Novel metronidazole-chalcone conjugates with potential to counter drug resistance in Trichomonas vaginalis

Article abstract of DOI:10.1016/j.ejmech.2014.03.076

Trichomoniasis is the most prevalent, curable sexually transmitted disease (STD), which increases risk of viral STDs and HIV. However, drug resistance has been developed by some strains of Trichomonas vaginalis against Metronidazole (MTZ), the FDA approve

Full text of DOI:10.1016/j.ejmech.2014.03.076

Accepted Manuscript
Novel metronidazole-chalcone conjugates with potential to counter drug resistance in
Trichomonas vaginalis
Amit Anthwal, U. Chinna Rajesh, M.S.M. Rawat, Bhavana Kushwaha, Jagdamba P.
Maikhuri, Vishnu L. Sharma, Gopal Gupta, Diwan S. Rawat
PII:
S0223-5234(14)00290-6
10.1016/j.ejmech.2014.03.076
EJMECH 6860
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 December 2013
Revised Date: 24 March 2014
Accepted Date: 27 March 2014
Please cite this article as: A. Anthwal, U.C. Rajesh, M.S.M. Rawat, B. Kushwaha, J.P Maikhuri, V.L.
Sharma, G. Gupta, D.S. Rawat, Novel metronidazole-chalcone conjugates with potential to counter drug
resistance in Trichomonas vaginalis, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.03.076.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Novel metronidazole-chalcone conjugates with potential to counter drug resistance in
Trichomonas vaginalis.
a, b
b
a,*
c
Amit Anthwal , U. Chinna Rajesh , M. S. M. Rawat Bhavana Kushwaha , Jagdamba P
,
c
d
c
b,*
Maikhuri , Vishnu L. Sharma , Gopal Gupta , Diwan S. Rawat
a
Department of Chemistry, H.N.B. Garhwal University (A Central University), Srinagar
(Garhwal), Uttarakhand-246174, India.
b
Department of Chemistry, University of Delhi, Delhi-110007, India.
c
d
Division of Endocrinology and Division of Medicinal & Process Chemistry, CSIR-Central
Drug Research Institute, Lucknow–226031, India.
Corresponding authors. Tel:+91 9411104325; E-mail address: msmrawat@gmail.com (M.S.M. Rawat) and Tel:
+
91-11-27662683; *E-mail: dsrawat@chemistry.du.ac.in
Abstract: Trichomoniasis is the most prevalent, curable sexually transmitted disease (STD),
which increases risk of viral STDs and HIV. However, drug resistance has been developed by
some strains of Trichomonas vaginalis against Metronidazole (MTZ), the FDA approved
drug against trichomoniasis. In the present study twenty two chalcone hybrids of
metronidazole have been synthesized in a quest to get new molecules with higher potential
against metronidazole-resistant Trichomonas vaginalis. All new hybrid molecules were found
active against Trichomonas vaginalis with varying levels of activity against MTZ-susceptible
and resistant strains. Eight compounds (4a, 4c, 4d, 4e, 4f, 4h, 4q and 4s) were found as
active as the standard drug with an MIC of 1.56 µg/ml against MTZ-susceptible strain.
However, compounds 4e, 4h and 4m were 4-times more active than MTZ against drug-
resistant Trichomonas vaginalis, amongst which 4e and 4h were most promising against both
susceptible and resistant strains.
Keywords: Trichomonas vaginalis, Metronidazole, Drug Resistance, HeLa cell line.
1

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1
. Introduction
Sexually Transmitted diseases have long been a big threat to mankind in developing as well
as developed countries [1-3]. Nearly 340 million new cases of curable STI’s are reported
every year world-wide and in which trichomoniasis is a major contributor. Metronidazole
(MTZ) is a drug of choice for trichomoniasis, which is also approved by the US-FDA [4].
However, the situation has been worsened by the resistance developed by microbes against
MTZ. The Trichomonas vaginalis infection damages the vaginal epithelium, which increases
the risk of women getting infected by HIV and chances of infected women transmitting HIV
to her sexual partner(s) are also increased considerably [5,6]. The Concept of hybrid
molecules is a rational approach to overcome the problem of drug resistance by introducing a
new pharmacophore in a biologically active molecule to get new molecule of therapeutic
importance [7,8]. In a quest to synthesize new hybrid molecules with better anti-trichomonas
activity, especially against the resistant strain, we planned to modify the metronidazole
framework at hydroxyl group. The rationale for selecting hydroxyl group was that due to its
presence MTZ metabolizes into 1-acetic acid metabolite, which results in loss of activity [9].
Many research groups have attempted to modify MTZ at hydroxyl group in order to get a
molecule with enhanced activity against Trichomonas vaginalis [10-14]. The chalcone is
another widely studied pharmacophore with wide range of biological activities. The
chalcones exhibit anticancer [15,16], anti-inflamatory [17,18], anti-oxidant [19,20] and
antimicrobial [21] activities. As a part of our ongoing research towards development of
biologically important molecules, we designed novel metronidazole-chalcone conjugates and
anticipated that if such an active pharmacophore is attached to metronidazole the resulting
hybrid molecules are likely to be more active especially against MTZ-resistant Trichomonas.,
we herein report synthesis and anti-trichomonas activity of 22 novel metronidazole-chalcone
conjugates. The structure of MTZ was modified at hydroxyl group to attach chalcone moiety
by covalent bond (figure1, scheme 1). The compounds (4a-4v) were synthesized by varying
substituents on aromatic ring of chalcone moiety; these compounds were screened for anti-
trichomonas activity against susceptible and resistant strains of Trichomonas vaginalis. The
safety of the most promising molecules was evaluated against human cervical (HeLa) cell
line.
2
. Results and discussion
.1. Chemistry
2
2

