Easily accessed nitroquinolones exhibiting potent and selective anti-tubercular activity

Article abstract of DOI:10.1016/j.ejmech.2021.113207

Nitro based DprE1 inhibitors exemplified by benzothiazinones have been reported to elicit potent anti-tubercular activity. Poor PK properties associated with benzothiazinones have inspired the discovery of alternative nitro based DprE1 inhibitors. Quinolone based antibiotics on the other hand have good PK properties. The potent anti-tubercular activity of nitro compounds and the good PK properties of the quinolones have elicited an interest in us to construct a new class of nitro containing compounds around the quinolone scaffold with the aim of identifying novel DprE1 inhibitors with potent anti-tubercular activity. Thus, we report herein the anti-tubercular activity of novel 6-nitroquinolone-3-carboxamide derivatives achieved using less than five cheap synthetic transformations. Among the 23 target compounds evaluated for anti-tubercular activity, 12 were active against Mtb─ exhibiting activity in the range of <0.244–31.865 μM. Compound 25 having a molecular weight of 399 Da and ClogP value of 2.7 is the most active (MIC₉₀: <0.244 μM) in this series. The SAR analyses suggest that anti-tubercular activity was influenced by substituents at position N-1 (R₂) and C-3 (R₃) of the quinolone ring. The activity data suggest that the nature of R₃ has a stronger influence on the SAR compared to R₂; with a fluorobenzyl and chlorobenzyl moiety at R₂ being the most favoured when R₃ is an aliphatic amine. Docking study confirms that compound 25 binds to the same hydrophobic pocket as does TCA1, and other nitro based DprE1 inhibitors, with its nitro group in close proximity with Cys387 residue.

Full text of DOI:10.1016/j.ejmech.2021.113207

European Journal of Medicinal Chemistry 213 (2021) 113207
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Easily accessed nitroquinolones exhibiting potent and selective anti-
tubercular activity
Phelelisiwe S. Dube a, Lesetja J. Legoabe a, Audrey Jordaan b, Omobolanle J. Jesumoroti a,
,
d
,
, *
Tendamudzimu Tshiwawa c, Digby F. Warner b e, Richard M. Beteck a
a Centre of Excellence for Pharmaceutical Sciences, North-West University, Potchefstroom 2520, South Africa
b SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology, University of Cape Town, Observatory, 7925, South Africa
c Faculty of Science, Department of Chemistry, Rhodes University, Grahamstown 6140, South Africa
d Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, 7701, South Africa
e Wellcome Centre for Infectious Diseases Research in Africa (CIDRI-Africa), Faculty of Health Sciences, University of Cape Town, Rondebosch, 7701, South
Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitro based DprE1 inhibitors exemplified by benzothiazinones have been reported to elicit potent anti-
tubercular activity. Poor PK properties associated with benzothiazinones have inspired the discovery of
alternative nitro based DprE1 inhibitors. Quinolone based antibiotics on the other hand have good PK
properties. The potent anti-tubercular activity of nitro compounds and the good PK properties of the
quinolones have elicited an interest in us to construct a new class of nitro containing compounds around
the quinolone scaffold with the aim of identifying novel DprE1 inhibitors with potent anti-tubercular
activity. Thus, we report herein the anti-tubercular activity of novel 6-nitroquinolone-3-carboxamide
derivatives achieved using less than five cheap synthetic transformations. Among the 23 target com-
pounds evaluated for anti-tubercular activity, 12 were active against Mtbe exhibiting activity in the
Received 19 August 2020
Received in revised form
24 November 2020
Accepted 12 January 2021
Available online 24 January 2021
Keywords:
Anti-tubercular
Nitro containing compounds
Quinolones
range of <0.244e31.865
m
M. Compound 25 having a molecular weight of 399 Da and ClogP value of 2.7 is
M) in this series. The SAR analyses suggest that anti-tubercular activity
the most active (MIC90: <0.244
m
DprE1 enzyme
was influenced by substituents at position N-1 (R2) and C-3 (R3) of the quinolone ring. The activity data
suggest that the nature of R3 has a stronger influence on the SAR compared to R2; with a fluorobenzyl
and chlorobenzyl moiety at R2 being the most favoured when R3 is an aliphatic amine. Docking study
confirms that compound 25 binds to the same hydrophobic pocket as does TCA1, and other nitro based
DprE1 inhibitors, with its nitro group in close proximity with Cys387 residue.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
million people fell ill with TB in 2018 and approximately 1.5 million
people died from it [4,6]. The current increase in the prevalence of
Mycobacterium tuberculosis (Mtb), the causative agent of
tuberculosis (TB), is a species of pathogenic bacterium belonging to
the family Mycobacteriaceace [1]. TB is a prehistoric disease that has
been one of the major scourges of humankind since times imme-
morial [2]. Although Mtb has been discovered since 1882 [3], today
TB still remains a major global health problem with one third of the
world’s population infected [4].
TB is due to noncompliance to first line TB treatment, poor efficacy
of second line drugs and co-infection with HIV (Human Immuno-
deficiency Virus) [4,7]. Studies have shown that a close association
exist between HIV infection and the development of active TB. The
probability of developing active TB is higher in people living with
HIV due to their compromised immune system, than in people who
are HIV negative [7,8].
TB is among the top ten causes of death and the leading cause
from a single pathogenic agent [5]. Globally, an estimated 10
Worldwide in 2018, an estimated 3.4% of new cases and 18% of
previously treated cases of TB were multidrug resistant (MDR-TB)
and extensively drug-resistant (XDR-TB), respectively. Overall,
there was an estimated 484 000 incident cases of MDR-TB and
about 214 000 deaths from MDR-TB in 2018 [4]. Thus, the devel-
opment of resistance to first line drugs (rifampicin, isoniazid,
* Corresponding author.
E-mail addresses: 25159194@nwu.ac.za, richmbi1@yahoo.com (R.M. Beteck).
https://doi.org/10.1016/j.ejmech.2021.113207
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

P.S. Dube, L.J. Legoabe, A. Jordaan et al.
European Journal of Medicinal Chemistry 213 (2021) 113207
ethambutol and pyrazinamide) by Mtb has worsened the man-
agement and treatment of TB, leading to the use of second line
drugs (aminoglycosides, fluoroquinolones, thioamides, bedaqui-
line, and delamanid) [9]. Moreover, the emergence of MDR-TB and
XDR-TB indicate not only the need for new diagnostic markers [4],
drugs amendment in second line treatment regimens [10], but also
emphasizes the search for new compounds exhibiting anti-
tubercular activity through a mode of action that differs from
currently deployed drugs [11].
into the early phases of clinical development [26,27]. Due to high
failure rates during clinical development [28], it is pertinent to
investigate other nitro-based compounds to eventually populate
the development pipeline. To this effect, our group decided to
investigate nitroquinolones as potential DprE1 inhibitors.
Quinolones represent an important class of organic molecules
that have been known to possess broad spectrum of pharmaco-
logical properties, good pharmacokinetic properties and have
attracted attention in medicinal chemistry [29,30]. Not only do they
possess a wide range of pharmacological activities, they also enjoy
synthetic tractability as there are several established protocols for
the synthesis of this scaffold [31].
We previously identified 6-nitroquinolone-3-carboxylate ester
with anti-tubercular activity as DprE1 inhibitors [32]. These com-
pounds, however, were not soluble in screening media and were
screened as suspensions. As a result, proper structure-activity
relationship could not be delineated for the said compounds. In
this study, we generated less lipophilic amide derivatives wherein
previous compounds were substituted with polar moieties of
different chain lengths and ring sizes at position C-3 of the qui-
nolone scaffold. Analogues wherein the benzenoid ring of the
quinolone template was substituted at position C-8 by either eCF3,
eNO2, eCN, eF where also synthesised, but only the analogue
bearing eCF3 (compound 3a) was soluble in screening media and
hence its data is reported herewith. All compounds in this study
selected for screening were soluble in screening media.
Nitro containing compounds, exemplified by benzothiazinones,
are new chemical entities (NCE) possessing selective and potent
inhibitory activity against Mtb. They exhibit a mode of action that is
completely different from other anti-tubercular agents currently in
the market [12]. Most notable, this compound class have been
validated to demonstrate anti-tubercular activity by acting as sui-
cide inhibitors of Decaprenylphosphoryl-
(DprE1) [13]. DprE1 is a flavoprotein and a sub unit of the enzyme
Decaprenylphosphoryl -ribose (DPR) epimerase which plays a vi-
b-D
-ribose 20- epimerase
D
tal role in Mtb cell wall synthesis [2,14]. The discovery of DprE1 as a
target of nitrobenzothiazinones (BTZs) gave an insight into these
agents’ mechanism of action [15]and has since inspired the inves-
tigation of other nitro-based compounds as DprE1 inhibitors and
potential anti-tubercular agents [16]]. In addition to BTZs, other
nitro based DprE1 inhibitors reported so far includes: nitro-
benzamides [17], dinitrobenzamides [18]], nitroquinaxoline [19],
trinitroxanthone [20], dinitrooxadiazole, dinitrotetrazole [21],
dinitrothiazole [22,23], nitrotriazole [24] (see Fig. 1).
