Accepted Article
Received Date : 09-Sep-2014 Revised Date
: 12-Oct-2014
Accepted Date : 17-Oct-2014 Article type
: Research Article
Design, Synthesis and Anti-HIV Evaluation of Novel Triazine Derivatives Targeting the Entrance Channel of the NNRTI Binding Pocket
Xuwang Chen,1 Qing Meng,1 Liyun Qiu,2 Peng Zhan,1 Huiqing Liu,3 Erik De Clercq,4 Christophe Pannecouque4 and Xinyong Liu 1,* 1
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road, 250012, Ji’nan, Shandong, P.R. China; 2
Department of Pharmacy, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013,
Shandong, P.R.China; 3
Institute of Pharmacology, School of Medicine, Shandong University, 44, West Culture Road, 250012,
Ji’nan, Shandong, P.R. China; 4
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
*
Corresponding author mail id: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12471 This article is protected by copyright. All rights reserved.
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Abstract A novel series of triazine derivatives targeting the entrance channel of the HIV-1 non-nucleoside reverse transcriptase inhibitor binding pocket (NNIBP) were designed and synthesized on the basis of our previous work. The results of a cell-based antiviral screening assay indicated that most compounds showed good to moderate activity against wild-type HIV-1 with EC50 values within the concentration range of 0.0078-0.16 µM (compound DCS-a4, EC50 = 7.8 nM). Some compounds displayed sub-micromolar activity against the K103N/Y181C resistant mutant strain (such as compound DCS-a4, EC50 = 0.65 µM). Molecular modeling studies confirmed that the new compounds could bind into the NNIBP similarly as the lead compound, and the newly introduced flexible heterocycles could occupy the entrance channel effectively. In addition, the preliminary structure-activity relationship and the RT inhibitory assay are presented in this paper.
Keywords: HIV-1, NNRTIs, Entrance channel, Antiviral activity, Molecular modeling
HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) have become an indispensable component of first-line drug regimens in the clinic with five drugs (nevirapine, delavirdine, efavirenz, etravirine and rilpivirine) approved by the US FDA for the treatment of AIDS. Especially, etravirine (TMC125) and rilpivirine (TMC278) which structurally belong to the diarylpyrimidine (DAPY) derivatives, display highly potent activity against wild-type and NNRTI-resistant mutant strains of HIV-1.[1, 2] Thus, the DAPY-type NNRTIs have been the research hotspot in structural modifications and optimizations during the past decade.[3]
X-ray crystallography and molecular modeling studies showed that the binding conformation of DAPYs resembled a prototypical horseshoe or “U” shape in the NNIBP, which contain three pharmacophoric groups: a hydrophobic center (A), hydrogen bond This article is protected by copyright. All rights reserved.
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donor/acceptor (B) and protein/solvent interface interaction domain (C) (Fig. 1).[4] The NH2 and Br groups on the 5,6-positions of the central pyrimidine ring of etravirine pointed to another open region, namely the entrance channel (D), which was surrounded by Lys101, Glu138 and Val179. However, the NH2 and Br groups did not display obvious interactions with the entrance channel probably because of the smaller volume.[4] Recently, novel HIV-1 NNRTIs targeting the entrance channel were reported by our research group[5] and Prof. W.L. Jorgensen’s group[6] with good antiviral activity against wild-type and NNRTI-resistant mutant strains of HIV-1, which further confirmed the rationale for design of the multi-sites binding NNRTIs to overcome the drug resistance based on the four pharmacophoric model (A-D).
In our previous studies, the multi-sites binding of piperidinylamino-diarylpyrimidine (pDAPY) derivatives was rationalized through a molecular hybridization strategy (Fig. 1).[5] Molecular modeling studies indicated that the pDAPY hybrid compounds could form hydrophobic interactions and hydrogen bond interactions with the non-nucleoside reverse transcriptase inhibitor binding pocket (NNIBP), and occupy the entrance channel simultaneously. Among the pDAPY derivatives, compound MD-c5 displayed the highest activity against wild-type and K103N/Y181C drug-resistant mutant strains of HIV-1 with EC50 values of 0.038 µM and 0.95 µM respectively. However, introduction of substituents (such as 4-SO2NH2-benzyl and 4-SO2CH3-benzyl) at the NH group of the piperidine ring decreased the antiviral activity in MT-4 cell cultures (Fig. 1) .[5]
Figure 1. The structures of the representative DAPY-type NNRTIs and pDAPYs.
To continue our research on these multi-sites binding NNRTIs targeting the entrance channel, a novel series of triazine derivatives was designed by combining the pharmacophoric groups of the drug etravirine and the pDAPY derivatives (Fig. 2).[7] This article is protected by copyright. All rights reserved.
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Moreover, on the basis of the above structure-activity relationship (SAR) analysis of pDAPY analogues and represented by the lead compound MD-c5, the N-benzyl piperidine moieties were modified and shortened using structural diverse substituents containing flexible heterocycles like morpholine, piperazine and piperidine. The newly designed compounds are expected to occupy the entrance channel more effectively and form multiple hydrogen bonds with the binding pocket of RT, in order to improve the activity against the drug-resistant mutant strains of HIV-1 (Fig. 2).
Figure 2. The design strategies of new triazine derivatives
Materials and Methods Chemistry All melting points were determined on a micro-melting point apparatus and are uncorrected. 1H-NMR (400MHz) and 13C-NMR (100MHz) spectra were obtained on a Brucker AV400 NMR-spectrometer in the indicated solvents. Infrared spectra (IR) were recorded with a Nexus 470FT-IR Spectrometer. Mass spectra were taken on a LC Autosampler Device: Standard G1313A instrument. TLC was performed on Silica Gel GF254 for TLC and spots were visualized by irradiation with UV light (254 nm). Flash column chromatography was performed on column packed with Silica Gel 60 (200-300 mesh). Solvents were reagent grade and, when necessary, were purified and dried by standard methods. The chemical materials were purchased from commercial suppliers and used without further purification.
Procedure for the synthesis of 2,4-dichloro-6-(mesityloxy)-1,3,5-triazine (S-2a) To an ice-bath cooled solution of cyanuric chloride S-1 (3.7 g, 20 mmol) in THF was added DIPEA (diisopropylethylamine, 3.9 g, 30 mmol). After that, a solution of This article is protected by copyright. All rights reserved.
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2,4,6-trimethylphenol (2.7 g, 20 mmol) in 20 mL THF was added dropwise to the above mixture and then stirred for 4 h at 0°C. After removal of THF under reduced pressure, water was added and the mixture was extracted with ethyl acetate. The combined organic phase was washed with brine and dried over anhydrous Na2SO4, filtered, and concentrated to give crude S-2a as a white solid without further purification.
Procedure for the synthesis of 4-(4,6-dichloro-1,3,5-triazin-2-yloxy)-3,5-dimethyl-benzo-nitrile (S-2b) Following the procedure in the preparation of S-2a, 4-hydroxy-3,5-dimethylbenzonitrile (2.9 g, 20 mmol) was reacted with cyanuric chloride S-1 (3.7 g, 20 mmol) in the presence of DIPEA (3.9 g, 30 mmol) to give crude S-2b as a white solid without further purification.
Procedure for the synthesis of 4-(4-chloro-6-(mesityloxy)-1,3,5-triazin-2-ylamino)-benzo-nitrile (S-3a) To a solution of S-2a (0.14 g) in 1,4-dioxane was added 4-amino-benzonitrile (60 mg, 0.5 mmol) and anhydrous K2CO3 (0.14 g, 1 mmol). The reaction mixture was stirred at 100°C for 6 h, and then dioxane was removed under reduced pressure. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. Purification on silica gel gave S-3a as a white solid. Yield: 83%, mp: 151-153°C; TLC Rf (EtOAc:petroleum ether 1:5) = 0.45; ESI-MS: m/z 366.4 [M+H]+.
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Procedure for the synthesis of 4-(4-chloro-6-(4-cyanophenylamino)-1,3,5-triazin-2-yloxy)3,5-dimethylbenzonitrile (S-3b) According to the procedure in the preparation of S-3a, the intermediate S-2b (0.15 g) was coupled with 4-amino-benzonitrile (60 mg, 0.5 mmol) in the presence of anhydrous K2CO3 (0.14 g, 1 mmol) to give S-3b as a white solid. Yield: 76%, mp: 262-264°C; TLC Rf (EtOAc:petroleum ether 2:5) = 0.45; ESI-MS: m/z 377.5 [M+H]+, 394.3 [M+NH4]+, 399.3 [M+Na]+.
Procedure for the synthesis of 4-(4-(mesityloxy)-6-(1-Boc-piperidin-4-ylamino)-1,3,5-triazin-2- ylamino)benzonitrile (S-4a) To a solution of S-3a (0.51 g, 1.4 mmol) and 4-amino-1-Boc-piperidine (0.4 g, 2 mmol) in dioxane was added anhydrous K2CO3 (0.28 g, 2 mmol) and water (5 mL). The mixture was stirred at 80°C for 8 h. After removal of the solvent under reduced pressure, water was added and the mixture was extracted with ethyl acetate. Combined organic layer was washed with saturated sodium chloride solution, and dried over anhydrous Na2SO4. Purification via flash column chromatography afforded intermediate S-4a as white solid. Yield: 77%, mp: 223-225°C; TLC Rf (EtOAc:petroleum ether 1:4) = 0.12; ESI-MS: m/z 530.3 [M+H]+, 552.5 [M+Na]+.
Procedure for the synthesis of 4-(4-(4-cyanophenylamino)-6-(1-methylpiperidin-4-ylamino)-1,3,5triazin-2-yloxy)-3,5-dimethylbenzonitrile (S-4b) Following the procedure for the preparation of S-4a, the intermediate S-3b (1.5 g, 4 mmol) was reacted with 4-amino-1-Boc-piperidine (1 g, 5 mmol) under the condition of anhydrous K2CO3 (0.69 g, 5 mmol) to give S-4b as a white solid. Yield: 80%, mp:
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215-217°C; TLC Rf (EtOAc:petroleum ether 2:5) = 0.14; ESI-MS: m/z 541.5 [M+H]+, 558.4 [M+NH4]+, 563.4 [M+Na]+.
