CN101537007A - Application of N-(thiofuran-2) pyrazolo (1, 5-a) pyridine-3-formanides compounds for preparing antineoplastic - Google Patents
Application of N-(thiofuran-2) pyrazolo (1, 5-a) pyridine-3-formanides compounds for preparing antineoplastic Download PDFInfo
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Abstract
The invention searches the novel micromolecule inhibitor pyrazolo (1, 5-a) miazines compounds of cyclin-dependent kinase CDK9 (cyclin-dependent kinase) through the virtual screening of a computer, biometrically measures activity thereof, and validates interaction mechanism. The invention specifically comprises the following steps: the three-dimensional crystal conformation of the cyclin-dependent kinase family member CDK9 is obtained in a way of homology modeling; and micromolecule three-dimensional database is screened with DOCK (molecular docking). The invention uses a MTT tumor cell growth inhibition test to biometrically measures the activity of the selected compounds, researches the selected compounds pyrazolo (1, 5-a) miazines with high activety in a way of molecular mechanism, validates the inhibiting effect of the compounds to the activity of CDK9 kinase, and clarifies the interaction mechanism of the compounds for inhibiting the external activity and the molecule of various malignancies such as lung cancer, osteosarcoma, oophoroma, cervical carcinoma, breast cancer, etc.
Description
Technical field
The present invention relates to a kind of application of compound of anti-malignant tumor, belong to the research field of antitumor drug.Specifically, the present invention relates to a kind of N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide and substituent thereof the purposes in suppressing multiple malignant tumor such as pulmonary carcinoma, osteosarcoma, ovarian cancer, cervical cancer, breast carcinoma.
Background technology
Malignant tumor is a big class disease of serious threat human health, and it forms main cause is the cellular abnormality hypertrophy that variation caused of cytogene level.Eukaryotic multiple fission is an accurate and complicated regulation process.Breeding is finished by cell cycle, and carrying out in order of cell cycle has its strict molecular regulation mechanism.Along with development of molecular biology, regulatory molecule that these are complicated and mechanism of action thereof are slowly found and are illustrated by human.The participation cell cycle regulating of having found at present mainly contains the three major types molecule: cell cycle dependant kinase (cyclin-dependent kinases, CDKs), cyclin (cyclins), cell cycle protein dependent kinase inhibitor (cyclin-dependent kinase inhibitors, CKIs), wherein CDK is in middle cardiac status.Cyclin can with corresponding CDK in 1: 1 mode in conjunction with forming complex, as the adjusting subunit of this complex the activity of corresponding C DK is carried out positivity and regulates, studies show that, not in conjunction with CDK and the equal non-activity of CDK monomer of cyclin.The CKI opposite with the cyclin effect then is the nucleoprotein that a class negativity is regulated CDK.
Statistics shows according to the study, and cyclin in 90% the human cancer of surpassing is arranged, CDK, and variation has taken place in related gene in CKI and the Rb approach, and wherein CDK and its corresponding cyclin's is not normal the most frequent, this shows its importance.CDK family has been found that at present 13 members (CDK1-CDK13) are divided into two classes by function difference in its born of the same parents: the CDKs of the CDKs of control cell cycle and control cell transcription.CDK9 belongs to the latter.CDK9 belongs to serine class kinases, it is called positivity transcriptional elongation factor b (P-TEFb) with corresponding cyclin (s) in conjunction with the complex that forms, this complex can phosphorylation rna plymerase ii (RNA polymerase II) and some negativity transcriptional elongation factors (NELF and N-TEFs) thus making to transcribe from initial position is extended, be to transcribe the key point that is prolonged.Discover CDK9 expression or (with) active can cause unusually in the cell that multiple protein is expressed or (with) its mRNA is unusual, and then cause the growth failure of cell, character is unusual, the cycle is unusual, proliferative disorder.
