CN101379058B - Pyrazole compounds that modulate the activity of CDK, GSK and aurora kinases - Google Patents

Abstract

The invention provides a compound of the formula (I): or a salt, solvate, tautomer or N-oxide thereof, wherein M is selected from a group D1 and a group D2: and R', E, A and X are as defined in the claims. Also provided are pharmaceutical compositions containing the compounds, processes for making the compounds and the use of the compounds in the prophylaxis or treatment of a disease state mediated by a CDK kinase, GSK-3 kinase or Aurora kinase.

Description

Regulate the pyrazole compound of CDK, GSK aurora kinase activity

Technical field

The present invention relates to suppress or the pyrazole compound of cell cycle regulation albumen (Cyclin) dependant kinase (CDK), glycogen synthase kinase (GSK) and aurora (Aurora) kinase activity, this compound is in treatment or prevent by the application in described kinase mediated morbid state or illness and have kinase inhibition or the new compound of regulation activity.The pharmaceutical composition and the new chemical intermediate that comprise described compound are also provided.

Background technology

Protein kinase has formed (a Hardie of big nation of the structure involved enzyme of the various signal transduction processes of wide region in responsible control cell, and Hanks G., S. (1995) The Protein KinaseFacts Book.I and II, Academic Press, San Diego, CA).Can for example, by the substrate (, albumen-tyrosine, albumen-serine/threonine, lipid etc.) of its phosphorylation these kinases be divided into some families.Identify suitable sequence motifs (for example, Hanks, S.K., Hunter, T., FASEB J., the 9:576-596 (1995) of each family conventionally and in these kinases families; The people such as Knighton, Science, 253:407-414 (1991); The people such as Hiles, Cell, 70:419-429 (1992); The people such as Kunz, Cell, 73:585-596 (1993); The people such as Garcia-Bustos, EMBO J., 13:2352-2361 (1994)).

Can carry out characterization to protein kinase with its regulation mechanism.These mechanism comprise, for example, the transphosphorylation, the protein-protein interaction that carry out from autophosphorylation effect, by other kinases, albumen-lipid interacts and albumen-polynucleotide interact.Each protein kinase may be regulated by more than one mechanism.

Kinases by adding phosphate-based many different cell processes that regulating in target protein, and said process comprises without limitation hyperplasia, differentiation, apoptosis, vigor, transcribes, translates and other signal transduction process.These phosphorylation events work with the form that can regulate and control or regulate the molecule ON/OFF of described target protein biological function to change.There is the phosphorylation of target protein as the response that stress wait various extracellular signals (hormone, neurotransmitter, GDF etc.), cell cycle events, environment or nutrition.The function of adequate proteins kinases in signal transduction pathway is can activate or deactivation (directly or indirectly), for example metabolic enzyme, adjusting albumen, acceptor, cytoskeletal protein, ionic channel or pump or transcription factor.In numerous disease, relate to the signal producing due to the controlled defectiveness of protein phosphorylation out of control, said disease comprises disease and illness and the vasculogenesis of for example inflammation, cancer, transformation reactions/asthma, immune disease and illness, central nervous system.

cell cycle protein dependent kinase

Eukaryotic cell fission process can be broadly divided into a series of external phases that are called as G1, S, G2 and M.Not gap and the sequential on the same group that the correct process that has shown each phase of cell cycle depends on its homologous protein companion of being called as the protein family of cell cycle protein dependent kinase (cdks) and being called as cyclin closely regulates.Cdks be can using sequence dependent background in the phosphorylation of homopolypeptide not with ATP the serine-threonine kinase albumen as cdc2 (the being also referred to as cdk1) homology of substrate.Cyclin is to be characterized as to comprise about 100 amino acid whose homologous regions albumen family of (be called as " cyclin white edge ", it can be used for the selectivity to specific cdk chaperone in conjunction with specific cdk chaperone and definition).

In the cell cycle, the regulation and control of various cdks and cyclin expression level, degradation rate and activation levels make circulation form a series of cdk/ cyclin complex bodys, and wherein said cdks has the enzyme activity.The formation control of these complex bodys going down to posterity of being undertaken by discrete cell cycle chechpoint (checkpoints), thereby fission process can be continued.Can not meet the pre-essential biological chemistry condition in given cell cycle chechpoint place, can not form required cdk/ cyclin complex body, may cause the apoptosis of cell cycle arrest and/or cell.As occurring in cancer, abnormal hyperplasia is usually to cause because lost correct cell cycle control.Therefore, the enzymic activity of inhibition cdk provides a kind of method that can stop and/or destroy the division of abnormal division cell.The diversity of cdks and cdk complex body, with and keying action in the mediated cell cycle very wide scope is provided can select possible therapeutic goal to it according to determined biochemical theory.

From the G1 of cell cycle in opposite directions advancing of S phase be mainly by associating and regulated by cdk2, cdk3, cdk4 and cdk6 with the member of D and E type cyclin.Described D-type cyclin seems to show and contributes to make to surmount G1 restriction point and go down to posterity, and wherein cdk2/ cyclin E complex body form is for being crucial from G1 to the transformation of S phase.Think and need cdk2/ cyclin A complex body by S phase and the process that enters into G2 subsequently.Mitotic division and to have triggered mitotic be to be regulated by the complex body of cdk1 and A and type B cell cyclin from G2 to the transformation of M phase.

G1 mutually in, retinoblastoma protein (Rb) and associated pocket albumen as p130 be the substrate of cdk (2,4, & 6)/cyclin complex body.Process by G1 is partly advanced by hyperphosphorylation, and therefore, Rb and p130 are by the deactivation of cdk (4/6)/cyclin-D complex body.The high phosphorylation of Rb and p130 caused transcription factor as the release of E2F and therefore produce pass through G1 and enter the mutually required gene of S-as the expression of the gene of cyclin E.The expression of cyclin E has promoted the formation of cdk2/ cyclin E complex body, and its further phosphorylation by Rb is increased or maintained E2F level.Described cdk2/ cyclin E complex body also phosphorylated cdna copies other required albumen, as NPAT, has shown to relate to this process in histone biosynthesizing.G1 advances and G1/S conversion is also that the Myc approach stimulating by mitogen regulates, and it is fed in cdk2/ cyclin E approach.Cdk2 is also relevant with the DNA infringement response approach of p53 mediation by the p53 adjusting of p21 level.P21 is the protein inhibitor of cdk2/ cyclin E, and therefore, it can block or postpone G1/S conversion.Therefore, cdk2/ cyclin E complex body may represent a kind of this time derive from Rb, Myc and p53 approach biological chemistry stimulate integrated point to a certain extent.Therefore,, for being intended to stop or recovering the treatment that cell cycle of abnormal division cell controls, Cdk2 and/or cdk2/ cyclin E complex body have represented good target.

The definite effect of not clear cdk3 in the cell cycle.Still also do not identify the cyclin companion of its homology, still, determined that the negative dominant form of cdk3 has postponed the cell in G1, thereby show that cdk3 plays a role in regulation and control G1/S conversion.

Although most cdks participate in the adjusting of cell cycle, there are indications that some member of cdk family also participates in other Biochemical processes.For example cdk5, it is that correct neuronal development is necessary and shown that it participates in some neuronal proteins as the phosphorylation of Tau, NUDE-1, synapsin 1, DARPP32 and Munc18/Syntaxin1A complex body.Neurone cdk5 is conventionally by being activated with p35/p39 protein binding.But Cdk5 activity may be passed through the combination of p25 (the brachymemma modification of p35) and be out of control.Local asphyxia, excitotoxicity (excitotoxicity) and beta amyloid peptide (amyloid peptide) can induce p35 to transform and cdk5 activity out of control subsequently to p25.Therefore, shown that p25 participates in neurodegenerative disease, as the pathogeny of Alzheimer's and be therefore interesting for resisting the target of therapy of these diseases.

Cdk7 has the nucleoprotein that cdc2 CAK is active and be combined with cyclin H.Cdk7 has been accredited as the component of TFIIH transcription complex (it has rna plymerase ii C-stub area (CTD) activity).Show that its bio-chemical pathway mediating by Tat-participates in the adjusting that HIV-1 transcribes.Cdk8 is in conjunction with cyclin C and shown that it participates in the phosphorylation of the CTD of rna plymerase ii.Equally, also show that cdk9/ cyclin-T1 complex body (P-TEFb complex body) participates in the prolongation control of rna plymerase ii.Activate and when HIV-1 is genomic to transcribe, also need PTEF-b by the interaction of itself and Cyclin T1 at viral trans-activator (transactivator) Tat.Therefore, Cdk7, cdk8, cdk9 and P-TEFb complex body may be the targets of antiviral therapy.

At molecular level, the adjusting of cdk/ cyclin complex body activity needs inhibition or the dephosphorylation event of a series of stimulations and phosphorylation.Cdk phosphorylation activates kinases (activating kinases) (CAKs) by one group of cdk and/or kinases carries out as wee1, Myt1 and Mik1.By Phosphoric acid esterase, as cdc25, (a & c), pp2a or KAP carry out in dephosphorylation effect.

Cdk/ cyclin complex body activity can further be regulated by two family's endogenous cell protein inhibitors: Kip/Cip family, or INK family.INK albumen and cdk4 and cdk6 specific binding.P16 ^ink4(being also referred to as MTS1) is sudden change or deleted potential tumor suppressor gene in many preinvasive cancers.Kip/Cip family comprises albumen as p21 ^{cip1, Waf1}, p27 ^kip1and p57 ^kip2.As previously discussed, p21 be induced by p53 and can deactivation cdk2/ cyclin (E/A) and cdk4/ cyclin (D1/D2/D3) complex body.In breast cancer, colorectal carcinoma and prostate cancer, observe low-level that atypical p27 expresses.On the contrary, shown that the excessively expression of cyclin E in solid tumor is relevant with patient's poor prognosis.The mistake expression that has shown cyclin D1 is relevant with the esophageal carcinoma, breast cancer, squamous cell cancer and nonsmall-cell lung cancer.

Cdks and the albumen relevant with it are summarized in the above coordinating and drive the keying action in cell cycle of proliferative cell.Also some bio-chemical pathways that cdks played to keying action are therein described.Therefore, may extremely wish to develop some use on generic target in the treatment proliferative disorders of the therapy of cdks, as the monotherapy of cancer.Think that Cdk inhibitor also can be used for treating other illness as virus infection, autoimmune disorder and neurodegenerative disease etc.When with other existing or new therapeutical agent coupling treatment, Cdk targeted therapies also provides some clinical benefits in the treatment of described before disease.Because not direct and DNA interacts and has therefore reduced the risk that secondary tumor forms, so target may have advantages of and be better than current many antineoplastic agents in the anti-cancer therapies of Cdk.

dispersivity large B cell lymphoid tumor (DLBCL)

Cell cycle progression is to regulate by the combined action of cyclin, cell cycle protein dependent kinase (CDKs) and CDK-inhibitor (CDKi) (it is the agent of negativity Cycle Regulation).P27KIP1 is the CDKi key in Cycle Regulation, and G1/S conversion needs its degraded.Although do not express p27KIP1 in the lymphocyte of hyperplasia, report that some aggressiveness B-cell lymphomas show abnormal p27KIP1 dyeing.In such lymphoma, find the abnormal high expression of p27KIP1.Clinical correlation to these discoveries is analyzed, and single argument and multivariate analysis all show that in this class tumour, high-caliber p27KIP1 expression is the mark of unfavorable prognosis.These results show that the abnormal expression of p27KIP1 in dispersivity large B cell lymphoid tumor (DLBCL) has disadvantageous clinical meaning, show that this abnormal p27KIP1 albumen may be by not bringing into play function with other Cycle Regulation thing protein-interacting.(Br.J.Cancer.1999 Jul；80(9)：1427-34。P27KIP1 unconventionality expression and with disadvantageous clinical effectiveness in dispersivity large B cell lymphoid tumor.SaezA, Sanchez E, Sanchez-Beato M, Cruz MA, Chacon I, Munoz E, Camacho FI, Martinez-Montero JC, Mollejo M, Garcia JF, Piris MA.Department of Pathology, Virgen de la Salud Hospital, Toledo, Spain.)

lymphocytic leukemia

B-cell lymphocytic leukemia (CLL) is modal leukemia on the Western Hemisphere, the new case nearly 10 who makes a definite diagnosis every year, 000 example (Parker SL, Tong T, Bolden S, Wingo PA:Cancer statistics, 1997.Ca.Cancer.J.Clin.47:5, (1997)).For the leukemia of other form, the overall prognosis bona of CLL, even if the patient in late period also has the average survival time was of 3 years.

With use before take alkylating agent (alkylator) as basic therapy compared with, increase fludarabine to symptomatic CLL patient and can produce the survival extended period that higher complete responsiveness (27% pair 3%) and the longer state of an illness do not develop (33 pairs 17 months) as initial therapy.Although obtaining afterwards complete clinical response in treatment is to improve the initial step of CLL survival, Most patients can not obtain to be alleviated completely or fludarabine is not responded.In addition, the all CLL patients that treat with fludarabine finally can be recurred, thereby while making to use separately, its effect is only as simple alleviating medicine (Rai KR, Peterson B, Elias L, Shepherd L, HinesJ, Nelson D, Cheson B, Kolitz J, Schiffer CA: the fludarabine being undertaken by the patients with chronic lymphocytic of not carrying out treatment before and the stochastic comparison (A randomized comparison of fludarabine and chlorambucil forpatients with previously untreated chronic lymphocytic leukemia) of Chlorambucil body.ACALGB SWOG, CTG/NCI-C and ECOG Inter-Group Study.Blood88:141a, 1996 (abstr 552, supplementary issues 1).Therefore, in the treatment of this disease, obtain more much progress if realize, need to determine to there is the drug-fast new promoting agent with new role mechanism of supplying the cytotoxicity of fludarabine and can cancelling inherent CLL r factor induction.

It is the p53 dysfunction that is characterised in that point mutation or karyomit(e) 17p13 disappearance to treatment difference in response and the poor factor of survival condition that the consistent prediction of most research causes CLL patient.In fact, in the case series of many independent agencies, the parafunctional CLL patient of verified these p53 does not respond alkylating agent or purine analogue treatment.Introducing can overcome in CLL with the suddenly change therapeutical agent of relevant resistance of p53 and will be major progress in this disease treatment.

Flavopiridol and CYC 202---inhibitor of cell cycle protein dependent kinase---have induced the apoptosis of the malignant cell that derives from B-cell lymphocytic leukemia (B-CLL) in vitro.

With Flavopiridol haptic stimutus the active and p27 (kip1) (negative regulator of cell cycle, it crosses expression in B-CLL) of Caspase-3 depend on cracking (the Blood.1998 Nov 15 of caspase; 92 (10): 3804-16.Flavopiridol has induced apoptosis (Flavopiridol inducesapoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence on functionalp53) the .Byrd JC in chronic lymphocytic leukemia cell in the situation that not having bcl-2 regulate sign or do not rely on functional p53 by activation caspase-3, Shinn C, Waselenko JK, Fuchs EJ, Lehman TA, NguyenPL, Flinn IW, Diehl LF, Sausville E, Grever MR).

aurora kinase

Recently, had been found that gang participates in the G2 of cell cycle and the novel serine/threonine kinase that is called as aurora kinase of M phase, and found that it is important mitotic division conditioning agent.

Although also do not illustrate the definite effect of aurora kinase, find its play a role (people such as Adams, TrendsCell Biol., 11:49-54 (2001) in Mitotic checkpoint control, karyomit(e) kinetics and division of cytoplasm.Aurora kinase is arranged in the body (mid-body) of extremely going up of the centrosome of division cells, bipolar spindle body and mitotic division organ.

Up to now, three class members (E.A.Nigg, Nat.Rev.Mol.Cell Biol.2:21-32, (2001)) of aurora kinase group in mammalian body, have been found.It is:

Aurora A (being also referred to as in the literature Aurora 2);

Aurora B (being also referred to as in the literature Aurora 1); With

Aurora C (being also referred to as in the literature Aurora 3).

Described aurora kinase has the catalysis region of high homology, (Katayama H, Brinkley WR, Sen S. but its N-terminal portions differs greatly; Aurora kinase: the effect (The Aurora kinase:role in cell transformation andtumorigenesis) in cell transformation and tumour generation; Cancer Metastasis Rev.2003 Dec; 22 (4): 451-64).

The substrate of having determined BTAK and B comprises kinesin-sample dynein, spindle body albumen, histone H 3 albumen, kinetochore protein and tumor suppressor protein p53.

Think that Aurora A kinases participates in spindle body and forms and become in early days and be positioned on centrosome mutually at G2, interim in the time of this section, its phosphorylation albumen relevant with spindle body (people such as Prigent, Cell, 114:531-535 (2003).The people such as Hirota, Cell, 114:585-598, (2003) find that the cell of disappearance Aurora A protein kinase can not start mitotic division.In addition, also have been found that the sudden change of Aurora A gene in (Adams, 2001) each kind or disintegrate to have caused mitotic division abnormal, comprise centrosome separation and maturation defect, spindle body distortion and chromosome segregation defect.

Aurora kinase in most of healthy tissuess conventionally with low expression level, but there is the tissue of somatoblast at high proportion, for example, except thymus gland and testis.In a word, the level that has been found that aurora kinase in many human cancers raise (people such as Giet, J.Cell.Sci.112:3591-361, (1999) and Katayama (2003).In addition, Aurora A kinases maps to the chromosome dyeing body 20q13 region that frequent discovery is amplified in many human cancers.

Therefore, for example, in human breast cancer, ovarian cancer and carcinoma of the pancreas, detect the remarkable expression excessively of AuroraA and (seen the people such as Zhou, Nat.Genet.20:189-193, (1998), the people such as Tanaka, Cancer Res., 59:2041-2044, and the people such as Han (1999), cancer Res., 62:2890-2896, (2002).

In addition, Isola, American Journal of Pathology 147,905-911 (1995) has reported that the amplification of Aurora aminoacyl site (20ql3) is with relevant without tubercle mammary cancer (node-negative breast cancer) patient's poor prognosis.

In mankind's bladder cancer, observe the amplification of Aurora-A and/or crossed the amplification expressed and show Aurora-A with dysploidy and invasive clinical behavior, see the people such as Sen, J.Natl.Cancer Inst, 94:1320-1329 (2002).

The carcinoma of the colon and rectum more than 50% (is shown in the people such as Bischoff, EMBO J., 17:3052-3065, and the people such as Takahashi (1998), Jpn.J.Cancer Res., 91:1007-1014 (2000)), ovarian cancer (is shown in the people Clin.Cancer Res. such as Gritsko, 9:1420-1426 (2003) and the tumor stomach (people such as Sakakura, British Journal of Cancer, the expression that has detected Aurora-A in 84:824-831 (2001) increases.

The people Cancer Research such as Tanaka, 59:2041-2044 (1999) finds have Aurora A to cross the sign of expression in the invasive duct adenocarcinoma of 94% mammary gland.

In kidney, uterine neck, neuroblastoma, melanoma, lymphoma, pancreas and prostate tumor cells system, find the high-caliber Aurora A kinases (people (1998) such as Bischoff, EMBO J., 17:3052-3065 (1998); The people J.Biol.Chem. such as Kimura, 274:7334-7340 (1999); The people such as Zhou, Nature Genetics, 20:189-193 (1998); The people such as Li, Clin Cancer Res.9 (3): 991-7 (2003)].

The expression level of Aurora-B in many human tumour cell lines is high, and said tumor cell line comprises leukemia cell [people such as Katayama, Gene 244:1-7)].The level of this kind of enzyme increases people such as [, J.Natl Cancer Inst., 91:1160-1162 (1999)] Katayama with the form of Duke ' s step function in primary carcinoma of the colon and rectum.

Although this kinases is tending towards being confined to the sexual cell in healthy tissues, but in some tumor cell lines, also detect the high level (seeing the people Journal of Biological Chemistry such as Kimura, 274:7334-7340 (1999)) of Aurora-3 (Aurora-C).The people such as Takahashi, Jpn J.Cancer Res.91:1007-1014 (2001)] in article, also report that Aurora-3 crosses and expresses in about 50% carcinoma of the colon and rectum.

About other report of the effect of aurora kinase in proliferative disorders can be referring to people such as Bischoff, Trends in Cell Biology 9:454-459 (1999); The people Journal ofCell Science such as Giet, the people Oncogene such as 112:3591-3601 (1999) and Dutertre, 21:6175-6183 (2002).

The people such as Royce have reported the expression of having noticed Aurora 2 genes (being called as STK15 or BTAK) in about 1/4th primary breast tumour.(Royce ME, Xia W, Sahin AA, Katayama H, Johnston DA, Hortobagyi G, Sen S, HungMC; STK15/Aurora-A in primary breast tumour expresses extremely relevant with core, but irrelevant with prognosis; Cancer.2004 Jan 1; 100 (1): 12-9).

Carcinoma of endometrium (EC) comprises the cancer of at least two types: endometrioid carcinoma (EECs) is the tumour relevant with oestrogenic hormon, and it is usually euploid and has good prognosis.Non-endometrioid carcinoma (NEECs; Slurries and hyaline cell form) irrelevant with oestrogenic hormon, and be usually aneuploid, and there is clinically aggressiveness.Also find that Aurora is amplified in 55.5% NEECs, but in any EECs, be not all amplified (P < or=0.001) (Moreno-Bueno G, Sanchez-Estevez C, Cassia R, Rodriguez-Perales S, Diaz-Uriarte R, Dominguez O, Hardisson D, AndujarM, Prat J, Matias-Guiu X, Cigudosa JC, Palacios J.Cancer Res.2003Sep 15; 63 (18): 5697-702).

The people such as Reichardt (Oncol Rep.2003 Sep-Oct; 10 (5): 1275-9) reported that the quantitative DNA analysis of finding Aurora in neurospongioma amplification by PCR shows five kinds (31%) (the 1x II level in 16 kinds of tumours of different WHO grades, 1x III level, 3x IV level) show the DNA cloning of Aurora 2 genes.The amplification of supposing Aurora 2 genes is nonrandom heredity variation in human glioma, and it plays a part certain in the genetic approach of tumour generation (tumourigenesis).

The people's such as Hamada result (Br.J.Haematol.2003 May; 121 (3): 439-47) also show that Aurora 2 is a kind of effectively material standed fors, show that it not only has disease activity, but also be the tumour origin (tumourigenesis) of non_hodgkin lymphoma.The retardance of the growth of tumour cell that the function that limits this gene has caused, it may be a kind of method for the treatment of non_hodgkin lymphoma.

(Clin Cancer Res.2003 Apr in the people's such as Gritsko research; 9 (4): 1420-6)), with 92 patients of suffering from Primary ovarian cancer kinase activity and the protein level of Aurora A.Vitro kinase analysis shows that Aurora A kinase activity raises in 44 routine cases (48%).In 52 samples (57%), detecting Aurora A protein level increases.The high protein level of AuroraA and kinase activity raise much relations.

Result (the Clin.Cancer Res.2003 Mar that the people such as Li obtain; 9 (3): 991-7) show Aurora A gene in pancreatic neoplasm and cancerous cell line by cross express, and show AuroraA cross express may carcinoma of the pancreas occur in play a role.

The mitotic kinase of its coding that equally, has shown Aurora A gene amplification and accompany is expressed and is increased in human bladder cancer with dysploidy and invasive clinical behavior.(J.Natl.Cancer Inst.2002 Sep 4；94(17)：1320-9)。

(Aurora-A crosses to express and makes to have overcome microtubule-kinetochore and adhere to outpost of the tax office Mol.Interv.2003 May for Dutertre S, Prigent C. for research that some groups carry out; 3 (3): 127-30 and Anand S, Penrhyn-Lowe S, Venkitaraman AR., Aurora-A amplification has overcome the mitotic spindle assembling outpost of the tax office, has induced the tolerance to taxol, Cancer Cell.2003 Jan; 3 (1): 51-62) show that excessively expressing of aurora kinase activity is relevant with the tolerance that current certain cancers is treated.For example, Aurora A crossing in mouse embryo fibroblasts expressed the susceptibility that can reduce the cytotoxic effect of these cells to Taxane derivative.Therefore, find that aurora kinase inhibitors is particularly useful for Current Therapy having been formed to the patient of tolerance.

Based on the work carried out up to now, imagination suppresses aurora kinase, particularly BTAK and aurora kinase B will be the effective means that stops tumor development.

The people such as Harrington (Nat Med.2004Mar; 10 (3): 262-7) inhibitor of verified aurora kinase has suppressed in vivo tumor growth and has induced tumour regression.In this research, aurora kinase inhibitors has been blocked cancer cell hyperplasia, and has triggered the necrocytosis that comprises leukemia, rectum colon and the mammary gland cell cancerous cell line in tying up to.In addition, also show that it can treat leukemia by cell death inducing in leukemia cell.VX-680 has effectively killed primary acute myelogenous leukemia (AML) cell (Andrews, Oncogene, 2005,24,5005-5015) that is difficult to treatment that derives from patient.

The cancer that may obey especially Aurora inhibitor comprises mammary cancer, bladder cancer, carcinoma of the colon and rectum, carcinoma of the pancreas, ovarian cancer, non_hodgkin lymphoma, neurospongioma and non-endometrial-like carcinoma of endometrium.Special leukemia of obeying Aurora inhibitor comprises acute myelogenous leukemia (AML), chronic Sui's cellularity leukemia (CML), B-cell lymphoma (amphicyte (Mantle cell)) and acute lymphoblastic leukemia (ALL).

glycogen synthase kinase

Glycogen synthase kinase-3 (GSK3) is a kind of serine-threonine kinase existing as two kinds of isoform (GSK3 α & beta GSK3 β) forms of generally expressing in human body.Imply that GSK3 is synthetic in fetal development, albumen, worked in hyperplasia, cytodifferentiation, microtubule dynamics, cell viability and apoptosis.Imply GSK3 involved in diseases state, as the process of diabetes, cancer, Alzheimer's, apoplexy, epilepsy, motor neuron and/or head trauma.Phylogenetic (Phylogenetically) GSK3 and cell cycle protein dependent kinase (CDKs) are closely related.

The total polypeptide substrate sequence that GSK3 identifies is (Ser/Thr)-X-X-X-(pSer/pThr), wherein X is any amino acid (being positioned on (n+1), (n+2), (n+3) position), and pSer and pThr are respectively phosphoric acid-Serine and phosphoric acid-Threonine (n+4).First Serine or Threonine on GSK3 phosphorylation (n) position.Show to be positioned at (n+4) thus the phosphoric acid-Serine on position or phosphoric acid-Threonine seemingly start GSK3 that to obtain the upset of maximum substrate necessary.The phosphorylation of GSK3 β on GSK3 α or Ser9 on Ser21 has caused the inhibition of GSK3.Mutagenesis and peptide competition research has produced the model that the phosphorylation N-end of GSK3 can be competed by automatically suppressing mechanism (autoinhibitory mechanism) and phosphoric acid-peptide substrates (S/TXXXpS/pT).Also have data to show that GSK3 α and GSK β can obtain ingenious adjusting by the phosphorylation of tyrosine 279 and 216 respectively.These residues have caused the reduction of kinase activity in body to the sudden change of Phe.All aspects that the X-ray crystalline texture of GSK3 β contributes to people that eye is expanded to GSK3 activation and regulates.

Thereby GSK3 has formed a part and its energy phosphorylation Glycogensynthase and the energy deactivation Glycogensynthase of Mammals insulin response approach.Therefore, thus think and be considered to resist II type or non-insulin-dependent diabetes mellitus (NIDDM) (NIDDM) by the synthetic rise of glycogen that suppresses the rise of the Glycogensynthase activity that produces of GSK3 and produce: a kind of wherein body tissue becomes the potential means of the illness of tolerance insulin stimulating.Regular Insulin is combined with extracellular insulin receptor and has been triggered the insulin response of cell in liver, fat or muscle tissue.It has caused phosphorylation and raising with backward IRS (IRS) albumen serous coat.The further phosphorylation of IRS albumen has caused phosphoinositide-3 kinases (PI3K) raising to this serous coat, it can discharge second messenger's phosphatidyl-4 alcohol radical (phosphatidylinosityl) 3 at this place, 4,5-triphosphate (PIP3).It has promoted 3-phosphoinositide-deopendent protein kinase 1 (PDK1) and protein kinase B (PKB or the Akt) colocalization (co-localisation) to this film, and at this place, PDK1 has activated PKB.Thereby PKB can phosphorylation also can suppress GSK3 α and/or GSK β by the phosphorylation of Ser9 or ser21 respectively.Then the inhibition of GSK3 has triggered the rise of Glycogensynthase activity.The similar cellular response of viewed response when the therapeutical agent that therefore, can suppress GSK3 can be induced with insulin stimulating.The another kind of substrate in vivo of GSK3 is eukaryotic protein synthesis initiator 2B (eIF2B).EIF2B is inactivated by phosphorylation and biosynthesizing that therefore can arrestin.Therefore, the inhibition of GSK3, the inhibition for example producing by deactivation " the Mammals target of rapamycin " albumen (mTOR) can upregulated protein biosynthesizing.Finally, have some signs to show that GSK3 activity is that protein kinase (MAPK) approach of the mitogen activation undertaken by the phosphorylation via GSK3 regulates, the phosphorylation of GSK3 is to be carried out as the protein kinase 1 (MAPKAP-K1 or RSK) of the protein kinase activation of mitogen activation by kinases.These data show that GSK3 activity may be stimulated and be regulated by mitogenesis, Regular Insulin and/or amino acid.

Also show that GSK3 β is the key ingredient in vertebrates Wnt signal transduction pathway.Show that this bio-chemical pathway is very crucial and is regulating the hyperplasia in healthy tissues for normal embryo development.The response stimulating as Wnt, GSK3 is suppressed.It can cause GSK3 substrate as the dephosphorylation of Axin (polyp of colon (APC) gene product of adenoma) and calcium Angiotensin Converting Enzyme (β-catenin).Abnormal adjusting and many related to cancer of Wnt approach are shown.Sudden change in APC and/or calcium Angiotensin Converting Enzyme is all very common in carcinoma of the colon and rectum and other tumour.Also shown that calcium Angiotensin Converting Enzyme is very important in cell adhesion.Therefore, GSK3 also can regulate cell adhesion process to a certain extent.Except the bio-chemical pathway being described, also have some data to show that GSK3 participates in regulating cell fission by phosphorylation and participates in transcription factor if c-Jun, CCAAT/ enhancer binding protein α (C/EBP α), c-Myc and/or other substrate are if the nf (NFATc) of activation T-cell, heat shock factor-1 (HSF-1) and c-AMP response element are in conjunction with the phosphorylation of albumen (CREB).Also show that GSK3 plays a part in adjusting apoptosis certain, but it has tissue specificity.The adjusting that GSK3 carries out apoptosis by proapoptosis (pro-apoptotic) mechanism may be relevant especially to the medical condition that neuronal cell apoptosis may occur.The example of these situations has head trauma, apoplexy, epilepsy, Alzheimer's and motor neuron, stein-leventhal syndrome, corticobasal sex change and Pick's disease.In test, show that GSK3 can the hyperphosphorylation albumen Tau relevant with microtubule in vitro.The hyperphosphorylation of Tau has destroyed the normal combination of itself and microtubule and can cause the formation of Tau silk in cell.Think that building up of these filaments finally caused neurone dysfunction and sex change.Therefore, the means of a kind of restriction and/or the effect of prevention neurodegeneration may be provided by suppressing phosphorylation that GSK3 suppresses Tau.

prior art

WO 02/34721 (Du Pont) discloses indeno [1, the 2-c] pyrazoles-4-ketone of a class as cell cycle protein dependent kinase inhibitor.

WO 01/81348 (Bristol Myers Squibb) described 5-sulfo--, sulfinyl-and sulfonyl pyrazole also [3,4-b]-pyridines as the application of cell cycle protein dependent kinase inhibitor.

WO 00/62778 (being also Bristol Myers Squibb) discloses a proteinoid tyrosine kinase inhibitor.

WO 01/72745A1 (Cyclacel) described 4-heteroaryl-pyrimidine class that 2-replaces with and preparation, comprise it pharmaceutical composition using and as the application of cyclin-dependant kinase (cdks) inhibitor and therefore produce its be used for the treatment of proliferative disorders as the application of cancer, leukemia, psoriasis etc.

WO 99/21845 (Agouron) has described for suppressing cell cycle protein dependent kinase (cdks), as the 4-aminothiazole derivs of CDK1, CDK2, CDK4 and CDK6.This invention also relates to treatment or the prophylactic applications of the pharmaceutical composition that comprises this compounds and treats the method for malignant tumour and other illness by this compounds of effective dosage.

WO 01/53274 (Agouron) discloses the compound of a class as CDK kinase inhibitor, and it comprises the phenyl ring with the acid amides-replacement being connected containing N heterocyclic group.

WO 01/98290 (Pharmacia & Upjohn) discloses the 3-aminocarboxyl-2-formamido group thiophene derivant of a class as kinases inhibitor.Narrate this compounds and there is multiple protein kinase activity.

WO 01/53268 and WO 01/02369 (Agouron) disclose some by arrestin kinases as cell cycle protein dependent kinase or Tyrosylprotein kinase regulates or the compound of inhibition of cell proliferation.

WO 00/39108 and WO 02/00651 (belonging to Du Pont Pharmaceuticals) have described a large heterocyclic compounds, and these compounds are trypsin-like serine proteases, especially the inhibitor of factor Xa and zymoplasm.Narrate that these compounds can be used as antithrombotics or for preventing thromboembolic states illness.

US 2002/0091116 (people such as Zhu), WO 01/1978 and WO 01/64642 disclose respectively not on the same group to the activated heterogeneous ring compound of factor Xa.

WO 03/035065 (Aventis) discloses the benzimidizole derivatives of a large class as kinases inhibitor, but it does not disclose it CDK kinases or GSK kinases are had to activity.

It is the connection heteroaryl compound of farnesyl transferase inhibitor that WO 97/36585 and US 5,874,452 (belonging to Merck) disclose.

WO 03/066629 (Vertex) discloses the benzimidazolyl-pyrazoles amine as GSK-3 inhibitor.

WO 97/12615 (Warner Lambert) discloses the benzimidazoles as 15-lipoxidase inhibitor.

WO 2004/54515 (SmithKline Beecham Corporation) discloses a class and has intended the benzimidazoles like thing as thrombopoietin.

WO 2004/41277 (Merck) discloses the amino-benzimidazoles of a class as androgen receptor modifier.

WO 2005/028624 (Plexxikon) discloses the framework (scaffolds) of the compound for having antagonism protein kinase activity.

Our early stage international patent application no PCT/GB2004/002824 (WO2005/002552) discloses the substituted 1H-benzimidazolyl-2 radicals-yl of a class as CDK, aurora kinase and GSK kinase inhibitor)-1H-pyrazoles-4-yl]-ureas.One in these compounds that concrete name examples has illustrated in WO2005/002552 is 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea.In the experimental section of WO 2005/002552,1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of free alkali form is described] preparation of-urea.

Summary of the invention

summary of the invention

The invention provides and there is cell cycle protein dependent kinase inhibition or adjusting activity and glycogen synthase kinase-3 (GSK3) inhibition or adjusting activity and/or aurora kinase inhibition or regulate active compound, and think that this compounds can be used for preventing or treating by described kinase mediated morbid state or illness.

Therefore, for example, think that compound of the present invention can be used for alleviating or reducing the incidence of cancer.

In first aspect, the invention provides the compound of formula (I)

Or its salt, solvate, tautomer or N-oxide compound,

Wherein M is selected from group D1 and group D2:

And wherein:

(A) in the time that M is group D1:

X is selected from O, NH and NCH ₃;

A is selected from a key and group NR ², wherein R ²hydrogen or methyl;

E is selected from a key, CH ₂, CH (CN) and C (CH ₃) ₂;

R ¹be selected from:

(i) cycloalkyl of 3 to 5 ring memberses that optionally replaced by hydroxyl, fluorine, amino, methylamino, methyl or ethyl;

(ii) comprise 1 or 2 and be selected from O, N, S and SO ₂heteroatomic ring member's the saturated heterocyclic group of 4 to 6 ring memberses, described heterocyclic group is optionally by C _1-4alkyl, amino or hydroxyl replace; But do not comprise unsubstituted 4-morpholinyl, unsubstituted tetrahydropyran-4-base, unsubstituted 2-pyrrolidyl and piperidin-4-yl unsubstituted and that 1-replaces;

(iii) 2 of following formula, the phenyl that 5-replaces:

Wherein (a) when X be NH or N-CH ₃time, R ³be selected from chlorine and cyano group; (b) in the time that X is O, R ³cN;

(iv) group CR ⁶r ⁷r ⁸, wherein R ⁶and R ⁷be selected from separately hydrogen and methyl, and R ⁸be selected from hydrogen, methyl, C _1-4alkyl sulfonyl ylmethyl, hydroxymethyl and cyano group;

(v) pyridazine-4-base that the substituting group that is optionally selected from methyl, ethyl, methoxyl group and oxyethyl group by one or two replaces;

(vi) substituted Imidazothiazole base, wherein said substituting group is selected from methyl, ethyl, amino, fluorine, chlorine, amino and methylamino; With

(vii) optionally substituted 1,3-dihydro-isoindole-2-base or optionally substituted 2,3-dihydro-indoles-1-base, is wherein selected from halogen, cyano group, amino, C at the optional substituting group described in various situations _1-4single-and dialkyl amido, CONH ₂or CONH-C _1-4alkyl C _1-4alkyl and C _1-4alkoxyl group, wherein said C _1-4alkyl and C _1-4alkoxyl group is optionally by hydroxyl, methoxyl group or amino replacement;

(viii) be optionally selected from hydroxyl, halogen, cyano group, amino, C by one or two _{1- 4}single-and dialkyl amido, CONH ₂or CONH-C _1-4alkyl, C _1-4alkyl and C _1-4the 3-pyridyl that the substituting group of alkoxyl group replaces, wherein said C _1-4alkyl and C _1-4alkoxyl group is optionally by hydroxyl, methoxyl group or amino replacement; But do not comprise compound 2-oxo-1,2-dihydro-Nicotinicum Acidum [3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-acid amides and 2,6-dimethoxy-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-niacinamide;

(ix) be optionally selected from halogen, cyano group, amino, C by one or two _1-4single-and dialkyl amido, CONH ₂or CONH-C _1-4alkyl C _1-4alkyl and C _1-4thiomorpholine or its S-oxide compound or S that the substituting group of alkoxyl group replaces, S-dioxide, wherein said C _1-4alkyl and C _1-4alkoxyl group is optionally by hydroxyl, methoxyl group or amino replacement; With

When E-A is NR ²time, R ¹also be selected from:

(x) 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, 3,4-difluorophenyl, 2,5-difluorophenyl, 3,5-difluorophenyl, 2,4,6-trifluorophenyl, 2-p-methoxy-phenyl, the chloro-2-p-methoxy-phenyl of 5-, cyclohexyl, unsubstituted 4-THP trtrahydropyranyl and tert-butyl;

(xi) group NR ¹⁰r ¹¹, wherein R ¹⁰and R ¹¹respectively C naturally _1-4alkyl or R ¹⁰and R ¹¹thereby be connected NR ¹⁰r ¹¹forming one optionally comprises and is selected from O, N, S and SO ₂second heteroatomic ring member's the saturated heterocyclic group of 4 to 6 ring memberses, described heterocyclic group is optionally by C _1-4alkyl, amino or hydroxyl replace;

(xii) optionally one or two is selected from hydroxyl, halogen, cyano group, amino, C _1-4single-and dialkyl amido, CONH ₂, CONH-C _1-4alkyl, C _1-4alkyl and C _{1 -4}the pyridone that the substituting group of alkoxyl group replaces, wherein said C _1-4alkyl and C _1-4alkoxyl group is optionally by hydroxyl, methoxyl group or amino replacement;

When E-A is C (CH ₃) ₂nR ²or CH ₂-NR ²time, R ¹also be selected from:

(xiii) unsubstituted 2-furyl and 2,6-difluorophenyl; With

When E-A is C (CH ₃) ₂nR ²time, R ¹also be selected from:

(xiv) unsubstituted phenyl; With

When E is CH ₂time, R ¹also be selected from:

(xv) unsubstituted tetrahydropyran-4-base; With

(B) in the time that M is group D2:

A is selected from a key and group NR ², wherein R ²hydrogen or methyl;

E is selected from a key, CH ₂, CH (CN) and C (CH ₃) ₂;

R ¹be selected from:

(xvi) the 3-furyl that the 2-of following formula replaces:

Wherein R ⁴and R ⁵identical or different and be selected from hydrogen and C _1-4alkyl, or R ⁴and R ⁵thereby be connected NR ⁴r ⁵formation one optionally comprises second and is selected from O, NH, NMe, S or SO ₂heteroatoms or 5-or 6-person's saturated heterocyclic group of group, described 5-or 6-person's saturated heterocyclic are optionally replaced by hydroxyl, fluorine, amino, methylamino, methyl or ethyl;

(xvii) the 2-furyl that the 5-of following formula replaces:

Wherein R ⁴and R ⁵identical or different and be selected from hydrogen and C _1-4alkyl, or R ⁴and R ⁵thereby be connected NR ⁴r ⁵formation one optionally comprises second and is selected from O, NH, NMe, S or SO ₂heteroatoms or 5-or 6-person's saturated heterocyclic group of group, described 5-or 6-person's saturated heterocyclic group are optionally replaced by hydroxyl, fluorine, amino, methylamino, methyl or ethyl; Prerequisite is that described compound is not 5-piperidin-1-yl methyl-furans-2-formic acid [3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-acid amides;

(xviii) group of following formula:

Wherein R ⁹hydrogen, methyl, ethyl or sec.-propyl; G is CH, O, S, SO, SO ₂or NH and described group are optionally selected from C by one, two or three _1-4alkyl, hydroxyl, C _1-4-oxyl, fluorine, amino, list-and two-C _1-4the substituting group of alkylamino replaces, and wherein said C _1-4alkyl and C _1-4-oxyl is separately optionally by hydroxyl, fluorine, amino, list-or two-C _1-4alkylamino replaces; With

(xix) 3 of following formula, the dibasic phenyl of 5-:

Wherein X is selected from O, NH and NCH ₃;

(C) in the time that M is group D1:

With X be O, A is group NR ², wherein R ²hydrogen, E is a key, and R ¹while being 2,6-difluorophenyl, the compound of formula (I) is a kind of acid salt, and it is selected from the salt forming with following acid, and said acid is selected from acetic acid, hexanodioic acid, alginic acid, xitix (for example L-AA), aspartic acid (for example ASPARTIC ACID), Phenylsulfonic acid, phenylformic acid, dextrocamphoric acid (for example (+) dextrocamphoric acid), capric acid, sad, carbonic acid, citric acid, cyclohexane sulfamic acid, dodecanoate, dodecyl sulphate, ethane-1,2-disulfonic acid, ethyl sulfonic acid, fumaric acid, tetrahydroxyadipic acid, gentisinic acid, glucoheptonic acid, D-glyconic acid, glucuronic acid (for example D-Glucose aldehydic acid), L-glutamic acid (for example Pidolidone), α ketoglutaric acid, oxyacetic acid, urobenzoic acid, hydrochloric acid, hydroxyethylsulfonic acid, isopropylformic acid, lactic acid (for example (+)-Pfansteihl and (±)-DL-LACTIC ACID), lactobionic acid, lauryl sulfonic acid, toxilic acid, oxysuccinic acid, (-)-L MALIC ACID, propanedioic acid, methylsulfonic acid, glactaric acid, naphthene sulfonic acid (for example naphthalene-2-sulfonic acid), naphthalene-1,5-disulfonic acid, nicotinic acid, oleic acid, vitamin B13, oxalic acid, palmitinic acid, flutter acid, phosphoric acid, propionic acid, sebacic acid, stearic acid, succsinic acid, sulfuric acid, tartrate (for example (+)-L-TARTARIC ACID), thiocyanic acid, toluenesulphonic acids (for example p-toluenesulphonic acids), valeric acid and xinafoic acids.

In one embodiment, group M be the group (A) of formula (I) above and (B) in defined group D1 or D2.

In another embodiment, group M is that the compound of group D1 and formula (I) is defined 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl in the group (C) of formula (I) above] acid salt of-urea.

In a specific embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] salt or the free alkali of-urea, and its lactic acid salt particularly.

The present invention also provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea with and the new application of salt (for example acid salt), solvate, tautomer or N-oxide compound.

The present invention particularly also provides:

Formula (I), (II), (III) or (XXX) or the application of the medicine of the morbid state that mediated by cell cycle protein dependent kinase or glycogen synthase kinase-3 for the preparation of prevention or treatment of the compound of its any group or embodiment or illness as herein defined.

Prevention or the morbid state that mediated by cell cycle protein dependent kinase or glycogen synthase kinase-3 for the treatment of or a method for illness, the method comprises to the individual administration that has these needs formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment as herein defined.

Alleviate or reduce a method for the incidence of the morbid state that mediated by cell cycle protein dependent kinase or glycogen synthase kinase-3 or illness, the method comprises to the individual administration that has these needs formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment as herein defined.

Treat and in Mammals, comprise abnormal Growth of Cells or the disease being caused by abnormal Growth of Cells or a method for illness, the quantity that the method comprises effectively suppressing abnormal cell growth is to described Mammals administration formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment as herein defined.

Alleviate or reduce a method for the incidence of the disease that comprises abnormal Growth of Cells in Mammals or caused by abnormal Growth of Cells or illness, the quantity that the method comprises effectively suppressing abnormal cell growth is to described Mammals administration formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment as herein defined.

Treat and in Mammals, comprise abnormal Growth of Cells or the disease being caused by abnormal Growth of Cells or a method for illness, the quantity that the method comprises effectively suppressing cdk kinases (as cdk1 or cdk2) or glycogen synthase kinase-3 activity to as described in Mammals administration formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment as herein defined.

Alleviate or reduce a method for the incidence of the disease that comprises abnormal Growth of Cells in Mammals or caused by abnormal Growth of Cells or illness, the quantity that the method comprises effectively suppressing cdk kinases (as cdk1 or cdk2) or glycogen synthase kinase-3 activity to as described in Mammals administration formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment as herein defined.

Suppress a method for cell cycle protein dependent kinase or glycogen synthase kinase-3, the method comprise by described kinases with suppress this kinase whose formula as herein defined (I), (II), (III) (XXX) or the compound of its any group or embodiment contact.

A kind of by with formula (I) as herein defined, (II), (III) (XXX) or the activity of the compound of its any group or embodiment inhibition cell cycle protein dependent kinase or glycogen synthase kinase-3 regulate the method for cell processes (for example cell fission).

As herein defined formula (I), (II), (III) (XXX) or the compound of its any group or embodiment be characterized as the application of the medicine of disease that aurora kinase (for example Aurora A kinases and/or Aurora B kinases) raises or illness for the preparation of prevention or treatment.

As herein defined formula (I), (II), (III) (XXX) or the compound of its any group or embodiment for the preparation of the application of the medicine of prevention or treatment cancer, said cancer is to be characterized as the cancer that aurora kinase (for example Aurora A kinases and/or Aurora B kinases) raises.

As herein defined formula (I), (II), (III) (XXX) or the compound of its any group or embodiment be selected from the application of the medicine of the patient's of the subgroup of the Ile31 modification with Aurora A gene cancer for the preparation of prevention or treatment.

Formula (I), (II), (III) or (XXX) or the application of having been made a definite diagnosis the medicine of the patient's of a forming section of the subgroup that becomes the Ile31 modification with Aurora A gene cancer for the preparation of prevention or treatment of the compound of its any group or embodiment as herein defined.

A kind of prevention or treatment are characterized as disease that aurora kinase (for example Aurora A kinases and/or Aurora B kinases) raises or the method for illness, and the method comprises administration formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment as herein defined.

