Synthesis and antiproliferative evaluation of novel 2-(4H-1,2,4-triazole-3-ylthio)acetamide derivatives as inducers of apoptosis in cancer cells

Article abstract of DOI:10.1016/j.ejmech.2016.05.017

In this study, a series of thiosemicarbazide derivatives 12–14, 1,2,4-triazol-3-thione derivatives 15–17 and compounds bearing 2-(4H-1,2,4-triazole-3-ylthio)acetamide structure 18–32 have been synthesized starting from phenolic compounds such as 2-naphthol, paracetamol and thymol. Structures and purity of the target compounds were confirmed by the use of their chromatographic and spectral data besides microanalysis. All of the synthesized new compounds 12–32 were evaluated for their anti-HIV activity. Among these compounds, three representatives 18, 19 and 25 were selected and evaluated by the National Cancer Institute (NCI) against the full panel of 60 human cancer cell lines derived from nine different cancer types. Antiproliferative effects of the selected compounds were demonstrated in human tumor cell lines K-562, A549 and PC-3. These compounds inhibited cell growth assessed by MTT assay. Compound 18, 19 and 25 exhibited anti-cancer activity with IC₅₀values of 5.96?μM (PC-3?cells), 7.90?μM (A549/ATCC cells) and 7.71?μM (K-562?cells), respectively. After the cell viability assay, caspase activation and Bcl-2 activity of the selected compounds were measured and the loss of mitochondrial membrane potential (MMP) was detected. Compounds 18, 19 and 25 showed a significant increase in caspase-3 activity in a dose-dependent manner. This was not observed for caspase-8 activity with compound 18 and 25, while compound 19 was significantly elevated only at the dose of 50?μM. In addition, all three compounds significantly decreased the mitochondrial membrane potential and expression of Bcl-2.

Full text of DOI:10.1016/j.ejmech.2016.05.017

European Journal of Medicinal Chemistry 121 (2016) 58e70
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and antiproliferative evaluation of novel 2-(4H-1,2,4-
triazole-3-ylthio)acetamide derivatives as inducers of apoptosis in
cancer cells
a
a
€
€
_
b
€
, *
b
Necla Kulaba s¸ , Esra Tatar , Ozlem Bing o€ l Ozakpınar , Derya Ozsavcı ,
c
c
a
Christophe Pannecouque , Erik De Clercq , Ilkay Küçükgüzel
Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Haydarpa s¸ a, 34668 Istanbul, Turkey
Marmara University, Faculty of Pharmacy, Department of Biochemistry, Haydarpa s¸ a, 34668 Istanbul, Turkey
KU Leuven, Department of Microbiology and Immunology, Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, B-3000 Leuven,
a
_
b
c
_
Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 January 2016
Received in revised form
In this study, a series of thiosemicarbazide derivatives 12e14, 1,2,4-triazol-3-thione derivatives 15e17
and compounds bearing 2-(4H-1,2,4-triazole-3-ylthio)acetamide structure 18e32 have been synthesized
starting from phenolic compounds such as 2-naphthol, paracetamol and thymol. Structures and purity of
the target compounds were confirmed by the use of their chromatographic and spectral data besides
microanalysis. All of the synthesized new compounds 12e32 were evaluated for their anti-HIV activity.
Among these compounds, three representatives 18, 19 and 25 were selected and evaluated by the Na-
tional Cancer Institute (NCI) against the full panel of 60 human cancer cell lines derived from nine
different cancer types. Antiproliferative effects of the selected compounds were demonstrated in human
tumor cell lines K-562, A549 and PC-3. These compounds inhibited cell growth assessed by MTT assay.
3
May 2016
Accepted 4 May 2016
Available online 7 May 2016
Keywords:
1,2,4-Triazoles
Thioethers
Anticancer activity
Apoptosis
Caspase-3
Caspase-8
Bcl-2
Compound 18, 19 and 25 exhibited anti-cancer activity with IC50 values of 5.96
mM (PC-3 cells), 7.90 mM
(A549/ATCC cells) and 7.71 M (K-562 cells), respectively. After the cell viability assay, caspase activation
m
and Bcl-2 activity of the selected compounds were measured and the loss of mitochondrial membrane
potential (MMP) was detected. Compounds 18, 19 and 25 showed a significant increase in caspase-3
activity in a dose-dependent manner. This was not observed for caspase-8 activity with compound 18
and 25, while compound 19 was significantly elevated only at the dose of 50
compounds significantly decreased the mitochondrial membrane potential and expression of Bcl-2.
2016 Elsevier Masson SAS. All rights reserved.
mM. In addition, all three
©
1
. Introduction
the cancers related to infectious agents, such as human papillo-
mavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV),
human immunodeficiency virus (HIV) and eleven biological agents
have been identified as group 1 carcinogens by the International
Agency for Research on Cancer Monographs. Most common cancers
in HIV-1-infected individuals are Kaposi sarcoma, lymphomas,
lung, colorectal and cervical carcinoma. The incidence and severity
of these cancers is greatly increased in immunosuppressed in-
dividuals [3]. Lung cancer is the main cause of cancer-related
deaths among HIV positive people, while colorectal cancer has
been identified as one of the tumors with an increasing incidence in
the HIV population [4]. Current therapies of cancer
include irradiation, chemotherapy and surgery. Although chemo-
therapy has been widely employed for various cancer treatments,
severe toxicity and resistance to conventional chemotherapeutic
Cancer is the second cause of death after cardiovascular diseases
and remains a major health problem worldwide [1]. It is charac-
terized by uncontrolled division and spread of abnormal cells that
have defects in normal cellular functions. Cancer development in-
cludes a multi-step process that contains induction of genetic
instability, abnormal expression of genes, abnormal signal trans-
duction, angiogenesis, metastasis, and immune abduction [2]. In-
fections with certain viruses, bacteria, and parasites have been
known as risk factors for several types of cancer in humans. Many of
*
Corresponding author.
E-mail address: ikucukguzel@marmara.edu.tr (I. Küçükgüzel).
_
http://dx.doi.org/10.1016/j.ejmech.2016.05.017
223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
0

