Design, synthesis, anti-mycobacterial and cytotoxic evaluation of C-4 functionalized 1,8-naphthalimide-heterocyclic hydrazide conjugates

Article abstract of DOI:10.1111/cbdd.13503

This manuscript discloses the design and synthesis of a series of C-4 functionalized 1,8-naphthalimide-heterocyclic hydrazide conjugates along with their anti-mycobacterial and cytotoxic evaluation. The present work assumes significance as it describes the first report on the amalgamation of C-4 substituted naphthalimides with various heterocyclic hydrazides. However, contrary to the rationale behind the synthesis of the conjugates, none of them inhibited the growth of Mycobacterium tuberculosis at the tested concentrations though these were non-cytotoxic towards the Vero kidney epithelial cell line.

Full text of DOI:10.1111/cbdd.13503

DR VIPAN KUMAR (Orcid ID : 0000-0002-6164-7161)
Article type : Research Article
Design, Synthesis, Anti-mycobacterial and Cytotoxic evaluation of C-4
functionalized 1,8-naphthalimide-heterocyclic hydrazide conjugates
Corresponding author mail-id: vipan_org@yahoo.com
Shalinia, Matt D. Johansenb, Laurent Kremerb,c, Vipan Kumar*a
a Department of Chemistry, Guru Nanak Dev University, Amritsar-143005, Punjab, India
b Institut de Recherche en Infectiologie de Montpellier (IRIM), CNRS, UMR 9004 Université de Montpellier, France
c
INSERM, IRIM, 34293 Montpellier, France
Abstract: This manuscript discloses the design and synthesis of a series of C-4 functionalized 1,8-
naphthalimide-heterocyclic hydrazide conjugates along with their anti-mycobacterial and cytotoxic
evaluation. The present work assumes significance as it describes the first report on the amalgamation
of C-4 substituted naphthalimides with various heterocyclic hydrazides. However, contrary to the
rationale behind the synthesis of the conjugates, none of them inhibited the growth of Mycobacterium
tuberculosis at the tested concentrations though these were non-cytotoxic towards the Vero kidney
epithelial cell line.
Keywords: Naphthalimides, Isoniazid, Heterocyclic hydrazides, Anti-mycobacterial, Cytotoxicity.
Introduction
Tuberculosis (TB) is an infectious disease caused by the most dangerous microbial pathogen
Mycobacterium tuberculosis (Mtb). It is the ninth leading cause of death worldwide, ranking
above HIV/AIDS. In 2017, 10.0 million new cases of TB were reported and there were 1.3
million deaths among HIV-negative people, and an additional 0.3 million deaths from TB
among HIV-positive people. This disease has engulfed many countries with strong evidence
showing African and Asian continents to be worst affected by TB along with the cases of TB-
HIV co-morbidity among patients.[1] Current treatment encompasses varied drugs classified
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into different categories, with the most common treatment regime consisting of a combination
of four first line drugs namely Isoniazid, Pyrazinamide, Rifampicin and Ethambutol. Along
with this, second line and third line drugs (Streptomycin, Ofloxacin, Kanamycin,
Capreomycin, Amikacin, Ethionamide, Linezolid, Cycloserine) are also in the queue to
combat TB.[2] There are 17 drugs in Phase I, II or III clinical trials, including eight new
compounds, two drugs that have received conditional regulatory approval based on Phase IIb
results.[3] Among these molecules, Delamanid and Bedaquiline are proved to be effective
drugs for multidrug-resistance (MDR)-TB patients and has been approved by U.S Food and
Drug Administration (FDA) in 2012 and by European Medicine Agency (EMA) in 2014, with
some countries having already started using them as shorter regimens for TB treatment.[4]
However, the nature of Mtb to become latent and resistant to available drugs has highlighted
TB as a global health emergency.[1] Consequently, there is a critical need to synthesize and
introduce more effective molecules to cure and eliminate TB.
Isoniazid (isonicotinic hydrazide, INH), because of high activity and low cost, has remained a
frontline drug in anti-TB chemotherapy.[5] However Mtb has developed resistance towards INH owing
to its conversion to N-acetylisoniazid by the enzyme N-acetyltransferase (NAT), which is present both
