Introduction

Human immunodeficiency virus type-1 (HIV-1) infects approximately 40 million individuals worldwide. Since 1981, 20 million people have died from this epidemic (UNAIDS/WHO, 2007). Although the introduction of highly active antiretroviral therapy (HAART) has dramatically decreased the morbidity and mortality resulting from the infection with HIV, the AIDS prevalence remains one of the world’s most serious health problems. In the research of anti-HIV agents, nonnucleoside reverse transcriptase inhibitors (NNRTIs) have gained a definitive and important place due to their unique antiviral potency, high specificity, and low toxicity. To date, more than 50 different NNRTIs have been reported. NNRTIs currently in clinical use have a low genetic barrier to resistance, and therefore, novel NNRTIs that are effective against the emerging drug-resistant viral strains are urgently needed (De Clercq, 2007; Jochmans, 2008; Ilina and Parniak, 2008).

Recently, from high-throughput screening (HTS) of compound libraries, a sulfanyltetrazole derivative (compound S1) was identified as a potent, broad-spectrum NNRTI lead of HIV-1 replication, which has a simple, yet distinctively different chemical structure from the other HIV-1 NNRTIs reported in the literature (Fig. 1) (O’Meara et al., 2007; Muraglia et al., 2006). Extensive structural modification and bioactivity research demonstrated that most derivatives showed submicromolar activity in cell assay and significant in vitro activity against the wild-type or double-mutant strain K103N+Y181C of HIV-1 RT (O’Meara et al. 2007; Muraglia et al., 2006; Gagnon et al., 2007).

figure b
Fig. 1
figure 1

SAR conclusions of lead compounds and the newly designed scaffold

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Notable features of molecular modeling and SAR conclusions of sulfanyltetrazole derivatives include: (1) The inhibitor’s amide carbonyl forms a key H-bond with the backbone N–H of K103, which is consistent with the good activity of these compounds against the K103 mutants. (2) The aryl group linked to the tetrazole core fits into the important hydrophobic pocket, where many key resistant mutations take place, which include Y188L, Y181C, F227C, and L100I. (3) The tetrazole portion of these inhibitors could simply be acting as a scaffold that orients the pharmacophores into the proper geometry for binding (O’Meara et al., 2007; Muraglia et al., 2006; Gagnon et al., 2007).

However, until now, we dis not know whether the integrity of “S–CH2–CO–NH” bridge between the tetrazole core and the anilide phenyl ring was a prerequisite structural requirement for the anti-HIV activity of sulfanyltetrazoles. Therefore, our design strategy was centered around changes of this flexible link, and in the designed molecules, the above-mentioned three fragments, which were considered to be necessary for conserving anti-HIV-1 activity, were left unchanged (Fig. 1).

It was noticed that many hydrazones (such as compounds HY-1 and BBNH) (Vicini et al., 2008; Borkow et al., 1997; Sluis-Cremer et al., 2002; Himmel et al., 2006), which were structurally related to our target compounds showed anti-HIV activities, so replacement of the “S–CH2–CO–NH” link of the lead compounds by a “S–CH2–CO–NH–N=C” link may improve such activity. Otherwise, molecular modeling of the lead compound also reveals that the anilide phenyl group is positioned near the binding pocket opening controlled by the Pro236 loop, which adjusts position based on the size of the NNRTI inhibitor bound (O’Meara et al. 2007; Muraglia et al., 2006; Gagnon et al., 2007), so lengthening of this flexible link should be tolerated.

Herein, to find potent HIV replication inhibitors and also to further establish the key structural requirements for the anti-HIV activity of sulfanyltetrazole derivatives, the synthesis of novel sulfanyltetrazole-hydrazones and their anti-HIV activities tested in MT-4 cells were reported.

Results and discussion

Chemistry

New N′-arylidene-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio] acetohydrazide derivatives (5a–t) were prepared as shown in Scheme 1. 1-(Naphthalen-1-yl)-1H- tetrazole-5-thiol (2) was synthesized by treating 1-isothiocyanatonaphthalene (1) with NaN3 in water (Lin et al., 2006). Compound 2 was reacted with ethyl 2-chloroacetate to afford the ethyl 2-(1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio)acetate (3), from which the 2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (4) was obtained by treatment with hydrazine hydrate. Condensation of 4 with various substituted benzaldehydes (including thiophene-2-carbaldehyde in 5a and furan-2-carbaldehyde in 5b) gave the corresponding hydrazones 5a–t (Table 1 and Scheme 1). The structures of the prepared compounds were confirmed by IR, 1H-NMR, 13C-NMR, and MS.

