CN105267220A - Application of 4-aminopyrimidine compounds - Google Patents

Abstract

The invention relates to the field of medicinal chemistry, in particular to application of 4-aminopyrimidine compounds, 4 compounds (1-4), medical application of medicinal compositions containing the compounds, and especially application of the medicinal compositions serving as Polo-like kinase 1 inhibitors.

Description

The purposes of 4-aminopyrimidine compounds

Technical field

The present invention relates to medicinal chemistry art, be specifically related to 4 compounds and the Pharmaceutical composition containing these compounds, and their medical application, particularly as the purposes of Polo sample kinases 1 (PLK1) inhibitor.

Background technology

In recent years, tumor surmounts cardiovascular disease, becomes the first dead disease in the whole world, and antitumor drug research has important science and realistic meaning.

Research finds, the unregulated cell growth that nearly all tumor all causes with cell cycle disorder, break up be obstructed, abnormal apoptosis is relevant.Tumour cell division frequency compared with normal cell is fast, and various regulation and control microtubule polymerization, centrosome copy, spindle is formed and the albumen usually overexpression of cytokinesis, and increased activity.A class important in traditional antineoplastic chemotherapy medicine is exactly by acting on tubulin, make tubulin polymerization or depolymerization, thus reaching interference tumour cell division, the object of Tumor suppression growth, as clinical widely used vinca medicine and taxanes medicine.But tubulin also has extremely important effect in normal cell, also participate in nerve synapse intracellular signaling, there is larger toxic and side effects in therefore traditional tubulin interacting agent, as paclitaxel has obvious toxicity to peripheral nervous system, their absorption distribution performance is also not ideal in addition.So now people turn one's attention to those overexpressions and can regulate and control tubulin function, affect the specific proteins of spindle effect, as microtubule kinesin (kinesin), Aurora A, Polo-like kinases (PLKs) etc. in tumor cell.

PLKs is serine/threonine kinases, structural conservation in multiple organism.3 members be closely related are comprised altogether in human cell, namely PLK1, PLK2 are (also referred to as Serum-InducibleKinase, Snk), PLK3 is (also referred to as FibroblastGrowthFactor-InducibleKinase, Fnk or Prk), also has the member that relatively far away in addition, i.e. PLK4 (also known as SNKakinKinase, Sak).The N that usual PLKs has high conservative holds protein serine/threonine territory (about 252 amino acid residues), simultaneously according to hypotype difference comprise 1 (PLK4) or 2 (PLK1-3) be positioned at C end conservative phosphoeptide binding site--polo-box (60-70 residue), two polo-box be together in series constitute polo-boxdomain (PBD).So far to the most study of PLK1, its function and regulatory mechanism are comparatively clear.

PLK1 mainly participates in the joint centrosome maturation that withers; Activation CDK1-cyclinB, to enter mitosis; Raise γ tubulin cyclic compounds, promote that bipolar spindle is formed, sister chromosome is separated; Promote complex (anaphase-promotingcomplex/cyclosome, APC/C) anaphase of cell division of phosphorylation, suppress early stage mitosis to suppress son (earlymitoticinhibitor, EMI-1), drive mitotic progression.Research finds, PLK1 can promote that in somatoblast, film is formed, and phosphorylation kinesin sample dynein MKLP1 and nucleardistributiongeneC (NUDC), participates in cytokinesis.In fact after cleaving phase PLK1 can promote that RhoGTP enzyme exchange factor Ect2 is positioned in the middle part of spindle, starts cytokinesis, and Ect2 activates the gathering that RhoA, RhoA trigger actomyosin contraction ring at cell cortex place, promotes cell intermediate recess to hang contracting simultaneously.Subcellular area positioning experiment shows, PLK1 is positioned centrosome, equatorial plate, centromere and cytokinesis place at different times.Between the G0 phase to S phase, expression and the activity of PLK1 rest on reduced levels, rise, reach peak in the M phase from the G2 phase.But PLK1 is not the necessary factor from the G2 phase to prophase of cell division, and when PLK1 is suppressed, then can extend the time be transitioned into needed for prometaphase (prometaphase) largely.

