CN112028836B

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

Info

Publication number: CN112028836B
Application number: CN202010940870.2A
Authority: CN
Inventors: 刘新泳; 姜向毅; 展鹏; 李敬; 黄伯世
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-09-09
Filing date: 2020-09-09
Publication date: 2021-12-07
Anticipated expiration: 2040-09-09
Also published as: CN112028836A

Abstract

The invention discloses a diaryl pyrimidine compound containing six-membered nitrogen heterocycle and a preparation method and application thereof. The compound has a structure shown in a general formula I. The invention also relates to a pharmaceutical composition containing the compound with the structure shown in the formula I. The activity screening experiment shows that the compound has good anti-HIV-1 activity, so the invention also provides the application of the compound in preparing anti-AIDS drugs.

Description

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

Technical Field

The invention relates to a derivative, a preparation method and application thereof, in particular to a diaryl pyrimidine derivative containing six-membered nitrogen heterocycle, a preparation method and application thereof, belonging to the technical field of medicines.

Background

Acquired Immune Deficiency Syndrome (AIDS) is a serious infectious disease that destroys the Human Immune system, mainly caused by Human Immunodeficiency Virus Type 1 (HIV-1). In the life cycle of HIV-1, Reverse Transcriptase (RT) is responsible for reverse transcription of single-stranded RNA carrying viral genetic information into double-stranded DNA, and is a key target for anti-AIDS drug design. Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs) are used as important components of HAART, have the advantages of high activity, strong selectivity, low toxicity and the like, and are always hot spots for research of anti-AIDS drugs.

Etravirine (ETravirine, ETR) and Rilpivirine (RPV) are the latest generation of HIV-1 marketed drugs, both of which belong to NNRTIs. However, the compound has low water solubility and oral bioavailability, and large oral dosage, and can easily cause serious toxic and side effects in clinical use. In addition, with their widespread use, a number of mutant strains against second-generation NNRTIs, such as E138A, E138K, Y181C, Y181I, and the like, have emerged clinically. Therefore, the research and development of new generation of high-efficiency anti-drug resistance NNRTIs is a hot spot field of the research of anti-AIDS drugs at present. Therefore, obtaining high-efficiency, broad-spectrum drug resistance and NNRTIs with good pharmacokinetic properties through reasonable structural modification is one of the important fields of current anti-AIDS drug research.

Disclosure of Invention

The invention provides a diaryl pyrimidine derivative containing six-membered nitrogen heterocycle and a preparation method thereof, and also provides a partial activity screening result and application of the compound.

The technical scheme of the invention is as follows:

mono-and hexa-nitrogen heterocycle-containing diaryl pyrimidine derivatives

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

wherein,

r is CN or CH ═ CHCN;

x is S, SO₂、CO、CH₂、CF₂、NCH₃Or NCHO.

Preferred derivatives of diarylpyrimidines containing six-membered nitrogen heterocycles according to the invention are one of the following:

preparation method of diaryl pyrimidine derivatives containing six-membered nitrogen heterocycle

The preparation method of the diaryl pyrimidine derivative containing the six-membered nitrogen heterocycle comprises the following steps: 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.

Application of triaryl pyrimidine derivative containing six-membered nitrogen heterocycle

The invention discloses an anti-HIV-1 activity screening result of a diaryl pyrimidine compound containing six-membered nitrogen heterocycle and the first application of the diaryl pyrimidine compound as an anti-HIV inhibitor. Experiments prove that the diaryl pyrimidine compound containing the hexa-nitrogen heterocycle can be used as non-nucleoside NNRTIs to prepare anti-HIV drugs. The invention also provides the application of the compound in the preparation of anti-HIV drugs.

anti-HIV-1 activity and toxicity test of target compound

Cell-level HIV-1 resistant wild strain III of diarylpyrimidine compounds containing six-membered nitrogen heterocycles synthesized according to the above method_BThe single mutants L100I, K103N, Y181C, Y188L, E138K, the double mutants K103N/Y181C (RES056) and F227L/V106A were screened for activity, and lamivudine (3TC), Nevirapine (NVP), Efavirenz (EFV), Etravirine (ETV) and zidovudine (AZT) were used as positive controls. Their anti-HIV-1 activity is shown in Table 1.

