Cytotoxicity and antimycobacterial properties of pyrrolo[1,2-a]quinoline derivatives: Molecular target identification and molecular docking studies

Article abstract of DOI:10.3390/antibiotics9050233

A series of ethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylates 4a-f and dimethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarboxylates 4g-k have been synthesized and evaluated for their anti-tubercular (TB) a

Full text of DOI:10.3390/antibiotics9050233

antibiotics
Article
Cytotoxicity and Antimycobacterial Properties of
Pyrrolo[1,2-a]quinoline Derivatives: Molecular Target
Identification and Molecular Docking Studies
Katharigatta N. Venugopala 1,2,*, Vijayakumar Uppar 3,† , Sandeep Chandrashekharappa 4,†
Hassan H. Abdallah 5 , Melendhran Pillay 6, Pran Kishore Deb 7 , Mohamed A. Morsy 1,8
Bandar E. Aldhubiab 1 , Mahesh Attimarad 1 , Anroop B. Nair 1 , Nagaraja Sreeharsha 1
,
,
,
Christophe Tratrat 1 , Abdulmuttaleb Yousef Jaber 7 , Rashmi Venugopala 9
,
Raghu Prasad Mailavaram 10 , Bilal A. Al-Jaidi 11 , Mahmoud Kandeel 12,13
,
Michelyne Haroun 1 and Basavaraj Padmashali 3
1
Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University,
Al-Ahsa 31982, Saudi Arabia; momorsy@kfu.edu.sa (M.A.M.); baldhubiab@kfu.edu.sa (B.E.A.);
mattimarad@kfu.edu.sa (M.A.); anair@kfu.edu.sa (A.B.N.); sharsha@kfu.edu.sa (N.S.);
ctratrat@kfu.edu.sa (C.T.); mharoun@kfu.edu.sa (M.H.)
2
3
4
Department of Biotechnology and Food Technology, Durban University of Technology,
Durban 4001, South Africa
Department of Chemistry, School of Basic Science, Rani Channamma University, Belagavi 591156, India;
vijay.uppar@gmail.com (V.U.); basavarajpadmashali@yahoo.com (B.P.)
Institute for Stem Cell Biology and Regenerative Medicine, National Center for Biological Sciences (NCBS),
Tata Institute of Fundamental Research (TIFR), Gandhi Krishi Vignana Kendra (GKVK) Campus,
Bangalore 560065, India; sandeepc@instem.res.in
5
6
7
Chemistry Department, College of Education, Salahaddin University, Erbil 44001, Iraq;
Hassan.Abdullah@su.edu.krd
Department of Microbiology, National Health Laboratory Services, KZN Academic Complex, Inkosi Albert
Luthuli Central Hospital, Durban 4001, South Africa; melendhra.pillay@nhls.ac.za
Faculty of Pharmacy, Philadelphia University, Amman 19392, Jordan; prankishore1@gmail.com (P.K.D.);
amyjaber@gmail.com (A.Y.J.)
