Dithiocarbamate-thiourea hybrids useful as vaginal microbicides also show reverse transcriptase inhibition: Design, synthesis, docking and pharmacokinetic studies

Article abstract of DOI:10.1016/j.bmcl.2014.12.062

Prophylactic prevention is considered as the most promising strategy to tackle STI/HIV. Twenty-five dithiocarbamate-thiourea hybrids (14-38) were synthesized as woman controlled topical vaginal microbicides to counter Trichomonas vaginalis and sperm along with RT inhibition potential. The four promising compounds (18, 26, 28 and 33) were tested for safety through cytotoxic assay against human cervical cell line (HeLa) and compatibility with vaginal flora, Lactobacillus. Docking study of most promising vaginal microbicide (33) revealed that it docked in a position and orientation similar to known reverse transcriptase inhibitor Nevirapine. The preliminary in vivo pharmacokinetics of compound 33 was performed in NZ-rabbits to evaluate systemic toxicity in comparison to Nonoxynol-9.

Full text of DOI:10.1016/j.bmcl.2014.12.062

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Dithiocarbamate–thiourea hybrids useful as vaginal microbicides
also show reverse transcriptase inhibition: Design, synthesis, docking
and pharmacokinetic studies q
Veenu Bala a,f, Santosh Jangir a, Dhanaraju Mandalapu a, Sonal Gupta a,f, Yashpal S. Chhonker c,f, Nand Lal a,
Bhavana Kushwaha b, Hardik Chandasana c,f, Shagun Krishna d, Kavita Rawat e, Jagdamba P. Maikhuri b,
Rabi S. Bhatta c,f, Mohammad I. Siddiqi d, Rajkamal Tripathi e, Gopal Gupta b, Vishnu L. Sharma a,f,
⇑
a Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Ext., Lucknow 226031, India
b Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
c Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
d Molecular & Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
e Toxicology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
f Academy of Scientific and Innovative Research (AcSIR), New Delhi 110001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Prophylactic prevention is considered as the most promising strategy to tackle STI/HIV. Twenty-five
dithiocarbamate–thiourea hybrids (14–38) were synthesized as woman controlled topical vaginal micro-
bicides to counter Trichomonas vaginalis and sperm along with RT inhibition potential. The four promising
compounds (18, 26, 28 and 33) were tested for safety through cytotoxic assay against human cervical cell
line (HeLa) and compatibility with vaginal flora, Lactobacillus. Docking study of most promising vaginal
microbicide (33) revealed that it docked in a position and orientation similar to known reverse transcrip-
tase inhibitor Nevirapine. The preliminary in vivo pharmacokinetics of compound 33 was performed in
NZ-rabbits to evaluate systemic toxicity in comparison to Nonoxynol-9.
Received 8 September 2014
Revised 15 December 2014
Accepted 19 December 2014
Available online xxxx
Keywords:
Dithiocarbamate
Thiourea
Non-nucleoside reverse transcriptase
inhibitor (NNRTI)
Spermicidal
Ó 2014 Elsevier Ltd. All rights reserved.
Microbicidal
Nonoxynol-9
The acquired immune deficiency syndrome (AIDS) epidemic
continues to spread throughout the world and especially imposes
a particular burden on women and girls. According to UNAIDS
2013 estimate nearly 52% of all individuals living with HIV are
now women of reproductive age (15–44 years).1 They acquire the
virus largely by heterosexual contacts, documented as the domi-
nant mode of this pandemic spread.2 However, male condoms
may reduce the risk of HIV infection by 80–90% but they are
beyond the control of women.3 Thus, women urgently need self
controlled methods to protect themselves against HIV infection.
In the absence of an effective prophylactic HIV vaccine, prevention
of new infections has become a priority. It was thought worthwhile
to integrate HIV prevention and reproductive health services
including unintended pregnancy protection for women as both
are related with unprotected sex. Topical vaginal microbicide is
one of the most promising female-controlled approaches effective
against STIs (Sexually transmitted infections) including HIV in con-
junction with pregnancy protection via sperm immobilization.4,5
A safe, efficacious and women friendly vaginal microbicide has
yet not been available because microbicides that are undergoing
preclinical and human clinical trials possess detergent action.