Procedure for the synthesis of 4-(4-(mesityloxy)-6-(piperidin-4-ylamino)-1,3,5-triazin-2-ylamino) benzonitrile (DSC-a1) TFA (1.5 mL, 20 mmol) was added dropwise under stirring to a solution of intermediate S-4a (1.5 g, 2.8 mmol) in CH2Cl2 at room temperature and the mixture was stirred for 10 h. Water was added, and the mixture was neutralized with 2N NaOH (aq) to pH = 7. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous Na2SO4, and filtered. After evaporation of the solvent, the obtained crude product was recrystallized from EtOH to give compound DSC-a1. Yield: 93%, mp: 247-249°C; TLC Rf (MeOH:EtOAc 1:10, add 2 drops of Et3N) = 0.26; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.81-10.01 (s, 1H), 7.74-7.91 (m, 3H), 7.61 (t, 2H, J = 8.8 Hz), 6.91 (s, 2H, PhH), 3.61-3.80 (m, 1H), 2.90-2.97 (m, 2H), 2.35-2.45 (m, 1H), 2.26 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3), 1.71-1.79 (m, 2H), 1.31-1.36 (m, 2H); 13C-NMR (100M, DMSO-d6, ppm) δ: 170.09, 166.51, 165.84, 165.39, 147.55, 144.81, 134.50, 133.10, 130.10, 129.45, 119.86, 103.79, 48.90, 45.69, 33.19, 20.77, 16.53; IR (KBr, cm-1): 3424 (υNH), 3269 (υNH), 2923 (υasCH3), 2220 (υC≡ N),
1578, 1509 (υC=N), 1196 (υC-N), 810 (ωC=N); ESI-MS: m/z 430.7 [M+H]+.
Procedure for the synthesis of 4-(4-(4-cyanophenylamino)-6-(piperidin-4-ylamino)-1,3,5-triazin2-yloxy)-3,5-dimethylbenzonitrile (DSC-b1) Following the procedure for the preparation of DSC-a1, the reaction of S-4b (2.7 g, 5 mmol) with TFA (2.7 mL, 36 mmol) afforded DSC-b1 as a white solid. Yield: 91%, mp: 166-168°C; TLC Rf (MeOH:EtOAc 1:5, add 2 drops of Et3N) = 0.14; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.94-10.17 (s, 1H), 8.60 (brs, 2H), 8.06 (d, 1H, J = 7.1 Hz), 7.83 (brs, 2H), 7.69 (s, 2H, PhH), 7.64 (d, 1H, J = 8.3 Hz), 4.33 (d, 1H, J = 4.5 Hz), 3.85-4.01 (m, 1H), This article is protected by copyright. All rights reserved.
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3.41-3.48 (m, 1H), 2.90-3.05 (m, 2H), 2.14 (s, 6H, 2 × CH3), 1.95-2.07 (m, 2H), 1.65-1.70 (m, 2H); IR (KBr, cm-1): 3424 (υNH), 2926 (υasCH3), 2226 (υC≡N), 1583, 1512 (υC=N), 1202 (υC-N), 811 (ωC=N); ESI-MS: m/z 441.6 [M+H]+.
Procedure for the synthesis of title compounds DSC-a2~a3 and DSC-b2~b3 Intermediate DSC-a1 (or DSC-b1, 0.5 mmol) was dissolved in anhydrous DMF in the presence of anhydrous K2CO3 (0.14 g, 1 mmol), followed by addition of appropriate substituted benzyl chloride (or 4-picolyl chloride hydrochloride) (0.5 mmol). The reaction mixture was stirred at room temperature for 10 h. The solvent was removed under reduced pressure, and water was added. After extraction with ethyl acetate, the organic phase was washed with saturated sodium chloride solution and dried over anhydrous Na2SO4. The product was further purified by flash column chromatography and then recrystallized in the mixture of ethyl acetate and petroleum ether to afford title compounds DSC-a2~a3 and DSC-b2~b3.
4-(4-(mesityloxy)-6-(1-(4-(methylsulfonyl)benzyl)piperidin-4-ylamino)-1,3,5-triazin-2-yl amino)benzonitrile (DSC-a2) White solid; yield: 61%, mp: 266-268°C; TLC Rf (EtOAc) = 0.23; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.83-10.09 (s, 1H), 7.80-7.91 (m, 5H), 7.50-7.73 (m, 3H), 6.91 (s, 2H, PhH), 3.20 (s, 3H, CH3), 3.56-3.59 (m, 2H), 2.94 (s, 2H), 2.25 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3), 1.95-1.99 (m, 2H), 1.80-1.95 (m, 2H), 1.52-1.55 (m, 2H); IR (KBr, cm-1): 3427 (υNH), 2923 (υasCH3), 2223 (υC≡N), 1579, 1510 (υC=N), 1409 (υasO=S=O), 1197 (υC-N), 1149 (υO=S=O), 1090 (υS=O), 811 (ωC=N); ESI-MS: m/z 598.4 [M+H]+.
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4-(4-(mesityloxy)-6-(1-(pyridin-4-ylmethyl)piperidin-4-ylamino)-1,3,5-triazin-2-ylamino) -benzonitrile (DSC-a3) White solid; yield: 55%, mp: 265-267°C; TLC Rf (MeOH:EtOAc 1:10) = 0.20; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.83-10.03 (s, 1H), 8.51 (d, 2H, J = 5.5 Hz, PyH), 7.78-7.89 (m, 3H), 7.59-7.65 (m, 2H), 7.32 (d, 2H, J = 5.4 Hz, PyH), 6.91 (s, 2H, PhH), 3.78 (brs, 1H), 3.52 (s, 2H), 2.75-2.82 (m, 2H), 2.25 (s, 3H, CH3), 2.07-2.10 (m, 2H), 2.04 (s, 6H, 2 × CH3), 1.76-1.84 (m, 2H), 1.50-1.56 (m, 2H); IR (KBr, cm-1): 3420 (υNH), 3260 (υNH), 2922 (υasCH3), 2221 (υC≡N), 1579, 1510 (υC=N), 1195 (υC-N), 811 (ωC=N); ESI-MS: m/z 521.5 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(1-(4-(methylsulfonyl)benzyl)piperidin-4-ylamino)-1,3,5-tr iazin-2-yloxy)-3,5-dimethylbenzonitrile (DSC-b2) White solid; yield: 58%, mp: 252-254°C; TLC Rf (EtOAc) = 0.13; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.93-10.11 (s, 1H), 7.75-7.90 (m, 4H), 7.63-7.66 (m, 2H), 7.68 (s, 2H, PhH), 7.54-7.59 (m, 2H), 3.80 (brs, 1H), 3.52-3.59 (m, 3H), 3.20 (s, 3H, CH3), 2.74-2.83 (m, 2H), 2.13 (s, 6H, 2 × CH3), 1.93-1.97 (m, 2H), 1.82-1.85 (m, 2H), 1.49-1.57 (m, 2H); IR (KBr, cm-1): 3415 (υNH), 2925 (υasCH3), 2226 (υC≡N), 1583, 1511 (υC=N), 1397 (υasO=S=O), 1200 (υC-N), 1149 (υO=S=O), 1089 (υS=O), 810 (ωC=N); ESI-MS: m/z 609.3 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(1-(pyridin-4-ylmethyl)piperidin-4-ylamino)-1,3,5-triazin2-yloxy)-3,5-dimethylbenzonitrile (DSC-b3) White solid; yield: 51%, mp: 263-265°C; TLC Rf (MeOH:EtOAc 1:10) = 0.15; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.92-10.10 (s, 1H), 8.51 (d, 2H, J = 5.7 Hz, PyH), 7.81-7.89 (m, 3H), 7.68 (s, 2H, PhH), 7.64 (d, 2H, J = 8.3 Hz, PhH), 7.31 (d, 2H, J = 5.6 Hz, PyH), 3.76 (brs, 1H), 3.52 (s, 2H), 2.74-2.82 (m, 2H), 2.13 (s, 6H, 2 × CH3), 2.05-2.08 (m, 2H), 1.72-1.85 (m, 2H), 1.52-1.58 (m, 2H); IR (KBr, cm-1): 3427 (υNH), 2926 (υasCH3), 2225 (υC≡ N),
1583, 1512 (υC=N), 1201 (υC-N), 810 (ωC=N); ESI-MS: m/z 532.4 [M+H]+.
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Procedure for the synthesis of title compounds DSC-a4, DSC-a5, DSC-a7, DSC-b4, DSC-b5 and DSC-b7
Intermediate S-3a (or S-3b, 0.5 mmol) was dissolved in the mixture of dioxane and water, followed by addition of appropriate substituent (0.5 mmol) and K2CO3 (0.14 g, 1 mmol). The reaction mixture was stirred at 80°C for 10 h. The solvent was removed under reduced pressure, and water was added. After extraction with ethyl acetate, the organic phase was washed with saturated sodium chloride solution and dried over anhydrous Na2SO4. The product was further purified by flash column chromatography and then recrystallized in the mixture of ethyl acetate and petroleum ether to afford title compounds DSC-a4, DSC-a5, DSC-a7, DSC-b4, DSC-b5 and DSC-b7.
4-(4-(mesityloxy)-6-(2-morpholinoethylamino)-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a4) White solid; yield: 68%, mp: 215-217°C; TLC Rf (EtOAc) = 0.15; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.95-10.04 (s, 1H), 7.85-7.89 (m, 2H), 7.63 (d, 3H, J = 8.1 Hz), 6.92 (s, 2H, PhH), 3.56 (brs, 2H), 3.51 (brs, 2H), 3.41 (d, 1H, J = 6.1 Hz), 3.22 (d, 1H, J = 6.1 Hz), 2.46 (d, 1H, J = 6.0 Hz), 2.39 (s, 2H), 2.32 (s, 1H), 2.25 (s, 5H), 2.04 (s, 6H, 2 × CH3); 13
C-NMR (100M, DMSO-d6, ppm) δ: 170.31, 169.98, 167.45, 165.87, 147.50, 144.71,
134.46, 133.21, 130.07, 129.46, 119.94, 103.86, 66.64, 57.44, 53.76, 53.61, 20.78, 16.50; IR (KBr, cm-1): 3420 (υNH), 2920 (υasCH3), 2223 (υC≡N), 1578, 1511 (υC=N), 1197 (υC-N), 812 (ωC=N); ESI-MS: m/z 460.5 [M+H]+.
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4-(4-(mesityloxy)-6-(piperazin-1-yl)-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a5) White solid; yield: 60%, mp: 176-178°C; TLC Rf (MeOH:EtOAc 1:5) = 0.21; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.02 (s, 1H), 7.73 (brs, 2H), 7.60 (brs, 2H), 6.93 (s, 2H, PhH), 3.71 (brs, 2H), 3.58 (brs, 2H), 2.70-2.74 (5H), 2.27 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3424 (υNH), 2921 (υasCH3), 2223 (υC≡N), 1578, 1508 (υC=N), 1198 (υC-N), 809 (ωC=N); ESI-MS: m/z 416.5 [M+H]+.
