Synthesis of some novel thiourea derivatives obtained from 5-[(4-aminophenoxy)methyl]-4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thiones and evaluation as antiviral/anti-HIV and anti-tuberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2007.04.010

As a continuation of our previous efforts on N-alkyl/aryl-N′-[4-(4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thione-5-yl)phenyl]thioureas 1-19 and N-alkyl/aryl-N′-[4-(3-aralkylthio-4-alkyl/aryl-4H-1,2,4-triazole-5-yl)phenyl]thioureas 20-22, a series of novel 5-[(4-aminophenoxy)methyl]-4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thiones 23-26 and several related thioureas, N-alkyl/aryl-N′-{4-[(4-alkyl/aryl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy]phenyl}thioureas 27-42 were synthesized for evaluation of their antiviral potency. Structures of the synthesized compounds were confirmed by the use of ¹H NMR, ¹³C NMR and HR-MS data. All compounds 1-42 were evaluated in vitro against HIV-1 (IIIB) and HIV-2 (ROD) strains in MT-4 cells, as well as other selected viruses such as HSV-1, HSV-2, Coxsackie virus B4, Sindbis virus and Varicella-zoster virus using HeLa, Vero, HEL and E6SM cell cultures, and anti-tuberculosis activity against Mycobacterium tuberculosis H37Rv. Compounds 4 and 5 showed weak activity against HSV-1, HSV-2 and TK^- HSV, whereas eight compounds showed marginal activity against Coxsackie virus B4. The most active derivative in this series was compound 38 which showed moderate protection against Coxsackie virus B4 with an MIC value of 16 μg/ml and a selectivity index of 5. This compound was also active against thymidine kinase positive Varicella-zoster virus (TK⁺ VZV, OKA strain) with an EC₅₀ value of 9.9 μg/ml. Compound 38 was the most active compound with 79% inhibition against M. tuberculosis H37Rv.

Full text of DOI:10.1016/j.ejmech.2007.04.010

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 381e392
http://www.elsevier.com/locate/ejmech
Original article
Synthesis of some novel thiourea derivatives obtained from 5-[(4-
aminophenoxy)methyl]-4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-
thiones and evaluation as antiviral/anti-HIV and anti-tuberculosis agents
a,
a
a
a
b
_
*
Ilkay Kuc¸ukguzel , Esra Tatar , Sx. Guniz Kuc¸ukguzel , Sevim Rollas , Erik De Clercq
¨ ¨ ¨ ¨
¨
¨
¨
a
_
Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Tibbiye cad. No. 49, Haydarpaxsa 34668 Istanbul, Turkey
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, 3000 Leuven, Belgium
Received 20 November 2006; received in revised form 30 March 2007; accepted 2 April 2007
Available online 13 May 2007
Abstract
As a continuation of our previous efforts on N-alkyl/aryl-N0-[4-(4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thione-5-yl)phenyl]thioureas
1e19 and N-alkyl/aryl-N0-[4-(3-aralkylthio-4-alkyl/aryl-4H-1,2,4-triazole-5-yl)phenyl]thioureas 20e22, a series of novel 5-[(4-aminophenoxy)-
methyl]-4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thiones 23e26 and several related thioureas, N-alkyl/aryl-N0-{4-[(4-alkyl/aryl-5-thioxo-
4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy]phenyl}thioureas 27e42 were synthesized for evaluation of their antiviral potency. Structures of
1
the synthesized compounds were confirmed by the use of H NMR, 13C NMR and HR-MS data. All compounds 1e42 were evaluated in vitro
against HIV-1 (IIIB) and HIV-2 (ROD) strains in MT-4 cells, as well as other selected viruses such as HSV-1, HSV-2, Coxsackie virus B4, Sind-
bis virus and Varicella-zoster virus using HeLa, Vero, HEL and E6SM cell cultures, and anti-tuberculosis activity against Mycobacterium tuber-
culosis H37Rv. Compounds 4 and 5 showed weak activity against HSV-1, HSV-2 and TKꢀ HSV, whereas eight compounds showed marginal
activity against Coxsackie virus B4. The most active derivative in this series was compound 38 which showed moderate protection against
Coxsackie virus B4 with an MIC value of 16 mg/ml and a selectivity index of 5. This compound was also active against thymidine kinase positive
Varicella-zoster virus (TKþ VZV, OKA strain) with an EC50 value of 9.9 mg/ml. Compound 38 was the most active compound with 79%
inhibition against M. tuberculosis H37Rv.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: 1,2,4-Triazole; Thioureas; Antiviral activity; Anti-tuberculosis activity
1. Introduction
inhibit HIV-1 RT competitively at the substrate binding site,
non-nucleoside reverse transcriptase inhibitors (NNRTIs)
exhibit their action by binding to a specific allosteric site,
thereby resulting in non-competitive inhibition of this enzyme
[1]. Examples of such non-nucleoside inhibitors of HIV-1 RT
may include widely diverging chemical classes such as dipyr-
idodiazepinones (nevirapine), bis-heteroarylpiperazines (dela-
virdine, U-90152), benzoxazinones (efavirenz; DMP-266),
TIBO derivatives (9-Cl TIBO), 1-[(2-hydroxyethoxy)-
methyl]-6-phenylthio)thymine (HEPT-S), a-anilinophenylace-
tamides (a-APAs) and phenylethylthiazolylthiourea (PETT)
derivatives (LY73497 and trovirdine HCl) (Fig. 1) [2,3].
A literature survey revealed that molecular modeling stud-
ies for the non-nucleoside inhibitor (NNI)-binding pocket of
Although combination therapies have been proven to de-
crease HIV-related mortality, there is still a need for develop-
ment of novel antiretroviral agents due to the emergence of
multi-drug resistance, which is a major challenge to successful
therapy for individuals infected with HIV. An essential step for
the replication of the virus involves reverse transcription of
retroviral RNA to proviral DNA by the enzyme reverse tran-
scriptase (RT). In contrast to nucleoside analogues which
* Corresponding author. Tel.: þ90 216 349 12 16; fax: þ90 216 345 29 52.
_
E-mail address: ikucukguzel@marmara.edu.tr (I. Kuc¸ukguzel).
¨ ¨
¨
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.04.010

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I. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 43 (2008) 381e392
382
CH3
H3C
S
H
NH
N
CH3
N
CH3
H
N
O
N
N
O
O
S
F3C
CH3
H3C
Cl
HN
N
Cl
N
O
O
N
N
CH3
N
N
H
O
N
H
Nevirapine
Delavirdine
Efavirenz
9-Cl TIBO
CH3
O
Br
NH
H3C
CH3
NH2
N
O
O
S
N
Cl
N
S
N
S
S
O
N
N
NH
H
N
H
N
HCl
H
Cl
O
H3C
H
OH
HEPT-S
Loviride (α-APA)
PETT (LY73497)
Trovirdine HCl (LY300046)
Fig. 1. Structures of the NNRTIs from different chemical classes.
HIV-1 RT might be an effective approach for new drug devel-
opment. The need for a butterfly-like conformation in order for
a compound to inhibit HIV-1 RT enzyme was first demon-
strated using X-ray crystallographic data obtained from nevir-
apine [4]. As a result of such pharmacophoric modeling
studies on the structures of existing NNRTIs, Bal et al. [5]
recently reported a 3D pharmacophoric distance map which
describes the structural requirements for a compound to
show anti-HIV activity via inhibition of HIV-1 RT [6].
Ahgren et al. [7] reported a new class of NNRTIs,
namely phenyl ethyl thiazolyl thiourea (PETT) analogues
(Fig. 1). Discovery of these derivatives as potent inhibitors
of HIV-1 was first reported by Bell et al. [8]. In this system-
atic search, pharmacophores essential for antiretroviral activ-
ity were identified. Disconnection of the bonds present in
rigid tricyclic nucleus of the TIBO derivative led to the gen-
eration of simpler, open-chained structures, PETT (LY
73497) and trovirdine (LY300046). Many PETTs and analo-
gous thiourea derivatives have been identified as NNRTIs
[9e17]. There is an increasing concern on thioureas as
some of them also have been reported as antiviral agents
against several Herpes viruses such as HSV [18], VZV
[19,20] and CMV [21e23].
their structure in the light of antiviral activity results of 1e22.
