5-Nitrofuran-2-yl derivatives: Synthesis and inhibitory activities against growing and dormant mycobacterium species

Article abstract of DOI:10.1016/j.bmcl.2008.12.088

Eighteen 5-nitrofuran-2-yl derivatives were prepared by reacting 5-nitro-2-furfural with various (sub)phenyl/pyridyl thiosemicarbazide using microwave irradiation. The compounds were tested for their in vitro activity against tubercular and various non-tubercular mycobacterium species in log-phase and 6-week-starved cultures. Compound N-(3,5-dibromopyridin-2-yl)-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide (4r) was found to be the most potent compound (MIC: 0.22 μM) and was 3 times more active than standard isoniazid (INH) and equally active as rifampicin (RIF) in log-phase culture of Mycobacterium tuberculosis H37Rv. In starved M. tuberculosis H37Rv, 4r inhibited with MIC of 13.9 μM and was found to be 50 times more active than INH and slightly more active than RIF.

Full text of DOI:10.1016/j.bmcl.2008.12.088

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1152–1154
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Nitrofuran-2-yl derivatives: Synthesis and inhibitory activities against
growing and dormant mycobacterium species
*
Dharmarajan Sriram , Perumal Yogeeswari, Prathiba Dhakla, Palaniappan Senthilkumar,
Debjani Banerjee, Thimmappa H. Manjashetty
Medicinal Chemistry & Antimycobacterial Research Laboratory, Pharmacy Group, Birla Institute of Technology & Science, Pilani, Hyderabad Campus,
Jawahar Nagar, Hyderabad, Andhra Pradesh 500 078, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Eighteen 5-nitrofuran-2-yl derivatives were prepared by reacting 5-nitro-2-furfural with various (sub)-
phenyl/pyridyl thiosemicarbazide using microwave irradiation. The compounds were tested for their
in vitro activity against tubercular and various non-tubercular mycobacterium species in log-phase
and 6-week-starved cultures. Compound N-(3,5-dibromopyridin-2-yl)-2-((5-nitrofuran-2-yl)methy-
Received 18 November 2008
Revised 17 December 2008
Accepted 20 December 2008
Available online 25 December 2008
lene)hydrazinecarbothioamide (4r) was found to be the most potent compound (MIC: 0.22 lM) and
was 3 times more active than standard isoniazid (INH) and equally active as rifampicin (RIF) in log-phase
culture of Mycobacterium tuberculosis H37Rv. In starved M. tuberculosis H37Rv, 4r inhibited with MIC of
Keywords:
5-Nitrofuran-2-yl derivatives
Thiosemicarbazones
Tuberculosis
13.9
lM and was found to be 50 times more active than INH and slightly more active than RIF.
Ó 2008 Elsevier Ltd. All rights reserved.
Dormant TB
Mycobacterium tuberculosis (MTB) causes more human deaths
than any other single infectious organism with an estimated eight
million new tuberculosis (TB) cases and two million fatalities each
year.1 MTB has two features that render it the deadliest infectious
disease to date: its high infectivity or virulence and its ability to
enter latency for subsequent reactivation, a phenomenon that
leads to deadliest synergy with acquired immune deficiency syn-
drome (AIDS).2 As a result TB is also the leading cause of death
for AIDS patients. Furthermore, the emergence of multi-drug resis-
tance TB (MDR-TB) is severely hampering TB treatment,3 and
therefore is an urgent need to develop novel TB chemotherapeutics.
A new TB treatment should offer at least one of three improve-
ments over the existing regimens: (a) shorten the total duration
of effective treatment and/or significantly reduce the total number
of doses needed to be taken under DOTS supervision; (b) improve
the treatment of MDR-TB, which cannot be treated with isoniazid
(INH) and rifampin (RIF) and/or (c) provide more effective treat-
ment of latent/dormant TB infection, which is essential for elimi-
nating TB. Earlier 5-nitrofuranayl-2-amide derivative was
reported for activity against MTB with minimum inhibitory con-
report synthesis of eighteen 5-nitrofuran-2-yl derivatives and its
in vitro activity against tubercular and various non-tubercular
mycobacterium species in log-phase and 6-week-starved cultures.
