Title of Invention	ARYL AND HETEROARYL UREA CHK1 INHIBITORS FOR USE AS RADIOSENSITIZERS AND CHEMOSENSITIZERS
Abstract	The present invention relates to a aryl- and heteroaryl-substituted urea compounds useful in the treatment of diseases and conditions related to DNA damage or lesions in DNA replication are disclosed. Methods of making the compounds and their use as therapeutic agents, for example, in treating cancer and other diseases characterized by detects in DNA replication, chromosome segregation, or cell division also are disclosed.

Title of Invention

ARYL AND HETEROARYL UREA CHK1 INHIBITORS FOR USE AS RADIOSENSITIZERS AND CHEMOSENSITIZERS

Abstract

The present invention relates to a aryl- and heteroaryl-substituted urea compounds useful in the treatment of diseases and conditions related to DNA damage or lesions in DNA replication are disclosed. Methods of making the compounds and their use as therapeutic agents, for example, in treating cancer and other diseases characterized by detects in DNA replication, chromosome segregation, or cell division also are disclosed.

Full Text	RYL AND HETEROARYL UREA CHKE INHINBOTORS FOR USE AS RADIOSENSITIZERS AND CHAMOSESITITZERS CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit o f U.S. provisional application Serial No. 60/273,124, filed March 2, 2001. TECHNICAL FIELD OF THE INVENTION The present invention relates to compounds useful for inhibiting enzymes chat maintain and repair the integrity of genetic material. More particularly, the present invention relates to a series of aryl- and heteroaryl-substituted urea compounds, methods of making the compounds, and their use as therapeutic agents, for example, in treating cancer and other diseases characterized by defects in deoxyribonucleic acid (DNA) replication, chromosome segregation, or cell division. BACKGROUND OF THE INVENTION An important and significant goal in healthcare is to discover and make available safer and more effective drugs for the treatment of cancer. Most chemotherapeutic agents act by disrupting DNA metabolism, DNA synthesis, DNA transcription, or microtubule spindle function, or by perturbing chromosomal structural integrity by introducing DNA lesions. These processes affect both normal and tumor cells. The maintenance of DNA integrity is essential to cell viability in normal cells, therefore, anticancer drugs have the lowest therapeutic index of any drug class. An individual cell creates an exact copy of its chromosomes, .,and then segregates each copy into two cells by a process called mitosis. The mototic cycle can be divided into three major events: DNA replication, chromosome segregation, and cell division. Cells have sensing mechanism to maintain the order of these steps with respect to one another and to ensure that each step is executed with high fidelity. The sensing mechanisms for these processes are referred to as "checkpoints" in L.H, Hartwell et al.. Science, Nov. 3, 1989, 246 (4530) :629-34. Cell cycle checkpoints have been reported to comprise at least three distinct classes of polypeptides- Each class of polypeptides acts sequentially in response to cell cycle signals or defects in chromosomal mechanisms (Carr, (1995) Science, 271:314-315). One family of proteins detects or senses DNA damage or abnormialities in the cell cycle. These sensors include Ataxia-Telangiectasia Mutated (Atm) and Ataxia-Telangiectasia Rad-related (Atr) (Keegan et al., (1996) Genes Dev. , 10:2423-2437). Another class of polypeptides amplify and transmit the signal detected by the detector and is exemplified by Rad53 (Allen et al . (1954) Genes Dev. , 8:2416-2488) and Chkl. In addition, cell cycle effectors, such as p53, mediate a cellular response, including, for example, arrest of mitosis and/or meiosis and apoptosis. DNA damage can be induced by drugs, radiation, or can be spontaneously generated during the course of normal metabolism. DNA damage checkpoints ensure that cells with unrepaired DNA lesions do not progress into the DNA synthesis phase or mitosis until chromosomal lesions have been removed. Cell —^'-^ cycle arrest can enhance the opportunity for DnA repair and increase the fidelity of cell divisi DNA damage can be recognized throughout the cell cycle. Checkpoints ensure that the growth of cells is arrested at multiple cell cycle phases. As a result, multiple cell cycle signaling pathways may result during sensitization of cells to DNA damaging agents. Much of the current understanding of the function of cell cycle checkpoints has been derived from the study of tumor-derived cell lines. In many cases, tumor cells have lost key cell cycle checkpoints (Hartwell et al. , Science, Dec. 16, 1994 ; 266(5192): 1821-8). It has been reported that a key step in the evolution of cells to a neoplastic state is the acquisition of mutations that inactivate cell cycle checkpoint pathways, such as p53. (Weinberg, R.A- (1995) Cell 81:323-330; Levine, A. J. (1997) Cell 88: 3234-331). Loss of these cell cycle checkpoints results in the inappropriate cycling of tumor cells in response to DNA damaging agents. When faced with cellular stresses, such as DNA damage, and cell cycle events with decreased fidelity, tumor cells have difficulty altering the kinetics of ceil cycle progression. Therefore, inhibition and disruption of additional DNA damage checkpoint pathways may further sensitize tumor cells to anticancer treatments, such as radiation and chemotherapy. Noncancerous tissue, which has intact cell cycle checkpoints, typically is insulated from temporary disruption of a single checkpoint pathway. Tumor cells, however, have defects in pathways controlling cell cycle progression such that the perturbation of additional cherkpoints, for example, the DNA damage checkpoint, renders them particularly sensitive to DNA damaging agents. For example, tumor cells that contain mutant p53 are defective both in the Gl DNA damage checkpoint and in the ability to maintain the G2 DNA damage checkpoint. (Bunz et al., Science, Nov. 20, 1998; 282(5393): 1497-501; Levine). Checkpoint inhibitors that target initiation of the G2 checkpoint or the S phase checkpoint are expected to further cripple the ability of these tumor cells to repair DNA damage and, therefore, selectively kill them over normal cells. Therefore, checkpoint inhibitors are expected to enhance the therapeutic index, which is a measure of the probability of tumor control relative to the probability of toxicity to normal tissue, of both radiation and systemic chemotherapy. The ability of checkpoint inhibitors to enhance the therapeutic index may be dependent upon tumor type. Tumors with cell cycle defects complementary to the DNA damage checkpoint pathways may be most sensitive to inhibitor drug treatment. In contrast, DNA-PK inhibitors, another distinct class of potential therapeutic agents, are expected to sensitize tumors independently of cell type. A systematic approach of applying checkpoint inhibitors and DNA-PK inhibitors also may be effective in the treatment of metastatic diseases that radi.ation therapy cannot target. The checkpoint proteins Atm and Atr are hypothesized to initiate a signal transduction pathway leading to cell cycle arrest in the presence of DNA damage or any block to DNA replication. Atm has been shown to play a role in a DNA damage checkpoint in response to ionizing radiation (IR). Patients lacking functional Atm develop the disease Atakia-Telangiectasia (A-T). Symptoms of A-T include ex- treme sensitivity to ionizing radiation (IR) , cerebellar degeneration, oculotaneous telangiectasias, gonadal deficiencies, immunodeficiencies and increased risk of cancer (Shiioh, Eur. J. Hum. Genet 1995; 3 (2) : 116-38) - Fibroblasts derived from these patients are thought to have defects in Gl, S, and G2 checkpoints and are defective in their response to IR (Kastan et al., Cell, Nov. 13, 1992; 71(4); 587-97; Scott et al., Int. J, Radiat. Biol., Dec, 1994; 66(5 Suppl): S157-63; and Beamish et al. , J. Biol. Chem. Aug. 26, 1993; 271(34):20486-93). Therefore, Atm may sense double-strand DNA damage caused by IR and radiomimetic drugs, and signal the cell cycle to arrest, such that damage can be repaired. Atr is a checkpoint protein stimulated by agents that cause double strand DNA breaks, single strand DNA breaks, and agents that block DNA radiation. Overexpression of Atr in muscle cells on iso-chromosome 3q results in a block to differentiation, abnormal centrosome numbers, chromosome instability, and abolishes the Gl arrest in response to IR (Smith et al., Nat. Genet., May 1993; 19(1): 39-46). Overexpression of a kinase inactive, dominant negative mutant of Atr sensitizes cells to IR, ultraviolet light (UV) , MMS, and cisplatin (Cliby et al., EMBO J, Jan. 2, 1998, 17(l):159-69 and Wright et al. , Proc. Nat'l Acad. Sci. U.S.A., June 23, 1998; 95 (13) :7445-50) . Cells containing overexpressed, mutant strain Atr also fail to arres. in 02 in response to IR. In addition, Atr is associated witn chromosomes in meiotic cells where DNA breaks and abnormal DNA structures persist as a result of the process of meiotic recombination** (Keegan et al. , Genes Dev. October 1, 1996; 10(19): 433-37) . Atr, like Atm, also senses DNA damage and agents that block DNA replication, as well as initiates a cell cycle arrest at G2 and S for DNA repair. Chkl is hypothesized to lie downstream from protein kinases Atm and/or Atr in the DNA damage checkpoint signal transduction pathway. {See, Sanchez et al., Science, 1997; 277:1497-1501; U.S. Patent No. 6,218,109.) In mammalian cells, Chkl is phosphorylated in response to agents that cause DNA damage including IR, UV, and hydroxyurea (Sanchez et al. , 1997; Lui et al. , Genes Dev. 2000; 14:1443-1459). The phosphorylation and activation of Chkl in mammalian cells is dependent on Atm (Chen et al., 1999) and Atr (Lui et al. , 2000). In the yeast S. poinbe, Chkl also appears to be involved in the response to IR and blocks to replication (3oddy et al. , 1998; Walworth et al, , 1993). Furthermore, Chkl has been shown to phosphorylate both weel (O'Connell et al. , EMSO J. 1997; 15:545-554) and Pdsl (Sanchez et al., Science 1999; 235:1166-1171) gene products known to be important in cell cycle control. These studies demonstrate that mammalian Chkl plays a role in both the Atm-dependent DNA damage checkpoint leading to arrest at S phase. However, a role for Chkl in the S phase replication checkpoint in mammalian cells has yet to be elucidated. Interestingly, Chkl knockout mice are embryonically lethal, thereby suggesting a role for Chkl in a developing organism in addition to its role in DNA damage checkpoints. Chkl may invoke a G2 arrest by phosphor-ylating and inactivating Cdc25C, the dual specificity phosphatase that normally dephosphorylates cyclin B/cdc2 as cells progress into mitosis (Fernery et al., Science, Sep. 5, 19S7; 277(5351}: 1495-7; Sanchez et al,; Matsuoka et al,; and Blasina et al., Curr. Sioi., Jan. 14, 1599; 9(1):1-1C). This mechanism of regulation of Cdc2 activity stimulates cell cycle arrest to prevent cells from entering mitosis in the presence of DNA damage or unreplicated DNA. SUMMARY OF THE INVENTION The present invention is directed to potent and selective chemosensitizing agents useful in the treatment of diseases and conditions related to DNA damage or lesions in DNA replication. The present compounds are inhibitors of the checkpoint kinase Chkl. In particular, aryl- and heteroaryl substituted urea comoounds have demonstrated siq-nificant activity for inhibiting Chkl. In one aspect, the present invention is directed to a method of inhibiting checkpoint kinase Chkl comprising the step of administering a compound of formula (I), or a composition containing the same, to an individual. Compounds of formula (I) have a structural formula: wherein: together to form an optionally substituted 3- to £-membered aliphatic ring; R6 is selected from the group consisting of halo and C1-6alkyl, and pharmaceutically acceptable salts or solvates thereof. In another aspect, the present invention is directed to aryl- and heteroaryl-disubstituted urea compounds having a structural formula (II) optionally substituted with from one to four sub-stituents selected from the group consisting of C1-6alkyl, aryl, N(R7),, OR7, N3, CN, C(O)R7 C1-2alk-ylenearyl, C1-3alkyleneN (R12) 2, and pharmaceutically acceptable salts or solvates thereof. Another aspect cf the present invention relates to carbamide-substituted heteroaryl groups having the structural formula (III) wherein W" is selected from the group consisting of heteroaryl, aryl, heterocycloalkyl, cycloalkyl,_. and C1-3 alkyl substituted with a heteroaryl or aryl group; R15 is selected from the group consisting of halo and C1-6alkyl. The present invention also is directed to pharmaceutical compositions containing one or more compounds of structural formula (II), to use of the compounds and compositions containing the compounds in therapeutic treatment of a disease or disorder, and ro methods of preparing the compounds and intermediates involved in the synthesis of the compounds of structural formula (II). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Radiation and most chemotherapeutic agents are therapeutically beneficial because they take advantage of inappropriate tumor cell proliferation. Cellular processes, such as DNA damage repair, and cell cycle checkpoints, protect tumor cells from the toxic effects of physical and chemical agents. , Treatments that modulate the underlying molecular mechanisms of cell cycle progression and resistance to DNA damage can potentiate tumor cell killing and enhance the therapeutic index of existing therapies. Most chemotherapeutic agents act by disrupting DNA metabolism.. Because these processes are shared by both normal and tumor cells, and because the maintenance of DNA integrity is essential to cell viability, anticancer drugs have the lowest therapeutic index of any drug class. By identifying and inhibiting cellular processes that tumor cells rely upon, the effectiveness of radiation and chemotherapy treatment regimens can be enhanced. The interruption of the DNA damage checkpoint protein function provides a novel means of killing tumor cells relative to normal cells. For example, Chkl ensures that cells with unrepaired DNA lesions caused by certain drugs or radiation do not progress through DNA synthesis phase or mitosis until chromosomal lesions have been removed. Accordingly, a tumor cell treated with a Chkl inhibitor in combination with a DNA damaging agent can kill using lower amounts of DNA damaging agent than tumor cells treated with the DNA damaging agent alone. Failure of cell cycle checkpoints in normal cells predisposes an individual to, or directly causes, many disease states, such as cancer, ataxia telangiectasia, embryo abnontalities, and various immunological defects associated with aberrant 3 and T cell development. The latter are associated with the pathological states of lupus, arthritis, and autoimmune diseases. intense research efforts, tnerefore, have focused on identifying cell cycle checkpoints and the proteins essential for the function of the checkpoints. Noncancerous tissue having intact cell checkpoints typically is insulated from temporary disruption of a single checkpoint pathway, such as the Chkl pathway- Tumor cells, however, have multiple defects in pathways controlling cell cycle progression such that perturbation of the DNA dam.age checkpoint can render cells particularly sensitive to DNA damaging agents. Therefore, checkpoint inhibitors are expected to enhance the therapeutic index, which is a measure of the probability of tumor control relative to the probability of toxicity to normal tissue to radiation and systemic chemotherapy. In contrast, other classes of inhibitors may not be amenable to combination chemotherapy because both normal and tumor tissue may be similarly sensitized. One aspect of the present invention is directed to a method of inhibiting Chkl, comprising the step of adminisuering a therapeutically effective amount of a compound of formula (I) , or a composition containing the same, to an individual. Compounds of formula (I) have a structural formula wherein X1 is null, -O-, -S-, -CK.-, or -N(R-).; X2 is -O-, -S-,.or -N(R-)-; Y is 0 or S; or =Y represents two hydrogen atoms attached to a common carbon atom; W is selected from the group consisting of heteroaryl, aryl, heterocycloalkyl, cycloalkyl, and C1-3 alkyl substituted with a heteroaryl or aryl group; and Z is selected from the group consisting of hydrogen, aryl, and heteroaryl; wherein said aryl groups of w and Z are optionally substituted with one to four substituents represented by R3, said heteroaryl groups of w and Z are optionally substituted with one to four substituents represented by R5, and said heterocycloalkyl and cycloalkyl groups of W are optionally substituted with one to two substituents represented by R6; R1 is selected from the group consisting of hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, and aryl; R6 is selected from the group consisting of halo and C1-6alkyl, and pharmaceutically acceptable salts or solvates thereof. Preferred compounds used in the method are those 1herein X1 and X2 are -N(H)-; Y is O or S; W is heteroaryl containing at least two heteroatoms selected from the group consisting of N, 0, and S, said ring is optionally substituted with from one to four substituents selected from the group consisting of C1-6alkyl, aryl, N(R3)2:., OR3, and halo, wherein R3 is as previously defined; Z is selected from the group consisting of: Additional preferred compounds of formula (I) are those wherein W is selected from the group consisting of pyridazine, pyrimidine, pyrazine, and triazine, optionally substituted with from one to four substituents selected from the group consisting of optionally substituted C1-6alkyl, aryl, N(R3),, OR3 Compounds preferred for use in the method also include those of formula (I) wherein J is se- The method of inhibiting Chkl also can be used to sensitize a tumor cell to a chemotherapeutic agent. As such, the present invention also is directed to a method of sensitizing a tumor cell to a chemotherapeutic agent corrprising administering a compound of formula (I), or a salt, solvate, or derivative thereof, or a composition comprising the same, to an individual. In another aspect, the present invention is directed to aryl- and heteroaryl-disubstituted urea compounds having a structural formula (II) optionally substituted with from one to four sub-stituents selected from the group consisting of C1-6alkyl, aryl, N(R7)2, OR7 N3, CN, C(O)R7, C1-3alk-ylenearyl, C1-3alkylene N(R12) n, Z' is selected from the group consisting of: and pharmaceutically acceptable salts cr solvares thereof. w optionally substituted with one to four substituents selected from the group consisting of C1-6alkyl, optionally substituted aryl, N(R3);, CF3, C(O)R7, N3, CN, C1-3alkylenearyl, C1-3alkyleneN(R12)., OR7, halo, wherein R7, Y and Z are as previously defined. More preferred compounds of formula (11) are those wherein: The term "alkylene" refers to an alkyl group having a substituent. For example, the term "C1-3alkyleneC (O)R" refers to an alkyl group containing one to three carbon atoms substituted with a -C(O)OR group. The alkylene group is optionally substituted with one or more of aryl, heteroaryl, and OR"', wherein R' is defined hereafter. The term "halo" or "halogen" is defined herein to include fluorine, bromine, chlorine, and iodine. When no substituent is indicated as attached to a carbon atom on a ring, it is understood that the carbon atom contains the appropriate number of hydrogen atoms. In addition, when nc substituent is indicated as attached to a carbonyl group or a nitrogen atom, for example, the substituent is understood to be hydrogen, e.g.. The abbreviation "Me" is methyl. The abbreviation to and C (O) is carbonyl (C=(O)) . The notation N(Rx)n, wherein x represents an alpha or numeric character, such as for example Ra, Rb, R4, R12, and the. like, is used to denote two Rx groups attached to a common nitrogen atom. When used in such notation, the Rx group can be the same or different, and is selected from the group as defined by the Rx group. The present invention also is directed to pharmaceutical compositions containing one or more compounds of structural formula (II) and (III), to use of the compounds and compositions containing the compounds in therapeutic treatment of a disease or disorder, and to methods of preparing the compounds and intermediates involved in the synthesis of "he compounds of structural formula (II) and (III) - Compounds useful for the method of the present invention have demonstrated activity in inhibiting Chkl in vitro. Compounds of the present invention have demonstrated selectivity for Chkl as against other protein kinases including Cdc2, Chk2, Atr, DNA-PK, PKA, and CaM KII. Compounds of the present invention can be used to potentiate the therapeutic effects of radiation and/or chemotherapeutics used in the treatment of cancers and other cell proliferation disorders in humans or animals. For example, compounds of the invention can be used to enhance treatment of tumors that are customarily treated with an antimetabolite, e.g., methotrexate or 5-fluorouracil (5-FU). The method of the present invention comprises administration of a Chkl inhibitor compound in combination with a chemotherapeutic agent that can effect single- or double-strand DNA breaks or that can block DNA replication or cell proliferation. Alternatively, the method of the present invention comprises administration of a Chkl inhibitor compound in combination with therapies that include use of an antibody, e.g., herceptin, that has activity in inhibiting the proliferation of cancer cells. Accordingly, cancers such as colorectal cancers, head and neck cancers, pancreatic cancers, breast cancers, gastric cancers, bladder cancers, vulvar cancers, leukemias, lymphomas, melanomas, renal cell carcinomas, ovarian cancers, brain tumors, osteo-sarcomas, and lung carcinomas, are susceptible to enhanced treatmenu in combination with the Chkl inhibitors of the invention. Tumors or neoplasms include growths of tissue cells wherein multiplication of cells is uncontrolled and progressive. Some such growths are benign, but others are termed "malignant," and can lead to death of the organism. Malignant neoplasms, or "cancers," are distinguished from benign growths in that, in addition to exhibiting aggressive cellular proliferation, can invade surrounding tissues and metastasize. Moreover, malignant neoplasms are characterized by shewing a greater loss of differentiation (greater "dedifferentiation") and organization relative to one another and surrounding tissues. This property is called "anaplasia." Neoplasms treatable by the present invention also include solid tumors, i.e., carcinomas and sarcomas. Carcinomas include malignant neoplasms derived from epithelial cells which infiitrace (i.e., invade) surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas derived from glandular tissue, or from tissues that form recognizable glandular structures. Another broad category of cancers includes sarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance, like embryonic connective tissue. The invention also enables treatment of cancers of the myeloid or lymphoid systems, including leukemias, lymphomas, and other cancers that typically are not present as a tumor mass, but are distributed in the vascular or lymphoreticular systems. Chkl activity is associated with various forms of cancer in, for exanrole, adult and pediatric oncology, growth of solid tumors/malignancies, myxoid and round cell carcinoma, locally advanced tumors, metastatic rancer, human soft tissue sarcomas, including Ewing's sarcoma, cancer metastases, including lymphatic metastases, squamous cell carcinoma, particularly of the head and neck, esophageal squamous cell carcinoma, oral carcinoma, blood cell malignancies, incluaing multiple myeloma, leukemias, including acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, and hairy cell leukemia, effusion lymphomas (body cavity based lymphomas), thymic lymphoma lung cancer (including small cell carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producing tumors, nonsmall cell cancers, breast cancer, including small cell carcinoma and ductal carcinoma), gastrointestinal cancers (including stomach cancer, colon cancer, colorectal cancer, and polyps associated with colorectal neoplasia), pancreatic cancer, liver cancer, urological cancers (including bladder cancer, such as primary superficial bladder tumors, invasive transitional cell carcinoma of the bladder, and muscle-invasive bladder cancer), prostate cancer, malignancies of the female genital tract (including ovarian carcinoma, primary peritoneal epithelial neoplasms, cervical carcinoma, uterine endometrial cancers, vaginal cancer, cancer of the vulva, uterine cancer and solid tumors in the ovarian follicle), malignancies of the male genital tract (including testicular cancer and penil cancer) , kidney cancer (including renal cell carcinma, brain cancer (including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, and metastatic tumor cell invasion in the central nervous system), bone cancers (including osteomas and osteosarcomas), skin cancers (including malignant melanoma, tumor progression of human skin keratino-cytes, and squamous cell cancer), thyroid cancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms' s tumors, gall bladder cancer, trophoblastic neoplasms, hemangiopericytoma, and Kaposi's sarcoma . Compounds of the present invention also can potentiate the efficacy of drugs in the treatment of inflammatory diseases. Examples of diseases that can benefit from combination therapy with compounds suitable for the method of the present invention are rheumatoid arthritis, psoriasis, vitiligo, Wegener's granulomatosis, and systemic lupus erythematosus (SLS). Treatment of arthritis. Wegener's granulomatosis, and SLE cften involves the use of immunosuppressive therapies, such as ionizing radiation, methotrexate, and cyclophosphamide. Such treatments typically induce, either direculy or indirectly, DNA damage. Inhibition of Chkl activity within the offending immune cells render the cells more sensitive to control by these standard treatments. Psoriasis and vitiligo commonly are treated with ultraviolet radiation (UV) in combination with psoralen. The present DNA damaging agents induce the killing effect of UV and psoralen, and increase the therapeutic index of this treatment regimen. In general, compounds useful in methods of the present invention potentiate control of inflammatory disease cells when in combination with currently used immunosuDDressive drugs. The present invention includes all possible stereoisomers and geometric isomers cf compounds of the present method and of structural formulae (I) , (11), and (III) . The present invention includes not only racemic compounds, but optically active isomers as well - When a compound of structural formulae (I), (II) , or (III) is desired as a single enantiomer, it can be obtained either by resolution of the final product or by stereospecific synthesis from either isomerically pure starting material or use of a chiral auxiliary reagent, for example, see Z. Ma et al., Tetrahedron: Asymmetry, 8(6), pages 883-8SS (1997) . Resolution cf the final product, an intermediate, or a starting material can be achieved by any suitable" method known in the