CN115093419A - Pyrimidone compound and preparation method and medical application thereof - Google Patents
Pyrimidone compound and preparation method and medical application thereof Download PDFInfo
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Abstract
The invention relates to a pyrimidone compound and a preparation method and application thereof, belonging to the technical field of pharmaceutical chemistry and pharmacotherapy. The compound, isomer, hydrate, solvate or pharmaceutically acceptable salt thereof shown in the formula I has good PAK5 inhibitory activity, can be used as a PAK5 inhibitor, and can be applied to preparation of drugs for diseases related to PAK 5.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical chemistry and pharmacotherapeutics, and particularly relates to a pyrimidinone compound. The compounds can be used for preparing medicines for diseases related to PAK 5. The invention also relates to a preparation method of the compounds and a pharmaceutical composition containing the compounds.
Background
p-21 activated kinases (PAKs) are important downstream effectors of the small G protein family members Cdc42 and Rac, and human-related PAKs family members are classified by sequence and structural homology into two classes, class I (PAK1, PAK2, PAK3) and class II (PAK4, PAK5, PAK 6). The class I PAKs and the class II PAKs have 50% of homology, the class II PAKs (PAK4, PAK5 and PAK6) have 80% of homology, and all have a conserved C-terminal kinase Domain and N-terminal regulatory Domain, and the N-terminal has 1 autophosphorylation region (P21 GTPase Binding Domain, PBD). Through analyzing the crystal structure of the PAKs protein, the discovery that: compared with the PAK kinase in class I, the PAKs kinase in class II has no PID inhibition area at the N terminal, so that the kinase activity is higher. The kinase activity of PAKs type II is mainly determined by its C-terminal kinase catalytic domain, while PAK5 is considered as an important mediator of tumor development, and from the viewpoint of each subtype structure, PAK5 shows its own function and mechanism in the process of tumor development.
The research on action mechanism shows that p21 activated kinase 5(PAK5) can be positioned in mitochondria, and the Ser112 site of phosphorylated BAD protein triggers the apoptosis resistance of tumor cells; phosphorylate the p65 subunit of NF-kB, promote the nuclear translocation of p65, further up-regulate the expression of cyclin D1, and promote the proliferation of breast cancer cells in vitro and in vivo; phosphorylated GATA-1 inhibits epithelial-mesenchymal transition of breast cancer cells; phosphorylated E47 inhibits colon cancer metastasis; the phosphorylation of p120-catenin-Ser288 complex causes cytoskeleton recombination, and promotes tumor cell movement.
The PAK5 small molecule inhibitor has good prospect for treating cancers related to high expression of PAK 5. Currently, there are few reports on PAK5 small molecule inhibitors, including PF-3758309 developed by Perey and GNE-2861 developed by Genetik.
Disclosure of Invention
The invention aims to provide a pyrimidinone compound based on the prior art, and pharmacological experiments prove that the compound has good PAK5 inhibitory activity, and particularly has strong antitumor activity on tumor cells such as renal cancer cells, liver cancer cells, colon cancer cells and breast cancer cells.
Another object of the present invention is to provide a process for the preparation of the above compound.
Another object of the present invention is to provide a pharmaceutical use of the above compound.
The technical scheme of the invention is as follows:
the invention relates to a compound, an isomer, a hydrate, a solvate or a pharmaceutically acceptable salt thereof with a structure shown as a general formula I,
wherein, the first and the second end of the pipe are connected with each other,
R 1 represents hydrogen or C 1 -C 6 An alkyl group;
R 2 represents hydrogen, halogen, cyano, trifluoromethyl, hydroxyl, amino, nitro, C 1 -C 6 Alkyl radical, C 1 -C 6 Alkoxy, phenyl, substituted phenyl, phenoxy, substituted phenoxy, benzyl or substituted benzyl, wherein the substituted phenyl, substituted phenoxy or substituted benzyl can be optionally mono-or polysubstituted by the following substituents: cyano, trifluoromethyl, hydroxy, amino, nitro, C 1 -C 4 Alkyl or C 1 -C 4 An alkoxy group;
l represents a covalent bond, -CH 2 -、-CH 2 CH 2 -、-NH-CH 2 -、-CH 2 -NH-, -NH-CO-or-CO-NH-;
R 3 represents halogen, hydroxy, C 1 -C 4 Alkoxy radical, C 1 -C 4 Alkylthio group, CH 3 -SO 2 -or CH 2 CH 3 -SO 2 -；
n represents an integer of 1 to 3.