Scheme 1
scheme 1

Reagents: (i) NaN3/H2O, HCl; (ii) NaOH/ClCH2COOEt, EtOH; (iii)N2H4 · H2O/EtOH; (iv) Aryl-CHO/EtOH, △

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Table 1 Anti-HIV activities, cytotoxicities, and selectivity indices of the new hydrazones 5a–t
figure c
Full size table

IR spectra of these new arylidene hydrazide derivatives 5 presented the N–H absorption at 3168–3196 cm−1, the C=O at 1667–1698 cm−1, and C=N at 1596–1613 cm−1 stretching bands. The 1H NMR spectra of the synthesized compounds were consistent with the presence of both E and Z geometric isomers. Two pairs of singlets associated with –S–CH2– and –CO–NH–protons, with ppm values in the range 4.63–4.75 and 4.24–4.32, 11.54–12.16 and 11.48–12.06, respectively. It is assumed that the N=CH double bond restricts rotation and gives rise to the formation of E and Z isomers with the E isomer dominating (Gürsoy et al., 1990; Yildir et al., 1995; Gürsoy et al., 1997; Mamolo et al., 2001). The 13C NMR spectra of 5a–t also revealed the presence of two isomers in DMSO-d6 as supported by the C=O, tetrazole-C, N=CH, and SCH2 carbon atoms resonating as double singlets at approximately 168.63–167.13 and 163.04–162.29, 156.81–156.40 and 156.50–151.29, 149.30–140.45 and 145.72–133.64, 36.48–35.57 and 36.25–35.05 ppm. The ESI-MS of target compounds showed the molecular ion at m/z (M+1) and m/z (M-195) with different intensities. The fragment at m/z (M-195) was probably formed via the breakage of the tetrazole-S bond.

Anti-HIV evaluation

Compounds 5a–t were evaluated for their in vitro anti-HIV-1 activity by using the IIIB strain for HIV-1 and the ROD strain for the HIV-2, and monitored by the inhibition of the virus-induced cytopathic effect in the human T-lymphocyte (MT-4) cells. The results are listed in Table 1. The compounds nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), and zidovudine (azidothymidine, AZT) were used as the reference drugs. Compounds 5q and 5r were found to be the only two compounds from the series inhibiting HIV-1 replication in cell culture. They showed EC50s of 29.62 μM (CC50 of 169.24 ± 23.83 μM) and 31.62 μM (CC50 >309.06 μM), and resulting in selectivity indices of 6 and >9, respectively.

Conclusions

We designed and synthesized a series of novel N′-arylidene-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazides, which were structurally confirmed by IR, 1H-NMR, 13C-NMR, and MS spectral analysis and evaluated for their inhibition of HIV-induced (HIV-1 IIIB, HIV-2 ROD) cytopathogenicity in MT-4 cell culture. The results showed that these derivatives exhibited deteriorated anti-HIV-1 activity, and none of them was active against HIV-2 replication.

Based on the chemical structure and the fact that compounds 5q and 5r inhibit HIV-1, but not HIV-2 replication, this molecule can be proposed to act as NNRTIs. The present results, together with previous SARs analysis of these kind of compounds (Muraglia et al., 2006; O’Meara et al. 2007), further demonstrated that the integrity of “S–CH2–CO–NH” link is prerequisite requirement for keeping sulfanyltetrazoles as potent and specific HIV-1 inhibitors. Probably, replacement of the “S–CH2–CO–NH” link with the “S–CH2–CO–NH–N=C” leads to notable changes of molecular flexibility and positional adaptability in the binding pocket of RT, which acutely influences the tight binding mode of NNRTI/RT. Although the pharmacological results are not very encouraging, the study provides useful information to further design newer sulfanyltetrazoles analogs as anti-HIV agents.

