Substituted carbamothioic amine-1-carbothioic thioanhydrides as novel trichomonicidal fungicides: Design, synthesis, and biology

Article abstract of DOI:10.1016/j.ejmech.2017.11.060

Sexually transmitted diseases like trichomoniasis along with opportunistic fungal infections like candidiasis are major global health burden in female reproductive health. In this context a novel non-nitroimidazole class of substituted carbamothioic amine-1-carbothioic thioanhydride series was designed, synthesized, evaluated for trichomonacidal and fungicidal activities, and was found to be more active than the standard drug Metronidazole (MTZ). Compounds were trichomonicidal in the MIC ranges of 4.77–294.1 μM and 32.46–735.20 μM against MTZ-susceptible and -resistant strains, respectively. Further, compounds inhibited the growth of at least two out of ten fungal strains tested at MIC of 7.50–240.38 μM. The most active compound (20) of this series was 3.8 and 9.5 fold more active than the MTZ against the two Trichomonas strains tested. Compound 20 also significantly inhibited the sulfhydryl groups present over Trichomonas vaginalis and was found to be more active than the MTZ in vivo. Further, a docking analysis carried out with cysteine proteases supported their thiol inhibiting ability and preliminary pharmacokinetic study has shown good distribution and systemic clearance.

Full text of DOI:10.1016/j.ejmech.2017.11.060

Accepted Manuscript
Substituted carbamothioic amine-1-carbothioic thioanhydrides as novel
trichomonicidal fungicides: Design, synthesis, and biology
Dhanaraju Mandalapu, Bhavana Kushwaha, Sonal Gupta, Shagun Krishna, Nidhi
Srivastava, Mahendra Shukla, Pratiksha Singh, Bhavana S. Chauhan, Ravi Goyani,
Jagdamba P. Maikhuri, Koneni V. Sashidhara, Brijesh Kumar, Renu Tripathi, Praveen
K. Shukla, Mohammad I. Siddiqi, Jawahar lal, Gopal Gupta, Vishnu L. Sharma
PII:
S0223-5234(17)30963-7
10.1016/j.ejmech.2017.11.060
EJMECH 9935
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 September 2017
Revised Date: 7 November 2017
Accepted Date: 22 November 2017
Please cite this article as: D. Mandalapu, B. Kushwaha, S. Gupta, S. Krishna, N. Srivastava, M. Shukla,
P. Singh, B.S. Chauhan, R. Goyani, J.P. Maikhuri, K.V. Sashidhara, B. Kumar, R. Tripathi, P.K. Shukla,
M.I. Siddiqi, J. lal, G. Gupta, V.L. Sharma, Substituted carbamothioic amine-1-carbothioic thioanhydrides
as novel trichomonicidal fungicides: Design, synthesis, and biology, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.060.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Substituted carbamothioic amine-1-carbothioic thioanhydrides as novel
trichomonicidal fungicides: Design, synthesis, and Biology#
Dhanaraju Mandalapu,a Bhavana Kushwaha,b Sonal Gupta,a,i Shagun Krishna,c Nidhi Srivastava,a Mahendra
Shukla,d Pratiksha Singh,e Bhavana S. Chauhan,g Ravi Goyani,h Jagdamba P. Maikhuri,b Koneni V. Sashidhara,a
Brijesh Kumar,f Renu Tripathi,g Praveen K. Shukla,e Mohammad I. Siddiqi,c Jawahar lal,d Gopal Gupta,b Vishnu L.
Sharmaa,i,*
b
c
aMedicinal & Process Chemistry Division, Endocrinology Division, Molecular and Structural Biology Division,
e
f
dPharmacokinetic & Metabolism Division, Microbiology Division, Sophisticated Analytical Instrument Facility,
gParasitology Division, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow-226031 (India),
hDepartment of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Raebareli
229 010, India, iAcademy of Scientific and Innovative Research (AcSIR), New Delhi-110001 (India).
Abstract:
Sexually transmitted diseases like trichomoniasis along with opportunistic fungal infections like
candidiasis are major global health burden in female reproductive health. In this context a novel
non-nitroimidazole class of substituted carbamothioic amine-1-carbothioic thioanhydride series
was designed, synthesized, evaluated for trichomonacidal and fungicidal activities, and was
found to be more active than the standard drug Metronidazole (MTZ). Compounds were
trichomonicidal in the MIC ranges of 4.77–294.1 µM and 32.46–735.20 µM against MTZ-
susceptible and -resistant strains, respectively. Further, compounds inhibited the growth of at
least two out of ten fungal strains tested at MIC of 7.50-240.38 µM. The most active compound
(20) of this series was 3.8 and 9.5 fold more active than the MTZ against the two Trichomonas
strains tested. Compound 20 also significantly inhibited the sulfhydryl groups present over
Trichomonas vaginalis and was found to be more active than the MTZ in vivo. Further, a
docking analysis carried out with cysteine proteases supported their thiol inhibiting ability and
preliminary pharmacokinetic study has shown good distribution and systemic clearance.
Keywords: Carbamothioic, Thioanhydrides, Trichomoniasis, Candidiasis, Thiol inhibition.
*Corresponding author: Medicinal and Process Chemistry Division, CSIR-Central Drug Research
Institute, Sector 10, Jankipuram ext., Lucknow, Uttar Pradesh 226031, India.
Tel.: 91-522-2772450; Ext. 4671; Fax: 91-522-2771941 E-mail address: vl_sharma@cdri.res.in.
#CDRI Communication No. 162/2017/VLS
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1. Introduction:
Sexually transmitted infections (STIs) have a major impact on reproductive health of millions of
people worldwide.[1, 2] Among the 30 infections identified to be sexually transmitted, incidence
and prevalence rate of trichomoniasis is higher than the remaining.[3] Trichomoniasis is a very
common, curable, non-viral sexually transmitted urogenital infection in humans caused by the
anaerobic, microaerophilic parasite Trichomonas vaginalis (TV).[4] Most of the cases in United
States and India occur among women of reproductive age[5] and a recent World Health
Organization (WHO) report estimates that more than 276 million people are afflicted with
trichomoniasis every year.[6] Women and men both can be affected but in men the symptoms are
mild and mostly go unreported, which results in its horizontal spread through heterosexual
contacts to sexual partners.[2] In men, the observed complications include urethral discharge,
dysuria, prostatitis, epididymitis, infertility and benign prostatic hyperplasia.[7-9] In women it is
often called “nuisance infection” and may cause endometritis, pyosalpinx, vaginitis, adnexitis,
preterm delivery, infertility, bacterial vaginosis and threat of cervical cancer.[10-13] However,
TV infections increase the risk of HIV and candidiasis transmissions through colonization of the
human urogenital tract epithelium.[14] In sexually active women opportunistic vulvovaginal
candidiasis, caused by Candida albicans has a high prevalence rate which often co-exists along
with TV.[15] The US-FDA approved drug Metronidazole (MTZ), a 5-nitroimidazole, has limited
efficacy against drug-resistant Trichomoniasis infections. For decades, the use of MTZ has been
notable for its effectiveness but the increasing drug-resistance and cross-resistance to MTZ and
other nitroimidazoles (NIs) has been limiting their application in recent times.[16, 17] In 21st
century the incidence of drug resistance cases augmented by 17 fold which are being treated
typically with increased and probably toxic doses of MTZ.[18, 19] Prolonged treatment or high
doses of MTZ may cause side effects such as headache, dry mouth, metallic taste, glossitis and
urticaria.[20] Since women are at the receiving end with long term consequences of STIs, lack of
effective clinical candidates necessitates the researchers to discover highly effective molecules,
preferably non-nitroimidazoles to treat TV and associated infections.[21] Reactive oxygen
species (ROS-) sensitive anaerobes TV and Candida albicans largely depend on cysteine to
overcome redox stress [22] making these infections very susceptible to thiol-inhibiting agents
which signifies thiol as vulnerable target for antitrichomonal and antifungal agents.
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Our previous research work has established that thiol modifying molecules (dithiocarbamates; a-
d) are effective against TV and fungal infections. [23-29] Dithiocarbamate (DTC) incorporated
MTZ hybrids (c) resulted with increased trichomonacidal and spermicidal activities than
MTZ.[30] The possibility of drug resistance to nitroimidazole class of compounds necessitates
the discovery of non-nitroimidazole compounds. Recently, disulfiram like compounds with two
DTCs linked together directly at sulfur atom (a) also yielded potent topical dual function
microbicides.[31] However like Disulfiram, (which is a drug with two DTC groups and is in
clinical use for more than 50 years [32]), these compounds underwent rapid cleavage of S-S
bond and failed to show up in pharmacokinetic evaluation after vaginal administration. Since
pharmacokinetic evaluation is a mandatory step in modern drug development, it was envisaged
that modification of S-S bond to -S- bond which might lead to the discovery of a new class of
compounds with effective anti-Trichomonas and antifungal activities, and with suitable stability
for pharmacokinetic evaluation.
In this present study we evaluated the new compounds against MTZ-susceptible and -resistant
TV infections and some fungi that infect the female vaginal tract. A novel series of molecules
with improved efficacy over the previous drug designs is being reported along with their in vivo
biological activities, fluorescence labelling of biological targets and safety evaluation against
human epithelium cells (HeLa).
(Figure 1)
2. Results and Discussion:
2.1 Chemistry
The general procedure for the synthesis of substituted carbamothioic amine-1-carbothioic
thioanhydrides (3-21, 25-29 and 33-38) has been depicted in scheme 1. Initially the intermediates
sodium substituted amine-1-carbodithioates (2a-2s, 24a-24e and 31a-31f) were synthesized by
the earlier reported methods.[28, 33] In general, various secondary amines (1a-1s) on reaction
with carbon disulfide and aq. sodium hydroxide yield corresponding carbodithioates (2a-2s).
(Scheme 1)
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Further sodium 4-(tert-butoxycarbonyl)piperazine-1-carbodithioate on reaction with different
alkyl halides gave tert-butyl 4-(substitutedthiocarbonothioyl)piperazine-1-carboxylate (22a-22e).
Furthermore Boc removal followed by reaction with carbon disulfide and aq. NaOH yielded
sodium 4-(substituted thiocarbonothioyl)piperazine-1-carbodithioates (24a-24e). Piperazine on
reaction with different substituted isothiocyanates/isocyanates in ethanol yielded different
urea/thiourea derivatives (31a-31f) which on reaction with carbon disulfide and aq. NaOH gave
sodium 4-(substituted carbomothioyl)piperazine-1-carbodithioates (32a-32f). Further
intermediate compounds 2a-2s, 24a-24e and 31a-31f were on reaction with
o
dimethylcarbamothioic chloride in acetonitrile at 0 C for 1-2 hr yielded the final desired
products (3-21, 25-29 and 33-38) in good yields.
2.2 Biological Evaluation
2.2.1 Anti-Trichomonas activity of synthesized compounds
All the synthesized compounds (3-21, 25-29 and 33-38) were evaluated for their anti-
Trichomonas activity against MTZ-susceptible, -resistant strains and safety against cervico
epithelium cells (HeLa). All the compounds were found to be active at MIC range of 4.77–
294.11 µM and 32.46–735.20 µM against MTZ-susceptible and -resistant strains, respectively,
where MTZ showed activity at MIC 18.24 and 365.40 µM. Among these four compounds (12,
20, 21 and 34) were 1 to 3.8 fold more active against -susceptible strain while twenty-six
compounds (3-5, 7-15, 17-21, 26, 28, 29, and 33-38) were 1.3 to 11.2 fold more active against -
resistant strain than the standard MTZ. The most active compound (20) was 3.8 and 9.5 fold
more active than the MTZ against two strains tested. It was also interesting to note that while the
standard drug MTZ was twenty folds less active in resistant strain than in susceptible, compound
20 was only 8-fold less active. This could be attributed to the entirely new drug design.
Structure activity relationship (SAR) study: The scaffold studied was substituted carbamothioic
amine-1-carbothioic thioanhydrides (3-21, 25-29 and 33-38), which contained four prototypes by
varying substituents at -NR1R2. Firstly, a single nitrogen amines with cyclic (3-7) and acyclic (8-
10) framework were synthesized and then a two nitrogen system i.e., substituted piperazines (11-
21) were incorporated at -NR1R2. Thereafter a dithiocarbamate (25-29) and a thiourea (33-38)
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group were integrated at N4-position of piperazine core. The anti-Trichomonas activity data
revealed that amine substituents -NR1R2 played vital role for the biological activity.
(Table 1) (Figure 2)
Cyclic (3-7) and acyclic (8-10) amines at -NR1R2 showed good to moderate activity (MIC 25.20-
112.61 µM) against -susceptible strain while against -resistant strain all the compounds except
compound 6 showed 1.5 to 3.8 fold better activity than MTZ irrespective of cyclic or acyclic
amines. The replacement of single nitrogen amines by a two nitrogen containing piperazines the
compounds exhibited better activity profile. Of the eleven compounds (11-21) four compounds
were comparable (12, 13 and 21) or more active (20) than MTZ at MIC 4.77-20.42 µM. The
activity data suggested that a 2-pyrimidyl (20) substituent at N4-position was more desirable over
alkyl (11, 13)/aryl (15-19)/carboxy (12, 14)/furoyl (21) substitutents. Against –resistant strain
except compound 16 remaining ten compounds were 1.3 to 9.5 times more active than MTZ.
Here again 2-pyrimidyl (20) substituent at N4-position was essential for resistant anti-
Tichomonas activity. The order of activity with substituents -NR1R2 was 2-pyrimidyl (20)>Boc
(12)>phenyl (15)> n-butyl (13)>methyl (11)>3-CF3 phenyl (17)>2,3-dichlorophenyl (19)>furoyl
(21)>ethoxycarbonyl (14)>3-cholorophenyl (18)>2-methoxyphenyl (16). In the scaffold
carbamothioic amine-1-carbothioic thioanhydride attempts were made to hybridize active
pharmacophores, dithiocarbamate (25-29) and thiourea (33-37) moieties at N4-position of
piperazine. Against MTZ-susceptible strain the incorporation of thiourea moiety was more
beneficial over dithiocarbamate as most active compound i.e., with phenyl thiourea (38, MIC
