Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/4965—Non-condensed pyrazines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
  • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
  • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
  • C07D241/14—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D241/20—Nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

• the present invention relates to compounds useful for inhibiting enzymes that maintain and re- pair the integrity of genetic material. More par ⁇ ticularly, 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 de ⁇ oxyribonucleic acid (DNA) replication, chromosome segregation, or cell division.
• DNA de ⁇ oxyribonucleic acid
• aberrantly proliferating cells means cell proliferation that deviates from the normal, proper, or expected course.
• aberrant cell proliferation includes inappropriate proliferation of cells wherein DNA or other cellular components have become damaged or defective.
• Aberrant cell proliferation also includes indications caused by, mediated by, or resulting in inappropriately high levels of cell division, inappropriately low levels of cell death (e.g., apoptosis) , or both.
• Such indications can be characterized, for example, by single or multiple local abnormal proliferations of cells, groups of cells or tissue(s), and include cancerous (benign or malignant) and noncancerous indications.
• cancerous benign or malignant
• noncancerous indications include all cancers (benign and malignant) involve some form of aberrant cell pro ⁇ liferation.
• Nonlimiting examples include carcinomas and sarcomas. Others are discussed below.
• Some noncancerous indications also involve aberrant cell proliferation.
• Nonlimiting examples of noncancerous indications involving aberrant cell proliferation include rheumatoid arthritis, psoriasis, vitiligo, Wegener's granulomatosis, and systemic lupus. Others are discussed below.
• DNA damaging agents are designed to kill aberrantly proliferating cells by disrupting vital cellular processes such as DNA metabolism, DNA synthesis, DNA transcription, and microtubule spindle formation. They also can oper ⁇ ate, for example, by introducing lesions into DNA that perturb chromosomal structural integrity. DNA damaging agents are designed and administered in ways that attempt to induce maximum damage and con ⁇ sequent cell death in aberrantly proliferating cells with a minimum damage to normal, healthy cells.
• DNA damaging agents include chemother- apeutics and radiation.
• chemother- apeutics and radiation.
• the effec- tiveness of DNA damaging agents in treating condi ⁇ tions involving aberrant cell proliferation have been less than desired, particularly in the treat ⁇ ment of cancer.
• the selectivity of such agents for aberrantly proliferating cells over healthy cells often is marginal.
• the chemotherapeutic agent called GemzarTM (gemcitabine, or 2 ',2' difluoro-2 1 - deoxycytidine) damages DNA by incorporating itself into DNA during synthesis. Left unrepaired, damaged DNA generally is rendered incapable of sustaining - A -
• cell cycle checkpoints detect the improperly made (or otherwise damaged) DNA.
• the activated cell cycle checkpoints trigger cell cycle arrest for a time sufficient to allow damaged DNA to be repaired.
• DNA-dam- aging agents such as chemotherapeutics, radiation, and other therapies.
• Other DNA-damaging agents cause tumor cells to arrest in S-phase. Tumor cells have been observed to resist certain chemotherapeutics simply by arresting in S phase while the chemotherapeutic agent is being administered.
• checkpoint activators DNA damaging agents that activate cell cycle checkpoints generally are referred to herein as "checkpoint activators.” DNA damaging agents that activate the checkpoint designated “Chkl” (pronounced “check- one") are referred to herein as “Chkl activators.” Inhibitors of such checkpoints, generally and spe ⁇ cifically, are referred to herein as “checkpoint inhibitors” and “Chkl inhibitors,” respectively.
• the cell cycle is structurally and func ⁇ tionally the same in its basic process and mode of regulation across all eukaryotic species.
• the mi ⁇ totic (somatic) cell cycle consists of four phases: the Gl (gap) phase, the S (synthesis) phase, the G2 (gap) phase, and the M (mitosis) phase.
• the Gl, S, and G2 phases are collectively referred to as inter- phase of the cell cycle.
• the Gl phase bio- synthetic activities of the cell progress at a high rate.
• the S phase begins when DNA synthesis starts, and ends when the DNA content of the nucleus of the cell has been replicated and two identical sets of chromosomes are formed.
• the cell then enters the G2 phase, which continues until mitosis starts.
• mitosis the chromosomes pair and separate, two new nuclei form, and cytokinesis occurs in which the cell splits into two daughter cells each receiving one nucleus con ⁇ taining one of the two sets of chromosomes.
• Cyto ⁇ kinesis terminates the M phase and marks the begin ⁇ ning of interphase of the next cell cycle.
• the se ⁇ quence in which cell cycle events proceed is tightly regulated, such that the initiation of one cell cycle event is dependent on the completion of the prior cell cycle event. This allows fidelity in the duplication and segregation of genetic material from one generation of somatic cells to the next.
• cell cycle checkpoints comprise at least three distinct classes of polypeptides, which act sequentially in response to cell cycle signals or defects in chromosomal mechanisms (Carr, A.M., Science, 272:314-315 (1996)) .
• the first class is a family of proteins that detect or sense DNA damage or abnormalities in the cell cycle. These sensors include Ataxia- telangiectasia Mutated protein (Atm) and Ataxia- Telangiectasia Rad-related protein (Atr) .
• the second class of polypeptides amplify and transmit the signal detected by the detector and is exempli ⁇ fied by Rad53 (Alen et al . Genes Dev. 8:2416-2488 (1994)) and Chkl.
• a third class of polypeptides includes cell cycle effectors, such as p53, that mediate a cellular response, for example, arrest of mitosis and apoptosis.
• Noncancerous tissue which has intact cell cycle checkpoints, typically is insulated from tem- porary disruption of a single checkpoint pathway.
• Tumor cells however, have defects in pathways con ⁇ trolling cell cycle progression such that the per ⁇ turbation of additional checkpoints renders them particularly sensitive to DNA damaging agents.
• 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 check ⁇ point (Bunz et al. , Science, 282:1497 501, 1998) .
• Atm and Atr initiate a signal transduction pathway leading to cell cycle arrest.
• Atm has been shown to play a role in a DNA damage checkpoint in response to ionizing radiation (IR) .
• IR ionizing radiation
• Atr is stimulated by agents that cause double strand DNA breaks, single strand DNA breaks, and agents that block DNA radia- tion.
• Chkl is a protein kinase that lies down ⁇ stream from Atm and/or Atr in the DMA damage check ⁇ point signal transduction pathway (Sanchez et al., Science, 277:1497 1501, 1997; U.S. Patent No. 6,218,109) .
• Chkl is phosphor- ylated in response to agents that cause DNA damage including ionizing radiation (IR) , ultraviolet (UV) light, and hydroxyurea (Sanchez et al. , supra; Lui et al., Genes Dev. , 24:1448 1459, 2000) .
• This phos- phorylation which activates Chkl in mammalian cells is dependent on Atm (Chen et al . , Oncogene, 28:249- 256, 1999) and Atr (Lui et al. , supra) .
• Chkl has been shown to phosphorylate both weel (O'Connell et al. , BMBO J., 16:545 554, 1997) and Pdsl (Sanchez et al. , Science, 286:1166
• Chkl invokes an S-phase arrest by phosphorylating Cdc25A, which reg ⁇ ulates cyclinA/cdk2 activity ( Xiao et al. , supra and Sorensen et al. , supra) . Chkl also invokes a G2 arrest by phosphorylating and inactivating Cdc25C, the dual specificity phosphatase that normally de- phosphorylates cyclin-B/cdc2 (also known as Cdkl) as cells progress from G2 into mitosis (Fernery et al. , Science, 277:1495 7, 1997; Sanchez et al. , supra,- Matsuoka et al.
• UCN-01 is a nonselective Chkl inhibitor.
• UCN-01 is toxic to cells at high doses.
• it nonspecifi- cally inhibits many cellular kinases and also inhib ⁇ its the Gl checkpoint (Tenzer and Pruschy, Curr. Med Chem. Anti-Cancer Agents, 3:35-46, 2003) .
• UCN-01 has been used in conjunction with cancer therapies, such as radiation, the anti-cancer agent camptothecin (Tenzer and Pruschy, supra) , and gemcitabine (Shi et al. , supra), with limited suc ⁇ cess.
• cancer therapies such as radiation, the anti-cancer agent camptothecin (Tenzer and Pruschy, supra) , and gemcitabine (Shi et al. , supra), with limited suc ⁇ cess.
• UCN-01 also has been used to potentiate the effects of temozolomide (TMZ) induced DNA mismatch repair (MMR) in glioblastoma cells (Hirose et al. , Cancer Res., 52:5843-5849, 2001) .
• TMZ temozolomide
• MMR DNA mismatch repair
• Caffeine a meth- ylxantriine
• Caffeine has also been used to enhance cytotox ⁇ icity of DNA-damaging agents, such as cis-platin and ionizing radiation, by mediating progression through the G2 checkpoint and thereby inducing cell death.
• DNA-damaging agents such as cis-platin and ionizing radiation
• the dose of caffeine used to accom ⁇ plish the cell cycle abrogation exceeds clinically acceptable levels and is not a viable therapeutic option.
• antisense nucleotides to Chkl kinase have been used to increase sensitivity to the topoisomerase inhibitor BNP1350 (Yin et al. , Bio- chem. Biophys. Res. Conwnun., 295:435-44, 2002), but demonstrate problems typically associated with anti- sense treatment and gene therapy.
• Chkl inhibitors have been disclosed in WO 02/070494, WO 04/014876, and WO 03/101444.
