Structure elaboration of isoniazid: synthesis, in silico molecular docking and antimycobacterial activity of isoniazid–pyrimidine conjugates

Article abstract of DOI:10.1007/s11030-019-10004-1

Abstract: Designing small molecule-based new drug candidates through structure modulation of the existing drugs has drawn considerable attention in view of inevitable emergence of resistance. A new series of isoniazid–pyrimidine conjugates were synthesize

Full text of DOI:10.1007/s11030-019-10004-1

Molecular Diversity
https://doi.org/10.1007/s11030-019-10004-1
ORIGINAL ARTICLE
Structure elaboration of isoniazid: synthesis, in silico molecular
docking and antimycobacterial activity of isoniazid–pyrimidine
conjugates
Hardeep Kaur1 · Lovepreet Singh1 · Kelly Chibale2 · Kamaljit Singh1
Received: 1 August 2019 / Accepted: 11 October 2019
© Springer Nature Switzerland AG 2019
Abstract
Designing small molecule-based new drug candidates through structure modulation of the existing drugs has drawn con-
siderable attention in view of inevitable emergence of resistance. A new series of isoniazid–pyrimidine conjugates were
synthesized in good yields and evaluated for antitubercular activity against the H37Rv strain of Mycobacterium tuberculosis
using the microplate Alamar Blue assay. Structure–anti-TB relationship profle revealed that conjugates 8a and 8c bearing
a phenyl group at C-6 of pyrimidine scafold were most active (MIC99 10 µM) and least cytotoxic members of the series.
In silico docking of 8a in the active site of bovine lactoperoxidase as well as a cytochrome C peroxidase mutant N184R
Y36A revealed favorable interactions similar to the heme enzyme catalase peroxidase (KatG) that activates isoniazid. This
investigation suggests a rationale for further work on this promising series of antitubercular agents.
Graphic abstract
µ
MIC99 10 M (H37RV)
Keywords Isoniazid · Pyrimidine · Conjugates · Tuberculosis · Drug resistance · Molecular docking · ADME
Abbreviations
MABA Microplate Alamar Blue assay
Hardeep Kaur and Lovepreet Singh have contributed equally to
WHO
SDG
INH
World Health Organization
Sustainable development goals
Isoniazid
this work.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-019-10004-1) contains
supplementary material, which is available to authorized users.
RIF
Rifampicin
PZA
EMB
PPE
Pyrazinamide
Ethambutol
* Kamaljit Singh
Polyphosphate ester
kamaljit.chem@gndu.ac.in
KatG
MTT
Heme (ferric) enzyme catalase peroxidase
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetra-
zoliumbromide
1
Department of Chemistry, UGC Centre of Advance Study-II,
Guru Nanak Dev University, Amritsar 143 005, India
2
South African Medical Research Council Drug Discovery
InhA
2-trans-enoyl-acyl carrier protein reductase
and Development Research Unit, Department of Chemistry
and Institute of Infectious Disease and Molecular Medicine,
University of Cape Town, Rondebosch 701, South Africa
ADME Adsorption, distribution, metabolism, excretion
Vol.:(0123456789)
1 3