Nonoxynol-9 (N-9), the only recommended microbicide for protec-
tion against sexual transmission of HIV-1 resulted in compromised
vagina owing to detergent action.6,7 Moreover, studies found that
with frequent use, N-9 causes mucosal erosion and local inflamma-
tion of the female reproductive tract, which increases susceptibil-
ity to HIV-1 and other viral infections. Thus, clinical safety
becomes a serious liability for vaginal products.8–10
Anti-HIV-1 potential of reverse transcriptase (RT) inhibitors in
genital tract secretions has already been established.11,12 So, it
was attempted to synthesize novel vaginal microbicides with RT
inhibition as best suited prophylactic approach against vaginal
q
CDRI Communication No. 8859.
Corresponding author. Tel.: +91 522 2772450x4671; fax: +91 522 2771941.
E-mail addresses: vlscdri1@rediffmail.com, vlscdri@gmail.com (V.L. Sharma).
⇑
http://dx.doi.org/10.1016/j.bmcl.2014.12.062
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Bala, V.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.062

2
V. Bala et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
HIV-1 transmission.13,14 The success of thiourea derivatives as
NNRTIs15,16 and ongoing research interest in the development of
dithiocarbamates (Fig. 1) as potent vaginal anti-trichomonal sper-
micides17–22 led us to hybridize these scaffolds in one chemical
entity (alkyl 4-(alkyl/arylcarbamothioyl)piperazine-1-carbodithio-
ate, Fig. 1) to achieve dual action. Anti-HIV spermicidal potential
of thiourea moiety has been recognized.23–25 Considering the
well-known ‘butterfly’ conformation of NNRTIs,26 like piperazine
derivative, Delavirdine (Fig. 1) these diverse scaffolds were synthe-
sized and evaluated for their in vitro RT inhibition assay to ascer-
tain their anti HIV-1 potential. Anti-Trichomonas and spermicidal
activity of synthesized compounds was also evaluated against
Trichomonas vaginalis and human sperm, respectively. The struc-
ture activity relationship (SAR) has also been discussed. The prom-
ising compounds were also assessed for their in vitro cytotoxicity
profile on vaginal flora (Lactobacillus) and human cervical epithe-
lium (HeLa) cells because they are intended to be used vaginally.
Pharmacokinetics of most potent compound versus N-9 was eval-
uated in female Newzealand (NZ) rabbits to examine its absorption
into systemic circulation and subsequent exposure in blood plasma
through vaginal wall. Moreover, a docking study was also carried
out to find a suitable correlation of most active compound with
NNRTIs activity and inhibition of the prospective receptor.
The compounds presented in this study namely alkyl 4-(alkyl/
aryl carbamothioyl) piperazine-1-carbodithioate (14–38) were
evaluated for anti-Trichomonas and spermicidal potential along
with RT inhibitory activity by using enzymatic RT assay (Table 1).
Trichomonas vaginalis causes punctuate hemorrhages giving HIV
open access to its target T-lymphocytes. These hemorrhages leak
the virus and cause them to concentrate in the infected area, which
increase HIV transmission by 2 to 3 fold.27 All the synthesized com-
pounds (14–38) except 25 and 36 exhibited anti-trichomonas activ-
ity ranging from 7.78–500
lg/mL. Remarkable activity (better than
N-9, MIC 20 g/mL) was observed in compounds 28, 33 and 14
l
which were active at 7.78
lg/mL, 15.56 lg/mL and 15.56 lg/mL,
respectively.
A potent contraceptive (spermicidal) activity in microbicides
would attract users, especially those at a high risk of acquiring
HIV/STD, and may improve compliance in usage. Spermicides capa-
ble of killing 100% human sperm almost instantaneously at physi-
ological concentrations in vitro are likely to provide adequate
pregnancy protection in vivo. Among synthesized twenty five com-
pounds, thirteen compounds (14, 16, 18, 23, 26–31, 33, 36 and 38)
exhibited spermicidal activity (Table 1) at 0.025–2% (w/v) concen-
tration and irreversibly immobilized 100% normal human sperma-
tozoa. Out of these thirteen compounds, four compounds (18, 26,
28 and 33) were active at concentration 0.025–0.05% comparable
or even more active than marketed spermicide N-9 (MEC, 0.05%).
Two compounds (18 and 33) demonstrated extremely potent
spermicidal activity at MEC 0.025%.