4-(4-(mesityloxy)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a7) White solid; yield: 57%, mp: 223-225°C; TLC Rf (EtOAc) = 0.16; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.03 (s, 1H), 7.71 (brs, 2H), 7.60 (brs, 2H), 6.94 (s, 2H, PhH), 3.77 (brs, 2H), 3.65 (brs, 2H), 2,36-2.33 (m, 4H), 2.27 (s, 3H, CH3), 2.21 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3415 (υNH), 2920 (υasCH3), 2219 (υC≡N), 1575, 1505 (υC=N), 1196 (υC-N), 809 (ωC=N); ESI-MS: m/z 430.5 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(2-morpholinoethylamino)-1,3,5-triazin-2-yloxy)-3,5-dime thylbenzonitrile (DSC-b4) White solid; yield: 70%, mp:228-230°C; TLC Rf (EtOAc) = 0.09; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.04-10.12 (s, 1H), 7.72-7.88 (m, 3H), 7.69 (s, 2H, PhH), 7.65-7.68 (m, 2H), 3.56 (t, 2H, J = 4.4 Hz), 3.52 (t, 2H, J = 4.4 Hz), 3.42 (d, 1H, J = 6.4 Hz), 3.19 (d, 1H, J = 6.5 Hz), 2.46 (t, 1H, J = 6.8 Hz), 2.39 (s, 2H), 2.28 (t, 1H, J = 6.8 Hz), 2.22 (s, 2H), 2.14 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3427 (υNH), 3270 (υNH), 2922 (υasCH3), 2226 (υC≡N), 1585, 1509 (υC=N), 1201 (υC-N), 811 (ωC=N); ESI-MS: m/z 471.4 [M+H]+.
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4-(4-(4-cyanophenylamino)-6-(piperazin-1-yl)-1,3,5-triazin-2-yloxy)-3,5-dimethyl-benzo nitrile (DSC-b5) White solid; yield: 63%, mp: 274-276°C; TLC Rf (MeOH:EtOAc 1:5) = 0.15; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.09 (s, 1H), 7.70 (s, 2H, PhH), 7.64 (brs, 4H), 3.72 (brs, 2H), 3.55 (brs, 2H), 2.69-2.75 (5H), 2.14 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3423 (υNH), 2924 (υasCH3), 2226 (υC≡N), 1585, 1496 (υC=N), 1199 (υC-N), 807 (ωC=N); ESI-MS: m/z 427.5 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)-3,5-dimeth ylbenzonitrile (DSC-b7) White solid; yield: 62%, mp: 259-261°C; TLC Rf (MeOH:EtOAc 1:10) = 0.28; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.11 (s, 1H), 7.70 (s, 2H, PhH), 7.65-7.60 (m, 4H), 3.77 (brs, 2H), 3.62 (brs, 2H), 2,37-2.32 (m, 4H), 2.21 (s, 3H, CH3), 2.13 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3423 (υNH), 2928 (υasCH3), 2224 (υC≡N), 1579, 1509 (υC=N), 1203 (υC-N), 807 (ωC=N); ESI-MS: m/z 441.4 [M+H]+.
Procedure for the synthesis of title compounds DSC-a6 and DSC-b6 To a solution of intermediate S-3a (or S-3b, 0.5 mmol) and 2-morpholinoethanol (65 mg, 0.5 mmol) in anhydrous THF was added NaH (40 mg, 1 mmol). The reaction mixture was stirred at 65°C for 18 h. After removal of the solvent under reduced pressure, water was added and the mixture was extracted with ethyl acetate. Combined organic layer was washed with saturated sodium chloride solution, and dried over anhydrous Na2SO4. The product was further purified by flash column chromatography and then recrystallized in the mixture of ethyl acetate and petroleum ether to afford title compounds DSC-a6 and DSC-b6.
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4-(4-(mesityloxy)-6-(2-morpholinoethoxy)-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a6) White solid; yield: 52%, mp: 163-165°C; TLC Rf (EtOAc) = 0.28; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.58 (s, 1H), 7.68 (brs, 4H), 6.96 (s, 2H, PhH), 4.41 (t, 2H, J = 5.7 Hz), 3.54 (t, 4H, J = 4.5 Hz), 2.63 (t, 2H, J = 5.7 Hz), 2.40 (s, 4H), 2.28 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3420 (υNH), 3291 (υNH), 2919 (υasCH3), 2217 (υC≡N), 1583, 1507 (υC=N), 1197 (υC-N), 809 (ωC=N); ESI-MS: m/z 461.4 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(2-morpholinoethoxy)-1,3,5-triazin-2-yloxy)-3,5-dimethylb enzonitrile (DSC-b6) White solid; yield: 50%, mp: 149-151°C; TLC Rf (EtOAc) = 0.28; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.65 (brs, 1H), 7.73 (brs, 6H), 4.41 (t, 2H, J = 5.7 Hz), 3.54 (t, 4H, J = 4.5 Hz), 2.63 (t, 2H, J = 5.7 Hz), 2.39 (brs, 4H), 2.04 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3431 (υNH), 2923 (υasCH3), 2226 (υC≡N), 1578, 1509 (υC=N), 1200 (υC-N), 814 (ωC=N); ESI-MS: m/z 472.4 [M+H]+.
Procedure for the synthesis of title compounds DSC-a8 and DSC-b8 To a solution of intermediate S-3a (or S-3b, 0.6 mmol) and 1-methylpiperidin-4-amine (68 mg, 0.6 mmol) in dioxane was added anhydrous K2CO3 (0.17 g, 1.2 mmol). The reaction mixture was stirred at 100°C for 16 h. After removal of the solvent under reduced pressure, water was added and the mixture was extracted with ethyl acetate. Combined organic layer was washed with saturated sodium chloride solution, and dried over anhydrous Na2SO4. The product was further purified by flash column chromatography and then recrystallized in the mixture of ethyl acetate and petroleum ether to afford title compounds DSC-a8 and DSC-b8.
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4-(4-(mesityloxy)-6-(1-methylpiperidin-4-ylamino)-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a8) White solid; yield: 54%, mp: 146-148°C; TLC Rf (MeOH:EtOAc 1:5) = 0.21; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.02-9.82 (s, 1H), 7.92-7.85 (m, 2H), 7.75-7.73 (m, 1H), 7.65-7.59 (m, 2H), 6.91 (s, 2H, PhH), 3.71-3.51 (m, 1H), 2.79-2.71 (m, 2H), 2.25 (s, 3H, CH3), 2.18 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3), 1.98-1.92 (m, 2H), 1.83-1.77 (m, 2H), 1.56-1.47 (m, 2H); IR (KBr, cm-1): 3420 (υNH), 2940 (υasCH3), 2223 (υC≡N), 1578, 1510 (υC=N), 1198 (υC-N), 811 (ωC=N); ESI-MS: m/z 444.6 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(1-methylpiperidin-4-ylamino)-1,3,5-triazin-2-yloxy)-3,5-d imethylbenzonitrile (DSC-b8) White solid; yield: 57%, mp: 163-165°C; TLC Rf (MeOH:EtOAc 1:5) = 0.14; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.10-9.92 (s, 1H), 7.85-7.82 (m, 3H), 7.68 (s, 2H, PhH), 7.65-7.62 (m, 2H), 3.71-3.45 (m, 1H), 2.79-2.72 (m, 2H), 2.19 (s, 3H, CH3), 2.13 (s, 6H, 2 × CH3), 2.02-1.96 (m, 1H), 1.83-1.80 (m, 2H), 1.73-1.68 (m, 1H), 1.53-1.47 (m, 2H); IR (KBr, cm-1): 3423 (υNH), 2938 (υasCH3), 2225 (υC≡N), 1581, 1513 (υC=N), 1201 (υC-N), 810 (ωC=N); ESI-MS: m/z 455.4 [M+H]+.
Procedure for the synthesis of title compounds DSC-a9 and DSC-b9 Intermediate S-3a (or S-3b, 0.6 mmol) and morpholine (52 mg, 0.6 mmol) were dissolved in the mixture of dioxane and water, followed by addition of K2CO3 (0.17 g, 1.2 mmol). The reaction mixture was stirred at 100°C for 13 h. The solvent was removed under reduced pressure, and water was added. After extraction with ethyl acetate, the organic phase was washed with saturated sodium chloride solution and dried over anhydrous Na2SO4. The product was further purified by flash column chromatography and then recrystallized in the mixture of ethyl acetate and petroleum ether to afford title compounds DSC-a9 and DSC-b9.
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4-(4-(mesityloxy)-6-morpholino-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a9) White solid; yield: 59%, mp: 210-212°C; TLC Rf (EtOAc:petroleum ether 1:4) = 0.13; 1
H-NMR (400M, DMSO-d6, ppm) δ: 10.07 (s, 1H), 7.72 (brs, 2H), 7.60 (brs, 2H), 6.94 (s,
2H, PhH), 3.76 (brs, 2H), 3.64 (brs, 6H), 2.27 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3428 (υNH), 2959 (υasCH3), 2226 (υC≡N), 1578, 1509 (υC=N), 1200 (υC-N), 814 (ωC=N); ESI-MS: m/z 417.6 [M+H]+.
4-(4-(4-cyanophenylamino)-6-morpholino-1,3,5-triazin-2-yloxy)-3,5-dimethyl-benzonitri le (DSC-b9) White solid; yield: 65%, mp: 285-287°C; TLC Rf (EtOAc:petroleum ether 2:5) = 0.24; 1
H-NMR (400M, DMSO-d6, ppm) δ: 10.14 (s, 1H), 7.70 (s, 2H, PhH), 7.64 (brs, 4H),
3.77-3.61 (m, 6H), 2.14 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3423 (υNH), 2925 (υasCH3), 2225 (υC ≡N
), 1583, 1511 (υC=N), 1201 (υC-N), 807 (ωC=N); ESI-MS: m/z 428.6 [M+H]+, 445.9
[M+NH4]+, 450.6 [M+Na]+.
