Design, synthesis, and anti-HIV-1 activity of 1-substituted 3-(3,5-dimethylbenzyl)triazine derivatives

Abstract

Background

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is an attractive target for the development of drugs used in the treatment of HIV-1 infection and acquired immune deficiency syndrome (AIDS). We have continued the search for novel anti-HIV-1 agents using the structure–activity relationships of the successful 1,3-disubstituted and 1,3,6-trisubstituted uracil-type HIV-1 RT inhibitors.

Methods

A series of new triazine analogs were synthesized using an established method. The anti-HIV-1 activities of these compounds were determined based on the inhibition of virus-induced cytopathogenicity in MT-4 cells. The cytotoxicity of the compounds was evaluated by assessing the viability of mock-infected cells.

Results

Some of the compounds showed good-to-moderate activities against HIV-1, with half-maximal effective concentrations (EC₅₀) in the submicromolar range. In particular, a dihydro-1-(4-aminobenzyl)triazine analog showed satisfactory anti-HIV-1 activity with an EC₅₀ of 0.110 µM and a selectivity index (SI) of 909. Furthermore, molecular modeling analyses were performed to explore the major interactions between HIV-1 RT and potent inhibitors. These results may be important for further development of this class of compounds as anti-HIV-1 agents.

Conclusion

The satisfactory anti-HIV-1 activity of triazine analogs may serve as the basis for further investigations of the behavior of this class of compounds against drug-resistant mutants.

Introduction

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is an attractive target for the development of drugs used in HIV-1 infection and AIDS treatment. It is a multifunctional enzyme that is crucial to viral replication. Currently, two functionally distinct classes of HIV-1 RT inhibitors (nucleoside and non-nucleoside) have been discovered and are used clinically. In particular, non-nucleoside RT inhibitors (NNRTIs), with a wide range of chemically diverse structures, have gained an important place in clinical use given their unique antiviral potency, generally low toxicity, and favorable pharmacokinetic properties.^1–3

Among the more than 50 different series of NNRTIs reported hitherto, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT, Figure 1), which possesses a uracil skeleton,^4,5 was the first compound shown to target HIV-1 RT specifically. Since HEPT discovery, related uracil derivatives have been synthesized. In particular, emivirine (Figure 1),^6–9 formerly known as MKC-442, was chosen as a candidate for clinical trials in AIDS patients, and is the best-known HEPT derivative. It also possesses an uracil skeleton. However, the phase III study was halted when emivirine was found to activate the liver enzyme cytochrome P450, which metabolizes protease inhibitors.¹⁰

Figure 1.

Structures of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) and emivirine.

We also continued the search for an anti-HIV-1 agent using the structure–activity relationships of the 1,3-disubstituted and 1,3,6-trisubstituted uracils.^11–16 We have demonstrated that the 3,5-dimethylbenzyl group at the N³-position and the amino group at the C6-position of the uracil skeleton play an important role in enhancing the anti-HIV-1 activity. Notably, substitution at the 1-position (with a benzyl or 4-picolyl group) of 6-amino-3-(3,5-dimethylbenzyl)uracil to obtain 1a–b (Table 1) yielded satisfactory anti-HIV-1 activity, with EC₅₀ values of 0.07 ± 0.01 µM and 0.03 ± 0.03 µM, respectively.^14,15 Furthermore, N¹-4-aminobenzyl-C6-aminouracil derivatives (1c, Table 1) exhibited superior activities, with EC₅₀ values of 0.010 ± 0.006 µM.¹⁶

Table 1.

Structures and anti-HIV-1 activity of 1-substituted-3-(3,5-dimethylbenzyl)uracil derivatives (la–c).

	Compound	R	EC50 (μM)	CC50 (μM)	SI
	Nevirapine	–	0.06 ± 0.01	>100	>1639
	la	benzyl	0.07 ± 0.01	46 ± 1	661
	lb	4-picolyl	0.03 ± 0.03	>100	>2863
	1c	4-aminobenzyl	0.010 ± 0.006	>20	>1923

In an ongoing effort to discover potential NNRTIs with high anti-HIV-1 activity and low cytotoxicity, we designed 22 structurally relevant, novel classes of 1-substituted 3-(3,5-dimethylbenzyl)triazine analogs (3a–c, 4a–p, and 5, Table 2), and 3-(4-fluorobenzyl)-1-(4-methoxybenzyl)triazine derivatives (6a–b, Table 2) by replacing the uracil skeleton with a triazine moiety, by means of using the biological isostere principle,^17–19 as shown in Figure 2. Moreover, it has been confirmed that emivirine bound to HIV-1 RT at the nevirapine-binding site in the X-ray co-crystal structure;²⁰ thus, preliminary structure–activity relationship studies and molecular modeling analyses using two types of prospective ligands (4c and 5, Table 2) were also performed to explore the major interactions between the nevirapine-binding site in HIV-1 RT and 4c or 5.

Table 2.

