Discovery of novel N-methyl carbazole tethered rhodanine derivatives as direct inhibitors of Mycobacterium tuberculosis InhA

Article abstract of DOI:10.1016/j.bmcl.2019.06.015

InhA (Enoyl-ACP reductase) plays a crucial role in the biosynthetic pathway of cell wall synthesis in Mycobacterium tuberculosis (Mtb). Isoniazid (INH) is an important first-line drug, which inhibits InhA. The rapid increase in resistance to INH and currently marketed drugs as well as emergence of MDR-TB and XDR-TB has complicated the diagnosis and treatment of Mtb with ever increasing threat to human kind. Herein, we report novel N-methyl carbazole derivatives as potential anti-TB compounds acting directly via InhA inhibition. All the synthesized final compounds were screened against Mtb virulent cell line H₃₇Rv and investigated the InhA enzyme inhibition. Interestingly, compound 9e displayed promising inhibition (91%) at 50 μM concentration and IC₅₀ of 2.82 μM against InhA. To understand the ligand receptor interaction between compound 9e and InhA, molecular docking and molecular dynamics experiments were performed. The computational results were in agreement with the observed experimental data. Further, the cytotoxicity studies on mammalian cells revealed that all the compounds were safe.

Full text of DOI:10.1016/j.bmcl.2019.06.015

Accepted Manuscript
Discovery of novel N-methyl carbazole tethered rhodanine derivatives as direct
inhibitors of Mycobacterium tuberculosis InhA
Mahamadhanif S. Shaikh, Ashish M. Kanhed, Balakumar Chandrasekaran,
Mahesh B. Palkar, Nikhil Agrawal, Christian Lherbet, Girish A. Hampannavar,
Rajshekhar Karpoormath
PII:
S0960-894X(19)30395-6
https://doi.org/10.1016/j.bmcl.2019.06.015
BMCL 26493
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
13 March 2019
31 May 2019
12 June 2019
Please cite this article as: Shaikh, M.S., Kanhed, A.M., Chandrasekaran, B., Palkar, M.B., Agrawal, N., Lherbet,
C., Hampannavar, G.A., Karpoormath, R., Discovery of novel N-methyl carbazole tethered rhodanine derivatives
as direct inhibitors of Mycobacterium tuberculosis InhA, Bioorganic & Medicinal Chemistry Letters (2019), doi:
https://doi.org/10.1016/j.bmcl.2019.06.015
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Discovery of novel N-methyl carbazole tethered rhodanine derivatives as
direct inhibitors of Mycobacterium tuberculosis InhA
a
a$
a$
Mahamadhanif S. Shaikh , Ashish M. Kanhed , Balakumar Chandrasekaran , Mahesh B.
a$
a
b
a
Palkar , Nikhil Agrawal , Christian Lherbet , Girish A. Hampannavar and Rajshekhar
Karpoormatha*
aDiscipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal (UKZN),
Westville, Durban 4001, South Africa.
bLaboratoire SPCMIB (UMR CNRS 5068), Université Paul Sabatier-Toulouse III, 118 route de Narbonne,
3
1062 Tou-louse Cedex 9, France ; ITAV‐USR3505, CNRS, Université Paul Sabatier-Toulouse III, Toulouse,
France.
*
Corresponding author: karpoormath@ukzn.ac.za (R. Karpoormath)
Fax: +27-312607792; Tel: +27-312607179, +27-721107207.
$Contributed equally.
1

ABSTRACT
InhA (Enoyl-ACP reductase) plays a crucial role in the biosynthetic pathway of cell
wall synthesis in Mycobacterium tuberculosis (Mtb). Isoniazid (INH) is an important first-
line drug, which inhibits InhA. The rapid increase in resistance to INH and currently
marketed drugs as well as emergence of MDR-TB and XDR-TB has complicated the
diagnosis and treatment of Mtb with ever increasing threat to human kind. Herein, we report
novel N-methyl carbazole derivatives as potential anti-TB compounds acting directly via
InhA inhibition. All the synthesized final compounds were screened against Mtb virulent cell
line H Rv and investigated the InhA enzyme inhibition. Interestingly, compound 9e
3
7
displayed promising inhibition (91%) at 50 µM concentration and IC of 2.82 µM against
5
0
InhA. To understand the ligand receptor interaction between compound 9e and InhA,
molecular docking and molecular dynamics experiments were performed. The computational
results were in agreement with the observed experimental data. Further, the cytotoxicity
studies on mammalian cells revealed that all the compounds were safe.
Key words:
Tuberculosis; Inh A inhibitor; Carbazole; Hybridization; GLIDE; Molecular dynamics.
2

