CN110156729B - Phenylpiperazine UBE2F small-molecule inhibitor and synthetic method thereof - Google Patents
Phenylpiperazine UBE2F small-molecule inhibitor and synthetic method thereof Download PDFInfo
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Abstract
The invention discloses a phenylpiperazine UBE2F micromolecule inhibitor and a synthetic method thereof, discloses a phenylpiperazine compound shown in a general formula I or pharmaceutically acceptable salt thereof, and further discloses a synthetic route of the general formula I and a synthetic method of each step. The phenylpiperazine compound is a targeted UBE2F micromolecule inhibitor, and can effectively inhibit the growth of tumor cells by preventing the G2/M process of the cell cycle and inducing apoptosis of the human tumor cells. Therefore, the compound is a novel specific anti-tumor drug by targeting UBE2F.
Description
Technical Field
The invention belongs to the technical field of medicines, particularly relates to the field of chemical drug synthesis and pharmacological activity, and particularly relates to a phenylpiperazine targeted UBE2F antitumor small-molecule inhibitor and a synthesis method thereof.
Background
The Neddylation pathway is a post-translational modification of a protein that covalently binds the ubiquitin-like molecule NEDD8 to the substrate protein molecule by activating a cascade of enzymatic reactions of enzyme E1 (NAE), coupling enzyme E2 (UBE 2M and UBE 2F) and ligase E3. Cullin-RING ligases (CRLs) are the largest superfamily of ubiquitin ligases E3, whose activity is dependent on the NEDD8 modification of the Cullin molecule. Studies have shown that aberrant regulation of CRLs E3 ubiquitin ligase can lead to abnormal proliferation of cells, genomic instability and tumor progression, and that overexpression is associated with a poor prognosis in a variety of tumors.
UBE2M E2 coupled enzyme catalyzes and regulates NEDD8 modification of Cullin1/2/3/4A/4B, while UBE2F E coupled enzyme specifically regulates NEDD8 modification of Cullin 5. CRL 5E 3 ubiquitin ligase can modify the pro-apoptotic protein NOXA through the K11 ubiquitin chain, thereby promoting NOXA degradation; inactivation of CRL 5E 3 ubiquitin ligase can promote accumulation of NOXA, thereby inducing NOXA-dependent apoptosis in tumor cells.
In tumor cells, aberrant activation of key molecules of the Neddylation pathway is common. Targeting key molecules in the Neddylation pathway is a new direction for developing novel antitumor drugs. At present, MLN4924 (also called Pevonedistat) as an NAE inhibitor enters clinical Ib-II stage, and shows remarkable anti-tumor curative effect. And inevitably exhibit large side effects due to its inhibition of the entire Neddylation pathway. Research shows that the activity of CRL 5E 3 ubiquitin ligase is closely related to the occurrence and development of lung cancer. The targeted UBE2F can selectively inhibit the activity of CRL 5E 3 ubiquitin ligase, induce the lung cancer cells to undergo apoptosis and simultaneously reduce the side effect of the compound as much as possible.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a phenylpiperazine UBE2F small molecule inhibitor which targets UBE2F, can selectively inhibit the activity of CRL 5E 3 ubiquitin ligase, induces lung cancer cells to undergo apoptosis and simultaneously reduces the side effect of the compound as much as possible.
The invention adopts the following technical scheme: a phenylpiperazine UBE2F small molecule inhibitor having a structure of formula I, or a salt having a structure of formula I:
wherein X and Y are selected from carbonyl and sulfonyl;
R 1 selected from methyl, phenyl, vinyl, 2-naphthyl, morpholinyl, indolyl, N, N-dimethyl, furyl, 4-nitrophenyl, 4-methyl-phenyl;
R 2 selected from furyl, phenyl, 5-N, N-dimethylaminonaphthyl, 4-methyl-phenyl, vinyl, 2-naphthyl, methyl, 2-pyridyl, 2-indolyl, N-dimethyl, morpholinyl, 3-bromo-phenyl, 4-nitro-phenyl, styryl, 2-pyridine-vinyl, 3-pyridine-vinyl, 4-pyridine-vinylA group, 4-methyl-styryl, 5-pyrimidinylyl, 5-methyl-furanylvinyl, 4-nitro-styryl, 2-naphthyl, 3-indolyl, 2-fluoro-styryl, 4-pyrazolyl-styryl, 2-chloro-styryl, 3-chloro-styryl, 4-fluoro-styryl, 3-trifluoromethyl-styryl, 1,2-dimethyl-3-indolynyl, 5-methyl-3-indolynyl, 6-bromo-3-indolynyl, 4-cyano-3-indolynyl, 5-nitro-3-indolynyl, 3-ethylindolyl, 4-aza-3-indolynyl, 5-azaindolynyl, 6-azaindolynyl, 7-azaindolynyl, 6-methoxy-3-indolynyl, 7-methoxy-3-indolynyl, 1-phenyl-3-indolynyl, 7-cyano-3-indolynyl.
The above structure was synthesized by the following scheme:
1) Preparation of e
4-fluoronitrobenzene and Boc protected piperazine are subjected to substitution reaction under alkaline conditions, after the reaction is completed, a compound b is obtained by extraction, drying and evaporation, the compound b is reduced by Pd/C in an alcoholic solvent, and is subjected to suction filtration and evaporation to obtain a compound C, the compound C is subjected to reaction with 3-chlorobenzoyl chloride in an alkaline organic solvent, and after extraction, drying and evaporation to obtain a compound d, the compound d is placed in a strong acid solvent, and the pH value is adjusted to obtain a compound e; the base used in the method can be potassium carbonate, sodium bicarbonate, triethylamine, DIPEA (N, N-diisopropylethylenediamine), sodium hydroxide and potassium hydroxide, the strong acid used in the method can be hydrochloric acid, sulfuric acid and trifluoroacetic acid, and the solvent used in the method can be water, dichloromethane, tetrahydrofuran, dioxane, methanol and ethanol;
2) h preparation method
b, removing a Boc protecting group under a strong acid condition, adjusting pH with alkali to obtain f, reacting the compound f with benzoyl chloride in an organic solvent under an alkaline condition, extracting, drying and evaporating to obtain a compound g, reducing the g in alcohol by using Pd/C, and performing suction filtration and evaporation to obtain a compound h; the base used in the method can be potassium carbonate, sodium bicarbonate, triethylamine, DIPEA, sodium hydroxide and potassium hydroxide, the strong acid used in the method can be hydrochloric acid, sulfuric acid and trifluoroacetic acid, and the solvent used in the method can be water, dichloromethane, tetrahydrofuran, dioxane, methanol and ethanol;
3) Preparation method of i
After a compound e or h is prepared, carrying out condensation reaction on T3P (1-propylcyclic phosphoric anhydride) serving as a condensing agent and corresponding carboxylic acid or reacting with sulfonyl chloride under an alkaline condition, extracting, drying, evaporating to dryness, and passing through a chromatographic column to obtain pure i; the base used in the process may be potassium carbonate, sodium bicarbonate, triethylamine, DIPEA, and the solvent used is dichloromethane, ethyl acetate, DMF (N, N-dimethylformamide), tetrahydrofuran, dioxane.
In the general formula I, X, Y and R 1 ，R 2 Selected from the following combinations:
the synthesis scheme is as follows:
the operation of the second scheme is that a compound f reacts with p-methyl benzoyl chloride under an alkaline condition, j is obtained after extraction, drying and evaporation, then reduction is carried out under a Pd/C condition, k is obtained after suction filtration and evaporation, l is obtained after purification, the k is reacted with Meldrum's acid, knoevenagel reaction is carried out on the l and various aromatic aldehydes under the alkaline condition, and m is obtained after column chromatography purification of a product; the base used in the process may be potassium carbonate, sodium bicarbonate, triethylamine, DIPEA, piperidine, and the solvent used is dichloromethane, ethyl acetate, DMF (N, N-dimethylformamide), tetrahydrofuran, dioxane, methanol, ethanol, toluene.
The invention has the beneficial effects that: the phenylpiperazine UBE2F small molecule inhibitor provided by the invention targets UBE2F, can selectively inhibit the activity of CRL 5E 3 ubiquitin ligase, induces lung cancer cells to apoptosis, and simultaneously reduces the side effect of the compound as much as possible.
Drawings
Table 1 shows: randomly selected different small molecules encompassed by the present invention have half inhibitory concentrations on the growth of human lung squamous carcinoma cells H2170;
FIG. 1 is a diagram: half inhibitory concentration (IC 50) of compound 47 small molecule inhibitor on human lung squamous carcinoma cells H2170, human lung adenocarcinoma cells H358 and H1650;
FIG. 2 is a diagram of: after the human lung squamous carcinoma cell H2170 and the human lung adenocarcinoma cell H1650 are treated by the compound 47 micromolecule inhibitor with different concentrations for 24 hours, a flow cytometer is used for detecting the cell cycle distribution and drawing a cell cycle distribution diagram;
after the human lung squamous carcinoma cell H2170 and the human lung adenocarcinoma cell H1650 are treated by the compound 47 small molecule inhibitor with different concentrations for 24 hours, western blot detects the change of apoptosis-related protein PARP, caspase3 and NOXA protein;
FIG. 3 is a diagram of: human lung squamous carcinoma cells H2170 and human lung adenocarcinoma cells H1650 are treated by 20 mu M compound 47 small molecule inhibitor for different time or different dose of compound 47 small molecule inhibitor for 24 hours, and then the level of modification of Cul5 protein neddylation and the level of protein NOXA as substrate protein and the level of modification of other cullin protein neddylation are detected by Western blot;
FIG. 4 is a diagram of: human squamous cell lung carcinoma cells H2170 Westernblot detects changes in UBE2F protein intracellularly after 24 hours of treatment with different concentrations of the small molecule inhibitor of Compound 47.
