CN101006075A - Bisarylurea derivatives useful for inhibiting CHK1 - Google Patents
Bisarylurea derivatives useful for inhibiting CHK1 Download PDFInfo
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- CN101006075A CN101006075A CNA2005800277583A CN200580027758A CN101006075A CN 101006075 A CN101006075 A CN 101006075A CN A2005800277583 A CNA2005800277583 A CN A2005800277583A CN 200580027758 A CN200580027758 A CN 200580027758A CN 101006075 A CN101006075 A CN 101006075A
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- C07—ORGANIC CHEMISTRY
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- 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
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- 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
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
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- 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
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- 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
Abstract
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 defects in DNA replication, chromosome segregation, or cell division also are disclosed. Formula (I).
Description
Technical field
The present invention relates to can be used for the compound of inhibitory enzyme, the integrity of genetic material is kept and repaired to this enzyme.More specifically, the present invention relates to a series of aryl-and the carbamide compound of heteroaryl-replacement, prepare the method for these compounds and they for example the defective in the duplicating of treatment cancer and other, chromosome segregation or cell fission with thymus nucleic acid (DNA) be the purposes that is used as therapeutical agent in the disease of feature.
Background technology
A large amount of different disease, situation and illnesss (hereinafter referred to as " indication ") are characterised in that the cell that relates to abnormality proliferation.With herein the time, " cell of abnormality proliferation " (or " unusual cell proliferation ") refers to break away from the cell proliferation of process normal, suitable or that expect.For example, unusual cell proliferation comprises DNA wherein or other cellular component is damaged or the unsuitable cell proliferation during defective.Unusual cell proliferation also comprises by unsuitable high-level cell fission, unsuitable low-level necrocytosis (for example, apoptosis) or the two and causes simultaneously, mediates or as its result's indication.This class indication can be for example is feature with the single or multiple local abnormality proliferation of cell, cell mass or tissue, comprises cancer (optimum or pernicious) and non-cancer indication.
According to definition, all cancers (optimum and pernicious) all relate to the abnormal cell proliferation of certain form.Limiting examples comprises cancer and sarcoma.Other is being discussed subsequently.Some non-cancer indication also relates to abnormal cell proliferation.The limiting examples that relates to the non-cancer indication of abnormal cell proliferation comprises rheumatoid arthritis, psoriasis, vitiligo, Wei Geneishi (Wegener ' s) granulomatosis and systemic lupus erythematosus.Being discussed below of other.
The method of indication that a kind of treatment relates to the cell of abnormality proliferation comprises uses dna damage reagent.These reagent are designed to by interrupting killing such as DNA metabolism, DNA is synthetic, DNA transcribes and the microtubule spindle body forms viable cell process the cell of abnormality proliferation.They also can for example work by DNA is introduced the damage of upsetting the chromosome structure integrity.Dna damage reagent is designed by this way and gives to attempting that cell induction to abnormality proliferation damages to greatest extent and subsequently necrocytosis, and normal healthy cell is only had minimal damage.
Up to the present, a large amount of different dna damage reagent are developed, other also just on stream.Dna damage reagent comprises chemotherapy and radiation.Regrettably, dna damage reagent can't reach requirement in the validity that treatment relates in the situation of abnormal cell proliferation, especially in the treatment cancer.This class reagent is normally qualified reluctantly to the selectivity (being sometimes referred to as therapeutic index) of the cell and the healthy cell of abnormality proliferation.
And all cells all has perception and repair mechanism, and it can work with cross-purpose with dna damage reagent.The perception mechanism that this class is called the cell cycle check point helps to keep the order in various cellular replication stages and guarantees that each step all finishes (Hartwell etal., Science, 246:629-634, (1989) with high frequency high fidelity; Weinert et al., Genes Dev., 8:652, (1994)).When cell detection when the dna damage, comprise detecting when having a mind to the inductive dna damage by dna damage reagent, some signal path active cells cycle checkpoint, cell replication cycle temporarily stops (" stagnation ").This stagnation makes the cell of abnormality proliferation repair their DNA if having time, and reaching usually is enough to make affected cell to continue the degree of survival and propagation.This undesirable reparation is tending towards destroying is enough to the inducing DNA damage to kill the effort of abnormality proliferation cell.
For example, be called Gemzar
TMChemotherapeutics (gemcitabine, or 2 ', 2 '-two fluoro-2 '-Deoxyribose cytidine) by in building-up process, himself being incorporated into DNA and damage dna.The damaged dna of not repairing that stays causes usually and can not survive.But in many targeted cells, (or damage) DNA of inappropriate generation is checked in the cell cycle check point.Cell cycle arrest for some time is triggered in activatory cell cycle check point, and this section period is enough to make that damaged dna is repaired.This is that the cell of abnormality proliferation is resisted a kind of mode such as the cell killing effect of dna damage reagent, radiation and the other therapies of chemotherapeutics in theory.
Other dna damage reagent causes that tumour cell stagnates in the S phase.Tumour cell is resisted some chemotherapeutics by being stuck in the S phase when being found in chemotherapeutics and being given simply.When medicine one was removed, dna damage just was repaired then, and cell cycle arrest stops, and cell carries out the remainder (Shi et al., Cancer Res.61:1065-72,2001) of cell cycle.Other therapeutical agent causes cell cycle arrest in other check point, comprises G1 and G2 (following description that will be more complete).The dna damage reagent of active cells cycle checkpoint is commonly called " check point activator " in this article.The dna damage reagent that the check point of " Chk1 " (reading to be " check point one ") is appointed as in activation is commonly called " Chk1 activator " in this article.The inhibitor of these check points is referred to as " check point inhibitor " in this article respectively or specifically is called " Chk1 inhibitor "
Therefore, expection suppresses various dna damages check point and helps to prevent upward inductive dna damage of cytothesis treatment, makes targeted cells to dna damage reagent sensitivity.Then, this sensitization is expected can increase the therapeutic index of these therapies.
In order to understand the present invention more completely, below will go through the effect of cell cycle in each stage and Chk1.
Spread all over all eucaryon species, the cell cycle is identical aspect its primary process and regulative mode on 26S Proteasome Structure and Function.Mitotic division (somatocyte) cell cycle is made up of following four-stage: G1 (gap) phase, S (synthesizing) phase, G2 (gap) phase and M (mitotic division) phase.G1, S and G2 phase are referred to as the interval of cell cycle.In the G1 phase, the biosynthesizing activity of cell is carried out with two-forty.The S phase when the synthetic beginning of DNA, S is final when nuclear dna content is replicated and forms the identical karyomit(e) of two covers ends.
Then, cell enters the G2 phase, and it lasts till that mitotic division begins.In mitotic division, chromosome pairing also separates, and two new nucleus form and the generation division of cytoplasm, and wherein cell fission is two daughter cells, and each acceptance contains the nuclear of one of two cover karyomit(e)s.Division of cytoplasm makes the final beginning that ends and indicate the interval of next cell cycle of M.The order that cell cycle events is carried out is regulated for strict, to such an extent as to the finishing of cell cycle before initial the depending on of a cell cycle events.This make from generation somatocyte to of future generation genetic material duplicate with separate fidelity of reproduction become possibility.
It is reported that the cell cycle check point comprises at least three kinds of different types of polypeptide, they are the defective in cell cycle signal or the chromosomal mechanism react (Carr, A.M., Science, 271:314-315, (1996)) in succession.The first kind is for detecting or perception dna damage or unusual gang's protein in the cell cycle.These susceptors comprise ataxia-telangiectasis mutain (Atm) and ataxia-telangiectasis Rad associated protein (Atr).The signal that the second class polypeptide amplifies and transmission is detected by detector, for example Rad53 (Alen et al., Genes Dev.8:2416-2488, (1994)) and Chk1.The 3rd class polypeptide comprises and mediates the cell cycle effector that for example mitotic division and apoptotic cells are replied, for example p53.
The understanding great majority of the function of cell cycle check point come from the research to tumour derived cell system at present.Under many circumstances, tumour cell has lost crucial cell cycle check point (Hartwell et al., Science 266:1821 28,1994).It is reported that the committed step of cell in the differentiation of tumprigenicity state is the sudden change that obtains to make cell cycle check point approach inactivation, relates to sudden change (Weinberg, R.A., Cell 81:323-330,1995 of p53 as those; Levine, A.J., Cell 88:3234 331,1997).Losing these cell cycle check points causes tumour cell to ignore dna damage and duplicate.
Non-tumprigenicity with intact cell cycle checkpoint organizes the single check point path of having avoided temporary transient usually to interrupt.But tumour cell has defective in the path of control cell cycle progression, to such an extent as to make them especially responsive to dna damage reagent to the upset of other check point.For example, the tumour cell that contains p53 sudden change G1 dna damage check point and keep the G2DNA damage inspection put two aspect (282:1497 501,1998 for Bunz et al., Science) defectiveness.The inhibitor expection of the check point that target G2 check point or S phase check point are initial can further weaken the ability of these tumour cell DNA plerosis damages, therefore be the candidate (Gesner that strengthens the therapeutic index of radiation and systemic chemotherapy, T., Abstract at SRI Conference:Protein Phosphorylation and Drug Discovery World Summit, March2003).
In the presence of any obstacle of dna damage or dna replication dna, check point albumin A tm and Atr start to cause the signal transduction pathway of cell cycle arrest.Atm has been proved in the dna damage check point of replying ionizing rays (IR) and has worked.Atr is caused the material and the material incentive of blocking dna radiating of double-stranded DNA fracture, single stranded DNA fracture.
Chk1 is a protein kinase, and it is arranged in downstream (Sanchez et al., Science, 277:1497-501,1997 of dna damage checkpoint signals transduction path Atm and/or Atr; U.S.Patent No.6,218,109).In mammalian cell, Chk1 replys the material that causes dna damage that comprises ionizing rays (IR), ultraviolet (UV) light and hydroxyurea and phosphorylation (Sanchez et al., supra; Lui et al., Genes Dev., 14:1448-59,2000).This phosphorylation of Chk1 depends on Atm (Chen et al., Oncogene, 18:249-56,1999) and Atr (Lui et al., aforementioned) in the activation mammalian cell.In addition, Chk1 is proved phosphorylation weel (O ' Connell et al., EMBO J., 16:545-554,1997) and Pds1 (Sanchez et al., Science, 286:1166-1171,1999), known their gene product is very important in cell cycle regulating.
These studies confirm that Mammals Chk1 works in the dna damage check point that the Atm that causes stagnating in the S phase relies on.(Feiioo et al., J.Cell Biol., 154:913-923,2001 are illustrated in the effect of Chk1 in S phase mammalian cell recently; Zhao et al., Proc.Nat.Acad.Sci.U.S.A., 99:14795-800,2002; Xiao et al., J Biol Chem., 278 (24): 21767-21773,2003; Sorensen et al., Cancer Cell, 3 (3): 247-58,2003), emphasized the effect of Chk1 aspect supervision DNA synthetic integrity.Chk1 regulates the active Cdc25A phosphorylation of cyclinA/cdk2 and causes that the S phase stagnates (Xiao etal., supra and Sorensen et al., aforementioned) by making.Chk1 is also by making Cdc25C phosphorylation and inactivation cause that G2 stagnates, wherein Cdc25C is the dual specificity phosphatase enzyme, and it makes cyclin-B/cdc2 (being also referred to as Cdk1) dephosphorylation (Fernery et al., Science usually when G2 enters mitotic division when cell, 277:1495 7,1997; Sanchez et al., supra; Matsuoka et al., Science, 282:1893-1897,1998; With Blasina et al., Curr.Biol., 9:1-10,1999).In both cases, to the active adjusting inducing cell of Cdk cycle arrest, enter mitotic division with the DNA cell that prevents there is dna damage or do not duplicate.
The cell cycle check point inhibitor of other kind works at G1 or G2/M phase.UCN-01, or 7-hydroxyl staurosporine, originally separated as non-specific kinase inhibitor, mainly act on protein kinase C, but it is found the activity that suppresses Chk1 recently and removes G2 cell cycle check point (Shi et al., aforementioned).Therefore, UCN-01 is non-selective Chk1 inhibitor, and thus, its pair cell when high dosage is toxic.When low dosage, it suppresses a lot of cell kinases non-specificly and suppresses G1 check point (Tenzer et al., Curr.Med Chem.Anti-Cancer Agents, 3:35-46,2003).
UCN-01 unites use with other cancer therapy, and for example radiation, carcinostatic agent camptothecine (Tenzer and Pruschy, aforementioned) and gemcitabine people such as (, aforementioned) Shi obtain limited success.In addition, UCN-01 also has been used to strengthen the effect (Hirose et al., CancerRes., 61:5843-5849,2001) that Temozolomide (TMZ) inductive dna mismatch is repaired (MMR) in the spongioblast oncocyte.Clinically, UCN-01 be not as expected for effective chemotherapeutics, may be because the failure of treatment plan and shortage cause (Grant and Roberts, Drug Resistance Updates to the evaluation of concrete key molecule target, 6:15-26,2003).So people such as Mack have reported that UCN-01 cell cycle dependency to cis-platinum in the non-small cell lung cancer cell system of cultivating strengthens, but do not identify the key cell cycle check point of UCN-01 target specifically.(Mack et al.，Cancer ChemotherPharmacol.，51(4)：337-348，2003)。
Exist several other be used to make the strategy of tumour cell to the chemotherapeutics sensitivity that influences the cell cycle.For example, give 2-aminopurine and removed many cells cycle checkpoint mechanism, for example mimosine inductive G1 stagnates or the hydroxyurea inductive S phase stagnates, cell is entered and process mitotic division (Andreassen et al., Proc Natl Acad Sci USA., 86:2272-2276,1992).Caffeine, a kind of methyl xanthine has been used to strengthen the cytotoxicity such as the dna damage material of cis-platinum and ionizing rays, and the G2 check point is passed in its mediation, and inducing cell death thus.(Bracey et al.，Clin.Cancer Res.，3：1371-1381，1997)。But, be used for realizing that the dosage of the caffeine that the cell cycle removes has surpassed acceptable clinically level, not that feasible treatment is selected.In addition, the kinase whose antisense nucleic acid of Chk1 has been used to increase susceptibility (Yin et al., Biochem.Biophys.Res.Commun., the 295:435-44 to topoisomerase enzyme inhibitor BNP1350,2002), still also show the problem relevant usually with antisense therapy and gene therapy.
The Chk1 inhibitor has been disclosed in WO 02/070494, WO 04/014876 and WO03/101444.Other Chk1 inhibitor comprises the di-aryl urea compounds that is described among the U.S. Patent Publication 2003-0069284A1, as aryl-and the carbamide compound of heteroaryl-replacement; Methyl xanthine and related compound (Fan et al., Cancer Res.55:1649-54,1995); Urea groups thiophene-based (WO 03/029241); N-pyrrolopyridinyl carboxyl acylamide (WO 03/028724); Antisense Chk1 oligonucleotide (WO 01/57206); Chk1 receptor antagonist (WO 00/16781); Heteroaromatic carboxamide derivative (WO 03/037886); Aminothiophene class (WO 03/029242); (indazolyl) benzimidazoles (WO 03/004488); Heterocycle-oxyimino-fluorenes class (WO02/16326); The derivative (scytonemin) (the 6th, 495, No. 586 United States Patent (USP)s) that contains Scytonema (scytoneman) skeleton; Heteroaryl benzamide class (WO 01/53274); Indazole compound (WO 01/53268); Indole carbazole class (people such as Tenzer, aforementioned); Look alkane derivatives (WO02/070515); Paullones (people such as Schultz, J.Med.Chem., Vol:42:2909-2919, (1999)); Indeno pyrazoles (WO 99/17769); Flavonoid (people such as Sedlacek, Int J.Oncol., 9:1143-1168, (1996)); The peptide derivant (WO98/53050) of the peptide ring of serine threonine kinases; Oxindole class (WO 03/051838).
But, still need badly in this area effectively and Chk1 inhibitor optionally.The present invention is devoted to these needs and other needs.
Summary of the invention
The present invention relates to the effective and selective depressant of check point kinase c hk1.Chk1 inhibitor of the present invention can be used for treating the indication that relates to abnormal cell proliferation, and can be in relating to dna damage or dna replication dna in the treatment of the indication of defective as chemical sensitization and radiosensitizer.
Therefore, one aspect of the present invention provides the compound of structural formula (I).These compounds can be used for suppressing in the method for Chk1, and this method comprises that the compound with the structural formula of significant quantity (I) gives individual step.