Tuberculosis (TB), a deadly infectious disease caused by the bacterium
Mycobacterium tuberculosis (Mtb) and is the prime cause of mortality across the globe with
increasing health and economic concerns especially in the developing nations. The WHO
global TB report 2017 stated that more than 200 countries carry active TB infection claiming
over 4,500 lives a day. To implement existing TB interventions, every year 2.5 billion USD is
required. Even after spending huge resources and funds, 490000 cases of multiple drug
1
resistance TB (MDR-TB) were reported in the year 2016. Further, the occurrence of co-
infection with the Human Immunodeficiency Virus (HIV) together with the increase in global
2
bacterial resistance has made TB as a re-emerging disease. The cell wall of the Mtb is
unusually constructed with almost 60% of lipids (Mycolic acids, C60-C90 long α-alkyl β-
hydroxylated fatty acids) that are essential for the survival of this bacterium. In Mtb, Enoyl-
ACP reductase (InhA) of the type II fatty acid synthase (FAS-II) system is mainly engaged in
the biosynthesis of these foremost components of mycobacterial cell wall.3
Isoniazid (INH) is a first line prodrug and Ethionamide (ETH), a second line prodrug
used for the treatment of TB and exhibit their versatile action via the InhA inhibition. For
active TB, these drugs are generally used along with rifampin, pyrazinamide and either
ethambutol or streptomycin, whereas for latent TB, INH is used alone.4,5 Few reports have
also mentioned the use of INH against other types of mycobacteria like M. kansasii, M.
5
xenopi and M. avium. The use of INH in the HIV carrying TB patients is also well
6
documented. INH and ETH being prodrugs, must be activated by some enzymes. A bacterial
catalase-peroxidase enzyme in Mtb known as KatG activates INH7 whereas the
8
monooxygenase ethA activates the ETH. These enzymes convert INH and ETH into
+
respective acyl radicals which immediately couples with NAD to form the NAD adduct. The
formed adduct binds tightly to the active-site of InhA and inhibits the natural Enoyl-AcpM
9
substrate and subsequently the action of FAS in Mtb. This action makes the InhA, one of the
best drug target for the treatment of TB. The resistance to these most important drugs is
reported mainly by the mutation in KatG and ethA in Mtb leading to the inability to activate
7
these prodrugs for their action. Thus, the development of resistance to several anti-TB drugs
including INH creates urgency for the discovery of new anti-TB agents.
The synthesis of novel carbazole derivatives and the investigation of their chemical
and biological behaviour have gained importance in recent decades for their biological,
medicinal and photoelectrical applications. Carbazole analogs from both natural and synthetic
origin are known to display wide range of pharmacological properties such as anticancer,
3

