HK1121747A - Protein kinase inhibitors - Google Patents

Description

Protein kinase inhibitors

Background

Technical Field

The present invention relates generally to compounds that inhibit protein kinase activity, and to compositions and methods related thereto.

Description of the related Art

Cancer (and other hyperproliferative diseases) is characterized by uncontrolled cell proliferation. This uncontrolled normal control of cell proliferation is often manifested as a result of genetic damage to cellular pathways that control cell cycle progression. The cell cycle is composed of DNA synthesis (S phase, cell division or mitosis (M phase), and a non-synthetic phase known as gap1(G1) and gap2 (G2).

Cells have some proteins that govern the transition of the cell cycle from one phase to another. For example, cyclins are a family of proteins whose concentration increases and decreases in the cell cycle. At appropriate times, the cyclins turn on different cyclin-dependent protein kinases (CDKs) which phosphorylate substrates essential for cell cycle progression. Specific CDKs activity at specific times is required both at the beginning of the cell cycle and during the coordination process. For example, CDK1 is the most prominent cell cycle regulator that coordinates control of M-phase activity. In any event, a number of other mitotic protein kinases have been identified which are involved In the M-phase, including polo, aurora and members of the NIMA (Never-In-Mitosis-a) family, as well as kinases involved In mitotic checkpoints, mitotic abscission (mitotic exit) and cytokinesis.

Aurora kinases are a family of oncogenic serine/threonine kinases that are concentrated in the mitotic apparatus (centrosomes, poles of bipolar spindles, or intermediates) and regulate centrosome separation, bipolar spindle assembly, and chromosome separation. Three human aurora kinase homologs (aurora-1, aurora-2, and aurora-3) have been identified. They all share a highly conserved catalytic region at the carboxy terminus, but their amino termini can be extended at various lengths without sequence similarity. Human aurora kinases are expressed in proliferating cells and are also overexpressed in many tumor cell lines, including breast, ovary, prostate, pancreas, and colon. Aurora-2 kinase acts as an oncogene and can transform both Rat1 fibroblasts and mouse NIH3T3 cells in vitro, and Aurora-2 can transform NIH3T3 cells that grow as tumors in nude mice in vivo. Excessive aurora-2 can drive cells to aneuploidy (abnormal number of chromosomes) by accelerating the loss of tumor suppressor genes and/or amplifying oncogenes, some events known to contribute to cell transformation. Cells with excess aurora-2 can circumvent mitotic checkpoints, which in turn can inappropriately activate proto-oncogenes. Upregulation of aurora-2 has been demonstrated in a number of pancreatic cancer cell lines. In addition, aurora-2 kinase antisense oligonucleotide treatment has been shown to cause cell cycle inhibition and increased apoptosis. Therefore, aurora-2 kinase is an attractive target for rational design of novel small molecule inhibitors for the treatment of cancer and other conditions.

Several quinazoline derivatives have been proposed for inhibiting protein kinase activity. For example, WO96/09294, WO96/33981 and EP0837063 describe the use of certain quinazoline compounds as inhibitors of receptor tyrosine kinases. Furthermore, WO01/21596 proposes the use of quinazoline derivatives for inhibiting aurora-2 kinase.

However, there is still a need for further improved inhibitors of protein kinase activity, such as inhibitors of aurora-2 kinase activity. The present invention fulfills these needs and provides several other related advantages.

Brief summary of the invention

The present invention relates generally to compounds having the following general structure (I):

including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein R₁、R₂、R₃、X、Z、L₂And w is as defined herein.

The compounds of the invention have a wide range of therapeutic applications and may be used in the treatment of diseases mediated at least in part by protein kinase activity, such as cancer. Thus, in one aspect of the invention, the compounds described herein are formulated as pharmaceutically acceptable compositions for administration to a subject in need thereof. In another aspect, the invention provides methods of treating or preventing a protein kinase mediated disease, such as cancer, in a patient in need of such treatment, comprising administering to the patient a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable composition comprising said compound. In certain embodiments, the protein kinase-mediated disease is an aurora-2 kinase-mediated disease.

In another aspect, the invention relates to inhibiting protein kinase activity in a biological sample, comprising contacting said biological sample with a compound described herein or a pharmaceutically acceptable composition comprising said compound. In certain embodiments, the protein kinase is an aurora-2 kinase.

Another aspect of the invention relates to a method of inhibiting protein kinase activity in a patient, comprising administering to said patient a compound described herein or a pharmaceutically acceptable composition comprising said compound. In certain embodiments, the protein kinase is an aurora-2 kinase.

These and other aspects of the invention will be apparent upon reference to the following detailed description and attached drawings. To this end, patents and other documents are cited herein to more clearly describe various aspects of the present invention. Each of these documents is incorporated herein by reference in its entirety.

Brief description of the drawings

FIG. 1 shows the in vivo anti-tumor activity of exemplary compounds of the invention.

Detailed description of the invention

The present invention relates generally to compounds useful as inhibitors of protein kinases and compositions and methods relating thereto. Such compounds of the invention have the following structure (I):

including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein:

x is NH, S or O;

z is CH or N;

R₁and R₂Are identical or different and are independently hydrogen, hydroxy, halo, -CN, -NO₂、-NH₂、-R、-OR、-SCH₃、-CF₃-C (═ O) OR, -OC (═ O) R, where R is alkyl OR substituted alkyl; or-O (CH)₂)_n-R_xWherein n is 2-4 and R_xIs N-methylpiperazine, morpholine or 2-methylpyrrolidine.

R₃Is hydrogen, -NH₂Alkyl, -CN, or-NO₂Or R is₃is-L₃-CyCl₃Wherein L is₃Is a direct bond, -S-or-NH-, and CyCl₃Is a carbocyclic, substituted carbonA ring, heterocycle or substituted heterocycle;

L₂are-C (═ S) NH-, -NHC (═ S) -, -NHC (═ S) NH-, -C (═ O) NH-, -NHC (═ O) -, -NHC (═ O) NH-, - (CH (O) NH-, - (CH)₂)_n-、-NH(CH₂)_n-、-(CH₂)_nNH-、-NH(CH₂)_nNH-、-C(＝S)NH(CH₂)_n-、-NHC(＝S)(CH₂)_n-、-(CH₂)_nC(＝S)NH(CH₂)_n-、-(CH₂)_nNHC(＝S)(CH₂)_n-、-NHC(＝O)-、-S(＝O)₂-、-S(＝O)₂NH-、-NHS(＝O)₂-wherein n is the same or different at each occurrence and is independently 1, 2, 3 or 4; and

w is-S (═ O)₂NHC(＝O)CH₃、-NHC(＝O)R_y、-NHS(＝O)₂R_yWherein R is_yIs alkyl or cycloalkyl, -NH₂、-NH₂HCl, and-S (═ O)₂-R_zWherein R is_zSelected from the group consisting of alkyl, substituted alkyl, amine, N-methylpiperazine, morpholine, and 2-methylpyrrolidine.

Unless otherwise indicated, the following terms used in the specification and claims have the meanings discussed below:

"alkyl" means a saturated straight or branched chain hydrocarbon group of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, for example, methyl, ethyl, propyl, 2-propyl, n-butyl, i-butyl, t-butyl, pentyl, hexyl and the like, preferably methyl, ethyl, propyl, or 2-propyl. Typical saturated straight chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; and saturated branched alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Typical saturated cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, -CH₂-cyclohexyl and the like; and unsaturated cyclic alkyl includes cyclopentenyl, cyclohexenyl, -CH₂Cyclohexenyl and the like. Cyclic alkyl radicals are also mentioned hereAre referred to as "cycloalkyl". Unsaturated alkyl groups contain at least one double or triple bond between adjacent carbon atoms (referred to as "alkenyl" or "alkynyl", respectively). Typical straight chain and branched alkenyl groups include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2, 3-dimethyl-2-butenyl, and the like; while typical straight and branched alkynyl groups include ethynyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

"alkylene" means a saturated straight chain divalent hydrocarbon group of 1 to 6 carbon atoms or a saturated branched divalent hydrocarbon group of 3 to 6 carbon atoms, for example, methylene, ethylene, 2-dimethylethylene, propylene, 2-methylpropylene, butylene, pentylene, etc., preferably methylene, ethylene, or propylene.

"cycloalkyl" refers to a saturated cyclic hydrocarbon group of 3 to 8 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

"alkoxy" refers to where R is_aA radical-OR which is an alkyl radical as defined above_aFor example, methoxy, ethoxy, propoxy, butoxy and the like.

"halogen" means fluorine, chlorine, bromine, or iodine, preferably fluorine and chlorine.

"haloalkyl" means an alkyl group substituted with one or more, preferably one, two or three, identical or different halogen atoms, e.g., -CH₂Cl、-CF₃、-CH₂CF₃、-CH₂CCl₃And the like.

"haloalkoxy" refers to wherein R is_bA radical-OR being a haloalkyl radical as defined above_bFor example, trifluoromethoxy, trichloroethoxy, 2-dichloropropoxy and the like.

"acyl" refers to wherein R_cIs hydrogen, alkyl, or a haloalkyl group as defined hereingroup-C (O) R_cFor example, formyl, acetyl, trifluoroacetyl, butyryl and the like.