Nitro-based DprE1 inhibitors are pro-drugs activated only once
inside Mtb, and hence the risk of cell toxicity is low [25]. Moreover,
this class of compounds are active against drug susceptible and
resistant forms of Mtb, and in some cases they show low nanomolar
(˂10 nM) activity against Mtb [2]. However, they also suffer from
poor drug-like properties such as short half-life, poor aqueous
solubility [25]. So far, only BTZ043 and PBTZ169 have progressed
2. Results and discussion
2.1. Chemistry
Our target compounds were obtained following a 3-step syn-
thetic route presented in Scheme 1. The first step (a) is the
condensation of diethyl ethoxymethylenemalonate (1) with
Fig. 1. Nitro containing compounds with anti-tubercular activity.
2

P.S. Dube, L.J. Legoabe, A. Jordaan et al.
European Journal of Medicinal Chemistry 213 (2021) 113207
appropriate nitroaniline (2) followed by intramolecular cyclisation
at higher temperatures of 245e250 ꢀC to obtain intermediate 3 in
60%e90% yields. The second step (b) is the nucleophilic substitu-
tion of benzyl halides by 3b. Intermediate 3b was treated with
potassium carbonate (K2CO3) to effect deprotonation of the sec-
ondary amine and enhance its nucleophilicity for subsequent
nucleophilic attack of primary benzyl halides. Intermediates 4a-f
were obtained in 50%e70% yields using this step (melting points
and other analytical data for these compounds are reported in ref
32). Compound 3a and other related analogues bearing a substit-
uent (F, CN, NO2, OCH3, CF3) at position 8 of the quinolone ring
failed to undergo nucleophilic substitution with organohalides.
This could be due to steric hindrance and/or electron-withdrawing
effect(s) of these substituents. In the final step (c), 4a-f were sub-
jected to aminolysis wherein they were treated with hydrophilic
amines under reflux to obtain amide derivatives 5e25 in 50e70%
overall yields. Aminolysis of 3a using this procedure was not
successful.
moiety (Ar-CH2-), this is evidence of successful N-alkylation of the
secondary amine at position N-1 by benzyl-halides. For the 13C
NMR spectra, the signals appearing at ca 174e176 (ketone) and
161e163 (amide) ppm of all the compounds are indicative of the
two carbonyl carbons present in all compounds. The signal at ca
38e39.33 ppm of all the compounds substituted with aliphatic
amines indicates the first methylene carbon of the amide moiety at
R3. This signal is evidence of the successful conversion of an ester to
an amide. This signal is absent in compound 5e8 since they contain
a hydroxamate at R3. Lastly the signal appearing at ca 55e56 ppm of
all the compounds is indicative of the methylene carbon of the
benzyl moiety at R2 and is also evidence of the successful N-
alkylation of the secondary amine by benzyl halides. IR confirmed
signature peaks corresponding to functional groups present in
target molecules. HRMS analyses confirms the expected molecular
ion for all compounds, except compound 8 wherein molecular ion
peak not found, probably unstable with the ionization method.
Reactions were monitored using thin layer chromatography
(TLC) and the synthesised quinolones were characterized using 1H
NMR, 13C NMR, IR and high resolution mass spectrometry (HRMS).
On the 1H NMR spectra, the signals of the respective protons of the
prepared derivatives were verified on the basis of their chemical
shifts, multiplicities and coupling constants. The signals appearing
at ca 9.73e9.79 ppm of all the compounds (except 5e8) is indica-
tive of the presence of an amide proton (-CONH-). In 5e8, the signal
at ca 11.47 ppm is assignable to the OH of the hydroxamate moiety.
The signal appearing at ca 5.95e6.3 ppm of all the synthesised
compounds is assignable to the methylene protons of the benzyl
2.2. Pharmacology
All target compounds (3a, 5e26) were screened in-vitro for anti-
tubercular activity against the gfp reporter strain of Mtb using
middlebrook 7H9 media supplemented with casitone, glucose and
tyloxpol. Rifampicin and isoniazid, a first line anti-tubercular
agents were included in the assay as a reference. Target com-
pounds were screened in a concentration range of 0.0002e10 mM
and a dose e response curve generated for each compound (see
ESI). The anti-tubercular activity is reported as the minimum con-
centration required to inhibit 90% (MIC90) of bacteria population.
Scheme 1. Synthesis of target compoundsaaReagents and conditions; (a) (i) ethanol, reflux, 12 h, (ii) diphenylether, 245e250 ꢀC, 10 min. (b) benzyl halide, DMF, K2CO3, reflux, 12 h.
(c) amine, reflux 24 h.
3

P.S. Dube, L.J. Legoabe, A. Jordaan et al.
European Journal of Medicinal Chemistry 213 (2021) 113207
Table 1
In vitro anti-tubercular activity, ClogP and structure of target compounds.
The MIC90 was evaluated on day 14 following incubation of Mtb
with target compounds. No precipitation was observed during
screening and compounds were soluble in screening media.
The anti-tubercular activity data is summarised in Table 1.
Thirteen out of the 23 compounds evaluated were active against
Entry
Clog Pa
R2
MIC90 (m
M)b
Day 14
3a
0.72
e
1.844
Mtb, exhibiting activity in the range <0.244e31.865
mM. Twelve out
of the 13 active compounds exhibit MIC90 values < 10
gives a high hit rate of >50% for this compound set.
mg/mL, which
Different substituents were appended at position N-1 (R2) and
C-3 (R3) of the quinolone ring to enable structure-activity rela-
tionship (SAR) analysis for these series. The activity data suggest
that the nature of the R3 showed a stronger influence on the SAR
than R1. R3 includes hydroxamate, N-(2-hydroxyethyl) ethyl-
enediaminyl 2-methoxyethylaminyl, 2-(2-aminoethoxy) ethanolyl
and 4-(2-aminoethyl) morpholinyl. With the exception of com-
5
6
7
8
2.67
2.51
1.94
1.23
>125
125
pound 10 (MIC90: 31.8
mM), all compounds wherein R3 is a
hydroxamate or 4-(2-aminoethyl) morpholinyl were inactive
against Mtb irrespective of the nature of R1; for example, com-
pound 5,7,8 and 11 were all inactive (MIC90 > 125 mM). Four out of
the five compounds wherein R3 is a 2-(2-aminoethoxy)ethanolyl
moiety showed anti-tubercular activity; the active compounds (12,
14e16) demonstrated good to moderate activity in the range of
8e16 mM. Apart from compound 19 (MIC90 > 125 mM), the subseries
wherein R3 is a N-(2-hydroxyethyl)ethylenediaminyl as seen in
compounds 17, 18 and 20 elicited potent anti-tubercular activity in
>125
>125
the range of 0.9e3.7
mM R3 in compounds 21e25 is 2-
methoxyethylaminyl moiety; two compounds in this subseries
exhibited sub micromolar activity, with compound 25
(MIC90 < 0.244 mM) being the most active compound in this study.
In compound 26 R3 is a carboxylic acid moiety, akin to most fluo-
roquinolones deployed in clinics as anti-tubercular agents. This
compound is, however, inactive (MIC90 > 125
analogues 14 (MIC90: 14.9 M) and 17 (MIC90: 3.6
moderate to potent anti-tubercular activity.
m
M) while its amide
m
m
M) exhibit
Overall, this study indicates that an aliphatic chain at position C-
3 promotes anti-tubercular activity better than a cyclic chain or
hydroxylamide at this position. This observation is evident when
comparing compounds 6 (MIC90; 125
m
M), 10 (MIC90; 31
mM), 12
9
3.67
3.51
2.94
>125
31.865
>125
(MIC90; 16 M) against compound 21 (MIC90; 0.6
m
mM). These
compounds all bear a 3-Cl-benzyl moiety as R2, but differ Only in
the nature of R3.