antibacterial and anti-TB activity.10,11 Similarly, literature indicated that the molecular
manipulation of rhodanine moiety resulted in potent compounds that exhibited a wide
spectrum of biological activities such as antimicrobial (including multidrug-resistant clinical
isolates), anti-TB (III, Fig. 1), antifungal, antidiabetic, anti-HIV and anticancer.12,13 Literature
also suggested that the rhodanine moiety is an important fragment for antimicrobial activity.14
Previously, N-methyl carbazole clubbed 1,2,3-triazoles (I, Fig. 1) were reported as promising
anti-mycobacterial agents.15
We are also aware of rhodanine moiety is classified into a substantial pan-assay
interfering compound (PAINS) that might confound the assay as well as may lead to apparent
activity results.16,17 The two main reasons reported for promiscuous behavior were a)
Formation of aggregates that non-specifically interact with the target which interferes with
biochemical assay and can be countered by performing the assays under high-detergent
conditions.18,19 b) Undergo facile reaction with nucleophiles via Michael addition to the
exocyclic double bonded sulfur, which provides high density of interaction sites for polar
2
0
interactions and hydrogen bonds. In order to identify PAINS and triage reactive compounds,
Dahlin et al. suggested two general approaches, namely knowledge-based and experimental-
based methods. The knowledge-based strategies included substructure filters, literature and
database searches, and consulting of experienced medicinal chemists, while the experimental-
based strategies included mechanistic experiments, thiol-based probes and multiple types of
counter-screens.21
However, the concept of PAINS was countered by some researchers as indicated in
recent literature reports that the criteria’s defining PAINS should not be considered as a
general criterion to exclude from screening compounds containing rhodanines and was
suggested that some special criteria such as target affinity and selectivity should be
employed.20, 22 Thus some researchers still consider rhodanines as privileged heterocycles and
building blocks for optimization and transformation into related simplified analogues and
fused heterocycles. The Michael acceptor functionality as well as low selectivity must be
confirmed experimentally and should not be just based on the presence of conjugated α,β-
unsaturated carbonyl. The positive aspects of Michael acceptors and multitarget properties
2
2
must be taken into consideration. Compounds showing potential biomolecular binding
properties could be further exploited in a useful way. It should be noted that the marketed
drugs like pioglitazone used for diabetes mellitus type-223 and epalrestat for diabetic
neuropathy contain rhodanine scaffold as core moiety, while epalrestat specifically contains
4

2
4
ene-rhodanine moiety. Further, rhodanine analogs have been recently reported for various
pharmacological activities in peer-reviewed medicinal chemistry journals.25-29
Earlier, our research group has reported a series of N-methyl carbazolo-thiazole
hybrid molecules (II, Fig. 1) as antimycobacterial agents against the virulent cell line of TB
and displayed moderate activity with a good cytotoxicity profile on mammalian Vero cell
1
0
line. Keeping in mind the activity profiles of those molecules, we envisaged to structurally
modify them to achieve better antimycobacterial activity. Hence, we attempted to synthesize
novel N-methyl carbazole tethered rhodanine derivatives (Fig. 1) using molecular
hybridization approach.
HO
O
S
Cl
O
N
NH
N
N
N
OCH3
OCH3
S
S
N
N
N
S
F3C
II
M. tuberculosis H Rv
CH3
III
3
7
M. tuberculosis H Rv
37
(MIC = 21 M)
(
MIC = 3.12 M)
N
N
N
Aryl/Heteroaryl
O
N
CH3
N
S
S
I
M. tuberculosis H Rv
37
(MIC = 30.6 M)
9
/ 10
N
CH3
Fig. 1. Design of N-methyl carbazole tethered rhodanine derivatives using molecular
hybridization approach.
The synthesis of N-methyl carbazole tethered rhodanine derivatives 5-((9-methyl-9H-
carbazol-6-yl)methylene)-3-(substituted phenyl)-2-thioxothiazolidin-4-one) (9a-9l) and 2-((9-
methyl-9H-carbazol-6-yl)methylene)-4-(5-substituted phenyl-1,3,4-thiadiazol-2-yl)-5-thioxo-
thiazolidin-4-one) derivatives (10a-10f) was achieved through an efficient and versatile
5