In vitro anti-HIV activity assays The anti-HIV activity and cytotoxicity of the compounds were evaluated against wild-type HIV-1 strain IIIB, a double RT mutant (K103N + Y181C) HIV-1 strain in MT-4 cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method as previously described.[1, 4, 8] Briefly, stock solutions (10 x final concentration) of test compounds were added in 25 µL volumes to two series of triplicate wells so as to allow simultaneous evaluation of their effects on mock-and HIV-infected cells at the beginning of each experiment. Serial 5-fold dilutions of test compounds (final 200 µL volume per well) were made directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot (Beckman instruments). Untreated control HIV-and mock-infected cell samples were included for each sample. HIV-1(IIIB)[9] stock (50 μL) at 100–300 CCID50 (cell culture infectious dose) or culture medium was added to either the infected or mock-infected wells of This article is protected by copyright. All rights reserved.
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the microtiter tray. Mock-infected cells were used to evaluate the effect of test compounds on uninfected cells in order to assess the cytotoxicity of the test compounds. Exponentially growing MT-4 cells were centrifuged for 5 min at 220 g and the supernatant was discarded. The MT-4 cells were resuspended at 6 x 105 cells/mL and 50 µL volumes were transferred to the microtiter tray wells. Five days after infection, the viability of mock-and HIV-infected cells was examined spectrophotometrically by the MTT assay.
The MTT assay is based on the reduction of yellow colored MTT (Acros Organics, Geel, Belgium) by mitochondrial dehydrogenase activity of metabolically active cells to a blue-purple formazan that can be measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer (Infinite M1000, Tecan), at two wavelengths (540 and 690 nm). All data were calculated using the median OD (optical density) values of tree wells. The 50% cytotoxic concentration (CC50) was defined as the concentration of the test compound that reduced the absorbance (OD540) of the mock-infected control sample by 50%. The concentration achieving 50% protection from the cytopathic effect of the virus in infected cells was defined as the 50% effective concentration (EC50).
Recombinant HIV-1 RT inhibitory assay The HIV-RT inhibition assay was performed by using an RT assay kit (Roche), and the procedure for assaying RT inhibition was performed as described in the kit protocol. Briefly, the reaction mixture consists of template/primer complex, 2’-deoxy-nucleotide-5’-triphosphates (dNTPs) and reverse transcriptase (RT) enzyme in the lysis buffer with or without inhibitors. After 1 h incubation at 37°C the reaction mixture was transferred to streptavidine-coated microtiter plate (MTP). The biotin labeled dNTPs that are incorporated in the template due to activity of RT were bound to streptavidine. The unbound dNTPs were washed using wash buffer and antidigoxigenin-peroxidase (DIG-POD) was
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added in MTP. The DIG-labeled dNTPs incorporated in the template was bound to anti-DIG-POD antibody. The unbound anti-DIG-POD was washed and the peroxide substrate (ABST) was added to the MTP. A colored reaction product was produced during the cleavage of the substrate catalyses by a peroxide enzyme. The absorbance of the sample was determined at OD 405 nm using microtiter plate ELISA reader. The resulting color intensity is directly proportional to the actual RT activity. The percentage inhibitory activity of RT inhibitors was calculated by comparing to a sample that does not contain an inhibitor. The percentage inhibition was calculated by formula as given below: % Inhibition = 100-[(OD 405 nm with inhibitor/OD 405 nm without inhibitor)×100].
Molecular simulation Molecular modeling was carried out with the Tripos molecular modeling packages SYBYL-X. All the molecules for docking were built using standard bond lengths and angles from SYBYL-X/base Builder and were then optimized using the Tripos force field. The flexible docking method, called Surflex-Dock, docks the ligand automatically into the ligand binding site of the receptor by using a protocol-based approach and an empirically derived scoring function. The protocol is a computational representation of a putative ligand that binds to the intended binding site and is a unique and essential element of the docking algorithm. The scoring function in Surflex-Dock, which contains hydrophobic, polar, repulsive, entropic, and salvation terms, was trained to estimate the dissociation constant (Kd) expressed in –log (Kd)2. Prior to docking, the protein was prepared by removing water molecules, the ligand, and other unnecessary small molecules from the crystal structure complex; simultaneously, polar hydrogen atoms were added to the protein. Surflex-Dock default settings were used for other parameters, such as the number of starting conformations per molecule, the size to expand search grid, the maximum number of rotatable bonds per molecule, and the maximum number of poses per ligand. During the docking procedure, all of the single bonds in residue side chains inside the defined RT binding pocket were regarded as rotatable or flexible, and the ligand was allowed to rotate on all single bonds and move This article is protected by copyright. All rights reserved.
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flexibly within the tentative binding pocket. The atomic charges were recalculated using the Kollman all-atom approach for the protein and the Gasteiger–Hückel approach for the ligand. The binding interaction energy was calculated to include van der Waals, electrostatic, and torsional energy terms defined in the Tripos force field. The 20-best-scoring ligand–protein complexes were kept for further analyses. The -log(Kd)2 values of the 20-best-scoring complexes, which represented the binding affinities of ligand with RT, presented a wide scope of functional classes. Therefore, only the highest-scoring 3D structural model of the ligand-bound RT was chosen to define the binding interaction.
Results and Discussion Chemistry The synthesis of the novel triazine derivatives was expeditiously carried out in our laboratory as depicted in Scheme 1. Firstly, the starting material 2,4,6-trichloro-1,3,5-triazine (S-1) was reacted with the appropriate 2,4,6-tri-substituted phenol and 4-amino-benzonitrile successively to construct the key intermediates S-3a and S-3b with satisfactory yields.[10] Then the chlorine atom of intermediates S-3a and S-3b were replaced by structurally diverse substituents containing morpholine, piperazine and piperidine heterocycles to give the target compounds DSC-a4~a9 and DSC-b4~b9. Meanwhile, the intermediates S-3a and S-3b were coupled with 4-amino-1-Boc-piperidine in a mixture of dioxane and water to afford the intermediates S-4a and S-4b, respectively. Then the Boc groups were removed with trifluoroacetic acid in dichloromethane at room temperature with high yields.[11] Finally, the exposed NH group of piperidine was reacted with substituted benzyl chloride or 4-picolyl chloride hydrochloride to obtain the compounds DSC-a2~a3 and DSC-b2~b3.
1
H-NMR, IR and ESI-MS of all the target compounds and 13C-NMR of representative
compounds from each sub-series were determined which were in agreement with the proposed structures. This article is protected by copyright. All rights reserved.
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Biological evaluation The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to evaluate the title compounds in a cell-based antiviral assay against HIV-1 (wild-type (IIIB) and a resistant mutant HIV-1 strain, containing K103N/Y181C in the RT) as reported previously[5, 7, 12-14]. nevirapine (NVP), delavirdine mesylate (DLV), efavirenz (EFV), etravirine (ETV) and zidovudine (AZT) were used as reference drugs, and the screening results are summarized in Table 1.
1) All the newly designed and synthesized compounds were active against wild-type HIV-1 with EC50 values within the concentration range of 0.0078-0.16 µM, and SI (selectivity index) values ranging from 25 to 1671. DSC-a4 was the most potent compound against wild-type and K103N/Y181C resistant mutant strain of HIV-1 with EC50 values of 7.8 nM and 0.65 µM, respectively. The activity of compound DSC-a4 against wild-type HIV-1 was 21.8 times more potent when compared to the reference drug NVP, 16.7 times more potent than DLV and comparable to EFV, AZT and ETV (in the same order of magnitude). The activity against K103N/Y181C resistant mutant strain was 3.8 times more potent than NVP, 55 times more potent than DLV and comparable to EFV.
Compared to our previous work (pDAPYs derivatives, the most active compound is MD-c5), compound DSC-a4 showed improved activity against both the wild-type and K103N/Y181C mutant resistant strains of HIV-1, which is in accordance withour design hypothesis.
2) Among compounds DSC-a1~a9 (R1 = Me sub-series), DSC-a2 was the least active compound probably due to its larger molecular weight and molecular volume, and the other compounds with smaller R2 substituents (DSC-a4~a9) showed higher activity against
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wild-type HIV-1. However, among the compounds DSC-b1~b9 (R1 = CN sub-series), DSC-b1 and DSC-b8 showed the lowest activities.
3) 2-morpholinoethylamino derivatives DSC-a4 and DSC-b4 displayed higher activity than the compounds with other substituents. Compounds DSC-a6 and DSC-b6 with 2-morpholinoethoxy group which was a little different from 2-morpholinoethylamino group (DSC-a4 and DSC-b4) showed clearly decreased activity against both wild-type and K103N/Y181C resistant mutant strain of HIV-1. These results indicate that a minor structural variation of the moiety targeting the entrance channel domain has a significant impact on the antiviral activity, and further structural modifications in this part possess the potential to improve the antiviral activity.
Subsequently, an HIV-1 RT inhibitory assay was performed via an ELISA assay using a commercial kit (Roche) to directly prove the binding target of the new triazine compounds,[5, 13]. The representative compound DSC-a4 displayed obvious inhibitory activity against the recombinant RT with an IC50 value 1.3 µM (Table 2).
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Table 1. Activity against wild-type (IIIB) and resistant mutant strain RES056 (K103N/Y181C, RT) of HIV-1 in MT-4 cell cultures using the MTT method.
IIIB Compds
DSC-a1
R2
-
K103N/Y181C
Ar
-
EC50 a (µM)
SI c
EC50 (µM)
SI
0.046 ± 0.0063
85
≥4.0
≤1
CC50 b (µM)
4.0 ± 0.34
DSC-a2
-
4-SO2Me-Ph
0.097 ± 0.047
82
>8.0
5.1
4.3
3.7
19
3.6
5.0
4.5
89
2.5 ± 1.0
>6
>15
DLV
-
-
0.13 ± 0.043
>227
>36
X1
>36
EFV
-
-
0.0074 ± 0.0026
>855
0.52 ± 0.022
>12
>6.3
ETV
-
-
>184
>4.6
>12594
>94
0.0041 ±
0.025 ± >1127
0.00021
0.0030 0.0074 ±
0.0072 ± AZT
-
>13066
0.00029
0.00087
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against
HIV-1-induced cytopathicity. bCC50: cytotoxic concentration of compound that reduces the normal uninfected cell viability by 50%. cSI: selectivity index, the ratio of CC50/EC50. X1 stands for ≥1 or
Received Date : 09-Sep-2014 Revised Date
: 12-Oct-2014
Accepted Date : 17-Oct-2014 Article type
: Research Article
Design, Synthesis and Anti-HIV Evaluation of Novel Triazine Derivatives Targeting the Entrance Channel of the NNRTI Binding Pocket
Xuwang Chen,1 Qing Meng,1 Liyun Qiu,2 Peng Zhan,1 Huiqing Liu,3 Erik De Clercq,4 Christophe Pannecouque4 and Xinyong Liu 1,* 1
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road, 250012, Ji’nan, Shandong, P.R. China; 2
Department of Pharmacy, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013,
Shandong, P.R.China; 3
Institute of Pharmacology, School of Medicine, Shandong University, 44, West Culture Road, 250012,
Ji’nan, Shandong, P.R. China; 4
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
*
Corresponding author mail id: [email protected]
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12471 This article is protected by copyright. All rights reserved.
