Molecular docking and in vitro antileishmanial evaluation of chromene-2-thione analogues

Article abstract of DOI:10.1021/ml200280r

Leishmaniases are an epidemic in various countries, and the parasite is developing resistance against available drugs. Thus, development of new drugs against Leishmania is an open area of investigation for synthetic organic chemists. To meet this challeng

Full text of DOI:10.1021/ml200280r

Letter
pubs.acs.org/acsmedchemlett
Molecular Docking and in Vitro Antileishmanial Evaluation of
Chromene-2-thione Analogues
Rajiv Kumar Verma,† Vijay Kumar Prajapati,‡ Girijesh Kumar Verma,† Deblina Chakraborty,§
Shyam Sundar,‡ Madhukar Rai,‡ Vikash Kumar Dubey,*,§ and Maya Shankar Singh*,†
†Department of Chemistry and Centre of Advanced Study, Faculty of Science, Banaras Hindu University, Varanasi-221005, India
‡Infectious Disease Research Laboratory, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India
§Department of Biotechnology, Indian Institute of Technology Guwahati, Assam-781039, India
S
* Supporting Information
ABSTRACT: Leishmaniases are an epidemic in various
countries, and the parasite is developing resistance against
available drugs. Thus, development of new drugs against
Leishmania is an open area of investigation for synthetic
organic chemists. To meet this challenge, a series of chromene-
2-thione derivatives have been synthesized and docked into the
active site of trypanothione reductase (TryR) enzyme required
for redox balance of the parasite. These were screened on
promastigote, axenic amastigote, and intracellular amastigote
stages of Leishmania donovani and found to show high levels of antileishmanial activity together with minimal toxicity to human
peripheral blood mononuclear cells. Compounds 3b and 3k were found to be the most active among the tested compounds.
Although the compounds show moderate antileishmanial activity, they identify a chemical space to design and develop drugs
based on these chromene-2-thione derivatives against the Leishmania parasite.
KEYWORDS: chromene-2-thione, visceral leishmaniasis, molecular docking, trypanothione reductase, Lipinsky rule
he Leishmaniases are a spectrum of neglected parasitic
diseases caused by different species of the genus
miltefosine are considered the best therapeutic solutions for
the treatment of VL.7 Considering the high toxicity and
incomplete efficacy of the current clinical drugs, the search for
new targets showing antileishmanial activity is very much
desirable and would be of great relevance to both synthetic and
medicinal chemists.
Coumarins (2H-chromene-2-ones) are among the best
known oxygenated heterocycles and are present as a structural
motif in numerous natural products. Interest in their chemistry
continues unabated because of the broad range of biological
activities displayed by this class of compounds.8−11 There are
several examples of coumarin derivatives isolated from various
families of plants, which have been tested and found to be
effective against the promastigote form of Leishmania parasite
within a range of 17−50 μg/mL for IC50 determination.12−15
Along with these naturally occurring coumarins, some synthetic
coumarins such as 4-(3, 4-dimethoxyphenyl)-6,7-dimethoxy-
2H-chromene-2-one have been shown to exhibit potent activity
against L. donovani amastigotes (IC50, 1.1 μM).15
T
Leishmania protozoan, which is transmitted to humans by the
bite of female phlebotomine sand fly. In the human body, the
parasites survive and multiply within phagolysosomes of
macrophages as intracellular amastigotes. Leishmaniasis is
endemic in 88 countries, and there are 500 000 new cases of
its visceral form each year, leading to 50 000 deaths.1
It is mainly manifested in three forms viz. visceral
leishmaniasis (VL), also known as kala-azar caused by
Leishmania donovani; cutaneous leishmaniasis (CL) caused by
Leishmania major, L. donovani, Leishmania tropica, and
Leishmania aethiopica; and muco-cutaneous leishmaniasis
(MCL) caused by Leishmania braziliensis. Among all
leishmaniases, the visceral form is the most severe.2 In the
absence of proper medication, it has a mortality rate of almost
100% independent of the immunological status of the
patient.3−5
At the beginning of the VL epidemic in India in early 1970s,
sodium stibogluconate (SSG) was the most effective anti-
leishmanial drug; however, over the years, its efficacy declined,
and currently, more than two-thirds of VL patients in the Bihar
state of India do not respond to SSG.6 Furthermore, the long
course of treatment of pentavalent antimony (SbV) often causes
side effects such as myalgia, pancreatitis, cardiac arrhythmia,
and hepatitis, leading to the reduction or cessation of treatment.
