Anti-tubercular activity and molecular docking studies of indolizine derivatives targeting mycobacterial InhA enzyme

Article abstract of DOI:10.1080/14756366.2021.1919889

A series of 1,2,3-trisubstituted indolizines (2a–2f, 3a–3d, and 4a–4c) were screened for in?vitro whole-cell anti-tubercular activity against the susceptible H37Rv and multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains. Compounds 2b–2d, 3a

Full text of DOI:10.1080/14756366.2021.1919889

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Anti-tubercular activity and molecular docking
studies of indolizine derivatives targeting
mycobacterial InhA enzyme
Katharigatta N. Venugopala, Sandeep Chandrashekharappa, Pran Kishore
Deb, Christophe Tratrat, Melendhran Pillay, Deepak Chopra, Nizar A. Al-
Shar’i, Wafa Hourani, Lina A. Dahabiyeh, Pobitra Borah, Rahul D. Nagdeve,
Susanta K. Nayak, Basavaraj Padmashali, Mohamed A. Morsy, Bandar
E. Aldhubiab, Mahesh Attimarad, Anroop B. Nair, Nagaraja Sreeharsha,
Michelyne Haroun, Sheena Shashikanth, Viresh Mohanlall & Raghuprasad
Mailavaram
To cite this article: Katharigatta N. Venugopala, Sandeep Chandrashekharappa, Pran
Kishore Deb, Christophe Tratrat, Melendhran Pillay, Deepak Chopra, Nizar A. Al-Shar’i, Wafa
Hourani, Lina A. Dahabiyeh, Pobitra Borah, Rahul D. Nagdeve, Susanta K. Nayak, Basavaraj
Padmashali, Mohamed A. Morsy, Bandar E. Aldhubiab, Mahesh Attimarad, Anroop B. Nair,
Nagaraja Sreeharsha, Michelyne Haroun, Sheena Shashikanth, Viresh Mohanlall & Raghuprasad
Mailavaram (2021) Anti-tubercular activity and molecular docking studies of indolizine derivatives
targeting mycobacterial InhA enzyme, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1,
1472-1487, DOI: 10.1080/14756366.2021.1919889
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 1472–1487
https://doi.org/10.1080/14756366.2021.1919889
RESEARCH PAPER
Anti-tubercular activity and molecular docking studies of indolizine derivatives
targeting mycobacterial InhA enzyme
Katharigatta N. Venugopalaa,b , Sandeep Chandrashekharappac, Pran Kishore Debd , Christophe Tratrata,
Melendhran Pillaye, Deepak Chopraf, Nizar A. Al-Shar’ig , Wafa Houranid, Lina A. Dahabiyehh , Pobitra Borahi,
Rahul D. Nagdevej, Susanta K. Nayakj, Basavaraj Padmashalik, Mohamed A. Morsya,l, Bandar E. Aldhubiaba
Mahesh Attimarada, Anroop B. Naira , Nagaraja Sreeharshaa,m, Michelyne Harouna, Sheena Shashikanthn,
Viresh Mohanlallb and Raghuprasad Mailavaramo
,
b
aDepartment of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa, Saudi Arabia; Department of
c
Biotechnology and Food Technology, Durban University of Technology, Durban, South Africa; Institute for Stem Cell Science and Regenerative
d
Medicine (inStem), Bangalore, India; Faculty of Pharmacy, Department of Pharmaceutical Sciences, Philadelphia University, Amman, Jordan;
eDepartment of Microbiology, National Health Laboratory Services, KZN Academic Complex, Inkosi Albert Luthuli Central Hospital, Durban,
f
g
South Africa; Department of Chemistry, Indian Institute of Science Education and Research Bhopal, Bhopal, India; Faculty of Pharmacy,
h
Department of Medicinal Chemistry and Pharmacognosy, Jordan University of Science and Technology, Irbid, Jordan; Department of
Pharmaceutical Sciences, School of Pharmacy, The University of Jordan, Amman, Jordan; Pratiksha Institute of Pharmaceutical Sciences,
Guwahati, India; Department of Chemistry, Visvesvaraya National Institute of Technology, Nagpur, India; Department of Chemistry, School of
i
j
k
l
Basic Science, Rani Channamma University, Belagavi, India; Faculty of Medicine, Department of Pharmacology, Minia University, El-Minia, Egypt;
mDepartment of Pharmaceutics, Vidya Siri College of Pharmacy, Bangalore, India; Department of Studies in Organic Chemistry, University of
n
o
Mysore, Mysore, India; Pharmaceutical Chemistry Division, Sri Vishnu College of Pharmacy, Bhimavaram, India
ABSTRACT
ARTICLE HISTORY
Received 29 December 2020
Revised 9 April 2021
Accepted 12 April 2021
A series of 1,2,3-trisubstituted indolizines (2a–2f, 3a–3d, and 4a–4c) were screened for in vitro whole-cell
anti-tubercular activity against the susceptible H37Rv and multidrug-resistant (MDR) Mycobacterium tuber-
culosis (MTB) strains. Compounds 2b–2d, 3a–3d, and 4a–4c were active against the H37Rv-MTB strain
with minimum inhibitory concentration (MIC) ranging from 4 to 32mg/mL, whereas the indolizines 4a–4c,
with ethyl ester group at the 4-position of the benzoyl ring also exhibited anti-MDR-MTB activity (MIC ¼
16–64 mg/mL). In silico docking study revealed the enoyl-acyl carrier protein reductase (InhA) and anthra-
nilate phosphoribosyltransferase as potential molecular targets for the indolizines. The X-ray diffraction
analysis of the compound 4b was also carried out. Further, a safety study (in silico and in vitro) demon-
strated no toxicity for these compounds. Thus, the indolizines warrant further development and may rep-
resent a novel promising class of InhA inhibitors and multi-targeting agents to combat drug-sensitive and
drug-resistant MTB strains.
KEYWORDS
Indolizine; mycobacterium
tuberculosis; InhA; docking;
X-ray crystal structure
1. Introduction
resistant tuberculosis (TDR-TB)6; the co-morbidities with acquired
immunodeficiency syndrome (AIDS)7,8 and the risks involved in
Tuberculosis (TB) is a communicable infectious disease and a
major cause of illness, particularly in low-income countries. It is
caused by the opportunistic bacillus Mycobacterium tuberculosis
(MTB) which primarily attacks the lungs (pulmonary) but may later
affect other parts (extra-pulmonary) of the body1. According to
the World Health Organisation (WHO), TB is considered as one of
the top 10 causes of death worldwide, and the leading cause of
death from a single infectious agent1. In 2019, TB resulted in
nearly 1.4 million deaths, including 208,000 deaths among human
immunodeficiency virus (HIV) positive patients2. HIV-infected
patients are 19 times more likely to develop TB than HIV-negative
subjects3,4. Several factors have contributed to the continuous
health threat of TB globally. This includes the development of
drug resistance such as multidrug-resistant tuberculosis (MDR-TB),
developing diabetes mellitus among TB patients9,10
.
Several therapeutics have been approved by the US Food and
Drug Administration (US-FDA) to enhance the treatment of TB.
Bedaquiline (approved in late 2012 under the FDA’s accelerated
review program) (Figure 1) was found to exhibit favourable anti-
TB effects and promising anti-TB action against MDR and XDR-TB
when combined with first-line or second-line anti-TB drugs, result-
ing in a shorter duration of treatment. However, bedaquiline suf-
fered from various adverse effects such as nausea, elongation of
QT interval, and drug interaction with Cytochrome P3A4 inducers
and inhibitors11. Delamanid (Figure 1) was the second anti-TB
agent approved by European Medicine Agency in late 2013.
Nevertheless, resistant MTB strains against both bedaquiline and
extensively drug-resistant tuberculosis (XDR-TB)5, and totally drug- delamanid have recently been reported, which acquired resistance
CONTACT Katharigatta N. Venugopala
Al-Ahsa 31982, Saudi Arabia; Pran Kishore Deb
19392, Jordan
kvenugopala@kfu.edu.sa
Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University,
prankishore1@gmail.com Department of Pharmaceutical Sciences, Philadelphia University, Amman
Supplemental data for this article can be accessed here.