synthetic route as illustrated in Scheme 1. In order to practically obtain the hybrid
compounds, we first attempted the reactions by conventional method i.e. reflux in ethanol
with a catalytic amount of piperidine for 24 h, but this resulted in very poor yields (10-18%).
In order to achieve higher yields, these reactions were carried out under controlled
microwave irradiation (CEM Discover, Explorer-12 Hybrid, Microwave conditions: 80 °C,
3
0 min at 150 psi) which resulted in the corresponding carbazolyl substituted rhodanine
derivatives 9a-9l and 10a-10f in considerably good yields (48–79 %).
Various rhodanine derivatives i.e. 3-substituted-phenyl-2-thioxothiazolidin-4-one (3a-
3
l) and 3-(5-substituted phenyl)-1,3,4-thiadiazol-2-yl)-2-thioxothiazolidin-4-one (5a-5f) were
3
0
rd
prepared as per the earlier reported method and were coupled to the 3 position of the
carbazole ring to yield final compounds 9a-9l and 10a-10f. Synthesis of various substituted
rhodanine derivatives was achieved by reacting bis(carboxylmethyl) trithiocarbonate (1) with
appropriately substituted aromatic/heteroaromatic amines (2a-2l) and 5-substituted phenyl-
1
,3,4-thiadiazol-2-amines (4a-4f) to obtain the desired intermediates 3a-3l and 5a-5f,
1
respectively. These intermediates were characterized and confirmed based on IR, H- and
1
3
-1
C-NMR data. In IR spectra, the appearance of 1757-1676 cm for C=O (carbonyl), 1180-
-1
1
100 cm for C=S and the absence of carboxylic acid group supported the formation of
1
compounds 3a-3l and 5a-5f. This was further substantiated from the H-NMR spectrum,
which showed the presence of a very distinct singlet at δ 4.4-4.17 ppm for -CH - (methylene),
2
while various aromatic/hetero-aromatic proton signals were observed between δ 8.53-6.40
1
3
ppm. In the C-NMR spectrum, the most characteristic carbon signals (C=S, C=O and CH )
2
appeared at around δ 206.45-197.25, 173.94-168.04 and 38.85-36.4 ppm respectively, thus
confirming the formation of the rhodanine nucleus. Synthesis of 9-methyl carbazole (7) was
achieved by N-methylation of commercially available carbazole (6) with iodomethane and
sodium hydride and its formation was confirmed by the absence of N-H stretching (IR) and
1
appearance of N-methyl protons ( H-NMR). Further, Vilsmayer-Hack formylation of 9-
methyl carbazole (7) in the presence of POCl and DMF yielded 9-methyl-carbazole-3-
3
carbaldehyde (8) in good yield and was confirmed by the presence of carbonyl stretching (IR)
1
and appearance of -CHO proton ( H-NMR).
6

Aryl/Heteroaryl
(
i)
Aryl/Heteroaryl
a-2l)
O
H N
2
N
S
S
O
O
S
S
(
2
HO
OH
S
Substituted aromatic/
(3a-3l)
Heteroaromatic amines
(1)
3-substituted phenyl-
2
-thioxothiazolidin-4-one
bis(carboxymethyl)
trithiocarbonate
5
-(substituted phenyl)-
-amino
,3,4-thiadiazoles
2
1
N
N
NH2
(i)
N
S
R
N
S
R
O
(
4a-4f)
N
S
S
(
5a-5f)
3-((5-substituted phenyl)-
1,3,4-thiadiazol-2-yl)-2-thioxo
thiazolidin-4-one
O
H
(iii)
(ii)
N
N
H
CH3
7)
-methyl carbazole
N
(
CH3
(
6)
9
(8)
9
H-carbazole
(iv)
Aryl/Heteroaryl
S
(
3a-3l)
O
N
(
5a-5f)
(iv)
S
N
N
S
N
CH3
O
(9a-9l)
N
R
Where.......
S
S
Aryl/Heteroaryl
N
CH3
OCH3
CF3
CH3
9
a :
b :
9
f :
9j :
COOCH3
(10a-10f)
0a : R = 2-F
N
O
H
9
1
9g :
OH
9k :
OCH3
10b : R = 4-Cl
10c : R = 2-Cl
9c :
OCH3
COOH
9h :
1
0d : R = 4-Br
9l :
Cl
OCH3
OCH3
OCH3
COOH
CH3
10e : R = 3-Cl
9d :
1
0f : R = 4-OCH3
9i :
O O
9e :
COOH
7

