CN114989049A - Vif inhibitor and application thereof in preparation of anti-HIV (human immunodeficiency virus) medicines - Google Patents

Vif inhibitor and application thereof in preparation of anti-HIV (human immunodeficiency virus) medicines Download PDF

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CN114989049A
CN114989049A CN202210651816.5A CN202210651816A CN114989049A CN 114989049 A CN114989049 A CN 114989049A CN 202210651816 A CN202210651816 A CN 202210651816A CN 114989049 A CN114989049 A CN 114989049A
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李锐
魏于全
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West China Hospital of Sichuan University
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Abstract

The invention relates to a Vif inhibitor and application thereof in preparing anti-HIV drugs, belonging to the technical field of chemical synthetic drugs. The invention solves the technical problem of providing a compound which has a novel structure and can be used as a Vif inhibitor. The structural formula of the compound is shown as formula I. The compound of the invention has good anti-HIV virus activity, low toxic and side effect and effective multiple drug resistance, can be used as a Vif inhibitor and is a new drug research for treating AIDS caused by HIVLays a foundation.
Figure DDA0003687982720000011

Description

Vif inhibitor and application thereof in preparation of anti-HIV (human immunodeficiency virus) medicines
Technical Field
The invention relates to a Vif inhibitor and application thereof in preparing anti-HIV drugs, belonging to the technical field of chemical synthetic drugs.
Background
After the first discovery of HIV-induced AIDS in the 80 s of the 20 th century, after more than 30 years of development, the HIV-induced AIDS becomes a global epidemic disease, 3200 ten thousand deaths have occurred in the world, and 3700 more than ten thousand of existing cases still exist, although the overall growth rate of the AIDS has a slow trend in recent years, the AIDS becomes a public health problem seriously threatening human health along with the accumulation of infected people.
To study the growth cycle of HIV-infected human immune cells, more than 40 anti-HIV drugs have been developed, which are mainly classified into four categories: viral entry inhibitors, reverse transcriptase inhibitors, integrase inhibitors and protease inhibitors. The four medicines can inhibit the replication of HIV to a certain extent, thereby prolonging the life cycle of patients and improving the quality of life. The best curative drugs at present are Protease Inhibitors (PIs) and two or more Reverse Transcriptase Inhibitors (RTIs) combined highly effective antiretroviral therapy (HAART) therapy, which can reduce drug resistance generated by single medication, inhibit virus replication to the maximum extent, recover damaged immune function partially or completely, and control the development of AIDS. However, this therapy is effective only in some patients, and a large number of AIDS patients still need to take medicines for a long time to prevent the deterioration of the disease. Serious side effects accumulated after long-term administration greatly reduce the compliance of the medicines, even new virus strains with multiple drug resistance appear, so that the development of new anti-HIV medicines with new action mechanisms, which are more effective, have lower toxic and side effects and are effective to multiple drug resistance, is urgently needed.
In the process of researching the replication of HIV infected human immune cells, the human immune system can secrete an endogenous APOBEC3G protein (apolipoprotein B mRNA editing enzyme catalyzes peptide 3G, APOBEC3G, A3G for short), the protein has the effect of cytidine deamination, and deoxycytidine is mutated into deoxyuridine in the negative chain of HIV-1 reverse transcription, so that G → A hypermutation which is lethal to viruses is generated. To combat the function of A3G in inhibiting viral replication, HIV encodes a specific accessory protein Vif (viral infectious agent) that is able to bind targetedly to the A3G protein and then recruit the ligands Cullin5 (cui 5), Elongin B, Elongin C, CBF- β to form the E3 ubiquitin ligase complex, polyubiquitination marker A3G, allowing A3G to undergo degradation via the proteasomal pathway, losing antiviral efficacy. A plurality of documents report that the interference of Vif-A3G axis can effectively protect the antiviral activity of A3G protein, a plurality of small molecular compounds are obtained through screening and optimization, and RN-18 compounds with the earliest development and the best effect are provided.
The structure of the prior reported RN-18 compound is as follows:
Figure BDA0003687982700000021
however, these compounds have remained in preclinical studies, and one important factor limiting their subsequent development is moderate antiviral efficacy, which is not sufficient to support studies as clinical candidates. Therefore, there is a need to investigate other novel compounds with high activity.
Disclosure of Invention
In view of the above defects, the technical problem to be solved by the present invention is to provide a compound which has a novel structure and can be used as a Vif inhibitor.
The structural formula of the compound is shown as the formula I:
Figure BDA0003687982700000022
the compound is obtained by further research on the basis of a compound Z or Z-1 researched in the earlier stage of the invention, and the research idea is as follows:
Figure BDA0003687982700000023
compared with RN-18, Z-1 has one more amino group on the B ring in the chemical structure, and the molecular simulation butt joint shows that one H of the amino group forms an intramolecular H bond with carbonyl O of an adjacent amide group, and one amino H forms an H bond with Gln136 residue of Vif protein, so that the activity is improved. Meanwhile, the molecular simulation docking result also shows that the nitro group of the C ring can have H bond effect with the amino acid Ala153 of the Vif protein, and the A ring substituted by methoxy extends into a hydrophobic pocket.
According to the structure-activity relationship, the nitro group at the para position of the C ring and the amino group on the B ring are beneficial to the activity of the compound, and the A ring is positioned in a hydrophobic pocket. In order to further improve the activity of the compound, the following compound optimization ideas are designed: the ring A is optimized and different substituents of the ring A are changed by keeping the ring C, the ring B and the substituents of the ring B unchanged; assaying the metabolites of compound Z-1 to increase activity by limiting the excessive metabolism of Z-1; change in bridged sulfone between ring B-ring C.
Researches show that the activity of the compound shown in the formula I can be obviously improved, and the activity of the original compound is improved by 20 times.
Further, the present invention also provides an isomer, a pharmaceutically acceptable salt or a hydrate of the compound of the present invention.
In one embodiment of the invention, the salt is a hydrochloride, sulfate, phosphate or nitrate. The salt can be a salt formed by amino of the compound in the formula I, or a salt formed by carboxyl of the compound in the formula I, or a salt formed by both amino and carboxyl.
The invention also provides application of the compound or isomer, pharmaceutically acceptable salt or hydrate of the invention in preparing Vif inhibitor.
The invention also provides the application of the compound or isomer, pharmaceutically acceptable salt or hydrate of the invention in preparing anti-HIV drugs. In one embodiment, the anti-HIV agent is an anti-HIV-type 1 virus agent or an anti-HIV-type 2 virus agent.
The invention also provides application of the compound or isomer, pharmaceutically acceptable salt or hydrate of the invention in preparing antitumor drugs.
The invention also provides application of the compound or isomer, pharmaceutically acceptable salt or hydrate of the invention in preparing anti-hepatitis drugs.
Compared with the prior art, the invention has the following beneficial effects:
the compound of the invention has good anti-HIV activity, low toxic and side effects, is effective to various drug-resistant strains, can be used as a Vif inhibitor, and lays a foundation for the research of new drugs for treating AIDS caused by HIV.
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FIG. 1 shows the results of analysis of the binding pattern of compound Z-1 of example 4 and compound 5a of the present invention, wherein (a) is the binding pattern of compound Z-1 to Vif protein; (b) is the binding pattern of compound 5a to Vif protein; (c) the structure of compound 5 a.
FIG. 2 shows the results of Western blot analysis of compound 5a in example 5, wherein a is the inhibition of A3G and Vif by compound 5 a; (b) the expression quantity of the Vif protein is shown; (c) the expression level is the expression level of A3G protein.
Detailed Description
The following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.
Synthesis of the Compound of example 1
1. Synthesis of Compounds 2a-2f, 3a-3j
The structures and the synthetic routes of the compounds 2a-2f and 3a-3j are as follows:
Figure BDA0003687982700000041
reaction reagents and conditions: (a) 4-nitrothiophenol, potassium carbonate, copper powder, cuprous oxide and ethylene glycol ethyl ether, and standing overnight at 95 ℃; (b) aniline, EDCI, tetrahydrofuran, Room Temperature (RT),5 h; (c) 30% aqueous hydrogen peroxide, glacial acetic acid, 55 ℃ and 6 h.
Synthesis of intermediate 1:
Figure BDA0003687982700000042
dissolving 2-bromo-5-aminobenzoic acid (1080mg, 5mmol) and 4-nitrothiophenol (775mg, 5mmol) in 15mL ethylene glycol ethyl ether, adding anhydrous K to the reaction solution 2 CO 3 (1380mg, 10mmol), copper powder (64mg, 1mmol) and cuprous oxide (72mg, 0.5mmol) were heated in a 95 ℃ oil bath to reflux overnight. After the reaction, the reaction mixture was cooled to room temperature, the pH of the reaction mixture was adjusted to about 5 to 6 with dilute hydrochloric acid, and the mixture was extracted with ethyl acetate (3X 25mL), and the organic layers were combined and washed with water 4 times and anhydrous Na 2 SO 4 Drying, concentrating, and purifying the crude product by column chromatography to give intermediate 1(774mg) with a yield of 46%.
