Metronidazole thiosalicylate conjugates: Synthesis, crystal structure, docking studies and antiamoebic activity

Article abstract of DOI:10.1016/j.bmcl.2012.06.083

Metronidazole thiosalicylate conjugates were synthesized and crystallised in order to discover new molecules having better efficacy than therapeutically administered drug metronidazole, used against Entamoeba histolytica. The three compounds (4-6) showed

Full text of DOI:10.1016/j.bmcl.2012.06.083

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5694–5699
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Metronidazole thiosalicylate conjugates: Synthesis, crystal structure,
docking studies and antiamoebic activity
Attar Salahuddin a, Subhash M. Agarwal b, Fernando Avecilla c, Amir Azam a,
⇑
a Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
b Institute of Cytology and Preventive Oncology (ICPO) I-7, Sector—39, Noida 201301, Uttar Pradesh, India
c Departamento de Química Fundamental, Universidade da Coruña, Campus da Zapateira s/n, 15071 A Coruña, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Metronidazole thiosalicylate conjugates were synthesized and crystallised in order to discover new mol-
ecules having better efficacy than therapeutically administered drug metronidazole, used against Ent-
amoeba histolytica. The three compounds (4–6) showed lower IC50 values than metronidazole on
HM1:IMSS strain of E. histolytica and displayed low cytotoxicity on MCF-7 cell line. In order to get an
insight into the mechanisms of action of these compounds, a homology model of E. histolytica thioredoxin
reductase (EhTHRase) was constructed and molecular docking was performed into the binding pocket to
identify the nature of interactions. The docking studies suggest that the improved inhibitory activity of
the newly synthesised metronidazole analogues could be due to involvement of the additional hydropho-
bic interactions in the binding mode. The result of the present study indicates the molecular fragments
that play an essential role in improving the antiamoebic activity.
Received 7 June 2012
Revised 23 June 2012
Accepted 26 June 2012
Available online 3 July 2012
Keywords:
Metronidazole
Amoebiasis
Entamoeba histolytica
MTT assay
Ó 2012 Elsevier Ltd. All rights reserved.
Thioredoxin reductase
Invasive amoebiasis is an emerging problem in the developed
East Asian countries men who had sex with men having co-infec-
tion of amoeba and HIV.1 It is well known that amoebeasis caused
by E. histolytica is transmitted by the ingestion of food or water
containing the cyst form of E. histolytica which is prevalent in trav-
elers and immigrants from endemic areas2 causing significant mor-
bidity and mortality.3 Incidence of this infection is further
increasing and now more than 50 million people are getting af-
fected causing 100,000 fatalities worldwide annually.4 Addition-
ally, the infection is not limited to amoebic dysentery but also
causes abscess in other body organs viz. liver4 and brain5 making
it more dreadful and life threatening.
two ways either by oxidative stress10 or by the formation of ad-
ducts of non-protein thiol or protein with the intermediate metab-
olites (Fig. 2). Recently, in E. histolytica it has been proved that
thioredoxin reductase is a target for nitroimidazole bearing drugs.9
In addition to accepted therapeutic efficacy clinical resistance to
nitroimidazole based drugs has been observed and documented.11
Besides, in many cases concern regarding carcinogenicity of metro-
nidazole has also been raised.12 To combat this neglected disease
and minimize clinical resistance there is continuous need of
designing and developing new compounds endowed with better
activity and low toxicity. In recent years discovery of the novel hy-
brid molecules against protozoal diseases have displayed enhanced
biological activities.13 In view of this we have designed and synthe-
sized some novel thiosalicylate analogs of metronidazole having
different oxidation states of sulfur. Our interest in metronidazole
analogs as an alternative to antiamoebic treatment is facilitated
by the fact that not only metronidazole is effective but also side
5-Nitroimidazole based drugs have been a boon to human
beings for the treatment of infections caused by bacteria and a
range of pathogenic protozoan parasites since 60 years. Presently,
Metronidazole, Tinidazole and Ornidazole (Fig. 1) are the highly
recommended drugs for the treatment of anaerobic protozoan
infections.6 The activity of 5-nitroimidazole drugs is ascribed to
the reduction at the nitro group that results either in the formation
of single-electron transfer reduction products, nitro radical anions
or further reduced highly reactive intermediates, nitrosoimidazole
or hydroxylamineimiadazoles.7
N
N
N
NO2
NO2
N
O
NO2
N
N
This reduction can either occur by reduced ferrodoxin8 or by
thioredoxin reductase.9 The damage to the cells mainly occurs in
Cl
S
OH
Cl
O
Ornidazole
Metronidazole Tinidazole
⇑
Corresponding author. Tel.: +91 11 26981717/3254; fax: +91 11 26980229.