The alkyl 4-(alkyl/arylcarbamothioyl)piperazine-1-carbodithio-
ate (14–38) were synthesized according to the strategy depicted in
Scheme 1. Sodium 4-(tert-butoxycarbonyl)piperazine-1-carbodi-
thioate (3) was synthesized by reaction of N-Boc piperazine with
carbon disulfide and sodium hydroxide. Reaction of
3
with
All the compounds inhibited the RT ranging 1.90–44.67% at
different alkyl halide in the presence of triethylamine in methanol
at rt yielded tert-butyl 4-(alkylthiocarbonothioyl)piperazine-1-
carboxylate (4–8). Treatment with trifluoroacetic acid (TFA) in
dichloromethane (DCM, 0–5 °C) and aqueous sodium bicarbonate
(NaHCO3) resulted in Boc deprotection and gave alkyl piperazine-
1-carbodithioate (9–13). Compounds 9–13 afforded alkyl
4-(alkyl/arylcarbamothioyl)piperazine-1-carbodithioate (14–38)
on treatment with isothiocyanates in ethanol at room temperature.
The structures of all newly synthesized compounds were
confirmed by 1H NMR, 13C NMR, IR, mass spectrometry
(ESMS and HRMS) and elemental analysis (see Supporting
information).
100 lg/mL concentration. The compounds that showed moderate
inhibitory activity (>30%) were 16, 18, 20, 29, 31, 33 whereas the
control NNRTIs marketed drug Nevirapine (NVP) showed 99.6%
inhibition. The enzyme assay results were visualized in combina-
tion with anti-trichomonas and spermicidal activity. The results
are summarized in Table 1 along with standard drug NVP.
This study included 1,4-disubstituted piperazine compounds
(14–38) having a substituted thiourea at the N1-position while
substituted dithiocarbamates at N4-position. The thiourea
substituents R1 were benzyl, phenyl, 3-pyridyl, phenethyl and ben-
zoyl while the dithiocarbamate substituents R2 have been propyl,
butyl, hexyl, octyl, and benzyl. The anti-Trichomonas activity
Br
O2N
S
N
S
N
H
N
H
N
N
S
N
R1
N
R2
H3C
Trovirdine as NNRTI(Thiourea scaffold)
Dithiocarbamate scaffold as microbicidal spermicide
R2
S
R1 NH
N
N
S
S
Designed Dithiocarbamate-thiourea Hybrid
O
S
O
HN
S
NH
HN
N
R1
R2
R1
HN
SH.
N
R2
N
N
O
Delavirdine as NNRTI (Piperazine derivative)
N-substituted dithiocarbamic acid amine salt as
microbicidal spermicide
Figure 1. Designing of dithiocarbamate–thiourea hybrids.
Please cite this article in press as: Bala, V.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.062

V. Bala et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
S
S
NH
R1
NH
(ii)
(i)
N
S
(iii)
HN
(1)
N
SNa
(3)
N
N
Boc
N
Boc
(2)
Boc
(4-8)
(iv)
S
S
R1
N
S
(v)
R1
H
N
N
S
N
R2
HN
S
(9-13)
(14-38)
Scheme 1. Synthesis of substituted dithiocarbamate–thiourea hybrids. Reagents and conditions: (i) di-tert-butyl dicarbonate, methanol, rt, 4 h; (ii) CS2, NaOH, ethyl acetate,
0–5 °C, 3 h; (iii) alkyl halide, triethyl amine, methanol, rt, 4–5 h; (iv) TFA, DCM, aq NaHCO3; (v) Isothiocyanate, ethanol, rt, 1–2 h.
Table 1
Anti-Trichomonas, spermicidal activity and RT inhibition assay of compounds 14–38
S
S
N
N
R1 NH
S
R2
Compound
Anti-Trichomonas activity MICa
(l
g/ml)
Spermicidal activity MECb (%)
% RT inhibitionc (100
lg/ml)
R1
R2
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Benzyl
Phenyl
3-Pyridyl
Phenethyl
Benzoyl
Benzyl
Propyl
Propyl
Propyl
Propyl
Propyl
Butyl
Butyl
Butyl
Butyl
Butyl
Hexyl
Hexyl
Hexyl
Hexyl
Hexyl
Octyl
Octyl
Octyl
Octyl
Octyl
15.56
500
125
250
62.5
500
125
250
250
125
250
>500
125
250
7.78
250
125
62.5
250
15.56
125
250
>500
62.5
62.5
1%
20.56
21.47
35.39
28.44
31.42
14.24
30.87
14.92
13.47
27.8
>2%
0.5%
>2%
0.025%
>2%
>2%
>2%
>2%
1%
>2%
>2%
0.05%
1%
Phenyl
3-Pyridyl
Phenethyl
Benzoyl
Benzyl
27.33
1.90
Phenyl
3-Pyridyl
Phenethyl
Benzoyl
Benzyl
26.14
14.84
23.19
36.46
23.96
44.67
15.67
41.29
9.11
0.05%
1%
1%
Phenyl
3-Pyridyl
Phenethyl
Benzoyl
Benzyl
2%
>2%
0.025%
>2%
>2%
2%
Benzyl
Benzyl
Benzyl
Benzyl
Benzyl
Phenyl
21.9
3-Pyridyl
Phenethyl
Benzoyl
25.77
11.85
12.35
99.6
>2%
1%
38
Nevirapine
Nonoxynol-9
20
0.05
a
b
c
MIC-Minimum inhibitory concentration
MEC-minimum effective concentration.
