Synthesis and anti-Candidal activity of N-(4-aryl/cyclohexyl)-2-(pyridine- 4-yl carbonyl) hydrazinecarbothioamide

Article abstract of DOI:10.1016/j.bmcl.2014.01.060

Eighteen N-(4-aryl/cyclohexyl)-2-(pyridine-4-yl carbonyl) hydrazinecarbothioamide derivatives were synthesized, evaluated against ten clinical isolates of Candida spp. and compared with itraconazole. Introduction of p-chloro (2c), p-iodo (2q), m-chloro (2

Full text of DOI:10.1016/j.bmcl.2014.01.060

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1299–1302
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-Candidal activity of N-(4-aryl/cyclohexyl)-
2-(pyridine-4-yl carbonyl) hydrazinecarbothioamide
b
b
a
Mashooq Ahmad Bhat a, , Abdul Arif Khan , Shahanavaj Khan , Mohamed A. Al-Omar ,
⇑
Mohammad Khalid Parvez c, Mohammed Salem Al-Dosari c, Abdullah Al-Dhfyan d
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia
b Department of Pharmaceutics, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia
c Department of Pharmacognosy, College of Pharmacy, King Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia
d Stem Cell & Tissue Re-Engineering Program, Research Center, King Faisal Specialized Hospital & Research Center, MBC-03, PO Box 3354, Riyadh 11211, Saudi Arabia.
a r t i c l e i n f o
a b s t r a c t
Article history:
Eighteen N-(4-aryl/cyclohexyl)-2-(pyridine-4-yl carbonyl) hydrazinecarbothioamide derivatives were
synthesized, evaluated against ten clinical isolates of Candida spp. and compared with itraconazole. Intro-
duction of p-chloro (2c), p-iodo (2q), m-chloro (2l) and o-nitro (2r) substitution at phenyl ring of thiosem-
icarbazide enhanced the anti-Candida activity. Compound (2c) bearing p-cholorophenyl ring was found to
be the most effective against Candida albicans ATCC 66027, Candida spp. 12810 (blood) and Candida spp.
Received 11 December 2013
Revised 15 January 2014
Accepted 21 January 2014
Available online 30 January 2014
178 (HVS) with MIC value of 0.09–0.78
lg/mL, whereas itraconazole exhibits the inhibitory activity with
Keywords:
Thiosemicarbazide
Isoniazid
In vitro anti-Candidal activity
Candida spp.
Cytotoxicity
MIC value of 0.04–1.56 g/mL against all tested strains. There is a correlation between anti-Candidal
l
activity and p-chloro substitution at phenyl ring of thiosemicarbazide. All synthesized compounds were
investigated for their potential cytotoxicity against non cancer cell line MCF-10A. The active compounds
2c, 2r and 2a were further investigated for their cytotoxic effects on three cancer cell lines; HT1080
(skin), HepG2 (liver) and A549 (lung). The active compounds showed minimal cytotoxic activity against
non cancer cell line and all three cancer cell lines. Moreover, compound 2c displaying better activity
against C. albicans ATCC66027 and Candida spp. [blood] compared to reference drug (itraconazole), rep-
resents a good lead for the development of newer, potent and broad spectrum anti-Candidal agents.
Ó 2014 Elsevier Ltd. All rights reserved.
Invasive fungal infections (IFIs) are life-threatening opportunis-
tic infections that are an increasingly important cause of morbidity
and mortality in patients, especially those with compromised
immune function and those hospitalized with serious underlying
diseases.1 The majority of these infections are caused by Candida
spp., with over 50% due to Candida albicans.2 These fungi are
responsible for various forms of disease, ranging from superficial
infections of the mucosal surfaces or skin to systemic infections,
which in most cases is life threatening.3 In general, for treatment
of an infection with Candida spp., amphotericin and azole drugs
are used, but these agents are not considered to satisfy medical
needs due to their toxicity, side effects, drug interactions, limited
routes, emergence of drug-resistant and drug-low-susceptible
strains.4 The most frequently implicated risk factors include treat-
ment with broad-spectrum antibiotics, use of central venous cath-
eters and implantable prosthetic devices, parenteral nutrition,
prolonged intensive care unit stay, hemodialysis and immunosup-
pression (including HIV infection, neutropenia, use of glucocorti-
costeroids, chemotherapeutic agents, and immunomodulators).5
Based on docking and molecular modeling studies it was pro-
posed that the possible target for antifungal activity of 4-arylthi-
osemicarbazides is the enzyme N-myristoyltransferase (NMT).
The ligand recognition process is connected with high-electron
density around the sulfur atom and geometry of NHANHAC(@S)ANH
core.6 In the course of our search for prototype antifungal agents
from the class of thiosemicarbazide derivatives, we have prelimin-
ary screened a series of compounds against clinical isolates of Can-
dida spp. and ATCC strains of Candida species. Among tested
compounds are 4-aryl/cyclohexyl thiosemicarbazides. Few other
aryl thiosemicarbazides that exhibit significant anti-Candidal activ-
ity have been reported in the literature.7–15 Those bearing isoquin-
oline ring are acting against several Candida species.16 Thus such
template could be seen as starting point for further optimization
of novel antifungal agents. In order to develop new potent anti-
fungal compounds, a new set of thiosemicarbazides (2a–r) based
on compound A (Fig. 1) were synthesized and in vitro anti-Candida
activity was studied. Pyridyl ring, a prominent scaffold present in
⇑
Corresponding author. Tel.: +966 1 4677343; fax: +966 1 4676220.
E-mail address: mashooqbhat@rediffmail.com (M.A. Bhat).
http://dx.doi.org/10.1016/j.bmcl.2014.01.060
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