Molecular Diversity
Modulator of
Activity and
contributor
towards ligand
binding
Multidrug-resistant diseases constitute a public health crisis
and a dastardly curse on humanity as the resistance to the
most frst-line drugs poses a serious challenge to the health
security, especially of the weaker section of population [1].
The situation exacerbates when the disease is communicable
and is asymptomatic during the initial phase, which often
delays onset of active treatment only to result in transmission
to others [2]. Tuberculosis (TB) is a contagious, infectious
disease caused by bacteria (Mycobacterium tuberculosis)
and is one of the major causes of death worldwide [3, 4]. In
2017 only, at least 1.6 million (including 0.3 million HIV
positive) people died from this disease. As per the estimate
of the World Health Organization (WHO) [5], there were
558,000 new cases of multidrug-resistant TB (MDR-TB)
with resistance to the most efective rifampicin. While “end
of TB” as an epidemic remains an aspiration, the initiatives
of the sustainable development goals (SDG) to end TB by
2030 needs to be supported to make it a reality [6, 7]. In this
direction, repurposing of existing drugs [8], derivatization of
the existing drugs [9] and screening libraries of new chemi-
cal entities [10] to fnd novel compounds, which could act
through diferent mechanisms against the TB bacterium, and
design of new drugs [11, 12] that can shorten and improve
TB treatment are the mainstay approaches used for fnding
drug leads. Molecular hybrids [13, 14], obtained by covalent
linking of two diferent pharmacophores, ofer advantages
in terms of solubility and efciency in formulation/delivery
and circumvent limitations of single drug or combination
therapy. Such drugs which promise dual mode of action are
expected to avoid/delay emergence of drug resistance.
Isoniazid (INH 1, Fig. 1) [15] is one of the most efective
frst-line anti-TB drugs, which is generally administered in
combination with rifampicin, pyrazinamide or ethambutol.
However, undesirable drug interactions in the combination,
Contributor
towards activity
and binding with
target
Fused Linker
Fig. 2 Design strategy of INH–pyrimidine conjugates and structure–
property indicators
mismatched pharmacokinetics and pharmacodynamics,
length of the therapy and side efects such as hepatotoxicity
constitute some of the key limitations [16, 17]. However,
the use of anti-TB molecular hybrids [18] (2, Fig. 1) based
on INH as one of the hybrid partners has been reported to
not only exhibit good anti-TB activity, but also circumvent
several limitations of the combination therapy. Meanwhile,
pyrimidines have reasonably broad spectrum of pharmaco-
logical activities such as antimalarial [19–23], anti-AIDS
[24, 25], antinociceptive [26, 27], antifungal [28, 29], anti-
cancer [30, 31] as well as antitubercular [32–34]. Some of
the molecular hybrids using pyridine sub-unit (3,4) [35, 36]
with good anti-TB activity are shown in Fig. 1. We hypoth-
esized elaborating structure of INH by linking INH with
pyrimidines to obtain small molecular conjugates would
not only provide new molecular hybrids, but also provide
an opportunity to evaluate their structure-dependent anti-
TB activity. Using the design strategy shown in Fig. 2, we
synthesized a series of compounds and evaluated their anti-
TB activity against H37Rv strains of M. tuberculosis using
the microplate Alamar Blue assay (MABA) and deduced
their structure–activity relationship profle. Additionally,
we have assayed the cytotoxicity of selected hybrids against
the mammalian Vero cell line. We also performed in silico
molecular docking of the most active compound in the active
sites of bovine lactoperoxidase (PDB ID: 3I6N) as well as a
cytochrome C peroxidase (PDB ID: 2V2E) mutant N184R
Y36A to understand their binding patterns.
The INH–pyrimidine conjugates were synthesized using
the protocol outlined in Scheme 1. The precursor alkyl
6-methyl/phenyl-2-oxo-4-substituted-1,2,3,4-tetrahydropy-
Fig. 1 Structures of isoniazid (INH, 1) and molecular hybrids: based
on INH (2), and pyrimidine core (3,4)
Scheme 1 Synthesis of INH–pyrimidine conjugates 8a–j
1 3