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1473
Figure 1. Chemical structures of approved anti-TB drugs (Bedaquiline, Delamanid and Pretomanid) and anti-TB compounds undergoing clinical trial (Q203, TBA-7371
and SQ109).
as a result of prolonged duration of therapy12,13. Pretomanid was novel chemical entities having multiple modes of action is of para-
the last drug candidate from the TB drug pipeline to be approved mount importance in the treatment of MDR, XDR, and TDR-
by the FDA in 2019 for the treatment of MDR and XDR TB TB infections.
(Figure 1)14,15
.
This study stems from our interest in designing new potential
For more than 40 years, there has been no significant active anti-TB therapeutics by focussing on structural modifications of
research in the arena of anti-TB drug discovery despite the fact the indolizine scaffold which has demonstrated potential anti-
that currently available drugs are considered cytotoxic and have mycobacterial activity as previously reported by our group21–23
low efficacy due to the emergence of resistant bacterial strains16. and other researchers24–27. Previously, we identified a series of
A recent call for the development of novel potential therapeutic poly-substituted indolizine A21, B22, and C23 as promising anti-TB
agents to overcome MDR, XDR and TDR-TB have led to the discov- agents against MDR-TB (Figure 2). Based on our previously
ery of a limited number of anti-TB drug candidates17. Among reported structure-activity relationship (SAR), the nature and the
them, TBA-7371 (an azaindole) and Q203 or telacebac (an imidazo- position of substituents on the indolizine scaffold were found to
pyridine amide) are very promising drug candidates for the poten- be very sensitive to retain the anti-mycobacterial activity against
tial treatment of MDR and XDR TB (Figure 1). However, susceptible MTB strain as well as rifampicin and isoniazid-resistant
considering the high attrition rate of drug candidates during clin- clinical isolates of MTB. Approximately, two-thirds of the synthes-
ical development, there is an urgent need to intensify and ised indolizines A, B, and C displayed moderate to good potency
broaden the scope of the search for novel anti-TB lead structures against susceptible H37Rv MTB strain, whilst half of the active
by utilising validated new mycobacterial drug targets besides derivatives demonstrated good inhibitory activity against MDR-
those targeted by current anti-TB therapies. A promising emerging MTB strain. Indolizine represents a privileged scaffold for the
approach to overcome MDR-TB is the development of multi-tar-
development of bioactive compounds. Several synthetic indoli-
geting compounds in which a single molecule has the ability to zines have also been reported to possess a broad spectrum of
bind to different biological targets18. This concept is known as
pharmacological activities such as analgesic, anti-inflammatory,
polypharmacology and has been proven promising in terms of anticancer, antidiabetic, antihistaminic, COX-2 inhibition, antileish-
efficacy, synergistic effect, and adverse events, and in preventing
manic, antimicrobial, antimutagenic, antioxidant, antiviral,
larvicidal, herbicidal and a7 nAChR inhibitors, anti-Alzheimer, anti-
both drug-drug interaction and resistance insurgence18. For
instance, SQ109, a drug candidate in the TB drug development schizophrenic, anticonvulsant and inhibitors of various
pipeline, exhibits its anti-TB activity by acting on multiple targets
enzymes28–30
.
(Figure 1). SQ109 is a well-known MmpL3 inhibitor that also
Despite the structural resemblance in the main bicyclic core of
shows inhibitory activity against MenA and MenG MTB enzymes our designed compounds with Q203 and TBA-7371, to date, no
as well19. It is worth mentioning that recently several natural indolizine-based anti-TB drug candidates have entered preclinical
products have also been reported as lead molecules against trials. With regards to the above considerations and as part of our
clinical MDR strains of MTB20. Therefore, the discovery of continuous interest in the discovery of new potential anti-TB

1474
K. N. VENUGOPALA ET AL.
Figure 2. Indolizine structures with anti-TB activity developed by our group.
drugs31–41 we report herein the anti-mycobacterial activity of a LC-MS, and elemental analysis as reported earlier from our
series of 1,2,3-trisubstituted indolizines of type D with the aim of research group42. The purity of the compounds was ꢀ99% as
expanding the current knowledge on our previously reported SAR
by exploring the influence of substituents at 1, 2 and 3-position
of the indolizine scaffold that might play a critical role in the anti-
TB activity against H37Rv strain ATCC 25177 and MDR strains of
MTB. Furthermore, the potential mechanism of action of the title
compounds is suggested based on their anti-tubercular profile in
parallel with the molecular modelling analyses. In addition, the
ADMET and toxicity characteristics of the most promising indoli-
zines are also presented.
measured by HPLC.
2.1.2. General procedure for the preparation of diethyl-3-(4-substi-
tutedbenzoyl)indolizine-1,2-dicarboxylates (4a–4c)
To a stirred solution of 1-(2-(4-fluoro/chloro/nitro-phenyl)-2-oxoe-
thyl)pyridin-1-ium bromide (1a/1b/1d) (0.0016 mol), in water
(10 mL), was added substituted diethyl 2-butynedioate
(0.0016 mol), stirred at 80 ꢁC for 3 h. Completion of reaction was
monitored on TLC. The reaction mixture was diluted with ethyl
acetate. The organic layer was separated, washed with brine and
dried under sodium sulphate. The crude compound was purified
by recrystallization method using hexane and ethyl acetate to
afford 69–83% yield of diethyl-3-(4-substitutedbenzoyl)indolizine-
1,2-dicarboxylate (4a–4c). The characterisation details of title com-
pounds (4a–4c) are reported below, and their spectral data are
shown in Figures S1–S9.
2. Materials and methods
2.1. Chemistry
The chemicals reported here were obtained from Sigma-Aldrich Co.
(St. Louis, MO, USA), while the solvents were obtained from Millipore
Sigma (Burlington, MA, USA). Thin-layer chromatography (TLC) using
silica gel (Sigma-Aldrich Co.) on aluminium foil was employed to
observe the chemical reactions; n-hexane and ethyl acetate (4:6)
were used as solvents. The reactions were visualised under an ultra-
violet (UV)-light/iodine chamber. B-545 was used to measure the
2.1.2.1. Diethyl-3-(4-fluorobenzoyl)indolizine-1,2-dicarboxylate (4a).
Appearance; Brown solid. Yield 72%, MP 138–139, FT-IR (KBr neat
cmꢂ1): 2985, 1733, 1695, 1635, 1608, 1228. 1H NMR (400 MHz,
CDCl3) d ¼ 9.51–9.48 (d, J ¼ 7.2 Hz, 1H), 7.78–7.70 (m, 3H),
7.16–7.11 (m, 2H), 6.82–6.77 (m, 1H), 4.36–4.30 (q, J ¼ 7.2 Hz, 2H),
3.76–3.68 (q, J ¼ 7.2 Hz, 2H), 1.37–1.33 (t, J ¼ 7.2 Hz, 3H), 1.13–1.11
(t, J ¼ 7.2 Hz, 3H). 13C-NMR (100 MHz CDCl3) d ¼ 183.77, 165.17,
164.12, 163.62, 163.23, 159.92, 141.51, 136.09, 136.02, 132.21,
131.59, 131.23, 129.89, 119.59, 115.62, 114.87, 110.18, 102.38,
97.63, 61.66, 60.28, 55.87, 14.32, 13.52. LC-MS (ESI, Positive): m/z:
(M þ H)þ: 384.38: Anal. calculated for: C21H18FNO5; C, 65.79; H,
4.73; N, 3.65; Found; C, 65.41; H, 4.50; N, 3.55.
€
melting points (Buchi, Labortechnik, Flawil, Switzerland). Fourier-
transform infrared (FT-IR) spectra were recorded on a Shimadzu FT-IR
spectrophotometer. Furthermore, 1H- and 13C-NMR spectra were
recorded on Bruker AVANCE III 400 MHz instruments using DMSO-d6
as a solvent. Chemical shifts (d) were recorded in parts per million
(ppm) downfield from tetramethylsilane, while the coupling con-
stants (J) were recorded in Hz. The splitting pattern was documented
as follows: s, singlet; d, doublet; q, quartette; m, multiplet. Liquid
chromatography–mass spectrometry (LC–MS; Agilent 1100 series)
was used to measure the mass spectra in conjunction with the MSD,
as well as 0.1% aqueous trifluoroacetic acid in an acetonitrile system
on the C18-BDS column. Then, elemental analysis was carried out
using the analyser FLASH EA 1112 CHN (Thermo Finnigan LLC, New
York, NY, USA). A single-crystal X-ray diffraction study was performed
using a Bruker KAPPA APEX II DUO diffractometer equipped with a
CCD detector; monochromated Mo Ka radiation (k ¼ 0.71073 Å) was
used. Data collection was carried out at 173(2) K using an Oxford
Cryostream cooling system featuring the Bruker Apex II software.
