Facile synthesis and antimycobacterial activity of isoniazid, pyrazinamide and ciprofloxacin derivatives

Article abstract of DOI:10.1111/cbdd.13836

Several rationally designed isoniazid (INH), pyrazinamide (PZA) and ciprofloxacin (CPF) derivatives were conveniently synthesized and evaluated in vitro against H37Rv Mycobacterium tuberculosis (M. tb) strain. CPF derivative 16 displayed a modest activity

Full text of DOI:10.1111/cbdd.13836

Received: 27 November 2020
DOI: 10.1111/cbdd.13836
Revised: 12 January 2021
Accepted: 28 January 2021
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R E S E A R C H L E T T E R
Facile synthesis and antimycobacterial activity of isoniazid,
pyrazinamide and ciprofloxacin derivatives
Shahinda S. R. Alsayed1
Hendra Gunosewoyo1
Shichun Lun2
Alan Payne3
William R. Bishai2,4
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1School of Pharmacy and Biomedical
Sciences, Faculty of Health Sciences,
Curtin University, Perth, WA, Australia
2Division of Infectious Disease, Department
of Medicine, Center for Tuberculosis
Research, Johns Hopkins School of
Medicine, Baltimore, MD, USA
3School of Molecular and Life Sciences,
Curtin University, Perth, WA, Australia
4Howard Hughes Medical Institute, Chevy
Chase, MD, USA
Abstract
Several rationally designed isoniazid (INH), pyrazinamide (PZA) and ciprofloxa-
cin (CPF) derivatives were conveniently synthesized and evaluated in vitro against
H37Rv Mycobacterium tuberculosis (M. tb) strain. CPF derivative 16 displayed
a modest activity (MIC = 16 µg/ml) and was docked into the M. tb DNA gyrase.
Isoniazid-pyrazinoic acid (INH-POA) hybrid 21a showed the highest potency in our
study (MIC = 2 µg/ml). It also retained its high activity against the other tested M.
tb drug-sensitive strain (DS) V4207 (MIC = 4 µg/ml) and demonstrated negligible
cytotoxicity against Vero cells (IC50 ≥ 64 µg/ml). Four tested drug-resistant (DR)
M. tb strains were refractory to 21a, similar to INH, whilst being sensitive to CPF.
Compound 21a was also inactive against two non-tuberculous mycobacterial (NTM)
strains, suggesting its selective activity against M. tb. The noteworthy activity of 21a
against DS strains and its low cytotoxicity highlight its potential to treat DS M. tb.
Correspondence
Hendra Gunosewoyo, School of Pharmacy
and Biomedical Sciences, Faculty of Health
Sciences, Curtin University, Perth, WA,
Australia.
Email: Hendra.Gunosewoyo@curtin.edu.au
K E Y W O R D S
William R. Bishai, Division of Infectious
Disease, Department of Medicine, Center
for Tuberculosis Research, Johns Hopkins
School of Medicine, Baltimore, MD, USA
Email. wbishai1@jhmi.edu
ciprofloxacin, hybrid molecules, indoleamides, isoniazid, pyrazinamide, tuberculosis
Funding information
Australian Research Council; National
Institutes of Health
1
INTRODUCTION
& Dick, 2012). Once the immune system of these latently
infected people become compromised due to, for instance,
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Tuberculosis (TB) is a highly contagious airborne dis-
ease that has existed for millennia and continues to pose
a major threat to human health. It is one of the oldest life-
threatening and leading deadliest diseases known to man-
kind, claiming more than 1 million lives annually throughout
the world (Barberis et al., 2017; WHO, 2020). TB is caused
by Mycobacterium tuberculosis (M. tb) organism that has
the ability to stay dormant for years, persisting in the host
body without any indication of disease, causing many peo-
ple to become symptom-free carriers (inactive TB; Boon
co-infection with HIV, this silent warfare of the bacteria will
ultimately transform into the active form of TB (Pawlowski
et al., 2012; Shankar et al., 2014). According to the World
Health Organisation (WHO), one quarter of the human popu-
lation harbour a latent M. tb infection with around 10 million
people falling ill with TB every year. In 2018, TB caused
an estimated 1.2 million deaths among HIV-negative people
in addition to 0.25 million deaths among HIV-positive pa-
tients. This inexorable burden ranks TB as the number one
cause of mortality/morbidity from a single infectious agent
Chem Biol Drug Des. 2021;97:1137–1150.
wileyonlinelibrary.com/journal/cbdd
© 2021 John Wiley & Sons Ltd.
1137
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(surpassing HIV/AIDS; WHO, 2020). The WHO directly
observed treatment, and short course (TB-DOTS) requires a
6-month minimum treatment for drug-sensitive (DS) TB with
the first-line anti-TB drugs divided into two phases. The first
2 months are the intensive phase treatment with a cocktail of
four drugs (Figure 1), including isoniazid (INH, 1), rifampin
(RIF), pyrazinamide (PZA, 3) and ethambutol (EMB). The
subsequent 4 months represent the continuation phase treat-
ment with INH and RIF to kill the dormant bacteria (Tiberi
et al., 2017; WHO, 2020). Poor patient compliance to the
lengthy duration of therapy, high pill count and drug side-
effects contributed to the emergence of drug-resistant (DR)
TB (Dheda et al., 2014; Lange et al., 2018; Seung et al., 2015;
Sotgiu & Migliori, 2015; Velayati et al., 2013). Hydrophilic
properties, charge, size and/or stability under physiological
conditions account for inefficient cellular penetration of
many anti-TB drugs and sub-optimal treatment. Accordingly,
high drug doses are prescribed to compensate for the reduced
penetration and bioavailability, taking its toll on the host's
vital organs, causing strong adverse effects. Therefore, highly
efficacious anti-TB drugs are urgently needed. A common
design tactic is to modify old drugs or existing compounds
with an established bioactivity in order to attain, ideally, an
enhanced anti-TB potency, efficacy against DR strains and
short duration of treatment.
Among the front-line TB antibiotics for treatment of DS
strains, INH 1 is a prodrug that requires activation by the
mycobacterial catalase-peroxidase enzyme (KatG) to the
reactive free radical form which then gets implicated in a
series of reactions, forming the isonicotinyl-NAD complex
2. This complex is a potent inhibitor to the enoyl-acyl car-
rier protein reductase InhA, a key enzyme in the biosynthe-
sis of mycolic acids (MAs; Vidossich et al., 2014). MAs are
the integral building blocks of the mycobacterial cell wall
and the primary mediators of the notorious impermeability
and hydrophobic characters of the outer coating (Alsayed
et al., 2019). Therefore, inhibiting InhA eventually leads to
collapsing of the mycobacterial outer coating that serves
as a protective permeability barrier from many antibiot-
ics (Vilcheze & Jacobs, 2019). However, serum concen-
tration of INH is greatly influenced by its acetylation via
N-acetyltransferase enzyme (NAT) which constitutes the main
metabolic pathway of INH in humans (Erwin et al., 2019).
