Design,synthesis,and anti-HIV-1 activity of 1-aromatic methyl-substituted 3-(3,5-dimethylbenzyl)uracil and N -3,5-dimethylbenzyl-substituted urea derivatives

Abstract

Background

A new series of 1-aromatic methyl-substituted 3-(3,5-dimethylbenzyl)uracil and N-3,5-dimethylbenzyl-substituted urea derivatives were synthesized and evaluated as non-nucleoside HIV-1 reverse transcriptase inhibitors.

Methods

A series of new 6-azido and 6-amino derivatives of 1-substituted-3-(3,5-dimethylbenzyl)uracils were synthesized using our previously reported method, and three acyclic derivatives were synthesized from urea. The anti-HIV-1 activities of these compounds were determined based on the inhibition of virus-induced cytopathogenicity in MT-4 cells. The cytotoxicities of the compounds were evaluated using the viability of mock-infected cells.

Results

Some of these compounds showed good-to-moderate activities against HIV-1 with half maximal effective concentration (EC₅₀) values in the submicromolar or subnanomolar range. Compared with emivirine, compound 6-amino-3-(3,5-dimethylbenzyl)-1-(4-aminobenzyl)uracil showed significant anti-HIV-1 activity with an EC₅₀ value of 10 nM and a high selectivity index of 1923. Preliminary structure–activity relationship studies and molecular modeling analyses were carried out to explore the major interactions between HIV-1 reverse transcriptase and the potent inhibitor 6-amino-3-(3,5-dimethylbenzyl)-1-(4-aminobenzyl)uracil; these results may be important for further development of this class of compounds as anti-HIV-1 agents.

Conclusion

The excellent activity of 6-amino-3-(3,5-dimethylbenzyl)-1-(4-aminobenzyl)uracil (EC₅₀: 0.010 ± 0.006 µM, SI: >1923) may serve as the basis for conducting further investigations on the behavior of this class of compounds against drug-resistant mutants.

Keywords

Anti-HIV-1 agents non-nucleoside reverse transcriptase inhibitors (NNRTIs)Uracil analogs Molecular modeling analysis HIV-1 reverse transcriptase (RT)

Introduction

In contrast to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) are highly specific. NNRTIs bind allosterically to the enzyme, thus making the catalytic reverse transcriptase (RT) subunit inactive.^1,2 Much effort has been made in the design and synthesis of NNRTIs, with more than 30 structurally different classes of NNRTIs reported.^3,4 One of the first NNRTIs, 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (Figure 1, HEPT), possesses the uracil skeleton.^5,6 Derivatives of HEPT have been investigated as NNRTIs for more than two decades. In particular, emivirine (Figure 1),^7–10 formerly known as MKC-442 and chosen as a candidate for clinical trials with AIDS patients, is the best-known HEPT derivative and also possesses the uracil skeleton.

Figure 1.

Structures of HEPT, emivirine, and l-substituted-3-(3,5-dimethylbenzyl)uracil derivatives (la–g).

However, the phase III study was halted when emivirine was found to activate the liver enzyme Cytochrome P450, which metabolizes protease inhibitors.¹¹ Although several NNRTIs, such as nevirapine, delavirdine, efavirenz, etravirine, and rilpivirine, have been approved by US Food and Drug Administration (FDA) for the treatment of HIV-1 infection, these molecules rapidly induce the development of drug-resistant HIV-1 strains. Therefore, we have actively investigated the development of an anti-HIV-1 agent using the structure–activity relationship (SAR) studies of 1,3-disubstituted and 1,3,6-trisubstituted uracils.^12–15 We have demonstrated that the 3,5-dimethylbenzyl group at the 3-position of the uracil skeleton plays an important role in the enhancement of anti-HIV-1 activity; notably, substitution at the 1-position (e.g. with a benzyl, cyanomethyl, or 4-picolyl group) of 3-(3,5-dimethylbenzyl)uracil to obtain 1a–c resulted in good antiviral activity against HIV-1.¹² Moreover, the azido or amino group introduced at the 6-position of 1-benzyl-3-(3,5-dimethylbenzyl)uracil 1d and 1e, respectively, resulted in satisfactory activity with EC₅₀ values of 0.067 ± 0.011 µM and 0.069 ± 0.006 µM, respectively.¹⁴ Furthermore, N¹-picolyl-C6-azido and N¹-picolyl-C6-amino-uracil derivatives, 1f and 1g, exhibited superior activities with EC₅₀ values of 0.050 ± 0.020 µM and 0.030 ± 0.030 µM, respectively. However, the EC₅₀ values of the abovementioned compounds 1a–g in our previous work were not satisfactory. Therefore, we evaluated newly synthesized 1,3,6-trisubstituted uracils with the aim of achieving greater anti-HIV-1 activity and lower cytotoxicity. In particular, as shown in Figure 2, we synthesized the 1-aromatic methyl-substituted derivatives of 6-azido or amino-3-(3,5-dimethylbenzyl)uracil to investigate the substituent effect by replacing the N¹-picolyl group of 1f or 1g with an appropriate aromatic-substituted methyl group such as 4-aminobenzyl, 2-furanylmethyl, and 4-pyrimidinylmethyl groups. Moreover, N-3,5-dimethylbenzyl-substituted urea derivatives with acyclic rings, as shown in Figure 2, were prepared to investigate whether the uracil skeleton is essential as the ligand to bind to HIV-1 RT. We also report the molecular-docking studies of some interesting ligands (7a and 7b) with the nevirapine-binding site in HIV-1 RT.

Figure 2.

Structural modification on l-substituted-3-(3,5-dimethylbenzyl)uracil derivatives If and lg.

Methods

Chemistry

The 1-substituted 6-azidouracil derivatives (5) and 1-substituted 6-aminouracil derivatives (6 and 7) were synthesized by alkylation at the 1-position of the uracil skeleton of the readily available 6-chlorouracil 2 using our previously reported method, as shown in Figure 3, Scheme 1.^12–15 First, 2 was reacted with appropriate alkyl halides (e.g. 4-nitrobenzyl bromide, 2-nitrobenzyl bromide, or 4-fluorobenzyl bromide) to afford the corresponding 1-alkylated products, 3. The Mitsunobu reaction of 3 treated with 3,5-dimethylbenzyl alcohol^14,15 in the presence of PPh₃ and TMAD (N,N,N',N'-tetramethylazodicarboxamide) yielded the 3-(3,5-dimethylbenzyl) congeners, 4. The nucleophilic substitution of the 6-chloro derivatives, 4, at the C6-position of the uracil moiety with sodium azide afforded the corresponding 6-azido derivatives, 5. The reduction of the 6-azido derivatives, 5 (except for 5e), with LiAlH₄ yielded the corresponding 6-amino derivatives, 6. The conversion of 5e was performed using NaBH₄/MeOH/THF¹⁶ to afford 6e, thus avoiding the excessive reduction of the cyanobenzyl group of 5e. The reactions of 6-azido-1-(4-nitrobenzyl)uracil derivative 5a and 6-azido-1-(2-nitrobenzyl)uracil derivative 5b with NaBH₄ and NiCl₂·6H₂O in the presence of MeOH and THF at 0°C afforded the corresponding diamino products (7a and 7b, respectively).¹⁷

Figure 3, Scheme 1.

Synthesis of 6-azido-l-substitueted-3-(3,5-dimethylbenzyl)uracil (5) and 6-amino-l-substitueted-3-(3,5-dimethylbenzyl)uracil (6 and 7). Reagents and conditions: (i) R₁X (X = Cl, Br), K₂CO₃, DMF, rt-70°C, 2–18 h, 25–75%; (ii) 3,5-(CH₃)₂BnOH, PPh₃, TMAD, THF, 50°C, 11–48 h, 60–84%; (iii) NaN₃, DMF, rt, 15–35 min, 65–98%; iv, LiAlH₄, THF, 0°C, 30 min, 69–74%; v, NaBH₄, MeOH, THF, 65°C, 30–50 min, 93–95%; vi, NaBH₄, NiCl₂-6H₂O, MeOH, THF, 0°C, 30 min, 80–90%.

1-(4-Hydroxybenzyl) or 1-(4-pyrimidinylmethyl)-substituted uracil derivatives (11, 12, 14, and 15) were synthesized from a previously reported intermediate,¹⁵ 6-chloro-3-(3,5-dimethylbenzyl)uracil 8. The alkylation of 8 afforded 1-substituted uracil derivatives 9 and 13 (Figure 4, Scheme 2); subsequent C6-azidation afforded products 10 and 14, respectively. The deprotection of the acetyl group of 10 with K₂CO₃/MeOH afforded C6-azido compound 11, whereas the reduction of 10 with LiAlH₄/THF afforded C6-amino derivative 12. The C6-amination of 14 with NaBH₄/MeOH/THF¹⁶ afforded 15.

Figure 4, Scheme 2.

Synthesis of 6-azido (or amino)-3-(3,5-dimethylbenzyl)-l-substituted uracils (11,12,14 and 15). Reagents and conditions: (a) 4-AcO-BnOH, PPh₃, TMAD, THF, 50°C, 12 h, 64%; (b) NaN₃, DMF, rt, 30 min, 89–96%; (c) K₂CO₃, MeOH, rt, 1 h, 78%; iv, LiAlH₄, THF, 0°C, 30 min, 95%; v, 4-(chloromethyl)pyrimidine, K₂CO₃, Nal, DMF, 18 h, 43%, vi, NaBH₄ MeOH, THF, 65°C, 55 min, 58%.