2.1.2.2. Diethyl-3-(4-chlorobenzoyl)indolizine-1,2-dicarboxylate (4b).
Appearance; Brown solid. Yield 74%, MP 137–138, FT-IR (KBr neat
cmꢂ1): 2989, 1736, 1696, 1637, 1605, 1227. 1H NMR (400 MHz,
CDCl3) d ¼ 9.53–9.49 (d, J ¼ 7.2 Hz, 1H), 7.76–7.71 (m, 3H),
7.15–7.10 (m, 2H), 6.82–6.77 (m, 1H), 4.37–4.30 (q, J ¼ 7.2 Hz, 2H),
3.72–3.68 (q, J ¼ 7.2 Hz, 2H), 1.37–1.33 (t, J ¼ 7.2 Hz, 3H), 1.13–1.10
(t, J ¼ 7.2 Hz, 3H). 13C-NMR (100 MHz CDCl3) d ¼ 183.51, 165.34,
164.15, 163.59, 163.30, 160.21, 141.49, 136.07, 135.89, 132.23,
131.28, 131.16, 129.91, 119.48, 115.49, 114.99, 110.24, 102.74,
97.69, 61.78, 60.36, 55.71, 14.42, 13.83. LC-MS (ESI, Positive): m/z:
(M þ H)þ: 400.2: Anal. calculated for: C21H18ClNO2; C, 63.09; H,
4.54; N, 3.50; Found; C, 62.91; H, 4.48; N, 3.45.
2.1.1. General synthetic procedure for the preparation of 1-(2-(sub-
stitutedphenyl)-2-oxoethyl)pyridin-1-ium bromides (1a-f) and ethyl
3-(substitutedbenzoyl)-2-methylindolizine-1-carboxylates (2a–2f
and 3a–3d)
Compounds 1a–1f, 2a–2f, and 3a–3d were synthesised, purified 2.1.2.3. Diethyl-3-(4-nitrobenzoyl)indolizine-1,2-dicarboxylate (4c).
by column chromatography, and well characterised by FT-IR, NMR, Appearance; Light green colour. Yield 70%, MP 111–112, FT-IR

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1475
(KBr neat cmꢂ1): 2983, 1739, 1700, 1649, 1603, 1225. 1H-NMR loopful of colonies into Middlebrook 7H9 broth containing glass
beads. The inoculum turbidity was adjusted to a McFarland num-
ber 1 standard and further diluted to 1:10 in M7H9 broth prior to
(400 MHz CDCl3) d ¼ 9.49–9.47 (d, J ¼ 7.2 Hz, 1H), 7.77–7.72 (m,
3H), 7.15–7.11 (m, 2H), 6.81–6.78 (m, 1H), 4.38–4.32 (q, J ¼ 7.2 Hz,
the addition of 100 mL to each of the test samples and drug-free
2H), 3.75–3.69 (q, J ¼ 7.2 Hz, 2H), 1.36–1.33 (t, J ¼ 7.2 Hz, 3H),
1.13–1.11 (t, J ¼ 7.2 Hz, 3H). 13C-NMR (100 MHz CDCl3) d ¼ 184.77,
wells. Growth control and a sterile control were also included for
each isolate. Sterile M7H9 broth was added to all perimeter walls
to avoid evaporation during the incubation. The plate was cov-
ered, sealed in a plastic bag, and incubated at 37 ꢁC. After 8 days
of incubation, 30 mL of 0.02% working solution of resazurin salt
was inoculated into each microtiter well. The plates were then
incubated overnight and read the following day. A positive reac-
tion resulted in a colour change from blue to pink owing to the
reduction of resazurin to rezarufin which confirmed MTB cell via-
bility/growth and, hence, drug resistance. The MICs were defined
as the minimum drug concentration to inhibit the growth of the
organism with no colour changes present in the well.
166.12, 165.08, 163.59, 163.29, 159.91, 142.47, 137.04, 136.03,
132.21, 131.25, 131.18, 129.95, 119.52, 116.59, 114.93, 109.15,
103.44, 98.59, 61.58, 60.29, 55.87, 14.31, 13.72. LC-MS (ESI,
Positive): m/z: (M þ H)þ: 411.2: Anal. calculated for: C21H18N2O7; C,
61.46; H, 4.42; N, 6.83; Found; C, 61.31; H, 4.30; N, 6.55.
2.2. Crystallography
Single-crystal X-ray diffraction data of the compound 4b was col-
lected on a Bruker KAPPA APEX II DUO diffractometer using
graphite-monochromated Mo-Ka radiation (v ¼ 0.71073 Å). Data
collection43 was carried out at 173(2) K. Temperature was con-
trolled by an Oxford Cryostream cooling system (Oxford Cryostat).
Cell refinement and data reduction were performed using the
SAINT program44. The data were scaled and absorption correction
was performed using SADABS44.
2.5. Safety studies (in vitro)
The safety of the tested indolizines was evaluated by MTT assay.
The
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
The structure was solved by direct methods using SHELXS-9745
and refined by the full-matrix least-squares method based on F2
using SHELXL-201446. All non-hydrogen atoms were refined aniso-
tropically. All hydrogen atoms, were placed in idealised positions
and refined in riding models with Uiso assigned 1.2 or 1.5 times
the Ueq of their parent atoms, and the C–H bond distances were
constrained to 0.95 Å for aromatic hydrogens and 1.00 Å for
methylene and methyl hydrogens. All geometrical calculations
were done using PLATON47. The program WinGx48 was used to
prepare molecular graphic images.
bromide) cytotoxicity assay was used to evaluate the cytotoxic
effect of the most promising compounds against peripheral blood
mononuclear cells (PBMCs) according to the described protocol50.
Cells were pipetted (90 mL of cell culture, 1 ꢃ 105 cells/mL) into
each well of 96-well microtiter plates, and the outer wells were
filled with PBS (phosphate buffer saline) in order to prevent the
medium from evaporation during incubation. Thereafter, plates
were incubated at 37 ꢁC for 24 h. Each well of the plate was then
treated with 10 mL of the compounds (1000–5 mg/mL). In the con-
trol wells, the negative control DMSO (dimethyl sulfoxide) and
media were added. Thereafter, the plates were incubated for
2 days at 37 ꢁC in a humidified incubator that contained a 5% CO2
atmosphere. After the incubation time, 20 mL of MTT reagent
(5 mg/mL) was further added to the individual well. The plate was
then incubated for a further 4 h at 37 ꢁC (5% CO2 incubator). The
media was then removed, and an aliquot of 100 mL DMSO was
added to each well in order to dissolve the formazan crystals that
were formed in metabolically active cells. After that, plates were
incubated for an extra hour. The absorbance of the formazan was
evaluated at 590 nm using an ELISA plate reader (Thermo
Scientific Multiskan GO).
2.3. Hirshfeld surface analysis and energy framework
calculation
The Hirshfeld surfaces analysis is a technique for visualisation and
investigation to illustrate the nature of intermolecular interactions.
The Hirshfeld surfaces and two-dimensional fingerprint plot for
compound 4b have been generated using the software Crystal
Explorer 17.5. This software has been also used to assess the inter-
action energies of the compound 4b.
2.4. Anti-tubercular activity
2.6. Molecular modelling
The antitubercular activity of the designed compounds 2a–2f,
3a–3d, and 4a–4c were evaluated against two types of MTB
strains, namely H37Rv and well characterised MDR strains using
the colorimetric Resazurin Microplate Assay (REMA) method49. A
100 mL of Middelbrook 7H9 broth was aseptically prepared and
dispensed in each of the wells of a 96 well flat-bottomed micro-
titer plate with lids (Lasec, South Africa). Each of the test com-
pounds was accurately weighed, dissolved in the appropriate
solvent, and filter sterilised using a 0.2 micron polycarbonate filter.
