Synthesis and biological evaluation of substituted N-alkylphenyl-3,5- dinitrobenzamide analogs as anti-TB agents

Article abstract of DOI:10.1039/c3md00366c

Here, a medicinal chemistry study of an N-alkylphenyl-3,5-dinitrobenzamide (DNB) scaffold as a potent anti-TB agent is presented. A series of chemical modifications were performed and forty-three new molecules were synthesized to study the structure-activity relationship (SAR) by evaluating against a sensitive strain (H₃₇Rv) of Mycobacterium tuberculosis (MTB). Potent DNB analogs 4b, 7a, 7c, 7d, 7j, 7r and 9a were further tested against resistant strains of MTB. Their intracellular as well as bactericidal potential was also evaluated. Cytotoxicity and in vivo pharmacokinetic studies suggested that DNB analogs have an acceptable safety index, in vivo stability and bio-availability. From the present work, two compounds 7a and 7d have shown nanomolar to sub micro-molar MIC in extracellular and intracellular assays.

Full text of DOI:10.1039/c3md00366c

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CONCISE ARTICLE
Synthesis and biological evaluation of substituted
N-alkylphenyl-3,5-dinitrobenzamide analogs as
anti-TB agents†
Cite this: DOI: 10.1039/c3md00366c
Gurunadham Munagala,ab Kushalava Reddy Yempalla,ab Sravan Kumar Aithagani,ab
Nitin Pal Kalia,c Furqan Ali,c Intzar Ali,c Vikrant Singh Rajput,ac Chitra Rani,ac
c
d
d
ac
*
Reena Chib, Rukmankesh Mehra, Amit Nargotra, Inshad Ali Khan,
ab
ab
*
*
Ram A. Vishwakarma
and Parvinder Pal Singh
Here, a medicinal chemistry study of an N-alkylphenyl-3,5-dinitrobenzamide (DNB) scaffold as a potent
anti-TB agent is presented. A series of chemical modifications were performed and forty-three new
molecules were synthesized to study the structure–activity relationship (SAR) by evaluating against a
sensitive strain (H37Rv) of Mycobacterium tuberculosis (MTB). Potent DNB analogs 4b, 7a, 7c, 7d, 7j, 7r
and 9a were further tested against resistant strains of MTB. Their intracellular as well as bactericidal
potential was also evaluated. Cytotoxicity and in vivo pharmacokinetic studies suggested that DNB
analogs have an acceptable safety index, in vivo stability and bio-availability. From the present work, two
compounds 7a and 7d have shown nanomolar to sub micro-molar MIC in extracellular and intracellular
assays.
Received 1st December 2013
Accepted 30th January 2014
DOI: 10.1039/c3md00366c
www.rsc.org/medchemcomm
used in conjunction with rst/second line anti-TB drugs
and other drugs used to treat secondary infections such as
cancer and HIV.1
Introduction
Tuberculosis (TB) remains a leading infectious disease world-
wide and infects about one-third of the world's population. The
World Health Organization (WHO) reported that TB caused
more than 8 million cases of illness and 1.4 million deaths
globally in 2011.1 Emergence of multidrug resistant TB (MDR-
TB) and extensively drug resistant TB (XDR-TB) has further
complicated the world situation.2–4 The 2012 WHO Global
tuberculosis report suggested that the situation is even worse in
India where the largest incidence of drug-resistant MTB has
appeared in the last two to three years. The current situation
necessitates the discovery and development of new anti-tuber-
culosis agents with low toxicity proles and having potency
against both drug-susceptible and drug-resistant MTB. In
addition to this, new chemical entities should be capable
of shortening the current duration of therapy and could be
Recently, the USFDA approved bedaquiline (also known as
TMC-207, developed by Johnson & Johnson), the rst drug
against MDR-TB which works by inhibiting ATP-synthase. The
approval of TMC-207 is being seen as a starting point in a new
era of TB treatment.5 Even though several other candidates such
as OPC-67683,6–8 PA-824,9,10 Moxioxacin,11 SQ109,12 Sutezolid,13
AZD5847 and Linezolid14 are presently being actively pursued in
Phase II/III clinical trials still there is a need for novel scaffolds
which should be effective against sensitive and resistant MTB.15
In search of novel anti-TB scaffolds, we initiated a discovery
aAcademy of Scientic and Innovative Research, CSIR-Indian Institute of Integrative
Medicine, Canal Road, Jammu 180 001, India. E-mail: ppsingh@iiim.res.in; ram@
iiim.res.in; iakhan@iiim.res.in; Fax: +91-191-2569333; Tel: +91-191-2569111
bMedicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal
Road, Jammu 180 001, India
cClinical Microbiology, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu 180 001, India
dDiscovery Informatics, CSIR-Indian Institute of Integrative Medicine, Canal Road,
Jammu 180 001, India
† Electronic supplementary information (ESI) available: Characterization data for
synthesized compounds and details regarding the pharmacokinetic studies are
provided. See DOI: 10.1039/c3md00366c
Fig.