Presently, amphotericin B and the oral anticancer drug
We have selected our title compounds 2H-chromene-2-
thione and benzo[f]chromene-2-thione for development and
assessment against L. donovani. The compounds have already
Received: November 24, 2011
Accepted: January 18, 2012
Published: January 18, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
been reported to exhibit profound antioxidant activities16 but
have not previously been tested against any Leishmania species.
Initially, the reaction of S,S-dimethyl trithiocarbonate with
substituted acetophenone 1 in the presence of NaH afforded
the β-oxodithioesters 2 in good yields (Scheme 1). Typical
synthetic strategies to obtain the title compounds in excellent
yields have been depicted in Scheme 1.
The enzyme (TryR) is a dimer consisting of two active sites.
Active sites are formed by the interaction of both chains. The
following is the description of residues around one of the active
sites.
The residues involved in bonding and nonbonding
interaction with ligands are as follows: Val-58, Lys-61, Leu-
62, and Thr-65 from the B chain and Phe-396, Thr-397, Pro-
398, Leu-399, Met-400, His-461, Pro-462, Thr-463, and Ser-
464 from the A chain. The residues involved in hydrogen
bonding with the ligands are as follows: Lys-61 from the B
chain and Leu-399, Met-400, and Ser-464 from the A chain.
These residues are found around the active sites Cys-52 and
Cys-57 of the B chain. As most of the ligands are surrounded by
the above residues, it can be concluded that these residues are
highly conserved and play important functional roles. Thus,
interaction of the ligands with these residues might be changing
the active site structure, which leads to inhibition of the
enzyme. Also, these ligands show low free energy of binding
against TryR; thus, they bind with high affinity. All of the
ligands mostly interact with residues of the A chain and very
few residues of the B chain. The interaction of ligands with the
enzyme is represented in Figure 2A. The figure shows that the
lead molecules dock at a position near the active site of TryR,
where the cofactor FAD (shown in red color) is bound. Ligand
3i shows the least free energy of binding followed by 3k, 3g, 3j,
3h, 3b, 3a, 3d, 3e, 3f, and 3c in that order.
Scheme 1. Typical Synthetic Strategy for the Synthesis of
2H-Chromene-2-thione and Benzo[f]chromene-2-thione
The protein contact potential was generated for all of the
docked conformations (Figure 2B). This depicted the whole
surface of the protein. Red and blue colors show negative and
positive charges, respectively. In this depiction, all of the ligands
were shown to be bound inside a cavity formed at the interface
of chain A and chain B, that is, the dimeric interface of the
enzyme. Although the docked conformation was different for
each ligand, the ligands are positioned at almost the same place,
thus showing a consensus pattern of interactions with the
residues near the active site. Figures showing interactions of
each ligand with the protein are submitted as Supporting
Information. It can be predicted that the inhibition modes of all
of these ligands could be similar, because of their interactions
with almost the same residues.
The β-oxodithioesters 2 were subjected to cyclocondensation
with various 2-hydroxy aromatic aldehydes in the presence of
InCl3 and urea to give chromene-2-thione in excellent yields
(Scheme 1 and Figure 1).17
Our initial testing of these compounds against promastigote
and amastigote stages of L. donovani showed that the
compounds are active in IC50 range of 37−927 μM against
axenic amastigotes, while IC50 was considerably higher (97−
4634 μM) against promastigotes (Table 2). The mean of the
IC50 values was found to be approximately five times lower
against amastigotes than promastigotes, demonstrating the
higher sensitivity of amastigotes to these compounds as
compared to promastigotes.
