A unique dithiocarbamate chemistry during design & synthesis of novel sperm-immobilizing agents

Article abstract of DOI:10.1039/c4ob00005f

1-Substituted piperazinecarbodithioates were obtained by an unusual removal of CS₂ in benzyl substituted dithiocarbamate derivatives under acid and basic conditions during design and synthesis of 1,4-(disubstituted) piperazinedicarbodithioates as double edged spermicides. A plausible mechanism for CS₂ removal has been proposed. All synthesized compounds were subjected to spermicidal, antitrichomonal and antifungal activities. Twenty-one compounds irreversibly immobilized 100% sperm (MEC, 0.06-31.6 mM) while seven compounds exhibited multiple activities. Benzyl 4-(2-(piperidin-1-yl)ethyl) piperazine-1-(carbodithioate) (18) and 1-benzyl 4-(2-(piperidin-1-yl)ethyl) piperazine-1,4-bis(carbodithioate) (24) exhibited appreciable spermicidal (MEC, 0.07 and 0.06 mM), antifungal (MIC, 0.069-0.14 and >0.11 mM) and antitrichomonal (MIC, 1.38 and 0.14 mM) activities. The probable mode of action of these compounds seems to be through sulfhydryl binding which was confirmed by fluorescence labeling of sperm thiols.

Full text of DOI:10.1039/c4ob00005f

Organic &
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PAPER
A unique dithiocarbamate chemistry during design
& synthesis of novel sperm-immobilizing agents†‡
Santosh Jangir,a Veenu Bala,a,d Nand Lal,a Lalit Kumar,a Amit Sarswat,a
Lokesh Kumar,b Bhavana Kushwaha,b Pratiksha Singh,c Praveen Kumar Shukla,c
Jagdamba Prasad Maikhuri,b Gopal Guptab and Vishnu Lal Sharma*a
Cite this: Org. Biomol. Chem., 2014,
12, 3090
1-Substituted piperazinecarbodithioates were obtained by an unusual removal of CS2 in benzyl substituted
dithiocarbamate derivatives under acid and basic conditions during design and synthesis of 1,4-(di-
substituted)piperazinedicarbodithioates as double edged spermicides. A plausible mechanism for CS2
removal has been proposed. All synthesized compounds were subjected to spermicidal, antitrichomonal
and antifungal activities. Twenty-one compounds irreversibly immobilized 100% sperm (MEC, 0.06–31.6 mM)
while seven compounds exhibited multiple activities. Benzyl 4-(2-(piperidin-1-yl)ethyl) piperazine-
1-(carbodithioate) (18) and 1-benzyl 4-(2-(piperidin-1-yl)ethyl)piperazine-1,4-bis(carbodithioate) (24)
exhibited appreciable spermicidal (MEC, 0.07 and 0.06 mM), antifungal (MIC, 0.069–0.14 and >0.11 mM)
and antitrichomonal (MIC, 1.38 and 0.14 mM) activities. The probable mode of action of these compounds
seems to be through sulfhydryl binding which was confirmed by fluorescence labeling of sperm thiols.
Received 2nd January 2014,
Accepted 6th March 2014
DOI: 10.1039/c4ob00005f
www.rsc.org/obc
survival of anaerobic microbes such as the STI, Trichomonas
vaginalis.9 Thus, sulfhydryl binding scaffolds have been uti-
Introduction
The current world population is expected to rise to 9.1 billion lized as viable option for the development of dually active
by year 20501 accompanied by an equally challenging rise in sperm immobilizing agents.10
number of sexually transmitted diseases (STD)2 and HIV infec-
In our ongoing efforts10–14 to design non-surfactant dual
tions.3 Hence there is a global need to control the human action vaginal spermicides (I–V, Fig. 1), the most viable phar-
population and spread of sexually transmitted infections (STI), macophore was found to be the dithiocarbamate (DTC) group
through the development of dually active agents capable of as it interacts with sulfhydryl groups present on spermatozoa
preventing conception and disease, and anti-STD vaginal and T. vaginalis.10 Moreover, the DTC group being a versatile
spermicides could be one of best choice. Furthermore the pharmacophore exhibits various biological activities i.e.,
spermicide currently on the market, nonoxynol-9 (N-9), microbicidal–spermicidal,15 fungicidal,16 antabuse,17 anti HIV18
increases the risk of transmission of these infections owing to activities, and being a very fascinating chemical appendage,
its surfactant action.4–7 Therefore, urgent efforts are warranted
to develop antimicrobial, non-detergent spermicidal agents,
preferably in a single chemical entity. The free sulfhydryls
modulate sperm membrane conformations and regulate
energy metabolism, which are vital for motility and viability.8
Moreover, sulfhydryl groups also play an important role in
aMedicinal & Process Chemistry Division, CSIR-Central Drug Research Institute,
Lucknow 226031, India. E-mail: vlscdri1@rediffmail.com, vlscdri@gmail.com;
Fax: +91-522-2771941; Tel: +91-522-2771940, Ext. 4671
bEndocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226031,
India
cMicrobiology Division, CSIR-Central Drug Research Institute, Lucknow 226031,
India
dAcademy of Scientific & Innovative Research, New Delhi, India
†CDRI Communication no. 8627.
‡Electronic supplementary information (ESI) available. See DOI:
Fig. 1 General structures of compounds studied (I–V) and envisaged
scaffold (VI).
10.1039/c4ob00005f
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it generates unique and interesting chemistry e.g., O–S
exchange,19,20 N-trifluoromethyl amine formation,21 elimin-
ation to give alkene,22 unusual epithio product.19
Consequently, it was thought worthwhile to design and syn-
thesize chemical entities with more than one dithiocarbamate
groups in a single framework. Since 4-(N-methyl)piperazine
carbothioic acid sodium salt23 has mild spermicidal activity
and piperazine itself has been designated as a privileged
scaffold,24 attempts were carried out to incorporate a dithiocar-
bamate group on both the nitrogen atoms of piperazine
moiety. Accordingly, 1,4-(disubstituted)piperazinedicarbo-
dithioate derivatives (VI, Fig. 1) were synthesized and evaluated
for spermicidal, antitrichomonal and antifungal activities. The
chemical synthesis, biological evaluation and structure–activity
relationship (SAR) are being discussed in this communication.
Scheme 2 Possible mechanism for synthesis of unusual product (14)
under acidic condition.
Chemistry
1,4-Piperazinedicarbodithioic acid esters were synthesized by
di-tert-butyl dicarbonate (DIBOC) protection and incorporation
of dithiocarbamate on the other nitrogen, followed by esterifi-
cation and deprotection, and incorporation of dithiocarbamate
on this free amine. Piperazine (1) was protected selectively at
one nitrogen (2) which on being reacted with carbon disulfide
under alkaline conditions gave dithiocarbamate sodium salt
(3). Compound (3) was reacted with alkyl halides to yield
carbodithioic esters (4–8), which were deprotected with TFA to
provide desired compounds (9–13), however, in the case of
benzyl chloride an unusual N-benzyl product (14) was isolated
in 20–30% yield (Scheme 1).
The formation of an unusual N-benzyl product (14) can be
explained (Scheme 2) on the basis of acid catalyzed N-depro-
tection of the tert-butyloxy carbonyl group.25,26 Under acidic
conditions N-deprotection occurred via intermediates II and
IIb to give the required product 9. The unusual product (14)
might have been formed when both the carbonyl and thiocar-
bonyl groups get protonated (I) and carbon disulfide is lost to
provide a benzyl cation,22,27 which in turn attacked the free
amine of IIa. The presence of a piperazine-1-carboxylic acid
fragment (IIc) in the mass spectrum further suggested this
mechanism.
The N-deprotected compounds (9, 10, 13) were subjected to
the incorporation of another dithiocarbamate group
(Scheme 3) to get the desired scaffold (VI, Fig. 1). The reaction
of these compounds with carbon disulfide under alkaline con-
ditions gave dithiocarbamate sodium salts (15, 19, 20), which
on being alkylated with alkyl halide in presence of triethyl
amine yielded desired 1,4-bisdithiocarbamate compounds
(21–32). Surprisingly, with S-benzyl compound (15) again
unusual products, benzyl 4-alkylpiperazine-1-carbodithioates
(14, 16–18) were isolated in 20–30% yields.
