Diversity oriented design of various hydrazides and their in vitro evaluation against Mycobacterium tuberculosis H37Rv strains

Article abstract of DOI:10.1016/j.bmcl.2011.06.074

Control and prevention of tuberculosis is a major challenge, as one-third of the world's population is infected with Mycobacterium tuberculosis. The resurgence of tuberculosis and the emergence of multidrug-resistance strains of mycobacteria, necessitate the search for new class of antimycobacterial agents. As a part of investigation of new antitubercular agents in this laboratory, we describe the syntheses of various hydrazides of comarins, quinolones and pyrroles and screening against M. tuberculosis (Mtb) H37_Rv by using rifampin as a standard drug. Among the designed molecules, the most prominent compounds 2a-g, 4a and 9a showed >90% GI at MIC <6.25 μg/mL. Finally, these studies suggests that compounds 2a-g, 4a and 9a may serve as promising lead scaffolds for further generation of new anti-TB agents.

Full text of DOI:10.1016/j.bmcl.2011.06.074

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4728–4731
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diversity oriented design of various hydrazides and their in vitro
evaluation against Mycobacterium tuberculosis H37Rv strains
Atul Manvar a, Abhay Bavishi b, Ashish Radadiya b, Jignesh Patel c, Vipul Vora d, Narshih Dodia e,
Kena Rawal c, Anamik Shah b,
⇑
a Institut für Organische und Biomolekulare Chemie, Georg-August-Universität Göttingen, 37077 Göttingen, Germany
b Medicinal Chemistry Laboratory, Department of Chemistry, Saurashtra University, Gujarat, Rajkot 360 005, India
c Alembic Research Center, Alembic Pharmaceuticals Limited, Gujarat, Vadodara 390 003, India
d Lupin Pharmaceuticals Limited, Ankleshwar, Gujarat, India
e Surya Pharmaceuticals Limited, Chandigarh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Control and prevention of tuberculosis is a major challenge, as one-third of the world’s population is
infected with Mycobacterium tuberculosis. The resurgence of tuberculosis and the emergence of multi-
drug-resistance strains of mycobacteria, necessitate the search for new class of antimycobacterial agents.
As a part of investigation of new antitubercular agents in this laboratory, we describe the syntheses of
various hydrazides of comarins, quinolones and pyrroles and screening against M. tuberculosis (Mtb)
H37Rv by using rifampin as a standard drug. Among the designed molecules, the most prominent com-
pounds 2a–g, 4a and 9a showed >90% GI at MIC <6.25 lg/mL. Finally, these studies suggests that com-
pounds 2a–g, 4a and 9a may serve as promising lead scaffolds for further generation of new anti-TB
agents.
Received 13 March 2011
Revised 29 May 2011
Accepted 16 June 2011
Available online 23 June 2011
Keywords:
Coumarin
Quinolone
Pyrrole
Hydrazides
Mtb
Ó 2011 Elsevier Ltd. All rights reserved.