Based on the experience with this type of molecules, a certain
degree of flexibility might be required for binding to HIV-1
RT. Following these observations, we have designed and syn-
thesized compounds 23e42 in which more flexibility is ascer-
tained by replacing the phenyl ring at the fifth position of
1,2,4-triazole ring with a phenoxymethyl moiety.
Tuberculosis is a chronic infection disease caused by several
species of mycobacteria. The incidence of tuberculosis is in-
creasing worldwide, partly due to poverty and inequity and
partly to the HIV/AIDS pandemic, which greatly increases the
risk of infectious diseases. During recent years, Mycobacterium
tuberculosis has developed increased resistance against drugs.
Some of these derivatives 1e3 and 21 have been reported to pos-
sess positive response against M. tuberculosis H37Rv [24].
Thiacetazone which possesses a thiosemicarbazone structure,
has been reported as a tuberculostatic agent. Doub and co-
workers [25] revealed anti-tuberculosis properties for a wide
number of phenylthioureas. Isoxyl (ISO), a thiourea (thiocar-
lide; 4,40-diisoamyloxythiocarbanilide), was reported to exhibit
potent activity against M. tuberculosis H37Rv (MIC, 2.5 mg/ml),
Mycobacterium bovis BCG (MIC, 0.5 mg/ml), Mycobacterium
avium (MIC, 2.0 mg/ml), and Mycobacterium aurum A1
(MIC, 2.0 mg/ml), resulting in complete inhibition of myco-
bacteria [26]. It was also reported that combination therapy
of INH (isonicotinic acid hydrazide) and ISO was more ef-
fective than monotherapy with either drug. In earlier studies,
3-thioxo/alkylthio-1,2,4-triazole derivatives [24] N-phenyl-N0-
[4-(5-alkyl/arylamino-1,3,4-thiadiazole-2-yl)phenyl]thioureas
[27] examined by the Tuberculosis Antimicrobial Acquisition
In a previous study [24], we have synthesized a series of
thioureas 1e22 (Fig. 2), in which a nitrogen-containing het-
erocycle is combined with thiourea moiety as in the case of
non-nucleoside reverse transcriptase inhibitors (NNRTIs)
phenylethylthiazolylthiourea (PETT) derivative LY 73497
and trovirdine (LY300046). The aim of the present work
was to investigate antiviral properties of 1e22 and to modify

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383
O
OH
O
O
O
O
CH3
a
H3C
N
H
H3C
N
H
I
b
O
O
H
N
H
N
O
NH2
O
O
N
H
c
O
N
H
R1
S
H3C
N
H
H3C
NH
II
IIIa-d
d
H
H
N
N
N
N
S
e
O
S
N
O
S
N
R1
R2
R1
N
H
N
H
H2N
23-26
27-42
IIIa (R1: CH3);IIIb (R1: C2H5); IIIc (R1: CH2CH=CH2); IIId (R1: C6H5);
23 (R1: CH3); 24 (R1: C2H5); 25 (R1: CH2CH=CH2); 26 (R1: C6H5);
27 (R1: CH3; R2: CH3); 28 (R1: CH3; R2: C2H5); 29 (R1: CH3; R2: CH2CH=CH2); 30 (R1: CH3; R2: C6H5);
31 (R1: C2H5; R2: CH3); 32 (R1: C2H5; R2: C2H5); 33 (R1: C2H5; R2: CH2CH=CH2); 34 (R1: C2H5; R2: C6H5);
35 (R1: CH2CH=CH2; R2: CH3); 36 (R1: CH2CH=CH2; R2: C2H5); 37 (R1: CH2CH=CH2; R2: CH2CH=CH2);
38 (R1: CH2CH=CH2; R2: C6H5); 39 (R1: C6H5; R2: CH3); 40 (R1: C6H5; R2: C2H5); 41 (R1: C6H5; R2:
CH2CH=CH2); 42 (R1: C6H5; R2: C6H5)
Scheme 1. Synthetic route to compounds 23e42. Reagents and conditions: (a) BreCH2eCOOEt/anhyd. K2CO3 e acetone, reflux; (b) NH2NH2.H2O, reflux;
(c) R1eNCS/EtOH, reflux; (d) NaOH (2 N), reflux; (e) R1eNCS/acetone, reflux.
and Coordinating Facility (TAACF), have been proved to
possess considerable inhibition against M. tuberculosis
H37Rv. These findings encouraged us to go further with
our ongoing studies on thiourea derivatives and to evaluate
anti-tuberculosis activity against M. tuberculosis H37Rv, be-
sides antiviral activity.
This work describes synthesis and a wide spectrum antiviral
activity screening of some novel 5-[(4-aminophenoxy)methyl]-
4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thiones 23e26
and several related thioureas, N-alkyl/aryl-N0-{4-[(4-alkyl/
aryl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methoxy]phe-
nyl}thioureas 27e42, together with thiourea derivatives 1e22
H
N
N
N
N
R3
S
S
N
R1
N
S
S
R3
R1
R2
R2
N
H
N
H
N
H
N
H
1-19
20-22
1 (R1: CH3; R2: CH3); 2 (R1: CH3; R2: C2H5); 3 (R1: CH3; R2: CH2CH=CH2); 4 (R1: CH3; R2: C6H11); 5 R1: (CH3; R2: C6H5);
6 (R1: C2H5; R2: CH2CH=CH2); 7 (R1: C2H5; R2: C6H11); 8 (R1: CH2CH=CH2; R2: CH3); 9 (R1: CH2CH=CH2; R2:
CH2CH=CH2); 10 (R1: CH2CH=CH2; R2: C6H5); 11 (R1: C6H11; R2: CH2CH=CH2); 12 (R1: C6H11; R2: C6H11); 13 (R1:
CH2CH2C6H5; R2: CH2CH=CH2); 14 (R1: C6H5; R2: CH3); 15 (R1: C6H5; R2: C2H5); 16 (R1: C6H5; R2: CH2CH=CH2); 17
(R1: C6H5; R2: C6H11); 18 (R1: C6H5; R2: C6H5); 19 (R1: C6H5; R2: C6H4Cl-para); 20 (R1: CH3; R2: CH3; R3: H); 21 (R1:
CH3; R2: CH2CH=CH2; R3: Cl); 22 (R1: C6H5; R2: CH2CH=CH2; R3: Cl)
Fig. 2. General structure and substituents of compounds 1e22, synthesized in a previous study [24].

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384
synthesized previously [24]. Anti-tuberculosis activity of
newly synthesized compounds 23e42 was also evaluated.
yielding the corresponding title compounds 1-alkyl/aryl-3-
{4-[(4-alkyl/aryl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)-
methoxy]-phenyl}thiourea derivatives 27e42 in yields be-
tween 41 and 88%. Synthetic route for compounds 23e46
is depicted in Scheme 1.
2. Chemistry
This type of reactions was reported to be performed in cer-
tain dry solvents or mixtures [28e30]. In the present study,
dry acetone [31] was tried and found to be useful [32,33]. How-
ever, the yields of thioureas 27e42 seemed to be affected by the
possible formation of several by-products such as urethans [25].
Another yield-limiting factor might be the side reaction of iso-
thiocyanates with heterocyclic nitrogen at the second position
of 1,2,4-triazoline ring present in the 23e26 series [30]. Never-
Synthesis of 23e46 required stepwise reactions starting
with etherification of N-(4-hydroxyphenyl)acetamide with
ethyl a-bromoacetate in the presence of anhydrous potassium
carbonate using dry acetone as reaction medium to give com-
pound I. The choice of N-(4-hydroxyphenyl)acetamide as the
starting compound provided a protected amino functionality
during the following two steps (Scheme 1) and allowed to in-
troduce different groups at R1 and R2 positions. Hydrazinoly-
sis of I with three-fold equimolar amount of hydrazine hydrate
gave 2-[4-(acetylamino)phenoxy]acetohydrazide II, which
was then reacted with alkyl or aryl isothiocyanates to yield
1-[2-(4-acetamidophenoxy)acetyl]-4-alkyl/aryl thiosemicarba-
zides IIIaed as described. Cyclocondensation of compounds
IIIaed to give 5-[(4-aminophenoxy)methyl]-4-methyl-2,4-
dihydro-3H-1,2,4-triazole-3-thione derivatives 23e26 was
performed by refluxing IIIaed in aqueous alkaline medium.
