Synthesis, biological evaluation and structure-activity relationship of 2-styrylquinazolones as anti-tubercular agents

Article abstract of DOI:10.1016/j.bmcl.2016.04.012

2-Styrylquinazolones are reported as a novel class of potent anti-mycobacterial agents. Forty-six target compounds have been synthesized using one pot reaction involving isatoic anhydride, amine, and triethyl orthoacetate followed by aldehyde to construct

Full text of DOI:10.1016/j.bmcl.2016.04.012

Accepted Manuscript
Synthesis, Biological Evaluation and Structure-Activity Relationship of 2-Styr-
ylquinazolones as Anti-tubercular Agents
Pradeep S. Jadhavar, Tejas M. Dhameliya, Maulikkumar D. Vaja, Dinesh
Kumar, Jonnalagadda Padma Sridevi, Perumal Yogeeswari, Dharmarajan
Sriram, Asit K. Chakraborti
PII:
S0960-894X(16)30373-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.04.012
BMCL 23774
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 February 2016
1 April 2016
6 April 2016
Please cite this article as: Jadhavar, P.S., Dhameliya, T.M., Vaja, M.D., Kumar, D., Sridevi, J.P., Yogeeswari, P.,
Sriram, D., Chakraborti, A.K., Synthesis, Biological Evaluation and Structure-Activity Relationship of 2-
Styrylquinazolones as Anti-tubercular Agents, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.04.012
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Graphical Abstract
Synthesis, Biological Evaluation and
Structure-Activity Relationship of 2-
Styrylquinazolones as Anti-tubercular
Agents
Leave this area blank for abstract info.
Pradeep S. Jadhavar,a Tejas M. Dhameliya,a Maulikkumar D. Vaja,a Dinesh Kumar,a Jonnalagadda Padma
Sridevi,b Perumal Yogeeswari,b Dharmarajan Sriramb and Asit K. Chakrabortia†
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER),
Sector 67, S. A. S. Nagar 160 062, Punjab, India.
bDepartment of Pharmacy, Birla Institute of Technology & Science – Pilani, Hyderabad Campus, Jawahar
Nagar, Hyderabad 500 078, India.

Bioorganic & Medicinal Chemistry Letters
Synthesis, Biological Evaluation and Structure-Activity Relationship of 2-
Styrylquinazolones as Anti-tubercular Agents
Pradeep S. Jadhavar,a Tejas M. Dhameliya,a Maulikkumar D. Vaja,a Dinesh Kumar,a Jonnalagadda Padma
Sridevi,b Perumal Yogeeswari,b Dharmarajan Sriram,b and Asit K. Chakrabortia*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S. A. S. Nagar, Punjab 160 062, India
bDepartment of Pharmacy, Birla Institute of Technology & Science – Pilani, Hyderabad Campus, Jawahar Nagar, Hyderabad 500 078, India.
*Corresponding author’s E.mail:akchakraborti@niper.ac.in; akchakraborti@rediffmail.com
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
2-Styrylquinazolones are reported as a novel class of potent anti-mycobacterial agents. Forty-six
target compounds have been synthesized using one pot reaction involving isatoic anhydride,
amine, and triethyl orthoacetate followed by aldehyde to construct the 2-styrylquinazolone
scaffold. The anti-mycobacterial potency of the compounds was determined
against H37Rv strain. Twenty-six compounds exhibited anti-Mtb activity in the range of 0.40-
6.25 µg/mL. Three compounds 8c, 8d and 8ab showed MIC of 0.78 µg/mL and were found to
be non-toxic (< 50% inhibition at 50 µg/mL) to HEK 293T cell lines with the therapeutic index
>64. The most potent compound 8ar showed MIC of 0.40 µg/mL with the therapeutic index
>125.An early structure activity relationship for this class of compounds has been established.
The computational studies indicate the possibility of these compounds binding to the penicillin
binding proteins (PBPs).