8.10 µM) at N4-position was 2.2 fold more active and rest of the compounds were less active
than standard MTZ. Again the same pattern of activity was observed with resistant strain as
compound 34 was 11.4 fold more active than MTZ. The order of activity among dithiocarbamate
containing compounds was benzyl (29)>butyronitrile (28)>ethyl (26)>butyl (27)>methyl (25)
while in thiourea derivatives it was phenyl (34)>cyclohexyl (37)>butyl (33)>phenethyl
(36)>benzyl (35). Furthermore a modification of thiourea moiety with urea moiety (38) resulted
in loss of activity in both the strains tested suggests it least favorable for desired activity.
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2.2.2 Antifungal activity of synthesized compounds
All the synthesized compounds (3-21, 25-29 and 33-38) were also evaluated for antifungal
activity against ten fungal strains including three opportunistic fungal strains and seven Candida
strains. The activity data showed that twelve compounds (3-7, 9-13, 20 and 21) were active
against all the ten fungal strains while remaining compounds inhibited the growth of at least two
fungal strains tested in the range of 7.50-240.38 µM. The amine substituents at -NR1R2 played a
significant role for antifungal activity as like in case of anti-Trichomonas activity. Substitutions
at -NR1R2 with cyclic/acyclic secondary amines (3-11) were found to be active at MIC 11.90-
240.38 µM where various substituted piperazine compounds (12-21) showed activity in a range
of MIC 8.66-163.39 µM. Further a DTC incorporated piperazine compounds (25-29) have shown
activity at MIC 7.50-147.05 µM while thiourea/urea hybridized compounds (33-38) exhibited
fungicidal activity at MIC 125.31-136.98 µM except compound 36 which was inactive in all the
ten strains tested. These fungicidal results suggested that in the prototype where DTC was
incorporated in piperazine core at -NR1R2 was more favorable over other three prototypes. It was
interesting to note that except one compound (36), all the remaining twenty nine compounds
were found to be active against at least two opportunistic fungal starins; cryptococcus
neoformans, sporothrix schenckii, trichophyton mentagrophytes at MIC 15.86-240.38 µM. One
compound 7 against Candida glabrata and four compounds (18, 26, 27 and 29) against Candida
parapsilosis had activity comparable to standard Flucanazole. Many volvovaginal infections
were associated with Candida infections which coheres to vaginal epithelium cells enhances the
transmission of STIs. Among compounds evaluated, twelve (3-7, 9-13, 20 and 21) have shown
activity against all the candida strains tested. Rest of the compounds except compounds 28 and
34-36 were active against at least one out of seven different candida strains evaluated.
(Table 2)
Since the compounds exhibited good activity against the protozoan STD-pathogen (T. vaginalis).
We also tested some of the promising structures (12, 20, 21, 34) against P. falciparum to observe
if the effects are universal to all kinds of protozoa or specific only to Trichomonas.
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These compounds were less active than standard antimalarial chloroquine (Supplementary Data
Table 1).
2.2.3 Thiol Inhibition by Active Compound 20
:
The lack of glutathione makes T. vaginalis to depend on cysteine to overcome redox stress,
making very susceptible to sulfhydryl manipulating agents.[22] The DTC compounds were
known to be sulfhydryl binding agents. With the presence of DTC in the most active compound
20 it was evaluated for its sulfhydryl binding ability. The inhibition of sulfhydryl groups over
Trichomonas vaginalis were localized qualitatively by fluorescence detection after labeling them
with mBBr dye. Motile Trichomonas vaginalis (control) and compound 20 treated Trichomonas
were digitally imaged for qualitative assessment. From figure 2, a significant inhibition of free
thiol fluorescence was observed with compound 20 treatment. In control group due to the
presence of higher number of free sulfhydryl groups the fluorescence intensities was higher than
in compound 20 treated groups. The decreased fluorescence indicates the interaction of
compound 20 with the sulfhydryls present over Trichomonas, which might be its
trichomonacidal mechanism.
(Figure 3)
2.2.4 Docking study
Further a docking study was also carried out with most active compound (20) to ascertain their
cysteine biosynthesis pathway. Cysteine is vital for all living life forms for protein synthesis, as a
precursor for glutathione and biomolecules also as a source for synthesis of iron-sulfur clusters.
This series of compounds possibly exhibit anti-Trichomonas activity by inhibiting the cysteine
biosynthesis pathway. To acquire knowledge about the binding mode of the most promising
compound 20 found in this study, we have performed the docking study with the homology
model of Cysteine Synthase (CS). The validation of the resulting model was done with the
Structural Analysis and Verification Server (SAVS) [42]. In the selected model the majority of
the residues (97%) occupy the most favourable region of Ramachandran plot and 2% and 1%
residue lie in additionally allowed region and generously allowed region [31]. This model was
used for docking studies. The Pyridoxal-5'-phosphate (PLP) binding site was taken for the
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docking of the most active compound. It was observed that compound 20 docked well in this
cavity with binding energy of -8.40 kcal/mol. The binding mode of docked complex is shown in
Figure 4. It was found that compound 20 is involved in forming a hydrogen bond with
catalytically important residue Lys43, which is supposed to interact covalently with PLP. The
docked complex was further stabilized by interactions with residues Gly178, Thr182, Thr179 and
Ser180 present in the active site.
(Figure 4)
2.2.5 In vivo anti-Trichomonas activity of compound 20:
Due to its better in vitro activity and safety against HeLa cells, compound 20 was further
screened for in vivo anti-Trichomonas activity. The in vivo efficacy of compound was evaluated
using the mouse abscess assay. Subcutaneous injection of live trichomonads resulted in a small
pustule of ~50 mm2 area on day-7 of injection (day-1 of treatment) in experimental and control
animals that grew to ~96.55 mm2 in area in controls but was reduced to ~1-2.5 mm2 after 5-days
of treatment with compound and MTZ (Figure 4). Thereafter the growth of abscess was
exponential in controls and it was >100 mm2 in area after 7 days. However, it was reduced to
~0.8–2.5 mm2 in compound-20 treated animals. On the day of autopsy (i.e. the day following
seven days of treatment), the abscess area measured ~96.5 mm2 in control animals and 2.35 mm2
in 50 mg of MTZ, 1.57 and 0.79 mm2 in 50 mg and 100 mg of compound-20 treated animals,
respectively.
(Figure 5)
2.2.6 Pharmacokinetics study
A preliminary pharmacokinetic study of compound 20 involved monitoring drug substance in
blood plasma. The animals tolerated the treatment well as no peculiarities in the animals’
behaviour were observed. Following 50 mg/kg oral dose of compound 20, peak serum
concentration Cmax= 47.1±2.8 ng/mL was achieved at 2 hr and could be monitored in serum up to
6 hr post dose. The concentration-time profile was analyzed using non-compartmental approach
and the calculated pharmacokinetic parameters are shown in Table 3. The pharmacokinetic
models were compared according to maximal correlation between observed and predicted
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concentration, minimal sum of squared residuals, Akaike’s Information Criterion (AIC) and
Schwarz Bayesian Criterion (SBC) [34, 35]. The volume of distribution (12.6±0.6 L/kg) was
larger than the total blood volume of rat (0.054 L/kg; [36]) and systemic clearance (4.9±0.2
L/h/kg) was higher than the total hepatic blood flow in rats (2.9 L/h/kg; [36]) indicating
extravascular distribution along with the extra hepatic elimination of the compound.
(Table 3)
3. Conclusion
Sexually transmitted diseases like trichomoniasis along with opportunistic fungal infections like
candidiasis are major global concern in female reproductive health now days. The increased drug
resistance to available 5-nitroimidazole drugs i.e., Metronidazole, Tinidazole necessitates the
researchers for new drug development. Developing non-nitro imidazole molecules to overcome
the drug resistance would be a useful strategy. In the present study we developed a novel series
of non-nitroimidazole molecules and evaluated their trichomonicidal and fungicidal efficacies.
All the compounds were found to be trichomonicidal at MIC range of 4.77–294.11 µM and
32.46–735.20 µM against MTZ-susceptible and -resistant strains, respectively. MTZ showed
activity at MIC of 18.24 and 365.40 µM respectively against the strains. Four compounds (12,
20, 21 and 34) were up to 3.8 fold more active against -susceptible strain and twenty-six
compounds (3-5, 7-15, 17-21, 26, 28, 29 and 33-38) were up to 11.2 fold more active against
resistant strain than the standard MTZ. The most active compound (20) was 3.8 and 9.5 fold
more active than MTZ against susceptible and resistant strains, respectively. The absence of nitro
group in this novel series of compounds could be the reason for the activity of compounds
against resistant trichomoniasis. Twenty-nine compounds were found to be active against at least
two opportunistic fungal strains at MIC 15.86-240.38 µM. Many vulvovaginal infections are
associated with Candida infections, which cohere to vaginal epithelium cells and enhance the
transmission of STIs. Among the compounds evaluated twelve (3-7, 9-13, 20 and 21) have
shown activity against all the ten candida strains tested at MIC 7.50-200.00 µM. Compound 20
significantly inhibited the free sulfhydryl groups presented over Trichomonas in vitro, and
demonstrated significant elimination of infection in in vivo assay at doses 50 mg/kg and 100
mg/kg compound. Most active compounds also have shown moderate antimalarial activity. A
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preliminary pharmacokinetic study has shown good distribution, systemic clearance of
compound 20 in SD-rats. The present study has identified promising structures that could
conquer the drug-resistance in Trichomonas and their further lead optimization may lead to the
identification of more effective non-nitro imidazole class of anti-microbial compounds.
Experimental Section
Chemistry
In general, all reagents and solvents were of commercial quality and were used without
further purification. All reactions were monitored by thin-layer chromatography (TLC)
using F254 silica gel plates with fluorescence (Aldrich). Melting points were determined in
open capillary tubes on an electrically heated block and are uncorrected. IR spectra (νmax
in cm-1) of the compounds were recorded on Perkin Elmer FT-IR RX1 PC
1
spectrophotometer. H NMR spectra were recorded on Bruker Supercon Magnet Avance
1
DPX-200/DRX-300 spectrometers (operating at 400 and 100 MHz, respectively, for H
and 13C) in deuterated solvents with TMS as internal reference (chemical shifts
in Hz.). Electrospray ionization mass spectra (ESI-MS) were recorded on Ion Trap LCQ
δ in ppm,
J
Advantage Max-IT (Thermo Electron Corporation). High-resolution mass spectra
(HRMS) were recorded on a 6520 Agilent Q Tof LC MS/MS (accurate mass). Elemental
analyses were performed on a 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. All final compounds were found to have >95% purity.
Intermediate compounds 2a-2s, 24a-24e and 31a-31f Compounds were synthesized
according to a previously reported procedure.[28, 33]
General procedure for the synthesis of dimethylcarbamothioic pyrrolidine-1-
carbothioic thioanhydride (3):
To a solution of sodium pyrrolidine-1-carbodithioate (2a; 2 equiv.) in 10 mL acetonitrile
o
at 0-5 C was added dimethylcarbamothioic chloride (1 equiv.) and stirred the contents
for 1-2 hr. After completion of reaction (as monitored by TLC) acetonitrile was
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evaporated and excess distilled water (3 x 30 mL), 20 mL of EtOAc were added to the
reaction mixture. Organic layer was separated, dried (over Na2SO4) and concentrated
under reduced pressure to get the title compound
81 °C; IR (KBr)
(cm-1): 3019, 1218; 1H NMR (400 MHz, CDCl3):
2H), 3.73 (t,
MHz, CDCl3):
3 in 79% yields as yellow solid. mp: 79-
ν
δ
3.89 (t,
J
= 7.0 Hz,
13
J
= 6.8 Hz, 2H), 3.54 (s, 3H), 3.47 (s, 3H), 2.12–2.00 (m, 4H); C (100
δ
186.9, 183.2, 55.0, 52.8, 45.0, 44.3, 26.3, 24.7; ESI-MS: m/z 235
(M+H+); HRMS (ESI): m/z calculated for C8H14N2S3 + H+ (M+H+): 235.0392. Found:
235.0389. Elemental analysis (%) for C8H14N2S3: Calcd.: C, 40.99; H, 6.02; N, 11.95;
Found, C, 40.63; H, 6.38; N, 11.79.
The compounds 4-21, 25-29 and 33-38 were synthesized by using a procedure similar to
described for compound 3.
Dimethylcarbamothioic piperidine-1-carbothioic thioanhydride (4)
The title compound (4) was synthesized from sodium piperidine-1-carbodithioate (2b; 2
equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 71% yield as yellow solid. mp:
1
90-92 °C; IR (KBr)
ν
(cm-1): 3019, 1214; H NMR (400 MHz, CDCl3):
δ
8.02 (s, 1H),
4.26 (bs, 2H), 3.89 (bs, 2H), 3.53 (s, 3H), 3.44 (s, 3H), 1.74–1.61 (m, 6H); 13C (100 MHz,
CDCl3): 187.6, 185.7, 54.0, 52.2, 44.9, 44.0, 26.3, 25.2, 23.8; ESI-MS: m/z 249
δ
(M+H+); HRMS (ESI): m/z calculated for C9H16N2S3 + H+ (M+H+): 249.0548. Found:
249.0549. Elemental analysis (%) for C9H16N2S3: Calcd.: C, 43.51; H, 6.49; N, 11.28;
Found, C, 43.15; H, 6.76; N, 11.39.
Dimethylcarbamothioic 3-methylpiperidine-1-carbothioic thioanhydride (5)
The title compound (5) was synthesized from sodium 3-methylpiperidine-1-
carbodithioate (2c; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 82% yield
1
as yellow solid. mp: 95-97 °C; IR (KBr)
CDCl3):
2.83 (m, 1H), 1.91–1.83 (m, 3H), 1.72–1.65 (m, 2H), 0.98 (bs, 3H); C (100 MHz,
ν
(cm-1): 3021, 1216; H NMR (400 MHz,
δ
5.24 (bs, 1H), 4.42–4.30 (m, 1H), 3.54–3.44 (m, 6H), 3.27–3.19 (m, 1H), 3.01–
13
CDCl3):
δ
187.3, 185.8, 59.8, 58.0, 53.5, 51.8, 44.9, 44.0, 32.4, 32.0, 31.1, 25.7, 24.4,
11

