Role of disulfide linkage in action of bis(dialkylaminethiocarbonyl)disulfides as potent double-Edged microbicidal spermicide: Design, synthesis and biology

Article abstract of DOI:10.1016/j.ejmech.2016.03.012

Trichomoniasis and candidiasis are amongst the most common morbidity-causing reproductive tract infections, generally treated by Metronidazole and Fluconazole respectively. Poor vaginal efficacy, drug-resistance and non-spermicidal nature limit their use as topical microbicidal contraceptives. Bis(dialkylaminethiocarbonyl)disulfides (4-38) were designed as dually active, non-surfactant molecules capable of eliminating Trichomonas vaginalis and Candida strains as well as irreversibly immobilizing 100% human sperm instantly, at doses non-cytotoxic to human cervical epithelial cells and vaginal microflora in vitro. Compounds 12, 16, 17 were fifty times more active than nonoxynol-9, OTC vaginal spermicide, and compounds 12 and 17 have shown remarkable in vivo activity in rabbit model. Most promising compound 17 has shown promise for further development as a double-edged vaginal microbicide due to their improved activity and safety along with notable in vivo trichomonicidal activity. Role of disulfide group was established by loss of spermicidal activity on chemical modifications (39-56). These disulfides might be targeting thiol groups present over cell membrane of human sperm and Trichomonas as shown by fluorescence labeling of free thiols.

Full text of DOI:10.1016/j.ejmech.2016.03.012

Accepted Manuscript
Role of disulfide linkage in action of bis(dialkylaminethiocarbonyl)disulfides as potent
double-Edged microbicidal spermicide: Design, synthesis and biology
Nand Lal, Santosh Jangir, Veenu Bala, Dhanaraju Mandalapu, Amit Sarswat, Lalit
Kumar, Ashish Jain, Lokesh Kumar, Bhavana Kushwaha, Atindra K. Pandey, Shagun
Krishna, Tara Rawat, Praveen K. Shukla, Jagdamba P. Maikhuri, Mohammad I.
Siddiqi, Gopal Gupta, Vishnu L. Sharma
PII:
S0223-5234(16)30184-2
10.1016/j.ejmech.2016.03.012
EJMECH 8437
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 December 2015
Revised Date: 2 March 2016
Accepted Date: 3 March 2016
Please cite this article as: N. Lal, S. Jangir, V. Bala, D. Mandalapu, A. Sarswat, L. Kumar, A. Jain, L.
Kumar, B. Kushwaha, A.K. Pandey, S. Krishna, T. Rawat, P.K. Shukla, J.P. Maikhuri, M.I. Siddiqi, G.
Gupta, V.L. Sharma, Role of disulfide linkage in action of bis(dialkylaminethiocarbonyl)disulfides as
potent double-Edged microbicidal spermicide: Design, synthesis and biology, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.012.
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Role
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disulfide
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bis(dialkylaminethiocarbonyl)disulfides as potent double-Edged
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microbicidal spermicide: Design, synthesis and biology
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Nand Lal, Santosh Jangir, Veenu Bala, Dhanaraju Mandalapu, Amit Sarswat, Lalit Kumar, Ashish
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Jain, Lokesh Kumar, Bhavana Kushwaha, Atindra K. Pandey, Shagun Krishna, Tara Rawat, Praveen
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K. Shukla, Jagdamba P. Maikhuri, Mohammad I. Siddiqi, Gopal Gupta, and Vishnu L. Sharma*
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Medicinal& Process Chemistry Division, Endocrinology Division, Microbiology Division,
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Molecular& Structural Biology Division,CSIR-Central Drug Research Institute, Lucknow-226031
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(India), Academy of Scientific & Innovative Research (AcSIR), New Delhi-110001 (India).
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Abstract:
Trichomoniasis and Candidiasis are amongst the most common morbidity-causing reproductive
tract infections, generally treated by Metronidazole and Fluconazole respectively. Poor vaginal
efficacy, drug-resistance and non-spermicidal nature limit their use as topical microbicidal
contraceptives. Bis(dialkylaminethiocarbonyl)disulfides (4–38) were designed as dually active,
non-surfactant molecules capable of eliminating Trichomonas vaginalis and Candida strains as
well as irreversibly immobilizing 100% human sperm instantly, at doses non-cytotoxic to human
cervical epithelial cells and vaginal microflorainvitro. Compounds 12, 16, 17 were fifty times
more active than nonoxynol-9, OTC vaginal spermicide, and compounds 12 and 17 have shown
remarkable in vivo activity in rabbit model. Most promising compound 17 has shown promise for
further development as a double-edged vaginal microbicide due to their improved activity and
safety along with notable in vivo trichomonicidal activity. Role of disulfide group was
established by loss of spermicidal activity on chemical modifications (39–56). These disulfides
might be targeting thiol groups present over cell membrane of human sperm and Trichomonasas
shown by fluorescence labeling of free thiols.
KEYWORDS: Dithiocarbamate, Disulfide, Disulfiram, Spermicide, Sulfhydryl, Contraceptive.
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* 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;
vlscdri@gmail.com
#
C.D.R.I. Communication No. 8720
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1. Introduction:
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Increasing sexually transmitted infections (STIs) along with the population explosion is a global
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challenge
that cannot be overlooked. Growing drug-resistance of Trichomonas to
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Metronidazole, and fungal strains to Fluconazole, is a cause of serious concern. Furthermore,
nonoxynol-9 (N-9), the OTC vaginal spermicide does not protect against STIs and HIV in
clinical situations but may in fact enhances their incidences due to its non-specific surfactant
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action. Most heterosexual women would like to reduce the risk of acquiring STIs and control
their fertility. Trichomoniasis, the most prevalent, non-viral STI, affects 250–350 million people
worldwide every year causing serious discomfort to women along with associated problems of
adverse pregnancy outcomes, pre-term delivery, low-birth weight infants, infertility, and cervical
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cancer. It is now well established that trichomoniasis extensively raises the vulnerability to
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HIV
and therefore controlling trichomoniasis alone could significantly reduce the incidence
of new HIV infections. Similarly, candidiasis caused by the fungus Candida albicans, is strongly
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associated with HIV-AIDS. In spite of increased ‘weapon store’ for antifungal agents, currently
available drugs do not suffice the growing demand of managing infections in complex patient
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populations.
One of the major problems is increased drug resistance mainly due to chronic
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antimycotic therapy in HIV-infected and other immuno-compromised patients. Usually, the
female partner shoulders the primary responsibility of STI and pregnancy protection during most
of the heterosexual contacts, including ‘vulnerable’ contacts amongst adolescents and
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promiscuous adults. As prevention of pregnancy and infection is better than abortion and cure
later, the best strategy would be to arrest the infection along with sperm in vagina during
transmission, and therefore there is a need to develop a topically active medication against STIs
(trichomoniasis and candidiasis) and sperm. Free thiol groups critically control the survival of
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predominantly anaerobic cells like Trichomonas vaginalis, Candida albicans
and
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spermatozoa. For example, the cleavage of disulfide bonds of sperm cell-specific hexokinase
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type 1 is associated with increased hexokinase activity and initiation of sperm motility. The
unique redox properties of protein thiols play an important role in enzyme catalysis, protein
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folding, and redox signaling, making it a key residue for chemical intervention
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manipulating cellular energy metabolism, motility and subsistence of Trichomonas, Candida and
sperm cells. Consequently, sulfhydryl binding agents can impede sperm, Trichomonas and
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Candida cells to achieve prophylactic contraception as exemplified by N-ethyl maleimide , a
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specific sulfhydryl alkylating agent, acrylophenone, quinolines and a variety of other thiol
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agents.
Dithiocarbamate (DTC) is a desirable pharmacophore in various medicinally significant
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compounds and is widely exploited in microbicidal spermicides,
fungicides
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agents. Thus, it was hypothesized that incorporating DTC and disulfide in a single
chemical entity can make it to interact with multiple targets (Trichomonas, fungi and sperm)
simultaneously. While investigating the DTC-disulfide hybrid framework, Disulfiram (DSF)
molecule (Figure 1) was found to be the most appropriate structure. Disulfiram is a FDA
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approved deterrent to alcohol abuse,which is current in clinical use. DSF was synthesized and
as expected it exhibited sperm immobilizing activity, which was mild. Encouragedby this
observation it was thought worthwhile to modify DSF framework and to synthesize bis(N-
substituted piperazinethiocarbonyl) disulfides (Figure 1) as safe, multi-targeting microbicidal
contraceptives.
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(Figure 1.)
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2. Results and Discussion:
2.1. Chemistry
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The compounds 4–38 have been synthesized according to Scheme 1 using different sodium
salts of dithiocarbamic acid (3). Secondary amine was reacted with carbon disulfide under
alkaline condition to furnish sodium dialkylcarbamodithioate (3) which was further treated with
sodium nitrite and hydrochloric acid at 0-5 °C in water to provide corresponding
bis(dialkylaminethiocarbonyl) disulfides (4–38, Table 1).
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(Scheme 1)
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2.2 Biological Evaluation
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2.2.1.Spermicidal activity
The spermicidal activity of compounds (4–38, Table 1) was evaluated in comparison to N-9.
Twenty-five compounds (4, 7–10, 12–18, 20–25, 28, 31 and 33–37) irreversibly immobilized
100% human sperm at concentration ranging from 1–0.001% (MEC) within 30 seconds. Six
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compounds (4, 12, 16–18 and 35) were found to be more potent than commercially available
spermicide N-9.
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The results of the effect on sperm motility of compounds (4–38, Table 1) propose that if -NR R
was dimethyl amine (4) the compound showed moderate spermicidal activity (MEC, 0.01%). If
methyl group was replaced by cyclohexyl (5) or benzyl (6), the activity was completely lost,
while introduction of cyclic amine (7–11) further decreases spermicidal activity. Among amines
with single nitrogen like pyrrolidine (7), piperidine (8), 4-methylpiperidine (10) and azepane (11)
activity remains unchanged in five (7) and six (8, 10) membered ring while decreases when ring
size increases to seven (11). Incorporation of one oxygen atom (9) into compound 8 increases
activity by 10 folds. Whereas introducing additional nitrogen atom in amino residue remarkably
increased spermicidal activity (12–18, MEC 1–0.001%). Among the alkyl substituted piperazines
(12–15) chain length determine the spermicidal activity i.e., bulkier the alkyl group lesser the
activity. While a substitution of the alkyl group with allyl (16), butyronitrile (17) and morpholino
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alkyl (18) retained high activity. Furthermore presence of aryl/heteroaryl group at NR R in this
framework decreased the spermicidal action (19–23, MEC 0.1 and 1%) while a benzoyl group
imparted mild activity (24, MEC 0.5%). A decrease or complete loss of spermicidal effect was
observed when carboxylate (25–27), mesyl (28), tosyl (29), alkyl/benzyl carbodithioate (30–34
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and 38) groups were introduced at NR R (Table 1). Interestingly replacement of alkyl
carbodithioate group with alkyl amino carbodithioate increased the spermicidal action (35–37,
MEC, 0.5–0.002%).
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(Table 1)
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A close look at structure activity relationship (SAR) of bis(dialkylaminethiocarbonyl) disulfide
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(4–38) revealed that small alkyl group at N position of piperazine (12, 16, 17, MEC 0.001%) is
desirable for sperm immobilization activity.
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2.2.2. Anti-Trichomonas activity
Seventeen compounds (4,6–13,16–18,23,25,33,35and38; Table 2) showed anti-trichomonal
activity against Metronidazole (MTZ) susceptible strains with MIC ranging from 3.125–100
µg/mL (MTZ = 2.0 µg/mL),while fourteen compounds (4, 6–13, 16–18, 25 and 38) among these
exhibited trichomonicidal action against resistant strain at MIC 3.125–100 µg/mL (MTZ = 50.0
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µg/mL). It is evident from the results (Table 2) that eleven compounds (4, 6, 7, 9–12, 16–18 and
25) illustrated better activity than MTZ against Trichomonas resistant strain. The comparison of
anti-Trichomonas activity against susceptible and resistant strains revealed that MTZ lost its
activity by 25 times against resistant strain while compounds (4, 7, 9, 10, 17and 25) had better
profile as there was no loss of activity.
