Synthesis of novel derivatives of 4-amino-3-(2-furyl)-5-mercapto-1,2,4- triazole as potential HIV-1 NNRTIs

Article abstract of DOI:10.3390/12082003

A series of 5-alkylthio (2a-d), 4-arylideneamino (3a-d) and 4-arylideneamino-5-alkylthio derivatives (4a-f) of 4-amino-3-(2-furyl)-5- mercapto-1,2,4-triazole (1) were synthesized by alkylation of the parent compound with alkyl halides and condensation with aldehydes, respectively. Sulfanyl dimers 5a-d and 4-iminomethyl dimer 6 were correspondingly prepared by reaction with alkane dibromides and 1,4-diformylbenzene. Mannich base 7 was also synthesized by aminomethylation of the 3-sulfanyltriazole 1 at the N ₁ position. The newly designed and synthesized substituted s-triazole derivatives were assayed for anti-HIV-1 activity by examination of their inhibition of HIV-1-induced cytopathogenicity in MT-4 cells and by determination of their inhibitory effect on HIV-1 reverse transcriptase. Compound 4e was found to be the most active inhibitor against HIV-1 replication in cell culture (EC₅₀ = 12 μM) and against HIV-1 reverse transcriptase (IC ₅₀ = 43.5 μM), which provided a good lead for further optimization.

Full text of DOI:10.3390/12082003

Molecules 2007, 12, 2003-2016
molecules
ISSN 1420-3049
© 2007 by MDPI
www.mdpi.org/molecules
Full Paper
Synthesis of Novel Derivatives of
4-Amino-3-(2-Furyl)-5-Mercapto-1,2,4-Triazole as
Potential HIV-1 NNRTIs
Jingde Wu 1, Xinyong Liu 1,*, Xianchao Cheng 1, Yuan Cao 1, Defeng Wang 1, Zhong Li 1,
Wenfang Xu 1, Christophe Pannecouque 2, Myriam Witvrouw 2 and Erik De Clercq 2
1
Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, No.44,
Wenhuaxi Road, Jinan, 250012, P.R. China
2
Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; E-
mail: erik.declercq@rega.kuleuven.be (Erik De Clercq)
* Author to whom correspondence should be addressed; E-mail: xinyongl@sdu.edu.cn;
Tel: (+86)-531-88382005; Fax: (+86)-531-88382264
Received: 21 July 2007; in revised form: 17 August 2007 / Accepted: 20 August 2007 / Published: 21
August 2007
Abstract: A series of 5-alkylthio (2a-d), 4-arylideneamino (3a-d) and 4-arylideneamino-
5-alkylthio derivatives (4a-f) of 4-amino-3-(2-furyl)-5-mercapto-1,2,4-triazole (1) were
synthesized by alkylation of the parent compound with alkyl halides and condensation with
aldehydes, respectively. Sulfanyl dimers 5a-d and 4-iminomethyl dimer 6 were
correspondingly prepared by reaction with alkane dibromides and 1,4-diformylbenzene.
Mannich base 7 was also synthesized by aminomethylation of the 3-sulfanyltriazole 1 at
the N1 position. The newly designed and synthesized substituted s-triazole derivatives were
assayed for anti-HIV-1 activity by examination of their inhibition of HIV-1-induced
cytopathogenicity in MT-4 cells and by determination of their inhibitory effect on HIV-1
reverse transcriptase. Compound 4e was found to be the most active inhibitor against HIV-
1 replication in cell culture (EC50 = 12 μM) and against HIV-1 reverse transcriptase (IC50 =
43.5 μM), which provided a good lead for further optimization.
Keywords: s-Triazole; Alkylation; Aminomethylation; Synthesis; HIV-1 NNRTIs

Molecules 2007, 12
2004
Introduction
The worldwide AIDS crisis continues, with some 43 million people infected by HIV-1 and 8,000
deaths every day in 2006. HIV-1 reverse transcriptase (RT) is a key enzyme in the HIV-1 replication
process and has been a key target for developing anti-HIV drugs. Non-nucleoside reverse transcriptase
inhibitors (NNRTIs) have found widespread use in HIV therapy in multidrug treatment regimes and in
highly active antiretroviral therapy (HAART) [1]. However, the use of NNRTIs in the clinic still
remains subject to the rapid emergence of drug resistance due to mutation of the HIV-RT enzyme and
this limits therapeutic options [2]. There is therefore an urgent need to develop new high potency drugs
against both the wild-type (WT) HIV-1 and the drug-resistant mutant strains [3].
In recent years a variety of potential candidates have been developed, especially new structural
NNRTI classes, in which some substituted five-member heterocyclic compounds have been reported to
be active in inhibition of HIV-1 replication targeting the RT step, e.g. capravirine, an imidazole
derivative (formerly known as S-1153 and AG-1549, Figure 1a), is one of the most promising NNRTIs
currently in clinical trials [4]. More recently, sulfanyltriazole derivatives (Figure 1b), have been
identified as potent HIV-1 NNRTIs with low nanomolar inhibitory activity for viruses with wild-type
or K103N/Y181C [5, 6]. In order to explore the structure-activity relationships (SAR) of this novel
scaffold, herein we describe the design, synthesis and anti-HIV-1 activities of a series of novel
substituted N4-arylideneaminotriazole derivatives (Figure 1c).
Figure 1.
NH2
X
N N
N
N
N
N
N
O
H
N
O
R
R
S
S
N
R2
S
O
N
N
R
Cl
R1
R1
Cl
a: Capravirine
b: Sulfanyltriazoles
c: Newly designed arylideneaminotriazoles
Results and Discussion
Compound 1, 4-amino-3-(2-furyl)-5-mercapto-4H-1,2,4-triazole, was prepared by a one-pot
reaction, which started with the reaction of 2-furoic acid hydrazide with carbon disulfide in ethanolic
potassium hydroxide, to form a potassium dithiocarbazinate intermediate, followed by a cyclo-
condensation with excess hydrazine to produce the s-triazole 1 as a white solid in good yield [7]. 2-
furoic acid hydrazide in turn was synthesized from the corresponding 2-furoic acid ester by reaction
with hydrazine hydrate. 5-Alkylthio derivatives 2a-d were prepared by the alkylation of compound 1
with alkyl halides in potassium hydroxide/ethanol solution (Scheme 1) [8, 9].
Schiff’s bases of 4-arylideneamino compounds 3a-d were obtained by the condensation reactions
of compound 1 with different aldehydes in refluxing ethanol solutions in the presence of trace amounts
of concentrated sulphuric acid [10]. The structures of these compounds 3 were confirmed by their 1H-

