WO2017096648A1 - Plk1抑制剂及其制备方法与应用 - Google Patents

Plk1抑制剂及其制备方法与应用 Download PDF

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WO2017096648A1
WO2017096648A1 PCT/CN2015/098880 CN2015098880W WO2017096648A1 WO 2017096648 A1 WO2017096648 A1 WO 2017096648A1 CN 2015098880 W CN2015098880 W CN 2015098880W WO 2017096648 A1 WO2017096648 A1 WO 2017096648A1
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formula
compound
cancer
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粟武
李红昌
房丽晶
刘科
张建超
潘正银
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Shenzhen Institute of Advanced Technology of CAS
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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

Definitions

  • the invention relates to a PLK1 inhibitor, a preparation method and application thereof, and belongs to the field of medicinal chemistry.
  • PLK1 is a member of the Polo-like kinase family and is a highly conserved serine/threonine protein kinase involved in centrosome maturation, spindle formation, and chromosome segregation during cell division, and plays an important role in the regulation of cell mitosis. effect.
  • antisense technology small RNA interference technology or small molecule inhibitors to knock out PLK1 gene expression in tumor cells or inhibit PLK1 activity can cause tumor cell growth inhibition and even apoptosis.
  • antisense oligonucleotides or small interfering RNA can specifically reduce the expression of PLK1, but have no significant effect on normal cells.
  • screening small chemical inhibitors of PLK1 from organically synthesized chemical small molecules or natural products such as ON01910, BI2536, HMN-214, GSK461364, etc., can inhibit PLK1 activity by competitive or non-competitive binding to ATP. These small molecule inhibitors have already entered the clinical research phase.
  • oligonucleotides are easily hydrolyzed by nucleases, and the effective time is short; small RNA interference technology also has problems of safety and stability.
  • both oligonucleotides and small RNAs are difficult to penetrate cell membranes, and the problem of transmembrane transport has been difficult to solve. Therefore, the main research direction at present is to screen chemical small molecule inhibitors of PLK1 from organically synthesized chemical small molecules or natural products.
  • small molecule kinase inhibitors currently successfully marketed for tumor therapy share a common weakness - resistance.
  • One of the objects of the present invention is to provide a novel class of PLK1 inhibitors.
  • Another object of the present invention is to provide a process for the preparation of the PLK1 inhibitor.
  • a further object of the invention is to provide the use of the PLK1 inhibitor or a composition comprising the PLK1 inhibitor.
  • the present invention provides a compound represented by the formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof:
  • R 1 to R 8 are each independently hydrogen or C 1-3 alkyl; preferably, R 1 to R 8 are both methyl
  • B is -(CH 2 ) n -CH 3 , and in B, the hydrogen atom on any CH 2 or CH 3 is substituted by C 1-3 alkyl, -OR 11 or -NR 10 R 11 ;
  • R 10 is a C 1-3 alkyl group
  • R 11 is C 1-6 alkyl or R 17 ;
  • R 9 or R 17 the hydrogen atom on any CH 2 is represented by 0 to 2 C 1-6 alkyl groups, -OH, -NH 2 , halogen, -OR 19 , -NHR 20 or -NR 20 R 21 Replace
  • X or Y are each independently O, NH or NR 22 ,
  • R 13 to R 15 are each independently H, halogen, C 1-3 alkyl
  • R 16 is C 1-6 alkyl; preferably, R 16 is methyl, ethyl, n-propyl, isopropyl, monofluoromethyl, difluoromethyl or trifluoromethyl;
  • R 18 is hydrogen, halogen, C 1-3 alkyl, carboxy or nitro, preferably R 18 is carboxy;
  • the R 19 to R 22 are each independently a C 1-3 alkyl group
  • p, p' are each independently selected from 0, 1, 2, 3 or 4; preferably, p, p' are each independently 1, 2, 3 or 4;
  • q, q', r, r', s, s' are each independently selected from 0, 1, 2, 3 or 4; preferably, q, q', r, r', s, s' are all 0;
  • t is selected from 0, 1, 2, 3 or 4; preferably, t is 1.
  • the compound represented by the formula (I) of the present invention is a DNA sequence in which the pyrrole-imidazole-based polyamide compound can specifically recognize and strongly bind to the PLK1 gene transcription promoter region shown in SEQ ID NO: 1, and inhibit PLK1. Transcription of the gene inhibits the expression of PLK1 protein, resulting in tumor cell growth inhibition or apoptosis.
  • the compound of the formula (I) of the present invention is capable of specifically transfecting the PLK1 gene transcription promoter sequence through the cell membrane and the nuclear membrane, and belongs to the class of polyamide compounds.
  • the pyrrole-imidazole-based polyamide represented by the general formula (I) of the present invention directly acts on the sequence of the PLK1 gene transcription promoter region, and is different from the small molecule inhibitor acting on the kinase protein molecule, thereby overcoming the small molecule kinase inhibition. Drug resistance problem.
  • the compound represented by the formula (I) a stereoisomer or a pharmaceutically acceptable salt thereof, the compound has a structure represented by the formula (II) to the formula (V). :
  • R 10 is methyl or trifluoromethyl
  • said R 12 is selected from the group consisting of NH 2 ,
  • said R 11 is selected from the group consisting of CH 3 , CF 3 , CH 2 COOH,
  • the compound of the formula (I) a stereoisomer or a pharmaceutically acceptable salt thereof according to the invention, the compound comprises:
  • the C 1-3 alkyl group of the present invention includes a methyl group, an ethyl group, a n-propyl group or an isopropyl group; and the C 1-6 alkyl group represents a linear or linear alkyl group having 1 to 6 carbon atoms.
  • the C 1-6 alkyl group represents a linear or linear alkyl group having 1 to 6 carbon atoms.
  • the C 1 ⁇ 6 alkyl or C 1 ⁇ 3 of the present invention is alkyl substituted with 0 to 3 halogen atoms as described above represents an alkyl group of C 1 ⁇ 3 alkyl or C 1 ⁇ 6 ⁇ 0 is hydrogen Three halogen atoms are substituted, for example, -CH 2 F, -CHF 2 , -CF 3 , -CH 2 FCH 3 , -CHF 2 CH 3 , -CF 2 CH 2 CH 3 , and the like.
  • the present invention provides a process for the preparation of the compound of the above formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein the method comprises the steps of:
  • A' is -(CH 2 ) m -, in A, the hydrogen atom on any CH 2 is replaced by -OP OH , -NHP NH ;
  • the A' is The R 12 is -OP OH or -NHP OH ; more preferably, A' is
  • the cleavage agent is NH 2 -(CH 2 ) n -CH 3 , and in the cleavage agent, the hydrogen atom on any CH 2 or CH 3 is substituted by -OR 11 or -NR 10 R 11 ;
  • the cutting agent is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the oxidizing agent is a combination of one or more of copper acetate, N-bromosuccinimide and oxygen;
  • each group in A' and the cleavage agent is the same as the present invention, and P OH and P NH respectively represent a hydroxy protecting group and an amino protecting group; preferably, the P OH and P NH are Boc, Fmoc, Cbz, Trt or Allyl, more preferably Fmoc.
  • step (c) in the method is replaced by step (c'), and further comprising step (c") after step (c');
  • R 24 is a hydroxyl group or a halogen
  • step (b) and step (c') the hydroxyl or amino group from which P OH or P NH is removed is protected with P OH ' or P NH ', said P OH ' or P NH 'is a hydroxyl protecting group or an amino protecting group which can withstand basic conditions; and after removing the P OH ' or P NH ' after the condensation reaction in the step (c"), the method of the formula (I) is obtained.
  • a compound preferably, the P OH ' or P NH ' is a tert-butoxycarbonyl group.
  • the formula (VI) is a formula (VI-1);
  • formula (VI-1) is prepared as follows:
  • the compound represented by the formula (2) is subjected to a condensation reaction with 4-tert-butoxycarbonylamino-1-methyl-1H-pyrrole-2-carboxylic acid after removing the tert-butoxycarbonyl protecting group, and repeating the de-tert-butylation
  • the compound represented by the formula (3) is obtained by an oxycarbonyl protecting group and a condensation reaction:
  • a compound represented by the formula (VI) is obtained by a condensation reaction of a compound represented by the formula (4) with 1-methyl-1H-imidazole-2-carboxylic acid.