There are many cancer therapy drugs at molecular target to occur in recent years, this class medicine with strong points, effect is remarkable, is called as " target agent ".Paul Ehrlich has just proposed the notion of targeted therapy (targeting therapy) before 100 years, the going deep into along with development of molecular biology and people to the research of tumor mechanism, target agent at specific carcinogenic mechanism has obtained very fast development in recent years, the carcinogenic cause of disease of these medicine direct aggressions, selectivity is strong, clinical trial result is remarkable, and side effect is light, and multiple medicines is united the effect that use can strengthen traditional chemotherapeutics often.Ten years in the past, the research of antitumor drug is generally at tumor cell molecular basis out of control, concentrate on the regulatory mechanism that recovers tumour cell cycle, thereby produced many direct or indirect inhibitor at different CDK, and at the inhibitor of Cell Cycle Checkpoints.
CDKs be oncotherapy have much potentiality target spot.Reported the CDK inhibitor more than 50 kinds at present, some of them have potential anti-tumor activity, some many CDK inhibitor has been developed to antitumor drug, and other has some carrying out clinical preceding or clinical experiment, and new CDK inhibitor also constantly is being developed.Inhibitor at cell cycle mainly contains following several big class at present, Flavopiridol (husband's degree of evening up), and the representative of first generation CDK inhibitor has been widely used in the research of antitumor clinical treatment.It is that a kind of alkaloid that obtains with separation in the India indigenous plant is a prototype, through the semi-synthetic a kind of artificial semi-synthetic flavone derivative that obtains.It has multiple anticancer mechanism, and it has inhibitory action to EGFR (EGF-R ELISA) tyrosine kinase and protein kinase A (PKA).Also have wide spectrum CDK inhibitory action simultaneously, can effectively suppress CDK1,2,4,6 and 7 in cell cycle role, its IC50 is between the 40-200nM.Research afterwards finds that again it also has inhibitory action to the activity of CDK9/cyclinT, can suppress to transcribe.Another main big class is a purine compound, comprises dimethylamino-purine, isopentennyladenine, Olomoucine (olomoucine), Roscovitine (a kind of CDK inhibitors of kinases) etc.They have in various degree inhibitory action respectively to CDK1 and CDK2, make cell be stuck in G1/S phase or G2/M phase, thus cell death inducing.The molecular mechanism of representative Roscovitine wherein is that it can be competitive in conjunction with the ATP site, and to people CDK2/cyclinE, CDK7/cyclinH, CDK9/cyclinT1 have high selection inhibitory action.Molecular pharmacology studies show that the rectum cancer cell of handling through Roscovitine can cause pRb (retinocytoma albumen) phosphorylation level to reduce.But it is big to show the effect toxic and side effects in clinical experiment, and separately medication presses down cancer characteristic and characteristics such as not obvious.Other CDK inhibitor, all at CDK1,2 or 4 equimolecular target spots are researched and developed as butyrolactone, Staurosporine class (D-82041 DEISENHOFEN), chloro-indole sulfonamides, thiazolamine class.There is report to have another kind of novel imidazoles [1,2-a] pyridines CDKs inhibitor also can effectively suppress CDK1 recently, 2 and 9, have good DEVELOPMENT PROSPECT.In addition, seldom to the report of the specific inhibitor achievement in research of novel targets CDK9.Trace it to its cause, except newer because of the CDK9 target spot, and in the past its research was biased toward beyond the treating AIDS, another major reason is that these have all greatly restricted the development that utilizes CDK9 to develop antitumor drug as target spot owing to also do not have the CDK9 crystal structure at present.
Therefore, be target spot at CDK9 at present, the needs exploitation is a kind of to suppress malignant tumor, especially medicine of multiple malignant tumor such as pulmonary carcinoma, osteosarcoma, ovarian cancer, cervical cancer, breast carcinoma and preparation method thereof.