During the ring closure, acetylamino group was hydrolyzed to
give a primary amino function which provided a nucleophilic
centre for the synthesis of disubstituted thiourea derivatives.
The final step comprised the reaction of the amines 23e26
with various alkyl or aryl isothiocyanates in dry acetone
1
theless, spectral findings such as the H NMR resonances at
13e14 ppm due to heterocyclic NHeCS function and the
lack of resonances attributable to NH2 function at around
4.68e4.80 ppm, supported the regioselectivity of this reaction.
All the new compounds 23e42 were characterized by their
melting points, and spectral data (1H NMR, 13C NMR and
HR-MS). These physical and spectral data of the synthesized
compounds are presented in Tables 1 and 2. The 1H NMR spec-
tra showed eOeCH2e chemical shifts at 4.75e5.24 ppm for
all compounds. Resonances due to the eNH2 function present
in 23e26 were observed at 4.68e4.80 ppm. These signals dis-
appeared in compounds 27e42 as evidence for the formation of
thiourea group and instead, resonances for thiourea NH groups
Table 1
Physical constants and HR-MS data for the synthesized compounds 23e42
NH
N
NH
N
S
O
S
N
O
S
N
R1
R2
R1
N
H
N
H
H2N
23-26
27-42
Compound
R1
R2
Yield (%)
M.p.(ꢁC)
Formula
log Pa
HReMS (m/z)
calculated
found
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
a
CH3
C2H5
e
e
76
52
88
72
46
41
64
71
39
39
72
46
63
57
74
51
41
47
82
77
143
160e161
117
C10H12N4OS
C11H14N4OS
C12H14N4OS
C15H14N4OS
C12H15N5OS2
C13H17N5OS2
C14H17N5OS2
C17H17N5OS2
C13H17N5OS2
C14H19N5OS2
C15H19N5OS2
C18H19N5OS2
C14H17N5OS2
C15H19N5OS2
C16H19N5OS2
C19H19N5OS2
C17H17N5OS2
C18H19N5OS2
C19H19N5OS2
C22H20N5OS2
1.58
2.01
2.14
2.80
1.98
2.39
2.51
3.39
2.37
2.76
2.88
3.72
2.51
2.89
2.98
3.84
3.12
3.49
3.60
4.43
236.0732 (Mþ)
250.0888 (Mþ)
262.0888 (Mþ)
298.0888 (Mþ)
309.0718 (Mþ)
323.0874 (Mþ)
335.0874 (Mþ)
372.0947 (MHþ)
323.0874 (Mþ)
337.1031 (Mþ)
349.1031 (Mþ)
385.1104 (MHþ)
335.0874 (Mþ)
349.1031 (Mþ)
361.1031 (Mþ)
398.1104 (MHþ)
372.0953 (MHþ)
386.1110 (MHþ)
397.1031 (Mþ)
434.1110 (Mþ)
236.0740 (EI)
250.0902 (EI)
262.0892 (EI)
298.0891 (EI)
309.0685 (EI)
323.0865 (EI)
335.0864 (EI)
372.0952 (FAB)
323.0873 (EI)
337.1030 (EI)
349.1001 (EI)
386.1094 (FAB)
335.0884 (EI)
349.1039 (EI)
361.1036 (EI)
398.1111 (FAB)
372.0913 (FAB)
386.1071 (FAB)
397.1013 (EI)
434.1124 (EI)
CH2CH]CH2
C6H5
CH3
e
e
CH3
162
200e202
140e141
164e166
183e185
188e190
153e155
218e220
180e182
142
CH3
CH3
C2H5
CH2CH]CH2
C6H5
CH3
CH3
C2H5
C2H5
C2H5
C2H5
CH2CH]CH2
C6H5
CH3
C2H5
CH2CH]CH2
CH2CH]CH2
CH2CH]CH2
CH2CH]CH2
C6H5
C2H5
110e113
135
CH2CH]CH2
C6H5
CH3
137e140
219e221
217e220
160
C6H5
C6H5
C2H5
CH2CH]CH2
C6H5
C6H5
120e122
Calculation of log P values were performed using ALOGPS 2.102 log P/log S calculation software http://www.vcclab.org.

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I. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 43 (2008) 381e392
385
Table 2
1H NMR spectral data of compounds 23e42
Compound 1H NMR (d)
23
24
3.43 (s, 3H, pN-CH3); 4.68 (b, 2H, Ar-NH2); 5.00 (s, 2H, eOCH2e); 6.48 (d, 2H, J ¼ 8.78 Hz, Ar-H); 6.72 (d, 2H, J ¼ 8.78 Hz, Ar-H).
1.25 (t, 3H, pNeCH2eCH3), 4.00 (q, 2H, pNeCH2eCH3); 4.72 (b, 2H, Ar-NH2); 5.00 (s, 2H, eOCH2e); 6.45 (d, 2H, J ¼ 8.78 Hz, Ar-H);
6.72, (d, 2H, J ¼ 8.78 Hz, Ar-H).
25
4.62 (s, 2H, NHeCH2eCH]CH2); 4.70e4.80 (b, 2H, Ar-NH2); 4.95 (s, 2H, eOCH2e); 5.0 (d, 1H, eCH2eCH]CH2, J ¼ 17.0 Hz, trans);
5.20 (d, 1H, eCH2eCH]CH2, J ¼ 10.5 Hz, cis); 5.78e5.95 (m, 1H, NHeCH2eCH]CH2); 6.45 (d, 2H, J ¼ 9. 00 Hz, Ar-H);
6.72 (d, 2H, J ¼ 8.70 Hz, Ar-H).
26
27
4.68 (b, 2H, Ar-NH2); 4.75 (s, 2H, eOCH2e); 6.35 (d, 2H, J ¼ 8.78 Hz, Ar-H); 6.45 (d, 2H, J ¼ 8.78 Hz, Ar-H); 7.40e7.50 (m, 5H, Ar-H).
2.89 (d, 3H, NHeCH3); 3.51 (s, 3H, pNeCH3) 5.22 (s, 2H, eOCH2e); 7.05 (d, 2H, J ¼ 8.78 Hz, o-OCH2); 7.25 (d, 2H, J ¼ 8.78 Hz, m-OCH2);
7.50 (s, 1H, thiourea NH) 9.40 (s, 1H, thiourea N0H); 13.90 (s, 1H, triazole NH).
28
29
1.10 (t, 3H, NHeCH2eCH3); 3.51 (m, 2H, Ar-NHeCSeNHeCH2eCH3); 3.57 (s, 3H, pNeCH3); 5.23 (s, 2H, eOCH2e); 7.05 (d, 2H,
J ¼ 8.78 Hz, o-OCH2); 7.27 (d, 2H, J ¼ 8.78 Hz, m-OCH2); 7.70 (s, 1H, thiourea NH); 9.30 (s, 1H, thiourea N0H); 13.98 (s, 1H, triazole NH).
3.46 (s, 3H, pNeCH3); 4.07 (s, 2H, eCH2eCH]CH2); 5.03e5.09 (m, 2H, eCH2eCH]CH2); 5.18 (s, 2H, eOCH2e); 5.82e6.00
(m, 1H, eCH2eCH]CH2); 7.02 (d, 2H, J ¼ 8.78 Hz, o-OCH2); 7.25 (d, 2H, J ¼ 8.78 Hz, m-OCH2), 7.70 (s, 1H, thiourea NH);
9.36 (s, 1H, thiourea N0H); 13.80 (s, 1H, triazole NH).