• Addi- tional Chkl inhibitors include diarylurea compounds, e.g., aryl- and heteroaryl-substituted urea com ⁇ pounds disclosed in U.S. Patent Publication No. 2003-0069284 Al; methylxanthines and related com ⁇ pounds (Fan et al.
• ureidothiophenes (WO 03/029241) ; N-pyrrolopyridinyl carboxamides (WO 0/28724) ; antisense Chkl nucleo ⁇ tides (WO 01/57206) ; Chkl receptor antagonists (WO 00/16781) ; heteroaromatic carboxamide derivatives (WO 03/037886) ; aminothiophenes (WO 03/029242) ; (indazolyl)benzimidazoles (WO 03/004488); hetero ⁇ cyclic-hydroxyimino-fluorenes (WO 02/16326) ; scyto- neman skeleton-containing derivatives (scytonemin) (U.S.
• the present invention relates to potent and selective inhibitors of the checkpoint kinase Chkl.
• the present Chkl inhibitors are useful in treating indications involving aberrant cell pro ⁇ liferation, and as chemosensitizing and radiosensi- tizing agents in the treatment of indications re ⁇ lated to DNA damage or lesions in DNA replication.
• one aspect of the present in ⁇ vention is to provide compounds of structural for ⁇ mula (I) .
• the compounds are useful in a method of inhibiting Chkl comprising a step of administering an effective amount of a compound of structural for ⁇ mula (I) to an individual.
• X 1 is null, -0-, -S-, -CH 2 -, or -N(R 1 )-;
• X 2 is -O-, -S-, or -N(R 1 )-;
• W is selected from the group consisting of heteroaryl, aryl, heterocycloalkyl, cycloalkyl, and Ci- 6 alkyl substituted with a heteroaryl or aryl group, wherein said aryl group W is optionally sub- stituted with one to four substituents represented by R 2 , said heteroaryl group W is optionally substi ⁇ tuted with one to four substituents represented by R 5 , and said heterocycloalkyl and cycloalkyl groups W are optionally substituted with one or two Cx.galkyl substituents;
• R 1 is selected from the group consisting of hydro, C 3. ._ 6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, and aryl;
• R 2 is selected from the group consisting of heteroaryl, halo, optionally substituted Ci_ 6 alkyl, C 2 -salkenyl, OCF 3 , NO 2 , CN, NC, N(R 3 ) 2 , OR 3 , CO 2 R 3 , C (O)N(R 3 ) 2/ C(O)R 3 , N(R 1 JCOR 3 , N(R 1 JC(O)OR 3 , N(R 1 )- C (0) Ci-galkyleneC(0)R 3 , N(R 1 )C(0) Ci_ 6 alkyleneC(0)OR 3 , N(R 1 ) C (0) Ci_ 6 alkylene0R 3 , N(R 1 )C(0) Ci_ 6 alkyleneNHC- (O)OR 3 , N(R 1 )C(O)Ci- 6 alkyleneSO 2 NR 3 , Cx.galkyleneOR 3 , and SR 3 ;
• R 3 is selected from the group consisting of hydro, C ⁇ salkyl, C 2 - 6 alkenyl, cycloalkyl, aryl, het ⁇ eroaryl, SO 2 R 4 , halo, Cx-galkyl substituted with one or more of halo, hydroxy, aryl, heteroaryl, hetero- cycloalkyl, N(R 4 ) 2/ and SO 2 R 4 , Ci-ealkylenearyl,
• R 4 is selected from the group consisting of null, hydro, Ci_ 6 alkyl, cycloalkyl, aryl, heteroaryl, Ci- 6 a-lkylenearyl, and SO 2 Ci- 6 alkyl, or two R 4 groups are taken together to form an optionally substituted 3- to 8-membered ring;
• R 6 is selected from the group consisting of hydro, C h alky!, C 2 _ 6 alkenyl, cycloalkyl, hetero- cycloalkyl, aryl, heteroaryl, SO 2 R 4 , Ci- 6 alkyl sub ⁇ stituted with one or more of halo, hydroxy, aryl, heteroaryl, heterocycloalkyl, N(R 4 ) 2 , and SO 2 R 4 , Ci_ 6 alkylenearyl, Cx-galkyleneheteroaryl, Ci- 6 alkylene- C 3 - 8 heterocycloalkyl, Ci_ s alkyleneSO 2 aryl, optionally substituted Ci_ 6 alkyleneN(R 4 ) 2/ OCF 3 , Ci_ 6 alkylene- N(R 4 ) 3 + , C 3 . 8 heterocycloalkyl, and CH(Ci_ 6 alkylene- N(R 4 ) 2 ) 2 ;
• R 7 and R 8 are selected from the group consisting of hydro, C; L - 6 alkyl, halo, OR 3 , N(R 3 J 2 , C(O)N(R 3 J 2 , C ⁇ alkylenearyl, CN, NO 2 , C(O)OR 11 , C(O)R 11 , and SR 11 ;
• R 9 is -C ⁇ C-R 10 or -CF 3 , or an R 8 and an R 9 group are taken together with the carbons to which they are attached to form a 5- or 6-membered carbo- cyclic aliphatic or aromatic ring system optionally containing one to three heteroatoms selected from the group consisting of O, NR 4 , and S;
• R 10 is selected from the group consisting of hydro, Ci_ 6 alkyl, aryl, Ci_ s alkylenearyl, hetero- aryl, and Ci_ 6 alkyleneheteroaryl;
• R 11 is selected from the group consisting of hydro, ⁇ Ci- 6 alkyl, C 2 - 6 alkenyl, aryl, C ⁇ - 3 alkylene- aryl, C 3 . 8 cycloalkyl, and C 2 _ 3 alkyleneC 3 _ 8 cycloalkyl;
• n is 1 or 2; or a pharmaceutically acceptable salt, or a prodrug, or a solvate thereof.
• a method comprises contacting a cell population com ⁇ prising aberrantly proliferating cells with at least one Chkl activator in an amount and for a time suf ⁇ ficient to substantially synchronize cell cycle arrest among the aberrantly proliferating cells.
• the cell popu ⁇ lation is contacted with at least one Chkl inhibitor in an amount and for a time sufficient to substan ⁇ tially abrogate the cell cycle arrest.
• X 1 is null, -0-, -S-, -CH 2 -, or
• X 2 is -O- , -S- , or -N (R 1 ) - ;
• W is selected from the group consisting of heteroaryl, aryl, heterocycloalkyl, cycloalkyl, and Ci_ 6 alkyl substituted with a heteroaryl or aryl group, wherein said aryl group W is optionally sub ⁇ stituted with one to four substituents represented by R 2 , said heteroaryl group W is optionally substi- tuted with one to four substituents represented by
• R 5 and said heterocycloalkyl and cycloalkyl groups W are optionally substituted with one or two Ci- 6 a-lkyl substituents,-
• R 1 is selected from the group consisting of hydro, Ci_ 6 alkyl, C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, and aryl;
• R 2 is selected from the group consisting of heteroaryl, halo, optionally substituted Ci_ 6 alkyl, C 2 - 6 alkenyl, OCF 3 , NO 2 , CN, NC, N(R 3 ) 2 , OR 3 , CO 2 R 3 , C(O)N(R 3 J 2 , C(O)R 3 , N(R 1 JCOR 3 , N(R 1 JC(O)OR 3 , N(R 1 )- C(O)Ci- 6 alkyleneC(O)R 3 , N(R 1 ) C(0)Ci_ 6 alkyleneC(0)OR 3 , N(R 1 )C(0)C(0)C(0)OR 3 , N(R 1 )C(0)Cx-ealkyleneOR 3 , N(R 1 )C(0)Ci_ 6 alkyleneNHC- (O)OR 3 , N(R 1 )C(O)Ci- 6 alkyleneSO 2 NR 3 , Cx-ealkyleneOR
• R 3 is selected from the group consisting of hydro, Ci_ 6 alkyl, C 2 _ 6 alkenyl, cycloalkyl, aryl, het ⁇ eroaryl, SO 2 R 4 , halo, Ci- 6 alkyl substituted with one or more of halo, hydroxy, aryl, heteroaryl, hetero ⁇ cycloalkyl, N(R 4 ) 2 , and SO 2 R 4 , Ci_ 6 alkylenearyl, Ci- 6 alkyleneheteroaryl, Ci-galkyleneCs-gheterocyclo- alkyl, Ci_ 6 alkyleneS0 2 aryl, optionally substituted Ci -6 alkyleneN(R 4 ) 2 , OCF 3 , C 1 .