Synthesis of compound 2 a:
Figure BDA0003687982700000043
intermediate 1(97mg, 0.3mmol) and 3-methoxyaniline were dissolved in 10mL of tetrahydrofuran, EDCI (65mg, 0.33mmol) was added to the reaction solution, and the reaction was allowed to react at room temperature for 5 hours. Concentrating under reduced pressure to remove tetrahydrofuran solvent, adding ethyl acetate and water for liquid separation, mixing the obtained organic layers, washing the organic layer with saturated salt water for 1 time, and collecting anhydrous Na 2 SO 4 Drying, concentrating, and purifying the crude product by column chromatography to obtain product 2a (65mg), with yield of 76% and purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.35(s,1H),8.12-8.00(d,J＝9.2Hz,2H),7.32(d,J＝5.7,1H),7.30-7.25(d,J＝9.2Hz,2H),7.25-7.19(d,J＝9.0Hz,1H),7.19-7.13(m,2H),6.93-6.88 (dd,J＝7.7,0.8Hz,1H),6.80-6.73(m,1H),6.67-6.59(m,1H),5.36(s,2H),3.70(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:159.86,147.33,145.29,140.68,131.18,129.79,127.60,124.41, 123.11,117.36,112.52,109.42,106.09,55.45.HRMS(ESI)calcd for C 20 H 17 N 3 O 4 S[M+Na] + m/z 418.0940,found:418.0800。
Synthesis of compound 2 b:
Figure BDA0003687982700000051
synthesis of Compound 2b referring to 2a, the starting material, 3-methoxyaniline, was replaced with 3-aminophenol, and the other treatments were identical, with a yield of 68.1% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.26(s,1H),9.32(s,1H),8.11-8.04(d,J＝9.1,2H), 7.31-7.25(d,J＝8.9,2H),7.22(m,2H),7.04(t,J＝8.0Hz,1H),7.01-6.96(d,J＝8.2Hz,1H), 6.92-6.87(dd,J＝7.9,0.84Hz,1H),6.75(dt,J＝8.4,1.8Hz,1H),6.45(dd,J＝7.9,2.3Hz,1H), 5.33(s,2H). 13 C NMR(101MHz,DMSO-d 6 )δ:164.25,157.96,150.46,147.62,147.12,140.62, 136.94,130.39,129.59,124.86,121.63,117.18,111.24,107.70.HRMS(ESI)calcd for C 19 H 15 N 3 O 4 S[M+Na]+m/z 404.0783,found:404.0674。
Synthesis of compound 2 c:
Figure BDA0003687982700000052
synthesis of Compound 2c referring to 2a, the starting material, 3-methoxyaniline, was replaced with 3, 4-dimethoxyaniline, and the other workup procedures were identical, with a yield of 78.3% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.20(s,1H),8.08(d,J＝9.0Hz,2H),7.34(d,J＝2.2Hz, 1H),7.28(d,J＝9.0Hz,2H),7.22(t,J＝7.9Hz,1H),7.12(dd,J＝8.7,2.3Hz,1H),6.88(dd,J＝13.9,2.3Hz,2H),6.76(d,J＝7.4Hz,1H),5.34(s,2H),3.70(s,3H),3.69(s,3H). 13 C-NMR(101 MHz,DMSO-d 6 )δ:163.87,150.44,148.95,147.58,147.19,145.75,136.97,133.27,130.35, 129.62,124.87,121.62,117.12,112.60,105.89(s),56.36,55.94.HRMS(ESI)calcd for C 21 H 19 N 3 O 5 S[M+H] + m/z 425.1045,found:425.1873。
Synthesis of compound 2 d:
Figure BDA0003687982700000061
synthesis of compound 2d referring to 2a, the starting material 3-methoxyaniline was replaced with 3, 4-difluoroaniline, the treatment was consistent, the yield was 65% and the purity was 97%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.60(s,1H),8.08(d,J＝8.9Hz,2H),7.85-7.76(m,1H), 7.35(m,2H),7.30-7.25(d,J＝9.0Hz,2H),7.23(d,J＝7.9Hz,1H),6.90(d,J＝8.2Hz,1H),6.79 (dd,J＝14.3,7.5Hz,1H),5.45(s,2H). 13 C-NMR(101MHz,DMSO-d 6 )δ:164.79,158.86,154.24, 150.42,147.44,147.11,137.07,129.66,127.13,126.49,124.80,121.35,119.66,117.22,112.52, 105.10,99.51.HRMS(ESI)calcd for C 19 H 13 F 2 N 3 O 3 S[M+Na] + m/z 424.0646,found:424.0538。
Synthesis of compound 2 e:
Figure BDA0003687982700000062
synthesis of Compound 2e referring to 2a, the starting material, 3-methoxyaniline, was replaced with 3, 5-dimethylaniline in a consistent workup procedure, in 61.0% yield and 99% purity.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.22(s,1H),8.11-8.04(d,J＝9.0Hz,2H),7.30-7.26(d,J ＝8.9Hz,2H),7.22(m,3H),6.90(dd,J＝8.2,0.8Hz,1H),6.76(dd,J＝7.6,0.8Hz,1H),6.70(s, 1H),5.33(s,2H),2.21(s,6H). 13 C-NMR(101MHz,DMSO-d 6 )δ:165.59,148.76,147.33,145.26, 139.36,137.92,131.11,127.82,127.65,127.52,125.52,124.40,123.13,117.95,117.34,21.55. HRMS(ESI)calcd for C 21 H 19 N 3 O 3 S[M+Na] + m/z 416.1147,found:416.1038。
Synthesis of compound 2 f:
Figure BDA0003687982700000071
synthesis of Compound 2f referring to 2a, the starting material 3-methoxyaniline was replaced with 3-bromo-2-methylaniline, and the other work-up procedures were identical, with a yield of 59.7% and a purity of 96%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.09(s,1H),8.09(d,J＝8.6Hz,2H),7.45(t,J＝7.2Hz, 2H),7.30(d,J＝8.3Hz,2H),7.24(t,J＝7.9Hz,1H),7.12(t,J＝8.0Hz,1H),6.93(d,J＝7.9Hz, 1H),6.80(dd,J＝6.8,4.1Hz,1H),5.39(s,2H),2.23(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ: 166.05,148.85,147.37,145.27,137.77,133.34,131.19,130.17,127.60,127.25,126.61,126.61, 125.08,124.47,123.46,117.50,18.68.HRMS(ESI)calcd for C 20 H 16 BrN 3 O 3 S[M+Na] + m/z 480.0096,found:479.9987。
Synthesis of compound 3 a:
Figure BDA0003687982700000072
compound 2a (64mg, 0.16mmoL) was dissolved in 5mL of glacial acetic acid and stirred at room temperature, 30% aqueous hydrogen peroxide (27.2mg, 0.8mmoL) was added to the solution, and the reaction solution was placed in an oil bath at 55 ℃ and heated under reflux for 6 hours. After completion of the reaction, the solvent was removed by concentration under reduced pressure, the aqueous layer (3X 25mL) was extracted with ethyl acetate, the organic layers were combined, and the organic layer was washed with saturated brine 1 time and anhydrous Na 2 SO 4 Drying, concentration and column chromatography of the crude product gave product 3a (30mg) in 46% yield and 97% purity.
1 H NMR(400MHz,CDCl 3 )δ:9.43(s,1H),8.36(d,J＝8.8Hz,2H),8.13-8.08(d,J＝8.8Hz, 2H),7.36(d,J＝9.1,Hz,2H),7.29(dd,J＝9.0,2.0Hz,2H),7.08(d,J＝2.5Hz,1H),6.83(d,J＝ 7.8Hz,1H),6.76(t,J＝7.5Hz,1H),5.47(s,2H),3.76(s,3H). 13 C-NMR(101MHz,CDCl 3 )δ: 164.42,159.89,150.46,147.37,140.79,136.98,130.45,129.68,124.89,121.54,117.13,112.83, 109.37,106.53,55.51.HRMS(ESI)calcd for C 20 H 17 N 3 O 6 S[M+Na] + m/z 450.0838,found: 450.0732。
Synthesis of compound 3 b:
Figure BDA0003687982700000081
synthesis of Compound 3b referring to 3a, the starting material, 3-methoxyaniline, was replaced with 4-methoxyaniline, and the other treatments were identical, with a yield of 43% and a purity of 96%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.26(s,1H),8.37(d,J＝8.3Hz,2H),8.19-8.07(d,J＝8.8 Hz,2H),7.57(m,2H),7.41-7.25(m,2H),7.08(d,J＝8.0Hz,1H),6.91(d,J＝15.7Hz,2H),5.45 (s,2H),3.77-3.74(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:163.88,156.08,150.45,147.39, 147.16,137.01,132.77,130.36,129.61,126.77,124.87,121.85,121.59,117.15,115.27,114.16, 55.72.HRMS(ESI)calcd for C 20 H 17 N 3 O 6 S[M+Na] + m/z 450.0838,found:450.0731。
Synthesis of compound 3 c:
Figure BDA0003687982700000082
synthesis of Compound 3c referring to 3a, the starting material, 3-methoxyaniline, was replaced with 3-aminophenol, and the other treatments were identical, with a yield of 42% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.30(s,1H),9.39(s,1H),8.39-8.33(d,J＝9.1Hz,2H), 8.16-8.10(d,J＝9.1Hz,2H),7.35(t,J＝7.9Hz,1H),7.32-7.25(m,2H),7.13-7.06(m,2H), 7.04-7.00(d,J＝8.1Hz,1H),6.54-6.49(dd,J＝8.5,1.8Hz,1H),5.45(s,2H). 13 C-NMR(101 MHz,DMSO-d 6 )δ:164.25,157.96,150.46,147.62,147.12,140.62,136.94,130.39,129.59, 124.86,121.63,117.18,111.24,107.70.HRMS(ESI)calcd for C 19 H 15 N 3 O 6 S[M+Na] + m/z 436.0682,found:436.0579。
Synthesis of compound 3 d:
Figure BDA0003687982700000083
synthesis of Compound 3d referring to FIG. 3a, the starting material, 3-methoxyaniline, was replaced with 4-aminophenol, and the other treatments were identical, with a yield of 51% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:8.79(s,1H),8.32-8.24(d,J＝9.1Hz,2H),7.99-7.90(d,J ＝9.1Hz,2H),7.67(d,J＝14.0Hz,1H),7.61(t,J＝7.9Hz,1H),7.54(dd,J＝7.7,1.0Hz,1H), 7.22(dd,J＝10.0,3.1Hz,1H),6.96-6.87(m,2H),6.15(dd,J＝6.9,3.4Hz,1H),6.07(dd,J＝10.0,2.1Hz,1H),5.75(s,1H). 13 C-NMR(101MHz,DMSO-d 6 )δ:184.58,162.48,155.17,148.83, 147.71,147.57,147.10,134.85,128.50,127.78,126.66,124.57,118.18,113.75,109.48,64.47. HRMS(ESI)calcd for C 19 H 15 N 3 O 5 S[M+K] + m/z 336.0732,found:436.0543。
Synthesis of compound 3 e:
Figure BDA0003687982700000091
