N-Alkyl/aryl-4-(3-substituted-3-phenylpropyl)piperazine-1-carbothioamide as dual-action vaginal microbicides with reverse transcriptase inhibition

Article abstract of DOI:10.1016/j.ejmech.2015.07.021

Abstract The growing population and health-care burden (due to STIs and HIV) imposes a particular economic crisis over resource-poor countries. Thus a novel approach as vaginal microbicides emerges as integrated tool to control both population and anti-ST

Full text of DOI:10.1016/j.ejmech.2015.07.021

European Journal of Medicinal Chemistry 101 (2015) 640e650
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
N-Alkyl/aryl-4-(3-substituted-3-phenylpropyl)piperazine-1-
carbothioamide as dual-action vaginal microbicides with reverse
*
transcriptase inhibition
Veenu Bala a , Dhanaraju Mandalapu , Sonal Gupta , Santosh Jangir ,
,
h
a
a
,
h
a
b
c
,
h
c
,
h
d
Bhavana Kushwaha , Yashpal S. Chhonker , Hardik Chandasana , Shagun Krishna ,
Kavita Rawat , Atul Krishna , Mala Singh , Satya N. Sankhwar , Praveen K. Shukla ,
Jagdamba P. Maikhuri , Rabi S. Bhatta , Mohammad I. Siddiqi , Rajkamal Tripathi ,
Gopal Gupta , Vishnu L. Sharma
Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
Molecular & Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
Toxicology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
Microbiology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
Department of Urology, King George Medical University, Lucknow 226003, India
Academy of Scientific and Innovative Research (AcSIR), New Delhi 110001, India
e
f
a
g
f
b
c
,
g
d
e
b
a, h, *
a
b
c
d
e
f
g
h
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 March 2015
Received in revised form
The growing population and health-care burden (due to STIs and HIV) imposes a particular economic
crisis over resource-poor countries. Thus a novel approach as vaginal microbicides emerges as integrated
tool to control both population and anti-STIs/HIV. Our continued efforts in this field led to the synthesis
of fifteen N-alkyl/aryl-4-(3-substituted-3-phenylpropyl) piperazine-1-carbothioamide (12e26) de-
rivatives as topical vaginal microbicides which were evaluated for anti-Trichomonas, spermicidal, anti-
fungal and reverse transcriptase (RT) inhibitory activities. All compounds were also tested for
preliminary safety through cytotoxicity assays against human cervical cell line (HeLa) and the vaginal
flora, Lactobacillus. Docking studies were performed to gain an insight into the binding mode and in-
teractions of the most promising compound 12 [oxo derivative], comprising of reverse transcriptase (RT)
19 June 2015
Accepted 10 July 2015
Available online 13 July 2015
Keywords:
Thiourea
NNRTI
Spermicide
Microbicide
Nonoxynol-9
inhibitory (72.30%), spermicidal (MEC 0.01%), anti-Trichomonas (MIC 46.72
mM) and antifungal (MIC 9.34
e74.8 M) activities, along with its hydroxyl (17) and O-alkylated 4-trifluoromethylphenoxy (22) de-
m
rivative, with similar activities. The stability of compound 12 in simulated vaginal fluid (SVF) and its
preliminary in vivo pharmacokinetics performed in female NZ-rabbits signifies its clinical safety in
comparison to marketed spermicide Nonoxynol-9.
©
2015 Elsevier Masson SAS. All rights reserved.
1
. Introduction
century [1] with ninety percent of growth taking place in developing
countries, which are currently under a lot of pressure for resources
[2]. The situation is further aggravated by the fact that an estimated
97 percent of unsafe abortions occur in developing countries [3],
mostly due to unintended pregnancies. Reproductive health prob-
lems related to pregnancy, childbirth and sexually transmitted in-
fections (STIs) including HIV/AIDS represent nearly one-fifth (18%) of
the global burden of disease (GBD) and have even higher share
among developing countries [4]. The growing health and economic
burdens of sexually transmitted infections (STIs), HIV, and popula-
tion growth in resource-poor countries have been integrated, and
According to the UN Department of Economic and Social Affairs,
the world population is posed to reach 11 billion by the end of this
*
CDRI Communication No. 9028.
*
Corresponding author. Medicinal and Process Chemistry Division, CSIR-Central
Drug Research Institute, Sector 10, Jankipuram Ext., Lucknow 226031, Uttar Pra-
desh, India.
E-mail addresses: vlscdri1@rediffmail.com, vlscdri@gmail.com (V.L. Sharma).
http://dx.doi.org/10.1016/j.ejmech.2015.07.021
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

V. Bala et al. / European Journal of Medicinal Chemistry 101 (2015) 640e650
641
vaginal microbicides come out as an emerging prophylactic
approach. A safe and effective woman controlled vaginal microbicide
is warranted as regular use of themarketed product Nonoxynol-9 (N-
Scheme 1 in which different isothiocyanates (2e6) were reacted
with piperazine (1) in ethanol at room temperature to yield
piperazine-1-carbothioic acid substituted amide (7e11) predomi-
9
) has been shown to damage cervicovaginal epithelium leading to
nantly
which
undergoes
further
reaction
with
3-
increased susceptibility towards STIs/HIV [5,6].
chloropropiophenone in the presence of triethyl amine to yield
N-alkyl/aryl-4-(3-oxo-3-phenylpropyl)-piperazine-1-
Hence, there is an urgent need of novel, safe and effective agents of
fertility control that simultaneously provide protection against STIs
such as the HIV, Trichomonas vaginalis (the most prevalent STI) and
Candida(thecommon opportunisticRTI, reproductive tractinfection).
In our ongoing efforts to target both sperm and Trichomonas simul-
taneously, several non-surfactant dually active molecules [7e9]
including benzenepropanamines [10] and selective serotonin reup-
take inhibitor (SSRI) antidepressants like fluoxetine and paroxetine
carbothioamide (12e16). These propanones were reduced to cor-
responding hydroxy compounds (17e21) with sodium borohydride
in methanol which were condensed with 4-chlorobenzotrifluoride
in dimethyl formamide in presence of sodium hydride to give N-
alkyl/aryl-4-(3-phenyl-3-(4-(trifluoromethyl)phenoxy)propyl)
piperazine-1-carbothioamide (22e26) in good yields.
[11] have already been reported. To further enhance the scope of our
2.2. Biological evaluation
research, it was envisaged to incorporate reverse transcriptase (RT)
inhibition in our new compounds to target HIV as well. Thiourea
scaffold is an established RT inhibitor [12a,12b] and we have recently
reported that dithiocarbamate-thiourea hybrids exhibit RT inhibition
along with spermicidal and anti-Trichomonas activities [13]. In order
to further optimize the RT inhibitory activity, thiourea moiety was
introduced in an already recognized, non-surfactant, dually-active
structural class of compounds i.e., benzenepropanamines (Fig. 1).
N-alkyl/aryl-4-(3-substituted-3-phenylpropyl)piperazine-1-
carbothioamide (12e26) derivatives were designed, synthesized
and evaluated for spermicidal, anti-Trichomonas, RT inhibition and
antifungal activities. Further structure activity relationship (SAR)
was discussed and subsequent safety assessment towards vaginal
flora (Lactobacillus) and cervical epithelium (HeLa cells) have also
been done. To evaluate stability in biological system and systemic
toxicity, the simulated vaginal fluid (SVF) stability and preliminary
pharmacokinetic study in female New Zealand (NZ) rabbits has also
been presented. Furthermore, a docking study was conducted to
find an appropriate association of structural modification with RT-
inhibitory activity.
2.2.1. Anti-Trichomonas activity
All the fifteen synthesized compounds (12e26, Table 1) were
subjected to anti-Trichomonas activity against both metronidazole
(MTZ)-susceptible and -resistant strains. Eleven compounds (except
18e20 and 22) were found active against MTZ-susceptible strain
with MIC 40.62e88.52
mM. Fourteen compounds (except 23) were
found active against MTZ-resistant strain with MIC
118.37e495.04
mM. In comparison, the standard drug MTZ displayed
MIC of 19.71
mM and 292.80 M against MTZ susceptible and
m
resistant strains while the marketed spermicide N-9 exhibited
minimum inhibitory concentration (MIC) of 81.16 M and 121.75
against MTZ-susceptible and resistant strains, respectively.
m
mM
2.2.2. Spermicidal activity
The spermicidal activity of compounds (12e26, Table 1) was
evaluated in comparison to marketed spermicide N-9. Among all
the synthesized compounds of this series, eleven (12e21 and 23)
immobilized 100% human sperms within 30 s at minimum effective
concentration (MEC) 0.01e1%. Four compounds (14, 17, 20, 23) have
shown spermicidal activity comparable to Nonoxynol-9 (MEC
0.05%) while two (12, 16) were found to be five times (MEC 0.01%)
more active than N-9.
2
. Results and discussion
2.1. Chemistry
2.2.3. HIV-1 RT (reverse transcriptase) inhibition
N-alkyl/aryl-4-(3-substituted-3-phenylpropyl)piperazin-1-
Fourteen compounds inhibited RT by 21.14e72.30% at 100 mg/mL
carbothioamides (12e26) have been synthesized according to
concentration. Compound 12 exhibited promising RT inhibitory
Fig. 1. General structure of synthesized Benzenepropanamine-thiourea hybrids.