Detailed Description
To better illustrate the present invention, specific examples are as follows:
example 1
1) b Synthesis
4-fluoronitrobenzene (10g, 70.87mmol), boc piperazine (111g, 59.06mmol), potassium carbonate (12.24g, 88.59mmol) and 100mL of acetonitrile are sequentially added into a 250mL round-bottom flask as a solvent, and the mixture is heated and refluxed for 5 hours; after cooling, the mixture is evaporated to dryness and 150mL of CH are added in 3 portions 2 Cl 2 Transferring, washing with water for three times, mixing organic phases, adding anhydrous magnesium sulfate, drying, evaporating to dryness to obtain b (14.9 g), with yield of 82.64%, 1 H NMR(400MHz,CDCl 3 )δ8.05(d,J＝7.6Hz,2H),7.13–6.69(m,2H),3.64(t,J＝7.1Hz,4H),3.25(t,J＝7.1Hz,4H),1.47(s,9H).
2) Synthesis of g
50mL CH 2 Cl 2 Adding b (13g, 42.3mmol) obtained in the previous step, and slowly dropwise adding 15mL of CF in 20min under ice-bath condition 3 COOH to the reaction system, and then stirring for 4 hours at normal temperature; evaporating to dryness after the reaction is completed, adding 25mL of water to dissolve the solid, dropwise adding 3mol/L NaOH to neutralize until the pH value is 10 to generate a large amount of yellow precipitate, and performing suction filtration on the turbid solution to obtain g (7.9 g) of yellow solid; the yield was 88.9%, 1 H NMR(400MHz,CDCl 3 )δ8.20–7.88(m,2H),7.14–6.86(m,2H),3.42–3.29(m,4H),3.21–3.06(m,4H),1.92(m,1H).
3) h Synthesis
In 100mL CH under ice bath conditions 2 Cl 2 Adding g (7g, 33.78mmol) and triethylamine (4.44g, 43.91mmol), gradually dropwise adding benzoyl chloride (5.46g, 38.85mmol), transferring to room temperature after dropwise addition, and stirring for 2h; after the reaction is completed, 50mL of water is added for quenching, and CH is added 2 Cl 2 50mL of solvent is washed by water (50 mL multiplied by 3), and the organic phase is added with anhydrous magnesium sulfate, dried and evaporated to dryness to obtain h (9.50 g); the yield is 90.33 percent
4) Synthesis of i
Adding h (8g, 25.70mmol) and palladium carbon (1.37 g, content 10%) in turn into 50mL ethanol, stirring at room temperature for 5h under hydrogen atmosphere, filtering to remove palladium after reaction is completeC, evaporating the filtrate to dryness to obtain i (6.5 g); the yield was 89.91%, 1 H NMR(400MHz,CDCl 3 )δ7.42(s,5H),6.85–6.77(m,2H),6.67–6.61(m,2H),3.74(d,4H),3.02(d,4H).
5) Synthesis of Compound 1
5mL CH under ice bath conditions 2 Cl 2 Adding 2-furancarboxylic acid (47.80mg, 0.426mmol) and T3P (169.63mg, 0.533mmol), stirring for 5min, dropwise adding triethylamine (53.95mg, 0.533mmol), adding i (100mg, 0.355mmol), and reacting at normal temperature for 4h; after the reaction was complete, 15mL of water was added for quenching, followed by 30mL of CH 2 Cl 2 After washing with water (50 mL. Times.3), the organic phase was dried over anhydrous magnesium sulfate and purified by column Chromatography (CH) 2 Cl 2 MeOH =20, 1,v/v) to give 1 (78.32 mg); the yield was 58.46%, 1 H NMR(400MHz,DMSO-d 6 )δ10.09(s,1H),7.95(m,2H),7.70–7.61(m,2H),7.60–7.40(m,8H),7.04–6.87(m,2H),3.77(s,4H),3.13(d,J＝9.7Hz,4H). 13 C NMR(100MHz,DMSO-d 6 )δ168.98,164.97,147.14,135.79,135.06,131.58,131.27,129.54,128.41,128.28,127.47,126.94,121.45,116.15,48.98.
EXAMPLE 2 Synthesis of Compound 2
5mL CH under ice bath conditions 2 Cl 2 Adding benzoic acid (52.09mg, 0.426mmol) and T3P (169.63mg, 0.533mmol), stirring for 5min, dropwise adding triethylamine (53.95mg, 0.533mmol), adding i (100mg, 0.355mmol), and reacting at room temperature for 4h; after the reaction was complete, 15mL of water was added for quenching, followed by 30mL of CH 2 Cl 2 After washing with water (50 mL. Times.3), the organic phase was dried over anhydrous magnesium sulfate and purified by column Chromatography (CH) 2 Cl 2 MeOH = 20; the yield was 52.55%, 1 H NMR(400MHz,DMSO-d 6 )δ10.09(s,1H),7.95(m,2H),7.70–7.61(m,2H),7.60–7.40(m,8H),7.04–6.87(m,2H),3.77(s,4H),3.13(d,J＝9.7Hz,4H). 13 C NMR(100MHz,DMSO-d 6 )δ168.98,164.97,147.14,135.79,135.06,131.58,131.27,129.54,128.41,128.28,127.47,126.94,121.45,116.15,48.98.
EXAMPLE 3 Synthesis of Compound 3
Synthesis method of compound 3Referring to the synthesis of compound 2 in example 2, the product was a white solid. The yield was 52.3%, 1 H NMR(400MHz,CDCl 3 )δ8.48(d,J＝8.5Hz,1H),8.35(d,J＝8.6Hz,1H),8.07(dd,J＝7.4,1.3Hz,1H),7.54(t,J＝8.1Hz,1H),7.46–7.33(m,6H),7.17(d,J＝7.5Hz,1H),6.87–6.76(m,2H),6.70–6.57(m,2H),3.69(d,4H),3.04(s,4H),2.87(s,6H). 13 C NMR(100MHz,CDCl 3 )δ170.42,152.00,149.00,135.45,134.26,130.56,130.32,129.89,129.73,129.69,128.55,128.47,127.10,124.93,123.12,118.69,116.96,115.14,49.63,45.42.
EXAMPLE 4 Synthesis of Compound 4
Synthesis of Compound 4 referring to the synthesis of Compound 2 in example 2, the product is a white solid. The yield was 62.5%, 1 H NMR(400MHz,DMSO-d 6 )δ9.99(s,1H),7.86(d,J＝8.1Hz,2H),7.69–7.59(m,2H),7.52–7.41(m,5H),7.32(d,J＝8.0Hz,2H),7.02–6.90(m,2H),3.77(s,4H),3.14(s,4H),2.38(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.01,164.81,147.07,141.24,135.76,132.14,131.63,129.54,128.81,128.42,127.49,126.93,121.46,116.14,48.99,20.95.
EXAMPLE 5 Synthesis of Compound 5
Synthesis of Compound 5 referring to the synthesis of Compound 2 in example 2, the product is a white solid. The yield was 71.6%, 1 H NMR(400MHz,DMSO-d 6 )δ9.97(s,1H),7.55(d,J＝8.5Hz,2H),7.46(m,5H),6.94(d,J＝8.4Hz,2H),6.41(m,1H),6.22(m,1H),5.70(m,1H),3.76(s,4H),3.13(s,4H). 13 C NMR(100MHz,DMSO-d 6 )δ168.97,162.58,146.98,135.78,131.98,131.49,129.53,128.40,126.92,126.08,120.32,116.33.
EXAMPLE 6 Synthesis of Compound 6
Synthesis of Compound 6 referring to the synthesis of Compound 2 in example 2, the product is a white solid. The yield was 35.2%, 1 H NMR(400MHz,DMSO-d 6 )δ9.99(s,1H),8.35(d,J＝2.0Hz,1H),8.08(m,2H),8.00(d,J＝8.1Hz,1H),7.77(m,1H),7.71–7.58(m,2H),7.42(m,5H),7.02–6.92(m,2H),6.84–6.73(m,2H),3.03(s,4H). 13 C NMR(100MHz,DMSO-d 6 )δ168.95,147.95,136.66,135.69,134.10,131.48,129.52,129.19,129.09,128.96,128.79,128.38,127.77,127.74,127.58,126.87,122.99,122.17,116.28,53.46,48.42.
EXAMPLE 7 Synthesis of Compound 7
Synthesis of Compound 7 referring to the synthesis of Compound 2 in example 2, the product is a white solid. The yield was 62.5%, 1 H NMR(400MHz,DMSO-d 6 )δ9.73(s,1H),7.52–7.39(m,7H),6.96–6.82(m,2H),3.75(s,4H),3.10(s,4H),2.00(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ168.97,167.63,146.65,135.77,131.96,129.53,128.41,126.92,120.07,116.38,49.07,23.76.
EXAMPLE 8 Synthesis of Compound 8
Synthesis of Compound 8 referring to the synthesis of Compound 2 in example 2, the product is a white solid. The yield was 56.3%, 1 H NMR(400MHz,DMSO-d 6 )δ10.47(s,1H),8.99–8.56(m,1H),8.19–7.98(m,2H),7.85–7.71(m,2H),7.65(d,1H),7.50–7.43(m,5H),7.02–6.91(m,2H),3.77(s,4H),3.16(s,4H). 13 CNMR(100MHz,DMSO-d 6 )δ168.98,161.86,150.05,148.31,147.29,138.02,135.78,130.73,129.53,128.41,126.93,126.64,122.12,121.19,116.15,48.90.