Department of Pharmacology, Faculty of Medicine, Minia University, El-Minia 61511, Egypt
Department of Public Health Medicine, University of KwaZulu-Natal, Howard College Campus,
Durban 4001, South Africa; rashmivenugopala@gmail.com
Department of Pharmaceutical Chemistry, Shri Vishnu College of Pharmacy, Vishnupur,
Bhimavaram 534 202, India; raghumrp@svcp.edu.in
Faculty of Pharmacy, Yarmouk University, Irbid 21163, Jordan; bilaljeaidi77@gmail.com
Department of Biomedical Sciences, College of Veterinary Medicine, King Faisal University,
Al-Ahsa 31982, Saudi Arabia; mkandeel@kfu.edu.sa
Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelsheikh University,
Kafrelsheikh 33516, Egypt
8
9
10
11
12
13
*
Correspondence: kvenugopala@kfu.edu.sa; Tel.: +966-1358-98842
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 April 2020; Accepted: 3 May 2020; Published: 7 May 2020
Abstract: A series of ethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylates
4a–f and dimethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarboxylates 4g–k
have been synthesized and evaluated for their anti-tubercular (TB) activities against H37Rv
(American Type Culture Collection (ATCC) strain 25177) and multidrug-resistant (MDR) strains of
Mycobacterium tuberculosis by resazurin microplate assay (REMA). Molecular target identification for
these compounds was also carried out by a computational approach. All test compounds exhibited
anti-tuberculosis (TB) activity in the range of 8–128
µg/mL against H37Rv. The test compound
dimethyl-1-(4-fluorobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarboxylate 4j emerged as the
Antibiotics 2020, 9, 233; doi:10.3390/antibiotics9050233
www.mdpi.com/journal/antibiotics

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most promising anti-TB agent against H37Rv and multidrug-resistant strains of Mycobacterium
tuberculosis at 8 and 16 g/mL, respectively. In silico evaluation of pharmacokinetic properties
µ
indicated overall drug-likeness for most of the compounds. Docking studies were also carried out to
investigate the binding affinities as well as interactions of these compounds with the target proteins.
Keywords: pyrrolo[1,2-a]quinoline; Mycobacterium tuberculosis; H37Rv; MDR-MTB; minimum
inhibitory concentration; cytotoxicity; computational studies; molecular target identification
1. Introduction
Tuberculosis (TB) continues to be one of the top causes of death worldwide. This infectious
disease is caused by the organism Mycobacterium tuberculosis (MTB) [1]. Approximately 1.3 million
deaths were caused by TB in 2018. In addition, people who are human immunodeficiency virus
(HIV)-positive found to be highly susceptible to TB infection. Approximately 300,000 deaths of
HIV-positive patients were due to TB infection [2]. In addition, the emergence of multidrug-resistant
TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) strains, which are highly resistant to
most of the currently available anti-TB drugs, trigger the urgent need for the development of novel
therapeutic agents to combat these resistant strains [3,4] as only a few drugs are available with
United States Food and Drug Administration (US FDA) approval (Figure 1). Reviews focused on
Mycobacterium tuberculosis and in the pursuit of developing novel anti-TB agents, we recently launched
a medicinal chemistry program aimed at developing novel, natural [5], cyclic depsipeptides [6] and
various heterocyclic scaffolds as potential anti-TB agents [ 13], including analogues of indolizine such
7–
as pyrrolo[1,2-a]quinoline and pyrrolo[1,2-a]isoquinoline, also known as 5,6-benzo-fused indolizine and
7,8-benzo-fused indolizine, respectively [14]. These scaffolds have attracted the attention of medical
chemists, as they exhibit a wide variety of pharmacological properties [15]. The pyrrolo[1,2-a]quinoline
derivatives possess antioxidant [16], anti-inflammatory [17,18], larvicidal [19], and antiviral [20]
activities. 4-Amino-pyrrolo[2,3-b]quinoline has also been tested as a potential treatment for Alzheimer’s
disease [21]. These pharmacological potentials of the pyrrolo[1,2-a]quinoline scaffold gave us the
impetus to further explore this scaffold in the quest of anti-TB drug discovery [5–12]. Herein, we
report the evaluation of some novel pyrrolo[1,2-a]quinoline derivatives as potential anti-TB agents
against H37Rv and MDR strains of Mycobacterium tuberculosis. Encouraging bioactivity of these tested
compounds against TB strains prompted us to elucidate their mechanism of action by employing
molecular docking calculations. The in silico pharmacokinetic profile (absorption, distribution,
metabolism, and excretion (ADME) properties) of these compounds was investigated to evaluate
their drug-likeness.
Figure 1. Anti-tubercular drugs 1, 2 and 3, which are approved in last 10 years [22].

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2. Results and Discussion
2.1. Chemistry
Construction of the title compounds 4a
–
k
was achieved by a two-step chemical synthesis
(Scheme 1). Purification was completed by column chromatography and the yield was 54–67%
after purification [19 23]. The purity of the compounds was ascertained by high-performance liquid
,
chromatography (HPLC) and was found to be >99%. The molecular structures of the title compounds
used for antitubercular properties are listed in Figure 2.
