Synthesis, antitubercular activity, and molecular modeling studies of analogues of isoliquiritigenin and liquiritigenin, bioactive components from Glycyrrhiza glabra

Article abstract of DOI:10.1007/s00044-015-1401-1

Isoliquiritigenin (ISL, 1) and liquiritigenin (LTG, 2) were isolated from the rhizomes of Glycyrrhiza glabra. In an attempt to develop potent and selective antituberculosis agents, a series of ISL analogues were synthesized mainly via acid- and base-catalyzed Claisen-Schmidt condensation reaction for their antitubercular activity. Compared to ISL (MIC = 25 μg/mL), analogues 5, 8, and 10 showed similar antitubercular activity, but, interestingly, 6, 7, and 15 exhibited twofold higher activity (MIC = 12.5 μg/mL) over ISL, against Mycobacterium tuberculosis. Among the LTG derivatives, LTG 4′-acetate and LTG-oxime were found to be as active (MIC = 25 μg/mL) as LTG. It is the first report on antimycobacterial activity of these ISL- and LTG-based derivatives. Molecular docking and in silico ADME studies revealed that compounds 6, 7, and 15 are potent inhibitors of M. tuberculosis H₃₇Rv alanine dehydrogenase and showed compliance with standard parameters of drug likeness.

Full text of DOI:10.1007/s00044-015-1401-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1401-1
ORIGINAL RESEARCH
Synthesis, antitubercular activity, and molecular modeling studies
of analogues of isoliquiritigenin and liquiritigenin, bioactive
components from Glycyrrhiza glabra
Rashmi Gaur1 Jay Prakash Thakur2 Dharmendra K. Yadav3
•
•
•
Deepak Singh Kapkoti1 Ram Kishor Verma4 Namita Gupta4
Feroz Khan3 Dharmendra Saikia2 Rajendra Singh Bhakuni1
•
•
•
•
•
Received: 10 July 2014 / Accepted: 29 June 2015
Ó Springer Science+Business Media New York 2015
Abstract Isoliquiritigenin (ISL, 1) and liquiritigenin
(LTG, 2) were isolated from the rhizomes of Glycyrrhiza
glabra. In an attempt to develop potent and selective
antituberculosis agents, a series of ISL analogues were
synthesized mainly via acid- and base-catalyzed Claisen–
Schmidt condensation reaction for their antitubercular
activity. Compared to ISL (MIC = 25 lg/mL), analogues
5, 8, and 10 showed similar antitubercular activity, but,
interestingly, 6, 7, and 15 exhibited twofold higher activity
(MIC = 12.5 lg/mL) over ISL, against Mycobacterium
tuberculosis. Among the LTG derivatives, LTG 40-acetate
and LTG-oxime were found to be as active (MIC = 25 lg/
mL) as LTG. It is the first report on antimycobacterial
activity of these ISL- and LTG-based derivatives. Molec-
ular docking and in silico ADME studies revealed that
compounds 6, 7, and 15 are potent inhibitors of M. tuber-
culosis H37Rv alanine dehydrogenase and showed com-
pliance with standard parameters of drug likeness.
Keywords Glycyrrhiza glabra Á Isoliquiritigenin Á
Liquiritigenin Á Derivative Á Mycobacterium tuberculosis
Introduction
Tuberculosis (TB) caused by Mycobacterium tuberculosis
infects\30 % of the global population. Nearly two million
people die with a global case fatality rate of 23 % and
reaching[50 % in some African countries. The worldwide
resurgence of TB is due to two major problems, first by the
acquired immunodeficiency syndrome (AIDS) epidemic
which started in the mid-1980s, and second, the outbreak of
multidrug-resistant tuberculosis (MDR-TB). Emergence of
drug-resistant strains of M. tuberculosis has led to
increased concern on current chemotherapy regimens. A
worldwide increase in the incidence of morbidity and
mortality from tuberculosis prompted WHO to declare this
disease a global emergency in the early 1990s (De Souza,
2006; Ballell et al., 2005; Janin, 2007).
Rashmi Gaur and Jay Prakash Thakur have contributed equally to this
work.
Although plant species have not yielded any antibacte-
rial compounds of comparable potency to the antibiotics
produced by microorganisms so far, many plant extracts
have been tested for activity against microorganisms in
anticipation that highly active compounds will be found
because natural products are a proven templates for the
development of new scaffolds of drugs (Cragg et al., 1997;
Newmann et al., 2000). Another important aspect that
strengthens the search of antimicrobial compounds from
natural products is the recent report about presence of
compounds that do not have antimicrobial activity per se,
but act as potent synergistic agents together with other
antimicrobials (Stermitz et al., 2000; Lechner et al., 2008).