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The results of trichomonacidal activity against MTZ susceptible strain revealed that if -NR R
was alkyl substituted acyclic amines (4–6), dimethylamine (4) and benzylmethyl amine (6) were
more preffered as their activity was comparable to standard drug MTZ. Whereas with cyclic
amines (7–11) the activity was significant and pyrrolidine (7) and morpholine (9) seemed to be
more desirable (MIC, 3.125 µg/mL) and an enhancement in ring size (8,11) resulted in decreased
activity. An addition of a methyl group (10) in compound 8at position 4 enhanced the activity by
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four fold. While a two nitrogen system i.e., alkyl substituted piperazine at -NR R (12-18)
smaller alkyl groups (12 and 16–18) were more desirable as the trichomonicidal activity was
retained. When alkyl group was replaced by aryl/heteroaryl (19–23), benzoyl (24), carboxylate
(25–27), mesyl/tosyl (28–29) and substituted carbodithioates (30–38) activity was decreased or
completely lost. The results suggested that smaller alkyl group in cyclic/acyclic amine and
piperazine framework was essential for anti-Trichomonas activity. The antitrichomonicidal
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activity against resistant strain of Trichomonas (Table 2) suggested that -NR R substitution (4–
38) had similar SAR as exhibited against susceptible strain. The small alkyl groups in one or two
nitrogen scaffolds were more desirable in cyclic and acyclic amines as four compounds (4, 7, 9
and 17;3.125 µg/mL) were sixteen fold more active than MTZ while compound 25 (6.25µg/mL),
6, 10–12 (12.5 µg/mL) and 16, 18 (25.0 µg/mL) were eight, four and two times more active
respectively. Compound 8 was equipotent and two compounds (13 and 38) were less active.
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(Table 2)
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The activity profile of compounds (4–38) against both the susceptible and resistant strains of
Trichomonas suggested that compounds (4, 6, 7, 9 and17) exhibiting activity (3.125 µg/mL)
comparable to MTZ (2 µg/mL) in susceptible strain were more effective against resistant strain.
MTZ lost the activity by twenty five times while in compounds 4, 7, 9and17 activity was not
decreased and in compound 6 the activity was lost by only four times. The result suggested that
dimethylamino (4), pyrrolidino (7), morpholino (9) and 4-(3-cyanoproyl)piperazine (17) groups
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in bis(dialkylaminethiocarbonyl) disulfides were more desirable to be effective against both
susceptible and resistant strains of Trichomonas. Further a docking study was also carried out
with less (13) and most active compounds (4 and 17) to ascertain their cysteine biosynthesis
pathway.
Cysteine is indispensably vital for all living life forms as an amino acid for protein synthesis, as a
precursor for glutathione and biomolecules such as coenzyme A, and as a source of sulphide for
synthesis of iron-sulphur clusters. This series of compounds containing bis(N-substituted
piperazinethiocarbonyl) disulfide moiety possibly exhibit anti-trichomonal activity by inhibiting
the cysteine biosynthesis pathway. To better understand the mechanism of inhibition of these
compoundsthe important component of cysteine biosynthesis pathway was explored.
The Trichomonas vaginalis cysteine synthase (TvCS) shows 44% of sequence identity with
template. The super imposition of the modeled complex with template showed a root-mean-
square deviation (RMSD) of 0.186Å.The validation of the resulting model was done with the
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Structural Analysis and Verification Server (SAVS) . The model in which the majority of the
residues (97%) occupy the most favorable region of Ramachandran plot and 2% and 1% residue
lie in additionally allowed region and generously allowed region respectively was selected for
further docking studies. Autodock4 tool was used to identify the possible binding site of the
enzyme for inhibitory activity of our molecules. The structure preparation and minimization
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before studies were performed with compounds using SYBYL 7.1 program package on silicon
graphics fuel work station with IRIX 6.5 operating system. The structure of the compounds were
prepared with the help of sketch module of Sybyl7.1 and geometry optimization was done using
MMFF94 force field with Powell energy minimization algorithm, Gasteiger-Huckel charges, and
0.001 kcal/(mol.Å) energy gradient convergence criterion.
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(Figure 2)
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In a recent study the analysis of the amino acid sequence of TvCS has been presented and it
revealed that TvCS contains all the active site residues identified for CS of S. Typhimurium and
A. thaliana. It was also suggested that the predicted active site lysine that covalently binds with
PLP is Lys43. However, residues Asn73, Gln144, His154, Gly178, Thr179, Ser180, Thr182, and
Ser259 are also connected to the co-factor with the help of hydrogen bonding. Therefore the
docking studies were performed by considering this active site including the above mentioned
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residues. The compounds docked well at the cavity that is suggested to involve in Pyridoxal-5'-
phosphate (PLP) binding. Figure 2 shows the binding mode of docked complexes of the most
active compounds with highest trichomonacidal activity. Most of the compounds in their docked
conformation are interacting with Lys43 residue that is predicted to binds covalently with PLP.
The compound 4 is also interacting with Gly178, Thr179, Ser180, Thr182 and Ser259. These
interactions are reported to play important role in PLP binding to the CS, mentioned in the study
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done by Westrop et al. The docked conformation of most and less potent molecules was
analyzed to see how they differ from each other based on their binding affinity with pocket
residues of PLP. Interestingly, in docking experiments, the less active compound 13 did not
mimic the active analogue compound 17 and 4 (Figure 2). It is not involved in formation of
hydrogen bond. Moreover, the thiocarbonyldisulfide moiety of compound 13 is protruding from
the PLP binding region (Figure 2). These studies inferred that compound 17 and 4 are more
potent compared to others due to its favorable H-bond interactions with Lys43 residue that may
be responsible for binding with PLP.
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2.2.3. Antifungal activity
The compounds (4–38) were screened against five fungal strains (Table 2) and thirty one of these
(4–26 and 28–35) inhibited the growth of one or more fungal strains at MIC 0.78–50 µg/mL
whereas eleven compounds (4, 7–13, 15, 25 and 35) were active against all the strains. The
subsequent SAR has been discussed on the basis of least MIC against any of the strain. The
results (Table 2) suggested that a secondary amine with one nitrogen was more preferred scaffold
as six compounds4, 6–8, 10 and 11 of the eight (4–11) had remarkable activity (MIC, 0.78–3.125
µg/mL) whereas compound 5 and 9 were active at MIC 12.5 µg/mL. Among two nitrogen
secondary amine derivatives i.e., piperazine scaffold (12–38) the activity decreased drastically as
only three compounds (14, 20 and 25) had activity at MIC 12.5 µg/mL and nine compounds (12,
16, 21, 22, 28, 30, 31, 33, 34) were marginally active (MIC 25 µg/mL) while rest of the
compounds were either active at highest tested concentration (MIC 50 µg/mL) or inactive. The
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activity data of compounds 4–6 implied that if -NR R was alkyl substituted acyclic amines the
smaller alkyl groups were most suitable for antifungal activity as dimethylamino derivative (4,
MIC, 0.78–12.5 µg/mL) exhibited significant activity. On the other hand, among cyclic amines
(7–11), pyrrolidine (7) and piperidine (8) were most active and an addition of oxygen atom (9),
methyl group (10) and enhancement of ring size (11) resulted in less active compounds.
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2.2.4. Safety and compatibility of promising compounds (4, 12, 16, 17, 18 and 35)
The overall assessment of biological profile of all the compounds suggested that five compounds
(4, 12, 16, 17, 18 and 35) of the series were most promising based on their spermicidal, anti-
Trichomonas and antifungal activities as compared to vaginal microbicide available in the
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market N-9. To assess the potential of these compounds for the choice as a candidate vaginal
microbicide, the safety (Table 3) against vaginal epithelium (HeLa cells) and flora
(Lactobacillus) was evaluated. The evaluated compounds (4, 12, 16, 17, 18 and 35; Table 3)
displayed better safety towards HeLa cells (IC 156-1414 µg/mL) and much better compatibility
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with Lactobacillus (IC50 361-3550 µg/mL) than N-9 (IC50 32.5 and 33.2 µg/mL respectively).
The selectivity index of compounds 4, 12, 16, 17, 18 and 35 was much higher (7–368) than that
of N-9 (0.64), with compounds 4 and 17 exhibiting the highest selectivity index of 188 and 368,
respectively.
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(Table 3)
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These compounds were three to forty three folds and eleven to ninety four folds safer against
HeLa cells and Lactobacillus than N-9 and therefore appeared apparently much safer for vaginal
use. Of these compounds 12, 16 and 17 were most potent spermicides but the shelf-life of
compound 16 was not good, therefore 12 and 17 were considered to be worthwhile in carrying
out the in vivo spermicidal activity (Table 4) in rabbit model.
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2.2.5. In vivo spermicidal activity of compound 12 and 17
Apparently a dose dependent reduction in pregnancy rate was evident in rabbits receiving
intravaginal instillation of compound 12 and 17, however the effect was more prominent with
compound 17. With compound 12 about 50–60% animals did not conceive at vaginal dose of
15–20 mg and with compound 17 this efficacy was 86–100% at 10-50 mg doses, while this was
only 20% in case of nonoxynol-9 (20 mg). The average litter size of animals receiving compound
12 before mating was reduced by 67%, 74% and 76% in 10, 15 and 20 mg groups, respectively.
With compound 17 the average litter size was diminished by 90%, 100% and 100% respectively
at doses 10, 25 and 50 mg groups. On the other hand, N-9 at 20 mg dose caused only 37%
reduction in average litter size of rabbits (Table 4).
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2.2.6. In vivo Anti- Trichomonas efficacy of compounds 4 and 17 in the mouse subcutaneous
abscess assay model
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The in vivo efficacy of compounds 4 and 17 was evaluated using the mouse abscess assay
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(Figure 3). Subcutaneous injection of live trichomonads resulted in a small pustule of ~70 mm
area on day-8 of injection (day-1 of treatment) in experimental and control animals that grew to
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~109 mm in area in controls but was reduced to ~15-25 mm after 5-days of treatment with
compounds 4, 17 and Metronidazole. Thereafter the growth of abscess was exponential in
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controls and 7 days after treatment it was ~190 mm in area while it was further subdued to ~5-
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10 mm in treated animals. On the day of autopsy (i.e. the day following seven days of
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treatment), the abscess was ~190 mm in area in untreated controls, 7.59 mm in metronidazole,
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4.45 mm in compound 4 and 5.75 mm in compound 17 treated groups. The spleen weight at
autopsy was ~0.4 g in control, ~0.59 g in saline treated, ~0.50 g in metronidazole treated, ~0.30 g
in compound 4 treated and ~0.30 g in compound 17 treated animals. From the results it was
found that compound 4 and 17 were found more potent than MTZ in In vivo.