In a preferred embodiment, R 1 Represents hydrogen, methyl, ethyl, isopropyl or tert-butyl.
In a more preferred embodiment, R 1 Represents hydrogen, methyl or ethyl.
In a preferred embodiment, R 2 Represents hydrogen, trifluoromethyl, fluorine, chlorine, cyano, methyl, ethyl, methoxy, ethoxy, phenoxy or benzyl.
In a more preferred embodiment, R 2 Represents hydrogen, trifluoromethyl or phenoxy.
In a preferred embodiment, R 3 Represents fluorine, chlorine, hydroxyl, methoxy, ethoxy, methylthio, ethylthio or CH 3 -SO 2 -。
In a more preferred embodiment, R 3 Represents fluorine, hydroxyl, methoxy, ethoxy, methylthio or CH 3 -SO 2 -。
In a preferred embodiment, L represents a covalent bond, -CH 2 -or-CH 2 CH 2 -。
In a preferred embodiment, n represents 1 or 2.
Further, the compound of formula I is preferably selected from the following compounds:
the invention discloses a preparation method of a compound shown in a general formula I, which comprises the following steps:
preferably, the reaction conditions are as follows: in the first step, the reaction conditions are TsOH, EtOH and reflux; in the second step, the reaction conditions are AIBN and CCl 4 And refluxing; in the third step, the reaction conditions are Et 3 N, MeOH and reflux.
These intermediates or the target compounds can be purified according to conventional isolation techniques and, if desired, converted into addition salts with pharmaceutically acceptable acids.
The invention also provides a pharmaceutical composition which takes the compound, the isomer, the hydrate, the solvate or the pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient and is assisted by pharmaceutically acceptable auxiliary materials. Wherein, the pharmaceutical composition can be tablets, capsules, granules, injections or sprays. The pharmaceutically acceptable carrier is selected from one or more of fillers, disintegrants, binders and lubricants, including but not limited to any and all solvents, dispersion media, coatings, absorption delaying agents and the like.
The pyrimidone compound provided by the invention has good PAK5 inhibitory activity, can be used as a PAK5 inhibitor and used for preparing medicines for diseases related to PAK5, wherein the diseases related to PAK5 are colorectal cancer, liver cancer, gastric cancer, cervical cancer, renal cancer, breast cancer or diabetes.
Unless otherwise indicated, the following terms used in the specification and claims have the meanings discussed below:
"pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:
(1) salifying with an acid by reaction of the free base of the parent compound with an inorganic or organic acid, including hydrochloric, hydrobromic, nitric, phosphoric, metaphosphoric, sulfuric, sulfurous, perchloric, and the like, the organic acid includes acetic acid, trifluoroacetic acid, propionic acid, acrylic acid, caproic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, ascorbic acid, camphoric acid, benzoic acid, hydroxybenzoic acid, γ -hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, cinnamic acid, dodecylsulfuric acid, gluconic acid, glutamic acid, aspartic acid, stearic acid, mandelic acid, succinic acid, glutaric acid, or malonic acid, and the like.
(2) The acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, an alkaline earth metal ion or an aluminum ion, or is complexed with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, quinine, or the like.
"pharmaceutical composition" refers to the combination of one or more of the compounds of the present invention, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, with another chemical ingredient, such as a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate the administration process to an animal.
"pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" refers to inactive ingredients in a pharmaceutical composition that do not cause significant irritation to an organism and do not interfere with the biological activity and properties of the administered compound, such as, but not limited to: calcium carbonate, calcium phosphate, various sugars (e.g., lactose, mannitol, etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic or methacrylic polymers, gelatin, water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated castor oil or polyethoxylated hydrogenated castor oil, sesame oil, corn oil, peanut oil, and the like.
"alkyl" means a saturated aliphatic radical of 1 to 20 carbon atoms, including straight and branched chain radicals (a numerical range referred to herein, e.g., "1 to 20", means that the radical, in this case alkyl, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms). More preferably, the alkyl group is a medium size alkyl group having 1 to 10 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, and the like. Preferably, alkyl is lower alkyl having 1 to 8 or 1 to 6 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl or tert-butyl and the like. Alkyl groups may be substituted or unsubstituted. When substituted alkyl, the substituent is preferably one or more, more preferably 1 to 3, most preferably 1 or 2 substituents.
"hydroxy" means an-OH group.
"nitro" means-NO 2 A group.
"cyano" means a-CN group.
"alkoxy" means-O- (unsubstituted alkyl) and-O- (unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.
"halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.
By adopting the technical scheme of the invention, the advantages are as follows:
the pyrimidinone compound provided by the invention has good PAK5 enzyme activity inhibition effect, can obviously inhibit the growth of breast cancer MCF-7 cell nude mouse transplanted tumor by blocking cell cycle and inhibiting tumor cell proliferation and migration processes, and therefore, can be used as a PAK5 inhibitor and applied to the treatment of PAK5 related diseases.
Drawings
FIG. 1 is a scratch test to examine the effect of compound Cpd.5 on the migratory capacity of breast cancer MCF-7 cells; in FIG. 1, A is a scratch test; in FIG. 1, B is the healing rate;
FIG. 2 is a flow cytometry analysis of the effect of compound Cpd.5 on breast cancer MCF-7 cell cycle distribution; in the six graphs in the first and second rows, the left peak represents G0/G1, the right peak represents G2/M, and the middle part represents S.
FIG. 3 is a graph showing the effect of compound Cpd.5 on proliferation of human breast cancer MCF-7 cell transplantable tumors in nude mice; in FIG. 3, A is the anatomical observation of nude mouse breast cancer MCF-7 cell transplantation tumor tissue; FIG. 3B is the tumor weight change curve of the nude mouse human breast cancer MCF-7 cell transplantation tumor; FIG. 3C is the tumor volume growth curve of nude mouse human breast cancer MCF-7 cell transplantation tumor;
FIG. 4 shows the docking results of compound Cpd.5 with PAK5 protein (PDB: 2F 57).
Detailed Description
The pyrimidinone compounds of the invention are further illustrated by the following examples, which are not intended to limit the invention in any way.
