Thienopyrimidines as novel inhibitors of mycobacterium tuberclosis: Synthesis and in-vitro studies

Article abstract of DOI:10.1002/ardp.201000394

A series of novel 5,6-unsubstituted thieno-[2,3-d]-pyrimidines has been synthesized and tested for growth inhibition of Mycobacterium tuberculosis H37Rv. Of twelve compounds synthesized eleven have shown antimycobacterial activity that differs in potency. Compounds 7b, 7c, 7d, 7e, 7f, and 7g exhibited good antimycobacterial activity. MIC values of the compounds tested were comparable with pyrazinamide. Six compounds which have shown good antimycobacterial activity were also subjected for cytotoxicity studies and were found to possess poor cytotoxicity. The study indicates the definite need for focusing attention on thienopyrimidines for further lead optimization.

Full text of DOI:10.1002/ardp.201000394

Arch. Pharm. Chem. Life Sci. 2011, 344, 459–465
459
Full Paper
Thienopyrimidines as Novel Inhibitors of Mycobacterium
tuberclosis: Synthesis and In-vitro Studies
P. Rashmi, Laxmi Venkatesh Gurachar Nargund, Kuntal Hazra, and J. N. Narendra Sharath Chandra
Department of Pharmaceutical Chemistry, Nargund College of Pharmacy, Bangalore, Karnataka, India
A series of novel 5,6-unsubstituted thieno-[2,3-d]-pyrimidines has been synthesized and tested for
growth inhibition of Mycobacterium tuberculosis H37Rv. Of twelve compounds synthesized eleven have
shown antimycobacterial activity that differs in potency. Compounds 7b, 7c, 7d, 7e, 7f, and 7g
exhibited good antimycobacterial activity. MIC values of the compounds tested were comparable
with pyrazinamide. Six compounds which have shown good antimycobacterial activity were also
subjected for cytotoxicity studies and were found to possess poor cytotoxicity. The study indicates the
definite need for focusing attention on thienopyrimidines for further lead optimization.
Keywords: Antimycobacterials / Mycobacterium tuberculosis / Thienopyrimidines
Received: December 18, 2010; Revised: January 16, 2011; Accepted: January 20, 2011
DOI 10.1002/ardp.201000394
Introduction
keen focus on a family of proteins which are involved in
modifications of mycolic acids, very long chain lipids and
these proteins have been demonstrated to be important in
the persistent phase of infection, many of which contribute
to its pathogenicity. Several studies indicate that functional
groups in the acyl chain of mycolic acids are important for
pathogenesis and persistence [2].
The World Health Organization has warned that the inci-
dence of drug resistant tuberculosis has reached a record
level, becoming a serious threat to global health [1]. HIV and
other immune suppressing conditions have dramatically
increased the risk of developing active tuberculosis and
HIV co-infection makes tuberculosis more difficult to diag-
nose and treat. Multi-drug resistant strains of tuberculosis
(TB) could become dominant forms of the disease in the next
few decades, adding heavy financial and medical burdens to
already struggling health systems.
Some reports have already indicated the usefulness
of thienopyrimidine as antitubercular drug [1, 2]. This
prompted us to continue our studies with the intention of
designing and synthesizing thienopyrimidines 7(a–l) and
evaluating them for their antitubercular activity.
In a series of studies into TB, scientists said ‘‘superbug’’
strains of the disease were have been gaining ground in some Results and discussion
countries and called for greater investment into research and
development of new drugs and possible vaccines. Scientists
pointed out that the combined impact of new drugs,
vaccines, and diagnostic tests could cut worldwide incidence
of TB by 94 percent by 2050, but the investment needed to
bring this about is falling way too short of the requirement.
This has created the need for developing simpler, economi-
cally viable molecules for tuberculosis. Priority for the dis-
covery of new anti-tuberculosis drugs interest has led to a
Compound 3 was formed (Scheme 1) by the condensation of
ethylcyanoactate with 2,5-dihydroxy-1,4-dithiane in presence
of triethylamine. Compound 4 was prepared through con-
densation reaction between formamide and compound (3)
followed by cyclization. IR and NMR spectra confirmed the
formation of compound 4. Compound 4 as it exists in two
tautomeric forms IR spectra showed the presence of ketone at
1660 cmꢀ1 and NMR spectra showed a broad peak which was
not prominent due to tautomerism. Absence of doublet due
to NH2 in IR spectra, absence of quartet and triplet due to
–CH2CH3 at 2-4 ppm in NMR spectra confirms the cyclization.