Antiviral activity of 3-(3,5-dimethylbenzyl)triazine analogs against HIV-1.

Compound	R1	R2	EC50 (μM)	CC50 (μM)	SI
3a	Bn	SMe	3.0 ± 1.0	45 ± 0.2	15
3b	4-MeO-Bn	SMe	6.0 ± 3.6	>37 ± 17	>6
3c	4-NH2-Bn	SMe	4.1 ± 0.3	88 ± 12	22
4a	Bn	NH2	0.19 ± 0.02	>100	>526
4b	4-MeO-Bn	NH2	0.25 ± 0.14	>100	>408
4c	4-NH2-Bn	NH2	0.068 ± 0.030	47 ± 7	691
4d	4-Me-Bn	NH2	0.31 ± 0.21	>100	323
4e	3,5-Mee-Bn	NH2	3.7 ± 0.3	>100	27
4f	4-F-Bn	NH2	0.24 ± 0.01	>100	>417
4g	2,6-F2-Bn	NH2	38 ± 18	>38 ± 18	>1
4h	4-NO2-Bn	NH2	0.7 ± 0.4	35 ± 15	50
4i	4-CN-Bn	NH2	0.35 ± 0.14	>100	>286
4j	4-CF3O-Bn	NH2	3.0 ± 1.0	>73 ± 27	>24
4k	4-CF3-Bn	NH2	1.8 ± 0.4	>67 ± 33	>37
41	4-Picolyl	NH2	0.34 ± 0.11	>100	>294
4m	2-Picolyl	NH2	0.58 ± 0.17	>100	>172
4n	3-Picolyl	NH2	1.2 ± 0.6	>100	>83
4o	CH2CN	NH2	60 ± 40	>60 ± 40	>1
4p	CH2CH2Ph	NH2	10.6 ± 7.5	48 ± 2	5
5	–	–	0.11 ± 0.05	>100	>909
6a	–	–	12 ± 8	>12 ± 8	>1
6b	–	–	60 ± 40	>60 ± 40	>1

EC₅₀, effective concentration; the concentration of compound required to protect the cell against viral cytopathogenicity by 50% in MT-4 cells; CC₅₀, cytotoxic concentration; the concentration of compound that reduces the normal uninfected MT-4 cell viability by 50%; SI, selectivity index (CC₅₀/EC₅₀).

Figure 2.

Design of 1,3,6-trisubstituted triazine derivatives.

Methods

Chemistry

3-(3,5-Dimethylbenzyl)-6-methylthio-1,3,5-triazine-2,4(1H,3H)-dione (2), 6-amino-3-(4-fluorobenzyl)-1-(4-methoxybenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (6a, Table 2), and its 6-hydroxy counterpart (6b, Table 2) were synthesized according to previous reports based on Balboni’s procedure.^21,22 The 1-substituted 6-methylthiotriazine derivatives (3) and 1-substituted 6-aminotriazine analogs (4) were essentially synthesized by alkylation at the 1-position of the triazine skeleton of the intermediate (2), using our previously reported method, as shown in Scheme 1.^14–16

Scheme 1.

Synthesis of 3-(3,5-dimethylbenzyl)triazine derivatives 3a–c, 4a–p, and 5. Reagents and conditions: i, R-X (X = Cl, Br), K₂CO₃, DMF, rt, 3–21 h, 35–98%; ii, liq. NH₃, MeOH, 100°C, 12–50 h, 30–92%; iii, NaBH₄, NiCl₂.6H₂O, MeOH, THF, 0°C, 30 min.

First, compound (2) was allowed to react with the appropriate alkyl halides (e.g. 4-nitrobenzyl bromide, 4-picolyl chloride, or 4-fluorobenzyl bromide) to yield the corresponding 1-alkylated products (3), 35–98% of which was treated with methanolic ammonia to give the 6-aminated products (4) in 30–92% yields. 1-(4-Aminobenzyl)-substituted triazine derivatives (3c, 4c, and 5) were synthesized from the 1-(4-nitrobenzyl)-substituted triazine analog 3h. Reduction of 3h with NaBH₄ and NiCl₂·6H₂O in the presence of MeOH and THF at 0°C afforded the 1-(4-aminobenzyl)-6-methylthiolated triazine analog 3c and dihydro-1-(4-aminobenzyl)triazine derivative 5 at 35% and 53%, respectively.²³ The C6-amination of 3c with methanolic ammonia gave 4c.

Anti-HIV-1 assay

MT-4 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL of penicillin G, and 100 mg/mL of streptomycin. The III_B strain of HIV-1 was used throughout the experiment. The virus was propagated and titrated in MT-4 cells. Virus stocks were stored at −80°C until use. The anti-HIV-1 activity of the test compounds was determined by the inhibition of virus-induced cytopathogenicity in MT-4 cells.²⁴ Briefly, MT-4 cells (1 × 10⁵ cells/mL) were infected with HIV-1 at a multiplicity of infection (MOI) of 0.1 and were cultured in the presence of various concentrations of the test compounds. After four-day incubation at 37°C in 5% CO₂, the number of viable cells was monitored by the water-soluble tetrazolium dye MTT. The cytotoxicity of the compounds was evaluated in parallel with their antiviral activity, based on the viability of mock-infected cells, as determined by the MTT method.