Keywords:
Tuberculosis
2-Styrylquinazolone
Anti-TB agents
H37Rv strain
SAR
2009 Elsevier Ltd. All rights reserved.
Human tuberculosis (TB) continues to be a major cause of
death every year, and the emergence of drug resistant strains of
Mycobacterium tuberculosis (Mtb) has reincarnated the demand
to discover and develop new drugs to combat with the deadly
pathogenic mycobacteria.1 The current year 2015 marks the 22nd
anniversary of the declaration of tuberculosis as a global health
emergency by WHO (World Health Organization).2 Despite
enormous efforts have been made in the hunt for new drugs, TB
still remains the first bacterial cause of mortality worldwide
causing an estimated 9.0 million new cases in 2013 and 1.5
million death, 360 000 of whom were HIV-positive.3 The first
line drugs such as isoniazid (INH), rifampin (R), pyrazinamide
(Z), streptomycin (S) and ethambutol (E) are used to treat active
TB and latent tuberculosis infection (LTBI) these suffer from one
or more serious side effects.4 For the treatment of drug-sensitive
TB, initially patients are treated with first-line four drugs (INH,
R, Z and E) for 2 months followed by 4 months of INH plus R
through directly observed treatment short course strategy
(DOTS) possessing a cure rate of >95%. The treatment of drug-
resistant TB requires 18-24 months or longer, involving the use
of more toxic and costly second-line medicines such as
ciprofloxacin (Cfx), para-amino salicylic acid (Pas), kanamycin
(Km), cycloserine (Dcs), ethionamide (Eto), amikacin (Amk),
capreomycin (Cm), thioacetazone (Thz).5 Notably, after 40 years
a new chemical entity, bedaquiline, has been approved by the
U.S. Food and Drug Administration (FDA) with the name Sirturo
in the end of 2012,6 and by the European Medicine Agency
(EMA) in 2014 for the treatment of MDR-TB patients.7 The
emergence of multi-drug resistant (MDR) and extensively drug-
resistant (XDR) strains of Mtb insists the need for new therapies,
which might have novel mechanism of action.8,9 Furthermore, the
resurgence in TB is alarming due to the development of
pathogenic synergy with HIV.5 Thus the discovery and
development of new anti-Mtb molecules continues to be the
perpetual interest to academia and pharma industry to tackle the
TB pandemic.
In continuation of our efforts to search for new anti-Mtb
scaffolds10 we were attracted by the findings on the identification
of the styrylquinazolones 1 and 2 (Figure 1) as the potent anti-
Mtb agents11 with high selectivity index (SI) under Tuberculosis
Antimicrobial Acquisition and Coordinating Facility (TAACF)
Program that encouraged us to synthesize various 2-
styrylquinazolones and evaluate their anti-Mtb potential to
establish the structure activity relationship (SAR).
Figure 1. Literature reported styryl quinazolones having anti-tubercular and
anti-bacterial activity.
In the present study we planned to explore the 2-
styrylquinazolones having the general structure as represented in
Figure 2, for evaluating their anti-Mtb potential. The goal of this

21
22
Ar1 = 4-OMe-C6H4, Ar2 = 4-OMe-C6H4
Ar1 = 4-Me-Ph-C6H4, Ar2 = 4-Cl-C6H4
Ar1 = 4-SMe-C6H4, Ar2 = 4-OCOCH3-
C6H4
Ar1 = 4-SMe-C6H4, Ar2 = 2-Furanyl
Ar1 = 2,3,4-tri-OMe-C6H4, Ar2 = 2-
Furanyl
60
67
8u
8v
study was to synthesize the diverse library of 2-
styrylquinazolones through variations in the C and D ring and to
establish the early SAR for this class of compounds.
23
24
25
71
75
74
8w
8x
8y
Ar1 = 2,3,4-tri-OMe-C6H4, Ar2 = 2-
26
75
8z
Figure 2.Designed 2-styrylquinazolones.
