CN115093419B - Pyrimidinone compound, preparation method and medical application thereof - Google Patents
Pyrimidinone compound, preparation method and medical application thereof Download PDFInfo
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Abstract
The invention relates to a pyrimidinone compound, a preparation method and application thereof, belonging to the technical fields of pharmaceutical chemistry and pharmacotherapeutics. The compound shown in the formula I, the isomer, the hydrate, the solvate or the pharmaceutically acceptable salt thereof has good PAK5 inhibition activity, can be used as a PAK5 inhibitor, and can be applied to preparing medicaments for diseases related to PAK 5.
Description
Technical Field
The invention belongs to the technical fields of pharmaceutical chemistry and pharmacotherapeutics, and particularly relates to a pyrimidinone compound. The compounds can be used for preparing medicines for treating 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 I (PAK 1, PAK2, PAK 3) and II (PAK 4, PAK5, PAK 6). Class I PAKs and class II PAKs have 50% homology, class II PAKs (PAK 4, PAK5, PAK 6) have 80% homology, both with a conserved C-terminal kinase domain and an N-terminal regulatory domain, with 1 autophosphorylation region at the N-terminal (P21 GTPase Binding Domain, PBD). By analyzing the crystal structure of the PAKs protein, the following steps are found: the N-terminal of type II PAKs kinase has no PID inhibition region relative to type I PAK kinase, so the kinase activity is higher. The kinase activity of type II PAKs is mainly determined by the kinase catalytic domain at the C-terminus, whereas PAK5 is considered as an important mediator of tumor development, and from each subtype structure, PAK5 shows its own functions and mechanisms during tumor development.
The research on the action mechanism shows that the p21 activated kinase 5 (PAK 5) can be positioned at mitochondria, and the Ser112 locus of the phosphorylated BAD protein triggers the anti-apoptosis of tumor cells; phosphorylating the p65 subunit of NF- κB to promote nuclear translocation of p65, thereby up-regulating the expression of cyclin CyclinD1 and promoting 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; phosphorylating the p120-catenin-Ser288 complex causes cytoskeletal recombination, promoting tumor cell motility.
PAK5 small molecule inhibitors hold good promise for the treatment of cancers involving high expression of PAK 5. At present, few reports about PAK5 small molecule inhibitors are included, including PF-3758309 developed by the pharmaceutical company of Buddha and GNE-2861 developed by the company of GeneTek.
Disclosure of Invention
The invention aims to provide a pyrimidinone compound based on the prior art, and pharmacological experiments prove that the pyrimidinone compound has good PAK5 inhibition activity and particularly has stronger anti-tumor activity on tumor cells such as kidney cancer cells, liver cancer cells, colon cancer cells, breast cancer cells and the like.
It is another object of the present invention to provide a process for the preparation of the above compounds.
It is a further object of the present invention to provide a pharmaceutical use of the above-mentioned compounds.
The technical scheme of the invention is as follows:
the invention relates to a compound with a structure shown in a general formula I, an isomer, a hydrate, a solvate or pharmaceutically acceptable salts thereof,
wherein, the liquid crystal display device comprises a liquid crystal display device,
R 1 represents hydrogen or C 1 -C 6 An alkyl group;
R 2 represents hydrogen, halogen, cyano, trifluoromethyl, hydroxy, amino, nitro, C 1 -C 6 Alkyl, C 1 -C 6 Alkoxy, phenyl, substituted phenyl, phenoxy, substituted phenoxy, benzyl or substituted benzyl, said substituted phenyl, substituted phenoxy or substituted benzyl optionally being mono-or polysubstituted with substituents as follows: 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, C 1 -C 4 Alkylthio, 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, hydroxy, methoxy, ethoxy, methylthio, ethylthio or CH 3 -SO 2 -。
In a more preferred embodiment, R 3 Represents fluorine, hydroxy, 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 synthetic route:
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 were Et 3 N, meOH and reflux.
These intermediates or target compounds can each be purified by conventional isolation techniques and converted, if desired, to addition salts with pharmaceutically acceptable acids.