Stock solutions of the test samples were aliquoted into cryovials
and stored at ꢂ20 ꢁC. A 100 mL of the test samples were added to
each of the wells containing Middlebrook 7H9 broth supple-
mented with 0.1% Casitone, 0.5% glycerol, and 10% OADC (oleic
acid, albumin, dextrose, and catalase). The test samples were then
serially diluted two folds directly in the broth of the microtiter
plate to the desired concentration ranging from 40–0.625 mg/mL.
Inoculums from clinical isolates were prepared fresh from
Molecular docking of the designed compounds into the active site
of proposed potential targets was performed using Accelrys
Discovery Studio 4.0 (from BIOVIA Software Inc.). The X-ray crystal
structures of mycobacterial enyol-ACP-reductase (InhA) and
anthranilate phosphoribosyltransferase (trpD) enzymes in complex
with their inhibitors (PDB accession codes 5G0S and 3R6C,
respectively) were retrieved from the RSCB Protein Data Bank.
Crystal complexes were prepared using the clean protein tool
and prepare protein protocol in DS to standardise atoms names,
correct connectivity and bond order, protonate the ionisable resi-
dues at a pH of 7.4, add missing residues and correct incomplete
ones, and remove water molecules. To validate the docking proto-
col, the co-crystallised ligand was extracted and re-docked into
the active site of the enzyme to ensure proper definition of the
binding site and to assess the accuracy of the docking algorithm
in reproducing the pose of the co-crystallised ligand. C-Docker
protocol was used to perform the docking studies of the tested
Middlebrook 7H11 agar plates by scraping and re-suspending indolizines into their potential targets. The ligands are docked

1476
K. N. VENUGOPALA ET AL.
into a rigid receptor while a set of ligand conformation is gener- 2-butynedioate in the presence of water with continuous stirring
at 80 ꢁC for 3 h resulted in the formation of title compounds
ated. Additional scoring functions PLP1, PLP2, Jain, and PMF were
2a–2f, 3a–3d, 4a–4c. The resulting title compounds were purified
using ethyl acetate and hexane as an eluent by column chroma-
tography, and the compound purity was found to be more than
99% with a satisfactory yield (69% to 83%). Synthesis, purification,
and characterisation details of compounds 1a–1f, 2a–2f, and
3a–3d were reported earlier from our research group42. The
chemical structures of the newly synthesised compounds (4a-4c)
have been ascertained with the help of spectroscopic techniques
such as FT-IR, NMR (1H and 13C), LC-MS, and elemental analysis. In
LC-MS, the molecular ion peaks of these novel compounds
(4a–4c) were in good agreement with their proposed molecular
masses. Elemental analysis results of the title compounds (4a–4c)
were within 0.4% of the calculated values.
utilised to ensure the optimal ligand orientation into the active
binding site of the target. The highest negative score of PLP1,
PLP2, Jain, and PMF51,52, indicating the strongest receptor-ligand
binding affinities, is considered for refining binding poses. The
binding energy calculation was performed using C-Docker proto-
col and in situ ligand minimisation protocol.
2.7. ADMET calculation (in silico)
The ADMET Descriptors protocol and the Toxicity Prediction
(TOPKAT) protocol in DS were used to calculate a range of absorp-
tion, distribution, metabolism, excretion, and toxicity (ADMET)
related properties including, aqueous solubility (AS), blood–brain
barrier (BBB) penetration, CYP2D6 inhibition, hepatotoxicity,
human intestinal absorption (HIA), plasma protein binding (PPB),
AlogP, and polar surface area (PSA); in addition, different toxicity
parameters including rodent carcinogenicity (for both male and
female rats and mice, CMR, CFR, CLM and CFM respectively), Ames
mutagenicity (AM), skin irritation (SI), ocular irritancy (OI), aerobic
biodegradability and developmental toxicity potential (AB) were
also calculated.
The primary goal of the current study was to investigate the
impact of the substitution pattern, in terms of nature and position
of substituents, at positions 1, 2, and 3 of the indolizine ring sys-
tem on the anti-tubercular activity of the resultant analogues. The
synthetic strategy for the development of the target compounds
involved a 1.3-dipolar [3 þ 2] cycloaddition as a key step, allowing
the introduction of the substituent in the diverse position of the
indolizine ring. The 1.3-dipolar [3 þ 2] cycloaddition of pyridinium
ylides with electron-deficient alkynes offered
a convenient
approach for the construction of an indolizine scaffold.
3. Results and discussion
3.1. Chemistry
3.2. Crystallography
The synthesis of the title compounds (2a–2f, 3a–3d, 4a–4c) is por-
trayed in Scheme 1. The intermediates (1a–1f) were obtained by
stirring a mixture of pyridine, and para-substituted phenacyl bro-
mides in a dry acetone medium at 5 h. Compounds 1a–1f on fur-
Compound 4b was crystallised in the triclinic centrosymmetric
space group P-1 with one molecule in the asymmetric unit. The
parameters for crystal data collection and structure refinement,
and the list of intramolecular and intermolecular interactions in
ther reaction with ethyl prop-2-ynoate/ethyl but-2-ynoate/diethyl the crystal structure of diethyl 3-(4-chlorobenzoyl)indolizine-1,2-
Scheme 1. Synthesis of 1,2,3-trisubstiuted indolizine derivatives (2a–2f, 3a–3d, 4a–4c): Reagents and conditions (i) pyridine, dry acetone, stir at room temperature,
5 h; (ii) ethyl propialate/ethy1 2-butynoate/diethy1 2- butynedioate water, stir 80ꢁC, 3 h

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1477
dicarboxylate (4b) are given in Table S1 and Table 1, respectively. shown in Figure 5. The contribution of individual intermolecular
The side chain ethoxy groups do not exhibit any orientational dis- interactions on the Hirshfeld surface can be identified through the
order. The molecular conformation is locked via C-HꢄꢄꢄO H-bonds
colour codes. If di is greater than de, it is shown by the dark sur-
(involving H1/O5 and H4/O2) forming pseudo-six membered rings
(Figure 3). The crystal packing is stabilised via C–HꢄꢄꢄO H-bonds
(involving H21 and O5), forming dimers, and these are connected
with centro symmetrically related dimers formed via C-HꢄꢄꢄO H-
bonds (involving H17/H18 with O3) via additional C–HꢄꢄꢄO H-
bonds (involving H3 and O3), thus leading to the overall forma-
tion of a “hexamer” in the solid-state (Figure 4). Thus, O3 functions
as a “trifurcated acceptor” which is a unique feature in the pack-
ing of this molecule in the crystal. In addition, the molecules are
connected via a pꢄꢄꢄp stacking motif, involving the 6-membered
ring (Cg1: centre of gravity of ring N1/C1-C5), the stacking dis-
face area that exists because of the acceptor of the hydrogen
bond. The red colour spot indicates shorter molecular contacts on
the dnorm surface, and the blue colour on the surface of the dnorm
reflects longer molecular contacts. The white colour on the dnorm
surface indicates the interaction around the van der Waals radii.
The red colour spot indicates the hydrogen bonding HꢄꢄꢄO con-
tacts in the dnorm surfaces, while the blue surface region repre-
sents the HꢄꢄꢄH contacts (Figure 5(a)). The characteristics of the de
surface appear as a relatively flat green region where the distan-
ces of contact are similar (Figure 5(b)). The strong hydrogen bond-
ing interactions present in the molecule (Figure 5(c)) are also
demonstrated by the adjacent highlighted red and yellow regions
on the shape index surface. Whereas, the HꢄꢄꢄH interactions
(Figure 5(d)) are shown by the blue curved and yellow regions on
the curvedness surfaces. The colour patches are mapped differ-
ently on the Hirshfeld surface depending on the closeness to the
neighbouring molecules (Figure 5(e)). The 2D fingerprint plots
indicate the sharp spike peak that reflects the strong hydrogen
bonding present in the molecule (Figure 6(a–h)). The fingerprint
tance being 3.654 Å. Finally,
a
short contact between
O1 ¼ C7ꢄꢄꢄC5–N1 of distance 3.359 Å, involving electrophilic C7 and
relatively less electrophilic C5 is present in the crystalline lattice
(Figure 4).
