Design, synthesis, and evaluation of 4-(substituted)phenyl-2-thioxo-3,4- dihydro-1H-chromino[4,3-d]pyrimidin-5-one and 4-(substituted)phenyl-3,4-dihydro- 1H-chromino[4,3-d]pyrimidine-2,5-dione analogs as antitubercular agents

Article abstract of DOI:10.1007/s00044-013-0850-7

This paper focuses on the design and antitubercular activity of molecules which are a hybrid of coumarin and pyrimidine nuclei. A set of 16 compounds, viz. 4-(substituted) phenyl-3,4-dihydro-1H-chromino[4,3-d]pyrimidine-2,5-diones and 4-(substituted)phenyl-2-thioxo-3,4-dihydro-1H-chromino[4,3-d]pyrimidin-5- ones have been synthesized using green chemistry principles and evaluated for antitubercular activity by microplate Alamar blue assay (MABA) and lumi-nescence- based low oxygen-recovery assay (LORA) with rifampicin as the standard. The required Mycobacterium tuberculosis H37Rv strain for LORA was cultured using a plasmid bearing an acetamidase promoter driving a bacterial luciferase gene for signal enhancement and were allowed to adapt to low oxygen content in the fermenter. Compounds 5d, 5e, and 5g demonstratedmaximumantitubercular activity,with % inhibition values of 58, 55, and 45 based on MABA and 62, 35 and 37 based on the LORA tests at 128 μM. To understand the relationship between structure and activity, recursive partitioning (RP) models were developed. Two different RP models were built, one based on the antitubercular activity and the other based on the toxicity of the molecules. The decision tree could identify descriptors that discriminate the active and inactive as well as toxic and less toxic 3,4-annelated coumarin analogs. This RP model will be utilized in further work to design more potent molecules. Springer Science+Business Media 2013.

Full text of DOI:10.1007/s00044-013-0850-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2564–2575
DOI 10.1007/s00044-013-0850-7
ORIGINAL RESEARCH
Design, synthesis, and evaluation of 4-(substituted)phenyl-
2-thioxo-3,4-dihydro-1H-chromino[4,3-d]pyrimidin-5-one
and 4-(substituted)phenyl-3,4-dihydro-1H-chromino[4,
3-d]pyrimidine-2,5-dione analogs as antitubercular agents
•
Premlata K. Ambre Raghuvir R. S. Pissurlenkar
•
•
Ravindra D. Wavhale Mushtaque S. Shaikh Vijay M. Khedkar
•
•
•
Baojie Wan Scott G. Franzblau Evans C. Coutinho
•
Received: 4 August 2012 / Accepted: 15 October 2013 / Published online: 26 October 2013
Ó Springer Science+Business Media New York 2013
Abstract This paper focuses on the design and antitubercular
activity of molecules which are a hybrid of coumarin and
pyrimidine nuclei. A set of 16 compounds, viz. 4-(substi-
tuted)phenyl-3,4-dihydro-1H-chromino[4,3-d]pyrimidine-2,5-
diones and 4-(substituted)phenyl-2-thioxo-3,4-dihydro-1H-
chromino[4,3-d]pyrimidin-5-ones have been synthesized using
green chemistry principles and evaluated for antitubercular
activity by microplate Alamar blue assay (MABA) and lumi-
nescence-based low oxygen-recovery assay (LORA) with rif-
ampicin as the standard. The required Mycobacterium
tuberculosis H37Rv strain for LORA was cultured using a
plasmid bearing an acetamidase promoter driving a bacterial
luciferase gene for signal enhancement and were allowed to
adapt to low oxygen content in the fermenter. Compounds 5d,
5e,and5g demonstratedmaximum antitubercular activity, with
% inhibition values of 58, 55, and 45 based on MABA and 62,
35 and 37 based on the LORA tests at 128 lM. To understand
the relationship between structure and activity, recursive par-
titioning (RP) models were developed. Two different RP
models were built, one based on the antitubercular activity and
the other based on the toxicity of the molecules. The decision
tree could identify descriptors that discriminate the active and
inactive as well as toxic and less toxic 3,4-annelated coumarin
analogs. This RP model will be utilized in further work to
design more potent molecules.
Keywords 3,4-Annelated coumarins Á
Antitubercular agents Á MABA Á LORA Á
Recursive partitioning (RP)
Introduction
Tuberculosis (TB) is the world’s second leading cause of
death as an infectious disease after acquired immune
deficiency syndrome (AIDS) (Jindani et al., 1980). The
current treatment of TB is an exceedingly lengthy therapy
spanning 6–9 months (Jarvis and Lamb, 1998), which
makes patient compliance difficult and is a frequent source
of drug-resistant strains (Marinis and Legakis, 1985; Cohen
and Tartasky, 1997). The need for a lengthy treatment is a
consequence of the presence of a population of persistent
bacilli that are not effectively eliminated by the current TB
drugs (Jarvis and Lamb, 1998; Kawakami et al., 2000).
Given the difficulties in successful completion of treatment
and the increasing incidences of drug-resistant strains,
additional targets for development of new drugs need to be
researched (Krajewski et al., 2005). Special efforts are
needed to find and develop new leads against the many
validated TB targets. New leads with novel modes of
action are urgently required to combat the drug-resistant
strains of M. tuberculosis and stop the emerging TB pan-
demic (Miyakawa et al., 1996).
Recognizing these needs, we have initiated efforts
toward synthesis and screening of diverse heterocyclic
entities that have been reported to be active. These are
quinolones (Garcia-Garcia et al., 2005), coumarins
(McKee et al., 1998; Manvar et al., 2008), imidazopyrans
(Stover et al., 2000), phenazines (Barry et al., 1957),
substituted triazoles (Shiradkar et al., 2007), phenothiaz-
enes (Amaral et al., 2001) pyrimidines, 1,2,3,4-
P. K. Ambre Á R. R. S. Pissurlenkar Á R. D. Wavhale Á
M. S. Shaikh Á V. M. Khedkar Á E. C. Coutinho (&)
Department of Pharmaceutical Chemistry, Bombay College of
Pharmacy, Mumbai 400 098, India
e-mail: evans@bcpindia.org
B. Wan Á S. G. Franzblau
Tuberculosis Research Centre, University of Illinois, Chicago,
IL, USA
123

Med Chem Res (2014) 23:2564–2575
2565
tetrahydropyrimidines (Desai et al., 2001; Manvar et al.,
2010), and pyranocoumarins fused with phenothiazine
(Virsdoia et al., 2010). Among this heterocyclic group,
there are reports of the naturally occurring derivatives of
coumarin-like calanolide-A (Spino et al., 1998; Xu et al.,
2004) with MIC value of 3.13 lg/ml, and coumarin-4-
acetic acid benzylidine hydrazides (Manvar et al., 2008)
with IC50 of 6.25 lg/ml as promising antitubercular agents.
The current work is a continuation of our ongoing search
for some new antitubercular agents. Taking into account
the fact that coumarins have been reported to possess
antitubercular activity and benzopyranopyrimidines are
interesting due to their antifungal and antibacterial activi-
ties (Kidwai et al., 2006), we hypothesize that their fusion
would bring about a synergism in the individual antitu-
bercular activities. Based on the information contained in
the steric and electrostatic contours in the CoMFA maps
reported in our earlier 3D-QSAR studies on coumarin
analogs (Manvar et al., 2008; Virsdoia et al., 2010), we
have designed, synthesized, and tested 16 molecular vari-
ants containing 4-(substituted)phenyl-3,4-dihydro-1H-chr-
omino[4,3-d]pyrimidine-2,5-diones and 4-(substituted)
phenyl-2-thioxo-3,4-dihydro-1H-chromino[4,3-d]pyrimidin-
5-ones and 2-thiopyrimidone nuclei. The compounds have
thioxo-3,4-dihydro-1H-chromino[4,3-d]pyrimidin-5-ones
as potential antitubercular agents.
Chemistry
Syntheses of 4-(substituted)phenyl-3,4-dihydro-1H-chromi-
no[4,3-d]pyrimidine-2,5-diones and 4-(substituted)phenyl-2-
thioxo-3,4-dihydro-1H-chromino[4,3-d]pyrimidin-5-ones
were carried out by a three component Biginelli pyrimidone
synthesis (Ryabukhin et al., 2011) which involved reaction
between a substituted aldehyde, 6-substituted-4-hydroxy
coumarin, urea (or thiourea), and an acid catalyst (such as
p-toluenesulfonic acid) (Scheme 1). The reaction was also
successfully worked out using microwave irradiation [dis-
cover microwave synthesizer (CEM)] of 850 W microwave
power in absence of a solvent for 2.5 min (Scheme 1) in
90 % yields or higher. The structures of the molecules were
confirmed by IR, 1H NMR, and 13C NMR spectra; details are
given in ‘‘Experimental’’ section. Spectral data (IR, 1H
NMR, and 13C NMR) of all the compounds are in full
agreement with the proposed structures.
