Azole-carbodithioate hybrids as vaginal anti-Candida contraceptive agents: Design, synthesis and docking studies

Article abstract of DOI:10.1016/j.ejmech.2013.09.007

Azole and carbodithioate hybrids were synthesized as alkyl 1H-azole-1-carbodithioates (7-27) and evaluated for spermicidal/microbicidal activities against human sperm, Trichomonas vaginalis and Candida species. Seventeen compounds (7-14, 16-18 and 20-25)

Full text of DOI:10.1016/j.ejmech.2013.09.007

Accepted Manuscript
Azole-carbodithioate hybrids as vaginal anti-Candida contraceptive agents: design,
synthesis and docking studies
Lalit Kumar, Nand Lal, Vikash Kumar, Amit Sarswat, Santosh Jangir, Veenu Bala,
Lokesh Kumar, Bhavana Kushwaha, Atindra K. Pandey, Mohammad I. Siddiqi,
Praveen K. Shukla, Jagdamba P. Maikhuri, Gopal Gupta, Vishnu L. Sharma
PII:
S0223-5234(13)00579-5
10.1016/j.ejmech.2013.09.007
EJMECH 6409
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 August 2013
Revised Date: 3 September 2013
Accepted Date: 5 September 2013
Please cite this article as: L. Kumar, N. Lal, V. Kumar, A. Sarswat, S. Jangir, V. Bala, L. Kumar, B.
Kushwaha, A.K. Pandey, M.I. Siddiqi, P.K. Shukla, J.P. Maikhuri, G. Gupta, V.L. Sharma, Azole-
carbodithioate hybrids as vaginal anti-Candida contraceptive agents: design, synthesis and docking
studies, European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.09.007.
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ACCEPTED MANUSCRIPT
Azole-carbodithioate hybrids as vaginal anti-Candida contraceptive agents: design,
synthesis and docking studies
Lalit Kumar, Nand Lal, Vikash Kumar, Amit Sarswat, Santosh Jangir, Veenu Bala, Lokesh Kumar,
Bhavana Kushwaha, Atindra K. Pandey, Mohammad I. Siddiqi, Praveen K. Shukla, Jagdamba P.
Maikhuri, Gopal Gupta, Vishnu L. Sharma
X
Y
R
N
R'
S
S
X, Y : N, CH
R : alkyl
R': alkyl, alkenyl

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Azole-carbodithioate hybrids as vaginal anti-Candida contraceptive agents:
design, synthesis and docking studies#
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Lalit Kumar,a Nand Lal,a Vikash Kumar,b Amit Sarswat,a Santosh Jangir,a Veenu Bala,a Lokesh
Kumar,c Bhavana Kushwaha,c Atindra K. Pandey,d Mohammad I. Siddiqi,b Praveen K. Shukla,d
Jagdamba P. Maikhuri,c Gopal Gupta,c Vishnu L. Sharmaa,*
aMedicinal & Process Chemistry Division, bMolecular & Structural Biology Division, cEndocrinology Division,
dFermentation & Technology Division, CSIR-Central Drug Research Institute, Lucknow-226031, India
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Abstract
Azole and carbodithioate hybrids were synthesized as alkyl 1H-azole-1-carbodithioates (7–
27) and evaluated for spermicidal/microbicidal activities against human sperm, Trichomonas
vaginalis and Candida species. Seventeen compounds (7-14, 16-18 and 20-25) showed
spermicidal activity at MEC 1.0% (w/v) and permanently immobilized 100% normal human
spermatozoa within ~30 second. Seventeen compounds (7–11, 13–18 and 20–25) exhibited
anti-Candida activity (IC50 1.26–47.69 µg/mL). All compounds were devoid of bactericidal
activity against four bacterial strains (50.00 µg/mL) and antiprotozoal activity against
Trichomonas vaginalis (200.00 µg/mL). Four promising compounds (10, 17, 20 and 22) have
better safety profile as compared to Nonoxynol-9 (N-9). Docking study was done to visualize
the possible interaction of designed scaffold with prospective receptor (Cyp51) of Candida
albicans.
Keywords
Anti-Candida, spermicides, imidazole, docking, carbon disulfide, dithiocarbamate
Corresponding author
*Tel: 91-522-2771940, Ext. 4671. Fax: 91-522-2771941.
E-mail: vlscdri1@rediffmail.com or vlscdri@gmail.com.
#CDRI communication no.: 208/2012/VLS
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1. Introduction
Approximately one-third of vulvovaginitis cases are attributable to Candida infection.
Candida species form a part of the lower genital tract flora in many healthy asymptomatic
women and reportedly ~50% female university students experience at least one physician-
diagnosed episode of vulvovaginal candidiasis (VVC) by age of 25. The estimated cost of
VVC is about 1 billion dollars in the US [1]. Fungal adhesion to the epithelium is a
prerequisite for colonization, and infections are characterized by the invasion of vaginal
epithelial cells. Additionally, hyphal formation damages epithelial tissue and contributes to
symptomatic vaginal infections [2], which may lead to subsequent attack by sexually
transmitted infections (STIs) and HIV.
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Please insert Figure 1.
Imidazoles, especially clotrimazole (1; Figure 1) and bifonazole (2; Figure 1) are highly
effective antifungal agents against mucosal infections caused by Candida albicans through
inhibiting ergosterol biosynthesis [3]. However, the emerging resistance against these drugs
[4, 5] has necessitated the search for new molecules.
In our enduring efforts to discover dually active vaginal microbicides [6-11], we have
reported that the hybridization of nitroimidazole structure with dithiocarbamate/phenylpropyl
groups leads to compounds with potent anti-Trichomonas, mild anti-Candida and spermicidal
activities [12, 13]. Moreover, metronidazole and other nitroimidazole antiprotozoals are
devoid of anti-Candida activity [14, 15] while similar compounds without a nitro group
possess antifungal efficacy [16-19]. A careful study of the antifungal structures (3-5; Figure
1) revealed that none of these had a nitro group. Hence, it was anticipated that hybridization
of various azoles lacking a nitro group with dithiocarbamate group (6; Figure 1) could lead to
the discovery of a chemical entity with potent anti-Candida, anti-Trichomonas and
spermicidal activities, since the dithiocarbamate group itself has been attributed with potent
activities against human sperm and Trichomonas [20]. The compounds thus synthesized were
evaluated in vitro against human sperm and various strains of Candida albicans and
Trichomonas vaginalis. The promising compounds were subsequently assessed for their
adverse effect on vaginal flora (Lactobacillus) and cervical epithelium (HeLa cells) for their
possible utility as vaginal microbicides. A docking study was also carried out to find a
suitable correlation between antifungal activity and inhibition of the prospective receptor.
