Effect of substituents on diarylmethanes for antitubercular activity

Article abstract of DOI:10.1016/j.ejmech.2006.09.020

Aminoalkyl derivatives of diarylmethanes were prepared using Grignard, Friedel-Crafts arylation and aminohydrochloride chain formation reactions. These series of compounds were evaluated against Mycobacterium tuberculosis H₃₇R_v and s

Full text of DOI:10.1016/j.ejmech.2006.09.020

European Journal of Medicinal Chemistry 42 (2007) 410e419
http://www.elsevier.com/locate/ejmech
Short communication
Effect of substituents on diarylmethanes for antitubercular activity*
a
a
a
a
Gautam Panda a, , Maloy Kumar Parai , Sajal Kumar Das , Shagufta , Manish Sinha ,
Vinita Chaturvedi b, Anil K. Srivastava b, Y.S. Manju b, Anil N. Gaikwad c,
Sudhir Sinha c
*
a Medicinal and Process Chemistry Division, Central Drug Research Institute, Chattar Manzil Palace, Lucknow-226001, UP, India
b Microbiology Division, Central Drug Research Institute, Lucknow-226001, UP, India
c Drug Target Discovery and Development Division, Central Drug Research Institute, Lucknow-226001, UP, India
Received 11 September 2006; received in revised form 26 September 2006; accepted 28 September 2006
Available online 15 November 2006
Abstract
Aminoalkyl derivatives of diarylmethanes were prepared using Grignard, FriedeleCrafts arylation and aminohydrochloride chain formation
reactions. These series of compounds were evaluated against Mycobacterium tuberculosis H37Rv and showed the activity in the range of 6.25e
25 mg/mL. Effect of heteroaryl, anthracenyl and phenanthrene groups on diarylmethane pharmacophores for antitubercular activity is described.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Antitubercular agents; Diarylmethanes; Phenanthrenes
Tuberculosis (TB) is a bacterial infection caused by Myco-
bacterium tuberculosis and is one of the major causes of
worldwide death [1]. It has been estimated that approximately
eight million people develop active disease and two million
die of the disease every year. However, there has been resur-
gence in the incidence of TB since 1985 and in 1993 WHO
declares TB as a global emergency [2]. The renaissance of
TB is related to the lack of the suitable therapeutic agents,
emergence of HIV AIDS epidemic and the development of
drug resistant strain [3]. However, no new drug has been intro-
duced in the market after the discovery of rifampicin [4].
Therefore there is an imperative need to develop novel an-
titubercular drugs for the management of tuberculosis, i.e.
having new mechanism of action and also will be able to min-
imize the chances of MDR strains with shorter duration of
therapy. In the recent years several new classes of compounds
such as derivatives of biphenyl methanones [5],
benzothiadiazine [6], oxazolidinones [7], azoles [8], benzyl-
purines[9], fluoroquinolones [10], benzo[h]chromene [11]
and benzo[a]anthracenes [11] have been reported as antituber-
cular agents. Recently we have also developed a new type of
antitubercular agents based on substituted diarylmethane scaf-
folds containing good amount of hydrophobicity. In this con-
text, the synthesis and antitubercular activity of aminoalkyl
and 2-hydroxy-aminoalkyl derivatives of phenanthrene
substituted diarylmethanes have been reported [12e15].
Therefore as a consequence of above facts and in continuation
of our previous work, we herein report the synthesis and anti-
tubercular activity of aminoalkyl derivatives of diarylmethanes
and substituted diarylmethanes.
Initially synthesis of aminoalkyl derivatives of unsubsti-
tuted diarylmethane derivatives was carried out. The reaction
of Grignard reagent 3 derived from 4-bromoanisole 2 with
4-benzyloxybenzaldehyde 1 furnished carbinol 4 which was
dehydrogenated under H2, Pd/C conditions. It was interesting
to find that hydroxyl functionality of 4 disappearing along
with deprotection of benzyl group to furnish diarylmethane
5. The reaction of 5 with two alkylamine hydrochloride chains
in the presence of K2CO3 and acetone led to the formation of
*
CDRI communication no. 6801.
* Corresponding author. Tel.: þ91 522 2364635; fax: þ91 522 2623405.
E-mail addresses: gautam_panda@lycos.com, bapi.rm@yahoo.com (G.
Panda).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.09.020

G. Panda et al. / European Journal of Medicinal Chemistry 42 (2007) 410e419
411
compounds 6a and 6b in 66% and 71% yields, respectively
(Scheme 1). By treatment of amines 6a and 6b with ethanolic
hydrogen chloride the corresponding salts 6a$HCl and
6b$HCl were prepared (Scheme 1).
resin (0.82 mmol/gm) in pyridine to furnish 25 and TBDMS
group on 25 was cleaved on solid support through treatment
with TBAF in THF to give 26 as phenolic derivative. Reaction
between 26 with three alkylaminohydrochloride chains in
presence of K2CO3 and DMF over resin furnished the com-
pound 27 which after cleavage with 1% TFA in DCM gave
the final hydroxy substituted phenolic derivatives 28aec in
good yields and purity, Scheme 3.
Further to synthesize aminoalkyl derivatives of substituted
diarylmethanes, Grignard reagent 3 was treated with commer-
cially available substituted heteroaryl or anthracenyl carbalde-
hydes to furnish carbinols 7, 8, 9 and 10 as enantiomeric
mixtures after column chromatography. Subsequent Friedele
Crafts arylation of carbinols 7, 8, 9 and 10 with phenol in the
presence of conc. H2SO4 or anhydrous AlCl3 provided 11, 12,
13 and 14 by nucleophilic attack of phenol through para carbon
atom of benzene ring. The reaction of 11, 12, 13 and 14 with 1-
(2-chloroethyl)-alkylamine hydrochlorides in the presence of
K2CO3 and acetone led to the formation of aminoalkyl deriva-
tives 15aee, 16aed, 17 and 18aed in good yields, respectively
(Scheme 2and Table 1). The ethanolic HCl salts of 15aee, 16ae
d, 17 and 18aed were found to be active against M. tuberculosis
H37Rv in vitro with MIC in the range of 6.25, 12.5 and 25 mg/mL
(Table 3). Further, by comparing the activity results of all diaryl-
methane derivatives i.e. pyridine, indole, anthracene and phen-
anthrene, it was observed that phenanthrene substituted
diarylmethane derivatives are active pharmacophore and were
selected for optimizing antitubercular activity, and thus further
synthetic transformations were performed.
