Indole chalcones: Design, synthesis, in vitro and in silico evaluation against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2020.112358

Indole chalcones were designed and synthesized as a promising set of compounds against H₃₇Rv strain of Mycobacterium tuberculosis. Within this library of compounds, (E)-1-(furan-3-yl)-3-(1H-indol-3-yl)prop-2-en-1-one (18), (E)-3-(1H-indol-3-yl)

Full text of DOI:10.1016/j.ejmech.2020.112358

Journal Pre-proof
Indole chalcones: Design, synthesis, in vitro and in silico evaluation against
Mycobacterium tuberculosis
Deepthi Ramesh, Annu Joji, Balaji Gowrivel Vijayakumar, Aiswarya Sethumadhavan,
Maheswaran Mani, Tharanikkarasu Kannan
PII:
S0223-5234(20)30328-7
DOI:
https://doi.org/10.1016/j.ejmech.2020.112358
EJMECH 112358
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 March 2020
Revised Date: 4 April 2020
Accepted Date: 16 April 2020
Please cite this article as: D. Ramesh, A. Joji, B.G. Vijayakumar, A. Sethumadhavan, M. Mani, T.
Kannan, Indole chalcones: Design, synthesis, in vitro and in silico evaluation against Mycobacterium
tuberculosis, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112358.
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2020 Published by Elsevier Masson SAS.

