CN112028836B - Diarylpyrimidine derivative containing six-membered nitrogen heterocycle and preparation method and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种含有六元氮杂环的二芳基嘧啶类化合物及其制备方法和应用。所述化合物具有通式I所示的结构。本发明还涉及含有式I结构化合物的药物组合物。活性筛选实验显示本发明化合物具有很好的抗HIV‑1活性，因此本发明还提供上述化合物在制备抗艾滋病药物中的应用。

The invention discloses a diarylpyrimidine compound containing a six-membered nitrogen heterocycle and a preparation method and application thereof. The compound has the structure shown in general formula I. The present invention also relates to pharmaceutical compositions containing compounds of formula I. Activity screening experiments show that the compound of the present invention has good anti-HIV-1 activity, so the present invention also provides the application of the above compound in the preparation of anti-AIDS drugs.

Description

Translated fromChinese

一种含有六元氮杂环的二芳基嘧啶类衍生物及其制备方法与 应用A kind of diarylpyrimidine derivatives containing six-membered nitrogen heterocycle and preparation method and application thereof

技术领域technical field

本发明涉及一种衍生物及其制备方法与应用，特别涉及一种含有六元氮杂环的二芳基嘧啶类衍生物及其制备方法与应用，属于医药技术领域。The invention relates to a derivative and a preparation method and application thereof, in particular to a diarylpyrimidine derivative containing a six-membered nitrogen heterocycle, a preparation method and application thereof, and belongs to the technical field of medicine.

背景技术Background technique

艾滋病(Acquired Immune Deficiency Syndrome,AIDS)是主要由人免疫缺陷病毒1型(Human Immunodeficiency Virus Type 1,HIV-1)引起的破坏人体免疫系统的严重传染性疾病。在HIV-1的生命周期中，逆转录酶(reverse transcriptase，RT)负责将携带病毒遗传信息的单链RNA逆转录成双链DNA，是抗艾滋病药物设计的关键靶标。非核苷类逆转录酶抑制剂(Non-nucleoside Reverse Transcriptase Inhibitors，NNRTIs)作为HAART的重要组成部分，具有活性高、选择性强、毒性低等优点，一直以来是抗艾滋病药物研究的热点。AIDS (Acquired Immune Deficiency Syndrome, AIDS) is a serious infectious disease mainly caused by Human Immunodeficiency Virus Type 1 (Human Immunodeficiency Virus Type 1, HIV-1) that destroys the human immune system. In the life cycle of HIV-1, reverse transcriptase (RT) is responsible for the reverse transcription of single-stranded RNA carrying viral genetic information into double-stranded DNA, which is a key target for anti-AIDS drug design. As an important part of HAART, Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs) have the advantages of high activity, strong selectivity and low toxicity.

依曲韦林(Etravirine，ETR)和利匹韦林(Rilpivirne，RPV)是最新一代HIV-1上市药物，二者均属于NNRTIs。但该类化合物水溶性和口服生物利用度很低，口服剂量大，在临床使用中容易引起了严重的毒副作用。此外，随着他们的广泛使用，目前在临床上已出现多种针对第二代NNRTIs的突变株，如E138A、E138K、Y181C以及Y181I等。因此，新一代高效抗耐药性NNRTIs的研发是目前抗艾滋病药物研究的热点领域。因此，通过合理的结构修饰得到高效、广谱抗耐药以及具有良好药代动力学性质的NNRTIs是当前抗艾滋病药物研究的重要领域之一。Etravirine (ETR) and Rilpivirne (RPV) are the latest generation of HIV-1 drugs on the market, both of which are NNRTIs. However, the water solubility and oral bioavailability of these compounds are very low, and the oral dose is large, which is likely to cause serious toxic and side effects in clinical use. In addition, with their widespread use, a variety of mutant strains targeting second-generation NNRTIs, such as E138A, E138K, Y181C, and Y181I, have appeared in clinical practice. Therefore, the development of a new generation of highly effective anti-drug-resistant NNRTIs is currently a hot field of anti-AIDS drug research. Therefore, obtaining NNRTIs with high efficiency, broad-spectrum anti-drug resistance and good pharmacokinetic properties through rational structural modification is one of the important fields of current anti-AIDS drug research.

发明内容SUMMARY OF THE INVENTION

本发明提供了一种含有六元氮杂环的二芳基嘧啶类衍生物及其制备方法，本发明还提供了上述化合物的部分活性筛选结果及其用途。The present invention provides a diarylpyrimidine derivative containing a six-membered nitrogen heterocycle and a preparation method thereof. The present invention also provides partial activity screening results of the above compounds and their uses.

本发明的技术方案如下：The technical scheme of the present invention is as follows:

一、含有六元氮杂环的二芳基嘧啶类衍生物1. Diarylpyrimidine derivatives containing six-membered nitrogen heterocycles

本发明的含有六元氮杂环的二芳基嘧啶类衍生物或其药学上可接受的盐，具有如下通式Ⅰ所示的结构：The six-membered nitrogen heterocycle-containing diarylpyrimidine derivatives or pharmaceutically acceptable salts thereof of the present invention have the structure shown in the following general formula I:

其中，in,

R为CN或CH＝CHCN；R is CN or CH=CHCN;

X为S、SO、SO₂、CO、CH₂、CF₂、NCH₃或NCHO。X is S, SO, SO2,_CO ,_CH2 ,_CF2 ,_NCH3 or NCHO.

根据本发明优选的，含有六元氮杂环的二芳基嘧啶类衍生物，是下列之一：Preferably according to the present invention, the diarylpyrimidine derivative containing six-membered nitrogen heterocycle is one of the following:

二、含有六元氮杂环的二芳基嘧啶类衍生物的制备方法2. Preparation method of diarylpyrimidine derivatives containing six-membered nitrogen heterocycle

含有六元氮杂环的二芳基嘧啶类衍生物的制备方法，步骤包括：首先，对硝基苄溴A与硫代吗啉在二氯甲烷中发生亲核取代反应得到化合物B，经氯化亚锡还原得到中间体C；以2,4-二氯嘧啶D为原料，在N,N-二甲基甲酰胺溶液中与3,5-二甲基-4-羟基苯腈或(E)-3,5-二甲基-4-羟基苯丙烯腈生成中间体E1或E2；中间体E1或E2与先前制备好的化合物C在醋酸钯催化下发生偶联反应得到目标化合物F1或F2；F1或F2被不同当量的间氯过氧苯甲酸氧化得到相应的终产物G1-4；The preparation method of diarylpyrimidine derivatives containing six-membered nitrogen heterocycle comprises the following steps: firstly, nucleophilic substitution reaction of p-nitrobenzyl bromide A and thiomorpholine in dichloromethane is carried out to obtain compound B; Reduction of stannous compound to obtain intermediate C; using 2,4-dichloropyrimidine D as raw material, in N,N-dimethylformamide solution with 3,5-dimethyl-4-hydroxybenzonitrile or (E )-3,5-dimethyl-4-hydroxyphenylacrylonitrile generates intermediate E1 or E2; intermediate E1 or E2 and previously prepared compound C undergo coupling reaction under the catalysis of palladium acetate to obtain target compound F1 or F2 ; F1 or F2 are oxidized by different equivalents of m-chloroperoxybenzoic acid to obtain the corresponding final product G1-4;