As can be seen from Table 1, the diarylpyrimidine compounds containing six-membered nitrogen heterocycles provided by the invention show good activity against HIV-1 wild strains, and the EC of the diarylpyrimidine compounds₅₀The range is between 0.028 and 0.0021 mu M, which is obviously superior to that of a control drug NVP (EC)₅₀0.15 μ M) and 3TC (EC)₅₀6.00 μ M). Among them, compound G2 (EC)₅₀0.0024 μ M) and G4 (EC)₅₀0.0021 muM) is particularly outstanding against HIV-1 wild strains, and is superior to all positive control drugs. In addition, the activity of the compound G4 on the single mutant strain K103N and Y181C is 0.0019 μ M and 0.0075 μ M respectively, which is better than that of the positive control ETR; the activities of Y181C on the single mutant L100I were 0.011. mu.M and 0.033. mu.M, respectively, comparable to the activity of ETR. And, compared to ETR (CC)₅₀>4.59 μ M) and EFV (CC)₅₀>6.34 μ M), all compounds showed lower cytotoxicity, CC thereof₅₀The range is 14.89-54.70 μ M. Therefore, the compounds have great research and development values and can be used for preparing candidate anti-HIV drugs for development.

From the results of the anti-HIV-1 experiment at the cellular level, the following structure-activity relationships can be obtained:

by comparing F1/F2, G1/G2, G3/G4 in pairs, R can be found₁The antiviral activity of the compound which is CH ═ CHCN is better than that of the compound which is R ═ CHCN₁A compound that is CN, but which also has increased cytotoxicity.

When R is₁At the same time, the antiviral activity sequence of the compound on HIV-1 wild strains and mutant strains is as follows: g4(X ═ SO)₂)≥G2(X＝SO)≥F2(X＝SO)；G3(X＝SO₂)≥G1(X＝SO) is equal to or more than F1(X is equal to SO). Cytotoxicity is regulated by the following rules: g4(X ═ SO)₂)≈G2(X＝SO)≥F2(X＝SO)；G3(X＝SO₂)≈G1(X＝SO)≥F1(X＝SO)。

Therefore, the diaryl pyrimidine compound containing the six-membered nitrogen heterocycle provided by the invention can be used as an HIV-1 inhibitor for preparing anti-AIDS drugs.

An anti-HIV-1 pharmaceutical composition contains the diaryl pyrimidine compound containing six-membered nitrogen heterocycle, pharmaceutically acceptable salt thereof and pharmaceutic adjuvants, and is prepared into medicines of different dosage forms.

Detailed Description

The following examples are given to aid in the understanding of the invention, but are not intended to limit the scope of the invention.

Example 1: preparation of 4- (4-nitrobenzyl) thiomorpholine (B)

4-Nitrobenzylbromide A (2.16g, 0.01mol) and thiomorpholine (1.03g,0.01mol) were dissolved in dichloromethane (20mL), and triethylamine (1.21g, 0.012mol) was added to the solution. The resulting mixture was stirred at room temperature until the reaction was complete as monitored by TLC. The reaction was quenched with 30mL of water, washed with saturated brine, and the organic layer was separated and dried over anhydrous Na₂SO₄Drying, filtering, and concentrating under reduced pressure. The residue was recrystallized from ethyl acetate/petroleum ether to give intermediate B. Pale yellow solid, yield 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)。

Example 2: preparation of 4- (thiomorpholinylmethyl) aniline (C)

