, thus promoting reaction at a specified site. Suitable examples of such groups unless otherwise specified are halogen atoms, mesyloxy, p-nitrobenzensulphonyloxy and tosyloxy groups.

0031<b>Optional</b> or <b>optionally</b> means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstances occurs and instances in which it does not. For example, "optionally substituted alkyl" includes "alkyl" and "substituted alkyl" as defined herein. 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 that are sterically impractical and/or synthetically non-feasible and/or inherently unstable.

0032<b>Substituted alkoxy</b> refers to alkoxy wherein the alkyl constituent is substituted (i.e., -O-(substituted alkyl)). One preferred substituted alkoxy group is "polyalkoxy" or -O-(optionally substituted alkylene)-(optionally substituted alkoxy), and includes groups such as -OCH₂CH₂OCH₃, and residues of glycol ethers such as polyethyleneglycol, and -O(CH₂CH₂O)_xCH₃, where x is an integer of about 2-20, preferably about 2-10, and more preferably about 2-5. Another preferred substituted alkoxy group is hydroxyalkoxy or -OCH₂(CH₂)_yOH, where y is an integer of about 1-10, preferably about 1-4.

0033<b>Substituted</b>- alkyl, aryl, and heteroaryl, which includes the substituted alkyl, aryl and heteroaryl moieties of any group containing an optionally substituted alkyl, aryl and heteroaryl moiety (e.g., alkoxy, aralkyl and heteroaralkyl), refer respectively to alkyl, aryl, and heteroaryl wherein one or more (up to about 5, preferably up to about 3) hydrogen atoms are replaced by a substituent independently selected from the group: <ul id="ul0004" list-style="dash" compact="compact"><li>R^a, -OR^b, -O(C₁-C₂ alkyl)O- (as an aryl substituent), -SR^b, -NR^bR^c, halogen, cyano, nitro, -COR^b, -CO₂R^b, -CONR^bR^c, -OCOR^b, -OCO₂R^b, -OCONR^bR^c, -NR^cCOR^b, -NR^cCO₂R^b, -NR^cCONR^bR^c, -CO₂R^b, -CONR^bR^c, -NR^cCOR^b, -SOR^a, -SO₂R^a, -SO₂NR^bR^c, and -NR^cSO₂R^a, where R^a is an optionally substituted C₁-C₆ alkyl, aryl, heteroaryl, aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl- group, R^b is H or optionally substituted C₁-C₆ alkyl, aryl, heteroaryl, aryl-C₁-C₄ alkyl-, or heteroaryl-C₁-C₄ alkyl- group; R^c is hydrogen or C₁-C₄ alkyl; where each optionally substituted R^a group and R^b group is independently unsubstituted or substituted with one or more substituents independently selected from C₁-C₄ alkyl, aryl, heteroaryl, aryl-C₁-C₄ alkyl-, heteroaryl-C₁-C₄ alkyl-, C₁-C₄ haloalkyl, -OC₁-C₄ alkyl, -OC₁-C₄ alkylphenyl, -C₁-C₄ alkyl-OH, -OC₁-C₄ haloalkyl, halogen, -OH, -NH₂, -C₁-C₄ alkyl-NH₂, -N(C₁-C₄ alkyl)(C₁-C₄ alkyl), -NH(C₁-C₄ alkyl), -N(C₁-C₄ alkyl)(C₁-C₄ alkylphenyl), -NH(C₁-C₄ alkylphenyl), cyano, nitro, oxo (as a substitutent for heteroaryl), -CO₂H, -C(O)OC₁-C₄ alkyl, -CON(C₁-C₄ alkyl)(C₁-C₄ alkyl), -CONH(C₁-C₄ alkyl), -CONH₂, -NHC(O)(C₁-C₄ alkyl), -NHC(O)(phenyl), -N(C₁-C₄ alkyl)C(O)(C₁-C₄ alkyl), -N(C₁-C₄ alkyl)C(O)(phenyl), -C(O)C₁-C₄ alkyl, -C(O)C₁-C₄ phenyl, -C(O)C₁-C₄ haloalkyl, -OC(O)C₁-C₄ alkyl, -SO₂(C₁-C₄ alkyl), - SO₂(phenyl), -SO₂(C₁-C₄ haloalkyl), -SO₂NH₂ -SO₂NH(C₁-C₄ alkyl), -SO₂NH(phenyl), - NHSO₂(C₁-C₄ alkyl), -NHSO₂(phenyl), and -NHSO₂(C₁-C₄ haloalkyl).</li></ul>

0034<b>Sulfanyl</b> refers to the groups: -S-(optionally substituted alkyl), -S-(optionally substituted aryl), -S-(optionally substituted heteroaryl), and -S-(optionally substituted heterocyclyl).

0035<b>Sulfinyl</b> refers to the groups: -S(O)-H, -S(O)-(optionally substituted alkyl), <ul id="ul0005" list-style="dash" compact="compact"><li>S(O)-optionally substituted aryl), -S(O)-(optionally substituted heteroaryl), -S(O)-(optionally substituted heterocyclyl); and -S(O)-(optionally substituted amino).</li></ul>

0036<b>Sulfonyl</b> refers to the groups: -S(O₂)-H, -S(O₂)-(optionally substituted alkyl), -S(O₂)-optionally substituted aryl), -S(O₂)-(optionally substituted heteroaryl), -S(O₂)-(optionally substituted heterocyclyl), -S(O₂)-(optionally substituted alkoxy), -S(O₂)-optionally substituted aryloxy), -S(O₂)-(optionally substituted heteroaryloxy), -S(O₂)-(optionally substituted heterocyclyloxy); and -S(O₂)-(optionally substituted amino).

0037<b>Pharmaceutically acceptable salts</b> refers to those salts that retain the biological effectiveness of the free compound and that are not biologically or otherwise undesirable, formed with a suitable acid or base, and includes pharmaceutically acceptable acid addition salts and base addition salts. <b>Pharmaceutically acceptable acid addition salts</b> include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and those derived from 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.

0038<b>Pharmaceutically acceptable base addition salts</b> 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. Base additioin salts also include those derived from pharmaceutically acceptable organic non-toxic bases, including salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

0039<b>Protecting group</b> has the meaning conventionally associated with it in organic synthesis, i.e. a group that selectively blocks one or more reactive sites in a multifunctional compound such that a chemical reaction can be carried out selectively on another unprotected reactive site and such that the group can readily be removed after the selective reaction is complete. A variety of protecting groups are disclosed, for example, in <nplcit id="ncit0009" npl-type="b"><text>T.H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley &amp; Sons, New York (1999</text></nplcit>), which is incorporated herein by reference in its entirety. For example, a hydroxy protected form is where at least one of the hydroxyl groups present in a compound is protected with a hydroxy protecting group. Likewise, amines and other reactive groups may similarly be protected.

0040<b>Solvate</b> refers to the compound formed by the interaction of a solvent and a compound of Formula I or salt thereof. Suitable solvates of the compounds of the Formula I are pharmaceutically acceptable solvates, such as hydrates, including monohydrates and hemi-hydrates.

0041Many of the compounds described herein contain one or more asymmetric centers (e.g. the carbon to which R₂ and R₂, are attached where R₂ differs from R_2') 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 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 and rotational isomers are also intended to be included.

0042When 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 crystallization; via formation of diastereoisomeric derivatives which may be separated, for example, by crystallization, 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 one enantiomer to the other by asymmetric transformation.

Compounds of the Present Invention

0043The present invention is directed to a class of novel compounds, which can be described as benzopyran-4-ones or chromen-4-ones, that are inhibitors of one or more mitotic kinesins. By inhibiting mitotic kinesins, but not other kinesins (e.g., transport kinesins), specific inhibition of cellular proliferation is accomplished. While not intending to be bound by any theory, 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. According to one embodiment of the invention, the compounds described herein inhibit the mitotic kinesin, KSP.

0044The compounds of the invention for inhibiting a human KSP kinesin contact a kinesin, particularly a human kinesin, preferably human KSP or fragments and variants thereof 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.

0045An object of the present invention is to develop inhibitors of mitotic kinesins, in particular KSP and especially human KSP, for the treatment of disorders associated with cell proliferation. Traditionally, dramatic improvements in the treatment of cancer, one type of cellular 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 compounds, compositions and methods described herein can differ in their selectivity and are preferably used to treat diseases of cellular proliferation, including, but not limited to cancer, hyperplasias, restenosis, cardiac hypertrophy, immune disorders, fungal disorders and inflammation.

0046Accordingly, the present invention relates to the use of the compounds represented by Formula I for the preparation of a medicament: <chemistry id="chem0002" num="0002"><img file="EP1511734B1_D0002.tif" /></chemistry> wherein: <ul id="ul0006" list-style="none" compact="compact"><li>R₁ is chosen from hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, and optionally substituted heteroaralkyl-;</li><li>R₂ and R₂, are independently chosen from hydrogen, optionally substituted alkyl-, optionally substituted alkoxy, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, and optionally substituted heteroaralkyl-; or R₂ and R₂, taken together form an optionally substituted 3- to 7-membered ring;</li><li>R₁₂ is selected from the group consisting of optionally substituted imidazolyl, optionally substituted imidazolinyl, -NHR₄; -N(R₄)(COR₃); -N(R₄)(SO₂R_3a); and - N(R₄)(CH₂R_3b);</li><li>R₃ is chosen from hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, optionally substituted heteroaralkyl-, R₁₅O- and R₁₇-NH-;</li><li>R_3a is chosen from optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, optionally substituted heteroaralkyl-, and R₁₇-NH-;</li><li>R_3b is chosen from hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, and optionally substituted heteroaralkyl-;</li><li>R₄ is chosen from hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted hetercyclyl-, and optionally substituted heteroaralkyl-;</li><li>R₅, R₆, R₇ and R₈ are independently chosen from hydrogen, acyl, optionally substituted alkyl-, optionally substituted alkoxy, halogen, hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl-, alkylsulfonamido-, alkylthio-, carboxyalkyl-, carboxamido-, aminocarbonyl-, optionally substituted aryl and optionally substituted heteroaryl-;</li><li>R₁₅ is chosen from optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, and optionally substituted heteroaralkyl-, and</li><li>R₁₇ is hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heteroaryl-, or optionally substituted heteroaralkyl-, including single stereoisomers, mixtures of stereoisomers; a pharmaceutically acceptable salt of a compound of Formula I; a pharmaceutically acceptable solvate of a compound of Formula I; or a pharmaceutically acceptable solvate of a pharmaceutically acceptable salt of a compound of Formula I.</li></ul>

0047When R₁₂ is an imidazole, R₁₂ has the formula: <chemistry id="chem0003" num="0003"><img file="EP1511734B1_D0003.tif" /></chemistry> wherein R₉ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl, optionally substituted aryl, optionally substituted aryl-C₁-C₄-alkyl -, optionally substituted heteroaryl-C₁-C₄-alkyl -, optionally substituted aryl-C₁-C₄-alkoxy -, optionally substituted heteroaryl-C₁-C₄-alkoxy -, optionally substituted heteroaryl-; and R₁₃ and R₁₃, are independently hydrogen, optionally substituted C₁-C₈ alkyl, optionally substituted aryl, or optionally substituted aryl-C₁-C₄-alkyl-

0048When R₁₂ is an imidazoline, R₁₂ has the formula <chemistry id="chem0004" num="0004"><img file="EP1511734B1_D0004.tif" /></chemistry> wherein R₉ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl, optionally substituted aryl, optionally substituted aryl-C₁-C₄-alkyl -, and optionally substituted heteroaryl-; and R₁₀, R_10', R₁₄, and R₁₄, are independently chosen from hydrogen, optionally substituted C₁-C₈ alkyl, optionally substituted aryl, and optionally substituted aryl-C₁-C₄-alkyl -,

0049In one embodiment, R₁ is chosen from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl-; R₂ and R₂, are independently chosen from hydrogen, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl-; or R₂ and R₂, taken together form an optionally substituted 3- to 7-membered ring, provided that if either R₂ or R_2' is hydrogen, then the other is not hydrogen; R₁₂ is selected from the group consisting of optionally substituted imidazolyl, optionally substituted imidazolinyl, -NHR₄; -N(R₄)(COR₃); -N(R₄)(SO₂R_3a); and - N(R₄)(CH₂R_3b); R₃ is chosen from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, R₁₅O- and R₁₇-NH-; R_3a is chosen from optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, and R₁₇-NH-; R_3b is chosen from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl-; R₄ is chosen from hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted hetercyclyl-, and optionally substituted heteroaralkyl-; R₅, R₆, R₇ and R₈ are independently chosen from hydrogen, acyl, optionally substituted alkyl, optionally substituted alkoxy, halogen, hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, optionally substituted aryl and optionally substituted heteroaryl-; R₁₅ is chosen from optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl-; and R₁₇ is chosen from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl, including single stereoisomers, mixtures of stereoisomers; a pharmaceutically acceptable salt of a compound of Formula I; a pharmaceutically acceptable solvate of a compound of Formula I; or a pharmaceutically acceptable solvate of a pharmaceutically acceptable salt of a compound of Formula I.

0050In another embodiment, R₂ and R₂, are hydrogen; and R₁ is chosen from optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl, provided however, that R₁ is not substituted phenyl; R₁₂ is selected from the group consisting of optionally substituted imidazolyl, optionally substituted imidazolinyl, -NHR₄; -N(R₄)(COR₃); -N(R₄)(SO₂R_3a); and - N(R₄)(CH₂R_3b); R₃ is chosen from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, R₁₅O- and R₁₇-NH-; R_3a is chosen from optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, optionally substituted heteroaralkyl, and R₁₇-NH-; R_3b is chosen from hydrogen, optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl-, and optionally substituted heteroaralkyl-; R₄ is chosen from hydrogen, optionally substituted alkyl-, optionally substituted aryl-, optionally substituted aralkyl-, optionally substituted heterocyclyl-, and optionally substituted heteroaralkyl-; R₅, R₆, R₇ and R₈ are independently chosen from hydrogen, acyl, optionally substituted alkyl, optionally substituted alkoxy, halogen, hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, optionally substituted aryl and optionally substituted heteroaryl-; R₁₅ is chosen from optionally substituted alkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl-; and R₁₇ is chosen from hydrogen, optionally substituted alkyl, optionally substituted aryl, optional ly substituted aralkyl, optionally substituted heteroaryl, and optionally substituted heteroaralkyl, including single stereoisomers, mixtures of stereoisomers; a pharmaceutically acceptable salt of a compound of Formula I; a pharmaceutically acceptable solvate of a compound of Formula I; or a pharmaceutically acceptable solvate of a pharmaceutically acceptable salt of a compound of Formula I. In a particularly preferred embodiment, where R₂ differs from R₂: the stereogenic center to which R₂ and R₂, are attached is of the R configuration.

<u>Nomenclature</u>

0051The compounds of Formula I can be named and numbered in the manner (e.g., using AutoNom version 2.1 or ISIS-DRAW, each of which utilizes the IUPAC system of nomenclature) described below. For example, the compound: <chemistry id="chem0005" num="0005"><img file="EP1511734B1_D0005.tif" /></chemistry> i.e., the compound according to Formula I where R₁ is benzyl, R₂ is propyl (particularly i - propyl), R₂. is hydrogen; R₁₂ is -N(R₄)(COR₃); R₃ is 3,4-dimethylphenyl-; R₄ is 3-aminopropyl-; R₅, R₆, and R₈ are hydrogen; and R₇ is cyano is named N-(3-amino-propyl)-N-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-3,4-dimethyl-benzamide.

0052Likewise, the compound: <chemistry id="chem0006" num="0006"><img file="EP1511734B1_D0006.tif" /></chemistry> i.e., the compound according to Formula I where R₁ is 3-methoxy-benzyl, R₂ is propyl (particularly i -propyl), R₂. is hydrogen; R₁₂ is substituted imidazoline; R₅, R₆, and R₈ are hydrogen; R₇ is chloro; R₉ is methylenedioxyphenyl-; R₁₀, R₁₀, R₁₄, and R_14' are hydrogen can be named 2-[1-(2-benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-chloro-3-(3 -methoxy-benzyl)-chromen-4-one.

Synthesis of the Compounds of Formula I

0053The compounds of Formula I can be prepared by following the procedures described with reference to the Reaction Schemes below or utilizing techniques well known in the art. See, for example, <nplcit id="ncit0010" npl-type="s"><text>Hirao et al. (1984) Synthesis 1076-1078</text></nplcit> and <nplcit id="ncit0011" npl-type="s"><text>Coppola et al. (1981) Synthesis 523-526</text></nplcit>.

0054Unless specified otherwise, the terms "solvent", "inert organic solvent" or "inert solvent" mean a solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran ("THF"), dimethylformamide ("DMF"), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, pyridine and the like]. Unless specified to the contrary, the solvents used in the reactions of the present invention are inert organic solvents.

0055The term "q.s." means adding a quantity sufficient to achieve a stated function, e.g., to bring a solution to the desired volume (i.e., 100%).

0056In general, esters of carboxylic acids may be prepared by conventional esterification procedures, for example alkyl esters may be prepared by treating the required carboxylic acid with the appropriate alkanol, generally under acidic conditions. Likewise, amides may be prepared using conventional amidation procedures, for example amides may be prepared by treating the relevant carboxylic acid with the appropriate amine. Alternatively, a lower-alkyl ester such as a methyl ester of the acid may be treated with an amine to provide the required amide, optionally in presence of trimethylalluminium following the procedure described in <nplcit id="ncit0012" npl-type="s"><text>Tetrahedron Lett. 48, 4171-4173, (1977</text></nplcit> Carboxyl groups may be protected as alkyl esters, for example methyl esters, which esters may be prepared and removed using conventional procedures, one convenient method for converting carbomethoxy to carboxyl is to use aqueous lithium hydroxide.

0057The salts and solvates of the compounds mentioned herein may as required be produced by methods conventional in the art. For example, if an inventive compound is an acid, a desired base addition salt can be prepared by treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary); an alkali metal or alkaline earth metal hydroxide; or the like. Illustrative examples of suitable salts include organic salts derived from amino acids such as glycine and arginine; ammonia; primary, secondary, and tertiary amines; such as ethylenediamine, and cyclic amines, such as cyclohexylamine, piperidine, morpholine, and piperazine; as well as inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

0058If a compound is a base, a desired acid addition salt may be prepared by any suitable method known in the art, including treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, such as glucuronic acid or galacturonic acid, alpha-hydroxy acid, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, or the like.

0059Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the examples hereinbelow. However, other equivalent separation or isolation procedures can, of course, also be used.

Brief Description Of Reaction Schemes

0060Reaction Scheme 1 illustrates a synthesis of compounds of formula 109, an intermediate in the synthesis of compounds of Formula I.

0061Reaction Scheme 2 illustrates a synthesis of compounds of Formula I wherein R₁₂ is -N(R₄)(COR₃).

0062Reaction Scheme 3 illustrates a synthesis of compounds of Formula I wherein R₇ is -OH.

0063Reaction Scheme 4 illustrates a synthesis of compounds of Formula I wherein R₇ is -OCH₃.

0064Reaction Scheme 5 illustrates another synthesis of compounds of Formula I wherein R₁₂ is -N(R₄)(COR₃).

0065Reaction Scheme 6 illustrates a synthesis of compounds of Formula I wherein R₁₂ is -N(R₄)(SO₂R_3a).

0066Reaction Scheme 7 illustrates a synthesis of compounds of Formula I wherein R₁₂ is -N(R₄)(CH₂R_3b).

0067Reaction Scheme 8 illustrates a synthesis of compounds of Formula I wherein R₁₂ is optionally substituted imidazolyl.

0068Reaction Scheme 9 illustrates another synthesis of compounds of Formula I wherein R₁₂ is optionally substituted imidazolyl.

0069Reaction Scheme 10 illustrates a synthesis of compounds of Formula I wherein R₁₂ is optionally substituted imidazolinyl.

0070Reaction Scheme 11 illustrates a second synthesis of compounds of Formula I wherein R₁₂ is optionally substituted imidazolinyl.

0071Reaction Scheme 12 illustrates a synthesis of compounds of Formula I wherein R₁₂ is -N(R₄)(COR₃) wherein R₃ is -OR₁₅.

0072Reaction Scheme 13 illustrates a synthesis of compounds of Formula I wherein R₁₂ is -N(R₄)(COR₃) wherein R₃ is -NHR₁₇.

0073Reaction Scheme 14 illustrates a synthesis of compounds of Formula 1407 which can be used as an intermediate in the synthesis of compounds of Formula I.

0074Reaction Scheme 15 illustrates a synthesis of compounds of Formula 1505 which can be used as an intermediate in the synthesis of compounds of Formula I.

Starting Materials

0075The optionally substituted compounds of Formula 101 are commercially available, e.g., from Aldrich Chemical Company, Milwaukee, WI. Other reactants are likewise commercially available or may be readily prepared by those skilled in the art using commonly employed synthetic methodology. <chemistry id="chem0007" num="0007"><img file="EP1511734B1_D0007.tif" /></chemistry>

Preparation of Compounds of Formula 103

0076Referring to Reaction Scheme 1, Step 1, about an equivalent of ethyl chloroformate is added over about one minute to a 0-5°C solution of a compound of Formula 101 (preferably wherein the amino protecting group, PG, is a Boc group) and a base such as triethylamine in a nonpolar, aprotic solvent such as THF. After about 15 minutes, a mixture of an excess of dimethylhydroxylamine hydrochloride (preferably about 1.2 equivalents) and a base such as triethylamine in a nonpolar, aprotic solvent such as THF is added over about 5 minutes. The product, a compound of Formula 103, is isolated and used without further purification.

Preparation of Compounds of Formula 105

0077Referring to Reaction Scheme 1, Step 2, a Grignard reagent is prepared by mixing a compound of formula R₁CH₂Br (generally about 3 equivalents) and magnesium turnings in a nonpolar, aprotic solvent such as diethyl ether. After about 1.5 hours, the Grignard reaction is generally complete. A solution of a compound of Formula 103 in a nonpolar, aprotic solvent such as ether, is added to the Grignard reagent. The temperature should be monitored and not allowed to exceed ∼30°C. The product, a compound of Formula 105, is isolated and purified.