H37Rv
Tuberculosis (TB) is a worldwide pandemic caused by different
mycobacterial species. According to the latest statistic, around
2 million people globally die annually by tuberculosis and 8 mil-
lion new cases each year, out of which developing countries have
significant contribution.1 Tuberculosis is the world’s second cause
of death after AIDS. Control and prevention of tuberculosis is
challenging and a major challenge for tuberculosis control is the
development of multidrug resistant tuberculosis (MDR-TB).2
Unfortunately, no new antibiotics or clinical candidates has been
developed in the past few decades.3 There are three front-line anti-
biotics, isoniazide, pyrazinamide, and rifampin and several second-
line antibiotics including ethionamide, para-aminosalicylic acid,
kanamycin, amikacin, capreomycin, ciprofloxacin, streptomycin,
etc. are used currently to cure tuberculosis.4 The existing treatment
of TB requires an exceedingly lengthy therapy and often needs a
cocktail of three or four different drugs.5 There are several demerits
of afore mentioned therapies such as prolonged treatment sche-
dule, host toxicity, ineffectiveness against resistance strains, etc.6
As a consequences of long term therapies have frequently led to
patients non-compliance and, in turn contributed to the MDR-TB
as well as extensively drug resistance tuberculosis (XDR-TB).7
The AIDS pandemic has led to an explosion of HIV-TB co-infection
of patients living with HIV/AIDS.8 The ever-increasing drug resis-
tance, toxicity and side effects of currently used antitubercular
drugs and disappearance of their antibacterial activity leads to a
pressing need for safer and prominent drug.9
To overcome the problems associated with the available treat-
ments, a wide network of organizations, donors and countries
has recently initiated the ‘Global Plan To STOP TB 2006–2015.’10
To accomplish these objectives, basic research at molecular level
is directed toward the identification of mechanism and validation
of new targets for drugs and new chemical entity.11 In this context,
we have initiated our synthetic program to work with various
privilege heterocyclic entities and its biological evaluation in
antitubercular therapeutic area12 and its 3D-QSAR analysis. Among
the heteroarenes, naturally occurring derivative of coumarin,
Calanolide A13 demonstrates potent antitubercular activity (MIC
3.13 lg/mL). In viewing such a prominent activity of Calanolide
A, we thought that functional group manipulations in coumarin
heterocycles may be a good starting point. As a part of coumarin
analogues, comarin-4-acetic acid benzylidene hydrazides14 and
4-arylaminocoumarins15 have been proved as highly active antitu-
bercular scaffolds. In order to understand the relationship between
structure and activity, 3D-QSAR analysis were also studies. In the
⇑
Corresponding author. Tel.: +91 281 2589609; fax: +91 281 2581013.
E-mail address: anamik_shah@hotmail.com (A. Shah).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.074

A. Manvar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4728–4731
4729
current communication, we decided to focus our attention on
syntheses and antitubercular evaluation against Mycobacterium
tuberculosis (Mtb) H37Rv strains of novel analogues of coumarin,
quinolone and pyrrole carbohydrazides. In the past few years, sev-
eral proposals intended at understanding the mechanism of action
of various drugs, particularly hydrazide functionality against
Mtb.16 Very recently, there are several reports in which hydrazide
scaffolds have been studied as potent antituberculars. As a part
of the studies, 4-quinolylhydrazones were tested against Mtb, most
of the compounds in the explored series expressed 100%
inhibition.17 Moreover, benzimidazole-5-carbohydrazides have
been in vitro evaluated against MDR-MTB.18 The hydrazones of
5-nitro-2-furoic acid has been evaluated against Mtb, the most
assay (MABA).25 The drugs in clinical use, rifampicin was used as a
reference. The compounds 2a–q (Table 1) was prepared with dif-
ferently substituted donor and acceptor groups in the core struc-
ture. In a structure–activity relationship (SAR) studied some
interesting trend that has been observed among the conjugates
and a number of compounds were found to be potent against
Mtb. The growth inhibition was observed in the range of 60–
100% at MIC <6.25 lM. The significantly active compound 2a hav-
ing 4-Br and electron acceptor 3-NO2 groups in the rings A and B,
respectively, exhibits 100% inhibition. Interestingly, the molecule
2b bearing 2-Ph group in the ring A and 3-NO2 in the ring B was
also observed as most potent (100% GI). The compound 2c having
donor and acceptor functionalities such as 2-Me, 3-Cl in the ring A
and 3-NO2 in the ring B, expressed 98% inhibition. From these find-
ings, it can be postulated that the presence of nitro group at C3 in
the ring B is significant for this specifically designed library. In or-
der to confirm the behavior and role played by NO2 group in the
ring B, more compounds were designed and evaluated by varying
the substitutions. Among the designed molecular diversity,
compounds 2j, 2k, and 2l has shown good activity (>70% GI) while
2n and 2p has shown <70% GI, indicating that alteration of the po-
sition of nitro at C2 in the ring B, affects the activity. It is quite
noteworthy that lacking of any substituents in compound 2e was
found highly active (97% inhibition). It can be postulated that in-
creased in potency of unsubstituted molecule 2e can be attributed
to combination of steric and electronic factors. In compound 2n,
the blending of halogenated acceptors such as Cl, F in the ring A
and 3-NO2 in the ring B have shown satisfactory reduction in po-
tency (70% GI), it indicates that Cl and F is not particularly favoured
for antitubercular activity in designed data set. If both the rings
were substituted with electron poor NO2 group 2q, it shows con-
siderably least potency, thus suggesting that modification of NO2
group at C3 position in the ring B is also preferable. Impressively,
if both the rings were decorated with Cl, compound 2d is highly ac-
tive analogue (98% GI). The compound bearing donor 3-Me in the
ring A and acceptor 2-NO2 in the ring B, compound 2f is found to
be significant biologically active (95% GI). Additionally, the alkyl
substituted quinazolyl-hyrazone 2g, was also found noticeably
promising.
