1,2,4-Triazole derivatives inhibiting the human immunodeficiency virus type 1 (HIV-1) in vitro

Article abstract of DOI:10.1002/1522-2675(200207)85:7<1883::AID-HLCA1883>3.0.CO;2-R

A novel series of selective 1,2,4-triazole nonnucleoside reverse transcriptase inhibitors (NNRTIs) is described. In MT-4 cells compound, 4f inhibited human immunodeficiency virus type 1 (HIV-1) induced cytopathology at an 1C₅₀ of 9.98 μM with a

Full text of DOI:10.1002/1522-2675(200207)85:7<1883::AID-HLCA1883>3.0.CO;2-R

Helvetica Chimica Acta Vol. 85 (2002)
1883
1,2,4-Triazole Derivatives Inhibiting the Human Immunodeficiency Virus
Type 1 (HIV-1) in vitro
by Irene M. Lagojaa)1), Christophe Pannecouque*b)1), Laura Musumecia), Matheus Froeyena),
Arthur Van Aerschota), Jan Balzarinib), Piet Herdewijna), and Erik De Clercqb)
a) Laboratory of Medicinal Chemistry
b) Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven
Dedicated to Professor Wolfgang Pfleiderer on the occasion of his 75th birthday
A novel series of selective 1,2,4-triazole nonnucleoside reverse transcriptase inhibitors (NNRTIs) is
described. In MT-4 cells compound, 4f inhibited human immunodeficiency virus type 1 (HIV-1) induced
cytopathology at an IC50 of 9.98 mm with a selectivity index of 18.6. The hypothetical docking model of RT/4f
derived from X-ray crystallographic structure of capravirine complex with HIV-1 RT links the activity profile to
the H-bonding network.
1. Introduction. Replication of human immunodeficiency virus type 1 (HIV-1) can
be reduced in HIV-1-infected patients with a combination of antiviral drugs targeted at
the reverse transcriptase (RT) and protease (PR) [1]. Unfortunately, due to the high
mutation rate of HIV, treatment, even drug combinations, select for drug-resistant HIV
variants [2]. There is a continuous need for new drugs that are active against clinical
drug-resistant mutant strains and/or aimed at new targets in the viral replicative cycle
[3].
The HIV RT is necessary for the conversion of the HIV single-stranded RNA to
double-stranded DNA [4]. Several clinically used drugs do have RT as a target. These
drugs either compete with the natural 2'-deoxynucleoside-5'-O-triphosphates or are
targeted at an allosteric, nonnucleoside binding site [5]. The latter compounds are
referred to as NNRTIs (nonnucleoside reverse transcriptase inhibitors). They
constitute a group of chemically dissimilar molecules that bind to a hydrophobic
pocket near, but distinct from, the polymerase active site of the p66 subunit. The
enzymatic activity of RT is inhibited by allosteric changes in the enzyme that cause a
distortion of the catalytic active-site aspartyl residues [6]. To the NNRTIs belongs
capravirine (S-1153), a 1,2,4,5-substituted imidazole derivative [7], which is able to
inhibit HIV-1 strains that are resistant to other NNRTIs. This ability to cope with
typical NNRTI-induced mutations is based, at least in part, on an extensive network of
H-bonds involving the main chain of residues 101, 103, and 236 of the p66 RT subunit
[8]. With the structure of capravirine as a lead compound, we synthesized a series of
1,2,4-triazole derivatives and determined their anti-HIV activity in a cell-based assay
[9].
1
)
Both authors contributed equally.

1884
Helvetica Chimica Acta Vol. 85 (2002)
The 3-ethyl-1-phenyl-5-(benzylsulfanyl)-1H-[1,2,4]triazole 4f had low cytotoxicity
(50% cytotoxic concentration (CC50: 185.20 mm)) and showed anti-HIV-1 activity at a
50% inhibitory concentration (IC50) of 9.98 mm (selectivity index 18.6). The selective
activity of this and related compounds, 4c and 4d, against HIV-1(IIIB) and their
inactivity against an NNRTI-resistant (RT K103N and Y181C) mutant, HIV-2(ROD),
and simian immunodeficiency virus (SIV(mac251)) clearly indicate that these
compounds qualify as NNRTIs and are targeted at HIV-1 RT.
2. Chemical Results and Discussion. Using capravirine as our lead substance, we
synthesized a new group of five-membered ring heterocycles, carrying two bulky
substituents in ortho to each other. The 1,2,4-triazole ring was selected as the
heterocyclic scaffold for the substituents because its chemistry is well-known. Indeed,
diverse biological applications have been reported for 5-alkyl-2-aryl-1,2,4-triazolidine-
3-thiones 1 [10] (Scheme 1), which possess a similar arrangement of substituents.
Scheme 1. Synthesis of 5-Alkyl-2-aryl-1,2,4-triazolidine-3-thiones 1, 3-Alkyl-1-aryl-4,5-dihydro-1H-1,2,4-tri-
azole-5-thiones 3, and 3-Alkyl-1-aryl-5-[(arylmethyl)sulfanyl]-1H-1,2,4-triazoles 4
In a one-pot reaction, 5-alkyl-2-aryl-1,2,4-triazolidine-3-thiones 1 can be obtained
in good yield from aldehydes, hydrazines, and KSCN [11]. To introduce a second bulky
substituent, a benzylation reaction was attempted. However, direct benzylation with
BnCl as a Mitsunobu reaction [12] yielded N-amino-N-phenylthiourea (2). Therefore,
the 1,2,4-triazolidine-3-thiones 1 were first oxidized with H2O2 to obtain the more-
stable aromatic 3-alkyl-1-aryl-4,5-dihydro-1H-1,2,4-triazole-5-thiones 3, which could
subsequently easily be benzylated to the corresponding 3-alkyl-1-aryl-5-[(arylmethyl)-
sulfanyl]-1H-1,2,4-triazoles 4 (Scheme 1).