Since solution phase synthesis of phenolic derivatives was
active with MIC ranging from 6.25 to 25 mg/mL against M. tu-
berculosis Tables 2 and 3), we wanted to explore the synthetic
methodology over solid phase to obtain hydroxy substituted
aminoalkyl derivatives. Towards this objective, Grignard re-
agent obtained from 9-bromo phenanthrene 19 was reacted
with 4-benzyloxy-benzaldehyde to furnish the carbinol 20
(87 %) which on treatment with phenol and conc. H2SO4
gave 22 as major product and 21 as minor one. Reaction of
22 with TBDMSCl/imidazole in DCM gave silyl derivative
23 which on hydrogenation over Pd/C gave the phenolic deriv-
ative 24. Compound 24 was loaded on 2-chlorotritylchloride
A closer look into the structureeactivity relationship in
these compounds shows that aminoalkyl derivatives of diaryl-
methane with indole 15aee, anthracenyl 17, phenanthrenyl
rings 18aed and 28aec showed the antitubercular activity
with MIC ranging from 6.25 to 25 mg/mL. However, amino-
alkyl derivatives of diarylmethane 6aeb and pyridine
substituted diarylmethanes 16aed exhibited MIC higher
than 25 mg/mL. It is interesting to note that increasing the
bulkiness of the substituents on diarylmethane resulted in bet-
ter activity. Compound 6aeb without substituent are having
lower order of activity whereas the activity increases in case
of 15aee, 17, 18aed and 28aec with indole, anthracene
and phenanthrene rings as substituents. It is noteworthy that
activity was partially dependent on the alkyl moiety on nitro-
gen atom. Compounds like 15a having dimethylaminoalkyl
chain is having MIC > 25 (MABA method) but 15b, 15c,
and 15e having diethylamine, pyrrolidine and azepane ring
are having MIC 12.5 whereas 15d having piperidine ring has
MIC 6.25 mg/mL. Thus, compounds having medium size ami-
noalkyl moiety gave better antitubercular activity.
Cytotoxicity of 15d, 18bed in VERO cell line as well as
mouse bone marrow macrophages at different concentrations
beginning from 10ꢀ MIC of the compounds was assayed
and is expressed as inhibitory concentration (IC50) [18]. On
the basis of IC50 values, selectivity index (SI) of 18b and
18c was found to be 20, indicating that these compounds
can be identified as useful leads (Table 4).
All the compounds synthesized (6aeb, 15aee, 16aed, 17,
18aed and 28aec) were evaluated for their antitubercular
CHO
C6H5
O
OCH2CH2NRR
OH
C6H5
O
1
+
a
b
c
OCH3
HO
X
OCH3
OCH3
OCH3
2, X = Br
Mg,
4
5
THF
6
3, X = MgBr
6
NRR%
6
6
HCl / ethanol
a
N(CH3)266
HCl
b
N
71
Scheme 1. Reagents and conditions: (a) Mg, THF, RT, 2 h, 73%; (b) Pd/C, H2, RT, 1 h, 95%; (c) ClCH2CH2NRR$HCl, K2CO3, acetone, 66e71%.

412
G. Panda et al. / European Journal of Medicinal Chemistry 42 (2007) 410e419
RRNH2CH2CO
OCH3
H3CO
HO
H3CO
OCH3
ClCH2CH2NRR.HCl
Phenol, 80°C
RT, 2-3 h
R
X
K2CO3, 65°C,
5-6h
R
conc. H2SO4 1h
or Anhy. AlCl3
R
11, 12, 13, 14
OH
7,8, 9, 10
15a-e, 16a-d, 17, 18a-d
2, X = Br
Mg,
THF
3, X = MgBr
+
RCHO
Scheme 2.
activity against M. tuberculosis H37Rv through Micro alamar
blue assay (MABA) [16], agar microdilution technique [17]
and standard BACTEC radiometric growth assay [17] and
their results are shown below (Table 3).
1.3. {2-[4-(4-Methoxy-benzyl)-phenoxy]-ethyl}-
dimethylamine 6a
A mixture of compound 5 (0.22 g, 1.028 mmol), anhydrous
K2CO3 (0.71 g, 5.14 mmol), 1-(2-chloroethyl)-dimethylamine
hydrochloride (0.284 g, 1.54 mmol) and dry acetone (20 mL)
was refluxed for 7 h. K2CO3 was filtered off and acetone
was removed. The residue was diluted with water and ex-
tracted with ethyl acetate. The organic layer was washed
with water, brine and dried over anhydrous Na2SO4. Column
chromatography over basic alumina and elution with 35%
ethyl acetate in hexane furnished compound 6a (0.2 g,
66.6%) as yellow semi-solid. IR (neat): 2928, 1501, 1256,
In conclusion we have reported the synthesis of the amino-
alkyl derivatives of diarylmethane and substituted diarylme-
thanes through Grignard, FriedeleCrafts arylation and
aminohydrochloride chain formation reactions and these com-
pounds were active in the range of 6.25e25 mg/mL against M.
tuberculosis H37Rv. Indole and phenanthrene substituted dia-
rylmethane derivatives showed promising activity. All these
results suggest that it will be interesting to prepare the analogs
of active molecules for finding new compounds that possess
better activity and bioavailability.
759 cmꢂ1 1H NMR (CDCl3, 200 MHz): d 7.10e7.04 (m,
.
4H), 6.85e6.79 (m, 4H), 4.05 (t, 2H, J ¼ 7 Hz), 3.85 (s,
2H), 3.77 (s, 3H), 2.74 (t, 2H, J ¼ 7 Hz), 2.34 (s, 6H). MS:
286 (Mþ þ H). Anal. Calcd for (C18H23NO2): C, 75.76; H,
8.12; N, 4.91. Found: C, 75.72; H, 8.18; N, 4.95.
1. Selected spectral data
1.1. (4-Benzyloxy-phenyl)-(4-methoxy-phenyl)-methanol 4
To a solution of 4-bromoanisole 2 (8.2 mL, 70.74 mmol) in
dry THF (30 mL) was added activated magnesium (1.93 g,
80.2 mmol) and was stirred at room temperature under dry ni-
trogen for 2 h. To Grignard reagent thus formed was added 4-
benzyloxybenzaldehyde 1 (5 g, 23.58 mmol) in THF (10 mL)
and stirring was continued for another 3e4 h. The reaction
mixture was quenched by gradual addition of saturated
NH4Cl (w10 mL) and THF was removed in vacuo. The mix-
ture was extracted thrice with ethyl acetate, washed with brine
and dried over sodium sulphate. It was concentrated and
charged over silica gel. Elution with 10% ethyl acetate in hex-
ane furnished carbinol product 4 (5.5 g, 73.3%) as white solid,
m.p. 93 ꢁC. IR (KBr): 3402, 1592, 1585, 1365, 1145,
1.4. 1-{2-[4-(4-Methoxy-benzyl)-phenoxy]-ethyl}-
piperidine 6b
As described for 6a, compound 5 (0.2 g, 1.02 mmol),
K2CO3 (0.71 g, 5.14 mmol) and 1-(2-chloroethyl)-piperidine
hydrochloride (0.22 g, 1.54 mmol) furnished 6b (0.19 g,
71.4%) as light yellow semi-solid. IR (neat): 3440, 1635,
1
1245, 770 cmꢂ1. H NMR (CDCl3, 200 MHz): d 7.10e7.04
(m, 4H), 6.85e6.79 (m, 4H), 4.07 (t, 2H, J ¼ 7 Hz), 3.85 (s,
2H), 3.79 (s, 3H), 2.75 (t, 2H, J ¼ 7 Hz), 2.52e2.47 (m,
4H), 1.62e1.55 (m, 4H), 1.45e1.43 (m, 2H). MS: 326
(Mþ þ H). Anal. Calcd for (C21H27NO2): C, 77.50; H, 8.36;
N, 4.30. Found: C, 77.59; H, 8.35; N, 4.35.
