CN114957249A - Irreversible covalent CDK inhibitor and preparation method and application thereof - Google Patents
Irreversible covalent CDK inhibitor and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an irreversible covalent CDK inhibitor and a preparation method and application thereof, wherein the inhibitor has a structure shown as a formula I. According to the invention, Michael addition electron acceptor groups such as acrylamide and cinnamamide are respectively introduced to the tail end of a Palbociclib piperazine group, and substituent groups with different electric effects are introduced to two sides of an unsaturated double bond of the acceptor group, so that the Michael addition reaction activity and the biological activity of the PAlbociclib piperazine group are adjusted. Researches show that the compounds can target proteins in an irreversible covalent bonding mode, have kinase inhibition activity and antiproliferative activity which are obviously superior to those of palbociclib, have good selectivity on CDK4/6, can effectively overcome drug resistance of the palbociclib, and have good application prospect in the aspect of treating malignant tumors.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to an irreversible covalent binding CDK inhibitor, and a preparation method and application thereof.
Background
Malignant tumors seriously threaten human health. For example, breast cancer, which is the most common cancer in women, is also a leading cause of cancer death in women worldwide.
Cyclin-dependent kinases (CDKs) belong to the serine/threonine protein kinase family, regulate the cell cycle, promote cell growth, proliferation and apoptosis. To date, the CDK family consists of 20 members (CDK1-20) that function in forming CDK/cyclin complexes with the associated cyclins. CDK4/6 interacts with cyclinD to form a complex, resulting in over-phosphorylation of Rb (cancer suppressor gene), mediating cell passage through G/S checkpoints and smooth progression to human S phase, thereby promoting cell proliferation. In 15-20% of breast cancer tissues, the expression level of cyclinD is obviously increased, and a G/S checkpoint mechanism is destroyed, so that the proliferation of breast cancer cells is promoted. Disruption of the CDK4/6-Rb-E2F pathway occurs in 90% of cancers. CDK4/6 is therefore an effective target for the development of therapeutics for intervention in cancer.
Palbociclib, the first cyclin-dependent kinase CDK4/6 inhibitor worldwide, was developed and developed by Pfizer corporation under the trade name Ibrance. Palbociclib reduces cell proliferation in Estrogen Receptor (ER) positive breast cancer cell lines by blocking progression of cells from G1 to S phase in the cell cycle. The united states Food and Drug Administration (FDA) accelerated approval of the combination of palbociclib and letrozole for use in advanced breast cancer, ER positive and HER2 negative in postmenopausal women, as an initial endocrine-based treatment of metastatic disease, at 2 months 2015. Because the palbociclib has the advantage of strong targeting in the aspect of breast cancer treatment, palbociclib is used as a first-line treatment medicament on the market and is widely applied clinically, but in the actual treatment process, part of patients still do not respond to the medicament and cannot obtain ideal curative effect; or develop resistance to the drug after a period of treatment.
Covalent inhibitors are a class of small molecule compounds that are capable of covalently binding to a particular target protein, thereby inhibiting its biological function. These inhibitors often contain functional groups such as acrylamide, β -lactam, sulfonyl fluoride, oxirane, etc., and can chemically react with specific amino acid residues in a target protein, such as cysteine residue, serine residue, lysine residue, and glutamic acid residue, to form covalent bonds, thereby inhibiting the biological functions of the corresponding protein. Compared with a non-covalent inhibitor, the most important advantage of the covalent inhibitor is that the covalent inhibitor has high affinity with the combination of target protein and relatively longer action time, because the combination of the covalent compound and the target is much firmer than that of the non-covalent drugs of the same kind, the anti-tumor activity of the drug molecules can be effectively improved, and the problem of drug resistance of the existing drugs is solved.
Based on this, designing irreversible covalent binding inhibitors is perhaps an effective means to improve the efficacy of existing palbociclib. In 2021, Rui Li et al reported Palbociclib acrylic acid derivatives (European Journal of Medicinal Chemistry,2021,219,113432), such as representative compound A1, whose bioactivity studies found that such derivatives were generally weak in activity, showed moderate inhibitory activity only against H1299 and MDA-MB-453 tumor cells, and were almost inactive against MDA-MB-231 and MCF-7. The reason for this is probably that the corresponding amino acid residue in the target (i.e., CDK4/6) is too reactive with the Michael addition electron acceptor in the compound to form an irreversible covalent bond and thus fail to exert the expected effect.