ACCEPTED MANUSCRIPT
18.7; ESI-MS: m/z 263 (M+H+); HRMS (ESI): m/z calculated for C10H18N2S3 + H+
(M+H+): 263.0705. Found: 263.0724. Elemental analysis (%) for C10H18N2S3: Calcd.: C,
45.76; H, 6.91; N, 10.67; Found, C, 45.99; H, 7.21; N, 10.80.
Dimethylcarbamothioic morpholine-4-carbothioic thioanhydride (6)
The title compound (6) was synthesized from sodium morpholine-4-carbodithioate (2d; 2
equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 78% yield as yellow solid. mp:
1
128-130 °C; IR (KBr)
ν
(cm-1): 3019, 1215, 1104; H NMR (400 MHz, CDCl3):
δ
4.31–
13
3.99 (m, 4H), 3.83 (bs, 4H), 3.52 (s, 3H), 3.42 (s, 3H); C (100 MHz, CDCl3):
δ
187.5,
186.5, 66.1, 52.9, 50.9, 44.9, 43.6; ESI-MS: m/z 251 (M+H+); HRMS (ESI): m/z
calculated for C8H14N2OS3 + H+ (M+H+): 251.0341. Found: 251.0334. Elemental analysis
(%) for C8H14N2OS3: Calcd.: C, 38.37; H, 5.64; N, 11.19; Found, C, 38.01; H, 5.98; N,
11.32.
Azepane-1-carbothioic dimethylcarbamothioic thioanhydride (7)
The title compound (
equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 85% yield as yellow solid. mp:
75-77 °C; IR (KBr) 4.14 (t, = 6.0
(cm-1): 3010, 1216; 1H NMR (400 MHz, CDCl3):
= 6.0 Hz, 2H), 3.54 (s, 3H), 3.47 (s, 3H), 1.91–1.84 (m, 4H), 1.64–1.61
187.2, 187.0, 55.0, 54.8, 45.0, 44.5, 28.0, 26.9, 26.4,
7) was synthesized from sodium azepane-1-carbodithioate (2e; 2
ν
δ
J
Hz, 2H), 3.87 (t,
J
13
(m, 4H); C (100 MHz, CDCl3):
δ
25.8; ESI-MS: m/z 263 (M+H+); HRMS (ESI): m/z calculated for C10H18N2S3 + H+
(M+H+): 263.0705. Found: 263.0702. Elemental analysis (%) for C10H18N2S3: Calcd.: C,
45.76; H, 6.91; N, 10.67; Found, C, 45.45; H, 7.22; N, 10.49.
Diethyl-1-carbothioic dimethylcarbamothioic thioanhydride (8)
The title compound (8) was synthesized from sodium diethylcarbamodithioate (2f; 2
equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 70% yield as yellow solid. mp:
1
84-86 °C; IR (KBr)
ν
(cm-1): 3020, 1211; H NMR (400 MHz, CDCl3):
δ
4.02–3.97 (m,
13
2H), 3.76–3.68 (m, 2H), 3.53 (s, 3H), 3.45 (s, 3H), 1.35–1.29 (m, 6H); C (100 MHz,
CDCl3):
δ
187.1, 186.5, 49.0, 48.9, 45.0, 44.4, 13.2, 11.1; ESI-MS: m/z 237 (M+H+);
12

