CN106866635B - PLK1 inhibitor and preparation method and application thereof - Google Patents
PLK1 inhibitor and preparation method and application thereof Download PDFInfo
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- CN106866635B CN106866635B CN201510916228.XA CN201510916228A CN106866635B CN 106866635 B CN106866635 B CN 106866635B CN 201510916228 A CN201510916228 A CN 201510916228A CN 106866635 B CN106866635 B CN 106866635B
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4164—1,3-Diazoles
- A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/4965—Non-condensed pyrazines
- A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention provides a PLK1 inhibitor and a preparation method and application thereof, wherein the PLK1 inhibitor is a compound shown as a general formula (I) and a stereoisomer or pharmaceutically acceptable salt thereof:
the definitions of all groups in the formula (I) are the same as the description. The PLK1 inhibitor can be specifically and high-strength combined with a DNA sequence in a PLK1 gene transcription promoter region shown in SEQ NO 1, inhibit the transcription of a PLK1 gene, inhibit the expression of PLK1 protein, cause the growth inhibition or apoptosis of tumor cells, can penetrate cell membranes and nuclear membranes and resist nuclease hydrolysis, and in addition, the drug resistance problem of a small molecule kinase inhibitor is overcome.
Description
Technical Field
The invention relates to a PLK1 inhibitor, a preparation method and application thereof, belonging to the field of pharmaceutical chemistry.
Background
PLK1 is a Polo-type kinase family member, is a highly conserved serine/threonine protein kinase, participates in the processes of centrosome maturation, spindle body formation, chromosome separation and the like in the cell division process, and plays an important role in regulating and controlling the cell mitosis process. The research shows that PLK1 is abnormally and highly expressed in various malignant tumors such as lung cancer, breast cancer, gastric cancer and the like, and the over-expression is one of the markers for poor prognosis of tumors, but the expression level is very low in normal cells and can not be even detected. Therefore, PLK1 is a widely interesting target in tumor diagnosis and therapy.
The antisense technology, the small molecule RNA interference technology or the small molecule inhibitor are used for knocking out the gene expressing PLK1 in the tumor cells or inhibiting the activity of PLK1, so that the growth inhibition and even the apoptosis of the tumor cells can be caused. It has been shown that antisense oligonucleotides or small interfering RNAs specifically reduce the expression of PLK1, but have no significant effect on normal cells. There have also been studies reporting that screening of chemical small molecule inhibitors of PLK1, such as ON01910, BI2536, HMN-214, GSK461364, etc., from organic synthetic chemical small molecules or natural products, which have partially entered clinical research, can inhibit PLK1 activity by competitive or non-competitive binding to ATP.
However, antisense oligonucleotides are susceptible to nuclease hydrolysis and have a short duration of action; the small molecule RNA interference technology also has the problems of safety and stability. In addition, oligonucleotides and small RNA are difficult to penetrate cell membranes, and the problem of transmembrane transport is difficult to solve. Therefore, the main research direction at present is to screen chemical small molecule inhibitors of PLK1 from organic synthetic chemical small molecules or natural products. However, small molecule kinase inhibitors that are currently successfully marketed for tumor therapy all have a common weakness-the development of resistance. Because tumor cells can generate mutation rapidly in the treatment process, the binding force of kinase to the small-molecule inhibitor is reduced, and the kinase is not sensitive to the treatment of the small-molecule inhibitor, so that the drug resistance is generated, and the chemical small-molecule inhibitor of PLK1 has the same drug resistance problem.
Therefore, the development of a novel PLK1 inhibitor is one of the technical problems to be solved in the art.
Disclosure of Invention
One of the objects of the present invention is to provide a novel class of PLK1 inhibitors.
The invention also aims to provide a preparation method of the PLK1 inhibitor.
It is a further object of the present invention to provide compositions containing said PLK1 inhibitor.
Still another object of the present invention is to provide the use of the PLK1 inhibitor or a composition comprising the PLK1 inhibitor.
To achieve the above objects, in one aspect, the present invention provides a compound represented by the general formula (I):
wherein:
R1～R8each independently is hydrogen or C1～3An alkyl group; preferably, R1～R8Are all methyl
A is- (CH)2)m-, in A, any CH2To a hydrogen atom of C1～3Alkyl, -OH, -NH2、-O-(C＝O)-R9or-NH- (C ═ O) -R9Substituted;
b is- (CH)2)n-CH3In B, any CH2Or CH3To a hydrogen atom of C1～3Alkyl, -OR11or-NR10R11Substituted;
preferably, A is
The R is12Selected from-OH, -NH2、-O-(C＝O)-R9or-NH- (C ═ O) -R9(ii) a More preferably, A is
Preferably, B is
R9Is composed of
R10Is C1～3An alkyl group;
R11is C1～6Alkyl or R17；
R17Is composed of
At R9Or R17In, any CH2Is substituted by 0 to 2C1～6Alkyl, -OH, -NH2Halogen, -OR19、-NHR20or-NR20R21Substituted;
x or Y are each independently O, NH or NR22，
R13～R15Each independently of the other being H, halogen, C1～3An alkyl group;
R16is C1～6An alkyl group; preferably, R16Is methyl, ethyl, n-propyl, isopropyl, monofluoromethyl, difluoromethyl or trifluoromethyl;
R18is hydrogen, halogen, C1～3Alkyl, carboxyl or nitro, preferably, R18Is a carboxyl group;
the R is19～R22Each independently is C1～3An alkyl group;
said C is1～6Alkyl or said C1～3Alkyl is substituted with 0-3 halogens, preferably 0-3 fluorines;
p, p' are each independently selected from 0, 1,2, 3 or 4; preferably, p' are each independently 1,2, 3 or 4;
q, q ', r ', s ' are each independently selected from 0, 1,2, 3 or 4; preferably, q ', r ', s ' are all 0;
t is selected from 0, 1,2, 3 or 4; preferably, t is 1.