in humans and mycobacteria. This metabolite lacks anti-mycobacterial property that eventually leads
to INH-resistance.[6] A number of derivatives and hybrids of INH have been reported where free NH2
has been protected to avoid N-acetylation.[7] Likewise, other hydrazides viz nicotinic hydrazides and
pyrazine-2-carbohydrazides have also been tested against Mtb, and the studies showed that their anti-
mycobacterial activity can be improved by hybridization with other pharmacophoric units. Thus these
hydrazides can be good precursors to produce a framework with better anti-tubercular potency.[8]
1,8-naphthalimide is an important heterocyclic framework that has received special attention
due to its crucial occurence in bioactive compounds and drugs in clinical trials. This recognition in
medicinal application is due to the -deficient conjugated planar structure that enables naphthalimide
derivatives to readily interact with various biological cations, anions, small molecules and
macromolecules like DNA, enzymes & receptors via non-covalent bonds. Furthermore, substitution
pattern on 1,8-naphthalimide central fragment determines its biological activity and 4-amino
substituent has proved to be critical for several chemical & biological applications.[9] Currently, some
naphthalimides like amonafide and mitonafide are used in clinical trials as anti-cancer agents while a
recent report has presented a combined in silico, in vitro and in vivo studies which revealed the anti-
tubercular potential of naphthalimides.[10] Moreover, several reports have also shown that
naphthalimide derivatives could effectively inhibit the growth of micro-organisms and possess vast
potential in the treatment of bacterial and fungal infections, further confirming the possibility of
naphthalimide utilisation in the medical field.[11] In particular, naphthalimide-heterocyclic ring
conjugates exhibited better antimicrobial activities against different bacterial strains compared to
reference drugs, chloromycin and norfloxacin.[12]
In the field of TB research & development, due to an increase in the number of
MDR/TDR strains, novel medical strategies focussing on the introduction of new molecules,
repurposing and derivatization of present drugs have been developed. In continuation,[13] the
present work describes design and synthesis of a library of hybrids of 1,8-naphthalimide with
three types of hydrazides viz isonicotinic hydrazide (INH), nicotinic hydrazide and pyrazine-2-
carbohydrazide in order to check their anti-mycobacterial potency. Our rationale considered
two major factors in order to have a potent anti-TB entity and is based on synergistic effect by
two pharmacophoric units: (a) clubbing naphthalimide with a nitrogen heterocycle (hydrazides
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of nicotinic acid, isonicotinic acid, pyrazinecarboxylic acid) that eventually brings possibility
of improving its anti-bacterial property and (b) protection of NH2 group of hydrazides by
introducing naphthalimide ring so as to evade N-acetylation, Figure 1. The further induction
of the alicyclic secondary amines into the designed hybrids is inspired from their presence in
various potent marketed anti-microbial drugs.[12d]
Results and Discussion:
The first set of target compounds 3a-e were synthesized via an initial heating of
1 and 2a
dissolved in minimum amount of anhydrous N-methyl-2-pyrrolidone (NMP) as solvent at
1500C for 1 h affording 3a and 3b. The compound 3b (R=Br) was used as precursor for the
synthesis of further conjugates 3c-e via heating 3b with secondary amine (piperidine/
morpholine/ hydroxyethyl piperazine) in anhydrous NMP at 1800C for 30 min, Scheme 1. The
structure of 3b was assigned on the basis of spectral data and analytical evidences. The
compound 3b, for example, showed a molecular ion peak at m/z 395.9941 [M+1]+ in its
1
1
HRMS along with characteristic signals in H and 13C NMR spectra. H NMR spectrum
exhibited a doublet at 8.87 (J=5.8 Hz) and a doublet of doublet at 7.92 (J=1.6, 4.5 Hz)
corresponding to the isonicotinyl ring protons along with a pair of doublets at 8.29 (J=7.8