"aryl" refers to an all-carbon monocyclic or fused polycyclic (i.e., rings sharing pairs of adjacent carbon atoms) of 6 to 12 carbon atoms with a fully conjugated pi-electron system. Non-limiting examples of aryl groups are phenyl, naphthyl and anthracenyl. The aryl group may be substituted or unsubstituted. When substituted, the aryl group is substituted with one or more, preferably one, two or three, more preferably one or two substituents independently selected from alkyl, haloalkyl, halo, hydroxy, alkoxy, mercapto, alkylthio, cyano, acyl, nitro, phenoxy, heteroaryl, heteroaryloxy, haloalkyl, haloalkoxy, carboxy, alkoxycarbonyl, amino, alkylamino or dialkylamino.

"heteroaryl" refers to a monocyclic or fused ring (i.e., a ring sharing pairs of adjacent atoms) containing one, two, three, or four ring atoms selected from N, O, or S, the remaining ring atoms being 5 to 12 ring atoms of C, and also having a fully conjugated pi-electron system. Non-limiting examples of unsubstituted heteroaryl groups are pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, purine, triazole, tetrazole, triazine, and carbazole. The heteroaryl group may be substituted or unsubstituted. When substituted, the heteroaryl is substituted with one or more, more preferably one, two or three, more preferably one or two substituents independently selected from alkyl, haloalkyl, halo, hydroxy, alkoxy, mercapto, alkylthio, cyano, acyl, nitro, haloalkyl, haloalkoxy, carboxy, alkoxycarbonyl, amino, alkylamino or dialkylamino.

"carbocyclic" refers to an aliphatic ring system having 3 to 14 ring atoms. The term "carbocyclic ring", whether saturated or partially unsaturated, also refers to optionally substituted rings. The term "carbocyclic" also includes aliphatic rings fused to one or more aromatic or non-aromatic rings, such as decahydronaphthyl or tetrahydronaphthyl, where the linking group or point of attachment is on the aliphatic ring.

"heterocycle" refers to a ring in which one, two or three ring atoms are selected from O, N, or S (O)_m(wherein m is an integer from 0 to 2), the remaining ring atoms being a saturated cyclic ring system of C having 3 to 14 ring atoms, wherein one or two C atoms may optionally be replaced by a carbonyl group. The heterocyclyl ring may optionally be independently substituted with one or more, preferably one, two, or three substituents selected from alkyl (wherein the alkyl may optionally be substituted with one or two substituents independently selected from carboxy or ester groups), haloalkyl, cycloalkylamino, cycloalkylalkyl, cycloalkylaminoalkyl, cycloalkylalkylaminoalkyl, cyanoalkyl, halo, nitro, cyano, hydroxy, alkoxy, amino, alkylamino, dialkylamino, hydroxyalkyl, carboxyalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylalkyl, heteroarylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, saturated or unsaturated heterocyclylamino, saturated or unsaturated heterocyclylaminoalkyl, and-COR_d(wherein R is_dIs alkyl). More specifically, the term heterocyclyl includes, without limitation, tetrahydropyranyl, 2-dimethyl-1, 3-dioxolane, piperidino, N-methylpiperidin-3-yl, piperazino, N-methylpyrrolidin-3-yl, pyrrolidino, morpholino, 4-cyclopropylmethylpiperazino, thiomorpholino-1-oxide, thiomorpholino-1, 1-dioxide, 4-ethoxycarbonylpiperazino, 3-oxopiperazino, 2-imidazolidinone, 2-pyrrolidone, 2-oxophomopiperazino, tetrahydropyrimidin-2-one, and derivatives thereof. In certain embodiments, the heterocyclic group is optionally substituted with one or two substituents independently selected from halo, alkyl substituted with carboxy, ester, hydroxy, alkylamino, saturated or unsaturated heterocyclic amino, saturated or unsaturated heterocyclic aminoalkyl, or dialkylamino.

"optional" or "optionally" means that the subsequently described event or circumstance does not necessarily occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, "a heterocyclic group optionally substituted with an alkyl" means that the alkyl is not necessarily present, and the description includes the case where the heterocyclic group is substituted with an alkyl and the case where the heterocyclic group is not substituted with an alkyl.

Finally, as used herein, the term "substituted" refers to any of the above groups (e.g., alkyl, aryl, heteroaryl, carbocycle, heterocycle, etc.) wherein at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent ("═ O"), two hydrogen atoms are replaced. "substituents" in the context of the present invention include halogen, hydroxy, oxo, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkoxy, thioalkyl, haloalkyl, hydroxyalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocycloalkyl, substituted heterocycloalkyl, -NR_eR_f、NR_eC(＝O)R_f、-NR_eC(＝O)NR_eR_f、-NR_eC(＝O)OR_f-NR_eSO₂R_f、-OR_e、-C(＝O)R_e-C(＝O)OR_e、-C(＝O)NR_eR_f、-OC(＝O)NR_eR_f、-SH、-SR_e、-SOR_e、-S(＝O)₂R_e、-OS(＝O)₂R_e、-S(＝O)₂OR_eWherein R is_eAnd R_fThe same or different and are independently hydrogen, alkyl, haloalkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substituted heterocycle, heterocycloalkyl, or substituted heterocycloalkyl.

In a more particular aspect of structure (I) above, X is NH and Z is CH.

In a more particular aspect of structure (I) above, R₁、R₂And R₃Selected from hydrogen, -NH₂、-OCH₃、-OH、-CF₃Halo, or-O (CH)₂)_n-R_xWherein n is 2-4 and R_xIs N-methylpiperazine, morpholine or 2-methylpyrrolidine.

In a more particular aspect of structure (I) above, L₂is-C (═ S) NH-or-C (═ S) NHCH₂-。

In a more particular aspect of structure (I) above, w is-S (═ O)₂NHC(＝O)CH₃or-S (═ O)₂-R₂Wherein R is_zIs selected from C₁-C₃Alkyl radical, C₁-C₃Substituted alkyl or amine.

In a more particular aspect of structure (I) above, w is-S (═ O)₂NHC(＝O)CH₃、-S(＝O)₂NH₂or-S (═ O)₂CH₃。

In a more particular aspect of structure (I) above, w is-S (═ O)₂NHC(＝O)CH₃。

In a more particular aspect of structure (I) above, R₁、R₂And R₃Selected from hydrogen, -NH₂、-OCH₃、-OH、-CF₃Halo, or-O (CH)₂)_n-R_xWherein n is 2-4 and R_xIs N-methylpiperazine, morpholine or 2-methylpyrrolidine, and w is-S (═ O)₂NHC(＝O)CH₃、-S(＝O)₂NH₂or-S (═ O)₂CH₃。

In a more particular aspect of structure (I) above, R₁And R₂Selected from hydrogen, halo, -CF₃or-OH, R₃Is hydrogen, and w is-S (═ O)₂NHC(＝O)CH₃、-S(＝O)₂NH₂or-S (═ O)₂CH₃。

In a more particular aspect of structure (I) above, X is NH, Z is CH, L₂is-C (═ S) NH-, andand the compound has the following structure (II):

in a more particular aspect of structure (II) above, R₁And R₂Is selected from-OCH₃、-OH、-CF₃Halo, or-O (CH)₂)_n-R_xWherein n is 2-4 and R_xIs N-methylpiperazine, morpholine or 2-methylpyrrolidine, and R₃Selected from hydrogen or-NH₂。

In a more particular aspect of structure (II) above, R₁And R₂Is selected from-OCH₃、-OH、-CF₃Or halo, and R₃Is hydrogen.

In a more particular aspect of structure (II) above, w is-S (═ O)₂NHC(＝O)CH₃or-S (═ O)₂-R_zWherein R is_zIs selected from C₁-C₃Alkyl radical, C₁-C₃Substituted alkyl or amine.

In a more particular aspect of structure (II) above, w is-S (═ O)₂NHC(＝O)CH₃、-S(＝O)₂NH₂or-S (═ O)₂CH₃。

In a more particular aspect of structure (II) above, R₁And R₂Is selected from-OCH₃、-OH、-CF₃Or halo, R₃Is hydrogen, and w is-S (═ O)₂NHC(＝O)CH₃、-S(＝O)₂NH₂or-S (═ O)₂CH₃。

In a more particular aspect of structure (II) above, R₁And R₂Is selected from-OCH₃、-OH、-CF₃Or halo, R₃Is hydrogen, and w is-S (═ O)₂NHC(＝O)CH₃、-S(＝O)₂NH₂Or-S(＝O)₂CH₃。

In a more particular aspect of structure (II) above, R₁And R₂Is methoxy, R₃Is hydrogen, w is-S (═ O)₂NHC(＝O)CH₃And the compound has the following structure (III):

in a more particular aspect of structure (II) above, R₁is-C1, R₂is-CF₃，R₃Is hydrogen, w is-S (═ O)₂NHC(＝O)CH₃And the compound has the following structure (IV):

in a more specific aspect of structure (I) above, compounds having the structure set forth in Table I below are provided.

TABLE I

Compounds having the same molecular formula but differing in their nature or order of atomic incorporation or spatial arrangement of atoms are referred to as "isomers". Isomers that differ in the arrangement of atoms in space are referred to as "stereoisomers". Stereoisomers that are not mirror images of each other are referred to as "diastereomers", and those that are not superimposable mirror images of each other are referred to as "enantiomers". When a compound has an asymmetric center, for example, in combination with four different groups, a pair of enantiomers may exist. One enantiomer can be characterized by the absolute configuration of the asymmetric center and can be described by the R-and S-sequencing rules of Cahn and Prelog (Cahn, R., Ingold, C, and Prelog, V.Angew.chem.78: 413-47, 1966; Angew.chem.Internal Ed.Eng.5: 385. 415, 511, 1966) or by the way in which the molecule rotates polarized light and is referred to as dextrorotatory or levorotatory (i.e., in (+) or (-) -isomeric form, respectively). A chiral compound may exist as individual enantiomers or mixtures thereof. Mixtures containing equal proportions of enantiomers are so-called "racemic mixtures".

