Imidazole derivatives as possible microbicides with dual protection

Article abstract of DOI:10.1016/j.ejmech.2009.10.021

Twenty seven derivatives (2-28) of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol were synthesized and evaluated for anti-trichomonas, spermicidal and antifungal activities. Twenty six compounds were active against Trichomonas vaginalis at MIC ranging from 1-42 μM and seven compounds (9,18,19,22,24,26,28) immobilized 100% human spermatozoa at 1% concentration (w/v). Twenty three compounds (2,3,5,8-26,28) exhibited antifungal activity at 25-50 μg/mL concentration. Seven compounds (9,18,19,22,24,26,28) showed significant anti-trichomonas and spermicidal activities and also exhibited mild antifungal activity. All the compounds were highly safe towards human cervical cell line (HeLa) as shown by the cell-viability assay of HeLa cells at 200 μg/mL concentration, whereas nonoxynol-9 (N-9, the marketed spermicidal microbicide) was highly cytotoxic. Therefore, it may be concluded that introduction of the pharmacophore responsible for spermicidal activity into a proven anti-trichomonas structure may lead to a potent dual function microbicide better and safer than N-9.

Full text of DOI:10.1016/j.ejmech.2009.10.021

European Journal of Medicinal Chemistry 45 (2010) 817–824
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Imidazole derivatives as possible microbicides with dual protectionq
,
b
b
Lalit Kumar a, Amit Sarswat a, Nand Lal a, Vishnu L. Sharma a , Ashish Jain , Rajeev Kumar ,
*
Vikas Verma b, Jagdamba P. Maikhuri b, Awanit Kumar c, Praveen K. Shukla c, Gopal Gupta b
a Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
b Division of Endocrinology, Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
c Division of Fermentation Technology, Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty seven derivatives (2–28) of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol were synthesized and
evaluated for anti-trichomonas, spermicidal and antifungal activities. Twenty six compounds were active
Received 24 January 2009
Received in revised form
23 July 2009
Accepted 15 October 2009
Available online 23 October 2009
against Trichomonas vaginalis at MIC ranging from 1–42
immobilized 100% human spermatozoa at 1% concentration (w/v). Twenty three compounds (2,3,5,8–26,28)
exhibited antifungal activity at 25–50 g/mL concentration. Seven compounds (9,18,19,22,24,26,28) showed
mM and seven compounds (9,18,19,22,24,26,28)
m
significant anti-trichomonas and spermicidal activities and also exhibited mild antifungal activity. All the
compounds were highly safe towards human cervical cell line (HeLa) as shown by the cell-viability assay of
HeLa cells at 200 mg/mL concentration, whereas nonoxynol-9 (N-9, the marketed spermicidal microbicide)
was highly cytotoxic. Therefore, it may be concluded that introduction of the pharmacophore responsible for
spermicidal activity into a proven anti-trichomonas structure may lead to a potent dual function microbicide
better and safer than N-9.
Keywords:
Imidazole derivatives
Metronidazole
Anti-trichomonas
Spermicidal
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
healthy human vagina is firmly resistant to HIV infection [18], still
w5 million new patients are added annually to w40 million living
The epidemics of AIDS and unwanted pregnancies appear to
thrive in the presence of overpopulation [1], poverty and other
sexually transmitted diseases (STDs) [2–4]. It is now well estab-
lished that trichomoniasis [5] (the most prevalent, non-viral STD)
significantly increases the vulnerability to HIV [6,7] and therefore
controlling trichomoniasis alone could significantly reduce the
incidence of new HIV infections. On the other hand, high number of
unintended pregnancies [8] may also indicate the need for newer,
cost-effective, safe and convenient contraceptives. A recent survey
[9] has shown that most women would prefer a product capable of
preventing HIV, pregnancy and STIs and almost half of respondents
would use contraceptive microbicides as a dual protection method.
Therefore, efforts are being undertaken to develop novel, dually-
active microbicidal agents [10–17]. Also since microbicides lacking
contraceptive activity may not be used with the required compli-
ance, especially during the ‘‘most vulnerable’’ sexual contacts
where the immediate worry of an unwanted pregnancy often
overwhelms the otherwise bigger fret of STD and/or HIV acquisition
[11], it would be worthwhile to develop dually active agents. The
with HIV, half of which are women [18]. This indicates, (a) high
prevalence of HIV in heterosexual contacts, and (b), increase in
number of women with compromised vaginal resistance caused by
vaginally applied chemical products and/or STD pathogens.
Currently all commercially available spermicidal microbicides [19]
have detergent ingredients that disrupt cell membranes. The most
widely used vaginal spermicide, nonoxynol-9 (N-9) has been
shown to damage the cervicovaginal epithelium because of its
membrane-disruptive properties, causing an acute inflammatory
tissue response, altered vaginal microflora, and enhanced risk of
opportunistic infections in the genitourinary tract. Such opportu-
nistic infections are known to enhance the susceptibility of the
ectocervical epithelium and the endocervical mucosa to HIV
infection. Hence, despite its ability to inactivate HIV in vitro, the
reported failure [20] of N-9 to prevent heterosexual vaginal trans-
mission of HIV in clinical settings has prompted the search for new
female-controlled, non-detergent, topical vaginal spermicidal
microbicides that are more effective as well as safer than N-9 [17].
Thus, a challenge is thrown open to chemists to design molecules
with spermicidal and anti-HIV/STI activities. Consequently, it was
thus thought worthwhile to introduce the pharmacophore
responsible for spermicidal activity into a proven structure with
anti-STI potential (and vice versa).
q
C.D.R.I. Communication No. 7691
* Corresponding author. Tel.: þ91 522 2612411x4320; fax: þ91 522 2623405.
E-mail address: vlscdri1@rediffmail.com (V.L. Sharma).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.021

818
L. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 817–824
Previously, some attempts have been made to modify metro-
2.2. Biology
The compounds synthesized (2–28) were evaluated for sper-
micidal, anti-trichomonas (Table 1) and antifungal activities (Table
2). All the compounds were active against Trichomonas vaginalis at
nidazole structure with a view to improve microbicidal activities
[21–23]. Accordingly, the authors were prompted to carry out
modifications in the structure of 2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethanol (Metronidazole, 1, Scheme 1, the currently used drug
for vaginal trichomoniasis [24]) with a view to introduce spermi-
cidal activity. The pharmacophores imparting spermicidal activity
have been reported to be dithiocarbamates [25], disulfide esters
[14], benzenepropanamine [26], selective serotonin reuptake
inhibitor (SSRI) antidepressants [27], thiourea derivatives [13],
acrylophenones [28] etc. It was planned to derivatize at hydroxyl
group of the side chain (Gen. Str I, Fig. 1), to introduce alkylamino
ester (Gen. Str II, Fig. 1) and dithiocarbamaoyl group (Gen. Str III,
Fig. 1) at position-2 of side chain. The compounds (2–28) syn-
thesised were screened for spermicidal, anti-trichomonas and
antifungal activities and also their safety was evaluated against
human cervical (HeLa) cell line.