Compound A1 structure of the invention
Disclosure of Invention
The purpose of the invention is as follows: in order to improve the curative effect of palbociclib and overcome the problem that drug resistance is generated in clinical use, the invention provides an irreversible covalent CDK inhibitor, wherein a substituent group of an acrylic acid/phenylacrylic acid structure is introduced to a piperazine ring of a palbociclib molecular structure, and substituent groups with different electrical effects and steric effects are introduced to a carbon-carbon double bond of the palbociclib molecular structure, so that the Michael addition reaction activity of the palbociclib molecular structure is regulated, the irreversible kinase inhibitory activity of the palbociclib molecular structure is enhanced, and particularly the irreversible covalent inhibitory activity of the palbociclib molecular structure on CDKs is enhanced; meanwhile, the structural fragment of the cinnamic acid has anti-tumor activity, and the compound has the anti-tumor activity and overcomes the drug resistance by the synergistic effect of the cinnamic acid and the structural fragment of the cinnamic acid.
The invention also provides a preparation method and application thereof in treating malignant tumors.
The technical scheme is as follows: in order to achieve the above object, the present invention provides an irreversible covalent binding CDK inhibitor, wherein the inhibitor has the following structure represented by formula I:
wherein R is
Any one of (1) or (b).
Preferably, the irreversible covalent binding CDK inhibitor has a structure selected from any one of:
compound 1-1:
compounds 1-2:
compounds 1-3:
compounds 1-4:
compounds 1-5:
compound 2-1:
compound 2-2:
compounds 2-3:
compounds 2-4:
the preparation method of the irreversible covalent CDK inhibitor comprises the following steps:
the palbociclib and the corresponding carboxylic acid are prepared by condensation reaction in the presence of a condensing agent and an alkaline reagent;
the synthetic route is shown in equation 1:
equation 1
Wherein R is
Any one of the above groups.
Wherein the condensing agent is any one of DCC, HATU or CDI.
Wherein the alkaline reagent is any one of DMAP, DIPEA or triethylamine.
Preferably, the mole ratio of the palbociclib, the carboxylic acid, the condensing agent and the alkaline agent is 1: 1-1.3: 1-1.5.
The invention discloses application of an irreversible covalent CDK inhibitor in preparation of a medicine for treating malignant tumors.
Wherein the malignant tumor comprises breast cancer and lung cancer, and further is Pabociclib-resistant breast cancer.
The pharmaceutical composition for treating malignant tumor according to the present invention, comprising the irreversible covalent-binding CDK inhibitor of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.
Wherein the composition of the irreversible covalent binding CDK inhibitor and the carrier is in the dosage form of capsules, powder, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.
The irreversible covalent CDK inhibitor designed by the invention contains a Michael addition electron acceptor in the structure, and is covalently bound with an electric Michael addition electron donor in a target structure, so that the activity of drug molecules is enhanced, and the irreversible covalent binding CDK inhibitor has a good application prospect in the aspect of treating malignant tumors.
Aiming at the problems that the palbociclib can generate drug resistance and has narrow anti-tumor spectrum and the like in clinical application, the invention introduces a substituent group with an acrylic acid/phenylpropenoic acid structure on a piperazine ring of a palbociclib molecular structure, and introduces substituent groups with different electrical effects and steric effects on a carbon-carbon double bond of the palbociclib molecular structure so as to adjust the Michael addition reaction activity of the palbociclib molecular structure, strengthen the irreversible kinase inhibition activity of the palbociclib molecular structure, and particularly strengthen the irreversible covalent inhibition activity of the palbociclib molecular structure on CDKs; meanwhile, the structural fragment of the cinnamic acid has anti-tumor activity, and the compound has the anti-tumor activity and overcomes the drug resistance by the synergistic effect of the cinnamic acid and the structural fragment of the cinnamic acid.
The amino acid residues of the existing palbociclib corresponding to the target CDK4/6 act through intermolecular force, the acting force is relatively weak, and drug resistance is easy to generate. The compound A1 introduces an acrylic acid structural fragment on a palbociclib side chain, and has the potential of forming covalent bond combination. However, previous studies found that the compound was generally less biologically active, exhibiting only moderate inhibitory activity against H1299 and MDA-MB-453 tumor cells, and little activity against MDA-MB-231 and MCF-7. Experiments of the invention find that the compound A1 has too weak reactivity with a Michael addition donor, and is difficult to form an irreversible covalent bond, so that the kinase inhibition activity is weak, and the drug resistance of palbociclib cannot be overcome. The compound of the invention has substituent groups on two sides of a carbon-carbon double bond, and can adjust the addition activity of the double bond and further adjust the anti-tumor activity. Whereas a1 does not have the above-described function. According to the invention, Michael addition electron acceptor groups such as acrylamide and cinnamamide are respectively introduced into the tail end of a side chain of Palbociclib, and substituent groups with different electrical effects are introduced into two sides of an unsaturated double bond of the acceptor group, so that the Michael addition reaction activity and the biological activity of the Palbociclib are regulated. Researches show that the compounds can target proteins in an irreversible covalent bonding mode, have kinase inhibition activity and antiproliferative activity which are obviously superior to those of palbociclib, have good selectivity on CDK4/6, can effectively overcome drug resistance of the palbociclib, and have good application prospect in the aspect of treating malignant tumors.