Hz) and 8.43 (J=7.8 Hz) representing naphthalimide ring protons. Appearance of peaks at
161.53, 161.57 and 164.35 corresponding to carbonyl carbons in 13C-NMR spectra further
corroborated the assigned structure.
In order to study structure-activity relationship of the target compounds, different
electronic environments were created by using varied types of reagent
target compounds viz 4a-e was synthesized by initially reacting and 2b and further following
afore mentioned procedures, Scheme 2. The structure of was again assigned on the basis of
2. Another set of the
1
4
spectral data and analytical evidences. Taking one of the derivative 4d, which showed
molecular ion peak at m/z 403.1381 [M+1]+ in its HRMS along with characteristic signals in
13
1
1H & C NMR spectrum. H NMR showed singlet at

9.13, a doublet of doublet at
(J=4.8, 7.9 Hz) corresponding to nicotinyl ring protons and a pair of doublets at 7.36 (J=8.2
Hz) and 8.44 (J=8.2 Hz) showing the presence of substituted naphthalimide ring protons. Its
13C NMR spectra showed characteristic absorptions at
161.81, 162.38 and 164.43 because of
carbonyls along with desired signals in aliphatic and aromatic regions
 7.60


.
In the same way, compounds 7a-e were synthesized by initially reacting 1 and 2c. While 2c, in
turn was prepared by esterification of 5 (commercially available) in dry CH3OH with catalytic amount
of conc. H2SO4 to get 6 which was further treated with hydrazine hydrate in ethanol [14], Scheme 3.
The compound 7a, showed molecular ion peak at m/z 319.0791 [M+1]+ and its 1H NMR showed two
multiplets at  7.90 and 8.53 corresponding to unsubstituted naphthalimide ring protons and two
doublets at  8.98 (J=2.48 Hz) and 9.23 (J=1.48 Hz) corresponding to pyrazinyl ring protons. 13C
NMR spectra showed peaks at  162.00 and 162.49 due to carbonyls along with requisite signals in
aromatic region.
The synthesized conjugates 3a-e, 4a-e and 7a-e along with their precursors were then evaluated
for their anti-mycobacterial activities against Mtb strain mc26230 keeping standard anti-TB drug INH
as a reference and the corresponding minimum inhibitory concentration (MIC99) values are enlisted in
Table 1. As evident from the data, all the compounds showed MIC99 values >200 g/mL. The whole
library of the synthesized hybrids was screened for their cytotoxicity against Vero kidney epithelial
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cell line and the results obtained are summarized in Table 1. As shown, cytotoxicity is described in
terms of inhibitory concentration (IC50) values. The data revealed that most of the conjugates were
non-cytotoxic (except 4a, 4b, 4c, 7c, 7e) with IC50 values >100 g/mL. Selectivity Index (SI) was also
calculated as a function of the IC50/MIC. Most of the compounds showed SI values >0.5 which was
low as compared to the reference drug, Isoniazid (SI >10,000).
Conclusion
In conclusion, we herein have synthesized C-4 substituted 1,8-naphthalimide-heterocyclic hydrazide
conjugates. The library of fifteen hybrids along with precursors was tested against Mtb strain
mc26230. The evaluation data revealed that although most of the scaffolds were non-cytotoxic against
the normal Vero kidney epithelial cell line, the amalgamation failed to elicit any favourable activity
from naphthalimide-nicotinic hydrazide/ pyrazine-2-carbohydrazide hybrids. Further, loss of activity
was observed among naphthalimide-Isoniazids prompting the need to optimize the chemistry during
the hybridization of these pharmacophoric units in an effort to maintain/improve the desired
properties.
Acknowledgements
The valuable support and financial assistance for this research by the Department of Science
and Technology, (DST) under the INSPIRE fellowship programme (Shalini, IF 160180) is
greatly acknowledged. Science and Engineering Research Board (SERB), New Delhi was
acknowledged by VK for financial assistance with grant no. EMR/2015/001687. MDJ
received financial support for his post-doctoral fellowship granted by Labex EpiGen Med, an
« Investissements d’avenir » program, (reference ANR-10-LABX-12-01).
Electronic Supplementary Information: General experimental section along with detailed
procedures of the synthesized hybrids and spectral data of all the compounds (3a-e, 4a-e, 7a-e) along
with scanned 1H and 13C NMR spectra of few representative compounds viz. 3a, 3b, 3c, 4c, 4d, 4e,7a,
7c are provided in the supplementary information file.
Conflicts of interest
The authors declare no conflict of interest
Figure 1: Rationale behind the design of target compounds taking one hybrid as a model structure.
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Scheme 1: Synthesis of naphthalimide-isonicotinic hydrazide (Isoniazid) hybrids (3a-3e).
Scheme 2: Synthesis of naphthalimide-nicotinic hydrazide hybrids (4a-4e).
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Scheme 3: Synthesis of naphthalimide-pyrazin-2-carbohydrazide hybrids (7a-7e).
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Table 1: In vitro anti-tubercular activities and cytotoxic evaluation of compounds (2b, 2c , 3a-e, 4a-e,
7a-e,) against mc26230 strain of M. Tuberculosis and Vero kidney epithelial cell line respectively.
Compound
MIC99
g/mL
IC50
g/mL)
Selectivity
Index (SI)
(
)
(
2b
2c
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
0.01
>100
>100
>100
>100
>100
>100
>100
64.79
59.67
47.64
>100
>100
>100
>100
45.7
>0.5
>0.5
3a
3b
3c
>0.5
>0.5
>0.5
3d
3e
>0.5
>0.5
4a
4b
4c
0.32395
0.29835
0.2382
>0.5
4d
4e
>0.5
7a
7b
7c
>0.5
>0.5
0.2285
>0.5
7d
7e
>100
71.05
>100
0.35525
>10,000
INH
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