Screening for in vitro antimycobacterial activity and three-dimensional quantitative structure-activity relationship (3D-QSAR) study of 4-(arylamino)coumarin derivatives

Article abstract of DOI:10.1111/j.1747-0285.2010.00997.x

The resurgence of tuberculosis and the emergence of multidrug-resistant strains of Mycobacteria necessitate the search for new classes of antimycobacterial agents. We have synthesized a small library of 50 analogues of 4-(arylamino)coumarins with various aromatic amines at the C₄-position of the coumarin scaffold. The compounds were evaluated for antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv with rifampicin as the standard. Of the molecules synthesized, compound 9 was found to be most potent with a minimum inhibitory concentration >6.25 lg/mL for 100% inhibition. In an effort to develop new and more effective molecules in this series, the relationship between structure and activity was investigated by comparative molecular field analysis. Various models were generated using comparative molecular field analysis alone and comparative molecular field analysis plus a hydropathy field (HINT). In all, eight models were generated with atom-fit and field-fit alignment strategies. The comparative molecular field analysis models (Models 3a and 4a) based on field-fit alignment were the best with statistically good correlation coefficients (r²) and cross-validated q². The values of r²_pred for the validation set were 0.469 and 0.516. Based on the comparative molecular field analysis contours, some insights into the structure-activity relationship of the compounds could be gained.

Full text of DOI:10.1111/j.1747-0285.2010.00997.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.00997.x
Chem Biol Drug Des 2010; 76: 412–424
Research Article
Screening for In Vitro Antimycobacterial Activity
and Three-Dimensional Quantitative
Structure–Activity Relationship (3D-QSAR) Study
of 4-(arylamino)coumarin Derivatives
Vijay Virsdoia1, Mushtaque S. Shaikh2,
(HINT). In all, eight models were generated with
atom-fit and field-fit alignment strategies. The com-
Atul Manvar3, Bhavik Desai4, Alpesh
parative molecular field analysis models (Models 3a
Parecha5, Raju Loriya6, Kinnari Dholariya6,
and 4a) based on field-fit alignment were the best
Gautam Patel7, Vipul Vora8, Kuldip
Upadhyay9, Karia Denish10, Anamik
Shah11,* and Evans C. Coutinho2,*
with statistically good correlation coefficients (r 2)
and cross-validated q 2. The values of r 2
for the
pred
validation set were 0.469 and 0.516. Based on the
comparative molecular field analysis contours,
some insights into the structure–activity relation-
ship of the compounds could be gained.
1Dishman Pharmaceuticals and Chemicals Ltd., API Division, Bavla,
Ahmedabad-382220, India
2Department of Pharmaceutical Chemistry, Bombay College of
Pharmacy, Kalina, Santacruz (E), Mumbai-400 098, India
3Jubilant Organosys, 1A, Sector 16-A, Noida-201 301,
Uttar Pradesh, India
Key words: 3D-QSAR, 4-(arylamino)coumarins, antitubercular
agents, CoMFA, HINT
4Beckman Coulter Inc,Webster, TX, USA
Abbreviations: 3D-QSAR, three-dimensional quantitative struc-
ture–activity relationship; CoMFA, Comparative molecular field analysis;
HINT, Hydropathic interactions; MIC, minimum inhibitory concentration;
MDR-TB, multidrug-resistant tuberculosis; XDR-TB, extensively drug-
resistance tuberculosis; TMPK, Thymidine monophosphate kinase.
5ATRI Pharma. Pvt. Ltd. Ankleshwar, India
6Scifinder Scholar, Pune, India
7US Vitamins, API Division, USA
8Lupin Pharma, Ankleshwar, India
9Torrent Research Center, Bhat, Gandhinagar-382428, Gujarat, India
10H & H.B.Kotak Science College, Gujarat, India
11Department of Chemistry, Saurashtra University, University Road,
Rajkot-360 005, Gujarat India
Received 3 November 2009, revised 3 March 2010 and accepted for
publication 5 May 2010
*Corresponding authors: Anamik Shah, anamik_shah@yahoo.com;
Evans C. Coutinho, evans@bcpindia.org
Control and prevention of tuberculosis (TB) is a major challenge as
one-third of the world's population is infected with Mycobacterium
tuberculosis. Nearly 2 million deaths because of TB occur every year
(1–3). According to a WHO report by the year 2020 nearly one billion
more people will be infected, and 70 million will die from TB if proper
steps are not taken to control it (4). TB is the world's second cause of
death from infectious diseases after acquired immune deficiency syn-
drome (AIDS) (5). Moreover, the association of TB with AIDS and the
emergence of drug-resistant TB strains have made available chemo-
therapeutic strategies increasingly ineffective (6).
The resurgence of tuberculosis and the emergence
of multidrug-resistant strains of Mycobacteria
necessitate the search for new classes of antimyco-
bacterial agents. We have synthesized
a small
library of 50 analogues of 4-(arylamino)coumarins
with various aromatic amines at the C4- position of
the coumarin scaffold. The compounds were evalu-
ated for antimycobacterial activity against Myco-
bacterium tuberculosis H37Rv with rifampicin as
the standard. Of the molecules synthesized, com-
pound 9 was found to be most potent with a mini-
mum inhibitory concentration >6.25 lg ⁄ mL for
100% inhibition. In an effort to develop new and
more effective molecules in this series, the rela-
tionship between structure and activity was inves-
tigated by comparative molecular field analysis.
Various models were generated using comparative
molecular field analysis alone and comparative
molecular field analysis plus a hydropathy field
Unfortunately, no new drugs or antibiotics have been developed in
the last 30 years against TB. There are three frontline drugs viz.
isoniazid, rifampicin, ethambutol, and several second-tier drugs that
include ethionamide, streptomycin, and p-aminosalicylic acid to treat
TB. However, many of these drugs have disadvantages, such as pro-
longed treatment schedules, host toxicity, ineffectiveness against
resistant strains. (5,7). The problem of TB is further complicated by
the emergence of multidrug-resistant TB (MDR-TB) (8–15) and
'extensively drug-resistance tuberculosis' (XDR-TB) that threaten TB
control globally (16,17).
412