Three acyclic derivatives (20aa, 20ab, and 20b) were synthesized from urea (Figure 5, Scheme 3).¹⁸ Urea 16 was refluxed with H₂O in the presence of benzylamine or 4-aminomethylpyridine to afford N-benzylurea 17a and N-(4-picolyl)urea 17b, respectively. Condensation of 17a and 17b with 3,5-dimethylbenzaldehyde in the presence of titanium(IV) isopropoxide and subsequent reduction with NaBH₄¹⁸ afforded the condensation products 18a and 18b, respectively. The treatment of N′-3,5-dimethylbenzyl derivatives 18a or 18b with trichloroacetyl isocyanate, followed by silica gel chromatography, afforded the intermediate 19aa and its regioisomer 19ab, or a single product 19b. Subsequently, 19aa, 19ab, and 19b were individually subjected to acidic methanolysis with MeOH/silica gel to afford 20aa, 20ab, and 20b;¹⁹ the regioisomers were identified using ¹H-NMR and COSY spectra.

Figure 5, Scheme 3.

Synthesis of IV-carbamoylurea derivatives (20aa, 20ab, and 20b). Reagents and conditions: (a) benzylamine, H₂O, 130°C, 2 h, 17a: 61% yield, 17b: 74% yield; (b) 3,5-dimethylbenzylaldehyde, titanium(iv) isopropoxide, THF, 50°C, 2 h, 18a: 79% yield, 18b: 20% yield; (c) trichloroacetylisocyanate, THF, rt, 1 h, 19aa: 32% yield, 19ab: 38% yield, 19b: 70% yield; (4) methanol, silica gel, 50°C, 40 h, 20aa: 74% yield, 20ab: 83% yield, 20b: 81% yield.

Anti-HIV-1 assay

MT-4 cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 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.²⁰ Briefly, MT-4 cells (1 × 10⁵ cells/mL) were infected with HIV-1 at a multiplicity of infection of 0.1 and were cultured in the presence of various concentrations of the test compounds. After 4-day incubation at 37°C in 5% CO₂, the number of viable cells was monitored by the water-soluble tetrazolium dye WST-8. 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 WST-8 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 3a–h

A solution of compound 2 (1.03 g, 7.0 mmol), appropriate alkyl halide (8.4 mmol) and K₂CO₃ (0.51 g, 3.68 mmol), in dry DMF (25.0 mL) was heated at RT –70°C. After 2–18 h stirring, the mixture was extracted with silica gel column chromatography (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 3a–h.

6-Chloro-1-(4-nitrobenzyl)uracil [3a]

Yield 75%; white crystal; ¹H NMR (400 MHz, DMSO-d₆): δ 11.81 (1H, brs, 3-NH), 8.22 (2H, d, J 8.8, 4-NO₂-Bn), 7.57 (2H, d, J 8.8, 4-NO₂-Bn), 6.05 (1H, s, H-5), 5.29 (2H, s, 4-NO₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 161.0, 150.4, 146.8, 146.4, 144.1, 127.6, 123.8, 102.7, 47.8; HRMS (ESI) Calcd for C₁₁H₈ClN₃NaO₄⁺ [M+Na]⁺: 304.00955. Found 304.02556; mp: 111.9–114.6°C.

6-Chloro-1-(2-nitrobenzyl)uracil [3b]

Yield 44%; white crystal; ¹H NMR (400 MHz, DMSO-d₆): δ 11.81 (1H, brs, 3-NH), 8.08 (1H, d, J 8.0, 2-NO₂-Bn), 7.78 (1H, dd, J 8.0 and 8.0, 2-NO₂-Bn), 7.61 (1H, dd, J 8.0 and 8.0, 2-NO₂-Bn), 7.39 (1H, d, J 8.0, 2-NO₂-Bn), 6.09 (1H, s, H-5), 5.47 (2H, s, 2-NO₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 161.1, 150.4, 147.0, 146.4, 134.7, 131.5, 128.8, 127.1, 125.3, 102.9, 46.1; HRMS (ESI) Calcd for C₁₁H₈ClN₃NaO₄⁺ [M+Na]⁺: 304.00955. Found 304.00970; mp: 145.9–147.5°C.

6-Chloro-1-(2,6-difluorobenzyl)uracil [3c]

Yield 21%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 8.66 (1H, brs, 3-NH), 7.30 (1H, m, 2,6-F₂-Bn), 6.93 (2H, m, 2,6-F₂-Bn), 5.89 (1H, s, H-5), 5.37 (2H, s, 2,6-F₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.4 (d, J 28), 160.4 (d, J 212), 159.9, 149.6, 147.6, 130.2, 111.9, 103.1, 39.0; HRMS (ESI) Calcd for C₁₁H₇ClF₂N₂NaO₂⁺ [M+Na]⁺: 295.00563. Found 295.00600; mp: 76.2–77.4°C.

6-Chloro-1-(4-fluorobenzyl)uracil [3d]

Yield 60%; white crystal; ¹H NMR (400 MHz, DMSO-d₆): δ 11.75 (1H, brs, 3-NH), 7.33 (2H, m, 4-F-Bn), 7.19 (2H, m, 4-F-Bn), 6.00 (1H, s, H-5), 5.14 (2H, s, 4-F-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 162.6, 161.0, 150.5, 146.6, 132.4 (d, J 12), 128.9 (d, J 36), 115.5 (d, J 84), 102.5, 47.5; HRMS (ESI) Calcd for C₁₁H₉ClFN₂O₂⁺ [M+H]⁺: 255.03311. Found 255.03365; mp: 93.4–95.0°C.

6-Chloro-1-(4-cyanobenzyl)uracil [3e]

Yield 59%; white crystal; ¹H NMR (400 MHz, DMSO-d₆): δ 11.78 (1H, brs, 3-NH), 7.84 (2H, d, J 8.4, 4-CN-Bn), 7.49 (2H, d, J 8.4, 4-CN-Bn), 6.04 (1H, s, H-5), 5.24 (2H, s, 4-CN-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 161.0, 150.5, 146.4, 142.0, 132.6, 127.3, 118.6, 110.2, 102.7, 47.9; HRMS (ESI) Calcd for C₁₂H₉ClN₃O₂⁺ [M+Na]⁺: 262.03778. Found 262.03830; mp: 131.0–132.0°C.

6-Chloro-1-(4-methoxybenzyl)uracil [3f]

Yield 47%; white crystal; ¹H NMR (400 MHz, DMSO-d₆): δ 11.78 (1H, brs, 3-NH), 7.22 (2H, d, J 8.8, 4-CH₃O-Bn), 6.92 (2H, d, J 8.8, 4-CH₃O-Bn), 5.97 (1H, s, H-5), 5.08 (2H, s, 4-CH₃O-Bn), 3.74 (3H, s, 4-CH₃O-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 160.9, 158.6, 150.5, 146.8, 128.3, 128.1, 114.0, 102.3, 55.1, 47.6; HRMS (ESI) Calcd for C₁₂H₁₂ClN₂O₃⁺ [M+H]⁺: 267.05310. Found 267.05375; mp: 123.5–124.8°C.

6-Chloro-1-[(2-furanyl)methyl]uracil [3g]

Yield 29%; brown crystal; ¹H NMR (400 MHz, CDCl₃): δ 9.25 (1H, brs, 3-NH), 7.38 (1H, dd, J 2.0 and 0.4, CH₂(furan-2-yl)), 6.41 (1H, d, J 3.2, CH₂(furan-2-yl)), 6.35 (1H, dd, J 3.2 and 2.0, CH₂(furan-2-yl)), 5.90 (1H, s, H-5), 5.23 (2H, s, CH₂(furan-2-yl)); ¹³C NMR (100 MHz, CDCl₃): δ 160.8, 149.8, 148.1, 147.5, 143.0, 110.7, 110.1, 103.0, 42.1; HRMS (ESI) Calcd for C₉H₇ClN₂NaO₃⁺ [M+Na]⁺: 249.00374. Found 249.00441; mp: 130.6–132.4°C.

6-Chloro-1-[(2-thiophenyl)methyl]uracil [3h]

Yield 70%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 9.25 (1H, brs, 3-NH), 7.38 (1H, dd, J 2.0 and 0.4, CH₂(thiophen-2-yl)), 6.41 (1H, d, J 3.2, CH₂(thiophen-2-yl)), 6.35 (1H, dd, J 3.2 and 2.0, CH₂(thiophen-2-yl)), 5.90 (1H, s, H-5), 5.23 (2H, s, CH₂(thiophen-2-yl)); ¹³C NMR (100 MHz, CDCl₃): δ 160.4, 149.7, 147.2, 136.4, 128.7, 126.9, 126.7, 103.0, 44.0; HRMS (ESI) Calcd for C₉H₇ClN₂NaO₂S⁺ [M+Na]⁺: 264.98090. Found 264.98138; mp: 138.2–139.4°C.

General procedure for the synthesis of 4a–h

A solution of compound 3a–h (1.93 mmol), triphenylphosphine (0.66 g, 2.51 mmol), 3,5-dimethylbenzylalcohol (0.28 g, 2.02 mmol), and TMAD (0.43 g, 2.51 mmol) in THF (16.3 mL) was stirred at 50°C. After 18 h stirring, the solution was filtered and concentrated to a small volume. The residual solution was purified by silica gel column chromatography to form 4a–h.