The formation of these unusual N-benzyl products
(14, 16–18) can be explained (Scheme 4) on the basis of the for-
mation of a triethylbenzyl ammonium salt (IIIb) in the pres-
ence of triethyl amine followed by loss of carbon disulfide.
Scheme 1 Reaction conditions for synthesis of alkyl piperazine-
1-carbodithioate; (a) DIBOC, CHCl3, TEA, 0–5 °C, 4 h; (b) CS2, NaOH,
EtOAc, 0–5 °C, 10 h; (c) alkyl halide, MeOH, TEA, rt, 3 h; (d) (i) TFA, DCM,
0–5 °C, 6 h; (ii) NaHCO3, H2O, 0–5 °C, 4 h.
Scheme 3 Reaction conditions for synthesis of 1-substituted and
1,4-(disubstituted) piperazinedicarbodithioate derivatives; (b) CS2, NaOH,
EtOAc, 0–5 °C, 10 h; (c) alkyl halide, MeOH, TEA, rt, 3 h.
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Anti-Trichomonas activity
Twenty compounds (9–13, 15, 16, 18–24 and 26–31) showed
antitrichomonal activity (Table 1) at MEC, 0.08–1.81 mM.
Eleven compounds (9, 10, 12, 13, 15, 19, 20, 24, 26, 27, and 31)
exhibited
moderate
antitrichomonal
activity
(MEC,
0.14–0.96 mM) while one compound (30) showed remarkable
activity at MEC, 0.08 mM. The standard Drug Metronidazole
demonstrated antitrichomonal activity at 0.018 mM.
Cytotoxicity to cervical epithelium (HeLa) and lactobacilli
Compounds 18 and 24 exhibited an IC50 of >1500 µM against
HeLa cells and lactobacilli (normal vaginal flora), in vitro. In
contrast N-9 displayed much lower IC50 against these cells
(82.3 and 35.0 µM, respectively).
Scheme 4 Possible mechanism for synthesis of unusual product
(14, 16–18) under basic conditions.
Structure–activity relationship (SAR)
S-Debenzylation is known to occur under basic conditions28–30
and the benzyl group is known to dance from one anionic
centre to other within the same molecule.31 The free amine
formed after the loss of carbon disulfide reacts with alkyl
halide and the carbodithioate anion of IIIa attacks the elec-
tron-deficient benzyl carbon to give unusual products (14,
16–18). Thus, loss of carbon disulfide and migration of the
benzyl group occurred simultaneously.
A structure–activity relationship (SAR) study revealed that
N-demethylation and S-esterification of the reference compound
(33) enhanced the spermicidal activity by two and a half fold
(9, 10, 13) and thirteen-fold (12) among alkyl piperazine-1-car-
bodithioates (9–13) whereas the activity was lost with S-mor-
pholinoethyl group (11). Two compounds (9, 13) exhibited
mild antifungal (MIC, 0.11–0.23 mM) and antitrichomonal
activity (MIC 0.50 and 0.57 mM). The N-alkylation of benzyl
4-piperazine-1-carbodithioate (9) with piperidinoethyl group (18)
gave highly potent spermicidal compound, which was 283 and
720 times more active than compound 9 and the reference
compound, 33 respectively. Whereas an N-allyl group (16),
N-benzyl (14) and N-butyl (17) groups were less desirable. The
incorporation of an additional carbodithioic acid group as
sodium salt in compound 9 and 13 retained the spermicidal
and antitrichomonal activity while the antifungal activity was
highly enhanced (15, 20).
Biological evaluation
Spermicidal activity
All compounds (except 11, 14 and 17) exhibited 100% spermi-
cidal activity (Table 1) at a minimum effective concentration
(MEC) ranging from 0.06–31.6 mM while reference compound
(33) and standard N-9 exhibited spermicidal activity at MEC,
50.4 and 0.8 mM respectively. Twenty one compounds (9, 10,
12, 13, 15, 16 and 18–32) were more potent than reference
compound 33. Additionally two compounds (18 and 24)
demonstrated extremely potent sperm immobilizing potential
and showed spermicidal activity at (MEC, 0.07 and 0.06 mM),
which were more active than commercially available spermi-
cide N-9.
The study also demonstrated that two alkyl variants at R1
and R2 played a significant role in sperm immobilization in
1,4-(disubstituted)
piperazinedicarbodithioate
derivatives
(21–32). When R1 was benzyl and R2 was varied from benzyl
(21), allyl (22), butyl (23) to piperidinoethyl (24), the spermici-
dal activity increased 24 > 23 > 21 > 22 and compound 24 was
840 times more active than the reference compound, (33) and
also exhibited antitrichomonal activity (MIC 0.14 mM), but the
antifungal activity was lost. Whereas with R1 as piperidinoethyl
and R2 as different alkyl groups (25–29), spermicidal, antitri-
chomonal and antifungal activities became moderate. Further,
with R1 as butyl and R2 with pyrrolidinoethyl (30), hydroxyethyl
(31) or allyl (32), the spermicidal activity was enhanced signifi-
cantly (19 times) for 30 and marginally (1.7 fold) for 31 and 32
with respect to 33 while antifungal and antitrichomonal
activity increased considerably (30).
Antifungal activity
Seven compounds (9, 13, 15, 18, 20, 26 and 30) showed anti-
fungal activity (Table 1) against one or more fungal strains viz.,
Candida albicans. Cryptococcus neoformans, Sporothrix
schenckii, Trichophyton mentagrophytes, Aspergillus fumigates,
Candida parapsilosis with minimum inhibitory concentration
(MIC) 0.002–0.23 mM. Compounds 13, 15 and 18 inhibited all
fungal strains while compounds 26 and 30 inhibited five and
compounds 9 and 20 inhibited four strains. Compound 15 was
found to be the most potent antifungal compound, which
inhibits all fungal strains at MIC, 0.002–0.14 mM. The stan-
Fluorescence labeling of sperm thiols
dard Fluconazole showed antifungal activity at MIC, 0.003 to To study the mode of action of the most active compounds (18
>0.104 mM.
and 24), free –SH groups were localized by fluorescence detec-
3092 | Org. Biomol. Chem., 2014, 12, 3090–3099
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Table 1 Biological activity of compounds (9–33)
Antifungal activitya (MICd, mM)
Spermicidal Anti-Trichomonas
activity
activity
Compd
–R1
–R2
H–
H–
1
2
3
4
5
6
(MEC, mM) (MEC SE in mM)
9
PhCH2–
0.20
0.20
>0.11
>0.11
0.20
>0.11
>0.11
0.20
19.8
18.3
0.50 0.055
0.91 0.009
10
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
11
12
H–
H–
>0.11
>0.11
>0.11
>0.11
—
1.81 0.15
0.96 0.05
3.9
13
14
CH3CH2CH2CH2– H–
0.11
>0.11
0.11
>0.11
0.23
>0.11
0.11
>0.11
0.23
>0.11
0.23
>0.11
22.9
—
0.57 0.37
—
PhCH2–
PhCH2–
15
PhCH2–
0.009
0.002
0.14
0.14
0.14
0.018 14.3
0.71 0.006
16
17
PhCH2–
PhCH2–
CH2vCHCH2–
CH3CH2CH2CH2– >0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
3.4
—
1.71 0.07
—
18
19
PhCH2–
0.069
0.069
>0.11
0.14
0.069
>0.11
0.14
0.14
0.07
26.9
1.38 0.087
0.67 0.020
>0.11
0.039
>0.11
>0.11
>0.11
20
CH3CH2CH2CH2–
0.005 >0.11
>0.11
0.15
0.039 31.6
0.79 0.067
21
22
23
PhCH2–
PhCH2–
PhCH2–
PhCH2–
CH2vCHCH2–
CH3CH2CH2CH2– >0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
11.9
13.6
2.6
1.19 0.075
1.36 0.12
1.30 0.095
24
25
PhCH2–
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
0.06
0.14 0.03
>0.11
0.11
21.7
22.4
12.7
12.8
—
26
27
28
0.056
>0.11
>0.11
0.11
>0.11
>0.11
0.11
>0.11
0.11
>0.11
>0.11
0.56 0.068
0.64 0.038
1.28 0.10
OHCH2CH2–
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
CH2vCHCH2–
29
30
CH3CH2CH2CH2– >0.11
>0.11
0.032
>0.11
0.032
>0.11
0.032
12.3
2.6
1.23 0.061
0.08 0.006
CH3CH2CH2CH2–
0.032
0.016 >0.11
31
32
CH3CH2CH2CH2– OHCH2CH2–
CH3CH2CH2CH2– CH2vCHCH2–
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
>0.11
29.5
29.9
0.74 0.026
—
33b
50.4
—
N-9c
Metronidazole
Fluconazole
0.8
—
0.018 0.006
—
0.003
0.006
0.006 >0.104 >0.104
0.006
—
a 1. Candida albicans; 2. Cryptococcus neoformans; 3. Sporothrix schenckii; 4. Trichophyton mentagrophytes; 5. Aspergillus fumigates; 6. Candida
parapsilosis (ATCC-22019). b Prepared by known procedure.23 c Spectrum Chemical Manufacturing Corp. (New Brunswick, N. J.). d Mean of three
replicates, SE value ranged from 0.00 to 0.03 mM.