Biological evaluation
1
been characterized by IR, H NMR, and 13C NMR spec-
troscopy and evaluated for antitubercular activity based on
microplate Alamar blue assay (MABA) and luminescence-
based low oxygen-recovery assay (LORA) assays; their
cytotoxicity measured in vitro on virulent cells. To gain
better insight into structure activity relationships, a recursive
partitioning (RP) model was developed using physico-
chemical, structural, electronic, spatial, thermodynamic, and
other descriptors (Blower et al., 2002; Rusinko et al., 2002;
Young and Hawkins, 2004; Choi et al., 2006; Strobl et al.,
2009).
The compounds were screened against M. tuberculosis
H37Rv strain using the radiometric BECTEC-12B system
(Becton Dickinson Diagnostic Instrument System, Sparks,
MD, USA) until a growth index (GI) of 800–999 was
reached. All compounds were also screened by the MABA
(Bartizal et al., 1997) and the LORA (Scott et al., 2007).
MABA checks cellular growth and viability of the bacteria
while LORA monitors actively replicating as well as non-
replicating persistence (NRP) bacteria. Compounds were
also checked for cytotoxicity on human virulent cell lines.
In both assays, rifampin was the standard drug. The results
are summarized in Table 1.
Results and discussion
Based on the structures, the molecules are divided into
two classes: the phenyl-2-thioxo-3,4-dihydro-1H-chromi-
no[4,3-d]pyrimidin-5-one (Class A, 4a–h) and the phenyl-
3,4-dihydro-1H-chromino[4,3-d]pyrimidine-2,5-dione (Class
B, 5a–h) with eight molecules per class. Majority of Class A
molecules are non-cytotoxic based on the human virulent cell
lines evaluation with exception of molecules 4g and 4h,
although these possess relatively good antitubercular activity
against H37Rv cells. The molecules belonging to Class B
(5a–h) inhibit growth of M. tuberculosis [50 %; however,
they have high cytotoxicity profiles.
Our earlier 3D-QSAR studies revealed that (Manvar et al.,
2008) steric or electronegative substitutions on the cou-
marin nucleus at the third, fourth, sixth, or seventh posi-
tions are favorable for activity, while no modifications are
allowed on the lactone ring of the coumarin nucleus. In
addition, the same results have also been reported by
Virsdoia et al., (2010) who showed that bulky substituent
at the fourth position of the coumarin increases activity,
while Manvar et al. (2008, 2010) have reported substituted
pyrimidones as potential antitubercular agents. With this
knowledge, we have designed a set of 16 compounds
with the coumarin ring fused to a thio or oxopyrimidine
core viz. 4-(substituted)phenyl-3,4-dihydro-1H-chromino
[4,3-d]pyrimidine-2,5-diones and 4-(substituted)phenyl-2-
It is interesting to note that the molecules with the
chromino[4,3-d]pyrimidine-2,5-dione nucleus (4a and 4e)
and molecules 5d, 5e, and 5f have acceptable cytotoxicity
and antitubercular activity profiles of which molecule 5d
ranks the highest with respect to its activity (MABA 58 %
123

2566
Med Chem Res (2014) 23:2564–2575
X
H
O
H
C
O
HN NH
R
X
R
R1
or
(i) (ii)
R1
H2N
NH2
O
O
O
O
1
2
3
Class A: 4a-h
Class B: 5a-h
Scheme 1 Synthesis of 4-(substituted)phenyl-2-thioxo-3,4-dihydro-
1H-chromino[4,3-d]pyrimidin-5-one (Class A, 4a–h) and 4-(substi-
tuted)phenyl-3,4-dihydro-1H-chromino[4,3-d]pyrimidine-2,5-diones
(Class B, 5a–h) analogs. (i) Conc. HCl, ethanol, reflux for 12 h; (ii)
MW; 850 W, 2.5 min; X = S/O
Table 1 Structural and experimental information for 4-(substituted)phenyl-2-thioxo-3,4-dihydro-1H-chromino[4,3-d]pyrimidin-2-ones and
4-(substituted)phenyl-3,4-dihydro-1H-chromino[4,3-d]pyrimidine-2,5-diones
X
2
1
3
HN
NH
4
R
9
R1
5
8
O
6
O
7
Class A: 4a-h
Class B: 5a-h
Sl. no. Class R
R1
X
Percentage yield
Microwave Conventional
Melting point (°C) MIC (% inhibition @ 128 lM)a IC50 [lM] vero
cell (cytotoxicity)
MABA
LORA
4a
4b
4c
4d
4e
4f
A
H
H
H
H
S
92
92
90
90
90
92
94
90
91
94
90
90
94
92
90
90
74
76
73
75
75
77
72
73
76
72
77
79
77
74
73
76
203–205
232–234
262–264
232–234
260–262
216–218
222–224
232–234
210–212
206–208
262–264
242–244
250–252
208–210
242–244
222–224
10
0
20
0
[128
[128
[128
[128
[128
[128
47.4
4-OCH3
3,4-OCH3
S
S
0
0
CH3 4-OCH3
S
0
1
CH3 3,4-OCH3
S
9
14
0
H
H
3-OCH3; 4-OH
3-Br
S
0
4g
4h
5a
5b
5c
5d
5e
5f
S
12
32
0
59
61
17
4
CH3 3-Br
S
47.6
B
H
H
H
H
O
O
O
O
O
O
O
O
84.7
4-OCH3
3,4-OCH3
8
119.2
[128
100.7
[128
[128
122.2
106.2
0
0
CH3 4-OCH3
58
55
37
45
7
62
35
1
CH3 3,4-OCH3
H
H
H
3-OCH3; 4-OH
5g
5h
a
2-OH
3-Br
37
15
% inhibition recorded at 128 lM concentration for all compounds, MIC of rifampin (standard) used 0.10 and 3.8 lM for MABA and LORA,
respectively, against M. tuberculosis H37Rv
and
LORA
62 %)
and
cytotoxicity
profile
a loss in the activity. A loss in the activity and simulta-
neously an increased in the cytotoxicity is noted when no
substituent is present on the 4-phenyl ring. Molecules that
have a methyl substitution at the ninth position of chromino
[4,3-d]pyrimidine-2,5-dione nucleus possess greater
(IC50 = 100.7 lM). The presence of electron donating
groups like methyl, methoxy, or hydroxyl on the 4-phenyl
ring on the chromino[4,3-d]pyrimidine-2,5-dione nucleus
impart greater antitubercular activity while bromine causes
123

Med Chem Res (2014) 23:2564–2575
2567
Fig. 1 RP tree generated with
moderate pruning: 1:1, 2:0, 3:0,
and 4:1, correspond to terminal
nodes 1–4 and each terminal
node corresponds to the value of
0 (less active) or 1 (active)
Table 2 Descriptors used to develop the RP models
antitubercular activity and are devoid of cytotoxicity
compared to the unsubstituted analogs.
Descriptor
Description
Structural
descriptor
The number of H-bond donor and acceptor,
number of rotatable bonds, molecular weight
Computational studies
Topological
descriptors
Wiener index (W), Zagreb index (Zagreb),
Hosoya index (Z), Kier and Hall molecular
connectivity index (v)
To understand structure–activity relationships, we have
built classification models based on the biological activity
and cytotoxicity using the technique of RP.