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Abstract A novel series of triazine derivatives targeting the entrance channel of the HIV-1 non-nucleoside reverse transcriptase inhibitor binding pocket (NNIBP) were designed and synthesized on the basis of our previous work. The results of a cell-based antiviral screening assay indicated that most compounds showed good to moderate activity against wild-type HIV-1 with EC50 values within the concentration range of 0.0078-0.16 µM (compound DCS-a4, EC50 = 7.8 nM). Some compounds displayed sub-micromolar activity against the K103N/Y181C resistant mutant strain (such as compound DCS-a4, EC50 = 0.65 µM). Molecular modeling studies confirmed that the new compounds could bind into the NNIBP similarly as the lead compound, and the newly introduced flexible heterocycles could occupy the entrance channel effectively. In addition, the preliminary structure-activity relationship and the RT inhibitory assay are presented in this paper.
Keywords: HIV-1, NNRTIs, Entrance channel, Antiviral activity, Molecular modeling
HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) have become an indispensable component of first-line drug regimens in the clinic with five drugs (nevirapine, delavirdine, efavirenz, etravirine and rilpivirine) approved by the US FDA for the treatment of AIDS. Especially, etravirine (TMC125) and rilpivirine (TMC278) which structurally belong to the diarylpyrimidine (DAPY) derivatives, display highly potent activity against wild-type and NNRTI-resistant mutant strains of HIV-1.[1, 2] Thus, the DAPY-type NNRTIs have been the research hotspot in structural modifications and optimizations during the past decade.[3]
X-ray crystallography and molecular modeling studies showed that the binding conformation of DAPYs resembled a prototypical horseshoe or “U” shape in the NNIBP, which contain three pharmacophoric groups: a hydrophobic center (A), hydrogen bond This article is protected by copyright. All rights reserved.
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donor/acceptor (B) and protein/solvent interface interaction domain (C) (Fig. 1).[4] The NH2 and Br groups on the 5,6-positions of the central pyrimidine ring of etravirine pointed to another open region, namely the entrance channel (D), which was surrounded by Lys101, Glu138 and Val179. However, the NH2 and Br groups did not display obvious interactions with the entrance channel probably because of the smaller volume.[4] Recently, novel HIV-1 NNRTIs targeting the entrance channel were reported by our research group[5] and Prof. W.L. Jorgensen’s group[6] with good antiviral activity against wild-type and NNRTI-resistant mutant strains of HIV-1, which further confirmed the rationale for design of the multi-sites binding NNRTIs to overcome the drug resistance based on the four pharmacophoric model (A-D).
In our previous studies, the multi-sites binding of piperidinylamino-diarylpyrimidine (pDAPY) derivatives was rationalized through a molecular hybridization strategy (Fig. 1).[5] Molecular modeling studies indicated that the pDAPY hybrid compounds could form hydrophobic interactions and hydrogen bond interactions with the non-nucleoside reverse transcriptase inhibitor binding pocket (NNIBP), and occupy the entrance channel simultaneously. Among the pDAPY derivatives, compound MD-c5 displayed the highest activity against wild-type and K103N/Y181C drug-resistant mutant strains of HIV-1 with EC50 values of 0.038 µM and 0.95 µM respectively. However, introduction of substituents (such as 4-SO2NH2-benzyl and 4-SO2CH3-benzyl) at the NH group of the piperidine ring decreased the antiviral activity in MT-4 cell cultures (Fig. 1) .[5]
Figure 1. The structures of the representative DAPY-type NNRTIs and pDAPYs.
To continue our research on these multi-sites binding NNRTIs targeting the entrance channel, a novel series of triazine derivatives was designed by combining the pharmacophoric groups of the drug etravirine and the pDAPY derivatives (Fig. 2).[7] This article is protected by copyright. All rights reserved.
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Moreover, on the basis of the above structure-activity relationship (SAR) analysis of pDAPY analogues and represented by the lead compound MD-c5, the N-benzyl piperidine moieties were modified and shortened using structural diverse substituents containing flexible heterocycles like morpholine, piperazine and piperidine. The newly designed compounds are expected to occupy the entrance channel more effectively and form multiple hydrogen bonds with the binding pocket of RT, in order to improve the activity against the drug-resistant mutant strains of HIV-1 (Fig. 2).
Figure 2. The design strategies of new triazine derivatives
Materials and Methods Chemistry All melting points were determined on a micro-melting point apparatus and are uncorrected. 1H-NMR (400MHz) and 13C-NMR (100MHz) spectra were obtained on a Brucker AV400 NMR-spectrometer in the indicated solvents. Infrared spectra (IR) were recorded with a Nexus 470FT-IR Spectrometer. Mass spectra were taken on a LC Autosampler Device: Standard G1313A instrument. TLC was performed on Silica Gel GF254 for TLC and spots were visualized by irradiation with UV light (254 nm). Flash column chromatography was performed on column packed with Silica Gel 60 (200-300 mesh). Solvents were reagent grade and, when necessary, were purified and dried by standard methods. The chemical materials were purchased from commercial suppliers and used without further purification.
Procedure for the synthesis of 2,4-dichloro-6-(mesityloxy)-1,3,5-triazine (S-2a) To an ice-bath cooled solution of cyanuric chloride S-1 (3.7 g, 20 mmol) in THF was added DIPEA (diisopropylethylamine, 3.9 g, 30 mmol). After that, a solution of This article is protected by copyright. All rights reserved.
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2,4,6-trimethylphenol (2.7 g, 20 mmol) in 20 mL THF was added dropwise to the above mixture and then stirred for 4 h at 0°C. After removal of THF under reduced pressure, water was added and the mixture was extracted with ethyl acetate. The combined organic phase was washed with brine and dried over anhydrous Na2SO4, filtered, and concentrated to give crude S-2a as a white solid without further purification.
Procedure for the synthesis of 4-(4,6-dichloro-1,3,5-triazin-2-yloxy)-3,5-dimethyl-benzo-nitrile (S-2b) Following the procedure in the preparation of S-2a, 4-hydroxy-3,5-dimethylbenzonitrile (2.9 g, 20 mmol) was reacted with cyanuric chloride S-1 (3.7 g, 20 mmol) in the presence of DIPEA (3.9 g, 30 mmol) to give crude S-2b as a white solid without further purification.
Procedure for the synthesis of 4-(4-chloro-6-(mesityloxy)-1,3,5-triazin-2-ylamino)-benzo-nitrile (S-3a) To a solution of S-2a (0.14 g) in 1,4-dioxane was added 4-amino-benzonitrile (60 mg, 0.5 mmol) and anhydrous K2CO3 (0.14 g, 1 mmol). The reaction mixture was stirred at 100°C for 6 h, and then dioxane was removed under reduced pressure. Water was added and the mixture was extracted with ethyl acetate. The organic phase was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. Purification on silica gel gave S-3a as a white solid. Yield: 83%, mp: 151-153°C; TLC Rf (EtOAc:petroleum ether 1:5) = 0.45; ESI-MS: m/z 366.4 [M+H]+.
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Procedure for the synthesis of 4-(4-chloro-6-(4-cyanophenylamino)-1,3,5-triazin-2-yloxy)3,5-dimethylbenzonitrile (S-3b) According to the procedure in the preparation of S-3a, the intermediate S-2b (0.15 g) was coupled with 4-amino-benzonitrile (60 mg, 0.5 mmol) in the presence of anhydrous K2CO3 (0.14 g, 1 mmol) to give S-3b as a white solid. Yield: 76%, mp: 262-264°C; TLC Rf (EtOAc:petroleum ether 2:5) = 0.45; ESI-MS: m/z 377.5 [M+H]+, 394.3 [M+NH4]+, 399.3 [M+Na]+.
Procedure for the synthesis of 4-(4-(mesityloxy)-6-(1-Boc-piperidin-4-ylamino)-1,3,5-triazin-2- ylamino)benzonitrile (S-4a) To a solution of S-3a (0.51 g, 1.4 mmol) and 4-amino-1-Boc-piperidine (0.4 g, 2 mmol) in dioxane was added anhydrous K2CO3 (0.28 g, 2 mmol) and water (5 mL). The mixture was stirred at 80°C for 8 h. After removal of the solvent under reduced pressure, water was added and the mixture was extracted with ethyl acetate. Combined organic layer was washed with saturated sodium chloride solution, and dried over anhydrous Na2SO4. Purification via flash column chromatography afforded intermediate S-4a as white solid. Yield: 77%, mp: 223-225°C; TLC Rf (EtOAc:petroleum ether 1:4) = 0.12; ESI-MS: m/z 530.3 [M+H]+, 552.5 [M+Na]+.
Procedure for the synthesis of 4-(4-(4-cyanophenylamino)-6-(1-methylpiperidin-4-ylamino)-1,3,5triazin-2-yloxy)-3,5-dimethylbenzonitrile (S-4b) Following the procedure for the preparation of S-4a, the intermediate S-3b (1.5 g, 4 mmol) was reacted with 4-amino-1-Boc-piperidine (1 g, 5 mmol) under the condition of anhydrous K2CO3 (0.69 g, 5 mmol) to give S-4b as a white solid. Yield: 80%, mp:
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215-217°C; TLC Rf (EtOAc:petroleum ether 2:5) = 0.14; ESI-MS: m/z 541.5 [M+H]+, 558.4 [M+NH4]+, 563.4 [M+Na]+.