The compound shown IN the general formula (I) is a pyrrole-imidazole polyamide compound which can be specifically identified and is combined with a DNA sequence IN a PLK1 gene transcription promoter region shown IN SEQ IN NO. 1 IN a high-strength manner, so that the transcription of a PLK1 gene is inhibited, the expression of a PLK1 protein is inhibited, and the growth inhibition or apoptosis of tumor cells is caused. The compound shown in the general formula (I) can penetrate through cell membranes AND nuclear membranes to specifically recognize PLK1 gene transcription promoter sequences, AND belongs to polyamide compounds, AND the prior art (Single-double pharmaceutical AND toxin analysis of pyrole-enzyme polyamino acids in microorganisms, Synold, Timothy W.; Xi, Bixin; Wu, Jun; et al, CANCER CHEMOTHERAPY AND PHMACATOLOGIY Volume:70Issue:4 Pages:617-625, AND Published: OCT2012) shows that the structure can resist nuclease hydrolysis, so that the compound shown in the general formula (I) can resist nuclease hydrolysis. Therefore, the pyrrole-imidazole polyamide compound shown in the general formula (I) overcomes the problems of short effective action time and transmembrane transport of oligonucleotide and small molecular RNA. In addition, the pyrrole-imidazole polyamide shown in the general formula (I) directly acts on a sequence in a transcription promoter region of PLK1 gene, and is different from a small molecule inhibitor acting on a kinase protein molecule, so that the drug resistance problem of the small molecule kinase inhibitor is overcome.
Preferably, in the compound represented by the general formula (I), the stereoisomer or the pharmaceutically acceptable salt thereof according to the present invention, the compound has a structure represented by formula (II) to formula (V):
preferably, R10Is methyl or trifluoromethyl;
preferably, said R is12Is selected from NH2、
Preferably, said R is11Is selected from CH3、CF3、CH2COOH、
Further preferably, in the compound represented by the general formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof according to the present invention, the compound includes:
said C of the invention1～3Alkyl includes methyl, ethyl, n-propyl or isopropyl; said C is1～6The alkyl group represents a straight-chain or straight-chain alkyl group having 1 to 6 carbon atoms, and includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, n-hexyl, and isohexyl groups.
Said C of the invention1～6Alkyl or said C1～3Alkyl substituted by 0 to 3 halogens represents C as described above1～3Alkyl or C1～6The hydrogen in the alkyl group being substituted by 0 to 3 halogen atoms, e.g. -CH2F、-CHF2、-CF3、-CH2FCH3、-CHF2CH3、-CF2CH2CH3And the like.
In another aspect, the present invention provides a method for preparing a compound represented by the general formula (I), a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein the method comprises the steps of:
(a) preparing a phenylhydrazine resin-supported resin of formula (IV);
formula (IV)
A' is- (CH)2)m-, in A, any CH2To hydrogen atom of-OPOH、-NHPNHSubstituted;
preferably, said A' is
The R is12is-OPOHor-NHPOH(ii) a More preferably, A' is
(b) Removing P in the formula (IV)OHOr PNH；
(c) Carrying out oxidation reaction and cutting reaction by using an oxidant and a cutting agent to prepare a compound shown in a formula (I);
the cutting agent is NH2-(CH2)n-CH3In the cleavage agent, any CH2Or CH3Hydrogen atom of (A) is-OR11or-NR10R11Substituted;
preferably, the cutting agent is
Preferably, the oxidant is one or more of copper acetate, N-bromosuccinimide and oxygen;
a' and the groups in the cleavage agent are as defined in the invention, POH、PNHRespectively represents a hydroxyl protecting group and an amino protecting group; preferably, said POH、PNHBoc, Fmoc, Cbz, Trt or Allyl, more preferably Fmoc.
According to a particular embodiment of the invention, in the process of the invention, between step (b) and step (c)To make room for
For removing POHOr PNHThe exposed hydroxyl or amino group undergoes a condensation reaction to form-O- (C ═ O) -R9or-NH- (C ═ O) -R9A step (2); the R is23Is hydroxyl or halogen.
According to a particular embodiment of the present invention, in the method of the present invention, step (c) of the method is replaced by step (c '), and further comprising step (c ") after step (c');
(c') carrying out an oxidation reaction and a cleavage reaction with an oxidizing agent and a cleavage agent; the cutting agent is NH2-(CH2)n-CH2N(R10)-(CH2)p’-YH，
(c') to
After condensation reaction of YH in the product obtained in step (c') to obtain said compound represented by formula (I); r24Is hydroxy or halogen;
optionally, between step (b) and step (c'), removing POHOr PNHExposed hydroxy or amino groups with POH' or PNH' protection, said POH' or PNH' is a hydroxyl-or amino-protecting group that can withstand basic conditions; and removing P after the condensation reaction in step (c')OH' or PNH' to obtain said compound of formula (I); preferably, said POH' or PNH' is tert-butoxycarbonyl.