Experimental

Chemistry

All melting points were determined on a micromelting point apparatus and are uncorrected. Infrared spectra (IR) were recorded with a Nexus 470FT-IR Spectrometer. 1H- and 13C-NMR spectra were obtained on a Brucker Avance-600 NMR-spectrometer in the indicated solvents. Chemical shifts are expressed in δ units and TMS as internal reference. Mass spectra were taken on a LC Autosampler Device: Standard G1313A instrument. TLC was performed on silica gel G for TLC (Merck) and spots were visualized by iodine vapors or by irradiation with UV light (254 nm). Solvents were reagent grade and, when necessary, were purified and dried by standard methods. Concentration of the reaction solutions involved the use of rotary evaporator at reduced pressure.

Preparation of 2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio] acetohydrazide (4)

A mixture of 1-isothiocyanatonaphthalene (1) (3.7 g, 20 mmol) and NaN3 2.0 g, (30 mmol) in 40 ml of H2O, was stirred at reflux for 4 h. After this time, the reaction mixture was cooled and filtered. The pH value of the filtrate was adjusted to 3.0 by concentrated hydrochloric acid, then the white solid that was formed was collected by filtration, washed with cold water, and dried under vacuum to give the desired 1-(naphthalen-1-yl)-1H-tetrazole-5-thiol (2) as a white power. The purity was good enough for use in the next step.

To a mixture of 2 (2.28 g, 10 mmol) and 2-chloroacetate (1.20 g, 10 mmol) in ethanol (60 ml), NaOH (0.40 g, 10 mmol) was added dropwise. The mixture was stirred at room temperature overnight or stirred under refluxing for 2 h. Ice water (400 ml) was added and the separated white solid 3 was filtered off, washed with water, and recrystallized from ethanol. Yield: 69.7%. M.p. 214–215°C.

To a solution of 6.30 g (20 mmol) of compound 3 in 30 ml of absolute ethanol, 2.0 g of 50% hydrazine monohydrate were added. The mixture was refluxed under stirring for 4 h. After cooling, the formed precipitate was collected by filtration and recrystallized from EtOH/DMF to give 4.46 g (74.3%) of 4 as white lamellar crystals, M.p. 188–189°C.

General procedure for the preparation of compounds (5a–t)

Compound 4 (0.6 g, 2.0 mmol) was added slowly into a stirred solution of aromatic aldehydes (2.1 mmol) in ethanol (20 ml). The reaction mixture was left to react under reflux for 2–4 h (checked by TLC). The solvent was evaporated and the formed product was collected by filtration, washed with cold ethanol, and crystallized from EtOH/DMF.

2-[1-(Naphthalen-1-yl)-1H-tetrazol-5-ylthio]-N′-(thiophen-2-ylmethylene)

Acetohydrazide (5a). White needle crystal. Yield: 25.3%. M.p. 228–229°C. IR (KBr, cm−1): 3168 (NH), 1667 (C=O), 1596 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.82, 11.76 (2s, 1H, CONH), 8.39–7.12 (m, 11H, CH=N, naphthalene and thiophene), 4.63, 4.26 (2s, 2H, CH2), 13C-NMR (DMSO-d6) δ: 167.81, 162.85 (C=O), 156.70, 156.44 (tetrazole-C), 142.85, 139.81 (C=N), 36.19, 35.95 (S-CH2). ESI-MS: m/z 395.3 (M+1), 199.4 (M-195). C18H14N6OS2 (394.07).

N′-(2-Furylmethylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]aceto hydrazide (5b). White square crystal. Yield: 26.4%. M.p. 211–212°C. IR (KBr, cm−1): 3177 (NH), 1676 (C=O), 1597 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.79, 11.72 (2s, 1H, CONH), 8.32–6.63 (m, 10H, CH=N, naphthalene and furan), 6.93 (dd, 1H, J1 = 3.6Hz, J2 = 3.3Hz), 4.65, 4.27 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 168.05, 162.97 (C=O), 156.65, 156.43 (tetrazole-C), 149.30, 145.72 (C=N), 36.43, 35.05 (S-CH2). ESI-MS: m/z 379.6 (M+1), 183.4 (M-195). C18H14N6O2S (378.09).