E-mail address: amir_sumbul@yahoo.co.in (A. Azam).
Figure 1. Antiprotozoal drugs Nitroimidazole core ring (red).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.083

A. Salahuddin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5694–5699
5695
Thioredoxin
reductase TrxR
N
N
O
O2
O2
-
N
Reactive oxygen species
Oxidative damage
NO2
N
N
O
Futile cycle
OH
OH
Ferrodoxin
Nitro radical
anion
R
S
N
SH
N
R
N
N
NH
N
O
Non-protein
thiols (NPT)
OH
OH
Nitroso
OH
Hydroxylamine
imidazole NPT adduct
Figure 2. Role of thioredoxin reductase in Nitroimidazole activation8–10
.
chains attached to the imidazole ring structure provides an oppor-
tunity to carry out various modifications. Moreover, previously we
have reported that different metronidazole analogues exhibit sig-
nificant antiamoebic activity.14
The primary alcoholic group of metronidazole (1) was con-
verted to the chloro group with thionyl chloride giving the hydro-
chloride salt of 1-(2-chloroethyl)-2-methyl-5-nitro-imidazole (2).
Nucleophilic substitution of methyl thiosalicylate (3) produced
the thioether linked metronidazole analogue (4). The thioether
linkage was easily regioselectively oxidised to sulfoxide (5) by so-
dium metaperiodate and to sulfone (6) by metachloroperbenzoic
acid (Scheme 1).15
Figure 3. Molecular structure of 2-(2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl-
thio) benzoate (4), showing the atomic numbering scheme. The ORTEP plot is at 30%
probability level.
The compound 4 crystallizes in the orthorhombic system, with
P212121 space group, and the compound 5 in the triclinic system,
ꢀ
with P 1 space group (Fig. 3, Fig. 4 and Table 1 Supplementary
data).16 The angles C(1)–S(1)–C(9) were found to be 102.88(15)°
in 4 and 95.79(7)° in 5, and the angles O(3)–S(1)–C(1) and O(3)–
S(1)–C(9) were found to be 105.93(7)° and 103.63(7)°, respectively,
in 5, all of them closer than S(sp3). These angles can have an expla-
nation for the presence of intramolecular non-bonded SÁ Á ÁO inter-
action which appear in the crystalline structures of 4 and 5. The
N
O
O
NO2
N
SH
.HCl
N
O
O
N
SOCl2
CHCl3
S
(79%)
NO2
NO2
3
N
N
K2CO3
ACN
1
4
2
OH
Cl
(98%)
m-CPBA
DCM
NaIO4
MeOH
N
N
N
N
NO2
NO2
O
O
O
O
O
S
O
S
O
(80%)
5
6
(86%)
Scheme 1. Synthesis of metronidazole-thiosalicylate conjugates. The percentage in bracket shows yield.

5696
A. Salahuddin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5694–5699
Figure 5. Relative in vitro antiamoebic activity.
All the thiosalicylate analogs evaluated for their antiamoebic
activity were relatively found more active than the standard drug
metronidazole. (Fig. 5)
To examine the effect of compounds 4, 5 and 6 as well as met-
ronidazole on cell proliferation, we studied their cytotoxicity on
human breast cancer MCF-7 cell line by MTT assay.21,22, A sub-
confluent population of MCF-7 cells was treated with increasing
concentrations of compounds and the number of viable cells was
measured after 48 h by MTT cell viability assay based on mitochon-
drial reduction of the yellow MTT tetrazolium dye to a highly blue
colored formazan product. This assay usually shows high correla-
tion with number of living cells and cell proliferation.
Figure 4. Molecular structure of 2-(2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethyl-
sulfinyl) benzoate (5), showing the atomic numbering scheme. The ORTEP plot is at
30% probability level.
Table 1
In vitro antiamoebic activity and cytotoxicity of compounds 4, 5, 6 and metronidazole
(MNZ)
Compound No.