active molecule having MIC 2.65
l
M.19 Furthermore, several guan-
ylhydrazones were screened against Mtb, the most prominent
compound exhibiting MIC 6.49
l
M.20 trans-Cinnamic acid have a
wide range of therapeutic importance,21 using molecular hybrid-
ization approach between isoniazide and trans-cinnamic acid, the
diverse set of molecules were in vitro evaluated. Among the
designed molecular diversity, the highly active analogue expressed
MIC 3.12 l
M.22
At the outset of our studies, the synthetic route to produce a di-
verse hydrazones of coumarins and quinolones are portrayed in
Schemes 1–3. The syntheses of carbohydrazones of pyrrole are de-
picted in Scheme 4. The corresponding hydrazones were prepared
by Meerwein arylations.23 The heteroarenes (4-hyroxycoumarins
and 4-hyroxyquinolones) were treated with corresponding diazo-
nium salts at lower temperature, leading to syntheses of molecu-
larly diverse hydrazones. The compounds 2a–q, 4a–f and 6a–f
were obtained in moderate to good yields. While pyrrole 8 was
treated with corresponding aldehydes in the presence of catalytic
amount of glacial acetic acid under refluxing conditions led to syn-
theses of compounds 9a–f.24 The structures of the compounds
were confirmed by 1H NMR and 13C NMR analysis. The antimyco-
bacterial activities of the compounds under study were assessed
at Tuberculosis Antimicrobial Acquisition and Coordinating Facility
(TAACF, USA) against Mtb H37Rv using the microplate alamar blue
R
N
R
N
The above findings of antimycobacterial activity prompted us to
design phenylhydrazono-chromenes (Table 2). Among the de-
signed molecules substituted with electron acceptor functional
group such as 4a having 4-NO2 and 4b having 4-Cl in the ring A,
showed 94% and 88% inhibitory activity towards Mtb, respectively.
However, the inclusion of various electron donors such as Me and
OMe in the ring A led to dramatically reduce the potency. The phe-
nyl ring when substituted by electron rich Me group (compounds
4c and 4e) showed 58% and 55% inhibitions, respectively. Whereas,
the phenyl ring bearing donor OMe (compound 4f), showed 55%
inhibitions. This observation indicates that the screenings of
various functional groups in the ring A are preferable. The unsub-
stituted analogue 4d, were found to be weakly potent. We can thus
O
O
N
H
N
Y
Y
(i)
X
40-65%
OH
R = H or Me
O
R = H or Me
2a-q
1
Scheme 1. Synthesis of 3-(2-phenylhydrazono)quinoline-2,4-(1H,3H)-dione deriv-
atives (2a–q). Regents and conditions: (i) ArN2Cl, 0–5 °C, 1.5 h.
O
O
O
O
N
H
N
(i)
X
observe
a strong relationship between the substituents that
49-54%
OH
O
4a-f
increase the electronic density.
3
In order to explore and evaluate more diverse compound
library, ring modification was carried out (Table 3). A small library
was screened in which phenyl ring was introduced as a side arm at
C6 position of the coumarin ring instead of benzo fusion. The
designed molecules 6a–f bearing various donor and acceptor
groups such as NO2, Me, Cl and F in the ring A was evaluated
against Mtb for their antitubercular activity. Unfortunately none
of the screened conjugates have shown considerable potency
(GI <25%). Hence we postulate that, introduction of bulky group
(6-phenyl in the coumarin heterocycle) is not compounds of
interest for Mtb. Furthermore, antitubercular activities of various
carbohydrazides of pyrrole are summarized in Table 4. The only
Scheme 2. Synthesis of 2-(2-phenylhydrazono)-1H-benzo[f]chromene-1,3(2H)-
diones (4a–f). Regents and conditions: (i) ArN2Cl, 0–5 °C, 1.5 h.