Intermediate 4- (4-nitrobenzyl) thiomorpholine B (1.0g) was dissolved in 20ml of anhydrous ethanol, followed by addition of stannous chloride dihydrate (5.0 g). The reaction mixture was stirred at room temperature under nitrogen blanket until the reaction was complete as monitored by TLC. To the mixture was added 2mol/L NaOH to adjust the pH to 7. The resulting white solid was filtered and washed with ethyl acetate. Saturated sodium chloride solution and ethyl acetate were added to the filtrate. Using anhydrous Na₂SO₄The organic layer was dried, filtered, concentrated under reduced pressure, and dried under vacuum to obtain 4- (thiomorpholinylmethyl) aniline C as an intermediate. Used directly in the next step without further purification. Yellow solid, yield: 52.2 percent. ESI-MS M/z 208.95(M + H)⁺,241.03(M+H)⁺，C₁₁H₁₆N₂S(208.10)。

Example 3: preparation of 4- ((2-chloropyrimidin-4-yl) oxy) -3, 5-dimethylbenzonitrile (E1)

2, 4-dichloropyrimidine D (1.49g, 0.01mol) and potassium carbonate (1.66g, 0.012mol) were dissolved in dimethylformamide (20mL), and 4-hydroxy-3, 5-dimethylbenzonitrile (1.47g, 0.01mol) was added to the solution, and stirred at 50 ℃ for 4h until the reaction was completed. Ice water (200mL) was added and the mixture was extracted with ethyl acetate (3X 50 mL). The combined organic layer was washed with saturated brine, and anhydrous Na was used₂SO₄Dried, filtered and concentrated under reduced pressure and finally recrystallized using ethyl acetate and petroleum ether to afford the pure intermediate 4- ((2-chloropyrimidin-4-yl) oxy) -3, 5-dimethylbenzonitrile E1. White solid, yield 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)。

Example 4: (E) preparation of (E) 3- (4- ((2-chloropyrimidin-4-yl) oxy) -3, 5-dimethylphenyl) acrylonitrile (E2)

The preparation method is the same as the above, except that the raw material is changed into (E) -3, 5-dimethyl-4-hydroxy-benzene acrylonitrile. White solid, yield 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)。

Example 5: preparation of 3, 5-dimethyl-4- ((2- ((4- (thiomorpholinmethyl) phenyl) amino) pyrimidin-4-yl) oxy) benzonitrile (F1)

4- ((2-Chloropyrimidin-4-yl) oxy) -3, 5-dimethylbenzonitrile E1(0.78g, 3.01mmol), 4- (thiomorpholinylmethyl) aniline C (0.50g, 3.40mmol), palladium acetate (0.034g, 0.15mmol), 4, 5-bis-diphenylphosphine-9, 9-dimethylxanthene (0.086g,0.15mmol) and cesium carbonate (1.46g,4.51mmol) were dissolved in 20mL of 1, 4-dioxane, protected with nitrogen, reacted at 90 ℃ for 10 hours and the reaction completion monitored by TLC. Cool to room temperature, filter the mixture and concentrate the filtrate under reduced pressure. And further purifying by silica gel chromatography with methanol/dichloromethane as eluent to obtain the target compound 3, 5-dimethyl-4- ((2- ((4- (thiomorpholinmethyl) phenyl) amino) pyrimidin-4-yl) oxy) benzonitrile F1. White solid, yield: 43.7%, melting point 173-.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)。

Example 6: (E) preparation of (E) -3- (3, 5-dimethyl-4- ((2- ((4- (thiomorpholinmethyl) phenyl) amino) pyrimidin-4-yl) oxy) phenyl) acrylonitrile (F2)

The procedure was as above except that the starting material was changed to (E) -3- (4- ((2-chloropyrimidin-4-yl) oxy) -3, 5-dimethylphenyl) acrylonitrile E2. White solid, yield: 41.7%, melting point: 197 ℃ and 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)。

Example 7: preparation of 3, 5-dimethyl-4- ((2- ((4- ((1- (1-oxidothiomorpholino) methyl) phenyl) amino) pyrimidin-4-yl) oxy) benzonitrile (G1)

