CN114456228B - Substituted glycine-3, 5-difluorophenylalanine peptide derivative and preparation method and application thereof - Google Patents
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Abstract
The invention provides a peptide derivative containing substituted glycine-3, 5-difluorophenylalanine, a preparation method and application thereof. The derivative has a structure shown in the following general formula I. The invention also relates to a preparation method of the derivative and application of the derivative as an HIV inhibitor in preparation of anti-AIDS drugs.
Description
Technical Field
The invention belongs to the technical field of synthesis and medical application of organic compounds, and particularly relates to a substituted glycine-3, 5-difluorophenylalanine peptide derivative, and a preparation method and application thereof.
Background
Acquired immunodeficiency syndrome (Acquired Immune Deficiency Syndrome, AIDS), also known as AIDS, is a chronic infectious disease caused by Human Immunodeficiency Virus (HIV) that is a hazard to human life and health. HIV belongs to RNA retrovirus and is divided into two subtypes of HIV-1/2, wherein HIV-1 has strong infectivity and high mortality and is a main pathogen of AIDS. HIV-2 is mainly limited to Western Africa, but with the continuous development of globalization and the increasing close communication of people, HIV-2 infection cases are also found in the United states, europe, south Africa, india and China, and attention should be paid. More than 30 drugs for treating AIDS are currently marketed, and the drugs respectively target reverse transcriptase, protease, integrase, CCR5 synergistic receptor and membrane fusion. However, due to the high mutation rate of HIV-1, drug resistance is easy to generate, and the single drug treatment effect is poor. "high efficacy antiretrotherapy" (Highly Active Antiretroviral Therapy, HAART) is effective in reducing HIV load in patients and improving life span and quality of life of patients, but the problems of serious adverse reactions, drug resistance, latent infection, and high cost caused by the long-term use of large amounts of drugs have led to the urgent discovery of new structural types of HIV inhibitors.
The construction of the compound library is an important part of drug development, and the progress of drug development can be greatly accelerated by applying a high-efficiency organic synthesis technology. Among them, multicomponent reactions have the advantages of simple operation, continuous reaction, atom economy, etc., and have received much attention to meet the needs of organic synthesis, construction of diverse compound libraries. The invention utilizes Ugi four-component reaction to construct a compound library and biological activity screening to find a class of substituted glycine-3, 5-difluorophenylalanine peptide derivatives with brand new structures, and part of the compounds have double inhibition effects on HIV-1 and HIV-2, show better selectivity on HIV-2 and have further research value.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a substituted glycine-3, 5-difluorophenylalanine peptide derivative and a preparation method thereof, and also provides an activity screening result of the compound serving as an HIV capsid protein inhibitor and application thereof.
The technical scheme of the invention is as follows:
1. substituted glycine-3, 5-difluorophenylalanine peptide derivative
The substituted glycine-3, 5-difluorophenylalanine peptide derivative or the pharmaceutically acceptable salt thereof has a structure shown in a general formula I:
Wherein, the liquid crystal display device comprises a liquid crystal display device,
R 1 the method comprises the following steps: hydrogen, methyl, ethyl, isopropyl substituted benzyl, cyclohexane methyl, cyclopropyl, benzyl; the substituent is selected from methyl, fluorine, chlorine, bromine, nitro, cyano, trifluoromethyl, methyl, methoxy and boric acid.
R 2 The method comprises the following steps: substituted phenyl, substituted benzyl, substituted naphthylmethyl, 2-methyl-1H-indole-3-methyl, 5-halo-1H-indole-3-methyl, phenylmethyl substituted with pinacol borate, 3- (trifluoromethyl) substituted 4,5,6, 7-tetrahydro-1H-indazole-1-methyl, 1, 5-dimethyl-3- (trifluoromethyl) -1H-pyrazole; the substituent is selected from methyl, fluorine, chlorine, bromine, nitro, cyano, trifluoromethyl, methyl, methoxy and boric acid.
According to the invention, R is preferably 1 Is fluoro-substituted benzyl, cyano-substituted benzyl, cyclohexane methyl and cyclopropylA benzyl group; r is R 2 Is naphthylmethyl, 2-methyl-1H-indole-3-methyl, 5-halo-1H-indole-3-methyl, pinacol borate substituted benzyl, 3- (trifluoromethyl) substituted 4,5,6, 7-tetrahydro-1H-indazole-1-methyl, 1, 5-dimethyl-3- (trifluoromethyl) -1H-pyrazole, boric acid substituted benzyl;
according to a further preferred embodiment of the invention, the substituted glycine-3, 5-difluorophenylalanine peptide derivative is one of the following compounds:
By "pharmaceutically acceptable salts" as used herein is meant salts of the compounds which are suitable for contact with the tissues of humans or lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit to risk ratio, generally water or oil soluble or dispersible, and effective for their intended use, within the scope of sound medical evaluation. Including pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, are contemplated herein and are compatible with the chemical nature of the compounds of formula I. A list of suitable salts is found in S.M. Birge et al, J.Pharm.Sci., pages 1977,66,1-19.
2. Preparation method of substituted glycine-3, 5-difluorophenylalanine peptide derivative
The preparation method of the substituted glycine-3, 5-difluorophenylalanine peptide derivative comprises the following steps: using Boc-L-phenylalanine (I-1) as a starting material and methylene dichloride as a solvent to generate an intermediate I-2 with N-methyl-4-aminoanisole through an amide condensation reaction; then the intermediate I-2 is dissolved in a proper amount of dichloromethane, and the Boc group is removed under the action of trifluoroacetic acid to obtain an intermediate I-3; then, reacting the intermediate I-3 with ammonium formate to obtain an intermediate I-4; the intermediate I-4 is dehydrated under the action of phosphorus oxychloride to obtain an intermediate isonitrile I-5, and then the I-5 reacts with acid, amine and aldehyde fragments through Ugi four components to obtain a target compound;
The synthetic route is as follows:
reagents and conditions: (i) N-methyl-4-aminoanisole, 1H-benzotriazole-1-yloxy tripyrrolidinyl hexafluorophosphate, N, N-diisopropylethylamine, dichloromethane, and transferring to room temperature at 0deg.C; (ii) trifluoroacetic acid, dichloromethane, room temperature; (iii) ammonium formate, acetonitrile, 90 ℃, reflux, 24h; (iv) dichloromethane, triethylamine, phosphorus oxychloride, 0 ℃ for 12 hours; (v) R is R 1 -CHO,R 2 -NH 2 ,R 3 -COOH, anhydrous methanol, 60 ℃,8h;
wherein R is 1 、R 2 As described in formula I above.
The room temperature of the invention is 20-30 ℃.
According to the preferred preparation method of the substituted glycine-3, 5-difluorophenylalanine peptide derivative, the specific steps are as follows:
(1) Adding Boc-L-phenylalanine (I-1) and 1H-benzotriazole-1-yl oxygen tripyrrolidine hexafluorophosphate into dichloromethane, and stirring for 30min under ice bath condition; then adding N, N-diisopropylethylamine and N-methyl-4-aminoanisole into the reaction liquid, removing the ice bath, transferring to room temperature, and monitoring by TLC; after the reaction is finished, the solvent is distilled off under reduced pressure, then saturated sodium bicarbonate solution is added into residues in a bottle, dichloromethane is used for extraction, an organic phase is separated, 1N HCl solution is added for washing, the organic phase is separated, saturated sodium chloride solution is added for washing, the organic phase is dried by anhydrous sodium sulfate, filtration and reduced pressure concentration are carried out on filtrate, and the obtained crude product is separated and purified by silica gel column chromatography to obtain an intermediate I-2;
(2) Adding the intermediate I-2 into dichloromethane, slowly dropwise adding excessive trifluoroacetic acid into the solution under stirring of ice bath condition, removing ice bath, transferring to room temperature, and monitoring by TLC; after the reaction is finished, the solvent is distilled off under reduced pressure, then saturated sodium bicarbonate solution is added to adjust the pH of the reaction solution to 7, and then dichloromethane solution is added for extraction; separating the organic phase, washing the organic phase with saturated sodium chloride solution for 3 times, drying the organic phase with anhydrous sodium sulfate, filtering, and evaporating the solvent under reduced pressure to obtain an intermediate I-3;
(3) Adding the intermediate I-3 and ammonium formate into acetonitrile solution, and heating at 90 ℃ for 10 hours; after the reaction is finished, extracting with water and ethyl acetate for three times, combining organic phases, filtering, evaporating the solvent under reduced pressure, and separating by silica gel column chromatography to obtain an intermediate I-4;
(4) Dissolving the intermediate I-4 in dichloromethane solution, adding triethylamine, slowly adding phosphorus oxychloride solution under ice bath condition, stirring at 0deg.C for 10 hr, quenching with ice water, extracting with dichloromethane, mixing organic phases, drying with anhydrous sodium sulfate, stirring with silica gel, and performing column chromatography to obtain intermediate I-5;
(5) Dissolving the corresponding substituted amine fragment and paraformaldehyde in anhydrous methanol solution, adding triethylamine, and stirring for 30 minutes at room temperature; subsequently adding the corresponding substituted acid fragment and stirring for 10 minutes at room temperature; then adding the isonitrile intermediate I-5, and adding the mixture into the mixture to reflux for 16 hours at 70 ℃; after the reaction is finished, adding saturated sodium bicarbonate solution into the residue in the bottle, extracting with dichloromethane, separating an organic phase, adding 1N HCl solution for washing, separating the organic phase, adding saturated sodium chloride solution for washing, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain the target product shown in the general formula 1.