O
O
O
O
O
N
H
N
(i)
Ph
Ph
X
49-57%
OH
6a-f
5
Scheme 3. Synthesis of 6-phenyl-3-(2-phenylhydrazono) chroman-2,4-dione
derivatives (6a–f). Regents and conditions: (i) ArN2Cl, 0–5 °C, 1.5 h.

4730
A. Manvar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4728–4731
Me
COMe
Me
Me
EtO2C
COMe
Me
Me
COMe
Me
(ii)
(i)
H2NHN
N
HN
N
H
8
N
H
7
40-67%
N
H
62%
O
O
9a-f
X
Scheme 4. Synthesis of 4-acetyl-N0-benzylidene-3,5-dimethyl-1H-pyrrole-2-carbohydrazides. Reagents and conditions: (i) NH2NH2ꢀH2O/EtOH (reflux), 5 h (ii) ArCHO, EtOH,
cat. AcOH (glacial).
Table 3
Table 1
The in vitro activity of compounds 6a–f against M. tuberculosis H37Rv strains
The in vitro activity of compounds 2a–q against M. tuberculosis H37Rv strains
O
O
R
2
H
N
N
O
N
2
3
3
4
H
N
Ph
N
Y
B
5
2
X
A
5
4
3
4
O
6
A
X
O
6
6a-f
5
2a-q
Compounds
X
GIa (%)
MICb
(l
g/mL)
C Log Pc
Compounds
Substitutions
Y
GIa (%)
MICb
g/mL)
C Log Pc
6a
6b
6c
6d
6e
6f
2-NO2
3-Me
2-Cl, 6-Cl
2-Cl, 3-F
4-F
25
23
23
19
18
17
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
4.097
4.853
5.780
5.210
4.497
4.853
(l
R
X
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
4-Br
2-Ph
2-Me, 3-Cl
4-Cl
3-NO2
3-NO2
3-NO2
4-Cl
100
100
98
98
97
95
94
89
85
82
80
79
70
70
64
61
60
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
2.643
3.668
2.728
3.463
2.037
3.035
3.412
3.086
3.343
3.249
2.279
3.086
3.249
2.636
2.493
2.493
1.523
2-Me
a
(GI) Growth inhibition of virulent strain of M. tuberculosis.
MIC of Rifampin: 0.015–0.125
inhibition).
H
H
b
lg/mL against M. tuberculosis H37Rv (97%
3-Me
2-NO2
4-NO2
H
2-NO2
H
c
C Log P is calculated on ChemDraw Ultra 12.0.
2-Cl, 3-Cl
2-Cl, 3-Cl
2-NO2
2-NO2
2-NO2
3-Cl
3-NO2
4-Cl
2-NO2
2-NO2
3-Cl
2-NO2
3-Cl, 4-Cl
3-Me
4-Cl, 5-F
2-NO2
4-Cl
Table 4
The in vitro activity of compounds 9a–f against M. tuberculosis H37Rv strains
O
Me
O
Me
Me
3-NO2
X
A
3
5
2
N
H
N
NH
a
(GI) Growth inhibition of virulent strain of M. tuberculosis.
MIC of Rifampin: 0.015–0.125
inhibition).
4
b
l
g/mL against M. tuberculosis H37Rv (97%
6
9a-f
c
C Log P is calculated on ChemDraw Ultra 12.0.
Compounds
X
GIa (%)
MICb
(
l
g/mL)
C Log Pc
9a
9b
9c
9d
9e
9f
4-Me
4-NO2
5-Cl
3-NO2
4-NMe2
5-NO2
92
32
29
21
18
18
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
2.945
2.189
3.159
2.189
2.681
2.189
Table 2
The in vitro activity of compounds 4a–f against M. tuberculosis H37Rv strains
O
O
H
N
2
A
5
a
3
4
N
(GI) Growth inhibition of virulent strain of M. tuberculosis.
MIC of Rifampin: 0.015–0.125 lg/mL against M. tuberculosis H37Rv (97%
inhibition).