CH3
N
CH3
N
O
O
Br
CH3
N
Br-
O
O
R
C2H5
C2H5
R1
O
O
(a)
O
+
R1
(b)
R
R
1
2
3a-f
4a-f
O
R1
(b)
CH3
O
CH3
O
N
CH3
O
O
R1
R
4g-k
R = 4-CN, 4-Br, 2-NO2, 4-H, 4-F, 3,5-CF3
R1 = H, COOCH3
Scheme 1. Synthetic scheme for the construction of ethyl 1-(substituted benzoyl)-5-methylpyrrolo
[1,2-a]quinoline-3-carboxylate 4a and dimethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline
analogues: Reagents and conditions: ( ) acetone, r.t., 30 min; ( ) DMF, K2CO3,
–f
-2,3-dicarboxylate 4g
–
k
a
b
30 min.

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Figure2. Chemical structure of ethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate
4a and dimethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarboxylate 4g
–f
–k
analogues tested for anti-tuberculosis (TB) activity against H37Rv strain and multidrug-resistant
Mycobacterium tuberculosis.
2.2. Antitubercular Activity
We conducted resazurin microplate assay to screen the title compounds 4a
activity against H37Rv and multidrug-resistant (MDR) strains of Mycobacterium tuberculosis. Minimum
inhibitory activity of the title compounds 4a is tabulated in Table 1. All 11 compounds tested exhibited
antitubercular activity against H37Rv strain, ATCC number 25,177, and only three compounds, 4f 4j
and 4k, exhibited anti-TB activity at 64, 16, and 32 g/mL, respectively, against multidrug-resistant
–k for antitubercular
–
k
,
,
µ
Mycobacterium tuberculosis, which was resistant to rifampicin (Rif) and isoniazid (Inh). The structure
activity relationship and molecular mechanics studies of the title compounds confirmed that
3,5-substitution on the benzoyl group at the first position of the indolizine nucleus is critical for
the activity.

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Table 1. Antitubercular activity of ethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-
carboxylate 4a and dimethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarboxylate
4g analogues against H37Rv and multidrug-resistant (MDR)– Mycobacterium tuberculosis (MTB)
strains of Mycobacterium tuberculosis.
–f
–k
MIC (µg/mL)
Compound Code
H37Rv *
MDR-MTB **
4a
4b
4c
4d
4e
4f
4g
4h
4i
64
32
32
8
20
16
32
128
128
8
NA
NA
NA
NA
NA
64
NA
NA
NA
16
4j
4k
32
32
* American Type Culture Collection (ATCC): 25177. ** These isolates are resistant to first-line antibiotics isoniazid
(0.2 g/mL) and rifampicin (1 g/mL). MIC, minimum inhibitory concentration; NA, not active (concentration
considered for screening was 0.2–128 µg/mL).
µ
µ
2.3. Toxicity Studies
The anti-TB compounds 4f
Overall, the three compounds exhibited no toxicity up to 250
cell lines. This result provides assurance about the safety of the compounds and encourages us to consider
4f 4j, and 4k as lead compounds for further refinement to achieve the most promising anti-TB agents
,
4j, and 4k (Table 1) were evaluated in safety studies via MTT assay.
g/mL across peripheral blood mononuclear
µ
,
against MDR strains of Mycobacterium tuberculosis.
2.4. Computational Studies
2.4.1. Target Identification
Potential protein targets that had one or more ligands with flexophore similarity value of 0.6 or
higher to one or more of the synthesized compounds were identified. Among those, five proteins had
five or more similarity matches between one of their co-crystal ligands and one of the synthesized
compounds. Figure 3 shows a plot of these proteins and the number of ligand similarity matches. As
shown, enzyme DprE1 had the most similarity matches. Five of the synthesized compounds, 4a–e,
displayed similarity with one or more of seven co-crystalized ligands of the DprE1 enzyme, with a total
of 15 similarity matches. This enzyme has an important role in the biosynthesis of the Mycobacterium
tuberculosis cell wall and is a potential target for developing anti-TB drugs.