4-(arylamino)coumarins: New Anti T.B. agents
The impact of ever-increasing drug resistance, the serious side
effects of some current anti-TB drugs and the lack of efficacy of
current treatments in immuno-suppressed patients combine to
make the development of new antimycobacterial agents an
urgent priority (8,18). There are several reports in the literature
of new structural classes being developed as antitubercular
agents. A series of C3 alkyl and arylalkyl-2,3-dideoxyhex-2-enopyr-
anosides with minimum inhibitory concentration (MIC) values
between 25.0 and 1.56 lg ⁄ mL against Mycobacterium tuberculo-
sis H37Rv(7) has been reported. Similarly, 5-acetylenic derivatives
of 2¢,3¢-dideoxyuridine and 3¢-fluoro-2¢,3¢-dideoxyuridine have been
tested for antimycobacterial activity against M. bovis, M. tubercu-
losis, and M. avium. The most promising compounds in this
series, 5-dodecynyl-2¢,3¢-dideoxyuridine and 5-tetradecynyl-3¢-fluoro-
2¢,3¢-dideoxyuridine showed excellent activity and inhibited the
growth of M. bovis and M. tuberculosis with MIC90 in the range
of 1–2 lg ⁄ mL, which is close to the reference drug rifampicin
(19). The derivatives of 5¢-O-[(N-acyl)sulfamoyl]adenosine were
evaluated against M. avium and M. tuberculosis, under iron-defi-
cient and iron-rich conditions. Of the various molecules in the
series, the 4-fluorosalicyl derivative was found to be the most
potent (MIC99 of 0.098 lM), while the 4-aminosalicyl derivative
was the first analogue that exhibited improved selectivity against
M. tuberculosis under iron-rich conditions (MIC99 of 25 lM) (20).
A series of 1-b-D-2¢-arabinofuranosyl and 1-(2¢-deoxy-2¢-fluoro-b-
D-ribofuranosyl)pyrimidine nucleosides with a variety of alkynyl,
alkenyl, alkyl, and halo substituents at the C5 position of the
uracil moiety was investigated against M. tuberculosis, M. bovis,
and M. avium. Among the designed nucleosides, 1-b-D-2¢-arabino-
furanosyl-5-dodecynyluracil, 1-(2¢-deoxy-2¢-fluoro-b-D-ribofuranosyl)-
5-dodecynyluracil, and 1-(2¢-deoxy-2¢-fluoro-b-D-ribofuranosyl)-5-tet-
radecynyluracil were the most potent against M. bovis and M. tuber-
culosis, respectively (21). Recently, thymidine monophosphate kinase
(TMPK) has been proposed as an attractive target for the develop-
ment of antitubercular agents. The newly developed (3-trifluorom-
ethyl-4-chlorophenyl)thiourea has a Ki of 0.6 lM and a selectivity
index of 600 against human TMPK. Moreover, it represents the first
TMPK inhibitor with good inhibitory activity on growing M. bovis
H37Rv strain. A three-dimensional quantitative structure–activity
relationship (3D-QSAR) study has been recently reported on 4-am-
inocoumarins tested as antiplatelet agents (39,40). To understand
the structural requirements for antitubercular versus antiplatelet
activity, the 3D-QSAR technique of comparative molecular field
analysis (CoMFA) (41,42) with and without inclusion of hydro-
pathic interactions (HINT) hydropathy fields (43) was also carried
out.
Methods and Materials
Chemistry
All chemicals and solvents were purchased from Spectorchem
(Mumbai, India) and SD Fine Chemicals (Mumbai, India) and used
without purification. For syntheses of the title compounds, a
monomode, open-vessel microwave synthesizer (Q-ProM model;
Questron Technologies Corporation, Mississauga, Ontario, Canada)
was used. Melting points were determined in open glass capil-
laries in a paraffin bath and are uncorrected. 1H NMR and 13C
NMR spectra were recorded on a Bruker AC 300 MHz FT-NMR
spectrophotometer using tetramethylsilane as the internal stan-
dard. Mass spectra were recorded on a JEOL SX 102 ⁄ DA-6000
by the FAB (fast atom bombardment) technique for ionization. FT-
IR spectra were recorded on a Shimadzu-8400 using KBr disks.
Thin layer chromatography (TLC) was carried out on Merck Kie-
selgel 60F254 (Merck 5549, USA) plates. The structures of the
molecules synthesized are given in Table 1, while all experimen-
tal details are briefly given in the supplementary material
(Table S1).
General method for the synthesis of 4-
(arylamino)coumarin derivatives (2–45)
Various 4 -hydroxycoumarins were prepared by earlier reported
method. Substituted 4-hydroxycoumarin (0.01 mol) and correspond-
ing amines (0.015 mol) were taken in a reaction vessel. The
reaction mixture was subjected to microwave irradiation (400 W)
at 140 ꢀC (Scheme 1). The reaction was monitored by TLC (ethy-
lacetate:hexane::3:2). After completion of the reaction as indi-
cated by TLC, acetone was added to the reaction mixture and
the solid mass obtained was filtered. The crude reaction mass
was finally purified by silica gel (60–100 mesh) column chroma-
tography employing ethyl acetate and hexane (3:2) as the mobile
phase (44–45).
(MIC99 = 20 lg ⁄ mL) and M. tuberculosis (MIC50
= 6.25 lg ⁄ mL)
strains (22).
Moreover, various structural classes, such as diarylmethanes (23),
oxazolyl thiosemicarbazones (24), pyrazolines (25), amphiphilic galac-
topyranosyl diamine and amino alcohols (26), isonicotinohydrazones
(27), triazoles (28) have been identified as promising antitubercular
leads. Calanolide A, a naturally occurring coumarin derivative and
an inhibitor of HIV-1 reverse transcriptase also have shown promis-
ing in vitro activity against M. tuberculosis with an MIC value of
3.13 lg ⁄ mL (29,30).
General methods for the synthesis of
phenothiazine derivatives (46–51)
Substituted 4-hydroxycoumarin (0.01 mol) and corresponding 2-ami-
nothiophenols (0.012 mol) were taken in a reaction vessel and sub-
jected to microwave irradiation (400 W) at 140 ꢀC for an
appropriate time (Schemes 2 and 3). The reaction was monitored by
TLC (chloroform:methanol::9:1). On completion, the reaction mixture
was allowed to cool. The reaction mass was washed with acetone
when the crude product separated out. This was purified by silica
gel (60–100 mesh) column chromatography using chloroform and
methanol (9:1) as the mobile phase (46,47).
We have been studying for quite some time heterocyclic com-
pounds with a variety of scaffolds as novel antitubercular agents
(31–37) and very recently, we have reported the in vitro antitu-
bercular activity and 3D-QSAR studies of coumarin-4-acetic acid
benzylidene hydrazides (38). In continuation of this work, the cur-
rent paper describes the syntheses of substituted 4-(arylami-
no)coumarins and coumarins fused with a benzothiazipine nucleus
and their in vitro antitubercular activity against M. tuberculosis
Chem Biol Drug Des 2010; 76: 412–424
413

Virsdoia et al.
Table 1: The structures and activities of 4-aminoarylcoumarine derivatives
R8
R7
R6
O
O
R5 HN
Ar
*R
Series I
Compounds
R5
R6
R7
R8
Ar
*Ro
*Rm
*Rp
bGI % (lg ⁄ mL)
pALogit
2
3
-H
-H
-H
-H
-H
-H
-H
-H
-CH3
-H
-H
-H
-H
-H
-CH3
-H
-H
-H
-H
-H
-H
-H
-H
-H
-CH3
-H
-H
-H
-H
-H
-H
-CH3
-H
-H
-H
-H
-H
-H
-H
-CH3
-H
-H
-H
-H
-H
-H
-H
-CH3
-H
-CH3
-H
-H
-H
-CH3
-H
-CH3
-H
-CH3
-CH3
-H
-H
-CH3
-H
-H
-CH3
-H
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-
-
-
-
-
-
-H
-H
-H
-
-
-
-
-
-
-H
-H
-H
-
-
-
-
-
65 (>6.25)
14 (>6.25)
38 (>6.25)
81 (>6.25)
10 (>6.25)
54 (>6.25)
100 (>6.25)
9 (>6.25)
4.97
3.93
4.55
5.29
3.73
4.75
ND
3.70
3.70
3.99
3.92
4.37
3.66
4.54
4.09
4.31
3.8
2.73
3.95
3.51
2.64
4.31
4.11
3.91
3.43
4.28
3.73
3.68
3.88
3.53
3.71
3.68
4.22
4.28
4.5
-CH3
-Cl
-H
-CH3
-H
-Cl
-H
-H
-H
-CH3
-H
-Cl
-H
-H
-H
-H
-CH3
-H
-H
-CH3
-CH3
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-Ph
-H
-H
-H
-H
-H
4
6
-Napha
-Napha
-Napha
-Napha
-Napha
-Napha
-Ph
7
8
-H
9
-CH3
-CH3
-CH3
-H
-H
-H
-CH3
-CH3
-CH3
-H
-H
-H
-H
-H
-H
-CH3
-CH3
-H
-CH3
-H
-H
-H
-H
-H
-H
-CH3
-H
-H
-H
-H
-H
-H
-H
12
13
14
15
16
17
19
20
21
22
23
24
25
26
27
28
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
-
9 (>6.25)
-H
-H
-H
-H
-H
-H
-H
di-Cl
-H
-H
-H
-H
-H
-H
-H
-H
-Cl
-Cl
-H
-H
-H
-H
-H
-H
-H
-CH3
-H
-OCH3
-CH3
-CH3
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-Cl
-Cl
-H
-H
-H
-H
-H
-H
-H
-Cl
-NO2
-H
-H
-H
-CH3
-H
-COOH
-COOH
-COOH
-COOH
-COOH
-COOH
-Br
-H
-Br
-F
-H
-F
-F
-F
-F
-F
-Cl
-H
-Cl
-OCH3
-OCH3
-Cl
18 (>6.25)
15 (>6.25)
33 (>6.25)
8 (>6.25)
41 (>6.25)
20 (>6.25)
28 (>6.25)
11 (>6.25)
1 (>6.25)
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
-Ph
18 (>6.25)
7 (>6.25)
1 (>6.25)
31 (>6.25)
22 (>6.25)
15 (>6.25)
4 (>6.25)
28 (>6.25)
11 (>6.25)
10 (>6.25)
15 (>6.25)
7 (>6.25)
-H
-H
-H
-H
-H
-CH3
-CH3
-H
-H
-H
-H
-H
-H
-H
10 (>6.25)
9 (>6.25)
-Cl
-H
-H
-H
-CH3
-H
-H
23 (>6.25)
30 (>6.25)
44 (>6.25)
50 (>6.25)
39 (>6.25)
51 (>6.25)
49 (>6.25)
4.6
-H
-H
-H
4.44
4.65
4.61
-H
-H
-Ph
-CH3
414
Chem Biol Drug Des 2010; 76: 412–424