642
V. Bala et al. / European Journal of Medicinal Chemistry 101 (2015) 640e650
Scheme 1. Reagents and conditions: (a) Ethanol, rt, 3e4 h; (b) 3-chloropropiophenone, triethyl amine, methanol, reflux, 3e4 h; (c) Methanol, NaBH
4
, 2 h; (d) 4-
chlorobenzotrifluoride, NaH, DMF, 6e7 h.
Table 1
RT inhibition, trichomonacidal and spermicidal activities of synthesized compounds (12e26).
Compd no.
Substituent (R)
% RT inhibition
at 100 mg/mL
Spermicidal MEC
mM
0.30
Mean concentration (m
M)a
a
%
Anti-Trichomonas MIC (MTZ susceptible)
Anti-Trichomonas MIC (MTZ resistant)
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Butyl
Phenyl
Cyclohexyl
Benzyl
Phenethyl
Butyl
Phenyl
Cyclohexyl
Benzyl
Phenethyl
Butyl
Phenyl
72.30
49.20
47.73
58.31
50.23
29.15
e
0.01
0.1
46.72 ± 7.76
88.52 ± 14.75
43.34 ± 7.20
85.14 ± 14.24
40.83 ± 6.78
46.44 ± 7.72
e
187.68 ± 31.28
354.10 ± 59.01
174.58 ± 29.09
340.59 ± 56.76
164.04 ± 27.34
186.56 ± 31.09
352.11 ± 58.68
173.61 ± 28.93
338.75 ± 56.45
163.18 ± 27.19
260.96 ± 43.49
e
2.83
1.39
2.72
0.05
0.1
0.01
0.05
0.1
0.26
1.49
2.82
24.32
47.18
45.30
59.62
21.14
24.32
51.03
38.4
1
0.05
0.1
27.70
1.35
2.61
173.13 ± 28.85
169.37 ± 28.22
40.62 ± 13.65
130.48 ± 21.74
50.10 ± 9.13
64.35 ± 10.79
60.91 ± 10.19
47.34 ± 8.64
19.71 ± 5.51
81.16 ± 0.03
e
>1
0.05
>20.87
1.00
Cyclohexyl
Benzyl
Phenethyl
>1
>1
>1
e
>19.80
>19.49
>18.97
e
495.04 ± 82.50
243.19 ± 40.53
118.37 ± 19.72
292.80 ± 48.80
121.75 ± 13.48
e
MTZ
N-9
Nevirapine
e
e
0.05
0.81
99.9
e
e
a
The experiments were carried out in triplicates.
activity at 72.30%. Another nine compounds (13e16, 20e22, 25, 26)
showed moderate activity (38.40e59.62%). The standard non-
nucleoside reverse transcriptase inhibitor (NNRTI), Nevirapine
at 200 mg/mL concentration by cell-viability assay. The compounds
didn't affect the viability of HeLa cells or growth of Lactobacilli
during 24 h of incubation and therefore appeared apparently much
safer for vaginal use in comparison to N-9.
(
NVP) showed 99.6% inhibition. The enzyme assay results were
visualized in combination with anti-Trichomonas, spermicidal
Table 1) and antifungal activities (Table 2).
(
2.3. Structure activity relationship (SAR)
2
.2.4. Antifungal activity
The antifungal activity data showed that five compounds
This study included N-alkyl/aryl-4-(3-substituted-3-phenylpropyl)
piperazin-1-carbothioamides (12e26) and the results of RT inhibition
activity (Table 1) among oxo derivatives (12e16) showed that the
(
12e15 and 25) were active against one or more fungal strains of
the all six strains tested at MIC 4.22e74.8 M, while N-9 was almost
inactive (MIC > 81.2 M) against them. Three compounds (12, 14
and 15) were active against three fungal strains out of six tested at
MIC 4.22e74.8 M. The standard Fluconazole showed antifungal
activity at MIC, 3.26 to >81.2 M (Table 2).
4
m
preference of alkyl/aryl chain at N -carbothioamide was butyl (12,
m
72.30%) > benzyl (15, 58.31%) > phenethyl (16, 50.23%) > phenyl (13,
49.20%) > cyclohexyl (14, 47.73%), while in the hydroxyl derivatives
benzyl (20, 47.18%) and phenethyl (21, 45.30%) were most desirable.
The alkylation of hydroxyl group with 4-benzotrifluoride (22e26)
suggested that butyl (22, 59.62%) and benzyl (25, 51.03%) groups were
most preferred. Oxo substituent at position-3 of propanamine chain
m
m
2
.2.5. Cytotoxicity assay against human cervical cell line (HeLa)
4
and compatibility with vaginal flora (Lactobacillus jenissi)
(12, 72.30%) was most suitable for butyl substituent in thiourea at N of
The compounds synthesized as vaginal microbicides may also
target other vaginal cells, such as the cervico-vaginal epithelia and
Lactobacilli. Hence all the synthesized compounds (12e26) were
evaluated for their safety towards human cervical cell line (HeLa,
Fig. 2a) and compatibility with vaginal flora, Lactobacillus (Fig. 2b)
piperazine followed by 4-trifluoromethylphenoxy (22, 59.62%) and
then hydroxyl (17, 29.15%) substitution. Similar trend was also fol-
lowed by phenyl (13 > 23 > 18) and benzyl (15 > 25 > 20) substitutions
in thiourea at N of piperazine. Whereas with cyclohexyl substituent
oxo group (14, 47.73%) was most preferred followed by hydroxyl (19,
4

V. Bala et al. / European Journal of Medicinal Chemistry 101 (2015) 640e650
643
Table 2
Antimicrobial activity of synthesized compounds (12e26).
Compd no.
Antifungal activity (MIC,
m
M)a
Candida
albicans
Cryptococcus
neofarmans
Sporothrix
schenckii
Trichophyton
mentagrophytes
Aspergillus
fumigates
Candida
parapsilosis
ATCC-22019
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
74.8
>81.2
34.7
9.34
17.6
8.6
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
6.53
9.34
17.6
4.33
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
6.53
67.9
8.47
4.22
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
3.26
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
12.1
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
>81.2
6.07
>81.2
6.53
>81.2
>81.2
>81.2
>81.2
Fluconazole
N-9
>81.2
>81.2
>81.2
a
The experiments were carried out in triplicates.
2
4.32%) and trifluoromethylphenoxy (24, 24.32%) groups. With phe-
piperazine and oxo at position-3 of propanamine chain was most
favorable for RT inhibition (12, 72.30%).
nethyl substituent the order of activity was found to be oxo (16,
5
0.23%) > hydroxyl (21, 45.30%) > trifluoromethylphenoxy (26,
The spermicidal activity data (Table 1) showed that oxo group
(12e16) at position-3 of propanamine chain was again most
4
38.40%). The data predicted that the combination of butyl at N of
desirable
over
hydroxyl
(17e21)
followed
by
tri-
fluoromethylphenoxy group (22e26). The order of preference of
4
substituent at N of piperazine among oxo compounds (12e16) was
butyl (12, MEC 0.01%) ¼ phenethyl (16) > cyclohexyl (14, MEC
0
.05%) > phenyl (13, MEC 0.1%) ¼ benzyl (15) whereas among
compounds with hydroxyl substituent butyl (17) and benzyl (20)
were more preferred at MEC 0.05%. The alkylation of hydroxyl
group with 4-benzotrifluoride (22e26, MEC >1%) resulted into loss
of activity except in phenyl substituted compound (23, MEC 0.05%).
4
It seems that structural combination of butyl thiourea at N posi-
tion of piperazine and oxo group at position-3 of propanamine
chain was most suitable for sperm immobilization (12, MEC 0.01%).
The better activity of the oxo-compounds may be attributed to the
fact that some benzenepropanaminones and acrylophenones have
been reported to be spermicidal [10,14].
The anti-Trichomonas activity results (Table 1) suggested that
oxo (12e16, MIC 40.83e88.52
22e26, MIC 47.34e130.48
substituted-3-phenylpropyl)piperazine-1-carbothioamides
12e26) were preferred over hydroxyl (17e21, MIC 40.62 to
500 M) as they displayed good activity against MTZ susceptible
strain while against resistant strain the order of preference was oxo
12e16) > hydroxyl (17e21) > trifluoromethylphenoxy (22e26).
mM) and trifluoromethylphenoxy
(
mM) groups in N-alkyl/aryl-4-(3-
(
>
m
(
Among oxo compounds (12e16) the descending order of activity
was found to be phenethyl (16), cyclohexyl (14), butyl (12), benzyl
(15) and phenyl (13) against both MTZ susceptible and resistant
strain. Within hydroxyl compounds (17e21) the preferred groups
4
in thiourea at N of piperazine were phenethyl (21) and butyl (17)
against MTZ susceptible and phenethyl (21) and cyclohexyl (19)
against resistant strain. When trifluoromethylphenoxy (22e26)
group was present at position-3 of propanamine chain then phe-
4
nethyl group in thiourea at N of piperazine (26) was most
preferred against both MTZ susceptible and resistant strain. The
comparison of activity of compounds (12e26) against MTZ sus-
ceptible and resistant against MTZ activity suggested that these
compounds were less active than MTZ in susceptible strain while
eight compounds (12, 14, 16, 17, 21, 22, 25, 26) exhibited better
activity against resistant strain. MTZ lost the activity by 15 fold from
Fig. 2. (a) Cytotoxicity of compounds (12e26) towards Human cervical cell lines
HeLa) and (b) Compatibility with normal vaginal flora, Lactobacillus.