Moreover, advances in identifying new antibiotics from
& Rajendra Singh Bhakuni
bhakunirs2000@gmail.com
1
Medicinal Chemistry Division, Central Institute of Medicinal
and Aromatic Plants (CSIR), P.O.-CIMAP, Kukrail Picnic
Spot Road, Lucknow 226015, U.P., India
2
Biotechnology Division, Central Institute of Medicinal
and Aromatic Plants (CSIR), Lucknow 226015, India
3
Metabolic and Structural Biology Department, Central
Institute of Medicinal and Aromatic Plants (CSIR),
Lucknow 226015, U.P., India
4
Analytical Chemistry Division, Central Institute of Medicinal
and Aromatic Plants (CSIR), Lucknow 226015, India
123

Med Chem Res
plant sources and expanding antibiotic chemical diversity
are providing crucial leads for new drugs.
from G. glabra against Staphylococcus aureus, Mycobac-
terium smegmatis, M. tuberculosis have been reported
(Mitscher et al., 1980; Gupta et al., 2008). Phenolics,
isoliquiritigenin (ISL, 1), and liquiritigenin (LTG, 2) were
previously reported from the roots of G. glabra (Gaur
et al., 2010, 2014).
Licorice is the name applied to the roots and stolons of
some Glycyrrhiza species. It is one of the oldest traditional
Chinese medicines, used as a tonic, anticoagulative,
expectorant, antitussive agent (Tang and Eisenbrand,
1992), in the treatment of gastric disease (Krausse et al.,
2004; Chin et al., 2007), and also used worldwide as a
natural sweetener as well as a flavoring additive in various
preparations. Glycyrrhizin, a major compound of Gly-
cyrrhiza glabra, is responsible for the sweetness and var-
ious biological properties of the roots (Nomura and Fukai,
1998). The plant contains a variety of compounds including
bioactive triterpenoids and phenols, which have mainly
antimicrobial, antioxidant, antiulcer, anti-inflammatory,
and antifungal activity. Antimicrobial activities of flavo-
noids, glabridin, and several glabridin analogues obtained
Chalcones (1,3-diaryl-2-propen-1-ones) with an enone
system between two aromatic rings (Fig. 1a) constitute an
important class of natural products which are considered as
precursors for the preparation of various flavonoids and
exhibit interesting pharmacological activities (Asl and
Hosseinzadeh, 2008; Gaur et al., 2014, 2015, Hans et al.,
2010; Lin et al., 2002). This prompted us to synthesize
derivatives of ISL and LTG in order to study their
antimicrobial effects. The indoles are widely distributed in
nature, and they possess a variety of significant biological
activities (Andreani et al., 2008; Biswal et al., 2012;
Fig. 1 a Basic skeleton of
a
O
O
O
chalcone derivatives responsible
for their biological activity.
b Antimycobacterial activity of
phenolics 1, 2, their analogues,
and indole-based chalcones
R
R
OH
OH
HO
N
H
N
H
3'
3'
4'
OH
2'
1'
4' OH
2'
1'
1
8
3
OH 7
6
O
5'
OH
O
2
HO
4
2
5
'
b
β
α
6'
6'
1
4
5
3
6
5
O
Isoliquiritigenin (1)
Liquiritigenin (2)
100 >100
>100 >100>100 >100>100
100
>100
100
90
80
70
60
50
40
30
20
10
0
25
25
25
25
12.5 12.5
25
25
2
25
1
12.5
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19
123

Med Chem Res
Grugni et al., 2006). So we have made changes in the
structure of ISL by substituting with indole moiety for
value addition.
according to the reported procedure (Guantai et al., 2011;
Bianco et al., 2003, 2004; Gaur et al., 2014; Ohkatsu and
Satoh, 2008) and their physical properties are described in
Table 1.
In continuation to our previous studies on bioactive
compounds from G. glabra (Gaur et al., 2010, 2014, 2015),
we tried to synthesize and explore the antimicrobial
potential of ISL and LTG derivatives (3–19) supported by
their in silico and in vitro activity studies (Fig. 1b) in the
present study.