The synthesis of 5-nitrofuran-2-yl derivatives was carried out in
three steps, as shown in Scheme 1. First, to a solution of (sub)ani-
lines/(sub)2-aminopyridines (1a–r) (0.01 mol) in ethanol (10 mL)
was added potassium hydroxide (0.01 mol) and carbon disulfide
(0.75 mL), and the mixture was stirred at 15–20 °C for 1 h to form
a potassium salt of dithiocarbamate (2a–r). To the stirred mixture
was added hydrazine hydrate (0.01 mol), and the stirring was con-
tinued at 80 °C for 1 h to obtain corresponding (sub)phenyl/pyridyl
thiosemicarbazide (3a–r) in 90% yield. Thiosemicarbazide deriva-
tives on condensation with 5-nitro-2-furfural in the presence of
glacial acetic acid afforded various thiosemicarbazones (4a–r) (Ta-
ble 1) in 62–86% yields. The reaction utilizes the micro wave irra-
diation 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–chloro-
form mixture. The purity of the compounds was checked by TLC
and elemental analyses, and the compounds of this study were
identified by spectral data. In the 1H NMR spectra the signals of
the respective protons of the prepared derivatives were verified
on the basis of their chemical shifts, multiplicities, and coupling
constants. The spectra of all the compounds showed a double dou-
blets in the range of 7.55–7.56 d ppm and 7.59–7.60 d ppm corre-
sponding to third and fourth position protons of the furan ring, a
singlet at 7.43 d ppm corresponding to methylene proton and a
D2O exchangeable singlet at 7.26 d ppm corresponding to NH pro-
centration (MIC)4 of 0.0002
ious thiosemicarbazones with lowest MIC of 0.05
l
g/mL; and we have also reported var-
l
g/mL.5,6 Given
the promising biological profile of 5-nitrofuranayl-2-amide deriva-
tive and thiosemicarbazones, we decided to design new derivatives
by combining both the pharmacophores. In the present study, we
* Corresponding author. Tel.: +91 09010534101.
E-mail address: drdsriram@yahoo.com (D. Sriram).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.088

D. Sriram et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1152–1154
1153
S
CS2
NH2NH2.H2O
NH
C
SK
NH2
KOH
R
R
X
X
1a-r
2a-r
S
C
H
S
C
NH
NHN
R
O
X
NH
NHNH2
5-Nitro-2-furfural
R
4a-r
X
NO2
3a-r
CH3COOH
MWI
Scheme 1. Synthetic protocol of compounds 4a–r.
Table 1
Physical constants and antimycobacterial activity
S
H
NHC
NHN
NO2
R
O
X
a
Compound
R
X
Yield (%)
MP (°C)
IC50
(l
M)
MIC in lM
MTBb
MSc
MMd
MFe
MPf
MVg
43.0
41.0
78.0
16.9
9.6
MKh
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
H
4-CH3
4-OCH3
2-Br
4-Cl
4-SO2 NH2
2,4-(CH3)2
2,6-(CH3)2
2,5-(CH3)2
2,6-(C2H5)2
2,4-(OCH3)2
2,4-(NO2)2
H
H
H
H
H
H
H
H
H
H
H
H
H
N
N
N
N
59.8
33.1
45.6
87.9
90.9
88.6
41.0
76.9
90.5
53.8
83.7
91.1
65.1
78.7
78.9
83.6
91.8
60.3
138–140
152–154
179–180
160–162
152–155
154–155
146–147
185–186
134–135
150–151
197–200
125–127
178–179
200–201
195–197
204–205
199–200
185–186
>215.2
>205.4
>195.1
>169.2
>192.4
<169.2
>196.3
>196.3
>196.3
>180.4
>178.3
<164.3
214.5
10.7
5.1
9.7
0.54
1.23
2.1
2.45
4.9
1.2
1.1
4.4
0.52
12.1
5.1
2.6
1.2
172.2
41.0
39.0
33.8
19.1
8.4
314.1
39.2
157.0
36.0
35.6
16.4
48.4
20.5
20.5
2.4
86.1
20.5
78.0
67.1
38.4
67.6
78.5
78.5
157.0
72.1
17.8
32.8
48.4
10.2
20.5
38.3
39.1
6.9
43.0
41.0
39.0
33.8
38.4
33.8
39.2
39.2
78.5
72.1
35.6
65.1
48.4
10.2
5.1
10.7
2.5
4.85
16.9
9.6
16.8
4.9
9.8
4.8
36.0
4.4
32.8
6.0
10.2
5.1
38.3
39.1
6.9
91.15
30.38
4.71
5.3
20.5
39.0
8.4
38.4
33.8
9.8
39.2
78.5
36.0
35.6
32.8
12.1
5.1
2.1
39.2
39.2
2.4
72.1
71.1
65.1
24.2
20.5
2.6
4.8
39.1
3.4
182.3
3.80
4.71
3-CH3
4-CH3
5-Cl
4,6-(CH3)2
3,5-Br2
<204.7
<204.7
191.8
<195.7
139.1
5.1
4.8
39.1
1.7
182.3
38.3
39.1
3.4
22.82
1.89
4.71
N
N
2.4
4.8
6.9
45.57
1.89
2.35
0.22
0.66
0.23
4.71
Isoniazid
Rifampicin
>455.8
>75.9
22.82
30.38
2.35
7.59
9.45
Ciprofloxacin
>188.5
a
Cytotoxicity in mammalian Vero cell lines.
b
c
d
e
f
M. tuberculosis.
M. smegmatis.
M. microti.
M. fortuitum.
M. phlei.
g
h
M. vaccae.
M. kansasii.
tons. The elemental analysis results were within (0.4%) of the the-
oretical values.
of bacterial growth. MICs of the synthesized compounds along
with the standard drugs for comparison are reported (Table 1).