According to a particular embodiment of the invention, in the process of the invention, the formula (VI) is formula (VI-1);
preferably, formula (VI-1) is prepared as follows:
(1) taking phenylhydrazine resin and 4-tert-butyloxycarbonyl amino-1-methyl-1H-pyrrole-2-carboxylic acid as raw materials, and repeatedly condensing and removing a tert-butyloxycarbonyl protecting group to prepare a compound shown in a formula (1):
(2) carrying out condensation reaction on a compound shown in a formula (1) and R-2- (9-fluorenylmethoxycarbonylamino) -4-tert-butyloxycarbonylaminobutyric acid to prepare a compound shown in a formula (2);
(3) removing the tert-butyloxycarbonyl protecting group from the compound shown in the formula (2), then carrying out condensation reaction with 4-tert-butyloxycarbonyl amino-1-methyl-1H-pyrrole-2-carboxylic acid, and repeatedly removing the tert-butyloxycarbonyl protecting group and carrying out condensation reaction to obtain the compound shown in the formula (3):
(4) carrying out condensation reaction on a compound shown in a formula (3) and 4-tert-butyloxycarbonylamino-1-methyl-1H-imidazole-2-carboxylic acid, and removing a tert-butyloxycarbonylamino protecting group to obtain a compound shown in a formula (4):
(5) the compound shown in the formula (4) and 1-methyl-1H-imidazole-2-carboxylic acid are subjected to condensation reaction to prepare the compound shown in the formula (VI-1).
The present invention is not limited to the conditions of the condensation reaction and the reaction for removing the t-butoxycarbonyl protecting group in the above-mentioned route.
In still another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of the compound represented by the general formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, or further comprising one or more pharmaceutically acceptable carriers or excipients.
In another aspect, the invention provides an application of the compound shown in the general formula (I), the stereoisomer or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing a medicament for treating or preventing cell hyperproliferation diseases.
Preferably, the hyperproliferative cell disease comprises one or more of colon cancer, rectal cancer, brain tumor, lung cancer, epidermal squamous carcinoma, bladder cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, colorectal cancer, renal cell carcinoma, gastric cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, non-hodgkin lymphoma, liver cancer, skin cancer, thyroid cancer, head and neck cancer, prostate cancer, glioma and nasopharyngeal carcinoma; more preferably, the hyperproliferative cell disease comprises breast cancer, lung cancer or stomach cancer.
In conclusion, the invention provides a novel PLK1 inhibitor, a preparation method and application thereof. The PLK1 inhibitor can specifically bind with high strength to DNA sequence in PLK1 gene transcription promoter region shown in SEQ NO. 1, inhibit transcription of PLK1 gene, inhibit expression of PLK1 protein, and cause tumor cell growth inhibition or apoptosis. And it is capable of passing through cell and nuclear membranes and is resistant to nuclease hydrolysis. In addition, the drug resistance problem of small molecule kinase inhibitors is overcome.
The abbreviations in the present invention have the following meanings:
PLK1 Polo-Like Kinase1, Porro-Like Kinase 1; BOC, t-butyloxycaronyl, tert-butyloxycarbonyl; fmoc is fluoroxylmethoxycarbonyl, fluorenylmethyloxycarbonyl; HOAt 1-hydroxy-7-azobenzotriazol; DMF is N, N-dimethylformamide; THF, tetrahydrofuran; DIEA is N, N-diisopropylethylamine; TFA is trifluoroacetic acid; PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate.
Drawings
FIG. 1 is a scheme for synthesis of compounds 1-5;
FIG. 2 is an HRMS profile of Compound 1;
figure 3 is an HRMS profile of compound 2;
figure 4 is the HRMS profile of compound 3;
FIG. 5 is an HNMS spectrum of a Herstein acid derivative Ht-2;
figure 6 is an HRMS profile of compound 4;
figure 7 is an HRMS profile of compound 5;
FIG. 8 is a graph showing the results of the experiment in which Compound 2 penetrated cell and nuclear membranes;
FIG. 9 is a graph showing the results of an experiment in which Compound 2 inhibited the expression of PLK1 protein in Hela cells;
FIG. 10 is a graph showing the results of an experiment in which Compound 2 inhibits Hela cell proliferation;
FIG. 11 is a graph showing the dose and frequency administered in the administration group of example 9;
FIGS. 12 to 14 are graphs showing the results of experiments conducted by Compound 2 of example 9 in inhibiting the proliferation of non-small cell lung cancer tumor cells in tumor-bearing mice.
Detailed Description
For a more clear understanding of the technical features, objects and advantages of the present invention, reference is now made to the following detailed description of the embodiments of the present invention taken in conjunction with the accompanying drawings, which are included to illustrate and not to limit the scope of the present invention.
FIG. 1 is a synthesis scheme of compounds 1-5, which is to prepare compounds 1-5 from Fmoc-protected phenylhydrazine resin as a raw material by multi-step method to obtain formula (1), then to prepare formula (2) -formula (6) in sequence, and finally to prepare compounds 1-5 according to formula (5) or formula (6).