Spectral data of compounds (5, 7, and 10)
4-Benzoyloxy-20,40-dimethoxychalcone (5) IR mmax (KBr):
1739 (ester CO), 1649 (chalcone CO), 1610, 1558, 1452,
;
1328, 1250, 1209, 1058 (aromatic) cm-1 1H NMR
(CDCl3, 300 MHz): d 3.85, 3.89 (s, 3H each, 29 OCH3),
6.50 (1H, d, J = 1.8 Hz, H-30), 6.57 (1H, dd, J = 8.7,
1.8 Hz, H-50), 7.25 (2H, d, J = 8.7 Hz, H-3, H-5), 7.49
(1H, d, J = 15.9 Hz, H-a), 7.56(2H, d, J = 7.8 Hz, H-400,
H-600), 7.64 (H-2, d, J = 8.7 Hz, 2H, H-6), 7.67 (1H, m,
H-500), 7.69 (1H, d, J = 15.9 Hz, H-b), 7.77 (1H, d,
J = 8.7 Hz, H-60), 8.20 (2H, d, J = 7.8 Hz, H-30, H-70);
13C NMR, DEPT (CDCl3, 75 MHz): d 55.98 (OCH3),
56.21 (OCH3), 99.07 (CH, C-30), 105.69(CH, C-50), 122.61
(CH, C-3, CH, C-5, C, C-10), 127.83 (CH, C-a), 129.05
(CH, C-2, CH, C-6), 129.69 (C, C-200), 129.86 (CH, C-400,
CH, C-600), 130.63 (CH, C-300, CH, C-700), 133.35 (CH,
C-60), 133.75 (C, C-1), 134.19 (CH, C-500), 141.25 (CH,
C-b), 152.55(C, C-4), 160.89 (C, C-20), 164.70 (C, C-40),
165.3 (C = O, C-100), 190.73 (C = O); ESI–MS, MeOH
(positive): m/z 389 [M ? H]?, 411 [M ? Na]? (negative):
m/z 387 [M – H]-, C24H20O5. CAS Registry Number:
619313-51-8.
Materials and methods
Extraction and isolation
Rhizomes of G. glabra L. (Fabaceae) were collected from
CIMAP research farm, Lucknow, India, in March 2011.
The plant was identified by a taxonomist at Botany and
Pharmacognosy Department, CIMAP, Lucknow, where a
voucher specimen #7401 was deposited. ISL and LTG
were isolated according to reported procedure (Gaur et al.,
2014).
HPLC analysis
HPLC analysis was performed using a Shimadzu LC-10AD
liquid chromatography equipped with two LC-10A pumps
controlled by a CBM-10 interface module, SPD-M10A VP
diode array detector, and a SIL-10ADVP auto injector. Data
were collected and analyzed using a Class LC-10 Work
Station. The samples were analyzed by using reverse-phase
chromatography on Waters Spherisorb ODS2 (250 9
4.6 mm i.d., 10 mm) column using binary gradient elution
with acetonitrile and water containing 0.1 % TFA mobile
phase (30:70) at a flow rate of 0.6 mL/min, a column tem-
perature of 25 °C, and UV detection at k 254 nm.
1-(500-Bromo-200, 400-dimethoxyphenyl)-2,3-dibromo-3-phenyl-
propan-1-one (7) A solution of bromine (0.03 mol,
1.5 mL) and chloroform (2 mL) was added drop wise with
continuous stirring at room temperature to a solution of 6
(0.268 g, 1 mmol) in chloroform (20 mL) in 30 min.
Evaporation of solvent afforded a semisolid, which was
washed with water several times, and triturated with light
petroleum ether 40–60 °C to give solid product, which was
crystallized in ethylacetate–hexane to yield 7 (in 90 %
yields). IR mmax (KBr): 3433, 1666 (CO), 1589, 1468, 1396,
Synthesis of ISL analogues
1271, 1215 (aromatic), 698 (C–Br) cm-1 1H NMR
;
Because of poor yield of ISL of the plant, its derivatives
(300 MHz, CDCl3): d 3.98 (3H, s, OCH3), 4.05 (3H, s,
(3–6, 8–15) were chemically synthesized (Scheme 1)
O
O
R2
R1
R2
a or
b
R1
3 +
+
R1
c/d
O
R2
H
CH3
O
4,5
3,7-15
4+e
OMe
Br
MeO
Br
6
Br
O
Scheme 1 General routes used for the chalcone synthesis. Reagents and conditions: (a) SOCl2–EtOH, RT, 2 h, 87 %; (b) NaOH–EtOH, RT,
2–4 h, 58–93 %; (c) Ac2O–C5H5N, RT, 0.5 h, 91 %; (d) PhCOCl–C5H5N, RT, 0.5 h, 96 %; (e) Br2, CH3COOH, RT
123

Med Chem Res
Table 1 Antimycobacterial activity of ISL, LTG, and their analogues against M. tuberculosis H37Rv strain by BACTEC-460 assay
MIC
Yield
(%)
Structure of compounds
Physical data
MP (°C)
(µg/mL)
OH
OH
O
HO
25
25
Yellow powder
208–210 0.00056
206–208 0.0006
1
2
OH
O
O
HO
Cream powder
Orange crystals
OH
OCH
H
CO
3
3
n.a.
