5-Nitro-2-furoic acid hydrazones: Design, synthesis and in vitro antimycobacterial evaluation against log and starved phase cultures

Article abstract of DOI:10.1016/j.bmcl.2010.06.096

Various 5-nitro-2-furoic acid hydrazones were synthesized and evaluated for in vitro activities against log and starved phase culture of two mycobacterial species and Mycobacterium tuberculosis (MTB) isocitrate lyase (ICL) enzyme inhibition studies. Among twenty one compounds, 5-nitro-N′-[(5-nitro-2- furyl)methylidene]-2-furohydrazide (4o) was found to be the most active compound in vitro with MICs of 2.65 and 10.64 μM against log- and starved-phase culture of MTB. Compound 4o also showed good enzyme inhibition of MTB ICL at 10 μM. The docking studies also confirmed the binding potential of the compounds at the ICL active site.

Full text of DOI:10.1016/j.bmcl.2010.06.096

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4313–4316
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Nitro-2-furoic acid hydrazones: Design, synthesis and in vitro
antimycobacterial evaluation against log and starved phase cultures
*
Dharmarajan Sriram , Perumal Yogeeswari, Devambatla Ravi Kumar Vyas, Palaniappan Senthilkumar,
Pritesh Bhat, Madala Srividya
Medicinal Chemistry & Antimycobacterial Research Laboratory, Pharmacy Group, Birla Institute of Technology & Science—Pilani, Hyderabad Campus, Jawahar Nagar,
Hyderabad—500 078, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Various 5-nitro-2-furoic acid hydrazones were synthesized and evaluated for in vitro activities against
log and starved phase culture of two mycobacterial species and Mycobacterium tuberculosis (MTB) isoci-
trate lyase (ICL) enzyme inhibition studies. Among twenty one compounds, 5-nitro-N0-[(5-nitro-2-
furyl)methylidene]-2-furohydrazide (4o) was found to be the most active compound in vitro with MICs
Received 1 April 2010
Revised 9 June 2010
Accepted 17 June 2010
Available online 20 June 2010
of 2.65 and 10.64 lM against log- and starved-phase culture of MTB. Compound 4o also showed good
enzyme inhibition of MTB ICL at 10
the compounds at the ICL active site.
l
M. The docking studies also confirmed the binding potential of
Keywords:
Antitubercular activity
Mycobacterium tuberculosis
5-Nitrofuranyl derivatives
Dormant tuberculosis
Ó 2010 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis (MTB) infects about 32% of the
world’s population. Every year, approximately 8 million of these
infected people develop active tuberculosis (TB) and almost 2 mil-
lion of these will die from the disease (WHO, 2005).1 Despite
40 years of anti-TB chemotherapy, tuberculosis remains one of
the leading infectious diseases worldwide. Among the main obsta-
cles to the global control of the disease are the HIV epidemic that
has dramatically increased the risk for developing active TB, the
increasing emergence of multi-drug resistant TB (MDR-TB) and
the recalcitrance of persistent infections to treatment with conven-
tional anti-TB drugs.2 The situation is exacerbated by the increas-
ing emergence of extensively drug-resistant (XDR) TB.3 Current
chemotherapy for TB largely relies on drugs that inhibit bacterial
metabolism with a heavy emphasis on inhibitors of the cell wall
synthesis.4 According to their mode of action, first and second line
TB drugs can be grouped as cell wall inhibitors (isonizid (INH), eth-
ambutol, ethionamide, cycloserine), nucleic acid synthesis inhibi-
tors (rifampicin (RIF), quinolones), protein synthesis inhibitors
(streptomycin, kanamycin) and inhibitors of membrane energy
metabolism (pyrazinamide). Existing TB drugs are therefore only
able to target actively growing bacteria through the inhibition of
cell processes such as cell wall biogenesis and DNA replication.