4-(arylamino)coumarins: New Anti T.B. agents
Table 1: (Continued )
O
O
R6
R5 HN
Ar
Series II
Compounds
R5
-H
R6
-H
Ar
bGI % (lg ⁄ mL)
pALogit
3.82
11
-Napha
11 (>6.25)
R8
R7
O
O
HN
Ar
*R
Series III
Compounds
R7
R8
Ar
*Ro
*Rm
*Rp
bGI % (lg ⁄ mL)
pALogit
5
10
18
29
-H
-H
-H
-H
-H
-H
-H
-H
-Ph
-Ph
-
-H
-H
-H
-
-H
-H
-H
-
-COOH
-COOH
7 (>6.25)
5 (>6.25)
26 (>6.25)
53 (>6.25)
3.64
3.45
4.27
4.74
-Napha
-Ph
-Ph
R8
O
R7
R6
Y
S
R5 HN
*R
Series IV
Compounds
Y
R5
-H
-CH3
-Cl
R6
R7
R8
Ar
*Ro
*Rm
*Rp
bGI % (lg ⁄ mL)
pALogit
46
47
48
49
O
O
NH
NH
-H
-H
-H
-Cl
-H
-CH3
-H
-H
-H
-Cl
-H
-Ph
-Ph
-Ph
-Ph
-H
-H
-H
-H
-Cl
-Cl
-H
-H
-H
-H
-H
-H
29 (>6.25)
36 (>6.25)
67 (>6.25)
8 (>6.25)
4.29
4.47
5.04
3.62
-H
-H
Chem Biol Drug Des 2010; 76: 412–424
415

Virsdoia et al.
Table 1: (Continued )
R8
O
O
O
O
S
R10
HN
R9
R5
*R
Series V
Compounds
R5
R8
R9
R10
Ar
*Ro
*Rm
*Rp
bGI % (lg ⁄ mL)
pALogit
50
51
-H
-H
-H
-H
-CH3
-CH3
-
-Ph
-Ph
-H
-H
-H
-H
-H
-H
78 (>6.25)
54 (>12.5)
5
4.53
-CH3
-Napha = a-naphthyl
*Ro ⁄ m ⁄ p positions with respect to –NH.
bGI = Growth inhibition of virulent strain of M. tuberculosis
The values in the parenthesis denote the MIC in lg ⁄ mL. MIC of Rifampin: 0.015–0.125 lg ⁄ mL against M. tuberculosis H37Rv (97% inhibition).
O
O
O
O
Microwave 400W, 140oC
3-6 min
R
R
Ar-NH2
OH
NH
Ar
4-Hydroxycoumarin 1
4-(arylamino)coumarins (2-45)
Scheme 1: Microwave-assisted synthesis of substituted 4-arylaminocoumarins (2–45).
NH2
O
O
S
O
O
SH
Microwave 400W, 140oC
6-9 min
R
R
R1
HN
OH
R1
R = Me or Cl
R1 = Cl
Compounds (46-49)
Scheme 2: Microwave-assisted synthesis of phenothiazine derivatives (46–49).
Antitubercular activity
(BACTEC 12B; Bectron Dickinson Diagnostic Instrument system,
Sparks, MD, USA) to achieve final concentration of
Antitubercular activity was determined using the BACTEC 460
system modified as described below. Stock solutions of test
compounds were prepared in dimethylsulfoxide (DMSO) in
1 mg ⁄ mL concentration and sterilized by passage through 0.22-
lm PFTE filters (Millex-FG; Millipore, Bedford MA, USA). Volumes
equivalent to 50 lL were added to 4 mL radiometric 7H12 broth
a
6.25 mg ⁄ mL. Controls received 50 mL DMSO. Rifampicin (Sigma
Chemical Co. St. Louis, MO, USA) was included as a positive
drug control. Rifampicin was solubilized and diluted in DMSO
and added to BACTEC-12 broth to achieve MIC inhibiting 99% of
the inoculum.
416
Chem Biol Drug Des 2010; 76: 412–424