91–92
70–71
90–91
87
91
96
O
3
OCOCH
3
OCH
H
CO
3
3
100
25
Light orange crystals
Dark orange crystals
O
4
OCOC
H
5
6
OCH
O
H
CO
3
3
5
OCH
O
H
CO
3
3
12.5
12.5
Yellow solid
65–66
93
90
6
OMe
Br
MeO
White shiny crystals
118–119
Br
O
Br
7
OH
HO
25
n.a.
25
Yellow powdery crystals
Yellow crystals
145–146
50–51
8
O
8
OCH
3
OCH
O
H
CO
3
3
92
83
9
CH
3
CH
3
OCH
H
CO
3
3
Yellow viscous
Viscous
O
10
NH
Cl
n.a.
n.a.
Yellow fluffy crystals
Yellow shiny crystals
180–181
110–111
60
85
O
11
12
NH
Cl
O
123

Med Chem Res
OCH3), 5.63 (1H, d, J = 11.4 Hz, H-3).6.13(1H, d,
J = 11.4 Hz, H-2), 6.51 (1H, s, H-300), 7.39–7.49 (5H, m,
H-20, H-30, H-40, H-50, H-60), 8.22 (1H, s, H-600); 13C and
DEPT NMR (75 MHz, CDCl3): d 50.90 (CH, C-3), 52.02
(CH, C-2), 56.89 (OCH3), 57.02 (OCH3), 96.62 (CH,
C-300), 104.33 (C, C-500), 118.86 (C, C-100), 128.72 (CH,
C-30, C-50), 129.21 (CH, C-20, C-60), 129.51 (CH, C-40),
137.05 (CH, C-600), 139.15 (C, C-10), 160.85 (C, C-200),
161.69 (C, C-400), 190.01 (C, C-1); HMBC correlations
(Fig. 2): C-1 with H-2/H-3/H-600, C-2 with H-3, C-3 with
H-2/H-20/H-60, C-10 with H-2/H-3/H-30, H-50, C-100 with
H-300/H-600, C-300 with 29 OCH3, C-500 with H-300/H-600;
ESI–MS, MeOH (positive): m/z 504 [M ? H]?, 526
[M ? Na]? (negative): m/z 503 [M - H]-, C15H15O3Br3.
was poured on ice (100 g) and neutralized with dilute
hydrochloric acid, which provided 11, 12 (Manna et al.,
1999).
General method for the synthesis of indole chalcones
(13–15) Chalcones 13, 14, and 15 were obtained by
reacting 3-acetylindole (1.59 g, 0.01 mol) and 2,
4-dimethoxyaldehyde (1.66 g, 0.01 mol), 4-hydroxyalde-
hyde (1.22 g, 0.01 mol), and o-tolualdehyde (1.20 g,
0.01 mol), respectively, by dissolving in methanol (35 ml),
and 1.0 mL SOCl2 was added at RT with constant stirring.
After completion of the reaction, it was poured on ice
(100 g). The reaction was completed in about 2 h, and the
solid obtained was separated by filtration, dried, and
crystallized in ethyl acetate hexane to provide respective
chalcones 13–15 (Manna et al., 1999; Kumar et al., 2010;
Gaur et al., 2015).
4-Isopropyl-20,40-dimethoxychalcone (10) IR mmax (KBr):
1653 (chalcone CO), 1605, 1463, 1327, 1254 (aromatics),
1212, 1162 (isopropyl) cm-1; 1H NMR (CDCl3, 300 MHz):
d 1.26 (6H, d, J = 6.9 Hz, H3-8, H3-9), 2.93 (1H, m, H-7),
3.86, 3.89 (s, 3H each, 29 OCH3), 6.49 (1H, d, J = 2.1 Hz,
H-30), 6.56 (1H, dd, J = 8.4, 2.1 Hz, H-50), 7.28 (2H, d,
J = 6.0 Hz, H-3, H-5), 7.48 (1H, d, J = 15.9 Hz, a-H),
7.53 (2H, d, J = 7.8 Hz, H-2, H-6), 7.67 (1H, d,
J = 15.9 Hz, b-H), 7.75 (1H, d, J = 8.4 Hz, H-60); 13C
NMR, DEPT (CDCl3, 75 MHz): d 24.22, 24.44 (CH3, C-8/
C-9), 34.26 (CH, C-7), 55.83 (OCH3), 56.12 (OCH3), 99.02
(CH, C-30), 105.65 (CH, C-50), 122.68 (C, C-10), 126.75
(CH, C-a), 127.66 (CH, C-2, C-6), 128.86 (CH, C-3, C-5),
133.08 (CH, C-60), 133.50 (C, C-1), 142.59 (CH, C-b),
151.70 (C, C-4), 160.81 (C, C-20), 164.56 (C, C-40), 191.06
(CO); ESI–MS, MeOH (positive): m/z 311 [M ? H]?, 333
[M ? Na]? (negative): m/z 309 [M - H]-, C20H22O3. CAS
Registry Number: 100246032-3.