EXAMPLE 1 preparation of Compound 1
(a) Swelling resin: a10 mL solid phase reactor was charged with 400mg of Fmoc-protected phenylhydrazine resin (0.66mmol/g, 0.264mmol) and 3mL of CH2Cl2Swelling the resin for 30min, removing CH2Cl2And is ready for use;
(b) removing Fmoc protecting groups: adding 3mL of 20% piperidine/DMF solution to the swollen resin from step (a), N2Bubbling and mixing evenly, after 10min, removing the solvent, and then adding 3mL of 20% piperidine/DMF solution, N2Bubbling and uniformly mixing, washing the resin with DMF (4X 3mL) after 10min, and then washing the resin with 3mL of anhydrous DMF for later use;
(c) amino acid condensation: 4-tert-Butoxycarbonylamino-1-methyl-1H-pyrroleDissolving 2-carboxylic acid (254mg, 1.056mmol) and triphosgene (BTC, 128mg, 0.433mmol) in 2mL of anhydrous THF, slowly adding collidine (488. mu.L, 3.696mmol) dropwise into the solution, reacting to generate a large amount of white precipitate immediately, adding the reaction for 3min, adding 2mL of DIEA/DMF solution (5%, v/v), completely removing the white precipitate, transferring the reaction solution to the phenylhydrazine resin subjected to protecting group removal in step (b), and N2Bubbling and uniformly mixing, carrying out condensation reaction for 0.5-1 h, pumping out reaction liquid, and washing resin with DMF (4X 3mL) for later use;
(d) removing the tert-butyloxycarbonyl protecting group: by CH2Cl2(2X 3mL) washing, CH extraction2Cl23.0mL of TFA/phenol/H was added2Removing the tert-butoxycarbonyl protecting group on the condensation product obtained in the step (b) by using a mixed solution of O (v: v: v ═ 92:5:2.5), removing the solvent after 2min, and adding 3.0mL of TFA/phenol/H again2The mixed solution of O (v: v: v ═ 92:5:2.5) was reacted for 20min with CH2Cl2(2X 3mL) and DMF (4X 3mL) and then 3mL of anhydrous DMF;
repeating the condensation and deprotection steps (c) and (d) until the synthesis of the peptide represented by the formula (1) supported on the phenylhydrazine resin is completed;
(e) condensation of γ -amino acids: dissolving R-2- (9-fluorenylmethoxycarbonylamino) -4-tert-butoxycarbonylaminobutyric acid (465mg, 1.056mmol) and triphosgene (128mg, 0.433mmol) in 2mL of anhydrous THF, slowly adding collidine (488 uL, 3.696mmol) dropwise into the solution to react to generate a large amount of white precipitate immediately, adding HOAt (144mg, 1.056mmol), adding 2mL of DIEA/DMF solution (5%, v/v), reacting for 5min, completely removing the white precipitate, and transferring the reaction solution to the straight-chain peptide (NH) loaded on phenylhydrazine resin shown in the formula (1)2-Py-Py-Py-Py-phenylhydrazine resin), N2Bubbling and uniformly mixing, carrying out condensation reaction for 0.5-1 h, pumping out reaction liquid, and washing resin with DMF (4X 3mL) to obtain the peptide shown as a formula (2) loaded on the phenylhydrazine resin;
(f) repeating the deprotection and condensation protection steps (d) and (c), wherein the synthesis of the peptide loaded on the phenylhydrazine resin shown in the formula (3) is completed;
(g) amino acid condensation: dissolving 4-tert-butyloxycarbonylamino-1-methyl-1H-imidazole-2-carboxylic acid (255mg, 1.056mmol) and triphosgene (128mg, 0.433mmol) in 1mL of anhydrous THF, slowly adding collidine (488. mu.L, 3.696mmol) dropwise to the solution to react immediately to generate a large amount of white precipitate, adding HOAt (144mg, 1.056mmol), adding 2mL of DIEA/DMF solution (5%, v/v), allowing the white precipitate to disappear completely, transferring the reaction solution to the peptide of formula (3) obtained in step (f), wherein the peptide is supported on phenylhydrazine resin, and N is N, N is2Bubbling and uniformly mixing, carrying out condensation reaction for 0.5-1 h, pumping out reaction liquid, and washing resin with DMF (4X 3mL) for later use;
(h) removing the tert-butyloxycarbonyl protecting group: by CH2Cl2(2X 3mL) washing, CH extraction2Cl23.0mL of TFA/phenol/H was added2Removing the tert-butoxycarbonyl protecting group from the product obtained in step (g) with a mixed solution of O (v: v: v ═ 92:5:2.5), removing the solvent after 2min, and adding 3.0mL of TFA/phenol/H again2The mixed solution of O (v: v: v ═ 92:5:2.5) was reacted for 20min with CH2Cl2Washing the resin with 2X 3mL of DMF (4X 3mL), and then washing the resin with 3mL of anhydrous DMF to obtain the peptide shown as the formula (4) loaded on the phenylhydrazine resin;
(i) condensation of the terminal amino acids: dissolving 1-methyl-1H-imidazole-2-carboxylic acid (132mg, 1.056mmol) and PyBOP (550mg, 1.056mmol) in 3mL of anhydrous DMF, adding DIEA (350. mu.L, 2.112mmol), reacting for 5min, and transferring the reaction solution to the phenylhydrazine-loaded phenylhydrazine of formula (4) obtained in step (H)In the peptide on the resin, N2Bubbling, uniformly mixing, carrying out condensation reaction for 2h, pumping out reaction liquid, and washing the resin with DMF (4X 3mL) to obtain the peptide shown as a formula (5) loaded on the phenylhydrazine resin;
(j) synthesis of Compound 1
After removing the Fmoc protecting group from the peptide of formula (5) supported on phenylhydrazine resin by the method of step (b), the resin was taken out, and 1mL of DMF, 200. mu.L of dimethylaminopropylamine and 10mg of Cu (OAc) were added2The reaction was shaken at room temperature for 12h, the resin filtered off and charged with 20mL CH2Cl2Washing the resin; the organic phase was concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile-H2Gradient elution of O (1% TFA) for 5min, 10% to 100% acetonitrile-H2Gradient elution with O (1% TFA) for 25min, retention time TRThe product was collected for 15min and lyophilized to give compound 1 as a pale yellow solid with HRMS as shown in figure 2.