The structural formula of formula (I) compound is:
X wherein
1For do not exist ,-O-,-S-, CH
2-or-N (R
1)-;
X
2For-O-,-S-or-N (R
1)-;
Y is O or S; Or=Y represents to be connected two hydrogen atoms on the identical carbon atoms;
The C that W is selected from heteroaryl, aryl, Heterocyclylalkyl, cycloalkyl and is replaced by heteroaryl or aryl
1-6Alkyl, wherein said aryl W are randomly individual with R by 1-4
2The substituting group of expression replaces, and described heteroaryl W is randomly individual with R by 1-4
5The substituting group of expression replaces, and described Heterocyclylalkyl and cycloalkyl W are randomly by one or two C
1-6Alkyl substituent replaces;
R
1Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, C
2-6Alkynyl and aryl;
R
2Be selected from heteroaryl, halogen, the optional C that replaces
1-6Alkyl, C
2-6Thiazolinyl, OCF
3, NO
2, CN, NC, N (R
3)
2, OR
3, CO
2R
3, C (O) N (R
3)
2, C (O) R
3, N (R
1) COR
3, N (R
1) C (O) OR
3, N (R
1) C (O) C
1-6Alkylidene group C (O) R
3, N (R
1) C (O) C
1-6Alkylidene group C (O) OR
3, N (R
1) C (O) C
1-6Alkylidene group OR
3, N (R
1) C (O) C
1-6Alkylidene group NHC (O) OR
3, N (R
1) C (O) C
1-6Alkylidene group SO
2NR
3, C
1-6Alkylidene group OR
3And SR
3
R
3Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, cycloalkyl, aryl, heteroaryl, SO
2R
4, halogen is by one or more halogens, hydroxyl, aryl, heteroaryl, Heterocyclylalkyl, N (R
4)
2And SO
2R
4The C that replaces
1-6Alkyl, C
1-6Alkylidene aryl, C
1-6The alkylidene group heteroaryl, C
1-6Alkylidene group C
3-8Heterocyclylalkyl, C
1-6Alkylidene group SO
2Aryl, the optional C that replaces
1-6Alkylidene group N (R
4)
2, OCF
3, C
1-6Alkylidene group N (R
4)
3 +, C
3-8Heterocyclylalkyl and CH (C
1-6Alkylidene group N (R
4)
2)
2, or two R
3Group lumps together and forms optional 3 to 8 yuan of aliphatic series rings that replace;
R
4Be selected from do not exist, hydrogen, C
1-6Alkyl, cycloalkyl, aryl, heteroaryl, C
1-6Alkylidene aryl and SO
2C
1-6Alkyl, or two R
4Group lumps together and forms optional 3 to 8 yuan of rings that replace;
R
5Be selected from C
1-6Alkyl, C
2-6Alkynyl, aryl, heteroaryl, Heterocyclylalkyl, N (R
3)
2, N (R
1) C (O) R
3, N (R
1) CO
2R
3, OR
3, halogen, N
3, CN, C
1-6Alkylidene aryl, C
1-6Alkylidene group N (R
3)
2, C (O) R
3, C (O) OR
3, C (O) N (R
3)
2, CF
3, and
R
6Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, cycloalkyl, Heterocyclylalkyl, aryl, heteroaryl, SO
2R
4, by one or more halogens, hydroxyl, aryl, heteroaryl, Heterocyclylalkyl, N (R
4)
2And SO
2R
4The C that replaces
1-6Alkyl, C
1-6Alkylidene aryl, C
1-6The alkylidene group heteroaryl, C
1-6Alkylidene group C
3-8Heterocyclylalkyl, C
1-6Alkylidene group SO
2Aryl, the optional C that replaces
1-6Alkylidene group N (R
4)
2, OCF
3, C
1-6Alkylidene group N (R
4)
3 +, C
3-8Heterocyclylalkyl and CH (C
1-6Alkylidene group N (R
4)
2)
2
R
7And R
8Be selected from hydrogen, C independently of one another
1-6Alkyl, halogen, OR
3, N (R
3)
2, C (O) N (R
3)
2, C
1-3Alkylidene aryl, CN, NO
2, C (O) OR
11, C (O) R
11And SR
11
R
9For-C ≡ C-R
10Or-CF
3, perhaps R
8And R
9Group forms 5 or 6 yuan of isocyclic aliphatic series or aromatic ring system with the carbon atom that they connected, and this system option ground contains 1-3 and is selected from O, NR
4Heteroatoms with S.
R
10Be selected from hydrogen, C
1-6Alkyl, aryl, C
1-6Alkylidene aryl, heteroaryl and C
1-6The alkylidene group heteroaryl;
R
11Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, aryl, C
1-3Alkylidene aryl, C
3-8Cycloalkyl and C
1-3Alkylidene group C
3-8Cycloalkyl;
N is 1 or 2;
Or be the acceptable salt of medicine or the prodrug or the solvate of formula (I) compound.
Another aspect of the present invention provides the pharmaceutical composition of the compound that contains one or more structural formulas (I), and the purposes of said composition in treatment disease or illness, wherein in vivo or stripped state the restraining effect of Chk1 is helped treatment or is of value to research or diagnosis.
Another aspect of the present invention provides sensitization is accepted the cell in the individuality of chemotherapy or radiotherapy owing to medical condition method, comprises compound and chemotherapeutics and radiotherapeutic agents one or both of of structural formula (I) are united individual administration.Non-limiting indication by this method treatment is a cancer.
The present invention provides the method that suppresses or prevent abnormal cell proliferation on the other hand.In one embodiment, this method comprises makes the cell mass that contains the abnormality proliferation cell contact for some time with at least a a certain amount of Chk1 activator, and described amount and time are enough to make the intercellular cell cycle arrest of abnormality proliferation basic synchronizationization.After in cell mass, obtaining the basic synchronization of cell cycle arrest, make cell mass contact for some time with at least a a certain amount of Chk1 inhibitor that is enough to remove basically cell cycle arrest.
Below to detailed description of preferred embodiments every aspect of the present invention is become apparent.
Detailed description of preferred embodiments
Compound of the present invention has following structural formula (I):
X wherein
1For do not exist ,-O-,-S-,-CH
2-, or-N (R
1)-;
X
2For-O-,-S-or-N (R
1)-;
Y is O or S; Perhaps=Y represents to be connected two hydrogen atoms on the identical carbon atoms;
The C that W is selected from heteroaryl, aryl, Heterocyclylalkyl, cycloalkyl and is replaced by heteroaryl or aryl
1-6Alkyl, wherein said aryl W are randomly individual with R by 1-4
2The substituting group of expression replaces, and described heteroaryl groups W is randomly individual with R by 1-4
5The substituting group of expression replaces, and described Heterocyclylalkyl and group of naphthene base W are randomly by one or two C
1-6Alkyl substituent replaces;
R
1Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, C
2-6Alkynyl and aryl;
R
2Be selected from heteroaryl, halogen, the optional C that replaces
1-6Alkyl, C
2-6Thiazolinyl, OCF
3, NO
2, CN, NC, N (R
3)
2, OR
3, CO
2R
3, C (O) N (R
3)
2, C (O) R
3, N (R
1) COR
3, N (R
1) C (O) OR
3, N (R
1) C (O) C
1-6Alkylidene group C (O) R
3, N (R
1) C (O) C
1-6Alkylidene group C (O) OR
3, N (R
1) C (O) C
1-6Alkylidene group OR
3, N (R
1) C (O) C
1-6Alkylidene group NHC (O) OR
3, N (R
1) C (O) C
1-6Alkylidene group SO
2NR
3, C
1-6Alkylidene group OR
3And SR
3
R
3Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, cycloalkyl, aryl, heteroaryl, SO
2R
4, halogen is by one or more halogens, hydroxyl, aryl, heteroaryl, Heterocyclylalkyl, N (R
4)
2And SO
2R
4The C that replaces
1-6Alkyl, C
1-6Alkylidene aryl, C
1-6The alkylidene group heteroaryl, C
1-6Alkylidene group C
3-8Heterocyclylalkyl, C
1-6Alkylidene group SO
2Aryl, the randomly C of Qu Daiing
1-6Alkylidene group N (R
4)
2, OCF
3, C
1-6Alkylidene group N (R
4)
3 +, C
3-8Heterocyclylalkyl and CH (C
1-6Alkylidene group N (R
4)
2)
2Or two R
3Group lumps together and forms optional 3 to 8 yuan of aliphatic series rings that replace;
R
4Do not exist for being selected from, hydrogen, C
1-6Alkyl, cycloalkyl, aryl, heteroaryl, C
1-6Alkylidene group heteroaryl and SO
2C
1-6Alkyl, or two R
4Group lumps together and forms optional 3 to 8 yuan of rings that replace;
R
5For being selected from C
1-6Alkyl, C
2-6Alkynyl, aryl, heteroaryl, Heterocyclylalkyl, N (R
3)
2, N (R
1) C (O) R
3, N (R
1) CO
2R
3, OR
3, halogen, N
3, CN, C
1-6Alkylidene aryl, C
1-6Alkylidene group N (R
3)
2, C (O) R
3, C (O) OR
3, C (O) N (R
3)
2, CF
3, and
R
6Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, cycloalkyl, Heterocyclylalkyl, aryl, heteroaryl, SO
2R
4, by one or more halogens, hydroxyl, aryl, heteroaryl, Heterocyclylalkyl, N (R
4)
2And SO
2R
4The C that replaces
1-6Alkyl, C
1-6Alkylidene aryl, C
1-6The alkylidene group heteroaryl, C
1-6Alkylidene group C
3-8Heterocyclylalkyl, C
1-6Alkylidene group SO
2Aryl, the optional C that replaces
1-6Alkylidene group N (R
4)
2, OCF
3, C
1-6Alkylidene group N (R
4)
3 +, C
3-8Heterocyclylalkyl, and CH (C
1-6Alkylidene group N (R
4)
2)
2
R
7And R
8Be selected from hydrogen, C independently of one another
1-6Alkyl, halogen, OR
3, N (R
3)
2, C (O) N (R
3)
2, C
1-3Alkylidene aryl, CN, NO
2, C (O) OR
11, C (O) R
11And SR
11
R
9For-C ≡ C-R
10Or-CF
3, or R
8And R
9The carbon atom that is connected with them lumps together and forms 5 or 6 yuan of isocyclic aliphatic series or aromatic ring system, and this system option ground contains 1-3 and is selected from O, NR
4Heteroatoms with S;
R
10Be selected from hydrogen, C
1-6Alkyl, aryl, C
1-6Alkylidene aryl, heteroaryl and C
1-6The alkylidene group heteroaryl;
R
11Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, aryl, C
1-3Alkylidene aryl, C
3-8Cycloalkyl and C
1-3Alkylidene aryl C
3-8Cycloalkyl;
N is 1 or 2;
Perhaps be the acceptable salt of medicine or the prodrug or the solvate of formula (I) compound.
The preferred compound of the present invention is such compound, wherein X
1And X
2For-N (H)-;
Y is O or S; And
W is the optional heteroaryl that replaces.
In one embodiment, W contains at least two heteroatomic heteroaryls that are selected from N, O and S, and described heteroaryl ring is randomly replaced by 1-4 substituting group, and described substituting group is selected from the optional C that replaces
1-6Alkyl, aryl, heteroaryl, N (R
3)
2, OR
3, C (O) N (R
3)
2, CO
2R
3, CN, CF
3And halogen, wherein R
3Define as the front.
Other preferred compound of structural formula (I) is that W is selected from pyridazinyl, pyrimidyl, pyrazinyl and triazinyl and randomly is selected from C by 1-4
1-6Alkyl, alkyl, heteroaryl, N (R
3)
2, C (O) N (R
3)
2, CO
2R
3, OR
3, CF
3The compound that substituting group replaced with halogen.
Other preferred compound of structural formula (I) is R wherein
6Be selected from the optional C that replaces
1-6Alkyl, C
1-6Alkylidene group N (R
4)
2, C
1-6Alkylidene group heteroaryl, C
1-6Alkylene heterocyclic alkyl and C
3-8The compound of Heterocyclylalkyl.In other preferred embodiment, R
6Be selected from C
1-6Alkyl, (CH
2)
1-6N (CH
3)
2, (CH2)
1-6N (CH
3),
In other preferred embodiment, W is selected from randomly and is selected from C by 1-4
1-6Alkyl, C
2-6Alkynyl, aryl, heteroaryl, CN, CO
2R
3, N (R
3)
2, OR
3, CF
3The following groups that substituting group replaced with halogen:
In a preferred embodiment, W is
Or
In most preferred embodiment, W is pyrazinyl and X
1And X
2N (H) respectively does for oneself.
In other preferred embodiment, W be 5 by R
5Pyrazine-2-base that base replaces, that is,
In most preferred embodiment, R
5Be CF
3, CH
3Or do not exist.Other most preferred embodiment comprises such compound, wherein R
7Be H; R
8Be H; R
9Be selected from-C ≡ CH and CF
3Perhaps R
8And R
9The carbon atom that is connected with them lumps together formation
Perhaps
In another most preferred embodiment, R
6Be selected from-(CH
2)
2N (CH
3)
2,
Terminology used here " alkyl " comprises and contains the straight chain of specifying carbonatoms and the alkyl of branching, typically is the propyl group and the butyl of methyl, ethyl and straight chain and branching.Except as otherwise noted, alkyl can contain nearly 20 carbon atoms.Term " alkyl " comprises " bridging alkyl ", i.e. C
6-C
16Two ring or multi-ring alkyls, for example norcamphyl, adamantyl, two ring [2.2.2] octyl groups, two ring [2.2.1] heptyl, two ring [3.2.1] octyl group or naphthane.Alkyl can randomly be substituted, for example by hydroxyl (OH), halogen, aryl, heteroaryl, cycloalkyl, Heterocyclylalkyl, amino (N (R
3)
2) and alkylsulfonyl (SO
2R
3) replacement, wherein R
3Define as the front.
Term " cycloalkyl " is defined as ring-type C
3-8Alkyl, for example cyclopropyl, cyclobutyl, cyclohexyl or cyclopentyl.Contain 1-3 and be independently selected from the heteroatoms of oxygen, nitrogen and sulphur on ring, the definition and the cycloalkyl of " Heterocyclylalkyl " are similar.Cycloalkyl and Heterocyclylalkyl can be saturated or the undersaturated loop systems of part, and it randomly for example is independently selected from C by 1-3
1-4Alkyl, C
1-3Alkylidene group OH, C (O) NH
2, NH
2, oxo (=O), the group of aryl, trifluoroacetyl group and OH replaces.Heterocyclylalkyl also can be randomly by C
1-6Alkyl, hydroxyl C
1-6Alkyl, C
1-3Alkylidene aryl or C
1-3Alkylidene group heteroaryl N-replaces.
Term " thiazolinyl " is except group contains carbon-to-carbon double bond, and definition is with " alkyl ".
Term " alkynyl " is except group contains carbon-to-carbon triple bond, and definition is with " alkyl ".
Term " alkylidene group " refers to have substituent alkyl.For example, term " C
1-6Alkylidene group C (O) OR " refer to the alkyl that contains 1-6 carbon atom that replaced by-C (O) OR group.Alkylidene group is randomly replaced by the substituting group of the optional alkyl substituent of one or more previous listed conducts.
Term " halogen " or " halogen " are defined as fluorine, bromine, chlorine and iodine here.
Separately or the term " aryl " of uniting use be defined as monocycle or polycyclic aromatic group here, preferred monocycle or bicyclic aromatic group, for example phenyl or naphthyl.Except as otherwise noted, aryl can be unsubstituted or by one or polysubstituted, especially is independently selected from for example halogen, C by 1-4
1-6Alkyl, C
2-6Thiazolinyl, OCF
3, NO
2, CN, NC, N (R
3)
2, OR
3, CO
2R
3, C (O) N (R
3)
2, C (O) R
3, N (R
1) COR
3, N (R
1) C (O) OR
3, N (R
1) C (O) OR
3, N (R
1) C (O) C
1-3Alkylidene group C (O) R
3, N (R
1) C (O) C
1-3Alkylidene group C (O) OR
3, N (R
1) C (O) C
1-3Alkylidene group NHC (O) OR
3, N (R
1) C (O) C
1-3Alkylidene group SO
2NR
3, C
1-3Alkylidene group OR
1And SR
3Group replace R wherein
1And R
3For as previously defined.Exemplary aryl includes, but are not limited to phenyl, naphthyl, tetralyl, chloro-phenyl-, tolyl, methoxyphenyl, trifluoromethyl, nitrophenyl, 2,4-methoxychlor phenyl or the like.Term " aryl C
1-3Alkyl " and " heteroaryl C
1-3Alkyl " be defined as and have C
1-3The aryl of alkyl substituent or heteroaryl.
Term " heteroaryl " is defined as monocycle or the bicyclic system that contains one or two aromatic ring and contain at least one nitrogen, oxygen or sulphur atom in aromatic ring here.Except as otherwise noted, heteroaryl can be unsubstituted or is selected from for example C by one or more (especially 1-4)
1-6Alkyl, aryl, heteroaryl, CF
3, CN, C (O) N (R
3)
2, CO
2R
2, N (R
3)
2, OR
3Replace with the substituting group of halogen, wherein R
3For as previously defined.The example of heteroaryl include but not limited to thienyl, furyl, pyridyl, _ azoles base, quinolyl, isoquinolyl, indyl, triazinyl, triazolyl, isothiazolyl, different _ the azoles base, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidyl, thiazolyl and thiadiazolyl group.
Term " hydrogen (hydro) " is defined as-H.
Term " hydroxyl " is defined as-OH.
Term " nitro " is defined as-NO
2
Term " cyano group " is defined as-CN.
Term " isocyano-" is defined as-NC.
Term " trifluoromethoxy " is defined as-OCF
3
Term " azido-" is defined as-N
3
Terminology used here " 3 to 8 yuan of rings " refers to isocyclic or heterocyclic aliphatic series or aromatic group, include but not limited to morpholinyl, piperazinyl, phenyl, thiophenyl, furyl, pyrryl, imidazolyl, pyrimidyl and pyridyl, it is randomly by one or more, and especially 1-3 the group as top aryl substituent example replaces.
The carbon content of the part of hydrocarbonaceous is by specifying carbon atom minimum value and peaked subscript to represent in this part, for example " C
1-6Alkyl " refer to have the alkyl group of 1-6 carbon atom, comprise end points.
In the structure of this paper, for the key of unsubstituted, substituting group is a methyl, for example
For
When no indicated substituting group is connected ring when going up carbon atom, should understand the hydrogen atom that this carbon atom contains suitable number.In addition, when no indicated substituting group was connected on carbonyl or the nitrogen-atoms, substituting group should be thought hydrogen, for example
Abbreviation " Me " is a methyl.Abbreviation CO and C (O) are carbonyl (C=O).
Symbol N (R
x)
2Be used to represent to be connected in two R of same nitrogen-atoms
xGroup, wherein x represents letter or number, for example R
a, R
b, R
3, R
4Deng.When using these symbols, R
xGroup can be identical or different, and be selected from by R
xThe defined group of group.