30
31
32
3.52 (s, 2H, pNeCH3); 5.24 (s, 2H, eOCH2); 7.05e7.47 (m, 9H, Ar-H); 9.67 (s, 1H, thiourea N0H); 9.72 (s, 1H, thiourea NH);
13.85 (s, 1H, triazole NH)
1.29 (t, 3H, pNeCH2CH3); 2.91 (d, 3H, NHeCH3); 4.03 (q, 2H, pNeCH2CH3); 5.17 (s, 2H, eOCH2); 7.05 (d, 2H, J ¼ 8.78 Hz, o-OCH2);
7.25 (d, 2H, J ¼ 8.78 Hz, m-OCH2); 7.51 (b, 1H, thiourea NH); 9.56 (b, 1H, thiourea N0H); 13.92 (s, 1H, triazole NH)
1.12 (t, 3H, NHeCH2eCH3); 1.27 (t, 3H, pNeCH2eCH3); 3.47 (m, 2H, NHeCH2eCH3); 4.03 (q, 2H, pNeCH2CH3); 5.22 (s, 2H, eOCH2);
7.06 (d, 2H, J ¼ 8.78 Hz, o-OCH2); 7.26 (d, 2H, J ¼ 8.78 Hz, m-OCH2); 7.51 (s, 1H, thiourea NH); 9.30 (s, 1H, thiourea N0H);
13.98 (s, 1H, triazole NH).
33
1.29 (t, 3H, pNeCH2eCH3); 4.03 (q, 2H, pNeCH2eCH3); 4.13 (s, 2H, eCH2eCH]CH2) 5.10 (d, 1H, eCH2eCH]CH2, J ¼ 17.0 Hz, trans);
5.24 (m, 3H, eOCH2e and eCH2eCH]CH2, cis); 5.80e6.00 (m, 1H, pNeCH2eCH]CH2); 7.06 (d, 2H, J ¼ 8.78 Hz, o-OCH2);
7.30 (d, 2H, J ¼ 8.78 Hz, m-OCH2); 7.67 (s, 1H, thiourea NH); 9.42 (s, 1H, thiourea N0H); 13.97 (s, 1H, triazole NH).
1.29 (t, 3H, pNeCH2eCH3); 4.08 (q, 2H, pNeCH2CH3); 5.24 (s, 2H, eOCH2); 7.04e7.47 (m, 9H, Ar-H); 9.66 and 9.71 (s, 2H, NHeCSeNH );
13.98 (s, 1H, triazole NH)
34
35
2.84 (d, 3H, NHeCH3); 4.65 (s, 2H, eCH2eCH]CH2); 5.04 (d, 1H, eCH2eCH]CH2, J ¼ 17.0 Hz, trans); 5.12 (s, 2H, eOCH2e); 5.17
(d, 1H, J ¼ 11.1 Hz, eCH2eCH]CH2, cis); 5.80e6.00 (m, 1H, eCH2eCH]CH2); 6.97 (d, 2H, J ¼ 8.78 Hz, o-OCH2); 7.20 (d, 2H, J ¼ 8.78 Hz,
m-OCH2), 7.45 (s, 1H, thiourea NH); 9.30 (s, 1H, thiourea N0H); 13.85 (s, 1H, triazole NH)
36
37
1.05 (m, 3H, NHeCH2eCH3); 3,42 (m, 2H, NHeCH2eCH3); 4.64 (s, 2H, eCH2eCH]CH2); 5.05 (d, 1H, eCH2eCH]CH2, J ¼ 17.57 Hz,
trans); 5.12 (s, 2H, eOCH2e); 5.17 (d, 1H, J ¼ 9.95 Hz, eCH2eCH]CH2, cis); 5.80e6.00 (m, 1H, eCH2eCH]CH2); 6.96 (d, 2H, J ¼ 8.78 Hz,
o-OCH2); 7.21 (d, 2H, J ¼ 8.78 Hz, m-OCH2); 7.45 (s, 1H, thiourea NH); 9.30 (s, 1H, thiourea N0H); 13.85 (s, 1H, triazole NH)
4.19 (s, 2H, NHeCH2eCH]CH2); 4.71 (d, 2H, pNeCH2eCH]CH2); 5,01e5.07 (d, 2H, pNeCH2eCH]CH2 and NHeCH2eCH]CH2
J ¼ 17.0 Hz, trans); 5.12 (s, 2H, eOCH2e); 5.15e5.19 (d, 2H, pNeCH2eCH]CH2 and NHeCH2eCH]CH2, J ¼ 11.1 Hz, cis); 5.80e6.00
(m, 2H, pNeCH2eCH]CH2 and NHeCH2eCH]CH2); 7.01 (d, 2H, J ¼ 8.78 Hz, o-OCH2); 7.26 (d, 2H, J ¼ 8.78 Hz, m-OCH2);
7.70 (s, 1H, thiourea NH); 9.40 (s, 1H, thiourea N0H); 13.96 (s, 1H, triazole NH)
38
4.71 (d, 2H, eCH2eCH]CH2); 5.05 (d, 1H, eCH2eCH]CH2, J ¼ 17.0 Hz, trans); 5.18 (s, 2H, eOCH2e); 5.21 (d, 1H, J ¼ 11.1 Hz,
pNeCH2eCH]CH2, cis); 5.80e6.00 (m, 1H, pNeCH2eCH]CH2); 7.02e7.47 (m, 9H, Ar-H); 9.66 and 9.71 (s, 2H, NH-CS-NH );
13.99 (s, 1H, triazole NH)
39
40
41
2.91 (d, 3H, NHeCH3); 4.96 (s, 2H, eOCH2e); 6.80 (d, 2H, J ¼ 8.78, o-OCH2); 7.18 (d, 2H, J ¼ 8.78, m-OCH2); 7.46e7.57 (m, 6H, Ar-H and
thiourea NH); 9.35 (b, 1H, thiourea N0H)
1.09 (t, 3H, eCH2eCH3); 3.44 (q, 2H, eCH2eCH3); 4.95 (s, 2H, eOCH2e); 6.80 (d, 2H, J ¼ 8.78 Hz, o-OCH2); 7.17 (d, 2H, J ¼ 8.78 Hz,
m-OCH2); 7.46e7.57 (m, 5H; Ar-H); 7.57 (s, 1H, thiourea NH); 9.25 (s, 1H, thiourea N0H); 13.97 (s, 1H, triazole NH)
4.97 (d, 2H, eCH2eCH]CH2); 5.10 (d, 1H, eCH2eCH]CH2, J ¼ 17.0 Hz, trans); 5.16 (d, 1H, J ¼ 11.1 Hz, eCH2eCH]CH2, cis); 5.82e5.95
(m, 1H, pNeCH2eCH]CH2); 6.83 (d, 2H, J ¼ 8.78 Hz, o-OCH2); 7.22 (d, 2H, J ¼ 8.78 Hz, m-OCH2); 7.29e7.59 (m, 5H, Ar-H); 7.69
(s, 1H, thiourea NH); 9.38 (s, 1H, Ar-thiourea N0H)
42
4.91 (s, 2H, eOCH2e); 6.76 (d, 2H, J ¼ 8.78 Hz, o-OCH2); 7.23 (d, 2H, J ¼ 8.78 Hz, m-OCH2); 7.04e7.51 (m, 10H, eC6H5);
9.56 (s, 1H, thiourea N0H); 9.63 (s, 1H, thiourea NH); 13.98 (s, 1H, triazole NH)
were observed at 7.50e9.72 ppm (R2-NHCSNHeC6H4OCH2e)
and 9.30e9.71 ppm (R2-NHCSNHeC6H4OCH2e). Triazole
NH resonances between 13.80 and 13.98 ppm, were observed
to be shifted to downfield because of strong intermolecular
hydrogen bonding as previously reported [34] The NeH protons
of compounds 23e26, 39 and 41 exchanged with deuterium in
the solvent used for obtaining the 1H NMR spectra. Remaining
chemical shifts were also recorded at expected values.
13C NMR data of the representative thiourea derivative 29
also supported the carbon framework of triazole ring and
thiourea moieties by displaying high accuracy between experi-
mental and calculated 13C chemical shifts (Table 3). Calculation
of the 13C NMR chemical shifts were performed using ACD/
CNMR Predictor software available online at ACD/I-Lab
Interactive Laboratory website (http://www.acdlabs.com/ilab).
As shown in Table 3, thiocarbonyl (C]S) carbon present in
triazoline ring and thiourea function resonated at 168.39 ppm
and 180.52 ppm, whereas these values were predicted as
168.40 ppm and 180.80 ppm, respectively. Remaining reso-
nanceswerealsoobservedtobeconsistentwithcalculatedvalues.