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Indole chalcones: Design, Synthesis, in vitro and in silico evaluation against
Mycobacterium tuberculosis
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Deepthi Ramesh , Annu Joji , Balaji Gowrivel Vijayakumar , Aiswarya Sethumadhavan ,
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Maheswaran Mani , Tharanikkarasu Kannan
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Department of Chemistry, Pondicherry University, Kalapet, Puducherry-605014, India.
Department of Microbiology, Pondicherry University, Kalapet, Puducherry-605014, India.
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Corresponding author: Tel: +91-413-265 4411; Fax: +91-413-265 6740.
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Email address: tharani.che@pondiuni.edu.in
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Abstract
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Indole chalcones were designed and synthesized as a promising set of compounds against
H Rv strain of Mycobacterium tuberculosis. Within this library of compounds, (E)-1-(furan-3-
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yl)-3-(1H-indol-3-yl)prop-2-en-1-one (18), (E)-3-(1H-indol-3-yl)-1-(thiophen-2-yl)prop-2-en-
1-one (20) and (E)-2-((1H-indol-2-yl)methylene)cyclopentan-1-one (24) displayed high anti-
tubercular activity at 50 µg/ml with MIC values of 210, 197 and 236 µM respectively. The in-
silico studies revealed that compound 18 exhibit binding modes similar to FAS-II inhibitors
like INH or Thiolactomycin against KasA protein. Cytotoxicity assay results suggest that the
compounds 18, 20 and 24 are non-cytotoxic to human megakaryocytes and murine B cells.
Keywords: Indole chalcones, Anti-tubercular, Mycobacterium tuberculosis, H Rv strain,
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Luciferase reporter mycobacteriophages (LRP), SARs, KasA protein, Cytotoxicity
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1.
Introduction
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Tuberculosis (Tb) is an airborne communicable ailment with high fatality rates ranking
above HIV/AIDS [1]. Out of 10 million new Tb infected people in 2018, there were
approximately 1.5 million deaths [1]. Though Tb became epidemic during the industrial
revolution, it was brought under control with the launch of BCG (Bacillus Calmette–Guérin)
vaccine in 1921 followed by antibiotics such as streptomycin (1943), isoniazid (1952) and
rifampicin (1963). Tb incidence also increased with HIV infection during the 1980s, and out of
10 million Tb deaths in 2018, 2,51,000 people were HIV positive [1]. To combat this disease,
presently four orally active antimicrobial agents such as isoniazid (INH), pyrazinamide (PZA),
rifampicin ( RIF) and ethambutol (EMB) are administered for two months succeeded by INH
RIF combination over a span of 4 months [2]. Even with all these efforts [3], Tb is widespread
and with emerging drug-resistance such as multi-drug-resistant Tb (MDR-Tb), extensively-
drug-resistant Tb (XDR-Tb) and totally-drug-resistant Tb (TDR-Tb) strains, this disease
increasingly hard to eradicate. The disease complexity, long period of the treatment,
practicality of drug sensitivity tests and lack of proper diagnosis are considerable challenges. In
addition to this, drugs such as INH and RIF show severe side effects such as hepatotoxicity. To
overcome these issues, several strategies like repurposing and revival of drugs such as
Clofazimine [4], structure and mechanism based drug design which involves genome
sequencing and identification of molecular targets [5], molecular hybridisation of active
pharmacophores [6] have been explored to find an ideal drug. An ideal Tb drug should fulfil
the criteria of improving the treatment of latent Tb, have zero interactions with HIV medicines,
lower dosage with improved efficacy, enhanced bioavailability and target both MDR and XDR
Tb strains [7]. A wide range of compounds have been screened to find a novel ideal drug for
curing Tb [2, 8]. In Tb drug discovery research, there are four promising types of targets.
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Tetrahydropyrans [9], diarylethers [10], methylthiazole [2], aminoproline [11], aryl amides
[12], piperazine indoleformamides [13], imidazopiperidines [14] etc. were used for targeting
Fatty acid synthase II (FASII) enoylacyl carrier protein reductase (InhA). Adamantyl ureas
[15], phenyl pyrroles [16], benzimidazoles [2], indolecarboxamides [17] etc. were used for
targeting Transmembrane transport protein large (MmpL3). Benzothiazinone [18],
benzothiazoles [19], 1,4-azaindoles [20], 4-aminoquinolone piperidine amides [21] etc. were
used for targeting Decaprenylphospho-beta-D-ribofuranose and phthalimide and quinoline
containing compounds were used for targeting 2-oxidase (DprE1) [2].
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Most of these compounds as well as commercial drugs have N-containing heterocyclic
moieties and they are one of the most sought-after pharmacophores for designing new and
efficient drugs in the pharmaceutical industry. Indole heterocycle is an important bioactive
compound and it serves as a crucial skeleton in naturally occurring alkaloids like tryptophan
(an amino acid), serotonin (a naturally-occurring neurotransmitter in humans), reserpine (a
tranquillizer isolated from the plant Rauvolfia serpentina), and indole 3-acetic acid (a
phytohormone) [22]. In addition, marine and bacterial indole alkaloids show anti-cancer [23],
anti-viral [24] , anti-bacterial [25], and anti-HIV [26] properties. Moreover, indole derivatives
are known for their role as antimicrobial [27, 28], antiviral [29], insecticidal [30], painkillers
[31], anti-inflammatory [32], depression medications [33], anti-tubercular [20, 34],
antineoplastic [35], antihypertensives [36], antioxidants [23], and anti-diabetic [33] agents. The
Food and Drug Administration (FDA) has even published a database highlighting the
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importance of N-containing heterocyclic compounds in 2015 and indole derivatives ranks 9
among the top 25 FDA approved drugs with 17 indole containing drugs in the market [37].
There are two active indole-based lead candidates, NITD-304 and NITD-349 (recognised as
MmpL3 inhibitors: see Fig. 1(a) for structure) which are in the clinical stage of evaluation for
drug-sensitive Tb strains [38]. MmpL3 is a transporter from Mycobacterial Membrane Protein
Large (MmpL) and carries mycolic acids as trehalose monomycolates (TMM) across the cell
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membrane for biosynthesis of Tb cell wall [39]. MmpL3 is a strategic drug target for MDR and
XDR Tb strains where treatment and survival options are limited. Similar to indole derivatives,
chalcones are also important pharmacophores which are (1,3-diarylprop-2-en-1-one) flavonoids
found in a number of natural products [40]. They impart strong colouration to plant pigments
and occur in many plant species such as Glycyrrhiza inflate [41], Angelica keiskei [42], and
Piper aduncum [43]. Chalcones can exist in E and Z forms with E isomer being more stable
[40] and showed antioxidant [44], anti-HIV [45], anti-alzheimer’s disease [46-48], antibacterial
[49], antileishmanial [48, 50], anticancer [51], antimalarial [52, 53], antiviral [48] and
antitubercular [48, 52] properties. The alkene bond fused with carbonyl group is accountable
for the bioactivity of chalcones, though the exact mechanism of activity is still under
investigation [40, 54]. Chalcones have inhibitory effects on various enzymes like trypsin and
topoisomerases due to complex formation of chalcone with the active sites of the enzymes [55].
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Considering the above aspects and in a quest to find novel anti-Tb agents, molecular
hybridisation in drug designing is considered in the present investigation by hybridising indole
and chalcone. Hybrid molecules can have modified selectivity, contrasting approaches of
action, lesser unwanted aftereffects, improved solubility and oral bioavailability [56, 57]. There
has been limited exploration of chalcones as antitubercular agents and indole chalcones, in
general, are not well reported. There are few reports of indole chalcones showing biological
activity [58-60] as shown in Fig. 1b. Till date, as per our knowledge, there are no reports on
using indole chalcones as antitubercular agents. Hence, in our endeavour to synthesize novel
antitubercular agents [61, 62], we have designed and synthesized indole-chalcone hybrids with
aromatic, heteroaromatic and fused rings. The basicity of the compounds was taken into
consideration for improved cell permeability. The compounds are analysed in vitro with H Rv
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strain of Mycobacterium tuberculosis (MTb) to discover their promising mycobacterial
properties and the results are presented in this paper.
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< Insert Fig. 1>
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2.
Experimental section
2.1.
Materials and Methods
Indole-3-carboxaldehyde was bought from Avra Synthesis, Hyderabad, India.
Acetophenones and piperidine were procured from Avra Synthesis, Hyderabad, India and
Sigma-Aldrich, USA. Absolute ethanol from Spectrochem Pvt. Ltd, Mumbai, India was used
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directly. Nuclear Magnetic Resonance spectra (NMR) were recorded in 400 MHz for H NMR
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and 101 MHz for C NMR on a Bruker Avance-400 spectrometer. The internal reference
compound used was trimethylsilane. Mass spectra were acquired using AGILENT
Technologies 6530B Accurate Mass QTOF-LC/MS. Thermo Nicolet 6700 spectrometer
recorded Fourier transform infrared spectra (FT-IR). Single crystal X-ray diffraction data were
acquired by Xcalibur Eos, Rigaku Oxford Diffraction instrument X-ray diffractometer of Mo-
Kα radiation (λ = 0.71073 Å). Empirical absorption was done using SCALE3 ABSPACK
scaling algorithm. The refinement was carried out by XL, in the Olex 2-1.2 package plus
structural solution by SHELXS-97. For cytotoxicity, the human megakaryocyte cell line Mo7e
(ACC 104) and Murine pro B cell line BA/F3 (ACC 300) were attained from German
Collection of Microorganisms and Animal Cell Cultures, DSMZ, Germany. Mo7e and BA/F3
cells were maintained in RPMI 1640 medium (Gibco, Waltham, MA USA) with 10 % fetal calf
serum (HiMedia, India) in the presence of 20 ng/ml human IL-3 and murine IL-3 (Peprotech
Asia, Rehovot, Israel) respectively.
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2.2.
Synthesis of indole chalcone derivatives
In an RB flask, 1H-indole-3-carboxaldehyde (1 mmol) and appropriate acetophenones
(1.2 mmol) were taken and 5 ml of ethanol and 5 drops of piperidine were added. The resulting
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solution was then refluxed at 70 C and TLC was used to track the reaction process. Upon
accomplishement of the reaction, the reaction mixture was transferred into cold water and
further neutralized utilizing 1N hydrochloric acid. The crude precipitate formed was filtered
out, dried and recrystallized from chloroform. All the indole chalcones (1 to 25) were
characterized using spectral techniques and the spectral data are listed in the supplementary
data.
2.3.
In vitro anti-Tb activity studies
2.3.1. Preparation of sample
The initial stock solution was made by the dissolution of 10mg of a sample in 1mL of
DMSO. The working stock solutions of 1 mg/mL and 0.5 mg/mL were prepared from this stock
solution by adding the required volume of Middlebrook 7H9 broth and further sterilized by
filtration using 0.45 µ filter.
2.3.2.
Luciferase reporter mycobacteriophages (LRP) assay
Four cryovials per set (two for control and two for 100 µg/mL and 50 µg/mL
concentrations) were taken. 400 µl of Middlebrook 7H9 broth was transferred into first two
vials and 350 µl in the third and fourth vial. About 50 µl of 1mg/mL of stock was added to the
3rd and 4th vial respectively to get a total concentration of 400 µl. 100 µl of M.Tb H Rv cell
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suspension was further introduced to the vials and incubated the vials at 37 C for 72 h. Then,
50 µl of phage phAE202 and 40 µl of 0.1M CaCl were introduced to all the vials (cell-phage
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mixture) and incubated at 37 C for 4 h. Post incubation, 100 µl of the cell-phage mixture was
moved to a luminometer cuvette. 100 µl of D-luciferin was introduced and thereafter the
relative light unit (RLU) was determined instantly in a luminometer (Berthold) at 10S
integration. The percentage of reduction in RLU of the test compared to control was calculated
by using the following equation,
ꢐꢈꢄꢅꢂꢈꢑ ꢊꢎꢏ − ꢒꢁꢓꢅ ꢊꢎꢏ
ꢀꢁꢂꢃꢁꢄꢅꢆꢇꢁ ꢈꢉ ꢊꢁꢋꢌꢃꢅꢍꢈꢄ ꢍꢄ ꢊꢎꢏ =
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ꢐꢈꢄꢅꢂꢈꢑ ꢊꢎꢏ
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Compounds with RLU reduction of 50% and above in comparison with control were
considered as active against MTb.
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2.4.
Molecular Docking
Docking was executed using AutoDock Vina v.1.1.2 [63] at maximum exhaustiveness of
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6AJH, 3IFZ, 4B6C, 1ZID, 4FDO, 1N40 ) from the RCSB Protein Data Bank (PDB) were used.
The structure was handpicked based on the highest resolution and lowest R factors. Using
AutoDock Tools v.1.5.6., extraneous solvent molecules and co-crystallized ligands were
removed from the protein. Only polar hydrogens were taken into account and gaussian and
gasteiger charges were assigned as preparation for docking. Energy minimized ligand files
were prepared using Perkin-Elmer Chem3D v15.0. Automation of the docking process was
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done with the use of PyRx-Python Prescription v0.8 [64]. Binding affinity values in kcal mol
were noted provided that root means square deviation (RMSD) values were below 2Å. Analysis
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of binding mode of the selected drug candidates post-docking was performed on BIOVIA
Discovery Studio Visualizer v.19.1.0 [65] to visualize the protein-ligand interactions.
2.5.
Cytotoxicity assay
Cell cytotoxicity assay was carried out using WST-1 (Roche, Basel, Switzerland) by
considering the manufacturer’s protocol. Mo7e and BA/F3 cells were plated in 96-well tissue
culture plate at a concentration of 20,000 cells per well in 100 µL of media. It was stimulated
with different concentrations of compounds 18, 20 and 24 for 24 h. Cells were also treated only
with DMSO to exclude solvent-induced cytotoxicity and 10 µL of WST-1 reagent was added
after incubation. The absorbance was measured against a background control as blank using
microplate (ELISA) reader at 440 nm. Statistical analysis was done using Graphpad Prism
v.6.0.1 (GraphPad Software, Inc., CA, US) and P value of <0.05 were deemed substantial.
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Results and Discussion
In a constant attempt to identify potential candidates against the virulent MTb, novel 2-
aminothiazole derivatives have been successfully synthesized in our lab and reported by our
research group [61, 62]. In continuation of this effort, in the extant research, a library of novel
indole-chalcones was designed, synthesized and screened against H Rv strain of MTb.
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Furthermore, in-silico anti-Tb activities against KasA protein present in MTb were carried out.
In our survey of existing literature, till date, indole chalcones have not been reported as active
compounds against Tb. Here, indole chalcone derivatives have been designed with hydrophilic
and lipophilic properties, and the indole core is retained in all the compounds as the active
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pharmacophoric fragment. The chalcone unit acts as the Michael acceptor and various groups
have been introduced in this unit for cell permeability and solubility.
3.1.
Design of indole chalcone derivatives
The pharmacokinetic properties of a drug should be known before synthesizing the drug
for better biological action. Hence, the indole chalcone compounds were designed using the
Lipinski [66] and Veber rules [67] for drug-likeness. Indole scaffold was retained in all the
compounds and substituted, fused, heteroaromatic rings were incorporated to induce
lipophilicity of indole chalcone derivatives. Additionally, aliphatic rings were also incorporated
for this purpose. It is widely accepted that by inducing lipophilicity, permeation of any drug
into the cell wall of Tb can be achieved. Table 1 shows the list of indole chalcones designed
for the present investigation. Molinspiration server [68] was used to gather the pharmacokinetic
properties of the indole chalcones and the properties are compiled in Table 2. According to the
data, the molar mass of the indole chalcones is less than 500 ranging from 211.26-347.42 which
indicates that they can be easily metabolised in comparison to larger molecules. The Log P
suggests the lipophilicity and values for indole chalcones are in the range of 2.49- 5.07. Log P
value of indole chalcones are in the recommended range, except for compound 5 which has
higher Log P values of 5.07. Interestingly, all the compounds possess 1-3 H-bond donors and 2-
5 H-bond acceptors. Out of 25 compounds designed, 24 are found to obey Lipinski Rule and
found to have drug-like character. Other parameters like topological polar surface area (TPSA)
and a sum of rotatable bonds were also evaluated. TPSA is associated with the hydrogen
bonding of the molecule and bioavailability of a drug. TPSA values are in the range of 32.86 to
78.69 and the count of rotatable bonds in the range of 1-5 which indicate promising oral
availability. All the compounds except 5 are found to have drug-like molecular (DLM)
properties and the probability to be lead candidates.
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< Insert Tables 1 and 2 >
3.2.
Absorption, Distribution, Metabolism and Excretion (ADME) properties
To be an efficient drug, the compound should have high biological activity in lower
effective concentration with low toxicity and should be active until the desired action takes
place. The drug discovery procedure takes the ADME properties of drug candidates into
consideration for better pharmacokinetic profile. The pharmacokinetic properties can be
calculated in silico using online databases like SwissADME (http://www.swissadme.ch/) [69]
and pkCSM (http://biosig.unimelb.edu.au/pkcsm/) [70]. The ADME properties of indole
chalcones are given in Table 3. Drug absorption was evaluated using solubility measurement
and intestinal permeability. The aqueous solubility of the compounds is given as the logarithm
of molar concentration and the solubility of designed compounds ranges from -2.90 to -6.045.
The compounds are moderately water-soluble due to the presence of lipophilic functionalities
aimed at improved cell permeability. As the absorption of an orally administrated drug occurs
mostly through the small intestine, the percentage absorption of the compounds was evaluated.
In general, Caco-2 permeability can predict the intake of oral drugs as Caco-2 from human
colon carcinoma resemble intestinal epithelial cells. It is important to mention that the
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compound should have Papp > 8 x10 cm/s for high permeability. Interestingly, all the
compounds show high cell permeability remarkably higher than the standard Tb drugs such as
INH and RIF. All the compounds except 16 show high intestinal absorption in the range of 90-
95% which is twice that of RIF. The presence of hydrophilic hydroxy group and higher
molecular weight of 16 renders the absorption of the drug by the intestine. INH shows 96%
intestinal absorption which is slightly higher than all the indole chalcones. However, 15
showed intestinal absorption similar to INH at 95%.
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The distribution profile of the drug was predicted using a volume of distribution (VDss),
fraction unbound and blood-brain barrier permeability. Higher VDss indicates better
distribution of the drug in the tissues than in plasma, and if Log VDss > 0.45 it shows the
greater distribution in the tissues. All the compounds are moderately distributed in the tissues,
they show better distribution than INH with compound 24 showing a higher range of 0.677.
RIF is highly distributed in tissues with a value of 1.49 which is much higher than all the
designed indole chalcones. Efficacy of drug calculated by fraction bound indicates that it is less
bound to blood proteins and is free to diffuse. The blood-brain barrier (BBB) permeability was
calculated by both SwissADME and pkSCM. The importance of BBB permeation is in
affecting the central nervous system as in tuberculosis meningitis. Trifluoromethyl derivatives
and nitro derivatives are unable to cross BBB similar to the standard drugs, INH and RIF. All
the compounds interact with cytochromes either as substrates or as inhibitors while INH and
RIF do not show any of these interactions. The total clearance of drugs (both hepatic and renal)
was also studied and all the indole chalcones show a lower total clearance of -0.091 – 1.045
logml/min/kg. The compound 16 shows a total clearance of 0.688 logml/min/kg similar to INH
and 17 is showing a total clearance of 1.045 logml/min/kg much higher than both INH and RIF.
It can be concluded that all the compounds show good ADME properties in comparison with
INH and RIF and can be considered as probable lead candidates.
<Insert Table 3>
3.3.
Synthesis and characterization of indole chalcones
As the compounds are found to possess DLM properties and show good ADME
properties, the designed indole chalcones are synthesized by aldol condensation as depicted in
Scheme 1. Using the literature procedure [40, 71], indole-3-carboxaldehyde was reacted with
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different substituted acetophenones to yield corresponding chalcones through Claisen–Schmidt
condensation. The synthesized compounds were characterized by spectroscopic techniques like
NMR, FT-IR and Mass spectrometry. The characteristic peak of C=C bond of chalcones
appears as doublets, between 7-8 ppm in 1H NMR spectra, and at 125 ppm and 147 ppm in
1
13C NMR spectra. The NH protons of indole appear between 10-12 ppm in H NMR spectra.
The disappearance of methyl protons from acetophenone further confirms the formation of the
expected product. In FT-IR spectra, the characteristic -NH stretching vibrations appeared
-1
around 3400 cm , stretching vibrations of -C=C in conjugation with C=O appeared around
-1
-1
1600 cm and carbonyl stretching vibrations appeared at 1700 cm . The mass spectroscopy
results show that the experimental molecular weight values are matching precisely with the
theoretical molecular weight values.
< Insert Scheme 1>
3.4.
Single crystal X-ray Diffraction analysis
The stereochemical arrangement of compounds can affect the drug-like properties and
hence the exact configuration of compounds should be determined for the present indole
chalcones as they can have either Z or E configuration. The single crystals of three compounds
(7, 11, 12) crystallized in DMSO were selected as representative compounds. The solved
single-crystal XRD patterns of the representative compounds are given in Fig. 2. The XRD data
show that C=O and C=C group exist in E configuration and compounds are planar. The
packing diagrams of 7, 11, 12 show the presence of intermolecular hydrogen bonding between
different crystals of the same compound. The hydrogen bonding exists between N of -NH
group of indoles of one crystal and O of -C=O of another crystal is at a distance of 1.977 Å,
2.021 Å, and 1.985 Å for 7, 11, 12. respectively. The presence of hydrogen bonding stabilizes
1
2