EXAMPLE 9 Synthesis of Compound 9
Synthesis of Compound 9 referring to the synthesis of Compound 2 in example 2, the product is a white solid. The yield was 39.5%, 1 H NMR(400MHz,CDCl 3 )δ9.72(s,1H),9.52(s,1H),7.71–7.65(m,4H),7.51–7.43(m,4H),7.47–7.40(m,4H),7.26–7.15(m,4H),6.99–6.94(m,2H),3.63(t,J＝7.1Hz,5H),3.20(t,J＝7.1Hz,5H). 13 C NMR(100MHz,CDCl 3 )δ170.10,161.26,145.33,136.01,135.53,134.56,131.72,130.94,128.92,127.59,127.41,124.40,122.81,122.09,114.70,112.28,110.27,49.01,46.37.
EXAMPLE 10 Synthesis of Compound 10
Synthesis of Compound 10 referring to the synthesis of Compound 2 in example 2, the product is a white solid. The yield was 58.3%, 1 H NMR(400MHz,DMSO-d 6 )δ9.49(s,1H),7.48–7.40(m,5H),7.12–7.06(m,2H),6.94–6.88(m,2H),3.60(d,4H),2.65(s,6H). 13 C NMR(100MHz,DMSO-d 6 )δ168.94,147.51,135.78,130.41,129.53,128.40,126.92,122.00,116.59,48.88,37.76.
EXAMPLE 11 Synthesis of Compound 11
Synthesis of Compound 11 referring to the synthesis of Compound 2 in example 2, the product is a white solid. The yield is 56.8 percent, 1 H NMR(400MHz,DMSO-d 6 )δ8.32(s,1H),7.49–7.40(m,5H),7.34–7.28(m,2H),6.90–6.84(m,2H),3.88–3.44(m,8H),3.41–3.36(m,4H),3.15(s,4H). 13 C NMR(100MHz,DMSO-d 6 )δ168.93,155.39,146.09,135.83,132.90,129.51,128.40,126.92,121.06,116.36,65.96,44.09.
EXAMPLE 12 Synthesis of Compound 12
Synthesis of Compound 12 referring to the Synthesis of Compound 2 in example 2, the product was a white solid. The yield was 62.5%, 1 H NMR(400MHz,DMSO-d 6 )δ10.40(s,1H),8.40–8.30(m,2H),8.22–8.11(m,2H),7.70–7.59(m,2H),7.51–7.39(m,5H),7.03–6.91(m,2H),3.63(d,4H). 13 C NMR(100MHz,DMSO-d 6 )δ168.97,163.19,148.97,147.47,140.73,135.79,130.99,129.54,129.01,128.41,126.94,123.47,121.53,116.07,48.83.
EXAMPLE 13 Synthesis of Compound 13
Synthesis of Compound 13 referring to the Synthesis of Compound 2 in example 2, the product was a white solid. The yield was 45.6%, 1 H NMR(400MHz,DMSO-d 6 )δ9.78(s,1H),7.59–7.52(m,2H),7.43(m,5H),7.31(d,J＝8.0Hz,2H),6.97–6.88(m,2H),6.84–6.77(m,2H),3.77–3.37(m,4H),3.06(s,4H),2.32(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ168.93,147.85,142.83,136.79,135.75,129.52,129.48,129.25,128.39,126.90,126.66,122.71,116.32,48.56,20.91.
EXAMPLE 14 Synthesis of Compound 14
Synthesis of Compound 14 referring to the Synthesis of Compound 2 in example 2, the product is a white solid. The yield was 45.8%, 1 H NMR(400MHz,DMSO-d 6 )δ10.19(s,1H),8.00(s,1H),7.91(d,J＝7.8Hz,1H),7.64(d,J＝8.2Hz,3H),7.56(t,J＝7.9Hz,1H),7.47(p,J＝6.0,4.6Hz,5H),6.97(d,J＝8.6Hz,2H),3.63(d,J＝111.5Hz,4H),3.25–3.03(m,4H). 13 C NMR(100MHz,DMSO-d 6 )δ168.98,163.43,147.30,136.99,135.78,133.14,131.20,131.11,130.30,129.54,128.41,127.23,126.93,126.29,121.51,116.10,48.89.
example 15
1) c Synthesis
B (8 g, 26.03mmol) and palladium carbon (1.38 g, content 10%) are sequentially added into 50mL ethanol, stirred for 5h at normal temperature under hydrogen atmosphere, and after complete reaction, the palladium carbon is removed by filtration, and the filtrate is evaporated to dryness to obtain c (6.2 g); the yield was 85.88%.
2) Synthesis of d
In 100mL CH under ice bath conditions 2 Cl 2 C (6 g, 21.63mmol) and triethylamine (3.28g, 32.45mmol) are added, then 3-chloro-benzoyl chloride (4.54g, 25.96mmol) is gradually added dropwise, and after the dropwise addition is completed, the mixture is transferred to room temperature and stirred for 2 hours; after the reaction is completed, 50mL of water is added for quenching, and CH is added 2 Cl 2 50mL of solvent is washed by water (50 mL multiplied by 3), the organic phase is added with anhydrous magnesium sulfate, dried and evaporated to dryness to obtain d (7.60 g); the yield was 84.47%, 1 H NMR(400MHz,DMSO-d 6 )δ10.17(s,1H),7.99(t,J＝1.9Hz,1H),7.90(m,1H),7.73–7.49(m,4H),7.05–6.89(m,2H),3.46(d,J＝10.3Hz,4H),3.06(t,J＝5.2Hz,4H),1.43(s,9H). 13 C NMR(100MHz,DMSO-d 6 )δ163.39,153.81,147.48,137.01,133.13,131.10,130.30,127.23,126.30,121.47,116.11,78.93,48.74,28.04.
3) e Synthesis of
50mL CH 2 Cl 2 Adding the above obtained d (7g, 16.83mmol), and slowly adding 6.5mL CF dropwise over 20min under ice bath condition 3 COOH to the reaction system, and then stirring for 4 hours at normal temperature; evaporating to dryness after the reaction is completed, adding 25mL of water to dissolve the solid, dropwise adding 3mol/L NaOH to neutralize until the pH value is 10 to generate a large amount of yellow precipitate, and performing suction filtration on the turbid solution to obtain the productYellow solid g (4.9 g); the yield was 92.19%.
4) Synthesis of Compound 15
In 5mL CH under ice bath conditions 2 Cl 2 Adding e (100mg, 0.316mmol) and triethylamine (48.06mg, 0.475mmol), then gradually dropwise adding acetyl chloride (29.83mg, 0.380mmol), and after dropwise adding, turning to room temperature and stirring for 2h; after the reaction is completed, 10mL of water is added for quenching, and CH is added 2 Cl 2 30mL of solvent, washing with water (15 mL. Times.3), adding anhydrous magnesium sulfate into the organic phase, drying, and evaporating to dryness to obtain 15 (89.2 mg); the yield was 78.54%, 1 H NMR(400MHz,DMSO-d 6 )δ10.18(s,1H),8.00(t,J＝1.9Hz,1H),7.91(m,1H),7.70–7.49(m,4H),7.04–6.87(m,2H),3.58(m,4H),3.09(m,4H),2.04(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ168.21,163.41,147.39,137.00,133.14,131.11,130.30,127.23,126.29,121.52,116.01,49.07,48.64,45.45,21.15.
EXAMPLE 16 Synthesis of Compound 16
Synthesis of Compound 16 referring to the synthesis of Compound 15 in example 15, the product is a white solid. The yield was 68.3%, 1 H NMR(400MHz,DMSO-d 6 )δ10.18(s,1H),8.00(t,J＝1.9Hz,1H),7.91(m,1H),7.67–7.61(m,3H),7.56(t,J＝7.9Hz,1H),7.04–6.94(m,2H),6.86(m,1H),6.15(m,1H),5.78–5.67(m,1H),3.70(m,4H),3.12(s,4H). 13 C NMR(100MHz,DMSO-d 6 )δ164.21,163.42,147.30,137.00,133.14,131.16,131.11,130.31,128.10,127.44,127.23,126.29,121.51,116.03,49.32,48.64.
EXAMPLE 17 Synthesis of Compound 17
Synthesis of Compound 17 referring to the synthesis of Compound 15 in example 15, the product is a white solid. The yield is 56.8 percent, 1 H NMR(400MHz,DMSO-d 6 )δ10.16(s,1H),8.49(s,1H),8.22(m,2H),8.09(d,J＝7.9Hz,1H),7.97(s,1H),7.88(d,J＝7.8Hz,1H),7.82–7.69(m,3H),7.59(m,4H),6.89(d,J＝8.7Hz,2H),3.23–3.03(m,8H). 13 C NMR(100MHz,DMSO-d 6 )δ163.43,146.85,136.93,134.46,133.12,131.99,131.77,131.39,131.12,130.29,129.39,129.33,129.09,128.86,127.86,127.71,127.21,126.27,122.85,121.44,116.28,48.30,45.85.