1 Structure of known dinitrobenzamide (DNB) based anti-
tubercular agents.
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programme and a diverse library of 20 000 drug-like molecules resistant (RifR) strain of MTB (data given in Fig. 2). This scaffold
procured from the Chembridge were screened against the inhibits novel anti-Tb targets and is effective against both
H37Rv strain of Mycobacterium tuberculosis (MTB), wherein one sensitive and resistant strains of MTB, but unfortunately not
of the hit series contains an N-(alkyl/alkylphenyl)-3,5-dini- much work has been done on this scaffold except for Christophe
trobenzamide scaffold. A literature survey revealed that N- et al.21 ndings where very closely related N-(substituted)-3,5-
(substituted)-3,5-dinitrobenzamide derivatives (A, Fig. 1) were dinitrobenzamide derivatives of formula C were evaluated in
known to have anti-TB activity16 and recently it was reported detail for anti-Tb activity. In this direction, we have started a
that the nitrobenzamide derivatives A, B and C resemble a very medicinal chemistry programme around the N-(alkyl/alkyl-
potent anti-TB candidate BTZ043 D in their mode of action and phenyl)-3,5-dinitrobenzamide scaffold and a series of chemical
inhibit the same target, heterodimeric decaprenyl-phosphor- modications were made to understand the SAR by evaluating
ibose-20-epimerase (DprE1).17–20 From our screening of the the synthesized compounds for anti-TB potential.
20 000 Chembridge library, eleven compounds containing the
N-(alkyl/alkylphenyl)-3,5-dinitrobenzamide skeleton showed
potent MIC against both sensitive (H37Rv) as well as rifampicin-
Results and discussion
Among the 11 identied hits, di-nitrobenzamide (DNB) analogs
3a and 4b have shown potent MIC against sensitive H37Rv and
RifR MTB. In order to re-validate these results, we rst synthe-
sized the most active hit 4b following Scheme 122 and conrmed
the activity results. We designed a medicinal chemistry strategy
for structural modication as presented in Fig. 3. As the most
potent hit 4b contains a chiral centre, we synthesized both
optically pure R- and S-enantiomers by coupling benzoyl chlo-
ride 6 with optically pure amines (Fig. 4). Between R- and S-
enantiomers, R-enantiomer 7a has shown more potent MIC
(0.09 mM) as compared to S-enantiomer 7b (11.66 mM).23
To know the effect of substituents present on the aromatic
ring B, a series of optically pure (both R- and S-) analogs from
corresponding un/substituted (F, Cl, OMe) amines 7c–i were
synthesized and tested against the H37Rv strain of MTB. MIC
data revealed that R-enantiomers (7e and 7g) were more active
than S-enantiomers (7f and 7h) except for uoro substituted
Scheme 1 Synthesis of the most active hit 4b.
Fig.
2 Hits identified from screening of 20 000 small drug-like
molecules.
Fig. 3 Strategy for functional SAR.
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ethyl 7t–u and n-propyl 7v diminished the activity (Fig. 4). This
activity pattern suggested that methyl is more preferable over
other alkyl groups.
One of the identied potent hit 3a contains an alkyl chain
instead of ring B and in order to know the role of aromatic ring
B, a series of six compounds 8a–f were synthesized wherein un/
substituted alkyl amines were placed instead of substituted
benzylamine (Fig. 5). None of the N-alkyl containing
compounds was found to be better than the N-benzyl contain-
ing compounds.
Furthermore, the effects of chiral centre and length of the
alkyl chain between N-atom and aromatic ring B were also
studied and a series of compounds 9a–h and 10a–e were
synthesized (Fig. 6 and 7). Activity data revealed that the
Fig. 5 Synthesis and screening result of N-alkyl DNB analogs 8(a–f).
