CN105878244A - CDK2 inhibitor and applications thereof - Google Patents

Abstract

The invention relates to the field of medicinal chemistry, and in particular to medical applications of compounds (1-20) and medical compositions containing the compounds, especially the applications as the CDK2 inhibitor. The structural formulas of the 20 compounds are shown in the description.

Description

CDK2 inhibitor and application thereof

Technical field

The present invention relates to medicinal chemistry art, be specifically related to 20 compounds and containing these compounds, Pharmaceutical composition and Their medical application, especially as the purposes of CDK2 inhibitor.

Background technology

Tumor is commonly encountered diseases and the frequently-occurring disease of serious harm human health, causes human death in having become as global range in recent years One of the main reasons.Predicting according to World Health Organization (WHO), to the year two thousand twenty, annual new cancer patient's number is up to 15,000,000. Meanwhile, the death toll of cancer also rapidly rises in the whole world, and the whole world in 2007 has 7,600,000 people and dies from cancer, to the year two thousand thirty Number of cancer deaths may increase to 13,200,000, and the task for the treatment of and prevention of tumour is the most arduous.

CDK is the serine/threonine protein kitase that a class is important, is the engine molecule of cell cycle operation.CDK activity is subject to Regulation and control to cyclin (Cyclin).Kinase activity the lowest during CDK individualism, only with corresponding Cyclin After in conjunction with, CDK just shows strong kinase activity.Have now been found that existence 13 kinds of CDK and 25 kinds of Cyclin, wherein wrap Include three kinds of interphase cell CDK (CDK2,4,6) and mitotic phase CDK1, Cyclin then can be divided into A, B, D, E tetra-class.CDK is combined with cyclin, activates CDK, activates substrate phosphorylation, drives each phase of cell cycle to enter Journey, thus cause growth and the propagation of cell.Research finds that multiple CDK is in high activity state in many tumor tissues, because of This, the CDK activity in selective suppression tumor cell likely can control its propagation.

It is the major reason of canceration owing to cell cycle is out of control, if it is possible to stoping cell cycle to enter the S phase, the exception of DNA is multiple System would not occur.CDK2 plays considerable role in cell cycle, if so can effectively suppress CDK2's Activity, it is possible to control the carrying out of cell cycle, and then reach the effect suppressing tumor cell proliferation out of control.Up to the present, The crystal complex structure having substantial amounts of CDK2 is the most resolved, understands different micromolecular inhibitor and CDK2's in depth for people Binding mode provides detailed structural information, also for carrying out Rational drug design based on structure further, develops new structure CDK2 inhibitor provide the foundation.

CDK2 is positioned people 12q13, the protein of coding 33ku.Can be combined with each other with Cyclin E and Cyclin A, point Do not play a role in G1/S phase and M phase.Cyclin E-CDK2 is that cell enters the Key kinases complex of S phase from G1. After Cyclin E with CDK2 is combined, its substrate protein of phosphorylation, as Retinoblastoma Protein (pRb), pRb family become Member P107, CDC6, be positioned the nucleoprotein P220 (NPAT) etc. in AT site, make DNA synthesis be carried out, cell by The G1 phase enters the S phase.But the kinase activity of Cyclin E-CDK2 is affected by strictly regulating and controlling.DNA is necessary as hereditary material Being copied in daughter cell exactly, when DNA sustains damage and mistake occurs, its duplication is not allowed to.This is Owing to higher eucaryotic cells exists a G1 time limit point, when activating some after DNA damage for Cyclin-CDK activity Inhibitive factor, including CIP/KIP (cyclin suppression albumen/kinase inhibition albumen) family member P21 (cip1), P27 (kip1) With P57 (kip2).The inhibitive factor of these kinase activities combines Cyclin E-CDK2 makes it lose kinase activity, it is impossible to phosphorylation Its substrate, the synthesis of DNA thus can not be activated.The kinase activity of CyclinE-CDK2 is to be subject to P21, P27 and P57 etc. Strict regulation and control.Cyclin E-CDK2 promotes cell to enter S after date, and the mission of its G1/S phase i.e. completes, by S phase kinases Associated proteins (Skp) 2-SCF mediates ubiquitination, by proteasomal degradation.