Molecules 2007, 12
2005
NMR spectra (DMSO-d6), in which two signals appeared at low field. The lower one (δ 13-12 ppm),
exchangeable with deuterium oxide, corresponds to the NH proton (tautomer of the SH one), and the
other one (δ 10-9 ppm) is attributed to the N=CH- group proton. Similar conditions to those described
for the preparation of compounds 2 and 3 were used for the preparation of 4-arylideneamino-5-
alkylthio derivatives 4a-f (Scheme 1).
Scheme 1.
SH
N
S
R1
N
1) NH3.H2O, CS2,
ClCH2COONa
N
N
R1CH2X
N
N
O
O
O
CONHNH2
NH2
NH2
2) NH2NH2.H2O
1
2a-d
OHC-R2
OHC-R2
SH
N
S
N
R1
N
R1CH2X
N
N
N
O
O
N=CH-R2
N=CH-R2
3a-d
4a-f
In order to increase the molecular diversity and analyze the structure-activity relationships, we also
synthesized some new symmetric dimeric derivatives. When 4-arylideneamino-3-(2-furyl)-5-mecapto-
1,2,4-triazoles 3 were mixed with half a mole of an alkyl dibromide and a molar equivalent of
potassium hydroxide in refluxing methanol, dimers of 1,2-{4-arylideneamino-5,5’-di(3-(2-furyl)-1,2,4-
triazolyl)}-dithioethane 5a-d were obtained as light yellow crystals in good yield (Scheme 2).
Scheme 2.
SH
S
(CH2)n
N
S
N
N
X-(CH2)n-X
N
N
N
N
N
N
O
O
O
R3
R3
R3
1 or 3
5a-d
5a n=2, R3=NH2
5b n=3, R3=NH2
5c n=2, R3=5-nitro-2-furylideneamino
5d n=3, R3=5-nitro-2-furylideneamino
Scheme 3.
SH
SH
N
HS
N
N
N
N
N
+
OHC
CHO
N
N
N
H
O
O
O
NH2
N C
H
C
N
1
6

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2006
4-Iminomethyl dimer 6 was prepared by the reaction of compound 1 with 1,4-diformylbenzene in
the refluxing ethanol (Scheme 3). Aminomethylation of compound 3 with formaldehyde and secondary
amines in ethanol media produced the Mannich base 7a in good yield (Scheme 4). An exception was
compound 7b, which was not obtained when the Mannich reaction was carried out under refluxing
conditions, and instead compound 8 was formed (Scheme 5). The structure of compound 8 was
1
confirmed by the H-NMR spectrum, in which no methylene protons signal from 1-dibutylamino
groups was found, as well as confirmed by the elemental analysis and IR spectra.
Scheme 4.
N
SH
S
N
N
CH2O / Piperidine
NO2
N
N
N
O
N
O
O
O
N=CH
N=CH
NO2
3a
7a
Scheme 5.
C4H9
N
N
C4H9
S
N
N
O
O
N=CH
NO2
SH
N
CH2O / NH(C4H9)2
NO2
N
7b
N
O
O
C4H9
C4H9
N=CH
N
N
N
3a
N
O
O
N=CH
NO2
8
The newly synthesized 5-alkylthio derivatives 2a-d, 4-arylideneamino ones 3a-d and the 4-
arylideneamino-5-alkylthio-1,2,4-triazoles were screened for their anti-HIV activities, including
inhibition of HIV-1-induced cytopathogenicity in MT-4 cells and determination of the inhibitory effect
on the HIV-1 RT. The experimental results are listed in Tables 1-4.

Molecules 2007, 12
Table 1. Anti-HIV-1 activity and cytotoxicity of 5-alkylthio-s-triazoles
2007
2a-d in MT-4 cell culture.
S
R1
N
N
N
O
NH2
2a-d
HIV-1 (IIIB)
EC50 (μM)a CC50 (μM)b
Compd.
R1
mp (°C)
SIc
2a
2b
2c
2d
Phenyl
201-203
175-178
235-236
156-158
>459.6
>413.9
>420.9
>466.4
>459.6
×1d
3-Methoxyphenyl
4-Cyanophenyl
Ethoxycarbonyl
>413.9
×1
>or=317.8
>466.4
<or×1
×1
a EC50: dose of compound required to achieve 50% protection of MT-4 cell from
HIV-1-induced cytotoxicity, as determined by the MTT [3-(4,5-dimethylthiazol-
b
2-yl)-2,5-diphenyltetrazolium bromide] method [11]. CC50: dose required to
reduce the viability of mock-infected cells by 50%, as determined by the MTT
method. c SI: selectivity index (CC50/ EC50). d The SI values: ×1 stand for ≥1or<1.
All data represent mean values for at least two separate experiments.
Table 2. Anti-HIV-1 activity and cytotoxicity of
4-arylideneamino-1,2,4-triazoles 3a-d.
SH
N
N
N
O
N=CH-R2
3a-d
HIV-1 (IIIB)
Compd.
R2
mp (°C)
EC50 (μM) CC50 (μM) SI
3a
3b
3c
3d
5-Nitro-2-furyl
198-201
216-218
225-227
226-228
68.3
--
170.8
--
3
4-Methoxyphenyl
3,4-Dimethoxyphenyl
4-Chlorophenyl
--
>281.8
>8.3
290.3
8.3
<1
<1