The generated N-acetylisoniazid metabolite is devoid of an-
ti-TB activity, leading to a significant decrease in the INH
bioavailability. Additionally, in M. tb, NAT is implicated in
the resistance mechanism to INH (Unissa et al., 2016). For
these reasons, chemically modifying the hydrazine unit in
INH via incorporating a functional group is commonly used
to avoid the N-acetylation process and thereupon improve the
drug bioavailability and the curative outcomes (Hu, Zhang,
et al., 2017). Moreover, appending lipophilic moieties to the
INH core can impart enhanced cell wall permeation to the
drug. Hence, INH analogues with greater lipophilicity have
emerged as potential anti-TB agents (Hu, Zhang, et al., 2017).
Like INH (Figure 1), PZA 3 is a prodrug which diffuses
into the M. tb granuloma, where it gets activated by the pyra-
zinamidase enzyme to the active form of the drug pyrazinoic
acid (POA, 4; Miotto et al., 2014). However, the exact mech-
anism of PZA is still ambiguous. After penetrating the TB
lesion, the active form of the drug POA accumulates inside
the bacillus and kills the bacterial cell in the acidic environ-
ment of the TB granuloma (Njire et al., 2016). On the other
hand, fluoroquinolones, such as ciprofloxacin (CPF, 5), have
become a mainstay in treating multi-drug-resistant (MDR)
TB. The mechanism of action of this class of antibiotics is
distinct from the first-line drugs in which they inhibit DNA
gyrases and in turn prevent bacterial DNA synthesis (Aldred
et al., 2016; Schluger, 2013). When CPF 5 was rendered more
hydrophobic by attaching alkyl substituents to the piperazine
FIGURE 1 Structures of INH,
isonicotinyl-NAD complex, POA, PZA and
ciprofloxacin

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NH, the resulting N-alkylated ciprofloxacins were more ac-
tive against M. tb than CPF 5 (Haemers et al., 1990).
13a,b and 16 were synthesized to investigate the effect of
these modifications on the in vitro biological activity com-
pared to the parent drug.
The present study entails a simple and efficient synthesis
of a number of INH, POA and CPF derivatives as well as their
in vitro anti-TB activity. All final compounds were screened
in vitro against M. tb H37Rv strain. The most potent com-
pound 21a in our study was further evaluated for its antimyco-
bacterial activity against another DS strain and four DR M. tb
strains in addition to Mycobacterium abscessus (M. abs) and
Mycobacterium avium (M. avium). In parallel, this compound
was also tested in Vero cells for cytotoxicity evaluation.
Our group has previously reported several indole-2-
carboxamides, such as 6, as potent anti-TB agents targeting
the mycobacterial membrane protein large 3 (MmpL3; Lun
et al., 2013; Onajole et al., 2013; Stec et al., 2016). MmpL3
is a crucial transporter implicated in the flipping and release
of the MAs precursors across the plasma membrane (Xu
et al., 2017). Thereafter, MAs get accumulated in the M. tb
cell envelope, forming a bilayer barrier, standing out as key
players in the infection process. Accordingly, we synthesized
and scrutinized the biological activity of two conjugates 18
and 20 in which we integrated INH and POA, respectively,
into the indoleamide architecture. Another hybrid conjugat-
ing both INH and POA 21a was also evaluated. The forego-
ing compounds design strategy is based on conjugating two
different pharmacophore moieties of diverse bioactive sub-
stances known to inhibit different targets in M. tb to develop
a new chemical entity. The new molecule might be capable
of simultaneously hitting different targets, exerting multi-
ple drug actions or one part can offset the adverse effects
caused by the other part. The aforementioned approach of
forming hybrids of bioactive molecules via conjugating effi-
cacious drug fragments is well-known in drug design (Panda
et al., 2016, 2019). Finally, we replaced the pyridine ring in
21a with a benzene ring, forming the analogous derivative
21b which is more lipophilic than INH-POA hybrid 21a to
compare their activities.
2
DESIGN
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The first design strategy was to incorporate an adamantane
ring as a hydrophobic moiety into the framework of INH, POA
and CPF. Adamantane is considered an add-on ‘lipophilic
bullet’ that has a multifaceted value in medicinal chemistry
(Wanka et al., 2013). The hydrophobic substituent constant
of this carbocyclic group is estimated as ᴨadamantyl = 3.1;
therefore integrating an adamantane motif into the structure
of highly water-soluble molecules can shift their ClogP to a
region that is more clinically useful (Liu et al., 2011). Hence,
our first approach is based on endowing the aforementioned
drugs with increased lipophilicity which in turn may enhance
their penetration through the mammalian host tissues and the
lipid-rich mycobacterial cell wall. Beyond enhancing the par-
tition coefficient, the adamantyl group may also improve the
stability of drugs and their pharmacokinetics and modulate
their therapeutic index (Liu et al., 2011; Wanka et al., 2013).
For instance, an inserted adamantane to the INH skeleton
might serve as a protecting group, chemically modifying the
hydrazine unit and blocking the N-acetylation process (Hu,
Zhang, et al., 2017). Towards this, five derivatives 10a,b,
3
CHEMISTRY
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The synthesis of target compounds 10a,b, 13a,b, 16, 18, 20
and 21a,b was accomplished as depicted in Schemes 1–4
SCHEME 1 Synthetic pathway for
compounds 10a,b

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using commercially available INH (1), POA (4), CPF (5)
and 4,6-difluoroindole-2-carboxylic acid (17). Adamantane
derivative 7 was esterified with methanol to give the diester
derivative 8. Subsequent mono hydrolysis with sodium hy-
droxide of the diester 8 yielded the carboxylic acid 9. INH
1 was then coupled with either 1-adamantanecarboxylic
acid or 9, following either amide coupling procedure A [1
-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochlo-
ride (EDC.HCl), hydroxybenzotriazole hydrate (HOBt) and
N,N-diisopropylethylamine (DIPEA)] or coupling condi-
tions B [EDC.HCl and 4-dimethylaminopyridine (DMAP)]
to give the requisite final compounds 10a,b (Scheme 1).
