CN114456228A - Substituted glycine-3, 5-difluorophenylalanine peptide derivative and preparation method and application thereof - Google Patents

Substituted glycine-3, 5-difluorophenylalanine peptide derivative and preparation method and application thereof Download PDF

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CN114456228A
CN114456228A CN202210157554.7A CN202210157554A CN114456228A CN 114456228 A CN114456228 A CN 114456228A CN 202210157554 A CN202210157554 A CN 202210157554A CN 114456228 A CN114456228 A CN 114456228A
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CN114456228B (en
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展鹏
姜向毅
刘新泳
李敬
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Shandong University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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 derivatives and application of the derivatives as HIV inhibitors in preparing anti-AIDS drugs.

Description

Substituted glycine-3, 5-difluorophenylalanine peptide derivative and preparation method and application thereof
Technical Field
The invention belongs to the technical field of organic compound synthesis and medical application, and particularly relates to a substituted glycine-3, 5-difluorophenylalanine peptide derivative and a preparation method and application thereof.
Background
Acquired immunodeficiency Syndrome (AIDS), also known as AIDS, is a chronic infectious disease that is harmful to human life and health caused by Human Immunodeficiency Virus (HIV). HIV belongs to RNA retrovirus and is divided into two subtypes of HIV-1/2, wherein HIV-1 has strong infectivity and high lethality rate and is the main pathogen of AIDS. HIV-2 is mainly limited to Western Africa, but with the continuous development of globalization and the increasing closeness of people communication, HIV-2 infection cases are also found in the United states, Europe, south Africa, India and China, and should be paid attention. More than 30 drugs for treating AIDS are currently on the market, targeting respectively reverse transcriptase, protease, integrase, CCR5 co-receptor and membrane fusion. However, because of the high mutation rate of HIV-1, drug resistance is easy to generate, and the single-drug treatment effect is poor. The Highly effective Antiretroviral Therapy (HAART) can effectively reduce the HIV load in a patient and improve the life span and the life quality of the patient, but the problems of serious adverse reaction, drug resistance, latent infection, high cost and the like caused by long-term use of a large amount of drugs make the discovery of HIV inhibitors with new structural types urgent.
The construction of a compound library is an important part of drug research and development, and the progress of drug research and development can be greatly accelerated by applying an efficient organic synthesis technology. Among them, the multicomponent reaction has the advantages of simple operation, continuous reaction, atom economy, etc., and meets the requirements of organic synthesis and construction of diverse compound libraries, and thus has received much attention. The invention utilizes the Ugi four-component reaction to construct a compound library and screens biological activity to discover a class of substituted glycine-3, 5-difluorophenylalanine peptide derivatives with a brand-new structure, wherein part of 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 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 derivatives
A substituted glycine-3, 5-difluorophenylalanine peptoid derivative or a pharmaceutically acceptable salt thereof has a structure shown in a general formula I:
Figure BDA0003513394380000021
wherein the content of the first and second substances,
R1comprises the following steps: hydrogen, methyl, ethyl, isopropyl substituted benzyl, cyclohexane methyl, cyclopropylA phenyl group, a benzyl group; the substituent is selected from methyl, fluorine, chlorine, bromine, nitro, cyano, trifluoromethyl, methyl, methoxy and boric acid.
R2Comprises the following steps: substituted phenyl, substituted benzyl, substituted naphthylmethyl, 2-methyl-1H-indole-3-methyl, 5-halogen-1H-indole-3-methyl, benzyl substituted by pinacol borate, 3- (trifluoromethyl) -substituted 4,5,6, 7-tetrahydro-1H-indazol-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 a preferred aspect of the invention, R1Is fluorine substituted benzyl, cyano substituted benzyl, cyclohexane methyl, cyclopropyl, benzyl; r2Is naphthylmethyl, 2-methyl-1H-indole-3-methyl, 5-halogen-1H-indole-3-methyl, benzyl substituted by pinacol borate, 4,5,6, 7-tetrahydro-1H-indazole-1-methyl substituted by 3- (trifluoromethyl), 1, 5-dimethyl-3- (trifluoromethyl) -1H-pyrazole, benzyl substituted by boric acid;
further preferred according to the invention is a substituted glycine-3, 5-difluorophenylalanine peptoid derivative which is one of the following compounds:
Figure BDA0003513394380000022
Figure BDA0003513394380000031
Figure BDA0003513394380000041
as used herein, "pharmaceutically acceptable salts" means salts of the compounds which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and lower animals without undue toxicity, irritation, and allergic response and the like, are commensurate with a reasonable benefit-to-risk ratio, are generally water or oil soluble or dispersible, and are effective for their intended use. Including pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which are contemplated herein and are compatible with the chemical nature of the compounds of formula I. A list of suitable salts is found on pages 1-19 of s.m. berge et al, j.pharm.sci.,1977, 66.
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: Boc-L-phenylalanine (I-1) is used as an initial raw material, dichloromethane is used as a solvent, and an intermediate I-2 is generated with N-methyl-4-aminoanisole through an amide condensation reaction; then, dissolving the intermediate I-2 in a proper amount of dichloromethane, and removing a Boc group 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; dehydrating the intermediate I-4 under the action of phosphorus oxychloride to obtain an intermediate isonitrile I-5, and then reacting the intermediate I-5 with acid, amine and aldehyde fragments through an Ugi four-component reaction to obtain a target compound;
the synthetic route is as follows:
Figure BDA0003513394380000042
reagents and conditions: (i) n-methyl-4-aminoanisole, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane are added, and the temperature is changed to room temperature at 0 ℃; (ii) trifluoroacetic acid, dichloromethane, room temperature; (iii) ammonium formate and acetonitrile, refluxing at 90 ℃ for 24 hours; (iv) dichloromethane, triethylamine, phosphorus oxychloride, at 0 ℃ for 12 h; (v) r1-CHO,R2-NH2,R3-COOH, anhydrous methanol, 60 ℃,8 h;
wherein R is1、R2As described above in formula I.
The room temperature of the invention is 20-30 ℃.