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Fig. 2 Fluorescence due to free thiols on human sperm treated with (A) control, (B) compound 18, (C) compound 24.
tion (after labeling with the thiols capturing dye mBBr) of
human sperm that were either motile (control) or immobilized
by compounds (18 and 24) treatment, and digitally imaged for
Experimental
Chemistry
qualitative assessment. It became clearly evident by visual In general, all reagents and solvents were commercial quality
assessment of fluorescence intensities that control sperm and were used without further purification. Melting points
(Fig. 2A) had remarkably higher number of free thiols as com- were determined in open capillary tubes on an electrically
pared with sperm immobilized by compounds 18 and 24 heated block and are uncorrected. IR spectra (νmax in cm−1) of
(Fig. 2B and 2C). Even though the difference was marked the compounds were recorded on Perkin Elmer’s FT-IR RX1 PC
throughout the structure of sperm, it was prominently notice- spectrophotometer. 1H NMR and 13C NMR spectra were
able in the tail region (principal piece). The diminished fluo- recorded on Bruker Supercon Magnet Avance/DPX-300 spec-
rescence of compounds (18 and 24) suggested the interaction trometers (300 MHz for 1H; 50, 75, 100 MHz 13C) in deuterated
with free thiol might be the mechanism of spermicidal action.
solvents with TMS as internal reference (chemical shifts δ in
ppm, J in Hz.). Electrospray Ionisation Mass spectra (ESI-MS)
were 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
Conclusions
It may be inferred that a benzyl dithiocarbamate at a nitrogen EA-1108 micro analyzer/Vario EL-III C H N S analyzer. All com-
of piperazine scaffold and a piperidinoethyl group (18) or an pounds were analyzed of C, H, N and the results obtained were
additional DTC group having piperidinoethyl group (24) at the within 0.4% of calculated values. The reaction progress was
other nitrogen were essential for sperm immobilization. The routinely monitored by thin layer chromatography (TLC) on
sodium dithiocarbamate group (15, 20) seems to be desirable precoated alumina/silica gel plates (Aldrich). Column chrom-
for high antifungal activity. The high activity of these com- atography was performed over Merck silica gel (60–120 Mesh).
pounds may be attributed to the interaction13 of a dithiocarba- All chemicals and solvents were procured from Sigma-Aldrich/
mate group with free sulfhydryl groups present over sperm Merck India Ltd.
membrane10 and Trichomonas.10 The remarkable antifungal
Synthesis of tert-butyl 4-(benzylthiocarbonothioyl)pipera-
activity might be due to the interaction of the DTC group with zine-1-carboxylate (4). To the mixture of sodium 4-(tert-butoxy-
Lanosterol 14α-demethylase (CYP-51), a prospective target in carbonyl)piperazine-1-carbodithioate (3, 10.0 g, 35.2 mmol),
Candida albicans.32–34 Compounds 18 and 24 of this series methanol (70.0 mL) and triethyl amine (7.33 mL, 52.1 mmol)
were found to be the most potent double-edged spermicides as was added benzyl chloride (4.0 g, 35.2 mmol) and stirred at
these were 11–13 times and 720–840 times more potent than room temperature for 3 h. The methanol from the reaction
N-9 and 33. These were also found to be much safer than N-9 mixture obtained was evaporated under reduced pressure.
in cytotoxicity assays. The mode of action of these compounds EtOAc (60.0 mL) was added to the reaction mixture, and the
was imaged (Fig. 2) by their interaction with free thiols on resulting solid salt was filtered off. The filtrate was washed
human sperm using a fluorescent thiol probe and a fluo- with water (2 × 20 mL). The organic layer was collected, dried
rescence microscope. A diminished fluorescence as compared over sodium sulfate and concentrated under reduced pressure
to control sperm suggested the sulfhydryl binding mechanism. to give the title compound (9.7 g, 78.5%) white solid; mp:
Thus, novel scaffolds, 1-substituted piperazinecarbodithioate 81–82 °C; IR (KBr) ν (cm−1): 2977, 2928, 1689, 1557, 1460,
1
(9–20) and 1,4-(disubstituted) piperazinedicarbodithioate 1223; H NMR (300 MHz, CDCl3): δ 7.40–7.24 (m, 5H), 4.57 (s,
(21–32) have evolved along with unique dithiocarbamate chem- 2H), 4.47–3.79 (m, 4H), 3.54 (t, J = 5.2 Hz, 4H), 1.47 (s, 9H);
istry. Further lead optimization is being carried out to arrive a 13C (50 MHz, CDCl3): δ 197.1 (CvS), 154.5 (CvO), 135.7,
better dually active spermicidal agents.
129.4, 128.7, 127.7, 80.6, 50.1, 42.9, 42.2, 28.4; ESI-MS: m/z 353
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(MH+); Anal. calcd for C17H24N2O2S2: C, 57.92; H, 6.86; N, 7.95; methane layer was separated and washed with water (2 ×
found, C, 58.14; H, 6.98; N, 8.17. 10 mL). The organic layer was collected, dried over sodium
The compounds (5–8) were prepared using a procedure sulfate, concentrated under reduced pressure and purified
similar to that described for compound 4. by column chromatography using silica (60–120 mesh) to give
tert-Butyl 4-((2-(piperidin-1-yl)ethylthio)carbonothioyl)- the title compound (9, 3.6 g, 54.5%) light yellow oil; IR (neat) ν
piperazine-1-carboxylate (5). The title compound was (cm−1): 3436, 2920, 2852, 1639, 1557, 1462, 1422, 1227; 1H
synthesized from sodium 4-(tert-butoxycarbonyl)piperazine- NMR (300 MHz, CDCl3): δ 7.39–7.23 (m, 5H), 4.57 (s, 2H),
1-carbodithioate (3) and 1-(2-chloroethyl)piperidine in 81.7% 4.33–3.91 (m, 4H), 2.92 (bs, 4H); 13C (75 MHz, CDCl3): δ 196.2
yield as off-white solid; mp: 92–94 °C; IR (KBr) ν (cm−1): 2974, (CvS), 135.8, 129.3, 128.5, 127.4, 52.3, 45.6, 41.9; ESI-MS: m/z
1
2859, 1690, 1222; H NMR (300 MHz, CDCl3): δ 4.10–4.05 (m, 253 (MH+); Anal. calcd for C12H16N2S2: C, 57.10; H, 6.39; N,
4H), 3.56–3.52 (m, 4H), 3.40–3.37 (m, 1H), 2.82–2.79 (m, 1H), 11.10; found, C, 57.26; H, 6.45; N, 10.98.
2.68–2.63 (m, 2H), 2.48 (bs, 4H), 1.59 (bs, 4H), 1.48 (s, 11H);
An unusual side product benzyl 4-benzylpiperazine-1-carbo-
13C (50 MHz, CDCl3): δ 197.9 (CvS), 154.6 (CvO), 80.7, 57.6, dithioate (14, 1.5 g, 23.1%) was also isolated as semisolid by
54.4, 42.8, 34.2, 28.5, 25.8, 24.3; ESI-MS: m/z 374 (MH+); Anal. using column chromatography.
calcd for C17H31N3O2S2: C, 54.66; H, 8.36; N, 11.25; found,
C, 54.58; H, 8.49; N, 11.36.