1300
M. A. Bhat et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1299–1302
O
H
N
NH
N
H
N
S
O
Compound A
CH3
Modulation of the Isoquinoline moiety
O
R
H
N
NH
Figure 2. ORTEP diagram of 2b at 50% probability.
N
H
N
S
absolute ethanol to yield N-(4-aryl/cyclohexyl)-2-(pyridin-4-yl-
carbonyl) hydrazinecarbothioamide (2a–r).21–24 The purity of the
compounds was checked by TLC and elemental analysis. The com-
pounds of this series were identified by spectral data. In the 1H
NMR spectra, the signals of the respective protons of the deriva-
tives were verified on the basis of chemical shift, multiplicity and
coupling constant. The spectra of all compounds showed D2O
exchangeable singlet at 7.9–10.1 ppm and 10.5–14.5 ppm corre-
sponding to NH protons and CONH protons.25 Analytical and spec-
tral data of the synthesized compounds were in good agreement
with composition of the synthesized compounds. The data of the
physiochemical properties of all the compounds is given in
(Table 1). Single crystal X-ray crystallography was obtained for
compound 2b (Fig. 2). All the compounds were screened for their
in vitro anti-Candidal activity against ten strains of Candida species
(Table 2).26 All the synthesized compounds were investigated for
their potential cytotoxicity against non cancer cell line MCF-10A.
The active compounds 2c, 2r and 2a were further investigated
for their cytotoxic effects on three cancer cell lines; HT1080 (skin),
HepG2 (liver) and A549 (lung).27,28
Compounds 2a-r
Figure 1. Structure of the lead compound A and newly synthesized compounds
(2a–r).
H
H
N
H
N
S
N
N
RNCS/ C2H5OH
R
H
N
H
N
H
N
O
O
(1a-r)
(2a-r)
Scheme 1. Synthetic protocol of the compounds (2a–r).
various bioactive molecules, has played a vital role in the develop-
ment of different medicinal agents.17–19 The anti-Candidal activity
of pyridine has also been reported.20 The pharmacophore was pre-
served and isoquinoline moiety was replaced by pyridine moiety of
the isoniazid.
The synthesis of isoniazid (INH) derivatives was carried out in
single step shown in (Scheme 1). Isoniazid was reacted with appro-
priate substituted phenyl/cyclohexyl isothiocyanate in presence of
In an initial screening program to determine potential
anti-Candida activities of thiosemicarbazides, compound A (MIC,
Table 1
Physical data of the synthesized compounds (2a–r)
N
H
N
S
R
N
H
N
H
O
(2a-r)
Compound
R
Molecular formula
C13H12N4OS
Yield %
mp (°C)
CLogPa
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
Phenyl
Cyclohexyl
4-Chloro phenyl
4-Methoxy phenyl
2,6-Dimethyl phenyl
3-Ethyl phenyl
70
75
70
72
80
75
70
65
80
75
70
65
70
75
60
65
60
70
200–202
223–225
210–212
235–237
290–292
220–222
238–240
256–258
260–262
278–280
268–270
290–292
270–272
245–247
290–292
170–172
210–212
248–250
1.37
1.68
2.36
1.32
2.29
2.37
1.83
0.70
1.83
1.83
1.83
2.36
1.32
1.32
0.79
1.86
2.80
1.83
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
13H18N4OS
13H11ClN4OS
14H14N4O2S
15H16N4OS
15H16N4OS
13H11N5O3S
17H15N7O3S2
14H14N4OS
14H14N4OS
14H14N4OS
13H11ClN4OS
14H14N4O2S
14H14N4O2S
15H15N5O2S
15H16N4O2S
13H11IN4OS
13H11N5O3S
4-Nitro phenyl
4-Sulfapyrimidine phenyl
4-Methyl phenyl
3-Methyl phenyl
2-Methyl phenyl
3-Chloro phenyl
2-Methoxy phenyl
3-Methoxy phenyl
4-Acetamido phenyl
4-Ethoxy phenyl
4-Iodo phenyl
2-Nitro phenyl
a
CLogP calculated by ACD/Chem Sketch.