EXAMPLE 18 Synthesis of Compound 18
Synthesis of Compound 18 referring to the synthesis of Compound 15 in example 15, the product is a white solid. The yield was 46.9%, 1 H NMR(400MHz,DMSO-d 6 )δ10.27(s,1H),7.98(d,J＝1.9Hz,1H),7.90(d,1H),7.65(d,5H),7.54(t,J＝7.8Hz,1H),7.48(d,J＝8.0Hz,2H),7.08(d,2H),3.26(t,J＝4.8Hz,4H),3.06(t,J＝4.8Hz,4H),2.41(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ163.54,143.87,136.84,133.13,131.61,131.21,130.32,129.90,128.04,127.64,127.27,126.35,125.46,121.42,117.16,49.06,45.37,21.02.
EXAMPLE 19 Synthesis of Compound 19
Synthesis of Compound 19 referring to the synthesis of Compound 15 in example 15, the product is a white solid. The yield was 68.5%, 1 H NMR(400MHz,DMSO-d 6 )δ10.21(s,1H),8.00(t,J＝1.9Hz,1H),7.92(m,1H),7.64(m,3H),7.55(t,J＝7.9Hz,1H),7.01–6.91(m,2H),3.58(m,4H),3.31(m,4H),3.18(t,J＝4.6Hz,4H),3.10(m,4H). 13 C NMR(100MHz,DMSO-d 6 )δ163.39,162.92,147.53,137.01,133.13,131.09,130.30,127.24,126.31,121.50,115.87,65.89,53.32,48.59,46.87,46.17,17.96,16.67,12.20.
EXAMPLE 20 Synthesis of Compound 20
5mL CH under ice bath conditions 2 Cl 2 Adding 2-indolecarboxylic acid (61.24mg, 0.380mmol) and T3P (151.13mg, 0.475mmol), stirring for 5min, dropwise adding triethylamine (48.06mg, 0.474mmol), adding e (100mg, 0.316mmol), and continuing to react for 4h at normal temperature; after the reaction was complete, 15mL of water was added for quenching, followed by 30mL of CH 2 Cl 2 After washing with water (50 mL. Times.3), the organic phase was dried over anhydrous magnesium sulfate and purified by column Chromatography (CH) 2 Cl 2 MeOH =20, 1,v/v) to give 1 (56.23 mg); the yield was 41.66%, 1 H NMR(400MHz,DMSO-d 6 )δ11.68–11.57(m,1H),10.19(s,1H),8.00(t,J＝1.9Hz,1H),7.91(d,J＝7.7Hz,1H),7.73–7.60(m,4H),7.56(t,J＝7.9Hz,1H),7.44(d,J＝8.2Hz,1H),7.20(t,J＝7.6Hz,1H),7.09–6.96(m,3H),6.85(d,J＝2.1Hz,1H),3.22(t,J＝5.2Hz,4H). 13 C NMR(100MHz,DMSO-d 6 )δ163.41,162.01,147.31,137.02,135.91,133.14,131.17,131.12,130.31,129.77,127.24,126.77,126.31,123.21,121.53,121.31,119.73,115.99,112.03,104.07,48.98.
EXAMPLE 21 Synthesis of Compound 21
Synthesis of Compound 21 referring to the synthesis of Compound 15 in example 15, the product is a white solid. The yield was 45.6%, 1 H NMR(400MHz,DMSO-d 6 )δ10.21(s,1H),8.00(d,J＝2.0Hz,1H),7.91(d,J＝7.7Hz,1H),7.67–7.62(m,3H),7.55(t,J＝7.9Hz,1H),7.00–6.94(m,2H),3.30(dd,J＝6.5,3.6Hz,4H),3.16(m,4H),2.81(s,6H). 13 C NMR(100MHz,DMSO-d 6 )δ163.42,147.13,136.98,133.13,131.36,131.12,130.30,127.24,126.31,121.49,116.20,53.38,48.67,45.95,17.98,16.68.
EXAMPLE 22 Synthesis of Compound 22
Synthesis of Compound 22 referring to the synthesis of Compound 20 in example 20, the product is a white solid. The yield was 72.3% by weight, 1 H NMR(400MHz,DMSO-d 6 )δ10.19(s,1H),8.00(t,J＝1.9Hz,1H),7.91(m,1H),7.87(d,J＝2.1Hz,1H),7.69–7.62(m,3H),7.56(t,J＝7.8Hz,1H),7.05(d,J＝3.3Hz,1H),7.02–6.95(m,2H),6.65(m,1H),3.19(m,4H). 13 C NMR(100MHz,DMSO-d 6 )δ163.41,158.25,147.26,146.96,144.73,137.01,133.14,131.20,131.11,130.30,127.23,126.30,121.51,116.00,115.69,111.30,48.96.
EXAMPLE 23 Synthesis of Compound 23
Synthesis of Compound 23 referring to the synthesis of Compound 20 in example 20, the product is a white solid. The yield was 53.2%, 1 H NMR(400MHz,DMSO-d 6 )δ10.18(s,1H),8.35–8.25(m,2H),7.99(t,J＝1.9Hz,1H),7.90(m,1H),7.76–7.69(m,2H),7.66–7.58(m,3H),7.56(t,J＝7.9Hz,1H),7.01–6.92(m,2H),3.62(d,4H),3.11(s,4H). 13 C NMR(100MHz,DMSO-d 6 )δ167.05,163.41,147.80,147.20,142.10,137.00,133.13,131.27,131.12,130.31,128.32,127.23,126.30,123.76,121.49,116.14,48.59,46.72.
EXAMPLE 24 Synthesis of Compound 24
Synthesis of Compound 24 the product was a white solid, as described for Compound 15 in example 15. The yield was 67.3%, 1 H NMR(400MHz,DMSO-d 6 )δ10.04(s,1H),7.60(t,J＝9.4,2.1Hz,4H),7.53(s,1H),7.49–7.40(m,8H),7.00–6.91(m,2H),6.81(d,1H),3.76(s,4H),3.13(s,4H). 13 C NMR(100MHz,DMSO-d 6 )δ168.93,147.96,136.68,135.71,134.11,131.49,129.51,129.19,
EXAMPLE 25 Synthesis of Compound 25
The synthesis of compound j, k was the same as h, i in example 1 to give compound k as a white solid; 5mL CH under ice bath conditions 2 Cl 2 Adding k (100mg, 0.338mmol), cinnamoyl chloride (67.68mg, 0.406mmol) and triethylamine (51.39mg, 0.507mmol), reacting in ice bath for 15min, and then transferring to normal temperature for further reaction for 2h; after the reaction was complete, 20mL of water was added and quenched, and 30mL of CH was added 2 Cl 2 Then, the organic phase was dried over anhydrous magnesium sulfate and passed through a Column (CH) 2 Cl 2 MeOH = 20) i.e. yellow solid 25 (73.31 mg); the yield is 50.67%, 1 H NMR(400MHz,DMSO-d 6 )δ10.07(s,1H),7.55(m,5H),7.48–7.38(m,3H),7.34(d,J＝7.9Hz,2H),7.27(d,J＝7.8Hz,2H),6.95(d,J＝8.8Hz,2H),6.81(d,J＝15.7Hz,1H),3.62(m,4H),3.12(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.59,163.42,147.43,139.94,139.73,135.32,133.35,132.32,130.08,129.46,129.37,128.08,127.59,122.97,120.71,116.89,55.38,49.52,21.39.
example 26
1) l Synthesis
Adding Meldrum's acid (2.93g, 20.31mmol) into 100mL of toluene solvent containing k (5 g, 16.93mmol), heating to 90 deg.C, stirring for 5h, cooling, precipitating solid, vacuum filtering the reaction system, washing with diethyl ether (20 mL × 3) for three times, and oven drying to obtain l (4.93 g); the yield was 75.89%.
2) Synthesis of Compound 26
Adding l (100mg, 0.262mmol), 3-pyridine benzaldehyde (33.70mg, 0.314mmol) and piperazine (2.23mg, 0.0262mmol) into 5mL of anhydrous toluene solvent, adding 100mg of anhydrous magnesium sulfate, heating to 90 deg.C, stirring for 10h, evaporating to dryness under reduced pressure, and passing through a chromatography Column (CH) 2 Cl 2 MeOH = 40) gave 26 (45.2 mg) as a yellow solid; the yield is 40.24 percent, 1 HNMR(400MHz,DMSO-d 6 )δ10.13(s,1H),8.81(d,J＝2.2Hz,1H),8.57(m,1H),8.02(m,1H),7.66–7.52(m,3H),7.48(m,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.59,162.98,150.71,149.64,147.54,139.73,136.59,134.49,133.35,132.12,131.12,129.37,127.59,124.93,124.50,120.77,116.87,49.47,21.39.
EXAMPLE 27 Synthesis of Compound 27
Synthesis of Compound 27 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 38.6%, 1 H NMR(400MHz,DMSO-d 6 )δ10.21(s,1H),8.64(d,J＝4.8Hz,1H),7.86(t,J＝7.7Hz,1H),7.59(m,4H),7.32(m,6H),6.96(d,J＝8.5Hz,2H),3.73(s,4H),3.12(s,4H). 13 C NMR(100MHz,DMSO-d 6 )δ169.59,163.17,153.41,150.34,147.53,139.73,139.17,137.70,133.35,132.23,129.37,127.59,126.64,124.93,124.59,120.71,116.84,55.38,49.48,21.39.