Molecular Diversity
Table 1 Structure of 5–8,
in vitro antitubercular activity
and cytotoxicity of INH–
pyrimidine conjugates 8a–j and
reference drugs
Entry Structure (Scheme 1)
8a–j
5–8 R1
R2
R3 Yield (%) CLogPa MIC99 (μM)b,c IC50 (μM)b,d SIe
1
2
3
4
5
6
7
8
a
b
c
d
e
f
Me
Et
iso-Pr
Et
Me
Et
Et
Et
iso-Pr
iso-Pr
(Figure 1)
–
–
–
Ph
Ph
Ph
Me
Me
Me
Me 88
Me 92
Me 84
Ph 90
Me 95
Me 96
Me 95
1.55 10
2.08 40
2.39 10
2.08 >160
0.04 >160
0.48 40
2.23 40
2.12 >160
2.14 40
2.14 80
−0.66 0.1423
−0.67 0.009
>275
>265
>255
nd
nd
nd
nd
>245
nd
nd
729
–
>27.5
>6.6
>25.5
nd
nd
nd
nd
>1.53
nd
nd
5123
–
g
h
i
p-F(C6H4)
p-MeO(C6H4) Me 90
m-NO2(C6H4) Me 86
p-NO2(C6H4) Me 92
9
10
11
12
13
14
j
1
–
–
–
–
RIFf
9f
−5.2
–
1.340
–
–
0.12
–
–
10f
b
c
aCalculated from Chem draw Ultra 11.0; Mean of three observations; MIC99 (μM) at day 14 in GAST/
d
e
Fe with H37Rv (microplate Alamar Blue); 50% cytotoxic concentration (Vero cell lines); SI is the ratio
of cytotoxicity (IC50 in μM) to MIC99 (in vitro activity against M. tuberculosis H37Rv) expressed in μM;
nd—not determined; fSee below
rimidine-5-carboxylates 5a–j were prepared through HCl
or polyphosphate ester (PPE)-catalyzed three-component
Biginelli condensation [37–43] of an alkyl acetoacetic ester,
urea and appropriate aldehyde in anhydrous EtOH. Oxida-
tion of 5a–j using nitric acid (aqueous 70% v/v) readily
furnished the corresponding alkyl 6-methyl/phenyl-2-oxo-
4-substituted-1,2-dihydropyrimidine-5-carboxylates 6a–j.
Chlorination of 6a–j with phosphorous oxychloride (POCl3)
yielded the corresponding alkyl 2-chloro-4-methyl/phenyl-
6-substituted pyrimidine-5-carboxylate 7a–j. Nucleophilic
substitution reaction of 7a–j with 1 (prepared from the com-
mercially available 4-cyanopyridine and hydrazine hydrate)
gave alkyl 4-methyl/phenyl-2-(2-isonicotinoylhydrazinyl)-
6-substituted pyrimidine-5-carboxylates 8a–j (INH–pyrimi-
dine conjugates) in 84–96% yield (Table 1). Structures of
5–8 were unambiguously established on the basis of spectral
(1H NMR, 13C NMR, MS, FTIR) data as well as microana-
lytical analysis (See Supporting Information).
cell wall in M. tuberculosis bacterium. In the current design
of conjugates (Fig. 2), it was expected that the structure
modifcation of 1 may infuence the physicochemical prop-
erties such as lipophilicity and/or difusion through the
lipid-rich bacterial cell wall and modulate the binding of
these conjugates in the binding domain of biological targets
and exert anti-TB activity. The designed INH–pyrimidine
conjugates 8a–j indeed showed high lipophilicity (cLogP:
0.04–2.39; Table 1) relative to INH as well as favorable
binding interactions similar to heme (ferric) enzyme catalase
peroxidase (KatG) endogenous to M. tuberculosis.
Conjugates 8a–j were tested for in vitro antitubercular
activity against H37Rv strains of M. tuberculosis using the
MABA assay and depicted (Table 1) good to moderate
activity in µM range, although the activity was less than the
standard drugs viz. 1, rifampicin (RIF) and kanamycin. Cor-
relating anti-TB activity with structure of these conjugates
revealed interesting trends. Replacing the C-5 ethyl ester
of 8b [MIC99 40 µM] with methyl (8a) or isopropyl ester
(8c) [8a=8c: MIC99 10 µM] (Entries 1–3, Table 1) led to
an increase in antitubercular activity. Similarly, when the
Isoniazid 1 exerts its anti-TB activity by inhibiting the
synthesis of mycolic acid, which is one of the essential
chemical pathways for the formation of the mycobacterial
1 3