ACCEPTED MANUSCRIPT
Elemental analysis (%) for C8H16N2S32: Calcd.: C, 40.64; H, 6.82; N, 11.85; Found, C,
40.50; H, 6.99; N, 11.61.
Dimethyl-1-carbothioic thioanhydride (9)
The title compound (9) was synthesized from sodium dimethylcarbamodithioate (2g; 2
equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 68% yield as yellow solid. mp:
1
89-91 °C; IR (KBr)
ν
(cm-1): 3019, 1218; H NMR (400 MHz, CDCl3):
δ
3.53 (s, 6H),
13
3.44 (s, 6H); C (100 MHz, CDCl3):
δ
187.3, 44.9, 44.0; ESI-MS: m/z 209 (M+H+);
HRMS (ESI): m/z calculated for C6H12N2S3 + H+ (M+H+): 209.0241. Found: 209.0282.
Elemental analysis (%) for C6H12N2S3: Calcd.: C, 34.59; H, 5.80; N, 13.44; Found, C,
34.37; H, 5.99; N, 13.56.
Dimethylcarbamothioic ethyl(methyl)-4-carbothioic thioanhydride (10)
The title compound (10) was synthesized from sodium ethyl(methyl)carbamodithioate
(
2h; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 84% yield as yellow
solid. mp: 85-87 °C; IR (KBr) 4.12–
(cm-1): 3010, 1210; 1H NMR (400 MHz, CDCl3):
4.03 (m, 1H), 3.82–3.77 (m, 1H), 3.55–3.37 (m, 9H), 1.31 (t,
MHz, CDCl3): 187.3, 187.1, 51.8, 51.5, 44.9, 44.0, 42.4, 41.1, 12.9, 10.7; ESI-MS: m/z
ν
δ
13
J
= 7.1 Hz, 3H); C (100
δ
223 (M+H+); Elemental analysis (%) for C7H14N2S3: Calcd.: C, 37.80; H, 6.35; N, 12.60;
Found, C, 37.54; H, 6.70; N, 12.84.
Dimethylcarbamothioic 4-methylpiperazine-1-carbothioic thioanhydride (11)
The title compound (11) was synthesized from sodium 4-methylpiperazine-1-
carbodithioate (2i; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 88% yield
as yellow solid. mp: 70-72 °C; IR (KBr)
CDCl3):
(100 MHz, CDCl3):
ν
(cm-1): 3011, 1218, 1167; 1H NMR (400 MHz,
13
δ
4.34–3.98 (m, 4H), 3.53 (s, 3H), 3.44 (s, 3H), 2.58 (s, 4H), 2.36 (s, 3H); C
δ
187.4, 186.7, 54.4, 53.9, 52.2, 50.5, 45.3, 44.9, 43.8; ESI-MS: m/z
264 (M+H+); HRMS (ESI): m/z calculated for C9H17N3S3 + H+ (M+H+): 264.0657. Found:
264.0656. Elemental analysis (%) for C9H17N3S3: Calcd.: C, 41.03; H, 6.50; N, 15.95;
Found, C, 41.25; H, 6.76; N, 15.59.
13