4-(arylamino)coumarins: New Anti T.B. agents
O
S
O
O
O
O
O
O
O
Microwave 400 W/140oC
8 min
NH
OH
+
NH2
SH
Scheme 3: Microwave-assisted synthesis of pyrano-coumaro-phenothiazine derivative (50–51).
Mycobacterium tuberculosis H37Rv strain (ATCC 27294; American
1
8
5
Y
O
2
type culture collection; Rockville, MD, USA) was cultured at 37 ꢀC
7
R1
on a rotary shaker in middle brook 7H9 broth (Difco Laboratories,
Detroit, MI, USA) supplemented with 0.2% v ⁄ v glycerol and 0.05%
v ⁄ v Tween 80 until the culture turbidity achieved an optical density
of 0.45–0.55 at 550 nm. Bacteria were pelleted by centrifugation,
washed twice, and resuspended in one-fifth of the original volume
in Dulbecco's phosphate-buffered saline [PBS, Irvine Scientific,
Santa Ana, (A)]. Large bacterial clumps were removed by passage
through an 8 mm filter (Malgene, Rochester, NY, USA), and aliquots
were frozen at )80 ꢀC. The cultures were thawed and an appropri-
ate dilution was performed such that a BACTEC-12B vial inoculated
with a 0.1 mL would reach a growth index (GI) of 999 in 5 days.
One-tenth of the diluted inoculums were used to inoculate 4 mL
fresh BACTEC 12B broth containing the test compounds. An addi-
tional control vial was included, which received a further 1:100
diluted inoculums (as well as 50 mL DMSO) for use in calculating
the MIC of rifampicin, respectively by established procedures.
3
6
4′
X
4
HN
5′
6′
1′
R′
8′
7′
Figure 1: A general representation of the structure of molecules
(X = S, Y = NH ⁄ O).
4-amino position, ab initio calculations were run on molecule 21
with the Gaussian software.b Geometry optimization was carried
out at both the AM1 and HF-3 ⁄ 21G level of theories. Based on
these calculations, the atom type for the N-atom was determined
as 'N3' i.e., sp3-pyramidal. The conformation of the aromatic ring
around the N-CAr bond was scanned using Systematic Search for
representative molecules with different substituents in the ortho
or meta-positions, and the global minimum conformation thus
determined was adopted for these and other molecules with simi-
lar substitutions.
Cultures were incubated at 37 ꢀC, and the growth of inhibition (GI)
determined daily until control cultures achieved a GI of 999. Assays
were usually completed in 5–8 days. Percent inhibition was defined
as 1-(GI of test sample ⁄ GI of control) 10. MIC of compound effect-
ing a reduction in daily change in GI, which was less than that
observed with a 1:100 diluted control culture on day the later
reached a GI of at least 30.
The geometries of all molecules, prior to the QSAR study, were
optimized by energy minimization by the conjugate gradient method
with the Tripos force field and the Gasteiger Hꢀckel charges for all
atoms, until a gradient 0.01 kcal ⁄ mol ⁄ ꢁ was reached. Gasteiger-
Hꢀckel charges were used for the QSAR studies.
Computational details
Comparative molecular field analysis was carried out using Sybyl
v.7.1 (Tripos Inc., USA)a running on a Pentium IV computer under
the Linux RedHat Enterprise 2.3.1.
Atom-fit-based alignment
The atoms of the 4-aminocoumarin ring are a common feature in
all molecules and this set was used for superimposition onto refer-
ence molecule 9. The alignment based on atom-fit strategy is
shown in Figure 2.
Ligand preparation
The antitubercular activity of the molecules considered in the
QSAR studies span a 3 log unit range (Table 1). The molecules
were built with the Sketch module in Sybyl. Figure 1 shows a
representative structure for these molecules; for the phenothiazine
derivatives, X represents a S-bridge between the coumarin and
the phenyl rings. To determine the correct geometry at the
Field-fit-based alignment
Field-fit alignment was also utilized for generating 3D-QSAR mod-
els. The steric and electrostatic fields were calculated around all
Chem Biol Drug Des 2010; 76: 412–424
417

Virsdoia et al.
into a training set (27 molecules) and a test set (9 molecules) by
means of chemical as well as biological diversity. Daylight finger-
prints of the molecules along with the pAlogit data were used to
separate the molecules into training and test sets based on the
Tanimoto similarity coefficient. Later on, few molecules were
swapped among the two datasets to get statistically sound models.
The final models were generated on a training set of 27 molecules
and a test set of six molecules.
Biological Data
For the QSAR study, the activity values were transformed as
followsc
Log activity (pALogit Þ ¼ ꢀlogc þ logit
ð1Þ
where 'c' is molar concentration = concentration (lg ⁄ mL) * 0.001 ⁄
(molecular weight); and
Figure 2: 4-Aminocoumarins aligned by the Atom-fit method.
logit ¼ log½% inhibition=ð100 ꢀ % inhibition)ꢁ:
ð2Þ
molecules with a grid spacing of 0.5 ꢁ. The molecules were then
aligned using the field-fit methodology with the fields around mole-
cule 9 as the reference. The alignment based on field-fit strategy
is shown in Figure 3.
CoMFA studies
In all CoMFA studies, default settings for the 3D cubic lattice, the
grid spacing, the probe atom, and the energy cut-offs were used.
Statically rotated alignments
The molecules previously aligned by atom-fit and field-fit method-
ologies were rotated in 3D space to generate additional orienta-
tion sets for QSAR studies. The STATIC ROTATE command in
Sybyl was used for generating such differently oriented sets. The
orientation that yielded statistically significant results was the
one obtained by rotating the molecules by 90ꢀ in the X, 180ꢀ in
the Y, and 270ꢀ in the Z directions (models 1a, 2a, 3a and 4a
in Table 2).
The CoMFA descriptors (steric and electrostatic fields) were used
as independent variables and the activity (as the negative loga-
rithm) values as the dependent variable in a partial least squares
(PLS) regression analysis (48) to derive 3D-QSAR models. The CoM-
FA models were generated from molecular alignments generated by
atom fit and field fit and their orientation in the 3D-grid optimized
with the static rotation command in Sybyl. The hydrophobic field
calculated using HINT (43) was used in conjunction with the CoMFA
fields to derive additional models.
Dataset
The set of 36 diverse molecules (Table S2 in supplementary infor-
mation) was used in the CoMFA study. The dataset was divided
The hydropathic fields were calculated with the HINT module in
Sybyl. The region defined for calculation of the CoMFA fields
was also used to describe the space in which the HINT fields
were generated. HINT calculates the hydrophobic interaction
between all atom pairs in a molecule using the following equa-
tion
X X
B ¼
where, bij = aiaj SiSj Rij Tij
bij
ð3Þ
bij = micro-interaction constant representing the attraction ⁄ interac-
tion between atoms i and j
ai = the hydrophobic atom constant for atom i
Si = the solvent accessible surface area for atom i
Rij = the functional distance behavior for the interaction between
atoms i and j
Figure 3: 4-Aminocoumarin derivatives aligned by the Field-fit
technique.
418
Chem Biol Drug Des 2010; 76: 412–424

