Design, synthesis, biological evaluation and molecular modelling studies of novel quinoline derivatives against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2009.02.026

We herein describe the synthesis and antimycobacterial activity of a series of 27 different derivatives of 3-benzyl-6-bromo-2-methoxy-quinolines and amides of 2-[(6-bromo-2-methoxy-quinolin-3-yl)-phenyl-methyl]-malonic acid monomethyl ester. The antimycob

Full text of DOI:10.1016/j.bmc.2009.02.026

Bioorganic & Medicinal Chemistry 17 (2009) 2830–2841
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, biological evaluation and molecular modelling studies
of novel quinoline derivatives against Mycobacterium tuberculosis q
Ram Shankar Upadhayaya a, Jaya Kishore Vandavasi a, Nageswara Rao Vasireddy a, Vivek Sharma a,
Shailesh S. Dixit a, Jyoti Chattopadhyaya b,
*
a Institute of Molecular Medicine, Pune 411 057, India
b Department of Bioorganic Chemistry, Biomedical Centre, Uppsala University, Box 581, SE-75123 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
We herein describe the synthesis and antimycobacterial activity of a series of 27 different derivatives of
3-benzyl-6-bromo-2-methoxy-quinolines and amides of 2-[(6-bromo-2-methoxy-quinolin-3-yl)-phenyl-
methyl]-malonic acid monomethyl ester. The antimycobacterial activity of these compounds was evalu-
ated in vitro against Mycobacterium tuberculosis H37Rv for nine consecutive days upon a fixed concentra-
Received 5 November 2008
Revised 11 February 2009
Accepted 15 February 2009
Available online 21 February 2009
tion (6.25 lg/mL) at day one in Bactec assay and compared to untreated TB cell culture as well as one
with isoniazide treated counterpart, under identical experimental conditions. The compounds 3, 8, 17
and 18 have shown 92–100% growth inhibition of mycobacterial activity, with minimum inhibitory con-
centration (MIC) of 6.25 lg/mL. Based on our molecular modelling and docking studies on well-known
Keywords:
ATP synthase
Diarylquinoline
diarylquinoline antitubercular drug R207910, the presence of phenyl, naphthyl and halogen moieties
seem critical. Comparison of docking studies on different stereoisomers of R207910 as well as com-
pounds from our data set, suggests importance of electrostatic interactions. Further structural analysis
of docking studies on our compounds suggests attractive starting point to find new lead compounds with
potential improvements.
Protein–ligand docking
Anti-tuberculosis drugs
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
HIV-negative individuals.4 The second is the emergence of drug-
resistant, multi-drug-resistant TB (MDR-TB)5 and recently devel-
Tuberculosis (TB) infection (Mycobacterium tuberculosis) has be-
come a serious worldwide problem, infecting in synergy with hu-
man immunodeficiency virus (HIV) infection.1 It is contagious,
transmitted through air and can infect different organs of the hu-
man body, although more than 75% of infection is caused in lungs.
It is estimated that, about one third of the world’s population is in-
fected with this disease.2 According to the World Health Organiza-
tion (WHO) approximately 8 million people are believed to have
contracted TB annually with almost 2 million deaths.2 Three devel-
opments have made the resurgence in TB especially alarming. The
first is its pathogenic synergy with HIV.3 The overall incidence of
TB in HIV-positive patients is 50 times than that of the rate for
oped extensive drug-resistant tuberculosis (XDR-TB).6 The third
is gradual understanding of the latent phase of this disease. Con-
sidering the world-wide TB problems, there is an urgent need to
develop relatively inexpensive new drugs to treat this deadly dis-
ease, especially the MDR- and XDR-TB strains.
The quinoline class of compounds has shown the promising
activity against resistant tuberculosis. Amongst these, diarylquino-
line R207910 (called as DARQ from here on), targeted to the proton
pump of bacterial ATP synthase has inhibited mycobacterial
growth very effectively.7 Based on sequence analysis of in vitro
mutants resistant to DARQ, it has been established that the target
of this putative drug molecule is the c subunit in the FO domain of
ATP synthase.7,8 Also, various mycobacterial species or mutants
harbouring different amino acids at locations Ala63 and Ile66 (sub-
unit c) were found to be resistant to DARQ.8 Based on these analy-
sis the binding site of DARQ has been proposed near Glu61, which
is a highly conserved acidic residue site in ATP synthase subunit c
family. It has been known for some time now that the correspond-
ing enzyme in Escherichia coli has an equivalent aspartic acid at this
location, which is involved in proton translocation in the enzyme,
along with another crucial residue, arginine (R186 in M. tuberculo-
sis and R210 in E. coli) from subunit a.9 Theoretical and structural
Abbreviations: ATP, adenosine triphosphate; MDR, drug-resistant; XDR, exten-
sive drug-resistant; MIC, minimum inhibitory concentration; DMSO, dimethyl
sulfoxide; TB, tuberculosis; WHO, World Health Organization; HIV, human immu-
nodeficiency virus; DARQ, diarylquinoline; EDCÁHCl, 1-ethyl-3-(3-dimethylamino-
propyl) carbodiimide hydrochloride; DBU, 1,8-diazabicyclo-[5.4.0] undec-7-ene;
TBAB, n-tetrabutylammonium bromide.
q
NOTE: All amino acid numbering corresponds to Mycobacterium tuberculosis ATP
synthase subunits a and c, unless otherwise stated.
* Corresponding author. Tel.: +46 18 471 45 77; fax: +46 18 554495.
E-mail address: jyoti@boc.uu.se (J. Chattopadhyaya).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.026

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 17 (2009) 2830–2841
2831
studies on DARQ has led to identification of an intramolecular
hydrogen-bond in RS stereoisomer, providing it an extra stability
over other isomers.10 Also, docking studies of DARQ (all four ster-
eoisomers) have suggested the important role of hydroxyl (ÀOH)
and N,N dimethyl (ÀN(CH3)2) moiety in binding to the target
protein.11
dures followed for all reported compounds along with the
respective yields are shown in Table 1. The % inhibition profile of
each compound against M. tuberculosis H37Rv in vitro is described
in Table 2.
2.1. Synthesis
Based on this available information, we have performed the
structural, chemical and functional analysis of DARQ in order to
shed some light on the origin of its antimycobacterial activity by
addressing which of the parts of the DARQ molecule is more
important as a pharmacophore for the anti-TB activity. Thus, we
have divided this molecule into four hemispheres as shown in Fig-
ure 1, and have synthesized new series of compounds12 based on
North-East (NE) and South-East (SE) hemispheres of DARQ
molecule.
For this purpose, the hydroxy group in the NE part, have been
substituted with different amines (compounds 3–20) and different
amides of a diacid (compounds 23–31), which can potentially act
on binding sites of ATP synthase with different binding ability.
The side chain with the N,N-dimethyl amino terminal of DARQ
was removed since, we assumed, both the amines and the amides
might act as binding sites to retain the potency of the compounds.
The naphthalene moiety of the SE part, which presumably acts as a
binding motif with its lipophilic nature, was substituted with dif-
ferent amines (3–20), and was replaced by the methyl ester group
in the amide class of compounds (23–31). The main reason behind
the change of SE oriented naphthalene moiety was to reduce the
bulk of the molecule, because once the total volume of the mole-
cule is reduced, the membrane penetration may become more
favourable, which may lead to an improved fit into the binding site.
We have also performed molecular modelling and docking studies
leading to identification of critical binding sites of the target ATP
synthase. Interactions between DARQ and its target protein, ATP
synthase, is important for its antimycobacterial activity, which in
turn would enable us to improve anti-TB activity of our new in-
house synthesized molecules.
The compounds were prepared using the synthetic sequence
illustrated in Scheme 1. 3-Benzyl-6-bromo-2-methoxy-quinoline
(1), was prepared according to literature procedure.13 It was bro-
minated at the benzylic centre using N-bromosuccinimide and
dibenzoyl peroxide to obtain 2 (81%). The series of compounds
3–16 were obtained in 10–59% yields by nucleophilic displacement
of bromine with a suitable amine (R–H), (Scheme 1) under the
influence of activated potassium carbonate in dry N,N-dimethyl-
formamide (Procedure A). The amines 17 (pyrazole derivative),
18 (6-amino coumarin derivative), 19 and 20 could not however
be synthesized using Procedure A, because of poorer acidic nature
of amino-proton of these relatively more basic amines. Hence we
employed a relatively more severe condition to generate the
respective conjugate base of these amines for bromo displacement
in compound 2. Thus, pyrazole derivative 17 was obtained by heat-
ing the bromo compound 2 with pyrazole in toluene at reflux in
presence of catalytic amount of phase transfer catalyst, n-tetrabu-
tylammonium bromide (TBAB) and 20% aqueous sodium hydroxide
solution (51%, Procedure B). We however got only a trace amount
of compound 18, by Procedure B. Hence we modified the procedure
by adding a organic base 1,8-diazabicyclo-[5.4.0] undec-7-ene
(DBU), a catalytic amount of TBAB, 6-amino coumarin hydrochlo-
ride and compound 2, in toluene at reflux to produce compound
18 in moderate yield (42%, Procedure C). Procedures A, B and C
failed completely to give compounds 19 and 20. Ethyl ester of
naphthyl acetic acid when however treated with sodium hydride
in dry tetrahydrofuran, followed by addition of bromo compound
2, gave a diastereomeric mixture of 19 and 20 (19%, Procedure D).
Table 1
2. Results
Procedures** used for the synthesis of compounds (3–31) and yields obtained
We have so far prepared 27 different 3-benzyl-6-bromo-2-
methoxy-quinoline derivatives and amides of 2-[(6-bromo-2-
Compd #
Procedure
Time (h)
Temperature
Yield (%)
3–16
17
A
A
B
A
B
C
A
D
A
D
E
E
E
F
2
2
80 °C
80 °C
110 °C
80 °C
110 °C
110 °C
80 °C
rt
80 °C
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
10–59
NR*
51
methoxy-quinolin-3-yl)-phenyl-methyl]-malonic
acid
mono-
12
2
12
12
2
6
2
6
16
16
16
16
16
16
16
16
16
16
16
methyl ester as two potential classes of compounds. Specific proce-
18
NR*
Traces#
42
19
20
NR*
10
NR*
9
23
24
25
27
25
14
NR*
15
26
E
F
E
E
F
NR*
14
27
28
29
30
31
13
14
30
28
F
F
Notes: NR = *No reaction. #Not isolated.