3.3. Hirshfeld surface analysis and energy framework
calculation
The Hirshfeld surfaces of the title compound 4b were mapped plot shows that the HꢄꢄꢄH contacts have a comparatively larger
over de, dnorm, shape index, curvedness, and fragment patches as contribution of 40.0% relative to other interactions and implies
Table 1. List of intramolecular and intermolecular interactions in the crystal structure of diethyl 3–(4-chlorobenzoyl)indolizine-1,2-dicarboxylate (4b).
Interaction Type
D–H (Å)
DꢄꢄꢄA (Å)
HꢄꢄꢄA (Å)
D–HꢄꢄꢄA (ꢁ)
Symmetry code
x, y, z
x, y, z
C1–H1ꢄꢄꢄO5
0.95
0.95
0.95
0.95
0.95
0.95
2.859
2.869
3.368
3.175
3.188
3.369
2.29
2.33
2.64
2.58
2.60
2.47
118
115
133
121
120
159
C4–H4ꢄꢄꢄO2
C3–H3ꢄꢄꢄO3
x, þy ꢂ 1, þz
C18–H18ꢄꢄꢄO3
C17–H17ꢄꢄꢄO3
C21–H21ꢄꢄꢄO5
ꢂx þ 1, ꢂy þ 2, ꢂz
ꢂx þ 1, ꢂy þ 2, ꢂz
ꢂx þ 1, ꢂy þ 1, ꢂz þ 1
Figure 3. The crystal structure of diethyl 3-(4-chlorobenzoyl)indolizine-1,2-dicarboxylate (4b). ORTEP drawn with 50% ellipsoidal probability. Intra-molecular C–HꢄꢄꢄOꢄH
bonds are shown as dotted lines.

1478
K. N. VENUGOPALA ET AL.
Figure 4. Crystal packing depicting C–HꢄꢄꢄOꢄH-bonds, p–p stacking interaction and O1 ¼ C7ꢄꢄꢄC5–N1 short contact in the compound 4b. Cg1 is the centre of gravity of
N1/C1–C5 ring. The dotted lines represent intermolecular interactions. Non-interacting hydrogens have been omitted for clarity.
Figure 5. Hirshfeld surfaces mapped with (a) dnorm (b) de (c) shape-index, (d) curvedness and (e) fragment patches for the compound 4b.
that van der Waals radii and hydrogen bonding play a key role in isoniazid were also used as positive controls (Table 2). The anti-
the crystal packing. The HꢄꢄꢄO contacts contribute 21.7% of the mycobacterial activity showed that compound 3a, substituted
total interactions. In addition, the percentage contribution in this
crystal structure of other intermolecular interactions are as follows:
CꢄꢄꢄH/HꢄꢄꢄC (14.8%), HꢄꢄꢄCl/ClꢄꢄꢄH (13.1%), CꢄꢄꢄC (3.5%), CꢄꢄꢄO/OꢄꢄꢄC
(3.2%), NꢄꢄꢄC/CꢄꢄꢄN (1.3%), OꢄꢄꢄCl/ClꢄꢄꢄO (1.1%), NꢄꢄꢄH/HꢄꢄꢄN (0.7%),
OꢄꢄꢄN/NꢄꢄꢄO (0.4%), ClꢄꢄꢄC/CꢄꢄꢄCl (0.1%).
with a fluorine atom at the 4-position of the benzoyl group and a
methyl group at the 2-position of the indolizine ring, is the most
potent molecule demonstrating the highest inhibitory action
against the susceptible H37Rv MTB strain with MIC value of 4 mg/
mL. Five Compounds (2b, 2d, 3d, 4a, and 4b) were found to be
equipotent, with a MIC value of 8 mg/mL. Compounds 2c, 3b, and
4c showed moderate anti-TB activity with a MIC value of 32 mg/
mL against the same strain. Meanwhile, no activity was observed
for the remaining indolizines against the H37Rv MTB strain.
The structure–activity relationship (SAR) analysis revealed the
presence of a hydrogen atom, a methyl or an ester substituent at
the 2-position of the indolizine core was favourable to attain
good anti-tubercular activity. The presence of the halogen and
the nitro group at the para position of the benzoyl ring were
found to be tolerated well to maintain the activity (except com-
pounds 2a and 3c) against the H37Rv MTB strain, whilst the sub-
Systematic and theoretical energy was measured in terms of
electrostatic, dispersion, and total energy using the program
Crystal Explorer software, which further facilitated to depict the
3D topological images for the compound 4b (detailed information
is provided in the supplementary section Figures S10-S13 and
Table S2).
3.4. Anti-tubercular activity
The anti-tubercular activity of the target compounds (2a–2f,
3a–3d, and 4a–4c) was evaluated (in vitro) against two different
MTB strains, namely susceptible H37Rv MTB strain, and rifampicin stitution with methyl and cyano group in compounds 2e and 2f,
and isoniazid-resistant MTB strain (Table 2). Rifampicin and respectively, completely abolished the activity against both the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1479
Figure 6. Two-dimensional fingerprint plot for the compound 4b showing the contributions of individual types of interactions: (a) all intermolecular contacts, (b) HꢄꢄꢄH
contacts, (c) OꢄꢄꢄH/HꢄꢄꢄO contacts, (d) CꢄꢄꢄH/HꢄꢄꢄC contacts, (e) ClꢄꢄꢄH/HꢄꢄꢄCl contacts, (f) CꢄꢄꢄC contacts, (g) CꢄꢄꢄO/OꢄꢄꢄC contacts, (h) NꢄꢄꢄC/CꢄꢄꢄN. The outline of the full fin-
gerprint is indicating in grey. The related surface patches associated with the specific contacts with the dnorm mapped are illustrated by surfaces to the left.
MTB strains. In the case of nitro derivatives, compounds 2d and 3b–3d) did not show activity against MDR-MTB except for com-
3d with a hydrogen and methyl group, respectively, at the 2-pos- pounds 2d and 3a, while the third series of compounds 4a–4c
ition of the indolizine ring showed four-fold increased activity as clearly inhibited the MDR-MTB strain. These findings suggest that
compared with the compound 4c with an ester group at the the three designed series are likely to be inhibiting different MTB
same position.
molecular drug targets; such that the first and second series of
indolizine derivatives are inhibiting a common target that is differ-
ent from that inhibited by the third series. Alternatively, the com-
pounds that retained activity against MDR-MTB strain (third series)
could be interacting with the same molecular target as those of
the first two series, yet, might be interacting differently with the
target binding site.
The structural alteration at the 2-position of the indolizine scaf-
fold resulted in a profound impact on the anti-mycobacterial
activity against MDR-MTB. The indolizine derivatives that were
active (2b–2d, 3a, 3b, and 3d) against the susceptible H37Rv
MTB strain were found to be inactive against MDR-MTB, except
for compounds 2d and 3a which exhibited 8-fold reduced MDR-
MTB activity compared to H37Rv MTB strain (Table 2). However,
The above findings and observations supported the view that
compounds 4a-4c with an ethyl ester group at the 4-position of the first and second series of indolizine were more likely to inhibit
the benzoyl ring showed moderate activity against MDR-TB, with the mycobacterial cell growth by interacting with the InhA
a two-fold decrease in potency as compared with the cellular enzyme. With regards to the mode of action of the ester indoli-
activity against susceptible H37Rv MTB strain. The anti-tubercular zine series 3, their differences in inhibitory activity between sus-
activity profile of the tested compounds against MDR-MTB pro- ceptible strains and clinical isolates of MDR-MTB strains suggested
vided a clue for their mode of action. Indeed, the results demon- that the derivatives 4a–4c were interacting with another molecu-
strated that the first and second series of indolizines (2a–2f and lar target. Thus, further molecular modelling study was carried out

1480
K. N. VENUGOPALA ET AL.
to elucidate the potential mode of action of the tested com- and also to identify the second molecular target for the ester
pounds as well as to rationalise their SAR as inhibitors of InhA
which represents one of the potential MTB therapeutic targets,
indolizine series 3.