Antiamoebic activity HM1:IMSS
Cytotoxicity MCF-7
IC50
(
lM)
S.D.
IC50 (
>250
>250
>250
>250
lM)
S.D.
The concentration range for all the compounds (4, 5, 6 & MNZ)
is mentioned in Figure 6, which illustrates that all the compounds
and metronidazole were non toxic in the concentration range of
4
5
6
MNZ
0.028
0.015
0.021
1.46
0.002
0.002
0.003
0.011
0.27
0.24
0.21
0.32
2.5–250 lM.
It was recently demonstrated by applying two-dimensional gel
electrophoresis and mass spectrometry that thioredoxin reductase
reduces metronidazole and other nitro compounds suggesting a
central role for this enzyme in the treatment of infections caused
by microaerophilic parasites, including E. histolytica.9 Therefore,
in order to offer insight into the mechanisms of action of the met-
ronidazole based analogs synthesized and crystallized (4, 5) we
undertook homology modeling of the amoebic enzyme and identi-
fied the interactions of inhibitors with the model.23,24 To build the
3D model of EhTHRase, a BLAST search was performed against the
Protein Data Bank and Yeast THRase (PDB code 3D8X) was selected
as the starting scaffold for model construction as it showed maxi-
mum homology (75%). The sequence alignment of the 3D8X with
EhTHRase sequence that was used for model construction is shown
in Figure 7.
sum of the van der Waals radii for S–O is 3.25 Å. The non-bonded
SÁ Á ÁO distances were found to be 2.760 Å, and 2.749 Å, respectively.
The quasi-rings adopt planar, conjugated five-membered struc-
tures, closed by the non-bonded sulfur and oxygen atoms. In 5,
s-cis–s-trans (synperiplanar-antiperiplanar) arrangement was
found around the C–C and C–S single bonds.17 The planarity of
the S(1), O(2), C(1), C(6) and C(7) moiety is better recognized in
the molecule of 5 than in 4 (mean deviations from planarity were
found to be, 0.1002 Å in 4, and 0.0532 Å in 5). Torsion angles for
S(1)–C(1)–C(6)–C(7) and C(1)–C(6)–C(7)–O(2) were found to be
7.3(4)° and 15.3(4)° in 4, and 6.2(2)° and 5.5(2)° in 5. This fairly
stable SÁ Á ÁO close contact might play an important role in the
low cytotoxicity of these compounds.18 The planes containing ben-
zyl and imidazole rings form a dihedral angles of 29.47(14)° in 4,
and 72.07(6)° in 5. In the crystal packing of 5, the distance between
the centers of imidazole rings is 3.633 Å, which indicate the pres-
Homology modeling was then carried out through Modeller
9v9.25 The final predicted structure was checked for main chain
conformation using PROCHECK,26 which showed that 92.3% of
the residues were in the ‘most favoured region’ and 7.7% in the
combined ‘allowed region’ as compared to 84.5% and 15.5%,
respectively for the template. No residue was found in the
ence of p–p stacking interactions between them. In 4, p–p stacking
interactions appear between imidazole and benzene rings and the
distance between the centers of the rings is 3.585 Å.
Experiments were carried out to determine the in vitro anti-
amoebic activity of the three compounds (4–6) and MNZ by mic-
rodilution method using HM1:IMSS strain of E. histolytica.19,20 All
the experiments were carried out in triplicate at each concentra-
tion level and repeated thrice. The data is presented in terms of
percent growth inhibition relative to untreated controls and plot-
ted as probit values as a function of drug concentration. The anti-
amoebic effect was compared with the most widely used
antiamoebic medication metronidazole which had a 50% inhibi-
tory concentration (IC50) of 1.46
oxide compound (5) was most active with IC50 = 0.015
sulfone compound (6) showed less activity than the Sulfoxide
with IC50 = 0.021 M. Among the three compounds the sulfide
lM in our experiments. The Sulf-
l
M. The
l
(4) was least active with IC50 = 0.028 lM. The results are summa-
rized in Table 1.
Figure 6. Cytotoxicity assessment by MTT Assay.

