Synthesis and biological evaluation of phaitanthrin congeners as anti-mycobacterial agents

Article abstract of DOI:10.1016/j.bmcl.2015.07.057

Abstract Natural alkaloid, tryptanthrin (indolo[2,1-b]quinazoline-6,12-dione) and its analogues are found to exhibit potent anti-tubercular activity against MDR-TB. A novel class of indolo[2,1-b]quinazolinones have been synthesized to evaluate their anti-

Full text of DOI:10.1016/j.bmcl.2015.07.057

Accepted Manuscript
Synthesis and biological evaluation of phaitanthrin congeners as anti-mycobac-
terial agents
Ahmed Kamal, B.V. Subba Reddy, B. Sridevi, A. Ravikumar, A.
Venkateswarlu, G. Sravanthi, J. Padma Sridevi, P. Yogeeswari, D. Sriram
PII:
S0960-894X(15)00761-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.07.057
BMCL 22946
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
28 April 2015
13 July 2015
18 July 2015
Please cite this article as: Kamal, A., Subba Reddy, B.V., Sridevi, B., Ravikumar, A., Venkateswarlu, A., Sravanthi,
G., Padma Sridevi, J., Yogeeswari, P., Sriram, D., Synthesis and biological evaluation of phaitanthrin congeners as
anti-mycobacterial agents, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2
015.07.057
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Synthesis and biological evaluation of phaitanthrin congeners as anti-
mycobacterial agents
a,c,
b,
a,b,c
a,c
b
Ahmed Kamal *, B. V. Subba Reddy *, B. Sridevi , A. Ravikumar , A.Venkateswarlu ,
c
d
d
d
G. Sravanthi , J. Padma Sridevi , P. Yogeeswari , D. Sriram
a
b
Medicinal Chemistry and Pharmacology, Natural Products Chemistry, CSIR-Indian
Institute of Chemical Technology, Hyderabad 500 007, India
c
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research, Hyderabad 500 037, India
d
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad
500078, India
Abstract
Natural alkaloid, tryptanthrin (indolo[2,1-b]quinazoline-6,12-dione) and its analogues are
found to exhibit potent anti-tubercular activity against MDR-TB. A novel class of indolo[2,1-
b]quinazolinones have been synthesized to evaluate their anti-mycobacterial activity. Enoyl-
acyl carrier protein reductase (InhA) of M. tuberculosis is one of the key enzymes and has
been validated as an effective anti-microbial target. In silico molecular docking study
demonstrates that the synthesized compounds exhibit high affinity for the M. tuberculosis
drug target InhA. Phaitanthrin is a natural product, which belongs to a family of tryptanthrin
and exhibits structural similarity except at position 6. Phaitanthrin derivatives are prepared by
modifying the keto functionality of tryptanthrin. These phaitanthrin congeners are found to
display promising anti-tubercular activity.
Keywords: Tryptanthrin, indolo[2,1-b]quinazoline-6,12-dione, MDR-TB, Enoyl-acyl carrier
protein reductase (InhA), In silico molecular docking study.
Corresponding authors Tel.: +91-40-27193157; fax: +91-40-27193189, e-mail:
ahmedkamal@iict.res.in (A. Kamal).
Tuberculosis (TB), which is acknowledged as a global health catastrophe, is a chronic
bacterial infection caused by Mycobacterium tuberculosis. Most infections in humans result
in asymptomatic, latent infection and about one in ten latent infections eventually progresses
1
to active disease. Hence, the discovery of new drugs is very much in need to combat

tuberculosis. Multidrug resistant (MDR) and extensively drug resistant (XDR) TB strains are
of concern for human society, the solution for growing emergence of resistance in the
pathogen is the development of new affordable drugs with high potency and low toxicity.
Indolo[2,1-b]quinazoline-6-12-dione or tryptanthrin is a versatile lead for designing
the potential drugs with diverse medical functions. Tryptanthrin is a potent and structurally
useful alkaloid isolated from the natural sources such as Phaius mishmensis and Isatis,
2
Calanthe, Wrightia, Couroupota and Strobilanthus species. In particular the orchid Phaius
mishmensis was reported to be a rich source of tryptanthrin based compounds and all those
3
shown in Fig. 1, have been isolated from this plant. Tryptanthrin consists of both quinazoline
and indole core structures and showed a variety of intriguing biological properties such as
4
antibacterial, antifungal, antiprotozoal and antiparasitic activities. Although systematic
synthesis and structure-activity relationship studies for anti-tubercular, COX inhibitory and
cytotoxic activity have led several promising candidates for further development, none of
5
them has yet been successfully launched for clinical usage. Structure of tryptanthrin is
comparatively simple, lacking asymmetric centres, it has a fairly low molecular weight and
has a structure which differs significantly from all previously established anti-tubercular
agents. Tryptanthrin proved rather more potent against M tb H37Rv (1 mg/L) and M. avium
(4 mg/L) than against M. smegmatis (6 mg/L) and exhibit same potency as that of well-
known anti-tubercular agents such as INH, streptomycin and ethambutol. One significant
implication of this is that tryptanthrin can be proposed to be operating by a molecular
mechanism different than that employed by most of the existing anti-tubercular agents and
6
might be useful in cases where existing agents would fail to cure patients.
The natural alkaloid, tryptanthrin represents a potential lead for new tuberculosis
drugs since tryptanthrin and its synthetic analogues possess potent in vitro activity against
Mycobacterium tuberculosis (Mtb). When tryptanthrin is tested against a panel of multidrug-
resistant strains of M. tuberculosis, it shows more potency (MICs of 0.5-1µg/mL) than
7
isoniazid (MICs 4-16µg/mL). Though tryptanthrin and its analogues are active against
MDR-TB, their cellular target is unknown. Therefore, tryptanthrin is an important class of
structural motif for the development of new drugs for the treatment of tuberculosis. As a part
of our interest on chemical and pharmacological studies of natural alkaloids, we have been
interested in compounds possessing the tryptanthrin skeleton due to their fascinating
biological activities.