**Procedure A: RH (1 equiv), K2CO3 (1.2 equiv), DMF, 80 °C, 2 h; 10–59%; Procedure
B: 20% aq NaOH (30 mL), pyrazole (1 equiv), TBAB (10 mol %), dry toluene, reflux,
12 h; 51%; Procedure C: 6-aminocoumarin hydrochloride (1 equiv), DBU (1 equiv),
TBAB (18 mol %), toluene, reflux, 12 h; 12%; Procedure D: dry THF, NaH (1.5 equiv),
naphthyl acetic acid ethyl ester (1.1 equiv), rt, 6 h, 19%; Procedure E: R in dry THF,
NaH (1.2 equiv), 1.5 h, 28 °C, added to 2 (1 equiv) in dry THF, 28 °C, 16 h, 13–27%;
Procedure F: RH (1.1 equiv), EDCÁHCl (1.2 equiv), HOBt (1.1 equiv), diisopropyl
amine (1.2 equiv), THF, rt, 16 h.
Figure 1. Four different Hemispheres of the molecular structure of highly active
R207910 (DARQ)7 is shown.

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R. S. Upadhayaya et al. / Bioorg. Med. Chem. 17 (2009) 2830–2841
Table 2
days upon single dose administration on the first day. From analy-
sis of graphs plotted for compounds 3 and 18, it can be seen that
the mycobacterial load, upon a single dose administration at day
one, never increase from day 1 to day 9, this observation reflects
interesting bactericidal nature of our tested compounds.
Based on our data it is clear that antimycobacterial activity of
the compounds 3, 8, 17 and 18 are comparable to the standard
drug isoniazid.18 Figure 2 reveals that compounds 8 and 17 can re-
duce the mycobacterial load up to a sterile condition, and no myco-
bacterial growth has been seen during the 9 days of study with
single dose treatment.
Another interesting observation emerged from our study is the
fact that the time taken to obtain the zero bacterial load under our
experimental condition is different for different compounds. Thus,
compounds 8 and 17 can clear the mycobacterial infection within 2
and 7 days, respectively, whereas the frontline drug, isonazid,18
under identical experimental condition (BACTEC 460 radiometric
method)16,17 can clear the mycobacterial infection within 9 days
to attain the same activity profile. We have observed through pre-
liminary BACTEC data that isoniazid has not shown zero growth of
M. tuberculosis. A possible reason could be that the M. tuberculosis
develops resistance when exposed to isoniazid for 9 days, which is
not the case with compound 8.
Description of the compounds synthesized for testing against M. tuberculosis, their %
inhibition and clog P
Compd no.
% Inhibition
clogP
Compd no.
% Inhibition
clogP
3
4
5
6
7
8
9
10
11
12
13
14
15
16
94.2
NT
NT
59
NT
100
53
69
14
18
16
4.69
5.50
6.00
5.06
5.25
7.43
6.10
4.82
4.47
6.03
5.49
4.11
7.30
4.57
17
18
19
20
23
24
25
26
27
28
29
30
31
Isoniazid
99.5
92.5
0
0
5
4.96
6.19
8.03
8.03
4.11
4.11
4.35
4.35
4.91
4.91
4.93
6.24
6.24
À0.8a
3
5
10
7
11
22
8
17
08
14
12
99
NT: Not tested.
a
From PubChem (http://pubchem.ncbi.nlm.nih.gov/). Actual formula and struc-
ture of compounds can be seen in Scheme 1.
Synthesis of compounds (23–31) were also started from the
bromo compound 2. Treatment of 2 with dimethyl malonate in
tetrahydrofuran using sodium hydride as a base gave 21 in excel-
lent yield (94%). Selective hydrolysis of one of the methyl esters in
21 was achieved by heating with aqueous potassium hydroxide in
methanol14 at reflux temperature to produce the compound 22 in
satisfactory yield (48%). Compound 22 upon treatment with pyr-
rolidine, 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydro-
chloride (EDCÁHCl), hydroxybenzotriazole (HOBt) and diisopropyl
amine in tetrahydrofuran (Procedure F) did not furnish compound
25–26. Compounds 23–28 were therefore prepared by treating
the corresponding amide of malonic acid methyl ester with so-
dium hydride in dry tetrahydrofuran followed by addition of com-
pound 2 in dry tetrahydrofuran (Procedure E). The compounds 29,
30 and 31 were synthesized in 14–30% yields by treating a suit-
able amine (R–H) with 22 using EDCÁHCl, HOBt and diisopropyl
amine in tetrahydrofuran (Procedure F).15 All the synthesized
compounds were fully characterized by 1H NMR, 13C NMR, distor-
tionless enhancement by polarization transfer (DEPT) and mass
spectroscopy.
The comparison of percentage (%) growth inhibition (Fig. 3)
ability of compounds with respect to standard drug isoniazid,
suggests that compounds 3, 17 and 18 are able to inhibit the
M. tuberculosis H37Rv 94.2% ( 0.2), 99.5% ( 0.4) and 92.5%
( 2.8), respectively, whereas compound 8 can inhibit the myco-
bacterial growth 100% ( 0). The mycobacterial activity profile
of 3, 8, 17 and 18 compounds suggests that each compound
has a bactericidal effect as there has been no growth in treated
control.
Five different concentrations (6.25, 3.25, 1.56, 0.78 and 0.39
lg/
mL) were chosen to determine the MIC of compounds 3, 8, 17 and
18 against H37Rv strain and was found to be 6.25 lg/mL for each
of them (Table 3).
2.4. Molecular modelling and docking
The docking of DARQ (RS stereoisomer) in the putative binding
site of ATP-synthase subunit a and c suggested that –OH and N,N-
dimethyl moieties may interact with the crucial residues R186 (a
subunit) and E61 (c subunit), as shown in Figure 4a. The distance
between –O:hydroxyl of DARQ and NH2 of R186 = 3.6 Å and –N:di-
methyl of DARQ and OE2 of E61 deprotonated = 4.3 Å. Only in the
case of RS stereoisomer these polar parts of DARQ came into the
vicinity of the polar areas of protein. The initial docking scores
(Intermolecular energies) of all four stereoisomers (SS, SR, RR and
RS) were comparable, but after forcefield based refinement, inter-
action energies (van der Waals and electrostatic contributions
only) between DARQ and protein was found to be lowest in the
case of RS stereoisomer (Table S1). Also, as observed in the RS ste-
reoisomer docked complex, the non-polar parts of DARQ molecule,
that is, naphthyl, phenyl and quinoline aromatic rings were making
effective van der Waals interaction with similar parts from the pro-
tein (Section 3).
Docking of some of the active and less active compounds from
our data set suggested similar interactions to the ones observed
for DARQ binding. The highly active compounds 3, 8, 17 and 18
docked into the same putative binding site of ATP synthase, are
shown in Figure 4 (compounds 3 and 18). In general all compounds
used in docking studies bind effectively in the binding site, but
split of docking intermolecular energies suggests that lacking elec-
trostatic contributions could be the reason of their moderate activ-
ity (Table S2 and Section 3).
2.2. Microbiology
All compounds 3–20, 23–31 and drug references were dissolved
in DMSO at a concentration of 6.25
used.
l
g/mL and stored at ꢀ4 °C until
2.3. Antimycobacterial activity
The compounds 3–20 and 23–31 were screened against M.
tuberculosis H37Rv (ATCC 27294) at the single concentration of
6.25 l
g/mL by BACTEC 460 radiometric method.16,17 It has been
found that 3-benzyl-6-bromo-2-methoxy-quinoline derivatives
(3–20) have displayed superior anti-TB activity (Table 2) than the
amide derivatives of 2-[(6-bromo-2-methoxy-quinolin-3-yl)-phe-
nyl-methyl]-malonic acid monomethyl ester (23–31). On the basis
of the calculated values of growth index (GI) and percentage TB
growth inhibition, the four compounds 3, 8, 17 and 18 were found
to show excellent antimycobacterial activity in the single dose
study (administered on day one). We have plotted the graphs for
growth index on the day basis for compounds 3, 8, 17 and 18 in
comparison with first-line tuberculosis treatment regimen, isonia-
zid18 (Figs. 2 and 3).