A kind of method of alleviating or reducing the incidence that is characterized as disease that aurora kinase (for example Aurora A kinases and/or Aurora B kinases) raises or illness, the method comprises administration formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment as herein defined.

A kind of prevention or treatment suffer from or suspect the method for the patient's who suffers from cancer cancer (or alleviating or reduce its incidence); The method comprises that (i) carries out diagnostic test to determine whether this patient has the Ile31 modification of Aurora A gene to patient; (ii) really have in the situation of described modification patient, there is as herein defined aurora kinase to thereafter this patient's administration and suppress active formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment.

A kind of prevention or treatment are characterized as the morbid state of aurora kinase (for example Aurora A kinases and/or Aurora B kinases) rise or the method for illness (or alleviating or reduce its incidence); The method comprises that (i) carries out diagnostic test with the flag sign surveying aurora kinase and raise with (ii) in this diagnostic test shows situation that aurora kinase raises to patient, has as herein defined aurora kinase to thereafter this patient's administration and suppresses active formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment.

A kind of prevention or treatment are characterized as (a) CDK kinases hyperactivity hyperkinesia; And/or (b) a kind of approach to the active sensitization of normal CDK; And/or (c) morbid state that cyclin E raises or the method for illness (or alleviate or reduce its incidence); The method comprises that (i) carries out diagnostic test to survey (a) and/or flag sign (b) and/or (c) to patient; (ii) show to exist in (a) and/or situation (b) and/or (c) at this diagnostic test, there is as herein defined the formula (I), (II), (III) of CDK kinase inhibiting activity or (XXX) or the compound of its any group or embodiment to thereafter this patient's administration.

Methods for the treatment of, medical use or the compound for applying, wherein by formula (I) as herein defined, (II), (III) (XXX) or the compound administration of its any group or embodiment for example, be confirmed as suffering from disease that a kind for the treatment of that is easily subject to carrying out with described compound affects or the patient subgroups of illness in (carrying out administration with treatment significant quantity) by any or multiple diagnostic test described here.

As herein defined formula (I), (II), (HI) (XXX) or the compound of its any group or embodiment for the preparation of prevention or treat the application of the medicine of morbid state described here.

For preventing or treat the formula as herein defined (I), (II), (III) of morbid state described here or (XXX) or the compound of its any group or embodiment.

Prevention or a method for the treatment of morbid state described here or illness (or alleviating or reduce its incidence), the method comprises to described Mammals drug treatment significant quantity formula (I), (II), (III) or (XXX) or the compound of its any group or embodiment as herein defined.

One comprises as herein defined formula (I), (II), (III) or (XXX) or its any group or the compound of embodiment and the pharmaceutical composition of pharmaceutically acceptable carrier.

A kind of for carry out the pharmaceutical composition of administration with aqueous solution form, this pharmaceutical composition is included in solubleness in water higher than 1mg/ml, typically higher than 5mg/ml, more typically higher than 15mg/ml, more typically higher than 20mg/ml and preferably higher than the formula as herein defined (I) of the salt form of 25mg/ml, (II), (III) or (XXX) or the compound of its any group or embodiment.

For the formula as herein defined (I) of medical science, (II), (III) or (XXX) or the compound of its any group or embodiment.

For above and here other local described any application and the defined compound here of method.

Be used for the treatment of the defined formula here (I), (II), (III) of B-cell lymphoma or (XXX) or the compound or its salt of its any group or embodiment (for example acid salt), solvate, tautomer or N-oxide compound.

Be used for the treatment of the defined formula here (I), (II), (III) of lymphocytic leukemia or (XXX) or the compound or its salt of its any group or embodiment (for example acid salt), solvate, tautomer or N-oxide compound.

Be used for the treatment of the defined formula here (I), (II), (III) of dispersivity large B cell lymphoid tumor or (XXX) or the compound or its salt of its any group or embodiment (for example acid salt), solvate, tautomer or N-oxide compound.

By give need such treatment patient's administration here defined formula (I), (II), (III) (XXX) or the compound or its salt of its any group or embodiment (for example acid salt), solvate, tautomer or N-oxide compound treat a B-cell lymphoma, the method for dispersivity large B cell lymphoid tumor or lymphocytic leukemia.

Be used for the treatment of leukemia, particularly defined formula here (I) recurrence or acute myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia and the chronic granulocytic leukemia of refractory, (II), (III) or (XXX) or the compound or its salt of its any group or embodiment (for example acid salt), solvate, tautomer or N-oxide compound.

For above and here other local described any application and 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of method] acid salt or free alkali and particularly its lactic acid salt of-urea.

general preferred parameter and definition

Unless particularly point out in context, otherwise general preferred parameter and definition is below applicable to part D1, D2, A, E, X, X ^aand R ¹to R ⁹, with and various group, sub-definite, embodiment and embodiment in various situations.

Unless need special instruction in context, otherwise any situation of formula (I) here of relating to is all understood to include any other group of compound in formula (II) to (VIII) and formula (I).

The rise of terminology used here aurora kinase be defined as comprising the expression of aurora kinase increase or cross express (comprising that gene amplification (being polygene copy) and the expression being caused by Transcription increase) and aurora kinase activity excessively and activate (comprising the activation being caused by sudden change).

Terminology used here " saturated " refers to the ring that wherein there is no multiple bond between annular atoms.

No matter its be use separately or as a kind of compound term as the part of "-oxyl ", terminology used here " alkyl " is a kind of comprising complete be to have the aliphatic series of carbon skeleton and the generic term of alicyclic (alicyclic) group.The example of alkyl comprises alkyl, cycloalkyl, cycloalkenyl, alkenyl, alkynyl, cycloalkylalkyl, cycloalkenyl alkyl.Specific alkyl is that saturated group is as alkyl and cycloalkyl.

The example of-oxyl comprises alkoxyl group, cycloalkyloxy, cycloalkenoxy, alkenyloxy, alkynyloxy group, cycloalkyl alkoxy, cycloalkenyl alkoxyl group.Concrete-oxyl is that saturated group is as alkoxyl group.

Here prefix " C used _1-n" (wherein n is integer) refer to the number of carbon atom in given group.Therefore, C _1-4alkyl comprises 1 to 4 carbon atom, and C _1-3-oxyl comprises 1 to 3 carbon atom, and the rest may be inferred.

C _1-4the example of alkyl comprises C _1-3alkyl or C _1-2alkyl, specific examples is to be selected from C ₁, C ₂, C ₃and C ₄any single value of alkyl or the combination of these values.

Term " alkyl " comprises straight chain and branched-chain alkyl.The example of alkyl has methyl, ethyl, propyl group, sec.-propyl, n-butyl, isobutyl-and tert-butyl.

The example of cycloalkyl has these groups derived from cyclopropane, tetramethylene and pentamethylene.

The example of alkenyl has vinyl (vinyl), 1-propenyl, 2-propenyl (allyl group), pseudoallyl, butenyl and fourth-Isosorbide-5-Nitrae-dialkylene.

The example of cycloalkenyl has cyclopropenyl radical and cyclobutene base.

The example of alkynyl has ethynyl and 2-propynyl (propargyl).

The example of cycloalkylalkyl and cycloalkenyl alkyl comprises cyclopropyl methyl.

The example of alkoxyl group has methoxyl group, oxyethyl group, n-propoxy-, iso-propoxy-, n-butoxy, isobutoxy and uncle-butoxy.

In the time that alkyl forms list-alkylamino or dialkyl amido a part of, this alkyl can be any in abovementioned alkyl example.Specific alkylamino and dialkyl amido are methylamino, dimethylamino, ethylamino, diethylamino, n-propyl group amino, isopropylamino, butyl amino, isobutylamino and iso-butyl amino.Specific alkyl-and dialkyl amido be methylamino and dimethylamino.

Terminology used here " saturated heterocyclic group " refers to the heterocyclic group that does not comprise multiple bond between adjacent annulus member.Described saturated heterocyclic group can comprise 1 or 2 heteroatomic ring member who is selected from O, S and N.

Based on context, described heterocyclic group for example can comprise, cyclic ether part (for example at tetrahydrofuran (THF) with in the situation of diox), cyclic thioether part (for example, in the situation of tetramethylene sulfide and dithiane), cyclic amine part (for example, in the situation of tetramethyleneimine), cyclic amide part (for example, in the situation of pyrrolidone), cyclic thioamide, ring-type thioester, ring-type urea (for example, in the situation of imidazolidin-2-one), cyclic ester part (for example, in the situation of butyrolactone), cyclic sulfones (for example, in the situation of tetramethylene sulfone and dioxy thiophene alkene (sulpholene)), cyclic sulphoxide, ring-type sulphonamide with and combination (for example thiomorpholine).

Unless illustrate, otherwise described saturated heterocyclic group typically is monocycle and conventionally comprised 4,5 or 6 ring memberses.

The particular instance of the saturated heterocyclic group that comprises 4 ring memberses is azetidinyl.

The example of the saturated heterocyclic group that comprises 5 ring memberses comprises tetramethyleneimine (for example 1-pyrrolidyl, 2-pyrrolidyl and 3-pyrrolidyl), pyrrolidone, tetrahydrofuran (THF) and tetramethylene sulfide.

The example of the saturated heterocyclic group that comprises 6 ring memberses comprises morpholine, thiomorpholine, thiomorpholine S-oxide compound, thiomorpholine S, and S-dioxide, piperidines (for example piperidino, 2-piperidyl, 3-piperidyl and 4-piperidyl), piperidone, diox, tetrahydropyrans (for example 4-THP trtrahydropyranyl), piperazinones, piperazine and N-alkylpiperazine class are as N methyl piperazine.

d1, D2, A, E, R in (A) of formula (I) and (B) group ¹ to R ⁹ specific embodiments with X and preferred version

In a general embodiment, M is group D1.

In another general embodiment, M is group D2.

X is selected from O, NH and NCH ₃.In a specific embodiment, X is O.

A is selected from a key and group NR ², wherein R ²hydrogen or methyl.

In one embodiment, A is a key.

In another embodiment, A is group NR ², wherein R ²hydrogen or methyl.

E is selected from a key, CH ₂, CH (CN) and C (CH ₃) ₂.

In a group of compound, E is a key.

In another group of compound, E is CH ₂.

In another group of these compounds, E is CH (CN).

In another group of compound, E is C (CH ₃) ₂.

In the time that M is group D1, R ¹can be selected from group (i), (ii), (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), (xi) and (xii).

When M is that group D1 and E-A are C (CH ₃) ₂nR ²or CH ₂-NR ²time, R ¹can also be selected from:

(xiii) unsubstituted 2-furyl and 2,6-difluorophenyl.

When M is that group D1 and E-A are C (CH ₃) ₂nR ²time, R ¹can also be selected from:

(xiv) unsubstituted phenyl.

When M is that group D1 and E are CH ₂time, R ¹can also be selected from:

(xv) unsubstituted tetrahydropyran-4-base.

Represent independent embodiment of the present invention at group (i) to the each group in the catalogue of (xv).

In embodiment (i), R ¹optionally by the cycloalkyl of 3 to 5 ring memberses of hydroxyl, fluorine, amino, methylamino, methyl or ethyl replacement.

Specific cycloalkyl is optionally substituted cyclopropyl and cyclobutyl, is more typically substituted cyclopropyl optionally.In a preferred embodiment, R ¹it is unsubstituted cyclopropyl.

In embodiment (ii), R ¹to comprise 1 or 2 to be selected from O, N, S and SO ₂heteroatomic ring member's the saturated heterocyclic group of 4 to 6 ring memberses, described heterocyclic group is optionally by C _1-4alkyl, amino or hydroxyl replace; But do not comprise unsubstituted 4-morpholinyl, unsubstituted tetrahydropyran-4-base, unsubstituted 2-pyrrolidyl and piperidin-4-yl unsubstituted and that 1-replaces.

In general preferred parameter and definition, state the example of saturated heterocyclic group in the above.

The particular instance of saturated heterocyclic group comprises:

The 5 Yuans rings (except unsubstituted 2-pyrrolidyl) that comprise the single heteroatomic ring member who is selected from O, N and S;

Comprise two 6 Yuans rings (except unsubstituted 4-morpholinyl) that are selected from the heteroatomic ring member of O, N and S.

Described saturated heterocyclic group can be substituted or not be substituted.In one embodiment, it is not substituted.In another embodiment, it is by one or two C _1-4alkyl, for example one or two methyl replaces.

A kind of specific saturated heterocyclic group is substituted tetrahydrofuran base (for example tetrahydrofuran (THF)-2 base and tetrahydrofuran (THF)-3-yl) optionally, more preferably unsubstituted tetrahydrofuran (THF) group.

In embodiment (iii), R ¹2 of following formula, the phenyl that 5-replaces:

Wherein (a) when X be NH or N-CH ₃time, R ³be selected from chlorine and cyano group; (b) in the time that X is O, R ³cN.

In a group of the compound in embodiment (iii), X is N-CH ₃and R ³be selected from chlorine and cyano group.

In another group of compound in embodiment (iii), X is O and R ³cN.

In embodiment (iv), R ¹group CR ⁶r ⁷r ⁸, wherein R ⁶and R ⁷be selected from separately hydrogen and methyl, and R ⁸be selected from hydrogen, methyl, C _1-4alkyl sulfonyl ylmethyl, hydroxymethyl and cyano group.

In embodiment (iv), R ¹particular instance be methyl, cyano methyl, HOCH ₂c (CH ₃) ₂-and 2-methyl sulphonyl ethyl.

In embodiment (iv), R ¹another particular instance be methyl and sec.-propyl.

In embodiment (v), R ¹it is the pyridazine-4-base that is optionally selected from the substituting group replacement of methyl, ethyl, methoxyl group and oxyethyl group by one or two.This pyridazinyl can be pyridazine-3-base or pyridazine-4-base, but pyridazine-4-base typically.Specific substituting group be methoxyl group and, for example, this pyridazinyl can carry two methoxy substitution bases.

In embodiment (vi), R ¹be substituted Imidazothiazole base, wherein said substituting group is selected from methyl, ethyl, amino, fluorine, chlorine, amino and methylamino.A kind of specific substituting group is methyl.

In embodiment (vii), R ¹optionally substituted 1,3-dihydro-isoindole-2-base or optionally substituted 2,3-dihydro-indoles-1-base, is wherein selected from halogen, cyano group, amino, C at the optional substituting group described in various situations _1-4single-and dialkyl amido, CONH ₂or CONH-C _1-4alkyl C _1-4alkyl and C _1-4alkoxyl group, wherein said C _{1 -4}alkyl and C _1-4alkoxyl group is optionally by hydroxyl, methoxyl group or amino replacement.

Specific substituting group is selected from methyl, ethyl, fluorine, chlorine (being preferably only positioned on the aromatic ring of this indoline or xylylenimine), CONH ₂, amino, methylamino, dimethylamino and methoxyl group.

In a group of the compound in embodiment (vii), its xylylenimine or indoline are not substituted separately.

In embodiment (viii), R ¹optionally to be selected from hydroxyl, halogen, cyano group, amino, C by one or two _1-4single-and dialkyl amido, CONH ₂or CONH-C _1-4alkyl, C _1-4alkyl and C _1-4the 3-pyridyl that the substituting group of alkoxyl group replaces, wherein said C _{1 -4}alkyl and C _1-4alkoxyl group is optionally by hydroxyl, methoxyl group or amino replacement; Prerequisite is that it can not form compound 2,6-dimethoxy-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-niacinamide or compound 2-oxo-1,2-dihydro-Nicotinicum Acidum [3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-acid amides.

In one embodiment, R ¹optionally to be selected from hydroxyl, halogen, cyano group, amino, C by one or two _1-4single-and dialkyl amido, CONH ₂or CONH-C _1-4alkyl, C _1-4alkyl and C _1-4the 3-pyridyl that the substituting group of alkoxyl group replaces, wherein said C _{1 -4}alkyl and C _1-4alkoxyl group is optionally replaced by hydroxyl, methoxyl group or amino, still, and wherein R ¹be 3-pyridyl, X is O, and A is that a key and E are keys, and its pyridyl has one or two and is selected from halogen, cyano group, amino, C _1-4single-and dialkyl amido, CONH ₂or CONH-C _1-4alkyl, C _1-4alkyl and C _2-4the substituting group of alkoxyl group, wherein said C _1-4alkyl and C _1-4alkoxyl group is optionally by hydroxyl, methoxyl group or amino replacement.

Specific substituting group is selected from methyl, ethyl, fluorine, chlorine, CONH ₂, amino, methylamino, dimethylamino and methoxyl group.Other specific substituting groups are selected from methyl, ethyl, fluorine, chlorine, CONH ₂, amino, methylamino and dimethylamino.

In a group of these compounds, its 3-pyridyl is not substituted.

In embodiment (ix), R ¹optionally to be selected from halogen, cyano group, amino, C by one or two _1-4single-and dialkyl amido, CONH ₂or CONH-C _1-4alkyl C _1-4alkyl and C _1-4thiomorpholine or its S-oxide compound or S that the substituting group of alkoxyl group replaces, S-dioxide, wherein said C _1-4alkyl and C _1-4alkoxyl group is optionally by hydroxyl, methoxyl group or amino replacement.

In a group of these compounds, its thiomorpholine or its S-oxide compound or S, S-dioxide is not substituted.

In embodiment (x), E-A is NR ²and R ¹be selected from: 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,4-difluorophenyl, 3,4-difluorophenyl, 2,5-difluorophenyl, 3,5-difluorophenyl, 2,4,6-trifluorophenyl, 2-p-methoxy-phenyl, the chloro-2-p-methoxy-phenyl of 5-, cyclohexyl, unsubstituted 4-THP trtrahydropyranyl and tert-butyl.

In embodiment (xi), E-A is NR ²and R ¹group NR ¹⁰r ¹¹, wherein R ¹⁰and R ¹¹respectively C naturally _1-4alkyl or R ¹⁰and R ¹¹thereby be connected NR ¹⁰r ¹¹forming one optionally comprises and is selected from O, N, S and SO ₂second heteroatomic ring member's the saturated heterocyclic group of 4 to 6 ring memberses, described heterocyclic group is optionally by C _1-4alkyl, amino or hydroxyl replace.

In this embodiment, a group of these compounds is R wherein ¹⁰and R ¹¹respectively C naturally _1-4the compound group of alkyl, particularly methyl.

Another group of these compounds is R wherein ¹⁰and R ¹¹thereby be connected NR ¹⁰r ¹¹forming one optionally comprises and is selected from O, N, S and SO ₂second heteroatomic ring member's the saturated heterocyclic group of 4 to 6 ring memberses, described heterocyclic group is optionally by C _1-4the compound group that alkyl, amino or hydroxyl replace.Its saturated heterocyclic group can be any in listed nitrogenous saturated heterocyclic group in general preferred parameter and definition in the above, but specific saturated heterocyclic group comprises pyrrolidyl, morpholinyl, piperazinyl and N-C _1-4alkyl-piperazinyl.Such group is not typically substituted or by one or two methyl substituted, and in a specific embodiment, it is not substituted.

In embodiment (xii), E-A is NR ²and R ¹optionally to be selected from hydroxyl, halogen, cyano group, amino, C by one or two _1-4single-and dialkyl amido, CONH ₂, CONH-C _1-4alkyl, C _1-4alkyl and C _1-4the pyriconyl that the substituting group of alkoxyl group replaces, wherein said C _1-4alkyl and C _1-4alkoxyl group is optionally by hydroxyl, methoxyl group or amino replacement.

Described pyridone can be replaced by N-, for example by alkyl as methyl substituted, and can be unsubstituted equally.

In embodiment (xiii), E-A is C (CH ₃) ₂nR ²or CH ₂-NR ²and R ¹be selected from unsubstituted 2-furyl and 2,6-difluorophenyl.

In embodiment (xiv), E-A is C (CH ₃) ₂nR ²and R ¹it is unsubstituted phenyl.

In embodiment (xv), E is CH ₂and R ¹it is unsubstituted tetrahydropyran-4-base.

In the time that M is group D2, R ¹can be selected from group (xvi), (xvii), (xviii) and (xix).

Group (xvi) to the each group in the catalogue of (xix) has represented the embodiment that the present invention is independent.

In embodiment (xvi), R ¹the 3-furyl of the 2-replacement of following formula:

Wherein R ⁴and R ⁵identical or different and be selected from hydrogen and C _1-4alkyl or R ⁴and R ⁵thereby be connected NR ⁴r ⁵formation one optionally comprises second and is selected from O, NH, NMe, S or SO ₂heteroatoms or 5-or 6-person's saturated heterocyclic group of group, this 5-or 6-person's saturated rings are optionally replaced by hydroxyl, fluorine, amino, methylamino, methyl or ethyl.In one embodiment, R ¹the 3-furyl of the 2-replacement of following formula:

Wherein R ⁴and R ⁵identical or different and be selected from hydrogen and C _1-4alkyl or R ⁴and R ⁵thereby be connected NR ⁴r ⁵formation one optionally comprises second and is selected from O, NH, NMe, S or SO ₂heteroatoms or 5-or 6-person's saturated heterocyclic group of group, this 5-or 6-person's saturated rings are optionally replaced by hydroxyl, fluorine, amino, methylamino, methyl or ethyl, still, wherein A is that a key and E are keys, R ⁴and R ⁵be not connected and make NR ⁴r ⁵form a kind of unsubstituted piperidines.

Superincumbent general preferred parameter and definitional part are stated specific saturated heterocyclic group, but specific saturated heterocyclic group comprises pyrrolidyl, morpholinyl, piperazinyl and N-C _1-4alkyl-piperazinyl.Such group is not typically substituted or is replaced by one or two methyl, and in a specific embodiment, it is not substituted.

Wherein R ⁴and R ⁵be selected from hydrogen and C _1-4the particular instance of the compound of alkyl is methylamino and dimethylamino, is more typically dimethylamino.

In embodiment (xvii), R ¹the 2-furyl of the 5-replacement of following formula:

Wherein R ⁴and R ⁵identical or different and be selected from hydrogen and C _1-4alkyl or R ⁴and R ⁵thereby be connected NR ⁴r ⁵formation one optionally comprises second and is selected from O, NH, NMe, S or SO ₂heteroatoms or 5-or 6-person's saturated heterocyclic group of group, described 5-or 6-person's saturated heterocyclic group are optionally replaced by hydroxyl, fluorine, amino, methylamino, methyl or ethyl; Prerequisite is that described compound does not form 5-piperidin-1-yl methyl-furans-2-formic acid [3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-acid amides.

In general preferred parameter and definitional part, specific saturated heterocyclic group is stated in the above, but specific saturated heterocyclic group comprises pyrrolidyl, morpholinyl, piperazinyl and N-C _1-4alkyl-piperazinyl.Such group is not typically substituted or is replaced by one or two methyl, and in a specific embodiment, it is not substituted.

In embodiment (xviii), R ¹the group of following formula:

Wherein R ⁹hydrogen, methyl, ethyl or sec.-propyl; G is CH, O, S, SO, SO ₂or NH and this group optionally by one, two or three are selected from C _1-4alkyl, hydroxyl, C _{1- 4}-oxyl, fluorine, amino, list-and two-C _1-4the substituting group of alkylamino replace and, wherein said C _1-4alkyl and C _1-4-oxyl is separately optionally by hydroxyl, fluorine, amino, list-or two-C _1-4alkylamino replaces.

In a group of the compound in embodiment (xix), G is selected from O and CH.

In embodiment (xviii), radicals R ¹typically be not substituted or replaced by one or two methyl, and not more typically being substituted.

In embodiment (xix), R ¹3 of following formula, the dibasic phenyl of 5-:

Wherein X ^abe selected from O, NH and NCH the same as X ₃.

X ^apreferably N-CH ₃.

Part R ¹the particular instance of-A-is as shown in table 1, and asterisk represents and radicals R ¹the tie point of carbonyl C=O in-E-A-C (=O)-NH-.

In table 1, preferred radicals R ¹-E-A-comprises A1, A4, A10, A11, A13, A20, A22, A23, A24, A29, A30, A31, A32, A38, A42, A43, A44, A46, A47, A49, A54 and A56.

In another embodiment, radicals R ¹-E-A is A57, A58 or A59.

Radicals R ¹a kind of preferred subset of-E-A-comprises A1, A4, A20, A24, A30, A44, A46 and A54.In its this subset, a kind of specific radicals R ¹-A-is group A24.

A group of the compounds of this invention is suc as formula shown in (II):

Wherein R ¹, E, A and X definition as described herein.

In formula (II), a subset of these compounds is that wherein X is the subset of O.

A subset of formula (II) compound can use formula (III) with and salt, particularly lactic acid salt represents:

In formula (III), a subset of these compounds is that wherein E is the subset of a key.

Another subset of formula (III) compound is that wherein E is CH ₂or C (CH ₃) ₂subset.

In particularly preferred embodiment of formula (III), E is a key, R ²h and R ¹it is defined cycloalkyl (i) here.In one embodiment, described cycloalkyl can be cyclopropyl or cyclobutyl.R ¹more preferably cyclopropyl.

For fear of doubt, should be understood that, unless in context, be illustrated, otherwise can be by radicals R ¹each general and concrete preferred parameter, embodiment and embodiment and radicals R ²and/or R ³and/or R ⁴and/or R ⁵and/or R ⁶and/or R ⁷and/or R ⁸and/or R ⁹and/or R ¹⁰and/or R ¹¹and/or D1 and/or D2 and/or A and/or E and/or X and/or X ^aand each general and concrete preferred parameter, embodiment and the embodiment of defined its any each group combine here, and all such combinations are all included in this application.

The various functional groups of common selected constitutional formula (I) compound and substituting group make the molecular weight of formula (I) compound be no more than 1000.More generally, the molecular weight of compound is lower than 750, for example, lower than 700, or lower than 650, or lower than 600, or lower than 550.Its molecular weight more preferably lower than 525 and, be for example 500 or lower.

In the following embodiments specific compound of the present invention is illustrated.

A kind of preferred compound of the present invention is 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea with and salt, solvate and tautomer.

(A) of formula (I) and (B) group with and the salt of the compound of group and embodiment, solvate, tautomer, isomer, N-oxide compound, ester, prodrug and isotropic substance

Unless stated otherwise, otherwise in the time relating to specific compound, also comprise its ion, salt, solvate and protected form, for example these materials as discussed below.

The compound of many formulas (I) can be with salt, and the form of for example acid salt exists, or, can be inorganic in some cases and salt organic bases, as carboxylate salt, sulfonate and phosphoric acid salt.All such salt all in scope of the present invention, and comprises the form of these compound salt in the time relating to the compound of formula (I).Described in the part before the application, unless in context, be illustrated, otherwise all quoting of formula (I) also thought and related to formula (II), (III) and defined its group here.

Salt of the present invention can be by conventional chemical process, as in pharmaceutical salts: character, select, and application (Pharmaceutical Salts:Properties, Selection, and Use), P.Heinrich Stahl (Editor), Camille G.Wermuth (Editor), ISBN:3-90639-026-8, Hardcover, the 388th page, the method described in August, 2002 is synthesized by the parent compound that comprises alkalescence or acidic moiety.Such salt normally can be by reacting these compounds of free acid or alkali form to be prepared with suitable alkali or acid at water or in organic solvent or in the mixture of the two; Conventionally use non-aqueous media as ether, ethyl acetate, ethanol, Virahol or acetonitrile.

Can form acid salt with the various acid of wide region, described acid can be both mineral acid, can be again organic acid.The example of acid salt comprises and is selected from acetic acid, 2,2-dichloro acetic acid, hexanodioic acid, alginic acid, xitix (for example L-AA), ASPARTIC ACID, Phenylsulfonic acid, phenylformic acid, 4-acetylamino benzoic acid, butyric acid, (+) dextrocamphoric acid, camphor-sulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, sad, styracin, citric acid, cyclohexane sulfamic acid, dodecyl sulphate, ethane-1,2-disulfonic acid, ethyl sulfonic acid, 2-ethylenehydrinsulfonic acid, formic acid, fumaric acid, tetrahydroxyadipic acid, gentisinic acid, glucoheptonic acid, D-glyconic acid, glucuronic acid (for example D-Glucose aldehydic acid), L-glutamic acid (for example Pidolidone), α ketoglutaric acid, oxyacetic acid, urobenzoic acid, Hydrogen bromide, hydrochloric acid, hydroiodic acid HI, hydroxyethylsulfonic acid, (+)-Pfansteihl, (±)-DL-LACTIC ACID, lactobionic acid, toxilic acid, oxysuccinic acid, (-)-L MALIC ACID, propanedioic acid, (±)-DL-amygdalic acid, methylsulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, vitamin B13, oxalic acid, palmitinic acid, flutter acid, phosphoric acid, propionic acid, L-Glutimic acid, Whitfield's ointment, 4-amino-Whitfield's ointment, sebacic acid, stearic acid, succsinic acid, sulfuric acid, tannic acid, (+)-L-TARTARIC ACID, thiocyanic acid, p-toluenesulphonic acids, undecylenic acid and valeric acid, and the salt of the acid of acylated amino and Zeo-karb formation.

This acid salt can also be selected from aspartic acid (for example D-ASP), carbonic acid, dodecanoate, the salt of isopropylformic acid, lauryl sulfonic acid, glactaric acid, naphthene sulfonic acid (for example naphthalene-2-sulfonic acid), toluenesulphonic acids (for example p-toluenesulphonic acids) and xinafoic acids.

The salt that one group of specific salt is formed by hydrochloric acid, hydroiodic acid HI, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succsinic acid, toxilic acid, oxysuccinic acid, hydroxyethylsulfonic acid, fumaric acid, Phenylsulfonic acid, toluenesulphonic acids, methylsulfonic acid, ethyl sulfonic acid, naphthene sulfonic acid, valeric acid, acetic acid, propionic acid, butyric acid, propanedioic acid, glucuronic acid and lactobionic acid is formed.

The salt that a group of these salt is formed by hydrochloric acid, acetic acid, hexanodioic acid, ASPARTIC ACID and DL-LACTIC ACID is formed.

Another group of these salt is made up of acetate, mesylate, esilate, DL-LACTIC ACID salt, adipate, D-Glucose aldehydic acid salt, D-Glucose hydrochlorate and hydrochloride.

Salt is as many in acid salt has advantages of is better than corresponding free alkali.For example, these salt have advantages of the free alkali that is better than below one or more, its:

More easily molten, therefore for example, better for i.v. administration (administration of being undertaken by input) and will there is the pharmacokinetics of improvement;

There is better stability (for example shelf lives improves);

There is better thermostability;

Alkalescence is lower and therefore better for i.v. administration;

There is preparation aspect;

There is the metabolisming property of improvement; With

Between patient, show lower clinical difference.

For the preparation of defined formula (I) here, (II), (III), (XXX) with and the preferred salt of liquid (for example moisture) composition of the compound of group and embodiment be such salt, it for example for example, has, higher than 25mg/ml liquid vehicle (water) in given liquid vehicle (water), more typically higher than 50mg/ml and preferably higher than the solubleness of 100mg/ml.

In another embodiment, for the preparation of formula as described herein (I), (II), (III), (XXX) with and the preferred salt of liquid (for example moisture) composition of the compound of group and embodiment be for example, to have higher than 1mg/ml in given liquid vehicle (water or Laemmli buffer system Laemmli), typically for example, higher than 5mg/ml liquid vehicle (water), more typically higher than 15mg/ml, more typically higher than 20mg/ml and preferably higher than the salt of the solubleness of 25mg/ml.

In another embodiment, preferred acid salt is mesylate, esilate, D-or Pfansteihl salt and hydrochloride.In a specific embodiment, said acid salt is lactic acid salt, particularly Pfansteihl salt or D-lactate, preferably Pfansteihl salt.

In one embodiment of the invention, provide one to contain with higher than 25mg/ml, typically higher than 50mg/ml and the formula as described herein (I) that preferably comprises salt form higher than the concentration of 100mg/ml, (II), (III), (XXX) with and the pharmaceutical composition of the aqueous solution of the compound of group and embodiment.

In another embodiment of the invention, provide one to contain with higher than 1mg/ml, typically for example, higher than 5mg/ml liquid vehicle (water), more typically higher than 15mg/ml, more typically higher than 20mg/ml and the formula as described herein (I) that preferably comprises salt form higher than the concentration of 25mg/ml, (II), (III), (XXX) with and the pharmaceutical composition of the aqueous solution of the compound of group and embodiment.

If described compound is negatively charged ion, or have can for the functional group of negatively charged ion, (for example ,-COOH can be-COO ^-), its can with suitable salt forming cation.The example of suitable inorganic cation comprises that alkalimetal ion is as Na without limitation ⁺and K ⁺, alkaline earth metal cation is as Ca ²⁺and Mg ²⁺, and other positively charged ion is as Al ³⁺.Suitable organic cations example comprises that ammonium ion (is NH without limitation ₄ ⁺) and substituted ammonium ion (for example NH ₃r ⁺, NH ₂r ₂ ⁺, NHR ₃ ⁺, NR ₄ ⁺).The example of some suitable substituted ammonium ions is these derived from ethamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, quadrol, thanomin, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethane and amino acid, as Methionin and arginic ion.An example of common quaternary ammonium ion is N (CH ₃) ₄ ⁺.

In the situation that comprises amine functionality at the compound of formula (I), it can form quaternary ammonium salt, for example can be according to the method that well known to a person skilled in the art by react to form quaternary ammonium salt with alkylating agent.Such quaternary ammonium compound is in the scope of formula (I).

The salt form of the compounds of this invention is pharmaceutically useful salt typically, and people such as Berge, 1977, " pharmaceutically useful salt (Pharmaceutically Acceptable Salts) ", J.Pharm.Sci., the 66th volume, the example to pharmacologically acceptable salt in 1-19 page is discussed.But pharmaceutically unacceptable salt also can be prepared as intermediate forms, then, can convert it into pharmaceutically useful salt.Can be used for the purifying of for example the compounds of this invention or such pharmaceutically unacceptable salt form of separation has also formed a part of the present invention.

The compound of the formula (I) that comprises amine functional group also can form N-oxide compound.While relating to the compound of the formula (I) that comprises amine functional group here, also comprise its N-oxide compound.

In the situation that comprises some amine Guan Nengtuan at compound, thereby one or more nitrogen-atoms can oxidized formation N-oxide compound.The particular instance of N-oxide compound is the N-oxide compound of tertiary amine or nitrogenous heterocyclic nitrogen-atoms.

Can be by for example, corresponding amine being processed to form N-oxide compound as hydrogen peroxide or peracid (peroxycarboxylic acid (peroxycarboxylic acid)) with oxygenant, see for example senior organic chemistry (Advanced Organic Chemistry), Jerry March, the 4th edition, Wiley Interscience, pages.More specifically, can use the method for L.W.Deady (Syn.Comm.1977,7,509-514) to prepare N-oxide compound, wherein this amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, react in as methylene dichloride at inert solvent.

The compound of formula (I) comprises all such forms can exist and relate to the compound of formula (I) with many different rotamerisms and tautomeric form time.For fear of query, wherein compound can exist and only specifically describing or represent in a kind of situation of form, formula (I) still comprises all other forms with a kind of form in some rotamerisms or tautomeric form.

For example, in the compound of formula (I), its benzoglyoxaline group can exist with any form in two kinds of tautomeric form A, A ', B and B ' below.For simply, general formula (I) has only been set forth A and A ' form, but this general formula should be counted as comprising whole four kinds of tautomeric forms.

Described pyrazole ring also can show tautomerism and can be so that two kinds of tautomeric form C and D exist below.

Other example of tautomeric form comprise for example ketone-, enol-and enolate (enolate)-form, for example it can be tautomerism pair below: ketone/enol (illustrated below), imines/enamine, acid amides/imino-alcohol, amidine/amidine, nitroso-group/oxime, thioketones/enethiol and nitro/aci-nitro.

Ketone enol enolate

The compound of formula (I) comprise one or more chiral centres and the situation that can exist with the form of two or more optical isomers in, unless illustrated in context, otherwise in the time relating to the compound of formula (I), comprise its all optical siomerism forms (for example enantiomer, epimer and diastereomer), these forms can be each optical isomer or mixture (for example racemic mixture) or two or more optical isomer forms.

For example, group A can comprise one or more chiral centres.Therefore, as E and R ¹while being all connected in the identical carbon atoms being positioned on this connector group A, said carbon atom normally chirality and therefore the compound of formula (I) will exist with a pair of enantiomeric forms (or existing in described compound in the situation of more than one chiral centre with more than one pair of enantiomeric forms).

Can with its optical activity to described optical isomer carry out characterization and discriminating (be expressed as by it+and-isomer, or d and l isomer) or can use Cahn, " R and S " nomenclature that Ingold and Prelog determine characterizes it according to its absolute stereo chemistry, see Advanced Organic Chemistry (Advanced Organic Chemistry), Jerry March, the 4th edition, John Wiley & Sons, New York, 1992, 109-114 page, and can be referring to Cahn, Ingold & Prelog, Angew.Chem.Int.Ed.Engl., 1966, 5, 385-415.

Can be with comprise that chiral chromatography (chromatography of carrying out) separates these optical isomers in interior many technology on chirality upholder, and such technology is well-known to those skilled in the art.

As the alternative selection of a kind of confession of chiral chromatography; these optical isomers can be by using chiral acid as (+)-tartrate, (-)-Pyrrolidonecarboxylic acid, (-)-bis--toluyl (toluloyl)-L-TARTARIC ACID, (+)-amygdalic acid, (-)-oxysuccinic acid and (-)-camphorsulfonic acid formation diastereo-isomerism salt; preferably with crystallization, this diastereomer is separated; then make this salt dissociate, thereby the enantiomer that obtains each free alkali separate.

In situation about existing with two or more optical isomer forms at the compound of formula (I), a kind of enantiomer in a pair of enantiomer may show the advantage that is better than another kind of enantiomer, for example, aspect biological activity, be better than another kind of enantiomer.Therefore, in certain embodiments, may wish only to use one in a pair of enantiomer as therapeutical agent, or only use one in many diastereomers as therapeutical agent.Therefore, the invention provides the composition that contains formula (I) compound with one or more chiral centres, wherein at least 55% the compound of (for example at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) formula (I) for example, exists with the form of single optical isomer (enantiomer or diastereomer).In a general embodiment, 99% or more (for example substantially all) of the total amount of compound of formula (I) can for example, exist with the form of single optical isomer (enantiomer or diastereomer).

Compound of the present invention comprises having the compound that one or more isotropic substances (isotopic) replace, and in the time relating to specific element, comprises all isotropic substances of this element in its scope.For example, in the time relating to hydrogen, comprise in its scope ¹h, ²h (D) and ³h (T).Equally, in the time relating to carbon and oxygen, in its scope, comprise respectively ¹²c, ¹³c and ¹⁴c and ¹⁶o and ¹⁸o.

Described isotropic substance can be to have radioactivity or cold.In one embodiment of the invention, described compound does not comprise radio isotope.Preferably this compounds for the treatment of application.But in another embodiment, described compound can comprise one or more radio isotope.Comprising such radioisotopic compound can be used in diagnostic environment.

Formula (I) also comprises that the ester of formula (I) compound that carries hydroxy-acid group or hydroxyl is as carboxylicesters and acyloxyate.The example of ester has the compound that comprises group-C (=O) OR, and wherein R is ester substituting group, for example C _1-7alkyl, C _3-20heterocyclic radical or C _5-20aryl, preferably C _1-7alkyl.Comprise without limitation-C of the particular instance of ester group (=O) OCH ₃,-C (=O) OCH ₂cH ₃,-C (=O) OC (CH ₃) ₃, and-C (=O) OPh.The example of acyloxy (oppositely ester (reverse ester)) can represent with-OC (=O) R, and wherein R is acyloxy substituting group, for example, and C _1-7alkyl, C _3-20heterocyclic radical or C _5-20aryl, preferably C _1-7alkyl.Comprise without limitation-OC of the particular instance of acyloxy (=O) CH ₃(acetoxyl group) ,-OC (=O) CH ₂cH ₃,-OC (=O) C (CH ₃) ₃,-OC (=O) Ph and-OC (=O) CH ₂ph.

Formula (I) also comprises the solvate (for example hydrate), complex body (the inclusion complex body or the inclusion compound that for example form as cyclodextrin with compound, or the complex body forming with metal) of any polymorphic forms of described compound, described compound and the prodrug of described compound." prodrug " refers to any compound that for example can be converted in vivo the compound of bioactive formula (I).

For example, some prodrugs are esters (for example, the unsettled ester of the acceptable metabolic of physiology) of active compound.Between metabilic stage, (C (=O) OR) is cleaved for this ester group, thereby produces described active medicine.Can be by esterification; for example in parent compound, the esterification of any carboxyl (C (=O) OH) forms such ester; wherein in suitable situation; can formerly protect any other reactive group in parent compound, then be gone if necessary protection.

The example of the unstable ester of such metabolic comprises those of following formula:

C(＝O)OR

Wherein R is:

C _1-7alkyl;

(for example ,-Me ,-Et ,-nPr ,-iPr ,-nBu ,-sBu ,-iBu ,-tBu);

C _1-7aminoalkyl group;

(for example, amino-ethyl; 2-(N, N-diethylamino) ethyl; 2-(4-morpholino) ethyl); With

Acyloxy-C _1-7alkyl;

(for example, acyloxy methyl;

Acyloxy ethyl;

Oxy acid methyl neopentyl;

Acetoxy-methyl;

1-acetoxyl group ethyl;

1-(1-methoxyl group-1-methyl) ethyl-carbonyl oxygen base (carbonxyloxy) ethyl;

1-(benzoyloxy) ethyl; Isopropoxy-carbonyl oxy-methyl;

1-isopropoxy-carbonyl oxygen base ethyl; Cyclohexyl-carbonyl oxy-methyl;

1-cyclohexyl-carbonyl oxygen base ethyl;

Cyclohexyloxy-carbonyl oxy-methyl;

1-cyclohexyloxy-carbonyl oxygen base ethyl;

(4-tetrahydro-pyran oxy) carbonyl oxy-methyl;

1-(4-tetrahydro-pyran oxy) carbonyl oxygen base ethyl;

(4-THP trtrahydropyranyl) carbonyl oxy-methyl; With

1-(4-THP trtrahydropyranyl) carbonyl oxygen base ethyl).

Some prodrugs also can be produced by enzyme activation described active compound, or in the time carrying out further chemical reaction, can obtain the compound (for example, as at ADEPT, GDEPT, in the situation of LIDEPT etc.) of described active compound.For example, described prodrug can be sugar derivatives or other glucosides conjugates, or can be amino acid ester derivative.

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl]-urea with and salt

It is a kind of that specifically formula (I)---the compound of group (A) is 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea.

Therefore, in a preferred embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] free alkali or the acid salt of-urea.

Can be by its derivative 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl that obtains described salt] free alkali of-urea has formula (XXX):

Can mention with its chemical name in this application the compound of formula (XXX)---1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea, or, for convenient, it can be called as the compound of " compounds X XX ", " compound of formula (XXX) " or embodiment 24.In these synonyms, refer to separately the compound shown in formula (XXX) above and there is 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] chemical name of-urea.

Relating to compound 1-cyclopropyl base-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea free alkali with and when acid salt, its scope comprise its all solvate, tautomer and isotropic substance and situation about allowing at context in N-oxide compound, other ionic species and prodrug.Therefore, in the time relating to the selective tautomer of formula (XXX), 1-cyclopropyl-3-[3-(6-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea is understood to refer to compound (XXX).

The acid salt of formula (XXX) can be selected from the salt forming with the various acid of wide region, and said acid not only comprises mineral acid but also comprise organic acid.The example of acid salt comprises and is selected from acetic acid, 2,2-dichloro acetic acid, hexanodioic acid, alginic acid, xitix (for example L-AA), aspartic acid (for example ASPARTIC ACID), Phenylsulfonic acid, phenylformic acid, 4-acetylamino benzoic acid, butyric acid, (+) dextrocamphoric acid (for example (+) dextrocamphoric acid), camphor-sulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, sad, carbonic acid, styracin, citric acid, cyclohexane sulfamic acid, dodecanoate, dodecyl sulphate, ethane-1,2-disulfonic acid, ethyl sulfonic acid, 2-ethylenehydrinsulfonic acid, formic acid, fumaric acid, tetrahydroxyadipic acid, gentisinic acid, glucoheptonic acid, D-glyconic acid, glucuronic acid (for example D-Glucose aldehydic acid), L-glutamic acid (for example Pidolidone), α ketoglutaric acid, oxyacetic acid, urobenzoic acid, Hydrogen bromide, hydrochloric acid, hydroiodic acid HI, hydroxyethylsulfonic acid, isopropylformic acid, lactic acid (for example (+)-Pfansteihl and, (-)-D-ALPHA-Hydroxypropionic acid), lactobionic acid, lauryl sulfonic acid, toxilic acid, oxysuccinic acid, (-)-L MALIC ACID, propanedioic acid, (±)-DL-amygdalic acid, methylsulfonic acid, glactaric acid, naphthene sulfonic acid (for example naphthalene-2-sulfonic acid), naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, vitamin B13, oxalic acid, palmitinic acid, flutter acid, phosphoric acid, propionic acid, L-Glutimic acid, Whitfield's ointment, 4-amino-Whitfield's ointment, sebacic acid, stearic acid, succsinic acid, sulfuric acid, tannic acid, tartrate (for example (+)-L-TARTARIC ACID), thiocyanic acid, toluenesulphonic acids (for example p-toluenesulphonic acids), undecylenic acid and valeric acid and xinafoic acids, and the salt of the acid of acylated amino and Zeo-karb formation.

The salt that one group of specific salt is formed by the acid that is selected from hydrochloric acid, hydroiodic acid HI, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succsinic acid, toxilic acid, oxysuccinic acid, hydroxyethylsulfonic acid, fumaric acid, Phenylsulfonic acid, toluenesulphonic acids, methylsulfonic acid, ethyl sulfonic acid, naphthene sulfonic acid, valeric acid, acetic acid, propionic acid, butyric acid, propanedioic acid, glucuronic acid and lactobionic acid is formed.

The salt that a group in these salt is formed by the acid that is selected from hydrochloric acid, acetic acid, hexanodioic acid, ASPARTIC ACID and D-or Pfansteihl is formed.

Another group of these salt is made up of acetate, mesylate, esilate, D-or Pfansteihl salt, adipate, D-Glucose aldehydic acid salt, maltonic acid and hydrochloride.In another embodiment, preferred acid salt is mesylate, esilate, D-or Pfansteihl salt and hydrochloride.

In a specific embodiment, said acid salt is DL-LACTIC ACID salt, particularly Pfansteihl salt or D-lactate, preferably Pfansteihl salt.

In another embodiment, the free alkali of formula (XXX) compound or salt are selected from Pfansteihl salt, free alkali dehydrate, esilate, free alkali and hydrochloride.

In another and preferred embodiment, the salt of formula (XXX) compound be selected from lactic acid salt and Citrate trianion with and composition thereof, and be more preferably selected from Pfansteihl salt and Citrate trianion with and composition thereof, particularly preferably Pfansteihl salt.State in more detail below and described and relate to 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the Pfansteihl salt of-urea and the particularly preferred embodiment of the present invention of Citrate trianion.

In another embodiment, the compound of formula (XXX) is free alkali.

Salt of the present invention, for example, as lactic acid salt (Pfansteihl salt) and Citrate trianion can be by conventional chemical method by parent compound 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yls]-urea synthesizes, said method is as in pharmaceutical salts: character, select, and application (Pharmaceutical Salts:Properties, Selection, and Use), P.Heinrich Stahl (Editor), Camille G.Wermuth (Editor), ISBN:3-90639-026-8, Hardcover, the 388th page, method described in August, 2002.Such salt conventionally can be by by parent compound 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea reacts to be prepared in water or in organic solvent or the mixture of the two with suitable acid; Conventionally use the medium of non-water as ether, ethyl acetate, ethanol, Virahol or acetonitrile.