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The novel metronidazole-chalcone conjugates were synthesized by multistep synthetic
protocol. In first step hydroxyl group of MTZ was tosylated with toluenesulphonyl-chloride
using pyridine as base and also as solvent to get 2-(2-methyl-5-nitro-1H-imidazol-1-
yl)ethyl4-methylbenzenesulfonate 1. Compound 1 on reaction with 4-hydroxyacetophenone
in presence of base K CO and DMF solvent yielded 1-(4-(2-(2-methyl-5-nitro-1H-imidazol-
2
3
1
-yl)ethoxy)phenyl)ethanone 3. Furthermore, compound 3 was reacted with various
substituted aldehydes in presence of base NaOH and methanol solvent to yield desired hybrid
molecules (4a-4v) in good yield as shown in scheme 1.
2
.2. Biology
All the chalcone-MTZ hybrids (4a-4v) exhibited anti-trichomonas activity against both the
MTZ susceptible (MIC 1.56 – 25µg/ml) and resistant (MIC 3.125 – 100 µg/ml) strains
whereas MTZ itself inhibited the growth of susceptible and resistant strain of T. vaginalis at
MIC of 1.56 and 12.50 µg/ml respectively (Table 1).
The effect of various electron withdrawing (4b-4q) and electron donating (4r-4u)
substituents in the aromatic ring of chalcone was studied. 1-{4-[2-(2-Methyl-5-nitro-
imidazol-1-yl)-ethoxy]-phenyl}-3-phenyl-propenone (4a), having unsubstituted aromatic ring
was as active as MTZ against the MTZ susceptible strain of T. vaginalis but four times less
active against resistant strain. The activity data against MTZ susceptible strain suggested that
an introduction of electron withdrawing (4b-4q), donating (4r-4u) or an additional phenyl
(4v) substituent in aromatic ring had marginal effect as seven compounds (4c-4f, 4h, 4q, 4s)
retained the activity while others were less active. As expected, all the compounds had higher
MIC values (2 – 16 times) against resistant T. vaginalis like MTZ (8 times). The substituents
in the aromatic ring played a significant role in exhibiting trichomonacidal activity against
resistant strain. Compound 4a having unsubstituted aromatic ring was found to be four times
less active (MIC 50 µg/ml) than MTZ against resistant T. vaginalis (MIC 12.50 µg/ml) while
the presence of a substituent enhanced the activity in eleven compounds, by two times (4c,4d,
4
f, 4i, 4l, 4n, 4q, 4t) and four times (4e, 4h, 4m). Whereas four compounds were equipotent
(4g, 4j, 4s, 4u) to MTZ and the activity decreased in six compounds (4b, 4k, 4o, 4p, 4r, 4v).
The electron withdrawing substituents seems to be more desirable for trichomonacidal
activity against resistant strain as compared to electron donating substituents as out of sixteen
compounds thirteen have been found to be more potent than MTZ. Among fluoro substituted
hybrids (4b-4f), 3,5-disubstitution (4e) enhanced the activity while in chloro (4g-4k) and
3

ACCEPTED MANUSCRIPT
bromo (4l-4n) compounds 3-substitution (4h, 4m) was most important. A trifluoromethyl
(
CF ) substitution in aromatic ring (4o, 4p) increased the activity as compared to compound
3
having unsubstituted aromatic ring (4a) but remained less active with respect to MTZ. 4-
Nitro substitution (4q) also increased the activity in comparison to 4a and MTZ by eight and
two folds respectively. Compounds with electron donating substituents (4r-4u) were more
active than 4a but only 3,5- dimethoxy compound (4t) was better than MTZ. A replacement
of aromatic ring with naphthyl ring (4v) was also not fruitful.
The hybridization of MTZ and chalcone scaffolds resulted into enhancement of the activity of
MTZ against resistant strain. Three compounds (4e, 4h, 4m) were four times more potent
than MTZ. The presence of an electron withdrawing substituent in aromatic ring was
detrimental for anti-trichomonas activity of these hybrid molecules. The activity of these
chalcone-MTZ hybrids may be attributed to the nitro group of MTZ scaffold [22] and the
alkylation of sulfhydryl groups[23] present over T. vaginalis via α,ß-unsaturated ketone
group of chalcone moiety as reported[24] for protein SH-groups being specifically modified
by α,ß-unsaturated compounds. Thus, it may be concluded that incorporation of chalcone
moiety in MTZ scaffold may overcome the MTZ resistance. However, the present study has
identified two very promising structures (4e, 4h) that could overcome the MTZ-resistance of
Trichomonas vaginalis by 4-times, without forfeiting activity against MTZ-susceptible strain
(Table-1) and safety against HeLa (Table-2), in vitro. The lead optimization of these might
lead to the identification of a candidate drug capable of overcoming the MTZ resistance of
Trichomonas.
3
. Experimental section
.1. Chemistry
3
All the chemicals used in synthesis were purchased from Sigma-Aldrich and were used as
such. Thin layer chromatography (Merck TLC silica gel 60 F ) was used to monitor the
2
54
progress of reactions. The compounds were purified when needed by silica gel column (60-
20 mesh). Melting points were determined on EZ-Melt automated melting point apparatus,
Stanford Research systems and are uncorrected. IR (chloroform/film) spectra were recorded
1
-1
using Perkin-Elmer FT-IR spectrophotometer and values are expressed as ʋmax cm . Mass
4