Benzyl 4-benzylpiperazine-1-carbodithioate (14). White
semisolid; IR (KBr) ν (cm−1): 2923, 2855, 1646, 1538, 1462,
1
tert-Butyl 4-((2-morpholinoethylthio)carbonothioyl)pipera- 1421, 1224; H NMR (300 MHz, CDCl3): δ 7.39–7.25 (m, 10H),
zine-1-carboxylate (6). The title compound was synthesized 4.56 (s, 2H), 4.32–3.92 (m, 4H), 3.53 (s, 2H), 2.94 (bs, 1H), 2.52
from sodium 4-(tert-butoxycarbonyl)piperazine-1-carbodithio- (bs, 3H); 13C (50 MHz, CDCl3): δ 196.4 (CvS), 137.3, 135.9,
ate (3) and 4-(2-chloroethyl)morpholine in 76.2% yield as 129.4, 129.2, 128.6, 128.4, 127.6, 127.5, 62.5, 52.4, 45.6, 42.2;
white solid; mp: 118–119 °C; IR (KBr) ν (cm−1): 2968, 2858, HRMS m/z calcd for C19H22N2S2 (MH+): 343.1224; found
1690, 1223; 1H NMR (300 MHz, CDCl3): δ 4.32–3.98 (m, 4H), 343.1258; Anal. calcd for C19H22N2S2: C, 66.62; H, 6.47; N,
3.73–3.70 (m, 4H), 3.61–3.47 (m, 6H), 2.74–2.66 (m, 2H), 8.18; found, C, 66.46; H, 6.52; N, 8.31.
2.55–2.50 (m, 4H), 1.48 (s, 9H); ESI-MS: m/z 376 (MH+); Anal.
calcd for C16H29N3O3S2: C, 51.17; H, 7.78; N, 11.19; found, C, similar to that described for compound 9.
51.35; H, 7.61; N, 11.07. 2-(Piperidin-1-yl)ethyl piperazine-1-carbodithioate (10). The
4-((2-(pyrrolidin-1-yl)ethylthio)carbonothioyl)- title compound was synthesized from tert-butyl 4-((2-(piperidin-
Compounds 10–13 were prepared using the procedure
tert-Butyl
piperazine-1-carboxylate (7). The title compound was syn- 1-yl)ethylthio)carbonothioyl)piperazine-1-carboxylate (5),
thesized from sodium 4-(tert-butoxycarbonyl)piperazine-1- TFA and sodium bicarbonate in 85.7% yield as light brown
carbodithioate (3) and 1-(2-chloroethyl)pyrrolidine in 82.5% solid; mp: 66–67 °C; IR (KBr) ν (cm−1): 3441, 2970, 2836, 1688,
1
yield as light yellow solid; mp: 75–76 °C; IR (KBr) ν (cm−1): 1220; H NMR (300 MHz, CDCl3): δ 4.49–3.94 (m, 4H), 3.50 (t,
2973, 2802, 1689, 1221; 1H NMR (300 MHz, CDCl3): δ 4.13 J = 7.5 Hz, 2H), 2.96–2.93 (m, 4H), 2.67 (t, J = 7.5 Hz, 2H), 2.51
(bs, 4H), 3.56–3.52 (m, 6H), 2.80 (t, J = 7.0 Hz, 2H), 2.60 (bs, (bs, 4H), 1.62–1.59 (m, 4H), 1.46–1.44 (m, 2H); 13C (100 MHz,
4H), 1.81–1.80 (m, 4H), 1.48 (s, 9H); 13C (100 MHz, CDCl3): CDCl3): δ 196.8 (CvS), 57.6, 54.3, 52.4, 51.6, 45.7, 33.8, 25.7,
δ 197.8 (CvS), 154.5 (CvO), 80.6, 54.8, 54.0, 50.1, 42.9, 36.0, 24.2; ESI-MS: m/z 274 (MH+); Anal. calcd for C12H23N3S2: C,
28.4, 23.5; ESI-MS: m/z 360 (MH+); Anal. calcd for 52.71; H, 8.48; N, 15.37; found, C, 52.59; H, 8.32; N, 15.19.
C16H29N3O2S2: C, 53.45; H, 8.13; N, 11.69; found, C, 53.61;
H, 8.27; N, 11.75.
2-Morpholinoethyl piperazine-1-carbodithioate (11). The
title compound was synthesized from tert-butyl 4-((2-morpho-
tert-Butyl 4-(butylthiocarbonothioyl)piperazine-1-carboxylate linoethylthio)carbonothioyl)piperazine-1-carboxylate (6), TFA
(8). The title compound was synthesized from sodium 4-(tert- and sodium bicarbonate in 84.2% yield as white solid; mp:
1
butoxycarbonyl)piperazine-1-carbodithioate (3) and 1-bromo- 54–55 °C; IR (KBr) ν (cm−1): 3402, 2963, 2846, 1638, 1224; H
butane in 79.5% yield as white solid; mp: 78–79 °C; IR (KBr) ν NMR (300 MHz, CDCl3): δ 4.31–3.87 (m, 4H), 3.74–3.71 (m,
1
(cm−1): 2937, 2855, 1643, 1219; H NMR (300 MHz, CDCl3): δ 4H), 3.52–3.43 (m, 3H), 3.03–3.00 (m, 3H), 2.71–2.66 (m, 2H),
4.13 (bs, 4H), 3.56–3.53 (m, 4H), 3.32 (t, J = 7.4 Hz, 2H), 2.54 (bs, 4H); ESI-MS: m/z 276 (MH+); Anal. calcd for
1.74–1.64 (m, 2H), 1.48 (s, 11H), 0.95 (t, J = 7.3 Hz, 3H); 13C C11H21N3OS2: C, 47.97; H, 7.68; N, 15.26; found, C, 47.76; H,
(100 MHz, CDCl3): δ 198.1 (CvS), 154.5 (CvO), 80.5, 49.9, 7.57; N, 15.19.
42.9, 37.0, 30.6, 28.4, 22.1, 13.7; ESI-MS: m/z 319 (MH+); Anal.
2-(Pyrrolidin-1-yl)ethyl piperazine-1-carbodithioate (12). The
calcd for C14H26N2O2S2: C, 52.79; H, 8.23; N, 8.80; found, C, title compound was synthesized from tert-butyl 4-((2-(pyrroli-
52.57; H, 8.14; N, 8.73. din-1-yl)ethylthio)carbonothioyl)piperazine-1-carboxylate (7),
Synthesis of benzyl piperazine-1-carbodithioate (9). To the TFA and sodium bicarbonate in 81.6% yield as light yellow oil;
mixture of tert-butyl 4-(benzylthiocarbonothioyl)piperazine-1- IR (neat) ν (cm−1): 3437, 2923, 2851, 1639, 1219; 1H NMR
carboxylate (4, 9.2 g, 26.2 mmol) and dichloromethane (300 MHz, CDCl3): δ 4.31–4.00 (m, 4H), 3.52 (t, J = 7.1 Hz, 2H),
(60.0 mL) was added 16% TFA in dichloromethane at (0–5 °C) 2.96–2.94 (m, 4H), 2.80 (t, J = 7.1 Hz, 2H), 2.60 (bs, 4H),
and stirred at room temperature for 6 h. A saturated solution 1.91–1.80 (m, 4H); ESI-MS: m/z 260 (MH+); Anal. calcd for
of sodium bicarbonate was added to the reaction mixture at C11H21N3S2: C, 50.93; H, 8.16; N, 16.20; found, C, 50.82; H,
(0–5 °C), and stirred at room temperature for 4 h. The dichloro- 8.05; N, 16.32.
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Butyl piperazine-1-carbodithioate (13). The title compound was filtered off. The filtrate was washed with water (2 × 5 mL).
was synthesized from tert-butyl 4-(butylthiocarbonothioyl) The organic layer was collected, dried over sodium sulfate,
piperazine-1-carboxylate (8), TFA and sodium bicarbonate in concentrated under reduced pressure and purified by column
87.0% yield as colourless oil; IR (neat) ν (cm−1): 3436, 2927, chromatography using silica (60–120 mesh) to give the usual
1
2863, 1641, 1227; H NMR (300 MHz, CDCl3): δ 4.28–3.98 (m, product 1-allyl 4-benzyl piperazine-1,4-bis(carbodithioate)
4H), 3.32 (t, J = 7.4 Hz, 2H), 2.96–2.93 (m, 4H), 1.74–1.64 (m, (22, 0.32 g, 52%) white solid (mp: 67–68 °C) with title compound
2H), 1.51–1.39 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H); 13C (100 MHz, (16, 0.14 g, 23.8%) an unusual side product as brown semi
CDCl3): δ 197.4 (CvS), 51.8, 45.7, 36.9, 30.7, 22.2, 13.7; solid; IR (KBr) ν (cm−1): 2922, 2846, 1641, 1564, 1462, 1422,
ESI-MS: m/z 219 (MH+); Anal. calcd for C9H18N2S2: C, 49.50; H, 1223; 1H NMR (300 MHz, CDCl3): δ 7.40–7.26 (m, 5H),
8.31; N, 12.83; found, C, 49.68; H, 8.45; N, 12.92.