The preparation method of the preferable substituted glycine-3, 5-difluorophenylalanine peptide derivative comprises the following specific steps:
(1) adding Boc-L-phenylalanine (I-1) and 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate into dichloromethane, and stirring for 30min under ice bath condition; adding N, N-diisopropylethylamine and N-methyl-4-aminoanisole into the reaction solution, removing the ice bath, transferring to room temperature, and monitoring by TLC; after the reaction is finished, evaporating the solvent under reduced pressure, adding a saturated sodium bicarbonate solution into the residue in the bottle, extracting with dichloromethane, separating an organic phase, adding a 1N HCl solution for washing, separating the organic phase, adding a 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 an intermediate I-2;
(2) adding the intermediate I-2 into dichloromethane, slowly dropwise adding excessive trifluoroacetic acid into the solution under the condition of ice bath and stirring, then removing the ice bath, transferring to room temperature, and monitoring by TLC; after the reaction is finished, evaporating the solvent under reduced pressure, adding a saturated sodium bicarbonate solution to adjust the pH of the reaction solution to 7, and then adding a dichloromethane solution for extraction; separating and taking an organic phase, washing the organic phase for 3 times by using a saturated sodium chloride solution, drying the organic phase by using anhydrous sodium sulfate, filtering, and evaporating the solvent to dryness under reduced pressure to obtain an intermediate I-3;
(3) adding the intermediate I-3 and ammonium formate into an acetonitrile solution, and heating for 10h at 90 ℃; after the reaction is finished, extracting the mixture for three times by using water and ethyl acetate, combining organic phases, filtering, decompressing and evaporating the solvent, and separating by using silica gel column chromatography to obtain an intermediate I-4;
(4) dissolving the intermediate I-4 in a dichloromethane solution, adding triethylamine, slowly adding a phosphorus oxychloride solution under an ice bath condition, stirring for 10 hours at 0 ℃, after the reaction is finished, quenching with ice water, extracting with dichloromethane, combining organic phases, drying with anhydrous sodium sulfate, mixing with silica gel, and carrying out column chromatography to obtain an intermediate I-5;
(5) dissolving the corresponding substituted amine fragment and paraformaldehyde in an anhydrous methanol solution, adding triethylamine, and stirring for 30 minutes at room temperature; then adding the corresponding substituted acid fragment, and stirring for 10 minutes at room temperature; then adding an isonitrile intermediate I-5, and adding reflux for 16 hours at 70 ℃; after the reaction is finished, adding a saturated sodium bicarbonate solution into the residue in the bottle, extracting with dichloromethane, separating an organic phase, adding a 1N HCl solution for washing, separating the organic phase, adding a 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 with the general formula 1.
3. Application of substituted glycine-3, 5-difluorophenylalanine peptide derivatives
The invention discloses an anti-HIV-1 activity screening result of a substituted glycine-3, 5-difluorophenylalanine peptide derivative and an application of the substituted glycine-3, 5-difluorophenylalanine peptide derivative as an HIV inhibitor for the first time. Experiments prove that the substituted glycine-3, 5-difluorophenylalanine peptoid derivative can be used as an HIV inhibitor for preparing anti-AIDS medicaments. The invention also provides application of the compound in anti-HIV drugs.
anti-HIV-1 Activity and toxicity test of the target Compound
A class of substituted glycine-3, 5-difluorophenylalanine peptoid derivatives synthesized according to the above procedure were tested at cellular level for anti-HIV activity and toxicity, and their anti-HIV-1 and anti-HIV-2 activity and toxicity data are shown in Table 1.
The newly synthesized substituted glycine-3, 5-difluorophenylalanine peptide derivative has better anti-HIV-1 activity. For example, the anti-HIV-1 activity of the object compounds I-7, I-9, I-19, I-21 is in the range of 2.53 to 2.94. mu.M, wherein the anti-HIV-1 activity (EC) of the object compound I-1950＝2.54μM,CC50>107.61) is particularly prominent. It is particularly emphasized that slight changes in the structure of some of the compounds have a significant effect on the activity, HIV-1 and HIV-2 selectivity, for example, compounds I-6 and I-7 differ by R2In contrast, I-6 has no anti-HIV-1/2 activity, and I-7 is an HIV-1 selective inhibitor; i-8 Only R compared with Compound I-62The substituent naphthyl has different substitution positions, and I-8 is an HIV-2 selective inhibitor. As another example, compound I-14 is also only R compared to compound I-132The difference in the substitution positions of the substituent naphthyl groups means that I-13 has little anti-HIV activity, and I-14 is an HIV-2 selective inhibitor. As another example, compound I-16 is R alone as compared to compound I-152The difference of one halogen atom in the substituent groups is that I-15 is an HIV-1 selective inhibitor and I-16 is an HIV-2 selective inhibitor. MakingZidovudine, a positive drug, has no selective inhibitory effect on HIV-1/2; nevirapine is an HIV-1 selective inhibitor, and has no HIV-1/2 double inhibition effect. Obviously, the biological activity of the substituted glycine-3, 5-difluorophenylalanine peptide derivative achieves unexpected effects and is worthy of further development.
The substituted glycine-3, 5-difluorophenylalanine peptide derivatives can be used as HIV-1 and HIV-2 inhibitors. In particular to the application of the compound as HIV-1 and HIV-2 inhibitors in preparing anti-AIDS drugs.
The invention relates to an anti-HIV-1 and anti-HIV-2 pharmaceutical composition, which comprises a class of peptide derivatives 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 a screening result of anti-HIV-1 and anti-HIV-2 activities of partial compounds and the first application thereof in the field of antivirus. Tests 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 to the application of the compound as HIV-1 and HIV-2 inhibitors in preparing anti-AIDS drugs.
Detailed Description
The invention will be understood by the following examples, which are given by way of illustration and are not intended to limit the scope of the invention.
Example 1: preparation of tert-butyl (S) - (3- (3, 5-difluorophenyl) -1- ((4-methoxyphenyl) (methyl) amino) -1-oxoprop-2-yl) carbamate (I-2)
Starting materials Boc-L-phenylalanine (I-1) (2.00g,6.64mmol) and 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate (3.46g,6.64mmol) were added to 20mL of dichloromethane and stirred under ice bath conditions for 30 min; then N, N-diisopropylethylamine (2.23mL,13.29mmol) and N-methyl-4-aminoanisole (610mg,4.43mmol) were added, the ice bath was removed and the mixture was stirred at room temperature and monitored by TLC; after 6h, the reaction was completed, the solvent was evaporated under reduced pressure, then 40mL of saturated sodium bicarbonate solution and 40mL of dichloromethane were added to the residue in the flask for extraction, the organic phase was separated and washed with 40mL of 1N HCl solution, the organic phase was separated and washed with 40mL of saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product of (S) - (3- (3, 5-difluorophenyl) -1- ((4-methoxyphenyl) (methyl) amino) -1-oxoprop-2-yl) carbamic acid tert-butyl ester (I-2) as an intermediate, which was 1.68g of crude product of (S) - (3- (3, 5-difluorophenyl) -1- ((4-methoxyphenyl) (methyl) amino) -1-oxoprop-2-yl) carbamic acid tert-butyl ester (I-2), as a yellow oil, with a yield of 60%.