N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
59
agents makes it a necessity to develop more selective drugs for
2. Results and discussion
cancer therapy. Thus, the development of potent and effective
novel antineoplastic drugs is one of the most important targets of
modern medicinal chemistry.
2.1. Chemistry
Apoptosis is programmed cell death to remove DNA-damaged,
virally infected, or other needless cells. It plays a key role as a
protective mechanism against carcinogenesis by eliminating
damaged cells [5,6]. Therefore, induction of apoptosis is a very
important chemopreventive strategy for cancer control. There are
two main apoptotic pathways: the extrinsic pathway (receptor
pathway) and the intrinsic pathway (mitochondrial pathway). The
extrinsic signaling pathway initiates apoptosis by transmembrane
receptor-mediated interactions. These involve death receptors that
are members of the tumor necrosis factor (TNF) receptor gene su-
perfamily, including FasL (Fatty acid synthetase ligand)/FasR (Fatty
The target compounds were synthesized performing a stepwise
reaction protocol as outlined in Scheme 1. Our convergent synthesis
strategy starts with preparation of N-aryl-2-chloroacetamides 1e5
by the reaction of substituted anilines with 2-chloroacetylchloride
in the presence of triethylamine [28e32]. Ethyl 2-[5-methyl-2-
(propan-2-yl)phenoxy]acetate 6, ethyl 2-[4-(acetylamino)phe-
noxy]acetate 7 and ethyl 2-(naphthalene-2-yloxy)acetate 8 were
obtained by the reaction of thymol, paracetamol and b-naphthol
with ethyl 2-bromoacetate, respectively [33,34]. N-Aryl-2-
chloroacetohydrazides 9e11 were prepared by heating com-
pounds 6e8 with hydrazine hydrate in methanol, respectively
[34e36]. Addition of the hydrazides 9e11 to benzylisothiocyanate
in ethanol then gave the corresponding thiosemicarbazides 12e14
[37]. Cyclocondensation of thiosemicarbazides 12 and 14 using
sodium hydroxide yielded the corresponding 5-(aryloxymethyl)-4-
benzyl-2,4-dihydro-3H-1,2,4-triazole-3-thiones 15 and 17, respec-
tively [33]. To avoid hydrolysis of acetamido moietyl, triethylamine
- ethanol was preferred to the use of aqueous sodium hydroxide in
the synthesis of N-{4-[(4-Benzyl-5-thioxo-4,5-dihydro-1H-1,2,4-
triazole-3-yl) methoxy]phenyl}acetamide 16 from the corre-
sponding thiosemicarbazide 13 [38]. As the final step, the 1,2,4-
triazoles 15e17 were reacted with 2-chloro-N-(4-substituted
phenyl)acetamides 1e5 in DMF to yield N-(substituted phenyl)-2-
(5-aryloxymethyl-4-benzyl-4H-1,2,4-triazole-3-ylthio)acetamide
derivatives 18e32 [27].
acid synthetase receptor), TNF-a (Tumor necrosis factor alpha)/
TNFR1 (Tumor necrosis factor receptor 1), Apo3L (Apo3 ligand)/
DR3 (Death receptor 3), Apo2L (Apo2 ligand)/DR4 (Death receptor
4
) and Apo2L/DR5 (Death receptor 5). TNF-a is on the cell mem-
brane surface that binds to FasL, thus procaspase-8 is triggered by
activation of FADD (Fas-associated death domain) in cell. The
mechanism of apoptosis is triggered when caspase-8 is activated.
Intrinsic pathway is defined by disruption of mitochondrial func-
tion. This involves activation of caspase-9 and caspase-3 as well as
formation of the apoptosome following the release of cytochrome
C. In this pathway, another family of proteins, Bcl-2 family, plays a
key role. The release of cytochrome C occurs as a result of induction
of mitochondrial outer membrane permeabilization (MOMP) by
Bcl-2. Thus, activated caspase-9 terminates the apoptotic phase by
activating the effector caspases, such as caspase-3 [7e10]. There-
fore, the defining of apoptosis inducers has become an interesting
approach for the discovery and development of potential anti-
cancer agents.
All synthesized compounds were checked for purity and iden-
tity using HPLC-UV/DAD and elemental analysis. All newly syn-
thesized compounds were characterized by their melting points,
1
13
FTIR, H NMR and HRMS spectral data, whereas C NMR spectra
The triazole ring is an important five-membered heterocycle
and isostere of imidazole, oxazole, pyrazole, thiazole, amide moiety
etc. in designing various types of new bioactive molecules. This
electron-rich aromatic structure easily binds to a variety of en-
zymes and receptors via weak interactions such as hydrogen bonds,
ion-dipole, hydrophobic interactions, van der Waals forces, etc.
Therefore compounds possessing triazole ring exhibit a broad
spectrum of biological activities. The ring system exists in two
structural isomers, the 1,2,3-triazole and the 1,2,4-triazole [11,12].
were recorded for some representative compounds 15e19, 22, 25,
27, 28, 32. H NMR, D O exchange, C NMR, HRMS and FTIR spectra
2
of selected compounds were given in Supplementary Data.
1
13
The IR spectra of compounds 12e14 have C]O, C]S and NeH
stretching
bands
of
acylthiosemicarbazide
group
at
ꢀ
1
ꢀ1
ꢀ1
1672e1699 cm , 1240e1269 cm and 3323e3210 cm , respec-
1
tively [39]. In the H NMR spectra, resonances assigned to the
1
2
4
N eH, N eH and N eH protons of thiosemicarbazides 12e14 were
detected at 9.71e10.12, 9.31e9.37 and 8.39e8.53 ppm, respectively
which are supported by the literatüre [40,41]. In addition, these NH
1,2,4-Triazoles have been reported to possess various biological
activities such as antimicrobial [13], anti-tuberculosis [14], anti-
inflammatory [15], analgesic [15] and anticancer activities [16].
The following 1,2,4-triazole derivatives are used in medicine:
ribavirin (antiviral) [12,17], fluconazole, itraconazole, voriconazole
protons of compounds 12 and 13, and NH proton of anilide group
1
for compound 13 were observed to exchange with D
2
O in the
H
NMR spectrum. Also the chemical shift of the benzylic protons,
-CH -, in the thiosemicarbazide derivatives were detected in the
2
(
antifungal) [18,19], alprazolam (tranquilizer), estazolam (hypnotic,
range of 4.64e4.71 ppm. In the IR spectra of compounds 15e17, the
disappearance of C]O stretching bands of the acylth-
iosemicarbazides, not revealing SeH stretching band at
sedative, tranquilizer) [20], trapidil (hypotensive) [17], anastrazole,
vorozole and letrozole (nonsteroidal competitive aromatase in-
hibitor) [17,19,21,22] (Fig. 1).
ꢀ1
~2600 cm
and detecting of C]S stretching band at
ꢀ
1
Among compounds with anti-HIV activity, 2-(4H-1,2,4-triazole-
-ylthio)acetamide derivatives have also been reported [23,24].
1254e1236 cm is an evidence for ring closure form of 1,2,4-
triazole-3-thione. These compounds of NeH bands frequency re-
3
ꢀ
1
Additionally, compounds bearing 2-(4H-1,2,4-triazole-3-ylthio)
acetamide structure with anticancer effects have been identified
such as compounds I and II which showed significant anticancer
activity against colon carcinoma cell line [25,26], compounds III
and IV exhibited inhibitor activity of the annexin A2-S100A10
protein [27], and compound V bearing (4H-1,2,4-triazole-3-ylthio)
ethanone structure displayed high antitumor activities against
HepG2, A549, PC-3M and MKN45 cell lines [19] (Fig. 2). In the light
of the literature findings, synthesis, characterization and evaluation
of anti-HIV activity of N-substituted phenyl-2-(5-aryloxymethyl-4-
benzyl-4H-1,2,4-triazole-3-ylthio)acetamide derivatives as poten-
tial anticancer agents were targeted in this study (Fig. 2).
gion at 3188e3084 cm were observed to be shifted to downfield
because of strong intermolecular hydrogen bonding as previously
reported [42]. Another support for 1,2,4-triazole-3-thione ring
closure was disappearance of NeH peak of thiosemicarbazide de-
rivatives at 8.39e10.12 ppm and the detection of signals between
2
13.90 and 14.02 ppm, due to the presence of triazole-N -H proton
[38], furthermore, triazole-N -H protons of compounds 15 and 16,
2
and NH proton of anilide group for compound 16 were observed to
1
13
exchange with D
2
O in the H NMR spectrum. C NMR spectra of
and thiocarbonyl
these compounds have resonances of triazole-C
5
(C]S) at 148.27e148.92 ppm and 169.11e169.28 ppm, respectively
[33]. The IR spectra of compounds 18e32 were characterized by the

60
N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
Fig. 1. Drugs bearing 1,2,4-triazole ring used in medicine.
ꢀ
1
presence of new C]O stretching band at 1707e1657 cm and
structures.
ꢀ1
CeSeC absorption band at 729e710 cm . Moreover, the absorp-
tion bands at 3350e3173 cm , which were attributed to the thi-
oacetamide group of NeH stretching vibrations [25,26]. For
ꢀ1
2
2
.2. Biological activity studies
ꢀ1
compounds 21, 26 and 31, the absorption bands at 1533e1495 cm
.2.1. Anti-HIV activity and cytotoxicity
ꢀ1
and 1327e1312 cm were assigned to the asymmetric and sym-
metric stretching vibrations of the NO group, respectively. For
compounds 18, 23 and 28, the stretching band of SO group was
detected at 1155e1152 cm . The IR spectra of synthesized thio-
ether derivatives from paracetamol 23e27 showed absorption
band at 1694 cm due to acetanilide group of C]O stretching
vibration. Additionally, the absorption band at 1713e1707 cm
was attributed to the ester group of C]O stretching vibration for
compounds 22, 27 and 32. In the H NMR spectra of compound
Anti-HIV activity and cytotoxicity data were obtained with the
synthesized compounds 12e32 using the strains HIV-1 (III ) and
B
HIV-2 (ROD) in an MT-4/MTT based assay [44] None of the tested
compounds showed antiviral activity at subtoxic concentrations
2
2
ꢀ
1
(see Supplementary Data Table S1).
ꢀ
1
ꢀ1
2.2.2. Screening of antiproliferative activity
1
Newly synthesized compounds 18, 19 and 25 were selected by
the National Cancer Institute (NCI) Developmental Therapeutic
Program (www.dtp.nci.nih.gov) for the in vitro cell line screening to
investigate their anticancer activity. The selection criteria guidance
is available online at the DTP web site (www.dtp.nci.nih.gov/docs/
misc/common_filesguidelines.html). The primary anticancer assay
was performed according to the US NCI protocol [45e49]. Selected
compounds 18, 19 and 25 were screened by the NCI for anticancer
activity against 60 human tumor cell lines. The anticancer
screening data at a concentration of 10 M are summarized in
Table 1. For NCI criteria, compounds, which reduce the growth of
any one of the cell lines to approximately 32% or less, are passed on
for evaluation in the full panel of cell lines over a 5-log dose range.
Compound 18 displayed antiproliferative activity against prostate
cancer, PC-3 (cell growth promotion 31.11%). Compound 19 showed
antiproliferative activity against three cancer cell lines (leukemia,
1
8e32, signals at about 4.05e4.22 ppm and 9.70e10.86 ppm that
were attributed to the thioacetamide group of eCH - protons and
NeH proton respectively while NeH peak of the 1,2,4-triazol-3-
thione disappeared [43]. In addition, NH protons of compounds
2
1
18, 19 and 25 were observed to exchange with D
2
O in the H NMR
13
spectrum. C NMR spectra of compounds 18, 19, 22, 25, 27, 28, 32
showed the presence of new carbonyl peak (C]O) due to thio-
acetamide group between 165.64 ppm and 167.02 ppm. High res-
olution mass spectra (HR-MS) confirmed the molecular weights
and empirical formula of compounds 18e32, with less than 5 mmu
bias between calculated and experimental m/z values of molecular
ion. The mass spectroscopic analysis of the compounds 18e32 was
studied under electron spray ionization. All the newly synthesized
compounds gave elemental analysis data consistent to assigned
ꢀ
5