Molecular Diversity
phenyl substituent at the C-6 position of the pyrimidine core
of the compounds 8a/8b was replaced with a methyl group,
while a decrease in antitubercular activity of the resultant
derivative 8e was observed (Entry 5, Table 1) compared
to the former, the activity of 8f was similar to that of 8b.
Switching the substituents at C-4 and C-6 positions of 8d
to make 8b showed a fourfold increase in activity (Table 1).
We further incorporated diferently substituted (p-fuoro/p-
methoxy/m-/p-nitro) aryl substituents at C-6 position of the
pyrimidine ring (Table 1). Thus, compared to 8b, introduc-
tion of a p-fuoro group in 8g (Entry 7, Table 1) did not show
change in activity, a p-methoxy substituent led to a fourfold
decrease (Table 1). Likewise, compared to 8c, one of the
most active members of the series, introducing a m-nitro-
phenyl (8i) or p-nitro phenyl (8j) substituents at C-6 position
of the pyrimidine core resulted in, respectively, two- and
fourfold decrease in the antitubercular activity, while their
activity was signifcantly superior to 8h (Entry 8, Table 1).
Among the nitro-substituted conjugates, the m-nitrophenyl-
substituted 8i (MIC99 40 µM) was twofold more active than
the corresponding p-nitrophenyl-substituted 8j (MIC99
80 µM). This brief structure–antitubercular activity profle
suggested that the variation of substituents at C-4 and C-6
positions of pyrimidine moiety modulates the antitubercular
activity of these conjugates. Cytotoxicity of selected com-
pounds was assayed against mammalian Vero cell line using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbro-
mide (MTT)-assay. The IC50 values were in μM concentra-
tion and are summarized in Table 1. The cytotoxicity data
revealed that the most potent compounds (8a and 8c) are
fairly non-toxic (Table 1) and also less cytotoxic than refer-
ence drug Emetine, although the cytotoxicity of the hybrids
was greater than 1. The ratios between cytotoxicity (IC50 in
μM) and in vitro antitubercular activity (MIC in μM) ena-
bled the determination of selectivity index (SI). The com-
pounds that exhibited SI values greater than 10 are consid-
ered non-toxic. Comparison of the selectivity index of potent
compounds also suggested that hybrids 8a and 8c had higher
SI values and were less cytotoxic. Thus, although none of
these conjugates was more active than the parent 1, more
structure diversifcation may lead to the refnement of the
molecular design and the attendant antitubercular activity.
The prodrug INH is activated by the heme (ferric)
enzyme catalase peroxidase (KatG) endogenous to M.
tuberculosis. Specifcally, upon activation with KatG, INH
is transformed into a potent antimycobacterial intermedi-
ate, which in combination with NADH forms an adduct
[44], which inhibits inhA (2-trans-enoyl-acyl carrier pro-
tein reductase) of M. tuberculosis. This leads to inhibition
of synthesis of mycolic acid, a major lipid of the bacterial
cell wall. However, the mechanism of activation is poorly
understood, partly because the binding interaction has not
been properly established. At the same time, mutation in
Phe254
Gln423
Glu118
Glu116
Fig. 3 Two- and three-dimensional docking poses of compound 8a
showing the interactions in the binding sites of bovine lactoperoxi-
dase (PDB ID: 3I6N)
KatG of M. tuberculosis is reported to be a major factor
of INH resistance. Consequently, most drug-resistant M.
tuberculosis strains are resistant to INH. Literature reports
[45] that INH is activated by mammalian lactoperoxidase
and mycobacterium KatG. Consequently, in this study, using
Schrodinger, LLC, New York, NY, 2019, we have conducted
docking studies of the most active member 8a of the series
with bovine lactoperoxidase (PDB ID: 3I6N) as well as a
cytochrome C peroxidase (PDB ID: 2V2E) mutant N184R
Y36A, as the active sites of the latter are very much similar
to KatG and are capable of activating INH. The pyrimidine
ring of the hybrid partner contributes in making π-stacking
interactions with Phe254 (ring-to-ring distance 5.36 Å). The
N2-H (next to CO) of the hydrazide moiety of INH unit
binds through hydrogen bonding to Glu118 (Glu118···H–N2:
2.64 Å) (Fig. 3), while N3–H of the hydrazide is hydrogen
bonded to Glu116 (Glu116-O···H–N3: 2.39 Å). Likewise,
N3 of the pyrimidine ring also makes hydrogen bonding
interaction with Glu116 (Glu116-O···H–N3-Pyr: 2.21 Å).
The ester carbonyl oxygen atom is hydrogen bonded with
a molecule of water (2.37 Å), the latter is further bonded,
1 3

Molecular Diversity
−4.348 and −5.732, respectively, while the Glide energies
were −27.699 and −23.605 kcal/mol, respectively.
Prediction of ADME parameters for all the members of
this series of hybrids was performed, and the results are
presented in Table S1 of Supporting Information. Except
QPlogHERG descriptor (prediction for HERG-K+channel
inhibition) and QPPMDCK for 8i and 8j, all other ADME
parameters were found to lie within the acceptable range.
In summary, a new series of INH–pyrimidine conjugates
were prepared as potential antitubercular conjugates against
H37Rv strains of M. tuberculosis. The compounds depicted
structure-dependent antitubercular activity. Two conjugates, 8a
and 8c, were identifed as the most active of this series. How-
ever, none of these compounds was more active than isoniazid,
and thus, more structure optimization is required for these new
conjugates. Cytotoxicity of selected compounds was assayed
against mammalian Vero cell line, and the most active members
were less cytotoxic than emetine used as the reference standard.
Heme
1.91Å
Asp146
Ser185
1.74Å
1.62Å
Arg184
Acknowledgements We gratefully acknowledge fnancial assistance
from CSIR, New Delhi (Project 02(0268)/16/EMR-II). We also thank
Ronnett Seldon and Dale Taylor for antimycobacterial and cytotoxicity
screening, respectively. KS thanks Schrodinger, India, for complimen-
tary license.
Author Contributions The manuscript was written through contribu-
tions of all authors. All authors have given approval to the fnal version
of the manuscript.
Fig. 4 Two- and three-dimensional docking poses of compound 8a
showing the interactions in the binding sites of cytochrome C peroxi-
dase (PDB ID: 2V2E) mutant N184R Y36A
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