In the initial log-phase culture screening against MTB, the com-
pounds showed good activity with MICs ranging from 0.22 to
The compounds were screened for their in vitro antimycobacte-
rial activity against MTB, and non-tubercular mycobacterial (NTM)
species like M. smegmatis ATCC 14468, M. microti MTCC 1727, M.
vaccae MTCC 997, M. phlei MTCC 1724, M. fortuitum MTCC 951,
and M. kansasii MTCC 3058 by agar dilution method similar to that
recommended by the National Committee for Clinical Laboratory
Standards7 for the determination of MIC in triplicate. The mini-
mum inhibitory concentration (MIC) is defined as the minimum
concentration of compound required to give complete inhibition
12.1
ity with MIC of <1
(MIC: 0.66 M). One compound 4r with MIC of 0.22
to be more active than rifampicin (RIF) (MIC: 0.23
compounds were more potent than ciprofloxacin (MIC of
4.71 M). Compound N-(3,5-dibromopyridin-2-yl)-2-((5-nitrofu-
ran-2-yl)methylene)hydrazinecarbothioamide (4r) was found to
l
M. Three compounds (4d, 4l, and 4r) showed excellent activ-
M and were found to be more active than INH
M was found
M). Twelve
l
l
l
l
l

1154
D. Sriram et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1152–1154
Table 2
it is considerably less active against non-growing than against
log-phase cells. All the six tested compounds were more potent
than INH and one compound (4r) was found to be more potent
Inhibitory activities of selected compounds against log-phase and 6-week-starved
mycobacterial cultures
Compound
MIC in
l
M against MTBa
Compound
MIC in
l
M against MSb
(with MIC of 13.9 lM) than RIF (MIC: 15.2 lM). 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 effec-
tive in eliminating persistent or dormant bacilli. This study re-
vealed that, these molecules active against slowly growing or
non-growing persistent bacilli are thought to be important to
achieve a shortened therapy. In the case of starved MS culture,
the tested compounds inhibited with MIC values ranging from
Log-phase Six-week-
Log-phase Six-week-
cells
starved cells
cells
starved cells
4d
4i
4j
4l
4p
4r
INH
Rifampin
0.54
1.1
1.2
33.8
153.9
36.0
4e
4f
4l
4p
4q
4r
INH
Rifampin
19.1
8.4
133.7
75.6
16.4
2.4
213.2
43.2
0.52
1.2
32.8
38.3
4.8
91.2
0.22
0.66
0.23
13.9
729.1
15.2
6.9
45.57
1.89
124.2
>729.1
22.6
43.2 to 213.2
729.1 M). Compound N-(5-chloropyridin-2-yl)-2-((5-nitrofuran-
2-yl)methylene)hydrazinecarbothioamide (4p) was found to be
most active compound with MIC of 43.2 M.
The compounds were further examined for toxicity (IC50) in a
mammalian Vero cell line till 62.5
g/mL concentrations.10 After
lM and were more potent than INH (MIC:
l
a
M. tuberculosis.
M. smegmatis.
b
l
l
be the most active compound in vitro with MICs of 0.22 lM against
72 h of exposure, viability was assessed on the basis of cellular
conversion of MTT into a formazan product and the results are re-
ported in Table 1. Eighteen compounds when tested showed IC50
MTB. With respect to structure–MTB activity relationships, pyr-
idylthiosemicarbazones were found to be more active than phenyl-
thiosemicarbazones. Among the phenyl or pyridyl ring
substituents, electron-withdrawing groups like nitro (4l), and hal-
ogen (4d, 4e, 4p, and 4r) enhanced the activity. Compounds with
electron-donating groups decreased the activity considerably (4b,
4c, 4g–k, 4n, 4o, and 4q). Among the methyl substituted phenyl-
and pyridylthiosemicarbazones derivatives, the di-substituted
compounds (4g–j and 4q) showed enhanced activity compared
with the mono-substituted derivative (4b, 4n, and 4o). All the
compounds were also screened for atypical mycobacteria (AM),
AM infection8 an illness caused by a type of mycobacterium other
than tuberculosis which cause a wide variety of infections such as
abscesses, septic arthritis, and osteomyelitis. They can also infect
the lungs, lymph nodes, gastrointestinal tract, skin, and soft tis-
sues. The rate of AM infections is rare, but it is increasing as the
AIDS population grows. Populations at risk include individuals
who have lung disease and weakened immune systems. The syn-
thesized compounds inhibited M. smegmatis (MS) with MICs rang-
values ranging from 139.1 to >215.2 lM. These results are impor-
tant as these compounds with their increased cytoliability, are
much less attractive in the development of a compounds for the
treatment of TB. This is primarily due to the fact that the eradica-
tion of TB requires a lengthy course of treatment, and the need for
an agent with a high margin of safety becomes a primary concern.