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61
62
63
64
65
66
67
2.2.7.Mode of action of bis(dialkylaminethiocarbonyl) disulfides:
2.2.7.1. Role of disulfide group in sperm immobilization
The activity of compounds (4–38) may be due to the disulfide linkage of the molecule as it has
been found that disulfide-sulfhydryl (S–S to SH) interconversion plays an important role for
4
9
motility, membrane integrity and fluidity of sperm and also very vital for the viability of
32
32
Candida and Trichomonas. Therefore, to establish the role of disulfide group, modifications
(Scheme 2) were carried out in bis(dialkylaminethiocarbonyl) disulfide scaffold. S-alkylated
compounds 39–44 and 45–56 were synthesized and evaluated for spermicidal activity. Alkyl 4-
alkylpiperazine-1-carbodithioates (39–44) were synthesized by reaction of sodium
dialkylcarbamodithioate (3) with alkyl halides in methanol at room temperature. While alkylene
bis(4-alkylpiperazine-1-carbodithioate) (45–56) were synthesized by reaction of 3 and
dihaloalkanes in acetonitrile at room temperature. Alkyl 4-alkylpiperazine-1-carbodithioates
(39–44, Table 5) were inactive at 1% concentration while compounds 45–56 (Table 6)
immobilized the sperm at MEC 0.5–1%. These results suggested that any modification at
9

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69
disulfide linkage resulted in 500 fold to 1000 fold decease or complete loss of activity. These
findings confirmed the role of disulfide group in spermicidal activity of this active scaffold.
2
2
2
70
71
72
(Scheme 2)
(Table 5)
(Table 6)
2
2
2
2
2
2
2
2
73
74
75
76
77
78
79
80
2.2.7.2. Mode of action of compound 12: Fluoroscence labeling of sperm thiols
To study the mode of action of bis(dialkylaminethiocarbonyl) disulfides, free –SH groups were
localized by fluorescence detection (after labeling with the thiols capturing dye mBBr) of human
sperm that were either motile (control) or immobilized by compound 12 treatment, and digitally
imaged for qualitative assessment. It became clearly evident by visual assessment of
fluorescence intensities that control sperm (Figure 4) had remarkably higher number of free
thiols as compared with sperm immobilized by compound 12 (Figure 4).
(Figure 4)
2
2
2
81
82
83
Even though the difference was marked throughout the structure of sperm, it was prominently
noticeable in the tail region (principal piece). The diminished fluorescence with compound 12
suggested the interaction with free thiol might be the mechanism of spermicidal action.
2
2
84
85
2.2.7.3. Mode of action of compound 4 and 17: Fluorescence labeling of Trichomonas thiols
(Figure 5)
2
2
2
2
2
2
2
2
2
86
87
88
89
90
91
92
93
94
The compounds were designed to target free thiols over the Trichomonas and the inhibition was
qualitatively assessed by fluorescence detection after labeling the free thiols with fluorimetric
thiol detector which specifically binds free thiols (Figure 5). Motile Trichomonasvaginalis
(control) and compounds 4 and 17-treated Trichomonas were digitally imaged for qualitative
assessment. It was clear from Figure 5 that there was reduction in free thiol fluorescence with
compound 4 and 17 treated Trichomonas. Fluorescence intensities were higher in controls, due to
the higher number of free thiols available than in compounds-treated samples. The decreased
fluorescence with the compounds treatment suggested the interaction of compound 4 and 17with
free thiols present over Trichomonas, which could be the mechanism of trichomonicidal action.
10

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95 3. Conclusion
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06
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11
12
13
14
15
16
17
18
Bis(dialkylaminethiocarbonyl) disulfide (4–38) designs were conceptualized, synthesized and
evaluated for spermicidal, antifungal and anti-Trichomonas activities. 4-Substituted piperazine
4
group with smaller alkyl chain at N position (12, 16, 17, MEC 0.001%) was most desirable for
spermicidal activity as these were fifty times more active than N-9. Whereas single nitrogen
secondary amines (4–11) were more appropriate for antifungal and anti- Trichomonas action,
though the activity was retained in two nitrogen secondary amines i.e., piperazine derivatives.
Eleven compounds were 2-16 fold more active than MTZ against resistant Trichomonas strain.
The findings suggested that bis(dialkylaminethiocarbonyl) disulfide is a very versatile scaffold.
Most promising compounds (4, 12, 16, 17, 18 and 35) were found to be highly safe and
compatible with cervico-vaginal epithelium and flora. Of these 12 and 17 exhibited remarkable
in vivo contraceptive activity in rabbit model with percentage efficacies of 62 and 100%,
respectively. Similarly the most active trichomonacidal compounds, 4 and 17 demonstrated
better in vivoactivities in mice model compared to the standard drug MTZ. Further a docking
study was carried out with less (13) and most active trichomonicidal compounds (4 and 17) to
determine their interaction with cysteine synthase. It was inferred that compound 4 and 17 were
more potent compared to other compounds due to their favorable H-bond interactions with
Lys43 residue, which may be responsible for binding with pyridoxal-5'-phosphate (PLP). The
role of disulfide group was also established by the loss of spermicidal activity on carrying out
chemical modification in this scaffold. These disulfide compounds might be imparting their
2
2
activity by interacting with sulfhydryl groups present over cell membrane of sperm and
33,48
Trichomonas
as
shown
by
fluorescence
labeling
of
thiols.
Thus
bis(dialkylaminethiocarbonyl) disulfide frame work has evolved as a potent lead for
development of double-edged vaginal microbicides.
3
3
19 4. Experimental
20
4.1 Chemistry
3
3
3
21
22
23
In general, 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
–
1
block and are uncorrected. IR spectra (νmax in cm ) of the compounds were recorded on Perkin
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34
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36
Elmer’s FT-IR RX1 PC spectrophotometer. H NMR & C NMR spectra were recorded on
1
BrukerSupercon Magnet Avance DRX-300 spectrometers (operating at 300 and 400 MHz for H;
1
3
50, 75, 100 and 125 MHz for C) in deuterated solvents with TMS as internal reference
(chemical shifts in ppm, J in Hz). Electrospray Ionisation Mass spectra (ESI-MS) were
δ
recorded on Thermo Lcq Advantage Max-IT and HR-DART MS were recorded on JEOL, JMS
T100LC Accu TOF. Elemental analyses were performed on Carlo Erba EA-1108 micro analyzer
/ Vario EL-III C H N S analyzer. All compounds were analyzed of C, H, N and the results
obtained were within ± 0.3% of calculated values. The reaction progress was routinely monitored
by thin layer chromatography (TLC) on pre-coated silica gel plates (Aldrich). Column
chromatography was performed over Merck silica gel (100–200 Mesh). All compounds were
1
13
characterized by TLC, H and C NMR, MS. Elemental analyses data meet the criteria of ≥95%
purity. All chemicals and solvents were procured from Sigma-Aldrich / Merck India Ltd. Sodium
3
0
dialkylcarbamodithioate (3) were prepared by known procedure.
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
Bis(dimethylaminothiocarbonyl) disulfide (4). The
mixture
of
sodium
dimethylcarbamodithioate (0.38 g, 2.7 mmol), sodium nitrite (0.19 g, 2.7 mmol, dissolved in 1
mL methanol) and 5 mL distilled water was stirred at 0–5 °C for 5 minutes. Then 1 mL
concentrated HCl was added drop wise and the reaction mixture was stirred at 0–5 °C for 20
minutes. White solid separated which was extracted with chloroform (10×2 mL). Combined
organic layer was washed with distilled water (5×2 mL) and dried over sodium sulfate. Sodium
sulfate was filtered off and filtrate was concentrated under reduced pressure. The crude product
was purified after recrystallization with ethanol to afford compound as white crystals (0.18 g,
–
1
1
56%); mp: 116–118 °C; IR (KBr) ν (cm ): 2977, 2943, 1587, 1473, 1430; H NMR (300 MHz,
1
3
CDCl3):
δ
3.63–3.61 (12H, m); C NMR (50 MHz, CDCl ):
δ
193.6 (C=S), 47.5, 42.1; ESI-MS:
3
+
(m/z) 241 (MH ); Anal. (%) calcd. for C H N S : C, 29.97; H, 5.03; N, 11.65; found, C, 29.89;
6
12
2 4
H, 5.13; N, 11. 73.
The following compounds (5–38) have been synthesized according to general procedure using
the corresponding sodium dialkylcarbamodithioate.
Bis(dicyclohexylaminothiocarbonyl) disulfide (5).The title compound was synthesized by
–
oxidation of sodium dicyclohexylcarbamodithioate in 56% yield as semi-solid; IR (KBr) ν (cm
1
1
): 2946, 1645, 1434; H NMR (300 MHz, CDCl ):
δ 5.35–4.90 (2H, m), 3.30–2.71 (2H, bs),
3
12

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3
3
3
3
3
3
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3
3
3
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3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
1.86–1.16 (40H, m); ESI-MS: (m/z) 513 (MH ); Anal. (%) calcd. for C26
H
44
N
2
S
4
: C, 60.88; H,
8.65; N, 5.46; found, C, 60.71; H, 8.82; N, 5.55.
Bis(benzylmethylaminothiocarbonyl) disulfide (6).The title compound was synthesized by
oxidation of sodium benzyl(methyl)carbamodithioate in 67% yield as white solid; mp: 150–152
–
1
1
°C; IR (KBr) ν (cm ): 2978, 2927, 1596, 1487, 1444; H NMR (300 MHz, CDCl ):
δ 7.37 (10H,
3
1
3
m), 5.40–3.30 (4H m) 3.54 (6H, s); C NMR (75 MHz, CDCl ):
δ
195.5 (C=S), 134.8, 134.3,
3
+
128.9, 128.3, 128.0, 127.8, 127.5, 62.1, 58.5, 45.4, 39.3; ESI-MS: (m/z) 393 (MH ); Anal. (%)
20 2 4
calcd. for C18H N S : C, 55.06; H, 5.13; N, 7.13; found, C, 55.14; H, 5.25; N, 7.03.
Bis(pyrrolidinylthiocarbonyl) disulfide (7).The title compound was synthesized by oxidation
of sodium pyrrolidine-1-carbodithioate in 69% yield as white solid; mp:136–138 °C; IR (KBr) ν
–
1
1
(cm ): 2948, 1657, 1434; H NMR (300 MHz, CDCl
3
):
δ 4.11–3.96 (8H, m), 2.16–2.02 (8H, m);
1
3
C NMR (75 MHz, CDCl ): δ188.2 (C=S), 56.0, 50.0, 25.6, 23.3; HRMS m/zcalcd. for
3
+
C H N S (MH ): 293.0275; found 293.0265; Anal. (%) calcd. for C H N S : C, 41.06; H,
10
16
2
4
10 16
2 4
5.51; N, 9.58; found, C, 41.15; H, 5.48; N, 9.51.
Bis(piperidinylthiocarbonyl) disulfide (8).The title compound was synthesized by oxidation of
sodium piperidine-1-carbodithioate in 71% yield as white solid; mp: 124–126 °C; IR (KBr) ν
–
1
1
(cm ): 2934, 1688, 1478, 1426; H NMR (300 MHz, CDCl ):
δ
4.24 (8H, bs), 1.77 (12H, m);
3
1
3
C NMR (50 MHz, CDCl3):
δ 191.7 (C=S), 52.0, 25.0, 23.2; HRMS m/zcalcd. for C H N S
12
20
2 4
+
(MH ): 321.0588; found 321.0573; Anal. (%) calcd. for C H N S : C, 44.96; H, 6.29; N, 8.74;
1
2
20
2 4
found, C, 44.86; H, 6.22; N, 8.79.
Bis(morpholinylthiocarbonyl) disulfide (9).The title compound was synthesized by oxidation
of sodium morpholine-4-carbodithioate in 70% yield as white solid; mp: 118–120 °C; IR (KBr) ν
–
1
1
(cm ): 2979, 2927, 1587, 1469, 1426; H NMR (400 MHz, CDCl
3
):
δ 4.23 (8H, s), 3.78–3.76
1
3
(8H, m); C NMR (50 MHz, CDCl ): δ 193.8 (C=S), 66.4, 52.7; HRMS m/zcalcd.
3
+
forC H N O S (MH ): 325.0173; found 325.0160; Anal. (%) calcd. for C H N O S : C,
1
0
16
2
2
4
10 16
2
2 4
37.01; H, 4.97; N, 8.63; found, C, 37.16; H, 4.75; N, 8.56.