High resolution mass spectra (HR-MS) confirmed the molec-
ular weights and empirical formula of compounds 23e42, with
less than 8 mmu bias between calculated and experimental m/z
values of either molecular or fragment ions (Table 1). Ionization

_
I. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 43 (2008) 381e392
386
Table 3
Assignment of 13C NMR spectrum of compound 29
mode was electron impact (EI) in most cases, whereas some
compounds 30, 34, 38e42, especially the ones with an aryl moi-
ety on terminal thiourea nitrogen, did not give molecular ion
peaks using this technique. These compounds were analyzed
using fast atomic bombardment (FAB) procedure giving exact
MHþ peaks instead of Mþ in 3-nitrobenzyl alcohol matrix.
Fragmentation pattern for a representative compound 29 which
is given in Scheme 2, also supported the expected structures.
First fragmentation was cleavage of thiourea moiety yielding
isothiocyanate fragment at m/z 278.0302 via allylamine loss,
or, formation of amine fragment at m/z 236.0713 by the expul-
sion of allyl isothiocyanate which was detected at m/z 99.0150.
Characteristic fragmentations for 1,2,4-triazoline ring were
also observed. Main fragmentation product was observed as
4-aminophenol radical cation, giving the base peak at m/z
109.0532. Other fragmentations were observed to be consistent
with the expected structures for compounds 23e42.
H
N
2
N
1
3
S
6
15
O
5
9
10
N
14
13
8
S
4
18
CH3
7
20
17
11
H2C
22
21
12
NH
19
NH
16
Carbon no.
Chemical shift (ppm)
Calculateda
Observed
3
168.40
148.55
37.67
168.39
149.07
30.89
5
7
8
10
60.82
61.05
155.56
115.51
122.20
132.84
180.80
47.39
155.12
115.61
126.59
134.05
180.52
41.01
11, 15
12, 14
13
17
20
3. Biological activity
21
22
132.11
121.89
129.13
124.39
3.1. Antiviral activity
a
Calculation of the 13C NMR chemical shifts were performed using ACD/
CNMR Predictor software available online at ACD/I-Lab Interactive Labora-
tory website http://www.acdlabs.com/ilab.
For each compound, the minimum inhibitory concentration
(MIC) and the minimal cytotoxic concentration (MCC) or the
50% cytotoxic concentration (CC50) were obtained. Thiourea
derivatives 1e22 were evaluated for their anti-HIV activity.
None of the synthesized compounds showed any specific
.
+
.
+
.
+
H2C
N
NH
N
NH
N
O
S
O
S
S
N
S
N
m/z 99.0143 (99.0150)
CH3
H2C
CH3
N
N
H
N
H
m/z 278.0296 (278.0302)
.
S
+
NH
N
m/z 335.0874 (335.0864)
O
S
N
CH3
m/z 236.0732 (236.0713)
H2N
NH
N
O
S
N
+
CH3
m/z 220.0539 (220.0550)
O
O
NH
N
S
+
+
S
H2C
.
+
+
H2C
N
N
H
N
N
H2N
CH3
H
O
N
m/z 108.0444 (108.0469)
m/z 128.0277 (128.0279)
S
m/z 207.0587 (207.0579)
CH3
H2C
N
N
.
+
H
H
.
+
NH
N
OH
m/z 276.1044 (276.1016)
S
+
H3C
N
H2N
H2N
CH3
m/z 129.0361 (129.0364)
m/z 109.0528 (109.0532)
(100%)
m/z 80.0495 (80.0513)
Scheme 2. HR-EI mass spectral fragmentation of 29 (experimental values in brackets).

_
I. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 43 (2008) 381e392
387
Table 4
Cytotoxicity and anti-HIV activity of compounds 1e42 in MT-4 cells
butterfly-like conformation as explained in a similar case by
Chaouni-Benabdallah et.al. [35].
Compound Anti-HIV activity
Cytotoxicity
CC50 (mg/ml)b
The compounds were also evaluated for in vitro antiviral
activity against Herpes simplex virus [HSV-1 (strain KOS),
thymidine kinase deficient (TKꢀ) strain of HSV-1 resistant
to acyclovir (ACVR), HSV-2 (G)], Varicella-zoster virus
(VZV) [OKA strain and a thymidine kinase deficient (TKꢀ)
VZV (07/1 strain)], Cytomegalovirus (CMV) [AD-169 and
Davis strains], Vaccinia virus (VV), Vesicular stomatitis virus
(VSV) in HEL and Embryonic skin muscle (E6SM) cell cul-
tures; VSV, Coxackie virus B4, Respiratory syncytial virus
(RSV) in HeLa cell cultures and Parainfluenza-3 virus, Sindbis
virus, Coxackie virus B4 and Punta Toro virus in Vero cell
cultures. Results are presented in Tables 5e9.
a
IC50 (mg/ml)
Max. protection (%)
HIV-1
(IIIB)
HIV-2
(ROD)
HIV-1
(IIIB)
HIV-2
(ROD)
1
>80.30
>82.37
>78.20
>8.82
>80.30
>82.37
>78.20
>8.82
1
0
0
80.30 ꢂ 7.50
82.37 ꢂ 5.10
78.20 ꢂ 14.73
8.82 ꢂ 2.34
42.80 ꢂ 21.31
56.97 ꢂ 20.30
2.58 ꢂ 0.31
80.90 ꢂ 14.71
65.43 ꢂ 5.43
19.40 ꢂ 8.34
10.06 ꢂ 1.78
1.71 ꢂ 1.06
13.03 ꢂ 0.49
94.40 ꢂ 18.99
91.27 ꢂ 13.13
55.13 ꢂ 29.95
3.99 ꢂ 0.85
28.57 ꢂ 6.77
10.29 ꢂ 2.02
75.40 ꢂ 16.83
116.00
2
3
3
9.5
8
1
4
0
5
>42.80
>56.97
>2.58
>42.80 35
>56.97 8.5
>2.58 21
10
0
6
7
3
8
>80.90
>65.43
>19.40
>10.06
>1.71
>80.90
>65.43
9
6.5
0
9
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
a
>19.40 30.5
0
As a result of antiviral screening of 1e42, none of the eval-
uated compounds showed specific antiviral effects (i.e., mini-
mal antivirally effective concentration less than one-fifth of
minimal cytotoxic concentration) against any of the viruses
examined. However, some of the compounds showed moder-
ate activity against certain viruses. Among the derivatives
screened, compound 4 which had a MCC value of 400 mg/ml,
showed weak activity against the herpes simplex family
[HSV-1 (KOS), HSV-2 (G) and HSV-1 TKꢀ (ACVR)] with the
MIC value of 48 mg/ml. Compound 5 also had a weak activity
against HSV-1 TKꢀ (ACVR) with the MIC value of 48 mg/ml
(Table 6). Compounds 33 and 42 possessed an MIC value of
48 mg/ml against Sindbis virus whereas MCC for these com-
pounds in HEL cell cultures were 400 and 80 mg/ml, respec-
tively (Table 7). Many thiourea derivatives from 27e42
series were observed to have weak activity against Coxackie
virus B4, but at a subtoxic concentration (Table 7). Com-
pounds 26, 29, 32, 33, 34, 37, 41 and 42 had the same MIC
value of 48 mg/ml, whereas their MCC were 400 mg/ml (selec-
tivity index ¼ 8.33) except for compounds 34 and 42 whose
MCC values were 80 mg/ml (selectivity index ¼ 5). Compound
38, bearing an allyl group at N-4 of 1,2,4-triazole ring and a
phenyl moiety at the terminal nitrogen of the thiourea, was
identified as the most active derivative and the MIC value
of this compound was 16 mg/ml against Coxackie virus B4,
whereas its MCC was 80 mg/ml (selectivity index ¼ 5). This
compound was also active against thymidine kinase positive
Varicella-zoster virus (TKþ VZV, OKA strain) with an EC50
value of 9.9 mg/ml; whereas it showed only a weak activity
against the thymidine kinase deficient (TKꢀ) VZV (07/1
strain) with an EC50 of 26.8 mg/ml (Table 9). On the other
hand, compound 38 possessed low toxicity on human embry-
onic lung (HEL) cells (MCC of >100 mg/ml).