art. Additionally, in situations where tautomers of the compounds of structural formulae (I), (II), and (III) are possible, the present invention is intended to include all tautomeric forms of the compounds. As demonstrated hereafter, specific stereoisomers can exhibit an exceptional ability to inhibit Chkl in combination with chemc-or radiotherapy with diminshed adverse effects typically associated with chemotherapeutic or radio-therapeutic treatments. Prodrugs of compositions of structural formulae (I), (II), and (III) also can be used as the compound and in the method of the present invention. It is well established that a prodrug approach, wherein a compound is derivatized into a form suitable for formulation and/or administration, and then is released as a drug in vivo, has been successfully employed to transiently (e.g., biore- versibly) alter the physicochemical properties of the compound (see, K. Bundgaard, Ed., Design of Prodrugs, Elsevier, Amsterdam, (1985) ; R.B. Silverman, The Organic Chemistry of Drug Design and Drug Action, Academic Press, San Diego, chapter 8, (1952); K.M. Hillgren et al, , Med. Res. Rev., 15, S3 (1595)) . Compounds of the present invention can contain several functional groups. The introduced functional groups, if desired or necessary, then can be modified to provide a prodrug for dose of formulation and/cr administration. Suitable prodrugs include, for example, acid derivatives, like amides, esters, and the like. It also is appreciated by those skilled in the art that N-oxides can be used as a prodrug. As used herein, the term pharmaceutically acceptable salts refers compounds of structural formula (I), (ID, and (III) which contain acidic moieties and form salts with suitable cations-Suitable pharmaceutically acceptable cations include alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) cations. The pharmaceutically acceptable salts of the compounds of structural formula (I), (II), and (III), which contain a basic center, are acid addition salts formed with pharmaceutically acceptable acids. Examples include the hydrochloride, hydro-bromide, sulfate or bisulfate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate, methanesulfonate, benzene sulphonate, and p-toluene-sulphonate salts. In light of the foregoing, any reference to compounds of the present inventioh appearing herein is intended to include compounds of structural formula (I), (II), and (III), as well as pharmaceutically acceptable salts and solvates thereof. The compounds of the present invention can be therapeutically administered as the neat chemical, but it is preferable to administer compounds of structural formula (I), (II), and (III) as a pharmaceutical composition or formulation. Accordingly, the present invention further provides pharmaceutical formulations comprising a compound of structural formula (I), (II) , and/or (III) , or pharmaceutically acceptable salts thereof, together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic and/or prophylactic ingredients. The carriers are "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Inhibition of the checkpoint kinase typically is measured using a dose-response assay in which a sensitive assay system is contacted with a compound of interest over a range of concentrations, including concentrauions at which no or minimal effect is observed, through higher concentrations at which partial effect is observed, to saturating concentrations at which a maximum effect is observed. Theoretically, such assays of the dose-response effect of inhibitor compounds can be described as a sigmoidal curve expressing a degree of inhibition as a function of concentration. The curve also theoretically passes through a point at which the concentration is sufficient to reduce activity of the checkpoint enzyme to a level that is 50% that of the difference between minimal and maximal enzyme activ-ity in the assay. This concentration is defined as the Inhibitory Concentration (50%) or IC50 value. Determination of IC50 values preferably are made using conventional biochemical (acellular) assay techniques or cell-based assay techniques. Comparisons of the efficacy of inhibitors often are provided with reference to comparative IC50 values, wherein a higher IC50 indicates that the test compound is less potent, and a lower IC50 indicates that the compound is more potent, than a reference compound. Compounds useful in the method of the present invention demonstrate an IC50 value of at least 0.1 nM when measured using the dose-response assay. Preferred compounds demonstrate an IC50 value of less than 10 μM. More preferred compounds demonstrate an IC50 value of less than 500 nM. Still more preferred compounds of the present invention demonstrate an IC50 value of less than 250 n.M, less than 100 nM, or less than 50 nM. Compounds and pharmaceutical compositions suitable for use in the present invention include those wherein the active ingredient is administered in an effective amount to achieve its intended purpose. More specifically, a "therapeutically effective amount" means an amount effective to inhibit development of, or to alleviate the existing symptoms of, the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. A "therapeutically effective dose" refers to that amount of the compound that results in achieving the desired effect. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cul-tures or experimental animals, e.g., for determining the LD50 {the dose lethal to 50% or tne population; and the ED50 (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio of LD50 to '50 Compounds that exhibit high therapeutic Tne indices (i.e., a toxic dose that is substantially higher than the effective dose) are preferred, data obtained can be used in forrriulating a dosage range for use in humans. The dosage of such compounds preferably lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized. The exact formulation, route of administration, and dosage is chosen by the individual physician in view of the patient's condition. . Dosage amount and interval can be adjusted individually to provide plasma levels of the active compound that are sufficient to maintain desired therapeutic effects. Pharmaceutical compositions of the invention can be formulated to include one or more cytokines, lymphokines, growth factors, or other hematopoietic factors which can reduce negative side effects that may arise from, or be associated with, administration of the pharmaceutical composition alone. Cytokines, lymphokines, growth factors, or other hematopoietic factors particularly useful in pharmaceutical compositions of the invention include, but are not limited to, M-CSF, GKi-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, III-6, IL-7, IL-8, IL-9. IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, TL-16, IL-17, IL-18, IFN, TNF, G-CSF, Meg-CSF, GM-CSF, thromboprotein, stem cell factor, erythropoietin, angiopoietins, including Ang-1, Ang-2, Ang-4, Ang-Y, and/or the human angiopoietin-like polypeptide, vascular endothelial growth factor (VEGF), angic-genin, bone morphogenic protein-1 (3MP-1) , 3Mr-2, BMP-3, BMP-4, BMP-5, BMP-6, EMP-7, BMP-6, BMP-S, BMP-IO, 3MP-11, BMP-12, 3MP-13, BMP-14, EMP-15, BMP receptor lA, BMP receptor IB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2, cytokine-induced neutrophil chemotactic factor 2, endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast growth factor (FGF) 4, FGF 5, FGF 6, FGF 7, FGF 6, FGF 8b, FGF 8c, FGF 9, FGF 10, FGF acidic, FGF basic, glial cell line-derived neutrophic factor receptor 1, glial cell line-derived neutrophic factor receptor 2, growth related protein, growth related protein, growth related protein, growth .related protein, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitorN'- factor receptor, nerve growth factor nerVe growth factor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growth factor 2, platelet-derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platfelet derived growth factor AB, platelet derived growth factor B chain, platelet derived growth factor 33, platelet derived growth factor receptor, platelet derived growth factor receptor, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor (TG?) , TGF, TGF 1, TGF 1.2, TGF 2, TGF, 3, TGF, 5, latent TG? I, TGF, binding protein I, TGF binding protein II, TGF binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, vascular endothelial growth factor, and chimeric proteins and biologically or immunologically active fragments thereof. The compounds useful according to the invention may be conjugated or linked to auxiliary moieties that promote any property of the compounds that may be beneficial in methods of therapeutic use. Such conjugates can enhance delivery of the compounds to a particular anatomical site or region of interest (e.g., a tumor), enable sustained therapeutic concentrations of the compounds in target cells, alter pharmacokinetic and pharmacodynamic properties of the compounds, and/or improve the therapeutic index or safety profile of the compounds. Suitable auxiliary moieties include, for example, amino acids, aligopeptides, or polypeptides, e.g. , antibodies such as monoclonal antibodies and other engineered antibodies,- and natural or synthetic ligands to receptors in target cells or tissues. Other suitable auxiliaries include fatty acid or lipid moieties, to promote biodistribution or uptake of the compound by target cells (see, e.g., Bradley et al. , Clin. Cancer Res. (2001) 7:3229. The therapeutic index of compositions comprising one or more compounds of the invention can be enhanced by conjugation of the compound(s) with antitumor antibodies as previously described (for example, Pieterse and McKinzie, Immunol. Rev. (1992) 129:57; Trail et al., Science (1993) 261:212; Rowlinscn-Bussa and Epenetos, Curr. Opin. Oncol. 1992; 4:1142). Tumor directed delivery of compounds of the invention would enhance the therapeutic benefit by minimizing potential nonspecific toxicities which can result from radiation treatment or chemotherapy. In another aspect, Chkl inhibitors and radioisotopes or chemotherapeutic agents can be conjugated to the same antibody molecule. Alternatively, Chkl inhibitor-conjugated tumor specific antibodies can be administered before, during, or after administration of chemotherapeutic-con3ugated antitumor antibody or radioimmunotherapy. Compounds of the present invention can enhance the therapeutic benefit cf radiation and chemotherapy treatment, including induction chemotherapy, primary (neoadjuvant) chemotherapy, and both adjuvant radiation therapy and adjuvant chemotherapy. In addition, radiation and chemotherapy are frequently indicated as adjuvants to surgery in the treatment of cancer. The goal of radiation and chemotherapy in the adjuvant setting is to reduce the risk of recurrence and enhance disease-free survival when the primary tumor has been controlled. Chemotherapy is utilized as a treatment adjuvant for colon, lung, and breast cancer, frequently when the disease is metastatic. Adjuvant radiation therapy is indicated in several diseases including colon, lung, and breast cancers as 'described above. 'For example, radiation frequently is used both pre- and posr-surgery as components of the treatment strategy for rectal carcinoma. Compounds for the present invention are therefore particularly useful following surgery in the treatment of cancer in combination with radio- and/or chemotherapy. A compound of the present invention also can radiosensitize a cell. The term "radiosensi-tize, " as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to human or other animal in a therapeuci-cally effective amount to increase the sensitivity of rhe cells to be radiosensitized to electromagnetic radiation and/or to promote the treatment of diseases that are treatable with electromagnetic radiation. Diseases that are treatable with electromagnetic radiation include neoplastic diseases, benign and malignant tumors, and"cancerous cells. Electromagnetic radiation treatment of other diseases not listed herein also is contemplated by the present invention. The terms "electromagnetic radiation" and "radiation" as used herein include, but are not limited to, radiation having the wavelength of 10-10 to 100 meters. Preferred embodiments of the present invention employ the electromagnetic radiation of: gamma-radiation (10-20 to 10-13 m) , X-ray radiation {10-1= to 10-9 m) , ultraviolet light (10 nm to 400 nm) , visible light (400 nm to 700 nm) , infrared radiation (700 nm to 1.0 mm) , and microwave radiation (1 mm to 30 cm) . Many cancer treatment protocols currently employ radiosensitizers activated by electromagnetic radiation, e.g., X-rays. Examples of X-ray-activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole. desmethylmisonidazole, pimonidasole, etanidazoie, nimorazole, mitomycin C, RSU 1069, SR 4233, EOS, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR) , 5-iododeoxyuridine (lUdR) , bromodeoxycytidine, flucro-deoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same. Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator cf the sensitizing agent. Examples of photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, PHOTO-FRIN'^, benzoporphyrin derivatives, NPe6, tin etio-pcrphyrin {SnET2), pheoborbide-a, bacteriochloro-phyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same. Radiosensitizers can be administered in conjunction with a therapeutically effective amount * of one ox more compounds in addition to the Chki inhibitor, such compounds including, but not limiited to, compounds that promote the incorporation cf radiosensitizers to the target cells, compounds that control the flow of therapeutics, nutrients, and/cr oxygen to the target cells, chemotherapeutic agents that act on the tumor with or without additional radiation, or other therapeutically effective compounds for treating cancer or other disease. Examples of additional therapeutic agents that can be used in conjunction with radiosensitizers include, but are not limited to, 5-fluorouracil C5-FU) , leucovorin, oxygen, carbogen, red cell transfusions, perfluorocarbons (e.g., FLUOSOLW-DA) , 2,3-DPG, BW12C, calcium channel blockers, pentoxifylline, antiangiogenesis compounds, hydralazine, and L-330. Chemotherapeutic agents that can be used include, but are not limited to, alkylating agents, antimetabolites, hormones and antagonists thereof, radioisotopes, antibodies, as well as natural products, and combinations thereof- For example, an inhibitor compound of the present invention can be adminisrered with antibiotics, such as doxorubicin and other anthracycline analogs, nitrogen muscards, such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin- independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH) . Other antineoplastic protocols include the use of an inhibitor compound with another treatment modality, e.g., surgery or radiation, also referred to herein as "adjunct anti-neoplastic modalities." Examples of .chemotherapeutic agents useful for the method of the present invention are listed in the following table. Examples of chematherapeutic agents that are particularly useful in conjunction with radio- sensitizers include, for example, adriamycin, camptothecin, carhoplatin, cisplatin, daunorubicin, doxorubicin, interferon (alpha, beta, gamma), inrer-leukin 2, irinotecan, docetaxel, paclitaxel, topotecan, and therapeutically effective analogs and derivatives of the same. As appreciated by persons skilled in the art, reference herein to treatment extends to prophylaxis, as well as to treatment of established diseases or s.ymptoms. It is further appreciated that the amount of a compound of the invention required for use in treatment varies with the nature of the condition being treated, and with the age and the condition of the patient, and is ultimately determined by the attendant physician or veterinarian. In general, however, doses employed for adult human treatment topically are in the range of 0.001 mg/kg to about 100 mg/kg per day. The desired dose can be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more subdoses per day. In practice, the physician determines the actual dosing regimen most suitable for an individual patient, and the dosage varies with the age, weight, and response of the particular patient. The above dosages are exemplary of the average case, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of the present invention. Formulations of the present invention can be administered in a standard manner for the treatment of the indicated diseases, such as orally, parenterally, transmucosally (e.g., sublingually or via buccal administration), topically, transdetmal-ly, rectally, via inhalation (e.g., nasal or deep lung inhalation) . Parenteral administration includes, but is not limited to intravenous, intraarterial, intraperitoneal, subcutaneous, intramascu-lar, intrathecal, and intraarticular. Parenteral administration also can be accomplished using a high pressure technique, like POWDERJECT'". For oral administration, including buccal administration, the composition can be in the form of tablets or lozenges formulated in conventional manner. For example, tablets and capsules for oral administration can contain conventional excipients such as binding agents (tor example, syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch, or polyvinylpyrrolidone), fillers (for example, lactose, sugar, microcrystalline cellulose, maize-starch, calcium phosphate, or sorbitol), lubricants (for example, magnesium stearate, stearic acid, talc, polyethylene glycol or silica), disintegrants (for example, potato starch or sodium starch glycolate), or wetting agents (for example, sodium lauryl sulfate) . The tablets can be coated according to methods well known in the art. Alternatively, the compounds of the present invention can be incorporated into oral liquid preparations such as aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, for example. Moreover, formulations containing these compounds can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can contain conventional additives, for example suspending agents, such as sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellu-lose, aluminum stearate gel, and .hydrogenated edible fats; emulsifying agents, such as lecithin, sorbitan monooleate, or acacia; nonaqueous vehicles (which can include edible oils), such as almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol; and preservatives, such as methyl or propyl p-hydroxybenzoate and sorbic acid. Such preparations also can be formulated as suppositories, e.g., containing conventional suppository bases, such as cocoa butter or other glycerides. Compositions for inhalation typically can be provided in the form of a solution, suspension, or emulsion that can be administered as a dry powder or in the form of an aerosol using a conventional propellant, such as dichlorodifluoromethane or trichlorofluoromethane. Typical topical and transdermal formulations comprise conventional aqueous or nonaqueous vehicles, such as eye drops, creams, ointments, lotions, and pastes, or are in the form of a medicated plaster, patch, or membrane. Additionally, compositions of the present invention can be formulated for parenteral administration by injection or continuous infusion. Formulations for injection can be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulation agents, such as suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use. A composition in accordance with the present invention also can be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example, subtutan-eously or intramuscularly) or by intramuscular in- Compounds suitable in the method include, but are not limited to: ulae (IV), (V), (VI), and (VII), can be prepared acccrding to the following synthetic scheme. In the scheme described below, it is understood' in the art that protecting groups can be employed where necessary in accordance with general principles of synthetic chemistry. These protecting groups are removed in the final steps of the synthesis under basic, acidic, or hydrogenolytic conditions which are readily apparent to those skilled in the art. 3y employing appropriate manipulation and protection of any chemical functionalities, synthesis of compounds of structural formulae (I), (II), and (III) not specifically set forth herein can be accomplished by methods analogous to the schemes set forth below. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification. All reactions and chromatography fractions were analyzed by thin-layer chromatography on 250-mm silica gel. plates, visualized with UV (ultraviolet) light and I2, by the formula HetAr-NH2, preferably under an inert atmosphere at room temperature (about 25oC), to afford a crude aryl pyrazine-disubstituted ureS compound. A more particular illustration for the preparation compounds of standard formulae (I) and (II) can include, for example, the following General Scheme 2 Step (1); TMS Diazomethane Esterification To a cooled (about 0°C), stirred solution of 4-amino-3-methcxyben2oic acid (5.0 g; 3 0 mmol) in dry methanol (15 0 mL) was added trimethylsilyl diazomethane (60 mL of 2.0 M solution in hexanes, 120 mmol) slowly over 1 hour. After stirring for 4 hours, the reaction was concentrated at reduced pressure, dissolved in ethyl acetate (200 mL), washed with 10% aqueous sodium carbonate and brine, then dried (MgSO4) , filtered, and concentrated in vacuo CO provide the desired ester as an off-whize solid (94% yield). Step (2) : D-Nitrophenvl Carbamate Procedure To a stirred, cooled (about 0°C) solurion of merhyi-3-amino-4-methoxy benzoate (5.0 g; 27.5 mmol) in dry dichloromethane (175 mL) was added pyridine (2.34 mL; 29 mmol) followed by 4-nitro-phenyl chloroformate (5.8 g; 29 mmol) under a nitrogen (N2) atmosphere. After stirring for S hours, the reaction was washed with 2N aqueous hydrochloric acid (2 X 200 mL), saturated aqueous sodium bicarbonate (2 X 200 mL) , and brine (200 mL) , then dried (MgSO4) and filtered. The filtered solution was diluted with ethyl acetate and hexanes (about 600 mL) until a precipitate formed. The solid was collected on a Buchner funnel with suction, and air dried to provide the desired carbamate as a white solid (70% yield). Step (3): Carbamate Coupling Procedure To a stirred solution of 4-methoxy-3-(4-nirro-phenoxycarbonylamino) -benzoic acid methyl ester (30 g; 8.7 mmol) in dry N-methyl pyrrolidine (50 mL) was added the amino pyrazine (0.84 g; 8.8 mmol) under a N2 atmosphere at room temperature. The reaction mixture was heated to 80oC for 6 hours, then allowed to cool to room temperature. Dilution with ethyl acetate (200 mL) and water (200 mL) provided the desired urea as a white solid (54% yield) . The following compounds were prepared using the general procedure described accompanying General Scheme 2, but substituting the R group below for the R group shown in General Scheme 2: Isocvanate Procedure; To a stirred solution of 2-methoxy-5-meth-yl-phenylisocyanate (43 mL; 0,3 mmol) in dry di-chicroechane (0.4 mL) was added 2-arr.inoquinoxaline (43.5 mg; 0.3 mmol) in a reaction vial under a nitrogen atmosphere. ' The vial was capped and heated to 80°C overnight (14 hours). The reaction mixture then was filtered, and the residue washed with di-chloromethane to provide the desired urea as a white solid Ol% yield) . The following compounds were prepared using the procedure described accompanying General Scheme 3, but substituting the Ar group in the table below for the Ar group in General Scheme 3: ethane {20 mL) . The reaction mixture was cooled to room temperature to precipitate the product, which was collected by filtration, washed with ethyl Example 4 Preparation of Compound 14 Example 5 Preparation of Compound 4 8 (S) -1- (2,2,2-trifluoroethanoyl)pyrrolidine-2-carboxylic acid [4-methoxy-3-(3-pyra2in-2-yl- ureido)phenyl] -amide A solution of 1-(5-amino-methoxyphenyl)-3-pyra2in-2-yl-urea (Compound 14, Example 4) (105 ma, 0.4 mmol) in dry pyridine (2 mL) at 0oC was treated with a solution of N-trifluoroacetyl-(S)-prolyl .chloride (0,1 M in dichloromethane, 4,5 mL, 0.45 mmol) and stirred 2 h at room temperature. The reaction was quenched with 1 N HCl (50 mL) and extracted with ethyl acetate (3 X 50 mL) . The Example 7 Preparation of Compound 16 Example 8 Preparation of Compound 42 N' [4-methoxy-3- (3-pyrazin-2-yl-ureido)phenyl] - methanesulfonamide A solution of 1-(5-amino-2-methoxyphenyl)-3-pyrazin-2-yl-urea (Compound 14, Example 4) (260 mg, 1 mmol) in dry pyridine (15 mL) was treated with methanesulfonyl chloride (0.08 mL, 1 mmol) and Example 9 Preparation of Compound 65 3-chloro-N- [3-methoxy-4- (3-pyrazin-2-yl-ureido)phenyl]-propionamide A solution of 1-{4-amino-2-methoxyphenyl)-3-pyra2in-2-yl-urea (Compound 32, Example 10) (259 mg, 1 mmol) in pyridine (3 mL) at 0oC was treated with chloroacetyl chloride (0.29 mL, 3 mmol). The suspension was warmed at 80°C until most solids dissolved, the reaction-mixture was cooled to room temperature and the product was precipitated with Example 13 Preparation of Compound 4 (cyclohexyl-methyl-aoiino) -N- [3-methoxy-4-(3-pyrazin-2-yl-ureido)phenyl] -propionamide LRMS (csi. positive) m/e 435.2 (M-1) Prepared according to the procedure of Compound 1S9 using C-(lH-ben2oinuda2oI-2-yl)-inethylaminc (26% yield). a solid and the mixture heated to 65 "C. After reaching temperaruie, the suspension gradually became a bright yellow solution. After about 4 hours a precipitate formed but the reaction was continued overnight. After cooling to RT, MeOH was removed by roiovap and the aqueous suspension diluted N-(3-Dimethylamino-propyl)-3-mcthoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzamide Compound 216: be made without departing from the spirit and scope thereof, and, thereore, only such limitation should be imposed as are indicated by the appended claims. WHAT IS CLAIMED IS: 1.Amethod inhibiting checkpoint kinease in a cell comprising a step of consisting the cell with an effective amount of a compound of formula f wherein X2 is null, -O-, -S-, -CH2-, or -N(R-)-; X- is-O-, -S-, or -N(R1)-; Y is O or S; or =Y representative two hydrogen atoms attached to a common carbon atom; W is selected from the group consisting of C1-3 alkyl substituted with a heterc-aryl Or aryl group; Z is selected from the group consisting of hydro, aryl, and heteroaryl; wherein said aryl croups of w and 2 are optionally substituted with one to four substicuents represented by R2, said heteroaryl groups of W and Z are optionally substituted vith one to four substit-uents represented by R-, and said heterocycloalkyl and cycloalkyl groups of V? are optionally substituted with one to two substituents represented by R5; carcinoma, an ovarian cancer, a brain tumor, an osteosarcoma, and a lung carcinoma. 27. A method od sensitising cells in an individual undergoing a chemotherapeutic or radio-' therapeutic treatment for a medical condition, com-prising administering a therapeutically effective amount of a compound of claim 19 in combination with a chemotherapeutic agent, a radiotherapeutically effective amount of a compound of claim 19 in combination with a chemotherapeutic agent, a radiotherapeutc agemt, or a mixture thereof to the individual. optionally substituted with from one to four sub-stituents selected from the croup consisting of 31. A method of inhibiting checkpoint kinase 1 in a cell substantially as herein described and exemplified. 32. A composition substantially as herein described and exemplified. Dated this 28 day of August 2003