780 cmꢂ1 1H NMR (CDCl3, 200 MHz): d 7.43e7.24 (m,
.
9H), 6.94e6.83 (m, 4H), 5.75 (s, 1H), 5.03 (s, 2H), 3.77 (s,
3H), 3.11 (bs, 1H). MS: 320 (Mþ). Anal. Calcd for
(C21H20O3): C, 78.73; H, 6.29. Found: C, 78.79; H, 6.35.
1.5. (1-Benzenesulfonyl-1H-indol-3-yl)-(4-methoxy-
phenyl)-methanol 7
1.2. 4-(4-Methoxy-benzyl)-phenol 5
As described for 4, compound 2 (1.31 mL, 10.52 mmol) in
dry THF (25 mL), magnesium (0.31 g, 12.842 mmol) and 1-
benzenesulfonyl-1H-indole-3-carbaldehyde (2.0 g, 7.01 mmol)
in THF (5 mL) furnished 7 (1.98 g, 72%) as brown semi-solid.
IR (neat): 3402, 2927, 1607, 1508, 1247, 1175, 1032,
The compound 4 (0.5 g, 0.84 mmol) was hydrogenated over
10% Pd/C (0.05 g) and after usual work-up and purification
furnished 5 (0.32 g, 95.8%) as white semi-solid. IR (neat):
1
3390, 1510, 1258, 759 cmꢂ1. H NMR (CDCl3, 200 MHz):
d 7.03e6.93 (m, 4H), 6.78e6.64 (m, 4H), 3.80 (s, 2H), 3.72
(s, 3H). MS: 214 (Mþ). Anal. Calcd for (C14H14O2): C,
78.48; H, 6.59. Found C, 78.42; H, 6.45.
767 cmꢂ1 1H NMR (CDCl3, 200 MHz): d 7.94e7.84
.
(m, 3H), 7.47e7.25 (m, 9H), 6.85 (m, 2H), 5.95 (s, 1H), 3.79
(s, 3H). MS: 393 (Mþ).

G. Panda et al. / European Journal of Medicinal Chemistry 42 (2007) 410e419
413
Table 1
Synthesis of aminoalkyl derivatives of substituted diarylmethane derivatives 15aee, 16aed, 17 and 18aed from carbinols 7, 8, 9 and 10, respectively
Entry Bromo compound RCHO
Carbinol
Alkylated product
Alkylated product
with chain
MeO
N
MeO
SO2
OHC
N
SO2
MeO
MeO
N
N
SO2
Br
O2S
1
15a, R= Me
15b, R= Et
OH
2
O
NRR
OH
7
15c, R-R= (CH2)4
15d, R-R= (CH2)5
15e, R-R= (CH2)6
11
MeO
N
MeO
MeO
N
MeO
N
OHC
Br
2
3
4
OH
O
N
NRR
2
2
2
16a, R= Me
16b, R= Et
16c, R-R= (CH2)4
8
OH
12
16d, R-R= (CH2)5
MeO
CHO
Br
OH
OH
MeO
MeO
MeO
OCH2CH2N(CH3)2
17
13
9
H3CO
H3CO
CHO
MeO
H3CO
Br
OCH2CH2NRR
OH
18a, R=Me
18b, R= Et
10
OH
14
18c, R-R= (CH2)4
18d, R-R= (CH2)5
1.6. (4-Methoxy-phenyl)-pyridin-3yl-methanol 8
(5 mL) furnished 8 (1.8 g, 45%) as white solid, m.p. 70 ꢁC.
IR (KBr): 3402, 1592, 1585, 1365, 1145, 780 cmꢂ1 1H
.
As described for 4, compound 2 (3.50 mL, 28.00 mmol) in
dry THF (30 mL), magnesium (0.896 g, 37.34 mmol) and pyr-
idine 3-carboxaldehyde (1.75 mL, 18.67 mmol) in THF
NMR (CDCl3, 200 MHz): d 8.43 (d, 1H, J ¼ 1.6 Hz), 8.29
(dd, 1H, J1 ¼ 1.3 Hz, J2 ¼ 4.8 Hz), 7.66 (d, 1H, J ¼ 7.8 Hz),
7.26e7.15 (m, 3H), 6.84 (d, 2H, J ¼ 8.6 Hz), 5.74 (s, 1H),

414
G. Panda et al. / European Journal of Medicinal Chemistry 42 (2007) 410e419
Table 2
Solid phase synthesis of aminoalkyl derivatives of diaryloxy
methanophenanthrenes
was neutralized with saturated aq. NaHCO3 and extracted
with ethyl acetate. The concentrated extract was subjected to
column chromatography on silica gel and elution with 15%
ethyl acetate in hexane furnished 11 (1.31 g, 62%) as brown
viscous oil. IR (neat): 3431, 2368, 1712, 1591, 1510, 1170,
Compound
Alkyl chain
Yield (%)
28a
28b
28c
ClCH2CH2N(CH3)2$HCl
ClCH2CH2N(CH2CH3)2$HCl
ClCH2CH2N(CH2)5$HCl
70
72
76
1
1105, 1025, 769 cmꢂ1. H NMR (CDCl3, 200 MHz): d 7.95
(d, 1H, J ¼ 8.2 Hz), 7.77 (d, 2H, J ¼ 8.6), 7.29e7.21 (m,
3H), 6.99e6.72 (m, 12H), 5.39 (s, 1H), 4.95 (bs, 1H), 3.79
(s, 3H). MS: 469 (Mþ).
3.76 (s, 3H). MS: 215 (Mþ). Anal. Calcd for (C13H13NO2): C,
72.54; H, 6.09; N, 6.51. Found: C, 72.59; H, 6.05; N, 6.84.