Molecules 2007, 12
Table 3. Anti-HIV-1 activity and cytotoxicity of 3,4,5-trisubstituted
2008
s-triazoles 4a-f.
S
R1
N
N
N
O
N=CH-R2
4a-f
HIV-1 (IIIB)
Compd.
R1
R2
EC50 (μM) CC50 (μM)
SI
4a
Phenyl
5-Nitro-2-furyl
5-Nitro-2-furyl
5-Nitro-2-furyl
5-Nitro-2-furyl
>6.7
--
6.7
<1
--
4b
3-Methoxyphenyl
4-Cyanophenyl
Ethoxycarbonyl
Phenyl
--
4c
>5.4
>3.2
6.0
<1
<1
21
4d
6.8
4e
3,4-Dimethoxyphenyl 12
260
4f
Ethoxycarbonyl
3,4-Dimethoxyphenyl >293.3
>or=293.3
683
<or×1
Nevirapine
−
0.03
22,767
Table 4. Inhibitory activity of substituted s-triazoles against HIV-1 RT.
Compd.
3a
4e
Nevirapine
IC50 (μM)a
325
43.5
4.5
a 50% of the inhibitory concentration of the substituted s-triazole
derivatives required to inhibit biotin deoxyuridine triphosphate
(biotin-dUTP) incorporation into the HIV-1 RT by 50%.
From the results, we determined that only compounds 4e and 3a exhibited significant inhibitory
activity against HIV-1 replication in MT-4 cell culture, with EC50 values of 12 μM and 68.3 μM,
respectively, and that most of the compounds had lost their effectiveness, compared with the prototype
sulfanyltriazole compounds, which is probably due to the substituents at positions 3, 4 and 5 of the
triazole heterocycle, which did not allow the molecules to bind to HIV-1 RT in a "butterfly-like"
conformation [12]. Compound 4e showed a moderate potency in the inhibition of HIV-1 RT, with an
IC50 value of 43.5 μM, which agrees well with the results obtained in the cell culture test, further
verifying the mechanism of action of this kind of compounds in targeting the NNRTI site of HIV-1
RT.
Conclusions
In summary, a series of 5-alkylthio- (2a-d), 4-arylideneamino- (3a-d) and 4-arylideneamino-5-
alkylthio derivatives (4a-f) of 4-amino-3-(2-furyl)-5-mercapto-1,2,4-triazole (1) were synthesized by
alkylation with alkyl halides and condensation with aldehydes, respectively. Other derivatives such as
sulfanyl dimers 5a-d, 4-iminomethyl dimer 6 and the Mannich base 7a were also prepared. The
1
structures of all newly synthesized compounds were assigned on the basis of elemental analysis, H-
NMR and IR spectra. Some substituted s-triazole derivatives were assayed for anti-HIV-1 activity by

Molecules 2007, 12
2009
inhibition of HIV-1-induced cytopathogenicity in MT-4 cells and by determination of the inhibitory
effect on the HIV-1 RT. The results showed that compound 4e was an active inhibitor against HIV-1
replication in cell culture (EC50 = 12 μM) and against HIV-1 RT (IC50 = 43.5 μM), which provides a
good lead for design and discovery of new high potent HIV-1 NNRTIs by structure-based molecular
modification.
Experimental
General
All melting points were measured on a Reichert-Jung Kofler melting point apparatus and are
1
uncorrected; Elementary analysis data were determined with a Heraeus CHN-RAPID instrument; H-
NMR spectra were recorded on a Bruker Avance 300 (300 MHz) or a Bruker Avance 600 (600 MHz)
spectrometer. The solvents used were DMSO-d6 or CDCl3, as indicated; chemical shifts δ are reported
in ppm units relative to the internal tetramethylsilane (TMS) standard. The coupling constants are
given in Hz, and the different signals are expressed using the standard abbreviations (s, singlet; d,
doublet; t, triplet; m, mutiplet; etc). Infrared spectra (IR) were recorded as KBr disks on a Shimadzu
IR-435 spectrometer. The assignment data are only the maximum value of the absorption peak. All
compounds were routinely checked by TLC. Merck silica gel 60 was used for flash column
chromatographic purification. Solvents were reagent grade and when necessary, were purified and
dried by standard methods. Concentration of the reaction solutions involved the use of a rotary
evaporator at reduced pressure.
General Procedure for the Preparation of Compounds 2
To a suspension of 4-amino-3-furyl-5-sulfanyl-1,2,4-triazole 1 (1 mmol) in ethanol (10 mL) was
added dropwise at 0°C an aqueous solution of potassium hydroxide (2 mol·L-1, 1 mmol). To this
mixture the appropriate alkyl halide (1.2 mmol) was added dropwise with vigorous stirring, the
temperature was allowed to raise to ambient (25°C) and the reaction was continued for 1-2 h. When
the reaction was complete (as checked by TLC), water (20 mL) was added, and a white precipitate was
formed, which was separated by filtration, washed with water, and recrystallized from the appropriate
solvent.
4-Amino-3-(2-furyl)-5-benzylthio-4H-1,2,4-triazole (2a). This compound was obtained as colorless
needles by recrystallization from ethanol; yield 73.2%; mp: 201-203°C; 1H-NMR (300 MHz, DMSO-
d6) δ: 7.88 (1H, d, J = 1.7 Hz, 5’-H-furan), 7.43-7.20 (6H, m, Ar-H), 6.68 (1H, d, J = 3.8 Hz, 3’-H-
furan), 6.11 (2H, s, NH2), 4.41 (2H, s, -CH2-S-); IR (cm-1): 3272, 3172 (υNH), 3127, 3109 (υ=CH), 3064,
3030 (Ar-H), 1648 (m, vC=N), 1602, 1583, 1462, 1431 (υArC=C), 1247 (s, υN-N=C), 696 (m, υC-S-C); Anal.
calcd. for C13H12N4OS: C, 57.21; H, 4.39; N, 20.81; Found: C, 57.33; H, 4.41; N, 20.47.
4-Amino-3-(2-furyl)-5-(3-methoxylbenzyl)thio-4H-1,2,4-triazole (2b). This compound was purified by
1
recrystallization from ethanol to give white needles; yield 67.3 %; mp: 175-178°C; H-NMR (300