thiazolyl
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Ar1 = Ph, Ar2 = 4-SMe-C6H4
Ar1 = Ph, Ar2 = 2,3,4-tri-OMe-C6H4
Ar1 = 4-OMe-C6H4, Ar2 = Ph
Ar1 = 4-OMe-C6H4, Ar2 = 4-Cl-C6H4
Ar1 = 4-OMe-C6H4, Ar2 = 2-thiazolyl
Ar1 = 4-OMe-C6H4, Ar2 = 2-Furanyl
Ar1 = 4-Me-C6H4, Ar2 = 4-OMe-C6H4
Ar1 = Ph, Ar2 = 2-thiazolyl
68
61
70
75
74
71
69
70
66
64
66
64
68
71
66
61
64
8aa
8ab
8ac
8ad
8ae
8af
During the progress of this work we were delighted to
observe that the styrylquinazolone
3 (Figure 1) has been
found to be potent antibacterial antibiotic.12
Recently we reported convenient synthesis of 2-
a
styrylquinazolones 8 through one-pot reaction involving isatoic
anhydride 4, aryl amine 5 and triethyl orthoacetate 6 at 120 0C to
form the intermediate 2-methyl-3-aryl-quinazolone followed by
treatment with aromatic aldehydes 7 (Scheme 1) to afford the
desired products.13
8ag
8ah
8ai
Ar1 = Ph, Ar2 = 2-Furanyl
Ar1 = 4-Cl-C6H4, Ar2 = Ph
Ar1 = 4-Cl-C6H4, Ar2 = 4-Cl-C6H4
Ar1 = 4-F-C6H4, Ar2 = Ph
Ar1 = 4-F-C6H4, Ar2 = 4-Cl-C6H4
Ar1 = 4-Br-C6H4, Ar2 = Ph
8aj
8ak
8al
Scheme 1. Synthesis of 2-styryl quinazolones (8).
8am
8an
8ao
8ap
8aq
The forty-six target compounds 8 having various substitutions
(electron donating/withdrawing) on C and D ring were
synthesized in the 54-79% yields (Table 1) following this
reported procedure.
Ar1 = 4-Br-C6H4, Ar2 = 4-F-C6H4
Ar1 = 4-F-C6H4, Ar2 = 4-Me- C6H4
Ar1 = 4-F-C6H4, Ar2 = 4-F-C6H4
Ar1 = 2,3,4-tri-OMe-C6H4, Ar2 = 4-SMe-
C6H4
Table 1. One pot synthesis of 2-stryrylquinazolones (8)a
Compd
No.
8a
Yieldb
(%)
65
44
63
8ar
Entry
2-Styrylquinazolones (8)
Ar1 = 4-SMe-C6H4, Ar2 = 2,3,4-tri-OMe-
1
2
3
4
5
6
Ar1 = Ph, Ar2 = Ph
Ar1 = 4-Me-C6H4, Ar2 = Ph
Ar1 = 4-SMe-C6H4, Ar2 = Ph
Ar1 = 2,3,4-tri-OMe-C6H4, Ar2 = Ph
Ar1 = Ph, Ar2 = 4-F-C6H4
Ar1 = 4-Me-C6H4, Ar2 = 4-F-C6H4
Ar1 = 2,3,4-tri-OMe-C6H4, Ar2 = 4-F-
C6H4
45
46
69
67
8as
8at
C6H4
72
8b
Ar1 = 4-Me-C6H4, Ar2 = 4-Me-C6H4
79
8c
aThe mixture of isatoic anhydride 4 (2.5 mmol), amine/NH4OAc (2.5 mmol)
and triethyl orthoacetate 6 (2.5 mmol) was heated under neat condition at 120
ºC for 5 h followed by addition of aldehyde 7 (2.5 mmol, 1 equiv) and
continued stirring for further 5 h.
66
8d
74
8e
68
8f
bIsolated yield of the 2-styrylquinazolone (8).
7
79
8g
The forty-six synthesized 2-styrylquinazolones (8a-8at) were
subjected to in vitro anti-Mtb activity test against M. tuberculosis
H37Rv (ATCC 27294 strain).14 The minimum inhibitory
concentration (MIC), minimum concentration in µg/mL of the
compound required for 99% inhibition of bacterial growth, of 8a
to 8at and those of the standard drugs (INH, R, E, Z and Cfx)
were determined in triplicate at pH 7.4 (Table 2). All of the
synthesized compounds showed MIC values in the micromolar
range (0.40 – >25 µg/mL). Twenty-two compounds exhibited
MIC in the range of 1.56–6.25 µg/mL, and the compounds 8c, 8d
and 8ab were found to be potent (MIC 0.78 µg/mL). The most
potent compound 8ar showed MIC of 0.40 µg/mL.