MIC ranging from 1.0–111.0
Metronidazole was active at 11.7
m
M (Table 1) whereas the standard drug
M. Seven compounds
m
(9,18,19,22,24,26,28) immobilized 100% human spermatozoa at 1%
concentration (w/v) (Table 1). Twenty three compounds (2,3,5,8–
26,28) exhibited antifungal activity at 50 mg/mL concentration
(Table 2). Metronidazole, and all of the compounds (except 5) were
highly safe towards human cervical cell line (HeLa) as shown by the
cell-viability assay of HeLa cells at 200
whereas N-9 was highly cytotoxic (Fig. 2).
mg/mL concentration,
The results indicated that on functionalization (2–10) at
hydroxyl group in 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol
(1) the anti-trichomonas activity was retained, and high spermicidal
activity (100% immobilization) in compound 9 suggested that we
have been able to introduce the dual activity as metronidazole (1)
showed no effect on sperm motility (Table 1). On the other hand,
compounds 2–8 exhibited moderate to appreciable anti-tricho-
monas activity but their spermicidal activity was negligible.
However, 5-nitroimidazoles bearing an arylsulfonylmethyl group
[21] and N-acetamide(sulfonamide)-2-methyl-4-nitro-1H-imidaz-
oles [22] have been reported to exhibit substantial activity against
T. vaginalis.
With a view to further enhance the spermicidal action in 2-(2-
methyl-5-nitro-1H-imidazol-1-yl)ethanol framework, alkylamino
side chain (11–18) and dithiocarbamoyl moieties (19–28) were
introduced at hydroxyl function. The compounds with alkylamino
side chain showed high anti-trichomonas activity. Compounds
(16,17) were seven to eight times more active than metronidazole
2. Results and discussion
2.1. Chemistry
Compounds (2–10, Scheme 1) were synthesized by the reactions
of appropriate reagents (a–e, Scheme 1) on hydroxyl group of 2-(2-
methyl-5-nitro-1H-imidazol-1-yl)ethanol (1, Scheme 1). Compounds
(11–18, Scheme 1) were prepared from 2-(2-methyl-5-nitro-1H-
imidazol-1-yl)ethyl acrylate (10, Scheme 1) on addition with
substituted azoles, substituted piperazines or N-methyl-3-phenyl-3-
(4-(trifluoromethyl)phenoxy)propan-1-amine in the presence of
triethylamine. Compounds (19–28, Scheme 1) were synthesized by
the reaction of compound 2 with sodium salt of substituted carbo-
dithioic acid in acetonitrile under reflux.
N
N
CH3
O2N
CH3
O2N
N
N
R
2: OMs
3: Cl
4: OCOCH3
5: OCOCH2Cl
R
2-5
SNa
O
a
b
6
S
N
N
CH3
O2N
N
N
O2N
CH3
N
CH3
O2N
c
e
N
OSO2CH3
N
O
OH
1
d
O
7
10
N
g
f
O2N
CH3
N
N
N
N
CH3
O2N
R1
R2
N
O2N
CH3
NR3R4
11: imidazol-1-yl
NR1R2
8: imidazol-1-yl
9: benzimidazo-1-yl
NR1R2
pyrrolidino
morpholino
N
8-9
19:
20:
R1
N
12: benzotriazol-1-yl
13: triazol-1-yl
14: 2-nitroimidazol-1-yl
15: 2-methyl-5-nitroimidazol-1-yl
16: 4-fluorophenylpiperazin-1-yl
17: 4-nitrophenylpiperazin-1-yl
R3
O
21: piperidino
22: dimethylamino
23: diethylamino
N
R4
8
S
R2
O
S
19-27
24: 4-(pyridin-2-yl)piperazin-1-yl
25: 4-(pyrimidin-2-yl)piperazin-1yl
11-1
18: N-methyl-3-phenyl-3-(4-(trifluoromethyl)
phenoxy)propan-1-amino
26: 4-(2-methoxyphenyl)piperazin-1-yl
27: 4-(4-nitrophenyl)piperazin-1-yl
28: 4-methylpiperazin-1-yl
Scheme 1. Reagents and conditions; a: CH3SO2Cl, Et3N, CH2Cl2, 0–5 ꢀC/ SOCl2, CHCl3(dry), reflux/ (CH3CO)2O, Et3N, CH2Cl2, DMAP, 0–5 ꢀC/ ClCH2COCl, Et3N, CH2Cl2, 0–5 ꢀC; b: CS2,
NaOH, MeOH, rt; c:(i) CH3SO2Cl, Et3N, CH2Cl2, 0–5 ꢀC; (ii) Py, 110-115ꢀC; d: (i) CH3SO2Cl, Et3N, CH2Cl2, 0–5 ꢀC; (ii) NR1R2, Et3N, toluene, reflux; e: ClCOCH2CH2Cl, Et3N, CH2Cl2, 0–5 ꢀC;
f: NR3R4, Et3N, CH2Cl2/ClCH2CH2Cl; g:(i) CH3SO2Cl, Et3N, CH2Cl2, 0–5 ꢀC; (ii) R1R2NC ¼ SSNa, CH3CN, reflux.

L. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 817–824
819
N
active as metronidazole and four compounds (19,24,26,28) had MIC
significantly better than metronidazole. Moreover, five compounds
(19,22,24,26,28) also immobilized 100% human spermatozoa and
therefore exhibited dual activity. Compound 22 showed remark-
able anti-trichomonas and spermicidal activities and also exhibited
mild antifungal activity. The notable activity in seven compounds
(9,18,19,22,24,26,28) may possibly be attributed to their high sulf-
hydryl affinity [14] resulting in interactions between sulfhydryl
group present on sperm membrane with benzimidazolyl side-
chain, benzenepropanamine [26] and dithiocarbamoyl group [16],
respectively.
N
N
CH3
O2N
O2N
CH3
N
N
CH3
O2N
N
R3
R2
N
S
O
2
N
R4
R
R1
S
O
Str. 1
Str. 2
Str.3
Fig. 1. General Structures of the compounds synthesized.
Therefore, it may be concluded that introduction of the
pharmacophore responsible for spermicidal activity into a proven
anti-trichomonas structure may lead to a potent dual function
spermicide, which can be better and safer than N-9.
and compound 18 immobilized 100% sperms at 1% concentration.
The addition of dithiocarbamoyl moiety further enhanced both the
activities. Three compounds 21–23 were ten to eleven times,
compound 20 six times and compound 27 was about four times as
Table 1
In-vitro Spermicidal activity and Anti-Trichomonas activity of the compounds (2–28) against human spermatozoa and Trichomonas vaginalis.