Full Text

RYL AND HETEROARYL UREA CHKE INHINBOTORS FOR USE AS RADIOSENSITIZERS AND CHAMOSESITITZERS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit o f U.S. provisional application Serial No. 60/273,124, filed March 2, 2001.
TECHNICAL FIELD OF THE INVENTION
The present invention relates to compounds useful for inhibiting enzymes chat maintain and repair the integrity of genetic material. More particularly, the present invention relates to a series of aryl- and heteroaryl-substituted urea compounds, methods of making the compounds, and their use as therapeutic agents, for example, in treating cancer and other diseases characterized by defects in deoxyribonucleic acid (DNA) replication, chromosome segregation, or cell division.
BACKGROUND OF THE INVENTION
An important and significant goal in healthcare is to discover and make available safer and more effective drugs for the treatment of cancer. Most chemotherapeutic agents act by disrupting DNA metabolism, DNA synthesis, DNA transcription, or microtubule spindle function, or by perturbing chromosomal structural integrity by introducing DNA lesions. These processes affect both normal and tumor cells. The maintenance of DNA integrity is essential to cell viability in normal cells, therefore, anticancer drugs have the lowest therapeutic index of any drug class.

An individual cell creates an exact copy of its chromosomes, .,and then segregates each copy into two cells by a process called mitosis. The mototic cycle can be divided into three major events: DNA replication, chromosome segregation, and cell division. Cells have sensing mechanism to maintain the order of these steps with respect to one another and to ensure that each step is executed with high fidelity. The sensing mechanisms for
these processes are referred to as "checkpoints" in L.H, Hartwell et al.. Science, Nov. 3, 1989, 246 (4530) :629-34.
Cell cycle checkpoints have been reported to comprise at least three distinct classes of polypeptides- Each class of polypeptides acts sequentially in response to cell cycle signals or defects in chromosomal mechanisms (Carr, (1995) Science, 271:314-315). One family of proteins detects or senses DNA damage or abnormialities in the cell cycle. These sensors include Ataxia-Telangiectasia Mutated (Atm) and Ataxia-Telangiectasia Rad-related (Atr) (Keegan et al., (1996) Genes Dev. , 10:2423-2437). Another class of polypeptides amplify and transmit the signal detected by the detector and is exemplified by Rad53 (Allen et al . (1954) Genes Dev. , 8:2416-2488) and Chkl. In addition, cell cycle effectors, such as p53, mediate a cellular response, including, for example, arrest of mitosis and/or meiosis and apoptosis.
DNA damage can be induced by drugs, radiation, or can be spontaneously generated during the course of normal metabolism. DNA damage checkpoints ensure that cells with unrepaired DNA lesions do not progress into the DNA synthesis phase or mitosis until chromosomal lesions have been removed. Cell

—^'-^
cycle arrest can enhance the opportunity for DnA repair and increase the fidelity of cell divisi DNA damage can be recognized throughout the cell cycle. Checkpoints ensure that the growth of cells is arrested at multiple cell cycle phases. As a result, multiple cell cycle signaling pathways may result during sensitization of cells to DNA damaging agents.
Much of the current understanding of the function of cell cycle checkpoints has been derived from the study of tumor-derived cell lines. In many cases, tumor cells have lost key cell cycle checkpoints (Hartwell et al. , Science, Dec. 16, 1994 ; 266(5192): 1821-8). It has been reported that a key step in the evolution of cells to a neoplastic state is the acquisition of mutations that inactivate cell cycle checkpoint pathways, such as p53. (Weinberg, R.A- (1995) Cell 81:323-330; Levine, A. J. (1997) Cell 88: 3234-331). Loss of these cell cycle checkpoints results in the inappropriate cycling of tumor cells in response to DNA damaging agents. When faced with cellular stresses, such as DNA damage, and cell cycle events with decreased fidelity, tumor cells have difficulty altering the kinetics of ceil cycle progression. Therefore, inhibition and disruption of additional DNA damage checkpoint pathways may further sensitize tumor cells to anticancer treatments, such as radiation and chemotherapy.
Noncancerous tissue, which has intact cell cycle checkpoints, typically is insulated from temporary disruption of a single checkpoint pathway. Tumor cells, however, have defects in pathways controlling cell cycle progression such that the perturbation of additional cherkpoints, for example, the DNA damage checkpoint, renders them particularly

sensitive to DNA damaging agents. For example, tumor cells that contain mutant p53 are defective both in the Gl DNA damage checkpoint and in the ability to maintain the G2 DNA damage checkpoint. (Bunz et al., Science, Nov. 20, 1998; 282(5393): 1497-501; Levine). Checkpoint inhibitors that target initiation of the G2 checkpoint or the S phase checkpoint are expected to further cripple the ability of these tumor cells to repair DNA damage and, therefore, selectively kill them over normal cells. Therefore, checkpoint inhibitors are expected to enhance the therapeutic index, which is a measure of the probability of tumor control relative to the probability of toxicity to normal tissue, of both radiation and systemic chemotherapy.
The ability of checkpoint inhibitors to enhance the therapeutic index may be dependent upon tumor type. Tumors with cell cycle defects complementary to the DNA damage checkpoint pathways may be most sensitive to inhibitor drug treatment. In contrast, DNA-PK inhibitors, another distinct class of potential therapeutic agents, are expected to sensitize tumors independently of cell type. A systematic approach of applying checkpoint inhibitors and DNA-PK inhibitors also may be effective in the treatment of metastatic diseases that radi.ation therapy cannot target.
The checkpoint proteins Atm and Atr are hypothesized to initiate a signal transduction pathway leading to cell cycle arrest in the presence of DNA damage or any block to DNA replication. Atm has been shown to play a role in a DNA damage checkpoint in response to ionizing radiation (IR). Patients lacking functional Atm develop the disease Atakia-Telangiectasia (A-T). Symptoms of A-T include ex-

treme sensitivity to ionizing radiation (IR) , cerebellar degeneration, oculotaneous telangiectasias, gonadal deficiencies, immunodeficiencies and increased risk of cancer (Shiioh, Eur. J. Hum. Genet 1995; 3 (2) : 116-38) - Fibroblasts derived from these patients are thought to have defects in Gl, S, and G2 checkpoints and are defective in their response to IR (Kastan et al., Cell, Nov. 13, 1992; 71(4); 587-97; Scott et al., Int. J, Radiat. Biol., Dec, 1994; 66(5 Suppl): S157-63; and Beamish et al. , J. Biol. Chem. Aug. 26, 1993; 271(34):20486-93). Therefore, Atm may sense double-strand DNA damage caused by IR and radiomimetic drugs, and signal the cell cycle to arrest, such that damage can be repaired.
Atr is a checkpoint protein stimulated by agents that cause double strand DNA breaks, single strand DNA breaks, and agents that block DNA radiation. Overexpression of Atr in muscle cells on iso-chromosome 3q results in a block to differentiation, abnormal centrosome numbers, chromosome instability, and abolishes the Gl arrest in response to IR (Smith et al., Nat. Genet., May 1993; 19(1): 39-46). Overexpression of a kinase inactive, dominant negative mutant of Atr sensitizes cells to IR, ultraviolet light (UV) , MMS, and cisplatin
(Cliby et al., EMBO J, Jan. 2, 1998, 17(l):159-69 and Wright et al. , Proc. Nat'l Acad. Sci. U.S.A., June 23, 1998; 95 (13) :7445-50) . Cells containing overexpressed, mutant strain Atr also fail to arres. in 02 in response to IR. In addition, Atr is associated witn chromosomes in meiotic cells where DNA breaks and abnormal DNA structures persist as a result of the process of meiotic recombination**
(Keegan et al. , Genes Dev. October 1, 1996; 10(19):

433-37) . Atr, like Atm, also senses DNA damage and agents that block DNA replication, as well as initiates a cell cycle arrest at G2 and S for DNA repair.
Chkl is hypothesized to lie downstream from protein kinases Atm and/or Atr in the DNA damage checkpoint signal transduction pathway. {See, Sanchez et al., Science, 1997; 277:1497-1501; U.S. Patent No. 6,218,109.) In mammalian cells, Chkl is phosphorylated in response to agents that cause DNA damage including IR, UV, and hydroxyurea (Sanchez et al. , 1997; Lui et al. , Genes Dev. 2000; 14:1443-1459). The phosphorylation and activation of Chkl in mammalian cells is dependent on Atm (Chen et al., 1999) and Atr (Lui et al. , 2000). In the yeast S. poinbe, Chkl also appears to be involved in the response to IR and blocks to replication (3oddy et al. , 1998; Walworth et al, , 1993). Furthermore, Chkl has been shown to phosphorylate both weel (O'Connell et al. , EMSO J. 1997; 15:545-554) and Pdsl (Sanchez et al., Science 1999; 235:1166-1171) gene products known to be important in cell cycle control. These studies demonstrate that mammalian Chkl plays a role in both the Atm-dependent DNA damage checkpoint leading to arrest at S phase. However, a role for Chkl in the S phase replication checkpoint in mammalian cells has yet to be elucidated. Interestingly, Chkl knockout mice are embryonically lethal, thereby suggesting a role for Chkl in a developing organism in addition to its role in DNA damage checkpoints.
Chkl may invoke a G2 arrest by phosphor-ylating and inactivating Cdc25C, the dual specificity phosphatase that normally dephosphorylates

cyclin B/cdc2 as cells progress into mitosis (Fernery et al., Science, Sep. 5, 19S7; 277(5351}: 1495-7; Sanchez et al,; Matsuoka et al,; and Blasina et al., Curr. Sioi., Jan. 14, 1599; 9(1):1-1C). This mechanism of regulation of Cdc2 activity stimulates cell cycle arrest to prevent cells from entering mitosis in the presence of DNA damage or unreplicated DNA.
SUMMARY OF THE INVENTION
The present invention is directed to potent and selective chemosensitizing agents useful in the treatment of diseases and conditions related to DNA damage or lesions in DNA replication. The present compounds are inhibitors of the checkpoint kinase Chkl. In particular, aryl- and heteroaryl substituted urea comoounds have demonstrated siq-nificant activity for inhibiting Chkl.
In one aspect, the present invention is directed to a method of inhibiting checkpoint kinase Chkl comprising the step of administering a compound of formula (I), or a composition containing the same, to an individual. Compounds of formula (I) have a structural formula:

wherein:

together to form an optionally substituted 3- to £-membered aliphatic ring;

R6 is selected from the group consisting of halo and C1-6alkyl,
and pharmaceutically acceptable salts or solvates thereof.
In another aspect, the present invention is directed to aryl- and heteroaryl-disubstituted urea compounds having a structural formula (II)

optionally substituted with from one to four sub-stituents selected from the group consisting of C1-6alkyl, aryl, N(R7),, OR7, N3, CN, C(O)R7 C1-2alk-ylenearyl, C1-3alkyleneN (R12) 2,

and pharmaceutically acceptable salts or solvates thereof.
Another aspect cf the present invention relates to carbamide-substituted heteroaryl groups having the structural formula (III)

wherein W" is selected from the group consisting of heteroaryl, aryl, heterocycloalkyl, cycloalkyl,_. and C1-3 alkyl substituted with a heteroaryl or aryl group;

R15 is selected from the group consisting of halo and C1-6alkyl.
The present invention also is directed to pharmaceutical compositions containing one or more compounds of structural formula (II), to use of the compounds and compositions containing the compounds in therapeutic treatment of a disease or disorder, and ro methods of preparing the compounds and intermediates involved in the synthesis of the compounds of structural formula (II).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Radiation and most chemotherapeutic agents are therapeutically beneficial because they take advantage of inappropriate tumor cell proliferation. Cellular processes, such as DNA damage repair, and cell cycle checkpoints, protect tumor cells from the toxic effects of physical and chemical agents. , Treatments that modulate the underlying molecular mechanisms of cell cycle progression and resistance to DNA damage can potentiate tumor cell killing and enhance the therapeutic index of existing therapies.
Most chemotherapeutic agents act by disrupting DNA metabolism.. Because these processes are shared by both normal and tumor cells, and because the maintenance of DNA integrity is essential to cell viability, anticancer drugs have the lowest therapeutic index of any drug class. By identifying and inhibiting cellular processes that tumor cells rely upon, the effectiveness of radiation and chemotherapy treatment regimens can be enhanced.
The interruption of the DNA damage checkpoint protein function provides a novel means of killing tumor cells relative to normal cells. For

example, Chkl ensures that cells with unrepaired DNA lesions caused by certain drugs or radiation do not progress through DNA synthesis phase or mitosis until chromosomal lesions have been removed. Accordingly, a tumor cell treated with a Chkl inhibitor in combination with a DNA damaging agent can kill using lower amounts of DNA damaging agent than tumor cells treated with the DNA damaging agent alone.
Failure of cell cycle checkpoints in normal cells predisposes an individual to, or directly causes, many disease states, such as cancer, ataxia telangiectasia, embryo abnontalities, and various immunological defects associated with aberrant 3 and T cell development. The latter are associated with the pathological states of lupus, arthritis, and autoimmune diseases. intense research efforts, tnerefore, have focused on identifying cell cycle checkpoints and the proteins essential for the function of the checkpoints.
Noncancerous tissue having intact cell checkpoints typically is insulated from temporary disruption of a single checkpoint pathway, such as the Chkl pathway- Tumor cells, however, have multiple defects in pathways controlling cell cycle progression such that perturbation of the DNA dam.age checkpoint can render cells particularly sensitive to DNA damaging agents. Therefore, checkpoint inhibitors are expected to enhance the therapeutic index, which is a measure of the probability of tumor control relative to the probability of toxicity to normal tissue to radiation and systemic chemotherapy. In contrast, other classes of inhibitors may not be amenable to combination chemotherapy because both normal and tumor tissue may be similarly sensitized.