N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
61
Fig. 2. Compounds bearing 2-(4H-1,2,4-triazole-3-ylthio)acetamide structure with anticancer effects.
SR; non-small cell lung cancer, A549/ATCC and NCI-HT460). For
non-small cell lung (A549/ATCC) cancer cell line, compound 19
showed cell growth promotion 27.93%. Compound 25 exhibited
antiproliferative activity against two cancer cell lines (leukemia, K-
DMSO (solvent)-treated samples. Whether or not compounds
caused cytotoxic effects in mouse embryonic fibroblast cell (NIH-
3T3) was also tested. Compounds showed no cytotoxicity effect on
NIH-3T3 cell, IC50 > 100 mg/ml (data not shown).
5
62; melanoma, UACC-62). For leukemia (K-562) cancer cell line,
compound 25 exhibited cell growth promotion 27.93%. The cell
viability and apoptosis assays (PC-3, A549 and K562) were studied
at the Marmara University Faculty of Pharmacy Department of
Biochemistry.
2
.2.4. Compounds 18, 19 and 25 induce apoptosis
Apoptosis is a complex process that involves a number of mol-
ecules and is classified into caspase-dependent or -independent
mechanisms. Caspase-dependent pathway can be further divided
into extrinsic or intrinsic pathway, as determined by involvement
of caspase-8 or caspase-9, respectively. Both pathways terminate in
activation of effector caspases such as caspase-3. To examine the
molecular mechanism of the observed cell apoptosis in compound
18, 19 and 25-induced apoptosis, we examined the status of acti-
vation of several critical apoptosis-related factors to elucidate the
apoptotic pathway(s) being activated by these compounds.
2.2.3. Cytotoxicity assay
To demonstrate that the newly synthesized compounds may
useful compounds with anticancer potential, firstly, we evaluated
their cytotoxicity toward cancer and normal cell lineages in this
study. We determined the cytotoxic effects of compound 18, 19 and
2
5 on cell survival using human prostate cancer cell, PC-3, human
non-small cell lung cancer, A549 and human chronic myelogenous
leukemia, K562. MTT assay was the antiproliferative assay
employed in this study. Based on the data gathered from this
experiment, the IC50 values for compound 18, 19 and 25 were
It is well documented two main protein families are known to be
crucial in the process of apoptosis: the Bcl-2 family as a determi-
nator of apoptosis and the other is the caspase family, an initiator of
apoptosis. Bcl-2 family members include anti-apoptotic (Bcl-2, Bcl-
xL) and pro-apoptotic proteins (Bax, Bak, Bid, Bim and Bad). Un-
balance between pro- and anti-apoptotic Bcl-2 family members is
5
.96
mM, 7.90 mM and 7.71 mM, respectively (see Supplementary
Data Fig. S37). No significant cell inhibitory effect was observed in

62
N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
Scheme 1. General procedure for the synthesis of compounds 18e32. Reagents and conditions: (i) ClCH
NH eNH $H O, MeOH, reflux; (iv) C CH NCS/EtOH reflux; (v) NaOH (aq), reflux, 10% HCl (for compounds 15 and 17) or TEA/EtOH, reflux (for compound 16); (vi) compounds 1e5/
DMF, K CO reflux.
2 2 2 5 2 3
COCl/DCM, TEA reflux; (ii) BrCH COOC H /K CO , acetone, reflux; (iii)
2
2
2
6
H
5
2
2
3
associated with the mitochondrial membrane depolarization that
leads to the activation of caspases [50,51].
ELISA and fluorospectrophotometric methods were employed to
detect the activation of caspase-8, and -3, Bcl-2 and MMP in the
Bcl-2 activity decreased as compared with the control group in dose
dependent manner (p < 0.001). On the other hand, cytoplasmic
caspase-3 activity elevated (p < 0.001). Added to all of our findings,
analysis of MMP using JC-1 staining in A-549 treated cells with
compound 19 corroborates with activation of caspase-dependent
apoptotic machinery (Fig. 6).
cytosolic fractions. Fig.
3 shows that significantly increased
caspase-3, decreased MMP and Bcl-2 levels in PC-3 cells following
stimulation with compound 18. Treatment of PC-3 cells with this
compound caused a significant increase in caspase-3 activity and
decrease in Bcl-2 levels compared to control in dose dependent
manner (p < 0.001). In contrast, there were no significant changes
in the activity of caspase-8. Moreover, MMP was disturbed
regardless of dose in compound-treated prostate cancer cells
Interestingly, compound 19 also stimulated significantly
extrinsic pathway by inducing caspase-8 activity at only 50
mM
dose (p < 0.01). Caspase-8 is an important apoptosis protein that is
activated initially in both the death receptor and the mitochondrial
pathway. Sequentially, caspase-8 activate Bid, a pro-apoptotic Bcl-2
family protein [55,56]. Although we did not measure Bid protein
levels, we deduced that increased activity of caspase-8 may cause
activation of Bid resulting in triggering inner cell death pathway.
These results point to different kinetics of apoptosis induction by
compound 19. Our results clearly demonstrated that compound 19
promoted loss of MMP, inhibited the expression of anti-apoptotic
Bcl-2 protein and induced activation of caspases-8, and -3 in
these cells, confirming the involvement of both intrinsic and
extrinsic apoptotic pathways in apoptosis.
Cisplatin (DDP) [cis-diamminedichloroplatinum (CDDP)] has
been one of the most widely used chemotherapeutic drugs for
advanced non-small cell lung cancer. Cisplatin-based combination
chemotherapy is the cornerstone for all stages of non-small lung
cancer. Using several cell lines including A549 cell line, Banerjee
et al. demonstrated that cisplatin has cytotoxic effect at increasing
(
p < 0.001). (Fig. 4). Based on the above data, compound 18 declined
Bcl-2 levels initiating a signaling cascade contributing to MMP loss
and resulting in an up-regulation of caspase-3. Thus, our data
suggested that compound 18 induced activation of intrinsic caspase
pathway in prostate cancer cell lines.
As the results of in vitro and clinical studies of its effect on
prostate cancer, docetaxel has been approved for the treatment of
hormone and drug resistant prostate cancer patients [52]. Accu-
mulating evidence supports that docetaxel induces apoptosis
significantly through inactivating the anti-apoptotic Bcl-2 protein
by its phosphorylation [53], increasing caspase3/7 activity in a dose
dependent (0.1e10 nM) manner [54]. It was also shown by our
study that apoptosis was induced in PC-3 cells with significant
cytotoxicity. Also, there was a concentration dependent increase in
caspase 3 enzyme activity and decrease Bcl-2 protein levels in
prostate carcinoma cells exposed to compound 18.
As shown in Fig. 5, similarly with compound 18, we determined
that compound 19 induced apoptosis in lung cancer cells via the
intrinsic pathway leading to caspase-3 release and subsequent
apoptotic death. Mitochondria are the main cellular component for
the intrinsic apoptotic pathway and plays a pivotal role in regu-
lating apoptosis. The permeabilization of the outer membrane and
alteration of the MMP (Djm) by the mitochondria initiating the
apoptotic cascade, releases soluble proapoptotic proteins and leads
to the activation of caspases including caspase-3 [55]. After the cells
were treated with compound 19, the polarization of the MMP and
doses (1e8 mM; IC50: 3 mM). Besides this, cisplatin altered the Bcl-2/
Bax ratio along with procaspase-9/cleaved caspase-9 ratio in tar-
geted cells which finally activated caspase-3 [57]. The findings are
consistent with our results. Moreover, compound 19 induced
extrinsic apoptotic pathway with increasing caspase 8 activity at
the highest dose.
To demonstrate that the induction of death in K562 leukemia
cancer cells following treatment with compound 25 was due to the
induction of apoptosis, caspase-3 and -8 activity, and Bcl-2 levels
and polarization of MMP were examined. After cells were exposed
to compound 25 (10, 20 and 50
mM) for 72 h, we found that
compound-induced apoptosis was accompanied by an increase of