Procedure for the synthesis of 4-(4-(mesityloxy)-6-(piperidin-4-ylamino)-1,3,5-triazin-2-ylamino) benzonitrile (DSC-a1) TFA (1.5 mL, 20 mmol) was added dropwise under stirring to a solution of intermediate S-4a (1.5 g, 2.8 mmol) in CH2Cl2 at room temperature and the mixture was stirred for 10 h. Water was added, and the mixture was neutralized with 2N NaOH (aq) to pH = 7. The organic layer was washed with saturated sodium chloride solution, dried over anhydrous Na2SO4, and filtered. After evaporation of the solvent, the obtained crude product was recrystallized from EtOH to give compound DSC-a1. Yield: 93%, mp: 247-249°C; TLC Rf (MeOH:EtOAc 1:10, add 2 drops of Et3N) = 0.26; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.81-10.01 (s, 1H), 7.74-7.91 (m, 3H), 7.61 (t, 2H, J = 8.8 Hz), 6.91 (s, 2H, PhH), 3.61-3.80 (m, 1H), 2.90-2.97 (m, 2H), 2.35-2.45 (m, 1H), 2.26 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3), 1.71-1.79 (m, 2H), 1.31-1.36 (m, 2H); 13C-NMR (100M, DMSO-d6, ppm) δ: 170.09, 166.51, 165.84, 165.39, 147.55, 144.81, 134.50, 133.10, 130.10, 129.45, 119.86, 103.79, 48.90, 45.69, 33.19, 20.77, 16.53; IR (KBr, cm-1): 3424 (υNH), 3269 (υNH), 2923 (υasCH3), 2220 (υC≡ N),
1578, 1509 (υC=N), 1196 (υC-N), 810 (ωC=N); ESI-MS: m/z 430.7 [M+H]+.
Procedure for the synthesis of 4-(4-(4-cyanophenylamino)-6-(piperidin-4-ylamino)-1,3,5-triazin2-yloxy)-3,5-dimethylbenzonitrile (DSC-b1) Following the procedure for the preparation of DSC-a1, the reaction of S-4b (2.7 g, 5 mmol) with TFA (2.7 mL, 36 mmol) afforded DSC-b1 as a white solid. Yield: 91%, mp: 166-168°C; TLC Rf (MeOH:EtOAc 1:5, add 2 drops of Et3N) = 0.14; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.94-10.17 (s, 1H), 8.60 (brs, 2H), 8.06 (d, 1H, J = 7.1 Hz), 7.83 (brs, 2H), 7.69 (s, 2H, PhH), 7.64 (d, 1H, J = 8.3 Hz), 4.33 (d, 1H, J = 4.5 Hz), 3.85-4.01 (m, 1H), This article is protected by copyright. All rights reserved.
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3.41-3.48 (m, 1H), 2.90-3.05 (m, 2H), 2.14 (s, 6H, 2 × CH3), 1.95-2.07 (m, 2H), 1.65-1.70 (m, 2H); IR (KBr, cm-1): 3424 (υNH), 2926 (υasCH3), 2226 (υC≡N), 1583, 1512 (υC=N), 1202 (υC-N), 811 (ωC=N); ESI-MS: m/z 441.6 [M+H]+.
Procedure for the synthesis of title compounds DSC-a2~a3 and DSC-b2~b3 Intermediate DSC-a1 (or DSC-b1, 0.5 mmol) was dissolved in anhydrous DMF in the presence of anhydrous K2CO3 (0.14 g, 1 mmol), followed by addition of appropriate substituted benzyl chloride (or 4-picolyl chloride hydrochloride) (0.5 mmol). The reaction mixture was stirred at room temperature for 10 h. The solvent was removed under reduced pressure, and water was added. After extraction with ethyl acetate, the organic phase was washed with saturated sodium chloride solution and dried over anhydrous Na2SO4. The product was further purified by flash column chromatography and then recrystallized in the mixture of ethyl acetate and petroleum ether to afford title compounds DSC-a2~a3 and DSC-b2~b3.
4-(4-(mesityloxy)-6-(1-(4-(methylsulfonyl)benzyl)piperidin-4-ylamino)-1,3,5-triazin-2-yl amino)benzonitrile (DSC-a2) White solid; yield: 61%, mp: 266-268°C; TLC Rf (EtOAc) = 0.23; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.83-10.09 (s, 1H), 7.80-7.91 (m, 5H), 7.50-7.73 (m, 3H), 6.91 (s, 2H, PhH), 3.20 (s, 3H, CH3), 3.56-3.59 (m, 2H), 2.94 (s, 2H), 2.25 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3), 1.95-1.99 (m, 2H), 1.80-1.95 (m, 2H), 1.52-1.55 (m, 2H); IR (KBr, cm-1): 3427 (υNH), 2923 (υasCH3), 2223 (υC≡N), 1579, 1510 (υC=N), 1409 (υasO=S=O), 1197 (υC-N), 1149 (υO=S=O), 1090 (υS=O), 811 (ωC=N); ESI-MS: m/z 598.4 [M+H]+.
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4-(4-(mesityloxy)-6-(1-(pyridin-4-ylmethyl)piperidin-4-ylamino)-1,3,5-triazin-2-ylamino) -benzonitrile (DSC-a3) White solid; yield: 55%, mp: 265-267°C; TLC Rf (MeOH:EtOAc 1:10) = 0.20; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.83-10.03 (s, 1H), 8.51 (d, 2H, J = 5.5 Hz, PyH), 7.78-7.89 (m, 3H), 7.59-7.65 (m, 2H), 7.32 (d, 2H, J = 5.4 Hz, PyH), 6.91 (s, 2H, PhH), 3.78 (brs, 1H), 3.52 (s, 2H), 2.75-2.82 (m, 2H), 2.25 (s, 3H, CH3), 2.07-2.10 (m, 2H), 2.04 (s, 6H, 2 × CH3), 1.76-1.84 (m, 2H), 1.50-1.56 (m, 2H); IR (KBr, cm-1): 3420 (υNH), 3260 (υNH), 2922 (υasCH3), 2221 (υC≡N), 1579, 1510 (υC=N), 1195 (υC-N), 811 (ωC=N); ESI-MS: m/z 521.5 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(1-(4-(methylsulfonyl)benzyl)piperidin-4-ylamino)-1,3,5-tr iazin-2-yloxy)-3,5-dimethylbenzonitrile (DSC-b2) White solid; yield: 58%, mp: 252-254°C; TLC Rf (EtOAc) = 0.13; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.93-10.11 (s, 1H), 7.75-7.90 (m, 4H), 7.63-7.66 (m, 2H), 7.68 (s, 2H, PhH), 7.54-7.59 (m, 2H), 3.80 (brs, 1H), 3.52-3.59 (m, 3H), 3.20 (s, 3H, CH3), 2.74-2.83 (m, 2H), 2.13 (s, 6H, 2 × CH3), 1.93-1.97 (m, 2H), 1.82-1.85 (m, 2H), 1.49-1.57 (m, 2H); IR (KBr, cm-1): 3415 (υNH), 2925 (υasCH3), 2226 (υC≡N), 1583, 1511 (υC=N), 1397 (υasO=S=O), 1200 (υC-N), 1149 (υO=S=O), 1089 (υS=O), 810 (ωC=N); ESI-MS: m/z 609.3 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(1-(pyridin-4-ylmethyl)piperidin-4-ylamino)-1,3,5-triazin2-yloxy)-3,5-dimethylbenzonitrile (DSC-b3) White solid; yield: 51%, mp: 263-265°C; TLC Rf (MeOH:EtOAc 1:10) = 0.15; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.92-10.10 (s, 1H), 8.51 (d, 2H, J = 5.7 Hz, PyH), 7.81-7.89 (m, 3H), 7.68 (s, 2H, PhH), 7.64 (d, 2H, J = 8.3 Hz, PhH), 7.31 (d, 2H, J = 5.6 Hz, PyH), 3.76 (brs, 1H), 3.52 (s, 2H), 2.74-2.82 (m, 2H), 2.13 (s, 6H, 2 × CH3), 2.05-2.08 (m, 2H), 1.72-1.85 (m, 2H), 1.52-1.58 (m, 2H); IR (KBr, cm-1): 3427 (υNH), 2926 (υasCH3), 2225 (υC≡ N),
1583, 1512 (υC=N), 1201 (υC-N), 810 (ωC=N); ESI-MS: m/z 532.4 [M+H]+.
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Procedure for the synthesis of title compounds DSC-a4, DSC-a5, DSC-a7, DSC-b4, DSC-b5 and DSC-b7
Intermediate S-3a (or S-3b, 0.5 mmol) was dissolved in the mixture of dioxane and water, followed by addition of appropriate substituent (0.5 mmol) and K2CO3 (0.14 g, 1 mmol). The reaction mixture was stirred at 80°C for 10 h. The solvent was removed under reduced pressure, and water was added. After extraction with ethyl acetate, the organic phase was washed with saturated sodium chloride solution and dried over anhydrous Na2SO4. The product was further purified by flash column chromatography and then recrystallized in the mixture of ethyl acetate and petroleum ether to afford title compounds DSC-a4, DSC-a5, DSC-a7, DSC-b4, DSC-b5 and DSC-b7.
4-(4-(mesityloxy)-6-(2-morpholinoethylamino)-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a4) White solid; yield: 68%, mp: 215-217°C; TLC Rf (EtOAc) = 0.15; 1H-NMR (400M, DMSO-d6, ppm) δ: 9.95-10.04 (s, 1H), 7.85-7.89 (m, 2H), 7.63 (d, 3H, J = 8.1 Hz), 6.92 (s, 2H, PhH), 3.56 (brs, 2H), 3.51 (brs, 2H), 3.41 (d, 1H, J = 6.1 Hz), 3.22 (d, 1H, J = 6.1 Hz), 2.46 (d, 1H, J = 6.0 Hz), 2.39 (s, 2H), 2.32 (s, 1H), 2.25 (s, 5H), 2.04 (s, 6H, 2 × CH3); 13
C-NMR (100M, DMSO-d6, ppm) δ: 170.31, 169.98, 167.45, 165.87, 147.50, 144.71,
134.46, 133.21, 130.07, 129.46, 119.94, 103.86, 66.64, 57.44, 53.76, 53.61, 20.78, 16.50; IR (KBr, cm-1): 3420 (υNH), 2920 (υasCH3), 2223 (υC≡N), 1578, 1511 (υC=N), 1197 (υC-N), 812 (ωC=N); ESI-MS: m/z 460.5 [M+H]+.
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4-(4-(mesityloxy)-6-(piperazin-1-yl)-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a5) White solid; yield: 60%, mp: 176-178°C; TLC Rf (MeOH:EtOAc 1:5) = 0.21; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.02 (s, 1H), 7.73 (brs, 2H), 7.60 (brs, 2H), 6.93 (s, 2H, PhH), 3.71 (brs, 2H), 3.58 (brs, 2H), 2.70-2.74 (5H), 2.27 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3424 (υNH), 2921 (υasCH3), 2223 (υC≡N), 1578, 1508 (υC=N), 1198 (υC-N), 809 (ωC=N); ESI-MS: m/z 416.5 [M+H]+.