We designed and synthesized a new series of tryptanthrin based molecules in which the keto
group at position 6 of the indolo[2,1-b]quinazoline core was replaced by other functional
groups. In this manuscript we report the synthesis and activity studies performed.
Results and Discussions
In this endeavor, we designed and synthesized a series of novel anti-tubercular agents, based
on indolo[2,1-b]quinazoline-6,12-dione and 11H-indeno[1,2-b]quinoxalin-11-one derived
natural products.
Scheme 1. Synthesis of indolo[2,1-b]quinazoline analogues. Reagents and conditions: a) N-
o
ethylpiperidine, DIC, pyridine, 24 h, 100 C; b) dimethylamine, ketone, neat, 30 °C, 12 h; c)
o
PhNHNH
2
, glacial AcOH, EtOH, 80 C, 12 h.

The synthesis of indolo[2,1-b]quinazoline-6,12-dione is shown in Scheme 1. Accordingly,
treatment of isatin (1) with isatoic anhydride (2) in the presence of N-ethyl piperidine and
o
diisopropylcarbodiimide(DIC) in dry pyridine at 100 C afforded the desired product 3 in
93% yield. Aldol condensation of 3 with ketones in the presence of dimethyl amine furnished
the β-hydroxyketones (4a-k). Finally, the condenstion of 4a-k with phenylhydrazine in
o
glacial acetic acid at 80 C afforded the corresponding indole derivatives (5a-j).

Table 1: Synthesis of indolo[2,1-b]quinazoline analogues (4a-4k) and (5a-5j):

Next, we prepared a new series of spiroindenoquinoxalines in good yields through a
condensation of β-hydroxyketones (9a-c) with phenylhydrazine under acidic conditions

(Scheme 2). Accordingly, treatment of o-phenylenediamine with ninhydrin in the presence of
acetic acid in ethanol gave the 11H-indeno[1,2-b]quinoxalin-11-one 8, which was then
Scheme 2: Synthesis of indenoquinoxaline derivatives. Reagents and conditions: a) glacial
o
o
AcOH, EtOH, 60 C, 12 h, 90%; b) dimethylamine, arylketone, neat, 25 C, 30 min; c) glacial
o
o
AcOH, HCl, 80 C, 30 min; d) ) PhNHNH
2
, EtOH, 80 C, 12 h.
2
2
2
2
2
2
subjected to aldol condensation with aryl ketones in the presence of dimethyl amine to afford
the β-hydroxyketones (9a-c). Upon treatment of β-hydroxyketone with phenylhydrazine in
o
the presence of catalytic amount of HCl in acetic acid at 80 C afforded the corresponding
pyrazole derivatives (10a-c).

Table2. Synthesis of indenoquinoxaline derivatives (9a-9c) and (10a-10c):
In-vitro MTB screening
These indolo[2,1-b]quinazoline-6,12-dione and 11H-indeno[1,2-b]quinoxalin-11-one
based molecules were screened for their in vitro anti-mycobacterial activity against M.
tuberculosis H37Rv (Mtb) by Agar dilution method for determination of the MIC values
along with standard drugs ethambutol and ciprofloxacin. Based on the results presented in
Table 3, some preliminary structure-activity relationship aspects were deduced. The
substituent present on aromatic ring of the phenacyl group had shown certain effects on
biological activity. Of various compounds, 2-aminophenacyl derivative 4f showed an
excellent anti-tubercular activity with MIC 3.99 µM, which is higher than ethambutol (MIC
1
5.3 µM) and ciprofloxacin (MIC 12.6 µM). Conversely, 4-aminophenacyl derivative 4i is
less effective than 4f. Furthermore, thiourea derivative 4k is less potent than free amino