3. Application of substituted glycine-3, 5-difluorophenylalanine peptide derivative
The invention discloses an anti-HIV-1 activity screening result of substituted glycine-3, 5-difluorophenylalanine peptide derivatives and first application of the anti-HIV-1 activity screening result as an HIV inhibitor. Experiments prove that the substituted glycine-3, 5-difluorophenylalanine peptide derivative can be used as an HIV inhibitor for preparing anti-AIDS drugs. The invention also provides application of the compound in anti-HIV medicaments.
anti-HIV-1 Activity and toxicity test of target Compounds
Cell level anti-HIV activity and toxicity tests were performed on a class of substituted glycine-3, 5-difluorophenylalanine peptide derivatives synthesized according to the above method, and their anti-HIV-1 and anti-HIV-2 activity and toxicity data are shown in Table 1.
The novel synthesized substituted glycine-3, 5-difluorophenylalanine peptide derivative shows better anti-HIV-1 activity. For example, the anti-HIV-1 activity of the target compounds I-7, I-9, I-19, I-21 is in the range of 2.53-2.94. Mu.M, wherein the anti-HIV-1 activity of the target compound I-19 (EC 50 ＝2.54μM,CC 50 >107.61 Especially. It is particularly emphasized that slight changes in the structure of part of the compounds have a significant effect on the activity, the selectivity of HIV-1 and HIV-2, for example, compounds I-6 and I-7 differ in that R 2 I-6 has no anti-HIV-1/2 activity, I-7 is an HIV-1 selective inhibitor; i-8 is simply R compared to Compound I-6 2 The substituent naphthyl is different in substituted position, and I-8 is an HIV-2 selective inhibitor. As another example, compound I-14 is also simply R as compared to compound I-13 2 The substituent naphthyl is different in substituted position, I-13 has little anti-HIV activity, and I-14 is an HIV-2 selective inhibitor. As another example, compound I-16 is simply R as compared to compound I-15 2 I-15 is HIV-1 selective inhibitor, and I-16 is HIV-2 selective inhibitor. Zidovudine as a positive drug has no selective inhibition of HIV-1/2; nevirapine is an HIV-1 selective inhibitor, and has no HIV-1/2 dual inhibition effect. Obviously, the bioactivity of the substituted glycine-3, 5-difluorophenylalanine peptide derivative achieves unexpected effects and deserves further development.
The peptide derivatives containing substituted glycine-3, 5-difluorophenylalanine can be used as HIV-1 and HIV-2 inhibitors. In particular, the compound is used as an HIV-1 and HIV-2 inhibitor for preparing anti-AIDS drugs.
An anti-HIV-1 and HIV-2 pharmaceutical composition comprises a peptide derivative containing substituted glycine-3, 5-difluorophenylalanine and one or more pharmaceutically acceptable carriers or excipients.
The invention provides a peptide derivative containing substituted glycine-3, 5-difluorophenylalanine and a preparation method thereof, and also provides screening results of partial compounds anti-HIV-1 and HIV-2 activity and first application thereof in the antiviral field. Experiments prove that the substituted glycine-3, 5-difluorophenylalanine peptide derivatives can be used as HIV-1 and HIV-2 inhibitors and have high application value. In particular, the compound is used as an HIV-1 and HIV-2 inhibitor for preparing anti-AIDS drugs.
Detailed Description
The invention will be further understood by the following examples, which are not intended to limit the scope of the invention.
Example 1: preparation of tert-butyl (S) - (3, 5-difluorophenyl) -1- ((4-methoxyphenyl) (methyl) amino) -1-oxopropan-2-yl) carbamate (I-2)
The starting material Boc-L-phenylalanine (I-1) (2.00 g,6.64 mmol), 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (3.46 g,6.64 mmol) was added to 20mL of dichloromethane and stirred for 30min under ice-bath conditions; n, N-diisopropylethylamine (2.23 mL,13.29 mmol) and N-methyl-4-aminoanisole (610 mg,4.43 mmol) were then added, the ice bath was removed and transferred to room temperature stirring, TLC monitoring; after 6h the reaction was completed, the solvent was distilled off under reduced pressure, then 40mL of saturated sodium bicarbonate solution, 40mL of dichloromethane were added to the residue in the bottle, the organic phase was taken out, 40mL of 1N HCl solution was added to wash, 40mL of saturated sodium chloride solution was taken out, the organic phase was added to wash, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the obtained crude product was separated by silica gel column chromatography (eluent EA: pe=1:8) to obtain 1.68g of crude product of tert-butyl (S) - (3, 5-difluorophenyl) -1- ((4-methoxyphenyl) (methyl) amino) -1-oxopropan-2-yl) carbamate (I-2) as an intermediate, yellow oil, yield 60%.
Spectral data:
1 H NMR(400MHz,DMSO-d 6 )δ7.31(d,J＝8.6Hz,2H,PhH),7.05(tt,J＝19.0,9.0Hz,4H,PhH),6.43(d,J＝7.0Hz,2H,PhH),4.14(td,J＝10.6,3.1Hz,1H,CH),3.80(s,3H,OCH 3 ),3.15(d,J＝7.8Hz,3H,NCH 3 ),2.87–2.73(m,1H,CH),2.69–2.57(m,1H,CH),1.29(s,9H,3(CH 3 )).
ESI-MS:m/z 443.78(M+Na).C 22 H 26 F 2 N 2 O 4 [420.4].
example 2: preparation of (S) -2-amino-3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-3)
Intermediate I-2 (500 mg,1.19 mmol) was added to 30mL of dichloromethane, and trifluoroacetic acid (410 mg,3.57 mmol) was then added slowly to the solution, stirred at room temperature, monitored by TLC; after completion of the reaction, the pH of the reaction mixture was adjusted to 7 with a saturated sodium hydrogencarbonate solution, 40mL of methylene chloride was added for extraction, the organic phase was separated, washed with a saturated sodium chloride solution (3X 20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2.36g of crude intermediate (S) -2-amino-N- (4-methoxyphenyl) -N-methyl-3-phenylpropionamide (3) as a yellow oil in 80% yield.
Spectral data:
1 H NMR(400MHz,DMSO-d 6 )δ7.07(d,J＝8.3Hz,2H),7.05–6.93(m,3H),6.57(h,J＝4.0Hz,2H),3.78(s,3H),3.35(dd,J＝7.6,5.9Hz,1H),3.09(s,3H),2.74(dd,J＝13.1,5.8Hz,1H),2.54–2.45(m,2H),1.82(s,2H).
example 3: preparation of intermediate (S) -3- (3, 5-difluorophenyl) -2-carboxamide-N- (4-methoxyphenyl) -N-methylpropanamide (I-4)
Intermediate I-3 (340 mg,1.06 mmol) was added to 10mL of acetonitrile, then ammonium formate (130 mg,2.12 mmol) was added to the solution, heated to reflux at 90℃for 24h, monitored by TLC; after 24h the reaction was completed, filtered, extracted with water, ethyl acetate (3X 10 mL) and the organic phases combined; 20mL of saturated sodium chloride solution was added, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by silica gel column chromatography (eluent EA: PE=1:8) to give crude product 1.68g of tert-butyl (I-4) carbamate as intermediate (S) - (3, 5-difluorophenyl) -1- ((4-methoxyphenyl)) methyl) amino) -1-oxopropan-2-yl) (yield 60%.
Spectral data:
1 H NMR(400MHz,DMSO-d 6 )δ8.47(d,J＝8.3Hz,1H),7.87(s,1H),7.22(d,J＝8.3Hz,2H),7.05(td,J＝8.9,4.6Hz,4H),6.50(h,J＝4.3Hz,2H),4.52(td,J＝8.8,4.5Hz,1H),3.80(s,3H),3.13(s,3H),2.88(dd,J＝13.6,4.6Hz,1H),2.67(dd,J＝13.7,9.4Hz,1H).