• alkyleneN(R 4 ) 3 + , C 3 - 8 het- erocycloalkyl, and CH(C ! _ 6 alkyleneN(R 4 ) 2 )2/ or two R 3 groups are taken together to form an optionally sub ⁇ stituted 3- to 8-membered aliphatic ring;
• R 4 is selected from the group consisting of null, hydro, Ci_ 6 alkyl, cycloalkyl, aryl, heteroaryl, Ci- 6 alkylenearyl, and SO 2 Ci- 6 alkyl, or two R 4 groups are taken together to form an optionally substituted 3- to 8-membered ring /
• R 5 is selected from the group consisting of Cx-galkyl, C 2 - 6 alkynyl, aryl, heteroaryl, heterocycloalkyl, N(R 3 ) 2/ N(R 1 JC(O)R 3 , N(R 1 JCO 2 R 3 , OR 3 , halo, N 3 , CN, Ci- S alkylenearyl, C 1 . s alkyleneN(R 3 ) 2 , C(O)R 3 , C(O)OR 3 , C(O)N(R 3 J 2 , CF 3 , and
• R s is selected from the group consisting of hydro, C;i . _ 6 alkyl, C 2 _ 6 alkenyl, cycloalkyl, hetero ⁇ cycloalkyl, aryl, heteroaryl, SO 2 R 4 , C ⁇ lky! sub ⁇ stituted with one or more of halo, hydroxy, aryl, heteroaryl, heterocycloalkyl, N(R 4 ) 2 , and SO 2 R 4 ,
• R 7 and R 8 are selected from the group consisting of hydro, C h alky!, halo, OR 3 , N(R 3 ) 2/ C(O)N(R 3 ) 2 , C ⁇ -aalkylenearyl, CN, NO 2 , C(O)OR 11 , C(O)R 11 , and SR 11 ;
• R 9 is -C ⁇ C-R 10 or -CF 3 , or an R 8 and an R 9 group are taken together with the carbons to which they are attached to form a 5- or 6- ⁇ nembered carbo- cyclic aliphatic or aromatic ring system optionally containing one to three heteroatoms selected from the group consisting of 0, NR 4 , and S;
• R 10 is selected from the group consisting of hydro, Ci- 6 alkyl, aryl, Ci_ 6 alkylenearyl, hetero- aryl, and Ci- ⁇ alkyleneheteroaryl;
• R 11 is selected from the group consisting of hydro, Ci- 6 alkyl, C 2 - 6 alkenyl, aryl, Ci- 3 alkylene- aryl, C 3 _ 8 cycloalkyl, and C- L -salkyleneCs-scycloalkyl; n is 1 or 2; or pharmaceutically acceptable salts, or prodrugs, or solvates thereof.
• Preferred compounds of the present inven ⁇ tion are those wherein X 1 and X 2 are -N(H)-; Y is 0 or S; and
• W is optionally substituted heteroaryl.
• W is heteroaryl containing at least two heteroatoms selected from the group con ⁇ sisting of N, 0, and S, said heteroaryl ring op- tionally substituted with one to four substituents selected from the group consisting of optionally substituted Ci- 6 alkyl, aryl, heteroaryl, N(R 3 ) 2 , OR 3 , C(O)N(R 3 J 2 , CO 2 R 3 , CN, CF 3 , and halo, wherein R 3 is as previously defined.
• W is selected from the group consisting of pyridazinyl, pyrimidinyl, pyra- zinyl, and triazinyl, optionally substituted with one to four substituents selected from the group consisting of Ci_ 6 alkyl, aryl, heteroaryl, N(R 3 ) 2 , C(O)N(R 3 ) 2, CO 2 R 3 , OR 3 , CF 3 , and halo.
• Additional preferred compounds of struc ⁇ tural formula (I) are those wherein wherein R 6 is selected from the group consisting of optionally substituted Ci_ 6 alkyl, Ci_ 6 alkyleneN(R 4 ) 2 , Ci_ 6 alkylene- heteroaryl, Ci- ⁇ alkyleneheterocycloalkyl, and C 3 _ 8 het- erocycloalkyl.
• R 5 is selected from the group consisting of Ci_ 6 alkyl, (CH 2 )L 6 N(CHs) 2 , (CH 2 )X-SNH(CH 3 ),
• W is se ⁇ lected from the group consisting of
• W is any substituent selected from the group consisting of Ci_ 6 alkyl, C 2 - ⁇ ⁇ alkynyl, aryl, heteroaryl, CN, CO 2 R 3 , N(R 3 ) 2 , OR 3 , CF 3 , and halo.
• W is any substituent selected from the group consisting of Ci_ 6 alkyl, C 2 - ⁇ ⁇ alkynyl, aryl, heteroaryl, CN, CO 2 R 3 , N(R 3 ) 2 , OR 3 , CF 3 , and halo.
• W is any substituent selected from the group consisting of Ci_ 6 alkyl, C 2 - ⁇ ⁇ alkynyl, aryl, heteroaryl, CN, CO 2 R 3 , N(R 3 ) 2 , OR 3 , CF 3 , and halo.
• W is any substituent selected from the group consisting of Ci_ 6 alkyl, C 2 - ⁇ ⁇ alkynyl,
• R 5 is CF 3 , CH 3 , or null.
• R 7 is H
• R 8 is H
• R 9 is selected from the group consisting of -C ⁇ CH and CF 3 ; or R 8 and R 9 are taken together with the carbons to which they are attached to form
• R 6 is selected from the group consisting of - (CH 2 ) 2 N(CH 3 ) 2 ,
• alkyl includes straight chained and branched hydrocarbon groups containing the indicated number of carbon atoms, typically methyl, ethyl, and straight chain and branched propyl and butyl groups. Unless otherwise indicated, the hydrocarbon group can contain up to 20 carbon atoms.
• the term “alkyl” includes "bridged alkyl,” i.e., a C 6 -C 16 bicyclic or polycyclic hydro ⁇ carbon group, for example, norbornyl, adamantyl, bicyclo [2.2.2] octyl, bicyclo [2.2.1]heptyl, bicyclo- [3.2.1] octyl, or decahydronaphthyl.
• Alkyl groups optionally can be substituted, for example, with hydroxy (OH), halo, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, amino (N(R 3 ) 2 ), and sulfonyl (SO 2 R 3 ) , wherein R 3 is as previously defined.
• cycloalkyl is defined as a cyclic C 3 - 8 hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, or cyclopentyl.
• Hetero ⁇ cycloalkyl is defined similarly as cycloalkyl, except the ring contains one to three heteroatoms independently selected from the group consisting of oxygen, nitrogen, and sulfur.
• Heterocycloalkyl groups optionally can be further N-substituted with C 1-6 - alkyl, hydroxyC;i . - 6 alkyl, or Ci_ 3 alkyleneheteroaryl .
• alkenyl is defined identically as “alkyl,” except the group contains a carbon- carbon double bond.
• alkynyl is defined identically as “alkyl,” except the group contains a carbon- carbon triple bond.
• alkylene refers to an alkyl group having a substituent.
• C 1 - 6 alkyleneC(O)OR refers to an alkyl group con ⁇ taining one to six carbon atoms substituted with a -C(O)OR group.
• the alkylene group is optionally substituted with one or more substituent previously listed as an optional alkyl substituent.
• halo or halogen is defined herein as fluorine, bromine, chlorine, and iodine.
• aryl alone or in combination, is defined herein as a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, e.g., phenyl or naphthyl. Unless otherwise indicated, an aryl group ' can be unsubsti- tuted or substituted with one or more, and in par ⁇ ticular one to four groups independently selected from, for example, halo, Ci_ 5 alkyl, C 2 - 6 alkenyl, OCF 3 , NO 2 , CN, NC, N(R 3 ) 2 , OR 3 , CO 2 R 3 , C(O)N(R 3 ) 2 , C(O)R 3 , N(R 1 ) COR 3 , N(R 1 JC(O)OR 3 , N(R 1 ) C (0)OR 3 , N(R 1 )C(0)C(0)OR 3 , N(R 1 )C(0)Ci-salkyleneC(0)R 3 , N(R 1
• arylCi_ 3 alkyl and “heteroarylCi- 3 alkyl” are defined as an aryl or heteroaryl group having a C ⁇ - 3 alkyl substituent.
• heteroaryl is defined herein as a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring. Unless otherwise indicated, a heteroaryl group can be unsubstituted or substituted with one or more, and in particular one to four, substituents selected from, for example, Ci_ 6 alkyl, aryl, hetero ⁇ aryl, CF 3 , CN, C(O)N(R 3 ) 2 , CO 2 R 2 , N(R 3 ) 2 , OR 3 , and halo, wherein R 3 is as previously defined.
• heteroaryl groups include, but are not limited to, thienyl., furyl, pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazinyl, triazolyl, iso- thiazolyl, isoxazolyl, imidizolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl .
• hydroxy is defined as -OH.
• nitro is defined as -NO 2 .
• cyano is defined as -CN.
• isocyano is defined as -NC.
• trifluoromethoxy is defined as -OCF 3 .
• zido is defined as -N 3 .
• 3- to 8-membered ring refers to carbocyclic and heterocyclic ali ⁇ phatic or aromatic groups, including, but not lim ⁇ ited to, morpholinyl, piperidinyl, phenyl, thio- phenyl, furyl, pyrrolyl, imidazolyl, pyrimidinyl, and pyridinyl, optionally substituted with one or more, and in particular one to three, groups exem- ⁇ plified above for aryl groups.
• Carbon atom content of hydrocarbon- containing moieties is indicated by a subscript designating the minimum and maximum number of carbon atoms in the moiety, e.g., "Ci-galkyl” refers to an alkyl group having one to six carbon atoms, inclu ⁇ sive.
• the substituent is methyl, for example,
• the carbon atom contains the appropriate number of hydrogen atoms.
• the sub- stituent is understood to be hydrogen, e.g.,
• R-C Il is R-C Il-H and R-N is R-NH 2
• Chkl inhibitor means any compound, known or after-discovered whether naturally occurring or synthetic, that is capable of at least partially abrogating cell cycle checkpoint activity of the Chkl protein. Abrogation of cell cycle checkpoint is achieved when the cellular checkpoint mecha ⁇ nism(s) is (are) overcome sufficiently to allow the cell to pass from the cell cycle phase in which it is halted to the next phase in the cell cycle or to allow the cell to pass directly to cell death. Abrogation of the cell cycle checkpoint permits cells to carry damage or imperfections to subsequent cell cycle phases, thereby inducing or promoting cell death. Cell death can occur by any mechanism, including apoptosis and mitotic catastrophe.