ACCEPTED MANUSCRIPT
spectral data were recorded in Thermo Finnigan LCQ Advantage max ion trap mass
1
13
spectrometer /data system. The H NMR and C NMR spectra were recorded on Jeol ECX
spectrospin at 400 MHz and 100 MHz respectively, in deuterated solvents with TMS as an
internal standard. Chemical shift values are recorded on δ ppm and the coupling constants J
are in Hz.
3
.1.1. 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl-4-methylbenzenesulfonate (1)
To a stirred solution of 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol (MTZ) (5g, 0.029 mol)
in anhydrous pyridine (75mL), para-toluenesulfonylchloride (11.13g, 0.0584 mol) was added
and the reaction mixture was stirred at room temperature for 12 hours. The solid precipitated
was filtered and crystallized from ethanol to give shining needle like crystals of compound 1
o
(
scheme 1). Yield 85% (pale yellow solid); mp 151 C; IR: 1596, 1526, 1460, 1430, 1366,
1
1
174, 898; H NMR (400 MHz, CDCl ): δ 2.45 (s, 3H, CH ), 2.52 (s, 3H, -CH ), 4.37 (t, 2H,
3
3
3
-NCH ), 4.54 (t, 2H, -OCH ), 7.30 (d, 2H, J=8.3 Hz, ArH), 7.60 (d, 2H, J=8.3 Hz, ArH), 7.79
2 2
1
3
(
s, 1H); C NMR (100 MHz, CDCl ): δ 13.91, 21.01, 44.61, 68.38, 127.21, 129.24, 131.50,
3
1
33.04, 138.03, 145.22, 151.57.
3
.1.2. 1-(4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)phenyl)ethanone (3)
To
a
stirred
solution
of
2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl-4-
methylbenzenesulfonate (1) (5g, 0.0153 mol) in DMF (50 mL) was added 4-
hydroxyacetophenone (2)(2.30g, 0.0169 mol) and reaction mixture was stirred overnight (12
o
hours) at room temperature (35-40 C). After completion of reaction, the solid was
precipitated by addition of water to reaction mixture. The precipitate was washed thoroughly
with water to remove DMF and other impurities. The product was recrystallized from
1
ethanol. Yield 74% (Pale brown solid); H NMR (400 MHz, CDCl ): δ 2.55 (s, 3H, CH ),
3
3
2
.63 (s, 3H, CH ), 4.39 (t, 2H, J=4.76 Hz, NCH ), 4.75 (t, 2H, J=4.76 Hz, OCH ), 6.85 (d,
3 2 2
2
H, J=9.52 Hz, ArH), 7.91 (d, 2H, J=9.52 Hz, ArH), 7.98 (s, 1H).
5

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3
.1.3. General procedure for synthesis of hybrid molecules (4a-4v):
To a stirred solution of 1-(4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)phenyl)ethanone
3) (200 mg, 0.6 mmol) in 10mL methanol corresponding substituted benzaldehyde /
(
napthaldehyde (1.1equiv.) was added , followed by addition of NaOH (4 equiv.) and reaction
mixture was stirred at room temperature for 3-4 hours. After completion of reaction
monitored by T.L.C., the product precipitated was filtered, washed with water. The crude
product obtained was recrystallized from methanol to get pure product (4a-4v) in good yield
(scheme 1).
3
.1.3.1. 1-{4-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-3-phenyl-propenone
-1
(
4a): Yield 68% (pale yellow solid); mp 185-186; IR (CHCl , cm ): 1658, 1602, 1468, 1362,
3
1
1
256, 1220,1179, 833, 764; H NMR (400 MHz, CDCl ): δ 2.65 (s, 3H), 4.26 (t, 2H, J=5.13
3
Hz), 4.76 (t, 2H, J=5.13 Hz), 6.91 (d, 2H, J=8.79 Hz), 7.41-7.43 (m, 3H), 7.51 (d, 1H,
J=16.11 Hz), 7.63-7.65 (m, 2H), 7.80 (d, 1H, J=16.11 Hz), 7.99 (d, 2H, J=5.86 Hz), 8.02 (s,
+
1
H); ESI-MS m/z 378.14 [M+1] , Anal. Calcd for C H N O : C, 66.83; H, 5.07; N, 11.13.
2
1
19
3
4
Found: C, 66.64; H, 5.19; N, 11.05.
3
.1.3.2. 3-(3-Fluoro-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
-1
propenone (4b): Yield 76% (pale brown solid); mp 147-148; IR (CHCl , cm ): 1659, 1600,
3
1
1
4
7
529, 1467, 1360, 1240, 1177, 1029, 830, 750; H NMR (400 MHz, CDCl ): δ 2.65 (s, 3H),
3
.42 (t, 2H, J=5.13 Hz), 4.76 (t, 2H, J=5.13 Hz), 6.91 (d, 2H, J=8.79 Hz), 7.09-7.14 (m, 1H),
.33 (d, 1H, J=9.52 Hz), 7.37-7.40 (m, 2H), 7.49 (d, 1H, J=15.38 Hz), 7.74 (d, 1H, J=15.38
+
Hz), 8.00 (s, 1H), 8.02 (d, 2H, J=8.79 Hz); ESI-MS m/z 396.18 [M+H] , Anal. Calcd for
C H FN O : C, 63.79; H, 4.59; N, 10.63. Found: C, 63.53; H, 4.64; N, 10.78.
21
18
3
4
3
.1.3.3. 3-(4-Fluoro-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
-1
propenone (4c): Yield 81% (pale yellow solid); mp168-169; IR (CHCl , cm ): 2925, 2864,
3
1
1
4
659, 1598, 1511, 1468, 1358, 1026, 825, 753; H NMR (400 MHz, CDCl ): δ 2.65 (s, 3H),
3
.42 (t, 2H, J=5.13 Hz), 4.76 (t, 2H, J=5.13 Hz), 6.90 (d, 2H, J=8.79 Hz), 7.09-7.13 (m, 2H),
6