The structure-activity relationship (SAR) analysis suggests that
the influence of substituents at position N-1 on anti-tubercular
activity depends on the substituents at position C-3 of the quino-
lone ring. Furthermore, the anti-tubercular activity also depends on
the position (meta or para) of the groups attached to the benzyl
substituent at position N-1 of quinolone core. The 4-fluorobenzyl
substituted compounds showed more activity than compounds
with a 3-fluorobenzyl unit substituted at position N-1. This is
10
11
evident when comparing compound 13 (MIC90, >125
compound 16 (MIC90, 8.077 M), 18 (MIC90, 3.703 M) against 20
(MIC90 0.919 M), and 22 (MIC90, 1.844 M) against 25 (MIC90
<0.244 M).
mM) against
m
m
m
m
,
m
Taking R2 and R3 together, this study suggests that R3 sub-
stituents showing optimal anti-tubercular activity could be main-
tained, while R2 could be modified further to improve on anti-
tubercular activity and/or modulate other needed drug-like prop-
erties during lead development. A graphical summary of SAR ana-
lyses is presented in Fig. 2.
12
2.62
16.043
Compound 25 being the most active compound in this study
was further evaluated in vitro against DprE1 mutant strain of Mtb.
This compound, however, exhibited reduced activity (MIC90
62.5 M) against DprE1 mutant compared to the wild type (MIC90
<0.24
pound 25.
:
:
m
m
M). This data suggests DprE1 as the likely target for com-
4

P.S. Dube, L.J. Legoabe, A. Jordaan et al.
European Journal of Medicinal Chemistry 213 (2021) 113207
Table 1 (continued)
Table 1 (continued )
Entry
Clog Pa
R2
MIC90
(m
M)b
Entry
Clog Pa
R2
MIC90 (m
M)b
Day 14
Day 14
13
2.05
>125
23
2.04
>125
14
1.34
14.996
24
25
2.04
2.75
>125
<0.244
15
16
1.34
2.05
14.01
8.077
26
INH
RF
3.01
e
e
e
e
>125
0.2
0.01
e
a
clog P values calculated with chem draw professionals.
b
MIC90
¼
determined in vitro against gfp Mtb strain, RF
¼
rifampicin,
INH ¼ isoniazid.
Compounds 3a, 16, 17, 18, 20, 21 and 25, all of which exhibited
potent anti-tubercular activity (MIC90, <10 M) were evaluated
m
in vitro against M. ulcerans e the causative agent for buruli ulcer.
The compounds were screened in triplicate at a concentration of
17
18
1.18
1.89
3.687
3.703
10
mg/ml. M. ulcerans were incubated for 8 days with the com-
pounds, then Alamar blue was added and the bacteria were incu-
bated for another 3 days. The remaining metabolic activity (%PC)
was calculated by dividing the blank-corrected fluorescence for
each test compound by that of the untreated control. None of these
compounds showed activity against M. ulcerans, all exhibiting %PC
> 90%. This results evinced that these compounds have intrinsic
and selective anti-tubercular activity. It is important to mention
that fluoroquinolones, exemplified by ciprofloxacin, have been re-
ported to exhibit equipotent activities against M. tuberculosis and
M. ulcerans [33].
19
20
1.18
1.89
>125
0.919
2.3. Molecular docking and theoretical drug-like properties
evaluation
We previously showed that hits emanating from this compound
class are not active against DprE1 mutant Mtb (DdprE1 C1
Tyr314His) e suggesting DprE1 as the most probable target. Based
on this, we subjected compound 25 to molecular docking studies
against DprE1 enzyme (PDB CODE: 4KW5). Docking confirms
compound 25 binds to the hydrophobic pocket wherein other nitro
based DprE1 inhibitors and TCA1 have been reported to bind [34].
Within this pocket, compound 25 makes three hydrogen bonds
with the enzyme via LYS134, SER228 and TRP230. The ligand also
made pi-pi interactions with the enzyme through TRY314 and
TRP230 (Fig. 3a).
Unlike TCA1 which forms a non-covalent bond with CYS387 (see
ESI), nitro based DprE1 inhibitors bind to the hydrophobic pocket
(active site) of DprE1 enzyme and are subsequently reduced to their
nitroso intermediates. The nitroso intermediate then forms a co-
valent adduct with a nearby CYS387. The docking pose of com-
pound 25 within the active site of DprE1 (Fig. 3b) shows the nitro
moiety of this ligand in close proximity (4.05 Å) with CYS387. This
suggests that in addition to the interactions seen in Fig. 3a, com-
pound 25 also makes covalent interaction with the enzyme.
Moreover, compound 25 have a good docking score of ꢁ7.60 kcal/
mol against the enzyme. All of these highlight DprE1 as the target
21
22
3.32
2.75
0.675
>125
(continued on next page)
5

P.S. Dube, L.J. Legoabe, A. Jordaan et al.
European Journal of Medicinal Chemistry 213 (2021) 113207
Fig. 2. Summary of property and SAR analyses of compounds investigated.
Fig. 3a. Binding interactions of compound 25 and DprE1 enzyme (PDB: 4KW5).
for compound 25.
Theoretical evaluation of the drug-likeness of the synthesised
compounds suggests that the compounds are likely to possess good
physico-chemical properties. The synthesised compounds exhibi-
ted optimal lipophilicity with ClogP values ranging from 0.72 to
3.67 and were all soluble in screening media. It is noteworthy to
6

P.S. Dube, L.J. Legoabe, A. Jordaan et al.
European Journal of Medicinal Chemistry 213 (2021) 113207
Fig. 3b. Docking pose of compound 25 in close proximity with CYS387.
mention that early in vitro studies have shown statistically signifi-
cant improvements in safety outcomes when compounds have
ClogP <3. This is a promising finding as for a long period of time, the
mean value of ClogP for approved drugs has been consistent at
2.3e2.6 [35]. Moreover, the target compounds followed the Lip-
inski’s rule of 5 with molecular weight less than 500 Da and lip-
ophilicity expressed as ClogP less than 5 [36], giving them the
potential to circumvent poor drug-like properties associated with
current nitro containing anti-tubercular agents. Lipophilicity and
molecular weight are often increased during hit and lead optimi-
zation. Thus, compounds with lower molecular weight and lip-
ophilicity are more likely to maintain drug-likeness during hit and
lead optimization which often involves structural incrementation
[34,37].
encouraging anti-tubercular activity (MIC90 1.84 mM), low molec-
ular weight (330 Da) and is worth further exploring.
4. Materials and methods
4.1. General information
The Chemicals and solvents used in this study were purchased
from various chemical vendors: Sigma-Aldrich (Pty) Ltd. (Johan-
nesburg, South Africa), Merck (Pty) Ltd. (Johannesburg, South Af-
rica) and were used without purification. The progress of the
reactions was monitored by thin layer chromatography (TLC) using
Merck 60F254 silica gel plates (Merck, Johannesburg, South Africa)
supported on aluminium and the plates were visualized under ul-
traviolet (UV254 and 366 nm) light or stained with iodine vapour.
1H and 13C NMR spectra were recorded on Bruker Biospin 600 MHz
spectrometer, and the chemical shifts are given in values referenced
to deuterated DMSO‑d6 and are reported in parts per million (ppm).
Chemical shifts for deuterated DMSO‑d6 appear at 2.5 ppm for 1H
and 39.5 ppm for 13C NMR spectra. Proton coupling patterns are
abbreviated as follows: s (singlet), d (doublet), t (triplet), q (quar-
tet), and m (multiplet). Coupling constants (J) are reported in Hz.
NMR data were analyzed using MestReNova Software, version
5.3.2e4936. Infrared (IR) spectra were recorded using a Bruker
Alpha-P FTIR instrument. Melting points (mp) were established
with a Büchi melting point B-545 instrument and were uncorrec-
ted. The High-resolution mass spectra (HRMS) were recorded by
means of a Bruker micrOTOF-Q II mass spectrometer using atmo-
spheric pressure chemical ionization (APCI) in positive ion mode.
3. Conclusion
The synthesised 6-nitroquinolone-3-carboxamides showed
potent and selective anti-tubercular activity against the gfp strain
of Mtb, exhibiting activity in the range of <0.244e31.865
mM.
Molecular docking studies show that this compound class bind
within the active pocket of DprE1 enzyme with a good docking
score of ꢁ7.60 kcal/mol. The compounds are likely to possess good
physico-chemical properties as they show optimal calculated lip-
ophilicity for drug molecules, with ClogP values ranging from 0.72
to 3.67 and molecular weight <500Da. These results are encour-
aging and suggest that this new class of compounds is worth
further exploring for potential anti-tubercular agents. The SAR
highlights that the substituents at position N-1(R2) of the quino-
lone ring depended on the substituents at position C-3(R3) of the
ring for anti-tubercular activity. This observation suggests that R2
could be derivatized further to generate compounds with superior
activity. Aliphatic amines at R3 are mostly favoured. Moreover, the
SAR favoured the fluorobenzyl moiety at R2, with the fluoride atom
influencing the activity more when at para position of the benzyl
unit. Compound 25 was the most active in this series
4.2. General synthetic procedure for the nitro-quinolone derivatives
Target compounds were obtained through three general pro-
cedures executed in a linear sequence as depicted in Scheme 1:
(a) To a round bottom flask containing 20 mL of ethanol,
commercially available nitro-aniline was added, followed by
diethylethoxymethylenemalonate (1. equiv). The flask was
stirred under reflux for 12 h. After completion as indicated by
(MIC90 < 0.244 mM). Furthermore, analogues of compound 3a did
not dissolve in DMSO, thus denying us structural activity analysis
around the benzenoid ring of the quinolone template. 3a showed
7

P.S. Dube, L.J. Legoabe, A. Jordaan et al.