• Chkl activator means any known or after- discovered agent having the ability to activate Chkl kinase activity in DNA repair and homeostasis at cell cycle checkpoints, and thus induce at least partial cell cycle arrest.
• Chkl activators include agents capable of arresting the cell cycle at any phase of the cell cycle, which phase may be referred to herein as the "target phase” for that activator.
• Target phases include any of the cell cycle phases except mitosis, i.e., the Gl phase, S phase, and G2 phase.
• Chkl activators useful in the invention include DNA damaging agents, such as chemotherapeu- tic agents and/or radiation. Suitable Chkl acti ⁇ vators also include radiotherapeutic agents, such as ionizing or ultraviolet radiation. Radiation Chkl activators include, but are not limited to, gamma- radiation, X-ray radiation, ultraviolet light, vis ⁇ ible light, infrared radiation, microwave radiation, and mixtures thereof.
• “Inhibiting aberrant cell proliferation” means to retard or eliminate the rate at which aberrantly proliferating cells proliferate. This inhibition can result either from a decreased rate of replication, an increased rate of cell death, or both. Cell death can occur by any mechanism, in- eluding apoptosis and mitotic catastrophe.
• Preventing aberrant cell proliferation means inhibiting aberrant cell proliferation prior to occurrence, or inhibiting the recurrence thereof.
• In vivo means within a living subject, as within an animal or human.
• agents can be used therapeutically in a subject to retard or eliminate the proliferation of aberrantly replicating cells. The agents also can be used as a prophylactic to prevent the occurrence or recurrence of aberrant cell proliferation or the manifestation of symptoms associated therewith.
• Such samples can be obtained by methods well known in the art.
• Exemplary biological fluid samples include blood, cerebrospinal fluid, urine, saliva.
• Exemplary tissue samples include tumors and biopsies thereof.
• the present compounds can be in numerous applications, both therapeutic and experimental.
• radiosensitizer as used here ⁇ in, is defined as a compound, administered to a human or other animal in a therapeutically effective amount to increase the sensitivity of cells to elec ⁇ tromagnetic radiation and/or to promote the treat ⁇ ment of diseases treatable with electromagnetic radiation.
• electromagnetic radiation and “radiation” as used herein include, but are not limited to, radiation having the wavelength of 10-20 to 100 meters.
• the present invention includes all possi ⁇ ble stereoisomers and geometric isomers of the com- pounds of structural formula (I) .
• the present in ⁇ vention includes not only racemic compounds, but optically active isomers as well.
• a compound of structural formula (I) 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 883-888 (1997) . Resolution of the final product, an intermediate, or a starting material can be achieved by any suitable method known in the art.
• the present invention is intended to include all tautomeric forms of the compounds.
• specific stereoisomers can exhibit an exceptional ability to inhibit Chkl in combination with chemo- therapeutic or radiotherapeutic treatments.
• Prodrugs of compounds of structural for ⁇ mula (I) also can be used as the compound in a method of the present invention. It is well es ⁇ tablished that a prodrug approach, wherein a com ⁇ pound is derivatized into a form suitable for for ⁇ mulation and/or administration, then released as a drug in vivo, has been successfully employed to transiently (e.g., bioreversibly) alter the physico- chemical properties of the compound (see, H. Bund- gaard, Ed. , “Design of Prodrugs, " Elsevier, Amster ⁇ dam, (1985) ; R.B. Silverman, "The Organic Chemistry of Drug Design and Drug Action," Academic Press, San Diego, chapter 8, (1992) ; K.M. Hillgren et al. , Med. Res. Rev., 15, 83 (1995)) .
• the compounds of the present invention can be therapeutically administered as the neat chem ⁇ ical, but it is preferable to administer compounds of structural formula (I) as a pharmaceutical com ⁇ position or formulation.
• the present inven- tion provides a pharmaceutical composition compris ⁇ ing a compound of the formula (I) together with a pharmaceutically acceptable diluent or carrier therefor.
• a process of preparing a pharmaceutical composition comprising admixing a compound of formula (I) with a pharmaceutically acceptable diluent or carrier therefor.
• the present invention further provides pharmaceutical formulations comprising a compound of structural formula (I) , or a pharmaceu ⁇ tically acceptable salt, prodrug, or solvate there- of, together with one or more pharmaceutically acceptable carriers and, optionally, other thera ⁇ Promotionic and/or prophylactic ingredients.
• the car ⁇ riers are "acceptable" in the sense of being com ⁇ patible with the other ingredients of the formula- tion and not deleterious to the recipient thereof.
• Inhibition of the checkpoint kinase typ ⁇ ically 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 concentrations at which no or minimal effect is observed, through higher concentrations at which partial effect is observed, to saturating con ⁇ centrations at which a maximum effect is observed.
• concentrations at which no or minimal effect is observed through higher concentrations at which partial effect is observed, to saturating con ⁇ centrations at which a maximum effect is observed.
• the curve also theo ⁇ retically passes through a point at which the con ⁇ centration 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 IC 50 value.
• Determination of IC 50 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 IC 50 values, wherein a higher IC 50 indicates that the test compound is less potent, and a lower IC 50 indicates that the compound is more potent, than a reference compound.
• Compounds of the present invention demon ⁇ strate an IC 50 value of less than 5 ⁇ M, and down to 0.1 nM, when measured using the dose-response assay.
• Preferred compounds demonstrate an IC 50 value of 500 nM or less. More preferred compounds of the present invention demonstrate an IC 50 value of less than 250 nM, less than 100 nM, less than 50 nM, or less than 20 nM.
• Preferred Chkl inhibitors of the invention are selective, i.e., demonstrate at least a 20-fold selectivity in inhibiting Chkl over the following protein kinases: protein kinase A, protein kinase C, cdc2, and pp60v-src. More preferred Chkl inhib ⁇ itors of the present invention preferably exhibit at least 75-fold selectivity in inhibiting Chkl over the following protein kinases: protein kinase A, protein kinase C, cdc2, and pp60v-src.
• Most pre ⁇ ferred Chkl inhibitors of the present invention demonstrate at least 75-fold selectivity against protein kinase A, protein kinase C, cdc2, pp60v-src, protein kinase B/Akt-1, p38MapK, ERKl, p70S6K, cdc2, cdk2, chk2, and the abl tyrosine kinase.
• "Fold selectivity" is defined as the IC 50 of the Chkl inhibitor for the comparison kinase divided by the IC 50 of the Chkl inhibitor for Chkl.
• the selective Chkl inhibitors do not func ⁇ tion as chemotherapeutic agents when administered alone.
• a nonselective Chkl inhibitor in contrast, can act as a chemotherapy agent by virtue of its ability to more substantially inhibit additional protein kinases or enzymes that are required for cell growth. This may result in additional cellular effects that lead to adverse side effects and/or a reduced therapeutic index.
• 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 pur ⁇ pose. More specifically, a "therapeutically effec- tive amount" means an amount sufficient to treat an individual suffering an indication, or to alleviate the existing symptoms of the indication. Deter ⁇ mination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
• compositions of the invention can be for ⁇ mulated to include cytokines, lymphokines, growth factors, other hematopoietic factors, or mixtures thereof, to reduce adverse side effects that can arise from, or be associated with, administration of the pharmaceutical composition alone.
• Cytokines, lymphokines, growth factors, or other hematopoietic factors particularly useful in pharmaceutical compo ⁇ sitions of the invention include, but are not limited to, M-CSF, GM-CSF, TNF, IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-IO, IL-Il, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNF, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, erythropoietin, angiopoietins, including Ang-1, Ang-2, Ang-4, Ang-Y, and/or the human angiopoietin-like polypeptide, vascular endo ⁇ thelial growth factor (VEGF) , angiogenin, bone morphogenic protein-1
• the compounds of structural formula (I) also can be conjugated or linked to auxiliary moieties that promote a beneficial property of the compound in a method 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 con ⁇ centrations 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 auxil- iary moieties include, for example, amino acids, oligopeptides, or polypeptides, e.g., antibodies such as monoclonal antibodies and other engineered antibodies; and natural or synthetic ligands to re- ceptors in target cells or tissues.
• auxiliaries include fatty acid or lipid moieties that promote biodistribution and/or uptake of the compound by target cells (see, e.g., Bradley et al. , Clin. Cancer Res. (2001) 7:3229) .
• Formulations of the present invention can be administered in a standard manner for the treat ⁇ ment of the indicated diseases, such as orally, parenterally, transmucosally (e.g., sublingually or via buccal administration) , topically, transdermal- Iy, rectally, via inhalation (e.g., nasal or deep lung inhalation) .
• Parenteral administration in ⁇ cludes, but is not limited to intravenous, intra ⁇ arterial, intraperitoneal, subcutaneous, intra ⁇ muscular, intrathecal, and intraarticular. Par- enteral administration also can be accomplished using a high pressure technique, like POWDERJECTTM.
• the composition can be in the form of tablets or lozenges formulated in conventional manner.
• tablets and capsules for oral administration can contain conventional excipients such as binding agents (for 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 accord ⁇ ing to methods well known in the art.
• 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.
• 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, frac ⁇ tionated coconut oil, oily esters, propylene glycol, and ethyl alcohol; and preservatives, such as methyl or propyl p-hydroxybenzoate and sorbic acid.
• 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
• 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 tri- chlorofluoromethane.
• Typical topical and trans ⁇ dermal 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.