A. Salahuddin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5694–5699
5697
Figure 7. Sequence alignment of EhTHRase with the template (Pdb id: 3D8X).
disallowed region. The PROCHECK result summary also showed
only 5 of 307 residues labeled while the torsion angles of the side
After the final model was built, the active site information was
obtained through superimposing 3-D structure of the EhTHRase
model with that of template protein 3D8X. Active site of modeled
thioredoxin reductase was constituted by amino acid residues Met
124, Gly 159, Gly 161, Ala 163, Arg 183, Arg 184 Arg 188 and Ile
246 corresponding to Met 126, Gly 161, Gly 163, Ser 165, Arg
185, Lys 186, Arg 190 and Ile 248 of template protein. Thus, the ac-
tive site forming residues were found to be mostly conserved
(Fig. 9).
Further, in order to decipher the possible interactions of the
crystal resolved inhibitors (4 and 5) with EhTHRase, the inhibitors
were docked in the binding pocket of the modeled protein using
GOLD v3.1.1 program.28 It was noted that GOLD score of 4 and 5
was 59.29 and 59.70 respectively, which is greater than metronida-
zole (antiamoebic drug in use) score value 39.33. This is in accor-
dance with our activity profile data, which indicated that both
compounds (4) and (5) have IC50 value lower than the standard
drug metronidazole. Further it is evident from the ligplot29 analysis
of docked complexes that the inhibitors place themselves nicely
into the active site of the enzyme and are mainly stabilized by
the hydrophobic interactions (Fig. 10a and 10b). The higher activity
of the synthesized metronidazole analogs compared to MNZ could
be partially due to involvement of these additional hydrophobic
interactions in the binding mode.
chain designated by
v1Àv2 plots showed only 6 labeled residues
out of 167. Also, it is established that the score for G-factors should
be above À0.50 for a reliable model. We observed that the G-factor
scores of the model was 0.02 for dihedral bonds, À0.14 for covalent
bonds and À0.14 overall. The distribution of the main chain bond
lengths and bond angles was 99.5% and 94.2% within limits. The
high quality of the structure is further evident by the fact that
according to VERIFY 3D27 98.39% of the residues have a score of
greater than 0.2, which indicates a good quality model. Further,
the root-mean-square deviation (RMSD) between the backbone
atoms of the template and the homology model was observed to
be 0.302 Å indicating reasonably good structural parameters of
the predicted structure (Fig. 8).
Specifically, the residues that interact with compound 4 and
contribute to the hydrophobic interactions are Met 124, Val 158,
Gly 159, Leu 181, His 182, Arg 184, Glu 210 and Ile 246, while
Figure 8. Structural superimposition of Ca trace of EhTHRase model (represented
in blue color) with known crystal structure (represented in red color). The root-
mean-square deviation (RMSD) between the template and the homology model
was 0.302 Å.
Figure 9. Overlay of the active site residues of the homology model with the
template 3D8X. Blue and red color sticks represents modeled and template proteins
respectively.

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A. Salahuddin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5694–5699
antiamoebic agents. It was found that 2-(2-(2-methyl-5-nitro-
1H-imidazol-1-yl) ethylsulfinyl) benzoate (5) shows promising
in vitro antiamoebic activity as well as low cytotoxicity. Further,
docking simulations indicated that the enhanced antiamoebic
activity could be due to additional hydrophobic interactions in
the binding site of E. histolytica thioredoxin reductase. These find-
ings provide us lead and encourage us to continue the efforts to-
wards the optimization of the efficacy profile of this structural
moiety for treatment of amoebiasis.
Acknowledgments
This work was supported by Council of Scientific and Industrial
Research (CSIR Grant # 01(2278)/08/EMR-II New Delhi, India). One
of us (A.S) acknowledges the Senior Research Fellowship from the
CSIR.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
06.083.
References and notes
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Bendesky, A.; Mene´ndez, D.; Ostrosky-Wegman, P. Mut. Res. 2002, 511, 133; (c)
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14. (a) Athar, F.; Husain, K.; Abid, M.; Agarwal, S. M.; Coles, S. J.; Hursthouse, M. B.;
Maurya, M. R.; Azam, A. Chem. Biodivers. 2005, 2, 1320; (b) Abid, M.; Agarwal, S.
M.; Azam, A. Eur. J. Med. Chem. 2008, 43, 2035.