试剂及条件：(i)硫代吗啉，三乙胺，二氯甲烷，室温；(ii)氯化亚锡，无水乙醇，氮气保护，室温；(iii)3,5-二甲基-4-羟基苯腈或(E)-3,5-二甲基-4-羟基苯丙烯腈，N,N-二甲基甲酰胺，碳酸钾，50℃；(iv)醋酸钯，4,5-双二苯基膦-9,9-二甲基氧杂蒽，碳酸铯，氮气保护，1,4-环氧六烷，90℃；(V)1.0或2.0当量的间氯过氧苯甲酸，二氯甲烷，室温。Reagents and conditions: (i) thiomorpholine, triethylamine, dichloromethane, room temperature; (ii) stannous chloride, absolute ethanol, nitrogen protection, room temperature; (iii) 3,5-dimethyl- 4-Hydroxybenzonitrile or (E)-3,5-dimethyl-4-hydroxyphenylacrylonitrile, N,N-dimethylformamide, potassium carbonate, 50°C; (iv) palladium acetate, 4,5 -Bisdiphenylphosphine-9,9-dimethylxanthene, cesium carbonate, nitrogen protection, 1,4-epoxyhexaane, 90°C; (V) 1.0 or 2.0 equivalents of m-chloroperoxybenzoic acid , dichloromethane, room temperature.

三、含有六元氮杂环的二芳基嘧啶类衍生物的应用3. Application of diarylpyrimidine derivatives containing six-membered nitrogen heterocycles

本发明公开了含有六元氮杂环的二芳基嘧啶类化合物的抗HIV-1活性筛选结果及其作为抗艾滋病毒抑制剂的首次应用。通过实验证明本发明所提供含有六元氮杂环的二芳基嘧啶类化合物的可作为非核苷类NNRTIs用于制备抗艾滋病毒药物。本发明还提供上述化合物在制备抗艾滋病毒药物中的应用。The invention discloses the screening results of anti-HIV-1 activity of diarylpyrimidine compounds containing six-membered nitrogen heterocycles and their first application as anti-HIV inhibitors. It is proved by experiments that the diarylpyrimidine compounds containing six-membered nitrogen heterocycles provided by the present invention can be used as non-nucleoside NNRTIs for preparing anti-HIV drugs. The present invention also provides the application of the above-mentioned compounds in the preparation of anti-HIV drugs.

目标化合物的抗HIV-1的活性和毒性实验Anti-HIV-1 Activity and Toxicity Test of Target Compounds

对按照上述方法合成的含有六元氮杂环的二芳基嘧啶类化合物进行了细胞水平的抗HIV-1野生株III_B，单突变株L100I、K103N、Y181C、Y188L、E138K以及双突变株K103N/Y181C(RES056)以及F227L/V106A的活性筛选，以拉米夫定(3TC)、奈韦拉平(NVP)、依法韦伦(EFV)、依曲韦林(ETV)和齐多夫定(AZT)为阳性对照。它们的抗HIV-1活性如表1所示。Cell-level anti-HIV-1 wild strain III_B , single mutant strain L100I, K103N, Y181C, Y188L, E138K and double mutant strain K103N were carried out on the diarylpyrimidine compounds containing six-membered nitrogen heterocycle synthesized according to the above method Activity screening of /Y181C (RES056) and F227L/V106A, using lamivudine (3TC), nevirapine (NVP), efavirenz (EFV), etravirine (ETV) and zidovudine (AZT) as positive control. Their anti-HIV-1 activities are shown in Table 1.

由表1可以看出，本发明所提供的含有六元氮杂环的二芳基嘧啶类化合物表现出了良好的抗HIV-1野生株活性，其EC₅₀范围在0.028～0.0021μM之间，明显优于对照药物NVP(EC₅₀＝0.15μM)和3TC(EC₅₀＝6.00μM)。其中，化合物G2(EC₅₀＝0.0024μM)和G4(EC₅₀＝0.0021μM)的对HIV-1野生株活性尤为突出，优于所有的阳性对照药物。此外，化合物G4对于单突变株K103N以及Y181C的活性分别为0.0019μM和0.0075μM，优于阳性对照ETR；对单突变株L100I，Y181C的活性分别为0.011μM和0.033μM，与ETR的活性相当。并且，相较于ETR(CC₅₀>4.59μM)以及EFV(CC₅₀>6.34μM)，所有的化合物展示出了较低的细胞毒性，其CC₅₀范围在14.89～54.70μM之间。因此该类化合物具有非常大的研究与开发的价值，可作为制备抗HIV的候选药物加以利用开发。It can be seen from Table 1 that the diarylpyrimidine compounds containing six-membered nitrogen heterocycles provided by the present invention exhibit good anti-HIV-1 wild strain activity, and their EC₅₀ ranges from 0.028 to 0.0021 μM. Significantly better than the control drugs NVP (EC₅₀ =0.15 μM) and 3TC (EC₅₀ =6.00 μM). Among them, compounds G2 (EC₅₀ =0.0024 μM) and G4 (EC₅₀ =0.0021 μM) had particularly outstanding activities against HIV-1 wild strain, which were superior to all positive control drugs. In addition, the activity of compound G4 against single mutant strain K103N and Y181C was 0.0019 μM and 0.0075 μM, respectively, which was better than that of the positive control ETR; the activity of compound G4 against single mutant strain L100I, Y181C was 0.011 μM and 0.033 μM, respectively, which was comparable to the activity of ETR. And, compared to ETR (CC₅₀ >4.59 μM) and EFV (CC₅₀ >6.34 μM), all compounds exhibited lower cytotoxicity, with CC₅₀ ranging from 14.89 to 54.70 μM. Therefore, such compounds have great research and development value, and can be utilized and developed as candidate drugs for preparing anti-HIV.

从细胞水平抗HIV-1实验结果可得到以下构效关系：The following structure-activity relationship can be obtained from the results of anti-HIV-1 experiments at the cellular level:

通过成对对比F1/F2，G1/G2，G3/G4，可以发现R₁为CH＝CHCN的化合物，抗病毒活性要优于R₁为CN的化合物，但是其细胞毒性也有所增加。By comparing F1/F2, G1/G2, G3/G4 in pairs, it can be found that the compound whose R₁ is CH=CHCN has better antiviral activity than the compound whose R₁ is CN, but its cytotoxicity is also increased.

当R₁相同时，化合物对HIV-1野生株及突变株的抗病毒活性顺序为：G4(X＝SO₂)≥G2(X＝SO)≥F2(X＝SO)；G3(X＝SO₂)≥G1(X＝SO)≥F1(X＝SO)。细胞毒性则有以下规律：G4(X＝SO₂)≈G2(X＝SO)≥F2(X＝SO)；G3(X＝SO₂)≈G1(X＝SO)≥F1(X＝SO)。When R₁ is the same, the sequence of the antiviral activity of the compounds against HIV-1 wild strain and mutant strain is: G4(X=SO₂ )≥G2(X=SO)≥F2(X=SO); G3(X=SO)₂ )≥G1(X=SO)≥F1(X=SO). Cytotoxicity has the following rules: G4(X=SO₂ )≈G2(X=SO)≥F2(X=SO); G3(X=SO₂ )≈G1(X=SO)≥F1(X=SO) .

