Potentiating metronidazole scaffold against resistant Trichomonas: Design, synthesis, biology and 3D-QSAR analysis

Article abstract of DOI:10.1021/ml200161t

Metronidazole (MTZ), the FDA-approved drug against Trichomonas vaginalis (TV), is being challenged seriously by drug resistance, while its inertness to sperm makes it ineffective as a vaginal contraceptive. Thirteen piperidine dithiocarbamate hybrids of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethane (8-20) were designed to potentiate the MTZ framework against drug resistance and sperm. New compounds were 1.2-12.1 times more effective against MTZ-susceptible and -resistant strains of TV. All of the compounds exhibited high safety toward cervical (HeLa) cells and Lactobacillus. Thirty-eight compounds were scrutinized by CoMFA and CoMSIA techniques of 3D quantitative structure-activity relationship. Good predictive r_pred² values for CoMFA and CoMSIA models reflected the robustness of the predictive ability. This was validated by designing five new analogues (46-50), which were potently microbicidal (3-10 and 10-20 times against MTZ-susceptible and -resistant TV, respectively) and spermicidal. This in vitro study may have significant clinical relevance, which could become evident in due course.

Full text of DOI:10.1021/ml200161t

Letter
pubs.acs.org/acsmedchemlett
Potentiating Metronidazole Scaffold against Resistant Trichomonas:
Design, Synthesis, Biology and 3D−QSAR Analysis†
Lalit Kumar,† Ashish Jain,‡ Nand Lal,† Amit Sarswat,† Santosh Jangir,† Lokesh Kumar,‡ Vishal Singh,‡
Priyanka Shah,§ Swatantra K. Jain,∥ Jagdamba P. Maikhuri,‡ Mohammad I. Siddiqi,§ Gopal Gupta,‡ and
Vishnu L. Sharma*,†
‡
§
†Medicinal & Process Chemistry Division, Endocrinology Division, and Molecular & Structural Biology Division,
CSIR-Central Drug Research Institute, Lucknow-226001, India
∥Department of Biotechnology, Jamia Hamdard University, New Delhi-110062, India
S
* Supporting Information
ABSTRACT: Metronidazole (MTZ), the FDA-approved drug against Trichomonas vaginalis
(TV), is being challenged seriously by drug resistance, while its inertness to sperm makes it
ineffective as a vaginal contraceptive. Thirteen piperidine dithiocarbamate hybrids of 2-(2-
methyl-5-nitro-1H-imidazol-1-yl)ethane (8−20) were designed to potentiate the MTZ
framework against drug resistance and sperm. New compounds were 1.2−12.1 times more
effective against MTZ-susceptible and -resistant strains of TV. All of the compounds
exhibited high safety toward cervical (HeLa) cells and Lactobacillus. Thirty-eight compounds
were scrutinized by CoMFA and CoMSIA techniques of 3D quantitative structure−activity
relationship. Good predictive rpred2 values for CoMFA and CoMSIA models reflected the robustness of the predictive ability. This
was validated by designing five new analogues (46−50), which were potently microbicidal (3−10 and 10−20 times against MTZ-
susceptible and -resistant TV, respectively) and spermicidal. This in vitro study may have significant clinical relevance, which
could become evident in due course.