Although EDC.HCl/HOBt-mediated coupling is typically
conducted at room temperature (rt) in the literature (Onajole
et al., 2013), even after 48 hr of stirring, very low yields
were obtained. Hence, we applied relatively harsh condi-
tions via heating EDC.HCl/HOBt amide coupling reactions
at 50°C for 18 hr which resulted in improved yield. Similar
EDC-coupling protocol was previously adopted in which
the reaction was heated at 45°C in a microwave (Bahde
et al., 2011). Alternatively, EDC.HCl/DMAP was also used
as coupling reagents which generally provided better yield
than EDC.HCl/HOBt in the derivatives in which both meth-
ods were employed. In Scheme 2, 1-adamantanecaboxylic
acid 11 was treated with hydrazine hydrate in the presence of
1,1′-carbonyldiimidazole (CDI) to render the hydrazide in-
termediate 12. Then, similar to 10a,b, under standard amide
coupling conditions A, compounds 13a,b were obtained via
reacting POA 4 with 1-adamantylamine and 12, respectively.
Both compounds 10a and 13a were previously reported,
but their synthetic protocols were different from ours and
they have never been evaluated against M. tb (Harikishore
et al., 2013; Naredla et al., 2013). On the other hand, the
synthesis of N-adamantyl CPF derivative 16 is delineated in
Scheme 3, accomplished in three steps. The pyrazine NH of
CPF 5 was initially protected using di-tert-butyl dicarbonate
(Boc)2O to form the N-Boc derivative 14 which was then
subjected to coupling procedure A with 1-adamantylamine to
provide the amide 15. The Boc group in the crude amide 15
was then cleaved using trifluoroacetic acid (TFA) to afford
the desired amine 16.
In the second part of our study, various conjugates were
synthesized as described in Scheme 4. For the preparation of
hybrid 18, amide coupling of 4,6-difluoroindole-2-carboxylic
acid 17 with INH 1 was conducted under coupling conditions
A. Compound 17 was initially converted into the hydrazide
derivative 19 using CDI and hydrazine hydrate, and was fol-
lowed by amide coupling of 19 with POA 4 to furnish con-
jugate 20. Treatment of INH 1 with POA 4 using coupling
method B delivered the desired hybrid 21a. This INH-POA
conjugate was previously prepared using different synthetic
strategies (Miniyar & Bhat, 1999; Panda et al., 2019). It was
evaluated against H37Rv M. tb strain, albeit without spec-
ifying its exact minimum inhibitory concentration (MIC)
(Miniyar & Bhat, 1999). Finally, analogue 21b was obtained
via reacting INH 1 with benzohydrazide following coupling
conditions A.
4
RESULTS AND DISCUSSION
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Final compounds 10a,b, 13a,b, 16, 18, 20 and 21a,b were
evaluated for their antimycobacterial activity in vitro against
M. tb H37Rv strain using the microplate alamarBlue assay
(MABA) to obtain their corresponding MIC values as
shown in Table 1. First, with the aim of increasing lipo-
philicity, an adamantyl group was appended to INH, PZA
and CPF, whereupon five compounds 10a,b, 13a,b and 16
were evaluated. Both adamantane-based INH derivatives
10a,b were less active (MIC > 64 and = 32 µg/ml, respec-
tively) than INH (MIC = 0.04 µg/ml) although they exhib-
ited ClogP values of 2.22 and 1.84, respectively, higher than
that of INH (ClogP = −0.67). It is noteworthy that the me-
thyl ester adamantane derivative 10b was more potent than
SCHEME 2 Synthetic pathway for
compounds 13a,b

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SCHEME 3 Synthetic pathway for
compound 16
SCHEME 4 Synthetic pathway for
compounds 18, 20 and 21a,b
PZA (MIC = 200 µg/ml [Hu, Wu, et al., 2017; Werngren
et al., 2012; Zhang et al., 2002]) and the unsubstituted ada-
mantane derivative 10a. PZA derivatives 13a (MIC = 32 µg/
ml) and 13b (MIC ≥ 32 µg/ml) were more potent compared
to PZA. The higher lipophilicity of 13a,b (ClogP = 2.33
and 1.78, respectively), emanated from the adamantyl motif,
compared to PZA (ClogP = −0.68) appeared to positively
modulate the activity of this lead. It is also conceded that
PZA displays poor activity against M. tb in vitro, not-
withstanding its potent effect in vivo (Gopal et al., 2019).
Additionally, our attempt to enhance the lipophilicity of CPF
resulted in compound 16 which exhibited moderate anti-TB
activity (MIC = 16 µg/ml) in comparison to its precursor
CPF (MIC = 0.25 µg/ml). Importantly, when docked into M.
tb DNA gyrase (PDB ID: 5BTC; Blower et al., 2016), the
keto-amide moiety of 16 was anchored in the gyrase bind-
ing pocket via hydrated magnesium ion bridge (Figure 2).
Several studies substantiated the importance of this bridge in
connecting the fluoroquinolones to the DNA gyrase enzyme,
foregrounding it as the primary conduit for the enzyme-
drug interactions (Aldred et al., 2013, 2014; Wohlkonig
et al., 2010). Two crucial amino acids Ser90 and Asp94 were
found to interact with this bridge, providing further support to
the hydrogen-bonding network (Figure 3; Aldred et al., 2016;
Blower et al., 2016; Wohlkonig et al., 2010). When 16 was
docked to the gyrase active site, the hydroxyl group of Ser90
was directly linked to the magnesium ion associated waters,
similar to CPF. Because of the absence of Ser90 in the wild

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TABLE 1 In vitro anti-TB activity of target compounds 10a,b, 13a,b, 16, 18, 20 and 21a,b as well as reference compounds INH, PZA, CPF
and 6
Analogue
10a
Structure
MICa (µg/ml)
ClogPb
>64
2.22
10b
32
1.84
13a
13b
16
≥32
32
2.33
1.78
2.99
16
18
20
>64
>64
2.37
1.96
21a
21b
2
−0.32
1.03
>64
INH 1
PZA 3
0.04
200
−0.67
−0.68
N
NH2
N
O
CPF 5
0.25
−0.72
(Continues)

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TABLE 1 (Continued)
Analogue
6
Structure
MICa (µg/ml)
ClogPb
0.004
5.74
aThe lowest concentration of drug causing at least 90% reduction of bacterial growth by the Microplate alamarBlue assay (MABA). The reported MIC values are an
average of three individual measurements.
bCalculated using ChemDraw 16.0.
(a)
(b)
Abbreviations:
DA: deoxyadenosine, DC: deoxycytidine, DG: deoxyguanosine,
DT: deoxythymidine, Arg: arginine, Asp: aspartic acid, Gly:
glycine, Ser: serine, PTR: O-phosphotyrosine.