Figure 10. Schematic 2D representation of interactions of (a) Compound 4 and (b)
Compound 5. Hydrogen bonds are shown with green dashed lines and hydrophobic
contacts by red arcs with radiating lines.
15. Synthesis Procedure
(a) Synthesis of 1-(2-chloroethyl)-2-methyl-5-nitro-1H-imidazole Hydrochloride
(2): To a stirred solution of Metronidazole (1) (5 g, 15.57 mmol) in 100 mL of
chloroform at room temperature was added thionyl chloride 10 mL. The
resulting mixture was stirred for 12 h and concentrated under vacuo to yield
hydrochloride salt of compound (2) Yield 98%; mp: 92–93 °C; Anal. calcd for
C6H10ClN3O2: C 37.61, H 18.50, N 21.93% found: C 37.89, H 18.22, N 22.2%; 1H
NMR (CDCl3) d (ppm): 8.0 (s,1H), 8.24 (4.64 t, 2H, J = 5.7 Hz), 3.90 (t, 2H,
J = 5.7 Hz), 2.58 (s, 3H), FAB-MS (m/z): [M++1] 192.
(b) Synthesis of methyl 2-(2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethylthio)
benzoate (4): To a stirred solution of methyl thiosalicylate (5 g, 29.72 mmol)
in 100 mL of acetonitrile was added potassium carbonate (12.3 g, 89.16 mmol),
the reaction mixture was then stirred for 15 min. at room temperature and
the NH1 atom of Arg 183 of the active site of the receptor forms
hydrogen bond with O3 of compound 4 (Fig. 10a). In case of Com-
pound 5, it interacts predominantly with amino acid residue Arg
184 through hydrogen bond and other non ligand residues that
shows involvement in hydrophobic interactions are Met 124, Val
158, Gly 159, Gly 160, Gly 161, Arg 183, Arg 188 and Ile 246.
(Fig. 10b). Previously too, studies from others30 as well as from
our group14b have demonstrated that 5-nitroimidazole analogues
having hydrophobic side chains show better antiamoebic activity
probably because these modifications enhance solubility and
membrane permeability of the compounds. Thus, the docking sim-
ulations corroborate the observed activity profile and confirm the
basis that has led to the development of newer molecules.
portion wise compound
2 (6.78 g, 29.72 mmol) was added. The reaction
mixture was then refluxed for 24 h and concentrated. The residue was
partitioned between ethylacetate and water, the organic layer was separated
and washed with brine, dried over anhydrous sodium sulphate and
concentrated to yield crude product which was crystallized in ethanol to
give yellow crystals of compound 4. Yield 79.8%; mp: 148–150 °C; Anal. calcd
for C14H15N3O4S: C, 52.33; H, 4.70; N, 13.08; S, 9.98, found: C, 52.11; H, 4.51; N,
13.2; S, 9.9%; IR mmax (cmÀ1): 1735 (C@O), (C–S–C), 1537 (NO2); 1H NMR
(CDCl3) d (ppm): 7.96 (d, 1H, J = 7.8 Hz), 7.91 (s,1H), 7.53-7.43 (m, 2H), 7.23 (d,
1H, J = 7.5 Hz) 4.55 (t, 2H, J = 6.9 Hz), 3.92 (s, 3H), 3.37 (t, 2H, J = 7.2 Hz), 2.44 (s,
To conclude, the present study describes the synthesis of metro-
nidazole thiosalicylate analogs (4–6) and their evaluation as

A. Salahuddin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5694–5699
5699
3H), 13C NMR (CDCl3) d (ppm): 166.7, 150.69, 138.32, 137.25, 133.31, 132.64,
131.57, 128.71, 126.13, 125.16, 52.32, 45.45, 31.64, 14.42. FAB-MS (m/z): [M+]
322.1 (100.0%) [M++1]323.09 (16.1%).
power microscope. The culture medium was removed by inverting the plate
and shaking gently. Plate was then immediately washed with sodium chloride
solution (0.9%) at 37 °C. This procedure was completed quickly and the plate
was not allowed to cool in order to prevent the detachment of amoeba. The
plate was allowed to dry at room temperature and the amoebae were fixed
with methanol and when dried, stained with (0.5%) aqueous eosin for 15 min.
