Development of 5-nitrothiazole derivatives: Identification of leads against both replicative and latent Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmcl.2012.10.060

Twenty eight 5-nitrothiazole derivatives were synthesized and evaluated for in vitro activities against Mycobacterium tuberculosis (MTB), cytotoxicity against HEK 293T. Among the compounds, 5-nitro-N-(5-nitrothiazol-2-yl)furan-2- carboxamide (20) was found to be the most active compound in vitro with MICs of 5.48 μM against log-phase culture of MTB and also non-toxic up to 100 μM.

Full text of DOI:10.1016/j.bmcl.2012.10.060

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7414–7417
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of 5-nitrothiazole derivatives: Identification of leads against
both replicative and latent Mycobacterium tuberculosis
Variam Ullas Jeankumar a, Manoj Chandran a, Ganesh Samala a, Mallika Alvala a, Pulla Venkat Koushik a,
Perumal Yogeeswari a, Elena G. Salina b, Dharmarajan Sriram a,
⇑
a Antitubercular Drug Discovery Laboratory, Department of Pharmacy, Birla Institute of Technology& Science - Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad 500 078, India
b Institution of the Russian Academy of Sciences A.N. Bach Institute of Biochemistry RAS, Moscow, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2012
Revised 25 September 2012
Accepted 13 October 2012
Available online 23 October 2012
Twenty eight 5-nitrothiazole derivatives were synthesized and evaluated for in vitro activities against
Mycobacterium tuberculosis (MTB), cytotoxicity against HEK 293T. Among the compounds, 5-nitro-N-(5-
nitrothiazol-2-yl)furan-2-carboxamide (20) was found to be the most active compound in vitro with
MICs of 5.48 lM against log-phase culture of MTB and also non-toxic up to 100 lM.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Antitubercular activity
Mycobacterium tuberculosis
Nitrothiazole derivatives
Dormant tuberculosis
Since 1993, when the World Health Organization (WHO) de-
clared tuberculosis (TB) a global emergency, there has been tre-
mendous progress in the fight against the disease. Directly
observed therapy short-course (DOTS), the recognized case man-
agement approach for TB, has been implemented in the 184 coun-
tries that account for 99 percent of all estimated TB cases. The
global burden of TB is falling slowly, and at least three regions in
the world are on track to achieve global targets for halving the
number of cases and deaths by 2015.1 Although TB is curable
and preventable, one of three people in the world is currently la-
tently infected with the TB bacterium without overt symptoms of
disease, and the active form of the disease kills 1.8 million people
annually. Co-infection with TB and HIV(TB/HIV) and a surge in
multidrug-resistant TB (MDR–TB) and extensively drug-resistant
TB (XDR–TB) are threatening to disrupt recent global successes in
TB control. The drugs currently used to treat TB were discovered
more than 40 years ago and do not meet the demands of today’s
TB epidemic. Specific and high effective anti-latent TB drugs are
still not found and traditional antibiotics such as isoniazid and
rifampin are currently being used for latent TB curing but the effec-
tiveness of such treatment of latent infection is rather controver-
lead to poor adherence among patients. Those who do not or can-
not complete their treatment may develop drug-resistant TB
strains that take up to two years to treat with second-line drugs,
often with severe side effects.4 There is an urgent need for novel
TB drug regimens that cure more rapidly, and for drugs that can
be safely taken concurrently with antiretroviral therapies.5
Recently Nitazoxanide (NTZ) and its deacetylated active metabolite
Tizaxonide (TIZ) were reported with its potency against killing
replicative and non-replicative Mycobacterium tuberculosis (MTB)
by a novel mechanism of action (Fig. 1).6 NTZ, a 5-nitro-thiazolyl
anti-parasitic drug kills MTB by disruption of MTB’s membrane
potential and pH homeostasis.7 In this paper we report the anti-
tubercular activity of various 5-nitrothiazolyl derivatives; which
are structural analogues of NTZ.
The general synthetic pathway followed to achieve the title
compounds is outlined in Figure 2 (Scheme 1 and 2). In the present
protocol (Scheme 1) the hydroxyl (–OH) group of various
2,3
sial.
TB requires treatment with multiple drugs, and existing regi-
mens require at least six months of monitored use to cure. They
are burdensome for patients and care providers alike, which can
⇑
Corresponding author.