Example 1
Preparation of 1-ethyl-6- (4- (methylsulfonyl) phenyl) -4- (3-phenoxyphenyl) -3,4,6, 7-tetrahydro-1H-pyrrolo [3,4-d ] pyrimidine-2, 5-dione (Compound Cpd.5):
the first step is as follows: the reaction conditions were TsOH, EtOH and reflux
A round-bottomed flask was charged with 3-phenoxybenzaldehyde 1a (10mmol), ethyl urea (12mmol), ethyl acetoacetate (1.26mL, 10mmol) and TsOH 4H 2 O (0.24g, 1mmol), added with absolute ethanol and stirred, heated to reflux at 78 ℃. The reaction was monitored by TLC. After about 4h the reaction was complete and the resulting precipitate was filtered and the crude product was purified by recrystallisation from the appropriate solvent to give 2.10g of intermediate (4a) as a white solid in 81% yield. The melting point is 205-206 ℃ (204-206 ℃); 1 H NMR(400MHz,CDCl 3 )δ8.03(s,1H),7.31-7.23(m,4H),5.72(s,1H),5.40(s,1H),4.07(dd,J 1 ＝7.2Hz,J 2 ＝5.2Hz,2H),2.34(s,3H),1.16(t,J＝7.2Hz,3H). 13 C NMR(100MHz,CDCl 3 )δ165.76,153.36,146.38,143.83,128.86,128.86,128.10,126.74,126.74,101.53,60.18,55.91,18.84。
the second step is that: the reaction conditions are AIBN and CCl 4 And reflux
The prepared ethyl 1-ethyl-6-methyl-2-oxo-4- (3-phenoxyphenyl) -1,2,3, 4-tetrahydropyrimidine-5-carboxylate intermediate 4a (10mmol) was added to CCl with vigorous stirring 4 (25mL) of the solution was heated to reflux, and 2.67g (15mmol) of NBS was added to the reaction solution in portions over 1.5 h. Refluxing was continued for 10 h. After completion of the reaction, the reaction was cooled to room temperature, and the precipitate was filtered. The filtrate was concentrated under reduced pressure and the residue was purified by column chromatography (PE: EA ═ 5:1) to give the bromo intermediate product (5 a).
The third step: the reaction conditions are Et 3 N, MeOH and reflux
The prepared ethyl 1-ethyl-6- (bromomethyl) -2-oxo-4- (3-phenoxyphenyl) -1,2,3, 4-tetrahydropyrimidine-5-carboxylate 5a (2.00mmol) and 4- (methylsulfonyl) aniline (2.20mmol) were dissolved in tetrahydrofuran, stirred well, triethylamine (0.57mL, 4.40mmol) was added, and the reaction was heated under reflux for about 4h, ending. After cooling to room temperature, the precipitate was removed by filtration, the filtrate was concentrated, diluted with ethyl acetate (10mL × 3), washed with dilute hydrochloric acid, saturated sodium bicarbonate and saturated brine (10mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain an oily crude product, which was subjected to column chromatography (PE: EA ═ 8:3) to obtain the objective compound cpd.5. 1 H NMR(400MHz,DMSO-d 6 )δ7.99-7.95(m,3H),7.88(s,1H),7.85(s,1H),7.43-7.34(m,3H),7.17-7.12(m,2H),7.05-7.00(m,3H),6.91–6.86(m,1H),4.95–4.76(m,2H),3.67-3.50(m,2H),3.17(s,3H),1.16(t,J＝7.2Hz,3H). 13 C NMR(100MHz,DMSO-d 6 )δ167.80,157.18,156.72,153.00,151.53,145.67,144.32,134.11,130.60,130.51,130.51,128.60,128.60,124.06,121.85,121.85,119.16,119.16,117.93,117.41,117.08,104.28,52.66,47.77,44.33,38.55,14.63.IR(KBr):3388,3101,2924,1770；HRMS(ESI)calcd for:C 27 H 25 N 3 O 5 SNa[M+Na] + :526.1413,found:526.1390.
EXAMPLE 2 preparation of Compound Cpd.1
Specific experimental procedure for compound cpd.1 referring to example 1, the synthetic route is as follows:
referring to the preparation method in example 1, compound cpd.1, white solid product, 0.512g, yield 65.07% was obtained. 1 H NMR(400MHz,DMSO-d 6 )δ9.38(s,1H),7.87(s,1H),7.38(q,J＝7.6Hz,3H),7.30(s,1H),7.17–7.05(m,4H),7.01(d,J＝7.7Hz,3H),6.88(d,J＝8.0Hz,1H),6.45(d,J＝7.4Hz,1H),5.24(s,1H),4.71(m,2H),3.07(s,1H),1.14(t,J＝7.2Hz,3H). 13 C NMR(100MHz,DMSO-d 6 )δ167.37,158.18,157.14,156.74,151.77,151.64,145.95,141.16,130.50,130.50,129.89,129.89,124.03,121.80,119.14,119.14,117.82,117.02,110.26,108.76,105.48,104.79,52.72,47.75,27.30,14.59.IR(KBr):3450(NH),3103,2980,1728,1232；HRMS(ESI)calcd for C 26 H 23 N 3 O 4 Na[M+Na] + :464.1586,found:464.1564.