One aspect of the present invention is directed to a method of inhibiting Chkl, comprising the step of adminisuering a therapeutically effective amount of a compound of formula (I) , or a composition containing the same, to an individual. Compounds of formula (I) have a structural formula

wherein X1 is null, -O-, -S-, -CK.-, or -N(R-).;
X2 is -O-, -S-,.or -N(R-)-;
Y is 0 or S; or =Y represents two hydrogen atoms attached to a common carbon atom;
W is selected from the group consisting of heteroaryl, aryl, heterocycloalkyl, cycloalkyl, and C1-3 alkyl substituted with a heteroaryl or aryl group; and
Z is selected from the group consisting of hydrogen, aryl, and heteroaryl;
wherein said aryl groups of w and Z are optionally substituted with one to four substituents represented by R3, said heteroaryl groups of w and Z are optionally substituted with one to four substituents represented by R5, and said heterocycloalkyl and cycloalkyl groups of W are optionally substituted with one to two substituents represented by R6;
R1 is selected from the group consisting of hydrogen, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, and aryl;

R6 is selected from the group consisting of halo and C1-6alkyl,
and pharmaceutically acceptable salts or solvates thereof.
Preferred compounds used in the method are those 1herein X1 and X2 are -N(H)-;
Y is O or S;
W is heteroaryl containing at least two heteroatoms selected from the group consisting of N, 0, and S, said ring is optionally substituted with from one to four substituents selected from the group consisting of C1-6alkyl, aryl, N(R3)2:., OR3, and halo, wherein R3 is as previously defined;
Z is selected from the group consisting
of:

Additional preferred compounds of formula (I) are those wherein W is selected from the group consisting of pyridazine, pyrimidine, pyrazine, and triazine, optionally substituted with from one to four substituents selected from the group consisting of optionally substituted C1-6alkyl, aryl, N(R3),, OR3

Compounds preferred for use in the method also include those of formula (I) wherein J is se-

The method of inhibiting Chkl also can be used to sensitize a tumor cell to a chemotherapeutic agent. As such, the present invention also is directed to a method of sensitizing a tumor cell to a chemotherapeutic agent corrprising administering a compound of formula (I), or a salt, solvate, or derivative thereof, or a composition comprising the same, to an individual.
In another aspect, the present invention is directed to aryl- and heteroaryl-disubstituted urea compounds having a structural formula (II)

optionally substituted with from one to four sub-stituents selected from the group consisting of C1-6alkyl, aryl, N(R7)2, OR7 N3, CN, C(O)R7, C1-3alk-ylenearyl, C1-3alkylene N(R12) n,

Z' is selected from the group consisting of:

and pharmaceutically acceptable salts cr solvares thereof.

w
optionally substituted with one to four substituents selected from the group consisting of C1-6alkyl, optionally substituted aryl, N(R3);, CF3, C(O)R7, N3, CN, C1-3alkylenearyl, C1-3alkyleneN(R12)., OR7, halo,

wherein R7, Y and Z are as previously defined.
More preferred compounds of formula (11) are those wherein:

The term "alkylene" refers to an alkyl group having a substituent. For example, the term "C1-3alkyleneC (O)R" refers to an alkyl group containing one to three carbon atoms substituted with a -C(O)OR group. The alkylene group is optionally substituted with one or more of aryl, heteroaryl, and OR"', wherein R' is defined hereafter.
The term "halo" or "halogen" is defined herein to include fluorine, bromine, chlorine, and iodine.

When no substituent is indicated as attached to a carbon atom on a ring, it is understood that the carbon atom contains the appropriate number of hydrogen atoms. In addition, when nc substituent is indicated as attached to a carbonyl group or a nitrogen atom, for example, the substituent is understood to be hydrogen, e.g..

The abbreviation "Me" is methyl. The abbreviation to and C (O) is carbonyl (C=(O)) .
The notation N(Rx)n, wherein x represents an alpha or numeric character, such as for example Ra, Rb, R4, R12, and the. like, is used to denote two Rx groups attached to a common nitrogen atom. When used in such notation, the Rx group can be the same

or different, and is selected from the group as defined by the Rx group.
The present invention also is directed to pharmaceutical compositions containing one or more compounds of structural formula (II) and (III), to use of the compounds and compositions containing the compounds in therapeutic treatment of a disease or disorder, and to methods of preparing the compounds and intermediates involved in the synthesis of "he compounds of structural formula (II) and (III) -
Compounds useful for the method of the present invention have demonstrated activity in inhibiting Chkl in vitro. Compounds of the present invention have demonstrated selectivity for Chkl as against other protein kinases including Cdc2, Chk2, Atr, DNA-PK, PKA, and CaM KII.
Compounds of the present invention can be used to potentiate the therapeutic effects of radiation and/or chemotherapeutics used in the treatment of cancers and other cell proliferation disorders in humans or animals. For example, compounds of the invention can be used to enhance treatment of tumors that are customarily treated with an antimetabolite, e.g., methotrexate or 5-fluorouracil (5-FU). The method of the present invention comprises administration of a Chkl inhibitor compound in combination with a chemotherapeutic agent that can effect single- or double-strand DNA breaks or that can block DNA replication or cell proliferation. Alternatively, the method of the present invention comprises administration of a Chkl inhibitor compound in combination with therapies that include use of an antibody, e.g., herceptin, that has activity in inhibiting the proliferation of cancer cells. Accordingly, cancers such as colorectal cancers,

head and neck cancers, pancreatic cancers, breast cancers, gastric cancers, bladder cancers, vulvar cancers, leukemias, lymphomas, melanomas, renal cell carcinomas, ovarian cancers, brain tumors, osteo-sarcomas, and lung carcinomas, are susceptible to enhanced treatmenu in combination with the Chkl inhibitors of the invention.
Tumors or neoplasms include growths of tissue cells wherein multiplication of cells is uncontrolled and progressive. Some such growths are benign, but others are termed "malignant," and can lead to death of the organism. Malignant neoplasms, or "cancers," are distinguished from benign growths in that, in addition to exhibiting aggressive cellular proliferation, can invade surrounding tissues and metastasize. Moreover, malignant neoplasms are characterized by shewing a greater loss of differentiation (greater "dedifferentiation") and organization relative to one another and surrounding tissues. This property is called "anaplasia."
Neoplasms treatable by the present invention also include solid tumors, i.e., carcinomas and sarcomas. Carcinomas include malignant neoplasms derived from epithelial cells which infiitrace (i.e., invade) surrounding tissues and give rise to metastases. Adenocarcinomas are carcinomas derived from glandular tissue, or from tissues that form recognizable glandular structures. Another broad category of cancers includes sarcomas, which are tumors whose cells are embedded in a fibrillar or homogeneous substance, like embryonic connective tissue. The invention also enables treatment of cancers of the myeloid or lymphoid systems, including leukemias, lymphomas, and other cancers that typically are not present as a tumor mass, but

are distributed in the vascular or lymphoreticular
systems.
Chkl activity is associated with various forms of cancer in, for exanrole, adult and pediatric oncology, growth of solid tumors/malignancies, myxoid and round cell carcinoma, locally advanced tumors, metastatic rancer, human soft tissue sarcomas, including Ewing's sarcoma, cancer metastases, including lymphatic metastases, squamous cell carcinoma, particularly of the head and neck, esophageal squamous cell carcinoma, oral carcinoma, blood cell malignancies, incluaing multiple myeloma, leukemias, including acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, and hairy cell leukemia, effusion lymphomas (body cavity based lymphomas), thymic lymphoma lung cancer (including small cell carcinoma, cutaneous T cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cancer of the adrenal cortex, ACTH-producing tumors, nonsmall cell cancers, breast cancer, including small cell carcinoma and ductal carcinoma), gastrointestinal cancers (including stomach cancer, colon cancer, colorectal cancer, and polyps associated with colorectal neoplasia), pancreatic cancer, liver cancer, urological cancers (including bladder cancer, such as primary superficial bladder tumors, invasive transitional cell carcinoma of the bladder, and muscle-invasive bladder cancer), prostate cancer, malignancies of the female genital tract (including ovarian carcinoma, primary peritoneal epithelial neoplasms, cervical carcinoma, uterine endometrial cancers, vaginal cancer, cancer of the vulva, uterine cancer and solid tumors in the ovarian follicle), malignancies of the male genital

tract (including testicular cancer and penil cancer) , kidney cancer (including renal cell carcinma, brain cancer (including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, and metastatic tumor cell invasion in the central nervous system), bone cancers (including osteomas and osteosarcomas), skin cancers (including malignant melanoma, tumor progression of human skin keratino-cytes, and squamous cell cancer), thyroid cancer, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, Wilms' s tumors, gall bladder cancer, trophoblastic neoplasms, hemangiopericytoma, and Kaposi's sarcoma . Compounds of the present invention also can potentiate the efficacy of drugs in the treatment of inflammatory diseases. Examples of diseases that can benefit from combination therapy with compounds suitable for the method of the present invention are rheumatoid arthritis, psoriasis, vitiligo, Wegener's granulomatosis, and systemic lupus erythematosus (SLS). Treatment of arthritis. Wegener's granulomatosis, and SLE cften involves the use of immunosuppressive therapies, such as ionizing radiation, methotrexate, and cyclophosphamide. Such treatments typically induce, either direculy or indirectly, DNA damage. Inhibition of Chkl activity within the offending immune cells render the cells more sensitive to control by these standard treatments. Psoriasis and vitiligo commonly are treated with ultraviolet radiation (UV) in combination with psoralen. The present DNA damaging agents induce the killing effect of UV and psoralen, and increase the therapeutic index of this treatment regimen. In general, compounds useful in methods of the present invention potentiate control of inflammatory disease

cells when in combination with currently used immunosuDDressive drugs.
The present invention includes all possible stereoisomers and geometric isomers cf compounds of the present method and of structural formulae (I) , (11), and (III) . The present invention includes not only racemic compounds, but optically active isomers as well - When a compound of structural formulae (I), (II) , or (III) is desired as a single enantiomer, it can be obtained either by resolution of the final product or by stereospecific synthesis from either isomerically pure starting material or use of a chiral auxiliary reagent, for example, see Z. Ma et al., Tetrahedron: Asymmetry, 8(6), pages 883-8SS (1997) . Resolution cf the final product, an intermediate, or a starting material can be achieved by any suitable" method known in the art. Additionally, in situations where tautomers of the compounds of structural formulae (I), (II), and (III) are possible, the present invention is intended to include all tautomeric forms of the compounds. As demonstrated hereafter, specific stereoisomers can exhibit an exceptional ability to inhibit Chkl in combination with chemc-or radiotherapy with diminshed adverse effects typically associated with chemotherapeutic or radio-therapeutic treatments.
Prodrugs of compositions of structural formulae (I), (II), and (III) also can be used as the compound and in the method of the present invention. It is well established that a prodrug approach, wherein a compound is derivatized into a form suitable for formulation and/or administration, and then is released as a drug in vivo, has been successfully employed to transiently (e.g., biore-

versibly) alter the physicochemical properties of the compound (see, K. Bundgaard, Ed., Design of Prodrugs, Elsevier, Amsterdam, (1985) ; R.B. Silverman, The Organic Chemistry of Drug Design and Drug Action, Academic Press, San Diego, chapter 8, (1952); K.M. Hillgren et al, , Med. Res. Rev., 15, S3 (1595)) .
Compounds of the present invention can contain several functional groups. The introduced functional groups, if desired or necessary, then can be modified to provide a prodrug for dose of formulation and/cr administration. Suitable prodrugs include, for example, acid derivatives, like amides, esters, and the like. It also is appreciated by those skilled in the art that N-oxides can be used as a prodrug.
As used herein, the term pharmaceutically acceptable salts refers compounds of structural formula (I), (ID, and (III) which contain acidic moieties and form salts with suitable cations-Suitable pharmaceutically acceptable cations include alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) cations. The pharmaceutically acceptable salts of the compounds of structural formula (I), (II), and (III), which contain a basic center, are acid addition salts formed with pharmaceutically acceptable acids. Examples include the hydrochloride, hydro-bromide, sulfate or bisulfate, phosphate or hydrogen phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate, gluconate, methanesulfonate, benzene sulphonate, and p-toluene-sulphonate salts. In light of the foregoing, any reference to compounds of the present inventioh appearing herein is intended to include compounds of