ACCEPTED MANUSCRIPT
4-(tert-Butoxycarbonyl)piperazine-1-carbothioic
thioanhydride (12)
The title compound
butoxycarbonyl)piperazine-1-carbodithioate (2j; 2 equiv.) and dimethylcarbamothioic
dimethylcarbamothioic
(12
)
was
synthesized
from
sodium
4-(tert
-
chloride (1 equiv.) in 85% yield as yellow solid. mp: 201-203 °C; IR (KBr)
ν
(cm-1):
1
3010, 1730, 1216, 1020; H NMR (400 MHz, CDCl3):
δ
4.29–3.97 (m, 4H), 3.64–3.61
187.5, 186.7,
13
(m, 4H), 3.53 (s, 3H), 3.42 (s, 3H), 1.49 (s, 9H); C (100 MHz, CDCl3):
δ
154.3, 80.6, 52.1, 50.6, 44.9, 43.6, 42.7, 28.3; ESI-MS: m/z 350 (M+H+); HRMS (ESI):
m/z calculated for C13H23N3O2S3 + H+ (M+H+): 350.1025. Found: 350.1029. Elemental
analysis (%) for C13H23N3O2S3: Calcd.: C, 44.67; H, 6.63; N, 12.02; Found, C, 44.39; H,
6.89; N, 12.15.
4-Butylpiperazine-1-carbothioic dimethylcarbamothioic thioanhydride (13)
The title compound (13) was synthesized from sodium 4-butylpiperazine-1-carbodithioate
(
2k; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 77% yield as yellow
1
solid. mp: 102-104 °C; IR (KBr)
ν
(cm-1): 3015, 1215, 1165; H NMR (400 MHz,
CDCl3):
δ
4.32–3.96 (m, 4H), 3.53 (s, 3H), 3.43 (s, 3H), 2.59 (bs, 4H), 2.42–2.38 (m,
13
2H), 1.53–1.45 (m, 2H), 1.40–1.33 (m, 2H), 0.94 (t,
J = 7.3 Hz, 3H); C (100 MHz,
CDCl3):
δ
187.4, 186.4, 57.6, 52.7, 52.1, 50.6, 44.9, 43.8, 28.9, 20.5, 13.9; ESI-MS: m/z
306 (M+H+); Elemental analysis (%) for C12H23N3S3: Calcd.: C, 47.17; H, 7.59; N, 13.75;
Found, C, 47.00; H, 7.86; N, 13.57.
4-(Ethoxycarbonyl) piperazine-1-carbothioic dimethylcarbamothioic thioanhydride
(14)
The title compound (14) was synthesized from sodium 4-(ethoxycarbonyl)piperazine-1-
carbodithioate (2l; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 80% yield
1
as yellow solid. mp: 112-114 °C; IR (KBr)
ν
(cm-1): 3011, 1736, 1216; H NMR (400
MHz, CDCl3):
δ
4.25–3.99 (m, 6H), 3.67–3.64 (m, 4H), 3.50 (s, 3H), 3.40 (s, 3H), 1.27 (t,
13
J
= 7.1 Hz, 3H); C (100 MHz, CDCl3):
δ
187.5, 186.8, 155.5, 61.8, 51.9, 50.6, 44.9,
14

ACCEPTED MANUSCRIPT
43.6, 42.9, 14.6; ESI-MS: m/z 322 (M+H+); Elemental analysis (%) for C11H19N3O2S3:
Calcd.: C, 41.10; H, 5.96; N, 13.07; Found, C, 41.00; H, 6.06; N, 13.10.
Dimethylcarbamothioic 4-phenylpiperazine-1-carbothioic thioanhydride (15)
The title compound (15) was synthesized from sodium 4-phenylpiperazine-1-
carbodithioate (2m; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 79% yield
as yellow solid. mp: 88-90 °C; IR (KBr)
ν
(cm-1): 3410, 1427, 1215, 1150; 1H NMR (400
MHz, CDCl3):
δ
7.33–7.29 (m, 2H), 6.95–6.92 (m, 3H), 4.46–4.11 (m, 4H), 3.54 (s, 3H),
13
3.45 (s, 3H), 3.39–3.37 (m, 4H); C (100 MHz, CDCl3):
δ 187.5, 186.5, 150.1, 129.3,
120.6, 116.3, 50.6, 49.1, 48.4, 44.9, 43.8; ESI-MS: m/z 326 (M+H+); HRMS (ESI): m/z
calculated for C14H19N3S3 + H+ (M+H+): 326.0814. Found: 326.0820. Elemental analysis
(%) for C14H19N3S3: Calcd.: C, 51.66; H, 5.88; N, 12.91; Found, C, 51.59; H, 6.11; N,
12.83.
4-(2-Methoxyphenyl)piperazine-1-carbothioic dimethylcarbamothioic thioanhydride
(16)
The title compound (16) was synthesized from sodium 4-(2-methoxyphenyl)piperazine-1-
carbodithioate (2n; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 81% yield
1
as yellow solid. mp: 170-172 °C; IR (KBr)
ν
(cm-1): 2925, 1468, 1225, 1026; H NMR
(400 MHz, CDCl3):
δ
7.09–7.05 (m, 1H), 6.98–6.91 (m, 3H), 4.51 (bs, 2H), 4.17–4.11 (m,
13
2H), 3.90 (s, 3H), 3.55–3.46 (m, 6H), 3.23 (s, 4H); C (100 MHz, CDCl3):
δ 187.5,
186.5, 152.2, 139.9, 123.8, 121.1, 118.5, 111.4, 55.4, 52.8, 50.9, 50.4, 49.8, 44.9, 43.8;
ESI-MS: m/z 356 (M+H+); HRMS (ESI): m/z calculated for C15H21N3OS3 + H+ (M+H+):
356.0920. Found: 356.0919. Elemental analysis (%) for C15H21N3OS3: Calcd.: C, 50.67;
H, 5.95; N, 11.82; Found, C, 50.50; H, 6.15; N, 11.78.
4-(2-(Trifluoromethyl)phenyl)piperazine-1-carbothioic
thioanhydride (17)
dimethylcarbamothioic
The
title
compound
(
17
)
was
synthesized
2o
from
2
sodium
equiv.)
4-(2-
and
(trifluoromethyl)phenyl)piperazine-1-carbodithioate
(
;
dimethylcarbamothioic chloride (1 equiv.) in 75% yield as yellow solid. mp: 95-97 °C; IR
15

ACCEPTED MANUSCRIPT
1
(KBr)
ν
(cm-1): 3020, 1429, 1215, 1028; H NMR (400 MHz, CDCl3):
δ
7.40 (t,
J
= 7.9
13
Hz, 1H), 7.16–7.06 (m, 3H), 4.48–4.16 (m, 4H), 3.54 (s, 3H), 3.46–3.45 (m, 7H); C
(100 MHz, CDCl3):
δ 187.5, 186.6, 150.2, 132.0, 131.7, 131.4, 131.0, 129.8, 125.5,
122.8, 118.8, 116.5, 116.5, 112.1, 112.1, 51.9, 50.4, 48.1, 44.9, 43.7; ESI-MS: m/z 394
(M+H+); HRMS (ESI): m/z calculated for C15H18F3N3S3 + H+ (M+H+): 394.0688. Found:
394.0692. Elemental analysis (%) for C15H18F3N3S3: Calcd.: C, 45.78; H, 4.61; N, 10.68;
Found, C, 45.47; H, 4.90; N, 10.50.
4-(3-Chlorophenyl)piperazine-1-carbothioic dimethylcarbamothioic thioanhydride
(18)
The title compound (18) was synthesized from sodium 4-(2-chlorophenyl)piperazine-1-
carbodithioate (2p; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 73% yield
1
as yellow solid. mp: 90-92 °C; IR (KBr)
ν
(cm-1): 3021, 1215, 1152, 758; H NMR (400
MHz, CDCl3):
δ
7.23–7.19 (m, 1H), 6.90–6.87 (m, 2H), 6.82–6.77 (m, 1H), 4.48–4.43
13
(m, 2H), 4.18–4.11 (m, 2H), 3.54 (s, 3H), 3.44 (m, 3H), 3.41–3.38 (m, 4H); C (100
MHz, CDCl3):
δ 187.5, 186.6, 151.0, 135.1, 130.3, 120.1, 115.9, 113.9, 51.1, 48.6, 48.5,
44.9, 43.7; ESI-MS: m/z 360 (M+H+); HRMS (ESI): m/z calculated for C14H18ClN3S3 +
H+ (M+H+): 360.0424. Found: 360.0424. Elemental analysis (%) for C14H18ClN3S32:
Calcd.: C, 46.71; H, 5.04; N, 11.67; Found, C, 46.90; H, 5.37; N, 11.50.
4-(2,3-Dichlorophenyl)piperazine-1-carbothioic
thioanhydride (19)
dimethylcarbamothioic
The title compound (19) was synthesized from sodium 4-(2,3-dichlorophenyl)piperazine-
1-carbodithioate (2q; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 80%
1
yield as yellow solid. mp: 235-237 °C; IR (KBr)
NMR (400 MHz, CDCl3): 7.25–7.17 (m, 2H), 6.96 (d/d,
4.51 (m, 2H), 4.18–4.11 (m, 2H), 3.55 (s, 3H), 3.45 (m, 3H), 3.22–3.20 (m, 4H); 13C (100
MHz, CDCl3): 187.5, 186.8, 149.9, 134.2, 127.7, 127.6, 125.5, 118.8, 52.8, 51.0, 50.4,
ν
(cm-1): 3019, 1423, 1215, 757; H
δ
J = 1.7 & 7.8 Hz, 1H), 4.52–
δ
44.9, 43.7; ESI-MS: m/z 394 (M+H+); HRMS (ESI): m/z calculated for C14H17Cl2N3S3 +
H+ (M+H+): 394.0034. Found: 394.0035. Elemental analysis (%) for C14H17Cl2N3S3:
Calcd.: C, 42.63; H, 4.34; N, 10.65; Found, C, 42.79; H, 4.60; N, 10.39.
16