E-mail address: dsriram@bits-hyderabad.ac.in (D. Sriram).
Figure 1. Structure of NTZ and TIZ.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.060

V. U. Jeankumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7414–7417
7415
Figure 2. General synthetic procedure.
Table 1
Physical constants and biological data of compounds 1–18
O
N
R1
R2
NH
NO2
S
OR4
R3
Compound
R1
R2
R3
R4
Yield in %
MP in oC
MTB MIC in
l
Ma
% Growth 25
l
mb
% Growth 100 l
mb
1
2
3
4
5
6
7
8
H
H
H
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl–
Cl–
Cl–
H
H
H
NO2–
NO2–
NO2–
(CH3)2CH–
(CH3)2CH–
(CH3)2CH–
H
–OH
69
55
60
65
55
73
65
58
55
67
72
75
66
70
60
65
71
66
—
163
157
173
178
169
171
177
175
160
191
158
165
164
159
157
186
201
179
—
>166.83
36.57
>123.82
20.85
18.28
15.47
74.81
66.45
28.52
84.66
74.11
15.64
>140.74
>125.85
>108.84
17.88
ND
100
ND
ND
83.46
ND
–COCH3
–COC6H5
–OH
–COCH3
–COC6H5
–OH
–COCH3
–COC6H5
–OH
–COCH3
–COC6H5
–OH
–COCH3
–COC6H5
–OH
–COCH3
–COC6H5
–COCH3
—
100
59.49
72.16
100
100
100
100
100
100
ND
ND
ND
29.36
45.93
ND
63.03
31.15
23.7
23.47
95.1
61.59
100
94.14
67.57
ND
ND
ND
7.1
20.56
ND
9
Cl–
10
11
12
13
14
15
16
17
18
NTZ
INH
Rif
CH3O–
CH3O–
CH3O–
NO2–
NO2–
NO2–
(CH3)2CH–
(CH3)2CH–
(CH3)2CH–
H
63.86
>110.25
162.71
0.72
H
—
—
ND
ND
ND
ND
ND
ND
—
—
—
—
—
—
—
—
—
0.48
a
Minimum inhibitory concentration against M. tuberculosis.
Cytotoxicity against HEK 293T.
b
commercially available substituted salicylic acids were first sub-
jected to acetylation/benzoylation to get the desired acetyl or ben-
zoyl derivative of the corresponding salicylic acid in quantitative
yield using well known procedures. The thus obtained product
was then coupled with 2-amino-5-nitrothiazole, synthesised in
quantitative yield from nitro methane via the bromination of
N,N-dimethyl-2-nitroetheneamine and subsequent treatment with
thiourea (as shown in scheme 2) by using a simple and efficient
protocol as previously described by Dah-Chieh8 to get the first
set of compounds in the library. Considering the low solubility
and low nucleophilicity of 2-amino-5-nitrothiazole, the coupling
was achieved by first converting the corresponding substituted sal-
icylic acid into the respective acid chloride and then coupling with
amine in THF or DMF as the solvent. The second set in the library
was achieved by either deacetylating or debenzoylating the above
obtained derivative using 6 N HCl in THF.9–11 Encouraged by the
promising anti-tubercular activity of the above synthesised deriv-
atives further work was initiated in our lab with the goal of obtain-
ing a lead series with tractable SAR and potencies better than the
above compounds and thus the second series (Scheme 2) in the li-
brary was designed and synthesised as shown in Table 1 and 2.
Both analytical and spectral data (1H NMR, 13C NMR, and mass
spectra) of all the synthesized compounds were in full agreement
with the proposed structures.12
All the new 5-nitrothiazole derivatives were screened for their
in-vitro anti-tubercular activity against Mycobacterium tuberculosis

7416
V. U. Jeankumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7414–7417
Table 2
Physical constants and biological data of compounds 19–28
19-27
28
Compound
R
Yield in %
65
MP in °C
MTB MIC in
l
Ma
% Growth 25
ND
l
mb
% Growth at 100
ND
l
mb
19
177
205.55
5.48
20
21
22
80
72
77
185
183
202
87.44
100
67.26
93.44
35.94
24.48
20.81
100
23
24
25
26
27
64
63
71
69
67
179
206
239
211
187
17.86
31.7
75
74.12
70.34
79.22
91.54
95.66
89.14
88.44
94.1
100
17.34
18.97
72.39
28
—
—
—
—
70
—
—
206
—
—
149.58
162.71
0.72
100
ND
ND
ND
43.33
ND
ND
NTZ
INH
Rif
—
—
0.48
ND
a
Minimum inhibitory concentration against M. tuberculosis.