structural formula (I), (II), and (III), as well as pharmaceutically acceptable salts and solvates
thereof.
The compounds of the present invention can be therapeutically administered as the neat chemical, but it is preferable to administer compounds of structural formula (I), (II), and (III) as a pharmaceutical composition or formulation. Accordingly, the present invention further provides pharmaceutical formulations comprising a compound of structural formula (I), (II) , and/or (III) , or pharmaceutically acceptable salts thereof, together with one or more pharmaceutically acceptable carriers and, optionally, other therapeutic and/or prophylactic ingredients. The carriers are "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
Inhibition of the checkpoint kinase typically is measured using a dose-response assay in which a sensitive assay system is contacted with a compound of interest over a range of concentrations, including concentrauions at which no or minimal effect is observed, through higher concentrations at which partial effect is observed, to saturating concentrations at which a maximum effect is observed. Theoretically, such assays of the dose-response effect of inhibitor compounds can be described as a sigmoidal curve expressing a degree of inhibition as a function of concentration. The curve also theoretically passes through a point at which the concentration is sufficient to reduce activity of the checkpoint enzyme to a level that is 50% that of the difference between minimal and maximal enzyme activ-ity in the assay. This concentration is defined as

the Inhibitory Concentration (50%) or IC50 value. Determination of IC50 values preferably are made using conventional biochemical (acellular) assay techniques or cell-based assay techniques.
Comparisons of the efficacy of inhibitors often are provided with reference to comparative IC50 values, wherein a higher IC50 indicates that the test compound is less potent, and a lower IC50 indicates that the compound is more potent, than a reference compound. Compounds useful in the method of the present invention demonstrate an IC50 value of at least 0.1 nM when measured using the dose-response assay. Preferred compounds demonstrate an IC50 value of less than 10 μM. More preferred compounds demonstrate an IC50 value of less than 500 nM. Still more preferred compounds of the present invention demonstrate an IC50 value of less than 250 n.M, less than 100 nM, or less than 50 nM.
Compounds and pharmaceutical compositions suitable for use in the present invention include those wherein the active ingredient is administered in an effective amount to achieve its intended purpose. More specifically, a "therapeutically effective amount" means an amount effective to inhibit development of, or to alleviate the existing symptoms of, the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
A "therapeutically effective dose" refers to that amount of the compound that results in achieving the desired effect. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cul-tures or experimental animals, e.g., for determining

the LD50 {the dose lethal to 50% or tne population; and the ED50 (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio of LD50 to

'50
Compounds that exhibit high therapeutic
Tne
indices (i.e., a toxic dose that is substantially higher than the effective dose) are preferred, data obtained can be used in forrriulating a dosage range for use in humans. The dosage of such compounds preferably lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed, and the route of administration utilized.
The exact formulation, route of administration, and dosage is chosen by the individual physician in view of the patient's condition. . Dosage amount and interval can be adjusted individually to provide plasma levels of the active compound that are sufficient to maintain desired therapeutic effects.
Pharmaceutical compositions of the invention can be formulated to include one or more cytokines, lymphokines, growth factors, or other hematopoietic factors which can reduce negative side effects that may arise from, or be associated with, administration of the pharmaceutical composition alone. Cytokines, lymphokines, growth factors, or other hematopoietic factors particularly useful in pharmaceutical compositions of the invention include, but are not limited to, M-CSF, GKi-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, III-6, IL-7, IL-8, IL-9. IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, TL-16, IL-17, IL-18, IFN, TNF, G-CSF, Meg-CSF, GM-CSF,

thromboprotein, stem cell factor, erythropoietin, angiopoietins, including Ang-1, Ang-2, Ang-4, Ang-Y, and/or the human angiopoietin-like polypeptide, vascular endothelial growth factor (VEGF), angic-genin, bone morphogenic protein-1 (3MP-1) , 3Mr-2, BMP-3, BMP-4, BMP-5, BMP-6, EMP-7, BMP-6, BMP-S, BMP-IO, 3MP-11, BMP-12, 3MP-13, BMP-14, EMP-15, BMP receptor lA, BMP receptor IB, brain derived neurotrophic factor, ciliary neutrophic factor, ciliary neutrophic factor receptor cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2, cytokine-induced neutrophil chemotactic factor 2, endothelial cell growth factor, endothelin 1, epidermal growth factor, epithelial-derived neutrophil attractant, fibroblast growth factor (FGF) 4, FGF 5, FGF 6, FGF 7, FGF 6, FGF 8b, FGF 8c, FGF 9, FGF 10, FGF acidic, FGF basic, glial cell line-derived neutrophic factor receptor 1, glial cell line-derived neutrophic factor receptor 2, growth related protein, growth related protein, growth related protein, growth .related protein, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitorN'- factor receptor, nerve growth factor nerVe growth factor receptor, neurotrophin-3, neurotrophin-4, placenta growth factor, placenta growth factor 2, platelet-derived endothelial cell growth factor, platelet derived growth factor, platelet derived growth factor A chain, platelet derived growth factor AA, platfelet derived growth factor AB, platelet derived growth

factor B chain, platelet derived growth factor 33, platelet derived growth factor receptor, platelet derived growth factor receptor, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor (TG?) , TGF, TGF 1, TGF 1.2, TGF 2, TGF, 3, TGF, 5, latent TG?
I, TGF, binding protein I, TGF binding protein II,
TGF binding protein III, tumor necrosis factor
receptor type I, tumor necrosis factor receptor type
II, urokinase-type plasminogen activator receptor,
vascular endothelial growth factor, and chimeric
proteins and biologically or immunologically active
fragments thereof.
The compounds useful according to the invention may be conjugated or linked to auxiliary moieties that promote any property of the compounds that may be beneficial in methods of therapeutic use. Such conjugates can enhance delivery of the compounds to a particular anatomical site or region of interest (e.g., a tumor), enable sustained therapeutic concentrations of the compounds in target cells, alter pharmacokinetic and pharmacodynamic properties of the compounds, and/or improve the therapeutic index or safety profile of the compounds. Suitable auxiliary moieties include, for example, amino acids, aligopeptides, or polypeptides, e.g. , antibodies such as monoclonal antibodies and other engineered antibodies,- and natural or synthetic ligands to receptors in target cells or tissues. Other suitable auxiliaries include fatty acid or lipid moieties, to promote biodistribution or uptake of the compound by target cells (see, e.g., Bradley et al. , Clin. Cancer Res. (2001) 7:3229.

The therapeutic index of compositions comprising one or more compounds of the invention can be enhanced by conjugation of the compound(s) with antitumor antibodies as previously described (for example, Pieterse and McKinzie, Immunol. Rev. (1992) 129:57; Trail et al., Science (1993) 261:212; Rowlinscn-Bussa and Epenetos, Curr. Opin. Oncol. 1992; 4:1142). Tumor directed delivery of compounds of the invention would enhance the therapeutic benefit by minimizing potential nonspecific toxicities which can result from radiation treatment or chemotherapy. In another aspect, Chkl inhibitors and radioisotopes or chemotherapeutic agents can be conjugated to the same antibody molecule. Alternatively, Chkl inhibitor-conjugated tumor specific antibodies can be administered before, during, or after administration of chemotherapeutic-con3ugated antitumor antibody or radioimmunotherapy.
Compounds of the present invention can enhance the therapeutic benefit cf radiation and chemotherapy treatment, including induction chemotherapy, primary (neoadjuvant) chemotherapy, and both adjuvant radiation therapy and adjuvant chemotherapy. In addition, radiation and chemotherapy are frequently indicated as adjuvants to surgery in the treatment of cancer. The goal of radiation and chemotherapy in the adjuvant setting is to reduce the risk of recurrence and enhance disease-free survival when the primary tumor has been controlled. Chemotherapy is utilized as a treatment adjuvant for colon, lung, and breast cancer, frequently when the disease is metastatic. Adjuvant radiation therapy is indicated in several diseases including colon, lung, and breast cancers as 'described above. 'For example, radiation frequently is used both pre- and

posr-surgery as components of the treatment strategy for rectal carcinoma. Compounds for the present invention are therefore particularly useful following surgery in the treatment of cancer in combination with radio- and/or chemotherapy.
A compound of the present invention also can radiosensitize a cell. The term "radiosensi-tize, " as used herein, is defined as a molecule, preferably a low molecular weight molecule, administered to human or other animal in a therapeuci-cally effective amount to increase the sensitivity of rhe cells to be radiosensitized to electromagnetic radiation and/or to promote the treatment of diseases that are treatable with electromagnetic radiation. Diseases that are treatable with electromagnetic radiation include neoplastic diseases, benign and malignant tumors, and"cancerous cells.
Electromagnetic radiation treatment of other diseases not listed herein also is contemplated by the present invention. The terms "electromagnetic radiation" and "radiation" as used herein include, but are not limited to, radiation having the wavelength of 10-10 to 100 meters. Preferred embodiments of the present invention employ the electromagnetic radiation of: gamma-radiation (10-20 to 10-13 m) , X-ray radiation {10-1= to 10-9 m) , ultraviolet light (10 nm to 400 nm) , visible light (400 nm to 700 nm) , infrared radiation (700 nm to 1.0 mm) , and microwave radiation (1 mm to 30 cm) .
Many cancer treatment protocols currently employ radiosensitizers activated by electromagnetic radiation, e.g., X-rays. Examples of X-ray-activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole.

desmethylmisonidazole, pimonidasole, etanidazoie, nimorazole, mitomycin C, RSU 1069, SR 4233, EOS, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR) , 5-iododeoxyuridine (lUdR) , bromodeoxycytidine, flucro-deoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same.
Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator cf the sensitizing agent. Examples of photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, PHOTO-FRIN'^, benzoporphyrin derivatives, NPe6, tin etio-pcrphyrin {SnET2), pheoborbide-a, bacteriochloro-phyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same.
Radiosensitizers can be administered in conjunction with a therapeutically effective amount
*
of one ox more compounds in addition to the Chki inhibitor, such compounds including, but not limiited to, compounds that promote the incorporation cf radiosensitizers to the target cells, compounds that control the flow of therapeutics, nutrients, and/cr oxygen to the target cells, chemotherapeutic agents that act on the tumor with or without additional radiation, or other therapeutically effective compounds for treating cancer or other disease. Examples of additional therapeutic agents that can be used in conjunction with radiosensitizers include, but are not limited to, 5-fluorouracil C5-FU) , leucovorin, oxygen, carbogen, red cell transfusions, perfluorocarbons (e.g., FLUOSOLW-DA) , 2,3-DPG, BW12C, calcium channel blockers, pentoxifylline, antiangiogenesis compounds, hydralazine, and L-330.

Chemotherapeutic agents that can be used include, but are not limited to, alkylating agents, antimetabolites, hormones and antagonists thereof, radioisotopes, antibodies, as well as natural products, and combinations thereof- For example, an inhibitor compound of the present invention can be adminisrered with antibiotics, such as doxorubicin and other anthracycline analogs, nitrogen muscards, such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like. As another example, in the case of mixed tumors, such as adenocarcinoma of the breast, where the tumors include gonadotropin-dependent and gonadotropin- independent cells, the compound can be administered in conjunction with leuprolide or goserelin (synthetic peptide analogs of LH-RH) . Other antineoplastic protocols include the use of an inhibitor compound with another treatment modality, e.g., surgery or radiation, also referred to herein as "adjunct anti-neoplastic modalities." Examples of .chemotherapeutic agents useful for the method of the present invention are listed in the following table.