1.7. Anthracen-9-yl-(4-methoxy-phenyl)-methanol 9
1.9. 4-[(4-Methoxy-phenyl)-pyridin-3-yl-methyl]-phenol12
As described for 4, compound 2 (16.15 g, 1.86 mmol) in
dry THF (20 mL), magnesium (2.06 g, 8.6 mmol) and anthra-
cene-9-carbaldehyde (5.94 g, 2.8 mmol) in THF (25 mL) fur-
nished 9 (6.0 g, 66%) as yellow semi-solid. IR (neat): 3510,
To a solution of carbinol 8 (2.6 g, 12.09 mmol) and phenol
(1.59 g, 16.93 mmol) in dry benzene (30 mL) was added alu-
minium trichloride (1.61 g, 12.09 mmol) and then the mixture
was heated at 80 ꢁC for 5 h. After cooling, the reaction mix-
ture was neutralized with saturated aq. NaHCO3 and extracted
with ethyl acetate. The concentrated extract was subjected to
column chromatography on silica gel and elution with 40%
ethyl acetate in hexane furnished 12 (1.8 g, 51.3%) as dark
yellow viscous oil. IR (neat): 3431, 1605, 1507, 1443, 1245,
2362, 1604, 1507, 1242, 1169, 732 cmꢂ1
.
1H NMR
(200 MHz, CDCl3): d 8.46 (s, 1H), 8.36 (d, 2H, J ¼ 9 Hz),
8.03 (d, 1H, J ¼ 7.8 Hz), 8.01 (d, 1H, J ¼ 9 Hz), 7.47e7.34
(m, 6H), 7.27 (d, 1H, J ¼ 9 Hz), 6.79 (d, 2H, J ¼ 10 Hz),
3.74 (s, 3H), 2.64 (d, 1H, J ¼ 5.4 Hz). MS: 314 (Mþ). Anal.
Calcd for (C22H18O2): C, 89.05; H, 5.77. Found: C, 89.12;
H, 5.64.
1
1172, 1028 cmꢂ1. H NMR (200 MHz, CDCl3): d 8.43e8.37
(m, 2H), 7.44e7.39 (m, 1H), 7.27e7.24 (m, 1H), 7.00 (d,
2H, J ¼ 8.6 Hz), 6.89e6.73 (m, 6H), 5.41 (s, 1H), 3.76 (s,
3H). MS: 292 (Mþ þ H). Anal. Calcd for (C19H17NO2): C,
78.33; H, 5.88; N, 4.81. Found: C, 78.56; H, 4.96; N, 4.75.
1.8. 4-[(1-Benzenesulfonyl-1H-indol-3-yl)-(4-methoxy-
phenyl)-methyl]-phenol 11
To a solution of carbinol 7 (1.77 g, 4.51 mmol) and phenol
(0.52 mL, 6.32 mmol) in dry benzene (20 mL) was added alu-
minium trichloride (0.601 g, 4.51 mmol) and the mixture was
heated at 80 ꢁC for 1 h. After cooling, the reaction mixture
1.10. 4-[9-Anthryl (4-methoxy-phenyl)methyl]phenol 13
As described for 11, carbinol 9 (2.85 g, 9.07 mmol) and
phenol (1.28 g, 13.61 mmol) in dry benzene (40 mL) furnished
13 (0.3 g, 15%) as a brown solid, m.p. 78 ꢁC. IR (KBr): 3431,
Table 3
In vitro antituberculosis activity of 6aeb, 15aee, 16aed, 17, 18aed and
28aec against M. tuberculosis H37Rv
1605, 1507, 1443, 1245, 1172, 1028 cmꢂ1
.
1H NMR
(200 MHz, CDCl3): d 8.44 (s, 1H), 8.14 (d, 2H, J ¼ 9 Hz),
8.00 (d, 2H, J ¼ 8.5 Hz), 7.45e7.20 (m, 4H), 7.14e6.90 (m,
4H), 6.97 (s, 1H), 6.77 (d, 2H, J ¼ 8 Hz), 6.69 (d, 2H,
J ¼ 8 Hz), 3.75 (s, 3H). MS: 390 (Mþ). Anal. Calcd for
(C28H22O2): C, 86.13; H, 5.68. Found: C, 86.31; H, 5.78.
Compound
MIC (mg/mL)
MABA
Microdilution
BACTEC
6a
6b
>25
>25
25
>25
>25
>12.5
12.5
12.5
6.25
12.5
>25
>25
>25
>25
12.5
25.0
6.25
6.25
6.25
12.5
12.5
12.5
ND
ND
15a
15b
15c
15d
15e
16a
16b
16c
16d
17
ND
ND
12.5
12.5
6.25
12.5
>25
>25
>25
>25
12.5
25
ND
6.25
12.5
ND
1.11. (2-{4-[(1-Benzenesulfonyl-1H-indol-3-yl)-
(4-methoxy-phenyl)-methyl]-phenoxy}-ethyl)-
dimethylamine 15a
12.5
12.5
12.5
ND
As described for 6a, compound 11 (0.175 g, 0.373 mmol),
K2CO3 (0.257 g, 1.86 mmol), 1-(2-chloroethyl)-dimethyl-
amine hydrochloride (0.064 g, 0.447 mmol) furnished 15a
(0.16 g, 81%) as orange viscous liquid. IR (neat): 3394,
18a
18b
18c
18d
28a
28b
28c
12.5
6.25
6.25
6.25
12.5
12.5
12.5
6.25
6.25
6.25
25
1
2361, 1592, 1463, 1242, 1033, 768 cmꢂ1. H NMR (CDCl3,
25
25
200 MHz): d 7.96 (s, 1H), 7.28e6.71 (m 16H), 6.46 (s, 1H),
5.48 (s, 1H), 3.96 (t, 2H, J ¼ 5.2 Hz), 3.70 (s, 3H), 2.65 (t,
2H, J ¼ 5.2 Hz), 2.26 (s, 6H). MS: 540 (Mþ).
ND means not done in that particular test system.

G. Panda et al. / European Journal of Medicinal Chemistry 42 (2007) 410e419
415
OBn
OBn
OCH2C6H5
OH
OBn
c
a
b
+
Br
OH
OH
\
OTBDMS
19
20
21
22
23
d
OH
h
O
O
O
OH
g
f
e
OCH2CH2NRR
OCH2CH2NRR
OH
OTBDMS
OTBDMS
28a-c
27
26
25
24
Scheme 3. Reagents and conditions: (a) Mg, 4-benzyloxybenzaldehyde, THF, RT, 2 h, 87%; (b) phenol, cat. H2SO4, benzene, 1 h, 52% (ortho/para ¼ 3:7); (c)
TBDMSCl, imidazole, DCM, 3e5 h, 87%; (d) 5% Pd/C, H2, ethyl acetate, 1e2 h, 87%; (e) tritylchloride resin, pyridine, 50 ꢁC, 8 h, 98%; (f) TBAF, THF,
0.5e1 h, 90%; (g) ClCH2CH2NRR$HCl, K2CO3, dry DMF, 48 h, then 1% TFA, DCM, 70e76%.