N′-(4-Chlorobenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5c). White needle crystal. Yield: 59.1%. M.p. 230–230°C. IR (KBr, cm−1): 3176 (NH), 1673 (C=O), 1596 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.92,11.83 (2s, 1H, CONH); 8.32–7.26 (m, 12H, CH=N, naphthalene and benzene), 4.71, 4.29 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 168.29, 163.15 (C=O), 156.64, 156.43 (tetrazole-C), 146.43, 143.37 (C=N), 36.30, 36.00 (S-CH2). ESI-MS: m/z 423.6 (M+1), 227.3 (M-195). C20H15ClN6OS (422.07).

N′-(3-Chlorobenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5d). White powder. Yield: 35.5%. M.p. 203–204°C. IR (KBr, cm−1): 3187 (NH), 1666 (C=O), 1611 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 12.01, 11.89 (2s, 1H, CONH), 8.32–7.25 (m, 11H, CH=N, naphthalene and benzene), 8.00 (s, 1H, PhH), 4.73, 4.30 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 168.42, 163.30 (C=O), 156.68, 156.45 (tetrazole-C), 146.04, 143.00 (C=N), 36.26, 35.97 (S-CH2). ESI-MS: m/z 423.5 (M+1), 227.4 (M-195). C20H15ClN6OS (422.07).

N′-(2-Chlorobenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5e). White lamellar crystal. Yield: 47.3%. M.p. 227–228°C. IR (KBr, cm−1): 3193 (NH), 1680 (C=O), 1598 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 12.12, 11.96 (2s, 1H, CONH), 8.58–7.41 (m, 10H, CH=N, naphthalene and benzene), 7.24 (d, 2H, J = 8.4Hz), 4.73, 4.29 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 168.39, 163.25 (C=O), 156.63, 156.43 (tetrazole-C), 143.60, 140.64 (C=N), 36.27, 35.98 (S-CH2). ESI-MS: m/z 423.5 (M+1), 227.3 (M-195). C20H15ClN6OS (422.07).

N′-(2-Fluorobenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5f). White needle crystal. Yield: 55.4%. M.p. 221–223°C. IR (KBr, cm−1): 3196 (NH), 1681 (C=O), 1608 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 12.02, 11.89 (2s, 1H, CONH), 8.41–7.25 (m, 12H, CH=N, naphthalene and benzene), 4.72, 4.30 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 168.33, 163.17 (C=O), 161.97, 160.40, 160.31, 156.65, 156.43 (tetrazole-C), 140.45, 137.49 (C=N), 36.19, 35.95 (S-CH2). ESI-MS: m/z 407.6 (M+1), 211.2 (M-195). C20H15FN6OS (406.1).

N′-(4-Fluorobenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5g). White solid. Yield: 58.6%. M.p. 248–250°C. IR (KBr, cm−1): 3176 (NH), 1675 (C=O), 1613 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.86, 11.78 (2s, 1H, CONH), 8.32–7.28 (m, 11H, N=CH, naphthalene and benzene), 7.25(d, 1H, J = 8.4Hz), 4.70, 4.29 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 168.22, 164.48 (C=O), 164.37, 163.06, 162.84, 162.73, 156.67, 156.45 (tetrazole-C), 146.62, 143.50 (C=N), 36.33, 36.01 (S-CH2). ESI-MS: m/z 407.6 (M+1), 211.2 (M-195). C20H15FN6OS (406.1).

N′-(4-Nitrobenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5h). White cubic crystal. Yield: 63.4%. M.p. 241–243°C. IR (KBr, cm−1): 3188 (NH), 1674 (C=O), 1613 (C=N), 1517, 1399 (NO2). 1H-NMR (DMSO-d6, ppm) δ: 12.16, 12.06 (2s, 1H, CONH), 8.32–7.25 (m, 11H, CH=N, naphthalene and benzene), 7.85 (d, 1H, J = 7.3Hz), 4.75, 4.32 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 168.63, 163.59 (C=O), 156.60, 156.40 (tetrazole-C), 148.30, 145.30 (C=N), 36.25, 36.00 (S-CH2). ESI-MS: m/z 434.6 (M+1), 238.2 (M-195). C20H15N7O3S (433.10).