  • the conditions of the condensation reaction and the de-tert-butoxycarbonyl protecting group in the above route are not limited in the present invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a compound of the formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, according to the present invention, or further comprising one or a variety of pharmaceutically acceptable carriers or Shape agent.
  • the present invention provides a compound represented by the above formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for the preparation of a medicament for treating or preventing a cell hyperproliferative disorder Application in medicine.
  • the cell hyperproliferative diseases include colon cancer, rectal cancer, brain tumor, lung cancer, epidermal squamous cell carcinoma, bladder cancer, pancreatic cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, colorectal cancer.
  • the present invention provides a novel PLK1 inhibitor and a preparation method and application.
  • the PLK1 inhibitor can specifically bind to the DNA sequence in the transcription promoter region of the PLK1 gene represented by SEQ:NO:1, inhibit the transcription of the PLK1 gene, inhibit the expression of PLK1 protein, and cause tumor cell growth inhibition or withering. Die. And it is able to cross cell membranes and nuclear membranes and resist nuclease hydrolysis. In addition, it overcomes the resistance problem of small molecule kinase inhibitors.
  • PLK1 Polo-Like Kinase1, Paul-like kinase 1; BOC: t-butyloxycarbonyl, tert-butoxycarbonyl; Fmoc: fluorenylmethyloxycarbonyl, fluorenylmethoxycarbonyl; HOAt: 1-hydroxy-7-azobenzotriazole; DMF: N,N-dimethylformamide; THF: tetrahydrofuran; DIEA: N,N-diisopropylethylamine; TFA: trifluoroacetic acid; PyBOP: benzotriazol-1-yloxy-hexafluorophosphate Pyrrolidinyl phosphorus.
  • Figure 1 is a synthetic route diagram of compounds 1 to 5;
  • FIG. 1 is a HRMS spectrum of Compound 1
  • FIG. 3 is a HRMS spectrum of Compound 2
  • FIG. 4 is a HRMS spectrum of Compound 3
  • Figure 5 is a HNMS spectrum of the Hurst acid derivative Ht-2
  • FIG. 6 is a HRMS spectrum of Compound 4.
  • Figure 8 is a graph showing the results of the experiment of the compound 2 penetrating the cell membrane and the nuclear membrane;
  • Figure 9 is a graph showing the results of inhibition of PLK1 protein expression in Hela cells by Compound 2;
  • Figure 10 is a graph showing the results of an experiment in which Compound 2 inhibits Hela cell proliferation
  • Figure 11 is a schematic view showing the dose and frequency of administration of the administration group of Example 9;
  • 1 is a synthetic route diagram of the compounds 1 to 5, which are prepared from a Fmoc-protected benzoquinone resin as a raw material, and the formula (1) is obtained in multiple steps, and then the formula (2) to the formula (6) are sequentially produced, and finally, according to the formula Compounds 5 to 5 were obtained by (5) or (6).
  • step (b) Removal of Fmoc protecting group: 3 mL of 20% piperidine/DMF solution was added to the resin after the swelling of step (a), and N 2 was bubbled and mixed. After 10 minutes, the solvent was removed, and then 3 mL of 20% piperidine was added. /DMF solution, N 2 was bubbled and mixed, after 10 min, the resin was washed with DMF (4 ⁇ 3 mL), and the resin was washed with 3 mL of anhydrous DMF, and set aside;
  • the resin was taken out, and 1 mL of DMF, 200 ⁇ L of dimethylaminopropylamine and 10 mg of Cu (OAc) were added.
  • the peptide supported on the phenylhydrazine resin represented by the formula (5) was obtained in the same manner as in Example 1, and the benzoquinone resin represented by the formula (5) was removed by the step (b) in Example 1. Fmoc protecting group in the peptide;
  • the Hurst acid derivative Ht-1 (539 mg, 1.056 mmol) and PyBOP (550 mg, 1.056 mmol) were dissolved in 3 mL of anhydrous DMF, DIEA (350 ⁇ L, 2.112 mmol) was added, and the reaction was carried out for 5 min. Except for the peptide loaded on the phenylhydrazine resin represented by formula (5) of Fmoc, N 2 was bubbled and mixed, and the condensation reaction was carried out for 1 hour.
  • Hurst acid derivative Ht-1 is as follows (for preparation, see J.AM. CHEM. SOC. 2004, 126, 3736-3747):
  • the peptide supported on the phenylhydrazine resin represented by the formula (5) was obtained in the same manner as in Example 1, and the benzoquinone resin represented by the formula (5) was removed by the step (b) in Example 1. Fmoc protecting group in the peptide;
  • Boc 2 O (243 ⁇ L, 1.056 mmol) was dissolved in 3 mL of anhydrous DMF, DIEA (350 ⁇ L, 2.112 mmol) was added, and the reaction solution was transferred to the phenylhydrazine resin supported by the formula (5) for removing Fmoc.
  • N 2 was bubbled and mixed, and the condensation reaction was carried out for 20 min.
  • the reaction solution was removed, and the resin was washed with DMF (4 ⁇ 3 mL) to obtain a Boc-protected peptide supported on the phenylhydrazine resin represented by the formula (6);
  • the resin obtained above was taken out, and 1 mL of DMF and 200 ⁇ L of N,N-bis(3-aminopropyl)methylamine were added thereto, and the mixture was shaken at 90 ° C for 1 hour, cooled to room temperature, and the resin was filtered off, and the resin was washed with 20 mL of CH 2 Cl 2 .
  • Ht-2-B (11.4 g, 44.2 mmol) and 4-(5-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)benzene-1,2
  • the diamine (Ht-2-A, 10 g, 31.0 mmol, prepared by Inorg. Chem. 1998, 37, 6018-6022) was dissolved in acetic acid (100 mL) and heated under reflux in an oil bath for 4 hours.
  • the acetic acid was evaporated under reduced pressure, cooled to room temperature and purified by column chromatography to yield The obtained product was dissolved in methanol (100 ml), magnetically stirred, nitrogen-protected, cooled in an ice water bath, and slowly added dropwise a solution of 3.1 g of sodium hydroxide in 50 ml of water, dropwise, and reacted at room temperature for 8 h. The reaction was complete by TLC, and methanol was evaporated. The pH was adjusted to be acidic, and the solid was precipitated, filtered, and dried to give a yellow-green solid Ht-2.
  • the peptide supported on the phenylhydrazine resin represented by the formula (5) was obtained in the same manner as in Example 1, and the benzoquinone resin represented by the formula (5) was removed by the step (b) in Example 1. Fmoc protecting group in the peptide;
  • the Hurst acid derivative Ht-1 (539 mg, 1.056 mmol) and PyBOP (550 mg, 1.056 mmol) in Example 2 were dissolved in 3 mL of anhydrous DMF, and DIEA (350 ⁇ L, 2.112 mmol) was added and reacted for 5 min.
  • the reaction solution was transferred to a peptide loaded on phenylhydrazine resin represented by formula (5) to remove Fmoc, N 2 was bubbled and mixed, condensation reaction was carried out for 1 hour, the reaction solution was removed, and the resin was washed with DMF (4 ⁇ 3 mL).
  • the peptide supported on the phenylhydrazine resin represented by the formula (5) was obtained in the same manner as in Example 1, and the benzoquinone resin represented by the formula (5) was removed by the step (b) in Example 1. Fmoc protecting group in the peptide;
  • the Hurst acid derivative Ht-1 (539 mg, 1.056 mmol) and PyBOP (550 mg, 1.056 mmol) in Example 2 were dissolved in 3 mL of anhydrous DMF, and DIEA (350 ⁇ L, 2.112 mmol) was added and reacted for 5 min.
  • the reaction solution was transferred to a peptide loaded on phenylhydrazine resin represented by formula (5) to remove Fmoc, N 2 was bubbled and mixed, condensation reaction was carried out for 1 hour, the reaction solution was removed, and the resin was washed with DMF (4 ⁇ 3 mL).