The synthesis, structure activity relationship (SAR) and docking study are being reported in
this communication.
2. Results and discussion
2.1. Chemistry
The synthetic route for the synthesis of alkyl 1H-azole-1-carbodithioate (7–27) has been
shown in Scheme 1. These compounds were synthesized by the reaction of azoles, carbon
disulfide and alkyl or alkenyl halide in the presence of a strong base (sodium hydride) at 0–5
°C. Reaction with imidazole, 2-methylimidazole and benzimidazole was completed
smoothly. When benzotriazole was used as reactant, an N-methylated product (27, Scheme 1)
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was obtained, instead of methyl 1H-benzotriazole-1-carbodithioate, probably due to the
presence of an additional nitrogen atom in the ring which decreased the basic strength at
reactive site [21].
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Please insert Scheme 1.
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2.2. Evaluation of biological activities
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2.2.1. Antifungal activity
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Fifteen compounds (9, 11–22, 25 and 27) exhibited antifungal activity (Table 1) against one
or more strains of fungi viz., Sporothrix schenckii, Trichophyton mentagrophytes, Aspergillus
fumigatus, Cryptococcus neoformans with IC50 ranging from 0.69–24.93 µg/mL. Compounds
17 and 22 inhibited four strains while compounds 9, 13 and 14 inhibited three strains of
fungi. Additionally, the growth of Sporothrix schenckii was inhibited by three compounds
(16, 22 and 27) and that of Trichophyton mentagrophytes was inhibited by two compounds
(11 and 25) at IC50 < 2.0 µg/mL. Compound 22 inhibited Cryptococcus neoformans with IC50
of 0.69 µg/mL while compound 11 also inhibited the growth of Aspergillus fumigatus with
IC50 6.21 µg/mL.
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Please insert Table 1
2.2.2. Anti-Candida activity
Seventeen compounds (7–11, 13–18 and 20–25) showed anti-Candida activity (Table 2)
against one or more Candida strains. Three compounds (10, 16 and 22) inhibited growth of
all the six Candida strains. Furthermore, the growth inhibition was observed in five and four
Candida strains by compounds 9, 11, 13, 17 and compound 14, respectively. Thirteen
compounds (7, 10, 11, 13-18, 20-22, 25) inhibited Candida albicans at IC50 ranging 1.26–
36.03 µg/mL and four out of thirteen compounds (10, 17, 20 and 22) had remarkable IC50
ranging from 1.26 to 1.43 µg/mL. Five compounds (8-10, 16 and 22) exhibited anti-Candida
activity against Candida parapsilosis with IC50 ranging 1.46–10.15 µg/mL. Compound 22
was found to be very active against Candida parapsilosis with low IC50 of 1.46 µg/mL.
Furthermore, anti-Candida data showed that compound 22 was the most active against PK3,
PK9, PK13 and PK 30 (four additional strains of Candida) with IC50 of 1.79, 3.33, 1.79 and
1.75 µg/mL, respectively.
The structure activity relationship indicated that among azoles, decreasing the alkyl chain
length and by combining phenyl ring with imidazole (imidazole was replaced by
benzimidazole) gave rise to the most active compound (22). Furthermore, imidazol-1-yl and
2-methylimidazol-1-yl with allyl (9, 16) or butyl (10, 17) form a good combination for anti-
Candida activity as these compounds generally inhibited all Candida strains at low IC50. On
the other hand, it was observed that on increasing the alkyl chain length, the anti-Candida
activity reduced (12, 18 and 26) in case of imidazol-1-yl, 2-methylimidazol-1-yl and
benzimidazol-1-yl moieties. The introduction of additional nitrogen in the imidazole ring
with allyl group (21) decreased the activity.
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Please insert Table 2
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2.2.3. Spermicidal activity
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All the compounds (except 27) showed spermicidal activity (Table 3) at 1.0% (w/v)
concentration and irreversibly immobilized 95-100% normal human spermatozoa. Out of the
total twenty one compounds, seventeen compounds (7–14, 16–18, 20–25) caused 100%
immobilization at 1.0% while three compounds 15, 19 and 26 caused 95% immobilization.
Five compounds (8, 10, 11, 18 and 22) demonstrated extremely potent sperm immobilizing
potential and immobilized 80-100% human sperm at 0.1% (w/v) concentration. Compound
18 was found to be most potent spermicide in this series.
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Please insert Table 3
2.2.4. Anti-Trichomonas activity
All the compounds (7–27) could not display trichomonacidal effect at 200.00 µg/mL (Table
3), the absence of nitro group attached with in azole ring may be the probable reason.
2.2.5. Antibacterial activity
Compounds (7–27) were also found to be inactive towards four bacterial strains such as E.
coli (ATCC 9637), Pseudomonas aeruginosa (ATCC BAA-427), Staphylococcus aureus
(ATCC 25923), Klebsiella pneumoniae (ATCC 27736) at 50.00 µg/mL (Table 1), which
indicated high fungicidal selectivity.
2.2.6. Cytotoxic assay using human cervical (HeLa) cell line and compatibility with
Lactobacillus
The four most active compounds (10, 17, 20 and 22) of this series displayed better safety
towards HeLa cells (IC50 48.70-85.45 µg/mL) and much better compatibility with
Lactobacillus (IC50 39.54-220.00 µg/mL) than Nonoxynol-9 (N-9) which showed IC50 37.00
and 21.00 µg/mL respectively (Table 4), and therefore appeared apparently much safer for
vaginal use. These compounds did not affect the viability of HeLa cells or growth of
Lactobacilli during 24 hours of incubation.
Please insert Table 4
A closer look to SAR revealed that sixteen compounds were dually active and possessed
spermicidal activity as well as antifungal activity against all the ten fungal strains (including
six Candida strains). However, these compounds failed to possess anti-Trichomonas activity,
plausibly due to the absence of nitro group in azole ring. Further structural optimization may
yield active compounds possessing multiple activities i.e., spermicidal, antifungal and anti-
Trichomonas activities.