Spectral data:
1H NMR(400MHz,DMSO-d6)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,OCH3),3.15(d,J＝7.8Hz,3H,NCH3),2.87–2.73(m,1H,CH),2.69–2.57(m,1H,CH),1.29(s,9H,3(CH3)).
ESI-MS:m/z 443.78(M+Na).C22H26F2N2O4[420.4].
example 2: preparation of (S) -2-amino-3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-3)
Intermediate I-2(500mg,1.19mmol) was added to 30mL of dichloromethane, and trifluoroacetic acid (410mg,3.57mmol) was slowly added to the solution, stirred at room temperature, monitored by TLC; after 1h, the reaction was completed, and then the reaction solution was adjusted to pH 7 with saturated sodium bicarbonate solution, extracted with 40mL of dichloromethane, the organic phase was separated, washed with saturated sodium chloride solution (3X 20mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2.36g of a crude product of intermediate (S) -2-amino-N- (4-methoxyphenyl) -N-methyl-3-hydrocinnamide (3) as a yellow oil in 80% yield.
Spectral data:
1H NMR(400MHz,DMSO-d6)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-carboxamido-N- (4-methoxyphenyl) -N-methylpropanamide (I-4)
Intermediate I-3(340mg,1.06mmol) was added to 10mL acetonitrile, then ammonium formate (130mg,2.12mmol) was added to the solution, heated to reflux at 90 ℃ for 24h, monitored by TLC; after 24h, the reaction is finished, the mixture is filtered, extracted by water and ethyl acetate (3X 10mL), and organic phases are combined; 20mL of a saturated sodium chloride solution was added and the organic phase was dried over anhydrous sodium sulfate, filtered, 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 1.68g of crude intermediate tert-butyl (S) - (3- (3, 5-difluorophenyl) -1- ((4-methoxyphenyl)) methyl) amino) -1-oxoprop-2-yl) carbamate (I-4) as a yellow oil with a yield of 60%.
Spectral data:
1H NMR(400MHz,DMSO-d6)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).C18H18F2N2O3[348.35].
example 4: preparation of intermediate (S) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methyl-2- (methyl-azabenzoyl) propylaminoisonitrile (I-5)
Intermediate I-4(410mg,1.18mmol) was added to 10mL of dichloromethane, then triethylamine (360mg,3.54mmol) was added to the solution, and phosphorus oxychloride (180mg,1.18mmol) was added slowly under ice bath conditions; stirring for 24h at 0 ℃ and monitoring by TLC; after 24h the reaction was complete, quenched with ice water, extracted with dichloromethane (3 × 15mL), the organic phases combined, dried over anhydrous sodium sulfate, filtered, the supernatant concentrated, and the supernatant taken up on silica gel and subjected to column chromatography (ethyl acetate: petroleum ether ═ 1:2) to afford intermediate (S) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methyl-2- (methyl-azaformyl) propylamine isonitrile (I-5)0.17g, a pale yellow solid, yield: 45 percent.
Spectral data:1H NMR(400MHz,DMSO-d6)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,OCH3),3.15(s,3H,NCH3),3.14–3.07(m,1H,CH),2.99(dd,J＝13.6,8.3Hz,1H,CH).ESI-MS:m/z332.2(M+1).C18H17F2N2O2[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 (18mg,0.20mmol) is dissolved in 6.0mL of anhydrous methanol, and 4-fluorobenzylamine (25mg, 0.20mmol) is dissolved in 6.0mL of anhydrous methanol, followed by addition of 2-naphthaleneacetic acid (40mg,0.20mmol), stirring at room temperature for 10 minutes, followed by addition of key intermediate I-5(50mg,0.15mmol), heating at 70 ℃ under reflux for 16 hours, monitored by TLC; after 16h, after the reaction is finished, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane are added into the residue in the bottle for extraction, an organic phase is separated and washed by 10mL of 1N HCl solution, an organic phase is separated and washed by 10mL of saturated sodium chloride solution, the organic phase is dried by anhydrous sodium sulfate and filtered, the filtrate is concentrated under reduced pressure, and the obtained crude product is separated by silica gel column chromatography (eluent EA: PE ═ 1:2) to obtain 0.01g of crude product of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (naphthalene-2-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropionamide (I-6) of the target product, namely (S) -3- (3, 5-difluorophenyl) -2- (2- (naphthalene-2-yl) acetamido) -N- (4-methoxyphenyl) -N-methylpropionamide (I-6), the yield is 11%, melting point: 112 ℃ and 114 ℃.
Spectral data:
1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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).C38H34F3N3O4[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 (20mg, 0.20mmol) and 4-fluorobenzylamine (25mg, 0.20mmol) are dissolved in 6.0mL of anhydrous methanol, triethylamine (20mg, 0.20mmol) is added and stirred at room temperature for 30 minutes, followed by addition of 2- (2-methyl-1H-indol-3-yl) acetic acid (35mg, 0.20mmol) and stirring at room temperature for 10 minutes, followed by addition of key intermediate I-5(50mg,0.15mmol), heated at 70 ℃ under reflux for 16 hours, monitored by TLC; after 16H, after the reaction was completed, 10mL of a saturated sodium bicarbonate solution and 10mL of dichloromethane were added to the residue in the flask and extracted, the organic phase was separated and washed with 10mL of a 1N HCl solution, the organic phase was separated and washed with 10mL of a saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was separated by silica gel column chromatography (eluent ethyl acetate: petroleum ether ═ 1:2), that was 0.009g of a crude product 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), pale yellow solid, yield 10.20%, melting point: 112.5-114.5 ℃.