Fig. 4 Synthesis and screening result of N-benzyl DNB analogs (a–v).
analogs where S-enantiomer 7c was more active than R-enan-
tiomer 7d. Among all these six analogs, both enantiomers of
uoro-substituted DNB analogs 7c and 7d had shown an MIC of
0.17 and 0.04 mM, respectively. The activity result suggested that
the presence of substituents on ring B plays a critical role in
modulating the anti-TB activity.
To know the role of the methyl group at ring A, we synthe-
sized a series of DNB analogs 7j–s without the methyl substit-
uent but with varying substituents at ring B and tested (the
result is given in Fig. 4). Again, R-enantiomers were found to be
more active than S-enantiomers except for uoro and methoxy
substituted analogs. Among all these synthesized analogs 7j–s,
three compounds 7j, 7n and 7r had shown a potent MIC of 0.05
mM, 0.17 mM and 0.19 mM, respectively. The activity results
suggested that the presence of the methyl group at aromatic
ring A is not very critical. On the other hand, replacement of the
Fig. 6 Synthesis and screening result of N-alkylphenyl DNB analogs
methyl group at the chiral centre with other groups such as 9(a–h).
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Fig. 7 Synthesis and screening result of N-phenyl DNB analogs
10(a–e).
Fig. 9 Screening of active hits against resistant strains.
presence of alkyl is also critical and the absence or increase in
the chain length lowers the activity. We also synthesized N,N-di-
substituted compound 11a which showed an MIC value of 1.52
mM (Fig. 8). The presence of N,N-di-substitution also lowers the
activity.
Among all the tested compounds, seven potent DNB analogs
4b, 7a, 7c, 7d, 7j, 7r and 9a were further screened against
rifampicin-resistant (RifR), isoniazid-resistant (INHR) and
multi-drug resistant (MDR) strains of MTB23 and results are
shown in Fig. 9. Four DNB analogs viz., 7a, 7d, 7j and 7r had
shown an MIC of <0.05 mM against RifR MTB and one derivative
7j had shown an MIC of <0.05 mM against INHR MTB. Two DNB
derivatives 7a and 7d had also shown a potent MIC against the
MDR strain of MTB.
Fig. 10 Bactericidal, intracellular and safety potential of active hits.
DNB analogs have shown bactericidal potential at their MIC
value (Fig. 10). As we know, tubercle bacilli survive and
multiply within macrophages and killing the intracellular
tubercle bacilli is the key requirement for efficient tubercu-
losis treatment, the seven DNB analogs were screened for
intracellular killing of MTB inside the macrophages.24 Five
DNB derivatives 4b, 7c, 7d, 7j and 7r have shown a sub-
micromolar MIC of 0.72, 0.71, <0.64, 0.80 and 0.76 respectively
in intracellular assay (Fig. 10).
The cytotoxic effects of seven potent DNB analogs have been
determined on the macrophage J-774 cell line25 (Fig. 10),
wherein none of the compounds had shown any cytotoxicity at
20 mg mlꢀ1 which suggested their high safety index.29
Fig.
analog 11a.
8 Synthesis and screening result of N,N-disubstituted DNB
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between N-atom and ring B decreased the activity, (iv) at the
chiral centre, the methyl group is preferred over H, Et- and n-
propyl groups and (v) the presence of N,N-disubstitution is not
preferred. In the case of resistant strains, DNB analogs having
the Me-group at the benzylic position are preferred. Moreover,
the SAR studies presented here also pave a way towards the
designing of more robust and potent analogs with a better PK
prole.
Conclusion
In summary, we have presented a rst medicinal chemistry
attempt towards the exploration of N-alkylphenyl-3,5-dini-
trobenzamide analogs as potent anti-TB agents. The DNB
derivatives possessed sub-micromolar to nanomolar potency
not only against extracellular (both sensitive and resistant MTB)
but also against intracellular assay (within macrophage). DNB
analogs also have an acceptable safety index. In vivo pharma-
cokinetic studies also suggested an interesting PK prole and
absolute intraperitoneal bioavailability. Presently, the efforts
towards the replacement of the nitro-group with other isosteres
as well as detailed investigation of this class of compounds is
undergoing in our institute and will be published in due course.