Preparation of Compounds of Formula 107

0078Referring to Reaction Scheme 1, Step 3, lithium bis(trimethylsilyl)amide (about 3.3 equivalents) is added slowly over ∼3 minutes to a -78°C solution of a compound of Formula 105 in a nonpolar, aprotic solvent such as THF. The reaction solution temperature should be monitored and the addition of base conducted at a rate sufficient to prevent the temperature from exceeding about -54°C. After the addition is complete, the resulting solution is maintained at -78°C for about 30 minutes. An acid chloride of Formula 106 (preferably, neat) is then added. The reaction solution is maintained at -78°C for about 30 minutes. The product is isolated and used without further purification.

0079A mixture of the above crude product, a base such as potassium carbonate, and a polar, aprotic solvent such as DMF is maintained at about room temperature for about 30 minutes. The product, a compound of Formula 107 is isolated and purified.

Preparation of Compounds of Formula 109

0080Referring to Reaction Scheme 1, Step 4, optionally, the protecting group, PG, may be removed from the amine. One of skill in the art will appreciate that the conditions for removal of the protecting group will vary with different protecting groups. Such conditions are well known in the art and can be found, e.g., in Greene et al. <i>supra.</i> When PG is Boc, it may be removed by treatment of a compound of Formula 107 with a mixture of aqueous TFA (preferably TFA:H₂O, 97.5:2.5) at room temperature. The product, a compound of Formula 109, is isolated and purified.

Preparation of Optimally Active Compounds

0081In compounds of the invention where R₂ differs from R_2', a particular stereo configuration (such as the (R) isomer) may be preferred at the stereogenic center to which R₂ and R_2' are attached. The optically active compound can be prepared by methods known in the art. For example, an amine of Formula 109 is dissolved in an inert organic solvent (such as IPA) and warmed to 60°C. In a separate vessel, a resolving agent (such as dibenzoyl-D-tartaric acid) is dissolved, preferably in the same warm solvent, and then quickly added (with agitation) to the warm amine solution. The reaction mixture is left to crystallize by cooling to room temperature over 16 hours under continuing agitation. The desired isomer, e.g., the (R) isomer of a compound of Formula 109, is isolated and purified.

0082For the sake of brevity in the remaining description of the synthesis of compounds of Formula I, it should be understood that either single isomer or a mixture of isomers may be employed to give the corresponding product. <chemistry id="chem0008" num="0008"><img file="EP1511734B1_D0008.tif" /></chemistry><chemistry id="chem0009" num="0009"><img file="EP1511734B1_D0009.tif" /></chemistry>

Preparation of Formula 203

0083Referring to Reaction Scheme 2, Step 1, to a solution of a compound of Formula 109 is added successively a slight excess (preferably about 1.2 equivalents) of an aldehyde comprising R₄, (i.e., a compound having the formula R₄^,CHO where R₄^,CH₂- is equivalent to R₄ and R₄ is as described above or is a protected precursor to such a substituent, e.g., (3-oxo-propyl)-carbamic acid <i>tert</i>-butyl ester) and a reducing agent such as sodium triacetoxyborohydride. The resulting mixture is stirred for several hours. The product, a compound of Formula 203 is isolated and purified.

Preparation of Formula 205

0084Referring to Reaction Scheme 2, Step 2, to a solution of a compound of Formula 203 and an amine base such as diisopropylethylamine in a nonpolar, aprotic solvent such as dichloromethane is added an R₃ acyl chloride (such as Cl-C(O)-R₃ where R₃ is as described above). The resulting solution is stirred under nitrogen at room temperature for several hours. The product, a compound of Formula 205 is isolated and purified.

Preparation of Formula 207

0085Optionally, any protecting groups on compounds of Formula 205 are then removed. For example, if R₄ comprises a protected amine wherein the protecting group is a Boc group, the Boc group can be removed by treatment of the compound of Formula 205 with an acid such as trifluoroacetic acid in a nonpolar, aprotic solvent such as dichloromethane, while maintaining the reaction at about room temperature. The reaction is monitored e.g., by TLC. Upon completion, the product, a compound of Formula 207 is isolated and purified. <chemistry id="chem0010" num="0010"><img file="EP1511734B1_D0010.tif" /></chemistry>

Preparation of Compounds of Formula 303

0086Referring to Reaction Scheme 3, Step 1, to a solution of a compound of Formula 301 in a nonpolar, aprotic solvent such as DMF is added sodium hydride. The resulting solution was stirred at about 45°C for about 5 minutes, then allyl alcohol (about 1.4 equivalents) is added via pipette. The resulting solution is stirred at about 45°C for about 12 hours and then cooled to room temperature. The product, a compound of Formula 303, is isolated and used without further purification.

Preparation of Compounds of Formula 305

0087Referring to Reaction Scheme 3, Step 2, to a room temperature solution of a compound of Formula 303, in an aprotic solvent such as acetonitrile, is added morpholine followed by Pd(PPh₃)₄. The resulting solution is stirred for about 5 minutes. The product, a compound of Formula 305, is isolated and purified. <chemistry id="chem0011" num="0011"><img file="EP1511734B1_D0011.tif" /></chemistry><chemistry id="chem0012" num="0012"><img file="EP1511734B1_D0012.tif" /></chemistry>

0088Referring to Reaction Scheme 4, a compound of Formula 401 is dissolved in 0.5 M sodium methoxide in methanol and heated to about 70°C. The temperature is maintained at about 70°C for about 12 hours and then cooled to room temperature. The product, a compound of Formula 403, is isolated and purified. <chemistry id="chem0013" num="0013"><img file="EP1511734B1_D0013.tif" /></chemistry><chemistry id="chem0014" num="0014"><img file="EP1511734B1_D0014.tif" /></chemistry>

Preparation of Compounds of Formula 503

0089Referring to Reaction Scheme 5, Step 1, to a solution of a compound of Formula 501 and a base such as triethylamine in a nonpolar, aprotic solvent such as CH₂Cl₂ at room temperature is added about an equivalent of an acid chloride of formula Cl-(CO)-CH₂R, over about 5 minutes. After about 30 minutes, the product, a compound of Formula 503, is isolated and used without any further purification.

Preparation of Compounds of Formula 505

0090Referring to Reaction Scheme 5, Step 2, AlCl₃ (about 1.3 equivalents) is added slowly over about 15 minutes to a compound of Formula 503 at about 140°C. After gas evolution has ceased, the reaction mixture is cooled to room temperature. The product, a compound of Formula 505, is isolated and purified.

Preparation of Compounds of Formula 507

0091Referring to Reaction Scheme 5, Step 3, a solution of a compound of Formula 505 and an amino acid of Formula 506 (preferably about 1.1 equivalents) in which the amine group has been suitably protected with a protecting group PG (preferably a Boc group), a coupling reagent such as HBTU (preferably about 1.2 equivalents), a base such as TEA, and a nonpolar, aprotic solvent such as CH₂Cl₂ is maintained at room temperature for about 5 hours. The product, a compound of Formula 507, is isolated and purified.

Preparation of Compounds of Formula 509

0092Referring to Reaction Scheme 5, Step 4, a mixture of ester of Formula 507 and a base such as potassium carbonate in a polar, aprotic solvent such as DMF is heated at about 140°C. After about 30 mins, the product, a compound of Formula 509, is isolated and purified.

Preparation of Compounds of Formula 511

0093Referring to Reaction Scheme 5, Step 5, the amine protecting group PG is then removed. When PG is Boc, this may be accomplished by treatment of a compound of formula 509 with aqueous acid (preferably, 97.5:2.5 TFA:H₂O) at room temperature for about one hour. The free amine is isolated and used without further purification.

0094The resulting product; an aldehyde comprising R_4' (i.e., a compound having the formula R_4'CHO where R_4'CH₂- is equivalent to R₄ and R₄ is as described above or is a protected precursor to such a substituent, e.g., (3-oxo-propyl)-carbamic acid <i>tert</i>-butyl ester; preferably, about 1.45 equivalents); a reducing reagent, such as Na(OAc)₃BH; and a nonpolar, aprotic solvent such as CH₂Cl₂ is maintained at room temperature for about 3 hours. The product, a compound of Formula 511, is isolated and purified.

Preparation of Compounds of Formula 513

0095Referring to Reaction Scheme 5, Step 6, to a solution of a compound of Formula 511, a base such as diisoproylethylamine, and a nonpolar, aprotic solvent such as CH₂Cl₂ at room temperature is added an acid chloride of the formula R₃COCl (preferably about 2 equivalents). After about 2.5 h, the product, a compound of Formula 513, is isolated and purified. <chemistry id="chem0015" num="0015"><img file="EP1511734B1_D0015.tif" /></chemistry><chemistry id="chem0016" num="0016"><img file="EP1511734B1_D0016.tif" /></chemistry>

0096Referring to Reaction Scheme 6, to a solution of a compound of Formula 203 and an amine base such as diisopropylethylamine in a nonpolar, aprotic solvent such as dichloromethane is added a compound having the formula Cl-S(O)₂-R_3a or O-(S(O)₂-R_3a)₂ where R_3a is as described above. The resulting solution is stirred under nitrogen at room temperature for several hours. The product, a compound of Formula 603 is isolated and purified. <chemistry id="chem0017" num="0017"><img file="EP1511734B1_D0017.tif" /></chemistry>

0097Referring to Reaction Scheme 7, to a solution of a compound of Formula 203 and an amine base such as diisopropylethylamine in a nonpolar, aprotic solvent such as dichloromethane is added a compound having the formula X-CH₂-R_3b where R_3b is as described above and X is Br, Cl, mesylate, or tosylate. The resulting solution is stirred under nitrogen at room temperature or with heat for several hours. The product, a compound of Formula 703 is isolated and purified. <chemistry id="chem0018" num="0018"><img file="EP1511734B1_D0018.tif" /></chemistry>

Preparation of Formula 803

0098Referring to Reaction Scheme 8, Step 1, to an optionally substituted compound of Formula 109 dissolved in a polar, aprotic solvent (such as DMF) in the presence of a base (such as potassium carbonate) is added one equivalent of an optionally substituted suitably protected aldehyde wherein such aldehyde further comprises a leaving group, preferably, a halide (such as bromoacetaldehyde dimethylacetal). The solution is heated at reflux, monitoring completion of the reaction (e.g., by TLC). The reaction mixture is cooled and the corresponding, optionally substituted compound of Formula 803 is isolated and purified.

Preparation of Formula 805

0099Referring to Reaction Scheme 8, Step 2, to an optionally substituted compound of Formula 803 in an inert solvent (such as dichloromethane) in the presence of about 1.5 molar equivalents of an amine base (such as triethylamine) is added about 1.5 molar equivalents of an R₉ acid chloride, such as, Cl-C(O)-R₉, where R₉ is as described herein. The reaction takes place, with stirring, at room temperature over a period of 4 to 24 hours. Completion is monitored, e.g., by TLC. The corresponding compound of Formula 805 is isolated and purified.

Preparation of Formula 807

0100Referring to Reaction Scheme 8, Step 3, a solution of a compound of Formula 805 and an excess of ammonium acetate in acetic acid is heated at reflux for 1-4 hours. Completion is monitored, e.g., by TLC. The corresponding compound of Formula 807 is isolated and purified. <chemistry id="chem0019" num="0019"><img file="EP1511734B1_D0019.tif" /></chemistry><chemistry id="chem0020" num="0020"><img file="EP1511734B1_D0020.tif" /></chemistry>

Preparation of Formula 903

0101Referring to Reaction Scheme 9, Step 1, a suspension of a compound of Formula 109, an alpha-haloketone reagent of the Formula R_13'(CO)CH₂X wherein X is a halide and R_13' is as described herein, and about an equivalent of a base, such as potassium carbonate in a polar, aprotic solvent such as DMF is stirred at room temperature. The reaction is diluted with water and the resulting compound, a compound of Formula 903, typically a sold, is used in the subsequent step without purification. Where the resulting compound is not a solid, it is isolated using standard procedures and then used in the subsequent step.

Preparation of Formula 905

0102Referring to Reaction Scheme 9, Step 2, a solution of the compound of Formula 903, about an equivalent of an amine base, such as triethylamine and about an equivalent of an acid chloride (such as a compound of Formula R₉-COCl) in an organic solvent such as methylene chloride is stirred at room temperature for several hours. Completion is monitored, e.g., by TLC. The corresponding compound of Formula 905 is isolated and purified.

Preparation of Formula 907

0103Referring to Reaction Scheme 9, Step 3, a solution of a compound of Formula 905 and an excess of ammonium acetate in acetic acid is heated at reflux using a Dean-Stark trap and condenser. Completion is monitored, e.g., by TLC. The corresponding compound of Formula 907 is isolated and purified.

Preparation of Formula 909

0104Referring to Reaction Scheme 9, Step 4, when R_13' comprises a protected aminoalkyl group, the amino protected group may be removed. For example, when the amino group is protected as the corresponding phthalimide, the protecting group is removed as follows. A solution of a compound of Formula 907 and an excess of anhydrous hydrazine in a polar, protic solvent such as ethanol is heated at reflux. The reaction is cooled to about 5°C and any precipitate is filtered off. The filtrate is concentrated in vacuo and purified to yield a compound of Formula 909. One of skill in the art will appreciate that other conditions may be used to remove other protecting groups. <chemistry id="chem0021" num="0021"><img file="EP1511734B1_D0021.tif" /></chemistry><chemistry id="chem0022" num="0022"><img file="EP1511734B1_D0022.tif" /></chemistry>

Preparation of Formula 1003

0105Referring to Reaction Scheme 10, Step 1, reductive amination of amines of Formula 109 with an optionally substituted, aldehyde-containing carbamic acid ester gives urethane intermediates. More specifically, to a solution of a compound of Formula 109 and an equivalent of a suitably protected aldehyde (<nplcit id="ncit0013" npl-type="s"><text>Seki et. al. Chem. Pharm. Bull. 1996, 44, 2061</text></nplcit>) in dichloromethane is added a slight excess of a reducing agent, such as sodium triacetoxyborohydride. The resultant cloudy mixture is maintained at ambient temperature. Completion is monitored, e.g., by TLC. The corresponding compound of Formula 1003 is isolated and used in the subsequent step without purification.

Preparation of Formula 1005

0106Referring to Reaction Scheme 10, Step 2, the amino protecting group PG is then removed. When PG is a Boc protecting group, this may be accomplished by the treatment of a solution of a compound of Formula 1003 in a nonpolar, aprotic solvent such as dichloromethane with a strong acid such as trifluoroacetic acid. The resultant solution is maintained at ambient temperature overnight and concentrated under reduced pressure. The residue is isolated to give a compound of Formula 1005 which was used in the subsequent step without purification.

Preparation of Formula 1007

0107Referring to Reaction Scheme 10, Step 3, to a solution of a compound of Formula 1005 in a nonpolar, aprotic solvent such as dichloromethane is added an excess, preferably about two equivalents of an amine base such as triethylamine, followed by about an equivalent or slight excess of an acid chloride of the formula R₉-CO-Cl. The resultant solution is stirred at ambient temperature for about 3 hours. Completion is monitored, e.g., by TLC. The corresponding compound of Formula 1007 is isolated and purified.

Preparation of Formula 1009

0108Referring to Reaction Scheme 10, Step 4, a solution of a compound of Formula 1007 in an excess of phosphorus oxychloride is heated at reflux. After 8 hours, the reaction mixture is allowed to cool to ambient temperature and concentrated under reduced pressure. The corresponding compound of Formula 1009 is isolated and purified. <chemistry id="chem0023" num="0023"><img file="EP1511734B1_D0023.tif" /></chemistry>

Preparation of Formula 1109

0109As an alternative to Steps 3 and 4 of Reaction Scheme 10, acylation of primary amines of Formula 1005, followed by acetic acid mediated cyclization, can proceed without isolation of the intermediate amides to provide the target compound of Formula 1109. This route is shown in Reaction Scheme 11.

0110More specifically, to a solution of a compound of Formula 1005 in a nonpolar, aprotic solvent such as dichloromethane is added an excess, preferably about two equivalents of an amine base, such as triethylamine, followed by about an equivalent of an acid chloride of the formula R₉-CO-Cl. The resultant solution is stirred at ambient temperature for 2 hours, then evaporated under reduced pressure. The resultant solid is treated with glacial acetic acid, then the resultant suspension is heated at reflux for about 48 hours. The reaction is cooled to ambient temperature then evaporated under reduced pressure. The corresponding compound of Formula 1109 is isolated and purified. <chemistry id="chem0024" num="0024"><img file="EP1511734B1_D0024.tif" /></chemistry>

0111Referring to Reaction Scheme 12, a compound of Formula 203 is reacted with a slight excess of a compound of the formula R₁₅O(CO)Cl in the presence of a base such as triethylamine in a nonpolar, aprotic solvent such as dichloromethane. The product, a compound of Formula 1203 is isolated and purified. <chemistry id="chem0025" num="0025"><img file="EP1511734B1_D0025.tif" /></chemistry>

0112Referring to Reaction Scheme 13, a compound of Formula 203 is treated with a slight excess of an isocyanate R₁₇-N=C=O in the presence of a base, such as triethylamine, in a nonpolar, aprotic solvent, such as dichloromethane. The product, a compound of Formula 1303, is isolated and purified. <chemistry id="chem0026" num="0026"><img file="EP1511734B1_D0026.tif" /></chemistry><chemistry id="chem0027" num="0027"><img file="EP1511734B1_D0027.tif" /></chemistry>

Preparation of Compounds of Formula 1403

0113Referring to Reaction Scheme 14, Step 1, a nonpolar, aprotic solvent, such as THF, and an excess of a solution of an optionally substituted vinyl magnesium bromide in a nonpolar, aprotic solvent (and more preferably, about three equivalents of a 1.0 M solution of an optionally substituted vinyl magnesium bromide in THF) is cooled to -78 °C while stirring under a nitrogen atmosphere. The mixture is treated dropwise with a solution of a compound of Formula 1401 in a nonpolar, aprotic solvent, such as THF over about 30 min. After the mixture is stirred for 30 min at -78 °C, the cooling bath is removed and the reaction mixture is warmed slowly to room temperature overnight (about 15 h). The product, a compound of Formula 1403, is isolated and purified.

Preparation of Compounds of Formula 1405

0114Referring to Reaction Scheme 14, Step 2, to a solution of a compound of Formula 1403 in an anhydrous, nonpolar, aprotic solvent, such as acetonitrile under an inert atmosphere, such as argon, is added about an equivalent of a compound of the Formula R_1'- X wherein R_1' is an optionally susbituted vinyl, optionally susbituted aryl, or optionally substituted heteroaryl and X is I, Br, or -OTf, and a base such as triethylamine followed by palladium (II) acetate (preferably, about 0.025 equivalents). The resulting solution is heated to about 80 °C. After about 15 h, the reaction mixture is allowed to cool to room temperature. The product, a compound of Formula 1405, is isolated and immediately purified.

Preparation of Compounds of Formula 1407

0115To a solution of a compound of Formula 1405 in a nonpolar, aprotic solvent such as ethyl acetate under nitrogen is added 10 wt % palladium on carbon. The nitrogen is replaced with a balloon of hydrogen and the flask is purged. After 3 h, the reaction flask is purged with nitrogen and filtered through a pad of celite (rinsing with a solvent such as ethyl acetate). The product, a compound of Formula 1407 is isolated and purified. <chemistry id="chem0028" num="0028"><img file="EP1511734B1_D0028.tif" /></chemistry>

Preparation of Compounds of Formula 1503

0116Referring to Reaction Scheme 15, Step 1, about one equivalent of carbonyldiimidazole is added slowly to a room temperature solution of a compound of Formula 1501 (preferably wherein the amino protecting group PG is Boc) in a nonpolar, aprotic solvent such THF. Afer about one hour, the product, a compound of Formula 1503, is isolated and used without further purification.

Preparation of Compounds of Formula 1505

0117Referring to Reaction Scheme 15, Step 2, a Grignard reagent is prepared from a compound of Formula R₁CH₂Br and magnesium turnings in a nonpolar, aprotic solvent such as THF. A solution of a compound of Formula 1503 in a nonpolar, aprotic solvent such as THF is cooled to about 0-5°C. The solution of the Grignard reagent is then added via syringe to the 0-5°C solution of the compound of Formula 1503. The temperature is monitored by internal thermometer and is not allowed to exceed about 15°C. The reaction mixture is maintained at about 0-5°C for about one hour. The product, a compound of Formula 1505, is isolated and purified.

Preferred Processes and Last Steps

0118A compound of Formula I is optionally contacted with a pharmaceutically acceptable acid or base to form the corresponding acid or base addition salt.

0119A pharmaceutically acceptable acid addition salt of a compound of Formula I is optionally contacted with a base to form the corresponding free base of Formula I. A pharmaceutically acceptable base addition salt of a compound of Formula I is optionally contacted with an acid to form the corresponding free acid of Formula I.

Preferred Compounds

Preferred R₁ when Either R₂ or R_2' is Not Hydrogen

0120When considering the compounds of Formula I, in a preferred embodiment when either one or both R₂ or R_2' is not hydrogen (more preferably, either one of R₂ or R_2' is not hydrogen), R₁ is selected from hydrogen, optionally substituted C₁-C₈ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aryl-C₁-C₄-alkyl-, and optionally substituted heteroaryl-C₁-C₄-alkyl- (more preferably optionally substituted aryl and optionally substituted aryl-C₁-C₄-alkyl-). In a more preferred embodiment R₁ is selected from hydrogen, optionally substituted C_1-C₄ alkyl, optionally substituted phenyl-C₁-C₄-alkyl-, optionally substituted naphthalenylmethyl, optionally substituted phenyl, and naphthyl. More preferably, R₁ is optionally substituted phenyl-C₁-C₄-alkyl- or optionally substituted heteroaryl-C₁-C₄-alkyl-.

0121In a most preferred embodiment R₁ is naphthyl, phenyl, bromophenyl, chlorophenyl, methoxyphenyl, ethoxyphenyl, tolyl, dimethylphenyl, chorofluorophenyl, methylchlorophenyl, ethylphenyl, phenethyl, benzyl, chlorobenzyl, methylbenzyl, methoxybenzyl, cyanobenzyl, hydroxybenzyl, dichlorobenzyl, dimethoxybenzyl, or naphthalenylmethyl. More preferably, R₁ is benzyl, cyanobenzyl, methoxybenzyl, or naphthalenylmethyl. Most preferably, R₁ is benzyl.