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2.3. Docking study
It is well documented that various azoles and non-azoles exhibit antifungal activity through
inhibition of the prospective receptor Cyp51, a member of the cytochrome P450 superfamily
[22-25]. In yeasts and fungi, P45014DM participates in ergosterol biosynthesis [25], which is
an essential requirement for fungal viability. Selective inhibition of Cyp51 causes depletion
of ergosterol and accumulation of lanosterol and some other 14-methyl sterols resulting in
growth inhibition of fungal cells. To visualize the interaction of designed scaffold with
Cyp51 of Candida albicans, docking studies were carried out. Docked conformations were
calculated using homology model of Candida albicans Cyp51, which were used in previous
studies as given in the supporting information [26, 27]. The four most active compounds (10,
17, 20 and 22) of this series were selected for the docking study and docked energy data is
provided in Table 5.
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Please insert Figure 2
Docking with compounds showed that there is common mode of interaction with Cyp51
through formation of co-ordinate bond between N atom of azole ring of inhibitors and Fe of
heme prosthetic group of receptor (Figure 2). Long alkyl chain of compounds 10 and 17 was
found to be close to hydrophobic residues Val509 and Val510. It has been reported that
Val509 and Val510 come under structurally selective residues of active site of Candida
albicans Cyp51 and interaction with these residues may have selectivity advantage to newly
designed antifungals [24]. Compound 20 showed lowest docking energy in agreement with in
vitro activity, which may be due to lack of alkyl chain. In case of compound 22, presence of
benzene ring stabilized the interaction by making hydrophobic contact with L376, which may
account for higher docked energy than compound 20. Other residues like Met306, Gly307,
and Thr311 and Pro375 were found to be in close contact with docked conformation of all
compounds.
Please insert Table 5
3. Conclusion
In this communication, hybrids of azole and carbodithioate scaffolds as alkyl 1H-azole-1-
carbodithioates (7–27) have been reported, which were evaluated for their spermicidal as well
as microbicidal activities against Trichomonas vaginalis and Candida species in vitro. Most
of the compounds exhibited significant anti-Candida activity and sixteen compounds (7-11,
13, 14, 16-18, 20-25) co-possessed spermicidal activity as well. While lack of nitro group in
azole scaffold imparted potent antifungal activity, carbodithioate group tendered appreciable
spermicidal effects though it failed to incorporate antiprotozoal (anti-Trichomonas) efficacy.
Our previous hybrid designs of nitro-azole and carbodithioate exhibited potent anti-
Trichomonas and spermicidal effects [12], lacking noticeable antifungal property. Hence, it is
apparent that in azole-carbodithioates hybrids, the presence of nitro group is the deciding
factor for presence of antifungal or antiprotozoal activities. The two activities fail to co-exist
due to the unique pharmacophoric properties of the nitro-group in azoles. Four promising
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compounds (10, 17, 20 and 22) of this series had a better safety profile as compared to N-9
and could be used as lead molecules for designing antimicrobial contraceptives. The
proposed docking analysis is noteworthy and can be utilized as a guideline for designing of
promising anti-Candida agents.
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4. Experimental section
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4.1. Chemistry
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In general, all reagents and solvents were of commercial quality and used without further
purification. Melting points were determined in open capillary tubes on an electrically heated
block and were uncorrected. IR spectra (νmax in cm-1) of the compounds were recorded on
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Perkin Elmer’s FT-IR RX1 PC spectrophotometer. H NMR & 13C NMR spectra were
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recorded on Bruker Supercon Magnet Avance/DRX-300 spectrometers (300 MHz for H;
75.4 MHz for 13C) in deuterated 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. 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 ± 0.4% 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 (60-120 Mesh). All
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compounds were characterized by TLC, H and 13C NMR and MS. Elemental analyses data
meet the criteria of ≥95% purity. All chemicals and solvents were procured from Sigma-
Aldrich/Merck India Ltd.
4.1.1. Synthesis of methyl 1H-imidazole-1-carbodithioate (7)
Into an ice-cooled suspension of sodium hydride (14.7 mmol, 60% in mineral oil) in THF
(5.0 mL) was added a solution of imidazole (0.5 gm) in THF (10.0 mL) dropwise in 5 min,
the mixture became dense by complete addition of imidazole. A solution of carbon disulfide
(11.0 mmol, 0.7 mL) in THF (5.0 mL) was added dropwise in 5 and then the reaction mixture
stirred at 0-5 °C for 15 min. The reaction mixture became clear and dark yellow into which a
solution of iodomethane (9.5 mmol, 0.6 mL) in THF (5.0 mL) was added dropwise in 2 min
then reaction mixture stirred for 10 min at 0-5 °C. Distilled water (2.0 mL) was added
carefully to quench excess of sodium hydride. Solvents were evaporated under reduced
pressure in rotavapor to provide dense reaction mixture. That was diluted with ethyl acetate
(10.0 mL), washed with distilled water (3 x 5.0 mL), and dried over anhydrous sodium
sulphate. Sodium sulphate was filtered off and washed with ethyl acetate (3 x 5.0 mL).
Combined filtrate was concentrated in rotavapor to provide crude oil that was purified by
column chromatography on silica-gel using chloroform as eluent to provide yellow oil.
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Yield 95%; Yellow oil; IR (neat): 2940, 1635, 1580 cm-1; ESI-MS (m/z): 159 (MH+); H
NMR (CDCl3, 300 MHz): δ 8.51 (s, 1H), 7.80 (s, 1H), 7.12 (s, 1H), 2.81 (s, 3H); Anal. Calcd.
for C5H6N2S2: C, 37.95; H, 3.82; N, 17.70; Found: C, 37.92; H, 3.80; N, 17.71.
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The following compounds 8–27 were prepared using a procedure described for compound 7
from the corresponding azoles, carbon disulfide and alkyl halide/alkenyl halide. All the
compounds were purified by column chromatography on silica-gel.
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4.1.2. 3-Cyanopropyl 1H-imidazole-1-carbodithioate (8)
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Eluent: 70% ethyl acetate-hexane; Yield 91%; Yellow oil; IR (neat): 2963, 2248, 1638, 1423
cm-1; ESI-MS (m/z): 212 (MH+); 1H NMR (CDCl3+CCl4, 300 MHz): δ 8.45 (s, 1H), 7.76 (s,
1H), 7.10 (s, 1H), 3.55 (t, 2H, J = 7.3 Hz), 2.53 (t, 2H, J = 6.9 Hz), 2.22–2.12 (m, 2H); 13C
NMR (CDCl3+CCl4, 75.5 MHz): δ 196.7 (C=S), 135.7, 132.0, 118.0, 117.8, 34.7, 24.0, 16.6;
Anal. Calcd. for C8H9N3S2: C, 45.47; H, 4.29; N, 19.89; Found: C, 45.42; H, 4.20; N, 19.88.