Many evidences show, PLK1 is a very attractive antineoplaston target.First, PLK1 is equal overexpression in kinds of tumors (breast carcinoma, ovarian cancer, colon cancer, cancer of pancreas, pulmonary carcinoma, carcinoma of endometrium, the cerebral tumor, skin carcinoma, head and neck cancer, esophageal carcinoma, gastric cancer, carcinoma of prostate), its expression is one of mark of poor prognosis in specific tumors, and in normal cell, the expression of (except growing multiplication extracellulars faster such as Placenta Hominis, spleen, ovary, testis) PLK1 is very low, sometimes even cannot measure.The composition activation of the second, PLK1 can induce the fibroblastic vicious transformation of NIH3T3.Three, PLK1 phosphorylation p53, makes the latter lose short apoptosis of tumor cells effect.4th, no matter be wild type or inactivation type (Lys82Metmutant), the overexpression of PLK1 all causes multinucleation.The expression of the 5th, high activity PLK1 (Thr210Aspmutant) can pass over DNA damage and causes the G2 phase to stagnate inspection.Importantly, the work of many scholars shows, oncocyte bipolar spindle can be caused to be formed and be obstructed, growth inhibited, even apoptosis with the PLK1 that antisense technology, siRNA technology or micromolecular inhibitor knock out in tumor cell.In Hela cell, inject specific antibody can the dazed and confused propagation of obvious T suppression cell, somatoblast is monopolar spindle phenomenon (referring to that chromosome condensation is showing a composition single centre core near the centrosome of separation), in 10 kinds of cell line, make the dominant negative gene of expressing viral PLK1, two kinds of cell lines can be caused " mitosis disaster " occurs.In contrast, the PLK1 knocked out in normal cell system does not show obvious cell cycle and is obstructed and growth inhibited, only poor growth is shown as expressed dominant negative PLK1 in normal epithelium cell, but centrosome maturation is normal, less trigger cell apoptosis, in addition, suppress PLK1 activity can form population of cells by inhibition tumor cell on soft agar, the tumor of Mus tumor deformity grafting model can also be suppressed to generate.

At present, the inhibitor that many companies have all carried out for PLK1 is studied, and increasing to the Research Literature of the Molecular biological function of PLKs, Patents also constantly occurs.The company such as Cyclacel, GlaxoSmithKline, Onconova, BoehringerIngelheim, SuperGen and Nippon Shinyaku Co., Ltd. (JP) Tokyo To, Japan all have developed oneself PLK1 inhibitor, and wherein B12536 has entered the clinical II phase and studies.But in general, current PLK1 inhibitor and structure type still few, and the PLK1 inhibitor of many reports is nonspecific inhibitor, as Wortmannin, Scytonemin, Staurosporine, morin, ON-01910 and HMN-214, only BI2536, GSK-461364 and LFM-A13 are PLK1 selective depressant.Because other subtype displays of PLKs goes out the effect that part Tumor suppression occurs, repair as PLK3 promotes when DNA damage to check, therefore studying PLK1 selective depressant becomes current hotspot.

Summary of the invention

The present invention constructs the Pharmacophore Model based on receptor according to the crystal complex of 14 PLK1 inhibitor, comprises 4 required characteristic sum 5 nonessential features.Feature must represent the most public binding mode of PLK1 inhibitor, nonessential feature corresponds to the structure diversity of inhibitor.This model can match with the avtive spot of PLK1 well, reflects that receptor is to compound structure and feature request fully.And this model also has stronger activity conformation identification ability, can be used for the binding mode of the PLK1 inhibitor studying new construction type.Retrospective analysis means are utilized to assess the virtual screening ability of this model, find that 4 feature must add that the performance of the nonessential feature that 3 non-hydrones are correlated with is best, enrichment factor up to 29.82, GH > 0.6, its good virtual screening ability of sufficient proof.4 new PLK1 inhibitor are obtained after using this model to carry out virtual screening.

The object of the invention is to, the medical application of above-claimed cpd or its pharmaceutically-acceptable salts and Pharmaceutical composition thereof is provided, especially in the purposes of preventing, delay or treating PLK1 participation or do not participate in the medicine of the disease, particularly tumor that mediate.

For achieving the above object, the invention provides the compound with structure as follows or its pharmaceutically acceptable salt:

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

According to the present invention, pharmaceutically acceptable salt comprises the acid-addition salts that compound 1 ~ compound 4 is formed with following acid: hydrochloric 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.Comprise the acid salt of inorganic base in addition, as: containing alkali metal cations, alkaline earth metal cation, ammonium cation salt.

Biological activity test result shows, compound provided by the present invention has PLK1 inhibition, has certain inhibitory action to the growth of tumor cell line simultaneously.The compounds of this invention can be used for treating various parenchymatous organ's cancer, comprising 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 etc., or leukemia, lymphatic cancer, can be wherein the cancer mediated by PLK1, also can be the cancer not relying on above-mentioned mechanism.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 ~ compound 4 pairs of PLK1 inhibit activities and the experimentation to tumor cell line inhibit activities

1.PLK1 inhibit activities is tested

1) experiment material: polo-likekinaseAssay/InhibitorScreeningKit, 96 orifice plates, 10Xwashbuffer, Kinasebuffer, 20XATP, Anti-Phospho-ThreoninePolyclonalPPT-07, HRP-conjugatedAnti-rabbitIgG, SubstrateReagent substrate reagent, StopSolution, PLKpositivecontrol (Cat#CY-E1163, every bottle of concentration: 2Unit/100 μ L, needs dilution to make concentration for 0.1mUnit/well before using).