Figure 3. Number of similarity matches for best identified potential protein targets for synthesized compounds.

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2.4.2. ADME Analysis
The result of ADME prediction from the SwissADME web server is presented in Table 2. For most
of the compounds, the cLogP value is in the optimum range, with the exception of compounds 4b 4i
and 4k, which have cLogP values exceeding the threshold of 5. This can cause low water solubility
,
,
and oral bioavailability. The number of rotatable bonds for all compounds, except compound 4k
is in the desired range of seven, or fewer, for oral bioavailability. Prediction of gastrointestinal (GI)
absorption shows high probability for most compounds; only compounds 4i and 4k were predicted
to have low GI absorption, most likely due to the high cLogP value and number of rotatable bonds.
Several compounds were predicted to have BBB permeability. All compounds except 4i were predicted
to be not potential P-glycoprotein (P-gp) substrates. Finally, the prediction of CYP isoform inhibition
indicates that CYP1A2 and CYP34A might be inhibited by some compounds, such as 4a and 4d, while
CYP2D6 was predicted to be not inhibited by any of the compounds. Overall, the compounds appear
to have druglike properties, as 8 out of the 11 compounds have zero violations of the Lipinski rule of
five conditions.
,
Table 2. Result of absorption, distribution, metabolism, and excretion (ADME) prediction from
SwissADME web server. GI, gastrointestinal; BBB, blood–brain barrier; CYP, cytochrome P.
GI
P-gp
Binding
CYP Inhibition
2D6
BBB
Permeable
Compound
Code
Rotatable
Bonds
Lipinski
Violations
cLogP 1
Absorption
1A2
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
Yes
34A
Yes
No
No
Yes
No
No
Yes
Yes
No
No
No
4a
4b
4c
4d
4e
4f
4.320
5.209
4.585
3.563
4.484
3.991
3.827
3.069
6.181
4.092
5.688
5
5
5
6
5
6
6
7
7
6
High
High
High
High
High
High
High
High
Low
Yes
Yes
Yes
No
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
0
1
0
0
0
0
0
0
1
0
2
4g
4h
4i
4j
High
Low
4k
8
1
cLogP was calculated using DataWarrior software [24].
2.4.3. Docking Studies
Docking is currently considered an essential routine calculation in any drug discovery study [25].
It gives better qualitative and quantitative insight into the binding affinity of the inhibitor at the active
site of the target enzyme [26,27]. Indeed, the calculated binding free energy, inhibition constant and
intramolecular interactions are the key parameters to compare the lists of inhibitors, which may be
hard to achieve experimentally. In this study, 11 compounds (shown in Table 1) were docked into
two proteins, PDB: Pks13 and DprE1, respectively. The docking free energy and calculated inhibition
constant of the docked compounds are listed in Table 3.
The parameters listed in Table 3 reveal the high affinity of these compounds against Pks13 protein
in comparison with DprE1 protein, as they showed higher binding affinity and less inhibition constant
in nanomolar units. Specifically, compound 4d showed the highest binding affinity against both the
proteins, which is in agreement with the experimental results, followed by compounds 4f and 4j and
the remaining compounds.
The high affinity of compounds 4d, 4f, and 4j is obviously attributed to the strong interactions
with the protein active site, as shown in Figures 4 and 5. Hydrogen bonds, pi–pi interactions, and van
der Waals forces are among these interactions.

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Table 3. Docking free energy (kcal/mol) and inhibition constant Ki of target compounds 4a–k.