Preferred R₁ when R₂ and R_2' Are Hydrogen

0122In an embodiment wherein R₂ and R_2' are both hydrogen, preferably R₁ is chosen from optionally substituted aryl, optionally substituted aryl-C₁-C₄-alkyl-, optionally substituted heteroaryl, and optionally substituted heteroaryl-C₁-C₄-alkyl-, provided however, that R₁ is not substituted phenyl. More preferably, R₁ is optionally substituted aryl-C₁-C₄-alkyl- or optionally substituted heteroaryl-C₁-C₄-alkyl-. More preferably, when R₂ and R_2' are both hydrogen, R₁ is selected from optionally substituted phenyl-C₁-C₄-alkyl, and optionally substituted naphthalenylmethyl. In a more preferred embodiment wherein R₂ and R_2' are both hydrogen, R₁ is chosen from benzyl, chlorobenzyl, methylbenzyl, methoxybenzyl, cyanobenzyl, hydroxybenzyl, dichlorobenzyl, dimethoxybenzyl, and naphthalenylmethyl. More preferably, R₁ is benzyl, cyanobenzyl, methoxybenzyl, or naphthalenylmethyl. More preferably, R₁ is benzyl.

Preferred R₂

0123When considering the compounds of Formula I and as will be appreciated by those skilled in the art, the compounds described herein possess a potentially chiral center at the carbon to which R₂ and R_2' are attached. The R₂ and R_2' groups may be the same or different; if different, the compound is chiral (i.e., has a stereogenic center). When R₂ and R_2' are different, in preferred embodiments R_2' is hydrogen and R₂ is other than hydrogen. The invention contemplates the use of pure enantiomers and mixtures of enantiomers, including racemic mixtures, although the use of a substantially optically pure enantiomer will generally be preferred. The term "substantially optically pure" or "enantiomerically pure" means having at least about 95% of the described enantiomer with no single impurity greater than about 1% and preferably, at least about 97.5% enantiomeric excess. In a preferred embodiment, the stereogenic center to which R₂ and R_2' are attached is of the R configuration.

0124In one embodiment, R₂ is optionally substituted C₁-C₄ alkyl, and R_2' is hydrogen or optionally substituted C₁-C₄ alkyl. More preferably, R_2' is hydrogen and R₂ is optionally substituted C₁-C₄ alkyl. In a most preferred embodiment R₂ is chosen from methyl, ethyl, propyl (particularly, c-propyl or i-propyl), butyl (particularly, t-butyl), methylthioethyl, methylthiomethyl, aminobutyl, (CBZ)aminobutyl, cyclohexylmethyl, benzyloxymethyl, methylsulfinylethyl, methylsulfinylmethyl, and hydroxymethyl, and R_2' is hydrogen. Especially preferred is when R_2' is hydrogen and R₂ is ethyl or propyl (particularly, c-propyl or i-propyl). More preferably, R₂ is i-propyl. More preferred is the embodiment wherein the stereogenic center to which R₂ and R_2' is attached is of the R configuration.

0125In another embodiment, both R₂ and R_2' are hydrogen.

Preferred R₃ Groups When R₁₂ is -N(R₄)(COR₃)

0126When considering the compounds of Formula I wherein R₁ is -N(R₄)(COR₃), in a preferred embodiment R₃ is selected from optionally substituted C₁-C₈ alkyl, optionally substituted aryl-C₁-C₄-alkyl-, optionally substituted heteroaryl-C₁-C₄-alkyl-, optionally substituted heteroaryl, optionally substituted aryl, R₁₅O- and R₁₇-NH-, R₁₅ is chosen from optionally substituted C₁-C₈ alkyl and optionally substituted aryl, and R₁₇ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl and optionally substituted aryl. Preferred R₃ are optionally substituted C₁-C₈ alkyl (e.g., C₁-C₈ alkyl substituted with lower-alkoxy), optionally substituted heteroaryl, and optionally substituted aryl.

0127In a more preferred embodiment, when R₃ is not R₁₇NH- or R₁₅O-, R₃ is chosen from phenyl; phenyl substituted with one or more of the following substituents: halo, C₁-C₄ alkyl, C₁-C₄ alkyl substituted with hydroxy (e.g., hydroxymethyl), C₁-C₄ alkoxy, C₁-C₄ alkyl substituted with C₁-C₄ alkoxy, nitro, formyl, carboxy, cyano, methylenedioxy, ethylenedioxy, acyl (e.g., acetyl), -N-acyl (e.g., N-acetyl) or trifluoromethyl; benzyl; phenoxymethyl-; halophenoxymethyl-; phenylvinyl-; heteroaryl-; heteroaryl- substituted with C₁-C₄ alkyl or C₁-C₄ alkyl substituted with halo (e.g., CF₃); C₁-C₄ alkyl substituted with C₁-C₄ alkoxy- and benzyloxymethyl-.

0128In a most preferred embodiment, when R₃ is not R₁₇NH- or R₁₅O-, R₃ is chosen from phenyl, halophenyl, dihalophenyl, cyanophenyl, halo(trifluoromethyl)phenyl, hydroxymethylphenyl, methoxyphenyl, ethoxyphenyl, carboxyphenyl, ethylphenyl, tolyl, methylenedioxyphenyl, ethlenedixoyphenyl, methoxychlorophenyl, dihydro-benzodioxinyl, methylhalophenyl, trifluoromethylphenyl, bis(trifluoromethyl)phenylbenzyl, furanyl, C₁-C₄ alkyl substituted furanyl, trifluoromethylfuranyl, C₁-C4 alkyl substituted trifluoromethylfuranyl, benzofuranyl, thiophenyl, C₁-C₄ alkyl substituted thiophenyl, benzothiophenyl, benzothiadiazolyl, pyridinyl, indolyl, methylpyridinyl, trifluoromethylpyridinyl, pyrrolyl, quinolinyl, picolinyl, pyrazolyl, C₁-C₄ alkyl substituted pyrazolyl, N-methyl pyrazolyl, C₁-C₄ alkyl substituted N-methyl pyrazolyl, C₁-C₄ alkyl substituted pyrazinyl, C₁-C₄ alkyl substituted isoxazolyl, benzoisoxazolyl, morpholinomethyl, methylthiomethyl, methoxymethyl, N-methyl imidazolyl, and imidazolyl. Yet more preferably, R₃ is tolyl, halophenyl, halomethylphenyl, hydroxymethylphenyl, methylenedioxyphenyl, formylphenyl or cyanophenyl.

0129In a more preferred embodiment, when R₃ is R₁₇NH-, R₁₇ is chosen from hydrogen, C₁-C₄ alkyl; cyclohexyl; phenyl; and phenyl substituted with halo, C₁-C₄ alkyl, C₁-C₄ alkoxy, or C₁-C₄ alkylthio.

0130In a most preferred embodiment, when R₃ is R₁₇NH-, R₁₇ is hydrogen isopropyl, butyl, cyclohexyl, phenyl, bromophenyl, dichlorophenyl, methoxyphenyl, ethylphenyl, tolyl, trifluoromethylphenyl or methylthiophenyl.

0131In an embodiment, wherein R₃ is R₁₅O-, R₁₅ is chosen from optionally substituted C₁-C₈ alkyl and optionally substituted aryl.

Preferred R_3aGroups when R₁₂ is -N(R₄)(SO₂R₃.)

0132Preferably, when R₁₂ is -N(R₄)(SO₂R_3a), R_3a is chosen from C₁-C₁₃ alkyl; phenyl; naphthyl; phenyl substituted with halo, C₁-C₄ alkyl, C₁-C₄ alkoxy, cyano, nitro, methylenedioxy, or trifluoromethyl; biphenylyl and heteroaryl. More preferably, R_3a is chosen from phenyl substituted with halo, C₁-C₄ alkyl, C₁-C₄ alkoxy, cyano, nitro, methylenedioxy, or trifluoromethyl and naphthyl.

Preferred R_3b Groups when R₁₂ is -N(R₄)(CH₂R_3b)

0133Preferably, when R₁₂ is -N(R₄)(CH₂R_3b), R_3b is chosen from C₁-C₁₃ alkyl; substituted C₁-C₄ alkyl; phenyl; naphthyl; phenyl substituted with carboxy, alkoxycarbonyl cyano, halo, C₁-C₄ alkyl-, C₁-C₄ alkoxy, nitro, methylenedioxy, or trifluoromethyl; biphenylyl, benzyl; and heterocyclyl.

0134Most preferably, R_3b is chosen from halophenyl, polyhalophenyl, methylhalophenyl, tolyl, dimethylphenyl, methoxyphenyl, dimethoxyphenyl, cyanophenyl, trifluoromethylphenyl, trifluoromethoxyphenyl, bis(trifluoromethyl)phenyl, carboxyphenyl, t-butylphenyl, methoxycarbonylphenyl, piperidinyl and naphthyl.

Preferred R₄ Groups when R₁₂ is -NHR₄, -N(R₄)(COR₃), or -N(R₄)(CH₂R_3b)

0135In a preferred embodiment when R₁ is NHR₄, -N(R₄)(COR₃), or-N(R₄)(CH₂R_3b), R₄ is chosen from hydrogen, optionally substituted C₁-C₁₃ alkyl, optionally substituted aryl, optionally substituted aryl-C₁-C₄-alkyl-, optionally substituted heterocyclyl, and optionally substituted heteroaryl-C₁-C₄-alkyl- (preferably hydrogen or optionally substituted C₁-C₁₃ alkyl).

0136More preferably, R₄ is chosen from hydrogen, C₁-C₄ alkyl; cyclohexyl; phenyl substituted with hydroxyl, C₁-C₄ alkoxy or C₁-C₄ alkyl; benzyl; heteroarylmethyl-; heteroarylethyl-; heteroarylpropyl-; and R₁₆-alkylene-, wherein R₁₆ is hydroxyl, di(C₁-C₄ alkyl)amino-, (C₁-C₄ alkyl)amino-, amino, C₁-C₄ alkoxy-, or N-heterocyclyl-, particularly pyrrolidino, piperidino or imidazolyl.

0137More preferably, R₄ is R₁₆-alkylene-, wherein R₁₆ is amino, C₁-C₄ alkylamino-, di(C₁-C₄ alkyl)amino-, C₁-C₄ alkoxy-, hydroxyl, or N-heterocyclyl. Preferably R₁₆ is amino.

0138In a most preferred embodiment when R₁₂ is NHR₄, -N(R₄)(COR₃), or-N(R₄)(CH₂R_3b), R₄ is chosen from hydrogen, 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, azetidinylethyl, azetidinylpropyl pyrrolidinylethyl, pyrrolidinylpropyl, piperidinylmethyl, piperidinylethyl, imidazolylpropyl, imidazolylethyl, (ethylpyrrolidinyl)methyl, (methylpyrrolidinyl)ethyl, (methylpiperidinyl)propyl, (methylpiperazinyl)propyl, furanylmethyl and indolylethyl.

0139More preferably, R₄ is aminoethyl, aminopropyl, aminobutyl, aminopentyl, aminohexyl, methylaminoethyl, methylaminopropyl, methylaminobutyl, methylaminopentyl, methylaminohexyl, dimethylaminoethyl, dimethylaminopropyl, dimethylaminobutyyl, dimethylaminopentyl, dimethylaminohexyl, ethylaminoethyl, ethylaminopropyl, ethylaminobutyl, ethylaminopentyl, ethylaminohexyl, diethylaminoethyl, diethylaminopropyl, diethylaminobutyyl, diethylaminopentyl, or diethylaminohexyl, most preferably aminopropyl.

Preferred R₄ Groups when R₁₂ is --N(R₄)(SO₂R_3a)

0140Preferably, when R₁₂ is -N(R₄)(SO₂R_3a), R₄ is chosen from C₁-C₄ alkyl, cyclohexyl; phenyl substituted with hydroxyl, C₁-C₄ alkoxy or C₁-C₄ alkyl; benzyl; heteroarylmethyl-; heteroarylethyl-; heteroarylpropyl-; heteroarylethyl-; heteroarylpropyl- and R₁₆-alkylene-, wherein R₁₆ is hydroxyl, di(C₁-C₄ alkyl)amino-, (C₁-C₄ alkyl)amino-, amino, C₁-C₄ alkoxy-, or N-heterocyclyl-, particularly pyrrolidino, piperidino or imidazolyl.

R₁₂ is an Imidazole

0141Preferably, when R₁₂ is an imidazole, R₁₂ has the formula: <chemistry id="chem0029" num="0029"><img file="EP1511734B1_D0029.tif" /></chemistry> wherein R₉ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl, optionally substituted aryl, optionally substituted aryl-C₁-C₄-alkyl -, optionally substituted heteroaryl-C₁-C₄-alkyl-, optionally substituted aryl-C₁-C₄-alkoxy -, optionally substituted heteroaryl-C₁-C₄-alkoxy -, optionally substituted heteroaryl-; and R₁₃ and R₁₃, are independently hydrogen, optionally substituted C₁-C₈ alkyl, optionally substituted aryl, or optionally substituted aryl-C₁-C₄-alkyl - (preferably optionally substituted aryl). More preferably, R₉ is phenyl substituted with C₁-C₄-alkyl, C₁-C₄-alkoxy-, and/or halo (especially C₁-C₄-alkyl and/or halo); phenyl; or benzyl. Yet more preferably, R₉ is tolyl; halophenyl; or halomethylphenyl.

0142In a preferred embodiment, R₁₃ is hydrogen and R_13' is substituted C₁-C₄ alkyl. More preferably, R₁₃ is hydrogen and R_13' is aminomethyl, aminoethyl, aminopropyl, acetylamino-methyl, acetylaminoethyl, benzyloxycarbonylamino-methyl or benzyloxycarbonylamino-ethyl.

R₁₂ is an Imidazoline

0143Preferably, when R₁₂ is an imidazoline, R₁₂ has the formula <chemistry id="chem0030" num="0030"><img file="EP1511734B1_D0030.tif" /></chemistry> wherein R₉ is chosen from hydrogen, optionally substituted C₁-C₈ alkyl, optionally substituted aryl, optionally substituted aryl-C₁-C₄-alkyl -, and optionally substituted heteroaryl-; and R₁₀, R₁₀, R₁₄, and R_14' are independently chosen from hydrogen, optionally substituted C₁-C₈ alkyl, optionally substituted aryl, and optionally substituted aryl-C₁-C₄-alkyl -. More preferably, R₉ is methylenedioxyphenyl; phenyl; phenyl substituted with C₁-C₄ alkyl, C₁-C₄ alkoxy, and/or halo; or benzyl. In a preferred embodiment, R₉ is methylenedioxyphenyl-; phenyl; or phenyl substituted with methoxy, halo and/or methyl (preferably halo and/or methyl, including tolyl), more preferably methylenedioxyphenyl or said substituted phenyls. In another preferred embodiment, R₁₀, R₁₀, R_14', and R₁₄ are independently hydrogen or optionally substituted alkyl (preferably optionally substituted C₁-C₄ alkyl). More preferably, R₁₀ and R₁₀, are independently selected from the group consisting of hydrogen or optionally substituted C₁-C₄ alkyl (and more particularly, methyl or aminoalkyl-) and R_14' and R₁₄ are hydrogen.

Preferred R₅, R₆, R₇, and R₈ Groups

0144When considering the compounds of Formula I, in other preferred embodiments R₅, R₆, R₇ and R₈ are independently chosen from hydrogen; acyl, alkyl; alkyl substituted with alkyl, alkoxy, halo, hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, lower-alkylaminocarbonyl- (e.g. methylaminocarbonyl- or ethylaminocarbonyl-), di(lower-alkyl)aminocarbonyl- (e.g. dimethylaminocarbonyl- or diethylaminocarbonyl-), aryl, or heteroaryl; alkoxy; alkoxy substituted with alkyl, acyl, alkoxy, halo, hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, lower-alkylaminocarbonyl- (e.g. methylaminocarbonyl- or ethylaminocarbonyl-), di(lower-alkyl)aminocarbonyl- (e.g. dimethylaminocarbonyl- or diethylaminocarbonyl-), aryl, or heteroaryl; halogen; hydroxyl; nitro; cyano; dialkylamino; alkylsulfonyl; alkylsulfonamido; alkylthio; carboxyalkyl; carboxamido; amidocarbonyl; aryl; aryl substituted with alkyl, acyl, alkoxy, halo, hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, lower-alkylaminocarbonyl- (e.g. methylaminocarbonyl- or ethylaminocarbonyl-), di(lower-alkyl)aminocarbonyl- (e.g. dimethylaminocarbonyl- or diethylaminocarbonyl-), aryl, or heteroaryl; heteroaryl or heteroaryl substituted with alkyl, acyl, alkoxy, halo, hydroxyl, nitro, cyano, dialkylamino, alkylsulfonyl, alkylsulfonamido, alkylthio, carboxyalkyl, carboxamido, aminocarbonyl, lower-alkylaminocarbonyl- (e.g. methylaminocarbonyl- or ethylaminocarbonyl-), di(lower-alkyl)aminocarbonyl- (e.g. dimethylaminocarbonyl- or diethylaminocarbonyl-), aryl, or heteroaryl.

0145More preferably, R₅, R₆, R₇, and R₈ are independently chosen from hydrogen, amino, alkylamino, hydroxyl, halogen (particularly chloro and fluoro), C₁-C₄ alkyl (particularly methyl), C₁-C₄ haloalkyl (particularly trifluoromethyl), C₁-C₄ alkoxy (particularly methoxy), C₁-C₄ haloalkoxy and cyano. More preferably, R₅, R₆, R₇, and R₈ are methoxy, hydrogen, cyano, or halo (especially Cl, F). Further preferred for each of the specific substituents: R₅ is amino, alkylamino, trifluoromethyl, hydrogen or halo; R₆ is hydrogen, alkyl (particulary, methyl) or halo; R₇ is hydrogen, halo, alkyl (particulary, methyl), alkoxy (particularly, methoxy), cyano, or trifluoromethyl; and R₈ is hydrogen or halo. Still further preferred are the compounds where only one of R₅, R₆, R₇, and R₈ is not hydrogen, especially R₇. More preferred are the compounds where R₅, R₆, and R₈ are hydrogen and R₇ is cyano, methoxy or halogen (especially Cl, F).

Preferred Salt Forms

0146Preferred compounds will generally be capable of forming acid addition salts (i.e., will comprise a site which reacts with a pharmaceutically acceptable acid to form an acid addition salt.) The present invention includes pharmaceutically acceptable acid addition salts of the compounds of Formula I. Acid addition salts of the present compounds are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic. Preferred salt forms are hydrochloric, phosphoric, and oxalic acid salts with the hydrochloric acid salt form being especially preferred.

0147The salts and/or solvates of the compounds of the formula (I) which are not pharmaceutically acceptable may be useful as intermediates in the preparation of pharmaceutically acceptable salts and/or solvates of compounds of formula (I) or the compounds of the formula (I) themselves, and as such form another aspect of the present invention.

Preferred Subgenus

0148In a particularly preferred subgenus of compounds of Formula I, R₁ is benzyl, halobenzyl, methoxybenzyl-, cyanobenzyl, or naphthalenylmethyl-; R₂ is ethyl or propyl; R_2' is hydrogen; R₅ is hydrogen; R₆ is hydrogen; R₇ is halo, cyano, methoxy or hydrogen; R₈ is hydrogen; and R₁₂ is -NR₄(COR₃) wherein R₃ is optionally substituted aryl (preferably, halophenyl, halomethylphenyl-, methylenedioxyphenyl-, methoxyphenyl-, ethoxyphenyl-, cyanophenyl- or phenyl substituted with lower-acyl or lower-alkylaminocarbonyl-, e.g. methylaminocarbonyl- or ethylaminocarbonyl-, or di(lower-alkyl)aminocarbonyl-, e.g. dimethylaminocarbonyl- or diethylaminocarbonyl-; and R₄ is R₁₆-alkylene- wherein R₁₆ is hydroxyl, di(C₁-C₄)alkylamino-, (C₁-C₄ alkyl)amino-, amino, pyrrolidino, piperidino, imidazolyl and morpholino (more preferably in such embodiments, R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or naphthalenylmethyl; and R₂ is propyl (especially i- or c-propyl).

0149In another particularly preferred subgenus of compounds of Formula I, R₁ is benzyl, halobenzyl, methoxybenzyl-, cyanobenzyl, or naphthalenylmethyl-; R₂ is ethyl or propyl; R_2' is hydrogen; R₅ is hydrogen; R₆ is hydrogen; R₇ is halo, cyano, methoxy or hydrogen; R₈ is hydrogen; R₁₂ is -NR₄(CH₂R_3b) wherein R₄ is R₁₆-alkylene- wherein R₁₆ is hydroxyl, di(C₁-C₄)alkylamino-, (C₁-C₄ alkyl)amino-, amino, pyrrolidino, piperidino, imidazolyl or morpholino.; and R_3b is optionally substituted aryl.

0150In a particularly preferred subgenus of compounds of Formula I, R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or naphthalenylmethyl; R₂ is chosen from ethyl or propyl; R_2' is hydrogen; R₅ is hydrogen; R₆ is hydrogen; R₇ is halo, cyano, methoxy or hydrogen; R₈ is hydrogen; and R₁₂ is optionally substituted imidazolinyl of the above formula wherein R_10, R_10', R₁₄ and R_14' are independently hydrogen or optionally substituted alkyl (preferably optionally substituted C₁-C₄ alkyl); and R₉ is optionally substituted phenyl (preferably, halophenyl, halomethylphenyl, tolyl, or methylenedioxyphenyl). More preferably in such embodiments R₁ is benzyl, methoxybenzyl, or cyanobenzyl; R₂ is propyl (especially i- or c- propyl); and R₁₆ is amino.