Inhibition (%) = observed inhibition(%) À negative control (%) of each tested compound. (All data presented over here are averages of three different observations).
Figure 2. (a) Cytotoxicity of compounds towards human cervical cell lines (HeLa). (b) Compatibility with normal vaginal flora (Lactobacillus).
Please cite this article in press as: Bala, V.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.062

4
V. Bala et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
results suggested that the alkyl substituents at N4-carbodithioate
group were preferred over benzyl substituent. The hexyl substitu-
ent at N4-position (R2) of piperazine resulted in most active com-
showed 30.87–44.67% RT inhibition at 100 lg/mL. The thiourea
moiety was thought responsible for RT inhibition. The activity with
respect to the substituent at N1-position (R1) of piperazine was of
the following order: 3-pyridyl (31, 44.67%) > benzoyl (33,
41.29%) > benzyl (29, 36.46%) > phenyl (20, 30.87%) > phenethyl
(17, 28.44%). It seems that structural combination of 3-pyridyl/
benzoyl/benzyl at N1 and octyl carbodithioate at N4-position was
most desirable for RT inhibition.
The structure of most active compounds (18 and 33) suggested
that benzoyl thiourea at N1 and propyl/octyl carbodithioate at
N4-position of piperazine were most required for sperm immobili-
zation. The vaginal microbicidal activity comparisons of these
compounds were made in view of all three activities together to
achieve the aim to arrive at a pharmacophore possessing multiple
pound 28 (MIC, 7.78
lg/mL) followed by propyl/octyl
substitution (compounds 14 and 33, 15.56
lg/mL). On the other
hand the benzoyl group at N1-thiourea substituent was most effec-
tive (18, 23, 28, 33 and 38). The results of spermicidal activity
showed that the preference of alkyl chain at N4-carbodithioate
was octyl > hexyl > propyl > benzyl > butyl whereas benzoyl was
most suitable group at N1-thiourea substituent. Two compounds
(26 and 28) were as active as N-9 (MEC, 0.05%) while two others
(18 and 33) exhibited two fold activity.
Among twenty five newly synthesized dithiocarbamate–thio-
urea hybrids (14–38), six compounds (16, 18, 20, 29, 31 and 33)
Figure 3. Morphological changes of HeLa cells exposed to compound 33 at 200
treated with N-9. (c) Cells treated with most promising compound 33.
l
g/mL for 24 h. (a) Control-HeLa cells incubated without any compound treatment (b) Cells
Figure 4. Docked conformations of compound 33 (pink) superimposed on NVP (Brown). NVP is displayed for comparison.
Please cite this article in press as: Bala, V.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.062

V. Bala et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
activities. The study resulted in twelve dually active compounds
(14, 16, 18, 23, 26-31, 33 and 38) of which the most promising com-
pound was octyl 4-(benzoylcarbamothioyl) piperazine-1-carbodi-
thioate (33) as it showed anti-Trichomonas (15.56 lg/mL),
spermicidal (0.025%) activity and RT inhibition (41.29%).