The compounds of the invention may have one or more asymmetric centers; thus, such compounds may exist as individual (R) -or (S) -stereoisomers or mixtures thereof. Unless otherwise indicated, the description or naming of a particular compound in the specification and claims includes both its individual enantiomers, as well as mixtures, racemates, and the like. Methods for determining the stereochemistry and separating stereoisomers thereof are well known in the art (for a discussion thereof see chapter 4, 4 th edition, j., John Wiley and Sons, new york city, 1992, of "ADVANCED ORGANIC CHEMISTRY" (ADVANCED ORGANIC CHEMISTRY) ".

The compounds of the present invention may exhibit tautomerism and structural isomerism. For example, the compounds described herein may take the E or Z configuration with respect to the double bond linking the 2-indolinone moiety to the pyrrole moiety or they may be a mixture of E and Z. The present invention includes any tautomeric or structurally isomeric form and mixtures thereof having the ability to modulate aurora-2 kinase activity and is not limited to any one tautomeric or structurally isomeric form.

It is believed that the compounds of the present invention may be metabolized by enzymes in an organism, such as a human, to produce metabolites that may modulate the activity of the protein kinase. Such metabolites are also within the scope of the present invention.

The compounds of the present invention can be prepared by one skilled in the art according to the following general reaction scheme and procedures described in more detail in the examples.

The chlorination of the (un) substituted 6-membered aromatic moiety may be carried out in the presence of sulfonyl chloride at about 0 ℃. The 4-chloro- (un) substituted benzene (2) may be nitrated with fuming nitric acid to give 1-chloro- (un) substituted-2-nitrobenzene (3), preferably at a temperature not exceeding about 25 ℃. Ethyl 2-cyano-2- (un) substituted-2-nitrophenyl) acetate (4) may be prepared by reacting compound 3 with ethyl cyanoacetate in the presence of potassium tert-butoxide in THF (to give compound 4 in 23% yield). In this step, the yield of compound 3 can be further optimized by reacting it in the presence of K2CO3 in DMF at a temperature of about 155 ℃ for 6 hours to obtain the ethyl cyano ester in high yield. The reduction of ester 4 can be carried out using known conditions with an excess of Zn powder (4-6 equivalents) to give ethyl 2-amino-5, 6-dimethoxy-1H-indole-3-carboxylate (5) without the production of N-hydroxy by-product.

The 2-amino-5, 6-dimethoxy-1H-indole-3-carboxylic acid ethyl ester (5) can be cyclized to the corresponding dihydro-4H-pyrimido [4, 5-b ] by heating in formamide and catalytic sodium methoxide at about 200-]Indole. Thionyl chloride and/or POCl may be used₃The dihydro-pyrimidine is converted to the 4-chloride (6) in good yield in a dioxane solvent. This 4-chloride can be used to prepare 4-piperazine (piprazine) -substituted tricyclic analogs as outlined in scheme 1. This 4-chloride can be reacted with piperazine (piprazine) in the presence of pyridine in a dioxane solvent at reflux temperature to give compound 8 in good yield. Can be obtained by reacting any cyclic ethyl ester in the presence of cyanoacetamide and anhydrous HCl₃Substitution at position(s) to give guanidine analog 10. These compounds can be cyclized in the presence of aqueous NaOH to 3-substituted tricyclic dihydro-pyrimidines.

Certain intermediates useful in the preparation of the target compounds are outlined in scheme 2 and described in detail in scheme 3. It is possible to use thiophosgene in the presence of CaCO in methylene chloride₃And water, to give isothiocyanate analog 13 in high yield. Compounds of formula I having a 4-substituted piperazine (piprazine analog) can be prepared by reacting compound 13 in the presence of pyridine and a dioxane solventAnd (4) preparation. Compound 14 was treated with 1-bromo-3-chloropropane and cesium carbonate in acetonitrile to give 1- (3-chloropropyloxy) -4-chloro-2-methoxybenzene 15. Various carbocyclic compounds such as N-methylpiperazine, morpholine and/or 2-methylpyrrolidine are reacted with compound 15 in acetonitrile to give compound 17 in high yield (scheme 2). Subsequently, it is subjected to nitration and under similar conditions, as described in scheme 1 for the preparation of compound 4, to prepare ethyl 2-cyano-2- (un) substituted-2-nitrophenyl) acetate.

Scheme 1

Flow chart 2

Flow chart 3

The compounds of the present invention or pharmaceutically acceptable salts thereof may be administered to human patients as such or in the form of pharmaceutical compositions wherein the above are mixed together with suitable carriers or excipients. Techniques for the formulation and administration of drugs can be found, for example, in REMINGTON' S pharma organic chemicals co.

"pharmaceutical composition" refers to a mixture of one or more compounds described herein, or a pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to aid in the administration of the compound to an organism.

"pharmaceutically acceptable excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the compound. Examples of excipients include, without limitation, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

By "pharmaceutically acceptable salts" is meant those salts which retain the biological potency and properties of the parent compound. Such salts may include: (1) acid addition salts obtained by reacting the free base of the parent compound with an inorganic acid such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, perchloric acid and the like, or with an organic acid such as acetic acid, oxalic acid, (D) -or (L) -malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, malonic acid and the like, preferably with hydrochloric acid or (L) -malic acid; or (2) salts formed by substitution with metal ions, e.g., alkali metal ions, alkaline earth metal ions, aluminum ions, when acidic protons are present in the parent compound; or a salt coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, etc.

The compounds of the invention may also act or be designed to act in the form of prodrugs. "prodrug" refers to a substance that is converted in vivo to the parent drug. Because of the ease of administration over the parent drug, prodrugs are often used in some cases. For example, a prodrug may be bioavailable when administered orally, whereas the parent drug is not bioavailable when administered orally. Prodrugs may also have improved solubility compared to the parent drug in the pharmaceutical composition. An example of a prodrug is a compound of the invention administered in the form of an ester (the "prodrug"), phosphate, amide, carbamate, or urea.

"therapeutically effective amount" refers to the amount of the compound administered that will alleviate one or more symptoms of the condition being treated to some extent. Where treatment of cancer is concerned, a therapeutically effective amount refers to an amount which has the following effect: (1) reducing the size of the tumor; (2) inhibiting tumor metastasis; (3) inhibiting tumor growth; and/or (4) alleviating one or more symptoms associated with cancer.

The term "protein kinase-mediated condition" or "disease" as used herein refers to any disease or other deleterious condition in which a known protein kinase plays a role. The term "protein kinase-mediated disorder" or "disease" also refers to such diseases or disorders that can be alleviated by treatment with a protein kinase inhibitor. Such disorders include, without limitation, cancer and other hyperproliferative disorders. In certain embodiments, the cancer is a cancer of colon, breast, stomach, prostate, pancreas, or ovarian tissue.

The term "Aurora-2 kinase-mediated disorder" or "disease" as used herein refers to any disease or other deleterious disorder in which Aurora is known to play a role. The term "Aurora-2 kinase-mediated disorder" or "disease" also refers to such diseases or disorders that can be alleviated by treatment with an Aurora-2 inhibitor.

As used herein, "administering" or "administering" refers to delivering a compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, to an organism for the purpose of preventing or treating a protein kinase mediated disorder.

Suitable routes of administration include, without limitation, oral, rectal, transmucosal or enteral administration or intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intravitreal, intraperitoneal, intranasal, or intraocular injection. In certain embodiments, preferred routes of administration are oral and intravenous.

Alternatively, the compounds may be administered locally rather than systemically, e.g., the compounds may be injected directly into a solid tumor, often in the form of a depot or sustained release formulation.

Furthermore, administration may also be in the form of a targeted drug delivery system, for example, administration may be carried out with liposomes coated with tumor-specific antibodies. Thus, the liposomes can be targeted to and taken up selectively by the tumor.

The pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical compositions used in the present invention may be prepared in any conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Suitable formulations depend on the chosen route of administration.

For injection, the compounds of the invention may be prepared as aqueous solutions, preferably in the form of solutions in physiologically compatible buffers such as Hanks 'solution, ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the active compounds may be formulated by bringing into association the compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient. Pharmaceutical preparations for oral use can be prepared with solid excipients, optionally grinding the resulting mixture, and processing the mixture of microparticles, if desired after addition of further suitable auxiliaries, to give tablets or dragee cores. Useful excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, for example, corn starch, wheat starch, rice starch, and potato starch, and also other substances such as gelatin, tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents such as cross-linked polyvinylpyrrolidone, agar, or alginic acid may be added. Salts such as sodium alginate may also be used.

Dragee cores may be provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, a transparent protective film (lacquer) solution, and a suitable organic solvent or solvent mixture. To identify or characterize combinations of different active compound doses, dyes or pigments may be added to the tablets or dragee coatings.

Pharmaceutical compositions for oral use include push-fit (push-fit) capsules made of gelatin, as well as sealed soft capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers may also be added to these formulations. Pharmaceutical compositions that may also be used include hard gelatin capsules. The capsules or pills can be packaged in brown glass or plastic bottles to protect the active compound from light. The container containing the active compound capsule formulation is preferably stored at controlled room temperature (15-30 ℃).