  • Hela cells in logarithmic growth phase were inoculated into 12-well plates at 1 mL per well (containing 50,000 cells), placed in a 37-degree, 5% carbon dioxide cell incubator for 24 hours; the original medium was aspirated.
  • Example 7 Compound 2 inhibits PLK1 protein expression in Hela cells
  • Hela cells in logarithmic growth phase were inoculated into 12-well plates at 1 mL per well (containing 50,000 cells), placed in a 37-degree, 5% carbon dioxide cell incubator for 24 hours; the original medium was aspirated.
  • the % trypsin was digested at room temperature for 1 min, the cells were resuspended in fresh DMEM medium, centrifuged at 3000 rpm for 3 minutes, and the cells were collected and washed twice with ice-cold PBS.
  • the PBS was removed, centrifuged at 5000 rpm for 3 minutes, RIPA lysate was added, incubated on ice for 2 hours, centrifuged at 13,000 rpm for 15 min, and the supernatant was collected.
  • the protein concentration was measured by, for example, the braford method, and the corresponding lysate was added to make the concentrations of the respective groups uniform.
  • 10% SDS-PAGE protein separation gel and 5% SDS-PAGE protein concentrate were prepared; 50 ⁇ g total protein 80v voltage was used to separate the sample, and 200mA constant current was transferred for 2h.
  • the membrane was immersed in a 5% skim milk blocking solution and blocked at room temperature for 1 hour. Add primary antibody PLK1 (dilution ratio 1:3000) and GAPDH (1:20000), shake slowly overnight at 4 degrees. Wash 3 times with PBST, add HRP-labeled secondary antibody (dilution ratio 1:3000), incubate for 1 hour at room temperature; then perform color development exposure in dark room; the results are shown in Figure 9, and compound 2 can be seen from Figure 9. It can effectively inhibit the expression of Plk1, and the inhibition efficiency is positively correlated with the drug concentration, and actin is used as an internal reference in the Western blotting experiment to prove that the protein loading in different samples is the same.
  • HeLa cells in logarithmic growth phase were inoculated into 6-well plates at 1 mL per well (containing 100,000 cells), placed in a 5% carbon dioxide cell incubator at 37 ° C for 24 hours; the original medium was aspirated.
  • the number of cells the results obtained as shown in Figure 10, can be seen from Figure 10 with the increase in drug concentration and prolonged action time, the number of Hela cells decreased significantly, indicating that the drug can effectively inhibit the proliferation of tumor cells.
  • Example 9 Compound 2 inhibits proliferation of non-small cell lung cancer tumor cells in tumor-bearing mice
  • Human A549 cells were seeded in 10 mm plastic culture dishes.
  • the complete medium was DMEM high glucose containing 10% FBS and 10% double antibody (chloramphenicol/streptomycin); the growth density of A549 cells was about 90%.
  • the medium was aspirated, digested with 0.25% trypsin at 37 ° C for 1 minute; 4 mL of complete medium was added to each 10 mm culture dish to stop trypsin activity; lightly blown, all the adherent cells were blown into suspension cells.
  • a 1 mL syringe was taken, 1 mL of A549 cell suspension was aspirated, and 2 million cells, 200 ⁇ l of cell suspension, were inoculated on the right side of each nude mouse. After inoculation of A549 cells, the nude mice were returned to the cage. When the tumor size is as long as about 200 mm 3 and the side length is 5 to 8 mm, administration is started.
  • Administration group 1 The dosage and frequency of administration are shown in Fig. 11; the dose of the drug is 1 mg/Kg body weight, and the concentration of the compound 2 after administration is about 20 ⁇ mol/mouse; the compound 2 is dissolved in physiological saline. (0.9% NaCl). From the first administration, the drug was given once every three days for a total of 7 times, and was not administered on the 21st day. The tumor samples were collected after the mice were sacrificed;