Depending upon the IC50 values of amastigotes and
promastigotes, we selected some derivatives for intracellular
macrophage susceptibility assay. The intracellular macrophage
susceptibility assay of compounds 3b [6-bromo-3-(4-methox-
ybenzoyl)-2H-chromene-2-thione], 3c [3-(furan-2-oyl)-8-me-
thoxy-2H-chromene-2-thione], 3h [3-(4-chlorobenzoyl)-8-me-
thoxy-2H-chromene-2-thione], and 3k [3-(thien-2-oyl)-benzo-
[f ]2H-chromene-2-thione] showed higher potency with IC50
values of 17, 26, 24, and 22 μM, respectively (Table 2).
The cytotoxicity of all of the synthesized compounds was
determined against PBMC (peripheral blood mononuclear
cells) at two concentrations (IC50 and twice that of IC50). None
of the compounds showed more than 40% killing at a
concentration twice that of the IC50 (Table 2). There is
Figure 1. Various synthesized potential conjugates against L. donovani.
Trypanothione reductase (TryR) is a key drug target enzyme
involved in the redox metabolism of the parasite, and inhibition
of TryR may disrupt the redox balance of the parasite leading to
parasite death.18−21 Thus, organic molecules may be designed,
which can bind the active site of TryR leading to its inhibition.
AutoDock 4.2 was used to perform molecular docking of
small molecules on macromolecular protein by treating the
ligand as conformationally flexible. Autodock 4.2 also has a free-
energy scoring function, which uses an AMBER force field to
estimate the free energy of binding of a ligand to its target. Our
in silico analysis suggests TryR as a possible target of these
compounds. These compounds show satisfactory docking with
acceptable statistics near the active site of TryR.22 Detailed
results of analysis (free energy of binding values for each ligand
along with the description of interacting residues) are presented
in Table 1.
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ACS Medicinal Chemistry Letters
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Table 1. Docking Statistics of the Compounds with TryR and Their Interaction with the Protein
binding
energy
entry
H-bond
possible hydrophobic interactions
3a
3b
−7.78
−8.09
2JK6:Met-400(A):NH
Pro-398, Phe-396, Glu-467, Glu-466, Thr-463 from A chain; Leu-62, Lys-61 from B chain
Val-58, Lys-61, Leu-62, Thr-65 from B chain; Phe-396, Thr-397, Pro-398, Leu-399, Met-400, His-461
and Pro-462 from A chain
3c
−6.82
Val-58, Lys-61, Leu-62, Thr-65 from B chain; Thr-463, Pro-398, Phe-396, Leu-399, Met-400 and Pro-462
from A chain
3d
3e
−7.63
−7.54
2JK6:Lys-61(B):HZ2
2JK6:Lys-61(B):HZ2
Val-58, Lys-61, Leu-62 from B chain; Phe-396, Pro-398, Leu-399, Thr-463 and Ser-464 from A chain
Val-58, Lys-61, Leu-62, Thr-65 from B chain; Phe-396, Pro-398, Met-400, His-461 and Pro-462 from A
chain
3f
−7.35
−8.22
−8.11
Lys-61, Leu-62, Thr-65, Phe-396, Pro-398, Met-400 and Pro-462
3g
3h
Leu-62, Thr-65 from B chain; Phe-396, Thr-397, Pro-398, Met-400, Pro-462 and Thr-463 from A chain
Val-58, Lys-61, Leu-62, Thr-65 from B chain; Phe-396, Pro-398, Met-400, Pro-462 and Thr-463 from A
chain
3i
3j
3k
−9.20
−8.17
−8.61
Val-58, Lys-61, Leu-62, Thr-65 from B chain; Phe-396, Leu-399, Met-400, Thr-463, Glu-466 and Glu-
467 from A chain
2JK6:Lys-61(B):HZ2 2JK6:Leu-
399(A):HN
Phe-396, Pro-462, Thr-463 and Ser-464 from A chain
2JK6:Ser-464(A):SH and OH
Val-58, Lys-61, Leu-62 from B chain; Phe-396, Pro-398, Leu-399 and Met-400 from A chain
Figure 2. (A) Binding of the 11 ligands to the dimer TryR (PDB ID: 2JK6). (B) A vacuum electrostatics depiction of TryR (PDB ID: 2JK6) bound
to all 11 ligands, showing protein contact potential.