The 4-hydroxythieno[2,3-d] pyrimidine 4 treated with potas-
sium carbonate in dry acetone to form potassium salt to
make the compound to exist in lactim form (lactam–lactim
Correspondence: P. Rashmi, Department of Pharmaceutical Chemistry,
Nargund College of Pharmacy, Banashankari III stage, Bangalore 560085,
Karnataka, India.
E-mail: rshvsh@gmail.com
Fax: 91-80-26421903
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

460
P. Rashmi et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 459–465
O
S
OH
(i)
CN
CH2
COOC2H5
O
CH3
S
NH2
HO
S
(1)
(2)
(3)
(ii)
O
O
CH3
O
N
NH
O
(iii)
N
S
N
S
(4)
(5)
(iv)
NH
NH
O
N
R
O
N
NH2
O
O
(v)
N
N
S
S
N
(6)
(7a-7l)
Scheme 1. (i) DMF, triethylamine, (ii) HCONH2, (iii) ClCH2COOC2H5, CH3ONa, dry acetone, (iv) NH2NH2 ꢁ H2O, ethanol, (v) R-CHO,
glacial acetic acid, ethanol.
tautomerism) which was then allowed to react with ethyl-
chloroacetate to form 5. Disappearance of –NH peak in
IR spectra and appearance of quartet at 4.16–4.21 ppm
and triplet at 1.21–1.25 confirms the formation of the
compound.
Peak at 3165.29 cmꢀ1 due to –NH and doublet at
3290.67 cmꢀ1 and 3178.79 cmꢀ1 due to –NH2 in IR spectra
as well as singlet at 4.2 ppm due to –NH2 and singlet at
9.1 ppm due to NH in NMR confirms the formation of com-
pound (6) obtained by treating compound 5 with hydrazine
hydrate confirms the structure. Compound 6 condensed with
different aromatic aldehydes in alcohol in presence of glacial
acetic acid to form Schiff bases 7(a–l). Disappearance of
doublet peak (–NH2) in IR spectra and singlet at 4.2 ppm
The compounds (7a-l) were evaluated against M. tuberculosis
H37Rv (ATCC 27294) using Middlebrook 7H9 broth.
The ability of compounds to inhibit the growth of
Mycobacterium species was determined by Ziehl-Neelsen stain-
ing. Pyrazinamide was used as standard. Eleven of twelve
synthesized compounds showed antimycobacterial activity
differing in potency. Compounds 7b, 7c, 7d, 7e, 7f, and 7g
exhibited good antimycobacterial activity. Compounds with
deactivating groups at o- and p-positions (7b, 7c, 7d, and 7e)
were found to be more potent antimycobacterials.
Compounds 7j and 7l with –OH (activating) group at p-
and m-position exhibited less potent antimycobacterial
activity compared to compounds with deactivating groups.
The compounds 7f and 7g showed good antimycobacterial
activity even though they have activating groups. The com-
pound 7h exhibited less potent antimycobacterial activity,
in spite of having two –OCH3 groups. 7i having –OH and
–OCH3 groups has shown weak antimycobacterial activity.
Compound 7k has not exhibited antimycobacterial activity.
–
due to –NH2, appearance of CH peak at 7.7 ppm in NMR
–
proved the structure of these compounds.
Table 1 shows the physicochemical properties and MIC
values against Mycobacterium tuberculosis H37Rv (ATCC
27294) and Table 2 shows spectral data.
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Arch. Pharm. Chem. Life Sci. 2011, 344, 459–465
Novel Thienopyrimidines as Antitubercular Agents
461
Table 1. Physical constants and in vitro antimycobacterial activities of compounds 3, 4, 5, 6, 7(a-l).
Sl. No
R
% Yield
Melting point, 8C
M. tuberculosis MICa, mM
7a
7b
7c
7d
7e
7f
7g
7h
7i
C6H5
43.69
94.57
54.56
74.23
80.12
78.1
86.24
89.13
89.44
84.25
81.56
58.1
288
272
154
240
285
225
264
279
281
317
305
236
–
320
70
64
70
71
66
62
266
293
137
–
274
60.97
C6H4-4-N(CH3)2
C6H4-6-Br
C6H4-6-NO2
C6H4-4-Cl
C6H4-4-OCH3
C6H4-3,4,5- OCH3
C6H4-3,4- OCH3
C6H4-3- OCH3-4-OH
C6H4-4-OH
C6H4-5-OH
C6H4-3-OH
Pyrazinamide
7j
7k
7l
–
a
MIC: minimum inhibitory concentration.