The structural research of CDKs helps our detailed understanding CDK activation under cyclin and ATP combines and phosphorylation Effect.The current crystal structure having parsed three kinds of CDKs: CDK2, CDK5 and CDK6.In the structural research of CDKs, Research with CDK2 is the most extensive.CDK2 is folded by typical protein kinase, and a small amount of N-end mainly has β-pleated sheet, spiral shell Rotation structure, substantial amounts of C-end mainly has alpha-helix.The binding site of ATP is just at the joint in the two region, ATP and CDK2 Combination interface is closed.The binding site of ATP can be divided into three regions: adenine, ribose and phosphate land Territory, adenine ring is positioned between Ala31 and Leu134, by Ile10, Ala31, Val64, Phe80, Glu81, Phe82, Leu83, Leu134 and Ala144 forms hydrophobic pocket.Except hydrophobic interaction, N6 and the N1 atom of adenine respectively with the carbonyl of Glu81 The NH of base and Leu83 forms hydrogen bond.When the hydroxyl of Asp86 and Gln131 forms hydrogen bond, the ribose ring of ATP and Val18 There is hydrophobic interaction.Triphosphate part forms hydrogen bond with Lys33, is connected with Asp145 and Asn132 under the mediation of magnesium ion.

Quickly growing of CDKs inhibitor, by the research to a large amount of crystal structures thus obtain CDK2 and part and interact Structural information, the target spot of all known CDK2 micromolecular inhibitors is ATP-binding site, with ATP with as side Formula interacts with CDK2.They, by forming hydrogen bond with Glu81 and Leu83, are formed hydrophobic with Ala31 and Leu134 Effect and ATP produce competitive inhibition.There is now a lot of different types of CDK2 inhibitor and obtain corresponding before clinical or clinical Achievement.Such as Flavopiridol/L868275 and UCN-01 of Clinical practice, treat the white blood of chronic lymphatic through being used as Sick rare medicine.CDKs is had higher selective inhibitor such as Roscovitine/CYC-202, BMS-387032/SNS-032 is recently also into a clinical phase and second phase experiment.AT-7519 is that Astex company uses based on broken The Mutiple Targets inhibitors of kinases that the drug discovery method of sheet obtains, it is possible to suppress multiple CDK kinases, such as: CDK1, CDK2, CDK4, CDK5 etc..Have been enter into clinical three phases research at present.One of reference inhibitor being used as drug design herein.

Summary of the invention

The present invention, with reference to existing CDK2 inhibitors of kinases AT-7519, utilizes MOE equimolecular simulation softward to analyze respectively The binding pattern of AT-7519 Yu CDK2, determines that avtive spot sets up Pharmacophore Model；Medicine based on molecular fragment is utilized to set Meter method, transforms pyrazole ring respectively according to avtive spot analysis result, obtains meeting binding pattern to three-dimensional structure database screening Compound, according to ADME prediction and principle of experience, from hit compound select target compound, through cell in vitro activity Screening, it is thus achieved that the CDK2 inhibitor of 20 novel structures.

It is an object of the invention to, it is provided that above-claimed cpd or its pharmaceutically-acceptable salts and the medical application of Pharmaceutical composition thereof, Especially in prevention, delay or treat CDK2 participation or be not involved in the disease of mediation, particularly treating the purposes of tumor.

For achieving the above object, the present invention provides and has compound or its pharmaceutically acceptable salt of structure as shown below:

The synthetic method of above compound can obtain from the pertinent literature delivered and patent.

According to the present invention, pharmaceutically acceptable salt includes the acid-addition salts that compound 1～compound 20 are formed with following acid: salt Acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-methyl benzenesulfonic acid, LOMAR PWA EINECS 246-676-2, citric acid, tartaric acid, lactic acid, Acetone acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid.Additionally include inorganic base Acid salt, such as: containing alkali metal cations, alkaline earth metal cation, ammonium cation salt.

Biological activity test result shows, compound provided by the present invention has CDK2 inhibition.The compounds of this invention can be used In treat various parenchymatous organ's cancers, including melanoma, hepatocarcinoma, renal carcinoma, pulmonary carcinoma, carcinoma of prostate, thyroid carcinoma, Skin carcinoma, colorectal carcinoma, cancer of pancreas, ovarian cancer, breast carcinoma, carcinoma of testis, osteocarcinoma, the brain cancer, the esophageal carcinoma, gastrointestinal cancer, Soft-tissue tumor, leukemia, lymphatic cancer etc., can be wherein the cancer mediated by CDK2, it is also possible to be not dependent on above-mentioned mechanism Cancer.Therefore, the present invention proposes, and the compounds of this invention can be used for the preparation of cancer therapy drug.

Detailed description of the invention

Embodiment 1

Compound 1～the compound 20 experimentation to CDK2 inhibitory activity

1.CDK2 inhibitory activity is tested

1) experiment material:

·CDK2/cyclinE Kinase

Kinase-Glo Plus reagent, including Kinase-Glo Substrate and Kinase-Glo Buffer

Substrate:Histone H derived peptide:HDB, H09-19T

Assay Buffer:25mM HEPES, 10 μMs of MgCl2,0.01%Triton X-100,100 μm/ml BSA, 2.5mM DTT, pH7.4

·ATP

384 orifice plates of White-opalescent

2) experimental procedure:

Testing compound all dissolves with DMSO, is made into the stock solution that concentration is 100 μMs.