Experimental MIC
Docking Free
Energy
Experimental MIC
Docking Free
Energy
Comp. Code
(µg/mL)
(µg/mL)
H37Rv *
Pks13
MDR-MTB
DprE1
4a
4b
4c
4d
4e
4f
4g
4h
4i
64
32
32
8
20
16
32
128
128
8
−10.91 (10.05 nM)
−9.73 (73.15 nM)
−11.18 (6.35 nM)
−11.40 (4.38 nM)
−9.44 (119.83 nM)
−9.16 (191.47 nM)
−10.40(23.84 nM)
−10.43 (22.68 nM)
−9.39 (131.39 nM)
−9.54 (101.68 nM)
−9.10 (212.01 nM)
NA
NA
NA
NA
NA
64
NA
NA
NA
16
−7.89 (1.63 µM)
−8.10 (1.15 µM)
−7.88 (1.67 µM)
−9.38 (133.64 nM)
−7.76 (2.05 µM)
−8.33 (782.97 nM)
−7.33 (4.22 µM)
−7.30 (4.48 µM)
−7.13 (5.96 µM)
−8.31 (810.31 nM)
−7.19 (5.40 µM)
4j
4k
32
32
* American Type Culture Collection (ATCC): 25177.
Compound 4d
Compound 4f
Compound 4j
Ligands overlap at the active site
Figure 4. Intramolecular interactions of compounds 4d
,
4f, and 4j with active site of Pks13 protein and
their overlap at the active site.

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Compound 4d
Compound 4f
Compound 4j
Ligands overlap at the active site
Figure 5. Intramolecular interactions of compounds 4d, 4f, and 4j with active site of DprE1 protein and
their overlap at the active site.
3. Materials and Methods
3.1. Chemistry of Compounds
Synthesis of a series of ethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate
and dimethyl 1-(substituted benzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarboxylate 4g–k
4a
–
f
analogues were achieved at 54–67% yield (Scheme 1 and Figure 2). The characterization of the title
compounds 4a-k is completed by instrumental techniques such as FT-IR, 1H-NMR, and 13C-NMR
(Figures S1–S33 are available as supplementary with experimental details) [19
,23]. The purity of
the compounds was confirmed by HPLC and it was found to be more than 99%. Physicochemical
characteristics of substituted pyrrolo[1,2-a]quinoline derivatives 4a–k are available in supplementary
material as Table S1.
3.2. Antitubercular Activity
3.2.1. Resazurin Microplate Assay
Anti-TB screening of test compounds 4a
–f and 4g–k was carried out against H37Rv and MDR
strains of Mycobacterium tuberculosis via the resazurin microplate assay (REMA) method, as described

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in our previous communication [8,28]. Testing was conducted on stored MDR-MTB cultures isolated
from patient sputum specimens; pure colonies from a single strain were retrieved from storage and
subcultured for further testing. Clinical isolates of a well-characterized MDR strain of Mycobacterium
tuberculosis with resistance to rifampicin (Rif) and isoniazid (Inh) were selected for testing. Mutations
within the rpoB gene and katG or inhA gene conferred resistance to Rif and Inh, respectively.
3.2.2. Determining Minimum Inhibitory Concentration
All the 11 test compounds 4a
which was performed 3 times against the H37Rv and MDR-TB strains, demonstrating resistance to
rifampicin (1 g/mL) and isoniazid (0.2 g/mL). The minimum inhibitory concentration (MIC) was
–f and 4g–k were assessed using the agar incorporation method,
µ
µ
determined [29]. The MTB reference strain H37Rv (American Type Culture Collection (ATCC) 25177)
and MDR-TB were cultured for a total of 3 weeks in Middlebrook 7H11 medium [30] and were then
supplemented with Oleic Albumin Dextrose Catalase (OADC) (0.005% v/v oleic acid, 0.2% w/v glucose,
0.085% w/v NaCl, 0.02% v/v catalase, and 0.5% 171 w/v bovine serum albumin (BSA)). Incubation was
set at 37 ◦C. The obtained cultures were utilized to prepare an inoculum in a sterile tube with 0.05%
Tween 80 and 4.5 mL of phosphate buffer with glass beads (5 mm in diameter) by vortexing. After this,
the cultures settled for a total of 45 min; the clear bacterial supernatant was standardized to McFarland
Number 1 using sterile water. The resulting bacterial concentration was approximately 1
forming units (CFU)/mL, which was then diluted with sterile water. Overall, 100 L of the dilution was
added to Middlebrook 7H10 agar plates containing 8–0.125 g/mL of the agent. The test compounds
(8
×
107 colony
µ
µ
µ
g/mL) were dissolved in distilled water and diluted to the required concentration before being
added to the agar medium. The test compound MICs were read 3 weeks following 37 ◦C incubation
and were regarded as the minimum drug concentration that could inhibit >99% growth of the bacterial
culture when compared to controls. The results of this evaluation are presented in Table 1.