The comparative data of antimycobacterial activity of com-
pounds 3 and 18 with isoniazid clearly shows that compounds 3
and 18 are able to clear the mycobacterial infection within nine

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 17 (2009) 2830–2841
2833
(ii or iii or
iv or v)
Br
Br
Br
(i)
Br
R1
N
O
N
3-20
O
N
O
2
1
3
4
5
: R1 = Imidazolyl (24%); : R1 = Pyridine 2-ylamine (26%); : R1 = 6-Methyl-pyridine-2-ylamine
6
7
(28%); : R1 = C-(Tetrahydro-furan-2-yl)-methylamine (32%); : R1 = C-(Thiophen-2-yl)-
8
9
methylamine (28%); : R1 = 1-(3-Trifluoromethyl-phenyl)-piperazinyl (49%); : R1 = Piperidinyl
10 11 12
(57%); : R1 = Morpholinyl (59%); : R1 = 5-Methyl-tetrazol-1-yl(50%); : R1 = Pyrrolidine-
13 14
2-carboxylic acid ethyl ester (11%); : R1 = Piperidine-4-carboxylic acid ethyl ester (15%);
:
15
16
R1 = C-Pyridin-4-yl-methylamine (17%); : R1 = Indolyl (10%); : R1 = 4-Nitro-imidazolyl
(46%); 17: R1 = Pyrazolyl (51%); 18: R1 = 6-Amino-chromen-2-one (42%); 19: R1 = Naphthalen-
1-yl-acetic acid ethyl ester (10%); 20: R1 = Naphthalen-1-yl-acetic acid ethyl ester (9%)
O
O
O
O
O
O
O
OH
Br
Br
(viii)
O
(vii)
2
O
O
N
N
21
22
(vi)
(ix)
O
Br
R2
O
O
N
23-31
24
23
25
28
: R2 = Morpholinyl (27%); : R2 = Morpholinyl (25%); : R2 = Pyrrolidinyl (14%);
26
27
: R2 = Pyrrolidinyl (15%); : R2 = Piperidinyl (14%); : R2 = Piperidinyl (13%);
29: R2 = Pyrazolyl (14%); 30: R2 = 1-(3-Trifluoromethyl-phenyl)-piperazinyl (30%);
31: R2 = 1-(3-Trifluoromethyl-phenyl)-piperazinyl (28%)
Scheme 1. Reagents and conditions: (i) NBS (1 equiv), dibenzoylperoxide (5 mol %), CCl4, reflux, 2 h; 81%; (ii) RH (1 equiv), K2CO3 (1.2 equiv), DMF, 80 °C, 2 h; 10–59%
(Procedure A, for 3–16); (iii) 20% aq NaOH (30 mL), pyrazole (1 equiv), TBAB (10 mol %), dry toluene, reflux, 12 h; 51% (Procedure B, for 17); (iv) 6-aminocoumarin
hydrochloride (1 equiv), DBU (1 equiv), TBAB (18 mol %), toluene, reflux, 12 h; 12% (Procedure C, for 18); (v) dry THF, NaH (1.5 equiv), naphthyl acetic acid ethyl ester
(1.1 equiv), rt, 6 h, 19%; (Procedure D, for 19 and 20); (vi) R = 3-morpholin-4-yl-3-oxo-propionic acid methyl ester (for 23 and 24), 3-oxo-3-pyrrolidin -1-yl-propionic acid
methyl ester35 (for 25 and 26), 3-oxo-3-piperidin-1-yl-propionic acid methyl ester36 (for 27 and 28); in dry THF, NaH (1.2 equiv), 1.5 h, 28 °C, added to 2 (1 equiv) in dry THF,
28 °C, 16 h, 13–27% (Procedure E); (vii) NaH (1.2 equiv), dimethyl malonate (1.4 equiv), THF, rt, 4 h; 94%; (viii) aq KOH (1 equiv), MeOH, reflux, 12 h; 48%; (ix) RH (1.1 equiv),
EDCÁHCl (1.2 equiv), HOBt (1.1 equiv), diisopropyl amine (1.2 equiv), THF, rt, 16 h, (Procedure F, for 29, 30 and 31).
2.5. Cytotoxicity
compound 18 the main binding site, the secondary amino func-
tionality was buried between the lipophilic portions thereby dis-
turbing the lipophilic–lypophobic balance of the molecule which
might have influenced its moderate antimycobacterial activity.
When we consider the structure of 8, it has a tetrahedral space
filling with the saturated piperizine ring, where it has nitrogens
to act as good binding sites as well as the substituted phenyl ring,
which may enhance the potency of the molecule by acting as a
binding motif. The phenyl ring also has a reasonable delocaliza-
tion of charges due to the presence of the polar trifluoromethyl
substitution at meta position, which provides a better lipophilic–
lypophobic balance, and it may favour better binding and penetra-
tion ability, thereby influencing the potency of the molecule. All
these explained properties makes the molecule 8 potential anti-
mycobacterial agent .
The most active compounds of our data set were also subjected
to cytotoxicity assay in two different concentrations at 1 and
10 lg/100 lL. Table 4 lists the percentages of cells viability after
24 and 72 h of addition of compounds. These results are encourag-
ing for further process of our work.
3. Discussion
On the basis of structure functional activity of all these com-
pounds, we assume that the compounds 3 and 17 bearing the
imidazole and the pyrazole moiety, respectively, may assist in
binding to the active sites favourably. The only difference is the
position of the ring nitrogens in these two heterocyclic amines,
which evidently do not perturb the lipophilic and lypophobic bal-
ance, and thereby showing comparable activity to isoniazid. For
The amides of 2-[(6-bromo-2-methoxy-quinolin-3-yl)-phenyl-
methyl]-malonic acid monomethyl ester (23–31) have not shown

2834
R. S. Upadhayaya et al. / Bioorg. Med. Chem. 17 (2009) 2830–2841
Figure 3. %TB growth inhibition shown for the compounds 3, 8, 17 and 18.
Comparison of percentage TB growth inhibition after 7–9 days with control
containing no bactericidal agent (left column) and 4 test compounds (3, 8, 17 and
18) in the middle, while the internal reference compound used in this study is
isoniazid. %TB growth inhibition of M. tuberculosis H37Rv and the corresponding
standard deviation (SD) of three consecutive readings for 3, 8, 17, 18 and isoniazid
are, 94.2% ( 0.2), 100% ( 0), 99.5% ( 0.4), 92.5% ( 2) and 99.3% ( 0.2).
Table 3
MIC of compounds against M. tuberculosis H37Rv
Compound
MIC (lg/mL)
3
8
17
18
6.25
6.25
6.25
6.25
All four stereoisomers of the DARQ molecule were modelled and
docked into the binding site. The binding site is flanked by trans-
membrane helices of subunit a and c of the ATP synthase homology
model. All 4 stereoisomers docked to the protein with almost sim-
ilar docking intermolecular energies (Table S2). Clustering (with
2 Å root mean square) was found better for stereoisomers SR, RR,
SS than RS, but scrutinizing the split of energies showed better
van der Waals and electrostatic energy balance in the case of RS
stereoisomer (Table S2).
Structural analysis suggested that the RS stereoisomer was
docking into a most acceptable pose. The –OH and –N(CH3)2 moi-
eties of DARQ were found to be in the vicinity of conserved polar
amino acids of subunit a and c (i.e., R186 and E61 of a and c sub-
unit, respectively). The hydrophobic phenyl moiety was placed in
the hydrophobic cavity (Leu68 and Phe65 of c subunit) of the pro-
tein and making stacking interactions with Phe65 (Fig. 4a). The
bulkier, bi-phenyl group was found to make stacking interactions
with Tyr64, however, was positioned in slightly polar area. We
suggest, the polar substituents at this location on DARQ may in-
crease its efficacy. Further, the quinoline moiety fills the void in
the protein and we propose that bromo-group on it would most
probably form some halogen bonding interactions and further sta-
bilize the system (unpublished results). These results in contrast to
previous docking study11 suggest the possible role of functional
groups of DARQ, other than hydroxyl and N,N-dimethyl.
Figure 2. The plot of three independent readings of antimycobacterial growth
index (GI), against M. tuberculosis H37Rv, are plotted against number of days (day 1
to day 9) for compounds 3, 8, 17 and 18 in comparison with isoniazid (internal
reference) upon a single dose administration (6.25 lg/mL) at day 1. The standard
deviation (SD) of GI in three consecutive readings for 3, 8, 17, 18 and isoniazid have
been calculated, please see Table S3 in Supplementary data for details.
The inhibitory effect of DARQ could be that while it sits between
the subunit–subunit interface with possible protein–ligand inter-
actions, consequently it does not allow the concomitant rotary
movements of the amino acids and the oligomeric C subunit ring.9
Also we assume that the N,N-dimethyl moiety could capture a pro-
ton and would not allow its transfer to the cytoplasmic side via
R186, owing to its comparative pKa. However, that scenario has
not been explicitly modelled in the present study. The possible
H-bonding between R186 and –OH moiety of DARQ and interaction
significant antimycobacterial activity. The reason may be the
unavailability of the binding aptitude of amides.
3.1. Docking studies
Our primary aim in this study was to analyse ligand–protein
interactions, based on which we could modify our compounds.

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 17 (2009) 2830–2841
2835
Figure 4. (a) Interactions of DARQ (RS) in the binding site of ATP synthase. (b) One active compound 3 from our data set in the binding site of ATP synthase. (c) Another active
compound 18 showing similar arrangement as DARQ molecule into the binding site. Arg186 (subunit a) and Glu61 (deprotonated and from subunit c) shown in blue and red,
respectively. Non-polar residues in white and polar in green. Ligands (DARQ, 3, 18) shown in orange licorice representation. Oxygen, nitrogen and bromine atoms are coloured
with red, blue and green schemes. Yellow ribbons representation for the transmembrane helices.
Table 4
not be critical for antitubercular activity, but they are needed any-
way for structural fine-tuning of the ligand in the binding site.
Cytotoxic effects of tested compounds on murine macrophage cells after 24 h and
72 h
Lowest energy selected conformation of comparatively smaller
Compound
10
24 h (%)
l
g/100
l
L
1
l
g/100
l
L
compound 3 docks in a similar manner (Fig. 4b), however, weaker
interactions between imidazole ring and protein and absence of
hydroxyl and flexible terminal amine could be the reason of its
moderate activity compared to DARQ. The imadazole functional
group could act as a potential proton acceptor but to a lesser extent
as compared to N,N-dimethyl, owing to its smaller pKa value.
Low energy docked pose of compound 8 was structurally simi-
lar to DARQ binding, however electrostatic interactions seems to
be missing (Table S2). Its piperazine moiety could act as a potential
proton acceptor and may have a role in antitubercular activity. The
compound 17 is almost similar to 3, with only heterocyclic ring dif-
ference. Another active compound 18, which is structurally similar
to original DARQ molecule sits in a similar arrangement with its
aromatic parts in stacking interaction with protein aromatics
(Fig. 4c). It has a slightly polar constituent in place of naphthyl
moiety of DARQ, which may interact with the Asn190 (subunit a)
and Asn67 (subunit c) that could be a factor for its moderate po-
tency, when its secondary amine is buried between two larger sub-
stituents. The van der Waals and relatively weaker electrostatic
interactions could be prominent for its activity. Similarly other ser-
72 h (%)
24 h (%)
72 h (%)
3
8
17
18
56
97
75
67
97
98
74
87
71
67
100
100
100
100
100
100
of DARQ molecule to all three subunits (two c and one a) could pro-
hibit the regular functioning of the enzyme. In contrast to previous
docking study,11 we suggest that arginine side chain need not flip
and still the inhibitory effect of DARQ could be attained.