N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
63
Table 1
fusion protein. Recent studies showed that, imatinib has cytotoxic
and apoptotic effects in a dose dependent manner on CML [58].
Baran et al., reported that proliferation rates of K562 cells were
Anticancer screening data in one dose at 10.00
m
M concentration.
Growth percent for
Panel
Cell line
compound
decreased with increasing
concentrations of imatinib
1
8
19
72.66
25
56.26
(0.5e600 nM; IC50: 310 nM). Also, they found that imatinib induced
apoptosis decreasing MMP and increasing caspase 3 activity. In our
study, compound 25 inhibited K562 cell proliferation in a dose
dependent manner. Additionally, we determined that the mecha-
nism of compound 25 einduced apoptosis is related to intrinsic
apoptotic pathway including increased caspase 3 activity, loss of
MMP and decreasing Bcl-2 levels.
A desirable property for any anticancer agent is selective toxicity
exhibited against the tumor cells compared with the cells/tissue
displaying dose-limiting toxicity. In this scenario, compound 18, 19
and 25 showed low cytotoxicity against normal cells and was
cytotoxic to several human tumor cell lines, inducing apoptosis
through the activation of the extrinsic and intrinsic apoptotic
pathways. These cellular effects make these compounds attractive
candidates drug for the treatment of cancer.
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
35.62
68.17
78.38
45.17
40.78
65.06
50.64
55.51
79.11
75.20
66.58
63.83
89.32
76.39
75.26
90.18
73.76
66.07
64.24
72.38
54.34
91.66
73.99
70.69
89.52
88.43
93.81
64.59
68.18
84.28
87.29
83.92
97.53
73.73
83.78
ꢀ6.63
27.93
69.61
81.19
78.05
88.01
80.57
72.48
17.74
90.37
63.84
28.93
64.71
72.21
97.69
78.50
69.97
90.26
70.93
80.31
75.19
81.24
74.26
71.22
Non-Small Cell Lung Cancer A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786e0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
Colon Cancer
78.25 105.21
89.65
52.92
76.65
70.03
76.92
65.88
88.56
88.14
85.37
80.34
85.58
53.25
60.37
93.87
56.39
58.98
70.90
80.24
92.76
92.33
3. Conclusion
In conclusion, anti-HIV activity of the synthesized new com-
pounds 12e32 were evaluated but a significant antiviral activity at
subtoxic concentrations was not observed. Among the synthesized
compounds, three representatives 18, 19 and 25 were selected and
evaluated by the National Cancer Institute (NCI) against the full
panel of 60 human cancer cell lines derived from nine different
cancer types. The compounds which exhibited good inhibition in
the primary screen were then evaluated for cell viability in the
second stage by using the mouse embryonic fibroblast cell line NIH-
CNS Cancer
Melanoma
93.63 103.08
40.88
64.43
75.57
99.05
88.46
33.36
93.85
43.52
65.98
72.25
76.25 102.78
80.87 108.08
89.26 112.62 102.43
50.27
88.97 105.47
3T3. Anticancer activity of these compounds were determined
against prostate cancer (PC-3), lung cancer (A549/ATCC) and leu-
kemia (K-562) cell lines at different concentrations. After the cell
viability assay, caspase activation and Bcl-2 activity of the selected
compounds were measured and the loss of mitochondrial mem-
brane potential (MMP) was detected.
90.50
69.83
98.36
32.51
83.17
51.25
90.09
74.65
88.77
99.39
41.06
77.13
94.04
69.40
63.86 101.90
33.38 101.35
93.18
67.27
69.08
Ovarian Cancer
Renal Cancer
Compound 18, 19 and 25 exhibited anti-cancer activity with IC50
values of 5.96
mM (PC-3 cells), 7.90 mM (A549/ATCC cells) and
92.00
84.79
68.20
7.71 M (K-562 cells), respectively. Cytotoxicity data gathered from
m
MT-4 cell based assay supported the data revealing compound 25
with highest inhibition potential against the K562 cell line among
compounds 18, 19 and 25. Compounds 18, 19 and 25 showed a
significant increase in caspase-3 activity in a dose-dependent
manner. The same situation was not observed for compounds 18
and 25 while caspase-8 activity of compound 19 was significantly
elevated only at the dose of 50 mM. In addition, all three compounds
significantly decreased the mitochondrial membrane potential and
expression of Bcl-2.
These results demonstrated that compound 19 triggers
apoptosis by using intrinsic and extrinsic pathways, while com-
pounds 18 and 25 induce apoptotic cell death by triggering intrinsic
pathway. Studies are on the way for optimization of apoptosis
inducing activity observed with the lead compounds identified in
this research. In the future studies, we will focus on understanding
of mechanisms underlying the apoptotic effect of 1,2,4-triazoles
with an amide moiety via thioether linkage.
108.89 108.74 110.14
92.26
74.73
75.95
81.12
38.90
77.97
41.65
93.88
91.06
82.08
58.57
72.46
70.60 100.92 101.04
81.59
93.95
42.69
31.11
82.34
50.05
69.08
86.16
65.83
33.29
70.45
56.49
71.92
53.63
71.92
90.54
75.65
75.67
75.46
97.42
78.58
72.35
85.66
87.12
52.82
54.24
72.63
70.15
75.00
97.92
72.38
69.62
77.83
Prostate Cancer
Breast Cancer
Bold values represent the cell lines selected for apoptosis studies.
caspase-3 activity and a decrease of Bcl-2 and polarization of MMP
in a dose-dependent manner (Fig. 7). The change in Bcl-2/MMP and
caspase-3 protein level indicated that compound 25 could induce
apoptosis through a mitochondria-mediated mechanism.
4
. Experimental
4.1. Chemistry
Imatinib is commonly used for the treatment of chronic mye-
logenous leukemia (CML), a hematological malignancy resulting
from a chromosomal translocation that generates the BCR/Abl
All solvents and reagents were obtained from commercial
sources and used without purification. All melting points ( C, un-
corrected) were determined using Schmelzpunktbestimmer SMP II
ꢁ

64
N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
þ
þþ
þþþ
p < 0.001 vs 10
m
M gp<0.05 ggp<0.01;
Fig. 3. Effect of compound 18 on apoptotic pathway of PC-3 cell line. *p < 0.05 **p < 0.01 ***p < 0.001 vs control. p < 0.05 p < 0.01
M compared to 50 M.
20
m
m
Fig. 4. Effect of compound 18 on PC-3 cell mitochondrial membrane potential (DJm). Cells staining green (JC-1 monomers) are cells with a disrupted DJm, while cells staining
orange (JC-1 aggregates) have a healthy DJm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
basic model melting point apparatus. Elemental analyses were
obtained using Leco CHNS-932 and are consistent with the
assigned structures. Infrared spectra were recorded on a Shimadzu
17e20 min) and subsequently degassed by ultrasonication, prior to
ꢀ
1
use. Solvent delivery was employed at a flow rate of 1 ml min
.
Detection of the analytes was carried out at 254 nm.
ꢀ1
FTIR 8400S and data are expressed in wavenumber
n (cm ). NMR
spectra were recorded on Brüker AVANCE-DPX 400 at 400 MHz for
H NMR; 300 MHz and 100 MHz for C NMR (Decoupled), the
4
.1.1. Synthesis of N-benzyl-2-(substituted aryl/naphthyloxyacetyl)
1
13
hydrazinecarbothioamide (12e14)
chemical shifts were expressed in
d
(ppm) downfield from tetra-
A mixture of compound 9, 10 or 11 (0.03 mol) and benzyliso-
thiocyanate (0.03 mol) in ethanol (40 ml) was heated under reflux
for 4 h. Then the solution was cooled, filtered and dried. The solid
product was recrystallized to give 12, 13 and 14, respectively.
methylsilane (TMS) using DMSO-d
6
as solvent. The liquid chro-
matographic system consists of an Agilent technologies 1100 series
instrument equipped with a quaternary solvent delivery system
and a model Agilent series G1315 A photodiode array detector. A
Rheodyne syringe loading sample injector with a 50 ml sample loop
4
.1.1.1. N-Benzyl-2-[[5-methyl-2-(propan-2-yl)phenoxy]acetyl]
was used for the injection of the analytes. Chromatographic data
were collected and processed using Agilent ChemStation Plus
software. The separation was performed at ambient temperature by
hydrazinecarbothioamide [12]. It was obtained as a white solid and
ꢁ
was recrystallized from methanol. HPLC t
yield 57%. UV (CH
R
(min): 12.33, Mp: 154 C,
3
OH) nm: 248 (ε: 18,807), 206 (ε: 50,182); IR
cm : 3310, 3258, 3210 (NeH), 1672 (C]O), 1252 (C]S), 1167, 1098
using a reversed phase. Nova-Pak C18 (3.9 600 ꢂ 150 mm, 5
mm
ꢀ1
particle size) column. All experiments were performed in gradient
mode. The mobile phase was prepared by mixing acetonitrile and
water (50:50 v/v during 0e5 min, 75:25 v/v during 5e8 min, 100:0
v/v during 8e14 min, 75:25 v/v during 14e17 min, 50:50 v/v during
1
(CeOeC); H NMR (400 MHz, DMSO-d
6
):
), 3.25 (m, 1H, -CH(CH
.66 (d, 2H, -NH-CH -C ), 6.60e7.22 (8H, Ar-H), 8.44 (s, 1H, -CS-
, exchangeable with D O), 9.35 (s, 1H, -NH-NH-CS-,
d
1.06 (d, 6H, -CH(CH
), 4.51 (s, 2H, -O-CH
3
)
2
),
-),
2
4
.14 (s, 3H, Ar-CH
3
3
)
2
2
2
6 5
H
NH-CH
2
2

N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
65
þ
þþ
þþþ
p < 0.001 vs 10 m
M gp<0.05 ggp<0.01;
Fig. 5. Effect of compound 19 on apoptotic pathway of A549 cell line. *p < 0.05 **p < 0.01 ***p < 0.001 vs control. p < 0.05 p < 0.01
M compared to 50 M.
20
m
m
Fig. 6. Effect of compound 19 on A549 cell mitochondrial membrane potential (DJm). Cells staining green (JC-1 monomers) are cells with a disrupted DJm, while cells staining
orange (JC-1 aggregates) have a healthy DJm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
exchangeable with D
with D O) ppm. Anal. Calcd. for C20
N,11.31; S, 8.63. Found: C, 63.65; H, 6.64; N, 11.31; S, 8.07.
2
O), 9.92 (s, 1H, -CO-NH-NH-, exchangeable
recrystallized from methanol. HPLC
t
R
(min): 10.59, Mp:
ꢁ
2
H
25
N
3
O
2
S: C, 64.66; H, 6.78;
180e181 C, yield 65%. UV (CH
3
OH) nm: 320 (ε: 1931), 250 (ε:
ꢀ
1
19,456), 217 (ε: 68,599); IR cm : 3323 (NeH), 1690 (C]O), 1269
1
(
(
8
C]S), 1213, 1109 (CeOeC); H NMR (400 MHz, DMSO-d
s, 2H, -O-CH -), 4.71 (s, 2H, -NH-CH -C ), 7.04e7.87 (12H, Ar-H),
.53 (s, 1H, -CS-NH-CH -), 9.37 (s, 1H, -NH-NH-CS-), 10.12 (s, 1H,
-CO-NH-NH-) ppm. Anal. Calcd. for C20H19N O S: C, 65.73; H, 5.24;
6
): d 4.67
2
2
6 5
H
4.1.1.2. N-(4-{2-[2-(Benzylcarbamothioyl)hydrazinyl]-2-oxoethoxy}
phenyl)acetamide [13]. It was obtained as a white solid and was
2
ꢁ
ꢀ
recrystallized from methanol. HPLC t
yield 56%. UV (CH OH) nm: 249 (ε: 31,915), 206 (ε: 42,180); IR cm
287, 3262, 3215 (NeH), 1699, 1657 (2 C]O), 1240 (C]S), 1209,
R
(min): 3.77, Mp: 189e191 C,
3 2
1
N,11.50; S, 8.77. Found: C, 66.21; H, 5.15; N, 11.47; S, 8.71.
3
:
3
1
1063 (CeOeC); H NMR (400 MHz, DMSO-d
6
):
-), 4.65 (d, 2H, -NH-CH
-, exchangeable with
O), 9.31 (s, 1H, -NH-NH-CS-, exchangeable with D O), 9.71 (s, 1H,
O), 10.02 (s, 1H, -CO-NH-Ar,
O) ppm. Anal. Calcd. for C18 S: C,
8.05; H, 5.41; N,15.04; S, 8.61. Found: C, 57.31; H, 5.28; N, 14.70; S,
d
1.92 (s, 3H, -NH-
4.1.2. Synthesis of 4-benzyl-5-(substituted aryl/
CO-CH
6
D
3
), 4.44 (s, 2H, -O-CH
.76e7.49 (9H, Ar-H), 8.45 (s, 1H, -CS-NH-CH
2
2
2 6 5
-C H ),
naphthyloxymethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(
15e17)
Compound 15 and 17 were prepared by refluxing 12 and 14
0.015 mol) in sodium hydroxide solution (2 N) for 4 h and followed
2
2
-
CO-NH-NH-, exchangeable with D
2
(
exchangeable with D
5
8
2
20 4 3
H N O
by treatment of sodium salts with HCl to give desired compounds.
Then, the solid product was recrystallized to give 15 and 17,
respectively. For compound 16, a mixture of compound 13
.37.
(0.015 mol) and triethylamine (6 ml) in ethanol (50 ml) was heated
4
.1.1.3. N-benzyl-2-[(naphthalen-2-yloxy)acetyl]hydrazinecarbo-
under reflux for 6 h and followed by treatment with HCl to give
thioamide [14]. It was obtained as a white solid and was
desired compounds. Then, the solid product was recrystallized to