Summary of the invention
The present invention is based on following principle: make up the three-dimensional crystalline structure that the method begin to adopt the homology mould to build obtains CDK9 from target spot, and based on this, adopt the DOCK method to dock with Specs company compound library micromolecule, by interactional Study on Molecular Mechanism between computer virtual screening, bioactivity screening and micromolecular compound and the target spot, seek the micromolecular inhibitor of the efficient special inhibition CDK9 kinase activity of energy.By illustrating and having verified that N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide is in the molecular mechanism that suppresses growth of tumour cell.We find first, N-of the present invention (thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide demonstrates efficient pharmacologically active in suppressing multiple malignant cell growth such as pulmonary carcinoma, osteosarcoma, ovarian cancer, cervical cancer, breast carcinoma, opens up a new way for seeking new effective cell cycle series antineoplastic medicament.
Before the present invention, N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-benzamide type chemical compound common chemical compound of a class just only of this purchase particularly, but we have studied its characteristic at anti-tumor aspect, and be deep into the interaction that molecular level is studied itself and CDK9.In addition, N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Methanamide and analog thereof can have been bought from the market, and it is more convenient to originate.
Therefore, first purpose of the present invention is to provide the purposes of a kind of N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide in suppressing multiple malignant tumor such as pulmonary carcinoma, osteosarcoma, ovarian cancer, cervical cancer, breast carcinoma.
In a specific embodiments, the invention provides chemical compound (being N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Methanamide) in the purposes of anti-malignant tumor as shown in the formula (I):
N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Methanamide
Wherein:
R
1, R
2, R
3Be C
1-C
6Straight or branched alkyl, C
1-C
4Perfluoroalkyl, C
1-C
4Alkanoyl, C
1-C
4Ester group, C
1-C
4Carboxyl, C
1-C
4Amide groups, C
1-C
4Thioether, C
1-C
4Diazanyl, C
1-C
4Hydrazides, halogen, sulfonic group, sulfanilamide ,-NO
2,-NH
2,-CHO ,-OH ,-H, phenyl and substituted-phenyl, various heterocycle and substituted heterocycle;
R
1’R
2’：(CH
2)
4、CH
2＝CH
2-CH
2＝CH
2；
R
2’R
3’：(CH
2)
4、CH
2＝CH
2-CH
2＝CH
2；
R
1', R
2', R
3' be-H, C
1-C
6Straight or branched alkyl, C
1-C
4Perfluoroalkyl, C1-C4 alkanoyl, C
1-C
4Ester group, C
1-C
4Carboxyl, C
1-C
4Amide groups, C
1-C
4Thioether, C
1-C
4Diazanyl, C
1-C
4Hydrazides, halogen, sulfonic group, sulfanilamide ,-NO
2,-NH
2,-CHO ,-OH, phenyl and substituted-phenyl, various heterocycle and substituted heterocycle;
R
1, R
2, R
3, R
1', R
2', R
3' heterocycle comprises furan, thiophene, pyrroles, oxazole, thiazole, imidazoles, isoxazole, isothiazole, pyrazoles, pyridine, pyrans, pyrimidine, pyridazine, pyrazine, triazine, benzofuran, benzothiophene, indole, quinoline, isoquinolin .alpha.-5:6-benzopyran, purine and other pyrimido imidazo ring systems, pteridine and other pyrimidine pyrazine ring system;
R
1, R
2, R
3, R
1', R
2', R
3' substituent group on phenyl and the heterocycle can be alkyl, thiazolinyl, alkynyl, alkoxyl, ether, thioether group, fluoroalkyl, carbonyl, ester group, carboxyl, amide groups, diazanyl, hydrazides, halogen, sulfonic group, sulfanilamide ,-NO
2,-NH
2,-CHO ,-OH, can be that single the replacement also can be polysubstituted;
The compound of Formula I pharmaceutically acceptable salt comprises hydrochlorate, phosphate, sulfate, acetate, maleate, citrate, benzene sulfonate, toluenesulfonate, fumarate, tartrate.
In another embodiment, described purposes can be to utilize N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide to suppress or treat purposes in the malignant tumor (preferred pulmonary carcinoma, osteosarcoma, ovarian cancer, cervical cancer, breast carcinoma etc.); Described purposes can be utilize N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide to prepare to suppress or the medicine of treatment malignant tumor (preferred pulmonary carcinoma, osteosarcoma, ovarian cancer, cervical cancer, breast carcinoma etc.) in purposes.