Materials

Instrumentation

¹H NMR and ¹³C NMR spectra were taken with an Ultrashield™ 400 Plus FT NMR System (BRUKER, Germany). Chemical shifts and coupling constants (J) were given in δ and Hz, respectively. Melting points were determined on a Yanaco MP-500D. High-resolution mass spectrometry was performed on an APEX IV mass spectrometer (BRUKER) with electrospray ionization mass spectroscopy (ESI-MS).

Compounds

General procedure for the synthesis of 3

A solution of compound 2 (41.6 mg, 0.15 mmol), appropriate alkyl halide (0.23 mmol) and K₂CO₃ (24.9 mg, 0.18 mmol), in dry DMF (0.25 mL) was stirred at room temperature. After 3–21 h stirring, the mixture was extracted with acetic-acid-ethylester (AcOEt). The organic extracts were washed with water and saturated sodium chloride solution, dried with sodium sulfate, and then evaporated. The residue was purified by silica gel column chromatography to afford 3.

1-Benzyl-3-(3,5-dimethylbenzyl)-6-methylthio-1,3,5-triazine-2,4(1H,3H)-dione (3a)

Yield 95%; white solid; ¹H NMR (400 MHz, CDCl₃): δ 7.31 (5H, m, Bn), 7.08 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 5.12 (2H, s, Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 2.54 (3H, s, SMe), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 170.1, 152.3, 150.5, 138.0, 136.0, 134.3, 129.6, 128.8, 128.3, 127.6, 126.7, 48.4, 45.6, 21.3, 15.3; HRMS (ESI) Calcd for C₂₀H₂₁N₃NaO₂S⁺ [M+Na]⁺: 390.12467. Found 390.12416; melting point (mp): 136.3–137.9°C.

3-(3,5-Dimethylbenzyl)-1-(4-methoxybenzyl)-6-methylthio-1,3,5-triazine-2,4(1H,3H)-dione (3b)

Yield 82%; white solid; ¹H NMR (400 MHz, CDCl₃): δ 7.29 (2H, d, J 8.4, 4-OMe-Bn), 7.07 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 6.85 (2H, d, J 8.4, 4-OMe-Bn), 5.05 (2H, s, 4-OMe-Bn), 5.02 (2H, s, 3,5-Me₂-Bn), 3.78 (3H, s, 4-OMe-Bn), 2.55 (3H, s, SMe), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 170.0, 159.6, 152.3, 150.5, 138.0, 136.0, 129.5, 129.5, 126.7, 126.3, 114.1, 55.3, 48.0, 45.6, 21.3, 15.3; HRMS (ESI) Calcd for C₂₁H₂₃N₃NaO₃S⁺ [M+Na]⁺: 420.13523. Found 420.13447; mp: 128.7–130.1°C.

1-(4-Aminobenzyl)-3-(3,5-dimethylbenzyl)-6-methylthio-1,3,5-triazine-2,4(1H,3H)-dione (3c)

Yield 35%; brown solid; ¹H NMR (400 MHz, CDCl₃): δ 7.17 (2H, d, J 8.4, 4-NH₂-Bn), 7.08 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 6.62 (2H, d, J 8.4, 4-NH₂-Bn), 5.03 (2H, s, 4-NH₂-Bn), 5.01 (2H, s, 3,5-Me₂-Bn), 3.71 (2H, brs, 4-NH₂-Bn), 2.56 (3H, s, SMe), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 170.0, 152.4, 150.5, 146.6, 138.0, 136.0, 129.6, 129.5, 127.0, 124.0, 115.0, 48.2, 45.6, 21.2, 15.3; HRMS (ESI) Calcd for C₂₀H₂₂N₄NaO₂S⁺ [M+Na]⁺: 405.13557. Found 405.13494; mp: 88.5–88.6°C.

General procedure for the synthesis of 4

Compound 3 (0.12 mmol) was dissolved in NH₃ (14.0 mL)/MeOH (3.0 mL), and then sealed and stirred for 12–50 h at 100°C. The mixture was evaporated, and the residue was purified by silica gel column chromatography (20–25% MeOH in CH₂Cl₂) to afford 4.