ESI-MS:m/z 349.3(M+1).C 18 H 18 F 2 N 2 O 3 [348.35].
example 4: preparation of intermediate (S) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methyl-2- (methyl-azaformyl) propylamine isonitrile (I-5)
Intermediate I-4 (410 mg,1.18 mmol) was added to 10mL of dichloromethane, and triethylamine (360 mg,3.54 mmol) was then added to the solution, followed by slow addition of phosphorus oxychloride (180 mg,1.18 mmol) under ice-bath conditions; stirring at 0deg.C for 24h, and monitoring by TLC; after 24h the reaction was completed, quenched with ice water, extracted with dichloromethane (3×15 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the supernatant concentrated, and stirred with silica gel to give intermediate (S) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methyl-2- (methyl-azaformyl) propanamine isonitrile (I-5) 0.17g as a pale yellow solid in yield by column chromatography (ethyl acetate: petroleum ether=1:2): 45%.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ7.26–7.10(m,3H,PhH),7.03(d,J＝8.8Hz,2H,PhH),6.76(hept,J＝4.2Hz,2H,PhH),4.48(dd,J＝8.3,5.8Hz,1H,CH),3.80(s,3H,OCH 3 ),3.15(s,3H,NCH 3 ),3.14–3.07(m,1H,CH),2.99(dd,J＝13.6,8.3Hz,1H,CH).ESI-MS:m/z332.2(M+1).C 18 H 17 F 2 N 2 O 2 [331.12].
example 5: preparation of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (naphthalen-2-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropanamide (I-6)
Paraformaldehyde (18 mg,0.20 mmol) is dissolved in 6.0mL of anhydrous methanol, and 4-fluorobenzylamine (25 mg,0.20 mmol) is dissolved in 6.0mL of anhydrous methanol, followed by addition of 2-naphthylacetic acid (40 mg,0.20 mmol), stirring at room temperature for 10 min, followed by addition of key intermediate I-5 (50 mg,0.15 mmol), heating reflux at 70 ℃ for 16h, TLC monitoring; after 16h the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and concentration of the filtrate under reduced pressure, and the obtained crude product was separated by silica gel column chromatography (eluent EA: PE=1:2) to give 0.01g of crude product of target product (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (naphthalen-2-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropanamide (I-6), as a pale yellow solid, yield 11%, melting point: 112-114 ℃.
Spectral data:
1 H NMR(400MHz,DMSO-d 6 )δ8.56(d,J＝8.2Hz,1H),7.93–7.86(m,1H),7.86–7.80(m,2H),7.69(d,J＝46.7Hz,1H),7.55–7.44(m,2H),7.39–7.32(m,1H),7.30–7.21(m,3H),7.19–7.09(m,3H),7.09–6.96(m,3H),6.60–6.47(m,2H),4.60(td,J＝8.8,7.9,5.1Hz,1H),4.53–4.20(m,2H),3.96(td,J＝27.6,27.1,13.4Hz,3H),3.84–3.77(m,3H),3.70(s,1H),3.21–3.11(m,3H),2.90(ddd,J＝19.1,14.3,4.3Hz,1H),2.75–2.62(m,1H). 13 C NMR(100MHz,DMSO-d 6 )δ171.76,170.90,169.49(d,J＝265.2Hz),162.51(dd,J＝245.8,13.4Hz),159.23,159.15,142.54(t,J＝9.4Hz),135.84,134.23(d,J＝3.1Hz),133.87,133.74,133.46,133.41,132.25,130.26(d,J＝8.2Hz),129.48(d,J＝8.1Hz),129.19,128.49,128.38,127.98,127.93,127.89,127.81,126.48,126.01,115.84(d,J＝21.4Hz),115.54(d,J＝21.3Hz),115.33,115.24,112.41(d,J＝24.4Hz),102.54,55.94,55.90,51.47,51.33,49.85,37.80,37.34.ESI-MS:m/z 654.70(M+1).C 38 H 34 F 3 N 3 O 4 [653.70].
example 6: preparation of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (2-methyl-1H-indol-3-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropanamide (I-7)
Paraformaldehyde (20 mg,0.20 mmol) and 4-fluorobenzylamine (25 mg,0.20 mmol) are dissolved in 6.0mL of anhydrous methanol, triethylamine (20 mg,0.20 mmol) is added and stirred at room temperature for 30 minutes, followed by 2- (2-methyl-1H-indol-3-yl) acetic acid (35 mg,0.20 mmol) which is stirred at room temperature for 10 minutes, followed by key intermediate I-5 (50 mg,0.15 mmol) which is heated at 70℃under reflux for 16 hours, TLC monitoring; after 16H the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and the filtrate was concentrated under reduced pressure, and the obtained crude product was separated by silica gel column chromatography (eluent ethyl acetate: petroleum ether=1:2) to give 0.009g of crude product of target product (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (2-methyl-1H-indol-3-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropanamide (I-7), light yellow solid, yield 10.20%, melting point: 112.5-114.5 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ10.76(d,J＝16.8Hz,1H),8.51(d,J＝8.1Hz,1H),7.34(d,J＝8.1Hz,1H),7.25(dd,J＝17.4,8.2Hz,3H),7.16(dd,J＝8.2,5.9Hz,2H),7.07(dd,J＝8.7,5.2Hz,3H),7.03–6.97(m,3H),6.89(t,J＝7.4Hz,1H),6.50(dd,J＝18.1,6.7Hz,2H),4.58(tt,J＝11.3,5.5Hz,1H),4.51–4.40(m,1H),4.18(d,J＝15.1Hz,1H),3.96(d,J＝17.6Hz,1H),3.87(d,J＝12.3Hz,1H),3.81(s,3H),3.72(d,J＝17.3Hz,1H),3.56(s,1H),3.15(d,J＝11.0Hz,3H),2.88(ddd,J＝23.4,13.6,4.2Hz,1H),2.74–2.60(m,1H),2.22(s,3H).ESI-MS:m/z657.71(M+1).C 37 H 35 F 3 N 4 O 4 [656.71].
example 7: preparation of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (naphthalen-1-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropanamide (I-8)
Paraformaldehyde (18 mg,0.20 mmol) and 4-fluorobenzylamine (25 mg,0.20 mmol) were dissolved in 6.0mL of anhydrous methanol, triethylamine (20 mg,0.20 mmol) was added and stirred at room temperature for 30 minutes, followed by 1-naphthaleneacetic acid (37 mg,0.20 mmol) stirred at room temperature for 10 minutes, then key intermediate I-5 (50 mg,0.15mmol,1.0 eq.) was added and heated under reflux at 70 ℃ for 16 hours, monitored by TLC; after 16H the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and the filtrate was concentrated under reduced pressure, and the crude product obtained was separated by silica gel column chromatography (eluent ethyl acetate: petroleum ether=1:2) to give 9mg of crude product of target product (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (2-methyl-1H-indol-3-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropanamide (I-8), light yellow solid, yield 10.20%, melting point: 112.5-114.5 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ8.59(d,J＝8.1Hz,1H),7.92–7.79(m,3H),7.52–7.41(m,3H),7.26(ddd,J＝14.5,12.3,7.2Hz,5H),7.18–7.12(m,2H),7.06(d,J＝8.6Hz,2H),6.94–6.83(m,1H),6.53(dt,J＝10.2,5.0Hz,2H),4.71–4.56(m,1H),4.51(t,J＝14.4Hz,1H),4.21–4.11(m,2H),4.05–3.93(m,3H),3.80(d,J＝7.6Hz,3H),3.13(s,3H),2.96–2.84(m,1H),2.74–2.64(m,1H). 13 C NMR(101MHz,DMSO-d 6 )δ171.75,170.87,168.54,163.70(d,J＝13.1Hz),163.06,161.32,159.23,142.49,135.87,134.35,133.75,133.10(d,J＝43.3Hz),130.25(d,J＝8.1Hz),129.57(d,J＝8.2Hz),129.21,128.43(d,J＝40.3Hz),127.59,127.50,126.36,126.17,126.04,125.94,125.89,125.77,125.09,115.91(d,J＝21.5Hz),115.54(d,J＝21.2Hz),115.31,112.49(d,J＝6.4Hz),112.31(d,J＝6.5Hz),102.49(t,J＝25.7Hz),55.91,51.67,49.92,49.27,37.83,37.45,37.28.ESI-MS:m/z 654.5(M+1).C 38 H 34 F 3 N 3 O 4 [653.70].