The compounds synthesized as vaginal microbicide with RT
often also target other cells of vagina, such as cervico-vaginal
epithelia and lactobacilli that are crucial in maintaining natural
barrier to invasions by pathogens, especially the HIV. The most
active compounds (18, 26, 28 and 33) were evaluated for their
safety towards human cervical cell line (HeLa) and compatibility
NVP was extracted from crystal structure and re-docked into the
active site of the crystal to obtain the conformation similar that
in crystal structure to validate our docking program. Autodock4.2
successfully reproduced the conformation of crystal structure. For
the docking simulations, the active site was selected by taking a
6.5 Å area around the co-crystallized ligand NVP.30–32
The docked complex of compound 33 is stabilized mainly by
considerable hydrophobic interactions. To study the binding mode
of compound 33, the docked conformation was compared with the
crystal structure of HIV-1 RT. The residues involved in hydrophobic
interaction include Leu100, Lys101, Val106, Val179, Tyr181,
Tyr188, and Leu234 from p66 subunit and Glu138 from p51 unit.
Figure 4 shows the binding mode of compound 33 comparable to
NVP. It is clear from the figure that compound 33 occupies almost
the same position as that of NVP in crystal structure. The benzoyl
moiety present in this compound formed a cavity with Tyr188,
Phe227, Trp229 and Leu234 residues, while the octyl group is ori-
ented towards Leu100, Lys103 and Val106 residues, which is nec-
essary for the stabilization of the complex.
Stability and quantification of the extent of drug substance
absorption after vaginal administration is essential in determining
the strategy of nonclinical pharmacology/toxicology development.
Simulated vaginal fluid (SVF, pH 4.2) was considered as a perfect
media to determine the stability of molecules that are intended for
intra-vaginal use.33 The compound 33 was incubated in SVF at a final
concentration 100 ng/mL for stability study. Samples were removed
at 0, 5, 15, 30, 45, and 60 min. The concentration of compound 33
with vaginal flora (Lactobacillus, Fig. 2) at 200 lg/mL concentration
by cell-viability assay. These compounds did not affect the viability
of HeLa cells or growth of Lactobacilli during 24 h of incubation and
therefore appeared apparently much safer for vaginal use. These
safety results support the utility of synthesized compounds as anti
HIV-1 microbicide. Comparative HeLa cell survival after treatment
with N-9 and most promising compound 33 is shown in Figure 3.
Docking study was performed to gain an insight into the bind-
ing mode and interactions of the most promising compound 33
with HIV-1 reverse transcriptase. The HIV-1 RT protein is a hetero-
dimeric protein that consists of two units p66 and p51 with a cat-
alytic and allosteric site.28 The inhibitors of HIV-1 RT can occupy
either the catalytic site or the allosteric site. The allosteric site is
the binding site of various NNRTIs including Nevirapine (NVP). Pre-
vious studies done on the NNRTIs showed that these inhibitors
occupy a hydrophobic pocket.29 For our docking studies, initially
Figure 5. Representative MRM ion-chromatograms of (a) blank plasma (compound 33), (b) compound 33 spiked in plasma, (2.12 min), (c) blank plasma (N-9), (d) N-9 spiked
in plasma, (e) PK sample at 1 h of compound 33 and (f) PK sample at 1 h of N-9.
Please cite this article in press as: Bala, V.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.062

6
V. Bala et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
was determined by LC–MS/MS. The stability data have shown that
compound 33 was stable in SVF up to 1.0 h. This behavior of com-
pound 33 is favorable for its vaginal administration and supports
its efficacy as vaginal microbicide with RT inhibition potential.
A preliminary vaginal pharmacokinetics study was carried out
to monitor drug substance in blood plasma. LC–MS/MS assay using
positive ion electro-spray ionization (ESI) in multiple reaction
monitoring (MRM) mode was found appropriate to develop selec-
tive, sensitive and reproducible analysis of compound 33, N-9 and
internal standard (IS) in rabbit plasma. The pharmacokinetic anal-
ysis was processed non-compartmental model using Phoenix Win-
Nonlin (version 6.3, Pharsight, MountainView, CA). The linear
trapezoidal method with linear interpolation was used to calculate
pharmacokinetic parameters. The mean peak plasma concentra-
tion (Cmax) 4.87 0.37 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 1.45 0.07 h. The systemic absorption of compound 33
was below the limit of quantitation (Fig. 5). After vaginal adminis-
tration, the presence of compound 33 was very less in systemic cir-
culation might be suggestive of minimal systemic adverse effects
or minimal toxic effect. In summary, together with pharmacologi-
cal findings, this study could provide useful clues and guidance,
such as dosage regimen and application strategy of compound 33
as a lead candidate to be used as vaginal microbicide.