In comparison, docking simulations on some of our synthesized
compounds reveal interesting facts. The docking intermolecular
energies and root mean square clustering (2 Å) for both active
and less active compounds were found to be not much different
from DARQ molecule, however, clearly the electrostatic part of
intermolecular energy was prominent only in the DARQ docking
simulations and specifically in RS stereoisomer (Table S2). This
suggests that only space-filling van der Waals interactions may

2836
R. S. Upadhayaya et al. / Bioorg. Med. Chem. 17 (2009) 2830–2841
ies compounds (3–20), does contain nitrogens, which are protonat-
able and could make interactions with binding site residues with
different strength. The reason for their varying activity could also
be associated with their delivery or octanol–water partitioning.
The compound 23/24 based on our docking studies fits well into
the binding site, but missing electrostatic interactions could be
the reason for its in vitro inactivity.
406, 408, 410. 13C NMR (100.6 MHz, CDCl3): 48.6, 54.0, 117.5,
126.2, 126.5, 128.29, 128.37, 128.47, 128.6, 129.7, 133.0, 137.5,
139.6, 144.6, 158.6.
5.1.2. Representative ProcedureA, for 3–16:Preparationof ( )-6-
bromo-3-(imidazol-1-yl-phenyl-methyl)-2-methoxy-quinoline
(3)
Overall, our preliminary docking studies suggest the phenyl
moiety of DARQ could be explored further to larger aromatics,
which could make stronger stacking interactions with protein ami-
no acid residues. Also, the halogen could be altered in second
round of modifications, which may improve the halogen bonding
interactions between the ligand and the protein. Amine and hydro-
xyl functionality seems to be critical, however, flexible side chains
with terminal amines and hydroxyl could be used in our com-
pounds, which could make lesser active compounds potent too.
A mixture of compound 2 (0.300 g, 0.74 mmol), imidazole
(0.05 g, 0.74 mmol) and potassium carbonate (0.20 g, 1.47 mmol)
in N,N-dimethylformamide (2 mL) were heated at 80 °C for 2 h.
The reaction mixture was poured into ice-water mixture and ex-
tracted with ethyl acetate. The organic layer was washed with
water, brine and dried over anhydrous sodium sulfate. Organic
layer was concentrated under vacuum to get crude product. The
crude product was purified by column chromatography on silica
gel (100–200 mesh), eluent: hexane–ethyl acetate (7:3, v/v) to af-
ford 3 (0.07 g, 24%) as off-white solid, mp 161–162 °C. 1H NMR
(400 MHz, CDCl3): d 3.97 (s, 3H), 6.82–6.88 (m, 2H), 7.08–7.11
(m, 3H), 7.29 (s, 1H), 7.34–7.38 (m, 3H), 7.41 (s, 1H), 7.67–7.73
(m, 2H), 7.76 (d, J = 1.6 Hz, 1H). 13C NMR (100.6 MHz, CDCl3): d
54.0, 59.3, 117.7, 119.2, 125.2, 125.9, 127.8, 128.62, 128.63,
128.9, 129.4, 129.7, 133.3, 135.9, 137.3, 137.4, 144.9. [M+H]+ = m/
e 394, 396.
4. Conclusion
In summary, compounds 3, 8, 17 and 18 exhibited moderate to
excellent inhibitory activity against M. tuberculosis. There is a great
similarity in these compounds as all compounds bearing a second-
ary heterocyclic amine or aryl piperazine except for 18. The aryl
piperizine substituted compound 8 was found to be most active,
even better than isoniazid under similar conditions. Although the
MIC of these compounds are not as good as the standard drug, iso-
niazid, the present results have however given us some insight on
the importance of the presence of a hydroxy group, the side chain
with the N,N-dimethyl amino terminus and/or the naphthalene
moiety, and why they are important for the better antitubercular
activity of these molecules. Docking studies speak for the impor-
tance of various functional groups of the DARQ molecule, which
could help in improving our compounds against TB infection. This
we consider as good starting points to improve the design of novel
class of anti-tuberculosis molecules.
5.1.3. N-((6-Bromo-2-methoxyquinolin-3-yl)(phenyl)methyl)
pyridin-2-amine (4)
Procedure A, yield 26%. Oily liquid. 1H NMR (400 MHz, CDCl3): d
3.99 (s, 3H), 5.22 (d, J = 6.0 Hz, 1H, D2O exchangeable), 6.14 (d,
J = 6.0 Hz, 1H), 6.30 (d, J = 8.0 Hz, 1H), 6.58–6.64 (m, 1H), 7.25–
7.40 (m, 6H), 7.62–7.69 (m, 2H), 7.83 (dd, J = 6.8, 2.0 Hz, 1H),
7.99 (s, 1H), 8.06 (d, J = 4.0 Hz, 1H). 13C NMR (100.6 MHz, CDCl3):
d 53.7, 55.7, 107.0, 113.7, 117.3, 126.5, 127.2, 127.4, 127.6, 128.5,
128.6, 129.6, 132.4, 134.5, 137.6, 140.8, 144.5, 148.3, 157.5,
160.4. [M+H]+ = m/e 420, 422.
5.1.4. [(6-Bromo-2-methoxy-quinolin-3-yl)-phenyl-methyl]-(6-
methyl-pyridin-2-yl)-amine (5)
Procedure A, yield 28%. Oily liquid. 1H NMR (400 MHz, CDCl3): d
2.60 (s, 3H), 4.08 (s, 3H), 6.04 (d, J = 7 Hz, 1H), 6.50–6.58 (m, 2H),
7.27–7.38 (m, 3H), 7.43–7.50 (m, 2H), 7.58–7.70 (m, 3H), 7.89 (d,
J = 1.0 Hz, 1H), 8.08 (s, 1H), 9.40 (br s, 1H, D2O exchangeable),
15.7 (br s, 1H, D2O exchangeable). 13C NMR (100.6 MHz, CDCl3):
d 24.2, 53.6, 55.9, 103.0, 113.2, 117.3, 126.8, 127.4, 128.4, 128.6,
129.6, 132.38, 132.42, 134.1, 134.4, 140.7, 141.9, 144.4, 144.5,
157.1, 157.2, 159.8, 160.3. [M+H]+ = m/e 434, 436.
5. Experimental
5.1. General methods
Purification and drying of reagents and solvents were carried
out according to literature procedure.19 Thin layer chromato-
graphic analyses were performed on E-Merck 60 F 254 precoated
aluminium thin layer chromatographic plates. All air-sensitive
reactions were carried out under a nitrogen atmosphere. Melting
points were determined on a Büchi melting point B-540 instru-
ment and are uncorrected. 1H spectra were recorded on a Bruker
Biospin 400 MHz spectrometer and TMS as an internal standard.
The values of chemical shifts are expressed in ppm and the cou-
pling constants (J) in Hertz (Hz). Mass spectra were recorded in
API 2000 LC/MS/MS system spectrometer and the IR spectra were
recorded in Perkin Elmer FT-IR spectrometer.
5.1.5. [(6-Bromo-2-methoxy-quinolin-3-yl)-phenyl-methyl]-
(tetrahydro-furan-2-ylmethyl)-amine (6)
Procedure A, yield 32%. White solid, mp 72–73 °C. 1H NMR
(400 MHz, CDCl3): d 1.52–1.65 (m, 2H, 1H, D2O exchangeable),
1.83–1.97 (m, 3H), 2.54–2.71 (m, 2H), 3.70–3.87 (m, 2H), 4.01 (s,
3H), 4.04–4.10 (m, 1H), 5.15 (d, J = 4.0 Hz, 1H), 7.17–7.23 (m,
1H), 7.25–7.30 (m, 2H), 7.37–7.44 (m, 2H), 7.60 (dd, J = 9.0,
2.0 Hz, 1H), 7.65 (d, J = 9.0 Hz, 1H), 7.87 (dd, J = 6.4, 2.0 Hz, 1H),
8.12 (s, 1H). 13C NMR (100.6 MHz, CDCl3): d 25.8, 29.2, 52.6, 53.5,
60.7, 67.9, 78.3, 117.0, 126.7, 127.1, 127.7, 127.8, 128.3, 128.4,
129.3, 129.5, 132.0, 133.9, 134.1, 142.2, 144.1, 160.4. [M+H]+ = m/
e 427, 429.
5.1.1. ( )-3-Bromo-3-(bromophenyl methyl)-2-methoxy-
quinoline (2)
A
mixture of 3-benzyl-6-bromo-2-methoxy-quinoline13 (1,
5.0 g, 15.24 mmol), N-bromosuccinimide (2.7 g, 15.24 mmol) and
dibenzoyl peroxide (0.18 g, 0.76 mmol) in carbon tetrachloride
was heated to reflux for 2 h. The reaction mixture was cooled to
room temperature, the solid separated was filtered, the filtrate
was concentrated under vacuum, the crude product was triturated
with hexane and dried to give the compound 2 (5.0 g, 81%) as an
off-white solid, mp 85–86 °C. 1H NMR (400 MHz, CDCl3): d 4.06
(s, 3H), 6.56 (s, 1H), 7.26–7.38 (m, 3H), 7.44–7.48 (m, 2H), 7.64–
7.69 (m, 2H), 7.87 (d, J = 4.0 Hz, 1H), 8.09 (s, 1H). [M+H]+ = m/e
5.1.6. [(6-Bromo-2-methoxy-quinolin-3-yl)-phenyl-methyl]-
thiophen-2-ylmethyl-amine (7)
Procedure A, yield 28%. Oily liquid, 1H NMR (400 MHz, CDCl3): d
2.1 (s, 1H, D2O exchangeable), 3.95 (s, 2H), 3.99 (s, 3H), 5.21 (s, 1H),
6.87–6.90 (m, 1H), 6.93–6.98 (m, 1H), 7.21–7.25 (m, 2H), 7.27–7.31
(m, 2H), 7.41–7.46 (m, 2H), 7.62 (dd, J = 8.8, 2.0 Hz, 1H), 7.67 (d,
J = 8.8 Hz, 1H), 7.87 (d, J = 2.0 Hz, 1H), 8.16 (s, 1H). 13C NMR

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 17 (2009) 2830–2841
2837
(100.6 MHz, CDCl3): d 46.5, 53.6, 59.8, 117.1, 124.4, 124.9, 126.6,
126.7, 127.3, 127.8, 128.39, 128.45, 128.6, 129.6, 132.2, 134.4,
141.7, 143.8, 144.2, 160.4. [M+H]+ = m/e 439, 441.