example 8: preparation of (S) -2- (2- (2- (5-bromo-1H-indol-3-yl) -N- (4-fluorobenzyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-9)
Paraformaldehyde (18 mg,0.20 mmol) and 4-fluorobenzylamine (25 mg,0.20 mmol) were dissolved in 6.0mL of anhydrous methanol, triethylamine (20 mg,0.20 mmol) was added and stirred at room temperature for 30 minutes, followed by 5-bromo-1H-indole-3-carboxylic acid (51 mg,0.20 mmol) stirred at room temperature for 10 minutes, then key intermediate I-5 (50 mg,0.15 mmol) was added, heated at 70℃under reflux for 16 hours, monitored by TLC; after 16H the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and the filtrate was concentrated under reduced pressure, and the crude product obtained was separated by column chromatography on silica gel (eluent ethyl acetate: petroleum ether=1:2) to give 0.011g of crude product of (S) -2- (2- (2- (5-bromo-1H-indol-3-yl) -N- (4-fluorobenzyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-9), 9.60% yield, melting point: 78-80 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ11.11(d,J＝2.4Hz,1H),8.57(t,J＝8.3Hz,1H),7.73–7.66(m,1H),7.31(dd,J＝13.5,8.5Hz,4H),7.23–7.17(m,4H),7.11–7.06(m,3H),6.56–6.47(m,3H),5.47(dd,J＝10.3,7.2Hz,1H),4.63–4.48(m,2H),4.19(dd,J＝15.0,6.6Hz,1H),4.05–3.91(m,2H),3.80(d,J＝1.5Hz,3H),3.63(d,J＝9.5Hz,2H),3.15(d,J＝9.5Hz,3H),2.90(td,J＝14.0,6.6Hz,1H),2.71(dd,J＝13.5,9.5Hz,1H). 13 C NMR(150MHz,DMSO-d 6 )δ171.90,170.86,168.41,162.54(dd,J＝246.1,13.2Hz),162.54(dd,J＝246.1,13.2Hz),159.23,142.50,135.86,135.34,134.32,130.18(d,J＝7.7Hz),129.75,129.41(d,J＝8.3Hz),129.16,125.99,125.91,123.92,123.86,121.79,121.64,115.83,115.69,115.50,115.36,115.31,115.26,113.74,113.71,112.46,112.43,112.33,112.30,111.50,108.51,102.47,102.30,55.91,51.55,49.92,49.15,37.79,37.34,30.37.ESI-MS:m/z 757.26(M+2Na),C 36 H 32 BrF 3 N 4 O 4 [721.58].
example 9: preparation of (S) -2- (2- (2- (5-bromo-1H-indol-3-yl) -N- (cyclohexylmethyl) acetamido) acetamide) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-10)
Paraformaldehyde (18 mg,0.20 mmol) and cyclohexylmethylamine (23 mg,0.20 mmol) were dissolved in 6.0mL of anhydrous methanol, triethylamine (20 mg,0.20 mmol) was added and stirred at room temperature for 30 minutes, followed by 5-bromo-1H-indole-3-carboxylic acid (500 mg,0.20 mmol) and stirred at room temperature for 10 minutes, then key intermediate I-5 (50 mg,0.15 mmol) was added, heated to reflux for 16 hours at 70℃and monitored by TLC; after 16H the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and the filtrate was concentrated under reduced pressure, and the obtained crude product was separated by silica gel column chromatography (eluent ethyl acetate: petroleum ether=1:2) to give the crude product of (S) -2- (2- (2- (5-bromo-1H-indol-3-yl) -N- (cyclohexylmethyl) acetamido) acetamide) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-10) 0.011g, white solid, yield 9.80%, melting point: 80-82 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ11.08(d,J＝6.4Hz,1H),8.51(t,J＝9.1Hz,1H),7.74–7.65(m,1H),7.31(dd,J＝8.5,4.0Hz,2H),7.26–7.13(m,2H),7.11–6.90(m,3H),6.52(d,J＝7.0Hz,2H),4.66–4.43(m,1H),4.06–3.86(m,2H),3.79(d,J＝3.8Hz,3H),3.77–3.67(m,1H),3.54(dd,J＝17.0,7.8Hz,1H),3.14(d,J＝9.6Hz,3H),3.12–3.03(m,1H),2.95(dd,J＝17.0,5.3Hz,1H),2.88(dd,J＝17.0,3.7Hz,1H),2.75(ddd,J＝26.8,13.3,8.3Hz,1H),1.59–1.56(m,1H),1.52–0.69(m,10H). 13 C NMR(100MHz,DMSO-d 6 )δ171.70,170.91,168.75,168.31,162.50(d,J＝232.9Hz),159.22,135.86,135.30(d,J＝4.8Hz),129.67(d,J＝2.3Hz),129.21,125.82,123.79,121.85,115.31,112.56(dd,J＝226.7,7.3Hz),112.49,112.25,108.77,102.48,55.92,52.81,51.50,51.02,37.79,37.24,36.96,36.04,30.71,30.48,30.35,26.52,25.79.ESI-MS:m/z 726.6(M+NH4 + ).C 36 H 39 BrF 2 N 4 O 4 [709.63].
example 10: preparation of (S) -2- (2- (N-cyclopropyl-2- (2-methyl-1H-indol-3-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-11)
Paraformaldehyde (20 mg,0.20 mmol) and cyclopropylamine (11 mg,0.20 mmol) are dissolved in 6.0mL of anhydrous methanol, triethylamine (20 mg,0.20 mmol) is added and stirred at room temperature for 30 minutes, followed by 2- (2-methyl-1H-indol-3-yl) acetic acid (40 mg,0.20 mmol) which is stirred at room temperature for 10 minutes, then key intermediate I-5 (50 mg,0.15 mmol) is added, heated to reflux at 70℃for 16 hours, monitored by TLC; after 16H the reaction was completed, 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by column chromatography on silica gel (eluent methanol: dichloromethane=1:40) to give 0.009g of crude product of target product (S) -2- (2- (N-cyclopropyl-2- (2-methyl-1H-indol-3-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-11), white solid, yield 9.80%, melting point: 98-100 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ10.71(s,1H),8.16(d,J＝8.2Hz,1H),7.34(d,J＝7.8Hz,1H),7.19(dd,J＝8.2,2.8Hz,3H),6.99(t,J＝8.1Hz,3H),6.93(t,J＝7.4Hz,1H),6.85(t,J＝7.2Hz,1H),6.47(d,J＝6.7Hz,2H),4.45(td,J＝8.8,4.6Hz,1H),3.94(d,J＝16.5Hz,1H),3.85(d,J＝17.0Hz,2H),3.76(d,J＝5.4Hz,3H),3.71(s,1H),3.10(d,J＝9.2Hz,3H),2.83(dd,J＝13.5,4.1Hz,1H),2.69–2.57(m,1H),2.26(s,3H),2.22–2.10(m,1H),1.33–1.11(m,2H),0.71–0.64(m,2H).ESI-MS:m/z 589.31(M+1).C 33 H 34 F 2 N 4 O 4 [588.66].
example 11: preparation of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (4- (pinacol borate) phenyl) acetamido) -N- (4-methoxyphenyl) -N-methylpropylamine (I-12)
Paraformaldehyde (20 mg,0.20 mmol) and 4-fluorobenzylamine (25 mg,0.20 mmol) were dissolved in 6.0mL of anhydrous methanol, triethylamine (20 mg,0.20 mmol) was added and stirred at room temperature for 30 minutes, followed by 4-phenylboronic acid pinacol ester (50 mg,0.20 mmol) which was stirred at room temperature for 10 minutes, then key intermediate I-5 (50 mg,0.15 mmol) was added and heated at 70℃under reflux for 16 hours, monitored by TLC; after 16h the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by column chromatography over silica gel (eluent methanol: dichloromethane=1:40) to give the crude product of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (4, 5-tetramethyl-1, 3, 2-dioxabenzaldehyde-2-yl) phenyl) acetamido) -N- (4-methoxyphenyl) -N-methylpropylamine (I-12) as a white solid in a yield of 0.011g, 10.5% melting point: 98-100 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ8.51(d,J＝8.3Hz,1H),7.59(t,J＝6.8Hz,2H),7.27–7.24(m,2H),7.22–7.19(m,2H),7.15(dd,J＝10.9,7.8Hz,4H),7.06–7.00(m,3H),6.50(t,J＝6.7Hz,2H),4.56(ddd,J＝17.0,10.9,6.0Hz,1H),4.42(dd,J＝14.5,7.6Hz,1H),4.18(d,J＝15.0Hz,1H),3.96–3.88(m,2H),3.82(d,J＝8.3Hz,3H),3.54(s,2H),3.14(d,J＝12.0Hz,3H),2.94–2.81(m,1H),2.73–2.61(m,1H),1.29(s,12H). 13 C NMR(150MHz,DMSO-d 6 )δ171.50,170.80,168.07,162.68,161.77,161.68,161.07,159.24,142.51,142.45,139.55,139.44,135.84,134.88,134.82,134.77,134.16(d,J＝3.0Hz),130.24(d,J＝8.1Hz),129.44(d,J＝8.2Hz),129.33,129.31,129.24,129.12,115.86,115.72,115.50(d,J＝21.3Hz),115.31,115.25,112.39(d,J＝24.6Hz),102.45,102.28,84.00,83.98,55.89,51.43,49.79,49.06,47.69,41.48,37.76,37.44,25.38,25.08.ESI-MS:m/z 730.16(M+1),752.50(M+Na).C 40 H 43 BF 3 N 3 O 6 [729.60].