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7
.43 (d, 1H, J=15.38 Hz), 7.61-7.65 (m, 2H), 7.76 (d, 1H, J=15.38 Hz), 7.99 (d, 2H, J=2.20
+
Hz), 8.02 (s, 1H); ESI-MS m/z 396.13 [M+1] , Anal. Calcd for C H FN O : C, 63.79; H,
2
1
18
3
4
4
.59; N, 10.63. Found: C, 63.55; H, 4.67; N, 10.75.
.1.3.4. 3-(3,4-Difluoro-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
3
-1
propenone (4d): Yield 83% (pale brown solid); mp162-163; IR (CHCl , cm ) : 2927, 2852,
3
1
1
4
6
658, 1601, 1515, 1468, 1260, 1180, 821, 754; H NMR (400 MHz, CDCl ): δ 2.65 (s, 3H),
3
.42 (t, 2H, J=5.13 Hz), 4.76 (t, 2H, J=5.13 Hz), 6.90 (d,d, 2H, J =2.20 Hz, J =6.59 Hz),
1
2
.97-7.00 (m, 1H), 7.33-7.39 (m, 2H), 7.42-7.45 (m, 1H), 7.69 (d,d, 1H, J =6.59 Hz, J =8.79
1
2
+
Hz), 7.98 (s, 2H), 8.01 (s, 1H); ESI-MS m/z 414.18 [M+H] , Anal. Calcd for C H F N O :
21
17
2
3
4
C, 61.02; H, 4.15; N, 10.17. Found: C, 61.14; H, 4.02; N, 10.28.
.1.3.5. 3-(3,5-Difluoro-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
3
-1
propenone (4e): Yield 70% (pale yellow solid); 148-149; IR (CHCl , cm ): 3057, 2986,
3
1
1
4
7
666, 1599, 1457, 1426,1215,1178,828,744; H NMR (400 MHz, CDCl ): δ 2.65 (s, 3H),
3
.43 (t, 2H, J=4.76 Hz), 4.77 (t, 2H, J=4.76 Hz), 6.85-6.88 (m, 1H), 6.91 (d, 2H, J=8.79 Hz),
.14 (d, 2H, J=6.59 Hz), 7.48 (d, 1H, J=15.38 Hz), 7.67 (d, 1H, J=15.38 Hz), 7.98 (d, 2H,
1
3
J=5.86 Hz), 8.01 (s, 1H); C-NMR (100 MHz, CDCl ): δ 14.61, 45.71, 66.78, 105.40,
3
1
1
1
3
10.90, 114.16, 123.79, 130.91, 131.36, 133.29, 138.22, 141.46, 151.60, 161.82, 164.43,
+
87.75; ESI-MS m/z 414.18 [M+1] , Anal. Calcd for C H F N O : C, 61.02; H, 4.15; N,
2
1
17
2
3
4
0.17. Found: C, 61.18; H, 4.07; N, 10.25.
.1.3.6. 3-(2,5-Difluoro-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
-1
propenone (4f): Yield 76% (pale yellow); mp 162-163; IR (CHCl , cm ): 3072, 2930, 1661,
3
1
1
601, 1480, 1251, 1179, 830, 753; H NMR (400 MHz, CDCl ): δ 2.65 (s, 3H), 4.43 (t, 2H,
3
J=4.76 Hz), 4.77 (t, 2H, J=4.76 Hz), 6.91 (d, 2H, J=8.79 Hz), 7.06-7.11 (m, 2H), 7.30-7.33
(m, 1H), 7.58 (d, 1H, J=16.11 Hz), 7.82 (d, 1H, J=16.11 Hz), 7.99 (d, 2H, J=3.66 Hz), 8.02
7

ACCEPTED MANUSCRIPT
+
(
s, 1H); ESI-MS m/z 414.18 [M+1] , Anal. Calcd for C H F N O : C, 61.02; H, 4.15; N,
2
1
17
2
3
4
1
3
0.17. Found: C, 61.15; H, 4.08; N, 10.30.
.1.3.7. 3-(2-Chloro-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
-1
propenone (4g): Yield 69% (pale yellow solid); mp 176-177; IR (CHCl , cm ): 3013, 2933,
3
1
1
4
7
658, 1594, 1517, 1428, 1252, 1167, 825,750; H NMR (400 MHz, CDCl ): δ 2.65(s,3H),
3
.42 (t, 2H, J=4.76 Hz), 4.76 (t, 2H, J=4.76 Hz), 6.91 (d, 2H, J=8.79 Hz), 7.31-7.34 (m, 2H),
.43-7.47 (m, 2H), 7.73-7.75 (m, 1H), 7.99 (s, 1H), 8.01 (d, 2H, J=8.79 Hz), 8.15 (d, 1H,
1
3
J=15.38 Hz); C-NMR (100 MHz, CDCl ): δ 14.64, 45.73, 66.75, 114.07, 124.35, 127.00,
3
1
1
4
3
27.60, 130.20, 130.94, 131.05, 131.57, 133.18, 133.32, 135.32, 138.29, 140.06, 151.62,
+
61.37, 188.43; ESI-MS m/z 412.10 [M+1] , Anal. Calcd for C H ClN O : C, 61.24; H,
2
1
18
3
4
.41; N, 10.20. Found: C, 61.37; H, 4.58; N, 10.05.
.1.3.8. 3-(3-Chloro-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
-1
propenone (4h): Yield 77% (pale brown solid); mp 159-160; IR (CHCl , cm ): 3057, 2986,
3
1
1
3
2
660, 1601, 1468, 1425, 1361, 1255, 1177, 831, 752; H NMR (400 MHz, CDCl ): δ 2.65 (s,
3
H), 4.43 (t, 2H, J=5.13 Hz), 4.77 (t, 2H, J=5.13 Hz), 6.91 (d, 2H, J=8.79 Hz), 7.35-7.37 (m,
H), 7.48-5.92 (m, 2H), 7.63 (s, 1H), 7.72 (d, 1H, J=15.38 Hz), 7.99 (s, 1H), 8.01 (d, 2H,
+
J=9.52 Hz). ESI-MS m/z 412.10 [M+1] , Anal. Calcd for C H ClN O : C, 61.24; H, 4.41;
2
1
18
3
4
N, 10.20. Found: C, 61.35; H, 4.51; N, 10.01.
.1.3.9. 3-(4-Chloro-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
3
-1
propenone (4i). Yield 69% (pale yellow solid); mp 173-174; IR (CHCl , cm ): 3123, 2922,
3
1
1
654, 1597, 1522, 1461, 1251, 1173, 1018, 810, 745; H NMR (400 MHz, CDCl ): δ 2.65 (s,
3
3
H), 4.42 (t, 2H, J=5.13 Hz), 4.76 (t, 2H, J=5.13 Hz), 6.90 (d, 2H, J=8.79 Hz), 7.39 (d, 2H,
J=8.79 Hz), 7.48 (d, 1H, J=15.38 Hz), 7.57 (d, 2H, J=8.05 Hz), 7.76 (d, 1H, J=15.38 Hz),
+
7
.99 (d, 2H, J=4.39 Hz), 8.01 (s, 1H); ESI-MS m/z 412.10 [M+1] , Anal. Calcd for
C H ClN O : C, 61.24; H, 4.41; N, 10.20. Found: C, 61.39; H, 4.52; N, 10.03.
21
18
3
4
8