This implies that current TB chemotherapy is characterized by an
efficient bactericidal activity but an extremely weak sterilizing
activity, defined as the ability to kill the slowly growing or slowly
metabolizing bacteria that persist after the growing bacteria have
been killed by bactericidal drugs. Sterilizing activity also describes
the ability to eliminate latent or ‘dormant’ bacteria that survive in-
side the host macrophages. This bias is hardly surprising as anti-TB
drugs have traditionally been identified by their ability to suppress
or kill replicating cultures of bacteria in vitro. The weak sterilizing
property of available TB drugs is one of the major drawbacks for
current TB chemotherapy.
Recently we reported some 5-nitrofuran-2-yl derivatives5 and
2-[3-(4-bromo-2-fluorobenzyl)-4-oxo-3,4-dihydro-1-phthalazinyl]-
acetic acid hydrazones6 with activity against both dormant and ac-
tively growing MTB. Tangallapally et al., reported some novel
nitrofuranyl amides with potent anti-TB activity.7 In this work
we have designed (Fig. 1) and synthesized novel 5-nitrofuranyl-
2-acid hydrazones and screened their activity against both growing
and dormant mycobacterial species.
5-Nitrofuroic acid (1) is converted to its ethyl ester (2) by
refluxing the acid 1 in absolute ethanol in presence of sulfuric acid.
Ethyl ester (2) (1 mol) on treatment with hydrazine hydrate
(2 mol) yielded acid hydrazide (3). This hydrazide which upon
reacted with various carbonyl compounds in presence of glacial
acetic acid at a pH 4–6 afforded the titled compounds 4a–u
(Scheme 1) in 52–89% yields.6 The reaction utilizes the microwave
irradiation in an unmodified domestic microwave oven at 80%
intensity with 30 s/cycle for 3 min. The resultant solid was washed
with dilute ethanol dried and recrystallized from ethanol–
chloroform mixture. The purity of the synthesized compounds
* Corresponding author.
E-mail addresses: dsriram@bits-hyderabad.ac.in, drdsriram@yahoo.com (D.
Sriram).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.096

4314
D. Sriram et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4313–4316
O
NO2
N
N
H
H
HN
N
N
CH2
Br
N
N
O
O
O2N
2: MIC: 0.0125 microgram/mL
O
F
1: MIC: 0.18 micromole
R1
R
NH
O
N
O
O2N
Figure 1. Development of 5-nitrofuranyl-2-acid hydrazones.
R
b
c
a
CONHN
CONHNH2
COOC2H5
COOH
R1
O
O
3
O
O
O2N
O2N
4a-u
O2N
O2N
2
1
Scheme 1. Reagents: (a) H2SO4, C2H5OH; (b) NH2NH2ꢁH2O; (c) CH3COOH.
was monitored by thin layer chromatography (TLC) and elemental
analyses and the structures were identified by spectral data.8
In the first phase the compounds were screened for their
in vitro antimycobacterial activity against log-phase cultures of
MTB, and Mycobacterium smegmatis ATCC 14468 (MS) by agar dilu-
tion method similar to that recommended by the National Com-
mittee for Clinical Laboratory Standards9 for the determination of
MIC in duplicate. The minimum inhibitory concentration (MIC) is
defined as the minimum concentration of compound required to
give complete inhibition of bacterial growth. MICs of the synthe-
sized compounds along with the standard drugs for comparison
are reported (Table 1). In the initial screening against log-phase
MTB, the newer compounds showed good activity with MICs rang-
and 2.7 times more potent than INH and RIF, respectively, against
log-phase culture of MC2.