Has the advantages that: compared with the prior art, the invention has the following advantages:
according to the invention, Michael addition electron acceptor groups such as acrylamide and cinnamamide are respectively introduced into the tail end of a side chain of Palbociclib, and substituent groups with different electrical effects are introduced into two sides of an unsaturated double bond of the acceptor group, so that the Michael addition reaction activity and the biological activity of the Palbociclib are regulated. Researches show that the compounds can target proteins in an irreversible covalent bonding mode, the Michael addition reaction activity of the compounds is strong, the kinase inhibition activity and the anti-proliferation activity of the compounds are obviously superior to those of palbociclib, wherein the inhibition activity of the compounds 1-2 to CDK6 is improved by 3 times than that of the palbociclib, the anti-proliferation activity to human breast cancer cells MDA-MB-231 is improved by 20 times, meanwhile, the activity to human non-small cell lung cancer A549 is also obviously improved, and the anti-tumor spectrum of the compounds is wider. In vitro experiments also show that the compound can effectively inhibit proliferation of palbociclib drug-resistant tumor cell lines, and prompt that the compound can overcome palbociclib drug resistance, and has a good application prospect in treating drug-resistant malignant tumors.
Drawings
FIG. 1 is a liquid phase diagram of compound 1-2 after incubation with mercaptoethanol for 12 h;
FIG. 2 is a mass spectrum of an addition product of compound 1-2 and mercaptoethanol;
FIG. 3 is a liquid phase diagram of compounds 2-4 incubated with mercaptoethanol for 12 h;
FIG. 4 is a mass spectrum of an addition product of compound 2-4 and mercaptoethanol;
FIG. 5 is a liquid phase diagram of Compound A1 after 24h incubation with mercaptoethanol;
FIG. 6 is a schematic representation of the interaction of compounds 1-2 with key amino acid residues of the target protein CDK6(PDB code:2 euf);
FIG. 7 is a 3D overlay of the effect of compound 1-2 (i.e., the green ligand in the figure), palbociclib (i.e., the red ligand in the figure) on key amino acid residues of the target protein CDK6(PDB code:2 euf);
FIG. 8 is a schematic representation of the interaction of compounds 2-4 with key amino acid residues of the target protein CDK6(PDB code:2 euf);
FIG. 9 is a 3D overlay of the effect of compounds 2-4 (i.e., the green ligand in the figure), palbociclib (i.e., the red ligand in the figure) on key amino acid residues of the target protein CDK6(PDB code:2 euf).
Detailed Description
The invention is further illustrated by the following figures and examples.
Materials, reagents and the like used in examples are commercially available unless otherwise specified.
HATU: 2- (7-azabenzotriazole) -N, N' -tetramethyluronium hexafluorophosphate;
DIPEA: n, N-diisopropylethylamine;
CDI: carbonyl imidazole;
DMAP: 4-dimethylaminopyridine;
DCC: dicyclohexylcarbodiimide.
Example 1
(E) Preparation of (E) -6-acetyl-2- ((5- (4- (but-2-enoyl) piperazin-1-yl) pyridin-2-yl) amino) -8-cyclopentyl-5-methylpyridon [2,3-d ] pyrimidin-7 (8H) -one (Compound 1-1)
Dissolving 2-butenoic acid in N, N-dimethylformamide, adding HATU under the condition of stirring, continuing to stir for 5 minutes, adding DIPEA into the reaction system, stirring for 3 minutes, then adding palbociclib (the molar ratio of 2-butenoic acid: HATU: DIPEA: palbociclib is 1.3: 1.5: 1.5: 1) into the solution, and heating and reacting for 6 hours at 55 ℃. The reaction progress was monitored by TCL and stopped until the point of the reactant had completely disappeared. Removing the solvent by using a rotary evaporator, adding dichloromethane and water for liquid separation and extraction, and taking an organic phase. After drying the organic phase over anhydrous sodium sulfate, the solvent was removed. A small amount of dichloromethane was added to the reaction mixture until the product was just dissolved. Then adding petroleum ether into the mixture, observing the precipitation of yellow solid, and filtering to obtain a crude product. Finally, the crude product is purified by sand making and column chromatography to obtain the product (eluent is dichloromethane/methanol-100: 1), and the yield is 82.3%.