European Journal of Medicinal Chemistry 213 (2021) 113207
TLC, the mixture was cooled to room temperature and the
ensuing precipitate filtered, dried and later poured into
boiling (250 ꢀC) diphenylether in a 2-neck round bottom
flask and heated at 245e250 ꢀC for 10 min. The cooled
mixture was washed with petroleum ether to obtain 3a-3b.
(b) Compound 3b was dissolved in DMF, subsequently treated
with potassium carbonate (K2CO3, 2. equiv), and different
benzyl halides (1. equiv). The reaction mixture was stirred
under reflux for 15 h. After reaction completion as indicated
by TLC analysis, the reaction mixture was evaporated to
dryness and deionized water poured in and stirred for
10 min. The resultant precipitate was filtered and dried to
obtain compound 4a-f in 80%e90% yields.
(c) Target compounds 5e25 were made following the procedure
reported by Beteck et al. (2019) [38]. Briefly, a gram of 4a-f
was then treated with the appropriate amine (5. equiv) in a
round bottom flask containing chloroform (20 mL) followed
by 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) (0.2 equiv).
The resultant mixture was refluxed for 2e3 days. After re-
action completion, 20 mL of chloroform was added and the
mixture washed with deionized water (20 mL ꢂ 4) in a
separating funnel. The organic phase was then evaporated to
dryness to obtain a crude which was recrystallization from
methanol to afford the corresponding target compounds
5e25 in 50e60% yields.
DMSO, d6)
d
11.47 (s, 1H, OH), 9.37 (s, 1H, AreH), 9.17 (s, 1H, AreH),
9.02 (d, J ¼ 2.7 Hz, 1H, AreH), 8.50 (dd, J ¼ 9.4, 2.7 Hz, 1H, AreH),
7.94 (d, J ¼ 9.4 Hz, 1H, AreH), 7.42 (s, 1H, NH), 7.39e7.35 (m, 2H,
AreH), 7.16 (d, J ¼ 6.3 Hz, 1H, AreH), 5.87 (s, 2H, CH2). 13C NMR
(151 MHz, DMSO, d6) d 174.78, 161.64, 150.22, 144.21, 142.87, 138.20,
133.86, 131.14, 128.33, 127.24, 127.03, 126.85, 125.35, 122.56, 120.12,
112.90, 55.86. HRMS (m/z, APCI) calcd for C17H12ClN3O5 374.0490
[Mþ1], found: 374.0490. IR (ATR) Vmax cmꢁ1: 3215.14(OeH stretch),
3056.31(AreH stretch), 1650.97(C]O stretch), 1601.63(C]O
stretch).
4.2.4. 1-(3-fluorobenzyl)-N-hydroxy 6-nitro-4-oxo-1,4-
dihydroquinolone-3-carboxamide, 7
Yellow powder, yield 61%, mp 245e246.1H NMR (600 MHz,
DMSO, d6)
d 11.47 (s, 1H, OH), 9.38 (s, 1H, AreH), 9.17 (s, 1H, AreH),
9.00 (dd, J ¼ 14.5, 2.7 Hz, 1H, AreH), 8.49 (dd, J ¼ 9.4, 2.7 Hz, 1H,
AreH), 7.93 (d, J ¼ 9.4 Hz, 1H, AreH), 7.43e7.36 (m, 1H, AreH),
7.19e7.11 (m, 2H, AreH, NH), 7.05 (d, J ¼ 7.8 Hz, 1H, AreH), 5.85 (d,
J ¼ 38.9 Hz, 2H, Ar-CH2). 13C NMR (151 MHz, DMSO, d6)
d 174.37,
161.39 (1JCF ¼ 244 Hz), 161.22, 149.79, 143.77, 142.48, 138.12 (2
JCF ¼ 15 Hz), 130.94 (3JCF ¼ 8.2 Hz), 126.78 (4JCF ¼ 26 Hz), 122.31,
122.13, 119.73, 114.83 (5JCF ¼ 7.2 Hz), 113.62, 113.47, 112.49, 55.57.
HRMS (m/z, APCI) calcd for C17H12FN3O5: 358.0834 [Mþ1], found:
358.0838. IR (ATR) Vmax cmꢁ1
:
3224.49(OeH stretch),
3056.47(AreH stretch),1772.84(C]O stretch), 1655.58(C]O
(d) In to a flask containing 0.5 g (1.32 mmol) of ethyl 1-(3-
cyanobenzyl)-6-nitro-4-oxo-1,4-dihydroquinolone-3-
carboxylate was added 30 mL of MeOH/THF mixture (1:1 v/
v), followed by LiOH (0.09 g, 3.97 mmol). The mixture was
stirred at room temperature for 12 h, after which 50 mL of
deionized water was added. The mixture was acidified to pH
1 by dropwise addition of concentrated HCl(aqu). The resul-
tant precipitate was filtered, dried and recrystallized from
ethanol to furnish compound 26.
stretch).
4.2.5. 1-(3-cyanobenzyl)-N-hydroxy-6-nitro-4-oxo-1,4-
dihydroquinolone-3-carboxamide, 8
Yellow powder, yield 50%, mp 250 ꢀC. 1H NMR (600 MHz, DMSO,
d6) d 11.49 (s, 1H, OH), 9.66 (s, 1H, AreH), 9.20 (s, 1H, AreH), 9.02 (d,
J ¼ 2.7 Hz, 1H, AreH), 8.48 (dd, J ¼ 9.4, 2.7 Hz, 1H, AreH), 7.94 (d,
J ¼ 9.4 Hz, 1H, AreH), 7.59 (d, J ¼ 7.8 Hz, 1H, AreH), 7.51 (s, 1H, NH),
7.38 (t, J ¼ 7.7 Hz, 1H, AreH), 7.31 (d, J ¼ 7.8 Hz, 1H, AreH), 5.80 (s,
2H, CH2)..13C NMR (151 MHz, DMSO, d6)
d 174.06, 161.00, 149.92,
149.65,143.52,142.20,134.90 133.59, 128.44,126.72,126.48,126.30,
124.66, 122.75, 121.85 119.61, 112.00, 55.92. HRMS: molecular ion
peak not found, probably unstable with the ionization method. IR
(ATR) Vmax cmꢁ1: 3370.35(OeH stretch), 3038.80(AreH stretch),
2957.52(CeH stretch), 1653.70(C]O stretch), 1602.93(C]O
stretch).
4.2.1. Ethyl 6-nitro-4-oxo-8-(trifluoromethyl)-1,4-
dihydroquinolone-3-carboxylate, 3a
Yellow powder, yield 70%, mp 236 ꢀC. 1H NMR (600 MHz,
DMSO‑d6)
4.18 (q, J ¼ 7.1 Hz, 2H, -OCH2CH3), 1.27 (t, J ¼ 7.1 Hz, 3H, -OCH2CH3).
13C NMR (151 MHz, DMSO)
173.05, 167.04, 158.00, 152.35, 139.70,
d 9.14 (s,1H, AreH), 8.74 (s, 1H, AreH), 8.43 (s, 1H, AreH),
d
130.36, 126.61, 124.99 (d[1],JCF ¼ 276 Hz), 122.09 (d[2],JCF ¼ 32 Hz),
4.2.6. 1(3-bromobenzyl)-N-(2-morpholinoethyl)-6-nitro-4-oxo-
1.4-dihydroquinolone-3-carboxamide, 9
110.68, 107.19, 59.14, 14.93. HRMS (m/z, APCI) calcd for
Mustard powder, yield 55%, mp 50 ꢀC. 1H NMR (600 MHz, DMSO,
C
13H9F3N2O5: 331.0408 [Mþ1], found: 331.0556. IR (ATR) Vmax
cmꢁ1: 3195.48 (NeH stretch), 3085.47 (AreH stretch), 2982.19
(CeH stretch), 1713.13 (C]O stretch), 1633.83 (C]O stretch).