EXAMPLE 3 preparation of Compound Cpd.2
The specific experimental procedure for compound cpd.2 is as follows, with reference to example 1:
referring to the preparation method in example 1, compound cpd.2 was obtained with a yield of 67%. 1 H NMR(400MHz,DMSO-d 6 )δ8.13(s,1H),7.79(d,J＝2.7Hz,1H),7.53(dd,J＝3.2,5.6Hz,2H),7.42-7.35(m,2H),7.32-7.23(m,2H),7.20–7.06(m,3H),7.03-6.95(m,2H),6.87(s,1H),5.22(s,1H),4.45(m,2H),4.14(m,2H),4.04(s,2H),1.03(t,J＝7.4Hz,3H). 13 C NMR(100MHz,DMSO-d 6 )δ168.67,161.36,157.14,156.76,151.91,151.72,146.13,134.69,131.79,131.70,130.54,130.48,130.06,129.98,124.01,121.72,119.11,117.85,116.84,116.02,115.87,115.81,115.66,104.11,52.80,47.20,42.04,38.20,14.55.IR(KBr):3390,3169,2972,1722；HRMS(ESI)calcd for C 27 H 24 FN 3 O 3 Na[M+Na] + :480.1699,found:480.1708.
EXAMPLE 4 preparation of Compound Cpd.3
Specific experimental procedure for compound cpd.3 referring to example 1, the synthetic route is as follows:
referring to the preparation in example 1, compound cpd.3 was obtained in 54% yield. 1 H NMR(400MHz,DMSO-d 6 )δ7.87(d,J＝1.8Hz,1H),7.65(d,J＝8.8Hz,1H),7.35(dd,J＝7.6,6.8Hz,1H),7.23(d,J＝8.8Hz,1H),7.12(t,J＝7.3Hz,1H),7.04–6.96(m,1H),6.86(dd,J＝8.0,1.8Hz,1H),5.23(s,1H),4.79-4.65(m,2H),3.34(s,2H),2.42(s,3H),1.13(t,J＝6.8Hz,3H). 13 C NMR(100MHz,DMSO-d 6 )δ167.34,157.17,156.74,151.76,151.74,145.91,137.72,131.57,131.57,130.55,130.49,130.49,127.80,124.03,121.81,121.81,119.16,118.83,118.83,117.84,117.04,104.67,52.75,47.68,38.42,16.16,14.62.IR(KBr):3332,3153,2978,1732；HRMS(ESI)calcd for:C 27 H 25 N 3 O 3 SNa[M+Na] + :494.1514,found:494.1523.
EXAMPLE 5 preparation of Compound Cpd.4
Specific experimental procedure for compound cpd.4 referring to example 1, the synthetic route is as follows:
referring to the preparation method in example 1, Compound Cpd.4 was obtained as a white solid in the yield of 65.07%, 0.512 g. 1 H NMR(400MHz,CDCl 3 )δ8.57(s,1H),7.28(d,J＝4.0Hz,4H),7.25(dd,J＝8.0,4.0Hz,1H),6.80(d,J＝8.0Hz,1H),6.74(s,1H),6.71(s,1H),6.03(s,1H),5.37(s,1H),3.97(s,2H),3.86(s,3H),3.85(s,3H),2.84(t,J＝6.0Hz,2H),2.75(t,J＝6.6Hz,2H). 13 C NMR(100MHz,CDCl 3 )δ165.38,152.64,149.03,148.60,147.61,143.96,131.71,128.64,128.64,127.85,126.56,126.56,120.60,111.92,111.43,99.52,59.94,55.94,55.87,51.12,47.70,35.73.IR(KBr,cm -1 ):v 3217,3063,2932,2832,1690,1543,1512,1458,1389,1312,1265,1234.MS(ESI)calcd for C 22 H 23 N 3 O 6 394.2[M+H] + ,found:394.8.