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4.1.3. Allyl 1H-imidazole-1-carbodithioate (9)
Eluent: 40% ethyl acetate-hexane; Yield 86%; Semisolid; IR (neat): 3113, 2960, 1637, 1531,
1468, 1368, 1275, 765 cm-1; ESI-MS (m/z): 185 (MH+); 1H NMR (CDCl3, 300 MHz): δ 8.47
(s, 1H), 7.78 (s, 1H), 7.10 (s, 1H), 5.99–5.85 (m, 1H), 5.42 (d, 1H, J = 16.9 Hz), 5.29 (d, 1H,
J = 10.0 Hz), 4.06 (d, 2H, J = 6.9 Hz); 13C NMR (CDCl3, 75.5 MHz): δ 197.2 (C=S), 135.7,
131.6, 129.7, 120.8, 117.8, 39.7; Anal. Calcd. for C7H8N2S2: C, 45.62; H, 4.38; N, 15.20;
Found: C, 45.52; H, 4.24; N, 15.11.
4.1.4. Butyl 1H-imidazole-1-carbodithioate (10)
Eluent: 5% ethyl acetate-hexane; Yield 71%; Yellow oil; IR (neat): 2963, 1634, 1446, 1369,
1218, 766 cm-1; ESI-MS (m/z): 201 (MH+); 1H NMR (CDCl3, 300 MHz): δ 8.49 (s, 1H), 7.80
(s, 1H), 7.10 (s, 1H), 3.40 (t, 2H, J = 7.4 Hz), 1.82–1.72 (m, 2H), 1.56–1.43 (m, 2H), 0.98 (t,
2H, J = 7.3 Hz); 13C NMR (CDCl3, 75.5 MHz): δ 198.5 (C=S), 135.7, 131.4, 117.8, 36.8,
29.5, 22.2, 13.7; Anal. Calcd. for C8H12N2S2: C, 47.97; H, 6.04; N, 13.98; Found: C, 47.99;
H, 6.14; N, 14.01.
4.1.5. Octyl 1H-imidazole-1-carbodithioate (11)
Eluent: 20% ethyl acetate-hexane; Yield 74%; Yellow oil; IR (neat): 2927, 2855, 1653, 1465,
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1368, 754 cm-1; ESI-MS (m/z): 257 (MH+); H NMR (CDCl3+CCl4, 300 MHz): δ 8.45 (s,
1H), 7.76 (s, 1H), 7.08 (s, 1H), 3.37 (t, 2H, J = 7.4 Hz), 1.83–1.73 (m, 2H), 1.46–1.44 (m,
2H), 1.29 (bs, 8H), 0.89–0.87 (m, 3H); 13C NMR (CDCl3+CCl4, 75.5 MHz): δ 197.1 (C=S),
134.7, 130.6, 116.9, 36.1, 31.0, 28.3, 28.2, 26.7, 21.8, 13.3; Anal. Calcd. for C12H20N2S2: C,
56.21; H, 7.86; N, 10.92; Found: C, 56.12; H, 7.84; N, 10.81.
4.1.6. Hexadecyl 1H-imidazole-1-carbodithioate (12)
Eluent: 70% ethyl acetate-hexane; Yield 55%; Yellow solid; mp 40-41 °C; IR (KBr): 2926,
2854, 1628, 1465, 1368, 761 cm-1; ESI-MS (m/z): 369 (MH+); 1H NMR (CDCl3, 300 MHz): δ
8.48 (s, 1H), 7.79 (s, 1H), 7.10 (s, 1H), 3.38 (t, 2H, J = 7.4 Hz), 1.83–1.73 (m, 2H), 1.56–1.41
(m, 2H), 1.26 (bs, 24H), 0.88 (t, 3H, J = 6.9 Hz); 13C NMR (CDCl3, 75.5 MHz): δ 198.5
(C=S), 135.7, 131.5, 117.8, 37.1, 32.0, 29.8, 29.7, 29.6, 29.5, 29.2, 29.0, 27.5, 22.8, 14.2;
Anal. Calcd. for C20H36N2S2: C, 65.16; H, 9.84; N, 7.60; Found: C, 65.24; H, 9.73; N, 7.61.
4.1.7. 2-(1,3-Dioxoisoindolin-2-yl)ethyl 1H-imidazole-1-carbodithioate (13)
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Eluent: 80% ethyl acetate-hexane; Yield 57%; Yellow solid; mp 152-153 °C; IR (KBr): 2946,
2850, 1715, 1613, 1467, 714 cm-1; ESI-MS (m/z): 318 (MH+); 1H NMR (CDCl3, 300 MHz): δ
8.44 (s, 1H), 7.88–7.86 (m, 2H), 7.76–7.74 (m, 3H), 7.09 (s, 1H), 4.13 (t, 2H, J = 6.4 Hz),
3.77 (t, 2H, J = 6.4 Hz); 13C NMR (CDCl3, 75.5 MHz): δ 196.8 (C=S), 168.0, 135.9, 134.4,
131.9, 131.7, 123.6, 118.0, 35.5, 34.9; Anal. Calcd. for C14H11N3O2S2: C, 52.98; H, 3.49; N,
13.24; Found: C, 52.92; H, 3.47; N, 13.21.
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4.1.8. Methyl-2-methyl 1H-imidazole-1-carbodithioate (14)
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Eluent: chloroform; Yield 88%; Semisolid; IR (neat): 2926, 2853, 1636, 1420, 761 cm-1; ESI-
MS (m/z): 173 (MH+); 1H NMR (CDCl3, 300 MHz): δ 7.57 (d, 1H, J = 1.5 Hz), 6.92 (d, 1H, J
= 1.4 Hz), 2.75 (s, 3H), 2.71 (s, 3H); 13C NMR (CDCl3, 75.5 MHz): δ 201.7 (C=S), 146.7,
127.5, 119.4, 21.1, 17.8; Anal. Calcd. for C6H8N2S2: C, 41.83; H, 4.68; N, 16.26; Found: C,
41.78; H, 4.84; N, 16.22.
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4.1.9. 3-Cyanopropyl-2-methyl 1H-imidazole-1-carbodithioate (15)
Eluent: 80% ethyl acetae-hexane; Yield 71%; Yellow oil; IR (neat): 2926, 2853, 2248, 1635,
1549, 763 cm-1; ESI-MS (m/z): 226 (MH+); 1H NMR (CDCl3+CCl4, 300 MHz). δ 7.57 (d, 1H,
J = 1.6 Hz), 6.90 (d, 1H, J = 1.6 Hz), 3.47 (t, 2H, J = 7.3 Hz), 2.72 (s, 3H), 2.53 (t, 2H, J =
6.9 Hz) 2.21–2.11 (m, 2H); 13C NMR (CDCl3+CCl4, 75.5 MHz): δ 199.2 (C=S), 147.3,
128.3, 119.1, 118.0, 35.8, 23.9, 18.6, 16.8; Anal. Calcd. for C9H11N3S2: C, 47.97; H, 4.92; N,
18.65; Found: C, 47.92; H, 4.91; N, 18.54.