  • Control group control mice were given only normal saline;
  • the body weight of the mice and the length and width of the tumor were weighed and recorded every three days before administration.
  • SPR surface plasmon resonance analysis using a Proteon XPR36 (Bio-Rad, California, USA) was used to determine the binding position and intensity of the DNA sequence of the transcription promoter region of the compound 1-5 and the PLK1 gene, wherein the transcription initiation of the PLK1 gene was performed.
  • the subregion has the following DNA sequence:
  • the binding of the compounds 2 to 5 to the target DNA is very strong, and the affinity reaches the nanomolar level, especially the compounds 2 to 4, wherein the compound 2 has the strongest binding ability to the target DNA, and the KD value reaches 2.04 ⁇ 10 -9 .

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Abstract

本发明提供PLK1抑制剂及其制备方法与应用，所述PLK1抑制剂具有通式(I)所示的化合物、其立体异构体或药学上可接受的盐，式(I)中的各基团的定义与说明书相同。本发明所述PLK1抑制剂能够特异性高强度的结合于SEQ:NO:1所示的PLK1基因转录启动子区域中的DNA序列，抑制PLK1基因的转录，抑制PLK1蛋白的表达，造成肿瘤细胞生长抑制或凋亡，并且其能够穿过细胞膜和核膜及抵抗核酸酶水解，此外，其还克服了小分子激酶抑制剂的耐药性难题。

Description

PLK1抑制剂及其制备方法与应用 技术领域
本发明涉及PLK1抑制剂及其制备方法与应用，属于药物化学领域。
背景技术
PLK1是Polo-类激酶家族成员，是一类高度保守的丝氨酸/苏氨酸蛋白激酶，参与细胞分裂过程中的中心体成熟、纺锤体形成以及染色体分离等过程，对细胞有丝分裂过程起重要的调控作用。研究发现，PLK1在肺癌、乳腺癌及胃癌等多种恶性肿瘤中异常高表达，其过表达也是肿瘤不良预后的标志之一，但在正常细胞中其表达水平很低，有时甚至无法测出。因此，PLK1在肿瘤诊断和治疗中是广受关注的靶点。
应用反义技术、小分子RNA干扰技术或小分子抑制剂敲除肿瘤细胞中表达PLK1的基因或抑制PLK1的活性，能造成肿瘤细胞生长抑制甚至凋亡。已有研究证明，反义寡核苷酸或小分子干扰RNA，能特异性地降低PLK1的表达，但对正常细胞没有明显影响。也有研究报道从有机合成的化学小分子或天然产物中筛选PLK1的化学小分子抑制剂，如ON01910、BI2536、HMN-214、GSK461364等，可通过与ATP竞争性或非竞争性结合来抑制PLK1活性，这些小分子抑制剂部分已进入临床研究阶段。
然而，反义寡核苷酸易被核酸酶水解，有效作用时间短；小分子RNA干扰技术也存在安全性、稳定性的问题。此外，寡核苷酸以及小分子RNA都难以穿透细胞膜，跨膜运输的问题一直难以解决。因此，目前主要的研究方向是从有机合成的化学小分子或天然产物中筛选PLK1的化学小分子抑制剂。然而，目前成功上市用于肿瘤治疗的小分子激酶抑制剂都存在一个共同的弱点—产生耐药性。由于肿瘤细胞能够在治疗过程中很快产生突变，从而使激酶对小分子抑制剂的结合力下降，使其对小分子抑制剂的治疗不敏感，因而产生耐药性，PLK1的化学小分子抑制剂具有同样的耐药性问题。
因此，开发新型的PLK1抑制剂是本领域亟需解决的技术问题之一。
发明内容
本发明的目的之一在于提供一类新型的PLK1抑制剂。
本发明的另一目的在于提供所述PLK1抑制剂的制备方法。
本发明的再一目的在于提供含有所述PLK1抑制剂的组合物。
本发明的再一目的在于提供所述PLK1抑制剂或含有所述PLK1抑制剂的组合物的应用。
为实现上述目的，一方面，本发明提供通式(I)所示的化合物、其立体异构体或药学上可接受的盐：
Figure PCTCN2015098880-appb-000001
其中：
R1～R8各自独立地为氢或C1～3烷基；优选地，R1～R8均为甲基
A为-(CH2)m-，在A中，任意CH2上的氢原子被C1～3烷基、-OH、-NH2、-O-(C＝O)-R9或-NH-(C＝O)-R9所取代；
B为-(CH2)n-CH3，在B中，任意CH2或CH3上的氢原子被C1～3烷基、-OR11或-NR10R11所取代；
优选地，所述A为
Figure PCTCN2015098880-appb-000002
所述R12选自-OH、-NH2、-O-(C＝O)-R9或-NH-(C＝O)-R9；更优选地，A为
Figure PCTCN2015098880-appb-000003
优选地，所述B为
Figure PCTCN2015098880-appb-000004
R9为
Figure PCTCN2015098880-appb-000005
R10为C1～3烷基；
R11为C1～6烷基或R17；
R17为
Figure PCTCN2015098880-appb-000006
Figure PCTCN2015098880-appb-000007
在R9或R17中，任意CH2上的氢原子被0～2个C1～6烷基、-OH、-NH2、卤素、-OR19、-NHR20或-NR20R21所取代；
X或Y各自独立地为O、NH或NR22，
R13～R15各自独立地为H、卤素、C1～3烷基；
R16为C1～6烷基；优选地，R16为甲基、乙基、正丙基、异丙基、一氟甲基、二氟甲基或三氟甲基；
R18为氢、卤素、C1～3烷基、羧基或硝基，优选地，R18为羧基；
所述R19～R22各自独立地为C1～3烷基；
所述C1～6烷基或所述C1～3烷基被0～3个卤素取代，优选被0～3个氟取代；
p、p’各自独立地选自0、1、2、3或4；优选地，p、p’各自独立地为1、2、3或4；
q、q’、r、r’、s、s’各自独立地选自0、1、2、3或4；优选地，q、q’、r、r’、s、s’均为0；
t选自0、1、2、3或4；优选地，t为1。
本发明通式(I)所示的化合物为吡咯-咪唑类聚酰胺化合物能够特异性识别且高强度的结合于SEQ IN NO:1所示的PLK1基因转录启动子区域中的DNA序列，抑制PLK1基因的转录，抑制PLK1蛋白的表达，造成肿瘤细胞生长抑制或凋亡。本发明通式(I)所示的化合物能够穿过细胞膜和核膜，特异性地识别PLK1基因转录启动子序列，并且其属于聚酰胺类化合物，现有技术(Single-dose pharmacokinetic and toxicity analysis of pyrrole-imidazole polyamides in mice,Synold,Timothy W.；Xi,Bixin；Wu,Jun；et al,CANCER CHEMOTHERAPY AND PHARMACOLOGY Volume:70 Issue:4 Pages:617-625,Published:OCT 2012)表明这类结构能够抵抗核酸酶水解，因此本发明通式(I)所示的化合物还能够抵抗核酸酶的水解。因此，本发明通式(I)所示的吡咯-咪唑类聚酰胺化合物克服了寡核苷酸以及小分子RNA有效作用时间短和跨膜运输的问题。此外，本发明通式(I)所示的吡咯-咪唑类聚酰胺直接作用于PLK1基因转录启动子区域中的序列，不同于小分子抑制剂作用于激酶蛋白分子，因此克服了小分子激酶抑制剂的耐药性难题。
优选地，在本发明所述的通式(I)所示的化合物、其立体异构体或药学上可接受的盐中，所述化合物具有式(II)～式(V)所示的结构：
Figure PCTCN2015098880-appb-000008
Figure PCTCN2015098880-appb-000009