Spatial descriptors Radius of gyration, molecular volume, area,
density, principal moment of inertia
RP model for antitubercular activity
Electronic
descriptors
Charge, Apol, HOMO, LUMO, dipole moment
Thermodynamic
descriptors
AlogP98, molecular refractivity
RP categorizes the molecules by deriving a binary decision
tree in which descriptors are used to split the data set into
smaller, homogeneous subsets. A 4-leaf RP decision tree
was obtained with 4 terminal and 3 non-terminal nodes for
the substituted chrominopyrimidine analogs based on 2D
descriptors (vide infra). At each decision point, the tree is
split into two branches, higher and lower responses,
according to the value of the descriptors. The terminal
nodes 1 and 4 contain molecules belonging to Actives class
(binary digit 1), while terminal nodes 2 and 3 contain
molecules belonging to Inactives class (binary digit 0). The
descriptors’ dipole moment, radius of gyration, and Apol
formed the decision points in the decision tree (Fig. 1). A
true response to any given split follows the branch to the
downside, while a false response to any given split follow
the branch to the upside in a decision tree.
moment of inertia, rotatable bonds, hydrogen bond accep-
tors, hydrogen bond donors, and AlogP98 (an alternate
method for calculating the o/w partition coefficient)
(Table 2). The dataset was categorized into two sets: the
Inactives (0) and the Actives (1). The molecules found to
be active by the MABA and LORA assays were classified
as actives and rest were classified as inactive. The Inactives
set had 11 molecules with \50 % inhibition, while the
Actives set had five molecules with [50 % inhibition (the
% inhibition being measured at 128 lM concentration).
The classification model was derived by variation of sta-
tistical parameters discussed in the ‘‘Materials and meth-
ods’’ section so as to improve the following two aspects.
‘‘Class % Observed Correct’’, the so-called intra class
prediction providing information on false negatives as well
as false positives, depending on the class to be examined;
The following 2D descriptors: were calculated using the
Cerius2 (Accelrys Inc., USA)—sum of atomic polariz-
abilities (Apol), dipole moment, radius of gyration, area,
molecular weight, molecular volume, density, principal
123

2568
Med Chem Res (2014) 23:2564–2575
Table 3 Statistical results of RP based on antitubercular activity
Table 5 Results of the cross-validation of the RP model for antitu-
bercular activity
Class No. of
%
Class %
Overall %
predicted
correct
Enrichment
molecules Molecules observed
correct
Class Number of
compound
%
Class % Overall % Enrichment
observed predicted
correct
Correct
1
2
11
5
68.75
31.25
100
100
100
100
1.46
3.20
1
2
11
5
68.75 72.73
31.25 80.00
88.89
57.14
1.29
1.83
Table 4 Antitubercular activity as predicted by the RP model
Table 6 Statistical results of the RP model based on the cytotoxicity
data
Sl.
no.
Activity RP-predicted
activity
Descriptors
Apol
Dipole
moment
Density
Class No. of
%
Class %
Overall %
predicted
correct
Enrichment
molecules Molecules observed
correct
4a
4b
4c
4d
4e
4f
0
0
0
0
0
0
1
1
0
0
0
1
1
0
1
0
0
0
0
0
0
0
1
1
0
0
0
1
1
0
1
0
13,509.62 3.10
14,415.84 4.14
15,322.06 2.25
14,977.26 4.76
15,883.48 2.98
14,808.80 4.01
13,487.40 8.70
14,048.82 10.05
12,191.82 3.10
13,098.04 4.12
14,004.26 2.29
13,659.46 4.77
14,565.68 2.86
13,491.00 4.02
12,584.78 5.33
12,169.60 8.64
1.20
1.20
1.20
1.18
1.18
1.22
1.41
1.38
1.18
1.18
1.18
1.16
1.16
1.20
1.21
1.40
1
2
12
4
75
25
100
100
100
100
1.33
4.00
The first primary split occurs on the dipole moment and
its value is 4.77. It is an electronic descriptor that indicates
the strength and orientation behavior of a molecule in an
electrostatic field and has been correlated to long-range
ligand–receptor recognition and binding. The dataset is
split into two sets with dipole moment above and below
4.77. The classification of the dataset based on dipole
moment is shown in Fig. 1.
4g
4h
5a
5b
5c
5d
5e
5f
The next split occurs with the sum of atomic polariz-
abilities (Apol) and its value is 12,180.71. The terminal
5g
5h
node
1 contains four molecules with Apol value
[12,180.71 and belong to ‘Actives’, while the remaining 1
molecule has a value below 12,180.71 forming the 2nd
terminal node (Fig. 1).
0 represents inactive while 1 represents active molecule
The next split is on density and its value is 1.17. The
terminal node 3 contains 10 molecules with density more
than 1.17 which is shown as ‘Inactives’ while the
remaining 1 molecule with density \1.17 forms the ter-
minal node 4 and is shown in ‘Actives’ (Fig. 1).
In order to avoid over fitting and to improve general-
ization of the model, internal validation using the tech-
nique of tenfold cross-validation which leaves out 10 % of
the molecules was carried out. An acceptable classification
percentage observed for the sets suggests the stable nature
of this model for external testing (Table 5). Considering
the size and other limitations in the data set, this predic-
tion rate is acceptable and the key descriptors determined
in the trial set are consistent with those observed in the
whole set.
‘‘Overall % Predicted Correct’’, the so-called overall
prediction, provides information on the accuracy of the
prediction when the whole set is predicted with the model;
enrichment factor (ER) for a specific class is the ratio of the
‘‘Overall % Predicted Correct’’ to the original percentage
of compounds belonging to that class. The statistical results
of the RP model and antitubercular activity predictions
from it are summarized in Tables 3 and 4, respectively.
All the 11 molecules of the Inactives set, were correctly
classified as True Negatives. So also the prediction was
100 % for Actives set, classifying all five molecules as
True Positives. The ER observed for Actives is 1.46, while
that for Inactives is 3.20. The number of true positives
among the predictions in each activity set is defined as
‘‘Overall % Predicted Correct’’. It is noteworthy that for
both sets (‘Actives’ and ‘Inactives’), the ‘‘Overall % Pre-
dicted Correct’’ was observed to be 100 %. The ER also
indicates that the final RP model could be used with con-
fidence for classifying a new library of molecules.
RP model for cytotoxicity (VCCT)
For building a RP model for cytotoxicity, the set of 16
substituted chrominopyrimidines was categorized into two
123

Med Chem Res (2014) 23:2564–2575
2569
been evaluated for toxicity on human virulent cell lines.
The classification model was derived by variation of
parameters discussed in the ‘‘Materials and methods’’
section seeking to improve the following parameters:
‘‘Class % Observed Correct’’, ‘‘Overall % Predicted
Correct’’, and ER. The statistical outcomes of the RP
model and cytotoxicity predictions from it are summarized
in Tables 6 and 7, respectively.
Table 7 The cytotoxicity of the molecules as predicted by the RP
model
Sl.
no.
VCCT RP-predicted
VCCT
Descriptors
Apol
Dipole- Radius of
gyration
mag
4a
4b
4c
4d
4e
4f
0
0
0
0
0
0
1
1
1
0
0
1
0
0
0
0
0
0
0
0
0
0
1
1
1
0
0
1
0
0
0
0
13,509.62 3.10
14,415.84 4.14
15,322.06 2.25
14,977.26 4.76
15,883.48 2.98
14,808.80 4.01
13,487.40 8.70
14,048.82 10.05
12,191.82 3.10
13,098.04 4.12
14,004.26 2.29
13,659.46 4.77
14,565.68 2.86
13,491.00 4.02
12,584.78 5.33
12,169.60 8.64
3.73
4.12
4.35
4.36
4.55
4.23
3.76
4.04
3.72
4.11
4.35
4.35
4.55
4.23
3.75
3.75
The molecules belonging to the ‘Non-toxic’ and
‘Toxic’ were correctly classified into their corresponding
classes of ‘True Positives’ and ‘True Negatives’ with
respective ER of 1.33 and 4.00. In a manner similar to
the RP model generated for antitubercular activity, a
5-leaf RP decision tree was obtained for the molecules
based on the 2D descriptors (Fig. 2). The first primary
split based on dipole moment with cutoff 8.67 places 14
molecules as ‘Non-toxic’ and 2 molecules as ‘Toxic’.
Further the secondary split based on radius of gyration
with cutoff as 3.73, places one molecule as ‘Toxic’ and
13 molecules in ‘Non-toxic’ set. The third split again
based on dipole moment at 4.77 places 10 molecules in
the ‘Non-toxic’ zone and subsequently the final split
based on Apol, with cutoff 12,841.41 places 9 molecules
as ‘Non-toxic’. The model was validated to avoid over
fitting and to improve generalization of the model. An
acceptable classification percentage observed for the
classes suggests reasonably good predictability (Table 8).
This accuracy in the predictions for the molecules sug-
gests the power of this model for prediction of toxicity
of an external set.