Cytotoxicity against HEK 293T.
b
16, 20–23, 25–26) inhibited MTB with MIC of less than 25 lM.
1,00E+07
1,00E+06
1,00E+05
1,00E+04
1,00E+03
1,00E+02
1,00E+01
1,00E+00
Twenty one compounds were found to be more potent than
standard drug NTZ. Compound 20 (5-nitro-N-(5-nitrothiazol-2-yl)-
furan-2-carboxamide) was found to be the most active compound
in vitro with MICs of 5.48 lM against log-phase culture of MTB and
was 29 times more potent than NTZ.
With respect to structure-TB activity relationship, we prepared
eighteen salicylanilides derivatives (1–18) and ten hetero-aryl
amide derivatives (19–27). Among salicylanilides derivatives, we
have done modification at o position of phenyl ring with hydroxyl,
acetoyl and benzoyl derivatives (R4), m-position with chloro, nitro,
isopropyl and hydrogen (R3), p-position with chloro and hydrogen
and 5th position with hydrogen (R2), chloro, methoxyl, nitro and
isopropyl groups (R1). Among p-chloro and 5-chloro derivatives
(1–3 vs 4–6) 5-chloro derivatives were found to be more active,
and they are also showed more promising than 3,5-dicholoro
derivatives (7–9). Methoxyl group at 5th position and isopropyl
group at 3rd and 5th position also retain activity (10–12, 16–18).
Introduction of nitro groups at 3rd and 5th position is detrimental
for activity (13–15). When compared to NTZ with MIC of
control
#28, 10 µg/ml Rif, 10 µg/ml
Inh, 10 µg/ml
#20, 10 µg/ml
Figure 3. The effectiveness of the compounds 20 and 28 for killing M. tuberculosis
‘non-culturable’ cells. ‘Non-culturable’ cells were washed and treated by 10 g/ml
of rifampicin (Rif), isoniazid (Inh) and compounds #20 and 28 for 7 days. Viability of
both treated and untreated ‘non-culturable’ cells was tested by the concentration of
cells which were able to recover from NC state by MPN assay.
l
H37Rv (ATCC27294) using an agar dilution method using drug
162.71 lM; introduction of p-chloro group (2) enhances 4.4 times
more active, 5-chloro group (5) enhances 8.9 times, 3,5-dichloro
groups (8) enhances 2.44 times, 5-methoxyl group (11) enhances
2.19 times, and 3,5-diisopryl groups (17) enhances 2.54 times
more activity. Whereas introduction of 3,5-dinitro groups (14)
makes the NTZ inactive. Among the substituent’s at o-position,
order of activity is benzoyl>acetoyl>hydroxyl, in 5-chloro, 5-meth-
oxyl and 3,5-diisopropyl derivatives. Among hetero-aryl amides
(19–27), 2-thienyl derivative (21) was found to be more potent
than 2-furanyl derivative (19). Introduction of 5-nitro group at
2-furanyl ring (20) enhances the activity 37.5 times, whereas
concentration from 50 lg/mL to 0.78 lg/mL in duplicates. The
minimum inhibitory concentration (MIC) was determined for each
compound. The MIC is defined as the minimum concentration of
compound required to completely inhibit the bacterial growth.
NTZ, Isoniazid and Rifampicin were used as a reference compound
and its MIC was found to be 50 lg/mL in our screening. The MIC
values of the synthesized compounds along with the standard drug
for comparison were reported in Table 1 and 2. Among the twenty
eight compounds screened twenty two compounds showed activ-
ity against MTB with MIC 650 lg/mL. Eleven compounds (4–6, 12,

V. U. Jeankumar et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7414–7417
7417
introduction of 5-nitro group at 2-thienyl group not showed any
pound 20 showed 32% inhibition at 100
lM with therapeutic index
appreciable activity. Introduction of phenyl ring at 5th position of
2-furanyl ring enhances the activity in many folds (MIC of 17.34–
of >20. Compound 16 was found to be cytotoxic in 25 lM itself.