6-Chloro-3-(3,5-dimethylbenzyl)-1-(4-nitrobenzyl)uracil [4a]

Yield 69%; clear oil; ¹H NMR (400 MHz, CDCl₃): δ 8.20 (2H, d, J 8.8, 4-NO₂-Bn), 7.45 (2H, d, J 8.8, 4-NO₂-Bn), 7.03 (2H, s, 3,5-Me₂-Bn), 6.91 (1H, s, 3,5-Me₂-Bn), 6.01 (1H, s, H-5), 5.34 (2H, s, 4-NO₂-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 160.2, 151.1, 147.7, 145.0, 142.6, 138.1, 135.9, 129.6, 128.2, 126.6, 124.1, 103.1, 49.2, 45.0, 21.3; HRMS (ESI) Calcd for C₂₀H₁₈ClN₃NaO₄⁺ [M+Na]⁺: 422.08780. Found 422.08734.

6-Chloro-3-(3,5-dimethylbenzyl)- 1-(2-nitrobenzyl)uracil [4b]

Yield 84%; brown oil; ¹H NMR (400 MHz, CDCl₃): δ 8.18 (1H, d, J 8.0, 2-NO₂-Bn), 7.62 (1H, dd, J 8.0 and 8.0, 2-NO₂-Bn), 7.50 (1H, dd, J 8.0 and 8.0, 2-NO₂-Bn), 7.08 (1H, d, J 8.0, 2-NO₂-Bn), 7.03 (2H, s, 3,5-Me₂-Bn), 6.91 (1H, s, 3,5-Me₂-Bn), 6.05 (1H, s, H-5), 5.60 (2H, s, 2-NO₂-Bn), 5.05 (2H, s, 3,5-Me₂-Bn), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 160.3, 151.0, 147.4, 145.5, 138.1, 135.9, 134.2, 131.7, 129.6, 128.5, 126.6, 126.4, 125.8, 103.1, 47.8, 45.0, 21.3; HRMS (ESI) Calcd for C₂₀H₁₈ClN₃NaO₄⁺ [M+Na]⁺: 422.08780. Found 422.08740.

6-Chloro-1-(2,6-difluorobenzyl)-3-(3,5-dimethylbenzyl)uracil [4c]

Yield 60%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.27 (1H, m, 2,6-F₂-Bn), 7.00 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 6.89 (2H, m, 2,6-F₂-Bn), 5.94 (1H, s, H-5), 5.39 (1H, s, 2,6-F₂-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.4 (d, J 28), 160.5, 159.9 (d, J 28), 150.9, 145.5, 137.9, 136.1, 130.0 (t, J 40), 129.4, 126.3, 111.7 (q, J 24), 102.7, 44.9, 39.8 (t, J 16), 21.3; HRMS (ESI) Calcd for C₂₀H₁₇ClF₂N₂NaO₂⁺ [M+Na]⁺: 413.08388. Found 413.08344; mp: 120.5–122.2°C.

6-Chloro-3-(3,5-dimethylbenzyl)- 1-(4-fluorobenzyl)uracil [4d]

Yield 84%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.27 (2H, m, 4-F-Bn), 7.00 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 6.89 (2H, m, 4-F-Bn), 5.94 (1H, s, H-5), 5.39 (2H, s, 4-F-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 163.7, 161.3, 160.4, 151.2, 145.5, 138.2 (d, J 52), 136.1, 129.6 (q, J 32), 126.5.5, 124.9, 115.9 (d, J 84), 102.7, 49.2, 44.9, 21.3; HRMS (ESI) Calcd for C₂₀H₁₉ClFN₂O₂⁺ [M+Na]⁺: 373.11136. Found 373.11074; mp: 99.0–99.9°C.

6-Chloro-1-(4-cyanobenzyl)- 3-(3,5-dimethylbenzyl)uracil [4e]

Yield 71%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.64 (2H, d, J 8.4, 4-CN-Bn), 7.40 (2H, d, J 8.4, 4-CN-Bn), 7.03 (2H, s, 3,5-Me₂-Bn), 6.91 (1H, s, 3,5-Me₂-Bn), 6.00 (1H, s, H-5), 5.29 (2H, s, 4-CN-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 160.2, 151.1, 145.1, 140.6, 138.1, 135.9, 132.7, 129.6, 128.0, 126.5, 118.3, 112.2, 103.0, 49.4, 45.0, 21.3; HRMS (ESI) Calcd for C₂₁H₁₈ClN₃NaO₂⁺ [M+Na]⁺: 402.09798. Found 402.09728; mp: 97.6–98.5°C.

6-Chloro-3-(3,5-dimethylbenzyl)- 1-(4-methoxybenzyl)uracil [4f]

Yield 83%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.28 (2H, d, J 8.8, 4-CH₃O-Bn), 7.03 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 6.83 (2H, d, J 8.8, 4-CH₃O-Bn), 5.93 (1H, s, H-5), 5.19 (2H, s, 4-CH₃O-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 3.79 (3H, s, 4-CH₃O-Bn), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 160.5, 159.5, 151.3, 145.7, 138.0, 136.2, 129.3, 127.5, 126.5, 124.8, 114.1, 102.5, 55.3, 49.4, 44.8, 21.3; HRMS (ESI) Calcd for C₂₁H₂₁ClN₂NaO₃⁺ [M+Na]⁺: 407.11329. Found 407.11293; mp: 96.4–97.2°C.

6-Chloro-3-(3,5-dimethylbenzyl)-1-[(2-furanyl) methyl]uracil [4g]

Yield 69%; brown crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.37 (1H, m, CH₂(furan-2-yl)), 7.04 (2H, s, 3,5-Me₂-Bn), 6.89 (1H, s, 3,5-Me₂-Bn), 6.38 (1H, d, J 3.2, CH₂(furan-2-yl)), 6.34 (1H, dd, J 3.2 and 2.0, CH₂(furan-2-yl)), 5.94 (1H, s, H-5), 5.23 (2H, s, CH₂(furan-2-yl)), 5.02 (2H, s, 3,5-Me₂-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 160.5, 150.8, 148.5, 145.3, 142.8, 138.0, 136.2, 129.5, 126.6, 110.6, 109.9, 102.7, 44.9, 42.9, 21.2; HRMS (ESI) Calcd for C₁₈H₁₇ClN₂NaO₃⁺ [M+Na]⁺: 367.08199. Found 367.08146; mp: 51.8–52.3°C.

6-Chloro-3-(3,5-dimethylbenzyl)-1-[(2-thiophenyl) methyl]uracil [4h]

Yield 68%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.28 (1H, dd, J 5.2 and 0.8, CH₂(thiophen-2-yl)), 7.16 (1H, d, J 3.2, CH₂(thiophen-2-yl)), 7.05 (2H, s, 3,5-Me₂-Bn), 6.97 (1H, dd, J 5.2 and 3.2, CH₂(thiophen-2-yl)), 6.90 (1H, s, 3,5-Me₂-Bn), 5.93 (1H, s, H-5), 5.39 (2H, s, CH₂(thiophen-2-yl)), 5.04 (2H, s, 3,5-Me₂-Bn), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 160.4, 150.9, 144.9, 138.0, 136.7, 136.1, 129.5, 128.6, 126.8, 126.6, 126.5, 102.7, 44.8, 44.7, 21.3; HRMS (ESI) Calcd for C₁₈H₁₇ClN₂NaO₂S⁺ [M+Na]⁺: 383.05915. Found 383.05870; mp: 94.9–95.5°C.

General procedure for the synthesis of 5a–h, 10, and 14

Compound 4a–h, 9, or 13 (0.58 mmol) was dissolved in dry DMF (4.0 mL), and NaN₃ (0.04 g, 0.67 mmol) was added to the solution, which was stirred for 30 min at room temperature. 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 (70% AcOEt in hexane) to form C6-azido derivatives.

6-Azido-3-(3,5-dimethylbenzyl)-1-(4-nitrobenzyl) uracil [5a]

Yield 90%; brownish crystal; ¹H NMR (400 MHz, CDCl₃): δ 8.19 (2H, d, J 8.4, 4-NO₂-Bn), 7.47 (2H, d, J 8.4, 4-NO₂-Bn), 7.03 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 5.60 (1H, s, H-5), 5.11 (2H, s, 4-NO₂-Bn), 5.05 (2H, s, 3,5-Me₂-Bn), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 160.0, 151.1, 150.3, 147.7, 142.9, 138.0, 136.2, 128.6, 126.4, 124.8, 124.0, 88.3, 46.2, 44.7, 21.3; HRMS (ESI) Calcd for C₂₀H₁₈N₆NaO₄⁺ [M+Na]⁺: 429.12817. Found 429.12714; mp: 143.6–146.7°C.

6-Azido-3-(3,5-dimethylbenzyl)-1-(2-nitrobenzyl) uracil [5b]

Yield 80%; colorless needle crystal; ¹H NMR (400 MHz, CDCl₃): δ 8.13 (1H, dd, J 8.4 and 1.6, 2-NO₂-Bn), 7.59 (1H, m, 2-NO₂-Bn), 7.48 (1H, m, 2-NO₂-Bn), 7.09 (1H, d, J 8.0, 2-NO₂-Bn), 7.03 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 5.63 (1H, s, H-5), 5.47 (2H, s, 2-NO₂-Bn), 5.06 (2H, s, 3,5-Me₂-Bn), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 161.0, 151.1, 150.6, 147.8, 138.0, 136.0, 134.0, 131.9, 129.4, 128.6, 126.9, 126.5, 125.5, 88.3, 44.7, 44.5, 21.3; HRMS (ESI) Calcd for C₂₀H₁₈N₆NaO₄⁺ [M+Na]⁺: 429.12817. Found 429.12757; mp: 127.6–128.3°C.