1.12. (2-{4-[(1-Benzenesulfonyl-1H-indol-3-yl)-
(4-methoxy-phenyl)-methyl]-phenoxy}-ethyl)-
diethylamine 15b
(CDCl3, 200 MHz): d 7.96 (d, 1H, J ¼ 8.2 Hz), 7.77 (d, 2H,
J ¼ 8.5 Hz), 7.65e7.31 (m, 3H), 7.09e6.08 (m, 12H), 5.39
(s, 1H), 4.08 (t, 2H, J ¼ 6.0 Hz), 3.78 (s, 3H), 2.89 (t, 2H,
J ¼ 5.9 Hz), 2.61e2.45 (m, 4H), 1.92e1.81 (m, 4H). MS:
567 (Mþ þ H).
As described for 6a, compound 11 (0.175 g, 0.373 mmol),
K2CO3 (0.257 g, 1.86 mmol) and 1-(2-chloroethyl)-diethyl-
amine hydrochloride (0.0765 g, 0.44 mmol) furnished 15b
(0.165 g, 78%) as yellow viscous oil. IR (neat): 3413, 2926,
1.14. 1-Benzenesulfonyl-3-{(4-methoxy-phenyl)-[4-(2-
piperidin-1-yl-ethoxy)-phenyl]-methyl}-1H-indole 15d
1
2360, 1607, 1507, 1245, 1033, 768 cmꢂ1. H NMR (CDCl3,
200 MHz): d 7.96 (d, 1H, J ¼ 8.2 Hz), 7.75 (d, 2H,
J ¼ 8.5 Hz), 7.55e7.32 (m, 3H), 7.18e6.71 (m, 12H), 5.42
(s, 1H), 3.97 (t, 2H, J ¼ 5.9 Hz), 3.70 (s, 3H), 2.79 (t, 2H,
J ¼ 5.9 Hz), 2.58e2.55 (m, 4H), 0.99 (t, 4H, J ¼ 7 Hz). MS:
569 (Mþ þ H).
As described for 6a, compound 11 (0.175 g, 0.373 mmol),
K2CO3 (0.257 g, 1.86 mmol) and 1-(2-chloroethyl)-piperidine
hydrochloride (0.088 g, 0.44 mmol) furnished 15d (0.15 g,
71%) as yellow viscous liquid. IR (neat): 3431, 2933, 2363,
1603, 1509, 1371, 1248, 1177, 753, 580 cmꢂ1 1H NMR
.
(CDCl3, 200 MHz): d 7.89 (d, 1H, J ¼ 8.2 Hz), 7.71 (d, 2H,
J ¼ 8.5 Hz), 7.38e7.35 (m, 3H), 7.01e6.71 (m, 12H), 5.32
(s, 1H), 4.00 (t, 2H, J ¼ 6.1 Hz), 3.71 (s, 3H), 2.69 (t, 2H,
J ¼ 6.1 Hz), 2.45e2.40 (m, 4H), 1.55e1.48 (m, 4H), 1.21e
1.14 (m, 2H). MS: 581 (Mþ þ H).
1.13. 1-Benzenesulfonyl-3-{(4-methoxy-phenyl)-[4-(2-
pyrrolidin-1-yl-ethoxy)-phenyl]-methyl}-1H-indole 15c
As described for 6a, compound 11 (0.175 g, 0.373 mmol),
K2CO3 (0.257 g, 1.86 mmol) and 1-(2-chloroethyl)-pyrrolidi-
neamine hydrochloride (0.075 g, 0.447 mmol) furnished 15c
(0.140 g, 67%) as brown viscous liquid. IR (neat): 3413,
1.15. 3-[[4-(2-Azepan-1-yl-ethoxy)-phenyl]-(4-methoxy-
phenyl)-methyl]-1-benzenesulfonyl-1H-indole 15e
2926, 2360, 1607, 1507, 1245, 1033, 768 cmꢂ1 1H NMR
.
As described for 6a, compound 11 (0.175 g, 0.373 mmol),
K2CO3 (0.257 g, 1.869 mmol) and 1-(2-Chloro-ethyl)-azepane
(0.088 g, 0.44 mmol) furnished 15e (0.151 g, 69%) as brown
viscous liquid. IR (neat): 3432, 2928, 2362, 1596, 1352,
Table 4
The cytotoxicity values and corresponding selectivity index (SI) of selected
compounds
766 cmꢂ1 1H NMR (CDCl3, 200 MHz): d 7.97 (d, 1H,
.
Compound
Cytotoxicity value (IC50
)
Selectivity index
J ¼ 8.2 Hz), 7.79 (d, 2H, J ¼ 8.5 Hz), 7.59e7.41 (m, 3H),
7.11e6.81 (m, 12H), 5.41 (s, 1H), 4.06 (t, 2H, J ¼ 6 Hz),
3.80 (s, 3H), 2.96 (t, 2H, J ¼ 6 Hz), 2.82e2.79 (m, 4H),
1.69e1.61 (m, 8H). MS: 595 (Mþ þ H).
15d
18b
18c
18d
62
125
125
62
10
20
20
10

416
G. Panda et al. / European Journal of Medicinal Chemistry 42 (2007) 410e419
1.16. (2-{4-[(4-Methoxy-phenyl)-pyridin-3-yl-methyl]-
1.20. (2-{4-[Anthracen-9-yl-(4-methoxy-phenyl)-methyl]-
phenoxy}-ethyl)-dimethylamine 16a
phenoxy}-ethyl)-dimethylamine 17
As described for 6a, compound 12 (0.2 g, 0.687 mmol),
K2CO3 (0.474 g, 3.43 mmol) and 1-(2-chloroethyl)-dimethyl-
amine hydrochloride (0.149 g, 1.03 mmol) furnished 16a as
yellow viscous oil (0.185 g, 74.59%). IR (neat): 3440, 1635,
As described for 6a, compound 13 (0.25 g, 0.641 mmol),
K2CO3 (0.443 g, 3.2 mmol) and 1-(2-chloroethyl)-dimethyl-
amine hydrochloride (0.138 g, 0.961 mmol) furnished 17
(0.2 g, 67%) as brown semi-solid. IR (neat): 3437, 3019,
1
1245, 770 cmꢂ1. H NMR (CDCl3, 200 MHz): d 8.33e8.30
2930, 1635, 1510, 1216, 761 cmꢂ1 1H NMR (CDCl3,
.
