Synthesis of various 3-nitropropionamides as Mycobacterium tuberculosis isocitrate lyase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2011.07.062

Various 3-nitropropionamides 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 22 compounds, 1-cyclopropyl-7-(3,5-dimethyl-4-(3-nitropropanoyl) piperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (22) was found to be the most active compound in vitro with MICs of 0.16 and 0.04 μM against log- and starved-phase culture of MTB. Compound 22 also showed good enzyme inhibition of MTB ICL with IC₅₀ of 0.10 ± 0.01 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.2011.07.062

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5149–5154
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of various 3-nitropropionamides as Mycobacterium tuberculosis
isocitrate lyase inhibitor
⇑
Dharmarajan Sriram , Perumal Yogeeswari, Swetha Methuku, Devambatla Ravi Kumar Vyas,
Palaniappan Senthilkumar, Mallika Alvala, Variam Ullas Jeankumar
Antitubercular Drug Discovery Laboratory, Department of Pharmacy, Birla Institute of Technology and Science-Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad 500 078, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Various 3-nitropropionamides 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 22 compounds, 1-cyclopropyl-7-(3,5-dimethyl-4-(3-nitropropa-
noyl)piperazin-1-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (22) was found
Received 31 March 2011
Revised 14 July 2011
Accepted 18 July 2011
Available online 26 July 2011
to be the most active compound in vitro with MICs of 0.16 and 0.04
lM against log- and starved-phase
culture of MTB. Compound 22 also showed good enzyme inhibition of MTB ICL with IC50 of
Keywords:
0.10 0.01
active site.
l
M. The docking studies also confirmed the binding potential of the compounds at the ICL
Antitubercular activity
Mycobacterium tuberculosis
Nitropropionic acid derivatives
Latent tuberculosis
Ó 2011 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis (MTB) claims more human lives
each year than any other bacterial pathogen. With approximately
3 million annual deaths in the 1990s, tuberculosis (TB) remains a
leading cause of mortality worldwide into the 21st century.1 It is
estimated that one-third of the world population harbor a latent
infection by the causative pathogen, MTB. One of the hallmarks
of TB is the persistent phase of infection. During this phase the bac-
teria are thought to be in a slow growing or non-growing state and
are recalcitrant to treatment by conventional anti-TB drugs.2,3 One
of the drawbacks of existing TB drugs is that they target actively
growing bacteria in cell processes such as cell wall biogenesis pro-
tein synthesis and chromosome replication. Patients who carry a
latent infection are at risk of reactivation of the disease and this
is one of the factors which cause a major obstacle to the global con-
trol of TB. Most of the available TB drugs were discovered by the
in vitro screening compounds against actively growing MTB under
rich nutrient and oxygen conditions. In an infection, however, MTB
is often in an environment where nutrients and oxygen are limit-
ing.4 Under these conditions, MTB is in a reduced state of growth
and can persist for long periods of time, avoiding being killed by
most TB drugs. This phenomenon of persistence is believed to be
the main reason why TB requires a lengthy therapy. A new agent
that targets the persistent state of growth would have a significant
impact on the treatment of TB by shortening the duration of
therapy.
To establish or maintain a persistent infection, MTB appears to
require the glyoxylate pathway to bypass the energy-generating
tricarboxylic acid cycle (TCA). The glyoxylate pathway uses isoci-
trate lyase (ICL) and malate synthase to incorporate carbon during
growth of microorganisms on acetate or fatty acids as the primary
carbon source.5 A recent study found that a double deletion of both
icl1 and icl2 resulted in complete impairment of mycobacterial
intracellular replication and rapid elimination of the bacteria from
the lungs, validating ICL and the glyoxylate pathway as a target for
compounds that would eliminate persistent bacteria.6 The require-
ment of ICL to a persistent infection makes it an attractive target
for drug discovery.5 3-Nitropropionate and bromopyruvate were
previously shown to inhibit mycobacterial ICL.7 In this work we
have reported synthesis of various 3-nitropropionamides and its
in vitro activities against log and starved phase culture of two
mycobacterial species and MTB ICL enzyme inhibition studies.