In a systemic effort to identify a non-detergent prophylactic
vaginal microbicide potentially capable of preventing sexual trans-
mission of HIV/STDs as well as providing fertility control, a series
of dithiocarbamate–thiourea hybrids as alkyl 4-(alkyl/aryl car-
bamothioyl)piperazine-1-carbodithioate (14–38) were synthesized
and evaluated for multiple activities (anti-Trichomonas, spermi-
cidal and RT inhibition). The intended dual action was achieved
and several compounds exhibited all the three activities with
remarkable safety against cervico-vaginal epithelial cells and vag-
inal flora. Among the safest structures, most promising compound
33 exhibited considerable RT inhibition, anti-Trichomonas and
spermicidal activities, which could be attributed to its utility as
vaginal microbicide. Proposed docking analysis of 33 is notable
and can be utilized as a guideline for designing of promising vagi-
nal microbicide with RT inhibition potential. Compound 33 was
also found pharmacokinetically safe in comparison to N-9 due to
least vaginal absorption to systemic circulation. Further lead opti-
mization is underway to develop novel vaginal microbicide pos-
sessing RT inhibitory activity in order to empower women to
deal independently with their reproductive health and fertility.
of compounds 14–38) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.12.
062.
References and notes
1. <http://www.unaids.org/en/media/unaids/contentassets/documents/epidemiology/
2013/gr2013/unaids_global_report_2013_en.pdf> (accessed on 8 May 2014).
2. <http://www.unaids.org/en/media/unaids/contentassets/documents/
unaidspublication/2004/GAR2004_en.pdf> (accessed on 8 May 2014).
3. Anderson, R. A.; Feathergill, K. A.; Diao, X. H.; Cooper, M. D.; Kirkpatrick, R.;
Herold, B. C.; Doncel, G. F.; Chany, C. J.; Waller, D. P.; Rencher, W. F.; Zaneveld,
L. J. J. Androl. 2002, 23, 426.
4. D’Cruz, O. J.; Zhu, Z.; Yiv, S. H.; Chen, C. L.; Waurzyniak, B.; Uckun, F. M.
Contraception 1999, 59, 319.
5. Bisika, T. J. Fam. Plan. Reprod. Health Care 2009, 35, 115.
6. Rosenstein, I. J.; Stafford, M. K.; Kitchen, V. S.; Ward, H.; Weber, J. N.; Taylor-
Robinson, D. J. Infect. Dis. 1998, 177, 1386.
7. Stafford, M. K.; Ward, H.; Flanagan, A.; Rosenstein, I. J.; Taylor-Robinson, D.;
Smith, J. R.; Weber, J.; Kitchen, V. S. Acquir. Immune Defic. Syndr. Human
Retrovirol. 1998, 17, 327.
8. Alt, C.; Harrison, T.; Dousman, L.; Fujita, N.; Shew, K.; Tran, T. T.; Shayesteh, S.;
Matsukawa, A.; Mirsalis, J.; D’Andrea, A. Curr. HIV Res. 2009, 7, 639.
9. Fichorova, R. N.; Bajpai, M.; Chandra, N.; Hsiu, J. G.; Spangler, M.; Ratnam, V.;
Doncel, G. F. Biol. Reprod. 2004, 71, 761.
10. Fichorova, R. N.; Zhou, F.; Ratnam, V.; Atanassova, V.; Jiang, S.; Strick, N.;
Neurath, A. R. Antimicrob. Agents Chemother. 2005, 49, 323.
11. Van Herrewege, Y.; Penne, L.; Vereecken, C.; Fransen, K.; van der Groen, G.;
Kestens, L.; Balzarini, J.; Vanham, G. AIDS Res. Hum. Retroviruses 2002, 18, 1091.
12. Zussman, A.; Lara, L.; Lara, H. H.; Bentwich, Z.; Borkow, G. AIDS 2003, 17, 653.
13. Balzarini, J.; Naesens, L.; Verbeken, E.; Laga, M.; Van Damme, L.; Parniak, M.;
Van Mellaert, L.; Anne, J.; De Clercq, E. AIDS 1998, 12, 1129.
14. Dezzutti, C. S.; James, V. N.; Ramos, A.; Sullivan, S. T.; Siddig, A.; Bush, T. J.;
Grohskopf, L. A.; Paxton, L.; Subbarao, S.; Hart, C. E. Antimicrob. Agents
Chemother. 2004, 48, 3834.