Scheme 1. Reagents and conditions: (i) bis(carboxymethyl) trithiocarbonate, substituted
aromatic/heteroaromatic amines (2a-2l and 4a-4f), H O, reflux, 100 C, 12-19 h; (ii) DMF,
2
NaH, CH I, stir, RT, 5 h; (iii) POCl , DMF, 0 °C, stir, 90 °C, 6 h, Na CO ; (iv) Compound 8,
3
3
2
3
rhodanine derivatives (3a-3l and 5a-5f), piperidine, ethanol, microwave irradiation, 30 mins,
0 ºC, 150 psi.
8
The final hybrids 9a-9l and 10a-10f were obtained by Knoevenagel condensation of 8
with 3-substituted-phenyl-2-thioxothiazolidin-4-one (3a-3l) and 3-(5-substituted phenyl)-
,3,4-thiadiazol-2-yl)-2-thioxothiazolidin-4-one (5a-5f), respectively. The IR spectra of the
hybrids presented bands for the C=O (carbonyl) and C=S groups of rhodanine moiety around
1
-1
-1
1
713.58-1678.41 cm and 1247.91-1221.96 cm , while the most prominent band appeared
-1
around 1605.94-1567.83 cm due to C=CH (arylidene) group. This observation was further
1
substantiated by the H-NMR of compounds 9a-9l and 10a-10f, displaying singlet peak
resonating around δ 8.06-7.91 ppm and δ 9.16-9.02 ppm, accounting for the CH=C
rd
(
arylidene) proton at 3 position of the carbazole nucleus. The disappearance of a distinct
singlet at around δ 4.4-4.17 ppm for the -CH - (methylene) protons of the rhodanine ring
2
evidently confirmed the formation of the desired final compounds. Moreover, the formation
of final derivatives was further confirmed by recording some of their mass spectra (HR-MS)
1
which displayed positive correlation with their anticipated molecular weights. IR, H-NMR,
13C-NMR and HR-MS spectra are provided in the supporting information.
These newly synthesized compounds were evaluated against H Rv for their anti-TB
3
7
potential and InhA inhibition as well as toxicity profile on mammalian cells. For our
1
0
previously reported compounds, the InhA inhibition activity was determined and the data is
presented in Table 1.
Table 1.
InhA inhibition data of the compounds 3 and 6a-0
Compound
code*
3
InhA inhibition
(% at 50 µM)
45
23
23
42
InhA inhibition
(% at 50 µM)
Compound code*
6h
6i
6j
6k
6l
6m
6n
6o
10
43
27
NI
42
58
42
30
6
6
6
6
6
6
6
a
b
c
d
e
f
Not soluble
10
30
17
g
*
Synthesis, characterization, M. tuberculosis H Rv and cytotoxicity evaluations results of
37
compounds 3 and 6a-0 were reported in our earlier publication.10
8

Anti-TB (in vitro) activity of all the final compounds was carried out at the Infectious
Disease Research Institute (IDRI) within the National Institute of Allergy and Infectious
Diseases (NIAID) screening program, Bethesda, MD, USA. All the hybrids were screened
against the virulent strain of M. tuberculosis H Rv grown under aerobic conditions by using
3
7
a dual read-out (OD590 and fluorescence) assay procedure.31-34 The results of the
antimycobacterial activity (MIC values) are presented in Table 2.
Table 2.
Antitubercular evaluation of 9a-9l and 10a-10f against Mtb H Rv and InhA along with
3
7
cytotoxicity data.
M. tuberculosis
InhA inhibition (% at
50 µM)
Cytotoxicity
IC50b (µM)
Compound
a
H Rv
3
7
9
a
6
>
100
235.2
NI
9
9
b
c
>100
50 (IC50 = 50 µM)
167.2
1
2
5
4
260.9 (5.2)c
9
d
>100
>100
185.0
262.7
9
1 (54% at 5 µM) IC = 2.82+
50
9
e
0
.36 µM
5
2
9
f
>100
277.2
6
2 (10% at 5 µM)
7 (12% at 5 µM)
289.3 (11.6)c
9
g
>100 (IC = 25 µM)
5
0
6
9
h
>100
210.1
296.4 (14.8)c
1
3
9
i
20 (IC =20 µM)
5
0
NI
9
j
>100
222.0
239.5 (3.2)c
3
2
9
k
>100 (IC = 75 µM)
5
0
NI
NI
9
l
>100
>100
>100
>100
>100
>100
>100
212.8
208.1
245.0
239.0
165.2
192.7
258.3
1
0a
0b
0c
0d
0e
0f
GEQ
3
5
5
7
3
2
1
1
1
Not soluble
1
3
4
1
> 50
88
---
a
MIC at which M. tuberculosis H Rv growth was completely inhibited and is derived from
3
7
b
the inflection point of the fitted curve to the lower asymptote (zero growth); Cytotoxicity
activity (IC ) was determined on mammalian Vero cell line. NI = No inhibition; Selectivity
c
50
index determined as the ratio cytotoxicity/IC50 (H Rv). The compounds presenting IC50
37
values >100 were not considered for the selectivity index calculation.
Among the tested series, compound 9i (MIC = 20 µM) with coumarin ring on
rhodanine nucleus displayed the best antimycobacterial activity with an IC 20 µM, followed
5
0
9