For administration by inhalation, the compounds for use in the present invention may be conveniently delivered in the form of a spray using pressurized packs or a nebulizer and a suitable propellant, such as, without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra-fluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be controlled by providing a valve to deliver a metered amount. For example, capsules and cartridges, e.g., gelatin capsules and cartridges, for use in an inhaler or insufflator may be prepared containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may also be prepared for parenteral administration, for example by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose vials, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of water-soluble forms of the active compounds, such as, without limitation, salts. Furthermore, it is also possible to prepare suspensions of the active compounds in a lipophilic matrix. Suitable lipophilic bases include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate and triglycerides, or materials such as liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension may optionally also contain suitable stabilizers and/or substances that increase the solubility of the compounds so that highly concentrated solutions can be prepared.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The compounds may also be prepared, for example, as rectal compositions such as suppositories or retention enemas using conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be prepared in depot formulations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. For such routes of administration, the compounds of the invention may be prepared with suitable polymeric or hydrophobic materials (e.g. in an emulsion using a pharmacologically acceptable oil), with ion exchange resins, or may be prepared as sparingly soluble derivatives, such as, without limitation, in the form of a sparingly soluble salt.

Non-limiting examples of pharmaceutically acceptable carriers for the hydrophobic compounds of the present invention are co-solvent systems comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase, such as VPD co-solvent systems. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v non-polar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300 prepared to the desired volume with anhydrous ethanol. The VPD cosolvent system (VPD: D5W) consisted of VPD diluted 1: 1 with 5% aqueous glucose. Such co-solvent systems dissolve hydrophobic compounds very well and, when administered systemically, have low toxicity of their own. The proportions of such co-solvent systems can naturally vary to a large extent, as long as their solubility and toxicity properties are not impaired. Furthermore, the nature of the co-solvent component may vary: for example, polysorbate 80 may be replaced by other non-polar surfactants of low toxicity, the fractional size of polyethylene glycol may be varied, other biocompatible polymers such as polyvinylpyrrolidone may be used in place of polyethylene glycol, and other sugars or polysaccharides may be used in place of glucose.

Alternatively, other delivery systems may be used to deliver the hydrophobic pharmaceutical compound. Liposomes and emulsions are well known examples of delivery matrices or carriers for hydrophobic drugs. In addition, certain organic solvents such as dimethyl sulfoxide can also be used, albeit often at the expense of higher toxicity.

In addition, the compounds may also be delivered using sustained release systems such as semipermeable matrices of hydrophobic solid polymers containing the therapeutic agent. Many sustained release materials have been identified and are well known to those skilled in the art. Depending on its chemical nature, sustained release capsules may release the compound over a period of weeks up to over 100 days. Depending on the chemical nature and biological stability of the therapeutic agent, additional strategies for protein stabilization may also be used.

The pharmaceutical compositions described herein may also contain suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, without limitation, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol.

Any of the protein kinase modulating compounds of the present invention may be provided in the form of a physiologically acceptable salt in which the compound to be protected may form a negatively or positively charged species. Examples of salts in which the compounds form positively charged moieties include, without limitation, quaternary amines (as defined elsewhere herein) such as salts of hydrochloride, sulfate, carbonate, lactate, tartrate, malate, maleate, succinate, and the like, wherein the nitrogen atom of the quaternary ammonium group is the nitrogen of a selected compound of the invention that has been reacted with a suitable acid. Wherein the compounds of the present invention form salts of negatively charged species including, without limitation, by reacting the carboxylic acid groups in the compounds with a suitable base (e.g., sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide (Ca (OH))₂) Etc.) to form sodium, potassium, calcium, and magnesium salts.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredient is included in an amount sufficient to achieve the desired purpose, e.g., in an amount sufficient to modulate protein kinase activity and/or to treat or prevent a condition associated with a protein kinase.

More specifically, a therapeutically effective amount refers to an amount of a compound that is effective to prevent, alleviate or ameliorate a disease or prolong the survival of the subject being treated.

The therapeutically effective amount is well within the skill of the art and can be determined, inter alia, in light of the detailed disclosure provided herein.

For any compound used in the methods of the invention, a therapeutically effective amount or dose can first be assessed by cell culture assays. Doses for animal models can then be formulated to obtain IC determined for inclusion in cell culture₅₀(i.e., the concentration of test compound that achieves half the maximum inhibition of protein kinase activity). This type of information can then be used to more accurately determine the dosage for a human.

Toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures performed in cell cultures or experimental animals, e.g., by determining the IC of the subject compound₅₀And LD₅₀(both are discussed elsewhere herein) to make the determination. The data obtained from these cell culture experiments and animal studies can be used to calculate a range of doses for use in humans. The dosage may vary depending on the dosage form employed and the route of administration employed. The exact formulation, route of administration and dosage can be selected by the individual physician in accordance with the patient's circumstances. (see, e.g., GOODMAN&Gimlan' S THE PHARMACOLOGICAL BASIS OF THERAPEUTIC, chapter 3,9 th edition, Hardman, J., and Limbard, L., edited by McGraw-Hill, New York City, 1996, page 46. )

The number and spacing of individual doses can be adjusted to provide plasma levels of the active agent sufficient to maintain kinase modulation. These plasma levels are referred to as the Minimum Effective Concentrations (MECs). The MEC will be different for each compound but can be estimated from in vitro data, such as the concentration required to achieve 50-90% kinase inhibition, which can be determined using the assays described herein. The dose required to obtain a MEC will depend on the individual characteristics and route of administration. Plasma concentrations were determined by HPLC analysis or biological assays.

The MEC value may be used to determine the dose interval. These compounds should be administered using a regimen that maintains plasma levels above MEC for 10-90% of the time, preferably 30-90% and most preferably 50-90%.

Currently, a therapeutically effective amount of a compound of the invention may be about 2.5mg/m per day²To 1500mg/m². Additional illustrative amounts range from 0.2 to 1000 mg/four times daily, from 2 to 500 mg/four times daily, and from 20 to 250 mg/four times daily.

In the case of topical administration or selective uptake, the effective local concentration of the drug is not related to the plasma concentration, and other procedures known in the art can be used to determine the correct dose number and interval.

The amount of composition administered will, of course, depend on the individual being treated, the severity of the affliction, the mode of administration, the judgment of the prescribing physician, and the like.

If desired, the compositions may be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The package may for example comprise a metal or plastic foil, such as a blister pack. The pack or dispenser device may have instructions for administration. The package or dispenser may also have a notice associated with the container in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the composition or of human or veterinary administration. Such notification may be, for example, a label approved by the U.S. food and Drug Administration for prescription drugs or an approved product insert. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container and labeled for treatment of the indicated indication. Suitable conditions indicated on the label may include treatment of tumors, inhibition of angiogenesis, treatment of fibrosis, diabetes, and the like.

As noted above, the compounds and compositions of the present invention find use in a number of protein kinase mediated diseases and conditions, including those mediated by aurora-2 kinase. Such diseases include, for example and without limitation, cancers such as lung cancer, NSCLC (non-small cell lung cancer), oat cell carcinoma, bone cancer, pancreatic cancer, skin cancer, dermatofibrosarcoma protruberans, head and neck cancer, cutaneous or intraocular cancer, uterine cancer, ovarian cancer, colorectal cancer, cancer of the anal region, gastric cancer, colon cancer, breast cancer, gynecological tumors (e.g., uterine sarcoma, cancer of the fallopian tube, endometrial cancer, cervical cancer, cancer of the vagina or cancer of the vulva), Hodgkin's disease, hepatocellular carcinoma, esophageal cancer, small bowel cancer, cancer of the endocrine system (e.g., cancer of the thyroid, pancreas, parathyroid or adrenal gland), sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer (particularly refractory to hormones), chronic or acute leukemia, solid tumors of childhood, eosinophilia, leukemia, Lymphocytic lymphomas, cancers of the bladder, cancers of the kidney or ureter (e.g., renal cell carcinoma, cancer of the renal pelvis), pediatric malignancies, neoplasms of the central nervous system (e.g., primary CNS lymphoma, spinal axis tumors, medulloblastoma, brain stem glioma, or pituitary adenoma), barrett's esophagus (pre-malignant syndrome), neoplastic skin diseases, psoriasis, mycoses (mycoses fungoides), and benign prostatic hypertrophy, diseases associated with diabetes such as diabetic retinopathy, retinal ischemia, and retinal neovascularization, cirrhosis, angiogenesis, cardiovascular diseases such as atherosclerosis, immune diseases such as autoimmune diseases, and kidney diseases.

The compounds of the invention may be combined with one or more other chemotherapeutic agents. The dosage of the compounds of the present invention may be adjusted for any drug-drug response. In one embodiment, the chemotherapeutic agent is selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, cell cycle inhibitors, enzymes, topoisomerase inhibitors such as CAMPTOSAR (irinotecan), biological response modifiers, anti-hormones, anti-angiogenic agents such as MMP-2, MMP-9, and COX-2 inhibitors, anti-androgens, platinum coordination complexes (cisplatin, etc.), substituted ureas such as hydroxyurea; methylhydrazine derivatives, for example, procarbazine; adrenocortical suppressants, e.g., mitotane, aminoglutethimide, hormones and hormone antagonists such as adrenocorticosteroids (e.g., prednisones), progestins (e.g., hydroxyprogesterone caproate), estrogenic agents (e.g., diethylstilbestrol), antiestrogens such as tamoxifen, androgens such as testosterone propionate, and aromatase inhibitors such as anastrozole, and AROMASIN (exemestane).