Compounds (str. 1)
R
Spermicidal activity
(percent sperm motility of
control at 1.0% concentration)
Anti-Trichomonas
activity (
M) (MIC ꢁ SE)
m
2
3
4
5
6
7
8
9
OMs
Cl
OAc
OCOCH2Cl
94.12
58.82
82.35
98.37
90.24
85.09
92.36
0
5.02 ꢁ 0.89
6.60 ꢁ 0.44
23.47 ꢁ 4.14
10.10 ꢁ 1.78
111.52 ꢁ 4.96
9.53 ꢁ 1.02
14.14 ꢁ 1.51
4.61 ꢁ 0.81
42.22 ꢁ 2.96
OC ¼ SSNa
Pyridinium methanesulfonate
imidazol-1-yl
benzimidazol-1-yl
acrylate
10
70.58824
Compounds (str. 2)
NR1R2
11
12
13
14
15
16
17
18
Imidazol-1-yl
Benzotrizol-1-yl
Triazol-1-yl
2-nitroimidazol-1-yl
2-methyl-5-nitroimidazol-1-yl
4-fluorophenylpiperazin-1-yl
4-nitrophenylpiperazin-1-yl
N-methyl-3-phenyl-3-(4-(trifluoromethyl)
phenoxy)propan-1-amino
100
95.734
100
10.67 ꢁ 3.01
27.62 ꢁ 0.32
21.26 ꢁ 0.57
9.25 ꢁ 0.99
20.60 ꢁ 1.71
1.54 ꢁ 0.82
1.45 ꢁ 0.77
11.70 ꢁ 0.21
100
88.23529
88.23529
82.35294
0
Compounds (str. 3)
NR3R4
pyrrolidino
19a
0
8.33 ꢁ 1.47
1.98 ꢁ 0.35
1.00 ꢁ 0.18
1.14 ꢁ 0.20
1.03 ꢁ 0.18
6.38 ꢁ 0.43
25.45 ꢁ 2.94
5.94 ꢁ 0.69
2.87 ꢁ 0.51
7.60 ꢁ 0.33
60.78 ꢁ 4.05
11.70 ꢁ 1.46
Inactive
20
morpholino
70.58824
70.58824
0
100
21
piperidino
dimethylamino
22a
23
diethylamino
4-(pyridin-2-yl)piperazin-1-yl
24a
0
25
4-(pyrimidin-2-yl)piperazin-1yl
4-(2-methoxyphenyl)piperazin-1-yl
94.11765
0
41.17647
0
0
26a
27
4-(4-nitrophenyl)piperazin-1-yl
28
4-methylpiperazin-1-yl
N-9
–
–
–
Metronidazole
Vehicle (Control)
100
100
a
These compounds were tested as D-(-)tartrate salt for spermicidal activity.

820
L. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 817–824
Table 2
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 precoated alumina/silica gel plates (Aldrich). Column
chromatography was performed over Merck silica gel
(60–120 mesh). All chemicals and solvents were procured from
Sigma-Aldrich/Merck India Ltd. 1-dialkylamino carbodithioic acid
sodium salts [25] were prepared by known procedures.
Antifungal activity against six different strains of fungi.
Compound
Antifungal activity (MIC in
m
g/mL)
4
1
2
3
5
6
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
N-9
>50
>50
>50
>50
>50
>50
25
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
>50
50
50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
–
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
25
>50
>50
>50
>50
50
50
50
50
50
50
50
50
50
50
50
50
50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
>50
50
3.1.1. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl
methanesulfonate (2)
50
50
>50
>50
>50
>50
>50
>50
>50
>50
25
Into a ice-cooled solution of 2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethanol (1, 0.011 mol) and triethylamine (0.017 mol) in dry
dichloromethane was added methane sulfonyl chloride (0.017 mol)
drop wise under stirring. The reaction mixture was further stirred
at 0–5 ꢀC for 2 h. The separated thick material was filtered and
washed with water (15 mL ꢃ 3) gave a cream coloured solid. The
solid was crystalized from acetonitrile/ethyl acetate (1:1, v/v). Yield
80%; Mp 139–140 ꢀC; IR (KBr): 3024, 2934, 1526, 1260, 745 cmꢂ1
;
>50
>50
>50
50
>50
>50
>50
50
50
25
>50
25
ESI-MS: m/z 250 (Mþ þ 1); 1H NMR (300 MHz, DMSO-d6,
d ppm):
d
8.05 (s, 1H), 4.65 (t, 2H, j ¼ 4.7 Hz), 4.55 (t, 2H, j ¼ 4.6 Hz), 3.14
25
50
50
25
>50
50
–
(s, 3H), 2.46 (s, 3H); Anal. Calcd. for C7H11N3O5S (249): C, 33.73; H,
4.45; N, 16.86; Found: C, 33.51; H, 4.25; N, 16.53.
>50
>50
>50
>50
>50
>50
0.5
>50
>50
>50
>50
>50
–
50
>50
50
>50
50
–
3.1.2. 1-(2-chloroethyl)-2-methyl-5-nitro-1H-imidazole (3)
Into a ice-cooled solution of 2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethanol (1, 0.003 mol) in dry chloroform (20 mL) was added
thionyl chloride (0.004 mol) dropwise under stirring. A sticky solid
separated out which was dissolved on heating the mixture to 65 ꢀC
in an oil bath. The reaction mixture was further heated at 65–70 ꢀC
for 3 h. Excess thionyl chloride was removed azeotropically. The
residue was taken into ethyl acetate (30 mL) and washed with
water (10 mL ꢃ 3). Organic layer was dried over sodium sulfate and
concentrated under reduce pressure to provide a brown solid. Yield
85%; Mp 78–79 ꢀC; IR (KBr): 3132, 2976, 1531, 1365, 1263,
756 cmꢂ1; ESI-MS: m/z 190 (Mþ þ 1), 192 (Mþ þ 3); 1H NMR
Fluconazole
1.0
2.0
1.0
2.0
1.0
1: Candida albicans; 2: Cryptococcus neoformans; 3: Sporothrix schenckii; 4: Tricho-
phyton mentagrophytes; 5: Aspergillus fumigatus; 6: Candida parapsilosis.
3. Experimental section
3.1. Chemistry
Melting points were determined in open capillary tubes on an
electrically heated block and are uncorrected. IR spectra (nmax in
cmꢂ1) of the compounds were recorded on Perkin Elmer’s FT-IR RX1
PC spectrophotometer.1H NMR & 13C NMR spectra were recorded on
Bruker Supercon Magnet Avance DPX-200/DRX-300 spectrometers
(operating at 200 and 300 MHz respectively for 1H; 50 and 75 MHz
respectively for 13C) in deuterated solvents with TMS as internal
(300 MHz, CDCl3,
d
ppm):
d
7.91 (s, 1H), 4.61 (t, 2H, j ¼ 5.6 Hz), 3.87
(t, 2H, j ¼ 5.6 Hz), 2.54 (s, 3H); Anal. Calcd.: for C6H8ClN3O2 (189):
C, 38.01; H, 4.25; N, 22.16; Found: C, 37.93; H, 4.16; N, 22.01.
3.1.3. Acetic acid 2-(2-methyl-5-nitro-1H-imidazol-1-yl)
ethyl ester (4)
In to an ice-cooled solution of 2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethanol (1, 0.015 mol) in dry dichloromethane (10 mL) was
added triethylamine (0.022 mol) and dimethylaminopyridine
reference (chemical shifts in
tion Mass spectra (ESI-MS) were recorded on a Micromass Quattro II
triple quadruple mass spectrometer. Elemental analyses were
d ppm, J in Hz.). Electronspray Ionisa-
Fig. 2. The viability of HeLa cells after 24 h incubation in 200
mg/mL of test compounds (Mean ꢁ SE of 3 estimations). MET (metronidazole); N-9 (nonoxynol-9).

L. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 817–824
821
(10 mol%) under stirring. Acetic anhydride (0.003 mol) was added
dropwise and the reaction mixture was further stirred for 1 h (0–
5 ꢀC). The reaction mixture was concentrated under reduced pres-
sure, diluted with water (15 mL) and extracted with ethyl acetate
(15 mL ꢃ 3). The combined organic phase was washed with water
(10 mL ꢃ 3) and dried over sodium sulfate. Sodium sulfate was
filtered off and washed with ethyl acetate (5 mL ꢃ 2). The combined
ethyl acetate layer was concentrated under reduce pressure to give
a light brown solid. Yield 88%; Mp 61–62 ꢀC; IR (KBr): 3124, 2963,
1741, 1528, 1263, 744 cmꢂ1; ESI-MS: m/z 213 (Mþ), 214 (Mþ þ 1); 1H
(10 mL) was added imidazole/benzimidazole (1 eq) portionwise at
room temperature. The reaction mixture was heated at 110–120 ꢀC
for 6 h under stirring in an oil bath. Toluene was distilled off under
reduced pressure and the crude material was taken in chloroform
(20 mL). The chloroform layer was washed with water (10 mL ꢃ 3)
and dried over sodium sulfate. Sodium sulfate was filtered off and
washed with chloroform (5 mL ꢃ 2). The filtrate was concentrated
under reduced pressure. The crude material was purified by column
chromatography over silica gel (60–120 mesh) with chloroform/
hexane as an eluent.
NMR (300 MHz, CDCl3,
d
ppm):
d
7.92 (s, 1H); 4.55 (t, 2H, j ¼ 5.2 Hz),
4.37 (t, 2H, j ¼ 5.1 Hz), 2.47 (s, 3H), 1.99 (s, 3H); Anal. Calcd. for
C8H11N3O4 (213): C, 45.07; H, 5.20; N,19.71; Found: C, 45.12; H, 5.39;
N, 19.61.
3.1.7.1. 1-(2-(1H-imidazol-1-yl)ethyl) 2-methyl-5-nitro-1H-imid-
azole (8). Light red solid; Yield 63%; Mp 158–159 ꢀC; IR (KBr): 3032,
2964, 1531, 1259, 736 cmꢂ1; ESI-MS: m/z 222 (Mþ þ 1), 154; 1H
NMR (300 MHz, CDCl3,
d ppm): d 7.97 (s, 1H), 7.26 (s, 1H), 7.03
3.1.4. Chloro-acetic acid 2-(2-methyl-5-nitro-1H-imidazol-1-yl)
ethyl ester (5)
(s, 1H), 6.60 (s, 1H), 4.54 (t, 2H, j ¼ 5.4 Hz), 4.36 (t, 2H, j ¼ 5.4 Hz),
1.85 (s, 3H); Anal. Calcd. for C9H11N5O2 (221): C, 48.86; H, 5.01; N,
31.66; Found: C, 48.58; H, 5.34; N, 31.82.
Into a ice-cooled solution of 2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethanol (1, 0.0011 mol) and triethylamine (0.0022 mol) in
dichloromethane (10 mL) was added chloroacetyl chloride
(0.0022 mol) dropwise under stirring. The reaction mixture was
further stirred at 0–5 ꢀC for 30 min. Dichloromethane was distilled
off and the residue was taken in ethyl acetate (20 mL). The ethyl
acetate layer was washed with water (10 mL ꢃ 3) and dried over
sodium sulfate. Sodium sulfate was filtered off and washed with
ethyl acetate (5 mL ꢃ 2). The filtrate was concentrated to give
a brown semisolid. Yield 71%; IR (KBr): 3021, 1747, 1534, 1364, 1262,
756 cmꢂ1; ESI-MS: m/z 248 (Mþ þ 1), 250 (Mþ þ 3); 1H NMR
3.1.7.2. 1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)
1H-benz-
imidazole (9). Light yellow solid; Yield 67%; Mp 210–211 ꢀC;
I.R.(KBr): 2922, 1519, 1257, 760 cmꢂ1; ESI-MS: m/z 272 (Mþ þ 1); 1H
NMR (300 MHz, CDCl3,
d ppm): d 8.00 (s, 1H), 7.83–7.79 (m, 1H),
7.64 (s, 1H), 7.33–7.30 (m, 2H), 7.23–7.20 (m, 1H), 4.69–4.61 (m, 4H),
1.70 (s, 3H); Anal. Calcd. for C13H13N5O2 (271): C, 57.56; H, 4.83; N,
25.82; Found: C, 57.87; H, 4.96; N, 25.49.
3.1.8. Acrylic acid 2-(2-methyl-5-nitro-1H-imidazol-1-yl)e
thyl ester (10)
(300 MHz, CDCl3,
d
ppm):
d
7.95 (s,1H), 4.62 (t, 2H, j ¼ 4.9 Hz), 4.52 (t,
2H, j ¼ 4.9 Hz), 4.01 (s, 2H), 2.53 (s, 3H); Anal. Calcd. for C8H10ClN3O4
To a ice-cooled solution of 2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethanol (1, 0.023 mol) and triethylamine (0.035 mol) in dry
dichloromethane (70 mL) was added a solution of 1,3-dichlor-
opropanone (0.046 mol) in chloroform (10 mL) under stirring in
2 h. The reaction mixture was allowed to attain room temper-
ature in 1 h under stirring. The reaction mixture was further
stirred for 3 h at room temperature. The reaction mixture was
concentrated and then diluted with chloroform (50 mL) and
water (20 mL). The two layers were stirred for 30 min and then
separated. Organic layer was washed with water (15 mL ꢃ 3) and
dried over sodium sulfate. Sodium sulfate was filtered off and
washed with chloroform (5 mL ꢃ 2) and the filtrate was
concentrated under reduce pressure. A yellowish solid was
formed. Yield 79%; Mp 42–43 ꢀC; IR (KBr): 3123, 2957, 1721,
1529, 1369, 1264, 742 cmꢂ1; ESI-MS: m/z 226 (Mþ þ 1); 1H NMR
(247): C, 38.80; H, 4.07; N, 16.97; Found: C, 38.52; H, 4.15; N, 17.13.
3.1.5. Sodium O-2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl
carbonodithioate (6)
The title compound was prepared by the known procedure [29].
To a ice-cooled solution of sodium hydroxide (0.0028 mol, 0.287 g)
in methanol (10 mL) was added 2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethanol (1, 0.0018 mol) in small portions under stirring. Carbon
disulfide was added dropwise at 0–5 ꢀC and the reaction mixture
was further stirred for 24 h at room temperature, during the course
of reaction pH maintained basic (10–12). Methanol was distilled off
and crude product was taken in acetone (10 mL) to give a precipitate.