The following are some of the pharmacological tests and results of representative compounds of the invention:
1. CCK-8 method for testing proliferation inhibitory activity of tumor cells
Counting the cells in logarithmic growth phase in the pancreatin digestion culture dish, counting the number of the counting plates, and paving 3000-6000 cells in each hole except the peripheral hole of the 96-well plate except the peripheral hole of which only 200 mu L of PBS is added; set 4 duplicate wells, add 100 μ Ι _ of complete medium of 10% FBS per well; after the cells were attached to the wall, the medium in the 96-well plate was discarded by inverting, the medium containing compounds of different concentrations was added, and the mixture was left at 37 ℃ with 5% CO 2 Culturing for 48h in an incubator; discarding the old culture medium in the 96-well plate after 48h, adding 90 mu L of serum-free culture medium and 10 mu L of CCK-8 solution into each well, uniformly mixing, and keeping out of the sun; putting the mixture into an incubator to continue incubation for 0.5, 1,2 and 4 hours; OD value was measured with a microplate reader at 450 nm. The experiment was repeated three times. Relative inhibition ratio (OD) Control group -OD Experimental group )/OD Control group ×100％。
Table 1 shows the activity data of the representative compounds 1 to 5 of the invention on four tumor cells (ACHN (human renal carcinoma cell), HepG2 (human liver carcinoma cell), HCT116 (human colon carcinoma cell) and MCF-7 (human breast carcinoma cell) 50 And (5) characterizing. 5-fluorouracil (5-Fu) and a pyrimidone nucleus compound are used as positive controls, wherein the structural formula of the pyrimidone nucleus compound is as follows:
as shown in Table 1, the compounds represented by the present application have good antitumor activity against tumor cells ACHN, MCF-7, HCT-116 and HepG2 cultured in vitro, and other compounds have similar effects. Wherein, the compound Cpd.5 has good proliferation inhibition activity (IC) on breast cancer cells MCF-7 50 2.7 ± 0.1 μ M), the antitumor activity is the best.
2. HTRF kit for detecting PAK5 enzyme activity inhibition
The PAK5 inhibitory activity was determined using the homogeneous time-resolved fluorescence (HTRF) STK-S2 kit (62ST2PEB, Cisbio). PAK5 protein was purchased from Abcam. Briefly, the kinase reaction was performed in 96-well microwell plates (Cisbio) in a reaction volume of 10. mu.L per well, containing the indicated concentrations of test compound, 5. mu.M peptide substrate, 1.5 ng/. mu.L PAK5, and 500. mu.M ATP in kinase buffer. After 20 min incubation at room temperature, the reaction was stopped by adding 5. mu.L of streptavidin-XL 665 and 5. mu.L of STK-antibody detection buffer. The plates were sealed and incubated at room temperature for 1 hour, and the resulting TRF energy transfer signal was then measured on an Envision-PerkinElmer. Fluorescence emission was measured at 620nm (antibody) and 665nm (XL 665). The emissivity of each pore was calculated (665/620), and the percent inhibition for each compound concentration was calculated according to the following formula: percent inhibition ═ max-test)/(max-min) × 100 ("max" denotes the proportion of no compound control and "min" denotes the proportion of no kinase control 50 Values were determined using GraphPad Prism 7.
Table 2 shows the activity data for the representative compounds of the invention, Cpd.1-Cpd.5, for PAK5, using IC for activity 50 And (5) characterizing.
The HTRF KinEASE kit is used for detecting the PAK5 kinase activity, and as can be seen from Table 2, the compounds represented by the application have good PAK5 inhibitory activity, and other compounds also have similar effects. Wherein, the enzyme activity inhibition ability of the compound Cpd.5 exceeds that of the pan-PAK inhibitor sunitinib, and the activity effect is optimal.