d6)
d
9.79 (t, J ¼ 5.2 Hz, 1H, NH), 9.15 (s, 1H, AreH), 9.04 (d,
J ¼ 2.4 Hz, 1H, AreH), 8.49 (dd, J ¼ 9.3, 2.4 Hz, 1H, AreH), 7.93 (d,
J ¼ 9.3 Hz, 1H, AreH), 7.57 (s, 1H, AreH), 7.53 (d, J ¼ 8.0 Hz, 1H,
AreH), 7.31 (t, J ¼ 7.8 Hz, 1H, AreH), 7.19 (d, J ¼ 7.7 Hz, 1H, AreH),
5.85 (s, 2H, Ar-CH2), 3.65e3.60 (m, 4H, 2 ꢂ CH2), 3.49 (dd, J ¼ 11.9,
6.0 Hz, 3H, morpholinyl-H), 2.45 (s, 5H, morpholinyl-H). 13C NMR
4.2.2. 1-(3-bromobenzyl)-N-hydroxy-6-nitro-4-oxo-1,4-
dihydroquinolone-3-carboxamide, 5
Yellow powder, yield 57%, mp 236 ꢀC$1H NMR (600 MHz, DMSO,
d6)
d
11.47 (s, 1H, OH), 9.38 (s, 1H, AreH), 9.16 (s, 1H, AreH), 9.01 (d,
(151 MHz, DMSO, d6) d 175.16, 162.65, 149.88, 143.42, 142.30, 137.67,
J ¼ 2.7 Hz, 1H, AreH), 8.50 (dd, J ¼ 9.4, 2.7 Hz, 1H, AreH), 7.94 (d,
J ¼ 9.4 Hz, 1H, AreH), 7.57 (s, 1H, NH), 7.51 (d, J ¼ 8.0 Hz, 1H, AreH),
7.30 (t, J ¼ 7.9 Hz, 1H, AreH), 7.19 (d, J ¼ 7.8 Hz, 1H, AreH), 5.87 (s,
130.57, 130.49, 128.98, 126.71, 126.37, 124.99, 121.74, 121.68, 119.27,
112.53, 65.80, 56.67, 54.96, 52.73, 35.27. HRMS (m/z, APCI) calcd for
C
23H23BrN4O5: 515.0925 [Mþ1], found: 515.0924. IR (ATR) Vmax
2H, Ar-CH2). 13C NMR (151 MHz, DMSO, d6)
d
174.05, 160.91, 149.48,
cmꢁ1: 3240.18(NeH stretch), 3080.37(AreH stretch), 2957.76(CeH
stretch), 1698.05(C]O stretch), 1683.20(C]O stretch).
143.48, 142.13, 137.69, 130.67, 130.52, 128.98, 126.52, 126.29, 124.98,
121.83, 121.78, 119.38, 112.17, 55.05. HRMS (m/z, APCI) calcd for
C
17H12BrN3O5: 418.0033 [Mþ1], found: 418.0026. IR (ATR) Vmax
4.2.7. 1-(3-chlorobenzyl)-N-(2-morpholinoethyl)-6-nitro-4-oxo-
1,4-dihydroquinolone-3-carboxamide, 10
cmꢁ1: 3205.91(OeH stretch), 3083.7(AreH stretch), 2900.78(CeH
stretch), 1653.73(C]O stretch), 1603.99(C]O stretch).
Yellow powder, yield 54%, mp 243e245 ꢀC. 1H NMR (600 MHz,
DMSO, d6)
d
9.77 (t, J ¼ 5.3 Hz, 1H, NH), 9.14 (s, 1H, AreH), 9.04 (d,
4.2.3. 1-(3-chlorobenzyl)-N-hydroxy-6-nitro-4-oxo-1,4-
dihydroquinolone-3-carboxamide, 6
J ¼ 3.5 Hz, 1H, AreH), 8.52e8.46 (m, 1H, AreH), 7.94 (d, J ¼ 9.0 Hz,
1H, AreH), 7.42 (s, 1H, AreH), 7.39e7.34 (m, 2H, AreH), 7.16 (d,
J ¼ 4.2 Hz, 1H, AreH), 5.85 (s, 2H, Ar-CH2), 3.62 (t, J ¼ 4.3 Hz, 4H,
Yellow powder, yield 59%, mp 263e265 ꢀC. 1H NMR (600 MHz,
8

P.S. Dube, L.J. Legoabe, A. Jordaan et al.
European Journal of Medicinal Chemistry 213 (2021) 113207
2 ꢂ CH2), 3.49 (dd, J ¼ 11.9, 6.0 Hz, 3H, morpholinyl-H), 2.45 (s, 5H,
1H, AreH), 7.62e7.48 (m, 2H, AreH), 5.90 (s, 2H, Ar-CH2), 4.62 (t,
J ¼ 5.4 Hz, 1H, OH), 3.62e3.44 (m, 8H, 4 ꢂ CH2)$13C NMR (151 MHz,
morpholinyl-H). 13C NMR (151 MHz, DMSO, d6)
d 174.76, 162.61,
149.91, 143.47, 142.27, 137.45, 133.15, 130.42, 127.61, 126.72, 126.40,
126.14, 124.66, 121.81, 119.29, 112.51, 65.83, 56.69, 55.07, 52.75,
35.29. HRMS (m/z, APCI) calcd for C23H23ClN4O5: 471.1430 [Mþ1],
found: 471.1425. IR (ATR) Vmax cmꢁ1: 3244.81(NeH stretch),
3080.55(AreH stretch), 2959.06(CeH stretch), 1656.22(C]O
stretch), 1610.88(C]O stretch).
DMSO, d6) d 175.18, 163.09, 150.45, 143.72, 142.54, 136.95, 131.71,
131.20, 130.16, 129.99, 127.10, 126.74, 122.06, 119.61, 118.27, 112.82,
111.74, 72.05, 69.03, 60.09, 55.35, 38.52. HRMS (m/z, APCI) calcd for
C
22H20N4O6: 437.1456 [Mþ1], found: 437.1422. IR (ATR) Vmax cmꢁ1
:
3190.88(OeH stretch), 3057.57(AreH stretch), 2929.13(CeH
stretch), 2227.07(C^N stretch), 1654.99(C]O stretch), 1608.84(C]
O stretch).
4.2.8. 1-(3-fluorobenzyl)-N-(2-morpholinoethyl)-6-nitro-4-oxo-
1,4-dihydroquinolone-3-carboxamide, 11
4.2.12. 1-(4-cyanobenzyl)-N-(2-(2-hydroxyethoxy) ethyl)-6-nitro-
4-oxo-1,4-dihydroquinolone-3-carboxamide, 15
Yellow powder, yield 60%, mp 246 ꢀC. 1H NMR (600 MHz, DMSO,
d6)
d
9.76 (t, J ¼ 5.3 Hz, 1H, NH), 9.13 (s, 1H, AreH), 9.03 (d,
Mustard powder, yield 53%, mp 240e242 ꢀC. 1H NMR (600 MHz,
J ¼ 2.3 Hz, 1H, AreH), 8.48 (dd, J ¼ 9.3, 2.3 Hz, 1H, AreH), 7.93 (d,
J ¼ 9.3 Hz, 1H, AreH), 7.39 (dd, J ¼ 3.3, 7.7 Hz, 1H, AreH), 7.15 (dd,
J ¼ 8.5, 9.4 Hz, 2H, AreH), 7.05 (d, J ¼ 7.7 Hz, 1H, AreH), 5.86 (s, 2H,
Ar-CH2), 3.62 (t, J ¼ 4.3 Hz, 4H, 2 ꢂ CH2), 3.49 (q, J ¼ 6.0 Hz, 3H,
morpholinyl-H), 2.44 (s, 5H, morpholinyl-H). 13C NMR (151 MHz,
DMSO, d6)
d
9.74 (t, J ¼ 5.5 Hz, 1H, NH), 9.14 (s, 1H, AreH), 9.04 (d,
J ¼ 2.7 Hz, 1H, AreH), 8.46 (dd, J ¼ 9.4, 2.7 Hz, 1H, AreH), 7.87 (d,
J ¼ 9.4 Hz, 1H, AreH), 7.83e7.80 (m, 2H, AreH), 7.44 (d, J ¼ 8.4 Hz,
2H, AreH), 5.95 (s, 2H, Ar-CH2), 4.51 (s, 1H, OH), 3.60 (t, J ¼ 5.6 Hz,
2H, CH2), 3.54 (dd, J ¼ 7.8, 5.3 Hz, 4H, 2 ꢂ CH2), 3.50 (dd, J ¼ 5.5,
DMSO, d6)
d
175.69, 163.56 (1JCF ¼ 244.0 Hz), 162.03, 150.85, 144.37,
5.6 Hz, 2H, CH2). 13C NMR (151 MHz, DMSO, d6)
d 174.81, 162.73,
143.25, 138.77, 138.72, 131.57 (3JCF ¼ 8.5 Hz), 127.48 (2JCF ¼ 56.3 Hz),
122.98 (4JCF ¼ 3.0 Hz), 122.74, 120.26, 115.39 (2JCF ¼ 21.1 Hz), 114.19
(3JCF ¼ 22.2 Hz), 113.49, 66.77, 57.64, 56.13, 53.69, 36.23. HRMS (m/z,
APCI) calcd for C23H23FN4O5: 455.1725 [Mþ1], found: 455.1724.