nd
by 9g bearing the hydroxyl (OH) group on the 2 position of aromatic nucleus indicated
good activity with IC 25 µM, while 9c (MIC = 50 µM), the 3,4-dimethoxy groups on
5
0
aromatic ring exhibited moderate inhibitory activity with an IC50 50 µM. The remaining
compounds in this series with halogen groups on the aromatic ring were found to be least
active. According to literature reports, an optimum lipophilicity is required to facilitate the
3
5
entrance of the molecules through lipid-enriched mycobacterial membrane. Hence, clog P
values of the most active compounds were calculated and the results presented no conclusive
outcome. However based on the calculated clog P values, 9e was one of the most polar
compounds due to the presence of carboxylic functional group, which could be responsible
for no cell permeability in the cell-based antimycobacterial inhibition. While compounds 9c,
9
g, 9i and 9k displayed favorable antimycobacterial activity, which could be attributed to the
presence of electron releasing lipophilic functionalities like dimethoxy, hydroxy and the
bulkier coumarine substituents, respectively. In addition, the functional group on the aromatic
ring attached to the rhodanine ring contributed to the lipophilicity and binding interactions.
Thus, poor antimycobacterial activity of compound 9e could be attributed to the hydrophilic,
polar carboxylic functional group on the aromatic ring of the rhodanine moiety.
To determine the potential InhA inhibition activity of these hybrids, an enzyme assay
3
6
was carried out in triplicate as per previously reported protocols. Assays were performed
using 2-trans-dodecenoyl-CoA as a substrate analogue and the percentage inhibition was
+
measured considering the conversion of the NADH into its oxidized form NAD at 340 nm.
GEQ compound, a well-known inhibitor of InhA, was used as reference37,38 and all the hybrid
molecules were tested at 50 μM in triplicate. The InhA inhibition activity data and the
cytotoxicity data of the new hybrids (9a-9l and 10a-10f) are represented in Table 2. From the
InhA inhibition data, compounds 9e, 9g and 9h exhibited higher (above 60%) inhibition at 50
μM i.e. 91%, 62% and 67%, respectively. For these three compounds, the assay was
performed at 5 μM concentration and specifically, 9e (54% inhibition) presented activity
above 50%. The IC value of 9e was determined using the 4-parameter curve-fitting software
5
0
XLFit (IDBS) and it was observed at higher potency of 2.82 μM (Fig. 2). Compound 9e
showed a very interesting activity against InhA enzyme but a very poor activity against Mtb
H Rv strain which could be due to the efflux pumps action or membrane permeability.
3
7
1
0