Yellow solid was filtered off and dried in vacuum desiꢂcc1ator. Yield
;
1H NMR
77%; Mp 250 ꢀd; IR (KBr): 1640, 1384, 1272, 674 cm
(300 MHz, D2O,
d
ppm):
d
7.31 (s, 1H), 3.97 (t, 2H, j ¼ 5.2 Hz), 3.75 (t,
(300 MHz, CDCl3,
d
ppm):
d
7.92 (s, 1H), 6.34 (d, 1H, j ¼ 15.8 Hz),
2H, j ¼ 5.1 Hz), 2.26 (s, 3H); Anal. Calcd. for C7H8N3NaO3S2 (269): C,
6.07–5.97 (m, 1H), 5.85 (d, 1H, j ¼ 9.0 Hz), 4.61–4.58 (m, 2H),
4.49–4.48 (m, 2H), 2.46 (s, 3H); Anal. Calcd. for C9H11N3O4
(225): C, 48.00; H, 4.92; N, 18.66; Found: C, 47.68; H, 5.24; N,
18.33.
31.22; H, 2.99; N, 15.60; Found: C, 31.39; H, 3.17; N, 15.91.
3.1.6. 1-[2-(2-methyl-5-nitro-1H-imidazol-1-yl)-ethyl]pyridinium
methanesulfonate (7)
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl methanesulfonate (2,
0.0006 mol) was added into excess of pyridine (2 mL) and the reaction
mixture was heated at reflux under stirring for 24 h. Pyridine was
distilled off under reduced pressure. The residual brown solid was
washed with ethyl acetate/hexane mixture (1:1; 10 mL ꢃ 2). Yield
3.1.9. Synthesis of 3-substituted-propionic acid 2-(2-methyl-5-
nitro-1H-imidazol-1-yl)ethyl ester (11–18, Scheme 1)
To a solution of acrylic acid 2-(2-methyl-5-nitro-1H-imidazol-1-
yl)-ethyl ester (10, 0.003 mol) in dichloromethane/dichloroethane
(10 mL)wasadded substituted diazoles, substitutedpiperazinesorN-
methyl-3-phenyl-3-(4-(trifluoromethyl)phenoxy)propan-1-amine
at room temperature under stirring. Reaction mixture was heated at
reflux under stirring for 5–8 h in an oil bath. Solvent was distilled off
and crude product was taken into ethyl acetate (20 mL). Organic
layer was washed with water (10 mL ꢃ 3) and dried over sodium
sulfate. Sodium sulfate was filtered off and washed with ethyl
acetate (5 mL ꢃ 2) and the filtrate was concentrated under reduce
pressure. Crude material was purified by column chromatography
using chloroform/hexane or ethyl acetate/hexane as eluent to give
the product.
82%; Mp 145–146 ꢀC; IR (KBr): 3444, 2929,1742,1650,1187, 778 cmꢂ1
;
1H NMR (300 MHz, D2O,
(m, 1H), 7.82 (s, 1H), 7.80–7.77 (m, 2H), 4.82 (t, 2H, j ¼ 5.5 Hz), 4.69
(t, 2H, j ¼ 5.4 Hz), 2.48(s, 3H),1.88 (s, 3H); Anal. Calcd. for C12H16N4O5S
(328): C, 43.90; H, 4.91; N, 17.06; Found: C, 43.53; H, 5.21; N, 17.31.
d
ppm):
d
8.48 (d, 2H, j ¼ 5.9 Hz), 8.36–8.31
3.1.7. Synthesis of 1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl)
1H-substituted azoles (8,9; Scheme 1)
To a solution of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl
methanesulfonate (1 eq) and triethylamine (1.5 eq) in toluene

822
L. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 817–824
3.1.9.1. 3-Imidazol-1-yl-propionic acid 2-(2-methyl-5-nitro-1H-imi-
dazol-1-yl)ethyl ester (11). Light yellow solid; Yield 77%; Mp 81–
82 ꢀC; IR (KBr): 3019,1744,1534, 1428, 1263, 758 cmꢂ1; ESI-MS: m/z
2.69 (t, 2H, j ¼ 6.8 Hz), 2.59 (d, 4H, j ¼ 4.9 Hz), 2.57 (s, 3H), 2.53–
2.47 (m, 2H); Anal. Calcd. for C19H24N6O6 (432): C, 52.77; H, 5.59; N,
19.43; Found: C, 52.51; H, 5.81; N, 19.79.
294 (Mþ þ 1); 1H NMR (300 MHz, CDCl3,
d ppm): d 7.93 (s, 1H), 7.47
(s, 1H), 7.03 (s, 1H), 6.88 (s, 1H), 4.54 (t, 2H, j ¼ 5.2 Hz), 4.40 (t, 2H,
j ¼ 5.1 Hz), 4.22 (t, 2H, j ¼ 6.4 Hz), 2.71 (t, 2H, j ¼ 6.4 Hz), 2.41 (s,
3H); Anal. Calcd. for C12H15N5O4 (293): C, 49.14; H, 5.16; N, 23.88;
Found: C, 49.42; H, 5.29; N, 23.68.
3.1.9.8. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(methyl(3-
phenyl-3-(4-(trifluoromethyl)phenoxy)propyl) amino)propanoate
(18). Light red semisolid; Yield 77%; IR (neat): 3020, 1740, 1616,
1529, 1326, 1216, 762 cmꢂ1; ESI-MS: m/z 535 (Mþ þ 1); 1H NMR
(300 MHz, CDCl3,
d
ppm):
d
7.85 (s, 1H), 7.39 (d, 2H, j ¼ 8.5 Hz),
3.1.9.2. 3-Benzotriazol-1-yl-propionic acid 2-(2-methyl-5-nitro-1H-
imidazol-1-yl)ethyl ester (12). Light yellow solid; Yield 51%; Mp
94–95 ꢀC; IR (KBr): 2969, 1742, 1530, 1364, 1263, 757 cmꢂ1; ESI-MS:
7.30–7.22 (m, 5H), 6.84 (d, 2H, j ¼ 8.5 Hz), 5.32–5.28 (m, 1H),
4.52–4.49 (m, 2H), 4.34–4.30 (m, 2H), 2.77–2.53 (m, 6H), 2.43
(s, 3H), 2.34 (s, 3H), 2.19–2.12 (m, 2H); 13C NMR (75 MHz, CDCl3,
m/z 345 (Mþ þ 1); 1H NMR (300 MHz, CDCl3,
d
ppm):
d
7.99 (d, 1H,
d ppm): d 171.3, 160.6, 150.3, 141.0, 138.6, 132.8, 128.8, 127.8, 126.8,
j ¼ 8.3 Hz), 7.81 (s, 1H), 7.54–7.43 (m, 2H), 7.35–7.30 (m, 1H), 4.81
126.75, 126.71, 126.1, 125.7, 123.1, 122.7, 122.5, 115.7, 78.0, 62.3, 53.4,
52.4, 44.8, 41.8, 36.1, 32.1, 14.1; Anal. Calcd. for C26H29F3N4O5 (534):
C, 58.42; H, 5.47; N, 10.48; Found: C, 58.73; H, 5.18; N, 10.76.