Spectral data:1H NMR(400MHz,DMSO-d6)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).C37H35F3N4O4[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)
Dissolving paraformaldehyde (18mg,0.20mmol) and 4-fluorobenzylamine (25mg, 0.20mmol) in 6.0mL of anhydrous methanol, adding triethylamine (20mg, 0.20mmol), stirring at room temperature for 30 minutes, then adding 1-naphthylacetic acid (37mg, 0.20mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(50mg,0.15mmol, 1.0eq.), heating at 70 ℃ for reflux for 16 hours, and monitoring by TLC; after 16H, after the reaction, adding 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane into the residue in the bottle for extraction, separating and washing an organic phase by adding 10mL of 1N HCl solution, separating and washing an organic phase by adding 10mL of saturated sodium chloride solution, drying the organic phase by using anhydrous sodium sulfate, filtering, decompressing and concentrating a filtrate, separating the obtained crude product by silica gel column chromatography (an eluent, namely ethyl acetate: petroleum ether ═ 1:2) to obtain 9mg of the crude product 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-8), pale yellow solid, yield 10.20%, melting point: 112.5-114.5 ℃.
Spectral data:1H NMR(400MHz,DMSO-d6)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).13C NMR(101MHz,DMSO-d6)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).C38H34F3N3O4[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 (18mg,0.20mmol) and 4-fluorobenzylamine (25mg, 0.20mmol) are dissolved in 6.0mL of anhydrous methanol, triethylamine (20mg, 0.20mmol) is added and stirred at room temperature for 30 minutes, followed by addition of 5-bromo-1H-indole-3-carboxylic acid (51mg, 0.20mmol) and stirring at room temperature for 10 minutes, followed by addition of key intermediate I-5(50mg,0.15mmol), heating at 70 ℃ for reflux for 16 hours, monitored by TLC; after 16H, after the reaction, adding 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane into the residue in the bottle for extraction, separating and washing an organic phase by adding 10mL of 1N HCl solution, separating and washing the organic phase by adding 10mL of saturated sodium chloride solution, drying the organic phase by using anhydrous sodium sulfate, filtering, decompressing and concentrating a filtrate, separating the obtained crude product by silica gel column chromatography (an eluent, ethyl acetate: petroleum ether ═ 1:2) to obtain 0.011g of the crude product of (S) -2- (2- (5-bromo-1H-indol-3-yl) -N- (4-fluorobenzyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-9), white solid, yield 9.60%, melting point: at the temperature of 78-80 ℃.
Spectral data:1H NMR(400MHz,DMSO-d6)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).13C NMR(150MHz,DMSO-d6)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),C36H32BrF3N4O4[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)
Dissolving paraformaldehyde (18mg,0.20mmol) and cyclohexylmethylamine (23mg, 0.20mmol) in 6.0mL of anhydrous methanol, adding triethylamine (20mg, 0.20mmol), stirring at room temperature for 30 minutes, then adding 5-bromo-1H-indole-3-carboxylic acid (500mg, 0.20mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(50mg,0.15mmol), heating at 70 ℃ under reflux for 16 hours, and monitoring by TLC; after 16H, after the reaction is finished, adding 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane into the residue in the bottle, extracting, separating and taking an organic phase, adding 10mL of 1N HCl solution for washing, separating and taking an organic phase, adding 10mL of saturated sodium chloride solution for washing, drying the organic phase by using anhydrous sodium sulfate, filtering, decompressing and concentrating the filtrate, and separating the obtained crude product by silica gel column chromatography (eluent ethyl acetate: petroleum ether ═ 1:2) to obtain 0.011g of 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-methylpropionamide (I-10), white solid, yield 9.80%, melting point: 80-82 ℃.
Spectral data:1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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+).C36H39BrF2N4O4[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)
Dissolving paraformaldehyde (20mg, 0.20mmol) and cyclopropylamine (11mg, 0.20mmol) in 6.0mL of anhydrous methanol, adding triethylamine (20mg, 0.20mmol), stirring at room temperature for 30 minutes, then adding 2- (2-methyl-1H-indol-3-yl) acetic acid (40mg,0.20mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(50mg,0.15mmol), heating at 70 ℃ under reflux for 16 hours, monitored by TLC; after 16H, after the reaction was completed, 10mL of a saturated sodium bicarbonate solution and 10mL of dichloromethane were added to the residue in the flask and extracted, the organic phase was separated and washed with 10mL of a 1N HCl solution, the organic phase was separated and washed with 10mL of a saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was subjected to silica gel column chromatography (eluent methanol: dichloromethane ═ 1:40) to give 0.009g of a crude product 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), white solid, yield 9.80%, melting point: 98-100 ℃.
Spectral data:1H NMR(400MHz,DMSO-d6)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).C33H34F2N4O4[588.66].
example 11: preparation of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4-fluorobenzyl) -2- (4- (pinacolato borate) phenyl) acetamido) -N- (4-methoxyphenyl) -N-methylpropanamine (I-12)
Dissolving paraformaldehyde (20mg, 0.20mmol) and 4-fluorobenzylamine (25mg, 0.20mmol) in 6.0mL of anhydrous methanol, adding triethylamine (20mg, 0.20mmol), stirring at room temperature for 30 minutes, then adding pinacol ester 4-phenylboronate (50mg, 0.20mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(50mg,0.15mmol), heating at 70 ℃ for reflux for 16 hours, and monitoring by TLC; after 16h, after the reaction, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane were added to the residue in the flask for extraction, the organic phase was separated and washed with 10mL of 1N HCl solution, the organic phase was separated and washed with 10mL of saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered, 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 obtain 0.011g of a crude product of (S) -3- (3, 5-difluorophenyl) -2- (2- (N- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxabenzaldehyde-2-yl) phenyl) acetamido) -N- (4-methoxyphenyl) -N-methylpropylamine (I-12) which is the target product, white solid, yield 10.5%, melting point: 98-100 ℃.