Second purpose of the present invention is to provide the purposes of substituent in suppressing malignant tumor of above-claimed cpd.
In a specific embodiments, provide the application of substituent aspect the inhibition tumor proliferation of a kind of N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide.
In another embodiment, provide the application of a kind of replacement N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide aspect multiple malignant tumor external activities such as inhibition pulmonary carcinoma, osteosarcoma, ovarian cancer, cervical cancer, breast carcinoma.
Also in another embodiment, provide the application of a kind of replacement N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide aspect inhibition cell cycle dependant kinase CDK9 kinase activity.
The 3rd goal of the invention of the present invention provides a kind of pharmaceutical composition for the treatment of malignant tumor or suppressing cell cycle dependant kinase CDK9 kinase activity.
In a specific embodiments, it comprises above-mentioned chemical compound and one or more pharmaceutically acceptable carriers, excipient or diluent.
The 4th goal of the invention of the present invention provides the method for new small molecule inhibitors N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide of screening CDK9.
In a specific embodiments, described method is to adopt homology mould construction method to obtain the three-dimensional crystal conformation of cell cycle dependant kinase family member CDK9, with DOCK (molecular docking) the micromolecule three-dimensional data base is screened; Suppress experiment by the mtt assay growth of tumour cell chemical compound of picking out is carried out biological activity determination, select highly active compound N-(thiophene-2) pyrazolo [1 then, 5-a] pyrimidine-3-benzamide type carries out Study on Molecular Mechanism, and the checking chemical compound is to the inhibitory action of CDK9 kinase activity.
In a specific embodiments, described homology mould build and DOCK screening step as follows:
In order to obtain the CDK9 three dimensional structure, this laboratory on the SGI graphics workstation, utilizes the homology mould of software I nsight II to build (MODELLER/HOMOLOGY) module according to the aminoacid sequence of known CDK9, CDK9 is carried out the homology mould of three dimensional structure and builds.Key step is as follows: the primary sequence with CDK9 is a probe, searches for its homologous protein with the BlastP program in the PDB storehouse, selects homology height and the known homologous protein of albumen type as template.With the homologous protein stack, determine SCR (structure conserved region) and LOOP district.Mould is built albumen and homologous protein sequence alignment, the sequence that can be complementary with homologous protein SCR district is that mould is built proteic SCR district, give mould then and build Protein S CR district space coordinates, then utilize the Modeler software kit, adopt data base query method, coordinate according to the SCR at the length of LOOP and two ends, search out suitable LOOP structure, the same method that adopts data base querying, build in the process directly from the reference protein mould of preparing for the doctoral qualifying examination at mould and to build proteic side chain, uncomfortable side chain is built by molecular mechanics method mould be rational structure comparatively.
Mould is built the Dynamics Optimization of back structure: utilize the Soak/Assembly program, whole albumen is added that two layers of thickness is 5
Hydrone, and outer molecule limited.Utilize Insight II/Discover3 software kit, at first initial model was optimized for 2000 steps, adopt altogether the rail gradient method to optimize up to ability RMS deviation then less than 0.5kcal/mol/ with steepest descent method
With molecule power blood balance 20ps, the simulation step-length is 1fs under 298K.The last conformation that obtains after the Dynamics Optimization obtains its final conformation with 5000 steps of molecular mechanics optimization.Last Profile-3D software is built proteic 18 ambient parameters to mould and is calculated, and reasonability score and normal conformation expection score are compared, and the reasonability that the back mould is built structure is optimized in checking.