4-(arylamino)coumarins: New Anti T.B. agents
Table 2: PLS statistics of CoMFA and CoMSIA 3D-QSAR models derived by atom-fit-based and field-fit alignments
Atom-fit alignment
CoMFA
Field-fit alignment
CoMFA
CoMFA + HINT
Model 2
CoMFA + HINT
Model 4
PLS statistics
Model 1
Model 1a
Model 2a
Model 3
Model 3a
Model 4a
n
27
6
27
5
0.47
0.93
0.41
0.98
–
56.9
0.169
27
6
27
5
27
6
27
6
27
6
27
6
PLS components
q2 (LOO)
0.41
0.88
0.50
0.97
–
0.38
0.94
0.38
0.98
–
0.35
0.90
0.51
0.97
–
0.46
0.98
0.49
0.99
–
0.58
0.99
0.47
0.99
0.377
245.4
0.077
0.40
0.97
0.52
0.99
–
0.56
0.98
0.52
0.99
0.305
187.6
0.087
r2
r2
pred
r2
BS
r2
YS
F
SE
40.2
0.219
51.8
0.163
39.1
0.201
151.8
0.097
121.4
0.108
Field contribution
Steric
Electrostatic
HINT
0.611
0.389
–
0.649
0.351
–
0.505
0.248
0.248
0.502
0.235
0.263
0.576
0.424
–
0.625
0.375
–
0.448
0.276
0.276
0.501
0.250
0.250
The models designated by letter 'a' refer to those deduced from datasets obtained with static rotation of particular alignments in 3D space.
CoMFA = Comparative molecular field analysis, HINT = Hydropathic interactions, n = number of molecules in training set, r2 = correlation coefficient, q2
(LOO) = cross-validated r2 with Leave-one-out method, r2 = Correlation coefficient for prediction of external datasets, r2 = Bootstrap Correlation coefficient,
pred
BS
F = F statistics, SE = standard error, PLS = partial least square analysis.
Tij = a discriminant function designed to keep the signs of interac-
tions consistent with the HINT convention that favorable interac-
tions are positive and unfavorable interactions are negative.
coumarin and this is then converted to the respective amines (57).
The 4-chlorocoumarins are strong skin irritants with several toxic
effects, so even a small-scale preparation is environmentally haz-
ardous. With the use of microwaves, it is possible to carry out this
reaction in a single step with a drastic reduction in reaction time.
This facile method has been optimized to prepare a small library of
50 compounds reported in this paper. The use of microwaves repre-
sents a far simpler and quicker mode of synthesis of 4-(arylami-
no)coumarins over methods reported in the literature. The method
involves irradiation of the reactants, the 4-hydroxycoumarin deriva-
tives, and the aryl amines in appropriate proportion, with 400 W
microwave power at 140 ꢀC in the absence of a solvent for an
appropriate time (Schemes 1, 2 and 3). All reactions were carried
out in a monomode open-vessel microwave synthesizer. The struc-
The models were internally evaluated by leave-one-out (LOO) cross-
validation. The optimal number of components was determined by
the SAMPLS (49) method and this was subsequently used to derive
the final QSAR models. In addition to the q2, the conventional cor-
relation coefficient r2 and standard errors (SE) were also computed,
the statistics of the models generated are shown in Table 2. The
models obtained were internally validated by bootstrapping (50–52),
Y-scrambling (53,54), and externally validated by calculating the
r2
for the validation set (test set).
pred
1
tures of the synthesized molecules were confirmed by H NMR, IR
Design of new molecules
spectra and mass spectra; details are given in supplementary data
(Table S1).
New molecules were designed based on a protocol published
recently (55). A library of 913 molecules was generated using the
CombiChem I utility in Cerius2 (Accelrys Inc., San Diego, CA, USA).
The generation of this library of molecules was guided by the CoM-
FA contours. Appropriate side chains were attached to molecules 6,
26 and 50. Molecules generated in this way were optimized with
the same protocol of energy minimization and then aligned as
described earlier. The activities of these molecules were predicted
using the best models 3a and 4a, respectively (Table 3). The ADME
and toxicity properties of the molecules were predicted with Quick-
Propd and the web-based PreADMET (56) tools to identify drugable
compounds.
Antitubercular activity
The compounds were screened against M. tuberculosis H37Rv
strain using the BECTEC 460 radiometric system. The results are
summarized in Table 1 with rifampin, as the standard. Of the
fifty compounds, compound 9 is the most potent showing 100%
inhibition of M. tuberculosis at 6.25 lg ⁄ mL, while compound 6 is
moderately active with 81% inhibition at the same concentration.
It is interesting to note that both bear a bulky naphthyl group at
the 4-position of the coumarin ring. The benzenoid part of the
coumarin skeleton in 9 is substituted with a 6-chloro and a 7-
methyl group, while in case of 6, it is unsubstituted. In contrast,
compounds with methyl substitutions at both the 5- and 8-posi-
tions on the coumarin ring (e.g., compounds 7, 12 and 13) are
nearly inactive. Compound 2 with a biaryl functionality at the 4-
position shows 65% inhibition and is more potent than closely
related analogues. When the biphenyl derivatives are additionally
Results and Discussion
Chemistry
The synthesis of 4-(arylamino)coumarins as reported in the literature
involves two steps. 4-Hydroxycoumarin is first converted to 4-chloro-
Chem Biol Drug Des 2010; 76: 412–424
419

Virsdoia et al.
Table 3: The predicted pALogit values for the designed molecules (A) Derivatives of molecules 6 (52–64) (B) Derivatives of molecules
26 (65–67) and (C) Derivatives of molecules 50 (68–79)
R1
O
O
O
O
O
O
R1
O
O
S
N
N
N
R2
R2
R2
R3
R3
A
B
C
Predicted activity
Model 3a
Substitution
R1
Molecules
Series
Model 4a
R2
R3
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
C
C
C
C
C
C
C
C
C
C
C
C
5.004
5.136
5.546
4.955
5.427
5.272
5.188
5.266
5.244
5.095
5.237
5.151
4.998
5.009
5.065
5.327
5.076
5.017
5.243
5.217
5.286
5.141
5.042
5.17
5.069
5.209
5.713
5.032
5.587
5.288
5.204
5.263
5.273
5.146
5.167
5.306
5.02
5.171
5.268
5.472
5.198
5.069
5.355
5.313
5.383
5.207
5.099
5.252
5.149
5.108
5.031
5.053
-COCH3
-H
-COCH3
-CH2Ph
-COOH
-CH2CH3
-CH=CH2
-Ph
-CH2Ph
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH2CH3
-CH=CH3
-H
-CH2Ph
-CH2CH3
-CH=CH2
-Ph
-COCH3
-CH2CH3
-CH2CH3
-CH2CH3
-H
-
-
-
-
-
-
-
-
-
-
-
-
-OPh
-CH3
-OCH3
-OH
-tBu
-
-COCH3
-COCH3
-COCH3
-CN
-H
-OCH3
-H
-NH2
-NH2
-NH2
-NH2
-OH
-F
-
-
-
-
-
-
-
-
-
-
-
-
-H
5.085
5.042
4.99
-OH
-OCH3
-SO2NH2
-SO2NH2
-CH2CH3
-CH=CH2
5.018
substituted with methyl groups on the coumarin ring, the activity
falls drastically e.g., compounds 3 and 4. No worthwhile activity
was observed in the series possessing an electron withdrawing –
COOH group (molecules 14–20). The activity of compounds bear-
ing a halogen substituent in the aromatic ring joined at the
4-position of the coumarin ring is variable with no clear-cut
pattern. In the phenothiazine series of compounds (molecules
46–50), compounds 50 and 48 show 78% and 67% inhibition,
respectively. From the activity range, it can be concluded that the
presence of the naphthyl group enhances potency, while methyl
substituents on the coumarin ring diminishes potency. Because
of the very interesting pattern of activity shown by these novel
compounds, the molecules were subjected to CoMFA studies to
probe the fine relationship between structure and activity that
exists for these compounds.
3D-QSAR study
The most interesting feature in these molecules is the geometry of
the 4-amino group. In an ideal situation, it must have a planar tri-
gonal geometry as it is backed by resonance to an aromatic system
as well as to the coumarin carbonyl group. However, preliminary
molecular mechanics calculations showed lack of planarity as the
hydrogen atom attached to the 4-amino group experiences severe
steric clash with the C5 hydrogen. To gain a better perspective of
the correct geometry, ab initio calculations were run on molecule
420
Chem Biol Drug Des 2010; 76: 412–424