The six compounds 7b, 7c, 7d, 7e, 7f, and 7g were tested for
cytotoxicity (IC50) using THP-1 (human, monocytes) cell line.
All six exhibited poor cytotoxicity to THP-1 cells. Table 3
shows the cytotoxicity to THP-1 cells and selective index (SI).
Synthesis
Synthesis of ethyl 2-aminothiophene-3-carboxylate (3)
[3, 4]
Triethylamine (50 m mol) was added dropwise over 10 min
to a mixture of 2,5-dihydroxy-1,4-dithiane (50 m mol), ethyl-
cyanoacetate (100 m mol), and dimethylformamide(40 mL).
The mixture was stirred for at 458C for 30 min, diluted with
0.4 M acetic acid, extracted with ether. The ethereal layer was
dried over sodium sulphate. The solvent removed and residue
cooled to get the product.
Conclusion
Though the MIC values obtained for H37Rv are large, the
compounds’ cytotoxicity concentration is thirty times more
than the MIC. This study proves that the class of thienopyr-
imidines can potentially serve as lead structure for further
optimization. In depth studies are underway and we hope to
develop therapeutic agents for TB which are of low cost and
were useful in developing countries.
Yield 66.5%, mp 448C, white crystals.
Synthesis of 4-hydroxythieno[2,3-d]pyrimidine (4) [5]
A mixture of ethyl-2-aminothiophene-3-carboxylate 3 (0.05 mol)
and formamide (20 mL) was refluxed for 8 h and allowed to cool
overnight and added water. The crystals filtered dried and recrys-
tallized with hot alcohol.
Yield 76.6%, mp 2608C, light brownish yellow colored crystals.
Experimental
Procedure for the synthesis of 4-(ethylacetoxy)thieno
[2,3-d]pyrimidine (5) [6–10]
General
The melting points were determined and are uncorrected. Infrared
spectra (KBr disc) were performed on FTIR-8300 Shimadzu and
A mixture of 4-hydroxy thieno[2,3-d]pyrimidine 4 (0.05 mol)
and anhydrous K2CO3 (0.1 mol) in excess of dry acetone
(100 mL) was stirred at reflux temperature for 4 h. To the stirred
suspension, a mixture of ethylchloroacetate (0.05 mol) in dry
acetone was added in dropwise over a period of 30 min at reflux
temperature, and the reflux continued for further 6 h. After
keeping the reaction mixture overnight, the excess of solvent
was removed to get the solid. The solid was recrystallized from
acetone.
the frequencies were expressed in cmꢀ1 1H-NMR spectra were
.
recorded on Bruker-Avance 400 MHz instrument with TMS
(0 ppm) as an internal standard; the chemical shifts (d) are
reported in ppm and coupling constants (J) are given in Hertz
(Hz). Signal multiplicities are represented by s (singlet), d (doublet),
t (triplet), m (multiplet), and br s (broad singlet). Completion of the
reaction and the purity of the compounds were checked on Merck
precoated silica gel 60 F-254. Yields were not optimized. All the
solvents and reagents were used without further purification.
Yield 74.64%, mp 1408C, buff color crystalline solid.
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462
P. Rashmi et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 459–465
Table 2. Spectral data of compounds 3, 4, 5, 6, 7(a-l).
Sl. No.