(dd, J = 8.8, 2.0 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.89 (d, J = 2.0 Hz,
1H), 8.22 (s, 1H). 13C NMR (100.6 MHz, CDCl3): d 13.6, 27.87,
27.93, 40.7, 50.8, 51.1, 53.0, 59.6, 67.0, 116.3, 126.2, 126.5, 127.7,
127.9, 128.2, 128.9, 131.4, 133.5, 140.3, 143.3, 159.9, 174.5.
[M+H]+ = m/e 484, 486.
5.1.7. 6-Bromo-2-methoxy-3-{phenyl-[4-(3-trifluoromethyl-
phenyl)-piperazin-1-yl]-methyl}-quinoline (8)
Procedure A, yield 49%. White solid, mp 182–183 °C. 1H NMR
(400 MHz, CDCl3): d 2.51–2.59 (m, 2H), 2.61–2.70 (m, 2H), 3.18–
3.30 (m, 4H), 4.03 (s, 3H), 4.77 (s, 1H), 7.0–7.1 (m, 3H), 7.17–7.22
(m, 1H), 7.26–7.36 (m, 3H), 7.38–7.46 (m, 2H), 7.61 (dd, J = 9.0,
2 Hz, 1H), 7.65 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 2 Hz, 1H), 8.27 (s,
1H). 13C NMR (100.6 MHz, CDCl3): d 48.7, 51.7, 53.7, 67.4, 111.81,
111.85, 115.67, 115.75, 117.2, 118.5, 126.7, 127.4, 128.2, 128.46,
128.54, 128.6, 129.5, 129.6, 132.2, 134.2, 140.4, 144.1, 151.3,
160.6. [M+H]+ = m/e 556, 558.
5.1.13. [(6-Bromo-2-methoxy-quinolin-3-yl)-phenyl-methyl]-
pyridin-4-ylmethyl-amine (14)
Procedure A, yield 17%. Reddish-yellow semi-solid. 1H NMR
(400 MHz, CDCl3): d 3.78 (s, 2H), 3.99 (s, 3H), 5.15 (s, 1H), 7.25–
7.37 (m, 5H), 7.40–7.43 (m, 2H), 7.63 (dd, J = 9.0, 2.0 Hz, 1H),
7.66 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 2.0 Hz, 1H), 8.06 (s, 1H), 8.51–
8.60 (m, 2H). 13C NMR (100.6 MHz, CDCl3): d 50.7, 53.6, 60.2,
117.2, 122.9, 126.5, 127.5, 127.7, 128.48, 128.51, 129.5, 132.3,
134.4, 141.4, 144.2, 149.2, 149.8, 160.3. [M+H]+ = m/e 435, 437.
5.1.8. 6-Bromo-2-methoxy-3-(phenyl-piperidin-1-yl-methyl)-
quinoline (9)
5.1.14. 6-Bromo-3-(indol-1-yl-phenyl-methyl)-2-methoxy-
quinoline (15)
Procedure A, yield 57%. Light-yellow solid, mp 150–152 °C. 1H
NMR (400 MHz, CDCl3): d 1.40–1.48 (m, 2H), 1.56–1.70 (m, 4H),
2.25–2.45 (m, 4H), 3.99 (s, 3H), 4.67 (s, 1H), 7.10–7.16 (m, 1H),
7.20–7.25 (m, 2H), 7.31–7.40 (m, 2H), 7.59 (dd, J = 8.8, 2.0 Hz,
1H), 7.63 (d, J = 8.8 Hz, 1H), 7.90 (d, J = 2.0 Hz, 1H), 8.24 (s, 1H).
13C NMR (100.6 MHz, CDCl3): d 24.7, 26.2, 53.2, 53.5, 68.1, 116.9,
126.86, 126.89, 128.2, 128.4, 128.6, 129.1, 129.6, 131.9, 134.3,
141.3, 143.9, 160.7. [M+H]+ = m/e 412, 414.
Procedure A, yield 10%. White solid, mp 232–233 °C. 1H NMR
(400 MHz, acetone-d6): d 3.99 (d, J = 9 Hz, 3H), 6.05 (s, 1H), 6.72
(d, J = 1.2 Hz, 1H), 6.87–6.91 (m, 1H), 7.07–7.15 (m, 1H), 7.16–
7.35 (m, 7H), 7.42 (d, J = 8.0 Hz, 1H), 7.64–7.78 (m, 3H), 7.89 (d,
J = 2.0 Hz, 1H). 13C NMR (100.6 MHz, acetone-d6): d 33.9, 45.1,
103.4, 108.4, 109.0, 110.7, 110.8, 113.5, 116.5, 118.2, 118.6,
118.7, 120.1, 120.5, 120.7, 121.4, 121.9, 123.7, 128.1, 129.2,
134.5, 152.8. [M+H]+ = m/e 444, 446.
5.1.9. 6-Bromo-2-methoxy-3-(morpholin-4-yl-phenyl-methyl)-
quinoline (10)
5.1.15. 6-Bromo-3-[(4-nitro-imidazol-)-1-yl-phenyl-methyl]-2-
methoxy-quinoline (16)
Procedure A, yield 59%. Light-yellow solid, mp 150–152 °C. 1H
NMR (400 MHz, CDCl3): d 2.34–2.4 (m, 2H), 2.47–2.49 (m, 2H),
3.71–3.85 (m, 4H), 4.01 (s, 3H), 4.70 (s, 1H), 7.17 (t, J = 7 Hz, 1H),
7.22–7.28 (m, 2H), 7.40 (d, J = 7.2 Hz, 2H), 7.60 (dd, J = 8.8, 2.0 Hz,
1H), 7.63 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 1.6 Hz, 1H), 8.25 (s, 1H).
13C NMR (100.6 MHz, CDCl3): d 52.6, 53.6, 67.1, 67.9, 117.1,
126.7, 127.3, 127.9, 128.3, 128.5, 128.6, 129.5, 132.1, 134.2,
140.1, 144.0, 160.5. [M+H]+ = m/e 414, 416.
Procedure A, yield 46%. White solid, mp 79–80 °C. 1H NMR
(400 MHz, CDCl3): d 4.01 (s, 3H), 6.87 (s, 1H), 7.10–7.14 (m, 2H),
7.37 (s, 1H), 7.40 (d, J = 1.4 Hz, 1H), 7.43–7.5 (m, 3H), 7.62 (d,
J = 1.4 Hz, 1H), 7.74 (s, 2H), 7.8 (s, 1H). 13C NMR (100.6 MHz,
CDCl3): d 54.2, 60.7, 118.1, 119.2, 122.9, 125.5, 127.5, 128.7,
129.4, 129.5, 129.8, 133.9, 135.5, 136.1, 136.7. [M+H]+ = m/e 439,
441.
5.1.16. ( )-6-Bromo-2-methoxy-3-(phenyl-pyrazol-1-yl-
5.1.10. 6-Bromo-2-methoxy-3-[(5-methyl-tetrazol-1-yl)-
phenyl-methyl]-quinoline (11)
methyl)-quinoline (17), Procedure B
20% aqueous solution of sodium hydroxide (30 mL) was added
to a mixture of compound 2 (2 g, 4.91 mmol), pyrazole (0.33 g,
4.91 mmol) and n-tetrabutyl ammonium bromide (0.13 g,
0.49 mmol) in dry toluene (20 mL) and heated to reflux for 2 h.
The reaction mixture was cooled to room temperature, diluted
with ethyl acetate and the organic layer was separated. The organic
layer was washed with water, brine, dried over anhydrous sodium
sulfate, filtered and concentrated under vacuum. The crude prod-
uct was purified by column chromatography on silica gel (100–
200 mesh) eluting with hexane–ethyl acetate (9:1) to afford 17
(1.0 g, 51%) as a white solid, mp 142–144 °C. 1H NMR (400 MHz,
CDCl3): d 3.96 (s, 3H), 6.29 (t, J = 2.1 Hz, 1H), 7.03 (s, 1H), 7.11–
7.19 (m, 2H), 7.26–7.31 (m, 2H), 7.32–7.38 (m, 3H), 7.61 (d,
J = 1.7 Hz, 1H), 7.65 (dd, J = 8.8, 2.0 Hz, 1H), 7.69 (d, J = 8.8 Hz,
1H), 7.75 (d, J = 2.0 Hz, 1H). 13C NMR (100.6 MHz, CDCl3): d 53.9,
63.7, 117.4, 125.6, 126.1, 128.2, 128.4, 128.5, 128.8, 129.75,
129.78, 136.1, 137.8, 140.1, 144.8, 159.7. [M+H]+ = m/e 394, 396.
Procedure A, yield 50%. White solid, mp 161–162 °C. 1H NMR
(400 MHz, CDCl3): d 2.58 (s, 3H), 3.98 (s, 3H), 6.93 (s, 1H), 7.22–
7.25 (m, 2H), 7.40–7.45 (m, 3H), 7.56 (s, 1H), 7.70 (s, 2H), 7.78 (s,
1H). 13C NMR (100.6 MHz, CDCl3): d 9.1, 54.2, 59.4, 117.9, 122.6,
125.9, 127.7, 128.7, 129.2, 129.4, 130.0, 133.6, 134.8, 138.2,
145.0, 152.0, 159.1. [M+H]+ = m/e 411, 413.