example 12: preparation of (S) -2- (2- (N- (4-cyanobenzene) -2- (naphthalen-2-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-13)
Paraformaldehyde (20 mg,0.22mmol,1.3 eq.) and 4-cyanobenzylamine (30 mg,0.20 mmol) are dissolved in 4.0mL of anhydrous methanol, triethylamine (20 mg,0.20 mmol) is added and stirred at room temperature for 30 minutes, followed by 2-naphthylacetic acid (40 mg,0.20 mmol) which is stirred at room temperature for 10 minutes, then key intermediate I-5 (50 mg,0.15 mmol) is added, heated to reflux for 16 hours at 70 ℃, monitored by TLC; after 16h the reaction was completed, 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and concentration of the filtrate under reduced pressure, and the resulting crude product was separated by column chromatography on silica gel (eluent methanol: dichloromethane=1:40) to give crude product of (S) -2- (2- (N- (4-cyanobenzene) -2- (naphthalen-2-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-13) in an amount of 0.008g, a white solid, yield 7.80%, melting point: 60-62 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ8.60(d,J＝8.3Hz,1H),7.86(dt,J＝14.0,6.9Hz,3H),7.76(t,J＝6.8Hz,2H),7.67(d,J＝22.8Hz,1H),7.49(p,J＝6.2,5.6Hz,2H),7.44–7.18(m,5H),7.13–6.91(m,3H),6.67–6.41(m,2H),4.79–4.48(m,2H),4.39(d,J＝16.0Hz,1H),4.20–3.95(m,2H),3.82(d,J＝19.2Hz,3H),3.70(dd,J＝16.1,6.3Hz,2H),3.15(d,J＝14.7Hz,3H),2.90(td,J＝15.1,14.5,6.6Hz,1H),2.70(dd,J＝13.6,10.0Hz,1H). 13 C NMR(150MHz,DMSO-d 6 )δ172.08,170.80,168.16,162.52(dd,J＝246.0,13.3Hz),159.25,144.11,142.49,135.84,133.75,133.43,132.86,132.66,132.30,129.15,128.88,128.49,128.27,128.14,128.07,127.99,127.97,127.93,127.90,127.82,126.48,126.42,126.02,119.27,115.34,115.25,112.40(d,J＝24.9Hz),110.30,102.50,102.32,55.94,52.12,51.49,50.56,50.17,37.80,37.41.ESI-MS:m/z661.12(M+1),683.52(M+Na).C 39 H 34 F 2 N 4 O 4 [660.72].
example 13: preparation of (S) -2- (2- (N- (4-cyanobenzene) -1- (naphthalen-1-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-14)
Paraformaldehyde (20 mg,0.22 mmol) and 4-cyanobenzylamine (30 mg,0.20 mmol) were dissolved in 4.0mL of anhydrous methanol, triethylamine (20 mg,0.20 mmol) was added and stirred at room temperature for 30 minutes, followed by 1-naphthaleneacetic acid (40 mg,0.20 mmol) stirred at room temperature for 10 minutes, then key intermediate I-5 (50 mg,0.15 mmol) was added, and heated at 70℃under reflux for 16 hours, monitored by TLC; after 16h the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and concentration of the filtrate under reduced pressure, and the resulting crude product was separated by column chromatography on silica gel (eluent methanol: dichloromethane=1:40) to give crude product of (S) -2- (2- (N- (4-cyanobenzene) -2- (naphthalen-2-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-14) in a yield of 0.012g as a pale yellow solid, 10.60% in melting point: 106-108 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ8.63(d,J＝8.1Hz,1H),7.90(d,J＝7.4Hz,1H),7.87–7.76(m,4H),7.53–7.45(m,2H),7.41(q,J＝10.3,8.8Hz,3H),7.34–7.20(m,3H),7.04(dd,J＝16.3,8.8Hz,2H),6.86(d,J＝9.4Hz,1H),6.51(t,J＝6.4Hz,2H),4.63–4.46(m,2H),4.39–4.16(m,2H),4.14–3.95(m,3H),3.80(d,J＝5.4Hz,3H),3.12(s,3H),2.89(td,J＝14.9,14.3,6.7Hz,1H),2.76–2.62(m,1H). 13 C NMR(100MHz,DMSO-d 6 )δ172.85,171.66,169.36,164.42,162.04(d,J＝13.0Hz),160.04,145.11,143.25(t,J＝9.3Hz),135.61(d,J＝212.5Hz),134.01,133.74(d,J＝12.6Hz),133.50,130.02,129.65,129.28(d,J＝32.1Hz),128.34,126.99,126.66(d,J＝18.0Hz),125.90,120.14,116.12,113.20(d,J＝24.4Hz),111.03,56.73,52.51,51.40,51.15,38.64,38.17,38.07.ESI-MS:m/z 636.54(M-Na).C 39 H 34 F 2 N 4 O 4 [660.72].
example 14: preparation of (S) -2- (2- (N- (4-cyanobenzene) -2- (5-fluoro-1H-indol-3-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-15)
Paraformaldehyde (30 mg,0.27 mmol) and 4-cyanobenzylamine (40 mg,0.27 mmol) were dissolved in 4.0mL of anhydrous methanol, triethylamine (30 mg,0.27 mmol) was added and stirred at room temperature for 30 minutes, followed by 2- (5-fluoro-1H-indol-3-yl) acetic acid (50 mg,0.27 mmol) and stirred at room temperature for 10 minutes, then key intermediate I-5 (70 mg,0.21 mmol) was added, heated to reflux at 70℃for 16 hours, and TLC monitoring was performed; after 16H the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and concentration of the filtrate under reduced pressure, and the obtained crude product was separated by silica gel column chromatography (eluent methanol: dichloromethane=1:40) to give 0.015g of crude product of (S) -2- (2- (N- (4-cyanobenzene) -2- (5-fluoro-1H-indol-3-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-15), pale yellow solid, yield 10.50%, melting point: 120-122 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ10.98(d,J＝15.4Hz,1H),8.57(d,J＝8.1Hz,1H),7.85–7.64(m,2H),7.37–7.31(m,2H),7.29–7.23(m,3H),7.19(dd,J＝14.2,2.3Hz,1H),7.09–6.96(m,3H),6.91(tq,J＝8.6,2.6Hz,1H),6.52(d,J＝6.0Hz,2H),4.67–4.45(m,2H),4.42–4.02(m,2H),4.02–3.91(m,1H),3.80(d,J＝2.8Hz,3H),3.75(s,1H),3.61(s,1H),3.14(d,J＝8.2Hz,3H),2.89(td,J＝15.5,14.6,6.6Hz,1H),2.69(ddd,J＝19.6,11.9,4.3Hz,1H).
example 15: preparation of (S) -2- (2- (2- (5-bromo-1H-indol-3-yl) -N- (4-cyanobenzene) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-16)
Paraformaldehyde (30 mg,0.27 mmol) and 4-cyanobenzylamine (40 mg,0.30 mmol) were dissolved in 4.0mL of anhydrous methanol, triethylamine (30 mg,0.27 mmol) was added and stirred at room temperature for 30 minutes, followed by 2- (5-bromo-1H-indol-3-yl) acetic acid (70 mg,0.27 mmol) and stirred at room temperature for 10 minutes, then key intermediate I-5 (70 mg,0.21 mmol) was added, heated to reflux at 70℃for 16 hours, and TLC monitoring was performed; after 16H the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration, and the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by column chromatography on silica gel (eluent methanol: dichloromethane=1:40) to give the desired product (S) -2- (2- (2- (5-bromo-1H-indol-3-yl) -N- (4-cyanophenyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-16) 0.017g, pale yellow solid, yield 11.20%, melting point: 120-122 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ11.08(d,J＝13.8Hz,1H),8.55(d,J＝8.1Hz,1H),7.73(dd,J＝12.1,8.2Hz,2H),7.63(d,J＝21.8Hz,1H),7.35–7.16(m,6H),7.09–6.91(m,3H),6.51(d,J＝5.9Hz,2H),4.66–4.43(m,2H),4.34(d,J＝15.9Hz,1H),4.19–3.88(m,2H),3.84–3.73(m,4H),3.62(s,1H),3.14(d,J＝10.0Hz,3H),2.89(td,J＝14.9,14.2,6.6Hz,1H),2.67(ddd,J＝17.7,13.6,9.5Hz,1H). 13 C NMR(100MHz,DMSO-d 6 )δ172.96,171.65,169.23,164.54(d,J＝13.4Hz),162.09(d,J＝13.3Hz),159.98(d,J＝10.2Hz),145.11,143.27,136.63,136.07(d,J＝8.0Hz),133.40,130.00,129.60,126.83,124.67,122.49(d,J＝21.0Hz),120.11,116.11,114.53,113.18(d,J＝24.7Hz),112.28,110.99,109.20,103.33,56.74,52.40,52.11,51.39,51.04,38.60,30.99.
example 16: preparation of (S) -2- (2- (N- (4-cyanophenyl) -2- (4- (4- (pinacol borate) phenyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-17)
Paraformaldehyde (30 mg,0.31 mmol) and 4-cyanobenzylamine (40 mg,0.30 mmol) were dissolved in 4.0mL of anhydrous methanol, triethylamine (30 mg,0.31 mmol) was added and stirred at room temperature for 30 minutes, followed by 4-phenylboronic acid pinacol ester (80 mg,0.31 mmol) which was stirred at room temperature for 10 minutes, then key intermediate I-5 (80 mg,0.24 mmol) was added and heated at 70℃under reflux for 16 hours, monitored by TLC; after 16h the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by column chromatography over silica gel (eluent methanol: dichloromethane=1:40) to give crude product of (S) -2- (2- (N- (4-cyanobenzene) -2- (4, 5-tetramethyl-1, 3, 2-dioxan-2-yl) phenyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-17) as a pale yellow solid in 11.20% yield, melting point: 46-48 ℃.