ESI-MS:[M+H] + ＝502.2561；
1 H NMR(600MHz,CDCl3)δ8.81(s,1H),8.21(d,J＝8.9Hz,1H),8.04(d,J＝2.6Hz,1H),7.33-7.37(m,1H),6.93(dq,J＝13.7,6.8Hz,1H),6.28-6.33(m,1H),5.86(dd,J＝17.9,8.9Hz,1H),3.81(d,J＝65.6Hz,4H),3.20–3.15(m,4H),2.55(s,3H),2.37(s,3H),2.07(s,3H),1.89-1.93(m,4H),1.26(d,J＝8.2Hz,4H),0.88(t,J＝6.9Hz,1H).
Example 2
(E) Preparation of (E) -6-acetyl-8-cyclopentyl-5-methyl-2- ((5- (4- (4,4, 4-trifluoro-2-enoyl) piperazin-1-yl) pyridin-2-yl) amino) pyridinyl [2,3-d ] pyrimidin-7 (8H) -one (compound 1-2)
Replacing 2-butenoic acid with 4,4, 4-trifluorobutenoic acid, using CDI as a condensing agent, DMAP as an alkali reagent, reacting 4,4, 4-trifluorobutenoic acid: CDI: DMAP: the mole ratio of palbociclib is 1.0: 1.0: 1.0: 1.0, the other methods are the same as example 1, and the yield is 80%.
ESI-MS:[M+H] + ＝570.2435；
1 H NMR(600MHz,DMSO)δ9.00(s,1H),8.01(s,1H),7.89(s,1H),7.77(d,J＝9.2Hz,1H),7.45(dd,J＝15.4,2.1Hz,1H),6.81(dd,J＝15.4,7.1Hz,1H),5.89–5.82(m,1H),3.79–3.72(m,5H),3.25(s,4H),2.44(s,3H),2.34(s,3H),2.26-2.19(m,2H),1.93(s,2H),1.84–1.76(m,2H),1.28–1.21(m,2H).
Example 3
(E) Preparation of (E) -6-acetyl-8-cyclopentyl-2- ((5- (4- (3-cyclopropylpropenyl) piperazin-1-yl) pyridin-2-yl) amino) -5-methylpyridine [2,3-d ] pyrimidin-7 (8H) -one (compounds 1-3)
The yield was 77.3% by the same method as in example 1 except that 3-cyclopropylacrylic acid was used instead of 2-butenoic acid, DCC was used as a condensing agent, and DMAP was used as an alkali agent.
ESI-MS:[M+H] + ＝542.2874；
1 H NMR(600MHz,CDCl 3 )δ8.81(s,1H),8.24(d,J＝8.6Hz,1H),8.02(s,1H),7.38(d,J＝7.8Hz,1H),6.42–6.38(m,1H),5.90–5.82(m,1H),3.82(d,J＝48.5Hz,4H),3.23–3.11(m,4H),2.55(s,3H),2.37(s,3H),2.09–2.06(m,1H),1.92–1.84(m,2H),1.60(s,4H),1.25(s,4H),0.93(td,J＝6.7,4.5Hz,2H),0.65(dd,J＝4.5,2.0Hz,1H).
Example 4
Preparation of 6-acetyl-8-cyclopentyl-2- ((5- (4- (2-fluoropropenyl) piperazin-1-yl) pyridin-2-yl) amino) -5-methylpyridine [2,3-d ] pyrimidin-7 (8H) -one (compounds 1-4)
The same procedure used in example 3 was repeated except that 2-fluoroacrylic acid was used instead of 3-cyclopropylacrylic acid, whereby the yield was 75%.
ESI-MS:[M+H] + ＝530.2142；
1 H NMR(600MHz,DMSO)δ8.88(s,1H),8.03(s,1H),7.80(s,1H),7.75(d,J＝9.2Hz,1H),7.45(dd,J＝15.4,2.1Hz,1H),6.81(dd,J＝15.4,7.1Hz,1H),5.89–5.82(m,1H),3.79–3.72(m,5H),3.25(s,4H),2.44(s,3H),2.34(s,3H),2.26-2.19(m,2H),1.93(s,2H),1.84–1.75(m,2H),1.28–1.20(m,2H).