2) experimental procedure:

Solution preparation washbuffer:100ml10Xwashbuffer adds 900ml deionized water, mixing.Kinasereactionbuffer:0.5ml20XATP adds in 9.5mlkinasebuffer, mixing.Testsample: mother liquid concentration 10 ^-2, be diluted to 10 with kinasebuffer ^-5m is for subsequent use.PLKpositivecontrol: draw 1 μ LPLKpositivecontrol (2Unit/100 μ L) and add in 1999 μ L deionized waters that to be made into concentration be that 10 μ Unit/ μ L working solutions are for subsequent use.

The 96 every holes of orifice plate add 10 μ Ltestsample (finalconc.10 ^-5m), every hole, solvent control hole adds 10 μ Lkinasebuffer, need operate on ice.

Every hole adds 80 μ Lkinasereactionbuffer, need operate on ice.

Every hole adds 10 μ LPLKpositivecontrol and starts reaction, mix homogeneously under room temperature.30-60min is hatched at plate Fresco Bag is sealed latter 30 DEG C.96 orifice plate operation tables are as follows:

Liquid light in plate patted and pour out, then every hole washbuffer washes 5 times, notices that every Kong Jun is full of washbuffer when washing, and washes rear liquid light and pats and knock down out or sucking-off.

Every hole adds 100 μ LAnti-Phospho-ThreoninePolyclonalPPT-07, is sealed by plate Fresco Bag, incubated at room temperature 30min.Discard residue antibody, original reagent bottle can not be refunded.

According to the 6th one step process hole flushing 5 times.

Every hole adds 100 μ LHRP-conjugatedAnti-rabbitIgG, and plate Fresco Bag is sealed, incubated at room temperature 30min.Discard remaining liq, original reagent bottle can not be refunded.

According to the 6th one step process hole flushing 5 times.

Every hole adds 100 μ LSubstrateReagent, incubated at room temperature 5-15min.

Add the order of SubstrateReagent in strict accordance with the 11st step, every hole adds 100 μ LStopSolution cessation reactions.

Adding within StopSolution30min, under 450nm wavelength, microplate reader reads the OD value in each hole, and calculates suppression ratio.

3) experimental result

(in table, compound numbers corresponds to compound numbers above)

2. tumor cell in vitro inhibit activities test

The compounds of this invention is in vitro to the inhibit activities of tumor cell line.

Adopt tetramethyl azo azoles salt (methylthiazolyltetrazolium, MTT) colorimetry tests the inhibit activities that the compounds of this invention is bred tumor cell in vitro, selected cell strain is human lung cancer cell A549, people's low differentiation gastric adenocarcinoma cells BGC-823, human liver cancer cells Hep G2 and human promyelocytic leukemia HL-60, positive control drug is amycin and hydroxy camptothecin.

During test, get and be in exponential phase of growth, one bottle, cell in good condition, add the tryptic digestive juice that concentration is 0.25%, digestion makes attached cell come off, and counting 2 ~ 4 × 104/ml, makes cell suspension.Obtained cell suspension is inoculated on 96 orifice plates, and 180 μ l/ holes, put constant temperature CO ₂24h is cultivated in incubator.Then change liquid, add test medicine, 20 μ l/ holes, cultivate 48h.Added by MTT in 96 orifice plates, 20 μ l/ holes, react 4h in incubator.Suck supernatant, add DMSO, 150 μ l/ holes, jolting 10min on plate shaker.

Be the optical density (OD value) in the every hole of mensuration, 570nm place at wavelength with enzyme-linked immunosorbent assay instrument, cell inhibitory rate=(negative control group OD value-tested material group OD value)/negative control group OD value × 100%.

Partial results is as shown in the table:

(in table, compound numbers corresponds to compound numbers above)

3. conclusion

PLK1 vitro inhibition activity shows, compound provided by the present invention has significant PLK1 inhibit activities; Part of compounds has the activity of extracorporeal suppression tumor cell strain.Because PLK1 has key effect in growth of tumour cell propagation, and have tumor cell in vitro inhibit activities to test support, compound provided by the present invention may be used for preventing or the treatment disease relevant with Polo sample kinases 1 inhibitor medicine in, in the medicine of especially tumor.

Claims (4)

1. compound 1 ~ compound 4 or its pharmaceutically acceptable salt purposes in the medicine for the preparation of prevention or the treatment disease relevant with Polo sample kinases 1 inhibitor.

2. a pharmaceutical composition, wherein containing claim 1 inclusion compound or its pharmaceutically acceptable salt and pharmaceutically acceptable carrier.

3. the purposes of claim 1, wherein relevant with Polo sample kinases 1 inhibitor disease is 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 or lymphatic cancer.

4. the purposes of claim 1, wherein pharmaceutically acceptable salt is the acid-addition salts that compound and hydrochloric 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 etc. are formed.