因此，本发明所提供的含有六元氮杂环的二芳基嘧啶类化合物可作为HIV-1抑制剂用于制备抗艾滋病药物。Therefore, the diarylpyrimidine compounds containing six-membered nitrogen heterocycles provided by the present invention can be used as HIV-1 inhibitors to prepare anti-AIDS drugs.

一种抗HIV-1的药物组合物，含有上述的含有六元氮杂环的二芳基嘧啶类化合物及其药学上可接受的盐与药用辅料，制成不同剂型的药物。An anti-HIV-1 pharmaceutical composition, comprising the above-mentioned diarylpyrimidine compound containing six-membered aza heterocycles, pharmaceutically acceptable salts and pharmaceutical excipients, to prepare medicines in different dosage forms.

具体实施方式Detailed ways

通过下述实施例有助于理解本发明，但是不能限制本发明的内容。The following examples help to understand the present invention, but do not limit the content of the present invention.

实施例1：4-(4-硝基苄基)硫吗啉(B)的制备Example 1: Preparation of 4-(4-nitrobenzyl)thiomorpholine (B)

将4-硝基苄基溴A(2.16g，0.01mol)和硫代吗啉(1.03g,0.01mol)溶于二氯甲烷(20mL)中，向该溶液中添加三乙胺(1.21g，0.012mol)。在室温下搅拌所得混合物，直到通过TLC监测反应完成。用30mL水淬灭反应，然后用饱和盐水洗涤，分离有机层，使用无水Na₂SO₄上干燥，过滤，减压浓缩。残渣用乙酸乙酯/石油醚重结晶得到中间体B。淡黄色固体，产率:92.1％。¹H NMR(400MHz,DMSO-d₆)δ8.19(d,J＝8.6Hz,2H,Ph-H),7.59(d,J＝8.6Hz,2H,Ph-H),3.64(s,2H,CH₂),2.67–2.60(m,8H,thiomorpholine-H)。ESI-MS:m/z 239.08(M+H)⁺,C₁₁H₁₄N₂O₂S(238.08)。4-Nitrobenzyl bromide A (2.16 g, 0.01 mol) and thiomorpholine (1.03 g, 0.01 mol) were dissolved in dichloromethane (20 mL), to this solution was added triethylamine (1.21 g, 0.012mol). The resulting mixture was stirred at room temperature until completion as monitored by TLC. The reaction was quenched with 30 mL of water, then washed with saturated brine, the organic layer was separated, dried_over anhydrous_Na2SO4 , filtered, and concentrated under reduced pressure. The residue was recrystallized from ethyl acetate/petroleum ether to give Intermediate B. Light yellow solid, yield: 92.1%.¹ H NMR (400 MHz, DMSO-d₆ ) δ 8.19 (d, J=8.6 Hz, 2H, Ph-H), 7.59 (d, J=8.6 Hz, 2H, Ph-H), 3.64 (s, 2H , CH₂ ), 2.67–2.60 (m, 8H, thiomorpholine-H). ESI-MS: m/z 239.08 (M+H)⁺ , C₁₁ H₁₄ N₂ O₂ S (238.08).

实施例2：4-(硫吗啉甲基)苯胺(C)的制备Example 2: Preparation of 4-(thiomorpholinemethyl)aniline (C)

将中间体4-(4-硝基苄基)硫吗啉B(1.0g)溶解于20ml无水乙醇中，然后添加二水合氯化亚锡(5.0g)。反应混合物在氮气保护下室温搅拌，直到通过TLC监测反应完成。向混合物中加入2mol/L NaOH以将pH值调节为7。将产生的白色固体过滤并用乙酸乙酯洗涤。滤液加入饱和氯化钠溶液和乙酸乙酯。使用无水Na₂SO₄干燥有机层，过滤，减压浓缩，真空干燥，最终得到中间体4-(硫吗啉甲基)苯胺C。直接在下一步中使用，无需进一步纯化。黄色固体，产率：52.2％。ESI-MS:m/z 208.95(M+H)⁺,241.03(M+H)⁺，C₁₁H₁₆N₂S(208.10)。The intermediate 4-(4-nitrobenzyl)thiomorpholine B (1.0 g) was dissolved in 20 ml of absolute ethanol and then stannous chloride dihydrate (5.0 g) was added. The reaction mixture was stirred at room temperature under nitrogen protection until completion as monitored by TLC. To the mixture was added 2 mol/L NaOH to adjust the pH to 7. The resulting white solid was filtered and washed with ethyl acetate. To the filtrate was added saturated sodium chloride solution and ethyl acetate. The organic layer was dried over anhydrous Na₂ SO₄ , filtered, concentrated under reduced pressure, and dried in vacuo to finally obtain the intermediate 4-(thiomorpholinemethyl)aniline C. Used directly in the next step without further purification. Yellow solid, yield: 52.2%. ESI-MS: m/z 208.95 (M+H)⁺ , 241.03 (M+H)⁺ , C₁₁ H₁₆ N₂ S (208.10).

实施例3：4-((2-氯嘧啶-4-基)氧基)-3,5-二甲基苯甲腈(E1)的制备Example 3: Preparation of 4-((2-chloropyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile (E1)

将2,4-二氯嘧啶D(1.49g，0.01mol)和碳酸钾(1.66g，0.012mol)溶于二甲基甲酰胺(20mL)中，向该溶液中加入4-羟基-3,5-二甲基苯甲腈(1.47g，0.01mol)，并在50℃下搅拌4h直到反应完成。加入冰水(200mL)，并用乙酸乙酯(3×50mL)萃取混合物。用饱和盐水洗涤合并的有机层，使用无水Na₂SO₄干燥，过滤并减压浓缩，最后使用乙酸乙酯和石油醚重结晶以提供纯中间体4-((2-氯嘧啶-4-基)氧基)-3,5-二甲基苯甲腈E1。白色固体,产率:88.4％。¹H NMR(400MHz,DMSO-d₆)δ8.70(d,J＝5.7Hz,1H,pyrimidine-H),7.75(s,2H,Ph-H),7.32(d,J＝5.7Hz,1H,pyrimidine-H),2.10(s,6H,Ph-CH₃×2)。ESI-MS:m/z 259.97(M+H)⁺,C₁₃H₁₀ClN₃O(259.05)。2,4-Dichloropyrimidine D (1.49 g, 0.01 mol) and potassium carbonate (1.66 g, 0.012 mol) were dissolved in dimethylformamide (20 mL) and to this solution was added 4-hydroxy-3,5 - Dimethylbenzonitrile (1.47 g, 0.01 mol) and stirred at 50°C for 4 h until the reaction was complete. Ice water (200 mL) was added and the mixture was extracted with ethyl acetate (3 x 50 mL). The combined organic layers were washed with saturated brine, dried over anhydrous Na₂ SO₄ , filtered and concentrated under reduced pressure, and finally recrystallized with ethyl acetate and petroleum ether to afford pure intermediate 4-((2-chloropyrimidine-4- group)oxy)-3,5-dimethylbenzonitrile E1. White solid, yield: 88.4%.¹ H NMR (400MHz, DMSO-d₆ ) δ 8.70 (d, J=5.7Hz, 1H, pyrimidine-H), 7.75 (s, 2H, Ph-H), 7.32 (d, J=5.7Hz, 1H) , pyrimidine-H), 2.10 (s, 6H, Ph-CH₃ ×2). ESI-MS: m/z 259.97 (M+H)⁺ , C₁₃ H₁₀ ClN₃ O (259.05).