8
9
Ar1 = 4-SMe-C6H4, Ar2 = 4-F-C6H4
Ar1 = Ph, Ar2 = 4-OMe-C6H4
Ar1 = 2,3,4-tri-OMe-C6H4, Ar2 = 4-OMe-
C6H4
70
60
8h
8i
10
65
8j
11
12
13
Ar1 = 4-SMe-C6H4, Ar2 = 4-OMe-C6H4
Ar1 = Ph, Ar2 = 4-F-C6H4
Ar1 = 4-Me-C6H4, Ar2 = 4-Cl-C6H4
Ar1 = 2,3,4-tri-OMe-C6H4, Ar2 = 4-Cl-
C6H4
Ar1 = 4-SMe-C6H4, Ar2 = 4-Cl-C6H4
Ar1 = 4-SMe-C6H4, Ar2 = 4-N,N-di-Me-
C6H4
64
67
71
8k
8l
8m
14
15
16
76
76
66
8n
8o
8p
The in vitro cell viability of the compounds with MIC ≤ 6.25
µg/mL were evaluated against HEK-293T (human embryonic
kidney) cell lines at 50 µg/mL concentration by using [(3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] MTT
assay. The % inhibitory cytotoxicity data are summarized in
Table 2 and graphically represented in Figure 3, along with the
MIC values of the respective compounds. In general, most of the
active compounds were found to be non-toxic (<50% inhibition)
and 8c, 8d, 8ab and 8ar turned out to be the most active
17
18
19
20
Ar1 = 4-SMe-C6H4, Ar2 = 2-thiazolyl
Ar1 = Ph, Ar2 = 2,6-di-OH-C6H4
Ar1 = Ph, Ar2 = 4-Phenylmethoxy-C6H4
Ar1 = 4-Cl-C6H4, Ar2 = 4-F-C6H4
73
54
68
65
8q
8r
8s
8t

a
compounds and promising anti-Mtb leads from this series with
therapeutic index of > 64 and >125 respectively.
99% inhibition of growth of M. tuberculosis H37Rv (ATCC 27294
strain).
b
% Inhibition at 50 µg/mL concentration determined against HEK
293T cell lines. ND: Not Determined.
The systematic variations on the C-ring keeping the
quinazolone nucleus and D ring intact have varying effects on the
anti-Mtb activity of the compounds. The substitution of
thiomethyl and methoxy groups at the 4-position of the C-ring
resulted in the active compounds 8cand 8ac having MIC of 0.78
µg/mL and 1.56 µg/mL respectively, more than its un-substituted
counterpart 8a (MIC of 3.125 µg/mL). While the substitution of
halides (8aj, 8al and 8an) and methyl (8b) group at the 4-
position gave less active compounds. The 3,4,5-trimethoxy
substitution on the C ring also resulted in the compound (8d)
with activity of 0.78 µg/mL. All the active compounds were
found to be safe with the selectivity index (SI) ranging from 16 to
>64 (Table 3, Figure 4).
Figure 3. Graphical representation of the anti-mycobacterial activity and
cytotoxicity profile of the synthesized compounds with MIC ≤ 12.5 µg/mL.
A complete SAR can be drawn considering the MIC
values and cytotoxicity data (Table 2).
Table 2. Biological evaluation of 8a-8at and a few standard
anti-TB drugs.
Figure 4. Ring C substitution on 2-styrylquniazolones.
HEK
293T
Inhibitionb
29.70
ND
HEK
293T
Inhibitionb
55.47
47.99
38.36
58.14
47.68
71.33
ND
Table 3. Effect of C-ring substitution on anti-TB activity.
MICa
MICa
Compd
No.
Compd
No.
% Inhibition
MIC
(g/mL)b
Compd
No.