X
b
O
6
c
C Log P is calculated on ChemDraw Ultra 12.0.
4a-f
Compounds
X
GIa (%)
MICb
(
lg/mL)
C Log Pc
4a
4b
4c
4d
4e
4f
4-NO2
4-Cl
5-Me, 6-Me
H
3-Me, 5-Me
4-OMe
94
88
64
62
58
55
<6.25
<6.25
<6.25
<6.25
<6.25
<6.25
4.139
4.353
4.588
3.644
4.638
3.559
hydrazides with electron donor substituted in the ring A, increased
in electron density, was found conformationally unfavorable. The
physicochemical parameter, C log P has been calculated from
ChemDraw Ultra 12.0. The thumb rule for C Log P values, to a drug
like molecules must be lowers than ‘5’ to by-pass the cell barrier.
The C Log P values seem to correlate to some extent with lipophil-
icity. Most of the compounds in the designed series showed C Log P
values less than 5.
a
(GI) Growth inhibition of virulent strain of M. tuberculosis.
MIC of Rifampin: 0.015–0.125 lg/mL against M. tuberculosis H37Rv (97%
inhibition).
b
c
C Log P is calculated on ChemDraw Ultra 12.0.
In conclusion, the presented work demonstrates the syntheses
and biological evaluation of various hydrazides as potent antimy-
cobacterial agents. Among the designed molecules, 2a–g, 4a and
9a has shown significant activity against M. tuberculosis H37Rv. In
addition, these new agents merit further studies to explore them
as potential chemotherapeutics for tuberculosis and for lead opti-
mization to design novel antitubercular agents.
compound possessing donor 4-Me group 9a, exhibited 92% inhibi-
tion. The compound having 5-NO2 group in the ring A showed least
potency (18% GI). The compounds bearing various donor and
acceptors 4-NO2, 5-Cl, 3-NO2, 4-NMe2 (9b–f) in the ring A were
found to be almost inactive against Mtb H37Rv strains. Pyrrole-

A. Manvar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4728–4731
4731
15. Virsodia, V.; Shaikh, M. S.; Manvar, A.; Desai, B.; Parecha, A.; Loriya, R.;
Dholariya, K.; Patel, G.; Vora, V.; Upadhyay, K.; Denish, K.; Shah, A.; Coutinho, E.
C. Chem. Biol. Drug Des. 2010, 76, 412.
16. (a) Shinkdikar, A. V.; Viswanathan, C. L. Bioorg. Med. Chem. Lett. 2005, 15, 1803;
(b) Timmins, G.; Master, S.; Rusnak, F.; Dereic, V. Antimicrob. Agents Chemother.
2004, 48, 3006; (c) Bagchi, M. C.; Maiti, B. C.; Mills, D.; Basak, S. C. J. Mol. Model.
2004, 10, 102.
17. Gemma, S.; Savini, L.; Altarelli, M.; Tripaldi, P.; Chiasserini, L.; Coccone, S. S.;
Kumar, V.; Camodeca, C.; Campiani, C.; Novellino, E.; Clarizio, S.; Delogu, G.;
Butini, S. Bioorg. Med. Chem. 2009, 17, 6063.
18. Camacho, J.; Barazarte, A.; Gamboa, N.; Rodrigues, J.; Rojas, R.; Vaisberg, A.;
Gilman, R.; Charris, J. Bioorg. Med. Chem. 2011. doi:10.1016/j.bmc.2011.01.050.
19. Sriram, D.; Yogeeswari, P.; Vyas, D.; Senthilkumar, P.; Bhat, P.; Srividya, M.
Bioorg. Med. Chem. Lett. 2010, 20, 4313.
Acknowledgements
The authors gratefully acknowledge Dr. Cecil Kwong, Tubercu-
losis Antimicrobial Acquisition and Coordinating Facility (TAACF),
USA for antitubercular screening and Department of Science and
Technology (DST) New Delhi, India for financial support under
the project entitled ‘National Facility for Drug Discovery through
New Chemical Entities (NCEs) Development and Instrumentation
Support to Small Manufacturing Pharma Enterprise’ under DPRS.