synthesis of Compound 3e referring to FIG. 3a, the starting material, 3-methoxyaniline, was replaced with 3, 4-dimethoxyaniline, and the other workup procedures were identical, with a yield of 47% and a purity of 96%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.21(s,1H),8.44-8.29(d,J＝9.1Hz,2H),8.10(d,J＝9.1Hz,2H),7.41-7.32(t,J＝7.8Hz,2H),7.31(d,J＝7.3Hz,1H),7.22(dd,J＝13.9,7.0Hz,1H), 7.09(t,J＝8.6Hz,1H),6.97-6.93(m,1H),5.45(s,2H),3.75(s,6H). 13 C-NMR(101MHz, DMSO-d 6 )δ:163.87,150.44,148.95,147.58,147.19,145.75,136.97,133.27,130.35,129.62, 124.87,121.62,117.12,112.60,105.89,56.36,55.94.HRMS(ESI)calcd for C 21 H 19 N 3 O 7 S [M+Na] + m/z 480.0944,found:480.0840。
Synthesis of compound 3 f:
Figure BDA0003687982700000092
synthesis of Compound 3f referring to 3a, the starting material 3-methoxyaniline was replaced with 3, 5-dimethoxyaniline and the other treatments were identical, with a yield of 51% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.41(s,1H),8.40-8.34(d,J＝8.8Hz,2H),8.13-8.10(d,J ＝8.8Hz,2H),7.36(t,J＝7.9Hz,1H),7.30(dd,J＝7.7,0.9Hz,1H),7.09(d,J＝3.7Hz,1H),6.95 (d,J＝7.2Hz,2H),6.28(d,J＝2.2Hz,1H),5.46(s,2H),3.74(d,J＝1.4Hz,6H). 13 C-NMR(101 MHz,DMSO-d 6 )δ:165.11,150.51,147.50,147.14,137.90,137.00,132.84,131.90,129.84, 129.71,127.55,126.41,125.03,124.99,121.74,121.38,117.40,49.07.HRMS(ESI)calcd for C 21 H 19 N 3 O 7 S[M+Na] + m/z 480.0944,found:480.0840。
Synthesis of Compound 3 g:
Figure BDA0003687982700000101
synthesis of 3g Compound 3-methoxyaniline was replaced with 4-chloro-2-fluoroaniline as the starting material according to 3a, and the other treatments were identical, with a yield of 37% and a purity of 97%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.53(s,1H),8.39(d,J＝8.5Hz,2H),8.16(d,J＝8.8Hz, 2H),8.04(t,J＝8.6Hz,1H),7.50(dd,J＝8.9,4.4Hz,1H),7.36(t,J＝8.0Hz,2H),7.27(d,J＝7.6 Hz,1H),7.07(d,J＝8.1Hz,1H),5.51(s,2H). 13 C-NMR(101MHz,DMSO-d 6 )δ:165.26,155.82, 153.33,150.50,147.68,147.11,137.07,130.62,129.56,126.82,126.38,124.93,121.60,120.81, 117.23,114.19.HRMS(ESI)calcd for C 19 H 13 ClFN 3 O 5 S[M+Na] + m/z 472.0248,found:472.0141。
Synthesis of compound 3 h:
Figure BDA0003687982700000102
3h Synthesis of Compound 3a, the starting material 3-methoxyaniline was replaced with 3-bromo-2-methylaniline in a consistent workup procedure with a 50% yield and 99% purity.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.22(s,1H),8.39(d,J＝8.9Hz,2H),8.19(d,J＝8.9Hz, 2H),7.74(d,J＝7.9Hz,1H),7.53-7.45(d,J＝7.8Hz,1H),7.36(t,J＝7.6Hz,1H),7.29(d,J＝7.2 Hz,1H),7.21(t,J＝8.0Hz,1H),7.08(d,J＝11.1Hz,1H),5.64-5.39(s,2H),2.40(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:165.11,150.51,147.78,147.22,137.90,137.00,132.84,130.51, 129.84,129.58,127.55,126.41,125.03,124.95,121.74,121.38,117.40,113.30,18.55.HRMS(ESI) calcd for C 20 H 16 BrN 3 O 5 S[M+Na] + m/z 511.9994,found:511.9890。
Synthesis of compound 3 i:
Figure BDA0003687982700000111
synthesis of Compound 3i referring to FIG. 3a, the starting material, 3-methoxyaniline, was replaced with 3, 5-dimethylaniline, and the other treatments were identical, with a yield of 47% and a purity of 96%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.24(s,1H),8.39-8.33(d,J＝8.8Hz,2H),8.16-8.09(d,J ＝8.7Hz,2H),7.35(t,J＝7.9Hz,1H),7.31-7.22(m,3H),7.08(d,J＝6.0Hz,1H),6.74(s,1H), 5.43(s,2H),2.27(s,6H). 13 C-NMR(101MHz,DMSO-d 6 )δ:164.26,150.43,147.56),147.17, 139.48,137.88,136.93,129.98,129.59,125.60,124.90,121.63,117.86,117.12,21.62.HRMS(ESI) calcd for C 21 H 19 N 3 O 5 S[M+H] + m/z 426.1045,found:426.1151。
Synthesis of compound 3 j:
Figure BDA0003687982700000112
the synthesis method of the compound 3j refers to 3a, the 3-methoxyaniline serving as the raw material is replaced by 3, 5-dimethylaniline, and other treatment methods are consistent, the yield is 43%, and the purity is 97%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.15(s,1H),9.03(s,1H),8.38-8.32(d,J＝8.8Hz,2H), 8.16-8.08(d,J＝8.9Hz,2H),7.35(t,J＝7.9Hz,1H),7.29(dd,J＝7.8,1.5Hz,2H),6.95-6.87(m, 2H),5.40(s,2H),3.75(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:163.74,150.45,147.64,147.12, 146.81,144.64,136.95,133.25,129.60,126.73,124.84,121.68,112.96,111.39,109.13,56.52. HRMS(ESI)calcd for C 20 H 17 N 3 O 7 S[M+Na] + m/z 466.0787,found:466.0678。
2. Synthesis of Compounds 4a-4e,5a-5e
The specific structure and the synthetic route are as follows:
Figure BDA0003687982700000121
reaction reagents and conditions: (a) aniline, EDCI, tetrahydrofuran, room temperature, 5h (b) 30% aqueous hydrogen peroxide, glacial acetic acid, 55 ℃,6 h.
Synthesis of compound 4 a:
Figure BDA0003687982700000122
synthesis of Compound 4a referring to 2a, the starting material, 3-methoxyaniline, was replaced with 3, 5-dimethylaniline, and the other treatments were identical, with a yield of 67% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.43(s,1H),9.19(s,1H),8.15-8.05(d,J＝8.9Hz,2H), 7.34-7.28(d,J＝9.0Hz,2H),7.26(d,J＝8.5Hz,1H),7.20(t,J＝7.9Hz,1H),6.88(d,J＝8.2Hz, 1H),6.74(t,J＝8.3Hz,1H),6.43(d,J＝2.5Hz,1H),6.30(dd,J＝8.5,2.5Hz,1H),5.36(s,2H), 3.65(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:165.51,156.53,153.47,149.13,147.27,145.19, 130.99,127.88,127.39,126.28,124.39,123.31,118.20,117.23,106.76,99.94,55.90.HRMS(ESI) calcd for C 20 H 17 N 3 O 5 S[M+Na] + m/z 434.0889,found:434.0781。
Synthesis of compound 4 b:
Figure BDA0003687982700000123
synthesis of Compound 4b referring to 2a, 3-methoxyaniline, a starting material, was replaced with 5-amino-4-methoxyphenol, and the other starting materials were dosed in the same amount as the treatment method, with a yield of 61% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.41(s,1H),9.19(s,1H),8.07(d,J＝13.0Hz,2H),7.30(d,J ＝6.7,4.9Hz,2H),7.26(d,J＝8.5Hz,1H),7.20(t,J＝7.9Hz,1H),6.88(d,J＝7.7Hz,1H),6.75 (d,J＝7.0Hz,1H),6.43(d,J＝2.4Hz,1H),6.30(dd,J＝8.5,2.4Hz,1H),5.36(s,2H),3.65(s, 3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:165.47,151.35,148.93,147.41,145.25,143.75,131.22, 127.59,124.44,123.44,117.53,112.86,111.33,110.69,56.70.HRMS(ESI)calcd for C 20 H 17 N 3 O 5 S [M+H] + m/z 412.0889,found:412.0960。
Synthesis of compound 4 c:
Figure BDA0003687982700000131
synthesis of Compound 4c referring to 2a, the starting material, 3-methoxyaniline, was replaced with 5-amino-4-methoxybenzoic acid, and the other treatments were identical, with a yield of 66% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.35(s,1H),8.24(s,1H),8.07(d,J＝13.8Hz,2H),7.69(d,J ＝8.2Hz,1H),7.32(d,J＝8.9Hz,2H),7.22(t,J＝7.9Hz,1H),6.96(d,J＝8.5Hz,1H),6.90(d,J ＝8.1Hz,1H),6.78(d,J＝8.8Hz,1H),5.40(s,2H),3.73(s,3H). 13 C-NMR(101MHz,DMSO-d 6 ) δ:165.72,154.11,148.89,147.40,145.26,131.23,127.66,127.46,127.30(s),126.64,124.98, 124.43,123.36,117.46,111.06,56.36.HRMS(ESI)calcd for C 21 H 17 N 3 O 6 S[M+Na] + m/z 462.0838, found:462.0729。
Synthesis of compound 4 d:
Figure BDA0003687982700000132
synthesis of Compound 4d referring to 2a, the starting material, 3-methoxyaniline, was replaced with 2, 5-dimethoxyaniline, and the other treatments were identical, with a yield of 66% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.21(s,1H),8.09(d,J＝8.9Hz,2H),7.51(d,J＝2.9Hz, 1H),7.31(d,J＝8.9Hz,2H),7.23(t,J＝7.9Hz,1H),6.91(dd,J＝8.5,3.5Hz,2H),6.77(d,J＝ 7.5Hz,1H),6.66(dd,J＝8.9,3.0Hz,1H),5.41(s,2H),3.67(s,3H),3.63(s,3H). 13 C-NMR(101 MHz,DMSO-d 6 )δ:165.65,153.37,148.86,147.50,145.09,131.26(s),128.05,127.27,123.91, 123.39,117.54,112.54,109.67,56.28.HRMS(ESI)calcd for C 21 H 19 N 3 O 5 S[M+H] + m/z 426.1045, found:426.1140。
Synthesis of compound 4 e:
Figure BDA0003687982700000141
synthesis of Compound 4e referring to 2a, the starting material, 3-methoxyaniline, was replaced with 5-fluoro-2-methoxyaniline, and the other treatments were identical, with a yield of 62% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.42(s,1H),8.08(d,J＝8.1Hz,2H),7.83(dd,J＝10.6,3.0 Hz,1H),7.31(d,J＝8.9Hz,2H),7.23(t,J＝7.9Hz,1H),6.98(dd,J＝16.9,8.5Hz,1H), 6.94-6.84(d,J＝8.5Hz,2H),6.77(d,J＝7.5Hz,1H),5.43(s,2H),3.67(s,3H). 13 C-NMR(101 MHz,DMSO-d 6 )δ:165.94,157.27,154.94,148.82,147.49,146.77,145.27,131.34,127.42, 126.93,124.43,123.32,117.55,112.50,109.63,56.67.HRMS(ESI)calcd for C 20 H 16 FN 3 O 4 S [M+Na] + m/z 436.0846,found:436.0737。
Synthesis of compound 5 a:
Figure BDA0003687982700000142
synthesis of Compound 5a referring to 3a, the starting material, 3-methoxyaniline, was replaced with 5-fluoro-2-methoxyaniline, and the other treatments were identical, with a yield of about 40% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.51(s,1H),9.15-8.98(s,1H),8.36(d,J＝8.6Hz,2H), 8.11(d,J＝8.3Hz,2H),7.35(dd,J＝9.3,5.5Hz,2H),7.31-7.20(d,J＝7.5Hz,1H),7.06(d,J＝ 7.9Hz,1H),6.88(d,J＝8.8Hz,1H),6.58(dd,J＝8.6,2.5Hz,1H),5.55(s,2H),3.7-3.55(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:164.54,151.48,150.45,147.70,147.18,145.03,136.98,130.45, 129.57,127.60,124.83,121.47,117.23,113.07,112.27,56.73.HRMS(ESI)calcd for C 20 H 17 N 3 O 7 S [M+H]+m/z 444.0787,found:444.0761。
Synthesis of compound 5 b:
Figure BDA0003687982700000151
synthesis of Compound 5b referring to 3a, the starting material, 3-methoxyaniline, was replaced with 5-amino-4-methoxyphenol, and the other treatments were identical, with a yield of 41% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.81(s,1H),8.42(d,J＝2.1Hz,1H),8.36(d,J＝8.9Hz, 2H),8.16(d,J＝8.9Hz,2H),7.83(dd,J＝8.5,2.0Hz,1H),7.36(t,J＝8.0Hz,1H),7.27(d,J＝ 7.7Hz,1H),7.18(d,J＝8.7Hz,1H),7.10-7.05(d,J＝7.9Hz,1H),5.56(s,2H),3.86(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:172.48,168.94,167.25,156.58,151.97,138.31,128.66,127.84, 126.37,124.78,123.82,118.90,114.70,113.87,112.47,56.41.HRMS(ESI)calcd for C 21 H 17 N 3 O 8 S [M+Na] + m/z 494.0736,found:494.0634。
Synthesis of compound 5 c:
Figure BDA0003687982700000152
synthesis of Compound 5c referring to 3a, the starting material, 3-methoxyaniline, was replaced with 2, 4-dimethoxyaniline, and the other workup procedures were identical, with a yield of 36% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.64(s,1H),8.39-8.32(d,J＝8.9Hz,2H),8.20-8.11(d, J＝8.9Hz,2H),7.48(d,J＝8.6Hz,1H),7.35(t,J＝7.9Hz,1H),7.28(dd,J＝7.8,1.1Hz,1H), 6.69-6.62(m,2H),6.61-6.58(d,J＝2.8Hz,1H),5.57(s,2H),3.80-3.77(s,6H). 13 C-NMR(101 MHz,DMSO-d 6 )δ:164.79,158.86,154.24,150.42,147.76,147.12,137.07,129.67,127.13, 126.50,124.80,121.66,119.66,117.22,112.52,105.10,99.51,56.24,55.90.HRMS(ESI)calcd for C 21 H 19 N 3 O 7 S[M+Na] + m/z 480.0944,found:480.0840。
Synthesis of compound 5 d:
Figure BDA0003687982700000161
synthesis of Compound 5d referring to 3a, the starting material, 3-methoxyaniline, was replaced with 2, 5-dimethoxyaniline, and the other original workup procedures were identical, with a yield of 36% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.59(s,1H),8.36(d,J＝8.9Hz,2H),8.15(d,J＝8.9Hz, 2H),7.49(d,J＝3.1Hz,1H),7.36(t,J＝7.9Hz,1H),7.29(d,J＝7.1Hz,1H),7.06(d,J＝8.0Hz, 1H),7.00(d,J＝9.0Hz,1H),6.76(dd,J＝9.3,4.7Hz,1H),5.55(s,2H),3.75(s,3H),3.72(s,J＝ 6.2Hz,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:164.71,153.52,150.44,147.67,147.18,146.04, 137.00,130.48,129.57,127.96,124.86,121.43,117.22,112.72,111.35,110.11,56.34.HRMS(ESI) calcd for C 21 H 19 N 3 O 7 S[M+Na] + m/z 480.0944,found:480.0840。
Synthesis of compound 5 e:
Figure BDA0003687982700000162
synthesis of Compound 5e referring to 3a, the starting material, 3-methoxyaniline, was substituted for 5-fluoro-2-dimethoxyaniline, and the other treatments were identical, with a yield of 39% and a purity of 97%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.76(s,1H),8.40-8.33(d,J＝8.9Hz,2H),8.18-8.12(d,J ＝8.9Hz,2H),7.86(dd,J＝10.4,3.1Hz,1H),7.36(t,J＝8.0Hz,1H),7.31-7.24(dd,J＝7.9,1.1 Hz,1H),7.09-7.03(m,2H),7.01(d,J＝3.2Hz,1H),5.53(s,2H),3.77(s,3H). 13 C-NMR(101 MHz,DMSO-d 6 )δ:165.01,150.46,147.59,147.19,136.99,130.55,130.17,129.56,124.90, 121.60,121.02,117.20,112.75,110.77,56.77.HRMS(ESI)calcd for C 20 H 16 FN 3 O 6 S[M+H] + m/z 446.0744,found:446.0616。
3. Synthesis of Compounds 6a-6c and Compound 7a
The specific structure and the synthetic route are as follows:
Figure BDA0003687982700000171
reaction reagents and conditions: (a) 3-amino group4-methoxyphenol, EDCI, tetrahydrofuran, room temperature reaction, 5h (b) bromine substituted alkyl alcohol, K 2 CO 3 DMF,60 ℃,5h (c) 30% aqueous hydrogen peroxide, glacial acetic acid, 55 ℃,6 h.
Synthesis of compound 6 a:
Figure BDA0003687982700000172
intermediate 4b (60mg, 0.15mmol) was dissolved in 10mL DMF and stirred with anhydrous potassium carbonate (41.4mg, 0.3mmol) followed by 2-bromoethanol (22.3mg, 0.18mmol) in a 60 ℃ oil bath and heated under reflux for 5h with the progress of the reaction monitored by TLC. Adding ethyl acetate and water for liquid separation after the reaction is finished, combining the obtained organic layers, washing the organic layer with saturated salt water for 3 times, and washing with anhydrous Na 2 SO 4 Drying, concentrating, and purifying the crude product by column chromatography to obtain product 6a (26mg) with yield of 38% and purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:8.14(d,J＝9.0Hz,2H),7.37(d,J＝8.9Hz,2H),6.97(m, 2H),6.85-6.58(m,2H),6.54(d,J＝7.6Hz,1H),6.37(d,J＝8.0Hz,1H),5.16(s,2H),3.94-3.62(s, 3H),3.57(t,J＝15.5Hz,2H),2.98(d,J＝16.0Hz,2H).HRMS(ESI)calcd for C 22 H 21 N 3 O 6 S [M+Na]+m/z 478.1151,found:478.1044。
Synthesis of compound 6 b:
Figure BDA0003687982700000173
synthesis of Compound 6b referring to 6a, the starting material, 2-bromoethanol, was substituted for 3-bromopropanol, and the other treatments were identical, with a yield of 36% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.27(s,1H),8.03(d,J＝9.1Hz,2H),7.37(d,J＝9.3Hz, 2H),6.91(m,4H),6.51(d,J＝7.6Hz,1H),6.38(d,J＝8.1Hz,1H),5.26(s,2H),4.83-4.17(m, 2H),4.10-3.85(m,1H),3.67(s,3H),3.51(t,J＝5.8Hz,2H),2.94(s,2H),1.79-1.63(t,J＝6.5Hz, 2H).HRMS(ESI)calcd for C 23 H 23 N 3 O 6 S[M+H] + m/z 492.1308,found:492.1205。
Synthesis of compound 6 c:
Figure BDA0003687982700000181
synthesis of Compound 6c referring to 6a, the starting material, 2-bromoethanol, was replaced with 4- (2-chloroethyl) morpholine hydrochloride, and the other treatments were identical, with a yield of 37% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.25(s,1H),8.03(d,J＝9.1Hz,2H),7.36(d,J＝9.3Hz, 2H),6.95(d,J＝16.8Hz,2H),6.76(s,2H),6.54(d,J＝7.7Hz,1H),6.40(d,J＝8.1Hz,1H),5.26 (s,2H),4.19(m,1H),3.86-3.59(s,3H),3.59-3.53(t,J＝4.6Hz 4H),3.17(d,J＝3.6Hz,1H), 3.11-2.86(t,J＝7.3Hz,2H),2.43-2.33(t,J＝4.1Hz,4H).HRMS(ESI)calcd for C 26 H 28 N 4 O 6 S [M+H]+m/z 525.1730,found:525.1806。
Synthesis of compound 7 a:
Figure BDA0003687982700000182
synthesis of Compound 7a referring to 3a, the starting material, 3-methoxyaniline, was substituted for ethyl 2- (3-amino-4-methoxyphenoxy) acetate in a consistent manner in other work-up procedures, with a yield of 34% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:8.26(d,J＝8.8Hz,2H),8.21(s,1H),8.09(d,J＝3.0Hz, 1H),8.04(d,J＝8.8Hz,2H),7.55(d,J＝7.8Hz,1H),7.37(t,J＝8.0Hz,1H),6.98(d,J＝8.2Hz, 1H),6.83(d,J＝8.9Hz,1H),6.70(dd,J＝8.9,3.0Hz,1H),4.64(s,2H),4.30(m,2H),3.79(s,3H), 1.32(t,J＝7.1Hz,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:169.08,163.75,152.09,150.36,146.31, 143.49,137.49,130.65,129.39,127.66,124.16,122.02,120.72,118.77,110.85,107.95(s),66.26, 61.38,56.29,14.21.HRMS(ESI)calcd for C 24 H 23 N 3 O 9 S[M+Na] + m/z 552.1155,found:552.1054。
4. Synthesis of Compounds 8a-8c,9a-9c
The specific structure and the synthetic route are as follows:
Figure BDA0003687982700000191
reaction reagents and conditions: (a) 3-amino-4-methoxyphenol, EDCI, tetrahydrofuran, room temperature reaction, 5h (b) bromo-substituted alcohol, K 2 CO 3 ,DMF,60℃,5h。
Synthesis of compound 9 a:
Figure BDA0003687982700000192
synthesis of Compound 9a referring to 3a, the starting material, 3-methoxyaniline, was substituted for 3-fluoro-dimethoxyaniline in a consistent workup procedure, 37% yield and 99% purity.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.15(s,1H),8.41-8.33(d,J＝9.1Hz,2H),8.20-8.13(d,J ＝8.9Hz,2H),7.77-7.69(dd,J＝7.5,2.2Hz,1H),7.36(t,J＝8.0Hz,1H),7.31-7.25(d,J＝8.0, 1.2Hz,1H),7.15(d,J＝6.1Hz,1H),7.13-7.03(m,2H),5.53(s,2H),3.86(s,3H). 13 C-NMR(101 MHz,DMSO-d 6 )δ:165.29,150.49,147.71,147.14,137.01,130.50,130.20,129.62,125.15, 124.90,123.93,121.54,121.34,120.88,117.25,61.82.HRMS(ESI)calcd for C 20 H 16 FN 3 O 6 S [M+Na] + m/z 468.0744,found:468.0641.