KEYWORDS: dithiocarbamate, metronidazole, piperidine, antitrichomonas, microbicidal, contraception
iseases like sexually transmitted infections (STIs)1 are
2
Dinsidious and diabolic and a global urgency that cannot
be overlooked. Each year, 340 million new cases of curable STIs
are reported, among which trichomoniasis has the highest
incidence, and the infected populations are at an elevated risk
of contracting viral sexually transmitted diseases (STDs) in-
cluding acquired immunodeficiency syndrome (AIDS).3 On the
other hand, of 208 million pregnancies that occurred globally in
2008, ∼41% were unintended,4 which indicates that contra-
ception remains an unmet need for many. As a direct con-
sequence of this, ∼46 million abortions are performed annually,
mostly in developing countries under unsafe conditions that
result in the death of ∼70 000 women each year.5 MTZ, the
FDA-approved drug6 against trichomoniasis, is nonspermicidal
and hence cannot offer pregnancy protection when used
vaginally. On the other hand, the emergence of resistance
against MTZ has made the situation grimmer. It would be
highly desirable to arrest Trichomonas along with sperm in the
vagina as soon as it is transmitted in semen; therefore, there is a
need to develop topically active medication against sperm and
Trichomonas vaginalis (TV) (MTZ also has a poor cure rate
when used vaginally7). The hybridization of two active ligands
leading to a novel class of molecules with a better biological
activity profile has been an approach in medicinal chemistry8,9
whereby a single chemical entity can be made to modulate
multiple targets simultaneously.10 Thus, it was thought worth-
while to modify the structure of MTZ (1, Figure 1) without
Figure 1. Structures of MTZ (1), dithiocarbamates (2), lead molecule
of previous work (3), and general structures of the synthesized
compounds (4−6).
altering the orientation of its active sites (e.g., the 5-nitro group,
which on being reduced to a nitro radical within the TV
parasite exhibits trichomonacidal activity11). The antimicro-
bial activity12 of dithiocarbamates (2, Figure 1) is well docu-
mented, and its incorporation into other chemical moieties has
resulted in compounds with appreciable antitrichomonas
activity (ATA).13−15 On the other hand, the metabolism11 of
MTZ into 1-acetic acid metabolite results in loss of activity.
Received: August 25, 2011
Accepted: December 16, 2011
Published: December 16, 2011
© 2011 American Chemical Society
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Letter
a
Scheme 1
a
Reagents and conditions: (a) Imidazole, (C6H5)3P, I2, toluene, reflux, 4 h. (b) Substituted piperidine, CS2, neat, 3−5 h. (c) Piperidin-4-one
monohydrate hydrochloride, Na2CO3, CS2, CH3CN, 0−5 °C to room temperature, 5 h. (d) NaBH4, MeOH, 0−5 °C, 2 h. (e) ClCOCHCl2/
ClSO2CH3/ ClCOCH2Cl, CH2Cl2, (C2H5)3N, 0−5 °C. (f) HNR1R2, (C2H5)3N, toluene, reflux. (g) NaSCSNR1R2, H2O−MeOH, 60 °C.
Therefore, to prevent this, compounds were synthesized by
substituting the hydroxyl group of ethyl side chain in MTZ
scaffold with various dithiocarbamates containing 4-substituted
piperidine moiety, as reported by us earlier (3, Figure 1).14
Various substitutions in piperidine moiety were explored (4 and 5;
Figure 1) to improve the lipophilicity (and hence bioavailability in
target cells through enhanced cell permeability), and an additional
dithiocarbamate group was introduced to augment the biologi-
cal activity further (6, Figure 1). Dithiocarbamates are capable
of reversing the MTZ resistance16 through their antioxidant
property17 and exhibit potent spermicidal activity through inter-
action with sulfhydryl groups present over sperm.18 Spermicidal
property could be considered as a highly complementary effect
since TV is transmitted along with spermatozoa that cause
unwanted pregnancies. The normal human vagina is naturally
protected against STDs by its low pH, which is growth inhibi-
tory for several pathogenic organisms, including Trichomonas.19
Infections normally occur when the vaginal pH is dis-
turbed, especially during the deposition of alkaline semen con-
taining sperm and STD pathogen(s); therefore, agents capable
of inactivating both are highly desirable. The new compounds
were evaluated in vitro against susceptible as well as MTZ-
resistant strains of TV, and their structure−activity relationship
(SAR) was studied by three-dimensional quantitative structure−
activity relationship (3D QSAR). The comparative molecular
field analysis (CoMFA) and comparative molecular similarity
indices analysis (CoMSIA) techniques were used by fitting
the parameters of the model to the activity data. To validate
our proposed models and the predictions done by QSAR, new
molecules were designed and evaluated for their biological
activities.