3.3. Safety Studies
The most promising anti-TB compounds 4f, 4j, and 4k from the series that exhibited anti-TB
activity against MDR strains of MTB were subjected to safety studies by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. MTT cytotoxicity assay evaluated the cytotoxic effects of
test compounds 4f, 4j, and 4k according to the described protocol [31].
3.4. Computational Studies
3.4.1. Target Identification
In order to identify the possible molecular target of the synthesized compounds, we adapted
ligand-based and structure-based in silico approaches. In the ligand-based approach, we searched
for ligands with known targets that had high similarity to the synthesized compounds, as similar
molecules are expected to bind to the same target [32–34]. In the first step, all ligands that are known
to interact with proteins from the Mycobacterium tuberculosis organism were retrieved from the Protein
Data Bank (PDB). The targets of these retrieved ligands are known and crystallized. In the second
step, each synthesized compound was used as a reference compound in a similarity query to find
compounds from the PDB-retrieved ligand data set that are similar to the reference compound. In order
to measure similarity, the flexophore pharmacophore descriptor was used. The advantages of using
this method are that it measures similarity in terms of the 3D-pharmacophore interaction points and
takes into account the flexibility of the molecules by generating a set of favorable conformations
rather than a single conformation. As the protein–ligand interactions are governed by the molecular
interactions represented by the molecule’s pharmacophore, the flexophore method is suitable in this
case [35]. Ligands from the PDB-retrieved ligand data set that display 0.6 or higher similarity with one
or more synthesized compounds were selected for further investigation. In the next step, the protein
targets of the selected ligands were examined. For each protein, the number of similarity matches was

Antibiotics 2020, 9, 233
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calculated as the total number of similarity queries with a value of 0.6 or higher between any of its
ligands and any of the synthesized compounds. The protein with the most similar ligands was selected
for further study.
3.4.2. ADME Analysis
The pharmacokinetic profiles of lead compounds are of prime importance. Early ADME
studies using in silico prediction can assist in deciding which lead compounds to select for further
development [26,36]. In this study, we used the SwissADME web server to predict important
ADME-related properties of the synthesized compounds [37]. The factors affecting oral bioavailability,
such as the number of rotatable bonds and Lipinski rule of five conditions, were calculated to
gain insight into the probability of oral bioavailability [38,39]. Also, the blood–brain barrier (BBB)
permeability was calculated, as this property might require an adjustment in order to avoid central
nervous system (CNS)-related adverse effects. The potential binding of the compounds to the efflux
pump P-glycoprotein (P-gp) was predicted, as this protein is known for efflux of a wide range of
molecules, including drug substances. This can lead to reduced bioavailability or resistance [40,41].
Finally, the potential inhibition of some important cytochrome P450 (CYP 450) isoforms was predicted,
as inhibition of one or more of these enzymes can cause drug–drug interactions [42].