Spectral data: 1 H NMR(400MHz,DMSO-d 6 )δ8.24(d,J＝8.1Hz,1H),7.60(d,J＝7.6Hz,2H),7.24(t,J＝8.0Hz,4H),7.02(d,J＝8.4Hz,3H),6.51(d,J＝6.8Hz,2H),4.47(tt,J＝10.9,5.4Hz,1H),4.01–3.88(m,3H),3.80(s,3H),3.71(d,J＝28.8Hz,1H),3.13(s,3H),2.87(dd,J＝13.5,4.2Hz,1H),2.70–2.66(m,1H),1.29(s,12H),0.74–0.58(m,4H). 13 C NMR(100MHz,DMSO-d 6 )δ170.76,168.07,162.49(dd,J＝246.0,13.4Hz),159.21,144.08,139.46,135.79,134.76,132.89,132.70,129.41,129.28,129.16,128.81,128.10,119.30,115.31,112.39(d,J＝24.6Hz),110.24,84.02,74.00,55.93,55.91,51.45,50.41,50.06,37.78,37.34,25.42,25.13.ESI-MS:m/z 759.56(M+Na).C 41 H 43 BF 2 N 4 O 6 [737.20].
example 17: preparation of (S) -2- (2- (N-cyclopropyl-2- (4- (4- (pinacol borate) phenyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-18)
Paraformaldehyde (40 mg,0.39 mmol) and cyclopropylamine (20 mg,0.39 mmol) were dissolved in 4.0mL of anhydrous methanol, triethylamine (40 mg,0.39 mmol) was added and stirred at room temperature for 30 minutes, followed by 4-phenylboronic acid pinacol ester (100 mg,0.39 mmol) and stirred at room temperature for 10 minutes, then key intermediate I-5 (100 mg,0.30 mmol) was added, heated at 70℃under reflux for 16 hours, monitored by TLC; after 16h the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and concentration of the filtrate under reduced pressure, and the resulting crude product was separated by column chromatography over silica gel (eluent methanol: dichloromethane=1:40) to give crude product of (S) -2- (2- (N-cyclopropyl-2- (4, 5-tetramethyl-1, 3, 2-dioxan-2-yl) phenyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-18) as a pale yellow solid in a yield of 11.20% melting point: 46-48 ℃.
Spectral data:
1 H NMR(400MHz,DMSO-d 6 )δ8.24(d,J＝8.1Hz,1H),7.60(d,J＝7.6Hz,2H),7.24(t,J＝8.0Hz,4H),7.02(d,J＝8.4Hz,3H),6.51(d,J＝6.8Hz,2H),4.47(tt,J＝10.9,5.4Hz,1H),4.01–3.88(m,3H),3.80(s,3H),3.71(d,J＝28.8Hz,1H),3.13(s,3H),2.87(dd,J＝13.5,4.2Hz,1H),2.70–2.66(m,1H),1.29(s,12H),0.74–0.58(m,4H).
13 C NMR(100MHz,DMSO-d 6 )δ173.20,170.91,168.57,162.50(dd,J＝245.7,13.4Hz),159.14,142.56,139.85,135.83,134.70,129.52,129.15,115.28,112.50,112.26,102.43,84.00,55.91,51.22,49.51,37.76,37.39,31.25,25.42,25.13,9.03,8.86.
ESI-MS:m/z 662.17(M+1),684.54(M+Na).C 36 H 42 BF 2 N 3 O6[661.31].
example 17: preparation of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (3- (trifluoromethyl) -4,5,6, 7-tetrahydro-1H-indazol-1-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropanamide (I-19)
Paraformaldehyde (40 mg,0.39 mmol) and 4-fluorobenzylamine (50 mg,0.39 mmol) were dissolved in 4.0mL of anhydrous methanol, triethylamine (40 mg,0.39 mmol) was added and stirred at room temperature for 30 minutes, followed by 2- (3- (trifluoromethyl) -4,5,6, 7-tetrahydro-1H-indazol-1-yl) acetic acid (100 mg,0.39 mmol) and stirred at room temperature for 10 minutes, then key intermediate I-5 (100 mg,0.30 mmol) was added, heated to reflux at 70℃for 16 hours, TLC monitoring; after 16H the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by column chromatography over silica gel (eluent methanol: dichloromethane=1:40) to give the crude product of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (3- (trifluoromethyl) -4,5,6, 7-tetrahydro-1H-indazol-1-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropanamide (I-19) as a pale yellow solid in a yield of 0.017g, 11.20% melting point: 46-48 ℃.
Spectral data:
1 H NMR(400MHz,DMSO-d 6 )δ8.61(d,J＝8.2Hz,1H),7.28–7.21(m,4H),7.15(t,J＝8.8Hz,2H),7.04(d,J＝8.9Hz,2H),7.01–6.95(m,1H),6.51(dd,J＝15.1,7.4Hz,2H),5.19(s,1H),4.99(s,2H),4.58–4.41(m,2H),4.05(d,J＝15.8Hz,2H),3.97–3.86(m,1H),3.79(d,J＝7.0Hz,3H),3.73–3.63(m,1H),3.13(d,J＝7.6Hz,3H),2.95–2.83(m,1H),2.67(dd,J＝13.6,9.6Hz,1H),2.49–2.28(m,3H),1.88–1.50(m,5H).
13 C NMR(100MHz,DMSO-d 6 )δ170.70,167.84,167.62,163.79,161.35,161.21,159.21,142.16,135.78,133.58,130.33,130.25,129.82,129.18,115.89,115.71,115.49,115.31,114.50,112.55,112.48,112.30,102.51,55.87,51.61,51.09,49.41,49.02,37.82,37.31,22.41,21.94,20.90,20.00.
example 18: preparation of (S) -2- (2- (N- (cyclohexylmethyl) -2- (5-methyl-3- (trifluoromethyl) -1H-pyrazol-1-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-20)
Paraformaldehyde (30 mg,0.31 mmol) and cyclohexylmethylamine (40 mg,0.31 mmol) were dissolved in 4.0mL of anhydrous methanol, triethylamine (30 mg,0.31 mmol) was added and stirred at room temperature for 30 minutes, followed by 2- (3- (trifluoromethyl) -4,5,6, 7-tetrahydro-1H-indazol-1-yl) acetic acid (70 mg,0.31 mmol), stirred at room temperature for 10 minutes, then key intermediate I-5 (80 mg,0.24 mmol) was added, heated at 70℃under reflux for 16 hours, TLC monitoring; after 16H the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by column chromatography over silica gel (eluent methanol: dichloromethane=1:40) to give the crude product of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (3- (trifluoromethyl) -4,5,6, 7-tetrahydro-1H-indazol-1-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropanamide (I-20) as a pale yellow solid in 11.20% yield, melting point: 46-48 ℃.
Spectral data:
1 H NMR(400MHz,DMSO-d 6 )δ8.64(d,J＝8.2Hz,1H),7.26(dd,J＝27.4,8.7Hz,2H),7.06–6.95(m,3H),6.51(dd,J＝17.3,5.6Hz,3H),5.23–4.92(m,2H),4.61–4.47(m,1H),4.13–3.91(m,2H),3.79(d,J＝6.2Hz,3H),3.14(d,J＝10.7Hz,5H),2.91(td,J＝15.9,14.8,6.8Hz,1H),2.71(dd,J＝13.6,9.5Hz,1H),2.14(d,J＝20.2Hz,4H),1.55–0.81(m,10H).
13 C NMR(100MHz,DMSO-d 6 )δ170.76,168.24,167.24,162.49(dd,J＝245.8,13.4Hz),159.22,142.47,135.80,129.21,129.13,115.33,115.25,112.54,112.30,103.91,102.50,55.87,53.12,51.72,51.57,49.95,37.82,37.28,35.98,30.62,30.52,26.47,25.77,25.72,10.90.
ESI-MS:m/z 662.36(M-1).C 33 H 38 F 5 N 5 O 4 [663.28].
example 19: preparation of (S) -2- (2- (N-benzyl-2- (2-methyl-1H-indol-3-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-21)
Paraformaldehyde (30 mg,0.27 mmol) and benzylamine (30 mg,0.27 mmol) were dissolved in 4.0mL of anhydrous methanol, triethylamine (30 mg,0.27 mmol) was added and stirred at room temperature for 30 minutes followed by 2- (2-methyl-1H-indol-3-yl) acetic acid (50 mg,0.27 mmol) and stirred at room temperature for 10 minutes, then key intermediate I-5 (70 mg,0.21 mmol) was added, heated at 70℃under reflux for 16 hours, monitored by TLC; after 16H the reaction was completed, 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and the filtrate was concentrated under reduced pressure, and the obtained crude product was separated by silica gel column chromatography (eluent methanol: dichloromethane=1:40) to give crude product 0.015g of (S) -2- (2- (N-benzyl-2- (2-methyl-1H-indol-3-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-21), pale yellow solid, yield 11.50%, melting point: 86-88 ℃.