(

644
V. Bala et al. / European Journal of Medicinal Chemistry 101 (2015) 640e650
susceptible to resistant strains while these compounds lost activity
by 2e4 times only.
The results of antifungal activity of N-alkyl/aryl-4-(3-
substituted-3-phenylpropyl)piperazine-1-carbothioamide (12e26,
use. The compound 12 was incubated in SVF at a concentration
200 ng/mL for stability study. Samples were removed at 0, 5, 15, 30,
45, and 60 min. The concentration of compound 12 was determined
by LC-MS/MS. The stability data have shown that compound 12 was
stable in SVF up to 1.0 h (Fig. 4). The SVF stability of marketed drug
N-9 was previously reported and found stable upto one hour [20].
This behavior of compound 12 is favorable for its vaginal admin-
istration and supports its efficacy as vaginal microbicide with RT
inhibition potential.
MIC 4.22e74.8
mM; Table 2) showed that the oxo derivatives
(12e16) presented better activity profile than hydroxyl/4-
trifluoromethylphenoxy derivatives (17e26).
The overall activity data showed that oxo derivatives (12e16)
were most preferred of which the most promising compound N-
butyl-4-(3-oxo-3-phenylpropyl) piperazin-1-carbothioamide (12)
exhibited good activity profile than N-9 as it showed RT inhibition
2.5.2. In-vivo pharmacokinetics
(
72.30%), anti-Trichomonas (MIC, 46.72
and MIC, 187.68 M against resistant strain), spermicidal (MEC,
.01%) and antifungal activity (MIC, 9.34e>81.2 M) against four
fungal strains.
m
M against MTZ susceptible
A preliminary vaginal pharmacokinetics study involves moni-
toring drug substance in blood plasma. Absorption into the sys-
temic circulation, and subsequent exposure in blood plasma should
be minimal to diminish systemic adverse effects or minimal toxic
effect. In vivo pharmacokinetic study was carried out after vaginal
administration compound 12 and N-9 at a dose of 33 mg/kg to
m
0
m
2
.4. Docking studies
female NZ-rabbit. The mean peak plasma concentration (Cmax
)
Docking study was performed to gain an insight into the binding
mode and interactions of the most promising compound, N-butyl-4-
3-oxo-3-phenylpropyl) piperazin-1-carbothioamide (12) i.e., oxo
3.66 ± 0.45 ng/mL of N-9 was achieved after vaginal dosing at 1.0 h.
The terminal half-life of plasma concentration of N-9 was
1.59 ± 0.16 h. The mean peak plasma concentration (Cmax)
(
derivative and its hydroxyl (17) and O-alkylated 4-
trifluoromethylphenoxy derivative (22) with HIV-1 RT. This protein
is a dimer and is constituted of two units namely p66 (chain A) and
p51 (chain B). Besides having a catalytic site it also has an allosteric
site which is the binding site of various NNRTIs including Nevirapine
and Delavirdine. It has been suggested by previous studies that these
inhibitors occupy the allosteric site which is formed by hydrophobic
residues [15,16]. Docking of compounds 12,17 and 22 was done with
the aim to explain the binding mode of these compounds into this
hydrophobic pocket. Nevirapine was redocked into the active site of
crystal structure for the validation of docking program used in this
study. The conformation of crystal structure was reproduced with
Autodock4.2. The active site region within the protein structure was
selected on the basis of previously reported structural information.
The most important amino acids involved in the hydrophobic in-
teractions with NNRTIs are Val106,Thr107, Tyr181, Tyr188, Phe227,
Trp229 from p66 subunit and Glu138 (p51 subunit) [17].
The interaction of compound 12 in the hydrophobic pocket has
been shown in Fig. 3. The benzene ring moiety occupies a hydro-
phobic aromatic-rich pocket formed mainly by the side chains of
Pro95.A, Tyr181.A, Tyr188.A and Glu138.B and the piperazine moiety
was oriented towards a cavity formed by Pro226.A, Phe227.A and
Leu234.A. The aliphatic chain attached with thiourea moiety inter-
acted hydrophobically with Val 106.A and Thr107.A. A hydrogen
bond is formed between aliphatic side chain attached with thiourea
group and the main-chain carbonyl oxygen of Lys101.A. A similar
hydrogen bond to the main-chain carbonyl oxygen of Lys101.A has
been observed for several other NNRTIs [18,19]. The compound 17
had similar binding mode as that of compound 12 with HIV-RT.
However the hydrogen bond that was observed between Lys101
and 12 has notbeen formed here. The binding mode of compound 22
depicted that it occupied an extended conformation because it has a
lower binding energy than 12.
4.43 ± 0.622 ng/mL of compound 12 was achieved after vaginal
dosing (Table 4). Compound 12 (Fig. 5) and N-9 both were not
significantly found in the in vivo plasma samples [20].
3. Conclusion
With a view to incorporate RT inhibitory activity into a micro-
bicide, N-alkyl/aryl-4-(3-substituted-3-phenylpropyl)piperazine-1-
carbothioamides (12e26) were designed, synthesized and evalu-
ated for their RT inhibition, spermicidal, anti-Trichomonas and
antimicrobial activity profile to be useful as vaginal microbicidal
spermicide. Five compounds (12e15, 23) in the series have been
identified as microbicidal spermicides with multiple activities. The
oxo derivatives of N-alkyl/aryl-4-(3-substituted-3-phenylpropyl)
piperazine-1-carbothioamides (12e16) were most preferred for
multiple activities. The most promising compound, N-butyl-4-(3-
oxo-3-phenylpropyl) piperazin-1-carbothioamide (12) exhibited
remarkable activity profile with RT inhibition (72.30%), anti-Tricho-
monas (MIC, 46.72
against resistant strain), spermicidal (MEC, 0.01%) and antifungal
activity (MIC, 9.34 e >81.2 M). The high safety against vaginal
mM against MTZ susceptible and MIC, 187.68 mM
m
epithelium (HeLa cells) and compatibility with vaginal flora (Lacto-
bacillus), SVF stability and negligible vaginal absorption supported
its suitability for topical vaginal application. The docking study has
revealed that compound 12 (oxo compound) interacted with HIV-1
RT in a manner similar to the standard drug Nevirapine. Compound
12 has a simple two step synthesis with good yield and can be
synthesized economically on pilot scale. The stability of the struc-
ture indicates that it can easily be formulated into various pre-coital,
vaginal products e.g. cream, gel and film. The SVF stability data
shows that the compound can remain active in vaginal milieu for up
to 60 min. Further in vivo studies would indicate the vaginal ab-
sorption of the compound which would decide its systemic toxicity
(if any). Furthermore, the in vivo studies would also indicate the
activity of this compound in preventing fertility and infection in
experimental animals after vaginal application before mating.
However these studies will be initiated shortly and the results
would be communicated later to a suitable biological journal. It may
be concluded that hybridization of benzenepropanamine and thio-
urea moiety provide novel lead with multiple activities including RT
inhibition. A further lead optimization (under progress) may result
into effective vaginal microbicides having spermicidal, anti-Tricho-
monas, antifungal and anti-HIV potential with enhanced safety to
cervico-vaginal cells in comparison to Nonoxynol-9.
The docking energies of compounds 12, 17 and 22 as predicted
by Auto dock have been given in Table 3. The docking energy of
these compounds and Nevirapine correlated well with their RT
inhibition.
2.5. Simulated vaginal fluid (SVF) stability and vaginal
pharmacokinetics of compound 12
2.5.1. Simulated vaginal fluid (SVF) stability
Simulated vaginal fluid (pH 4.2) was considered as a perfect
medium to determine the stability of molecules for intra-vaginal

V. Bala et al. / European Journal of Medicinal Chemistry 101 (2015) 640e650
645
Fig. 3. Interaction of Compound 12 (Pink), compound 17 (Magenta) and compound 22 (Purple) with HIV-RT. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
4
. Experimental
internal reference (chemical shifts d in ppm, J in Hz.). Elemental
analyses were performed on Carlo Erba EA-1108 micro analyzer/
Vario EL-III C, H, N analyzer. All compounds were analyzed of C, H, N
and the results obtained were within ±0.4% of calculated values.
The reaction progress was routinely monitored by thin layer
4
.1. General procedure
All reagents and solvents were commercial quality and were
used without further purification. Melting points were determined
in open capillary tubes on an electrically heated block and are
ꢀ
1
uncorrected. IR spectra (
nmax in cm ) of the compounds were
1
recorded on Perkin Elmer's FT-IR RX1 PC spectrophotometer.
H
NMR spectra were recorded on Bruker Supercon Magnet Avance
DPX-200/DRX-300 spectrometers (operating at 400 and 100 MHz
respectively for 1H and C) in deuterated solvents with TMS as
13
Table 3
Docking energies of compounds 12, 17 and 22 predicted by Auto dock.
Compound
Docking energy (kcal/mol)
% RT inhibition
Nevirapine
ꢀ8.73
ꢀ8.22
ꢀ7.57
ꢀ7.84
99.60
72.30
29.15
59.62
1
1
2
2
7
2
Fig. 4. % Conc. Remaining vs Time plot for SVF stability of compound 12.

646
V. Bala et al. / European Journal of Medicinal Chemistry 101 (2015) 640e650
ꢂ
(cm ): 3389, 2927, 1386, 1208; 1H NMR
ꢀ1
Table 4
82e84 C; IR (KBr)
n
Pharmacokinetic profile of compound 12 and Nonoxynol-9 after vaginal adminis-
tration in NZ rabbit.