0151In a particularly preferred subgenus of compounds of Formula I, R₁ is benzyl, halobenzyl, methoxybenzyl, cyanobenzyl, or naphthalenylmethyl; R₂ is chosen from ethyl or propyl; R_2' is hydrogen; R₅ is hydrogen; R₆ is hydrogen; R₇ is halo, cyano, methoxy or hydrogen; R₈ is hydrogen; and R₁₂ is optionally substituted imidazole of the above formula wherein R₁₃ is hydrogen and R_13' is hydrogen or optionally substituted alkyl (preferably optionally substituted C₁-C₄ alkyl); and R₉ is optionally substituted aryl (preferably, halophenyl, halomethylphenyl, tolyl, or methylenedioxyphenyl). More preferably, R₁₃ is hydrogen and R_13' is aminomethyl, aminoethyl aminopropyl, acetylamino-methyl, acetylaminoethyl, benzyloxycarbonylamino-methyl or Benzyloxycarbonylamino-ethyl. More preferably in such embodiments R₁ is benzyl, methoxybenzyl, or cyanobenzyl; R₂ is propyl (especially i- or c- propyl); and R₁₆ is amino.

0152When R₁ is -N(R₄)(SO₂R_3a), R₁ is most preferably chosen from C₁-C₄ alkyl, benzyl, substituted benzyl and substituted phenyl; R₂ is C₁-C₄ alkyl; R_2' is hydrogen; R_3a is chosen from substituted phenyl and naphthyl; R₄ is R₁₆-alkylene-; R₇ is hydrogen, fluoro, methyl or chloro; R₅, R₆ and R₈ are hydrogen; and R₁₆ is chosen from hydroxyl, di(C₁-C₄)amino, (C₁-C₄ alkyl)amino, amino, pyrrolidino, piperidino, imidazolyl and morpholino.

0153When R₁₂ is -NHR₄ or -N(R₄)(CH₂R_3b), R₁ is preferably chosen from hydrogen, optionally substituted C₁-C₄ alkyl, optionally substituted benzyl, optionally substituted phenyl, and optionally substituted naphthalenylmethyl; R₂ is optionally substituted C₁-C₄ alkyl and R_2' is hydrogen; R_3b is chosen from optionally substituted alkyl; optionally substituted phenyl; biphenylyl, optionally substituted aralkyl; and optionally substituted heterocyclyl; and R₄ is chosen from hydrogen, optionally substituted C₁-C₄ alkyl; cyclohexyl; optionally substituted phenyl; optionally substituted benzyl; heterocyclyl; heteroarylmethyl; heteroarylethyl; and heteroarylpropyl. More preferably, R₄ is R₁₆-alkylene-, wherein R₁₆ is hydroxyl, di(C₁-C₄)alkylamino-, (C₁-C₄ alkyl)amino-, amino, C₁-C₄ alkoxy-, or N-heterocyclyl.

0154When R₁₂ is -NHR₄ or -N(R₄)(CH₂R_3b), R₁ is most preferably chosen from C₁-C₄ alkyl, optionally substituted benzyl, and optionally substituted phenyl (more preferably optionally substituted benzyl, e.g., benzyl, cyanobenzyl); R₂ is optionally substituted C₁-C₄ alkyl (more preferably propyl, especially i- or c-propyl); R_2' is hydrogen; R_3b is chosen from optionally substituted phenyl, optionally substituted heterocyclyl and naphthyl; R₄ is chosen from hydrogen, optionally substituted benzyl, optionally substituted heterocyclyl and R₁₆-alkylene-; R₆ and R₇ are chosen from halo, cyano, methoxy or hydrogen; R₅ and R₈ are hydrogen; and R₁₆ is chosen from di(C₁-C₄ alkylamino)-, (C₁-C₄ alkyl)amino-, amino, pyrrolidinyl, piperidinyl, imidazolyl and morpholinyl.

0155In a particularly preferred subgenus of compounds of Formula I, R₁ is benzyl, halobenzyl (especially Cl-benzyl and F-benzyl), methoxybenzyl-, cyanobenzyl, or naphthalenylmethyl-; R₂ is ethyl or propyl; R_2' is hydrogen; R₅ is hydrogen; R₆ is hydrogen; R₇ is halo, cyano, methoxy or hydrogen; R₈ is hydrogen; and R₁₂ is -NH4 wherein R₄ is hydrogen (more preferably in such embodiments, R₁ is benzyl, halobenzyl, cyanobenzyl; and R₂ is propyl, especially i- propyl or c-propyl).

0156When R_3b is present, it is most preferably chosen from phenyl substituted with one or more halo, methyl, methoxy, cyano, trifluoromethyl, trifluoromethoxy, carboxy, and or methoxycarbonyl groups [e.g., halophenyl, polyhalophenyl, tolyl, dimethylphenyl, methoxyphenyl, dimethoxyphenyl, cyanophenyl, trifluoromethylphenyl, trifluoromethoxyphenyl, bis(trifluoromethyl)phenyl, carboxyphenyl, t-butylphenyl, methoxycarbonylphenyl]; piperidinyl and naphthyl.

0157Particularly preferred compounds include: <ul id="ul0007" list-style="none" compact="compact"><li>N-(3-Amino-propyl)-N-{1-[3-(3-cyano-benzyl)-7-hydroxy-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;</li><li>4-Acetyl-N-(3-amino-propyl)-N-[1-(3-benzyl-7-methoxy-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;</li><li>2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-l-yl]-2-methyl-propyl}-3-benzyl-4-oxo-4H-chromene-7-carbonitrile;</li><li>3-Benzyl-2-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-4-oxo-4H-chromene-7-carbonitrile;</li><li>Benzo[1,3]dioxole-5-carboxylic acid (3-amino-propyl)-{1-[7-chloro-3-(3-cyanobenzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;</li><li>N-(3-Amino-propyl)-N-{1-[7-chloro-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;</li><li>N-(3-Amino-propyl)-N-[1-(7-chloro-3-naphthalen-1-yl methyl-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-2-methoxy-acetamide;</li><li>4-Acetyl-N-(3-amino-propyl)-N-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;</li><li>N-(3-Amino-propyl)-N-{1-[7-chloro-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;</li><li>Benzo[1,3]dioxole-5-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>Benzo[1,3]dioxole-5-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>3-Benzyl-2-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-hydroxy-chromen-4-one;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-3-fluoro-4-methyl-benzamide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-methoxy-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-3-fluoro-4-methyl-benzamide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-methoxy-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-4-methyl-benzamide;</li><li>3-Benzyl-2-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-methoxy-chromen-4-one;</li><li>3-Benzyl-7-fluoro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,4-dimethyl-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-chromen-4-one;</li><li>3-Benzyl-2-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-fluoro-chromen-4-one;</li><li>3-Benzyl-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,4-dimethyl-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-7-cyano-chromen-4-one;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-4-methyl-benzamide;</li><li>4-Acetyl-N-(3-amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;</li><li>N-(3-Amino-propyl)-3-fluoro-N-{1-[7-fluoro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl }-4-methyl-benzamide;</li><li>4-Acetyl-N-(3-amino-propyl)-N-{1-[7-chloro-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-benzamide;</li><li>(2-{1-[-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-2-p-tolyl-1H-imidazol-4-yl}-ethyl)-carbamic acid benzyl ester;</li><li>2-[1-(2-Benzo[1,3]dioxol-5-yl-4,4-dimethyl-4,5-dihydro-imidazol-1-yl)-2-methylpropyl]-3-benzyl-7-cyano-chromen-4-one;</li><li>4-Acetyl-N-(3-amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-chlor-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-3-fluoro-4-methyl-benzamide;</li><li>N-(3-Amino-propyl)-N-{1-[7-fluoro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methoxy-benzamide;</li><li>Benzo[1,3]dioxole-5-carboxylic acid (3-amino-propyl)-{1-[7-fluoro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl -amide;</li><li>2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-3-benzyl-7-chloro-chromen-4-one;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-hydroxy-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-3-fluoro-4-methyl-benzamide;</li><li>Benzo[1,3]dioxole-5-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-hydroxy-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide;</li><li>N-(3-Amino-propyl)-N-{1-[7-fluoro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-2-methoxy-acetamide;</li><li>N-(3-Amino-propyl)-N-{1-[7-fluoro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;</li><li>N-(3-Amino-propyl)-N-[(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-cyclopropylmethyl]-4-methyl-benzamide;</li><li>2,3-Dihydro-benzo[1,4]dioxine-6-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>N-(3-Amino-propyl)-3-fluoro-N-{1-[7-cyano-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;</li><li>3-Benzyl-7-chloro-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one;</li><li>3-Benzyl-7-fluoro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-chromen-4-one;</li><li>2-[1-(4-Aminomethyl-2-p-tolyl-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one;</li><li>3-Benzyl-7-methoxy-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one;</li><li>2-[1-(2-Benzo[1,3]dioxol-5 -yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one;</li><li>4-Acetyl-N-(3-amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;</li><li>4-Acetyl-N-(3-amino-propyl)-N-{1-[7-cyano-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-benzamide;</li><li>3-Benzyl-7-chloro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-chromen-4-one;</li><li>3-Benzyl-7-fluoro-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one;</li><li>2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-fluoro-chromen-4-one;</li><li>N-(3-Amino-propyl)-N-{1-[7-cyano-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;</li><li>Benzo[1,3]dioxole-5-carboxylic acid (3-amino-propyl)-{1-[7-cyano-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;</li><li>3-(2-{1-[2-(3-Fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methyl-propyl}-7-methoxy-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;</li><li>3-{7-Chloro-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-4-oxo-4H-chromen-3-ylmethyl}-benzonitrile;</li><li>3-{7-Methoxy-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-4-oxo-4H-chromen-3-ylmethyl}-benzonitrile;</li><li>3-{7-Fluoro-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-4-oxo-4H-chromen-3-ylmethyl}-benzonitrile;</li><li>N-(3-Amino-propyl)-N-{1-[7-chloro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;</li><li>N-(3-Amino-propyl)-N-{1-[3-(3-cyano-benzyl)-7-methoxy-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;</li><li>N-(3-Amino-propyl)-N-{1-[3-(3-cyano-benzyl)-7-methoxy-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;</li><li>3-{2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-fluoro-4-oxo-4H-chromen-3-ylmethyl}-benzonitrile;</li><li>3-Benzyl-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methylpropyl}-4-oxo-4H-chromene-7-carbonitrile;</li><li>2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-4-oxo-4H-chromene-7-carbonitrile;</li><li>3-(7-Chloro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methylpropyl}-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;</li><li>3-{2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-7-methoxy-4-oxo-4H-chromen-3-ylmethyl}-benzonitrile;</li><li>3-(7-Fluoro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methylpropyl}-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;</li><li>Benzo[1,3]dioxole-5-carboxylic acid (3-amino-propyl)-{1-[7-cyano-3-(3-cyanobenzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;</li><li>Benzo[1,3]dioxole-5-carboxylic acid (3-amino-propyl)-{1-[3-(3-cyano-benzyl)-7-methoxy-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;</li><li>Benzo[1,3]dioxole-5-carboxylic acid (3-amino-propyl)-{1-[3-(3-cyano-benzyl)-7-fluoro-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;</li><li>N-(3-Amino-propyl)-N-{1-[3-(3-cyano-benzyl)-7-fluoro-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;</li><li>Benzo[1,3]dioxole-5-carboxylic acid (3-amino-propyl)-{1-[7-chloro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-amide;</li><li>N-(3-Amino-propyl)-N-{1-[7-cyano-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;</li><li>3-Benzyl-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-4-oxo-4H-chromene-7-carbonitrile;</li><li>2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-methoxy-chromen-4-one;</li><li>N-(3-Amino-propyl)-N-{1-[7-chloro-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-2-methoxy-acetamide;</li><li>N-(3-Amino-propyl)-N-{1-[7-cyano-3-(3-cyano-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;</li><li>N-(3-Amino-propyl)-N-{1-[3-(3-cyano-benzyl)-7-fluoro-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-3-fluoro-4-methyl-benzamide;</li><li>2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-l-yl]-2-methyl-propyl}-7-chloro-3-(3-methoxy-benzyl)-chromen-4-one;</li><li>2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methylpropyl}-3-benzyl-7-fluoro-chromen-4-one;</li><li>2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methylpropyl}-3-benzyl-4-oxo-4H-chromene-7-carbonitrile;</li><li>2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-3-benzyl-7-fluoro-chromen-4-one;</li><li>2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methylpropyl}-7-chloro-3-(3-methoxy-benzyl)-chromen-4-one;</li><li>2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methylpropyl}-3-benzyl-7-chloro-chromen-4-one;</li><li>3-Benzyl-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methylpropyl}-7-methoxy-chromen-4-one;</li><li>3-(2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-7-chloro-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;</li><li>3-(2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methylpropyl-7-chloro-4-oxo-4H-chromen-3 -ylmethyl)-benzonitrile;</li><li>2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-7-fluoro-3-(3-methoxy-benzyl)-chromen-4-one;</li><li>2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methylpropyl}-3-(3-cyano-benzyl)-4-oxo-4H-chromene-7-carbonitrile;</li><li>N-(3-Amino-propyl)-3-fluoro-N-{1-[7-hydroxy-3-(3-methoxy-benzyl)-4-oxo-4H-chromen-2-yl]-2-methyl-propyl}-4-methyl-benzamide;</li><li>2-{1-[4-(2-Amino-ethyl)-2-p-tolyl-imidazol-1-yl]-2-methyl-propyl}-3-(3-methoxy-benzyl)-4-oxo-4H-chromene-7-carbonitrile;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-4-ethoxy-benzamide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-6-trifluoromethyl-nicotinamide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-6-trifluoromethyl-nicotinamide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-isonicotinamide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-4-cyano-benzamide;</li><li>4-Acetylamino-N-(3-amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-6-trifluoromethyl-nicotinamide;</li><li>Benzo[1,2,3]thiadiazole-5-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>Benzo[1,2,3]thiadiazole-5-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>4-Acetylamino-N-(3-amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-benzamide;</li><li>Benzo[1,2,3]thiadiazole-5-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-nicotinamide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-4-methoxy-benzamide;</li><li>Benzo[1,2,3]thiadiazole-5-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-Methyl-pyrazine-2-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>N-(3-Amino-propyl)-N-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-3-dimethylamino-benzamide;</li><li>2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1-yl)-2-methyl-propyl]-3-(3-methoxy-benzyl)-4-oxo-4H-chromene-7-carbonitrile;</li><li>7-Chloro-3-(3-methoxy-benzyl)-2-[2-methyl-1-(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one;</li><li>7-Chloro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methylpropyl}-3-(3-methoxy-benzyl)-chromen-4-one;</li><li>2-[1-(2-Benzo[1,3]dioxol-5-yl-4,5-dihydro-imidazol-1yl)-2-methyl-propyl]-7-chloro-3-(3-methoxy-benzyl)-chromen-4-one;</li><li>7-Fluoro-3-(3-methoxy-benzyl)-2-[2-methyl-1(2-p-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one;</li><li>7-Fluoro-2-{1-[2-(3-fluoro-4-methyl-phenyl)-4,5-dihydro-imidazol-1-yl]-2-methylpropyl}-3-(3 -methoxy-benzyl)-chromen-4-one;</li><li>2-{1-[-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methylpropyl}-7-fluoro-3-(3-methoxy-benzyl)-chromen-4-one;</li><li>2-{1-[4-(2-Acetylamino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methylpropyl}-3-(3-methoxy-benzyl)-4-oxo-4H-chromene-7-carboxylic acid amide;</li><li>3-(2-{1-[4-(2-Amino-ethyl)-2-(3-fluoro-4-methyl-phenyl)-imidazol-1-yl]-2-methylpropyl}-7-fluoro-4-oxo-4H-chromen-3-ylmethyl)-benzonitrile;</li><li>N-{1-[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-2-p-tolyl-1H-imidazol-4-ylmethyl -acetamide;</li><li>Benzo[b]thiophene-2-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>1-Methyl-1H-indole-2-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-tert-Butyl-2-methyl-2H-pyrazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>2,5-Dimethyl-2H-pyrazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-tert-Butyl-2-methyl-2H-pyrazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>2,5-Dimethyl-2H-pyrazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>2,5-Dimethyl-2H-pyrazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-Methyl-2H-pyrazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-Methyl-2H-pyrazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>Furan-2-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>Furan-2-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>2,5-Dimethyl-furan-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>2,5-Dimethyl-furan-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-Methyl-thiophene-2-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-Methyl-thiophene-2-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-Methyl-isoxazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-Methyl-2-trifluoromethyl-furan-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-Methyl-isoxazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-Methyl-isoxazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>Benzo[c]isoxazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>Benzo[c]isoxazole-3-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>1-Methyl-1H-pyrrole-2-carboxylic acid (3-amino-propyl)-[1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>1-Methyl-1H-imidazole-4-carboxylic acid (3-amino-propyl)-[(R)-1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>N-(3-Amino-propyl)-N-[(R)-1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methylpropyl]-3-dimethylamino-benzamide;</li><li>5-Methyl-2-trifluoromethyl-furan-3-carboxylic acid (3-amino-propyl)-[(R)-1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>5-Methyl-isoxazole-3-carboxylic acid (3-amino-propyl)-[(R)-1-(3-benzyl-7-cyano-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>1-Methyl-1H-imidazole-4-carboxylic acid (3-amino-propyl)-[(R)-1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>1-Methyl-1H-pyrrole-2-carboxylic acid (3-amino-propyl)-[(R)-1-(3-benzyl-7-fluoro-4- - oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide;</li><li>Benzo[c]isoxazole-3-carboxylic acid (3-amino-propyl)-[(R)-1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide; and</li><li>5-Methyl-isoxazole-3-carboxylic acid (3-amino-propyl)-[(R)-1-(3-benzyl-7-fluoro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-amide.</li></ul>

Utility, Testing and Administration

General Utility

0158Once made, the compounds of the invention find use in a variety of applications involving alteration of mitosis. As will be appreciated by those skilled in the art, mitosis may be altered in a variety of ways; that is, one can effect 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.

0159In a preferred embodiment, the compounds of the invention are used to inhibit mitotic spindle formation, thus causing prolonged cell cycle arrest in mitosis. By "inhibit" in this context is meant decreasing or interfering with mitotic spindle formation or causing mitotic spindle dysfunction. 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.

0160The compounds of the invention are useful to bind to, and/or inhibit the activity of, a mitotic kinesin, KSP. In a preferred embodiment, the KSP is human KSP, although the compounds may be used to bind to or inhibit the activity of KSP kinesins from other organisms. In this context, "inhibit" means either increasing or decreasing spindle pole separation, causing malformation, i.e., splaying, of mitotic spindle poles, or otherwise causing morphological perturbation of the mitotic spindle. Also included within the definition of KSP for these purposes are variants and/or fragments of KSP. See <patcit id="pcit0002" dnum="US6437115B"><text>U.S. Patent 6,437,115</text></patcit>. The compounds of the invention have been shown to have specificity for KSP.

0161The compounds of the invention are used to treat cellular proliferation diseases. Such disease states which can be treated by the compounds, compositions and methods provided herein include, but are not limited to, cancer (further discussed below), autoimmune disease, fungal disorders, arthritis, graft rejection, inflammatory bowel disease, cellular proliferation induced after medical procedures, including, but not limited to, surgery, angioplasty, and the like. Treatment includes inhibiting cellular proliferation. It is appreciated that in some cases the cells may not be in an abnormal state and still require treatment. Thus, in one embodiment, the invention herein includes application to cells or individuals afflicted or subject to impending affliction with any one of these disorders or states.

0162The compound and positions 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 compounds and compositions of the invention include, but are not limited to: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, 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, leiomyoma, 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), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (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), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma); Hematologic: blood (myeloid 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 above identified conditions.

Testing

0163For assay of KSP-modulating activity, generally either KSP or a compound according to the invention is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g., a microtiter plate, an array, etc.). The insoluble support may be made of any composition to which the sample 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 convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, Teflon™, etc. Microtiter plates and arrays 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 sample is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the sample and is nondiffusable. Preferred methods of binding include the use of antibodies (which do not sterically block either 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 sample, 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.

0164The compounds of the invention may be used on their own to inhibit the activity of a mitotic kinesin, particularly KSP. A compound of the invention is combined with KSP and the activity of KSP is assayed. Kinesin (including KSP) 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.

0165Methods of performing motility assays are well known to those of skill in the art. (See e.g., <nplcit id="ncit0014" npl-type="s"><text>Hall, et al. (1996), Biophys. J., 71: 3467-3476</text></nplcit>, <nplcit id="ncit0015" npl-type="s"><text>Turner et al., 1996, AnaL Biochem. 242 (1):20-5</text></nplcit>; <nplcit id="ncit0016" npl-type="s"><text>Gittes et al., 1996, Biophys. J. 70(1): 418-29</text></nplcit>;<nplcit id="ncit0017" npl-type="s"><text> Shirakawa et al., 1995, J. Exp. BioL 198: 1809-15</text></nplcit>; <nplcit id="ncit0018" npl-type="s"><text>Winkelmann et al., 1995, Biophys. J. 68: 2444-53</text></nplcit>; <nplcit id="ncit0019" npl-type="s"><text>Winkelmann et al., 1995, Biophys. J. 68: 72S</text></nplcit>.)

0166Methods known in the art for determining ATPase hydrolysis activity also can be used. Preferably, solution based assays are utilized. <patcit id="pcit0003" dnum="US6410254B"><text>U.S. Patent 6,410,254</text></patcit> describes such assays. Alternatively, conventional methods are used. For example, P_i release from kinesin can be quantified. Preferably, 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 µL of the reaction mixture is quenched in 90 µL of cold 0.3 M 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 µL 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 is read at an absorbance of 650 nm. If phosphate standards were used, absorbance readings can be converted to mM P_i and plotted over time. Additionally, ATPase assays known in the art include the luciferase assay.

0167ATPase activity of kinesin motor domains also can be used to monitor the effects of agents and are well known to those skilled in the art. A TPase assays of kinesin are performed in the absence of microtubules. Alternatively, the ATPase assays are performed in the presence of microtubules. Different types of agents can be detected in the above assays. Preferably the effect of a agent is independent of the concentration of microtubules and ATP. Alternatively, the effect of the agents on kinesin ATPase can be decreased by increasing the concentrations of ATP, microtubules or both. Alternatively, the effect of the agent is increased by increasing concentrations of ATP, microtubules or both.