66
N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
þ
þþ
þþþ
p < 0.001 vs 10
m
M gp<0.05 ggp<0.01;
Fig. 7. Effect of compound 25 on apoptotic pathway of K562 cell line. *p < 0.05 **p < 0.01 ***p < 0.001 vs control. p < 0.05 p < 0.01
M compared to 50 M.
20
m
m
give 16.
3
yield 67%. UV (CH OH) nm: 320 (ε: 1924), 258 (ε: 19,819), 213 (ε:
ꢀ
1
5
6,840); IR cm : 3084 (NeH), 1632 (C]N), 1236 (C]S), 1103
1
(CeOeC); H NMR (400 MHz, DMSO-d
6
):
2
d 5.20 (s, 2H, -O-CH -),
4.1.2.1. 4-Benzyl-5-[[5-methyl-2-(propan-2-yl)phenoxy]methyl]-2,4-
5
.30 (s, 2H, -N-CH -C H ), 6.95e7.75 (12H, Ar-H), 14.02 (s, 1H, -NH
2 6 5
dihydro-3H-1,2,4-triazole-3-thione [15]. It was obtained as a white
solid and was recrystallized from methanol. HPLC t
Mp: 141e143 C, yield 90%. UV (CH
13
3
at the triazole moiety) ppm; C NMR (100.6 MHz, CDCl -d): d 47.01,
R
(min): 13.97,
OH) nm: 258 (ε: 18,488), 205 (ε:
ꢁ
60.82, 105.86, 121.64, 121.96, 125.22, 125.94, 126.22, 127.01, 127.51,
127.81, 128.19, 129.11, 134.57, 135.88, 153.23, 148.42 (triazole-C5),
3
ꢀ
1
5
2,638); IR cm : 3092 (NeH), 1614 (C]N), 1254 (C]S), 1165, 1094
1
169.23 (C]S) ppm. Anal. Calcd. for C19H15N OS: C, 69.14; H, 4.93;
(
CeOeC); H NMR (400 MHz, DMSO-d
.14 (s, 3H, Ar-CH ), 2.65e2.68 (m, 1H, -CH(CH
CH -), 5.23 (s, 2H, -N-CH -C ), 6.65e7.25 (8H, Ar-H), 13.95 (s,
H, ¼NeNH-, exchangeable with D
CDCl -d): 21.57, 23.35, 26.36, 47.53, 60.82, 112.47, 123.10, 126.66,
27.89, 128.76, 129.28, 134.46, 134.64, 136.93, 154.39, 148.92 (tri-
azole-C ), 169.28(C]S) ppm. Anal. Calcd. for C20 OS: C, 67.96;
H, 6.56; N,11.89; S, 9.07. Found: C, 67.41; H, 6.40; N, 11.71; S, 8.79.
6
):
d
0.87 (d, 6H, -CH(CH
3
)
2
),
3
N,12.09; S, 9.23. Found: C, 68.54; H, 4.89; N, 11.99; S, 8.43.
2
3
3 2
)
), 4.97 (s, 2H, -O-
2
2
6 5
H
1
3
1
2
O) ppm. C NMR (100.6 MHz,
4.1.3. Synthesis of 2-{[3-(substituted aryl/naphthyloxymethyl)-4-
benzyl-4,5-dihydro-1H-1,2,4-triazol-5-yl]sulfanyl}-N-(4-substituted
phenyl)acetamide (18e32)
3
d
1
5
H
23
N
3
Compound 15, 16 or 17 (0.001 mol) was dissolved in DMF
(10 ml) and anhydrous potassium carbonate (0.002 mol) was
added. The solution was stirred to 1 h at room temperature. Com-
pounds 1e5 dissolved in DMF were added to this solution (3 ml).
Then the reaction medium was heated by refluxing in oil bath for
4
.1.2.2. N-{4-[(4-benzyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)
methoxy]phenyl}acetamide [16]. It was obtained as a white solid
and was recrystallized from methanol. HPLC t (min): 4.43, Mp:
OH) nm: 256 (ε: 35,248), 207 (ε: 41,257);
IR cm : 3268, 3239, 3188 (NeH), 1640 (C]O), 1603 (C]N), 1240
ꢁ
6 h in the range of 50e60 C. At the end of this time, the solution
R
ꢁ
2
20 C, yield 58%. UV (CH
3
was poured onto ice, the resulting precipitate filtered and washed
with water. Then compounds 18e32 were recrystallized.
ꢀ1
1
(
C]S), 1206, 1036 (CeOeC); H NMR (400 MHz, DMSO-d
s, 3H, -NH-CO-CH ), 4.90 (s, 2H, -O-CH -), 5.19 (s, 2H, -N-CH
), 6.59e7.32 (9H, Ar-H), 9.69 (s, 1H, -CO-NH-Ar, exchangeable
with D O), 13.90 (s, 1H, eNH at the triazole moiety, exchangeable
with D -d): 24.40, 46.96,
O) ppm; 13C NMR (100.6 MHz, CDCl
0.84, 115.10, 121.18, 127.72, 128.27, 128.99, 134.10, 135.70, 153.23,
48.27 (triazole-C ), 168.58 (CH CONH), 169.11(C]S) ppm. Anal.
Calcd. for C18 S: C, 61.00; H, 5.12; N,15.81; S, 9.05. Found: C,
0.21; H, 5.06; N, 15.56; S, 8.78.
6
): d 1.88
(
3
2
2
-
4
4
.1.3.1. 2-{[3-[[5-Methyl-2-(propan-2-yl)phenoxy]methyl)-4-benzyl-
,5-dihydro-1H-1,2,4-triazol-5-yl]sulfanyl}-N-(4-sulfamoylphenyl)
6 5
C H
2
acetamide [18]. It was obtained as a white solid and was recrys-
ꢁ
2
3
d
tallized from methanol. HPLC t
UV (CH
(min): 11.89, Mp: 107 C, yield 71%.
R
6
1
ꢀ1
3
OH) nm: 261 (ε: 24,062), 205 (ε: 54,706); IR cm : 3302,
3
3
3
273 (NeH), 1686 (C]O), 1595 (C]N), 1155 (S]O), 1094 (CeOeC),
H
18
N
4
O
2
1
7
16 (CeS); H NMR (400 MHz, DMSO-d
.17 (s, 3H, Ar-CH ), 2.41e2.42 (m, 1H, -CH(CH
CH -), 5.15 (s, 2H, -N-CH -C ), 5.26 (s, 2H, -O-CH
(12H, Ar-H), 7.18 (s, 2H, -SO -NH , exchangeable with D
1H, -CO-NH-, exchangeable with D
CDCl -d): 21.60, 23.26, 26.02, 37.99, 47.99, 60.83, 112.68, 119.35,
6
):
d
0.85 (d, 6H, -CH(CH
), 4.12 (s, 2H, -S-
-), 6.65e7.70
O), 10.58 (s,
2
O) ppm; C NMR (100.6 MHz,
3 2
) ),
6
2
3
3 2
)
2
2
H
6 5
2
4
.1.2.3. 4-Benzyl-5-[(naphthalen-2-yloxy)methyl]-2,4-dihydro-3H-
2
2
2
13
1,2,4-triazole-3-thione [17]. It was obtained as a white solid and
ꢁ
was recrystallized from methanol. HPLC t
R
(min): 12.14, Mp: 204 C,
3
d