3
3
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17
18
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23
24
25
26
27
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30
the crystal structure in crystal packing and in the case of 7 and 12, in a single unit-cell, the
compounds are arranged in head to tail overlap with a length of 3.370 Å and 3.406 Å
respectively. The compound 11 shows a displaced head to tail overlap in a unit cell and the
crystal parameters of the three compounds are given in Table 4.
<Insert Fig. 2 and Table 4>
3.5.
In vitro antimycobacterial activity of indole chalcones
After successful synthesis and characterization of indole chalcones, they were analyzed
for in vitro antitubercular activity against H Rv strain of MTb. Here, minimum inhibitory
3
7
concentration (MIC) of indole-chalcones that yield 50% inhibition using LRP assay was
considered to possess antitubercular activity. Rifampicin, the Group D Tb drug is considered
the reference compound in the present investigation. The results of in vitro analysis are
presented in Table 2 along with the pharmacokinetic analysis data. The activity was afflicted
by the presence of different substituents in conjugation with chalcones. The different
heterocyclic substituents showed moderate to good activity with 65% to 85% inhibition having
MIC values from 155 µM to 189 µM.
The substituted phenyl indole chalcone, 1 with fluorine substitution shows moderate
activity with less than 55% inhibition at both 100 µg/ml and 50 µg/ml concentrations with MIC
value of 188 µM. The possibility of fluorine being an active substituent was further explored by
introducing trifluoromethyl groups in para and meta positions of phenyl ring (2 and 3). The
trifluoromethyl groups were inactive with less than 30% inhibition of mycobacterium.
Similarly, the introduction of chloro group (4) in para position of phenyl ring did not show
much difference in activity with only 50% inhibition at 100 µg/ml and the inhibition further
decreased with lowering of concentration. The attachment of Cl group at ortho-para positions
1
3