On the other hand, the invention provides one and prepare 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] acid salt of-urea, for example, as the method for lactic acid salt (Pfansteihl salt) and Citrate trianion, the method comprises formation a kind of 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] solution of-urea free alkali in solvent (typically being organic solvent) or solvent mixture, thus with sour, this solution is processed to the precipitation that forms described acid salt.

Described acid can be added into be arranged in the form that is dissolved in the solution of the mixable solvent of solvent wherein with described free alkali.It can be a kind of its acid salt insoluble solvent therein that free alkali is dissolved in solvent wherein at first.Or, the solvent that free alkali is dissolved in wherein at first can be that a kind of described acid salt at least can be partially dissolved in solvent wherein, subsequently, add wherein the described acid salt lower different solvents of solvability therein, thereby described salt is precipitated out from this solution.

Form acid salt at another kind, for example, in the method for lactic acid salt (Pfansteihl salt) and Citrate trianion, by 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea is dissolved in and comprises volatile acid optionally comprising in the solvent of cosolvent, thereby form a kind of acid salt solution with volatile acid, then, the solution of gained concentrated or evaporate to isolate described salt.Another example of the acid salt that can prepare in this way is acetate.

On the other hand, the invention provides a kind of defined 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl here that forms] acid salt of-urea, for example, as the method for lactic acid salt (Pfansteihl salt) and Citrate trianion, the method comprises to be processed the compound of formula (XXX) with defined organic acid here or mineral acid in organic solvent:

Thereby obtain 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] acid salt of-urea and organic acid or mineral acid, and optionally the acid salt forming is thus separated.

In the time forming, described salt can be precipitated out conventionally from described organic solvent, therefore can by isolate solid from solution, for example, separate to be separated by filtration.

Can a kind of salt form of the present invention be changed into free alkali by method well known to the skilled person and optionally convert it into the form of another kind of salt.For example, can be by making this salts solution for example, by a post that comprises amine stationary phase (a Strata-NH ₂post) form free alkali.Or, can with sodium bicarbonate to described salt the solution in water process, thereby this salt is decomposed and is settled out free alkali.Then, can make free alkali and another kind of sour combination by one of other local described method or here above.

Preferred salt for example, has many advantages as for example lactic acid salt of acid salt (Pfansteihl salt) and Citrate trianion.For example, described salt will have advantages of below one or more, its:

More easily molten, particularly therefore its solubleness in the aqueous solution be improved and

For example, for i.v. administration (administration of being undertaken by input), be more suitable for;

Make to control and therefore for i.v., administration is more suitable for the pH of solution;

Antitumour activity may be improved; With

Therapeutic index may be improved.

Other advantage of these salt is it:

There is better stability, for example thermostability (for example shelf lives is improved);

There is preparation aspect; With

There are better physicochemical property.

Because have good solubleness and have better solubleness in water in buffering system, therefore lactic acid salt of the present invention (for example Pfansteihl salt) is advantageous particularly.

For example, be for example, to have higher than 1mg/ml in given liquid vehicle (water) for the preparation of the preferred salt of liquid (moisture) pharmaceutical composition, typically for example, higher than 5mg/ml liquid vehicle (water), more typically higher than 15mg/ml, more typically higher than 20mg/ml and preferably higher than the acid salt (as lactic acid salt) of the solubleness of 25mg/ml.

The aqueous solution (for example pharmaceutical dosage forms) of this salt has represented another aspect of the present invention.Can such solution be cushioned or it does not cushioned.In solution, this salt ordinary solution is from 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl that forms proton form]-urea and one or more counter ion.Therefore, on the other hand, the present invention also provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of proton form] aqueous solution of-urea and one or more counter ion and optional one or more other counter ion (for example derived from other salt as the counter ion of sodium-chlor or buffer reagent).

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the salt pharmaceutically useful salt typically of-urea, people such as Berge, 1977, " pharmaceutically useful salt (Pharmaceutically Acceptable Salts) ", J.Pharm.Sci., the 66th volume, the example to pharmacologically acceptable salt in 1-19 page is discussed.But pharmaceutically unacceptable salt also can be prepared as intermediate forms, then can convert it into pharmaceutically useful salt.Therefore, its such pharmaceutically unacceptable salt form has also formed a part of the present invention.

Compound 1-cyclopropyl base-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea also can form N-oxide compound.Can form N-oxide compound with aforesaid method.

, compound 1-cyclopropyl base-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl the same with other compound of the present invention]-urea with and acid salt also can exist and in the time relating to this compound, comprise in this application all such forms with many different tautomeric forms.

More specifically, at 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea with and salt in, its benzimidazolyl-can be taked determined two kinds of tautomeric form A below " and B " in any.For simplicity, general formula (I) has been set forth form A ", comprise all tautomeric forms but this formula should be regarded as.

Therefore, in the time relating to a kind of alternative tautomeric form, 1-cyclopropyl-3-[3-(6-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea obviously also relates to-1H-pyrazoles-4-yl with 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)] compound that-urea is identical.

Its pyrazole ring also can show tautomerism and its can be with two kinds of tautomeric form C below " and D " form exist.

In addition, the cis of this urea and transoid conformation also can be as follows.

Relating to 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea with and when salt, also comprise that it has the modification that one or more isotropic substances replace, and in the time relating to a kind of element-specific, be included in all isotropic substances of this element within the scope of it.For example, in the time relating to hydrogen, comprise in its scope ¹h, ²h (D) and ³h (T).Equally, in the time relating to carbon and oxygen, in its scope, comprise respectively ¹²c, ¹³c and ¹⁴c and ¹⁶o and ¹⁸o.

Described isotropic substance can be to have radioactivity or cold.In one embodiment of the invention, described compound does not comprise radio isotope.Preferably this compounds for the treatment of application.But in another embodiment, described compound can comprise one or more radio isotope.Comprising such radioisotopic compound can be used in diagnostic environment.

Relating to 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea with and also comprise its any polymorphic forms, solvate (for example hydrate), complex body (the inclusion complex body or the inclusion compound that for example form as cyclodextrin with compound, or the complex body forming with metal) when salt.

lactic acid salt and Citrate trianion, its mixture and crystallization

, 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl apparent as the part before the application] the preferred salt of-urea is the acid salt forming with lactic acid (more preferably Pfansteihl), citric acid or its mixture.

For convenience's sake, can be called as respectively lactic acid salt, Pfansteihl salt and Citrate trianion here with the salt of lactic acid, Pfansteihl and citric acid formation.

In a specific embodiment, said salt is Pfansteihl salt or D-lactate, preferably Pfansteihl salt.

In another embodiment, said salt is the salt forming with citric acid.

More specifically, said salt is the mixture of Pfansteihl salt and Citrate trianion.

In the time of solid state, lactic acid salt of the present invention (particularly Pfansteihl salt) or Citrate trianion can be crystallization or amorphous or its mixture.

In-individual embodiment, said lactic acid salt (particularly Pfansteihl salt) or Citrate trianion are unbodied.

In the time of amorphous solid, not having the three-dimensional structure and the position of molecule for other molecule in amorphous form that are conventionally present in crystalline form is random substantially, see the people J.Pharm.Sci. (1997) such as such as Hancock, 86,1).

In another embodiment, described lactic acid salt (particularly Pfansteihl salt) or Citrate trianion are crystallization substantially, be that it can be 50% to 100% crystallization, and it can be more specifically at least 50% crystallization, or at least 60% crystallization, or at least 70% crystallization, or at least 80% crystallization, or at least 90% crystallization, or at least 95% crystallization, or at least 98% crystallization, or at least 99% crystallization, or at least 99.5% crystallization, or at least 99.9% crystallization, for example 100% crystallization.

In another embodiment, it is 50% to 100% crystallization that described lactic acid salt (particularly Pfansteihl salt) or Citrate trianion are selected from, for example at least 50% crystallization, at least 60% crystallization, at least 70% crystallization, at least 80% crystallization, at least 90% crystallization, at least 95% crystallization, at least 98% crystallization, at least 99% crystallization, at least 99.5% crystallization, at least 99.9% crystallization, for example lactic acid salt of 100% crystallization (particularly Pfansteihl salt) or Citrate trianion.

More preferably these 95% to 100% crystallizations of described lactic acid salt (particularly Pfansteihl salt) or Citrate trianion, for example at least 98% crystallization, or at least 99% crystallization, or at least 99.5% crystallization, or at least 99.6% is crystallization or at least 99.7% crystallization or at least 99.8% crystallization or at least 99.9% crystallization, for example salt of 100% crystallization (or can be selected from these salt).

An example of the basic salt for crystallization is the crystalline salt forming with Pfansteihl.

Another example of the basic salt for crystallization is the crystalline salt forming with citric acid.

Salt of the present invention can for example, by solvation (aquation) or can be not for example, by solvation (anhydrous) in the time of solid state.

In one embodiment, described salt is not for example, by solvation (anhydrous).

Not not the crystalline salt that defined and lactic acid (particularly Pfansteihl) forms here by another example of the salt of solvation.

Terminology used here " anhydrous " is also not precluded within on described salt or in described salt, has the possibility (crystal of for example described salt) of some water.For example, can there be some water to be for example present in, on the surface of this salt (salt crystal), or can have a small amount of water to be for example present in, in salt body (crystal).The common per molecule compound of anhydrous form comprises lower than 0.4 molecular water, and more preferably per molecule compound comprises lower than 0.1 molecular water, for example 0 molecular water.

In another embodiment, described lactic acid salt (particularly Pfansteihl salt) or Citrate trianion are by solvation.Described salt by the situation of aquation in, it can comprise and is for example up to three molecular crystal water, more generally comprises and is up to two molecular waters, for example a part water or two molecular waters.Can also form non-stoichiometric hydrate, the water molecule number wherein existing is lower than 1 or be not integer.For example, while wherein existing lower than 1 molecular water, can there is for example 0.4 or 0.5 or 0.6 or 0.7 or 0.8 or 0.9 molecular water in per molecule compound.

Other solvate comprises that alcoholate is as ethylate and Virahol compound.

In one embodiment, described lactic acid salt (particularly Pfansteihl salt) by solvation for example by water and/or alcohol solvent.

Described Pfansteihl salt and Citrate trianion can according to the part before the application and here other local described method be prepared.

The advantage of described Pfansteihl salt and Citrate trianion is included in the general advantage described in the application's previous section.But the advantageous particularly part of lactic acid salt of crystallization of the present invention is also it:

Non-hygroscopic;

Anhydrous and do not form hydrate;

Exist and think that it does not show polymorphic with monocrystalline form;

It is crystallization;

Stable storing;

There is narrow fusing point and form can not occur in the time analyzing with DSC and change;

In water, there is good solubility; With

In buffering system, there is better solubleness.

Therefore, described Pfansteihl salt exists can not form hydrate and form can not change under exemplary process (handling), processing and condition of storage stable crystalline forms.

Terminology used here ' stable ' or ' stability ' comprise chemical stability and solid-state (physics) stability.Term ' chemical stability ' refers to described compound and can be stored under general condition of storage with the form or the dosage form (pharmaceutically acceptable carrier, thinner or auxiliary agent are blended in-rise as described herein with example in said preparation for they) that separate, and chemical degradation or decomposition occur or do not occur hardly.' solid-state stability ' refers to described compound and can or be stored under general condition of storage with solid preparation form (pharmaceutically acceptable carrier, thinner or auxiliary agent mix as described herein with example in said preparation for they) with the solid form of separation, there is or not occur hardly solid-state conversion (for example hydration, dehydration, solvation (solvatisation), desolvation (desolvatisation), crystallization, recrystallization or solid-state inversion of phases).

Described Pfansteihl salt and Citrate trianion with and composition thereof in water, there is good solubleness and therefore can be used for the aqueous solution that preparation comprises salt described in relative high density.Therefore, in another embodiment, provide and comprised concentration higher than 1mg/ml, typically for example, higher than 5mg/ml liquid vehicle (water or buffering system), more typically higher than 15mg/ml, more typically higher than 20mg/ml and preferably higher than 25mg/ml 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the Pfansteihl salt of-urea or the aqueous solution of Citrate trianion or its mixture (being for example pharmaceutical compositions).In this embodiment, particularly preferably comprise (i) described Pfansteihl salt or (ii) aqueous solution of the mixture of described Pfansteihl salt and Citrate trianion (being for example pharmaceutical compositions).

The aqueous solution of described Pfansteihl salt or Citrate trianion or its mixture can be take pH as 2 to 6, and for example 2 to 5, and be more specifically 4 to 6, the form of the aqueous solution as 4 to 5 provides.

The aqueous solution of described Pfansteihl salt or Citrate trianion or its mixture can be cushioned or not be cushioned, and still, in one embodiment, it is cushioned, for example, to the pH of above-mentioned scope.

Preferred buffer reagent is that these can be buffered to described solution about 4.5 pH and nonvolatile these buffer reagents under this solution lyophilize condition used.

Forming in the context of salt with Pfansteihl, preferred buffer reagent is the buffer reagent being formed by citric acid, and it is proofreaied and correct to correct pH by NaOH or HCl, and for example pH value of solution is about 4.5.Under this pH and in described citrate buffer solution, described free alkali has the solubleness of about 80mg/ml.

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl that the aqueous solution of described Pfansteihl salt or Citrate trianion or its mixture comprises proton form]-urea and Pfansteihl salt and/or Citrate trianion counter ion.For example can also exist other counter ion and these counter ion to derive to derive from tension regulator if salt solution (being muriate counter ion) and/or buffer reagent are as citrate buffer agent.For example, by described Pfansteihl salt with have in the situation of aqueous solution of citrate buffer agent, will there are Pfansteihl salt and Citrate trianion counter ion, the character of described Citrate trianion counter ion depends on the pH of this solution.In addition, the aqueous solution of Pfansteihl salt or Citrate trianion or its mixture for example can also comprise one or more other vehicle that conventionally can find in I.V. preparation, as tension regulator (example be have been described in detail in American Pharmacopeia (the United States Pharmacopeia) and NF (the National Formulary) and the example comprises that hexasaccharide (hexose sugars) is as glucose, dextrose (D-Glucose)).

Therefore, in another embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of protonated form]-urea and one or more be selected from Pfansteihl salt and Citrate trianion with and composition thereof the aqueous solution of counter ion; And this solution also optionally comprise (i) one or more other counter ion as chloride ion and/or (ii) one or more I.V. vehicle for example, as tension regulator (hexasaccharide is as glucose, preferably D-Glucose).

The described aqueous solution especially can for example, form by lactic acid salt being dissolved in citrate ion (citrate buffer agent) solution or Citrate trianion being dissolved in the solution of lactate ions.Described lactic acid salt and citrate ion can be with 10: 1 or lower, for example 10: 1 to 1: 10, more preferably less than 8: 1, or lower than 7: 1, or lower than 6: 1, or lower than 5: 1 or lower than 4: 1 or lower than 3: 1 or lower than 2: 1 or lower than 1: 1, the more especially lactic acid salt of 1: 1 to 1: 10: Citrate trianion ratio is present in described solution.In one embodiment, described lactic acid salt and citrate ion were with 1: 1 to 1: 10, for example 1: 1 to 1: 8, or 1: 1 to 1: 7 or 1: 1 to 1: 6 or 1: 1 to 1: 5, for example lactic acid salt of about 1: 4.4: Citrate trianion ratio is present in described solution.

Can be by the application's this section and other local described each aqueous solution lyophilize here, thereby obtain a kind of solid preparation, when needed, carry out to be easily reassembled as the aqueous solution (being preferably sterile solution) by adding water (being preferably sterilized water) or comprising I.V. vehicle as the aqueous medium of salt solution and/or dextrose.

Therefore, the present invention also provides the freeze-dried preparation that comprises defined Pfansteihl salt here or Citrate trianion or its mixture (being for example pharmaceutical compositions), for example, in the time being dissolved in water, wherein said preparation has 2 to 6, for example 2 to 5, and 4 to 6 as 4 to 5 pH more especially.

In another embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl that comprises protonated form]-urea and one or more be selected from Pfansteihl salt and Citrate trianion take and composition thereof the freeze-dried preparation (for example, as pharmaceutical compositions) of counter ion; And said preparation also optionally comprise (i) one or more other counter ion as chloride ion and/or (ii) one or more I.V. vehicle for example, as tension regulator (hexasaccharide as glucose, preferably D-Glucose).

In each freeze-dried preparation, the ratio of Pfansteihl salt and citrate ion can be the ratio described in the aqueous solution above.

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl]-urea with and the crystalline structure of salt

As described above, 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] lactic acid salt (particularly Pfansteihl salt) of-urea or Citrate trianion can be amorphous or be crystallization substantially.In a specific embodiment, described lactic acid salt (particularly Pfansteihl salt) or Citrate trianion are crystallization substantially, and term " substantially for crystallization " has implication as defined above.1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] lactic acid salt (particularly Pfansteihl salt) of-urea is particularly substantially for crystallization.

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] free alkali of-urea can be also amorphous or be crystallization substantially.In a specific embodiment, described free alkali is crystallization substantially, and term " being crystallization substantially " has implication as defined above.In one embodiment, 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] free alkali of-urea exists with dihydrate crystalline form.

Crystal and crystalline structure described here have formed another aspect of the present invention.

As implied above, think that lactic acid salt of the present invention exists with the monocrystalline form with characteristic described here.This crystalline form has represented a kind of preferred embodiment of the present invention.But if really there is other crystalline form, scope of the present invention is not repelled these crystalline forms.

Therefore, at described 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] lactic acid salt (particularly Pfansteihl salt) of-urea is substantially in the situation for crystallization, monocrystalline form (for example here the crystalline form of institute's definition and feature) may be preponderated, but also can exist the quantity of a small amount of other crystalline form and these other crystalline forms to be preferably negligible quantity.

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] crystalline form (for example here the crystalline form of institute's definition and feature) of-urea (or its salt) comprises-1H-pyrazoles-4-yl less than or equal to approximately 5% weight 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)] other crystalline form of-urea (or its salt), particularly comprise other crystalline form (or its salt) less than or equal to approximately 1% weight.

In a preferred embodiment, the invention provides the basic monocrystalline form that comprises described salt for crystallization (for example here the crystalline form of institute's definition and feature) and not higher than 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of any other crystalline form of salt described in 5% weight] salt of-urea (for example as herein defined lactic acid salt as Pfansteihl salt).

For example, other crystalline form in described monocrystalline form (here the crystalline form of institute's definition and feature) is preferably lower than 4%, or lower than 3%, or lower than 2%, and particularly comprise other crystalline form less than or equal to approximately 1% weight.Other for example, in described monocrystalline form (here the crystalline form of institute's definition and feature) contained crystalline form is more preferably lower than 0.9%, or lower than 0.8%, or lower than 0.7%, or lower than 0.6%, or lower than 0.5%, or lower than 0.4%, or lower than 0.3%, or lower than 0.2%, or lower than 0.1%, or lower than 0.05%, or lower than 0.01% weight, other crystalline forms of for example 0% weight.

Can with many technology come described in characterization crystal with and crystalline structure, described technology comprises monocrystalline X-radiocrystallography, X-ray powder diffraction (XRPD), dsc (DSC) and infrared spectroscopy, for example fourier transform infrared spectroscopy (FTIR).Can with gravimetric analysis steam absorption research (gravimetric vapour sorption studies) and also can with XRPD to described crystal the behavior under different moisture levels condition analyze.

Can measure by X-radiocrystallography the crystalline structure of compound, said X-radiocrystallography can be according to conventional methods as described herein and basic crystal theory (Fundamentals ofCrystallography), C.Giacovazzo, H.L.Monaco, D.Viterbo, F.Scordari, G.Gilli, G.Zanotti and M.Catti, (International Union ofCrystallography/Oxford University Press, 1992ISBN 0-19-855578-4 (p/b), 0-19-85579-2 (h/b)) described in these methods carry out.This technology comprises X-ray diffraction analysis and the explanation of monocrystalline.

Measure 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl by X-radiocrystallography] crystalline structure of-urea free alkali dihydrate and Pfansteihl salt---see respectively the following examples 69 and 71.

Table 2 and 4 in embodiment 69 and 71 respectively with crystallography message file (Crystallographic Information File) (CIF) form (see Hall, Allen andBrown, Acta Cryst. (1991) .A47,655-685; Http:// www.iucr.ac.uk/iucr-top/cif/home.html) provided 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea with and coordinate (coordinate) data of the crystal of Pfansteihl salt.Or those skilled in the art also can use or preferably use another kind of file layout for example, as PDB file layout (with the form of EBIMacromolecular Structure Database (Hinxton, UK) consistent form).But, it is evident that, with different file layouts represent or to these table in coordinate (coordinate) process also in scope of the present invention.Numeral deviation (s.u., stardard uncertairty (standard uncertainty)) in these table brackets.In Figure 4 and 5, described Lactated crystalline structure is illustrated.

In one embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the dihydrate free alkali of-urea, its be crystallization and (i) there is the defined crystalline structure of coordinate in table 2 here; And/or (ii) wherein said crystal belongs to and has lattice parameter a=7.66 (10), b=15.18 (10),

β=98.53 (2) °, the monclinic spacer P2 of α=γ=90 ° ₁/ n (#14).

In another embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the Pfansteihl salt of-urea, its be crystallization and there is the defined crystalline structure of coordinate in table 4 here.

In another embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the Pfansteihl salt of-urea, its be crystallization and there is the crystalline structure described in Figure 4 and 5.

In another embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the Pfansteihl salt of-urea, it is crystallization, has and belongs to orthogonal (orthorhombic) spacer P2 ₁2 ₁2 ₁(#19) and under 97 (2) K, there is a=9.94 (10), b=15.03 (10),

the crystalline structure of the lattice parameter of α=β=γ=90 °.

In another embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the Pfansteihl salt of-urea, its be crystallization and at room temperature there is a=10.08 (10), b=15.22 (10)

the lattice parameter of α=β=γ=90 °.

Therefore, in another embodiment, the invention provides be crystallization and:

(a) there is the crystalline structure described in Figure 4 and 5; And/or

(b) there is the defined crystalline structure of coordinate of table 4 here; And/or

(c) under 97 (2) K, there is a=9.94 (10), b=15.03 (10),

the lattice parameter of α=β=γ=90 °; And/or

(d) at room temperature there is a=10.08 (10), b=15.22 (10), the lattice parameter of α=β=γ=90 °; And/or

(e) there is the orthorhombic space group of belonging to P2 ₁2 ₁2 ₁(#19) crystalline structure; 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the Pfansteihl salt of-urea.

Another kind of selectable mode is to analyze the crystalline structure of compound with the solid state technology of X-ray powder diffraction (XRPD).Can be according to conventional methods introduction (Introductionto X-ray Powder Diffraction) Ron Jenkins and Robert L.Snyder (the JohnWiley & Sons of the described and X-ray powder diffraction of method (seeing embodiment 70 and 72) as described herein, New York, 1996) these methods, are carried out XRPD.In XRPD diffractogram, exist the peak (relative with random background noise) of defined to show that described compound has certain degree of crystallinity.

The X-diffraction powder style of compound is carried out to characterization by diffraction angle (2 θ) and spacing (d) parameter of X-ray diffraction spectrum.At Bragg ' s equation, in n λ=2d Sin θ, relate to these parameters, (wherein n=1; The wavelength of λ=negative electrode used; D=spacing; And θ=diffraction angle).Due to the characteristic of these data, so here, spacing, diffraction angle and overall style (overall pattern) are very important for the evaluation of crystal in X-ray powder diffraction.Because it may change according to direction, granularity and the measuring condition of crystal growth, so should not carry out strict interpretation to relative intensity.In addition, described diffraction angle is commonly referred to as the angle that one meets the scope of 2 θ ± 0.2 °.Described peak refers to main peak and except above-mentioned these peaks, also comprises these peaks of the intermediate value that is not more than diffraction angle.

Can be with XRPD to 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] Pfansteihl salt and the free alkali form of-urea carry out characterization.In various situations, according to diffraction angle (2 θ), spacing (inter planarspacing) (d) and/or relative intensity powder x-ray diffraction style is represented.Table 3,5 and 6 in embodiment 70 and 72 has represented-1H-pyrazoles-4-yl corresponding to 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)] spacing (d) value of the X-ray diffraction spectrum of the diffraction angle value of free alkali, Pfansteihl salt and the dihydrate free alkali form of-urea.

Therefore, 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea with and salt have substantially as the Fig. 3,6 in embodiment 70 and 72,7 or 8 and/or table 3,5 or 6 as shown in X-ray powder diffraction style.

Therefore, in one embodiment, the invention provides show comprise be positioned at Fig. 3,6,7 or 8 and/or table 3 and/or table 5 and/or table 6 shown in 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of X-ray powder diffraction style at peak under the identical diffraction angle of these diffraction angle of X-ray powder diffraction pattern] crystal of-urea free alkali or its Pfansteihl salt, and wherein all peaks optionally have identical relative intensity.More specifically, the crystal of described salt is those crystal with basic X-ray powder diffraction style as shown in Fig. 3,6,7 or 8.

In a preferred embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl with the ray powder diffraction of X-substantially as shown in Figure 6 style] crystal of-urea Pfansteihl salt.

In another embodiment, the invention provides and show 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl that is positioned at the peak under the diffraction angle identical with these diffraction angle of the ray powder diffraction of X-shown in Fig. 6 style] the basic Pfansteihl salt for crystallization of-urea.Described peak preferably has the relative intensity identical with the relative intensity at peak in Fig. 6.

The present invention also provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl with the ray powder diffraction of X-substantially as shown in Figure 6 style] the basic Pfansteihl salt for crystallization of-urea.

Can use the peak existing under diffraction angle (2 θ) and spacing (d), and preferably by the intensity shown in table 5 in embodiment 72, the X-ray powder diffraction style of described Pfansteihl salt be carried out to characterization.

Therefore, the invention provides cyclopropyl-3-[3-(6-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl that shows the X-ray powder diffraction style with the characteristic peak under the diffraction angle (2 θ ± 1.0 degree as ± 0.2 degree, particularly ± 0.1 degree) that is arranged in embodiment 72 tables 5] crystal of-urea Pfansteihl salt.

The present invention also provides to have to show and has been positioned at 17.50,18.30,19.30,19.60, with 21.85 ± 1.0 degree as ± 0.2 degree, cyclopropyl-3-[3-(6-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of X-ray powder diffraction style of the main peak under diffraction angle 2 θ of particularly ± 0.1 degree] crystal of-urea Pfansteihl salt.Can also be with being positioned at 12.40,15.20,15.60,17.50,18.30,18.50,19.30,19.60,21.85 in X-ray diffraction style, and peaks under 27.30 ± 1.0 degree 2 θ further carry out characterization to described crystal.

Cyclopropyl-3-[3-(6-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] crystal of-urea Pfansteihl salt can also use by the distance d between the crystal face of embodiment 72 tables 5

the characteristic X-ray powder diffraction pattern representing carries out qualitative.

In another embodiment, the invention provides cyclopropyl-3-[3-(6-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the Lactated crystal of-urea, it has and comprises to be positioned at 5.06, 4.85, 4.60, 4.53, the X-ray powder diffraction style of the characteristic peak occurring with the form (d) of spacing of lattice (lattice spacing) of powder x-ray diffraction under 4.07, and it more specifically comprises to be positioned at 7.13, 5.83, 5.68, 5.06, 4.85, 4.79, 4.60, 4.53, 4.07, the other characteristic peak occurring with the spacing of lattice (lattice spacing) of the powder x-ray diffraction under 3.26 dusts form (d).

In another embodiment, the invention provides to have to be characterized as to exist and be positioned at 17.50, 18.30, 19.30, 19.60, with 21.85 degree, more specifically be positioned at 12.40, 15.20, 15.60, 17.50, 18.30, 18.50, 19.30, 19.60, 21.85, with the main peak under the diffraction angle (2 θ) of 27.30 degree, with 5.06, 4.85, 4.60, 4.53, with 4.07, be more specifically 7.13, 5.83, 5.68, 5.06, 4.85, 4.79, 4.60, 4.53, 4.07, 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl with the X-ray powder diffraction style of the spacing (d) of 3.26 dusts] the basic Pfansteihl salt for crystallization of-urea.

In another embodiment, the invention provides to have to be characterized as have that to be positioned at described diffraction angle (2 θ) and spacing (d) lower and more preferably have 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of the X-ray powder diffraction style at the peak of the intensity shown in embodiment 72 tables 5]-urea be the Pfansteihl salt of crystallization substantially.

The present invention also provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl that shows the X-ray powder diffraction style with the characteristic peak under the diffraction angle (2 θ ± 1.0 degree as ± 0.2 degree, particularly ± 0.1 degree) that is positioned at table 2] crystal of-urea free alkali.

In another embodiment, the invention provides 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] crystal of-urea free alkali, it has the X-ray powder diffraction style under the spacing of lattice (d) that a kind of characteristic peak comes across table 2.

Can also carry out characterization to crystalline salt of the present invention with dsc (DSC).

With DSC to described Pfansteihl salt carried out analyze and its at 190 ℃, show a peak (fusing point and decomposition).

Therefore, on the other hand, the invention provides and be anhydrous and in the time analyzing with DSC, at 190 ℃, show a kind of 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of endotherm(ic)peak] the Pfansteihl salt of-urea.

The present invention shows the peak that is positioned under the diffraction angle identical with these diffraction angle of X-ray powder diffraction style shown in Fig. 6,7 or 8 and near 190 ℃, also shows 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl with the endotherm(ic)peak of decomposing according to heat analysis (DSC)] the Pfansteihl salt of-urea.

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] free alkali of-urea shows some peaks and near 193 ℃, also shows with the exothermic peak of decomposing according to heat analysis (DSC) under the diffraction angle identical with these diffraction angle of the ray powder diffraction of X-shown in Fig. 3 and/or table 2 style.

Can analyze steam absorption (GVS) method by standard weights, for example the method described in the part E of embodiment 68 is analyzed in the behavior under high humidity salt of the present invention.

Described Pfansteihl salt under the condition of high relative humidity can with stable anhydrous crystal forms form exist and under such condition its crystalline structure can not change.

Can use infrared spectroscopy, for example FTIR is to 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] salt of-urea further carries out qualitative.The infrared spectra (KBr disk method (disc method)) of Pfansteihl salt comprises and is positioned at 3229,2972 and 1660cm ^-1characteristic peak.

Therefore, in another embodiment, the invention provides in the time analyzing by KBr disk method, show to comprise and be positioned at 3229,2972 and 1660cm ^-11-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of infrared spectra of the characteristic peak at place] the Pfansteihl salt (being preferably basic crystallization) of-urea.

As apparent from paragraph above, can carry out qualitative to Pfansteihl salt of the present invention with many different the physical-chemical parameters.Therefore, in a preferred embodiment, the invention provides be crystallization and be characterised in that 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl with any or multiple (any combination) or all parameters below] the Pfansteihl salt of-urea, i.e. this salt:

(a) there is the crystalline structure described in Figure 4 and 5; And/or

(b) there is the defined crystalline structure of coordinate of table 4 in embodiment 71 here; And/or (c) under 97 (2) K, there is a=9.94 (10), b=15.03 (10),

the lattice parameter of α=β=γ=90 °; And/or

(d) at room temperature there is a=10.08 (10), b=15.22 (10),

the lattice parameter of α=β=γ=90 °; And/or

(e) there is the orthorhombic space group of belonging to P2 ₁2 ₁2 ₁(#19) crystalline structure; And/or

(f) there is the existence of being characterised in that and be positioned at 17.50, 18.30, 19.30, 19.60, with the main peak under the diffraction angle (2 θ) of 21.85 degree, and more specifically also have and be positioned at 12.40, 15.20, 15.60, 17.50, 18.30, 18.50, 19.30, 19.60, 21.85, with the main peak and/or 5.06 under 27.30 degree, 4.85, 4.60, 4.53, with 4.07 spacing (d), and more specifically also have 7.13, 5.83, 5.68, 5.06, 4.85, 4.79, 4.60, 4.53, 4.07, X-ray powder diffraction style with the spacing (d) of 3.26 dusts, and/or

(g) show and be positioned at the peak under the diffraction angle identical with these diffraction angle of X-ray powder diffraction style shown in the table 5 of Fig. 6 or embodiment 72 and optionally, wherein said peak has the relative intensity identical with the relative intensity at peak in Fig. 6 or table 5; And/or

(h) there is X-ray powder diffraction style substantially as shown in Figure 6; And/or

(i) be anhydrous and in the time analyzing with DSC, at 190 ℃, show a kind of endotherm(ic)peak; And/or

(j), in the time analyzing by KBr disk method, show to comprise and be positioned at 3229,2972 and 1660cm ^-1the infrared spectra of the characteristic peak at place.

the compound of the group (C) of formula (I)

In a group (being the group (C) of formula (I)) of formula (I) compound, M is group D1; X is O; A is group NR ², wherein R ²hydrogen; E is a key; R ¹it is 2,6-difluorophenyl; With compound be by the selected group of acid salt that form of acid.

Therefore, in one embodiment, the invention provides some 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] acid salt of-urea, these salt are and are selected from acetic acid, hexanodioic acid, alginic acid, xitix (for example L-AA), aspartic acid (for example ASPARTIC ACID), Phenylsulfonic acid, phenylformic acid, dextrocamphoric acid (for example (+) dextrocamphoric acid), capric acid, sad, carbonic acid, citric acid, cyclohexane sulfamic acid, dodecanoate, dodecyl sulphate, ethane-1,2-disulfonic acid, ethyl sulfonic acid, fumaric acid, tetrahydroxyadipic acid, gentisinic acid, glucoheptonic acid, D-glyconic acid, glucuronic acid (for example D-Glucose aldehydic acid), L-glutamic acid (for example Pidolidone), α ketoglutaric acid, oxyacetic acid, urobenzoic acid, hydrochloric acid, hydroxyethylsulfonic acid, isopropylformic acid, lactic acid (for example (+)-Pfansteihl and (±)-DL-LACTIC ACID), lactobionic acid, lauryl sulfonic acid, toxilic acid, oxysuccinic acid, (-)-L MALIC ACID, propanedioic acid, methylsulfonic acid, glactaric acid, naphthene sulfonic acid (for example naphthalene-2-sulfonic acid), naphthalene-1,5-disulfonic acid, nicotinic acid, oleic acid, vitamin B13, oxalic acid, palmitinic acid, flutter acid, phosphoric acid, propionic acid, sebacic acid, stearic acid, succsinic acid, sulfuric acid, tartrate (for example (+)-L-TARTARIC ACID), thiocyanic acid, toluenesulphonic acids (for example p-toluenesulphonic acids), the salt that the acid of valeric acid and xinafoic acids forms.

In one embodiment, described acid salt is by the diacid of selecting oneself, alginic acid, xitix (for example L-AA), aspartic acid (for example ASPARTIC ACID), phenylformic acid, dextrocamphoric acid (for example (+) dextrocamphoric acid), capric acid, sad, carbonic acid, cyclohexane sulfamic acid, dodecanoate, dodecyl sulphate, ethane-1, 2-disulfonic acid, tetrahydroxyadipic acid, gentisinic acid, glucoheptonic acid, D-glyconic acid, L-glutamic acid (for example Pidolidone), α ketoglutaric acid, oxyacetic acid, urobenzoic acid, isopropylformic acid, lauryl sulfonic acid, glactaric acid, naphthalene-1, 5-disulfonic acid, nicotinic acid, oleic acid, vitamin B13, oxalic acid, palmitinic acid, flutter acid, sebacic acid, stearic acid, tartrate (for example (+)-L-TARTARIC ACID), the acid of thiocyanic acid and xinafoic acids forms.

In another embodiment, described acid salt forms by being selected from acetic acid, hexanodioic acid, xitix, aspartic acid, citric acid, DL-LACTIC ACID, fumaric acid, glyconic acid, glucuronic acid, urobenzoic acid, hydrochloric acid, L-glutamic acid, DL-oxysuccinic acid, p-toluenesulphonic acids, methanesulfonic (mesylate), ethyl sulfonic acid (esilate), sebacic acid, stearic acid, succsinic acid and tartaric acid.

In another embodiment, described acid salt is formed by the diacid of selecting oneself, xitix, aspartic acid, glyconic acid, urobenzoic acid, L-glutamic acid, sebacic acid, stearic acid and tartaric acid.

In another specific embodiment, described compound is the acid salt forming with hydrochloric acid.

Preferred salt is such salt, and it for example for example, has, higher than 25mg/ml liquid vehicle (water) in the liquid vehicle providing (water), more typically higher than 50mg/ml and preferably higher than the solubleness of 100mg/ml.Such salt is for liquid form, for example advantageous particularly by injecting or input the administration of carrying out.

The present invention provides one to comprise with higher than 25mg/ml on the other hand, typically for example, higher than 50mg/ml and the composition of the aqueous solution that preferably comprises salt described here higher than the concentration of 100mg/ml (pharmaceutical composition).

Have higher than the salt of the present invention of 25mg/ml solubleness and comprise D-Glucose aldehydic acid salt, mesylate, esilate and DL-LACTIC ACID salt, three kinds of salt below have the solubleness that exceedes 100mg/ml.

Therefore,, in a specific embodiment, provide 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] mesylate of-urea.

In another specific embodiment, provide 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] esilate (esilate) salt of-urea.

In another specific embodiment, provide 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the DL lactic acid salt of-urea.In one embodiment, described lactic acid salt is Pfansteihl salt.

Free alkali or parent compound that the compound (being acid salt) of the group (C) of formula of the present invention (I) can be derived from wherein have formula (IA):

The salt of compound (IA) can be amorphous or crystallization.

In one embodiment, described salt is amorphous form.

In another embodiment, described compound is crystalline form.

Described compound can be for example, by solvation (anhydrous) or by solvation.

In one embodiment, described salt is not by solvation.

In another embodiment, said salt is by solvation, for example, by aquation.

Described compound by the situation of aquation in, it for example can comprise, and is up to three molecular crystal water, more generally comprises and is up to two molecular waters, for example a part water or two molecular waters.

Salt of the present invention has the 1-of being better than (2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] advantage of the free alkali form of-urea.

For example, described salt has higher solubleness and therefore for example, for more favourable for injection or input (i.v. input) application of parenteral formulation in water.Salt of the present invention also has one or more and is selected from other advantages below:

Improve pharmacokinetics character;

Stability is better, for example, improved the shelf lives;

Alkalescence reduces, thereby makes it be more suitable for i.v. application;

There is preparation aspect;

There is the metabolisming property of improvement; With

Clinical difference between patient reduces.

Described salt can use (A) to formula (I) and (B) any method described in the application's previous section that the salt of group's compound is described be prepared.

The compound of the group (C) of formula (I) typically is pharmaceutically useful salt.But pharmaceutically unacceptable salt also can be prepared as intermediate forms, then can convert it into pharmaceutically useful salt.For example can be used for the purifying of the compounds of this invention or such pharmaceutically unacceptable salt form of separation has also formed a part of the present invention.

The compound of the group (C) of formula (I) can exist and in the time relating to compound of the present invention, comprise all such forms with many different tautomeric forms.For fear of query, wherein compound can exist and only specifically describing or represent in a kind of situation of form, the application still comprises all other forms with a kind of form in some rotamerisms or tautomeric form.

For example, in compound of the present invention, described benzoglyoxaline group can be taked following two kinds of tautomeric form A recited above " and B " in any.

Described pyrazole ring also can show tautomerism and can be with two kinds of tautomeric form C below " ' and D " ' exist.

Compound of the present invention comprises having the compound that one or more isotropic substances replace, and in the time relating to a kind of element-specific, is included in all isotropic substances of this element within the scope of it.For example, in the time relating to hydrogen, comprise in its scope ¹h, ²h (D) and ³h (T).Equally, in the time relating to carbon and oxygen, in its scope, comprise respectively ¹²c, ¹³c and ¹⁴c and ¹⁶o and ¹⁸o.

Described isotropic substance can be to have radioactivity or cold.In one embodiment of the invention, described compound does not comprise radio isotope.Preferably this compounds for the treatment of application.But in another embodiment, described compound can comprise one or more radio isotope.Comprising such radioisotopic compound can be used in diagnostic environment.

The present invention also comprises the prodrug of any polymorphic forms of described compound and complex body (the inclusion complex body or the inclusion compound that for example form with the material such as cyclodextrin, or the complex body forming with metal) and described compound." prodrug " refers to any compound that for example can change in vivo biologically active cpds of the present invention.

biologic activity

Compound of the present invention has cell cycle protein dependent kinase inhibition or adjusting activity and glycogen synthase kinase-3 (GSK3) to be suppressed or regulates activity and/or aurora kinase inhibition or adjusting active, and therefore thinks that it can be used for prevention or treats by described kinase mediated morbid state or illness.

Therefore, for example, think that compound of the present invention will can be used for alleviating or reducing the incidence of cancer.

More specifically, formula (I) with and the compound of group be the inhibitor of cell cycle protein dependent kinase.For example, compound of the present invention is to CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK7 kinases, and the cell cycle protein dependent kinase that is particularly selected from CDK1, CDK2, CDK3, CDK4, CDK5 and CDK6 has activity.

Preferred compound is to suppress one or more to be selected from CDK1, CDK2, CDK4 and CDK5, the kinase whose compound of CDK of for example CDK1 and/or CDK2.

In addition, CDK4, CDK8 and/or CDK9 may be also interested.

1-ring-propyl group-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] lactic acid salt of-urea or Citrate trianion are to CDK2, CDK4, CDK5, CDK6 and CDK9 kinases, and particularly CDK2 has activity.

Compound of the present invention also has activity to glycogen synthase kinase-3 (GSK-3).

Compound of the present invention also has activity to aurora kinase.The preferred compound of the present invention is that these have the IC lower than 0.1 μ M ₅₀the compound of value.

Particularly 1-ring-propyl group-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] lactic acid salt or the Citrate trianion of-urea be the inhibitor of aurora kinase and for example suppress Aurora A and/or Aurora B.

Compare with CDK2 with CDK1, chemical compound lot of the present invention is to the Aurora A kinases a kind of preferred embodiment of the present invention that shown selectivity and such compounds represented.For example, the IC of many compound antagonism Aurora A of the present invention ₅₀value is the IC of antagonism CDK1 and CDK2 ₅₀1/10th to one of percentage.

Because the activity of its adjusting or inhibition CDK and aurora kinase and glycogen synthase kinase, so expect that it can be used for providing a kind of and stops the cell cycle of abnormal division cell or recover the means that its cell cycle is controlled.Therefore, expect that described compound can be used for treatment or prevents proliferative disease as cancer.Also think that compound of the present invention can be used for treatment such as virus infection, II type or non-insulin-dependent diabetes mellitus (NIDDM), autoimmune disorder, head trauma, apoplexy, epilepsy, neurodegenerative disease as the illness Alzheimer's, motor neuron, stein-leventhal syndrome, corticobasal sex change and for example autoimmune disorder of Pick's disease and neurodegenerative disease.

Think and can use the group in morbid state and the illness of the compounds of this invention to comprise virus infection, autoimmune disorder and neurodegenerative disease.

CDKs at cell cycle, apoptosis, transcribe, play a role in the adjusting of differentiation and CNS function.Therefore, CDK inhibitor can be used for treating such disease, wherein has the disorder of hyperplasia, apoptosis or for example cancer of differentiation illness.Specifically, RB+ve tumour may be responsive especially to CDK inhibitor.RB-ve tumour also may be to CDK inhibitor sensitivity.

Example that can repressed cancer comprises cancer without limitation, for example bladder cancer, mastocarcinoma, colorectal carcinoma (for example carcinoma of the colon and rectum is as adenocarcinoma of colon and adenoma of colon), kidney, epidermal carcinoma, liver cancer, lung cancer, for example gland cancer, the cancer of small cell lung cancer and nonsmall-cell lung cancer, oesophagus, gall-bladder, ovary, pancreas is the cancer of exocrinosity carcinoma of the pancreas, stomach, uterine neck, Tiroidina, prostate gland or skin for example, for example squamous cell cancer; The hematopoiesis tumour of Lymphoid tissue system, for example leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, He Jiejin lymphomas, non_hodgkin lymphoma, hair cell lymphoma or Burkett ' s lymphoma; The hematopoiesis tumour of myeloid lineage, for example acute and chronic granulocytic leukemia, myelodysplastic syndrome or promyelocytic leukemia; Thyroid follcular carcinoma (thyroid follicularcancer); The tumour of interstitial origin, for example fibrosarcoma or habdomyosarcoma; The tumour of central or peripheral nervous system, for example astrocytoma, neuroblastoma, neurospongioma or schwannoma; Melanoma; Spermocytoma; Teratoma; Osteosarcoma; Xeroderma pitmentosum; Keratoctanthoma; Thyroid follcular carcinoma; Or Kaposi's sarcoma.

Described cancer can be the cell cycle protein dependent kinase to any or multiple CDK1 of being selected from, CDK2, CDK3, CDK4, CDK5 and CDK6, for example, one or more are selected from the CDK kinases of CDK1, CDK2, CDK4 and CDK5, the cancer of the inhibition sensitivity of for example CDK1 and/or CDK2.

Can determine whether a kind of specific cancer is the cancer of the inhibition sensitivity of cell cycle protein dependent kinase or aurora kinase by the Growth of Cells test as described in embodiment 79 and 80 below or the method described in the part that title is " diagnostic method " that can be used in.

Also known CDKs apoptosis, hyperplasia, break up and transcribe in play a role, therefore, CDK inhibitor may also can be used for the disease for the treatment of below except cancer; Virus infection, for example simplexvirus, poxvirus, Epstein-Barr virus, sindbis virus (Sindbis virus), adenovirus, HIV, HPV, HCV and HCMV; Prevention HIV infected individuals AIDS development; Chronic inflammatory disease, for example glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel and the autoimmune diabetes of systemic lupus erythematosus, autoimmunization mediation; Cardiovascular disorder is cardiac hypertrophy, restenosis, atherosclerosis for example; Neurodegenerative illness, for example Alzheimer's, dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, Duchenne-Arandisease (atropy) and the cerebellar degeneration relevant with AIDS-; Glomerulonephritis; Myelodysplastic syndrome, local ischemic damage, apoplexy and the reperfusion injury relevant with myocardial infarction, arrhythmia, atherosclerosis, toxin-induced or with alcohol relevant hepatic diseases, blood disease, for example, chronic anaemia and aplastic anemia; The degenerative disease of Musculoskeletal, for example, the rhinosinusitis of osteoporosis and sacroiliitis, aspirin sensitive, cystic fibrosis, multiple sclerosis, ephrosis and cancer pain.

Also find that some cell cycle protein dependent kinase inhibitors can be used for and other carcinostatic agent coupling.For example, cell cycle protein dependent kinase inhibitor flavopiridol and other carcinostatic agent one are used from combination therapy.

Therefore, being used for the treatment of in pharmaceutical composition of the present invention, application or the method for the disease that comprises abnormal cell growth or illness, the described disease that comprises abnormal cell growth or illness is cancer in one embodiment.

One group of cancer comprises human breast carcinoma (for example primary mammary tumor, without tubercle mammary cancer, the invasive duct adenocarcinoma of breast, non-endometrioid mammary cancer); And lymphoma mantle cell.In addition, other cancer also has carcinoma of the colon and rectum and carcinoma of endometrium.

Another group's cancer comprises mammary cancer, ovarian cancer, colorectal carcinoma, prostate cancer, the esophageal carcinoma, squamous cell carcinoma and nonsmall-cell lung cancer.