However, with propanal as the starting material to obtain the 5-Et analogues, the
reaction was not straightforward. Reaction of propanal with phenylhydrazine and
KSCN did not lead to a single product. An unseparable 1:1 mixture of two compounds
with the same molecular weight was obtained. Likewise, following the oxidation step,
no separation was possible. In the past [11], a structure with an endocyclic S-atom has
been considered a possible product of the ring closure of hydrazones and KSCN, but
was later ruled out for chemical reasons. Here, a 1:1 mixture of 5-ethyl-2-phenyl-1,2,4-
triazolidine-3-thione (1c) and 5-ethyl-3-phenyl-1,3,4-thiadiazolidin-2-imine (5) was

Helvetica Chimica Acta Vol. 85 (2002)
1885
obtained and oxidized to the corresponding mixture of 3-ethyl-4,5-dihydro-1-phenyl-
1H-1,2,4-triazole-5-thione (3c) and 5-ethyl-3-phenyl-1,3,4-thiadiazol-2(3H)-imine (6)
(Scheme 2).
Scheme 2. Synthesis of the 1,2,4-Triazolidine-3-thione and 1,3,4-Thiadiazolidine-2-imine Derivatives 1c and 5,
Respectively, and Their Oxidation Products 3c and 6, Respectively.
A triazole derivative could be obtained by a further rearrangement of 6. Indeed,
through a Dimroth rearrangement [13], 6 could be transformed into 5-ethyl-2,3-
dihydro-2-phenyl-1H-1,2,4-triazole-3-thione (7) (Scheme 3).
Scheme 3. Dimroth Rearrangement of 1,3,4-Thiadiazol-2(3H)-imine 6.
The two isomers 3c and 7 could be separated by column chromatography. The
position of the NH was determined by way of NOE-difference spectroscopy.
Irradiation of the NH gave rise to a positive NOE enhancement of the Et as well as
the Ph moiety for 7, whereas, in case of 3c, only a positive NOE enhancement for the Et
group could be observed. Benzylation of 3c yielded the corresponding 3-ethyl-1-
phenyl-5-[(phenylmethyl)sulfanyl]-1H-1,2,4-triazole (4c), whereas, in the case of 7
under the same reaction conditions (NaH, benzyl chloride), the corresponding 5-ethyl-
2,3-dihydro-1-[(2-methylphenyl)methyl]-2-phenyl-1H-1,2,4-triazole-3-thione (8) could
be obtained.
We also decided to synthesize an example of a 1,2,4-triazole derivative with a
different substitution pattern on the heterocyclic ring. The Ph ring remained in a-
position with respect to the benzylsulfanyl substituent, but, in contrast to the previous
series, it was substituted at N(3) instead of N(2). This alters the ring geometry. The
isomeric 4,5-dihydro-4-phenyl-1H-1,2,4-triazole-5-thione (9) [14] were obtained by

1886
Helvetica Chimica Acta Vol. 85 (2002)
cyclization of 4-(phenylsulfanyl)semicarbazide with HCOOEt. Subsequent benzyla-
tion yielded 3-[(arylmethyl)sulfanyl]-4-phenyl-4H-1,2,4-triazoles 10.
3. Biological Results and Discussion. We evaluated a series of substituted 1,2,4-
triazole derivatives for their potential to inhibit the in vitro replication of HIVand SIV
in a cell-culture model for acute infection. The antiviral activity and cytotoxicity data
are presented in Table 1.
Table 1. Inhibition of HIV-1, HIV-2, and SIV Replication in MT-4 Cells by Triazole Derivatives
Compound
IC50 [mm]a)
HIV-1
CC50 [mm]b)
HIV-2
ROD
SIV
mac251
IIIB
NNRTIrc)
1a
3c
4a
4b
4c
4d
4e
4f
7
8
10a
10b
10c
> 42.02
> 243.58
> 60.70
> 31.41
ND
ND
ND
ND
> 72.65
> 76.24
> 58.50
> 185.20
ND
> 118.64
ND
> 152.15
ND
> 42.02
> 243.58
> 60.70
> 31.41
> 72.65
> 76.24
> 58.50
> 185.20
> 243.6
> 118.64
> 201.54
> 152.15
> 87.41
> 42.02
> 243.58
> 60.70
> 31.41
> 72.65
> 76.24
> 58.50
> 185.20
> 243.6
> 118.64
> 201.54
> 152.15
> 87.41
42.02 Æ 19.19
> 243.58
60.70 Æ 15.33
31.41 Æ 15.88
72.65 Æ 14.82
76.24 Æ 19.88
58.50 Æ 14.38
185.20 Æ 80.57
17.30 Æ 0.85
16.88 Æ 4.94
> 40.62 (25 37%)d)
9.98 Æ 4.37
> 243.6
> 243.6
46.63 Æ 21.33
118.61 Æ 3.20
201.54 Æ 36.99
152.15 Æ 51.28
87.41 Æ 10.20
> 201.54
> 152.15(15 42%)d)
> 87.41
a) 50% Inhibitory concentration, or concentration required to inhibit the viral cytopathic effect by 50% in MT-4
cells. Data represent average values Æ SD for at least two independent experiments. b) 50% Cytotoxic
concentration, or concentration that reduced MT-4 cell viability by 50%. c) NNRTI-Resistant HIV-1 strain.
d) Percent inhibition of virus replication at the indicated concentration is mentioned between parentheses.