4-(arylamino)coumarins: New Anti T.B. agents
21 with the Gaussian software.b Calculations were run on struc-
tures with both pyramidal and planar trigonal geometries for the 4-
amino group. The calculations revealed that the N-atom adopts a
state that is between these two ideal geometries. As no such atom
type has been defined in the Tripos force field, the atom type for
the N-atom was set to be 'N3' i.e., sp3-pyramidal.
Analysis of the contours for CoMFA models
The CoMFA contours (Figures 4, 5, and 6) were analyzed around
the most active, moderately active, and the least active molecules
50, 3 and 26, respectively. The steric contours indicate bulky
groups attached to the 7-position on the aromatic ring of the cou-
marin ring are strongly favored. The C6-position on the coumarin
ring should be unsubstituted as indicated by the small yellow con-
tour near this position (Figure 6). This is aptly borne by the fact
that the alkyl and aryl groups decrease the activity of molecules 3
and 26, respectively compared to molecule 50, which does not
have a substituent at the C6-position (Figure 5).
The next issue was to fix the geometry at the N-C4 bond between
the amine N and the C4 atom on the coumarin ring (Figure 1). As
mentioned previously, certain molecules have an S-bridge between
the aromatic ring and the 3-coumarin position. The activities of
these constrained cyclic amines (e.g., molecule 50) are higher than
the corresponding non-cyclic amines; this indicates that the confor-
mation attained by such systems must be considered in building
the conformation of the other molecules in the series. This fact
was used to fix the conformation around the N-C4 bond for the
open chain molecules. The constrained arylaminocoumarins were
first subjected to complete minimization, and the final conforma-
tions achieved for these molecules were adopted for the other non-
cyclic arylaminocoumarins. Further, Systematic conformational
search was executed on representative molecules to derive the
optimal conformation to be used for the CoMFA studies.
The electrostatic contours (Figure 4) depict that electronegative
groups on the aromatic ring will improve activity. For molecules
with a phenothiazine ring (e.g., molecule 50), electronegative
groups are favored at the meta-positions to the amino functionality
i.e., 5¢ and 7¢-positions, while the para-position i.e., 6¢-position
needs an electropositive group to improve activity. The same pat-
tern is followed in the case of molecules with N-phenyl groups. It
is interesting to find that electronegative group para to the amino
group have a detrimental effect on the activity and indeed this
position needs to be filled with electropositive groups. This is the
main reason why molecules 3 and 26 with electronegative carboxyl
and fluoro groups have greatly diminished activities.
Comparative molecular field analysis alone and in conjunction with
HINT hydropathy fields was carried out. CoMFA models 3a and 4a
with additional HINT fields were the most robust, with correlation
coefficients (r2) of 0.987 and 0.983 and cross-validated r2 (q2) of
0.578 and 0.556, respectively (Table 2). Both these models were
As mentioned earlier, some 3D-QSAR studies have been carried
out on 4-aminocoumarins with regard to antiplatelet activity
(39,40). One of these studies was carried out on a diverse set of
2- ⁄ 4-aminocoumarins, coumarins, and benzocoumarins substituted
with 1-piperazine, and pyrimidine, pyrido[1,2-a]pyrimidine and thia-
zo[1,2-a]pyrimidines analogues. The difference between these mol-
ecules and the present series is the presence of an aromatic
group on 4-amino functionality. For antiplatelet activity, bulky sub-
stituents are required at the 7- and 8-positions of the coumarin
ring but are disfavored in the 4¢-position of the 4-piperazine
group. In strong contrast, for antitubercular activity, bulky groups
are preferred at the 7-position, and further, this site seems to be
encompassed by a large hydrophobic pocket. Similarly, comparison
tested for their predictive ability on the validation set. The r2
for
pred
the various models are presented in Table 2 and the best value is
0.522, indicating that most models have decent predictive
powers. Further the robustness of the models was gauged by cross-
validation using bootstrapping (50–52) carried out with 100 runs.
The values of r2 are close to the related r2 values. To check the
BS
redundancy and chance correlation, the activity values were ran-
domized (Y-scrambling) (53–55) and the regression coefficients were
calculated for models 3a and 4a. These r2 calculated on random-
YS
ized activity were significantly smaller (0.377 and 0.305 for models
3a and 4a, respectively) indicating low degree of redundancy in the
training set.
Molecules 8, 21 and 39 were found to be outliers; their activities
were not well predicted by any of the models. One of the reasons
for the poor prediction may be an inadequate consideration of the
conformation of these molecules in the development of the models.
Incorporation of the HINT hydropathy field to the CoMFA models
gave a significant improvement in the quality of the models. The
statistical data in Table 2 indicate that the hydrophobic property
along with steric and electrostatic fields is necessary to explain the
activity of these molecules. The contribution of the HINT hydropathy
field was seen to be equal to the electrostatic field.
Graphical interpretation of the results
The CoMFA contour maps were generated using scalar products of
coefficients and standard deviation (STDEV*COEFF) set at 80% and
20% for favored and disfavored levels, respectively.
Figure 4: Comparative molecular field analysis electrostatic con-
tours displayed around molecule 50.
Chem Biol Drug Des 2010; 76: 412–424
421

Virsdoia et al.
partition coefficient in biological fluids, and membranes like skin
and Blood Brain Barrier (BBB), apparent Caco-2 cell permeability,
MDCK cell permeability, binding to human serum albumin, metabo-
lism, human oral absorption, IC50 value for blockage of HERG K+
channels, and central nervous system activity. For details of the
essential ADMET properties, the reader is referred to the supple-
mentary material. A few molecules with the best activity and the
satisfactory ADMET profile are enlisted in Table 3. Their respective
ADMET data (Table S3 in supplementary data) show that most mol-
ecules have satisfactory drug-like attributes. Toxicity data show that
only 28 molecules are predicted to be non-carcinogenic and six
were devoid of mutagenic effects.
Conclusions
Figure 5: Comparative molecular field analysis contours dis-
played around molecules 3 and 26.
A small library of 4-(arylamino)coumarins was synthesized using
microwave-assisted synthesis in good yields and short reaction
times. All compounds were screened for activity against M. tuber-
culosis H37Rv strain. A 3D-QSAR analysis of this library of com-
pounds was carried out using CoMFA alone and CoMFA in
conjunction with a hydrophobic field evaluated with HINT to map
the structural features responsible for antitubercular activity. Inclu-
sion of the HINT hydropathy field to the CoMFA fields improved the
quality of the models. Models obtained by field-fit alignment were
better in terms of statistics than those with database ⁄ atom-fit
alignment. Analysis of the CoMFA contours provided details on the
fine relationship linking structure and activity and offered ideas for
structural modifications that can improve the activity. There is still
a good scope for optimization of these molecules.
Acknowledgments
The authors are thankful to DST (program FIST, Sanction No.
SR ⁄ FST ⁄ CSI-072 ⁄ 2003, Dt. 24 ⁄ 12 ⁄ 2003) and to UGC (SAP program,
Sanction No. 540 ⁄ 6 ⁄ DRS ⁄ 2004, SAP-I, Dt. 26 ⁄ 03 ⁄ 2004) for their
generous financial and instrumentation support. Special thanks are
due to 'National Facility for Drug Discovery through New Chemical
entities (NCE's) Development & Instrumentation Support to Small
Manufacturing Pharma Enterprises' Program under Drug & Pharma
Research Support (DPRS) jointly funded by Department of Science
& Technology (DST, sanction letter no. VI ⁄ D&P ⁄ 188 ⁄ 06-07 ⁄ , TDT,
dated 30 ⁄ 03 ⁄ 07) New Delhi, Government of Gujarat Industries Com-
missionerate (Sanction Letter No. IC ⁄ SSI ⁄ R&D ⁄ SU ⁄ 07 ⁄ 3279 ⁄ 168,
dated 17 ⁄ 01 ⁄ 07) & Saurashtra University (Sanction Letter No.PL-
G ⁄ UGC ⁄ MS ⁄ 238 ⁄ 20006, dated 19 ⁄ 09 ⁄ 06), Rajkot. The authors are
also grateful to Government of Gujarat for PG Diploma's for Instru-
mentation & Patentization Financial Grant (Sanction Letter KVT ⁄ U-
NI ⁄ CHABA ⁄ 2008 ⁄ 20690-207).
Figure 6: Comparative molecular field analysis steric contours
displayed around molecule 50.
of the electrostatic contours reveals a favor for electronegative
groups at the 4-position for antitubercular activity. Thus, there are
clear distinctions between the steric and electrostatic attributes in
4-aminocoumarins that separate antiplatelet from antitubercular
activity.
Design of new molecules
The end purpose of all medicinal chemistry endeavors is to design
and synthesize better molecules. The molecules should not only be
potent but also have a good safety profile. Thus, it is necessary to
consider ADMET properties right at the design stage itself. With
this idea, around 913 molecules were designed and their activity
predicted using the best CoMFA models. The 135 molecules pre-
dicted to be highly active were then screened for various ADMET
properties like aqueous solubility (QPlogS), partition coefficient in
different solvents ⁄ media (QPlogPoct, QPlogPw, QPlogPo ⁄ w, CIQPlogS),
Authors are also thankful to the Dr. Cecil Kwong, Tuberculosis Anti-
microbial Acquisition and Coordinating Facility (TAACF), USA, for an-
titubercular screening. The computational facilities at BCP were
provided by the All India Council for Technical Education through
grant F. No. 8022 ⁄ RID ⁄ NPROJ ⁄ RPS-5 ⁄ 2003-04 ⁄ . The Department of
Science and Technology is also thanked for infrastructure facilities
through the FIST program (SR ⁄ FST ⁄ LS1-163 ⁄ 2003).
422
Chem Biol Drug Des 2010; 76: 412–424