" Chk1 inhibitor " refers to remove at least in part the active any compound of the proteic cell cycle check point of Chk1, and it is for producing naturally or synthetic is known or the compound found afterwards.When the check point mechanism that has fully overcome cell, make cell enter the next stage of cell cycle, or make and then realize the releasing of cell cycle check point when cell directly enters necrocytosis from its cell cycle phase that stops.The releasing of cell cycle check point makes cell to damage or defective is brought follow-up cell cycle phase into, induces thus or promotes necrocytosis.Any mechanism that comprises natural death of cerebral cells and mitotic division disaster all can cause necrocytosis.
" Chk1 activator " refers to any reagent known or that find afterwards, and it can activate the Chk1 kinases in the activity aspect DNA reparation and the running balance at the cell cycle check point, and is induced to the small part cell cycle arrest thus.The Chk1 activator comprises can make cell cycle arrest at the reagent in any stage of cell cycle, and this stage can be called " the target stage " of activator here.The target stage comprises any cell cycle outside the mitotic division, i.e. G1 phase, S phase and G2 phase.Can be used for Chk1 activator of the present invention and comprise dna damage reagent, such as chemotherapeutics and/or radiation.The Chk1 activator that is fit to also comprises radiotherapeutic agents, such as ionization or uv-radiation.Radioactivity Chk1 activator includes but not limited to gamma-radiation, x-ray radiation, UV-light, visible light, ir radiation, microwave radiation and combination thereof.
" inhibition abnormal cell proliferation " refers to delay or eliminate the speed of the propagation of this abnormality proliferation cell.This restraining effect causes the reduction of multiple-copy rate or the increase of necrocytosis speed, or the two all has.Necrocytosis can be caused by any mechanism that comprises natural death of cerebral cells and mitotic division disaster.
" prevent abnormal cell proliferation " and refer to before abnormal cell proliferation takes place its inhibition or suppress its recurrence.
" in vivo " refer in vivo, in animal or human's body.In this article, be used in patient's body to the reagent treatability, to delay or to eliminate the propagation of unusual replicating cell.This reagent also can be used as the appearance of prophylactic agent with the generation that prevents abnormal cell proliferation or recurrence or the symptom relevant with it.
" exsomatize " and refer in vitro.Isolated cells group's example comprises vitro cell culture and such as the biological sample of the liquid of taking from the human or animal or tissue sample.This sample can obtain by means commonly known in the art.Exemplary biological fluid comprises blood, celiolymph, urine, saliva.Exemplary tissue samples comprises tumour and its slicer.In this article, this compound can be used in the extensive application such as treatment and experiment.
Terminology used here " radiation-sensitizing agents " is defined as people or other animal with the administration of treatment significant quantity to increase cell to the susceptibility of electromagnetic radiation and/or improve the treatment of diseases of available electromagnetic radiation treatment.
Terminology used here " electromagnetic radiation " and " radiation " include but not limited to that wavelength is 10-20 to 100 meter radiation.
The present invention includes all possible steric isomer and the geometrical isomer of structural formula (I) compound.The present invention not only comprises racemic compound, also comprises the optically active isomer.When structural formula (I) compound need be as single enantiomer, it can be obtained by the fractionation of end product, or pure starting raw material or use chiral auxiliary(reagent) from isomery obtain by stereospecificity is synthetic, for example referring to Z.Ma et al., Tetrahedron:Asymmetry, 8 (6), pages 883-888 (1997).The fractionation of end product, intermediate or starting raw material can be finished by any currently known methods in this area.In addition, when may there be tautomer in the compound of structural formula (I), the present invention was intended to the tautomeric forms that comprises that this compound is all.As illustrated in after this, specific steric isomer can demonstrate the ability of uncommon inhibition Chk1 when combining with chemotherapy or radiotherapy in the treatment.
The prodrug of the compound of structural formula (I) also can be used as the compound in the inventive method.Confirm that fully the prodrug approach has been successfully used to the physics-chem characteristic of (biological example reversibly) change compound temporarily, wherein compound deriving is to be suitable for preparing and/or the form of administration, then in vivo as drug release (referring to, H.Bund-gaard, Ed., " Design of Prodrugs, " Elsevier, Amsterdam, (1985); R.B.Silverman, " The Organic Chemistryof Drug Design and Drug Action, " Academic Press, San Diego, chapter8, (1992); K.M.Hillgren et al., Med.Res.Rev., 15,83 (1995)).
Compound of the present invention can comprise one or more functional groups.If expectation or needs, the functional group can be modified so that prodrug to be provided.The prodrug that is fit to for example comprises acid derivative, as acid amides or ester.Those skilled in the art will appreciate that also the N-oxide compound also can be used as prodrug.
Terminology used here " the acceptable salt of medicine " refers to contain acidic moiety and forms the compound that has the structural formula (I) that is fit to cationic salt.The acceptable positively charged ion of medicine that is fit to comprises basic metal (for example, sodium or potassium) and alkaline-earth metal (for example calcium or magnesium) positively charged ion.In addition, contain basic center structural formula (I) compound the acceptable salt of medicine for and the acid salt that forms of the acceptable acid of medicine.Limiting examples comprises hydrogenchloride, hydrogen bromide, vitriol, dithionate, phosphoric acid salt, hydrophosphate, acetate, benzoate, succinate, fumarate, maleate, lactic acid salt, Citrate trianion, tartrate, gluconate, metilsulfate, benzene sulfonate and tosilate.According to noted earlier, any The compounds of this invention that appears at herein of here mentioning is intended to comprise compound and the acceptable salt of its medicine or the solvate of structural formula (I).
The compounds of this invention can be as the administration of pure chemistry product in treatment, but the compound of preferred structure formula (I) is with the form administration of pharmaceutical composition or preparation.Therefore, the invention provides the compound that comprises formula (I) and the pharmaceutical composition of medicine acceptable diluent or carrier thereof.The method of pharmaceutical compositions also is provided, and it comprises that the compound of formula (I) can be accepted diluent or carrier with its medicine to be mixed.
Therefore, the present invention also provides pharmaceutical preparation, and it comprises structural formula (I) compound or the acceptable salt of its medicine, prodrug or its solvate, together with one or more medicine acceptable carriers, and optional other therapeutic and/or preventative composition.Carrier means compatible with other composition of preparation and harmless to its recipient for " acceptable ".
The kinase whose restraining effect of check point typically using dosage effect measuring method detects, wherein make sensitive detection system and finite concentration scope be concerned about that compound contacts, this concentration range comprises does not observe or only observes the MIN concentration of doing the time spent, the saturation concentration of the higher concentration when observing partial action when observing maximum effect.In theory, the detection of this dosage effect effect to inhibitor compound can be described with suppressing the sigmoid curve that kilsyth basalt is shown the function of concentration.In theory, this curve is also through the point of 50% the level that the check point enzymic activity is enough to be reduced in this detection enzymic activity minimum value and maximum value difference.This concentration is called inhibition concentration (50%) or IC
50Value.IC
50The mensuration of value is preferably used conventional biological chemistry (acellular) determination techniques or based on the determination techniques of cell.
Usually with reference to IC
50The usefulness of relatively coming the comparison inhibitor of value is wherein compared with control compound, higher IC
50The value representation test compounds is renderd a service lower, lower IC
50The value representation test compounds is renderd a service higher.When using dosage effect assay method was measured, compound exhibits of the present invention was lower than the IC of 5 μ M
50Value, and be low to moderate 0.1nM.Preferred compound shows 500nM or lower IC
50Value.The present invention more preferably compound exhibits is lower than 250nM, is lower than 100nM, is lower than 50nM or is lower than the IC of 20nM
50Value.
The preferred Chk1 inhibitor of the present invention is for optionally, promptly with respect to following protein kinase, the inhibition of Chk1 demonstrated at least 20 times selectivity: protein kinase A, protein kinase C, cdc2 and pp60v-src.The preferred Chk1 inhibitor of the present invention preferably shows at least 75 times selectivity to the inhibition of Chk1 with respect to following protein kinase: protein kinase A, protein kinase C, cdc2 and pp60v-src.The most preferred Chk1 inhibitor of the present invention shows at least 75 times selectivity to the inhibition of Chk1 with respect to following protein kinase: protein kinase A, protein kinase C, cdc2, pp60v-src, protein kinase B/Akt-1, p38MapK, EPK1, p70S6K, cdc2, cdk2, chk2 and ab1 Tyrosylprotein kinase." times selectivity " is defined as the Chk1 inhibitor to contrasting kinase whose IC
50Value is divided by the IC of Chk1 inhibitor to Chk1
50Value.
When individually dosed, selectivity Chk1 inhibitor does not play chemotherapeutics.On the contrary, nonselective Chk1 inhibitor can be used as chemotherapeutics, and this is because its required other protein kinase of cell growth inhibiting or active ability of enzyme more fully.This may cause causing other cytosis of the therapeutic index of adverse side effect and/or reduction.
Be suitable for compound used in this invention and pharmaceutical composition comprise activeconstituents wherein with the significant quantity administration to obtain those of its intended purposes.More specifically, " treatment significant quantity " amount of referring to be enough to treat the amount of the individuality of suffering from indication or alleviating the existing symptom of this indication.Determine the treatment significant quantity fully within those skilled in the art's limit of power, especially according to detailed disclosing that this paper provided.
Except the Chk1 inhibitor, pharmaceutical composition of the present invention also can be formulated as and comprise cytokine, lymphokines, somatomedin, other Hemopoietic factor, or its mixture, so that reduce pharmaceutical composition issuable or relative adverse side effect when individually dosed.The cytokine that in pharmaceutical composition of the present invention, is particularly useful, lymphokine, somatomedin or other hematopoietic factor include but not limited to M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNF, G-CSF, Meg-CSF, GM-CSF, the thrombosis element, STEM CELL FACTOR, erythropoietin, angiogenesis hormone (comprises Ang-1, Ang-2, Ang-4, Ang-Y and/or people's angiogenesis hormone sample polypeptide), vascular endothelial growth factor (VEGF), angiogenin, bone morphogenetic protein-1 (BMP-1), BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, bmp receptor IA, bmp receptor IB, Brain Derived Neurotrophic Factor, ciliary neurotrophic factor, the neutrophilic chemotactic factor 1 of cntf receptor cytokine induction, the neutrophilic chemotactic factor 2 of cytokine induction, the neutrophilic chemotactic factor 2 of cytokine induction, endothelial cell growth factor (ECGF), endothelin-1, epidermal growth factor, come from epithelial neutrophilic granulocyte and attract thing, fibroblast growth factor (FGF) 4, FGF5, FGF6, FGF7, FGF8, FGF8b, FGF8c, FGF9, FGF10, acid FGF, basic FGF, come from the neutrophil leucocyte factor acceptor 1 of glial cell line, come from the neutrophil leucocyte factor acceptor 2 of glial cell line, the protein that growth is relevant, the protein that growth is relevant, the protein that growth is relevant, the protein that growth is relevant, heparin-bounding Urogastron, pHGF, hepatocyte growth factor receptor, insulin-like growth factor I, rhIGF-1, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukaemia inhibitory factor, the leukaemia inhibitory factor acceptor, nerve growth factor, trk C, neurotrophic factor-3, neurotrophic factor-4, placenta growth factor, placenta growth factor 2, platelet-derived endothelial cell growth factor (ECGF), Thr6 PDGF BB, Thr6 PDGF BB A chain, Thr6 PDGF BB AA, Thr6 PDGF BB AB, Thr6 PDGF BB B chain, Thr6 PDGF BB BB, platelet derived growth factor receptor, platelet derived growth factor receptor, the pre-B growth-stimulating factor, STEM CELL FACTOR, the STEM CELL FACTOR acceptor, transforming growth factor (TGF), TGF, TGF1, TGF1.2, TGF2, TGF3, TGF5, abeyance TGF1, TGF, conjugated protein I, the conjugated protein II of TGF, the conjugated protein III of TGF, I type Tumor Necrosis Factor Receptors, II type Tumor Necrosis Factor Receptors, urokinase type plasminogen-activating factor acceptor, vascular endothelial growth factor, with and chimeric protein and biology or immunologic competence fragment.
The compound of structural formula (I) can be to grip altogether or be connected with slave part also, and this slave part is improvement compound beneficial characteristics in treatment usefulness method.This is gripped altogether and to specific anatomical site or target area (for example can strengthen compound, tumour) conveying, can make compound in target cell, keep treatment concentration, change the pharmacokinetics and the pharmacodynamic profiles of compound, and/or improve the therapeutic index or the security of compound.The slave part that is fit to for example comprises amino acid, oligopeptides or polypeptide, and for example, antibody is as monoclonal antibody and other engineered antibody; And the natural or synthetic ligands of acceptor in target cell or the tissue.Other subsidiary that is fit to comprises and improves bio distribution and/or target cell to the lipid acid of the picked-up of compound or lipid part (for example, referring to Bradley et al., Clin.Cancer Res. (2001) 7:3229).
Preparation of the present invention can be specified the standard manner administration of disease with treatment, for example by oral, parenteral, through mucous membrane (for example, hypogloeeis or through the oral cavity), local, through skin, rectum, suction (for example, intranasal or dark lung suck) administration.Administered parenterally includes but not limited in vein, artery, intraperitoneal, subcutaneous, muscle, the sheath and intra-articular administration.Administered parenterally also can use pressure technique such as POWDERJECT
TMFinish.
For the oral administration that comprises orally administering, composition can be the tablet or the lozenge form of usual manner preparation.For example, the tablet and the capsule that are used for oral administration can comprise conventional excipients, for example tackiness agent (for example, syrup, Sudan Gum-arabic, gelatin, sorbyl alcohol, tragacanth, starch mucus or polyvinylpyrrolidone), weighting agent (for example, lactose, sucrose, Microcrystalline Cellulose, W-Gum, calcium phosphate or sorbyl alcohol), lubricant (for example, Magnesium Stearate, stearic acid, talcum, polyoxyethylene glycol or silica), disintegrating agent (for example, yam starch or sodium starch glycollate) or wetting agent (for example, Sodium Lauryl Sulphate BP/USP).Tablet can carry out dressing according to method well-known in the art.
Perhaps, compound for example of the present invention can be incorporated in in the oral liquid such as water-based or oily suspensions, solution, emulsion, syrup or elixir.And the preparation that contains these compounds can exist by drying products, prepares again with water or other carrier that is fit to before using.This type of liquid preparation can comprise conventional additives, for example, suspension agent is as the edible fat of sorbitol syrups, methylcellulose gum, glucose/sucrose syrup, gelatin, Natvosol, Vltra tears, carboxy methyl cellulose, aluminium stearate gel and hydrogenant; Emulsifying agent is as Yelkin TTS, single oleic acid sorbitan ester or Sudan Gum-arabic; Nonaqueous carrier (it can comprise edible oil) is as Prunus amygdalus oil, fractionated coconut oil, oily ester, propylene glycol and ethylene glycol; And sanitas, as methyl p-hydroxybenzoate or propyl ester and Sorbic Acid.
Said preparation also can be formulated as suppository, for example, contains conventional suppository base, as Oleum Cocois or other glyceryl ester.The composition that is generally used for sucking can solution, the form of suspension or emulsion provides, and it can be used as the dried powder administration or uses conventional propellant such as Refrigerant 12 or trichlorofluoromethane with the aerosol form administration.Typical part and preparation capable of permeating skin comprise conventional water-based or nonaqueous carrier, such as eye drop, creme, ointment, lotion and paste, perhaps are the form of paste, patch or the film of pastille.
In addition, composition of the present invention can be formulated as and be suitable for by injection or the continuous enteron aisle external administration that injects.The preparation that is used for injecting can be suspension, solution or the emulsion form of oiliness or aqueous carrier, and can contain the prescription agent, such as suspending agent, stablizer and/or dispersion agent.Perhaps, activeconstituents can be powder type, prepares again with suitable carriers (for example, aseptic apyrogenic water) before use.
Composition of the present invention also can be formulated as depot formulations.This long-acting preparation can be through implanting (for example, subcutaneous or intramuscular) or passing through administered intramuscular.Therefore, compound of the present invention can be with suitable polymers or hydrophobic material (emulsion in for example acceptable oil), ion exchange resin is prepared or as indissoluble derivative (for example, difficulty soluble salt).
Concerning the animal doctor used, formula (I) compound or the acceptable salt of its medicine, prodrug or solvent can carry out administration as suitable acceptable preparation according to common animal doctor's practice.The animal doctor can easily determine the dosage and the route of administration of suitable particular animals.The animal of available The compounds of this invention and method treatment includes but not limited to the animal in pet, domestic animal, displaying animal and the zoological park.
Synthetic method
Compound of the present invention can be by following synthetic route preparation.Starting raw material can obtain or pass through literature method used for a long time well known to those skilled in the art to prepare from commercial source.Radicals X, R
1, R
3, R
4, R
5, R
6, R
7, R
8, R
9As defined above.
Route 1
As shown in Scheme 1, by with alkaline purification, then add R such as salt of wormwood, triethylamine or sodium hydride
6X can become the compound of formula 1 compound of formula 2, and wherein X is halogen, methanesulfonates or tosylate.The example of this reaction solvent for use comprises DMF, THF, CH
2Cl
2And composition thereof.This is reflected at and carries out about 15 minutes to 12 hours between 0 ℃ and 100 ℃.
Perhaps, the compound of formula 1 can with formula R
6The compound of X, wherein X is a hydroxyl, the gained mixture is handled with triphenylphosphine and di-isopropyl azodicarboxylate in such as the solvent of THF, so that the compound of formula 2 to be provided.
Available hydrogen is handled the compound of formula 2 in the presence of the catalyzer that carries palladium or Raney nickel such as platinum oxide, carbon, or in the presence of metallic zinc to handle this compound, so that the compound of formula 3 to be provided such as the acid source of saturated ammonium chloride or hydrochloride aqueous solution.The example of solvent for use comprises methyl alcohol, ethanol, ethyl acetate or its mixture in this reaction.Reaction is usually in room temperature or be lower than and carried out under the room temperature 1-12 hour.