6-Amino-1-benzyl-3-(3,5-dimethylbenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4a)

Yield 53%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 7.30 (5H, m, Bn), 7.10 (2H, brs, 6-NH₂), 6.58 (1H, s, 3,5-Me₂-Bn), 6.83 (2H, s, 3,5-Me₂-Bn), 5.07 (2H, s, Bn), 4.83 (2H, s, 3,5-Me₂-Bn), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 155.9, 153.6, 151.2, 137.3, 137.1, 135.6, 128.4, 128.3, 127.3, 126.3, 124.8, 45.0, 44.2, 20.8; HRMS (ESI) Calcd for C₁₉H₂₀N₄NaO₂⁺ [M+Na]⁺: 359.14785. Found 359.14730; mp: 196.1–197.2°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(4-methoxybenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4b)

Yield 58%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 7.72 (2H, s, 6-NH₂), 7.21 (2H, d, J 8.4, 4-OMe-Bn), 6.90 (2H, d, J 8.4, 4-OMe-Bn), 6.86 (1H, s, 3,5-Me₂-Bn), 6.82 (2H, s, 3,5-Me₂-Bn), 4.98 (2H, s, 4-OMe-Bn), 4.82 (2H, s, 3,5-Me₂-Bn), 3.73 (3H, s, 4-OMe-Bn), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 158.7, 155.9, 153.6, 151.3, 137.4, 137.2, 128.4, 128.2, 127.6, 124.9, 113.9, 55.1, 44.5, 44.3, 20.9; HRMS (ESI) Calcd for C₂₀H₂₂N₄NaO₃⁺ [M+Na]⁺: 389.15841. Found 389.15798; mp: 237.9–239.0°C.

6-Amino-1-(4-aminobenzyl)-3-(3,5-dimethylbenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4c)

Yield 86%; pale yellow solid; ¹H NMR (400 MHz, CD₃OD): δ 6.93 (2H, d, J 8.4, 4-NH₂-Bn), 6.83 (2H, s, 3,5-Me₂-Bn), 6.78 (1H, s, 3,5-Me₂-Bn), 6.57 (2H, d, J 8.4, 4-NH₂-Bn), 4.88 (2H, s, 4-NH₂-Bn), 4.86 (2H, s, 3,5-Me₂-Bn), 2.15 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CD₃OD): δ 158.3, 157.3, 152.9, 148.9, 139.1, 138.3, 129.9, 129.0, 126.6, 125.0, 116.6, 61.5, 46.3, 21.4; HRMS (ESI) Calcd for C₁₉H₂₁N₅NaO₂⁺ [M+Na]⁺: 374.15875. Found 374.15808; mp: 227.4–228.4°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(4-methylbenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4d)

Yield 80%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 7.75 (2H, brs, NH₂), 7.14 (4H, m, 4-Me-Bn), 6.86 (2H, s, 3,5-Me₂-Bn), 6.83 (1H, s, 3,5-Me₂-Bn), 5.01 (2H, s, 4-Me-Bn), 4.82 (2H, s, 3,5-Me₂-Bn), 2.27 (3H, s, 4-Me-Bn), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 156.0, 153.6, 151.3, 137.4, 137.2, 136.6, 132.7, 129.0, 128.4, 126.5, 124.9, 44.8, 44.3, 20.9, 20.6; HRMS (ESI) Calcd for C₂₀H₂₂N₄NaO₂⁺ [M+Na]⁺: 373.16350. Found 373.16299; mp: 243.0–244.6°C.

6-Amino-1-(3,5-dimethylbenzyl)-3-(3,5-dimethylbenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4e)

Yield 81%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 7.71 (2H, brs, NH₂), 6.91 (1H, s, 3,5-Me₂-Bn), 6.86 (1H, s, 3,5-Me₂-Bn), 6.84 (2H, s, 3,5-Me₂-Bn), 6.81 (2H, s, 3,5-Me₂-Bn), 4.98 (2H, s, 3,5-Me₂-Bn), 4.83 (2H, s, 3,5-Me₂-Bn), 2.23 (6H, s, 3,5-Me₂-Bn), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 156.0, 153.7, 151.3, 137.5, 137.5, 137.2, 135.5, 128.8, 128.4, 124.8, 124.0, 44.9, 44.3, 20.9; HRMS (ESI) Calcd for C₂₁H₂₄N₄NaO₂⁺ [M+Na]⁺: 387.17915. Found 387.17857; mp: 267.3–268.6°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(4-fluorobenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4f)

Yield 62%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 7.78 (2H, s, NH₂), 7.31 (2H, m, 4-F-Bn), 7.18 (2H, m, 4-F-Bn), 6.86 (1H, s, 3,5-Me₂-Bn), 6.83 (2H, s, 3,5-Me₂-Bn), 5.03 (2H, s, 4-F-Bn), 4.82 (2H, s, 3,5-Me₂-Bn), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 161.8 (d, J 246), 155.9, 153.6, 151.3, 137.4, 137.2, 132.0, 128.8 (d, J 8), 128.4, 124.9, 115.2 (d, J 21), 44.5, 44.3, 20.9; HRMS (ESI) Calcd for C₁₉H₁₉FN₄ NaO₂⁺ [M+Na]⁺: 377.13843. Found 377.13797; mp: 235.5–236.2°C.