M. A. Bhat et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1299–1302
1301
Table 2
Anti-Candidal activity of the compounds (2a–r) as MIC values (
l
g/mL)
Compound C. tropicalis
C. parapsilosis C. albicans
Candida sp.
[HVS*]
Candida sp.
[HVS] 11972 [HVS] 178
Candida sp. Candida sp.
Candida sp.
[urine]
Candida sp.
[urine]
Candida sp.
[blood]
ATCC 66029 ATCC 22019
ATCC
[urine] 300
66027
13184
12341
12485
12810
2a
2b
2c
2d
2e
2f
2g
2h
2i
1.6
0.39
>50
0.19
>50
0.78
>50
>50
>50
>50
>50
>50
0.78
>50
>50
0.19
>50
0.19
6.25
0.04
0.19
>50
0.09
>50
0.78
>50
>50
>50
>50
>50
>50
0.19
>50
>50
0.39
>50
0.39
1.6
12.5
>50
0.78
>50
6.25
>50
>50
>50
>50
>50
>50
6.25
>50
>50
1.6
6.25
>50
0.19
>50
12.5
>50
>50
>50
>50
>50
>50
0.39
>50
>50
0.78
>50
0.19
6.25
0.02
6.25
>50
0.19
>50
6.25
>50
>50
>50
>50
>50
>50
6.25
>50
>50
0.19
>50
0.09
0.39
0.19
0.19
>50
0.39
>50
0.78
>50
>50
>50
>50
>50
>50
0.78
>50
>50
1.6
1.6
1.6
0.78
>50
0.09
>50
1.6
>50
>50
>50
>50
>50
>50
0.09
>50
>50
1.6
>50
0.19
>50
0.78
>50
>50
>50
>50
>50
>50
1.6
>50
>50
0.19
>50
0.19
0.19
0.19
>50
0.19
>50
6.25
>50
>50
>50
>50
>50
>50
0.78
>50
>50
1.6
>50
0.39
>50
6.25
>50
>50
>50
>50
>50
>50
6.25
>50
>50
1.6
2j
2k
2l
2m
2n
2o
2p
2q
2r
>50
1.6
0.78
0.39
>50
1.6
1.6
>50
0.19
6.25
0.09
>50
0.78
0.39
0.04
>50
0.39
0.39
0.25
IT#
1.56
0.09
*
HVS: High Vaginal Swab.
IT: itraconazole.
#
Table 3
In vitro cytotoxicity of the compounds against cancer cell lines
25 lg/mL against a fluconazole) was chosen as the first hit for fur-
ther derivatization.6 Replacement of the isoquinoline nucleus by
pyridine and preserving the pharmacophore [A(C@O)ANHANHA
(C@S)ANHAR] was studied. Surprisingly when the R was changed
by electron donating groups (compounds 2d, 2f, 2i, 2j, 2k, 2m and
2n), the anti-Candidal activity was lost. Insertion of cyclohexy
group, 2b also led to the loss of activity. These disappointing re-
sults led us to switch to the electron withdrawing groups. Com-
pounds 2c, 2q, 2l, 2o and 2r achieved the strongest anti-Candida
activity. The best activity was observed when phenyl was replaced
by p-chlorophenyl group, compound 2c presents a good anti-Can-
dida activity with a selectivity oriented towards C. albicans ATCC
Compound
IC50 (lg/mL)
HT 1080 (Skin)
HepG2 (Liver)
A549 (Lung)
2c
2r
2a
540.12
833.4
684.14
201.63
1200.5
409.55
715.83
142
409.55
813
2042.5
182
Itraconazole
the compounds (2a–r) were evaluated against non cancer cell line;
MCF-10A (non-tumorigenic epithelial cell line) for their cytotoxic
properties using WST-1 assay. None of the active compounds
showed any cytotoxicity to non cancer cell line up to highest con-
66027 and Candida spp. 12810 [blood] (MIC, 0.09 lg/mL). In partic-
ular, this compound showed similar to better activity than refer-
ence drug for several Candida strains. The compound with p-iodo
substitution on the phenyl group was found to be more active
centration of (IC50 >300 lM), indicating a high selectivity of anti-
against Candida spp. 178 [HVS] with (MIC, 0.09
pound with m-chloro substitution on phenyl group was found to
be active against Candida spp. 12810 [blood] with (MIC, 0.09 g/
mL) whereas itraconazole exhibits inhibitory activity with (MIC,
0.04–1.56 g/mL). The compound 2c bearing p-chloro substitution
lg/mL) and com-
Candidal activity. The compounds 2c, 2r and 2a were evaluated