6-Azido-1-(2,6-difluorobenzyl)- 3-(3,5-dimethylbenzyl)uracil [5c]

Yield 98%; brown crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.25 (1H, m, 2,6-F₂-Bn), 6.99 (2H, s, 3,5-Me₂-Bn), 6.88 (2H, m, 2,6-F₂-Bn), 6.87 (1H, s, 3,5-Me₂-Bn), 5.56 (1H, s, H-5), 5.17 (1H, s, 2,6-F₂-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 2.26 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.5 (d, J 28), 161.3, 160.0 (d, J 32), 150.8 (d, J 28), 137.9, 136.4, 129.9 (t, J 40), 129.2, 126.2, 111.8 (d, J 24), 111.5 (t, J 12), 88.2, 44.6, 36.8 (t, J 12), 21.3; HRMS (ESI) Calcd for C₂₀H₁₇F₂N₅NaO₂⁺ [M+Na]⁺: 420.12425. Found 420.12268; mp: 96.8–99.8°C.

6-Azido-3-(3,5-dimethylbenzyl)- 1-(4-fluorobenzyl)uracil [5d]

Yield 86%; brown crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.34 (2H, m, 4-F-Bn), 7.02 (2H, s, 3,5-Me₂-Bn), 7.01 (2H, m, 4-F-Bn), 6.89 (1H, s, 3,5-Me₂-Bn), 5.56 (1H, s, H-5), 5.05 (2H, s, 3,5-Me₂-Bn), 4.99 (2H, s, 4-F-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 163.7, 161.3 (d, J 40), 151.2, 150.6, 137.9, 136.4, 131.6 (d, J 12), 130.0 (d, J 24), 129.3, 126.3, 115.8 (d, J 84), 88.1, 46.3, 44.6, 21.3; HRMS (ESI) Calcd for C₂₀H₁₈FN₅NaO₂⁺ [M+Na]⁺: 402.13367. Found 402.13242; mp: 78.8–82.5°C.

6-Azido-1-(4-cyanobenzyl)-3-(3,5-dimethylbenzyl) uracil [5e]

Yield 96%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.62 (2H, d, J 8.0, 4-CN-Bn), 7.41 (2H, d, J 8.0, 4-CN-Bn), 7.02 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 5.59 (1H, s, H-5), 5.07 (2H, s, 4-CN-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 161.0, 151.1, 150.3, 141.0, 138.0, 136.3, 132.6, 129.4, 128.4, 126.4, 118.4, 112.1, 88.2, 46.5, 44.7, 21.3; HRMS (ESI) Calcd for C₂₁H₁₈N₆NaO₂⁺ [M+Na]⁺: 409.13834. Found 409.13718; mp: 131.3–133.4°C.

6-Azido-3-(3,5-dimethylbenzyl)- 1-(4-methoxybenzyl)uracil [5f]

Yield 87%; brown crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.30 (2H, d, J 8.4, 4-CH₃O-Bn), 7.03 (2H, s, 3,5-Me₂-Bn), 6.88 (1H, s, 3,5-Me₂-Bn), 6.85 (2H, d, J 8.4, 4-CH₃O-Bn), 5.54 (1H, s, H-5), 5.05 (2H, s, 3,5-Me₂-Bn), 4.97 (2H, s, 4-CH₃O-Bn), 3.79 (3H, s, 4-CH₃O-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 161.3, 159.4, 151.2, 150.8, 137.9, 136.5, 129.6, 129.3, 127.9, 126.3, 114.1, 88.0, 55.3, 46.5, 44.6, 21.3; HRMS (ESI) Calcd for C₂₁H₂₁N₅NaO₃⁺ [M+Na]⁺: 414.15366. Found 414.15233; mp: 93.4–98.7°C.

6-Azido-3-(3,5-dimethylbenzyl)-1-[(2-furanyl) methyl]uracil [5g]

Yield 65%; brown oil; ¹H NMR (400 MHz, CDCl₃): δ 7.35 (1H, dd, J 1.6 and 0.8, CH₂(furan-2-yl)), 7.03 (2H, s, 3,5-Me₂-Bn), 6.88 (1H, s, 3,5-Me₂-Bn), 6.35 (1H, d, J 3.2, CH₂(furan-2-yl)), 6.32 (1H, dd, J 3.2 and 1.6, CH₂(furan-2-yl)), 5.56 (1H, s, H-5), 5.03 (2H, s, 3,5-Me₂-Bn), 5.01 (2H, s, CH₂(furan-2-yl)), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 161.2, 150.8, 150.5, 148.8, 142.7, 137.9, 136.5, 129.3, 126.5, 110.6, 109.6, 88.1, 44.5, 39.9, 21.3; HRMS (ESI) Calcd for C₁₈H₁₇N₅NaO₃⁺ [M+Na]⁺: 374.12236. Found 374.12183.

6-Azido-3-(3,5-dimethylbenzyl)- 1-[(2-thiophenyl)methyl]uracil [5h]

Yield 86%; brown oil; ¹H NMR (400 MHz, CDCl₃): δ 7.26 (1H, dd, J 5.2 and 0.8, CH₂(thiophen-2-yl)), 7.14 (1H, d, J 3.6, CH₂(thiophen-2-yl)), 7.04 (2H, s, 3,5-Me₂-Bn), 6.95 (1H, dd, J 5.2 and 3.6, CH₂(thiophen-2-yl)), 6.88 (1H, s, 3,5-Me₂-Bn), 5.54 (1H, s, H-5), 5.17 (2H, s, CH₂(thiophen-2-yl)), 5.04 (2H, s, 3,5-Me₂-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 161.1, 150.8, 150.2, 137.9, 137.1, 136.4, 129.3, 128.4, 126.7, 126.4, 126.4, 88.1, 44.5, 41.6, 21.3; HRMS (ESI) Calcd for C₁₈H₁₇N₅NaO₂S⁺ [M+Na]⁺: 390.09952. Found 390.09913.

1-[4-(Acetoxy)benzyl]-6-azido-3- (3,5-dimethylbenzyl)uracil [10]

Yield 96%; yellow crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.35 (2H, d, J 8.8, 4-AcO-Bn), 7.06 (2H, d, J 8.8, 4-AcO-Bn), 7.04 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 5.96 (1H, s, H-5), 5.24 (2H, s, 4-AcO-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 2.29 (3H, s, 4-AcO-Bn), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 169.4, 161.2, 151.2, 150.7, 150.4, 138.0, 136.4, 133.3, 129.3, 129.3, 126.3, 121.9, 88.0, 46.3, 44.6, 21.3, 21.1; HRMS (ESI) Calcd for C₂₂H₂₁N₅NaO₄⁺ [M+Na]⁺: 442.14858. Found 442.14776; mp: 39.6–41.5°C.

6-Azido-3-(3,5-dimethylbenzyl)- 1-(4-pyrimidinylmethyl)uracil [14]

Yield 89%; brown oil; ¹H NMR (400 MHz, CDCl₃): δ 9.13 (1H, s, 4-pyrimidinylmethyl), 8.69 (1H, d, J 5.2, 4-pyrimidinylmethyl), 7.20 (1H, d, J 5.2, 4-pyrimidinylmethyl), 7.01 (2H, s, 3,5-Me₂-Bn), 6.89 (1H, s, 3,5-Me₂-Bn), 5.64 (1H, s, H-5), 5.13 (2H, s, 4-pyrimidinylmethyl), 5.05 (2H, s, 3,5-Me₂-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 163.5, 161.2, 158.9, 157.2, 151.1, 150.7, 137.9, 136.3, 129.3, 126.2, 118.4, 88.1, 47.1, 44.6, 21.3; HRMS (ESI) Calcd for C₁₈H₁₇N₇NaO₂⁺ [M+Na]⁺: 386.13359. Found 386.13924.

General procedure for the synthesis of 6a–d, 6f–h, and 12

Compound 5a-h or 10 (0.64 mmol) was dissolved in dry THF (10.0 mL) under nitrogen atmosphere. To this stirred solution, we carefully added LiAlH₄ (0.02 g, 0.77 mmol) at 0°C. Stirring was continued at 0°C for 5 min, and the reaction quenched by the addition of AcOEt (5.0 mL) until no effervescence was observed. Aqueous 1N HCl (2.2 mL) was then added, and the product was extracted with AcOEt. The combined 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 (10% MeOH in AcOEt) to form C6-amino analogs.

6-Amino-3-(3,5-dimethylbenzyl)- 1-(4-nitrobenzyl)uracil [6a]

Yield 71%; yellowish crystal; ¹H NMR (400 MHz, CDCl₃): δ 8.12 (2H, d, J 8.4, 4-NO₂-Bn), 7.30 (2H, d, J 8.4, 4-NO₂-Bn), 6.93 (2H, s, 3,5-Me₂-Bn), 6.84 (1H, s, 3,5-Me₂-Bn), 5.13 (2H, s, 4-NO₂-Bn), 5.07 (2H, s, 3,5-Me₂-Bn), 4.99 (1H, brs, 6-NH₂), 4.93 (1H, s, H-5), 2.23 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.9, 153.7, 151.8, 147.6, 142.5, 138.0, 137.1, 129.1, 127.2, 125.8, 124.2, 78.6, 45.5, 44.4, 21.3; HRMS (ESI) Calcd for C₂₀H₂₀N₄NaO₄⁺ [M+Na]⁺: 403.13768. Found 403.13612; mp: 109.3–112.2°C.