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3
.1.3.10. 3-(2,4-Dichloro-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
-1
propenone (4j): Yield 73% (pale yellow solid); mp 192-193; IR (CHCl , cm ): 2924, 2864,
3
1
1
658, 1602, 1470, 1373, 1255, 1179, 828, 754; H NMR (400 MHz, CDCl ): δ 2.65 (s, 3H),
3
4
.42 (t, 2H, J=4.76 Hz), 4.77 (t, 2H, J=4.76 Hz), 6.91 (d, 2H, J=8.79 Hz), 7.30 (d,d, 1H,
J =2.20 Hz, J =5.86 Hz), 7.42-7.47 (m, 2H), 7.67 (d, 1H, J=8.79 Hz), 7.99 (s, 2H), 8.01 (s,
1
2
+
1
H), 8.07 (d, 1H, J=15.38 Hz); ESI-MS m/z 446.06 [M+1] , Anal. Calcd for C H Cl N O :
21 17 2 3 4
C, 56.52; H, 3.84; N, 9.42. Found: C, 56.38; H, 3.96; N, 9.29.
3
.1.3.11. 3-(2,6-Dichloro-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
-1
propenone (4k): Yield 79% (pale brown); mp 212-213; IR (CHCl , cm ): 3003, 2927,
3
1
1
666,1597, 1472, 1250, 1174, 828, 750; H NMR (400 MHz, DMSO-d ): δ 2.52 (s, 3H), 4.42
6
(t, 2H, J=5.13 Hz), 4.72 (t, 2H, J=5.13 Hz), 5.88-5.91 (m, 1H), 7.00 (d, 2H, J=8.79 Hz), 7.05
(d, 1H, J=8.79 Hz), 7.25-7.29 (m, 1H), 7.40 (d, 2H, J=8.05 Hz), 7.90 (d, 2H, J=8.79 Hz),
+
8
.03 (s, 1H); ESI-MS m/z 446.06 [M+1] , Anal. Calcd for C H Cl N O : C, 56.52; H, 3.84;
2
1
17
2
3
4
N, 9.42. Found: C, 56.40; H, 3.92; N, 9.31.
3
.1.3.12. 3-(2-Bromo-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
-1
propenone (4l): Yield 82% (pale yellow solid); mp 167-168; IR (CHCl , cm ): 3018, 1658,
3
1
1
4
7
2
6
1
600, 1464, 1361, 1214, 1023, 973, 826, 747; H NMR (400 MHz, CDCl ): δ 2.65 (s, 3H),
3
.42 (t, 2H, J=5.13 Hz), 4.76 (t, 2H, J=5.13 Hz), 6.91 (d, 2H, J=8.79 Hz), 7.25-7.26 (m, 1H),
.34-7.41 (m, 2H), 7.64 (d, 1H, J=8.05 Hz), 7.72 (d, 1H, J=9.52 Hz), 7.99 (s, 1H), 8.01 (d,
1
3
H, J=8.79 Hz), 8.10 (d, 1H, J=15.38 Hz); C-NMR (100 MHz, CDCl ): δ 14.62, 45.70,
3
6.72, 114.05, 124.57, 125.71, 127.61, 127.76, 130.93, 131.18, 131.49, 133.28, 133.41,
+
34.90, 138.20, 142.50, 151.61, 161.36, 188.37;ESI-MS m/z 456.11 [M+1] , Anal. Calcd for
C H BrN O : C, 55.28; H, 3.98; N, 9.21. Found: C, 55.39; H, 3.85; N, 9.32.
21
18
3
4
3
.1.3.13. 3-(3-Bromo-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
-1
propenone (4m): Yield 65% (pale yellow solid); mp162-163; IR (CHCl , cm ): 3069, 2925,
3
9