6-Amino-1-(2,6-difluorobenzyl)-3-(3,5-dimethylbenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4g)

Yield 30%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 10.97 (2H, brs, NH₂), 7.29 (2H, m, 2,6-F₂-Bn), 6.92 (2H, s, 3,5-Me₂-Bn), 6.87 (1H, s, 2,6-F₂-Bn), 6.87 (1H, s, 3,5-Me₂-Bn), 4.90 (2H, s, 2,6-F₂-Bn), 4.84 (2H, s, 3,5-Me₂-Bn), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 161.0 (dd, J 248 and 7), 152.2, 150.1, 137.2, 135.9, 130.2 (dd, J 10 and 10), 129.5, 126.5, 111.8 (dd, J 19 and 6), 110.3 (dd, J 16 and 16), 44.3, 38.0 (dd, J 4 and 4), 20.9; HRMS (ESI) Calcd for C₁₉H₁₈F₂N₄ NaO₂⁺ [M+Na]⁺: 395.12900. Found 395.12877; mp: 252.9–254.8°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(4-nitrobenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4 h)

Yield 61%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 8.22 (2H, d, J 8.4, 4-NO₂-Bn), 7.85 (2H, s, NH₂), 7.51 (2H, d, J 8.4, 4-NO₂-Bn), 6.85 (2H, s, 3,5-Me₂-Bn), 6.85 (1H, s, 3,5-Me₂-Bn), 5.18 (2H, s, 4-NO₂-Bn), 4.82 (2H, s, 3,5-Me₂-Bn), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 155.9, 153.7, 151.2, 146.8, 143.6, 137.3, 137.2, 128.4, 127.6, 125.0, 123.6, 45.1, 44.4, 20.9; HRMS (ESI) Calcd for C₁₉H₁₉N₅NaO₄⁺ [M+Na]⁺: 404.13293. Found 404.13250; mp: 256.2–257.2°C.

6-Amino-1-(4-cyanobenzyl)-3-(3,5-dimethylbenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4i)

Yield 69%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 7.83 (2H, d, J 7.6, 4-CN-Bn), 7.80 (2H, brs, NH₂), 7.42 (2H, d, J 7.6, 4-CN-Bn), 6.85 (1H, s, 3,5-Me₂-Bn), 6.84 (2H, s, 3,5-Me₂-Bn), 5.13 (2H, s, 4-CN-Bn), 4.82 (2H, s, 3,5-Me₂-Bn), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 155.9, 153.7, 151.3, 141.5, 137.4, 137.2, 132.4, 128.4, 127.3, 125.0, 118.7, 110.1, 45.2, 44.4, 20.9; HRMS (ESI) Calcd for C₂₀H₁₉N₅NaO₂⁺ [M+Na]⁺: 384.14310. Found 384.14269; mp: 264.9–266.4°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(4-trifluoromethoxybenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4j)

Yield 92%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 7.81 (2H, brs, NH₂), 7.36 (2H, m, 4-OCF₃-Bn), 7.36 (2H, m, 4-OCF₃-Bn), 6.87 (1H, s, 3,5-Me₂-Bn), 6.82 (2H, s, 3,5-Me₂-Bn), 5.07 (2H, s, 4-OCF₃-Bn), 4.81 (2H, s, 3,5-Me₂-Bn), 2.21 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 155.9, 154.1, 151.3, 137.4, 137.2, 135.3, 128.4, 128.4, 124.9, 121.2, 117.3, 44.6, 44.3, 20.8; HRMS (ESI) Calcd for C₂₀H₁₉F₃N₄NaO₃⁺ [M+Na]⁺: 443.13015. Found 443.12966; mp: 246.8–247.9°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(4-trifluoromethylbenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4k)

Yield 56%; white needle; ¹H NMR (400 MHz, DMSO-d₆): δ 7.82 (2H, brs, NH₂), 7.72 (2H, d, J 8.0, 4-CF₃-Bn), 7.46 (2H, d, J 8.0, 4-CF₃-Bn), 6.85 (1H, s, 3,5-Me₂-Bn), 6.83 (2H, s, 3,5-Me₂-Bn), 5.14 (2H, s, 4-CF₃-Bn), 4.82 (2H, s, 3,5-Me₂-Bn), 2.21 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 155.9, 153.6, 151.3, 147.5, 140.7, 137.3, 137.2, 128.4 (q, J 40), 127.2, 125.4 (q, J 3), 124.9, 45.0, 44.3, 20.8; HRMS (ESI) Calcd for C₂₀H₁₉F₃N₄NaO₂⁺ [M+Na]⁺: 427.13523. Found 427.13472; mp: 232.0–232.7°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(4-picolyl)-1,3,5-triazine-2,4(1H,3H)-dione (4l)