(
400 MHz, CDCl ): d 2.91 (4H, bs), 2.13e2.09 (4H, m), 2.04 (1H, s),
.74e1.62 (4H, m), 1.46e1.40 (2H, m),1.38e1.37 (4H, m); ESI-MS: m/
3
1
þ
Parameters
max (ng/mL)
Compound 12
4.43 ± 0.622
N-9
z 228 (MþH ); Elemental analysis (%) for C11
21 3
H N S: Calcd.: C,
C
3.66 ± 0.45
58.11; H, 9.31; N, 18.48; Found, C, 57.94; H, 9.45; N, 18.62.
AUC0-∞ (hr ng/mL)
14.65 ± 0.58
0.75 ± 0.0
12.89 ± 4.13
1.0 ± 0.0
T
max
4
.1.4. Piperazine-1-carbothioic acid benzylamide (10)
The title compound was synthesized from piperazine (1) and
V
d
/F (L/kg)
Cl/F (L/h/kg)
1/2 (h)
15,049.45 ± 677.11
2317.121 ± 90.33
4.67 ± 2.27
7931.32 ± 3611.91
3976.12 ± 2439.78
1.59 ± 0.16
t
benzyl isothiocyanate (5) in 77% yield as yellow solid; mp
ꢂ
ꢀ1
1
73e75 C; IR (KBr)
n (cm ): 3433, 3019, 1323, 1216; H NMR
Abbreviation: Cmax: Maximum concentration achieved in plasma, AUC: area under
the curve from 0 to ∞ hr, V : volume of distribution, Cl: clearance, t1/2: terminal half
life, MRT: Mean residence time (Each value represents mean ± SD, n ¼ 3).
(400 MHz, CDCl ):
3
d
7.36e7.26 (5H, m), 4.88e4.87 (2H, m), 3.79 (4H,
d
þ
t, J ¼ 5.1 Hz), 2.91 (4H, t, J ¼ 5.1 Hz); ESI-MS: m/z 235 (MþH );
17 3
Elemental analysis (%) for C12H N S: Calcd.: C, 61.24; H, 7.28; N,
17.85; Found, C, 61.02; H, 7.35; N, 18.01.
4.1.5. Piperazine-1-carbothioic acid phenethylamide (11)
The title compound was synthesized from piperazine (1) and
phenethyl isothiocyanate (6) in 69% yield as white solid; mp
ꢂ
ꢀ1
1
6
9e71 C; IR (KBr)
n (cm ): 3435, 2958, 1325, 1217; H NMR
(
400 MHz, CDCl ): 7.34e7.20 (5H, m), 3.97e3.92 (2H, m), 3.69 (4H,
3
d
t, J ¼ 3.8 Hz), 2.96 (2H, t, J ¼ 5.1 Hz), 2.86 (4H, t, J ¼ 3.8 Hz), 1.69 (1H,
þ
s); ESI-MS: m/z 249 (MþH ); Elemental analysis (%) for C13
19 3
H N S:
Calcd.: 62.61; H, 7.68; N, 16.85; Found, C, 62.46; H, 7.52; N, 16.96.
4.1.6. N-Butyl-4-(3-oxo-3-phenylpropyl)piperazine-1-
carbothioamide (12)
The mixture of piperazine-1-carbothioic acid butylamide (7, 1 g,
4
.98 mmol), triethyl amine (0.75 g, 7.46 mmol) and methanol
Fig. 5. Pharmacokinetic profile of compound 12 in rabbit after vaginal administration.
ꢂ
(20 mL) was heated to 60e65
C.
A
solution of 3-
chloropropiophenone (0.76 g, 4.48 mmol) in methanol was added
dropwise in 2 h. The reaction mixture was further stirred under
reflux for 2 h. After completion of the reaction, methanol was
distilled off, water (5 mL) was added and extracted with dichloro
methane (5 mL ꢁ 3). Combined organic layers were washed with
water (5 mL ꢁ 3), dried over anhydrous sodium sulphate and
concentrated under reduced pressure. The crude material was
purified by column chromatography over silica gel (100e200 mesh)
with ethyl acetate/hexane (1:4) as an eluent to yield the title
chromatography (TLC) on pre-coated alumina/silica gel plates
(
Aldrich).
4.1.1. Piperazine-1-carbothioic acid butylamide (7)
To the mixture of piperazine (1, 2 g, 23.26 mmol) in ethanol
(
25 mL) was added butyl isothiocyanate (2, 0.95 g, 11.63 mmol)
under stirring at rt for 3 h. Ethanol was distilled off under reduced
pressure and ethyl acetate (50 mL) was added. The ethyl acetate
layer was washed with water (5 mL ꢁ 3) and dried over sodium
sulfate. Sodium sulfate was filtered off and the filtrate was
concentrated under reduced pressure. Crude was purified by col-
umn chromatography over silica gel (100e200 mesh) with chlo-
roform/methanol as an eluent to get the title compound 7 (1.8 g,
ꢀ
1
compound 12 as yellow liquid (1.45 g, 87%); IR (neat)
3
d
n
(cm ):
383, 2956, 1681, 1383, 1216; H NMR (400 MHz, CDCl ):
7.97e7.95 (2H, m), 7.60e7.56 (1H, m), 7.50e7.46 (2H, m), 3.81(4H,
1
3
t, J ¼ 5.1 Hz), 3.68e3.63 (2H, m), 3.20 (2H, t, J ¼ 7.2 Hz), 2.88 (2H, t,
J ¼ 7.2 Hz), 2.56 (4H, t, J ¼ 5.1 Hz), 1.64e1.57 (2H, m), 1.41e1.35 (2H,
ꢀ
1
1
1
3
7
7%) as yellow liquid; IR (neat)
NMR (400 MHz, CDCl ): 3.80e3.77 (4H, m), 3.69e3.64 (2H, m),
.94e2.91 (4H, m), 1.64e1.57 (2H, m), 1.41e1.36 (2H, m), 0.95 (3H, t,
n (cm ): 3440, 2963, 1327, 1216; H
m), 0.95 (3H, t, J ¼ 7.3 Hz); C (100 MHz, CDCl
3
): d 198.9, 182.1,
3
d
1
36.8, 128.7, 128, 52.8, 52.6, 47.2, 46.1, 36.1, 31.4, 20.2, 14.2, 13.9;
HRMS (ESI): m/z calculated for C18
34.1953. Found: 334.1922. Elemental analysis (%) for C18H N
þ
þ
2
H
27
N
3
OS þ H (MþH ):
þ
J ¼ 7.3 Hz); ESI-MS: m/z 202 (MþH ); Elemental analysis (%) for
S: Calcd.: C, 34.69; H, 7.51; N, 14.87; Found, C, 34.73; H,
.39; N, 14.96.
3
27 3
OS:
9 19 3
C H N
7
Calcd.: C, 64.83; H, 8.16; N, 12.60; Found, C, 64.72; H, 7.98; N, 12.46.
The compounds 13e16 were prepared using a procedure similar
to described for compound 12.
The compounds 8e11 were prepared using a procedure similar
to described for compound 7.
4
.1.7. N-Phenyl-4-(3-oxo-3-phenylpropyl)piperazine-1-
4
.1.2. Piperazine-1-carbothioic acid phenylamide (8)
The title compound was synthesized from piperazine (1) and
carbothioamide (13)
The title compound was synthesized from piperazine-1-
carbothioic acid phenylamide (8) and 3-chloropropiophenone in
phenyl isothiocyanate (3) in 67% yield as fluffy white solid; mp:
6
ꢂ
ꢀ1
1
ꢀ1
6e68 C; IR (KBr)
n
(cm ): 3400, 3019, 1342, 1215; H NMR
ꢂ
7
3
d
4% yield as light yellow solid; mp 86e88 C; IR (KBr)
n
(cm ):
NMR (400 MHz, CDCl ):
8.00e7.93 (4H, m), 7.60e7.57 (2H, m), 7.50e7.46 (3H, m),
.33e7.32 (1H, m), 3.94e3.92 (2H, m), 3.25e3.17 (6H, m), 2.86e2.80
(
400 MHz, CDCl ):
3
d
7.34e7.28 (4H, m), 7.15e7.11 (1H, m), 3.89 (4H,
1
394, 3019, 1682, 1313, 1216;
H
3
þ
t, J ¼ 4.9 Hz), 2.90 (4H, t, J ¼ 4.9 Hz); ESI-MS: m/z 222 (MþH );
15 3
Elemental analysis (%) for C11H N S: Calcd.: C, 59.69; H, 6.83; N,
7
18.99; Found, C, 59.66; H, 7.03; N, 18.83.
13
(4H, m); C (100 MHz, CDCl
3
þ DMSO):
d 197.4, 180.5, 139.7, 139.4,
135.4, 131.6, 127.2, 126.8, 126.7, 126.5, 51.5, 51.4, 51.2, 46.8, 34.6,
þ
4.1.3. Piperazine-1-carbothioic acid cyclohexylamide (9)
34.5; ESI-MS: m/z 354 (MþH ); Elemental analysis (%) for
OS: Calcd.: C, 67.96; H, 6.56; N, 11.89; Found, C, 68.01; H,
6.66; N, 11.93.