Spectral data:1H NMR(400MHz,DMSO-d6)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).13C NMR(150MHz,DMSO-d6)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).C40H43BF3N3O6[729.60].
example 12: preparation of (S) -2- (2- (N- (4-cyanophenyl) -2- (naphthalen-2-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-13)
Paraformaldehyde (20mg, 0.22mmol, 1.3eq.) and 4-cyanobenzylamine (30mg, 0.20mmol) were dissolved in 4.0mL of anhydrous methanol, triethylamine (20mg, 0.20mmol) was added and stirred at room temperature for 30 minutes, followed by addition of 2-naphthylacetic acid (40mg,0.20mmol) and stirring at room temperature for 10 minutes, followed by addition of key intermediate I-5(50mg,0.15mmol), heating at 70 ℃ under reflux for 16 hours, monitored by TLC; after 16h, after the reaction was completed, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane were added to the residue in the flask for extraction, the organic phase was separated and washed with 10mL of 1N HCl solution, the organic phase was separated and washed with 10mL of saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered, 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 obtain 0.008g of crude product of (S) -2- (2- (N- (4-cyanophenyl) -2- (naphthalen-2-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-13) as a target product as a white solid, yield 7.80%, melting point: 60-62 ℃.
Spectral data:1H NMR(400MHz,DMSO-d6)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).13C NMR(150MHz,DMSO-d6)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).C39H34F2N4O4[660.72].
example 13: preparation of (S) -2- (2- (N- (4-cyanophenyl) -1- (naphthalen-1-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-14)
Dissolving paraformaldehyde (20mg, 0.22mmol) and 4-cyanobenzylamine (30mg, 0.20mmol) in 4.0mL of anhydrous methanol, adding triethylamine (20mg, 0.20mmol), stirring at room temperature for 30 minutes, subsequently adding 1-naphthylacetic acid (40mg,0.20mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(50mg,0.15mmol), heating at 70 ℃ for reflux for 16 hours, and monitoring by TLC; after 16h, after the reaction was completed, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane were added to the residue in the flask for extraction, the organic phase was separated and washed with 10mL of 1N HCl solution, the organic phase was separated and washed with 10mL of saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered, 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 obtain 0.012g of crude product of (S) -2- (2- (N- (4-cyanophenyl) -2- (naphthalen-2-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-14) as a pale yellow solid, yield 10.60%, melting point: 106-108 ℃.
Spectral data:1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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).C39H34F2N4O4[660.72].
example 14: preparation of (S) -2- (2- (N- (4-cyanophenyl) -2- (5-fluoro-1H-indol-3-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-15)
Dissolving paraformaldehyde (30mg, 0.27mmol) and 4-cyanobenzylamine (40mg, 0.27mmol) in 4.0mL of anhydrous methanol, adding triethylamine (30mg, 0.27mmol), stirring at room temperature for 30 minutes, subsequently adding 2- (5-fluoro-1H-indol-3-yl) acetic acid (50mg, 0.27mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(70mg, 0.21mmol), heating at 70 ℃ under reflux for 16 hours, and monitoring by TLC; after 16H, after the reaction was completed, 10mL of a saturated sodium bicarbonate solution and 10mL of dichloromethane were added to the residue in the flask and extracted, the organic phase was separated and washed with 10mL of a 1N HCl solution, the organic phase was separated and washed with 10mL of a saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was subjected to silica gel column chromatography (eluent methanol: dichloromethane ═ 1:40) to give 0.015g of a crude product of (S) -2- (2- (N- (4-cyanophenyl) -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:1H NMR(400MHz,DMSO-d6)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-cyanophenyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-16)
Dissolving paraformaldehyde (30mg, 0.27mmol) and 4-cyanobenzylamine (40mg, 0.30mmol) in 4.0mL of anhydrous methanol, adding triethylamine (30mg, 0.27mmol), stirring at room temperature for 30 minutes, subsequently adding 2- (5-bromo-1H-indol-3-yl) acetic acid (70mg, 0.27mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(70mg, 0.21mmol), heating at 70 ℃ under reflux for 16 hours, and monitoring by TLC; after 16H, after the reaction, adding 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane into the residue in the bottle for extraction, separating an organic phase, adding 10mL of 1N HCl solution for washing, separating an organic phase, adding 10mL of saturated sodium chloride solution for washing, drying the organic phase with anhydrous sodium sulfate, filtering, decompressing and concentrating the filtrate, separating the obtained crude product by silica gel column chromatography (eluent methanol: dichloromethane ═ 1:40) to obtain 0.017g of (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), pale yellow solid, yield 11.20%, melting point: 120-122 ℃.
Spectral data:1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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- (pinacolato borate) phenyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropaneamide (I-17)
Dissolving paraformaldehyde (30mg, 0.31mmol) and 4-cyanobenzylamine (40mg, 0.30mmol) in 4.0mL of anhydrous methanol, adding triethylamine (30mg, 0.31mmol), stirring at room temperature for 30 minutes, subsequently adding pinacol ester 4-phenylboronate (80mg, 0.31mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(80mg, 0.24mmol), heating at 70 ℃ for reflux for 16 hours, and monitoring by TLC; after 16h, after the reaction, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane are added into the residue in the bottle for extraction, the organic phase is separated and washed by 10mL of 1N HCl solution, the organic phase is separated and washed by 10mL of saturated sodium chloride solution, the organic phase is dried by anhydrous sodium sulfate, filtered, the filtrate is concentrated under reduced pressure, and the obtained crude product is subjected to silica gel column chromatography (eluent methanol: dichloromethane ═ 1:40) to obtain 0.017g of the crude product of the target product (S) -2- (2- (N- (4-cyanobenzene) -2- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxybenzaldehyde-2-yl) phenyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamide (I-17) Light yellow solid, yield 11.20%, melting point: 46-48 ℃.
Spectral data:1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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).C41H43BF2N4O6[737.20].
example 17: preparation of (S) -2- (2- (N-cyclopropyl-2- (4- (4- (pinacolato borate) phenyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropanamine (I-18)
Dissolving paraformaldehyde (40mg, 0.39mmol) and cyclopropylamine (20mg, 0.39mmol) in 4.0mL of anhydrous methanol, adding triethylamine (40mg, 0.39mmol), stirring at room temperature for 30 minutes, then adding pinacol ester of 4-phenylboronic acid (100mg, 0.39mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(100mg, 0.30mmol), heating at 70 ℃ under reflux for 16 hours, and monitoring by TLC; after 16h, after the reaction, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane are added into the residue in the bottle for extraction, an organic phase is separated and washed by 10mL of 1N HCl solution, an organic phase is separated and washed by 10mL of saturated sodium chloride solution, the organic phase is dried by anhydrous sodium sulfate, filtered, filtrate is concentrated under reduced pressure, and the obtained crude product is subjected to silica gel column chromatography separation (eluent methanol: dichloromethane ═ 1:40) to obtain 0.017g of the crude product of (S) -2- (2- (N-cyclopropyl-2- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxybenzaldehyde-2-yl) phenyl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-18), pale yellow solid, yield 11.20%, melting point: 46-48 ℃.