(g/mL)
(g/mL)
Entry
R
at 50 µg/mL
HEK 293T
29.70
SI
3.125
>25
3.125
0.78
1.56
3.125
1.56
6.25
12.5
3.125
12.5
12.5
>25
8a
8b
8c
8d
8e
8f
8aa
8ab
8ac
8ad
8ae
8af
8ag
8ah
8ai
1
2
3
4
5
6
7
8
H
4-Me
3.125
> 25
0.78
0.78
1.56
12.5
25
>16
-
8a
8b
NDa
0.78
0.78
25
38.70
26.54
ND
4-SMe
3,4,5-tri-OMe
4-OMe
4-Cl
38.70
>64
>64
>32
-
8c
26.54
8d
38.36
NDa
NDa
8ac
8aj
8al
8an
>25
ND
6.25
12.5
12.5
12.5
>25
46.12
ND
8g
8h
8i
4-F
-
66.29
ND
4-Br
6.25
24.86
>8
ND
aND: Not determined.
ND
ND
8j
8aj
8ak
8al
ND
ND
8k
8l
The systemic variations of the above seven compounds by
varying the D ring substitution was next studied to check the
effect on the activity of the compounds. Substitutions were
introduced on 8a, 8c, 8d and 8ac which have shown activity in
the range of 0.78 to 3.125 µg/mL to get more potent and selective
compounds (Figure 5).
12.5
25
ND
25
ND
ND
12.5
6.25
6.25
6.25
>25
ND
8m
8n
8o
8p
8q
8r
8s
8am
8an
8ao
8ap
8aq
8ar
8as
8at
INH
R
3.125
12.5
12.5
6.25
6.25
12.5
6.25
1.56
3.125
6.25
6.25
3.125
6.25
38.90
ND
24.86
18.66
21.06
ND
ND
29.00
18.12
ND
0.40
>25
15.41
ND
34.00
33.60
28.20
35.80
40.00
42.07
58.77
3.125
0.098
0.197
1.56
6.25
1.56
14.27
ND
8t
Figure 5. Ring D substitutions on 2-styrylquniazolones.
8u
8v
8w
8x
8y
8z
The substitution by halide (8e and 8l) or alkoxy group (8i and
8s) at the 4 position resulted in decrease in activity (Table 4,
Figure 6). 4-Thiomethtyl substitution resulted in the compound
(8aa) with equal activity. 3,4,5-Trimethoxy substitution gave the
more potent compound (8ab) with MIC of 0.78 µg/mL and
selectivity >64. The 2,6-di-hydroxy substitution yielded
compound (8r) with MIC value of 6.25 µg/mL and is less potent
than the parent compound 8a. Replacement of D-ring with 5-
ND
ND
E
ND
Z
ND
Cfx

membered heterocyclic rings like furan-2-yl (8ai) and thiofuran-
2-yl (8ah) did not improve the activity. However, 8ah has shown
activity equal to the parent compound 8a.
Replacement of D-ring with 5-membered heterocyclic rings like
furan-2-yl (8x) and thiofuran-2-yl (8q) did not improve the
activity. 4-Acetyloxy substitution yielded compound (8w) with
the activity of 6.25 µg/mL less than that of the parent compound
8c. None of the substituents was able to increase the activity of
the parent compound 8c.
Figure 6. Study of ring D substitution on 8a.
Table 4. Effect of D-ring substitution on anti-TB activity of 8a.
Figure 8. Study of ring D substitution on 8c.
% Inhibition
MIC
(g/mL)b
Compd
No.
Entry
R
at 50 µg/mL
HEK 293T
NDa
SI
Table 6. Effect of D-ring substitution on anti-TB activity of 8c.
% Inhibition
MIC
Compd
No.
Entry
R
at 50 µg/mL
HEK 293T
NDa
SI
1
2
3
4
5
6
7
4-F
4-Cl
25
-
-
8e
8l
(g/mL)b
12.5
12.5
12.5
3.125
0.78
6.25
NDa
NDa
NDa
1
2
3
4
4-F
4-Cl
12.5
12.5
>25
-
-
-
-
4-OCH3
-
8h
8o
8k
8p
8i
NDa
NDa
NDa
4-OCH2Ph
4-SMe
-
8s
4-OCH3
4-N, N-di-Me
55.47
<16
>64
>8
8aa
8ab
8r
12.5
3,4,5-tri-OMe
2,6-di-OH
47.99
18.12
5
6
6.25
6.25
40.00
29.00
>8
>8
8x
8q
8
9
12.5
NDa
-
8ai
3.125
66.29
<16
8ah
7
8
4-OCOCH3
6.25
>25
35.80
NDa
-
-
8w
aND: Not determined.