6-Amino-3-(3,5-dimethylbenzyl)-1-(2-nitrobenzyl) uracil [6b]

Yield 67%; yellowish crystal; ¹H NMR (400 MHz, CDCl₃): δ 8.11 (1H, dd, J 8.4 and 1.2, 2-NO₂-Bn), 7.63 (1H, m, 2-NO₂-Bn), 7.50 (1H, m, 2-NO₂-Bn), 7.24 (1H, d, J 8.0, 2-NO₂-Bn), 7.00 (2H, s, 3,5-Me₂-Bn), 6.86 (1H, s, 3,5-Me₂-Bn), 5.50 (2H, s, 2-NO₂-Bn), 5.04 (1H, s, H-5), 5.04 (2H, s, 3,5-Me₂-Bn), 4.71 (2H, brs, 6-NH₂), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.5, 152.9, 152.0, 147.6, 137.9, 137.2, 134.5, 130.7, 129.1, 127.4, 126.1, 126.1, 125.7, 79.1, 44.4, 43.2, 21.3; HRMS (ESI) Calcd for C₂₀H₂₀N₄NaO₄⁺ [M+Na]⁺: 403.13768. Found 403.13663; mp: 177.1–181.6°C.

6-Amino-3-(3,5-dimethylbenzyl)- 1-(2,6-difluorobenzyl)uracil [6c]

Yield 89%; yellowish crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.23 (1H, m, 2,6-F₂-Bn), 6.97 (2H, s, 3,5-Me₂-Bn), 6.91 (2H, m, 2,6-F₂-Bn), 6.83 (1H, s, 3,5-Me₂-Bn), 5.20 (1H, s, 2,6-F₂-Bn), 5.02 (2H, s, 3,5-Me₂-Bn), 4.97 (1H, s, H-5), 4.78 (2H, brs, 6-NH₂), 2.25 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.7, 162.4 (d, J 28), 159.9 (d, J 28), 153.3, 151.6, 137.8, 137.3, 130.3 (t, J 40), 128.8, 125.7, 112.0 (q, J 28), 111.0, 79.1, 44.3, 21.2; HRMS (ESI) Calcd for C₂₀H₁₉F₂N₃NaO₂⁺ [M+Na]⁺: 394.13375. Found 394.13294; mp: 53.1–57.2°C.

6-Amino-3-(3,5-dimethylbenzyl)- 1-(4-fluorobenzyl)uracil [6d]

Yield 84%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.21 (2H, m, 4-F-Bn), 7.04 (2H, m, 4-F-Bn), 7.01 (2H, s, 3,5-Me₂-Bn), 6.86 (1H, s, 3,5-Me₂-Bn), 5.07 (2H, s, 4-F-Bn), 5.05 (2H, s, 3,5-Me₂-Bn), 5.00 (1H, s, H-5), 4.55 (1H, brs, 6-NH₂), 2.26 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 163.8, 162.7, 153.6, 152.1, 137.9, 137.3, 130.8 (d, J 12), 129.0, 128.2 (d, J 32), 126.1, 116.4 (d, J 84), 79.4, 45.6, 44.3, 21.3; HRMS (ESI) Calcd for C₂₀H₂₀FN₃NaO₂⁺ [M+Na]⁺: 376.14318. Found 376.14182; mp: 166.8–168.4°C.

6-Amino-3-(3,5-dimethylbenzyl)- 1-(4-methoxybenzyl)uracil [6f]

Yield 88%; yellowish oil; ¹H NMR (400 MHz, CDCl₃): δ 7.14 (2H, d, J 8.8, 4-CH₃O-Bn), 6.99 (2H, s, 3,5-Me₂-Bn), 6.87 (1H, s, 3,5-Me₂-Bn), 6.86 (2H, d, J 8.8, 4-CH₃O-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 5.02 (2H, s, 4-CH₃O-Bn), 4.97 (1H, s, H-5), 4.71 (1H, brs, 6-NH₂), 3.78 (3H, s, 4-CH₃O-Bn), 2.25 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.8, 159.6, 154.1, 152.2, 137.8, 137.4, 128.9, 127.7, 127.0, 126.0, 114.7, 79.0, 55.3, 45.8, 44.3, 21.3; HRMS (ESI) Calcd for C₂₁H₂₃N₃NaO₃⁺ [M+Na]⁺: 388.16316. Found 388.16225.

6-Amino-3-(3,5-dimethylbenzyl)-1-[(2-furanyl) methyl]uracil [6g]

Yield 53%; red-brown crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.37 (1H, m, CH₂(furan-2-yl)), 7.04 (2H, s, 3,5-Me₂-Bn), 6.86 (1H, s, 3,5-Me₂-Bn), 6.45 (1H, d, J 3.2, CH₂(furan-2-yl)), 6.37 (1H, dd, J 3.2 and 2.0, CH₂(furan-2-yl)), 5.05 (2H, s, 3,5-Me₂-Bn), 5.04 (1H, s, H-5), 5.01 (2H, s, CH₂(furan-2-yl)), 4.89 (1H, brs, 6-NH₂), 2.26 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.6, 153.6, 151.6, 148.9, 142.7, 137.8, 137.3, 129.0, 126.3, 111.2, 110.1, 78.0, 44.3, 39.2, 21.3; HRMS (ESI) Calcd for C₁₈H₁₉N₃NaO₃⁺ [M+Na]⁺: 348.13186. Found 348.13110; mp: 69.8–74.8°C.

6-Amino-3-(3,5-dimethylbenzyl)- 1-[(2-thiophenyl)methyl]uracil [6h]

Yield 79%; yellow crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.26 (1H, m, CH₂(thiophen-2-yl)), 7.01 (1H, d, J 3.6, CH₂(thiophen-2-yl)), 7.00 (2H, s, 3,5-Me₂-Bn), 6.94 (1H, dd, J 5.2 and 3.6, CH₂(thiophen-2-yl)), 6.85 (1H, s, 3,5-Me₂-Bn), 5.21 (2H, s, CH₂(thiophen-2-yl)), 5.04 (2H, s, 3,5-Me₂-Bn), 4.97 (1H, s, H-5), 4.82 (1H, brs, 6-NH₂), 2.25 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.7, 153.6, 151.7, 137.9, 137.7, 137.2, 129.0, 127.0, 126.4, 126.4, 125.9, 79.4, 44.3, 41.6, 21.3; HRMS (ESI) Calcd for C₁₈H₁₉N₃NaO₂S⁺ [M+Na]⁺: 364.10902. Found 364.10866; mp: 72.3–78.5°C.

6-Amino-3-(3,5-dimethylbenzyl)- 1-(4-hydroxybenzyl)uracil [12]

Yield 95%; colorless oil; ¹H NMR (400 MHz, CD₃OD): δ 6.95 (2H, d, J 8.4, 4-HO-Bn), 6.76 (2H, s, 3,5-Me₂-Bn), 6.72 (1H, s, 3,5-Me₂-Bn), 6.62 (2H, d, J 8.4, 4-HO-Bn), 4.89 (2H, s, 3,5-Me₂-Bn), 4.89 (2H, s, 4-HO-Bn), 4.86 (1H, s, H-5), 2.11 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CD₃OD): δ 165.2, 158.2, 157.1, 153.5, 138.9, 138.8, 129.6, 129.0, 127.6, 126.3, 116.5, 77.1, 46.2, 45.0, 21.4; HRMS (ESI) Calcd for C₂₀H₂₁N₃NaO₃⁺ [M+Na]⁺: 374.14751. Found 374.14671.

6-Amino-1-(4-cyanobenzyl)- 3-(3,5-dimethylbenzyl)uracil [6e]

To a refluxing mixture of compound 5e (0.08 g, 0.20 mmol) and NaBH₄ (0.008 g, 0.20 mmol) in THF (3.0 mL), MeOH (0.3 mL) was added over a period of 15 min. The mixture was the allowed to cool to room temperature, sat. NaHCO₃ aq (5.0 mL) was added, and the solution was extracted with AcOEt, washed with saturated aqueous sodium chloride solution, dried with sodium sulfate, and then evaporated. The residual solution was purified by silica gel column chromatography (20% MeOH in AcOEt) to form a white crystal 6e (0.07 g, 0.19 mmol, 95%). ¹H NMR (400 MHz, CDCl₃): δ 7.67 (2H, d, J 8.0, 4-CN-Bn), 7.35 (2H, d, J 8.0, 4-CN-Bn), 7.04 (2H, s, 3,5-Me₂-Bn), 6.89 (1H, s, 3,5-Me₂-Bn), 5.18 (2H, s, 4-CN-Bn), 5.06 (1H, s, H-5), 5.05 (2H, s, 3,5-Me₂-Bn), 4.27 (1H, brs, 6-NH₂), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.4, 152.7, 151.9, 140.4, 137.9, 137.0, 133.1, 129.2, 127.0, 126.3, 118.1, 112.5, 80.1, 45.9, 44.4, 21.3; HRMS (ESI) Calcd for C₂₁H₂₀N₄NaO₂⁺ [M+Na]⁺: 383.14785. Found 383.14664; mp: 128.5–131.6°C.

General procedure for the synthesis of 7a,b

Compound 5a,b (0.20 mmol) was dissolved in the 3:1 mixture of dry MeOH (3.0 mL) and dry THF (1.0 mL), and NaBH₄ (0.03 g, 0.92 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 (20% MeOH in AcOEt) to form diamino derivative 7a,b.