The invention also provides a pharmaceutical composition, which takes the compound, isomer, hydrate, solvate or pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient, and is assisted with pharmaceutically acceptable auxiliary materials. Wherein the pharmaceutical composition can be tablet, capsule, granule, injection or spray. 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 retarders, and the like.
The pyrimidinone compound provided by the invention has good PAK5 inhibition activity, can be used as a PAK5 inhibitor and is used for preparing medicaments 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" means those salts which retain the biological effectiveness and properties of the parent compound. Such salts include:
(1) Salified with acids obtained by reaction of the free base of the parent compound with inorganic acids including hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid, perchloric acid, and the like, or with organic acids including 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, gamma-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, malonic acid, and the like.
(2) The acidic proton present in the parent compound is replaced by a metal ion or a salt formed by complexation with an organic base such as alkali metal ion, alkaline earth metal ion or aluminum ion, and an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, quinine, etc.
"pharmaceutical composition" means that one or more of the compounds of the present invention, or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof, is admixed with another chemical ingredient, such as a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate the process of administration to animals.
"pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" refers to an inactive ingredient in a pharmaceutical composition that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the compound being administered, 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 groups (the numerical ranges mentioned herein, e.g., "1 to 20", refer to such groups, which in this case are alkyl groups, which 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, t-butyl, pentyl, and the like. Preferably, the alkyl group is a lower alkyl group having 1 to 8 or 1 to 6 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, etc. 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, cyclopentoxy, 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 inhibit proliferation and migration processes of tumor cells by blocking cell cycle, and can obviously inhibit the growth of breast cancer MCF-7 cell nude mice transplantation tumor, so that the pyrimidinone compound can be used as a PAK5 inhibitor and applied to the treatment of PAK5 related diseases.
Drawings
FIG. 1 is a scratch assay to examine the effect of compound Cpd.5 on the ability of breast cancer MCF-7 cells to migrate; FIG. 1A is a scratch test; b in fig. 1 is the healing rate;
FIG. 2 is a graph of the effect of flow cytometry detection compound Cpd.5 on the cell cycle distribution of breast cancer MCF-7; in the six figures in the first and second rows, the left peak represents G0/G1, the right peak represents G2/M, and the middle portion represents S.
FIG. 3 is the effect of compound Cpd.5 on proliferation of nude mouse human breast cancer MCF-7 cell transplantation tumor; FIG. 3A is an anatomic view of the tissue of a nude mouse human breast cancer MCF-7 cell transplantation tumor; FIG. 3B is a graph showing the tumor weight change curve of the nude mouse human breast cancer MCF-7 cell transplantation tumor; FIG. 3C is a tumor volume growth curve of a nude mouse human breast cancer MCF-7 cell transplantation tumor;
FIG. 4 is the DOCK result of compound Cpd.5 with PAK5 protein (PDB: 2F 57).
Detailed Description
The pyrimidinones according to 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: the reaction conditions are TsOH, etOH and reflux
Into a round bottom flask was charged 3-phenoxybenzaldehyde 1a (10 mmol), ethylurea (12 mmol), ethyl acetoacetate (1.26 mL,10 mmol) and TsOH.4H 2 O (0.24 g,1 mmol) was added to absolute ethanol and stirred, and heated at 78 ℃. The reaction was monitored by TLC. After about 4h the reaction was complete, the resulting precipitate was filtered and the crude product was purified by recrystallisation from an appropriate solvent to give 2.10g of intermediate (4 a) 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。
and a second step of: the reaction conditions are AIBN and CCl 4 And reflux flow
The prepared ethyl 1-ethyl-6-methyl-2-oxo-4- (3-phenoxyphenyl) -1,2,3, 4-tetrahydropyrimidine-5-carboxylate intermediate 4a (10 mmol) was added to CCl with vigorous stirring 4 (25 mL) solution, warmed to reflux, and 2.67g (15 mmol) of NBS was added to the reaction solution in portions over 1.5 h. Reflux was continued for 10h. After the reaction was completed, 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 brominated intermediate product (5 a).