FIGURE 2 2D representation of the putative binding interactions of ciprofloxacin (a) and compound 16 (b) with DNA gyrase [Colour figure
can be viewed at wileyonlinelibrary.com]
type (WT) M. tb gyrase, it was proved that the critical water–
metal ion bridge that govern the interaction between the fluo-
roquinolones and the enzyme is primarily anchored by one
amino acid Asp94 (Aldred et al., 2016). This is in accord-
ance with previous results showing fluoroquinolone resist-
ance in M. tb brought forth by various mutations at Asp94 in
GyrA which could be attributed to the disrupted bridge func-
tion (Aldred et al., 2016; Maruri et al., 2012). Unlike CPF,
the water-mediated magnesium ion network that bridges the
quinolone core of 16 to the enzyme did not seem to interact
with Asp94 (Figure 2). Compound 16 was also aligned in
a slightly different fashion from CPF in the gyrase binding
pocket as portrayed in Figure 3. All of these may contribute
to the reduced activity of 16 compared to CPF.
The first two hybrids, comprising the 4,6-difluoroindole
nucleus linked to INH or POA to give 18 and 20, respec-
tively, showed a dramatic loss of activity (MIC > 64 µg/ml)
compared to the original agents. The inactivity of 18 and 20
relative to the 4,6-difluoroindoleamide 6 (MIC = 0.004 µg/
ml) is likely ascribed to the diminished lipophilicity of these
two analogues (ClogP = 2.37 and 1.96, respectively) com-
pared to 6 (ClogP = 5.74). In addition, extending the amide
linker of the indoleamide analogues in our previous study
was unfavourable (Alsayed et al., 2020), resonating with the
reduced activities of 18 and 20. On the other hand, tether-
ing INH with POA, which generated conjugate 21a, led to
the most active compound in our current study (MIC = 2 µg/
ml). In fact, this hybrid was previously reported to exhibit
>70% growth inhibition at 3 µg/ml against the highly viru-
lent M. tb Erdman strain (Panda et al., 2019). Evidently, this
Next, we conjugated pharmacophoric units of differ-
ent anti-TB substances with distinct mechanism of actions.

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hybrid exhibited higher anti-TB activity than PZA but less
than INH. Meanwhile, the homologous derivative 21b, in
which we replaced the pyridine moiety with benzoic acid,
was bereft of anti-TB activity (MIC > 64 µg/ml) despite its
increased lipophilicity compared to 21a (ClogP = 1.03 and
−0.32). The discrepancy between the activities of 21a and
21b could be attributed to the presence/lack of the INH prod-
rug moiety, respectively. In other words, removal of the INH
pharmacophore in 21b likely accounted for its attenuated
activity compared to 21a, as both compounds retained PZA
which displayed poor in vitro activity (MIC = 200 µg/ml [Hu,
Wu, et al., 2017; Werngren et al., 2012; Zhang et al., 2002]).
Indeed, this was further corroborated by the previous find-
ings of Judge et al in which when they coupled INH with
benzoic acid, the resulting N'-benzoylisonicotinohydrazide
compound showed some potency against the H37Rv strain
(Judge et al., 2013). Overall, it seems that lipophilicity is not
the sole driving force for anti-TB activity in our study. The
higher anti-TB activity demonstrated by the more hydrophilic
anti-TB drugs, currently in the market, compared to our rel-
atively lipophilic analogues, gives validity to the notion that
the positive correlation between lipophilicity and anti-TB ac-
tivity is not a clear-cut phenomenon (Machado et al., 2018;
Piccaro et al., 2015; Tong et al., 2017).
The most potent INH-POA hybrid 21a, together with CPF
as a positive control, were further evaluated for their cytotox-
icity against mammalian cells. In addition, these two com-
pounds were tested for their antimycobacterial activity against
another DS and four DR M. tb strains, as well as M. abs and
M. avium (Table 2). Both 21a and CPF exhibited the same
high IC50 against Vero cells (IC50 ≥ 64 µg/ml), indicating their
limited cytotoxicity. We further assessed 21a and CPF against
a panel of clinical isolates of M. tb, originally procured from
TB patients. Compound 21a retained its activity against DS
M. tb strain (V4207), albeit less active than CPF (MIC = 4 and
0.25 µg/ml, respectively). This hybrid was stripped of its po-
tency when tested against two multi-drug-resistant (MDR) M.
tb strains (V2475, KZN494) and two extensively drug-resistant
(XDR) M. tb strains (R506, TF274). Notably, the anti-TB ac-
tivity of CPF against the tested MDR strains was 8-fold higher
than the XDR ones (MIC = 0.25 and 2 µg/ml, respectively).
Panda et al evaluated hybrid 21a against tuberculous and non-
tuberculous mycobacterial (NTM) strains, wherein it exhib-
ited activity at 20 µg/ml concentration against Mycobacterium
bovis (M. bovis) and at 10 µg/ml against Mycobacterium mari-
num (M. marinum) and Mycobacterium fortuitum (M. fortu-
itum; Panda et al., 2019). Hence, in line with our pursuit to
scrutinize the antimycobacterial activity of 21a, we further
evaluated both 21a and CPF against another two NTM strains,
namely M. abs and M. avium. Surprisingly, compound 21a
was devoid of activity against both strains (MIC > 64 µg/ml).
CPF, however, displayed a 32-fold surge in potency against M.
avium compared to M. abs (MIC = 0.25 and 8, respectively).
This in turn suggests the selective activity of 21a against DS
M. tb.
FIGURE 3 Close-up view of the DNA gyrase active site
(retrieved from PDB ID: 5BTC) in complex with CPF (magenta) and
compound 16 (light brown, docked in silico in the active site), showing
their overlay and different alignment. Green circled is the chelation/
hydrogen-bonding network, designating the water/magnesium ion
bridge (red/yellow spheres) coordinating the keto acid in CPF. The
putative binding profile of CPF also shows an increased support of the
hydrogen bond interactions by Ser90 and Asp94 [Colour figure can be
viewed at wileyonlinelibrary.com]
TABLE 2 Cytotoxicity [IC50 (µg/ml)] against Vero cells of 21a and CPF (positive control) and their activity [MIC (µg/ml)] on selected clinical
isolates of M. tb and two NTM strains
IC50
M. tb
V4207/
DS
V2475/
MDRb
KZN494/
MDRb
R506/
XDRc
TF274/
XDRc
Vero cells
SIa
M. abs
M. avium
21a
CPF
≥64
≥64
≥32
≥256
4
0.25
>32
0.25
>32
0.25
>32
2
>32
2
>64
8
>64
0.25
aSelectivity index (SI) = IC50 (Vero)/MIC(H37Rv).
bResistant to INH and rifampin (RIF).
cResistant to INH, RIF, levofloxacin, ofloxacin and kanamycin.