IR(ATR) Vmax cmꢁ1: 3234.51(NeH stretch), 3054.69(AreH stretch),
2921.24(CeH stretch), 1655.36(C]O stretch), 1610.45(C]O
stretch).
150.09, 143.56, 142.32, 140.62, 132.38, 127.04, 126.77, 126.43, 121.83,
119.28, 117.94, 112.66, 110.43, 71.79, 68.76, 59.88, 55.38, 39.30.
HRMS (m/z, APCI) calcd for C22H20N4O6: 437.1456 [Mþ1], found:
437.1416. IR (ATR) Vmax cmꢁ1: 3525.44(OeH stretch), 3245.71(NeH
stretch),
3055.31(AreH
stretch),
2864.73(CeH
stretch),
2228.30(C^N stretch), 1655.37(C]O stretch), 1609.55(C]O
stretch).
4.2.9. 1-(3-chlorobenzyl)-N-(2-(2-hydroxyethoxy) ethyl)-6-nitro-
4-oxo-1,4-dihydroquinolone-3-carboxamide, 12
4.2.13. 1-(4-fluorobenzyl)-N-(2-(2-hydroxyethoxy) ethyl)-6-nitro-
4-oxo-1,4-dihydroquinolone-3-carboxamide, 16
Mustard powder, yield 50%, mp 50 ꢀC. 1H NMR (600 MHz, DMSO,
Yellow powder, yield 55%, mp 193e194 ꢀC. 1H NMR (600 MHz,
d6)
d
10.19 (t, J ¼ 5.3 Hz, 1H, NH), 9.56 (s, 1H, AreH), 9.45 (d,
DMSO, d6)
d
9.78 (t, J ¼ 5.5 Hz, 1H, NH), 9.15 (s, 1H, AreH), 9.02 (d,
J ¼ 2.5 Hz, 1H, AreH), 8.91 (dd, J ¼ 9.4, 2.5 Hz, 1H, AreH), 8.36 (d,
J ¼ 9.4 Hz, 1H, AreH), 7.80 (dd, J ¼ 9.9, 4.3 Hz, 3H, AreH), 7.58 (d,
J ¼ 4.0 Hz, 1H, AreH), 6.27 (s, 2H,Ar-CH2), 5.00 (t, J ¼ 5.4 Hz, 1H,
OH), 4.02e3.89 (m, 9H, NH, 4 ꢂ CH2)$13C NMR (151 MHz, DMSO, d6)
J ¼ 2.7 Hz, 1H, AreH), 8.48 (dd, J ¼ 9.4, 2.7 Hz, 1H, AreH), 7.98 (d,
J ¼ 9.4 Hz, 1H, AreH), 7.34 (dd, J ¼ 8.6, 5.4 Hz, 2H, AreH), 7.19 (t,
J ¼ 8.8 Hz, 2H, AreH), 5.83 (s, 2H, Ar-CH2), 4.61 (t, J ¼ 5.4 Hz, 1H,
OH), 3.60e3.47 (m, 8H, 4 ꢂ CH2). 13C NMR (151 MHz, DMSO, d6)
d
175.72, 163.70, 150.93, 144.42, 143.22, 138.38, 134.10, 131.36,
d
175.80, 163.86 (1JCF ¼ 240 Hz), 161.59, 150.97, 144.53, 143.35,
128.55, 127.64, 127.37, 127.10, 125.60, 122.72, 120.27, 113.37, 72.68,
132.17 (4JCF ¼ 3.0 Hz), 129.56 (3JCF ¼ 8.3 Hz), 127.77, 127.45, 116.47
(2JCF ¼ 21.7 Hz), 116.54, 116.40, 113.41, 72.85, 69.82, 60.89, 56.13,
39.32. HRMS (m/z, APCI) calcd for C21H20FN3O6: 430.1409 [Mþ1],
found: 430.1389. IR (ATR) Vmax cmꢁ1: 3403.74(OeH stretch),
3299.53(NeH stretch), 3042.47(AreH stretch), 2890.23(CeH
stretch), 1660.78(C]O stretch), 1610.17(C]O stretch).
69.65, 60.74, 56.05, 39.17. HRMS (m/z, APCI) calcd for C21H20ClN4O5:
446.1113 [Mþ1], found: 446.1076. IR (ATR) Vmax cmꢁ1
:
3206.72(NeH stretch), 3040.02(AreH stretch), 2893.26(CeH
stretch), 1716.78(C]O stretch), 1698.40(C]O stretch).
4.2.10. 1-(3-fluorobenzyl)-N-(2-(2-hydroxyethoxy) ethyl)-6-nitro-
4-oxo-1,4-dihydroquinolone-3-carboxamide, 13
4.2.14. 1-(3-cynobenzyl)-N-(2-(2-hydroxyethyl) amino) ethyl)-6-
nitro-4-oxo-1,4-dihydroquinolone-3-carboxamide, 17
Yellow powder, yield 52%, mp 198e200 ꢀC. 1H NMR (600 MHz,
DMSO, d6)
d
9.76 (t, J ¼ 5.5 Hz, 1H, NH), 9.13 (s, 1H, AreH), 9.00 (d,
Mustard powder, yield 50%, mp 51 ꢀC. 1H NMR (600 MHz, DMSO,
J ¼ 2.8 Hz, 1H, AreH), 8.46 (dd, J ¼ 9.4, 2.8 Hz, 1H, AreH), 7.91 (d,
J ¼ 9.4 Hz, 1H, AreH), 7.40e7.35 (m, 1H, AreH), 7.18e7.09 (m, 2H,
AreH), 7.03 (d, J ¼ 7.8 Hz, 1H, AreH), 5.85 (s, 2H, Ar-CH2), 4.60 (t,
J ¼ 5.2 Hz, 1H, OH), 3.57e3.45 (m, 8H, 4 ꢂ CH2). 13C NMR (151 MHz,
d6)
d
9.76 (d, J ¼ 5.5 Hz, 1H, NH), 9.14e9.01 (m, 2H, AreH), 8.47 (d,
J ¼ 7.5 Hz, 1H, AreH), 7.84 (dd, J ¼ 7.5, 8.4 Hz, 3H, AreH), 7.57 (s, 2H,
AreH), 5.89 (s, 2H, Ar-CH2-), 4.49 (s, 1H, OH), 3.46 (d, J ¼ 4.5 Hz, 2H,
CH2), 3.43 (d, J ¼ 4.5 Hz, 2H, CH2), 2.73 (s, 2H, CH2), 2.63 (s, 2H, CH2).
DMSO, d6)
d
175.50, 163.84 (1JCF ¼ 244 Hz), 161.80, 150.74, 144.17,
13C NMR (151 MHz, DMSO, d6)
d 175.16, 163.02, 150.36, 143.71,
143.39, 138.90 (2JCF ¼ 25 Hz), 131.74 (3JCF ¼ 8.3 Hz), 127.79
142.55, 136.98, 131.74, 131.23, 130.18, 130.02, 127.11, 126.72, 122.09,
119.59, 118.30, 113.02, 111.77, 60.29, 55.35, 51.32, 48.43, 39.80.
HRMS (m/z, APCI) calcd for C22H21N5O5: 436.1615 [Mþ1], found:
(3JCF
¼
8 Hz), 123.12, 122.87, 120.48, 115.63, 115.49, 114.43
(4JCF ¼ 5 Hz), 113.48, 72.85, 69.82, 60.89, 56.32, 39.33. HRMS (m/z,
APCI) calcd for C21H20FN3O6: 430.1409 [Mþ1], found: 430.1373. IR
(ATR) Vmax cmꢁ1: 3384.88(OeH stretch), 3195.40(NeH stretch),
3059.15(AreH stretch), 2916.73(CeH stretch), 1659.41(C]O
stretch), 1610.81(C]O stretch).
436.1609. IR (ATR) Vmax cmꢁ1
:
3226.80(OeH stretch),
3046.23(AreH stretch), 2839.12(CeH stretch), 2225.26(C^N
stretch), 1654.25(C]O stretch), 1608.51(C]O stretch).