The synthetic protocol of 3-nitropropionamides are depicted in
Scheme 1 and briefly, equimolar ratio of 3-nitropropionic acid,
dicyclohexylcarbodiimide, and corresponding amines were dis-
solved in dichloromethane (DCM), dimethyl formamide or
DCM–methanol mixture depending upon solubility of amines.
The reaction mixture exposed to microwave irradiation of 420 W
for 2–3 min. The reaction is monitored by TLC and after completion
of the reaction precipitated dicyclohexyl urea was removed by fil-
tration and filtrate is evaporated to gives target compounds 1–22
in 46–86% yield. The purity of compounds was checked by TLC
and elemental analyses. Both analytical and spectral data (1H
NMR, 13C NMR, and mass spectra) of all the synthesized com-
pounds were in full agreement with the proposed structures.8
⇑
Corresponding author.
E-mail address: dsriram@bits-hyderabad.ac.in (D. Sriram).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.062

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D. Sriram et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5149–5154
O
N+
NH
O
-O
X
N+
OH
DCC, MWI
O
1-11
R
-O
RNH2 or RR1NH
O
R
O
N+
N
-O
R1
12-22
O
Scheme 1. Synthetic protocol for 3-nitropropinamides.
Lipophilicity of the synthesized derivatives and that of the parent
compound 3-nitropropionic acid, are expressed in terms of their
log P values (Table 1). These values were computed with a routine
method called calculated log P (C log P) using ChemBioDraw Ultra
11.0 software.
The compounds were screened for their in vitro antimycobacte-
rial activity initially against log-phase cultures of M. tuberculosis
H37Rv (OD 600), and Mycobacterium smegmatis ATCC 14468 (MS)
by agar dilution method similar to that recommended by the Na-
tional Committee for Clinical Laboratory Standards for the determi-
nation of MIC in duplicate.9 The minimum inhibitory concentration
(MIC) is defined as the minimum concentration of compound re-
quired to give complete inhibition of bacterial growth. MICs of the
synthesized compounds along with the standard drugs for compar-
ison are reported (Table 1). In the initial screening against log-phase
MTB, 3-nitropropionamides showed good activity with MICs rang-
1-cyclopropyl-7-(3,5-dimethyl-4-(3-nitropropanoyl)piperazin-1-
yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (22) was found to be the most active compound in vitro with
MICs of 0.16 lM against log-phase culture of MTB and was 4.5 times
more potent than standard first line anti-TB drug INH. With respect
to structure-MTB activity, for the phenyl substituted compounds(1–
9) compound 1 without any substituent in phenyl ring displayed an
MIC of 257.48 lM serves as the standard for potency comparison.
The activity slightly improved by the introduction of electron donat-
ing groups (2 and 3) like methyl and methoxyl group. Whereas
introduction of electron withdrawing groups (3–6) such as chloro,
bromo, and fluoro enhances activity approximately 2 times. Surpris-
ingly introduction di-substitution with both halogen and methyl
groups (8 and 9) enhances activity to five folds with MIC ranging
from 43.51–51.51 lM. Similar kind of SAR found in pyridyl substi-
tuted amides (10 and 11) also. Compounds with open chain second-
ing from 0.16 to 287
activity with MIC of <5
0.72 M) two compounds (20 and 22) were found to be more active
and all the compounds were more active than parent 3-nitroprop-
l
M. Four compounds (19–22) showed excellent
ary amides showed very poor activity (12 and 13) with MIC of
l
M. When compared to isoniazid (INH) (MIC:
>250
lM. Compounds (19–22) bearing fluoroquinolone moiety
l
showed excellent activity with MIC of <4
lM. All the compounds
were also screened for another mycobacterial species MS, the syn-
ionic acid (MIC: >419.92 lM). Among 22 compounds screened,
thesized compounds inhibited MS with MICs ranging from 0.08 to
Table 1
Physical constants and antimyobacterial activities of 3-nitropropionamides
O
N+
R
-O
O
No.