3
Structure
IR nmax, cmꢀ1, KBr
NMR (CDCl3) d ppm
3412.19, 3304.17(NH2), 1654.98
–
7.06–7.08 (d, H, CH (H1)), 7.25–7.26 (d, H, CH
(H2)), 7.32 (s, 2H, NH2), 4.42–4.48 (q, 2H, CH2),
1.33–1.36 (t, 3H, CH3)
O
–
(C O), 1521.98, 1490 (C C)
–
–
O
S
NH2
–
4
5
3285 (N–H), 1660 (C O), 1590,
–
7.26–7.27 (d, H, CH (H1)), 7.44–7.46 (d, H, CH
(H2)), 7.99 (s, H, CH (H3)), 5–6 (brs, H, OH)
–
O
1520 (C C)
–
NH
S
N
–
–
1746 (C O), 3081.98–3097.42
–
(Ar–CH), 1568, 1579.51,
1551.49 (C C), 1667.29 (C N)
7.21–7.22 (d, H, SCH¼), 7.41–7.42 (d, H, CH–),
–
O
N
7.90 (s, H, NCHN), 4.66 (s, 2H, OCH2),
4.16–4.21 (q, 2H, CH2), 1.21–1.25 (t, 3H, CH3)
–
–
–
–
O
N
S
–
6
3290.67, 3178.79 (NH2), 3165.29
–
7.30–7.31 (d, H SCH¼), 7.36–7.37 (d, H CH–),
–
8.17 (s, H, NCHN), 4.63 (s, 2H, OCH2), 7.7 (s, H,
NH), 2.5 (s, 2H, NH2)
H
N
(NH), 1685.8 (C O), 3047.63–3109.35
–
(Ar–CH), 1566, 1535.39, (C C),
O
NH2
–
–
O
–
1660.77 (C N)
–
N
S
N
–
7a
3223.16 (NH), 1697.41 (C O), 1651.
–
–
12 (C N)
–
7.63–7.68 (d, H, SCH¼, J ¼ 5.8), 7.40–7.42
H
N
–
(d, H, CH–, J ¼ 5.8), 8.41 (s, H, NCHN),
–
5.23 (s, 2H, OCH2), 8.05 (s, H, CONH), 7.75
O
N
–
(s, H, N CH) 7.4–7.73 (m, 5H, ArH)
–
O
N
S
N
–
7b
3238.59 (NH), 1678.13 (C O),
–
–
1612.54 (C N)
–
7.60–7.62 (d, H, SCH¼, J ¼ 5.78), 7.4–7.43 (d,
H
N
–
H, CH–, J ¼ 5.78), 8.39 (s, H, NCHN), 5.18 (s,
–
O
N
N
2H, OCH2), 7.9 (s, H, CONH), 7.90 (s, H, NNCH),
6.73 (d, 2H, ArH, J ¼ 7.89), 7.52 (d, 2H, ArH,
J ¼ 7.89), 2.49 (s, 6H, N–CH3)
O
N
N
S
–
7c
3238.59 (NH), 1678.13 (C O),
–
7.60–7.61 (d, H, SCH¼, J ¼ 5.8), 7.39–7.40
H
N
–
–
1612.54 (C N), 582.52 (C–Br)
–
(d, H, CH–, J ¼ 5.8), 8.35 (s, H, NCHN),
–
5.06 (s, 2H, OCH2), 10.58 (s, H, CONH),
O
N
N
O
–
7.7 (s, H, N CH), 7.42–7.70 (m, 4H, ArH)
–
N
Br
S
–
7d
3228.39 (NH), 1678.13 (C O),
–
–
1645.24 (C N)
–
7.66–7.68 (d, H, SCH¼, J ¼ 5.8), 7.41–7.42
H
N
–
(d, H, CH–, J ¼ 5.8), 8.44 (s, H, NCHN), 5.2
–
(s, 2H, OCH2), 8.44 (s, H, CONH), 8.06
O
N
N
O2N
O
–
(s, H, N CH), 7.69–8.13 (m, 4H, ArH)
–
N
S
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Arch. Pharm. Chem. Life Sci. 2011, 344, 459–465
Table 2. (continued )
Novel Thienopyrimidines as Antitubercular Agents
463
Sl. No.