The compounds which showed good activity against log-phase
culture of MTB and MC2 were further screened against six-week-
starved cells of MTB and MC2 according to the literature procedure
in duplicate and MICs were reported in Table 1.11 Several in vitro
model systems have been proposed to mimic the conditions found
in the human chronic tuberculosis lesion (a granuloma), including
oxygen starvation12 nutrient deprivation13 and rifampicin-induced
persistence.14 Development of a screen under carbon-starvation
conditions is feasible and less challenging than for oxygen depriva-
tion. Prolonged deprivation of nutrients results in a marked slow-
ing of bacterial growth and concomitant phenotypic antibiotic
resistance.15 As bacteria can easily grow upon being returned to
nutrient-rich media, this model allows easy quantification of anti-
biotic effectiveness. Against MTB, nine compounds were tested and
they inhibited starved culture of MTB with MIC’s ranging from
ing from 2.65 to 48.22
l
M. Two compounds (4k and 4o) showed
M. When compared to INH
(MIC: 0.72 lM) and RIF (MIC: 0.48 lM) all the compounds were
excellent activity with MIC of <5
l
less active. Compound 5-nitro-N0-[(5-nitro-2-furyl)methylidene]-
2-furohydrazide (4o) was found to be the most active compound
10.64 to 91.49
lM (Table 1). INH and RIF had poor activity against
in vitro with MIC’s of 2.65
lM against MTB. With respect to struc-
starved cells with MICs of >91.14 and >15.18 lM. All the nine
ture-MTB activity, substituents with electron-withdrawing groups
like nitro and halogens on the benzaldehyde, actophenone and
furanyl derived acid hydrazones (4a–u) enhanced the activity. Sim-
ilarly substituents like hydroxyl, and methoxy groups reduce the
activity. With respect to the carbimino terminal, the order of activ-
ity was found to be (sub)acetophenone > (sub)benzaldehyde.
Among furanyl derivatives (4n and 4o) introduction of nitro group
at 5th position enhances the activity ꢀ9 times. All the compounds
were also screened for atypical mycobacteria MC2 which infects
lungs.10 The synthesized compounds inhibited MC2 with MICs
tested compounds were more potent than INH and one compound
was found to be more potent than RIF. The presence of persistent
and dormant MTB is thought to be the cause for the lengthy TB
chemotherapy, since the current TB drugs are not effective in elim-
inating persistent or dormant bacilli. Therefore, these drugs active
against slowly growing or non-growing persistent bacilli are
thought to be important to achieve a shortened therapy. In the case
of starved MC2 culture, the all the tested compounds inhibited with
MICs ranging from 5.30 to 48.22
INH (MIC: >91.14 M). Five compounds were found to be more po-
tent than RIF (MIC: >15.18
M). Compound N0-[1-(4-bromophe-
lM and were more potent than
l
ranging from 0.68 to 192.0
l
M and 10 compounds were more po-
M) and one compound was more po-
M). Compound 4o was found to be 67
l
tent than INH (MIC: 45.57
l
nyl)ethylidene]-5-nitro-2-furohydrazide (4r) was found to be
most active compound with MIC of 4.43 against starved MS.
tent than RIF (MIC: 1.89
l

D. Sriram et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4313–4316
4315
Table 1
Physical constants and in vitro antimycobacterial and cytotoxicity of the compounds
H
CH3
R
H
R
CONHN
CONHN
CONHN
O
O
O
O2N
O
4a-m
O2N
O2N
4p-u
4n-o
R
MIC in log phasea
MIC in starved phase MTBa
MIC in acetate for MC2
a
a
Entry
R
Yield (%)
Mp (°C)
CC50
MTB
48.22
MC2
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
H
4-NO2
4-Cl
4-Br
4-F
4-OH
4-CH3
4-OCH3
3-Br
2-NO2
2-CF3
4-OH,3-OCH3
2,6-(Cl)2
H
5-NO2
H
89
88
84
89
86
86
84
60
77
89
89
88
87
76
87
87
71
52
66
76
85
197
237
214
180
224
>250
198
120
156
174
170
228
220
182
227
176
218
170
152
168
194
NT
NT
NT
NT
NT
NT
>228.74
NT
NT
205.57
>191.01
NT
NT
NT
212.52
>228.74
>196.52
>177.50
214.64
>217.57
NT
192
NT
NT
NT
NT
NT
NT
91.49
NT
NT
41.11
19.10
NT
NT
NT
10.64
45.74
19.65
17.75
21.46
43.51
NT
48.22
20.55
10.65
36.97
45.09
45.42
22.87
21.60
36.97
10.29
9.56
20.48
38.09
25.08
5.30
45.74
19.65
4.43
20.55
21.28
18.48
22.54
>45.42
11.45
21.60
18.48
10.29
4.76
40.97
19.04
25.08
2.65
11.45
9.84
82.23
42.56
73.94
90.19
>45.42
45.74
43.20
36.97
10.29
4.76
81.94
76.18
50.16
0.68
45.74
19.65
17.75
21.46
43.51
>43.21
45.57
1.89
4q
4r
4s
4t
4-NO2
4-Br
4-F
4-CH3
2-OH
8.88
5.35
10.89
>43.21
0.72
42.92
43.51
>43.21
>91.14
>15.18
4u
Isoniazid
Rifampicin
>455.73
>75.94
>91.14
>15.18
0.48
a
CC50 and MIC in (
l
M), NT indicates not tested.