ACCEPTED MANUSCRIPT
1
2
853, 1658, 1601, 1527, 1467, 1423, 1254, 1218, 1176, 829, 748; H NMR (400 MHz,
CDCl ): δ 2.64 (s, 3H), 4.42 (t, 2H, J=5.13 Hz), 4.76 (t, 2H, J=5.13 Hz), 6.91 (d, 2H, J=8.79
3
Hz), 7.30 (d, 1H, J=8.05 Hz), 7.49 (d, 1H, J=15.38 Hz), 7.53 (d, 2H, J=7.32 Hz), 7.70 (d, 1H,
J=15.38 Hz), 7.78 (s, 1H), 7.99 (d, 2H, J=5.86 Hz), 8.02 (s, 1H); ESI-MS m/z 456.11 [M+1]
+
,
Anal. Calcd for C H BrN O : C, 55.28; H, 3.98; N, 9.21. Found: C, 55.37; H, 3.87; N,
21 18 3 4
9
3
.33.
.1.3.14. 3-(4-Bromo-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
-1
propenone (4n): Yield 79% (pale yellow solid); mp159-160; IR (CHCl , cm ): 3126, 2881,
3
1
1
4
657, 1599, 1524, 1465, 1255, 1177, 812, 750; H NMR (400 MHz, CDCl ): δ 2.64 (s, 3H),
3
.42 (t, 2H, J=5.13 Hz), 4.76 (t, 2H, J=5.13 Hz), 6.90 (d, 2H, J=9.52 Hz), 7.48 (d, 1H, J=5.86
Hz), 7.51-7.56 (m, 4H), 7.72 (d, 1H, J=16.11 Hz), 7.99 (d, 2H, J=2.20 Hz), 8.01 (s, 1H); ESI-
+
MS m/z 456.11 [M+1] , Anal. Calcd for C H BrN O : C, 55.28; H, 3.98; N, 9.21. Found:
2
1
18
3
4
C, 55.39; H, 3.83; N, 9.37.
.1.3.15. 1-{4-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-3-(2-trifluoromethyl-
3
-1
phenyl)-propenone (4o): Yield 78% (pale brown solid); mp 208-209; IR (CHCl , cm ):
3
1
3
043, 2968, 1662, 1602, 1468, 1321, 1255, 1171, 1119, 1065, 1019, 825, 751; H NMR (400
MHz, CDCl ): δ 2.65 (s, 3H), 4.42 (t, 2H, J=4.76 Hz), 4.76 (t, 2H, J=4.76 Hz), 6.91 (d, 2H,
3
J=8.79 Hz), 7.38 (d, 1H, J=15.38 Hz), 7.50 (t, 1H, J=8.05 Hz), 7.60 (t, 1H, J=8.05 Hz), 7.73
(d, 1H, J=7.32 Hz), 7.81 (d, 1H, J=8.05 Hz), 7.98 (s, 2H), 8.00 (s, 1H), 8.09 (d, 1H, J=15.38
+
Hz); ESI-MS m/z 446.12 [M+1] , Anal. Calcd for C H F N O : C, 59.33; H, 4.07; N, 9.43.
22
18
3
3
4
Found: C, 59.45; H, 3.98; N, 9.32.
.1.3.16. 1-{4-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-3-(4-trifluoromethyl-
3
-1
phenyl)-propenone (4p): Yield 64% (pale yellow solid); mp 161-162; IR (CHCl , cm ):
3
1
3
043, 2968, 1662, 1602, 1468, 1321, 1255, 1171, 1119, 1065, 1019, 825, 751; H NMR (400
MHz, CDCl ): δ 2.65 (s, 3H), 4.43 (t, 2H, J=5.13 Hz), 4.77 (t, 2H, J=5.13 Hz), 6.92 (d, 2H,
3
1
0

ACCEPTED MANUSCRIPT
J=8.79 Hz), 7.57 (d, 1H, J=16.11 Hz), 7.67 (d, 2H, J=7.69 Hz), 7.73 (d, 2H, J=8.79 Hz), 7.79
+
(
d, 1H, J=16.11 Hz), 8.00 (d, 2H, J=8.42 Hz), 8.03 (s, 1H); ESI-MS m/z 446.12 [M+1] ,
Anal. Calcd for C H F N O : C, 59.33; H, 4.07; N, 9.43. Found: C, 59.47; H, 3.96; N, 9.35.
2
2
18
3
3
4
3
.1.3.17. 1-{4-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-3-(4-nitro-phenyl)-
-1
propenone (4q): Yield 74% (pale yellow solid); mp 231-232; IR (CHCl ,cm ): 2920, 2852,
3
1
1
3
663, 1599, 1517, 1473, 1344, 1256, 1179, 831, 753; H NMR (400 MHz, CDCl ): δ 2.65 (s,
3
H), 4.42 (t, 2H, J=5.13 Hz), 4.76 (t, 2H, J=5.13 Hz), 6.90 (d, 2H, J=8.79 Hz), 7.61 (d, 1H,
J=16.11 Hz), 7.77-7.82 (m, 3H), 7.99 (s, 1H), 8.02 (d, 2H, J=8.79 Hz), 8.28 (d, 2H, J=8.79
+
Hz); ESI-MS m/z 423.12 [M+1] , Anal. Calcd for C H N O : C, 59.71; H, 4.30; N, 13.26.
2
1
18
4
6
Found: C, 59.86; H, 4.20; N, 13.41.
.1.3.18. 3-(4-Methoxy-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-
3
-1
propenone (4r): Yield 65%(pale yellow solid); mp 169-170; IR (CHCl , cm ): 2926, 2851,
3
1
1
3
6
655, 1597, 1511, 1467, 1256, 1172, 1030, 825, 753; H NMR (400 MHz, CDCl ): δ 2.65 (s,
3
H), 3.86 (s, 3H), 4.42 (t, 2H, J=4.76 Hz), 4.76 (t, 2H, J=4.76 Hz), 6.90 (d, 2H, J=8.79 Hz),
.94 (d, 2H, J=8.79 Hz), 7.39 (d, 1H, J=15.38 Hz), 7.60 (d, 2H, J=8.05 Hz), 7.77 (d, 1H,
+
J=15.38 Hz), 8.00 (d, 3H, J=8.79 Hz); ESI-MS m/z 408.15 [M+1] , Anal. Calcd for
C H N O : C, 64.86; H, 5.20; N, 10.31. Found: C, 64.94.71; H, 5.08; N, 10.45.
22
21
3
5
3
.1.3.19. 3-(3,4-Dimethoxy-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-
-1
phenyl}-propenone (4s): Yield 73% (pale brown solid); mp136-137; IR (CHCl , cm ): 3014,
3
1
2
927, 2844, 1654, 1598, 1513, 1466, 1259, 1027, 828, 754; H NMR (400 MHz, CDCl ): δ
3
2
.65 (s, 3H), 3.93 (s, 3H), 3.95 (s, 3H), 4.42 (t, 2H, J=5.13 Hz), 4.76 (t, 2H, J=5.13 Hz), 6.91
(d, 3H, J=9.52 Hz), 7.15 (s, 1H), 7.22-7.24 (m, 1H), 7.36 (d, 1H, J=15.38 Hz), 7.75 (d, 1H,
+
J=15.38 Hz), 7.99 (d, 2H, J=2.93 Hz), 8.02 (s, 1H); ESI-MS m/z 438.23 [M+1] , Anal. Calcd
for C H N O : C, 63.15; H, 5.30; N, 9.61. Found: C, 63.28; H, 5.19; N, 9.48.
2
3
23
3
6
1
1