Spectral data:
1H NMR(400MHz,DMSO-d6)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).
13C NMR(100MHz,DMSO-d6)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).C36H42BF2N3O6[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 (40mg, 0.39mmol) and 4-fluorobenzylamine (50mg, 0.39mmol) are dissolved in 4.0mL of anhydrous methanol, triethylamine (40mg, 0.39mmol) is added and stirred at room temperature for 30 minutes, followed by addition of 2- (3- (trifluoromethyl) -4,5,6, 7-tetrahydro-1H-indazol-1-yl) acetic acid (100mg, 0.39mmol) and stirring at room temperature for 10 minutes, followed by addition of key intermediate I-5(100mg, 0.30mmol), heating at 70 ℃ for reflux for 16 hours, monitored by TLC; after 16H, after the reaction is finished, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane are added into the residue in the bottle for extraction, an organic phase is separated and washed by 10mL of 1N HCl solution, an organic phase is separated and washed by 10mL of saturated sodium chloride solution, the organic phase is dried by anhydrous sodium sulfate and filtered, the filtrate is concentrated under reduced pressure, and the obtained crude product is subjected to silica gel column chromatography (eluent methanol: dichloromethane ═ 1:40) to obtain a 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) Product 0.017g, light yellow solid, yield 11.20%, melting point: 46-48 ℃.
Spectral data:
1H NMR(400MHz,DMSO-d6)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).
13C NMR(100MHz,DMSO-d6)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-methylpropanamine (I-20)
Dissolving paraformaldehyde (30mg, 0.31mmol) and cyclohexylmethylamine (40mg, 0.31mmol) in 4.0mL of anhydrous methanol, adding triethylamine (30mg, 0.31mmol), stirring at room temperature for 30 minutes, then adding 2- (3- (trifluoromethyl) -4,5,6, 7-tetrahydro-1H-indazol-1-yl) acetic acid (70mg, 0.31mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(80mg, 0.24mmol), heating at 70 ℃ for reflux for 16 hours, TLC monitoring; after 16H, after the reaction is finished, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane are added into the residue in the bottle for extraction, an organic phase is separated and washed by 10mL of 1N HCl solution, an organic phase is separated and washed by 10mL of saturated sodium chloride solution, the organic phase is dried by anhydrous sodium sulfate and filtered, the filtrate is concentrated under reduced pressure, and the obtained crude product is subjected to silica gel column chromatography (eluent methanol: dichloromethane ═ 1:40) to obtain a 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) Product 0.017g, light yellow solid, yield 11.20%, melting point: 46-48 ℃.
Spectral data:
1H NMR(400MHz,DMSO-d6)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).
13C NMR(100MHz,DMSO-d6)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).C33H38F5N5O4[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)
Dissolving paraformaldehyde (30mg, 0.27mmol) and benzylamine (30mg, 0.27mmol) in 4.0mL of anhydrous methanol, adding triethylamine (30mg, 0.27mmol), stirring at room temperature for 30 minutes, then adding 2- (2-methyl-1H-indol-3-yl) acetic acid (50mg, 0.27mmol), stirring at room temperature for 10 minutes, then adding key intermediate I-5(70mg, 0.21mmol), heating at 70 ℃ for reflux for 16 hours, monitored by TLC; after 16H, after the reaction is finished, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane are added into the residue in the bottle for extraction, an organic phase is separated and washed by 10mL of 1N HCl solution, an organic phase is separated and washed by 10mL of saturated sodium chloride solution, the organic phase is dried by anhydrous sodium sulfate and filtered, the filtrate is decompressed and concentrated, and the obtained crude product is separated by silica gel column chromatography (eluent methanol: dichloromethane ═ 1:40) to obtain 0.015g of crude product 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:
1H NMR(400MHz,DMSO-d6)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).C37H36F2N4O4[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)
Dissolving paraformaldehyde (30mg, 0.27mmol) and benzylamine (30mg, 0.27mmol) in 4.0mL of anhydrous methanol, adding triethylamine (30mg, 0.27mmol), stirring at room temperature for 30 minutes, adding 2-naphthylacetic acid (50mg, 0.27mmol), stirring at room temperature for 10 minutes, adding key intermediate I-5(70mg, 0.21mmol), heating at 70 ℃ for reflux for 16 hours, and monitoring by TLC; after 16h, after the reaction was completed, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane were added to the residue in the flask for extraction, the organic phase was separated and washed with 10mL of 1N HCl solution, the organic phase was separated and washed with 10mL of saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product, which was subjected to silica gel column chromatography (eluent methanol: dichloromethane ═ 1:40) to give 0.014g of a crude product of (S) -2- (2- (N-benzyl-2- (naphthalen-2-yl) acetamido) -3- (3, 5-difluorophenyl) -N- (4-methoxyphenyl) -N-methylpropylamine (I-22), a pale yellow solid, yield 10.50%, melting point: 46-48 ℃.
Spectral data:
1H NMR(600MHz,DMSO-d6)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).
13C NMR(100MHz,DMSO-d6)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).C38H35F2N3O4[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-oxyethyl) (4-fluorobenzyl) amino) -2-oxyethyl) phenyl) boronic acid (I-23)
Dissolving paraformaldehyde (40mg, 0.39mmol) and 4-fluorobenzylamine (50mg, 0.39mmol) in 4.0mL of anhydrous methanol, adding triethylamine (40mg, 0.39mmol), stirring at room temperature for 30 minutes, adding 4-boronic acid phenylacetic acid (70mg, 0.39mmol), stirring at room temperature for 10 minutes, adding key intermediate I-5(100mg, 0.30mmol), heating at 70 ℃ for reflux for 16 hours, and monitoring by TLC; after 16h, after the reaction was completed, 10mL of saturated sodium bicarbonate solution and 10mL of dichloromethane were added to the residue in the flask for extraction, the organic phase was separated and washed with 10mL of 1N HCl solution, the organic phase was separated and washed with 10mL of saturated sodium chloride solution, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting crude product was subjected to silica gel column chromatography (eluent methanol: dichloromethane ═ 1:40) to obtain 0.017g of a crude product of (S) - (4- (2- ((3- (3, 5-difluorophenyl) -1- ((4-methoxyphenyl) (methyl) amino) -1-oxypropylene-2-yl) amino) -2-oxyethyl) (4-fluorobenzyl) amino) -2-oxyethyl) phenyl) boronic acid (I-23), white solid, yield 12.0%, melting point: 123.6-125.8 ℃.