Example 5
Preparation of 6-acetyl-8-cyclopentyl-2- ((5- (4- (2-cyanopropenyl) piperazin-1-yl) pyridin-2-yl) amino) -5-methylpyridine [2,3-d ] pyrimidin-7 (8H) -one (compounds 1-5)
The same procedure used in example 3 was repeated except for using 2-cyanoacrylic acid in place of 3-cyclopropylacrylic acid, whereby the yield was 75%.
ESI-MS:[M+H] + ＝538.2007；
1 H NMR(600MHz,DMSO)δ9.05(s,1H),8.22(s,1H),7.89(s,1H),7.77(d,J＝9.2Hz,1H),7.45(dd,J＝15.4,2.1Hz,1H),6.81(dd,J＝15.4,7.1Hz,1H),5.89–5.82(m,1H),3.77–3.70(m,5H),3.29(s,4H),2.44(s,3H),2.34(s,3H),2.26-2.19(m,2H),1.93(s,2H),1.84–1.76(m,2H),1.28–1.21(m,2H).
Example 6
Preparation of 6-acetyl-2- ((5- (4-cinnamoylpiperazin-1-yl) pyridin-2-yl) amino) -8-cyclopentyl-5-methylpyridon [2,3-d ] pyrimidin-7 (8H) -one (compound 2-1)
The method is the same as the example 2 except that the method takes the phenylacrylic acid as the raw material and the triethylamine replaces DMAP, and the yield is 76.5 percent.
ESI-MS:[M+H] + ＝578.2874；
1 H NMR(600MHz,CDCl 3 )δ8.83(s,1H),8.43(s,1H),8.25(d,J＝9.1Hz,1H),8.05(s,1H),7.72(d,J＝15.4Hz,1H),7.55(s,1H),7.54(s,1H),7.39(s,1H),7.38(s,1H),6.92(d,J＝15.4Hz,1H),5.83-5.92(m,1H),3.90(d,J＝38.1Hz,4H),3.26–3.21(m,4H),2.55(s,3H),2.38(s,3H),2.36–2.31(m,2H),2.08(dd,J＝8.0,5.6Hz,2H),1.92–1.85(m,2H),1.70(dd,J＝10.5,5.4Hz,2H).
Example 7
(E) Preparation of (E) -6-acetyl-8-cyclopentyl-2- ((5- (4- (3- (3, 4-dimethoxyphenyl) acryloyl) piperazin-1-yl) pyridin-2-yl) amino) -5-methylpyridinyl [2,3-d ] pyrimidin-7 (8H) -one (compound 2-2)
Compound 2-2 was prepared in the same manner as in example 2, using 3, 4-dimethoxyphenylacrylic acid as the starting material, at a yield of 81.3%.
ESI-MS:[M+H] + ＝638.3085；
1 H NMR(600MHz,CDCl 3 )δ8.83(s,1H),8.43(s,1H),8.25(d,J＝9.1Hz,1H),8.05(s,1H),7.72(d,J＝15.4Hz,1H),7.55(s,1H),7.54(s,1H),7.39(s,1H),7.38(s,1H),6.92(d,J＝15.4Hz,1H),5.83-5.92(m,1H),3.90(d,J＝38.1Hz,4H),3.26–3.21(m,4H),2.55(s,3H),2.38(s,3H),2.36–2.31(m,2H),2.03-2.10(m,2H),1.92–1.85(m,2H),1.60-1.72(m,2H).
Example 8
(E) Preparation of (E) -6-acetyl-8-cyclopentyl-2- ((5- (4- (3- (4-hydroxy-3-methoxyphenyl) acryloyl) piperazin-1-yl) pyridin-2-yl) amino) -5-methylpyridin [2,3-d ] pyrimidin-7 (8H) -one (compound 2-3)
Compound 2-3 was prepared in the same manner as in example 2 using 3-methoxy-4-hydroxyphenyl acrylic acid as the starting material, with a yield of 79.5%.