(m, 2H), 7.25 (d, 1H, J ¼ 7.8 Hz), 7.08e7.04 (m, 1H),
6.90e6.68 (m, 8H), 5.32 (s, 1H), 3.91 (t, 2H, J ¼ 6 Hz),
3.63 (s, 3H), 2.59 (t, 2H, J ¼ 6 Hz), 2.20 (s, 6H). MS: 363
(Mþ þ H). Anal. Calcd for (C23H26N2O2): C, 76.21; H, 7.23;
N, 7.73. Found C, 76.35; H, 7.34; N, 7.85.
200 MHz): d 8.26 (d, 1H, J ¼ 8 Hz), 8.21e7.90 (m, 2H),
7.73 (d, 1H, J ¼ 8 Hz), 7.15e6.62 (m, 13H), 5.00 (s, 1H),
4.24 (t, 2H, J ¼ 6.2 Hz), 3.77 (s, 3H), 2.84 (t, 2H,
J ¼ 6.2 Hz), 2.39 (s, 6H). MS: 462 (Mþ þ H). Anal. Calcd
for (C32H31NO2): C, 83.26; H, 6.77; N, 3.03. Found: C,
83.29; H, 6.85; N, 2.95.
1.17. Diethyl-(2-{4-[(4-methoxy-phenyl)-pyridin-3-yl-
methyl]-phenoxy}-ethyl)-amine 16b
1.21. (4-Benzyloxy-phenyl)-phenanthren-9-yl-
methanol 20
As described for 6a, compound 12 (0.2 g, 0.687 mmol),
K2CO3 (0.474 g, 3.43 mmol) and 1-(2-chloroethyl)-diethyl-
amine hydrochloride (0.176 g, 1.03 mmol) furnished 16b as
yellow viscous oil (0.170 g, 65.38%). IR (neat): 3440, 1635,
As described for 4, compound 19 (2 g, 7.78 mmol) in dry
THF (30 mL), magnesium (0.205 g, 8.55 mmol) and 1
(0.825 g, 3.89 mmol) in THF (5 mL) furnished 20 (1.32 g,
87%) as yellow semi-solid. IR (neat): 3452, 1597, 1582,
1
1245, 770 cmꢂ1. H NMR (CDCl3, 200 MHz): d 8.37e8.33
1367, 1148, 785 cmꢂ1
.
1H NMR (CDCl3, 200 MHz):
(m, 2H), 7.29 (d, 1H, J ¼ 7.7 Hz), 7.18e6.72 (m, 9H), 5.35
(s, 1H), 3.97 (t, 2H, J ¼ 6 Hz), 3.68 (s, 3H), 2.78 (t, 2H,
J ¼ 6 Hz), 2.55 (q, 4H, J ¼ 7.1 Hz), 1.00 (t, 6H, J ¼ 7.2 Hz).
MS: 391 (Mþ þ H). Anal. Calcd for (C25H30N2O2): C,
76.89; H, 7.74; N, 7.17. Found C, 76.75; H, 7.64; N, 7.29.
d 8.71e8.69 (m, 2H), 8.01e7.89 (m, 3H), 7.65e7.49 (m,
4H), 7.38e7.25 (m, 7H), 6.93 (d, J ¼ 8.5 Hz, 2H), 6.50 (s,
1H), 5.02 (s, 2H), 2.31 (bs, 1H). MS: 390 (Mþ). Anal. Calcd
for (C28H22O2): C, 86.13; H, 5.68. Found C, 86.54; H, 6.73.
1.18. 3-{(4-Methoxy-phenyl)-[4-(2-pyrrolidin-1-yl-
ethoxy)-phenyl]-methyl}-pyridine 16c
1.22. 2-[(4-Benzyloxy-phenyl)-phenanthren-9-yl-
methyl]-phenol 21
As described for 6a, compound 12 (0.2 g, 0.687 mmol),
K2CO3 (0.474 g, 3.43 mmol) and 1-(2-chloroethyl)-pyrroli-
dine hydrochloride (0.174 g, 1.03 mmol) furnished 16c as yel-
low viscous oil (0.165 g, 62.26%). IR (neat): 3440, 1635,
As described for 11, carbinol 20 (2.0 g, 5.01 mmol) and
phenol (0.63 g, 7.6 mmol) in dry benzene (30 mL) furnished
21 (0.360 mg, 30%) as dark orange solid, m.p. 90 ꢁC. IR
1
(KBr): 2926, 1628, 1453, 1259, 1051, 785 cmꢂ1. H NMR
1
1245, 770 cmꢂ1. H NMR (CDCl3, 200 MHz): d 8.32e8.30
(CDCl3, 200 MHz): d 8.66 (d, 1H, J ¼ 8.2 Hz), 8.62 (d, 1H,
J ¼ 8 Hz), 7.99 (d, 1H, J ¼ 8 Hz), 7.57e7.37 (m, 10H), 7.16
(d, 1H, J ¼ 7.8 Hz), 7.14e7.04 (m, 3H), 7.14 (d, 2H,
J ¼ 8.4 Hz), 7.00 (s, 1H), 6.96 (d, 2H, J ¼ 8.4 Hz), 6.37 (s,
1H), 4.98 (s, 2H), 4.87 (bs, 1H). MS: 466 (Mþ). Anal. Calcd
for (C34H26O2): C, 87.52; H, 5.62. Found: C, 87.25; H, 5.73.
(m, 2H), 7.27 (d, 1H, J ¼ 7.8 Hz), 7.07e6.67 (m, 9H), 5.31
(s, 1H), 3.95 (t, 2H, J ¼ 6 Hz), 3.61 (s, 3H), 2.75 (t, 2H,
J ¼ 6 Hz), 2.49e2.47 (m, 4H), 1.65e1.58 (m, 4H). MS: 389
(Mþ þ H). Anal. Calcd for (C25H28N2O2): C, 77.29; H, 7.26;
N, 7.21. Found C, 77.35; H, 7.34; N, 7.09.
1.19. 3-{(4-Methoxy-phenyl)-[4-(2-piperidin-1-yl-
ethoxy)-phenyl]-methyl}-pyridine 16d
1.23. 4-[(4-Benzyloxy-phenyl)-phenanthren-9-yl-
methyl]-phenol 22
As described for 6a, compound 12 (0.2 g, 0.687 mmol),
K2CO3 (0.474 g, 3.43 mmol) and 1-(2-chloroethyl)-piperidine
hydrochloride (0.19 g, 1.03 mmol) furnished 16d as yellow
viscous oil (0.18 g, 65.2%). IR (neat): 3440, 1635, 1245,
As described for 11, carbinol 20 (2.0 g, 5.01 mmol) and
phenol (0.63 g, 7.6 mmol) in dry benzene (30 mL) furnished
22 (0.849 g, 70%) as dark orange solid, m.p. 85 ꢁC. IR
1
770 cmꢂ1
.