Compound 3, 5-dimethyl-4- ((2- ((4- (thiomorpholinmethyl) phenyl) amino) pyrimidin-4-yl) oxy) benzonitrile F1(0.15g,0.33mmol) was dissolved in 20mL of dichloromethane, followed by slow addition of 3-chloroperbenzoic acid (0.081g,0.33mmol, 70% purity). The resulting mixture was stirred at room temperature for 4 hours until it was passedTLC monitoring was complete. 20mL of saturated sodium bisulfite solution was added to the mixture and extracted with dichloromethane (3X 20 mL). The combined organic layers were washed with saturated brine and anhydrous Na₂ SO₄Drying, filtration and concentration under reduced pressure, and final recrystallization from ethyl acetate and petroleum ether to afford the pure target compound 3, 5-dimethyl-4- ((2- ((4- ((1- (1-oxidothiomorpholino) methyl) phenyl) amino) pyrimidin-4-yl) oxy) benzonitrile G1. as a yellow solid in 79.2% yield, melting point: 176-.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)。

Example 8: (E) preparation of (E) -3- (3, 5-dimethyl-4- ((2- ((4- ((1-oxidothiomorpholino) methyl) phenyl) amino) pyrimidin-4-yl) oxy) phenyl) acrylonitrile (G2)

The procedure was as above except that the starting material was changed to (E) -3- (3, 5-dimethyl-4- ((2- ((4- (thiomorpholinmethyl) phenyl) amino) pyrimidin-4-yl) oxy) phenyl) acrylonitrile F2. White solid, yield: 75.4%, melting point: 176 ℃ and 178 ℃.¹H NMR(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)。

Example 9: 4- ((2- ((4- (((1, 1-Oxothiomorpholino) methyl) phenyl) amino) pyrimidin-4-yl) oxy) -3, 5-dimethylbenzonitrile (G3)

The procedure is as in example 7, except that 2 equivalents of 3-chloroperbenzoic acid are used. White solid, yield: 68.6%, melting point: 117 ℃ and 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)。

Example 10: (E) -3- (4- ((2- ((4- (((1, 1-Oxothiomorpholino) methyl) phenyl) amino) pyrimidin-4-yl) oxy) -3, 5-dimethylphenyl) acrylonitrile

The procedure is as in example 8, except that 2 equivalents of 3-chloroperbenzoic acid are used. White colour (Bai)Color solid, yield: 71.2%, melting point: 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/z 489.89(M+H)⁺,C₂₆H₂₇N₅O₃S(489.18)。

Example 11: anti-HIV Activity test (MT-4 cell model)

See (R) Pauwels R, et al, J. Virol. methods.1988,20,309, Pannecouque C, et al, nat Protocols2008,3,427.

Interpretation of terms: MT-4 cells: human acute lymphoblastic leukemia cells; MTT assay: MTT is 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazole bromide; trade names are thiazole blue; DMSO, DMSO: dimethyl sulfoxide (DMSO).

Principle of testing

Because the HIV-infected MT-4 cells can be diseased within a certain period of time (5-7 days), a solution of a compound to be detected with a proper concentration is added into the suspension of the HIV-infected MT-4 cells, and after a period of culture (5-7 days), the activity of the MT-4 cells is measured by an MTT (methyl thiazolyl tetrazolium) analysis method, so that the concentration of a drug (EC) for protecting 50% of the cells from cytopathic diseases is obtained (the concentration of the drug is the same as that of the drug (EC)₅₀) The anti-HIV activity of the target compound can be obtained. Simultaneously obtaining the concentration (CC) of the target compound which can cause 50 percent of cells not infected by HIV to generate pathological changes₅₀). Principle of MTT assay: MTT, i.e., bromo-3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium nitrogen, binds to live intracellular succinate dehydrogenase and does not react with dead cells. At present, the MTT method is an enzyme analysis method for rapidly reflecting the cell activity.

Test materials and methods

(1) HIV-1 wild type strain III_BSingle mutants L100I, K103N, Y181C, Y188L, E138K and double mutants K103N/Y181C (RES056) and F227L/V106A: provided by the institute of microbiology and immunology, Rega institute of Leuven university, belgium.

(2) MT-4 cells: provided by the institute of microbiology and immunology, Rega institute of Leuven university, belgium.

(3) MTT: purchased from Sigma, usa.

(4) Sample treatment: the samples were dissolved in DMSO to give appropriate concentrations just before use and diluted 5-fold with double distilled water, 5 dilutions each.