3,4,5-tri-OMe
8as
aND: Not determined.
The substitution by halogen atom (8f and 8m) at the 4
position did not improve the activity (Table 5, Figure 7).
Alkoxy group (8ag) at the 4 position resulted in some
improvement in the activity with MIC value of 12.5 µg/mL.
While the methyl substitution (8at) increased the potency to
3.125 µg/mL. Overall the substitution tried on 8b didn’t
significantly alter the activity.
Effect of D-ring substitution on 8ac (Figure 9) on the
anti-TB activity and selectivity of the resultant compounds
is demonstrated in Table 7. The substitution by halogen
atom (8ad) and alkoxy group (8u) at the 4 position resulted
in compounds with MIC values of 3.125 µg/mL and 1.56
µg/mL respectively. Replacement of D-ring with 5-
membered heterocyclic rings like furan (8af) and thiofuran
(8ae) did not improve the activity. None of the substituents
was able to increase the activity of the parent compound 8ac
as all the substitutions tried gave equipotent or less potent
compounds compared to the parent compound.
Figure 7. Study of ring D substitution on 8b.
Table 5. Effect of D-ring substitution on anti-TB activity of 8b.
% Inhibition
MIC
(g/mL)b
Compd
No.
Entry
R
at 50 µg/mL
HEK 293T
NDa
SI
Figure 9. Study of ring D substitution on 8ac.
1
2
3
4
4-F
4-Cl
>25
25
-
-
8f
Table 7. Effect of D-ring substitution on anti-TB activity of
NDa
NDa
8ac.
8m
8ag
8at
% Inhibition
4-OCH3
4-Me
12.5
3.125
-
MIC
(g/mL)b
Compd
No.
Entry
R
at 50 µg/mL
HEK 293T
SI
14.27
>16
aND: Not determined.
The substitution by halogen atom (8h and 8o), alkoxy group
(8k and 8as), and N,N-dimethylamine group (8p) at the 4
position resulted in decrease in activity (Table 6, Figure 8).

1
2
Cl
3.125
1.56
58.14
33.60
<16
>32
1
2
3
4-F
4- Cl
6.25
3.125
12.5
46.12
38.90
NDa
>8
>16
-
8ad
8u
8g
8n
8j
OCH3
4- OCH3
3
4
6.25
1.56
71.33
47.68
<8
8af
8ae
4
3.125
42.07
>16
8y
>32
5
6
6.25
0.40
58.77
15.41
<8
8z
aND: Not determined.
4-SMe
>125
8ar
Effect of D-ring substitution on 8aj and 8al (Figure 10)
on the anti-TB activity and selectivity is shown in Table 8.
Halogen substitution (8am and 8t) led to some improvement
in activity.
aND: Not determined.
Recently styrylquinazolines have been reported as new
antibiotics and their site of action has been identified to be the
PBPs (Pbp2a and Pbp1) present in S. aureus.12 The PBPs are
required for the final stages of cell-wall formation in bacteria and
are also molecular targets for β-lactam antibiotics.15,16 The high
resolution (1.8 Å) S. aureus PBP2a crystal structure (1VQQ) was
reported.17 Active site of this crystal structure was used to
identify the styrylquinazoline antibiotics.12 In order to get further
insight on the Mtb activity of the styrylquinazolines described
under our investigation in this report we planned to understand
the putative targets of these styrylquinazolines thorough
computational studies. We performed blast search FASTA
sequence of S. aureus PBP2a (1VQQ) against the PDB protein
and found that the M. tuberculosis PBPa (3UN7) has 22%
sequence identity, 51% sequence similarity, and an E- value
(expectation value) of 1e-12. Although the sequence identity is
low, the active site of these PBPs are conserved.12,16 The protein
3UN7 is in apo form and its co-crystallised structures with
antibiotics imipenem [3UPN], penicillin G[3UPO], and
ceftriaxone [3UPP] are reported in the PDB reflecting that these
antibiotics are bound to its active site.16 To validate the similarity
of the active sites, we have performed the sequence alignment
(Figure 12) of these two proteins and compared their active
sites.17,18
A comparison19 of the key nine amino acid residues of the
active site of PBP2a with that of PBPa revealed that seven of
these amino acid residues are identical, while the Met641 in
PBP2a is replaced by Leu467 in PBPa which is considered to be
similar. The only dissimilarity is that the Tyr446 in PBP2a is
replaced by Glu268 in PBPa (Figure 12). Thus, out of the nine
amino acid residues of the active site, seven are identical, one is
similar, and one is dissimilar. This suggested that their active
sites can be considered to be similar. Therefore we have used the
active site of M. tuberculosis PBPa for docking our compounds.