3.5. Toxicity study
Table 2. In vitro anti-mycobacterial activity of 1,2,3-trisubstituted indolizine
derivatives (2a–2f, 3a–3d, and 4a–4c) against H37Rv and MDR strains of
The promising activities of the designed compounds are very
encouraging to undertake prospective optimisation cycles.
However, it is highly recommended to optimise the ADMET prop-
erties for a lead compound early in the development stage to
reduce later problems due to unfavourable ADMET characteris-
tics53,54. Based on the in silico calculated ADME and toxicity
parameters (Table 3), all the compounds are showing acceptable
ADMET profile which makes them worthy of further optimisation
towards achieving the desired drug-like properties.
Mycobacterium tuberculosis.
O
O
N
R1
O
R2
(2a-2f, 3a-3d, 4a-4c)
MIC (mg/mL)
Moreover, the toxicity profile for compounds 2d, 3a, 4a, 4b,
and 4c that showed promising activity against MDR-MTB was fur-
ther assessed against peripheral blood mononuclear cells (PBMCs)
and no signs of toxicity were observed with concentrations up to
256 mg/mL.
Entry
R1
R2
H37Rva
MDR-MTBb
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
4a
4b
H
H
H
H
H
H
CH3
CH3
CH3
F
Cl
Br
NO2
CH3
CN
F
Cl
Br
NO2
F
Cl
NA
8
32
NA
NA
NA
64
NA
NA
32
NA
NA
NA
16
8
NA
NA
4
32
NA
8
3.6. Computational molecular modelling
A molecular docking study was carried out to gain insight into
the mechanism of action of the indolizine derivatives (2a–2f,
3a–3d, and 4a–4c). In light of the MTB inhibitory action, InhA
appeared to be a potential molecular target for the title com-
pounds; therefore these compounds were docked against InhA
enzyme in an attempt to corroborate their mode of action with
their observed anti-mycobacterial activity.
The binding feature common to all InhA inhibitors is the active
participation of NAD, as a cofactor, contributing strong interaction
with substrates through the H-bonding involvement from its
CH3
CO2CH2CH3
CO2CH2CH3
CO2CH2CH3
8
8
16
64
4c
NO2
32
<1
<0.2
Rifampicin
Isoniazid
ꢀ1
ꢀ0.2
aAmerican Type Culture Collection (ATCC): 25177; bthese isolates were found to be
resistant to the first line antibiotics, rifampicin (1 lg/mL), and isoniazid (0.2 lg/mL).
MDR-MTB: multidrug-resistant strains of Mycobacterium tuberculosis; MIC: min-
imum inhibitory concentration; NA: not active at the concentration range
(0.2–64 lg/mL).
Table 3. Calculated ADMET descriptors and toxicity parameters of indolizine derivatives (2a–2f, 3a–3d, and 4a–4c).
Compounds
MIC (mg/mL)
ADMET descriptorsa
Toxicity parametersb
AB DTP CMR CFR CLM CFM
MDR-
CYP2D6
Index Code H37Rv MTB AS BBB inhibition Hepatotoxicity HIA PPB AlogP
PSA
AM
SI
OI
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
4a
4b
4c
NA
8
32
8
NA
NA
4
32
NA
8
NA
NA
NA
64
NA
NA
32
NA
NA
NA
16
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
3
1
2
1
1
1
2
2
1
4
True
True
False
False
False
False
True
False
False
False
False
False
True
True
False
False
True
False
False
True
False
False
True
True
False
True
0
0
0
0
0
0
0
0
0
0
0
0
1
True 3.767 48.88
True 4.226 48.88
0
0
0
0.156 0.051
0.002 0.05
0
0
0
0
0
0
0
0
0.001
0.001
0.001
0.008
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.248
0
0
0.976
0
0
0.001
0
0
0.188 0.996
0
0
1
1
True 4.31
48.88
0
0
0
0
0.05 0.012
0.84 0.001
0.036
0.359
0.999
1
1
1
True 3.456 91.703 0.062
True 4.048 48.88
True 3.441 71.815 0.945
True 4.254 48.88
True 4.712 48.88
True 4.796 48.88
True 3.942 91.703 0.012
True 3.972 75.11
true 4.43 75.11
0
0
0
0
0
0
0
0
0
0.731 0.447
0.028 0.468
0.976 0.001
0.99 0.001
1
0.989
0.861
9
0.006 0.476 0.985 0.992 0.001
0.984 0.129 0.956 0.008
10
11
12
13
0
8
8
32
0
0
0.993 0.034 0.487 0.117 0.008
0.478 0.04 0.521 0.239 0.008
1
16
64
0.599
True 3.661 117.933 0.069 0.004 0.642
1
0.068 0.117
0.001 0.806
Aqueous solubility (AS)
Blood brain barrier (BBB) penetration
Human intestinal absorption (HIA)
Level
Value
Drug-likeness
Level
Value
Description
Level
Description
0
1
2
3
4
5
log(Sw) <ꢂ8.0
Extremely low
0
1
2
3
4
Very High Brain-Blood ratio greater than 5:1
0
1
2
3
Good absorption
Moderate absorption
Low absorption
ꢂ8.0 <log(Sw) <ꢂ6.0 No, very low, but possible
ꢂ6.0 <log(Sw) <ꢂ4.1 Yes, low
High
Brain-Blood ratio between 1:1 and 5:1
Medium
Low
Brain-Blood ratio between 0.3:1 and 1:1
Brain-Blood ratio less than 0.3:1
ꢂ4.1 <log(Sw) <ꢂ2.0 Yes, good
Very low absorption
ꢂ2.0 <log(Sw) ꢅ0.0
0.0 <log(Sw)
Yes, optimal
No, too soluble
Undefined Outside 99% confidence ellipse
Probability values
Probability level
Low probability
Description
0.0–0.30
Such a chemical is not likely to produce a positive response in an experimental assay
Greater than 0.30 but less than 0.70
Greater than 0.70
Intermediate probability
High probability
Likely to produce a positive response in an experimental assay
aKey to the above calculated ADMET descriptors.
bKey to the above calculated toxicity parameters.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1481
ribose hydroxyl moiety. Additionally, the residue LYS 165 partici- pose of 2d showed three hydrogen bond contacts with active
pates in the binding network by interacting with the hydroxyl site’s residues in the “in” conformation (pdb 5G0S), while only one
oxygen of the NAD ribose through hydrogen bonding, and the with the “out” conformation (pdb 5G0U) (Figure 8). The binding
residue ILE 91 is engaged in H-bonding with the amide functional- mode of 2d revealed that the benzoyl ring occupies the same
ity of the nicotinamide part of NAD. These residues greatly con- location in both the crystal involving H-bond interaction with the
tributed to the stability of the cofactor. Hence, the latter tends to co-factor NAD but differ on the orientation of the indolizine ring
play a critical role in molecular interaction with the substrate and (Figure 8). In light of the comparative docking analysis the InhA
other residues in the InhA binding domain. Consequently, the crystal structure of 5G0S, “in” conformation, appeared as more
presence of NAD during a docking study may have a profound appropriate to investigate the binding patterns. Hence, docking of
effect not only on the ligand interaction but also on the occu- the remaining derivatives was performed with the Tyr 158-in con-
pancy size of the active site that may affect ligand orientation. formation InhA active site (PDB 5G0S). The docking results,
Besides, a great majority of reported InhA inhibitors make a stable reported in Table 4, indicated that the active indolizines 2b–2d,
hydrogen bonding to the backbone hydroxyl group of the main 3a, 3b, and 4a–4c displayed greater binding energy as compared
chain TYR 158, responsible for blocking the enoyl-acyl carrier pro- to that of inactive congeners 2a, 2e, 2f, and 3c. In general, the
tein reductase function55. The role of TYR 158 in the catalytic predicted docking energy of indolizines was consistent with the
mechanism of InhA has been the focus of many studies which observed MTB inhibitory activity of the tested compounds, except
revealed that its side chain can adopt different conformations for the compounds 2d, 3d, and 4c with a nitro substitution
when direct InhA inhibitors bind the catalytic site. Mainly two con- (Figure 9). However, the order of the potency in the second indoli-
zine series was 3a (F) > 3b (Cl) ꢆ 3c (Br) which correlates with
formations were identified, an “in” and “out” conformations. The
the docking scores. A similar correlation between the activity and
docking scores was also observed in the first indolizine series in
which the order of bioactivity was 2b (Cl) > 2c (Br) ꢆ 2a (F), 2f
(CN), 2e (CH3). Among the compounds 2a and 3a, the most active
compound 3a (F) displayed a higher binding energy score than
the inactive compound 2a (F). The compound 2b (Cl) being more
active than compound 3b (Cl) demonstrated a higher energy
score. A similar trend was also observed for the bromine deriva-
tives 2c and 3c. The nitro compounds 2d and 3d were equipotent
and showed comparable docking scores. As for the third series,
the indolizines 4a–4c showed very high docking scores. The
former is resembling the substrate-NAD-InhA ternary complex,
and the latter resembles the NAD-InhA binary complex (Figure 7);
however, in the majority of available InhA-inhibitor complexes
TYR158 is in the “in” conformation and the inhibitor is forming
interactions with the cofactor56–58. However, a novel binding
mode of a benzimidazole-based InhA inhibitor has been recently
disclosed, occupying an extended hydrophobic pocket formed by
the amino acid residues Phe149, Met155, Pro156, Ala157, and
Ile215 in the in-Tyr 128 conformation57. Certain InhA inhibitors
were also reported to co-crystallize with Tyr 158-out active site
residue59–65
.