As these synthesized compounds showed activity against dor-
Table 2
Inhibitory activities of selected compounds and 3-nitro-
propionic acid against MTB ICL
mant mycobacterium, we decided to explore the possible mecha-
nism by screening some compounds against MTB ICL enzyme. ICL
is an important enzyme in the glyoxylate cycle during carbohy-
drate starvation in MTB it catalyzes the cleavage of isocitrate to
glyoxylate and succinate, allowing the organisms to survive on
acetate or fatty acids.16 The glyoxylate cycle is not present in high-
er animals, and due to its necessity for survival for the persistent
phase of the infection, ICL is considered an ideal drug target for
persistent MTB. Several small-molecule inhibitors have been de-
scribed17 as MTB ICL inhibitors; however, none has been developed
as a drug for MTB. Isocitrate lyase activity was determined at 37 °C
by measuring the formation of glyoxylate-phenylhydrazone in the
presence of phenylhydrazine and isocitrate lyase at 324 nm based
on the method described.18 The compounds were screened with a
Compd
% Inhibition (lM)
4g
4j
19.82 (10)
46.80 (10)
30.12 (10)
86.8 (10)
34.86 (10)
48.90 (10)
73.12 (10)
28.13 (10)
32.54 (10)
63.2 (100)
4k
4o
4p
4q
4r
4s
4t
3-NP
single concentration of 10
screened compounds along with the standard MTB ICL inhibitor
3-nitropropionic acid (3-NP) (at 100 M) for comparison are
reported (Table 2). All the nine compounds inhibited MTB ICL with
percentage inhibition ranging from 19.82% to 86.8% at 10 M. Two
compounds (4o, 4r) showed more than 50% inhibition and all these
compounds were found to be more potent than standard 3-NP at
the dose level tested. Further investigation could provide lead com-
pounds for drug development against persistent tuberculosis.
l
M and percentage inhibition of the
Cys191 mutated to Ser191) and bromopyruvate (PDB entry 1F8M)
were used for docking. Docking was carried out using GOLD,20 which
uses the genetic algorithm (GA). For each of the 100 independent GA
runs, a maximum number of 100,000 GA operations were per-
formed, whereby all variables for the GA were set to their default
values. Default cutoff values of 2.9 Å (dH-X) for hydrogen bonds
and 6.0 Å for van der Waals were employed. When the top three
solutions attained rmsd values within 1.5 Å, GA docking was termi-
nated. The GOLD docking run showed very strong electrostatic and
hydrophobic pattern of interaction for the ligand (Fig. 2), very well
comparable to interaction pattern observed with both inhibitors
bromopyruate and 3-nitropropionic acid crystallized with MTB
ICL, indicating a high affinity to the enzyme. The following interac-
tions were observed for ligand docked to mutated protein (Cys 191
to Ser 191, PDB entry 1F8I), nitro group of nitrofuroic acid moiety
l
l
Some compounds were further examined for cytotoxicity (CC50
)
in a mammalian Vero cell line at single concentration of 62.5 g/
l
mL. After 72 h of exposure, viability was assessed on the basis of
cellular conversion of MTT into a formazan product using the Pro-
mega Cell Titer 96 non-radioactive cell proliferation assay.19 Most
of the compounds were not cytotoxic to Vero cells.