7e
Structure
IR nmax, cmꢀ1, KBr
NMR (CDCl3) d ppm
–
3172.1 (NH), 1732.11 (C O), 1639.47
–
7.62–7.63 (d, H, SCH¼, J ¼ 5.8), 7.4–7.48
H
N
–
(C N)
–
–
(d, H, CH–, J ¼ 5.8), 8.4 (s, H, NCHN), 5.23
–
O
N
N
(s, 2H, OCH2), 8.04 (s, H, CONH), 11.85
(s, H, NNCH), 7.50–7.52 (d, 2H, ArH, J ¼ 8.55),
7.75–7.77 (d, 2H, ArH, J ¼ 8.55)
O
N
Cl
S
–
7f
3259.09 (NH), 1671.83 (C O),
–
–
1656.65 (C N)
–
7.63–7.65 (d, H, SCH¼, J ¼ 5.8), 7.4–7.42 (d, H,
H
N
–
–
CH–, J ¼ 5.8), 8.4 (s, H, NCHN), 5.2 (s, 2H,
O
N
OCH2), 8.14 (s, H, CONH), 7.96 (s, H, NNCH),
6.99–7.02 (d, H, ArH, J ¼ 8.84), 7.65–7.68
(m, 2H, ArH), 3.79 (s, 3H, OCH3)
O
N
O
S
N
–
7g
7h
7i
3189.09 (NH), 1670.33 (C O),
–
–
1583.65 (C N)
–
7.61–7.63 (d, H, SCH¼, J ¼ 5.79), 7.41–7.42
H
N
–
O
(d, H, CH–, J ¼ 5.79), 8.42 (s, H, NCHN), 5.24
–
O
N
(s, 2H, OCH2), 9.6 (s, H, CONH), 7.96 (s, H,
NNCH), 7.04 (s, 2H, ArH), 3.8 (s, 6H, OCH3), 3.6
(s, 3H, OCH3)
O
N
O
O
S
N
–
3186.51 (NH), 1674.27 (C O),
–
7.61–7.63 (d, H, SCH¼, J ¼ 5.8), 7.40–7.42
H
N
–
1597.11 (C N)
–
–
O
O
(d, H, CH–, J ¼ 5.8), 8.41 (s, H, NCHN), 5.23
–
O
N
N
(s, 2H, OCH2), 9.70 (s, H, CONH), 7.97 (s, H,
NNCH), 7.22–7.34 (d, H, ArH, J ¼ 8.6), 7.00–
7.22 (m, 2H, ArH), 3.79 (d, 6H, OCH3)
O
N
S
3292.60 (OH), 3198.08 (NH), 1687.77
–
7.61–7.62 (d, H, SCH¼, J ¼ 5.8), 7.4–7.42 (d, H,
H
N
–
–
O
(C O), 1666.55 (C N)
–
CH–, J ¼ 5.8), 8.41 (s, H, NCHN), 5.25 (s, 2H,
–
–
O
N
OCH2), 9.4 (s, H, CONH), 7.09 (s, H, NNCH),
6.81–6.83 (d, H, ArH, J ¼ 8.13), 7.098–7.119
(m, 2H, ArH), 3.82 (s, 3H, OCH3), 9.4 (s, H, OH)
O
N
OH
S
N
7j
3209.66 (OH), 3146 (NH), 1701.27
–
7.61–7.62 (d, H, SCH¼, J ¼ 5.8), 7.4–7.41 (d, H,
H
N
–
–
–
(C O), 1648.48 (C N)
–
CH–, J ¼ 5.8), 8.4 (s, H, NCHN), 5.18 (s, 2H,
–
O
N
OCH2), 7.94 (s, H, CONH), 7.99 (s, H, NNCH),
6.81–6.83 (d, 2H, ArH, J ¼ 8.64), 7.54–7.56 (d,
2H, ArH, J ¼ 8.64), 9.89 (s, H, OH)
O
N
OH
S
N
7k
3248.69 (OH), 3159 (NH), 1711.17
–
7.61–7.62 (d, H, SCH¼, J ¼ 5.8), 7.4–7.41
H
N
–
–
(C O), 1668.48 (C N)
–
(d, H, CH–, J ¼ 5.8), 8.4 (s, H, NCHN),
–
–
5.20 (s, 2H, OCH2), 8.41 (s, H, CONH),
O
N
O
N
7.21 (s, H, NNCH), 6.84–6.9 (m, 3H, ArH),
7.73–7.75 (d, 2H, ArH, J ¼ 7.8), 10.04 (s, H, OH)
OH
S
N
7l
3253.69 (OH), 3172.1 (NH), 1732.11
–
7.61–7.63 (d, H, SCH¼, J ¼ 5.8), 7.4–7.42
H
N
–
–
OH
(C O), 1639.47 (C N)
–
(d, H, CH–, J ¼ 5.8), 8.4 (s, H, NCHN),
–
–
O
N
N
5.20 (s, 2H, OCH2), 7.96 (s, H, CONH), 7.94 (s, H,
NNCH), 6.83–6.9 (d, 2H, ArH, J ¼ 8.64), 7.09–
7.24 (d, 2H, ArH, J ¼ 8.64), 9.60 (s, H, OH)
O
N
S
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Table 3. Cytotoxicity and selective index.