Anti-HIVactivity was observed for compounds 4c, 4d, 4f, and 8, of which the former
three compounds, 4c, 4d, and 4f, are structurally related. The basis of these three
molecules is 3-alkyl-5-(benzylsulfanyl)-1-phenyl-1H-1,2,4-triazole. From comparison
of the in vitro antiretroviral activity of this subgroup of molecules, it appears that a m-Cl
substituent on the 5-phenylsulfanyl moiety is deleterious for the antiretroviral activity
(4a vs. 4d, and 4b vs. 4c). m-Me vs. a m-Cl substitution (compounds 4c and 4d, resp.)
gave similar anti-HIV activity and cytotoxicity. Comparison of the 3-Me vs. the 5-Et
derivative (4c vs. 4e) revealed that the 3-Me compound was slightly more active and
slightly less cytotoxic. Comparison of 4f and 4e indicated that 3-methylation of the
benzylsulfanyl group rendered the compound less active. Comparing 3c with its
benzylated derivative 4f clearly established the necessity of the benzyl moiety for anti-
HIV activity. Displacement of the 1-aryl moiety to the 4-position gave the triazole
derivatives inactive (4c and 4d vs. 10c and 10b).
When the 1,2,4-triazole derivatives were evaluated for their inhibitory activity
against HIV-1 RT, compound 4f was inhibitory at an IC50 of 156 and 128 mm in the
presence of poly(rA ¥ dT) and poly(rC ¥ dG), respectively, as the template/primer
(Table 2). Compounds 4e, 10b, and 8 inhibited the reaction by 50% at concentrations of

Helvetica Chimica Acta Vol. 85 (2002)
1887
Table 2. Inhibitory Activity of Triazole Derivatives against Recombinant HIV-1 RT with Poly (rC ¥ dG) or Poly
(rA ¥ dT) as the Template/Primer, and [3H]dGTP and [3H]dTTP as the Radiolabeled Substrate
Test compounds
IC50 [mm]a)
Poly (rA ¥ dT)
(dTTP)
Poly (rC ¥ dG)
(dGTP)
4c
4d
4e
4f
> 710 (8%)b)
> 633 (21%)
> 646 (37%)
156
> 710 (24%)
> 633 (39%)
> 646 (45%)
128
8
10b
> 646 (45%)
710
594
632
a) 50% Inhibitory concentration or compound concentration required to inhibit HIV-1 RT activity by 50%.
b) Percent enzyme inhibition at the indicated concentration is mentioned between parentheses.
ca. 600 700 mm. Thus, the most efficient inhibitor of HIV-1, i.e., 4f, was also most
inhibitory to HIV-1 RT. Compound 4c afforded slight inhibition of the RT reaction at
the highest concentration tested (ca. 710 mm).
4. Computer-Aided Ligand Design.
The crystal structure of HIV-1 RT with
capravirine (pdb entry 1EP4) was used as starting point [8]. All H2O molecules were
removed as well as the S-1153 inhibitor. The structure 4f was drawn in Macromodel and
optimized in the AM1 force field [15]. We used a flexible superposition program that
can vary the dihedral angles of molecule 4f, superimposing this molecule onto the
reference molecule capravirine with the SEAL program [16] and optimizing the
resulting score with an adaptive simulated annealing optimizer [17] (Fig. 1,a). The final
structure was then put in the NNRTI site of RT. The topology and amber force field
parameters for 4f were constructed based on existing similar parameter values in the
amber5 software [18]. Electrostatic charges were calculated by means of the GAMESS
[19] and RESP methods [20].
The complex was then energy-minimized, and the results were examined with
RasMol [21] (Fig. 1,b). We assume that molecules 10x bind in the same way as
molecules 4x, as the ring systems of both molecule series can be superimposed
completely. Following superposition, we obtain an apparent swap of N(2) with C(3) of
the triazole ring.
The capravirine-RT complex is stabilized by two H-bonds from the carbamate
group of capravirine to P236 main-chain O-atom and K103 backbone N-atom. As our
molecules 4x and 10x lack a carbamate group, which could contribute to the binding of
the new inhibitors, a lower affinity is expected compared to capravirine (Fig. 2). A
third H-bond is observed from the five-ring N-atom N(2) to K101 main-chain CO,
mediated by a H2O molecule (distance N(2) ¥¥¥ K101.N 4.09 ä). In our RT-4f model,
N(2) of the triazole ring may possibly make an analogous H2O-mediated H-bond with
K101 backbone O-atom (distance 4f N(2) ¥¥¥ K101.O 4.64 ä). However, this interaction
with N(2) is also possible for the 10x series with N(1) taking the position of C(3)
(distance 4f C(3) ¥¥¥ K101.O 4.47 ä). An additional H2O-mediated H-bond might be
formed from N(1) to the Y318 OH group. Based on the distances (4f N(1) ¥¥¥ Y318.OH

1888
Helvetica Chimica Acta Vol. 85 (2002)
Fig. 1. a) Flexible-seal fit of compound 4f onto capravirine, b) model of compound 4/7 bound to the NNRTI site
of HIV-1 RT. The distances from the atom N(2) to K101.O and Y318.OH are indicated with dashed lines.