In to the situation of the activated compound of aurora kinase, think that the particular instance that can use the present invention to suppress the cancer of the compound of aurora kinase comprises:

Human breast carcinoma (for example primary mammary tumor, without tubercle mammary cancer, the invasive duct adenocarcinoma of breast, non-endometrioid gland breast cancer);

Ovarian cancer (for example Primary ovarian cancer);

Carcinoma of the pancreas;

Human bladder cancer;

Carcinoma of the colon and rectum (for example primary carcinoma of the colon and rectum);

Tumor stomach;

Kidney;

Cervical cancer;

Neuroblastoma;

Melanoma;

Lymphoma;

Prostate cancer;

Leukemia;

Non-endometrial-like carcinoma of endometrium;

Neurospongioma; With

Non_hodgkin lymphoma.

Aurora inhibitor may useful especially cancer comprise mammary cancer, bladder cancer, carcinoma of the colon and rectum, carcinoma of the pancreas, ovarian cancer, non_hodgkin lymphoma, neurospongioma and non-endometrial-like carcinoma of endometrium.

Aurora inhibitor may the specific cancer of useful especially a small group be mammary cancer, ovarian cancer, colorectal carcinoma, liver cancer, cancer of the stomach and prostate cancer.

Another group's cancer that Aurora inhibitor may be particularly suitable for treatment is hematology cancer, particularly leukemia.Therefore, in another embodiment, the compound of formula (I) is used to treat hematology cancer, particularly leukemia.Specific leukemia is selected from acute myelogenous leukemia (AML), chronic with cellularity leukemia (CML), B-cell lymphoma (amphicyte) and acute lymphoblastic leukemia (ALL) (or being called as acute lymphoblastic leukemia).In one embodiment, described leukemia is selected from acute myelogenous leukemia, myelodysplastic syndrome and chronic granulocytic leukemia recurrence or difficult medical treatment.

One group of cancer comprises human breast carcinoma (for example primary mammary tumor, without tubercle mammary cancer, the invasive duct adenocarcinoma of breast, non-endometrioid mammary cancer); And lymphoma mantle cell.In addition, other cancer also has carcinoma of the colon and rectum and carcinoma of endometrium.

Another group's cancer comprises the hematopoiesis tumour of Lymphoid tissue system, for example leukemia, lymphocytic leukemia, lymphoma mantle cell and B-cell lymphoma (as dispersivity large B cell lymphoid tumor).

A kind of specific cancer is lymphocytic leukemia.

Another kind of specific cancer is lymphoma mantle cell.

Another kind of specific cancer is dispersivity large B cell lymphoid tumor.

Also think compound of the present invention, and particularly these have aurora kinase and suppress active compound and particularly can be used for treatment or prevention and exist aurora kinase level to raise about or be characterised in that the type of cancer of aurora kinase level rising, for example related cancer in the application's beginning part.

Can measure the activity of the compounds of this invention as cell cycle protein dependent kinase, aurora kinase and glycogen synthase kinase-3 inhibitors with the test described in embodiment below, and can use IC ₅₀value is determined the activity level that given compound shows.The preferred compound of the present invention is that these have lower than 1 μ M, more preferably less than the IC of 0.1 μ M ₅₀the compound of value.

the advantage of compound of the present invention

Compound of the present invention (for example compound of embodiment 24,62,63 and 64) has advantages of many compounds of the prior art that are better than.For example, compound of the present invention (seeing Table A) is particularly to Aurora A and the kinase whose selectivity of B and effect enhancing.

table A: 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline -2-yl)-1H-pyrazoles-4-yl] inhibition to aurora kinase in vitro of-urea

Aurora kinase	IC 50(nM)
		Aurora-A	52%, under 3nM
Aurora-B	58%, under 3nM

Vitro kinase activity is to measure according to the scheme described in embodiment 75 and 76.

Compound of the present invention other be better than prior art compound be also advantageous in that it has different susceptibility (seeing table B and embodiment 81 below) to P450 enzyme.

table B:1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline -2-yl)-1H-pyrazoles-4-yl]-urea is to expressed Cytochrome P450 isoform in-vitro Inhibitory Effect

P450 hypotype	IC50(μM)
		CYP1A2	＞10
CYP2D6	＞10
		CYP3A4	＞10
CYP2C9	＞10
		CYP2C19	＞10

In addition, other advantages that are better than prior art compound of compound of the present invention are that it shows some improvement in drug metabolism and pharmacokinetics properties.Compound of the present invention particularly has the plasma proteins combination of reduction.In tested all kinds, the compound of embodiment 24,62,63 and 64 and the combination of plasma proteins all quite restraining, its scope be from rat plasma 61% in mice plasma 82%.It has advantages of these compounds can to obtain in body circulation more free drug to reach suitable site of action and brings into play its therapeutic action.For tumour, the free fraction increase of performance pharmacotoxicological effect may cause effect of improving, thereby makes to reduce dosage.

Compound of the present invention (for example embodiment 24,62,63 and 64) is in hyperplasia and bacterium colony generation (clonogenic) test (for example, in the test described in embodiment 79 and 80), also show the cytoactive of improvement at antagonism wide region solid tumor cell aspect being, thereby show that it has the antitumour activity of improvement (table C).These data show that the treatment of carrying out with these compounds, compared with normal cell, has different effects to tumour cell.In the outpost of the tax office (checkpoint) compromise tumour cell, owing to suppressing to have destroyed mitotic division by aurora kinase, suppressed division of cytoplasm and having walked around the spindle body outpost of the tax office, so compounds for treating has caused multinucleation.Show that this multinucleation seems to have caused necrocytosis.On the contrary, in the normal outpost of the tax office competent cell (competent cells) of processing with compound, in compound treatment after 24 hours, a few cell is by multinucleation or death, there is on the contrary the cell of higher proportion to experience reversible G2/M inhibition, once and remove compound, reenter its cell cycle.These differences of its effect can reflect such practical work, if there is not chromosome segregation accurately, normal cell has the outpost of the tax office that stops its cell cycle in position, as the postmitotic p53-dependency outpost of the tax office.For tumour cell, there are not these outposts of the tax office, thereby mitotic division is proceeded and multinucleation occurred.

table 3:1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline -2-yl)-1H-pyrazoles-4-yl] restraining effect of-urea to the formation of tumour cell bacterium colony

^*the expression of+expression wild type p53;-represent not have p53 expression or p53 there is no function.

In addition, the solubleness of the salt form of the compounds of this invention in the aqueous solution is improved and shows better physicochemical property, for example, have lower logD.

the preparation method of formula (I) compound

In this section, unless stated otherwise, otherwise as please all other parts in basis, in the time relating to formula (I), also comprise its all other groups and embodiment of formula (II), (III), (XXX) and definition here.

The compound of formula (I) can be prepared according to synthetic method well known to the skilled person.

For example, wherein A is that the compound of the formula (I) of a key (, wherein A and carbonyl form a kind of amido linkage) can be by by the compound of formula (X):

With carboxylic acid R ¹-E-CO ₂h or its reactive derivatives react to be prepared under the standard conditions that form acid amides.

Coupled reaction between carboxylic acid and amine (X) can be carried out in the case of existing the reagent that forms peptide bond common type.The example of such reagent comprise 1,3-dicyclohexyl carbodiimide (DCC) (people such as Sheehan, J.Amer.Chem Soc.1955, 771067), 1-ethyl-3-(3 '-dimethylaminopropyl)-carbodiimide (EDC) (people such as Sheehan, J.Org.Chem., 1961,26,2525), take urea (uronium) as basic coupler as O-(7-azepine benzo triazol-1-yl)-N, N, N ', N '-tetramethyl-urea hexafluorophosphate (HATU) (L.A.Carpino, J.Amer.Chem.Soc., 1993 1154397) with take phosphorus (phosphonium) as basic coupler as 1-benzo-triazole oxygen base three (pyrrolidino) phosphorus hexafluorophosphate (PyBOP) (people such as Castro, Tetrahedron Letters, 1990 31, 205).Advantageously use (people such as Konig, Chem.Ber., 103,708,2024-2034) will be combined with 1-hydroxyl azepine benzotriazole (HOAt) or I-hydroxybenzotriazole (HOBt) take carbodiimide as basic coupler.Preferred coupling reagent comprises the EDC and the DCC that combine with HOAt or HOBt.

Described coupled reaction normally non-aqueous aprotic solvent as acetonitrile, diox, dimethyl sulfoxide (DMSO), methylene dichloride, dimethyl formamide or N-Methyl pyrrolidone in, or carry out in the aqueous solvent optionally with one or more mixable cosolvent.This reaction can at room temperature be carried out, or in the lower situation of the reactivity of reactant (for example, in the situation of the phenyl amines as poor in the electronics of sulfuryl amine group with electron-withdrawing group), this reaction can be carried out at the temperature of suitable rising.Can there is the alkali that interference can not occur in this reaction, for example tertiary amine is as carried out in the situation of triethylamine or DIPEA.

For alternative selection, can use the reactive derivative of described carboxylic acid, for example acid anhydrides or chloride of acid as altogether.With reactive derivative as reacting normally of carrying out of acid anhydrides by by as described in amine and acid anhydrides at room temperature in the situation that having alkali as pyridine, stirred.

The amine of formula (X) can be by being also originally prepared the nitro-compound of corresponding formula (XI) under standard conditions.This reduction for example can complete by room temperature carry out catalytic hydrogenation at polar solvent in as ethanol or dimethyl formamide in the situation that existing catalyzer to drape over one's shoulders carbon as palladium.

Nitro-the compound of formula (XI) can pass through nitro-pyrazole carboxylic acid of formula (XII):

With 4-morpholine-4-ylmethyl-benzene-1,2-diamine reactant (forming wherein M is the compound of D1) or 4,5-dimethoxy-benzene-1,2-diamine reactant (forming wherein M is the compound of D2) is prepared.

Reaction between described diamines and carboxylic acid (XII) can be as mentioned above in the situation that having reagent as DCC or EDC in the situation that there is HOBt, under described before acid amides coupling condition, carry out, thereby obtain intermediate o-amino-phenyl-acid amides (not showing), then make its cyclisation, thereby form benzoglyoxaline ring.Last cyclisation step normally by heating to carry out under refluxing in the situation that there is acetic acid.

In route 1, narrated a kind of illustrative reaction scheme, it has shown that wherein M is the preparation of formula (X) compound of group D1.

Route 1

In can embodiment part below, find the representative condition of each step in route 1.

Wherein M is that the compound of group D2 can be prepared by similar mode, just with 4,5-dimethoxy-benzene-1, and the diamines (XVI) in 2-diamines replacement route 1.

Therein A be the another kind of formula (I) compound of a key synthetic in, can also be by diamines---4-morpholine-4-ylmethyl-benzene-1,2-diamines and 4,5-dimethoxy-benzene-1,2-diamines reacts with the carboxylic acid that A is wherein the formula (XVII) of a key, thus the compound of the formula of obtaining (I).

Described diamines and carboxylic acid (XVII) react can with the condition of the conditional likelihood of preparing nitro-compound (XI) recited above under carry out.The carboxylic acid of formula (XVII) can be prepared by the reaction sequence shown in route 2.

As shown in route 2; can be by reacting with thionyl chloride substituted or unsubstituted 4-nitro-3-pyrazole carboxylic acid (XVIII) esterification; thereby obtain chloride of acid intermediate, then by itself and ethanol synthesis, thereby form ethyl ester (XIX).Or, can in the situation that there is an acidic catalyst (an one example is thionyl chloride), react to carry out described esterification by alcohol and carboxylic acid shown in general.This reaction at room temperature uses esterifying alcohol (for example ethanol) to carry out as solvent typically.Then, can drape over one's shoulders carbon by its nitroreduction with palladium according to standard method, thereby obtain amine (XX).By amine (XX) and suitable carboxylic acid R ¹-E-CO ₂h is becoming to carry out coupling under the condition of acid amides with described these conditions are above same or analogous, thereby obtains acid amides (XXI).Then, can make the hydrolysis of ester group of acid amides (XXI) in as methyl alcohol at the mixable solvent of polar water as sodium hydroxide with alkali metal hydroxide, this hydrolysis is normally at room temperature carried out.

Route 2

Wherein A is NR ²the compound of formula (I) can be prepared with the standard method of synthetic urea.For example, this compounds can pass through by the aminopyrazole compound of formula (X) and by the formula R of suitable replacement ¹the isocyanic ester of-E-N=C=O reacts to be prepared in as DMF at polar solvent.Described reaction is at room temperature carried out easily.

Another kind of alternative mode is that the urea of formula (I) can pass through the amine of formula (X) and formula R ¹-E-NH ₂amine in the situation that there is carbonyl dimidazoles (CDI), react to be prepared.This reaction for example, is undertaken by heating (using microwave heater) to the temperature that is up to approximately 150 ℃ in as THF at polar solvent typically.

Not using CDI, can there is not the alkali that disturbs as completed at room temperature or lower temperature in as methylene dichloride at solvent triethylamine in the case of existing by triphosgene (two (trichloromethyl) carbonic ether) in the reaction that described two kinds of amine couplings form described urea yet.

Instead the another kind of CDI is selected, and also can replace triphosgene with phosgene.

In described many reactions, may one or more radical protections must be got up prevent on the undesirable position of this molecule, react in the above.Blocking group (Protective Groups in Organic Synthesis) (T.Green and P.Wuts in organic synthesis; The 3rd edition; John Wiley and Sons, 1999) in, can find the example of blocking group and functional group is protected and de-protected method.

Hydroxyl for example can be for example with ether (OR) or ester (OC (=O) R), for example, with tert-butyl ether; Benzyl, diphenyl-methyl (diphenyl methyl) or trityl (trityl group) ether; Trimethyl silyl or tert-butyl dimetylsilyl ether; Or ethanoyl ester (OC (=O) CH ₃,-OAc) form protect.Aldehydes or ketones base for example can be respectively with acetal (R-CH (OR) ₂) or ketal (R ₂c (OR) ₂) form protect, wherein carbonyl (> C=O) is converted to diether (> C (OR) by reacting with for example primary alconol ₂).Can there is this aldehydes or ketones base of easily regenerating that is hydrolyzed sour in the situation that with a large amount of excessive water.Amido for example can be with acid amides (NRCO-R) or urethane (NRCO-OR), for example, with: methane amide (NHCO-CH ₃); Benzyloxy acid amides (NHCO-OCH ₂c ₆h ₅,-NH-Cbz); With uncle-butoxy acid amides (NHCO-OC (CH ₃) ₃,-NH-Boc); 2-xenyl-2-propoxy-acid amides (NHCO-OC (CH ₃) ₂c ₆h ₄c ₆h ₅-NH-Bpoc), with 9-fluorenyl methoxy acid amides (NH-Fmoc), with 6-nitro black false hellebore (veratryl) oxygen base acid amides (NH-Nvoc), with 2-trimethylsilylethoxy) acid amides (NH-Teoc), with 2; 2,2-, tri-chloroethoxy acid amides (NH-Troc), protect with allyloxy acid amides (NH-Alloc) or with the form of 2 (phenyl sulfonyl) oxyethyl group acid amides (NH-Psec).For for example cyclic amine of amine and heterocycle N-H group, other blocking group comprises that tosyl group (tosyl) and methylsulfonyl (mesyl) and benzyl are as p-methoxy-benzyl (PMB).Carboxylic acid group can be with ester, for example, with: C _1-7alkyl ester (for example, methyl esters; Uncle-butyl ester); C _1-7haloalkyl ester (for example, C _1-7tri haloalkyl ester); Three C _1-7alkyl silyl-C _1-7alkyl ester; Or C _5-20aryl-C _1-7alkyl ester (for example, benzyl ester; Nitrobenzyl ester); Or with acid amides, for example, protect with the form of methane amide.Thiol group for example can be with thioether (SR), for example, with: benzyl thioether; Acetylamino methyl ether (S-CH ₂nHC (=O) CH ₃) form protect.

The acid salt of (C) group of composition formula (I) can be at synthetic parent compound 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] be formed during-urea, or can be by the free alkali of parent compound is changed into required salt, or form by a kind of salt of parent compound being changed into the another kind of required salt of parent compound.Parent compound 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea (compound of formula (IA)) can be prepared by the method described in route 3 below.

As shown in route 3, by 3,4-dinitro carboxylic acid (XIII) (a kind of compound that can obtain by commercial sources) changes into morpholide (morpholide) (XXI).Can be by acid (XIII) being changed into reactive derivative as the acid amides of chloride of acid as described in forming with standard method.For example, can, by heating, then form described chloride of acid by removing excessive thionyl chloride with methylbenzene azeotropic together with excessive thionyl chloride under the reflux temperature of this thionyl chloride.

Can combine with boron trifluoride dinitrobenzene benzyl morpholine (XXIII) is processed morpholide (XXI) is reduced as sodium borohydride by the reductive agent with suitable.The temperature that this reduction reaction is reducing in as tetrahydrofuran (THF) at anhydrous solvent typically is for example carried out at the temperature of 0-5 ℃.Then, can under standard conditions, this dinitrobenzene benzyl morpholine (XXIII) be reduced into diamino benzyl morpholine (XXIV), for example, can reduce by the catalytic hydrogenation of at room temperature carrying out in as ethanol at polar solvent in the situation that existing catalyzer to drape over one's shoulders carbon as palladium.

Then, this diamino benzyl morpholine (XXIV) is reacted with 4-nitropyrazole-3-carboxylic acid that can obtain by commercial sources, thereby form nitropyrazole base-benzoglyoxaline (XXV).Can be by using peptide coupling reagent if promote to form with aromatic amine O-(benzotriazole-1-yl)-N of amido linkage, N, N ', N '-tetramethyl-urea a tetrafluoro borate (TBTU) first forms a kind of amido linkage and forms described nitropyrazole base-benzoglyoxaline (XXV) between described carboxylic acid and diamino benzyl compounds (XXIV).Then, by heating in excessive Glacial acetic acid, for example, at the temperature of about 65 ℃, heat to make this midbody acid amide (not shown) cyclisation to form nitro-pyrazolyl-benzoglyoxaline (XXV).

Can under standard conditions, this nitropyrazole base-benzoglyoxaline (XXV) be reduced into corresponding amine (XXVI), for example, can reduce by the catalytic hydrogenation of at room temperature carrying out in as ethanol or dimethyl formamide at polar solvent in the situation that existing catalyzer to drape over one's shoulders carbon as palladium.

Also can this amine (XXVI) be changed into urea (IA) again with the standard method of synthetic urea, for example can pass through amine (XXVI) and 2,6-difluorophenyl-isocyanic ester polar solvent as THF at room temperature or at lower temperature, for example at the temperature of 0-5 ℃, react to transform.

Can prepare acid salt of the present invention with the free alkali form of urea (IA).

Route 3

Salt of the present invention can be by ordinary method as in pharmaceutical salts: character, select, and application (Pharmaceutical Salts:Properties, Selection, and Use), P.HeinrichStahl (Editor), Camille G.Wermuth (editor), ISBN:3-90639-026-8, Hardcover, the 388th page, the method described in August, 2002 is prepared by described free alkali.For example, described salt can be by reacting described free alkali to be prepared in water or organic solvent or in the mixture of the two with suitable acid; Conventionally use non-aqueous media as ether, ethyl acetate, methyl alcohol, ethanol, Virahol or acetonitrile.

On the other hand, the invention provides one and prepare 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] method of acid salt of-urea, the method comprises formation 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] solution of-urea free alkali in the mixture of solvent (being generally organic solvent) or solvent, this solution is processed with acid, thereby formed the precipitation of described acid salt.

Described acid is added into be arranged in the form that is dissolved in the solution of the mixable solvent of solvent wherein with described free alkali conventionally.

It can be a kind of its acid salt insoluble solvent therein that described free alkali starts to be dissolved in solvent wherein.Or, the solvent that described free alkali starts to be dissolved in wherein can be the solvent that a kind of described acid salt dissolves therein at least partly, add wherein subsequently described acid salt more insoluble different solvents therein, thereby this salt is precipitated out from solution.

For example, in a kind of method of preparation salt of the present invention, described free alkali is dissolved in the first solvent (it can be that ethyl acetate or ethyl acetate and alcohol are as the mixture of methyl alcohol), then add wherein the solution (for example strong solution or saturated solution) of sour example hydrochloric acid in the second solvent (it can be that ether is as ether Huo dioxin (dioxin)), thereby form the precipitation of described acid salt, then collect this precipitation, for example, collect by filtration.

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl] preparation method of-urea

In our embodiment and superincumbent route 1 and 3 of earlier application WO 2005/002552, disclosing [3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-acid amides can be with comprising that the step order of step is prepared below:

(i) by 4-morpholine-4-ylmethyl-benzene-1,2-diamines and 4-nitro-1H-pyrazoles-3-carboxylic acid in the situation that there is 1-ethyl-3-(3 '-dimethylaminopropyl)-carbodiimide (EDC) and I-hydroxybenzotriazole (HOBt) at N, in dinethylformamide (DMF), react, thereby obtain 5-morpholine-4-ylmethyl-2-(4-nitro-1H-pyrazole-3-yl) 1H-benzoglyoxaline; With

(ii) by draping over one's shoulders with palladium that charcoal is processed under hydrogen atmosphere and by its nitroreduction;

Or

(i) by 4-amino-1H-pyrazole-3-carboxylic ester and suitable carboxylic acid in the situation that there is 1-ethyl-3-(3 '-dimethylaminopropyl)-carbodiimide (EDC) and I-hydroxybenzotriazole (HOBt) at N, in dinethylformamide (DMF), react or it is reacted in the situation that there is triethylamine with suitable chloride of acid, thereby forming 4-acid amides-1H-pyrazole carboxylic acid; With

(ii) by 4-morpholine-4-ylmethyl-benzene-1,2-diamines reacts in the situation that there is 1-ethyl-3-(3 '-dimethylaminopropyl)-carbodiimide (EDC) and I-hydroxybenzotriazole (HOBt) with suitable 4-acid amides-1H-pyrazole carboxylic acid in dimethyl formamide (DMF), thereby obtains [3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-acid amides.

Have been found that now except this nitro-pyrazole compound and described diamines being reacted and then its nitroreduction being become to amine; or by outside this acid amides-pyrazoles and described diamine reactant; this amino-pyrazoles can also be reacted with described diamines, condition is that the amino of this amino-pyrazol is suitably protected.Then, can be by the product cyclisation of this reaction, thus form benzoglyoxaline.In addition, also find to complete the operation of removing amine protecting group group and being cyclized into benzoglyoxaline by a step.

Therefore, on the other hand, the invention provides the method for a kind of preparation formula (XXVII) or compound or its salt (XXVIII):

or

The method comprises:

(i) be a kind of compound of formula (XXIX) of amine-blocking group by PG wherein:

(ii) with the compound of formula (XXXI):

In organic solvent, in the situation that having coupler as EDC and HOBt, react:

Formula (XXVIII) is the regional isomer (regioisomer) of (XXVII).

Described amine-blocking group PG can be any blocking group known when amine groups being protected under the condition used of method in the above, sees the people such as such as Green recited above.Therefore, for example, nitrogen can be with acid amides (NCO-R) or urethane (NCO-OR), for example, with: methane amide (NCO-CH ₃); Benzyloxy acid amides (NCO-OCH ₂c ₆h ₅,-NH-Cbz); With uncle-butoxy acid amides (NCO-OC (CH ₃) ₃, N-Boc); 2-xenyl-2-propoxy-acid amides (NCO-OC (CH ₃) ₂c ₆h ₄c ₆h ₅n-Bpoc), 9-fluorenyl methoxy acid amides (N-Fmoc), 6-nitro black false hellebore oxygen base acid amides (N-Nvoc), 2-trimethylsilylethoxy) acid amides (N-Teoc), 2; the form of 2,2-, tri-chloroethoxy acid amides (N-Troc), allyloxy acid amides (N-Alloc) or 2-(phenyl sulfonyl) oxyethyl group acid amides (N-Psec) is protected.Other blocking group of amine comprises that benzyl is as p-methoxy-benzyl (PMB).Preferably amine protecting group group is urethane (NCO-OR), for example, and benzyloxy acid amides (NCO-OCH ₂c ₆h ₅,-NH-Cbz), or uncle-butoxy acid amides (NCO-OC (CH ₃) ₃, N-Boc); Or allyloxy acid amides (N-Alloc).In one embodiment, described blocking group PG is blocking group APG, and it is the amine protecting group group that can be removed under acidic conditions.Such group comprises urethane.A kind of particularly preferred urethane blocking group is the uncle-butoxy carbonyl that can be removed under acidic conditions.

In one embodiment; then; from formula (XXVII) or compound (XXVIII), remove described blocking group PG and replace blocking group, APG, thereby the formula of formation (XXVIIa) or compound (XXVIIIa).

The compound of a kind of particularly preferred formula (XXIX) is the compound of formula (XXXII) below:

The present invention also provides the new chemical intermediate itself of formula (XXXII).

The present invention also provides formula (XXVII) or new chemical intermediate itself (XXVIII), for example below formula (XXVIIa) or new chemical intermediate (XXVIIIa).Therefore, the invention provides 4-amino-1H-pyrazoles-3-carboxylic acid (2-amino-4-morpholine-4-ylmethyl-phenyl)-acid amides as new chemical intermediate or 4-amino-1H-pyrazoles-3-carboxylic acid (2-amino-5-morpholine-4-ylmethyl-phenyl)-acid amides with and protection form.(the new chemical intermediate of (XXVII) is [3-(2-amino-4-morpholine-4-ylmethyl-phenyl amino formyl radical)-1H-pyrazoles-4-yl]-carboxylamine uncle-butyl ester to a kind of particularly preferred formula.The new chemical intermediate of a kind of particularly preferred formula (XXVIII) is [3-(2-amino-5-morpholine-4-ylmethyl phenyl amino formyl radical)-1H-pyrazoles-4-yl]-carboxylamine uncle-butyl ester.

In the time that blocking group PG is uncle-butoxy carbonyl, the total recovery of described method is higher than 85%.In addition, described method be also advantageous in that it uses relatively simple and cheap reagent and solvent and another to be also advantageous in that product is easy to purifying.

On the other hand, the invention provides the method that one is prepared 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine or its salt, the method comprises:

(i) formula (XXVIIa) or compound (XXVIIIa) are processed in solvent with acid:

or

Optionally it is heated; With

(ii) this reaction is neutralized.

Described amine-blocking group APG can be to relating to formula (XXVII) or (XXVIII) any known blocking group used when the defined amine groups of compound is protected above, and it is removed under method condition used in the above.

In step (i), can carry out with heating with sour reacting, for example can be heated to the temperature of 80 to 100 ℃.The solvent that can carry out therein step (i) is alcoholic solvent, and it can be for example ethanol.

In step (i); described blocking group preferably can be by processing with acid the group being removed; as Boc group, can select described acid, thereby as long as it is suitable for, by protonated described intermediate, its carbonyl is activated to carry out described cyclization.Suitable acid comprises that strong acid is as sulfuric acid, methylsulfonic acid or hydrochloric acid, and a kind of specific acid is hydrochloric acid.

After the reaction of step (i) completes, (for example can disappear and judge by parent material (XIIIa)), can neutralize described reaction.

In step (ii), use the alkali that interference can not occur." can there is not the alkali of interference " and refer to the alkali that can not react with prepared compound as sodium carbonate in term herein.Step (ii) is at room temperature carried out typically.

In step (ii), this reaction is neutralized and is for example neutralized to this reaction to be neutralized agent saturated and be positioned under 8.5 pH with neutralizing agent.

After step (ii), can be by described compound and carbonylation agent as 1,1 '-carbonyl dimidazoles (CDI) or phosgene equivalent react, and then with cyclopropylamine, it are processed.Phosgene equivalent comprises triphosgene or phosgene.Preferred carbonylation agent is 1,1 '-carbonyl dimidazoles (CDI).

Another kind of alternative mode is, described urea can pass through described amino-pyrazol---3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine, thereby for example react in THF and obtain ring-type urea at solvent in the situation that having alkali as pyridine with phenyl chloroformate, then with cyclopropylamine, it is processed to be prepared, or can be by by described amino-pyrazol and cyclopropyl isocyanic ester, (it can be reset and be made (as US 4 by the Curtius of cyclopropane-carboxylic acid trinitride, 313, 755 and US 4, 299, described in 778) react to be prepared.

Therefore, the present invention is that one is prepared 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl on the other hand] method of-urea or its salt, the method comprises:

(i) by formula, (compound of (XXVIIa) is processed in solvent with acid, optionally it is heated;

(ii) by this reaction neutralization;

(iii) product of step (ii) is reacted with carbonylation agent;

(iv) product of step (iii) is reacted with cyclopropylamine.

Step (iii) refluxing typically, for example, to the highest approximately 100 ℃, be more typically up to and carry out at 70-75 ℃.In step (iii), this reaction can be carried out in as tetrahydrofuran (THF) at polar aprotic solvent.Carbonylation agent can be compound as 1,1 '-carbonyl dimidazoles (CDI) or phosgene equivalent are as triphosgene or phosgene.Preferred carbonylation agent is 1,1 '-carbonyl dimidazoles (CDI).

Step (iv) is typically in heating, for example, be heated to be up to carry out at the temperature of approximately 100 ℃.

After step (iv), can carry out salt conversion or recrystallization (for example using 2-propyl alcohol or ethanol as solvent) to increase its purifying and to provide a kind of crystalline form to its product.

Step (iii) has above provided midbody compound and/or its regional isomer (XXXIIIa) of formula (XXXIII):

Then, formula (XXXIII) and intermediate (XXXIIIa) (can be separated if necessary) are reacted with cyclopropylamine, thus the compound of the formula of obtaining (XXX).

Therefore, on the other hand, the invention provides one and prepare the method for defined formula (XXX) compound here, the method comprises reacts formula (XXXIII) or compound (XXXIIIa) with cyclopropylamine, and optionally forms thereafter the acid salt of formula (XXX) compound.This reaction typically polar aprotic solvent as N-Methyl pyrrolidone in, preferably raise temperature as higher than 80 ℃, more typically higher than 90 ℃, for example, at the temperature of 95 ℃ to 105 ℃, carry out.

Also can with aforesaid method prepare part A in its Chinese style (I) be NH group the compound of defined other formula (I) here with and group.

Therefore the part A that, the present invention provides one to prepare in defined its Chinese style (I) here is on the other hand the method for formula (I) compound of NH group; The method comprises the compound of (i) formula (XXXIII) and/or its regional isomer (XXXIIIa), or (ii) compound and/or its regional isomer (XXXIVa) of formula (XXXIV):

With formula R ¹-E-NH ₂compound react, preferably react in as N-Methyl pyrrolidone at polar aprotic solvent, preferably in the temperature raising as higher than 80 ℃, more typically higher than 90 ℃, for example at the temperature of 95 ℃ to 105 ℃, react, and optionally form thereafter the acid salt of formula (I) compound.

The present invention also provides formula (XXXIII), (XXXIIIa), (XXXIV) and new chemical intermediate (XXXIVa).

In another embodiment, prepare the method for 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine or its salt in the above or prepare 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl above] formula in the method for-urea or its salt (compound of (XXVIIa) can be with comprising that the method for step is prepared below:

(i) be the amine-blocking group that can remove with acid by PG wherein---the compound of the formula (XXIX) of APG;

(ii) in the situation that having coupler as EDC and HOBt, react in organic solvent with the compound of formula (XXXI).

Thereby method described here optionally has described salt recrystallization is obtained to a kind of crystalline form, the example other step of crystalline form as herein defined.

purification process

Compound separation of the present invention out and to it can be carried out to purifying by many many methods well known to the skilled person, the example of these class methods comprises that chromatographic technique for example, as column chromatography (dodging column chromatography) and HPLC.Preparation property LC-MS be a kind of for to little organic molecule as described herein compound carry out standard and the effective means of purifying.Can change that crude product material is separated better and improve the detection of MS to sample liquid phase chromatography (LC) and the method for mass spectroscopy (MS).To comprise and change post, volatility eluent and properties-correcting agent and gradient the optimization of preparation property gradient LC method.These methods are well-known when preparation property LC-MS method is optimized in prior art, then with it, compound are carried out to purifying.At Rosentreter U, Huber U.; Best fraction in preparation property LC/MS is collected; J CombChem.; 2004; 6 (2), 159-64 and Leister W, Strauss K, Wisnoski D, Zhao Z, Lindsley C., for the foundation (Development of acustom high-throughput preparative liquid chromatography/massspectrometer platform for the preparative purification and analyticalanalysis of compound libraries) of the preparation purifying of compound library and the self-defining high yield property the prepared liquid chromatography/mass spectrometry instrument platform of analytical analysis; J Comb Chem.; 2003; 5 (3); In 322-9, these class methods are described.

Experimental section is below described this type systematic of one that compound is carried out to purifying by preparation property LC-MS, but those skilled in the art will recognize that the alternative means that can use these system and methods described here.Particularly, can be that basic method replaces inversion method described here in order to the positive property prepared LC.Great majority preparation property LC-MS system is utilized anti-phase LC and volatile acidic properties-correcting agent, and this is because the method very effectively and because of its eluent can be sprayed mass compatible with positive ion electrospray for micromolecular purifying.Or can be with using other chromatographic solution for example positive LC, or as the carrying out of being summarized in above-mentioned analytical procedure buffering moving phase, alkaline properties-correcting agent etc. to as described in compound carry out purifying.

recrystallization

Formula (I) compound with and salt, particularly 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea with and the recrystallization method of salt can be undertaken by method well known to the skilled person, for example can be referring to (P.Heinrich Stahl (Editor), Camille G.Wermuth (Editor), ISBN:3-90639-026-8, pharmaceutical salts handbook: character, select, and application (Handbook ofPharmaceutical Salts:Properties, Selection, and Use), the 8th chapter, publisher: Wiley-VCH).In the time directly separating from this reaction mixture, the product being obtained by organic reaction is seldom pure.If described compound (or its salt) is solid, can carry out purifying and/or crystallization to it by the solvent recrystallization with suitable.Good recrystallization solvent should dissolve a proper amt material to be purified at elevated temperatures, but at lower temperature, only can dissolve a small amount of described material.It at low temperatures should dissolved impurity or dissolved impurity not at all.Finally, described solvent can be easily removed from the product that this has been purified.This often means that described solvent has relatively low fusing point and those skilled in the art by the recrystallization solvent of understanding for predetermined substance, if or can not obtain for information about time, can test some solvents.In order to obtain the good yield of the material being purified, dissolve all described impuritys with the hot solvent of minimum.In practice, thus use higher than the solvent of aequum 3-5% and make this solution can be by unsaturated.If described impure compound is included in impurity insoluble in this solvent, can, by removing by filter these impurity, then make this solution crystallization.In addition,, if described impure compound comprises the coloring matter that is not the natural trace containing of described compound, can, by adding a small amount of decolorizing charcoal to be removed, filter and make its crystallization to it in this hot solution.When cooling this solution, conventionally automatically there is crystallization.If crystallization does not occur automatically for it, can carry out induced crystallization by this solution being cooled to lower than the temperature of room temperature or by the monocrystalline (crystal seed) that adds pure substance.Can also be by using anti-solvent (anti-solvent) carry out recrystallization and/or its yield is optimized.In this case, compound is dissolved in suitable solvent at elevated temperatures, filters, then add wherein other solvent that required compound has low solubility therein to help its crystallization.Then, conventionally go out this crystallization by isolated by vacuum filtration, washing, is then dried it, for example, it is dried in baking oven or is dried by dry (desiccation).

Crystallization comprises that with other example of method (it comprises evaporation step by steam crystallization, for example in the test tube sealing or airflow, evaporate), with by melt crystallization (crystallization technique handbook (Crystallization Technology Handbook), the 2nd edition, A.Mersmann edits, and 2001).

Particularly can be for example, using the compound recrystallization of formula (I) (using 2-propyl alcohol or ethanol as solvent) to increase its purity and to obtain a kind of crystalline form.

Conventionally the crystal being obtained is analyzed as X-ray powder diffraction (XRPD) or X-ray crystalline diffraction by X-ray diffraction method.

pharmaceutical preparation

Although described active compound or its salt can be administered alone, but it preferably for example, carries out administration with the form of the pharmaceutical composition (preparation) that comprises at least one active compound of the present invention and one or more pharmaceutically useful carriers, auxiliary agent, vehicle, thinner, weighting agent, buffer reagent, stablizer, sanitas, lubricant or other material well known to the skilled person, said pharmaceutical composition also optionally comprises other treatment or preventative dose; The reagent of for example reduction or alleviation some side effects relevant with chemotherapy.The particular instance of such reagent comprises that time length of antiemetic and prevention or the reduction neutropenia relevant with chemotherapy and prevention for example, because red blood corpuscle or white cell level reduce the reagent of the complication causing, erythropoietin (EPO), rHuGM-CSF (GM-CSF) and granulocyte colony-stimulating factor (G-CSF).

Therefore, the present invention also provides as the method for pharmaceutical composition as defined above and pharmaceutical compositions, the method comprises at least one active compound as defined above and one or more pharmaceutically useful carriers, vehicle, buffer reagent, auxiliary agent, stablizer or other material, and material is admixed together as described herein carries out.

Terminology used here " pharmaceutically useful " relates within the scope of rational medical judgment, be applicable to for example, contact with the tissue of individual (people) and can not produce excessive toxicity, stimulation, atopic reaction or other problem or complication, have compound, material, composition and/or the formulation of rational interests/risk ratio.Each carrier, vehicle etc. must be also " acceptable " in meaning that can be compatible with other composition of described preparation.

Therefore, the present invention provide on the other hand pharmaceutical compositions defined formula here (I) compound with and group suc as formula (II) and compound (III) and group thereof.

Described pharmaceutical composition can be for being suitable in oral, parenteral, part, nose, any form of eyes, ear, rectum, intravaginal or percutaneous dosing.At described composition, for the situation of parenteral admin, it can be configured to for intravenously, intramuscular, intraperitoneal, subcutaneous administration or for being directly delivered to target organ or tissue by injection, input or other transfer mode.Described transmission can be undertaken and can be by passive transmission or by utilizing suitable front pump to carry out by bolus injection (bolus injection), short-term input or input for a long time.

The pharmaceutical preparation that is applicable to parenteral admin comprises and can comprise oxidation inhibitor, buffer reagent, bacteriostatic agent and make said preparation and water-based and the non-aqueous aseptic parenteral solution of the solute that required taker's blood etc. oozes; With water-based and the non-aqueous sterile suspension that can comprise suspensoid and thickening material.At R.G.Strickly, solvability vehicle (SolubilizingExcipients in oral and injectable formulations) in oral and injection formulations, PharmaceuticalResearch, the 21st volume (2) 2004, the example to these materials in 201-230 page is described.In addition, its can also comprise cosolvent, ORGANIC SOLVENT MIXTURES, cyclodextrin complexing agent, emulsifying agent (being used to form and stable emulsion), be used to form liposome liposome component, be used to form polymer gel can gel (gellable) polymkeric substance, freezing drying protective agent and the combination for the reagent that especially the described activeconstituents of stable meltable form and the blood that makes described preparation and required taker etc. ooze.Described preparation may reside in single dose or multi-dose container, for example, in the ampoule and bottle of sealing, and it can be stored under lyophilize (lyophilized) condition, only need just to add wherein sterile liquid carrier before use, as water for injection.

If the distance between the pH value of the pKa of described medicine and described preparation is enough far away, can be by regulating pH that ionizable drug molecule is dissolved to required concentration.For intravenously and intramuscular administration, acceptable pH scope is 2-12, but for subcutaneous administration, acceptable pH scope is 2.7-9.0.The pH of described solution controls by the salt form of medicine, strong acid/alkali example hydrochloric acid or sodium hydroxide, or control by buffer agent solution, described buffer agent solution comprises the buffered soln being formed by glycine, Citrate trianion, acetate, maleate, succinate, Histidine, phosphoric acid salt, three (hydroxymethyl) aminomethane (TRIS) or carbonate without limitation.

In injection formulations, usually use the combination of the aqueous solution and water-miscible organic solvent/tensio-active agent (being cosolvent).Water-miscible organic solvent and tensio-active agent used in injection formulations comprise propylene glycol, ethanol, Liquid Macrogol, poly(oxyethylene glycol) 400, glycerine, N,N-DIMETHYLACETAMIDE (DMA), METHYLPYRROLIDONE (NMP without limitation; Pharmasolve), dimethyl sulfoxide (DMSO) (DMSO), Solutol HS 15, Cremophor EL, Cremophor RH 60 and polysorbate80.Conventionally (but not always being) can dilute such preparation before injection.

Propylene glycol, PEG 300, ethanol, Cremophor EL, Cremophor RH 60 and polysorbate80 are the mixable organic solvent of complete water and tensio-active agent and couplings each other used in the injectable formulation that can obtain by commercial sources.IV inject or IV input before, conventionally by least 2 times of the organic formulations dilutions of gained.

Or, can increase its solubleness in water by forming molecular complex with cyclodextrin.

Liposome is the closed spherical capsule by the overall diameter moisture core composition and that have < 100 μ m (overall diameter) of outside lipid bilayer (bilayer) film and inside.According to its hydrophobicity level, if medicine is encapsulated or be inserted in liposome, liposome can the hydrophobic medicine of solubilising appropriateness.If drug molecule becomes the integral part of bimolecular lamellar lipid membrane, liposome also can this hydrophobic drug of solubilising, and in this case, described hydrophobic drug is dissolved in the lipid part of lipid bilayer.A kind of typical Liposomal formulation comprises water, the phosphatide of have-5-20mg/ml, isotonic agent, pH 5-8 buffer reagent, and optional cholesterol.

Described preparation may reside in single dose or multi-dose container, for example, in the ampoule and bottle of sealing, and it can be stored under lyophilize (lyophilised) condition, only need just to add wherein sterile liquid carrier before use, as water for injection.

Described pharmaceutical preparation can be by being prepared the compound of formula (I) or its acid salt lyophilize.Lyophilize refers to a kind of process of composition freeze-dried.Therefore lyophilize and lyophilize (lyophilisation) can be used as synonym use here.A kind of typical process is by described compound dissolution, makes the preparation clarification of gained, is for example applicable to, in cryodesiccated container (bottle) by its sterile filtration and by its aseptic transferring to.In the situation of bottle, can with lyo-stoppers by its part beyond the Great Wall.Said preparation can be cooled to and freeze and by its lyophilize under standard conditions, then its sealing built, thereby form a kind of stable, dry lyophily (lyophile) preparation.Described composition has low residual moisture content conventionally, for example lower than this lyophile weight 5% for example lower than 1% weight.

This freeze-dried preparation for example can comprise other vehicle, thickening material, dispersion agent, buffer reagent, oxidation inhibitor, sanitas and tension regulator.Typical buffer reagent comprises phosphoric acid salt, acetate, Citrate trianion and glycine.The example of oxidation inhibitor comprises xitix, sodium bisulfite, sodium metabisulfite, MTG (monothioglycerol), thiocarbamide, butylated hydroxytoluene, butylated BHA and edetate.Sanitas can comprise phenylformic acid with and salt, Sorbic Acid with and alkyl ester, phenol, butylene-chlorohydrin, benzylalcohol, thimerosal, benzalkonium chloride and the cetylpyridinium chloride of salt, p-hydroxy-benzoic acid.If desired, can carry out adjustment of tonicity with described before buffer reagent and dextrose and sodium-chlor.

In Freeze Drying Technique, conventionally promote this process and/or provide volume (bulk) and/or mechanical integrity for lyophilize cake (lyophilized cake) with swelling agent (bulking agents).Swelling agent refers in the time of lyophilize together with described compound or its salt, can provide the lyophilize cake of physically stable, better freezing dry process and rebuild rapidly and completely can be freely water-soluble solid particulate thinner.Can also solution etc. be oozed with swelling agent.

Described water-soluble swelling agent can be the conventional any pharmaceutically useful inert solids of lyophilize.Such swelling agent comprises that for example carbohydrate is as glucose, maltose, sucrose and lactose; Polyvalent alcohol is as sorbyl alcohol or N.F,USP MANNITOL; Amino acid is as glycine; Polymkeric substance is as polyvinylpyrrolidone; With polysaccharide as dextran.

The weight of swelling agent is generally approximately 1 to approximately 5 with the ratio of the weight of active compound, and for example approximately 1 to approximately 3, for example, in approximately 1 to 2 scope.

Or it can be provided with solution form, described solution can be strong solution and can be sealed in suitable bottle.Can by filter or suitable stage of process for preparation to described bottle with and inclusion autoclaving these formulations are carried out to sterilizing.Before transmission, may need provided preparation further dilute or prepare, for example need to be diluted in suitable aseptic input packing.

Can be prepared by sterilized powder, particle and tablet solution and the suspension of (Extemporaneous) injection immediately.

In preferred embodiment of the present invention, described pharmaceutical composition is for being suitable for i.v. administration, for example, by injecting or input the form of carrying out administration.

The present invention also can comprise aseptic pharmaceutically acceptable water-based or non-aqueous solution, dispersion, suspension or emulsion for the pharmaceutical composition of parenteral injection and for being just rearranged into before use the sterilized powder of aseptic injectable solution or dispersion.The example of suitable water-based and non-aqueous carrier, thinner, solvent or matrix (vehicles) comprise water, ethanol, polyvalent alcohol (as glycerine, propylene glycol, polyoxyethylene glycol etc.), carboxymethyl cellulose with and suitable mixture, vegetables oil (as sweet oil) and injectable organosilane ester as ethyl oleate.For example can by with coating material as Yelkin TTS, in the situation of dispersion by maintaining required granularity and by maintain suitable mobility with tensio-active agent.

Composition of the present invention can also comprise auxiliary agent as sanitas, wetting agent, emulsifying agent and dispersion agent.Can be by comprising various antibacterial agents and anti-mycotic agent, such as nipagin esters (paraben), butylene-chlorohydrin, phenol xitix etc. guarantee to prevent the effect of microorganism.Also wish to comprise that isotonic agent is as carbohydrate, sodium-chlor etc.Can postpone the reagent that absorbs and cause the absorption that extends injectable drug form as aluminum monostearate and gelatin by comprising.

If compound solubleness unstable or in aqueous medium in aqueous medium is low, it can be configured to the form of the enriched material that is arranged in organic solvent.Then, can use system this enriched material be diluted to lower concentration, and its time in short-term during administration is enough stable.Therefore, provide on the other hand a kind of pharmaceutical composition that comprises the non-aqueous solution being formed by one or more organic solvents completely, its like this administration or more generally before administration with suitable IV vehicle (salt solution, dextrose; Buffering or be not cushioned) it is diluted to (the solubilising vehicle (Solubilizing excipientsin oral and injectable formulations) in oral and injection formulations, Pharmaceutical Research, 21 (2), 2004, p201-230).The example of solvent and tensio-active agent has propylene glycol, PEG300, PEG400, ethanol, N,N-DIMETHYLACETAMIDE (DMA), METHYLPYRROLIDONE (NMP, Pharmasolve), glycerine, Cremophor EL, Cremophor RH 60 and polysorbate.Specific non-aqueous solution is made up of the propylene glycol of 70-80% and the ethanol of 20-30%.A kind of specific non-aqueous solution is made up of 70% propylene glycol and 30% ethanol.Another kind of solution is 80% propylene glycol and 20% ethanol.Conventionally diluted at least 2-times by these solvent couplings and conventionally inject (bolus) or IV input at IV before.The typical amounts that IV injects preparation is～50% for glycerine, propylene glycol, PEG300, PEG400, and is～20% for ethanol.The typical amounts of IV input preparation is～15% for glycerine, is 3%, and for propylene glycol, PEG300, PEG400 and ethanol, is～10% for DMA.

In preferred embodiment of the present invention, described pharmaceutical composition is for being suitable for i.v. administration, for example, by injecting or input the form of carrying out administration.For intravenous administration, described solution can be with itself form administration, or can before administration, be injected in infusion bag (comprising pharmaceutically useful vehicle, as 0.9% salt solution or 5% dextrose).

In another preferred embodiment, described pharmaceutical composition can be for being suitable for the form of subcutaneous (s.c.) administration.