(5) Positive control drug: lamivudine (3TC), Nevirapine (NVP), Efavirenz (EFV), Etravirine (ETV) and zidovudine (AZT).

(6) The test method comprises the following steps: diluting the sample, adding into suspension of HIV-infected MT-4 cell, measuring cell activity by MTT colorimetric method after a period of time, recording absorbance (A) value at 590nm in enzyme labeling instrument, and calculating EC₅₀、CC₅₀And an SI.

(7) MTT staining method: after adding the sample for incubation for a period of time, 20. mu.L of MTT solution (5mg/mL) was added to each well, incubation was continued for several hours, the staining solution was discarded, 150. mu.L of DMSO was added to each well, mixed well, and absorbance was recorded at 590nm in a microplate reader.

The specific operation is as follows: dissolving the compound in DMSO or water, diluting with phosphate buffer solution, and mixing 3 × 10 solutions⁵MT-4 cells were preincubated with 100. mu.L of compound solutions at various concentrations for 1h at 37 ℃. Then, 100. mu.L of a virus dilution of appropriate concentration was added to the mixture, and the cells were incubated at 37 ℃ for 1 h. After three washes, the cells were resuspended in culture medium with or without compound, respectively. Cells were then incubated at 5% CO₂Incubate at 37 ℃ for 7 more days in the environment and supplement the stock culture with culture medium with or without compound on the third day after infection. The procedure was repeated twice for each culture condition. Cytopathic effects on the virus were monitored daily with a reverse optical microscope. In general, this experimentThe virus dilutions used in (1) often develop cytopathic effects the fifth day after viral infection. The inhibitory concentration of the drug is that concentration at which the drug produces 50% inhibition of viral cytopathic effects while not being directly toxic to cells (EC)₅₀) And (4) showing. It is to be emphasized that, when the compound is poorly water soluble and requires DMSO to dissolve, the volume-specific concentration of DMSO relative to water is typically less than 10% (final DMSO concentration in MT-4 cell culture medium is less than 2%). Since DMSO can affect the antiviral activity of the test compound, the antiviral activity of a solution containing the same concentration of DMSO should also be run in parallel to the control blank. In addition, the final DMSO concentration (1/1000) was much lower than that required to affect HIV-1 replication in MT-4 cells.

In vitro anti-HIV-1 (III) of a target Compound_B) And HIV-1 clinical common double-mutation and single-mutation drug-resistant strain activity screening data are provided by the institute of microbiology and immunology of Rega research institute of Leuven university, Belgium, all activity data are measured by at least two independent and parallel experiments, and the results are shown in Table 1.

anti-HIV-1 (III) Compounds of Table 1_BAnd clinically common double-mutation and single-mutation drug-resistant strains) activity and cytotoxicity

Note:^a EC₅₀(ii) a concentration of a compound that protects 50% of MT-4 cells infected with HIV-1 from cytopathic effects;^b CC₅₀the concentration of the compound of interest that causes lesions in 50% of cells not infected with HIV.

Fourth, conclusion

As can be seen from Table 1, the diaryl pyrimidine derivatives containing six-membered nitrogen heterocycles provided by the invention are a series of non-nucleoside HIV-1 inhibitors with novel structures, and most compounds show excellent activity for inhibiting HIV-1 wild strains and mutant strains. Among them, compound G2 (EC)₅₀0.0024 μ M) and G4 (EC)₅₀0.0021 muM) is particularly outstanding against HIV-1 wild strains, and is superior to all positive control drugs. Compound G4 forThe activities of the single mutants K103N and Y181C were 0.0019. mu.M and 0.0075. mu.M, respectively, which are superior to the positive control ETR. And, compared to ETR (CC)₅₀>4.59 μ M) and EFV (CC)₅₀>6.34 μ M), all compounds showed lower cytotoxicity, CC thereof₅₀The range is 14.89-54.70 μ M. Therefore, the compounds have great research and development values and can be used for preparing candidate anti-HIV drugs for development.

Claims

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;

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.