• compositions of the present invention can be formulated for parenteral adminis- tration by injection or continuous infusion.
• mulations for injection can be in the form of sus ⁇ pensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulation agents, such as suspending, stabilizing, and/or dispersing agents.
• the active ingredient can be in powder form for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) before use.
• a composition of the present invention also can be formulated as a depot preparation.
• Such long acting formulations can be administered by implantation (for example, subcutaneously or intra ⁇ muscularly) or by intramuscular injection.
• the compounds of the invention can be for- mulated with suitable polymeric or hydrophobic materials (e.g., an emulsion in an acceptable oil), ion exchange resins, or as sparingly soluble derivatives (e.g., a sparingly soluble salt) .
• a compound of formula (I) is administered as a suitably acceptable formulation in accordance with normal veterinary practice.
• the veterinarian can readily determine the dosing regimen and route of adminis ⁇ tration that is most appropriate for a particular animal .
• Animals treatable by the present compounds and methods include, but are not limited to, pets, livestock, show animals, and zoo specimens.
• the compounds of the present invention can be prepared by the following synthetic schemes.
• compounds of formula 1 can be converted to compounds of formula 2 by treatment with a base, such as potassium carbonate, triethylamine, or sodium hydride, followed by the addition of R 6 X, wherein X is a halide, mesylate, or tosylate.
• a base such as potassium carbonate, triethylamine, or sodium hydride
• R 6 X wherein X is a halide, mesylate, or tosylate.
• solvents used in this re ⁇ action include DMF, THF, CH 2 Cl 2 , and mixtures there ⁇ of.
• the reaction is conducted at temperatures be ⁇ tween 0 0 C and 100 0 C for about 15 minutes to 12 hours.
• compounds of formula 1 can be admixed with a compound of formula R 6 X, wherein X is hydroxyl, and the resulting mixture is treated with triphenylphosphine and diisopropylazodicar- boxylate in a solvent, such as THF, to provide com ⁇ pounds of formula 2.
• Compounds of formula 2 can be treated with hydrogen gas in the presence of a catalyst such as platinum oxide, palladium on carbon, or Raney nickel, or treated with an acid source, such as sat ⁇ urated aqueous ammonium chloride or aqueous hydrogen chloride in the presence of zinc metal, to provide compounds of formula 3.
• a catalyst such as platinum oxide, palladium on carbon, or Raney nickel
• an acid source such as sat ⁇ urated aqueous ammonium chloride or aqueous hydrogen chloride in the presence of zinc metal
• solvents used in this reaction include methanol, ethanol, ethyl acetate, or mixtures thereof.
• the reaction general ⁇ ly is conducted at room temperature or below for periods of one to twelve hours.
• Compounds of formula 5 can be prepared by combining compounds of formula 3 with compounds of formula 4 (prepared as described in Scheme 2) .
• compounds of formula 4 can be prepared by treating a compound of formula 6 with a base, such as DIEA, and diphenyl phophoryl azide.
• a base such as DIEA
• diphenyl phophoryl azide A typical solvent for this reaction is THF, and the reaction is performed behind a blast shield at 20°C to 80°C for one to twelve hours.
• Scheme 3 shows an alternative synthesis of compounds of formula 5.
• Compounds of formula 3 are treated with compounds of formula 7, which is pre ⁇ pared according to Scheme 4.
• One solvent that can be used is DMF, and the reaction temperature is maintained between room temperature and 60 0 C over a one- to twelve-hour time period.
• compounds of formula 7 can be prepared from compounds of formula 8 by treatment with an aryl chloroformate, such as phenyl chloroformate or p-nitrophenyl chloroformate, in the presence of a base, such as pyridine.
• aryl chloroformate such as phenyl chloroformate or p-nitrophenyl chloroformate
• solvents used in this reaction include CH 2 Cl 2 or pyridine, at temperatures of 0 0 C to room tempera ⁇ ture.
• Scheme 5 shows an alternative approach to compounds of formula 5.
• Compounds of formula 9 are converted to compounds of formula 2 by treatment with an alcohol in the presence of a base, such as sodium hydride, potassium bis (trimethylsilyl) amide, or n-butyllithium.
• a base such as sodium hydride, potassium bis (trimethylsilyl) amide, or n-butyllithium.
• solvents used in this reaction include THF or diethyl ether.
• the reaction typically is performed at temperatures be ⁇ tween -15 0 C and room temperature for about 1 to 6 hours.
• Compounds of formula 2 are converted to com ⁇ pounds of formula 5 following procedures described in Scheme 1.
• Scheme 6 shows an alternative synthesis of compounds of formula 5.
• Compounds of formula 3 can be converted to compounds of formula 10 following procedures described in Scheme 4.
• Compounds of formula 10 can be converted to compounds of formula 5 following procedures described in Scheme 1.
• Step 1 3- (4-Bromo-2-nitro-phenoxymeth- yl) -1-methyl-piperidine
• Step 2 1-Methyl-3- (2-nitro-4-trimethyl- silanylethynyl-phenoxymethyl) -piperidine
• 3- (4-bromo-2- nitro-phenoxymethyl) -1-methyl-piperidine 910 mg, 2.76 mmol
• trimethylsilylacetylene 543 mg, 5.5 mmol
• dichlorobis (triphenylphosphine) - palladium(II) 39 mg, 0.55 mmol
• copper(I) iodide 42 mg, 0.22 mmol
• DBU 1-Methyl-3- (2-nitro-4-trimethyl- silanylethynyl-phenoxymethyl) -piperidine
• Step 4 1- [2- (1-Methyl-piperidin-3-yl- methoxy) -5-trimethylsilanylethynyl-phenyl] -3- (5- methyl-pyrazin-2-yl) -urea
• Step 5 1- [5-Ethynyl-2- (1-methyl-piper ⁇ idin-3-ylmethoxy) -phenyl] -3- (5-methyl-pyrazin-2-yl) - urea
• 1- [2- (1-methyl- piperidin-3-ylmethoxy) -5-trimethylsilanylethynyl- phenyl] -3- (5-methyl-pyrazin-2-yl) -urea 43 mg, 0.095 mmol
• potassium fluoride 27 mg, 0.47 mmol
• the reaction was refluxed for 1.5 hours, cooled to room temperature, and partitioned between ethyl acetate (30 mL) and H 2 O (30 mL) .
• the organics were isolated, dried (MgSO 4 ) , filtered, and concen ⁇ trated to yield the product as a tan solid (35 mg, 97%) .
• Step 1 Dimethyl- [2- (2 ⁇ nitro-4-trimeth- ylsilanylethynyl-phenoxy) -ethyl] -amine
• Step 2 2- (2-Dimethylamino-ethoxy) -5-tri- methylsilanylethynyl-phenylamine
• Step 4 1- [2- (2-Dimethylamino-ethoxy) -5- ethynyl-phenyl] -3- (5-methyl-pyrazin-2-yl) -urea
• Step 1 3- (2-Nitro-4-trimethylsilanyl- ethynyl-phenoxymethyl) -pyridine Prepared according to the procedure of Compound 1 Step 2 using 3- (4-bromo-2-nitro-phenoxy- methyl) -pyridine (prepared as in Compound 1 Step 1, from 4-.bromo-2-nitro phenol and 3-pyridine meth- anol) .
• Step 2 2- (Pyridin-3-ylmethoxy) -5-tri- methylsilanylethynyl-phenylamine
• Step 4 1- [5-Ethynyl-2- (pyridin-3-ylmeth- oxy) -phenyl] -3- (5-methyl-pyrazin-2-yl) -urea
• Step 2 l-Methyl-3- (3-nitro-5, 6, 7, 8- tetrahydro-naphthalen-2-yloxymethyl) -piperidine Prepared according to the procedure for Compound 1 Step 1 using 3-nitro-5, 6,7, 8-tetrahydro- naphthalen-2-ol (see above) and (1-methyl-piperidin- 3-yl) -methanol.
• Step 3 3- (l-Methyl-piperidin-3-ylmeth- oxy) -5,6,7, 8-tetrahydro-naphthalen-2-ylamine
• Step 4 1- [3- (1-Methyl-piperidin-3-yl- methoxy) -5,6,7, 8-tetrahydro-naphthalen-2-yl] -3- (5- methyl-pyrazin-2-yl) -urea
• Step 1 3-Nitro-5, 6,7, 8-tetrahydro-naph- thalen-2-ol
• Step 2 l-Methyl-2- (3-nitro-5, 6, 7, 8- tetrahydro-naphthalen-2-yloxymethyl) -piperidine
• Step 3 3- (1-Methyl-piperidin-2-ylmeth ⁇ oxy) -5,6,7, 8-tetrahydro-naphthalen-2-ylamine
• Step 4 1- [3- (l-Methyl-piperidin-2-yl- methoxy) -5, 6, 7, 8-tetrahydro-naphthalen-2-yl] -3- (5- methyl-pyrazin-2-yl) -urea - 6S -
• Step 4 (S) -3- ⁇ 4-Trifluoromethyl-2- [3- (5- methyl-pyrazin-2-yl) -ureido] -phenoxymethyl ⁇ -piper- idine-1-carboxylic acid tert-butyl ester
• 5-methyl-pyra- zine-2-carbonyl azide (1.14 g, 7 mmol) in toluene (20 mL) was placed in a preheated oil bath at 90 0 C for 15 minutes.