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MHz, DMSO-d6) δ: 7.91 (1H, d, J = 1.5 Hz, 5’-H-furan), 7.24-6.82 (5H, m, Ar-H), 6.71 (1H, d, J = 3.5
Hz, 3’-H-furan), 6.14 (2H, s, NH2), 4.40 (2H, s, -CH2-S-), 3.37 (3H, s, OCH3); IR (cm-1): 3280, 3182
(υNH), 3130, 3109 (υ=CH), 3051, 3010 (Ph-H), 2957, 2939 (υCH3), 1641 (m, vC=N), 1608, 1583, 1465,
1433 (υC=C), 1272 (s, υN-N=C), 709 (m, υC-S-C); Anal. calcd. for C14H14N4O2S: C, 55.40; H, 4.75; N,
18.80; Found: C, 55.51; H, 4.67; N, 18.53.
4-Amino-3-(2-furyl)-5-(4-cyanobenzyl)thio-4H-1,2,4-triazole (2c). This compound was purified by
1
recrystallization from ethanol to afford white needles; yield 83%; mp: 235-236°C; H-NMR (300
MHz, DMSO-d6) δ: 7.87 (1H, d, J = 1.2 Hz, 5’-H-furan), 7.76 (2H, AB, d, J = 8.7 Hz, 3,5-H2-Ph), 7.62
(2H, AB, d, J = 8.7 Hz, 2,6-H2-Ph), 7.21 (1H, d, J = 3.6 Hz, 3’-H-furan), 6.68 (1H, dd, J1 = 1.2 Hz, J2
= 3.6 Hz, 4’-H-furan), 6.14 (2H, s, NH2), 4.48 (2H, s, SCH2); IR (cm-1): 3378, 3290 (d, υNH, m), 2225
(υCN, s), 1603, 1517, 1463, 1432, 1414 (s), 1250, 1114, 1017 (s), 978, 902, 748 (s), 556; Anal. calcd.
for C14H11N5OS: C, 56.55; H, 3.73; N, 23.55; Found: C, 55.98; H, 3.77; N, 23.65.
4-Amino-3-(2-furyl)-5-(ethoxylcarbonyl)methylthio-4H-1,2,4-triazole (2d). This compound was
purified by recrystallization from ethanol to give white needles; yield 71%; mp: 156-158°C; 1H-NMR
(300 MHz, DMSO-d6) δ: 7.88 (1H, d, J = 1.5 Hz, 5’-H-furan), 7.21 (1H, d, J = 3.6 Hz, 3’-H-furan),
6.69 (1H, dd, J1 = 1.5 Hz, J2 = 3.6 Hz, 4’-H-furan), 4.44 (2H, s, -CH2-S- ), 4.10 (2H, q, J = 7.5 Hz,
OCH2), 1.18 (3H, t, J = 7.5 Hz, CH3); IR (cm-1): 3322 (υNH), 3154, 3128 (υ=CH), 2993, 2972 (υCH3),
1739 (s, υc=o), 1629, 1415, 1380, 1304, 1176 (s), 1024, 997, 898; Anal. calcd. for C10H12N4O3S: C,
44.77; H, 4.51; N, 20.88. Found: C, 44.82; H, 4.47; N, 20.96.
General Procedure for the Preparation of Compounds 3
A mixture of 4-amino-3-furyl-5-sulfanyl-1,2,4-triazole 1 (0.1 mmol) with an aromatic aldehyde in
ethanol/water (2:1, 40 mL), was refluxed for about 3 h (checked by TLC). When the reaction solution
had cooled down, the crude product was precipitated, collected by filtration and recrystallized from an
appropriate solvent.
4-(5-Nitrofurylidene)amino-3-(2-furyl)-5-sulfanyl-4H-1,2,4-triazole (3a). This compound was
prepared by the condensation of compound 1 with 5-nitro-2-furaldehyde diacetate in sulfuric acid-
ethanol-water (0.1:1:2) solution, and purified by recrystallization from acetone/ethanol (4:1) to give
yellow needles; yield 98.3%; mp: 198-201°C; 1H-NMR (300 MHz, DMSO-d6) δ: 14.39 (1H, s, NH or
SH), 10.34 (1H, s, -N=CH), 8.01 (1H, d, J = 1.5 Hz, 5’-H-furan), 7.88 (1H, AB, d, J1 = 3.5 Hz, 4’’-H-
furan), 7.75 (1H, AB, d, J2 = 3.5 Hz, 3’’-H-furan), 7.28 (1H, d, J = 3.0 Hz, 3’-H-furan), 6.78 (1H, dd,
J1 = 1.5 Hz, J2 = 3.0 Hz, 4’-H-furan); IR (cm-1): 3109 (s, υ=CH), 2977, 2936 (w, υCH), 1623 (s, υC=N),
1536 (s, υasNO2), 1450 (s, υC=C), 1349 (υNO2, s), 1274 (υN-N=C, s), 968 (s), 721 (υC-S-C, m); Anal. calcd.
for C11H7N5O4S: C, 43.28; H, 2.31; N, 22.94. Found: C, 43.35; H, 2.33; N, 23.05.
4-(4-Methoxybenzylidene)amino-3-(2-furyl)-5-sulfanyl-4H-1,2,4-triazole (3b). This compound was
prepared by the reaction of compound 1 with 4-methoxyphenyl aldehyde and purified by
recrystalization from ethanol to obtain white needles; yield 72%; mp: 216-218°C; 1H-NMR (600 MHz,