The pharmaceutical dosage form that is suitable for oral administration comprises tablet, capsule, capsule sheet (caplets), pill, lozenge, syrup, solution, powder, particle, elixir and suspension, Sublingual tablet, wafer or patch and cheek patch.

The pharmaceutical composition of contained (I) compound can be prepared according to known technology, referring to for example, and Remington ' s Pharmaceutical Sciences, Mack PublishingCompany, Easton, PA, USA.

Therefore, tablet composition can contain the unitary dose of active compound and inert diluent or carrier as sugar or sugar alcohol etc.; Lactose, sucrose, sorbyl alcohol or N.F,USP MANNITOL; And/or not if sodium carbonate, calcium phosphate, calcium carbonate or Mierocrystalline cellulose or derivatives thereof are if methylcellulose gum, ethyl cellulose, Vltra tears and starch are as W-Gum derived from sugared thinner.These tablets for example can also contain such as bonding agent and granulation agent, for example, for example, for example, for example, if polyvinylpyrrolidone, disintegrating agent (expandable cross-linked polymer is as cross-linked carboxymethyl cellulose), lubricant (stearate), sanitas (parabens), oxidation inhibitor (BHT), buffer reagent (phosphoric acid salt or citrate buffer agent) and effervescent are as the standard analysis Citrate trianion/bicarbonate mixture.Such vehicle is well-known and does not here need to discuss in detail.

Capsule preparations can be hard capsule or soft capsule class preparation and the active ingredient that can comprise solid, semisolid or liquid form.Gelatine capsule can be synthesized or be formed derived from the equivalent of plant by animal gelatin or its.

Described solid dosage (such as tablet, capsule etc.) can, by dressing or not by dressing, still have a kind of dressing, for example protectiveness film clothing (for example wax or paint film (varnish)) or controlled release coat conventionally.Described dressing (for example Eudragit ^tMtype polymkeric substance) can be designed as desired location in gi tract and discharge active ingredient.Therefore, can select dressing, to degrade under some pH condition, thereby optionally in stomach or ileum or duodenum, discharge described compound in gi tract.

There is not dressing, or except dressing, described medicine can also be present in and comprise in the solid substrate of controlling release reagent, for example delayed release reagent of said control release reagent, and it can be suitable for selectivity under various acidity in gi tract or alkaline condition and discharge described compound.For example, or the dressing that described substrate material or retardance discharge can be taked the form of erodible polymkeric substance (maleic anhydride polymer), this material is etched during by gi tract in described formulation basic continous.Select as another kind, described active compound can be formulated in the transfer system that described compound perviousness control release can be provided.Perviousness discharges with other delayed release or extended release preparation and can be prepared according to method well known to the skilled person.

Described pharmaceutical composition comprises about 1% to about 95%, the activeconstituents of preferably approximately 20% to approximately 90%.Pharmaceutical composition of the present invention can be for example unit dosage, for example the form of ampulla, bottle, suppository, dragee (drag é es), tablet or capsule.

Pharmaceutical composition for oral administration can obtain like this, by activeconstituents and solid carrier are combined, if necessary, by gained granulating mixture, if then needed or necessary, this mixture is processed into tablet, dragee core or capsule adding after suitable vehicle.Can also be blended into and make the activeconstituents can be with in the amount diffusion or d/d plasticity carrier of being measured.

Composition for topical application comprises ointment, emulsifiable paste, spraying, patch, gel, liquid drops and inset (for example intraocular inset).This based composition can be prepared according to known method.

Composition for parenteral admin exists with the form of aseptic water-based or oily solution or fine suspensions conventionally, or can be for existing by the interim sterilized powder form of cutting apart very carefully building of sterile water for injection.

The example that is used for the preparation of rectum or intravaginal administration comprises vaginal suppository and suppository, and it for example can be formed by the effigurate plastic type of the tool that comprises described active compound (moldable) or waxy substance.

For can take by the composition of inhalation can inhalation of dust composition or the form of liquid or powder spray, and can carry out administration with the standard form that uses powder inhalator device or aerosol diverting device.Such device is well-known.For the administration of being undertaken by suction, described powder formulation comprises active compound and inert solid powdery thinner conventionally as lactose.

Described pharmaceutical preparation can be to be positioned at unitary package, and the form that is generally " patient packs (patient packs) " that comprise whole therapeutic process in blister packing is presented in patient.Separate from a kind of packing in enormous quantities pharmacist in patient's the situation of medicine supply, patient packing has advantages of and is better than tradition prescription, patient always approach be contained in described patient's packing at the packing interpolation thing being conventionally missed in patient's prescription.Show to be mingled with packing interpolation thing and improved the compliance of patient to doctor's indication.

Compound of the present invention exists with unit dosage forms conventionally, and same, conventionally comprises the compound that is enough to provide required biologically active level.For example, can comprise 0.1 milligram to 2 grams activeconstituents, for example 1 nanogram to 2 milligram activeconstituents for the preparation of oral administration.In this scope, the specific sub-ranges (sub-ranges) of compound is that 0.1 milligram to 2 grams activeconstituents (is more generally as 10 milligrams to 1 gram, for example, 50 milligrams to 500 milligrams or 1 microgram to 20 milligram (for example 1 microgram to 10 milligram, for example 0.1 milligram to 2 milligrams activeconstituents).

For oral compositions, unit dosage can comprise 1 milligram to 2 grams, more typically comprises 10 milligrams to 1 gram, and for example 50 milligrams to 1 gram, for example active compound of 100 milligrams to 1 gram.

Described active compound will be delivered medicine to and be needed its patient (for example human or animal patient) with the quantity that is enough to obtain required therapeutic action.

methods for the treatment of

Think that the compound of defined formula (I), (II), (III), (XXX) and each group can be used for prevention or treats the various disease states or the illness that are mediated by cell cycle protein dependent kinase, glycogen synthase kinase-3 and aurora kinase here.Narrate the example of such morbid state or illness in the above.

Described compound is delivered medicine to the individuality that needs such administration conventionally, for example human or animal patient, preferably people.

Described compound conventionally with treatment or prevent useful and conventionally nontoxic quantity by administration.But, for example, some situation (suffering from the situation of life-threatening disease), the benefit of Medicine-feeding type (I) compound may be more important than the shortcoming of any toxic action or side effect, in this case, think and wish with these compounds of quantity administration with appropriate toxicity.

Described compound can be by long term administration to maintain useful therapeutic action or can be only by short-term administration.Or it can carry out administration with pulse or continuous mode.

The typical per daily dose of described compound can be per kilogram of body weight 100 piks to 100 milligram, it is more typically 5 nanograms to 25 mg/kg body weight, and be more typically 10 nanograms to 15 mg/kg body weight (for example 10 nanograms to 10 milligram, and be more typically 1 microgram/kg body weight to 20 mg/kg body weight, as 1 microgram to 10 mg/kg body weight), but in the situation of needs, can the higher or lower dosage of administration.

Described compound (for example compound 1-cyclopropyl base-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea or its salt as lactic acid salt or Citrate trianion) can be by administration every day or can be for example every 2 or 3 or 4 or 5 or 6 or 7 or 10 or 14 or 21 or 28 days repeat administrations.But, last, will be matched and will be considered by attending doctor with the character that is treated disease or physiology illness by the type of the compound quantity of administration and composition therefor.

Compound 1-cyclopropyl base-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea or its salt comprises with 1mg/m as an example of the per daily dose scheme of lactic acid salt (particularly Pfansteihl salt) or Citrate trianion ²/ sky-100mg/m ²/ day, particularly 1mg/m ²/ sky-10mg/m ²/ day, more especially 3-6mg/m ²/ sky (is equivalent to 2.5-5mg free alkali/m ²/ day) initial dose or with 2.5mg/m ²/ sky-1.5g/m ²/ day, particularly 25mg/m ²/ sky-600mg/m ²/ day, more especially 200-500mg/m ²/ sky is as 250mg/m ²/ sky or 45-200mg/m ²/ sky is as 45-150mg/m ²/ sky or 56-185mg/m ²/ sky (is equivalent to 45-150mg free alkali/m ²/ day) Lactated effective dose gives said compound (for example carrying out administration with the form of Pfansteihl salt), but in the situation of needs, can use higher or lower dosage.

In the specific administration time table of one (schedule), patient was by continuous (accontinuous) IV input 2 hours to 120 hours, for example 2 to 96 hours, the particularly 1-cyclopropyl-3-[3-of 24 to 72 hours (5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea or its salt is as lactic acid salt (particularly Pfansteihl salt) or Citrate trianion, and with required interval as repeated treatment every one to three week.

More specifically, patient can be by continuous IV input 24 hours every days 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea or its salt is as lactic acid salt (particularly Pfansteihl salt) or Citrate trianion input 5 days, and repetitive therapy week about, or input 48 hours and every two weeks repetitive therapy or input 72 hours and every three weeks repetitive therapy.

In the specific administration time table of another kind, the form that patient injects with each 2 hours IV was every 1,2 or 3 week, carry out once a day one week input 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea or its salt as lactic acid salt (particularly Pfansteihl salt) or Citrate trianion or every 1,2 or 3 week once, each 2 hours.

Can be by a kind of dosage regimen with the phase of not treating frequently as every one to two week, continuous IV input gave higher dosage as 1.5g/m in 24 to 48 hours ²/ day.Can be by the dosage regimen with longer continuing medication (still still thering is the administration/not administration of circulation) as every two to three weeks 48 to 72 hours, continuous IV inputted to give lower dosage.

Particularly, compound or 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of formula (I ')]-urea or its all salt as lactic acid salt or Citrate trianion, particularly lactic acid salt can be with 250mg/m ²/ day, every 3 weeks, the continuous IV input mode of 72 hours delivered medicine to patient.

In another embodiment, compound or 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of formula (I ')]-urea or its all salt can be delivered medicine to patient with the treatment cycle of a kind of five days as lactic acid salt or Citrate trianion, particularly lactic acid salt.

Finally, will match and will be considered by attending doctor to the quantity of drug compound and the type of composition therefor with the character that is treated disease or physiological conditions.

Here the compound of defined formula (I), (II), (III), (XXX) and group can be with the form of unique therapeutical agent by administrations, or it can carry out administration with conjoint therapy with one or more other compounds that is used for the treatment of particular disease states, and described particular disease states is ND cancer as hereinbefore defined for example.Can be comprised without limitation topoisomerase enzyme inhibitor, alkylating agent, metabolic antagonist, DNA bonding agent, microtubule inhibitors (target is in the reagent of tubulin) by other therapeutical agent of administration or use (no matter be parallel administration or with different timed interval administrations) or the example of therapy together from the compounds of this invention, specific example has cis-platinum, endoxan, Dx, Rinotecan, fludarabine, 5FU, taxanes, ametycin and radiotherapy.

Can comprise monoclonal antibody and signal transduction inhibitor from other example of the therapeutical agent of the compound administration together of defined formula (I), (II), (III), (XXX) here (no matter be parallel administration or with different timed interval administrations).

For the situation that CDK or Aurora inhibitor are combined with other therapies, can and carry out two or more treatments by different approach with each different administration time table.

The compound of formula (I) with a kind of, two kinds, three kinds, four kinds or multiple (preferably one or both, more preferably a kind of) in the situation of other therapeutical agent administration in conjoint therapy, can be by (in identical or different pharmaceutical preparation) or the order administration simultaneously of described compound.When by order administration, it can be for example, with the very near timed interval (in 5-10 minute) of distance or for example, with long period interval (interval 1,2,3,4 or a few hours, or even in the situation of needs with the longer timed interval of interval) carry out administration, the character of accurate dosage and described therapeutical agent () matches.

Compound of the present invention can also be with non-chemotherapeutic treatment as radiotherapy, photodynamic therapy, gene therapy; Use is combined in operation together with dietary control.

For with the conjoint therapy of other chemotherapeutics for, the compound of formula (I) and a kind of, two kinds, three kinds, four kinds or multiple other therapeutical agent for example can together be formulated in a kind of formulation that comprises two kinds, three kinds, four kinds or multiple therapeutical agent.Or each therapeutical agent can independently be prepared and be existed with the form of the test kit optionally with its operation instruction.

Those skilled in the art will be understood described dosage regimen and be used conjoint therapy by his or her general knowledge.

diagnostic method

Before the compound of Medicine-feeding type (I), can screen to determine that whether disease that this patient suffers from or may just suffer from or illness be disease or the illness of easily benefiting from the impact of the treatment that Aurora and/or the activated compound of cell cycle protein dependent kinase are carried out to patient.

For example, can determine whether this patient suffers from or illness or for example cancer of disease that may suffer from are that a kind of being characterized as causes CDKs hyperactivity hyperkinesia or a kind of gene unconventionality of the sensitization towards the approach to normal CDK activity or illness or the disease of protein abnormal expression to picking up from patient's biological sample analysis.Cause such abnormal example of the activation of CDK2 signal or sensitization to comprise that cyclin E raises (Harwell RM, Mull BB, Porter DC, Keyomarsi K.; J Biol Chem.2004 Mar 26; 279 (13): 12695-705) or p21 or p27 lose or have CDC4 modification (Rajagopalan H, Jallepalli PV, Rago C, Velculescu VE, Kinzler KW, Vogelstein B, a Lengauer C.; Nature.2004Mar 4; 428 (6978): 77-81).Have the rise of CDC4 variation or cyclin E, the tumour of particularly crossing expression or p21 or p27 forfeiture may be responsive especially to CDK inhibitor.Or or in addition, illness or the disease that can also determine whether patient suffers from or may just suffer to picking up from patient's biological sample analysis, for example cancer is to be characterized as illness or the disease that aurora kinase raises and therefore may be responsive especially to Aurora inhibitor.Term raises and comprises the expression of rising or cross and express, and comprises that gene amplification (being polygene copy) and the expression being caused by Transcription increase, and excessively active and activation, comprises the activation being caused by sudden change.

Therefore, can carry out the flag sign that a kind of diagnostic test is crossed expression, raised or activate to survey aurora kinase to patient, or can carry out a kind of diagnostic test to patient, to survey the rise of cyclin E or the forfeiture of p21 or p27 or to occur the flag sign of CDC4 varient.Term diagnosis comprises screening.Mark comprises genetic marker, comprises and for example measures the sudden change that DNA forms to determine Aurora or CDC4.Terms tag also comprises the characteristic that is characterised in that Aurora or cyclin E (comprising the mRNA level of enzymic activity, enzyme level, enzyme state (be for example phosphorylated or be not phosphorylated) and above-mentioned albumen) raise.The tumour with cyclin E rise or p21 or p27 forfeiture may be responsive especially to CDK inhibitor.Before treatment, can preferably screen tumour with regard to the rise of cyclin E or the forfeiture of p21 or p27.Therefore, can carry out diagnostic test to patient, to survey the flag sign of the rise of cyclin E or the forfeiture of p21 or p27.

Described diagnostic test normally carries out with the biological sample that is selected from tumor biopsy sample, blood sample (separation of Exfoliated tumor cells and enrichment), ight soil examination of living tissue (stool biopsies), saliva, chromosome analysis, Pleural fluid, peritoneal fluid or urine.

Have been found that (referring to people such as Ewart-Toland, (Nat Genet.2003 Aug; 34 (4): 403-12) susceptibility of the cancer of the individuality of a part of subgroup that), forms the Ile31 modification with STK gene (gene of BTAK) to some form increases.Therefore, think that such individuality of suffering from cancer suppresses active compound and is benefited had aurora kinase by use.Therefore, to suffering from or suspecting that the patient who suffers from cancer screens to determine whether he or she becomes a part for Ile31 modification subgroup colony.In addition also find, (the people such as Rajagopalan (Nature.2004 March 4; 428 (6978): 77-81)), in people's carcinoma of the colon and rectum and carcinoma of endometrium, in CDC4 (being also referred to as Fbw7 or Archipelago), there are some sudden change (people such as Spruck, Cancer Res.2002 August, 15; 62 (16): 4535-9).Determine that the individuality that carries sudden change in CDC4 may mean that this patient is particularly suitable for treating with CDK inhibitor.Before treatment, can preferably screen the tumour that has CDC4 modification.This screening process will relate to direct Sequencing conventionally, oligonucleotide microarray is analyzed or mutant specific antibody.

Having the activation mutant of Aurora or the tumour of Aurora rise (described Aurora comprises its any isoform) may be responsive especially to Aurora inhibitor.Before treatment, preferably filter out the tumour that there is the rise of Aurora or there is the Aurora of Ile31 modification (people such as Ewart-Toland, Nat Genet.2003 August; 34 (4): 403-12).The people such as Ewart-Toland have determined the common genetic variant (causing aminoacid replacement F31I) of a class in STK15, and it is preferably exaggerated and with dysploidy to a certain degree in human colon's tumour.These results play an important role consistent with the Ile31 modification of STK15 in human cancer susceptibility.Particularly, shown that this polymorphism in Aurora A is the genetic modifier fir that forms mammary cancer people such as (, Carcinogenesis, 2004,25 (11), 2225-2230) Sun.

Aurora A gene shine is to karyomit(e) 20q13 region, and it is for example usually amplified in breast, bladder, colon, ovary, carcinoma of the pancreas in many cancers.The patient who suffers from the tumour with this gene amplification may be to suppress to target the treatment sensitivity especially of aurora kinase.

Sudden change to for example Aurora isoform of albumen and rise and karyomit(e) 20q13 amplification is determined and the method analyzed is well known to a person skilled in the art.Some screening methods comprise that standard method is as reverse transcriptase-polymerase chain reaction (RT-PCR) or in situ hybridization without limitation.

In the screening of being undertaken by RT-PCR, by generating the cDNA copy of mRNA, then with PCR, this cDNA is increased the mRNA level in tumour is assessed.Method, Primer selection and the amplification condition of pcr amplification is all well known by persons skilled in the art.Nucleic acid operation and PCR carry out with standard method, as for example Ausubel, F.M. wait people to edit, popular scheme (Current Protocols in MolecularBiology in molecular biology, 2004, John Wiley & Sons Inc., or Innis, M.A. wait people to edit, PCR scheme: methods and applications guide (PCR Protocols:a guide to methods andapplications), 1990, Academic Press, carrying out like that described in San Diego.People such as Sambrook, 2001, the 3rd edition, molecular cloning: laboratory manual (MolecularCloning:A Laboratory Manual), is also described the reaction and the operation that relate to nucleic acid in Cold Spring Harbor Laboratory Press.Or, can use the RT-PCR test kit (for example Roche MolecularBiochemicals) that can obtain by commercial sources, or can use the US patent 4 being introduced into as a reference here, 666,828,4,683,202,4,801,531,5,192,659,5,272,057,5,882,864 and 6, method described in 218,529.

The example that is used for the hybridization in situ technique that mrna expression is assessed is fluorescence in situ hybridization (FISH) (seeing Angerer, 1987 Meth.Enzymol., 152:649).

In situ hybridization generally includes key step below: (1) fixes analyzed tissue; (2) this sample is carried out to prehybridization processing to increase the accessibility of target Nucleotide, and reduce non-specific binding; (3) mixture of nucleic acid is hybridized to the nucleic acid in biological structure or tissue; (4) post-hybridization washing to be to remove nucleic acid fragment not combined in described hybridization, and (5) survey the nucleic acid fragment of being hybridized.Conventionally probe used in such application is carried out to mark, for example, with radio isotope or fluorescent flag, it is carried out to mark.Preferred probe sufficiently long, for example, approximately 50,100 or 200 Nucleotide is to approximately 1000 or more Nucleotide, to guarantee carrying out specific cross with target Nucleotide () under stringent condition.Be described in Ausubel for the standard method of carrying out FISH, F.M. wait people to edit, popular scheme (Current Protocolsin Molecular Biology) in molecular biology, 2004, the fluorescence in situ hybridization of John Wiley & Sons Inc and John M.S: technology summary (Fluorescence in Situ Hybridization:TechnicalOverview) .Bartlett, the molecular diagnosis of cancer, method and scheme (Molecular Diagnosisof Cancer, Methods and Protocols), the 2nd edition, ISBN:1-59259-760-2, in March, 2004, pps.077-088, Series:Methods in MolecularMedicine.

Or other method of solid-phase immunoassay, Western blotting, 2 dimension sds polyacrylamide gel electrophoresis, ELISA, flow cytometry and the detection differential protein well known in the prior art that can carry out by the immunohistochemistry of tumor sample, with microtiter plate is assessed the protein product of being expressed by mRNAs.These detection methods will comprise use site specific antibody.All such the well-known technology that those skilled in the art will recognize that the forfeiture of rise for surveying cyclin E or p21 or p27 or survey the saltant type of CDC4 modification, the upper Aurora of mediation of Aurora can be used for situation of the present invention.

Therefore, all these technology also all can be used for determining and are particularly suitable for the tumour for the treatment of with compound of the present invention.

Have the saltant type of CDC4 or the rise of cyclin E, the tumour of particularly crossing the forfeiture of expression or p21 or p27 may be responsive especially to CDK inhibitor.Before treatment, can, preferably with regard to the rise of cyclin E, particularly cross and express (Harwell RM, MullBB, Porter DC, Keyomarsi K.; J Biol Chem.2004 Mar 26; 279 (13): 12695-705) or the forfeiture of p21 or 27 or CDC4 modification (Rajagopalan H, JallepalliPV, Rago C, Velculescu VE, Kinzler KW, Vogelstein B, Lengauer C.; Nature.2004 Mar 4; 428 (6978): 77-81) tumour is screened.

The treatment that can select the patient who suffers from lymphoma mantle cell (MCL) to use compound of the present invention to carry out with the diagnostic test summarized here.MCL is a kind of unique clinical pathology entity of non_hodgkin lymphoma, it is characterized in that the little extremely medium sized lymphocytosis of the coexpression with CD5 and CD20, it is a kind of clinical course of invasive and refractory, and usually has t (11; 14) (q13; Q32) transposition.Excessively expressing of the cyclin D1 mRNA finding in lymphoma mantle cell (MCL) is a kind of diagnostic flag of key.The people such as Yatabe (Blood.2000 April 1; 95 (7): 2253-61) propose cyclin D1-positive (positivity) and should be counted as the one in MCL standard conditions, and should on the basis of this New Terms, probe into the innovative treatments of this disease that is difficult to treatment.The people such as Jones (J Mol Diagn.2004May; 6 (2): 84-9) developed real-time (real-time), the quantitative reverse transcription PCR test that a kind of cyclin D1 (CCND1) for assisted diagnosis lymphoma mantle cell (MCL) is expressed.The people such as Howe (Clin Chem.2004 Jan; 50 (1): 80-7) carrying out cell cycle protein D 1 mrna expression with the RT-PCR of real-time quantitative assesses and finds to diagnose the MCL in blood, marrow and tissue with the quantitative RT-PCR of the cyclin D1 mRNA that is standardized to CD19 mRNA.Or, the treatment that can select the patient who suffers from mammary cancer to use CDK inhibitor to carry out with the diagnostic test summarized above.Tumour cell common overexpressing cell cyclin E and shown that cyclin E is crossed and expresses people such as (, Cancer Res, 2000,60,481-489) Harwell in mammary cancer.Therefore, mammary cancer particularly can be treated with CDK inhibitor provided here.

antimycotic application

The compound that the present invention provides defined formula (I), (II), (III), (XXX) here on the other hand using and group as the application of anti-mycotic agent.

Here the compound of defined formula (I), (II), (III), (XXX) with and group can be used for (being for example used for the treatment of Mammals as people) in animal medicine, or can be used for treating plant (for example, for agricultural and gardening), or can be used as general anti-mycotic agent, for example, as sanitas and sterilizing agent.

In one embodiment, the invention provides for prevent or treat Mammals as the compound of the defined formula here (I) of people's fungi infestation, (II), (III), (XXX) with and group.

Here the compound that defined formula (I), (II), (III) be also provided with and group for the preparation of prevention or treatment Mammals as the application of the medicine of people's fungi infestation.

For example, compound of the present invention can deliver medicine to suffers from or has the human patients of suffering from infection risk, described infection be by, in other organisms, the local fungal infection that Candida, Trichophyton, sporule genus or Epidermophyton etc. cause or the mucosal infections (for example mycodermatitis and vaginal candidiasis) being caused by Candida albicans cause.Compound of the present invention also can be used for treatment or the whole body fungi infestation that caused by for example Candida albicans, Cryptococcus neoformans, flavus, Aspergillus fumigatus, Coccidiodies, Paracoccidioides, Histoplasma or blastomycete of prevention by administration.

On the other hand, the invention provides the antifungal composition for agricultural (comprising gardening) application, its compound that comprises defined formula (I), (II), (III), (XXX) here with and group and the upper acceptable diluent or carrier of agricultural.

The present invention also provides a kind of method for the treatment of having animal (comprising that Mammals is as people), plant or the seed of fungi infestation, it comprise with significant quantity here defined formula (I), (II), (III), (XXX) with and the compound of group to said animal, plant or seed, or process the position of said plant or seed.

The present invention also provides a kind of method that fungi infestation in plant or seed is processed, it comprise with the compound that comprises defined formula (I), (II), (III), (XXX) here of antimycotic significant quantity with and the antifungal composition of group described plant or seed are processed.

Can select that inhuman CDK enzyme is had to specific these compounds of the present invention by different shaker tests.Specific effect can be used as antimycotic or antiparasitic in the compound of the CDK of eukaryote pathogenic agent enzyme.The kinase whose inhibitor of Candida CDK, CKSI can be used for treating moniliosis.Anti-mycotic agent can be used for resisting the infection of institute's define styles above, or usually betides weak or immunosuppressed patient as suffered from leukemia and lymphadenomatous patient, accept the people of immunosuppressive therapy and having procatarxis implementations as the opportunistic infection in the patient of diabetes or AIDS and nonimmune inhibition patient.

Can screen and can be used for mould fungus inhibition disease as the material of at least one fungi related in moniliosis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, torulosis, chromomycosis, coccidioidomycosis, conidiosporosis, histoplasmosis, mycetoma, rhinosporidosis, nocardiosis, pseudactinomycosis, penicilliosis, monoliasis or sporotrichosis with test described in the prior art.Can determine the anti-mycotic agent that has therapeutic value in the treatment of being carried out as Aspergillus fumigatus, flavus, AN, Aspergillus nidulans or terreus clone's CDK gene pairs aspergillosis by yeast using by different shaker tests, or be in the situation of mucon-nycosis in described fungal infection, described CDK test can be derived from yeast as rhizopus arrhizus, rhizopus oryzae, absidia corymbifera, absidia rasmosa or small trichophytin.The source of other CDK enzyme comprises pathogenic agent Pneumocystis carinii.

For example, can carry out external assessment to the anti-mycotic activity of described compound by measuring minimum inhibition concentration (M.I.C.), described minimum inhibition concentration is that specified microorganisms is in the concentration of its lower described test compound that can not grow in appropriate media.In practice, a series of agar plates (it has the test compound of being sneaked into certain concentration separately) are inoculated with the type culture of for example Candida albicans, then each plate is cultivated suitable period at 37 ℃.Then, check that these plates exist or do not have the growth of fungi and note suitable M.I.C. value.Or, can in liquid culture, carry out a kind of turbidity test and can in embodiment 64, find the scheme that the example of this test is summarized.

Can carry out administration to the mouse of having used fungi, the bacterial strain of for example Candida albicans or flavus to carry out inoculation by intraperitoneal or intravenous injection or oral administration carries out assessing in body to described compound under a series of dosage levels.The growth (by histology or by monitoring from infecting recovery fungi) of the fungi infestation in the mouse group that can undertaken treating and treating by monitoring is assessed compound activity.Dosage level (the PD of 50% provide protection can be provided the lethal effect of described infection according to complanation compound described in it ₅₀) described activity is measured.

For the mankind's antimycotic application, here the compound of defined formula (I), (II), (III), (XXX) with and group can be administered alone or with put into practice the pharmaceutical carrier of the selecting administration that is mixed according to required route of administration and standard pharmaceutical.Therefore, for example, it can be used on title is above that the preparation described in " pharmaceutical preparation " part carries out oral administration, parenteral admin, intravenous administration, intramuscular administration or subcutaneous administration.

For the oral and parenteral admin that human patients is carried out, when by oral or parenteral administration, the dosage level of antifungal compound of the present invention can be 0.01 to 10mg/kg (with divided doses), and it especially depends on the effect of described compound.The tablet of described compound or capsule for example can comprise 5mg to 0.5g active compound, depend on the circumstances, and it at every turn can be single by administration, two or more.Attending doctor in any situation will determine that for each patient optimal actual dose (significant quantity) and this dosage change the age along with particular patient, body weight and response.

Or described antifungal compound can be with the form of suppository or vaginal suppository by administration, or it can carry out topical application with the form of lotion, solution, emulsifiable paste, ointment or face powder.For example, it can be blended in the emulsifiable paste being made up of the aqueous emulsion of polyoxyethylene glycol or whiteruss; Or it can be blended into by Chinese wax or paraffinum molle alba base in ointment that form, that can have as required such stablizer and sanitas with 1 to 10% concentration.

Except above-mentioned treatment application, with the anti-mycotic agent that such differentiates that shaker test is established also can be used as sanitas in food for example, for promoting feed additive that domestic animal body weight increases or the disinfectant preparation for xenobiotic matter is processed, for example, for hospital equipment and room are purified.In a similar manner, parallel to it to Mammals CDK and insect CDK, as the restraining effect of Drosophilia CDK5 gene compares (people (1994) the FEBS Lett 356:317-21 such as Hellmich), make from compound described here, to select the inhibitor that can distinguish people/Mammals and insect enzyme.Therefore, the present invention obviously considers application and the preparation of the compounds of this invention in sterilant, as for controlling such as fruit bat of insect.

In another embodiment, some in theme CDK inhibitor can be according to for mammalian enzyme, the inhibition specificity of plant CDK ' s being selected.For example, plant CDK can be formulated in the telling test that uses one or more people's fermentoids to select to have these compounds of maximum selectivity for suppressing plant enzyme.Therefore, the present invention particularly considers the preparation for the theme CDK inhibitor of agricultural application, as the preparation of the forms such as defoliating agent.

For agricultural and gardening object, compound of the present invention can be configured to the composition forms use that is suitable for application-specific and expection object.Therefore, described compound can be applied with the form of face powder or particle, seed dressing (seed dressing), the aqueous solution, dispersion or emulsion, dips, spraying, aerosol or fumigant.These compositions can also be to dispersible powder, particle or cereal (grains) or to provide for the form of the enriched material that dilutes before use.This based composition can comprise known and in agricultural and gardening acceptable these conventional carriers, thinner or auxiliary agent and its can be prepared according to routine operation.Described composition can also comprise other activeconstituents, for example, has weeding or kills compound or other mycocide of insect active.Described compound and composition can be applied in many ways; for example it can directly be applied on leaf, stem, branch, seed or the root of plant or can be applied in soil or other growth medium; and it not only can be used for eliminate disease, but also preventability protective plant or seed are not under fire.For example, described composition can comprise the activeconstituents of 0.01 to 1% weight.For the application in field, the suitable applications rate of activeconstituents can be 50 to 5000g/ hectares.

The compound that the present invention also comprises defined formula (I), (II), (III), (XXX) here with and group control make the fungi of timber corruption and process soil that plant grows therein, for the rice terrace of rice transplanting or for the application of the water that pours into.The compound that the present invention also considers defined formula (I), (II), (III), (XXX) here with and group's application of not affected by fungi infestation for the protection of the cereal storing and other non-plant position.

brief Description Of Drawings

Fig. 1 is 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl described in embodiment 69 below] the thermal ellipsoid figure (thermal ellipsoid plot) of the free alkali dihydrate of-urea.

Fig. 2 has represented 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl as described in embodiment 69 below] package bundle (packing) figure of the free alkali dihydrate of-urea.

Fig. 3 has represented 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl as described in embodiment 70 below] the XRPD style of the free alkali of-urea.

Fig. 4 has represented 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl as described in embodiment 71 below] the Pfansteihl salt thermal ellipsoid figure of-urea.

Fig. 5 has represented 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl as described in embodiment 71 below] the package bundle figure of the Pfansteihl salt of-urea.

Fig. 6 has represented 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl as described in embodiment 72 below] beginning of Pfansteihl salt and the XRPD style of stability test sample of-urea.

Fig. 7 has represented 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl as described in embodiment 72 below] beginning of free alkali (FB1) and the XRPD style of stability test sample of-urea.

Fig. 8 has represented 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl as described in embodiment 72 below] beginning of free alkali dihydrate (FB2) and the XRPD style of stability test sample of-urea.

Embodiment

embodiment

With reference now to the particular described in embodiment below, the present invention is carried out to non-limitative illustration.

In these embodiments, use abbreviation below.

AcOH acetic acid

Uncle BOC-butoxy carbonyl

CDI 1,1-carbonyl dimidazoles

DMAW90 solvent mixture: DCM:MeOH, AcOH, H ₂o (90: 18: 3: 2)

DMAW120 solvent mixture: DCM:MeOH, AcOH, H ₂o (120: 18: 3: 2)

DMAW240 solvent mixture: DCM:MeOH, AcOH, H ₂o (240: 20: 3: 2)

DCM methylene dichloride

DMF dimethyl formamide

DMSO dimethyl sulfoxide (DMSO)

EDC 1-ethyl-3-(3 '-dimethylaminopropyl)-carbodiimide

Et ₃n triethylamine

EtOAc ethyl acetate

Et ₂o ether

HOAt 1-hydroxyl azepine benzotriazole

HOBt I-hydroxybenzotriazole

MeCN acetonitrile

MeOH methyl alcohol

SiO ₂silicon-dioxide

TBTU N, N, N ', N '-tetramethyl--O-(benzotriazole-1-yl) urea tetrafluoro boron

Hydrochlorate

THF tetrahydrofuran (THF)

analyzing LC-MS system and method describes

In these embodiments, with the liquid chromatography and the mass spectrum that use system described below and operational condition, prepared compound is carried out to characterization.There are different isotopic atoms and having quoted from the situation of single quality in existence, the quality that compound is quoted from be single isotropic substance (monoisotopic) quality ( ³⁵cl; ⁷⁹br etc.).As described below, use some systems, and these systems are furnished with quite similar operational condition and move under these conditions.Also operational condition used is described below.

waters Platform LC-MS system:

HPLC system: Waters 2795

Mass detector: Micromass Platform LC

PDA detector: Waters 2996 PDA

acid analysis condition:

Eluent A:H ₂o (0.1% formic acid)

Eluent B:CH ₃cN (0.1% formic acid)

Gradient: 5-95% eluent B in 3.5 minutes

Flow velocity: 0.8ml/min

Post: Phenomenex Synergi 4 μ MAX-RP 80A, 2.0x50mm

alkalescence analysis condition:

Eluent A:H ₂o (uses NH ₄oH is adjusted to the 10mM NH of pH=9.2 ₄hCO ₃buffer reagent)

Eluent B:CH ₃cN

Gradient: 05-95% eluent B in 3.5 minutes

Flow velocity: 0.8ml/min

Post: Phenomenex Luna C18 (2) 5 μ m 2.0x50mm

polarity analysis condition:

Eluent A:H ₂o (0.1% formic acid)

Eluent B:CH ₃cN (0.1% formic acid)

Gradient: 00-50% eluent B in 3 minutes

Flow velocity: 0.8ml/min

Post: Phenomenex Synergi 4 μ MAX-RP80A, 2.0x50mm

lipotropy analysis condition:

Eluent A:H ₂o (0.1% formic acid)

Eluent B:CH ₃cN (0.1% formic acid)

Gradient: 55-95% eluent B in 3.5 minutes

Flow velocity: 0.8ml/min

Post: Phenomenex Synergi 4 μ MAX-RP 80A, 2.0x50mm

long-term acid analysis condition:

Eluent A:H ₂o (0.1% formic acid)

Eluent B:CH ₃cN (0.1% formic acid)

Gradient: 05-95% eluent B in 15 minutes

Flow velocity: 0.4ml/min

Post: Phenomenex Synergi 4 μ MAX-RP 80A, 2.0x150mm

long-term alkaline analysis condition:

Eluent A:H ₂o (uses NH ₄oH is adjusted to the 10mM NH of pH=9.2 ₄hCO ₃buffer reagent)

Eluent B:CH ₃cN

Gradient: 05-95% eluent B in 15 minutes

Flow velocity: 0.8ml/min

Post: Phenomenex Luna C18 (2) 5 μ m 2.0x50mm

platform MS condition:

Capillary voltage: 3.6 kV (being 3.40kV on negativity ES)

Cone (Cone) voltage: 25V

Source temperature: 120 ℃

Sweep limit: 100-800amu

Ionization mode: cation electrodeposition spraying or

Anionic electrodeposition spraying or

The spraying of cation A MP.AMp.Amp anionic electrodeposition

waters Fractionlynx LC-MS system:

HPLC system: 2767 automatic sampler-2525 binary gradient pumps

Mass detector: Waters ZQ

PDA detector: Waters 2996 PDA

the acidic conditions of analyzing:

Eluent A:H ₂o (0.1% formic acid)

Eluent B:CH ₃cN (0.1% formic acid)

Gradient: 5-95% eluent B in 4 minutes

Flow velocity: 2.0ml/min

Post: Phenomenex Synergi 4 μ MAX-RP 80A, 4.6x50mm

polarity analysis condition:

Eluent A:H ₂o (0.1% formic acid)

Eluent B:CH ₃cN (0.1% formic acid)

Gradient: 00-50% eluent B in 4 minutes

Flow velocity: 2.0ml/min

Post: Phenomenex Synergi 4 μ MAX-RP 80A, 4.6x50mm

lipotropy analysis condition:

Eluent A:H ₂o (0.1% formic acid)

Eluent B:CH ₃cN (0.1% formic acid)

Gradient: 55-95% eluent B in 4 minutes

Flow velocity: 2.0ml/min

Post: Phenomenex Synergi 4 μ MAX-RP 80A, 4.6x50mm

fractionlynx MS condition:

Capillary voltage: 3.5kV (being 3.2kV on negativity ES)

Cone voltage: 25V (being 30V on negativity ES)

Source temperature: 120 ℃

Sweep limit: 100-800amu

Ionization mode: cation electrodeposition spraying or

Anionic electrodeposition spraying or

The spraying of cation A MP.AMp.Amp anionic electrodeposition

(Mass Directed) purifying LC-MS system of quality guiding

Preparation property LC-MS be for to little organic molecule as described herein compound carry out the effective standard method of purifying.Can change with to better crude product being separated and improving the detection of MS to sample liquid chromatography (LC) and mass spectrum (MS) method.The optimization that preparation property gradient LC method is carried out comprises change post, volatility eluent and properties-correcting agent and gradient.These methods that property prepared LC-MS method is optimized are well known in the prior art, and then with it, described compound are carried out to purifying.At Rosentreter U, Huber U.; Optimum fraction in preparation property LC/MS is collected (Optimal fraction collecting in preparativeLC/MS); J Comb Chem.; 2004; 6 (2), 159-64 and Leister W, Strauss K, Wisnoski D, Zhao Z, Lindsley C., for the foundation (Development of a custom high-throughput preparative liquidchromatography/mass spectrometer platform for the preparativepurification and analytical analysis of compound libraries) of the usual high-throughput property the prepared liquid chromatography/mass spectrometry instrument platform of the preparation purifying to compound library and analytical analysis; J CombChem.; 2003; 5 (3); In 322-9, these class methods are described.

To being described for this type systematic of one that compound is carried out to purifying by preparation property LC-MS, still those skilled in the art will recognize that and can use substituting of described these system and methods below.Particularly, can be that basic method replaces inversion method described here in order to the positive property prepared LC.Most preparation property LC-MS systems are utilized anti-phase LC and volatile acidic properties-correcting agent, and this is because the method very effectively and because of described eluent can be sprayed mass compatible with cation electrodeposition for micromolecular purifying.Or can be with using other chromatographic solution for example positive LC, or as the carrying out of being summarized in above-mentioned analytical procedure buffering moving phase, alkaline properties-correcting agent etc. to as described in compound carry out purifying.

preparation property LC-MS system:

waters Fractionlynx system:

Hardware:

2767 Dual Loop automatic sampler/fraction collectors (Fraction Collector)

2525 prepare pump

CFO (post fluid tissue device (column fluidic organiser)), selects for post

RMA, as (the Waters reagent manager) of make up pump

Waters ZQ mass spectrograph

Waters 2996 Photo diode-array detectors

Waters ZQ mass spectrograph

Software:

Masslynx 4.0

Waters MS operational conditions:

Capillary voltage: 3.5kV (being 3.2kV on negativity ES)

Cone voltage: 25V

Source temperature: 120 ℃

Multiplier: 500V

Sweep limit: 125-800amu

Ionization mode: cation electrodeposition spraying or

Anionic electrodeposition spraying

agilent 1100 LC-MS preparation systems:

Hardware:

Automatic sampler: 1100 series " prepALS "

Pump: for the preparation of 1100 series " PrepPump " of property flow gradient with for 1100 series " QuatPump " to prepare the pump properties-correcting agent that flows

UV detector: 1100 series " MWD " multiwavelength detector

MS detector: 1100 series " LC-MSD VL "

Fraction collector: 2x " Prep-FC "

Make Up pump: " Waters RMA "

Agilent Active splitter

Software:

Chemstation：Chem32

Agilent MS operational conditions:

Capillary voltage: 4000V (being 3500V on negativity ES)

Fragmentor/Gain：150/1

Dry gas flow velocity: 13.0L/min

Gas temperature: 350 ℃

Atomizer pressure: 50psig

Sweep limit: 125-800amu

Ionization mode: cation electrodeposition spraying or

Anionic electrodeposition spraying

chromatographic condition:

Post:

1. low pH chromatography:

Phenomenex Synergy MAX-RP，10μ，100x21.2mm

(or for the stronger compound of polarity, using Thermo Hypersil-Keystone HyPurity Aquastar, 5 μ, 100x21.2mm)

2. high pH chromatography:

Phenomenex Luna C18(2)，10μ，100x21.2mm

(or using Phenomenex Gemini, 5 μ, 100x21.2mm)

Eluent:

1. low pH chromatography:

solvent orange 2 A:h ₂o+0.1% formic acid, pH～1.5

solvent B:cH ₃cN+0.1% formic acid

2. high pH chromatography:

solvent orange 2 A:h ₂o+10mM NH ₄hCO ₃+ NH4OH, pH=9.2

solvent B:cH ₃cN

3. compensation solvent (Make up solvent):

MeOH+0.2% formic acid (for the chromatography of two types)

Method:

According to the trace of analyzing, select optimal preparative chromatography type.A kind of typical convention is the analysis LC-MS that operation is used the chromatogram type (low or high pH) that is best suited for compound structure.In the time that trace analysis shows good chromatogram, select the suitable preparation method of same type.For low and high pH chromatography, typical operational conditions is:

flow velocity:24ml/min

gradient:all gradients are all used 95%A+5%B conventionally for initial 0.4 minute.Then, according to analyze trace, select a kind of 3.6 minutes gradient with obtain good separation (for example for a little earlier retain compound, with 5% to 50%B; Use 35% to 80%B etc. for the compound of medium reservation)

washing:in the time that finishing, described gradient carries out the washing step of 1.2 minutes

reequilibrate:ensuing in service, move 2.1 minutes reequilibrate steps to prepare described system

composition (Make Up) flow velocity:1ml/min

Solvent:

All compounds are all dissolved in 100%MeOH or 100%DMSO conventionally.

According to provided information, those skilled in the art can carry out purifying to compound described here by preparation property LC-MS.

Unless otherwise indicated, otherwise the parent material of each embodiment can obtain by commercial sources.

embodiment 1

5-cyano group-2-methoxyl group-N-[3-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline -2-yl)-1H-pyrazoles-4-yl]-benzamide synthetic

synthesizing of 1A. (3,4-dinitrobenzene-phenyl)-morpholine-4-base-ketone (methanone)

The mixture of 3,4-dinitrobenzoic acid (10.0g) and thionyl chloride (30ml) is heated 2 hours under refluxing, be cooled to envrionment temperature and by removing excessive thionyl chloride with methylbenzene azeotropic.Resistates is absorbed in THF (100ml) and at 0 ℃ and in this mixture, adds morpholine (4.1ml) and Et simultaneously ₃n (7.2ml).This mixture is stirred 3 hours, add wherein water (100ml), then extract with EtOAc.By organic moiety salt water washing, dry (MgSO ₄) and it is concentrated under vacuum.By resistates MeOH recrystallization, obtain (3,4-dinitrobenzene-phenyl)-morpholine-4-base-ketone (8.23g) of yellow solid form.

( ¹H NMR(300MHz，DMSO-d ₆)δ8.3(d，1H)，83(s，1H)，8.0(d，1H)，3.7-3.5(m，8H)).

synthesizing of 1B. (3,4-diamino-phenyl)-morpholine-4-base-ketone

By (3,4-dinitrobenzene-phenyl) jolting 10 hours at ambient temperature under hydrogen atmosphere of-morpholine-4-base-ketone (1.0g) and the mixture of 10%Pd/C (150mg) in MeOH (30ml), then by its with plug of celite filter and by its decrement under vacuum, obtain (3,4-diamino-phenyl)-morpholine-4-base-ketone (900mg).

( ¹HNMR(300MHz，DMSO-d ₆)δ6.6(s，1H)，6.5(s，2H)，4.8(s，1.5H)，4.6(s，1.5H)，4.1(s，1H)，3.6(m，4H)，3.4(m，4H)).

1C.4-morpholine-4-ylmethyl-benzene-1,2-diamines synthetic

In mixture to (3,4-dinitrobenzene-phenyl)-morpholine-4-base-ketone (2.84g) in anhydrous THF (50ml), add NaBH ₄(954mg), then drip wherein BF ₃.Et ₂o (3.2ml).This mixture is stirred 3 hours at ambient temperature, then by adding MeOH to be extinguished.By this mixture vacuum decrement, between EtOAc and water, distribute, by organic moiety salt water washing, dry (MgSO ₄) and it is concentrated under vacuum.Resistates is carried out to purifying by flash column chromatography, carry out wash-out with EtOAc, obtain 4-(3,4-dinitrobenzene-benzyl)-morpholine (1.08g).

By 4-(3,4-dinitrobenzene-benzyl) jolting 4 hours at ambient temperature under hydrogen atmosphere of-morpholine (550mg) and the mixture of 10%Pd/C (75mg) in MeOH (10ml), then by its with plug of celite filter and by its decrement under vacuum, obtain 4-morpholine-4-ylmethyl-benzene-1 as the main ingredient of mixture, 2-diamines (483mg).

1D.5-morpholine-4-ylmethyl-2-(4-nitro-1H-pyrazole-3-yl) 1H-benzene and imidazoles is synthetic

By 4-morpholine-4-ylmethyl-benzene-1,2-diamines (2.30g, 11.1mmol), 4-nitro-1H-pyrazoles-3-formic acid (1.57g, 10.0mmol), EDC (2.13g, 11.1mmol) and the mixture of HOBt (1.50g, 11.1mmol) in dry DMF (25ml) stir at ambient temperature 24 hours.By this mixture vacuum decrement, resistates dissolving crude product is heated 3 hours in AcOH (40ml) and by it under refluxing.Under vacuum, remove desolventizing and resistates is carried out to purifying with flash column chromatography, the MeOH that is arranged in EtOAc with 0-20% carries out wash-out, obtains 5-morpholine-4-ylmethyl-2-(4-nitro-1H-pyrazole-3-yl) IH-benzoglyoxaline of yellow solid form.(1.0g，61％)。(LC/MS：R _t 1.83，[M+H]+329)。

1E.3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles synthesizing of-4-base amine

Under nitrogen atmosphere, palladium is draped over one's shoulders to carbon (10%, 0.08g) and joins 5-morpholine-4-ylmethyl-2-4-nitro-1H-pyrazole-3-yl) in the solution of 1H-benzoglyoxaline (0.82g, 2.5mmol) in DMF (30ml).By the jolting 4 hours under hydrogen atmosphere of this mixture, then use diatomite filtration, wash with MeOH.By filtrate vacuum concentration, obtain 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (530mg, 71%) of brown solid.(LC/MS：R _t 1.94，[M+H]+299)。

1F.5-cyano group-2-methoxyl group-benzoic synthetic

To 2-hydroxyl-5-cyano group-methyl benzoate (2.g, 5.6mmol), K ₂cO ₃in (4.68g, 16.8mmol) mixture in acetone (50ml), add methyl iodide (0.7ml, 5.6mmol).Then, by this reaction heated overnight at 65 ℃, form a kind of solid, it is leached while hot and it is washed with acetone, obtain 5-cyano group-2-methoxyl group-methyl benzoate (0.45g).This crude product is dissolved in THF (5ml), then with the LiOH (0.108g 0.26mmol) that is arranged in water (5ml), it is processed and it is at room temperature stirred and spent the night.This reaction is also extracted it with EtOAc (x 2) with 2M HCl acidifying.By dry organic moiety (MgSO ₄) and by its decrement under vacuum, obtain 5-cyano group-2-methoxyl group-phenylformic acid (0.277g).(acid LC/MS:R _t2.92, [M+H] ⁺178).