4g
4h
5a
5b
5c
5d
5e
5f
5g
5h
0 represents non-toxic while 1 represents toxic molecule
sets, the ‘Non-toxic’ (binary digit 0) set containing 12
molecules with cytotoxicity \100 lM (IC50) and the sec-
ond ‘Toxic’ (binary digit 1) set containing four molecules
whose cytotoxicity is[100 lM (IC50). The molecules have
Fig. 2 RP tree generated with
moderate pruning: 1:1, 2:1, 3:0,
4:0, and 5:1 correspond to
terminal nodes 1–5 and each
terminal node corresponds to
the value of 0 (less toxic) or 1
(toxic)
123

2570
Med Chem Res (2014) 23:2564–2575
Experimental
Table 8 Results of the cross-validation of the RP model for
cytotoxicity
An earlier 3D-QSAR study (Manvar et al., 2008) con-
ducted on coumarin-4-acetic acid benzylidene hydrazides
and 4-(arylamino)coumarins was the starting point in the
design of new and more potent coumarin analogs. Promi-
nent steric contours were observed on the phenyl ring
of the benzylidene hydrazide moiety as well as on the
4-(arylamino)coumarin ring which indicated that substitu-
ents such as methoxy and bromo would improve the anti-
tubercular activity.
Class No. of
%
Class %
Overall %
predicted
correct
Enrichment
molecules Molecules observed
correct
1
2
12
4
75
25
75.0
50.0
81.8
40.0
1.09
1.60
i
( )
O
O
O
O
O
+
H
O
R
The wealth of information in the CoMFA steric and
electrostatic contours was used to design some new cou-
marins analogs by cyclising the coumarin ring at the third
and fourth positions with urea and thiourea groups to
H
O
H
O
R
H
1
Scheme 2 Synthesis of 6-substituted-4-hydroxycoumarin analogs.
Zinc chloride, phosphorus oxychloride, 60–65 °C, 35 h
produce
4-(substituted)phenyl-3,4-dihydro-1H-chromi-
no[4,3-d]pyrimidine-2,5-diones and 4-(substituted)phenyl-
2-thioxo-3,4-dihydro-1H-chromino[4,3-d]pyrimidine-5-
ones.
Conclusions
A set of 16 molecules belonging to 4-(substituted)phenyl-
3,4-dihydro-1H-chromino[4,3-d]pyrimidine-2,5-diones and
4-(substituted)phenyl-2-thioxo-3,4-dihydro-1H-chromino
[4,3-d]pyrimidin-5-ones were synthesized using micro-
wave irradiation in 90–96 % yield, higher than that
obtained by the conventional method (72–79 %). All
molecules were assayed against M. tuberculosis H37Rv
strain using rifampicin as the standard by the MABA and
LORA procedures to determine the MIC values. The
molecules were also evaluated for cytotoxicity; conducted
on human virulent cell lines. Based on results from the
MABA and LORA studies, molecules with the phenyl-3,4-
dihydro-1H-chromino[4,3-d]pyrimidine-2,5-dione nucleus
are more potent than those with phenyl-2-thioxo-3,4-
dihydro-1H-chromino[4,3-d]pyrimidin-5-one nucleus. The
former class is also identified as non-cytotoxic, compared
to the latter. Electron donating groups like methyl/methoxy
when placed at the nineth position of the 3,4-annelated
coumarin nucleus and a 4-methoxy group on the 4-phenyl
ring of the chromino core (e.g., 5d, 5e, 5g) improve
potency in terms of inhibition of M. tuberculosis H37Rv
strain and further such derivative are also seen to be non-
cytotoxic. A RP model has been developed and is able to
accurately classify molecules as active/inactive or toxic/
non-toxic.
Chemistry
All compounds were characterized and their structures
confirmed by spectral properties. Solvents and reagents
used for synthesis were of laboratory grade. All reactions
were monitored by thin layer chromatography using Merck
precoated silica plates (GF254). Melting points were
recorded in open capillaries on an electrically heated
Thermonik melting point apparatus and are uncorrected;
boiling points were recorded in a Thiel’s tube. IR spectra
were recorded (KBr disk method) on a JASCO FT-IR 5300
1
spectrophotometer. H NMR spectra were recorded on a
Bruker AN 300 MHz instrument and 13C NMR were
recorded on a Bruker Advanced II 400 MHz instrument
(for compounds 4a–4c, 4e–4h, 5a–5c, 5e, 5g, 5h) and
Varian Mercury Plus 400 MHz instrument (for 4d, 5d, 5f).
Spectra Chemical shifts are reported in parts per million
(ppm) down field from tetramethylsilane (TMS) as the
internal standard. The synthetic outline is given in the
schemes and detailed procedures are given below.
General method for synthesis of 6-substituted-4-
hydroxycoumarin (Scheme 2)
A mixture of 1.35 g (14.5 mmol) of phenol, 5.95 g
(43.7 mmol) of anhydrous ZnCl2, 4 ml (0.043 mol) of
phosphorus oxychloride, and 1.50 g (0.014 mol) of ma-
lonic acid was heated to 60–65 °C. On completion of the
reaction (30–35 h), the reaction mixture was cooled and
poured on to 50–60 ml cold water and kept aside for 2–3 h
till the product precipitated out. The precipitate was filtered
and dissolved in saturated solution (*50 ml) of sodium
bicarbonate and then acidified with 10 % glacial acetic acid
The molecules were synthesized based on the hypothesis
that a fusion of the coumarin and pyrimidin-2,5-dione
nuclei both of which cores have been shown to be active
against tuberculosis, would give potent antitubercular
agents. Indeed the fusion of chromino nucleus with the
pyrimidine framework has revealed a novel core and some
promising molecules. Molecules 5d, 5e, 5g are worthy of
being considered for further development.
123

Med Chem Res (2014) 23:2564–2575
2571
to pH 2–3 with continuous stirring; the precipitated crude
compound (1) was crystallized using aq. methanol (yield
60 %).
104.53, 113.49, 115.97, 116.65, 123.72, 123.98, 127.54,
129.28, 132.12, 151.95, 157.59, 163.89, 165.36.
4-(3,4-Dimethoxy)phenyl-2-thioxo-3,4-dihydro-1H-chro-
mino[4,3-d]pyrimidin-5-one (4c) IR (KBr, cm-1): 3,439
(N–H), 2,935 (aromatic C–H stretch), 1,664 (C=O), 1,564
General procedure for the synthesis of 4-
(substituted)phenyl-3,4-dihydro-1H-chromino[4,3-
d]pyrimidine-2,5-dione (4a–h) and 4-(substituted)phenyl-
2-thioxo-3,4-dihydro-1H-chromino[4,3-d]pyrimidin-2-one
(5a–h) (Table 1; Scheme 1)
1
(aromatic C=C bending), 1,248 (C=S), 1,140 (C–O). H
NMR (CDCl3, d ppm): 3.73 (s, 3H, (O)–C–H), 3.87 (s, 3H,
(O)–C–H), 6.11 (s, 1H, (NH)–C–H), 6.71–6.81 (m, 3H,
Ar–H), 7.64–8.03 (m, 4H, Ar–H), 11.44 (s, 2H, NH). 13C
NMR (DMSO-d6, d ppm): 35.36, 55.40, 55.58, 104.47,
111.01, 111.35, 111.95, 116.98, 118.73, 123.73, 123.85,
130.63, 131.97, 147.23, 148.55, 152.02, 164.22, 165.13.
(i) Conventional approach:
A mixture of 600 mg
(1.5 mEq) of substituted 4-hydroxycoumarin (1),
250 mg (1 mEq) of substituted benzaldehyde (2), and
144 mg (1 mEq) of urea or thiourea (3) in 20 ml of
ethanol containing 4 drops of concentrated hydro-
chloric acid was heated under reflux for 12 h or until
completion of the reaction. The mixture was kept
aside for several hours at room temperature and the
crude product (4a–h or 5a–h) which precipitated out
was filtered, washed with cold methanol (3 9 10 ml)
dried and re-crystallized from aqueous methanol.
(ii) Microwave-assisted approach (Kidwai et al., 2006)
(Scheme 1): A mixture of 600 mg (1.5 mEq) of
substituted 4-hydroxycoumarin, 250 mg (1 mEq) of
substituted benzaldehyde and 144 mg (1 mEq) of
urea or thiourea with a catalytic amount of 4-meth-
ylbenzenesulfonic acid was placed in a 25-ml round
bottom flask and irradiated with microwave at 850 W
for 2.5 min [discover microwave synthesizer (CEM)].