4.2.15. 1-(3-fluorobenzyl)-N-(2-(2-hydroethyl) amino) ethyl-6-
nitro-4-oxo-1,4-dihydroquinolone-3-carboxamide,18
4.2.11. 1-(cyanobenzyl)-N-(2-(hydroxyethoxy) ethyl)-6-nitro-4-
oxo-1,4-dihydroquinolone-3-carboxamide, 14
Mustard powder, yield 51%, mp 191 ꢀC. 1H NMR (600 MHz,
Mustard powder, yield 55%, mp 234 ꢀC. 1H NMR (600 MHz,
DMSO, d6)
d
9.75 (t, J ¼ 5.5 Hz, 1H, NH), 9.14 (s, 1H, AreH), 9.01 (d,
DMSO, d6)
d
9.79 (t, J ¼ 5.4 Hz, 1H, NH), 9.16 (s, 1H, AreH), 9.02 (d,
J ¼ 2.7 Hz, 1H, AreH), 8.48 (dd, J ¼ 9.4, 2.7 Hz, 1H, AreH), 7.93 (t,
J ¼ 10.0 Hz, 1H, AreH), 7.39 (td, J ¼ 8.0, 6.3 Hz, 1H, AreH), 7.20e7.10
(m, 2H, AreH), 7.05 (t, J ¼ 8.1 Hz, 1H, AreH), 5.85 (s, 2H, Ar-CH2),
J ¼ 4.3 Hz, 1H, AreH), 8.47 (dd, J ¼ 9.4, 4.3 Hz, 1H, AreH), 7.88 (t,
J ¼ 11.5 Hz, 1H, AreH), 7.83 (s, 1H, AreH), 7.78 (dd, J ¼ 7.9, 6.5 Hz,
9

P.S. Dube, L.J. Legoabe, A. Jordaan et al.
European Journal of Medicinal Chemistry 213 (2021) 113207
4.47 (s, 1H, OH), 3.50e3.40 (m, 5H, NH, 2 ꢂ CH2), 2.75e2.60 (m, 4H,
161.11, 150.05, 143.47, 142.31, 137.83 (4JCF ¼ 3.3 Hz), 130.66
(3JCF ¼ 8.3 Hz), 126.71, 126.44, 122.04, 121.79, 119.40, 114.55
(2JCF ¼ 24 Hz),113.35 (2JCF ¼ 15 Hz),112.37, 70.27, 57.63, 55.24, 37.98.
HRMS (m/z, APCI) calcd for C20H18FN3O5: 400.1303 [Mþ1], found:
2 ꢂ CH2). 13C NMR (151 MHz, DMSO, d6)
d 175.03, 162.99
(1JCF ¼ 239 Hz), 161.39, 150.23, 143.71, 142.59, 138.12 (3JCF ¼ 15 Hz),
130.95 (3JCF ¼ 8.4 Hz), 126.98(4JCF ¼ 4.4 Hz), 122.32, 122.08, 119.64,
114.83 (2JCF ¼ 25 Hz), 114.69, 113.63 (2JCF ¼ 25 Hz), 112.88, 60.28,
55.50, 51.32, 48.41, 38.82. HRMS (m/z, APCI) calcd for C21H21FN4O5:
400.1324. IR (ATR) Vmax cmꢁ1
:
3239.65(NeH stretch),
3059.86(AreH stretch), 2930.21(CeH stretch), 1665.89(C]O
stretch), 1613.55(C]O stretch).
429.1569 [Mþ1], found: 429.1569. IR (ATR) Vmax cmꢁ1
:
3244.57(OeH stretch), 3054.94(AreH stretch), 2840.71(CeH
stretch), 1658.89(C]O stretch), 1612.45(C]O stretch).
4.2.20. 1-(3-cyanobenzyl)-N-(2-methoxyethyl)-6-nitro-4-oxo-1,4-
dihydroquinolone-3-carboxamide, 23
4.2.16. 1-(4-cynobenzyl)-N-(2-(2-hydroxyethyl) amino) ethyl)-6-
nitro-4-oxo-1,4-dihydroquinolone-3-carboxamide, 19
Mustard powder, yield 55%, mp 348e350 ꢀC. 1H NMR (600 MHz,
DMSO, d6)
d
9.97 (t, J ¼ 5.2 Hz,1H. NH), 9.85 (t, J ¼ 5.3 Hz,1H, AreH),
Yellow powder, yield 61%, mp 238e239 ꢀC. 1H NMR (600 MHz,
9.27 (t, J ¼ 3.7 Hz, 1H, AreH), 9.14e9.12 (m, 1H, AreH), 8.26 (dd,
J ¼ 9.2, 2.4 Hz, 1H, AreH), 7.92e7.88 (m, 1H, AreH), 7.84 (s, 1H,
AreH), 7.77 (dd, J ¼ 4.6, 3.2 Hz, 1H, AreH), 7.56 (dd, J ¼ 9.2, 5.5 Hz,
1H, AreH), 5.90 (s, 2H, Ar-CH2), 3.70e3.52 (m, 4H, 2 ꢂ CH2), 3.33 (s,
DMSO, d6)
d
9.71 (t, J ¼ 5.5 Hz, 1H, NH), 9.16e9.11 (m, 1H, AreH),
9.04 (t, J ¼ 5.8 Hz, 1H, AreH), 8.45 (dd, J ¼ 9.4, 2.8 Hz, 1H, AreH),
7.87 (dd, J ¼ 9.4, 5.5 Hz, 1H, AreH), 7.81 (d, J ¼ 8.4 Hz, 2H, AreH),
7.44 (t, J ¼ 8.0 Hz, 2H, AreH), 5.94 (s, 2H, Ar-CH2), 4.36 (s, 1H, OH),
3.52e3.37 (m, 5H, NH, 2 ꢂ CH2), 2.77e2.73 (m, 2H, CH2), 2.67e2.62
3H, CH3). 13C NMR (151 MHz, DMSO, d6)
d 175.26, 175.21, 163.06,
148.77, 143.43, 139.02, 137.02, 136.84, 136.71, 130.89, 129.99, 129.71,
127.01, 121.66, 119.94, 119.81, 117.95, 70.40, 70.36, 57.62, 37.97.
HRMS (m/z, APCI) calcd for C21H18N4O5: 407.1350 [Mþ1], found:
(m, 2H, CH2). 13C NMR (151 MHz, DMSO, d6)
d 175.06,162.91, 150.28,
143.82, 142.60, 140.93, 132.66, 127.32, 127.05, 126.68, 122.12, 118.22,
113.13, 110.71, 60.35, 55.65, 51.32, 48.42, 38.91. HRMS (m/z, APCI)
calcd for C22H21N5O5: 436.1615 [Mþ1], found: 436.1625. IR (ATR)
407.1366. IR (ATR) Vmax cmꢁ1
:
3213.77(NeH stretch),
3038.80(AreH stretch), 2874.49(CeH stretch), 2229.39(C^N
stretch), 1655.22(C]O stretch), 1608.88(C]O stretch).
Vmax cmꢁ1
: 3220.88 (OeH stretch), 3047.22(AreH stretch),
2836.25(CeH stretch), 2228.99(C^N stretch), 1658.05(C]O
stretch).
4.2.21. 1-(4-cyanobenzyl)-N-(2- methoxyethyl)-6-nitro-4-oxo-1,4-
dihydroquinolone-3-carboxamide, 24
4.2.17. 1-(4-fluorobenzyl)-N-(2(2-hydroxyethyl) amino) ethyl)-6-
nitro-4-oxo-1,4-dihydroquinolone-3-carboxamide, 20
Yellow powder, yield 60%, mp 257 ꢀC. 1H NMR (600 MHz, DMSO,
Yellow powder, yield 50% mp 50 ꢀC. 1H NMR (600 MHz, DMSO,
d6)
d
9.73 (t, J ¼ 5.1 Hz, 1H, NH), 9.14 (s, 1H, AreH), 9.04 (d,
d6)
d
9.75 (s, 1H, NH), 9.14 (s, 1H, AreH), 9.02 (s, 1H, AreH), 8.48 (d,
J ¼ 2.6 Hz, 1H, AreH), 8.50e8.40 (m, 1H, AreH), 7.84 (dd, J ¼ 5.4,
8.8 Hz, 3H, AreH), 7.45 (t, J ¼ 10.2 Hz, 2H, AreH), 5.95 (s, 2H, Ar-
CH2), 3.57e3.49 (m, 4H, 2 ꢂ CH2), 3.32 (s, 3H, CH3). 13C NMR
J ¼ 9.2 Hz, 1H, AreH), 7.97 (d, J ¼ 9.3 Hz, 1H, AreH), 7.33 (s, 2H,
AreH), 7.19 (t, J ¼ 8.3 Hz, 2H, AreH), 5.83 (s, 2H, Ar-CH2), 4.47 (s, 1H,
OH), 3.50e3.40 (m, 5H, NH, 2 ꢂ CH2), 2.87e2.62 (m, 4H, 2 ꢂ CH2).