4.1.10. Allyl 2-methyl 1H-imidazole-1-carbodithioate (16)
Eluent: chloroform; Yield 90%, Semisolid; IR (neat): 2928, 2856, 1637, 1437, 760 cm-1. ESI-
MS (m/z): 199 (MH+); 1H NMR (CDCl3+CCl4, 300 MHz): δ 7.56 (d, 1H, J = 1.6 Hz), 6.88 (d,
1H, J = 1.6 Hz), 5.97–5.84 (m, 1H), 5.41 (dd, 1H, J = 1.2, 16.9 Hz), 5.28 (dd, 1H, J = 10.0,
1.2 Hz), 3.97 (d, 2H, J = 7.0 Hz), 2.71 (s, 3H); 13C NMR (CDCl3+CCl4, 75.5 MHz): δ 199.7
(C=S), 146.9, 129.9, 127.9, 120.8, 119.2, 41.0, 18.3; Anal. Calcd. for C8H10N2S2: C, 48.45;
H, 5.08; N, 14.13; Found: C, 48.12; H, 5.24; N, 14.34.
4.1.11. Butyl 2-methyl 1H-imidazole-1-carbodithioate (17)
Eluent: 2% methanol-chloroform; Yield 64%; Semisolid; IR (neat): 2963, 1634, 1446, 1369,
1
1218, 766 cm-1; ESI-MS (m/z): 215 (MH+); H NMR (CDCl3+CCl4, 300 MHz): δ 7.55 (d,
1H, J = 1.6 Hz), 6.87 (d, 1H, J = 1.6 Hz), 3.30 (t, 2H, J = 7.4 Hz), 2.70 (s, 3H), 1.81–1.71 (m,
2H), 1.56–1.43 (m, 2H), 0.99 (t, 3H, J = 7.3 Hz); 13C NMR (CDCl3+CCl4, 75.5 MHz): δ
199.8 (C=S), 145.7, 126.7, 118.3, 36.9, 28.4, 21.4, 17.2, 12.8; Anal. Calcd. for C9H14N2S2: C,
50.43; H, 6.58; N, 13.07; Found: C, 50.42; H, 6.44; N, 13.10.
4.1.12. Octyl 2-methyl 1H-imidazole-1-carbodithioate (18)
Eluent: chloroform; Yield 82%; Yellow oil; IR (neat): 2927, 2856, 1637, 1460, 763 cm-1;
ESI-MS (m/z): 271 (MH+); 1H NMR (CDCl3, 300 MHz): δ 7.57 (d, 1H, J = 1.7 Hz), 6.90 (d,
1H, J = 1.7 Hz), 3.30 (t, 2H, J = 7.4 Hz), 2.70 (s, 3H), 1.82–1.72 (m, 2H), 1.50–1.41 (m, 2H),
1.29 (bs, 8H), 0.89 (t, 3H, J = 6.8 Hz); 13C NMR (CDCl3, 75.5 MHz): δ 201.3 (C=S), 146.7,
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127.6, 119.5, 38.3, 31.9, 29.8, 29.2, 27.3, 22.7, 18.0, 14.2; Anal. Calcd. for C13H22N2S2: C,
57.73; H, 8.20; N, 10.36; Found: C, 57.70; H, 8.21; N, 10.25.
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4
4.1.13. Hexadecyl 2-methyl 1H-imidazole-1-carbodithioate (19)
5
Eluent: chloroform; Yield 51%; Yellow oil; IR (neat): 2926, 2854, 1635, 1460, 765 cm-1;
ESI-MS (m/z): 383 (MH+); 1H NMR (CDCl3, 300 MHz): δ 7.57 (d, 1H, J = 1.6 Hz), 6.90 (d,
1H, J = 1.6 Hz), 3.30 (t, 2H, J = 7.4 Hz), 2.71 (s, 3H), 1.82–1.72 (m, 2H), 1.46–1.41 (m, 2H),
1.26 (m, 24H), 0.87 (t, 3H, J = 6.8 Hz); 13C NMR (CDCl3, 75.5 MHz): δ 201.3 (C=S), 146.7,
127.6, 119.5, 38.3, 32.0, 29.8, 29.8, 29.8, 29.7, 9.5, 29.5, 29.2, 29.2, 27.3, 22.8, 18.1, 14.2;
Anal. Calcd. for C21H38N2S2: C, 65.91; H, 10.01; N, 7.32; Found: C, 66.12; H, 10.12; N, 7.40.
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4.1.14. Methyl 1H-1,2,4-triazole-1-carbodithioate (20)
Eluent: 80% chloroform-hexane; Yield 58%; Yellow crystalline solid; mp 45-46 °C; IR
(KBr): 2923, 2853, 1619, 1369, 766 cm-1; ESI-MS (m/z): 160 (MH+); 1H NMR (CDCl3+CCl4,
300 MHz): δ 9.14 (s, 1H), 8.04 (s, 1H), 2.75 (s, 3H); 13C NMR (CDCl3+CCl4, 75.5 MHz): δ
199.5 (C=S), 153.5, 143.1, 19.7; Anal. Calcd. for C4H5N3S2: C, 30.17; H, 3.16; N, 26.39;
Found: C, 30.32; H, 3.34; N, 26.28.
4.1.15. Allyl 1H-1,2,4-triazole-1-carbodithioate (21)
Eluent: 70% chloroform-hexane; Yield 35%; Oil; IR (neat) 2925, 2854, 1637, 1437, 760 cm-
1; ESI-MS (m/z): 186 (MH+); 1H NMR (CDCl3, 300 MHz): δ 9.13 (s, 1H), 8.04 (s, 1H), 5.99–
5.86 (m, 1H), 5.43 (dd, 1H, J = 1.0, 16.9 Hz), 5.29 (dd, 1H, J = 10.0, 0.7 Hz), 3.98 (d, 2H, J =
6.9 Hz); 13C NMR (CDCl3, 75.5 MHz): δ 197.8 (C=S), 153.4, 143.2, 129.9, 120.9, 39.3;
Anal. Calcd. for C6H7N3S2: C, 38.90; H, 3.81; N, 22.68; Found: C, 38.95; H, 3.82; N, 22.97.