Examples of alkylating agents that the above methods may be combined with include, without limitation, fluorouracil (5-FU) alone or in further combination with folinic acid; other pyrimidine analogs such as UFT, capecitabine, gemcitabine and cytarabine, alkyl sulfonates, e.g., busulfan (for the treatment of chronic myelogenous leukemia), improsulfan and piposulfan; aziridines, for example, benzotepa, carboquone, metotepipa and uretepa; ethylene and methyl ether amines, such as hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; and nitrogen mustards, for example chlorambucil (for treating chronic lymphocytic leukemia, primary macroglobulinemia and non-hodgkin's lymphoma), cyclophosphamide (for treating hodgkin's disease, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, wilms tumor and rhabdomyosarcoma), estramustine, ifosfamide, novembichin, pomatemustine and uracil mustard (for treating primary thrombocytosis, non-hodgkin's lymphoma, hodgkin's disease and ovarian cancer); and triazines, for example, dacarbazine (for the treatment of soft tissue sarcoma).

Examples of antimetabolite chemotherapeutic agents with which the above methods may be combined include, without limitation, folic acid analogs such as methotrexate (for the treatment of acute lymphocytic leukemia, choriocarcinoma, mycosis fungoides, breast cancer, head and neck cancer, and osteosarcoma) and pteropterin; and purine analogs such as mercaptopurine and thioguanine which have been found to be useful in the treatment of acute myeloid leukemia, acute lymphocytic leukemia and chronic myeloid leukemia.

Examples of natural product-based chemotherapeutic agents that the above methods may be combined with include, without limitation, vinca alkaloids, e.g., vinblastine (for treatment of breast and testicular cancer), vincristine, and vindesine; etoposide, e.g., etoposide and teniposide, both of which are useful in the treatment of testicular cancer and kaposi's sarcoma; antibiotic chemotherapeutic agents, for example, daunorubicin, doxorubicin, epirubicin, mitomycin (for treating gastric, cervical, colon, breast, bladder, and pancreatic cancer), dactinomycin, temozolomide, plicamycin, bleomycin (for treating skin, esophageal, and genitourinary tract cancers); and enzymatic chemotherapeutic agents such as L-asparaginase.

Examples of useful COX-II inhibitors include Vioxx, CELEBREX (celecoxib), valdecoxib, paracoxib, rofecoxib, and Cox 189.

In WO96/33172 (published as 24/10/1996), WO96/27583 (published as 7/1996), European patent application 97304971.1 (published as 8/7/1997), European patent application 99308617.2 (published as 29/10/1999), WO98/07697 (published as 26/1998), WO98/03516 (published as 29/1998), WO98/34918 (published as 13/1998), WO98/34915 (published as 13/1998), WO98/33768 (published as 6/1998), WO98/30566 (published as 16/1998), European patent application 606,046 (published as 13/1994), European patent application 931,788 (published as 1999/7/28), WO 90/055 (published as 6/1998), WO 5231/1990) and WO 5221/10/1999, Examples of useful matrix metalloproteinase inhibitors are described in WO99/52889 (published as 10/21/1999), WO99/29667 (published as 6/17/1999), PCT International application PCT/IB98/01113 (published as 7/21/1998), European patent application 99302232.1 (published as 3/25/1999), British patent application 9912961.1 (published as 6/3/1999), US5,863,949 (published as 1/26/1999), US5,861,510 (published as 1/19/1999), and European patent application 780,386 (published as 6/25/1997), which are all incorporated herein by reference in their entirety. Preferred MMP-2 and MMP-9 inhibitors are those that inhibit little or no MMP-1. More preferred are those which selectively inhibit MMP-2 and/or MMP-9 relative to other matrix metalloproteinases (i.e., MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).

Some specific examples of MMP inhibitors useful in the present invention are AG-3340, RO32-3555, RS13-0830, and a compound selected from the group consisting of: 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (1-hydroxycarbamoyl-cyclopentyl) -amino ] -propionic acid; 3-exo-3- [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -8-oxa-bicyclo [3.2.1] octane-3-carboxylic acid hydroxyamide; (2R, 3R)1- [4- (2-chloro-4-fluoro-benzyloxy) -benzenesulfonyl ] -3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide; 4- [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -tetrahydro-pyran-4-carboxylic acid hydroxyamide; 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (1-hydroxycarbamoyl-cyclobutyl) -amino ] -propionic acid; 4- [4- (4-chloro-phenoxy) -benzenesulfonylamino ] -tetrahydro-pyran-4-carboxylic acid hydroxyamide; (R)3- [4- (4-chloro-phenoxy) -benzenesulfonylamino ] -tetrahydro-pyran-3-carboxylic acid hydroxyamide; (2R, 3R)1- [4- (4-fluoro-2-methylbenzyloxy) -benzenesulfonyl ] -3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide; 3- [ [ (4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (1-hydroxycarbamoyl-1-methyl-ethyl) -amino ] -propionic acid, 3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (4-hydroxycarbamoyl-tetrahydro-pyran-4-yl) -amino ] -propionic acid, 3-exo-3- [4- (4-chloro-phenoxy) -benzenesulfonylamino ] -8-oxa-bicyclo [3.2.1] octane-3-carboxylic acid hydroxyamide, 3-endo-3- [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -8-oxa-bicyclo [3.2.1] octane-3- A formic acid hydroxyamide; and (R)3- [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -tetrahydro-furan-3-carboxylic acid hydroxyamide; and pharmaceutically acceptable salts and solvate compounds of these compounds.

Other anti-angiogenic agents, other COX-II inhibitors, and other MMP inhibitors may also be used in the present invention.

The compounds of the present invention may also be combined with other signal transduction inhibitors, such as agents that can inhibit the EGFR (epidermal growth factor receptor) response, such as EGFR antibodies, EGF antibodies, and molecules that are EGFR inhibitors; VEGF (vascular endothelial growth factor) inhibitors; and erbB2 receptor inhibitors, such as organic molecules or antibodies that bind to the erbB2 receptor, such as HERCEPTIN (Genentech, inc., South San Francisco, CA). EGFR inhibitors are described, for example, in WO95/19970 (published as 1995 at 27), WO98/14451 (published as 1998 at 4 and 9), WO98/02434 (published as 1998 at1 and 22), and U.S. Pat. No. 5,747,498 (published as 1998 at 5 and 5), and such substances can be used in the present invention as described herein.

EGFR-inhibitors include, but are not limited to, monoclonal antibody C225 and anti-EGFR 22Mab (Imclone Systems, Inc., New York, NY), compound ZD-1839(AstraZeneca), BIBX-1382(Boehringer Ingelheim), MDX-447(Medarex Inc., Annandale, NJ), and OLX-103(Merck & Co., Whitehouse State, NJ), as well as EGF fusion toxin (Seragen, Hopkinton, MA).

These and other EGFR-inhibitors can be used in the present invention. VEGF inhibitors, such as SU-5416 and SU-6668(Sugen Inc., South San Francisco, Calif.) may also be combined with the compounds of the present invention. In, for example, WO01/60814A3 (published as 8/23/2001), WO99/24440 (published as 5/20/1999), PCT International application PCT/IB99/00797 (published as 5/3/1999), WO95/21613 (published as 8/17/1995), WO99/61422 (published as 12/2/1999), US5,834,504 (published as 11/10/1998), WO01/60814, WO98/50356 (published as 11/12/1998), US5,883,113 (published as 3/16/1999), US5,886,020 (published as 3/23/1999), US5,792,783 (published as 8/11/1998), WO99/10349 (published as 3/5964/1999), WO 2/32856 (published as 1997/6312/1997), WO 8/96 (published as 546/1998), WO 096/22592 (published as 1998/22512/22592/1998), WO 093/22592 (published as 22512/22592/1998), WO 2/63856 (published as 2256/22512/22592/22522), VEGF inhibitors are described in WO99/16755 (published as 4/8/1999), and WO98/02437 (published as 1/22/1998), which are all incorporated herein by reference. Other examples of some specific VEGF inhibitors for use in the present invention are IM862(Cytran inc., Kirkland, WA); anti-VEGF monoclonal antibodies of Genentech, inc; and angiozyme (a synthetic Ribozyme from Ribozyme) (Boulder, CO) and Chiron (Emeryville, CA). These and other VEGF inhibitors may be used in the present invention as described herein. In addition, inhibitors of The pErbB2 receptor, such as GW-282974(GlaxoWellcome pic), and monoclonal antibodies AR-209(Aronex pharmaceuticals Inc., The Woodlands, TX) and 2B-1(Chiron) may also be combined with The compounds of The present invention, for example, combinations of these substances shown in WO98/02434 (published as 1/22/1998), WO99/35146 (published as 7/15/1999), WO99/35132 (published as 7/15/1999), WO98/02437 (published as 1/22/1998), WO97/13760 (published as 4/17/1997), WO95/19970 (published as 7/27/1995), US5,587,458 (published as 12/24/1999), and US5,877,305 (published as 3/2/1999) are incorporated herein by reference in their entirety. ErbB2 receptor inhibitors for use in the present invention are also described in US6,284,764 (release date 9/4/2001), which is incorporated herein by reference in its entirety. In accordance with the present invention, erbB2 receptor inhibitor compounds and substances, as well as other compounds and substances that inhibit the erbB2 receptor, described in the above-mentioned PCT applications, US patents, and US provisional applications, may be used with the compounds of the present invention.