After the completion of the reaction, the resulting
sticky material was treated with a few drops of
methanol 4–5 times and then filtered. The product
was then purified by recrystallization from aqueous
methanol.
4-(4-Methoxy)phenyl-9-methyl-2-thioxo-3,4-dihydro-1H-
chromino-[4,3-d]pyrimidin-5-one (4d) IR (KBr, cm-1):
3,210 (N–H), 2,930 (aromatic C–H stretch), 1,672 (C=O),
1,575 (aromatic C=C bending), 1,267 (C=S), 1,109 (C–
1
O). H NMR (DMSO-d6, d ppm): 2.44 (s, 3H, (Ar)–C–H),
3.79 (s, 3H, (O)–C–H), 6.03 (s, 1H, (NH)–C–H), 6.84–7.
26 (m, 4H, Ar–H), 7.41–7.83 (m, 3H, Ar–H), 11.33 (s,
1H, NH), 11.55 (s, 1H, NH). 13C NMR (DMSO-d6, d
ppm): 20.44, 54.95, 104.44, 113.57, 115.84, 117.00, 123.
43, 127.73, 130.70, 132.96, 133.22, 150.23, 157.44, 164.
47, 165.07.
4-(3,4-Dimethoxy)phenyl-9-methyl-2-thioxo-3,4-dihydro-1H-
chromino-[4,3-d]pyrimidin-5-one (4e) IR ((KBr, cm-1):
3,270 (N–H), 2,953 (aromatic C–H stretch), 1,664 (C=O),
1,574 (aromatic C=C bending), 1,267 (C=S), 1,143 (C–O).
1H NMR (DMSO-d6, d ppm): 2.46 (s, 3H, (Ar)–C–H), 3.73
(s, 3H, (O)–C–H), 3.86 (s, 3H, (O)–C–H), 6.69–6.79 (m,
3H, Ar–H), 6.05 (s, 1H, (NH)–C–H), 7.30–7.41 (m, 2H,
Ar–C–H), 7.78–7.84 (m, 2H, Ar–C–H), 11.33 (s, 1H, NH),
11.57 (s, 1H, NH). 13C NMR (DMSO-d6, d ppm): 20.50,
35.35, 55.41, 55.60, 104.46, 110.98, 111.40, 115.78, 116.
38, 118.70, 123.33, 130.26, 133.02, 133.34, 147.30, 148.
59, 150.15, 163.92, 165.39.
4-Phenyl-2-thioxo-3,4-dihydro-1H-chromino[4,3-d]pyrim-
idin-5-one (4a) IR (KBr, cm-1): 3,402 (N–H), 1,668 (C=
1
O), 1,568 (aromatic C=C bending), 1,278 (C=S). H NMR
(CDCl3, d ppm): 6.10 (s, 1H, (NH)–C–H), 7.23–7.39 (m,
5H, Ar–H), 7.61 (m, 2H, Ar–H), 8.00 (m, 2H, Ar-H), 11.30
(s, 1H, NH), 11.52 (s, 1H, NH). 13C NMR (CDCl3, d ppm):
36.20, 103.92, 105.66, 116.65, 124.40, 124.90, 126.49,
126.89, 128.65, 132.87, 135.22, 152.31, 152.53, 164.59,
165.80, 166.80, 169.30.
4-(4-Hydroxy-3-methoxy)phenyl-9-methyl-2-thioxo-3,4-
dihydro-1H-chromino-[4,3-d]pyrimidin-5-one (4f) IR
(KBr, cm-1): 3,497 (O–H), 3,070 (N–H), 2,970 (aromatic
C–H stretch), 1,666 (C=O), 1,564 (aromatic C=C bending),
1,269 (C=S), 1,128 (C–O). 1H NMR (CDCl3, d ppm): 3.75
(s, 3H, (O)–C–H), 5.61 (s, 1H, (Ar)–O–H), 6.07 (s, 1H,
(NH)–C–H), 6.73–6.86 (m, 3H, Ar–C–H), 7.63–8.05 (m,
4H, Ar–C–H), 11.31 (s, 1H, NH), 11.53 (s, 1H, NH). 13C
NMR (DMSO-d6, d ppm): 35.21, 55.65, 104.55, 110.62,
115.10, 115.95, 116.26, 118.87, 123.65, 124.09, 127.13,
132.14, 144.97, 147.33, 151.85, 163.79, 165.99.
4-Anisyl-2-thioxo-3,4-dihydro-1H-chromino[4,3-d]pyrimi-
din-5-one (4b) IR (KBr, cm-1): 3,072 (N–H), 2,837
(aromatic C–H stretch), 1,670 (C=O), 1,510 (aromatic C=C
1
bending), 1,259 (C=S), 1,163 (C–O). H NMR (CDCl3, d
ppm): 3.80 (s, 3H, (O)–C–H), 6.05 (s, 1H, (NH)–C–H), 6.
86–7.26 (m, 4H, Ar–H), 7.63–8.05 (m, 4H, Ar–H), 11.49
(s, 2H, NH). 13C NMR (DMSO-d6, d ppm): 34.95, 54.79,
123

2572
Med Chem Res (2014) 23:2564–2575
4-(3-Bromo)phenyl-2-thioxo-3,4-dihydro-1H-chromino-[4,3-
d]pyrimidin-5-one (4g) IR (KBr, cm-1): 3,069 (N–H),
2,719 (aromatic C–H stretch), 1,664 (C=O), 1,564 (aro-
4-(4-Methoxy)phenyl-9-methyl-3,4-dihydro-1H-chromi-
no[4,3-d]pyrimidine-2,5-dione (5d) IR (KBr, cm-1):
3,260 (N–H), 2,930 (aromatic C–H stretch), 1,624 (C=O),
1,575 (aromatic C=C bending), 1,149 (C–O). 1H NMR
(DMSO-d6, d ppm): 2.45 (s, 3H, (Ar)–C–H), 3.79 (s, 3H,
(O)–C–H), 6.02 (s, 1H, (NH)–C–H), 6.85–7.29 (m, 4H,
Ar–C–H), 7.41 (s, 1H, Ar–C–H), 7.78–7.83 (m, 2H, Ar–C–
H), 11.33–11.55 (s, 2H, NH). 13C NMR (DMSO-d6, d
ppm): 20.45, 54.97, 104.48, 113.60, 115.87, 116.91, 123.
43, 127.74, 130.57, 133.02, 133.28, 150.22, 157.48, 164.
38, 165.11.
1
matic C=C bending), 1,267 (C=S), 763 (C–Br). H NMR
(CDCl3, d ppm): 6.06 (s, 1H, (NH)–C–H), 7.17–7.34 (m,
4H, Ar–C–H), 7.64–8.05 (m, 4H, Ar–C–H), 11.29–11.57
(s, 2H, NH). 13C NMR (DMSO-d6, d ppm): 35.55, 103.84,
116.00, 116.83, 121.83, 123.83, 123.92, 125.63, 128.82,
129.23, 129.95, 132.15, 141.40, 152.06, 164.43, 165.13.
4-(3-Bromo)phenyl-9-methyl-2-thioxo-3,4-dihydro-1H-chr-
omino[4,3-d]pyrimidin-5-one (4h) IR (KBr, cm-1):
3,412 (N–H), 2,918 (aromatic C–H stretch), 1,658 (C=O),
1,577 (aromatic C=C bending), 1,269 (C=S), 785 (C–Br).
1H NMR (DMSO-d6, d ppm): 2.77 (s, 3H, (Ar)–C–H), 6.40
(s, 1H, (NH)–C–H), 6.67–6.86 (m, 4H, Ar–C–H), 7.20–7.
43 (m, 3H, Ar–C–H), 8.06 (s, 2H, NH). 13C NMR (DMSO-
d6, d ppm): 20.44, 45.13, 104.90, 115.24, 115.82, 121.13,
122.85, 126.80, 129.47, 129.75, 130.61, 132.81, 142.83,
150.23, 160.74, 161.35.
4-(3,4-Dimethoxy)phenyl-9-methyl-3,4-dihydro-1H-chromi-
no[4,3-d]pyrimidine-2,5-dione (5e) IR (KBr, cm-1):
3,280 (N–H), 2,930 (aromatic C–H stretch), 1,664 (C=O),
1,564 (aromatic C=C bending), 956 (C–Br). 1H NMR
(DMSO-d6, d ppm): 2.44 (s, 3H, (Ar)–C–H), 3.72 (s, 3H,
(O)–C–H), 3.86 (s, 3H, (O)–C–H), 6.04 (s, 1H, (NH)–C–
H), 6.68–6.78 (m, 2H, Ar–C–H), 7.30–7.41 (s, 2H, Ar–C–
H), 7.77–7.83 (m, 2H, Ar–C–H), 11.32–11.56 (s, 2H, NH).