The title compound was synthesized from piperazine (1) and
20 23 3
C H N
cyclohexyl isothiocyanate (4) in 73% yield as yellow solid; mp

V. Bala et al. / European Journal of Medicinal Chemistry 101 (2015) 640e650
647
4.1.8. N-Cyclohexyl-4-(3-oxo-3-phenylpropyl)piperazine-1-
18 29 3
C H N OS: Calcd.: C, 64.44; H, 8.71; N, 12.52; Found, C, 64.30; H,
carbothioamide (14)
8.89; N, 12.38.
The title compound was synthesized from piperazine-1-
carbothioic acid cyclohexylamide (9) and 3-chloropropiophenone
4.1.12. N-Phenyl-4-(3-hydroxy-3-phenylpropyl)piperazine-1-
carbothioamide (18)
The title compound was synthesized from 4-(3-oxo-3-
phenylpropyl)-N-phenylpiperazine-1-carbothioamide (13) in 92%
ꢀ
1
in 89% yield as colorless liquid; IR (neat)
n
(cm ): 3429, 2928,
7.97e7.94 (2H, m),
1
1683, 1328, 1216; H NMR (400 MHz, CDCl
3
):
d
7
3
2
.60e7.55 (1H, m), 7.49e7.46 (2H, m), 3.79 (4H, t, J ¼ 5.2 Hz),
ꢀ
1
.20e3.17 (3H, m), 2.87 (2H, t, J ¼ 7.3 Hz), 2.55 (4H, t, J ¼ 5.1 Hz),
.13e2.09 (2H, m), 1.73e1.62 (4H, m), 1.44e1.39 (2H, m), 1.20e1.15
yield as colorless liquid; IR (neat)
n
(cm ): 3678, 3391, 2953, 1316,
7.36e7.31 (7H, m), 7.29e7.21
(3H, m), 4.87e4.84 (1H, m), 2.70e2.53 (8H, m), 1.85 (4H, t,
1
1216; H NMR (400 MHz, CDCl
3
):
d
(
1
2
2H, m); 13C (100 MHz, CDCl
3
):
28.6, 128.3, 128.1, 128.0, 54.3, 53.1, 52.8, 47.1, 44.9, 38.7, 36.2, 33.1,
5.6, 25.0; HRMS (ESI): m/z calculated for C20
d 199.1, 180.9, 136.9, 133.2, 128.7,
13
J ¼ 5.5 Hz); C (100 MHz, CDCl
3
):
d 186.1, 144.5, 135.8, 129.5, 128.7,
128.4, 127.7, 127.3, 125.6, 75.3, 56.6, 52.7, 50.1, 42.3, 34.1; ESI-MS: m/
þ
29
H N
3
OS þ H
þ
þ
(
C
MþH ): 360.2110. Found: 360.2098. Elemental analysis (%) for
z 355 (MþH ); Elemental analysis (%) for C20
25 3
H N OS: Calcd.: C,
H
20 29
N
3
OS: Calcd.: C, 66.81; H, 8.13; N, 11.69; Found, C, 66.67; H,
67.57; H, 7.09; N, 11.82; Found, C, 67.81; H, 6.83; N, 11.97.
7.84; N, 11.53.
4.1.13. N-Cyclohexyl-4-(3-hydroxy-3-phenylpropyl)piperazine-1-
4.1.9. N-Benzyl-4-(3-oxo-3-phenylpropyl)piperazine-1-
carbothioamide (19)
carbothioamide (15)
The title compound was synthesized from piperazine-1-
carbothioic acid benzylamide (10) and 3-chloropropiophenone in
The title compound was synthesized from N-cyclohexyl-4-(3-
oxo-3-phenylpropyl)piperazine-1-carbothioamide (14) in 97%
ꢂ
ꢀ1
yield as light yellow solid; mp 92e94 C; IR (KBr)
n
d
(cm ): 3352,
7.36e7.35 (5H,
ꢂ
ꢀ1
1
9
2
0% yield as yellow solid; mp 110e112 C; IR (KBr)
971, 1682, 1325, 1216; H NMR (400 MHz, CDCl
n
d
(cm ): 3433,
3335, 2934, 1344, 1217; H NMR (400 MHz, CDCl
3
):
1
3
):
7.96e7.95 (2H,
m), 5.42 (1H, s), 4.95e4.92 (1H, m), 3.86e3.84 (4H, m), 2.73e2.51
(7H, m), 2.12e2.07 (2H, m), 1.92e1.89 (2H, m), 1.74e1.63 (4H, m),
m), 7.60e7.56 (1H, m), 7.49e7.46 (2H, m), 7.36e7.33 (5H, m),
.91e4.89 (2H, m), 3.90e3.89 (4H, m), 3.22e3.19 (2H, m), 2.88 (2H,
13
4
3
1.26e1.12 (4H, m); C (100 MHz, CDCl ): d 181.1, 144.8, 144.5, 128.4,
13
t, J ¼ 8.0 Hz), 2.58e2.57 (4H, m); C (100 MHz, CDCl
3
þ DMSO):
198.9, 182.2, 138.0, 136.9, 133.4, 128.9, 128.8, 128.2, 128.1, 127.8,
2.8, 52.7, 50.5, 47.5, 36.2; HRMS (ESI): m/z calculated for
128.3, 127.2, 125.6, 73.4, 56.9, 56.8, 54.5, 53.1, 52.6, 47.2, 33.9, 33.2,
þ
d
31.0, 25.7, 25.0; HRMS (ESI): m/z calculated for C20
31
H N
3
OS þ H
þ
5
C
(MþH ): 362.2266. Found: 362.2252. Elemental analysis (%) for
þ
þ
21
25
H N
3
OS þ H (MþH ): 368.1797. Found: 368.1792. Elemental
OS: Calcd.: C, 68.63; H, 6.86; N, 11.43;
Found, C, 68.52; H, 6.65; N, 11.64.
C
20
H
31
N
3
OS: Calcd.: C, 66.44; H, 8.64; N, 11.62; Found, C, 66.36; H,
25 3
analysis (%) for C21H N
8.72; N, 11.57.
4.1.14. N-Benzyl-4-(3-hydroxy-3-phenylpropyl)piperazine-1-
4.1.10. N-Phenethyl-4-(3-oxo-3-phenylpropyl)piperazine-1-
carbothioamide (20)
carbothioamide (16)
The title compound was synthesized from piperazine-1-
carbothioic acid phenethylamide (11) and 3-chloropropiophenone
The title compound was synthesized from N-benzyl-4-(3-oxo-3-
phenylpropyl)piperazine-1-carbothioamide (15) in 96% yield as
ꢂ
ꢀ1
white solid; mp 106e108 C; IR (KBr)
n
(cm ): 3780, 3431, 2957,
7.35e7.26 (10H, m), 5.69
ꢂ
ꢀ1
1
in 76% yield as yellow solid; mp 128e130 C; IR (KBr)
n
(cm ):
):
1327, 1216; H NMR (400 MHz, CDCl
3
):
d
1
3
429, 2928, 1683, 1328, 1216; H NMR (400 MHz, CDCl
3
(1H, s), 4.93 (1H, t, J ¼ 5.8 Hz), 4.87e4.86 (2H, m), 3.87 (4H, t,
13
d
7.96e7.94 (2H, d, J ¼ 7.76 Hz), 7.58 (1H, t, J ¼ 7.3 Hz), 7.49e7.45
J ¼ 5.0 Hz), 2.73e2.50 (6H, m), 1.91e1.87 (2H, m); C (100 MHz,
CDCl ): 182.4, 144.5, 137.9, 128.9, 128.4, 128.2, 127.9, 127.2, 125.6,
75.4, 56.8, 52.7, 50.5, 47.4, 33.9; HRMS (ESI): m/z calculated for
(
2H, m), 7.33e7.30 (2H, m), 7.25e7.20 (3H, m), 3.96e3.91 (2H, m),
3
d
3
2
d
.71 (4H, t, J ¼ 4.9 Hz), 3.17 (2H, t, J ¼ 7.1 Hz), 2.95 (2H, t, J ¼ 6.8 Hz),
13
þ
þ
.85 (2H, t, J ¼ 7.2 Hz), 2.51 (4H, t, J ¼ 5.0 Hz); C (100 MHz, CDCl
3
):
C
21
H N
27 3
OS þ H (MþH ): 370.1953. Found: 370.1916. Elemental
198.0, 182.0, 138.9, 128.8, 128.7, 126.6, 48.5, 47.2, 47.0, 46.9, 45.6,
analysis (%) for C21 OS: Calcd.: C, 68.26; H, 7.36; N, 11.37;
27 3
H N
þ
4
5.6, 45.0, 35.2; ESI-MS: m/z 382 (MþH ); Elemental analysis (%)
Found, C, 68.12; H, 7.25; N, 11.45.
27 3
for C22H N OS: Calcd.: C, 69.26; H, 7.13; N, 11.01; Found, C, 69.31;
H, 7.03; N, 11.19.