Accordingly, the binding mode of the indolizine 2d was inves- equipotent indolizines 4a (F) and 4b (Cl) showed comparable
tigated by docking it into the active site of the InhA enzyme utilis- docking scores, whilst the nitro derivative 4c showing a higher
ing crystal structures of the in and out conformations, namely docking score was four-fold less potent than the compounds 4a
5G0S and 5G0U respectively Figure 8.
and 4b.
The observed MTB inhibitory activity pattern of the indolizines
The docking results revealed that the estimated binding energy
of 2d was ꢂ107.46 kcal/mol and ꢂ144.02 kcal/mol with the InhA can be accounted for their distinctive feature of binding mode
binding site 5G0U (Tyr 158-out) and 5G0S (Tyr 158-in) respectively, into the InhA active site. The key binding interaction of the title
indicating that the indolize 2d interacted more favourably with compounds was highlighted in Figures 9 and 10. The binding
the in Tyr 158 conformation active residue. Indeed, the binding poses of the title compounds showed that the benzoyl ring is
Figure 7. The two reported “in and out” conformations of Tyr158 in mycobacterial InhA enzyme. PDB ID for the in conformation is 5G0S, and that for the out con-
formation is 5G0U.

1482
K. N. VENUGOPALA ET AL.
Figure 8. Comparative binding mode of indolizine 2d into InhA binding domain with (a) in-Tyr 158 (PDB ID: 5G0S) and (b) out-Tyr 158 conformations (PDB ID: 5G0U),
respectively. The ligand, NAD and the receptor were represented as sticks in solmon, yellow and cyan colours respectively. The hydrogen bonds are shown as green
dotted lines.
Table 4. Docking results of the indolizine derivatives 2a–2f, 3a–3d and 4a–4c into the InhA binding domain (PDB 5G0S).
Residues Interaction
Binding E.
Entry
R1
R2
(Kcal/mol)
H-Bond (Dist. Å, atom)
Pi-interaction
2a
2b
2c
2d
H
H
H
H
F
Cl
Br
ꢂ114.75
ꢂ128.17
ꢂ120.18
ꢂ144.02
Tyr 158 ( 2.71, F)
Met 98 (2.32, CO benzoyl)
Met 98 (2.37, CO benzoyl)
Met 98 (2.13, CO benzoyl)
Tyr 158 (2.56, NO2)
Phe 97 (Pi–Pi)
Phe 97 (Pi–Pi)
Phe 97 (Pi–Pi)
Met 103 (Pi–S)
NO2
NAD (2.00, NO2)
2e
2f
3a
H
H
CH3
CH3
CN
F
ꢂ97.47
ꢂ99.48
ꢂ152.37
Met 98 (2.27, CO benzoyl)
NAD (2.19, F)
Phe 97 (Pi–Pi)
Met 103 (Pi–S)
Phe 97 (Pi–Pi)
Met 103 (Pi–S)
3b
CH3
Cl
ꢂ124.20
Met 98 (2.15, CO benzoyl)
3c
3d
CH3
CH3
Br
NO2
ꢂ104.21
ꢂ147.40
Met 98 (2.13, CO benzoyl)
Tyr 158 (2.55, NO2)
NAD (1.99, NO2)
Phe 97 (Pi–Pi)
Met 103 (Pi–S)
4a
CO2CH2CH3
F
ꢂ165.58
Met 98 (2.32, CO benzoyl)
Tyr 158 (3.53, F)
Phe 97 (Pi–Pi)
Met 103 (Pi–S)
NAD (2.82, F)
4b
4c
CO2CH2CH3
CO2CH2CH3
Cl
ꢂ165.95
ꢂ182.03
Met 98 (2.45, CO benzoyl)
Phe 97 (Pi–Pi)
Met 103 (Pi–S)
NO2
Met 98 (2.04, CO benzoyl)
Tyr 158 (2.93, NO2)
NAD (2.01, NO2)
Met 98 (1.97)
Native ligand
ꢂ186.54
Phe 149 (Pi ꢂ Pi)
Gln 100 (2.41)
Tyr 158 (2.82)
NAD (2.06 )
located near to the co-factor binding site with a sandwich orienta- alteration from the rotation of the benzoyl group could provide a
tion between the nicotinamide ring of NAD and the main chain rationale for the lack of potency observed for these derivatives.
Tyr 158 which is consistent with the binding mode of known InhA However, the interaction of the residue Met 98 with the native lig-
inhibitors. The active indolizines 2b–2d, 3a, 3b, and 4a–4c were and and certain InhA inhibitors is reported as well61,62. It was also
predicted to adopt a similar conformation in the binding domain demonstrated that interacting with the Met 98 residue through H-
by forming strong hydrogen bonding with the residue Met 98 bonding was the key interaction to retain the MTB inhibitory
and the carbonyl moiety of the benzoyl group, whereas the car- activity when the main chain Tyr 158 was not involved in any
bonyl moiety of the benzoyl group for the inactive indolizines 2a, hydrogen bonding with the ligand.59 Derivatives 2d, 3a, 3d, 4a,
2e, 2f, and 3c was orientated away from the residue Met 98 pre- and 4c were able to make additional hydrogen bonding contacts
venting the H-bonding interaction (Figure 9). The conformation with residues in the active site. For instance, the para nitrobenzoyl

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1483
Figure 9. Predicted binding interaction of indolizines (2a, 2b, 2d, 3a, 3b, 3d, 4a–4c) with InhA binding domain (PDB 5G0S). Ligand and receptor were represented as
solmon and cyan respectively. Hydrogen bonding contact is shown with green dotted lines. p–p, p–sulphur and hydrophobic interactions are shown with magenta,
gold and violet, respectively. For displaying key bingind interaction, NAD has been omitted.
indolizines 2d, 3d, and 4c showed two H-bonding interactions lack of hydrophobic interaction with the residues Phe 97 and Met
between the nitro group and the residue Tyr 158 and the hydro- 103 for the compound 4c (NO2) provides an explanation for its
gen atom of the hydroxyl group from the NAD ribose (Figure 10). reduced potency in comparison with its nitro congeners 2d and
Similarly, the derivative 4a having a fluorine atom in the para pos- 3d (Figure 9).
ition of the benzoyl moiety showed a moderate hydrogen bond-
The molecular modelling analysis indicated that the observed
ing contact with the hydroxyl group of the NAD ribose and a MTB potency of indolizines was in good agreement with the pre-
weak H-bond with the residue Tyr 158, while compound 3a dem- dicted binding mode and binding energy predictions. These com-
onstrated only one strong H-bond with the hydroxyl of the NAD putational results strongly support our assumption that the
ribose but exempting from any H-bond interaction with the resi- molecular target of the designed indolizine is enoyl-acyl carrier
due Tyr 158 (Figure 10). The strength of H-bonding involvement protein reductase, and the key interactions with the residues Met
with NAD ribose may explain the greater potency observed for 98 and Phe 91 were highlighted as being mainly responsible for
the derivative 3a (2.19 Å) as compared to the compound the observed activity of the compounds. These findings could pro-
4a (2.82 Å).