实施例4：(E)-3-(4-((2-氯嘧啶-4-基)氧基)-3,5-二甲基苯基)丙烯腈(E2)的制备Example 4: Preparation of (E)-3-(4-((2-chloropyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile (E2)

制备方法同上，只不过把原料换为(E)-3,5-二甲基-4-羟基苯丙烯腈。白色固体,产率:84.7％。¹H NMR(400MHz,DMSO-d₆)δ8.67(d,J＝5.7Hz,1H,pyrimidine-H),7.62(d,J＝16.7Hz,1H,CH＝),7.52(s,2H,Ph-H),7.25(d,J＝5.7Hz,1H,pyrimidine-H),6.45(d,J＝16.7Hz,1H,CH＝),2.07(s,6H,Ph-CH₃×2)。ESI-MS:m/z 286.3(M+H)⁺,C₁₅H₁₂ClN₃O(285.07)。The preparation method is the same as above, except that the raw material is replaced with (E)-3,5-dimethyl-4-hydroxybenzeneacrylonitrile. White solid, yield: 84.7%.¹ H NMR(400MHz, DMSO-d₆ )δ8.67(d,J=5.7Hz,1H,pyrimidine-H),7.62(d,J=16.7Hz,1H,CH=),7.52(s,2H, Ph-H), 7.25 (d, J=5.7 Hz, 1H, pyrimidine-H), 6.45 (d, J=16.7 Hz, 1H, CH=), 2.07 (s, 6H, Ph-CH₃ ×2). ESI-MS: m/z 286.3 (M+H)⁺ , C₁₅ H₁₂ ClN₃ O (285.07).

实施例5：3,5-二甲基-4-((2-((4-(硫吗啉甲基)苯基)氨基)嘧啶-4-基)氧基)苯甲腈(F1)的制备Example 5: Preparation of 3,5-dimethyl-4-((2-((4-(thiomorpholinemethyl)phenyl)amino)pyrimidin-4-yl)oxy)benzonitrile (F1) preparation

将4-((2-氯嘧啶-4-基)氧基)-3,5-二甲基苯甲腈E1(0.78g，3.01mmol)，4-(硫吗啉甲基)苯胺C(0.50g，3.40mmol)，醋酸钯(0.034g，0.15mmol)，4,5-双二苯基膦-9,9-二甲基氧杂蒽(0.086g,0.15mmol)以及碳酸铯(1.46g,4.51mmol)溶于20mL 1,4-二氧六环中，氮气保护，90℃反应10小时，通过TLC监测反应完成。冷却至室温，过滤混合物并减压浓缩滤液。以甲醇/二氯甲烷为洗脱剂，经硅胶层析进一步纯化，得目标化合物3,5-二甲基-4-((2-((4-(硫吗啉甲基)苯基)氨基)嘧啶-4-基)氧基)苯甲腈F1。白色固体,产率：43.7％,熔点:173-175℃。¹H NMR(400MHz,DMSO-d₆)δ9.62(s,1H,NH),8.40(d,J＝5.4Hz,1H,pyrimidine-H),7.76(s,2H,Ph-H),7.33–7.16(m,2H,Ph-H),6.96(d,J＝7.2Hz,2H,Ph-H),6.57(d,J＝5.4Hz,1H,pyrimidine-H),3.38(s,2H,CH₂),2.60(s,8H,thiomorpholine-H),2.12(s,6H,Ph-CH₃×2)。¹³C NMR(101MHz,DMSO)δ168.31,161.01,160.08,154.05,139.32,133.23,133.03(Ph-C×4),129.13(Ph-C×2),119.09(Ph-C×2),118.75,108.85,97.62,62.61,54.76(thiomorpholine-C×2),27.63(thiomorpholine-C×2),16.29(Ph-CH₃×2)。ESI-MS:m/z 432.06(M+H)⁺,C₂₄H₂₅N₅OS(431.18)。4-((2-Chloropyrimidin-4-yl)oxy)-3,5-dimethylbenzonitrile E1 (0.78 g, 3.01 mmol), 4-(thiomorpholinemethyl)aniline C (0.50 g, 3.40 mmol), palladium acetate (0.034 g, 0.15 mmol), 4,5-bisdiphenylphosphine-9,9-dimethylxanthene (0.086 g, 0.15 mmol) and cesium carbonate (1.46 g, 4.51 mmol) was dissolved in 20 mL of 1,4-dioxane, under nitrogen protection, and reacted at 90° C. for 10 hours. The completion of the reaction was monitored by TLC. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated under reduced pressure. Further purification by silica gel chromatography with methanol/dichloromethane as eluent affords the target compound 3,5-dimethyl-4-((2-((4-(thiomorpholinemethyl)phenyl)amino ) pyrimidin-4-yl)oxy)benzonitrile F1. White solid, yield: 43.7%, melting point: 173-175°C.¹ H NMR (400MHz, DMSO-d₆ ) δ 9.62 (s, 1H, NH), 8.40 (d, J=5.4 Hz, 1H, pyrimidine-H), 7.76 (s, 2H, Ph-H), 7.33 –7.16(m,2H,Ph-H),6.96(d,J=7.2Hz,2H,Ph-H),6.57(d,J=5.4Hz,1H,pyrimidine-H),3.38(s,2H,_CH2 ), 2.60 (s, 8H, thiomorpholine-H), 2.12 (s, 6H, Ph-_CH3 x 2).¹³ C NMR(101MHz, DMSO)δ168.31,161.01,160.08,154.05,139.32,133.23,133.03(Ph-C×4),129.13(Ph-C×2),119.09(Ph-C×2),118.75,108.85 , 97.62, 62.61, 54.76 (thiomorpholine-C × 2), 27.63 (thiomorpholine-C × 2), 16.29 (Ph-CH₃ × 2). ESI-MS: m/z 432.06 (_M +H)⁺ ,_C24H25N5OS (_431.18 ).

实施例6：(E)-3-(3,5-二甲基-4-((2-((4-(硫吗啉甲基)苯基)氨基)嘧啶-4-基)氧基)苯基)丙烯腈(F2)得制备Example 6: (E)-3-(3,5-Dimethyl-4-((2-((4-(thiomorpholinomethyl)phenyl)amino)pyrimidin-4-yl)oxy) Phenyl) acrylonitrile (F2) was prepared