优选地，R10为甲基或三氟甲基；
优选地，所述R12选自NH2、
Figure PCTCN2015098880-appb-000010
Figure PCTCN2015098880-appb-000011
优选地，所述R11选自CH3、CF3、CH2COOH、
Figure PCTCN2015098880-appb-000012
Figure PCTCN2015098880-appb-000013
进一步优选地，在本发明所述的通式(I)所示的化合物、其立体异构体或药学上可接受的盐中，所述化合物包括：
Figure PCTCN2015098880-appb-000014
Figure PCTCN2015098880-appb-000015
Figure PCTCN2015098880-appb-000016
本发明所述C1～3烷基包括甲基、乙基、正丙基或异丙基；所述C1～6烷基表示碳原子数为1～6的直链或直链烷基，包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、仲戊基、正己基或异己基等。
本发明所述C1～6烷基或所述C1～3烷基被0～3个卤素取代表示如上所述的C1～3烷基或C1～6烷基中的氢被0～3个卤素原子取代，例如-CH2F、-CHF2、-CF3、-CH2FCH3、-CHF2CH3、-CF2CH2CH3等。
另一方面，本发明提供所述的通式(I)所示的化合物、其立体异构体或药学上可接受的盐的制备方法，其中，所述方法包括如下步骤：
(a)制备苯肼树脂负载的式(IV)树脂；
式(IV)
A’为-(CH2)m-，在A中，任意CH2上的氢原子被-OPOH、-NHPNH所取代；
优选地，所述A’为
Figure PCTCN2015098880-appb-000017
所述R12为-OPOH或-NHPOH；更优选地，A’为
Figure PCTCN2015098880-appb-000018
Figure PCTCN2015098880-appb-000019
(b)脱除式(IV)中的POH或PNH；
(c)以氧化剂及切割剂进行氧化反应及切割反应以制得式(I)所示的化合物；
所述切割剂为NH2-(CH2)n-CH3，在切割剂中，任意CH2或CH3上的氢原子被-OR11或-NR10R11所取代；
优选地，所述切割剂为
Figure PCTCN2015098880-appb-000020
优选地，所述氧化剂为醋酸铜、N-溴代丁二酰亚胺和氧气中的一种或多种的组合；
A’及切割剂中各基团的定义与本发明相同，POH、PNH分别表示羟基保护基及氨基保护基；优选地，所述POH、PNH为Boc、Fmoc、Cbz、Trt或Allyl，更优选Fmoc。
根据本发明的具体实施方案，在本发明方法中，在步骤(b)与步骤(c)之间以
Figure PCTCN2015098880-appb-000021
对脱除POH或PNH暴露的羟基或氨基进行缩合反应以形成-O-(C＝O)-R9或-NH-(C＝O)-R9的步骤；所述R23为羟基或卤素。
根据本发明的具体实施方案，在本发明方法中，所述方法中的步骤(c)被步骤(c’)所替代，并且在步骤(c’)之后进一步包括步骤(c”)；
(c’)以氧化剂及切割剂进行氧化反应及切割反应；所述切割剂为 NH2-(CH2)n-CH2N(R10)-(CH2)p’-YH，
(c”)以
Figure PCTCN2015098880-appb-000022
对步骤(c’)所得产物中的YH进行缩合反应后以制得式(I)所示的所述的化合物；R24为羟基或卤素；
选择性地，在步骤(b)与步骤(c’)之间，将脱除POH或PNH暴露的羟基或氨基以POH’或PNH’进行保护，所述POH’或PNH’为可耐受碱性条件的羟基保护基或氨基保护基；并在步骤(c”)进行缩合反应之后脱除POH’或PNH’以制得式(I)所示的所述的化合物；优选地，所述POH’或PNH’为叔丁氧羰基。
根据本发明的具体实施方案，在本发明方法中，所述式(VI)为式(VI-1)；
Figure PCTCN2015098880-appb-000023
优选地，式(VI-1)按如下路线制备：
(1)以苯肼树脂以及4-叔丁氧羰基氨基-1-甲基-1H-吡咯-2-羧酸为原料经重复缩合及脱叔丁氧羰基保护基制得式(1)所示的化合物：
Figure PCTCN2015098880-appb-000024
(2)以式(1)所示的化合物与R-2-(9-芴甲氧羰基氨基)-4-叔丁氧羰基氨基丁酸进行缩合反应制得式(2)所示的化合物；
Figure PCTCN2015098880-appb-000025
(3)式(2)所示的化合物脱除叔丁氧羰基保护基后与4-叔丁氧羰基氨基-1-甲基-1H-吡咯-2-羧酸进行缩合反应，重复脱叔丁氧羰基保护基及缩合反应制得式(3)所示的化合物：
Figure PCTCN2015098880-appb-000026
(4)以式(3)所示的化合物与4-叔丁氧羰基氨基-1-甲基-1H-咪唑-2-羧酸进行缩合反应并脱除叔丁氧羰基保护基得式(4)所示的化合物：
Figure PCTCN2015098880-appb-000027
(5)以式(4)所示的化合物与1-甲基-1H-咪唑-2-羧酸进行缩合反应制得式(VI-1)所示的化合物。
本发明对上述路线中缩合反应以及脱叔丁氧羰基保护基反应的条件不作限定。
再一方面，本发明提供药物组合物，其包含有效剂量的本发明所述的通式(I)所示的化合物、其立体异构体或药学上可接受的盐，或进一步包含一种或多种药学上可接受的载体或赋 形剂。
再一方面，本发明提供所述的通式(I)所示的化合物、其立体异构体或药学上可接受的盐或所述的药物组合物在制备治疗或预防细胞过度增殖性疾病的药物中的应用。
优选地，所述的细胞过度增殖性疾病包括结肠癌、直肠癌、脑瘤、肺癌、表皮鳞癌、膀胱癌、胰腺癌、乳腺癌、卵巢癌、宫颈癌、子宫内膜癌、结直肠癌、肾细胞癌、胃癌、食管腺癌、食管鳞状细胞癌、非霍奇金淋巴瘤、肝癌、皮肤癌、甲状腺癌、头颈癌、前列腺癌、神经胶质瘤及鼻咽癌中的一种或多种；更优选地，所述的细胞过度增殖性疾病包括乳腺癌、肺癌或胃癌。
综上所述，本发明提供了一种新型的PLK1抑制剂以及制备方法与应用。该PLK1抑制剂能够特异性高强度的结合于SEQ:NO:1所示的PLK1基因转录启动子区域中的DNA序列，抑制PLK1基因的转录，抑制PLK1蛋白的表达，造成肿瘤细胞生长抑制或凋亡。并且其能够穿过细胞膜和核膜及抵抗核酸酶水解。此外，其还克服了小分子激酶抑制剂的耐药性难题。
本发明中的缩写具有如下意义：
PLK1:Polo-Like Kinase1,保罗样激酶1；BOC:t-butyloxycarbonyl，叔丁氧羰基；Fmoc:fluorenylmethyloxycarbonyl，芴甲氧羰基；HOAt:1-羟基-7-偶氮苯并三氮唑；DMF:N,N-二甲基甲酰胺；THF:四氢呋喃；DIEA:N,N-二异丙基乙胺；TFA:三氟乙酸；PyBOP:六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷。
附图说明
图1为化合物1～5的合成路线图；
图2为化合物1的HRMS图谱；
图3为化合物2的HRMS图谱；
图4为化合物3的HRMS图谱；
图5为赫斯特酸衍生物Ht-2的HNMS图谱；
图6为化合物4的HRMS图谱；
图7为化合物5的HRMS图谱；
图8为化合物2穿透细胞膜与核膜的实验结果图；
图9为化合物2抑制Hela细胞PLK1蛋白表达实验结果图；
图10为化合物2抑制Hela细胞增殖的实验结果图；
图11为实施例9给药组的给药剂量和频率示意图；
图12～图14为实施例9化合物2抑制非小细胞肺癌肿瘤细胞在荷瘤小鼠中的增殖实验结果图。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例及附图对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。
图1为化合物1～5的合成路线图，其由Fmoc保护的苯肼树脂为原料，经多步制得式(1)，随后依次制得式(2)～式(6)，最后根据式(5)或式(6)制得化合物1～5。
实施例1 化合物1的制备
(a)树脂溶胀：于一个10mL的固相反应器中加入400mg Fmoc保护的苯肼树脂(0.66mmol/g，0.264mmol)及3mL CH2Cl2，将树脂溶胀30min，抽除CH2Cl2，备用；
(b)脱除Fmoc保护基：将3mL 20％哌啶/DMF溶液加入步骤(a)溶胀后的树脂中，N2鼓泡混匀，10min后，抽除溶剂，再加入3mL 20％哌啶/DMF溶液，N2鼓泡混匀，10min后，用DMF(4×3mL)洗涤树脂，再用3mL无水DMF洗涤树脂，备用；
(c)氨基酸缩合：将4-叔丁氧羰基氨基-1-甲基-1H-吡咯-2-羧酸(254mg，1.056mmol)和三光气(BTC，128mg，0.433mmol)溶于2mL无水THF，向该溶液中缓慢滴加三甲基吡啶(collidine，488μL，3.696mmol)，反应立即产生大量白色沉淀，加完反应3min，再加入2mLDIEA/DMF溶液(5％,v/v)，白色沉淀完全消失，将该反应液转移到步骤(b)脱除保护基的苯肼树脂中，N2鼓泡混匀，缩合反应0.5～1h，抽除反应液，用DMF(4×3mL)洗涤树脂，备用；