HRMS (ESI) m/z theoretical calculation C55H68N21O9[M+H]+1166.5503 found 1166.5508.
EXAMPLE 2 preparation of Compound 2
Preparing the peptide represented by the formula (5) supported on phenylhydrazine resin according to the same procedure as in example 1, and removing the Fmoc protecting group from the peptide represented by the formula (5) supported on phenylhydrazine resin according to the procedure (b) in example 1;
the herceptic acid derivative Ht-1(539mg, 1.056mmol) and PyBOP (550mg, 1.056mmol) were dissolved in 3mL of anhydrous DMF, DIEA (350. mu.L, 2.112mmol) was added thereto, reaction was carried out for 5min, the reaction solution was transferred to Fmoc-removed peptide represented by formula (5) supported on phenylhydrazine resin, N2Bubbling, uniformly mixing, carrying out condensation reaction for 1h, pumping out reaction liquid, and washing the resin with DMF (4X 3 mL); the resin was removed and 1mL DMF, 200. mu.L dimethylaminopropylamine and 10mg Cu (OAc) were added2The reaction was shaken at room temperature for 12h, the resin filtered off and charged with 20mL CH2Cl2Washing the resin; the organic phase is concentrated and the organic phase is concentrated,the residue was purified by semi-preparative HPLC: 10% acetonitrile-H2Gradient elution of O (1% TFA) for 5min, 10% to 100% acetonitrile-H2Gradient elution with O (1% TFA) for 25min, retention time TRThe product was collected and lyophilized at 18.5min to give compound 2 as a pale yellow solid with HRMS as shown in figure 3.
HRMS (ESI) m/z theoretical calculation C84H96N27O11[M+H]+1658.7777 found 1658.7774.C84H97N27O11 2+；
The structure of the herceptic acid derivative Ht-1 is as follows (see J.AM. CHEM. SOC.2004,126, 3736-3747):
EXAMPLE 3 preparation of Compound 3
Preparing the peptide represented by the formula (5) supported on phenylhydrazine resin according to the same procedure as in example 1, and removing the Fmoc protecting group from the peptide represented by the formula (5) supported on phenylhydrazine resin according to the procedure (b) in example 1;
reacting Boc2O (243. mu.L, 1.056mmol) was dissolved in 3mL of anhydrous DMF, DIEA (350. mu.L, 2.112mmol) was added, and the reaction solution was transferred to Fmoc-free peptide of formula (5) supported on phenylhydrazine resin, N2Bubbling, mixing uniformly, and carrying out condensation reaction for 20 min. The reaction solution was aspirated off, and the resin was washed with DMF (4X 3mL) to obtain Boc-protected peptide represented by formula (6) supported on phenylhydrazine resin;
taking out the resin obtained above, adding 1mL of DMF, 200 mu L N, N-bis (3-aminopropyl) methylamine, shaking at 90 ℃ for reaction for 1h, cooling to room temperature, filtering the resin, and adding 20mL of CH2Cl2Washing the resin; the organic phase was concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile-H2Gradient elution of O (1% TFA) for 5min, 10% to 100% acetonitrile-H2Gradient elution with O (1% TFA) for 25min, retention time TRCollecting the product for 16min, and freeze-drying to obtain a light yellow solid for later use;
4mg (3. mu. mol) of the above solid was dissolved in 0.5mL of anhydrous DMF, and the Herstellic acid derivative Ht-2(2.7mg, 6. mu. mol), PyBOP (3.1mg, 6. mu. mol) and DIEA (5. mu.L, 30. mu. mol) were added, reacted with shaking at room temperature for 2h, and purified by semi-preparative HPLC: 10% acetonitrile-H2Gradient elution of O (1% TFA) for 5min, 10% to 100% acetonitrile-H2Gradient elution with O (1% TFA) for 25min, retention time TRCollecting the product for 18min, and freeze-drying to obtain a solid; this solid was dissolved in 1mL CH2Cl21mL TFA was added under ice bath, reacted at 0 ℃ for 1h, 20mL cold ether was added, and the precipitate was collected by centrifugation and purified by semi-preparative HPLC: 10% acetonitrile-H2Gradient elution of O (1% TFA) for 5min, 10% to 100% acetonitrile-H2Gradient elution with O (1% TFA) for 25min, retention time TRThe product was collected for 17min and lyophilized to give compound 3 as a pale yellow solid with HRMS as shown in figure 4.
HRMS (ESI) m/z theoretical calculation C83H95N28O10[M+2H]2+822.3926, found 822.3934.