Fig. 2. Determination of IC of compound 9e against InhA.
5
0
All the final compounds were further evaluated for cytotoxicity on a mammalian Vero
cell line.39 The cellular conversion of MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-
tetrazolium bromide] into a formazan product was used to evaluate cytotoxicity. After 72 h of
exposure, the viability of the cells was assessed using the Promega Cell Titre 96 non-
radioactive cell proliferation assay. The IC values were determined and are presented in
5
0
Table 2. All the novel carbazolyl substituted derivatives displayed an interesting cytotoxicity
profile with IC values ranging from 165.2-296.4 µM. Among the series, 9i displayed least
5
0
cytotoxicity (IC = 296.4 µM) followed by 9c, 9e, 9f, 9g and 10f (IC > 250 µM). None of
5
0
50
the final compounds exhibited substantial cytotoxic activity against the human Vero cell line
at concentrations < 100 µM. These results are particularly important, as the compounds with
their increased cyto-viability could be very attractive in developing new chemical entities
(NCEs) to treat chronic infectious diseases such as TB, principally due to long treatment
regimen required. Thus, there is an urgent need to develop new anti-TB agents with a high
margin of safety.
In order to understand the ligand-enzyme interactions between compound 9e and
4
0
41
InhA (PDB ID: 4TZK) active-site, a molecular docking study was performed using Glide
program of Schrodinger. A Grid was generated and validated by docking the existing co-
crystallized ligand in PDB 4TZK. The docking results exhibited similar pose besides crucial
molecular interactions with the active-site residues as reported in the literature. The docked
complex presented a characteristic, strong hydrogen bonding interaction between the
carbonyl oxygen (C=O) of pyrrolidinone and –OH group of Tyr158. Further, a distinctive
halogen bond was observed between the chlorine and –HN of Gly104 as presented in original
PDB report. All crucial molecular interactions were reproduced that subsequently validated
1
1

the protocols for further docking of the synthesized hybrid compounds into the active-site of
the protein. Interestingly, the most active compound 9e exhibited essential interactions with
InhA. The carbazole moiety of 9e buried inside the hydrophobic pocket of the protein core
consisting of Met103, Gly104, Phe149, Pro193, Met199, Leu207, Ala211 and Ile215, while
the rhodanine nucleus stabilized within the active-site by interacting with hydrophobic
residues (Phe97 and Ala198) as well as exhibiting a weak hydrogen bond with Met98
(2.77Å). The carboxylic acid group in 9e contributed towards the orientation of the entire
ligand in the active-site of the enzyme by orienting itself into the solvent accessible area of
the protein cavity. Similarly, the phenyl ring in 9e provided the stability to the ligand inside
the active-site through hydrophobic interactions involving residues of Ile16, Phe41, Phe97
and Ala198. The molecular interactions of 9e with the active-site residues are presented in
Fig. 3.
Fig. 3. The docked pose (thick-tube model, magenta colour) of compound 9e into the active-
site showing crucial interactions.
To confirm the validity and stability of the proposed docking results, the dynamic
stability of 9e within the enzyme was examined in the complex over 10 ns duration by using
Maestro-Desmond Tool.42,43 The post dynamic analyses were carried out to understand the
stability of the ligand-protein complex which was determined by average root mean square
deviation (RMSD).44,45 Over the period of time, RMSD-P, RMSF-P and RMSD-L
(P=Protein, L=Ligand) were scrutinized to support our docking study. The initial pose of
ligand-protein complex was considered as a reference frame to calculate these properties. The
RMSD-P was basically calculated to understand the large-scale movements in the protein
when the ligand is present in the active-site. The RMSD-P for the complex was observed in
the range of 0.6 to 1.8 Å. This observation suggested that 9e in the active-site of the protein
has not influenced the stability of protein back-bone throughout the simulation run. Further,
1
2