Secondary structure in the three dimensional structure of CDK9 is shown, observation beta wherein folds the distribution with the alpha spiral, the active region search is carried out on proteinic surface, the active region that searches shown one by one compare, select possible simultaneously in conjunction with document and active region feature in conjunction with the active region.All histidine His are changed the PDB file after the CDK9 Dynamics Optimization into Hid, the cysteine Cys that generates disulfide bond is changed into Cyx, after the Insight II simulation and the ownership field of force, be stored as the receptor.mol2 file, use for DOCK.The acceptor molecule surface has the DMS program to generate, and reuse SPHGEN generates the negative-appearing image of avtive spot, and negative-appearing image is piled up by the ball of different sizes and formed, as the representative of active area feature.Use the possible conformation of part in the conformation searching method search database of setting out then, simulate the model of action of part automatically according to the characteristics in negative-appearing image district based on molecule anchor point fragment, and according to the best in theory model of action of energy marking record.Write the dock.in file at last, determine the position of each Parameter File, operation DOCK simulation and screening under LINUX.
The 5th goal of the invention of the present invention provides a kind of CDK9 three-dimensional crystalline structure and the mould construction method thereof that is obtained by said method.
The 6th goal of the invention of the present invention provides a kind of pharmaceutical composition that is used for the treatment of malignant tumor, and it comprises arbitrary chemical compound and one or more pharmaceutically acceptable carriers, excipient or diluent in the general formula I as active component; Perhaps, comprise prepared chemical compound and one or more pharmaceutically acceptable carriers, excipient or the diluent of building by above-mentioned homology mould of screening technique as active component.
Description of drawings
Fig. 1: the concrete C-21 of the representative chemical compound of highly active N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-benzamide type substitutive derivative has carried out Determination of biological activity, enlarges the scope that its experiment in vitro is used.What abscissa was represented is that the various tumor cell line H-1299 that preserve my chamber are Lung Squamous Carcinoma Cells system, A549 is that another kind of Lung Squamous Carcinoma Cells is, HT1080 is an osteosarcoma cell line, the MCF-7 breast cancer cell line, Hela is a cervical cancer tumer line, and SKOV3 is an ovarian cancer cell line; Vertical coordinate represents to adopt C-21 that above-mentioned tumor cell line is handled the IC that the back is drawn
50Value (the drug level value when tumor cell is killed and wounded by half)
Fig. 2: the C-21 structural formula of compound, it is that general formula I is at R
1Trifluoromethyl replaces, R
2The benzene dimethoxy replaces, R
2' R
3': (CH
2)
4Replace R
1' N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-a kind of concrete substitutive derivative of 3-benzamide type of obtaining after replacing of ethoxycarbonyl
Fig. 3: C-21 is to the western blot figure of the inhibition of the zymogenesis of CDK9 experiment, the negative control treatment of PBS wherein, and use the C-21 of 2,5,10,20,40 μ M to handle cell pyrolysis liquid respectively.PSer2CTD represents the phosphorylation degree of 2 serines of the big subunit C-terminal of rna plymerase ii (that is to say the phosphorylation site of CDK9) after drug treating, can be found out by the depth of black splotch among the figure.Fig. 4: Compound C-21 and CDK9 crystal structure combination pattern, the molecular structural formula that wherein colored bar graph is C-21 and all the other blackish green structures are that the three-dimensional crystalline structure mould of CDK9 is built figure, wherein the blackish green part of entity is its kinase activity district.Three viride nitens colo(u)r streaks are represented three hydrogen bonds that C-21 and CDK9 kinase activity district form among the figure.
The specific embodiment:
The present invention will be further described below in conjunction with embodiment.