4.1.15. N-Phenethyl-4-(3-hydroxy-3-phenylpropyl)piperazine-1-
carbothioamide (21)
4
.1.11. N-Butyl-4-(3-hydroxy-3-phenylpropyl)piperazine-1-
The title compound was synthesized from N-phenethyl-4-(3-
oxo-3-phenylpropyl)piperazine-1-carbothioamide (16) in 89%
carbothioamide (17)
ꢂ
ꢀ1
To the solution of N-butyl-4-(3-oxo-3-phenylpropyl)piperazine-
-carbothioamide (12, 1 g, 3 mmol) in methanol was added sodium
yield as white solid; mp 88e90 C; IR (KBr)
n
(cm ): 3486, 3433,
7.36e7.31 (6H, m),
1
1
3021, 1330, 1216; H NMR (400 MHz, CDCl
3
):
d
borohydride (0.22 g, 6 mmol) in small portions within 15e20 min
under stirring at 0e5 C. It was further stirred at rt for 2 h. After
completion of the reaction, methanol was distilled off, water
7.24e7.20 (4H, m), 4.93 (1H, t, J ¼ 5.7 Hz), 3.97e3.92 (2H, m), 3.77
ꢂ
13
(5H, bs), 2.96 (2H, t, J ¼ 6.7 Hz), 2.72e2.46 (8H, m); C (100 MHz,
3
CDCl ): d 182.2, 144.5, 139.0, 128.9, 128.8, 128.4, 127.3, 126.8, 125.6,
(
(
(
15 mL) was added and extracted with dichloromethane
15 mL ꢁ 3). Combined organic layers were washed with water
5 mL ꢁ 3), dried over sodium sulphate and concentrated under
75.4, 56.8, 52.6, 47.2, 47.0, 35.2, 33.9; HRMS (ESI): m/z calculated for
þ
þ
C
22
H
29
N
3
OS þ H (MþH ): 384.2110. Found: 384.2096. Elemental
analysis (%) for C22 OS: Calcd.: C, 68.89; H, 7.62; N, 10.96;
29 3
H N
reduced pressure to yield the title compound 17 as white solid
Found, C, 68.72; H, 7.56; N, 11.14.
ꢂ
ꢀ1
(
0.98 g, 98%); mp 112e114 C; IR (KBr)
n
(cm ): 3357, 2957, 1336,
1
1
216, 668; H NMR (400 MHz, CDCl
3
):
d
7.36e7.33 (5H, m), 5.50 (1H,
4.1.16. Butyl 4-(3-phenyl-3-(4-(trifluoromethyl)phenoxy)propyl)
piperazine-1-carbothioamide (22)
s), 4.95 (1H, t, J ¼ 5.7 Hz), 3.86 (4H, t, J ¼ 4.9 Hz), 3.69e3.64 (2H, m),
2
(
1
2
.76e2.51 (6H, m), 1.92e1.88 (2H, m), 1.65e1.57 (2H, m), 1.42e1.36
A solution of butyl 4-(3-hydroxy-3-phenylpropyl)piperazine-1-
carbothioamide (17, 0.5 g, 1.04 mmol) in anhydrous dry DMF
(5 mL) was added into a suspension of sodium hydride (0.051 g,
1
3
2H, m), 0.95 (3H, t, J ¼ 7.3 Hz); C (100 MHz, CDCl
3
):
d 182.4, 144.5,
28.4, 127.3, 125.6, 75.4, 59.0, 56.9, 52.7, 47.2, 46.2, 45.1, 33.9, 31.4,
9.8, 20.3, 14.0; HRMS (ESI): m/z calculated for C18
þ
ꢂ
H
29
N
3
OS þ H
2.08 mmol, 60% in wax) in dry DMF (5 mL) at 0e5 C in 30 min.
þ
(
MþH ): 336.2110. Found: 336.2098. Elemental analysis (%) for
Reaction mixture was further stirred at rt for 30 min, heated at

648
V. Bala et al. / European Journal of Medicinal Chemistry 101 (2015) 640e650
ꢂ
n H
(cm ): 3431, 2962, 1327, 1216; 1
ꢀ1
8
0e85 C for 2 h. The reaction mixture was allowed to cool to rt and
yield as yellow oil; IR (neat)
NMR (400 MHz, CDCl ): 7.36e7.30 (6H, m), 7.24e7.20 (6H, m),
a solution of 4-chloro benzotrifluoride (0.35 g, 1.9 mmol) in dry
3
d
DMF (3 mL) was added drop wise. The reaction mixture was stirred
at 110e115 C for 3 h, poured over ice and it was extracted with
6.22e6.20 (2H, m), 4.93 (1H, t, J ¼ 5.7 Hz), 3.65e3.62 (2H, m), 3.47
ꢂ
13
(4H, s), 2.84e2.63 (2H, m), 2.33e2.12 (8H, m); C (100 MHz,
chloroform (20 mL ꢁ 2). The organic layer was washed with brine
CDCl
3
):
d
185.6, 161.2, 144.5, 138.1, 128.5, 127.3, 125.6, 123.6, 119.9,
þ
(
5 mL ꢁ 5) followed by water (5 mL) and dried over sodium sulfate.
87.1, 58.2, 52.7, 47.1, 36.9, 31.6; ESI-MS: m/z 528 [MþH ]; Elemental
Sodium sulfate was filtered off and concentration of the filtrate
under reduced pressure gave a residue which was purified by col-
umn chromatography (silica gel, 100e200 mesh, 10% meth-
32 3 3
analysis calculated (%) for C29H F N OS: Calcd.: C, 66.01; H, 6.11; N,
7.96; Found: C, 65.83; H, 6.34; N, 8.14.
4.2. Biological materials and methods
4.2.1. Spermicidal activity
anoleCHCl
3
) to give 17 as yellow liquid (yield 62%); IR (neat)
n
):
ꢀ
1
1
(
cm ): 3430, 3019, 1351, 1216; H NMR (400 MHz, CDCl
3
d
8.21e8.18 (2H, m), 7.60e7.56 (5H, m), 7.16 (2H, t, J ¼ 5.1 Hz),
5
.21e5.18 (1H, m), 3.48e3.46 (4H, m), 3.35e3.33 (2H, m), 2.96 (2H,
Spermicidal activity was determined by MEC i.e., the minimum
effective spermicidal concentration as previously described in
standard procedure [21]. Briefly, the test compounds were dis-
solved in a minimum volume of DMSO and diluted with physio-
logical saline (0.85% NaCl in distilled water) to make a 1.0% test
solution. 0.05 mL of liquefied human semen was added to 0.25 mL
of test solution and vortexed for 10 s at low speed. A drop of the
mixture was then placed on a microscope slide, covered with a
cover glass and examined under a phase contrast microscope in five
fields of vision. The percentage of motile spermatozoa was deter-
mined by visual scoring in the next 60 s and recorded (Table 1). The
concentration of compound capable of immobilizing 100% cells and
keeping them immotile even after dilution with 1.0 mL nutrient
t, J ¼ 5.1 Hz), 2.36 (6H, t, J ¼ 5.1 Hz), 1.64e1.57 (2H, m), 1.41e1.35
13
(
2H, m), 0.97e0.93 (3H, m); C (100 MHz, CDCl
3
): d 182.5, 162.6,
1
44.5, 128.2, 127.3, 125.4, 123.4, 121.5, 114.5, 83.9, 59.0, 56.8, 52.7,
þ
4
7.2, 46.2, 33.9, 31.4, 20.3, 13.9; ESI-MS: m/z 480 [MþH ];
Elemental analysis calculated (%) for C25 OS: Calcd.: C,
2.61; H, 6.73; N, 8.76; Found: C, 62.42; H, 6.49; N, 8.63.
32 3 3
H F N
6
4.1.17. Phenyl 4-(3-phenyl-3-(4-(trifluoromethyl)phenoxy)propyl)
piperazine-1-carbothioamide (23)
The title compound was synthesized from phenyl 4-(3-hydroxy-
3
-phenylpropyl)piperazine-1-carbothioamide (18) in 59% yield as
ꢀ
1
1
yellow oil; IR (neat)
n
(cm ): 3434, 3020, 1325, 1216; H NMR
ꢂ
(
400 MHz, CDCl
3
):
d
8.30e8.28 (2H, m), 7.79e7.76 (6H, m),
medium (Hanks Balanced Salt Solution) and incubating at 37 C for
7
.56e7.55 (3H, m), 7.18e7.15 (2H, m), 6.36 (1H, t, J ¼ 5.1 Hz), 4.60
30 min in a CO
reference control.
2
incubator was recorded as MEC. N-9 was used as
(
1H, t, J ¼ 7.2 Hz), 3.51e3.48 (4H, m), 3.30e3.26 (2H, m), 2.66 (4H, t,
1
3
J ¼ 5.1 Hz), 2.17 (2H, t, J ¼ 5.0 Hz); C (100 MHz, CDCl
3
): d 186.0,
161.5, 144.5, 135.9, 129.5, 128.7, 128.5, 127.7, 127.3, 125.6, 123.6,
4.2.2. Anti-Trichomonas activity
1
22.1, 113.1, 75.7, 56.7, 52.7, 49.7, 42.4, 34.1, 31.0; ESI-MS: m/z 500
In vitro drug susceptibility assays were carried out using the
standard procedure [22]. Stock solutions (100.0 mg/mL) of test
compounds were prepared in DMSO. These stock solutions were
serially diluted with TYM medium to obtain concentration up to
þ
[MþH ]; Elemental analysis calculated (%) for C27
H
28
3
F N
3
OS:
Calcd.: C, 64.91; H, 5.65; N, 8.41; Found: C, 64.84; H, 5.48; N, 8.29.