4.1.16. Methyl 1H-benzo[d]imidazole-1-carbodithioate (22)
Eluent: 50% chloroform-hexane; Yield 84%; Yellow solid; mp 62-64 °C; IR (KBr): 2925,
1
2856, 1675, 1599 cm-1; ESI-MS (m/z) 209 (MH+); H NMR (CDCl3, 300 MHz): δ 8.86 (s,
1H), 8.53 (dd, 1H, J = 2.3, 5.6 Hz), 7.81–7.77 (m, 1H), 7.43–7.26 (m, 2H), 2.83 (s, 3H); 13C
NMR (CDCl3, 75.5 MHz): δ 199.0 (C=S), 145.4, 141.7, 132.9, 125.8, 125.2, 121.1, 115.7,
20.0; Anal. Calcd. for C9H8N2S2: C, 51.89; H, 3.87; N, 13.45; Found: C, 51.62; H, 3.64; N,
13.34.
4.1.17. 3-Cyanopropyl 1H-benzo[d]imidazole-1-carbodithioate (23)
Eluent: 90% chloroform-hexane. Yield 62%; Yellow oil; IR (neat): 2963, 2364, 1626, 1499,
1
768 cm-1; ESI-MS (m/z): 262 (MH+); H NMR (CDCl3, 300 MHz): δ 8.85 (s, 1H), 8.53 (dd,
1H, J = 2.3, 5.6 Hz), 7.82–7.76 (m, 1H), 7.43–7.39 (m, 2H), 3.57 (t, 2H, J=7.2 Hz), 2.55 (t,
2H, J = 6.9 Hz), 2.23–2.14 (m, 2H); 13C NMR (CDCl3, 75.5 MHz): δ 196.6 (C=S), 145.2,
141.7, 132.9, 126.1, 125.6, 121.1, 118.5, 115.8, 34.8, 23.9, 16.6; Anal. Calcd. for
C12H11N3S2: C, 55.14; H, 4.24; N, 16.08; Found: C, 55.35; H, 4.47; N, 16.24.
4.1.18. Allyl 1H-benzo[d]imidazole-1-carbodithioate (24)
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Eluent: 40% chloroform-hexane. Yield 87%; Yellow oil; IR (neat): 2924, 2852, 1635, 1458,
1
765 cm-1; ESI-MS (m/z): 235 (M+); H NMR (CDCl3, 300 MHz): δ 8.85 (s, 1H), 8.55–5.52
3
(m, 1H), 7.80–7.77 (m, 1H), 7.43–7.35 (m, 2H), 6.03–5.89 (m, 1H), 5.45 (d, 1H, J = 17.0
Hz), 5.31 (d, 1H, J = 10.0 Hz), 4.10 (d, 2H, J = 6.9 Hz). 13C NMR (CDCl3, 75.5 MHz): δ
197.2 (C=S), 145.4, 141.7, 132.9, 130.0, 125.8, 125.3, 121.1, 120.9, 115.8, 39.9; Anal. Calcd.
for C11H10N2S2: C, 56.38; H, 4.30; N, 11.95; Found: C, 56.61; H, 4.54; N, 11.84.
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4.1.19. Butyl 1H-benzo[d]imidazole-1-carbodithioate (25)
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Eluent: 60% chloroform-hexane. Yield 75%; Yellow semisolid; IR (neat): 2960, 2928, 2858,
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1653, 1447, 763 cm-1; ESI-MS (m/z): 251 (MH+); H NMR (CDCl3, 300 MHz): δ 8.87 (s,
1H), 8.56–8.53 (m, 1H), 7.81–7.77 (m, 1H), 7.43–7.35 (m, 2H), 3.45 (t, 2H, J = 7.5 Hz),
1.84–1.76 (m, 2H), 1.59–1.49 (m, 2H), 1.0 (t, 3H, J = 7.3 Hz); 13C NMR (CDCl3, 75.5 MHz):
δ 198.5 (C=S), 145.4, 141.7, 133.0, 125.7, 125.2, 121.1, 115.9, 37.0, 29.5, 22.3, 13.7; Anal.
Calcd. for C12H14N2S2: C, 57.56; H, 5.64; N, 11.19; Found: C, 57.38; H, 5.43; N, 11.26.
4.1.20. Octyl 1H-benzo[d]imidazole-1-carbodithioate (26)
Eluent: 40% chloroform-hexane. Yield 55%, Yellow semisolid. IR (KBr): 2926, 2856, 1656,
1447, 763 cm-1. ESI-MS (m/z): 307 (MH+). 1H NMR (CDCl3, 300 MHz): δ 8.87 (s, 1H), 8.55
(dd, 1H, J = 2.2, 6.0 Hz), 7.80 (dd, 1H, J=3.4, 6.6 Hz), 7.43–7.35 (m, 2H), 3.44 (t, 2H, J=7.4
13
Hz), 1.88–1.85 (m, 2H), 1.55–1.49 (m, 2H), 1.47 (bs, 8H), 0.89 (t, 3H, J = 4.3, 2.3 Hz). C
NMR (CDCl3, 75.5 MHz): δ 198.6 (C=S), 145.4, 141.7, 133.0, 125.7, 125.2, 121.1, 115.9,
37.3, 31.9, 29.8, 29.2, 27.5, 22.8, 14.2. Anal. Calcd. for C16H22N2S2: C, 62.70; H, 7.24; N,
9.14; Found: C, 62.56; H, 7.34; N, 9.27.
4.1.21. 1-Methyl-1H-benzo[d][1,2,3]triazole (27)
Eluent: 50% chloroform-hexane. Yield 67%; Light green solid; mp 70-71 °C. IR (KBr):
2962, 2856, 762 cm-1; ESI-MS (m/z): 134 (MH+); 1H NMR (CDCl3+CCl4, 300 MHz): δ 8.02
(d, 1H, J = 8.3 Hz), 7.46–7.42 (m, 2H), 7.38–7.30 (m, 1H), 4.28 (s, 3H); 13C NMR
(CDCl3+CCl4, 75.5 MHz,): δ 146.1, 133.6, 127.3, 109.1, 123.8, 120.2, 34.1; Anal. Calcd. for
C7H7N3 C, 63.14; H, 5.30; N, 31.56; Found: C, 63.21; H, 5.25; N, 31.61.