Helvetica Chimica Acta Vol. 85 (2002)
1889
Fig. 2. Structure analogy of capravirine vs. triazole derivatives 4
4.66 ä, 4f C(3) ¥¥¥ Y318.OH 5.38 ä), this is more likely the case for the 4x series than
for the 10x series of molecules, which could partially explain the differences in binding
strength and activity.
5. Conclusions. We synthesized a series of [1,2,4] triazole derivatives based on the
structure of capravirine. Some of these compounds inhibited the HIV replication in
vitro, based on an NNRTI type of action.
Experimental Part
General. For all reactions, anal. grade solvents were used. Compound 9 was prepared according to the
procedure described in [14]. TLC was performed on TLC aluminium sheets (Merck, silica gel 60 F254) and silica
(200 425 mesh) was used for column chromatography (CC). M.ps. were determined with a B¸chi SMP-20 cap.
melting-point apparatus. NMR Spectra were recorded on a Varian Gemini-200 spectrometer (1H: 200 MHz, 13C:
50 MHz; 13C and 1H signals refer to TMS; all NH/OH protons were assigned by exchange with D2O. Exact mass
measurements were performed on a quadrupole time-of-flight mass spectrometer (Q-Tof-2, Micromass,
Manchester, UK) equipped with a standard electrospray-ionization (ESI) interface; samples were infused in
i-PrOH/H2O 1:1 at 3 ml/min.
Synthesis 5-Alkyl-2-aryl-1,2,4-triazolidine-3-thiones (1): General Procedure. To a stirred soln. of an aldehyde
(2.5 mmol) in AcOH (10 ml), hydrazine (2.5 mmol) or hydrazine hydrochloride was added. After 30 min,
KSCN (0.37 g, 3.8 mmol) was added at r.t. After stirring for 5 h at 308, the mixture was checked with TLC
(CH2Cl2/MeOH 99 :1). After adding 50 ml of H2O, the precipitate was filtered off and washed with H2O. Mostly,
recrystallization from MeOH afforded pure products.
2-(3-Chlorophenyl)-5-methyl-1,2,4-triazolidine-3-thione (1a). Yield 55.5%. M.p. 128 1308 (MeOH). Rf
(CH2Cl2/MeOH 9 :1) 0.83. 1H-NMR ((D6)DMSO): 1.34 (d, J ˆ 6, Me); 5.19 (q, J ˆ 5.8, HÀC(5)); 7.00 (t, J ꢀ 8.8,
HÀC(4), HÀC(6) of C6H4); 7.06 (s, HÀC(2) of C6H4); 7.32 (t, J ꢀ 8, HÀC(5) of C6H4); 9.16 (s, NH); 10.58
(s, NH). 13C-NMR ((D6)DMSO): 22.4 (Me); 114.5, 115.4, 122.2, 130.9, 133.8, 152.5 (C(6), C(5), C(4), C(2),
‡
C(3), C(1) of C6H4); 78.6, 177.8 (C(5), 3-CˆS). HR-MS: 228.0369 ([M ‡ 1] C6H10ClN3S; calc. 228.0362).
5-Methyl-2-phenyl-1,2,4-triazolidine-3-thione (1b). Yield 61.4%. M.p. 169 1718. Rf (CH2Cl2/MeOH 9 :1)
0.83. 1H-NMR ((D6)DMSO): 1.33 (d, J ˆ 5.8, Me); 5.05 (q, J ˆ 5.8, HÀC(5)); 7.00 (dd, J ˆ 5.4, 4.2, HÀC(3),
HÀC(5) of Ph); 7.26 7.33 (HÀC(2), HÀC(4), HÀC(6) of Ph); 7.03 (s, NH); 8.94 (s, NH); 10.31 (s, NH).
13C-NMR ((D6)DMSO): 21.4 (Me); 116.3, 123.1, 129.4, 151.4 (C(2), C(6); C(4); C(3), C(5); C(1) of Ph); 79.3,
‡
177.7 (C(5), 3-CˆS). HR-MS: 194.0748 ([M ‡ 1] , C9H11N3S; calc. 194.0752).
Oxidation Reaction: General Procedure. To a stirred soln. of 1 (100 mg, 0.52 mmol) in acetone (5 ml), H2O2
(ca. 0.1 mmol) was added. After stirring for 1 h at r.t., the mixture was checked by TLC (CH2Cl2/MeOH 99 :1).
After removing the solvent under reduced pressure, the product was either purified by CC or directly used for
further reactions.
1-(3-Chlorophenyl)-4,5-dihydro-3-methyl-1H-1,2,4-triazole-5-thione (3a). Yield 80%. M.p. 144 1468. Rf
(CH2Cl2/MeOH 99 :1) 0.77. 1H-NMR ((D6)DMSO): 2.55 (s, Me); 7.34 7.48 (m, 4 arom. H); 8.42 (s, NH).

1890
Helvetica Chimica Acta Vol. 85 (2002)
13C-NMR ((D6)DMSO): 13.2 (Me); 122.4, 124.7, 129.2, 130.4, 135.1, 137.7 (C(6), C(2), C(4), C(5), C(1), C(3) of
‡
‡
C6H4); 154.3, 159.2 (C(5), CˆS). HR-MS: 226.0238 ([M ‡ 1] , C9H9ClN3S ; calc. 226.0206).