1H NMR (CDCl3, 200 MHz): d 8.36e8.31 (m,
(KBr): 2932, 1621, 1443, 1252, 1041, 790 cmꢂ1. H NMR
2H), 7.29 (d, 1H, J ¼ 7.8 Hz), 7.19e6.72 (m, 9H), 5.35 (s,
1H), 4.02 (t, 2H, J ¼ 6 Hz), 3.67 (s, 3H), 2.71 (t, 2H,
J ¼ 6 Hz), 2.48e2.43 (m, 4H), 1.56e1.49 (m, 4H), 1.37e
1.39 (m, 2H). MS: 403 (Mþ þ H). Anal. Calcd for
(C26H30N2O2): C, 77.58; H, 7.51; N, 6.96. Found C, 77.65;
H, 7.60; N, 6.85.
(CDCl3, 200 MHz): d 8.70 (d, 1H, J ¼ 8.2 Hz), 8.64 (d, 1H,
J ¼ 8 Hz), 8.01 (d, 1H, J ¼ 8 Hz), 7.58e7.32 (m, 10H), 7.22
(d, 1H, J ¼ 7.8 Hz), 7.15e7.06 (m, 3H), 7.07 (s, 1H), 6.88
(d, 2H, J ¼ 8.4 Hz), 6.73 (d, 2H, J ¼ 8.4 Hz), 6.12 (s, 1H),
5.01 (s, 2H). MS: 466 (Mþ). Anal. Calcd for (C34H26O2): C,
87.52; H, 5.62. Found: C, 87.59; H, 6.70.

G. Panda et al. / European Journal of Medicinal Chemistry 42 (2007) 410e419
417
1.24. {4-[(4-Benzyloxy-phenyl)-phenanthren-9-yl-
methyl]-phenoxy}-tert-butyl-dimethyl-silane 23
4. Representative chain reaction on solid phase and
cleavage from resin beads
To a solution of 22 (0.500 g, 1.07 mmol), and imidazole
(0.109 g, 1.60 mmol) in dry DCM (10 mL), TBDMSCl
(0.241 g, 1.60 mmol) was added and stirred at room tempera-
ture for 8 h. After extraction and column chromatography on
silica gel, 22 furnished the silyl derivative 23 (0.540 g, 87%)
A mixture of compound 26 (0.06 g, 0.049 mmol), anhy-
drous K2CO3 (0.203 g, 1.47 mmol), aminohydrochloride chain
(1.47 mmol) and dry DMF (15 mL) was refluxed for 48 h.
K2CO3 was filtered off and DMF was removed. The resin
beads were washed with DMF, methanol, water, methanol,
DCM and ether. It was dried under vacuo. Treatment of whole
resin beads 27 with 1% TFA in DCM gave the hydroxy
substituted alkylamine derivatives 28aec.
1
as white semi-solid. H NMR (CDCl3, 200 MHz): d 8.60 (d,
1H, J ¼ 8.2 Hz), 8.57 (d, 1H, J ¼ 8.2 Hz), 7.88 (d, 1H,
J ¼ 8 Hz), 7.45e7.15 (m, 8H), 7.06 (s, 1H), 6.92 (d, 2H,
J ¼ 8.4 Hz), 6.88 (d, 2H, J ¼ 8.2 Hz), 6.81 (d, 2H,
J ¼ 8.1 Hz), 6.71 (d, 2H, J ¼ 8.6 Hz), 6.57 (d, 2H,
J ¼ 8.4 Hz), 5.95 (s, 1H), 4.83 (s, 2H), 0.78 (s, 9H), 0.09 (s,
6H). MS: 580 (Mþ). Anal. Calcd for (C40H40O2Si): C,
82.71; H, 6.94. Found: C, 83.00; H, 6.83.
4.1. 4-{[4-(2-Dimethylamino-ethoxy)-phenyl]-
phenanthren-9-yl-methyl}-phenol 28a
Dark yellow semi-solid. IR (neat): 3323, 2937, 1623, 1529,
1
1460, 1249, 1052 cmꢂ1. H NMR (CDCl3, 200 MHz): d 8.60
1.25. 4-{[4-(tert-Butyl-dimethyl-silanyloxy)-phenyl]-
phenanthren-9-yl-methyl}-phenol 24
(d, 1H, J ¼ 8.2 Hz), 8.58 (d, 1H, J ¼ 8.2 Hz), 7.98 (d, 1H,
J ¼ 8 Hz), 7.60e7.41 (m, 5H), 7.06 (s, 1H), 6.96e6.90 (m,
4H), 6.70e6.66 (m, 4H), 6.04 (s, 1H), 4.08 (t, 2H,
J ¼ 7 Hz), 2.82 (t, 2H, J ¼ 7 Hz), 2.42 (s, 6H). MS: 448
(Mþ þ H). Anal. Calcd for (C31H29NO2): C, 83.19; H, 6.53;
N, 3.13. Found: C, 83.25; H, 6.63; N, 3.12.
The silyl derivative 23 (0.490 g, 0.84 mmol) was hydroge-
nated over Pd/C (0.015 g) and after usual work-up and purifi-
cation furnished 24 (0.360 g, 87%) as pale yellow semi-solid.
1H NMR (CDCl3, 200 MHz): d 8.61 (d, 1H, J ¼ 8.2 Hz), 8.57
(d, 1H, J ¼ 8.2 Hz), 7.88 (d, 1H, J ¼ 8 Hz), 7.45e7.18 (m,
5H), 6.93 (s, 1H), 6.85e6.78 (m, 4H), 6.59e6.53 (m, 4H),
5.93 (s, 1H), 4.30 (s, 1H), 0.79 (s, 9H), 0.01 (s, 6H). MS:
490 (Mþ). Anal. Calcd for (C33H34O2Si): C, 80.77; H, 6.98.
Found: C, 80.89; H, 6.90.
4.2. 4-{[4-(2-Diethylamino-ethoxy)-phenyl]-
phenanthren-9-yl-methyl}-phenol 28b
Dark yellow semi-solid. IR (neat): 3420, 2972, 1613, 1502,
1
1473, 1240, 1031 cmꢂ1. H NMR (CDCl3, 200 MHz): d 8.62
(d, 1H, J ¼ 8.2 Hz), 8.60 (d, 1H, J ¼ 8.2 Hz), 8.00 (d, 1H,
J ¼ 8 Hz), 7.64e7.51 (m, 5H), 7.01 (s, 1H), 7.05e6.97 (m,
4H), 6.80e6.73 (m, 4H), 6.11 (s, 1H), 4.08 (t, 2H,
J ¼ 7 Hz), 2.84 (t, 2H, J ¼ 7 Hz), 2.75 (q, 4H, J ¼ 7 Hz),
1.11 (t, 6H, J ¼ 7 Hz). MS: 476 (Mþ þ H). Anal. Calcd for
(C33H33NO2): C, 83.33; H, 6.99; N, 2.94. Found: C, 83.25;
H, 6.93; N, 2.99.