Derivatization of LTG derivatives
The plant derived LTG was derivatized to four analogues as
per Scheme 2. LTG-oxime (16), LTG 7,40-diacetate (17),
LTG 40-acetate (18), and LTG 7,40-dibenzoate (19) were
obtained by acetylation and benzoylation of LTG according
to reported procedure (Gaur et al., 2010, 2014, 2015).
Bioevaluation
BACTEC radiometric susceptibility assay
Mycobacterium tuberculosis H37Rv (ATCC 27294) used in
this screening was obtained from the National JALMA
Institute for Leprosy and other Mycobacterial Diseases
General method for the synthesis of indole chalcones 1-(200-
Chlorophenyl)-3-indolyl-2-propen-1-one (11) and 1-(400-
chlorophenyl)-3-indolyl-2-propen-1-one (12). Chalcones
11 and 12 were obtained by condensing indole-3-carbox-
aldehyde (1.45 g, 0.01 mol) with 2-chloroacetophenone
(1.54 g, 0.01 mol) and 4-chloroacetophenone (1.54 g,
0.01 mol), respectively, by dissolving in methanol (35 ml)
and 40 % NaOH–methanol (5 ml) at RT with constant
stirring (Scheme 1). After completion of the reaction, it
¨
(ICMR), Agra, India, and maintained on Lowenstein–Jen-
sen media slant at 37 °C. After 21 days of incubation,
bacterial cells were scraped from slants and transferred in
1.0 mL of BACTEC diluting fluid and made the complete
homogenized suspension by vortexing with glass beads
(2 mm diameter). The suspension was allowed to stand for
a few minutes to permit sedimentation of the bacterial
clumps if any. The turbidity of the homogenous suspension
was adjusted to McFarland standard 1.0 with diluting fluid.
A BACTEC 12B vial (Becton–Dickinson) was injected
with 0.1 mL of this suspension. This vial was used as
primary inoculum after the growth index (GI) reached a
value of about 500 (approximately 1.0 9 106 cfu/mL).
Briefly, 0.1 mL of bacterial suspension from the primary
inoculum culture vial (GI 500) was injected into test
compound-containing vials using 1.0 mL insulin syringe.
To comply with 1 % proportion method, 0.1 mL of pri-
mary inoculum was added to 9.9 mL BACTEC diluting
fluid to obtain 1:100 dilutions. From this, 0.1 mL was
injected into two 12B media vials and used as control. The
H
H
H
OMe
MeO
H
Br
O
H
Br
Br
H
H
H
Fig. 2 HMBC correlation diagram of analogue of ISL derivative (7)
123

Med Chem Res
OH
OCOCH3
OH
OCOCH
3
CH3COO
OH
O
HO
O
O
O
HO
HO
Reflux, 3h
Reflux, 2h
Acetylation, rt
f
O
O
O
Liquiritigenin (2)
Liquiritigenin-4'-acetate (19)
Liquiritigenin-7,4'-diacetate (17)
I
soliquiritigenin (1)
h
OH
Benzoylation
g
O
HO
OCOC
H
5
6
O
H C OCO
5
6
NOH
LTG-Oxime (16)
O
Liquiritigenin-7,4'-dibenzoate (18)
Scheme 2 Reagents and conditions: (f) Ac2O-C5H5N, RT, 0.5 h, (g) PhCOCl-C5H5N, RT, 0.5 h, (h) NH2OHÁHCl, CH3COONa, EtOH, reflux
vials were incubated at 37 °C, and the GI was recorded
every 24 h in a BACTEC 460 TB instrument (Becton–
Dickinson). Once the GI of the control vial (1:100) reached
30, then the GI values of the test (compounds containing)
vials were compared with that of control vials based on
difference in growth (DGI). The result was interpreted as
follows: If the difference (called as DGI) of the current GI
from previous day GI in the case of drug containing vials is
lower than the DGI of 1:100 control vial for the same
period, then the test compound is termed as active against
MTB or otherwise inactive. The twofold serial dilution
technique was used to assess the minimum inhibitory
concentration (MIC) of a test compound.
of the other parameters were assigned their default values
(Yadav et al., 2012, 2013a, b).