Yield 69%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 8.52 (2H, m, 4-picolyl), 7.80 (2H, brs, NH₂), 7.22 (2H, m, 4-picolyl), 6.84 (2H, s, 3,5-Me₂-Bn), 6.84 (1H, s, 3,5-Me₂-Bn), 5.08 (2H, s, 4-picolyl), 4.82 (2H, s, 3,5-Me₂-Bn), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 155.9, 153.7, 151.2, 149.7, 144.9, 137.4, 137.2, 128.4, 124.9, 121.3, 44.5, 44.3, 20.9; HRMS (ESI) Calcd for C₁₈H₁₉N₅NaO₂⁺ [M+Na]⁺: 360.14310. Found 360.14257; mp: 227.9–228.6°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(2-picolyl)-1,3,5-triazine-2,4(1H,3H)-dione (4m)

Yield 58%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 8.51 (1H, m, 2-picolyl), 7.80 (1H, m, 2-picolyl), 7.32 (2H, m, 2-picolyl), 6.85 (1H, s, 3,5-Me₂-Bn), 6.83 (2H, s, 3,5-Me₂-Bn), 5.16 (2H, s, 2-picolyl), 4.81 (2H, s, 3,5-Me₂-Bn), 2.21 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 156.6, 154.5, 153.8, 151.4, 148.9, 137.4, 137.1, 136.8, 128.3, 124.8, 122.6, 121.5, 46.6, 44.2, 20.9; HRMS (ESI) Calcd for C₁₈H₁₉N₅NaO₂⁺ [M+Na]⁺: 360.14310. Found 360.14233; mp: 188.7–189.6°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(3-picolyl)-1,3,5-triazine-2,4(1H,3H)-dione (4n)

Yield 79%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 8.51 (2H, m, 3-picolyl), 7.63 (1H, m, 3-picolyl), 7.38 (1H, m, 3-picolyl), 6.84 (2H, s, 3,5-Me₂-Bn), 6.84 (1H, s, 3,5-Me₂-Bn), 5.08 (2H, s, 3-picolyl), 4.82 (2H, s, 3,5-Me₂-Bn), 2.50 (3H, s, SMe), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 155.8, 153.6, 151.3, 148.6, 148.3, 137.4, 137.2, 134.3, 131.5, 128.4, 124.9, 123.5, 44.3, 43.2, 20.9; HRMS (ESI) Calcd for C₁₈H₁₉N₅NaO₂⁺ [M+Na]⁺: 360.14310. Found 360.14238; mp: 265.2–266.4°C.

6-Amino-1-cyanomethyl-3-(3,5-dimethylbenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (4o)

Yield 59%; brown solid; ¹H NMR (400 MHz, DMSO-d₆): δ 9.01 (2H, brs, NH₂), 6.88 (2H, s, 3,5-Me₂-Bn), 6.87 (1H, s, 3,5-Me₂-Bn), 4.81 (2H, s, cyanomethyl), 4.66 (2H, s, 3,5-Me₂-Bn), 2.22 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 178.0, 158.5, 148.1, 137.3, 137.1, 128.4, 125.1, 49.7, 43.6, 20.9; HRMS (ESI) Calcd for C₁₄H₁₅N₅NaO₂⁺ [M+Na]⁺: 308.11180. Found 308.11123; mp: 286.1–287.8°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(4-phenethyl)-1,3,5-triazine-2,4(1H,3H)-dione (4p)

Yield 55%; white solid; ¹H NMR (400 MHz, DMSO-d₆): δ 7.79 (2H, brs, NH₂), 7.27 (5H, m, phenethyl), 6.87 (1H, s, 3,5-Me₂-Bn), 6.84 (2H, s, 3,5-Me₂-Bn), 4.78 (2H, s, 3,5-Me₂-Bn), 4.02 (2H, t, J 7.2, phenethyl), 2.85 (2H, t, J 7.2, phenethyl), 2.24 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 155.8, 153.8, 151.0, 137.7, 137.5, 137.1, 128.9, 128.4, 128.2, 126.4, 125.1, 44.2, 43.4, 32.8, 20.9; HRMS (ESI) Calcd for C₂₀H₂₂N₄NaO₂⁺ [M+Na]⁺: 373.16350. Found 373.16296; mp: 208.8–210.5°C.

Dihydro-1-(4-aminobenzyl)-3-(3,5-dimethylbenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (5)