0168Compounds that inhibit the biochemical activity of KSP in vitro may then be screened in vivo. In vivo screening methods include assays of cell cycle distribution, cell viability, or the presence, morphology, activity, distribution, or number 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, <patcit id="pcit0004" dnum="US6437115A"><text>U.S. Patent 6,437, 115</text></patcit>. Microscopic methods for monitoring spindle formation and malformation are well known to those of skill in the art (see, e.g., <nplcit id="ncit0020" npl-type="s"><text>Whitehead and Rattner (1998), J. Cell Sci. 111:2551-61</text></nplcit>; <nplcit id="ncit0021" npl-type="s"><text>Galgio et al, (1996) J. Cell Biol., 135:399-414</text></nplcit>).

0169The compounds of the invention inhibit the KSP kinesin. One measure of inhibition is IC₅₀, defined as the concentration of the compound at which the activity of KSP is decreased by fifty percent relative to a control. Preferred compounds have IC₅₀'s of less than about 1 mM, with preferred embodiments having IC₅₀'s of less than about 100 µM, with more preferred embodiments having IC₅₀'s of less than about 10 µM, with particularly preferred embodiments having IC₅₀'s of less than about 1 µM, and especially preferred embodiments having IC₅₀'s of less than about 100 nM, and with the most preferred embodiments having IC₅₀'s of less than about 10 nM. Measurement of IC₅₀ is done using an ATPase assay such as described herein.

0170Another measure of inhibition is K_i. For compounds with IC₅₀'s less than 1 µM, the K_i or K_d is defined as the dissociation rate constant for the interaction of the compounds described herein with KSP. Preferred compounds have K_i's of less than about 100 µM, with preferred embodiments having K_i's of less than about 10 µM, and particularly preferred embodiments having K_i's of less than about 1 µM and especially preferred embodiments having K_i's of less than about 100 nM, and with the most preferred embodiments having K_i's of less than about 10 nM.

0171The K_i for a compound is determined from the IC₅₀ based on three assumptions and the Michaelis-Menten equation. 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: <maths id="math0001"><math display="block"><mi mathvariant="normal">V</mi><mo mathvariant="normal">=</mo><msub><mi mathvariant="normal">V</mi><mi>max</mi></msub><mo>⁢</mo><msub><mi mathvariant="normal">E</mi><mn mathvariant="normal">0</mn></msub><mo>⁢</mo><mfenced open="[" close="]"><mi mathvariant="normal">I</mi><mo mathvariant="normal">-</mo><mfrac><mrow><mfenced><msub><mi mathvariant="normal">E</mi><mn mathvariant="normal">0</mn></msub><mo mathvariant="normal">+</mo><msub><mi mathvariant="normal">I</mi><mn mathvariant="normal">0</mn></msub><mo mathvariant="normal">+</mo><mi>Kd</mi></mfenced><mo mathvariant="normal">-</mo><msqrt><msup><mfenced><msub><mi mathvariant="normal">E</mi><mn mathvariant="normal">0</mn></msub><mo mathvariant="normal">+</mo><msub><mi mathvariant="normal">I</mi><mn mathvariant="normal">0</mn></msub><mo mathvariant="normal">+</mo><mi>Kd</mi></mfenced><mn mathvariant="normal">2</mn></msup><mo mathvariant="normal">-</mo><mn mathvariant="normal">4</mn><mspace width="1em" /><msub><mi mathvariant="normal">E</mi><mn mathvariant="normal">0</mn></msub><mo>⁢</mo><msub><mi mathvariant="normal">I</mi><mn mathvariant="normal">0</mn></msub></msqrt></mrow><mrow><mn mathvariant="normal">2</mn><mo>⁢</mo><msub><mi mathvariant="normal">E</mi><mn mathvariant="normal">0</mn></msub></mrow></mfrac></mfenced></math><img file="EP1511734B1_D0031.tif" /></maths> where V is the observed rate, V_max is the rate of the free enzyme, I₀ is the inhibitor concentration, E₀ is the enzyme concentration, and K_d is the dissociation constant of the enzyme-inhibitor complex.

0172Another measure of inhibition is GI₅₀, defined as the concentration of the compound that results in a decrease in the rate of cell growth by fifty percent. Preferred compounds have GI₅₀'s of less than about 1 mM; those having a GI₅₀ of less than about 20 µM are more preferred; those having a GI₅₀ of less than about 10 µM more so; those having a GI₅₀ of less than about 1 µM more so; those having a GI₅₀ of less than about 100 nM more so; and those having a GI₅₀ of less than about 10 nM even more so. Measurement of GI₅₀ is done using a cell proliferation assay such as described herein. Compounds of this class were found to inhibit cell proliferation.

0173In vitro potency of small molecule inhibitors is determined, for example, by assaying human ovarian cancer cells (SKOV3) for viability following a 72-hour exposure to a 9-point dilution series of compound. Cell viability is determined by measuring the absorbance of formazon, a product formed by the bioreduction of MTS/PMS, a commercially available reagent. Each point on the dose-response curve is calculated as a percent of untreated control cells at 72 hours minus background absorption (complete cell kill).

0174Anti-proliferative compounds that have been successfully applied in the clinic to treatment of cancer (cancer chemotherapeutics) have GI₅₀'s that vary greatly. For example, in A549 cells, paclitaxel GI₅₀ is 4 nM, doxorubicin is 63 nM, 5-fluorouracil is 1 µM, and hydroxyurea is 500 µM (data provided by National Cancer Institute, Developmental Therapeutic Program, http://dtp.nci.nih.gov/). Therefore, compounds that inhibit cellular proliferation, irrespective of the concentration demonstrating inhibition, may be useful.

0175To 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 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.

0176The determination of the binding of the compound of the invention to KSP may be done in a number of ways. The compound is labeled, for example, with a fluorescent or radioactive moiety, and binding is determined directly. For example, this may be done by attaching all or a portion of KSP to a solid support, adding a labeled test compound (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.

0177By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles such as magnetic particles, chemiluminescent tag, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal.

0178Sometimes, only one of the components is labeled. For example, the kinesin proteins may be labeled at tyrosine positions using ¹²⁵I, or with fluorophores. Alternatively, more than one component may be labeled with different labels; using ¹²⁵I for the proteins, for example, and a fluorophor for the antimitotic agents.

0179The compounds of the invention may also be used as competitors to screen for additional drug candidates. "Candidate 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 assays herein include candidate agents which do not bind the cellular proliferation protein in its endogenous native state termed herein as "exogenous" agents. Preferably exogenous agents further exclude antibodies to KSP.

0180Candidate 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 functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

0181Candidate 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, and/or amidification to produce structural analogs.

0182Competitive screening assays may be done by combining KSP and a drug candidate in a first sample. A second sample comprises a compound of the present invention, 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 a drug candidate capable of binding to KSP and potentially inhibiting 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.

0183Preferably the binding of the candidate agent to KSP is determined through the use of competitive binding assays. 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.

0184In 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.

0185Incubation 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.

0186Preferably, 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 inhibiting, the activity of KSP. 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.

0187Alternatively, 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 with a lack of competitor binding, may indicate the candidate agent is capable of binding to KSP.

0188Inhibition is tested by screening for candidate agents capable of inhibiting 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. 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 screening 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.

0189Alternatively, differential screening may be used to identify drug candidates that bind to the native KSP, but cannot bind to modified KSP.

0190Positive 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.

0191A 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.

Administration

0192Accordingly, the compounds of the invention are administered to cells. By "cells" herein is meant any cell in which mitosis or meiosis can be altered. By "administered" herein is meant administration of a therapeutically effective dose of a compound 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, 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, route of administration, age, body weight, general health, sex, diet, time of administration, nature of the formulation, drug interaction, and the precise condition requiring treatment and its severity may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. However, an effective amount of a compound of Formula I for the treatment of neoplastic growth (typically by intravenous administration), for example colon or breast carcinoma, will generally be in the range of 0.1 to 100 (including 1 to 100) mg/m² of surface area of the recipient per dose on a once a week to once a month schedule and usually in the range of 2 to 30 mg/m² of surface area of the recipient per dose on a once a week to once a month schedule. An effective amount of a salt, solvate, or solvate of a salt of a compound of Formula I may be determined as a proportion of the effective amount of the compound of Formula I per se. It is envisaged that similar dosages would be appropriate for treatment of the other conditions referred to herein.

0193A "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 a mammal, and in the most preferred embodiment the patient is human.

0194Compounds of the invention having the desired pharmacological activity may be administered, preferably as a pharmaceutically acceptable composition comprising an pharmaceutical excipient, 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.%.

0195The agents may be administered alone or in combination with other treatments, i.e., radiation, or other chemotherapeutic agents such as the taxane class of agents that appear to act on microtubule formation or the camptothecin class of topoisomerase I inhibitors. When used, other chemotherapeutic agents may be administered before, concurrently, or after administration of a compound of the present invention. In one aspect of the invention, a compound of the present invention is co-administered with one or more other chemotherapeutic agents. By "co-administer" it is meant that the present compounds are administered to a patient such that the present compounds as well as the co-administered compound may be found in the patient's bloodstream at the same time, regardless when the compounds ate actually administered, including simultaneously.

0196The administration of the compounds and compositions of the present invention can be done in a variety of ways, 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 compound or composition may be directly applied as a solution or spray.

0197Pharmaceutical dosage forms include a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutical excipients. As is known in the art, pharmaceutical excipients are secondary ingredients which function to enable or enhance the delivery of a drug or medicine in a variety of dosage forms (e.g.: oral forms such as tablets, capsules, and liquids; topical forms such as dermal, opthalmic, and otic forms; suppositories; injectables; respiratory forms and the like). Pharmaceutical excipients include inert or inactive ingredients, synergists or chemicals that substantively contribute to the medicinal effects of the active ingredient. For example, pharmaceutical excipients may function to improve flow characteristics, product uniformity, stability, taste, or appearance, to ease handling and administration of dose, for convenience of use, or to control bioavailability. While pharmaceutical excipients are commonly described as being inert or inactive, it is appreciated in the art that there is a relationship between the properties of the pharmaceutical excipients and the dosage forms containing them.

0198Pharmaceutical excipients suitable for use as carriers or diluents are well known in the art, and may be used in a variety of formulations. See, e.g., <nplcit id="ncit0022" npl-type="b"><text>Remington's Pharmaceutical Sciences, 18 th Edition, A. R. Gennaro, Editor, Mack Publishing Company (1990</text></nplcit>); <nplcit id="ncit0023" npl-type="b"><text>Remington: The Science and Practice of Pharmacy, 20th Edition, A. R. Gennaro, Editor, Lippincott Williams &amp; Wilkins (2000</text></nplcit>); <nplcit id="ncit0024" npl-type="b"><text>Handbook of Pharmaceutical Excipients, 3rd Edition, A. H. Kibbe, Editor, American Pharmaceutical Association, and Pharmaceutical Press (2000</text></nplcit>); and <nplcit id="ncit0025" npl-type="b"><text>Handbook of Pharmaceutical Additives, compiled by Michael and Irene Ash, Gower (1995</text></nplcit>).

0199Oral solid dosage forms such as tablets will typically comprise one or more pharmaceutical excipients, which may for example help impart satisfactory processing and compression characteristics, or provide additional desirable physical characteristics to the tablet. Such pharmaceutical excipients may be selected from diluents, binders, glidants, lubricants, disintegrants, colors, flavors, sweetening agents, polymers, waxes or other solubility-retarding materials.

0200Compositions for intravenous administration will generally comprise intravenous fluids, i.e., sterile solutions of simple chemicals such as sugars, amino acids or electrolytes, which can be easily carried by the circulatory system and assimilated. Such fluids are prepared with water for injection USP.

0201Fluids used commonly for intravenous (IV) use are disclosed in Remington, the Science and Practice of Pharmacy [full citation previously provided], and include: <ul id="ul0008" list-style="none" compact="compact"><li>alcohol (e.g., in dextrose and water ("D/W") [e.g., 5% dextrose] or dextrose and water [e.g., 5% dextrose] in normal saline solution ("NSS"); e.g. 5% alcohol);</li><li>synthetic amino acid such as Aminosyn, FreAmine, Travasol, e.g., 3.5 or 7; 8.5; 3.5, 5.5 or 8.5 % respectively;</li><li>ammonium chloride e.g., 2.14%;</li><li>dextran 40, in NSS e.g., 10% or in D5/W e.g., 10%;</li><li>dextran 70, in NSS e.g., 6% or in D5/W e.g., 6%;</li><li>dextrose (glucose, D5/W) e.g., 2.5-50%;</li><li>dextrose and sodium chloride e.g., 5-20% dextrose and 0.22-0.9% NaCl;</li><li>lactated Ringer's (Hartmann's) e.g., NaCl 0.6%, KCl 0.03%, CaCl₂ 0.02%;</li><li>lactate 0.3%;</li><li>mannitol e.g., 5%, optionally in combination with dextrose e.g., 10% or NaCl e.g., 15 or 20%;</li><li>multiple electrolyte solutions with varying combinations of electrolytes, dextrose, fructose, invert sugar Ringer's e.g., NaCl 0.86%, KCl 0.03%, CaCl₂ 0.033%;</li><li>sodium bicarbonate e.g., 5%;</li><li>sodium chloride e.g., 0.45, 0.9, 3, or 5%;</li><li>sodium lactate e.g., 1/6 M; and</li><li>sterile water for injection The pH of such fluids may vary, and will typically be from 3.5 to 8 such as known in the art.</li></ul>

0202The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention.

0203All anhydrous solvents were purchased from Aldrich Chemical Company in SureSeal® containers. Reagents were added and aqueous extractions performed with single or multichannel pipettors. Filtrations were performed using Whatman/Polyfiltronics 24 well, 10 mL filtration blocks. Evaporation of volatile materials from the array was performed with a Labconco Vortex-Evaporator or by sweeping with a 4 x 6 nitrogen manifold.

Example 1

Synthesis of Compounds

0204<chemistry id="chem0031" num="0031"><img file="EP1511734B1_D0032.tif" /></chemistry><chemistry id="chem0032" num="0032"><img file="EP1511734B1_D0033.tif" /></chemistry>

a) 3-Phenyl-propionic acid 3-chloro-phenyl ester

0205To a solution of 3-chlorophenol (<b>1</b>, 50.1 g, 0.3 mol), triethylamine (TEA, 85 mL), and CH₂Cl₂ (500 mL) at 23°C was added hydrocinnamoyl chloride (<b>2</b>, 31 mL, 0.3 mol) over 5 minutes. After 30 minutes, the reaction mixture was concentrated in vacuo. The crude slurry was then dissolved in 10:1 hexanes:EtOAc (300 mL) and washed with I N NaOH (100 mL) and brine (100 mL). The organic layer was dried (MgSO₄) and filtered through a plug of silica gel (10:1 hexanes:EtOAc rinse). The eluent was concentrated to afford 78 g of a slightly yellow oil, which was used without any further purification.

b) 1-(4-Chloro-2-hydroxy-phenyl)-3-phenyl-propan-1-one

0206AlCl₃ (52 g, 0.39 mol) was added slowly over 15 minutes to ester <b>3</b> (78 g, 0.3 mol) at 140°C. After an additional 15 minutes gas evolution ceased and the reaction mixture was poured into a 1L beaker and allowed to cool to RT. The resulting solid was dissolved in CH₂Cl₂ (100 mL) and slowly quenched with <b>1</b> N HCl (200 mL). This mixture was diluted with EtOAc (600 mL) and the layers were separated. The organic layer was washed with brine (100 mL), dried (MgSO₄), filtered, and concentrated. The crude oil was dissolved in 20:1 hexanes:EtOAc (500 mL) and flushed through a plug of silica gel (100% hexanes; 20:1 hexanes:EtOAc rinse). The filtrate was concentrated to provide a slightly brown oil, which was purified by flash column chromatography (50:1 hexanes:EtOAc; 40:1 hexanes:EtOAc; 30:1 hexanes:EtOAc; 20:1 hexanes:EtOAc) to yield 31.2 g (40%) of <b>4</b> as a white solid.

c) 2-tert-Butoxycarbonylamino-3-methyl-butyric acid 5-chloro-2-(3-phenyl-propionyl)-phenyl ester

0207A solution of phenol <b>4</b> (23.18g, 89.1 mmol), BOC-D-Valine (<b>5</b>, 21.29 g, 98.05 mmol), HBTU (40.57 g, 107 mmol), TEA (37 mL, 265 mmol), and CH₂Cl₂ (155 mL) were maintained at 23°C for 5 hours. The reaction mixture was diluted with EtOAc (500 mL) and washed with saturated aqueous NH₄Cl (2 x 100 mL) and brine (100 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (100% hexanes; 50:1 hexanes:EtOAc; 40:1 hexanes:EtOAc; 30:1 hexanes:EtOAc; 20:1 hexanes:EtOAc; 10:1 hexanes:EtOAc) to yield 35.7 g (87%) of <b>6</b> as a yellow oil.

d) [1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-carbamic acid tert-butyl ester

0208A mixture of ester <b>6</b> (35.55 g, 79.17 mmol), K₂CO₃ (21.8 g, 158.4 mmol), and DMF (264 mL) was placed into a 140°C oil bath. After 30 mins, the reaction mixture was quenched with H₂O (300 mL) and extracted with Et₂O (3 x 200 mL). The organic layer was washed with brine (100 mL), dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (10:1 hexanes:EtOAc) to yield 3.5 g (10.2%) of 7.

e) 2-(1-Amino-2-methyl-propyl)-3-benzyl-7-chloro-chromen-4-one

0209Chromenone <b>7</b> (1.8 g, 4.18 mmol) and TFA:H₂O (97.5:2.5, 30 mL) was maintained at 23°C for 1h. The reaction mixture was concentrated. The residue was dissolved in EtOAc (100 mL) and washed with 1 N NaOH (25 mL) and brine (25 mL). The organic layer was dried (MgSO₄), filtered, and concentrated to provide a colorless oil, which was used without further purification.

f) N-{2-[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propylamino]-1,1-dimethyl-ethyl}-4-methyl-benzamide

0210Chromenone <b>8</b> (72 mg, 0.22 mmol), aldehyde <b>9</b> (65 mg, 0.32 mmol), Na(OAc) ₃BH (184 mg, 0.87 mmol), and CH₂Cl₂ (1 mL) was maintained at 23°C for 3 h. The reaction mixture was diluted with EtOAc (20 mL) and washed with 1 N NaOH (5 mL) and brine (5 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (3:1 hexanes:EtOAc) to yield 80 mg (70%) of <b>10</b> as a white solid.

g) 3-Benzyl-7-chloro-2-[1-(4,4-dimethyl-2-p-tolyl-4,5-dihydro-imidazol-1-yl)-2-methylpropyl]-chromen-4-one

0211Chromenone <b>10</b> (80 mg, 0.15 mmol), POCl₃ (0.1 mL, 1.1 mmol), and PhMe (1 mL) were heated to 110°C. After 3.5h, an additional portion of POCl₃ (0.1 mL, 1.1 mmol) was added. After 1h, the reaction mixture was diluted with EtOAc (20 mL) and washed with 1 N NaOH (10 mL) and brine (10 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (20:1 CHCl₃:MeOH) to yield 50 mg (65%) of <b>11</b> as a white solid.

Example 2

Synthesis of Compounds

0212<chemistry id="chem0033" num="0033"><img file="EP1511734B1_D0034.tif" /></chemistry>

a) Preparation of {3-[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propylamino]-propyl}-carbamic acid tert-butyl ester

0213Chromenone <b>8</b> (420 mg, 1.24 mmol), aldehyde <b>12</b> (280 mg, 1.6 mmol), NaCN(OAc) ₃BH (790 mg, 3.7 mmol), and CH₂Cl₂ (4.1 mL) was maintained at 23°C for 3 h. The reaction mixture was diluted with EtOAc (20 mL) and washed with 1 N NaOH (5 mL) and brine (5 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (5:1 hexanes:EtOAc; 3:1 hexanes:EtOAc) to yield 460 mg (75%) of <b>13</b> as a viscous oil.

b) Preparation of {3-[[1-(3-Benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-(4-methyl-benzoyl)-amino]-propyl}-carbamic acid tert-butyl ester

0214To a solution of chromenone <b>13</b> (1.3 g, 2.6 mmol), diisoproylethylamine (DIEA, 1.8 mL), and CH₂Cl₂ (7.5 mL) at 23°C was addedp-toluoyl chloride (0.7 mL, 5.22 mmol). After 2.5 h, the reaction mixture was diluted with EtOAc (100 mL) and washed with saturated aqueous NaHCO₃ (2 x 20 mL) and brine (20 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (3:1 hexanes:EtOAc) to yield 1.43 g (89%) of <b>14</b> as a colorless oil.

c) Preparation of N-(3-Amino-propyl)-N-[1-(3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide

0215Chromenone <b>14</b> (1.43 g, 2.32 mmol) and TFA:H₂O (97.5:2.5, 30 mL) was maintained at 23°C for 1h. The reaction mixture was concentrated. The residue was dissolved in EtOAc (100 mL) and washed with 1 N NaOH (25 mL) and brine (25 mL). The organic layer was dried (MgSO₄), filtered, and concentrated to provide a white solid which was deemed >95% pure by ¹H NMR and LCMS analysis.

0216Using procedures analogous to those set forth above in Example 2, the following compound was prepared. <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="48mm" /><colspec colnum="2" colname="col2" colwidth="28mm" /><thead><row><entry valign="top">Structure</entry><entry valign="top">LRMS (MH) <i>m</i>/<i>z</i></entry></row></thead><tbody><row><entry><chemistry id="chem0034" num="0034"><img file="EP1511734B1_D0035.tif" /></chemistry></entry><entry valign="middle">471.2</entry></row></tbody></tgroup></table></tables>

Example 3

Synthesis of compounds

0217<chemistry id="chem0035" num="0035"><img file="EP1511734B1_D0036.tif" /></chemistry>

0218Carbonyldiimidazole (9.14 g, 56.37 mmol) was added slowly to a r.t. solution of BOC-D-Valine (<b>5</b>, 12.25 g, 56.37 mmol) and THF (185 mL). Afer 1 h, the solution was washed with 50% aqueuous NaCl (200 mL), followed by brine (2 x 200 mL). The organic layer was dried (MgSO₄), filtered, and concentrated to provide a white solid, which was used without further purification.