Take testing compound, be diluted to 20 μ L with assaybuffer, obtain the mensuration concentration that compound concentration is 10 μMs.

4*ATP:ATP obtains 4*working solution after diluting with assay buffer.

4*Substrate: substrate obtains 4*working solution after diluting with assay buffer.

2.5*CDK2/cyclin E Enzyme:CDK2/cyclin E kinases obtains after diluting with assay buffer 2.5*working solution。

In 384 orifice plates of White-opalescent, the every hole of experimental port adds the testing compound that 1 μ L mensuration concentration is 10 μMs, empty White hole (ZPE) and negative control hole (HPE) add 1 μ L 10%DMSO solution.

Testing compound experimental port and the every hole of ZPE add 4 μ L 2.5*CDK2/cyclin E kinases, add 1 μ L in HPE hole assay buffer。

With 1000rpm centrifugation 1min on centrifuge, mixed liquor is made to mix.

2*ATP-substrate mixtures. is obtained after mixing isopyknic 4*ATP and 4*Substrate

Every hole adds 5 μ L 2*ATP-substrate mixtures, and the cumulative volume making reaction mixture is 10 μ L.

With 1000rpm centrifugation 1min on centrifuge, mixed liquor is made to mix.

1 hour is hatched under the conditions of 384 orifice plates are placed in 30 DEG C.

Every hole adds 10 μ L Kinase Glo Plus reagent, with 1000rpm centrifugation 1min on centrifuge, makes mixed liquor After mixing, under the conditions of being subsequently placed in 27 DEG C, hatch 20min.

Envision reads luminous intensity, calculates suppression ratio.

3) experimental result

(in table, compound numbers is corresponding to compound numbers above)

2. tumor cell in vitro inhibitory activity test

1) experiment material:

Breast cancer cell MDA-MB-231, tetramethyl azo side indigo plant MTT, modified form RPMI1640 culture medium, 10% tire Ox blood serum, 0.25% trypsin, 96 orifice plates, U.S. BIO-RAD 680 type microplate reader, CO2 constant incubator.

2) experimental procedure:

Take the logarithm the cell of trophophase, MDA-MB-231 tumor cell suspension is adjusted to density be 5*10⁴/ mL, with 5*10³Individual/hole is inoculated on 96 orifice plates, and 10 μ L are inoculated in every hole.It is placed in 37 DEG C, 5%CO₂Saturated humidity incubator In make cell attachment, hatch 12-24 hour.

Adding the 100 μ L culture medium containing variable concentrations compound in every hole, each compound is with 3 Concentraton gradient.Each Concentration sets four multiple holes, is not added with during the hole reading of cell making blank, adds cell but is not added with the hole of compound and makees negative control, AT-7519 is as positive control.

Continue at 37 DEG C, 5%CO₂After hatching 48h in saturated humidity incubator, every hole adds the MTT dye of 10Ml 0.5% Color liquid, hatches 4h.

After Li Xin, discarding the MTT solution in hole and culture medium, then every hole adds 100 μ L DMSO, and vibrate 2min, First is made to be completely dissolved.

The absorbance OD value measuring every hole at wavelength 570nm is used in microplate reader.

Calculate inhibitory rate of cell growth and IC₅₀Value:

Suppression ratio (%)=(negative control hole mean OD value-experimental port mean OD value)/(negative control hole mean OD value-blank Hole average 0D value) * 100%

3) experimental result:

3. conclusion

CDK2 vitro inhibition activity shows, compound provided by the present invention has significant CDK2 inhibitory activity；Portion Differentiation compound has the activity of extracorporeal suppression tumor cell strain.Owing to CDK2 has key in growth of tumour cell is bred Property effect, and have tumor cell in vitro inhibitory activity experiment support, compound provided by the present invention may be used for prevention or Treat in the medicine of the disease relevant with CDK2 inhibitor, especially in the medicine of tumor.

Claims (4)

1. compound 1～compound 20 (structure is as follows with numbering) or its pharmaceutically acceptable salt preparation for prevention or treatment and Purposes in the medicine of the disease that CDK2 inhibitor is relevant.

2. a pharmaceutical composition, wherein contains claim 1 inclusion compound or its pharmaceutically acceptable salt and pharmaceutically can connect The carrier being subject to.

3. the purposes of claim 1, wherein relevant with CDK2 inhibitor disease is cancer.

4. the purposes of claim 1, wherein pharmaceutically acceptable salt is compound and hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, first sulphur Acid, benzenesulfonic acid, p-methyl benzenesulfonic acid, LOMAR PWA EINECS 246-676-2, citric acid, tartaric acid, lactic acid, acetone acid, acetic acid, maleic acid or amber The acid-addition salts that amber acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid etc. are formed.