N′-(3-Methylbenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5i). White lamellar crystal. Yield: 75.2%. M.p. 251–252°C. IR (KBr, cm−1): 3182 (NH), 1673 (C=O), 1611 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.84, 11.74 (2s,1H, CONH), 8.32–7.32 (m, 11H, N=CH, naphthalene and benzene), 7.25 (d, 1H, J = 6.3Hz), 4.71, 4.29 (2s, 2H, CH2), 2.50, 2.34 (2s, 3H, -CH3). 13C-NMR (DMSO-d6) δ: 168.16, 163.00 (C=O), 156.73, 156.46 (tetrazole-C), 147.78, 144.80 (C=N), 36.27, 36.05 (S-CH2), 21.38, 21.33 (CH3). ESI-MS: m/z 403.6 (M+1), 207.3 (M-195). C21H18N6OS (402.13).

N′-(4-Methoxybenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5j). White needle crystal. Yield: 59.7%. M.p. 231–233°C. IR (KBr, cm−1): 3180 (NH), 1663 (C=O), 1606 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.74, 11.66 (2s, 1H, CONH), 8.32–6.99 (m, 9H, CH=N, naphthalene and benzene), 7.86 (d, 1H, J = 7.2Hz), 7.00 (d, 2H, J = 8.7Hz, PhH), 4.69, 4.27 (2s, 2H, CH2), 3.79 (2s, 3H, OCH3). 13C-NMR (DMSO-d6) δ: 167.95, 162.75 (C=O), 161.39,1 61.25 (OCH3), 156.73, 156.48 (tetrazole-C), 147.59, 144.51 (C=N), 40.18 (OCH3), 36.40, 36.03 (S-CH2). ESI-MS: m/z 419.5 (M+1), 223.4 (M-195). C21H18N6O2S (418.12).

N′-(2,4-Dimethoxybenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5k). White power. Yield:59.4%. M.p. 239–241°C. IR (KBr, cm−1): 3175 (NH), 1658 (C=O), 1612 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.71, 11.59 (2s, 1H, CONH), 8.44–6.60 (m, 10H, N=CH, naphthalene and benzene), 7.25 (dd, 1H, J1 = 3.3Hz, J2 = 11.5Hz), 4.68, 4.24 (2s, 2H, CH2), 3.83 (m, 6H, –OCH3). 13C-NMR (DMSO-d6) δ: 167.80, 163.06 (C=O), 162.89, 162.50, 159.65, 159.55 (=C–O), 156.74, 156.48 (tetrazole-C), 143.32, 140.40 (C=N), 56.28, 56.23, 55.92 (OCH3), 36.19, 35.95 (S–CH2). ESI-MS: m/z 449.5 (M+1), 471.5 (M+Na), 253.4 (M-195). C22H20N6O3S (448.13).

N′-(2,3-Dimethoxybenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5l). White needle crystal. Yield: 58.2%. M.p. 256–258°C. IR (KBr, cm−1): 3175 (NH), 1675 (C=O), 1598 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.91, 11.74 (2s, 1H, CONH), 8.47–7.11 (m, 10H, N=CH, naphthalene and benzene), 7.26 (d, 1H, J = 8.4Hz), 4.71, 4.28 (2s, 2H, CH2), 3.79 (m, 6H, OCH3). 13C-NMR (DMSO-d6) δ: 167.13, 162.94 (C=O), 162.94, 162.78, 153.13, 153.10 (=C–O), 156.70, 153.14 (tetrazole-C), 143.28, 140.46 (C=N), 56.50, 56.21 (OCH3), 36.34, 36.24 (S–CH2). ESI-MS: m/z 449.5 (M+1), 471.5 (M+Na), 253.4 (M-195). C22H20N6O3S (448.13).