EXAMPLE 28 Synthesis of Compound 28
Synthesis of 28 see the procedure for the synthesis of 26 in example 26, the product is a yellow solid. The yield was 45.3% by weight, 1 H NMR(400MHz,DMSO-d 6 )δ10.21(s,1H),8.64(d,J＝5.2Hz,2H),7.69–7.43(m,5H),7.34(d,J＝7.8Hz,2H),7.27(d,J＝7.7Hz,2H),7.06–6.91(m,3H),3.66(m,4H),3.13(s,4H),2.36(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.59,162.65,150.87,147.65,142.52,139.73,137.36,133.35,131.96,129.37,127.59,127.44,122.15,120.82,116.84,49.42,21.39.
EXAMPLE 29 Synthesis of Compound 29
Synthesis of Compound 29 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 42.4%, 1 H NMR(400MHz,DMSO-d 6 )δ10.01(s,1H),7.55(dd,J＝32.2,8.2Hz,5H),7.40–7.16(m,6H),6.95(d,J＝8.6Hz,2H),6.75(d,J＝15.6Hz,1H),3.62(m,4H),3.12(s,4H),2.34(s,6H). 13 C NMR(100MHz,DMSO-d 6 )δ169.59,163.59,147.39,139.90,139.86,139.72,133.35,132.58,132.38,130.06,129.37,128.06,127.59,121.92,120.68,116.89,49.52,21.43,21.39.
EXAMPLE 30 Synthesis of Compound 30
Synthesis of Compound 30 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 65.3%, 1 H NMR(400MHz,DMSO-d 6 )δ10.20(s,1H),9.17(s,1H),9.06(s,2H),7.68–7.48(m,3H),7.34(d,J＝7.7Hz,2H),7.27(d,J＝7.7Hz,2H),7.05–6.85(m,3H),3.73(s,4H),3.12(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.59,162.58,158.81,155.98,147.65,139.73,133.35,133.28,131.96,129.37,127.59,126.65,120.85,116.85,49.44,21.39.
EXAMPLE 31 Synthesis of Compound 31
Synthesis of Compound 31 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 52.1%, 1 H NMR(400MHz,DMSO-d 6 )δ9.98(s,1H),7.56(d,J＝8.8Hz,2H),7.33(d,J＝7.8Hz,2H),7.31–7.20(m,3H),6.93(d,J＝8.6Hz,2H),6.70(d,J＝3.2Hz,1H),6.52(d,J＝15.4Hz,1H),6.24(d,J＝3.2Hz,1H),3.73(s,4H),3.11(s,4H),2.34(d,J＝4.9Hz,6H). 13 C NMR(100MHz,DMSO-d 6 )δ169.59,163.50,154.70,150.17,147.32,139.72,133.35,132.48,129.37,127.59,127.07,120.49,118.68,116.88,116.12,109.41,49.54,21.38,13.99.
EXAMPLE 32 Synthesis of Compound 32
Synthesis of Compound 32 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield is 35.6 percent, 1 H NMR(400MHz,DMSO-d 6 )δ10.21(s,1H),8.32–8.25(m,2H),7.93–7.84(m,2H),7.70–7.55(m,3H),7.52–7.21(m,6H),7.06–6.92(m,3H),3.77(s,4H),3.22–3.04(m,4H). 13 C NMR(100MHz,DMSO-d 6 )δ169.47,162.69,148.02,141.95,137.48,136.30,132.07,131.10,130.04,129.11,128.91,127.44,124.74,124.63,120.79,116.87,49.44.
EXAMPLE 33 Synthesis of Compound 33
Synthesis of Compound 33 referring to the synthesis of Compound 25 in example 26, the product is a white solid. The yield was 65.33%, 1 H NMR(400MHz,DMSO-d 6 )δ9.73(s,1H),7.47–7.39(m,2H),7.33(d,J＝7.9Hz,2H),7.26(d,J＝7.9Hz,2H),6.94–6.85(m,2H),3.71(s,4H),3.08(s,4H),2.35(s,3H),1.99(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.58,168.07,147.17,139.71,133.36,132.49,129.36,127.58,120.55,116.88,49.64,24.28,21.39.
EXAMPLE 34 Synthesis of Compound 34
Synthesis of Compound 34 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 56.2%, 1 H NMR(400MHz,DMSO-d 6 )δ10.10(s,1H),8.13(s,1H),8.04–7.89(m,3H),7.82–7.67(m,2H),7.66–7.52(m,4H),7.39–7.22(m,4H),7.03–6.86(m,3H),3.74(s,4H),3.13(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.60,163.46,147.46,139.90,139.73,133.88,133.51,133.36,132.91,132.34,129.37,129.33,129.09,128.79,128.15,127.59,127.45,127.23,124.01,123.40,120.71,116.90,55.37,49.51,21.39.
EXAMPLE 35 Synthesis of Compound 35
Synthesis of Compound 35 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 41.5%, 1 H NMR(400MHz,DMSO-d 6 )δ11.63(s,1H),9.83(s,1H),7.96(d,J＝7.1Hz,1H),7.81(d,J＝2.8Hz,1H),7.72(d,J＝15.6Hz,1H),7.59(d,J＝8.5Hz,2H),7.51–7.44(m,1H),7.34(d,J＝7.7Hz,2H),7.27(d,J＝7.8Hz,2H),7.24–7.18(m,2H),6.95(d,J＝8.7Hz,2H),6.78(d,J＝15.7Hz,1H),3.65(m,4H),3.13(m,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.59,164.94,147.05,139.72,137.91,134.49,133.37,133.00,131.26,129.37,127.59,125.32,122.76,120.87,120.31,117.04,116.77,112.70,49.07,21.39.
EXAMPLE 36 Synthesis of Compound 36
Synthesis of Compound 36 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield is 35.6 percent, 1 H NMR(400MHz,DMSO-d 6 )δ10.09(s,1H),7.88–7.67(m,1H),7.65–7.51(m,3H),7.46(m,1H),7.30(m,5H),7.09(d,J＝8.6Hz,1H),6.74(d,J＝15.8Hz,3H),3.62(d,4H),3.12(s,4H),2.35(s,3H),1.74–1.22(m,2H). 13 C NMR(100MHz,DMSO-d 6 )δ170.05,165.28,162.67,160.15,145.48,141.33,135.18,133.91,133.19,133.11,130.65,130.57,129.72,129.64,129.39,129.11,125.53,125.50,122.80,122.51,122.31,121.86,121.83,116.17,115.97,114.60,48.93,46.30,21.35.
EXAMPLE 37 Synthesis of Compound 37
Synthesis of Compound 37 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 36.3%, 1 H NMR(400MHz,DMSO-d 6 )δ10.06(s,1H),8.59(d,J＝2.6Hz,1H),7.94(d,J＝8.2Hz,2H),7.79(s,1H),7.74(d,J＝8.3Hz,2H),7.63–7.54(m,3H),7.34(d,J＝7.8Hz,2H),7.28(s,2H),6.96(d,J＝8.5Hz,2H),6.82(d,J＝15.7Hz,1H),6.59(t,J＝2.2Hz,1H),3.62(m,4H),3.12(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.59,163.41,147.44,141.86,140.71,139.73,138.95,133.35,133.09,132.32,129.37,128.31,127.59,122.80,120.69,119.04,116.89,108.71,55.38,49.53,21.39.
EXAMPLE 38 Synthesis of Compound 38
Synthesis of Compound 38 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 41.3%, 1 H NMR(400MHz,DMSO-d 6 )δ10.17(s,1H),7.91–7.73(m,2H),7.68–7.52(m,3H),7.49–7.23(m,6H),6.91(m,3H),3.62(m,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.58,162.87,147.57,139.73,135.13,133.82,133.35,133.13,132.06,131.51,130.52,129.37,128.31,128.08,127.59,126.07,120.78,116.84,55.38,49.43,21.39.
EXAMPLE 39 Synthesis of Compound 39
Synthesis of 39 see the procedure for the synthesis of 26 in example 26, the product is a yellow solid. The yield was 45.6%, 1 H NMR(400MHz,DMSO-d 6 )δ10.08(s,1H),7.69(s,1H),7.63–7.40(m,6H),7.39–7.22(m,4H),6.96(d,J＝8.5Hz,2H),6.86(d,J＝15.7Hz,1H),3.62(m,4H),3.12(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.59,163.05,147.52,139.73,138.28,137.65,134.17,133.34,132.15,131.29,129.66,129.37,127.76,127.59,126.52,124.72,120.72,116.87,99.99,49.47,21.39.
EXAMPLE 40 Synthesis of Compound 40
Synthesis of Compound 40 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 39.6%, 1 H NMR(400MHz,DMSO-d 6 )δ10.06(s,1H),7.93–7.82(m,1H),7.76–7.62(m,3H),7.61–7.46(m,4H),7.34(d,J＝7.7Hz,2H),7.27(d,J＝7.7Hz,2H),6.95(d,J＝8.5Hz,2H),6.80(d,J＝15.7Hz,1H),3.62(m,4H),3.12(s,4H). 13 C NMR(100MHz,DMSO-d 6 )δ169.99,164.16,159.08,156.56,145.48,141.33,141.19,135.18,133.89,131.79,131.76,129.48,129.40,129.37,129.11,129.04,123.34,123.14,122.80,119.94,117.27,117.07,114.65,48.73,45.23,21.35.
EXAMPLE 41 Synthesis of Compound 41
Synthesis of Compound 41 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was found to be 56.2%, 1 H NMR(400MHz,DMSO-d 6 )δ10.05(s,1H),7.68(m,2H),7.62–7.51(m,3H),7.38–7.24(m,6H),6.95(d,J＝8.7Hz,2H),6.74(d,J＝15.7Hz,1H),3.73(s,4H),3.12(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.58,163.35,147.45,139.73,138.73,133.35,132.28,131.98,130.29,130.20,129.37,127.59,122.87,120.71,116.88,116.55,116.33,55.38,49.51,21.39.