3
3
3
3
3
3
3
3
3
3
3
3
3
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3
3
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3
31
32
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49
50
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52
53
54
55
56
of phenyl moiety (as in 5) showed an insignificant increase with more than 50% inhibition at
both concentrations with MIC value of 164 µM. The activity of halo-substituents was further
studied by introducing bromo substituent at the para position as in compound 6. Surprisingly,
the compound 6 showed 65% inhibition in 50 µg/ml concentration. The bromo group emerged
as the most active group among the halo substituted compounds with MIC value of 155 µM.
The inhibitory potential of halo substituted compounds are as follows: 4-Br>2,4 di-Cl> 4-F> 4-
Cl> CF . The analysis shows that two compounds (5 and 6) hindered the growth of
3
Mycobacterium at a concentration not more than 50 µg/ml. The introduction of the nitro group
at the meta position (7) had no effect with compound showing merely 50% inhibition albeit at a
higher concentration of 100 µg/ml. Furthermore, the bioisosteres of different substituents were
considered for in vitro analysis The bioisostere of fluorine substituent, a hydroxy group (8)
showed MIC value of 189 µM at 50 µg/ml with activity similar to the compound 1. However,
another bioisostere, amino group (9) shows no activity with less than 50% inhibition. The
bioisostere of Cl, a methyl group (compound 10) also showed similar activity with only 50%
inhibition at 100 µg/ml concentration. Methoxy substitution as in 11 and 12 is also inactive
against the bacterium. Fig. 3a shows the correlation between phenyl substituents and
mycobacterial activity of indole chalcone derivatives. We observed that, based on resonance,
the presence of electron-withdrawing groups in substituted phenyl ringsof indole chalcones
augmented the antimycobacterial inhibition. Also, from the decreasing inhibitory potential of
halo substituted compounds, it can be concluded that there has been an influence of the size of
substituents on activity. All the halogens by means of inductive effect can attract the electrons
from other atoms and the inductive effect will create a dipole moment within the compound.
This can enhance the water solubility and enables the interaction of a drug with the biological
target. The exception of hydroxyl group being active despite being an electron-donating group
can be associated with its H-bond forming ability with the target. Here 7 compounds show
promising inhibition at 100ug/ml with more than 50% inhibition of Mycobacterium, and 4
1
4