3. Compound Cpd.5 inhibits breast cancer cell MCF-7 migration
The effect of compound cpd.5 on tumor cell migration was investigated using a scratch test, the results of which are shown in fig. 1. MCF-7 cells were treated with 1. mu.M and 5. mu.M of compound Cpd.5 and the positive control drug 5-FU, respectively, and the migration of MCF-7 cells was photographed at 0h and 24h for each group.
The experimental results show that: the mobility of MCF-7 cells treated by the 5-FU group and the compound Cpd is lower than that of a blank Control group at 24h, the capability of inhibiting cell migration is further enhanced along with the increase of the administration concentration, and the mobility of the MCF-7 cells is gradually reduced and even better than that of the Control drug 5-FU. The difference was statistically significant compared to the control group (. about.. about.p < 0.0001). At a concentration of 5 μ M, the mobility of MCF-7 cells in the compound cpd.5 group was significantly lower than that of 5-FU, indicating that the compound cpd.5 was able to inhibit the migration of MCF-7 in breast cancer cells, with statistical differences (× P < 0.0001).
4. Compound Cpd.5 induces MCF-7 cycle arrest in breast cancer cells
MCF-7 cells were treated with different concentrations of compound Cpd.5(0.1, 0.5, 1. mu.M), pyrimidinone nuclei and 5-fluorouracil for 36h before cell cycle detection using PI single staining in combination with flow cytometry, and the histogram showed the percentage of cell cycle distribution.
As can be seen from FIG. 2, the MCF-7 cells in the blank control group were mostly localized in G 0 /G 1 In the meantime, the ratio of each cell cycle cell is not changed greatly (P) after the action of pyrimidone mother nucleus and low concentration (0.1-0.5 mu M) compound Cpd.5>0.05). After high concentration (1. mu.M) of compound Cpd.5, G was compared to the blank 0 /G 1 The proportion of cells in the phase is significantly reduced, G 2 Increased cell proportion in the M phase (P)<0.05), indicating that compound cpd.5 induces G2/M phase arrest in MCF-7 cells.
5. Research on antitumor activity of compound Cpd.5 in vivo
Establishing MCF-7 nude mouse transplantation tumor model by using 4-6 week-old female SPF-level BALB/cA-nu nude mice, wherein tumors begin to form in about 30 days, and the tumor volume reaches 100mm 3 Then, the administration was started by dividing into four groups at random, followed by observation for 20 days, and weighing every other dayBody weight and tumor formation volume of nude mice were measured, and after 20 days, the mice were sacrificed and tumors were removed (fig. 3A).
No death or adverse symptoms were observed in the mice during the administration period, and no significant change in body weight was observed in the nude mice of the compound Cpd.5 administration group compared to the control group, indicating that the compound was not toxic in vivo. Under the condition of the same administration dosage, the tumor inhibition rate of the compound Cpd.5 reaches 52.8 percent, is improved by 6.9 percent compared with that of a positive control medicament 5-FU, and embodies that the compound Cpd.5 has good in-vivo anti-tumor activity.
6. Molecular docking results
The molecular docking results of compound cpd.5 with PAK5 protein are shown in fig. 4: the compound cpd.5 may interact with both the hinge region and the DFG region of the PAK5 protein. Wherein, the carbonyl group at the 2-position in the structure of the 4-phenyl-3, 4,6, 7-tetrahydro-1H-pyrrolo [3,4-d ] pyrimidine-2, 5-diketone can form a hydrogen bond with the key amino acid residue leu526 in the hinge region; the mesyl on the side chain at position 6 can form a hydrogen bond with Asp586 in the DFG region, thereby inhibiting the enzyme activity of PAK 5.