Molecules 2007, 12
2011
DMSO-d6) δ: 12.84 (1H, s, NH or SH), 10.34 (1H, s, -N=CH), 7.66 (1H, d, J = 1.7 Hz, 5’-H-furan),
7.45 (2H, d, J = 6.7 Hz, 2,6-H2-Ph), 7.43 (2H, d, J = 6.7 Hz, 3,5-H2-Ph), 7.20 (1H, d, J = 3.7 Hz, 3’-H-
furan), 6.65 (1H, dd, J1 = 1.7 Hz, J2 = 3.7 Hz, 4’-H-furan), 3.99 (3H, s, OCH3); IR (cm-1, KBr): 3101
(m, υAr-H), 2974, 2932 (υ=C-H), 1629, 1569 (υC=N), 1453 (υC-O); Anal. calcd. for: C14H12N4O2S: C,
55.99; H, 4.03; N, 18.65. Found: C, 55.82; H, 4.04; N, 18.56.
4-(3,4-Dimethoxylphenylidene)amino-3-(2-furyl)-5-sulfanyl-4H-1,2,4-triazole (3c). This compound
was prepared by the reaction of compound 1 with 3,4-dimethoxylphenyl aldehyde and purified by
1
recrystallization from ethanol to give white needles; yield 56.1%; mp: 225-227°C; H-NMR (600
MHz, DMSO-d6) δ: 12.26 (1H, s, NH or SH), 9.88 (1H, s, -N=CH), 7.64 (1H, d, J = 1.8 Hz, 5’-H-
furan), 7.56-7.20 (3H, m, Ph-H), 7.01 (1H, d, J = 8.1 Hz, 3’-H-furan), 6.58 (1H, dd, J1 = 1.8 Hz, J2 =
8.1 Hz, 4’-H-furan), 4.00 (3H, s, OCH3), 3.97 (3H, s, OCH3); IR (cm–1): 3137, 3115 (m, νAr-H), 2963,
2933 (νCH3), 1604 (νC=C), 1275 (m, νN-N=C); Anal. calcd. for C15H14N4O3S: C, 54.54; H, 4.27; N, 16.96.
Found: C, 54.27; H, 4.13; N, 17.03.
4-(4-Chlorobenzylidene)amino-3-(2-furyl)-5-sulfanyl-4H-1,2,4-triazole (3d). This compound was
prepared by the reaction of compound 1 with 4-chlorophenyl aldehyde, purified by recrystallization
1
from ethanol, and obtained as light yellow needles, yield 77%, mp: 226-228°C; H-NMR (600 MHz,
DMSO-d6) δ: 11.16 (1H, s, NH or SH), 10.40 (1H, s, -N=CH), 7.68 (1H, d, J = 1.3 Hz, 5’-H-furan),
7.52 (2H, d, J = 7.1 Hz, 2,6-H2-Ph), 7.46 (2H, d, J = 7.1 Hz, 3,5-H2-Ph), 6.99 (1H, d, J = 2.9 Hz, 3’-H-
furan), 6.58 (1H, dd, J1 = 1.3 Hz, J2 = 2.9 Hz, 4’-H-furan); IR (cm–1): 3081 (m, υAr-H), 2963, 2933 (υ=C-
H), 1626, 1562, 1594 (υC=N, C=C); Anal. calcd. for C13H9ClN4OS: C, 51.23; H, 2.98; N, 18.38. Found:
C, 51.14; H, 2.96; N, 18.42.
General Procedure for the Preparation of Compounds 4
To a suspension of 4-(arylidene)amino-3-furyl-5-sulfanyl-1,2,4-triazole 3 (1 mmol) in ethanol (10
mL) in ice bath was added dropwise an aqueous solution of KOH (2 mol·L-1, 1 mmol) with vigorous
stirring. When the solid was dissolved, an alkyl halide (1.2 mmol) was added dropwise, and the
temperature was raised to ambient (25°C) and kept there for 1-2 h. When the reaction was complete (as
indicated by TLC), it was then cooled and a white precipitate was formed, which was separated by
filtration, washed with water and recrystallized from a suitable solvent.
4-(5-Nitrofurylidene)amino-3-(2-furyl)-5-benzylthio-4H-1,2,4-triazole (4a). This compound was
1
purified by recrystallization from ethanol to obtain yellow needles; yield 66.4%; mp: 159-161°C; H-
NMR (300 MHz, DMSO-d6) δ: 8.82 (1H, s, -N=CH), 7.93 (1H, d, J = 1.5 Hz, 5’-H-furan), 7.83 (1H,
AB, d, J1 = 3.6 Hz, 4’’-H-furan), 7.65 (1H, AB, d, J2 = 3.6 Hz, 3’’-H-furan), 7.34-7.22 (5H, m, H5-Ph),
7.02 (1H, d, J = 3.6 Hz, 3’-H-furan), 6.71 (1H, dd, J1 = 1.5 Hz, J2 = 3.6 Hz, 4’-H-furan), 4.43 (1H, s,
SCH2); IR (cm-1): 3123, 3027 (Ar-H, w), 1572, 1495, 1435, 1347 (s), 1275, 1255, 1014, 964, 743, 710;
Anal. calcd. for C15H14N4O3S: C, 54.68; H, 3.31; N, 17.71; Found: C, 54.80; H, 3.35; N, 17.85