3
3
3
3
3
3
3
3
3
3
3
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3
57
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68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
compounds show inhibition at even lower concentration of 50 µg/ml. Among phenyl
substituted indole chalcones, compound 6 emerged as a promising candidate with MIC of 155
µM.
The library was further expanded by introducing heterocyclic scaffolds substituting
phenyl ring. Different acetyl pyridines were introduced (13, 14, 15) which showed promising
activity. Among these, the presence of 3-pyridine (14) showed higher activity with 70%
inhibition at 100 µg/ml and all the pyridine derivatives showed MIC value of 201 µM. We
noticed that the replacement of the pyridine ring with hydroxy phenyl piperazine (16) increased
the activity at 100 µg/ml and displayed moderate inhibition of 65% at 50 µg/ml. The compound
16 displayed lower MIC value of 143 µM when compared to the pyridine substituents. This
may be due to the presence of an additional -OH group capable of forming H-bonding. The
introduction of 1-phenyl imidazole (17) in place of the 3-pyridine system did not affect the
activity. Both 14 and 17 showed similar activity against H Rv strain with MIC of 17 being
3
7
slightly lower at 159 µM. In case of 17, lowering the concentration from 100 µg/ml to 50 µg/ml
had no perceivable effect in bacterial growth inhibition. The presence of furan (18) showed
exceptionally high activity with more than 85% inhibition in both 100 µg/ml and 50 µg/ml
concentrations. The most striking aspect of compound 18 is the negligible difference of 2%
inhibition with change in concentration from 100 µg/ml to 50 µg/ml . A similar trend was
observed for 2-thiophene moiety (20), with a slight difference of 5% in inhibition with change
in concentration from higher to lower value. However, 3-thiophene (21) when compared to 20
showed lower inhibition of 60%. Both 20 and 21 showed lower MIC values of 197 µM
compared to furan (18) counterpart. Surprisingly, one of the more prominent heterocyclic rings,
2-pyrrole (19) showed disappointing results with no inhibition even at higher concentration.
Higher activity among N-containing rings is well connected to their increasing basicity. The
basicity order and activity of N-rings are as follows: Piperazine>Imidazole>Pyridine>Pyrrole.
Among the three pyridine derivatives, 3-pyridyl derivative (14) showed higher activity
1
5