2. Loading of 24 on resin
A mixture of compound 24 (0.200 g, 0.40 mmol), 2-chlor-
otritylchloride resin (0.24 g, 0.20 mmol) and pyridine (5 mL)
was refluxed at 50 ꢁC for 8 h. The solvent was removed and
the resin beads were washed with pyridine, methanol, DCM
and ether. Treatment of few resin beads 25 with 1% TFA in
DCM gave the authentic starting material 24.
4.3. 4-{Phenanthren-9-yl-[4-(2-piperidin-1-yl-ethoxy)-
phenyl]-methyl}-phenol 28c
3. Cleavage of TBDMS group of 25 with TBAF on resin
Dark yellow semi-solid. IR (neat): 3395, 2935, 1610, 1513,
1
The resin bound compound 25 (0.030 g, 0.024 mmol) was
treated with 1 M TBAF in THF (0.036 mL, 0.036 mmol) at
room temperature for 24 h. The solvent was removed and
the resin beads were washed with pyridine, methanol, DCM
and ether. Treatment of few resin beads 26 with 1% TFA in
DCM gave 4-[(4-hydroxy-phenyl)-phenanthren-9-yl-methyl]-
phenol as dark orange solid, m.p. 112 ꢁC. IR (KBr): 3323,
1453, 1247, 1076 cmꢂ1. H NMR (CDCl3, 200 MHz): d 8.62
(d, 1H, J ¼ 8.2 Hz), 8.60 (d, 1H, J ¼ 8.2 Hz), 8.02 (d, 1H,
J ¼ 8 Hz), 7.70e7.51 (m, 5H), 7.10 (s, 1H), 7.00e6.90 (m,
4H), 6.80e6.70 (m, 4H), 6.09 (s, 1H), 4.08 (t, 2H,
J ¼ 7 Hz), 2.79 (t, 2H, J ¼ 7 Hz), 2.53 (t, 4H, J ¼ 7 Hz),
1.67e1.60 (m, 6H). MS: 488 (Mþ þ H). Anal. Calcd for
(C34H33NO2): C, 83.74; H, 6.82; N, 2.87. Found: C, 83.63;
H, 6.90; N, 2.98.
1602, 1505, 1441, 1365, 1230, 1170 cmꢂ1
.
1H NMR
(CDCl3, 200 MHz): d 8.72 (d, 1H, J ¼ 8.2 Hz), 8.65 (d, 1H,
J ¼ 8.2 Hz), 8.02 (d, 1H, J ¼ 9 Hz), 7.69e7.48 (m, 5H), 7.14
(s, 1H), 7.01 (d, 4H, J ¼ 8.2 Hz), 6.75 (d, 4H, J ¼ 8.2 Hz),
6.12 (s, 1H), 4.67 (bs, 2H). 13C NMR: d 154.4, 139.1, 136.4,
131.8, 131.6, 131.2, 130.2, 129.1, 128.7, 127.0, 126.8,
126.5, 125.6, 123.4, 122.7, 115.7, 109.9, 52.2. MS: 376
(Mþ). Anal. Calcd for (C27H20O2): C, 86.14; H, 5.36. Found:
C, 86.45; H, 5.78.
5. Antimycobacterial activity
5.1. Agar micro dilution method
Drug susceptibility and determination of MIC of the test
compounds/drugs against M. tuberculosis H37Rv were per-
formed by agar microdilution method where serial two-fold

418
G. Panda et al. / European Journal of Medicinal Chemistry 42 (2007) 410e419
dilutions of each test compound were added into 7H10 agar
and M. tuberculosis H37Rv was used as test organism. MIC
is the concentration of the compound that completely inhibits
the growth and colony forming ability of M. tuberculosis.
In 24 well plate, 3 mL middle brook 7H11 agar medium
with OADC supplement is dispensed in each well. The test
compound is added to the middle brook medium agar before
in duplicate so that final concentration of test compound in
each well is 25, 12.5, 6.25, 3.125 and 1.56 mg/mL. The known
CFU of H37Rv culture was dispensed on top of agar in each
well in negative pressure biosafety hood. The plates are then
incubated at 37 ꢁC/5% CO2 incubator. The concentration at
which complete inhibition of colonies was observed was taken
as MIC of test drug.
containing different concentrations of test compound/known
toxic compound/DMSO. After 24 h incubation (37 ꢁC, 5%
CO2) 20 mL MTS reagent (Promega Kit) is added and absor-
bance is read after 2 h at 490 nm. Absorbance shown by
DMSO containing wells is taken as 100% survivors. A com-
pound is considered toxic if it causes ꢃ50% inhibition at con-
centration 10-fold higher than its MIC (minimum
concentration which completely inhibits growth of M. tubercu-
losis H37Rv).
Acknowledgements
Department of Science and Technology (SR/FTP/CSA-05/
2002), New Delhi, India supported this research project. Ma-
loy, Sajal and Shagufta thank CSIR for providing fellowships
(CSIR-NET-JRF).
5.2. BACTEC method
Stock solution of the test compounds prepared in DMSO at
1 mg/mL was sterilized by passage through 0.22 m filters.
Fifty microliters were added to 4 mL radiometric 7H12 broth
(BACTEC 12B; Becton Dickinson Diagnostic Instrument Sys-
tem, US) to achieve final concentrations. Controls received
50 mL DMSO. Isoniazid and rifampin (Sigma Chemical Co.
St. Louis, MO) were included as positive drug control. In
BACTEC method, 104e105 CFU/mL of M. tuberculosis
H37Rv was inoculated in 4 mL fresh BACTEC 12B broth con-
taining the test compounds. An additional control was inocu-
lated with 1:100 dilution of the inoculum to represent 1% of
the bacterial population (102e103 CFU/mL). The vials were
incubated at 37 ꢁC and GI readings were recorded daily until
the GI in 1:100 control had reached 30. The concentration of
the drug producing final GI reading lower than those in 1:100
control was considered to have inhibited more than 90% of the
bacteria and was defined as the MIC.
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M. tuberculosis, H37Ra was used as a suitable surrogate for
the virulent H37Rv strain. The standard antitubercular agents
Rifamycin, isoniazid, p-aminosalicylic acid, ethambutol and
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