ACCEPTED MANUSCRIPT
Dimethylcarbamothioic 4-(pyrimidin-2-yl)piperazine-1-carbothioic thioanhydride
(20)
The title compound (20) was synthesized from sodium 4-(2,3-dichlorophenyl)piperazine-
1-carbodithioate (2r; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 84%
1
yield as yellow solid. mp: 180-182 °C; IR (KBr)
NMR (400 MHz, CDCl3): 8.36 (d, = 4.7 Hz, 2H), 6.59 (t,
(m, 8H), 3.54 (s, 3H), 3.45 (m, 3H); 13C (100 MHz, CDCl3):
ν
(cm-1): 3019, 1500, 1427, 1215; H
δ
J
J
= 4.7 Hz, 1H), 4.39–4.03
δ
187.4, 186.6, 161.1, 157.8,
110.7, 52.0, 50.7, 44.9, 43.7, 42.5; ESI-MS: m/z 328 (M+H+); HRMS (ESI): m/z
calculated for C12H17N5S3 + H+ (M+H+): 328.0719. Found: 328.0718. Elemental analysis
(%) for C12H17N5S3: Calcd.: C, 44.01; H, 5.23; N, 21.38; Found, C, 44.30; H, 5.55; N,
21.09.
4-(furan-2-carbonyl)piperazine-1-carbothioic dimethylcarbamothioic thioanhydride
(21)
The title compound (21) was synthesized from sodium 4-(furan-2-yl)piperazine-1-
carbodithioate (2s; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 77% yield
as yellow solid. mp: 84-86 °C; IR (KBr)
ν
(cm-1): 3017, 1422, 1216, 1090; 1H NMR (400
MHz, CDCl3):
δ
7.52 (d,
J
= 0.7 Hz, 1H), 7.13–7.11 (m, 1H), 6.54–6.52 (m, 1H), 4.36–
13
4.03 (m, 8H), 3.53 (s, 3H), 3.43 (m, 3H); C (100 MHz, CDCl3):
δ 187.6, 186.8, 159.0,
147.4, 144.2, 117.5, 111.6, 51.8, 50.7, 45.6, 44.9, 43.5; ESI-MS: m/z 418 (M+H+);
Elemental analysis (%) for C13H17N3O2S3: Calcd.: C, 45.46; H, 4.99; N, 12.23; Found, C,
45.29; H, 5.33; N, 12.00.
Dimethylcarbamothioic
thioanhydride (25)
4-(methylthiocarbonothioyl)piperazine-1-carbothioic
The
title
compound
(
25
)
was
synthesized
24a
from
2
sodium
equiv.)
4-
(methylthiocarbonothioyl)piperazine-1-carbodithioate
(
;
and
dimethylcarbamothioic chloride (1 equiv.) in 77% yield as yellow solid. mp: 235-237 °C;
IR (KBr)
ν
(cm-1): 3022, 1215; 1H NMR (400 MHz, CDCl3):
δ
4.35–4.11 (m, 8H), 3.53–
17

ACCEPTED MANUSCRIPT
13
3.42 (m, 6H), 2.70 (s, 3H); C (100 MHz, CDCl3):
δ 199.0, 187.6, 186.5, 50.4, 48.5,
45.0, 43.5, 20.0; ESI-MS: m/z 340 (M+H+); Elemental analysis (%) for C10H17N3S5:
Calcd.: C, 35.37; H, 5.05; N, 12.37; Found, C, 35.09; H, 5.31; N, 12.12.
4-(Ethylthiocarbonothioyl)piperazine-1-carbothioic
thioanhydride (26)
dimethylcarbamothioic
The
title
compound
(
26
)
was
synthesized
24b
from
2
sodium
equiv.)
4-
(ethylthiocarbonothioyl)piperazine-1-carbodithioate
(
;
and
dimethylcarbamothioic chloride (1 equiv.) in 70% yield as yellow solid. mp: 98-100 °C;
IR (KBr)
3.53–3.42 (m, 6H), 3.33 (q,
MHz, CDCl3):
ν
(cm-1): 3015, 1218, 1150; 1H NMR (400 MHz, CDCl3):
= 7.4 & 14.8 Hz, 2H), 1.38 (t, J = 7.4 Hz, 3H); C (100
δ
4.43–4.18 (m, 8H),
13
J
δ
198.1, 187.6, 186.5, 50.3, 48.0, 45.0, 43.5, 31.5, 13.6; ESI-MS: m/z 354
(M+H+); Elemental analysis (%) for C11H19N3S5: Calcd.: C, 37.36; H, 5.42; N, 11.88;
Found, C, 37.45; H, 5.75; N, 11.70.
4-(Butylthiocarbonothioyl)piperazine-1-carbothioic
thioanhydride (27)
dimethylcarbamothioic
The
title
compound
(
27
)
was
synthesized
24c
from
2
sodium
equiv.)
4-
(butylthiocarbonothioyl)piperazine-1-carbodithioate
(
;
and
dimethylcarbamothioic chloride (1 equiv.) in 76% yield as yellow solid. mp: 85-87 °C; IR
1
(KBr)
ν
(cm-1): 3022, 1220, 1129; H NMR (400 MHz, CDCl3):
δ
4.34–4.18 (m, 8H),
3.53–3.42 (m, 6H), 3.33 (t,
J
= 7.4 Hz, 2H), 1.75–1.67 (m, 2H), 1.51–1.42 (m, 2H), 0.96
13
(t,
J = 7.3 Hz, 3H); C (100 MHz, CDCl3): δ 198.4, 187.6, 50.4, 45.0, 43.5, 37.0, 30.5,
22.1, 13.6; ESI-MS: m/z 382 (M+H+); HRMS (ESI): m/z calculated for C13H23N3S5 + H+
(M+H+): 382.0568. Found: 382.0567. Elemental analysis (%) for C13H23N3S5: Calcd.: C,
40.91; H, 6.07; N, 11.01; Found, C, 40.70; H, 6.30; N, 11.13.
4-((3-cyanopropylthio)carbonothioyl)piperazine-1-carbothioic
dimethylcarbamothioic thioanhydride (28)
The
title
compound
(
28
)
was
synthesized
from
24d
sodium
4-((3-
cyanopropylthio)carbonothioyl)piperazine-1-carbodithioate
(
;
2
equiv.) and
18