Table 2. Antileishmanial Activity and Cytotoxicity of
Chromene-2-thione Conjugates
antimony gluconate. The single oral drug miltefosine available
for VL has limitations of a long half-life and toxicity issues.
Paromomycin also shows adverse events during treatment. The
antifungal drug amphotericin B deoxycholate requires long
hospitalization and adverse effects such as renal toxicity.23 The
lipid formulation of amphotericin B is the best of the available
drug options for VL patients, but it is expensive. So, in the
effort to develop an antileishmanial drug for the treatment of
VL, we have tested these compounds against promastigote and
amastigote stages of L. donovani, and the experimental
observations showed that compounds 3b and 3k were found
to be the most active. The increased potency of the compounds
in the intracellular macrophage susceptibility assay leaves room
for further exploration of the metabolism of the chemical
compounds within the macrophage. Inhibition of TryR by
compounds may lead to an imbalance in the parasite's redox
potential, which weakens the parasite's defense against the
oxidative pressure that they experience within the macrophage,
and thus, an increased potency is obtained.
IC50 (μM)
cytotoxicity (%)
axenic
entry amastigotes promastigotes
intracellular
amastigotes
at 2 ×
at IC50
IC50
3a
3b
3c
3d
3e
3f
502
198
65
1398
450
16.63
32.91
12.31
10.72
10.95
11.08
9.40
14.82
40.51
25.96
17.57
6.66
17
26
1224
1030
4634
4300
744
524
83
927
446
73
19.52
33.14
20.22
26.96
27.79
24.63
24.40
3g
3h
3i
48
24
22
0.46
221
37
335
15.11
12.03
28.84
16.02
3j
2774
97
3k
36
a
3l
0.061
0.465
a
Compound 3l is amphotericin B.
The ligands were also checked for compliance to the
Lipinsky rule of five, and the results are summarized in Table 3.
The rule states that a molecule likely to be developed as an
orally active drug candidate should show no more than one
violation of the following four criteria: It should not have more
than five hydrogen bond donors, it should not have more than
apparently only a weak or no correlation between log IC50 and
binding energies. This may be due to differences in the
bioavailability of these compounds.
Treatment options for VL are limited. A high level of parasite
resistance has been developed for the available drug sodium
245
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ACS Medicinal Chemistry Letters
Letter
Funding
Table 3. Molinspiration Calculation of Properties for the
Lipinsky Rule
a
This work was carried out under financial support from the
Council of Scientific and Industrial Research (Grant 01(2260)/
08/EMR-II), the Department of Science and Technology
(Grant SR/S1/OC-66/2009), New Delhi, and partly supported
by NIAID, NIH Grant No. 1P50AI074321. R.K.V. is grateful to
CSIR, New Delhi, for a senior research fellowship; G.K.V. is
thankful to UGC, New Delhi, for a junior research fellowship;
and V.K.P. is thankful to the Indian Council of Medical
Research for a senior research fellowship.
entry nViol natoms miLog P
MW
nON nOHNH nrotb
acceptable
range→
<5
<500
<10
<5
3a
0
0
0
0
0
0
0
0
0
0
0
23
22
20
19
19
20
22
22
25
22
22
3.805
3.87
326.37
379.27
290.34
339.21
355.27
306.40
314.40
334.82
346.40
306.34
322.41
4
3
4
3
2
3
3
3
3
3
2
0
0
0
0
0
0
0
0
0
0
0
4
3
3
2
2
3
3
3
3
2
2
3b
3c
3d
3e
3f
2.627
3.071
3.713
3.269
3.818
4.048
4.956
4.156
4.798
Notes
The authors declare no competing financial interest.
3g
3h
3i
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■
Corresponding Author
*Tel: +91-361-2582203. Fax: +91-361-2582249. E-mail:
vdubey@iitg.ernet.in (V.K.D.). Tel: +91-542-6702502. Fax:
+91-542-2368127. E-mail: mssinghbhu@yahoo.co.in (M.S.S.).
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