Bis(4-methylpiperidinylthiocarbonyl) disulfide (10).The title compound was synthesized by
oxidation of sodium 4-methylpiperidine-1-carbodithioate in 68% yield as white solid; mp: 128–
–
1
1
130 °C; IR ν(KBr) (cm ): 2929, 1587, 1480, 1437; H NMR (300 MHz, CDCl ):
δ 5.39–4.88
3
1
3
(4H, m), 3.34–3.33 (4H, m) 1.83–1.80 (6H, m), 1.46–1.39 (4H, m), 1.01–1.00 (6H, m); C NMR
13

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3
3
3
3
3
3
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4
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4
4
4
4
4
4
4
4
4
4
4
84
85
86
87
88
89
90
91
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96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
(75 MHz, CDCl3):
δ
192.6 (C=S), 54.9, 52.0, 51.8, 34.0, 33.4, 30.8, 21.2; ESI-MS: (m/z) 349
+
(MH ); Anal. (%) calcd. for C14
8.12.
24 2 4
H N S : C, 48.23; H, 6.94; N, 8.04; found, C, 48.15; H, 6.79; N,
Bis(azepanylthiocarbonyl) disulfide (11).The title compound was synthesized by oxidation of
–
sodium azepane-1-carbodithioate in 65% yield as white solid; mp: 112–114 °C; IR (KBr) ν (cm
1
1
3
): 2934, 1599, 1492, 1421; H NMR (300 MHz, CDCl ): δ 4.21–4.15 (8H, m), 2.04–2.01 (4H
1
3
m), 1.94–1.86 (4H, m), 1.65–1.61 (8H, m); C NMR (75 MHz, CDCl ):
δ 193.2 (C=S), 58.1,
3
+
53.6, 28.0, 26.5, 26.3, 25.7; HRMS m/zcalcd. for C H N S (MH ): 349.0876; found 349.0901;
14
24
2 4
24 2 4
Anal. (%) calcd. for C14H N S : C, 48.23; H, 6.94; N, 8.04; found, C, 48.31; H, 6.84; N, 8.16.
Bis(4-butyl-1-piperazinylthiocarbonyl) disulfide (12).The title compound was synthesized by
oxidation of sodium 4-butylpiperazine-1-carbodithioate in 61% yield as yellow solid; mp: 105–
–
1
1
107 °C; IR (KBr) ν (cm ): 2957, 1586, 1473, 1430; H NMR (300 MHz, CDCl
3
): δ 4.29 (8H,
bs), 2.60 (8H, bs), 2.39 (4H, t, J = 7.2 Hz), 1.53–1.46 (4H, m), 1.43–1.33 (4H, m), 0.93 (6H, t, J
13
= 7.2 Hz); C NMR (75 MHz, CDCl ):
δ 193.3 (C=S), 57.8, 52.7, 39.5, 28.9, 20.6, 14.0; HRMS
3
+
m/zcalcd. for C H N S (MH ): 435.1745; found 435.1739; Anal. (%) calcd. for C H N S : C,
1
8
34
4
4
18 34
4 4
49.73; H, 7.88; N, 12.89; found, C, 49.53; H, 7.98; N, 12.80.
Bis(4-octyl-1-piperazinylthiocarbonyl) disulfide (13).The title compound was synthesized by
oxidation of sodium 4-octylpiperazine-1-carbodithioate in 55% yield as white solid; mp: 93–95
–
1
1
°C; IR (KBr) ν (cm ): 2930, 1585, 1471, 1430; H NMR (300 MHz, CDCl
3
): δ 4.29 (8H, bs),
2.60 (8H, bs), 2.38 (4H, t, J = 7.3 Hz), 1.49 (4H, bs), 1.28 (20H, bs), 0.88 (6H, t, J = 6.9 Hz);
1
3
C NMR (75 MHz, CDCl ): 193.4 (C=S), 58.2, 52.8, 31.8, 29.5, 29.3, 27.5, 26.8, 22.7, 14.1;
3
+
ESI-MS: (m/z) 547 (MH ); Anal. (%) calcd. for C H N S : C, 57.09; H, 9.21; N, 10.24; found,
26
50
4 4
C, 57.39; H, 9.18; N, 10.04.
Bis(4-hexadecyl-1-piperazinylthiocarbonyl) disulfide (14).The title compound was
synthesized by oxidation of sodium 4-hexadecylpiperazine-1-carbodithioate in 56% yield as off
–
1
1
white solid; mp: 95–97 °C; IR (KBr) ν (cm ): 2926, 1586, 1471, 1430; H NMR (300 MHz,
CDCl3): 4.43–4.29 (8H, m), 2.60–2.49 (8H m), 2.40–2.35 (4H, m), 1.49 (4H, m), 1.25 (52H,
m), 0.88 (6H, t, J = 7.2 Hz); C NMR (75 MHz, CDCl
δ
1
3
3
):
δ
193.3 (C=S), 58.1, 52.7, 31.9, 29.6,
+
29.6, 29.5, 29.5, 29.3, 27.4, 26.8, 22.6, 14.1; ESI-MS: (m/z) 771 (MH ); Anal. (%) calcd. for
C H N S : C, 65.40; H, 10.71; N, 7.26; found, C, 65.55; H, 10.61; N, 7.19.
42
82
4 4
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Bis(4-admentyl-1-piperazinylthiocarbonyl) disulfide (15).The title compound was synthesized
by oxidation of sodium 4-adamentylpiperazine-1-carbodithioate in 59% yield as white solid; mp:
–
1
1
125–127 °C; IR (KBr) ν (cm ): 2927, 1590, 1433, 1374; H NMR (300 MHz, CDCl ):
δ
4.27–
3
1
3
3.58 (8H, m), 2.59–1.54 (24H, m), 1.25 (12H, bs), 1.07 (2H, bs); C NMR (75 MHz, CDCl ):
δ
3
193.5 (C=S), 58.2, 32.0, 29.8, 29.7, 29.7, 29.6, 29.4, 27.5, 26.9, 22.8, 14.2; ESI-MS: (m/z) 591
+
(MH ); Anal. (%) calcd. for C H N S : C, 60.97; H, 7.85; N, 9.48; found, C, 61.15; H, 8.10; N,
3
0
46
4 4
9.25.
Bis(4-allyl-1-piperazinylthiocarbonyl) disulfide (16). The title compound was synthesized by
oxidation of sodium 4-allylpiperazine-1-carbodithioate in 65 % yield as light yellow solid; mp:
–
1
1
105–107 °C; IR (KBr) ν (cm ): 2919, 1588, 1471, 1425; H NMR (300 MHz, CDCl ):
δ 5.91–
3
1
3
5.78 (2H, m), 5.25–5.18 (4H, m), 4.30 (8H, bs), 3.05 (4H, d, J = 6.5 Hz), 2.63–2.60 (8H, m); C
NMR (100 MHz, CDCl3):δ 193.5 (C=S), 134.2, 118.8, 61.1, 54.0, 52.5, 51.3; HRMS m/zcalcd.
+
for C H N S (MH ): 403.1119; found 403.1115; Anal. (%) calcd. for C H N S : C, 47.72; H,
1
6
26
4
4
16 26
4 4
6.51; N, 13.91; found, C, 47.96; H, 6.31; N, 13.74.
Bis[4-(3-cyanoproyl)-1-piperazinylthiocarbonyl] disulfide (17). The title compound was
synthesized by oxidation of sodium 4-(3-cyanopropyl)piperazine-1-carbodithioate in 62% yield
–
1
1
as off white solid; mp: 108–110 °C; IR (KBr) ν (cm ): 2951, 1585, 1473, 1429; H NMR (400
MHz, CDCl ): 4.23 (8H, bs), 2.55 (8H, s), 2.47(4H, t, J = 6.6 Hz), 2.39 (4H, t, J = 7.0 Hz),
3
δ
1
3
3
1.82–1.75 (4H, m); C NMR (75 MHz, CDCl ): δ 193.5 (C=S), 119.5, 55.6, 52.5, 51.2, 22.6,
+
14.9; HRMS m/zcalcd. for C H N S (MH ): 457.1337; found 457.1337; Anal. (%) calcd. for
1
8
28
6 4
C H N S : C, 47.34; H, 6.18; N, 18.40; found, C, 47.45; H, 6.10; N, 18.26.
18
28
6 4
Bis[4-(2-morpholinoethyl)-1-piperazinylthiocarbonyl] disulfide (18). The title compound was
synthesized by oxidation of sodium 4-(2-morpholinoethyl)piperazine-1-carbodithioate in 60%
–
1
1
yield as white solid; mp: 110–112 °C; IR ν (KBr) (cm ): 2955, 1589, 1471, 1430; H NMR (300
1
3
MHz, CDCl
3
):
δ 4.28 (8H, bs), 3.72–3.69 (8H m), 2.67–2.47 (24H, m); C NMR (75 MHz,
CDCl ): δ 193.5 (C=S), 66.9, 56.4, 56.3, 55.4, 55.0, 54.1, 53.3, 53.0, 51.4; ESI-MS: (m/z) 549
3
+
(MH ); Anal. (%) calcd. for C H N O S : C, 48.14; H, 7.35; N, 15.31; found, C, 48.32; H,
2
2
40
6
2 4
7.21; N, 15.45.
Bis[4-(4-fluorophenyl)-1-piperazinylthiocarbonyl] disulfide (19).The title compound was
synthesized by oxidation of sodium 4-(4-fluorophenyl)piperazine-1-carbodithioate in 69% yield
15

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58
59
60
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62
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65
66
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68
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71
72
73
as white solid; mp: 140–142 °C; IR (KBr) ν (cm ): 2924, 1586, 1474, 1429; H NMR (300
1
3
MHz, CDCl
NMR (75 MHz, CDCl
ESI-MS: (m/z) 511 (MH ); Anal. (%) calcd. for C H F N S : C, 51.74; H, 4.74; N, 10.97;
3
):
δ
7.04–6.99 (4H, m), 6.94–6.89 (4H, m), 4.46 (8H m), 3.32–3.29 (8H, m); C
3
): 193.8 (C=S), 159.5, 147.0, 147.0, 118.7, 118.6, 116.1, 115.8, 50.3;
δ
+
2
2
24
2
4 4
found, C, 51.69; H, 4.81; N, 10.87.
Bis[4-(2-methoxyphenyl)-1-piperazinylthiocarbonyl] disulfide (20).The title compound was
synthesized by oxidation of sodium 4-(2-methoxyphenyl)piperazine-1-carbodithioate in 60%
–
1
1
yield as white solid; mp: 125–127 °C; IR (KBr) ν (cm ): 2998, 1592, 1474, 1429; H NMR (300
1
3
MHz, CDCl3):
MHz, CDCl ):
δ
δ
7.08–6.88 (8H, m), 4.47 (8H m), 3.89 (6H, s), 3.24 (8H, bs); C NMR (75
193.6 (C=S), 152.2, 139.9, 123.8, 121.1, 118.6, 111.4, 55.5, 50.3; ESI-MS:
3
+
30 4 2 4
(m/z) 535 (MH ); Anal. (%) calcd. for C24H N O S : C, 53.90; H, 5.65; N, 10.48; found, C,
53.98; H, 5.52; N, 10.39.