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4-(5-Nitrofurylidene)amino-3-(2-furyl)-5-(3-methoxylbenzyl)thio-4H-1,2,4-triazole (4b). Separated by
flash column chromatography using as eluant cyclohexane/ethyl acetate (1:3), and further purified by
recrystallization from ethanol /petroleum ether (bp: 90°C) to give yellow needles; yield 49.2%; mp:
1
77-79°C; H-NMR (300 MHz, DMSO-d6) δ: 8.82 (1H, s, =CH), 7.93 (1H, d, J = 1.5 Hz, 5’-H-furan),
7.84 (1H, d, J = 4.0 Hz, 3’’-H-furan), 7.64 (1H, d, J = 4.0 Hz, 4’’-H-furan), 7.16 (1H, m, Ph-H), 7.01
(1H, d, J = 3.6 Hz, 3’-H-furan), 6.87-6.82 (3H, m, Ph-H), 6.70 (1H, dd, J1 = 1.5 Hz, J2 = 3.6 Hz, 4’-H-
furan), 4.37 (2H, s, SCH2), 3.65 (3H, s, OCH3); IR (cm–1): 3091 (νAr-H, s), 1532, 1492, 1441, 1349 (s),
1267, 967; Anal. calcd. for C15H14N4O3S: C, 53.52; H, 3.78; N, 16.42; Found: C, 52.82; H, 3.85; N,
17.01.
4-(5-Nitrofurylidene)amino-3-(2-furyl)-5-(4-cynobenzyl)thio-4H-1,2,4-triazole (4c). This compound
was purified by recrystallization from ethanol to give yellow needles; yield 78%; mp: 216-218°C; 1H-
NMR (300 MHz, DMSO-d6) δ: 8.85 (1H, s, =CH), 7.92 (1H, d, J = 1.5 Hz, 5’-H-furan), 7.84 (1H, d, J
= 4.0 Hz, 4’’-H-furan), 7.73 (2H, d, J = 8.2 Hz, 2,6-H2-Ph), 7.65 (1H, d, J = 4.0 Hz, 3’’-H-furan), 7.52
(2H, d, J = 8.2 Hz, 3,5-H2-Ph), 7.01 (1H, d, J = 3.6 Hz, 3’-H-furan), 6.70 (1H, dd, J1 = 1.5 Hz, J2 = 3.6
Hz, 4’-H-furan), 4.48 (1H, s, SCH2); IR (cm–1): 2226 (νCN), 1531, 1436, 1349, 1272, 1021, 965, 810,
754, 738; Anal. calcd. for C19H12N6O4S: C, 54.28; H, 2.88; N, 19.99. Found: C, 54.40; H, 2.95; N,
20.10.
4-(5-Nitro-2-furylmethylidene)amino-3-(2-furyl)-5-(ethoxylcarbonyl)methylthio-4H-1,2,4-triazole
(4d). This compound was purified by recrystallization from ethanol to give yellow needles; yield 80%;
mp: 139-140°C; 1H-NMR (300 MHz, DMSO-d6) δ: 8.94 (1H, s, =CH), 7.93 (1H, d, J = 1.5 Hz, 5’-H-
furan), 7.86 (1H, d, J = 3.6 Hz, 4’’-H-furan), 7.71 (1H, d, J = 3.6 Hz, 3’’-H-furan), 7.06 (1H, d, J = 3.0
Hz, 3’-H-furan), 6.72 (1H, dd, J1 = 1.5 Hz, J2 = 3.0 Hz, 4’-H-furan), 4.08 (2H, q, J = 7.2 Hz, OCH2),
4.07 (2H, s, SCH2), 1.13 (3H, t, J = 7.2 Hz, CH3); IR (cm–1): 1736 (νC=O, s), 1530, 1442, 1349, 1307,
1278, 1188, 1028, 766; Anal. calcd. for C15H13N5O6S: C, 46.04; H, 3.35; N, 17.89; Found: C, 45.87; H,
3.39; N, 18.08.
4-(3,4-Dimethoxylphenylidene)amino-3-(2-furyl)-5-benzylthio-4H-1,2,4-triazole (4e). This compound
was separated by flash column chromatography using as eluant hexane/ethyl acetate (3:2) and further
1
purified by recrystallization from ethanol to give white crystals; yield 48.1%; mp: 100-102°C; H-
NMR (300 MHz, DMSO-d6) δ: 8.62 (1H, s, =CH), 7.89 (1H, d, J = 1.5 Hz, 5’-H-furan), 7.48-7.12 (8H,
m, Ar-H), 7.01 (1H, d, J = 3.6 Hz, 3’-H-furan), 6.70 (1H, dd, J1 = 1.5 Hz, J2 = 3.6 Hz, 4’-H-furan),
4.40 (2H, s, SCH2), 3.85 (3H, s, OCH3), 3.82 (3H, s, OCH3); IR (cm-1): 3100 (ν=CH), 3025 (Ph-H),
2957, 2935 (νCH3), 1598, 1576, 1513 (s), 1274 (νN-N=C, s), 1138 (s), 1026, 1020 (s), 784, 704 (νC-S-C);
Anal. calcd. for C22H20N4O3S: C, 62.84; H, 4.79; N, 13.32; Found: C, 62.96; H, 4.83; N, 13.28.
4-(3,4-Dimethoxylphenylidene)amino-3-(2-furyl)-5-(ethoxylcarbonyl)methylthio-4H-1,2,4-triazole
(4f). This compound was purified by recrystallization from acetone/cyclohexane (1:1) to give white
1
crystals; yield 33.4%; mp: 196-199°C; H-NMR (300 MHz, DMSO-d6) δ: 8.62 (1H, s, N=CH), 7.89
(1H, d, J = 1.5 Hz, 5’-H-furan), 7.31 (1H, m, Ph-H), 7.22 (1H, d, J = 3.0 Hz, 3’-H-furan), 7.01-6.86