And a third step of: the reaction conditions were Et 3 N, meOH and reflux
Prepared ethyl 1-ethyl-6- (bromomethyl) -2-oxo-4- (3-phenoxyphenyl) -1,2,3, 4-tetrahydropyrimidine-5-carboxylate 5a (2.00 mmol) and 4- (methylsulfonyl) aniline (2.20 mmol) were dissolved in tetrahydrofuran, stirred uniformly, triethylamine (0.57 ml,4.40 mmol) was added, heated and refluxed for about 4 hours, and the reaction was completed. Cooling to room temperature, filtering to remove precipitate, concentrating the filtrate, adding ethyl acetate (10 mL×3) for dilution, washing with dilute hydrochloric acid, saturated sodium bicarbonate and saturated saline (10 mL×3), combining organic phases, drying over anhydrous sodium sulfate, and distilling under reduced pressure to obtain crude product as an oil, and subjecting to column chromatography (PE: EA=8:3) to obtain the target 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
For a specific experimental procedure for compound cpd.1, reference is made to example 1, which is synthesized as follows:
referring to the preparation method in example 1, compound cpd.1 was obtained as a white solid product, 0.512g, yield 65.07%. 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
For a specific experimental procedure for compound cpd.2, reference is made to example 1, which is synthesized as follows:
referring to the production method in example 1, compound cpd.2 was obtained in 67% yield. 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
For a specific experimental procedure for compound cpd.3, reference is made to example 1, which is synthesized as follows:
referring to the preparation method 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
For a specific experimental procedure for compound cpd.4, reference is made to example 1, which is synthesized as follows:
referring to the preparation method in example 1, compound cpd.4 was obtained as a white solid product, 0.512g, yield 65.07%. 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 present invention:
1. testing proliferation inhibitory Activity of tumor cells by CCK-8 method
Cells in logarithmic growth phase in the pancreatin digestion culture dish are counted by a counting plate, and 3000-6000 cells are paved in each of the other 96-well plates except that 200 mu L of PBS is added to the peripheral holes; 4 duplicate wells were set, and 10% FBS in complete medium 10 was added to each well0 μL; after the cells are attached, the culture medium in the 96-well plate is discarded by reverse buckling, the culture medium containing the compounds with different concentrations is added, and the mixture is placed at 37 ℃ and 5 percent CO 2 Culturing in an incubator for 48 hours; after 48 hours, the old culture medium in the 96-well plate is discarded, 90 mu L of serum-free culture medium and 10 mu L of CCK-8 solution are added into each well, and the mixture is uniformly mixed and is operated in a dark place; placing the mixture into an incubator for continuous incubation for 0.5,1,2 and 4 hours; OD value was measured by a microplate reader at 450 nm. The experiment was repeated three times. Relative inhibition = (OD Control group -OD Experimental group )/OD Control group ×100％。
Table 1 shows the activity data of the present invention representing compounds 1-5 for four tumor cells (ACHN (human kidney cancer cell), hepG2 (human liver cancer cell), HCT116 (human colon cancer cell) and MCF-7 (human breast cancer cell), activity use IC 50 Characterization. Taking 5-fluorouracil (5-Fu) and a pyrimidinone mother nucleus compound as positive controls, wherein the pyrimidinone mother nucleus compound has the following structural formula:
as can be seen from Table 1, the compounds represented in 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 μm), the antitumor activity was optimal.
2. HTRF kit for detecting PAK5 enzyme activity inhibition effect
PAK5 inhibition activity was determined using the Homogeneous Time Resolved Fluorescence (HTRF) STK-S2 kit (62 ST2PEB, cisbio). PAK5 protein was purchased from Abcam. Briefly, kinase reactions were performed in 96-well microwell plates (Cisbio) with 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. Incubation at room temperature 20After minutes, the reaction was stopped by adding 5. Mu.L of streptavidin-XL 665 and 5. Mu.L of detection buffer for STK-antibody. Plates were sealed and incubated for 1 hour at room temperature, and then the resulting TRF energy transfer signal was measured on an Envision-Perkinelmer. Fluorescence emissions were measured at 620nm (antibody) and 665nm (XL 665). Emissivity of each well was calculated (665/620) and percent inhibition for each compound concentration was calculated according to the following formula: percent inhibition = (max-test)/(max-min) ×100 ("max" represents the proportion of no compound control, "min" represents the proportion of no kinase control. IC 50 Values were determined using GraphPad Prism 7.