In this work we identified some novel 5-nitrothiazolyl deriva-
72.39 lM), substituents in phenyl ring; presence of para chloro,
tives with activity against both replicative and dormant MTB.
bromo, nitro and methyl group are beneficial for activity. Com-
pound 20 (5-nitro-N-(5-nitrothiazol-2-yl)furan-2-carboxamide)
Acknowledgments
was found to be most promising molecule with MICs of 5.48 lM
and replacement of its 5-nitrothiazole moiety with 6-nitrobenz-
thiazole leads to 5-nitro-N-(6-nitrobenzo[d]thiazol-2-yl)furan-2-
The authors are thankful to Department of Biotechnology (BT/01/
COE/05/06/01), Government of India for their financial assistances.
carboxamide (28). Compound 28 (MIC of 149.58 lM) was 27.2
times less potent than 20, but slightly more potent than NTZ.
References and notes
Compounds 20 and 28 were also examined for their activity
against dormant MTB bacilli at 10 lg/mL. To obtain dormant cells,
1. World Health Organization, 2011/2012 Tuberculosis Global Facts, Geneva,
Switzerland (2011).
M. tuberculosis bacilli were grown in potassium-deficient Sauton’s
medium supplemented with ADC and 0.05% of Tween-80 (37 °C,
200 rpm). ‘Dormancy’ was detected by inability of the cells to form
colonies onto agar solidified Sauton’s medium. Resuscitation of
both treated and untreated NC cells was performed in liquid Sau-
ton’s medium, with the concentration of cells recovered from dor-
mancy being estimated by Most Probable Numbers (MPN) assay
and with the use of statistical approaches.13 It was found that after
treatment with compounds dormant cells were less able to recover
from dormancy (Fig. 3). Both compounds caused a ꢀ1-log decrease
2. Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. Nature 2011, 469,
483.
3. Chao, M. C.; Rubin, E. J. Annu. Rev. Microbiol. 2010, 64, 293.
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12. (a) 5-Nitro-N-(5-nitrothiazol-2-yl)furan-2-carboxamide (20): yellowish brown
solid; yield (80%). 1H NMR (300 MHz, DMSO-d6) dH7.81 (d, J = 3.8, 1H, ArH),
7.88 (d, J = 3.8, 1H, ArH), 8.69 (s, 1H, -thiazole). 13CNMR (75MHz, CDCl3): dc
162.3, 156, 152.5, 145.3, 141.6, 141.3, 119.1, 113.1. Anal. Calcd for
C8H4N4O6S:C33.81; H, 1.42; N, 19.71. Found: C, 33.88, H, 1.39; N, 19.68. MS
m/z: 283.94 [M+].
in the viability of dormant cells after incubation with 10 lg/ml for
7 days. Although this effect is quite a modest one the activity of
these compounds exceeds the effectiveness of isoniazid and is
comparable to rifampicin in relation to dormant M. tuberculosis ba-
cilli. These compounds may be regarded as the prominent com-
pound for the development of derivatives which are more
effective for dormant MTB cells and latent tuberculosis.
Compounds which showed reasonable anti-TB activity were
also tested for in-vitro cytotoxicity against HEK293Tcells at 25
and 100 lM concentrations by (4,5-dimethylthiazol-2-yl)-2,5-
(b) 4-Chloro-2-(5-nitrothiazol-2-ylcarbamoyl)phenyl benzoate (6): yellow
solid; yield (73%).1H NMR (300 MHz, DMSO-d6) dH7.67–8.16 (m,8H, ArH),
8.63 (s, 1H, -thiazole). 13CNMR (75MHz, CDCl3): dc 166.4, 162.8, 144.8, 143.2,
136.5, 134.1, 133.8, 131.4, 129.8, 128.1, 127.3, 126.4, 122.6. Anal. Calcd for
C17H10ClN3O5S:C, 50.57; H, 2.50; N, 10.41. Found: C, 50.53; H, 2.51; N,
diphenyltetrazolium bromide (MTT) assay. Percentage growth of
cells was reported in Table 1,2. The most promising anti-TB com-
10.39.MS m/z: Found [M+m/z:%] 403.04, 100; 405.04, 37.1.
13. deMan, J. C. J. Appl. Microbiol. 1975, 1, 67.