(t, 2H, j ¼ 6.5 Hz), 4.46 (t, 2H, j ¼ 5.0 Hz), 4.34 (t, 2H, j ¼ 4.9 Hz), 3.04
(t, 2H, j ¼ 6.5 Hz), 2.34 (s, 3H); 13C NMR (75 MHz, CDCl3,
d ppm):
d
170.0, 150.7, 145.9, 138.5, 133.2, 127.6, 124.0, 120.0, 109.4, 63.2,
44.9, 43.1, 34.0, 14.3; Anal. Calcd. for C15H16N6O4 (344): C, 52.32; H,
4.68; N, 24.41; Found: C, 52.09; H, 4.99; N, 24.25.
3.1.10. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl dialkylamino-
1-carbodithioate (19–28, Scheme 1)
To a solution of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl
methanesulfonate (2, 0.002 mol) in acetonitrile (20 mL) was added
dialkylamino carbodithioic acid sodium salt (0.002 mol) at room
temperature. The reaction mixture was heated at reflux in an oil
bath under stirring for 6–9 h. The reaction mixture was concen-
trated and diluted with ethyl acetate (25 mL). The ethyl acetate
layer was washed with water (10 mL ꢃ 3) and dried over sodium
sulfate. Sodium sulfate was filtered off and washed with ethyl
acetate (5 mL ꢃ 2). The filtrate was concentrated under reduce
pressure to provide the crude product which was purified by
column chromatography over silica gel with ethylacetate/hexane as
eluent. The free base (1 eq) was dissolved in methanol (20 mL) and
a solution of D-(-) tartaric acid (1 eq) in methanol (10 mL) was
added with stirring at room temperature. The reaction mixture was
further stirred overnight at room temperature. Adding dry ethyl
ether precipitated the tartrate salts (19,22,24,26).
3.1.9.3. 3-[1,2,4]Triazol-1-yl-propionic acid-2-(2-methyl-5-nitro-1H-
imidazol-1-yl)ethyl ester (13). Yellow solid; Yield 55%; Mp 57–
58 ꢀC; IR (KBr): 3018, 1743, 1533, 1365, 1265, 760 cmꢂ1; ESI-MS: m/z
295 (Mþ þ 1); 1H NMR (300 MHz, CDCl3,
d ppm): d 8.07 (s, 1H), 7.89
(s, 1H), 7.86 (s, 1H), 4.54 (t, 2H, j ¼ 5.1 Hz), 4.41 (t, 4H, j ¼ 5.6 Hz),
2.87 (t, 2H, j ¼ 6.1 Hz), 2.42 (s, 3H); 13C NMR (75 MHz, CDCl3,
d
ppm): d 169.8, 151.9, 150.3, 143.5, 138.0, 132.8, 62.8, 44.6, 44.3,
33.5, 14.0; Anal. Calcd. for C11H14N6O4 (294): C, 44.90; H, 4.80; N,
28.56; Found: C, 44.61; H, 4.98; N, 28.38.
3.1.9.4. 3-(4-Nitro-1H-imidazol-1-yl)-propionic acid-2-(2-methyl-5-
nitro-1H-imidazol-1-yl)ethyl ester (14). Light yellow solid; Yield
68%; Mp 108–109 ꢀC; IR (KBr): 3083, 2965, 1730, 1522, 1267,
744 cmꢂ1; ESI-MS: m/z 339 (Mþ þ 1); 1H NMR (300 MHz, CDCl3,
d
ppm):
d
8.02 (s,1H), 7.92 (s,1H), 7.58 (s,1H), 4.61 (t, 2H, j ¼ 4.9 Hz),
4.45 (t, 2H, j ¼ 5.0 Hz), 4.33 (t, 2H, j ¼ 6.0 Hz), 2.84 (t, 2H, j ¼ 6.1 Hz),
2.48 (s, 3H); Anal. Calcd. for C12H14N6O6 (338): C, 42.61; H, 4.17; N,
24.84; Found: C, 42.38; H, 4.28; N, 24.69.
3.1.10.1. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl pyrrolidine-1-
carbodithioate (19). Brown solid; Yield 86%; Mp 138–139 ꢀC, IR
(KBr): 3020,1529,1436,1216, 760, cmꢂ1; ESI-MS: m/z 300 (Mþ), 301
3.1.9.5. 3-(2-Methyl-5-nitro-1H-imidazol-1-yl)-propionic acid 2-(2-
methyl-5-nitro-1H-imidazol-1-yl)ethyl ester (15). White solid; Yield
81%; Mp 207–208 ꢀC; IR (KBr): 3055, 2899, 1742, 1506, 1260,
750 cmꢂ1; ESI-MS: m/z 353 (Mþ þ 1); 1H NMR (300 MHz, CDCl3,
(Mþ þ 1); 1H NMR (300 MHz, CDCl3,
d ppm): d 7.95 (s, 1H), 4.67
(t, 2H, j ¼ 6.9 Hz), 3.93 (t, 2H, j ¼ 6.9 Hz), 3.70–3.62 (m, 4H), 2.59
(s, 3H), 2.15–1.96 (m, 4H); 13C NMR (75 MHz, CDCl3,
d ppm): 190.8,
151.4, 138.5, 133.4, 55.5, 50.9, 45.3, 34.9, 26.2, 24.4, 14.9; Anal. Calcd.
for C11H16N4O2S2 (300): C, 43.94; H, 5.32; N, 18.64; Found: C, 43.69;
H, 5.54; N, 18.46; Tartrate salt mp 92–93 ꢀC.
d
ppm):
d
7.94 (s, 1H), 7.91 (s, 1H), 4.61 (t, 2H, j ¼ 4.9 Hz), 4.45 (t, 2H,
j ¼ 5.0 Hz), 4.22 (t, 2H, j ¼ 6.3 Hz), 2.83 (t, 2H, j ¼ 6.3 Hz), 2.49 (s,
3H), 2.46 (s, 3H); Anal. Calcd. for C13H16N6O6 (352): C, 44.32; H,
4.58; N, 23.85; Found: C, 44.02; H, 4.68; N, 23.65.
3.1.10.2. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl morpholine-4-
carbodithioate (20). Off white solid; Yield 76%; Mp 127–128 ꢀC; IR
(KBr): 3018, 1530, 1264, 762 cmꢂ1; ESI-MS: m/z 316 (Mþ), 317
3.1.9.6. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl-3-(4-(4–fluo-
rophenyl)piperazin-1-yl)propanoate (16). Yellow solid; Yield 81%;
Mp 89–90 ꢀC; IR (KBr): 2955, 1730, 1514, 1268, 744 cmꢂ1; ESI-MS:
(Mþ þ 1); 1H NMR (300 MHz, CDCl3,
d ppm): d 7.94 (s, 1H), 4.60 (t,
2H, j ¼ 6.9 Hz), 4.43–4.23 (m, 2H), 4.01–3.89 (m, 2H), 3.75–3.68 (m,
6H), 2.57 (s, 3H); Anal. Calcd. for C11H16N4O3S2 (316): C, 41.76; H,
5.10; N, 17.71; Found: C, 41.68; H, 5.33, N, 17.63.
m/z 406 (Mþ þ 1); 1H NMR (200 MHz, CDCl3,
d ppm): d 7.89 (s, 1H),
6.95–6.77 (m, 4H), 4.57 (t, 2H, j ¼ 5.2 Hz), 4.39 (t, 2H, j ¼ 5.2 Hz),
3.06–3.01 (m, 4H), 2.69–2.61 (m, 2H), 2.58–2.53 (m, 4H), 2.49
(s, 3H), 2.46–2.42 (m, 2H); 13C NMR (75 MHz, CDCl3,
d
ppm):
d
171.3,
3.1.10.3. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl piperidine-1-
carbodithioate (21). Light yellow solid; Yield 76%; Mp 109–110 ꢀC;
IR (KBr): 3020, 1529, 1363, 1216, 761, cmꢂ1; ESI-MS: m/z 314 (Mþ),
158.7, 155.5, 150.3, 147.8, 138.6, 132.8, 117.8, 117.7, 115.6, 115.3, 62.4,
53.2, 50.0, 44.8, 32.0, 14.2; Anal. Calcd. for C19H24FN5O4 (405): C,
56.29; H, 5.97; N, 17.27; Found: C, 56.60; H, 6.21; N, 17.22.