N′-(3,4-Dimethoxybenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5m). White needle crystal. Yield: 61.2%. M.p. 244–246°C. IR (KBr, cm−1): 3172 (NH), 1675 (C=O), 1600 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.73, 11.66 (2s, 1H, CONH), 8.31–7.00 (m, 10H, N=CH, naphthalene and benzene), 7.25 (d, 1H, J = 8.4Hz), 4.68, 4.27 (2s, 2H, CH2), 3.79, 3.75 (2s, 6H, OCH3). 13C-NMR (DMSO-d6) δ: 168.00, 162.79 (C=O), 162.75, 151.09, 149.41 (C–O), 156.81, 151.29 (tetrazole-C), 147.94, 144.65 (C=N), 56.50, 56.01, 55.88, 55.83 (OCH3), 36.25, 36.05 (S-CH2). ESI-MS: m/z 449.6 (M+1), 471.5 (M+Na), 253.4 (M-195). C22H20N6O3S (448.13).

N′-(4-Hydroxy-3-methoxybenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5n). White needle crystal. Yield: 74.8%. M.p. 248–249°C. IR (KBr, cm−1): 3170 (NH), 1668 (C=O), 1597 (C=N), 3072 (OH). 1H-NMR (DMSO-d6, ppm) δ: 11.68, 11.61 (2s, 1H, CONH), 9.60, 9.58 (2s, 1H, OH), 8.31–6.81 (m, 11H, CH=N, naphthalene and benzene), 4.67, 4.27 (2s, 2H, CH2), 3.80 (m, 6H, OCH3) 13C-NMR (DMSO-d6) δ: 167.30, 162.03 (C=O), 156.26, 155.91 (tetrazole-C), 148.75, 147.78, 147.64 (C–OH, C–OCH3), 144.40, 133.64 (C=N), 55.37, 55.34 (OCH3), 35.57, 35.44 (S-CH2). ESI-MS: m/z 435.6 (M+1), 239.4 (M-195). C21H18N6O3S (434.12).

N′-(2-Hydroxy-5-methoxybenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5o). White needle crystal. Yield: 49.8%. M.p. 239–241°C. IR (KBr, cm−1): 3442 (OH), 3209 (NH), 1698 (C=O), 1610 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 12.02, 11.72 (2s, 1H, CONH), 10.52, 9.40 (2s, 1H, OH), 8.44-6.83 (m, 11H, N=CH, naphthalene and benzene), 4.69, 4.29 (2s, 2H, CH2), 3.82, 3.80 (d, 3H, OCH3) 13C-NMR (DMSO-d6) δ: 167.93, 162.90 (C=O), 156.71, 156.43 (tetrazole-C), 148.47, 147.49, 146.47 (C–OH, C–OCH3), 141.82, 134.23 (C=N), 56.33, 56.28 (OCH3), 36.46, 35.80 (S-CH2). ESI-MS: m/z 435.6 (M+1), 457.5 (M+Na), 239.3 (M-195). C21H18N6O3S (434.12).

N′-(2-Hydroxybenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5p). White needle crystal. Yield: 43.3%. M.p. 213–215°C. IR (KBr, cm−1): 3440 (OH), 3181 (NH), 1672 (C=O), 1613 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 12.06, 11.71 (d, 1H, CONH), 10.90, 10.09 (2s, 1H, OH), 8.42–6.86 (m, 11H, CH=N, naphthalene and benzene), 7.56 (d, 1H, J = 6.2 Hz), 4.70, 4.29 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 167.90, 162.92 (C=O), 157.69, 156.91 (C–OH), 156.70, 156.43 (tetrazole-C), 147.59, 141.89 (C=N), 36.48, 35.76 (S-CH2). ESI-MS: m/z 405.7 (M+1), 209.4 (M-195). C20H16N6O2S (404.11).

N′-(3-Hydroxybenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5q). White needle crystal. Yield: 75.%. M.p. 241–242°C. IR (KBr, cm−1): 3288 (NH), 1657 (C=O), 1611 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.80, 11.72 (2s, 1H, CONH), 9.68, 9.64 (2s, 1H, –OH), 8.32–6.83 (m, 12H, N=CH, naphthalene and benzene), 4.71, 4.28 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 168.12, 163.00 (C=O), 162.78, 158.13 (C–OH), 156.69, 156.46 (tetrazole-C), 147.80, 144.91 (C=N), 36.47, 36.25 (S-CH2). ESI-MS: m/z 405.7 (M+1), 209.4 (M-195). C20H16N6O2S (404.11).