Molecules 2007, 12
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(2H, m, Ph-H), 6.68 (1H, dd, J1 = 1.5 Hz, J2 = 3.0 Hz, 4’-H-furan), 4.91 (2H, s, SCH2), 4.13 (2H, q, J
= 7.2 Hz, OCH2), 3.69 (3H, s, OCH3), 3.57 (3H, s, OCH3), 1.13 (3H, t, J = 7.2 Hz, CH3); IR (cm-1):
3146 (ν=CH, m), 3020 (Ph-H, m), 2955 (νCH3, w), 1740 (νC=O, s), 1517 (s), 1450, 1450, 1414 (m), 1271
(νN-N=C, s), 1032, 1013, 754 (m); Anal. calcd. for C19H20N4O5S: C, 54.80; H, 4.84; N, 13.45. Found: C,
54.65; H, 4.87; N, 13.53.
General Procedure for the Preparation of Compounds 5
To a suspension of 4-(arylidene)amino/amino-3-furyl-5-sulfanyl-1,2,4-triazole 1 or 3 (1 mmol) in
ethanol (10 mL) in an ice bath was added dropwise an aqueous solution of KOH (2 mol·L-1, 1 mmol)
with vigorous stirring. When the solid was dissolved, the appropriate alkyl dihalide (0.5 mmol) was
added dropwise and the temperature was allowed to raise at 25°C for 1 h, then the reaction solution
was refluxed for 1-2 h. (checked by TLC). The product was separated by flash column
chromatography with suitable eluents and further purified by recrystallization from an appropriate
solvent.
1,2-bis[(4-Amino-3-(2-furyl)-4H-1,2,4–triazol)-5-yl]dithioethane (5a). This compound was prepared
by the reaction of compound 1 with 1,2-dibromoethane under reflux for 2 days, and purified by
1
recrystallization from ethanol to obtain a yellow solid; yield 52%; mp: 289-291°C; H-NMR (600
MHz, DMSO-d6) δ: 7.90 (2H, d, J = 1.5 Hz, 5’-H-furan), 7.24 (2H, d, J = 3.4 Hz, 3’-H- furan), 6.71
(2H, dd, J1 = 1.8 Hz, J2 = 3.4 Hz, 4’-H-furan), 6.15 (4H, s, NH2), 3.55 (4H, t, J = 19.8 Hz); IR (cm-1):
3329, 3258 (m, νN-H), 3181, 3134 (ν=CH), 2977, 2947 (w, νCH2), 1628 (s, νC=N), 1526 (νasNO2), 1432,
1416 (s, νC=C, νN-N=C), 1018, 898, 751 (s), 738 (s); Anal. calcd. for C14H14N8O2S2: C, 43.07; H, 3.61; N,
28.70. Found: C, 43.01; H, 3.63; N, 28.64.
1,3-bis[(4-Amino-3-(2-furyl)-4H-1,2,4–triazol)-5-yl]dithiopropane (5b). This compound was prepared
from compound 1 reacted with 1,3-dibromopropane under reflux for 2 day, and purified by
1
recrystallization from ethanol to obtain a yellow solid; yield 62%; mp: 297-299°C; H-NMR (600
MHz, DMSO-d6) δ: 7.89 (2H, d, J = 1.8 Hz, 5’-H-furan), 7.23 (2H, d, J = 3.4 Hz, 3’-H-furan), 6.70
(2H, dd, J1 = 1.8 Hz, J2 = 3.4 Hz, 4’-H-furan), 6.13 (4H, s, NH2), 3.29 (4H, t, J = 18.8 Hz, CH2-S),
2.13 (2H, m, CH2); IR (cm-1): 3327, 3284 (m, νN-H), 3174, 3130 (ν=CH), 2977, 2947 (νCH2), 1635, 1432,
1416 (s, νC=C, νN-N=C), 1021, 1009, 756 (s), 740 (s); Anal. calcd. for C15H16N8O2S2: C, 44.54; H, 3.99;
N, 27.70; Found: C, 44.56; H, 4.01; N, 27.78.
1,2-bis[(4-(5-Nitro-2-furylmethylidene)amino-3-(2-furyl)-4H-1,2,4-triazol)-5-yl]dithioethane
(5c).
This compound was prepared from the reaction of compound 3a with dibromoethane, and purified by
1
recrystallization from ethanol to give a yellow solid; yield 61.2%; mp: 220°C; H-NMR (600 MHz,
DMSO-d6) δ: 8.89 (2H, s, -N=CH), 7.92 (2H, d, J = 1.8 Hz, 5’-H-furan), 7.83 (2H, d, J = 3.9 Hz, 4’’-
H-furan), 7.68 (2H, d, J = 3.9 Hz, 3’’-H-furan), 7.02 (2H, d, J = 3.6 Hz, 3’-H-furan), 6.70 (2H, dd, J1 =
1.8 Hz, J2 = 3.6 Hz, 4’-H-furan), 3.54 (4H, s, CH2-S); IR (cm-1): 3113, 3009 (ν=CH), 2923 (νCH), 1568,
1437 (νC=C), 1526 (νasNO2), 1349 (s, νsNO2), 1258 (s, νN-N=C), 713 (νC-S-C); Anal. calcd. for
C24H16N10O8S2: C, 45.28; H, 2.53; N, 20.00; Found: C, 45.26; H, 2.60; N, 19.98.