Spectral data:
1 H NMR(400MHz,DMSO-d 6 )δ10.78(d,J＝15.8Hz,1H),8.53(d,J＝8.1Hz,1H),7.38(d,J＝7.8Hz,1H),7.29(d,J＝8.7Hz,2H),7.26–7.22(m,3H),7.16–7.09(m,2H),7.07(d,J＝8.4Hz,2H),7.04–6.97(m,3H),6.94–6.88(m,1H),6.59–6.44(m,2H),4.62(ddd,J＝16.5,10.3,5.7Hz,1H),4.54–4.44(m,1H),4.21(d,J＝15.2Hz,1H),4.04–3.89(m,1H),3.87(d,J＝9.5Hz,1H),3.77(d,J＝7.6Hz,1H),3.59(s,1H),3.16(d,J＝11.9Hz,3H),2.89(ddd,J＝23.5,13.7,4.5Hz,1H),2.68(ddd,J＝28.5,13.7,9.6Hz,1H),2.23(d,J＝4.2Hz,3H).
ESI-MS:m/z 637.42(M-1).C 37 H 36 F 2 N 4 O 4 [638.27].
example 20: preparation of (S) -2- (2- (N-benzyl-2- (naphthalen-2-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-22)
Paraformaldehyde (30 mg,0.27 mmol) and benzylamine (30 mg,0.27 mmol) were dissolved in 4.0mL of anhydrous methanol, triethylamine (30 mg,0.27 mmol) was added and stirred at room temperature for 30 minutes followed by 2-naphthylacetic acid (50 mg,0.27 mmol), stirred at room temperature for 10 minutes, then key intermediate I-5 (70 mg,0.21 mmol) was added and heated at 70℃under reflux for 16 hours, monitored by TLC; after 16h the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration and concentration of the filtrate under reduced pressure, and the resulting crude product was separated by column chromatography on silica gel (eluent methanol: dichloromethane=1:40) to give 0.014g of crude product of target product (S) -2- (2- (N-benzyl-2- (naphthalene-2-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-22), light yellow solid, yield 10.50%, melting point: 46-48 ℃.
Spectral data:
1 H NMR(600MHz,DMSO-d 6 )δ8.53(d,J＝8.3Hz,1H),7.89(s,1H),7.83(dd,J＝15.8,8.8Hz,2H),7.63(s,1H),7.52–7.45(m,2H),7.38–7.32(m,2H),7.29(dd,J＝8.0,6.1Hz,3H),7.26–7.23(m,1H),7.15(dd,J＝21.0,7.2Hz,3H),7.07–6.97(m,3H),6.56–6.46(m,2H),4.60(td,J＝9.2,8.1,4.6Hz,1H),4.34(dd,J＝133.6,15.0Hz,2H),4.00–3.92(m,2H),3.87(d,J＝17.5Hz,1H),3.80(d,J＝8.4Hz,3H),3.70(s,1H),3.15(d,J＝19.0Hz,3H),2.89(ddd,J＝27.5,13.7,4.3Hz,1H),2.72–2.64(m,1H).
13 C NMR(100MHz,DMSO-d 6 )δ171.70,170.81,168.18,163.80,163.67,161.22,159.22,138.01,135.85,133.91,133.42,132.25,129.21,129.14,128.84,128.48,128.40,128.15,127.94,127.89,127.80,127.57,127.41,126.49,126.01,115.34,115.25,112.54,112.30,102.53,55.94,55.91,51.49,49.77,49.56,47.60,37.82,37.31.
ESI-MS:m/z 658.38(M+1),636.05(M+Na).C 38 H 35 F 2 N 3 O 4 [636.05].
example 21: preparation of (S) - (4- (2- ((2- ((3- (3, 5-difluorophenyl) -1- ((4-methoxyphenyl) (methyl) amino) -1-oxopropan-2-yl) amino) -2-oxoethyl) (4-fluorobenzyl) amino) -2-oxoethyl) phenyl) boronic acid (I-23)
Paraformaldehyde (40 mg,0.39 mmol) and 4-fluorobenzylamine (50 mg,0.39 mmol) are dissolved in 4.0mL of anhydrous methanol, triethylamine (40 mg,0.39 mmol) is added and stirred at room temperature for 30 minutes, then 4-boronic acid phenylacetic acid (70 mg,0.39 mmol) is added and stirred at room temperature for 10 minutes, then key intermediate I-5 (100 mg,0.30 mmol) is added and heated at 70℃for 16 hours under reflux, TLC monitoring; after 16h the reaction was completed, then 10mL of saturated sodium bicarbonate solution, 10mL of dichloromethane were added to the residue in the bottle, the organic phase was separated, 10mL of 1N HCl solution was added to wash, 10mL of saturated sodium chloride solution was added to wash the organic phase, the organic phase was dried over anhydrous sodium sulfate, filtration, and the filtrate was concentrated under reduced pressure, and the resulting crude product was separated by column chromatography over silica gel (eluent methanol: dichloromethane=1:40) to give crude product (S) - (4- (2- ((2- ((3- (3, 5-difluorophenyl) -1- ((4-methoxyphenyl) (methyl) amino) -1-oxypropane-2-yl) amino) -2-oxyethyl) (4-fluorobenzyl) amino) -2-oxyethyl) phenyl) boronic acid (I-23) as a white solid in a yield of 12.0%, melting point: 123.6-125.8 ℃.
Spectral data:
1 H NMR(400MHz,DMSO-d 6 )δ8.52(d,J＝8.2Hz,1H),7.98(s,2H),7.73(d,J＝7.6Hz,2H),7.27(d,J＝8.5Hz,2H),7.23–7.17(m,3H),7.14–7.11(m,3H),7.07–6.98(m,3H),6.53(d,J＝7.1Hz,2H),4.63–4.50(m,1H),4.42(d,J＝14.8Hz,1H),4.20(d,J＝15.0Hz,1H),3.92(dd,J＝16.7,6.9Hz,1H),3.81(s,3H),3.75(d,J＝7.6Hz,1H),3.52(s,2H),3.15(d,J＝11.2Hz,3H),2.90(dt,J＝17.7,8.8Hz,1H),2.75–2.61(m,1H).
13 C NMR(150MHz,DMSO-d 6 )δ171.69,170.79,168.11,163.35(d,J＝13.3Hz),161.92(dd,J＝243.3,17.8Hz),161.72(d,J＝13.3Hz),159.24,142.53,142.47,137.90,135.86,134.56,134.49,134.22,130.22(d,J＝8.1Hz),129.50(d,J＝8.2Hz),129.14,128.75,128.69,115.81(d,J＝21.3Hz),115.49(d,J＝21.3Hz),115.34,115.29,112.39(d,J＝24.4Hz),102.47,102.30,55.93,51.42,49.85,49.05,47.65,37.79,37.42.
example 5 in vitro anti-HIV Activity test of target Compounds (MT-4 cells) experiments
Term interpretation: MT-4 cells: human acute lymphoblastic leukemia cells; MTT assay: MTT is 3- (4, 5-dimethyl thiazole-2) -2, 5-diphenyl tetrazolium bromide, and the trade name is thiazole blue; DMSO: dimethyl sulfoxide.
Principle of testing
Since MT-4 cells infected by HIV are diseased within a certain period of time (5-7 days), a solution of a compound to be detected with a proper concentration is added into an MT-4 cell suspension infected by HIV, and after a period of time (5-7 days) of culture, the viability of MT-4 cells is measured by MTT assay to obtain a drug concentration (EC) for protecting 50% of cells from cytopathy 50 ) Thus obtaining the anti-HIV activity of the target compound. At the same time, the concentration of the target compound (CC) which lesions 50% of HIV-uninfected cells was obtained 50 ) Select coefficients (selectivity index, si=cc are calculated 50 /EC 50 )。
Principle of MTT assay: MTT, i.e., bromo-3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium, can bind to living intracellular succinate dehydrogenase without reacting with dead cells. The current MTT method is an enzyme analysis method which rapidly reflects the cell viability.
Test materials and methods
(1)HIV-1(III B ) HIV-2 (ROD) strain: supplied by the institute of microbiology and immunology, university of Leuven, belgium;
(2) MT-4 cells: supplied by the institute of microbiology and immunology, university of Leuven, belgium;
(3) MTT: purchased from Sigma, usa;
(4) Sample treatment: dissolving the sample in DMSO to prepare proper concentration before using, and diluting with double distilled water for 5 times, wherein each dilution is 5 times;
(5) Positive control: zidovudine, nevirapine;
(6) The testing method comprises the following steps: diluting the sample, adding into suspension of HIV-infected MT-4 cells, determining cell activity by MTT colorimetric method after a period of time, recording absorbance (A) value at 590nm in a microplate reader, and calculating EC 50 、CC 50 SI;
(7) MTT staining method: after incubation for a period of time with the addition of the sample, 20. Mu.L of MTT solution (5 mg/mL) was added to each well, incubation was continued for several hours, the staining solution was discarded, and 150. Mu.L of DMSO was added to each well, mixed well, and absorbance was recorded at 590nm in a microplate reader.