N′-(4-Hydroxybenzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5r). White needle crystal. Yield: 74.3%. M.p. 258–259°C. IR (KBr, cm−1): 3169 (NH), 1671 (C=O), 1605 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.64, 11.57 (2s, 1H, CONH), 9.55 (s, 1H, –OH), 8.32–6.81 (m, 11H, N=CH, naphthalene and benzene), 7.25 (d, 1H, J = 8.4Hz), 4.67, 4.26 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 167.81, 162.77 (C=O), 162.59, 160.00, 159.87 (C–OH), 156.73, 156.47 (tetrazole-C), 148.02, 144.94 (C=N), 36.43, 36.25 (S-CH2). ESI-MS: m/z 405.7 (M+1), 209.4 (M-195). C20H16N6O2S (404.11).

N′-(4-(Dimethylamino)benzylidene)-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5s). White cubic crystal. Yield: 69.6%. M.p. 253–255°C. IR (KBr, cm−1): 3165 (NH), 1669 (C=O), 1606 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.54, 11.48 (2s,1H, CONH), 8.32–7.24 (m, 10H, N=CH, naphthalene and benzene), 6.74 (d, 2H, J = 8.4Hz), 4.67, 4.25 (2s, 2H, CH2), 2.97 (s, 6H, NCH3). 13C-NMR (DMSO-d6) δ: 167.56, 162.29 (C=O), 156.79, 156.50 (tetrazole-C), 156.50, 152.05, 151.94 (C–N(Me)2), 145.47, 134.23 (C=N), 40.31 (NCH3), 36.48, 36.11 (S-CH2). ESI-MS: m/z 432.6 (M+1), 236.3 (M-195). C22H21N7OS (431.15).

N′-Benzylidene-2-[1-(naphthalen-1-yl)-1H-tetrazol-5-ylthio]acetohydrazide (5t). White needle crystal. Yield: 58.7%. M.p. 225–226°C. IR (KBr, cm−1): 3183 (NH), 1670 (C=O), 1607 (C=N). 1H-NMR (DMSO-d6, ppm) δ: 11.86, 11.77 (2s, 1H, CONH), 8.32–7.25 (m, 12H, N=CH, naphthalene and benzene), 7.26 (d, 1H, J = 8.4Hz), 4.71, 4.29 (2s, 2H, CH2). 13C-NMR (DMSO-d6) δ: 168.22, 163.04 (C=O), 156.69, 156.46 (tetrazole-C), 147.74, 144.64 (C=N), 36.34, 36.04 (S-CH2). ESI-MS: m/z 389.5 (M+1), 193.5 (M-195). C20H16N6OS (388.11).

Anti-HIV activity assays

Given the multitude of enzymes and steps of the HIV multiplication cycle targeted by antiviral compounds and hydrazones considered to be popular fragments in bioactive anti-HIV agents, to evidence any potential antiviral activity, we chose to evaluate our compounds in cell-based, rather than cell-free, enzymatic assays. In our opinion, the former are better suited for optimization studies of a given lead compound.

The anti-HIV activity and cytotoxicity were evaluated against wild-type HIV-1 strain IIIB and HIV-2 (ROD) in MT-4 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method (Pauwels et al., 1988; Pannecouque et al., 2008). MT-4 cells were suspended in culture medium at 1×105 cells/ml and infected with HIV at a multiplicity of infection (MOI) of 0.02. Immediately after viral infection, 100 μl of the cell suspension was placed in each well of a flat-bottomed microtiter tray containing various concentrations of the test compounds. Stock solutions of the test compounds were prepared in DMSO at a concentration of 10 mg/ml. After 4 days of incubation at 37°C, the number of viable cells was determined using the MTT method. Compounds were tested in parallel for cytotoxic effects in uninfected MT-4 cells.

The 50% effective antiviral concentration (EC50) was defined as the compound concentration required to protect 50% of the virus-infected cells against viral cytopathicity. The 50% cytotoxic concentration (CC50) was defined as the compound concentration required to reduce the viability of mock-infected cells by 50%. The greater than symbol (>) is used to indicate the highest concentration at which the compounds were tested and still found to be noncytotoxic. Average EC50 and CC50 values for at least two separate experiments were presented.