Embodiment 1:MMT surveys the inhibitory action to tumor cell of chemical compound
Adopt conventional mtt assay to measure the proliferation inhibition activity of each chemical compound to tumor cell line.The take the logarithm H1299 cell of trophophase of preliminary activity experiment, adjusting cell concentration with the RPMI1640 culture fluid that contains 10% serum is 1 * 10
5/ ml is inoculated in 96 well culture plates, and every hole 100 μ l at 37 ℃, cultivate 24h under the 5%CO2 condition.The grouping dosing, each concentration is established three parallel holes, processed group adds the medicine of variable concentrations, negative control group adds isopyknic PBS, and behind the cultivation 48h, every hole adds the MTT 20 μ l of 5mg/ml, 37 ℃ are continued to abandon supernatant behind the cultivation 4h, every hole adds 150 μ l DMSO, and vibration detects 570nm optical density (OD) value to precipitation dissolving fully on microplate reader.Calculate the growth of tumour cell suppression ratio.Mapping can obtain dose-effect curve to the growth of tumour cell suppression ratio at the variable concentrations with same medicine, obtains the half-inhibition concentration IC of this medicine
50
Further activity experiment still adopts mtt assay, goes on foot all kinds of solid tumor cells that the highest active C-21 chemical compound is used for preserving this chamber among the result in the selection to be, measures its suppression ratio, obtains the IC of this medicine to each tumor cell line
50
Experimental result:
12 chemical compounds significantly suppress the propagation (IC of tumor cell in 27 chemical compounds
50＜50 μ moL), the half-inhibition concentration (IC of 1 chemical compound wherein
50) below 20 μ moL, the IC50 of each chemical compound sees the following form.Select the highest wherein active C-21 chemical compound further to study.The C-21 pair cell is the proliferation inhibition rate such as the following table of all kinds of solid tumor cell such as A459, Hela, HT1080, SKOV3, MCF-7 system
Turbid phenomenon appears in " * " in the last table
We have further carried out Determination of biological activity to highly active C-21 chemical compound, enlarge the scope that its experiment in vitro is used.The result shows that it has the effect of growth obvious suppression, its IC to multiple malignant cells such as pulmonary carcinoma, osteosarcoma, ovarian cancer, cervical cancer, breast carcinoma
50Between 10-30 μ M, and most IC
50＞20 μ M.As shown in Figure 1.
Further provide structural formula such as Fig. 2 of C-21
Embodiment 2:C-21 is to the inhibition experiment of the zymogenesis of CDK9
This research is selected to suppress the highest active C-21 chemical compound (a kind of N-(thiophene-2) pyrazolo [1, the 5-a] pyrimidine-concrete substitutive derivative of 3-benzamide type) and is further carried out Molecular Study.
The take the logarithm H1299 cell of trophophase is pressed the operation of RIPA cell pyrolysis liquid description, severally obtains fresh cell pyrolysis liquid down through ultrasonic again, and the BCA test kit is quantitative.Regulate cell pyrolysis liquid and CTD protein liquid concentration to suitable, add the medicine C-21 of variable concentrations, concussion mixing (can add a certain amount of ATP liquid simultaneously), 37 ℃ of reaction 4h get and carry out the 10%SDS-PAGE electrophoresis in right amount.After electrophoresis is finished, press 0.65mA/cm according to the gel area
2Energized, electrotransfer 3 hours.After shifting end, PBST washes PVFD film 15min, and the room temperature gentleness is shaken, and puts into 4 ℃ of jogs of confining liquid (5% defatted milk) and spends the night.Film is encapsulated in the plastic foil by 0.1ml/cm
2Add the diluent of monoclonal antibody of the rna plymerase ii of anti-phosphorylation, gentleness is shaken 2h under the room temperature.Get film PBST and wash 3 times, each 10min is encapsulated in another clean plastic foil by 0.1ml/cm again
2Add the sheep anti-mouse igg diluent, gentleness is shaken 2h under the room temperature.Discard two and resist, PBST washes 3 times, each 10min.Press BeyoECL Plus description at last in scotography.
Its result such as Fig. 3.Isopyknic PBS is used in the wherein negative contrast of PBS (cell isotonic solution) respectively, and the C-21 of 2,5,10,20,40 μ M handles cell pyrolysis liquid.PSer2CTD represents the phosphorylation degree of 2 serines of the big subunit C-terminal of rna plymerase ii, can be found out by the depth of immune marking band among the figure.The phosphorylation degree of rna plymerase ii weakens with the increasing of drug dose as seen from the figure, illustrates thus between the decline degree of kinase activity of CDK9 and the drug level to be certain dose-dependence.