Table 2 shows the activity data for compounds Cpd.1-Cpd.5 of the invention for PAK5, active use IC 50 Characterization.
The HTRF KinEASE kit is used for detecting the activity of the PAK5 kinase, and as can be seen from table 2, the compounds represented by the application have good PAK5 inhibitory activity, and other compounds have similar effects. Wherein, the enzyme activity inhibition capability 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 studied using a scratch assay, 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 5-FU, respectively, as positive control, and the migration of MCF-7 cells was observed in each of the groups at 0h and 24 h.
The experimental results show that: both the 5-FU group and compound cpd. the group treated MCF-7 cells had lower mobility at 24h than the Control group, and their ability to inhibit cell migration was further enhanced with increasing dosing concentration, with progressive decrease in mobility of MCF-7 cells, even better than the Control 5-FU. The differences compared to the control group were statistically significant (×p < 0.0001). At a concentration of 5. Mu.M, the mobility of the MCF-7 cells of compound Cpd.5 group was significantly lower than that of 5-FU, indicating that compound Cpd.5 was able to inhibit the migration of MCF-7 cells of breast cancer, the differences were statistically significant (P < 0.0001).
4. Compound Cpd.5 induces MCF-7 cycle arrest in breast cancer cells
MCF-7 cells were treated with various concentrations of compound Cpd.5 (0.1, 0.5, 1. Mu.M), pyrimidinone nuclei and 5-fluorouracil for 36h and the cell cycle was examined by PI single-dye combined flow cytometry and the histogram showed the percentage of cell cycle distribution.
As can be seen from FIG. 2, the MCF-7 cells of the blank group are mostly located at G 0 /G 1 At stage, after the pyrimidinone mother nucleus and the compound Cpd.5 with low concentration (0.1-0.5 mu M), the cell proportion of each cell cycle is not changed greatly (P)>0.05). After action of Cpd.5 at high concentration (1. Mu.M), G relative to the control group 0 /G 1 The cell proportion in the phase is obviously reduced, G 2 Increased cell ratio in the/M phase (P)<0.05 Compound cpd.5 induced G2/M phase arrest of MCF-7 cells.
5. In vivo anti-tumor Activity study of Compound Cpd.5
Establishing an MCF-7 nude mice transplantation tumor model by using 4-6 week old female SPF-grade BALB/cA-nu nude mice, and starting to form tumors about 30 days, wherein the tumor volume reaches 100mm 3 After the start of the administration, the mice were randomly divided into four groups, and the weight and the tumor formation volume of the nude mice were weighed every other day by day for 20 days, and after 20 days, the mice were sacrificed and the tumors were removed (fig. 3A).
During the dosing period, mice did not see death and adverse symptoms, and there was no significant change in body weight of nude mice in the compound cpd.5 dosed group compared to the placebo 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 group reaches 52.8 percent, which is improved by 6.9 percent compared with the positive control medicine group 5-FU, thus showing that the compound Cpd.5 has good in vivo anti-tumor activity.
6. Molecular docking results
The molecular docking of compound cpd.5 with PAK5 protein is shown in fig. 4: compound cpd.5 may interact with both the hinge and DFG regions of the PAK5 protein. Wherein, the carbonyl group at the 2-position in the 4-phenyl-3, 4,6, 7-tetrahydro-1H-pyrrolo [3,4-d ] pyrimidine-2, 5-dione structure can form a hydrogen bond with the key amino acid residue leu526 of the hinge region; the methanesulfonyl group in the 6-position side chain can form a hydrogen bond with the Asp586 of the DFG region, thereby inhibiting the enzymatic activity of PAK 5.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments may be modified or some technical features may be replaced equivalently; such modifications and substitutions do not depart from the spirit of the invention.
Claims (4)
1. A compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:
2. a pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof as a major active ingredient, in combination with a pharmaceutically acceptable carrier.
3. Use of a compound of claim 1, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of a PAK 5-related disorder.
4. The use according to claim 3, 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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