Supplementary data
20. Bairwa, R.; Kakwani, M.; Tawari, N. R.; Lalchandani, J.; Ray, M. K.; Ranjan, M. G.
R.; Degani, M. S. Bioorg. Med. Chem. Lett. 2010, 20, 1623.
21. (a) Bezerra, D. P.; Castro, F. O.; Alves, A. P. N. N.; Pessoa, C.; Moraes, M. O.;
Silveira, E. R.; Lima, M. A. S.; Elmiro, F. J. M.; Costa-Lotufo, L. V. Braz. J. Med. Biol.
Res. 2006, 39, 801; (b) Naz, S.; Ahmad, S.; Rasool, S. A.; Sayeed, S. A.; Siddiqi, R.
Microbiol. Res. 2006, 161, 43; (c) Rastogi, N.; Goh, K. S.; Horgen, L.; Barrow, W.
W. FEMS Immunol. Med. Microbiol. 1998, 21, 149.
22. Carvalho, S. A.; Da Silva, E. F.; De Souza, M. V. N.; Lourenço, M. C. S.; Vicente, F.
R. Bioorg. Med. Chem. Lett. 2008, 18, 538.
23. Shen, G.; Yu, X. –M.; Blagg, B. S. J. Bioorg. Med. Chem. Lett. 2004, 14, 5903.
24. Murineddu, G.; Loriga, G.; Gavini, E.; Peana, A. T.; Mulé, A. C.; Pinna, G. A. Arch.
Pharm. Pharm. Med. Chem. 2001, 334, 393.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.074.
References and notes
1. WHO Global tuberculosis control: a short update to the 2009 report http://
www.who.int/tb/publications/global_report/2009/update/en/index.html.
2. Kumar, K.; Awasthi, D.; Lee, S.-Y.; Zanardi, I.; Ruzsicska, B.; Knudson, S.; Tonge,
P. J.; Slayden, R. A.; Ojima, I. J. Med. Chem. 2011, 54, 374.
3. Maresca, A.; Carta, F.; Vullo, D.; Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem.
Lett. 2009, 19, 4929.
4. (a) Lugo, M. –T.; Bewley, C. A. J. Med. Chem. 2008, 51, 2606; (b) Janin, Y. L.
Bioorg. Med. Chem. 2007, 15, 2479. and references cited therein.
5. Blumberg, H. M.; Burman, W. J.; Chaisson, R. E.; Daley, C. L.; Etkind, S. C.;
Friedman, L. N.; Fujiwara, P.; Grzemska, M.; Hopewell, P. C.; Iseman, M. D.;
Jasmer, R. M.; Koppaka, V.; Menzies, R. I.; O’Brien, R. J.; Reves, R. R.; Reichman,
L. B.; Simone, P. M.; Starke, J. R.; Vernon, A. A. Am. J. Respir. Crit. Care Med. 2003,
167, 603.
6. Saquib, M.; Gupta, M. K.; Sagar, R.; Prabhakar, Y. S.; Shaw, A. K.; Kumar, R.;
Maulik, P. R.; Gaikwad, A. N.; Sinha, S.; Srivastava, A. K.; Chaturvedi, V.;
Srivastava, R.; Srivastava, B. S. J. Med. Chem. 2007, 50, 2942.
25. Antimycobacterial assay: Antitubercular activity was determined using the
modified BACTEC 460 system in which stock solutions as test compounds were
prepared in dimethylsulfoxide (DMSO) at 1 mg/mL and sterilized by passage
through 0.22 lm PFTE filters (Millex-FG, Millepore, Bedford, MA) 50 mL was
added to 4 mL radiometric 7H12 broth (BACTEC-12B; Bectron Dickinson
Diagnostic Instrument system, Sparks, MD) to achieve final concentration of
6.25 lg/mL. Controls received 50 lL DMSO. Rifampin (Sigma Chemicals Co., St.