R
Yield (%)
Mp (°C)
c log P
MIC in lM
Active phase
MTBb
Starved phase
MS MTB
MSa
1
2
3
4
NH
84
67
65
73
197–199
188–190
154–155
174–175
0.81
1.37
0.95
0.99
128.73
120.07
111.50
54.67
257.48
240.14
223.00
109.34
>257.48
>240.14
>223.00
13.69
64.36
60.03
55.75
27.33
H3C
H3CO
Cl
NH
NH
NH

D. Sriram et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5149–5154
5151
Table 1 (continued)
No.
R
Yield (%)
Mp (°C)
c log P
MIC in lM
Active phase
MTBb
Starved phase
MS MTB
MSa
5
6
Br
NH
NH
82
78
138–140
204–205
1.11
0.67
91.54
183.09
117.82
91.54
45.77
58.91
F
117.82
235.64
H3C
NH
7
86
55
83
59
161–162
133–135
118–120
161–162
1.17
1.49
1.84
0.70
224.98
206.04
87.07
224.98
51.51
>224.98
206.04
43.53
74.99
51.51
43.53
CH3
NH
8
Cl
Br
CH3
NH
9
43.53
H3C
H3C
10
>239.00
239.00
239.00
59.75
54.43
NH
N
Cl
NH
11
12
52
83
148–149
238–240
1.03
0.40
217.75
287.02
108.87
108.87
N
H3C
N
>287.02
>287.02
>287.02
>247.21
>167.59
H3C
H3C
N
13
14
52
51
220–221
90–92
1.46
2.70
>247.21
167.59
>247.21
>167.59
>247.21
>167.59
H3C
C6H5
N
C6H5
C2H5
15
16
N
N
50
78
120–122
113–115
0.53
1.43
58.07
58.07
116.14
29.03
47.47
N
N
189.90
189.90
>189.90
(continued on next page)

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D. Sriram et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5149–5154
Table 1 (continued)
No.
R
Yield (%)
Mp (°C)
c log P
MIC in lM
Active phase
MTBb
Starved phase
MS MTB
MSa
F3C
17
18
56
46
230–232
164–165
2.62
1.72
75.46
150.92
75.46
37.73
90.14
N
N
C6H5H2C
HOOC
N
N
>180.29
>180.29
>180.29
O
N
F
19
20
21
70
85
74
222–223
238–239
224–226
1.34
1.39
2.01
7.44
0.09
6.91
1.85
1.80
3.44
0.95
0.09
1.72
0.47
0.09
1.72
N
C2H5
O
N
HOOC
F
N
O
N
N
HOOC
F
CH3
N
N
C2H5
O
F
N
HOOC
F
CH3
22
62
231–232
3.37
0.08
0.16
0.08
0.04
N
N
OCH3
N
CH3
3-Nitropropionic acid
Isoniazid
Ciprofloxacin
À0.59
>419.92
45.57
4.71
>419.92
0.72
NT
>419.92
>364.67
2.35
>419.92
>364.67
NT
a
Mycobacterium smegmatis.
Mycobacterium tuberculosis, NT indicates not tested.
b
287.02
l
M and four compounds were more potent than INH (MIC:
M). Compound 22 was found to be more potent with MIC
lM and was 569 times more potent than INH against log-
All the compounds were further screened against six-week
nutrient starved cells of MTB according to the literature proce-
dure10 in duplicate and MICs were reported in Table 1. Against dor-
mant MTB synthesized novel 3-nitropropionic acid derivatives
45.57
l
of 0.08
phase culture of MS.