Embodiment 3: Compound C-21 and CDK9 crystal structure combination pattern
According to the aminoacid sequence of known CDK9, on the SGI graphics workstation, utilize the homology mould of software I nsight II to build (MODELLER/HOMOLOGY) module, CDK9 is carried out the homology mould of three dimensional structure and build.Key step is as follows: the primary sequence with CDK9 is a probe, searches for its homologous protein with the BlastP program in the PDB storehouse, selects homology height and the known homologous protein of albumen type as template.With the homologous protein stack, determine SCR (structure conserved region) and LOOP district.Mould is built albumen and homologous protein sequence alignment, the sequence that can be complementary with homologous protein SCR district is that mould is built proteic SCR district, give mould then and build Protein S CR district space coordinates, then utilize the Modeler software kit, adopt data base query method, coordinate according to the SCR at the length of LOOP and two ends, search out suitable LOOP structure, the same method that adopts data base querying, build in the process directly from the reference protein mould of preparing for the doctoral qualifying examination at mould and to build proteic side chain, uncomfortable side chain is built by molecular mechanics method mould be rational structure comparatively.
The secondary structure of CDK9 is shown, observation beta wherein folds the distribution with the alpha spiral, active region search is carried out on proteinic surface, the active region that searches is shown one by one compare, the while is selected possible in conjunction with the active region in conjunction with document and active region feature.All histidine His are changed the PDB file after the CDK9 Dynamics Optimization into Hid, the cysteine Cys that generates disulfide bond is changed into Cyx, after the Insight II simulation and the ownership field of force, be stored as the receptor.mol2 file, use for DOCK.The acceptor molecule surface has the DMS program to generate, and reuse SPHGEN generates the negative-appearing image of avtive spot, and negative-appearing image is piled up by the ball of different sizes and formed, as the representative of active area feature.Use the possible conformation of part in the conformation searching method search database of setting out then, simulate the model of action of part automatically according to the characteristics in negative-appearing image district based on molecule anchor point fragment, and according to the best in theory model of action of energy marking record.Write the dock.in file at last, determine the position of each Parameter File, operation DOCK simulation and screening under LINUX.
The binding mode figure that mutually combines when Fig. 4 is micromolecular compound C-21 and CDK9 three-dimensional crystalline structure DOCK.The kinase activity district of C-21 and CDK9 has formed three hydrogen bonds altogether as shown in Figure 4, and pyrazolo [1,5-a] pyrimidine structure stretches into inside, active region fully in the mode of unfolding, and its nitrogen-atoms and 103 phenylalanine form a hydrogen bond.Other 106 cysteine respectively form a hydrogen bond with the oxygen atom of amide and the sulphur atom of thiophene respectively, fine from scheming analysis C-21 and CDK9 at energy and structure coupling, really can effectively stop combining of CDK9 active region and its downstream substrate, thereby the CTD section phosphorylation level of rna plymerase ii is descended.
In sum, by can further proving of molecular pharmacology experiment: N-of the present invention (thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide demonstrates obvious suppression kinds of tumor cells growth effect, its Study on Molecular Mechanism has verified that mould is built and DOCK result's correctness, embody efficient, characteristics such as molecular target is determined and specific aim is extremely strong, analog after it is optimized for further exploitation becomes drug candidate and has made the preparation on the mechanism, also for providing new approaches with other types antitumor drug drug combination.