13C NMR (DMSO-d6, d ppm): 20.51, 55.39, 55.57, 104.43,
110.95, 111.36, 115.78, 116.49, 118.69, 123.34, 130.45,
132.97, 133.27, 147.24, 148.56, 150.16, 164.01, 165.32.
4-Phenyl-3,4-dihydro-1H-chromino[4,3-d]pyrimidine-2,5-
dione (5a) IR (KBr, cm-1): 3,435 (N–H), 3,069 (aro-
matic C–H stretch), 1,658 (C=O), 1,566 (aromatic C=C
bending). 1H NMR (CDCl3, d ppm): 6.06 (s, 1H, (NH)–C–
H), 7.18–7.34 (m, 5H, Ar–C–H), 7.41–7.64 (m, 2H, Ar–C–
H), 8.02–8.06 (m, 2H, Ar–C–H), 11.29 (s, 1H, NH), 11.56
(s, 1H, NH). 13C NMR (DMSO-d6, d ppm): 35.69, 104.22,
115.93, 117.03, 123.80, 123.84, 125.76, 126.50, 127.71,
128.04, 129.29, 132.00, 138.38, 152.04, 162.97, 164.42,
165.23.
4-(4-Hydroxy-3-methoxy)phenyl-3,4-dihydro-1H-chromi-
no[4,3-d]pyrimidine-2,5-dione (5f) IR (KBr, cm-1):
3,497 (O–H), 3,069 (N–H), 2,939 (aromatic C–H stretch),
1,668 (C=O), 1,564 (aromatic C=C bending), 1,151(C–O).
1H NMR (CDCl3, d ppm): 3.74 (s, 1H, O–H), 5.60 (s, 3H,
(O)–C–H), 6.06 (s, 1H, (NH)–C–H), 6.68–6.85 (m, 3H,
Ar–C–H), 7.62–8.03 (m, 4H, Ar–C–H), 11.29–11.52 (s,
2H, NH). 13C NMR (DMSO-d6, d ppm): 35.59, 55.86, 104.
74, 111.75, 115.29, 116.10, 117.35, 119.23, 123.79, 123.
99, 129.65, 132.11, 144.98, 147.52, 152.10, 164.33, 164.
84.
4-(4-Methoxy)phenyl-3,4-dihydro-1H-chromino[4,3-d]pyrim-
idine-2,5-dione (5b) IR (KBr, cm-1): 3,069 (N–H), 2,835
(aromatic C–H stretch), 1,670 (C=O), 1,564 (aromatic C=C
1
bending), 1,178 (C–O). H NMR (CDCl3, d ppm): 3.80 (s,
4-(2-Hydroxy)phenyl-3,4-dihydro-1H-chromino[4,3-d]pyrim-
idine-2,5-dione (5g) IR (KBr, cm-1): 3,393 (O–H), 3,065
(N–H), 1,672 (C=O), 1,560 (aromatic C=C bending), 1,105
3H, (O)–C–H), 6.05 (s, 1H, (NH)–C–H), 6.87–7.41 (m, 4H,
Ar–C–H), 7.63–8.05 (m, 4H, Ar–C–H), 11.31–11.52 (s,
2H, NH). 13C NMR (DMSO-d6, d ppm): 35.19, 54.92, 104.
54, 113.57, 116.04, 117.30, 123.79, 123.95, 127.77, 130.
66, 132.11, 152.06, 157.44, 164.45, 164.90.
1
(C–O). H NMR (CDCl3, d ppm): 3.74 (s, 1H, O–H), 5.37
(s, 1H, (NH)–C–H), 7.12–7.30 (m, 3H, Ar–C–H), 7.42–7.
62 (m, 4H, Ar–C–H), 8.02–8.14 (s, 1H, Ar–C–H), 10.41 (s,
2H, NH). 13C NMR (DMSO-d6, d ppm): 48.65, 113.83,
116.02, 116.13, 116.29, 122.20, 122.50, 123.64, 124.23,
125.09, 128.09, 128.57, 131.82, 132.15, 151.98, 152.21,
160.40.
4-(3,4-Dimethoxy)phenyl-3,4-dihydro-1H-chromino[4,3-d]
pyrimidine-2,5-dione (5c) IR (KBr, cm-1): 3,461 (N–H),
2,837 (aromatic C–H stretch), 1,664 (C=O), 1,564 (aro-
1
matic C=C bending), 1,140 (C–O). H NMR (CDCl3, d
ppm): 3.73 (s, 3H, (O)–C–H), 3.87 (s, 3H, (O)–C–H), 6.08
(s, 1H, (NH)–C–H), 6.71–7.80 (m, 3H, Ar–C–H), 7.63–8.
06 (m, 4H, Ar–C–H), 11.31–11.53 (s, 2H, NH). 13C NMR
(DMSO-d6, d ppm): 35.36, 55.40, 55.58, 104.47, 111.01,
111.35, 115.94, 116.98, 118.73, 123.73, 123.85, 130.63,
131.97, 147.23, 148.55, 152.02, 164.22, 165.13.
4-(3-Bromo)phenyl-3,4-dihydro-1H-chromino[4,3-d]pyrim-
idine-2,5-dione (5h) IR (KBr, cm-1): 3,280 (N–H), 2,930
(aromatic C–H stretch), 1,670 (C=O), 1,510 (aromatic C=C
1
bending), 1,163 (C–O), 956 (C–Br). H NMR (CDCl3, d
ppm): 6.06 (s, 1H, (NH)–C–H), 7.18–7.34 (m, 4H, Ar–C–
H), 7.64–8.06 (m, 4H, Ar–C–H), 11.29–11.56 (s, 2H, NH).
123

Med Chem Res (2014) 23:2564–2575
2573
13C NMR (DMSO-d6, d ppm): 35.57, 103.84, 116.00, 116.
86, 121.82, 123.83, 123.91, 125.66, 128.81, 129.24, 129.
97, 132.15, 141.49, 152.06, 164.45, 165.10.
(0.22 mm pore size) and stored at -80 °C. Alamar blue
solution was purchased from Alamar Biosciences/Accumed,
Westlake, OH, USA. Black, clear-bottomed, 96-well micro-
plates (black view plates) were purchased from Packard
Instrument Company, Meriden, CT, USA, in order to mini-
mizebackgroundfluorescence. Fluorescencewasmeasuredin
a Cytofluor II microplate fluorometer purchased from
Perseptive Biosystems, Framingham, MA, USA.
Biological evaluation
Two methods were used for in vitro evaluation of anti-M.
tuberculosis activity viz. (a) microplate Alamar blue assay
(MABA) (Bartizal et al., 1997; Scott et al., 2007) and
(b) luminescence-based low oxygen-recovery assay
(LORA) (Scott et al., 2007).
Alamar blue susceptibility test (MABA) Antimicrobial
susceptibility testing was performed in black, clear-bot-
tomed, 96-well microplates in order to minimize back-
ground fluorescence. Outer perimeter wells were filled with
sterile water to prevent dehydration in experimental wells.
Initial drug dilutions were prepared in either dimethyl
sulfoxide or distilled deionized water and subsequent two-
fold dilutions were performed in 0.1 ml of 7H9GC (no
Tween 80) in the microplates. BACTEC 12B-passaged
innocula were initially diluted 1:2 in 7H9GC and 0.1 ml
was added to the wells. Frozen innocula were initially
diluted 1:20 in BACTEC 12B medium followed by a 1:50
dilution in 7H9GC. Wells containing drug only were used to
detect auto-fluorescence of compounds. Additional control
wells consisted of bacteria only (B) and medium only (M).
Plates were incubated at 37 °C. Starting at day 4 of incu-
bation, 20 ml of Alamar Blue solution and 12.5 ml of 20 %
Tween 80 were added to one B well and one M well, and
plates were re-incubated at 37 °C. Wells were observed at
12 and 24 h for a color change from blue to pink and for a
reading of 50,000 fluorescence units (FU). Fluorescence
was measured in a Cytofluor II microplate fluorimeter in
bottom-reading mode with excitation at 530 nm and emis-
sion at 590 nm. If the B wells became pink by 24 h, reagent
was added to the entire plate. If the well remained blue or
50,000 FU was measured, additional M and B wells were
tested daily until a color change occurred, at which time
reagents were added to all remaining wells. Plates were then
incubated at 37 °C, and results were recorded at 24-h post-
reagent addition. Visual MICs were defined as the lowest
concentration of drug that prevented a color change. For
fluorometric MICs, a background subtraction was per-
formed on all wells with a mean of triplicate micro-wells.