EXAMPLE 42 Synthesis of Compound 42
Synthesis of Compound 42 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 49.3%, 1 H NMR(400MHz,DMSO-d 6 )δ10.11(s,1H),7.97(s,1H),7.93(d,J＝7.8Hz,1H),7.76(d,J＝7.8Hz,1H),7.70(d,J＝7.8Hz,1H),7.64(d,J＝15.8Hz,1H),7.59(d,J＝8.9Hz,2H),7.34(d,J＝8.0Hz,2H),7.27(d,J＝7.8Hz,2H),6.95(s,2H),6.91(s,1H),3.62(m,4H),3.12(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.58,162.97,147.55,139.73,138.12,136.54,133.35,132.14,131.89,130.61,130.39,129.37,127.59,125.86,125.18,124.32,120.69,116.88,55.38,21.39.
EXAMPLE 43 Synthesis of Compound 43
Synthesis of Compound 43 referring to the synthesis of Compound 26 in example 26, the product is a yellow solid. The yield was 45.6%, 1 H NMR(400MHz,DMSO-d 6 )δ10.06(s,1H),7.82(s,1H),7.67–7.49(m,5H),7.41(t,J＝7.8Hz,1H),7.34(d,J＝7.7Hz,2H),7.27(d,J＝7.8Hz,2H),6.95(d,J＝8.5Hz,2H),6.84(d,J＝15.7Hz,1H),3.73(s,4H),3.12(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.58,163.03,147.52,139.73,138.19,137.92,133.35,132.55,132.14,131.56,130.60,129.37,127.59,126.92,124.72,122.75,120.70,116.87,55.38,49.47,21.39.
EXAMPLE 44 Synthesis of Compound 44
1) 44a Synthesis
Slowly dripping phosphorus oxychloride (633.54mg, 4.13mmol) into DMF (302.04mg, 4.13mmol) under the ice bath condition, stirring for 15min under the ice bath condition, slowly dripping 1,2-dimethylindole (500mg, 3.44mmol) dissolved in 5mL of DMF, and then stirring for 3h at normal temperature; after the reaction is completed, 5mL of water is added, the mixture is shaken and a white solid appears, and after suction filtration, the dried solid is the compound 44a (378.2 mg) with the yield of 63.37%.
2) Synthesis of Compound 44
Synthesis of Compound 44 the product was a yellow solid, as seen in the synthesis of Compound 26 in example 26. The yield was 53.2%, 1 H NMR(400MHz,DMSO-d 6 )δ9.75(s,1H),7.85(m,1H),7.68(d,J＝15.5Hz,1H),7.52(d,J＝8.9Hz,2H),7.47–7.41(m,1H),7.27(d,J＝8.1Hz,2H),7.20(d,J＝7.9Hz,2H),7.18–7.15(m,1H),7.15–7.08(m,1H),6.93–6.85(m,2H),6.70(d,J＝15.6Hz,1H),3.66(s,3H),3.49(m,4H),2.47(s,3H),2.28(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.07,164.61,146.49,141.54,139.22,137.23,132.85,132.56,128.87,127.08,125.03,121.59,120.58,119.73,119.35,116.54,115.45,110.03,108.04,29.75,20.89,10.24.
EXAMPLE 45 Synthesis of Compound 45
45a synthesis method see 44a synthesis procedure in example 44, and compound 45 synthesis method see 26 synthesis procedure in example 26, and the product is yellow solid. The yield was 37.6%, 1 H NMR(400MHz,DMSO-d 6 )δ11.43(d,J＝2.7Hz,1H),9.75(s,1H),7.68(d,J＝3.5Hz,2H),7.63(s,1H),7.53(d,J＝8.9Hz,2H),7.31–7.27(m,2H),7.26(d,J＝2.0Hz,1H),6.97(m,1H),6.90–6.85(m,2H),6.66(d,J＝15.6Hz,1H),3.55(m,4H),3.04(s,4H),2.41(s,3H),2.28(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.08,164.53,146.52,139.21,135.77,134.30,132.85,132.53,130.97,129.12,128.87,127.08,125.08,123.76,119.86,119.73,116.52,115.74,111.94,111.76,59.71,49.18,21.43,20.88.
EXAMPLE 46 Synthesis of Compound 46
Synthesis of Compound 46 referring to the synthesis of Compound 45 in example 45, the product is a yellow solid. The yield was 53.6%, 1 H NMR(400MHz,DMSO-d 6 )δ11.78–11.71(m,1H),9.86(s,1H),7.90(d,J＝8.6Hz,1H),7.85(d,J＝2.2Hz,1H),7.71(d,J＝15.8Hz,1H),7.67(d,J＝1.8Hz,1H),7.62–7.57(m,2H),7.37–7.34(m,2H),7.33(d,J＝2.0Hz,1H),7.27(d,J＝7.9Hz,2H),6.98–6.93(m,2H),6.78(d,J＝15.7Hz,1H),3.73(s,4H),3.11(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.08,164.19,146.61,139.21,138.28,133.29,132.84,132.36,131.48,128.86,127.08,123.83,123.14,121.52,119.84,117.12,116.52,114.94,114.89,112.31,49.14,20.89.
EXAMPLE 47 Synthesis of Compound 47
Synthesis of 47 see the Synthesis of 45 in example 45, the product is a yellow solid. The yield was 53.1%, 1 H NMR(400MHz,DMSO-d 6 )δ12.15(d,J＝2.7Hz,1H),9.84(s,1H),8.36–8.30(m,2H),7.96(d,J＝2.8Hz,1H),7.70(d,J＝15.7Hz,1H),7.59(d,J＝8.8Hz,2H),7.34(d,J＝7.9Hz,2H),7.29–7.24(m,3H),6.99–6.93(m,2H),6.82(d,J＝15.8Hz,1H),3.62(m,4H),3.14(m,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.08,164.10,149.55,146.65,143.60,139.22,133.44,132.84,132.29,130.84,128.86,128.09,127.08,119.86,117.51,117.10,116.58,116.53,110.99,49.13,20.88.
EXAMPLE 48 Synthesis of Compound 48
Synthesis of Compound 48 referring to the synthesis of Compound 45 in example 45, the product is a yellow solid. The yield was 45.1%, 1 H NMR(400MHz,DMSO-d 6 )δ12.26–12.06(m,1H),9.85(s,1H),8.42(s,1H),8.03(d,J＝2.6Hz,1H),7.73(d,J＝15.8Hz,1H),7.68–7.63(m,1H),7.59(m,3H),7.38–7.32(m,2H),7.27(d,J＝7.9Hz,2H),7.00–6.93(m,2H),6.87(d,J＝15.8Hz,1H),3.63(m,4H),3.12(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.08,163.96,146.70,139.22,132.91,132.84,132.61,132.18,128.87,127.08,125.16,125.05,124.51,120.52,119.89,118.13,116.52,113.66,112.82,102.54,54.87,49.12.
EXAMPLE 49 Synthesis of Compound 49
Synthesis of Compound 49 referring to the synthesis of Compound 45 in example 45, the product is a yellow solid. The yield is 48.6 percent, 1 H NMR(400MHz,DMSO-d 6 )δ12.29(s,1H),10.06(s,1H),8.87(d,J＝2.2Hz,1H),8.13(m,1H),8.10(s,1H),7.78(d,J＝15.8Hz,1H),7.67(d,J＝9.0Hz,1H),7.64–7.54(m,2H),7.34(d,J＝8.1Hz,2H),7.27(d,J＝7.8Hz,2H),7.00–6.93(m,2H),6.87(d,J＝15.8Hz,1H),3.63m,4H),3.12(s,4H),2.36(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.09,163.87,146.72,141.68,140.58,139.23,134.07,132.83,132.56,132.18,128.87,127.08,123.98,120.05,118.13,117.71,116.55,116.47,114.22,112.87,49.09,20.88.
EXAMPLE 50 Synthesis of Compound 50
Synthesis of Compound 50 referring to the synthesis of Compound 26 in example 26, the product is a white solid. The yield was 75.3%, 1 H NMR(400MHz,DMSO-d 6 )δ10.77(s,1H),9.76(s,1H),7.57(d,J＝7.9Hz,1H),7.52–7.44(m,2H),7.37–7.30(m,3H),7.27(d,J＝7.9Hz,2H),7.12(d,J＝2.3Hz,1H),7.10–7.04(m,1H),7.03–6.87(m,3H),3.11(s,4H),3.01(t,J＝7.6Hz,2H),2.64(m,2H),2.35(s,3H). 13 CNMR(100MHz,DMSO-d 6 )δ169.10,164.11,149.53,146.65,143.60,139.23,133.46,132.81,132.27,130.84,128.87,128.11,127.08,119.87,117.48,117.10,116.59,116.52,110.98,49.13,20.88.
EXAMPLE 51 Synthesis of Compound 51
Synthesis of Compound 51 the product was a yellow solid as described for Compound 45 in example 45. The yield was 56.3%, 1 H NMR(400MHz,DMSO-d 6 )δ11.74(s,1H),10.00(s,1H),8.48(m,1H),8.03(s,1H),7.85(m,1H),7.73(d,J＝15.3Hz,1H),7.69–7.62(m,2H),7.47(d,J＝15.3Hz,1H),7.34(d,J＝8.1Hz,2H),7.30–7.21(m,3H),6.95–6.90(m,2H),3.62(m,4H),3.10(s,4H),2.35(s,3H). 13 CNMR(100MHz,DMSO-d 6 )δ170.05,165.13,145.48,142.87,141.39,139.81,135.07,133.86,133.01,129.84,129.37,129.15,129.11,122.90,120.84,118.97,117.81,114.71,110.98,48.85,45.46,21.36.