(d)脱除叔丁氧羰基保护基：用CH2Cl2(2×3mL)洗涤，抽除CH2Cl2，加入3.0mL TFA/苯酚/H2O(v:v:v＝92:5:2.5)混合溶液脱除步骤(b)所得缩合产物上的叔丁氧羰基保护基，2min后抽除溶剂，再次加入3.0mL TFA/苯酚/H2O(v:v:v＝92:5:2.5)混合溶液反应20min，用CH2Cl2(2×3mL)及DMF(4×3mL)洗涤树脂，再用3mL无水DMF洗涤树脂，备用；
重复上述缩合及脱保护步骤(c)和(d)，直至完成式(1)所示的负载在苯肼树脂上的肽的合成；
Figure PCTCN2015098880-appb-000028
(e)γ-氨基酸的缩合：将R-2-(9-芴甲氧羰基氨基)-4-叔丁氧羰基氨基丁酸(465mg，1.056mmol)和三光气(128mg，0.433mmol)溶于2mL无水THF，向该溶液中缓慢滴加三甲基吡啶(488μL，3.696mmol)，反应立即产生大量白色沉淀，加完反应1min，加入HOAt(144mg，1.056mmol)，再加入2mL DIEA/DMF溶液(5％,v/v)，反应5min，白色沉淀完全消失，将该反 应液转移到式(1)所示的负载在苯肼树脂上的直链肽(NH2-Py-Py-Py-Py-苯肼树脂)中，N2鼓泡混匀，缩合反应0.5～1h，抽除反应液，用DMF(4×3mL)洗涤树脂，得到式(2)所示的负载在苯肼树脂上的肽；
Figure PCTCN2015098880-appb-000029
(f)重复脱保护及缩合护步骤(d)和(c)，其中，直至完成得到式(3)所示的负载在苯肼树脂上的肽的合成；
Figure PCTCN2015098880-appb-000030
(g)氨基酸缩合：将4-叔丁氧羰基氨基-1-甲基-1H-咪唑-2-羧酸(255mg，1.056mmol)和三光气(128mg，0.433mmol)溶于1mL无水THF，向该溶液中缓慢滴加三甲基吡啶(488μL，3.696mmol)，反应立即产生大量白色沉淀，加完反应3min，加入HOAt(144mg，1.056mmol)，再加入2mL DIEA/DMF溶液(5％,v/v)，白色沉淀完全消失，将该反应液转移到步骤(f)所得式(3)所示的负载在苯肼树脂上的肽中，N2鼓泡混匀，缩合反应0.5～1h，抽除反应液，用DMF(4×3mL)洗涤树脂，备用；
(h)脱除叔丁氧羰基保护基：用CH2Cl2(2×3mL)洗涤，抽除CH2Cl2，加入3.0mL TFA/苯酚/H2O(v:v:v＝92:5:2.5)混合溶液脱除步骤(g)所得产物上的叔丁氧羰基保护基，2min后抽除溶剂，再次加入3.0mL TFA/苯酚/H2O(v:v:v＝92:5:2.5)混合溶液反应20min，用CH2Cl2(2×3mL)及DMF(4×3mL)洗涤树脂，再用3mL无水DMF洗涤树脂，得式(4)所示的负载在苯肼树脂上的肽；
Figure PCTCN2015098880-appb-000031
(i)末端氨基酸的缩合：将1-甲基-1H-咪唑-2-羧酸(132mg，1.056mmol)和PyBOP(550mg，1.056mmol)溶于3mL无水DMF，加入DIEA(350μL，2.112mmol)，反应5min，将该反应液转移到步骤(h)所得的式(4)所示的负载在苯肼树脂上的肽中，N2鼓泡混匀，缩合反应2h，抽除反应液，用DMF(4×3mL)洗涤树脂，得到式(5)所示的负载在苯肼树脂上的肽；
Figure PCTCN2015098880-appb-000032
(j)化合物1的合成
采用步骤(b)中的方法脱除式(5)所示的负载在苯肼树脂上的肽中的Fmoc保护基后，将树脂取出，加入1mL DMF、200μL二甲氨基丙胺及10mg Cu(OAc)2，室温振摇反应12h，将树脂滤除，并用20mL CH2Cl2洗涤树脂；将有机相浓缩，残留物用半制备型HPLC纯化：10％乙腈-H2O(含1％的TFA)等梯度洗脱5min，10％至100％的乙腈-H2O(含1％的TFA)梯度洗脱25min，保留时间TR＝15min，收集产物，冷冻干燥，得到淡黄色固体化合物1，其HRMS如图2所示。
HRMS(ESI)m/z:理论计算值C55H68N21O9[M+H]+1166.5503实测:1166.5508。
实施例2 化合物2的制备
按实施例1同样的步骤制得式(5)所示的负载在苯肼树脂上的肽，并采用实施例1中的步骤(b)脱除式(5)所示的负载在苯肼树脂上的肽中的Fmoc保护基；
将赫斯特酸衍生物Ht-1(539mg，1.056mmol)和PyBOP(550mg，1.056mmol)溶于3mL无水DMF，加入DIEA(350μL，2.112mmol)，反应5min，将该反应液转移到脱除Fmoc的式(5) 所示的负载在苯肼树脂上的肽中，N2鼓泡混匀，缩合反应1h，抽除反应液，用DMF(4×3mL)洗涤树脂；将树脂取出，加入1mL DMF、200μL二甲氨基丙胺及10mg Cu(OAc)2，室温振摇反应12h，将树脂滤除，并用20mL CH2Cl2洗涤树脂；将有机相浓缩，残留物用半制备型HPLC纯化：10％乙腈-H2O(含1％的TFA)等梯度洗脱5min，10％至100％的乙腈-H2O(含1％的TFA)梯度洗脱25min，保留时间TR＝18.5min，收集产物，冷冻干燥，得到淡黄色固体化合物2，其HRMS如图3所示。
HRMS(ESI)m/z:理论计算值C84H96N27O11[M+H]+1658.7777,实测:1658.7774.C84H97N27O11 2+；
赫斯特酸衍生物Ht-1的结构如下(制备方法参见J.AM.CHEM.SOC.2004,126,3736-3747)：
Figure PCTCN2015098880-appb-000033
实施例3 化合物3的制备
按实施例1同样的步骤制得式(5)所示的负载在苯肼树脂上的肽，并采用实施例1中的步骤(b)脱除式(5)所示的负载在苯肼树脂上的肽中的Fmoc保护基；
将Boc2O(243μL，1.056mmol)溶于3mL无水DMF，加入DIEA(350μL，2.112mmol)，将该反应液转移到脱除Fmoc的式(5)所示的负载在苯肼树脂上的肽中，N2鼓泡混匀，缩合反应20min。抽除反应液，用DMF(4×3mL)洗涤树脂，得到Boc保护的式(6)所示的负载在苯肼树脂上的肽；
Figure PCTCN2015098880-appb-000034
将以上所得树脂取出，加入1mL DMF、200μL N,N-双(3-氨丙基)甲胺，90℃振摇反应1h，冷至室温，将树脂滤除，并用20mL CH2Cl2洗涤树脂；将有机相浓缩，残留物用半制备型HPLC纯化：10％乙腈-H2O(含1％的TFA)等梯度洗脱5min，10％至100％的乙腈-H2O(含1％的TFA)梯度洗脱25min，保留时间TR＝16min，收集产物，冷冻干燥，得到淡黄色固体，备用；
取上述固体4mg(3μmol)溶于0.5mL无水DMF，加入赫斯特酸衍生物Ht-2(2.7mg，6μmol)、PyBOP(3.1mg，6μmol)和DIEA(5μL，30μmol)，室温振摇反应2h，用半制备型HPLC纯化：10％乙腈-H2O(含1％的TFA)等梯度洗脱5min，10％至100％的乙腈-H2O(含1％的TFA)梯度洗脱25min，保留时间TR＝18min，收集产物，冷冻干燥得固体；将该固体溶于1mL CH2Cl2，冰浴下加入1mL TFA，0℃反应1h，加入20mL冷的乙醚，离心收集沉淀，用半制备型HPLC纯化：10％乙腈-H2O(含1％的TFA)等梯度洗脱5min，10％至100％的乙腈-H2O(含1％的TFA)梯度洗脱25min，保留时间TR＝17min，收集产物，冷冻干燥，得到淡黄色固体产物化合物3，其HRMS如图4所示。
HRMS(ESI)m/z:理论计算值C83H95N28O10[M+2H]2+822.3926,实测822.3934.
赫斯特酸衍生物Ht-2的结构及合成路线如下：
Figure PCTCN2015098880-appb-000035
将Ht-2-B(11.4g,44.2mmol)与4-(5-(4-甲基哌嗪-1-基)-1H-苯并[d]咪唑-2-基)苯-1,2-二胺(Ht-2-A，10g,31.0mmol，制备方法参见Inorg.Chem.1998,37,6018-6022)溶于乙酸(100mL)中，油浴加热回流反应4小时。减压蒸除乙酸，冷却至室温，柱层析纯化，得到草绿色固体。将所得产物溶于甲醇(100ml)，磁力搅拌，氮气保护，冰水浴冷却，缓慢滴加3.1g氢氧化钠的50ml水溶液，滴毕，室温反应8h，TLC检测反应完全，蒸除甲醇，稀盐酸调pH酸性，析出固体，过滤，干燥，得黄绿色固体Ht-2，其HNMR分别如图5所示。