against three cancer celines; HT1080 (skin), HepG2 (Liver), A549
(Lung) using MTT assay (Table 3). The biological study indicated
that screened compounds showed minimal cytotoxicity (IC50
l
l
>400 lg/mL), against all three cancer cell lines.
on phenyl group was found to be most potent derivative of the
series against all strains of Candida spp. This could result from
increased lipophilicity associated with chloro phenyl group. The
biological results indicate that C. albicans ATCC 66027 was more
susceptible to compounds 2a, 2c, 2e, 2l, 2o and 2q (MIC, 0.09–
In conclusion we focused on the synthesis of thiosemicarbazide
derivatives of isoniazid (2a–r) which were screened in vitro against
ten strains of Candida spp. The compounds bearing p-chlorophenyl,
p-iodophenyl, m-chlorophenyl, o-nitrophenyl and p-acetamido
substitution were more active than the compounds bearing meth-
ylphenyl, methoxyphenyl, cyclohexyl, sulfonamidophenyl and eth-
oxyphenyl against all Candida spp. Compound 2c was the most
effective compound against C. albicans ATCC66027 and Candida
0.78
da tropicalis ATCC 66029 was equally susceptible to compounds 2c,
2o, 2q and 2r (MIC, 0.19 g/mL) as to itraconazole (0.19 g/mL).
Candida spp. 178 [blood] was more susceptible to compounds 2c,
2o and 2q (MIC, 0.09–0.19 g/mL) as compared to itraconazole
(MIC, 0.19 g/mL). It is apparent that there is a positive correlation
lg/mL) as compared to itraconazole (MIC, 1.56 lg/mL). Candi-
l
l
spp. [blood] (MIC, 0.09 lg/mL). This outcome confirms that p-
l
chlorophenyl substitution have a considerable influence on anti-
Candidal activity. The active compounds showed lowest cytotoxic-
ity against tested three cancer cell lines and non cancer cell line.
Based on the reported results, compounds 2c, 2q, 2l, 2r and 2o
could also be used in association with azole derivatives to evaluate
their antifungal activity. Moreover, compound 2c displaying better
activity against C. albicans ATCC66027 and Candida spp. [blood]
compared to reference drug (itraconazole), represents a good lead
for the development of newer, potent and broad spectrum anti-
Candidal agents.
l
between anti-Candidal activity and electronegative functional
groups like chloro, iodo and nitro. The hydrophobicity balance
was already proved to be important in such series to obtains anti
Candida activity.29 From a pharmacological point of view it is
important for the studied compounds to exhibit high bioactivity
and at the same time show no or low cytotoxicity effects, other-
wise the activity might just be due to general toxicity, which dis-
qualifies the compound as a drug or lead molecule candidate. All

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M. A. Bhat et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1299–1302
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Supplementary data
Supplementary data (Crystallographic Data Center (CCDC) num-
ber 959803 contains crystallographic data for the structure 2b.
Copies of the data can be obtained, free of charge, on application
to CCDC 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44 1223
336033; e-mail: deposit@ccdc.cam.ac.uk/http://www.ccdc.cam.
ac.uk). associated with this article can be found, in the online ver-
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