• Step 3 (R) -3- (2-Amino-4-trifluoromethyl- phenoxymethyl) -piperidine-1-carboxylic acid tert- butyl ester Prepared according to Compound 4 Step 3 using (R) -3- (4-trifluoromethyl-2-nitro-phenoxymeth- yl) -piperidine-1-carboxylic acid tert-butyl ester.
• Step 4. (R) -3- ⁇ 4-Trifluoromethyl-2- [3- (5- methyl-pyrazin-2-yl) -ureido] -phenoxymethyl ⁇ -piper- idine-1-carboxylic acid tert-butyl ester
• the organic layer was washed with brine, dried over MgSO 4 , filtered, and dried under reduced pressure.
• the product was purified using a Biotage 4OM car- tridge, eluting with hexane/ethyl acetate (500 mL 1/1), then CH 2 Cl 2 /MeOH/NH 4 OH (98/8/2, 500 mL) to yield a light yellow oil.
• Step 2 2- (l-Methyl-piperidin-4-yloxy) -5- trifluoromethyl-phenylamine Prepared according to Compound 2 Step 2 using l-methyl-4- (2-nitro-4-trifluoromethyl-phen- oxy) -piperidine.
• Step 3 1- [2- (1-Methyl-piperidin-4-yl- oxy) -5-trifluoromethyl-phenyl] -3- (5-methyl-pyrazin- 2-yl) -urea
• Step 1 3- (2-Nitro-4 ⁇ trifluoromethyl- phenoxymethyl) -piperidine-1-carboxylic acid tert- butyl ester Prepared according to Compound 1 Step 1 using 2-nitro-4-trifluoromethyl-phenol and 3-hy- droxymethyl-piperidine-1-carboxylic acid tert-butyl ester.
• Step 2. 3- (2-Amino-4-trifluoromethyl- phenoxymethyl) -piperidine-1-carboxylic acid tert- butyl ester
• the product was extracted with ethyl acetate (3 x 50 mL) , The organic layer was washed with brine, dried over MgSO 4 , filtered, and dried under reduced pressure.
• the product was purified by Biotage 40M cartridge eluting with CH 2 Cl 2 /MeOH/NH 4 OH (90/8/2) to yield a yellow solid.
• Step 2 2- (1-Methyl-piperidin-3-ylmeth- oxy) -5-trifluoromethyl-phenylamine Prepared according to Compound 4 Step 3 using l-methyl-3- (2-nitro-4-trifluoromethyl-phenoxy- tnethyl) -piperidine.
• Step 3 1- [2- (1-Methyl-piperidin-3-yl- methoxy) -5-tri£luoromethyl-phenyl3 -3- (5-methyl- pyrazin-2-yl) -urea
• Step 2 7- (Pyridin-3-ylmethoxy) -2,3-di ⁇ hydro-benzo[1,4]dioxin-6-ylamine Prepared from 3- (7-nitro-2,3-dihydro ⁇ benzo [1,4]dioxin-6-yloxymethyl) -pyridine hydro ⁇ chloride salt (266 mg, 0.82 tnmol) according to the method of Compound 2 Step 2. The product was isolated as a purple oil which was used immediately in the next reaction.
• Step 3 1- (5-Methyl-pyrazin-2-yl) -3- [7- ( ⁇ yridin-3-ylmethoxy) -2,3-dihydro-benzo[1,4] dioxin- 6-yl] -urea
• the final product was prepared according to the procedure of Compound 1 Step 4 from 7- (pyridin-3-ylmethoxy) -2,3-dihydro-benzo [1,4] dioxin- 6-ylamine and 5-methyl-pyrazin-2-carbonyl azide and was isolated as a tan solid.
• Step 1 Dimethyl- [2- (7-nitro-2,3-dihydro- benzo[1,4] dioxin-6-yloxy) -ethyl] -amine
• Step 2 7- (2-Dimethylamino-ethoxy) -2,3- dihydro-benzo [1,4]dioxin-6-ylamine
• Step 3 1- [7- (2-Dimethylamino-ethoxy) - 2,3-dihydro-benzo [1,4]dioxin-6-yl] -3- (5-methyl- pyrazin-2-yl) -urea
• the final compound was prepared according to the method of Compound 1 Step 4, from 5-methyl- pyrazin-2-carbonyl azide and 7- (2-dimethylamino- ethoxy) -2,3-dihydro-benzo [1,4] dioxin-6-ylamine.
• the product was isolated as a tan solid.
• Step 1 3-Nitro-5, 6,7, 8-tetrahydro-naph- thalen-2-ol
• Step 2 Dimethyl- [2- (3-nitro-5, 6,7, 8- tetrahydro-naphthalen-2-yloxy) -ethyl] -amine
• Step 3 3- (2-Dimethylamino-ethoxy) - 5,6,7, 8-tetrahydro-naphthalen-2-ylamine
• Step 4 1- [3- (2-Dimethylamino-ethoxy) - 5, 6, 7, 8-tetrahydro-naphthalen-2-yl] -3- (5-methyl- pyrazin-2-yl) -urea
• Compounds of the present invention can be used to potentiate the therapeutic effects of radi ⁇ ation and/or a chemotherapeutic agent used in the treatment of cancers and other cell proliferation indications involving eukaryotic cells, including those in humans and other animals.
• 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) .
• an antimetabolite e.g., methotrexate or 5- fluorouracil (5-FU)
• the present com ⁇ pounds inhibit aberrantly proliferating cells, both cancerous and noncancerous.
• Use of compounds of the present invention can result in partial or complete regression of aberrantly proliferating cells, i.e., the partial or complete disappearance of such cells from the cell population.
• the method of the invention can be used to slow the rate of tumor growth, decrease the size or number of tumors, or to induce partial or complete tumor regression.
• the invention can be used in vivo or ex vivo where no aberrant cell pro ⁇ liferation has been identified or where no aberrant cell proliferation is ongoing, but where aberrant cell proliferation is suspected or expected, respec ⁇ tively. Moreover, the invention also can be used wherever aberrant cell proliferation has been pre ⁇ viously treated to prevent or inhibit recurrence of the same.
• the "cell population comprising aberrantly proliferating cells" refers to any cell population where no aber ⁇ rant cell proliferation has been identified or is ongoing, but where aberrant cell proliferation is suspected or expected, respectively, and/or any cell population previously treated for aberrant cell pro- liferation to prevent or inhibit recurrence of the same.
• One method of the present invention com ⁇ prises administration of a therapeutically effective amount of a present Chkl inhibitor compound in com ⁇ bination with a chemotherapeutic agent that can effect single- or double-strand DNA breaks or that can block DNA replication or cell proliferation.
• a method of the present invention comprises administration of a therapeutically effec ⁇ tive amount of at least one of the present Chkl inhibitor compounds in combination with therapies that include use of an antibody, e.g., herceptin, that has activity in inhibiting the proliferation of cancer cells.
• cancers for example, colorectal cancers, head and neck cancers, pancre ⁇ atic cancers, breast cancers, gastric cancers, bladder cancers, vulvar cancers, leukemias, lymph ⁇ omas, melanomas, renal cell carcinomas, ovarian cancers, brain tumors, osteosarcomas, and lung car ⁇ cinomas, are susceptible to enhanced treatment by administration of a present Chkl inhibitor in com ⁇ bination with a chemotherapeutic agent or an anti ⁇ body.
• Cancers include tumors or neoplasms which are growths of tissue cells wherein multiplication of cells is uncontrolled and progressive. Some such growths are benign, but others are termed "malig ⁇ nant," and can lead to death of the organism.
• Malignant neoplasms are distinguished from benign growths in that, in addition to exhibit- ing aggressive cellular proliferation, can invade surrounding tissues and metastasize. Moreover, malignant neoplasms are characterized by showing a greater loss of differentiation (greater "dediffer- entiation") and organization relative to one another and surrounding tissues. This property is called “anaplasia. "
• Cancers treatable by the present invention also include solid tumors, i.e., carcinomas and sar- comas.
• Carcinomas include malignant neoplasms de ⁇ rived from epithelial cells which infiltrate (i.e., invade) surrounding tissues and give rise to metas ⁇ tases.
• Adenocarcinomas are carcinomas derived from glandular tissue, or from tissues that form recog- nizable glandular structures.
• Another broad cate ⁇ gory of cancers includes sarcomas, which are tumors whose cells are embedded in a fibrillar or homogen ⁇ eous substance, like embryonic connective tissue.
• the present invention also enables treatment of can- cers of the myeloid or lymphoid systems, including leukemias, lymphomas, and other cancers that typ ⁇ ically are not present as a tumor mass, but are dis ⁇ tributed in the vascular or lymphoreticular systems.
• Chkl activity is associated with various forms of cancer in, for example, adult and pediatric oncology, growth of solid tumors/malignancies, myxoid and round cell carcinoma, locally advanced tumors, metastatic cancer, human soft tissue sar ⁇ comas, including Ewing's sarcoma, cancer metastases, including lymphatic metastases, squamous cell car ⁇ cinoma, particularly of the head and neck, esopha- geal squamous cell carcinoma, oral carcinoma, blood cell malignancies, including multiple myeloma, leu- kemias, including acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leu- kemia, 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,
• Compounds of the present invention also can potentiate the efficacy of drugs in the treat ⁇ ment of inflammatory diseases.
• diseases that can benefit from combination therapy with com ⁇ pounds suitable for the method of the present inven- tion are rheumatoid arthritis, psoriasis, vitiligo, Wegener's granulomatosis, and systemic lupus eryth ⁇ ematosus (SLE) .
• Immunosuppressive therapies such as ionizing radi- ation, methotrexate, and cyclophosphamide.