D. Sriram et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5149–5154
5153
Table 2
compounds showed good activity. Among the compounds
screened, 22 was found to be the most active compound in vitro
ICL inhibitory studies and cytotoxicity of 3-nitropropionamides
with MICs of 0.04 lM against starved-phase culture of MTB and
Compounds
IC50
(lM)
CC50 (lM)
was 4 times more potent towards starved-phase MTB than log-
phase MTB. 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 eliminating persistent or
dormant bacilli. Therefore, these drugs active against slowly grow-
ing or non-growing persistent bacilli are thought to be important
to achieve a shortened therapy. In starved MS culture the tested
compounds inhibited growth with MIC ranging from 0.04 to
4
5
9
21.32 0.80
32.46 0.42
36.80 0.31
12.60 0.21
41.21 1.2
30.18 0.6
0.20 0.04
0.12 0.01
1.46 0.04
0.10 0.01
116.0 1.1
NT
NT
NT
NT
NT
NT
>148.74
>144.53
>138.14
>127.42
<524.90
15
16
17
19
20
21
22
3-NP
>287 lM and most of the compounds were more potent than stan-
dard 3-nitropropionic acid and INH.
NT indicates not tested.
Several mechanisms may be involved in the killing of non-
growing cells. The lack of efficacy of INH (which targets cell wall
mycolic acid biosynthesis) in starved cells suggests that cell wall
synthesis is not important during starvation, a phenomenon that
has been commonly observed in other bacteria.11 Drugs could tar-
get processes that are critical for survival even when bacteria are
not replicating, such as transcription, allowing drugs like rifampin
to retain some activity. Non-growing cells might have alterations
in their cell walls that result in changed permeability to antibiotics.
Finally, a drug might associate with the bacteria during incubation
and not be removed by washing, thus manifesting its antibacterial
effects during outgrowth. Any or all of these mechanisms might be
functional in vivo, where a persistent antibacterial effect might
lead to more-rapid clearance of infection during treatment. The
strategy for survival of TB during chronic stages of infection is
thought to involve a metabolic shift in the bacteria’s carbon source
to C2 substrates generated by the b-oxidation of fatty acids.12 Un-
der these conditions, glycolysis is decreased and the glyoxylate
shunt is significantly up-regulated allowing anaplerotic mainte-
nance of the TCA cycle.13 The glyoxylate shunt converts isocitrate
to succinate and glyoxylate, catalyzed by the enzyme ICL, followed
by the addition of acetyl-CoA to glyoxylate to form malate by ma-
late synthase. It has been shown that expression of ICL is up-regu-
lated under certain growth conditions14 and during infection of
macrophages by Mycobacterium spp.15 Furthermore, ICL is required
for the survival of bacteria in activated macrophages but not in
Table 3
Computational data for the compounds
Molecule Affinity Moldock Hydrogen
Interacting amino acid
residues
score
bonding
energy
11
21
À32.26 À107.37 À19.29
À31.89 À83.07 À15.57
Ser191, Gly192, His193, Tyr89,
Ser91, Gly92, Trp93
Tyr89, Ser91, Gly92, Trp93,
His180, Ser191, Gly192,
Arg228, Asn313, Ser317
Tyr89, Ser91, Trp93, His180,
Ser191, Gly192, His193,
Asn313, Ser315, Ser317
Ser315, Ser317, Ser191,
His193, Asn313, Leu194
Ser315, Ser191, Gly192,
His193, Asn313, Leu194
22
À30.9
À107.71 À18.86
3-NP
4
À30.43 À89.75
À14.21
À29.98 À103.65 À15.16
showed good activity with MICs ranging from 0.04 to >287
Four compounds (19–22) showed excellent activity with MIC of
<2 M. When compared to standard MTB ICL inhibitor 3-nitro-
propionic acid (MIC: >419.92 M) 19 compounds were found to
lM.
l
l
be more active. When compared to INH (MIC: >364.67) which is
not active against dormant MTB, most of the synthesized
Figure 1. Figure depicting binding site of 3-nitropropionate in ICL.