Because the synthesized compounds are intended to be used
vaginally and dually active agents are the need of the present
time,20 these compounds were also evaluated for spermicidal
effect against human sperm and for safety toward cervico-
vaginal epithelium and microflora.
Compounds 8−11 (Scheme 1) were synthesized in one pot
directly from iodo derivative (7), substituted piperidine, and
carbon disulfide under solvent free condition by reported
method.21 Similarly, compound 12 was prepared directly from
7, piperidone monohydrate hydrochloride, sodium carbonate,
and carbon disulfide in an acetonitrile−water mixture (2:1),
which was then reduced by using sodium borohydride to
hydroxyl compound 13. Compound 13 was dichloroacetylated,
mesylated, and chloroacetylated to provide compounds 14,
15, and 16, respectively, by using usual reaction conditions.
Substitution of chloro group of 16 by cyclic amine in toluene
provided compounds 17 and 18 (Scheme 1),15 whereas
substitution by sodium salt of carbodithioic acid in methanol−
water mixture (1:1) at 60 °C gave compounds 19 and 20 in
good yield.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl-4-substituted pi-
peridine-1-carbodithioates (8−20) were found to be active
against MTZ-susceptible as well as MTZ-resistant strains of
TV (Table 1). Eleven compounds (8, 10−15, and 17−20)
were 3.6−8.6 times more active as compared to MTZ, while
compound 9 was equipotent and 16 was 4 times less active
against susceptible strain of TV. On the other hand, all of the
13 compounds showed 1.2−12.1 times more efficacy against
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ACS Medicinal Chemistry Letters
Letter
along with 25 compounds (21−45) reported14 earlier by us
were investigated by 3D-QSAR techniques.
Table 1. In Vitro Antitrichomonas, Spermicidal Activities,
and Safety Data of the Compounds 8−20
In the present QSAR study, CoMFA and CoMSIA models
were derived from the 34 ligands in the training set (Table S1
in the Supporting Information). In comparison to CoMFA, the
CoMSIA methodology provides a more detailed three-
dimensional information in terms of hydrophobic and hydro-
gen bond interactions. Using CoMSIA analysis, we achieved
comparatively good correlation for combination of four mole-
cular descriptors: steric, electrostatic, hydrophobic, and hydro-
gen bond acceptor fields. Cross-validated coefficient (leave-
one-out) q2 of 0.692 with five optimal number of component
and 0.603 with four optimal number of component and
noncross-validated r2 of 0.925 with standard error of estimate
(SEE) of 0.148 and 0.882 with SEE of 0.183 were obtained for
CoMFA and CoMSIA models, respectively. Good predictive
MIC μmol
IC50 (μg/mL)
c
SI
cytotoxicity
against
HeLa
compatibility
a
b
ATA
ATA
(at 1%)
(%)
with
d
compd [susceptible] [resistant]
Lactobacillus
8
3.24
12.4
64.8
49.5
24.1
26.9
30.5
30.3
56.7
61.3
100
100
510
>1000
>1000
>1000
>1000
930
>1000
>1000
>1000
>1000
380
9
10
11
12
13
14
15
16
17
18
19
20
MTZ
N-9
1.68
1.90
1.89
1.41
1.53
<100
<100
<100
100
350
100
>1000
>1000
930
740
12.4
49.3
1.37
100
>1000
590
246
100
54.9
54.7
96.7
37.7
<100
<100
<100
<100
<100
100
760
>1000
>1000
>1000
>1000
>1000
33.0
2
1.36
2.41
2.35
840
rpred values for the test set (Table S2 in the Supporting
>1000
980
Information) of 0.679 and 0.732 for CoMFA and CoMSIA
models, respectively, reflect the predictive ability of CoMFA
and CoMSIA models. The statistical results are summarized in
Table S3 in the Supporting Information. Figure S2 (Supporting
Information) shows the plot of experimental versus predicted
values for CoMFA and CoMSIA models. These results indicate
that 3D QSAR models could be used reliably to design new
antitrichomonas agents. Figure S3a,b (Supporting Information)
obtained from CoMFA and CoMSIA models is comparable in
terms of steric and electrostatic requirements. It seems from
Figure S3a in the Supporting Information that the presence of
an optimal size of the steric group around piperidine ring plays
an important role in ATA. Although CoMFA and CoMSIA
models disfavor the presence of more bulky groups around the
piperidine ring after a certain distance, some flexible molecules
with bulkier groups like 11, 17, 18, 33, and 34 are found to be
very active, as these tend to fold into a smaller size during the
energy minimization. Figure S3c (Supporting Information)
obtained from CoMSIA contour analyses provides additional
information in terms of the effect of hydrophobic and hydrogen
bond acceptor substituents on ATA for compound 38.