Synthesis of compound 9 b:
Figure BDA0003687982700000193
synthesis of Compound 9b referring to 3a, the starting material, 3-methoxyaniline, was substituted for 3-fluoro-dimethoxyaniline, and the other workup procedures were identical, with a yield of 37% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.84(s,1H),8.41(d,J＝8.0Hz,2H),8.21(d,J＝8.8Hz, 2H),7.73(dd,J＝8.6,6.6Hz,1H),7.40(t,J＝7.9Hz,1H),7.33(d,J＝7.7Hz,1H),7.11(d,J＝ 8.0Hz,1H),7.06(dd,J＝11.6,5.8Hz,1H),6.89(td,J＝8.6,2.7Hz,1H),5.59(s,2H),3.85(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:164.89,159.73,150.45,147.73,147.09,137.09,130.48,129.63, 126.79,124.84,123.17,121.42,117.29,106.85,100.54,56.67.HRMS(ESI)calcd for C 20 H 16 FN 3 O 6 S[M+Na] + m/z 468.0744,found:468.0640。
Synthesis of compound 9 c:
Figure BDA0003687982700000201
synthesis of Compound 9c referring to FIG. 3a, the starting material, 3-methoxyaniline, was substituted for 6-fluoro-dimethoxyaniline in a consistent workup procedure with a yield of 38% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.09(s,1H),8.35(d,J＝8.9Hz,2H),8.18(d,J＝8.9Hz, 2H),7.35(m,3H),7.07(d,J＝7.9Hz,1H),7.01-6.96(m,1H),6.94(d,J＝8.8Hz,1H),5.69(s, 2H),3.84(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ:165.94,157.27,154.94,148.82,147.49, 146.77,145.27,131.34,127.42,126.93,124.43,123.32,117.55,112.50,109.63,56.67.HRMS(ESI) calcd for C 20 H 16 FN 3 O 4 S[M+Na] + m/z 436.0846,found:436.0737。
5. Synthesis of Compounds 11a-11h,12a-12h
The specific structure and the synthetic route are as follows:
Figure BDA0003687982700000202
reaction reagents and conditions: (a) ammonia/methanol solution, toluene, 60 ℃,4h. (b) sulfamic acid, NaClO 2 Tetrahydrofuran, 0 ℃ -room temperature (c) orthoMethoxyaniline derivatives, EDCI, tetrahydrofuran, RT,5h. (d) 4-nitrothiophenol, triethylamine, n-butanol, 120 ℃ overnight.
Synthesis of intermediate 10:
Figure BDA0003687982700000211
4, 6-dichloro-5-pyrimidinecarbaldehyde (1050mg, 6mmol) was dissolved in toluene, 7mol/L ammonia/methanol solution (12 mmol) was added and placed in an oil bath at 60 ℃ for reaction for 4 hours, and no starting material remained as monitored by TLC. The reaction solution was concentrated under reduced pressure to give a crude product of 4-amino-6-chloropyrimidinecarboxaldehyde (800 mg). Sulfamic acid (593mg, 6mmol) and 4-amino-6-chloropyrimidine-5-acetaldehyde were weighed out and mixed into 15mL of tetrahydrofuran, and the mixture was stirred in an ice bath for 10 minutes. Sodium chlorite (900mg, 10.2mmol) was weighed, dissolved in 2mL of water, and slowly added dropwise to the reaction solution, after the dropwise addition was completed, the reaction solution was moved to room temperature for reaction, and the progress of the reaction was monitored by TLC. The treatment method comprises the following steps: the reaction mixture was concentrated under reduced pressure, extracted with dichloromethane (3X 25mL), the organic layers were combined, and the organic layer was washed with saturated brine 1 time and anhydrous Na 2 SO 4 Drying, concentration and purification of the crude product by column chromatography gave product 10(600mg) in about 67% yield.
Synthesis of Compounds 11 a-h:
intermediate 10(80mg, 0.46mmol) and the o-anisidine derivative were dissolved in 10mL of tetrahydrofuran, EDCI (98mg, 0.5mmol) was added to the reaction solution, and the reaction was stirred at room temperature for 5 hours. Concentrating under reduced pressure to remove tetrahydrofuran solvent, separating with ethyl acetate and water, mixing the obtained organic layers, washing the organic layer with saturated salt water for 1 time, and collecting anhydrous Na 2 SO 4 Drying, concentrating, and separating and purifying the crude product by column chromatography to obtain the product 11 a-h. .
Synthesis of compound 12 a:
Figure BDA0003687982700000212
compound 11a (65mg, 0.25mmol) and 4-nitrothiophenol were weighed(41.8mg, 0.27mmol) was dissolved in 5mL of n-butanol, and triethylamine (50.5mg, 0.5mmol) was added and the mixture was put in an oil bath at 120 ℃ overnight for reaction. Concentrating the reaction solution under reduced pressure after the reaction is completed, separating with ethyl acetate and water, combining the obtained organic layers, washing the organic layer with saturated salt water for 1 time, and collecting anhydrous Na 2 SO 4 Drying, concentrating, and purifying the crude product by column chromatography to obtain the product 12a with a yield of 46% and a purity of 97%.
1 H-NMR(400MHz,CDCl 3 )δ:9.88(s,1H),8.23(s,1H),8.20(d,J＝2.5Hz,2H),7.99(m, 1H),7.74(d,J＝8.3Hz,2H),7.15(dd,J＝11.3,4.2Hz,1H),7.07(d,J＝7.5Hz,2H),6.96(t,J＝ 7.5Hz,1H),3.81(s,3H). 13 C-NMR(101MHz,DMSO-d 6 )δ160.46,157.79,147.31,140.52, 134.27,127.14,125.88,124.17,123.83,120.70,113.62,112.00,56.30.HRMS(ESI)calcd for C 18 H 15 N 5 O 4 S[M+H] + m/z 398.0845,found:398.0919。
Synthesis of compound 12 b:
Figure BDA0003687982700000221
synthesis of Compound 12b referring to 12a, the starting material, 2-methoxyaniline, was substituted for 3-fluoro-2-methoxyaniline, and the other treatments were identical, with a yield of 41% and a purity of 97%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.24(s,1H),8.25-8.21(d,J＝2.9Hz,2H),8.20(s,1H), 7.96(d,J＝7.6Hz,1H),7.75(m 2H),7.13(d,J＝7.5,Hz,2H),7.11-7.09(m,1H),3.90-3.81(s, 3H).13C NMR(101MHz,DMSO-d 6 )δ163.83,160.82,160.40,157.89,147.34,140.46,135.22, 127.27,124.20,123.95,119.44,113.74,49.07.HRMS(ESI)calcd for C 18 H 14 FN 5 O 4 S[M+H] + m/z 416.0751,found:416.0832。
Synthesis of compound 12 c:
Figure BDA0003687982700000222
synthesis of Compound 12c referring to 12a, the starting material, 2-methoxyaniline, was substituted for 4-fluoro-2-methoxyaniline, and the other treatments were identical, with a yield of 43% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.94(s,1H),8.24-8.19(d,J＝3.1Hz,2H),7.88(d,J＝5.3 Hz,2H),7.69(d,J＝8.0Hz,2H),7.53(m,1H),7.06(s,1H),7.01(dd,J＝10.9,2.6Hz,1H),6.79 (dd,J＝8.6,2.6Hz,1H),3.81(s,3H).HRMS(ESI)calcd for C 18 H 14 FN 5 O 4 S[M+H] + m/z 416.0751, found:416.0826。.
Synthesis of compound 12 d:
Figure BDA0003687982700000223
synthesis of Compound 12d referring to 12a, the starting material, 2-methoxyaniline, was substituted for 5-fluoro-2-methoxyaniline, and the other treatments were identical, with a yield of 39% and a purity of 97%.