5.91–5.78 (m, 1H), 5.24–5.18 (m, 2H), 4.57 (s, 2H), 4.41–3.94
Synthesis of sodium 4-(benzylthiocarbonothioyl)piperazine- (m, 4H), 3.03 (d, J = 6.6 Hz, 2H), 2.54 (bs, 4H); 13C (50 MHz,
1-carbodithioate (15). Benzyl piperazine-1-carbodithioate (9, CDCl3): δ 196.4 (CvS), 135.9, 134.0, 129.4, 128.6, 127.5, 118.9,
3.61 g, 14.3 mmol) was dissolved in ethyl acetate (75 mL) and 61.1, 52.3, 50.7, 42.2; HRMS m/z calcd for C15H20N2S2 (MH+):
to this, aqueous sodium hydroxide (0.86 g, 21.4 mmol, 30%) 293.1146; found 293.1141; Anal. calcd for C15H20N2S2: C,
was added whilst keeping the temperature below 5 °C. Carbon 61.60; H, 6.89; N, 9.58; found, C, 61.48; H, 6.74; N, 9.46.
disulfide (1.2 mL, 28.6 mmol) dissolved in ethyl acetate
The unusual side products (14, 17 and 18) were isolated
(20 mL) was added dropwise with stirring below 5 °C. The reac- using a procedure similar to that described for compound (16)
tion mixture was stirred at room temperature for a further 10 h with usual products (21, 23 and 24). Compounds (25–32) were
to furnish a white solid. The solvent was distilled off and the also synthesized by a similar method.
crude was recrystallised in methanolic ether to obtain sodium
4-(benzylthiocarbonothioyl)piperazine-1-carbodithioate
Benzyl 4-butylpiperazine-1-carbodithioate (17). The title
compound was synthesized from sodium 4-(benzylthiocarbo-
(83.7%) as a white powder; mp: >250 °C; IR (KBr) ν (cm−1): nothioyl)piperazine-1-carbodithioate (15) and 1-bromobutane
1
2918, 2850, 1643, 1509, 1468, 1417, 1221; H NMR (300 MHz, in 25.7% yield as yellow oil; IR (neat) ν (cm−1): 2953, 2814,
DMSO-d6): δ 7.13–7.04 (m, 5H), 4.29–4.28 (m, 2H), 4.15 (bs, 1632, 1553, 1465, 1425, 1220; 1H NMR (300 MHz, CDCl3):
2H), 3.92–3.87 (m, 2H), 3.63–3.57 (m, 4H); ESI-MS: m/z 351 δ 7.39–7.25 (m, 5H), 4.56 (s, 2H), 4.38–3.92 (m, 4H), 2.50 (bs,
(MH+); Anal. calcd for C13H15N2NaS4: C, 44.54; H, 4.31; N, 4H), 2.37–2.34 (m, 2H), 1.50–1.43 (m, 2H), 1.38–1.29 (m, 2H),
7.99; found, C, 44.36; H, 4.22; N, 7.87.
The following compounds (19–20) were prepared using a 136.0, 129.4, 128.6, 127.5, 57.9, 52.6, 42.1, 29.0, 20.6, 14.0;
procedure similar to that described for compound (15).
ESI-MS: m/z 309 (MH+); Anal. calcd for C16H24N2S2: C, 62.29;
Sodium 4-((2-(piperidin-1-yl)ethylthio)carbonothioyl)piper- H, 7.84; N, 9.08; found, C, 62.36; H, 7.92; N, 9.23.
azine-1-carbodithioate (19). The title compound was Benzyl 4-(2-(piperidin-1-yl)ethyl) piperazine-1-carbodithioate
0.92 (t, J = 5.5 Hz, 2H); 13C (50 MHz, CDCl3): δ 196.3 (CvS),
synthesized from 2-(piperidin-1-yl)ethyl piperazine-1-carbo- (18). The title compound was synthesized from sodium
dithioate (10), carbon disulfide and sodium hydroxide in 4-(benzylthiocarbonothioyl)piperazine-1-carbodithioate (15) and
85.4% yield as white solid; mp: 205–206 °C; IR (KBr) ν (cm−1): 2-chloroethylpiperidine in 28.5% yield as light yellow solid;
1
2932, 2867, 1641, 1215; H NMR (300 MHz, DMSO-d6): δ 4.15 mp: 78–79 °C; IR (KBr) ν (cm−1): 2927, 2853, 1639, 1526, 1452,
(bs, 4H), 3.92–3.67 (m, 4H), 3.14 (t, J = 7.2 Hz, 2H), 2.26 (bs, 1404, 1210; 1H NMR (300 MHz, CDCl3): δ 7.38–7.24 (m, 5H),
3H), 2.14 (bs, 3H), 1.23–1.11 (m, 6H); ESI-MS: m/z 372 (MH+); 4.56 (s, 2H), 4.33–3.93 (m, 4H), 2.56–2.45 (m, 12H), 1.61–1.58
Anal. calcd for C13H22N3NaS4: C, 42.02; H, 5.97; N, 11.31; (m, 4H), 1.45–1.44 (m, 2H); 13C (50 MHz, CDCl3): δ 196.4
found, C, 42.14; H, 6.07; N, 11.45.
(CvS), 135.9, 129.4, 128.6, 127.6, 56.4, 55.2, 55.0, 53.0, 50.8,
Sodium 4-(butylthiocarbonothioyl)piperazine-1-carbodithio- 50.0, 42.2, 25.6, 24.1; HRMS m/z calcd for C19H29N3S2 (MH+):
ate (20). The title compound was synthesized from butyl 364.1881; found 364.1875; Anal. calcd for C19H29N3S2: C,
piperazine-1-carbodithioate (13), carbondisulfide and sodium- 62.77; H, 8.04; N, 11.56; found, C, 62.56; H, 7.91; N, 11.42.
hydroxide in 89.2% yield as white solid; mp: >250 °C; IR (KBr)
Dibenzyl piperazine-1,4-bis(carbodithioate) (21). The title
ν (cm−1): 2962, 2931, 1638, 1216; 1H NMR (300 MHz, compound was synthesized from sodium 4-(benzylthiocarbo-
DMSO-d6): δ 4.39 (bs, 4H), 4.16–3.90 (m, 4H), 3.24 (t, J = 7.4 Hz, nothioyl)piperazine-1-carbodithioate (15) and benzyl chloride
2H), 1.63–1.56 (m, 2H), 1.41–1.33 (m, 2H), 0.88 (t, J = 7.3 Hz, in 51.2% yield as white solid; mp: 124–126 °C; IR (KBr) ν
3H); ESI-MS: m/z 317 (MH+); Anal. calcd for C10H17N2NaS4: (cm−1): 2923, 2851, 1640, 1562, 1455, 1401, 1215; 1H NMR
C, 37.95; H, 5.41; N, 8.85; found, C, 38.12; H, 5.55; N, 8.97.
(300 MHz, CDCl3): δ 7.39–7.24 (m, 10H), 4.57 (s, 4H), 4.32–4.20
Synthesis of benzyl 4-allylpiperazine-1-carbodithioate (m, 8H); 13C (75 MHz, CDCl3): δ 197.5 (CvS), 135.6, 129.4,
(16). To the mixture of sodium 4-(benzylthiocarbonothioyl)- 128.7, 127.8, 48.7, 42.2; HRMS m/z calcd for C20H22N2S4
piperazine-1-carbodithioate (15, 0.58 g, 1.64 mmol), methanol (MH+): 419.0744; found 419.0754; Anal. calcd for C20H22N2S4:
(20.0 mL) and triethyl amine (0.34 mL, 2.46 mmol) was added C, 57.38; H, 5.30; N, 6.69; found, C, 57.56; H, 5.45; N, 6.78.