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5
CONCLUSION
C18 100A (21.2 mm × 150 mm) column, with detection at
254 and 280 nm on a Shimadzu SPD-20A detector, flow
rate = 25.0 ml/min. Method 1: 5–50% acetonitrile/H2O in
15 min; 50–50% acetonitrile/H2O in 10 min; 50–5% acetoni-
trile/H2O in 10 min. Method 2: 20–50% acetonitrile/H2O in
15 min; 50–70% acetonitrile/H2O in 10 min; 70–20% acetoni-
trile/H2O in 10 min. Both solvents contained 0.05 vol% of
trifluoroacetic acid (TFA). Purities of final compounds were
established by analytical HPLC, which was carried out using
Waters 1525 binary pump, 717 plus autosampler, and 2487
dual wavelength absorbance detector, with a Phenomenex
Luna® 5 µm C18(2) 100 Å (150 × 4.6 mm) column. Analytical
HPLC method: flow rate = 1 ml/min; gradient elution over
30 min. Gradient: 100% H2O to 100% acetonitrile in 10 min;
100% acetonitrile in 10 min; 100% acetonitrile to 100% H2O
in 10 min. Both solvents again incorporated 0.05% TFA. The
purity of all tested compounds was >95% as determined by
the HPLC method described above.
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In summary, we rationally designed, concisely synthesized and
evaluated the anti-TB activity of several INH, PZA and CPF
derivatives against H37Rv DS M. tb strain. The adamantane-
dependent modulation of the preceding drugs resulted in en-
hanced lipophilicity (higher ClogP) in compounds 10a,b, 13a,b,
and 16. The noteworthy activity of the adamantane-derived INH
compound 10b set this derivative forth for future modifications
in which the integrated methyl ester functional group can serve
as a building block amenable to further structure changes. The
higher lipophilicity of 13a,b, with respect to the reference stand-
ard PZA was likely responsible for their improved potency.
CPF derivative 16 manifested modest activity and was docked
into M. tb DNA gyrase enzyme. In derivatives 18, 20 and 21a,
pharmacophores from various anti-TB ligands working through
different mechanisms were conjugated. INH-POA hybrid 21a
displayed the most potent activity in our study which was rep-
licated against V4207 DS M. tb strain. Compound 21a and
CPF showed low cytotoxicity in mammalian cells (Vero Cells).
Unlike CPF, when tested against four MDR- and XDR-TB
strains and two NTM strains, 21a was devoid of activity. This
in turn suggests the selective activity of 21a against DS M. tb
without any significant effects on the tested DR M. tb strains and
the NTM strains M. abs and M. avium. These findings provide
valuable information for future elaboration on the adamantane-
based compounds, especially the methyl ester derivative 10b, to
attain higher potency and drug-like molecules. Additionally, the
low cytotoxicity of hybrid 21a and its high in vitro potency merit
further studies on its in vivo anti-TB activity against DS strains.
6.1.2
General procedure for amide coupling
|
(Method A)
To a solution of the appropriate carboxylic acid (1 equiv.)
in anhydrous dimethylformamide (DMF, 10 ml/mmol),
hydroxybenzotriazole hydrate (HOBt, 2 equiv.) and 1-et
hyl-3-(3-(dimethylaminopropyl)carbodiimide
hydrochlo-
ride (EDC·HCl, 2 equiv.) were added at room temperature
(rt). After stirring for 10 min, the corresponding amine (1.5
equiv.) and N,N-diisopropylethylamine (DIPEA, 3 equiv.)
were added, and the reaction mixture was stirred at 50°C for
18 hr. After this time, NaHCO3 solution (25 ml) was added,
and the mixture was extracted with EtOAc (3 × 25 ml). The
combined organic layers were washed with NaHCO3 solution
(5 × 25 ml), brine (1 × 25 ml), dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure. The resi-
due was purified by flash chromatography using dichlo-
romethane/methanol (DCM/MeOH) gradient prior to further
preparative HPLC purification unless otherwise stated.
6
EXPERIMENTAL DETAILS
Chemistry
|
6.1
|
6.1.1
General information
|
1H NMR and 13C NMR spectra were recorded on a Bruker
Avance III spectrometer at 400 and 100 MHz, respectively,
with TMS as an internal standard. Standard abbreviations in-
dicating multiplicity were as follows: s = singlet, d = dou-
blet, dd = doublet of doublets, t = triplet, td = triplet of
doublets, q = quadruplet, m = multiplet and br = broad.
HRMS experiments were performed on a Thermo Scientific
Q-Exactive Orbitrap mass spectrometer. TLC was carried
out on Analtech silica gel TLC plates (200 µm, 20 × 20 cm).
Flash chromatography was performed using a Teledyne Isco
CombiFlash Rf system with RediSep columns or manu-
ally using SiliCycle SiliaFlash® P60 Silica Gels [40–63 µm
(230–400 mesh)]. Final compounds were purified by pre-
parative HPLC unless otherwise stated. The preparative
HPLC employed a Phenomenex Luna® Omega 5 μm Polar
6.1.3
General procedure for amide coupling
|
(Method B)
To a stirred solution of carboxylic acid (1 mmol) in a 1:1
mixture of tetrahydrofuran (THF) and DCM, EDC.HCl
(1.2 mmol), the corresponding amine (1.2 mmol) and
4-dimethylaminopyridine (DMAP, 0.3 mmol) were added
and the reaction mixture was stirred at room temperature for
72 hr. The solvent was then removed under vacuum, and the
residue was purified by flash chromatography using DCM/
MeOH gradient prior to further HPLC purification unless
otherwise stated.

1146
RESEARCH LETTER
|
6.1.4
General procedure for amide coupling
(s, 3H), 1.88 (d, J = 2.6 Hz, 6H), 1.70 (d, J = 2.2 Hz, 6H); 13
C
NMR (DMSO-d6) δ 176.7, 164.0, 149.6, 141.5, 122.5, 40.1,
39.0, 36.5, 28.0; HRMS (ESI) m/z calcd for C17H21N3O2
([M + H]+) m/z 300.1707; found 300.1700.
|
(Method C)
A
mixture of carboxylic acid (1 mmol) and
1,1′-carbonyldiimidazole (CDI) (1.5 mmol) in anhydrous
DMF (10 ml) was stirred at rt for 2 hr, followed by the addi-
tion of hydrazine hydrate solution (5 mmol) and stirring was
continued for 16 hr at rt. Water (25 ml) was then added to the
reaction mixture and the formed precipitate was filtered off,
washed with water (4 × 25 ml) and dried. The crude product
was used directly in the next step without additional purifica-
tion as they were already pure according to the crude 1H/13C
NMR spectra.
Methyl-3-(2-isonicotinoylhydrazine-1-carbonyl)
adamantane-1-carboxylate (10b)
The title compound was obtained from reacting INH and
compound 9 following method A or B. After flash chroma-
tography, the product was further purified via crystallization
from diethyl ether. White solid, yield: 30% (method A) and
45% (method B). 1H NMR (DMSO-d6) δ 10.52 (s, 1H), 9.67
(s, 1H), 8.76 (dd, J = 4.6, 1.3 Hz, 2H), 7.77 (dd, J = 4.5,
1.5 Hz, 2H), 3.61 (s, 3H), 2.11 (s, 2H), 1.96 (s, 2H), 1.91–
1.73 (m, 8H), 1.65 (s, 2H); 13C NMR (DMSO-d6) δ 177.0,
176.0, 164.4, 150.9, 140.1, 121.7, 52.0, 41.0, 40.3, 38.04,
37.99, 35.3, 27.9; HRMS (ESI) m/z calcd for C19H23N3O4
([M + H]+) m/z 358.1761; found 358.1754.