N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
67
122.69, 126.31, 127.11, 127.27, 128.68, 129.26, 134.21, 134.35, 136.63,
123.01, 126.06, 126.56, 127.27, 129.11, 129.50, 130.91, 133.54, 134.39,
1
38.87, 142.01, 154.69, 152.67 (triazole-C and ), 166.64
3
C
5
137.00, 142.61, 154.56, 152.96 (triazole-C
5
), 154.03 (triazole-C
3
),
þ
þ
(
SCH
2
CONH) ppm. HRMS (ESI ) (m/z): calculated for C28
H
32
N
5
O
4
S
2
166.46 (ArCOOC
calculated for C31
Anal. Calcd. for C31
2
H
H
5
), 167.02 (SCH
2
CONH) ppm. HRMS (ESI ) (m/z):
þ
þ
(
MþH) 566.1890, found 566.1893. Anal. Calcd. for C28
H
31
N
5
O
S
4 2
:
N
35 4
O
4
S (MþH) 559.2374, found 559.2374.
C, 59.45; H, 5.52; N,12.38; S,11.34. Found: C, 58.78; H, 5.63; N,12.61;
S, 11.28.
H
34
N
4
O
4
S: C, 66.64; H, 6.13; N, 10.03; S, 5.74.
Found: C, 66.70; H, 6.07; N, 10.03; S, 5.70.
4
4
.1.3.2. 2-{[3-[[5-Methyl-2-(propan-2-yl)phenoxy]methyl]-4-benzyl-
,5-dihydro-1H-1,2,4-triazol-5-yl] sulfanyl}-N-(4-chlorophenyl)acet-
4.1.3.6. 2-[(3-{[4-(Acetylamino)phenoxy]methyl}-4-benzyl-4,5-
dihydro-1H-1,2,4-triazol-5-yl) sulfanyl]-N-(4-sulfamoylphenyl)acet-
amide [19]. It was obtained as a brown solid and was recrystallized
amide [23]. It was obtained as a white solid and was recrystallized
ꢁ
ꢁ
from methanol. HPLC t
R
(min): 15.49, Mp: 172 C, yield 64%. UV
from methanol. HPLC t
(CH
R
(min): 3.51, Mp: 230e232 C, yield 62%. UV
OH) nm: 255 (ε: 37,989), 206 (ε: 54,723); IR cm : 3296, 3250,
ꢀ1
ꢀ1
(
(
(
(
2
1
(
1
1
CH
3
OH) nm: 252 (ε: 26,574), 206 (ε: 59,709); IR cm : 3231, 3173
3
NeH), 1676 (C]O), 1595 (C]N), 1163 (CeCl), 1093 (CeOeC), 719
3229, 3188 (NeH), 1674 (C]O), 1593 (C]N), 1152 (S]O), 1099
1
1
CeS); H NMR (400 MHz, DMSO-d
s, 3H, Ar-CH ), 2.71 (m,1H, -CH(CH
H, -N-CH -C ), 5.22 (s, 2H, -O-CH
0.36 (s, 1H, -CO-NH-, exchangeable with D
300 MHz, DMSO-d ): 20.91, 22.68, 25.22, 37.29, 46.93, 59.94,
12.77, 120.61, 121.85, 125.57, 126.52, 127.06, 127.82, 128.65, 128.69,
33.28, 135.11, 135.82, 137.67, 154.38, 151.18 (triazole-C ), 152.23
6
):
d
0.81 (d, 6H, -CH(CH
), 4.05 (s, 2H, -S-CH -), 5.12 (s,
-), 6.71e7.57 (12H, Ar-H),
3
)
2
), 2.14
(CeOeC), 710 (CeS); H NMR (400 MHz, DMSO-d
-NH-CO-CH ), 4.11 (s, 2H, -S-CH -), 5.11 (s, 2H, -N-CH
(s, 2H, -O-CH
6
):
d
1.91 (s, 3H,
H
6 5
3
3
)
2
2
3
2
2
-C ), 5.21
2
6
H
5
2
2
-), 6.69e7.73 (13H, Ar-H), 9.72 (s, 1H, -CH
ppm. HRMS (ESI ) (m/z): calculated for C26
567.1479, found 567.1478. Anal. Calcd. for C26
2
-CO-NH-Ar)
1
3
þ
þ
2
O) ppm; C NMR
H
27
N
O
6
O
5
S
2
(MþH)
6
d
H
26
N
6
5
2
S : C, 55.11; H,
4.62 N, 14.83; S, 11.32. Found: C, 54.05; H, 4.58; N, 14.36; S, 10.78.
5
þ
(
triazole-C
3
), 165.64 (SCH
2
CONH) ppm. HRMS (ESI ) (m/z): calcu-
4.1.3.7. 2-[(3-{[4-(Acetylamino)phenoxy]methyl}-4-benzyl-4,5-
dihydro-1H-1,2,4-triazol-5-yl)sulfanyl]-N-(4-chlorophenyl)acetamide
þ
lated for C28
Calcd. for C28
H
H
30ClN
29ClN
4
O
2
S (MþH) 521.1773, found 521.1774. Anal.
4
O
2
S: C, 64.54; H, 5.61; N, 10.75; S, 6.15. Found:
[24]. It was obtained as a brown solid and was recrystallized from
ꢁ
C, 64.24; H, 5.54; N, 10.62; S, 6.58.
methanol. HPLC t
R
(min): 9.04, Mp: 218 C, yield 68%. UV (CH
3
OH)
ꢀ1
nm: 251 (ε: 48,085), 205 (ε: 71,938); IR cm : 3281, 3258, 3188
4
4
.1.3.3. 2-{[3-[[5-Methyl-2-(propan-2-yl)phenoxy]methyl]-4-benzyl-
,5-dihydro-1H-1,2,4-triazol-5-yl]sulfanyl}-N-(4-bromophenyl)acet-
(NeH), 1657 (C]O), 1597 (C]N), 1175 (CeCl), 1094 (CeOeC), 712
1
(CeS); H NMR (400 MHz, DMSO-d
6
):
d
1.91 (s, 3H, -NH-CO-CH
), 5.21 (s, 2H, -O-CH
-CO-NH-Ar), 10.44 (s, 1H,
3
),
-),
amide [20]. It was obtained as a white solid and was recrystallized
from methanol. HPLC t
4.07 (s, 2H, -S-CH
2
-), 5.11 (s, 2H, -N-CH
2
-C
6
H
5
2
ꢁ
R
(min): 15.76, Mp: 180 C, yield 78%. UV
6.73e7.50 (13H, Ar-H), 9.71 (s, 1H, -CH
2
ꢀ
1
þ
(
(
(
(
CH
3
OH) nm: 254 (ε: 21,564), 205 (ε: 55,092); IR cm : 3302, 3258
3
CH eCOeNH-Ar) ppm. HRMS (ESI ) (m/z): calculated for
NeH), 1674 (C]O), 1601 (C]N), 1157 (CeBr), 1094 (CeOeC), 719
C
C
26
H
H
25ClN
24ClN
5
O
O
3
S (MþH)þ 522.1361, found 522.1361. Anal. Calcd. for
1
CeS); H NMR (400 MHz, DMSO-d
s, 3H, Ar-CH ), 2.69e2.71 (m, 1H, -CH(CH
.12 (s, 2H, -N-CH -C ), 5.22 (s, 2H, -O-CH
2
6
):
d
0.81 (d, 6H, -CH(CH
), 4.05 (s, 2H, -S-CH
-), 6.62e7.43 (12H, Ar-
3
)
2
), 2.14
26
5
3
S: C, 59.82; H, 4.63 N, 13.42; S, 6.14. Found: C, 59.69;
3
3
)
2
2
-),
H, 4.62; N, 13.29; S, 6.27.
5
2
6
H
5
þ
H), 10.35 (s, 1H, -CO-NH-) ppm. HRMS (ESI ) (m/z): calculated for
4.1.3.8. 2-[(3-{[4-(Acetylamino)phenoxy]methyl}-4-benzyl-4,5-
dihydro-1H-1,2,4-triazol-5-yl)sulfanyl]-N-(4-bromophenyl)acet-
amide [25]. It was obtained as a white solid and was recrystallized
þ
C
C
5
28
H
30BrN
29BrN
4
O
O
2
S (MþH) 565.1267, found 565.1267. Anal. Calcd. for
28
H
4
2
S: C, 59.47; H, 5.17; N, 9.91; S, 5.67. Found: C, 59.30; H,
ꢁ
.10; N, 9.84; S, 5.49.
from methanol. HPLC t
CH
(NeH), 1657 (C]O), 1597 (C]N), 1171 (CeBr), 1072 (CeOeC), 710
R
(min): 9.45, Mp: 223e224 C, yield 82%. UV
ꢀ
1
(
3
OH) nm: 252 (ε: 49,467), 206 (ε: 72,689); IR cm : 3254, 3187
4
4
.1.3.4. 2-{[3-[[5-Methyl-2-(propan-2-yl)phenoxy]methyl]-4-benzyl-
,5-dihydro-1H-1,2,4-triazol-5-yl] sulfanyl}-N-(4-nitrophenyl)acet-
1
(CeS); H NMR (400 MHz, DMSO-d
4.07 (s, 2H, -S-CH -CO-), 5.11 (s, 2H, -N-CH
CH -), 6.73e7.45 (m, 13H, Ar-H), 9.71 (s, 1H, -CH
exchangeable with D O), 10.37 (s, 1H, CH eCOeNH-Ar, exchange-
O) ppm; C NMR (300 MHz, DMSO-d ): 23.77, 37.33,
47.09, 59.97, 114.78, 115.10, 120.31, 121.01, 126.98, 127.90, 128.65,
131.61, 133.33, 135.09, 138.08, 152.97, 151.13 (triazole-C ), 151.97
6
):
d
1.91 (s, 3H, -NH-CO-CH
-C ), 5.21 (s, 2H, -O-
-CO-NH-Ar,
3
),
amide [21]. It was obtained as a yellow solid and was recrystallized
from methanol. HPLC t
2
2
6 5
H
ꢁ
R
(min): 14.54, Mp: 155 C, yield 78%. UV
2
2
ꢀ
1
(
CH
3
OH) nm: 314 (ε: 15,750), 205 (ε: 53,537); IR cm : 3277, 3208
), 1101 (CeOeC),
0.84 (d, 6H, -CH(CH ),
), 4.15 (s, 2H, -S-CH -),
-), 6.57e8.20 (12H, Ar-
2
3
1
3
(NeH), 1707 (C]O), 1616 (C]N), 1510, 1327 (eNO
2
able with D
2
6
d
1
7
2
5
21 (CeS); H NMR (400 MHz, DMSO-d
.16 (s, 3H, Ar-CH ), 2.73 (m, 1H, -CH(CH
.15 (s, 2H, -N-CH -C ), 5.25 (s, 2H, -O-CH
6
):
d
3 2
)
3
3
)
2
2
5
2
6
H
5
2
þ
(triazole-C
(ESI ) (m/z): calculated for C26
found 566.0856. Anal. Calcd. for C26
3
), 165.66 (SCH
2
CONH), 167.78 (CH
25BrN
24BrN
12.36; S, 5.66. Found: C, 54.82; H, 4.24; N, 12.22; S, 5.42.
3
CONH) ppm. HRMS
þ
þ
H), 10.86 (s, 1H, -CO-NH-) ppm. HRMS (ESI ) (m/z): calculated for
H
H
5
O
3
S (MþH) 566.0856,
þ
C
C
5
28
H
H
30
N
N
5
O
O
4
S (MþH) 532.2013, found 532.2022. Anal. Calcd. for
5 3
O S: C, 55.13; H, 4.27 N,
28
29
5
4
S: C, 63.26; H, 5.50; N, 13.17; S, 6.03. Found: C, 62.95; H,
.44; N, 13.05; S, 5.91.
4
.1.3.9. 2-[(3-{[4-(Acetylamino)phenoxy]methyl}-4-benzyl-4,5-
4.1.3.5. Ethyl 4-[({[3-{[5-methyl-2-(propan-2-yl)phenoxy]methyl}-4-
dihydro-1H-1,2,4-triazol-5-yl)sulfanyl]-N-(4-nitrophenyl)acetamide
benzyl-4,5-dihydro-1H-1,2,4-triazol-5-yl] sulfanyl}acetyl)amino]ben-
zoate [22]. It was obtained as a white solid and was recrystallized
[26]. It was obtained as a yellow solid and was recrystallized from
ꢁ
methanol. HPLC t
R
(min): 7.81, Mp: 231 C, yield 45%. UV (CH
3
OH)
ꢁ
from methanol. HPLC t
CH
R
(min): 15.13, Mp: 166 C, yield 50%. UV
nm: 314 (ε: 13,128), 245 (ε: 21,723), 228 (ε: 22,649), 205 (ε: 47,628);
ꢀ1
(
3
OH) nm: 533 (ε: 3401), 273 (ε: 30,492), 207 (ε: 68,985); IR
cm : 3234 (NeH), 1711, 1682 (2 C]O), 1599 (C]N), 1101 (CeOeC),
IR cm : 3287, 3269, 3202 (NeH), 1694, 1659 (C]O), 1601 (C]N),
ꢀ
1
1
1495, 1312 (eNO
2
), 1109 (CeOeC), 716 (CeS); H NMR (400 MHz,
1.91 (s, 3H, -NH-CO-CH ), 4.14 (s, 2H, -S-CH -), 5.11 (s,
), 5.21 (s, 2H, -O-CH -), 6.73e8.16 (13H, Ar-H), 9.70
1
7
1
-
5
21 (CeS); H NMR (400 MHz, DMSO-d
.22 (t, 3H, -OeCH eCH ), 2.16 (s, 3H, Ar-CH
CH(CH ), 4.11 (s, 2H, -S-CH
.14 (s, 2H, -N-CH -C
6
):
d
0.84 (d, 6H, -CH(CH
), 2.72e2.76 (m, 1H,
-), 4.17e4.23 (q, 2H, -O-CH
3
)
2
),
DMSO-d
6
):
d
3
2
2
3
3
2H, -N-CH
2
-C
6
H
5
2
3
)
2
2
2
eCH
3
),
(s, 1H, -CH
(ESI ) (m/z): calculated for C26
533.1602. Anal. Calcd. for C26
2
-CO-NH-Ar), 10.85 (s, 1H, CH
3
eCOeNH-Ar) ppm. HRMS
þ
þ
2
6
H
5
), 5.25 (s, 2H, -O-CH
2
-), 6.56e7.92 (12H, Ar-
-d):
14.61, 21.60, 23.29, 26.23, 37.01, 48.30, 60.95, 61.08, 112.66, 119.15,
25
H N
6
O
5
S (MþH) 533.1602, found
13
H), 10.56 (s, 1H, -CO-NH-) ppm; C NMR (100.6 MHz, CDCl
3
24 6 5
H N O S: C, 58.64; H, 4.54 N, 15.78; S,
d
6.02. Found: C, 59.50; H, 4.68; N, 15.65; S, 6.07.