0219(2-Bromoethyl)benzene (2.51 mL, 18.38 mmol), magnesium turnings (477 mg, 19.62 mmol), and THF (20 mL) were heated to 60°C for 1 hour, then allowed to cool to r.t. A solution of <b>16</b> (2.0 g, 9.19 mmol) and THF (20 mL) was cooled to 0-5°C. The solution of the phenethyl magnesium chloride was then added via syringe to the 0-5°C solution of the valine imidazole. The temperature was monitored by internal thermometer and was not allowed to exceed 15°C. The reaction mixture was maintained at 0-5°C for 1 hour. The reaction mixture was quenched with saturated aqueous NH₄Cl (20 mL), and diluted with EtOAc (100 mL). The layers were separated and the organic layer was washed with brine (30 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (10:1 hexanes:EtOAc; 5:1 hexanes:EtOAc) to yield 1.15 g (41 %) of <b>17</b>. LRMS (MH-tBuOCO) <i>m</i>/<i>z</i> 206.1.

0220LHMDS (1.M in THF, 9.53 mL, 3.3 equiv) was added slowly via syringe to a - 78°C solution of ketone <b>17</b> (882 mg, 2.89 mmol). After the addition was complete the resulting light orange solution was maintained at - 78°C for 40 mins. Neat 4-chloro-2-fluorobenzoyl chloride (<b>18</b>, 460 µL, 2.89 mmol ) (assumed density to be 1.20 g/mL) was added dropwise via syringe. The reaction solution turned to an orange color and was maintained for 40 mins. The reaction solution was quenched with saturated aqueous NH₄Cl (20 mL), and diluted with EtOAc (50 mL). The layers were separated and the organic layer was washed with brine (30 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (10:1 hexanes:EtOAc) to yield 1.15 g (86%) of <b>19</b>. LRMS (MH-HF) <i>m</i>/<i>z</i> 442.1.

0221A mixture of <b>19</b> (1.15 g, 2.49 mmol), K₂CO₃ (420 mg, 3.04 mmol), and DMF (12 mL) was maintained at r.t. for 30 mins. The yellow reaction solution was quenched with brine (50 mL), and diluted with Et₂O (50 mL). The layers were separated and the organic layer was washed with brine (2 x 50 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (10:1 hexanes:EtOAc) to yield <b>20</b>. LRMS (MH) m/z 442.1.

0222Using methods analogous to those described above in Examples 2 and 3, the compounds shown below were prepared. <chemistry id="chem0036" num="0036"><img file="EP1511734B1_D0037.tif" /></chemistry><tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="10mm" /><colspec colnum="2" colname="col2" colwidth="22mm" /><colspec colnum="3" colname="col3" colwidth="26mm" /><colspec colnum="4" colname="col4" colwidth="39mm" /><thead><row><entry valign="top"><b>R₇</b></entry><entry valign="top"><b>R</b></entry><entry valign="top"><b>R'</b></entry><entry valign="top"><b>Stereochemistry (R/S)</b></entry></row></thead><tbody><row><entry>Cl</entry><entry>H</entry><entry>H</entry><entry>3/1</entry></row><row><entry>Cl</entry><entry namest="col2" nameend="col3" align="left">-CH₂CMe₂N=C(<i>p</i>-tol)-</entry><entry>3/1</entry></row><row><entry>Cl</entry><entry>H₂N(CH₂)₃-</entry><entry><i>p</i>-toluoyl</entry><entry>3/1</entry></row><row><entry>Cl</entry><entry>H₂N(CH₂)₃-</entry><entry>MeOCH₂C(O)-</entry><entry>3/1</entry></row><row><entry>Cl</entry><entry>H₂N(CH₂)₃-</entry><entry><i>p</i>-Br-phenyl-</entry><entry>3/1</entry></row><row><entry>Cl</entry><entry>H₂N(CH₂)₃-</entry><entry><i>p</i>-toluoyl</entry><entry>R</entry></row><row><entry>Cl</entry><entry>H₂N(CH₂)₃-</entry><entry><i>p</i>-toluoyl</entry><entry>S</entry></row><row><entry>Cl</entry><entry namest="col2" nameend="col3" align="left">-CH₂CMe₂N=C(p-tol)-</entry><entry>R</entry></row><row><entry>Cl</entry><entry namest="col2" nameend="col3" align="left">-CH₂CMe₂N=C(p-tol)-</entry><entry>S</entry></row><row><entry>F</entry><entry>H</entry><entry>H</entry><entry>3/1</entry></row><row><entry>F</entry><entry>H₂N(CH₂)₃-</entry><entry><i>p</i>-toluoyl</entry><entry>3/1</entry></row><row><entry>F</entry><entry>H₂N(CH₂)₃-</entry><entry>MeOCH₂C(O)-</entry><entry>3/1</entry></row><row><entry>F</entry><entry namest="col2" nameend="col3" align="left">-CH₂CMe₂N=C(p-tol)-</entry><entry>3/1</entry></row></tbody></tgroup></table></tables>

Example 4

Alternative Methods for Preparation of Compounds

0223<chemistry id="chem0037" num="0037"><img file="EP1511734B1_D0038.tif" /></chemistry>

0224Ethylchloroformate (11.0 mL, 115 mmol) was added over 1 minute to a 0-5°C solution of BOC-D-Valine (<b>5</b>, 25.0 g, 115 mmol), triethylamine (16.0 mL, 115 mmol), and THF (145 mL) under N₂. The internal temperature of the reaction solution rose to 9°C. After 15 mins, a mixture of dimethylhydroxylamine hydrochloride (13.46 g, 138 mmol), triethylamine (32.0 mL, 230 mmol), and THF (110 mL) was added over 5 minutes. The internal temperature rose to 17°C. Upon completion of addition, the ice/H₂O bath was removed and the reaction solution maintained at 23°C for 1 hour. The reaction solution was then concentrated. The crude residue was dissolved in EtOAc (200 mL) and washed with 1 N HCl (200 mL) and brine (100 mL). The organic layer was dried (MgSO₄), filtered, and concentrated to provide 30 g (~100%) of <b>21</b> as a colorless oil, which was used without further purification.

0225(2-Bromoethyl)benzene (38.0 mL, 273 mmol), magnesium turnings (7.0 g, 289 mmol), and Et₂O (500 mL) were mixed in a 1L round-bottom flask equipped with a reflux condenser at 23°C under a N₂ atmosphere. After ~10 mins the reaction mixture begins to exotherm and the reaction mixture was allowed to progress to reflux with intermittent cooling with an ice/H₂O bath. After 1.5 hour, the Grignard reaction was complete and the solution had cooled to 23°C. A solution of <b>21</b> (18.0 g, 82.7 mmol) and Et₂O (200 mL) was added via cannula to the 20°C solution of the phenethylmagnesium bromide. The temperature was monitored by internal thermometer and was not allowed to exceed ~30°C. The reaction mixture temperature was monitored by an internal thermometer and regulated (20-30°C) with an ice/H₂O bath. After 1 h at 23°C, the reaction mixture was quenched by pouring into 1 N HCl (300 mL). The layers were separated and the organic layer was washed with brine (100 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (10:1 hexanes:EtOAc) to yield 13.4 g (53%) of <b>17</b>. LRMS (MH-tBuOCO) <i>m</i>/<i>z</i> 206.1.

0226Lithium bis(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 94.0 mL, 3.3 equiv) was added slowly over ~3 minutes via syringe to a - 78°C solution of ketone <b>17</b> (8.74 g, 28.62 mmol) and THF (100 mL). The reaction solution temperature was monitored by an internal thermometer, and addition of the base was done at a rate sufficient to prevent the temperature from exceeding -54°C. After the addition was complete the resulting solution was maintained at -78°C for 30 mins. Neat 4-chloro-2-fluorobenzoyl chloride (<b>18</b>, 4.58 mL, 28.62 mmol) (assumed density to be 1.20 g/mL) was added drop-wise over ~1 minute via syringe (temperature rose from -78°C to -59°C). The reaction solution was maintained at - 78°C for 30 mins. The reaction solution was quenched with 1 N HCl (100 mL). The layers were separated and the organic layer was washed with brine (100 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was used without further purification.

0227A mixture of the above crude product, K₂CO₃ (4.75 g, 34.34 mmol) and DMF (100 mL) was maintained at 23°C for 30 mins. The reaction mixture was quenched by addition of Et₂O (200 mL) and brine (200 mL). The layers were separated and the organic layer was washed with brine (2 x 200 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue absorbed onto silica gel (CH₂Cl₂) and was purified by flash column chromatography (10:1 hexanes:EtOAc) to yield 11.5 g (91% for 2 steps) of <b>20</b> as a white solid. LRMS (MH) <i>m</i>/<i>z</i> 442.1.

Example 5

0228<chemistry id="chem0038" num="0038"><img file="EP1511734B1_D0039.tif" /></chemistry>

0229Methylmagnesium chloride (82.5 mL, 3.0 M in THF, 247 mmol) was added over 5 mins to a 0-5°C solution of <b>21</b> (17.95 g, 69.0 mmol) and THF (200 mL) under an N₂ atmosphere. The reaction mixture temperature was monitored by an internal thermometer, and addition of the Grignard was done at a rate sufficient to prevent the temperature from exceeding 19°C. Upon complete addition the cooling bath was removed and the reaction mixture was maintained at 23°C for 2 hours. The reaction mixture was then quenched with 1 N HCl (100 mL). The layers were separated and the organic layer was washed with brine (100 mL). The organic layer was dried (MgSO₄), filtered, and concentrated to provide 12.5 g (84%) of 22 as a white solid (>95% pure by ¹H NMR), which was used without further purification.

0230LHMDS (128 mL, 1.0 M in THF) was added via syringe over 3 minutes to a - 78°C solution of ketone <b>22</b> (12.5 g, 58.1 mmol) and THF (200 mL). The reaction solution temperature was monitored by an internal thermometer, and addition of the base was done at a rate sufficient to prevent the temperature from exceeding -58°C. After 30 minutes, a solution of α-bromo-<i>m</i>-tolunitrile (<b>23</b>, 12.5 g, 63.9 mmol) and THF (50 mL) was added over 30 seconds (temperature rose from -78 to -60°C). The cooling bath was immediately replaced by a ice/H₂O bath and the reaction solution was maintained at ~0°C for 20 minutes. The reaction solution was quenched with 1 N HCl (100 mL). The layers were separated and the organic layer was washed with brine (100 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (10:1 hexanes:EtOAc) to yield 11.6 g (60%) of <b>24</b>. Inspection of the ¹H NMR revealed that <b>24</b> was only ~80% pure, but no further purification was performed. LRMS (MH-tBuOCO) <i>m</i>/<i>z</i> 231.1.

0231Lithium bis(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 28.4 mL, 3.3 equiv) was added slowly via syringe to a - 78°C solution of ketone <b>24</b> (2.84 g, 8.6 mmol) and THF (40 mL). The reaction solution temperature was monitored by an internal thermometer, and addition of the base was done at a rate sufficient to prevent the temperature from exceeding - 48°C. After the addition was complete the resulting solution was maintained at -78°C for 30 mins. Neat 4-chloro-2-fluorobenzoyl chloride (<b>18</b>, 1.38 mL, 8.6 mmol ) (assumed density to be 1.20 g/mL) was added dropwise via syringe. The reaction solution turned to an orange color and was maintained for 30 mins. The reaction solution was quenched with 1 N HCl (20 mL). The layers were separated and the organic layer was washed with brine (20 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was used without further purification.

0232A mixture of the above crude product, K₂CO₃ (1.43 g, 10.34 mmol) and DMF (43 mL) was maintained at 23°C for 1 hour. The reaction mixture was quenched by addition of Et₂O (100 mL) and brine (200 mL). The layers were separated and the organic layer was washed with brine (2 x 200 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue absorbed onto silica gel (CH₂Cl₂) and was purified by flash column chromatography (5:1 hexanes:EtOAc) to yield 2.7 g (67% for 2 steps) of <b>25</b>. Inspection of the ¹H NMR revealed that <b>25</b> was only ~80% pure, but no further purification was performed. LRMS (MH) <i>m</i>/<i>z</i> 467.1.

0233Chromenone <b>25</b> (2.71 g, 5.80 mmol) and TFA:H₂O (97.5:2.5, 25 mL) was maintained at 23°C for 1h. The reaction mixture was concentrated. The residue was dissolved in EtOAc (100 mL) and washed with 1 N NaOH (25 mL) and brine (25 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (1:1 hexanes:EtOAc; 1:2 hexanes:EtOAc; 1:4 hexanes:EtOAc) to yield 1.20 g (56%) of <b>26</b>. LRMS (MH) <i>m</i>/<i>z</i> 367.1.

Example 6

0234<chemistry id="chem0039" num="0039"><img file="EP1511734B1_D0040.tif" /></chemistry>

0235Lithium bis(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 23.5 mL, 3.3 equiv) was added slowly via syringe to a - 78°C solution of ketone <b>17</b> (2.18 g, 7.14 mmol) and THF (20 mL). The reaction solution temperature was monitored by an internal thermometer, and addition of the base was done at a rate sufficient to prevent the temperature from exceeding - 50°C. After the addition was complete the resulting solution was maintained at -78°C for 30 mins. Neat 2,4-difluorobenzoyl chloride (<b>27</b>, 1.05 mL, 8.57 mmol) was added dropwise via syringe. The reaction solution turned to an orange color and was maintained for 30 mins. The reaction solution was quenched with 1 N HCl (20 mL). The layers were separated and the organic layer was washed with brine (20 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (10:1 hexanes:EtOAc) to yield 2.16 g (68%) of <b>28</b>. Inspection of the ¹H NMR revealed that <b>28</b> was only ~85% pure, but no further purification was performed.

0236A mixture of <b>28</b> (2.16 g, 4.9 mmol), K₂CO₃ (812 mg, 5.86 mmol), and DMF (24 mL) was maintained at 23°C for 1 hour. The reaction mixture was quenched with brine (50 mL), and diluted with Et₂O (50 mL). The layers were separated and the organic layer was washed with brine (2 x 50 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (10:1 hexanes:EtOAc) to yield 1.25 g (60%) of <b>29</b>. LRMS (MH) <i>m</i>/<i>z</i> 426.2.

Example 7

0237<chemistry id="chem0040" num="0040"><img file="EP1511734B1_D0041.tif" /></chemistry>

0238Lithium bis(trimethylsilyl)amide (LHMDS, 1.0 M in THF, 47 mL, 3.3 equiv) was added slowly via syringe to a - 78°C solution of ketone <b>17</b> (4.37 g, 14.31 mmol) and THF (55 mL). The reaction solution temperature was monitored by an internal thermometer, and addition of the base was done at a rate sufficient to prevent the temperature from exceeding -50°C. After the addition was complete the resulting solution was maintained at - 78°C for 30 mins. A solution of 4-cyano-2-fluorobenzoyl chloride (<b>30</b>, 1.38 mL, 8.6 mmol) and THF (5 mL) was added rapidly via syringe (temperature rose from -78 to -50°C). The reaction solution turned to a dark red color and was maintained for 30 mins. The reaction solution was quenched with 1 N HCl (20 mL). The layers were separated and the organic layer was washed with brine (20 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was used without further purification.

0239A mixture of the above crude product, K₂CO₃ (2.40 g, 17.17 mmol) and DMF (70 mL) was maintained at 23°C of 1 hour. The reaction mixture was quenched by addition of Et₂O (200 mL) and brine (200 mL). The layers were separated and the organic layer was washed with brine (2 x 200 mL). The organic layer was dried (MgSO₄), filtered, and concentrated. The resulting residue was purified by flash column chromatography (10:1 hexanes:EtOAc) to yield 4.95 g (80% for 2 steps) of 31. LRMS (MH) <i>m</i>/<i>z</i> 433.2.

Example 8

0240<chemistry id="chem0041" num="0041"><img file="EP1511734B1_D0042.tif" /></chemistry>

02417-Fluoro-Chromenone <b>32</b> (96 mg, 0.19 mmol) was dissolved in 0.5 M sodium methoxide in methanol (10 mL) and heated to 70°C. The temperature was maintained at 70°C for 12 hours and then cooled to room temperature. The solvent was removed under reduced pressure. To the remaining residue was added EtOAc (20 mL) and water (20 mL). The layers were separated and the aqueous phase was extracted with additional EtOAc (3 x 15 mL). The organic phases were combined, washed with brine (25 mL) and dried (Na₂SO₄). Concentration <i>in vacuo</i> gave an amorphous white solid which was purified using flash column chromatography (20:1 DCM:MeOH) to provide 92 mg (94%) of <b>33</b>. LRMS (MH) m/z 509.3.

Example 9

0242<chemistry id="chem0042" num="0042"><img file="EP1511734B1_D0043.tif" /></chemistry>

0243Compound <b>29</b> (608 mg, 1.43 mmol) was dissolved into a solution of 0.5 M sodium methoxide in methanol (40 mL). The mixture was heated to reflux for 3 hours (or until LC-MS indicated no starting material left). Then the solvent was evaporated. The residue was dissolved in dichloromethane (200 mL) and water (200 mL) was added, in which pH of the mixed solution was adjusted to 9 by adding 2N HCl solution. The collected organic layers were dried by sodium sulfate. After evaporation of solvents, the final compound <b>34</b> (580 mg, 92%) was dried under vacuum.

Example 10

0244<chemistry id="chem0043" num="0043"><img file="EP1511734B1_D0044.tif" /></chemistry>

0245To a solution of <b>32</b> (56 mg, 0.11 mmol) in DMF (5 mL) was added NaH (5 mg of 60% dispersion in mineral oil, 0.15 mmol). The resulting solution was stirred at 45°C for 5 mins., then allyl alcohol was added (10 µL, 0.15 mmol) via pipette. The resulting solution was stirred at 45°C for 12 hours and then cooled to room temperature. EtOAc (50 mL) and water (15 mL) were added and the layers were separated. The aqueous phase was extracted with EtOAc (3 x 15 mL). The organic phases were combined, washed with water (2 x 30 mL) and brine (2 x 30 mL), dried (Na₂SO₄) and concentrated under reduced pressure to yield 35 as an amorphous solid, which was used without further purification (54 mg, 91 %). LRMS (MH) m/z 535.0. [00237] To a room temperature solution of <b>35</b> (54 mg, 0.10 mmol) and MeCN (5 mL) was added morpholine (44 µL, 0.50 mmol), followed by Pd(PPh₃)₄ (5 mg, 10%). The resulting solution was stirred for 5 minutes and then concentrated under reduced pressure. The remaining residue was purified by flash column chromatography using stepwise elution (20:1 DCM:MeOH, 10:1 DCM:MeOH) to provide 41 mg of <b>36</b> as an off-white solid (81%). LRMS (MH) <i>m</i>/<i>z</i> 495.2.

Example 11

Preparation of 3-Benzyl-7-chloro-2-[2-methyl-1-(2-<i>p</i>-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one

a) {2-[1-(3-Benzyl-7-chloro-4-oxo-4

H

-chromen-2-yl)-2-methyl-propylamino]-ethyl}-carbamic acid

tert

-butyl ester

0246To a solution of 2-(1-amino-2-methyl-propyl)-3-benzyl-7-chloro-chromen-4-one (6.0 g, 18 mmol) and (3-oxoethyl)carbamic acid <i>tert</i>-butyl ester (3.6 g, 23 mmol) in methylene chloride (150 mL) was added sodium triacetoxyborohydride (7.4 g, 35 mmol). The reaction mixture was stirred at room temperature for 16 hours, at which time it was diluted with 1 N sodium hydroxide (150 mL) and stirred vigorously for 2 hours. The organic layer was washed with 1 N sodium hydroxide (100 mL) and brine (100 mL), dried over magnesium sulfate, and concentrated. The residue was purified by flash chromatography (0→50% ethyl acetate/hexanes) to give 5.7 g (67%) of the title compound. MS(ES+) m/e 485 [M+H]⁺.

b) 2-[1-(2-Amino-ethylamino)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one

0247A solution of {2-[1-(3-benzyl-7-chloro-4-oxo-4<i>H</i>-chromen-2-yl)-2-methyl-propylamino]-ethyl}-carbamic acid <i>tert</i>-butyl ester (5.7 g, 12 mmol) in 4:1 methylene chloride/trifluoroacetic acid (250 mL) was maintained at room temperature for 1.5 hours, at which time it was concentrated. The residue was dissolved in methylene chloride (200 mL), washed with 10% sodium carbonate, saturated sodium bicarbonate, and brine, dried over magnesium sulfate, and concentrated. The residue (4.2 g, 91% yield) was used without futher puification.

c)

N

-{2-[1-(3-Benzyl-7-chloro-4-oxo-4

H

-chromen-2-yl)-2-methyl-propylamino]-ethyl}-4-methyl-benzamide

0248To a cooled (0°C) solution of 2-[1-(2-amino-ethylamino)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one (4.1 g, 11 mmol) and triethylamine (2.2 mL, 17 mmol) in methylene chloride (100 mL) was added a solution of <i>p</i>-toluyl chloride (1.7 g, 11 mmol) in methylene chloride (20 mL). The reaction was maintained at 0 °C for 2 hours, at which time it was diluted with ether (250 mL). The resultant solution was washed with 1 N hydrogen chloride (2 x 200 mL), saturated sodium bicarbonate (200 mL) and brine (150 mL), dried over magnesium sulfate, and concentrated. The residue was purified by flash chromatography (0→60% ethyl acetate/hexanes) to give 2.8 g (50%) of the title compound. MS(ES+) m/e 503 [M+H]⁺.

d) 3-Benzyl-7-chloro-2-[2-methyl-1-(2-

p

-tolyl-4,5-dihydro-imidazol-1-yl)-propyl]-chromen-4-one

0249A mixture of <i>N</i>-{2-[1-(3-benzyl-7-chloro-4-oxo-4<i>H</i>-chromen-2-yl)-2-methyl-propylamino]-ethyl}-4-methyl-benzamide (2.8 g, 5.5 mmol) and phosphorus oxychloride (9.4 mL, 100 mmol) in toluene (60 mL) was heated at 85 °C for 7 hours, then heated at reflux for 1 hour. The reaction was concentrated and the residual phosphorus oxychloride removed by toluene azeotrope. The residue was diluted with ethyl acetate (100 mL) and washed with saturated sodium bicarbonate (100 mL) and brine (100 mL), dried over magnesium sulfate, and concentrated. The resultant residue was purified by flash chromatography (0→8% methanol/methylene chloride) to give 1.4 g (50%) of the title compound. MS(ES+) m/e 485 [M+H]⁺.