Results and discussion
Chemistry
ISL (1) and LTG (2) were isolated from the chloroform/ethyl
acetate fraction of rhizomes, identified by spectral analysis of
their IR, 1H, 13C NMR, DEPT-135 and electrospray ionization
mass spectroscopy and comparison with the data reported in
literature (Gaur et al., 2010, Gaur et al., 2014). Because of low
yield of ISL (1) in the plant, we followed a direct synthetic
approach for other analogues (Scheme 1). 20,40-Dimethoxy-4-
hydroxychalcone (3) was synthesized via acid-catalyzed
Claisen–Schmidt condensation by adding thionyl chloride to a
mixture of 2, 4-dimethoxyacetophenone and 4-hydroxyben-
zaldehyde in ethanol at room temperature (Jaypal et al., 2010).
The resulted chalcone 3 was further acetylated to 4-acetoxy-
20,40-dimethoxychalcone (4) and benzoylated to derivative,
4-benzoyloxy-20,40-dimethoxychalcone (5). 20,40-Dimethoxy-
chalcone (6) and 20,40-dihydroxychalcone (8) were obtained
by reacting 2, 4-dimethoxyacetophenone/2, 4-dihydroxyace-
tophenone and benzaldehyde in the presence of ethanolic
NaOH. 20,40-Dimethoxychalcone (6) gave a tribromo product
7 by electrophilic substitution reaction in the benzene ring and
addition reaction at the chalcone double bond by adding
bromine in acetic acid at room temperature (Scheme 1).
The chalcone 8, reported from G. astragalin, has shown
its presence (TLC) in the ethyl acetate fraction of G. glabra
roots. The ISL derivative, 4-isopropyl-20,40-dimethoxychal-
cone (10) was synthesized by reacting 2, 4-dimethoxyace-
tophenone and 4-isopropylbenzaldehyde. Indole-based
chalcones 11–15 were synthesized using respective alde-
hydes and ketones in acidic and basic conditions
(Scheme 1). The new active chalcones 7 and 15 were
Molecular docking studies
To find the possible bioactive conformations of chalcone
derivatives, the Sybyl X 1.3 software (Certara, USA)
interfaced with Surflex–Dock program was used to dock
the compounds into the active site of the known antitu-
bercular target protein alanine dehydrogenase (PDB ID:
2VOE) (Tripathi and Ramachandran, 2008) due to avail-
ability of experimental 3D protein crystallographic struc-
tural data in the RCSB Protein Data Bank (PDB; www.
rcsb.org/). The program automatically docks ligand into the
binding pocket of a target protein by using a protocol-based
algorithm and empirically produced scoring function. The
protocol is a very important and necessary factor for
docking algorithm and works as a computational repre-
sentation of proposed ligand that interacts into the binding
site. Surflex–Dock scoring function has several factors that
play an important role in the legend–receptor interaction, in
terms of hydrophobic, polar, repulsive, entropies, and sal-
vation, and it is a worldwide well-established and recog-
nized method. The most standard docking protocols have
ligand flexibility in the docking process, while counting the
protein as a rigid structure. During the docking process, all
123

Med Chem Res
characterized based on their 1D (1H, 13C, DEPT), 2D
(COSY, HSQC, HMBC) NMR correlations, IR, and mass.
The present study deals with the isolation, identification
of ISL and LTG from G. glabra, and synthesis of their
derivatives for antituberculer evaluation. It is the first
report on antimycobacterial activity of ISL derivatives
3–10, indole-based chalcones 11–15, and LTG derivatives
16–19. 20,40-Dimethoxychalcone (6) and new chalcones
1-(500-bromo-200,400-dimethoxyphenyl)-2,3-dibromo-3-phenyl-
propan-1-one (7) and trans-1-indolyl-3-(20-methylphenyl)-
2-propen-1-one (15) possessed two times better activities
over the parent compound (1).
based chalcones (11–15), trans-1-indolyl-3-(20-methyl-
phenyl)-2-propen-1-one (15) possessed antitubercular
activity twofold higher (MIC = 12.5 lg/mL) over the
parent chalcone ISL (1). In the indole chalcones 11–15, the
deoxygenation and methylation in the phenyl ring might be
responsible for the activity in trans-1-indolyl-3-(20-
methylphenyl)-2-propen-1-one (15). A recent study repor-
ted that ISL (1) possessed MIC value 25 lg/mL (Chok-
chaisiri et al., 2009) in agreement with the data of this
study.