Bis[4-(4-nitro-2-(trifluoromethyl)phenyl)-1-piperazinylthiocarbonyl] disulfide (21). The title
compound was synthesized from sodium 4 (4-nitro-2-(trifluoromethyl)phenyl)piperazine-1-
–
1
carbodithioate in 56% yield as white solid; m.p: 146–148 °C; IR (KBr)ν (cm ): 2925, 2854,
1
1535, 1468, 1425,1327,1221; H NMR (300 MHz, CDCl ):
δ
8.14 (2H, d, J = 1.4 Hz), 7.75 (2H,
3
13
dd, J = 8.64, 1.8 Hz), 7.26–7.20 (2H, m), 4.49 (8H, bs), 3.38 (8H, t, J = 4.9 Hz); C NMR (100
MHz, CDCl
3
):
δ
198.6 (C=S), 156.8, 142.8, 128.0, 124.6, 124.5, 124.4, 124.4, 122.9, 52.6, 51.5,
+
50.5, 45.4; ESI-MS: m/z 701 (MH ); Anal. (%) calcd. for C H F N O S : C, 41.14; H, 3.16; N,
2
4
22
6
6
4 4
11.99; found, C, 41.37; H, 3.19; N, 12.15.
Bis(4-pyridin-2-yl-1-piperazinylthiocarbonyl) disulfide (22).The title compound was
synthesized by oxidation of sodium 4-(pyridin-2-yl)piperazine-1-carbodithioate in 61% yield as
–
1
1
white-yellow solid; mp: 168–170 °C; IR (KBr) ν (cm ): 2944, 1657, 1561, 1474; H NMR (300
MHz, CDCl
3
):
δ
8.19–8.14 (2H, m); 7.57–7.47 (2H, m); 6.72–6.67 (4H, m); 4.41 (8H, bs), 3.78
+
(8H, bs); ESI-MS: (m/z) 477 (MH ); Anal. (%) calcd. for C H N S : C, 50.39; H, 5.07; N,
2
0
24
6 4
17.63; found, C, 50.55; H, 5.32; N, 17.41.
Bis(4-pyrimidin-2-yl-1-piperazinylthiocarbonyl) disulfide (23).The title compound was
synthesized by oxidation of sodium 4-(pyrimidin-2-yl)piperazine-1-carbodithioate in 62% yield
–
1
as white solid; mp: 125–127 °C; IR (KBr) ν (cm ): 2991, 2861, 1586, 1552, 1481, 1420, 1352,
1
1221; H NMR (300 MHz, CDCl ):
δ
8.35 (4H, d, J = 4.7 Hz), 6.59–6.56 (2H, m), 4.38 (8H, bs),
3
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00
01
02
03
4.06–4.03 (8H, m); ESI-MS: (m/z) 479 (MH ); Anal. (%) calcd. for C18
H
22
N
8
S
4
: C, 45.16; H,
4.63; N, 23.41; found, C, 45.29; H, 4.42; N, 23.24.
Bis(4-benzoyl-1-piperazinylthiocarbonyl) disulfide (24). The title compound was synthesized
from sodium 4-benzoylpiperazine-1-carbodithioate in 52% yield as white solid; m.p: 150–152
–
1
1
°C; IR (KBr) ν (cm ): 2969, 1742, 1530, 1364, 1263, H NMR (300 MHz, CDCl ):
δ 7.44–7.42
3
1
3
(10H, m), 4.32–3.70 (16H, m); C NMR (75 MHz, CDCl ): 197.5, 163.4, 135.6, 133.5, 132.1,
3
+
129.4, 129.1, 128.7, 127.9, 127.8, 50.1, 42.2; ESI-MS: m/z 531 (MH ); Anal. (%) calcd. for
C H N O S : C, 54.31; H, 4.94; N, 10.56; found, C, 54.12; H, 5.15; N, 10.69.
24
26
4
2 4
Bis(4-ethoxycarbonyl-1-piperazinylthiocarbonyl) disulfide (25).The title compound was
synthesized by oxidation of sodium 4-(ethoxycarbonyl)piperazine-1-carbodithioate in 61% yield
–
1
as white solid; mp: 120–122 °C; IR (KBr) ν (cm ): 2978, 2901, 1686, 1479, 1418, 1282, 1162,
1
1126; H NMR (300 MHz, CDCl ):
δ 4.29 (8H, bs), 4.22–4.15 (4H, q, J = 7.1 Hz), 3.70–3.67
3
1
3
(8H, m), 1.29 (6H, t, J = 7.1 Hz); C NMR (75 MHz, CDCl ):
δ
194.0 (C=S), 155.2 (C=O), 62.0,
3
+
52.0, 43.2, 14.6; ESI-MS: (m/z) 468 (MH ); Anal. (%) calcd. for C16
H
26
N
4
O
4
S
4
: C, 41.18; H,
5.62; N, 12.01; found, C, 41.24; H, 5.41; N, 12.28.
Bis(4-isobutoxycarbonyl-1-piperazinylthiocarbonyl) disulfide (26). The title compound was
synthesized from sodium 4-(isobutoxycarbonyl)piperazine-1-carbodithioate in 50% yield as
–
1
1
white solid; m.p 130-132 °C; IR (KBr) ν (cm ): 2927, 2866, 1639, 1455, 1412; H NMR (300
MHz, CDCl3): 4.30 (8H, bs), 3.92 (4H, d, J = 6.6 Hz), 3.69 (8H, bs), 2.02–1.89 (2H, m), 0.95
δ
1
3
(12H, d, J = 6.7 Hz); C NMR (50 MHz, CDCl ):
δ
194.0, 155.2, 62.0, 51.8, 49.2, 24.9, 14.7;
3
+
ESI-MS: m/z 523(MH ); Anal. (%) calcd. for C H N O S : C, 45.95; H, 6.56; N, 10.72; found,
2
0
34
4
4 4
C, 46.12; H, 6.65; N, 10.85.
Bis(4-tert-butoxycarbonyl-1-piperazinylthiocarbonyl) disulfide (27).The title compound was
synthesized by oxidation of sodium 4-(tert-butoxycarbonyl)piperazine-1-carbodithioate in 69%
–
1
1
yield as white solid; mp: 163-165 °C; IR (KBr) ν (cm ): 2976, 2843, 1690, 1228; H NMR (300
1
3
MHz, CDCl3):
193.9 (C=S), 154.4 (C=O), 80.7, 52.3, 43.1, 28.4;ESI-MS: (m/z) 545 (M + Na); Anal. (%)
calcd. for C H N O S : C, 45.95; H, 6.56; N, 10.72; found, C, 46.14; H, 6.42; N, 10.61.
δ4.27 (8H, bs), 3.64–3.61 (8H, m), 1.48 (18H, s); C NMR (50 MHz, CDCl ):
3
δ
2
0
34
4
4 4
Bis(4-methylsulfonyl-1-piperazinylthiocarbonyl) disulfide (28). The title compound was
synthesized from sodium 4-(methylsulfonyl)piperazine-1-carbodithioate in 46% yield as white
17

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20
21
22
23
24
25
26
27
28
29
30
31
32
33
solid; m.p: 138–140 °C; IR (KBr) ν (cm ): 2969, 1742, 1530, 1364, 1263, H NMR (300 MHz,
CDCl3): 4.41 (4H, t, J = 5.2 Hz), 3.96 (4H, t, J = 5.4 Hz), 3.50 (4H, t, J = 5.1 Hz), 3.24 (4H, t,
δ
1
3
3
J = 5.3 Hz), 2.84 (6H s); C NMR (50 MHz, CDCl ): δ194.0, 62.0, 51.8, 43.2; ESI-MS: m/z 479
+
(MH ); Anal. (%) calcd. for C H N O S : C, 30.11; H, 4.63; N, 11.70; found, C, 30.40; H,
1
2
22
4
4 6
4.90; N, 11.52.
Bis(4-tosyl-1-piperazinylthiocarbonyl) disulfide (29). The title compound was synthesized
from sodium 4-tosylpiperazine-1-carbodithioate in 48% yield as white solid; m.p: 140–142 °C;
–
1
1
IR (KBr) ν (cm ): 2969, 1742, 1530, 1364, 1263, H NMR (300 MHz, CDCl ):
δ
7.64–7.61 (4H,
3
m), 7.35–7.26 (4H, m), 4.38–4.32 (5H, m), 3.92–3.89 (2H, m), 3.27–2.97 (9H, m), 2.43 (6H, s);
1
3
3
C NMR (100 MHz, CDCl ): δ198.5, 146.7, 128.9, 128.8, 122.9, 48.9, 45.3, 22.7; ESI-MS: m/z
+
653 (M + Na); Anal. (%) calcd. for C H N O S : C, 45.69; H, 4.79; N, 8.88; found, C, 45.58;
2
4
30
4
4 6
H, 4.95; N, 8.70.
Bis(4-butylthiocarbonothioyl-1-piperazinylthiocarbonyl) disulfide (30).The title compound
was synthesized by oxidation of sodium 4-(butylthiocarbonothioyl)piperazine-1-carbodithioate
–
1
in 56% yield as light green solid; mp: 172–174 °C; IR (KBr) ν (cm ): 2927, 2866, 1639, 1216;
1
3
H NMR (300 MHz, CDCl ): δ4.40–4.39 (16H, m), 3.33 (4H, t, J = 7.4 Hz ), 1.75–1.66 (4H, m),
1
3
3
1.52–1.40 (4H, m), 0.95 (6H, t, J = 7.3 Hz ); C NMR (50 MHz, CDCl ): δ198.7 (C=S), 194.0
+
(C=S), 48.7, 37.2, 30.6, 22.2, 13.7;ESI-MS: (m/z) 587 (MH ); Anal. (%) calcd. for C H N S :
2
0
34
4 8
C, 40.92; H, 5.84; N, 9.54; found, C, 41.10; H, 5.97; N, 9.69.
Bis(4-hexylthiocarbonothioyl-1-piperazinylthiocarbonyl) disulfide (31).The title compound
was synthesized by oxidation of sodium 4-(hexylthiocarbonothioyl)piperazine-1-carbodithioate
–
1
1
in 69% yield as yellow solid; mp:158–160 °C; IR (KBr) ν (cm ): 2923, 2860, 1628, 1214; H
NMR (300 MHz, CDCl ): 4.40–4.39 (16H, m), 3.50 (4H, t, J = 7.4 Hz), 2.68–2.63 (4H, m),
3
δ
1
3
3
2.47 (8H, bs), 1.59–1.57 (8H, m), 1.45–1.43 (4H, m); C NMR (75 MHz, CDCl ): δ198.6
+
(C=S), 194.0 (C=S), 57.4, 54.4, 48.9, 34.6, 25.9, 24.4; ESI-MS: (m/z) 643 (MH ); Anal. (%)
calcd. for C H N S : C, 44.82; H, 6.58; N, 8.71; found, C, 44.65; H, 6.72; N, 8.86.
2
4
42
4 8
Bis(4-heptylthiocarbonothioyl-1-piperazinylthiocarbonyl) disulfide (32).The title compound
was synthesized by oxidation of sodium 4-(heptylthiocarbonothioyl)piperazine-1-carbodithioate
–
1
1
in 55% yield as white solid; mp: 169–171 °C; IR (KBr) ν (cm ): 2930, 2854, 1639, 1216; H
NMR (300 MHz, CDCl3): 4.41–4.39 (16H, m), 3.32 (4H, t, J = 7.4 Hz), 1.76–1.74 (4H, m),
δ
18

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34
35
36
37
38
39
40
41
42
43
44
45
46
47
1.42–1.25 (16H, m), 0.91–0.86 (6H, m); C NMR (125 MHz, CDCl3):
δ
198.7 (C=S), 193.9
+
(C=S), 48.8, 48.1, 37.5, 31.7, 29.0, 28.9, 28.5, 22.6, 14.1; ESI-MS: (m/z) 671 (MH ); Anal. (%)
calcd. for C H N S : C, 46.53; H, 6.91; N, 8.35; found, C, 46.74; H, 6.78; N, 8.46.