1 H-NMR(400MHz,DMSO-d 6 )δ:8.44(d,J＝4.5Hz,1H),8.28-8.16(m,3H),8.03(d,J＝ 10.0Hz,1H),7.72(d,J＝8.1Hz,2H),7.60-7.46(m,1H),7.06(dd,J＝9.0,5.2Hz,1H),6.95(dd, J＝8.6,3.0Hz,1H),3.80(s,3H).HRMS(ESI)calcd for C 18 H 14 FN 5 O 4 S[M+H] + m/z 416.0751, found:416.0834。
Synthesis of compound 12 e:
Figure BDA0003687982700000231
synthesis of Compound 12e referring to 12a, the starting material, 2-methoxyaniline, was substituted for 5-chloro-2-methoxyaniline, and the other treatments were identical, with a yield of 38% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.07(s,1H),8.20(d,J＝7.0Hz,3H),7.73(d,J＝8.7Hz, 2H),7.19(dd,J＝8.8,2.6Hz,1H),7.14(s,1H),7.08(d,J＝8.8Hz,1H),3.82(s,3H). 13 C NMR (101MHz,DMSO)δ163.75,160.76,160.39,157.86,149.50,147.29,140.54,134.13,128.63, 124.88,124.19,122.55,113.74,113.28,56.69.HRMS(ESI)calcd for C 18 H 14 ClN 5 O 4 S[M+H] + m/z 432.0455,found:432.0534。
Synthesis of compound 12 f:
Figure BDA0003687982700000232
synthesis of Compound 12f referring to 12a, the starting material, 2-methoxyaniline, was substituted for 6-fluoro-2-methoxyaniline, and the other treatments were identical, with a yield of 46% and a purity of 96%.
1 H-NMR(400MHz,DMSO-d 6 )δ:10.02(s,1H),8.23(d,J＝9.0Hz,2H),8.19-7.94(m,1H), 7.74(d,J＝8.2Hz,2H),7.33(dd,J＝21.9,10.9Hz,1H),6.98(d,J＝8.3Hz,1H),6.91(t,J＝8.9 Hz,1H),3.98-3.81(s,3H).HRMS(ESI)calcd for C 18 H 14 FN 5 O 4 S[M+H] + m/z 416.0751,found: 416.0826。
Synthesis of Compound 12 g:
Figure BDA0003687982700000241
synthesis of 12g Compound with reference to 12a, the starting material, 2-methoxyaniline, was substituted for 3-methoxy-4 aminophenol, and the other treatments were identical, giving a yield of 47% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.68(s,1H),9.46(s,1H),8.26-8.15(d,J＝8.8Hz,3H), 7.75(d,J＝8.5Hz,2H),7.54(d,J＝12.6Hz,1H),7.09-6.83(m,2H),6.48(d,J＝1.8Hz,1H), 6.37(dd,J＝8.5,1.7Hz,1H),3.80-3.70(s,3H).13C NMR(101MHz,DMSO)δ160.49,157.71, 156.54,153.22,147.33,140.49,134.36,125.11,124.16(s),118.14,113.55,106.67,99.99(s),56.07. HRMS(ESI)calcd for C18H15N5O5S[M+H]+m/z 414.0794,found:414.0871.
Synthesis of compound 12 h:
Figure BDA0003687982700000242
synthesis of Compound 12h referring to 12a, the starting material, 2-methoxyaniline, was substituted for 4-methoxy-3-aminophenol, and the other treatments were identical, with a yield of 51% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.75(s,1H),9.02(s,1H),8.19(d,J＝12.0Hz,3H),7.74(d, J＝8.7Hz,2H),7.62(s,1H),7.07(s,2H),6.87(d,J＝8.8Hz,1H),6.52(dd,J＝8.8,2.9Hz,1H), 3.73(s,3H). 13 C NMR(101MHz,DMSO)δ160.99,160.44,157.77 151.41,147.30,140.56,134.23, 127.94,127.27,125.04,124.16,113.71,113.21,111.47,110.66,57.05.HRMS(ESI)calcd for C 18 H 15 N 5 O 5 S[M+H] + m/z 414.0794,found:414.0873。
6. Synthesis of Compounds 13a-13c,14a-14c
The specific structure and the synthetic route are as follows:
Figure BDA0003687982700000243
reaction reagents and conditions: (a) o-methoxyaniline derivatives, EDCI, tetrahydrofuran, RT,5h (b) 4-nitrothiophenol, triethylamine, n-butanol, 120 ℃.
The compound 2-chloro-6-methylnicotinic acid (97mg, 0.3mmoL) and 4-amino-3-methoxyphenol were dissolved in 10mL of tetrahydrofuran, EDCI (65mg, 0.33mmoL) was added, and the reaction was stirred at room temperature for 5 hours. Concentrating under reduced pressure to remove tetrahydrofuran solvent, separating with ethyl acetate and water, mixing the obtained organic layers, washing the organic layer with saturated salt water for 1 time, and collecting anhydrous Na 2 SO 4 Drying, concentration and purification of the crude product by column chromatography gave the product 13a-13c (80mg) in 66% yield.
Synthesis of compound 14 a:
Figure BDA0003687982700000251
compound 13a (65mg, 0.25mmol) and 4-nitrothiophenol (41.8mg, 0.27mmol) were weighed out and dissolved in 5mL of n-butanol, and triethylamine (50.5mg, 0.5mmol) was added thereto and placed in an oil bath at 120 ℃ overnight for reaction. Concentrating under reduced pressure to remove reaction solution after reaction, separating with ethyl acetate and water, mixing the obtained organic layers, washing the organic layer with saturated salt water for 1 time, and collecting anhydrous Na 2 SO 4 Drying, concentrating, and purifying the crude product by column chromatography to obtain 14a with a yield of 47% and a purity of 99%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.63(s,1H),9.05(s,1H),8.19(d,J＝8.9Hz,2H),7.95(d, J＝7.8Hz,1H),7.65(d,J＝8.8Hz,2H),7.47(s,1H),7.28(d,J＝7.9Hz,1H),6.87(d,J＝9.6Hz, 1H),6.53(dd,J＝7.3,3.6Hz,1H),3.71(s,3H),2.39(s,3H).13C NMR(101MHz,DMSO)δ 165.05,160.46,153.19,151.29,146.77,144.01,143.24,137.37,132.93,130.25,127.70,124.10, 113.09,111.71,110.80,56.82,24.31.HRMS(ESI)calcd for C20H17N3O5S[M+H]+m/z 412.0889,found:412.0963。
Synthesis of compound 14 b:
Figure BDA0003687982700000252
compound 14b synthesis method referring to 14a, the starting material 4-methoxy-3-aminophenol was substituted for 5-fluoro-2-methoxyaniline, and the other treatments were identical, with a yield of 49% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.89(s,1H),8.18(d,J＝8.9Hz,2H),7.99(d,J＝7.8Hz, 1H),7.82(dd,J＝10.5,2.7Hz,1H),7.66(d,J＝8.9Hz,2H),7.30(d,J＝7.9Hz,1H),7.09(dd,J＝9.1,5.2Hz,1H),7.02-6.96(m,1H),3.81(s,3H),2.41(s,3H).13C NMR(101MHz,DMSO-d 6 )δ 165.45,160.64,154.92,153.28,147.26,146.80,143.09,137.59,132.94,129.94,128.19,124.11, 121.55,112.77,56.83,24.34.HRMS(ESI)calcd for C 20 H 16 FN 3 O 4 S[M+H] + m/z 414.0846,found: 414.0925。
Synthesis of compound 14 c:
Figure BDA0003687982700000261
synthesis of Compound 14c referring to 14a, the starting 4-methoxy-3-aminophenol was substituted for 5-chloro-2-methoxyaniline, and the other workup procedures were identical, with a yield of 47% and a purity of 98%.
1 H-NMR(400MHz,DMSO-d 6 )δ:9.91(s,1H),8.22-8.15(d,J＝8.9Hz,2H),7.98(d,J＝ 7.5Hz,1H),7.95(d,J＝1.9Hz,1H),7.71-7.63(d,J＝9.0Hz,2H),7.30(d,J＝7.9Hz,1H),7.21 (dd,J＝8.8,2.6Hz,1H),7.13-7.08(d,J＝8.9Hz,1H),3.81(s,3H),2.38(s,3H).13C NMR(101 MHz,DMSO-d 6 )δ165.47,160.65,153.34,149.98,146.80,143.07,137.59,132.95,129.82,128.37, 125.33,124.15,122.82,121.53,113.40,56.61,24.34.HRMS(ESI)calcd for C 20 H 16 ClN 3 O 4 S [M+H] + m/z 430.0550,found:430.0614。
EXAMPLE 2 in vitro anti-HIV-1 Activity Studies
1. HIV-1 infectivity titration
Virus is expressed by Johnson&The method described by Byington was modified for titration as follows: HIV-1 stock solution was added to a 96-well plate for 4-fold dilution, and 10 concentration gradients were set, 6 duplicate wells per concentration, and 6 control wells were set. 50 μ L (4X 10) of C8166 cells were added to each well 5 mL), final volume per well was 200 μ L. At 37 ℃ with 5% CO 2 The incubator is used for 2 days. Day 3 was supplemented with 100. mu.L of fresh RPMI-1640 complete medium, and HIV-1-induced Cytopathic effect (CPE) in each well was observed under an inverted microscope on day seven, as determined by whether syncytia (Syncytium) were formed in each well; according to Reed&TCID of Muench method calculation virus 50 (50％Tissue culture infection dose)。
2. Inhibition experiment of compound on experimental strain HIV-1IIIB cytopathic effect (CPE)
Mix 8X 10 5 mu.L/well of 50. mu.L/mL C8166 cells were inoculated into a solution containing 100. mu.L of the diluted drug96 well cell culture plates, then 50. mu.L of HIV-1 was added IIIB Or HIV-1 RF Adding 1300TCID to the supernatant 50 Per well, 3 replicate wells were set. Setting normal cell control hole, AZT and Z-1 as positive drug control, and placing at 37 deg.C and 5% CO 2 The incubator of (4) was cultured for 3 days, the formation of syncytia was counted under an inverted microscope (100X), and EC was calculated 50 。
3. Inhibition of HIV-1IIIB replication in H9 cells
2 x 10 to 5 Infection of NL4-3(MOI 0.03) with H9 cells/mL at 37 ℃ with 5% CO 2 Incubating in an incubator for 2 hours; cells were washed 2 times with PBS, resuspended in RPMI-1640 complete medium, and the cell number was adjusted to 2X 10 5 Per mL; the test compound was diluted 5-fold in equal ratio (initial final concentration 200. mu.g/mL, total 6 concentrations) in RPMI-1640 complete medium (containing 10% FBS) in 96-well culture plates in 3-fold-wells per dilution, 100. mu.L of the cells were inoculated into 96-well plates containing 100. mu.L of the compound at different concentrations, incubated overnight, and HIV-1 was added IIIB . Then taking HIV-1 IIIB Supernatant in 1500 TCID 50 Per well, infect for 2 hours, wash out virus and medicine, add the medicine of the dilution gradient again, AZT is the positive drug control. At 37 ℃ with 5% CO 2 The incubator of (1) was cultured for 3 days, supplemented with 100. mu.L of a medium containing the same drug concentration on day 4, and the supernatant was collected on day 7 and inactivated with 0.5% Triton-X100. ELISA method for detecting inhibition effect of compound on HIV-1 replication by adopting capture p24 antigen, EC 50 Concentration of compound at which 50% inhibition of p24 occurred.