But the compound of the compound through type 3 of formula 5 and the compound of formula 4 (as preparation as described in the route 2) chemical combination and preparing.The example of solvent for use comprises toluene, benzene and dimethylbenzene in this reaction.Be reflected at and carried out under 60-100 ℃ 5-12 hour.
Route 2
As shown in Scheme 2, the compound of formula 4 can prepare by using the compound such as the alkaline purification formula 6 of DIEA and phenylbenzene phosphinylidyne (phophoryl) trinitride.The typical solvent of this reaction is THF, and this is reflected under 20-80 ℃ and the blast shield and carried out 1-12 hour.
Route 3
The another kind of synthetic method of the compound of route 3 display types 5.The compound of formula 3 is used the compound treatment of the formula 7 for preparing according to route 4.A kind of spendable solvent is DMF, in 1-12 hour time temperature of reaction is remained between room temperature and 60 ℃.
Route 4
As shown in Scheme 4, can be in the presence of alkali, by coming the compound of preparation formula 7 with the compound of handling formula 8 such as the aryl esters chloroformate of benzene chloro-formic ester or p-nitrophenyl chloroformate such as pyridine.The example of this reaction solvent for use comprises 0 ℃ of CH to room temperature
2Cl
2Or pyridine.
Route 5
The another kind of preparation method of the compound of route 5 display types 5.By handling with alcohol in the presence of such as the alkali of sodium hydride, two (trimethyl silyl) acid amides sylvite or n-Butyl Lithium, the compound of formula 9 becomes the compound of formula 2.The example that is used for the solvent of this reaction comprises THF or ether.Reaction typically-15 ℃ to the room temperature about 1-6 hour.Continue to use the compound that the step of describing in the route 1 becomes the compound of formula 2 formula 5.
Route 6
The another kind of synthetic method of the compound of route 6 display types 5.Adopt the step of describing in the route 4 compound of formula 3 can be changed into the compound of formula 10.Adopt the step of describing in the route 1 compound of formula 10 can be changed into the compound of formula 5.
Concrete and the non-limiting instance of the compound of structural formula (I) below is provided, and the method for illustrating among its synthetic U.S. Patent Application Publication 2003-0069284 A1 (incorporating this paper by reference at this) according to following and while pending trial is carried out.
Be used in being abbreviated as in following synthesizing: hour (h), water (H
2O), sal epsom (MgSO
4), hydrochloric acid (HCl), dimethyl sulfoxide (DMSO) (DMSO), diisopropyl azo-2-carboxylic acid (DIAD), methylene dichloride (CH
2Cl
2), chloroform (CHCl
3), methyl alcohol (MeOH), ammonium hydroxide (NH
4OH), deuterated chloroform (CDCl
3), tetrahydrofuran (THF) (THF), N-Methyl pyrrolidone (NMP), acetate (AcOH), ethyl acetate (EtOAc), ethanol (EtOH), ether (Et
2O), yellow soda ash (Na
2CO
3), sodium bicarbonate (NaHCO
3), nitric acid (HNO
3), hydrochloric acid (HCl), sodium-chlor (NaCl), sodium sulfate (Na
2SO
4), dimethyl formamide (DMF), 1,8-diazabicylo-[5.4.0] 11 carbon-7-alkene (DBU) and N, N-diisopropylethylamine (DIEA).
Intermediate 1:
5-methyl-pyrazine-2-carbonyl azide thing
Under condition under room temperature and the nitrogen, (25 grams, 181mmol) (31.7ml 181mmol), obtains brown solution to the adding of the suspension in 540ml THF DIEA to the 5-methyl-pyrazine-2-carboxylic acid that stirs.Then under blast shield dropwise to add diphenylphosphine acylazide thing (39.2ml, 181mmol) solution in 50ml THF in 1 hour.Allowing reactant stir spends the night.Then reactant at room temperature rotary evaporation to small volume and at Et
2O (1L) and H
2Distribute between the O (1L).H
2The O layer is with 2 * 250ml Et
2O strips, and the organism that merges washs 2 * 1L with saturated sodium bicarbonate.Organism drying (MgSO
4), filter and be concentrated into solid and determine, with it with Et
2O smashs to pieces and obtains yellow solid (15 grams, 50%).Can place 20mlEt by crude product with 1 gram
2Among the O, and at room temperature handle several minutes, be separated to purer compound with 1-2 gram decolorizing carbon.After filtering and concentrating, this material is a pure white, and the TLC that carries out in EtOAc proves homogeneity.The rate of recovery is generally 65%.
Compound 1:
1-[5-ethynyl-2-(1-methyl-piperidines-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazine-2-yl)-urea
Step 1:
3-(4-bromo-2-nitro-phenoxymethyl)-1-methyl-piperidines
Under nitrogen atmosphere, to the 1-methyl piperidine-3-methyl alcohol that stirs (1g, 5.3mmol), 2-nitro-4-bromo-phenol (1.15g, 5.3mmol) and triphenylphosphine (1.39g, 5.3mmol) solution in dry THF (25ml) drip the diisopropyl azo-2-carboxylic acid (1.04mL, 5.3mmol).The mixture that obtains at room temperature stirred 12 hours, used ethyl acetate (75ml) dilution then and (2 * 50ml) wash with salt solution.The organic layer MgSO that merges
4Drying, filtration and concentrating under reduced pressure.Crude product is through chromatographic column (silica gel) purifying and use the CH that contains 5%MeOH
2Cl
2Eluant solution obtains colorless oil.
Step 2:
1-methyl-3-(2-nitro-4-trimethylammonium-silylation ethynyl-phenoxymethyl)-piperazine Pyridine
Under room temperature and nitrogen, to 3-(4-bromo-2-nitro-phenoxymethyl)-1-methyl-piperidines (910mg that stirs, 2.76mmol) solution in 40ml benzene adds trimethyl silyl acetylene (543mg, 5.5mmol), two (triphenylphosphine)-palladium (the II) (39mg of dichloro, 0.55mmol), cupric iodide (I) (42mg, 0.22mmol) and DBU (1.26 grams, 8.3mmol).With reaction mixture reflux 6 hours.After being cooled to room temperature, reaction mixture filter with benzene and with filtrate at ethyl acetate (100ml) and H
2Distribute between the O (100ml).Make organism drying (MgSO
4), filter, concentrate and at 95/5 CH
2Cl
2Carry out chromatography among the/MeOH, the product that obtains wishing.
Step 3:
2-(1-methyl-piperidines-3-ylmethoxy)-5-TMS ethynyl-aniline
Under refluxad (1.25g, 3.72mmol) solution in 20ml AcOH adds iron powder (1.4g/25g/ atom) in batches to 1-methyl-3-(2-nitro-4-TMS ethynyl-phenoxymethyl)-piperidines that stirs.After 1 hour, reaction mixture cools off slightly, with 50ml EtOAc dilution, and filters, and washes solid with EtOAc then.The filtrate evaporate to dryness, residue is at EtOAc (50ml) and saturated NaHCO
3Distribute (50ml).Water extracts with 50ml EtOAC, the 100ml salt water washing of the organic layer of merging, dry (MgSO
4), filter and simmer down to oily matter (100%).
Step 4:
1-[2-(1-methyl-piperidines-3-base-methoxyl group)-5-TMS ethynyl-benzene Base]-3-(5-methyl-pyrazine-2-yl)-urea
To the 5-methyl-pyrazine-2-carbonyl azide thing (163mg that stirs; 1.0mmol) solution in toluene (4ml) (be heated in advance 90 ℃ 15 minutes) adds 1-methyl-3-(2-nitro-4-TMS ethynyl-phenoxymethyl)-piperidines (305mg; 1mmol).Mixture is cooled to 65 ℃ and stirred 12 hours.Then reaction mixture is cooled to room temperature and filtration, obtains required material.
Step 5:
1-[5-ethynyl-2-(1-methyl-piperidines-3-ylmethoxy)-phenyl]-3-(the 5-methyl- Pyrazine-2-yl)-urea
In nitrogen and under the reflux conditions, to 1-[2-(1-methyl-piperidines-3-ylmethoxy)-5-TMS ethynyl-phenyl]-3-(5-methyl-pyrazine-2-yl)-urea (43mg, 0.095mmol) solution in the 5ml absolute ethanol add Potassium monofluoride (27mg, 0.47mmol).Reaction refluxed 1.5 hours, was cooled to room temperature, and at ethyl acetate (30ml) and H
2Distribute between the O (30ml).Separation of organic substances, dry (MgSO
4), filter and concentrate, obtain brown solid (35mg, 97%).
1H-NMR(400MHz，CDCl
3)δ：11.31(brs，1H)，8.56(s，1H)，8.23(s，1H)，8.17(s，1H)，7.59(brs，1H)，7.16(d，1H)，6.78(d，1H)，3.86(m，2H)，3.12(m，1H)，2.96(s，1H)，2.82(m，1H)，2.52(s，3H)，2.34(m，1H)，2.20(s，3H)，1.97(m，1H)，1.91-1.63(m，4H)，1.07(m，1H)。LRMS (apci, positive ion (positive)) m/e 380.5 (M+1).
Compound 2:
1-[2-(2-dimethylamino-oxyethyl group)-5-ethynyl-phenyl]-3-(5-methyl-pyrazine-2-yl)-urea
Step 1:
Dimethyl-[2-(2-nitro-4-TMS ethynyl-phenoxy group)-ethyl]- Amine
Use [2-(4-bromo-2-nitro-phenoxy group-ethyl] dimethyl-amine (it prepare as compound 1 step 1, from 4-bromo-2-nitrophenols and N, N-dimethylaminoethanol) to prepare according to the method for compound 1 step 2.
Step 2:
2-(2-dimethylamino-oxyethyl group)-5-TMS ethynyl-aniline
Dimethyl-[2-(2-nitro-4-TMS ethynyl-phenoxy group)-ethyl]-amine (1mmol) is dissolved among the 1ml MeOH, adds the 0.5ml saturated ammonium chloride, adds zinc powder (5mmol) then.Mixture stirred 10 minutes, used EtOAc (50ml) and yellow soda ash (50ml, 10% aqueous solution) dilution then.Organic layer MgSO
4Drying is filtered and is under reduced pressure concentrated to obtain limpid oily matter (97%).
Step 3:
1-[2-(2-dimethylamino-oxyethyl group)-5-TMS ethynyl-phenyl]-3- (5-methyl-pyrazine-2-yl)-urea
Method preparation according to compound 1 step 4.
Step 4:
1-[2-(2-dimethylamino-oxyethyl group)-5-ethynyl-phenyl]-3-(5-methyl-pyrazine- The 2-yl)-urea
Method according to compound 1 step 5 prepares end product (81mg, 97%).
1H-NMR(400MHz，CDCl
3)δ：10.68(brs，1H)，8.57(s，1H)，8.51(s，1H)，8.08(s，1H)，7.95(s，1H)，7.18(dl 1H)，6.82(d 1H)，4.18(m，2H)，2.99(s，1H)，2.82(m，2H)，2.52(s，3H)，2.39(s，6H)。LRMS (apci, positive ion (positive)) m/e340.5 (M+1).
Compound 3:
1-[5-ethynyl-2-(pyridin-3-yl methoxyl group)-phenyl]-3-(5-methyl-pyrazine-2-yl)-urea
Step 1:
3-(2-nitro-4-TMS-ethynyl-phenoxymethyl)-pyridine
Use 3-(4-bromo-2-nitro-phenoxy methyl)-pyridine (it prepares as compound 1 step 1, from 4-bromo-2-nitrophenols and 3-piconol) to prepare according to the method for compound 1 step 2.
Step 2:
2-(pyridin-3-yl methoxyl group)-5-TMS ethynyl-aniline
Method preparation according to compound 1 step 3.
Step 3:
1-(5-methyl-pyrazine-2-base-3-[2-(pyridin-3-yl methoxyl group)-5-trimethyl silane Ethyl-acetylene base-phenyl]-urea
Method preparation according to compound 1 step 4.
Step 4:
1-[5-ethynyl-2-(pyridin-3-yl methoxyl group)-phenyl]-3-(5-methyl-pyrazine-2- Base)-urea
Method according to compound 1 step 5 prepares end product.
1H-NMR(400MHz，d
6-DMSO)δ：10.16(s，IH)，8.79(s，1H)，8.64(d，1H)，8.56(brs，1H)，8.37(s，1H)，7.96(d，1H)，7.50(d，1H)，7.28(brs，1H)，7.18(m，2H)，5.26(s，2H)，4.02(s，1H)，2.30(s，3H)。LRMS (apci, positive ion) m/e360.4 (M+1).
Compound 4:
1-[3-(1-methyl-piperidines-3-ylmethoxy)-5,6,7,8-tetrahydrochysene-naphthalene-2-yl]-3-(5-methyl-pyrazine-2-yl)-urea
Step 1:
3-nitro-5,6,7,8-naphthane-2-alcohol
With 5,6,7, (2.0g 13.5mmol) is dissolved in CHCl to 8-tetrahydrochysene-naphthalene-2-alcohol
3(45ml) and in the acetic acid (22.5ml).Dropwise add dense HNO
3(0.87ml, acetic acid 13.7mmol) (22.5ml) solution.Stir after 20 hours, mixture water (70ml) dilutes and uses Na
2CO
3Be neutralized to pH10.CHCl is separated and used to water layer
3Washing.MgSO is used in the organic layer water and the salt water washing that merge
4Drying is filtered and is concentrated under vacuum.Crude product uses 1: 10 chromatographic separation of EtOAc/ hexane on silicon-dioxide, then at SiO
2Last use hexane/ether (75: 1) carries out the chromatographic separation second time.
Step 2:
1-methyl-3-(3-nitro-5,6,7,8-tetrahydrochysene-naphthalene-2-yloxymethyl)-piperidines
Method according to compound 1 step 1 is used 3-nitro-5,6,7,8-tetrahydrochysene-naphthalene-2-alcohol (on seeing) and the preparation of (1-methyl-piperidines-3-yl) methyl alcohol.
Step 3:
3-(1-ethyl-piperidines-3-ylmethoxy)-5,6,7,8-tetrahydrochysene-naphthalene-2-base amine
With Pd (OH)
2(catalytic amount) prepared in EtOH (20ml) by 1-methyl-3-(3-nitro-5,6,7,8-tetrahydrochysene-naphthalene-2-yloxymethyl)-piperidines (1mmol).Mixture under atmospheric pressure stirred 16 hours.Catalyzer removes by filter with C salt, and filtrate obtains required material through concentrating under reduced pressure.
Step 4:
1-[3-(1-methyl-piperidines-3-base-methoxyl group)-5,6,7,8-tetrahydrochysene-naphthalene-2-yl]-3-(5- Methyl-pyrazine-2-yl)-urea
Method according to compound 1 step 4 is used 3-(1-methyl-piperidines-3-ylmethoxy)-5,6,7,8-tetrahydrochysene-naphthalene-2-base amine and 5-methyl-pyrazine-2 carbonyl azide compound.
1H NMR(400MHz，CDCl
3)δ：10.98(brds，1H)，8.45(brds，1H)，8.36-8.30(m，1H)，8.20(s，1H)，8.02(s，1H)，6.54(s，1H)，3.91-3.79(m，2H)，3.27-3.14(m，1H)，2.96-2.84(m，1H)，2.56-2.46(m，4H)，2.51(s，3H)，2.43-2.29(m，1H)，2.30(s，3H)，2.11-1.99(m，1H)，1.93-1.83(m，2H)，1.83-1.69(m，6H)，1.18-1.04(m，1H)。LRMS (apci, positive ion) m/e 410.3 (M+1).
Compound 5:
1-[3-(1-methyl-piperidines-2-ylmethoxy)-5,6,7,8-tetrahydrochysene-naphthalene-2-yl]-3-(5-methyl-pyrazine-2-yl)-urea
Step 1:
3-nitro-5,6,7,8-tetrahydrochysene-naphthalene-2-alcohol
The method of describing according to compound 4 steps 1 prepares.
Step 2:
1-methyl-2-(3-nitro-5,6,7,8-tetrahydrochysene-naphthalene-2-yloxymethyl)-piperidines
Method according to compound 1 step 4 is used 3-nitro-5,6,7, pure and mild (1-methyl-piperidines-2-the yl)-methyl alcohol preparation of 8-tetrahydrochysene-naphthalene-2-.
Step 3:
3-(1-methyl-piperidines-2-ylmethoxy)-5,6,7,8-tetrahydrochysene-naphthalene-2-base amine
Use 1-methyl-2-(3-nitro-5,6,7,8-tetrahydrochysene-naphthalene-2-yloxymethyl 1)-piperidines preparation according to the method for compound 4 steps 3.
Step 4:
1-[3-(1-methyl-piperidines-2-base-methoxyl group)-5,6,7,8-tetrahydrochysene-naphthalene-2-yl]-3-(5- Methyl-pyrazine-2-yl)-urea
Method according to compound 1 step 4 is used 3-(1-methyl-piperidines-2-ylmethoxy)-5,6,7,8-tetrahydrochysene-naphthalene-2-base amine and 5-methyl-pyrazine-2-carbonyl azide thing preparation.Crude product is recrystallization from absolute ethanol.
1H NMR(400MHz，d
6-DMSO)δ：10.06(s，1H)，10.02-9.87(brd，1H)，8.66(s，1H)，8.16(s，1H)，7.89(s，1H)，6.69(s，1H)，4.55-4.47(m，1H)，2.89-2.81(m，1H)，2.71-2.60(m，7H)，2.43(s，3H)，2.30(s，3H)，2.07-1.97(m，1H)，1.85-1.74(m，1H)，1.74-1.58(m，7H)，1.57-1.45(m，1H)。LRMS (apci, positive ion) m/e 410.5 (M+1).
Compound 6:
(S)-1-(5-methyl-pyrazine-2-yl)-3-[2-(piperidines-3-ylmethoxy)-5-trifluoromethyl-phenyl]-urea
Step 1.