The compounds of the present invention may also be used with other agents for the treatment of cancer, including, without limitation, agents that enhance anti-tumor immune responses, such as CTLA4 (cytotoxic lymphocyte antigen 4) antibodies, and other agents that block CTLA 4; and antiproliferative agents such as other inhibitors of farnesyl protein transferase, for example as described in the references cited in the "background" section of US patent 6,258,824B 1.

The above methods may also be performed in conjunction with radiation therapy, wherein the amount of the compound of the present invention in combination with the radiation therapy is effective to treat the above conditions.

Techniques for using radiation therapy are known in the art, and these techniques can be used for the combination therapies described herein. Administration of a compound of the invention in such combination therapy may be determined as described herein.

The invention may be further understood in view of the following non-limiting examples.

Examples

EXAMPLE 1 chemical Synthesis of kinase inhibitors

¹H NMR spectra were recorded on a Varian 400 spectrometer with solvent as internal standard. Chemical shifts are expressed in ppm (δ). Unless otherwise specified, proton NMR chemical shift values are in deuterated CDCl₃Or DMSOd 6. ESI Mass Spectra (MS) were obtained on VG-Quattro II and PE-SEIEX (API) mass spectrometers. Thin layer chromatography was performed with fluorescent indicator on Merck Kieselgel silica60 plates coated with a 250 μm layer. UV light (λ 254nm) and or iodine vapor was used to visualize the components. Flash column chromatography was performed on 70-230 mesh 60 Å silica gel and on a combiflash (teledyne isco) using a RediSep flash column. All solvents used were the best anhydrous grade obtained from Aldrich. Analytical HPLC was performed on a Waters Breeze system with the following conditions and quoted as retention time in minutes (RT). The column used was a symmetrical C185 μm, 4.6X 150mm column (WAT 045905). All experiments with moisture sensitive compounds were performed under dry nitrogen or argon. Unless otherwise specified, the starting materials were obtained commercially (Aldrich, Fluka, Lancaster and TCI) and were the best grade and used without further purification. Where applicable, the organic solvent is replaced with anhydrous Na₂SO₄Drying and subjecting to Yamamoto RE500 rotary evaporator under 15-20mmHgIt is evaporated.

Example 2-preparation of 4-chloro-1, 2-dimethoxy-benzene 2 in scheme 1

One with thermometer, CaCl₂A500 mL three-necked flask with a guard tube and dropping funnel was placed at0 deg.C, 25g (23.06mL, 1 eq.) of veratrole 1 was added thereto, and then 24.42g (14.53mL, 1 eq.) of sulfonyl chloride was added dropwise thereto. When completely added, the reaction mixture was allowed to reach room temperature after 1 hour, it was distilled under reduced pressure (125 ℃ C. 1300 ℃ C.), and the resulting yellow oil was collected and dried to give Compound 2(27.8g, 89.6%) as a yellow liquid.

EXAMPLE 3 preparation of 1-chloro-4, 5-dimethoxy-2-nitrobenzene 3

In a 500mL three-necked flask equipped with a thermometer and a dropping funnel was charged 27.8g (1 equivalent) of 1, 4-chloro-1, 2-dimethoxybenzene 2, and 30.43g (3 equivalents, 20.4mL) of fuming nitric acid was added dropwise thereto while keeping the temperature thereof at not more than 25 ℃. When added completely, the reaction mixture was allowed to stand for 1.5 hours and the resulting solid compound 3 was treated with water, the yellow solid was filtered off and washed with water and dried (31.3g, 89.4%) to give some yellow solid.

EXAMPLE 4 preparation of ethyl 4-2-cyano-2- (4, 5-dimethoxy-2-nitrophenyl) acetate Prepare for

Potassium tert-butoxide 32.28g (2 equiv.) was added to an ice-cold solution of ethyl cyanoacetate 32.54g (30.61mL, 2 equiv.) in THF (250mL) and stirred for 15 min. To the white suspension was added 31.30g (1 equivalent) of Compound 3 (1-chloro-4, 5-dimethoxy-2-nitrobenzene), after which the reaction mixture was heated under reflux for 24 hours. The cooled reaction mixture was poured into water and extracted into ether and the solvent was evaporated. The crude compound ethyl 2-cyano-2- (4, 5-dimethoxy-2-nitrophenyl) acetate 4 was purified by flash column chromatography as a thick yellow oil (9.5g, 22.6%) before use in the next step.

EXAMPLE 5 preparation of ethyl 2-amino-5, 6-dimethoxy-1H-indole-3-carboxylate 5

A solution of 49.5g (1 eq) of ethyl 2-cyano-2- (4, 5-dimethoxy-2-nitrophenyl) acetate in 50mL of AcOH was reacted with 8.44g (4 eq) of Zn powder by heating at 65 ℃ for 12 hours. The reaction mixture was cooled and filtered with a filter aid, washed well with AcOH and the filtrate was concentrated to give a residue which was treated with water and extracted into dichloromethane which was purified by column chromatography (4.4g, 55%) as a brown solid.

Example 6-6, 7-dimethoxy-3H-pyrimido [4, 5-b]Preparation of indol-4 (9H) -one 6 Prepare for

A solution of ethyl 2-amino-5, 6-dimethoxy-1H-indole-3-carboxylate (5)4.4g (1 eq), NaOMe (900mg), and formamide (50ml) in N₂Followed by heating at 2200 ℃ for 2 hours. The solution was cooled, stored for 2.5 days and filtered. The solid separated from the formamide was filtered and washed with water, and dried to obtain compound 6(6, 7-dimethoxy-4-piperazin-1-yl-9, 9 a-dihydro-4 aH-pyrimido [4, 5-b) as a dark brown solid]Indole) purified by flash column chromatography as a dark brown solid (2.8g (70%).

Example 7-4-chloro-6J-dimethoxy-9, 9 a-dihydro-4 aH-pyrimido [4, 5-b]Indole Indole 7

The 4-chloro-tricyclic and quinazoline constructs were synthesized using literature methods (Pandey, A. et al, J.Med. chem.2002, 45: 3772-93; Matsuno, K. et al, J.Med. chem.2002, 45: 3057-66; Matsuno, K. et al, J.Med. chem.2002, 45: 4513-23; and Venugopalan, B. et al, J.heterocyclic. chem.1988, 25: 1633-39). Mixing Compound 6(2.8g) and POCl₃(20mL) and p-dioxane 65mThe suspension of L was heated under reflux for 6 hours. The resulting mixture was cooled and the solvent was evaporated. The crude product was purified by column chromatography eluting with 1% MeOH in DCM to give compound 7 as a pale yellow solid (2.2g, 73.3%).

Example 8-6, 7-dimethoxy-4- (piperazin-1-yl) -9H-pyrimido [4.5-b]Indoles 8

Compound 7 was dissolved in p-dioxane (50ml), to which piperazine (piprazine) (3.9g) was added at room temperature under argon, followed by pyridine (5 ml). The reaction mixture was heated to reflux for 16 hours and then allowed to cool. The solvent was removed under vacuum and the resulting crude product was purified by flash column chromatography using a solvent system of DCM and 10% MeOH. Compound 8 obtained after purification was a partially white solid (3.9g, 66.10%).

Example 9-preparation of N-acetyl-4-isothiocyanato-benzenesulfonamide 13 in scheme 2

The unsubstituted amine and or N-acetyl-4-amino-benzenesulfonamide are dissolved in DCM25mL and added to 0.934g CaCO dissolved in 15mL of water₃And 0.534ml of thiophosgene. The reaction mixture was stirred overnight. The resulting mixture was extracted into DCM and dried to give compound 13 as a white solid (0.462g, 38.6%).

Example 10-4- (6-chloro-7-trifluoromethyl-9H-pyrimido [4, 5-b ] in Table 1]Indoles -4-yl) -piperazine-1-thiocarboxylic acid (4-acetylsulfamoyl-benzene Preparation of the Compound 1

To the stirred compound; to a solution of 6-chloro-4- (piperazin-1-yl) -7- (trifluoromethyl) -9H-pyrimido [4, 5-b ] indole (prepared in a similar manner to that given in example 8) in DCM was added compound 13, to which was then added pyridine. The resulting reaction mixture was stirred at room temperature for 12 hours. After complete addition, the solvent was evaporated off. The crude product was purified by column chromatography using DCM and a solvent system of 5% MeOH (0.108g, 97%) as a white solid.

Example 11-4- (6, 7-dimethoxy-9H-pyrimido [4, 5-b ] in Table 1]Indole-4- Preparation of (4-acetylsulfamoyl-phenyl) -amide of yl) -piperazine-1-thiocarboxylic acid, compound 2 And (4) preparing.

To a stirred solution of compound 8 (prepared as described in example 8) in DCM was added compound 13, followed by pyridine. The resulting reaction mixture was stirred at room temperature for 12 hours. After complete addition, the solvent was evaporated off. The crude product was purified by column chromatography using a solvent system of DCM and 5% MeOH (0.043g, 59.1%, white solid form).

Example 12-4- (6-chloro-9H-pyrimido [4, 5-b ] in Table 1]Indol-4-yl) -piperazines Preparation of (4-acetylsulfamoyl-phenyl) -1-thiocarboxylic acid amide, compound 3

To a stirred solution of compound 6-chloro-4- (piperazin-1-yl) -9H-pyrimido [4, 5-b ] indole (prepared in a similar manner to that given in example 8) in DCM was added compound 13, to which was then added pyridine. The resulting reaction mixture was stirred at room temperature for 12 hours. After complete addition, the solvent was evaporated off. The crude product was purified by combiflash corporation using a solvent system of DCM and 10% MeOH (0.12g, 63.3%) as a white solid.