5154
D. Sriram et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5149–5154
Figure 2. Figure depicting binding of 22 to ICL at the active site of 3-nitropropionate.
resting macrophages.6 It has also been demonstrated that ICL is
important for survival of MTB in the lungs of mice during the per-
sistent phase of infection (2–16 weeks), but is not essential during
the acute phase (0–2 weeks) of infection.6
As these synthesized compounds showed activity against dor-
mant mycobacterium, we decided to explore the possible mecha-
nism by screening some compounds against MTB ICL enzyme
(Table 2).16 Among 10 compounds screened, three compounds
References and notes
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(19, 20, and 22) inhibited ICL with IC50 of less than 1
10 screened compounds showed better activity (IC50 0.1–
36.80 M) than standard 3-nitropropionic acid (IC50 116 M).
Four compounds were further examined for cytotoxicity (CC50
in a mammalian Vero cell line at single concentration of 62.5 g/
lM. All the
7. Kumar, R.; Bhakuni, V. Proteins 2008, 72, 892.
8. Spectroscopic data for representative compound 22 is given below. 1H NMR
(300 MHz, DMSO-d6) d (ppm): 0.28–0.46 (m, 4H, cyclopropyl), 1.32 (m, 1H,
cyclopropyl), 1.15 (d, 6H, –CH3 J = 9.8 Hz), 2.86 (t, 2H, –CH2 J = 7.1 Hz), 3.44–
3.51 (m, 4H, –CH2), 3.53 (s, 3H, –OCH3), 4.02 (m, 2H, –CH), 4.42 (t, 2H, –CH2
J = 7.4 Hz), 6.71 (s, 1H, C5-H), 7.95 (s, 1H, C2-H), 12.00 (s, 1H, COOH); 13C NMR
(DMSO-d6) d (ppm): 176.4 (1C@O of 4-quinolone), 171.1 (1C@O of amide),,
166.2 (1C@O of 3-COOH), 158.6 (1CH of 6-benzene), 146.9 (1C of 2-quinolone),
146.3 (1CH of 8-benzene), 132.5 (1CH of 7-benzene), 125.9 (1C of 8a-
quinolone), 123.8 (1C of 4a-quinolone), 109.3 (1C of 3-quinolone), 104.5
(1CH of 4-benzene), 72.0 (3rd CH2 of nitropropanoyl), 55.8 (1C of methoxy),
54.4 (2ÂC of piperazine), 53.1(2ÂC of piperazine), 36.1(1ÂC of cyclopropane),
28.6 (2nd CH2 of nitropropanoyl), 18.7 (2ÂC of methyl), 7.7 (2ÂC of
cyclopropane). Anal (C23H27FN4O7) C, H, N.
l
l
)
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. Most of
the compounds were not cytotoxic to Vero cells (Table 2). Com-
pound 22 was not toxic up to 127.42
of 3185 for starved MTB. CC50 of standard anti-TB compound INH
is >455.73 M.
lM with selectivity index
l
9. National Committee for Clinical Laboratory Standards. Antimycobacterial
Susceptibility Testing for Mycobacterium tuberculosis. Proposed standard
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7161.
In this study we have designed and developed various small
molecule inhibitors of MTB ICL based on 3-nitripropionate. A series
of derivatives of 3-nitropropionate were synthesized and docked
into catalytic core of ICL and coordinates were taken from PDB
ID: 1F8I. Docking studies using Molegro Virtual Docker 2.2.5 pro-
gram,17 showed that 4, 11, 21, 22 has given better affinity, Moldock
score, hydrogen bonding energy compared to 3-nitropropionate
(Table 3). Moreover the interacting amino acid residues were also
almost same (Figs. 1 and 2).
15. Graham, J. E.; Clark-Curtiss, J. E. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11554.
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D.; Bhat, P.; Manjashetty, T. H. Chem. Biol. Drug Des. 2010, 75, 381.
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Acknowledgments
The authors are thankful to University Grant Commission
[F. No. 36-61/2008 (SR)], Government of India for their financial
assistance.