15. D’Cruz, O. J.; Uckun, F. M. J. Antimicrob. Chemother. 2006, 57, 411.
16. D’Cruz, O. J.; Uckun, F. M. Biol. Reprod. 1999, 60, 1419.
17. Jain, A.; Lal, N.; Kumar, L.; Verma, V.; Kumar, R.; Singh, V.; Mishra, R. K.;
Sarswat, A.; Jain, S. K.; Maikhuri, J. P.; Sharma, V. L.; Gupta, G. Antimicrob. Agents
Chemother. 2011, 55, 4343.
18. Kumar, L.; Sarswat, A.; Lal, N.; Sharma, V. L.; Jain, A.; Kumar, R.; Verma, V.;
Maikhuri, J. P.; Kumar, A.; Shukla, P. K.; Gupta, G. Eur. J. Med. Chem. 2010, 45,
817.
19. Maikhuri, J. P.; Dwivedi, A. K.; Dhar, J. D.; Setty, B. S.; Gupta, G. Contraception
2003, 67, 403.
20. Kiran Kumar, S. T.; Kumar, L.; Sharma, V. L.; Jain, A.; Jain, R. K.; Maikhuri, J. P.;
Kumar, M.; Shukla, P. K.; Gupta, G. Eur. J. Med. Chem. 2008, 43, 2247.
21. Jangir, S.; Bala, V.; Lal, N.; Kumar, L.; Sarswat, A.; Kushwaha, B.; Singh, P.;
Shukla, P. K.; Maikhuri, J. P.; Gupta, G.; Sharma, V. L. Org. Biomol. Chem. 2014,
12, 3090.
22. Kumar, L.; Lal, N.; Kumar, V.; Sarswat, A.; Jangir, S.; Bala, V.; Kushwaha, B.;
Pandey, A. K.; Siddiqi, M. I.; Shukla, P. K.; Maikhuri, J. P.; Gupta, G.; Sharma, V. L.
Eur. J. Med. Chem. 2013, 70, 68.
23. D’Cruz, O. J.; Dong, Y.; Uckun, F. M. Biochem. Biophys. Res. Commun. 2003, 302,
253.
24. D’Cruz, O. J.; Venkatachalam, T. K.; Mao, C.; Qazi, S.; Uckun, F. M. Biol. Reprod.
1959, 2002, 67.
25. You, J. C.; Han, G. H.; Lee, C. H.; Song, D. N.; Chung, K. H.; WO 2011159137,
2011.
Acknowledgments
26. Schafer, W.; Friebe, W. G.; Leinert, H.; Mertens, A.; Poll, T.; von der Saal, W.;
Zilch, H.; Nuber, B.; Ziegler, M. L. J. Med. Chem. 1993, 36, 726.
27. Sorvillo, F.; Smith, L.; Kerndt, P.; Ash, L. Emerg. Infect. Dis. 2001, 7, 927.
28. Rawal, R. K.; Kumar, A.; Siddiqi, M. I.; Katti, S. B. J. Mol. Model. 2007, 13, 155.
29. Das, K.; Clark, A. D., Jr.; Lewi, P. J.; Heeres, J.; De Jonge, M. R.; Koymans, L. M.;
Vinkers, H. M.; Daeyaert, F.; Ludovici, D. W.; Kukla, M. J.; De Corte, B.; Kavash,
R. W.; Ho, C. Y.; Ye, H.; Lichtenstein, M. A.; Andries, K.; Pauwels, R.; De Bethune,
M. P.; Boyer, P. L.; Clark, P.; Hughes, S. H.; Janssen, P. A.; Arnold, E. J. Med. Chem.
2004, 47, 2550.
30. Sybyl, Version 7.1, Tripos, St. Louis, MO, 2005.
31. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D.
S.; Olson, A. J. J. Comput. Chem. 2009, 30, 2785.
32. Das, K.; Martinez, S. E.; Bauman, J. D.; Arnold, E. Nat. Struct. Mol. Biol. 2012, 19,
253.
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 (S.J., D.M., H.C.), and
UGC (N.L.) for research fellowships. This study was partially sup-
ported by DHR, ICMR, New Delhi, Government of India (GAP
00155) and CSIR-Network project PROGRAM, CSIR, New Delhi,
India (BSC 0101).
Supplementary data
33. Owen, D. H.; Katz, D. F. Contraception 1999, 59, 91.
Supplementary data (supplementary data like scan copies of 1H
NMR spectra of 2–13 and 1HNMR, 13C NMR and HRMS spectral data
Please cite this article in press as: Bala, V.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.062