Fig. 11 In vivo PK studies of DNB derivatives 7a and 7d.
Authors' contribution
PPS, IAK and RAV participated in the design and execution of
this study and moreover draing of the manuscript was done by
PPS. GM, KR and SKA performed the chemical syntheses. NPK,
FA, IA, VR, CR and RC performed and interpreted bio-activity
data. RM and AN helped in the selection of the 20 000 drug like
commercial library by selecting various lters of drug-likeness
with the help of bio-informatics tools.
Fig. 12 Structure–activity relationship of DNB analogs wrt the H37Rv
strain of MTB.
In order to know the in vivo stability, in vivo pharmacoki-
netics (PK) of two potent hits 7a and 7d were evaluated in male
Balb mice via intraperitoneal (IP) and intravenous (IV) routes of
administration at a dose of 2.5 and 1 mg kgꢀ1, respectively
(Fig. 11). PK study revealed that both the compounds achieved
maximum plasma concentration Cmax within half-an-hour and
is comparatively higher than their in vitro MIC value but their
half-lives were found to be short. Both the DNB derivatives had
shown good absolute intraperitoneal bioavailability (7a, 49%;
7d 35%, detailed discussion and PK parameters are provided in
part B of the ESI†). This is the rst report regarding the phar-
macokinetic (PK) studies of this class of compounds which
suggests that the DNB scaffold is quite stable under in vivo
conditions. We are presently pursuing the studies towards the
oral PK-properties and other pre-clinical parameters that will be
reported in due course.
Acknowledgements
Funding was provided by CSIR-OSDD with research grant # HCP
0001. GM, KR, SKA, IA, VR, CR and NPK thank CSIR, UGC and
ICMR for their Fellowship. IIIM Communication no. IIIM/1578/
2013.
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– general procedure: to a solution of acid 5 (2.6 mmol) in
benzene (10 ml) thionyl chloride (2 ml of a 2.0 M CH2Cl2
solution; 4.0 mmol) was added. The reaction was stirred
under reux for 5 h and concentrated to give crude acid
chloride as a yellow solid. This yellow solid was dissolved
in dry CH2Cl2 (10 ml) and then added dropwise to the
corresponding anilines (2.6 mmol) in pyridine at 0 ꢁC.
Aer being stirred at room temperature for 4 h, the
reaction mixture was concentrated. The resulting mixture
was partitioned between ethyl acetate and water, the
organic layer was separated and washed with brine, dried
over sodium sulphate and ltered. The ltrate was
concentrated under reduced pressure. The residue was
puried by silica gel column chromatography using
EtOAc–hexane as eluent and characterized by NMR and
mass spectroscopy (details along with spectra are given in
the ESI).†
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That
Covalently
Modify
the
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USA), M. tuberculosis MDR (resistant to isoniazid and
rifampicin), M. tuberculosis INHR (resistant to isoniazid)
and M. tuberculosis RifR (resistant to rifampicin) using the
micro-broth dilution method.26,27 The bacterial strains were
grown for 10 to 15 days in Middlebrook 7H9 broth (Difco
Laboratories, Detroit, Mich.) supplemented with 0.5% (v/v)
glycerol, 0.25% (v/v) Tween 80 (Himedia, Mumbai, India),
and 10% ADC (albumin dextrose catalase, Becton
MIC to 8XMIC were added in the media and the MTB
infected cell lines were incubated at 37 C with 5% CO2 for
ꢁ
4 days with a daily change of compounds containing
media. Intracellular bacteria, which have been protected
from this treatment, were quantied by host-cell lysis in a
mild detergent such as 0.25% SDS, and plated on to
Middlebrook 7H10 agar supplemented with 0.5% (v/v)
glycerol, 0.25% (v/v) Tween 80, and 10% OADC at
ꢁ
Dickinson, Sparks, MD) under shaking conditions at 37 C
incubated at 37 ꢁC for
3 weeks for viable colony
in 5% CO2 to facilitate exponential-phase growth of the
formation.28 The activity of the compound was dened as
the minimum concentration of the compound exhibiting
2-log killing of intracellular MTB with respect to the
untreated control.
organism.