Among LTG (2) and its derivatives, LTG-oxime (16)
and LTG 40-acetate (18) were found to be as active as the
parent flavonoid 2 (MIC = 25 lg/mL). LTG 7,40-diacetate
(17) was moderately active (MIC = 100 lg/mL), whereas
LTG 7,40-dibenzoate (19) showed no activity at 100 lg/mL
concentration. Rifampicin was used as a positive control.
ISL and LTG were also previously reported as anti-in-
flammatory (Kobayashi et al., 1995; Lin et al., 2002), hep-
atoprotective (Gaur et al., 2010), antimycobacterial, and
antitumor agents (Jaypal et al., 2010). However, this is the
first report on antimycobacterial activity of their derivatives.
The in vitro antimycobacterial activity of ISL, LTG, and
their derivatives was evaluated against M. tuberculosis
H37Rv strain by BACTEC-460 radiometric susceptibility
assay. Table 1 shows the minimal inhibitory concentration
(MIC) values of all 19 test compounds. The MICs of ISL
and its derivatives, 4-benzoyloxy-20,40-dimethoxychalcone
(5), 20,40-dihydroxychalcone (8), and 4-isopropyl-20,40-
dimethoxychalcone (10), were 25 lg/mL, whereas chalcone
20,40- dimethoxychalcone (6) (MIC, minimal inhibitory
concentrations = 12.5 lg/mL) and its new derivative 1-(500-
bromo-200,400-dimethoxyphenyl)-2,3-dibromo-3-phenylpropan-
1-one (7) (MIC = 12.5 lg/mL) were approximately twice
as active as ISL (1) (MIC = 25 lg/mL). Derivative 4 was
moderately active with MIC value 100 lg/mL. No activity
was detected at 100 lg/mL concentrations in derivatives
20,40-dimethoxy 4-hydroxychalcone (3) and 4, 20,40-
trimethoxychalcone (9). This shows that ether groups in
ISL analogues 5, 8, and 10 had a minimal effect on anti-
tubercular activity, but the presence of methoxy groups at
position 20,40 and absence of OH group (deoxygenation) at
position 4 in ISL analog 8 and a bromine derivative of ISL
(6) showed enhancement in the activity. Among indole-
Molecular docking revealed high binding affinity
The aim of the molecular docking study was to show the
binding site interactions and active conformation and to
elucidate the possible mechanism of action. In the studied
work, the orientation and binding affinity (in terms of
Sybyl total docking score) of active compounds 6, 7, and
15 on known tuberculosis target alanine dehydrogenase
(PDB: 2VOE) were explored. The results of docking sug-
gest that active compounds 6, 7, and 15 bind well to the
target alanine dehydrogenase. The molecular interactions
were measured in terms of Sybyl total docking score and
Fig. 3 Molecular docking studies of active compounds against antituberculosis target alanine dehydrogenase of M. tuberculosis H37Rv (PDB
ID: 2VOE). Compounds 6, 7, and 15 docked well on alanine dehydrogenase with total docking score of 5.5193 (a), 4.9948 (b), and 4.1852 (c)
123

Med Chem Res
Table 2 Docking scores (total score) of ISL (1), LTG (2) and their analogues against antituberculosis target alanine dehydrogenase (PDB ID:
2VOE)
Compound Total scorea Binding site amino acids within 4 A
H-bond forming
residue
Length of No. of
˚
H-bond A H-bond
˚
1
4.8134
CYS-212, GLY-213, ARG-185, ASN-188, GLY-189, GLY-191,
ARG-214, ARG-185, ILE-186, ASN-188, GLY-189M MET190,
GLY-191, PHE-211, ARG-214, ILE-215
GLY-189
ASN-188
ARG-185
ASN-188
ARG-185
2.0
1.9
1.9
1.9
1.9
3
2
6
4.8439
5.5193
GLY-189, ARG-214, ARG-185, ILE-186, ASN-188, GLY-189,
PHE-211, ILE-215, CYS-212, GLY-213
2
2
CYS-212, GLY-213, ARG-185, ASN-188, GLY-189, GLY-191,
PHE-211, ARG-214, ARG-185, ILE-186, ASN-188, GLY-189,
MET-190, GLY-191, PHE-211, ARG-214
ARG-214 ARG-214 1.9
2.2
7
4.9948
6.7638
CYS-212, GLY-213, GLY-189, GLY-191, ARG-214, ARG-185,
ASN-188, GLY-189, PHE-211, ARG-214
ARG-214
ARG-214
ARG-214
ARG-214
GLY-189
ARG-214
2.0
2.2
2.1
1.7
2.0
2.4
2.5
2
3
15
PHE-211, CYS-212, GLY-213, ARG-214, ARG-185, ASN-188,
GLY-189, PHE-211, ILE-215, CYS-212, ASN-188, GLY-189,
GLY-191, PHE-211, ARG-214
Rifampcin 4.1852
GLU-210, PHE-211, CYS-212, PHE-211, CYS-212, GLY-213,
ARG-214, ASN-188, GLY-189, PHE-211, CYS-212, GLY-213,
ARG-214, ASN-188, CYS-212, ARG-214, ASN-188, GLY-189,
ARG-214, ASN-188, PHE-211, CYS-212, ARG-214
2
a
Surflex–Dock scores (total scores) were expressed in -log10 (Kd) units to represent binding affinities
revealed the binding site interacting amino acid residues of
the target (Fig. 3).