The stained plate was washed once with tap water, then twice with distilled
(c) Synthesis of methyl 2-(2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethylsulfinyl)
benzoate (5): To a stirred solution of compound 4 (4 g, 12.4 mmol) in methanol
30 mL was added sodium metaperiodate (3.98 g, 18.61 mmol) the reaction
mixture was stirred at room temperature for 4 h. The reaction mixture was
concentrated and the residue was diluted with water and extracted with
dichloromethane (DCM), the combined organic extracts were washed with
brine and concentrated to yield crude compound 5 which was purified by
column (DCM/MeOH) chromatography to give pure product which was
crystallized in 30% DCM:hexane. Yield 80%; mp: 178–180 °C; Anal. calcd for
water and then allowed to dry. 200 ll portions of 0.1 N sodium hydroxide
solution was added to each well to dissolve the protein and release the dye. The
optical density of the resulting solution in each well was determined at 490 nm
with a micro plate reader. The% inhibition of amoebal growth was calculated
from the optical densities of the control and test wells and plotted against the
logarithm of the dose of the drug tested. Linear regression analysis was used to
determine the best fitting line from which the IC50 value was found.
21. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
22. Cytotoxicity: MCF-7 cells were cultured and maintained as a monolayer in
Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10%
of fetal calf serum (Sigma) and 1% antibiotics penicillin/streptomycin/
Neomycin. All cells were cultured at 37 °C in a 100% humidity atmosphere
C
14H15N3O5S: C, 49.84; H, 4.48; N, 12.46; O, 23.71; S, 9.50; found: C, 49.8; H,
5.2; N, 12.59; S, 9.47%; IR mmax (cmÀ1): 1743 (C@O), 1052 (S@O), 1533 (NO2);
1H NMR (CDCl3) d (ppm): 8.21–8.23 (dd, 1H, J = 7.5 & 0.6 Hz), 8.09-8.12 (dd,1H
J = 7.8 & 1.2 Hz), 7.99 (s, 1H), 7.80–7.86 (dt, 1H J = 7.8 &1.2 Hz), 7.57–7.63 (dt,
1H J = 7.8 &1.2 Hz), 4.73–4.76 (m, 2H), 3.87 (s, 3H), 3.61–3.71 (m, 1H), 3.01–
3.09 (m, 1H), 2.68 (s, 1H) 13C NMR (CDCl3) d (ppm): 165.75, 150.94, 146.76,
138.81, 134.01, 133.43, 131.22, 130.78, 126.52, 124.62, 56.52, 52.89, 41.03,
14.54. FAB-MS (m/z): [M+] 338.02, [M++1] 339.08 (17.1%).
and 5% CO2. The effect of compounds 4, 5,
6 and the standard drug
(d) Synthesis of methyl 2-(2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethylsulfonyl)
benzoate (6): To a stirred solution of compound 4 (2 g, 6.2 mmol) in 25 mL of
DCM was added slowly m-Chloro perbenzoic acid (2.24 g, 13.02 mmol), the
reaction mixture was then stirred for 4–5 h and precipitate was obtained and
filtered. The filtrate was then washed once with saturated sodium bicarbonate
and then with brine, dried over anhydrous sodium sulphate and concentrated
to yield crude compound 6 which was purified with column chromatography
(DCM/MeOH) Yield 86.5%; mp: 115–116 °C; Anal. calcd for C14H15N3O6S: C,
47.59; H, 4.28; N, 11.89; S, 9.07 found: C, 47.53; H, 4.55; N, 11.7; S, 9.1;%; IR
mmax (cmÀ1): 1752 (C@O), 1315 1150 (SO2), 1530 (NO2); 1H NMR (CDCl3) d
(ppm): 7.98 (d, 2H, J = 7.2 Hz), 7.93 (s,1H), 7.46-7.54 (m, 2H), 7.25 (d, 1H J =
7.4 Hz), 4.82 (t, 2H J = 8.8 Hz), 4.07 (t, 2H, J = 8.8 Hz), 3.94 (s, 3H), 2.64 (s, 3H)
13C NMR (CDCl3) d (ppm): 167.09, 151.1, 138.32, 137.3, 134.3, 133.23, 132.99,
131.56, 130.38, 130.24, 55.13, 53.37, 39.71, 14.34. FAB-MS (m/z): [M+] 354
(100.0%), [M++1] 355.08 (16.3%), [M++2] 356.07 (4.2%).