EXAMPLE 52 Synthesis of Compound 52
Synthesis of 52 see the procedure for the synthesis of 45 in example 45, the product is a yellow solid. The yield was 41.2%, 1 H NMR(400MHz,DMSO-d 6 )δ11.79(s,1H),10.05(s,1H),8.52(m,1H),8.07(d,J＝2.9Hz,1H),7.94–7.87(m,2H),7.79(d,J＝15.3Hz,1H),7.69(d,J＝8.7Hz,2H),7.51(d,J＝15.3Hz,1H),7.38(d,J＝7.7Hz,2H),7.34–7.25(m,4H),6.97(d,J＝8.8Hz,2H),3.77(s,4H),3.21–3.03(m,4H),2.39(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ170.05,165.08,145.48,144.46,143.61,141.39,139.92,135.07,133.86,132.63,129.37,129.11,128.50,123.95,122.90,120.42,114.71,110.15,106.19,48.85,45.46,21.36.
EXAMPLE 53 Synthesis of Compound 53
Synthesis of Compound 53 referring to the synthesis of Compound 45 in example 45, the product is a yellow solid. The yield was 47.1%, 1 H NMR(400MHz,DMSO-d 6 )δ12.10(s,1H),9.89(s,1H),8.83(s,1H),8.30(d,J＝5.5Hz,1H),8.04(s,1H),7.90(d,J＝5.5Hz,1H),7.74(d,J＝15.7Hz,1H),7.62–7.56(m,2H),7.34(d,J＝8.1Hz,2H),7.27(d,J＝7.9Hz,2H),6.98–6.93(m,2H),6.84(d,J＝15.7Hz,1H),3.72(s,4H),3.12(s,4H),2.35(s,3H),1.99(s,1H). 13 C NMR(100MHz,DMSO-d 6 )δ169.09,164.02,146.66,139.23,139.13,135.13,134.28,133.64,132.90,132.83,132.26,129.16,128.87,127.08,119.87,118.04,116.51,114.52,111.70,59.72,49.11,43.68,22.16,20.89,20.73,14.05.
EXAMPLE 54 Synthesis of Compound 54
Synthesis of Compound 54 referring to the synthesis of Compound 45 in example 45, the product is a yellow solid. The yield was 51.3%, 1 H NMR(400MHz,DMSO-d 6 )δ11.54(s,1H),9.85(s,1H),7.87(d,J＝8.7Hz,1H),7.72(m,2H),7.63(d,J＝8.6Hz,2H),7.38(d,J＝7.7Hz,2H),7.31(d,J＝7.8Hz,2H),7.06–6.96(m,3H),6.89(m,1H),6.78(d,J＝15.7Hz,1H),3.85(s,3H),3.67(m,4H),3.15(s,4H),2.40(s,3H). 13 CNMR(100MHz,DMSO-d 6 )δ169.60,164.99,156.57,147.04,139.72,138.88,134.53,133.37,133.01,130.28,129.37,127.59,121.07,120.30,119.46,117.04,116.47,112.83,110.76,95.87,55.72,49.68,21.39.
EXAMPLE 55 Synthesis of Compound 55
Synthesis of 55 see the procedure for the synthesis of 45 in example 45, the product is a yellow solid. The yield was 62.4%, 1 H NMR(400MHz,DMSO-d 6 )δ11.74(d,J＝2.8Hz,1H),9.82(s,1H),7.77–7.68(m,2H),7.62–7.56(m,2H),7.54(d,J＝8.1Hz,1H),7.34(d,J＝8.0Hz,2H),7.27(d,J＝7.9Hz,2H),7.13(t,J＝7.9Hz,1H),6.97–6.91(m,2H),6.80(d,J＝7.8Hz,1H),6.75(d,J＝15.6Hz,1H),3.94(s,3H),3.62(d,J＝80.2Hz,4H),3.11(s,4H),2.35(s,3H). 13 C NMR(101MHz,CDCl 3 )δ174.35,169.69,151.80,151.66,144.45,139.36,138.13,137.75,135.33,134.11,132.62,132.33,131.58,126.35,125.09,121.77,121.59,118.03,108.24,60.50,54.43,26.13.
EXAMPLE 56 Synthesis of Compound 56
Synthesis of 56 see the procedure for the synthesis of 45 in example 45, the product is a yellow solid. The yield was 31.2%, 1 H NMR(400MHz,DMSO-d 6 )δ9.95(s,1H),8.13(s,1H),8.11–8.05(m,1H),7.79(d,J＝15.8Hz,1H),7.70–7.53(m,8H),7.50–7.45(m,1H),7.36–7.32(m,4H),7.27(d,J＝7.9Hz,2H),7.01–6.89(m,3H),3.72(s,4H),3.12(s,4H),2.35(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.15,164.12,146.68,139.27,138.07,136.64,132.90,132.84,132.77,132.23,129.92,128.88,127.25,127.08,126.05,124.22,123.51,121.68,120.54,119.93,118.15,116.52,113.72,111.16,59.75,49.11,28.96,20.88.
EXAMPLE 57 Synthesis of Compound 57
Synthesis of Compound 57 referring to the synthesis of Compound 45 in example 45, the product is a yellow solid. The yield was 49.2%, 1 H NMR(400MHz,DMSO-d 6 )δ9.94(s,1H),8.29(d,J＝8.1Hz,1H),7.97(s,1H),7.77(d,J＝15.8Hz,1H),7.72(d,J＝7.4Hz,1H),7.60(d,J＝8.7Hz,2H),7.34(m,4H),7.25(d,J＝7.8Hz,2H),6.97–6.92(m,2H),6.88(d,J＝15.9Hz,1H),3.79–3.62(m,4H),3.10(s,4H),2.33(s,3H). 13 C NMR(100MHz,DMSO-d 6 )δ169.16,164.02,146.71,139.26,136.87,132.74,132.57,132.15,131.80,128.86,127.37,127.07,125.88,125.37,120.53,119.99,118.32,116.95,116.50,113.26,94.73,49.09,20.86.
EXAMPLE 58 Synthesis of Compound 58
Synthesis of Compound 58 the product was a white solid, as seen in the synthesis of Compound 45 in example 45. The yield is 85.1 percent, 1 H NMR(400MHz,CDCl 3 )δ7.37–7.32(m,2H),7.30–7.26(m,2H),7.22(d,J＝8.1Hz,2H),6.98–6.85(m,3H),3.77(m,4H),3.19(s,4H),2.39(s,3H). 13 C NMR(100MHz,CDCl 3 )δ170.59,151.00,140.00,132.68,129.23,129.11,127.25,120.58,116.71,49.79,21.38.
the structural formulae of the above compounds 1 to 58 are shown in the following table:
biological test example 1: tumor cell proliferation inhibition assay
The small molecule inhibitors of the invention were tested for their activity in inhibiting human tumor cell proliferation using the CCK8 method (MCE, USA) or the ATPLite method (Perkin-Elmer, USA).
The cell strains adopt a human lung squamous carcinoma cell strain H2170 and human lung adenocarcinoma cell strains H358 and H1650. The medium was RPMI1640 (Gibco, USA) +10% FBS (fetal bovine serum, gibco, USA) + double antibody.
Preparing a reagent: the stock solution was 20mM, the solvent was DMSO (Sigma-Aldrich, USA), stored at-20 deg.C, and was diluted to the desired concentration using cell culture medium.
The CCK8 detection method comprises the following steps: the concentration of the added solution is 5 multiplied by 10 per hole of a 96-hole plate 4 Cell suspension 100. Mu.L/mL, in cell incubator (37 ℃,5% CO) 2 ) And (5) culturing. After 24 hours, 100. Mu.L of medium containing small molecule reagents was added to give final concentrations of 0,0.0046,0.0137,0.0412,0.1235,0.3704,1.1111,3.3333, 10 and 30. Mu.M, respectively. Three wells were set, incubation was continued for 72 hours and the medium solution was discarded, 100. Mu.L of a medium containing 10% CCK8 solution was added to each well, and after 1 hour of incubation, OD at 450nm was measured using a microplate reader SpectraMax iD 3. Calculation of half inhibitory concentration of cells IC 50 The value is obtained.
ATPLite detection method: the concentration of the added solution is 5 multiplied by 10 per hole of a 96-hole plate 4 Cell suspension 100. Mu.L/mL, in cell incubator (37 ℃,5% CO) 2 ) And (5) culturing. After 24 hours, 100. Mu.L of medium containing small molecule reagents was added to give final concentrations of 0,0.0046,0.0137,0.0412,0.1235,0.3704,1.1111,3.3333, 10 and 30. Mu.M, respectively. Three wells were set, incubation was continued for 72 hours and the medium solution was discarded, 50. Mu.L of ATPlute reagent was added to each well, and after shaking at room temperature at 700 rpm for 2 minutes, the plate was transferred to an opaque white plate. After dark treatment at room temperature for 10 minutes, chemiluminescence was measured using a microplate reader SpectraMax iD 3. Calculation of half inhibitory concentration of cells IC 50 The value is obtained.