Spectral data:
1H NMR(400MHz,DMSO-d6)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).
13C NMR(150MHz,DMSO-d6)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 (MT-4 cells) of the Compounds of interest
Interpretation of terms: MT-4 cells: human acute lymphoblastic leukemia cells; MTT assay: MTT is 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide, and the trade name is thiazole blue; DMSO, DMSO: dimethyl sulfoxide (DMSO).
Principle of testing
Because the HIV-infected MT-4 cells can be 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 the suspension of the HIV-infected MT-4 cells, and after a period of culture (5-7 days), the activity of the MT-4 cells is measured by an MTT (methyl thiazolyl tetrazolium) analysis method, so that the concentration of a drug (EC) for protecting 50% of the cells from cytopathic diseases is obtained (the concentration of the drug is the same as that of the drug (EC)50) The anti-HIV activity of the target compound can be obtained. Simultaneously obtaining the concentration (CC) of the target compound which can cause 50 percent of cells not infected by HIV to generate pathological changes50) Calculating the selection coefficient (SI ═ CC)50/EC50)。
Principle of MTT assay: MTT, i.e., bromo-3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium nitrogen, binds to live intracellular succinate dehydrogenase and does not react with dead cells. At present, the MTT method is an enzyme analysis method for rapidly reflecting the cell activity.
Test materials and methods
(1)HIV-1(IIIB) HIV-2(ROD) strain: provided by the institute of microbiology and immunology, Rega institute of university of Leuven, belgium;
(2) MT-4 cells: provided by the institute of microbiology and immunology, Rega institute of university of Leuven, belgium;
(3) MTT: purchased from Sigma, usa;
(4) sample treatment: dissolving a sample in DMSO to prepare a proper concentration before use, and diluting by 5 times with double distilled water, wherein each dilution is 5 dilutions;
(5) positive control: zidovudine, nevirapine;
(6) the test method comprises the following steps: diluting the sample, adding into suspension of HIV-infected MT-4 cell, measuring cell activity by MTT colorimetric method after a period of time, recording absorbance (A) value at 590nm in enzyme labeling instrument, and calculating EC50、CC50And a SI;
(7) MTT staining method: after adding the sample for incubation for a period of time, 20. mu.L of MTT solution (5mg/mL) was added to each well, incubation was continued for several hours, the staining solution was discarded, 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 solution, and mixing 3 × 10 solutions5MT-4 cells were preincubated with 100. mu.L of compound solutions at various concentrations for 1h at 37 ℃. Then, 100. mu.L of a virus dilution of appropriate concentration was added to the mixture, and the cells were incubated at 37 ℃ for 1 h. After three washes, the cells were resuspended in culture medium with or without compound, respectively. Cells were then incubated at 5% CO2Incubate at 37 ℃ for 7 more days in the environment and supplement the stock culture with culture medium with or without compound on the third day after infection. The procedure was repeated twice for each culture condition. Cytopathic effects on the virus were monitored daily with a reverse optical microscope. Generally, the virus dilutions used in this experiment often developed cytopathic effects the fifth day after viral infection. The inhibitory concentration of the drug is that concentration at which the drug produces 50% inhibition of viral cytopathic effects while not being directly toxic to cells (EC)50) And (4) showing. It is to be emphasized that, when the compound is poorly water soluble and requires DMSO to dissolve, the volume-specific concentration of DMSO relative to water is typically less than 10% (final DMSO concentration in MT-4 cell culture medium is less than 2%). Since DMSO can affect the antiviral activity of the test compound, the antiviral activity of a solution containing the same concentration of DMSO should also be run in parallel to the control blank. In addition, the final DMSO concentration (1/1000) was much lower than that required to affect HIV replication in MT-4 cells.
In vitro anti-HIV-1 (III) of a target CompoundB) And HIV-2(ROD) Activity screening data were provided by the institute of microbiology and immunology, university of Leuven, Belgium, Rega, all activity data were obtained in at least two independent, parallel experiments, and the results are shown in Table 1.
TABLE 1 substituted glycine-3, 5-difluorophenylalanine peptoid derivatives anti-HIV activity, toxicity and selection index (MT-4 cells)
Figure BDA0003513394380000221
Figure BDA0003513394380000222
Figure BDA0003513394380000231
aEC50: concentration of compound that protects 50% of HIV-1 infected cells from cytopathic effects;
bCC50: (ii) concentration of compound that causes lesions in 50% of cells not infected with HIV-1;
cand (3) SI: coefficient of selectivity, CC50/EC50The ratio of (A) to (B);
and (3) analyzing an experimental result: as shown in Table 1, the newly synthesized substituted glycine-3, 5-difluorophenylalanine peptide derivatives of the invention have better anti-HIV-1 activity. For example, the anti-HIV-1 activity of the object compounds I-7, I-9, I-19, I-21 is in the range of 2.53 to 2.94. mu.M, wherein the anti-HIV-1 activity (EC) of the object compound I-1950＝2.54μM,CC50>107.61) is particularly prominent. It is worth mentioning that slight changes in the structure of some of the compounds have a significant effect on the activity, HIV-1 and HIV-2 selectivity, for example, compounds I-6 and I-7 differ by R2In contrast, I-6 has no anti-HIV-1/2 activity, and I-7 is an HIV-1 selective inhibitor; i-8 Only R compared with Compound I-62The substituent naphthyl has different substitution positions, and I-8 is an HIV-2 selective inhibitor. As another example, compound I-14 is also only R compared to compound I-132The difference in the substitution positions of the substituent naphthyl groups means that I-13 has little anti-HIV activity, and I-14 is an HIV-2 selective inhibitor. As another example, compound I-16 is R alone as compared to compound I-152The difference of one halogen atom in the substituent groups is that I-15 is an HIV-1 selective inhibitor and I-16 is an HIV-2 selective inhibitor. Zidovudine as a positive drug has no selective inhibitory effect on HIV-1/2; nevirapine is an HIV-1 selective inhibitor, and has no HIV-1/2 double inhibition effect. Obviously, takeThe biological activity of the 3, 5-difluorophenylalanine analog peptide derivative instead of glycine achieves unexpected effects, and is worthy of further development.