The crystal structure of MTB ICL bound with inhibitor, 3-nitro-
propionate was taken from the Protein Data Bank (PDB entry 1F8I
forms multicentred trifurcated H-bonding with Arg 228 and Tyr 89
(ARG228:NH12ꢁ ꢁ ꢁNFA:O11, 2.27 ÅA; ARG228:NH11ꢁ ꢁ ꢁNFA:O11,
0

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D. Sriram et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4313–4316
0
2.87 ÅA). The hydrazinehydrazide group (N–NH) forms a bifurcated
0
H-bond with Ser 191 and His 193 (SER191:HOꢁ ꢁ ꢁNFA:N14, 2.00 ÅA;
0
HIS193:HD1ꢁ ꢁ ꢁNFA:N14, 2.38 ÅA) The oxygen of nitrofuraldehyde
group forms H-bond with Ser 317 (SER317:HOꢁ ꢁ ꢁNFA:O20). All most
all the amino acid residues involved in the interaction with the
ligands were common compared to the interaction observed with
the enzyme crystallized with 3-nitropropionic acid except for
residues Tyr 89, Ser 191 which are involved in H-bond and Leu
194, Trp 283 which forms hydrophobic interactions with the ligand.
Acknowledgements
The authors are thankful to University Grant Commission [F.No.
36-61/2008 (SR)], Government of India for their financial
assistances.
References and notes
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D.; Bhat, P.; Veugopal, B.; Pavan, V. V. S.; Manjashetty, T. H. Med. Chem. 2009, 5,
422.
7. Tangallapally, R. P.; Yendapally, R.; Lee, R. E.; Lenaerts, A. J. M.; Lee, R. E. J. Med.
Chem. 2005, 48, 8261.
8. Spectroscopic data for representative compound 4o is given below. 5-Nitro-N0-
[(5-nitro-2-furyl)methylidene]-2-furohydrazide: 1H NMR (300 MHz, CDCl3) dH 7,2
(s, 1H), 7.43 (s, 1H), 7.32 (dd, 1H), 7.46 (dd, 1H), 8.01 (dd, 1H), 8,52 (dd, 1H). 13
C
NMR (75 MHz, CDCl3) dC 111.3, 111.4, 116.1, 116.2, 134.6, 150.9, 151.8, 152.0,
153.9, 166.3.
9. National Committee for Clinical Laboratory Standards. Antimycobacterial
Susceptibility Testing for Mycobacterium tuberculosis. Proposed standard
M24-T. National Committee for Clinical Laboratory Standards, Villanova, Pa.,
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10. Schreiber, J.; Burkhardt, U.; Rusch-Gerdes, S.; Amthor, M.; Richter, E.; Zugehor,
M.; Rosahl, W.; Ernst, M. Pneumologie 2001, 55, 238.
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653.
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Figure 2. (A) Ligand docked inside the active site pocket of the MTB ICL Cys191
muteated to Ser191. (B) Binding mode of ligand with the MTB ICL (Cys191 mutated
to Ser191) only the residues in the active site were shown (residues within a
0
distance of less than 11.0 ÅA are shown), hydrogen bonds are displayed by dotted
green lines; Mg2+ is shown as black sphere.
16. McKinney, J. D.; Bentrup, K. H.; Muñoz-Elías, E. J.; Miczak, A.; Chen, B.; Chan, W.
T.; Swenson, D.; Sacchettini, J. C.; Jacobs, W. R.; Russell, D. G. Nature 2000, 406,
735.
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7161.
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19. Gundersen, L. L.; Nissen-Meyer, J.; Spilsberg, B. J. Med. Chem. 2002, 45, 1383.
20. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. J. Mol. Biol. 1997, 267,
727.
0
0
2.53 ÅA; TYR89:OHꢁ ꢁ ꢁNFA:O10, 2.39 ÅA). The oxygen of nitrofuroic
acid group forms H-bond with Arg228 (ARG228:NH12ꢁ ꢁ ꢁNFA:O5,
0
1.99 ÅA). The carboxyl group of nitrofuroic acid forms multicentred
H-bond with Ser191, Gly 192, and His193 (SER191:HOꢁ ꢁ ꢁNFA:O12,
0
0
1.68 ÅA; GLY192:HNꢁ ꢁ ꢁNFA:O12, 1.42 ÅA; HIS193:HNꢁ ꢁ ꢁNFA:O12,