HNMR(DMSO-d6，400MHz)：2.44(s,3H),2.79(s,2H),3.23(s,2H),6.96(m,1H),7.05(brs,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)+]。
实施例4 化合物4的制备
按实施例1同样的步骤制得式(5)所示的负载在苯肼树脂上的肽，并采用实施例1中的步骤(b)脱除式(5)所示的负载在苯肼树脂上的肽中的Fmoc保护基；
将实施例2中的赫斯特酸衍生物Ht-1(539mg，1.056mmol)和PyBOP(550mg，1.056mmol)溶于3mL无水DMF，加入DIEA(350μL，2.112mmol)，反应5min，将该反应液转移到脱除Fmoc的式(5)所示的负载在苯肼树脂上的肽中，N2鼓泡混匀，缩合反应1h，抽除反应液，用DMF(4×3mL)洗涤树脂；将树脂取出，加入1mL DMF、200μL N,N-双(3-氨丙基)甲胺及10mgCu(OAc)2，室温振摇反应12h，将树脂滤除，并用20mL CH2Cl2洗涤树脂；将有机相浓缩，残留物用半制备型HPLC纯化：10％乙腈-H2O(含1％的TFA)等梯度洗脱5min，10％至100％的乙 腈-H2O(含1％的TFA)梯度洗脱25min，保留时间TR＝17min，收集产物，冷冻干燥，得固体，备用；
将3.4mg上述所得固体(2μmol)溶于0.5mL无水DMF，加入间苯二甲酸(3.3mg，20μmol)、PyBOP(10.4mg，20μmol)和DIEA(50μL，300μmol)，室温振摇反应2h，用半制备型HPLC纯化：10％乙腈-H2O(含1％的TFA)等梯度洗脱5min，10％至100％的乙腈-H2O(含1％的TFA)梯度洗脱25min，保留时间TR＝18.0min，收集产物，冷冻干燥，得到淡黄色产物化合物4；其HRMS如图6所示。
HRMS(ESI)m/z:理论计算值C94H106N28O14[M+2H]2+925.4216,实测925.4209。
实施例5 化合物5的制备
按实施例1同样的步骤制得式(5)所示的负载在苯肼树脂上的肽，并采用实施例1中的步骤(b)脱除式(5)所示的负载在苯肼树脂上的肽中的Fmoc保护基；
将实施例2中的赫斯特酸衍生物Ht-1(539mg，1.056mmol)和PyBOP(550mg，1.056mmol)溶于3mL无水DMF，加入DIEA(350μL，2.112mmol)，反应5min，将该反应液转移到脱除Fmoc的式(5)所示的负载在苯肼树脂上的肽中，N2鼓泡混匀，缩合反应1h，抽除反应液，用DMF(4×3mL)洗涤树脂；将树脂取出，加入1mL DMF、200μL N,N-双(3-氨丙基)甲胺及10mgCu(OAc)2，室温振摇反应12h，将树脂滤除，并用20mL CH2Cl2洗涤树脂；将有机相浓缩，残留物用半制备型HPLC纯化：10％乙腈—H2O(含1％的TFA)等梯度洗脱5min，10％至100％的乙腈—H2O(含1％的TFA)梯度洗脱25min，保留时间TR＝17min，收集产物，冷冻干燥，得固体，备用；
将3.4mg上述所得固体(2μmol)溶于0.5mL无水DMF，加入实施例3中的赫斯特酸衍生物Ht-2(2.7mg，6μmol)、PyBOP(3.1mg，6μmol)和DIEA(5μL，30μmol)，室温振摇反应2h，用半制备型HPLC纯化：10％乙腈-H2O(含1％的TFA)等梯度洗脱5min，10％至100％的乙腈-H2O(含1％的TFA)梯度洗脱25min，保留时间TR＝18.5min，收集产物，冷冻干燥，得到淡黄色产物化合物5；其HRMS如图7所示。
HRMS(ESI)m/z:理论计算值C112H124N34O12[M+2H]2+1068.5064,实测1068.5052。
实施例6 化合物2穿透细胞膜与核膜的实验
取处于对数生长期的Hela细胞,按每孔1mL(含5万个细胞)接种于12孔板中，置于37度，5％二氧化碳细胞培养箱中培养24小时；吸除原培养基，分别加入含有不同浓度化合物2的DMEM高糖培养基中，使每孔的药物浓度分别为0μM、2μM、10μM、50μM；将细胞重新放入培养箱中孵育24-72小时，荧光显微镜下观察药物在细胞中的定位，所得结果如图8所示，从图8中可以看出10μM浓度化合物2处理Hela细胞24和48小时后,能够有效到达细胞。
实施例7 化合物2抑制Hela细胞PLK1蛋白表达实验
取处于对数生长期的Hela细胞，按每孔1mL(含5万个细胞)接种于12孔板中，置于37度，5％二氧化碳细胞培养箱中培养24小时；吸除原培养基，分别加入含有不同浓度化合物2的DMEM高糖培养基中，使每孔的药物浓度分别为0μM、2μM、10μM、50μM；将细胞重新放入培养箱中孵育72小时；吸除培养基，用0.25％胰蛋白酶室温消化1min，用新鲜DMEM培养基重悬细胞，3000rpm离心3分钟，收集细胞，用冰冷的PBS清洗两次。
去除PBS，5000rpm离心3分钟，加RIPA裂解液，冰上孵育2小时，4度13000rpm离心15min，收集上清液，利用比如braford法测量蛋白浓度，加相应裂解液，使各组蛋白浓度一致。配置10％的SDS-PAGE蛋白分离胶和5％的SDS-PAGE蛋白浓缩胶；取50μg总蛋白80v电压跑胶分离样品，200mA恒流转膜2h。
将膜浸泡于5％脱脂牛奶封闭液，室温封闭1小时。加一抗PLK1(稀释比1：3000)和GAPDH(1：20000)，4度慢摇过夜。用PBST清洗3次，加HRP标记的二抗(稀释比1：3000)，室温1小时孵育；然后在暗室进行显色曝光；所得结果如图9所示，从图9中可以看出化合物2能够有效地抑制Plk1的表达，且抑制效率与药物浓度呈正相关，并且以肌动蛋白作为Western印迹实验中的内参，以证明不同样品中的蛋白检测时上样量相同。
实施例8 化合物2抑制Hela细胞增殖的实验
取处于对数生长期的Hela细胞，按每孔1mL(含10万个细胞)接种于6孔板中，置于37℃，5％二氧化碳细胞培养箱中培养24小时；吸除原培养基，分别加入含有不同浓度化合物2的DMEM高糖培养基中，使每孔的药物浓度分别为0μM、2μM、10μM、50μM；将细胞重新放入培养箱中孵育48小时或72小时，然后统计最终的细胞数量，所得结果如图10所示，从图10中可以看出随着药物浓度的升高和作用时间的延长，Hela细胞的数量明显减少，说明药物能有效抑制肿瘤细胞的增殖作用。
实施例9 化合物2抑制非小细胞肺癌肿瘤细胞在荷瘤小鼠中的增殖实验
将人A549细胞接种在10mm塑料培养皿中，完全培养基使用DMEM高糖，其含10％FBS和10％双抗(氯霉素/链霉素)；待A549细胞生长密度达到90％左右，吸除培养基，用0.25％胰蛋白酶(trypsin)于37℃消化1分钟；每个10mm培养皿加入完全培养基4mL，终止胰蛋白酶活性；轻轻吹打，将贴壁细胞全部吹散为悬浮细胞；在离心机中，用1000rpm的速度离心3分钟，去除上清液，用PBS重悬A549细胞；用血球计数板计算细胞浓度，加适量PBS，将细胞浓度调整至1.0×107个/ml
取1mL注射管，吸取1mL A549细胞悬浮液，在每只裸鼠的右侧腋下接种200万细胞，即200μl细胞悬浮液；接种完A549细胞后，将裸鼠放回饲养笼。待肿瘤大小长到约200mm3，边长5～8mm时，开始进行给药。
给药1组：给药剂量和频率示意图如图11所示；药物的剂量为1mg/Kg体重，换算后化合物2的给药浓度约为20μmol/只小鼠；将化合物2溶解在生理盐水中(0.9％NaCl)。从第一次给药起，每三天给一次药，一共持续7次，第21天不给药，处死小鼠后收集肿瘤样本；
给药2组：其给药剂量与给药频率与给药1组相同，其目的在于重复和确认给药1组的可靠性；
对照组：对照组小鼠只给予生理盐水；
每三天在给药前，称量并记录小鼠体重和肿瘤的长宽。肿瘤体积的计算公式为V＝0.5×长×宽×宽；给药后的第21天，处死裸鼠，收集肿瘤样本；化合物2对肿瘤增殖的抑制效果如图12～14所示，从图12～14中可以看出，与对照组相比，给药组裸鼠体内接种的人A549肺癌细胞移植瘤体积和重量都显著降低，说明以图11方式进行间隔连续给药后，能明显抑制肿瘤细胞在裸鼠体内的增殖。
实施例10
采用SPR(surface plasmon resonance analysis using a Proteon XPR36(Bio-Rad,California,USA))对化合物1～5与PLK1基因转录启动子区域DNA序列结合位置以及强度进行测定，其中，所述PLK1基因转录启动子区域具有如下DNA序列：
Figure PCTCN2015098880-appb-000036
测试结果表明化合物1-5均能与上述DNA序列中灰色部分发生特异性结合，并且化合物2～5与该DNA序列的结合强度结果如表1所示：
化合物 化合物2 化合物3 化合物4 化合物5
KD(M) 2.04×10-9 2.24×10-9 4.57×10-9 1.44×10-8
从上表中可以看出化合物2～5与靶标DNA的结合非常强，亲和力达到纳摩尔水平，特别是化合物2～4，其中化合物2的与靶标DNA的结合力最强，KD值达到2.04×10-9。