68
N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
ꢁ
R 3
t (min): 13.04, Mp: 177e178 C, yield 54%. UV (CH OH) nm: 319 (ε:
4
4
.1.3.10. Ethyl 4-[{[(3-{[4-(Acetylamino)phenoxy]methyl}-4-benzyl-
,5-dihydro-1H-1,2,4-triazol-5-yl)sulfanyl]acetyl}amino]benzoate
ꢀ1
19,636), 307 (ε: 20,598), 222 (ε: 75,378); IR cm : 3350, 3214
(NeH), 1690 (C]O), 1597 (C]N), 1533, 1327 (eNO ), 1098
4.18 (s, 2H,
[27]. It was obtained as a white solid and was recrystallized from
2
ꢁ
1
methanol. HPLC t
nm: 258 (ε: 27,294), 205 (ε: 49,503); IR cm : 3262, 3235, 3192
R
(min): 8.71, Mp: 217 C, yield 43%. UV (CH
3
OH)
(CeOeC), 729 (CeS); H NMR (400 MHz, DMSO-d
6
): d
ꢀ1
-S-CH -), 5.26e5.36 (m, 4H, -N-CH -C and -O-CH
2
2
6
H
5
2
-), 7.04e8.16
þ
(NeH), 1707, 1694, 1663 (C]O), 1601 (C]N), 1105 (CeOeC), 721
(16H, Ar-H), 10.88 (s, 1H, -CO-NH-Ar) ppm. HRMS (ESI ) (m/z):
1
þ
(
1
-
6
CeS); H NMR (400 MHz, DMSO-d
.91 (s, 3H, -NH-CO-CH ), 4.11 (s, 2H, -S-CH
O-CH -CH ), 5.11 (s, 2H, -N-CH -C ), 5.21 (s, 2H, -O-CH
.68e7.90 (13H, Ar-H), 9.71 (s, 1H, -CH -CO-NH-Ar), 10.59 (s, 1H,
eCOeNH-Ar) ppm; C NMR (100.6 MHz, CDCl -d): 14.85,
6
):
d
1.22 (t, 3H, -CO-CH
-CO-), 4.17e4.23 (q, 2H,
-),
2
-CH
3
),
calculated for C28
Anal. Calcd. for C28
H
H
24
N
5
O
4
S (MþH) 526.1544, found 526.1546.
3
2
23
N
5
O
4
S: C, 63.99; H, 4.41 N, 13.32; S, 6.10.
2
3
2
6
H
5
2
Found: C, 64.47; H, 4.44; N, 13.32; S, 5.96.
2
13
CH
3
3
d
4.1.3.15. Ethyl 4-[{[(4-benzyl-5-[(naphthalen-2-yloxy)methyl]-4H-
2
1
4.45, 38.14, 47.90, 60.71, 60.96, 115.27, 119.06, 121.06, 125.25,
27.66, 128.55, 129.26, 130.84, 134.03, 135.54, 143.63, 153.62, 151.92
1,2,4-triazol-3-yl) sulfanyl]acetyl}amino]benzoate [32]. It was ob-
tained as a white solid and was recrystallized from methanol. HPLC
ꢁ
(
(
triazole-C
SCH CONH), 168.41 (CH
5
), 152.56 (triazole-C
3
), 165.86 (ArCOOC
2
H
5
), 166.65
t
R
(min): 13.55, Mp: 187e188 C, yield 46%. UV (CH
3
OH) nm: 320 (ε:
þ
ꢀ1
2
3
CONH) ppm. HRMS (ESI ) (m/z): calcu-
1251), 275 (ε: 27,060), 213 (ε: 61,930); IR cm : 3252, 3185 (NeH),
1713, 1684 (C]O), 1601 (C]N), 1092 (CeOeC), 723 (CeS); H NMR
þ
1
lated for C29
Calcd. for C29
6
H
30
N
5
O
5
S (MþH) 560.1962, found 560.1967. Anal.
H
29
N
5
O
5
S: C, 62.24; H, 5.22 N, 12.51; S, 5.73. Found: C,
(400 MHz, DMSO-d
CH -), 4.18e4.23 (q, 2H, -O-CH
and -O-CH -), 7.04e7.85 (16H, Ar-H), 10.60 (s, 1H, -CO-NH-Ar)
ppm; C NMR (100.6 MHz, CDCl -d): 14.61, 37.02, 48.53, 60.84,
6
):
d
2 3
1.23 (t, 3H, -CO-CH -CH ), 4.15 (s, 2H, -S-
3.96; H, 5.20; N, 12.88; S, 5.56.
2
2
-CH ), 5.26e5.36 (m, 4H, -N-CH -
3
2
C
6
H
5
2
13
4
.1.3.11. 2-({4-Benzyl-5-[(naphthalen-2-yloxy)methyl]-4H-1,2,4-
3
d
triazol-3-yl}sulfanyl)-N-(4- sulfamoylphenyl)acetamide [28]. It was
obtained as a white solid and was recrystallized from methanol.
61.08, 105.46, 119.18, 121.58, 122.06, 125.87, 125.96, 126.95, 127.34,
127.94, 129.10, 129.50, 130.93, 133.44, 134.77, 142.58, 154.23, 152.59
ꢁ
HPLC t
3
(
(
5
R
(min): 10.32, Mp: 178e180 C, yield 62%. UV (CH
3
OH) nm:
(triazole-C
3
), 153.12 (triazole-C
5
), 166.46 (ArCOOC
2
H
5
), 166.97
ꢀ1
þ
20 (ε: 1248), 262 (ε: 26,701), 211 (ε: 64,435); IR cm : 3312, 3202
NeH), 1684 (C]O), 1597 (C]N), 1155 (S]O), 1096 (CeOeC), 723
CeS); 1H NMR (400 MHz, DMSO-d
): 4.15 (s, 2H, -S-CH -),
.26e5.37 (m, 4H, -N-CH -C and -O-CH -), 7.04e7.76 (18H, Ar-H
and eSO -NH ), 10.60 (s, 1H, -CO-NH-Ar) ppm;
100.6 MHz, CDCl -d): 38.13, 48.14, 60.77, 105.58, 119.35,
21.63,121.76, 125.27, 125.80, 126.03, 126.88, 127.16, 127.27, 127.75,
28.61, 129.26, 134.57, 138.97, 142.05, 153.29, 152.33 (triazole-C ),
(SCH
2
CONH) ppm. HRMS (ESI ) (m/z): calculated for C31
29
H N
4
O
4
S
þ
(MþH) 553.1904, found 553.1905. Anal. Calcd. for C31
28 4 4
H N O S: C,
6
d
2
67.37; H, 5.11 N, 10.14; S, 5.80. Found: C, 68.46; H, 5.14; N, 10.29; S,
5.90.
2
6
H
5
2
1
3
2
2
C NMR
(
1
1
3
d
4.2. Biological methods
3
4.2.1. In vitro antiviral assays
4.2.1.1. Inhibition of HIV-induced cytopathicity in MT-4 cells.
Evaluation of the antiviral activity of the compounds against HIV-1
þ
1
52.70 (triazole-C
calculated for C28
Anal. Calcd. for C28
5
), 166.60 (SCH
2
CONH) ppm. HRMS (ESI ) (m/z):
þ
26
H N
5
O
4
S
2
(MþH) 560.1421, found 560.1419.
H
25
N
5
O
4
S
2
: C, 60.09; H, 4.50 N, 12.51; S, 11.46.
B
strain III and HIV-2 strain ROD in MT-4 cells was performed using
Found: C, 61.46; H, 5.05; N, 12.66; S, 11.54.
the MTT assay as previously described [44,59]. Stock solutions
(
10 ꢂ final concentration) of test compounds were added in 25
mL
4
.1.3.12. 2-({4-Benzyl-5-[(naphthalen-2-yloxy)methyl]-4H-1,2,4-
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
compounds were made directly in flatbottomed 96-well microtiter
trays using a Biomek 3000 robot (Beckman Instruments, Fullerton,
CA). Untreated control HIV and mock-infected cell samples were
triazol-3-yl}sulfanyl)-N-(4-chlorophenyl)acetamide [29]. It was ob-
tained as a brown solid and was recrystallized from methanol. HPLC
ꢁ
t
4
R
(min): 13.88, Mp: 183 C, yield 59%. UV (CH
3
OH) nm: 320 (ε:
ꢀ1
425), 252 (ε: 24,567), 210 (ε: 67,155); IR cm : 3250, 3183 (NeH),
672 (C]O), 1597 (C]N), 1175 (CeCl), 1094 (CeOeC), 727 (CeS);
H NMR (400 MHz, DMSO-d
H, -N-CH -C and -O-CH
1
1
6
):
d
4.11 (s, 2H, -S-CH
2
-), 5.26e5.36 (m,
included for each sample. HIV-1(III
B
) [60] or HIV-2 (ROD) [61] stock
4
-
(
2
6
H
5
2
-), 7.04e7.76 (16H, Ar-H), 10.40 (s, 1H,
þ
(50 L) at 100e300 CCID50 (cell culture infectious dose) or culture
m
CO-NH-Ar) ppm. HRMS (ESI ) (m/z): calculated for C28
H24ClN
4
O
2
S
medium was added to either the infected or mock-infected wells of
the microtiter tray. Mock-infected cells were used to evaluate the
effect of test compounds on uninfected cells in order to assess the
cytotoxicity of the test compounds. Exponentially growing MT-
þ
MþH) 515.1303, found 515.1304. Anal. Calcd. for C28
H23ClN O S:
4 2
C, 65.30; H, 4.50 N, 10.88; S, 6.23. Found: C, 64.04; H, 4.41; N, 10.51;
S, 6.06.
4
cells [62] were centrifuged for 5 min at 220 g and the supernatant
5
4
.1.3.13. 2-({4-Benzyl-5-[(naphthalen-2-yloxy)methyl]-4H-1,2,4-
was discarded. The MT-4 cells were resuspended at 6 ꢂ 10 cells/
triazol-3-yl}sulfanyl)-N-(4-bromophenyl)acetamide [30]. It was ob-
mL, and 50- L volumes were transferred to the microtiter tray
m
tained as a white solid and was recrystallized from methanol. HPLC
wells. Five days after infection, the viability of mock- and HIV-
infected cells was examined spectrophotometrically by the MTT
assay. The MTT assay is based on the reduction of yellow colored 3-
ꢁ
t
R
(min): 14.16, Mp: 191 C, yield 70%. UV (CH
3
OH) nm: 320 (ε:
ꢀ
1
1595), 255 (ε: 22,474), 210 (ε: 60,851); IR cm : 3296, 3246 (NeH),
1
672 (C]O), 1597 (C]N), 1175 (CeBr), 1092 (CeOeC g.b.), 725
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
1
(
CeS); H NMR (400 MHz, DMSO-d 4.11 (s, 2H, -S-CH
6
):
d
2
-),
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial dehydro-
genase of metabolically active cells to a blue-purple formazan that
can be measured spectrophotometrically. The absorbances were
read in an eight-channel computer-controlled photometer (Infinite
M1000, Tecan, Mechelen, Belgium), at two wavelengths (540 and
690 nm). All data were calculated using the median OD (optical
density) value of three 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
5
.26e5.36 (m, 4H, -N-CH -C and -O-CH -), 7.04e7.76 (16H, Ar-
2
6
H
5
2
þ
H), 10.40 (s, 1H, -CO-NH-Ar) ppm. HRMS (ESI ) (m/z): calculated for
þ
C
C
4
28
H
24BrN
23BrN
4
O
O
2
S (MþH) 559.0798, found 559.0798. Anal. Calcd. for
28
H
4
2
S: C, 60.11; H, 4.14 N,10.01; S, 5.73. Found: C, 59.77; H,
.13; N, 9.84; S, 5.62.
4
.1.3.14. 2-({4-Benzyl-5-[(naphthalen-2-yloxy)methyl]-4H-1,2,4-
triazol-3-yl}sulfanyl)-N-(4-nitrophenyl)acetamide [31]. It was ob-
tained as a yellow solid and was recrystallized from methanol. HPLC