Examples of chematherapeutic agents that are particularly useful in conjunction with radio-

sensitizers include, for example, adriamycin, camptothecin, carhoplatin, cisplatin, daunorubicin, doxorubicin, interferon (alpha, beta, gamma), inrer-leukin 2, irinotecan, docetaxel, paclitaxel, topotecan, and therapeutically effective analogs and derivatives of the same.
As appreciated by persons skilled in the art, reference herein to treatment extends to prophylaxis, as well as to treatment of established diseases or s.ymptoms. It is further appreciated that the amount of a compound of the invention required for use in treatment varies with the nature of the condition being treated, and with the age and the condition of the patient, and is ultimately determined by the attendant physician or veterinarian. In general, however, doses employed for adult human treatment topically are in the range of 0.001 mg/kg to about 100 mg/kg per day. The desired dose can be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more subdoses per day. In practice, the physician determines the actual dosing regimen most suitable for an individual patient, and the dosage varies with the age, weight, and response of the particular patient. The above dosages are exemplary of the average case, but there can be individual instances in which higher or lower dosages are merited, and such are within the scope of the present invention.
Formulations of the present invention can be administered in a standard manner for the treatment of the indicated diseases, such as orally, parenterally, transmucosally (e.g., sublingually or via buccal administration), topically, transdetmal-ly, rectally, via inhalation (e.g., nasal or deep

lung inhalation) . Parenteral administration includes, but is not limited to intravenous, intraarterial, intraperitoneal, subcutaneous, intramascu-lar, intrathecal, and intraarticular. Parenteral administration also can be accomplished using a high pressure technique, like POWDERJECT'".
For oral administration, including buccal administration, the composition can be in the form of tablets or lozenges formulated in conventional manner. For example, tablets and capsules for oral administration can contain conventional excipients such as binding agents (tor example, syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch, or polyvinylpyrrolidone), fillers (for example, lactose, sugar, microcrystalline cellulose, maize-starch, calcium phosphate, or sorbitol), lubricants (for example, magnesium stearate, stearic acid, talc, polyethylene glycol or silica), disintegrants (for example, potato starch or sodium starch glycolate), or wetting agents (for example, sodium lauryl sulfate) . The tablets can be coated according to methods well known in the art.
Alternatively, the compounds of the present invention can be incorporated into oral liquid preparations such as aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, for example. Moreover, formulations containing these compounds can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can contain conventional additives, for example suspending agents, such as sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellu-lose, aluminum stearate gel, and .hydrogenated edible

fats; emulsifying agents, such as lecithin, sorbitan monooleate, or acacia; nonaqueous vehicles (which can include edible oils), such as almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol; and preservatives, such as methyl or propyl p-hydroxybenzoate and sorbic acid.
Such preparations also can be formulated as suppositories, e.g., containing conventional suppository bases, such as cocoa butter or other glycerides. Compositions for inhalation typically can be provided in the form of a solution, suspension, or emulsion that can be administered as a dry powder or in the form of an aerosol using a conventional propellant, such as dichlorodifluoromethane or trichlorofluoromethane. Typical topical and transdermal formulations comprise conventional aqueous or nonaqueous vehicles, such as eye drops, creams, ointments, lotions, and pastes, or are in the form of a medicated plaster, patch, or membrane.
Additionally, compositions of the present invention can be formulated for parenteral administration by injection or continuous infusion. Formulations for injection can be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulation agents, such as suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use.
A composition in accordance with the present invention also can be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example, subtutan-eously or intramuscularly) or by intramuscular in-

Compounds suitable in the method include, but are not limited to:

ulae (IV), (V), (VI), and (VII), can be prepared
acccrding to the following synthetic scheme. In the scheme described below, it is understood' in the art that protecting groups can be employed where necessary in accordance with general principles of synthetic chemistry. These protecting groups are removed in the final steps of the synthesis under basic, acidic, or hydrogenolytic conditions which are readily apparent to those skilled in the art. 3y employing appropriate manipulation and protection of any chemical functionalities, synthesis of compounds of structural formulae (I), (II), and (III) not specifically set forth herein can be accomplished by methods analogous to the schemes set forth below.
Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification. All reactions and chromatography fractions were analyzed by thin-layer chromatography on 250-mm silica gel. plates, visualized with UV (ultraviolet) light and I2,

by the formula HetAr-NH2, preferably under an inert atmosphere at room temperature (about 25oC), to afford a crude aryl pyrazine-disubstituted ureS compound.

A more particular illustration for the preparation compounds of standard formulae (I) and (II) can include, for example, the following General Scheme 2
Step (1); TMS Diazomethane Esterification
To a cooled (about 0°C), stirred solution of 4-amino-3-methcxyben2oic acid (5.0 g; 3 0 mmol) in dry methanol (15 0 mL) was added trimethylsilyl diazomethane (60 mL of 2.0 M solution in hexanes, 120 mmol) slowly over 1 hour. After stirring for 4 hours, the reaction was concentrated at reduced pressure, dissolved in ethyl acetate (200 mL), washed with 10% aqueous sodium carbonate and brine, then dried (MgSO4) , filtered, and concentrated in

vacuo CO provide the desired ester as an off-whize solid (94% yield).
Step (2) : D-Nitrophenvl Carbamate Procedure
To a stirred, cooled (about 0°C) solurion of merhyi-3-amino-4-methoxy benzoate (5.0 g; 27.5 mmol) in dry dichloromethane (175 mL) was added pyridine (2.34 mL; 29 mmol) followed by 4-nitro-phenyl chloroformate (5.8 g; 29 mmol) under a nitrogen (N2) atmosphere. After stirring for S hours, the reaction was washed with 2N aqueous hydrochloric acid (2 X 200 mL), saturated aqueous sodium bicarbonate (2 X 200 mL) , and brine (200 mL) , then dried (MgSO4) and filtered. The filtered solution was diluted with ethyl acetate and hexanes (about 600 mL) until a precipitate formed. The solid was collected on a Buchner funnel with suction, and air dried to provide the desired carbamate as a white solid (70% yield).
Step (3): Carbamate Coupling Procedure
To a stirred solution of 4-methoxy-3-(4-nirro-phenoxycarbonylamino) -benzoic acid methyl ester (30 g; 8.7 mmol) in dry N-methyl pyrrolidine (50 mL) was added the amino pyrazine (0.84 g; 8.8 mmol) under a N2 atmosphere at room temperature. The reaction mixture was heated to 80oC for 6 hours, then allowed to cool to room temperature. Dilution with ethyl acetate (200 mL) and water (200 mL) provided the desired urea as a white solid (54% yield) .

The following compounds were prepared using the general procedure described accompanying

General Scheme 2, but substituting the R group below for the R group shown in General Scheme 2:

Isocvanate Procedure;
To a stirred solution of 2-methoxy-5-meth-yl-phenylisocyanate (43 mL; 0,3 mmol) in dry di-chicroechane (0.4 mL) was added 2-arr.inoquinoxaline (43.5 mg; 0.3 mmol) in a reaction vial under a nitrogen atmosphere. ' The vial was capped and heated to 80°C overnight (14 hours). The reaction mixture then was filtered, and the residue washed with di-chloromethane to provide the desired urea as a white solid Ol% yield) .
The following compounds were prepared using the procedure described accompanying General Scheme 3, but substituting the Ar group in the table below for the Ar group in General Scheme 3:

ethane {20 mL) . The reaction mixture was cooled to room temperature to precipitate the product, which was collected by filtration, washed with ethyl

Example 4 Preparation of Compound 14

Example 5 Preparation of Compound 4 8

(S) -1- (2,2,2-trifluoroethanoyl)pyrrolidine-2-carboxylic acid [4-methoxy-3-(3-pyra2in-2-yl-
ureido)phenyl] -amide
A solution of 1-(5-amino-methoxyphenyl)-3-pyra2in-2-yl-urea (Compound 14, Example 4) (105 ma, 0.4 mmol) in dry pyridine (2 mL) at 0oC was treated with a solution of N-trifluoroacetyl-(S)-prolyl .chloride (0,1 M in dichloromethane, 4,5 mL, 0.45 mmol) and stirred 2 h at room temperature. The reaction was quenched with 1 N HCl (50 mL) and extracted with ethyl acetate (3 X 50 mL) . The

Example 7 Preparation of Compound 16

Example 8 Preparation of Compound 42

N' [4-methoxy-3- (3-pyrazin-2-yl-ureido)phenyl] -
methanesulfonamide
A solution of 1-(5-amino-2-methoxyphenyl)-3-pyrazin-2-yl-urea (Compound 14, Example 4) (260 mg, 1 mmol) in dry pyridine (15 mL) was treated with methanesulfonyl chloride (0.08 mL, 1 mmol) and

Example 9
Preparation of Compound 65

3-chloro-N- [3-methoxy-4- (3-pyrazin-2-yl-ureido)phenyl]-propionamide
A solution of 1-{4-amino-2-methoxyphenyl)-3-pyra2in-2-yl-urea (Compound 32, Example 10) (259 mg, 1 mmol) in pyridine (3 mL) at 0oC was treated with chloroacetyl chloride (0.29 mL, 3 mmol). The suspension was warmed at 80°C until most solids dissolved, the reaction-mixture was cooled to room temperature and the product was precipitated with

Example 13 Preparation of Compound 4

(cyclohexyl-methyl-aoiino) -N- [3-methoxy-4-(3-pyrazin-2-yl-ureido)phenyl] -propionamide

LRMS (csi. positive) m/e 435.2 (M-1)

Prepared according to the procedure of Compound 1S9 using C-(lH-ben2oinuda2oI-2-yl)-inethylaminc (26% yield).

a solid and the mixture heated to 65 "C. After reaching temperaruie, the suspension gradually became a bright yellow solution. After about 4 hours a precipitate formed but the reaction was continued overnight. After cooling to RT, MeOH was removed by roiovap and the aqueous suspension diluted

N-(3-Dimethylamino-propyl)-3-mcthoxy-4-[3-(5-methyl-pyrazin-2-yl)-ureido]-benzamide

Compound 216:

be made without departing from the spirit and scope thereof, and, thereore, only such limitation
should be imposed as are indicated by the appended claims.

WHAT IS CLAIMED IS:
1.Amethod inhibiting checkpoint kinease in a cell comprising
a step of consisting the cell with an effective amount of a compound of
formula

f wherein X2 is null, -O-, -S-, -CH2-, or
-N(R-)-;
X- is-O-, -S-, or -N(R1)-;
Y is O or S; or =Y representative two hydrogen
atoms attached to a common carbon atom;
W is selected from the group consisting of
C1-3 alkyl substituted with a heterc-aryl Or aryl group;
Z is selected from the group consisting of hydro, aryl, and heteroaryl;
wherein said aryl croups of w and 2 are optionally substituted with one to four substicuents represented by R2, said heteroaryl groups of W and Z are optionally substituted vith one to four substit-uents represented by R-, and said heterocycloalkyl and cycloalkyl groups of V? are optionally substituted with one to two substituents represented by R5;

carcinoma, an ovarian cancer, a brain tumor, an
osteosarcoma, and a lung carcinoma.

27. A method od sensitising cells in an
individual undergoing a chemotherapeutic or radio-' therapeutic treatment for a medical condition, com-prising administering a therapeutically effective
amount of a compound of claim 19 in combination with
a chemotherapeutic agent, a radiotherapeutically effective
amount of a compound of claim 19 in combination with
a chemotherapeutic agent, a radiotherapeutc agemt,
or a mixture thereof to the individual.

optionally substituted with from one to four sub-stituents selected from the croup consisting of

31. A method of inhibiting checkpoint kinase 1 in a cell substantially
as herein described and exemplified.
32. A composition substantially as herein described and exemplified.
Dated this 28 day of August 2003

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Patent Number

227445

Indian Patent Application Number

1354/CHENP/2003

PG Journal Number

10/2009

Publication Date

06-Mar-2009

Grant Date

07-Jan-2009

Date of Filing

28-Aug-2003

Name of Patentee

ICOS CORPORATION

Applicant Address

22021 20TH AVENUE S.E, BOTHELL, WASHINGTON 98201,

Inventors:

#	Inventor's Name	Inventor's Address
1	GAUDINO, JOHN, JOSEPH	4224 PRAIRIE FIRE CIRCLE, LONGMONT, COLORADO 80503,
2	COOK, ADAM, WADE	3381 LARKSPUR DRIVE, LONGMONT, COLORADO 80503,
3	BURGESS, LAURENCE, EDWARD	5562 HIGH COUNTRY COURT, BOULDER, COLORADO 80301,
4	KEEGAN, KATHLEEN, S	5812 W MERCER WAY, MERCER ISLAND, WASHINGTON 98040,
5	KESICKI, EDWARD, A	2504 208TH PLACE SE, BOTHELL, WASHINGTON 98021,
6	COWEN, SCOTT, DOUGLAS	2119 MALLARD PLACE, LONGMONT, COLORADO 80504,

PCT International Classification Number

C07D213/30

PCT International Application Number

PCT/US02/06452

PCT International Filing date

2002-03-01

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/273,124	2001-03-02	U.S.A.