Metronidazole on cell proliferation was measured by using an MTT based
assay.18 Exponentially growing viable cells were plated at 1.2 Â 104 cells per
well into 96-well plates and incubated for 48 h before the addition of the
compounds/metronidazole. Stock solutions of compounds were initially
dissolved in 20% (v/v) DMSO and further diluted with fresh complete
medium. The growth-inhibitory effects of the compounds were measured
using standard tetrazolium MTT assay. After 48 h of incubation at 37 °C and 5%
CO2, the medium was removed and 25
medium was added to each well. The plates were incubated at 37 °C for 4 h. At
the end of the incubation period, the medium was removed and 100 L DMSO
lL of MTT (5 mg/mL) in serum free
l
added to all wells. The metabolized MTT product dissolved in DMSO was
quantified by reading the absorbance at 570 nm with a reference wavelength
of 655 nm in an ELISA plate reader (Labsystems Multiskan RC, Helsinki,
Finland. All assays were performed in triplicate. Percent viability was defined
as the relative absorbance of treated versus untreated control cells.
23. Homology modeling of Entamoeba histolytica Thioredoxin reductase (EhTHRase):
E. histolytica Thioredoxin reductase (EhTHRase) full amino acid sequence of
314 residues was retrieved from Entrez database (Accession: EAL50345.1) and
identification of homologues was carried out by performing PDB database
search using BLAST (http://blast.ncbi.nlm.nih.gov). An appropriate template,
PDB id: 3D8X (Thioredoxin reductase from Yeast) was identified based on the
e-value and sequence identity. The template and the target sequences were
then aligned. The alignment contains residues numbered 5 to 313 of the target
protein. The first 4 and last 1 amino acids are not present in the model, as the
X-ray crystal structure 3D8X does not contain the equivalent amino acids. Thus
the model is made up of residues 5–313. Modeller9v925 which models protein
tertiary structure by satisfaction of spatial restraints was used for protein
structure modeling. The modeled structures were ranked on the basis of
molpdf scores generated by modeler and the one with least score was selected
for model validation. Thereafter, the goodness of predicted EhTHRase model
was assessed using PROCHECK26, which checks the stereochemical quality of a
protein structure by analyzing overall and residue by residue geometry. The
protein was further subjected to VERIFY3D27, which derives a ‘3D-1D’ profile
based on the local environment of each residue, described by the statistical
preferences for: the area of residue which is buried, the fraction of side-chain
area that is covered by polar atoms (oxygen and nitrogen) and the local
secondary structure (alpha, beta, loop). Further, in order to assess the
reliability of the modeled structure of EhTHRase, we calculated the root
mean square deviation (RMSD) by superimposing it on the known template
structure.
24. Molecular docking: The binding mode and biomolecular interactions between the
crystals resolved inhibitors (4 and 5) and modeled EhTHRase was analyzed using
GOLD v3.1.1 program.28 The GOLD 3.1.1 (Genetic Optimization for Ligand
Docking) from Cambridge Crystallographic Data Centre, UK, uses genetic
algorithm to explore the full range of ligand conformational flexibility with
partial flexibility of the protein. Active site radiuses were taken as equivalent
positions in the template structure obtained by structural superimposition of
template-target structure. Standard default settings, consisting of population
size-100, number of islands-5, selection pressure-1.1, niche size-2, migrate-2,
cross over-95, number of operations-100,000 were adopted for docking of each
moleculetotheprotein. Also, 100 dockingconformations(poses)weregenerated
and the best docked conformation was selected based on the Goldscore ranking,
for further analysis. Finally, Ligplot29 was used to map the hydrogen and
hydrophobic interaction of the docked inhibitor to the modeled structure.
25. Sali, A.; Blundell, T. L. J. Mol. Biol. 1993, 234, 779.
16. X-ray crystal structure determination: Three-dimensional X-ray data were
collected on a Bruker SMART Apex CCD diffractometer at 100(2) K, using a
graphite monochromator and Mo-K radiation (k = 0.71073 Å) by the /-
x
scan
method. Reflections were measured from a hemisphere of data collected of
frames each covering 0.3 ° in . Of the 13972 in 4 and 18584 in 5 reflections
a
x
measured, all of which were corrected for Lorentz and polarization effects, and
for absorption by semi-empirical methods based on symmetry-equivalent and
repeated reflections, 2152 in 4 and 2720 in 5 independent reflections exceeded
the significance level |F|/r(|F|)>4.0. Complex scattering factors were taken
from the program package SHELXTL.16 The structures were solved by direct
methods and refined by full-matrix least-squares methods on F2. The non-
hydrogen atoms were refined with anisotropic thermal parameters in all cases.