4
.1.18. Cyclohexyl 4-(3-phenyl-3-(4-(trifluoromethyl)phenoxy)
0.1 mg/mL in 48-well plates. DMSO/TYM was used as vehicle in
4
propyl)-pipera-zine-1-carbothioamide (24)
The title compound was synthesized from cyclohexyl 4-(3-
hydroxy-3-phenylpropyl)piperazine-1-carbothioamide (19) in 62%
control wells. Parasites (5 ꢁ 10 trophozoites/L) were added to
ꢂ
these wells and incubated anaerobically at 37 C. Cells were
checked for viability at different time intervals from 3 to 48 h under
the microscope at 200ꢁ magnification. Viability of the cells was
determined by trypan blue exclusion assay. Minimum concentra-
tion of the test agent at which all cells were found dead in 48 h was
considered as its MIC. The experiment was repeated three times to
confirm the MIC.
ꢀ1
1
yield as yellow oil; IR (neat)
NMR (400 MHz, CDCl ):
7.39e7.29 (9H, m), 5.40 (1H, t, J ¼ 4.8 Hz),
.80e3.93 (8H, m), 3.51e3.48 (4H, m), 1.79e1.62 (4H, m), 1.45e1.37
7H, m); 13C (100 MHz, CDCl
): 186.2, 159.8, 140.6, 123.8, 121.8,
n (cm ): 3458, 3018, 1312, 1215; H
3
d
4
(
3
d
118.5, 111.5, 75.4, 58.3, 53.4, 50.9, 50.5, 44.6, 29.1, 20.8, 14.1; ESI-MS:
þ
m/z 506 [MþH ]; Elemental analysis calculated (%) for
C
6
27
H F
34 3
N
3
OS: Calcd.: C, 64.13; H, 6.78; N, 8.31; Found: C, 64.32; H,
4.2.3. RT assay
.54; N, 8.39.
The anti HIV-1 RT activity of synthesized compounds was
measured by using non radioactive colorimetric ELISA RT Assay kit
(Roche) [23], and the procedure for assaying RT inhibition was
performed as described elsewhere [24a,24b]. The detection and
quantification of synthesized DNA as a parameter for RT activity
that incorporate digoxigenin and biotin-labeled dUTP in the DNA
strand. The synthesized DNA strand is separated from free biotin
labeled dUTP by streptovidin coated plates. The peroxidase conju-
gated anti DIG-POD antibodies bind to the digoxigenin-labeled DNA
that is incorporated in synthesized DNA. Further, the peroxidase
substrate ABTS is added. The peroxidase enzyme catalyzes the
cleavage of the substrate, producing a colored reaction product.
4
.1.19. Benzyl 4-(3-phenyl-3-(4-(trifluoromethyl)phenoxy)propyl)
piperazine-1-carbothioamide (25)
The title compound was synthesized from benzyl 4-(3-hydroxy-
3
-phenylpropyl)piperazine-1-carbothioamide (20) in 57% yield as
ꢀ
1
1
yellow oil; IR (neat)
n
(cm ): 3431, 2839, 1328, 1235; H NMR
(
400 MHz, CDCl
3
):
d
7.87e7.84 (4H, m), 7.65e7.60 (4H, m),
7
.54e7.49 (4H, m), 6.69e6.63 (2H, m), 4.93 (1H, t, J ¼ 6.8 Hz), 4.46
(
2H, s), 3.84e3.78 (4H, m), 2.79e2.69 (4H, m), 2.04 (4H, t,
13
J ¼ 9.6 Hz); C (100 MHz, CDCl
3
): d 184.3, 160.2, 142.2, 135.9, 129.4,
128.6, 127.5, 123.9, 122.2, 111.1, 84.0, 57.9, 53.5, 51.2, 49.9, 42.1, 29.7,
þ
2
8.0; ESI-MS: m/z 514 [MþH ]; Elemental analysis calculated (%)
Briefly, the reaction mixture consists of 20 mL each of template/
primer complex with dNTPs, reverse transcriptase (RT) enzyme in
for C28
H
30
F
3
N
3
OS: Calcd.: C, 65.48; H, 5.89; N, 8.18; Found: C, 65.33;
H, 6.04; N, 8.42.
the lysis buffer with or without inhibitors to make a total volume of
ꢂ
60
mL was incubated at 37 C for 1 h. As a negative control, 20
mL
4
.1.20. Phenethyl 4-(3-phenyl-3-(4-(trifluoromethyl)phenoxy)
lysis buffers without RT were used. The activity of compounds was
measured at 100 g/mL and Nevirapine was used as a positive
propyl)-piperazine-1-carbothioamide (26)
m
The title compound was synthesized from phenethyl 4-(3-
hydroxy-3-phenylpropyl)piperazine-1-carbothioamide (21) in 61%
control. The reaction mixture was transferred in streptavidine-
coated micro-titre plate (MTP) and incubated for 1 h at 37 C.
ꢂ

V. Bala et al. / European Journal of Medicinal Chemistry 101 (2015) 640e650
649
After incubation, plates were washed 5 times with 250
buffer to remove the unbound dNTPs and 200
m
L washing
was performed on an API-4000-Q-Trap mass spectrometer from
Applied Biosystems (Foster City, CA) equipped UFLC from Shimadzu
mL of anti-
digoxigenin-peroxidase (Anti-DIG-POD, 200 mU/mL) diluted in
conjugate dilution buffer was added. After 1 h, plates were washed
again, 200 mL ABTS substrate solution/well was added and incu-
bated at room temperature until sufficient color was developed.
Optical Density (OD) was measured by ELISA reader at 405 nm
Co. (Columbia, MD). A Thermo Accucore C18 (150 ꢁ 4.6 mm, 5
mm)
column was used for separation of compound 12, N-9 and internal
standard (IS) phenacetin. The system was calibrated in isocratic
mode with a mobile phase. The mass spectrometric detection of the
compound 33, N-9 and phenacetin was performed in the multiple
reactions monitoring mode (MRM) using electrospray ionization
(ESI) source in positive mode. The source temperature and ion
(
reference wavelength: 490 nm). The resulting color intensity is
directly proportional to the actual RT activity. The percentage
inhibitory activity of RT inhibitors was calculated by using the
formula shown below.
ꢂ
spray voltage were set at 400 C and 5500 V, respectively. The
detection and quantification of analytes was performed using the
multiple reactions monitoring (MRM) mode using ion pre-
cursor/product ion combinations. The total analysis time was
3.5 min per sample. All the raw data were processed with PE SCIEX
Analyst Software (Version 1.4.1) from Applied Biosystems.
ðOD 405 nm with inhibitorÞ ꢁ 100
%RT Inhibition ¼ 100 ꢀ
ðOD 405 nm without inhibitorÞ
Calibration standards (CS) were prepared by spiking working
standard solution into 50
tion range of 0.195e100.0 ng/mL for both analyte. Quality control
QC) samples at four different concentrations were prepared in a
similar manner with appropriate working stock solution. The CS
and QCs samples were prepared by spiking 5 L of appropriate
calibration stock into 45 L of blank plasma and 200 L acetonitrile
of IS solution (containing 200 ng/mL phenacetin) were added,
mixed for 5 min on a cyclomixer (Spinix Tarsons, Kolkata, India) and
then centrifuged at 13,000 rpm (Sigma Centrifuge, Germany) for
mL rabbit plasma to obtain a concentra-
4
.2.4. Antifungal activity
The minimum inhibitory concentration (MIC) of compounds
(
were determined by broth micro-dilution technique as per guide
lines of National Committee for Clinical Laboratory Standards using
RPMI 1640 media buffered with MOPS [3-(N-Morpholino)pro-
panesulfonic acid] [25]. Starting inoculums of test culture was
m
m
m
3
ꢂ
1
e5 ꢁ 10 CFU/mL. Micro-titre plates were incubated at 35 C. MICs
were recorded after 48 h of incubation (Table 2).
2
2
0 min. Pharmacokinetic study samples (50
00 L of IS solution and taken for processing. Samples were mixed
L of
clear supernatant was transferred into auto-sampler vial and
analyzed by injecting 10 L supernatant to LC-MS/MS method. The
mL) were spiked with
4
.2.5. Cytotoxicity towards human cervical (HeLa) cell line by
m
lactate dehydrogenase release assay [21]
properly and centrifuged at 13,000 rpm for 20 min and 100
m
4
HeLa cells seeded at a density of 5 ꢁ 10 per well in 96-well
plates were incubated with either the culture medium containing
dilutions of test compounds, or vehicle (control), for 24 h. There-
m
method was validated to meet the acceptance criteria of FDA
guidelines for the bioanalytical method validation. Blood samples
after 5 mL of MTT solution (5 mg/mL in buffer, pH 7.4) was added to
each well. The formazan crystals formed inside the viable cells were
solubilized in DMSO, and the optical density at 540 nm (OD540) was
recorded in a microplate reader (Microquant; BioTek, USA).
(200 mL) from each rabbit were collected in heparinized tubes from
marginal ear vein at 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, 6.0 and 8.0 h
post-dosing. The in vivo pharmacokinetics study in rabbits was
approved by the Institutional Animal Ethical committee (IAEC
approval No. IAEC/2012/91 Ns).
4.2.6. Effect on Lactobacillus acidophillus in vitro
The effect of compounds exhibiting potent spermicidal activity
on Lactobacillus acidophilus was determined by following the
method published earlier [26]. Spores of Lactobacillus jensenii (ATCC
Acknowledgment
2
5258, strain 62G) were grown in 6% Rogosa SL broth medium (Hi
We acknowledge Mrs. Tara Rawat (Senior Technical Officer) for
technical assistance and SAIF Division for spectral data. We
acknowledge ICMR (V.B, S.G and Y.S), CSIR (D.M, S.J, H.C) for
research fellowships. This study was supported by DHR, ICMR, New
Delhi, Government of India (GAP 00155) and CSIR-Network project
PROGRAM, CSIR, New Delhi, India (BSC 0101).