>10.06
>1.71
>13.03
4
3
9
0
1
>13.03
>94.40
>91.27
>55.13
>3.99
0
>94.40 23.5
>91.27 5.5
0
3
>55.13 13.5
>3.99 16
>28.57 25
0
3
>28.57
>10.29
>75.40
0
>10.29
>75.40
4.5
2.5
0.5
1.5
2.5
6
31
6
>116.00 >116.00
5
>65.30
>125
>125
>65.30
>125
>125
0
65.30 ꢂ 50.9
ꢃ119
4
8
>125
>125
>125
>125
>125
>125
3.5
2
9
2.5
1
>125
ꢃ112
>112
>107
>107
>125
>125
>114
1
1.5
1.5
3.5
1.5
9.5
3
2
ꢃ107
3
ꢃ107
>98.90
>68.10
>88.30
>72.30
>27.20
>101
>87.80
>77.90
>91.10
>71.40
6.5
2
ꢃ87.80
76.78 ꢂ 8.60
94.18 ꢂ 5.63
75.88 ꢂ 4.71
31.93 ꢂ 3.86
108.25 ꢂ 5.44
90.03 ꢂ 10.82
71.50 ꢂ 4.18
23.20 ꢂ 4.04
ꢃ83.50
8
2
>31.40 26.5
25.5
3.5
2.5
1
>110
>86.20
>69.70
1
4
3
>76.50
>66.50
>20.20
>20.30 19
22.5
2
>83.50 >113
0.5
1
>15.00
>92.30 >103
>19.40
11
0.5
4.5
22.03 ꢂ 6.27
107.08 ꢂ 12.87
44.15 ꢂ 10.36
1.5
5.5
>30.60
>41.50
Concentration required to protect MT-4 cells against the cytopathogenicity
of HIV by 50%.
b
Concentration required to reduce MT-4 cell viability by 50%.
activity against HIV-1 (IIIB) or HIV-2 (strain ROD) in MT-4
cells. Based on the experience with this type of molecules, it
was considered that a certain degree of flexibility might be re-
quired for binding to HIV-1 RT. Following these observations,
we have designed and synthesized compounds 27e42, in
which more flexibility is ascertained by replacing the phenyl
ring on the fifth position of 1,2,4-triazole ring with a phenoxy-
methyl moiety. Anti-HIV screening results of compounds
1e42 are given in Table 4. However, they were also
found inactive, probably due to their inability to exist in
3.2. Anti-tuberculosis activity
Compounds II, IIIaed and 23e42 were also tested for in
vitro anti-tuberculosis activity against M. tuberculosis
H37Rv using the BACTEC 12B medium in a broth microdilu-
tion assay, the Microplate Alamar Blue Assay (MABA)
[36,37]. Rifampicin was used as the standard in the antimyco-
bacterial assays. Triazoles 24e26 and thiourea derivatives 27,
28, 31, 32 and 36 were completely inactive against

_
I. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 43 (2008) 381e392
388
Table 5
Cytotoxicity and antiviral activity of compounds 1e42 in HeLa cell cultures
Table 6
Cytotoxicity and antiviral activity of compounds 1e42 in HEL and E6SM cell
cultures
Compound Minimum
cytotoxic
Minimum inhibitory concentrationb (mg/ml)
Compound Minimum
cytotoxic
Minimum inhibitory concentrationb (mg/ml)
Vesicular
stomatitis
virus
Coxsackie
virus B4
Respiratory
syncytial
virus
concentrationa
(mg/ml)
Herpes
simplex
virus-1
(KOS)
Herpes Vaccinia Vesicular Herpes
stomatitis simplex
concentrationa
(mg/ml)
simplex virus
virus-2
(G)
virus
virus-1
TKꢀ
KOS
1
400
400
400
80
>80
>80
>80
>16
>16
>80
>400
>80
>80
>80
>16
>3.2
>80
>400
>80
>400
>80
>80
>16
>16
>80
>16
>400
>400
>400
>400
>400
>80
>80
>400
>80
>80
>80
80
>80
>80
>80
>80
>80
>16
>16
>80
>400
>80
>80
>80
>16
>3.2
>80
>400
>80
>400
>80
>80
>16
>16
>80
>16
>400
>400
>400
>400
>400
>80
>80
>400
>80
>80
>80
>80
>80
>80
>80
>16
>80
>80
>80
>16
2
3
>80
>16
ACVr
4
5
80
>16
>80
1
400
ꢃ400
400
240
240
240
240
240
48
240 >80
240 >400
>80
240
6
400
ꢃ400
400
400
400
80
2
7
>400
>80
>80
3
240
48
240
80
>80
>80
>80
>80
>80
>80
>80
>16
>16
240
48
8
4
400
400
9
5
>80
240
>80
240
>80
240
>80
>16
>16
>80
240
>80
240
>80
>16
>16
48
240
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
>80
>16
6
400
400
7
>80
240
>80
240
16
>3.2
>80
8
400
400
ꢃ400
ꢃ80
ꢃ400
400
80
9
ꢃ80
80
>80
>16
>16
>3.2
>80
240
240
>400
>80
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Brivudin
>16
>16
>3.2
>80
240
80
16
>400
>80
>80
>3.2
>3.2
>3.2
400
400
>80
>80
>80
>80
>80
>80
80
80
>16
>16
ꢃ3.2
ꢃ80
ꢃ16
ꢃ80
ꢃ16
ꢃ16
80
>3.2
>80
>16
>80
>16
>16
>16
>16
400
>3.2
>3.2
>3.2
>3.2
>80
>16
>80
>16
>16
>16
>16
80
>80
>16
>80
>16
>16
>16
>16
80
>80
>16
>80
>16
>16
>16
>16
>80
>16
>80
>16
>16
>16
>16
400
80
>80
>16
>400
ꢃ400
>400
>400
>400
400
400
>400
400
400
400
400
400
400
400
80
>400
>400
>400
>400
>400
>80
ꢃ16
>400
>400
>400
>400
>400
>400
>400
400
240 >400
240 >400
>400
>400
>400
>400
>400
>400
>80
>400
>400
>400
>80
>400
>400
240
240
240
240
>80
>400 >400
>400 >400
>400 >400
>400 >400
>400 >400
>400
>400
>400
240
>400
>80
>80
240
240
240
240
>80
>80
240
>80
>400
240
>80
>80
>80
>400
240
>80
>80
>80
>16
>80
>80
>80
>16
>80
>80
>400
>400
>400
400
>400 >400
240 >400
240 >400
>80
240
240
>16 (80)
>80
>80
>80
>400
>400
240
>80
>80
>80
>400
>400
240
400
400
400
80
>400
>400
>400
400
>400 >400
>400 >400
240 >400
>80
>16 (80)
>80
>400
>400
240
>80
>400
>400
240
>80
80
>80
>400
>400
240
>400
>400
>400
400
>400 >400
>400 >400
Brivudin
(S)-DHPA >400
ꢃ400
>400
>400
48
>400
>400
240
>400
>400
9.6
240
>80
240
>80
Ribavirin
>400
>80
>80
80
>80
>400
48
a
Required to cause a microscopically detectable alteration of normal cell
>400
0.0768
9.6
3.2 >400
48 240
morphology.
b
Ribavirin >400
Acyclovir >400
Gancyclovir >100
a
48
Required to reduce virus-induced cytopathogenicity by 50%.
0.384
0.032
0.384 >400 >400
0.032 >100 >100
48
2.4
Required to cause a microscopically detectable alteration of normal cell
morphology.
M. tuberculosis H37Rv at 6.25 mg/mL, whereas the remaining
compounds exhibited varying degrees of inhibition in the pri-
mary screen. None of the tested compounds were considered
for further evaluation as they exhibited less than 90% inhibi-
tion in the primary screen (MIC > 6.25 mg/mL). Compound
38 was the most active compound with 79% inhibition against
M. tuberculosis H37Rv. Compounds 30, 33, 34 and 37 also
had positive responses exhibiting inhibition percentages of
70, 75, 76 and 59, respectively.
b
Required to reduce virus-induced cytopathogenicity by 50%.