制备方法同上，只不过把原料换为(E)-3-(4-((2-氯嘧啶-4-基)氧基)-3,5-二甲基苯基)丙烯腈E2。白色固体,产率：41.7％,熔点：197-199℃。¹H NMR(400MHz,DMSO-d6)δ9.58(s,1H,NH),8.38(d,J＝5.5Hz,1H,pyrimidine-H),7.67(d,J＝16.7Hz,1H,CH＝),7.54(s,2H,Ph-H),7.35–7.22(m,2H,Ph-H),6.92(d,J＝7.7Hz,2H,Ph-H),6.51(d,J＝5.4Hz,1H,pyrimidine-H),6.48(d,J＝16.7Hz,1H,CH＝),3.36(s,2H,CH₂),2.58(s,8H,thiomorpholine-H),2.09(s,6H,Ph-CH₃×2)。¹³C NMR(101MHz,DMSO)δ168.69,160.76,160.16,152.36,150.53,139.41,131.80,131.02(Ph-C×2),129.17(Ph-C×2),128.67(Ph-C×2),119.35(Ph-C×2),118.70,97.61,96.71,62.69,54.76(thiomorpholine-C×2),27.70(thiomorpholine-C×2),16.57(Ph-CH₃×2)。ESI-MS:m/z 458.15(M+H)⁺,C₂₆H₂₇N₅OS(457.19)。The preparation method is the same as above, except that the raw material is replaced with (E)-3-(4-((2-chloropyrimidin-4-yl)oxy)-3,5-dimethylphenyl)acrylonitrile E2. White solid, yield: 41.7%, melting point: 197-199°C.¹ H NMR(400MHz, DMSO-d6)δ9.58(s,1H,NH),8.38(d,J=5.5Hz,1H,pyrimidine-H),7.67(d,J=16.7Hz,1H,CH= ), 7.54(s, 2H, Ph-H), 7.35–7.22(m, 2H, Ph-H), 6.92(d, J=7.7Hz, 2H, Ph-H), 6.51(d, J=5.4Hz) , 1H, pyrimidine-H), 6.48(d, J=16.7Hz, 1H, CH=), 3.36(s, 2H, CH₂ ), 2.58(s, 8H, thiomorpholine-H), 2.09(s, 6H, Ph-CH₃ ×2).¹³ C NMR(101MHz, DMSO)δ168.69,160.76,160.16,152.36,150.53,139.41,131.80,131.02(Ph-C×2),129.17(Ph-C×2),128.67(Ph-C×2),119.35 (Ph-C×2), 118.70, 97.61, 96.71, 62.69, 54.76 (thiomorpholine-C×2), 27.70 (thiomorpholine-C×2), 16.57 (Ph-CH₃ ×2). ESI-MS: m/z_458.15 (M+H)⁺ ,_C26H27N5OS (_457.19 ).

实施例7：3,5-二甲基-4-((2-((4-((1-(1-氧化硫代吗啉代)甲基)苯基)氨基)嘧啶-4-基)氧基)苄腈(G1)的制备Example 7: 3,5-Dimethyl-4-((2-((4-((1-(1-oxidothiomorpholino)methyl)phenyl)amino)pyrimidin-4-yl) Preparation of oxy)benzonitrile (G1)

将化合物3,5-二甲基-4-((2-((4-(硫吗啉甲基)苯基)氨基)嘧啶-4-基)氧基)苯甲腈F1(0.15g,0.33mmol)溶解于20mL二氯甲烷中，然后缓慢加入3-氯过苯甲酸(0.081g,0.33mmol,纯度为70％)。将所得混合物在室温搅拌4小时直至通过TLC监测完成。将20mL的饱和亚硫酸氢钠溶液加入到混合物中，并用二氯甲烷(3×20mL)萃取。合并的有机层用饱和食盐水洗涤，用无水Na₂ SO₄干燥，过滤并减压浓缩，最后使用乙酸乙酯和石油醚重结晶以提供纯的目标化合物3,5-二甲基-4-((2-((4-((1-(1-氧化硫代吗啉代)甲基)苯基)氨基)嘧啶-4-基)氧基)苄腈G1。黄色固体，产率：79.2％,熔点：176-178℃。¹H NMR(400MHz,DMSO-d₆)δ10.10(s,1H,NH),8.59(d,J＝6.8Hz,1H,pyrimidine-H),7.78(s,2H,Ph-H),7.26(d,J＝8.0Hz,2H,Ph-H),7.00(d,J＝8.0Hz,2H,Ph-H),6.72(d,J＝6.9Hz,1H,pyrimidine-H),3.57(s,2H,CH₂),3.11(s,4H,thiomorpholine-H),2.84(s,4H,thiomorpholine-H),2.14(s,6H,Ph-CH₃×2)。¹³C NMR(151MHz,DMSO)δ168.31,161.01,160.08,154.06,139.42,133.24,133.04(Ph-C×2),131.07(Ph-C×2),129.14(Ph-C×2),119.13(Ph-C×2),118.80,108.84,97.65,61.57,46.14(thiomorpholine-C×2),44.03(thiomorpholine-C×2),16.28(Ph-CH₃×2)。ESI-MS:m/z 447.99(M+H)⁺,C₂₄H₂₅N₅O₂S(447.17)。Compound 3,5-dimethyl-4-((2-((4-(thiomorpholinemethyl)phenyl)amino)pyrimidin-4-yl)oxy)benzonitrile F1 (0.15 g, 0.33 mmol) was dissolved in 20 mL of dichloromethane, then 3-chloroperbenzoic acid (0.081 g, 0.33 mmol, 70% purity) was added slowly. The resulting mixture was stirred at room temperature for 4 hours until completion as monitored by TLC. 20 mL of saturated sodium bisulfite solution was added to the mixture and extracted with dichloromethane (3 x 20 mL). The combined organic layers were washed with saturated brine, dried over anhydrous Na₂ SO₄ , filtered and concentrated under reduced pressure, and finally recrystallized using ethyl acetate and petroleum ether to provide pure target compound 3,5-dimethyl-4 -((2-((4-((1-(1-thiomorpholino)methyl)phenyl)amino)pyrimidin-4-yl)oxy)benzonitrile G1. Yellow solid, yield: 79.2%, melting point: 176-178° C.¹ H NMR (400 MHz, DMSO-d₆ ) δ 10.10 (s, 1H, NH), 8.59 (d, J=6.8 Hz, 1H, pyrimidine-H), 7.78 ( s,2H,Ph-H),7.26(d,J=8.0Hz,2H,Ph-H),7.00(d,J=8.0Hz,2H,Ph-H),6.72(d,J=6.9Hz, 1H,pyrimidine-H),3.57(s,2H,CH₂ ),3.11(s,4H,thiomorpholine-H),2.84(s,4H,thiomorpholine-H),2.14(s,6H,Ph-CH₃ × 2)^.13C NMR(151MHz,DMSO)δ168.31,161.01,160.08,154.06,139.42,133.24,133.04(Ph-C×2),131.07(Ph-C×2),129.14(Ph-C×2), 119.13(Ph-C×2),118.80,108.84,97.65,61.57,46.14(thiomorpholine-C×2),44.03(thiomorpholine-C×2),16.28(Ph-_CH3 ×2).ESI-MS:m /z 447.99 (M+H)⁺ , C₂₄ H₂₅ N₅ O₂ S (447.17).