ESI-MS:[M+H] + ＝624.2929；
1 H NMR(600MHz,CDCl 3 )δ8.82(s,1H),8.21(d,J＝9.0Hz,1H),8.06(d,J＝2.8Hz,1H),7.66(d,J＝15.3Hz,1H),7.36(dd,J＝9.1,2.9Hz,1H),7.12(dd,J＝8.2,1.8Hz,1H),7.01(d,J＝1.8Hz,1H),6.93(d,J＝8.2Hz,1H),6.75(d,J＝15.3Hz,1H),5.84-8.91(m,1H),3.94(s,3H),3.87-3.91(m,4H),3.49(s,1H),3.25–3.20(m,4H),2.55(s,3H),2.37(s,3H),2.35(dd,J＝14.0,5.7Hz,2H),2.11–2.03(m,2H),1.92–1.85(m,2H).
Example 9
(E) Preparation of (E) -2- (4- (6- ((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7, 8-dihydropyridin [2,3-d ] pyrimidin-2-yl) amino) pyridin-3-yl) piperazine-1-carbonyl) -3-benzeneacrylonitrile
Compounds 2-4 were prepared in 56% yield from 2-cyano-3-phenylacrylic acid by the same method as example 1.
ESI-MS:[M+H] + ＝603.2827；
1H NMR(600MHz,CDCl3)δ8.90(s,1H),8.23(d,J＝9.0Hz,1H),8.14(d,J＝2.6Hz,1H),7.91(s,1H),7.90(d,J＝1.4Hz,1H),7.81(s,1H),7.54–7.51(m,1H),7.50(s,1H),7.49–7.47(m,1H),7.37(dd,J＝9.1,2.9Hz,1H),5.91–5.84(m,1H),3.90–3.88(m,4H),3.30–3.25(m,4H),2.54(s,3H),2.38(s,3H),2.36–2.33(m,2H),2.09-2.05(m,2H),1.90–1.86(m,2H),1.71–1.67(m,2H).
Example 10
CDK kinase and tumor cell inhibitory Activity assay for Compounds of the invention
The inhibitory activity of the compound disclosed by the invention, palbociclib and compound A1 on CDK kinase (CDK4/6/9/12) and tumor cells is respectively determined by using a kinase kit and an MTT method.
Kinase activity assay: test compound and compound a1 were dissolved in 100% DMSO, respectively, to prepare 250.0 μmol/L stock solutions. Solutions of 1, 10, 50, 250, 1250nM (final test concentration) were prepared by dilution with 1x kinase base buffer. Blank DMSO and palbociclib were selected as negative and positive controls, respectively. The test compound solution was transferred to the target plate with the dispenser Echo 550, the test compound solution was added to each well of the 96-well plate, and then 10.0. mu.L of 0.1. mu.g/mL enzyme solution was added to each well, followed by incubation at room temperature for 10 min. Next, 10.0. mu.L of a mixture of substrate and ATP was added to each well and incubated in an incubator at 37 ℃ for 60 min. To each well was added 25.0. mu.L of reaction stop buffer. Reading data by a Caliper instrument, calculating inhibition rate, drawing a regression curve and measuring IC 50 The value is obtained.
MTT cell assay: MDA-MB-231 and A549 cells in logarithmic growth phase at 1X 10 5 cells/mL were seeded in 96-well plates and allowed to adhere overnight. After the cells were attached to the wall, the drug was administered for testing. First, a target compound to be tested and compound A1 were dissolved in dimethyl sulfoxide (DMSO) to prepare a solution of 2 mmol/L. Then the culture medium is respectively diluted to 1.25, 2.5, 5, 10, 20 and 40 mu mol/L. In addition, cells without drug treatment served as negative control group, and palbociclib served as positive control. Drugs at different concentrations were added to the test wells and the cells were incubated for 72h in an incubator at 37 ℃. Followed by addition of 10. mu.L of 5 mg/mL MTT solution and incubation for an additional 4 h. Removing supernatant after 4h, adding 150 mu L DMSO substituted culture medium into the supernatant to fully dissolve the formazan precipitate, measuring the OD value absorbance value of each well with a full-wavelength microplate reader at 490nm wavelength, calculating the cell growth inhibition rate, and calculating the required IC through SPSS18 software or formula 50 The value is obtained.
The formula for calculating the inhibition rate is as follows:
percent inhibition%
The results are shown in table 1 below:
table 1, inhibitory activity of the compounds disclosed in the present invention and palbociclib, compound a1 on CDK kinase (CDK4/6/9/12) and tumor cells.
As can be seen from the results in Table 1, the positive control Palbociclib showed potent inhibitory activity, IC, against CDK4, 6 50 Values were 26, 38nM, respectively, with little activity against CDK9, 12, suggesting that it is a CDK4, 6 selective inhibitor. Similarly, the compounds disclosed in the present invention also showed good CDK4, 6 selective inhibitory activity, especially compound 1-2 and compound 2-4, which activity was significantly enhanced compared to palbociclib. Compound A1 was the least active among the tests, and its IC 50 Values of 59, 112nM, respectively, suggest a decrease in target activity.