(S)-3-methylol-piperidines-1-carboxylic acid tert-butyl ester
Use (S)-piperidines-1 according to WO 02-070494, the preparation of the 3-dicarboxylic acid 1-tert-butyl ester.
Step 2.
(S)-3-(4-trifluoromethyl-2-nitro-phenoxymethyl)-piperidines-1-carboxylic acid tert-butyl ester
Use (S)-3-methylol-piperidines-1-carboxylic acid tert-butyl ester and 2-nitro-4-trifluoromethyl-phenol preparation according to compound 1 step 1.
Step 3.
(S)-3-(2-amino-4-trifluoromethyl-phenoxymethyl)-piperidines-1-carboxylic acid tertiary butyl Ester
To (S)-3-(4-trifluoromethyl-2-nitro-phenoxymethyl)-piperidines-1-carboxylic acid tertiary butyl ester that stirs (4.04g, 10mmol) solution in ethanol (30ml) add the Pearlman catalyzer (421mg, 3mmol).Reaction is with hydrogen (balloon) purge 3 times and stirred 12 hours.Reactant filters and drying under reduced pressure with C salt.This material adopts Biotage 40M column purification, with hexane/ethyl acetate (3/1) wash-out, is cured as the oily matter of white solid subsequently.
Step 4.
(S)-3-{4-trifluoromethyl-2-[3-(5-methyl-pyrazine-2-yl)-urea groups]-the phenoxy group first Base }-piperidines-1-carboxylic acid tert-butyl ester
(1.14g, 7mmol) solution in toluene (20ml) places the oil bath 15 minutes that is preheated to 90 ℃ with 5-methyl-pyrazine-2-carbonyl azide thing of stirring.(2.62g, 7mmol), reaction is cooled to 65 ℃ and stirred 12 hours to add (S)-3-(2-amino-4-trifluoromethyl-phenoxymethyl)-piperidines-1-carboxylic acid tert-butyl ester.Reactant is cooled to room temperature and concentrating under reduced pressure.Solid uses Biotage 40M column purification, uses hexane/ethyl acetate (1/1) wash-out to obtain amorphous solid.
Step 5.
(S)-1-(5-methyl-pyrazine-2-yl)-3-[2-(piperidines-3-ylmethoxy)-5-fluoroform Base-phenyl]-urea
To (S)-3-{4-trifluoromethyl-2-[3-(5-methyl-pyrazine-2-the yl)-urea groups that stirs]-phenoxymethyl }-(560mg, 1.1mmol) solution in two _ alkane (2ml) adds HCl (two _ alkane solution of 4ml 4M sol.) to piperidines-1-carboxylic acid tertiary butyl ester.Stir after 3 hours, the reactant concentrating under reduced pressure is to obtain 400mg (89%) light yellow solid.
1H-NMR(400MHz，d
6-DMSO)δ：10.60(s，1H)，9.20(s，2H)，8.79(s，1H)，8.60(s，1H)，8.35(s，1H)，7.40(d，1H)，7.20(s，1H)，4.15(m，2H)，3.50(d，1H)3.30(d，1H)，2.90(m，2H)，2.40(s，3H)，1.40-2.00(m，4H)。LRMS (apci, positive ion) m/e410.30 (M+1).
Compound 7:
(R)-1-(5-methyl-pyrazine-2-yl)-3-[2-(piperidines-3-ylmethoxy)-5-trifluoromethyl-phenyl]-urea
Step 1.
(R)-3-methylol-piperidines-1-carboxylic acid tert-butyl ester
Use (R)-piperidines-1 according to WO 02/070494, the preparation of the 3-dicarboxylic acid 1-tert-butyl ester.
Step 2.
(R)-3-(4-trifluoromethyl-2-nitro-phenoxymethyl)-piperidines-1-carboxylic acid tert-butyl ester
Use (R)-3-methylol-piperidines-1-carboxylic acid tert-butyl ester and 2-nitro-4-trifloro methyl phenol preparation according to compound 1 step 1.
Step 3.
(R)-3-(2-amino-4-trifluoromethyl-phenoxymethyl)-piperidines-1-carboxylic acid uncle fourth fat
Use (R)-3-(4-trifluoromethyl-2-nitro-phenoxymethyl)-piperidines-1-carboxylic acid tert-butyl ester preparation according to compound 4 steps 3.
Step 4.
(R)-3-{4-trifluoromethyl-2-[3-(5-methyl-pyrazine-2-yl)-urea groups]-the phenoxy group first Base }-piperidines-1-carboxylic acid tert-butyl ester
Use (R)-3-(2-amino-4-trifluoromethyl-phenoxymethyl)-piperidines-1-carboxylic acid tert-butyl ester and 5-methyl-pyrazine-2-carbonyl azide thing preparation according to compound 1 step 4.
Step 5.
(R)-1-(5-methyl-pyrazine-2-yl)-3-[2-(piperidines-3-ylmethoxy)-5-fluoroform Base-phenyl]-the urea hydrochloride
Use (R)-3-{4-trifluoromethyl-2-[3-(5-methyl-pyrazine-2-yl)-urea groups according to compound 6 steps 5]-phenoxymethyl }-piperidines-1-carboxylic acid tert-butyl ester preparation.
1H-NMR(400MHz，d
6-DMSO)δ：10.50(s，1H)，9.08(s，2H)，8.78(s，1H)，8.60(s，1H)，8.25(s，1H)，7.40(d，1H)，7.20(d，1H)，4.10(m，2H)，3.70(m，1H)，3.50(m，1H)，3.22(d，1H)，2.90(m，2H)，2.40(s，3H)，1.95(d，1H)，1.80(m，2H)，1.45(m，1H)。LRMS (apci, positive ion) m/e 410.3 (M+1).
Compound 8:
1-[2-(1-methyl-piperidin-4-yl oxygen base)-5-trifluoromethyl-phenyl]-3-(5-methyl-pyrazine-2-yl) urea
Step 1.
1-methyl-4-(2-nitro-4-trifluoromethyl-phenoxy group)-piperidines
With 2-nitro-4-trifluoromethyl-phenol (2.07g, 10mmol), 1-methyl-piperidines-4-alcohol (1.21g, 10.5mmol) and triphenylphosphine (2.75g is 10.5mmol) with 30ml THF dilution and place nitrogen.Reaction mixture is cooled to 0 ℃, drips DIAD (2.12g, the solution in 1ml THF 10.5mmol) then.Reaction mixture was stirred 12 hours, be warming up to room temperature.Reactant dilutes with ethyl acetate (150ml) and yellow soda ash (10% aqueous solution of 150ml).Organic layer is washed with salt, uses MgSO
4Drying is filtered and drying under reduced pressure.Product uses the Biotage40M column purification, with hexane/ethyl acetate (500ml 1/1) and CH subsequently
2Cl
2/ MeOH/NH
4(98/8/2,500ml) wash-out obtains light yellow oil to OH.
Step 2.
2-(1-methyl-piperidin-4-yl oxygen base)-5-trifluoromethyl-aniline
Use 1-methyl-4-(2-nitro-4-trifluoromethyl-phenoxy group)-piperidines preparation according to compound 2 steps 2.
Step 3.
1-[2-(1-methyl-piperidin-4-yl-oxygen base)-5-trifluoromethyl-phenyl]-3-(the 5-methyl- Pyrazine-2-yl)-urea
Use 2-(1-methyl-piperidin-4-yl oxygen base)-5-trifluoromethyl-aniline and 5-methyl-pyrazine-2-carbonyl azide thing preparation according to compound 1 step 4.
1H-NMR(400MHz，CDCl
3)δ：8.65(s，1H)，8.45(s，1H)，8.15(s，1H)，7.22(d，1H)，6.95(d，1H)，4.45(m，1H)，2.95(m，2H)，2.55(s，3H)，2.45(s，3H)，1.85-2.30(m，6H)。LRMS (apci, positive ion) m/e 410.3 (M+1).
Compound 9:
1-(5-methyl-pyrazine-2-yl)-3-[2-(piperidines-3-ylmethoxy)-5-trifluoromethyl-phenyl]-urea
Step 1.
3-(2-nitro-4-trifluoromethyl-phenoxymethyl)-piperidines-1-carboxylic acid uncle fourth fat
Use 2-nitro-4-trifluoromethyl-phenol and 3-methylol-piperidines-1-carboxylic acid tert-butyl ester preparation according to compound 1 step 1.
Step 2.
3-(2-amino-4-trifluoromethyl-phenoxymethyl)-piperidines-1-carboxylic acid uncle fourth fat
Use 3-(2-nitro-4-trifluoromethyl-phenoxymethyl)-piperidines-1-carboxylic acid tert-butyl ester preparation according to compound 6 steps 3.
Step 3.
3-{2-[3-(5-methyl-pyrazine-2-yl)-urea groups]-4-trifluoromethyl-phenoxymethyl }- Piperidines-1-carboxylic acid tert-butyl ester
Use 3-(2-amino-4-trifluoromethyl-phenoxymethyl)-piperidines-1-carboxylic acid tert-butyl ester and 5-methyl-pyrazine-2-carbonyl azide thing preparation according to compound 1 step 4.
Step 4.
1-(5-methyl-pyrazine-2-yl)-3-[2-(piperidines-3-ylmethoxy)-5-trifluoromethyl- Phenyl]-the urea hydrochloride.
Use 3-{2-[3-(5-methyl-pyrazine-2-yl)-urea according to compound 6 steps 5]-4-trifluoromethyl-phenoxymethyl)-piperidines-1-carboxylic acid tert-butyl ester preparation.
1H-NMR(400MHz，d
6-DMSO)δ：10.41(s，1H)，8.90(m，2H)，8.79(s，1H)，8.60(s，1H)，8.25(s，1H)，7.40(d，1H)，7.25(d，1H)，4.15(m，2H)，2.40(s，3H)，1.20-3.80(m，9H)。LRMS (apci, positive ion) m/e 410.3 (M+1).
Compound 10:
1-[2-(1-methyl-piperidines-3-ylmethoxy)-5-trifluoromethyl-phenyl]-3-(5-methyl-pyrazine-2-yl)-urea
Step 1.
1-methyl-3-(2-nitro-4-trifluoromethyl-phenoxymethyl)-piperidines
With 2-nitro-4-trifluoromethyl-phenol (2.07g, 10mmol) (1-methyl-piperidines-3-yl)-methyl alcohol (1.36g, 10.5mmol) and triphenylphosphine (2.75g is 10.5mmol) with 30ml THF dilution and place nitrogen.Reactant is cooled to 0 ℃, drips DLAD (2.12g, 10.5mmol) solution in 2ml THF then.Reaction mixture was stirred 12 hours.Reaction mixture ethyl acetate (100ml) and HCl (50ml, 2N) dilution.(alkalize to pH=12 with solid sodium hydroxide then by 2 * 50ml) washings with ethyl acetate for water layer.(3 * 50ml) extract product with ethyl acetate.Organic layer is washed with salt, uses MgSO
4Drying is filtered and drying under reduced pressure.Product is used CH through the Biotage40M column purification
2Cl
2/ MeOH/NH
4OH (90/8/2) wash-out obtains yellow solid.
Step 2.
2-(1-methyl-piperidines-3-base-methoxyl group)-5-trifluoromethyl-aniline
Use 1-methyl-3-(2-nitro-4-trifluoromethyl-phenoxymethyl)-piperidines according to compound 4 steps 3
Step 3.
1-[2-(1-methyl-piperidines-3-base-methoxyl group)-5-trifluoromethyl-phenyl]-3-(5-first Base-pyrazine-2-yl)-urea
Use 2-(1-methyl-piperidines-3-ylmethoxy)-5-trifluoromethyl-aniline and 5-methyl-pyrazine-2-carbonyl azide thing preparation according to compound 1 step 4.
1H-NMR(400MHz，CDCl
3)δ：11.40(s，1H)，8.78(s，1H)，8.20-8.40(m，3H)，7.28(d，1H)，6.95(d，1H)，3.95(m，2H)，3.20(m，1H)，2.85(m，1H)，2.50(s，3H)，1.00-2.40(m，10H)。LRMS (apci, positive ion) m/e 424.4 (M+1).
Compound 11:
1-(5-methyl-pyrazine-2-yl)-3-[7-(pyridin-3-yl methoxyl group)-2,3-dihydro-benzo [1,4] two _ English-6-yl]-urea
Step 1:
3-(7-nitro-2,3-dihydro-benzo [1,4] two _ English-6-yloxymethyl)-pyridine hydrochloric acid Salt
In nitrogen and under the room temperature, to 7-nitro-2,3-dihydro-benzo [1,4] (197mg is 1mmol) (according to Bourlot et al., J.Med.Chem. for two _ English-6-alcohol, 1998,41 (17), 3140 and Besson et al., Tetrahedron, 1995,51, the method preparation of 3197-3204) (97 μ L 1mmol), then add triphenylphosphine (288mg to the solution adding pyridin-3-yl-methyl alcohol in 2.4ml THF, 1.1mmol) and drip DIAD (216uL, 1.1mmol).After stirring is spent the night, distribute by the rotary evaporation concentrated reaction mixture and between ethyl acetate and water.Compound does not all dissolve at each layer, isolates and washs with ethyl acetate with hydrochloride by filtering.
Step 2:
7-(pyridin-3-yl methoxyl group)-2,3-dihydro-benzo [1,4] two _ English-6-base amine
According to the method for compound 2 steps 2 from 3-(7-nitro-2,3-dihydro-benzo [1,4] two _ English-6-yloxymethyl)-pyridine hydrochloride (266mg, 0.82mmol) preparation.Product is separated as purple oily matter, is directly used in next reaction.
Step 3:
1-(5-methyl-pyrazine-2-yl)-3-[7-(pyridin-3-yl methoxyl group)-2,3-dihydro-benzene And [1,4] two _ English-6-yl]-urea
By 7-(pyridin-3-yl methoxyl group)-2,3-dihydro-benzo [1,4] two _ English-6-base amine and 5-methyl-pyrazine-2-carbonyl azide thing prepare end product and are separated as the brown solid according to the method for compound 1 step 4.
1H-NMR(400MHz，d
6-DMSO)δ：10.22(brs，1H)，9.98(s，1H)，8.77(s，1H)，8.62(d，1H)，8.57(s，1H)，7.93(d，1H)，7.76(s，1H)，7.44(m，2H)，6.78(s，1H)，5.17(s，2H)，4.20(s，4H)，2.36(s，3H)。LRMS (apci, positive ion) m/e 394.0 (M+1).
Compound 12:
1-[7-(2-dimethylamino-oxyethyl group)-2,3-dihydro-benzo [1,4] two _ English-6-yl]-3-(5-methyl-pyrazine-2-yl)-urea
Step 1:
Dimethyl-[2-(7-nitro-2,3-dihydro-benzo [1,4] two _ English-6-base oxygen base)-second Base] amine
Method according to compound 1 step 1 is used N, N-dimethylethanolamine and 7-nitro-2,3-dihydro-benzo [1,4] two _ English-6-alcohol preparation.Product is separated as yellow oil.
Step 2:
7-(2-dimethylamino-oxyethyl group)-2,3-dihydro-benzo [1,4] two _ English-6-base amine
At room temperature, to the dimethyl that stirs-[2-(7-nitro-2,3-dihydro-benzo [1,4] two _ English-6-base oxygen base)-ethyl]-(151mg, 0.56mmol) solution in 5.6ml 95% ethanol adds Pearlman catalyzer (40mg) to amine.Use hydrogen to make suspension, remain on then in 1 the atmospheric hydrogen through 3 vacuum/purge circulations.After stirring is spent the night, use GF/F filter paper and 95% ethanol by removing by filter catalyzer, filtrate is concentrated into clear and bright oily matter, it slowly becomes purple.This material directly is used to next reaction.
Step 3:
1-[7-(2-dimethylamino-oxyethyl group)-2,3-dihydro-benzo [1,4] two _ English-6- Base]-3-(5-methyl-pyrazine-2-yl)-urea
According to the method for compound 1 step 4, from 5-methyl-pyrazine-2-carbonyl azide thing and 7-(2-dimethylamino-oxyethyl group)-2,3-dihydro-benzo [1,4] two _ English-6-base amine prepares final compound.Product is separated as the brown solid.
1H-NMR (400MHz, d
6-DMSO) δ: 10.32 (brs, 1H), 10.03 (s, 1H), 9.51 (brs, 1H), 8.96 (s, 1H), 8.19 (s, 1H), 7.65 (s, 1H), 6.64 (s, 1H), 4.25 (m, 2H), 4.19 (s, 4H), 3.59 (m, 2H), 2.79 (s, 6H), 2.36 (s, 3H) .LRMS (apci, positive ion) m/e 374.4 (M+1).
Compound 13:
1-[3-(2-dimethylamino-oxyethyl group)-5,6,7,8-tetrahydrochysene-naphthalene-2-yl]-3-(5-methyl-pyrazine-2-yl)-urea
Step 1:
3-nitro-5,6,7,8-tetrahydrochysene-naphthalene-2-alcohol
Referring to compound 4 steps 1.
Step 2:
Dimethyl-[2-(3-nitro-5,6,7,8-tetrahydrochysene-naphthalene-2-base oxygen base)-ethyl]-amine
Method according to compound 1 step 1 is used 3-nitro-5,6,7,8-tetrahydrochysene-naphthalene-2-alcohol preparation.
Step 3:
3-(2-dimethylamino-oxyethyl group)-5,6,7,8-tetrahydrochysene-naphthalene-2-base amine
Use dimethyl-[2-(3-nitro-5,6,7,8-tetrahydrochysene-naphthalene-2-base oxygen base)-ethyl]-amine preparation according to the method for compound 1 step 3.