3-bromoprop-1-ene (0.14 g, 1.64 mmol) and stirred at room
1-Allyl 4-benzyl piperazine-1,4-bis(carbodithioate) (22). The
temperature for 3 h. Methanol from the reaction mixture title compound was synthesized from sodium 4-(benzylthiocar-
obtained was evaporated under reduced pressure. EtOAc bonothioyl)piperazine-1-carbodithioate (15) and 3-bromoprop-
(15.0 mL) was added to the reaction mixture, and the solid salt 1-ene in 52.0% yield as off-white solid; mp: 67–68 °C; IR (KBr)
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ν (cm−1): 2979, 2857, 1638, 1539, 1458, 1406, 1213; 1H NMR
1-(2-Hydroxyethyl)4-(2-(piperidin-1-yl)ethyl)piperazine-1,4-
(300 MHz, CDCl3): δ 7.40–7.25 (m, 5H), 5.99–5.85 (m, 1H), bis(carbodithioate) (27). The title compound was synthesized
5.36–5.16 (m, 2H), 4.58 (s, 2H), 4.23–4.21 (m, 8H), 4.01 (d, J = from sodium 4-((2-(piperidin-1-yl)ethylthio)carbonothioyl)-
7.0 Hz, 2H); 13C (50 MHz, CDCl3): δ 197.4 (CvS), 197.2 (CvS), piperazine-1-carbodithioate (19) and 2-bromoethanol in 76.3%
135.6, 132.1, 129.4, 128.7, 127.7, 119.1, 48.6, 42.1, 40.3; yield as white solid; mp: 75–76 °C; IR (KBr) ν (cm−1): 3436,
ESI-MS: m/z 369 (MH+); Anal. calcd for C16H20N2S4: C, 52.13; 2930, 2851, 1640, 1217; 1H NMR (300 MHz, CDCl3): δ 4.51–4.20
H, 5.47; N, 7.60; found, C, 52.26; H, 5.61; N, 7.72.
(m, 8H), 3.90 (t, J = 5.9 Hz, 2H), 3.61 (t, J = 5.9 Hz, 2H), 3.52 (t,
1-Benzyl 4-butyl piperazine-1,4-bis(carbodithioate) (23). The J = 7.4 Hz, 2H), 3.05 (bs, 1H), 2.69 (t, J = 7.4 Hz, 2H), 2.52 (bs,
title compound was synthesized from sodium 4-(benzylthiocar- 4H), 1.62–1.60 (m, 4H), 1.47–1.41 (m, 2H); 13C (50 MHz,
bonothioyl)piperazine-1-carbodithioate (15) and 1-bromobu- CDCl3): δ 198.0 (CvS), 197.9 (CvS), 61.2, 57.4, 54.3, 48.8,
tane in 56.3% yield as off-white solid; mp: 63–64 °C; IR (KBr) ν 39.5, 34.0, 29.7, 25.6, 24.2; HRMS m/z calcd for C15H27N3OS4
(cm−1): 2965, 2812, 1639, 1543, 1456, 1401, 1213; 1H NMR (MH+): 394.1115; found 394.1120; Anal. calcd for C15H27N3OS4:
(300 MHz, CDCl3): δ 7.39–7.25 (m, 5H), 4.58 (s, 2H), 4.25 (bs, C, 45.77; H, 6.91; N, 10.67; found, C, 45.62; H, 6.85; N, 10.79.
8H), 3.32 (t, J = 7.4 Hz, 2H), 1.74–1.64 (m, 2H), 1.51–1.39 (m,
1-Allyl
4-(2-(piperidin-1-yl)ethyl)piperazine-1,4-bis(carbo-
2H), 0.94 (t, J = 7.3 Hz, 3H); 13C (50 MHz, CDCl3): δ 198.3 dithioate) (28). The title compound was synthesized from
(CvS), 197.3 (CvS), 135.6, 129.3, 128.6, 127.7, 48.5, 42.1, 37.0, sodium
4-((2-(piperidin-1-yl)ethylthio)carbonothioyl)pipera-
30.5, 22.1, 13.7; HRMS m/z calcd for C17H24N2S4 (MH+): zine-1-carbodithioate (19) and 3-bromoprop-1-ene in 79.8%
385.0901; found 385.0895; Anal. calcd for C17H24N2S4: C, yield as off-white solid; mp: 88–89 °C; IR (KBr) ν (cm−1): 2932,
1
53.08; H, 6.29; N, 7.28; found, C, 52.86; H, 6.11; N, 7.43.
2802, 1637, 1210; H NMR (300 MHz, CDCl3): δ 5.99–5.86 (m,
1-Benzyl 4-(2-(piperidin-1-yl)ethyl)piperazine-1,4-bis(carbo- 1H), 5.36–5.17 (m, 2H), 4.27 (bs, 8H), 4.02 (d, J = 6.9 Hz, 2H),
dithioate) (24). The title compound was synthesized from 3.50 (t, J = 7.3 Hz, 2H)), 2.65 (t, J = 7.3 Hz, 2H), 2.47 (bs, 4H),
sodium 4-(benzylthiocarbonothioyl)piperazine-1-carbodithio- 1.60–1.57 (m, 4H), 1.45–1.44 (m, 2H); 13C (75 MHz, CDCl3): δ
ate (15) and 2-chloroethylpiperidine in 49.7% yield as off-white 198.1 (CvS), 197.1 (CvS), 132.1, 119.0, 57.3, 54.3, 48.8, 40.2,
solid; mp: 97–98 °C; IR (KBr) ν (cm−1): 2933, 2851, 1640, 1552, 34.3, 25.8, 24.2; ESI-MS: m/z 390 (MH+); Anal. calcd for
1
1464, 1425, 1226; H NMR (300 MHz, CDCl3): δ 7.40–7.26 (m, C16H27N3S4: C, 49.32; H, 6.98; N, 10.78; found, C, 49.51; H,
5H), 4.58 (s, 2H), 4.39–4.27 (m, 8H), 3.51 (t, J = 7.3 Hz, 2H), 7.12; N, 10.85.
2.67 (t, J = 7.3 Hz, 2H), 2.50 (bs, 4H), 1.60–1.59 (m, 4H),
1-Butyl
4-(2-(piperidin-1-yl)ethyl)piperazine-1,4-bis(carbo-
1.46–1.44 (m, 2H); 13C (50 MHz, CDCl3): δ 198.2 (CvS), 197.5 dithioate) (29). The title compound was synthesized from
(CvS), 135.6, 129.4, 128.7, 127.8, 57.4, 54.4, 48.7, 42.2, 34.4, sodium
4-((2-(piperidin-1-yl)ethylthio)carbonothioyl)pipera-
25.9, 24.3; HRMS m/z calcd for C20H29N3S4 (MH+): 440.1323; zine-1-carbodithioate (19) and 1-bromobutane in 82.5% yield
found 440.1315; Anal. calcd for C20H29N3S4: C, 54.63; H, 6.65; as light yellow solid; mp: 91–92 °C; IR (KBr) ν (cm−1): 2935,
1
N, 9.56; found, C, 54.51; H, 6.42; N, 9.45.
2865, 1638, 1218; H NMR (300 MHz, CDCl3): δ 4.40–4.14 (m,
Bis(2-(piperidin-1-yl)ethyl) piperazine-1,4-bis(carbodithio- 8H), 3.53 (t, J = 7.3 Hz, 2H), 3.32 (t, J = 7.4 Hz, 2H), 2.69 (t, J =
ate) (25). The title compound was synthesized from sodium 4- 7.3 Hz, 2H), 2.52 (bs, 4H), 1.75–1.60 (m, 6H), 1.52–1.42 (m,
((2-(piperidin-1-yl)ethylthio)carbonothioyl)piperazine-1-carbo- 4H), 0.95 (t, J = 7.3 Hz, 3H); 13C (50 MHz, CDCl3): δ 198.4
dithioate (19) and 2-chloroethylpiperidine in 79.1% yield as (CvS), 198.2 (CvS), 57.4, 54.4, 48.6, 37.1, 34.3, 30.6, 25.8,
white solid; mp: 133–135 °C; IR (KBr) ν (cm−1): 2926, 2857, 24.3, 22.2, 13.8; HRMS m/z calcd for C17H31N3S4 (MH+):
1658, 1249; 1H NMR (300 MHz, CDCl3): δ 4.28 (bs, 8H), 3.50 (t, 406.1479; found 406.1473; Anal. calcd for C17H31N3S4: C,
J = 7.3 Hz, 4H), 2.65 (t, J = 7.3 Hz, 4H), 2.49–2.47 (m, 8H), 50.33; H, 7.70; N, 10.36; found, C, 50.10; H, 7.59; N, 10.21.