• immunosuppressive therapies such as ionizing radi- ation, methotrexate, and cyclophosphamide.
• Such treatments typically induce, either directly or in ⁇ directly, DNA damage.
• Inhibition of Chkl activity within the offending immune cells render the cells more sensitive to control by these standard treat- ments.
• Psoriasis and vitiligo commonly are treated with ultraviolet radiation (UV) in combination with psoralen.
• UV ultraviolet radiation
• the present DNA damaging agents induce the killing effect of UV and psoralen, and increase the therapeutic index of this treatment regimen.
• compounds useful in methods of the present invention potentiate control of inflammatory disease cells when in combination with currently used immunosuppressive drugs.
• the present invention also can be used in methods of treating noncancerous proliferating cells.
• Such conditions include, but are not limited to, atherosclerosis, restenosis, vasculitis, neph ⁇ ritis, retinopathy, renal disease, proliferative skin disorders, psoriasis, keloid scarring, actinic keratosis, Stevens-Johnson Syndrome, rheumatoid arthritis (RA) , systemic-onset juvenile chronic arthritis (JCA) , osteoporosis, systemic lupus eryth- matosis, hyperproliterative diseases of the eye in ⁇ cluding epithelial down growth, proliferative vit- reoretinopathy (PVR) , diabetic retropathy, Hemangio- proliferative diseases, ichthyosis, or papillomas.
• atherosclerosis restenosis, vasculitis, neph ⁇ ritis, retinopathy, renal disease,
• Noncancerous conditions treatable by the present invention also include inflammation and inflammatory diseases, conditions, or disorders.
• indications include, but are notlimited to, rheumatoid arthritis, psoriasis, vitiligo, Wegener's granulomatosis, and systemic lupus erythematosus (SLE) .
• Treatment of arthritis, Wegener's granulomatosis, and SLE often involves the use of immunosuppressive therapies, such as ionizing radiation, methotrexate, and cyclophosphamide.
• Psoriasis and vitiligo commonly are treated with ultraviolet radiation (UV) in combination with psoralen. Such treatments typically induce, either directly or indirectly, DNA damage.
• UV ultraviolet radiation
• Chkl activity within the offending immune cells renders the cells more sensitive to control by these standard treatments.
• Chkl inhibitors useful in the invention optionally can be used to potentiate control of inflammatory disease cells when administered in combination with immunosuppres ⁇ sive drugs.
• Chkl activator e.g., a chemotherapeu- tic agent
• Chkl inhibitor e.g., a Chkl inhibitor according to the present invention.
• at least one Chkl activator is administered at a dose and for a time sufficient to induce substantial synchroniza ⁇ tion of cell cycle arrest in proliferating cells.
• At least one Chkl inhibitor is administered to abrogate the cell cycle arrest and induce therapeutic cell death.
• the method is useful with any Chkl activator, and finds application in treating or preventing cancerous and noncancerous aberrant cell proliferation.
• the Chkl inhibitor is a selec ⁇ tive Chkl inhibitors.
• a population of aberrantly proliferating cells can be contacted with one Chkl inhibitor or can be contacted with more than one
• Chkl inhibitor If more than one Chkl inhibitor is used, the Chkl inhibitors can be coadministered or administered at separate times as determined by the attending physician or laboratory technician.
• a population of aberrantly proliferating cells also can be contacted with one Chkl activator or can be contacted with more than one Chkl acti ⁇ vator. If more than one Chkl activator is used, the Chkl activators can be coadministered or adminis ⁇ tered at separate times as determined by the attend- ing physician or laboratory technician.
• the present invention can be applied to cell populations ex vivo.
• the present compounds can be used ex vivo to determine the opti- al schedule and/or dosing of administration of a Chkl inhibitor for a given indication, cell type, patient, and other parameter. Information gleaned from such use can be used for experimental purposes or in the clinic to set protocol for in vitro treat ⁇ ment. Other ex vivo uses for which the invention is suited are apparent to those skilled in the art.
• a compound of the present invention also can radiosensitize a cell.
• Diseases ' that are treat- ble with electromagnetic radiation include neoplas ⁇ tic diseases, benign and malignant tumors, and can- cerous cells.
• Electromagnetic radiation treatment of other diseases not listed herein also is contem ⁇ plated by the present invention.
• Preferred embodiments of the present invention employ the electromagnetic radiation of: gamma-radiation (10- 20 to 10-13 m) , X-ray radiation (10-12 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) .
• radiosensitizers activated by electromagnetic radiation, e.g., X-rays.
• X-ray-acti ⁇ vated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR) , 5- iododeoxyuridine (IUdR) , bromodeoxycytidine, fluoro- deoxyuridine (FUdR) , hydroxyurea, cis-platin, and therapeutically effective analogs and derivatives of the same.
• metronidazole misonidazole
• desmethylmisonidazole pimonidazole
• etanidazole nimorazole
• Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator of the sensitizing agent.
• photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, PHOTOFRIN ® , benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2) , pheoborbide-a, bacteriochlorophyll-a, naph- thalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and deriva- tives of the same.
• Radiosensitizers can be administered in conjunction with a therapeutically effective amount of one or more compounds in addition to the Chkl inhibitor, such compounds including, but not limited to, compounds that promote the incorporation of radiosensitizers to the target cells, compounds that control the flow of therapeutics, nutrients, and/or oxygen to the target cells, chemotherapeutic agents that act on the tumor with or without additional radiation, or other therapeutically effective com- pounds for treating cancer or other disease.
• Radiosensitizers include, but are not limited to, 5-fluorouracil (5-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-BSO.
• 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 prod ⁇ ucts, and combinations thereof.
• an inhibitor compound of the present invention can be administered with antibiotics, such as doxorubicin and other anthracycline analogs, nitrogen mustards, such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cis-platin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like.
• antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards, such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cis-platin, hydroxyurea, taxol and its natural and synthetic derivatives, and the like.
• antibiotics such as doxorubicin and other anthracycline analogs
• nitrogen mustards such as cyclophosphamide
• pyrimidine analogs such as 5-fluorouracil, cis-platin
• hydroxyurea taxol and its natural and synthetic derivatives, and the like.
• anti- neoplastic 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.”
• Additional chemotherapeutic agents useful in the invention in ⁇ clude hormones and antagonists thereof, radioiso- topes, antibodies, natural products, and combina ⁇ tions thereof. Examples of chemotherapeutic agents useful for the method of the present invention are listed in the following table.
• chemotherapeutic agents that are particularly useful in conjunction with radio- sensitizers include, for example, camptothecin, carboplatin, cis-platin, daunorubicin, doxorubicin, interferon (alpha, beta, gamma) , irinotecan, hydroxyurea, chlorambucil, 5-fluorouracil (5-FU) , methotrexate, 2-chloroadenosine, fludarabine, azacytidine, gemcitabine, pemetrexed, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, and therapeutically effective analogs and derivatives of the same.
• compounds of the present invention are useful in combination with gemcitabine, alone or further with paclitaxel.
• Compounds of the present invention also are useful in combination with pemetrexed, alone or further with cis platin, carboplatin, or other platins.
• a present Chkl inhibitor also can be administered in combination with gemcitabine and pemetrexed.
• a present Chkl inhibitor administered in combination with gemcitabine can be useful in the treatment of, for example, pancreatic carcinoma, leiomyosarcoma of the uterus, bone sarcoma, metastatic nonsmall cell lung cancer, extremity and trunk soft tissue sarcoma, renal cell cancer, adenocarcinoma, and Hodgkin's disease.
• a present Chkl inhibitor administered with pemetrexed can be useful in the treatment of mesothelioma.
• reference herein to treatment extends to pro- phylaxis, as well as to treatment of established diseases or symptoms. Reference to treatment also refers to the reduction of the rate of proliferation or the reduction of recurrence of the treated indi- cation. 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.
• doses administered for adult human treatment typically are in the range of 0.001 mg/kg to about 100 mg/kg per day.
• the de ⁇ sired 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.
• 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 individual instances can exists wherein higher or lower dosages are merited, and such are within the scope of the present inven- tion.
• a present Chkl inhibitor can likewise occur at any dose and time sufficient to achieve substantial abrogation of the cell cycle checkpoint. Typically, though not necessarily, such times include up to about 72 to about 96 hours, depending upon various factors. In some embodiments, it is desirable or necessary to administer Chkl inhibitor over a period of up to about several weeks or more, as determined by the attending physician or technician. Thus, a present Chkl inhibitor typically can be administered for up to about 1 hour, up to about 2 hours, up to about 3 hours, up to about 4 hours, up to about 6 hours, up to about 12 hours, up to about 18 hours, up to about 24 hours, up to about 48 hours, or up to about 72 hours.
• Chkl inhibitors of the present invention can be administered over a plurality of doses.
• the Chkl inhibitor can be given at a frequency of: four doses delivered as one dose per day at four-day intervals (q4d x 4) ; four doses delivered as one dose per day at three-day intervals (q3d x 4) ; one dose delivered per day at five-day intervals (qd x 5) ; one dose per week for three weeks (qwk3); five daily doses, with two days rest, and another five daily doses (5/2/5) ; or, any dose regimen determined to be appropriate for the circumstance.
• PCR-amplified fragment was cloned into pCI-Neo as an EcoRI-SalI fragment (Invitrogen, Carlsbad, CA) , then subcloned as an EcoRI-NotI fragment into pFastBacI (Gibco-BRL, Bethesda, MD) .