Example 13-preparation of 1- (3-Chloropropoxy) -4-chloro-2-methoxybenzene 15 according to scheme 2 Prepare for

The compound 4-chloro-2-methoxyphenol 14, cesium carbonate and 1-bromo-3-chloropropane were heated under reflux in acetonitrile for 1 hour. The reaction mixture was cooled and the solvent was evaporated. The residue obtained was dissolved in water (20ml) and extracted into DCM. The DCM layer was washed with brine and dried. The solvent was evaporated and the resulting solid was treated with ether, and the solid was collected to give compound 15(7.34g, 99%) as a pale yellow oil.

Example 14-1- (3- (4-chloro-2-methoxyphenoxy) propyl) -4-methyl in scheme 2 Preparation of the radical piperazine 17

Compound 15 was dissolved in acetonitrile and N-methylpiperazine (2 equivalents) was added thereto, and the resulting reaction mixture was heated to 70 ℃ for 8 hours. The reaction mixture was cooled and the solvent was evaporated. The residue was treated with ether and the precipitated solid was filtered off and dried to give a pale yellow-brown solid (5.9g, 63.2%) as a pale yellow-brown solid.

Example 15-1- (3- (4-chloro-2-methoxy-5-nitrophenoxy) propyl) -4-methyl Preparation of piperazine 18

Acetic acid was slowly added to the nitric acid at 5 ℃. To this mixture was added compound 17 in powder form and stirred for 15 minutes. The resulting reaction mixture was warmed to room temperature and stirred overnight. The solvent was evaporated, the viscous liquid poured into ice water and NaHCO₃The solution dilutes it. The resulting mixture was evaporated and purified by silica column chromatography eluting with 5% MeOH in dichloromethane (1.8g, 52.1%) as a yellow solid.

EXAMPLE 16 preparation of ethyl 2-cyano-2- (4-chloro-2-nitrophenyl) acetate

The compound 7- (3- (4-methylpiperazin-1-yl) propoxy) -6-methoxy-4- (piperazin-1-yl) -9H-pyrimido [4, 5-b ] indole was prepared using a method similar to that given for compounds 4,5, 6 and 7 (schemes 1 and 2).

Example 17 inhibition of Aurora-2 kinase Activity by MP277 and MP300

Exemplary compounds MP277 (Structure IV) and MP300 (Structure III) were evaluated in an aurora-2 kinase inhibition assay.

In this assay, luciferase-derived Light Units (LU) are measured by luminometer and kinase activity is determined by quantifying the amount of ATP remaining in solution after a kinase reaction. The percent inhibition of each compound was determined by comparing the luminometer reading of the drug-treated reaction with a control containing no drug (DMSO control) and a control containing no Aurora-2 enzyme (ATP control) using the following equation:

recombinant aurora-2 kinase (Upstate, Lake plasmid, NY) produced in sf9 cells was reacted in a 50 μ l reaction at 30 ℃ with 62.5 μm Kenpeptide (Calbiochem, San Diego, Calif.), 3 μm ATP (Invitrogen, Carlsbad, Calif.) and kinase reaction buffer (40mM Tris-HCl, 10mM MgCl. RTM.)₂And 0.1. mu.g/. mu.l Bovine Serum Albumin (BSA)) for 2 hours. This reaction is carried out in the presence of a drug that has been previously diluted with DMSO to the desired concentration. After incubation, 50 μ l of Kinase-Glo ® (Promega, Inc., Madison, Wis.) solution was added to each reaction mixture and allowed to equilibrate for 10 minutes at room temperature. The Kinase-Glo solution contains luciferase and luciferin, which react with ATP to produce light. Luciferase-derived Light Units (LU) were measured using a luminometer (Thermo-Electron, Vantaa, Finland) and the kinase activity was determined by quantifying the amount of ATP remaining in the solution after the kinase reaction.

Determination of the drug concentration (IC) of the exemplary Compounds MP277 and MP300 whose 50% aurora-2 kinase Activity is inhibited₅₀). IC of MP277₅₀The content of the active carbon was 0.049. mu.M,and IC of MP300₅₀< 0.005. mu.M. Such inhibitory activity of MP277 and MP300 is unexpectedly high, in particular, for example, compared to the significantly lower activity levels observed for structurally related compounds like those in which the structural groups present on MP277 and MP300 are:

a compound substituted by one of the following structures:

thus, the inhibitory activity of exemplary compounds of the invention, such as MP277 and MP300, against aurora-2 kinase is significantly higher than that of other structurally related compounds.

Example 18-MP277 induced cytotoxicity of cancer cells

To evaluate the cytocidal power against cancer cell lines, an in vitro cytotoxicity assay was performed. Tumor cell lines used were purchased from the American Type Culture Collection and were determined as follows: panc-1 (pancreas), MiaPaCa-2 (pancreas), MCF-7 (breast), HT-29 (colon), U2-OS (osteosarcoma), OVCAR-3 (ovary), HepG2 (hepatocellular carcinoma) and TT (medullary thyroid). The Cell-Titer-Glo non-radioactive Cell proliferation assay (Promega corp., Madison, W1) was used for this assay. First, these cells were cultured in RPMI1640 medium (Cat #21870-076, Invitrogen corporation) supplemented with 300mg/L L-glutamine, 100 units/ml penicillin, 100. mu.g/ml streptomycin, and 10% fetal bovine serum. All cell lines were cultured at 37 ℃ in a humidified thermostat with an atmosphere of 5% CO 2.

On day 0, these cells were plated at a density of 2000 to 10000 cells per well, depending on their growth rate, into 0.09mL of medium in a 96-well Microlite TCT microtiter plate (7418, Thermo Labsystems, Franklin, Mass.). On day 1, 10 μ l serial dilutions of each compound were added to the plate in triplicate. After culturing it at 37 ℃ for 4 days in a humidified thermostat, these cells were lysed in Cell-Titer-Glo reagent (which also contains luciferase). The luciferase reaction uses ATP released from lysed cells to generate light, the intensity of which is linear to the amount of ATP. Thus, the amount of light generated is a reflection of the number of cells remaining in the well after treatment with the drug. This luminescence was measured with a Luminoskan luminometer (Thermo electron corp., Vantaa, Finland). Data are expressed as percent control cell survival calculated from luminescence corrected for background. Percent survival of cells was determined by dividing the mean luminescence of treated cells by the mean luminescence of controls and multiplying by 100.

Calculated IC of MP277 for the following cell lines₅₀The value: panc-1, MiaPaCa-2, MCF-7, HT-29, U2-OS, OVCAR-3, HepG2 and TT are as follows: 40.67. mu.M, 66.59. mu.M, 22.46. mu.M, 14.65. mu.M, 25.93. mu.M, 24.97. mu.M, 7.83. mu.M and 51.67. mu.M. As noted above, the level of activity of MP277 is unexpectedly high relative to the level of activity observed for structurally related compounds.

Example 19 in vivo inhibition of tumor growth by MP277

To evaluate the efficacy of MP277 on tumor cells in vivo systems, a xenograft study was performed in mice. Will be 1 × 10⁷One HT-29 human colon cancer cell was injected subcutaneously into 16 Nu/Nu athymic nude mice (Charles river laboratories, Wilmington, Mass.). According to the formula ((Wide)²Length)/2 to measure tumor volume. Growing the tumor volume to about 100mm³(day 0), at which time, the mice were randomized into two groups: 8 mice were treated with 25mg/kgMP277, and 8 additional mice were givenAn equal volume of drug matrix was used. For this study, the drug matrix used was 60% propylene glycol, 30% polyethylene glycol 300, 10% ethanol with 150mg/mL 2-hydroxypropyl- β -cyclodextrin. Each mouse received 0.1ml of the drug or matrix intraperitoneally according to a schedule of q.d.x5 for two weeks with a two-day rest period between each week. During the duration of this study, no significant toxicity was noted with either the drug or the matrix. In this way, MP277 was found to be effective in inhibiting tumor growth in vivo, the results of which are shown in FIG. 1.

Example 20-assay with Aurora-2 kinase and cancer cell-based cytotoxicity assay Assays to determine the Activity of exemplary Compounds

The exemplified compounds described herein were evaluated using an aurora-2 kinase inhibition assay, particularly the assay described in example 17 above. The compounds tested in this assay included compounds 1, 2, 3, 4, 8, 26, 34, 42, 107 and 115 described in table 1 above.

Briefly, the Light Units (LU) produced by luciferase are measured using a luminometer and kinase activity is determined by quantifying the amount of ATP remaining in solution after a kinase reaction. The percent inhibition of each compound was determined by comparing the luminometer reading of the drug-treated reaction with a control containing no drug (DMSO control) and a control containing no Aurora-2 enzyme (ATP control) using the following equation:

recombinant aurora-2 kinase (NY) produced in sf9 cells was reacted in a 50 μ l reaction at 30 ℃ with 62.5 μm Kenprox peptide (Calbiochem, San Diego, Calif.), 3 μm ATP (lnvitrogen, Carlsbad, Calif.) and kinase reaction buffer (40mM Tris-HCl, 10mM MgCl. RTM.)₂And Bovine Serum Albumin (BSA) 0.1. mu.g/. mu.l 2And (4) hours. This reaction is carried out in the presence of a drug that has been previously diluted with DMSO to the desired concentration. After incubation, 50 μ l of kinase-Glo ® (Promega, inc., Madison, W1) solution was added to each reaction mixture and allowed to equilibrate for 10 minutes at room temperature. The Kinase-Glo solution contains luciferase and luciferin, which react with ATP to produce light. Luciferase-derived Light Units (LU) were measured using a luminometer (Thermo-Electron, Vantaa, Finland) and the kinase activity was determined by quantifying the amount of ATP remaining in the solution after the kinase reaction. IC of test Compounds₅₀The values are as in the following Table 2 "IC₅₀Under the heading A2K ".