11.7
292
>1000
33.0
a
ATA, antitrichomonas activity against MTZ-susceptible clinical
b
isolate of T. vaginalis. ATA, antitrichomonas activity against MTZ-
c
resistant (ATCC 50143) strain of T. vaginalis. SI, sperm immo-
bilization (normal human spermatozoa). Lactobacillus jensenii (ATCC
d
25258).
the MTZ-resistant strain of TV. Interestingly, the least active
compound (16) was comparable to MTZ in its activity against
the resistant strain. Six compounds (8, 9, and 13−16) com-
pletely immobilized human sperm at 1% concentration, while
the rest were inactive (Table 1). The results of spermi-
cidal activity indicated that a tetrahedral carbon at position 4 of
piperidine with smaller groups (8, 9, and 13−16) was preferred
over a longer chain at position 4 of the piperidine moiety
(10, 11, and 17−20) and trigonal carbon (12). All of the
compounds exhibited a high safety profile in comparison to
nonoxynol-9 (N-9, the OTC spermicide for vaginal applica-
tion) toward both HeLa cells and Lactobacillus with IC50 values
ranging from 350 to >1000 μg/mL (Table 1). MTZ is reported
to inhibit Lactobacillus growth partially at 1000−4000 μg/mL
and totally at ≥5000 μg/mL but was found to have no effect at
concentrations of 500−1000 μg/mL.22
On the basis of the structural guidelines derived from
CoMFA and CoMSIA models and their contour maps, a set of
five compounds (46−50) were designed, and their activities
were predicted (Table 2). We synthesized compounds (46−50)
having methyl and ethyl groups as substituents around the
piperidine ring due to less bulkyness nature of these group,
according to Scheme 1.
With a view to further optimize the biological activities, these
nitroimidazolyl-4-substituted piperidine-1-carbodithioates (8−20)
Table 2. Predicted and Experimental ATA by CoMFA and CoMSIA and in vitro Antitrichomonas, Spermicidal Activities, and
Safety Data of the Compounds 46−50
a
PA [susceptible]
MIC μmol
IC50 (μg/mL)
b
c
d
e
EA
ATA
ATA
SI (at 1.0%)
cytotoxicity against
HeLa
compatibility with
f
compd CoMFA CoMSIA
[susceptible]
[susceptible]
[resistant]
(%)
Lactobacillus
46
5.752
5.716
5.623
6.839
6.721
5.870
5.719
5.615
5.905
5.895
5.72
5.72
5.99
5.44
5.74
1.90
1.90
1.03
3.65
1.83
11.7
15.2
15.2
14.3
14.6
29.2
292
100
100
170
170
>1000
>1000
>1000
>1000
>1000
>1000
33.0
47
48
100
190
49
100
94.0
130
50
100
MTZ
N-9
<100
100
>1000
33.0
a
b
c
PA, predicted ATA. EA, experimental ATA against susceptible strain of TV. ATA, antitrichomonas activity against MTZ-susceptible strain of TV.
d
e
f
ATA, antitrichomonas activity against MTZ-resistant (ATCC 50143) strain of TV. SI, sperm immobilization. Lactobacillus jensenii (ATCC
25258).