6-Amino-3-(3,5-dimethylbenzyl)- 1-(4-aminobenzyl)uracil [7a]

Yield 80%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.01 (2H, d, J 8.4, 4-NH₂-Bn), 7.00 (2H, d, J 8.4, 4-NH₂-Bn), 6.85 (2H, s, 3,5-Me₂-Bn), 6.61 (1H, s, 3,5-Me₂-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 4.97 (2H, s, 4-NH₂-Bn), 4.91 (1H, s, H-5), 4.65 (2H, brs, 6-NH₂), 3.76 (2H, brs, 4-NH₂-Bn), 2.26 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 162.8, 154.2, 152.2, 146.5, 137.8, 137.5, 128.9, b127.7, 126.0, 124.5, 115.7, 79.0, 46.0, 44.3, 21.3; HRMS (ESI) Calcd for C₂₀H₂₂N₄NaO₂⁺ [M+Na]⁺: 373.16350. Found 373.16279; mp: 167.5–168.9°C.

6-Amino-3-(3,5-dimethylbenzyl)- 1-(2-aminobenzyl)uracil [7b]

Yield 80%; yellowish needle crystal; ¹H NMR (400 MHz, CD₃OD): δ 6.96 (1H, m, 2-NH₂-Bn), 6.88 (1H, d, J 7.2, 2-NH₂-Bn), 6.80 (2H, s, 3,5-Me₂-Bn), 6.74 (1H, s, 3,5-Me₂-Bn), 6.70 (1H, d, J 7.6, 2-NH₂-Bn), 6.61 (1H, m, 2-NH₂-Bn), 4.91 (2H, s, 2-NH₂-Bn), 4.91 (2H, s, 3,5-Me₂-Bn), 4.86 (1H, s, H-5), 2.14 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CD₃OD): δ 165.1, 157.1, 135.7, 154.8, 138.9, 138.8, 129.8, 129.6, 128.0, 126.3, 122.7, 120.6, 118.7, 76.9, 45.1, 42.6, 21.4; HRMS (ESI) Calcd for C₂₀H₂₂N₄NaO₂⁺ [M+Na]⁺: 373.16350. Found 373.16246; mp: 157.3–162.2°C.

1-[4-(Acetoxy)benzyl]-6-chloro-3- (3,5-dimethylbenzyl)uracil [9]

A solution of compound 8 (0.26 g, 1.00 mmol), triphenylphosphine (0.34 g, 1.30 mmol), 4-(acetoxy)benzyl alcohol (0.28 g, 2.02 mmol), and TMAD (0.22 g, 1.30 mmol) in THF (8.5 mL) was stirred at 50°C. After 24 h stirring, the solution was filtered and concentrated to a small volume. The residual solution was purified by silica gel column chromatography (50% AcOEt in hexane) to form syrup 9 (0.26 g, 0.64 mmol, 64%). ¹H NMR (400 MHz, CDCl₃): δ 7.35 (2H, d, J 8.8, 4-AcO-Bn), 7.06 (2H, d, J 8.8, 4-AcO-Bn), 7.04 (2H, s, 3,5-Me₂-Bn), 6.90 (1H, s, 3,5-Me₂-Bn), 5.96 (1H, s, H-5), 5.24 (2H, s, 4-AcO-Bn), 5.04 (2H, s, 3,5-Me₂-Bn), 2.29 (3H, s, 4-AcO-Bn), 2.28 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 169.3, 160.5, 151.2, 150.5, 145.6, 138.0, 136.1, 132.9, 129.5, 128.9, 126.5, 122.0, 102.7, 49.3, 44.9, 21.3, 21.1; HRMS (ESI) Calcd for C₂₂H₂₁ClN₂NaO₄⁺ [M+Na]⁺: 435.10821. Found 435.10700.

6-Azido-3-(3,5-dimethylbenzyl)- 1-(4-hydroxybenzyl)uracil [11]

Compound 10 (0.06 g, 0.15 mmol) was dissolved in dry MeOH (5.0 mL), and K₂CO₃ (0.42 g, 3.00 mmol) was added to the solution, which was stirred for 30 min at room temperature. 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 AcOEt to form a red-brown crystal 11 (0.04 g, 0.12 mmol, 78%). ¹H NMR (400 MHz, CDCl₃): δ 7.17 (2H, d, J 8.8, 4-HO-Bn), 7.02 (2H, s, 3,5-Me₂-Bn), 6.86 (1H, s, 3,5-Me₂-Bn), 6.74 (2H, d, J 8.8, 4-HO-Bn), 6.16 (1H, brs, 4-HO-Bn), 5.14 (1H, s, H-5), 5.05 (2H, s, 3,5-Me₂-Bn), 4.97 (2H, s, 4-HO-Bn), 2.25 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 163.3, 161.3, 155.8, 151.2, 137.9, 136.8, 129.7, 129.2, 126.2, 115.5, 78.2, 57.0, 45.0, 44.4, 21.3; HRMS (ESI) Calcd for C₂₀H₁₉N₅NaO₃⁺ [M+Na]⁺: 400.13801. Found 400.11876; mp: 79.6–83.8°C.

6-Chloro-3-(3,5-dimethylbenzyl)- 1-(4-pyrimidinylmethyl)uracil [13]

A solution of compound 8 (0.27 g, 1.00 mmol), 4-(chloromethyl)pyrimidine (0.22 g, 1.72 mmol), K₂CO₃ (0.24 g, 1.72 mmol), and NaI (0.03 g, 0.20 mmol) in DMF (4.0 mL) was stirred at room temperature. After 48 h stirring, the solution was extracted with AcOEt, washed with saturated aqueous sodium chloride solution, dried with sodium sulfate, and then evaporated. The residual solution was purified by silica gel column chromatography (50% AcOEt in hexane) to form a brown crystal 13 (0.16 g, 0.44 mmol, 44%). ¹H NMR (400 MHz, CDCl₃): δ 9.15 (1H, s, 4-pyrimidinylmethyl), 8.70 (1H, d, J 5.2, 4-pyrimidinylmethyl), 7.22 (1H, d, J 5.2, 4-pyrimidinylmethyl), 7.01 (2H, s, 3,5-Me₂-Bn), 6.89 (1H, s, 3,5-Me₂-Bn), 6.03 (1H, s, H-5), 5.34 (2H, s, 4-pyrimidinylmethyl), 5.04 (2H, s, 3,5-Me₂-Bn), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 163.2, 160.4, 159.0, 157.3, 151.1, 154.6, 138.0, 135.9, 129.4, 126.3, 118.3, 102.7, 49.9, 44.9, 21.3; HRMS (ESI) Calcd for C₁₈H₁₇ClN₄NaO₂⁺ [M+Na]⁺: 379.09322. Found 379.09275; mp: 97.1–98.2°C.

6-Amino-3-(3,5-dimethylbenzyl)- 1-(4-pyrimidinylmethyl)uracil [15]

To a refluxing mixture of compound 14 (0.07 g, 0.21 mmol) and NaBH₄ (0.008 g, 0.21 mmol) in THF (3.0 mL), MeOH (0.3 mL) was added over a period of 15 min. The mixture was then allowed to cool to room temperature, sat. NaHCO₃ aq (5.0 mL) was added, and the solution was extracted with AcOEt, washed with saturated aqueous sodium chloride solution, dried with sodium sulfate, and then evaporated. The residual solution was purified by silica gel column chromatography (20% MeOH in AcOEt) to form a yellowish crystal 15 (0.04 g, 0.12 mmol, 58%). ¹H NMR (400 MHz, CDCl₃): δ 9.14 (1H, d, J 0.8, 4-pyrimidinylmethyl), 8.78 (1H, d, J 5.2, 4-pyrimidinylmethyl), 7.59 (1H, dd, J 5.2 and 0.8, 4-pyrimidinylmethyl), 7.00 (2H, s, 3,5-Me₂-Bn), 6.85 (1H, s, 3,5-Me₂-Bn), 5.86 (1H, brs, 6-NH₂), 5.08 (1H, s, H-5), 5.07 (2H, s, 3,5-Me₂-Bn), 4.99 (2H, s, 4-pyrimidinylmethyl), 2.25 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 163.2, 162.7, 158.7, 158.4, 154.6, 151.9, 137.9, 137.2, 129.1, 126.1, 121.6, 79.9, 47.8, 44.3, 21.3; HRMS (ESI) Calcd for C₁₈H₁₉N₅NaO₂⁺ [M+Na]⁺: 360.14310. Found 360.14230; mp: 85.8–89.0°C.

General procedure for the synthesis of 17a and 17b

A mixture of urea 16 (4.32 g, 72.0 mmol) and benzylamine or 4-aminomethylpyridine (60.0 mmol) in H₂O (30.0 mL) was stirred at 130 °C. After 12 h stirring, the solution was filtered and concentrated to a small volume. The residual solution was purified by silica gel column chromatography to form a benzylated or picolylated product.

N-Benzylurea [17a]

Yield 61%; white crystal; ¹H NMR (400 MHz, DMSO-d₆): δ 7.19–7.32 (5H, m, Ph), 6.39 (1H, t, J 6.0, NH-CH₂-Ph), 5.50 (2H, s, NH₂), 4.16 (2H, d, J 6.0, NH-CH₂-Ph); ¹³C NMR (100 MHz, DMSO-d₆): δ 158.7, 140.9, 128.2, 127.0, 126.5, 42.8; HRMS (ESI) Calcd for C₈H₁₀N₂NaO [M+Na]⁺: 173.06853. Found 173.06964; mp 146.2–149.2°C.