4. Experimental protocols
4-(Acetylamino)phenol (Paracetamol) was a gift from
Drogsan Pharmaceuticals. Ethyl bromoacetate, hydrazine
hydrate, methyl-, ethyl-, allyl- and phenyl-isothiocyanate were

_
I. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 43 (2008) 381e392
389
Table 7
Cytotoxicity and antiviral activity of compounds 1e42 in Vero cell cultures
Compound
Minimum cytotoxic
concentrationa (mg/ml)
Minimum inhibitory concentrationb (mg/ml)
Parainfluenza-3 virus
Reo virus-1
Sindbis virus
Coxsackie virus B4
Punta Toro virus
1
400
400
400
80
>80
>80
>80
>16
>80
>80
>80
>80
>80
>16
>16
>3.2
>80
>80
>80
>400
>80
>16
>16
>16
>16
>16
>3.2
>16
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>16
>80
>80
>80
>16
>80
>80
>80
>16
>80
>80
>80
>80
>80
>16
>16
>3.2
>80
>80
>80
>400
>80
>16
>16
>16
>16
>16
>3.2
>16
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>16
>80
>80
>80
>16
>80
>80
>80
>16
>80
>80
>80
>80
>80
>16
>16
>3.2
>80
>80
>80
>400
>80
>16
>16
>16
>16
>16
>3.2
>16
>80
>80
>80
>80
>80
>80
>80
>80
48
>80
>80
>80
>16
>80
>80
>80
>80
>80
>16
>16
>3.2
>80
>80
>80
>400
>80
>16
>16
>16
>16
>16
>3.2
>16
>80
48
>80
>80
>80
>16
>80
80
2
3
4
5
400
ꢃ80
400
ꢃ80
ꢃ80
80
6
7
>80
>80
>80
>16
>16
>3.2
>80
>80
>80
>400
>80
>16
>16
>16
>16
>16
>3.2
>16
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>80
>16
>80
>80
>80
>16
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
ꢃ16
16
ꢃ80
ꢃ80
ꢃ80
ꢃ400
400
80
ꢃ16
80
80
ꢃ16
16
80
400
400
400
400
400
400
400
400
400
ꢃ80
400
400
400
80
>80
>80
48
>80
>80
48
48
48
>80
>80
>80
>80
>16
>80
>80
>80
>16 (48)
>80
80
48
16
400
400
400
80
>80
80
48
>16 (48)
Brivudin
(S)-DHPA
Ribavirin
a
>400
>400
>400
>400
>400
48
>400
>400
48
>400
>400
240
>400
>400
>400
>400
>400
48
Required to cause a microscopically detectable alteration of normal cell morphology.
Required to reduce virus-induced cytopathogenicity by 50%.
b
purchased from Fluka. Sodium hydroxide, anhydrous potas-
4.1. Ethyl(4-acetamidophenoxy)acetate (I)
sium carbonate and all solvents were supplied from Merck.
1H and 13C NMR spectra were obtained using a Bruker
AVANC-DPX 400 instrument. HR-EI-mass spectra were per-
formed using a Jeol JMS-700 instrument.
SMILES were generated from the structures using the ACD/
ChemSketch version 8.0 molecular editor (http://www.acdlabs.
com) and then log P values were calculated using ALOGPS 2.
102 log P/log S calculation software [38, 39]. The calculated
log P values for all the compounds are given in Table 1.
4-(Acetylamino)phenol (0.04 mol) was refluxed in acetone
containing anhydrous K2CO3 (0.06 mol) for 4 h; then ethyl
bromoacetate (0.042 mol) was added dropwise for 1 h and
after that the reaction mixture was heated for 8 h. At the
end of this duration the flask content was filtered and evapo-
rated to get the crude product. The crude product was recrys-
tallized from ethanol. M.p. 100e103 ꢁC (lit. 103 ꢁC) [40],
yield 63%.

_
I. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 43 (2008) 381e392
390
Table 8
Cytotoxicity and antiviral activity of compounds 23e42 against Cytomegalo-
virus (CMV) in human embryonic lung (HEL) cells
2.84 (d, 3H, eNHCH3); 3.32 (s, DMSOeH2O); 4.48 (s, 2H,
eOCH2e); 6.87 (d, 2H, J ¼ 9.37 Hz, m-NHAc); 7.45 (d,
2H, J ¼ 8,78 Hz, o-NHAc); 7.94 (s, 1H, eCSNHeCH3);
9.24 (s, 1H, eCONHNHe), 9.78 (s, 1H, eCONHeCH3);
10.00 (s, 1H, eCONHNHe). HR-MS (EIþ), m/z (calculated/
found): 296.0943/296.0953.
a
Compound Antiviral activity EC50
(mg/ml)
Cytotoxicity
(mg/ml)
AD-169 strain Davis strain Cell morphology Cell growth
(MCC)b
(CC50)c
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
>100
>100
>100
>100
>100
>100
>100
63
>100
>100
>100
>100
>100
>100
>100
49
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
ꢃ100
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
42
4.3.2. 1-[2-(4-Acetamidophenoxy)acetyl]-4-ethyl
thiosemicarbazide IIIb
C13H18N4O3S;. yield %87. M.p. 182e184 ꢁC. 1H NMR
(DMSO-d6) d: 1.97 (s, 3H, eCOCH3); 2.46 (m, DMSO);
3.59 (d, 3H, eNHCH2e); 3.32 (s, DMSOeH2O); 4.54 (s,
2H, eOCH2e); 6.95 (d, 2H, J ¼ 9.37 Hz, m-NHAc); 7.47
(d, 2H, J ¼ 8,78 Hz, o-NHAc); 7.95 (s, 1H, eCSNHeCH3);
9.18 (s, 1H, eCONHNHe), 9.82 (s, 1H, eCONHeCH3);
10.05 (s, 1H, eCONHNHe).
63
49
>100
55
45
45
41
37
>100
>100
100
>100
>100
77
4.3.3. 1-[2-(4-Acetamidophenoxy)acetyl]-4-allyl
thiosemicarbazide IIIc
C14H18N4O3S; yield 87%. M.p. 196e200 ꢁC. 1H NMR
(DMSO-d6) d: 1.96 (s, 3H, eCOCH3); 2.46 (m, DMSO);
4.06 (b, 2H, eCH2eCH]CH2); 3.32 (s, DMSOeH2O);
4.49 (s, 2H, eOCH2e); 5.01 (d, 1H, J ¼ 10.54 Hz, eCH2e
CH]CH2 cis); 5.09 (d, 1H, J ¼ 17.57 Hz, eCH2eCH]CH2
trans); 5.71e5.83 (m, 1H, eCH2eCH]CH2); 6.86 (d, 2H,
J ¼ 9.37 Hz, m-NHAc); 7.43 (d, 2H, J ¼ 8,78 Hz, o-NHAc);
8.15 (s, 1H,eCSNHeCH3); 9.30 (s, 1H, eCONHNHe);
9.78 (s, 1H, eCONHeCH3); 10.03 (s, 1H, eCONHNHe).
45
45
>100
>100
>100
>20
>100
>100
>100
37
>50
>50
>50
>50
Ganciclovir
Cidofovir
a
0.64
0.075
1.9
0.64
>400
>400
87
ꢃ18
Effective concentration required to reduce virus plaque formation by 50%.
Virus input was 20 plaque forming units (PFU).
b
Minimum cytotoxic concentration that causes a microscopically detectable
alteration of cell morphology.
c
4.3.4. 1-[2-(4-Acetamidophenoxy)acetyl]-4-phenyl
thiosemicarbazide IIId
Cytotoxic concentration required to reduce cell growth by 50%.
C17H18N4O3S; yield 85%. M.p. 190e192 ꢁC (lit.190e
191 ꢁC) [40]. 1H NMR (DMSO-d6) d: 1.97 (s, 3H, eCOCH3);
2.47 (m, DMSO); 3.31 (s, DMSOeH2O); 4.54 (s, 2H, e
OCH2e); 6.90 (d, 2H, J ¼ 8.78 Hz, m-NHAc); 7.11e7.16,
7.28e7.39 (m, 5H, Ar-H) 7.45 (d, 2H, J ¼ 9.37 Hz, o-NHAc);
9.63 (s, 2H, eNHCSNH-Ar); 9.78 (s, 1H, eCONHeCH3);
10.22 (s, 1H, eCONHNHe).