Louis, MO) was included as a positive drug control. Rifampicin was solubilized
and diluted in DMSO and added to BACTEC-12 broth to achieve a range of
concentration for determination of minimum inhibitory concentration (MIC,
lowest concentration inhibiting 99% of the inoculums, MIC value of rifampicin
is 0.25 g/mL at 95% inhibition of H37Rv strain). M. tuberculosis H37Rv strain
(ATCC 27294; American type culture collection, Rockville, MD) was cultured at
37 °C on a rotary shaker in middle brook 7H9 broth (Difco Laboratories, Detroit,
MI) supplemented with 0.2 v/v glycerol and 0.05% v/v Tween 80, until the
culture turbidity achieved an optical density of 0.45–0.55 at 550 nm. Bacteria
were pelleted by centrifugation, washed twice and resuspended in one fifth of
the original volume in Dulbecoo’s phosphate buffered saline [PBS, Irvine
Scientific, Santa Ana (A)]. Large bacterial clumps were removed by passage
7. Pathak, A. K.; Pathak, V.; Suling, W. J.; Riordan, J. R.; Gurcha, S. S.; Besra, G. S.;
Reynolds, R. C. Bioorg. Med. Chem. 2009, 17, 872.
8. Bloom, B. R.; Murray, C. J. Science 1992, 257, 1055.
9. Horita, Y.; Takii, T.; Chiba, T.; Kuroishi, R.; Maeda, Y.; Kurono, Y.; Inagaki, E.;
Nishimura, K.; Yamamoto, Y.; Abe, C.; Mori, M.; Onozaki, K. Bioorg. Med. Chem.
Lett. 2009, 19, 6313.
10. http://www.stoptb.org/ (accessed July 2007).
11. Ventura, C.; Martins, F. J. Med. Chem. 2008, 51, 612.
through an 8 lm filter (Malgene, Rochester, NY) and aliquots were frozen at
ꢁ80 °C. Cultures were prepared and an appropriate dilution performed such
that a BACTEC-12B vial inoculated with 0.1 mL would reach a growth index
(GI) of 999 in five days. One tenth of the diluted inoculum was used to
inoculate 4 mL of fresh ACTEC-12B broth containing the test compounds. An
additional control vial was included which received a further 1:100 diluted
12. (a) Dodia, N.; Shah, A. Indian J. Heterocycl. Chem. 1999, 9, 139; (b) Dodia, N.;
Shah, A. Indian J. Heterocycl. Chem. 2000, 10, 69; (c) Virsodia, V.; Pissurlenkar, R.
R.; Manvar, D.; Dholakia, C.; Adlakha, P.; Shah, A.; Coutinho, E. C. Eur. J. Med.
Chem. 2008, 43, 2103; (d) Gaveriya, H.; Desai, B.; Vora, V.; Shah, A. Indian J.
Pharm. Sci. 2002, 64, 59; (e) Kharkar, P. S.; Desai, B.; Gaveria, H.; Varu, B.; Loriya,
R.; Naliapara, Y.; Shah, A.; Kulkarni, V. M. J. Med. Chem. 2002, 45, 4858; (f)
Desai, B.; Sureja, D.; Naliapara, Y.; Shah, A.; Saxena, A. K. Bioorg. Med. Chem.
2001, 9, 1993; (g) Manvar, A.; Pissurlenkar, R. R. S.; Virsodia, V.; Upadhyay, K.;
Manvar, D.; Mishra, A.; Acharya, H.; Parecha, A.; Dholakia, C.; Shah, A.;
Coutinho, E. C. Mol. Divers. 2010, 14, 285.
inoculum (as well as 50 lL DMSO) for use in calculating the MIC of rifampicin,
respectively, by established procedures. Cultures were incubated at 37 °C and
the Growth of Inhibition (GI) determined daily until control cultures achieved a
GI of 999. Assays were usually completed in 5-8 days. Percent inhibition was
defined as 1-(GI of test sample/GI of control) 100. Minimum inhibitory
concentration of compound effecting a reduction in daily change in GI, which
was less than that observed with a 1:100 diluted control culture on day the
later reached a GI of at least 30.
13. Nayyar, A.; Jain, R. Curr. Med. Chem. 2005, 12, 1873.
14. Manvar, A.; Malde, A.; Verma, J.; Virsodia, V.; Mishra, A.; Upadhyay, K.;
Acharya, H.; Coutinho, E.; Shah, A. Eur. J. Med. Chem. 2008, 43, 2395.