N-(4-picolyl)urea [17b]

Yield 74%; white crystal; ¹H NMR (400 MHz, DMSO-d₆): δ 8.47–8.48 (2H, m, picolyl), 7.22–7.23 (2H, m, picolyl), 6.53 (1H, t, J 6.0, NH-CH₂-), 5.63 (2H, s, NH₂), 4.19 (2H, d, J 6.0, NH-CH₂-); ¹³C NMR (100 MHz, DMSO-d₆): δ 158.7, 150.1, 149.4, 121.9, 41.8; HRMS (ESI) Calcd for C₇H₉N₃NaO [M+Na]⁺: 174.06378. Found 174.06470; mp 187.0–187.2°C.

General procedure for the synthesis of 18a and 18b

A mixture of 3,5-dimethylbenzaldehyde (0.40 mL, 3.0 mmol), benzylurea 17a or 17b (1.5 mmol), and titanium (IV) isopropoxide (0.50 mL, 1.7 mmol) were slurred in THF (2.0 mL). This slurry was stirred at 50°C under nitrogen. After 2 h stirring, the solution was then cooled to 0°C, and NaBH₄ (0.06 g, 1.5 mmol) was added. The ice bath was removed, and the resulting slurry was allowed to stir for 30 min. The slurry was again cooled to 0°C and quenched by dropwise addition of 1 N HCl aq (6.0 mL), the solution was extracted with AcOEt, washed with saturated aqueous sodium chloride solution, dried with sodium sulfate, and then evaporated. The residual solution was purified by silica gel column chromatography to form a crystal 18a or 18b (0.32 g, 1.18 mmol, 79%).

N-benzyl-N′-(3,5-dimethylbenzyl)urea [18a]

Yield 79%; white crystal; ¹H NMR (400 MHz, DMSO-d₆): δ 7.20–7.32 (5H, m, Ph), 6.84 (3H, s, 3,5-Me₂-Bn), 6.40 (1H, t, J 6.0, NH-CH₂-Ph), 6.37 (1H, t, J 6.0, NH-CH₂-Ph), 4.22 (2H, d, J 6.0, NH-CH₂-Ph), 4.16 (2H, d, J 6.0, NH-CH₂-Ph), 2.23 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 158.1, 141.0, 140.7, 137.1, 128.2, 127.9, 126.9, 126.5, 124.7, 42.9, 42.9, 20.9; HRMS (ESI) Calcd for C₁₇H₂₀N₂NaO [M+Na]⁺: 291.14678. Found 291.14575; mp 146.0–147.6°C.

N′-3,5-dimethyl-N-(4-picolyl)urea [18b]

Yield 20%; white crystal; ¹H NMR (400 MHz, DMSO-d₆): δ 8.47–8.48 (2 H, m, picolyl), 7.22–7.24 (2 H, m, picolyl), 6.84 (3 H, s, 3,5-Me₂-Bn), 6.50–6.56 (2 H, m, NH),4.25 (2 H, d, J 6.4, NH-CH₂), 4.15 (2 H, d, J 6.0, NH-CH₂), 2.24 (6 H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 158.04, 150.16, 149.35, 140.57, 137.08, 127.92, 124.73, 121.86, 42.91, 41.99, 20.90; HRMS (ESI) Calcd for C₁₆H₁₉N₃NaO₂ [M+Na]⁺: 292.14203. Found 292.14097; mp 138.1–138.8°C.(

N-benzyl-N′-3,5-dimethylbenzyl-N-trichloroacetylcarbamoylurea [19aa] and N-benzyl-N′-3,5-dimethylbenzyl-N′-trichloroacetylcarbamoylurea [19ab]

To a solution of compound 18a (0.13 g, 0.5 mmol) in THF (5.0 mL), trichloroacetyl isocyanate (0.24 mL, 2.0 mmol) was added dropwise. After 1 h stirring, the solution was filtered and concentrated to a small volume. The residual solution was purified by silica gel column chromatography (50% t-butyl methyl ether in CH₂Cl) to form milky white oils 19aa (0.07 g, 0.16 mmol, 32%) and 19ab (0.09 g, 0.19 mmol, 38%), respectively.

Compound 19aa

¹H NMR (400 MHz, CDCl₃): δ 7.18–7.39 (5H, m, Ph), 6.85 (1H, s, 3,5-Me₂-Bn), 6.82 (2H, s, 3,5-Me₂-Bn), 5.11 (2H, s, N-CH₂-Ph), 4.28 (2H, d, J 4.8, NH-CH₂-Ph), 2.16 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 161.0, 157.1, 152.1, 138.3, 138.1, 137.1, 128.5, 128.2, 127.3, 126.2, 124.6, 92.2, 44.9, 43.5, 20.9.

Compound 19ab

¹H NMR (400 MHz, CDCl₃): δ 6.95–7.28 (5H, m, Ph), 6.94 (1H, s, 3,5-Me₂-Bn), 6.79 (2H, s, 3,5-Me₂-Bn), 4.91 (2H, s, N-CH₂-Ph), 4.34 (2H, d, J 5.2, NH-CH₂-Ph), 2.23 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 156.5, 151.2, 139.5, 136.2, 134.9, 130.3, 128.9, 128.0, 124.4, 92.2, 47.0, 45.1, 21.2.

N′-3,5-dimethylbenzyl-N′-trichloroacetylcarbamoyl-N-(4-picolyl)urea [19b]

To a solution of compound 18b (0.29 g, 1.06 mmol) in THF (15.0 mL), trichloroacetyl isocyanate (0.15 mL, 1.27 mmol) was added dropwise. After 10 min stirring, the solution was filtered and concentrated to a small volume. The residual solution was purified by silica gel column chromatography (20% MeOH in AcOEt) to form yellowish crystal 19b (0.34 g, 0.74 mmol, 70%). ¹H NMR (400 MHz, DMSO-d₆): δ 9.03 (1H, t, J 6.0, NH-CH₂), 8.47–8.48 (2H, m, picolyl), 7.25–7.26 (2H, m, picolyl), 6.79–6.88 (3H, m, 3,5-Me₂-Bn), 4.89 (2H, s, N-CH₂), 4.39 (2H, d, J 6.0, NH-CH₂), 2.20 (6H, s, 3,5-Me₂-Bn); mp 186.1–186.9°C.

General procedure for the synthesis of 20aa, 20ab, and 20b

A mixture of urea 19aa, 19ab, or 19b (0.046 mmol) and silica gel (0.5 g) in MeOH 5.0 mL) was stirred at 50°C. After 18 h stirring, the solution was filtered and concentrated to a small volume. The residual solution was purified by silica gel column chromatography to form 20aa, 20ab, or 20b (6.54 g, 43.6 mmol, 61%).

N-benzyl-N-carbamoyl-N′-(3,5-dimethylbenzyl) urea [20aa]

Yield 74%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.26–7.39 (5H, m, Ph), 6.88 (1H, s, 3,5-Me₂-Bn), 6.75 (2H, s, 3,5-Me₂-Bn), 5.02 (2H, s, N-CH₂-Ph), 4.36 (2H, d, J 5.2, NH-CH₂-Ph), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 157.5, 156.1, 138.2, 137.9, 136.7, 129.3, 129.0, 128.0, 126.3, 125.0, 47.0, 44.7, 21.2; HRMS (ESI) Calcd for C₁₈H₂₁N₃NaO₂ [M+Na]⁺: 334.15260. Found 334.15086; mp 123.5–124.6°C.

N-benzyl-N′-carbamoyl-N′-3,5-dimethylbenzylurea [20ab]

Yield 83%; white crystal; ¹H NMR (400 MHz, CDCl₃): δ 7.17–7.32 (5H, m, Ph), 6.91 (1H, s, 3,5-Me₂-Bn), 6.84 (2H, s, 3,5-Me₂-Bn), 4.93 (2H, s, N-CH₂-Ph), 4.44 (2H, d, J 5.2, NH-CH₂-Ph), 2.27 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, CDCl₃): δ 157.8, 156.6, 139.0, 138.2, 136.6, 129.7, 128.6, 127.3, 127.3, 124.0, 46.9, 44.7, 21.3; HRMS (ESI) Calcd for C₁₈H₂₁N₃NaO₂ [M+Na]⁺: 334.15260. Found 334.15074; mp 118.1–119.5°C.

N′-carbamoyl-N′-3,5-dimethylbenzyl-N- (4-picolyl)urea [20b]

Yield 81%; white crystal; ¹H NMR (400 MHz, DMSO-d₆): δ 8.96 (1H, t, J 5.6, NH-CH₂), 8.37–8.38 (2H, m, picolyl), 7.22 (2H, s, NH₂), 7.06–7.07 (2H, m, picolyl), 6.81 (1H, s, 3,5-Me₂-Bn), 6.71 (2H, s, 3,5-Me₂-Bn), 4.82 (2H, s, N-CH₂), 4.29 (2H, d, J 5.6, NH-CH₂), 2.17 (6H, s, 3,5-Me₂-Bn); ¹³C NMR (100 MHz, DMSO-d₆): δ 157.0, 156.1, 149.4, 148.8, 138.1, 137.2, 128.1, 123.9, 121.8, 44.9, 42.5, 20.9; HRMS (ESI) Calcd for C₁₇H₂₀N₄NaO₂ [M+Na]⁺: 335.14785. Found 335.14757; mp 151.2–153.3°C.