4. Toxicity test of Compounds on C8166 cells
The test compounds were placed in 96-well culture plates and diluted 5-fold at equal ratios in RPMI-1640 complete medium (containing 10% FBS), set at 6 concentrations, each concentration set at 3 replicate wells, and 100. mu.L of medium was added per well. Blank control wells were also set. Add 100. mu.L of 4X 10 to each well 5 C8166 cells/mL. Standing at 37 deg.C for 5% CO 2 The incubator of (2) is cultured for 3 days, and the cytotoxicity of the cells is detected by adopting an MTT colorimetric method. OD value was measured with an ELx800 microplate reader, and the wavelength of measurement was set as595nm and a reference wavelength of 630 nm. Calculating to obtain CC 50 Value (50% cytoxic concentration).
5. Toxicity test of Compound on H9 cells
The prepared compound to be tested is placed on a 96-well culture plate and cultured by RPMI-1640 complete medium (containing 10% FBS), 5-fold equal-ratio dilution is carried out, 6 concentration gradients are set, each concentration is provided with 3 repeated wells, 100 mu L of medium is added into each well, and meanwhile, blank control wells are arranged. Add 100. mu.L of 2X 10 to each well 5 mL of H9 cells. At 37 ℃ with 5% CO 2 The culture box is cultured for 3 days, the culture medium containing the same drug concentration is supplemented by 100 mu L on the 4 th day, and the cytotoxicity is detected by adopting an MTT colorimetric method on the 7 th day. OD was measured with an ELx800 microplate reader, and the measured wavelength was 595nm and the reference wavelength was 630 nm. Calculating to obtain CC 50 The value is obtained.
6. Results of antiviral Activity of Compounds
According to the SFDA 'non-clinical pharmacodynamic research technical guide principle (2006)', the internationally universal test method for the anti-HIV activity of the medicine is adopted to respectively carry out CC of human immune cells C8166 and H9 50 And EC 50 The assay was performed with compounds having CC50, EC50 and Therapeutic Index (TI) for C8166 cells and H9 cells as shown in table 1:
CC of Compounds of Table 1 against C8166 cells and H9 cells 50 、EC 50 And Therapeutic Index (TI)
Figure BDA0003687982700000271
Figure BDA0003687982700000281
Figure BDA0003687982700000291
Note: CC (challenge collapsar) 50 ，EC 50 In units of (μ M); AZT: zidovudine; 3 TC: lamivudine; NA does notAnd (4) determining.
The structures of compounds Z and Z-1 are as follows:
Figure BDA0003687982700000292
7. study on structure-activity relationship
Z, Z-1 is taken as a reference compound, AZT and 3TC are taken as positive controls, the synthesized compound is respectively used for carrying out antiviral activity determination on human immune cells C8166 and H9, and the structure-activity relationship is comprehensively analyzed:
firstly, a substituent group of a ring A of a lead compound Z-1 is investigated, whether a substituent group and a bridging sulfur atom on the ring A are oxidized or not is investigated to influence the activity, the investigated substituent groups are respectively methoxy, methyl, hydroxyl, fluorine atom and bromine atom, the substitution positions are ortho-position, meta-position, para-position and polysubstitution, the synthesized compound has 2a-f, and the antiviral activity of the compound is analyzed. By changing the substitution position of Z-1 methoxy group, the activity of meta-substituted 2a decreases, while meta-hydroxy-substituted 2b is analyzed for its EC 50 The value was 0.38. mu.M, the activity was almost maintained; further analysis of the multiple substitution of the methoxy group, methyl group and halogen atom on the A ring gave compounds 2c, 2d, 2e and 2f, respectively, and found that the activity of 3, 4-dimethoxy-substituted 2c, 3, 5-dimethyl-substituted 2e and 2-methyl-3-bromo-substituted 2f on the A ring disappeared and the activity of 3, 4-difluoro-substituted 2d slightly decreased. Oxidizing the sulfur atoms of the compound 2 series into sulfone to obtain a compound 3a-j, wherein the antiviral activities of the methoxy meta-substituted 3a and the para-substituted 3b of the A ring disappear, and the activities of the hydroxyl meta-substituted 3d and the para-substituted 3c are reduced; the activity of 3e substituted by 3, 4-dimethoxy is reduced, and the activity of 3f substituted by 3, 5-dimethoxy is disappeared; 3g of 2-fluoro-4-chloro-substituted activity decreased; the activity of 2-methyl-3-bromo substitution for 3h disappeared; a decrease in 3, 5-dimethyl-substituted 3i activity; the 3j activity of the 3-hydroxy 4-methoxy substitution disappears, which initially suggests that the methoxy substitution at the ortho position of the A ring helps to maintain activity, and the meta hydroxy substitution does not reduce activity.
Next, the influence of the position of the hydroxyl group and other substituents on the activity is examined, the examined substituents are hydroxyl, carboxyl, methoxy and fluorine atoms, the substitution positions are 4-position and 5-position, the compounds 4a-e are synthesized, and the structure-activity relationship of the compounds is further analyzed. Comparing 4-hydroxy substituted 4a and 5-hydroxy substituted 4b, 4b was found to be 0.47. mu.M active, 50-fold that of 4 a; subsequent replacement of the 5-hydroxy group by a carboxy, methoxy, fluorine atom gave compounds 4c, 4d, 4e, respectively, which were found to be slightly less active than 4b, but all at a low micromolar level, in the order of activity 5-hydroxy substituted 4b > 5-carboxy substituted 4c > 5-fluorine atom substituted 4e > 5-methoxy substituted 4 d. Further, oxidation of the sulfur atom of the compound 4a-e to sulfone gave a compound 5a-e, and it was found that the activity of 5-hydroxy-substituted 5a was increased to 0.07. mu.M, the activity of 5-carboxy-p-substituted 5b, 4-methoxy and 5-methoxy-substituted 5c, 5d was slightly decreased, and the activity of 5-fluoro-substituted 5e was slightly decreased. From the above-mentioned influence of the position of the substituent on the A ring and the kind of the substituent on the activity, it is known that the substitution at the 5-position contributes to the enhancement of the activity, and the activity is the most preferable when the substitution at the 5-position is a hydroxyl group.
Through preliminary investigation, the methoxy group is required to be in the ortho position of a benzene ring, and the hydroxyl group is required to be in the 5 position at the same time, so that the activity of the compound is facilitated, so that in order to improve the water solubility of the compound 5a, a substitution reaction is further carried out at the hydroxyl position, a water-soluble group is introduced, and the activity of the compound is investigated at the same time, so that the compounds 6a-c and 7a are obtained. Analysis shows that the activity of 6a substituted by hydroxy ethanol, 6c substituted by hydroxy propanol and 6c substituted by morpholinylethyl is lost, and the activity of compound 7a obtained by oxidizing the sulfur atom of 6c is also lost, which indicates that the introduction of a water-soluble group on the 5-hydroxy group is not beneficial to the activity of the compound.
After the A ring substituent optimization is considered, the A ring and the B ring are further optimized from the perspective of the metabolite of Z-1. In the early stage, through an identification test on a Vif inhibitor Z-1 metabolite, the half-life of Z-1 is found to be short, meanwhile, one more hydroxyl group is arranged on a benzene ring of the metabolite, but the position of the hydroxyl group cannot be determined, so that the hydroxylation of the A-ring and B-ring blocking compounds is optimized. Firstly, introducing fluorine atoms on an A ring to block metabolic sites; in addition, subsequent antiviral activity studies were conducted by changing the B-ring benzene ring to a heterocyclic ring.
Compounds 9a-c were obtained by introducing a fluorine atom into ring a, and compared with 3-fluoro substituted 9a, 4-fluoro substituted 9b, 6-fluoro substituted 9c, it was found that the activities 9a, 9c were lost and the activity of 9b was slightly decreased; therefore, the method of blocking metabolic sites by fluorine atoms does not help to improve the activity. Next, the B-ring aniline ring was replaced with a pyrimidine ring and a pyridine ring, and the influence of the change in the substituent of the a ring on the activity of the compound was examined. Firstly, analyzing the compounds 12a-h with the B ring as the pyrimidine ring, wherein the bridged sulfur atom is not oxidized, the A ring still fixes the methoxyl group at the ortho position, the investigated substituent groups are hydrogen atom, fluorine atom, chlorine atom and hydroxyl, and the substitution positions are ortho, meta and para positions of the methoxyl group. The substituent of the A ring is 12a of hydrogen, 12b of 3-fluorine substitution, and the activity is reduced; the activities of 4-fluoro-substituted 12c, 5-fluoro-substituted 12d, 5-chloro-substituted 12e, 6-fluoro-substituted 12f, 4-hydroxy-substituted 12g, and 5-hydroxy-substituted 12h were almost disappeared. And analyzing the compounds 14a-c with pyridine ring as ring B, wherein the sulfur atom of the bridge bond is still not oxidized, the environment-friendly A has ortho-methoxy, and the substituent comprises hydroxyl, fluorine atom, chlorine atom and methoxy 5 position. The activity of 5-hydroxy substituted 14a, 5-fluoro substituted 14b and 5-chloro substituted 14c was found to be reduced compared to the Z-1 ratio. Indicating that the activity of the compound is reduced by the pyrimidine ring and the pyridine ring of the B ring.