4,5-Dihydro-3-methyl-1-phenyl-1H-1,2,4-triazole-5-thione (3b): Yield 97.5%. M.p. 105 1078. Rf (CH2Cl2/
MeOH 99 :1): 0.85. 1H-NMR ((D6)DMSO): 2.43 (s, Me); 7.35 7.37 (m, 5 arom. H); 8.35 (s, NH); 13C-NMR
((D6)DMSO): 13.0 (Me); 124.5, 129.3, 129.6, 136.8 (C(2), C(6); C(4); C(3), C(5); C(1) of Ph); 153.9, 158.8
‡
(C(5), 3-CˆS). HR-MS: 192.0598 ([M ‡ 1] , C9H10N3S; calc. 192.0595).
3-Ethyl 4,5-dihydro-1-phenyl-1H-1,2,4-triazole-5-thione (3c): Yield 41.6%. M.p. 188 1918. Rf (AcOEt/
hexane 2 :1) 0.79. Exact mass (C10H11N3S) [M ‡ H] : Calc. 206.0752, found: 206.0758. 1H-NMR ((D6)DMSO):
‡
1.22 (t, J ˆ 7.4, Me); 2.63 (q, J ˆ 7.4, CH2); 7.55 (d, J ˆ 6, HÀC(2), HÀC(6) of Ph); 7.24 7.38 (m, HÀC(8),
HÀC(4), HÀC(5) of Ph); 8.36 (s, NH). 13C-NMR ((D6)DMSO): 12.2 (Me); 21.5 (CH2); 124.5, 129.2, 129.5,
‡
136.8 (C(2), C(6); C(4); C(3), C(5); C(1) of Ph); 152.7, 158.8 (C(5), CˆS). HR-MS: 206.0758 ([M ‡ 1] ,
‡
C10H11N3S ; calc. 206.0752).
Benzylation Reaction: General Procedure. To a stirred soln. of 3 or 7 (1.55 mmol) in MeCN (10 ml), NaH
(100 mg, 50% suspension in H2O) and, after 1 h, the corresponding BnCl (1.55 mmol) was added. The mixture
was left overnight at r.t., and then evaluated by TLC (CH2Cl2/MeOH 97:3). After removing the solvent, the
product was purified by CC (CH2Cl2/MeOH 97:3).
1-(3-Chlorophenyl)-5-{[(3-chlorophenyl)methyl]sulfanyl}-3-methyl-1H-1,2,4-triazole (4a). Yield 62.6%.
‡
Oil. Rf (CH2Cl2/MeOH 99 :1) 0.51. Exact mass (C16H14N3SCl2) [M ‡ H] : Calc. 350.0285, found: 350.0302.
1H-NMR (CDCl3): 2.49 (s, Me); 4.31 (s, CH2); 7.19 7.45 (m, 8 arom. H). 13C-NMR (CDCl3): 13.1 (Me); 35.2
(CH2); 121.9, 124.4, 129.1, 130.3, 135.0, 138.0 (C(6), C(2), C(4), C(5), C(1), C(3) of ClC6H4), 127.1, 127.3, 128.6,
129.6, 133.9, 139.5 (C(6), C(2), C(5), C(4), C(3), C(1) of ClC6H4CH2); 153.2 (C(3), 159.7 (C(5)). HR-MS:
‡
‡
350.0302 ([M ‡ 1] ; C16H14Cl2N3S ; calc. 350.0285).
1-(3-Chlorophenyl)-3-methyl-5-{[(3-methylphenyl)methyl]sulfanyl}-1H-1,2,4-triazole (4b). Yield 63.5%.
Oil. Rf (CH2Cl2/MeOH 95 :5) 0.83. 1H-NMR (CDCl3): 2.31 (s, MeC6H4); 2.47 (s, MeÀC(3)); 4.34 (s, CH2);
7.02 7.47 (m, 8 arom. H). 13C-NMR (CDCl3): 13.0 (MeÀC(3)); 21.0 (MeÀC6H4); 35.8 (CH2); 121.8, 124.2,
129.1, 130.2, 134.9, 138.0 (C(6), C(2), C(4), C(5), C(1), C(3) of ClC6H4); 125.9, 127.9, 128.2, 128.4, 136.9, 137.8
‡
(C(6), C(2), C(4), C(5), C(3), C(1) of MeC6H4); 152.9 (C(3), 160.3 (C(5)). HR-MS: 330.0832 ([M ‡ 1] ,
‡
C17H17ClN3S ; calc. 330.0832).
3-Methyl-5-{[(3-methylphenyl)methyl]sulfanyl}-1-phenyl-1H-1,2,4-triazole (4c). Yield 60%. Oil. Rf
(CH2Cl2/MeOH 99 :1) 0.41. 1H-NMR (CDCl3): 2.32 (s, MeC6H4); 2.49 (MeÀC(3)); 4.36 (s, CH2); 7.19 7.26
(m, 5 H); 7.38 7.51 (m, 5 H). 13C-NMR (CDCl3): 13.0 (C(3)); 21.19 (MeC6H4); 36.0 (CH2); 124.4, 129.4, 129.8,
137.0 (C(2), C(6); C(4); C(3), C(5); C(1) of Ph); 126.2, 128.1, 128.3, 129.8, 137.1, 137.3 (C(6), C(2), C(4), C(5),
‡
‡
C(3), C(1) of MeC6H4); 153.1 (C(3)); 160.0 (C(5)). HR-MS: 296.1234 ([M ‡ 1] C17H18N3S ; calc. 296.1221).