1.61–1.44 (m, 12H); 13C (50 MHz, CDCl3): δ 198.2 (CvS), 57.5,
1-Butyl 4-(2-(pyrrolidin-1-yl)ethyl)piperazine-1,4-bis(carbo-
54.4, 48.7, 34.5, 26.0, 24.3; HRMS m/z calcd for C20H36N4S4 dithioate) (30). The title compound was synthesized from
(MH+): 461.1901; found 461.1910; Anal. calcd for C20H36N4S4: sodium 4-(butylthiocarbonothioyl)piperazine-1-carbodithioate
C, 52.13; H, 7.87; N, 12.16; found, C, 52.36; H, 7.62; N, 12.05.
(20) and 2-chloroethylpyrrolidine in 85.2% yield as yellow
1-(2-(Piperidin-1-yl)ethyl)4-(2-(pyrrolidin-1-yl)ethyl)pipera- solid; mp: 75–76 °C; IR (KBr) ν (cm−1): 2922, 2865, 1642, 1211;
zine-1,4-bis(carbodithioate) (26). The title compound was 1H NMR (300 MHz, CDCl3): δ 4.26 (bs, 8H), 3.53 (t, J = 7.1 Hz,
synthesized from sodium 4-((2-(piperidin-1-yl)ethylthio)- 2H), 3.32 (t, J = 7.4 Hz, 2H), 2.82 (t, J = 7.1 Hz, 2H), 2.61 (bs,
carbonothioyl)piperazine-1-carbodithioate (19) and 2-chloro- 4H), 1.81 (4H, bs), 1.75–1.65 (m, 2H), 1.52–1.39 (m, 2H), 0.95
ethylpyrrolidine in 82.7% yield as yellow solid; mp: (t, J = 7.3 Hz, 3H); HRMS m/z calcd for C16H29N3S4 (MH+):
136–138 °C; IR (KBr) ν (cm−1): 2927, 2801, 1640, 1227; 1H NMR 392.1323; found 392.1315; Anal. calcd for C16H29N3S4: C,
(300 MHz, CDCl3): δ 4.28–4.18 (m, 8H), 3.54–3.47 (m, 4H), 2.80 49.06; H, 7.46; N, 10.73; found, C, 49.23; H, 7.62; N, 10.85.
(t, J = 7.0 Hz, 2H), 2.65 (t, J = 7.3 Hz, 2H), 2.59 (bs, 4H),
1-Butyl 4-(2-hydroxyethyl)piperazine-1,4-bis(carbodithioate)
2.49–2.47 (m, 4H), 1.85–1.80 (m, 4H), 1.61–1.43 (m, 6H); 13C (31). The title compound was synthesized from sodium 4-
(50 MHz, CDCl3): δ 198.2 (CvS), 57.4, 54.7, 54.4, 54.1, 48.8, (butylthiocarbonothioyl)piperazine-1-carbodithioate (20) and
36.3, 34.4, 25.9, 24.3, 23.5; ESI-MS: m/z 447 (MH+); Anal. calcd 2-bromoethanol in 84.1% yield as white solid; mp: 78–79 °C;
for C19H34N4S4: C, 51.08; H, 7.67; N, 12.54; found, C, 51.26; H, IR (KBr) ν (cm−1): 3437, 2922, 2851, 1634, 1219; 1H NMR
7.82; N, 12.65.
(300 MHz, CDCl3): δ 4.29 (bs, 8H), 3.90 (t, J = 5.9 Hz, 2H), 3.61
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(t, J = 5.9 Hz, 2H), 3.33 (t, J = 7.4 Hz, 2H), 2.33 (bs, 1H), were carried out using the standard procedure. Stock solutions
1.75–1.65 (m, 2H), 1.52–1.39 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H); (100 µg mL−1) of test compounds were prepared in DMSO.
13C (50 MHz, CDCl3): δ 198.4 (CvS), 197.7 (CvS), 61.3, 48.5, These stock solutions were serially diluted with TYM medium
39.3, 37.1, 30.5, 22.1, 13.7; ESI-MS: m/z 339 (MH+); Anal. calcd to obtain concentrations up to 0.1 µg mL−1 in 48-well plates.
for C12H22N2OS4: C, 42.57; H, 6.55; N, 8.27; found, C, 42.75; H, DMSO/TYM was used as vehicle in control wells. Parasites (5 ×
6.68; N, 8.39.
104 trophozoites L−1) were added to these wells and incubated
1-Allyl 4-butyl piperazine-1,4-bis(carbodithioate) (32). The anaerobically at 37 °C. Cells were checked for viability at
title compound was synthesized from sodium 4-(butylthiocar- different time intervals from 3 to 48 h under a microscope at
bonothioyl)piperazine-1-carbodithioate (20) and 3-chloroprop- 40× magnification. Viability of the cells was determined by
1-ene in 78.1% yield as off-white solid; mp: 59–60 °C; IR (KBr) trypan blue exclusion assay. Minimum conc. of the test agent
ν (cm−1): 2963, 2870, 1639, 1211; 1H NMR (300 MHz, CDCl3): δ at which all cells were found dead in 48 h was considered its
6.10–5.86 (m, 1H), 5.36–5.17 (m, 2H), 4.27 (bs, 8H), 4.03 (d, J = MIC. The experiment was repeated three times to confirm the
7.0 Hz, 2H), 3.33 (t, J = 7.4 Hz, 2H), 1.75–1.62 (m, 2H), MIC (Table 1).
1.52–1.42 (m, 2H), 0.95 (t, J = 7.3 Hz, 3H); 13C (75 MHz,
Cytotoxicity of compounds toward human cervical (HeLa)
CDCl3): δ 198.5 (CvS), 197.3 (CvS), 132.2, 119.1, 48.7, 40.3,
cells10
37.1, 30.6, 22.2, 13.7 ; HRMS m/z calcd for C13H22N2S4 (MH+):
335.0744; found 335.0741; Anal. calcd for C13H22N2S4: C, HeLa cells seeded at a density of 5 × 104 per well in 96-well
46.67; H, 6.63; N, 8.37; found, C, 46.85; H, 6.77; N, 8.49.
plates were incubated with either the culture medium contain-
ing dilutions of test compounds, or vehicle (control), for 24 h.
Thereafter 5 µL of MTT solution (5 mg mL−1 in buffer, pH 7.4)
was added to each well. The formazan crystals formed inside
the viable cells were solubilized in DMSO, and the optical
density at 540 nm (OD540) was recorded in a microplate reader
(Microquant; BioTek, USA).
Biology
Spermicidal activity11
The spermicidal assay was adapted from a 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 solu-
tion 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 60 seconds and
recorded (Table 1). The lowest concentration of compound
which immobilized 100% human sperm irreversibly in all the
three semen samples from different individuals is given in
Table 1.
Compatibility of compounds with Lactobacillus10
Spores of Lactobacillus jensenii (ATCC 25258, strain 62G) were
procured from ATCC and grown in 6% Rogosa SL broth
medium (Hi Media, India) containing 0.132% acetic acid at
37 °C in microwell plates. Serial dilutions of test compounds
were added to experimental wells, and vehicle was added to
control wells in triplicate. Approximately 1000 CFU of L. jensenii
were inoculated into each well. The plates were incubated at
37 °C in a humidified atmosphere containing 5% CO2 for
24 h. At the end of the experiment, the cultures were mixed
thoroughly, 100 µL from each well was transferred to the
corresponding well of a 96-well plate, and the numbers of lacto-
bacilli were estimated by measuring the turbidity (OD610) in a
microplate reader.
Antifungal activity13
The MICs of compounds were determined by broth micro-
dilution technique as per the guidelines of the National Com-
mittee for Clinical Laboratory Standards using RPMI
1640 media buffered with MOPS [3-(N-morpholino)propanesul-
fonic acid]. Starting inoculums of test culture was 1–5 × 103
CFU mL−1. Micro titer plates were incubated at 35 °C. MICs
were recorded after 48 h of incubation (Table 1).