The structure and the synthetic route of the herceptic acid derivative Ht-2 are as follows:
Ht-2-B (11.4g,44.2mmol) and 4- (5- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) benzene-1, 2-diamine (Ht-2-A, 10g,31.0mmol, prepared as described in Inorg. chem.1998,37, 6018. sup. 6022) were dissolved in acetic acid (100mL) and reacted in an oil bath under heating and refluxing for 4 hours. And (4) distilling off acetic acid under reduced pressure, cooling to room temperature, and purifying by column chromatography to obtain a grass green solid. Dissolving the obtained product in methanol (100ml), magnetically stirring, protecting with nitrogen, cooling in ice water bath, slowly adding dropwise 50ml aqueous solution of 3.1g sodium hydroxide, reacting at room temperature for 8h, detecting by TLC to completely react, evaporating to remove methanol, adjusting pH with dilute hydrochloric acid, precipitating solid, filtering, and drying to obtain yellowish green solid Ht-2, wherein HNMR is shown in FIG. 5.
HNMR(DMSO-d6，400MHz)：2.44(s,3H),2.79(s,2H),3.23(s,2H),6.96(m,1H),7.05(br s,1H),7.47(m,1H),7.69-7.73(m,2H),8.08(m,1H),8.33-8.49(m,2H),8.83(s,1H)，12.73(br s,1H),13.41(br s,1H)。MS(ES):453.2[(M+H)+]。
EXAMPLE 4 preparation of Compound 4
Preparing the peptide represented by the formula (5) supported on phenylhydrazine resin according to the same procedure as in example 1, and removing the Fmoc protecting group from the peptide represented by the formula (5) supported on phenylhydrazine resin according to the procedure (b) in example 1;
the herceptic acid derivative Ht-1(539mg, 1.056mmol) and PyBOP (550mg, 1.056mmol) in example 2 were dissolved in 3mL of anhydrous DMF, DIEA (350. mu.L, 2.112mmol) was added, reaction was carried out for 5min, and the reaction solution was transferred to Fmoc-removed peptide of formula (5) supported on phenylhydrazine resin, N2Bubbling, uniformly mixing, carrying out condensation reaction for 1h, pumping out reaction liquid, and washing the resin with DMF (4X 3 mL); the resin was removed and 1mL of DMF, 200. mu. L N, N-bis (3-aminopropyl) methylamine and 10mg of Cu (OAc) were added2The reaction was shaken at room temperature for 12h, the resin filtered off and charged with 20mL CH2Cl2Washing the resin; the organic phase was concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile-H2Gradient elution of O (1% TFA) for 5min, 10% to 100% acetonitrile-H2Gradient elution with O (1% TFA) for 25min, retention time TRCollecting the product for 17min, and freeze-drying to obtain solid for later use;
3.4mg of the solid obtained above (2. mu. mol) was dissolved in 0.5mL of anhydrous DMF and added with isophthalic acid (3.3mg, 20. mu. mol), PyBOP (10.4mg, 20. mu. mol) and DIEA (50. mu.L, 300. mu. mol), reacted at room temperature with shaking for 2h and purified by semi-preparative HPLC: 10% acetonitrile-H2Gradient elution of O (1% TFA) for 5min, 10% to 100% acetonitrile-H2Gradient elution with O (1% TFA) for 25min, retention time TRCollecting the product after 18.0min, and freeze-drying to obtain a light yellow product, namely compound 4; the HRMS is shown in fig. 6.
HRMS (ESI) m/z theoretical calculation C94H106N28O14[M+2H]2+925.4216, found 925.4209.
EXAMPLE 5 preparation of Compound 5
Preparing the peptide represented by the formula (5) supported on phenylhydrazine resin according to the same procedure as in example 1, and removing the Fmoc protecting group from the peptide represented by the formula (5) supported on phenylhydrazine resin according to the procedure (b) in example 1;
the herceptic acid derivative Ht-1(539mg, 1.056mmol) and PyBOP (550mg, 1.056mmol) in example 2 were dissolved in 3mL of anhydrous DMF, DIEA (350. mu.L, 2.112mmol) was added, reaction was carried out for 5min, and the reaction solution was transferred to Fmoc-removed peptide of formula (5) supported on phenylhydrazine resin, N2Bubbling, uniformly mixing, carrying out condensation reaction for 1h, pumping out reaction liquid, and washing the resin with DMF (4X 3 mL); the resin was removed and 1mL of DMF, 200. mu. L N, N-bis (3-aminopropyl) methylamine and 10mg of Cu (OAc) were added2The reaction was shaken at room temperature for 12h, the resin filtered off and charged with 20mL CH2Cl2Washing the resin; the organic phase was concentrated and the residue was purified by semi-preparative HPLC: 10% acetonitrile-H2Gradient elution of O (1% TFA) for 5min, 10% to 100% acetonitrile-H2Gradient elution with O (1% TFA) for 25min, retention time TRCollecting the product for 17min, and freeze-drying to obtain solid for later use;
3.4mg of the solid obtained above (2. mu. mol) was dissolved in 0.5mL of anhydrous DMF, and the herceptic acid derivatives Ht-2(2.7mg, 6. mu. mol), PyBOP (3.1mg, 6. mu. mol) and DIEA (5. mu.L, 30. mu. mol) from example 3 were added, reacted with shaking at room temperature for 2h, and purified by semi-preparative HPLC: 10% acetonitrile-H2Gradient elution of O (1% TFA) for 5min, 10% to 100% acetonitrile-H2Gradient elution with O (1% TFA) for 25min, retention time TRCollecting the product after 18.5min, and freeze-drying to obtain a light yellow product, namely compound 5; the HRMS is shown in fig. 7.
HRMS (ESI) m/z theoretical calculation C112H124N34O12[M+2H]2+1068.5064, found 1068.5052.