ACCEPTED MANUSCRIPT
3
.1.3.20. 3-(2,5-Dimethoxy-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-
-1
phenyl}-propenone (4t): Yield 81% (yellow solid); mp107-108; IR (CHCl , cm ): 3007,
3
1
2
2
6
950, 1654, 1596, 1461, 1422, 1358, 1168, 1032, 829, 744; H NMR (400 MHz, CDCl ): δ
3
.65 (s, 3H), 3.82 (s, 3H), 3.86 (s, 3H), 4.42 (t, 2H, J=4.76 Hz), 4.76 (t, 2H, J=4.76 Hz), 6.86-
.91 (m, 3H), 6.94 (d,d, 1H, J =2.93 Hz, J =5.86 Hz), 7.15 (d, 1H, J=2.93 Hz), 7.55 (d, 1H,
1
2
1
3
J=16.11 Hz), 7.99 (s, 2H), 8.01 (s, 1H), 8.05 (d, 1H, J=16.11 Hz); C-NMR (100 MHz,
CDCl ): δ 14.51, 45.65, 55.69, 55.96, 66.62, 112.29, 113.64, 113.89, 116.93, 122.48, 124.30,
3
1
4
5
3
30.72, 131.93, 133.17, 138.22, 139.48, 151.60, 153.11, 153.31, 161.08, 188.96; ESI-MS m/z
+
38.23 [M+1] , Anal. Calcd for C H N O : C, 63.15; H, 5.30; N, 9.61. Found: C, 63.27; H,
2
3
23
3
6
.17; N, 9.43.
.1.3.21. 3-(3,4-Dimethyl-phenyl)-1-{4-[2-(2-methyl-5-nitro-imidazol-1-yl)-ethoxy]-
-1
phenyl}-propenone (4u): Yield 77% (pale brown); mp164-165; IR (CHCl , cm ): 2929,
3
1
1
2
656, 1601, 1469,1425, 1252, 1178, 820, 752; H NMR (400 MHz, CDCl ): δ 2.30 (s, 6H),
3
.65 (s, 3H), 4.42 (t, 2H, J=5.13 Hz), 4.77 (t, 2H, J=5.13 Hz), 6.91 (d, 2H, J=8.79 Hz), 7.18
(d, 1H, J=7.32 Hz), 7.37-7.41 (m, 2H), 7.46 (d, 1H, J=16.11 Hz), 7.76 (d, 1H, J=16.11 Hz),
+
7
.99 (d, 2H, J=3.66 Hz), 8.02 (s, 1H); ESI-MS m/z 406.12 [M+H] , Anal. Calcd for
C H N O : C, 68.13; H, 5.72; N, 10.36. Found: C, 68.27; H, 5.81; N, 10.48.
23
23
3
4
3
.1.3.22. 1-{4-[2-(2-Methyl-5-nitro-imidazol-1-yl)-ethoxy]-phenyl}-3-naphthalen-1-yl-
-1
propenone (4v): Yield 71 % (pale brown solid); mp 221-222; IR (CHCl , cm ): 3055, 3009,
3
1
1
3
4
654, 1598, 1524, 1466, 1251, 1176, 1022, 903, 752; H NMR (400 MHz, CDCl ): δ 2.65 (s,
3
H), 4.43 (t, 2H, J=5.13 Hz), 4.76 (t, 2H, J=5.13 Hz), 6.92 (d, 2H, J=8.79 Hz), 7.50-7.62 (m,
H), 7.88-7.94 (m, 3H), 7.99 (s, 1H), 8.06 (d, 2H, J=8.79 Hz), 8.25 (d, 1H, J=8.05 Hz), 8.65
+
(
d, 1H, J=15.38 Hz); ESI-MS m/z 428.15 [M+1] , Anal. Calcd for C H N O : C, 70.25; H,
2
5
21
3
4
4
3
.95; N, 9.83. Found: C, 70.11; H, 4.81; N, 9.97.
.2. Biology
1
2

ACCEPTED MANUSCRIPT
3
.2.1.Anti-Trichomonal Assays
Clinical isolates of metronidazole-susceptible T. vaginalis collected at Post Graduate Institute
of Medical Research and Education, Chandigarh, India, were obtained from the laboratory of
Divya Singh [25], and a metronidazole-resistant strain of T. vaginalis (CDC085 [ATCC
5
0143]) was procured from the American Type Culture Collection (ATCC, USA). Both
strains were cultured under partial anaerobic condition in TYM medium (0.1% K HPO4,
2
0
.06% KH PO , 0.5% NaCl, 0.5% glucose, 2.0% yeast extract, 0.2% L-lysine, 0.15%
2 4
tryptone [pH 6.8]) supplemented with 10% (V/V) heat-inactivated fetal bovine serum, 2%
vitamin mixture, 100 U of penicillin/ml, and a 100-µg/ml streptomycin solution in 15-ml
screw-cap sterile tubes, followed by incubation at 37°C in a CO incubator.
2
The T. vaginalis parasites to be used in drug susceptibility assays were grown in TYM
medium for 1 day following regular subculturing and were in the log phase of growth. In
vitro drug susceptibility assays were carried out according to the standard procedure [26].
Stock solutions (10.0 mg/ml) of test compounds were prepared in dimethyl sulfoxide
(DMSO) and diluted with TYM medium to obtain a concentration of 100 µg/ml, and then
further serially diluted with the same medium to 0.78 µg/ml in a 48-well plate. DMSO
3
(
0.05%) in TYM was used as vehicle in control wells. Parasites (5 X 10 trophozoites/well)
were added to these wells and incubated anaerobically at 37°C. Trophozoite growth and
viability in drug-containing wells were monitored by trypan blue staining and cell number
score on a daily basis, in comparison to the control. Assay results were clearly defined after
2
4 h in terms of the MIC (the lowest concentration of compound at which all trophozoites
became nonviable). Metronidazole (Sigma-Aldrich) was used as reference standard.
.2.2.Cytotoxicity of compounds toward human cervical (HeLa) cells.
3
HeLa cells were procured from National Centre for Cell Sciences, Pune, India, and grown in
Dulbecco modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with fetal bovine
1
3