4-(4-(mesityloxy)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a7) White solid; yield: 57%, mp: 223-225°C; TLC Rf (EtOAc) = 0.16; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.03 (s, 1H), 7.71 (brs, 2H), 7.60 (brs, 2H), 6.94 (s, 2H, PhH), 3.77 (brs, 2H), 3.65 (brs, 2H), 2,36-2.33 (m, 4H), 2.27 (s, 3H, CH3), 2.21 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3415 (υNH), 2920 (υasCH3), 2219 (υC≡N), 1575, 1505 (υC=N), 1196 (υC-N), 809 (ωC=N); ESI-MS: m/z 430.5 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(2-morpholinoethylamino)-1,3,5-triazin-2-yloxy)-3,5-dime thylbenzonitrile (DSC-b4) White solid; yield: 70%, mp:228-230°C; TLC Rf (EtOAc) = 0.09; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.04-10.12 (s, 1H), 7.72-7.88 (m, 3H), 7.69 (s, 2H, PhH), 7.65-7.68 (m, 2H), 3.56 (t, 2H, J = 4.4 Hz), 3.52 (t, 2H, J = 4.4 Hz), 3.42 (d, 1H, J = 6.4 Hz), 3.19 (d, 1H, J = 6.5 Hz), 2.46 (t, 1H, J = 6.8 Hz), 2.39 (s, 2H), 2.28 (t, 1H, J = 6.8 Hz), 2.22 (s, 2H), 2.14 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3427 (υNH), 3270 (υNH), 2922 (υasCH3), 2226 (υC≡N), 1585, 1509 (υC=N), 1201 (υC-N), 811 (ωC=N); ESI-MS: m/z 471.4 [M+H]+.
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4-(4-(4-cyanophenylamino)-6-(piperazin-1-yl)-1,3,5-triazin-2-yloxy)-3,5-dimethyl-benzo nitrile (DSC-b5) White solid; yield: 63%, mp: 274-276°C; TLC Rf (MeOH:EtOAc 1:5) = 0.15; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.09 (s, 1H), 7.70 (s, 2H, PhH), 7.64 (brs, 4H), 3.72 (brs, 2H), 3.55 (brs, 2H), 2.69-2.75 (5H), 2.14 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3423 (υNH), 2924 (υasCH3), 2226 (υC≡N), 1585, 1496 (υC=N), 1199 (υC-N), 807 (ωC=N); ESI-MS: m/z 427.5 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-yloxy)-3,5-dimeth ylbenzonitrile (DSC-b7) White solid; yield: 62%, mp: 259-261°C; TLC Rf (MeOH:EtOAc 1:10) = 0.28; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.11 (s, 1H), 7.70 (s, 2H, PhH), 7.65-7.60 (m, 4H), 3.77 (brs, 2H), 3.62 (brs, 2H), 2,37-2.32 (m, 4H), 2.21 (s, 3H, CH3), 2.13 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3423 (υNH), 2928 (υasCH3), 2224 (υC≡N), 1579, 1509 (υC=N), 1203 (υC-N), 807 (ωC=N); ESI-MS: m/z 441.4 [M+H]+.
Procedure for the synthesis of title compounds DSC-a6 and DSC-b6 To a solution of intermediate S-3a (or S-3b, 0.5 mmol) and 2-morpholinoethanol (65 mg, 0.5 mmol) in anhydrous THF was added NaH (40 mg, 1 mmol). The reaction mixture was stirred at 65°C for 18 h. After removal of the solvent under reduced pressure, water was added and the mixture was extracted with ethyl acetate. Combined organic layer was washed with saturated sodium chloride solution, and dried over anhydrous Na2SO4. The product was further purified by flash column chromatography and then recrystallized in the mixture of ethyl acetate and petroleum ether to afford title compounds DSC-a6 and DSC-b6.
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4-(4-(mesityloxy)-6-(2-morpholinoethoxy)-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a6) White solid; yield: 52%, mp: 163-165°C; TLC Rf (EtOAc) = 0.28; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.58 (s, 1H), 7.68 (brs, 4H), 6.96 (s, 2H, PhH), 4.41 (t, 2H, J = 5.7 Hz), 3.54 (t, 4H, J = 4.5 Hz), 2.63 (t, 2H, J = 5.7 Hz), 2.40 (s, 4H), 2.28 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3420 (υNH), 3291 (υNH), 2919 (υasCH3), 2217 (υC≡N), 1583, 1507 (υC=N), 1197 (υC-N), 809 (ωC=N); ESI-MS: m/z 461.4 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(2-morpholinoethoxy)-1,3,5-triazin-2-yloxy)-3,5-dimethylb enzonitrile (DSC-b6) White solid; yield: 50%, mp: 149-151°C; TLC Rf (EtOAc) = 0.28; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.65 (brs, 1H), 7.73 (brs, 6H), 4.41 (t, 2H, J = 5.7 Hz), 3.54 (t, 4H, J = 4.5 Hz), 2.63 (t, 2H, J = 5.7 Hz), 2.39 (brs, 4H), 2.04 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3431 (υNH), 2923 (υasCH3), 2226 (υC≡N), 1578, 1509 (υC=N), 1200 (υC-N), 814 (ωC=N); ESI-MS: m/z 472.4 [M+H]+.
Procedure for the synthesis of title compounds DSC-a8 and DSC-b8 To a solution of intermediate S-3a (or S-3b, 0.6 mmol) and 1-methylpiperidin-4-amine (68 mg, 0.6 mmol) in dioxane was added anhydrous K2CO3 (0.17 g, 1.2 mmol). The reaction mixture was stirred at 100°C for 16 h. After removal of the solvent under reduced pressure, water was added and the mixture was extracted with ethyl acetate. Combined organic layer was washed with saturated sodium chloride solution, and dried over anhydrous Na2SO4. The product was further purified by flash column chromatography and then recrystallized in the mixture of ethyl acetate and petroleum ether to afford title compounds DSC-a8 and DSC-b8.
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4-(4-(mesityloxy)-6-(1-methylpiperidin-4-ylamino)-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a8) White solid; yield: 54%, mp: 146-148°C; TLC Rf (MeOH:EtOAc 1:5) = 0.21; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.02-9.82 (s, 1H), 7.92-7.85 (m, 2H), 7.75-7.73 (m, 1H), 7.65-7.59 (m, 2H), 6.91 (s, 2H, PhH), 3.71-3.51 (m, 1H), 2.79-2.71 (m, 2H), 2.25 (s, 3H, CH3), 2.18 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3), 1.98-1.92 (m, 2H), 1.83-1.77 (m, 2H), 1.56-1.47 (m, 2H); IR (KBr, cm-1): 3420 (υNH), 2940 (υasCH3), 2223 (υC≡N), 1578, 1510 (υC=N), 1198 (υC-N), 811 (ωC=N); ESI-MS: m/z 444.6 [M+H]+.
4-(4-(4-cyanophenylamino)-6-(1-methylpiperidin-4-ylamino)-1,3,5-triazin-2-yloxy)-3,5-d imethylbenzonitrile (DSC-b8) White solid; yield: 57%, mp: 163-165°C; TLC Rf (MeOH:EtOAc 1:5) = 0.14; 1H-NMR (400M, DMSO-d6, ppm) δ: 10.10-9.92 (s, 1H), 7.85-7.82 (m, 3H), 7.68 (s, 2H, PhH), 7.65-7.62 (m, 2H), 3.71-3.45 (m, 1H), 2.79-2.72 (m, 2H), 2.19 (s, 3H, CH3), 2.13 (s, 6H, 2 × CH3), 2.02-1.96 (m, 1H), 1.83-1.80 (m, 2H), 1.73-1.68 (m, 1H), 1.53-1.47 (m, 2H); IR (KBr, cm-1): 3423 (υNH), 2938 (υasCH3), 2225 (υC≡N), 1581, 1513 (υC=N), 1201 (υC-N), 810 (ωC=N); ESI-MS: m/z 455.4 [M+H]+.
Procedure for the synthesis of title compounds DSC-a9 and DSC-b9 Intermediate S-3a (or S-3b, 0.6 mmol) and morpholine (52 mg, 0.6 mmol) were dissolved in the mixture of dioxane and water, followed by addition of K2CO3 (0.17 g, 1.2 mmol). The reaction mixture was stirred at 100°C for 13 h. The solvent was removed under reduced pressure, and water was added. After extraction with ethyl acetate, the organic phase was washed with saturated sodium chloride solution and dried over anhydrous Na2SO4. The product was further purified by flash column chromatography and then recrystallized in the mixture of ethyl acetate and petroleum ether to afford title compounds DSC-a9 and DSC-b9.
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4-(4-(mesityloxy)-6-morpholino-1,3,5-triazin-2-ylamino)benzonitrile (DSC-a9) White solid; yield: 59%, mp: 210-212°C; TLC Rf (EtOAc:petroleum ether 1:4) = 0.13; 1
H-NMR (400M, DMSO-d6, ppm) δ: 10.07 (s, 1H), 7.72 (brs, 2H), 7.60 (brs, 2H), 6.94 (s,
2H, PhH), 3.76 (brs, 2H), 3.64 (brs, 6H), 2.27 (s, 3H, CH3), 2.04 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3428 (υNH), 2959 (υasCH3), 2226 (υC≡N), 1578, 1509 (υC=N), 1200 (υC-N), 814 (ωC=N); ESI-MS: m/z 417.6 [M+H]+.
4-(4-(4-cyanophenylamino)-6-morpholino-1,3,5-triazin-2-yloxy)-3,5-dimethyl-benzonitri le (DSC-b9) White solid; yield: 65%, mp: 285-287°C; TLC Rf (EtOAc:petroleum ether 2:5) = 0.24; 1
H-NMR (400M, DMSO-d6, ppm) δ: 10.14 (s, 1H), 7.70 (s, 2H, PhH), 7.64 (brs, 4H),
3.77-3.61 (m, 6H), 2.14 (s, 6H, 2 × CH3); IR (KBr, cm-1): 3423 (υNH), 2925 (υasCH3), 2225 (υC ≡N
), 1583, 1511 (υC=N), 1201 (υC-N), 807 (ωC=N); ESI-MS: m/z 428.6 [M+H]+, 445.9
[M+NH4]+, 450.6 [M+Na]+.