Compound 3h (0.085 g, 0.21 mmol) was dissolved in the 3:1 mixture of dry MeOH (3.0 mL) and dry THF (1.0 mL), and NaBH₄ (0.033 g, 1.00 mmol) and NiCl₂· 6H₂O (0.08 g, 0.32 mmol) were added to the solution, which was stirred for 30 min at 0°C. The mixture was extracted with AcOEt, washed with saturated aqueous sodium chloride solution, dried with sodium sulfate, and then evaporated. The residue was purified by silica gel column chromatography (AcOEt) to give compound 3c (0.028 g, 0.07 mmol, 35%, the structural assignment was described above) and 5 (0.038 g, 0.11 mmol, 53%), respectively. Compound 5: yield 53%; brown solid; ¹H NMR (400 MHz, CDCl₃): δ 7.04 (2H, d, J 8.4, 4-NH₂-Bn), 6.97 (2H, s, 3,5-Me₂-Bn), 6.87 (1H, s, 3,5-Me₂-Bn), 6.67 (1H, brs, NH), 6.61 (2H, d, J 8.4, 4-NH₂-Bn), 4.88 (2H, s, 4-NH₂-Bn), 4.43 (2H, s, 3,5-Me₂-Bn), 4.24 (2H, d, J 2.0, triazine-CH₂), 3.72 (2H, brs, 4-NH₂-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 154.5, 153.2, 146.3, 138.1, 137.8, 129.5, 128.9, 125.8, 125.2, 115.2, 53.3, 49.0, 44.0, 21.3; HRMS (ESI) Calcd for C₁₉H₂₂N₄NaO₂⁺ [M+Na]⁺: 361.16350. Found 361.16325; mp: 65.7–67.8°C.

6-Amino-3-(4-fluorobenzyl)-1-(4-methoxybenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (6a)

White solid; ¹H NMR (400 MHz, DMSO-d₆): δ 8.03 (2H, s, 6-NH₂), 7.34 (2H, m, 4-F-Bn), 7.21 (2H, d, J 8.4, 4-OMe-Bn), 7.13 (2H, m, 4-F-Bn), 6.90 (2H, d, J 8.4, 4-OMe-Bn), 4.97 (2H, s, 4-OMe-Bn), 4.88 (2H, s, 4-F-Bn), 3.73 (3H, s, 4-OMe-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 161.2 (d, J 241), 158.7, 155.8, 153.3, 151.2, 133.7 (d, J 3), 129.7 (d, J 8), 128.2, 127.4, 115.0 (d, J 22), 113.8, 55.0, 44.5, 43.7; HRMS (ESI) Calcd for C₁₈H₁₇FN₄NaO₃⁺ [M+Na]⁺: 379.11769. Found 379.11696; mp: 233.4–234.6°C.

3-(4-Fluorobenzyl)-6-hydroxy-1-(4-methoxybenzyl)-1,3,5-triazine-2,4(1H,3H)-dione (6b)

White solid; ¹H NMR (400 MHz, DMSO-d₆): δ 11.88 (1H, s, 6-OH), 7.39 (2H, m, 4-F-Bn), 7.26 (2H, d, J 8.8, 4-OMe-Bn), 7.14 (2H, m, 4-F-Bn), 6.87 (2H, d, J 8.8, 4-OMe-Bn), 4.85 (2H, s, 4-OMe-Bn), 4.80 (2H, s, 4-F-Bn), 3.72 (3H, s, 4-OMe-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 161.4 (d, J 242), 158.5, 150.1, 148.7., 148.7, 132.7 (d, J 3), 129.6 (d, J 9), 129.1, 128.4, 115.0 (d, J 21), 113.6, 55.0, 44.0, 43.8; HRMS (ESI) Calcd for C₁₈H₁₆FN₃NaO₄⁺ [M+Na]⁺: 380.10171. Found 380.12046; mp: 157.1–158.1°C.

Results

Biological activity

The antiviral activities of the 6-methylthio (3a–c), 6-amino (4a–p), and dihydro (5) derivatives of 1-substituted-3-(3,5-dimethylbenzyl)triazine and 6-amino (6a) and 6-hydroxy (6b) analogs of 1-(4-methoxybenzyl)-3-(4-fluorobenzyl)triazine were determined by examining the inhibitory effects of these compounds on HIV-1-induced cytopathogenicity and cell viability in MT-4 cells. The cytotoxicity of these compounds, which were dissolved in dimethyl sulfoxide at concentrations up to 100 µM, was evaluated based on the viability of mock-infected cells, as determined by the MTT method. Some of the compounds in the series of 6-amino derivatives of 1-benzylated-3-(3,5-dimethylbenzyl)triazine (4a–k) exhibited good-to-moderate anti-HIV-1 activity with EC₅₀ values ranging from 0.068 µM to 3.7 µM, except for the 1-(2,6-difluorobenzyl)triazine derivative 4g, which had an EC₅₀ value of 38 ± 18 µM (Table 2). Among these compounds, the 6-amino derivative of N¹-(4-aminobenzyl)triazine 4c showed satisfactory anti-HIV-1 activity, with an EC₅₀ value of 0.068 ± 0.030 µM, and exhibited moderate values of CC₅₀ (47 ± 7 µM) and selectivity index (SI) (691). The introduction of the p-amino group of the benzyl skeleton at the N¹ position in triazine increased the activity of the compounds compared to the activity of the unsubstituted compound 4a (EC₅₀ = 0.19 ± 0.02 µM). A similar result was obtained for the N¹-(4-aminobenzyl)uracil analog 1c (EC₅₀ = 0.010 ± 0.006 µM), compared to the unsubstituted compound 1a (EC₅₀ = 0.07 ± 0.01 µM) (Table 1).¹⁶