Percent inhibition defined as the lowest drug concentration
effecting an inhibition of 90 % was considered as MIC.
Microplate Alamar blue assay (MABA)
MABA is a sensitive, rapid, inexpensive, and non-radio-
metric method that offers the potential for screening, with or
without analytical instrumentation, a large number of anti-
microbial compounds against slow-growing mycobacteria.
The Alamar blue oxidation–reduction dye is a general
indicator of cellular growth and/or viability; the blue, non-
fluorescent, oxidized form becomes pink and fluorescent
upon reduction by the reducing power of living cells to
convert resazurin to the fluorescent molecule, resorufin.
Growth can therefore be measured with a spectrophotom-
eter or determined by a visual color change thereby gen-
erating a quantitative measure of viability and cytotoxicity.
Materials and methods
Bacterial strains and growth conditions Mycobacterium
tuberculosis H37Rv ATCC 27294 (H37Rv) was obtained
from the American Type Culture Collection (Rockville,
MD, USA). H37Rv innocula was first passaged in radio-
metric 7H12 broth (BACTEC 12B; Becton Dickinson
Diagnostic Instrument Systems, Sparks, NV, USA) until
the GI reached 800–999.
H37Rv was grown in 100 ml Middlebrook 7H9 broth
(Difco, Detroit, MI, USA) supplemented with 0.2 % (v/v)
glycerol (Sigma Chemical Co., Saint Louis, MO, USA),
10 % (v/v) oleic acid, albumin, dextrose, catalase (OADC;
Difco), and 0.05 % (v/v) Tween 80 (Sigma, USA). The
complete medium is referred to as 7H9GC-Tween. The
culture was incubated in 500-ml nephelometer flasks on a
rotary shaker (New Brunswick Scientific, Edison, NJ,
USA) at 150 rpm and 37 °C until it reached an optical
density of 0.4–0.5 at 550 nm. Bacteria were washed and
suspended in 20 ml of phosphate-buffered saline and pas-
sed through 8-mm-pore-size filter to eliminate clumps.
Luminescence-based LORA
Screening for new antimicrobial agents is routinely con-
ducted only against actively replicating bacteria. However,
it is now widely accepted that a physiological state of NRP
is responsible for antimicrobial tolerance in many bacterial
infections. In tuberculosis, the key to shortening the
6-month regimen lies in targeting this NRP subpopulation.
Chemicals and media Rifampicin (RMP) and Clofaz-
amine (CLF) were purchased from Sigma Chem. Co., USA.
They were solubilized according to the manufacturer’s rec-
ommendations and stock solutions were filter sterilized
123

2574
Med Chem Res (2014) 23:2564–2575
Therefore, a high-throughput, luminescence-based LORA
was developed to screen antimicrobial agents against NRP
M. tuberculosis.
oxygen. The number of CFU was estimated by plating
dilutions of aliquots on Dubos oleic-albumin agar plates in
triplicate and incubating the cultures at 37 °C. The colonies
were enumerated every week for 5 weeks. The cells were
harvested at the 22nd day, a time when the A570 and DOC
readings indicated achievement of the desired growth
phase (NRP-2). 50 ml aliquots of bacterial culture samples
were centrifuged (2,7009g, 30 min, 4 °C), washed once
with pre chilled phosphate-buffered saline (PBS; pH 7.4),
suspended in 1 ml of PBS, and stored at -80 °C.
Materials and methods
Construction of plasmid luxAB The recombinant shuttle
vector pFCA-luxAB was constructed by inserting the luxAB
gene from pSMT1 into pFPCA1, which uses the acetamidase
promoter to provide an enhanced signal. The luxAB genes
were derived by PCR amplification with primers P1 (TAG
GATCCTAAGAAAGATGAAATTTGGAAACTTCC) and
P2 (TTCTTTAAATTACGAGTGGTATTTGACGATGTT
GG). The amplified PCR product was cloned into the pGEM-
T easy vector (Promega). After an Escherichia coli clone
containing pGEMT-luxAB was obtained, the isolated plas-
mid was extracted and digested with BamHI and EcoRI.
Following the ligation reaction with pFPCA1, the resulting
plasmid, pFCA-luxAB, was used to transform E. coli. The
plasmid was isolated, and the structure was confirmed by
restriction enzyme digestion and sequencing analysis.
In vitro LORA and conventional aerobic culture assay
Prior to use, the cultures were thawed, diluted in Middle
brook 7H12 broth (Middle brook 7H9 broth containing
1.0 mg/ml casitone, 5.6 g/ml palmitic acid, 5.0 mg/ml
bovine serum albumin, and 4.0 g/ml filter-sterilized cata-
lase), and sonicated for 15 s. For LORA, the microplate
cultures were placed under anaerobic conditions (oxygen
concentration, \0.16 %) using an Anoxomat model
WS-8080 (MART Microbiology) and three cycles of evacu-
ation and filling with a mixture of 10 % H2, 5 % CO2, and the
balance N2. An anaerobic indicator strip was placed inside the
chamber to visually confirm the removal of oxygen. The
plates were incubated at 37 °C for 10 days and then trans-
ferred to an ambient gaseous condition (5 % CO2-enriched
air) incubator for a 28 h ‘‘recovery.’’ The numbers of CFU
(determined by subculture onto Middle brook 7H11 agar)
during the 10-day incubation did not increase and remained
essentially unchanged. On day 11 (after the 28 h aerobic
recovery), 100-l culture was transferred to white 96-well
microtiter plates for determination of luminescence. For the
conventional assay, the microplate cultures were placed in an
incubator under ambient gaseous conditions (5 % CO2 enri-
ched air) for 7 days and 100-l culture was transferred to white
96-well microtiter plates for determination of luminescence. A
10 % solution of n-decanal aldehyde (Sigma) in ethanol was
freshly diluted tenfold in PBS, and 100 l was added to each
well with an auto-injector. Luminescence was measured in a
Victor2 multi label reader (Perkin-Elmer Life Sciences) using
a reading time of 1 s. The MIC defined as the lowest drug
concentration effecting growth inhibition of 90 % relative to
the growth for the drug-free controls.
Electroporation of plasmid pFCA-luxAB into M. tubercu-
losis H37Rv ATCC 27294 Mycobacterium tuberculosis
H37Rv ATCC 27294 was obtained from the American
Type Culture Collection (Manassas, VA, USA). After
5–7 days of culture in 200 ml Middlebrook 7H9 medium
supplemented with oleic acid–albumin–dextrose–catalase,
the cells were washed and transformed by mixing at least
1.0 g of purified plasmid and incubating at room tempera-
ture for 30 min, followed by electroporation. The transfor-
mants were cultured on Middlebrook 7H11 agar containing
20 g/ml kanamycin for 4 weeks. Selected colonies were
transferred to 100 l of Middlebrook 7H9 broth and sonicated
at 30 W for 20 s. (model S3000; Misonix Inc.) at ambient
temperature prior to the measurement of the luminescence.
Growth conditions For the fermenter culture, recombi-
nant H37Rv (pFCAluxAB) was grown to NRP phase 2
(NRP-2) in 300 ml of Dubos Tween albumin broth (Becton
Dickinson) in a BioStat Q fermenter (B. Braun Biotech) to
mimic the Wayne oxygen-limited culture with a headspace
ratio (HSR) of 0.5 and agitated at a stir rate of 120 rpm
with no detectable perturbation of the surface of the med-
ium, as described previously. The fermenter culture was
operated and maintained within a bio-safety level-3 labo-
ratory. The dissolved oxygen concentration (DOC) was
continuously monitored with an In-gold oxygen sensor
probe. The optical densities of the cultures at 570 nm
(A570 values), the numbers of relative light units (RLUs)
and the CFU levels were determined at 3-day intervals.
Bacterial samples were removed through a silicone septum
with a syringe in order to preclude the introduction of
Computational studies
RP analysis was carried out using the CSAR RP method
and the decision tree was generated from the results of RP.