5-{[(3-Chlorophenyl)methyl]sulfanyl}-3-methyl-1-phenyl-1H-1,2,4-triazole (4d): Yield 66%. Oil; Rf
(CH2Cl2/MeOH 97:3) 0.26. 1H-NMR (CDCl3): 2.47 (s, Me); 4.31 (s, CH2); 7.19 7.49 (m, 9 H). 13C-NMR
(CDCl3): 12.9 (Me), 35.3 (CH2); 124.2, 127.3, 129.3, 139.7 (C(2), C(6); C(3), C(5); C(4); C(1) of Ph), 127.2,
128.6, 129.2, 129.6, 133.9, 139.7 (C(6), C(2), C(5), C(4), C(3), C(1) of ClC6H4); 153.2 (C(3)); 159.3 (C(5)). HR-
‡
MS: 316.0690 ([M ‡ 1] , C16H15ClN3S; calc. 316.0675).
3-Ethyl-5-{[(3-methylphenyl)methyl]sulfanyl}-1-phenyl-1H-1,2,4-triazole (4e). Yield 65%. Oil. Rf (CH2Cl2/
MeOH 99 :1) 0.82. 1H-NMR (CDCl3): 1.38 (t, J ˆ 6.6, Me); 2.31 (s, MeC6H4); 2.80 (q, J ˆ 6.6, CH2); 4.44
(s, 2 H); 7.08 7.27 (m, 4 H); 7.35 7.50 (m, 5 arom. H of Ph). 13C-NMR (CDCl3): 12.5 (Me); 21.2 (MeC6H4);
21.8 (CH2), 37.9 (C6H4CH2); 123.9, 129.2, 129.9, 136.1 (C(4); C(2), C(6); C(3), C(5), C(1) of Ph); 124.1, 124.9,
126.2, 128.3, 129.4, 129.9 (C(6), C(5), C(4), C(2), C(1), C(3) of MeC6H4); 152.7 (C(3)); 160.2 (C(5)). HR-MS:
‡
‡
310.1395 ([M ‡ 1] , C18H20N3S ; calc. 310.1378).
3-Ethyl-1-phenyl-5-[(phenylmethyl)sulfanyl]-1H-1,2,4-triazole (4f). Yield 60%. Oil. Rf (CH2Cl2/MeOH
99 :1) 0.52. 1H-NMR (CDCl3): 1.30 (t, J ˆ 7.6, Me); 2.77 (q, J ˆ 7.6, CH2); 4.40 (s, CH2); 7.26 7.48 (m, 10 H).
13C-NMR (CDCl3): 12.4 (Me); 20.1 (CH2); 36.24 (PhCH2); 124.9, 129.5, 130.1, 137.5 (C(2), C(6); C(4); C(3),
C(5); C(1) of Ph); 127.3, 128.5, 128.9, 129.2 (C(2), C(6); C(4); C(3), C(5); C(1) of PhCH2); 152.9 (C(3); 160.0
‡
‡
(C(5)). HR-MS: 296.1237 ([M ‡ 1] , C17H18N3S ; calc. 296.1221).
5-Ethyl-2,3-dihydro-1-[(3-methylphenyl)methyl]-2-phenyl-1H-1,2,4-triazole-3-thione (8). Yield 43.5%. Oil.
Rf (CH2Cl2/MeOH 99 :1) 0.95. 1H-NMR (CDCl3): 1.35 (t, J ˆ 7.6, Me); 2.17 (s, Me); 2.80 (q, J ˆ 7.6, CH2); 4.36
(s, CH2); 7.09 7.51 (m, 9 H). 13C-NMR (CDCl3): 12.4 (Me); 20.1 (CH2); 21.3 (MeC6H4); 30.8 (C6H4CH2); 124.8,
126.5, 129.8, 142.5 (C(2), C(6); C(3), C(5); C(4); C(1) of Ph); 126.2, 128.3, 128.8, 129.2, 137.9, 138.0 (C(6), C(5),
‡
‡
C(4), C(2), C(3), C(1) of MeC6H4); 158.4 (C(5)); 160.2 (C(3)). HR-MS: 310.1386 ([M ‡ 1] , C18H19N3S ; calc.
310.1378).

Helvetica Chimica Acta Vol. 85 (2002)
1891
4-Phenyl-3-[(phenylmethyl)sulfanyl]-4H-1,2,4-triazole (10a). Yield 85.4%. M.p. 88 918. Rf (CH2Cl2/
MeOH 97 :3) 0.72. 1H-NMR (CDCl3): 4.44 (s, CH2); 7.17 7.30 (m, 7 arom. H); 7.42 7.44 (m, 3 arom. H); 8.26
(s, HÀC(5)). 13C-NMR (CDCl3): 37.1 (CH2); 124.8, 128.7, 129.4, 135.9 (C(2), C(6); C(3), C(5); C(4); C(1) of
Ph); 127.4, 128.2, 128.8, 132.9 (C(4); C(2), C(6); C(3), C(5); C(1) of Bn); 144.1, 150.1 (C(5), C(3)). HR-MS:
‡
‡
268.0920 ([M ‡ 1] , C15H14N3S ; calc. 268.0908).
3-{[(3-Methylphenyl)methyl]sulfanyl}-4-phenyl-4H-1,2,4-triazole (10b). Yield 75.4%. Oil. Rf (CH2Cl2/
MeOH 97:3) 0.92. 1H-NMR (CDCl3): 2.27 (s, Me); 4.43 (s, CH2); 7.06 7.22 (m, 6 arom. H); 7.43 7.47
(m, 3 arom. H); 8.30 (s, HÀC(5)). 13C-NMR (CDCl3): 20.9 (Me); 37.3 (CH2); 124.9, 129.0, 129.6, 135.7 (C(2),
C(6); C(3), C(5); C(4); C(1) of Ph)), 125.4, 128.0, 128.3, 128.8, 136.9, 138.1 (C(6), C(2), C(4), C(5), C(5), C(1)
‡
‡
of MeC6H4); 144.2, 150.5 (C(5), C(3). HR-MS: 282.1062 ([M ‡ 1] , C16H16N3S ; calc. 282.1065).