4-(arylamino)coumarins: New Anti T.B. agents
Mycobacterium bovis, and Mycobacterium avium by novel dide-
oxy nucleosides. J Med Chem;50:4766–4774.
References
20. Qiao C., Gupte A., Boshoff H.I., Wilson D.J., Bennett E.M., Somu
R.V. et al. (2007) 5¢-O-[(N-acyl)sulfamoyl]adenosines as antituber-
cular agents that inhibit MbtA: an adenylation enzyme required
1. Dye C., Scheele S., Dolin P., Pathania V., Raviglione M.C. (1999)
Consensus statement. Global burden of tuberculosis: estimated
incidence, prevalence, and mortality by country. WHO Global
Surveillance and Monitoring Project. J Amm Med Assoc;
282:677–686.
for siderophore biosynthesis of the mycobactins.
Chem;50:6080–6094.
J Med
21. Johar M., Manning T., Tse C., Desroches N., Agrawal B., Kunim-
oto D.Y. et al. (2007) Growth inhibition of Mycobacterium bovis,
Mycobacterium tuberculosis and Mycobacterium avium in vitro:
effect of 1-beta-D-2¢-arabinofuranosyl and 1-(2¢-deoxy-2¢-fluoro-
beta-D-2¢-ribofuranosyl) pyrimidine nucleoside analogs. J Med
Chem;50:3696–3705.
22. Van Daele I., Munier-Lehmann H., Froeyen M., Balzarini J., Van
Calenbergh S. (2007) Rational design of 5¢-thiourea-substituted
alpha-thymidine analogues as thymidine monophosphate kinase
inhibitors capable of inhibiting mycobacterial growth. J Med
Chem;50:5281–5292.
23. Panda G., Parai M.K., Das S.K., Shagufta, Sinha M., Chaturvedi
V. et al. (2007) Effect of substituents on diarylmethanes for an-
titubercular activity. Eur J Med Chem;42:410–419.
24. Gupta R.A., Gupta A.K., Soni L.K., Kaskhedikar S.G. (2007) Ratio-
nalization of physicochemical characters of oxazolyl thiosemicar-
bazone analogs towards multi-drug resistant tuberculosis: a
QSAR approach. Eur J Med Chem;42:1109–1116.
2. Frieden T.R., Sterling T.R., Munsiff S.S., Watt C.J., Dye C. (2003)
Tuberculosis. Lancet;362:887–899.
3. WHO. (2006) Tuberculosis Facts. In: Tuberculosis Facts.
4. WHO. (2004) Tuberculosis Fact Sheet. In: Tuberculosis Fact
Sheet.
5. Guerrin-Tran E., Thiolet J.M., Rousseau C., Henry S., Poirier C.,
Che D. et al. (2006) An evaluation of data quality in a network
for surveillance of Mycobacterium tuberculosis resistance to
antituberculosis drugs in Ile-de-France region-2001–2002. Eur J
Epidemiol;21:783–785.
6. Somoskovi A., Parsons L.M., Salfinger M. (2001) The molecular
basis of resistance to isoniazid, rifampin, and pyrazinamide in
Mycobacterium tuberculosis. Respir Res;2:164–168.
7. Saquib M., Gupta M.K., Sagar R., Prabhakar Y.S., Shaw A.K., Ku-
mar R. et al. (2007) C-3 alkyl ⁄ arylalkyl-2,3-dideoxyhex-2-enopyr-
anosides as antitubercular agents: synthesis, biological
evaluation, and QSAR study. J Med Chem;50:2942–2950.
8. Pilheu J.A. (1998) Tuberculosis 2000: problems and solutions. Int
J Tuberc Lung Dis;2:696–703.
25. Ali M.A., Shaharyar M., Siddiqui A.A. (2007) Synthesis, struc-
tural activity relationship and anti-tubercular activity of novel
pyrazoline derivatives. Eur J Med Chem;42:268–275.
9. Espinal M.A. (2003) The global situation of MDR-TB. Tuberculo-
sis (Edinb);83:44–51.
26. de Almeida M.V., Le Hyaric M., Amarante G.W., Silva Lourenco
M.C., Lima Brandao M.L. (2007) Synthesis of amphiphilic galac-
topyranosyl diamines and amino alcohols as antitubercular
agents. Eur J Med Chem;42:1076–1083.
10. Peloquin C.A., Berning S.E. (1994) Infection caused by Mycobac-
terium tuberculosis. Ann Pharmacother;28:72–84.
11. Murray J.F. (1998) Tuberculosis and HIV infection: a global per-
spective. Respiration;65:335–342.
27. Swamy B.N., Suma T.K., Rao G.V., Reddy G.C. (2007) Synthesis
of isonicotinoylhydrazones from anacardic acid and their in vitro
12. Gordin F.M., Nelson E.T., Matts J.P., Cohn D.L., Ernst J., Benator
D. et al. (1996) The impact of human immunodeficiency virus
infection on drug-resistant tuberculosis. Am J Respir Crit Care
Med;154:1478–1483.
activity against Mycobacterium smegmatis. Eur
J Med
Chem;42:420–424.
28. Shiradkar M., Suresh Kumar G.V., Dasari V., Tatikonda S., Akula
K.C., Shah R. (2007) Clubbed triazoles: a novel approach to an-
titubercular drugs. Eur J Med Chem;42:807–816.
29. McKee T.C., Covington C.D., Fuller R.W., Bokesch H.R., Young S.,
Cardellina I.J. et al. (1998) Pyranocoumarins from tropical spe-
cies of the genus Calophyllum: a chemotaxonomic study of
extracts in the National Cancer Institute collection. J Nat
Prod;61:1252–1256.
30. Spino C., Dodier M., Sotheeswaran S. (1998) Anti-HIV coumarins
from Calophyllum seed oil. Bioorg Med Chem Lett;8:3475–3478.
31. Dodia N., Shah A. (1999) Single step synthesis and antimicro-
bial, antitubercular screening of substituted2,4-dihydroquinolines.
Ind J Heterocyl Chem;9:139–142.
32. Dodia N., Shah A. (2000) Synthesis and antitubercular activity of
some substituted 3-nitro and 3-bromo-4-hydroxy-2-quinolones.
Ind J Hterocyl Chem;10:69–70.
13. Moss A.R., Alland D., Telzak E., Hewlett D. Jr, Sharp V., Chiliade
P. et al. (1997) A city-wide outbreak of a multiple-drug-resistant
strain of Mycobacterium tuberculosis in New York. Int J Tuberc
Lung Dis;1:115–121.
14. Maher D., Raviglione M. (2005) Global epidemiology of tubercu-
losis. Clin Chest Med;26:167–182, v.
15. Frieden T.R., Munsiff S.S. (2005) The DOTS strategy for controlling
the global tuberculosis epidemic. Clin Chest Med;26:197–205.
16. No authors listed (2006) Editorial:XDR-TB-a Global Threat.
Lancet; 368:964.
17. Wright A., Bai G., Barrera L., Boulahbal F., Martꢂn-Casabona N.,
Gilpin C. et al. (2006) Emergence of Mycobacterium tuberculosis
with Extensive Resistance to Second-Line Drugs–Worldwide,
2000–2004. Morbidity and Mortality Weekly Report (MMWR);
55:301–5.
18. He X., Alian A., Stroud R., de Montellano P.R.O. (2006) Pyrroli-
dine carboxamides as a novel class of inhibitors of enoyl acyl
carrier protein reductase from Mycobacterium tuberculosis. J
Med Chem;49:6308–6323.
33. Desai B., Sureja D., Naliapara Y., Shah A., Saxena A.K. (2001)
Synthesis and QSAR studies of 4-substitutedphenyl-2,6-dimethyl-
3,5-bis-N-(substitutedphenyl)carbamoyl-1,4-dihydropyridines
as
potential antitubercular agents. Bioorg Med Chem;9:1993–1998.
19. Rai D., Johar M., Srivastav N.C., Manning T., Agrawal B., Kun-
imoto D.Y. et al. (2007) Inhibition of Mycobacterium tuberculosis,
Chem Biol Drug Des 2010; 76: 412–424
423