derivative 4f but more effective than other derivatives having 4-ethylphenacyl 4h and 4-
fluorophenacyl 4d groups. In addition 4j, 5a, 5c, 5f, 5g, 9c and 10c also showed significant
MIC values, which are lower than that of ciprofloxacin, whereas the remaining compounds
showed moderate activity with MIC value similar to that of ethambutol. The presence of
bromo substituent on phenacyl ring (4e) drastically reduced the activity than the fluoro
substituent on phenacyl ring (4d) may be due to the lipophilicity of fluorine. Overall, the
phenacyl derivatives of tryptanthrin are considerably active than indole substituted
derivatives. However, the corresponding 4-cyano phenyl substituent on indole moiety 5c
found to exhibit potent activity than 4-cyano phenacyl substitutent 4c. Among
indenoquinoxaline derivatives, 9c and 10c are more active than others in the series of
compounds (9a-c and 10a-c). Among them, 10c is more active than 9c due to rigidity in
spirocyclic system. In case of indenoquinoxaline derivatives, 4-aminophenacyl 9c is more
active than 2-aminophenacyl 9b. Similarly, 10c showed significant activity than 10b. The
synthesis of several analogs demonstrates that the activity totally depends on the substitution
pattern of the aromatic ring.
Table 3. Anti-mycobacterial activity of compounds 4a-k, 5a-j, 9a-c and 10a-c against M.
tuberculosis H37Rv strain (ATCC-27294).
Compound
ClogP
MIC(µM)
4
a
2.55
2.88
2.16
2.77
3.49
2.49
2.32
16.91
16.23
15.90
8.85
111.89
3.99
4b
4
c
4d
4
4
4
e
f
g
16.69
4h
4.35
7.88
4
i
1.83
4.35
3.59
3.57
4.87
3.91
4.31
5.02
5.74
16.32
11.86
5.98
8.58
29.38
7.06
27.74
56.43
6.18
4
j
4k
5
a
5b
5c
5d
5e
5
f

5
g
3.65
3.91
3.65
4.23
4.23
1.96
2.57
7.06
5.51
7.30
12.32
14.16
14.43
17.01
8.45
12.41
56.85
7.17
5h
5
9
i
a
9b
9
c
1
1
1
0a
0b
0c
Ethambutol
15.3
Ciprofloxacin
12.6
ClogP was calculated using QikProp model of version 6.0, Schrödinger, 2013.
2
.3 Molecular docking studies:
In order to gain more insight, these new series of phaitanthrin congeners were docked
to validate whether the synthesized compounds have comparable binding mode in active site
of enoyl-acyl carrier protein reductase (InhA) of M .tuberculosis, which is one of the key
enzymes and has been validated as an effective anti-microbial target. The synthesized
8
compounds were docked within the active site of InhA crystal structure (PDB code: 4U0J)
9
using Glide 6.0(Schr dinger, 2013). As all the synthesized compounds are racemic
mixtures, docking studies were carried out for both the isomers. During the binding studies it
was observed that all synthesized compounds exhibit hydrogen bonding interaction with Tyr
158. As depicted in Figure 2, Compound 4f showed interactions with active site residues Ile
21, Gly 96, Phe 97, Met 98, Met 103, Phe 149, Met 155, Pro 156, Ala 157, Met 161, Gly
192, Pro 193, Thr 196, Ala 198, Met 199, Ile 194, Ile 202, Leu 207, Ile 215, Leu 218. While
in compound 4k, apart from π-cation interactions of side chain phenyl ring with Lys 165 and
NH- group of thiourea in the side chain formed H-bonding with Gly 14 was additionally
observed.
-

Figure 2.a) Three-dimensional diagram displays the binding poses for compound 4f
interactions at active site of InhA. b) Key residues within a 4.5 Å sphere of 4f in the binding
pocket are shown. c) Compound 4k interactions within the active site of InhA. d) Hydrogen
bonding of 4k with Gly 14, Tyr 158 and π- cation with Lys 165 (dark green dashed lines) in
active site. Pink and yellow dashed lines represent H bonding with amino acid side chain, H
bonding with amino acid backbone respectively.
In summary, a new series of indolo[2,1-b]quinazolinedione and indenoquinoxaline
derivatives were synthesized and screened for their in vitro anti-mycobacterial activity. A
number of compounds have shown good in vitro anti-mycobacterial activity. Among various
congeners of phaitanthrin, 2-aminophenyl 4f and its thiourea 4k are identified as lead
molecules for TB. In addition, 5f, 5c, 10c, 5g, 4h, 9c, 5a and 4d have also shown promising
activity thus paving the way for the discovery of newer anti-tubercular potential agent. In
silico molecular modelling studies were performed to examine the interactions of phaitanthrin
congeners with InhA of M. tuberculosis and these interactions indicated the stability of
phaitanthrin congeners in the active residue. Thus, enhanced affinity of phaitanthrin

congeners to binding site of InhA may lead to the synthesis of anti-tubercular agents that are
likely to be capable of combating MDR strains of M. tuberculosis.
Acknowledgements
The authors, B.S and A.R acknowledge the National Institute of Pharmaceutical
Education and Research (NIPER) -Hyderabad, Department of Pharmaceuticals,
Ministry of Chemicals and Fertilizers, Govt. of India for the award of research
fellowship.
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Graphical Abstract
Synthesis and biological evaluation of phaitanthrin congeners as
anti-mycobacterial agents