N. Kulaba s¸ et al. / European Journal of Medicinal Chemistry 121 (2016) 58e70
69
the cytopathic effect of the virus in infected cells was defined as the
4.2.2.5. Statistical analysis. The data were reported as mean-
s ± standard deviations and analysed by one-way analysis of vari-
ance (ANOVA) followed by the Tukey's multiple comparison tests
using GraphPad Prism 5. Differences between means at p < 0.05
level were considered significant.
5
0% effective concentration (EC50).
4.2.2. Anticancer activity
4.2.2.1. Cell culture. Human leukemic cell line, K562 (ATCC, CCL-
2
43), human lung adenocarcinoma cell lineA549 (ATCC, CCL-185),
Acknowledgments
human prostate adenocarcinoma cell line, PC-3 (ATCC, CRL-1435)
were maintained in DMEM (Dulbecco's Modified Eagle Medium)
We thank the Division of Cancer Research, National Cancer
Institue (NCI), Bethesda, MD, for the anticancer activity screening.
The authors are grateful to Dr. Jürgen Gross from the Institute of
Organic Chemistry, University of Heidelberg, for his generous help
on obtaining ESI mass spectra of the compounds. The authors wish
to thank Kristien Erven and Kris Uyttersprot for dedicated technical
assistance for the evaluation of the anti-HIV activity.
supplemented with 10% FBS (Fetal bovine serum), 1%
and penicillin/streptomycin (Gibco) at 37 C in a humidified incu-
L
-Glutamine
ꢁ
2
bator with 5% CO .
4
(
(
.2.2.2. Cell viability assay. Cell viability was determined by the 3-
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTT) assay. Briefly, the cells (1 ꢂ 10 cells/well) were seeded onto
6-well plates and incubated overnight. Then, the cells were
treated with different concentrations (5e100 M) of compounds
4
9
Appendix A. Supplementary data
m
for 48 h. After the incubation period, MTT was added into each well
to a final concentration of 0.5 mg/ml and incubated for 4 h. The
culture medium was then removed and 100 mL of the SDS buffer
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.05.017.
was added to solubilize the purple formazan product. Absorbances
at wavelengths of 570 and 630 nm were measured by a microplate
reader (Biotek, Winooski, VT).
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