5.1.11. 1-[(6-Bromo-2-methoxy-quinolin-3-yl)-phenyl-
methyl]-pyrrolidine-2-carboxylic acid ethyl ester (12)
Procedure A, yield 11%. Light-yellow semi-solid. 1H NMR
(400 MHz, CDCl3): d 1.05 (t, J = 7.2 Hz, 3H), 1.78–2.0 (m, 3H),
2.10–2.16 (m, 1H), 2.45–2.54 (m, 1H), 3.05–3.15 (m, 1H), 3.46–
3.54 (m, 1H), 3.74–3.92 (m, 2H), 4.00 (s, 3H), 5.12 (s, 1H), 7.15–
7.25 (m, 3H), 7.40 (d, J = 6.8 Hz, 2H), 7.60 (dd, J = 8.8, 2.0 Hz, 1H),
7.62 (d, J = 8.8 Hz, 1H), 7.94 (d, J = 2.0 Hz, 1H), 8.36 (s, 1H). 13C
NMR (100.6 MHz, CDCl3): d 14.0, 23.7, 30.3, 52.8, 53.5, 60.1, 63.5,
65.2, 117.0, 126.8, 127.3, 128.0, 128.4, 128.6, 129.0, 129.6, 132.0,
134.8, 141.0, 144.0, 160.1, 174.8. [M+H]+ = m/e 470, 472.
5.1.17. ( )-6-{[(6-Bromo-2-methoxy-quinolin-3-yl)-phenyl-
methyl]-amino}-chromen-2-one (18), Procedure C
5.1.12. 1-[(6-Bromo-2-methoxy-quinolin-3-yl)-phenyl-
methyl]-piperidine-4-carboxylic acid ethyl ester (13)
A mixture of 2 (2 g, 4.91 mmol), 6-aminocoumarin hydrochlo-
ride (0.97 g, 4.91 mmol), 1,8-diazabicyclo-[5.4.0]undec-7-ene
(0.74 g, 4.91 mmol), and n-tetrabutylammonium bromide (0.31 g,
0.98 mmol) in dry toluene (20 mL) were heated under reflux for
14 h. The reaction mixture was cooled to room temperature,
poured into water, diluted with ethyl acetate and the organic layer
was separated. The organic layer was washed with water followed
Procedure A, yield 15%. Light-yellow semi-solid, 1H NMR
(400 MHz, CDCl3): d 1.24 (t, J = 7.2 Hz, 3H), 1.76–1.90 (m, 4H),
1.92–2.00 (m, 2H), 2.26–2.33 (m, 1H), 2.70–2.82 (m, 1H), 2.90–
3.0 (m, 1H), 3.99 (s, 3H), 4.12 (q, J = 7.2 Hz, 2H), 4.70 (s, 1H),
7.12–7.18 (m, 1H), 7.20–7.25 (m, 2H), 7.35–7.40 (m, 2H), 7.59

2838
R. S. Upadhayaya et al. / Bioorg. Med. Chem. 17 (2009) 2830–2841
by brine, dried over anhydrous sodium sulfate, filtered and concen-
trated under vacuum. The crude product was purified by column
chromatography on silica gel (100–200 mesh) eluting with hex-
ane–ethyl acetate (9:1, v/v) to afford 18 (1.0 g, 42%) as a pale-yel-
low solid, mp 88–89 °C. 1H NMR (400 MHz, CDCl3): d 4.02 (s, 3H),
4.33 (d, J = 3.6 Hz, 1H), 5.77 (d, J = 3.6 Hz, 1H), 6.32 (d, J = 9.5 Hz,
1H), 6.44 (d, J = 2.8 Hz, 1H), 6.80 (dd, J = 9.0, 2.8 Hz, 1H), 7.13 (d,
J = 9.0 Hz, 1H), 7.29–7.38 (m, 5H), 7.48 (d, J = 5.6 Hz, 1H), 7.65
(dd, J = 9.0, 2.0 Hz, 1H), 7.70 (d, J = 9.0 Hz, 1H), 7.82 (d, J = 2 Hz,
1H), 7.98 (s, 1H). 13C NMR (100.6 MHz, CDCl3): d 53.8, 57.8,
109.3, 116.8, 117.5, 117.6, 118.7, 119.3, 126.5, 126.6, 127.6,
128.0, 128.5, 128.9, 132.7, 134.5, 140.6, 143.4, 143.6, 144.6,
147.1. [M+H]+ = m/e 486, 488.
128.15, 128.18, 128.31, 129.3, 132.0, 134.0, 139.0, 143.2, 159.7,
167.46, 167.50. [M+H]+ = m/e 458, 460.
5.1.20. 2-[(6-Bromo-2-methoxyquinolin-3-yl)-phenyl-methyl]-
malonic acid monomethyl ester (22)
Compound 21 (3.0 g, 6.55 mmol) was added to a stirred solution
of potassium hydroxide (0.36 g, 6.55 mmol) in water–methanol
(1:4, 25 mL) and heated to reflux for 12 h. The volatiles were re-
moved under reduced pressure, poured into ice-water and ex-
tracted with diethyl ether. The aqueous layer was separated,
acidified with 15% hydrochloric acid solution and extracted with
chloroform. The organic layer was washed with brine, dried over
anhydrous sodium sulfate, and concentrated under vacuum to ob-
tain the pure 22 (1.40 g, 48%) as a sticky solid. 1H NMR (400 MHz,
DMSO-d6): d 3.53 (s, 3H), 3.55 (s, 2H), 3.97 (s, 3H), 4.00 (s, 2H),
4.50–4.57 (m, 2H), 5.05–5.08 (d, 2H), 7.12–7.20 (m, 5H), 7.25–
7.31 (m, 3H), 7.60–7.62 (m, 3H), 7.81–7.84 (m, 3H), 13.00 (br s,
2H, D2O exchangeable), (Diastereomeric mixture in 3:2 ratio by
1H NMR spectroscopy). 13C NMR (100.6 MHz, DMSO-d6): d 43.5,
43.6, 52.2, 52.3, 53.7, 55.5, 55.6, 116.5, 126.4, 126.8, 126.9, 127.7,
128.0, 128.1, 128.2, 128.26, 128.3, 128.4, 129.2, 129.3, 132.1,
133.9, 134.0, 139.5, 139.7, 143.1, 159.8, 159.9, 168.1, 168.3.
[M+H]+ = m/e 444, 446.
5.1.18. ( )-3-(6-Bromo-2-methoxy-quinolin-3-yl)-2-naphtha
len-1-yl-3-phenyl-propionic acid ethyl ester (19 and 20), Proce
dure D
Sodium hydride (0.031 g, 0.737 mmol) was added to an ice-cold
solution of ethyl ester of naphthyl acetic acid (0.115 g, 0.54 mmol)
in tetrahydrofuran (4 mL) at 0 °C and stirred at room temperature
for 0.5 h. To this reaction mixture was added the solution of 2
(0.20 g, 0.49 mmol) in tetrahydrofuran (4 mL) at 0 °C and stirred
at room temperature for 6 h. The reaction mixture was poured into
ice-water and extracted into ethyl acetate. The organic layer was
washed with brine, dried over anhydrous sodium sulfate, filtered
and concentrated under vacuum. The crude product was purified
by column chromatography on silica gel (100–200 mesh) eluting
with hexane–ethyl acetate (9:1, v/v) to afford 19 (0.05 g, 19%) as
a light yellow solid, mp 141–142 °C.
5.1.21. Representative Procedure E, for 23, 24, 25, 26, 27 and 28:
Preparation of methyl 3-(6-bromo-2-methoxyquinolin-3-yl)-2-
(morpholine-4-carbonyl)-3-phenylpropanoate (23 and 24)
3-Morpholin-4-yl-3-oxo-propionic acid methyl ester20 (0.225 g,
1.22 mmol) was dissolved in dry THF (3 mL) and sodium hydride
(0.035 g, 1.47 mmol) was added fractionwise and stirred at 28 °C
for 1.5 h. And then compound 2 (0.500 g, 1.22 mmol) was dissolved
in dry THF (2 mL) and added to the reaction mixture. Reaction was
stirred at 28 °C for 16 h. and monitored by tlc. Reaction was
quenched by adding water and THF was removed on rotatory evap-
orator, extracted with chloroform (2 Â 20 mL). Organic layer was
washed with water, brine and dried over sodium sulfate and con-
centrated to get the crude product. This was purified by column
chromatography on silica gel (230–400 mesh) eluent: 30% ethyl
acetate in hexane to afford the 23 and 24.
Compound 23, Upper spot: (0.054 g, 27% yield). White solid, mp
206-208 °C. 1H NMR (400 MHz, CDCl3): d 3.31–3.35 (m, 1H), 3.37–
3.44 (m, 1H), 3.49–3.58 (m, 5H), 3.60–3.63 (m, 2H), 3.67–3.72 (m,
1H), 3.78–3.83 (m, 1H), 4.00 (s, 3H), 4.88 (d, J = 11.8 Hz, 1H), 5.23
(d, J = 11.8 Hz, 1H), 7.13–7.18 (m, 1H), 7.20–7.25 (m, 2H), 7.30–
7.34 (m, 2H), 7.60–7.66 (m, 2H), 7.78 (s, 1H), 7.81 (s, 1H). 13C
NMR (100.6 MHz, CDCl3): d 42.8, 46.5, 46.6, 52.1, 52.6, 53.7, 66.6,
66.8, 117.3, 126.4, 127.0, 127.4, 128.34, 128.37, 128.5, 129.1,
132.3, 134.4, 139.4, 144.1, 160.2, 164.9, 168.4. [M+H]+ = m/e 513,
515.
Compound 19, Upper spot: (10%). 1H NMR (400 MHz, CDCl3): d
0.91 (t, J = 7.0 Hz, 3H), 3.80–3.94 (m, 2H), 3.95 (s, 3H), 5.50 (d,
J = 12.3 Hz, 1H), 5.57 (d, J = 12.3 Hz, 1H), 7.20–7.26 (m, 1H), 7.28–
7.34 (m, 3H), 7.44–7.50 (m, 4H), 7.52–7.55 (m, 2H), 7.58–7.64
(m, 3H), 7.70 (d, J = 7.2 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H), 8.49 (d,
J = 8.6 Hz, 1H). 13C NMR (100.6 MHz, CDCl3): d 13.8, 47.4, 49.8,
53.6, 60.9, 116.9, 122.9, 125.46, 125.53, 125.7, 126.1, 126.4,
127.0, 127.4, 128.0, 128.2, 128.4, 128.7, 129.02, 129.08, 131.9,
132.0, 132.5, 133.8, 134.3, 140.8, 143.7, 160.4, 172.5.