ꢂ
Media, India) containing 0.132% acetic acid at 37 C. Briefly, Rogosa
SL broth medium was distributed in a 48-well plate (500m l/well).
Serial dilutions of test compounds were added to experimental
wells, and vehicle was added to control wells in triplicate.
Approximately, 1000 CFU of L. jensenii were inoculated into each
ꢂ
well and the plates were incubated at 37 C in a humidified at-
mosphere containing 5% CO
the cultures were mixed thoroughly, 100
2
for 24 h. At the end of the experiment,
L from each well was
Appendix A. Supplementary data
m
transferred to the corresponding well of a 96-well plate, and the
numbers of lactobacilli were estimated by measuring the turbidity
1
1
Supplementary data (scan copies of H NMR spectra of 7e11, H
13
NMR and C NMR spectral data of compounds 12e26, ESI-Mass
spectra of 12, 15, 17e20 and HRMS spectra of 12, 14, 15, 17,
9e21) related to this article can be found at http://dx.doi.org/10.
(
OD610) in a microplate reader. The average number of Lactobacilli
control ¼ 100) is given in Fig. 2.
(
1
1016/j.ejmech.2015.07.021.
4.2.7. Docking studies
Compound 12 was drawn using the sketch module of Sybyl7.1
References
[
27]. It was energy minimized using the MMFF94 force field and
charges MMFF94 with 1000 iterations. The crystal structure of HIV-
reverse transcriptase (RT) enzyme with DNA and a non-
nucleoside inhibitor nevirapine (NVP) was retrieved from PDB
Accession code: 3V81) [15]. The docking study of the compound 12
was done using Autodock4.2 software [28].
[
[
[
[
[
1] http://www.ibtimes.co.uk/global-population-increase-11-billion-2100-africa-
478415 (accessed 06.01.15).
2] http://www.un.org/en/development/desa/news/population/world-
urbanization-prospects-2014.html (accessed 06.01.15).
3] http://www.who.int/reproductivehealth/topics/unsafe_abortion/article_
unsafe_abortion.pdf (accessed 06.01.15).
4] J.J. Speidel, D.C. Weiss, S.A. Ethelston, S.M. Gilbert, Philos. Trans. Roy. Soc. B
1
(
364 (2009) 3049e3065.
4
.2.8. Pharmacokinetics and stability studies [29]
5] I.J. Rosenstein, M.K. Stafford, V.S. Kitchen, H. Ward, J.N. Weber, D. Taylor-
The method development, validation and all sample analysis
Robinson, J. Infect. Dis. 177 (1998) 1386e1390.

650
V. Bala et al. / European Journal of Medicinal Chemistry 101 (2015) 640e650
[
[
[
[
6] L. Van Damme, G. Ramjee, M. Alary, B. Vuylsteke, V. Chandeying, H. Rees,
D.I. Stuart, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 3984e3989.
[17] R.K. Rawal, A. Kumar, M.I. Siddiqi, S.B. Katti, J. Mol. Model. 13 (2007) 155e161.
[18] J.H. Chan, J.S. Hong, R.N. Hunter 3rd, G.F. Orr, J.R. Cowan, D.B. Sherman,
S.M. Sparks, B.E. Reitter, C.W. Andrews 3rd, R.J. Hazen, M. St Clair, L.R. Boone,
R.G. Ferris, K.L. Creech, G.B. Roberts, S.A. Short, K. Weaver, R.J. Ott, J. Ren,
A. Hopkins, D.I. Stuart, D.K. Stammers, J. Med. Chem. 44 (2001) 1866e1882.
[19] G. Barreiro, C.R. Guimaraes, I. Tubert-Brohman, T.M. Lyons, J. Tirado-Rives,
W.L. Jorgensen, J. Chem, Inf. Model. 47 (2007) 2416e2428.
[20] Y.S. Chhonker, H. Chandasana, V. Bala, L. Kumar, V.L. Sharma, G. Gupta,
R.S. Bhatta, J. Chromatogr. B 965 (2014) 127e132.
[21] R.K. Jain, J.P. Maikhuri, S.T. Kiran Kumar, V.L. Sharma, A.K. Dwivedi, K. Mitra,
V.K. Bajpai, G. Gupta, Hum. Reprod. 22 (2007) 708e716.
P. Sirivongrangson, L. Mukenge-Tshibaka, V. Ettiegne-Traore, C. Uaheowitchai,
S.S. Karim, B. Masse, J. Perriens, M. Laga, Lancet 360 (2002) 971e977.
7] V. Bala, S. Jangir, V. Kumar, D. Mandalapu, S. Gupta, L. Kumar, B. Kushwaha,
Y.S. Chhonker, A. Krishna, J.P. Maikhuri, P.K. Shukla, R.S. Bhatta, G. Gupta,
V.L. Sharma, Bioorg. Med. Chem. Lett. 24 (2014) 5782e5786.
8] S. Jangir, V. Bala, N. Lal, L. Kumar, A. Sarswat, B. Kushwaha, P. Singh,
P.K. Shukla, J.P. Maikhuri, G. Gupta, V.L. Sharma, Org. Biomol. Chem. 12 (2014)
3090e3099.
9] L. Kumar, N. Lal, V. Kumar, A. Sarswat, S. Jangir, V. Bala, B. Kushwaha,
A.K. Pandey, M.I. Siddiqi, P.K. Shukla, J.P. Maikhuri, G. Gupta, V.L. Sharma, Eur.
J. Med. Chem. 70 (2013) 68e77.
[
10] S.T. Kiran Kumar, V.L. Sharma, M. Kumar, P.K. Shukla, P. Tiwari, R.K. Jain,
[22] J.A. Upcroft, P. Upcroft, Antimicrob. Agents Chemother. 45 (2001) 1810e1814.
[23] S.-L. Cao, Y. Han, C.-Z. Yuan, Y. Wang, Z.-K. Xiahou, J. Liao, R.-T. Gao, B.-B. Mao,
B.-L. Zhao, Z.-F. Li, X. Xu, Eur. J. Med. Chem. 64 (2013) 401e409.
[24] (a) S. Fatima, A. Sharma, R. Saxena, R. Tripathi, S.K. Shukla, S.K. Pandey,
R.P. Tripathi, Eur. J. Med. Chem. 55 (2012) 195e204;
J.P. Maikhuri, D. Singh, G. Gupta, M.M. Singh, Bioorg. Med. Chem. 14 (2006)
6593e6600.
[
11] V.S. Kumar, V.L. Sharma, P. Tiwari, D. Singh, J.P. Maikhuri, G. Gupta,
M.M. Singh, Bioorg. Med. Chem. Lett. 16 (2006) 2509e2512.
[
12] (a) O.J. D'Cruz, Y. Dong, F.M. Uckun, Biochem. Biophys. Res. Comm. 302 (2003)
(b) V. Murugesan, V.S. Tiwari, R. Saxena, R. Tripathi, R. Paranjape, S. Kulkarni,
N. Makwana, R. Suryawanshi, S.B. Katti, Bioorg. Med. Chem. 19 (2011)
6919e6926.
253e264;
(
b ) J.C. You, G.H. Han, C.H. Lee, D.N. Song, K.H. Chung, (2011) Korean Patent,
WO2011159137 A2, http://www.google.com/patents/WO2011159137A2?
cl¼en.
[25] L. Kumar, N. Lal, V. Kumar, A. Sarswat, S. Jangir, V. Bala, B. Kushwaha,
A.K. Pandey, M.I. Siddiqi, P.K. Shukla, J.P. Maikhuri, G. Gupta, V.L. Sharma, Eur.
J. Med. Chem. 70 (2013) 68e77.
[26] A. Jain, N. Lal, L. Kumar, V. Verma, R. Kumar, L. Kumar, V. Singh, R.K. Mishra,
A. Sarswat, S.K. Jain, J.P. Maikhuri, V.L. Sharma, G. Gupta, Antimicrob. Agents
Chemother. 55 (2011) 4343e4351.
[27] Sybyl, Version 7.1, Tripos, Inc., St. Louis, MO, 2005.
[28] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, J. Comput. Chem. 30 (2009) 2785e2791.
[
13] V. Bala, S. Jangir, D. Mandalapu, S. Gupta, Y.S. Chhonker, N. Lal, B. Kushwaha,
H. Chandasana, S. Krishna, K. Rawat, J.P. Maikhuri, R.S. Bhatta, M.I. Siddiqi,
R. Tripathi, G. Gupta, V.L. Sharma, Bioorg. Med. Chem. Lett. 25 (2015)
8
81e886.
14] J.P. Maikhuri, A.K. Dwivedi, J.D. Dhar, B.S. Setty, G. Gupta, Contraception 67
2003) 403e408.
15] K. Das, S.E. Martinez, J.D. Bauman, E. Arnold, Nat. Struct. Mol. Biol. 19 (2012)
53e259.
16] R.M. Esnouf, J. Ren, A.L. Hopkins, C.K. Ross, E.Y. Jones, D.K. Stammers,
[
[
[
(
2
[29] Human Services, Food and Drug Administration Center for Drug Evaluation
and Research Center (CDER), Center for Veterinary Medicine, 2001.