6.1.5
Preparation of intermediates and final
|
compounds 8, 9, 10a,b, 12, 13a,b, 14, 16, 18–
20 and 21a,b
Dimethyl adamantane-1,3-dicarboxylate (8)
Adamantane-1-carbohydrazide (12)
To a solution of 1,3-adamantanedicarboxylic acid in metha-
nol, 0.5 ml of conc. H2SO4 was added and the reaction mix-
ture was refluxed for 16 hr and then concentrated in vacuo.
The residue was slowly quenched with saturated NaHCO3
solution, followed by extraction with DCM (3 × 25 ml). The
combined organic layers were washed with brine (1 × 25 ml),
dried over anhydrous Na2SO4, filtered and evaporated under
vacuum. 1H NMR data of 8 matched that reported in the lit-
This compound was obtained from 1-adamantanecarboxylic
acid employing method C and its 1H NMR data matched that
reported in the literature (Seliverstova et al., 2018). White
solid, yield: 78%. 1H NMR (DMSO-d6) δ 8.67 (s, 1H), 4.10 (s,
2H), 1.94 (s, 3H), 1.75 (d, J = 2.7 Hz, 6H), 1.71–1.58 (m, 6H).
N-(1-adamantyl)pyrazine-2-carboxamide (13a)
The title compound was synthesized from POA and
1-adamantylamine according to general procedure A and its
1H NMR data matched that reported in the literature (Naredla
et al., 2013). The purity of the compound was >95% after
1
erature (Averina et al., 2014). White solid, yield: 98%. H
NMR (DMSO-d6) δ 3.59 (s, 6H), 2.12–2.03 (m, 2H), 1.90 (s,
2H), 1.84–1.70 (m, 8H), 1.63 (s, 2H).
1
flash chromatography. White solid, yield: 76%. H NMR
3-(Methoxycarbonyl)adamantane-1-carboxylic acid (9)
To a solution of compound 8 (1 mmol) in dry THF, a solu-
tion of NaOH (1.1 mmol) in dry methanol was added dropwise
under an argon atmosphere. After 24 hr of stirring at rt, the re-
action mixture was concentrated in vacuo, diluted with water
(1 × 25 ml) and washed with DCM (1 × 25 ml). The aqueous
layer was subsequently acidified with HCl until the pH value
dropped to 1–2 and extracted with DCM (4 × 25 ml). The com-
bined organic layers were dried over anhydrous Na2SO4 filtered
and evaporated under reduced pressure. The crude product was
used for the next reaction without further purification. White
(DMSO-d6) δ 9.16 (s, 1H), 8.81 (s, 1H), 8.63 (s, 1H), 7.74 (s,
1H), 2.08 (s, 9H), 1.67 (s, 6H).
N'-(adamantane-1-carbonyl)pyrazine-2-carbohydrazide
(13b)
The title compound was obtained from POA and compound
12 employing method A and further purified via crystalli-
zation from ethanol after flash chromatography. Buff solid,
yield: 50%. 1H NMR (DMSO-d6) δ 10.48 (s, 1H), 9.54 (s, 1H),
9.16 (d, J = 1.3 Hz, 1H), 8.91 (d, J = 2.4 Hz, 1H), 8.77 (dd,
J = 2.3, 1.5 Hz, 1H), 2.00 (s, 3H), 1.88 (d, J = 2.1 Hz, 6H), 1.70
(s, 6H); 13C NMR (DMSO-d6) δ 176.5, 162.5, 148.3, 144.9,
144.1, 144.0, 40.1, 39.0, 36.5, 28.0; HRMS (ESI) m/z calcd
for C16H20N4O2 ([M + H]+) m/z 301.1659; found 301.1651.
1
solid, yield: 70%. H NMR (CDCl3) δ 3.67 (s, 3H), 2.17 (s,
2H), 2.06 (s, 2H), 1.88 (s, 8H), 1.69 (s, 2H); 13C NMR (CDCl3)
δ 183.3, 177.2, 51.8, 40.9, 40.8, 39.5, 37.9, 37.7, 35.3, 27.7.
N'-(adamantane-1-carbonyl)isonicotinohydrazide (10a)
The title compound was synthesized from INH and
1-adamantanecarboxylic acid according to general procedure
A. White solid, yield: 87%. 1H NMR (DMSO-d6) δ 10.57 (s,
1H), 9.59 (s, 1H), 8.82 (s, 2H), 7.86 (d, J = 5.8 Hz, 2H), 2.01
7-(4-(tert-butoxycarbonyl)piperazin-1-yl)-1-cyclopropyl-
6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(14)
Commercially available CPF (2 mmol) was dissolved in
20 ml of water:dioxane (1:1) containing 4 ml of 2.0 M

RESEARCH LETTER
1147
|
aqueous NaOH solution. Di-tert-butyl dicarbonate
(Boc2O, 3 mmol) was then added, and the reaction mix-
ture was stirred at rt until completion (48 hr). Three quar-
ters of the solvent was evaporated in vacuo, followed by
acidification with aqueous 2.0 M HCl solution and the
formed precipitate was filtered off, extensively washed
J = 25.9, 4.4 Hz); HRMS (ESI) m/z calcd for C15H10F2N4O2
([M + H]+) m/z 317.0845; found 317.0839.
4,6-difluoro-1H-indole-2-carbohydrazide (19)
This
compound
was
synthesized
from
4,6-difluoroindole-2-carboxylic acid (17) according to
1
1
with water (2 × 50 ml) and dried. H NMR data of the
method C. Buff solid, yield: 74%. H NMR (DMSO-d6) δ
product matched that reported in the literature (Tehler
et al., 2013). White solid, yield: 99%. 1H NMR (CDCl3) δ
8.78 (s, 1H), 8.05 (d, J = 12.9 Hz, 1H), 7.37 (d, J = 7.1 Hz,
1H), 3.67 (t, J = 5.0 Hz, 4H), 3.60–3.43 (m, 1H), 3.29 (t,
J = 5.1 Hz, 4H), 1.50 (s, 9H), 1.43–1.36 (m, 2H), 1.24–
1.18 (m, 2H).