3-{[(3-Chlorophenyl)methyl]sulfanyl}-4-phenyl-4H-1,2,4-triazole (10c). Yield 70%. Oil. Rf (CH2Cl2/
MeOH 95 :5) 0.81. 1H-NMR (CDCl3): 4.43 (s, CH2); 7.19 7.32 (m, 6 arom. H); 7.46 7.50 (m, 3 arom. H);
8.30 (s, HÀC(5)). 13C-NMR (CDCl3): 36.6 (CH2); 125.1, 129.1, 129.6, 134.3 (C(2), C(6); C(3), C(5); C(4); C(1)
of Ph); 127.3, 127.9, 129.5, 129.8, 133.1, 138.4 (C(6), C(5), C(4), C(2), C(1), C(3) of C6H4CH2); 144.4, 150.1
‡
‡
(C(5), C(3)). HR-MS: 302.0511 ([M ‡ 1] , C15H13ClN3S ; calc. 302.0519).
Synthesis of 5-ethyl-2,3-dihydro-2-phenyl-1H-1,2,4-triazole-3-thione (7) by a Dimroth Rearrangement. A
soln. of 500 mg (2.43 mmol) of the mixture 1f/6 in aq. NaOH (25 ml, 10%) was heated for 5 h at 808. After this
time, TLC (AcOEt/hexane 2 :1) revealed only two spots: the isomer 1f, which had not been involved in the
Dimroth rearrangement (higher Rf), and the newly formed heterocycle 7 at lower Rf. After adding H2O and the
extraction with CH2Cl2, the solvent was removed, and the product was purified by CC (AcOEt/hexane 2 :1).
Yield 28%. Oil. Rf (AcOEt/hexane 2 :1) 0.68. 1H-NMR (CDCl3): 1.38 (t, J ˆ 7.6, Me); 2.86 (q, J ˆ 7.6, CH2); 7.36
(t, J ˆ 7.4, HÀC(4) of Ph); 7.36 (dd, J ˆ 7.6, 8.2, HÀC(3), HÀC(5) of Ph); 7.66 (t, J ˆ 8.2, HÀC(2), HÀC(6) of
Ph); 8.44 (s, NH). 13C-NMR (CDCl3): 12.4 (Me); 21.8 (CH2); 119.9, 127.8, 129.7, 141.0 (C(2), C(6); C(4); C(3),
C(5); C(1) of Ph), 164.5 (C(5)); 177.2 (CˆS).
Viruses and Cells. The origin of the HIV-1(IIIB) virus stock has been described in [22]. HIV-1 and
HIV-2(ROD) [23] stocks were obtained from the culture supernatant of HIV-1- or HIV-2-infected MT-4 cells,
resp. [24]. Simian immunodeficiency virus (SIV(mac251)) was originally isolated by Daniel et al. [25] and was
obtained from C. Bruck (Smith-Kline-RIT, Rixensart, Belgium). SIV(mac251) stocks were prepared from the
supernatant of SIV-infected MT-4 cells. S0561945 is an HIV-1(IIIB) strain, possessing the K103N and Y181C
mutations in its RT gene, resulting in resistance towards nonnucleoside RT inhibitors (NNRTIs) [26].
Antiviral Activity and Cytotoxicity Assays. The inhibitory effects of a series of substituted imidazole and
1,2,4-triazole derivatives on HIV-1, HIV-2, and SIV replication were monitored by measuring the viability of
MT-4 cells at 5 d after infection. Cytotoxicity of the compounds was determined in parallel by measuring the
viability of mock-infected cells on day 5. The number of viable cells was quantified semi-automatically by a
tetrazolium-based colorimetric method with 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium (MTT), as
described by Pauwels et al. [9].
Reverse Transcriptase (RT) Assays. For determination of the 50% inhibitory concentration (IC50) of the test
compounds against HIV-1 RT, the RNA-dependent DNA polymerase assay was performed as follows: the
reaction mixture (50 ml) contained 50 mm Tris ¥ HCl (pH 7.8), 5 mm DTT, 300 mm glutathione, 500 mm EDTA,
150 mm KCl, 5 mm MgCl2, 1.25 mg of bovine serum albumin, a fixed concentration of the labelled substrate
[3H]dGTP or [3H]dTTP (ca. 6 mm), a fixed concentration of the template/primer poly (rC ¥ oligo-dG) or poly
(A ¥ oligo-dT) (0.1 mm; Amersham Pharmacia Biotech), 0.06% Triton X-100, 5 ml of inhibitor solution
(containing various concentrations (10-fold dilutions) of the compounds), and 5 ml of the RT preparations. The
reaction mixtures were incubated at 378 for 30 min; then 200 ml of yeast RNA (2 mg/ml) and 1 ml of CCl2COOH
(5% (v/v)) in sat. phosphate buffer were added. The solns. were kept on ice for at least 15 min; then the acid-
insoluble material was filtered over Whatman GF/C glass-fiber filters and washed with 5% CCl3COOH in H2O
and EtOH. The filters were then analyzed for radioactivity in a liquid scintillation counter (Canberra Packard,
Zellik, Belgium). The IC50 value for each test compound was determined as the compound concentration that
inhibited HIV RT activity by 50%.