The above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A compound, isomer, hydrate, solvate or pharmaceutically acceptable salt thereof shown in formula I,
wherein the content of the first and second substances,
R 1 represents hydrogen or C 1 -C 6 An alkyl group;
R 2 represents hydrogen, halogen, cyano, trifluoromethylRadical, hydroxyl, amino, nitro, C 1 -C 6 Alkyl radical, C 1 -C 6 Alkoxy, phenyl, substituted phenyl, phenoxy, substituted phenoxy, benzyl or substituted benzyl, wherein the substituted phenyl, substituted phenoxy or substituted benzyl can be optionally mono-or polysubstituted by the following substituents: cyano, trifluoromethyl, hydroxy, amino, nitro, C 1 -C 4 Alkyl or C 1 -C 4 An alkoxy group;
l represents a covalent bond, -CH 2 -、-CH 2 CH 2 -、-NH-CH 2 -、-CH 2 -NH-, -NH-CO-or-CO-NH-;
R 3 represents halogen, hydroxy, C 1 -C 4 Alkoxy radical, C 1 -C 4 Alkylthio group, CH 3 -SO 2 -or CH 2 CH 3 -SO 2 -；
n represents an integer of 1 to 3.
2. A compound, isomer, hydrate, solvate or pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 Represents hydrogen, methyl, ethyl, isopropyl or tert-butyl; preferably, R 1 Represents hydrogen, methyl or ethyl.
3. A compound, isomer, hydrate, solvate or pharmaceutically acceptable salt thereof according to claim 1, wherein R 2 Represents hydrogen, trifluoromethyl, fluorine, chlorine, cyano, methyl, ethyl, methoxy, ethoxy, phenoxy or benzyl; preferably, R 2 Represents hydrogen, trifluoromethyl or phenoxy.
4. A compound, isomer, or pharmaceutically acceptable salt thereof according to claim 1, wherein R 3 Represents fluorine, chlorine, hydroxyl, methoxy, ethoxy, methylthio, ethylthio or CH 3 -SO 2 -; preferably, R 3 Represents fluorine, hydroxyl, methoxy, ethoxy, methylthio or CH 3 -SO 2 -。
5. A compound, isomer, hydrate, solvate or pharmaceutically acceptable salt thereof according to claim 1, wherein L represents a covalent bond, -CH 2 -or-CH 2 CH 2 -; n represents 1 or 2.
6. The compound, isomer, hydrate, solvate or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the group consisting of:
7. a process for the preparation of a compound according to claim 1, wherein the synthetic route is as follows:
8. a pharmaceutical composition comprising a compound, isomer, hydrate, solvate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 as an active ingredient or a main active ingredient, in association with a pharmaceutically acceptable carrier.
9. Use of a compound, isomer, hydrate, solvate or pharmaceutically acceptable salt thereof according to any of claims 1-6 for the manufacture of a medicament for the treatment of a disease associated with PAK 5.
10. The use according to claim 9, wherein the PAK 5-related disease is colorectal cancer, liver cancer, gastric cancer, cervical cancer, renal cancer, breast cancer or diabetes.
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Non-Patent Citations (4)
| Title |
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| ALTAF, REEM ET AL.,: "Synthesis, Biological Evaluation, 2D-QSAR, and Molecular Simulation Studies of Dihydropyrimidinone Derivatives as Alkaline Phosphatase Inhibitors", 《ACS OMEGA》, vol. 7, no. 8, pages 7140 * |
| MOSTAFA, AMANY S.ET AL.,: "Synthesis and In Vitro Activity of Pyrrolo[3,4-d]pyrimidine-2,5-diones as Potential Non-nucleoside HCV Inhibitors", 《CURRENT ENZYME INHIBITION》, vol. 12, no. 2, pages 171 * |
| PEREZ, ROLANDO ET AL.,: "Traceless Solid-Phase Synthesis of Bicyclic Dihydropyrimidones Using Multidirectional Cyclization Cleavage", 《JOURNAL OF COMBINATORIAL CHEMISTRY》, vol. 4, no. 5, pages 4 - 6 * |
| REGISTRY: "1381398-11-3等", 《STN》, pages 1 - 92 * |
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