The IC50 values of compound 4r was found to be 139.1 lM and
showed selectivity index (IC50/MIC) of 632.2.
Screening of the antimycobacterial activity of these 5-nitrofu-
ran-2-yl derivatives, identified N-(3,5-dibromopyridin-2-yl)-2-
((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide (4r) as a
new lead endowed with high activity towards log-phase and
starved MTB, and NTM. The present study reveals the importance
of these compounds effective for the treatment of TB, and NTM
infections. In conclusion, it has been shown that the potency, selec-
tivity, and low cytotoxicity of these compounds make them valid
leads for synthesizing new compounds that possess better activity
with low cytotoxicity. Further structure–activity and mechanistic
studies should prove fruitful.
ing from 2.4 to 172.2
than INH (MIC: 45.57
iotii the compounds showed activity with MICs ranging from 6.9 to
l
M and 13 compounds were more potent
lM). With regard to activity against M. micr-
157.0
22.82
l
l
M and four compounds were more potent than INH (MIC:
M). M. vaccae was inhibited by the synthesized compounds
Acknowledgments
with MICs ranging from 2.1 to 78.0
more potent than INH (MIC: 182.3
inhibited M. phlei (MP) with MICs ranging from 2.5 to 39.1
and were more potent than INH (MIC: 91.15 M). Against M. fortu-
itum the compounds showed activity with MICs ranging from 3.4
to 78.5 M and three compounds were more potent than INH
(MIC: 22.82 M). The compounds were also screened against M.
kansasi and were inhibited with MICs ranging from 1.7 to
39.2 M and all compounds were more potent than INH (MIC:
182.3 M). Compound 4r inhibited all the eight mycobacterium
l
l
M and all 18 compounds were
M). All the compounds also
The authors are thankful to Department of Biotechnology (BT/
01/COE/05/06/01), Government of India for their financial
assistances.
lM
l
References and notes
l
l
1. WHO Global Tuberculosis Control Report, 2008.
2. Bates, I.; Fenton, C.; Gruber, J.; Lalloo, D.; Lara, A.; Squire, S.; Theobald, S.;
Thomson, R.; Tolhurst, R. Lancet Infect. Dis. 2004, 4, 368.
3. Zignol, M.; Hosseini, M. S.; Wright, A.; Weezenbeek, C. L.; Nunn, P.; Watt, C. J.;
Williams, B. G.; Dye, C. J. Infect. Dis 2006, 194, 479.
4. Lee, R.; Tangallapally, R. P.; McNeil, M. R.; Anne, L. WO 2005/007625.
5. Sriram, D.; Yogeeswari, P.; Thirumurugan, R.; Kumar, P. R. J. Med. Chem. 2006,
49, 3448.
6. Sriram, D.; Yogeeswari, P.; Prathiba, D.; Senthilkumar, P.; Debjani, B. Bioorg.
Med. Chem. Lett. 2007, 17, 1888.
7. National Committee for Clinical Laboratory Standards. Antimycobacterial
susceptibility testing for Mycobacterium tuberculosis. Proposed standard M24-
T. National Committee for Clinical Laboratory Standards, Villanova, PA, 1995.
8. Katoch, V. M. Ind. J. Med. Res. 2004, 120, 290.
l
l
species with MIC ranging from 0.22 to 6.9
lM and was more potent
than INH.
The compounds which showed good activity against log-phase
culture of MTB and MS were further screened against 6-week-
starved cells of MTB and MS according to the literature procedure.9
Against MTB, six compounds were tested and they inhibited
starved culture of MTB with MICs ranging from 13.9 to 153.9
(Table 2). INH had poor activity against starved cells with MIC of
729.1
M. As previously observed9 RIF retained activity, although
lM
9. Xie, Z.; Siddiqi, N.; Rubin, E. R. Antimicrob. Agents Chemother. 2005, 49, 4778.
10. Gundersen, L. L.; Nissen-Meyer, J.; Spilsberg, B. J. Med. Chem. 2002, 45, 1383.
l