In cell experiments, the positive control Palbociclib showed strong inhibitory activity on CDK4 and CDK6 on two tumor cells, and IC 50 The values are 4.72 and 3.07 mu M respectively, the cell inhibition activity of the compound disclosed by the invention is obviously improved, the activity of all the compounds is better than that of a positive control, particularly the compound 1-2 and the compound 2-4 have the inhibition activity on MDA-MB-231 cells which is improved by 20 times and 5 times respectively compared with Palbociclib, and the inhibition activity on A549 is improved by 5 times, and the design idea of introducing a high-reactivity Michael addition receptor is successful. In the assay, compound A1 was the least active, with little activity against MDA-MB-231 and IC against A549 cells 50 The value was also only 13.2 μ M, significantly higher than the positive control and the compound of the invention.
Example 11
In vitro inhibition activity of Palbociclib drug-resistant cell line
The compounds 1-2 and 2-4 were selected as representative compounds, and together with palbociclib and compound a1, MTT experiments were performed (MTT method as in example 10) to determine the inhibitory activity against pabociclib-resistant MDA-MB-231 cells, so as to determine the potential of the test compounds to overcome tumor resistance, with the test results as shown in table 2 below:
TABLE 2 inhibitory Activity of Compounds 1-2, 2-4, A1 and Pabociclib against Pabociclib-resistant MDA-MB-231 cell lines.
a Drug resistance index (MDA-MB-231 cell IC) resistant to Pabociclib 50 Value/non-drug resistant MDA-MB-231 cell IC 50 Value of
From the results in table 2, it can be seen that the activity of palbociclib against drug-resistant strains decreases sharply, and the drug resistance index thereof reaches 10 or more; a1 likewise showed no significant activity against drug-resistant strains. The compounds 1-2 and 2-4 of the invention still show very good inhibitory activity on drug-resistant strains, and the IC thereof 50 The values respectively reach 0.218 and 0.720 mu M, and no drug resistance phenomenon is seen, which indicates that the tested compound can well avoid the cross drug resistance with the palbociclib. This is likely related to the irreversible covalent binding mode of action of such compounds.
Example 12
Michael addition Activity test of Compounds of the present invention
The formation of covalent addition products was determined by HPLC (column: Agilent zorbax sb-Aq C18 (standard: 4.6X 250mm, particle size: 5 μ M), column temperature 25 ℃, sample introduction amount of 5 μ L, mobile phase: methanol: water 10:90, v/v, flow rate of 0.8ml/min, detection wavelength 215nm), selecting compounds 1-2(1 μ M) and 2-4(1 μ M) having better in vitro activity, incubating in pH7.0 buffer solution at 37 ℃ for 12h with excess mercaptoethanol, and determining the Michael addition reaction target by simulating the effect of the compounds on protein. The test results are shown in fig. 1 to 5.
As can be seen from FIGS. 1 to 4, after the compounds 1-2 and 2-4 and the Michael addition donor mercaptoethanol are co-cultured for 12h, Michael addition products can be generated, and the structures of the Michael addition products are confirmed by mass spectrometry, which indicates that the two compounds have better Michael addition reaction activity. As can be seen from FIG. 5, although compound A1 also contains acrylamide structure, no obvious addition product is formed even after 24h of co-culture with mercaptoethanol, indicating that its covalent binding activity is weak, probably because of the lack of electron-withdrawing groups (such as strong electron-withdrawing groups) on both sides of the carbon-carbon double bond in A1, resulting in insufficient electrophilic addition ability. The detection result is consistent with the in vitro kinase inhibition activity and the cell inhibition activity, and indirectly proves that the irreversible covalent binding activity of the tested compound is closely related to the biological activity of the tested compound.
Example 13
Simulated docking studies of Compounds 1-2, 2-4 with CDK6
After confirming that the compounds 1-2 and 2-4 have stronger Michael addition reaction activity in vitro, in order to further verify whether the compounds 1-2 and 2-4 have the potential of covalent binding with the amino acid residues of the target protein, the binding and action patterns of the compounds 1-2 and 2-4 and the target protein CDK6(PDB code:2euf) are simulated by using MOE (molecular Operating environment) software, and the docking results are shown in FIGS. 6-9.