The specific operation is as follows: dissolving the compound in DMSO or water, diluting with phosphate buffer, and concentrating 3×10 5 MT-4 cells were preincubated with 100. Mu.L of compound solution at various concentrations for 1h at 37 ℃. To this mixture, 100. Mu.L of a suitable concentration of virus diluent was then added and the cells were incubated for 1h at 37 ℃. After three washes, the cells were resuspended in culture medium with or without compound, respectively. The cells were then exposed to 5% CO 2 In the environment, incubation was carried out at 37℃for a further 7 days, and the stock culture was supplemented with medium with or without compound on the third day after infection. The procedure was repeated twice for each culture condition. Cytopathic effects on viruses are daily performed with reverse opticsAnd (5) monitoring by a microscope. Generally, the viral dilutions used in this experiment often develop cytopathic effects the fifth day after viral infection. The drug inhibitory concentration is the concentration at which the drug produces 50% inhibition of the viral cytopathic effect while not directly toxic to the cells (EC) 50 ) And (3) representing. It is emphasized that when compounds are poorly water soluble and require DMSO to be dissolved, the DMSO volume to volume concentration is typically less than 10% relative to water (DMSO final concentration in MT-4 cell culture medium is less than 2%). Since DMSO can affect the antiviral activity of the test compounds, antiviral activity in comparison to blank experiments with DMSO solutions of the same concentration should also be run in parallel. In addition, the final DMSO concentration (1/1000) was far lower than that required to affect HIV replication in MT-4 cells.
In vitro anti-HIV-1 (III) of target compounds B ) And HIV-2 (ROD) activity screening data were provided by the institute of microbiology and immunology, university of Leuven, belgium, and all activity data were measured in at least two independent, parallel experiments, the results of which are shown in Table 1.
TABLE 1 anti-HIV Activity, toxicity and selection index (MT-4 cells) of substituted Glycine-3, 5-difluorophenylalanine peptide derivatives
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a EC 50 : compound concentration that protects 50% of HIV-1 infected cells from cytopathic effects;
b CC 50 : a concentration of compound that lesions 50% of HIV-1 uninfected cells;
c SI: selectivity coefficient, CC 50 /EC 50 Is a ratio of (2);
analysis of experimental results: as shown in Table 1, the novel synthesized substituted glycine-3, 5-difluorophenylalanine peptide derivative shows better anti-HIV-1 activity. For example, the anti-HIV-1 activity of the target compounds I-7, I-9, I-19, I-21 is in the range of 2.53-2.94. Mu.M, wherein the anti-HIV-1 activity of the target compound I-19 (EC 50 ＝2.54μM,CC 50 >107.61 Especially. It is worth mentioning that slight changes in the structure of part of the compounds have a significant effect on the activity, the selectivity of HIV-1 and HIV-2, for example, compounds I-6 and I-7 differ in that R 2 I-6 has no anti-HIV-1/2 activity, I-7 is an HIV-1 selective inhibitor; i-8 is simply R compared to Compound I-6 2 The substituent naphthyl is different in substituted position, and I-8 is an HIV-2 selective inhibitor. As another example, compound I-14 is also simply R as compared to compound I-13 2 The substituent naphthyl is different in substituted position, I-13 has little anti-HIV activity, and I-14 is an HIV-2 selective inhibitor. As another example, compound I-16 is simply R as compared to compound I-15 2 I-15 is HIV-1 selective inhibitor, and I-16 is HIV-2 selective inhibitor. Zidovudine as a positive drug has no selective inhibition of HIV-1/2; nevirapine is an HIV-1 selective inhibitor, and has no HIV-1/2 dual inhibition effect. Obviously, the bioactivity of the substituted glycine-3, 5-difluorophenylalanine peptide derivative achieves unexpected effects and deserves further development.
Claims (5)
1. Comprising a substituted glycine-3, 5-difluorophenylalanine peptide derivative, or a pharmaceutically acceptable salt thereof, characterized by being one of the following:
2. the process for producing a peptide derivative containing a substituted glycine-3, 5-difluorophenylalanine as claimed in claim 1, comprising the steps of: using Boc-L-phenylalanine (I-1) as a starting material and methylene dichloride as a solvent to generate an intermediate I-2 with N-methyl-4-aminoanisole through an amide condensation reaction; then the intermediate I-2 is dissolved in a proper amount of dichloromethane, and the Boc group is removed under the action of trifluoroacetic acid to obtain an intermediate I-3; then, reacting the intermediate I-3 with ammonium formate to obtain an intermediate I-4; the intermediate I-4 is dehydrated under the action of phosphorus oxychloride to obtain an intermediate isonitrile I-5, and then the I-5 reacts with acid, amine and aldehyde fragments through Ugi four components to obtain a target compound;
The synthetic route is as follows:
reagents and conditions: (i) N-methyl-4-aminoanisole, 1H-benzotriazole-1-yloxy tripyrrolidinyl hexafluorophosphate, N, N-diisopropylethylamine, dichloromethane, and transferring to room temperature at 0deg.C for 8H; (ii) trifluoroacetic acid, dichloromethane, room temperature, 4h; (iii) ammonium formate, acetonitrile, 90 ℃, reflux, 24h; (iv) Dichloromethane, triethylamine, phosphorus oxychloride, 0 ℃ for 12 hours; (v) R is R 1 -CHO，R 2 -NH 2 ，R 3 -COOH, anhydrous methanol, 60 ℃,8h;
wherein R is 1 、R 2 As indicated in the corresponding position in the above claim 1.
3. The process for producing a peptide derivative containing a substituted glycine-3, 5-difluorophenylalanine as claimed in claim 2, comprising the steps of:
(1) Adding Boc-L-phenylalanine (I-1) and 1H-benzotriazole-1-yl oxygen tripyrrolidine hexafluorophosphate into dichloromethane, and stirring for 30min under ice bath condition; then adding N, N-diisopropylethylamine and N-methyl-4-aminoanisole into the reaction liquid, removing the ice bath, transferring to room temperature, and monitoring by TLC; after the reaction is finished, the solvent is distilled off under reduced pressure, then saturated sodium bicarbonate solution is added into residues in a bottle, dichloromethane is used for extraction, an organic phase is separated, 1NHCl solution is added for washing, the organic phase is separated, saturated sodium chloride solution is added for washing, the organic phase is dried by anhydrous sodium sulfate, filtration and reduced pressure concentration are carried out on filtrate, and the obtained crude product is separated and purified by silica gel column chromatography to obtain an intermediate I-2;
(2) Adding the intermediate I-2 into dichloromethane, slowly dropwise adding excessive trifluoroacetic acid into the solution under stirring of ice bath condition, removing ice bath, transferring to room temperature, and monitoring by TLC; after the reaction is finished, the solvent is distilled off under reduced pressure, then saturated sodium bicarbonate solution is added to adjust the pH of the reaction solution to 7, and then dichloromethane solution is added for extraction; separating the organic phase, washing the organic phase with saturated sodium chloride solution for 3 times, drying the organic phase with anhydrous sodium sulfate, filtering, and evaporating the solvent under reduced pressure to obtain an intermediate I-3;
(3) Adding the intermediate I-3 and ammonium formate into acetonitrile solution, and heating at 90 ℃ for 10 hours; after the reaction is finished, extracting with water and ethyl acetate for three times, combining organic phases, filtering, evaporating the solvent under reduced pressure, and separating by silica gel column chromatography to obtain an intermediate I-4;
(4) Dissolving the intermediate I-4 in dichloromethane solution, adding triethylamine, slowly adding phosphorus oxychloride solution under ice bath condition, stirring at 0deg.C for 10 hr, quenching with ice water, extracting with dichloromethane, mixing organic phases, drying with anhydrous sodium sulfate, stirring with silica gel, and performing column chromatography to obtain intermediate I-5;
(5) Dissolving the corresponding substituted amine fragment and paraformaldehyde in anhydrous methanol solution, adding triethylamine, and stirring for 30 minutes at room temperature; subsequently adding the corresponding substituted acid fragment and stirring for 10 minutes at room temperature; then adding the isonitrile intermediate I-5, and adding the mixture into the mixture to reflux for 16 hours at 70 ℃; after the reaction is finished, adding saturated sodium bicarbonate solution into the residue in the bottle, extracting with dichloromethane, separating an organic phase, adding 1NHCl solution for washing, separating the organic phase, adding saturated sodium chloride solution for washing, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the obtained crude product by silica gel column chromatography to obtain the target product.
4. The use of a peptide derivative containing substituted glycine-3, 5-difluorophenylalanine as defined in claim 1 for the preparation of a medicament for the treatment and prevention of aids.
5. An anti-HIV pharmaceutical composition comprising a substituted glycine-3, 5-difluorophenylalanine peptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, and one or more pharmaceutically acceptable carriers or excipients.
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