EXAMPLE 6 experiment of Compound 2 penetrating cell and nuclear membranes
Taking Hela cells in logarithmic growth phase, inoculating the Hela cells into a 12-well plate according to 1mL (containing 5 ten thousand cells) per well, and culturing for 24 hours in a 37-degree 5% carbon dioxide cell incubator; absorbing the original culture medium, and adding into DMEM high-sugar culture medium containing compound 2 with different concentrations respectively to make the drug concentration in each well respectively 0 μ M, 2 μ M, 10 μ M, and 50 μ M; the cells were placed in the incubator again and incubated for 24-72 hours, and the localization of the drug in the cells was observed under a fluorescence microscope, and the results are shown in FIG. 8. it can be seen from FIG. 8 that Hela cells were efficiently reached after being treated with Compound 2 at a concentration of 10. mu.M for 24 and 48 hours.
EXAMPLE 7 experiment of Compound 2 for inhibiting the expression of PLK1 protein in Hela cells
Taking Hela cells in logarithmic growth phase, inoculating the Hela cells into a 12-well plate according to 1mL (containing 5 ten thousand cells) per well, and culturing for 24 hours in a 37-degree 5% carbon dioxide cell incubator; absorbing the original culture medium, and adding into DMEM high-sugar culture medium containing compound 2 with different concentrations respectively to make the drug concentration in each well respectively 0 μ M, 2 μ M, 10 μ M, and 50 μ M; putting the cells into the incubator again for incubation for 72 hours; the medium was aspirated, digested with 0.25% trypsin for 1min at room temperature, cells were resuspended in fresh DMEM medium, centrifuged at 3000rpm for 3min, cells were collected and washed twice with ice-cold PBS.
PBS is removed, centrifuged at 5000rpm for 3 minutes, RIPA lysate is added, incubated on ice for 2 hours, centrifuged at 13000rpm for 15 minutes at 4 degrees, the supernatant is collected, the protein concentration is measured, for example, by the braford method, and the corresponding lysate is added to make the concentration of each histone uniform. Preparing 10% SDS-PAGE protein separation gel and 5% SDS-PAGE protein concentration gel; samples were isolated by running the gel at a voltage of 80v for 50. mu.g total protein and then rotated at 200mA for 2 h.
The membrane was soaked in 5% skim milk blocking solution and blocked at room temperature for 1 hour. Add primary anti-PLK 1 (dilution ratio 1: 3000) and GAPDH (1: 20000) and shake slowly at 4 ℃ overnight. Washing with PBST for 3 times, adding secondary HRP-labeled antibody (dilution ratio 1: 3000), and incubating at room temperature for 1 hour; then carrying out color development exposure in a dark room; the obtained results are shown in fig. 9, and it can be seen from fig. 9 that compound 2 can effectively inhibit the expression of Plk1, the inhibition efficiency is in positive correlation with the drug concentration, and actin is taken as an internal reference in Western blot experiments to prove that the loading amount is the same when protein is detected in different samples.
EXAMPLE 8 experiment of Compound 2 for inhibition of Hela cell proliferation
Taking Hela cells in logarithmic growth phase, inoculating the Hela cells into a 6-well plate according to 1mL (containing 10 ten thousand cells) per well, and culturing in a 5% carbon dioxide cell incubator at 37 ℃ for 24 hours; absorbing the original culture medium, and adding into DMEM high-sugar culture medium containing compound 2 with different concentrations respectively to make the drug concentration in each well respectively 0 μ M, 2 μ M, 10 μ M, and 50 μ M; the cells are put into the incubator again for incubation for 48 hours or 72 hours, then the final cell number is counted, the obtained result is shown in figure 10, and the figure 10 shows that the number of Hela cells is obviously reduced along with the increase of the concentration of the drug and the prolonging of the action time, which indicates that the drug can effectively inhibit the proliferation of the tumor cells.
EXAMPLE 9 experiment of Compound 2 for inhibiting proliferation of non-Small cell Lung cancer tumor cells in tumor-bearing mice
Human a549 cells were seeded in 10mm plastic culture dishes using DMEM high glucose in complete medium containing 10% FBS and 10% diabody (chloramphenicol/streptomycin); when the growth density of the A549 cells reaches about 90%, absorbing the culture medium, and digesting with 0.25% trypsin (trypsin) at 37 ℃ for 1 minute; adding 4mL of complete culture medium into each 10mm culture dish, and stopping the trypsin activity; lightly blowing and beating to blow off all adherent cells into suspension cells; centrifuging at 1000rpm for 3min in a centrifuge, removing the supernatant, and resuspending A549 cells with PBS; calculating cell concentration with blood counting plate, adding appropriate amount of PBS, and adjusting cell concentration to 1.0 × 107Per ml
Taking a 1mL injection tube, sucking 1mL of A549 cell suspension, and inoculating 200 ten thousand cells, namely 200 mul of cell suspension, in the right armpit of each nude mouse; after inoculation of a549 cells, nude mice were returned to the cage. The size of the tumor to be treated is about 200mm3And when the side length is 5-8 mm, the administration is started.