0250Following procedures analogous to those set forth herein in Examples 1, 3, and/or 4, the following compounds were prepared: <chemistry id="chem0044" num="0044"><img file="EP1511734B1_D0045.tif" /></chemistry><tables id="tabl0003" num="0003"><table frame="all"><tgroup cols="4" rowsep="0"><colspec colnum="1" colname="col1" colwidth="10mm" /><colspec colnum="2" colname="col2" colwidth="35mm" /><colspec colnum="3" colname="col3" colwidth="24mm" /><colspec colnum="4" colname="col4" colwidth="16mm" colsep="0" /><thead><row><entry rowsep="1" align="center" valign="top"><b>R₇</b></entry><entry rowsep="1" align="center" valign="top"><b>R₁</b></entry><entry rowsep="1" align="center" valign="top"><b>R₉</b></entry><entry rowsep="1" align="center" valign="top"><b>[M+H]⁺</b></entry></row></thead><tbody><row rowsep="1"><entry align="center" valign="middle">CN</entry><entry align="center" valign="middle">3-MeO-Ph-CH₂-</entry><entry align="center" valign="middle">3,4-piperonyl</entry><entry colsep="1" align="center" valign="middle">536</entry></row><row rowsep="1"><entry align="center" valign="middle">Cl</entry><entry align="center" valign="middle">3-MeO-Ph-CH₂-CH₂-</entry><entry align="center" valign="middle">4-Me-Ph</entry><entry colsep="1" align="center" valign="middle">515</entry></row><row rowsep="1"><entry align="center" valign="middle">Cl</entry><entry align="center" valign="middle">3-MeO-Ph-CH₂-CH₂-</entry><entry align="center" valign="middle">3-F-4-Me-Ph</entry><entry colsep="1" align="center" valign="middle">533</entry></row><row rowsep="1"><entry align="center" valign="middle">Cl</entry><entry align="center" valign="middle">3-MeO-Ph-CH₂-</entry><entry align="center" valign="middle">3,4-piperonyl</entry><entry colsep="1" align="center" valign="middle">545</entry></row><row rowsep="1"><entry align="center" valign="middle">F</entry><entry align="center" valign="middle">3-MeO-Ph-CH₂-CH₂-</entry><entry align="center" valign="middle">4-Me-Ph</entry><entry colsep="1" align="center" valign="middle">499</entry></row><row rowsep="1"><entry align="center" valign="middle">F</entry><entry align="center" valign="middle">3-MeO-Ph-CH₂-CH₂-</entry><entry align="center" valign="middle">3-F-4-Me-Ph</entry><entry colsep="1" align="center" valign="middle">517</entry></row></tbody></tgroup></table></tables>

Example 12

Preparation of 2-[(R)-1-(4-Aminomethyl-2-p-tolyl-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one

a) 2-[(R)-1-(3-Phthalimido-2-oxo-propylamino)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one

0251To 2-((R)-1-amino-2-methyl-propyl)-3-benzyl-7-chloro-chromen-4-one (0.5 g, 1.5 mMol) and K₂CO₃ (0.21 g, 1.5 mMol) in DMF (10 mL) was added N-(3-bromo-2-oxopropyl)-phthalimide (0.45 g, 1.5 mMol) (<nplcit id="ncit0026" npl-type="s"><text>Nair et al.; J. Org. Chem.; 40; 1975; 1745</text></nplcit>). The reaction was stirred at RT for 3 h, concentrated under vacuum, taken up in EtOAc, washed with water, brine, dried (Na₂SO₄) and evaporated to give the title compound (0.88 g, 100%) as a yellow solid: MS (ES) m/e 543.2 (M + H)⁺.

b) 2-{(R)-1-[N-Toluoyl-(3-phthalimido-2-oxo-propyl)amino]-2-methyl-propyl}-3-benzyl-7-chloro-chromen-4-one

0252To 2-[(R)-1-(3-phthalimido-2-oxo-propylamino)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one (0.88 g, 1.5 mmol) in CH₂Cl₂ (10 mL) was added Et₃N (0.23 mL, 1.6 mMol) and toluoyl chloride (0.21 mL, 1.6 mMol). The reaction was stirred at RT for 18 h, concentrated under vacuum, taken up in EtOAc, washed with 1 N HCl, brine, dried (MgSO₄) and evaporated to dryness. Purification by flash chromatography on silica gel (5-15% EtOAc/hexane) followed by trituration with pet. ether, filtration and drying under vacuum gave the title compound (0.89 g, 90%) as a white solid: MS (ES) m/e 661.2 (M + H)⁺.

c) N-{1-[(R)-1-(3-Benzyl-7-chloro-4-oxo-chromen-2-yl)-2-methyl-propyl]-2-p-tolyl-1H-imidazol-4-ylmethyl -phthalimide

0253To 2-{(R)-1-N-toluoyl-(3-phthalimido-2-oxo-propyl)amino]-2-methylpropyl}-3-benzyl-7-chloro-chromen-4-one (0.88 g, 1.3 mMol) and NH₄OAc (5.13 g, 66.8 mMol) was added HOAc (15 mL). The reaction was stirred and heated to reflux (155 °C oil bath) for 2.5 h, cooled to RT and concentrated under vacuum. The remaining residue was taken up in EtOAc, washed with water, brine, dried (MgSO₄) and evaporated to dryness. Purification by flash chromatography on silica gel ( 50% EtOAc/hexane) followed by trituration with (1:2) Et₂O/ pet. ether, filtration and drying under vacuum gave the title compound (0.51 g, 61%) as a tan solid: ¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J = 8.6 Hz, I H), 7.89 (2d, 2 H), 7.73 (2d, 2 H), 7.53 (d, J = 1.8 Hz, 1 H), 7.52 (s, 1 H), 7.39 (d, J = 8.0 Hz, 2 H), 7.36 (dd, 1 H), 7.32 (d, J = 8.0 Hz, 2 H), 7.14 (m, 3 H), 6.74 (d, J = 1.5 Hz, 1 H), 6.72 (s, 1 H), 5.04 (d, J = 8.8 Hz, 1 H), 4.94 (s, 2 H), 4.01 (d, J = 15.5 Hz, 1 H), 2.63 (m, 1H), 2.46 (s, 3 H), 2.44 (d, J = 15.5 Hz, 1 H), 0.95 (d, J = 6.6 Hz, 3 H), 0.29 (d, J = 6.7 Hz, 3 H); MS (ES) m/e 642.0 (M + H)⁺.

d) 2-[(R)-1-(4-Aminomethyl-2-p-tolyl-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one

0254To N-{1-[(R)-1-(3-benzyl-7-chloro-4-oxo-3,4-dihydro-chromen-2-yl)-2-methyl-propyl]-2-p-tolyl-1H-imidazol-4-ylmethyl}-phthalimide (0.50 g, 0.78 mMol) in EtOH (15 mL) was added hydrazine monohydrate (0.12 mL, 2.5 mMol). The reaction was stirred at RT for 72 h, filtered through a pad of Celite^® to remove the insoluble precipitate, rinsed with EtOH and evaporated to dryness. Purification by flash chromatography on silica gel [5 - 10% (5% NH₄OH in MeOH)/CH₂Cl₂] gave the title compound (364 mg, 91%) as a white solid foam: ¹H NMR (400 MHz, CDCl₃) δ 8.14 (d, J = 8.6 Hz, 1 H), 7.63 (d, J = 1.8 Hz, 1 H), 7.37 (m, 6 H), 7.15 (m, 3 H), 6.71 (d, J = 1.7 Hz, 1 H), 6.69 (s, 1 H), 5.00 (d, J = 10.9 Hz, 1 H), 4.07 (d, J = 15.5 Hz, 1 H), 3.92 (d, 1 H), 3.88 (d, 1 H), 2.72 (br s, 2 H), 2.65 (m, 1 H), 2.49 (s, 3 H), 2.48 (d, J = 15.5 Hz, 1 H), 0.93 (d, J = 6.6 Hz, 3 H), 0.30 (d, J = 6.7 Hz, 3 H); MS (ES) m/e 512.2 (M + H)⁺.

0255Following procedures analogous to those set forth above, the following compounds were prepared: <chemistry id="chem0045" num="0045"><img file="EP1511734B1_D0046.tif" /></chemistry><tables id="tabl0004" num="0004"><table frame="all"><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="15mm" /><colspec colnum="2" colname="col2" colwidth="39mm" /><colspec colnum="3" colname="col3" colwidth="27mm" /><colspec colnum="4" colname="col4" colwidth="25mm" /><thead><row><entry align="center" valign="top"><b>R₇</b></entry><entry align="center" valign="top"><b>R₁</b></entry><entry align="center" valign="top"><b>R₉</b></entry><entry align="center" valign="top"><b>[M+H]⁺</b></entry></row></thead><tbody><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry>4-Me-Ph</entry><entry align="center">510</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry>3-F-4-Me-Ph</entry><entry align="center">528</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry>3-F-4-Me-Ph</entry><entry align="center">535</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry>3-F-4-Me-Ph</entry><entry align="center">544</entry></row><row><entry align="center">Cl</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry>4-Me-Ph</entry><entry align="center">556</entry></row><row><entry align="center">Cl</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry>3-F-4-Me-Ph</entry><entry align="center">574</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry>4-Me-Ph</entry><entry align="center">517</entry></row><row><entry align="center">Cl</entry><entry align="center">3-CN-Ph-CH₂-</entry><entry>4-Me-Ph</entry><entry align="center">551</entry></row><row><entry align="center">Cl</entry><entry align="center">3-CN-Ph-CH₂-</entry><entry>3-F-4-Me-Ph</entry><entry align="center">569</entry></row><row><entry align="center">F</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry>4-Me-Ph</entry><entry align="center">540</entry></row><row><entry align="center">CN</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry>4-Me-Ph</entry><entry align="center">547</entry></row><row><entry align="center">CN</entry><entry align="center">3-CN-Ph-CH₂-</entry><entry>3-F-4-Me-Ph</entry><entry align="center">560</entry></row><row><entry align="center">F</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry>3-F-4-Me-Ph</entry><entry align="center">588</entry></row><row><entry align="center">F</entry><entry align="center">3-CN-Ph-CH₂-</entry><entry>3-F-4-Me-Ph</entry><entry align="center">553</entry></row></tbody></tgroup></table></tables>

Example 13

Preparation of N-{1-[(R)-1-(3-Benzyl-7-chloro-4-oxo-3,4-dihydro-chromen-2-yl)-2-methyl-propyl]-2-p-tolyl-1H-imidazol-4-ylmethyl}-acetamide

0256To 2-[(<i>R</i>)-1-(4-aminomethyl-2-p-tolyl-imidazol-1-yl)-2-methyl-propyl]-3-benzyl-7-chloro-chromen-4-one (0.17 g, 0.33 mmol) in CH₂Cl₂ (5 mL) was added with stirring, pyridine (27 uL, 0.33 mMol) and Ac₂O (63 uL, 0.67 mMol). After stirring at RT for 4 h the reaction was concentrated under vacuum. The remaining residue was triturated with (1:1) Et₂O/pet. ether, filtered and dried under vacuum to give the title compound (163mg, 89%) as a white solid: ¹HNMR (400 MHz, CDCl₃) δ 8.14 (d, J = 8.6 Hz, 1 H), 7.59 (d, J = 1.8 Hz, 1 H), 7.43 (m, 6 H), 7.17 (m, 3 H), 6.84 (br s, 1 H), 6.71 (d, J = 2.2 Hz, 1 H), 6.69 (s, 1 H), 5.07 (d, J = 10.9 Hz, 1 H), 4.50 (2d, 1 H), 4.43 (2d, 1 H), 4.07 (d, J = 15.5 Hz, 1 H), 2.66 (m, 1 H), 2.51 (s, 3 H), 2.48 (d, J = 15.5 Hz, 1 H), 2.00 (s, 3 H), 0.95 (d, J = 6.6 Hz, 3 H), 0.34 (d, J = 6.7 Hz, 3 H); MS (ES) m/e 554.4 (M + H)⁺.

Preparatory Example

0257<chemistry id="chem0046" num="0046"><img file="EP1511734B1_D0047.tif" /></chemistry>

0258A mixture of 4-cyano-2-fluorobenzoic acid (1, 25 g), DMF (0.1 mL), and thionyl chloride (50 mL) in a 250 mL round-bottom flask equipped with a reflux condenser under N₂ atmosphere was heated to reflux in a 90°C oil bath for 30 mins. The reflux condenser was replaced with a distillation head and the excess thionyl chloride was distilled from the reaction pot. The oil bath temperature was increased to 120°C to facilitate distillation. After 2 hours, the reaction vessel was cooled to 23°C and placed under reduced preasure (~25 Torr). The reaction vessel was then heated to 90°C for 20 mins, then to 130°C for 90 minutes to remove any excess remaining thionyl chloride. The reaction vessel was then cooled to 23°C and placed under high vacuum (~0.2 Torr) The reaction vessel was equipped with a clean distillation head and collection flask. The vessel was placed into a 137°C oil bath and the product was distilled from the reaction pot (b.p. = 107°C @ ~0.2 Torr). Upon cooling the distillate solidified, providing 2 as a white solid in close to quantitative yield.

0259A mixture of 4-chloro-2-fluorobenzoic acid (3, 25 g), DMF (0.1 mL), and thionyl chloride (50 mL) in a 250 mL round-bottom flask equipped with a reflux condenser under N₂ atmosphere was heated to reflux in a 90°C oil bath for 30 mins. The reflux condenser was replaced with a distillation head and the excess thionyl chloride was distilled from the reaction pot. The oil bath temperature was increased to 120°C to facilitate distillation. After 2 hours, the reaction vessel was cooled to 23°C and placed under reduced pressure (~25 Torr). The reaction vessel was then heated to 90°C for 20 mins, then to 120°C for 90 minutes to remove any excess remaining thionyl chloride. The reaction vessel was then cooled to 23°C and then equipped with a clean distillation head and collection flask. The vessel was placed into a 160°C oil bath and the product was distilled from the reaction pot (b.p. = 124°C @ ~25 Torr), furnishing 4 was a colorless oil in near quantitative yield.

Preparatory Example

0260<chemistry id="chem0047" num="0047"><img file="EP1511734B1_D0048.tif" /></chemistry>

0261Piperonyloyl Chloride (1, 7.8 g, 42 mmol) was added to a solution of 2-amino-2-methyl-1-propanol (4 ml, 42 mmol), CH₂Cl₂ (200 mL), and triethylamine (11.7 ml, 84 mmol) at room temperature. The reaction solution was concentrated after 45 minutes, and the resulting residue was diluted with EtOAc (40 mL) and washed with brine (30 mL). The organic layer was dried (MgSO₄), filtered, and concentrated to yield a light brown oil. The crude material (2, 8.8g) was used without further purification.

0262Tetrapropylammonium perruthenate (TPAP, 638 mg, 1.8 mmol) was added portion-wise to a solution of 2 (8.6 g, 36.3 mmol), CH₂Cl₂ (73 ml, 2mL/mmol), 4-methyl-morpholine N-oxide (6.4 g, 54.5 mmol), and molecular sieves, 4Å activated powder (18g, 500mg/mmol) at 0°C under N₂. The reaction was allowed to warm to r.t. after 15 minutes. After 1 hour the reaction was complete (TLC) and was filtered through silca, eluted with EtOAc (100 mL), and the filtrate was concentrated. This yielded 7 g of off-white solid (3). The material was recrystallized: EtOAc (30 mL), MeOH (10 mL), and Hexanes (1 mL) were added portion wise with heating and sonication. This was brought to a boil after which hexane (100 mL) was added while cooling. Crystals immediately began to precipitate as the solution was cooled. The mixture was filtered and the crystals were washed with hexane (10 mL) to provide 4.88 g (57 %) of 3 as off-white fluffy crystals.

Preparatory Example

0263<chemistry id="chem0048" num="0048"><img file="EP1511734B1_D0049.tif" /></chemistry>

0264A mixture of 2-amino-2-methyl-1-propanol (4 ml, 42 mmol), CH₂Cl₂ (200 mL), 4-acetyl benzoic acid (1, 6.9 g, 42 mmol), EDC (1-(3-dimethylaminopropyl)-3-eythylcarbodiimide HCl, 12.1 g, 63 mmol), HOBT (N-hydroxybenzotriazole H₂O, 6.4 g, 42 mmol), and Hunig's base (diisopropylethylamime, 22 mL, 126 mmol) was stirred at room temperature for 18 hours. Upon completion (TLC, LC/MS) the reaction mixture was concentrated, and the resulting residue was diluted with EtOAc (50 mL) and washed with NaHCO₃ (2 x 40 mL) and brine (40 mL). The organic layer was dried (MgSO₄), filtered, and the filtrate was concentrated. The crude material was purified by flash column chromatography (4:1:1 hexanes:EtOAc:CH₂Cl₂; 1:1:1 hexanes:EtOAc:CH₂Cl₂; 1:1 EtOAc:CH₂Cl₂) to remove bisacylated material. Alcohol 2 was obtained in 54 % yield (5.36 g). Tetrapropylammonium perruthenate (TPAP, 387 mg, 1.1 mmol) was added portion-wise to a solution of 2 (5.36 g, 22.8 mmol), CH₂Cl₂ (46 mL, 2mL/mmol). 4-methyl-morpholine N-oxide (4 g, 34.2 mmol), and molecular sieves, 4A activated powder (11.4g, 500mg/mmol) at 0°C under N₂. The reaction was allowed to warm to r.t. after 15 minutes. After I hour the reaction was complete (TLC) and was filtered through silca, which was eluted with EtOAc (100 mL), and the filtrate was concentrated. This yielded 4.4 g of light pink solid (3). The material was recrystallized: 1:1 Hexanes:EtOAc (5 mL), CH₂Cl₂ (20 mL), and MeOH (10 mL) were added portion-wise with heating and sonication. This was brought to a boil after which hexane (100 mL) was added while cooling. Crystals immediately began to precipitate as the solution was cooled. The mixture was filtered and the crystals were washed with hexane (10 mL), affording 2.46 g (46 %) of 3 as off-white fluffy crystals.

Preparatory Example

0265<chemistry id="chem0049" num="0049"><img file="EP1511734B1_D0050.tif" /></chemistry>

0266A solution of 4-acetyl benzoic acid (500 mg) in oxalyl chloride (5 mL) was heated to reflux for 2 hours. Any remaining oxalyl chloride was evaporated by rot-vap, and the residue was dried by vacuum. The yield of product was quantitative.

0267Preparatory Example <chemistry id="chem0050" num="0050"><img file="EP1511734B1_D0051.tif" /></chemistry>

((<i>R</i>)-1-Isopropyl-2-oxo-but-3-enyl)-carbamic acid <i>tert</i>-butyl ester

0268Tetrahydrofuran (THF, 100 mL) and a 1.0M solution of vinyl magnesium bromide in THF (360 mL, 360 mmol, 3.1 equiv) was cooled to -78 °C while stirring under a nitrogen atmosphere. The mixture was treated dropwise with a solution of [(<i>R</i>)-(methoxymethyl-carbamoyl)-methyl-propyl]-carbamic acid <i>tert</i>-butyl ester (30.3 g, 116 mmol, 1 equiv) in THF (50 mL) over 30 min. After the resultant dark yellow mixture was stirred for 30 min at -78 °C, the cooling bath was removed and the reaction mixture was warmed slowly to room temperature overnight (15 h). The reaction mixture was poured slowly into an ice-chilled solution of 1N aqueous hydrochloric acid (700 mL) and then warmed to room temperature. The organics were extracted with (3 x 600 mL) ethyl acetate, dried over sodium sulfate, filtered, and concentrated in vacuo. Purification by flash column chromatography (5-10% ethyl acetate/hexanes) provided the product as a white solid (16.8 g, 64%). ESMS [M+H]⁺; 228.4. <chemistry id="chem0051" num="0051"><img file="EP1511734B1_D0052.tif" /></chemistry><b>[(<i>R</i>)-(<i>E</i>)-1-Isopropyl-4-(3-methoxy-phenyl)-2-oxo-but-3-enyl]-carbamic acid <i>tert</i>-butyl ester</b>

0269To a solution of ((<i>R</i>)-1-Isopropyl-2-oxo-but-3-enyl)-carbamic acid <i>tert</i>-butyl ester (13.54 g, 59.6 mmol) in dry acetonitrile (150 mL) under argon, was added 3-iodoanisole (13.96 g, 59.6 mmol), triethylamine (9.1 mL, 65.6 mmol) followed by palladium (II) acetate (335 mg, 1.49 mmol). The resulting clear yellow solution was heated to 80 °C. Upon heating, the reaction darkened and the precipitation of palladium black occurred. After 15 h, the reaction mixture was allowed to cool to room temperature, quenched with water (150 mL) and diluted with ether (150 mL). The ether layer was washed with brine (100 mL) and the combined aqueous layers were extracted with ether (two 50 mL portions). The extracts were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was immediately purified by silica gel chromatography (9:1 hexanes/EtOAc) to provide 17.6 g (88%) of [(<i>R</i>)-(<i>E</i>)-1-Isopropyl-4-(3-methoxy-phenyl)-2-oxo-but-3-enyl]-carbamic acid <i>tert-</i>butyl ester as a yellow oil. MS(ES+) m/e 334.0 [M+H]⁺.