ACCEPTED MANUSCRIPT
dimethylcarbamothioic chloride (1 equiv.) in 69% yield as yellow solid. mp: 91-93 °C; IR
1
(KBr)
3.54–3.42 (m, 8H), 2.51 (t,
MHz, CDCl3):
ν
(cm-1): 3020, 2231, 1216; H NMR (400 MHz, CDCl3):
δ
4.37–4.12 (m, 8H),
13
J
= 7.2 Hz, 2H), 2.12 (q, J = 7.1 & 14.2 Hz, 2H); C (100
δ
196.6, 187.6, 187.3, 51.2, 50.1, 45.0, 43.5, 35.0, 28.8, 16.3; ESI-MS: m/z
393 (M+H+); Elemental analysis (%) for C13H20N4S5: Calcd.: C, 39.77; H, 5.13; N, 14.27;
Found, C, 39.51; H, 5.41; N, 14.12.
4-(Benzylthiocarbonothioyl)piperazine-1-carbothioic
thioanhydride (29)
dimethylcarbamothioic
The
title
compound
(
29
)
was
synthesized
24e
from
2
sodium
equiv.)
4-
(benzylthiocarbonothioyl)piperazine-1-carbodithioate
(
;
and
dimethylcarbamothioic chloride (1 equiv.) in 80% yield as yellow solid. mp: 83-85 °C; IR
1
(KBr)
ν
(cm-1): 3410, 1639, 1442, 1216; H NMR (400 MHz, CDCl3):
δ
7.40 (d,
J
= 7.5
Hz, 2H), 7.36–7.29 (m, 3H), 4.60 (s, 2H), 4.35–4.13 (m, 8H), 3.54–3.53 (m, 3H), 3.45–
13
3.42 (m, 3H); C (100 MHz, CDCl3):
δ 197.4, 187.6, 186.5, 135.5, 129.3, 128.6, 127.7,
50.1, 45.0, 43.5, 42.1; ESI-MS: m/z 416 (M+H+); HRMS (ESI): m/z calculated for
C16H21N3S5 + H+ (M+H+): 416.0412. Found: 416.0412. Elemental analysis (%) for
C16H21N3S5: Calcd.: C, 46.23; H, 5.09; N, 10.11; Found, C, 46.01; H, 5.28; N, 10.25.
4-(Butylcarbamothioyl)piperazine-1-carbothioic
thioanhydride (33)
dimethylcarbamothioic
The title compound (33) was synthesized from sodium 4-(butylcarbamothioyl)piperazine-
1-carbodithioate (32a; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 76%
1
yield as yellow solid. mp: 108-110 °C; IR (KBr)
ν
(cm-1): 3018, 1216, 1192; H NMR
(400 MHz, CDCl3):
δ
5.48 (s, 1H), 4.35–4.08 (m, 8H), 3.71–3.66 (m, 2H), 3.53 (s, 3H),
13
3.43 (s, 3H), 1.68–1.60 (m, 2H), 1.46–1.36 (m, 2H), 0.97 (t,
J = 7.3 Hz, 3H); C (100
MHz, CDCl3):
δ
187.4, 186.5, 181.9, 50.6, 50.0, 46.1, 45.0, 43.6, 31.3, 20.1, 13.8; ESI-
MS: m/z 365 (M+H+); HRMS (ESI): m/z calculated for C13H24N4S4 + H+ (M+H+):
365.0957. Found: 365.0948. Elemental analysis (%) for C13H24N4S4: Calcd.: C, 42.82; H,
6.63; N, 15.37; Found, C, 42.59; H, 6.88; N, 15.09.
19

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Dimethylcarbamothioic
thioanhydride (34)
4-(phenylcarbamothioyl)piperazine-1-carbothioic
The
title
compound
(
34
)
was
synthesized
32b
from
2
sodium
equiv.)
4-
(phenylcarbamothioyl)piperazine-1-carbodithioate
(
;
and
dimethylcarbamothioic chloride (1 equiv.) in 83% yield as yellow solid. mp: 82-84 °C; IR
(KBr)
(cm-1): 3410, 1490, 1216, 1194, 1127; 1H NMR (400 MHz, CDCl3+DMSO-d6):
8.83 (s, 1H), 7.14–7.13 (m, 4H), 6.99 (bs, 1H), 4.15–3.92 (m, 8H), 3.32–3.23 (m, 6H);
13C (100 MHz, CDCl3+DMSO-d6):
192.1, 191.1, 187.1, 145.3, 133.1, 130.7, 130.1,
ν
δ
δ
55.8, 55.2, 51.9, 49.7, 48.4; ESI-MS: m/z 385 (M+H+); HRMS (ESI): m/z calculated for
C15H20N4S4 + H+ (M+H+): 385.0644. Found: 385.0635. Elemental analysis (%) for
C15H20N4S4: Calcd.: C, 46.84; H, 5.24; N, 14.57; Found, C, 46.50; H, 5.49; N, 14.40.
4-(Benzylcarbamothioyl)piperazine-1-carbothioic
thioanhydride (35)
dimethylcarbamothioic
The
title
compound
(
35
)
was
synthesized
32c
from
2
sodium
equiv.)
4-
(benzylcarbamothioyl)piperazine-1-carbodithioate
(
;
and
dimethylcarbamothioic chloride (1 equiv.) in 83% yield as yellow solid. mp: 218-220 °C;
1
IR (KBr)
ν
(cm-1): 3410, 1639, 1402, 1216, 1185; H NMR (400 MHz, CDCl3):
δ
7.35–
7.27 (m, 4H), 6.20–6.07 (m, 1H), 4.87–4.86 (m, 2H), 4.27–4.08 (m, 8H), 3.50–3.39 (m,
6H); 13C (100 MHz, CDCl3):
187.4, 186.5, 182.0, 137.6, 128.8, 128.0, 50.2, 46.3, 45.0,
δ
43.6, 30.9; ESI-MS: m/z 399 (M+H+); HRMS (ESI): m/z calculated for C16H22N4S4 + H+
(M+H+): 399.0800. Found: 399.0791. Elemental analysis (%) for C16H22N4S4: Calcd.: C,
48.21; H, 5.56; N, 14.05; Found, C, 48.02; H, 5.85; N, 14.29.
Dimethylcarbamothioic
thioanhydride (36)
4-(phenethylcarbamothioyl)piperazine-1-carbothioic
The
title
compound
(
36
)
was
synthesized
32d
from
2
sodium
equiv.)
4-
(phenethylcarbamothioyl)piperazine-1-carbodithioate
(
;
and
dimethylcarbamothioic chloride (1 equiv.) in 70% yield as yellow solid. mp: 85-87 °C; IR
(KBr) 7.36–7.31
(cm-1): 3410, 1639, 1402, 1216, 1185; 1H NMR (400 MHz, CDCl3):
ν
δ
(m, 2H), 7.26–7.22 (m, 3H), 5.54–5.53 (m, 1H), 4.38–3.93 (m, 10H), 3.52–3.41 (m, 6H),
20

ACCEPTED MANUSCRIPT
13
3.00–2.96 (m, 2H); C (100 MHz, CDCl3):
δ 187.4, 186.5, 181.8, 138.7, 128.7, 126.7,
50.6, 50.0, 47.0, 45.9, 45.0, 43.7, 35.1; ESI-MS: m/z 413 (M+H+); HRMS (ESI): m/z
calculated for C17H24N4S4 + H+ (M+H+): 413.0957. Found: 413.0946. Elemental analysis
(%) for C17H24N4S4: Calcd.: C, 49.48; H, 5.86; N, 13.58; Found, C, 49.32; H, 6.11; N,
13.31.
4-(Cyclohexylcarbamothioyl)piperazine-1-carbothioic
thioanhydride (37)
dimethylcarbamothioic
The
title
compound
(
37
)
was
synthesized
32e
from
2
sodium
equiv.)
4-
(cyclohexylcarbamothioyl)piperazine-1-carbodithioate
(
;
and
dimethylcarbamothioic chloride (1 equiv.) in 70% yield as yellow solid. mp: 238-240 °C;
IR (KBr) 5.32–5.30 (m, 1H),
(cm-1): 3019, 1216, 1190; 1H NMR (400 MHz, CDCl3):
ν
δ
4.36–4.06 (m, 8H), 3.68–3.60 (m, 1H), 3.53–3.42 (m, 6H), 2.14–2.09 (m, 2H), 1.76–1.65
13
(m, 3H), 1.48–1.38 (m, 2H), 1.15–1.24 (m, 3H); C (100 MHz, CDCl3):
δ 187.5, 186.5,
180.7, 54.6, 50.6, 49.9, 45.8, 45.0, 43.6, 33.0, 25.5, 24.9; ESI-MS: m/z 391 (M+H+);
HRMS (ESI): m/z calculated for C15H26N4S4 + H+ (M+H+): 391.1113. Found: 391.1123.
Elemental analysis (%) for C15H26N4S4: Calcd.: C, 46.12; H, 6.71; N, 14.34; Found, C,
46.20; H, 6.99; N, 14.06.
4-(Cyclohexylcarbamoyl)piperazine-1-carbothioic
thioanhydride (38)
dimethylcarbamothioic
The
title
compound
(
38
)
was
synthesized
32f
from
2
sodium
equiv.)
4-
(cyclohexylcarbamoyl)piperazine-1-carbodithioate
(
;
and
dimethylcarbamothioic chloride (1 equiv.) in 73% yield as yellow solid. mp: 109-111 °C;
1
IR (KBr)
ν
(cm-1): 3020, 1215, 1150; H NMR (400 MHz, CDCl3):
δ
4.30 (s, 3H), 4.14–
4.02 (m, 2H), 3.67–3.60 (m, 4H), 3.54–3.53 (m, 3H), 3.45–3.43 (m, 3H), 1.98–1.96 (m,
2H), 1.74–1.71 (m, 2H), 1.64 (s, 1H), 1.43–1.35 (m, 2H), 1.30 (s, 1H), 1.27–1.08 (m,
13
3H); C (100 MHz, CDCl3):
δ 187.5, 186.7, 156.5, 51.6, 50.4, 49.6, 44.9, 43.7, 42.6,
33.9, 25.6, 25.0; ESI-MS: m/z 418 (M+H+); HRMS (ESI): m/z calculated for
C15H26N4OS3 + H+ (M+H+): 375.1341. Found: 375.1348. Elemental analysis (%) for
C15H26N4OS3: Calcd.: C, 48.10; H, 7.00; N, 14.96; Found, C, 48.22; H, 7.31; N, 14.64.
21