实施例8：(E)-3-(3,5-二甲基-4-((2-((4-((1-氧化硫代吗啉代)甲基)苯基)氨基)嘧啶-4-基)氧基)苯基)丙烯腈(G2)的制备Example 8: (E)-3-(3,5-Dimethyl-4-((2-((4-((1-thiomorpholino)methyl)phenyl)amino)pyrimidine- Preparation of 4-yl)oxy)phenyl)acrylonitrile (G2)

制备方法同上，只不过把原料换为(E)-3-(3,5-二甲基-4-((2-((4-(硫吗啉甲基)苯基)氨基)嘧啶-4-基)氧基)苯基)丙烯腈F2。白色固体，产率：75.4％,熔点：176-178℃。¹HNMR(400MHz,DMSO-d₆)δ10.07(s,1H,NH),8.57(d,J＝6.6Hz,1H,pyrimidine-H),7.70(d,J＝16.7Hz,1H,CH＝),7.55(s,2H,Ph-H),7.31(d,J＝8.0Hz,2H,Ph-H),6.95(d,J＝8.0Hz,2H,Ph-H),6.66(d,J＝6.8Hz,1H,pyrimidine-H),6.50(d,J＝16.6Hz,1H,CH＝),3.54(s,2H,CH₂),3.10(s,4H,thiomorpholine-H),2.81(s,4H,thiomorpholine-H),2.11(s,6H,Ph-CH₃×2)。¹³C NMR(151MHz,DMSO)δ168.69,160.74,160.16,152.35,150.58,139.55,131.81,131.77,130.84(Ph-C×2),129.21(Ph-C×2),128.68(Ph-C×2),119.41(Ph-C×2),118.77,97.66,96.71,61.63,46.14(thiomorpholine-C×2),44.03(thiomorpholine-C×2),16.56(Ph-CH₃×2)。ESI-MS:m/z 474.18(M+H)⁺,C₂₆H₂₇N₅O₂S(473.19)。The preparation method is the same as above, except that the raw material is replaced with (E)-3-(3,5-dimethyl-4-((2-((4-(thiomorpholinemethyl)phenyl)amino)pyrimidine-4 -yl)oxy)phenyl)acrylonitrile F2. White solid, yield: 75.4%, melting point: 176-178°C.¹ HNMR (400MHz, DMSO-d₆ )δ10.07(s,1H,NH),8.57(d,J=6.6Hz,1H,pyrimidine-H),7.70(d,J=16.7Hz,1H,CH= ),7.55(s,2H,Ph-H),7.31(d,J＝8.0Hz,2H,Ph-H),6.95(d,J＝8.0Hz,2H,Ph-H),6.66(d,J =6.8Hz, 1H, pyrimidine-H), 6.50(d, J=16.6Hz, 1H, CH=), 3.54(s, 2H, CH₂ ), 3.10(s, 4H, thiomorpholine-H), 2.81(s , 4H, thiomorpholine-H), 2.11 (s, 6H, Ph-CH₃ ×2).¹³ C NMR (151MHz, DMSO) δ168.69, 160.74, 160.16, 152.35, 150.58, 139.55, 131.81, 131.77, 130.84(Ph-C×2), 129.21(Ph-C×2), 128.68(Ph-C×2) , 119.41(Ph-C×2), 118.77, 97.66, 96.71, 61.63, 46.14(thiomorpholine-C×2), 44.03(thiomorpholine-C×2), 16.56(Ph-CH₃ ×2). ESI-MS: m/z_474.18 (M₊ H)⁺ ,_C26H27N5O2S (_473.19 ).

实施例9：4-((2-((4-(((1,1-氧化硫代吗啉代)甲基)苯基)氨基)嘧啶-4-基)氧基)-3,5-二甲基苄腈(G3)Example 9: 4-((2-((4-(((1,1-thiomorpholino)methyl)phenyl)amino)pyrimidin-4-yl)oxy)-3,5- Dimethylbenzonitrile (G3)

制备方法同实施例7，只不过使用2个当量的3-氯过苯甲酸。白色固体，产率：68.6％,熔点：117-119℃。¹H NMR(600MHz,DMSO-d₆)δ9.62(s,1H,NH),8.40(d,J＝5.5Hz,1H,pyrimidine-H),7.75(s,2H,Ph-H),7.28(s,2H,Ph-H),7.00(d,J＝7.1Hz,2H,Ph-H),6.56(d,J＝5.4Hz,1H,pyrimidine-H),3.54(s,2H,CH₂),3.09(s,4H,thiomorpholine-H),2.82(s,4H,thiomorpholine-H),2.11(s,6H,Ph-CH₃×2)。¹³C NMR(151MHz,DMSO)δ168.31,161.02,160.06,154.05,139.54,133.24,133.05(Ph-C×2),130.91(Ph-C×2),129.09(Ph-C×2),119.14(Ph-C×2),118.85,108.85,97.70,59.76,50.81(thiomorpholine-C×2),50.43(thiomorpholine-C×2),16.28(Ph-CH₃×2)。ESI-MS:m/z 463.89(M+H)⁺,C₂₄H₂₅N₅O₃S(463.17)。The preparation method is the same as that of Example 7, except that 2 equivalents of 3-chloroperbenzoic acid are used. White solid, yield: 68.6%, melting point: 117-119°C.¹ H NMR (600MHz, DMSO-d₆ ) δ 9.62(s, 1H, NH), 8.40(d, J=5.5Hz, 1H, pyrimidine-H), 7.75(s, 2H, Ph-H), 7.28 (s, 2H, Ph-H), 7.00 (d, J=7.1Hz, 2H, Ph-H), 6.56 (d, J=5.4Hz, 1H, pyrimidine-H), 3.54 (s, 2H, CH₂ ), 3.09(s, 4H, thiomorpholine-H), 2.82(s, 4H, thiomorpholine-H), 2.11(s, 6H, Ph-CH₃ ×2).¹³ C NMR(151MHz,DMSO)δ168.31,161.02,160.06,154.05,139.54,133.24,133.05(Ph-C×2),130.91(Ph-C×2),129.09(Ph-C×2),119.14(Ph-C×2) -C × 2), 118.85, 108.85, 97.70, 59.76, 50.81 (thiomorpholine-C × 2), 50.43 (thiomorpholine-C × 2), 16.28 (Ph-CH₃ × 2). ESI-MS: m/z_463.89 (_M +H)⁺ ,_C24H25N5O3S (_463.17 ).

实施例10：(E)-3-(4-((2-((4-(((1,1-氧化硫代吗啉代)甲基)苯基)氨基)嘧啶-4-基)氧基)-3,5-二甲基苯基)丙烯腈Example 10: (E)-3-(4-((2-((4-(((1,1-oxythiomorpholino)methyl)phenyl)amino)pyrimidin-4-yl)oxy base)-3,5-dimethylphenyl)acrylonitrile

制备方法同实施例8，只不过使用2个当量的3-氯过苯甲酸。白色固体，产率：71.2％,熔点：102-104℃。¹H NMR(400MHz,DMSO-d₆)δ9.59(s,1H,NH),8.38(d,J＝5.5Hz,1H,pyrimidine-H),7.67(d,J＝16.6Hz,1H,CH＝),7.53(s,2H,Ph-H),7.34(d,J＝4.3Hz,2H,Ph-H),6.98(d,J＝7.4Hz,2H,Ph-H),6.51(d,J＝5.4Hz,1H,pyrimidine-H),6.48(d,J＝16.7Hz,1H,CH＝),3.53(s,2H,CH₂),3.09(s,4H,thiomorpholine-H),2.81(s,4H,thiomorpholine-H),2.10(s,6H,Ph-CH₃×2)。¹³C NMR(101MHz,DMSO)δ168.70,160.74,160.17,152.31,150.55,139.70,133.17,131.80,130.67(Ph-C×2),129.24(Ph-C×2),128.69(Ph-C×2),119.40(Ph-C×2),118.85,97.72,96.76,59.82,50.80(thiomorpholine-C×2),50.38(thiomorpholine-C×2),16.56(Ph-CH₃×2)。ESI-MS:m/z489.89(M+H)⁺,C₂₆H₂₇N₅O₃S(489.18)。The preparation method is the same as that of Example 8, except that 2 equivalents of 3-chloroperbenzoic acid are used. White solid, yield: 71.2%, melting point: 102-104°C.¹ H NMR (400MHz, DMSO-d₆ )δ9.59(s,1H,NH),8.38(d,J=5.5Hz,1H,pyrimidine-H),7.67(d,J=16.6Hz,1H,CH =), 7.53(s, 2H, Ph-H), 7.34(d, J=4.3Hz, 2H, Ph-H), 6.98(d, J=7.4Hz, 2H, Ph-H), 6.51(d, J=5.4Hz, 1H, pyrimidine-H), 6.48(d, J=16.7Hz, 1H, CH=), 3.53(s, 2H, CH₂ ), 3.09(s, 4H, thiomorpholine-H), 2.81( s, 4H, thiomorpholine-H), 2.10 (s, 6H, Ph-CH₃ ×2).¹³ C NMR(101MHz, DMSO)δ168.70, 160.74, 160.17, 152.31, 150.55, 139.70, 133.17, 131.80, 130.67(Ph-C×2), 129.24(Ph-C×2), 128.69(Ph-C×2) , 119.40(Ph-C×2), 118.85, 97.72, 96.76, 59.82, 50.80(thiomorpholine-C×2), 50.38(thiomorpholine-C×2), 16.56(Ph-CH₃ ×2). ESI-MS: m/z_489.89 (_M +H)⁺ ,_C26H27N5O3S (_489.18 ).