Results

Biological activity

The antiviral activities of the 6-azido (5a–h, 11, and 14) and 6-amino (6a–h, 7a–b, 12, and 15) derivatives of 1-substituted-3-(3,5-dimethylbenzyl)uracils and N-3,5-dimethylbenzyl-substituted urea derivatives 20aa–20b were determined by examining the inhibitory effects of these compounds on the HIV-1-induced cytopathogenicity and cell viability in MT-4 cells. The cytotoxicities of these compounds, which were dissolved in dimethyl sulfoxide at concentrations up to 20 µM, were evaluated based on the viability of mock-infected cells, as determined by the MTT method. Emivirine (MKC-442) was also tested as a positive control. As shown in Table 1, almost every compound in the series of 6-azido and 6-amino derivatives of 1-substituted-3-(3,5-dimethylbenzyl)uracils exhibited good-to-moderate anti-HIV-1 activity with EC₅₀ values ranging from 10 nM to 2.0 µM. Almost every 6-azido derivative exhibited considerably less anti-HIV-1 activity than the 6-amino derivatives; for example, 5a (6-azidouracil with a 4-nitrobenzyl group at the N¹ position) was approximately 10 times less potent (EC₅₀ = 0.37 ± 0.24 µM, SI = 27; SI: selectivity index) than its 6-amino counterpart, 6a (EC₅₀ = 0.068 ± 0.005 µM, SI >294).

Table 1.

Antiviral activity of 3-(3,5-dimethylbenzyl)uracil derivatives against HIV-1.




Compound	R₁	R₂	EC₅₀(µM)^a	CC₅₀(µM)^b	SI^c

Emivirine	–	–	0.013 ± 0.001	>20	>1539
5a	4-NO₂-Bn	N₃	0.37 ± 0.24	13.6 ± 3.8	>27
5b	2-NO₂-Bn	N₃	0.94 ± 0.16	> 20	>21
5c	2,6-F₂-Bn	N₃	0.10 ± 0.01	>20	>192
5d	4-F-Bn	N₃	0.23 ± 0.01	>20	>87
5e	4-CN-Bn	N₃	0.13 ± 0.05	>20	>156
5f	4-MeO-Bn	N₃	0.18 ± 0.07	>20	>111
5g	CH₂ (furan-2-yl)	N₃	0.12 ± 0.04	>20	>176
5h	CH₂ (thiophen-2-yl)	N₃	0.12 ± 0.01	>20	>174
6a	4-NO₂-Bn	NH₂	0.068 ± 0.005	>20	>294
6b	2-NO₂-Bn	NH₂	0.35 ± 0.13	>20	>58
6c	2,6-F2-Bn	NH₂	0.042 ± 0.012	>20	>476
6d	4-F-Bn	NH₂	0.037 ± 0.008	>20	>548
6e	4-CN-Bn	NH₂	0.099 ± 0.012	>20	>203
6f	4-MeO-Bn	NH₂	0.096 ± 0.001	>20	>209
6g	CH₂ (furan-2-yl)	NH₂	0.038 ± 0.009	>20	>533
6h	CH₂ (thiophen-2-yl)	NH₂	0.093 ± 0.004	>20	>215
7a	4-NH₂-Bn	NH₂	0.010 ± 0.006	>20	>1923
7b	2-NH₂-Bn	NH₂	2.0 ± 1.1	>20	>10
11	4-HO-Bn	N₃	1.8 ± 0.2	>20	>11
12	4-HO-Bn	NH₂	0.043 ± 0.014	>20	>465
14	CH₂ (pyrimidin-4-yl)	N₃	1.8 ± 0.6	>20	>11
15	CH₂ (pyrimidin-4-yl)	NH₂	1.8 ± 0.7	>20	>11
20aa	–	–	>20	>20	–
20ab	–	–	>20	>20	–
20b	–	–	>20	>20	–

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).

Subsequently, the 6-amino derivatives were further investigated for SAR studies. Among the compounds shown in Table 1, the 6-amino derivatives of N¹-(2,6-difluorobenzyl)uracil (6c), N¹-(4-fluorobenzyl)uracil (6d), N¹-[(2-furanyl)methyl]uracil (6g), N¹-(4-aminobenzyl)uracil (7a), and N¹-(4-hydroxybenzyl)uracil (12) showed satisfactory anti-HIV-1 activities with EC₅₀ values of 0.042 ± 0.012 µM, 0.037 ± 0.008 µM, 0.038 ± 0.009 µM, 0.010 ± 0.006 µM, and 0.043 ± 0.014 µM, respectively. The introduction of fluorine atom(s) on the benzene ring of the benzyl group at the N¹ position in uracil (6c and 6c) made the compounds more active than the unsubstituted compound 1e (EC₅₀ = 0.069 ± 0.006 µM), as reported in our previous study.¹⁵ Moreover, the introduction of polar groups at the para-position of the benzyl group (e.g. the amino group in 7a or hydroxyl group in 12) increased the anti-HIV-1 activity. In particular, 7a exhibited excellent activity with an EC₅₀ value of 0.010 ± 0.006 µM and an SI value of>1923, which are high and comparable to those of the positive control emivirine (EC₅₀ = 0.013 ± 0.001 µM, SI >1539). However, the corresponding regioisomer of 7a, N¹-(2-aminobenzyl)uracil derivative 7b, exhibited decreased antiviral activity with an EC₅₀ value of 2.0 ± 1.1 µM, which was almost two orders of magnitude lower than the EC₅₀ value of 7a (0.010 ± 0.006 µM). The probable cause of this decreased anti-HIV-1 activity is described in detail in the next paragraph, “Molecular modeling analysis.” Unfortunately, in the case of N-3,5-dimethylbenzyl-substituted urea derivatives 20aa–20b, no significant anti-HIV-1 activity was observed (EC₅₀ >20 µM and CC₅₀ >20 µM), indicating that the conversion of the uracil skeleton into the acyclic derivative may decrease the binding affinity of these compounds to the HIV-1 RT; that is, the uracil skeleton plays an important role in exerting HIV-1 activity in the 1-aromatic methyl-substituted 3-(3,5-dimethylbenzyl)uracil series.

Discussion

Molecular modeling analysis

The X-ray co-crystal structure (PDB: 1VRT) of HIV-1 RT with nevirapine was taken from PDB (1VRT)² and used for docking studies. A docking model consisting of ligand 7a, which showed the most promising anti-HIV-1 activity, 7b, which is the regioisomer of 7a, or 1-benzyluracil derivative 1e bound to the HIV-1 RT was constructed by a conformational search using MacroModel (ver. 9.1). AMBER* was used as the force field, and more than 3,000 conformers of the corresponding ligands were optimized. Figure 6a shows the molecular docking of 7a 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.²¹ The 6-amino group of 7a is hydrogen bonded to the amide group of the Lys101 residue (NH^…O = C). Moreover, the 3,5-dimethylbenzyl moiety, which was oriented around the hydrophobic area, enhanced the π–π stacking of the benzene rings of the Tyr181 and Tyr188 residues. Moreover, 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. The results of this calculation were the same as that for the unsubstituted 1-benzyluracil 1e (data not shown) or 1-(4-picolyl)uracil 1g;¹⁵ however, 7a with a 4-aminobenzyl group at the N¹ position was more potent (EC₅₀ = 0.010 ± 0.006 µM, Table 1) than 1e (0.069 ± 0.006 µM) or 1g (0.030 ± 0.030 µM).^14,15 Based on these results, we believe that the water solubility of the 4-aminobenzyl group of 7a may result in the formation of hydrogen bonds between the nitrogen atom of this amino group and water molecules outside the HIV-1 RT, which would stabilize ligand 7a, and thus improve the anti-HIV-1 activity.^21,22

Figure 6.

(a) Molecular docking of N¹-4-aminobenzyl derivative 7a into the allosteric site of HIV-1 RT (PDB code: 1VRT); (b) molecular docking of N¹-2-aminobenzyl derivative 7b into the allosteric site of HIV-1 RT (PDB code: 1VRT); (c) superimposition of the docked conformation of 7a (green) and 7b (magenta) in the HIV-1 RT (PDB code: 1VRT).

On the other hand, the regioisomer 7b, with severely decreased anti-HIV-1 activity (2.0 ± 1.1 µM, Table 1) was also docked with the HIV-1 RT, and the resulting most stable conformation is shown in Figure 6b. The conformation of ligand 7b was substantially different from that of 7a because of steric repulsion between the amino groups at the ortho-position on the benzene ring and the 3,5-dimethylbenzyl group at the N³ position of uracil, or steric repulsion between the amino group at the ortho-position on the benzene ring and HIV-1 RT itself. Therefore, hydrophobic interactions between the HIV-1 RT and the 3,5-dimethylbenzyl group at the N³ position decreased. Moreover, it may be difficult to stabilize the hydrogen bonding between the amino group at the uracil C6-position and the amide group of the Lys101 residue (NH^…O = C), which plays an important role for the exertion of anti-HIV-1 activity.^23–28 Figure 6c shows the overlay of the docked conformations of 7a (green) and its regioisomer 7b (magenta) bound to the HIV-1 RT at the nevirapine-binding site, showing that the most stable conformations of 7a and 7b differ substantially. Thus, the activity of 7b against HIV-1 is decreased compared with that of 7a.

Footnotes

Funding

This research was partially supported by a Grant-in-Aid for Young Scientists (B), No. 24790123, from the Japan Society for the Promotion of Science (JSPS).

Conflict of interest

The authors indicated no potential conflict of interest.

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