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Biological Materials and Methods:
Anti-Trichomonas assay
Metronidazole-susceptible T. vaginalis strain was a clinical isolate obtained from the
laboratory of Divya Singh [37] and metronidazole-resistant strain of T. vaginalis
(CDC085 [ATCC 50143]) was procured from American Type Culture Collection. In vitro
drug susceptibility of Trichomonas vaginalis was assayed as detailed earlier.[16] Briefly,
parasites were incubated at 37°C in the presence of test compounds or MTZ, serially
diluted from a stock-solution in DMSO using the culture medium, in 48-well culture
plates. 0.05% DMSO in culture media (the highest concentration of DMSO in test wells)
was used as vehicle in control wells. Cell viability was checked after 48 h by trypan blue
exclusion assay. The minimum concentration of the test agent at which all cells were
found dead was considered as its MIC. MTZ (the most widely used drug against
Trichomonas vaginalis) was procured from Sigma-Aldrich, and used as reference
standard. All experiments were repeated three times (Table 1).
Antifungal assay
The MIC of compounds were determined by broth micro-dilution technique as per the
guidelines of National Committee for Clinical Laboratory Standards using RPMI 1640
media buffered with MOPS [3-(N-Morpholino)propanesulfonic acid]. Starting inoculums
of test culture was 1–5 x 103 CFU/mL. Micro titer plates were incubated at 35 °C. MICs
were recorded after 48h of incubation. All experiments were repeated three times.[22]
(Table 2).
Cytotoxicity of compounds toward human cervical (HeLa) cells
The cytotoxic effect of test compounds were evaluated in an in vitro model of
cervicovaginal epithelium (HeLa) cells, using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,
5-diphenyl tetrazolium bromide] assay.[24] Cells seeded in 96 well plates were incubated
in culture medium (DMEM with 10% fetal calf serum) for 24 h at 37°C in a 5% CO2–
95% air atmosphere. After 24 h, the culture medium was replaced with fresh medium
containing serial dilutions of test compounds starting with 1000 µg/ml in experimental
22

ACCEPTED MANUSCRIPT
wells and 0.05% DMSO in culture medium in control wells. After incubation for another
24 h, 5 µL of 5 mg/mL MTT solution in PBS [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). All experiments were repeated three times. (Table 1)
Qualitative estimation of inhibition of free sulfhydryl groups on Trichomonas
vaginalis
The effect of test compound on Trichomonas free sulfhydryl groups was examined and
imaged by a method published earlier with slight modification [38] using a fluorescence
microscope, after labelling with the fluorometric thiol detector using a thiol –detection
assay kit (Cayman). Trichomonas vaginalis was treated with the vehicle or the test
compound at MIC and incubated for 24 h at 37°C. After incubation, trichomonads were
pelleted at 700 xg for 10 min at 4°C and washed 2-3 times with PBS. Thereafter 50 µL
fluorometric thiol detector (pre-diluted 100x with dilution buffer), was added and
incubated in the dark. A drop of this sample was taken on a microscope slide, covered
with a coverslip and imaged on a Nikon Eclipse 80i microscope equipped with
epifluoresence illumination, using the UV-1A filter. Exposure times were the same for all
samples.
Subcutaneous abscess assay for measuring anti-Trichomonal efficacy of test compounds in
the mouse model
The subcutaneous abscess assay of Krieger et. al. [39] was used. Briefly, the parasites (T.
vaginalis) were cultured under partial anaerobic condition in TYM medium and on attaining
concentration of approximately 2x106 cells/mL (in ~48 hrs) trichomonads were harvested from
the culture by centrifugation at 250 xg for 10 min and then re-suspended in sterile saline. Six
week old mice were inoculated subcutaneously with T. vaginalis (50 µL of 2 x 106 organisms per
mL) into the left hind flank. Control animals were injected with sterile saline only. Five groups
were used for each experiment (n=3). The abscess / lesion formation was determined by
palpation 7 days after injection, and measured daily thereafter. Fine needle biopsy specimens
23

ACCEPTED MANUSCRIPT
were taken from the lesion and examined microscopically to ensure infection. Infected animals
were then treated with compounds (orally) with a dose of 50 mg/kg and 100 mg/kg for 7 days
and abscess size measured longitudinally and the area calculated as πr2. MTZ was used as
positive control. The assay was approved by the Institutional Animal Ethics Committee.28
Docking study
The sequence of Trichomonas vaginalis cysteine synthase (TvCS) was retrieved from
Uniprot (A2GMG5). Since crystal structure of TvCS is not available, therefore a
homology model was constructed using crystal structure of cysteine synthase from
Escherichia coli (PDB ID-2BHS) [40] as template with the help of MODELLER package.
[41] All docking studies were carried using AUTODOCK4.2 Package.51 For molecular
visualization and structure manipulation Chimera was used. [42]
In vivo pharmacokinetic assay
The pharmacokinetic studies of compound 20 was carried out in young and healthy male
Sprague Dawley rats weighing 250±25 g obtained from laboratory animal division, CSIR-CDRI,
Lucknow. The animals were housed in plastic cages in standard laboratory conditions with a
regular 12 hr day-night cycle. Standard pelleted laboratory chow (Goldmohar Laboratory Animal
Feed, Lipton India Ltd, Chandigarh, India) and water were allowed ad libitum. The rats were
acclimatized to this environment for at least five days before conducting the experiment. The
oral dose pharmacokinetic study was conducted in overnight fasted (12-16 hr) rats (n = 4 per
time point). All experiments, euthanasia and disposal of carcasses were carried out as per the
guidelines of Local Ethics Committee for animal experimentation. Suspension formulations
containing 12.5 mg/mL of compound was prepared separately by triturating the compound, gum
acacia (1% w/v) and water (drop wise addition) in mortar and pestle. A single 50 mg/kg oral
dose was given to conscious rats using rat feeding needle. Blood samples were withdrawn at
various predefined times up to 24 hr post dose. Serum samples were harvested and stored at -80
°C until analysis. A Shimadzu UFLC pump (LC-20AD) with online degasser (DGU-20A3), an
auto-sampler (SIL-HTc) with a temperature-controlled peltier-tray and a triple quadrupole API
4000 mass spectrometer (Applied Biosystems, Toronto, Canada) was used for analysis.
Chromatographic separation was made on a Discovery HS C-18 column (5 µm, 50x4.6 mm id)
24

ACCEPTED MANUSCRIPT
preceded with a guard column (5 µm, 20 x 4.0 mm, id) packed with the same material with
mobile phase [acetonitrile: aqueous ammonium acetate buffer (0.01M; pH, 4.5) (80:20, %v/v)]
pumped at a flow rate of 0.6 mL/min under isocratic condition. The mobile phase was degassed
by ultra sonication for 15 min before use. LC-MS/MS system was equilibrated for
approximately 20 min before commencement of analysis. Total analysis time was 3 min per
sample. The mass spectral analysis was performed in positive ionization mode at 5500 V using
multiple reaction monitoring technique to monitor the transitions m/z 328.3→ m/z 206.9 for
compound 20 and m/z 180.1 →m/z 138.1 for phenacetin (internal standard). Data acquisition and
quantitation were performed using analyst software (version 1.4.2; AB Sciex, Toronto, Canada).
The method utilizes 50 µL of serum. For sample clean up protein precipitation was used. The
method showed linearity over the range of 1- 200 ng/mL with recovery of >50% and acceptable
accuracy and precision [43] [FDA, Guidance for Industry: Bioanalytical Method Validation].
Supporting Information:
1H NMR, 13C NMR spectra of the compounds, HRMS spectra and supplementary were available
free of charge via internet at ……………..
ACKNOWLEDGEMENTS
We acknowledge Mrs. Tara Rawat (Senior Technical Officer) for technical assistance and
SAIF Division for providing spectral data. We are grateful to CSIR (D.M., M.S.), and
ICMR (B.K., S.G., S.K., N.S.) 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).
Abbreviations:
STDs, sexually transmitted disease; TV, Trichomonas vaginalis; DTC, dithiocarbamate; SAR,
structure activity relationship; MTZ, Metronidazole; NI, nitroimidazoles; HeLa, epithelium cells;
3D-QSAR, 3D-quantitative structure–activity relationship; SAR, structure–activity relationship;
MIC, minimum inhibitory concentration; IC50, half maximal inhibitory concentration; CoMFA,
comparative molecular field analysis; PLS, partial least squares; LDH, lactate dehydrogenase;
DMEM, dulbecco’s modified eagle’s medium; TLC, thin layer chromatography.
25

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