to confirm the binding stability of 9e, RMSD-L was computed and observed in the range of
.2 to 1.2 Å which indicated the good stability of the ligand with respect to the protein and its
0
binding pocket. For the entire duration of the simulation run, no major deviation in RMSD
was observed (Fig. 4a). The structural integrity of the protein and the residual mobility of the
ligand were quantified in terms of RMSF-P. For almost all residues including loop residues
except the terminal residues of the protein, the RMSF-P was below 2.05 Å whereas the
terminals showed the RMSF-P was up to 3.3 Å (Fig. 4b). In the protein-ligand stability, the
interactions were also evaluated over a period of the simulation time, which indicated that H-
bond was maintained only for 10% of the simulation time, either with Met98 or Gln100 with
the H-bond distance ≤ 2.5 Å and donor angle ≥120° and acceptor angle ≥ 90° indicating that
H-bond interactions in the docking study was weak. Further, for 76% of the simulation time,
Tyr158 residue was involved in hydrophobic interactions which played a valid role in the
stability of ligand-protein complex (Fig. S1). The ligand RMSF was observed in between 1.0
to 2.5 Å explaining the range of ligand atom positions over the simulation time period. The
other ligand parameters like rGyr, MolSA, SASA and PSA were observed in the acceptable
range (Fig. S2).
a
b
Fig. 4. (a) Protein and Ligand (9e) RMSD, blue line indicates protein RMSD whereas pink
line indicates ligand RMSD; (b) Protein RMSF over the simulation time of 10 ns.
1
3

In summary, novel carbazole hybrid derivatives were synthesized and evaluated for
their anti-TB and InhA inhibition activity. Compounds 9c, 9g, 9i and 9k exhibited the
moderate anti-TB activity considering the IC value below 100 µM against the virulent TB
5
0
cell line H Rv. Compound 9e showed promising InhA inhibition with an IC value of 2.82
3
7
50
µM. To understand the molecular interactions of the most active compound 9e with the
active-site, molecular docking was carried out which was further supported by the molecular
dynamics. The cytotoxicity (IC ) assessment against the human Vero cell line using MTT
5
0
assay demonstrated that the newly synthesized compounds were non-cytotoxic. These
exciting results have led to the identification of carbazole hybrids as a new class of InhA
direct inhibitor and will further assist the scientific community to develop safer yet potential
anti-TB agents, which is the need of the hour.
Supporting Information
All experimental data, spectral images and figures associated with molecular dynamics are
provided in supporting information.
Disclosure statement
Authors hereby declare that there are no financial/commercial conflicts of interest.
Acknowledgements
Authors are grateful to Discipline of Pharmaceutical Sciences, College of Health
Sciences, University of KwaZulu-Natal (UKZN), Durban, South Africa for providing access
to necessary facilities. Author BC gratefully acknowledge National Research Foundation
(NRF), South Africa (SA) for the research funding in the form of NRF-Innovation Post-
Doctoral Research Fellowship (Grant No. 99546). RK is also thankful to NRF-SA for
funding this project (Grant No. 103728 and 112079). Authors also sincerely thank Centre for
High Performance Computing (CHPC), Cape Town, South Africa for computational
resources. Authors express heartfelt thanks to Mr. Dilip Jagjivan and Dr. Caryl Janse Van
Rensburg (UKZN, South Africa) for their assistance in NMR and HR-MS experiments.
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Graphical Abstract
Discovery of novel N-methyl carbazole tethered rhodanine derivatives as
direct inhibitors of Mycobacterium tuberculosis InhA
a
a$
a$
Mahamadhanif S. Shaikh , Ashish M. Kanhed , Balakumar Chandrasekaran , Mahesh B.
a$
a
b
a
Palkar , Nikhil Agrawal , Christian Lherbet , Girish A. Hampannavar , and Rajshekhar
Karpoormatha*
Synthesis, spectral studies antimycobacterial, InhA enzyme inhibition and cytotoxicity evaluation of
novel carbazole hybrid compounds are described.
COOH
O
N
S
S
N
9e
M. tb H37 Rv MIC = 20 µM
inhA inhibition 91% at 50 µM
IC50 = 2.82 µM
MD
Docking
RESEARCH HIGHLIGHTS
Novel N-methyl carbazole tethered rhodanines were synthesized and characterized.

1
9





All of them were evaluated against Mtb virulent cell line and InhA enzyme inhibition.
9e displayed promising inhibition at IC 2.82 µM against InhA.
5
0
Cytotoxicity data suggested that the compounds are safe.
Molecular docking and molecular dynamics supported the in vitro results.
2
0