实施例11：抗HIV活性实验(MT-4细胞模型)Example 11: Anti-HIV activity assay (MT-4 cell model)

参见①Pauwels R,et al.J.Virol.Methods.1988,20,309.②Pannecouque C,etal.Nat Protocols2008,3,427.See ① Pauwels R, et al. J. Virol. Methods. 1988, 20, 309. ② Pannecouque C, et al. Nat Protocols 2008, 3, 427.

术语解释：MT-4细胞：人急性淋巴母细胞白血病细胞；MTT分析法：MTT即为3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐；商品名:噻唑蓝；DMSO：二甲基亚砜。Explanation of terms: MT-4 cells: human acute lymphoblastic leukemia cells; MTT assay: MTT is 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide ; Trade name: thiazole blue; DMSO: dimethyl sulfoxide.

测试原理Test principle

由于HIV感染的MT-4细胞在一定时间内(5-7天)会发生病变，因此向HIV感染的MT-4细胞悬浊液中加入适当浓度的待检测化合物溶液，经过一段时间(5-7天)的培养后，用MTT分析法测定MT-4细胞活力，得到保护50％细胞免于细胞病变的药物浓度(EC₅₀)即可得出目标化合物的抗HIV的活性。同时得到目标化合物使50％未感染HIV的细胞发生病变的浓度(CC₅₀)。MTT分析法原理：MTT即溴化-3-(4,5-二甲基-2-噻唑基)-2,5-二苯基四唑氮,可与活的细胞内琥珀酸脱氢酶相结合，而不与死亡细胞发生反应。目前MTT法是一种快捷反映细胞活力的酶分析方法。Since HIV-infected MT-4 cells will develop lesions within a certain period of time (5-7 days), an appropriate concentration of the compound to be tested is added to the HIV-infected MT-4 cell suspension, and after a period of time (5-7 days) After 7 days) of culture, the MT-4 cell viability was measured by MTT assay, and the anti-HIV activity of the target compound was obtained by obtaining the drug concentration (EC₅₀ ) that protects 50% of the cells from cytopathic changes. At the same time, the concentration (CC₅₀ ) of the target compound that causes lesions of 50% of HIV-uninfected cells is obtained. Principle of MTT analysis method: MTT is bromide-3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium nitrogen, which can interact with live intracellular succinate dehydrogenase. binds without reacting with dying cells. At present, MTT method is a rapid enzymatic analysis method to reflect cell viability.

测试材料和方法Test Materials and Methods

(1)HIV-1野生株III_B，单突变株L100I、K103N、Y181C、Y188L、E138K以及双突变株K103N/Y181C(RES056)以及F227L/V106A：由比利时Leuven大学Rega研究院微生物与免疫学研究所提供。(1) HIV-1 wild strain III_B , single mutant strains L100I, K103N, Y181C, Y188L, E138K and double mutant strains K103N/Y181C (RES056) and F227L/V106A: by the Institute of Microbiology and Immunology, University of Leuven, Belgium Provided.

(2)MT-4细胞：由比利时Leuven大学Rega研究院微生物与免疫学研究所提供。(2) MT-4 cells: provided by the Institute of Microbiology and Immunology, Rega Institute, University of Leuven, Belgium.

(3)MTT：购自美国Sigma公司。(3) MTT: purchased from Sigma Company in the United States.

(4)样品处理：样品临用前溶于DMSO配成适当浓度，并用双蒸水作5倍稀释，各5个稀释度。(4) Sample treatment: The sample was dissolved in DMSO to make an appropriate concentration before use, and diluted 5 times with double distilled water, each with 5 dilutions.

(5)阳性对照药：拉米夫定(3TC)、奈韦拉平(NVP)、依法韦伦(EFV)、依曲韦林(ETV)和齐多夫定(AZT)。(5) Positive control drugs: lamivudine (3TC), nevirapine (NVP), efavirenz (EFV), etravirine (ETV) and zidovudine (AZT).

(6)测试方法：样品稀释后加入到HIV感染MT-4细胞悬浊液中，经过一段时间后用MTT比色法测定细胞活力，于酶标仪中，在590nm下记录吸光度(A)值，计算出EC₅₀、CC₅₀以及SI。(6) Test method: The sample was diluted and added to the suspension of HIV-infected MT-4 cells. After a period of time, the cell viability was measured by MTT colorimetry, and the absorbance (A) value was recorded at 590 nm in a microplate reader. , calculate EC₅₀ , CC₅₀ and SI.

(7)MTT染色法：加入样品培养一段时间后，再向每孔加入MTT溶液(5mg/mL)20μL，继续培养若干小时，弃染色液，并向每孔加入150μL的DMSO，充分混合，于酶标仪中，在590nm下记录吸光度。(7) MTT staining method: After adding the sample and culturing for a period of time, add 20 μL of MTT solution (5 mg/mL) to each well, continue to culture for several hours, discard the staining solution, and add 150 μL of DMSO to each well, mix well, and put In a microplate reader, record the absorbance at 590 nm.