We are grateful to Cindy Heens, Kristien Erven, Liesbet De Dier, and Lizette van Berckelaer for their
excellent technical assistance, and to Christiane Callebaut for dedicated editorial help. We thank Prof. Jef
Rozenski for the mass spectrometry. I. Lagoja is a research associate of the Rega Foundation. We thank ISEP/
FORTIS and the European Commission for financial support. Results of this research are subject of a patent
application (I. M. Lagoja, C. Pannecouque, A. Van Aerschot, P. Herdewijn, and E. De Clercq, Patent appl. PCT/
EPOI/02140, 24.02.2001).

1892
Helvetica Chimica Acta Vol. 85 (2002)
REFERENCES
[1] E. De Clercq, J. Med. Chem. 1995, 38, 2491.
[2] a) J. M. Coffin, Science 1995, 267, 483 489; b) A.-M. Vandamme, K. Van Vaerenbergh, E. De Clercq,
Antiviral Chem. Chemother. 1998, 9, 187.
[3] a) E. De Clercq, Rev. Med. Virol. 2000, 10, 255; b) J. P. Moore, M. Stevenson, Nat. Rev. Mol. Cell Biol. 2000,
1, 40 49.
[4] R. A. Katz, A.-M. Skalka, Annu. Rev. Biochem. 1994, 63, 133.
¬
[5] H. Jonckheere, J. Anne, E. De Clercq, Med. Res. Rev. 2000, 20, 129.
[6] R. Esnouf, J. Ren, C. Ross, Y. Jones, D. Stammers, D. Stuart, Nat. Struct. Biol. 1995, 2, 303.
[7] T. Fujiwara, A. Sato, M. El-Farrash, S. Miki, K. Abe, Y. Isaka, M. Kodama, Y. Wu, L. B. Chen, H. Harada,
H. Sugimoto, M. Hatanaka, Y. Hinuma, Antimicrob. Agents Chemother. 1998, 42, 1340.
[8] J. Ren, C. Nichols, L. E. Bird, T. Fujiwara, H. Sugimoto, D. I. Stuart, D. K. Stammers, J. Biol. Chem. 2000,
275, 14316.
[9] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Herdewijn, J. Desmyter, E. De Clercq, J. Virol.
Methods 1988, 20, 309.
[10] a) W. Lunkenheimer, G. Jaeger, F. Hoffmeister, Deutsches Patent DT2440378 A1, 1974; b) E. S. H. El
Ashry, L. F. Awad, M. Winkler, J. Chem. Soc., Perkin Trans., 2000, 829, 834.
[11] H. Schildknecht, G. Renner, Liebigs Ann. Chem. 1972, 761, 189.
[12] D. L. Comins, G. Jianhua, Tetrahedron. Lett. 1994, 35, 2819.
[13] A. Sitte, R. Wessel, H. Paul, Monatsh. Chemie 1975, 106, 1291.
[14] M. Pesson, S. Dupin, G. Polmans, Bull. Soc. Chim. Fr. 1961, 1581.
[15] F. Mohamadi, N. G. J. Richards, W. C. Guida, R. Liskamp, M. Liton, C. Caufield, G. Chang, T. Hendrickson,
W. C. Still, J. Comput. Chem. 1990, 11, 440.
[16] S. K. Kearsley, G. M. Smith, Tetrahedron Comput. Methodol. 1990, 3, 615.
[17] L. Ingber, Math. Comp. Modelling 1993, 18, 29.
[18] W. D. Cornell, P. Cieplak, C. I. Bayly, I. R. Gould, K. M. Mertz, D. M. Ferguson, D. C. Spellmeyer, T. Fox,
J. W. Caldwell, P. A. Kollman, J. Am. Chem. Soc. 1995, 117, 5179.
[19] M. W. Schmidt, K. K. Baldridge, J. A. Boatz, S. T. Elbert, M. S. Gordon, J. H. Jensen, S. Koseki, N.
Matsunaga, K. A. Nguyen, S. Su, T. L. Windus, M. Dupuis, J. A. Montgomery, J. Comput. Chem. 1993, 14,
1347.
[20] C. I. Bayly, P. Cieplak, W. D. Cornell, P. A. Kollman, J. Phys. Chem. 1993, 97, 10269.
[21] R. A. Sayle, E. J. Milner-White, TIBS 1995, 20, 374.
[22] M. Popovic, M. G. Sarngadharan, E. Read, R. C. Gallo, Science 1984, 224, 497.
¬
[23] F. Barre-Sinoussi, J. C. Chermann, M. T. Nugeyre, S. Chamaret, J. Gruest, C. Dauguet, C. Axler-Blin, F.
¬
Vezinet-Brun, C. Rouzioux, W. Rozenbaum, L. Montagnier, Science 1983, 220, 868.
[24] S. Harada, Y. Koyanagi, N. Yamamoto, Science 1985, 229, 563.
[25] M. D. Daniel, N. L. Letvin, P. K. Sehgal, G. Hunsmann, D. K. Schmidt, N. W. King, R. C. Desrosiers, J. Gen.
Virol. 1987, 68, 3183.
[26] M. Witvrouw, W. Pluymers, N. Neamati, T. Burke, G. Pais, E. De Clercq, C. Pannecouque, Z. Debyser,
Abstracts of 14th International Conference on Antiviral Research, Seattle, Washington, USA; Antiviral
Res. 2001, 50, No. 1: A49 abstract No. 33.
Received May 6, 2002