3
3
3
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3
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99
00
01
02
03
04
05
06
07
08
followed by 4-pyridyl (13) and 2-pyridyl (15) derivatives. In 3-pyridyl derivative (14), C=O
group in meta position acts as a deactivating substituent while in the case of 4-pyridyl and 2-
pyridyl derivatives, C=O group in para and ortho positions respectively act as a slightly
activating substituent. This can decrease the inductive effect of C=O group thereby decreasing
the basicity of pyridine derivatives. Hence, the basicity order of pyridine derivatives follows
the order: 3-pyridyl derivative (14) > 4-pyridyl (13) > 2-pyridyl (15) which is reflected in their
activity. Aliphatic piperazine (16) is showing high basicity and hence shows 76% inhibition
followed by aromatic counterparts. Pyrrole (19) being least basic shows no inhibition and
similarly, furan (18) shows more activity than thiophene due to its higher basicity. As the ring
size decreases, the activity is found to increase. The bioisosteres of pyridine (furan, thiophene
and imidazole) showed higher activity and emerged as promising scaffolds in drug
development. Fig. 3b shows the effect of heterocyclic substitution on the antimycobacterial
activity of indole chalcones.
Moving onto fused heterocyclic systems, introduction of 1,3- benzodioxole (22) adjacent
to chalcone moiety has shown promising inhibition with better MIC value of 171 µM. 22
shows good inhibition at 100 µg/ml and a moderate growth inhibition of 66% at 50 µg/ml .
Napthyl group (23) however showed no inhibition. We have introduced two cyclic keto
functionals adjacent to chalcones to study their influence in activity. Aliphatic cyclopentanone
group (24) showed relatively higher result with more than 85% inhibition with MIC value of
236 µM. The replacement of cyclopentanone with larger cyclohexanone moiety (25), however,
reduced the inhibition to 53% with MIC of 221 µM. The effect of fused heterocyclic and
aliphatic systems on the anti-mycobacterial activity is shown in Fig. 3c. Heterocyclic
compounds showed Log P values in the range of 2.49 - 3.68. The relatively low Log P values
indicate the higher lipophilicity of compounds and hence easier cell permeability. The effect of
Log P values on the MIC of MTb is given in Fig. 4.
1
6

4
4
4
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18
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20
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22
23
24
25
26
27
28
29
30
31
32
33
34
<Insert Fig. 3 and Fig. 4 >
The structure activity relationship is explained in terms of bioisosteres which are used in
drug design for improving pharmacological activity, improved target selectivity and reducing
the side effects. The different bioisosteres employed in the current study are described in Table
5 with corresponding Log P and MIC values. The substituted phenyl indole chalcone, 1 with
fluorine substitution shows moderate activity with MIC value of 188 µM and Log P value of
3.65. When fluorine is replaced by its bioisosteres, hydroxy (8) and amino (9) groups, there is a
decrease in Log P values with decreasing inhibition of bacterial growth. A similar trend is
followed when chloro (4) and bromo (6) substitutions are replaced with hydroxy group. In both
cases, electron withdrawing groups in para position of phenyl ring emerged as more potent for
inhibiting the bacterial growth. Electron donating group in para position instead of electron
withdrawing group shows diminishing inhibition as seen by replacing chlorine with its
bioisostere methyl (10). The presence of dichloro substitution as in compound 5 is more
efficient than p-chloro substitution despite the violation of Lipinski rule regarding Log P. The
trifluoromethyl group, the bioisostere of halogens was inactive with less than 30% inhibition
compared to 1, 4, 6. The bromo group (6) emerged as the most active group among the halo
substituted compounds and their bioisosteres with MIC value of 155 µM which can be
attributed to electron withdrawing efficiency and larger size of bromo substitution. Different
acetyl pyridines (13, 14, 15) showed promising activity with 3-pyridine (14) showing higher
activity with 70% inhibition at 100 µg/ml and all the pyridine derivatives showed MIC value of
201 µM. The pyridine ring was replaced with their bioisosteres furan and thiophene to check
their inhibitory potential. The presence of furan (18) showed exceptionally high activity with
>85% inhibition at both concentrations with a difference of 2% in inhibition from change in
concentration from 100 µg/ml to 50 µg/ml. 2-thiophene moiety (20) showed similar trend but
with slight difference of 5% in inhibition rate with change in concentration. However, 3-
1
7

4
4
4
4
4
4
4
4
4
4
4
4
4
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35
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40
41
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45
46
47
48
49
50
51
52
53
thiophene (21) showed lower inhibition than all other bioisosteres of pyridine except for 2-
pyridine derivative (15). Furan (18) shows more activity due to its higher basicity and as the
ring size decreases, the activity is found to increase.
< Insert Table 5 >
From the above discussions, it can be summarized that the heterocyclic compounds
showed higher activity in LRP assay and turned out to be promising scaffolds for further
modifications. The activity was associated with the size and basicity of the heterocyclic rings.
Five membered rings show better activity than six-membered rings owing to their small size
and hence better cell permeability. Among all the compounds, 18, 24 and 20 emerged as most
active compounds with higher inhibition of mycobacterium and MIC values ranging from 170
µM to 210 µM. These compounds exhibit higher inhibition at 50 µg/ml which is similar to
clinically used PZA showing anti-Tb activity of 50 µg/ml at pH 5.5 and 400 ug/ml at pH 5.95
[72]. The studies reveal that indole-chalcone compounds are promising starting points for drug
candidates and must be further explored for their potential .
3.6.
Docking studies
4
4
4
4
4
4
4
54
55
56
57
58
59
60
The successful synthesis and antitubercular properties of some of the indole chalcones
prompted us to explore the binding of the compounds to plausible molecular targets. The
aforementioned studies helped to identify active lead compounds, 18, 20, 24. Molecular
docking studies were performed to recognize the potential target for inhibiting MTb. In order to
pin down the mode of binding as well as binding affinities, a suitable protein target should first
be selected. Several well-known anti-tubercular drug-receptors were selected for blind docking
and are listed as follows: mycolic acid transporters (PDB: 6AJH) [73], DNA gyrase (PDB:
1
8