Group 1 of administration: a schematic of the dose and frequency of administration is shown in fig. 11; the dosage of the drug is 1mg/Kg body weight, and the converted administration concentration of the compound 2 is about 20 mu mol/mouse; compound 2 was dissolved in physiological saline (0.9% NaCl). Once every three days for a total of 7 doses from the first dose, no dose was given on day 21, and tumor samples were collected after sacrifice of the mice;
control group: control mice were given saline only;
mice body weights and tumor length and width were weighed and recorded every three days prior to dosing. The formula for calculating the tumor volume is V ═ 0.5 × length × width; on day 21 post-dose, nude mice were sacrificed and tumor samples were collected; the tumor proliferation inhibition effect of the compound 2 is shown in fig. 12-14, and as can be seen from fig. 13-14, compared with the control group, the tumor volume and weight of the human A549 lung cancer cell transplanted in vivo inoculated in the nude mice of the administration group are both significantly reduced, which shows that the tumor cell proliferation in the nude mice can be significantly inhibited after the intermittent continuous administration in the mode of fig. 11.
Example 10
The combination position and strength of the DNA sequences of the transcription promoter regions of the compounds 1-5 and PLK1 genes are measured by SPR (surface plasmon resonance (Bio-Rad), California and USA), wherein the transcription promoter region of the PLK1 gene has the following DNA sequences: gggcgctcccatggtgccgcgcggcgGGCGGGtttggattttaaatccccgcggCCAATCAGtggcgcgcaggcttttgtaacgttcccagcGCCGCGtttgaattcggggaggagcgGAGCGGTGCGGAGGCtctgctcggatcgaggtctgcagcgcagcttcgggagcATGAGTGCTGCAGTGACTGCAG
Test results show that the compounds 1-5 can be specifically combined with the gray part in the DNA sequence, and the results of the combination strength of the compounds 2-5 and the DNA sequence are shown in Table 1:
| compound (I) | |
|
|
|
| KD(M) | 2.04×10-9 | 2.24×10-9 | 4.57×10-9 | 1.44×10-8 |
From the above table, it can be seen that the compounds 2-5 have very strong binding with the target DNA, and the affinity reaches nanomolar level, especially the compounds 2-4, wherein the compound 2 has the strongest binding force with the target DNA, and the KD value reaches 2.04 × 10-9。
Claims (13)
1. A compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof:
wherein: the compound represented by the formula (I) has a structure represented by the formulae (II) to (V):
The R is12Is selected from
Or
R10Is C1～3Alkyl or trifluoromethyl;
R11is C1～6Alkyl or R17；
R17Is composed of
Or
At R17In, any CH2Is substituted by 0 to 2C1～6Alkyl, -OH, -NH2Halogen, -OR19、-NHR20or-NR20R21Substituted;
each Y is independently O, NH or NR22，
R13～R15Each independently of the other being H, halogen, C1～3An alkyl group;
R16is C1～6An alkyl group; r18Is hydrogen, halogen, C1～3Alkyl, carboxyl or nitro;
said C is1～6Alkyl or said C1～3The alkyl is substituted by 0-3 halogens;
p' is selected from 0, 1,2, 3 or 4;
q ', r ', s ' are each independently selected from 0, 1,2, 3 or 4; t is selected from 0, 1,2, 3 or 4;
the R is19～R22Each independently being a C1-3 alkyl group.
2. The compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein R16Is methyl, ethyl, n-propyl, isopropyl, monofluoromethyl, difluoromethyl or trifluoromethyl.
3. The compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein R18Is a carboxyl group.
4. The compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein C is1～6Alkyl or said C1～3The alkyl group is substituted with 0 to 3 fluorine groups.
5. The compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein p' is 1,2, 3 or 4.
6. The compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein q ', r ', s ' are all 0.
7. The compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein t is 1.
8. The compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof according to claim 1, wherein R is11Is selected from CH3、CF3、CH2COOH、
Or
9. A compound of general formula (I), a stereoisomer or pharmaceutically acceptable salt thereof, according to claim 1, which comprises:
10. A pharmaceutical composition comprising an effective dose of a compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 9, or further comprising one or more pharmaceutically acceptable carriers or excipients.
11. Use of a compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, or a pharmaceutical composition according to claim 10, for the manufacture of a medicament for the treatment or prevention of a hyperproliferative cell disorder.
12. The use of claim 11, wherein the hyperproliferative cell disorder comprises one or more of colon cancer, rectal cancer, brain tumor, lung cancer, epidermal squamous carcinoma, bladder cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, colorectal cancer, renal cell carcinoma, gastric cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, non-hodgkin's lymphoma, liver cancer, skin cancer, thyroid cancer, head and neck cancer, prostate cancer, glioma, and nasopharyngeal carcinoma.
13. The use according to claim 12, wherein the hyperproliferative disease comprises breast cancer, lung cancer or stomach cancer.
Priority Applications (2)
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| CN201510916228.XA CN106866635B (en) | 2015-12-10 | 2015-12-10 | PLK1 inhibitor and preparation method and application thereof |
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| JP6044935B2 (en) * | 2013-03-11 | 2016-12-14 | 学校法人日本大学 | New PI polyamide |
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| Title |
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| Activity of a Py–Im Polyamide Targeted to the Estrogen Response Element;Nicholas G. Nickols;《Mol Cancer Ther.》;20130226;675-684 * |
| An ab-initio study of pyrrole and imidazole arylamides;ARA M. ABRAMYAN, et al.;《J. Serb. Chem. Soc.》;20131231;1789-1795 * |
| Microwave Assisted Synthesis of Py-Im Polyamides;James W. Puckett,et al.;《Org. Lett.》;20120511;2774-2777 * |
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