3.4.3. Docking Studies
The antitubercular target compounds shown in Table 1 were selected for further theoretical
investigation using docking calculations. GaussView and Gaussian09 software were used to build the
starting structures of potential compounds [43]. Each structure was optimized using the molecular
mechanics method, then the AM1 semi-empirical method was implemented in gaussian09 code to
get the structures at their energy ground state. Two target proteins were tested theoretically; Pks13
thioesterase and decaprenylphosphoryl-Beta-d-ribose oxidase. The crystal structure of Pks13 and
DprE1 were downloaded from the RCSB Protein Databank with PDB codes 5V3Y [44] and 5P8K [45],
respectively. The protein structure was cleaned from water and other co-crystalized molecules. The
Autodock4 program (Molinspiration database) was used to prepare the structures of the ligand
compounds and the protein macromolecule and to perform the docking calculations. Polar hydrogens
and Kollman united atom- type charges were added to neutralize the protein structure. Autogrid4
was used to prepare the force field parameters with grid box dimensions of 60
separation equal to 0.375 Å. The Lamarckian genetic algorithm, which is considered one of the best
docking algorithms, was used to run 250 docking runs for the 11 inhibitors and control ligands. The
×
60
×
60 with point
docked conformations were clustered and ranked according to the binding or docking free energy (∆G).
The protein inhibitor intermolecular interactions at the active site were visualized using Discovery
studio 5.0 visualizer.
4. Conclusions
In summary, a series of compounds 4a
activity. The test compound 4j emerged as the most promising anti-TB compound against MDR strains
of Mycobacterium tuberculosis. In vitro cytotoxicity evaluation of 4f 4j, and 4k confirmed the safety
of the compounds up to 250 g/mL. Prediction of ADME properties showed that most compounds,
–k with [1,2-a]quinoline scaffolds were tested for anti-TB
,
µ
except 4f and 4j, have good druglike properties and potential oral bioavailability. Docking calculations
were used to study the binding affinity of the target compounds and to elucidate the intermolecular
interactions at the protein active site, which supported the experimental findings.
Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/5/
233/s1, Figure S1: FT-IR of ethyl-1-(4-cyanobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4a);
1
Figure S2: H- NMR of ethyl-1-(4-cyanobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4a); Figure S3:
13C-NMR of ethyl-1-(4-cyanobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4a); Figure S4: FT-IR
of ethyl-1-(4- bromobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4b); Figure S5: 1H-NMR of

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ethyl-1-(4-bromobenzoyl) -5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4b); Figure S6: 13C-NMR of ethyl-1
-(4-bromobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4b); Figure S7: FT-IR of ethyl-1-(4- fluorobenz
oyl) -5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4c); Figure S8: 1H-NMR of ethyl-1-(4-fluorobenzoyl)-5-
methylpyrrolo[1,2-a]quinoline-3-carboxylate (4c); Figure S9: 13C-NMR of ethyl-1-(4-fluorobenzoyl)-5-methylpyrr
olo[1,2-a]quinoline-3-carboxylate (4c); Figure S10: FT-IR of ethyl-5-methyl-1-(2-nitrobenzoyl) pyrrolo[1,2-a]quino
line-3-carboxylate (4d); Figure S11: 1H-NMR of ethyl-5-methyl-1-(2-nitrobenzoyl)pyrrolo[1,2-a]quinoline-3-
carboxylate (4d); Figure S12: 13C-NMR of ethyl-5-methyl-1-(2-nitrobenzoyl)pyrrolo[1,2-a]quinoline-3-carboxylate
(
4d); Figure S13: FT-IR of ethyl-1-benzoyl-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4e); Figure S14: 1H-