具体操作如下：将化合物用DMSO或水溶解后用磷酸盐缓冲液稀释，将3×10⁵MT-4细胞与100μL不同浓度的化合物溶液在37℃共同预孵育1h。然后向该混合物中加入100μL适当浓度的病毒稀释液，将细胞于37℃孵育1h。洗涤三次后，将细胞再次分别悬浮于含有或不含化合物的培养基质中。接着将细胞在5％CO₂环境中，于37℃下再孵育7天，并于感染后的第三天用含有或不含化合物的培养基质补充原培养液。每种培养条件都重复操作两次。对病毒的细胞病变作用每天都用反向光学显微镜监控。一般来讲，本实验中所用的病毒稀释液常常会在病毒感染后第五天发生细胞病变。药物抑制浓度以药物对病毒致细胞病变作用产生50％抑制作用而同时对细胞无直接毒性的浓度(EC₅₀)表示。值得强调的是，当化合物水溶性较差，需要用DMSO才能溶解时，DMSO体积比浓度相对于水来讲，一般会低于10％(DMSO在MT-4细胞培养介质中最终浓度小于2％)。因为DMSO能影响测试化合物抗病毒活性，对含有相同浓度DMSO溶液的抗病毒活性对比空白实验也应该平行操作进行。另外，DMSO最终浓度(1/1000)远远低于影响HIV-1在MT-4细胞中复制所需浓度。The specific operations are as follows: the compounds are dissolved in DMSO or water and then diluted with phosphate buffer, and 3×10⁵ MT-4 cells are pre-incubated with 100 μL of compound solutions of different concentrations at 37° C. for 1 h. Then 100 μL of virus dilution solution of appropriate concentration was added to the mixture, and the cells were incubated at 37° C. for 1 h. After three washes, cells were resuspended in culture medium with or without compound, respectively. The cells were then incubated for an additional 7 days at 37°C in a 5%_CO2 environment, and the stock medium was supplemented with culture medium with or without compound on the third day after infection. Each culture condition was repeated twice. The cytopathic effect on the virus was monitored daily with reverse light microscopy. In general, the virus dilutions used in this experiment often developed cytopathic effects on the fifth day after virus infection. The inhibitory concentration of the drug was expressed as the concentration (EC₅₀ ) at which the drug produced 50% inhibition of the cytopathic effect of the virus without direct toxicity to cells. It is worth emphasizing that when the compound is poorly water-soluble and requires DMSO to dissolve, the volume concentration of DMSO relative to water is generally lower than 10% (the final concentration of DMSO in MT-4 cell culture medium is less than 2%). ). Because DMSO can affect the antiviral activity of the test compound, the antiviral activity comparison of the solution containing the same concentration of DMSO should also be performed in parallel. In addition, the final concentration of DMSO (1/1000) was far below the concentration required to affect HIV-1 replication in MT-4 cells.

目标化合物的体外抗HIV-1(III_B)及HIV-1临床常见双、单突变耐药株活性筛选数据由比利时Leuven大学Rega研究院微生物与免疫学研究所提供，所有的活性数据都经过至少两次独立、平行的实验测得，结果见表1。The in vitro anti-HIV-1(III_B ) and HIV-1 clinical common double and single mutation drug-resistant strains of the target compounds were provided by the Institute of Microbiology and Immunology, Rega Institute, University of Leuven, Belgium. All activity data were at least Two independent, parallel experiments were measured, and the results are shown in Table 1.

表1化合物抗HIV-1(III_B及临床常见双、单突变耐药株)活性和细胞毒性Table 1 Activity and cytotoxicity of compounds against HIV-1 (III_B and common clinical double and single mutation drug-resistant strains)

注：^a EC₅₀:保护50％感染HIV-1的MT-4细胞免于细胞病变的化合物浓度；^b CC₅₀:目标化合物使50％未感染HIV的细胞发生病变的浓度。Notes:^a_EC50 : concentration of compound that protects 50% of HIV-1 infected MT-4 cells from cytopathic;^b CC50: concentration of target compound that causes₅₀ % of HIV-uninfected cells to become cytopathic.

四、结论4. Conclusion

由表1可以看出，本发明所提供的含有六元氮杂环的二芳基嘧啶类衍生物是一系列结构新颖的非核苷类HIV-1抑制剂，绝大部分化合物表现出优秀的抑制HIV-1野生株和突变株的活性。其中，化合物G2(EC₅₀＝0.0024μM)和G4(EC₅₀＝0.0021μM)的对HIV-1野生株活性尤为突出，优于所有的阳性对照药物。化合物G4对于单突变株K103N以及Y181C的活性分别为0.0019μM和0.0075μM，优于阳性对照ETR。并且，相较于ETR(CC₅₀>4.59μM)以及EFV(CC₅₀>6.34μM)，所有的化合物展示出了较低的细胞毒性，其CC₅₀范围在14.89～54.70μM之间。因此该类化合物具有非常大的研究与开发的价值，可作为制备抗HIV的候选药物加以利用开发。As can be seen from Table 1, the diarylpyrimidine derivatives containing six-membered nitrogen heterocycles provided by the present invention are a series of novel non-nucleoside HIV-1 inhibitors, and most of the compounds show excellent inhibition. Activity of HIV-1 wild and mutant strains. Among them, compounds G2 (EC₅₀ =0.0024 μM) and G4 (EC₅₀ =0.0021 μM) had particularly outstanding activities against HIV-1 wild strain, which were superior to all positive control drugs. The activity of compound G4 against single mutant strain K103N and Y181C was 0.0019 μM and 0.0075 μM, respectively, which was better than that of the positive control ETR. And, compared to ETR (CC₅₀ >4.59 μM) and EFV (CC₅₀ >6.34 μM), all compounds exhibited lower cytotoxicity, with CC₅₀ ranging from 14.89 to 54.70 μM. Therefore, such compounds have great research and development value, and can be utilized and developed as candidate drugs for preparing anti-HIV.

Claims (5)

1. The diaryl pyrimidine derivative containing the six-membered nitrogen heterocycle or the pharmaceutically acceptable salt thereof has a structure shown as the following general formula I:

wherein,

r is CN or CH ═ CHCN;

x is SO or SO₂。

2. A six membered nitrogen heterocycle containing diarylpyrimidine derivatives as claimed in claim 1, wherein the pharmaceutically acceptable salt of said compound is sodium, hydrochloride, sulfate, tartrate or citrate.

3. A process for the preparation of six-membered nitrogen heterocycle containing diarylpyrimidine derivatives as claimed in claim 1, comprising the steps of:

firstly, carrying out nucleophilic substitution reaction on p-nitrobenzyl bromide A and thiomorpholine in dichloromethane to obtain a compound B, and reducing by stannous chloride to obtain an intermediate C; 2, 4-dichloropyrimidine D is taken as a raw material, and is reacted with 3, 5-dimethyl-4-hydroxybenzonitrile or (E) -3, 5-dimethyl-4-hydroxybenzeneacrylonitrile in an N, N-dimethylformamide solution to generate an intermediate E1 or E2; carrying out coupling reaction on the intermediate E1 or E2 and the previously prepared compound C under the catalysis of palladium acetate to obtain a target compound F1 or F2; f1 or F2 is oxidized by m-chloroperoxybenzoic acid with different equivalent weights to obtain a corresponding final product G1-4;

reagents and conditions: (i) thiomorpholine, triethylamine, dichloromethane, room temperature; (ii) stannous chloride, absolute ethyl alcohol and nitrogen protection at room temperature; (iii)3, 5-dimethyl-4-hydroxybenzonitrile or (E) -3, 5-dimethyl-4-hydroxybenzeneacrylonitrile, N-dimethylformamide, potassium carbonate, 50 ℃; (iv) palladium acetate, 4, 5-bis (diphenylphosphine) -9, 9-dimethyl xanthene, cesium carbonate, nitrogen protection, 1, 4-epoxy hexaalkane, 90 ℃; (V)1.0 or 2.0 equivalents of m-chloroperoxybenzoic acid, dichloromethane, room temperature.

4. Use of a biaryl pyrimidine derivative containing a six-membered nitrogen heterocycle as defined in claim 1 in the preparation of a medicament against HIV.

5. A pharmaceutical composition comprising a diarylpyrimidine derivative containing a six-membered nitrogen heterocycle according to claim 1 and one or more pharmaceutically acceptable carriers or excipients.