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1,3-bis[(4-(5-Nitro-2-furylmethylidene)amino-3-(2-furyl)-4H-1,2,4-triazol)-5-yl]dithiopropane (5d).
This compound was prepared from the reaction of compound 3a with dibromopropane, purified by
1
recrystallization from ethanol and obtained as yellow solid; yield 61%; mp: 220°C; H-NMR (600
MHz, DMSO-d6) δ: 8.92 (2H, s, -N=CH), 7.93 (2H, d, J = 1.7 Hz, 5’-H-furan), 7.84 (2H, d, J = 4.0 Hz,
4’’-H-furan), 7.70 (2H, d, J = 4.0 Hz, 3’’-H-furan), 7.04 (2H, d, J = 3.4 Hz, 3’-H-furan), 6.72 (2H, dd,
J1 = 1.7 Hz, J2 = 3.4 Hz, 4’-H-furan), 3.30 (4H, t, J = 7.0 Hz, CH2-S), 2.1 (2H, m, CH2 ); IR (cm-1):
3150, 3112 (w, ν=CH), 2913 (νCH), 1527, 1437 (νC=C, C=N), 1527 (νasNO2), 1350 (s, νsNO2); Anal. calcd. for
C25H18N10O8S2: C, 46.15; H, 2.79; N, 21.53. Found: C, 46.22; H, 2.81; N, 21.56.
Preparation of 1,4-bis[(4-Iminomethyl-3-(2-furyl)-5-mercapto-4H-1,2,4-triazole -4-yl] phene (6). The
preparation of compound 6 by the reaction of compound 3a with 1,4-diformylbenzene in refluxing
ethanol for 2 h used the same conditions of compounds 3a-d and the product was obtained as a white
solid; yield 61%; mp: 264-265°C; 1H-NMR (600 MHz, DMSO-d6) δ: 14.30 (2H, s, NH or SH), 10.04
(2H, s, -N=CH), 8.17 (4H, s, Ph-H4), 7.99 (2H, d, J = 1.7 Hz, 5’-H-furan), 7.18 (2H, d, J = 3.4 Hz, 3’-
H-furan), 6.72 (2H, dd, J1 = 1.7 Hz, J2 = 3.4 Hz, 4’-H-furan); IR (cm-1): 3423 (νNH), 3087, 3015 (m,
ν=CH), 2965, 2992, 2923 (w, νCH), 1626 (νC=N), 1527, 1452 (νC=C, C=N), 1266 (s), 969, 749; Anal. calcd.
for C20H14N8O2S2: C, 51.94; H, 3.05; N, 24.23. Found: C, 51.87; H, 3.02; N, 24.29.
Preparation of 4-[2-(5-nitro)furylmethylidene]amino-3-(2-furyl)-5-mercapto-piperidylmethyl-4H-
1,2,4-triazole (7a). Compound 3a (10 mmol) was dissolved in a mixed ethanol and dioxane solvent
(2:1, 90 mL), then formaldehyde (40%, 1.5 mL) and piperidine (10 mmol) in ethanol (100 mL) were
added to this solution. The mixture was stirred for 1-2 h and then kept overnight at room temperature.
The solid was collected by filtration and recrystallized from a mixture of ethanol and dioxane (2:1) to
1
yield the title compound 7a; yield: 80%; mp: 195-197°C; H-NMR (600 MHz, DMSO-d6) δ: 10.28
(1H, s, N=CH), 8.00 (1H, d, J = 1.7 Hz, 5’-H-furan), 7.84 (1H, d, J = 4.0 Hz, 3’’-H-furan), 7.71 (1H, d,
J = 4.0 Hz, 4’’-H-furan), 7.29 (1H, d, J = 3.5 Hz, 3’-H-furan ), 6.78 (1H, dd, J1 = 1.7 Hz, J2 = 3.5 Hz,
4’-H-furan), 5.12 (2H, s, N-CH2-N), 2.74 (4H, t, J = 5.2 Hz, 3,5-H4-piperidine), 1.50 (4H, d, J = 4.7
Hz, 2,6-H4-piperidine), 1.34 (2H, d, J = 5.3 Hz, 4-H2-piperidine); IR (cm-1): 3141, 3120 (w, ν=CH),
2942, 2930 (w, νCH2), 1621, 1530, 1497, 1459, 1434, 1426 (νC=C, C=N), 1349, 1303, 1277, 764; Anal.
calcd. for C17H18N6O4S: C, 50.74; H, 4.51; N, 20.88. Found: C, 50.80; H, 4.49; N, 20.9.
Preparation of 4-[2-(5-nitro)furylmethylidene]amino-3-(2-furyl)-5-mercapto-(bis-n-butylamino)-4H-
1,2,4-triazole (8). Compound 3a (10 mmol) was dissolved in a mixed ethanol and dioxane solvent
(2:1, 90 mL), then formaldehyde (40%, 1.5 mL) and di-n-butylamine (10 mmol) in ethanol (100 mL)
were added to this solution. The mixture was refluxed under stirring for 1 h. The white solid formed
was collected by filtration and recrystallized from a mixture of ethanol and dioxane (1:1) to give the
1
title compound 8; yield: 78%; mp: 214-215°C; H-NMR (600 MHz, DMSO-d6) δ: 11.20 (1H, s,
N=CH), 7.84 (1H, d, J = 1.7 Hz, 5’-H-furan), 7.84 (1H, d, J = 4.0 Hz, 3’’-H-furan), 7.83 (1H, d, J =
4.0 Hz, 4’’-H-furan), 7.52 (1H, d, J = 3.5 Hz, 3’-H-furan ), 6.68 (1H, dd, J1 = 1.7 Hz, J2 = 3.5 Hz, 4’-
H-furan), 2.81 (4H, t, J = 7.6 Hz, N-(CH2-)2), 1.54 (4H, m, 2CH2), 1.36 (4H, m, 2CH2), 0.92 (6H, t, J =
7.4 Hz, 2CH3); IR (cm-1): 3110 (w, ν=CH), 2962, 2934, 2862 (w, νCH2), 1530, 1494, 1450 (νC=C
, C=N),

Molecules 2007, 12
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1374, 1350, 1253; Anal. calcd. for C19H24N6O4: C, 56.99; H, 6.04; N, 20.99. Found: C, 57.12; H, 5.99;
N, 21.02.
Anti-HIV-1 assays in MT-4 cells culture
Activity of the compounds against HIV-1 (IIIB strain) multiplication in acutely infected cells was
based on the inhibition of virus-induced cytopathogenicity in MT-4 cells. Briefly, culture medium (50
μL) containing 1×104 cells was added to each well of flat-bottomed microtiter trays containing culture
medium (50 μL) with or without various concentrations of the tested compounds and then a HIV-1
suspension (20 μL) containing 100 CCID50 (50% cell culture infective dose) was added. After 5 days
of incubation at 37°C, the number of viable cells was determined by the MTT method. The cytotoxity
of the compounds was evaluated in parallel with their antiviral activity, based on the viability of mock-
infected cells as monitored by the MTT method.
HIV-1 RT inhibition assay
Inhibition of HIV-1 RT was developed using nucleotides linked to microtiter plate with
colorimetric detection of incorporated biotin-dUTP into homopolymer template primers [13]. The
incorporated quantities of the biotin-dUTP into the enzyme represented the activity of HIV-1 RT. IC50
values corresponded to the concentration of the substituted s-triazole derivatives required to inhibit
biotin-dUTP incorporation into the HIV-1 RT by 50%.
Acknowledgements
The financial support of this work by the National Natural Science Foundation of China
(NSFC30371686), Ministry of Science and Technology of China for the Key Project of International
Cooperation (2003DF000033) and NSF Shandong Province (Y2003C11) is gratefully acknowledged.
We also thank State New Drug Screening Center of Shanghai, China, for assistance with the anti-HIV-
1 RT assay.
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Sample Availability: Samples of the compounds are available from the authors.
© 2007 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.