All hydrogen atoms were left to refine freely. A final difference Fourier map
showed no residual density outside: 0.197 and À0.341 in 4 and 0.554 and
À0.419 in compound 5 e.ÅÀ3. CCDC No. 872750 for compound 4 and 872751
for 5 contain the supplementary crystallographic data for this Letter. These
data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/
retrieving.html, or from the Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033; or e-mail:
deposit@ccdc.cam.ac.uk.
17. Ángyán, J. G.; Poirier, R. A.; Kucsman, Á.; Csizmadia, I. G. J. Am. Chem. Soc. 1987,
109, 2237.
18. Nagao, Y.; Hirata, T.; Goto, S.; Sano, S.; Kakehi, A.; Iizuka, K.; Shiro, M. J. Am.
Chem. Soc. 1998, 120, 3104.
19. Wright, C. W.; O_Neill, M. J.; Phillipson, J. D.; Warhurst, D. C. Antimicrob. Agents
Chemother. 1988, 32, 1725; (b) Diamond, L. S.; Harlow, D. R.; Cunnick, C. C.
Trans. R Soc. Trop. Med. Hyg. 1978, 72, 431; (c) Gillin, F. D.; Reiner, D. S.;
Suffness, M. Antimicrob. Agents Chemother. 1982, 22, 342; (d) Keene, A. T.;
Harris, A.; Phillipson, J. D.; Warhurst, D. C. Plant. Med. 1986, 278.
20. In vitro antiamoebic assay: Compounds 4, 5 and 6 were screened in vitro for
antiamoebic activity against HM1:IMSS strain of E. histolytica by microdilution
method. E. histolytica trophozoites were cultured in culture tubes by using
Diamond TYIS-33 growth medium. The test compounds (1 mg) were dissolved
in DMSO (40
solutions of the compounds were prepared freshly before use at
concentration of 1 mg/mL. Twofold serial dilutions were made in the wells of
96-well microtiter plate (costar). Each test includes metronidazole as
lL, level at which level no inhibition of amoeba occurs). The stock
a
a
standard amoebicidal drug, control wells (culture medium plus amoebae)
and a blank (culture medium only). All the experiments were carried out in
triplicate at each concentration level and repeated thrice. The amoeba
suspension was prepared from
a
confluent culture by pouring off the
26. Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. J Appl. Cryst.
1993, 26, 283.
27. Lüthy, R.; Bowie, J. U.; Eisenberg, D. Nature 1992, 5, 83.
28. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267,
727.
medium at 37 °C and adding 5 mL of fresh medium, chilling the culture tube
on ice to detach the organisms from the side of flask. The number of amoeba/
mL was estimated with the help of a heamocytometer, using trypan blue
exclusion to confirm the viability. The suspension was diluted to 105 organism/
ml by adding fresh medium and 170
l
L of this suspension was added to the
29. Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. Protein Eng. 1995, 8, 127.
30. (a) Upcroft, J. A.; Campbell, R. W.; Benakli, K.; Upcroft, P.; Vanelle, P. Antimicrob.
Agents Chemother. 1999, 43, 73; (b) Upcroft, J. A.; Dunn, L. A.; Wright, J. M.;
Benakli, K.; Upcroft, P.; Vanelle, P. Antimicrob. Agents Chemother. 2006, 50, 344;
(c) Busatti, H. G.; Vieira, A. E.; Viana, J. C.; Silva, H. E.; Souza-Fagundes, E. M.;
Martins-Filho, O. A.; Alves, R. J.; Gomes, M. A. Parasitol. Res. 2007, 102, 145.
test and control wells in the plate so that the wells were completely filled (total
volume, 340
l
L). An inoculum of 1.7 Â 104 organisms/well was chosen so that
confluent, but not excessive growth, took place in control wells. Plate was
sealed and gassed for 10 min with nitrogen before incubation at 37 °C for 72 h.
After incubation, the growth of amoeba in the plate was checked with a low