2
6
46
4 8
Bis(4-octylthiocarbonothioyl-1-piperazinylthiocarbonyl) disulfide (33).The title compound
was synthesized by oxidation of sodium 4-(octylthiocarbonothioyl)piperazine-1-carbodithioate in
–
1
1
62% yield as pale yellow solid; mp: 147–149 °C; IR (KBr) ν (cm ): 2923, 2855, 1643, 1219; H
NMR (300 MHz, CDCl3): 4.66–4.24 (16H, m), 3.32 (4H, t, J = 7.4 Hz), 1.76–1.66 (4H, m),
1.44–1.28 (20H, m), 0.90–0.86 (6H, m); C NMR (100 MHz, CDCl + DMSO-d ):
δ
1
3
δ
197.6
3
6
(C=S), 193.2 (C=S), 47.2, 36.8, 36.5, 31.0, 28.4, 28.2, 27.8, 21.9, 13.4; ESI-MS: (m/z) 699
+
(MH ); Anal. (%) calcd. for C H N S : C, 48.09; H, 7.21; N, 8.01; found, C, 47.93; H, 7.38; N,
2
8
50
4 8
8.16.
Bis(4-decylthiocarbonothioyl-1-piperazinylthiocarbonyl) disulfide (34).The title compound
was synthesized by oxidation of sodium 4-(decylthiocarbonothioyl)piperazine-1-carbodithioate
–
1
in 65% yield as light yellow solid; mp:138–140 °C; IR (KBr) ν (cm ): 2926, 2856, 1635, 1218;
1
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
6
H NMR (300 MHz, DMSO-d ): δ4.49–4.46 (4H, m), 4.38–4.05 (8H, m), 3.80 (4H, t, J = 5.7
Hz), 3.25 (4H, t, J = 7.4 Hz), 1.68–1.58 (4H, m), 1.41–1.24 (28H, m), 0.87–0.83 (6H, m);ESI-
+
MS: m/z 755 (MH ); Anal. (%) calcd. for C H N S : C, 50.88; H, 7.74; N, 7.42; found, C,
3
2
58
4 8
51.03; H, 7.56; N, 7.28.
Bis[4-((2-(prrrolidin-1-yl)ethylthio)carbonothioyl)-1-piperazinylthiocarbonyl]
disulfide
(35).The title compound was synthesized by oxidation of sodium 4-((2-(pyrrolidin-1-
yl)ethylthio)carbonothioyl)piperazine-1-carbodithioate in 63% yield as white solid; mp: 140–145
–
1
1
°d; IR (KBr) ν (cm ): 2921, 2863, 1642, 1219; H NMR (300 MHz, CDCl ): δ4.40–4.39 (16H,
3
1
3
m), 3.53 (4H, t, J = 7.0 Hz ), 2.82 (4H, t, J = 7.0 Hz), 2.60 (8H, bs), 1.80 (8H, bs ); C NMR (75
3
MHz, CDCl ): δ198.5 (C=S), 194.0 (C=S), 54.7, 54.1, 50.8, 48.9, 36.4, 29.7, 23.6;ESI-MS: (m/z)
+
669 (MH ); Anal. (%) calcd. for C24
6.14; N, 12.33.
40 6 8
H N S : C, 43.08; H, 6.03; N, 12.56; found, C, 42.93; H,
Bis[4-((2-(piperidin-1-yl)ethylthio)carbonothioyl)-1-piperazinylthiocarbonyl]
disulfide
(36).The title compound was synthesized by oxidation of sodium 4-((2-(pireidin-1-
yl)ethylthio)carbonothioyl)piperazine-1-carbodithioate in 57% yield as white solid; mp:182–184
–
1
1
°C; IR (KBr) ν (cm ): 2926, 2854, 1638, 1217; H NMR (300 MHz, CDCl
3
): δ4.40–4.39 (16H,
m), 3.50 (4H, t, J = 7.3 Hz), 2.66 (4H, t, J = 7.3 Hz), 2.47 (8H, bs), 1.59–1.57 (8H, m), 1.45–
19

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78
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80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
1.43 (4H, m); C NMR (75 MHz, CDCl ):
δ
198.6 (C=S), 194.0 (C=S), 57.4, 54.4, 48.9, 34.6,
3
+
25.9, 24.4; ESI-MS: (m/z) 697 (MH ); Anal. (%) calcd. for C26
H
44
N
6
8
S : C, 44.79; H, 6.36; N,
12.05; found, C, 44.92; H, 6.14; N, 12.29.
Bis[4-((2-(morpholin-1-yl)ethylthio)carbonothioyl)-1-piperazinylthiocarbonyl]
disulfide
(37).The title compound was synthesized by oxidation of sodium 4-((2-(morpholin-1-
yl)ethylthio)carbonothioyl)piperazine-1-carbodithioate in 54% yield as white solid; mp:170–172
–
1
1
°C; IR (KBr) ν (cm ): 2923, 2858, 1639, 1215; H NMR (300 MHz, CDCl
3
): δ4.41–4.39 (16H,
1
3
m), 3.73–3.70 (8H, m), 3.51 (4H, t, J = 7.2 Hz), 2.70 (4H, t, J = 7.2 Hz), 2.55–2.52 (8H, m); C
NMR (125 MHz, CDCl3): δ198.2 (C=S), 193.9 (C=S), 66.9, 57.0, 53.4, 49.4, 34.3;ESI-MS: (m/z)
+
701 (MH ); Anal. (%) calcd. for C H N O S : C, 41.11; H, 5.75; N, 11.99; found, C, 41.27; H,
24
40
6
2 8
5.91; N, 12.13.
Bis(4-benzylthiocarbonothioyl-1-piperazinylthiocarbonyl) disulfide (38).The title compound
was synthesized by oxidation of sodium 4-(benzylthiocarbonothioyl)piperazine-1-carbodithioate
–
1
in 61% yield as white solid; mp:135–137 °C; IR (KBr) ν (cm ): 2918, 2850, 1639, 1534, 1494,
1
1452, 1219; H NMR (300 MHz, CDCl
3
):
δ7.39–7.25 (10H, m), 4.58 (4H, s), 4.39–4.38 (16H,
1
3
m); C NMR (75 MHz, CDCl ):
δ197.5 (C=S), 135.6, 129.4, 128.7, 127.7, 48.6, 42.2; ESI-MS:
3
+
(m/z) 655 (MH ); Anal. (%) calcd. for C H N S : C, 47.67; H, 4.62; N, 8.55; found, C, 47.89;
2
6
30
4 8
H, 4.75; N, 8.31.
Propyl 4-butylpiperazine-1-carbodithioate (39). To the mixture of sodium 4-butylpiperazine-
1-carbodithioate (2.0 g, 8.33 mmol), triethyl amine (1.21 g, 12.49 mmol) and methanol (10 mL)
was added propyl bromide (1.02 g, 8.33 mmol) drop wise through dropping funnel over 30
minutes at room temperature. The above reaction mixture was further stirred at room temperature
for one hour. The reaction mixture was concentrated over rotavapor and 50 mL ethyl acetate was
added to it. Ethyl acetate layer was washed with distilled water (5 x 3 mL) and dried over sodium
sulfate. Sodium sulfate was filtered off and filtrate was concentrated under reduced pressure. The
crude product was purified over column chromatography (100–200 mesh) using MeOH/CHCl
3
–
1
as eluent to afford compound as yellow liquid (0.70 g, 65%); IR (Neat) ν (cm ): 3019, 2400,
1
1632, 1523, 1215; H NMR (400 MHz, CDCl ):
δ 4.34–3.96 (4H, m), 3.30–3.27 (2H, m), 2.51
3
(4H, t, J = 5.1 Hz), 2.38–2.34 (2H, m), 1.76–1.70 (2H, m), 1.51–1.44 (2H, m), 1.38–1.29 (2H,
1
3
3
m), 1.02 (3H, t, J = 7.4 Hz), 0.92 (3H, t, J = 7.3 Hz); C NMR (100 MHz, CDCl ): δ 197.3,
20

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21
22
23
24
25
58.0, 52.7, 49.9, 39.2, 29.0, 22.2, 20.7, 14.1, 13.6; ESI-MS m/z: 261 (MH ); Anal. (%) calcd. for
C H N S : C, 55.34; H, 9.29; N, 10.76; found C, 54.52; H, 9.46; N, 10.85.
12
24
2 2
The following compounds (40–44) were prepared using a procedure similar to that described for
compound 39 from the corresponding sodium dialkylcarbamodithioate and haloalkane.
Hexyl 4-butylpiperazine-1-carbodithioate (40). The title compound was synthesized from
sodium 4-butylpiperazine-1-carbodithioate and hexyl bromide in 62% yield as yellow liquid; IR
–
1
1
(Neat) ν (cm ): 3020, 2932, 2401, 1634, 1466; H NMR (400 MHz, CDCl
3
): δ 4.34–3.74 (4H,
m), 3.29 (2H, t, J = 7.5 Hz), 2.52–2.49 (4H, m), 2.38–2.34 (2H, m), 1.71–1.66 (2H, m), 1.49–
1
3
1.28 (10H, m), 0.94–0.88 (6H, m); C NMR (100 MHz, CDCl ):
δ
197.3, 58.0, 52.7, 51.1, 37.3,
3
+
31.4, 29.0, 28.8, 28.6, 22.6, 20.7, 14.1, 14.1; ESI-MS m/z: 303 (MH ); Anal. (%) calcd. for
C H N S : C, 59.55; H, 9.99; N, 9.26; found C, 59.63; H, 10.14; N, 9.33.
15
30
2 2
Propyl 4-allylpiperazine-1-carbodithioate (41). The title compound was synthesized from
sodium 4-allylpiperazine-1-carbodithioat and propyl bromide in 87% yield as yellow liquid;IR
–
1
1
(Neat) ν (cm ): 3434, 2968, 2402, 1423, 1218; H NMR (400 MHz, CDCl ):
δ
5.89–5.79 (1H,
3
m), 5.23–5.16 (2H, m), 4.35–3.97 (4H, m), 3.30–3.26 (2H, m), 3.04–3.01 (2H, m), 2.52 (4H, t, J
1
3
3
= 5.1 Hz), 1.77–1.70 (2H, m), 1.04–1.00 (3H, m); C NMR (100 MHz, CDCl ): δ 197.4, 134.3,
+
118.7, 61.2, 52.4, 49.8, 39.2, 22.1, 13.6; ESI-MS m/z: 245 (MH ); Anal. (%) calcd. for
C H N S : C, 54.05; H, 8.25; N, 11.46; found C, 54.03; H, 8.53; N, 11.41.
11
20
2 2
Hexyl 4-allylpiperazine-1-carbodithioate (42). The title compound was synthesized from
sodium 4-allylpiperazine-1-carbodithioate and hexyl bromide in 80% yield as yellow liquid; IR
–
1
1
(Neat) ν (cm ): 3079, 2859, 2402, 1641, 1463; H NMR (400 MHz, CDCl ):
δ
5.88–5.84(1H,
3
m), 5.23–5.17 (2H, m), 4.69–3.97 (4H, m), 3.29 (2H, t, J = 7.5 Hz), 3.04–3.01 (2H, m), 2.53
1
3
(4H, t, J = 5.1 Hz), 1.71–1.64 (2H, m), 1.42–1.28 (6H, m), 0.90–0.87 (3H, m); C NMR (100
MHz, CDCl3): 197.5, 134.3, 118.7, 61.2, 52.4, 51.0, 37.4, 31.4, 28.8, 28.6, 22.6, 14.1; ESI-MS
m/z: 287 (MH ); Anal. (%) calcd. for C H N S : C, 58.69; H, 9.15; N, 9.78; found C, 58.53; H,
δ
+
1
4
26
2 2
9.25; N, 9.83.