A bacterial suspension was prepared by
suspending M. tuberculosis growth in normal saline
containing 0.5% Tween 80 and turbidity was adjusted to 1
McFarland standard which is equivalent to 1.0 ꢂ 107 CFU 25 Cytotoxicity assay: the cyto-toxicities of the synthesized
mlꢀ1. The 2-fold serial dilutions of compounds were
compounds were evaluated for mouse macrophage (J-774)
cell lines using MTT assay, in a 96 well plate format. Cells
were incubated in Roswell Park Memorial Institute (RPMI)
containing 10% fetal calf serum (FCS) and supplemented
with 75 mg per litre penicillin, 100 mg per litre
streptomycin, 110 mg per litre sodium pyruvate, 2.38 g per
litre HEPES, 0.05 mM 2 b-mercaptoethanol, and 2 g per
litre NaHCO3. Ten 2-fold serial dilutions of N-alkylphenyl-
3,5-dinitrobenzamide (DNB) analogs starting from 20 mg
mlꢀ1 were added in the plate, and the plate was incubated
prepared in Middle brook 7H9 (Difco laboratories) for M.
tuberculosis in100 ml per well in 96-well
U bottom
microtitre plates (Tarson, Mumbai, India). The above-
mentioned bacterial suspension was further diluted 1 : 50
in the growth media and 100 ml volume of this diluted
inoculum was added to each well of the plate resulting in
the nal inoculum of 1.0 ꢂ 106 CFU mlꢀ1 in the well and
the nal concentrations of compounds ranged from 0.015
to 32 mg mlꢀ1. The plates were incubated at 37 ꢁC for 3-
weeks in 5% CO2. The plates were read visually and the
minimum concentration of the compound showing no
turbidity was recorded as MIC. Minimum bactericidal
concentration (MBC) was determined by plating 10 ml
volume from each well showing no turbidity on
Middlebrook 7H10 agar supplemented with 0.5% (v/v)
glycerol, 0.25% (v/v) Tween 80, and 10% OADC (oleic acid,
albumin dextrose catalase, Becton Dickinson, Sparks, MD,
USA).
ꢁ
for 24 h at 37 C in a CO2 incubator. Aer the completion
of
incubation
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) was added and cells
ꢁ
were further incubated for 3 h at 37 C in a CO2 incubator.
Formation
of
formazan
salt
by
mitochondrial
dehydrogenases and was determined by using a Elisa
reader at 450 nm (Multiskan Spectrum; Thermo Electron
Corporation, USA). The percentage cytotoxicity was
calculated with respect to the untreated cells.
24 Intracellular killing activity: The intracellular killing potential 26 R. Maccari, R. Ottana, F. Monforte and M. G. Vigorita, In
of the N-alkylphenyl-3,5-dinitrobenzamide (DNB) analogs
was determined on macrophage J774 cell lines. The cells
were grown and maintained using RPMI media
Vitro Antimycobacterial Activities of 2-Monosubstituted
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supplemented with 10% foetal calf serum (FCS). About 106 27 R. J. Wallace, D. R. Nash, L. C. Steele and V. Steingrube,
macrophage cells were cultured in each well of a 24 well
plate and kept at 37 ꢁC with 5% CO2 for 24 h. Aer
formation of monolayer cells and before transfection,
Susceptibility testing of slowly growing mycobacteria by a
microdilution MIC method with 7H9 broth, J. Clin.
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macrophage cells were washed with growth medium 28 A. Kalita, I. Verma and G. K. Khuller, Role of human
without antibiotics and kept for 1 h at 37 ꢁC. The
monolayer was transfected with M. tuberculosis H37Rv at a
multiplicity of infection (MOI) of 10. Plates were incubated
neutrophil peptide-1 as
antituberculosis chemotherapy, J. Infect. Dis., 2004, 150,
a
possible adjunct to
1476.
at 37 ꢁC with 5% CO2 for 2 h. The media was decanted 29 G. C. Mangalindan, M. T. Talaue, L. J. Cruz, S. G. Franzblau,
and fresh RPMI media containing amikacin 50 mg mlꢀ1
was added and the plate was incubated at 37 ꢁC with 5%
CO2 for 48 h. The antibiotic supplemented media was
changed daily. Serial dilutions of the test compounds from
L. B. Adams, A. D. Richardson, C. M. Ireland and
G. P. Concepcion, Agelasine F from a Philippine Agelas sp.
Sponge Exhibits In Vitro Antituberculosis Activity, Planta
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