[(<i>R</i>)-(<i>Z</i>)-4-(3-Cyano-phenyl)-1-isopropyl-2-oxo-but-3-enyl]-carbamic acid <i>tert</i>-butyl ester

0270Following the procedure described for [(<i>R</i>)-(<i>E</i>)-1-isopropyl-4-(3-methoxyphenyl)-2-oxo-but-3-enyl]-carbamic acid <i>tert</i>-butyl ester with 3-iodobenzonitrile (5.50 g, 24.0 mmol, I equiv) afforded the title compound as a yellow solid (7.4 g of ~90% purity material). ESMS [M+H]⁺: 329.2. <chemistry id="chem0052" num="0052"><img file="EP1511734B1_D0053.tif" /></chemistry>

[(<i>R</i>)-(<i>E</i>)-1-Isopropyl-4-(3-methoxy-phenyl)-2-oxo-butyl]-carbamic acid <i>tert</i>-butyl ester

0271To a solution of [(<i>R</i>)-(<i>E</i>)-1-Isopropyl-4-(3-methoxy-phenyl)-2-oxo-but-3-enyl]-carbamic acid tert-butyl ester (17.6 g, 52.9 mmol) in ethyl acetate (450 mL) under nitrogen was added 10 wt % palladium on carbon (300 mg). The nitrogen was replaced with a balloon of hydrogen and the flask was purged. After 3 h, the reaction flask was purged with nitrogen and filtered through a pad of celite (rinsing with ethyl acetate). The filtrate was concentrated under reduced pressure and the residue was purified by silica gel chromatography (9:1 hexanes/EtOAc) to provide 16.2 g (91 %) of [(<i>R</i>)-(<i>E</i>)-1-Isopropyl-4-(3-methoxy-phenyl)-2-oxo-butyl]-carbamic acid <i>tert</i>-butyl ester as a colorless oil. MS(ES+) m/e 336.4 [M+H]⁺. [α]_D²⁰ = +19.1 (<i>c</i> = 0.755, MeOH). ¹H NMR (400 MH₂, CDCl₃) δ 7.21 (m, 1H), 6.80-6.77 (m, 2H), 6.75 (s, 1H), 5.13 (d, <i>J</i>= 8.4 Hz, 1H), 4.28 (dd, <i>J</i>= 8.8, 4.4 Hz, 1H), 3.81 (s, 3H), 2.93-2.88 (m, 2H), 2.85-2.76 (m, 2H), 2.14 (m, 1H), 1.46 (s, 9H), 1.00 (d, <i>J</i>= 6.8 Hz, 3H), 0.75 (d, <i>J</i> = 6.8 Hz, 3H). <chemistry id="chem0053" num="0053"><img file="EP1511734B1_D0054.tif" /></chemistry>

[(<i>R</i>)-4-(3-Cyano-phenyl)-1-isopropyl-2-oxo-butyl]-carbamic acid <i>tert</i>-butyl ester

0272Palladium on carbon (740 mg of 10% wt/wt Pd/C) was added to a degassed solution of ((<i>R</i>)-(<i>Z</i>)-4-(3-cyano-phenyl)-1-isopropyl-2-oxo-but-3-enyl]-carbamic acid <i>tert-</i>butyl ester (7.4 g, 22.5 mmol, 1 equiv) in methanol (200 mL) at room temperature. The reaction mixture was thoroughly degassed and backfilled with hydrogen gas from a balloon. Hydrogenation proceeded at atmospheric pressure for 2.5 h. The (degassed) reaction mixture was then diluted with diethyl ether (300 mL), filtered through Celite, and washed with additional ether (2 x 100 mL). Upon concentration in vacuo, the residue was purified by flash column chromatography (20% ethyl acetate-hexanes) to provide the ketone product as a white solid (5.9 g, 79%). ESMS [M+H]⁺: 331.2. ¹H NMR (400 MHz, CDCl₃) δ 7.51 (m, 2H), 7.46 (m, 1H), 7.41 (m, 1H), 5.07 (m, 1H), 4.24 (m, 1H), 2.98 (m, 2H), 2.86 (m, 2H), 2.11 (m, 1H), 1.45 (s, 9H), 0.98 (d, 3H, <i>J</i> = 6.76 Hz), 0.74 (d, 3H, <i>J</i> = 6.78 Hz). [α]_D =+24.74 (c = 0.95, CH₃OH).

Example 14

0273Following procedures analogous to those set forth above, the following compounds were prepared: <chemistry id="chem0054" num="0054"><img file="EP1511734B1_D0055.tif" /></chemistry><tables id="tabl0005" num="0005"><table frame="all"><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="13mm" /><colspec colnum="2" colname="col2" colwidth="35mm" /><colspec colnum="3" colname="col3" colwidth="56mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><thead><row><entry align="center" valign="top"><b>R₇</b></entry><entry align="center" valign="top"><b>R₁</b></entry><entry align="center" valign="top"><b>R₃</b></entry><entry align="center" valign="top"><b>[M+H]⁺</b></entry></row></thead><tbody><row><entry align="center">F</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">3-F-4-Me-Ph-</entry><entry align="center">549</entry></row><row><entry align="center">F</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">4-MeO-Ph-</entry><entry align="center">547</entry></row><row><entry align="center">F</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">4-Me-Ph-</entry><entry align="center">531</entry></row><row><entry align="center">F</entry><entry align="center">3-MeO-Ph-CH₂-CH₂-</entry><entry align="center">3,4-piperonyl-</entry><entry align="center">561</entry></row><row><entry align="center">F</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">Methoxymethyl-</entry><entry align="center">485</entry></row><row><entry align="center">CN</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">4-Ac-Ph-</entry><entry align="center">566</entry></row><row><entry align="center">CN</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">4-Me-Ph-</entry><entry align="center">538</entry></row><row><entry align="center">CN</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">3,4-piperonyl-</entry><entry align="center">568</entry></row><row><entry align="center">CN</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">3-F-4-Me-Ph-</entry><entry align="center">556</entry></row><row><entry align="center">Cl</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">3-F-4-Me-Ph-</entry><entry align="center">565</entry></row><row><entry align="center">Cl</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">4-Me-Ph-</entry><entry align="center">547</entry></row><row><entry align="center">Cl</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">Methoxymethyl-</entry><entry align="center">501</entry></row><row><entry align="center">Cl</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">3,4-piperonyl-</entry><entry align="center">577</entry></row><row><entry align="center">MeO-</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">3-F-4-Me-Ph-</entry><entry align="center">561</entry></row><row><entry align="center">MeO-</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">4-Me-Ph-</entry><entry align="center">543</entry></row><row><entry align="center">MeO-</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">3,4-piperonyl</entry><entry align="center">573</entry></row><row><entry align="center">OH</entry><entry align="center">3-MeO-Ph-CH₂-</entry><entry align="center">3-F-4-Me-Ph-</entry><entry align="center">547</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">4-OEt-Ph-</entry><entry align="center">538.2</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">6-trifluoromethyl-3-pyridyl</entry><entry align="center">563.2</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">2-benzo[b]thiophene</entry><entry align="center">550.2</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(5-t-butyl-2-methyl-2H-pyrazole)</entry><entry align="center">554.4</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(2,5-dimethyl-2H-pyrazole)</entry><entry align="center">512.4</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">6-trifluoromethyl-3-pyridyl</entry><entry align="center">556.0</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">2-furyl</entry><entry align="center">477.0</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(5-t-butyl-2-methyl-2H-pyrazole)</entry><entry align="center">547.2</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">4-CN-Ph-</entry><entry align="center">529.2</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">4-AcNH-Ph-</entry><entry align="center">561.4</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">6-trifluoromethyl-3-pyridyl-</entry><entry align="center">573.0</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">5-benzo[1,2,3]thiadiazole-</entry><entry align="center">562.2</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">2-furyl-</entry><entry align="center">494.4</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(2,5-dimethyl-2H-pyrazole)-</entry><entry align="center">522.0</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">4-pyridyl-</entry><entry align="center">488.4</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(2,5-dimethyl-2H-pyrazole)-</entry><entry align="center">505.4</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">4-AcNH-Ph-</entry><entry align="center">544.4</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">5-benzo[1,2,3]thiadiazole-</entry><entry align="center">545.2</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">2-(1-methyl-1H-indole)-</entry><entry align="center">547.4</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-pyridyl-</entry><entry align="center">505.2</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">4-OMe-Ph-</entry><entry align="center">517.2</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(2,5-dimethyl-furan)-</entry><entry align="center">512.2</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(2,5-dimethyl-furan)-</entry><entry align="center">505.2</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">2-(5-methyl-thiophene)-</entry><entry align="center">514.2</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">2-(5-methyl-thiophene)-</entry><entry align="center">507.0</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">2-(1-methyl-1H-pyrrole)-</entry><entry align="center">497.4</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">5-benzo[1,2.3]thiadiazole-</entry><entry align="center">550.0</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">6-Me-3-pyridyl-</entry><entry align="center">518.2</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">2-(1-methyl-1H-pyrrole)-</entry><entry align="center">506.0</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">2-(5-methyl-pyrazine)-</entry><entry align="center">520.2</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(5-methyl-isoxazole)-</entry><entry align="center">508.2</entry></row><row><entry align="center">Cl</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-benzo[c]isoxazole-</entry><entry align="center">544.2</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">4-(1-methyl-1H-imidazole)-</entry><entry align="center">498.1</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-N(CH₃)₂-Ph-</entry><entry align="center">537.4</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(5-methyl-2-trifluoromethyl-furan)-</entry><entry align="center">566.4</entry></row><row><entry align="center">CN</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(5-methyl-isoxazole)-</entry><entry align="center">499.6</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">4-(1-methyl-1H-imidazole)-</entry><entry align="center">491-4</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">2-(1-methyl-1H-pyrrole)-</entry><entry align="center">490.4</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-benzo[c]isoxazole-</entry><entry align="center">528.4</entry></row><row><entry align="center">F</entry><entry align="center">Ph-CH₂-</entry><entry align="center">3-(5-methyl-isoxazole)-</entry><entry align="center">492.4</entry></row></tbody></tgroup></table></tables>

Example 15

0274A pharmaceutical composition for intravenous administration is prepared in the following manner. 1 mg/mL (as free base) IV solution with the vehicle being pH 5.0, 50 mM sodium acetate buffer containing 3.5% (w/v) mannitol: <tables id="tabl0006" num="0006"><table frame="bottom"><tgroup cols="2" colsep="0" rowsep="0"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="35mm" /><thead><row><entry valign="top">Composition*</entry><entry valign="top">Unit Formula (mg/mL)</entry></row><row><entry valign="top">Compound of Example 2</entry><entry valign="top" /></row></thead><tbody><row><entry>(free base)</entry><entry>1.000</entry></row><row><entry>Glacial Acetic Acid</entry><entry>1.081</entry></row><row><entry>Sodium Acetate Trihydrate</entry><entry>4.355</entry></row><row><entry>Mannitol, pyrogen free</entry><entry>35.000</entry></row><row><entry>Water for Injection (WFI)</entry><entry>q.s. to 1 mL</entry></row></tbody></tgroup><tgroup cols="2" rowsep="0"><colspec colnum="1" colname="col1" colwidth="42mm" /><colspec colnum="2" colname="col2" colwidth="35mm" /><tbody><row><entry namest="col1" nameend="col2" align="justify">*All components other than the active compound are USP or Ph, Eur.</entry></row></tbody></tgroup></table></tables>

0275A suitable compounding vessel is filled to approximately 75% of the bulk solution volume with WFI. The glacial acetic acid (1.081 g), sodium acetate trihydrate (4.355 g), mannitol (35.000 g), and active (1.000 g) are weighed and individually added to the compounding vessel. After the additions, the ingredients are dissolved in the mixture by stirring with a mixer. The pH of the bulk solution is measured and adjusted to 5.0 with 5N NaOH or 5N glacial acetic acid. The solution is brought to its final volume (1 liter) with WFI. Where the active compound is a pharmaceutically acceptable salt, hydrate or salt of a hydrate, e.g., a monochloride salt or a hydrate of a monochloride salt, the amount of the active equivalent to the free base is added.

Example 16

Preparation of a hydrochloride salt of N-(3-Aminopropyl)-N-[(R)-1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]4-methyl-benzamide

0276In one embodiment, a hydrochloric acid salt of N-(3-Aminopropyl)-N-[(R)-1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide, such as prepared in accordance with Example 2, is formed in the following manner. An HCl salt is suitably prepared by reacting the free base (e.g. dissolved in a suitable solvent, such as one or more of TBME, THF, and ethylacetate) with hydrochloric acid, suitably aqueous hydrochloric acid or otherwise in the presence of water. The mixture is suitably seeded with HCl salt to assist crystallization. The resulting salt may be isolated using conventional techniques such as filtration and drying (suitably washing the salt with a solvent, such as one or more of TBME, THF, and ethylacetate).

0277In a particular embodiment, the salt is prepared as follows: <ol id="ol0001" compact="compact"><li>1. Dissolve the free base into 10 volumes of TBME and 5 volumes of THF, dissolution suitably conducted at room temperature;</li><li>2. Add aqueous HCl, e.g. 6.0 M or 12.0 M, suitably 1.1 eq, e.g. dropwise;</li><li>3. Suitably seed the solution with crystals of HCl salt, e.g. prepared according to preparation B below;</li><li>4. Suitably stir until crystals form;</li><li>5. Isolate the crystals, suitably by filtration, washing with a suitable solvent such as TBME, and drying.</li></ol>

0278In more particular embodiments a salt is prepared as follows:

Preparation A

0279To a vial fitted with a stir bar add 500 mg of N-(3-Aminopropyl)-N-[(R)-1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide, and 3 ml TBME, and dissolve the free base in the TBME at room temperature. Bubble in HCl gas for about 30 seconds until a white solid forms. Filter and rinse the solids twice with 500 µl TBME. Transfer the solids to a test tube, add 4 ml TBME and a stir bar, and stir at room temperature (the salt does not dissolve). Place the test tube containing the mixture in an Argonaut RS10 heating block (Argonaut Technologies, Foster City, California), or an equivalent, and temperature cycle as follows: <ol id="ol0002" compact="compact"><li>1. 30 min at room temperature</li><li>2. ramp to 40C over 30 min and hold at 40C for 30 min</li><li>3. ramp to room temperature over 30 min and hold at room temperature for 30 min</li><li>4. ramp to 10C over 30 min and hold at 10C for 30 min</li><li>5. ramp to room temperature over 30 min and hold at room temperature for 30 min</li><li>6. repeat 2 to 5 as desired By 20 hours cycling some solvent evaporates, a white powder forms on the sides and a melt-like solid forms in the bottom of the test tube. Add 2 ml TBME. After 15 days, filter the light yellow solids and wash twice with about 0.5 ml TBME then twice with 1 ml hexane. Dry in a vacuum oven at room temperature for 12-24 hours to obtain about 440 mg HCl salt.</li></ol>

0280<figref idref="f0001">Figures 1</figref> and <figref idref="f0002">2</figref> represent a XRPD and MDSC scan, respectively, of a salt prepared according to Preparation A.

Preparation B

0281Dissolve 4.23 g of N-(3-Aminopropyl)-N-[(R)-1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]-4-methyl-benzamide in 5 volumes (21 ml) of THF. Add 10 volumes (42 ml) of TBME (tert-butyl methyl ether), then 1.1 eq of 12N HCl (750 µl) in one portion. Seed with HCl salt such as prepared in Preparation A (e.g. in 3 additions of about 10 mg each staggered over about 10 minutes). Stir the mixture overnight, e.g. for about 12-24 hours. Filter the mixture, wash the solids with TBME, and dry the solids in a vacuum oven at room temperature overnight, e.g. for about 12-24 hours, to yield about 4.3 g of HCl salt.

0282<figref idref="f0003">Figure 3</figref> represents an XRPD scan of a salt prepared according to Preparation B.

0283The present invention includes crystalline N-(3-Aminopropyl)-N-[(R)-1-3-benzyl-7-chloro-4-oxo-4H-chromen-2-yl)-2-methyl-propyl]4-methyl-benzamide hydrochloride salt having characteristic X-ray diffraction peaks at about 4.8, 9.7, 14.7, 17.9, 18.3, 20.1, 20.9, 22.5, 23.2, 23.8, 26. and 26.9 degrees 2 theta.

Example 17

Inhibition of Cellular Viability in Tumor Cell Lines Treated with KSP Inhibitors.

Materials and Solutions:

0284<ul id="ul0009" list-style="bullet" compact="compact"><li>Cells: SKOV3, Ovarian Cancer (human).</li><li>Media: Phenol Red Free RPMI + 5% Fetal Bovine Serum + 2mM L-glutamine.</li><li>Colorimetric Agent for Determining Cell Viability: Promega MTS tetrazolium compound.</li><li>Control Compound for max cell kill: Tapotecan, 1 µM. <b>Procedure: Day 1 - Cell Plating:</b></li></ul>

0285Adherent SKOV3 cells are washed with 10mLs of PBS followed by the addition of 2mLs of 0.25% trypsin and incubation for 5 minutes at 37°C. The cells are rinsed from the flask using 8 mL of media (phenol red-free RPMI+ 5%FBS) and transferred to fresh flask. Cell concentration is determined using a Coulter counter and the appropriate volume of cells to achieve 1000 cells/100µL is calculated. 100 µL of media cell suspension (adjusted to 1000 cells/100 µL) is added to all wells of 96-well plates, followed by incubation for 18 to 24 hours at 37°C, 100% humidity, and 5% CO₂. allowing the cells to adhere to the plates.

Procedure: Day 2 - Compound Addition:

0286To one column of the wells of an autoclaved assay block are added an initial 2.5 µL of test compound(s) at 400X the highest desired concentration. 1.25 µL of 400X (400µM) Topotecan is added to other wells (optical density's from these wells are used to subtract out for background absorbance of dead cells and vehicle). 500 µL of media without DMSO are added to the wells containing test compound, and 250 µL to the Topotecan wells. 250 µL of media + 0.5% DMSO is added to all remaining wells, into which the test compound(s) are serially diluted. By row, compound-containing media is replica plated (in duplicate) from the assay block to the corresponding cell plates. The cell plates are incubated for 72 hours at 37°C, 100% humidity, and 5% CO₂.

Procedure: Day 4 - MTS Addition and OD Reading:

0287The plates are removed from the incubator and 40 µl MTS / PMS is added to each well. Plates are then incubated for 120 minutes at 37°C, 100% humidity, 5%CO₂, followed by reading the ODs at 490nm after a 5 second shaking cycle in a ninety-six well spectrophotometer.

Data Analysis

0288The normalized % of control (absorbance- background) is calculated and an XLfit is used to generate a dose-response curve from which the concentration of compound required to inhibit viability by 50% is determined. The compounds of the present invention show activity when tested by this method as described above.

Example 18

Enantiomer Separation

0289An enriched 3:1 R:S mixture of chromenone enantiomers was separated into its pure enantiomers by chiral chromatography with the following conditions: Column - Chiralpak AD, 250 x 4.6 mm (Diacel Inc.). Sample-22.5 mg/ml in 1:1 i-PrOH:hexanes. Conditions - 40 min at isocratic 50% <i>i</i>-PrOH in Hexanes, (S)-enantiomer elutes at 18.35 min, (R)-enantiomer elutes at 26.87 min. The (R)-enantiomer was significantly more potent than the (S)-enantiomer of the compound of Example 2.

Example 19

Monopolar Spindle Formation following Application of a KSP Inhibitor

0290Human tumor cells Skov-3 (ovarian) were plated in 96-well plates at densities of 4,000 cells per well, allowed to adhere for 24 hours, and treated with various concentrations of the chromenone compounds for 24 hours. Cells were fixed in 4% formaldehyde and stained with antitubulin antibodies (subsequently recognized using fluoresonfly-labeled secondary antibody) and Hoechst dye (which stains DNA).

0291Visual inspection revealed that the 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 causes mitotic arrest with arrested cells displaying monopolar spindles.

Example 20

Inhibition of Cellular Proliferation in Tumor Cell Lines Treated with KSP Inhibitor

0292Cells were plated in 96-well plates at densities from 1000-2500 cells/well of a 96-swell plate 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 T₀. A tetrazolium-based assay using the reagent 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Patent No.<patcit id="pcit0005" dnum="US5185450A"><text> 5,185,450</text></patcit>) (see Promega product catalog #G3580, CellTiter 96® AQ_ueous 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 growth inhibition.

0293The 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.

0294A Gi₅₀ was calculated by plotting the concentration of compound in µM vs the percentage of cell growth in treated wells. The Gi₅₀ calculated for the compounds is the estimated concentration at which growth is inhibited by 50% compared to control, i.e., the concentration at which: <maths id="math0002"><math display="block"><mn>100</mn><mo>×</mo><mfenced open="[" close="]"><mfenced><msub><mi>Treated</mi><mn>48</mn></msub><mo>−</mo><msub><mi mathvariant="normal">T</mi><mn>0</mn></msub></mfenced><mo>/</mo><mfenced><msub><mi>Control</mi><mn>48</mn></msub><mo>−</mo><msub><mi mathvariant="normal">T</mi><mn>0</mn></msub></mfenced></mfenced><mo>=</mo><mn>50</mn></math><img file="EP1511734B1_D0056.tif" /></maths> wherein Treated₄₈ is the value at 48 hours for the treated cells and Control is the value at 48 hours for the control population.

0295All concentrations of compounds are tested in duplicate and controls are averaged over 12 wells. A very similar 96-well plate layout and Gi₅₀ calculation scheme is used by the National Cancer Institute (see <nplcit id="ncit0027" npl-type="s"><text>Monks, et al., J. NatI. Cancer Inst. 83:757-766 (1991</text></nplcit>)). However, the method by which the National Cancer Institute quantitates cell number does not use MTS, but instead employs alternative method.

0296Compounds of Examples 1-13 above inhibited cell proliferation in human ovarian tumor cell lines (SKOV-3).

Example 21

Calculation of IC₅₀:

0297Measurement of a compound's IC₅₀ 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 µM 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 (Sigma P0294), 100 nM KSP motor domain, 50 µg/ml microtubules, 1 mM DTT (Sigma D9779), 5 µM 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 compound are made in a 96-well microtiter plate (Corning Costar 3695) using Solution 1. Following serial dilution each well has 50 µl of Solution 1. The reaction is started by adding 50 µl 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 IC₅₀ determination the data acquired is fit by the following four parameter equation using a nonlinear fitting program (e.g., Grafit 4): <maths id="math0003"><math display="block"><mi mathvariant="normal">y</mi><mo mathvariant="normal">=</mo><mfrac><mi>Range</mi><mrow><mn mathvariant="normal">1</mn><mo mathvariant="normal">+</mo><msup><mfenced><mfrac><mi mathvariant="normal">x</mi><msub><mi>IC</mi><mn mathvariant="normal">50</mn></msub></mfrac></mfenced><mi mathvariant="normal">s</mi></msup></mrow></mfrac><mo mathvariant="normal">+</mo><mi>Background</mi></math><img file="EP1511734B1_D0057.tif" /></maths> where y is the observed rate and x is the compound concentration.