Propyl 4-(3-cyanopropyl)piperazine-1-carbodithioate (43). The title compound was
synthesized from sodium 4-(3-cyanopropyl)piperazine-1-carbodithioate and propyl bromide in
–
1
1
55% yield as yellow liquid; IR (Neat) ν (cm ): 3444, 3019, 2400, 1637, 1466, 1215; H NMR
(400 MHz, CDCl3): 4.35–3.94 (4H, m), 3.30–3.26 (2H, m), 2.53–2.49 (6H, m), 1.87–1.80 (2H,
m), 1.79–1.70 (2H, m), 1.59 (2H, m), 1.02 (3H, t, J = 7.4 Hz); C NMR (100 MHz, CDCl ):
δ
13
δ
3
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197.6, 119.6, 55.8, 52.5, 50.8, 49.7, 39.2, 29.7, 22.7, 22.1, 14.9, 13.6; ESI-MS m/z: 272 (MH );
Anal. (%) calcd. for C H N S : C, 53.10; H, 7.80; N, 15.48; found C, 53.34; H, 8.01; N, 15.27.
1
2
21
3 2
Hexyl 4-(3-cyanopropyl)piperazine-1-carbodithioate (44). The title compound was
synthesized from sodium 4-(3-cyanopropyl)piperazine-1-carbodithioate and hexyl bromide in
–
1
1
58% yield as yellow liquid; IR (Neat) ν (cm ) 3020, 2931, 2401, 1637, 1424; H NMR (400
MHz, CDCl3) 4.31–3.86 (4H, m), 3.31–3.27 (2H, m), 2.53–2.48 (4H, m), 1.86 (2H, t, J = 6.8
Hz), 1.82–1.80 (2H, m), 1.73–1.70 (4H, m), 1.69–1.65 (2H, m), 1.60–1.55 (4H, m), 0.90–0.86
δ
1
3
(3H, m); C NMR (100 MHz, CDCl
3
)
δ
197.7, 121.1, 57.7, 55.8, 52.5, 37.4, 31.4, 28.8, 28.6,
+
22.7, 22.6, 15.0, 14.1; ESI-MS m/z: 314 (MH ); Anal. (%) calcd. for C H N S : C, 57.46; H,
1
5
27
3 2
8.68; N, 13.40; found C, 57.34; H, 8.51; N, 13.27.
Synthesis of methylene bis(4-methylpiperazine-1-carbodithioate) (45). The mixture of 4-
methylpiperazine-1-carbodithioate (2.21 gm, 11.16 mmol) and diiodomethane (0.3 mL, 3.72
mmol) in CH CN (20 mL) was stirred at room temperature for overnight. The reaction mixture
3
was concentrated under reduced pressure, crude product was treated with water (10 mL) and
extracted withEtOAc (10 x 3 mL). EtOAc layer was washed with water (5 x 3 mL) and
combined organic layers were dried on anhydrous sodium sulfate, filtered, and concentrated. The
crude product was purified over column chromatography (100–200 mesh) using MeOH/CHCl3
–
1
as eluent to afford compound as white solid (0.76 g, 56%); mp: 140–142 °C; IR (KBr) ν (cm )
1
2929, 2799, 1658; H NMR (300 MHz, CDCl ):
δ
5.42 (2H, s), 4.32 (4H, bs), 3.90 (4H, bs), 2.48
): 195.7 (C=S), 54.3, 51.3, 49.9, 45.6; ESI-
: C, 42.82; H, 6.63; N, 15.37; found C,
3
1
3
(8H, bs), 2.31 (6H, s); C NMR (75 MHz, CDCl
3
δ
+
MS: (m/z)365 (MH ); Anal. (%) calcd. for C13
H
24
N
4
S
4
42.96; H, 6.75; N, 15.42.
The following compounds (46–56) were prepared using a procedure similar to that described for
compound 45 from the corresponding sodium dialkylcarbamodithioate and dihaloalkane.
Ethane-1,2-diyl bis(4-methylpiperazine-1-carbodithioate) (46). The title compound was
synthesized from sodium 4-methylpiperazine-1-carbodithioate and 1,2-dibromoethane in 53%
–
1
1
yield as white solid; mp: 150–152 °C; IR (KBr) ν (cm ): 2931, 2854, 1630; H NMR (300 MHz,
1
3
CDCl3):
δ
4.35 (4H, bs), 3.96 (4H, bs), 3.68 (4H, s), 2.51–2.48 (8H, m), 2.33 (6H, s); C NMR
+
(75 MHz, CDCl3):
δ
196.2 (C=S), 54.4, 51.2, 50.0, 45.7, 35.8; ESI-MS: (m/z)379 (MH ); Anal.
(%) calcd. for C H N S : C, 44.41; H, 6.92; N, 14.80; found C, 44.32; H, 7.16; N, 14.72.
1
4
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Propane-1,3-diyl bis(4-methylpiperazine-1-carbodithioate) (47). The title compound was
synthesized from sodium 4-methylpiperazine-1-carbodithioate and 1,2-dibromopropane in 53%
–
1
1
yield as white solid; mp: 120–122 °C; IR (KBr) ν (cm ): 2927, 2853, 1654; H NMR (300 MHz,
CDCl3):
δ
4.32–3.97 (8H, m), 3.43 (4H, t, J = 7.2 Hz), 2.50–2.47 (8H, m), 2.32 (6H, s), 2.18–
+
2.09 (2H, m); ESI-MS: (m/z) 393 (MH ); Anal. (%) calcd. for C15
H
28
N
4
4
S : C, 45.88; H, 7.19; N,
14.27; found C, 45.67; H, 7.21; N, 14.47.
Methylene bis(4-allylpiperazine-1-carbodithioate) (48). The title compound was synthesized
from sodium 4-allylpiperazine-1-carbodithioate and diiodomethane in 54% yield as white solid;
–
1
1
mp: 125–127 °C; IR (KBr) ν (cm ): 2908, 2804, 1646; H NMR (300 MHz, CDCl ):
δ
5.88–
3
5.79 (2H, m), 5.42 (2H, s), 5.23–5.17 (4H, m), 4.34 (4H, bs), 3.90–3.89 (4H, m), 3.03 (4H, d, J =
1
3
3
6.5 Hz), 2.52 (8H, bs,); C NMR (75 MHz, CDCl ): δ 195.8 (C=S), 134.2, 118.8, 61.1, 52.3,
+
51.6, 50.1, 45.7; ESI-MS: (m/z)417 (MH ); Anal. (%) calcd. for C17
H
28
N
S
4 4
: C, 49.00; H, 6.77;
N, 13.45; found C, 49.25; H, 6.67; N, 13.59.
Ethane-1,2-diyl bis(4-allylpiperazine-1-carbodithioate) (49). The title compound was
synthesized from sodium 4-allylpiperazine-1-carbodithioate and diiodoethane in 46% yield as
–
1
1
white solid; mp: 140–142 °C; IR (KBr) ν (cm ): 2925, 2856, 1659; H NMR (300 MHz, CDCl
3
+ CCl4):
δ 5.87–5.78 (2H, m), 5.21–5.16 (4H, m), 4.36–3.89 (8H, m), 3.63 (4H, s), 3.02 (4H, d, J
1
3
= 6.4 Hz), 2.52 (8H, bs); C NMR (75 MHz, CDCl + CCl ):
δ
196.2 (C=S), 134.6, 118.8, 61.4,
3
4
+
30 4 4
52.6, 51.0, 35.9; ESI-MS: (m/z)431 (MH ); Anal. (%) calcd. for C18H N S : C, 50.19; H, 7.02;
N, 13.01; found C, 50.21; H, 7.32; N, 13.30.
Propane-1,3-diyl bis(4-allylpiperazine-1-carbodithioate) (50). The title compound was
synthesized from sodium 4-allylpiperazine-1-carbodithioate and diiodopropane in 50% yield as
–
1
1
white solid; mp: 108–110 °C; IR (KBr) ν (cm ): 2923, 2807, 1632; H NMR (300 MHz, CDCl
3
+ CCl4):
δ 5.89–5.77 (2H, m), 5.23–5.17 (4H, m), 4.33–3.97 (8H, m), 3.42 (4H, t, J = 7.2 Hz),
1
3
3.03 (4H, d, J = 6.5 Hz), 2.54–2.51 (8H, m), 2.18–2.08 (2H, m); C NMR (75 MHz, CDCl
3
+
+
CCl
4
): δ 196.3 (C=S), 134.5, 118.6, 61.3, 52.5, 50.9, 50.0, 35.9, 28.2; ESI-MS: (m/z)445 (MH );
Anal. (%) calcd. for C H N S : C, 51.31; H, 7.25; N, 12.60; found C, 51.38; H, 7.27; N, 12.63.
1
9
32
4 4
Methylene bis(4-butylpiperazine-1-carbodithioate) (51). The title compound was synthesized
from sodium 4-butylpiperazine-1-carbodithioate and diiodomethane in 50% yield as white solid;
-1
1
mp: 116–118 °C; IR (KBr) ν (cm ): 2933, 2847, 1642; H NMR (300 MHz, CDCl + CCl ):
δ
3
4
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5.38 (2H, s), 4.29–3.89 (8H, m), 2.50 (8H, bs), 2.37–2.32 (4H, m), 1.48–1.29 (8H, m), 0.92 (6H,
1
3
t, J = 7.2 Hz); C NMR (75 MHz, CDCl
3
+ CCl
4
):
δ 195.6 (C=S), 57.9, 52.7, 51.5, 50.2, 45.6,
+
29.1, 20.7, 14.2; ESI-MS: (m/z)449 (MH ); Anal. (%) calcd. for C H N S : C, 50.85; H, 8.09;
1
9
36
4 4
N, 12.48; found C, 50.76; H, 8.21; N, 12.29.
Ethane-1,2-diyl bis(4-butylpiperazine-1-carbodithioate) (52). The title compound was
synthesized from sodium 4-butylpiperazine-1-carbodithioate and diiodoethane in 46% yield as
–
1
1
white solid; mp: 120–122 °C; IR (KBr) ν (cm ): 2927, 2821, 1639; H NMR (300 MHz, CDCl
+ CCl ):
(4H, m), 1.37–1.30 (4H, m), 0.95–0.90 (6H, m); C NMR (75 MHz, CDCl
3
4
δ 4.28-3.96 (8H, m), 3.64 (4H, s), 2.52–2.49 (8H, m), 2.37–2.33 (4H, m), 1.49–1.44
13
3
+ CCl
4
):
δ
195.9
+
(C=S), 58.0, 52.8, 51.3, 50.3, 35.8, 29.1, 20.8, 14.2; ESI-MS: (m/z)463 (MH ); Anal. (%) calcd.
for C H N S : C, 51.90; H, 8.28; N, 12.11; found C, 52.11; H, 8.35; N, 12.20.
2
0
38
4 4
Propane-1,3-diyl bis(4-butylpiperazine-1-carbodithioate) (53). The title compound was
synthesized from sodium 4-butylpiperazine-1-carbodithioate and diiodopropane in 40% yield as
–
1
1
white solid; mp: 80–82 °C; IR (KBr) ν (cm ): 2926, 2816, 1643; H NMR (300 MHz, CDCl +
3
CCl
2.17–2.07 (2H, m), 1.50–1.42 (4H, m), 1.38–1.30 (4H, m), 0.93 (6H, t, J = 7.2 Hz); C NMR
(75 MHz, CDCl + CCl ): 196.4 (C=S), 58.0, 52.8, 50.6, 35.9, 29.1, 28.3, 20.8, 14.2; ESI-MS:
(m/z) 477 (MH ); Anal. (%) calcd. for C H N S : C, 52.90; H, 8.46; N, 11.75; found C, 52.88;
4
): δ 4.29–3.97 (8H, m), 3.40 (4H, t, J = 7.1 Hz), 2.52–2.49 (8H, m), 2.38–2.33 (4H, m),
1
3
δ
3
4
+
2
1
40
4 4
H, 8.35; N, 11.55.