315 (Mþ þ 1); 1H NMR (300 MHz, CDCl3,
d ppm): d 7.96 (s, 1H), 4.62
(t, 2H, j ¼ 6.8 Hz), 4.33–4.25 (m, 2H), 3.91–3.87 (m, 2H), 3.71 (t, 2H,
j ¼ 6.9 Hz), 2.58 (s, 3H), 1.82–1.60 (m, 6H); Anal. Calcd. for
C12H18N4O2S2 (314): C, 45.84; H, 5.77; N, 17.82; Found: C, 46.17; H,
6.04; N, 17.42.
3.1.9.7. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 3-(4-(4-nitro-
phenyl)piperazin-1-yl)propanoate (17). Dark yellow solid; Yield
66%; Mp 115–116 ꢀC; IR (KBr): 2926, 2823, 1738, 1596, 1526, 1248,
750 cmꢂ1; ESI-MS: m/z 433 (Mþ þ 1); 1H NMR (300 MHz, CDCl3,
d
ppm):
d
8.11 (d, 2H, j ¼ 9.2 Hz), 7.92 (s, 1H), 6.81 (d, 2H, j ¼ 9.3 Hz),
3.1.10.4. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl dimethylcarba-
modithioate (22). Yellow solid; Yield 79%; Mp 109–110 ꢀC; IR (KBr):
4.63 (t, 2H, j ¼ 5.2 Hz), 4.43 (t, 2H, j ¼ 5.2 Hz), 3.40–3.37 (m, 4H),

L. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 817–824
823
3020, 1530, 1261, 786, cmꢂ1; ESI-MS: m/z 274 (Mþ), 275 (Mþ þ 1);
3.2. Biology
1H NMR (300 MHz, CDCl3,
d ppm): d 7.93 (s, 1H), 4.58 (t, 2H,
j ¼ 7.0 Hz), 3.66 (t, 2H, j ¼ 6.9 Hz), 3.55 (s, 3H), 3.36 (s, 3H), 2.56 (s,
3.2.1. Spermicidal activity
3H); 13C NMR (50 MHz, CDCl3,
d
ppm): 195.3, 151.5, 138.6, 133.5,
d
Spermicidal test was adapted from the standard procedure [30].
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% solution. A spermicidal test was
performed with each compound solution and for this purpose
0.05 mL of liquefied human semen was added to 0.25 mL of test
solution and vortexed for 10 s at low speed. A drop of the mixture
was then placed on a microscope slide, covered with a cover glass
and examined under a phase contrast microscope in five fields of
vision. The percentage of motile spermatozoa was determined by
visual scoring in the next 30–40 s and recorded (Table 1). N-9 was
used as reference control.
45.8, 45.2, 41.8, 36.0, 14.9; Anal. Calcd. for C9H14N4O2S2 (274): C,
39.40; H, 5.14; N, 20.42; Found: C, 39.66; H, 5.36; N, 20.66; Tartrate
salt mp 88–89 ꢀC.
3.1.10.5. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl dieth-
ylcarbamodithioate (23). Light yellow solid; Yield 70%; Mp 65–
66 ꢀC; IR (KBr): 2926, 1526, 1267, 739 cmꢂ1; ESI-MS: m/z 303
(Mþ þ 1); 1H NMR (CDCl3, 300 MHz,
d ppm): d 7.99 (s, 1H), 4.63 (t,
2H, j ¼ 6.9 Hz), 4.07–4.00 (m, 2H), 3.76–3.74 (m, 2H), 3.68 (t, 2H,
j ¼ 6.9 Hz), 2.57 (s, 3H), 1.28 (t, 6H, j ¼ 7.9 Hz); 13C NMR (75 MHz,
CDCl3, d ppm): 193.7, 151.4, 138.5, 133.4, 50.15, 47.18, 45.2, 35.6, 14.9,
12.8, 11.8; Anal. Calcd. for C11H18N4O2S2 (302): C, 43.69; H, 6.00; N,
18.53; Found: C, 43.71; H, 6.22, 18.66.
3.2.2. Anti-Trichomonas activity
T. vaginalis parasites to be used in drug susceptibility assays
were grown in TYM medium [31] supplemented with 10% Fetal Calf
Serum (FCS), vitamin mixture and 100 U/mL penicillin/strepto-
mycin mixtures, at 37 ꢀC in 15 mL tubes for one day following
regular subculturing, and were in the log phase of growth. The
cultures routinely attained a concentration of 2 ꢃ 107 cells/mL in
48 h. An inoculum of 1 ꢃ104 cells per tube was used for mainte-
nance of the culture. In vitro drug susceptibility assays were carried
3.1.10.6. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(pyridin-2-
yl)piperazine-1-carbodithioate (24). Yellow solid; Yield 65%; Mp
144–145 ꢀC; IR (KBr): 3021, 1597, 1522, 1216, 764 cmꢂ1; ESI-MS: m/z
d ppm): d 8.18–8.16 (m,
1H), 7.93 (s, 1H), 7.54–7.48 (m, 1H), 6.69–6.60 (m, 2H), 4.59 (t, 2H,
393 (Mþ þ 1); 1H NMR (300 MHz, CDCl3,
j ¼ 6.9 Hz), 4.54–4.32 (m, 2H), 4.13–3.93 (m, 2H), 3.73–3.68 (m, 6H),
2.55 (s, 3H); 13C NMR (75 MHz, CDCl3,
d ppm): d 194.8, 158.0, 151.0,
147.6, 138.2, 137.6, 133.1, 113.9, 106.8, 44.7, 44.1, 35.0, 14.5; Anal.
Calcd. for C16H20N6O2S2 (392): C, 48.96; H, 5.14; N, 21.41; Found;
C, 48.77; H, 5.38; N, 21.19; Tartrate salt hygroscopic.
out using the standard procedure [32]. Stock solutions (100 mg/mL)
of test compounds were prepared in DMSO. These stock solutions
were serially diluted with TYM medium to obtain concentrations
up to 0.1 mg/mL in 48-well plates. DMSO/TYM was used as vehicle
3.1.10.7. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(pyrimidin-
2-yl) piperazine-1-carbodithioate (25). Yellow solid; Yield 78%; Mp
179–180 ꢀC; IR (KBr): 3020, 1586, 1529, 1261, 761 cmꢂ1; ESI-MS: m/
in control wells. Parasites (5 ꢃ104 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 200ꢃ magnification. Viability of the cells was
determined by trypan blue exclusion assay. Minimum concentra-
tion of the test agent at which all cells were found dead in 48 h was
considered as its minimum inhibitory concentration (MIC).