Compound 20, Lower spot: (9%). 1H NMR (400 MHz, CDCl3): d
0.92 (t, J = 7.0 Hz, 3H), 3.87–3.98 (m, 2H), 4.06 (s, 3H), 5.37 (d,
J = 12.3 Hz, 1H), 5.55 (d, J = 12.2 Hz, 1H), 6.80–6.90 (m, 3H), 7.02–
7.06 (m, 2H), 7.38–7.50 (m, 3H), 7.63–7.70 (m, 3H), 7.75 (d,
J = 8.0 Hz, 1H), 7.82–7.84 (m, 1H), 7.97 (d, J = 1.8 Hz, 1H), 8.20–
8.25 (m, 2H). 13C NMR (100.6 MHz, CDCl3): d 13.9, 47.8, 53.9,
60.9, 117.2, 123.0, 125.29, 125.33, 126.1, 126.3, 126.5, 127.7,
128.0, 128.4, 128.6, 128.7, 129.1, 129.3, 132.0, 132.2, 132.7,
133.4, 133.7, 139.4, 144.1, 160.6, 172.6.
5.1.19. ( )-2-[(6-Bromo-2-methoxyquinolin-3-yl)-phenyl-
methyl]-malonic acid dimethyl ester (21)
Sodium hydride (0.014 g, 0.58 mmol) was added in portions to a
stirred solution of dimethyl malonate (0.08 g, 0.67 mmol) in anhy-
drous tetrahydrofuran (2 mL) at 0 °C and allowed to warm up to
Compound 24, Lower spot: (0.047 g, 25%). Off-white solid, mp
188–190 °C. 1H NMR (400 MHz, CDCl3): d 2.95–3.05 (m, 1H),
3.23–3.32 (m, 2H), 3.34–3.44 (m, 1H), 3.48–3.62 (m, 7H), 3.99 (s,
3H), 4.72 (d, J = 12.0 Hz, 1H), 5.24 (d, J = 12.0 Hz, 1H), 7.15–7.25
(m, 5H), 7.60–7.67 (m, 2H), 7.80–7.87 (m, 2H). 13C NMR
(100.6 MHz, CDCl3): d 42.6, 45.5, 46.6, 51.6, 52.7, 53.7, 66.2, 66.5,
117.2, 126.2, 127.2, 127.6, 128.44, 128.48, 128.53, 129.2, 132.3,
133.5, 139.5, 144.1, 160.4, 165.4, 168.4. [M+H]+ = m/e 513, 515.
room temperature during 0.5 h. The solution of
2 (0.20 g,
0.49 mmol) in tetrahydrofuran (2 mL) was added to the reaction
mixture and stirred at room temperature for 4 h. The volatiles were
removed under vacuum, poured into ice-water mixture, extracted
with dichloromethane, the organic layer was washed with water,
brine, dried over anhydrous sodium sulfate, filtered and concen-
trated under vacuum. The crude product was triturated with n-
pentane and dried to give the product 21 (0.20 g, 94% yield) as a
sticky mass. 1H NMR (400 MHz, CDCl3): d 3.54 (s, 3H), 3.56 (s,
3H), 4.02 (s, 3H), 4.53 (d, J = 12.0 Hz, 1H), 5.12 (d, J = 12.0 Hz,
1H), 7.13–7.19 (m, 1H), 7.20–7.25 (m, 2H), 7.28–7.32 (m, 2H),
7.59–7.65 (m, 2H), 7.83–7.86 (m, 2H). 13C NMR (100.6 MHz,
CDCl3): d 43.6, 52.3, 52.4, 53.6, 55.0, 116.4, 126.3, 126.9, 127.4,
5.1.22. 3-(6-Bromo-2-methoxy-quinolin-3-yl)-3-phenyl-2-
(pyrrolidine-1-carbonyl)-propionic acid methyl ester (25 and
26)
Procedure E. 25, Upper spot: Yield 14%. Off-white solid, mp
196–198 °C, 1H NMR (400 MHz, CDCl3): d 1.43–1.49 (m, 2H),
1.67–1.83 (m, 2H), 3.14–3.21 (m, 1H), 3.34–3.40 (m, 1H), 3.54 (s,
3H), 3.58–3.64 (m, 1H), 3.76–3.82 (m, 1H), 4.00 (s, 3H), 4.73 (d,
J = 12.0 Hz, 1H), 5.22 (d, J = 12.0 Hz, 1H), 7.11–7.16 (m, 1H), 7.18–

R. S. Upadhayaya et al. / Bioorg. Med. Chem. 17 (2009) 2830–2841
2839
7.24 (m, 2H), 7.34 (d, J = 7.6 Hz, 2H), 7.57–7.65 (m, 2H), 7.79 (d,
5.1.25. 3-(6-Bromo-2-methoxy-quinolin-3-yl)-3-phenyl-2-[4-
(3-trifluoromethyl-phenyl)-piperazine-1-carbonyl]-propionic
acid methyl ester (30 and 31)
J = 8.0 Hz, 2H). 13C NMR (100.6 MHz, CDCl3): d 23.7, 25.5, 44.1,
45.8, 46.3, 52.1, 53.6, 54.4, 116.4, 126.4, 126.7, 128.1, 128.4,
128.5, 129.2, 131.9, 133.4, 139.7, 143.1, 160.0, 163.3, 168.2.
[M+H]+ = m/e 497, 499.
Procedure F. 30, Upper spot: Yield 30%. Off-white solid, mp
195–196 °C. 1H NMR (400 MHz, CDCl3): d 2.94–3.02 (m, 2H),
3.08–3.22 (m, 2H), 3.44–3.52 (m, 1H), 3.55 (s, 3H), 3.73–3.84 (m,
2H), 3.85–3.99 (m, 1H), 4.02 (s, 3H), 4.79 (d, J = 12.0 Hz, 1H), 5.27
(d, J = 12.0 Hz, 1H), 6.93–7.06 (m, 2H), 7.11 (d, J = 8.0 Hz, 2H),
7.19 (t, J = 4.0 Hz, 2H), 7.25–7.30 (m, 2H), 7.34 (m, 1H), 7.60–7.66
(m, 2H), 7.91–7.98 (m, 2H). 13C NMR (100.6 MHz, CDCl3): d 42.1,
45.7, 46.6, 48.9, 49.3, 52.4, 52.7, 53.7, 112.78, 112.82, 112.87,
112.91, 116.86, 116.90, 116.94, 117.0, 117.3, 119.4, 125.5, 126.4,
127.1, 127.5, 128.37, 128.39, 128.5, 129.1, 139.4, 144.1, 150.8,
160.2, 164.8, 168.4. [M+H]+ = m/e 656, 658.
Compound 26, Lower spot: Yield 15%. Off-white solid, mp 196–
198 °C. 1H NMR (400 MHz, CDCl3): d 1.51–1.57 (m, 2H), 1.66–1.82
(m, 2H), 2.95–3.06 (m, 1H), 3.12–3.20 (m, 1H), 3.31–3.4 (m, 1H),
3.53 (s, 3H), 3.58–3.68 (m, 1H), 3.97 (s, 3H), 4.52 (d, J = 12.0 Hz,
1H), 5.23 (d, J = 12.0 Hz, 1H), 7.10–7.20 (m, 3H), 7.20–7.23 (m,
1H), 7.26–7.29 (m, 1H), 7.61–7.68 (m, 2H), 7.8–7.9 (m, 2H). 13C
NMR (100.6 MHz, CDCl3): d 23.6, 25.2, 43.7, 45.4, 46.6, 52.1,
53.5, 53.7, 116.4, 126.4, 126.8, 128.0, 128.2, 128.3, 128.4, 129.3,
131.9, 133.9, 139.2, 143.1, 160.0, 164.4, 168.5. [M+H]+ = m/e 497,
499.
31, Lower Spot: Yield 28%. Off-white solid, mp 194–196 °C. 1H
NMR (400 MHz, CDCl3): d 2.53–2.57 (m, 1H), 2.68–2.79 (m, 1H),
3.01–3.23 (m, 3H), 3.48–3.62 (m, 4H), 3.67–3.81 (m, 2H), 4.00 (s,
3H), 4.79 (d, J = 12.0 Hz, 1H), 5.27 (d, J = 12.0 Hz, 1H), 6.95–6.99
(m, 2H), 7.11 (d, J = 7.2 Hz, 2H), 7.20 (t, J = 7.2 Hz, 2H), 7.21–7.28
(m, 2H), 7.34 (t, J = 8.0 Hz, 1H), 7.62–7.66 (m, 2H), 7.85–7.88 (m,
2H). 13C NMR (100.6 MHz, CDCl3): d 41.4, 43.7, 45.2, 47.5, 47.7,
50.1, 52.3, 53.8, 54.9, 111.39, 111.43, 111.5, 115.2, 115.24, 116.5,
119.1, 126.4, 126.7, 128.2, 128.5, 128.6, 129.8, 130.0, 132.0,
133.8, 139.5, 143.1, 150.7, 160.1, 165.1, 168.5. [M+H]+ = m/e 656,
658.