Claims (7)
1, the application in the medicine of N-(thiophene-2) pyrazolo [1, the 5-a] pyrimidine-3-Carbox amide with following general structure (I) aspect preparation anti-malignant tumor propagation;
Wherein:
R
1, R
2, R
3Be C
1-C
6Straight or branched alkyl, C
1-C
4Perfluoroalkyl, C
1-C
4Alkanoyl, C
1-C
4Ester group, C
1-C
4Carboxyl, C
1-C
4Amide groups, C
1-C
4Thioether, C
1-C
4Diazanyl, C
1-C
4Hydrazides, halogen, sulfonic group, sulfanilamide, NO
2, NH
2,-CHO ,-OH ,-H, phenyl and substituted-phenyl, various heterocycle and substituted heterocycle;
R
1’R
2’：(CH
2)
4、CH
2＝CH
2-CH
2＝CH
2；
R
2’R
3’：(CH
2)
4、CH
2＝CH
2-CH
2＝CH
2；
R
1', R
2', R
3' be-H, C
1-C
6Straight or branched alkyl, C
1-C
4Perfluoroalkyl, C
1-C
4Alkanoyl, C
1-C
4Ester group, C
1-C
4Carboxyl, C
1-C
4Amide groups, C
1-C
4Thioether, C
1-C
4Diazanyl, C
1-C
4Hydrazides, halogen, sulfonic group, sulfanilamide, NO
2, NH
2,-CHO ,-OH ,-H, phenyl and substituted-phenyl, various heterocycle and substituted heterocycle;
R
1, R
2, R
3, R
1', R
2', R
3' substituent group on the phenyl can be alkyl, thiazolinyl, alkynyl, alkoxyl, ether, thioether group, fluoroalkyl, carbonyl, ester group, carboxyl, amide groups, diazanyl, hydrazides, halogen, sulfonic group, sulfanilamide, NO
2, NH
2,-CN ,-CHO ,-OH, can be that single the replacement also can be polysubstituted;
R
1, R
2, R
3, R
1', R
2', R
3' heterocycle comprises furan, thiophene, pyrroles, oxazole, thiazole, imidazoles, isoxazole, isothiazole, pyrazoles, pyridine, pyrans, pyrimidine, pyridazine, pyrazine, triazine, benzofuran, benzothiophene, indole, quinoline, isoquinolin .alpha.-5:6-benzopyran, purine and other pyrimido imidazo ring systems, pteridine and other pyrimidine pyrazine ring system;
R
1, R
2, R
3, R
1', R
2', R
3' replacement on the heterocycle can be alkyl, thiazolinyl, alkynyl, alkoxyl, ether, thioether group, fluoroalkyl, carbonyl, ester group, carboxyl, amide groups, diazanyl, hydrazides, halogen, sulfonic group, sulfanilamide, NO
2, NH
2,-CN ,-CHO ,-OH, can be that single the replacement also can be polysubstituted;
The compound of Formula I pharmaceutically acceptable salt comprises hydrochlorate, phosphate, sulfate, acetate, maleate, citrate, benzene sulfonate, toluenesulfonate, fumarate, tartrate.
2, the application of the replacement N-of claim 1 (thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide aspect the inhibition tumor proliferation.
3, the application of the replacement N-of claim 1 (thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide aspect multiple malignant tumor external activities such as inhibition pulmonary carcinoma, osteosarcoma, ovarian cancer, cervical cancer, breast carcinoma.
4, the application of the replacement N-of claim 1 (thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide aspect inhibition cell cycle dependant kinase CDK9 kinase activity.
5, a kind of method of screening the new small molecule inhibitors of cell cycle dependant kinase CDK9 comprises:
Adopt homology mould construction method to obtain the three-dimensional crystal conformation of cell cycle dependant kinase family member CDK9, the micromolecule three-dimensional data base is screened with DOCK (molecular docking);
Suppress experiment by the mtt assay growth of tumour cell chemical compound of picking out is carried out biological activity determination;
Select highly active chemical compound pyrazolo [1,5-a] miazines then and carry out Study on Molecular Mechanism, the checking chemical compound is to the inhibitory action of CDK9 kinase activity;
Wherein the micromolecular inhibitor that is screened is N-(thiophene-2) pyrazolo [1,5-a] pyrimidine-3-Carbox amide.
6, a kind of pharmaceutical composition that is used for the treatment of malignant tumor, it comprises as chemical compound arbitrary in the claim 1 to 4 of active component and one or more pharmaceutically acceptable carriers, excipient or diluent.
7, a kind of pharmaceutical composition that is used for the treatment of malignant tumor, it comprise as active component by claim 5 prepared chemical compound and one or more pharmaceutically acceptable carriers, excipient or diluent.
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