4
4
4
4
4
4
4
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4
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74
75
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77
78
79
80
81
82
83
84
85
86
3IFZ) [74], GyrB ATPase (PDB: 4B6C) [75], long-chain enoyl-acyl carrier protein reductase
(PDB: 1ZID) [76], oxidoreductase DprE1 (PDB: 4FDO) [77], cytochrome P450 (PDB: 1N40)
[78], β-ketoacyl synthase KasA (PBD: 2WGD) [79].
The in silico results which best fit the in-vitro observations (KasA protein, PDB: 2WGD)
were analyzed post-docking to identify interacting amino acid groups and were compared with
previously studied mechanisms. KasA protein (PDB ID: 2WGD) inhibition is long known to
suppress the MTb disease [80]. KasA protein is essential to the cell wall synthesis of MTb and
inhibition of this protein by a molecule could be an indicator of its nature as an anti-Tb agent.
The in silico results of the compounds against KasA protein are given in Table 6. Compound
-1
20 displayed a binding affinity of -6.5 kcal mol and it shows interactions with GLU-40
(electrostatic interactions of N on GLY with indole rings of 20), SER-41 (H-bonding of NH of
20 to C=O of SER), GLU-224 (H-bonding of GLU OH with C=O of 20) and LEU-371
(hydrophobic interactions with thiophene present in 20). Compound 24 has a binding strength
-1
of -6.9 kcal mol and it shows interactions with GLU-40 (electrostatic interaction of indole
ring with N of GLU), LEU-371 and ARG-225 (both show hydrophobic interactions with
-1
cyclopentanone). Similarly, compound 18 shows binding affinity of -7.9 kcal mol and
interacts with THR-313 (H-bonding of C=O of 18 and OH on THR), HIS-311 (electrostatic and
hydrophobic interaction between furan of 18 and imidazole of HIS). VAL-278, ALA-215,
ALA-279, PRO-280 and ILE-317 also showed hydrophobic interactions with indole moiety
present in 18. Post analysis it was noted that compounds 20 and 24 interacted with two of the
same amino acids namely GLU-40 and LEU-371. What was most interesting was that 18
showed high binding affinity to KasA protein as well as its binding mode was similar to known
FAS-II inhibitors such as INH and Thiolactomycin [80]. The compound 18 displayed the
highest inhibition of MTb among the synthesized compounds and the specific interactions of 18
with KasA protein are shown in Fig. 5. The interactions of 18 with HIS-311 (a member of the
1
9

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06
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10
11
12
catalytic triad), VAL-278, ALA-279 and PRO-280 and its proximity to PHE-404 which is a
gatekeeper to the acyl channel present in KasA protein are the likely the causes for its efficacy
in MTb inhibition. Due to this, compound 18 may prove to be a useful candidate against INH
resistant MTb strains and could prove to be a valuable starting point for new anti-tubercular
drugs.
<Insert Table 6 and Fig. 5>
3.7.
Plausible mechanism of action
The metal chelation is important in Tb infection as MTb needs iron to grow inside the
host organism. The bacteria take iron from the host and hence, withholding the supply of iron
can, in turn, reduce the multiplication of bacteria. Iron chelators can reduce bacterial growth
either by withholding iron or by forming free radicals which may be toxic to bacteria [81, 82].
The iron chelator should be permeable to the cell membrane and hence, the presence of
lipophilic functional groups is important. In chalcone, the carbonyl oxygen and other
heteroatoms can act as electron donors to form complexes with metal atoms. The properties of
chalcones such as smaller size, higher charge density, higher stability and presence of electron-
donating heteroatoms in the ring allow for the chelation of metals. This can have a considerable
role in the improved activity of chalcones. The importance of metal chelation can be explained
by overtone concept of cell permeability and Tweedy’s theory of chelation. As stated by
overtone theory, cellular membranes are made up of lipids and allow the entry of lipophilic
molecules only. Chalcones can chelate metal ions reducing the polarity of metal ions by the
overlap of ligand orbital and metal orbital. The π-electrons are delocalised on chelating ring
augmenting the complex to enter into the cell membranes. This can block the binding sites for
metal which is important for the bacterium itself [83], hence increasing antimycobacterial
2
0

5
5
5
5
13
14
15
16
properties of the metal chalcone complexes. The presence of heterocycles in conjugation with
the carbonyl oxygen further strengthens the possibility of metal chelation by donating the lone
pair of electrons. The possible binding modes of chalcones with metal [84] are shown in Fig. 6.
5
17
<Insert Fig. 6>
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
3.8.
Evaluation of cytotoxicity
Compounds (E)-1-(furan-3-yl)-3-(1H-indol-3-yl)prop-2-en-1-one (18), (E)-3-(1H-indol-
3-yl)-1-(thiophen-2-yl)prop-2-en-1-one (20) and (E)-2-((1H-indol-2-yl)methylene)cyclopentan-
1-one (24) were tested for cytotoxicity in human megakaryocyte cell line (Mo7e) and Murine
pro B cell line (BA/F3). Compounds which inhibit more than 50% cell growth were considered
to be cytotoxic to cells. We observed that more than 80% cell viability in a human
megakaryocyte cell line, Mo7e when treated with compounds 18, 20 and 24. Similarly, in
murine Pro B cell line BA/F3, cell viability of more than 60% was observed when treated with
compounds 18, 20 and 24. Overall the results suggest that the compounds 18, 20 and 24 are not
cytotoxic to human megakaryocytes and murine B cells. Percentage cell viability of Mo7e and
BA/F3 cells against test compounds 18, 20 and 24 is given in Fig. 7.
<Insert Fig. 7>
5
33
5
5
5
5
34
35
36
37
4.
Conclusion
A library of indole chalcone derivatives was synthesized and its potency against H Rv
3
7
strain of M.Tb is studied. Among these, 3 compounds , (E)-1-(furan-3-yl)-3-(1H-indol-3-
2
1