The results are shown in Table 1 and FIG. 1.
Table 1: the small molecule inhibitor of the invention has the advantages of high activity, high stability and low cost, and can be used for treating human lung squamous carcinoma cells H2170 IC 50 Value of
| Sample name | IC 50 Value (μ M) | Sample name | IC 50 Value (μ M) |
| Compound 13 | 69.8 | Compound 35 | 9.9 |
| |
91.1 | Compound 36 | 109.8 |
| |
8.8 | Compound 41 | 9.0 |
| Compound 31 | 12.9 | |
1.8 |
| Compound 32 | 9.4 | Compound 48 | 11.6 |
Table 1 shows the inhibition of the growth of human lung squamous carcinoma cells H2170 by randomly selecting different small molecules encompassed by the present invention. Inoculating lung cancer cell H2170 into 96-well plate at 5000/well, treating with small molecule inhibitor with concentration gradient (each concentration is 3 multiple wells) for 72 hr, detecting cell growth with CCK8 kit, and calculating half Inhibition Concentration (IC) of tumor growth 50 )
FIG. 1 shows that compound 47 small molecule inhibitor inhibits the growth of human lung cancer cells.Lung cancer cells H2170, H358, and H1650 were seeded at 5000/well in 96-well plates, treated with compound 47 small molecule inhibitor in concentration gradient (3 multiple wells per concentration) for 72 hours, and cell growth was measured using ATPLite kit to calculate half Inhibitory Concentration (IC) for tumor growth 50 )
The above experimental results show that the compounds comprised by the present invention, selected randomly, have good activity of inhibiting the growth of lung cancer cells, especially compound 47,IC 50 The value is less than 10 mu M in different lung cancer cell lines, and the structure of the compound can be determined by analyzing the structural formula of the compound, or the salt with the structure of the general formula I can be used as a potential antitumor small molecule inhibitor.
The mechanism of action of the substance having the structure of formula I will be described below by taking compound 47 as an example.
Biological test example 2: experiment for inducing cell cycle G2/M phase retardation and apoptosis
The influence of the small molecule inhibitor on the cell cycle process and the cell apoptosis is detected, and the flow cytometry and the Western blot method are adopted.
The cell strain adopts a human lung squamous carcinoma cell strain H2170 and a human lung adenocarcinoma cell strain H1650. The medium was RPMI1640 (Gibco, USA) +10% FBS (fetal bovine serum, gibco, USA) + double antibody.
Preparing a reagent: the stock solution was 20mM in DMSO as a solvent, stored at-20 deg.C, dissolved at room temperature for use, and diluted to the desired concentration using cell culture medium.
The flow cytometry detection method comprises the following steps: lung cancer cells were seeded at a density of 50-60% in a 60mm cell culture dish, in a cell culture incubator (37 ℃,5% 2 ) And (5) culturing. After 24 hours, medium containing small molecule reagents was added to final concentrations of 2.5,5, 10 and 20 μ M, respectively, and solvent DMSO was used as a negative control. After further incubation for 24 hours cells were harvested by trypsinization, fixed overnight in 70% alcohol, stained with propidium iodide/ribonuclease staining solution (PI/RNase) (BD Biosciences, UK) for 15 minutes and cell cycle was examined by Cytoflex S flow cytometry.
The Western blot detection method comprises the following steps: the lung cancer cells are planted in the density of 50-60%60mm cell culture dish, in cell culture chamber (37 ℃,5% CO) 2 ) And (5) culturing. After 24 hours, adding culture media containing small molecular reagents, wherein the final concentrations are 2.5,5, 10 mu M and 20 mu M respectively, taking a solvent DMSO as a negative control, continuously culturing for 24 hours, then harvesting cells, and detecting the apoptosis level by using a Western blot method. The antibodies used were PARP (Cell Signaling Technology, USA), caspase-3 (Cell Signaling Technology, USA), NOXA (Merck Millipore, germany), beta-Actin (Sigma-Aldrich, USA), respectively. The results are shown in FIG. 2.
FIG. 2 is a graph showing that small molecule inhibitors of Compound 47 induce cell cycle G2/M phase arrest and apoptosis in a dose-dependent manner. Treating lung cancer cells H2170 or H1650 with compound 47 small molecule inhibitor for 24 hr, fixing overnight, staining with PI/RNase staining solution, and detecting cell cycle with Cytoflex S flow cytometer; levels of apoptosis were measured using PARP, caspase-3 and NOXA antibodies. beta-Actin is used as a reference for the loading amount.
The experimental results show that the compound with the structure shown in the general formula I can induce the G2/M phase retardation of the lung cancer cell cycle and the apoptosis of the lung cancer cells, and can be used as a potential anti-tumor small molecule inhibitor.
Biological test example 3: cullin neddylation modification inhibition assay
The activity of the small molecule inhibitor for inhibiting neddylation modification of the cullin molecules in the lung cancer cells is detected, and a Western blot method is adopted.
The cell strain adopts a human lung squamous carcinoma cell strain H2170 and a human lung adenocarcinoma cell strain H1650. The medium was RPMI1640 (Gibco, USA) +10% FBS (fetal bovine serum, gibco, USA) + double antibody.
Preparing a reagent: the stock solution was 20mM in DMSO as a solvent, stored at-20 deg.C, dissolved at room temperature for use, and diluted to the desired concentration using cell culture medium.
The Western blot detection method comprises the following steps: lung cancer cells were seeded at a density of 50-60% on 60mm cell culture dishes and incubated in a cell incubator (37 ℃,5% 2 ) And (5) culturing. After 24 hours, medium containing small molecule reagents was added to final concentrations of 0.3,1,3, 10 and 20 μ M, respectively, with solvents DMSO and MLN4924 (0.3 μ M) as the negative, respectivelyAnd (4) sex and positive control, continuously culturing for a certain time, harvesting cells, and detecting neddylation level of a plurality of cullin proteins by using a Western blot method. The antibodies used were respectively the Cul1 antibody (Santa Cruz, USA), the Cul2 antibody (Santa Cruz, USA), the Cul3 antibody (Cell Signaling Technology, USA), the Cul4A antibody (Proteintech, USA), the Cul4B antibody (Proteintech, USA), the Cul5 antibody (Santa Cruz, USA), NOXA (Merck Millipore, germany), the β -Actin antibody (Sigma-Aldrich, USA). The results are shown in FIG. 3.
FIG. 3 is a graph showing that compound 47 small molecule inhibitors inhibit the neddylation modification of Cul5 protein in a time and dose dependent manner, inducing the accumulation of its substrate protein NOXA. Lung cancer cells H2170 or H1650 were treated with compound 47 small molecule inhibitor and neddylation modification levels and substrate NOXA protein levels of multiple cullins were determined using specific cullin antibodies and NOXA antibodies. beta-Actin is used as a reference for the loading amount. And SE: short exposure; LE: and (4) long exposure.
The above experimental results show that the compounds of the present invention having the structure of formula I specifically inhibit the neddylation modification of Cul5 protein in a time and dose dependent manner, inducing the accumulation of its substrate protein NOXA, while having no effect on neddylation of other cullins. Suggesting that the target point of action is NEDD 8E 2 coupled enzyme UBE2F.
Biological test example 4: UBE2F protein inhibition assay
The influence of the small molecule inhibitor on the UBE2F protein level in the lung cancer cells is detected by adopting a Western blot method. The cell line was human lung squamous carcinoma cell line H2170. The medium was RPMI1640 (Gibco, USA) +10% FBS (fetal bovine serum, gibco, USA) + double antibody.
Preparing a reagent: the stock solution was 20mM in DMSO as a solvent, stored at-20 deg.C, dissolved at room temperature for use, and diluted to the desired concentration using cell culture medium.
The Western blot detection method comprises the following steps: lung cancer cells were seeded at a density of 50-60% on 60mm cell culture dishes and incubated in a cell incubator (37 ℃,5% 2 ) And (4) culturing. After 24 hours, medium containing small molecule reagents was added to final concentrations of 0.3,1,3, 10 and 20. Mu.M, respectively, and solvents DMSO and MLN4924 (0.3. Mu.M) were negative, respectivelyAnd positive control, continuously culturing for 24 hours, harvesting cells, and detecting UBE2F protein level by using a Western blot method. The antibodies used were UBE2F antibody (Abcam, UK), UBE2M (Cell Signaling Technology, USA) and beta-Actin antibody (Sigma-Aldrich, USA), respectively. The results are shown in FIG. 4.
FIG. 4 is a graph showing that small molecule inhibitors of Compound 47 selectively reduce the levels of UBE2F protein in cells. Lung cancer cells H2170 were tested for protein levels using UBE2F and UBE2M antibodies 24 hours after treatment with compound 47 small molecule compound. beta-Actin is used as a reference for the loading amount. And SE: short exposure; LE: and (4) long exposure.
The above experimental results show that the compounds of the invention having the structure of general formula I selectively reduce levels of UBE2F protein in a dose-dependent manner, while UBE2M, a family member of UBE2F, has no effect and is a specific inhibitor targeting UBE2F.
Claims (3)
1. A phenylpiperazine UBE2F small molecule inhibitor, characterized by having the structure of formula I, or a salt having the structure of formula I:
wherein X, Y are carbonyl;
R 1 is 4-methyl-phenyl;
R 2 is 7-azaindolevinyl.
2. A method for synthesizing the small molecule inhibitor of claim 1, comprising the steps of:
3. a pharmaceutical composition comprising the small molecule inhibitor of claim 1.
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