Claims (7)

1. The peptide derivative containing substituted glycine-3, 5-difluorophenylalanine or pharmaceutically acceptable salt thereof is characterized by having a structure shown in a general formula I:
Figure FDA0003513394370000011
wherein the content of the first and second substances,
R1comprises 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;
R2comprises the following steps: substituted phenyl, substituted benzyl, substituted naphthylmethyl, 2-methyl-1H-indole-3-methyl, 5-halogen-1H-indole-3-methyl, benzyl substituted by pinacol borate, 3- (trifluoromethyl) -substituted 4,5,6, 7-tetrahydro-1H-indazol-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.
2. The substituted glycine-3, 5-difluorophenylalanine-containing peptoid derivative of claim 1, wherein R is1Is fluorine substituted benzyl, cyano substituted benzyl, cyclohexane methyl, cyclopropyl, benzyl; r is2Is naphthylmethyl, 2-methyl-1H-indole-3-methyl, 5-halogen-1H-indole-3-methyl, benzyl substituted by pinacol borate, 4,5,6, 7-tetrahydro-1H-indazole-1-methyl substituted by 3- (trifluoromethyl), 1, 5-dimethyl-3- (trifluoromethyl) -1H-pyrazole and benzyl substituted by boric acid.
3. The substituted glycine-3, 5-difluorophenylalanine-containing peptoid derivative according to claim 1 or 2, which is one of the following:
Figure FDA0003513394370000021
4. the process for producing a peptide derivative containing substituted glycine-3, 5-difluorophenylalanine according to claim 1 or 2, comprising the steps of: Boc-L-phenylalanine (I-1) is used as an initial raw material, dichloromethane is used as a solvent, and an intermediate I-2 is generated with N-methyl-4-aminoanisole through an amide condensation reaction; then, dissolving the intermediate I-2 in a proper amount of dichloromethane, and removing a Boc group 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; dehydrating the intermediate I-4 under the action of phosphorus oxychloride to obtain an intermediate isonitrile I-5, and then reacting the intermediate I-5 with acid, amine and aldehyde fragments through an Ugi four-component reaction to obtain a target compound;
the synthetic route is as follows:
Figure FDA0003513394370000031
reagents and conditions: (i) n-methyl-4-aminoanisole, 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane are added, and the temperature is changed to room temperature at 0 ℃; (ii) trifluoroacetic acid, dichloromethane, room temperature; (iii) ammonium formate and acetonitrile, refluxing at 90 ℃ for 24 hours; (iv) dichloromethane, triethylamine, phosphorus oxychloride, at 0 ℃ for 12 h; (v) r1-CHO,R2-NH2,R3-COOH, anhydrous methanol, 60 ℃,8 h;
wherein R is1、R2As described above in formula I.
5. The method for preparing the peptide derivative containing substituted glycine-3, 5-difluorophenylalanine according to claim 4, comprising the following steps:
(1) adding Boc-L-phenylalanine (I-1) and 1H-benzotriazole-1-yloxytripyrrolidinyl hexafluorophosphate into dichloromethane, and stirring for 30min under ice bath condition; adding N, N-diisopropylethylamine and N-methyl-4-aminoanisole into the reaction solution, removing the ice bath, transferring to room temperature, and monitoring by TLC; after the reaction is finished, evaporating the solvent under reduced pressure, adding a saturated sodium bicarbonate solution into the residue in the bottle, extracting with dichloromethane, separating an organic phase, adding a 1N HCl solution for washing, separating the organic phase, adding a 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 an intermediate I-2;
(2) adding the intermediate I-2 into dichloromethane, slowly dropwise adding excess trifluoroacetic acid into the solution under the condition of ice bath and stirring, then removing the ice bath, transferring to room temperature, and monitoring by TLC; after the reaction is finished, evaporating the solvent under reduced pressure, adding a saturated sodium bicarbonate solution to adjust the pH of the reaction solution to 7, and then adding a dichloromethane solution for extraction; separating and taking an organic phase, washing the organic phase for 3 times by using a saturated sodium chloride solution, drying the organic phase by using anhydrous sodium sulfate, filtering, and evaporating the solvent to dryness under reduced pressure to obtain an intermediate I-3;
(3) adding the intermediate I-3 and ammonium formate into an acetonitrile solution, and heating for 10h at 90 ℃; after the reaction is finished, extracting the mixture for three times by using water and ethyl acetate, combining organic phases, filtering, decompressing and evaporating the solvent, and separating by using silica gel column chromatography to obtain an intermediate I-4;
(4) dissolving the intermediate I-4 in a dichloromethane solution, adding triethylamine, slowly adding a phosphorus oxychloride solution under an ice bath condition, stirring for 10 hours at 0 ℃, after the reaction is finished, quenching with ice water, extracting with dichloromethane, combining organic phases, drying with anhydrous sodium sulfate, mixing with silica gel, and carrying out column chromatography to obtain an intermediate I-5;
(5) dissolving the corresponding substituted amine fragment and paraformaldehyde in an anhydrous methanol solution, adding triethylamine, and stirring for 30 minutes at room temperature; then adding the corresponding substituted acid fragment, and stirring for 10 minutes at room temperature; then adding an isonitrile intermediate I-5, and adding reflux for 16 hours at 70 ℃; after the reaction is finished, adding a saturated sodium bicarbonate solution into the residue in the bottle, extracting with dichloromethane, separating an organic phase, adding a 1N HCl solution for washing, separating the organic phase, adding a 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 with the general formula 1.
6. The use of the peptide derivatives containing substituted glycine-3, 5-difluorophenylalanine according to claim 1 or 2 for the preparation of a medicament for the treatment and prevention of aids.
7. An anti-HIV pharmaceutical composition comprising a substituted glycine-3, 5-difluorophenylalanine peptoid derivative or a pharmaceutically acceptable salt thereof according to claim 1 or 2 and one or more pharmaceutically acceptable carriers or excipients.
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