4.2. 2-(4-Acetamidophenoxy)acetohydrazide (II)
Compound II was prepared by refluxing ethyl[4-(acetyla-
mino)phenoxy]acetate (I) (0.015 mol) with hydrazine hydrate
(80%) (0.045 mol) in ethanol for 3 h. The reaction mixture
was allowed to cool and the precipitate washed with ice-cold
water and recrystallized from ethanol. M.p. 194e196 ꢁC
(lit. 194e195 ꢁC) [40], yield 80%.
4.4. General procedure for the synthesis of 5-[(4-
aminophenoxy)methyl]-4-methyl/ethyl/allyl/phenyl-2,4-
dihydro-3H-1,2,4-triazole-3-thiones (23e26)
4.3. General procedure for the synthesis of 1-[2-(4-
acetamidophenoxy)acetyl]-4-alkyl/aryl
thiosemicarbazides (IIIaed)
Compounds 23e26 were prepared by refluxing IIIaed
(0.01 mol) in sodium hydroxide solution (2 N) for 4 h and
followed by treatment of sodium salts with HCl to give desired
compounds. Compounds 23e26 were recrystallized from
ethanol.
2-(4-Acetamidophenoxy)acetohydrazide (II) (0.018 mol)
was dissolved in boiling ethanol and equimolar amounts of
methyl-isothiocyanate (IIIa), ethyl-isothiocyanate (IIIb), allyl-
isothiocyanate (IIIc) and phenyl-isothiocyanate (IIId) were
added and refluxed for 4 h. The flask content was allowed to
cool and the filtered precipitates were washed with boiling
ethanol.
4.5. General procedure for the synthesis of N-alkyl/aryl-
N0-{4-[(4-alkyl/aryl-5-thioxo-4,5-dihydro-1H-1,2,4-
triazol-3-yl)methoxy]phenyl}thioureas (27e42)
4.3.1. 1-[2-(4-Acetamidophenoxy)acetyl]-4-methyl
thiosemicarbazide IIIa
C12H16N4O3S; yield 87%. M.p. 222e226 ꢁC. 1H NMR
(DMSO-d6) d: 1.97 (s, 3H, eCOCH3); 2.46 (m, DMSO);
Compounds 27e42 (0.0015 mol) were prepared by reflux-
ing 23e26 in dry acetone for 6 h with equimolar amounts of
appropriate isothiocyanates. The reaction mixtures were then
evaporated and the crude products of 26e29, 31, 32, 35e38,

_
I. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 43 (2008) 381e392
391
Table 9
Cytotoxicity and antiviral activity of compounds 23e42 against varicella-zos-
ter virus (VZV) in human embryonic lung (HEL) cells
growing MT-4 cells [44] were centrifuged for 5 minutes at
1000 rpm and the supernatant was discarded. The MT-4 cells
were resuspended at 6 ꢄ 105 cells/ml, and 50 ml volumes
were transferred to the microtiter tray wells. Five days after
infection, the viability of mock- and HIV-infected cells was
examined spectrophotometrically by the MTT assay.
Compound
Antiviral activity
a
EC50 (mg/ml)
Cytotoxicity
(mg/ml)
TKþ VZV
OKA strain
TKꢀ VZV
07/1 strain
Cell morphology
(MCC)b
Cell growth
c
(CC50)
The MTTassay is based on the reduction of yellow coloured
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial de-
hydrogenase of metabolically active cells to a blue-purple for-
mazan that can be measured spectrophotometrically. The
absorbances were read in an eight-channel computer-controlled
photometer (Multiscan Ascent Reader, Labsystems, Helsinki,
Finland), at two wavelengths (540 and 690 nm). All data were
calculated using the median OD (optical density) value of tree
wells. The 50% cytotoxic concentration (CC50) was defined
as the concentration of the test compound that reduced the
absorbance (OD540) of the mock-infected control sample by
50%. The concentration achieving 50% protection from the
cytopathic effect of the virus in infected cells was defined as
the 50% effective concentration (EC50).
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
>100
>100
>100
>100
>100
>100
>100
33
>100
>100
>100
>100
>100
>100
>100
38
>100
>100
>100
>100
>100
>100
>100
ꢃ100
>100
>100
>100
ꢃ100
>100
>100
>100
ꢃ100
>100
>100
>100
>100
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
42
>100
64
41
88
58
42
34
29
>100
64
41
>100
64
46
9.9
26.8
>100
>100
>100
39
>100
>100
>100
26
>50
>50
>50
>50
4.6.2. Antiviral assays
The antiviral assays, other than HIV-1, were based on inhi-
bition of virus-induced cytopathicity in either E6SM cells
(HSV-1, HSV-2, VV, VSV), human embryonic lung (HEL)
cells [Varicella-zoster virus VZV), HCMV], HeLa cells (Re-
spiratory syncytial virus) or Vero cells (Coxsackie B4 virus,
Parainfluenza-3 virus, Sindbis virus, Punta Toro virus, Reovi-
rus-1), following previously established procedures [45e47].
Briefly, confluent cell cultures in microtiter 96-well plates were
inoculated with 100 CCID50 of virus, one CCID50 being the
virus dose required to infect 50% of the cell cultures.
After a 1 h virus adsorption period, residual virus was re-
moved, and the cell cultures were incubated in the presence
of varying concentrations (400, 200, 100, . mg/ml) of the
test compounds. Viral cytopathicity was recorded as soon as
it reached completion in the control virus-infected cell cultures
that had not been treated with the test compounds.
Acyclovir
Brivudin
a
0.17
0.02
16
25
400
400
200
128
Effective concentration required to reduce virus plaque formation by 50%.
Virus input was 20 plaque forming units (PFU).
b
Minimum cytotoxic concentration that causes a microscopically detectable
alteration of cell morphology.
c
Cytotoxic concentration required to reduce cell growth by 50%.
42 were recrystallized from ethanol, 30, 33, 34, 39 were
washed with boiling ethanol and 40, 41 were recrystallized
from methanol.
4.6. In vitro antiviral assays
4.6.1. Inhibition of HIV-induced cytopathicity in MT-4 cells
Evaluation of the antiviral activity of the compounds
against HIV-1 strain IIIB and HIV-2 strain (ROD) in MT-4
cells was performed using the MTT assay as previously de-
scribed [41]. Stock solutions (10 ꢄ final concentration) of
test compounds were added in 25 ml volumes to two series
of triplicate wells so as to allow simultaneous evaluation of
their effects on mock- and HIV-infected cells at the beginning
of each experiment. Serial five-fold dilutions of test com-
pounds were made directly in flat-bottomed 96-well microtiter
trays using a Biomek 2000 robot (Beckman instruments, Full-
erton, CA). Untreated control HIV- and mock-infected cell
samples were included for each sample.
HIV-1(IIIB) [42] or HIV-2 (ROD) [43] stock (50 ml) at
100e300 CCID50 (cell culture infectious dose) or culture me-
dium was added to either the infected or mock-infected wells
of the microtiter tray. Mock-infected cells were used to evalu-
ate the effect of test compound on uninfected cells in order to
assess the cytotoxicity of the test compound. Exponentially
4.7. In vitro evaluation of antimycobacterial activity
against M. tuberculosis H37Rv
Primary screening was conducted at 6.25 mg/ml against
M. tuberculosis H37Rv in BACTEC 12B medium using the
BACTEC 460 radiometric system [36]. Compounds effecting
<90% inhibition in the primary screening (MIC > 6.25 mg/ml)
were not evaluated further. Compounds demonstrating at least
90% inhibition in the primary screening were re-tested at
lower concentration (MIC) in a broth microdilution assay Ala-
mar Blue. The MIC was defined as the lowest concentration
inhibiting 99% of the inoculum.
Acknowledgements
The authors are grateful to Dr. Jurgen Gross from the Insti-
¨
tute of Organic Chemistry, University of Heidelberg, for his

_
I. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 43 (2008) 381e392
392
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Med. Chem. Lett. 13 (2003) 2929e2932.
generous help on obtaining HR-EI/FAB mass spectra of the
synthesized compounds. We also thank Dr. Joseph A. Maddry
from the Tuberculosis Antimicrobial Acquisition and Coordi-
nating Facility (TAACF), National Institute of Allergy and
Infections Diseases Southern Research Institute, GWL Han-
sen’s Disease Centre, Colorado State University, Birmingham,
AL, USA, for the in vitro evaluation of antimycobacterial
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