Virsdoia et al.
34. Gaveriya H., Desai B., Vora V., Shah A. (2002) Synthesis and an-
titubercular activity studies of some unsymmetrical 1,4-dihydro-
pyridines. Ind J Pharm Sci;64:59–62.
35. Kharkar P.S., Desai B., Gaveria H., Varu B., Loriya R., Naliapara
Y. et al. (2002) Three-dimensional quantitative structure-activity
relationship of 1,4-dihydropyridines as antitubercular agents. J
Med Chem;45:4858–4867.
36. Virsodia V., Pissurlenkar R.R., Manvar D., Dholakia C., Adlakha
P., Shah A. et al. (2008) Synthesis, screening for antitubercular
activity and 3D-QSAR studies of substituted N-phenyl-6-methyl-
2-oxo-4-phenyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxami des. Eur
J Med Chem;43:2103–2115.
37. Virsodia V., Manvar A., Upadhyay K., Loriya R., Karia D., Jaggi
M. et al. (2009) Synthesis of 1-(2,6-dichlorophenyl)-3-methylene-
1,3-dihydroindol-2-one derivatives and in vitro anticancer evalua-
3D-QSAR in drug design; theory methods and applications. The
Netherlands: ESCOM Lieden; p. 523–550.
49. Bush B.L., Nachbar R.B. Jr (1993) Sample-distance partial least
squares: PLS optimized for many variables, with application to
CoMFA. J Comput Aided Mol Des;7:587–619.
50. Richard D., Cramer R.D. III, Bunce J.D., Patterson D.E., Frank I.E.
(1988) Crossvalidation, bootstrapping, and partial least squares
compared with multiple regression in conventional QSAR stud-
ies. Quant Struct-Act Relat;7:18–25.
51. Gunter B. (1994) Bootstrapping. Infect Control Hosp Epidemi-
ol;15:543–547.
52. Shao J. (1996) Bootstrap Model Selection. J Am Stat As-
soc;91:655–665.
53. Tropsha A., Gramatica P., Gombar V.K. (2003) The Importance of
Being Earnest: validation is the Absolute Essential for Success-
ful Application and Interpretation of QSPR Models. QSAR Comb
Sci;22:69–77.
tion against SW620 colon cancer cell line. Eur
Chem;44:1355–1362.
J Med
38. Manvar A., Malde A., Verma J., Virsodia V., Mishra A., Upadhy-
ay K. et al. (2008) Synthesis, anti-tubercular activity and 3D-
QSAR study of coumarin-4-acetic acid benzylidene hydrazides.
Eur J Med Chem;43:2395–2403.
39. Roma G., Cinone N., Di Braccio M., Grossi G., Leoncini G., Si-
gnorello M.G. et al. (2000) Synthesis, antiplatelet activity and
comparative molecular field analysis of substituted 2-amino-4H-
pyrido[1,2-a]pyrimidin-4-ones, their congeners and isosteric ana-
logues. Bioorg Med Chem;8:751–768.
40. Roma G., Braccio M.D., Carrieri A., Grossi G., Leoncini G., Grazia
Signorello M. et al. (2003) Coumarin, chromone, and 4(3H)-pyri-
midinone novel bicyclic and tricyclic derivatives as antiplatelet
agents: synthesis, biological evaluation, and comparative molec-
ular field analysis. Bioorg Med Chem;11:123–138.
41. Cramer R.D., Patterson D.E., Bunce J.D. (1988) Comparative
molecular field analysis (CoMFA). 1 Effect of shape on binding
of steroids to carrier proteins. J Am Chem Soc;110:5959–
5967.
54. Rucker C., Rucker G., Meringer M. (2007) y-Randomization
and its variants in QSPR ⁄ QSAR. J Chem Inf Model;47:2345–
2357.
55. Manvar A.T., Pissurlenkar R.R., Virsodia V.R., Upadhyay K.D.,
Manvar D.R., Mishra A.K. et al. (2010) Synthesis, in vitro antitu-
bercular activity and 3D-QSAR study of 1,4-dihydropyridines. Mol
Divers;14:285–305.
56. Lee S.K., Lee I.H., Kim H.J., Chang G.S., Chung J.E., No K.T.
(2003) The PreADME approach: Web-based program for rapid
prediction of physico-chemical, drug absorption and drug-like
properties. Massachusetts, USA: Blackwell Publishing.
57. Zagorevskii V.A., Savelev V.L., Meshcheryakova L.M. (1970) On
synthesis of 4-aminocoumarins and on mechanism of halogen
substitution in 4- chlorocoumarin. Khim Geterotsikl Soed;8:1019–
1023.
Notes
aSybyl version 7.1 Tripos Inc. USA.
42. Cramer R.D. 3rd, Patterson D.E., Bunce J.D. (1989) Recent
advances in comparative molecular field analysis (CoMFA). Prog
Clin Biol Res;291:161–165.
43. Kellogg G.E., Semus S.F., Abraham D.J. (1991) HINT: a new
method of empirical hydrophobic field calculation for CoMFA. J
Comput-Aid Mol Des;5:545–552.
bGaussian 03, Gaussian Inc., Wallingford CT, USA.
cCoMFA and QSAR Manual S, Associates Inc., 1699 S Hanley Rd.,
St. Louis, MO 631444, USA.
dQuickProp. Schrodinger LLC.
44. Chavda M., Shah A., Bhatt S., Deo K., Kundu P. (2003) Antimi-
crobial screening and single step synthesis of some arylamino
coumarins. Ind J Chem;42B:1502–1507.
45. Shah A.K., Bhatt N.S., Raval R.V., Thakor V.M. (1984) Synthesis
of 4-hydroxycoumarins. Current Science;53:1241–1242.
46. Shah A., Naliapara Y., Sureja D., Motohashi N., Kurihara T.,
Kawase M. et al. (1998) Biological activity of 6,12-dihydro-1-
Supporting Information
Additional Supporting Information may be found in the online ver-
sion of this article:
Table S1. Synthetic and spectral data of the compounds.
benzopyrano
Res;18:61–63.
[3,4-b][1,4]
benzothiazin-6-ones.
Anticancer
Table S2. The experimental and predicted pALogit values for the
training and test set molecules used in 3D-QSAR studies.
47. Shah A., Naliapara Y., Sureja D., Motohashi N., Kawase M.,
Miskolci C. et al. (1998) 6,12-Dihydro-1-benzopyrano [3, 4-b] [1,
4] benzothiazin-6-ones: Synthesis and MDR reversal in tumor
cells. Anticancer Res;18:3001–3004.
Table S3. ADMET data for the designed compounds.
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
48. Wold S., Johansson E., Cocchi M. (1993) PLS-partial least
squares projections to latent structures in 3D-QSAR in drug
design; theory methods and applications. In: Kubinyi M.H., edi-
tor. PLS-partial least squares projections to latent structures in
424
Chem Biol Drug Des 2010; 76: 412–424