Compound MICa, mM IC50b, mM
Procedure with THP-1 culture
The cell suspension from the confluent culture flask was
transferred to sterile tubes, centrifuged at 2000 rpm for
10 min and cell pellet was separated. Known volume of media
was added to the pellet and cells were resuspended and cell
count was adjusted to 1.0 ꢂ 105 cells/mL using RPMI medium
containing 10% FBS. To each well of the 96 well microtitre
plate, 0.1 mL of the diluted cell suspension (approximately
10 000 cells) was added. After 2 h, 100 mL of different test
concentrations of test drugs were added on to the partial
monolayer in microtitre plates. The plates were then incu-
bated at 378C for 3 days in 5% CO2 atmosphere, and micro-
scopic examination was carried out and observations
were noted every 24 h interval. After 72 h, 10 mL of MTT
(5mg/mL) in PBS was added to each well. The plates were gently
shaken and incubated for 3 h at 378C in 5% CO2 atmosphere.
Microtitre plates were centrifuged at 2000 rpm for 15 min and
supernatant was removed. 100 mL of propanol was added and
the plates were gently shaken to solublise the formed forma-
zan. The absorbance was measured using a microplate reader
at a wavelength of 540 nm. The percentage growth inhibition
was calculated using the following formula and inhibit cell
SIc, mM
7b
7c
7d
7e
7f
70
64
70
71
66
62
2492.90
2387.80
2415.90
2264.80
2454.70
2019.90
35.61
37.31
34.51
31.89
37.19
32.58
7g
a
MIC: Minimum inhibitory concentration. b IC50: Concentration
of test drug needed to inhibit cell growth by 50%. c SI: Selectivity
index calculated by dividing the IC50 by the MIC.
Procedure for synthesis of thieno[2,3-d]pyrimidin-4-
acetoxyhydrazide (6) [6]
To a suspension of 5 (0.01 mol) in 40 ml ethanol 0.015 mol of
hydrazine hydrate was added and the reaction mixture was
refluxed for 4 h. The resulting mixture was allowed to cool
and filtered. The solid obtained dried and recrystallized with
hot water.
Yield 64.2%; 257–2608C; yellowish crystalline solid.
concentration of test drug needed to growth by 50% (IC50
)
values is generated from the dose-response curves for each
cell line.
General procedure for synthesis of N-substituted-2-
(thieno[2,3-d]pyrimidine-4-yloxy)acetohydrazides
(7a–7l) [11]
% Growth Inhibition
Equimolar (0.03 mol) quantity of 4 and different aldehydes
was refluxed in 25 mL alcohol for 2–3 h in the presence of a
few drops of glacial acetic acid. Solvent was evaporated and the
product was poured to water, filtered and dried. The crude solid
was recrystallized in appropriate solvent systems to give the
products.
Mean OD of individual tested group
¼
ꢂ 100
Mean OD of control group
Selectivity index
A selectivity index can be calculated by dividing the IC50
by the MIC: If the SI is >10, the compound is then evaluated
further [13].
Antitubercular activity
Authors are thankful to Principal, Nargund College of Pharmacy,
for providing the facilities for research work and IISc. Bangalore,
for providing NMR spectral data and also providing docking studies
data.
The ability of compounds to inhibit the growth of Mycobacterium
species grown in Middlebrook 7H9 broth and standard strain of
Mycobacterium tuberculosis H37Rv (ATCC 27294) was determined by
Ziehl-Neelsen stain. The basal medium is prepared according to
manufacturer’s instructions (Hi-Media) and sterilized by auto-
claving. 4.5 mL of broth is poured into each one of the sterile
bottles. To this, 0.5 mL of ADC supplement is added. This supple-
ment contains catalase, dextrose and bovine serum albumin
fraction. Then a stock solution of the compound is prepared
(10 mg/mL). From this appropriate amount of solution is trans-
ferred to media bottles to achieve final concentrations of 25, 50,
and 100 mg/mL. Finally, 10-mL suspension of Mycobacterium tuber-
culosis strain (100 000 organisms/mL, adjusted by McFarland’s
turbidity standard) is transferred to each of the tubes and incu-
bated at 378C. Along with this, one growth control without
compound and standard drug controls were also set up. The
bottles are inspected for growth twice a week for a period of three
weeks. The appearance of turbidity is considered as growth and
indicates resistance to the compound. The growth is confirmed
by making a smear from each bottle and performing a Ziehl-
Neelsen stain.
The authors have declared no conflict of interest.
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