Another important contribution to the cellular activity of the potential
bioactive compounds is the hydrophobic interaction implicated by design approach.
the residues Phe 97 and Met 103 (Figure 9 and Table 4), Where We next focussed on the prospective identification of the cellu-
vide a valuable guide to designing more potent indolizines as
InhA inhibitors through structure-based
a
Phe 97 formed T-shaped pi-stacking interaction with the indolizine lar target of the ester derivatives 4a–4c. According to the cellular
ring, except for compound 4c, and the residue Met 103 was activity profile against MDR MTB, the ester indolizine series 4a–4c,
observed to interact with the indolizine ring through pi-sulfur identified as potential InhA inhibitors, may have more than one
interactions with the exception of compounds 2b and 4c. The molecular MTB target. In the quest to identify the second

1484
K. N. VENUGOPALA ET AL.
Figure 10. Predicted binding interaction of indolizines (4a, 4c) with InhA binding domain (PDB 5G0S). Ligand, NAD and receptor were represented as solmon, yellow
and cyan respectively. Hydrogen bonding contact is shown with green dotted lines.
Table 5. Docking results of the indolizine derivatives 4a–4c into the anthranilate phosphoribosyl binding domain (PDB 3R6C).
Residues Interaction
Binding E.
Entry
R1
R2
F
(Kcal/mol)
H-Bond (Dist. Å, atom)
Hydrophobic
4a
CO2Et
ꢂ170.37
Arg 193 (2.09, CO benzoyl)
Ans 138 (2.41, CO ester)
Gly 107 (3.58, F)
Thr 108 (3.51, F)
Arg 193 (3.40, Pi-cation)
Gly 107 (3.22, Cl)
Arg 193 (3.31, Pi-cation)
4b
4c
CO2Et
CO2Et
Cl
ꢂ166.81
ꢂ140.06
Arg 193 (2.16, CO benzoyl)
Ans 138 (2.24, CO ester)
Ans 138 (2.15, CO ester)
NO2
Figure 11. Predicted binding interaction of indolizines (4a–4d) with anthranilate phosphoribosyl binding domain (PDB 3R6C). Ligand and receptor were represented as
solmon and cyan respectively. Hydrogen bonding contact is shown with green dotted lines. Halogen bonding and hydrophobic intreactions are shown with cyan and
violet respectively.
potential target for the indolizines 4a–4c, again, computational involvement of halogen bonding interaction with the receptor.
molecular docking studies were carried out. Based on the MTB With regards to the above considerations, a large number of
activity of derivatives 4a–4c against both susceptible and resistant known TB molecular targets were screened. The molecular target
MTB strains, we hypothesised that the presence of the ester func- anthranilate phosphoribosyltransferase appeared to be the appro-
tional group at 2-position of the indolizine may strongly contrib- priate enzyme target for indolizines 4a–4c. The docking scores,
ute to the bioactivity through the formation of hydrogen bonding presented in Table 5, were also found to be consistent with the
contact. Furthermore, it has been observed that the indolizine 4a MTB activity of the compounds in which the equipotent indoli-
and 4b, having halogen substituent in the para position of the zines 4a and 4b displayed similar binding energy, while the less
benzoyl moiety, had exhibited identical anti-TB activity and were active indolizine 4c showed a lower docking score. The binding
more active than the nitro derivative 4c, indicative of probable interaction of the indolizines 4a–4c with the anthranilate

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1485
phosphoribosyl enzyme was illustrated in Figure 11. The most
active compounds 4a and 4b were found to adopt similar orienta-
tion in the anthranilate phosphoribosyl binding domain showing
two strong hydrogen bonding contacts between the carbonyl
group of benzoyl and ester at 2-position of indolizine ring with
the amino acid residues Arg 193 and Asn 138, respectively.
Among the numerous hydrophobic interactions, the fluorine atom
of compound 4a showed halogen interaction with the residues
Gly 107 and Thr 108, similarly, the chlorine atom of compound 4b
exhibited interaction with the residue Gly 107. Both indolizines 4a
and 4b were also involved in pi-cation interaction between the
benzoyl ring of the indolizine and the residue Arg 193. The bind-
ing mode of the indolizine 4c differed from its congeners demon-
strating only one H-bond contact between the residue Asn 138
Acknowledgements
The authors extend their appreciation to the Deputyship for
Research & Innovation, Ministry of Education in Saudi Arabia for
funding this research work through the project number 1058,
King Faisal University, Kingdom of Saudi Arabia for support and
encouragement; the Department of Microbiology, National Health
Laboratory Services, Inkosi Albert Luthuli Central Hospital, Durban
South Africa for providing the facility to carry out in vitro anti-TB
activity and Dr. Hong Su, Center for Supramolecular Chemistry
Research, Department of Chemistry, University of Cape Town,
South Africa, for single-crystal X-ray data collection. D.C. thanks
IISER Bhopal for research facilities and infrastructure. R.D.N and
S.K.N. thank the Department of Chemistry, VNIT, Nagpur for sup-
and the carbonyl of ester group at the 2-position of indolizine port and encouragement. The authors wish to thank Mr. Tameem
ring, explaining the lower observed MTB activity. However,
although the above computational results reasonably explained
the observed anti-tubercular activities of the tested compounds,
yet, they need to be experimentally verified and validated via bio-
molecular in vitro testing. Thus, it could be the subject of pro-
spective research due to its importance in confirming the in silico
results that is imperative to guide prospective optimisation of
these lead compounds towards designing more potent and select-
ive drug candidates.
M. Alyahian for providing technical support in this project.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This research was funded by the Deputyship for Research &
Innovation, Ministry of Education of Saudi Arabia (Research Project
Number 1058).
4. Conclusion
In the current study, three novel series of 1,2,3-trisubstituted indo-
lizine derivatives (2a–2f, 3a–3d, and 4a–4c) were evaluated for
their anti-TB activity against susceptible and MDR-MTB strains. The
majority of tested compounds showed good to excellent anti-
tubercular activity with MIC ranging from 4 to 64 mg/mL. The
inhibition profile of indolizines and molecular docking strongly
supported that InhA is the principal drug target for the investi-
gated compounds. Molecular modelling insight indicated that the
conformation changes resulting from the benzoyl group rotation
provided a rationale for the MTB cellular activity of the indolizines
as InhA inhibitor. Furthermore, the cellular activity profile of the
ester indolizine derivatives (4a-4c) against both the strains
revealed their inhibitory action in multiple molecular targets in
which anthranilate phosphoribosyltransferase might be a potential
additional drug target. Our results highlighted the importance of
indolizines as a novel promising class of multi-targeting agents for
MTB with favourable toxicity profile. Such compounds might serve
as attractive leads for further development of potential drug can-
didates for combating both the drug-sensitive and drug-resistant
tuberculosis strains.
The compound 4b was crystallised in a triclinic centrosymmet-
ric crystal system with space group P-1. In stabilising the molecu-
lar structure, the C–HꢄꢄꢄO and other short contact have played an
important role. Hirschfeld surface analysis with 2D fingerprint
plots was performed to provide insight into the stability of the
crystal structure in order to understand and visualise the contribu-
tion of distinct intermolecular interactions. Systematic and theoret-
ical energy was measured in terms of electrostatic, dispersion, and
total energy using the program Crystal Explorer software, which
further depicts 3D topological images.
ORCID
Katharigatta N. Venugopala
0680-1549
Pran Kishore Deb
Nizar A. Al-Shar’i
Lina A. Dahabiyeh
Bandar E. Aldhubiab
http://orcid.org/0000-0003-
http://orcid.org/0000-0002-8650-2874
http://orcid.org/0000-0002-1599-5704
http://orcid.org/0000-0002-4688-7052
http://orcid.org/0000-0002-2684-4186
Anroop B. Nair
Raghuprasad Mailavaram
http://orcid.org/0000-0003-2850-8669
http://orcid.org/0000-0001-6126-5860
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