Step 4:
1-[3-(2-dimethylamino-oxyethyl group)-5,6,7,8-tetrahydrochysene-naphthalene-2-yl]-3-(the 5-methyl- Pyrazine-2-yl)-urea
Method according to compound 1 step 4 is used 3-(2-dimethylamino-oxyethyl group)-5,6,7,8-tetrahydrochysene-naphthalene-2-base amine and 5-methyl-pyrazine-2-carbonyl azide thing preparation.
1H NMR(400MHz，CDCl
3)δ：10.26(brds，1H)，8.74(s，1H)，8.45(brds，1H)，8.07(s，1H)，7.99(s，1H)，6.60(s，1)，4.17-4.10(m，2H)，2.99-2.89(m，2H)，2.77-2.66(m，4H)，2.54-2.44(m，9H)，1.81-1.73(m，4H)。LRMS (apci, positive ion) m/e 370.3 (M+1).
Methods of treatment
Compound of the present invention can be used for strengthening the radiation that is used in cancer and the treatment of other cell proliferation indication and/or the effect of chemotherapeutics, and wherein these cell proliferation indications relate to eukaryotic cell, comprise the cell in people and other animal.For example, compound of the present invention can be used for strengthening to the tumor treatment of giving repeated exhortations to talk endlessly such as the ammonia first usually or the metabolic antagonist of 5 FU 5 fluorouracil (5-FU) is treated.In general, compound of the present invention suppresses the cell of abnormality proliferation, comprise cancer with non-cancer.
The use of The compounds of this invention can cause partially or completely disappearing of abnormality proliferation cell, i.e. this class cell part or completely dissolve from cell mass.Therefore, for example, when the cell mass of abnormality proliferation is a tumour cell, compound of the present invention can be used for delaying the speed of tumor growth, reduces the size or the quantity of tumour, and/or induces tumor regression partially or completely.
In all embodiments, when also not having abnormal cell proliferation to be identified or do not have abnormal cell proliferation to carry out, but suspect or when expecting to have abnormal cell proliferation, compound of the present invention can be distinguished in vivo or exsomatize and use.In addition, the present invention also can be before abnormal cell proliferation uses during by treatment, to prevent or to suppress its recurrence.In these and related embodiment, " cell mass that comprises the abnormality proliferation cell " refers to wherein not have abnormal cell proliferation to be identified or carries out, but suspect or expect to have any cell mass of abnormal cell proliferation, and/or before abnormal cell proliferation is treated to prevent or to suppress any cell mass of its recurrence.
A kind of method of the present invention comprises Chk1 inhibitor compound of the present invention and the chemotherapeutics Combined Preparation with the treatment significant quantity, and this chemotherapeutics can be realized list or double-stranded DNA fracture or dna replication dna capable of blocking or cell proliferation.Perhaps, a kind of method of the present invention comprises the Chk1 inhibitor compound at least a of the present invention of treatment significant quantity and comprises the therapy Combined Preparation that uses antibody, wherein said antibody has the activity that suppresses cancer cell propagation, for example Trastuzumab (herceptin).Correspondingly, responsive such as the cancer of colorectal carcinoma, incidence cancer, carcinoma of the pancreas, mammary cancer, cancer of the stomach, bladder cancer, carcinoma vulvae, leukemia, lymphoma, melanoma, renal cell carcinoma, ovarian cancer, brain tumor, osteosarcoma and lung cancer to the enhancing treatment that the Combined Preparation by Chk1 inhibitor of the present invention and chemotherapeutics or antibody carries out.
Cancer comprises tumour or vegetation, and they are histiocytic grower, wherein the exhibition of the multiplication of cell not broken hair out of hand.Some these class growers are benign, and other are called " virulent ", and can cause organism death.Malignant growth, or the difference of " cancer " and optimum grower is, except showing cell proliferation fast, near tissue and shifting also can invading.In addition, being characterized as relative to each other of malignant growth demonstrates the forfeiture differentiation (bigger " dedifferenting ") largely and the forfeiture sense of organization with near tissue.This character is known as " anaplasia ".
Can also comprise solid tumor by the cancer of the present invention's treatment, i.e. cancer knurl and sarcoma.The cancer knurl comprises and comes from epithelial malignant growth that tissue also causes shifting near its infiltration (that is invasion and attack).Gland cancer is the cancer knurl that comes from glandular tissue, or comes from the tissue that forms identifiable glandular structure.The cancer of another big class comprises sarcoma, and it is the tumour in the material (as embryonic connective tissue) of cell is embedded into fibrillar or homogeneous.The present invention also makes treatment spinal cord or lymphoid cancer become possibility, these cancers comprise leukemia, lymphoma and other do not show as lump usually but be dispersed in vascular or lymphoreticular system in cancer.
Chk1 is active relevant with the cancer of pediatric oncology with for example being grown up of various ways, the grower that comprises solid tumor/malignant tumour, myxoma and round cell carcinoma, local late tumor, metastatic carcinoma, people soft tissue sarcoma, comprise You Wenshi (Ewing ' s) sarcoma, metastatic carcinoma, comprise the lymph transfer, squamous cell carcinoma, especially the head and neck, the esophagus squamous cell carcinoma, oral carcinoma, blood cell malignancies, comprise multiple myeloma, leukemia, comprise acute lymphoblastic leukemia, acute nonlymphocytic leukemia, lymphocytic leukemia, chronic granulocytic leukemia and hairy cell leukemia, exudative lymphoma (based on the lymphoma of body cavity), thymic lymphoma knurl lung cancer (comprises small cell carcinoma, cutaneous T cell lymphoma, hodgkin's (Hodgkin ' s) lymphoma, the non-Hodgkin lymphomas, adrenocortical carcinoma, produce the tumour of ACTH, non-small cell carcinoma, breast cancer, comprise small cell carcinoma and duct carcinoma), gastrointestinal cancer (comprises cancer of the stomach, colorectal carcinoma, colorectal carcinoma, the polyp that the colorectum tumorigenesis is relevant), the pancreas cancer, liver cancer, the urinary tract cancer (comprises bladder cancer, primary bladder surface tumour for example, the intrusion transsitional cell carcinoma of bladder, muscle invasive bladder cancer), prostate cancer, the female genital tract malignant tumour (comprises ovarian cancer, primary peritonaeum epithelium vegetation, cervical cancer, carcinoma of endometrium, carcinoma of vagina, the carcinoma of vulva, uterus carcinoma and the solid tumor in ovarian follicle), male genetic road malignant tumour (comprising carcinoma of testis and penile cancer), kidney (comprising renal cell carcinoma), the cancer of the brain (comprises the endogenous character cerebral tumor, neuroblastoma, the stellate cell cerebral tumor, neurospongioma, invade the metastatic tumor cell of central nervous system), osteocarcinoma (comprising osteoma and osteosarcoma), skin carcinoma (comprises malignant melanoma, human skin keratinocyte tumor progression and squamous cell carcinoma), thyroid carcinoma, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, the kidney parent cell (Wilms ' s) knurl, carcinoma of gallbladder, trophoderm vegetation, Hemangiopericytoma and Ka Boxishi (Kaposi ' s) sarcoma.Therefore, the administration of Chk1 inhibitor of the present invention can expect that meeting strengthens treatment plan.
Compound of the present invention also can be strengthened the validity of medicine in the treatment diseases associated with inflammation.The example of disease that can benefit from the compound combination therapy that is suitable for the inventive method is rheumatic arthritis, psoriasis, vitiligo, wegner's granulomatosis, and systemic lupus erythematosus (SLE).The treatment of sacroiliitis, wegner's granulomatosis and systemic lupus erythematosus often related to utilize immunosuppressant therapy, for example ionizing rays, methotrexate and endoxan.This treatment is directly or indirectly inducing DNA damage usually.Active inhibition causes cell to be easier to be controlled by these standard treatments to Chk1 in the immunocyte that damages.Psoriasis and vitiligo are usually with ultraviolet radiation (UV) and psoralene combination therapy.Dna damage reagent of the present invention is induced the killing effect of ultraviolet ray and psoralene, increases the therapeutic index of this treatment plan.Usually, when combining, can be used for compound of the present invention and strengthened control the inflammatory diseases cell with the immunosuppressive drug of present use.
Except above disclosed cancer, the present invention also can be used in the method for the non-cancer proliferating cells of treatment.These situations comprise but not in atherosclerosis, restenosis, vasculitis, ephritis, retinopathy, ephrosis, hyperproliferative skin disease is levied, psoriasis, the keloid scar, actinic keratosis, ectrodermosis erosiva pluriorificialis, rheumatic arthritis (RA), the juvenile chronic arthritis (JCA) of health outbreak, osteoporosis, systemic lupus erythematosus, comprise that epidermis is grown in interior transition proliferative eye disease downwards, proliferative vitreoretinopathy (PVR), diabetic retinopathy, the vascular proliferation disease, ichthyosis, or papilloma.
Can also comprise inflammation and diseases associated with inflammation, situation or symptom by the non-cancer proterties condition of the present invention's treatment.The example of these indications includes but not limited to rheumatic arthritis, psoriasis, vitiligo, wegner's granulomatosis and systemic lupus erythematosus (SLE).The treatment of sacroiliitis, wegner's granulomatosis and SLE often related to utilize immunosuppressive therapy, for example ionizing rays, methotrexate and endoxan.Psoriasis and vitiligo are usually with ultraviolet radiation (UV) and psoralene combination therapy.This treatment is directly or indirectly inducing DNA damage usually.Active inhibition causes cell to be easier to be controlled by these standard treatments to Chk1 in the immunocyte that damages.Usually, when with the immunosuppressive drug Combined Preparation of present use, can be used for Chk1 inhibitor of the present invention and can randomly be used to strengthen control the inflammatory diseases cell.
A kind ofly preferably be described in people such as Keegan with the method for Chk1 inhibitor of the present invention administration in the 60/503rd, No. 925 U.S. Provisional Application of submitting on September 17th, 2003, its disclosure is incorporated this paper into by reference in its entirety.The method of this inhibition abnormal cell proliferation relates on time with Chk1 activator (for example, chemotherapeutics) and Chk1 inhibitor of the present invention administration.In this method, dosage and the duration administration of at least a Chk1 activator to be enough to induce cell cycle arrest synchronous basically in the proliferative cell.When basically the stage of finishing is synchronous, with the administration of at least a Chk1 inhibitor, to eliminate cell cycle arrest and inductive treatment cell death.This method can be used with any Chk1 activator, and in treatment or prevent being applied in the abnormal cell proliferation of cancer and non-cancer.
Preferably, this Chk1 inhibitor is a selectivity Chk1 inhibitor.The cell mass of abnormality proliferation can contact with a kind of Chk1 inhibitor or can contact with multiple Chk1 inhibitor.If multiple Chk1 inhibitor is used, then these Chk1 inhibitor administration simultaneously or at the time administration that separates, this is by attending doctor or laboratory technician's decision.
The cell mass of abnormality proliferation can contact with a kind of Chk1 activator or can contact with multiple Chk1 activator.If multiple Chk1 activator is used, then these Chk1 activators administration simultaneously or at the time administration that separates, this is by attending doctor or laboratory technician's decision.
The present invention can be used to the isolated cells group.For example, compound of the present invention can exsomatize and use to determine that the Chk1 inhibitor is to given indication, cell type, patient's time of administration table and/or dosage and other parameter.The information of collecting from this use can be used to experiment purpose or set the external treatment scheme clinically.Other stripped purposes that the present invention is fit to it will be apparent to those skilled in the art that.
The also radiation-curable sensitized cell of compound of the present invention.The disease of available electromagnetic radiation treatment comprises neoplastic disease, optimum and malignant tumour and cancer cells.
The present invention also pays close attention to the electromagnetic radiation treatment of other disease that excludes this paper.The preferred embodiments of the invention are used following electromagnetic radiation: gamma-radiation (10-20 to 10-30m), X-radiation (10-12 to 10-9m), UV-light (10nm to 400nm), visible light (400nm to 700nm), ir radiation (700nm to 1.0mm) and microwave radiation (1mm to 30cm).
Current, many cancer treatment plans use by electromagnetic radiation (as the X-ray) activatory radiosterilization.The example of X ray activatory radiosterilization includes but not limited to following material: effective analogue and derivative thereof are gone up in metronidazole, misonidazole, demethyl misonidazole, Pimonidazole, etanidazole, Nimorazole (nimorazole), ametycin, RSU1069, SR4233, EO9, RB6145, niacinamide, 5-bromouracil deoxyribose (BUdR), idoxuridine (IUdR), bromine Deoxyribose cytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cis-platinum and treatment.
The photodynamic therapy of cancer (PDT) is used the radioactivation agent of visible light as sensitizing agent.The example of light power radiosterilization includes but not limited to following material: effective analogue and its derivative are gone up in hematoporphyrin derivative, PHOTOFRIN_, benzoporphyrin derivative, NPe6, mesoetioporphyrin tin (SnET2), pheoborbide-a, bacteriochlorophyll-a, naphthalene phthalocyanine, phthalocyanine, Phthalocyanine Zinc and treatment.
Radiosterilization can go up one or more compound Combined Preparation except that the Chk1 inhibitor of significant quantity with treatment, these compounds include but not limited to, promote radiosterilization in conjunction with compound, control therapeutical agent, nutrition and/or the oxygen flow of target cell to the compound of target cell, chemotherapeutics when having or do not have other radiation effect in tumour, or other is used for the treatment of compounds effective on cancer or other treatment of diseases.Can include but not limited to 5 FU 5 fluorouracil (5-FU), formyl tetrahydrofolic acid, oxygen, carbogen, red blood corpuscle blood transfusion, perfluoro-carbon (for example FLUOSOLW-DA), 2 with the example that radiosterilization is united other therapeutical agent of use, 3-DPG, BW12C, calcium ion channel blockor, pentoxifylline, inhibition vasculogenesis compound, hydralazine and L-BSO.
Spendable chemotherapeutics includes but not limited to alkylating agent, metabolic antagonist, hormone and antagonist thereof, radio isotope, antibody and natural product and combination thereof.For example, inhibitor compound of the present invention can use with antibiotics (as Dx and other anthracene nucleus analogue), nitrogen mustards (as endoxan), pyrimidine analogue (as 5 FU 5 fluorouracil), cis-platinum, hydroxyurea, taxol and natural and synthesis of derivatives or the like.As another example, for mixed tumor, for example wherein tumour comprises the mammary cancer that relies on gonad-stimulating hormone and do not rely on gonadotroph, described compound can with Leuprolide or goserelin (the synthetic peptide analogs of LH-RH) Combined Preparation.Other anti-knurl scheme comprises makes inhibitor compound use with another therapeutic modality, and this therapeutic modality for example is operation or radiation, and below it is also referred to as " auxiliary anti-knurl mode ".Can be used for other chemotherapeutics of the present invention and comprise hormone and antagonist, radio isotope, antibody, natural product and combination thereof.The example that can be used on the therapeutical agent in the method that adopts The compounds of this invention is listed in the following table.
| Alkylating agent | Mustargen | Chlormethine |
| Endoxan | Ifosfamide | Melphalan |
| Chlorambucil | Nitrosourea | Carmustine (BCNU) |
| Lomustine (CCNU) | Me-CCNU (methyl--CCNU) | Aziridine/methyl-trimeric cyanamide |
| Tretamine (TEM) | Triethylenethio-hosphopramide | (plug is for group) |
| Altretamine | (HMM, altretamine) | Alkylsulfonate |
| Busulfan | Triazine | Dacarbazine (DTIC) |
| Metabolic antagonist | Folacin | Rheumatrex |
| Trimetrexate | Pyrimidine analogue | 5 FU 5 fluorouracil |
| Fluorodeoxyuridine | Gemcitabine | Cytosine arabinoside |
| (AraC, cytosine arabinoside) | The 5-azacitidine | 2,2 '-difluoro deoxidation-cytidine |
| Purine analogue | Ismipur | The 6-Tioguanine |
| Azathioprine | 2 '-deoxycoformycin | (pentostatin) |
| Red hydroxyl nonyl-VITAMIN B4 (EHNA) | Fludarabine phosphate | 2-chlorodeoxyadenosine |
| (CldAdo, 2-CdA) | Many target spots antifol | I type topoisomerase enzyme inhibitor |
| Camptothecine | Hycamtin | Rinotecan |
| Natural product | Anti-mitosis medicine | Paclitaxel |
| Vincaleucoblastine | Vinealeucoblastine(VLB) (VLB) | Vincristine(VCR) |
| Vinorelbine | Taxotere _(Docetaxel) | Estramustine phosphate |
| Estracyte Epipodophylotoxins | Etoposide | Teniposide |
| Microbiotic | actimomycin D | Daunomycin (daunorubicin) |
| Dx (Zorubicin) | mitoxantroneidarubicin | Bleomycin splicamycin (Plicamycin) |
| Ametycin | Gengshengmeisu | Enzyme |
| L-asparagine acid amides enzyme | Biological response modifier | Interferon-' alpha ' |
| IL-2 | G-CSF | GM-CSF |
| Differentiation agent | The tretinoin derivative | Radiosensitizer |
| Metronidazole | Misonidazole | The demethyl misonidazole |
| Pimonidazole | Etanidazole | Naxogin |
| RSU1069 | EO9 | RB6145 |
| SR4233 | Nicotinamide | 5-bromouracil deoxyribose |
| 5-iododeoxyuridine | Bromodeoxyribouridine | All ingredients |
| Platinum coordination complex | Cis-platinum | Carbon platinum |
| Oxaliplatin | Amerantrone | Mitoxantrone |
| The urea that replaces | Hydroxyurea | The Procarbazine derivative |
| N-methyl hydrazine (MIH) | Procarbazine | The adrenal cortex inhibitor |
| Mitotane (o, p '-DDD) | ainoglutethimide | Cytokine |
| Interferon, rabbit-(α, beta, gamma) | Interleukin II hormone and antagonist | Adrenocorticosteroids/antagonist |
| Prednisone and equivalent | Dexamethasone | ainoglutethimide |
| Progestin | Hydroxyprogesterone caproate bp 98 | Medroxyprogesterone acetate |
| Magace | Oestrogenic hormon | Diethylstilbestrol |
| Lynoral/equivalent | Antiestrogen | Tamoxifen |
| Male sex hormone | Testosterone | Fluoxymesterone/equivalent |
| Antiandrogen | Drogenil | Gonadotropin releasing hormone analogues |
| Hormone analogs | Leuprolide | The non-steroid antiandrogen |
| Drogenil | Photosensitizers, | Hematoporphyrin derivative |
| Phytochrome | Benzoporphyrin derivative | Npe6 |
| Tin mesoetioporphyrin (SnET2) | pheoboride-a | Bacteriochlorophyll II-a |
| The phthalocyanine naphthalene | The phthalein cyanine compound | Phthalocyanine zinc |
| Growth factor receptor antagonist | The EGFR antagonist | The HER-2 antagonist |
Especially the example that can be used for uniting with radiosterilization use comprises for example camptothecine, carboplatin, cis-platinum, daunorubicin, Dx, Interferon, rabbit (α, β, γ), Rinotecan, hydroxyurea, Chlorambucil, 5 FU 5 fluorouracil (5-FU), methotrexate, 2-chloroadenine nucleosides, fludarabine, U-18496, gemcitabine, pemetrexed, interleukin-22, Rinotecan, Docetaxel, Paclitaxel, effective analogue and derivative thereof are gone up in Hycamtin and treatment.