(151 MHz, DMSO, d6) d 175.11, 162.91, 150.37, 143.85, 142.60, 140.90,
13C NMR (151 MHz, DMSO, d6)
d
174.96, 162.94,
132.66, 127.32, 127.04, 126.71, 122.11, 119.56, 118.22, 112.90, 110.71,
70.57, 57.89, 55.67, 38.29. HRMS (m/s, APCI) calcd for C21H20FN3O6:
150.08(1JCF ¼ 244 Hz), 143.69, 142.54, 131.06, 128.75 (3JCF ¼ 8.0 Hz),
126.97, 126.61(4JCF ¼ 3.8 Hz), 122.06, 119.72, 115.74(2JCF ¼ 15 Hz),
115.60, 112.81, 60.27, 55.30, 51.31, 48.40, 38.81. HRMS (m/z, APCI)
calcd for C21H21FN4O5: 429.1569 [Mþ1], found: 429.1590. IR (ATR)
407.1350 [Mþ1], found: 407.1339. IR (ATR) Vmax cmꢁ1
:
3240.71(NeH stretch),3056.34(AreH stretch), 2932.14(CeH
stretch), 2227.21(C^N stretch), 1663.15(C]O stretch), 1610.46(C]
O stretch).
Vmax cmꢁ1
: 3205.68 (NeH stretch), 3043.47(AreH stretch),
2918.49(CeH stretch), 1655.96(C]O stretch), 1610.71(C]O
stretch).
4.2.22. 1-(4-fluorobenzyl)-N-(2-methoxyethyl)-6-nitro-4-oxo-
1,4-dihydroquinolone-3-carboxamide, 25
4.2.18. 1-(3-chlorobenzyl)-N-(2-methoxyethyl)-6-nitro-4-oxo-1,4-
dihydroquinolone-3-carboxamide, 21
Yellow powder, yield 60%, mp 50 ꢀC. 1H NMR (600 MHz, DMSO,
d6)
d
9.78 (t, J ¼ 5.4 Hz, 1H, NH), 9.19e9.11 (m, 1H, AreH), 9.02 (d,
Mustard powder, yield 60% mp 194e196 ꢀC. 1H NMR (600 MHz,
J ¼ 2.7 Hz, 1H, AreH), 8.48 (dd, J ¼ 9.4, 2.7 Hz, 1H, AreH), 7.98 (t,
J ¼ 9.9 Hz, 1H, AreH), 7.38e7.29 (m, 2H, AreH), 7.24e7.14 (m, 2H,
AreH), 5.82 (s, 2H, Ar-CH2), 3.55e3.47 (m, 4H, 2 ꢂ CH2), 3.31 (s, 3H,
DMSO, d6)
d
9.78 (t, J ¼ 5.3 Hz, 1H, NH), 9.15 (s, 1H, AreH), 9.01 (t,
J ¼ 5.3 Hz, 1H, AreH), 8.49 (dd, J ¼ 9.4, 2.8 Hz, 1H, AreH), 7.93 (d,
J ¼ 9.4 Hz, 1H, AreH), 7.45e7.33 (m, 3H, AreH), 7.15 (dd, J ¼ 7.6,
4.9 Hz, 1H, AreH), 5.86 (s, 2H, Ar-CH2), 3.59e3.46 (m, 4H, 2 ꢂ CH2),
CH3). 13C NMR (151 MHz, DMSO, d6)
d
175.43,163.03 (1JCF ¼ 243 Hz),
161.20, 150.59, 144.15, 142.96, 131.77, 129.75 (3JCF ¼ 8.3 Hz), 127.38,
127.07(4JCF ¼ 3 Hz), 122.48, 120.17, 116.74(2JCF ¼ 22 Hz), 112.99,
70.96, 58.32, 55.74, 38.66. HRMS (m/z, APCI) calcd for C20H18FN3O5:
3.31 (s, 3H, CH3). 13C NMR (151 MHz, DMSO, d6)
d 175.72, 163.67,
150.96, 144.35, 143.19, 138.40, 134.11, 131.36, 128.54, 127.61, 127.39,
127.08, 125.57, 122.71, 120.28, 113.29, 71.17, 58.24, 55.94, 38.87.
HRMS (m/z, APCI) calcd for C20H18ClN3O5: 416.1008 [Mþ1], found:
416.1028. IR (ATR) Vmax cmꢁ1: 3084.41(AreH stretch), 2879.66(CeH
stretch), 1656.23(C]O stretch), 1610.60(C]O stretch).
400.1303, [Mþ1] found: 400.1306. IR (ATR) Vmax cmꢁ1
:
3246.88(NeH stretch), 3077.70(AreH stretch), 2960.02(CeH
stretch), 1664.22(C]O stretch), 1613.24(C]O stretch).
4.2.19. 1-(3-fluorobenzyl)-N-(2-methoxyethyl) 6-nitro-4-oxo-1,4-
dihydroquinolone-3-carboxamide, 22
4.2.23. 1-(3-cyanobenzyl)-6-nitro-4-oxo-1,4-dihydroquinolone-
3-carboxylic acid, 26
Yellow powder, yield 65% mp 237e239 ꢀC. 1H NMR (600 MHz,
Yellow powder, yield 68%, mp 265e267 ꢀC. 1H NMR (600 MHz,
DMSO, d6)
d
9.78 (t, J ¼ 5.1 Hz,1H, NH), 9.14 (d, J ¼ 4.6 Hz,1H, AreH),
DMSO‑d6)
AreH), 8.55 (d, J ¼ 8.5 Hz, 1H, AreH), 7.97e7.53 (m, 5H, AreH), 5.98
(s, 2H, Ar-CH2). 13C NMR (151 MHz, DMSO)
178.23, 165.59, 152.88,
145.00, 143.28, 137.14, 132.46, 132.01, 130.78, 130.58, 128.31, 126.53,
122.24, 120.99, 118.89, 112.40, 110.31, 56.56. HRMS (m/z, APCI) calcd
for C18H12N3O5: 350.0771, [Mþ1] found: 350.0742.
d 14.38 (s, 1H, COOH), 9.38 (s, 1H, AreH), 9.03 (s, 1H,
9.02 (d, J ¼ 2.5 Hz, 1H, AreH), 8.48 (dd, J ¼ 9.3, 2.5 Hz, 1H, AreH),
7.90 (dd, J ¼ 2.5, 9.4 Hz, 1H, AreH), 7.39 (dd, J ¼ 10.2, 7.7 Hz, 1H,
AreH), 7.22e7.09 (m, 2H, AreH), 7.05 (d, J ¼ 7.7 Hz, 1H, AreH), 5.87
(s, 2H, Ar-CH2), 3.51 (dt, J ¼ 7.0, 5.1 Hz, 4H, 2 ꢂ CH2), 3.31 (s, 3H,
d
CH3). 13C NMR (151 MHz, DMSO, d6)
d
174.81, 162.75 (1JCF ¼ 242 Hz),
10

P.S. Dube, L.J. Legoabe, A. Jordaan et al.
European Journal of Medicinal Chemistry 213 (2021) 113207
4.3. In vitro antimycobacterial assay
Appendix A. Supplementary data
The In vitro anti-tubercular evaluation was done following the
procedure by Beteck et al. (2019) [39]. Briefly, a 10 mL culture of
Mtb pMSp12: GFP was grown to an optical density (0D600) of
0.6e0.7. The cultures were grown in a middlebrook 7H9 medium
supplemented with 0.03% casitone, 0.4% glucose, and 0.05% tylox-
apol and diluted at 1:500, prior to inoculation. The compounds to
be tested were then reconstituted to a concentration of 10 mM in
DMSO. Two-folds serial dilution of the test compounds were plated
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113207.
Data availability statement
Dose-response curves and characterization data (1H,13C and
HRMS spectra) for compounds 3a, and 5e26 are available in the
supplementary file. Samples of compounds can be obtained from
corresponding author upon reasonable request.
in 96 well plates and 50 mL of the diluted Mtb culture was then
added to each well. Rifampicin (2 ꢂ MIC) was used as a minimum
growth control for assay and 5% DMSO as the maximum growth
control. The micro titre plates were sealed in a secondary container
and incubated at 37 ꢀC with 5% CO2 and humidification. Fluores-
cence readings (excitation 485 nM; emission 520 nM) obtained for
the individual wells were measured using a plate reader (FLUOstar
OPTIMA, BMG LABTECH) at day 14. The raw fluorescence data was
analyzed using the CDD Vault from Collaborative Drug Discovery, in
which data was normalized to the minimum and maximum inhi-
bition controls to generate a dose response curve (% inhibition).
Using the Levenberg-Marquardt damped least squares method, the
minimum inhibitory concentration (MIC) was calculated. The
lowest concentration of drug/test compound that inhibited growth
of more than 90% of the bacterial population was considered the
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