4.2. Biology
4.2.1. Antifungal/ Anti-Candida activity [11]
The MIC of compounds were determined by broth micro-dilution technique as per the
guidelines of National Committee for Clinical Laboratory Standards using RPMI 1640 media
buffered with MOPS [3-(N-Morpholino)propanesulfonic acid]. Starting inoculum of test
culture was 1–5 x 103 CFU/mL. Micro titre plates were incubated at 35 °C. MICs, IC50 were
recorded spectrophotometrically at 492 nm after 48 h of incubation (Table 1 and 2).
4.2.2. Spermicidal activity [7]
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
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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 60 seconds and recorded (Table 3).
4.2.3. Anti-Trichomonas activity [11]
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Trichomonas vaginalis parasites to be used in drug susceptibility assays were grown in TYM
(Trypticase Yeast-Extract Maltose) medium supplemented with 10% FCS (fetal calf serum),
vitamin mixture and 100.0 U/mL penicillin/streptomycin, at 37 °C in 15.0 mL tubes for one
day, followed by regular subculturing, and were in the log phase of growth. The cultures
routinely attained a concentration 2 x 107 cells/mL in 48 h. Inoculum of 1 x 104 cells per tube
was used for maintenance of the culture. In vitro drug susceptibility assays were carried out
using the standard procedure. Stock solutions (100.0 µg/mL) of test compounds were
prepared in DMSO. These stock solutions were serially diluted with TYM medium to obtain
concentration upto 0.1 µg/mL in 48-well plates. DMSO/TYM was used as vehicle in control
wells. Parasites (5 x 104 trophozoites/L) were added to these wells and incubated
anaerobically at 37 °C. Cells were checked for viability at different time intervals from 3 to
48 h under the microscope at 200X 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 3)
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4.2.4. Antibacterial activity [11]
The MIC of each test compound was determined against five bacterial isolates by broth
microdilution technique as per guidelines of National Committee for Clinical Laboratory
Standards. MIC was measured in 96 well tissue culture plate (Cellstar Greiner Bio One,
Germany) using Mueller Hinton broth media (Sigma Chemical Co.). The inoculum of the test
cultures was maintained at 1-5 ×103 cfu/mL. Micro-titer plates were incubated at 35 °C in a
moist, dark chamber, and MIC and IC50 were recorded spectrophotometrically (Softmax pro®
4.3, Versamax microplate reader at 492 nm, Molecular Devices) after 48 h (Table 1).
4.2.5. Cytotoxicity towards human cervical (HeLa) cell line by lactate dehydrogenase –
release assay [11]
A colorimetric assay for lactate dehydrogenase (LDH) release was used for the evaluation of
the cytotoxicity of spermicidal compounds against the HeLa cell line (Table 4).
Exponentially growing HeLa cells were seeded into 96-well tissue culture plates at a density
of 2 ×104 cells per well (in triplicate). After 24 h incubation in a CO2 incubator at 37 °C in
5% CO2 , 95% air atmosphere, the culture medium [Dulbecco’s modified Eagle’s medium
(DMEM)] was replaced with 100.0 ꢀL 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 ꢀL 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
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(Promega, Madison, WI, USA) by following the instructions of the manufacturer. Optical
densities at 490 nm were measured in a micro-plate reader (ꢀQuant, Bio-Tek, USA).
4.2.6. Effect on Lactobacillus acidophillus in vitro [11]
The effect of compounds exhibiting potent spermicidal activity on Lactobacillus acidophillus
was determined by following the method published earlier [11]. Briefly, Rogosa SL agar
plates (7.5%; containing 0.132% acetic acid), prepared with (experimental) or without
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(control) the addition of spermicidal compounds, were inoculated with L. acidophillus (∼70
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spores/10 cm2) and incubated at 37 °C in 5% CO2 and 95% air for 72 h. The number and size
of colonies were 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 4).
4.3. Molecular modelling and docking studies
Minimized 3D structure of inhibitors except Fluconazole was generated by Sybyl7.1 [28] and
used for docking study. Docking was carried out with inhibitors (Fluconozole, compounds
10, 17, 20, 22) using Autodock 3 [29]. Bound Fluconozole coordinate information was
obtained from 3LD6 [30] and docking was performed to investigate that whether its binding
to C. albicans CYP51 is similar or not. After getting the conformation close to co-crystallized
Fluconazole, docking with other inhibitors was carried out. Heme group was processed
separately by manually assigning the +3 charge on Fe and co-ordinate information was
finally merged with receptor. Grid box was centred on Heme group and Lamarckian genetic
algorithm [31] was used for calculation of 50 docking pose for each compound (Table 5).
Acknowledgments
We acknowledge Mrs. Tara Rawat (Technical Officer) for technical assistance and SAIF
Division for spectral data. We acknowledge UGC (L.K., N.L., and L.K.), CSIR (A.S., and
S.J.), and ICMR (V.B. and V.K.) for research fellowships. This study was partially supported
by a grant from the Ministry of Health and Family Welfare, Government of India.
Supplementary data
1
Supplementary data like scan copies of H NMR and 13C NMR spectra can be found at
http://dx.doi.org/
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Chauhan, Med. Chem. Commun. 3 (2012) 1104-1110.
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1
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Captions
3
4
Figure 1. Chemical structures of known antifungal agents 1-5 and azole-carbodithioate
hybrids 6
5
6
Scheme 1. Synthetic strategy for azole-carbodithioate hybrids 7–27
Table 1. In vitro Antifungal and Antibacteriala activity of compounds 7-27
Table 2. In vitro anti-Candida activity of compounds 7–27
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Table 3. Sperm immobilization and anti-Trichomonas activity of compounds 7–27
Table 4. Toxicity of compounds 10, 17, 20, 22
Table 5. Docked energy and IC50 of compounds 10, 17, 20, 22
Figure 2. Docked conformation of compounds 10 (a), 17 (b), 20 (c) and 22 (d) into active site
of Candida albicans Cyp51. Protein residues are shown in hot pink color with both stick and
surface representaions. All docked compounds are shown in green color. Only polar
hydrogens are shown for clarity.
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Table 1.