In summary, the ortho position of the methoxy group on the A ring, while the para position of the hydroxy group on the methoxy group, contributes to the activity of the compound. Through a series of optimization, the compounds 2b, 4c and 5a are found to have better antiviral activity, wherein the compound 5a has the best treatment effect and the EC (effective dose) on H9 cells infected by HIV (human immunodeficiency virus) 50 Reaches 0.07 mu M, which is improved by 5 times compared with the control compound Z-1.
EXAMPLE 3 inhibition of HIV-1 Vif-induced degradation of APOBEC3G
1. Experimental methods
TREX-hVif-15 is derived from 293T cell line and stably expresses HIV-1Vif by a Tet On expression system. In the experiment, HIV-1Vif expression is induced by transfecting EYFP-A3G plasmid and adding DOX, and whether the drug has an inhibiting effect on Vif-induced A3G degradation is detected in the presence of the drug. Fluorescence was detected if the drug had a degrading effect on HIV-1Vif or a protecting effect on A3G, whereas no fluorescence was detected.
Inoculation of 2X 10 in 24-well plates 5 Pore TREX-hVif-15 cell, thinAfter about 60% of the cells were confluent, EYFP-N1-hA3G plasmid was transfected by Lipofectamine 2000, and after 6h, the solution was changed, and DOX (1. mu.g/ml) and various concentrations of drug were added to set up a negative control and a positive control. And (3) observing the fluorescence intensity under a fluorescence microscope after 48 hours, collecting cells, detecting the total amount of the cells expressing yellow fluorescent protein by using a flow cytometer, and analyzing the effect of the medicament on the degradation process of A3G induced by Vif.
2. Protective results of the Compounds on the degradation of A3G
The results of the fluorescence screening experiment mainly performed on compounds with the anti-HIV virus inhibitory activity of H9 cells below 10 μ M are shown in Table 2:
TABLE 2 degradation protection ratio of A3G by the compound
Figure BDA0003687982700000311
Figure BDA0003687982700000321
The compound can protect the degradation of A3G to a certain degree through the protection rate of the compound on A3G, wherein the compound with good antiviral activity has a certain protection effect on A3G.
EXAMPLE 4 binding Pattern analysis of Compound 5a
In order to better predict the binding pattern of compound 5a to VIf protein, the binding pattern was constructed manually and autonomously, and the dominant conformation was found by energy minimization, and the most stable conformation was further found by molecular dynamics simulation. The specific implementation method comprises the following steps: selecting a crystal complex 4N9F from a PDB library, keeping the binding mode of Z-1 and the crystal complex 4N9F unchanged, replacing Z-1 with 5a, adopting Build Fragment of discovery studio to construct a proper angle between 5a and protein by adjusting the optimal spatial position of the crystal complex, obtaining 100 possible conformations by rotating the included angle between the proteins, performing energy minimization MM2 on 100 conformations, calculating the energy change of the system, selecting the system with the minimum energy to perform molecular dynamics simulation, and selecting a stable conformation according to the RMSD value, wherein the stable conformation is shown in figure 1.
The docking results show that four hydrogen bond interactions are formed between the compound 5a and Vif, and the B ring of the compound 5a is embedded into a hydrophobic cavity formed by Tyr148, Ile155 and pro 157; the oxygen of the methoxyl group forms a hydrogen bond with Tyr148, and the hydrogen of the hydroxyl group forms a hydrogen bond with Cys 133; the amino group of the central B ring forms two hydrogen bond interactions with Gln136 and Tyr 148. Through comparison with the binding mode of the compound Z-1, the hydroxyl of the 5a A ring and Cys133 form hydrogen bonds to replace the hydrogen bonding action of the A ring methoxy and Cys133, and probably because the change of the hydrogen bonding action causes the compound 5a to have better antiviral activity than the Z-1.
Example 5 Western blotting experiment
1. Experimental methods
The plasmid pcDNA3.1-h A3G-HA is used for transfecting TREX-hVif-15 cells, liquid is changed once every 6 hours, DOX (0.1 mu g/ml) and different drug concentrations are added, and negative and positive control groups are set. Extracting total cell protein after 48 hours, and detecting the expression quantity of A3G-HA and Vif proteins by western blotting.
The protein is extracted by using the Biyuntian protein extraction kit, and the experimental method is carried out according to the instruction. The protein sample with the determined concentration was added to 5 × Loading Buffer. Boiling in boiling water for denaturation for 5min, cooling on ice, and loading.
2. Results of the experiment
To further verify whether compound 5a protects the A3G protein by antagonizing the Vif protein, we next performed a Western blot experiment (figure 2). As can be seen from the b-graph in fig. 2, compound 5a can antagonize Vif protein, the Vif protein level decreases in a concentration-dependent manner as the concentration of compound 5a increases, and the antagonism of Vif by compound 5a at a concentration of 50 μ M is almost equivalent to Z-1 (in fig. 2, the b-graph is a statistical graph of the a-graph). Meanwhile, we tested the protection of compounds 5a and Z-1 on protein A3G in the presence of both A3G and Vif, and it can be seen from the c diagram of FIG. 2 that the amount of protein A3G is increased with the increase of the concentration of compound 5a, and the protection efficiency of compound 5a on protein A3G is equivalent to that of compound Z-1 at the same concentration (50. mu.M). Therefore, Western experiments show that the compound 5a antagonizes Vif protein and has better A3G protecting effect than that of the control compound Z-1.
Example 65 a inhibition experiment of drug-resistant Strain replication in TZM-bl cells
1. Experimental methods
In 96-well cell culture plates, 2X 10 cells were used 5 Per mL of TZM-bl cells plated first, 100. mu.L of each well was added at 37 ℃ with 5% CO 2 The incubator of (1) was cultured overnight. Additional 100 μ L of compound containing different dilution concentrations and 100 μ L of virus dilution were added to each well: pNL4-3 gp41(36G)V38A,N42T (MOI＝0.03)、HIV-1 4755-5 (MOI＝0.06)、HIV-1 A17 (MOI＝0.03)、 HIV-1 L10R/M46I/L63P/V82T/I84V (MOI 0.03), pYU2G140S/Q148H (MOI 0.03), positive control wells and cell control wells containing no compound were placed at 37 ℃, 5% CO 2 And (5) culturing. After 48 hours, the culture supernatant was discarded, and the cells were washed 2 times with 200. mu.L of PBS per well, and then lysed for 30min at 4 ℃ by adding 100. mu.L of cell lysate per well. Transferring 80 μ L/well of cell lysate to a white 96-well plate, adding a luciferase substrate prepared in advance, adding 80 μ L/well, detecting relative fluorescence units (RLU) of each well by a FLEX Station 3 microplate reader, and calculating EC 50 。
2. Results of the experiment
To further verify the inhibitory effect of the compound on clinical drug-resistant strains, we also completed the inhibitory effect of compound 5a on various drug-resistant strains, and the inhibitory results are shown in table 3.
Table 3. summary of compound activity against resistant virus strains.
Figure BDA0003687982700000331
From the above results, it was found that Compound 5a inhibits the drug-resistant strain pNL4-3 against the fusogenic agent gp41(36G)V38A,N42T Nucleoside reverse transcriptase inhibitor drug-resistant strain HIV-1 4755-5 Integrase-inhibiting drug-resistant strain pYU2 G140S/Q148H Protease inhibitor-resistant strain HIV-1 L10R/M46I/L63P/V82T/I84V Has a good inhibiting effect on the surface of the steel plate,probably due to the different antiviral mechanisms of compound 5a with fusogenic enzyme inhibitors, nucleoside reverse transcriptase inhibitors, integrase inhibitors, protease inhibitors; meanwhile, the non-nucleoside reverse transcriptase inhibitor drug-resistant strain is also found to have certain resistance to the compound 5a, probably because the Vif inhibitor can also bind to a non-nucleoside active site of the reverse transcriptase while acting on Vif.
Note: the calculation formula in the embodiment is as follows:
drawing an inhibition rate response curve according to the experimental result, and according to Reed&The Muench method calculates the 50% Effective Concentration (EC) of the compound for inhibiting viruses 50 ) 50% inhibitory cell growth concentration (CC) 50 ) And Therapeutic Index (TI) against HIV-1 activity: TI ═ CC 50 /EC 50 。
1) Cell growth survival rate (%) (experimental well OD value/control well OD value × 100;
2) HIV-1 cytopathic inhibition (%) ═ 1-experimental well syncytia/control well syncytia) × 100;
3) HIV-1 replication inhibition (%) was 100- (experimental well OD value-blank well OD value)/(control well syncytia number-blank well OD value) × 100.

Claims (8)

1. A compound of formula I:
Figure FDA0003687982690000011
2. an isomer, pharmaceutically acceptable salt or hydrate of the compound of claim 1.
3. The isomer, pharmaceutically acceptable salt or hydrate according to claim 2, wherein said salt is hydrochloride, sulfate, phosphate or nitrate.
4. Use of a compound according to claim 1 or an isomer, a pharmaceutically acceptable salt or hydrate according to any one of claims 2 to 3 for the preparation of a Vif inhibitor.
5. Use of a compound according to claim 1 or an isomer, pharmaceutically acceptable salt or hydrate according to any one of claims 2 to 3 for the preparation of an anti-HIV medicament.
6. Use according to claim 5, characterized in that: the anti-HIV drug is an anti-HIV-1 type virus drug or an anti-HIV-2 type virus drug.
7. Use of the compound according to claim 1 or the isomer, pharmaceutically acceptable salt or hydrate according to any one of claims 2 to 3 for the preparation of an antitumor drug.
8. Use of the compound of claim 1 or the isomer, pharmaceutically acceptable salt or hydrate of any one of claims 2 to 3 for the preparation of an anti-hepatitis medicament.
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