studied compounds 6, 7, and 15 binds well with the M.
tuberculosis H37Rv target alanine dehydrogenase through
hydrogen and electrostatic bonds and this could be the
reason for higher molecular stability and activity (Table 2).
For comparison of binding affinity and docking score,
known antituberculosis drug rifampicin was selected for
docking simulation. The docking results of rifampicin with
that of targetalanine dehydrogenase of M. tuberculosis
H37Rv showed a significant binding affinity as indicated by
docking score 4.1852 (Table 2). This docking result was
considered as positive control parameter and later was used
to compare the significance level of active compounds 6, 7,
and 15 on the basis of their docking scores. Parent com-
pounds ISL (1) and LTG (2) also showed a high binding
affinity as indicated by docking score of 4.8134 and
4.8439, respectively, and revealed the formation of
hydrogen bond between the ligand and receptor. The
docking results of compound 6 showed a high docking
score indicated by total score of 5.5193 and formed the two
Assessment through electronic pharmacokinetic
parameters
The pharmacokinetics parameters such as ADME/Tox
(absorption, distribution, metabolism, excretion, and toxi-
city) are important descriptors for human therapeutic use of
drugs. The ADME descriptors were calculated by Qikprop
¨
v.3.2 software (Schrodinger, USA, trial version) and
evaluated for compliance with their standard ranges.
Screening of active parent compounds, namely ISL (1) and
LTG (2), and their analogues, namely compounds 6, 7, and
15 for compliance with standard electronic ADME
parameters, e.g., log S for aqueous solubility, log IC50 for
˚
hydrogen bonds of length 1.9 and 2.2 A to the binding site
`
HERG (the human Ether-a-go-go-Related Gene)
basic residue arginine (Arg-214) (Fig. 3a). Similarly,
docking results of compound 7 also showed a high binding
affinity as indicated by total docking score 4.9948 and
K? Channel (potassium ion channel) blockage, apparent
Caco-2 (colon cancer cell line) permeability, log BB for
brain/blood barrier, apparent MDCK (Madin–Darby canine
kidney) membrane permeability, log Kp for skin perme-
ability, no. of metabolic reactions, log Khsa serum protein
binding, topological polar surface area (TPSA), predicted
percent (%) of human oral absorption, and predicted human
oral absorption through qualitative model, was evaluated.
The compound distribution in human was evaluated by
˚
formed the two hydrogen bonds of length 2.0 and 2.2 A to
the basic residue arginine (Arg-214) (Fig. 3b). Likewise,
compound 15 docking results also showed higher binding
affinity than rifampicin, as indicated by total docking score
of 6.7638 and formed three hydrogen bonds of length 2.1,
˚
1.7, and 2.0 A to the basic residue arginine (ARG-214) and
glycine (GLY-189) (Fig. 3c). Docking results showed that
123

Med Chem Res
following factors, e.g., log BB, permeability (apparent
Caco-2 and MDCK permeability, and log Kp for skin
permeability), the volume of distribution and plasma pro-
tein binding was evaluated by log Khsa. ADME results
showed that compound 7 violates the standard range for
aqueous solubility logs. Compounds 6, 7, and 15 showed
Caco-2 permeability and log BB for brain/blood barrier.
Compounds 6, 7, and 15 showed apparent MDCK perme-
ability and underlimit TPSA value for oral bioavailability
(Table 3).
Conclusions
The present study has shown that the ISL-type chalcones as
well as LTG-derived flavonoids could be used as possible
leads for the control of TB infections. Therefore, plants are
excellent sources or information centers of diverse molecules
to be modified for the discovery of human therapeutics.
Acknowledgments The authors are grateful to the, Director,
CIMAP for providing necessary facilities for this work. Financial
support from the National Medicinal Plant Board (NMPB), New
Delhi, is duly acknowledged. We acknowledge the Council of Sci-
entific and Industrial Research (CSIR), New Delhi, for financial
support through networking project GENESIS (BSC0121) at CSIR-
CIMAP, Lucknow.
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