5
5
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63
yl)prop-2-en-1-one (18), (E)-3-(1H-indol-3-yl)-1-(thiophen-2-yl)prop-2-en-1-one (20) and (E)-
2-((1H-indol-2-yl)methylene)cyclopentan-1-one (24) were found to be potential drug
candidates against tuberculosis with MIC of 210, 197, 236 µM respectively. The activity is
linked to the size of the heterocyclic ring and their ability to chelate metal atoms vital to
mycobacterium. The docking studies run to comprehend the binding manner of indole
chalcones indicate that compound 18 showed binding modes similar to FAS-II inhibitors like
INH or Thiolactomycin against KasA protein. Cytotoxicity assay results suggest that the
compounds 18, 20 and 24 are noncytotoxic to human megakaryocytes and murine B cells.
Compound 20 is showing high potential against MTb with lower cytotoxicity. Overall, the
studies reveal that the compounds show potential as an important drug backbone against Tb.
The structural modifications for enhancement in anti-mycobacterial activity is worth exploring
and is under progress in our laboratory.
Acknowledgement
Deepthi Ramesh thanks DST for DST-INSPIRE fellowship (JRF). Authors thank University
Grants Commission (UGC), New Delhi for their financial support under Special Assistance
Program, stage DSA-I to Department of Chemistry, Pondicherry University (No. F.540/6/DSA-
1/2016/(SAP-1) dated 31-10-2018). Authors thank S. Anbarasu, Sathyabama University,
Chennai, India for mycobacterial studies, Central Instrumentation Facility (CIF), Pondicherry
University for spectral analysis. The authors also thank Binoy Saha, Arjith Saha, Department of
Chemistry, Pondicherry University for XRD characterization, and Ramkishore Matsa,
Department of Chemistry, Pondicherry University for his timely help.
2
2

5
5
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5
64
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72
Figures and Scheme legends
5
5
73
74
Fig. 1. (a) Indole drugs in a clinical trial for MDR-Tb (b) Biologically active indole-chalcones
in literature.
5
5
75
76
Fig. 2. Single-crystal XRD data. The ORTEP diagrams (a, d, g), Packing diagrams (b, e, h) and
Hydrogen bond interaction diagrams (c, f, i) of compounds 7, 11, 12.
5
5
77
78
Fig. 3. Correlation diagram of percentage inhibition with differently substituted indole
chalcones.
5
79
Fig. 4. Effect of Log P values of indole chalcones against the MIC values.
5
5
80
81
Fig. 5. Post docking analysis (a) KasA protein interaction with 18 (b) Ligand receptor
interactions of KasA and 18 (c) 2-D interaction diagram of 18 with KasA.
5
5
5
82
83
84
Fig. 6. (a) and (b) show two different binding modes of chalcones with metal, (c) shows a
binding mode of chalcone with heterocyclic groups and (d) shows the presence of back
bonding in heterocyclic chalcones causing chelation increasing lipophilicity and activity.
5
5
85
86
Fig. 7. Percentage cell viability of Mo7e and BA/F3 cells against test compounds 18, 20 and
24. (a) Mo7e cells and (b) BA/F3 cells were stimulated with indicated concentrations of
2
3

5
5
87
88
compounds 18, 20 and 24 for 24 h. Cytotoxicity of cells was studied using WST-1 assay. Data
are represented as mean ± s.e.m. P value is calculated using student t-test. *P < 0.05.
5
89
Scheme
1:
Synthesis
of
indole
chalcone
derivatives.
2
4

Table 1: Library of Indole chalcone derivatives
Category I: Substituted phenyl indole chalcone derivatives (1-12)
OH
O
N
H
8
Category II: Six-membered heterocyclic indole chalcones (13-16)
N
O
N
H
1
5
Category III: Five-membered heterocyclic indole chalcones (17-21)
HN
O
N
H
1
9
2
5

Category IV: Fused indole chalcones (22-23)
Category V: Aliphatic indole chalcones (24,25)
2
6

5
5
90
91
Table 2: Pharmacokinetic analysis and in vitro mycobacterial analysis result of indole
chalcones
Lipinski’s Rule of 5
Veber Rule
No. of
% Inhibition
MIC
against
MTb
(µM)
188
Mol.
Wt
H
H
No of
TPSA
100
50
Code. Log P
rotatabl
2
donor acceptor violations
(Å )
µg/ml µg/ml
e bonds
1
2
3
4
5
6
7
8
9
.
.
.
.
.
.
.
.
.
3.65
4.68
4.68
4.46
5.07
4.59
3.72
3.30
2.83
4.23
3.81
3.43
2.49
2.49
2.61
3.10
3.11
2.73
2.94
265.29
315.29
315.29
281.74
316.19
326.19
292.29
263.30
262.31
261.32
277.32
307.35
248.28
248.28
248.28
347.42
313.36
237.26
236.27
1
1
1
1
1
1
1
2
3
1
1
1
1
1
1
2
1
1.
2
2
2
2
2
2
2
5
3
3
2
3
4
3
3
3
5
4
3
3
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
32.86
32.86
32.86
32.86
32.86
32.86
78.69
53.09
58.88
32.86
42.10
51.33
45.75
45.75
45.75
59.57
50.69
46.00
48.65
3
4
4
3
3
3
4
3
3
3
4
5
3
3
3
3
4
3
3
54.25 50.26
31.17 29.68
26.31 24.52
50.65 48.65
55.16 52.26
65.86 64.86
51.73 48.70
> 317
> 317
>355
164
155
> 342
189
55.12
48.7
54.9
37.24
> 381
> 382
> 360
> 325
201
1
1
1
1
1
1
1
1
1
1
0.
1.
2.
3.
4.
5.
6.
7.
8.
9.
50.85 41.68
36.68 27.12
31.69 27.05
64.2
58.35
70.72 63.09
62.05 53.10
76.44 63.83
70.73 63.57
87.67 85.34
49.37 26.38
201
201
143
159
210
> 423
2
7

2
2
2
2
2
2
0.
1.
2.
3.
4.
5.
3.68
3.37
3.98
4.94
2.96
3.47
253.33
253.33
291.31
297.36
211.26
225.29
1
1
1
1
1
1
2
2
4
2
2
2
0
0
0
0
0
0
32.86
32.86
51.33
32.86
32.86
32.86
3
3
3
3
1
1
87.18 81.97
63.38 62.05
75.72 66.76
197
197
171
41.83
30.8
> 336
236
86.34 84.29
61.97 53.07
221
1
.4
2 µg/ml)
.4
2 µg/ml)
INH
RIF
-0.96 137.14
3
6
4
0
3
68.01
1
5
> 99%
> 99%
(
(
2
2.62
822.95
16
220.16
5
92
5
5
93
94
Note: Pharmacokinetic analysis are obtained from the Molinspiration server
(http://www.molinspiration.com)
5
95
2
8