ACCEPTED MANUSCRIPT
serum (10%), and antibiotics (a penicillin-streptomycin mixture [100 U/ml]). The cultures
were maintained in a CO incubator at 37°C in a 5% CO –95% air atmosphere. An MTT (3-
2
2
[
4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide)-based colorimetric assay for
evaluation of the cytotoxicity of drug formulations against the human cervical cell line
4
(
HeLa) was used [26]. Cells seeded at a density of 5 X 10 per well in 96-well plates were
incubated in culture medium (DMEM with 10% fetal calf serum) for 24 h at 37°C in a 5%
CO2–95% air atmosphere. After 24 h, the culture medium was replaced with fresh medium
containing dilutions of test compounds (starting with 1.0 mg/ml) in experimental wells and
0
.05% DMSO in culture medium in control wells. After incubation for another 24 h, 5 µL 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 optical density at 540 nm
(
OD ) was recorded in a microplate reader (Microquant; BioTek, USA).
540
Acknowledgements
DSR thanks University Grants Commission (41-20282012(SR), 2012), New Delhi, India and
University of Delhi, Delhi, India for financial support. AA and UCR are thankful to
University Grants Commission for research fellowship. Authors are thankful to USIC,
University of Delhi for spectral data.
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List of figure captions:
Figure 1: Metronidazole-chalcone conjugates
Tables
Table 1: The anti-Trichomonas activity of chalcone-MTZ hybrids (4a-4v)
Entry
Ar
Compound
MIC against T. vaginalis
Mtz-Susceptible
µg/ml)
Mtz-Resistant
(µg/ml)
50
(
1
2
3
4
5
C
3-FC
4-FC
3,4-FC
3,5-FC
6
H
5
4a
4b
4c
4d
4e
1.56
25
H
100
6
4
H
1.56
1.56
1.56
6.25
6.25
3.125
6
4
H
H
6
3
6
3
1
7

ACCEPTED MANUSCRIPT
6
7
8
9
2,5-FC
6
H
3
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
1.56
3.125
1.56
6.25
6.25
6.25
3.125
3.125
6.25
6.25
3.125
1.56
3.125
1.56
12.5
6.25
6.25
1.56
6.25
12.5
3.125
6.25
12.5
25
6.25
3.125
6.25
25
25
6.25
25
12.5
12.5
12.5
25
2-ClC
3-ClC
4-ClC
H
4
6
H
H
6
6
4
4
1
0
1
2
3
4
5
6
7
8
9
0
1
2
2,4-ClC
2,6-ClC
6
H
3
1
1
1
1
1
1
1
1
1
2
2
2
6
H
3
2-BrC
3-BrC
4-BrC
H
4
H
4
H
4
6
6
6
2- CF C H
3 6 4
4-CF
C H
6 4
3
4-NO
C H
2
6 4
4-OMeC H
3,4-OMeC
2,5-OMeC
3,4-MeC
1-naphthyl
-
6
4
H
H
6
3
6
3
H
4u
4v
-
6
3
MTZ
12.5
Table 2: The cyto-toxicity of the most promising molecules, 4e & 4h against human
cervical (HeLa) cell line in vitro
Compound
IC against
50
HeLa (µg/ml)
4
4
e
>1000
h
>1000
MTZ
>1000
Figures and Schemes
Figure 1
1
8

ACCEPTED MANUSCRIPT
N
N
o
K CO , DMF, 35-40 C, 8-10 h
NO2
NO2
2
3
N
NO2
p-TsCl
N
N
Pyridine, RT, 12 h
O
N
O
HO
O
TsO
HO
Metronidazole
2
3
1
N
NO2
N
Ar CHO
Ar
NaOH
NO2
N
O
Methanol, r.t.
2-3 hours
N
O
O
O
3
Where Ar = 4a, C H
4l, 2-BrC6H4
6
5
4
4
4
4
4
4
4
4
4
4
b, 3-FC H
4m, 3-BrC H
6
4
4
6
4
c, 4-FC H
d, 3,4-FC H
e, 3,5-FC H
f, 2,5-FC H
g, 2-ClC H
h, 3-ClC H
i, 4-ClC H
j, 2,4-ClC H
6
4n, 4-BrC H
6
4
6
3
4o, 2-CF C H
3 6
4
6
3
4
4
4
p, 4-CF C H
3 6
4
6
3
q, 4-NO C H
6 4
2
6
4
r, 4-OMeC H
6 4
6
4
4s, 3,4-OMeC6H3
4t, 2,5-OMeC H
6
4
6
3
6
3
k, R=2,6-ClC H
6
3
4u, 3,4-MeC H
6
3
4v, 1-naphthyl
Scheme 1
1
9

ACCEPTED MANUSCRIPT
Research Highlights
•
•
•
•
All hybrid molecules were found active against susceptible and resistant strain of T.
vaginalis.
Eight compounds were found as potent as standard drug against metronidazole
susceptible strain of T. vaginalis.
Five, seven and three compounds were found equipotent, 2-fold and 4-fold active than
standard drug respectively.
In vitro cytotoxicity of two most active compounds is much above than MIC values.

ACCEPTED MANUSCRIPT
Supporting Information:
1
H NMR spectra of compound 4e
1
3
C NMR spectra of compound 4e

ACCEPTED MANUSCRIPT
1
H NMR spectra of compound 4g
1
3
C NMR spectra of compound 4g

ACCEPTED MANUSCRIPT
1
H NMR spectra of compound 4i
1
H NMR spectra of compound 4t

ACCEPTED MANUSCRIPT
1
3
C NMR spectra of compound 4t