According to the present invention, compound of the present invention can be only with gemcitabine or add that also Paclitaxel unites use.Compound of the present invention also can be only with pemetrexed or add that also cis-platinum, carboplatin or other platinum class unite use.Chk1 inhibitor of the present invention also can with gemcitabine and pemetrexed Combined Preparation.
Can be used for for example treatment of leiomyosarcoma, osteosarcoma, metastatic nonsmall-cell lung cancer, four limbs and soft tissues of trunk's sarcoma, renal cell carcinoma, gland cancer and the Hokdkin disease in carcinoma of the pancreas, uterus with the Chk1 inhibitor of the present invention of gemcitabine Combined Preparation.Can be used for the treatment of mesothelioma with the Chk1 inhibitor of the present invention of pemetrexed administration.
One skilled in the art can appreciate that treatment mentioned in this article prolongs and prevent and to the treatment of established disease or symptom.Mention that treatment also refers to reduce the recurrence of the indication that multiplication rate or minimizing treat.Will be understood that also The compounds of this invention required consumption in treatment becomes with the character of treatment situation and patient's age and situation, this is by monitoring doctor or animal doctor's final decision.
Yet, for adult treatment, usually dosage at 0.001mg/kg to about 100mg/kg scope of every day.Required dosage can be easily with the single dose administration, or in proper spacing with multiple dose administration, for example every day is with two, three, four or sub-doses (subdose) administration more frequently.In fact, the doctor determines the dosage of actual the most suitable individual patient, and this dosage becomes with age, body weight and concrete reaction.Above-mentioned dosage is exemplary generalized case, but Special Circumstances also can exist, and wherein with the higher or lower dosage of employing, and such situation all is within the scope of the invention.
Similarly, cell mass can occur in any dosage and time with contacting of Chk1 inhibitor of the present invention, as long as can be enough to realize removing basically the cell cycle check point.Usually, but not necessarily, such period comprises that this depended on multiple factor up to about 72 to about 96 hours.In some embodiments, wish or must several approximately weeks of as many as or above during in give Chk1 inhibitor, this is determined by attending doctor or technician.Thereby Chk1 inhibitor of the present invention usually can the administration as many as about 1 hour, about 2 hours of as many as, about 3 hours of as many as, about 4 hours of as many as, about 6 hours of as many as, as many as be up to about 12 hours, about 18 hours of as many as, about 24 hours of as many as, about 48 hours of as many as, or about 72 hours of as many as.The scope that it will be understood by those skilled in the art that herein statement is only for exemplary, and within the scope of being explained and outside scope and subrange also within the scope of the invention.
Chk1 inhibitor of the present invention can dosage the multiple administration.For example, the dosed administration that the Chk1 inhibitor can be such: four times dosage is sent a day (q4d * 4) as dose every four days; Every three days four times dosage is sent one day (q3d * 4) as dose; Every five days dosage is once sent one day (qd * 5); Weekly dosage continues three weeks (qwk3); Five per daily doses were had a rest two days, another five per daily doses (5/2/5); Or conform with situation and any dosage of determining.
Embodiment
Embodiment 1
Determine the IC of Chk1 inhibitor
50Value
The cDNA of people Chk1 is identified and is cloned, described in No. 99/11795 international application published of WO of submitting on September 4th, 1998 before.The FLAG_ label is inserted the N-terminal of total length Chk1 with frame.5 ' primer contains EcoRI site, Kozak sequence, and coding is used to utilize M2 antibody (Sigma, Saint Louis, IL) FLAG of protein affinity purification
_Label.3 ' primer contains the SalI site.Pcr amplified fragment as the EcoRI-SalI fragment cloned into pCI-Neo (Invitrogen, Carlsbad, CA), then as EcoRI-NotI fragment subclone advance pFastBacI (Gibco-BRL, Bethesda, MD).Described in Gibco-BRL Bac-to-Bac handbook, carry out the preparation of recombinant baculovirus, and use it for to infect and be grown in the CCM3 substratum (UT) the Sf-9 cell in is added with FLAG so that express for HyClone Laboratories, Logan
_The Chk1 albumen of label.
Be added with FLAG from frozen block purifying through the SF9 of baculovirus infection cell
_The Chk1 of label.The refrigerated cell lump is mixed with the molten born of the same parents' damping fluid of isopyknic 2X, and this damping fluid contains Tris-HCl pH7.5,200mM NaCl, 50mM B-glycerophosphate, 25mM NaF, the 4mM MgCl of 100mM
2, 0.5mM EGTA, 0.2% tween (TWEEN)
_-20,2mM vanadic acid sodium, 2mM DTT and protease inhibitor cocktail (Complete mini, Boehringer Mannheim 2000 catalog#1836170).Then, cell is with the free pestle homogenate of Dounce homogenizer 20 times, and with 48, centrifugal 1 hour of 400xg.With the solution pre-wash of M2 affinity post with 10 column volumes, 50mM glycine (pH3.5) was 20mMTris (pH7.5) then before this, and 150mM NaCl three times in turn, finishes with Tris NaCl washing.Subsequently, with 20mM Tris pH7.5,150mM NaCl, 0.1% tween of 25 column volumes
_-20, the complete small-sized proteolytic enzyme sheet washing pillar of 1mMEGTA, 1mM EDTA and 1X.In 4 hours, clarifying lysate is attached to the M2 affinity resin in batches at 4 ℃ then.Then pour the mixture of resin and lysate into pillar, and collect effluent.With 20mM Tris (pH7.5), 150mM NaCl and the 3mM N-octyl group glucosides washing of resin with 10 column volumes.Then contain 0.5mg/mL FLAG with 6 column volumes
_Cold 20mM Tris (pH7.5), 150mM NaCl, 3mM N-octyl group glucosides wash-out from pillar of peptide (Sigma, 2000 Catalog#_F3290) are added with FLAG
_The Chk1 of label.Collect three components and analyzed and whether had the Chk1 that is added with the FLAG label.
Protein kinase is used in the mensuration of Chk1 kinase activity, this mensuration comprises the FLAG of 100ng purifying
_The Cdc25C peptide (H-leu-tyr-arg-ser-pro-ser-met-pro-glu-asn-leu-asn-arg-ar g-arg-arg-OH) (SEQID NO:1) of-Chk1 (150pmol ATP/min), 20 μ m, 4 μ m ATP, 2 μ Ci[
32P] γ-ATP, 20mM Hepes pH7.2,5mMMgCl
2, 0.1%NP40 and 1mM DTT.This mensuration is used for determining the IC of The compounds of this invention
50Reaction is initial by the reaction mixture that adding contains ATP, at room temperature carries out 10min.By adding phosphoric acid (final concentration of 150mM) termination reaction, and with reactant transfer to the phosphorylated cotton disk.With the phosphorylated cotton disk with 150mM phosphoric acid washing five times and air-dry.The interpolation scintillation solution is also counted disk in the Wallac scintillometer.The IC of determined Chk1 inhibitor of the present invention
50Value is for about 8 to about 500nM.
Embodiment 2
Selectivity
With respect to one or more other protein kinases, promptly DNA-PK, Cdc2, casein kinase i (CKI), Chk2, p38 map kinase, ERK kinases, protein kinase A (PKA) and/or calcium-calmodulin protein kinase ii (CaM KII) are tested Chk1 selection of inhibitors of the present invention.Except Chk2, all these kinase whose mensuration programs were existing in the literature in the past to be described, and comprises the U.S. Patent application 08/184,605 that U.S. Patent Publication 2002-016521 A1 and on January 21st, 1994 submit to, incorporates them into this paper by reference.
Compound is as follows to the determination of activity of Chk2: at room temperature, and at 4mM ATP, 1mCi[
32P] γ-ATP, 20mM Hepes pH7.5,5mM MgCl
2Existence is hatched the Chk2 that is added with the His label of 128ng purifying 20 minutes with the Chk1 inhibitor of as many as 100mM down with 0.25%NP40.With the phosphoric acid termination reaction of final concentration 150mM, 5/8 reaction mixture is transferred to the phosphorylated cotton disk.With disk with 150mM phosphoric acid washing five times and air-dry.The interpolation scintillator also utilizes in the Wallac beta-particle counter tube radiocounting.
P38 MAP kinase, ERK kinases, PKA, CaM KII and Cdc2 be available from NewEngland Biolabs, and utilize 4-50 μ M ATP and measure up to the Chk1 inhibitor concentration of 100 μ M according to shop instruction.The test of all inhibitor all shows with respect to other enzyme at least 100 times selectivity to Chk1.
Embodiment 3
Chk1 of the present invention suppresses Chk1 function in the agent inhibit cell
In order to determine that Chk1 inhibitor of the present invention suppresses the Chk1 function in cell, can in molecular assay, test inhibitor based on cell.Because Mammals Chk1 has been proved at external phosphorylation Cdc25C, this points out it to bear in the response dna damage and regulates cyclinB/cdc2, strengthens the active ability of CyclinB/cdc2 so can analyze the Chk1 inhibitor.This experiment can design as follows: hatched 7 hours with 800rads irradiation BeLa cell and at 37 ℃.Because these cells are p53 function feminine gender, they only are stuck in G2.Then, the nocodazole is added to the concentration of 0.5 μ g/mL, and cell was hatched 15 hours at 37 ℃.The purpose that adds the nocodazole is to capture any G2 of passing to stagnate the cell that enters M.At last,, added the Chk1 inhibitor 8 hours, results, dissolved cell and with albumen at the antibody mediated immunity precipitation equivalent of CyclinB1 (NewEngland Biolabs) by producer suggestion.Then determine the cdc2 kinase activity relevant (Yu et al., JBiol Chem.1998 November 11 with CyclinB by measuring histone h1 kinase activity assay IPs; 273 (50): 33455-64).
In addition, can determine the ability of Chk1 inhibitor elimination IR inductive G2 dna damage of the present invention check point by using the mitotic index determination experiment.With HeLa cell (about 1 * 10
6) handle in a manner described.Cell with the PBS washing once, then is resuspended among the 75mM KCl of 2.5mL by centrifugal collection, and recentrifuge.Then with the cold acetic acid of cell: fix in the methyl alcohol (1: 3), and hatched 20 minutes on ice in the 3mL prepared fresh.Cell is precipitated, and the sucking-off stationary liquid is resuspended among the PBS of 0.5mL.By with transfer pipet with 100 μ L fixed cell transfer to microscope with preparing karyomit(e) mitosis metaphase on the glass slide and with the submergence of 1mL stationary liquid.Then that slide glass is air-dry, with Wright's stain (Sigma) dyeing 1 minute, then once and once with 50% methanol wash with water washing.Determine mitotic cell to have condensation and to lack nuclear membrane.
Embodiment 4
Chk1 inhibitor of the present invention strengthens killing of cancer therapy pair cell
In order to prove that The compounds of this invention makes the killing effect sensitivity of target cell to dna damage reagent to the restraining effect of Chk1, cell can be hatched and is exposed to radiation or chemical dna damage reagent in the presence of Chk1 inhibitor of the present invention.Under 37 ℃, containing 5%CO
2The humidification insulation can in, will be seeded in cell in the 96 hole microwell plates with the density in the every hole of 1000-2000 and be grown among the RMPI1640 that contains 10%FBS, 100U/mL penicillin and 100 μ g/mL Streptomycin sulphates 18 hours.The cell of test can comprise interested any cell or clone, for example 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, PC3, HL-60, K562 and MOLT4.All specified cells mean following human cell line:
| HeLa | Adenocarcinoma of the uterine cervix |
| ACHN | Renal adenocarcinoma |
| 786-0 | Renal adenocarcinoma |
| HCT116 | Colorectal carcinoma |
| SW620 | Colorectal carcinoma, nodus lymphoideus transferring rate |
| HT-29 | Colorectum gland cancer |
| Colo205 | Adenocarcinoma of colon |
| SK-MEL-5 | Melanoma |
| SK-MEL-28 | Malignant melanoma |
| A549 | Lung cancer |
| H322 | Bronchovesicular cancer (broncholoalveolar carcinoma) |
| OVCAR-3 | Adenocarcinoma ovaries |
| SK-OV-3 | Adenocarcinoma ovaries |
| MDA-MB-231 | Mammary cancer |
| MCF-7 | Mammary cancer |
| PC-3 | Prostate cancer, from metastatic tumor to bone |
| HL-60 | Acute promyelocytic leukemia |
| K562 | Chronic granulocytic leukemia |
| MOLT4 | Acute lymphoblastic leukemia; The T lymphoblast |
Cell is handled with the substratum that only contains chemotherapeutics or with the substratum that contains chemotherapeutics and Chk1 inhibitor.Before growing with measurement, cell was hatched about 5 days by the picked-up of measuring the 3H-thymidine.Chemotherapeutics comprises Etoposide, Dx, cis-platinum, Chlorambucil, 5 FU 5 fluorouracil (5-FU).The growth-inhibiting of cell to 90% required drug level of untreated control cells is defined as GI
90
Compound of the present invention can be tested other the metabolic antagonist that comprises methotrexate, hydroxyurea, 2-chloroadenine nucleosides, fludarabine, U-18496 and gemcitabine, to assess the ability that it strengthens these reagent killing effect.Unite enhancing by assessment and gemcitabine and kill the HT29 colorectal carcinoma, compound of the present invention can be compared mutually.
In addition, can test the ability of Chk1 inhibitor enhancing of the present invention by radiation destroys.
Embodiment 5
Animal tumor model
In mouse, strengthen the ability of passing through dna damage reagent kill tumor in order to test Chk1 inhibitor of the present invention, set up the xenotransplantation tumor model that adopts colon tumor cell system.5 FU 5 fluorouracil (5-FU) or gemcitabine can be used as dna damage reagent.HT29 and Colo205 (human colon carcinoma) and H460 and Calu-6 (non-small cell carcinoma) cell are used in 6-8 week female thymus gland Balb/c in age (nu/nu) mouse and cultivate the xenotransplantation tumour.Mouse is raised in bioclean Laminar Flow Room, freely absorbs sterile food and water.At 5%CO
2Add in the wet environment, clone grows to subconfluence in RPMI 1640 substratum that are supplemented with 10%FBS, 100U/mL penicillin, 100 μ g/mL Streptomycin sulphates and 1.5mM L-glutaminate.The unicellular suspension of preparation in CMF-PBS, and cell concn is adjusted to 1 * 10
8Cell/mL.Amount to inoculation 1 * 10 in right side of mice or right leg subcutaneous (s.c.)
7Cell (100 μ L).
When tumour reaches 75-100cm
3When (usually inoculation back 7-11 days), with mouse at random (5-15 mouse/group) be divided into four treatment group uses.Tumour is with vernier caliper measurement, and gross tumor volume uses the following formula estimation that draws by rule of thumb: gross tumor volume (cm
3)=length of tumor (cm) * tumour width (cm) * tumor thickness (cm)/3.3.Treatment comprises i) with 160mg/kg intraperitoneal (i.p) injection gemcitabine.In mouse, observe delaying of tumor growth with the gemcitabine treatment.Expectation can reduce gross tumor volume and prolongs life to mouse with the treatment of 160mg/kg gemcitabine Combined with Oral Chk1 inhibitor.Every other day monitor the tumour size at experimental session.
Obviously, under the condition that does not break away from spirit and scope of the invention, can make many modifications and variations to the present invention described above, therefore, the present invention is only limited by appending claims.