In vitro anti-HIV activity assays The anti-HIV activity and cytotoxicity of the compounds were evaluated against wild-type HIV-1 strain IIIB, a double RT mutant (K103N + Y181C) HIV-1 strain in MT-4 cell cultures using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) method as previously described.[1, 4, 8] Briefly, stock solutions (10 x final concentration) of test compounds were added in 25 µL volumes to two series of triplicate wells so as to allow simultaneous evaluation of their effects on mock-and HIV-infected cells at the beginning of each experiment. Serial 5-fold dilutions of test compounds (final 200 µL volume per well) were made directly in flat-bottomed 96-well microtiter trays using a Biomek 3000 robot (Beckman instruments). Untreated control HIV-and mock-infected cell samples were included for each sample. HIV-1(IIIB)[9] stock (50 μL) at 100–300 CCID50 (cell culture infectious dose) or culture medium was added to either the infected or mock-infected wells of This article is protected by copyright. All rights reserved.
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the microtiter tray. Mock-infected cells were used to evaluate the effect of test compounds on uninfected cells in order to assess the cytotoxicity of the test compounds. Exponentially growing MT-4 cells were centrifuged for 5 min at 220 g and the supernatant was discarded. The MT-4 cells were resuspended at 6 x 105 cells/mL and 50 µL volumes were transferred to the microtiter tray wells. Five days after infection, the viability of mock-and HIV-infected cells was examined spectrophotometrically by the MTT assay.
The MTT assay is based on the reduction of yellow colored MTT (Acros Organics, Geel, Belgium) by mitochondrial dehydrogenase activity of metabolically active cells to a blue-purple formazan that can be measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer (Infinite M1000, Tecan), at two wavelengths (540 and 690 nm). All data were calculated using the median OD (optical density) values of tree wells. The 50% cytotoxic concentration (CC50) was defined as the concentration of the test compound that reduced the absorbance (OD540) of the mock-infected control sample by 50%. The concentration achieving 50% protection from the cytopathic effect of the virus in infected cells was defined as the 50% effective concentration (EC50).
Recombinant HIV-1 RT inhibitory assay The HIV-RT inhibition assay was performed by using an RT assay kit (Roche), and the procedure for assaying RT inhibition was performed as described in the kit protocol. Briefly, the reaction mixture consists of template/primer complex, 2’-deoxy-nucleotide-5’-triphosphates (dNTPs) and reverse transcriptase (RT) enzyme in the lysis buffer with or without inhibitors. After 1 h incubation at 37°C the reaction mixture was transferred to streptavidine-coated microtiter plate (MTP). The biotin labeled dNTPs that are incorporated in the template due to activity of RT were bound to streptavidine. The unbound dNTPs were washed using wash buffer and antidigoxigenin-peroxidase (DIG-POD) was
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added in MTP. The DIG-labeled dNTPs incorporated in the template was bound to anti-DIG-POD antibody. The unbound anti-DIG-POD was washed and the peroxide substrate (ABST) was added to the MTP. A colored reaction product was produced during the cleavage of the substrate catalyses by a peroxide enzyme. The absorbance of the sample was determined at OD 405 nm using microtiter plate ELISA reader. The resulting color intensity is directly proportional to the actual RT activity. The percentage inhibitory activity of RT inhibitors was calculated by comparing to a sample that does not contain an inhibitor. The percentage inhibition was calculated by formula as given below: % Inhibition = 100-[(OD 405 nm with inhibitor/OD 405 nm without inhibitor)×100].
Molecular simulation Molecular modeling was carried out with the Tripos molecular modeling packages SYBYL-X. All the molecules for docking were built using standard bond lengths and angles from SYBYL-X/base Builder and were then optimized using the Tripos force field. The flexible docking method, called Surflex-Dock, docks the ligand automatically into the ligand binding site of the receptor by using a protocol-based approach and an empirically derived scoring function. The protocol is a computational representation of a putative ligand that binds to the intended binding site and is a unique and essential element of the docking algorithm. The scoring function in Surflex-Dock, which contains hydrophobic, polar, repulsive, entropic, and salvation terms, was trained to estimate the dissociation constant (Kd) expressed in –log (Kd)2. Prior to docking, the protein was prepared by removing water molecules, the ligand, and other unnecessary small molecules from the crystal structure complex; simultaneously, polar hydrogen atoms were added to the protein. Surflex-Dock default settings were used for other parameters, such as the number of starting conformations per molecule, the size to expand search grid, the maximum number of rotatable bonds per molecule, and the maximum number of poses per ligand. During the docking procedure, all of the single bonds in residue side chains inside the defined RT binding pocket were regarded as rotatable or flexible, and the ligand was allowed to rotate on all single bonds and move This article is protected by copyright. All rights reserved.
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flexibly within the tentative binding pocket. The atomic charges were recalculated using the Kollman all-atom approach for the protein and the Gasteiger–Hückel approach for the ligand. The binding interaction energy was calculated to include van der Waals, electrostatic, and torsional energy terms defined in the Tripos force field. The 20-best-scoring ligand–protein complexes were kept for further analyses. The -log(Kd)2 values of the 20-best-scoring complexes, which represented the binding affinities of ligand with RT, presented a wide scope of functional classes. Therefore, only the highest-scoring 3D structural model of the ligand-bound RT was chosen to define the binding interaction.
Results and Discussion Chemistry The synthesis of the novel triazine derivatives was expeditiously carried out in our laboratory as depicted in Scheme 1. Firstly, the starting material 2,4,6-trichloro-1,3,5-triazine (S-1) was reacted with the appropriate 2,4,6-tri-substituted phenol and 4-amino-benzonitrile successively to construct the key intermediates S-3a and S-3b with satisfactory yields.[10] Then the chlorine atom of intermediates S-3a and S-3b were replaced by structurally diverse substituents containing morpholine, piperazine and piperidine heterocycles to give the target compounds DSC-a4~a9 and DSC-b4~b9. Meanwhile, the intermediates S-3a and S-3b were coupled with 4-amino-1-Boc-piperidine in a mixture of dioxane and water to afford the intermediates S-4a and S-4b, respectively. Then the Boc groups were removed with trifluoroacetic acid in dichloromethane at room temperature with high yields.[11] Finally, the exposed NH group of piperidine was reacted with substituted benzyl chloride or 4-picolyl chloride hydrochloride to obtain the compounds DSC-a2~a3 and DSC-b2~b3.
1
H-NMR, IR and ESI-MS of all the target compounds and 13C-NMR of representative
compounds from each sub-series were determined which were in agreement with the proposed structures. This article is protected by copyright. All rights reserved.
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Biological evaluation The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to evaluate the title compounds in a cell-based antiviral assay against HIV-1 (wild-type (IIIB) and a resistant mutant HIV-1 strain, containing K103N/Y181C in the RT) as reported previously[5, 7, 12-14]. nevirapine (NVP), delavirdine mesylate (DLV), efavirenz (EFV), etravirine (ETV) and zidovudine (AZT) were used as reference drugs, and the screening results are summarized in Table 1.
1) All the newly designed and synthesized compounds were active against wild-type HIV-1 with EC50 values within the concentration range of 0.0078-0.16 µM, and SI (selectivity index) values ranging from 25 to 1671. DSC-a4 was the most potent compound against wild-type and K103N/Y181C resistant mutant strain of HIV-1 with EC50 values of 7.8 nM and 0.65 µM, respectively. The activity of compound DSC-a4 against wild-type HIV-1 was 21.8 times more potent when compared to the reference drug NVP, 16.7 times more potent than DLV and comparable to EFV, AZT and ETV (in the same order of magnitude). The activity against K103N/Y181C resistant mutant strain was 3.8 times more potent than NVP, 55 times more potent than DLV and comparable to EFV.
Compared to our previous work (pDAPYs derivatives, the most active compound is MD-c5), compound DSC-a4 showed improved activity against both the wild-type and K103N/Y181C mutant resistant strains of HIV-1, which is in accordance withour design hypothesis.
2) Among compounds DSC-a1~a9 (R1 = Me sub-series), DSC-a2 was the least active compound probably due to its larger molecular weight and molecular volume, and the other compounds with smaller R2 substituents (DSC-a4~a9) showed higher activity against
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wild-type HIV-1. However, among the compounds DSC-b1~b9 (R1 = CN sub-series), DSC-b1 and DSC-b8 showed the lowest activities.
3) 2-morpholinoethylamino derivatives DSC-a4 and DSC-b4 displayed higher activity than the compounds with other substituents. Compounds DSC-a6 and DSC-b6 with 2-morpholinoethoxy group which was a little different from 2-morpholinoethylamino group (DSC-a4 and DSC-b4) showed clearly decreased activity against both wild-type and K103N/Y181C resistant mutant strain of HIV-1. These results indicate that a minor structural variation of the moiety targeting the entrance channel domain has a significant impact on the antiviral activity, and further structural modifications in this part possess the potential to improve the antiviral activity.
Subsequently, an HIV-1 RT inhibitory assay was performed via an ELISA assay using a commercial kit (Roche) to directly prove the binding target of the new triazine compounds,[5, 13]. The representative compound DSC-a4 displayed obvious inhibitory activity against the recombinant RT with an IC50 value 1.3 µM (Table 2).
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Table 1. Activity against wild-type (IIIB) and resistant mutant strain RES056 (K103N/Y181C, RT) of HIV-1 in MT-4 cell cultures using the MTT method.
IIIB Compds
DSC-a1
R2
-
K103N/Y181C
Ar
-
EC50 a (µM)
SI c
EC50 (µM)
SI
0.046 ± 0.0063
85
≥4.0
≤1
CC50 b (µM)
4.0 ± 0.34
DSC-a2
-
4-SO2Me-Ph
0.097 ± 0.047
82
>8.0
5.1
4.3
3.7
19
3.6
5.0
4.5
89
2.5 ± 1.0
>6
>15
DLV
-
-
0.13 ± 0.043
>227
>36
X1
>36
EFV
-
-
0.0074 ± 0.0026
>855
0.52 ± 0.022
>12
>6.3
ETV
-
-
>184
>4.6
>12594
>94
0.0041 ±
0.025 ± >1127
0.00021
0.0030 0.0074 ±
0.0072 ± AZT
-
>13066
0.00029
0.00087
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell cultures against
HIV-1-induced cytopathicity. bCC50: cytotoxic concentration of compound that reduces the normal uninfected cell viability by 50%. cSI: selectivity index, the ratio of CC50/EC50. X1 stands for ≥1 or