Metronidazole (Sigma-Aldrich), the anti-trichomonas drug pres-
ently available in the market, was used as reference standard. The
experiment was repeated three times to confirm the MIC (Table 1).
z 394 (Mþ þ 1); 1H NMR (300 MHz, CDCl3,
d ppm): d 8.33–8.31 (m,
2H), 7.93 (s, 1H), 6.58–6.54 (m, 1H), 4.60 (t, 2H, j ¼ 6.9 Hz), 4.50–
4.31 (m, 2H), 4.19–3.94 (m, 6H), 3.72 (t, 2H, j ¼ 6.9 Hz), 2.56 (s, 3H);
13C NMR (75 MHz, CDCl3,
d ppm): d 195.3, 161.3, 158.0, 151.4, 138.6,
133.5, 111.0, 45.2, 43.1, 35.4, 14.9; Anal. Calcd. for C15H19N7O2S2
(393): C, 45.79; H, 4.87; N, 24.92; Found: C, 45.64; H, 4.84; N, 25.13.
3.1.10.8. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-(2-methox-
yphenyl)piperazine-1-carbodithioate (26). Yellow solid; Yield 70%;
Mp 131–132 ꢀC; IR (KBr): 3020,1594, 1530, 1216, 760 cmꢂ1; ESI-MS:
3.2.3. Antifungal activity
The MIC of compounds were determined [33] by broth micro-
dilution technique as per guide lines of National Committee for
Clinical Laboratory Standards using RPMI 1640 media buffered with
MOPS [3-(N-Morpholino)propanesulfonic acid]. Starting inoculums
of test culture was 1–5 ꢃ103 CFU/mL. Micro titer plates were
incubated at 35 ꢀC. MICs were recorded after 48 h of incubation
(Table 2).
m/z 422 (Mþ þ 1); 1H NMR (200 MHz, CDCl3,
d ppm): d 7.93 (s, 1H),
7.09–7.00 (m, 1H), 6.93–6.85 (m, 3H), 4.60 (t, 2H, j ¼ 6.9 Hz),
4.53–4.42 (m, 2H), 4.19–3.95 (m, 2H), 3.86 (s, 3H), 3.70 (t, 2H,
j ¼ 6.9 Hz), 325–3.04 (m, 4H), 2.56 (s, 3H); Anal. Calcd. for
C18H23N5O3S2 (421): C, 51.29; H, 5.50; N, 16.61; Found: C, 51.50; H,
5.33; N, 16.49; Tartrate salt mp 99–100 ꢀC.
3.1.10.9. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl
4-(4-nitro-
3.2.4. Cyto-toxicity assay using human cervical (HeLa) cell line
The cyto-toxicity of compounds were assessed [12] against
human cervical (HeLa) cell line using the MTT (3-[4,5-dimethyl
thiazol-2-yl]-2,5-diphenyltetrazolium bromide) based colorimetric
assay. HeLa cells (procured from National Centre for Cell Science,
Pune, India) seeded at a density of 5.0 ꢃ 104 per well in 96 well
plates were incubated in culture medium (DMEM with 10% FCS) for
24 h at 37 ꢀC in 5%CO2/95% air atmosphere. After 24 h, culture
medium was replaced with fresh medium containing dilutions of
phenyl)piperazine-1-carbodithioate (27). Orange solid; Yield 75%;
Mp 199–200 ꢀC; IR (KBr): 3114, 2919, 1595, 1318, 1254, 745 cmꢂ1
;
ESI-MS: m/z 437 (Mþ þ 1); 1H NMR (300 MHz, TFA-d,
d ppm): d 8.04
(d, 2H, j ¼ 8.6 Hz), 7.82 (s, 1H), 7.48 (d, 2H, j ¼ 8.7 Hz), 4.54 (t, 2H,
j ¼ 6.2 Hz), 4.40–4.21 (m, 4H), 3.55–3.48 (m, 6H), 2.52 (s, 3H); Anal.
Calcd. for C17H20N6O4S2 (436): C, 46.78; H, 4.62; N, 19.25; Found: C,
46.61; H, 4.75; N, 19.06.
3.1.10.10. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 4-methyl-
piperazine-1-carbodithioate (28). Brown solid; Yield 65%; Mp
105–106 ꢀC; IR (KBr): 3020, 1531, 1217, 761 cmꢂ1; ESI-MS: m/z 330
test compounds (0–200
m
g/mL). Vehicle (10% DMSO) was added to
0
m
g/mL (control) wells. After incubation for another 24 h, 10 m
L of
MTT solution (5 mg/mL in PBS, pH 7.4) was added to each well. The
formazan crystals formed inside the viable cells were solubilized in
DMSO and the OD was recorded at 540 nm in a microplate reader
(Microquant, Bio-Tek, USA). Cyto-toxicity at the highest concen-
(Mþ þ 1); 1H NMR (300 MHz, CDCl3,
d ppm): d 7.89 (s,1H), 4.61–4.49
(m, 2H), 4.45–4.24 (m, 2H), 4.04–3.82 (m, 2H), 3.64 (t, 2H, 6.9 Hz),
2.53–246 (m, 7H), 2.34(s, 3H). Anal. Calcd. for C12H19N5O2S2 (328): C,
43.75; H, 5.81; N, 21.26; Found: C, 43.66; H, 5.65; N, 21.32.
tration tested (200 mg/mL) has been displayed in Fig. 2.

824
L. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 817–824
[14] A.K. Dwivedi, V.L. Sharma, N. Kumaria, S.T.V.S. Kiran Kumar, P.K. Srivastava,
Acknowledgements
H. Ansari, J.P. Maikhuri, G. Gupta, J.D. Dhar, R. Roy, B.S. Joshi, P.K. Shukla,
M. Kumar, S. Singh, Bioorg. Med. Chem. 15 (2007) 6642–6648.
[15] V. Lakshmi, A. Saxena, S.K. Mishra, R. Raghubir, M.N. Srivastava, R.K. Jain,
J.P. Maikhuri, G. Gupta, Arch. Med. Res. 39 (2008) 631–638.
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M. Kumar, P.K. Shukla, G. Gupta, Eur. J. Med. Chem. 43 (2008) 2247–2256.
[17] S. Solanki, W.D. Potter, L. Anderson, US Patent 2005131238 (2005).
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Authors are thankful to Mrs. Tara Rawat for her technical
assistance. L.K. and N.L. are thankful to the University Grants
Commission, New Delhi, A.S. and A.K. is thankful to the Council of
Scientific and Industrial Research, New Delhi, A.J. and R.K. are
thankful to the Indian Council of Medical Research, New Delhi for
the grant of research fellowships. We are also grateful to the staff
members of SAIF Division, CDRI, for spectral data.
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