Fluorescent labeling of sperm thiols
Free thiols on human sperm (after treatment with vehicle, and
two most promising compounds 18 and 24) were 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 18 and 24 at
MEC, as well as equal volume of saline (Control) in parallel
and incubated for 15 min at room temperature. After incu-
Antitrichomonal activity10
Trichomonas vaginalis parasites to be used in drug suscepti- bation, sperm were pelleted at 700g for 10 min and washed
bility assays were grown in TYM medium supplemented with 2–3 times with fresh PBS. To the pelleted sperm in 1 mL PBS,
10% FCS, vitamin mixture and 100 U mL−1 penicillin/strepto- 0.5 mM (final concentration) mBBr was added and incubated
mycin, at 37 °C in 15 mL tubes for one day, followed by regular for 15 min in the dark. After incubation the sperm was pel-
subculturing, and were in the log phase of growth. The cul- leted and washed with PBS, finally dissolved in 200 μL PBS. A
tures routinely attained a concentration of 2 × 107 cells mL−1 drop of this sample was then put on a microscope slide,
in 48 h. Inoculums of 1 × 104 cells per tube were used for covered with a cover glass and imaged using the UV1A filter on
maintenance of the culture. In vitro drug susceptibility assays a Nikon Eclipse 80i microscope equipped with epifluorescence
3098 | Org. Biomol. Chem., 2014, 12, 3090–3099
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Paper
illumination. Exposure times were the same for all samples. 15 A. K. Dwivedi, V. L. Sharma, N. Kumaria, K. Kumar,
The experimental results have been given in Fig. 2.
G. Gupta, J. P. Maikhuri, J. D. Dhar, P. Kumar, A. H. Ansari,
P. K. Shukla, M. Kumar, R. Roy, K. P. Madhusudanan,
R. C. Gupta, P. Srivastava, R. Pal and S. Singh, Indian Pat,
IN 245185, 2009.
Acknowledgements
16 R. D. Sousa, C. Thurier, C. Len, Y. Pouilloux, J. Barrault and
F. Jerome, Green Chem., 2011, 13, 1129–1132.
17 D. Chen and Q. P. Dou, Expert Opin. Ther. Targets, 2008, 12,
739–748.
18 A. Spallarossa, S. Cesarini, A. Ranise, O. Bruno, S. Schenone,
P. La Colla, G. Collu, G. Sanna, B. Secci and R. Loddo,
Eur. J. Med. Chem., 2009, 44, 1650–1663.
We acknowledge Ms Tara Rawat (S.T.O.) for technical assist-
ance and SAIF Division, for spectral data. We are grateful to
CSIR (S.J. and A.S.), UGC (L.K., N.L. and L.K.), and ICMR (V.B.)
for research fellowships. This study was partially supported
by a grant from the Ministry of Health and Family Welfare,
Government of India.
19 S. T. V. S. Kiran Kumar, V. L. Sharma and A. K. Dwivedi,
J. Heterocycl. Chem., 2006, 43, 1–5.
20 N. Lal, L. Kumar, A. Sarswat, S. Jangir and V. L. Sharma,
Org. Lett., 2011, 13, 2330–2333.
21 Y. Hagooly, J. Gatenyo, A. Hagooly and S. Rozen, J. Org.
Chem., 2009, 74, 8578–8582.
22 H. Dillen, S. Chambon, T. J. Cleij, L. Lutsen, D. Vanderzande
and P. J. Adriaensens, Macromolecules, 2011, 44, 4711–
4720.
23 R. P. Tripathi, A. R. Khan, B. S. Setty and A. P. Bhaduri, Acta
Pharm., 1996, 46, 169–176.
24 H. Takahashi, T. Ishioka, Y. Koiso, M. Sodeoka and
Y. Hashimoto, Biol. Pharm. Bull., 2000, 23, 1387–
1390.
25 V. V. Sureshbabu and N. Narendra, in Amino Acids, Peptides
and Proteins in Organic Chemistry, ed. A. B. Hughes,
Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, 2011, vol.
4, pp. 14–16.
Notes and references
1 http://www.un.org/News/Press/docs/2005/pop918.doc.htm
(accessed on 19/03/13), 2013.
2 http://www.cdc.gov/std/stats/STI-Estimates-Fact-Sheet-Feb2013.
pdf (accessed on 19/03/13), 2013.
3 http://www.unaids.org/en/media/unaids/contentassets/
documents/epidemiology/2012/gr2012/20121120_UNAIDS_
Global_Report_2012_en.pdf (accessed on 19/03/13), 2013.
4 J. Stephenson, J. Am. Med. Assoc., 2000, 284, 949.
5 R. E. Roddy, L. Zekeng, K. A. Ryan, U. Tamoufe and
K. G. Tweedy, J. Am. Med. Assoc., 2002, 287, 1117–1122.
6 L. Van Damme, G. Ramjee, M. Alary, B. Vuylsteke,
V. Chandeying, H. Rees, P. Sirivongrangson, L. Mukenge-
Tshibaka, V. Ettiegne-Traore, C. Uaheowitchai, S. S. Karim,
B. Masse, J. Perriens and M. Laga, Lancet, 2002, 360, 971–
977.
26 D. I. Eneanya, J. R. Bianchine, D. O. Duran and
B. D. Andresen, Annu. Rev. Pharmacol. Toxicol., 1981, 21,
575–596.
27 Y. Zhou, J. He, C. Li, L. Hong and Y. Yang, Macromolecules,
2011, 44, 8446–8457.
28 P. Manna, R. Singh(late), K. K. Narang and S. K. Manna,
Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem., 2005,
44, 1880–1886.
7 O. J. D’Cruz and F. M. Uckun, Contraception, 2001, 64, 113–123.
8 B. T. Storey, Mol. Hum. Reprod., 1997, 3, 203–213.
9 F. D. Gillin, D. S. Reiner, R. B. Levy and P. A. Henkart, Mol.
Biochem. Parasitol., 1984, 13, 1–12.
10 A. Jain, N. Lal, L. Kumar, V. Verma, R. Kumar, L. Kumar,
V. Singh, R. K. Mishra, A. Sarswat, S. K. Jain, J. P. Maikhuri,
V. L. Sharma and G. Gupta, Antimicrob. Agents Chemother.,
2011, 55, 4343–4351.
29 A. K. Pandey, R. Singh and V. K. Verma, Synthesis, 1982,
1068–1070.
30 J. E. Corrie, J. R. Hlubucek and G. Lowe, J. Chem. Soc.,
Perkin Trans. 1, 1977, 1421–1423.
31 L. E. Kaim, L. Grimaud and S. R. Purumandla, Synlett,
2011, 13, 1816–1820.
32 K. Chauhan, M. Sharma, P. Singh, V. Kumar, P. K. Shukla,
M. I. Siddiqi and P. M. S. Chauhan, MedChemComm, 2012,
3, 1104–1110.
33 A. C. Karaburun, Z. A. Kaplancikli, N. Gundogdu-
Karaburun and F. Demirci, Lett. Drug Des. Discovery, 2011,
8, 811–815.
34 S. M. Monti, A. Maresca, F. Viparelli, F. Carta, G. De
Simone, F. A. Muhlschlegel, A. Scozzafava and C. T. Supuran,
Bioorg. Med. Chem. Lett., 2012, 22, 859–862.
11 R. K. Jain, J. P. Maikhuri, S. T. Kiran Kumar, V. L. Sharma,
A. K. Dwivedi, K. Mitra, V. K. Bajpai and G. Gupta, Hum.
Reprod., 2007, 22, 708–716.
12 A. K. Dwivedi, V. L. Sharma, N. Kumaria, S. T. Kiran
Kumar, P. K. Srivastava, A. H. Ansari, J. P. Maikhuri,
G. Gupta, J. D. Dhar, R. Roy, B. S. Joshi, P. K. Shukla,
M. Kumar and S. Singh, Bioorg. Med. Chem., 2007, 15,
6642–6648.
13 S. T. Kiran Kumar, L. Kumar, V. L. Sharma, A. Jain,
R. K. Jain, J. P. Maikhuri, M. Kumar, P. K. Shukla and
G. Gupta, Eur. J. Med. Chem., 2008, 43, 2247–2256.
14 L. Kumar, A. Jain, N. Lal, A. Sarswat, S. Jangir, L. Kumar,
V. Singh, P. Shah, S. K. Jain, J. P. Maikhuri, M. I. Siddiqi,
G. Gupta and V. L. Sharma, ACS Med. Chem. Lett., 2012, 3,
83–87.
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