5.1.23. 3-(6-Bromo-2-methoxy-quinolin-3-yl)-3-phenyl-2-
(piperidine-1-carbonyl)-propionic acid methyl ester (27 and 28)
Procedure E. 27, Upper spot: Yield 14%. Off-white solid, mp
218–220 °C. 1H NMR (400 MHz, DMSO-d6): d 1.21–1.29 (m, 2H),
1.50–1.60 (m, 4H), 3.38–3.43 (m, 2H), 3.43 (s, 1H), 3.96 (s, 3H),
5.10 (s, 2H), 7.13–7.19 (m, 1H), 7.21–7.28 (m, 2H), 7.39–7.45 (m,
2H), 7.60–7.65 (m, 1H), 7.68–7.74 (m, 1H), 7.97 (s, 1H), 8.37 (s,
1H). 13C NMR (100.6 MHz, DMSO-d6): d 23.8, 25.4, 26.2, 42.8,
44.3, 46.4, 51.9, 52.1, 53.7, 116.5, 126.4, 126.8, 128.1, 128.5,
128.7, 129.0, 132.0, 133.3, 139.7, 143.1, 160.1, 163.5, 168.5.
[M+H]+ = m/e 511, 513.
5.2. Biological analysis
Compound 28, Lower spot: Yield 13%. Off-white solid, mp 209–
211 °C. 1H NMR (400 MHz, DMSO-d6): d 1.18–1.24 (m, 1H), 1.28–
1.33 (m, 2H), 1.36–1.47 (m, 3H), 1.51–1.70 (m, 1H), 3.05–3.11
(m, 1H), 3.29–3.43 (m, 2H), 3.45 (s, 3H), 3.96 (s, 3H), 5.05–5.17
(m, 2H), 7.13–7.17 (m, 1H), 7.22 (t, J = 8.0 Hz, 2H), 7.26–7.31 (m,
2H), 7.65 (d, J = 9.0 Hz, 1H), 7.72 (dd, J = 9.0, 2.0 Hz, 1H), 8.02 (d,
J = 2.0 Hz, 1H), 8.60 (s, 1H). 13C NMR (100.6 MHz, DMSO-d6): d
24.3, 25.4, 26.1, 43.6, 45.4, 47.3, 51.9, 52.6, 53.7, 117.1, 126.3,
126.9, 128.0, 128.3, 128.5, 129.2, 132.2, 133.6, 139.8, 144.1,
160.5, 164.7, 168.8. [M+H]+ = m/e 511, 513.
5.2.1. Cytotoxicity assay
Cellular viability in the presence and absence of test compounds
was determined by MTT (3-(4,5-dimethylthiazol-2yl)-2,5-di-
methyl tetrazolium bromide; Sigma–Aldrich) microcultured tetra-
zolium assay.21,22 The cells (monocytic cell line, THP-1) were
plated in flat-bottomed 96-well plates (10,000 cells/100 lL) and
cultured in controlled atmosphere (5% CO2 at 37 °C). Cells were
cultured in the presence of medium along with DMSO (live con-
trols). Different concentrations of compounds (1
10 g/100 L) were added to the cells. After 24 h and 72 h, stock
MTT solution (5 mg/mL) was added to the culture. Cells were again
kept in CO2 incubator for 4 h. After 4 h, 100 L isopropanol was
lg/100 lL and
l
l
5.1.24. Representative Procedure F, for 29, 30 and 31:
Preparation of 3-(6-bromo-2-methoxy-quinolin-3-yl)-3-
phenyl-2-(pyrazole-1-carbonyl)-propionic acid methyl ester
(29)
l
added and mixed 5–6 times. The absorbance was read at 540 nm
in a plate reader (Bio-Rad—450). The results were represented as
percentage cell viability (Table 4). All the experiments were carried
out in triplicates and the readings were the mean of three readings.
To a stirred solution of compound 22 (0.600 g, 1.35 mmol), in
dry tetrahydrofuran (10 mL) was added N-hydroxybenzotriazole
(0.100 g, 1.48 mmol), pyrazole (0.100 g, 1.48 mmol), EDCÁHCl
(0.309 g, 1.62 mmol) and diisopropyl amine (0.22 mL, 1.62 mmol)
at 0 °C and stirred at room temperature for 16 h. The volatiles
were removed under reduced pressure, poured into ice-water
and extracted with chloroform. The organic layer was washed
with brine, dried over anhydrous sodium sulfate and concentrated
under vacuum. The crude product was purified by column chro-
matography on silica gel (230–400 mesh) eluent: hexane–ethyl
acetate (7:3) to get pure product 29 as a diastereomeric mixture.
Yield 14%. Off-white solid, mp 180–182 °C. 1H NMR (400 MHz,
CDCl3): d 3.46 (s, 3H), 3.50 (s, 3H), 3.98 (s, 3H), 4.05 (s, 3H),
5.35 (d, J = 3.6 Hz, 1H), 5.38 (d, J = 4.0 Hz, 1H), 5.95 (d,
J = 12.0 Hz, 1H), 6.05 (d, J = 12.0 Hz, 1H), 6.37–6.41 (m, 1H),
6.42–6.46 (m, 1H), 7.04–7.10 (m, 1H), 7.11–7.16 (m. 2H), 7.16–
7.21 (m, 1H), 7.23–7.28 (m, 1H), 7.29–7.32 (m, 3H), 7.36–7.40
(m, 2H), 7.60–7.65 (m, 2H), 7.65–7.68 (m, 1H), 7.76 (s, 1H), 7.83
(s, 1H), 7.86 (s, 2H), 7.96 (s, 1H), 8.07 (dd, J = 10.4, 3.2 Hz, 2H).
13C NMR (100.6 MHz, CDCl3): d 29.7, 45.1, 52.8, 52.9, 53.0,
110.5, 117.1, 126.2, 127.0, 127.1, 128.3, 128.4, 128.5, 128.7,
129.2, 132.3, 133.5, 134.4, 138.8, 144.1, 144.6, 160.2, 165.8,
167.6. [M+H]+ = m/e 494, 496.
5.3. Computational methods
5.3.1. Homology modelling
Subunit a (gi:54040843) sequence of the M. tuberculosis ATP
synthase was queried in BLAST23 search at NCBI (http://
www.ncbi.nlm.nih.gov/). All parameters were used with their de-
fault values during the BLAST search. The obtained sequences were
aligned using CLUSTALx24 multiple sequence alignment methodol-
ogy (aln1). At the same time, corresponding subunit a sequence of
the E. coli ATP synthase was also subjected to BLAST search. Ob-
tained sequences were aligned using secondary structure defini-
tion in CLUSTALx for the transmembrane helix regions as known
from solved-structure of ATP synthase a/c subunits from E. coli
(PDB Id: 1C17)(aln2). The pre-aligned M. tuberculosis sequences
(aln1) were then added to the structure-sequence alignment of
E. coli sequences (aln2) using profile-to-profile methodology in
CLUSTALx to yield alignment (aln3). Same process was repeated
for the subunit c of M. tuberculosis. From the larger alignment,
the sequences of subunit a and c of M. tuberculosis and E. coli were
extracted. This extracted alignment was used for generating
homology models of combined subunit a–c complex, where

2840
R. S. Upadhayaya et al. / Bioorg. Med. Chem. 17 (2009) 2830–2841
E. coli a–c subunit sequences were used as template sequences for
generating the homology model of M. tuberculosis target se-
quences. Finally, the homology model consisted of 2 c-subunits
and 1 a-subunit having inter-subunit contacts. MODELLER25 was
used for generating the homology models. On visual inspection,
models were found to be reasonable and root mean square devia-
In large, we did not perform molecular dynamics simulations
over the complex or the homology model, for which we assume
usually deteriorate the system, specially in the absence of good
forcefield parameterisation. The general forcefield parameterisa-
tion for this diarylquinoline class of compounds will be used in fu-
ture to obtain better energetics and structural and interaction
analysis.
tion for Ca atoms in transmembrane helix regions was found to be
1.20 Å (for a subunit) and 0.84 Å (for c subunit) between template
structure and homology model. The model with lowest value of
MODELLER OBJECTIVE FUNCTION was selected and was energy
minimised (Conjugate Gradients Minimisation for 2000 steps in
NAMD26) to remove the steric clashes. The critical residues of the
subunit a and c were kept in pre-defined protonation states during
the whole process. Glu-61 (subunit c) protonated: Glu-61 (subunit
c) deprotonated: Arg 186 (subunit a): protonated. Satisfaction of
such charges in the membrane domain is absolutely necessary, ow-
ing to high free-energy cost of putting a charge in membrane
dielectric. We also assume here that proton translocation mecha-
nism of the ATP synthase of both E. coli and M. tuberculosis is same.
All structural analysis was performed using VMD.34 Simulations
were run on
a workstation having AMD Opteron Processor
(2.4 GHz Dual Core) running with Red Hat Enterprise Linux 5.
Acknowledgement
We thank Dr. Anjlina Wali, Institute of Molecular Medicine, for
cytotoxicity screening of compounds.
Supplementary data
Supplementary data (characterization data (1H NMR, 13C NMR,
DEPT) for all compounds and Tables S1, S2 and S3 are included)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.02.026.
5.3.2. Molecular modelling
DARQ molecule and its four configurations were sketched in
ChemOffice27 and were subjected to energy minimisation. Flexible
terminal amine of RS stereoisomer was subjected to conforma-
tional analysis to obtain an intramolecular H-bond. Other com-
pounds were sketched and were subjected to conformational
analysis and subsequent energy minimisation in Ghemical.28 The
Gasteiger–Marsili29 partial atomic charge model was used for
DARQ. The charges were made into CHARMM30 topology files using
Paratool.31 Various parameters were constructed ad hoc for bond
lengths, angles and dihedrals of the DARQ to be used in further
analysis. As we are to compare the energetics of different stereo-
isomers of same compound (DARQ), we assume the inconsistencies
in calculations cancel out.
References and notes
1. (a) World Health Organization. In Anti-tuberculosis Drug Resistance in the World.
The WHO/IUALTD Global project on Anti-Tuberculosis Drug Resistance
Surveillance. World Health Organization (Publication
Geneva, Switzerland, 1997.; (b) Nakata, K.; Honda, T. N.; Weiden, M.; Keicho,
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Selected protein–ligand complex (only DARQ molecule) were fur-
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minimisation in NAMD to refine the system as well as remove ste-
ric clashes. The interaction energies were calculated between the
ligand and protein (comprising of van der Waals and electrostatic
terms) of the minimised complexes.
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