Antifungal activity (IC50 in µg/mL) againstb
Compound
Azole
R
Ss
Tm
Af
Cn
methyl
>50
>50
4.96
>50
ND
>50
ND
>50
>50
>50
ND
6.21
>50
>50
>50
>50
7
cyanopropyl
allyl
8
4.43
>50
5.75
3.79
>50
9
butyl
10
11
12
13
octyl
1.48
18.43
6.15
decahexyl
ethylphthalimide
>50
4.17
>50
28.59
methyl
cyanopropyl
allyl
4.41
>50
1.50
5.71
4.47
>50
11.68
24.93
>50
>50
>50
9.10
>50
3.35
5.57
>50
>50
14
15
16
17
18
19
18.10
23.79
>50
butyl
2.02
>50
octyl
decahexyl
7.07
>50
methyl
allyl
4.34
5.78
>50
>50
>50
>50
>50
>50
20
21
methyl
cyanopropyl
allyl
1.54
ND
ND
>50
>50
2.42
ND
24.39
>50
>50
>50
>50
0.69
>50
>50
>50
>50
22
23
24
25
26
>50
1.40
>50
butyl
octyl
1.58
0.08
>50
>50
>50
27
Fluconazole.
0.06
0.38
0.46
2
3
4
5
aCompounds (7–27) showed MIC at >50 µg/mL against four bacterial strains: E. coli (ATCC 9637),
Pseudomonas aeruginosa (ATCC BAA-427), Staphylococcus aureus (ATCC 25923), Klebsiella
pneumonia. bSs: Sporothrix schenckii; Tm: Trichophyton mentagrophytes; Af: Aspergillus fumigatus;
Cn: Cryptococcus neoformans.
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Table 2.
Anti-Candida activity (IC50 in µg/mL) againsta
Compound
Azole
R
Ca
Cp
PK3
>50
>50
6.79
5.41
4.61
>50
PK9
>50
PK13 PK30
methyl
5.75
>50
>50
1.29
5.72
>50
5.45
7.24
>50
5.27
2.09
1.94
>50
>50
>50
>50
>50
>50
7
cyanopropyl
allyl
43.49 >50
10.77
1.80
1.53
1.06
>50
8
3.94
6.17
7.26
3.50
9
butyl
10
11
12
13
14
15
16
17
18
19
20
21
octyl
31.39 7.68
>50 >50
decahexyl
ethylphthalimide
methyl
11.09 30.95 19.53 16.02
37.29 >50 36.82 25.95
41.03 47.69 >50
cyanopropyl
allyl
36.03 >50
>50
2.10
2.12
>50
>50
2.21
1.34
7.21
>50
1.43
2.53
10.15 3.61
7.26
7.64
4.53
5.09
butyl
>50
>50
>50
>50
>50
4.69
>50
>50
octyl
19.03 >50
>50 >50
decahexyl
methyl
31.17 >50
38.03 >50
N
N
allyl
>50
45.49 >50 >50
N
methyl
1.26
1.46
1.79
3.33
3.86
1.79
>50
1.75
22
cyanopropyl
allyl
>50
>50
6.39
>50
>50
>50
>50
>50
>50
>50
23
24
25
40.09 39.32 >50
21.80 37.37 >50
butyl
octyl
>50
>50
0.13
>50
>50
0.46
>50
>50
0.21
>50
>50
-
>50
>50
-
>50
>50
-
26
27
Fluconazole
2
3
4
5
aCa: Candida albicans, Cp: Candida parapsilosis (cp ATCC- 22019), PK3: Candida albicans
(MTCC-183), PK9: Candida albicans (patient isolate CDRI), PK13: Candida albicans (ATCC-
10231), PK30: Candida albicans (patient isolate)
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Table 3.
Compound
Percent sperm immobilizationtion
Anti-Trichomonas activity
(MEC in µg/mL)
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
37.4
1.0% (w/v)
0.1% (w/v)
0%
92-95%
0%
100%
100%
100%
100%
100%
100%
100%
100%
95%
100%
100%
100%
95%
100%
100%
100%
100%
100%
100%
95%
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
80%
80%
0%
0%
0%
0%
0%
0%
100%
0%
0%
0%
80%
0%
0%
0%
0%
0%
100%
0%
23
24
25
26
27
0%
100%
0%
Nonoxynol-9
Metronidazole
2.0
3
4
5
6
7
8
9
10
11
12
13
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Table 4.
HeLa cell (IC50 in
µg/mL)
Lactobacillus acidophillus (IC50 in
Compound Structure
µg/mL)
48.70
85.45
53.69
45.64
10
17
220.0
39.54
N
N
N
20
S
S
N
73.60
37.00
45.63
21.00
22
N
S
S
Nonoxynol-
9
3
4
5
6
7
Table 5.
Anti-Candida activity Docked energy
Compound
Structure
(IC50 in µg/mL)
(in Kcal/mole)
1.29
-7.78
10
17
20
1.34
1.43
-8.17
-5.74
N
N
S
N
S
N
1.26
0.13
-7.62
-7.30
22
N
S
S
Fluconazole
8
9
10
11
12
13
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Figure 1.
3
4
Cl
N
N
N
N
5
6
7
8
9
Bifonazole (2)
Clotrimazole (1)
10
11
N
N
Cl
H
Cl
Cl
12
13
14
15
16
17
18
19
20
21
O
N
O
H
Cl
O
O
Cl
N
N
N
Ketoconazole (3)
O
Econazole (4)
F
X
F
Y
R
N
N
N
22
X, Y : N, CH
N
R`
N
N
S
S
23
Figure 2.
HO
R, R`: alkyl
N
24
Azole-Carbodithioate Hybrids (6)
Fluconazole (5)
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7
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Scheme 1.
N
7: R = methyl
8: R = cyanopropyl
9: R = allyl
N
10: R = butyl
11: R = octyl
12: R = decahexyl
13: R = ethylphthalimide
S
7-13
S R
N
N
N
N
a
N
N
H
CH3
N
CH3
N
N
a
H
CH3
S R
S
a
N
N
R X
14-19
27
14: R = methyl
15: R = cyanopropyl
16: R = allyl
17: R = butyl
18: R = octyl
N
27: R = methyl
N
H
N
a
N
a
H
N
19: R = decahexyl
N
N
H
N
N
22: R = methyl
23: R = cyanopropyl
24: R = allyl
25: R = butyl
26: R = octyl
N
N
R = alkyl, alkenyl
X = halide
S
S R
S
S R
20: R = methyl
21: R = allyl
20, 21
22-26
4
5
6
7
8
9
Reagent and conditions; a: CS2, NaH, THF, 0-5 °C - rt, 3-5 h.
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Research Highlights
ꢀAzole and carbodithioate hybrids were synthesized.
ꢀ Compounds were evaluated for spermicidal/microbicidal activity.
ꢀ Sixteen compounds were dually active against sperm and Candida.
ꢀ Promising compounds were safer than Nonoxynol-9.
ꢀ Docking study provided guidelines for designing anti-Candida agents.