12.04 (s, 1H), 9.86 (s, 1H), 7.16 (s, 1H), 7.01 (dd, J = 9.4,
1.4 Hz, 1H), 6.87 (td, J = 10.4, 1.9 Hz, 1H), 4.52 (s, 2H); 13
C
NMR (DMSO-d6) δ 160.8, 159.5 (dd, J = 238.4, 12.3 Hz),
156.1 (dd, J = 248.5, 15.5 Hz), 138.0 (dd, J = 15.2, 13.2 Hz),
132.0 (d, J = 3.3 Hz), 113.6 (dd, J = 21.8, 0.7 Hz), 98.0, 95.6
(dd, J = 29.7, 23.3 Hz), 95.0 (dd, J = 25.9, 4.5 Hz).
N-(1-adamantyl)-1-cyclopropyl-6-fluoro-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide
(16)
4,6-difluoro-N'-(pyrazine-2-carbonyl)-1H-indole-2-
carbohydrazide (20)
The title compound was obtained from POA and interme-
diate 19 employing method A or B. The product was fur-
ther crystallized from DCM to attain >95% purity. Light
buff solid, yield: 32% (method A) and 70% (method B). 1H
NMR (DMSO-d6) δ 12.22 (br s, 1H), 10.72 (br s, 2H), 9.23
(s, 1H), 8.94 (d, J = 1.8 Hz, 1H), 8.81 (s, 1H), 7.41 (s, 1H),
7.07 (d, J = 9.0 Hz, 1H), 6.92 (t, J = 10.1 Hz, 1H); 13C NMR
(DMSO-d6) δ 162.9, 160.1, 159.9 (dd, J = 239.1, 12.1 Hz),
156.3 (dd, J = 249.2, 15.6 Hz), 148.6, 144.6, 144.2, 144.1,
138.3 (dd, J = 15.2, 13.0 Hz), 131.0 (d, J = 3.2 Hz), 113.6
(d, J = 21.8 Hz), 99.9, 96.0 (dd, J = 29.7, 23.3 Hz), 95.2 (dd,
J = 25.9, 4.3 Hz); HRMS (ESI) m/z calcd for C14H9F2N5O2
([M + H]+) m/z 318.0797; found 318.0793.
Compound 14 and 1-adamantylamine were reacted follow-
ing general procedure A. The residue obtained after evapo-
rating the EtOAc extract in vacuo was dissolved in 20 ml
DCM, followed by the addition of 4 ml TFA. The reaction
mixture was stirred for 12 hr and concentrated in vacuo.
NaHCO3 solution was then added to the residue, followed
by extraction with DCM (3 × 50 ml). The combined organic
phases were washed with brine (1 × 25 ml), dried over an-
hydrous Na2SO4, filtered and concentrated under reduced
pressure. The residue was purified by flash chromatography
using DCM/MeOH gradient prior to further HPLC purifica-
1
tion. White solid, yield: 40%. H NMR (DMSO-d6) δ 9.89
(s, 1H), 8.59 (s, 1H), 7.82 (d, J = 13.6 Hz, 1H), 7.45 (d,
J = 7.5 Hz, 1H), 3.80–3.61 (m, 1H), 3.18 (t, J = 4.4 Hz, 4H),
2.90 (s, 4H), 2.04 (s, 9H), 1.66 (s, 6H), 1.32–1.24 (m, 2H),
1.14–1.03 (m, 2H); 13C NMR (DMSO-d6) δ 174.8, 163.0,
152.9 (d, J = 247.0 Hz), 147.1, 143.5 (d, J = 10.6 Hz),
138.8, 121.9 (d, J = 6.9 Hz), 111.9 (d, J = 22.6 Hz), 111.4,
107.0, 51.0, 47.0 (d, J = 4.2 Hz), 43.2, 41.9, 36.5, 35.5, 29.3,
8.0; HRMS (ESI) m/z calcd for C27H33FN4O2 ([M + H]+)
m/z 465.2660; found 465.2659.
N'-isonicotinoylpyrazine-2-carbohydrazide (21a)
The title compound was prepared from POA and INH em-
ploying method B and further purified via recrystallization
from diethyl ether after flash chromatography. White solid,
1
yield: 66%. H NMR (DMSO-d6) δ 10.96 (s, 2H), 9.22 (s,
1H), 8.94 (d, J = 2.3 Hz, 1H), 8.79 (d, J = 6.0 Hz, 3H), 7.82
(d, J = 5.8 Hz, 2H); 13C NMR (DMSO-d6) δ 164.7, 162.8,
150.9, 148.6, 144.5, 144.2, 144.1, 140.0, 121.9; HRMS (ESI)
m/z calcd for C11H9N5O2 ([M + H]+) m/z 244.0829; found
244.0824.
4,6-difluoro-N'-isonicotinoyl-1H-indole-2-
carbohydrazide (18)
The title compound was obtained from commercially avail-
able 4,6-difluoroindole-2-carboxylic acid (17) and INH
N'-benzoylpyrazine-2-carbohydrazide (21b)
The title compound was obtained from benzohydrazide and
1
1
employing method A. White solid, yield: 64%. H NMR
POA employing method A. White solid, yield: 41%. H
(DMSO-d6) δ 12.22 (s, 1H), 10.95 (s, 1H), 10.77 (s, 1H), 8.87
(s, 2H), 7.92 (d, J = 4.9 Hz, 2H), 7.39 (d, J = 2.0 Hz, 1H),
7.07 (dd, J = 9.3, 1.7 Hz, 1H), 6.95 (td, J = 10.4, 1.9 Hz, 1H);
13C NMR (DMSO-d6) δ 164.5, 160.4, 160.0 (dd, J = 239.3,
12.1 Hz),156.3 (dd, J = 249.2, 15.6 Hz), 149.9, 140.9, 138.4
(dd, J = 15.2, 12.9 Hz), 130.8 (d, J = 3.2 Hz), 122.5, 113.6
(d, J = 21.7 Hz), 99.9, 96.0 (dd, J = 29.7, 23.2 Hz), 95.2 (dd,
NMR (DMSO-d6) δ 10.85 (s, 1H), 10.60 (s, 1H), 9.22 (d,
J = 1.2 Hz, 1H), 8.94 (d, J = 2.4 Hz, 1H), 8.80 (dd, J = 2.4,
1.5 Hz, 1H), 7.93 (d, J = 7.1 Hz, 2H), 7.61 (t, J = 7.3 Hz,
1H), 7.53 (t, J = 7.4 Hz, 2H); 13C NMR (DMSO-d6) δ 167.2,
163.3, 148.3, 144.4, 144.3, 143.7, 132.8, 132.2, 129.2, 127.9;
HRMS (ESI) m/z calcd for C12H10N4O2 ([M + H]+) m/z
243.0877; found 243.0870.