1G.5 cyano group-2-methoxyl group-N-[3-(5-morpholine-4-ylmethyl-1H-benzo imidazoles-2-yl)-1H-pyrazoles-4-yl] synthetic (the chloride of acid method) of-benzamide

By 5-cyano group-2-methoxyl group-phenylformic acid (embodiment 1F) (40mg, 0.22mmol) be dissolved in DCM (5ml), then drip wherein oxalyl chloride (34.4mg, 0.264mmol), then add wherein DMF (1).This reaction mixture is stirred 1 hour at ambient temperature, and vacuum decrement, then evaporates (x 2) again with toluene.By 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (100mg, 0.33mmol), 5-cyano group-2-methoxyl group-Benzoyl chloride and diisopropylethylamine (1.83 μ l, the 0.9mmol) mixture in THF (5ml) stirs and makes it be warmed to room temperature and reaches 2 hours at 0 ℃.Then, by this reaction mixture vacuum concentration.Resistates is carried out to purifying [SiO with flash column chromatography ₂, 5-7%MeOH-DCM], obtain 5-cyano group-2-methoxyl group-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-benzamide (12mg).(acid LC/MS:R _t2.02min[M-H]+458).

embodiment 2

6-methyl-imidazo [2.1-b] thiazole-5-formic acid [3-(5-morpholine-4-ylmethyl- 1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-acid amides synthetic

By 6-methyl-imidazo [2.1-b] thiazole-5-formic acid (Bionet) (61mg, 0.33mmol), 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (100mg, 0.33mmol), EDC (77mg, 0.39mmol) and HOAt (54mg, mixture 0.39mmol) stirs 1 hour in DMF (3ml) at 80 ℃, then it is stirred 20 hours at ambient temperature.Distribute between EtOAc and saturated NaHCO by this mixture vacuum decrement and by resistates.By organic moiety salt water washing, dry (MgSO ₄) and by its decrement under vacuum.Resistates is carried out to purifying with preparation property LC/MS, obtain 6-methyl-imidazo [2.1-b] thiazole-5-formic acid [3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-acid amides (29mg).(alkaline LC/MS:R _t2.56[M+H] ⁺463).

embodiment 3

2-cyano group-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl]-ethanamide synthetic

By cyano group-acetic acid (23mg, 0.28mmol), 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (70%, 100mg, 0.23mmol), the mixture of TBTU (89mg, 0.28mmol) and DMF (2ml) stirs and spends the night at 25 ℃.Then, by this mixture vacuum-evaporation.Carry out purifying by flash column chromatography method, carry out wash-out with DCM-6%MeOH/DCM, obtain 2-cyano group-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of yellow solid form]-ethanamide (65mg, 77%).(LC/MS (acid process/final compound): R _t4.61, [M+H] ⁺366).

embodiment 4

2-cyano group-2-cyclopropyl-N-[3-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline -2-yl)-1H-pyrazoles-4-yl]-ethanamide

synthesizing of 4A. cyano group-cyclopropyl-acetic acid

1N NaOH (3.26ml, 3.26mmol) is joined in the solution of cyano group-cyclopropyl-ethyl acetate (0.5g, 3.26mmol) in THF (15ml).It being stirred at 25 ℃ after 4 hours, by this reaction mixture vacuum-evaporation, it is dissolved in water (20ml) and by adding 1N HCl solution (3.26ml) to neutralize it again.Then, this mixture is extracted to (3x20ml) with EtOAc, and merge, dry (Na ₂sO ₄) organism vacuum-evaporation, obtain the impure cyano group-cyclopropyl-acetic acid of oily matter form of clarification.By the preparation for embodiment 4B in the situation that not carrying out any purifying of this material.

4B.2-cyano group-2-cyclopropyl-N-[3-(5-morpholine-4-ylmethyl-1H-benzene and imidazoles-2-yl)-1H-pyrazoles-4-yl]-ethanamide

The product of embodiment 4A is reacted according to the method described in embodiment 3 with 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine, and difference is at DCM and saturated NaHCO by this crude product ₃between the aqueous solution, distribute, then by using Et ₂o development is carried out purifying to it.LC/MS (acid process) R _t1.79[M+H] ⁺406.

embodiment 5-14

According to the method for embodiment 1,2 and 3, it is carried out to the improvement shown in some following tables, the compound of Preparation Example 5 to 14.

embodiment 15

n-[3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] synthesizing of-racemize-2-morpholine methane amide-trifluoroacetate

15A.5,6-dimethoxy-2-(4-nitro-1H-pyrazole-3-yl)-1H-benzo synthesizing of imidazoles

To EDC (4.81g, 25mmol), HOBt (3.40g, 25mmol) and triethylamine (4.67g, 46mmol) in the solution in DMF (100ml), add 4-nitro-lH-pyrazoles-3-formic acid (3.63g, 23.09mmol) and 4,5-dimethoxy-benzene-1,2-diamines dihydrochloride (5.06g, 20.99mmol) also at room temperature stirs this mixture to spend the night.Under vacuum except desolventizing and by the solid of gained at EtOAc (50ml) and saturated NaHCO ₃between the aqueous solution (50ml), distribute.Form precipitation and by its filtering.By filtrate water, then wash with ether, then by itself and MeOH azeotropic together with toluene, obtain 4-nitro-1H-pyrazoles-3-formic acid (2-amino-4,5-dimethoxy-phenyl)-acid amides (2.35g, 36%).4-nitro-1H-pyrazoles-3-formic acid (2-amino-4,5-dimethoxy-phenyl)-acid amides (2.35g, 7.65mmol) is dissolved in acetic acid (150ml) and by it and at 140 ℃, is refluxed 5 hours.Make this solution cooling and under vacuum except desolventizing.The solid of gained is distributed between EtOAc (25ml) and salt solution (25ml).Organic layer is separated to dry (MgSO ₄), filter and under vacuum, remove desolventizing, obtain 5,6-dimethoxy-2-(4-nitro-1H-pyrazole-3-yl)-1H-benzoglyoxaline (2.08g, 94%).

15B.3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4- synthesizing of base amine

By 5,6-dimethoxy-2-(4-nitro-1H-pyrazole-3-yl)-1H-benzoglyoxaline (2.08g, 7.2mmol) and 10% palladium drape over one's shoulders mixture under chamber moderate pressure the hydrogenation of carbon (200mg) in ethanol (150ml) and DMF (50ml) and spend the night.This reaction mixture is removed to desolventizing with diatomite filtration and under vacuum.By the solid of gained and methyl alcohol together with toluene azeotropic and under vacuum except desolventizing.This crude product material is carried out to purifying by flash column chromatography method, with DCM: MeOH: acetic acid: water (120: 18: 3: 2) [DMAW120], then uses DCM: MeOH: acetic acid: water (90: 18: 3: 2) (DMAW90) carry out wash-out.Product fraction merged and under vacuum, remove desolventizing, obtaining 3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (～1g, 53%).

15C.N-[3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles- 4-yl]-racemize-4-BOC-2-morpholine methane amide synthetic

To EDC (125mg, 0.54mmol) and HOAt (74mg, 0.54mmol) in the solution in DMF (2ml), add 3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (117mg, 0.45mmol) (embodiment 15B) and (racemize)-BOC-2-carboxyl morpholine (125mg, 0.54mmol), this mixture is at room temperature stirred and spent the night.Then, this mixture is distributed between EtOAc and water.Then, organic layer is washed with saturated sodium bicarbonate aqueous solution, salt solution continuously, be then dried (MgSO ₄).This solution vaporising under vacuum is extremely dried and resistates is carried out to purifying [SiO with flash column chromatography ₂gradient elution: EtOAc-hexane (1: 1)-EtOAc-MeOH (80: 20)], obtain N-[3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of colorless solid form]-racemize-4-BOC-2-morpholine methane amide (65mg).(LC/MS (acid process): R _t2.65min, [M+H] ⁺473).

15D.N-[3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles- 4-yl]-racemize-2-morpholine methane amide-trifluoroacetate synthetic

By N-[3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-racemize-4-BOC-2-morpholine methane amide (65mg, 0.14mmol) and phenylmethylether (60 μ l, 0.56mmol) be dissolved in trifluoroacetic acid and methylene dichloride (1: 1; In mixture 2ml).It was at room temperature placed after 3 hours, this mixture is evaporated to dry, obtain N-[3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of colorless solid form]-racemize-2-morpholine methane amide-trifluoroacetate (73mg) (LC/MS (acid process): R _t1.42min, [M-H ⁺] ^-371.

embodiment 16

n-[3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] synthesizing of-racemize-4 sec.-propyl-2-morpholine methane amide

To the N-[3-(5 that is arranged in MeCN (1ml), 6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-racemize-2-morpholine methane amide-trifluoroacetate (embodiment 15D) (34mg, 0.07mmol) and K ₂cO ₃in (20mg, 0.14mmol), add 2-iodopropane (17 μ l, 0.15mmol).This mixture is stirred at 80 ℃ about 48 hours, thereafter, this mixture is concentrated and resistates is carried out to purifying [SiO by flash column chromatography method ₂gradient elution: DCM: MeOH (98: 2) is to DCM: MeOH: dense NH ₃water (90: 10: 1)], obtain N-[3-(5,6 dimethoxys-1H-benzoglyoxaline also-2-yl)-1H-pyrazoles-4-yl of colourless gummy form]-racemize-4-sec.-propyl-2-morpholine methane amide (12mg) (LC/MS (alkaline method): R _t2.52min[M+H] ⁺415).

embodiment 17

n-[3-(5,6-dimethoxy-H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] synthesizing of-racemize-1-methyl-piperidines-3-methane amide

To 3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (65mg, 0.25mmol) (embodiment 15B) (racemize)-1-methyl-piperidines-3-formic acid-hydrochloride (50mg, 0.27mmol) and diisopropyl ethyl amine (50 μ l, 0.27mmol) in the solution in DMF (1ml), add TBTU (97mg, 0.30mmol).This mixture is at room temperature stirred about 16 hours, thereafter, add wherein the 1N NaOH aqueous solution (1ml) and this mixture is stirred 1 hour again.Then, this mixture vaporising under vacuum is extremely dried and resistates is carried out to purifying (SiO by flash column chromatography method ₂, use DCM: MeOH (98: 2) to DCM: MeOH; Dense NH ₃water (70: 30: 3) gradient elution), obtain N-[3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of colourless gummy form]-racemize-1-methyl-piperidines-3-methane amide (20mg) (LC/MS (alkaline method): R _t2.35min, (M+H)+385).

embodiment 18

the chloro-N-of 3-(5,6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4 synthesizing of-Ji-5-(4-methyl-piperazine-1-yl)-benzamide

synthesizing of the chloro-5-of 18A.3-(4-methyl-piperazine-1-yl)-benzonitrile

The fluoro-5-chloro-benzonitrile of 3-(1g, 6.4mmol) is dissolved in DMSO (20ml), then adds wherein K ₂cO ₃(1.3g, 9.6mmol) and 1-methylpiperazine (1.4ml, 12.8mmol).This reaction mixture is heated 20 hours at 80 ℃.Ether is joined in this crude product material (10ml), then with 1N HCl, it is carried out to acidifying.Precipitation is leached from this crude product reaction mixture, obtain the chloro-5-of 3-(4-methyl-piperazine-1-yl)-benzonitrile (1.4g, yield the is 93%) (LC/MS:R of white solid form _t1.83[M+H] ⁺236, acid process).

the chloro-5-of 18B.3-(4-methyl-piperazine-1-yl)-benzoic synthetic

In the chloro-5-of 3-(4-methyl-piperazine-1-yl)-benzonitrile (1.4g, 5.9mmol) being dissolved in ethanol (10ml), add 2M NaOH (20ml) and this reaction mixture is heated 20 hours under refluxing.By this mixture vacuum decrement this crude product is acidified to pH 6 with 1N HCl, then by it at EtOAc and H ₂between O, distribute.Organic layer vacuum-evaporation, to dry, is obtained to the title compound (LC/MS:R of 0.7g white solid form _t1.67, [M+H] ⁺256, acid process).

the chloro-N-[3-of 18C.[3-(5.6-dimethoxy-1H-benzimidazolyl-2 radicals-yl)-1H -pyrazoles-4-yl]-5-(4-methyl-piperazine-1-yl)-benzamide synthetic

Described compound is to use the method similar to embodiment 15C to be prepared, but replace (racemize)-BOC-2-carboxyl morpholine as reagent in 15C with the chloro-5-of 3-(4-methyl-piperazine-1-yl)-phenylformic acid (200mg, 0.78mmol).This crude product is carried out to purifying [SiO by flash column chromatography method ₂, carry out wash-out with DMAW240-90, obtain the title compound (LC/MS:R of the filbert solid form of 92mg (yield is 25%) _t2.07[M+H] ⁺496).

embodiment 19-21

According to the method described in embodiment 15, the improvement shown in it is carried out, the compound of Preparation Example 19-21.

embodiment 22

the chloro-2-methoxyl group-N-{3-[5-of 5-(4-methyl-piperazine-1-ylmethyl)-1H- benzimidazolyl-2 radicals-yl]-1H-pyrazoles-4-yl }-benzamide synthetic

synthesizing of 22A. (3,4-dinitrophenyl)-(4-methylpiperazine-1-yl)-ketone

By 3,4-dinitrobenzoic acid (50g, 0.24mol) reflux under at SOCl ₂(160ml) heating 6 hours in.Then, that this mixture vacuum-evaporation is extremely dry.This product is dissolved in THF and is cooled to 5 ℃.Drip N methyl piperazine (26.2ml, 0.24mol) and Et to the form with the solution that is arranged in THF (50ml) in this solution ₃n (42ml).It was at room temperature stirred after the night, this solution is poured in water (1.5L) and by it and is stirred 0.5 hour at about 5 ℃.Collect the solid precipitation forming and be dried, obtaining (3,4-dinitrophenyl)-(4-the methylpiperazine-1-yl)-ketone (40g) of yellow solid form.

synthesizing of 22B.1-(3,4-diamino benzyl)-4-methylpiperazine

In cold soln (5 ℃) to (3,4-dinitrophenyl)-(4-methylpiperazine-1-yl)-ketone (12.2g, 0.041mol) in THF, drip NaBH ₄, then drip wherein BF ₃.OEt ₂solution keeps its temperature lower than 5 ℃ simultaneously.Make this mixture in 2 hours to room temperature, then it is at room temperature stirred 2 hours again.Then, in this mixture, add MeOH (causing boiling) carefully, continued to stir 10 minutes, then that this mixture is concentrated.By resistates at EtOAc and saturated NaHCO ₃between the aqueous solution, distribute.By organic layer water, salt water washing, is then dried (MgSO ₄).By this solution for vacuum evaporation and by resistates SiO ₂flash column chromatography method is carried out purifying, with DCM: MeOH (98: 2) to DCM: MeOH: dense NH ₃water (90: 10: 1) gradient elution, obtains a kind of orange crystalline solid (3.7g).Used MeOH recrystallization, obtained 1-(3,4-dinitrobenzene the benzyl)-4-methylpiperazine (1g) of orange crystalline solid form.

synthesizing of 22C.1-(3,4-diamino benzyl)-4-methylpiperazine

1-(3,4-dinitrobenzene benzyl)-4-methylpiperazine (1g) is dissolved in DMF: MeOH (1: 1,20ml) and by its together with 10%Pd/C (50mg) at H ₂under atmosphere, stir 6 hours.Then, this mixture filtered and it is evaporated, obtaining a kind of dark solid, it is not carried out directly using in the situation of any other purifying.

the chloro-2-methoxyl group-N-{3-[5-4-of 22D.5-methyl-piperazine-1-ylmethyl) -1H-benzimidazolyl-2 radicals-yl]-1H-pyrazoles-4-yl }-benzamide synthetic

4-(the chloro-2-methoxyl group-benzoyl-amido of 5-)-1H-pyrazoles-3-formic acid (1.17g), this crude product diamines, 1-(3,4-diamino benzyl)-4-methylpiperazine (0.87g) and TBTU (1.52g) are dissolved in DMF (15ml) and are stirred about 16 hours.Then, this mixture is evaporated to dry, obtains a kind of dark solid.This dark solid (100mg) is dissolved in AcOH (4ml) and this mixture is heated 3 hours at 80 ℃.Carry out purifying (SiO by this reaction mixture vacuum-evaporation and by resistates by flash column chromatography method ₂, carry out wash-out with DMAW120), obtain the chloro-2-methoxyl group-N-{3-[5-of 5-(4-methyl-piperazine-1-ylmethyl)-1H-benzimidazolyl-2 radicals-yl of diacetin form]-1H-pyrazoles-4-yl }-benzamide (35mg).(LC/MS (acid process/final compound): R _t6.63[M+H] ⁺480).

embodiment 23

1-(fluoro-benzyl-the 3-[3-of 2,6-bis-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline -2-yl)-1H-pyrazoles-4-yl]-urea synthetic

By 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (embodiment 1E), (100mg, 0.33mmol) and CDI (217mg, 1.34mmol) the mixture microwave radiation in THF (2ml) (150 ℃, 150W) 15 minutes.Then, add wherein the fluoro-benzylamine of 2,6-bis-(384mg, 2.68mmol) and by the radiation 15 minutes again under identical condition of this reaction mixture.After cooling, this heterogeneous mixture is filtered, filtrate is concentrated and resistates is carried out to purifying (SiO by column chromatography ₂, with DCM: MeOH: AcOH: H ₂o (240: 20: 3: 2) (DMAW240 to) (120: 18: 3: 2) (DMAW120) gradient elution, obtain 1-(the fluoro-benzyl of 2,6-bis-)-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea (30mg 19%).(acid LC/MS:R _t1.84[M+H] ⁺468).

embodiment 24-34

According to the general method described in embodiment 23, but it is carried out to the improvement shown in some following tables, the compound of Preparation Example 24 to 34.

embodiment 35

1-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles- 4-yl]-3-pyridin-3-yl-urea synthetic

By 3-aminopyridine (31.5mg, 0.33mmol), Et ₃the mixture of N (0.195ml, 1.32mmol) in DCM (3ml) is cooled to 0 ℃, then uses triphosgene (85mg, 0.28mmol) to process it.This reaction is stirred 1 hour at ambient temperature, then add wherein 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (100mg, 0.33mmol) and it is stirred to this reaction at ambient temperature and carry out completely.This mixture is processed 30 minutes with the 2M NaOH that is arranged in MeOH, then by its decrement under vacuum.Resistates is carried out to purifying [SiO by flash column chromatography method ₂, 2-20%MeOH/DCM], then use DCM, then develop with ether, obtain 1-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-3-pyridin-3-yl-urea (20mg).(alkaline LC/MS:R _t2.29, [M+H] ⁺419).

embodiment 36

thiomorpholine-4-formic acid [3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl) -1H-pyrazoles-4-yl]-acid amides synthetic

Phosgene (20% toluene solution) (0.3ml) is joined to 3-(in the solution of 5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-y)-1H-pyrazoles-4-base amine (100mg, 0.33mmol) in toluene/DCM (1: 1) mixture at 0 ℃.This reaction is stirred 1 hour at ambient temperature, then blow excessive phosgene off with nitrogen steam flow.Add wherein thiomorpholine (35mg, 0.33mmol) and this reaction is stirred 1 hour at ambient temperature, then it being stirred 1 hour at 60 ℃.Then, carry out purifying by this mixture vacuum concentration and by resistates with preparation property LC/MS, obtain thiomorpholine-4-formic acid [3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-acid amides (polarity LC/MS:R _t2.58, [M+H]+428).

embodiment 37

1-(4-fluorophenyl)-1-methyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzo imidazoles-2-yl)-1H-pyrazoles-4-yl]-urea synthetic

Prepare title compound method used similar to the method described in embodiment 35, just replace 3-aminopyridine with the fluoro-methylphenylamine of 4-, and this reaction is carried out 2 hours at 50 ℃.This crude product is separated from chilled reaction mixture with precipitation forms, then with flash column chromatography, it is carried out to purifying [SiO ₂, with DCM: MeOH: AcOH: water (240: 20: 3: 2) carry out wash-out].The product of gained is developed with ether, is obtained 1-(4-fluorophenyl)-1-methyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of colorless solid form]-urea (3mg).(acid LC/MS:R _t2.12, [M+H] ⁺450).

embodiment 38-43

According to the method described in embodiment 35 and 37, it is carried out to the improvement shown in following table, the compound of Preparation Example 38 to 43.

embodiment 44

1-(4-fluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals- base)-1H-pyrazoles-4-yl]-urea synthetic

Add 4-fluorophenyl isocyanic ester and by room temperature stir about 16 hours of this mixture to 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazol-1-yl amine (embodiment 1E) (100mg, 0.33mmol) that is arranged in THF (2ml).Add wherein resin-carried trimethylol aminomethane (tris-amine) (800mg, 4mmol/g) and this mixture is stirred 4 hours again.By removing by filter resin, filtrate is used to 1N KOH (2ml, MeOH: THF; 1: 3) process and by about this solution stirring 16 hours.Then, by this mixture at EtOAc and H ₂between O, distribute.Water layer is further extracted with EtOAc, then by merged organic fraction salt water washing, dry (MgSO ₄) and be evaporated to dry.This solid-state dissolving crude product is developed in DCM and with hexane, collected and obtain a kind of solid by filtration.This solid is carried out to purifying [SiO by flash column chromatography method ₂etOAc-MeOH (90: 10)], obtain 1-(4-fluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of yellow solid form]-urea (30mg, 20%) (LC/MS (acid process): R _t2.01min, [M-H ⁺] ^-434).

embodiment 45-56

According to the method described in embodiment 44, just its condition is carried out to the improvement shown in following table, the compound of Preparation Example 45 to 56.

embodiment 57-59

According to the general method described in embodiment 23, just it is carried out to the improvement shown in following table, the compound of Preparation Example 57 to 59.

embodiment 60

1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline -2-yl)-1H-pyrazoles-4-yl]-urea-hydrochloride synthetic

synthesizing of 60A. (3,4-dinitrobenzene-phenyl)-morpholine-4-base-ketone

The mixture of 3,4-dinitrobenzoic acid (1mol.eq.) and thionyl chloride (9.2mol.eq.) is heated 6 hours under refluxing, be cooled to envrionment temperature and by removing excessive thionyl chloride with methylbenzene azeotropic.Resistates is absorbed in THF (8vol.), then at 0-5 ℃, in this mixture, adds morpholine (1.0mol.eq.) and Et simultaneously ₃n (1.1mol.eq.).This mixture is stirred 1 hour at ambient temperature, be then poured in water (25vol.).This mixture is cooled to 3-7 ℃ and placed 0.5 hour, and during this period, product displays with precipitation forms.By filtering collecting precipitation, water washs and is dried it, obtains 3 of yellow solid form, 4-dinitrobenzene-phenyl)-morpholine-4-base-ketone (75%).

( ¹HNMR(300MHz，DMSO-d ₆)δ8.3(d，1H)，8.3(s，1H)，8.0(d，1H)，3.7-3.5(m，8H).

synthesizing of 60B.4-(3,4-dinitrobenzene-benzyl)-morpholine

At 0-5 ℃, in the mixture in (25vol.), add NaBH at anhydrous tetrahydro furan (THF) to (3,4-dinitrobenzene-phenyl)-morpholine-4-base-ketone (1mol.eq.) ₄(2mol.eq.), then drip wherein BF ₃.Et ₂o (1.01mol.eq.), maintains its temperature at 0-5 ℃.Then, this mixture is stirred 3 hours at ambient temperature, then by adding methyl alcohol by its quencher.Then, by this mixture vacuum decrement, at ethyl acetate and saturated NaHCO ₃between the aqueous solution, distribute.This mixture is stirred 30 minutes rapidly, then carry out layer and separate.By organic layer water and salt water washing in succession, then by its decrement under vacuum.By product methanol crystallization, obtain 4-(3,4-dinitrobenzene-benzyl)-morpholine (85%).(LC/MS (alkaline method): R _t2.80, [M+H] ⁺268).

60C.4-morpholine-4-ylmethyl-benzene-1,2-diamines synthetic

By 4-(3,4-dinitrobenzene-benzyl)-morpholine (1mol.eq.) and the mixture of 5%Pd/C (0.05wt.eq.) in IMS (33vol.) stir at 0-5 ℃, with hydrogen, this container purified to (charged) simultaneously.This mixture is carefully warmed to 15-20 ℃ until (< 24 hours) carried out completely in this reaction in the situation that stirring.This mixture is filtered and filtrate is evaporated to dry, obtain 4-morpholine-4-ylmethyl-benzene-1,2-diamines (90%).This material is directly used in to next step.(LC/MS (alkaline method): R _t1.64, [M-N (CH ₂cH ₂) ₂o ^-] ⁺121).

60D.5-morpholine-4-ylmethyl-2-(4-nitro-1H-pyrazole-3-yl) 1H- synthesizing of benzoglyoxaline

By 4-morpholine-4-ylmethyl-benzene-1,2-diamines (1mol.eq.) and 4-nitro-1H-pyrazoles-3-formic acid (1mol.eq.) be dissolved in dimethyl formamide (DMF) (10vol.) in.Add wherein O-(benzotriazole-1-yl)-N, N, N ', N '-tetramethyl-urea a tetrafluoro borate (TBTU) (1.2mol.eq.) also stirs this mixture 24 hours at ambient temperature.By this mixture vacuum concentration until can't see solvent and be distilled out again.Then, resistates is dissolved in glacial acetic acid (10vol.) and by it and heats～12 hours at 65 ℃.By this mixture vacuum concentration, then it is dissolved in water (6vol.) at 75 ℃.This dark solution was cooled to 0-5 ℃ in 2 hours, during this period, forms a kind of solid.This solid is leached and ethyl acetate for aqueous filtrate (4vol.) and tetrahydrofuran (THF) (2vol.) are diluted.In this mixture that is carrying out stirring, slowly add solid NaHCO ₃until do not observe further boiling and its pH reaches 6.8.Then, this mixture is stirred to and observes precipitation.After this mixture is left standstill to 2 hours at 0-5 ℃, by solid collected by filtration water (2vol.) and ethyl acetate (2vol.), it is washed, be dried, obtain 5-morpholine-4-ylmethyl-2-(4-nitro-1H-pyrazole-3-yl) 1H-benzoglyoxaline (40%) of brown solid.(LC/MS (alkaline method): R _t1.93, [M-H ⁺] ^-327).

60E.3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles synthesizing of-Ji amine

Under nitrogen atmosphere, add 5%Pd/C (0.1 wt.eq.) to 5-morpholine-4-ylmethyl-2-(4-nitro-1H-pyrazole-3-yl) the 1H-benzoglyoxaline (1mol.eq.) that is arranged in DMF (36vol.).This reaction vessel is stirred 24 hours at ambient temperature with hydrogen cleaning and by it.Then,, by this mixture diatomite filtration, use methanol wash.By filtrate vacuum concentration, obtain 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (90%) of brown solid.(LC/MS (alkaline method): R _t1.94, [M-H ⁺] ^-297.This product is applied in the situation that not carrying out any purifying.

60F.1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzo imidazoles-2-yl)-1H-pyrazoles-4-yl]-urea-hydrochloride synthetic

At 0-5 ℃, stir simultaneously, in 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl) mixture of-1H-pyrazoles-4-base amine (1mol.eq.) in THF (10vol.), add isocyanic acid 2,6-difluoro phenyl ester (1.3mol.eq.).Then, this mixture is stirred 16 hours at ambient temperature, thereafter, this mixture is processed with the 1M KOH aqueous solution (4vol.).It is being stirred after 2 hours again, then by this mixture vacuum concentration and by it at ethyl acetate and saturated NaHCO ₃between the aqueous solution, distribute.Organic layer is washed to dry (MgSO with saturated brine ₄), be evaporated to dryly, then resistates is carried out to purifying [SiO by flash column chromatography method ₂, use CH ₂cl ₂-MeOH (98: 2)-(90: 10) gradient elution], obtain 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea.

the recrystallization of 60G. free alkali and characterization

After processing by silicon-dioxide chromatogram as described in embodiment 60E, product is dissolved in minimum hot ethyl acetate, filter and make it cooling.Obtain thus the free alkali of fine crystallization solid form.

Compound 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea has the physical-chemical parameters below.

PKa value-3.42,6.92 & 10.97

logP-3.24

logP _ion-0.36

logD(pH＝6)2.27

(pH＝6.5)2.68

(pH＝7.4)3.11

the formation of 60H. hydrochloride

This product is dissolved in to the solution in ether in ethyl acetate and with excessive saturated HCl to be processed.Collect the precipitation of gained by filtration, with ether washing and it is dried, obtain 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of colorless solid form]-urea-hydrochloride (59%).(LC/MS (acid process): R _t1.80, [M+H] ⁺454).

By for example, substituting described hydrogenchloride with other acid (DL lactic acid, ethyl sulfonic acid and methylsulfonic acid) and changing as required solvent composition, can prepare 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] other salt of-urea.

the solubleness comparison of 60J. free alkali and hydrochloride

Measure and compared the solubleness of described free alkali and hydrochloride.Find the solubleness < 0.001mg/ml of described free alkali under pH 7.4 (be cushioned the aqueous solution), and the solubleness of its hydrochloride under pH7.1 (being arranged in the aqueous solution that has carried out buffering) is 0.093mg/ml.Therefore, the solubleness of its hydrochloride is significantly better than the solubleness of its free alkali.

embodiment 61

1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline -2-yl)-1H-pyrazoles-4-yl] mensuration of-urea acid salt solubleness

With operation described below by 1-(2,6-difluorophenyl)-N-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] free alkali and the various acid of-urea combines, so that the solubleness of gained acid salt is assessed.

operation

In the bottle of a 8ml, add described free alkali (59mg, 0.13mmol) and water (0.59ml).To adding suitable acid (1eq., 0.13mmol) in this bottle and by the jolting 16 hours at ambient temperature of this bottle.,, this bottle is estimated thereafter.If observe a kind of homogeneous solution, finish experiment, and can show that the salt that formed thus has the conclusion higher than the solubleness of 100mg/ml.

If still there is solid residue, add wherein again 0.59ml water also by this bottle jolting 4 hours.If form a kind of homogeneous solution at this one-phase, can show that this salt has the conclusion higher than the solubleness of 50mg/ml.

If at this moment still have solid residue, add wherein again 1.18ml water and this bottle is carried out to jolting at ambient temperature.If form a kind of homogeneous solution, can show that the solubleness of this salt is higher than the conclusion of 25mg/ml.If still have solid residue, can show that the solubleness of this salt is lower than the conclusion of 25mg/ml.

By making its salts solution by a Strata-NH ₂post and its free alkali is reproduced.

Experimental result as in the table below.

According to the result shown in this table, can show that mesylate, esilate and DL-LACTIC ACID salt particularly can be used for preparing aqueous liquid composition, for example, for the conclusion of the composition of parenteral admin.

embodiment 62

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl] free alkali and the salt of-urea

Can be by method described below or similar method, by the compound of embodiment 24---1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea is with free alkali or acid salt isolated in form out.

free alkali

Processing by silicon-dioxide chromatogram after (seeing embodiment 24), the product of embodiment 24 is dissolved in the hot MeOH of minimum volume, filter and make it cooling.After～16 hours, collect the product of colourless crystallization solid form.

hydrochloride (general operation)

After processing by silicon-dioxide chromatogram, its product (2.05g) is dissolved in to MeOH: EtOAc (1: 10; 100ml), also with 4N HCl dioxane solution (1.1mol.eq.), it is processed.Collect the precipitation of gained and be dried by filtration, obtaining 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea hydrochloride (1.5g).This product is dissolved in the MeOH of minimum volume, then uses Et ₂o develops, until sustainable several seconds of a kind of turbidity.After cooling night by it, collect the product of colourless crystallization solid form.

mesylate

Collect the product of colourless crystallization solid form with above-mentioned general operation, just replace hydrochloric acid with methylsulfonic acid.

other salt

Expection can be prepared interested other salt with above-mentioned general operation.

embodiment 63

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl] free alkali of-urea and the solubility test of salt

With operation described below by the compound of embodiment 24---1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea and various acid combines that the solubleness of gained acid salt is assessed.

operation

To the free alkali (50mg, 0.131mmol) and the water (0.5ml) that add the compound of embodiment 24 in the bottle of a 8ml.In this bottle, add suitable acid (1eq., 0.131mmol) also by this bottle jolting 14-16 hour at ambient temperature.,, this bottle is estimated thereafter.If observe a kind of homogeneous solution, finish experiment, and can show that the salt that formed thus has the conclusion higher than the solubleness of 100mg/ml.

If still there is solid residue, add wherein again 0.5ml water also by this bottle jolting 6 hours.If form a kind of homogeneous solution at this one-phase, can show that this salt has the conclusion higher than the solubleness of 50mg/ml.

If at this moment still have solid residue, add wherein again 1ml water and this bottle is carried out to jolting at ambient temperature.If form a kind of homogeneous solution, can show that the solubleness of this salt is higher than the conclusion of 25mg/ml.If still have solid residue, can show that the solubleness of this salt is lower than the conclusion of 25mg/ml.

By making its salts solution by a Strata-NH ₂post and its free alkali is reproduced.

Experimental result as in the table below.

According to the result shown in this table, can show that acetate, mesylate, esilate, DL-LACTIC ACID salt, adipate, D-Glucose aldehydic acid salt, maltonic acid and hydrochloride particularly can be used for preparing aqueous liquid composition, for example, for the conclusion of the composition of parenteral admin.

From collected up to now data, showing compound of the present invention and particularly 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] free alkali and the salt (particularly Pfansteihl salt) of-urea has advantages of many prior art compounds that are better than.Particularly, such advantage comprises the advantage below one or more:

Improve the solubleness in the aqueous solution;

There are better physicochemical property, particularly there is lower logD;

To the susceptibility difference of P450 enzyme;

Drug metabolism and pharmacokinetics character are improved;

Improve stability, for example, improved the shelf lives and/or improved thermostability;

Reduce required dosage;

Improve therapeutic goal, and the effect of Aurora A and B particularly;

Improve hyperplasia and bacterium colony and produced the cytoactive in (clonogenic) test;

Improve antitumour activity; With

Improve therapeutic index.

embodiment 64

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl] the Lactated preparation of-urea

To 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] add Pfansteihl (166mg, 1.85mmol) in-solution of urea (0.7g, 1.83mmol) in EtOAc-MeOH.This mixture is stirred at ambient temperature, then by its decrement under vacuum.EtOH (20mL) recrystallization seething with excitement by use carries out purifying to this solid, after dry, obtain 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea, Pfansteihl salt (0.48g).

embodiment 65

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl] Pfansteihl salt synthetic of-urea

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the Pfansteihl salt of-urea can be prepared with the route of synthesis shown in route below.

the 1st step: (3,4-dinitrobenzene-phenyl)-morpholine-4-base-ketone synthetic

By 3,4-dinitrobenzoic acid (10g, 47mmol, 1eq.) and solution for the thionyl chloride (4.5mL of DMF (0.1mL) in THF (100mL), 62mmol, 1.3eq.) process, be then heated and reflux 2.5 hours.This mixture, in cooled on ice, was then added wherein to triethylamine (10mL, 71mmol, 1.1eq.) in 20 minutes, keep 5 ℃ of its internal temperature < simultaneously.In 15 minutes, in the dense thick yellow suspension of gained, add morpholine (6.2mL, 71mmol, 1.5eq), keep 10 ℃ of its internal temperature < simultaneously.Remove ice bath and make this mixture be warmed to room temperature.After 15 minutes, then add wherein a morpholine (1mL, 11mmol, 0.24eq.) and this mixture is stirred to a night.Carry out cooling by the dilution of this mixture water (250ml) and with ice to it.Suction filtration goes out a kind of beige solid, is used another part of cold water (25ml) washing, and vacuum-drying, obtains title compound (12.7g, 96%).

synthesizing of the 2nd step: 4-(3,4-dinitrobenzene-benzyl)-morpholine

Sodium borohydride (3.36g, 89mmol, 2.1eq.) is ground, be placed in the flask of nitrogen flush and be suspended in THF (120mL).Being cooled to～0 ℃ time, add wherein boron trifluoride etherate (11.3mL, 89mmol, 2.1eq.) by injection.This reaction slight exotherm is also noticed some hydrogen of generation.Disposable 4-(3, the 4-dinitrobenzoyl) morpholine (11.91g, 42mmol, 1.0eq.) that adds wherein solid form, then use a THF (20mL) to clean this container.Remove ice bath and this suspension is at room temperature stirred to 3 hours, and then carrying out cooling on ice to it.Add wherein carefully methyl alcohol (100mL) (generation hydrogen), then make this mixture reflux 1 hour.By this mixture vacuum concentration, then resistates is distributed between ethyl acetate (100ml) and the saturated solution of sodium bicarbonate/water (100mL) of 1: 1.Organic phase is separated, and water (50mL) then uses salt solution (100ml) wash and it is dried to (MgSO ₄).By initial supercarbonate for washings ethyl acetate (50mL) extract for the second time, then this extract is washed with extracting the identical aqueous solution used for the first time, be then dried (MgSO ₄), merge, concentrated, obtain 10.97g crude product material.With methyl alcohol (45mL, 10mL washings) recrystallization, obtain title compound (9.34g, 83%).

the 3rd step: 4-morpholine-4-ylmethyl-benzene-1,2-diamines synthetic

4-(3,4-dinitrobenzene benzyl) morpholine (21g, 101mmol) is suspended in ethanol (0.9L) and this container is purified with nitrogen.10% palladium is draped over one's shoulders to charcoal (1.05g) to be suspended in ethanol (25ml) and to be joined in this substrate.In cooled on ice, then its atmosphere is changed to this mixture into hydrogen.Make this mixture be warmed to 15-20 ℃ and it is continued under environmental stress to hydrogenation 2 days.By this container nitrogen purge, then by this mixture diatomite filtration, use ethanol (0.3L) to wash in batches.Concentrated, obtain title compound (15.8g, 97%).

the 4th step: 4-nitro-1H-pyrazoles-3-methyl-formiate synthetic

In the reaction vessel of being furnished with digital thermometer and stirring rod of a 20L, put into 4-nitro-1H-pyrazoles-3-formic acid (1.117Kg, 7.11mol, 1wt) and methyl alcohol (8.950L, 8vol).This reaction mixture is stirred under nitrogen, be cooled to 0 to 5 ℃, in 180 minutes, add wherein thionyl chloride (0.581L, 8.0mol, 0.52vol) and make the mixture of gained be warmed to 18 to 22 ℃ and it is stirred a night at this temperature, thereafter ¹h NMR analyzes (d ₆-DMSO) show to react completely.By this reaction mixture concentrating under reduced pressure at 40 to 45 ℃, resistates is processed with toluene and it is reduced pressure at 40 to 45 ℃ under concentrated (3x2.250L again, 3x2vol), obtain 4-nitro-1H-pyrazoles-3-methyl-formiate (1.210Kg, 99.5%th) of pale solid form.

the 5th step: 4-amino-1H-pyrazoles-3-methyl-formiate synthetic

Under nitrogen, be furnished with to a 20L and in the reaction vessel of digital thermometer and stirring rod, put into palladium and drape over one's shoulders carbon (10% wet paste, 0.170Kg, 0.14wt).At one independently in container, by 4-nitro-1H-pyrazoles-3-methyl-formiate (1.210Kg, 7.07mol, 1wt) slurries in ethanol (12.10L, 10vol) are warmed to 30 to 35 ℃ and dissolve and under nitrogen, this solution joined in described catalyzer to complete.After purging by nitrogen-hydrogen order, introduce hydrogen atmosphere and this reaction mixture is maintained to 28 to 30 ℃ until use ¹h NMR analyzes (d ₆-DMSO) observe react completely (5 to 10 hours).Purging after circulation, this reaction mixture being positioned under nitrogen is filtered and solution decompression is concentrated, obtaining 4-amino-1H-pyrazoles-3-methyl-formiate (0.987Kg, 98.9%th).

the 6th step: uncle 4--butoxy carbonyl amino-1H-pyrazoles-3-formic acid synthetic

To 4-amino-1H-pyrazoles-3-methyl-formiate (50.0g, 355mmol) in the mixture in diox (500mL), add the 2M NaOH aqueous solution (213mL, 426mmol), this mixture is heated to 50 ℃ and stirred 5 hours.Then, in this mixture, add (BOC) ₂o (81.4g, 373mmol), Yong diox cleans (100ml) and this mixture is reheated to 5 hours at 50 ℃ it, then it is stirred 14 hours at ambient temperature.Under vacuum, remove diox and add wherein water (1L).The pH that makes this mixture with the dense HCl aqueous solution is for～2 and collect formed solid by filtration, and it is dried on filter.By with toluene (x3) azeotropic with come further this solid to be dried, obtain uncle 4--butoxy carbonyl amino-1H-pyrazoles-3-formic acid (70.0g, 87%) of purple solid form again in vacuum drying oven.

the 7th step: [3-(2-amino-4-morpholine-4-ylmethyl-phenyl amino formyl radical)-1H -pyrazoles-4-yl]-carboxylamine uncle-butyl ester synthetic

By uncle 4--butoxy carbonyl amino-1H-pyrazoles-3-formic acid (10.0g, 44.1mmol), 4-morpholine-4-ylmethyl-benzene-1,2-diamines (10.0g, 48.5mmol), EDC (10.14g, 52.9mmol) and HOBt (7.15g, 52.9mmol) mixture in DMF (150mL) stirs 20 hours at ambient temperature, then under vacuum, removes most of solvent.By resistates at EtOAc (150ml) and saturated NaHCO ₃between the aqueous solution (150mL), distribute, carry out layer and separate and by organic moiety salt water washing, use MgSO ₄be dried and by its decrement under vacuum, obtain [3-(2-amino-4-morpholine-4-ylmethyl-phenyl amino formyl radical)-1H-pyrazoles-4-yl]-carboxylamine uncle-butyl ester (17.6g, 96%) of brown solid.LC/MS analyzes and shows comprise～its two-acid amides of 15% of this product. ¹the level that shows this material in H NMR is about 5%.In step subsequently by the cracking of two-acid amides.

the 8th step: 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrrole synthesizing of azoles-4-base amine

By [3-(2-amino-4-morpholine-4-ylmethyl-phenyl amino formyl radical)-1H-pyrazoles-4-yl]-carboxylamine uncle-butyl ester (12.0g; 28.8mmol) at 85 ℃, heat 14 hours with the mixture of the 2M HCl aqueous solution (50mL), then make it to be cooled to envrionment temperature.Add wherein carefully solid Na ₂cO ₃until the pH of this mixture be～8.5 and this solution by saturated.Form a kind of gumminess liquid of dark color.To make mixture precipitation and solvent is come down in torrents out.In remaining resistates, add EtOH (60mL), this mixture is heated 1 hour under refluxing, then by its heat filtering, it is washed to remove inorganic residues with EtOH (2x20mL).By filtrate decrement under vacuum, obtain a kind of vitreous solid, then by it at Et ₂stir 1 hour in O (60mL) and collect the red-purple powder of gained and by its vacuum-drying by filtration, obtain 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (6.8g, 80%, purity is～90%).

the 9th step: 7-morpholine-4-ylmethyl-2,4-dihydro-1,2,4,5a, 10-pentaaza-ring penta diene is (cyclopenta) [a] fluorenes-5-ketone also

To 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (3.2g that is carrying out at ambient temperature stirring, 10.7mmol) in the mixture in anhydrous THF (50mL), add 1,1 '-carbonyl dimidazoles (1.78g, 11mmol).This mixture is heated 14 hours under refluxing, be then cooled to envrionment temperature.Collect formed solid by filtration, with THF (20ml) washing and by its vacuum-drying, obtain 7-morpholine-4-ylmethyl-2 of pink solid form, 4-dihydro-1,2,4,5a, 10-pentaaza-cyclopenta [a] fluorenes-5-ketone (2.34g, 67%).

the 10th step: 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline- 2-yl)-1H-pyrazoles-4-yl]-urea synthetic

To 7-morpholine-4-ylmethyl-2,4-dihydro-1,2,4,5a, adds cyclopropylamine (6.9mL, 99mmol) in the mixture of 10-pentaaza-cyclopenta [a] fluorenes-5-ketone (10.7g, 32.9mmol) in NMP (65mL).This mixture is heated 5 hours at 100 ℃.LC/MS analyze show～75% be converted into product, therefore, then add wherein a cyclopropylamine (2.3mL, 33mmol), this mixture is heated 4 hours at 100 ℃, be then cooled to envrionment temperature.By the dilution of this mixture water (100ml), extract with EtOAc (100mL).By the saturated NH of organic moiety ₄the Cl aqueous solution (2x50ml) and salt solution (50ml) wash, and then will again use EtOAc (3x100mL) to extract containing water section.By merged organic moiety MgSO ₄be dried and it concentrated under vacuum, obtain 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of brownish glass shape solid form]-urea, (9.10g).

the 11st step: 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline- 2-yl)-1H-pyrazoles-4-yl]-urea, Pfansteihl salt synthetic

To 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea (9.10g, 24mmol) EtOAc-iPrOH (1: 1, in solution 90mL), add Pfansteihl (2.25g, 25mmol).This mixture is stirred 24 hours at ambient temperature, then by its decrement under vacuum.With toluene (100ml) and Et ₂o (100ml) makes resistates provide continuous slurries, collects the solid of gained and is dried (8.04g).