Cerius2 molecular modeling software was used to calculate
the 2D descriptors for the molecules. The correlation
matrices were built for the descriptors and those with zero
variance as well as containing 95 % of zero values were
eliminated. These remaining descriptors formed the inde-
pendent variables (X), while the biological activity/toxicity
123

Med Chem Res (2014) 23:2564–2575
2575
Kidwai M, Saxena S, Mohan R (2006) Environmentally benign
synthesis of benzopyranopyrimidines. Russ J Org Chem 42:52.
doi:10.1134/s107042800601009x
Krajewski WW, Jones TA, Mowbray SL (2005) Structure of
Mycobacterium tuberculosis glutamine synthetase in complex
with a transition-state mimic provides functional insights. Proc
Natl Acad Sci USA 102:10499–10504
Manvar A, Malde A, Verma J, Virsodia V, Mishra A, Upadhyay K,
Acharya H, Coutinho E, Shah A (2008) Synthesis, anti-
tubercular activity and 3D-QSAR study of coumarin-4-acetic
acid benzylidene hydrazides. Eur J Med Chem 43:2395
Manvar AT, Pissurlenkar RR, Virsodia VR, Upadhyay KD, Manvar
DR, Mishra AK, Acharya HD, Parecha AR, Dholakia CD, Shah
AK, Coutinho EC (2010) Synthesis, in vitro antitubercular
activity and 3D-QSAR study of 1,4-dihydropyridines. Mol
Divers 14:285. doi:10.1007/s11030-009-9162-8
served as the dependent variable (Y) for the analysis. The
CARTTM (classification and regression trees) method was
applied to generate the decision tree classification model.
The active and inactive classes (toxic and less toxic for the
toxicity model) were given equal weight and the splits were
scored using Twoing rule scoring function. The pruning
factor was varied between 0 and 3. Maximum tree depth
(layers \10) was also varied between 5 and 10, while the
default values were set for maximum number of generic
splits (30) and the number of knots per variable (20). The
decision tree model was internally validated by cross-val-
idation with the number of cross-validation groups set to
10.
Marinis E, Legakis NJ (1985) In-vitro activity of ciprofloxacin against
clinical isolates of mycobacteria resistant to antimycobacterial
drugs. J Antimicrob Chemother 16:527
McKee TC, Covington CD, Fuller RW, Bokesch HR, Young S,
Cardellina IJ, Kadushin MR, Soejarto DD, Stevens PF, Cragg
GM, Boyd MR (1998) Pyranocoumarins from tropical species of
the genus Calophyllum: a chemotaxonomic study of extracts in
the National Cancer Institute collection. J Nat Prod 61:1252.
doi:10.1021/np980140anp980140a
Acknowledgments The computational, synthetic, and evaluation
studies were possible due to the grants received from the All India
Council for Technical Education (F. nos. 8022/RID/NPROJ/RPS-5/
2003-04 and 8023/BOR/RID/RPS-181/2008-09), Department of
Biotechnology (File no. BT/PR11810/BRB/10/690/2009), Council for
Scientific Research (File no. 01/2399/10/EMRII) New Delhi and
University of Mumbai (F. no. APD/237/157/2010).
Miyakawa Y, Ratnakar P, Rao AG, Costello ML, Mathieu-Costello O,
Lehrer RI, Catanzaro
A (1996) In vitro activity of the
References
antimicrobial peptides human and rabbit defensins and porcine
leukocyte protegrin against Mycobacterium tuberculosis. Infect
Immun 64:926
Amaral L, Kristiansen JE, Viveiros M, Atouguia J (2001) Activity of
phenothiazines against antibiotic-resistant Mycobacterium tuber-
culosis: a review supporting further studies that may elucidate
the potential use of thioridazine as anti-tuberculosis therapy.
J Antimicrob Chemother 47:505
Barry VC, Belton JG, Conalty ML, Denneny JM, Edward DW,
O’Sullivan JF, Twomey D, Winder F (1957) A new series of
phenazines (rimino-compounds) with high antituberculosis
activity. Nature 179:1013
Bartizal K, Gill CJ, Abruzzo GK, Flattery AM, Kong L, Scott PM,
Smith JG, Leighton CE, Bouffard A, Dropinski JF, Balkovec J
(1997) In vitro preclinical evaluation studies with the echino-
candin antifungal MK-0991 (L-743,872). Antimicrob Agents
Chemother 41:2326
Rusinko A III, Young SS, Drewry DH, Gerritz SW (2002) Optimi-
zation of focused chemical libraries using recursive partitioning.
Comb Chem High Throughput Screen 5:125
Ryabukhin SV, Plaskon AS, Boron SY, Volochnyuk DM, Tolmachev
AA (2011) Aminoheterocycles as synthons for combinatorial
Biginelli reactions. Mol Divers. doi:10.1007/s11030-010-9253-6
Scott GM, Weinberg A, Rawlinson WD, Chou S (2007) Multidrug
resistance conferred by novel DNA polymerase mutations in
human cytomegalovirus isolates. Antimicrob Agents Chemother
51:89. doi:AAC.00633-06
Shiradkar M, Suresh Kumar GV, Dasari V, Tatikonda S, Akula KC,
Shah R (2007) Clubbed triazoles: a novel approach to antitu-
bercular drugs. Eur J Med Chem 42:807
Blower P, Fligner M, Verducci J, Bjoraker J (2002) On combining
recursive partitioning and simulated annealing to detect groups of
biologically active compounds. J Chem Inf Comput Sci 42:393
Choi SY, Shin JH, Ryu CK, Nam KY, No KT, Park Choo HY (2006)
The development of 3D-QSAR study and recursive partitioning
of heterocyclic quinone derivatives with antifungal activity.
Bioorg Med Chem 14:1608
Spino C, Dodier M, Sotheeswaran S (1998) Anti-HIV coumarins from
Calophyllum seed oil. Bioorg Med Chem Lett 8:3475
Stover CK, Warrener P, VanDevanter DR, Sherman DR, Arain TM,
Langhorne MH, Anderson SW, Towell JA, Yuan Y, McMurray
DN, Kreiswirth BN, Barry CE, Baker WR (2000) A small-
molecule nitroimidazopyran drug candidate for the treatment of
tuberculosis. Nature 405:962. doi:10.1038/35016103
Cohen FL, Tartasky D (1997) Microbial resistance to drug therapy: a
review. Am J Infect Control 25:51
Desai B, Sureja D, Naliapara Y, Shah A, Saxena AK (2001) Synthesis
and QSAR studies of 4-substituted phenyl-2,6-dimethyl-3, 5-bis-
N-(substituted phenyl)carbamoyl-1,4-dihydropyridines as poten-
tial antitubercular agents. Bioorg Med Chem 9:1993
Garcia-Garcia A, Galvez J, de Julian-Ortiz JV, Garcia-Domenech R,
Munoz C, Guna R, Borras R (2005) Search of chemical scaffolds
for novel antituberculosis agents. J Biomol Screen 10:206
Jarvis B, Lamb HM (1998) Rifapentine. Drugs 56:607
Jindani A, Aber VR, Edwards EA, Mitchison DA (1980) The early
bactericidal activity of drugs in patients with pulmonary
tuberculosis. Am Rev Respir Dis 121:939
Kawakami K, Namba K, Tanaka M, Matsuhashi N, Sato K, Takemura
M (2000) Antimycobacterial activities of novel levofloxacin
analogues. Antimicrob Agents Chemother 44:2126
Strobl C, Malley J, Tutz G (2009) An introduction to recursive
partitioning: rationale, application, and characteristics of classi-
fication and regression trees, bagging, and random forests.
Psychol Methods 14:323
Virsdoia V, Shaikh MS, Manvar A, Desai B, Parecha A, Loriya R,
Dholariya K, Patel G, Vora V, Upadhyay K (2010) Screening for
in vitro antimycobacterial activity and three dimensional quanti-
tative structure–activity relationship (3D QSAR) Study of 4
(arylamino) coumarin derivatives. Chem Bio Drug Design 76:412
Xu Z-Q, Barrow WW, Suling WJ, Westbrook L, Barrow E, Lin Y-M,
Flavin MT (2004) Anti-HIV natural product (?)-calanolide A is
active against both drug-susceptible and drug-resistant strains of
Mycobacterium tuberculosis. Bioorg Med Chem 12:1199
Young SS, Hawkins DM (2004) Using recursive partitioning analysis
to evaluate compound selection methods. Methods Mol Biol
275:317
123