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ACS Medicinal Chemistry Letters
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The biological assay demonstrated that the newly designed
compounds possessed potent ATA comparable to the predicted
ATA (Table 2). In comparison to the standard drug MTZ, the
activity increased by 3−10-fold against susceptible strain and
10−20 times against the resistant strain. All of these com-
pounds (46−50) irreversibly immobilized the human sperm at
1% concentration and exhibited a high degree of safety toward
cervical epithelium (HeLa) and normal vaginal microflora
(Lactobacillus). Among them, 2-(2-methyl-5-nitro-1H-imidazol-
1-yl)ethyl-4-methylpiperidine-1-carbodithioate (48) was the
most active compound and that was 10 and 20 times more
potent than MTZ against susceptible and resistant strains of
TV, respectively. It also irreversibly immobilized 100% human
sperm at 1% (MEC) in ∼30 s and exhibited a high degree of
safety against HeLa cells and Lactobacillus.
ASSOCIATED CONTENT
* Supporting Information
■
S
Tables of training, test set data, and statistical results; plot of
experimental vs predicted values for CoMFA and CoMSIA
models; contour analysis from CoMFA and CoMSIA models;
1
experimental procedure; H, 13C NMR; and HRMS spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 91-522-2612411, ext. 4320. Fax: 91-522-2623405. E-mail:
vlscdri1@rediffmail.com or vlscdri@gmail.com.
■
Funding
We acknowledge UGC (L.K., N.L., and L.K.), CSIR (A.S., S.J.,
and P.S.), and ICMR (A.J.) for fellowships. This study was
partially supported by a grant from the Ministry of Health and
Family Welfare, Government of India.
The hybridization of MTZ framework with dithiocarba-
mate group led to the design of nitroimidazolyl-4-substituted
piperidine-1-carbodithioates (8−20) with highly enhanced
ATA that could defeat the infection's resistance to MTZ and
complementary spermicidal activity. The 3D QSAR analysis of
these compounds along with 25 compounds (21−45) reported
Notes
The authors declare no competing financial interest.
†C.DRI Communication No. [8176].
earlier14 by means of CoMFA and CoMSIA models and their
2
contour maps was found to have good predictive abilities, rpred
=
ACKNOWLEDGMENTS
We acknowledge Tara Rawat (Technical Officer) and SAIF
Division, for spectral data.
■
0.679 and 0.732 obtained from CoMFA and CoMSIA,
respectively. 3D QSAR models could describe the steric, elec-
trostatic, hydrophobic, and hydrogen bond acceptor require-
ments for the compounds to be active against trichomonas
species. Analyses reveal that steric field at the position around
the piperidine ring has a major influence on the ATA. A yellow
contour, present nearby this group on the other side of the
green contour, recommends not putting sterically increas-
ing bulk in this direction (Figure S3a,b in the Supporting
Information). The QSAR analysis resulted in the design of five
new compounds (46−50) with highly improved ATA without
forfeiting the complementary spermicidal activity. The
augmented microbicidal activity and desirable sperm immobi-
lizing capacity of these nitroimidazolyl carbodithioates (8−20
and 46−50) might be attributable to their additional capability
to interact18 with free thiol groups, which play an impor-
tant role in the survival of predominantly anaerobic cells like
spermatozoa23 and TV.24 The nucleophilic character of
the sulfur atom and the unique redox properties of the thiol
group make it a key residue for enzyme catalysis, protein
folding, and redox signaling and regulation,25 which are
important for cellular energy metabolism, motility, and
subsistence. The undoing of MTZ resistance (Table 2)
may again be attributed to the redox properties of dithio-
carbamate group17 since the reduction26,27 of the nitro
group of MTZ to cytotoxic nitro radical is inhibited in
resistant TV.28,29
This study highlights a novel method for potentiating the
MTZ framework against susceptible and resistant TV strains.
The incorporation of spermicidal activity into these improved
drug candidates may have serious implications in their possible
use as topical prophylactic contraceptives. The good statistical
correlation indicated that 3D QSAR analysis along with contour
mapping could reasonably be extended to the design of novel
lead compounds incorporating essential pharmacophores that
are closely associated with desired biological responses. Fur-
ther lead optimization may result into a potent dual active
microbicidal spermicide.
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