IPrOH (200mL) recrystallization seething with excitement by use and this solid is carried out to purifying, after dry, obtain 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of beige solid form]-urea, Pfansteihl salt (5.7g).

embodiment 66

the 1st step: the preparation of (3,4-dinitrophenyl)-morpholine-4-base-ketone

3,4-dinitrobenzoic acid (1.000Kg, 4.71mol, 1.0wt), tetrahydrofuran (THF) (10.00L, 10.0vol) and dimethyl formamide (0.010L, 0.01vol) are joined to a flask being arranged under nitrogen atmosphere.At 20 to 30 ℃, add wherein thionyl chloride (0.450L, 6.16mol, 0.45vol) and this reaction mixture is heated to 65 to 70 ℃.With ¹h NMR analyzes (d ₆-DMSO) measure it and whether react completely, conventionally need 3 hours.This reaction mixture is cooled to 0 to 5 ℃ and at 0 to 10 ℃, add wherein triethylamine (1.25L, 8.97mol, 1.25vol).At 0 to 10 ℃, in this reaction mixture, add morpholine (0.62L, 7.07mol, 0.62vol) and these slurries are stirred 30 minutes at 0 to 10 ℃.With ¹h NMR analyzes (d ₆-DMSO) measure it and whether react completely.This reaction mixture is warmed to 15 to 20 ℃ and also adds wherein water (4.00L, 4.0vol).Then, this mixture is joined in the flange flask of a 40L who comprises 15 to 25 ℃ of water (21.00L, 21.0vol) so that product precipitation.By cooling the inclusion of this flask and make its at 0 to 5 ℃ aging 1 hour, then pass through solid collected by filtration.Wash filter cake with water (4x5.00L, 4x5.0vol), the pH that finds last washings is pH 7.With ¹h NMR analyzes this wet cake and whether has triethylamine hydrochloride.By this filter cake at 40 to 45 ℃ vacuum-drying until KF shows its water-content < 0.2%w/w, obtain (3 of yellow solid form, 4-dinitrophenyl)-morpholine-4-base-ketone (1.286Kg, 97.0%, KF 0.069%w/w).

the preparation of the 2nd step: 4-(3,4-dinitrobenzene-benzyl)-morpholine

(3,4-dinitrophenyl)-morpholine-4-base-ketone (0.750Kg, 2.67mol, 1.0wt) and tetrahydrofuran (THF) (7.50L, 10.0vol) are joined to one to be arranged in the flask under nitrogen atmosphere and to be cooled to 0 to 5 ℃.At 0 to 5 ℃, add wherein boron trifluoride etherate (0.713L, 5.63mol, 0.95vol) and this suspension stirred at this temperature 15 to 30 minutes.In 90 to 120 minutes, be divided into 6 equal portions and add wherein sodium borohydride (0.212Kg, 5.60mol, 0.282wt).(adding the heat release of noticing a kind of delay after the 1st part for 10 to 15 minutes.Once you begin heat release, that this reaction mixture is again cooling, add wherein again other parts with the intervals of 10 to 15 minutes, thereby this reaction is cooled at every turn between adding).This reaction mixture is stirred 30 minutes at 0 to 5 ℃.With ¹h NMR analyzes (d ₆-DMSO) measure it and whether react completely.At 0 to 10 ℃, drip wherein methyl alcohol (6.30L, 8.4vol) with this reaction mixture of quencher (produce rapidly gas, produce some foams).By this by quencher reaction mixture at 0 to 10 ℃, stir 25 to 35 minutes, be then warmed to and by it at 20 to 30 ℃ (heat release produces gas/ether in the time of dissolution of solid) stir until the generation of gas is slack-off.This mixture is heated to 65 to 70 ℃ and it is stirred 1 hour at this temperature.This mixture is cooled to 30 to 40 ℃ and by its vacuum concentration at 40 to 45 ℃, obtains 4-(3,4-dinitrobenzene-benzyl)-morpholine (0.702Kg, the 98.4%) crude product of yellow/orange solid form.

4-(3,4-dinitrobenzene-benzyl)-morpholine (2.815kg, 10.53mol, 1.0wt) and methyl alcohol (12.00L, 4.3vol) are joined and is arranged in the flask under nitrogen atmosphere and is heated to 65 to 70 ℃.Maintain this temperature until it dissolves completely.Then, this mixture is cooled to 0 to 5 ℃ and by its at this temperature aging 1 hour.By filtering, solid is separated.By methyl alcohol (2x1.50L for filter cake, 2x0.5vol) washing and by its vacuum-drying at 35 to 45 ℃, obtain the 4-(3 of yellow solid form, 4-dinitrobenzene-benzyl)-morpholine (2.353Kg, take the input amount of the 2nd step as basic yield is as 83.5%, the total material dropping into take the 1st step is as basic total recovery is as 82.5%).

the 3rd step: 4-morpholine-4-base-methyl-benzene-1, the preparation of 2-diamines

By 4-(3,4-dinitrobenzene-benzyl)-morpholine (0.800Kg, 2.99mol, 1.0wt) and ethanol (11.20L, 14.0vol) join in a suitable flask and by it and stir at 15 to 25 ℃, and carry out the circulation of three vacuum/nitrogen purgings.Make 10% palladium drape over one's shoulders carbon (10%Pd/C, 50% wet mashed prod, 0.040Kg, 0.05wt weight in wet base) becomes slurries and is joined in this reaction in ethanol (0.80L, 1.0vol).This mixture is cooled to 10 to 20 ℃ and carry out three vacuum/nitrogen and purge circulation.Carry out the circulation of three vacuum/hydrogen purges and this reaction is stirred under hydrogen atmosphere at 10 to 20 ℃.With ¹h NMR analyzes (d ₆-DMSO) measure it and whether react completely, conventionally need 14 to 20 hours.Carrying out three vacuum/nitrogen purges circulation and this reaction mixture is filtered with glass microfiber paper under nitrogen.By washing with alcohol (3x0.80L for filter cake, 3x1.0vol) and merged filtrate and washings are concentrated into dry under vacuum at 35 to 45 ℃, obtain 4-morpholine-4-base-methyl-benzene-1 of brown solid, 2-diamines (0.611Kg 98.6%).

the 4th step: the preparation of 4-nitro-1H-pyrazoles-3-methyl-formiate

4-nitro-1H-pyrazoles-3-formic acid (1.00kg, 6.37mol, 1.0wt) and methyl alcohol (8.00L, 8.0vol) are joined in a flange flask of being furnished with mechanical stirrer, condenser and thermometer.This suspension is cooled under nitrogen to 0 to 5 ℃ and at this temperature, add wherein thionyl chloride (0.52L, 7.12mol, 0.52vol).This mixture was warmed to 15 to 25 ℃ in 16 to 24 hours.With ¹h NMR analyzes (d ₆-DMSO) measure it and whether react completely.By this mixture vacuum concentration at 35 to 45 ℃.In resistates, add toluene (2.00L, 2.0vol) and under vacuum, remove toluene at 35 to 45 ℃.With toluene (2.00L, 2.0vol) repetition azeotropic twice, obtain 4-nitro-1H-pyrazoles-3-methyl-formiate (1.071Kg, 98.3%) of pale solid form.

the 5th step: the preparation of 4-amino-1H-pyrazoles-3-methyl-formiate

The suspension of 4-nitro-1H-pyrazoles-3-methyl-formiate (1.084Kg, 6.33mol, 1.0wt) and ethanol (10.84L, 10.0vol) is heated to 30 to 35 ℃ and hold it at this temperature until dissolve completely.Under nitrogen, 10% palladium is draped over one's shoulders to carbon (10%Pd/C wet mashed prod, 0.152Kg, 0.14wt) and join one independently in flask and carry out three vacuum/nitrogen and purge circulation.Solution by 4-nitro-1H-pyrazoles-3-methyl-formiate in ethanol joins in described catalyzer and carries out three vacuum/nitrogen and purges circulation.Carry out the circulation of three vacuum/hydrogen purges and this reaction is placed under hydrogen atmosphere.This reaction mixture is stirred at 28 to 30 ℃, until ¹h NMR analyzes (d ₆-DMSO) think that it reacts completely.This mixture is filtered under nitrogen and by its vacuum concentration at 35 to 45 ℃, obtain 4-amino-1H-pyrazoles-3-methyl-formiate (0.883Kg, 98.9%) of red-purple solid form.

the 6th step: the preparation of uncle 4--butoxy carbonyl amino-1H-pyrazoles-3-formic acid

By 4-amino-1H-pyrazoles-3-methyl-formiate (1.024Kg, 7.16mol, 1.0wt) He diox (10.24L, 10.0vol) join in a flange flask of being furnished with mechanical stirrer, condenser and thermometer.At 15 to 25 ℃, add wherein 2M aqueous sodium hydroxide solution (4.36L, 8.72mol, 4.26vol) and this mixture is heated to 45 to 55 ℃.Its temperature is maintained to 45 to 55 ℃, until use ¹h NMR analyzes (d ₆-DMSO) measure time show that it reacts completely.At 45 to 55 ℃, add wherein and connect two carbonic acid di-tert-butyls (Boc acid anhydrides, 1.667Kg, 7.64mol, 1.628wt) and this mixture is stirred 55 to 65 minutes. ¹h NMRIPC analyzes (d ₆-DMSO) show to exist 9% unreacted intermediate.At 55 ℃, then add wherein some to connect two carbonic acid di-tert-butyls (Boc acid anhydrides, 0.141Kg, 0.64mol, 0.14wt) and this mixture is stirred 55 to 65 minutes.With ¹h NMR analyzes (d ₆-DMSO) measure it and whether react completely.At 35 to 45 ℃, under vacuum, remove diox and add water (17.60L, 20.0vol) in resistates.With 2M aqueous hydrochloric acid (4.30L, 4.20vol), its pH is adjusted to pH 2 and this mixture is filtered.Filter cake water (10.00L, 9.7vol) is sized mixing 20 to 30 minutes and starch this mixture and filter.Heptane (4.10L, 4.0vol) for filter cake is washed and is layered on dry 16 to 20 hours of pad upper driving (pulled).Make this solid and toluene (5x4.00L, 5x4.6vol) azeotropic together, then by its vacuum-drying at 35 to 45 ℃, obtain uncle 4--butoxy carbonyl amino-1H-pyrazoles-3-formic acid (1.389Kg, 85.4%) of red-purple solid form.

the 7th step: [3-(2-amino-4-morpholine-4-ylmethyl-phenyl amino formyl radical)-1H- pyrazoles-4-yl] preparation of-carboxylamine uncle-butyl ester

By uncle 4--butoxy carbonyl amino-1H-pyrazoles-3-formic acid (0.750Kg, 3.30mol, 1.0wt), 4-morpholine-4-base-methyl-benzene-1,2-diamines (0.752Kg, 3.63mol, 1.0wt) and N, N '-dimethyl formamide (11.25L, 15.0vol) joins in a flange flask of being furnished with mechanical stirrer and thermometer under nitrogen.At 15 to 25 ℃, add wherein I-hydroxybenzotriazole (HOBT, 0.540Kg, 3.96mol, 0.72wt).At 15 to 25 ℃, add wherein N-(3-dimethylaminopropyl)-N '-ethyl carbodiimide (EDC, 0.759Kg, 3.96mol, 1.01wt) and this mixture stirred at this temperature 16 to 24 hours.With ¹whether H NMR analyzes to measure it and reacts completely.By this reaction mixture vacuum concentration at 35 to 45 ℃.Resistates is distributed between ethyl acetate (7.50L, 10.0vol) and saturated sodium bicarbonate aqueous solution (8.03L, 10.7vol) and carry out layer and separate.Salt solution (3.75L, 5.0vol) for organic phase is washed, be dried (1.00Kg, 1.33wt) and it is filtered with magnesium sulfate.By ethyl acetate (1.50L, 2.0vol) washing for filter cake.The vacuum concentration at 35 to 45 ℃ by merged filtrate and washings; obtain [3-(2-amino-4-morpholine-4-ylmethyl-phenyl amino formyl radical)-1H-pyrazoles-4-yl]-carboxylamine uncle-butyl ester (1.217Kg, 88.6%) of Vandyke brown solid form.

the 8th step: 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles the preparation of-4-base amine

By [3-(2-amino-4-morpholine-4-ylmethyl-phenyl amino formyl radical)-1H-pyrazoles-4-yl]-carboxylamine uncle-butyl ester (1.350Kg; 3.24mol; 1.0wt) and ethanol (6.75L, 5.0vol) join in a flange flask of being furnished with mechanical stirrer, condenser and thermometer.At 15 to 30 ℃, under nitrogen, add wherein concentrated hydrochloric acid aqueous solution (1.10L, 13.2mol, 0.80vol), then this inclusion is heated to 70 to 80 ℃ and it is kept at this temperature 16 to 24 hours.At 70 to 80 ℃, add wherein second part of hydrochloric acid (0.11L, 1.32mol, 0.080vol) and this reaction is reheated to 4 hours.Whether it reacts completely to analyze mensuration with HPLC.This reaction mixture is cooled to 10 to 20 ℃ and at this temperature, add wherein salt of wormwood (1.355Kg, 9.08mol, 1.0wt) in batches.This suspension is stirred to and stops producing gas, then it is filtered.Ethanol (1.35L, 1.0vol) for filter cake is washed and retain filtrate.At 15 to 25 ℃,, modulate 20 to 40 minutes filter cake furnishing slurries with ethanol (4.00L, 3.0vol), this mixture is filtered.By ethanol (1.35L, 1.0vol) washing for filter cake and by merged all filtrates vacuum concentration at 35 to 45 ℃.In resistates, add ethanol (4.00L, 3.0vol) and under vacuum, remove ethanol at 35 to 45 ℃.In resistates, add tetrahydrofuran (THF) (5.90L, 4.4vol) and it is stirred 10 to 20 minutes at 15 to 25 ℃.By the solution filter of gained, by tetrahydrofuran (THF) (1.35L, 1.0vol) washing for filter cake and by merged filtrate vacuum concentration at 35 to 45 ℃.In this enriched material, add tetrahydrofuran (THF) (5.40L, 4.0vol) and under vacuum, remove tetrahydrofuran (THF) at 35 to 45 ℃.In enriched material, add tetrahydrofuran (THF) (5.40L, 4.0vol) and at 35 to 45 ℃ under vacuum, remove tetrahydrofuran (THF), obtain the required product of red-purple form of foam---3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (0.924Kg, 95.5%, 82.84% (obtaining with the calculating of HPLC area)).

the 9th step: 7-morpholine-4-ylmethyl-2,4-dihydro-1,2,4,5a, 10-pentaaza-ring penta 2 alkene is the preparation of [a] fluorenes-5-ketone also

By 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine (0.993Kg, 3.33mol, 1.0wt) and tetrahydrofuran (THF) (14.0L, 15.0vol) join in a flange flask of being furnished with mechanical stirrer, condenser and thermometer.These inclusion are stirred under nitrogen at 15 to 25 ℃ and add wherein 1,1 '-carbonyl dimidazoles (0.596Kg, 3.67mol, 0.60wt).Then, these inclusion are heated to 60 to 70 ℃ and it is stirred at this temperature 16 to 24 hours.Whether it reacts completely to analyze detection with TLC.This mixture is cooled to 15 to 20 ℃ and by its filtration.Tetrahydrofuran (THF) (4.00L, 4.0vol) for filter cake is washed and is spread out (pulled) dry 15 to 30 minutes.By the vacuum-drying at 35 to 45 ℃ of these solids, obtain 7-morpholine-4-ylmethyl-2 of red-purple solid form, 4-dihydro-1,2,4,5a, 10-pentaaza-cyclopenta [a] fluorenes-5-ketone (0.810Kg, 75.0% (theoretical value), 92.19% (obtaining with the calculating of HPLC area)).

the 10th step: 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals -yl)-1H-pyrazoles-4-yl] preparation of-urea

By 7-morpholine-4-ylmethyl-2,4-dihydro-1,2,4,5a, 10-pentaaza-cyclopenta [a] fluorenes-5-ketone (0.797Kg, 2.46mol, 1.0wt) and 1-Methyl-2-Pyrrolidone (2.40L, 3.0vol) join in a flange flask of being furnished with mechanical stirrer, condenser and thermometer.At 15 to 30 ℃, under nitrogen, add wherein cyclopropylamine (0.279Kg, 4.88mol, 0.351wt).These inclusion are heated to 95 to 105 ℃ and it is stirred at this temperature 16 to 24 hours.With ¹whether H NMR analyzes to survey it and reacts completely.This reaction mixture is cooled to 10 to 20 ℃ and add wherein ethyl acetate (8.00L, 10.0vol) and saturated sodium-chloride water solution (2.50L, 3.0vol), this mixture is stirred 2 to 5 minutes and carry out layer separation.Organic phase and saturated sodium-chloride water solution (5.00L, 6.0vol) are stirred 25 to 35 minutes together, this mixture is filtered and ethyl acetate (0.40L, 0.5vol) for filter cake is washed.Retain this filter cake, filtrate is transferred in a separating funnel and to it and carried out layer separation.This process is repeated 3 times again, remained solid and organic phase are merged and by this mixture at 35 to 45 ℃ vacuum concentration to dry.During being dissolved in to propan-2-ol (8.00L, 10.0vol) at 45 to 55 ℃, this enriched material also adds wherein activated carbon (0.080Kg, 0.1wt).This mixture is stirred 30 to 40 minutes at 45 to 55 ℃, then by its heat filtering at 45 to 55 ℃.Propan-2-ol (0.40L, 0.5vol) for filter cake is washed.In merged filtrate, add activated carbon (0.080L, 0.1wt), washing, and this mixture is stirred 30 to 40 minutes at 45 to 55 ℃.By this mixture heat filtering propan-2-ol (0.40L, 0.5vol) for filter cake is washed at 45 to 55 ℃.The vacuum concentration at 35 to 45 ℃ by filtrate and washings.At 25 to 35 ℃, in enriched material, add ethyl acetate (8.00,10.0vol) and water (2.20L, 3.0vol) and this mixture is stirred 1 to 2 minute.Carry out layer separate and by organic phase vacuum concentration at 35 to 45 ℃.To adding ethyl acetate (4.00L, 5.0vol) in resistates and by its vacuum concentration at 35 to 45 ℃.In resistates, add ethyl acetate (4.00L, 5.0vol) and this mixture is stirred 2 to 20 hours at 15 to 25 ℃.By cooling this mixture 0 to 5 ℃ and by its at this temperature aging 90 to 120 minutes, then it is filtered.Ethyl acetate (0.80L, 1.0vol) for filter cake is washed and spread out dry 15 to 30 minutes.By the vacuum-drying at 35 to 45 ℃ of these solids, obtain 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of brown solid]-urea (0.533Kg, 56.8%, 93.20% (obtaining with the calculating of HPLC area)).

The product of the 9th step to some batches is processed in this way, and the details of each batch of starting raw material and product quantity is described in table 1A.

table 1A-forms by urea the yield that step-10th step obtains

Batch	7-morpholine-4-ylmethyl-2,4-dihydro-1,2,4,5a, the input amount (g) of 10-pentaaza-cyclopenta [a] fluorenes-5-ketone	1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] input amount (g) of-urea	The chemical purity that HPLC area shows
				1	680	442 55.2% (theoretical values), 64.9%w/w	91.80
2	882	487 47.0% (theoretical values), 56.6%w/w	91.21
				3	879	445 43.0% (theoretical values), 50.6%w/w	91.66
4	797	533 56.8% (theoretical values), 66.8%w/w	93.20

the 11st step: 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzoglyoxaline- 2-yl)-1H-pyrazoles-4-yl] preparation of-urea Pfansteihl salt

By 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea (1.859Kg, 4.872mol, 1.0wt), propan-2-ol (9.00L, 5.0vol) and ethyl acetate (8.00L, 4.5vol) join in a flange flask of being furnished with mechanical stirrer and thermometer.These inclusion stirred under nitrogen and at 15 to 25 ℃, add wherein Pfansteihl (0.504Kg, 5.59mol, 0.269wt), then adding a row ethyl acetate scavenging solution (0.90L, 0.5vol).This mixture is stirred 120 to 140 minutes at 15 to 25 ℃.By filtering to isolate solid, ethyl acetate (2x2.00L, 2x1.0vol) for filter cake is washed and spread out dry 20 to 40 minutes.Filter cake is dissolved in ethanol (33.00L, 17.7vol) at 75 to 85 ℃, is cooled to 65 to 70 ℃ and make the clarification of this solution by filtering with glass microfiber paper.By cooling filtrate 15 to 25 ℃ and make its at this temperature aging 2 to 3 hours.Separate by the solid having filtered crystallization, by ethanol (2x1.00L, 2x0.5vol) washing filter cake for and spread out and be dried at least 30 minutes.By the vacuum-drying at 35 to 45 ℃ of this solid, obtain 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl of dark pink homogeneous solid form]-urea Pfansteihl salt (1.386Kg, 58.7% (theoretical value), 99.47% (obtaining with the calculating of HPLC area)).

This Lactated infrared spectra (KBr disk method) comprises and is positioned at 3229,2972 and 1660cm ^-1under characteristic peak.

Do not wish to be bound by any theory, think that these infrared peaks can be attributed on the construction package of this salt as follows like that:

embodiment 67

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl] crystalline free base of-urea and synthesizing of crystalline salt form

a.1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals- base)-1H-pyrazoles-4-yl] preparation of-urea free alkali

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the crude product sample of-urea free alkali is to make as summarized in embodiment 60, first with silica gel column chromatography, it is carried out to purifying, with EtOAc-MeOH (98: 2-80: 20) carry out wash-out.Then,, by obtained free alkali sample recrystallization from hot methanol, obtain 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] crystalline material of-urea free alkali.

b.1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals- base)-1H-pyrazoles-4-yl] preparation of-urea free alkali dihydrate

By 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the crude product sample dissolution of-urea free alkali in THF, then by its vacuum concentration to minimum volume (～4 volumes).In this solution, drip water (2-4 volume) until this solution becomes muddy.Add wherein a small amount of THF so that this solution again becomes clarification and this mixture is left standstill to a night, obtain a kind of crystalline material, it is air-dry, obtain 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea free alkali dihydrate.

c.1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals- base)-1H-pyrazoles-4-yl] preparation of-urea hydrochloride

By 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the crude product sample dissolution of-urea free alkali in minimum MeOH, then with EtOAc, it is diluted.At 0 ℃, to the HCl (4M dioxane solution) that slowly adds 1.1 equivalents in this solution.After adding, collect the solid being precipitated out by filtration from solution.To adding MeOH in this solid and by mixture decrement under vacuum.In order to remove the remaining MeOH of trace, by the water evaporation in resistates, then it is dried under 60 ℃/0.1mbar, obtain described hydrochloride.

d.1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals- base)-1H-pyrazoles-4-yl] preparation of-urea esilate

To 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] add 1 equivalent ethyl sulfonic acid in the-solution of urea free alkali in MeOH-EtOAc.This mixture is stirred at ambient temperature, then by its decrement under vacuum.Resistates is absorbed in MeOH and to adding Et in this solution ₂o.Make this mixture leave standstill 72 hours, collect formed solid and be dried by filtration, obtain 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea esilate.

e.1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals- base)-1H-pyrazoles-4-yl] preparation of-urea mesylate

To 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] (67 μ are l) to add 1 equivalent methylsulfonic acid in the-solution of urea free alkali (394mg) in MeOH-EtOAc.Collect formed solid by filtration, it is washed with EtOAc.This dissolution of solid, in minimum hot MeOH, is made it to cooling, then use Et ₂o develops it.This solid is left standstill to 72 hours, then by filtering, it is collected, it is washed with MeOH, obtain 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea mesylate.

embodiment 68

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl] characteristic of-urea free alkali and salt

To 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] various forms of-urea carries out characterization.By determining for the selected form of characterization of mainly polymorphic and salt-stable category being investigated.For the further selected salt of characterization is Pfansteihl salt, free alkali dihydrate, esilate, free alkali and hydrochloride.

A. dsc (DSC):

On the TA instrumentQ1000 that is furnished with 50position automatic sampler (auto-sampler), collect differential thermogram.The standard of energy and temperature correction is indium.These samples are heated to 250 ℃ with the speed of 10 ℃/min from 10.On these samples, maintain the nitrogen purging of 30ml/min.(unless stated otherwise, otherwise) used 2 to 10mg samples and all samples to be all encased in and covers in the foraminate aluminium dish of tool.

Identify	Fusing point (℃)
		Pfansteihl salt	190℃
Free alkali dihydrate	Desolventizing compound (at 110 ℃ of peaks of locating)
		Esilate	Do not observe (being up to 350 ℃)
Free alkali	193℃
		Hydrochloride	190℃

B. thermogravimetric analysis (TGA):

On TA Instruments Q500, collect differential thermogram.Sample is heated with the speed of 10 ℃/min.On these samples, maintain the nitrogen purging of 100ml/min.Conventionally 5-20mg sample is loaded on tared (tared) opening aluminium dish.

identify	observe
		pfansteihl salt	lose 1.7% unconjugated solvent, at 190 ℃, in the situation that thering is degraded, melted.
free alkali dihydrate	weightless (before degraded) 4.1%w/w (be equivalent to 1 and work as water gaging)
		esilate	lose 4% unconjugated solvent, do not have other significantly can recognize feature
free alkali	lose 1.7% unconjugated solvent, at 193 ℃, in the situation that thering is degraded, melt
		hydrochloride	lose 5.4% unconjugated solvent, at 190 ℃, in the situation that thering is degraded, melt

C. PLM

On the Leica LM/DM microscope with the digital camera of catching image, sample is studied.A small amount of sample is placed in to the immersion oil that is arranged on slide glass and it is covered with cover glass.As far as possible each particulate separated and use the ratio of enlargement of 50-500x with the part crossed polars being connected with λ wave plate, it to be checked.

Identify	Observe
		Pfansteihl salt	Irregular crystalline particle
Free alkali dihydrate	Irregular crystalline particle
		Esilate	Irregular crystalline particle
Free alkali	Needle crystals particle
		Hydrochloride	Irregular crystalline particle

D.XRPD (X-ray powder diffraction)

D5000

With CuK alpha-ray (40kV, 40mA), θ-θ protractor, automatically disperse and accept slit, the secondary monochromator of graphite (graphite secondary monochromator) and scintillometer and carry out XRPD research on Siemens D5000 diffractometer.In continuous sweep mode, during by the step of the step-length of 0.02 ° of 2 θ or 0.005 ° of 2 θ and 1 second, (step time) collects data at 2 ° to the angular range of 30 ° of 2 θ.

With the sample of the flat form of the powder preparation of accepting of grinding, under envrionment conditions, move these samples.It is 12mm that about 25-50mg sample is gently filled into diameter, the degree of depth is (to be cut into zero-background (510) silicon wafer (wafer) (The Gem Dugout of polishing in the cavity of 0.5mm, 1652 Princeton Drive, Pennsylvania StateCollege, PA 16803, USA)).

All XRPD analyze and carry out with Diffrac Plus XRD Commander software v2.3.1.

Bruker AXS C2 GADDS diffractometer (for the sample being reclaimed by GVS)

With CuK alpha-ray (40kV, 40mA), automatically XYZ platform, for laser video microscope (laser video microscope) and the HiStar 2-dimensional plane detector of sampling location automatically, on Bruker AXS C2 GADDS diffractometer, obtain the X-ray powder diffraction style of sample.X-ray optics is single by what be connected with the pinhole collimator (pinhole collimator) of 0.3mm

many layer mirror (multilayer mirror) forms.

Analyzer, the effective scale of X-bundle of rays on sample is about 4mm.Adopt θ-θ continuous sweep mode, the detector distance of a sample is 20cm, and it has provided effective 2 θ scopes of 3.2-29.8 °.The typical irradiation time of sample (exposure time) is 120s.

With the sample of the flat form of the powder preparation of accepting of grinding.Thereby about 1-2mg sample is pressed onto lightly and on a slide glass, obtains a kind of flat surface.

Record the XRPD trace of described Pfansteihl salt and free alkali.Its trace shows good signal to noise ratio, and represents that it is a kind of crystalline material.

E. gravimetric analysis steam absorption (Gravimetric Vapour Sorption) is (GVS):

All samples are all to move on the Hiden IGASorp moisture absorption analyzer of operation CFRSorp software.Sample size is about 10-25mg.As described below water adsorption/the desorption isotherm of carrying out like that.Under room humidity and temperature (approximately 40%RH, 25 ℃), sample loaded and unloaded, using thereafter XRPD (using Bruker AXS C2 GADDS system) to analyze it.

Standerd isotherm operation is the single loop starting under 40%RH.

Humidity ladder is as follows:

40，50，60，70，80，90

85，75，65，55，45，35，25，15，5，0

10，20，30，40

(i) Pfansteihl salt

The GVS thermoisopleth of Pfansteihl salt shows that its sample does not show moisture absorption behavior and do not form hydrate.Carrying out after GVS experiment, the XRPD trace of sample is consistent with inputted material, shows not undergo phase transition at experimental session.

(ii) free alkali

At experimental session, the weight of the sample room between 0%R.H～95%R.H differs about 9%.It shows that this sample is hygroscopic in essence.

embodiment 69

with X-ray diffraction to 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzene and imidazoles-2-yl)-1H-pyrazoles-4-yl]-urea dihydrate freing alkali crystal structure enters the mensuration of row

Be colourless and be that size is 0.2x0.2x0.2mm for the crystal of described diffraction experiment ³irregularly shaped.It is by the aqueous solution precipitation of esilate being obtained with THF in liquid-liquid diffusion experiment.Compare the equivalence (equivalence) of the phase homomorphism of determining such sample and made by free alkali (water is as the scope of anti-solvent solvent for as such as ethanol of alcohols, and ketone is if methyl ethyl ketone and ethers are as THF and diox) by the X-ray powder diffraction style to two kinds of samples.With the CuK alpha-ray that derives from Rigaku rotating anode RU3HR

osmium blueness, with burnt optics (Osmic blue confocal optics), AFC91/4 χ protractor and Rigaku Jupiter CCD detector, is collected crystallographic data under 101 (2) K.In four ω scanning, collect image, one of them scanning is positioned under 2 θ=30 ° and three scannings are positioned under 2 θ=90 °, detector to crystal apart from 67mm.Carry out data gathering and image processed and measured with Dtrek with CrystalClear software.Although uptake factor is medium (μ=0.82mm ^-1) data, but with 4 ^thorder Fourier absorbs to correct data is corrected to compensate glue and crystal clamper micromount (micromount) absorption.Find that this crystal belongs to monclinic spacer P2 ₁/ n (#14), has a=7.66 (10), b=15.18 (10),

β=98.53 (2) °, the lattice parameter of α=γ=90 °.Numeral deviation (s.u., stardard uncertairty) in bracket.

Crystalline structure is resolved by the direct method of carrying out in SHELXS-97.With SHELXL-97 from in the intensity data of 2822 the unique reflections altogether in the analytical range (resolutionrange) of (3.85 < θ < 60.01), extract 274 crystallographic parameters.Final statistics parameter is: wR2=0.2416 (all data), R _f=0.0866 (data of I > 2 σ (I)) and goodness of fit S=1.145.

In this asymmetry unit, find to exist a part free alkali and two molecular waters.The elementary composition of this asymmetry unit is C ₁₉h ₂₆n ₇o ₄and the density of this crystal calculating is 1.36Mg/m ³.In the time determining the heteroatomic position of being combined with hydrogen atom with Fo-Fc disparity map (difference maps) inspection, hydrogen atom betides on geometrical plane (geometrical ground).The position of hydrogen atom and thermal parameter are compressed on corresponding non-hydrogen atom.Simulate the thermal motion (seeing Fig. 1) of non-hydrogen atom by the anisotropic hot factor.

This crystalline structure comprises (N22-H...N14 in a molecule

) and seven intermolecular hydrogen bondings (seeing Fig. 2).1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea molecule is connected one to the other in chain along b crystalline axis direction by two H-keys: N7-H...O24

and N25-H...N2

the benzoglyoxaline part that derives from two chains with

distance be stacked into together.1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] network of-urea molecule formed the bag being occupied by four water moleculess, and these four water moleculess are symmetrical between two with respect to symmetry centre.Three H-keys are by 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea molecule connects together with water molecules, and one and 1 ^stwater molecules (O1W1-H...N16 ) be connected and remain two and 2 ^ndwater molecules (N1-H...O1W2

and O1W2-H...O19

) be connected.Water molecules participates in interaction each other: O1W1-H...O1W2 by two other H-key

and O1W2-H...O1W1

The thermal ellipsoid presentation of the described structure producing by X-ray diffraction institute is provided in Fig. 1, and its package bundle figure is provided in Fig. 2.

Composition 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] coordinate of atom of structure of-urea free alkali dihydrate is as described in Table 2.Numeral deviation (s.u., stardard uncertairty) in bracket.

table 2

_cell_length_a 7.662(10)

_cell_length_b 15.184(10)

_cell_length_c 17.711(10)

_cell_angle_alpha 90.00

_cell_angle_beta 98.53(2)

_cell_angle_gamma 90.00

_cell_measurement_temperature 101(2)

loop_

_atom_site_label

_atom_site_type_symbol

_atom_site_fract_x

_atom_site_fract_y

_atom_site_fract_z

_atom_site_U_iso_or_equiv

_atom_site_adp_type

_atom_site_occupancy

_atom_site_symmetry_multiplicity

_atom_s ite_calc_flag

N1 N 0.4468(4) 0.0332(2) 0.71441(19) 0.0274(9) Uani 1 1 d

H1 H 0.5453 0.0189 0.6973 0.033 Uiso 1 1 calc

N2 N 0.3749(4) -0.01642(19) 0.76559(19) 0.0253(8) Uani 1 1 d

C3 C 0.2277(5) 0.0286(2) 0.7751(2) 0.0237(9) Uani 1 1 d

C4 C 0.2074(6) 0.1060(2) 0.7308(2) 0.0246(9) Uani 1 1 d

C5 C 0.3539(5) 0.1058(3) 0.6923(2) 0.0254(10) Uani 1 1 d

H5 H 0.3822 0.1490 0.6572 0.030 Uiso 1 1 calc

C6 C 0.1101(5) -0.0035(2) 0.8265(2) 0.0213(9) Uani 1 1 d

N7 N 0.1457(5) -0.0752(2) 0.87205(19) 0.0268(9) Uani 1 1 d

H7 H 0.2403 -0.1087 0.8758 0.032 Uiso 1 1 calc

C8 C 0.0015(6) -0.0852(2) 0.9119(2) 0.0251(10) Uani 1 1 d

C9 C -0.0262(6) -0.1443(2) 0.9695(2) 0.0266(10) Uani 1 1 d

H9 H 0.0553 -0.1898 0.9865 0.032 Uiso 1 1 calc

C10 C -0.1833(5) -0.1319(2) 1.0008(2) 0.0258(10) Uani 1 1 d

C11 C -0.3006(6) -0.0649(3) 0.9758(2) 0.0295(10) Uani 1 1 d

H11 H -0.4052 -0.0590 0.9982 0.035 Uiso 1 1 calc

C12 C -0.2704(6) -0.0064(3) 0.9194(2) 0.0321(11) Uani 1 1 d

H12 H -0.3527 0.0387 0.9023 0.039 Uiso 1 1 calc

C13 C -0.1115(6) -0.0163(2) 0.8878(2) 0.0261(10) Uani 1 1 d

N14 N -0.0434(4) 0.03474(19) 0.83324(19) 0.0254(8) Uani 1 1 d

C15 C -0.2143(5) -0.1900(2) 1.0676(2) 0.0263(10) Uani 1 1 d

H15A H-0.1009 -0.1979 1.1018 0.032 Uiso 1 1 calc

H15B H -0.2963 -0.1593 1.0970 0.032 Uiso 1 1 calc

N16 N -0.2871(5) -0.2772(2) 1.04532(18) 0.0268(8) Uani 1 1 d

C17 C -0.4708(6) -0.2702(3) 1.0075(2) 0.0303(10) Uani 1 1 d

H17A H -0.4749 -0.2350 0.9602 0.036 Uiso 1 1 calc

H17B H -0.5421 -0.2395 1.0416 0.036 Uiso 1 1 calc

C18 C -0.5484(6) -0.3603(3) 0.9879(2) 0.034 4(11) Uani 1 1 d

H18A H -0.6723 -0.3540 0.9631 0.041 Uiso 1 1 calc

H18B H -0.4814 -0.3896 0.9513 0.041 Uiso 1 1 calc

O19 O -0.5428(4) -0.41359(18) 1.05435(16) 0.0343(8) Uani 1 1 d

C20 C -0.3636(6) -0.4216(3) 1.0925(3) 0.0344(11) Uani 1 1 d

H20A H -0.2914 -0.4518 1.0584 0.041 Uiso 1 1 calc

H20B H -0.3617 -0.4580 1.1390 0.041 Uiso 1 1 calc

C21 C -0.2855(6) -0.3338(3) 1.1140(2) 0.0287(10) Uani 1 1 d

H21A H -0.3537 -0.3048 1.1503 0.034 Uiso 1 1 calc

H21B H -0.1626 -0.3413 1.1397 0.034 Uiso 1 1 calc

N22 N 0.0659(4) 0.16310(19) 0.72860(18) 0.0242(8) Uani 1 1 d

H22 H -0.0267 0.1453 0.7484 0.029 Uiso 1 1 calc

C23 C 0.0617(5) 0.2451(2) 0.6976(2) 0.0247(9) Uani 1 1 d

O24 O 0.1870(4) 0.27405(17) 0.66702(16) 0.0304(8) Uani 1 1 d

N25 N -0.0851(4) 0.2937(2) 0.70242(19) 0.0270(8) Uani 1 1 d

H25 H -0.0807 0.3509 0.6948 0.032 Uiso 1 1 calc

C26 C -0.2479(6) 0.2563(3) 0.7194(3) 0.0320(11) Uani 1 1 d

H26 H -0.3061 0.2121 0.6820 0.038 Uiso 1 1 calc

C27 C -0.3687(6) 0.3144(3) 0.7561(2) 0.0346(11) Uani 1 1 d

H27A H -0.4974 0.3069 0.7404 0.041 Uiso 1 1 calc

H27B H -0.3304 0.3757 0.7681 0.041 Uiso 1 1 calc

C28 C -0.2705(6) 0.2417(3) 0.8022(3) 0.0370(11) Uani 1 1 d

H28A H -0.3387 0.1896 0.8144 0.044 Uiso 1 1 calc

H28B H -0.1716 0.2585 0.8421 0.044 Uiso 1 1 calc

O1W1 O -0.0371(4) -0.37 444(18) 0.97522(18) 0.0392(8) Uani 1 1 d

H1W1 H 0.0243 -0.4072 1.0168 0.047 Uiso 1 1 d

H2W1 H -0.1218 -0.3425 0.9983 0.047 Uiso 1 1 d

O1W2 O 0.1516(4) -0.4721(2) 1.1013(2) 0.0421(9) Uani 1 1 d

H1W2 H 0.113(7) -0.509(4) 1.067(3) 0.051 Uiso 1 1 d

H2W2 H 0.2534 -0.4527 1.0856 0.051 Uiso 1 1 d

embodiment 70

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl] mensuration of-urea free alkali XRPD style

With marble mortar to grinding gently for the sample of X-ray powder diffraction (XRPD) data gathering and these samples load to crystallography kapillary and (derive from HamptonResearch, quartz or glass mould 10, diameter be 0.4 or 0.7mm) in.At room temperature, with the CuK alpha-ray that derives from Rigaku rotating anode RU3HR

osmium blueness is collected diffraction pattern with burnt optics (Osmic blue confocal optics), 1/4 χ protractor and Rigaku HTC imaging plate detector.When In service inspection device rotates the crystal distance to 250mm to φ axle, collect 2D image.Control data gathering and these 2D images are changed into 1D figure (2 θ-intensity) (average intensity with the spacing of 0.01 ° or 0.02 ° 360 ° of 3-30 ° of 2 angle, θ scope interior orientation 0 < χ <) with Datasqueeze with CrystalClear software.In house, carries out the processing of 1D XRPD style and visual by AstexXRPD program.

XRPD style between the not syncrystallization batch and the relative intensity at peak do not change, its with have that only a kind of crystalline form is consistent.

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl is provided in Fig. 3] the XRPD style of the FB1 form of-urea free alkali and listed the details of its main peak in table 3.

2 θ of table 3. main peak, d-spacing (spacing) and relative intensity.

embodiment 71

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl] mensuration of-urea lactic acid salt crystalline structure

Determine 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] the monocrystalline form of-urea Pfansteihl salt.Diffraction experiment crystal used is that the size by being obtained by ethanol evaporation is 0.1x0.1x0.1mm ³colourless prism.Under 97K, with the CuK alpha-ray that derives from Rigaku rotating anode RU3HR

osmium blueness is collected crystallographic data with burnt optics (Osmic blue confocal optics), AFC9 1/4 χ protractor and Rigaku Jupiter CCD detector.In 5 ω scanning, collect image with detector, single pass is positioned under 2 θ=15 ° and four scanning is positioned under 2 θ=90 °, detects the crystal distance to 67 mm.Carry out data gathering and image processed and measured with Dtrek with CrystalClear software.Although uptake factor is medium (μ=0.78mm ^-1), but with 4 ^thorder Fourier absorbs to correct data is corrected to compensate glue and crystal clamper (micromount (micromount)) absorption.Find that this crystal belongs to orthorhombic space group P2 ₁2 ₁2 ₁(#19), there is a=9.94 (10), b=15.03 (10),

the lattice parameter of α=β=γ=90 °.Numeral deviation (s.u., stardard uncertairty) in bracket.Carry out once of short duration room temperature and scan to check lattice parameter and symmetry.Find its symmetry and lattice parameter identical with symmetry under 97 (2) K also similar to it (room temperature a=10.08, b=15.22,

).

Crystalline structure is resolved by the direct method of carrying out in SHELXS-97.Select absolute configuration, to mate Pfansteihl salt configuration used in crystallization experiment.With SHELXL-97 from

in the intensity data of 3417 the unique reflections altogether in the analytical range (resolution range) of (4.01 < θ < 58.92), extract 308 crystallographic parameters.Final statistics parameter is: wR2=0.2275 (all data), R _f=0.0817 (data of I > 2 σ (I)) and goodness of fit S=1.076.

In this asymmetry unit, find to exist the protonated free alkali of a part and a part Pfansteihl salt anionic.The elementary composition of this asymmetry unit is C ₂₂h ₂₉n ₇o ₅and the density of this crystal calculating is 1.30Mg/m ³.In the time determining the heteroatomic position of being combined with hydrogen atom with Fo-Fc disparity map (difference maps) inspection, hydrogen atom betides on geometrical plane (geometrical ground).The position of hydrogen atom and thermal parameter are compressed on corresponding non-hydrogen atom.Simulate the thermal motion (seeing Fig. 4) of non-hydrogen atom by the anisotropic hot factor.

This crystalline structure comprises (N22-H...N14 in a molecule

) and seven intermolecular hydrogen bondings, it has formed complex body 3D network (seeing Fig. 5).In intermolecular H-key two, N7-H...O24

and N25-H...N2

by 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea molecule is connected in the chain of c crystalline axis direction.Pfansteihl salt anionic is by H-key O3L-H...O1L be connected in the chain of a crystalline axis direction.The H-key of two bifurcateds connects 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-uronium and Pfansteihl salt anionic.Protonated morpholine nitrogen-atoms and two carboxyl oxygen atoms interaction (N16-H...O1L

and N16-H...O2L

), and pyrazoles N1 nitrogen is the H donor (N1-H...O2L of O2L and O3L

n1-H...O3L

).

The thermal ellipsoid presentation of the described structure producing by X-ray diffraction institute is provided in Fig. 4, and its package bundle figure is provided in Fig. 5.

Composition 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl] coordinate of the atom of-Lactated structure of urea is as described in Table 4.Numeral deviation (s.u., stardard uncertairty) in bracket.

table 4

_cell_length_a 9.941(10)

_cell_length_b 15.034(10)

_cell_length_c 16.175(10)

_cell_angle_alpha 90.00

_cell_angle_beta 90.00

_cell_angle_gamma 90.00

_cell_measurement_temperature 97(2)

loop_

_atom_site_label

_atom_site_type_symbol

_atom_site_fract_x

_atom_site_fract_y

_atom_site_fract_z

_atom_site_U_iso_or_equiv

_atom_site_adp_type

_atom_site_occupancy

_atom_site_symmetry_multiplicity

_atom_site_calc_flag

N1 N 0.9111(5) 0.4310(3) 0.5668(2) 0.0509(12) Uani 1 1 d

H1 H 0.9653 0.3878 0.5824 0.061 Uiso 1 1 calc

N2 N 0.8702(5) 0.4971(3) 0.6177(2) 0.0503(12) Uani 1 1 d

C3 C 0.7902(5) 0.5479(3) 0.5704(3) 0.0395(11) Uani 1 1 d

C4 C 0.7795(6) 0.5130(3) 0.4891(3) 0.0431(12) Uani 1 1 d

C5 C 0.8601(5) 0.4380(3) 0.4893(3) 0.0449(12) Uani 1 1 d

H5 H 0.8766 0.3991 0.4441 0.054 Uiso 1 1 calc

C6 C 0.7254(5) 0.6280(3) 0.6003(3) 0.0404(12) Uani 1 1 d

N7 N 0.7166(4) 0.6504(3) 0.6825(2) 0.0428(10) Uani 1 1 d

H7 H 0.7473 0.6201 0.7250 0.051 Uiso 1 1 calc

C8 C 0.6485(5) 0.7316(3) 0.6840(3) 0.0413(11) Uani 1 1 d

C9 C 0.6136(5) 0.7875(3) 0.7496(3) 0.0443(12) Uani 1 1 d

H9 H 0.6337 0.7722 0.8052 0.053 Uiso 1 1 calc

C10 C 0.5477(6) 0.8667(3) 0.7300(3) 0.0482(12) Uani 1 1 d

C11 C 0.5166(6) 0.8863(3) 0.6481(3) 0.0495(13) Uani 1 1 d

H11 H 0.4708 0.9403 0.6364 0.059 Uiso 1 1 calc

C12 C 0.5495(6) 0.8304(3) 0.5826(3) 0.0508(13) Uani 1 1 d

H12 H 0.5264 0.8449 0.5272 0.061 Uiso 1 1 calc

C13 C 0.6186(5) 0.7510(3) 0.6021(3) 0.0428(12) Uani 1 1 d

N14 N 0.6671(4) 0.6851(3) 0.54 97(2) 0.0434(10) Uani 1 1 d

C15 C 0.5154(6) 0.9337(3) 0.7949(3) 0.0529(14) Uani 1 1 d

H15A H 0.4767 0.9027 0.8434 0.064 Uiso 1 1 calc

H15B H 0.4462 0.9749 0.7733 0.064 Uiso 1 1 calc

N16 N 0.6353(5) 0.9869(3) 0.8225(3) 0.0504(11) Uani 1 1 d

H16 H 0.6962 0.9472 0.8458 0.060 Uiso 1 1 calc

C17 C 0.7050(7) 1.0325(4) 0.7543(4) 0.0652(16) Uani 1 1 d

H17A H 0.6420 1.0734 0.7260 0.078 Uiso 1 1 calc

H17B H 0.7370 0.9882 0.7135 0.078 Uiso 1 1 calc

C18 C 0.8234(7) 1.0844(4) 0.7881(4) 0.0732(18) Uani 1 1 d

H18A H 0.8887 1.0426 0.8130 0.088 Uiso 1 1 calc

H18B H 0.8689 1.1157 0.7421 0.088 Uiso 1 1 calc

O19 O 0.7835(5) 1.1470(3) 0.8481(3) 0.0804(14) Uani 1 1 d

C20 C 0.7191(8) 1.1040(4) 0.9155(4) 0.0724(19) Uani 1 1 d

H20A H 0.6921 1.1492 0.9568 0.087 Uiso 1 1 calc

H20B H 0.7835 1.0629 0.9423 0.087 Uiso 1 1 calc

C21 C 0.5984(6) 1.0533(4) 0.8886(4) 0.0619(16) Uani 1 1 d

H21A H 0.5299 1.0950 0.8668 0.074 Uiso 1 1 calc

H21B H 0.5591 1.0218 0.9366 0.074 Uiso 1 1 calc

N22 N 0.7055(5) 0.5524(3) 0.4260(2) 0.0455(10) Uani 1 1 d

H22 H 0.6642 0.6028 0.4368 0.055 Uiso 1 1 calc

C23 C 0.6930(6) 0.5175(4) 0.3483(3) 0.0475(13) Uani 1 1 d

O24 O 0.7394(4) 0.4431(2) 0.32976(19) 0.0524(10) Uani 1 1 d

N25 N 0.6245(5) 0.5675(3) 0.2934(2) 0.0506(11) Uani 1 1 d

H25 H 0.5979 0.5428 0.2468 0.061 Uiso 1 1 calc

C26 C 0.5929(6) 0.6602(3) 0.3080(3) 0.0512(13) Uani 1 1 d

H26 H 0.6709 0.7017 0.3144 0.061 Uiso 1 1 calc

C27 C 0.4712(6) 0.6964(4) 0.2675(3) 0.0580(15) Uani 1 1 d

H27A H 0.4182 0.6557 0.2321 0.070 Uiso 1 1 calc

H27B H 0.4743 0.7589 0.2481 0.070 Uiso 1 1 calc

C28 C 0.4692(7) 0.6806(4) 0.3585(3) 0.0642(17) Uani 1 1 d

H28A H 0.4156 0.6298 0.3794 0.077 Uiso 1 1 calc

H28B H 0.4718 0.7331 0.3954 0.077 Uiso 1 1 calc

C1L C 0.7508(6) 0.8367(4) 0.9477(3) 0.0521(14) Uani 1 1 d

O1L O 0.6267(5) 0.8403(3) 0.9593(3) 0.0793(14) Uani 1 1 d

O2L O 0.8130(4) 0.8862(3) 0.8976(2) 0.0595(11) Uani 1 1 d

C2L C 0.8308(7) 0.7682(4) 0.9940(4) 0.0692(17) Uani 1 1 d

H2L H 0.7934 0.7082 0.9802 0.083 Uiso 1 1 calc

O3L O 0.9655(5) 0.7716(3) 0.9651(4) 0.0935(17) Uani 1 1 d

H3L H 1.0127 0.7353 0.9918 0.140 Uiso 1 1 calc

C3L C 0.8189(9) 0.7814(7) 1.0854(5) 0.108(3) Uani 1 1 d

H3L1 H 0.7804 0.7279 1.1106 0.162 Uiso 1 1 calc

H3L2 H 0.7603 0.8324 1.0966 0.162 Uiso 1 1 calc

H3L3 H 0.9082 0.7925 1.1088 0.162 Uiso 1 1 calc

embodiment 72

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)- 1H-pyrazoles-4-yl] stability of-urea salt under 40 ℃ of 75%RH

With marble mortar, about 15mg is ground gently for the sample that carries out stability study and by it form with thin layer transfer to Petri dish.Then, these samples are placed into and are comprised in the sealed vessel of saturated NaCl solution with excessive undissolved NaCl.This container be put in the thermostatted remaining at 40 ℃ again and provide the environment of 40 ℃ and ≈ 75% relative humidity (RH) for it.With regular time interval, these samples are analyzed with X-ray powder diffraction (XRPD).