Claims (10)

  1. 通式(I)所示的化合物、其立体异构体或药学上可接受的盐：
    Figure PCTCN2015098880-appb-100001
    其中：
    R1～R8各自独立地为氢或C1～3烷基；优选地，R1～R8均为甲基
    A为-(CH2)m-，在A中，任意CH2上的氢原子被C1～3烷基、-OH、-NH2、-O-(C＝O)-R9或-NH-(C＝O)-R9所取代；
    B为-(CH2)n-CH3，在B中，任意CH2或CH3上的氢原子被C1～3烷基、-OR11或-NR10R11所取代；
    优选地，所述A为
    Figure PCTCN2015098880-appb-100002
    所述R12选自-OH、-NH2、-O-(C＝O)-R9或-NH-(C＝O)-R9；更优选地，A为
    Figure PCTCN2015098880-appb-100003
    优选地，所述B为
    Figure PCTCN2015098880-appb-100004
    R9为
    Figure PCTCN2015098880-appb-100005
    R10为C1～3烷基；
    R11为C1～6烷基或R17；
    R17为
    Figure PCTCN2015098880-appb-100006
    Figure PCTCN2015098880-appb-100007
    在R9或R17中，任意CH2上的氢原子被0～2个C1～6烷基、-OH、-NH2、卤素、-OR19、-NHR20或-NR20R21所取代；
    X或Y各自独立地为O、NH或NR22，
    R13～R15各自独立地为H、卤素、C1～3烷基；
    R16为C1～6烷基；优选地，R16为甲基、乙基、正丙基、异丙基、一氟甲基、二氟甲基或三氟甲基；
    R18为氢、卤素、C1～3烷基、羧基或硝基，优选地，R18为羧基；
    所述R19～R22各自独立地为C1～3烷基；
    所述C1～6烷基或所述C1～3烷基被0～3个卤素取代，优选被0～3个氟取代；
    p、p’各自独立地选自0、1、2、3或4；优选地，p、p’各自独立地为1、2、3或4；
    q、q’、r、r’、s、s’各自独立地选自0、1、2、3或4；优选地，q、q’、r、r’、s、s’均为0；
    t选自0、1、2、3或4；优选地，t为1。
  2. 根据权利要求1所述的通式(I)所示的化合物、其立体异构体或药学上可接受的盐，其中，所述化合物具有式(II)～式(V)所示的结构：
    Figure PCTCN2015098880-appb-100008
    Figure PCTCN2015098880-appb-100009
    优选地，R10为甲基或三氟甲基；
    优选地，所述R12选自NH2、
    Figure PCTCN2015098880-appb-100010
    Figure PCTCN2015098880-appb-100011
    优选地，所述R11选自CH3、CF3、CH2COOH、
    Figure PCTCN2015098880-appb-100012
    Figure PCTCN2015098880-appb-100013
  3. 根据权利要求1所述的通式(I)所示的化合物、其立体异构体或药学上可接受的盐，所述化合物包括：
    Figure PCTCN2015098880-appb-100014
    Figure PCTCN2015098880-appb-100015
    Figure PCTCN2015098880-appb-100016
  4. 权利要求1～3中任一项所述的通式(I)所示的化合物、其立体异构体或药学上可接受的盐的制备方法，其中，所述方法包括如下步骤：
    (a)制备肼树脂负载的式(VI)，优选所述肼树脂包括苯肼树脂；
    Figure PCTCN2015098880-appb-100017
    A’为-(CH2)m-，在A中，任意CH2上的氢原子被-OPOH、-NHPNH所取代；
    优选地，所述A’为
    Figure PCTCN2015098880-appb-100018
    所述R12为-OPOH或-NHPOH；更优选地，A’为
    Figure PCTCN2015098880-appb-100019
    Figure PCTCN2015098880-appb-100020
    (b)脱除式(VI)中的POH或PNH；
    (c)以氧化剂及切割剂进行氧化反应及切割反应以制得式(I)所示的所述的化合物；
    所述切割剂为NH2-(CH2)n-CH3，在切割剂中，任意CH2或CH3上的氢原子被-OR11或-NR10R11所取代；
    优选地，所述切割剂为
    Figure PCTCN2015098880-appb-100021
    优选地，所述氧化剂为醋酸铜、N-溴代丁二酰亚胺和氧气中的一种或多种的组合；
    A’及切割剂中各基团的定义与权利要求1～3中任一项相同，POH、PNH分别表示羟基保护基及氨基保护基；优选地，所述POH、PNH为Boc、Fmoc、Cbz、Trt或Allyl，更优选Fmoc。
  5. 根据权利要求4所述的方法，其中，在步骤(b)与步骤(c)之间以
    Figure PCTCN2015098880-appb-100022
    对脱除POH或PNH暴露的羟基或氨基进行缩合反应以形成-O-(C＝O)-R9或-NH-(C＝O)-R9的步骤；所述R23为羟基或卤素。
  6. 根据权利要求4或5所述的方法，其中，所述方法中的步骤(c)被步骤(c’)所替代，并且在步骤(c’)之后进一步包括步骤(c”)；
    (c’)以氧化剂及切割剂进行氧化反应及切割反应；所述切割剂为NH2-(CH2)n-CH2N(R10)-(CH2)p’-YH，
    (c”)以
    Figure PCTCN2015098880-appb-100023
    对步骤(c’)所得产物中的YH进行缩合反应后以制得式(I)所示的所述的化合物；R24为羟基或卤素；
    选择性地，在步骤(b)与步骤(c’)之间，将脱除POH或PNH暴露的羟基或氨基以POH’或PNH’进行保护，所述POH’或PNH’为可耐受碱性条件的羟基保护基或氨基保护基；并在步骤(c”)进行缩合反应之后脱除POH’或PNH’以制得式(I)所示的所述的化合物；优选地，所述POH’或PNH’为叔丁氧羰基。
  7. 根据权利要求4所述的方法，其中，所述式(VI)为式(VI-1)；
    Figure PCTCN2015098880-appb-100024
    优选地，式(VI-1)按如下路线制备：
    (1)以苯肼树脂以及4-叔丁氧羰基氨基-1-甲基-1H-吡咯-2-羧酸为原料经重复缩合及脱叔丁氧羰基保护基制得式(1)所示的化合物：
    Figure PCTCN2015098880-appb-100025
    (2)以式(1)所示的化合物与R-2-(9-芴甲氧羰基氨基)-4-叔丁氧羰基氨基丁酸进行缩合反应制得式(2)所示的化合物；
    Figure PCTCN2015098880-appb-100026
    (3)式(2)所示的化合物脱除叔丁氧羰基保护基后与4-叔丁氧羰基氨基-1-甲基-1H-吡咯-2-羧酸进行缩合反应，重复脱叔丁氧羰基保护基及缩合反应制得式(3)所示的化合物：
    Figure PCTCN2015098880-appb-100027
    (4)以式(3)所示的化合物与4-叔丁氧羰基氨基-1-甲基-1H-咪唑-2-羧酸进行缩合反应并脱除叔丁氧羰基保护基得式(4)所示的化合物：
    Figure PCTCN2015098880-appb-100028
    (5)以式(4)所示的化合物与1-甲基-1H-咪唑-2-羧酸进行缩合反应制得式(VI-1)所示的化合物。
  8. 药物组合物，其包含有效剂量的权利要求1～3中任一项所述的通式(I)所示的化合物、其立体异构体或药学上可接受的盐，或进一步包含一种或多种药学上可接受的载体或赋形剂。
  9. 权利要求1～3中任一项所述的通式(I)所示的化合物、其立体异构体或药学上可接受的盐或权利要求8所述的药物组合物在制备治疗或预防细胞过度增殖性疾病的药物中的应用。
  10. 根据权利要求9所述的应用，其中，所述的细胞过度增殖性疾病包括结肠癌、直肠癌、脑瘤、肺癌、表皮鳞癌、膀胱癌、胰腺癌、乳腺癌、卵巢癌、宫颈癌、子宫内膜癌、结直肠癌、肾细胞癌、胃癌、食管腺癌、食管鳞状细胞癌、非霍奇金淋巴瘤、肝癌、皮肤癌、甲状腺癌、头颈癌、前列腺癌、神经胶质瘤及鼻咽癌中的一种或多种；优选地，所述的细胞过度增殖性疾病包括乳腺癌、肺癌或胃癌。
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