Methylene bis(4-(3-cyanopropyl)piperazine-1-carbodithioate) (54). The title compound was
synthesized from sodium 4-(3-cyanopropyl)piperazine-1-carbodithioate and diiodomethane in
–
1
1
65% yield as white solid; mp: 130–132 °C; IR (KBr) ν (cm ): 2944, 2822, 2246, 1647; H NMR
(300 MHz, CDCl + CCl ): 5.36 (2H, s), 4.28–3.91 (8H, m), 2.53–2.41 (16H, m), 1.87–1.78
(4H, m); C NMR (75 MHz, CDCl + CCl ): 195.7 (C=S), 119.1, 55.8, 52.6, 50.0, 45.7 , 22.8,
15.0; ESI-MS: (m/z)471 (MH ); Anal. (%) calcd. for C H N S : C, 48.48; H, 6.42; N, 17.85;
3
4
δ
1
3
δ
3
4
+
1
9
30
6 4
found C, 48.55; H, 6.32; N, 17.62.
Ethane-1,2-diyl bis(4-(3-cyanopropyl)piperazine-1-carbodithioate) (55). The title compound
was synthesized from sodium 4-(3-cyanopropyl)piperazine-1-carbodithioate and diiodoethane in
–
1
1
51% yield as white solid; mp: 152–154 °C; IR (KBr) ν (cm ): 2929, 2814, 2248, 1632; H NMR
(300 MHz, CDCl ): 4.33–3.96 (8H, m), 3.68 (4H, s), 2.55–2.43 (16H, m), 1.89–1.80 (4H, m);
3
δ
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C NMR (75 MHz, CDCl3): δ 196.3 (C=S), 119.6, 55.8, 52.5, 50.1, 35.8, 22.7, 15.0; ESI-MS:
+
(m/z)485 (MH ); Anal. (%) calcd. for C20
H
32
N
6
S
4
: C, 49.55; H, 6.65; N, 17.34; found C, 49.75;
H, 6.39; N, 17.19.
Propane-1,3-diyl bis(4-(3-cyanopropyl)piperazine-1-carbodithioate) (56). The title
compound was synthesized from sodium 4-(3-cyanopropyl)piperazine-1-carbodithioate and
–
1
diiodopropane in 56% yield as white solid; mp: 118–120 °C; IR (KBr) ν (cm ): 2942, 2823,
1
2248, 1646; H NMR (300 MHz, CDCl + CCl ):
δ 4.34–4.05 (8H, m), 3.40 (4H, t, J = 7.1 Hz),
3
4
1
3
2.55–2.42 (16H, m), 2.16–2.07 (2H, m), 1.88–1.79 (4H, m); C NMR (75 MHz, CDCl
3
+ CCl
4
):
196.5 (C=S), 119.1, 55.9, 52.6, 50.7, 35.9, 28.2, 22.7, 15.0; ESI-MS: (m/z)499 (MH ); Anal.
(%) calcd. for C H N S : C, 50.57; H, 6.87; N, 16.85; found C, 50.68; H, 6.94; N, 16.75.
+
δ
2
1
34
6 4
4.2. Biological materials and methods
3
2
4.2.1. Spermicidal activity
Spermicidal assay was adapted from the standard procedure. Briefly, the test compounds were
dissolved in a minimum volume of DMSO and diluted with physiological 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 seconds 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
determined by visual scoring in the next 30 seconds and recorded (Table 1).
3
2
7
7
7
7
7
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7
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7
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38
39
40
41
42
43
44
45
4.2.2. Anti-Trichomonas activity
Trichomonas vaginalis parasites to be used in drug susceptibility assays were grown in TYM
medium supplemented with 10% FCS, vitamin mixture and 100 U/mL penicillin/streptomycin, at
37 °C in 15 mL tubes for one day, followed by regular subculturing, and were in the log phase of
7
growth. The cultures routinely attained a concentration of 2 x 10 cells/mL in 48h. Inoculums of
4
1 x 10 cells per tube were used for maintenance of the culture. In vitro drug susceptibility assays
were carried out using the standard procedure. Stock solutions (100 µg/mL) of test compounds
were prepared in DMSO. These stock solutions were serially diluted with TYM medium to
obtain concentration up to 0.1 µg/mL in 48-well plates. DMSO/TYM was used as vehicle in
4
control wells. Parasites (5 x 10 trophozoites/L) were added to these wells and incubated
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anaerobically at 37 °C. Cells were checked for viability at different time intervals from 3 to 48 h
under the microscope at 40X magnification. Viability of the cells was determined by trypan blue
exclusion assay. Minimum concentration 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
(Table 2)
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56
4.2.3. Antifungal activity
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
3
MOPS [3-(N-Morpholino)propanesulfonic acid]. Starting inoculums of test culture was 1–5 x 10
CFU/mL. Micro titer plates were incubated at 35 °C. MICs were recorded after 48h of incubation
(Table 2).
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4.2.4. Cytotoxicity towards human cervical (HeLa) cell line by lactate dehydrogenase –
3
0
release assay
A colorimetric assay for lactate dehydrogenase (LDH) release was used for the evaluation of the
cytotoxicity of spermicidal compounds against the HeLa cell line. Exponentially growing HeLa
4
cells were seeded into 96-well tissue culture plates at a density of 2 ×10 cells per well (in
2 2
triplicate). After 24 h incubation in a CO incubator at 37 °C in 5% CO , 95% air atmosphere,
the culture medium [Dulbecco’s modified Eagle’s medium (DMEM)] was replaced with 100.0
µL of fresh medium containing serially diluted spermicidal compounds. Control wells contained
the medium only. Culture plates were incubated for another 5 h, and then 50.0 µL of the
supernatant from each well of the assay plate was pipetted into the corresponding well of a flat-
bottom 96-well plate. Colour reaction for LDH assay and IC50 measurement for cytotoxicity
were performed using CytoTox-96 kit (Promega, Madison, WI, USA) by following the
instructions of the manufacturer. Optical densities at 490 nm were measured in a micro-plate
reader (µQuant, Bio-Tek, USA) (Table 3).
3
0
7
7
7
7
71
72
73
74
4.2.5. Effect on Lactobacillus acidophillusin vitro
The effect of compounds exhibiting potent spermicidal activity on Lactobacillus acidophillus
was determined by following the method published earlier. Briefly, Rogosa SL agar plates
(7.5%; containing 0.132% acetic acid), prepared with (experimental) or without (control) the
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addition of spermicidal compounds, were inoculated with L. acidophillus (∼70 spores/10 cm )
and incubated at 37 °C in 5% CO and 95% air for 72 h. The number and size of colonies were
2
recorded at the end of the experiment. The average colony size (% of control) was multiplied by
the colony number and divided by 100 to arrive at the data presented. The aver-age colony size
for the control was taken as 100% (Table 3).
4
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4.2.6. Spermicidal activity (in vivo)
All animal experiments were conducted in accordance and as per the approval of the Institutional
Animal Ethics Committee of CSIR-Central Drug Research Institute (approval No.
IAEC/2014/93). The rabbits were housed in stainless steel cages and kept in uniform husbandry
conditions of temperature (25-26ºC), relative humidity (50-70%) and light/dark cycle (12/12 h).
Young, adult female Belgian rabbits were given test compound through vaginal instillation. The
test compounds were incorporated in K-Y-Jelly (Johnson & Johnson) through geometrical
dilution. Pure K-Y-Jelly was used in control groups. Two mL of test/control jelly containing the
test compounds at indicated doses (Table 4) was instilled 10–12 cm deep into the vagina of each
rabbit with a catheter attached to the gavage needle of a syringe with the animal held in supine
position. The animal was released after 5 min. Treated females were hand-mated once with a
proven buck. Each buck was allowed one mating. Mating was re-confirmed by presence of
sperm in vaginal smear. The mated rabbits were then kept in separate cages and allowed to
complete gestation of 30-34 days. The pregnancies and litter size was recorded at completion of
the gestation (Table 4).
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4.2.7. Anti-Trichomonalactivity (in vivo)
All animal experiments were conducted in accordance and as per the approval of the Institutional
Animal Ethics Committee of CSIR-Central Drug Research Institute (approval No.
IAEC/2014/94). The mice were housed in polypropylene cages and kept in uniform husbandry
conditions of temperature (25-26ºC), relative humidity (50-70%) and light/dark cycle (12/12 h).
The subcutaneous abscess assay of Krieger et al. (1983) was used. Briefly, the parasites (T.
vaginalis) were cultured under partial anaerobic condition in TYM medium andon attaining
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concentration of approximately 2x10 cells/ml (in ~48 hrs); trichomonads were harvested from
the culture by centrifugation at 250xg for 10 min and then re-suspended in sterile saline. Six-
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week-old mice were inoculated subcutaneously with T. vaginalis (50 µl of 2 x 10 organisms per
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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
were taken from the lesion and examined microscopically to ensure infection.Infected animals
were then treated with compounds (subcutaneously) with a dose of 5.0 µg in 50 µL of saline
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daily for 7 days and abscess size measured longitudinally and the area calculated as πr .
Metronidazole was used as positive control. Control injections of sterile saline did not result in
abscess formation.
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4.2.8. Fluorescent labeling of sperm thiols
Free thiols on human sperm (after treatment with vehicle, and two most promising compound 12
was examined and imaged using a fluorescence microscope, after labeling with the thiols
capturing dye mBBr. The semen sample (0.5 ml) was treated with 2.5 ml of compound 12 at
MEC, as well as equal volume of saline (Control) in parallel and incubated for 15 min at room
temperature. After incubation, sperm were pelleted at 700×g for 10 min and washed 2–3 times
with fresh PBS. To pelleted sperm in 1 ml PBS, 0.5mM (final concentration) mBBr was added
and incubated for 15 min in the dark. After incubation sperm were pelleted and washed with
PBS, finally dissolved in 200 µl PBS. A drop of this sample was then taken on a microscope
slide, covered with a cover glass and imaged using the UV1A filter on a Nikon Eclipse 80i
microscope equipped with epifluorescence illumination. Exposure times were the same for all
samples.
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4.2.9. Qualitative estimation of inhibition of free sulfhydryl groups on
Trichomonasvaginalis
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The effect of test compound on Trichomonas free sulfhydryl groups was examined and imaged
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by a method published earlier with slight modification 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 for 5 min in the dark. A drop of this sample was taken
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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.
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4.2.10. 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
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constructed usingcrystal structure of cysteine synthase from Escherichia coli (PDB ID-2BHS)
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as template with the help of MODELLER package. All docking studies were carried using
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AUTODOCK4.2 Package. For molecular visualization and structure manipulation Chimera was
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used.
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ASSOCIATED CONTENT
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Supporting Information: H NMR spectra of all the compounds, C NMR spectra of forty eight
compounds except (5, 22, 23, 34 and 37) and HRMS data of compounds7, 8, 9, 11, 12, 16 and
19. This material is available free of charge via internet at ……….
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ACKNOWLEDGMENT
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We acknowledge SAIF Division, for providing spectral data. We are grateful to CSIR (S.J., D.M.
and A.S.), UGC (L.K., N.L. and L.K.), and ICMR (V.B., B.K., S.K. and A.J.) for research
fellowships. This study was supported by a grant from the Department of Health Research
(DHR), Indian Council of Medical Research, New Delhi, India, tenable at the Centre for Drug
Discovery and Development in Reproductive Health at CSIR-CDRI, Lucknow, India
(GAP0155).
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ABBREVIATIONS
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STIs, sexually transmitted infections; N-9, nonoxynol-9; DTC, dithiocarbamate; DSF,
disulfiram; SAR, structure activity relationship; MTZ, metronidazole; MOPS, [3-(N-
morpholino)propanesulfonic acid]; LDH, lactate dehydrogenase; DMEM,dulbecco’s modified
eagle’s medium; HCG, human chorionic gonadotrophin; TLC, thin layer chromatography.
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