To load to for the sample that carries out XRPD data gathering in crystallography kapillary (derive from Hampton Research, made by quartz, diameter=0.4mm).At room temperature, with the CuK alpha-ray that derives from Rigaku rotating anode RU3HR

osmium blueness is collected diffraction pattern with burnt optics (Osmic blue confocal optics), 1/4 χ protractor and Rigaku HTC imaging plate detector.When In service inspection device rotates the crystal distance to 250mm to φ axle, collect 2D image.Control data gathering and with Datasqueeze, these 2D images are changed into 1D with CrystalClear software and scheme (2 θ-intensity) (average intensity with the spacing of 0.01 ° 360 ° of 3-30 ° of 2 angle, θ scope interior orientation 0 < χ <).In house, carries out the processing of 1D XRPD style and visual by AstexXRPD program.

In the time being exposed under 40 ℃ and 75%RH, in 1-2 month, the style of the XRPD of lactic acid salt, free alkali (FB1) and dihydrate free alkali (FB2) does not change.Lactic acid salt, free alkali (FB1) and dihydrate free alkali (FB2) in Fig. 6-8, are provided in the time starting and the XRPD style of sample when stability experiment.

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl is provided in Fig. 6] the Pfansteihl salt XRPD style of the free alkali of-urea and listed the details of its main peak in table 5.

table 5

1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl is provided in Fig. 8] the XRPD style of the dihydrate free alkali FB2 form of-urea free alkali and listed the details of its main peak in table 6.

table 6

biologic activity

embodiment 73

the CDK2/ cyclin A kinase inhibiting activity test (IC of activation ₅₀ ) measurement

The kinase inhibiting activity of the compounds of this invention is tested by scheme below.

By the CDK2/ cyclin A of activation (people such as Brown, Nat.Cell Biol., 1, pp438-443,1999; Lowe, E.D., waits people Biochemistry, 41, pp15625-15634,2002) use 2.5X concentration to test damping fluid (50mM MOPS pH 7.2,62.5mM β-glycerophosphate, 12.5mM EDTA, 37.5mM MgCl ₂112.5mM ATP, 2.5mM DTT, 2.5mM sodium orthovanadate, 0.25mg/ml bovine serum albumin) be diluted to 125pM, and by 10 these materials of μ l and 10 μ l histone substrate mixture (60 μ l ox histone h1s (Upstate Biotechnology, 5mg/ml), 940 μ l H ₂o, 35 μ Ci γ ³³p-ATP) mix and it is joined in 96 orifice plates together with the various dilutions of 5 μ l test compounds in DMSO (being up to 2.5%).This reaction is carried out 2 to 4 hours, then used excessive ortho-phosphoric acid (5 μ l, 2%) to stop this reaction.On Millipore MAPH filter plate, isolated the γ not still being blended in described histone h1 by phosphorylation histone h1 ³³p-ATP.The hole of described MAPH plate is moistening with 0.5% ortho-phosphoric acid, then with micropore vacuum filtration unit, the result of reacting in these holes is filtered.After filtration, by 200 μ l 0.5% ortho-phosphoric acid washed twice for resistates.After this filter is dried, add wherein 20 μ l Microscint 20 scintillation solutions, then it is counted 30 seconds on Packard Topcount.

Calculate the inhibition % to CKD2 activity and it is mapped to determine by the active CDK2 concentration (IC that suppresses 50% required test compound ₅₀).

The compound of embodiment 1,10,11,18,20,22,30,31,32,46,47 and 54 has the IC lower than 1 μ M in described CDK2 test ₅₀value, and the compound of embodiment 44,45,48,51 and 53 has the IC lower than 10 μ M ₅₀value.

embodiment 74

measurement (the IC of the CDK1/ cell periodic protein B kinase inhibiting activity test of activation ₅₀ )

The test of CDK1/ cell periodic protein B is identical with CDK2/ cyclin A test above, just uses CDK1/ cell periodic protein B (Upstate Discovery) and this enzyme is diluted to 6.25nM.

The compound of embodiment 1,4,6,10,11,13,22,42,47 and 54 has the IC lower than 1 μ M in CDK1 test ₅₀value, and the compound of embodiment 3,8,9,16,17,20,24,28,29,31,32,34,39,41,45,46,48,49,50,51,52,53 and 56 has the IC lower than 10 μ M ₅₀value, and the compound of embodiment 2,23,26,27,33,37 and 43 has the IC lower than 50 μ M ₅₀value.

embodiment 75

aurora A kinase assay

Can (DELFIA) measure Aurora A kinase activity with using dissociating of the derivative biotinylated peptide of GSK3-to amplify group of the lanthanides fluroimmunoassay (Dissociative Enhanced Lanthanide Fluoro ImmunoAssay).With the fluorescence of time explanation at λ _ex=337nm, λ _emunder=620nm, by the anti-rabbit igg antibody of phosphoric acid-specificity primary antibody (primaryantibody) and europium-mark, the quantity of produced phosphorylated peptide is carried out quantitatively.

kinase reaction:

In 96 orifice plates with 0.5nM Aurora A (UpstateDiscovery), 3 μ M Biotin-CGPKGPGRRGRRRTSSFAEG, 15 μ M ATP and various dilutions, 0.1mg/ml BSA, 0.001%Brij-35,0.5% glycerine, 0.2mM EDTA, the 10mM MgCl of compound in 10mM MOPS (pH 7.0) for the total reaction volume of 25 μ l ₂, 0.01% beta-mercaptoethanol and 2.5%DMSO set up test reaction.This reaction is at room temperature carried out 60 minutes, then comprised 100mM EDTA, 0.05%Surfact-Amps20 (Pierce) and 1x and be arranged in the Blocker of TBS with 100 μ l ^tMthe stop buffer of BSA (Pierce) stops this reaction.

detecting step:

Then, described reaction mixture transferred in the 96-orifice plate (Pierce) of Neutravidin-coating and cultivated 30 minutes to catch by biotinylated peptide.With after 200 μ l TBST damping fluids/hole washing 5 times, in all holes, add anti-phosphoric acid-(Ser/Thr)-AKT substrate antibody (CeU Signalling Technology) and Eu-N ₁the mixture of anti-rabbit igg (PerkinElmer) is also placed 1 hour.Carrying out again after once washing step, in all holes, adding DELFIA to strengthen solution (Perkin Elmer).Cultivating after 5 minutes, on the board-like reader of Fusion, these holes are counted.

In superincumbent test, the compound of embodiment 1 to 56 all has the IC lower than 1 μ M ₅₀value.The hydrochloride of embodiment 60H has the IC of 0.0025 μ M ₅₀value.

embodiment 76

aurora B kinase assay

kinase reaction:

In 96 orifice plates with 5nM AuroraB (ProQinase), 3 μ M Biotin-CGPKGPGRRGRRRTSSFAEG, 15 μ M ATP and various dilutions, 0.1mg/ml BSA, 0.025%Surfact-Amps 20, the 5mM MgCl of compound in 25mM TRIS (pH 8.5) for the total reaction volume of 25 μ l ₂, 1mM DTT, & 2.5%DMSO set up test reaction.This reaction is at room temperature carried out 90 minutes, then comprised 100mM EDTA, 0.05%Surfact-amps20 (Pierce) and 1x and be arranged in the Blocker of TBS with 100 μ l ^tMthe stop buffer of BSA (Pierce) stops this reaction.

As described in AuroraA, carry out detection steps.

In Aurora B test, the hydrochloride of embodiment 60H shows 57% restraining effect under the concentration of 0.003 μ M.

embodiment 77

the test of GSK3-B kinase inhibiting activity

GSK3-β (Upstate Discovery) is used to 25mM MOPS, pH 7.00,25mg/ml BSA, 0.0025%Brij-35,1.25% glycerine, 0.5mM EDTA, 25mMMgCl ₂, 0.025% beta-mercaptoethanol, 37.5mM ATP is diluted to 7.5nM and 10 μ l is mixed with 10 μ l substrate mixture.For GSK3-β, described substrate mixture is that 12.5 μ M are positioned at 1ml and have 35 μ Ci γ ³³phosphoric acid-Glycogensynthase peptide-2 (Upstate Discovery) in the water of P-ATP.Enzyme is joined in 96 orifice plates together with the various dilutions of 5 μ l test compounds in DMSO (being up to 2.5%) with substrate.Make this reaction carry out 3 hours (GSK3-β), then use excessive ortho-phosphoric acid (5 μ l, 2%) to stop this reaction.This filter operation is identical with the CDK2/ cyclin A test of activation above.

embodiment 78

cDK Choice tests

78A. option A

Can test the kinase whose kinase inhibiting activity of many differences the compounds of this invention with the general approach described in embodiment 3 above, but described scheme is carried out to improvement described below.

At 20mM MOPS pH 7.0,1mM EbTA, 0.1% γ-mercaptoethanol, 0.01%Brij-35,5% glycerine, in 1mg/ml BSA becomes kinase dilution 10x active redundancy liquid.A unit equals at 30 ℃, and per minute is sneaked into 1nmol phosphoric acid salt with the ATP final concentration of 100uM in 0.1mg/ml histone h1 or CDK7 peptide substrate.

The substrate (except CDK7) of all CDK test is all histone h1, before use, is diluted to 10X active redundancy liquid with 20mM MOPS pH 7.4.The substrate of CDK7 is the specific peptide that is diluted to 10X active redundancy liquid with deionized water.

cDK1/ cell periodic protein B, CDK2/ cyclin A, CDK2/ cyclin the examination of E, CDK3/ cyclin E, CDK5/p35, CDK6/ cyclinD3 test operation:

With the end reaction volume of 25 μ l, by 8mM MOPS pH7.0 for described enzyme (5-10mU), 0.2mM EDTA, 0.1mg/ml histone h1,10mM acetic acid Mg and [γ- ³³p-ATP] (specific activity is approximately 500cpm/pmol, and concentration is desired concn) cultivate.By adding Mg ²⁺[γ- ³³p-ATP] start this reaction.It was at room temperature cultivated after 40 minutes, by adding 5 μ l 3% phosphoric acid solutions to stop this reaction.This reflecting point of 10ml is washed to P30filter mat and in 75mM phosphoric acid to washing in 5 minutes 3 times, in methyl alcohol, washing once, is then dried and it is counted.

the test operation of CDK7/ cyclin H/MAT1

With the end reaction volume of 25 μ l, by described enzyme (5-10mU) and 8mM MOPS pH7.0,0.2mM EDTA, 500 μ M peptides, 10mM acetic acid Mg and [γ- ³³p-ATP] (specific activity is about 500cpm/pmol, and concentration is desired concn) cultivate together.By adding Mg ²+ [γ- ³³p-ATP] start this reaction.It was at room temperature cultivated after 40 minutes, by adding 5 μ l 3% phosphoric acid solutions to stop this reaction.This reflecting point of 10ml washed in 75mM phosphoric acid to washing in 5 minutes to P30filtermat and by it 3 times and it is washed once in methyl alcohol, be then dried and it is counted.

78A. option b

Inhibition activity to these enzymes in Upstate Discovery Ltd is analyzed.In enzyme buffer liquid (as in the table below), under 10x final concentration, these enzymes are prepared.Then, as table described in, by these enzymes test damping fluid in various substrates with ³³p-ATP (～500cpm/pmol) cultivates together.

By adding Mg/ATP to start this reaction.Make this reaction at room temperature proceed 40 minutes, then stop this reaction with 5 μ l 3% phosphoric acid solutions.10 these reaction mixtures of μ l are transferred to and in filtermatA or P30 filtermat and by it, washed three times in 75mM phosphoric acid and wash once in methyl alcohol, be then dried to carry out scintillation counting.

Under the concentration being discussed in more detail below, in duplicate per-cent activity is compared with the control tested and calculated to test compound to all kinase whose effects.In the high situation of restraining effect, measure its IC ₅₀.

Enzyme	Enzyme buffer liquid	Test damping fluid	Substrate	ATP concentration (μ M)
					Cdk3	A	A	0.1mg/ml histone h1	200
Cdk6	A	A	0.1mg/ml histone h1	200
					Cdk7	A	A	500 μ M peptides	90
Cdk9	A	A	100μM KTFCGTPEYLAPEVRREPRI LSEEEQEMFRDFDYIADWC	45

Enzyme buffer liquid used is:

A:20mM MOPS pH 7.0,1mM EDTA, 0.1% beta-mercaptoethanol, 0.01%Brij-35,5% glycerine, 1mg/ml BSA

Test damping fluid used is:

A:8mM MOPS pH 7.0,0.2mM EDTA, 10mM acetic acid Mg

embodiment 79

anti-proliferative activity

Can measure by measuring described compound cytostatic ability in much clone the anti-proliferative activity of the compounds of this invention.The restraining effect of cell growth is to measure with AlamarBlue test (Nociari, M.M, Shalev, A., Benias, P., Russo, C.Journalof Immunological Methods 1998,213,157-167).The basis of described method is the ability that great-hearted cell is reduced into resazurin (resazurin) its fluorescence-causing substance resorufin (resorufin).For each hyperplasia test, cell is coated with and is plated on 96 orifice plates and makes it to recover 16 hours, then add wherein inhibitor compound and it is cultivated 72 hours again.In the time that this incubation period finishes, add wherein 10% (v/v) Alamar Blue and it is cultivated 6 hours again, then under 535nM ex/590nM em, fluorescence-causing substance is measured.In the situation of non-proliferative raji cell assay Raji, cell is maintained to 96 hours under merging, then add inhibitor compound, then cultivated 72 hours.Test to measure the number that has vigor cell with above-mentioned Alamar Blue.In addition, also record any metamorphosis.These clones can derive from ECACC (European Collection of cell Cultures).

In the test of use HCT-116 clone, the hydrochloride of embodiment 60H has the IC of 0.070 μ M ₅₀.

Particularly use the HCT-116 clone (ECACCReference:91091005) of derived from human colorectal carcinoma to test compound of the present invention.

Find that chemical compound lot of the present invention has the IC lower than 25 μ M in this test ₅₀value and preferred compound have the IC lower than 1 μ M ₅₀value.Or, find that chemical compound lot of the present invention has lower than the Cmin of observing polyploidy or multinucleation under it of 10 μ M and preferred compound and has the IC lower than 100nM ₅₀value.

Find compound 1-cyclopropyl base-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea has the IC lower than 1 μ M in this test ₅₀value.In addition, also find that it has the Cmin of observing polyploidy or multinucleation under it lower than 100nM.

embodiment 80

a. general bacterium colony forms testing program

In bacterium colony generation (clonogenic) test, to the various processing of carrying out with compound, the effect of the tumor cell line to adhesion is assessed.

Cell is inoculated in 6 or 24 hole tissue culturing plates and makes it to recover 16 hours with the concentration of the relevant substratum of 75 to 100 cell/ml.

Compound or matrix contrast (DMSO) are joined in these holes in duplicate, obtain 0.1% DMSO final concentration.Adding after compound, making colony growth 10 to 14 days to carry out optimum discrete enumeration with it.These bacterium colonies are fixed and it is dyeed in 2ml 0.4%w/v Viola crystallina in 2ml Carnoys fixing agent (25% acetic acid, 75% methyl alcohol).Colony number in each hole is counted.With Prism Graphpad software, calculate IC50 value by S shape dosage-response (slope is variable) IC50 curve.

b.1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals- base)-1H-pyrazoles-4-yl]-urea bacterium colony form testing program

Producing in (clonogenic) test with 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl at bacterium colony] the various processing carried out of-urea assess the effect of A2780, A549, HCT 116, HCT 116N7, HT-29, MCF7, MIA-Pa-Ca-2, SW620 clone.

Cell is inoculated in 6 or 24 hole tissue culturing plates and makes it to recover 16 hours with the concentration of the relevant substratum of 75 to 100 cell/ml.

Clone	Substratum	Remarks
			HCT 116	DMEM+10％FBS+GLUTAMAXI
HCT 116N7	DMEM+10％FBS+GLUTAMAXI+ 0.4mg/ml G418
			HT-29	McCoy ' 5a+10%FBS+2mM L-glutamine
SW620	L-15+10％FBS+GLUTAMAXI	CO 2Atmosphere
			A2780	RPMI 1640+2mM glutamine+10%FBS
A549	DMEM+10％FBS+GLUTAMAXI
			MCF7	EMEM+10%FBS+2mM L-glutamine+1%NEAA
MIA-Pa-Ca-2	DMEM+l0％FBS+GLUTAMAXI

By 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea or matrix contrast (DMSO) joins in these holes in duplicate, obtains 0.1% DMSO final concentration.Adding after compound, making colony growth 10 to 14 days to carry out optimum discrete enumeration with it.These bacterium colonies are fixed and it is dyeed in 2ml 0.4%w/v Viola crystallina in 2ml Carnoys fixing agent (25% acetic acid, 75% methyl alcohol).Colony number in each hole is counted.Only write down and show from individual cells hyperplasia to about 50 cells or the more cellulous many cells bacterium colony of being permitted cellulous bacterium colony (comprising successfully the whole cell cycle of division of cytoplasm).Do not record single multinuclear (polyploid) cell.With Prism Graphpad software, calculate IC by S shape dosage-response (slope is variable) IC50 curve ₅₀value.

Be in the part of " advantage of the compounds of this invention " at title, to the results are described in table C of these tests.

embodiment 81

measure the effect to Cytochrome P450

Measure the compound of embodiment 24 effect to CYP450s 1A2,2C9,2C19,3A4 and 2D6 with the Pan Vera Vivid Cyp450 screening reagent box that derives from Invitrogen (Paisley, UK).CYPs is that the form of the baculosomes that comprises CYP450 and NADPH reductase enzyme is provided.These substrates are the substrates with intense fluorescence (fluorescent Vivid).

Final reaction mixture is as follows:

1A2

100mM potassiumphosphate, pH 8,1% methyl alcohol, 2 μ M 1A2 Blue vivid substrates, 100 μ MNADP ⁺, 4nM CYP450 1A2,2.66mM G-6-P salt, 0.32U/ml G-6-P salt desaturase.

2C9

50mM potassiumphosphate, pH 8,1% methyl alcohol, 2 μ M Green vivid substrates, 100 μ MNADP ⁺, 8nM CYP450 2C9,2.66mM G-6-P salt, 0.32U/ml G-6-P salt desaturase.

2C19

50mM potassiumphosphate, pH 8,1% methyl alcohol, 8 μ M Blue vivid substrates, 100 μ MNADP ⁺, 4nM CYP450 2C19,2.66mM G-6-P salt, 0.32U/ml G-6-P salt desaturase.

3A4

100mM potassiumphosphate, pH 8,1% methyl alcohol, 10 μ M 3A4 Blue vivid substrates, 100 μ MNADP ⁺, 2.5nM CYP450 3A4,2.66mM G-6-P salt, 0.32U/ml G-6-P salt desaturase.

2D6

100mM potassiumphosphate, pH 8,1% methyl alcohol, 5 μ M 2D6 Blue vivid substrates, 100 μ MNADP ⁺, 5nM CYP450 2D6,2.66mM G-6-P salt, 0.32U/ml G-6-P salt desaturase.

With timed interval of 30s on Molecular Devices Spectramax Gemini reader to fluorescence monitoring 20 minutes.Excite with emission wavelength is 390nm and 460nm for 1A2,2C19 and 3A4, is 390nm and 485nm, and is 485nm and 530nm for 2D6 for 2C9.By carrying out curve determination initial stage speed.

In methyl alcohol, test compound is prepared and under the concentration of 10 μ M to it effect to CYP450s test.

pharmaceutical preparation

embodiment 82

(i) tablet formulation

The tablet composition of contained (I) compound is admixed together and compressed in known manner and form a kind of tablet and be prepared as the Magnesium Stearate of lubricant by the lactose (BP) using 50mg compound and 197mg as thinner and 3mg.

(ii) capsule preparations

Capsule preparations is by by the compound of 100mg formula (I) and 100mg lactose is admixed together and the mixture of gained is filled in the opaque hard gelatin capsule of standard and is prepared.

(iii) injectable preparation I

By injection carry out administration parenteral composition can by for example, by the compound of formula (I) (salt form) thus being dissolved in the activity compound concentration that obtains 1.5% weight in the water that comprises 10% propylene glycol is prepared.Then, by filtering, this solution is carried out to sterilizing, fill it in ampoule and by its sealing.

(iv) injectable Formulation II

Be by will be for example, at the compound of water dissolution type (I) (salt form) (2mg/ml) and N.F,USP MANNITOL (50mg/ml) for the parenteral composition injected, this solution sterilization is filtered and filled it in sealable 1ml bottle or ampoule, be prepared.

(v) injectable Formulation III

The preparation being used for by injecting or input i.v. transmission can for example, by being dissolved in the water the compound of formula (I) (salt form) to be prepared with the concentration of 20mg/ml.Then, by the sealing of this bottle and by autoclaving, it is carried out to sterilizing.

(vi) injectable preparation IV

The preparation being used for by injecting or input i.v. transmission can for example, for example, by being dissolved in the compound of formula (I) (salt form) in the water that comprises buffer reagent (0.2M acetate, pH4.6) and being prepared with the concentration of 20mg/ml.Then, by the sealing of this bottle and by autoclaving, it is carried out to sterilizing.

(vii) freeze-dried preparation I

The aliquots containig of defined prepared here formula (I) compound or its salt is put in 50mL bottle and by its lyophilize.During lyophilize, by the freezing scheme of a step (at-45 ℃), said composition is freezing.Its temperature is risen to-10 ℃ so that its annealing (annealing), then its temperature is reduced at-45 ℃, it is carried out freezing, then at+25 ℃ dry about 3400 minutes for the first time, then, if temperature to 50 ℃, gathers and is dried for the second time with the step increasing.Be set to 80 millitorrs (millitor) at the pressure between dry epoch for the first time and for the second time.

(viii) freeze-dried preparation II

The aliquots containig of defined prepared here formula (I) compound or its salt is put in 50mL bottle and by its lyophilize.During lyophilize, by the freezing scheme of a step (at-45 ℃), said composition is freezing.Its temperature is risen to-10 ℃ so that its annealing (annealing), then its temperature is reduced at-45 ℃, it is carried out freezing, then at+25 ℃ dry about 3400 minutes for the first time, then, if temperature to 50 ℃, gathers and is dried for the second time with the step increasing.Be set to 80 millitorrs (millitor) at the pressure between dry epoch for the first time and for the second time.

(ix) for the freeze-dried preparation III of i.v. administration

By by 1-cyclopropyl-3-[3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl]-urea Pfansteihl salt is dissolved in 0.02M sodium hydroxide or hydrochloric acid take the concentration of 12.86mg/ml and calibrates to pH and in 4.5 citrate buffer solution, prepare a kind of aqueous solution that has carried out buffering.

This buffered soln, in the case of being filled into carrying out removing the filtration of particulate matter in a kind of container (as 1 class vial), is then sealed its part to (for example filling in row sealing with Florotec).If this compound and preparation are enough stable, by autoclaving suitable time at 121 ℃, said preparation is carried out to sterilizing.If said preparation is stable not, can not carry out autoclaving, can carry out sterilizing with suitable filter to it and it is filled in sterile vials under aseptic condition.With the suitable cycle by this solution lyophilize, described periodic ring for example:

Freezing-in 2 hours, to be refrigerated to-40 ℃ and it is kept 3 hours at-40 ℃.

Be dried-in 8 hours ,-30 ℃, keep 7 hours from-40 ℃ to-30 ℃ and by it for the first time.

For the second time dry-in 4 hours to+30 ℃ and it is kept to 8-10 hour at+30 ℃.

After this lyophilize end cycle, bottle is backfilled to normal atmosphere with nitrogen, beyond the Great Wall stopper be fixed (for example, with aluminium folder (crimp)).For intravenous administration, the solid that this can the be lyophilized pharmaceutically useful vehicle of recombinating, in 0.9% salt solution or 5% dextrose.This solution can carry out administration like this, or it can be injected in infusion bag (comprising pharmaceutically useful vehicle as 0.9% salt solution or 5% dextrose) before administration.

(vii) subcutaneous injection preparation

Be by the compound of formula (I) is mixed with the Semen Maydis oil of pharmaceutical grades for the composition of subcutaneous administration, obtain that the concentration of 5mg/ml is prepared.By said composition sterilizing and fill it in suitable container.

embodiment 83

the mensuration of anti-mycotic activity

Can carry out by scheme below the anti-mycotic activity of mensuration formula (I) compound.

Described compound being resisted to the activity of one group of fungi including Candida parpsilosis, Oidium tropicale, Candida albicans-ATCC 36082 and Cryptococcus neoformans tests.At 4 ℃, test microorganism is maintained on Sabourahd dextrose agar slant culture (slants).By making yeast there is amino acid (Difco on going barrel at 27 ℃, Detroit, Mich.) and the yeast of 0.05M morpholine propanesulfonic acid (MOPS)-nitrogen basis broth culture (YNB) (pH 7.0) in growth one prepare yesterday single state (Singlet) suspension of each organism.Then, by centrifugal this suspension, use 0.85%NaCl washed twice, the cell suspension that then this has been carried out to washing carries out 4 seconds sound places (Branson ultrasonoscope, 350 types, Danbury, Conn.).Single state (singlet) blastospore is counted in hematimeter and be adjusted to required concentration with 0.85%NaCl.

Carry out the activity of determination test compound by the modification of meat soup microdilution technology.Test compound is diluted to the ratio of 1.0mg/ml with DMSO, then with have MOPS (with fluconazole in contrast) YNB meat soup (pH 7.0) be diluted to 64 μ g/ml, thereby obtain the working solution of each compound.Use 96-orifice plate, its 1st hole and the 3rd to 12 holes are prepared with YNB meat soup, ten times of dilutions of described compound solution are prepared into (concentration range is 64 to 0.125 μ g/ml) in the 2nd to 11 holes.Using the 1st hole as aseptic contrast with the blank of spectrophotometer measurement test.Using the 12nd hole as growth control.With the material in each hole in 10 the 2nd to 11 holes of μ l, microtiter plate is inoculated that (final inoculum size is 10 ⁴individual organism/ml).The plate that these have been carried out to inoculation is cultivated 48 hours at 35 ℃.By these turbine mixer (Vorte-Genie 2Mixer for plate, Scientific Industries, Inc., Bolemia, N.Y.) stir after 2 minutes, by measuring 420nm (Automatic MicroplateReader, DuPont Instruments, Wilmington, Del.) under optical density metric measurement IC50 value.IC50 terminal is defined as compared with control wells, shows the lowest concentration of drug that growth is reduced to about 50% (or more).For turbidity test, when it is defined in this concentration, 50% lowest concentration of drug (IC50) of the turbidity < in this hole contrast.Minimum molten born of the same parents' concentration (MCC) be by by all holes from described 96-orifice plate subspecies (sub-culturing) to Sabourahd dextrose agar (SDA) plate, it is cultivated at 35 ℃ 1 to 2 day, then check what its vigor was measured.

embodiment 84

for the control of whole plant fungi infestation in body being carried out to the scheme of biological evaluation

By the compound dissolution of formula (I) in acetone, subsequently with acetone by its serial dilution, thereby obtain a kind of desired concn scope.According to pathogenic agent used, final processing volume is by adding 9 volume 0.05% tween 20s ^tMthe aqueous solution or 0.01%Triton X-100 ^tMobtain.

Then,, by scheme below, the compounds of this invention is assessed the activity of tomato wilt (Phytophthora infestans) with described composition.Make tomato (cultivar Rutgers) seed at few soil (soil-less), grow until its growth of seedling is high to 10-20cm in basic potting mixtures take peat.Then, with test compound, these plants are sprayed to runoff (run-off) with the ratio of 100ppm.After 24 hours, by spraying test plant is inoculated with the moisture sporocyst suspension of Phytophthorainfestans, and it is placed a night in dew chamber (dew chamber).Then, these plants are transferred in greenhouse until show ill on the control plant of processing.

Also tested the activity of Powdery Mildew (Ervsiphe vraminis), wheat (cultivar Monon), the Leaf Blotch (Septoria tritici) of wheat and the Glume Blotch (Leptosphaerianodorum) of wheat of leaf rust (handle rest fungus handle), the wheat of the compounds of this invention antagonism wheat by similar scheme.

equivalent

Providing above-described embodiment is for the present invention will be described, these embodiment can not be regarded as and will carry out any restriction to scope of the present invention.It is evident that, can carry out many modifications and variations that do not depart from principle of the present invention with the particular of the present invention described in embodiment to described above.All such modifications and variations are all included in this application.

Claims (87)

1. the compound of formula (I):

Or its salt or tautomer,

Wherein M is group:

Wherein:

X is selected from O, NH and NCH ₃; With

R ¹-E-A-is selected from the group described in lower list 1, and wherein asterisk represents and radicals R ¹the tie point of carbonyl C=O in-E-A-C (=O)-NH-:

2. compound as claimed in claim 1, wherein X is O.

3. compound as claimed in claim 1, wherein said radicals R ¹-E-A-is selected from A1, A4, A10, A11, A13, A20, A22, A23, A24, A29, A30, A31, A32, A38, A42, A43, A44, A46, A47, A49, A54 and A56.

4. compound as claimed in claim 1, wherein said radicals R ¹-E-A-is selected from A1, A4, A20, A24, A30, A44, A46 and A54.

5. compound as claimed in claim 1, wherein said radicals R ¹-E-A-is selected from A24, A45, A51, A57 and A58.

6. compound as claimed in claim 1, wherein said radicals R ¹-E-A-is group A24.

7. compound as claimed in claim 1, wherein said compound is compound 1-cyclopropyl base-3-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea or its salt.

8. compound as claimed in any of claims 1 to 7 in one of claims, it is salt form.

9. compound as claimed in claim 8, wherein said compound is the form that is selected from the salt of acetate, mesylate, esilate, DL-LACTIC ACID salt, adipate, D-Glucose aldehydic acid salt, maltonic acid and hydrochloride.

10. compound as claimed in claim 7, it is free alkali form.

11. compounds as claimed in claim 10, it is crystallization.

The dihydrate of the free alkali of 12.1-cyclopropyl-3-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea, its be crystallization and (i) there is the defined crystalline structure of coordinate in table 2; (ii) wherein said crystal belongs to monclinic spacer P2 ₁/ n (#14), has lattice parameter a=7.66 (10), b=15.18 (10),

β=98.53 (2) °, α=γ=90 °.

13. compounds as claimed in claim 7, its be selected from lactic acid salt and Citrate trianion with and composition thereof salt form.

14. compounds as claimed in claim 13, it is Pfansteihl salt.

15. compounds as claimed in claim 13, it is Citrate trianion.

16. compounds as claimed in claim 13, it is the mixture of Pfansteihl salt and Citrate trianion.

17. compounds as claimed in claim 13, wherein said lactic acid salt or Citrate trianion are crystallization.

18. as the compound of claim 17, and wherein said salt is 50% to 100% crystallization.

19. compounds as claimed in claim 17, wherein said lactic acid salt or Citrate trianion are those salt, it is 95% to 100% crystallization.

20. compounds as described in any one in claim 17-19, wherein said lactic acid salt is Pfansteihl salt.

21. compounds as described in any one in claim 13 to 20, it is anhydrous.

22. compounds as claimed in claim 17, this compound is the Pfansteihl salt of 1-cyclopropyl-3-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea, its be crystallization and be characterised in that the parameter having below, i.e. this salt:

(a) there is the crystalline structure described in Figure 4 and 5; With

(b) there is the defined crystalline structure of coordinate in the table 4 of embodiment 71; With

(c) under 97 (2), there is K a=9.94 (10), b=15.03 (10),

the lattice parameter of α=β=γ=90 °; With

(d) at room temperature there is a=10.08 (10), b=15.22 (10), the lattice parameter of α=β=γ=90 °; With

(e) there is the orthorhombic space group of belonging to P2 ₁2 ₁2 ₁(#19) crystalline structure; With

(f) have and be characterised in that the X-ray powder diffraction style that has the main peak under the diffraction angle (2 θ) that is positioned at 12.40,15.20,15.60,17.50,18.30,18.50,19.30,19.60,21.85 and 27.30 degree and/or there is the spacing (d) of 7.13,5.83,5.68,5.06,4.85,4.79,4.60,4.53,4.07 and 3.26 dusts; With

(g) show and be positioned at the peak under the diffraction angle identical with these diffraction angle of X-ray powder diffraction style shown in Fig. 6 of embodiment 72 or table 5 and optionally, wherein said peak has the relative intensity identical with peak in Fig. 6 or table 5; With

(h) be anhydrous and in the time carrying out DSC, at 190 ℃, show an endotherm(ic)peak; With

(i), in the time analyzing by KBr disk method, show one and comprise and be positioned at 3229,2972 and 1660cm ^-1under the infrared spectra of characteristic peak.

23. 1 kinds of aqueous solution, it is to comprise Pfansteihl salt or Citrate trianion or its mixture of 1-cyclopropyl-3-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea higher than the concentration of 1mg/ml, the described aqueous solution is pharmaceutical compositions.

24. as the aqueous solution of claim 23, the described aqueous solution is pharmaceutical compositions, and it is to comprise Pfansteihl salt or Citrate trianion or its mixture of 1-cyclopropyl-3-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea higher than the concentration of 25mg/ml.

25. aqueous solution as claimed in claim 23, it comprises (i) described Pfansteihl salt or (ii) mixture of described Pfansteihl salt and Citrate trianion.

26. aqueous solution as described in any one in claim 23-25, its pH is 2 to 6.

27. aqueous solution as claimed in claim 26, its tool pH is 4 to 5.

28. aqueous solution as claimed in claim 26, it is cushioned.

29. aqueous solution as claimed in claim 28, lactic acid salt and citrate buffer agent that it comprises 1-cyclopropyl-3-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea.

30. as the aqueous solution of claim 29, and this solution has about 4.5 pH value of solution.

31. 1 kinds of aqueous solution, 1-cyclopropyl-3-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea that it contains protonated form and one or more be selected from Pfansteihl salt and Citrate trianion with and composition thereof counter ion; And optionally (i) one or more other counter ion and/or (ii) one or more vehicle.

32. as the aqueous solution of claim 31, and wherein said one or more other counter ion are chloride ions.

33. as the aqueous solution of claim 31 or 32, and wherein said one or more vehicle are tension regulators.

34. aqueous solution as claimed in claim 31, wherein said lactic acid salt and citrate ion are with 10:1 or lower lactic acid salt: Citrate trianion molar ratio is present in described solution.

35. aqueous solution as claimed in claim 34, wherein said lactic acid salt and citrate ion are with the lactic acid salt of 1:1 to 1:10: Citrate trianion molar ratio is present in described solution.

36. aqueous solution as claimed in claim 35, wherein said lactic acid salt and citrate ion are with the lactic acid salt of 1:4.4: Citrate trianion molar ratio is present in described solution.

37. 1 kinds of freeze-dried preparations, it is by forming aqueous solution lyophilize as defined in any one in claim 33 to 35.

38. 1 kinds of freeze-dried preparations, 1-cyclopropyl-3-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea that it comprises protonated form and one or more be selected from Pfansteihl salt and Citrate trianion with and composition thereof counter ion; And optionally (i) one or more other counter ion and/or (ii) one or more vehicle, described freeze-dried preparation is pharmaceutical compositions.

39. as the freeze-dried preparation of claim 38, and wherein said one or more other counter ion are chloride ions.

40. as the freeze-dried preparation of claim 38 or 39, and wherein said one or more vehicle are tension regulators.

41. compounds as defined in any one in claim 1 to 22 are the application by the medicine of morbid state that cell cycle protein dependent kinase or glycogen synthase kinase-3 mediated or illness for the preparation of prevention or treatment.

42. compounds as defined in any one in claim 1 to 22 are for the preparation of the purposes of the medicine of the incidence for alleviating or reduce the morbid state that mediated by cell cycle protein dependent kinase or glycogen synthase kinase-3 or illness.

43. compounds as defined in any one in claim 1 to 22 comprise abnormal Growth of Cells or the purposes by the medicine of the abnormal disease that Growth of Cells caused or illness for the preparation of being used for the treatment of in Mammals.

44. compounds as defined in any one in claim 1 to 22 are for the preparation of the purposes of the medicine of the method for suppressing cell cycle protein dependent kinase or glycogen synthase kinase-3.

45. compounds as defined in any one in claim 1 to 22 are for the preparation of being characterized as disease that aurora kinase raises or the application of the medicine of illness for preventing or treating.

46. compounds as defined in any one in claim 1 to 22 are for the preparation of the application for preventing or treat the medicine of cancer, and described cancer is to it is characterized by the cancer that aurora kinase raises.

47. compounds as defined in any one in claim 1 to 22 are for the preparation of the application for preventing or treat the medicine of the cancer of the patient subgroups that is selected from the Ile31 modification with Aurora A gene.

48. compounds as defined in any one in claim 1 to 22 are for the preparation of the purposes of the medicine for alleviating or reduce the method that is characterized as the disease of aurora kinase rise or the incidence of illness.

49. 1 kinds of pharmaceutical compositions, it comprises compound as defined in any one in claim 1 to 22 and pharmaceutically useful carrier.

50. for carry out the pharmaceutical composition of administration with aqueous solution form, the compound as defined in any one in claim 1 to 22 that this pharmaceutical composition comprises salt form, and the solubleness of described salt in water is higher than 1mg/ml.

51. for carry out the pharmaceutical composition of administration with aqueous solution form, the compound as defined in any one in claim 1 to 22 that this pharmaceutical composition comprises salt form, and the solubleness of described salt in water is higher than 25mg/ml.

52. 1 kinds of pharmaceutical compositions, it comprises a kind of aqueous solution, the compound as defined in any one in claim 1 to 22 that the described aqueous solution comprises salt form, the concentration of described salt is higher than 100mg/ml.

53. 1 kinds of pharmaceutical compositions, it comprises a kind of aqueous solution, the compound as defined in any one in claim 1 to 22 that the described aqueous solution comprises salt form, the concentration of described salt is higher than 1mg/ml.

54. pharmaceutical compositions as described in any one in claim 49 to 53, wherein said salt is selected from acetate, mesylate, esilate, DL-LACTIC ACID salt, adipate, D-Glucose aldehydic acid salt, D-Glucose hydrochlorate and hydrochloride.

55. pharmaceutical compositions as described in any one in claim 49 to 54, wherein said salt is Pfansteihl salt.

56. pharmaceutical compositions as described in any one in claim 49 to 51, it is a kind of liquid composition.

57. pharmaceutical compositions as described in any one in claim 49 to 51, it is the dried forms for being dissolved in the water.

58. pharmaceutical compositions as described in any one in claim 49 to 57, it is suitable for parenteral admin.

59. pharmaceutical compositions as claimed in claim 58, it is suitable for carrying out parenteral admin by injection.

60. pharmaceutical compositions as claimed in claim 58, it is suitable for carrying out parenteral admin by input.

61. pharmaceutical compositions as described in any one in claim 49 to 57, it is the form that is suitable for i.v. administration.

62. pharmaceutical compositions as described in any one in claim 49 to 57, it is the form that is suitable for oral administration.

63. prepare as a method for the defined compound of any one in claim 1 to 22, the method comprises the compound of formula (X)

With:

(i) carboxylic acid R ¹-E-CO ₂h reacts under the condition that forms acid amides, is the compound of the formula (I) of a key thereby obtain wherein A; Or

(ii) formula R ¹the isocyanic ester of-E-N=C=O reacts under the condition that forms urea, is the compound of the formula (I) of NH thereby obtain wherein A; Or

(iii) formula R ¹-E-NR ²the reagent of the formation urea that the amine of H comprises carbonyl in existence, reacting, is NR thereby obtain wherein A ²the compound of formula (I).

Prepare the method for 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-base amine or its salt for 64. 1 kinds, the method comprises:

(i) by formula (XXVIIa) or compound (XXVIIIa):

Process in solvent with acid, optionally it is heated; With

(ii) by this reaction neutralization.

Prepare the method for 1-cyclopropyl-3-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea or its salt for 65. 1 kinds, the method comprises:

(i) compound of formula as defined in claim 64 (XXVIIa) is processed in solvent with acid, optionally it is heated;

(ii) by this reaction neutralization;

(iii) product of step (ii) is reacted with carbonylation agent; With

(iv) product of step (iii) is reacted with cyclopropylamine.

66. methods as described in claim 65, wherein said carbonylation agent is 1,1'-carbonyl dimidazoles, triphosgene or phosgene.

Prepare the method for 1-cyclopropyl-3-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea for 67. 1 kinds; The method comprises formula (XXXIII) or compound (XXXIIIa):

React with cyclopropylamine, and optionally form acid salt thereafter.

Prepare the method for formula as defined in any one in claim 1 to 22 (I) compound for 68. 1 kinds, the part A in wherein said formula (I) is group NH; The method comprises the compound of formula (XXXIII) and/or its regional isomer (XXXIIIa):

With formula R ¹-E-NH ₂compound react, and optionally form thereafter the acid salt of formula (I) compound.

69. 1 kinds of pharmaceutical compositions, it comprises compound as defined in any one in claim 1 to 22 and pharmaceutically acceptable carrier, and this pharmaceutical composition is suitable for parenteral admin.

70. pharmaceutical compositions as described in claim 69, it is for carrying out administration with aqueous solution form.

71. 1 kinds of pharmaceutical compositions, it comprises a kind of aqueous solution, and the described aqueous solution is to comprise compound as defined in any one in claim 25 to 32 higher than the concentration of 25mg/ml.

72. pharmaceutical compositions as described in claim 71, it is to comprise described compound higher than the concentration of 100mg/ml.

Prepare 1-(2 for 73. 1 kinds, 6-difluorophenyl) method of acid salt of-N-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-urea, the method comprises formation 1-(2,6-difluorophenyl)-N-［ 3-(5-morpholine-4-ylmethyl-1H-benzimidazolyl-2 radicals-yl)-1H-pyrazoles-4-yl ］-solution of urea free alkali in the mixture of solvent or solvent, this solution is processed with acid, thereby be settled out a kind of acid salt.

74. methods as described in claim 73, wherein said acid is added into be arranged in the form that is dissolved in the solution of the mixable solvent of solvent wherein with free alkali.

75. the method as described in claim 73, it is a kind of wherein its acid salt insoluble solvent therein that wherein said free alkali is dissolved in solvent wherein at first.

76. methods as described in claim 73, are wherein dissolved in described free alkali in the first solvent, then add wherein the solution of acid in the second solvent, thereby form the precipitation of its acid salt, then collect this precipitation.

77. compounds as claimed in claim 43 are for the preparation of the purposes of medicine, and in wherein said Mammals, comprising abnormal Growth of Cells or the disease being caused by abnormal Growth of Cells or illness is cancer.

78. compounds described in claim 77 are for the preparation of the purposes of medicine, wherein said disease or illness are cancers, and described cancer is selected from cancer, stomach, uterine neck, Tiroidina or the prostatic cancer of bladder cancer, mastocarcinoma, colorectal carcinoma, kidney, epidermal carcinoma, liver cancer, lung cancer, oesophagus, gall-bladder, ovary, pancreas; The hematopoiesis tumour of Lymphoid tissue system, the hematopoiesis tumour that described Lymphoid tissue is is selected from leukemia, B-cell lymphoma, T-cell lymphoma, He Jiejin lymphomas, non_hodgkin lymphoma, hair cell lymphoma or Burkett's lymphoma; The hematopoiesis tumour of myeloid lineage, it is myelodysplastic syndrome; The tumour of interstitial origin, the tumour of described interstitial origin is selected from fibrosarcoma or rhabdosarcoma; The tumour of central or peripheral nervous system, the tumour of described central or peripheral nervous system is selected from astrocytoma, neuroblastoma, neurospongioma or schwannoma; Melanoma; Spermocytoma; Teratoma; Osteosarcoma; Xeroderma pitmentosum; Keratoctanthoma; Or Kaposi's sarcoma.

79. compounds as described in claim 77 are for the preparation of the purposes of medicine, and wherein said disease or illness are the cancers of skin.

80. if the compound of claim 77 is for the preparation of the purposes of medicine, and wherein said disease or illness are thyroid follcular carcinomas.

81. compounds described in claim 78 are for the preparation of the purposes of medicine, and wherein said disease or illness are leukemia.

82. compounds described in claim 81 are for the preparation of the purposes of medicine, and wherein said leukemia is selected from acute myelogenous leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia or chronic granulocytic leukemia recurrence or difficult medical treatment.

83. compounds described in claim 77 are for the preparation of the purposes of medicine, and wherein said morbid state or illness are cancers, and described cancer is selected from mammary cancer, ovarian cancer, colorectal carcinoma, prostate cancer, the esophageal carcinoma, squamous cell carcinoma or nonsmall-cell lung cancer.

84. compounds described in claim 77 are for the preparation of the purposes of medicine, and wherein said morbid state or illness are cancers, and described cancer is selected from B-cell lymphoma or lymphocytic leukemia.

85. compounds as described in claim 77 are for the preparation of the purposes of medicine, and wherein said morbid state or illness are cancers, and described cancer is dispersivity large B cell lymphoid tumor.

86. compounds described in claim 77 are for the preparation of the purposes of medicine, wherein said morbid state or illness are cancers, and described cancer is selected from mammary cancer, bladder cancer, carcinoma of the colon and rectum, carcinoma of the pancreas, ovarian cancer, non_hodgkin lymphoma, neurospongioma or non-endometrial-like carcinoma of endometrium.

87. compounds described in claim 77 are for the preparation of the purposes of medicine, and wherein said cancer is selected from B-cell lymphoma, T-cell lymphoma, He Jiejin lymphomas, non_hodgkin lymphoma, hair cell lymphoma or Burkett ' s lymphoma.

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