• Recombinant baculovirus was prepared as described in the Gibco-BRL Bac-to-Bac manual and used to infect Sf-9 cells grown in CCM3 medium (HyClone Laboratories, Logan, UT) for expression of FLAG ® -tagged Chkl protein.
• FLAG -tagged Chkl was purified from frozen pellets of baculovirus-infected SF9 cells. Frozen cell pellets were mixed with an equal volume of 2X lysis buffer containing 100 mM Tris-HCl pH 7.5, 200 mM NaCl, 50 mM B-glycerophosphate, 25 mM NaF, 4 mM MgCl 2 , 0.5 mM EGTA, 0.2% TWEEN ⁇ -20, 2 mM sodium van ⁇ adate, 2 mM DTT, and a cocktail of protease inhib ⁇ itors (Complete mini, Boehringer Mannheim 2000 catalog #1836170) .
• 2X lysis buffer containing 100 mM Tris-HCl pH 7.5, 200 mM NaCl, 50 mM B-glycerophosphate, 25 mM NaF, 4 mM MgCl 2 , 0.5 mM EGTA, 0.2% TWEEN ⁇ -20
• the cleared lysate then was bound to M2 affinity resin in batch at 4 0 C for 4 hours.
• the mixture of resin and lysate then was poured into a column and the flow through collected.
• the resin was washed with 10 column volumes of 20 mM Tris pH 7.5, 150 mM NaCl, and 3 mM N-octyl gluco- side.
• FLAG " -tagged Chkl then was eluted from the column with 6 column volumes of cold 20 mM Tris pH 7.5, 150 mM NaCl, 3 mM N-octyl gluc ⁇ side containing 0.5 mg/mL FLAG ® peptide (Sigma, 2000 Catalog # F- 3290) .
• the protein kinase was used in an assay for Chkl kinase activity that includes 100 ng purified FLAG ⁇ -Chkl (150 pmol of ATP/min) , 20 ⁇ m Cdc25C peptide (H-leu-tyr-arg-ser-pro-ser-met-pro- glu-asn-leu-asn-arg-arg-arg-arg-OH) (SEQ ID NO: 1) , 4 ⁇ m ATP, 2 ⁇ Ci [ 32 P] ⁇ -ATP, 20 mM Hepes pH 7.2, 5 mM MgCl 2 , 0.1% NP40, and 1 mM DTT.
• This assay was used to determine IC 50 of compounds of the present invention. Reactions were initiated by the addition of ATP-containing reaction mix and carried out at room temperature for 10 min. Reactions were stopped by the addition of phosphoric acid (150 mM final concentration) and transferred to phosphocellulose discs. The phosphocellulose discs were washed five times with 150 mM phosphoric acid and air-dried. Scintillation fluid was added and discs were counted in a Wallac scintillation counter. Chkl inhibitors of the present invention that were subjected to the assay have measured IC 50 values of about 8 to about 500 nM.
• Activity of the compounds against Chk2 was assayed as follows: 128 ng of purified His-tagged Chk2 was incubated with up to 100 mM Chkl inhibitor in the presence of 4 mM ATP, 1 mCi [ 32 P] ⁇ -ATP, 20 mM Hepes pH 7.5, 5 mM MgCl 2 , and 0.25% NP40 for 20 minutes at room temperature. Reactions were stopped with a final concentration of 150 mM phosphoric acid, and 5/8 of the reaction mixture was trans ⁇ ferred to phosphocellulose discs. The discs were washed five times with 150 mM phosphoric acid, and air-dried. Scintillant was added and radioactivity was counted using a Wallac beta counter.
• p38 MAP kinase, ERK kinase, PKA, CaM KII, and Cdc2 were purchased from New England Biolabs, and assays were performed according to the manufac ⁇ turer's instructions using 4-50 ⁇ M ATP and testing Chkl inhibitor concentrations as high as 100 ⁇ M. All inhibitors tested showed at least a 100-fold selectivity for Chkl over the other enzymes.
• Chkl Inhibitors of the Invention Inhibit Chkl Function in Cells
• Chkl inhibitors of the invention inhibit Chkl function in cells
• inhib ⁇ itors can be tested in molecular cell-based assays. Because mammalian Chkl has been shown to phosphor- ylate Cdc25C in vitro,' suggesting that it negatively regulates cyclin B/cdc2 in response to DNA damage, the ability of the Chkl inhibitors to enhance the activity of CyclinB/cdc2 can be analyzed. The experiment can be designed as follows: HeLa cells are irradiated with 800 rads and incubated for 7 hours at 37 0 C. Because these cells are functionally p53 negative, they arrest exclusively in G2.
• nocodazole is added to a concentration of 0.5 ⁇ g/mL and the cells are incubated for 15 hours at 37°C.
• the addition of nocodazole is designed to trap any cells that progress through the G2 arrest into M.
• a Chkl inhibitor is added for 8 hours, the cells harvested, lysed and immunoprecipitated equal amounts of protein with an antibody to Cyclin Bl (New England Biolabs) as suggested by the manufac ⁇ turer. IPs then are analyzed for CyclinB-associated cdc2 kinase activity by assaying histone Hl kinase activity (Yu et al. , J Biol Chem. , Dec. 11, 1998; 273 (50) :33455-64) .
• the ability of the subject is assessed for CyclinB-associated cdc2 kinase activity by assaying histone Hl kinase activity (Yu et al. , J Biol Chem. , Dec
• Chkl inhibitors to abrogate the IR-induced G2 DNA damage checkpoint can be established using mitotic index assay experiments.
• HeLa cells (approximately IxIO 5 ) are treated as described above. Cells are harvested by centrifugation, washed once with PBS, then resuspended in 2.5 mL of 75 mM KCl and centrifuged again. The cells then are fixed in 3 mL of freshly prepared cold acetic acid:methanol (1:3) and incubated on ice for 20 minutes. Cells are pelleted, fix solution aspirated and resuspended in 0.5 mL of PBS.
• Mitotic spreads are prepared by pipeting 100 ⁇ L of the fixed cells onto a glass microscope slide and flooding the sample with 1 mL of fix solution. Slides then are air dried, stained with Wright's stain (Sigma) for 1 minute, followed by one wash with water and one wash with 50% methanol. The presence of condensed chromosomes and lack of nuclear envelope identifies mitotic cells.
• Example 4
• Chkl Inhibitors of the Present Invention Enhance Killing of Cells by Cancer Treatments
• cells can be incubated in the presence of a present Chkl inhibitor and exposed to either irradiation or a chemical DNA-damaging agent.
• Cells plated at a density of 1000-2000 per well in 96-well microtitre plates are grown in RMPI 1640 containing 10% FBS, 100 U/mL penicillin and 100 ⁇ g/mL strepto ⁇ mycin for 18 hours at 37°C in a humidified incubator with 5% CO 2 - Cells tested can include any cells or cell lines of interest, such as HeLa, ACHN, 786-0, HCT116, SW620, HT29, Colo205, SK-MEL-5, SK-MEL-28, A549, H322, OVCAR-3, SK-OV-3, MDA-MB-231, MCF-7, PC- 3, HL-60, K562, and M0LT4. All cell line desig ⁇ nations refer to the following human cell lines:
• Cells are treated with media containing chemotherapeutic drugs alone or chemotherapeutic drugs and a Chkl inhibitor. Cells are incubated for approximately 5 days before growth is measured by determination of levels of 3H-thymidine uptake.
• Chemotherapeutic drugs include etoposide, doxorubicin, cis-platin, chlorambucil, 5-fluorour- acil (5-FU) .
• the drug concentration necessary to inhibit cell growth to 90% of untreated control cells is defined as the GI 90 .
• Compounds of the present invention can be tested with additional antimetabolites, including methotrexate, hydroxyurea, 2-chloroadenosine, fludarabine, azacytidine, and gemcitibine to assess therein ability to enhance killing of the agents.
• Compounds of the present invention can be compared to one another by assessing enhanced killing of HT29 colorectal carcinoma in combination with gemcitibine.
• xenograft tumor models using colon tumor cell lines are established.
• 5- fluorouracil (5-FU) or gemcitabine can be used as DNA damaging agents.
• HT29 and Colo205 (human colon carcinoma) and H460 and Calu-6 (nonsmall cell car ⁇ cinoma) cells can be used to propagate xenograft tumors in 6-8 week old female thymic Balb/c (nu/nu) mice. Mice are maintained in a laminar airflow cabinet under pathogen-free conditions and fed sterile food and water ad libitum.
• Cell lines are grown to subconfluence in RPMI 1640 media supple ⁇ mented with 10% FBS, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, and 1.5 mM L-glutamine in a 5% CO 2 humidified environment.
• Single cell suspensions are prepared in CMF-PBS, and cell concentration adjusted to IxIO 8 cells/mL.
• Mice are inoculated subcutane- ously (s.c.) on the right flank or right leg with a total of IxIO 7 cells (100 ⁇ L) .
• Mice are randomized (5-15 mice/group) into four treatment groups and used when tumors reach a volume of 75-100 cm 3 (usually 7-11 days post-inocula ⁇ tion) .
• Treatment consists of i) 100 ⁇ L intra ⁇ peritoneal (i.p) injection of gemcitabine at 160 mg/kg. A delay in tumor growth is observed in the mice treated with gemcitabine.
• Treatment of mice with both 160 mg/kg gemcitabine in combination with oral administration of Chkl inhibitors is expected to reduce tumor volumes and prolong life. Tumor size is monitored every other day for the duration of the experiment.

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