NMR of ethyl-1-benzoyl-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4e); Figure S15: 13C-NMR of ethyl-1-
benzoyl-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4e); Figure S16: FT-IR of dimethyl-1-benzoyl-5-methylp
yrrolo[1,2-a]quinoline-2,3-dicarboxylate (4f); Figure S17: 1H-NMR of dimethyl-1-benzoyl-5-methylpyrrolo[1,2-a]
quinoline-2,3-dicarboxylate (4f); Figure S18: 13C-NMR of dimethyl-1-benzoyl-5-methylpyrrolo[1,2-a]quinoline-2,3-
dicarboxylate (4f); Figure S19: FT-IR of dimethyl-1-(4-cyanobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicar
boxylate (4g); Figure S20: 1H-NMR of dimethyl-1-(4-cyanobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarbo
xylate (4g); Figure S21: 13C-NMR of dimethyl-1-(4-cyanobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarbox
ylate (4g); Figure S22: FT-IR of dimethyl-5-methyl-1-(2-nitrobenzoyl)pyrrolo[1,2-a]quinoline-2,3-dicarboxylate
(
(
4h); Figure S23: 1H-NMR of dimethyl-5-methyl-1-(2-nitrobenzoyl)pyrrolo [1,2-a]quinoline-2,3-dicarboxylate
4h); Figure S24: 13C-NMR of dimethyl-5-methyl-1-(2-nitrobenzoyl)pyrrolo[1,2-a]quinoline-2,3-dicarboxylate (4h);
Figure S25: FT-IR of ethyl-1-(3,5-bis(trifluoromethyl)benzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate (4i);
Figure S26: 1H-NMR of ethyl-1-(3,5-bis(trifluoromethyl)benzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxylate
(
4i); Figure S27: 13C-NMR of ethyl-1-(3,5-bis(trifluoromethyl)benzoyl)-5-methylpyrrolo[1,2-a]quinoline-3-carboxy
late (4i); Figure S28: FT-IR of dimethyl-1-(4-fluorobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarboxylate
(
4j); Figure S29: 1H-NMR of dimethyl-1-(4-fluorobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarboxylate (4j);
Figure S30: 13C-NMR of dimethyl-1-(4-fluorobenzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarboxylate (4j);
Figure S31: FT-IR of dimethyl-1-(3,5-bis(trifluoromethyl)benzoyl)-5-methylpyrrolo[1,2-a]quinoline-2,3-dicarboxy
late (4k); Figure S32: 1H-NMR of dimethyl-1-(3,5-bis(trifluoromethyl)benzoyl)-5-methylpyrrolo[1,2-a]quinoline-
2,3-dicarboxylate (4k); Figure S33: 13C-NMR of dimethyl-1-(3,5-bis(trifluoromethyl)benzoyl)-5-methylpyrrolo[1,
2-a]quinoline-2,3-dicarboxylate (4k); Table S1: Physicochemical characteristics of substituted pyrrolo[1,2-a]quino
line derivatives 4a–k.
Author Contributions: Conceptualization, K.N.V., S.C., H.H.A., and M.P.; Methodology, K.N.V., V.U., S.C., H.H.A.,
M.P., P.K.D., M.A.M., M.A., A.B.N., N.S., C.T., A.Y.J., R.V., R.P.M., B.A.A.-J., M.K., M.H., and B.P.; Software, K.N.V.,
H.H.A., P.K.D., and M.K.; Validation, K.N.V., H.H.A., and M.A.M.; Formal analysis, K.N.V., V.U., S.C., M.P.,
M.A., N.S., A.Y.J., B.A.A.-J., and M.H.; Investigation, K.N.V., H.H.A., M.P., P.K.D., A.B.N., A.Y.J., R.P.M., and B.P.;
Resources, K.N.V., S.C., P.K.D., B.E.A., C.T., R.V., and B.A.A.-J.; Data curation, K.N.V., S.C., M.P., M.A.M., and
C.T.; Writing—original draft preparation, K.N.V., V.U., S.C., H.H.A., M.P., P.K.D., M.A.M., B.E.A., M.A., N.S., C.T.,
A.Y.J., R.V., R.P.M., M.K., M.H., and B.P.; Writing—review and editing, K.N.V., H.H.A., P.K.D., B.E.A., A.B.N., and
R.V.; Visualization, K.N.V., and H.H.A.; Supervision, K.N.V.; Project administration, K.N.V.; Funding acquisition,
K.N.V., M.P., M.A.M., B.E.A., M.A., A.B.N., N.S., and C.T. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the Deputyship for Research & Innovation, Ministry of Education in Saudi
Arabia (Research Project Number 1058).
Acknowledgments: 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. The authors wish
to thank Tameem M. Alyahian for providing technical support in this project.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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