1148
RESEARCH LETTER
|
tuberculosis gyrase: Enhancing drug activity against wild-type and
resistant gyrase. Proceedings of the National Academy of Sciences
of the United States of America, 113(7), E839–E846. https://doi.
org/10.1073/pnas.1525055113
6.2
Biology
|
MIC was determined using Microplate alamarBlue assay
(MABA) as previously reported (Collins & Franzblau, 1997;
Pieroni et al., 2011). Modified MABA was used to determine
the MIC of PZA. Briefly, 7H9 (Difco™ Middlebrook 7H9
Broth, BD) without Tween 80 but containing 10% OADC
(BBL™ Middlebrook OADC Enrichment, BD) was prepared
fresh with the pH value was adjusted to 5.45. Assay was
set up as previously reported (Collins & Franzblau, 1997;
Pieroni et al., 2011). After 15 days of incubation, 15 µl of
7H9 (pH = 10.75) was added to each well and immedi-
ately followed by addition of 32.5 µl of alamar blue. Plate
was incubated overnight before reading. MABA format was
also used in the cytotoxicity evaluation on Vero Cells (Lun
et al., 2013).
Aldred, K. J., Breland, E. J., Vlckova, V., Strub, M. P., Neuman, K.
C., Kerns, R. J., & Osheroff, N. (2014). Role of the water-metal
ion bridge in mediating interactions between quinolones and
Escherichia coli topoisomerase IV. Biochemistry, 53(34), 5558–
5567. https://doi.org/10.1021/bi500682e
Aldred, K. J., McPherson, S. A., Turnbough, C. L. Jr., Kerns, R. J., &
Osheroff, N. (2013). Topoisomerase IV-quinolone interactions are
mediated through a water-metal ion bridge: Mechanistic basis of
quinolone resistance. Nucleic Acids Research, 41(8), 4628–4639.
https://doi.org/10.1093/nar/gkt124
Alsayed, S. S. R., Beh, C. C., Foster, N. R., Payne, A. D., Yu, Y., &
Gunosewoyo, H. (2019). Kinase targets for mycolic acid bio-
synthesis in Mycobacterium tuberculosis. Current Molecular
Pharmacology, 12(1), 27–49. https://doi.org/10.2174/1874467211
666181025141114
Alsayed, S. S. R., Lun, S., Luna, G., Beh, C. C., Payne, A. D., Foster,
N., Bishai, W. R., & Gunosewoyo, H. (2020). Design, synthesis, and
biological evaluation of novel arylcarboxamide derivatives as anti-
tubercular agents. RSC Advances, 10(13), 7523–7540. https://doi.
org/10.1039/C9RA10663D
Averina,E.B.,Sedenkova,K.N.,Bakhtin,S.G.,Grishin,Y.K.,Kutateladze,
A. G., Roznyatovsky, V. A., Rybakov, V. B., Butov, G. M., Kuznetsova,
T. S., & Zefirov, N. S. (2014). symm-Tetramethylenecyclooctane:
En route to polyspirocycles. Journal of Organic Chemistry, 79(17),
8163–8170. https://doi.org/10.1021/jo501380y
Bahde, R. J., Appella, D. H., & Trenkle, W. C. (2011). A one-pot prepa-
ration of N-2-mercaptobenzoyl-amino amides. Tetrahedron Letters,
52(32), 4103–4105. https://doi.org/10.1016/j.tetlet.2011.05.115
Barberis, I., Bragazzi, N. L., Galluzzo, L., & Martini, M. (2017). The
history of tuberculosis: From the first historical records to the
isolation of Koch's bacillus. Journal of Preventive Medicine and
Hygiene, 58(1), E9–E12.
Blower, T. R., Williamson, B. H., Kerns, R. J., & Berger, J. M. (2016).
Crystal structure and stability of gyrase-fluoroquinolone cleaved
complexes from Mycobacterium tuberculosis. Proceedings of the
National Academy of Sciences of the United States of America,
113(7), 1706–1713. https://doi.org/10.1073/pnas.1525047113
Boon, C., & Dick, T. (2012). How Mycobacterium tuberculosis goes to
sleep: The dormancy survival regulator DosR a decade later. Future
Microbiology, 7(4), 513–518. https://doi.org/10.2217/fmb.12.14
Collins, L., & Franzblau, S. G. (1997). Microplate alamar blue assay
versus BACTEC 460 system for high-throughput screening of com-
pounds against Mycobacterium tuberculosis and Mycobacterium
avium. Antimicrobial Agents and Chemotherapy, 41(5), 1004–1009.
https://doi.org/10.1128/aac.41.5.1004
6.3
Molecular docking protocol
|
In silico molecular modelling analysis was undertaken
using the Molecular Operating Environment MOE software
version 2008.10 (Chemical Computing Group, Montreal,
Canada). The X-ray crystal structure of M. tb DNA gy-
rase in complex with CPF (5BTC; Blower et al., 2016)
was retrieved from the protein data bank (PDB). The bind-
ing pocket was ready for docking after 3D protonating
the enzyme, whereby hydrogens and partial charges were
added to the system for optimization. In order to validate
the docking protocol, the co-crystallized CPF was docked
into the active site. Next, the structure of 16 was drawn in
ChemDraw Ultra 16.0, saved as.mol file, opened inside the
MOE program, 3D protonated and geometrically optimized
using MMFF94x forcefield with gradient 0.05. Compound
16 was then docked into the same binding site of CPF using
MOE-DOCK function, employing Triangle Matcher place-
ment method and scored using London dG scoring method-
ology. Then, molecular mechanics forcefield was applied to
relax the generated poses which were further ranked using
London dG scoring function and the top 30 poses were re-
tained. The best-ranked pose with the lowest binding free
energy (i.e. the smallest docking score) was selected.
ACKNOWLEDGMENTS
Dheda, K., Gumbo, T., Gandhi, N. R., Murray, M., Theron, G., Udwadia,
Z., Migliori, G. B., & Warren, R. (2014). Global control of tuber-
culosis: From extensively drug-resistant to untreatable tuberculo-
sis. The Lancet Respiratory Medicine, 2(4), 321–338. https://doi.
org/10.1016/S2213-2600(14)70031-1
Erwin, E. R., Addison, A. P., John, S. F., Olaleye, O. A., & Rosell, R.
C. (2019). Pharmacokinetics of isoniazid: The good, the bad, and
the alternatives. Tuberculosis (Edinb), 116S, S66–S70. https://doi.
org/10.1016/j.tube.2019.04.012
Gopal, P., Gruber, G., Dartois, V., & Dick, T. (2019). Pharmacological
and molecular mechanisms behind the sterilizing activity of
SSRA is grateful for the support of Curtin International
Postgraduate Research Scholarship (CIPRS). WRB is
thankful for the support of NIH grants AI 37856 and HL
133190. HG acknowledges the ARC Discovery Early Career
Researcher Award DE160100482.
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Bishai WR, Gunosewoyo H. Facile synthesis and
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