In contrast, three types of 6-methylthiolated derivatives (3a–c) exhibited considerably less anti-HIV-1 activity than their 6-amino counterparts (4a–c); for example, 3a (N¹-benzyl-6-methylthiotriazine) was approximately 16 times less potent (EC₅₀ = 3.0 ± 1.0 µM, SI = 15) than its N¹-benzyl-6-aminotriazine analog, 4a (EC₅₀ = 0.19 ± 0.02 µM, SI > 526). The 6-(4-picolyl), 6-(2-picolyl), and 6-(3-picolyl) derivatives 4l–n showed moderate anti-HIV-1 activity with EC₅₀ values from 0.34 µM to 1.2 µM.

Additionally, the dihydro-N¹-4-aminobenzyltriazine analog 5, in which the 6-methylthio group of the triazine system of 3c was removed and hydrogenated, showed not only comparable anti-HIV-1 activity (EC₅₀ = 0.11 ± 0.05 µM) but also low cytotoxicity with CC₅₀ and SI values of > 100 µM and > 909, respectively.

Unfortunately, the 6-(cyanomethyl) and 6-(ethylphenyl) analogs (4o–p) and the 6-amino or 6-hydroxy derivatives of N³-(4-fluorobenzyl)-N¹-(4-methoxybenzyl)triazine (6a–b) hardly demonstrated anti-HIV-1 activity, indicating that the N¹-benzyl or picolyl skeleton as well as the N³-3,5-dimethylbenzyl system plays an important role in the anti-HIV-1 activity of the triazine series.

Discussion

Molecular modeling analysis

The X-ray co-crystal structure (PDB: 1VRT) of HIV-1 RT with bound nevirapine was taken from PDB² for use in docking studies. A docking model comprising ligand 4c or 5, which showed the most promising anti-HIV-1 activity, bound to HIV-1 RT, was constructed by a conformational search using MacroModel (ver. 9.1). AMBER* was used as the force field, and more than 3000 conformers of the corresponding ligands were optimized. Figure 3(a) and (b) shows the molecular docking of 4c and 5, respectively, in the allosteric site of HIV-1 RT. This docking structure was found to be significantly different from that of the nevirapine-binding site in the HIV-1-emivirine complex.²⁰ The 6-amino group of 4c is hydrogen bonded to the amide group of the Lys101 residue (NH^…O = C), while the 6-methylen moiety of the reduced triazine analog 5 is not hydrogen bonded to the Lys101 residue. Moreover, the 3,5-dimethylbenzyl moiety of 4c or 5, which was oriented around the hydrophobic area, enhanced the π–π stacking of the benzene rings of the Tyr181 and Tyr188 residues. A CH–π interaction was observed between the methyl group of the 3,5-dimethylbenzyl moiety and the indole skeleton of the Trp229 residue, or between the benzene rings of the 3,5-dimethylbenzyl moiety and the isobutyl groups of the Leu234 residue. In particular, the result of this calculation of HIV-1 RT with bound 4c was almost the same as that for N¹-benzyluracil (1a), N¹-4-picolyluracil (1b), or N¹-4-aminobenzyluracil (1c).^15,16 As to the comparison of the triazine ring with an uracil skeleton for anti-HIV-1 activity, for instance, triazine analog 4a was less potent (EC₅₀ = 0.19 ± 0.02 µM, Table 2) than the uracil counterpart 1c (0.07 ± 0.01 µM, Table 1), suggesting that nitrogen atom at the N⁵ position of triazine ring might contribute to the decrease in the anti-HIV-1 activity.

Figure 3.

(a) Molecular docking of 6-amino-N¹-4-aminobenzyl analog 4c into the allosteric site of HIV-1 RT (PDB code: 1VRT); (b) Molecular docking of dihydro-N¹-4-aminobenzyl derivative 5 into the allosteric site of HIV-1 RT (PDB code: 1VRT); (c) Superimposition of the docked conformation of 4c (cyan) and 5 (gray) in HIV-1 RT (PDB code: 1VRT).

Figure 3(c) shows the overlay of the docked conformations of the 6-amino analog 4c (cyan) and dihydro-triazine derivative 5 (gray) bound to HIV-1 RT at the nevirapine-binding site, showing that the most stable conformations of 4c and 5 were substantially similar.

In conclusion, we designed 22 structurally relevant, novel classes of 1-substituted 3-(3,5-dimethylbenzyl)triazine analogs, and 3-(4-fluorobenzyl)-1-(4-methoxybenzyl)triazine derivatives. The satisfactory anti-HIV-1 activity of triazine analogs may serve as the basis for further investigations of the behavior of this class of compounds against drug-resistant mutants.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: partially supported by a Grant-in-Aid for Young Scientists (B), No. 24790123, from the Japan Society for the Promotion of Science (JSPS).