The results show that the compounds 1-2 and 2-4 can effectively enter the active pocket of CDK6, the optimal binding conformation of the compounds has higher coincidence with the optimal conformation of palbociclib, and the compounds 1-2 and 2-4 are taken as derivatives of palbociclib, and the action mode of the compounds and target proteins is similar to that of the palbociclib. Specifically, in the pyrimidopyridone structure of compounds 1-2, the pyrimidone carbonyl group and the acetone carbonyl group can form strong hydrogen bonding with Lys43 and Glu21, respectively, and the piperazine and acrylamide structural fragments thereof are located in the solvent region of the protein, and stabilize the ligand-receptor complex through hydrophobic interaction (see fig. 6 and fig. 7 for details). Similarly, compounds 2-4 primarily utilized arylamine and cyano groups to interact with Lys43, Ala23, Val101, etc., residues, while their acrylamide moieties also extended outside the pocket (see figures 8, 9 for details). It should be noted thatWhether the compound is 1-2 or 2-4, the acrylamide structural fragment is very close to Thr106 and Thr107 residues of CDK, and the distance between the hydroxyl group of the Thr106 and Thr107 residues and the carbon-carbon double bond of the compound 1-2 and 2-4 is measured by the "measure" functional module of MOE, and the value is found to be
Completely within the bonding range, indicating that the 1-2, 2-4 carbon-carbon double bond of the compound is likely to undergo covalent addition with Thr106, Thr 107. In combination with the results of the aforementioned enzymatic activity, cellular activity and in vitro addition experiments, it can be further concluded that the disclosed compounds are irreversible covalent CDK4/6 selective inhibitors and have stronger irreversible binding force, thus the biological activity is also stronger and the drug resistance is favorably overcome.
Claims (10)
1. An irreversible covalent binding CDK inhibitor, wherein said inhibitor has the structure of formula I:
wherein R is
Any one of (1) or (b).
2. A method of preparing an irreversible covalent binding CDK inhibitor according to claim 1, comprising the steps of:
the palbociclib is prepared by condensation reaction of palbociclib and corresponding carboxylic acid in the presence of a condensing agent and an alkaline reagent;
the synthetic route is shown in equation 1:
wherein R is
Any one of (1) or (b).
3. The method of preparing an irreversible covalent binding CDK inhibitor according to claim 2 wherein the condensing agent is any one of DCC, HATU or CDI.
4. The method of claim 2, wherein the basic agent is any one of DMAP, DIPEA, or triethylamine.
5. The method for preparing an irreversible covalent CDK inhibitor according to claim 2, wherein the molar ratio of palbociclib, carboxylic acid, condensing agent, and alkaline agent is 1:1 to 1.3:1 to 1.5.
6. Use of an irreversible covalent binding CDK inhibitor of claim 1 in the preparation of a medicament for the treatment of a malignancy.
7. The use of claim 6, wherein the malignancy comprises breast cancer, lung cancer and Pabociclib-resistant breast cancer.
8. The use of claim 6, wherein the malignancy is Pabociclib-resistant breast cancer.
9. A pharmaceutical composition for the treatment of malignant tumors, comprising the irreversible covalent binding CDK inhibitor of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.
10. Pharmaceutical composition according to claim 9, wherein the composition of the irreversible covalent binding CDK inhibitor to a carrier is preferably in the form of a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.
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| WO2018106870A1 (en) * | 2016-12-08 | 2018-06-14 | Icahn School Of Medicine At Mount Sinai | Compositions and methods for treating cdk4/6-mediated cancer |
| CN110548151A (en) * | 2018-06-01 | 2019-12-10 | 四川大学 | Design method of covalent drug |
| CN114644643A (en) * | 2020-12-21 | 2022-06-21 | 南京大学 | Twin drug and synthesis method and application thereof |
| CN114907337A (en) * | 2021-02-08 | 2022-08-16 | 四川大学 | Covalent inhibitors targeting CDK4 or CDK6 and uses thereof |
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| WO2018106870A1 (en) * | 2016-12-08 | 2018-06-14 | Icahn School Of Medicine At Mount Sinai | Compositions and methods for treating cdk4/6-mediated cancer |
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| CN110548151A (en) * | 2018-06-01 | 2019-12-10 | 四川大学 | Design method of covalent drug |
| CN114644643A (en) * | 2020-12-21 | 2022-06-21 | 南京大学 | Twin drug and synthesis method and application thereof |
| CN114907337A (en) * | 2021-02-08 | 2022-08-16 | 四川大学 | Covalent inhibitors targeting CDK4 or CDK6 and uses thereof |
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