In addition, to further evaluate the cytotoxic activity of the exemplary agents on cell lines, an in vitro cytotoxicity assay was conducted, which was essentially as described above in example 18. The compounds tested in this assay included compounds 1, 2, 3, 4, 8, 26, 34, 42, 107 and 115 described in table 1 above.

Briefly, the tumor cell lines used were purchased from the American Type CultureCollection and were determined as follows: panc-1 (pancreas), MiaPaCa-2 (pancreas), MCF-7 (breast), HT-29 (colon), U2-OS (osteosarcoma), OVCAR-3 (ovary), HepG2 (hepatocellular carcinoma) and TT (medullary thyroid), PC-3 (prostate) and A549 (lung). The Cell-Titer-Glo non-radioactive Cell proliferation assay (Promega corp., Madison, W1) was used for this assay. First, these cells were cultured in RPMI1640 medium (Cat #21870-076, Invitrogen Corporation) supplemented with 300mg/L L-glutamine, 100 units/ml penicillin, 100. mu.g/ml streptomycin and 10% fetal bovine serum. All cell lines were incubated at 37 ℃ with 5% CO₂The atmosphere was incubated in a humidified thermostat.

On day 0, these cells were plated at a density of 2000 to 10000 cells per well, depending on their growth rate, into 0.09mL of medium in a 96-well Microlite TCT microtiter plate (7418, Thermo Labsystems, Franklin, Mass.). On day 1, 10 μ l serial dilutions of each compound were added to the plate in triplicate. After culturing it at 37 ℃ for 4 days in a humidified thermostat, these cells were lysed in Cell-Titer-Glo reagent (which also contains luciferase). The luciferase reaction uses ATP released from lysed cells to generate light, the intensity of which is linear to the amount of ATP. Thus, the amount of light generated is a reflection of the number of cells remaining in the well after treatment with the drug. This luminescence was measured with a Luminoskan luminometer (Thermo electron corp., Vantaa, Finland). Data are expressed as percent control cell survival calculated from luminescence corrected for background. Percent survival of cells was determined by dividing the mean luminescence of treated cells by the mean luminescence of controls and multiplying by 100.

Calculated IC of each test compound against various cancer cell lines₅₀The values are shown in table 2 below:

TABLE 2

Cell-based IC₅₀Value (μ M)

Compound #	IC50A2K	Panc-1	MiaPaCa-2	MCF-7	HT-29	U2-Os	OVCAR-3	HepG2	Pc-3	A549	TT
												1	0.49	37.50	18.61	14.60	5.01	13.15	13.07	25.97	52 29	53.78	11.00
2	0.005	197.66	172.64	125.59	93.60	71.72	29.40	294.69	164.04	194 06	63.97
												3	0.074	35.76	22.11	208.85	29.80	20.83	47.98	49.55	47.29	37 03	13.20
4	0.023	136.13	85.19	248.94	103 20	36.29	79.04	88.00	159.70	72.69
												8	0.018			83.10	27.31		82 37	81.79
26	0.317	138.89	8.92	14.66	176.45	6.95	156.29	4.38	22.31	129.83
												34	2.09	125 32	9.82	76.91	138 37	27.01	110.86	81.93	21.93	186.24	99.13
42	4.97	216.22	182.29	4.62	139.94	41.33	85.06	98.49	189.38	160.03	128.76
												107	0.586	300.00	107.78	＞300	＞300	8.70	175.57	172.93	2.33	51 52	146.65
115	0.743	49.82	55.35	87.84	97.42	50.37	174.69	57.93	75.68	149.05	36.63

Any US patents, US patent application publications, US patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the application data sheet are incorporated herein by reference in their entirety.

From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.

Claims (20)

1. A compound having the following structure (I):

including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein:

x is NH, S or O;

z is CH or N;

R₁and R₂Are the same or different and independentGround is hydrogen, hydroxy, halo, -CN, -NO₂、-NH₂、-R、-OR、-SCH₃、-CF₃-C (═ O) OR, -OC (═ O) R, where R is alkyl OR substituted alkyl; or-O (CH)₂)_n-R_xWherein n is 2-4 and R_xIs N-methylpiperazine, morpholine or 2-methylpyrrolidine;

R₃is hydrogen, -NH₂Alkyl, -CN, or-NO₂Or R is₃is-L₃-CyCl₃Wherein L is₃Is a direct bond, -S-or-NH-, and Cycl₃Is a carbocyclic ring, a substituted carbocyclic ring, a heterocyclic ring or a substituted heterocyclic ring;

L₂are-C (═ S) NH-, -NHC (═ S) -, -NHC (═ S) NH-, -C (═ O) NH-, -NHC (═ O) -, -NHC (═ O) NH-, - (CH (O) NH-, - (CH)₂)_n-、-NH(CH₂)_n-、-(CH₂)_nNH-、-NH(CH₂)_nNH-、-C(＝S)NH(CH₂)_n-、-NHC(＝S)(CH₂)n-、-(CH₂)_nC(＝S)NH(CH₂)_n-、-(CH₂)_nNHC(＝S)(CH₂)_n-、-NHC(O)-、-S(O)₂-、-S(＝O)₂NH-、-NHS(＝O)₂-wherein n is the same or different at each occurrence and is independently 1, 2, 3 or 4; and

w is-S (O)₂NHC(O)CH₃、-NHC(O)R_y、-NHS(O)₂R_yWherein R is_yIs alkyl or cycloalkyl, -NH₂、-NH₂HCl, and-S (O)₂-R_zWherein R is_zSelected from the group consisting of alkyl, substituted alkyl, amine, N-methylpiperazine, morpholine, and 2-methylpyrrolidine.

2. The compound of claim 1, wherein X is NH and Z is CH.

3. The compound of claim 1, wherein R₁、R₂And R₃Selected from hydrogen, -NH₂、-OCH₃、-OH、-CF₃Halo, or-O (CH)₂)_n-R_xWherein n is 2-4 and R_xIs N-methylpiperazine, morpholine or 2-methylpyrrolidine.

4. The compound of claim 1, wherein L₂is-C (═ S) NH-.

5. The compound of claim 1, wherein w is-S (═ O)₂NHC(＝O)CH₃。

6. The compound of claim 1, wherein w is-S (═ O)₂NHC(＝O)CH₃、-S(O)₂NH₂or-S (O)₂CH₃。

7. The compound of claim 1, wherein R₁、R₂And R₃Selected from hydrogen, -NH₂、-OCH₃、-OH、-CF₃Halo, or-O (CH)₂)_n-R_xWherein n is 2-4 and R_xIs N-methylpiperazine, morpholine or 2-methylpyrrolidine and w is-S (O)₂NHC(O)CH₃、-S(O)₂NH₂or-S (O)₂CH₃。

8. The compound of claim 1, wherein R₁And R₂Selected from hydrogen, halo, -CF₃or-OH, R₃Is hydrogen and w is-S (O)₂NHC(O)CH₃、-S(O)₂NH₂or-S (O)₂CH₃。

9. The compound of claim 1, wherein X is NH, Z is CH, L₂is-C (═ S) NH-, and the compound has the following structure (II):

10. the compound of claim 9, wherein R₃Is hydrogen and R₁And R₂Is selected from-OCH₃、-OH、-CF₃Halo, or-O (CH)₂)_n-R_xWherein n is 2-4 and R_xIs N-methylpiperazine, morpholine or 2-methylpyrrolidine.

11. The compound of claim 9, wherein R₁And R₂Is selected from-OCH₃、-OH、-CF₃Or halo, and R₃Is hydrogen.

12. The compound of claim 9, wherein w is-S (═ O)₂NHC(＝O)CH₃、-S(＝O)₂NH₂or-S (═ O)₂CH₃。

13. The compound of claim 9, wherein R₁And R₂Is selected from-OCH₃、-OH、-CF₃Or halo, R₃Is hydrogen, and w is-S (O)₂N HC(＝O)CH₃、-S(＝O)₂NH₂or-S (═ O)₂CH₃。

14. The compound of claim 9, wherein R₁And R₂Is selected from-OCH₃、-OH、-CF₃Or halo, R₃Is hydrogen, and w is-S (═ O)₂NHC(＝O)CH₃、-S(＝O)₂NH₂or-S (═ O)₂CH₃。

15. The compound of claim 9, wherein R₁And R₂Is methoxy, R₃Is hydrogen, w is-S (═ O)₂NHC(＝O)CH₃And the compound has the following structure(III)：

16. The compound of claim 9, wherein R₁is-Cl, R₂is-CF₃，R₃Is hydrogen, w is-S (═ O)₂NHC(＝O)CH₃And the compound has the following structure (IV):

17. a composition comprising a compound according to any one of claims 1-16 and a pharmaceutically acceptable excipient.

18. A method of treating a protein kinase-mediated disease, comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 17.

19. The method of claim 18, wherein the protein-kinase mediated disease is cancer.

20. The method of claim 18, wherein the cancer is a cancer of the pancreas, breast, ovary, colon, liver, thyroid, prostate, lung, or bone.