Figure 10. Study of ring D substitution on 8aj, 8al and 8am.
Table 8. Effect of D-ring substitution on anti-TB activity of
8aj, 8al and 8an.
% Inhibition
MIC
(g/mL)b
Compd
No.
Entry
R
X
at 50 µg/mL
HEK 293T
NDa
SI
1
2
3
4
4-Cl
4-F
F
Cl
12.5
6.25
>25
-
8am
8t
34.0
NDa
>8
-
4- Cl
4-F
Cl
8ak
8ap
4-Me
6.25
21.06
>8
aND: Not determined.
Effect of D-ring substitution on 8d (Figure 11) on the anti-
Mtb activity and selectivity is shown in Table 9.The substitution
by halogen atom (8g and 8n) and alkoxy group (8j) at the 4
position resulted in decrease in activity. Replacement of D-ring
with 5-membered heterocyclic rings such as furan (8y) and
thiofuran (8z) did not improve the activity. Substitution by
methylthio group (8ar) gave the most potent compound from the
series with MIC of 0.40 µg/mL.
The 3D QSAR techniques such as CoMFA/CoMSIA and
molecular docking provide a deeper understanding of the
structure activity correlations that would serve as a predictive
model for further optimization of the lead structure.20 Docking
analysis was performed on our active molecules to identify key
amino acid residues involved in making interactions with the
PBPa active site of 3UPN. The GOLD program21 was used to
carry out this analysis. The docked pose of the most potent
molecule 8d is shown in Figure 13. The hydrogen bonding
interactions were found with the key active site residues Ser222,
Gly221 and Asn283 with high gold score of 58 (Figure 13).
Methoxy group of 8d interacts with the Ser222 and Gly221;
whilst the quinazolone oxygen interacts with the Asn283. This
analysis indicates the possibility of these molecules binding to
the PBPs. However, further experimental proofs are needed to
confirm this.
Figure 11. Study of ring D substitution on 8d.
Table 9. Effect of D-ring substitution on anti-TB activity of 8d.
% Inhibition
MIC
(g/mL)b
Compd
No.
Entry
R
at 50 µg/mL
HEK 293T
SI

class of compounds has been also established. The computational
study indicates that these compounds might act through PBPs as
that of styrylquinazolone antibiotics.
Acknowledgments
The author DK thanks CSIR, New Delhi for the awards of Research
Associate fellowship and MDV thanks UGC-RGNF, New Delhi for
Junior Research Fellowship.
References and notes
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The present work reveals new 2-styrylquinazolones with
potent anti-Mtb activity. Forty-six compounds were synthesized
using one pot reaction of isatoic anhydride, amine and triethyl
orthoacetate followed by treatment of aromatic aldehyde. These
were evaluated for in vitro anti-Mtb activity. Twenty-six
compounds displayed good in vitro anti-mycobacterial activity
ranging from 0.40-6.25 µg/mL. The most potent compounds 8c,
8d, 8ab (MIC, 0.78 µg/mL) and 8ar (MIC,0.40 µg/mL) were
highly selective with therapeutic index >64 and >125
respectively. These compounds have shown potency better than
that of the standard drugs E, Z and Cfx. The early SAR for this

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