Sequence table
<110>Icos Corporation
＜120〉be used to suppress the compound of CHK1
<130>27866/39987A
＜140〉wait to transfer the possession of
<141>2005-06-24
<150>US 60/583,080
<151>2004-06-24
<160>1
<170>PatentIn version 3.3
<210>1
<211>16
<212>PRT
＜213〉artificial
<220>
＜223〉synthetic peptide
<400>1
Leu Tyr Arg Ser Pro Ser Met Pro Glu Asn Leu Asn Arg Arg Arg Arg
1 5 10 15
Claims (32)
1. the compound that has following structural formula
X wherein
1For do not exist ,-O-,-S-, CH
2-or-N (R
1)-;
X
2For-O-,-S-or-N (R
1)-;
Y is O or S; Or=Y represents to be connected two hydrogen atoms on the common carbon atom;
The C that W is selected from heteroaryl, aryl, Heterocyclylalkyl, cycloalkyl and is replaced by heteroaryl or aryl
1-6Alkyl, wherein said aryl W are randomly individual with R by 1-4
2The substituting group of expression replaces, and described heteroaryl W is randomly individual with R by 1-4
5The substituting group of expression replaces, and described Heterocyclylalkyl and cycloalkyl W are randomly by one or two C
1-6Alkyl substituent replaces;
R
1Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, C
2-6Alkynyl and aryl;
R
2Be selected from heteroaryl, halogen, the optional C that replaces
1-6Alkyl, C
2-6Thiazolinyl, OCF
3, NO
2, CN, NC, N (R
3)
2, OR
3, CO
2R
3, C (O) N (R
3)
2, C (O) R
3, N (R
1) COR
3, N (R
1) C (O) OR
3, N (R
1) C (O) C
1-6Alkylidene group C (O) R
3, N (R
1) C (O) C
1-6Alkylidene group C (O) OR
3, N (R
1) C (O) C
1-6Alkylidene group OR
3, N (R
1) C (O) C
1-6Alkylidene group NHC (O) OR
3, N (R
1) C (O) C
1-6Alkylidene group SO
2NR
3, C
1-6Alkylidene group OR
3And SR
3
R
3Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, cycloalkyl, aryl, heteroaryl, SO
2R
4, halogen is by one or more halogens, hydroxyl, aryl, heteroaryl, Heterocyclylalkyl, N (R
4)
2And SO
2R
4The C that replaces
1-6Alkyl, C
1-6Alkylidene aryl, C
1-6Alkylidene group heteroaryl, C
1-6Alkylidene group C
3-8Heterocyclylalkyl, C
1-6Alkylidene group SO
2Aryl, the optional C that replaces
1-6Alkylidene group N (R
4)
2, OCF
3, C
1-6Alkylidene group N (R
4)
3 +, C
3-8Heterocyclylalkyl and CH (C
1-6Alkylidene group N (R
4)
2)
2, or two R
3Base lumps together and forms optional 3 to 8 yuan of aliphatic series rings that replace;
R
4Be selected from do not exist, hydrogen, C
1-6Alkyl, cycloalkyl, aryl, heteroaryl, C
1-6Alkylidene aryl and SO
2C
1-6Alkyl, or two R
4Group lumps together and forms optional 3 to 8 yuan of rings that replace;
R
5Be selected from C
1-6Alkyl, C
2-6Alkynyl, aryl, heteroaryl, Heterocyclylalkyl, N (R
3)
2, N (R
1) C (O) R
3, N (R
1) CO
2R
3, OR
3, halogen, N
3, CN, C
1-6Alkylidene aryl, C
1-6Alkylidene group N (R
3)
2, C (O) R
3, C (O) OR
3, C (O) N (R
3)
2, CF
3, and
R
6Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, cycloalkyl, Heterocyclylalkyl, aryl, heteroaryl, SO
2R
4, by one or more halogens, hydroxyl, aryl, heteroaryl, Heterocyclylalkyl, N (R
4)
2And SO
2R
4The C that replaces
1-6Alkyl, C
1-6Alkylidene aryl, C
1-6The assorted virtue of alkylidene group, C
1-6Alkylidene group C
3-8Heterocyclylalkyl, C
1-6Alkylidene group SO
2Aryl, the optional C that replaces
1-6Alkylidene group N (R
4)
2, OCF
3, C
1-6Alkylidene group N (R
4)
3 +, C
3-8Heterocyclylalkyl and CH (C
1-6Alkylidene group N (R
4)
2)
2
R
7And R
8Be selected from hydrogen, OR independently of one another
3, C
1-6Alkyl, halogen, N (R
3)
2, C (O) N (R
3)
2, C
1-3Alkylidene aryl, CN, NO
2, C (O) OR
11, C (O) R
11And SR
11
R
9For-C ≡ C-R
10Or-CF
3, perhaps R
8And R
9The carbon atom that group is connected with them lumps together and forms 5 or 6 yuan of isocyclic aliphatic series or aromatic ring system, and this system option ground contains 1-3 and is selected from O, NR
4Heteroatoms with S;
R
10Be selected from hydrogen, C
1-6Alkyl, aryl, C
1-6Alkylidene aryl, heteroaryl and C
1-6The alkylidene group heteroaryl;
R
11Be selected from hydrogen, C
1-6Alkyl, C
2-6Thiazolinyl, aryl, C
1-3Alkylidene aryl, C
3-8Cycloalkyl and C
1-3Alkylidene group C
3-8Cycloalkyl;
N is 1 or 2;
The perhaps acceptable salt of the medicine of described compound, or prodrug, or solvate.
2. according to the compound of claim 1, wherein
X
1And X
2For-N (H)-;
Y is O or S;
W is at least two the heteroatomic heteroaryls that are selected from N, O and S that contain of optional replacement.
3. according to the compound of claim 2, R wherein
6Be selected from the optional C that replaces
1-6Alkylidene group N (R
4)
2, C
1-6Alkylidene group heteroaryl, C
1-6Alkylene heterocyclic alkyl and C
3-8Heterocyclylalkyl.
4. according to the compound of claim 3, R wherein
6Be selected from-CH
2CH
3,-(CH
2)
1-6N (CH
3)
2,-(CH
2)
1-6NH (CH
3),
With
5. according to the compound of claim 2, wherein W is selected from pyridazinyl, pyrimidyl, pyrazinyl and triazinyl, randomly is selected from the optional C that replaces by 1-4
1-6Alkyl, C
2-6Alkynyl, aryl, heteroaryl, N (R
3)
2, C (O) N (R
3)
2, CO (O) R
3, OR
3, CF
3, CN and halogen substituting group replace.
6. according to the compound of claim 2, wherein W is selected from
With
7. according to the compound of claim 2, wherein W is selected from
With
8. according to the compound of claim 2, wherein W is a pyrazinyl.
9. compound according to Claim 8, wherein W is by R at 5
5Pyrazine-2-base that substituting group replaces.
10. according to the compound of claim 9, R wherein
5Be selected from CF
3, CH
3Do not exist.
11. according to the compound of claim 1, wherein R
7And R
8Be hydrogen.
12. according to the compound of claim 1, wherein R
9For-C ≡ CH or-CF
3
13. according to the compound of claim 1, wherein R
8And R
9The carbon atom that is connected with them lumps together formation
Or
14. according to the compound of claim 3, wherein R
6Be selected from-(CH
2)
2N (CH
3)
2,
With
15. one kind comprises the compound of claim 1 and the composition of medicine acceptable carrier.
16. be selected from the compound of following group:
1-[5-ethynyl-2-(1-methyl-piperidines-3-ylmethoxy)-phenyl]-3-(5-methyl-pyrazine-2-yl)-urea;
1-[2-(2-dimethylamino-oxyethyl group)-5-ethynyl-phenyl]-3-(5-methyl-pyrazine-2-yl)-urea;
1-[5-ethynyl-2-(pyridin-3-yl methoxyl group)-phenyl]-3-(5-methyl-pyrazine-2-yl)-urea;
1-[3-(1-methyl-piperidines-3-ylmethoxy)-5,6,7,8-tetrahydrochysene-naphthalene-2-yl]-3-(5-methyl-pyrazine-2-yl)-urea;
1-[3-(1-methyl-piperidines-2-ylmethoxy)-5,6,7,8-tetrahydrochysene-naphthalene-2-yl]-3-(5-methyl-pyrazine-2-yl)-urea;
(S)-1-(5-methyl-pyrazine-2-yl)-3-[2-(piperidines-3-ylmethoxy)-5-trifluoromethyl-phenyl]-urea;
(R)-1-(5-methyl-pyrazine-2-yl)-3-[2-(piperidines-3-ylmethoxy)-5-trifluoromethyl-phenyl]-urea;
1-[2-(1-methyl-piperidin-4-yl oxygen base)-5-trifluoromethyl-phenyl]-3-(5-methyl-pyrazine-2-yl) urea;
1-(5-methyl-pyrazine-2-yl)-3-[2-(piperidines-3-ylmethoxy)-5-trifluoromethyl-phenyl]-urea;
1-[2-(1-methyl-piperidines-3-ylmethoxy)-5-trifluoromethyl-phenyl]-3-(5-methyl-pyrazine-2-yl)-urea;
1-(5-methyl-pyrazine-2-yl)-3-[7-(pyridin-3-yl methoxyl group)-2,3-dihydro-benzo [1,4] two _ English-6-yl]-urea;
1-[7-(2-dimethylamino-oxyethyl group)-2,3-dihydro-benzo [1,4] two _ English-6-yl]-3-(5-methyl-pyrazine-2-yl)-urea; And
1-[3-(2-dimethylamino-oxyethyl group)-5,6,7,8-tetrahydrochysene-naphthalene-2-yl]-3-(5-methyl-pyrazine-2-yl)-urea.
17. a method that suppresses check point kinases 1 in the cell comprises the step that described cell is contacted with the compound of the claim 1 of significant quantity.
18. method that makes the cell sensitization in the individuality, described individuality is accepted chemotherapy or radiotherapy in the treatment because of medical indications, and described method comprises with the compound of claim 1 of treatment significant quantity and chemotherapeutics, radiotherapeutic agents or its mixture to described individual Combined Preparation.
19., also comprise with cytokine, lymphokine, somatomedin, other Hemopoietic factor or its mixture to described individual administration according to the method for claim 18.
20. according to the method for claim 18, wherein said chemotherapeutics is selected from alkylating agent, metabolic antagonist, hormone or its antagonist, radio isotope, antibody and composition thereof.
21. according to the method for claim 18, wherein said radiotherapeutic agents is selected from gamma-radiation, X-optical radiation, UV-light, visible light, ir radiation and microwave radiation.
22. according to the method for claim 18, wherein said situation is the cancer that is selected from colorectal carcinoma, incidence cancer, carcinoma of the pancreas, mammary cancer, cancer of the stomach, bladder cancer, carcinoma vulvae, leukemia, lymphoma, melanoma, renal cell carcinoma, ovarian cancer, cerebral tumor, osteosarcoma and lung cancer.
23. according to the method for claim 18, wherein said situation is for being selected from myxoma and round cell carcinoma, local late tumor, metastatic carcinoma, ewing's sarcoma, metastatic carcinoma, lymphatic metastasis, squamous cell carcinoma, the esophagus squamous cell carcinoma, oral carcinoma, multiple myeloma, acute lymphoblastic leukemia, acute nonlymphocytic leukemia, lymphocytic leukemia, chronic granulocytic leukemia, hairy cell leukemia, exudative lymphoma (based on the lymphoma of body cavity), thymic lymphoma knurl lung cancer, small cell carcinoma, cutaneous T cell lymphoma, hodgkin's lymphomas, the non-Hodgkin lymphomas, adrenocortical carcinoma, produce the tumour of ACTH, non-small cell carcinoma, mammary cancer, small cell carcinoma, duct carcinoma, cancer of the stomach, colorectal carcinoma, colorectal carcinoma, the polyp that the colorectum tumorigenesis is relevant, the pancreas cancer, liver cancer, bladder cancer, primary bladder surface tumour, the intrusion transsitional cell carcinoma of bladder, muscle invasive bladder cancer, prostate cancer, ovarian cancer, primary peritonaeum epithelium vegetation, cervical cancer, carcinoma of endometrium, carcinoma of vagina, carcinoma vulvae, uterus carcinoma and the solid tumor in ovarian follicle, carcinoma of testis, penile cancer, renal cell carcinoma, the endogenous character cerebral tumor, neuroblastoma, the stellate cell cerebral tumor, neurospongioma, invade the metastatic tumor cell of central nervous system, osteoma and osteosarcoma, malignant melanoma, human skin keratinocyte tumor progression, squamous cell carcinoma, thyroid carcinoma, retinoblastoma, neuroblastoma, peritoneal effusion, malignant pleural effusion, mesothelioma, the nephroblastoma, carcinoma of gallbladder, trophoderm vegetation, the cancer of Hemangiopericytoma and Kaposi sarcoma.
24. according to the method for claim 18, wherein said treatment is to being selected from the inflammation administration of rheumatoid arthritis, psoriasis, vitiligo, Wei Geneishi granuloma and systemic lupus erythematous.
25. according to the method for claim 18, wherein the described compound of claim 1 has at least 20 times selectivity with respect to protein kinase A, protein kinase C, cdc2 and pp60v-src inhibition Chk1.
26. according to the method for claim 18, the compound of wherein said claim 1 has at least 75 times selectivity for protein kinase A, protein kinase C, cdc2 and pp60v-src to the inhibitory phase of Chk1.
27. according to the method for claim 18, the compound of wherein said claim 1 has at least 100 times selectivity for protein kinase A, protein kinase C, cdc2 and pp60v-src to the inhibitory phase of Chk1.
28. method that suppresses abnormal cell proliferation, comprise allowing the cell mass that contains the abnormality proliferation cell contact so that the cell cycle arrest basic synchronization between the described abnormality proliferation cell, and contact with the compound of claim 1 so that described cell cycle arrest fundamental solution removes with the described cell mass of relief with the Chk1 activator.
29. according to the method for claim 28, wherein said Chk1 activator comprises at least a chemotherapeutics.
30. according to the method for claim 28, wherein said Chk1 activator comprises ionization or uv-radiation.
31. according to the method for claim 28, wherein said ionization radiation and radiation-sensitizing agents, photosensitizers or its mixture are united and are used.
32. according to the method for claim 28, the cell of wherein said abnormality proliferation is non-cancer sexual cell.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US58308004P | 2004-06-25 | 2004-06-25 | |
| US60/583,080 | 2004-06-25 |
Publications (1)
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|---|---|
| CN101006075A true CN101006075A (en) | 2007-07-25 |
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| CNA2005800277583A Pending CN101006075A (en) | 2004-06-25 | 2005-06-24 | Bisarylurea derivatives useful for inhibiting CHK1 |
Country Status (9)
| Country | Link |
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| EP (1) | EP1765808A1 (en) |
| JP (1) | JP2008504283A (en) |
| KR (1) | KR20070028591A (en) |
| CN (1) | CN101006075A (en) |
| AU (1) | AU2005267185A1 (en) |
| BR (1) | BRPI0512593A (en) |
| CA (1) | CA2571424A1 (en) |
| MX (1) | MX2007000259A (en) |
| WO (1) | WO2006012308A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107849025A (en) * | 2015-07-23 | 2018-03-27 | 伊莱利利公司 | For treating the CHK1/2 inhibitor of neuroblastoma and/or soft tissue sarcoma |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1778648A2 (en) * | 2004-08-19 | 2007-05-02 | ICOS Corporation | Compounds useful for inhibiting chk1 |
| ES2356068T3 (en) | 2005-03-29 | 2011-04-04 | Icos Corporation | DERIVATIVES OF HETEROARIL UREA USEFUL TO INHIBIT CHK1. |
| GB201008005D0 (en) | 2010-05-13 | 2010-06-30 | Sentinel Oncology Ltd | Pharmaceutical compounds |
| WO2023287839A2 (en) * | 2021-07-13 | 2023-01-19 | Brown University | Urinary tract cancer treatment guided by mutational landscape |
| CA3233080A1 (en) * | 2021-09-28 | 2023-04-06 | Pioneer Hi-Bred International, Inc. | Seedling germination and growth conditions |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01163723A (en) * | 1987-12-21 | 1989-06-28 | Nippon Telegr & Teleph Corp <Ntt> | Organic nonlinear optical material |
| JPH09249628A (en) * | 1996-03-12 | 1997-09-22 | Tanabe Seiyaku Co Ltd | Derivatives of phenol |
| ATE297383T1 (en) * | 1997-12-22 | 2005-06-15 | Bayer Pharmaceuticals Corp | INHIBITION OF P38 KINASE USING SYMMETRIC AND ASYMMETRIC DIPHENYL UREASES |
| UA76977C2 (en) * | 2001-03-02 | 2006-10-16 | Icos Corp | Aryl- and heteroaryl substituted chk1 inhibitors and their use as radiosensitizers and chemosensitizers |
| JP2005503400A (en) * | 2001-09-13 | 2005-02-03 | ベーリンガー インゲルハイム ファーマシューティカルズ インコーポレイテッド | Method for treating cytokine-mediated diseases |
| US20030236287A1 (en) * | 2002-05-03 | 2003-12-25 | Piotrowski David W. | Positive allosteric modulators of the nicotinic acetylcholine receptor |
| US7202244B2 (en) * | 2002-05-29 | 2007-04-10 | Millennium Pharmaceuticals, Inc. | Chk-1 inhibitors |
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2005
- 2005-06-24 KR KR1020077001927A patent/KR20070028591A/en not_active Application Discontinuation
- 2005-06-24 MX MX2007000259A patent/MX2007000259A/en not_active Application Discontinuation
- 2005-06-24 CA CA002571424A patent/CA2571424A1/en not_active Abandoned
- 2005-06-24 EP EP05788966A patent/EP1765808A1/en not_active Withdrawn
- 2005-06-24 WO PCT/US2005/022515 patent/WO2006012308A1/en active Application Filing
- 2005-06-24 AU AU2005267185A patent/AU2005267185A1/en not_active Abandoned
- 2005-06-24 CN CNA2005800277583A patent/CN101006075A/en active Pending
- 2005-06-24 BR BRPI0512593-6A patent/BRPI0512593A/en not_active IP Right Cessation
- 2005-06-24 JP JP2007518316A patent/JP2008504283A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107849025A (en) * | 2015-07-23 | 2018-03-27 | 伊莱利利公司 | For treating the CHK1/2 inhibitor of neuroblastoma and/or soft tissue sarcoma |
Also Published As
| Publication number | Publication date |
|---|---|
| CA2571424A1 (en) | 2006-02-02 |
| MX2007000259A (en) | 2007-07-20 |
| AU2005267185A1 (en) | 2006-02-02 |
| KR20070028591A (en) | 2007-03-12 |
| EP1765808A1 (en) | 2007-03-28 |
| WO2006012308A1 (en) | 2006-02-02 |
| BRPI0512593A (en) | 2008-03-25 |
| JP2008504283A (en) | 2008-02-14 |
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