Synthesis and anti-angiogenic activity of benzothiazole, benzimidazole containing phthalimide derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.10.106

Benzothiazole and benzimidazole containing phthalimide derivatives (NK037, NK041, NK042, NK0139A and NK0148) have been synthesized and their anti-angiogenic activity was evaluated using ex vivo egg yolk angiogenesis model. A comparative study with pure thalidomide (NKTA) has also been performed to describe the efficacy of these derivatives in blocking angiogenesis. NK037, NK041 and NK042 were equally potent in blocking egg yolk angiogenesis and the anti-angiogenesis effect was higher than NKTA suggesting the efficacy of these three derivatives in blocking angiogenesis when compare to control. Other two derivatives NK0139A and NK0148 showed effect less than NKTA and stronger than control in ex vivo angiogenesis.

Full text of DOI:10.1016/j.bmcl.2012.10.106

Bioorganic & Medicinal Chemistry Letters 23 (2013) 287–290
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-angiogenic activity of benzothiazole, benzimidazole
containing phthalimide derivatives
Shunmugam Nagarajan a, Syamantak Majumder a, Upendra Sharma b, Saranya Rajendran a,
Neeraj Kumar b, Suvro Chatterjee a, , , Bikram Singh b, ,
⇑
⇑
a Vascular Biology Lab, AU-KBC Research Centre, MIT Campus, Anna University, Chennai, Tamil Nadu 600 044, India
b Natural Plant Products Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176 061, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Benzothiazole and benzimidazole containing phthalimide derivatives (NK037, NK041, NK042, NK0139A
and NK0148) have been synthesized and their anti-angiogenic activity was evaluated using ex vivo egg
yolk angiogenesis model. A comparative study with pure thalidomide (NKTA) has also been performed to
describe the efficacy of these derivatives in blocking angiogenesis. NK037, NK041 and NK042 were
equally potent in blocking egg yolk angiogenesis and the anti-angiogenesis effect was higher than NKTA
suggesting the efficacy of these three derivatives in blocking angiogenesis when compare to control.
Other two derivatives NK0139A and NK0148 showed effect less than NKTA and stronger than control
in ex vivo angiogenesis.
Received 24 August 2012
Revised 20 October 2012
Accepted 23 October 2012
Available online 1 November 2012
Keywords:
Thalidomide
Anti-angiogenesis
Phthalimide
Ó 2012 Published by Elsevier Ltd.
Angiogenesis is the process of generating new capillary blood
vessels which play an important role both in health (wound heal-
ing,1 female reproductive cycle2) and disease (cancer,3 rheumatoid
arthritis,4 diabetic retinopathy5). The growth and maintenance of
solid tumors is highly dependent on neovascularization and can
be regulated by agents that interfere with either the stimulation
or proliferation of endothelial cells.6 As a result, the control of angi-
ogenesis continues to be an attractive area for the development of
novel therapeutic agent.5
Thalidomide (NKTA) is a sedative and/or hypnotic drug much
used in the 1950s, which was withdrawn from clinics as its terato-
genicity was discovered.7 However, basic research on thalidomide
(NKTA) did not come to a halt, and in earlier 1990s thalidomide
(NKTA) proved to be an anti-angiogenic agent.8 It has, since, been
established to be effective for the treatment of various diseases,
including graft-versus-host disease, cancers, AIDS, and other angi-
ogenesis-dependent disorders.9 The United States Food and Drug
Administration (FDA) approved thalidomide (NKTA) for the treat-
ment of Erythema Nodosum Leprosum (ENL) in 1998.10 Further-
more, many clinical studies of thalidomide (NKTA) for the
treatment of multiple myeloma, breast cancer, prostate cancer,
and other conditions are on-going in the U.S. The side effects asso-
ciated with clinically effective thalidomide (NKTA) treatment has
led to the design and synthesis of more potent analogues with re-
duced toxicity.11
Derivatives of nitrogen and sulfur containing heterocyclic com-
pounds such as benzimidazole, benzothiazole and benzothiadiaz-
ole have found number of application as anticancer agent.12 We
postulated that phthalimide derivatives of these heterocyclic com-
pounds can be more active than thalidomide (NKTA) with less side
effect. Recently, Yeh et al. reported the therapeutic effects of can-
tharidin derivatives with these type of heterocyclic compounds
without bridging ether oxygen on human hepatocellular carci-
noma cells.13 Kok et al. have evaluated the anticancer activity of
benzothiazole substituted phthalimide derivatives towards three
human cancer cell lines and demonstrated the potential of these
compounds as anticancer agents.14
Thalidomide (NKTA) has been shown to inhibit angiogenesis
potentially in vivo.8 Our previous study have also showed thalido-
mide (NKTA) mediated inhibition of angiogenesis by interacting
with soluble guanyl cyclase.15 The present study aims to synthe-
size structurally modified phthalimide derivatives and compara-
tively evaluate their anti-angiogenic activity.
Phthalimide derivatives were synthesized using ionic liquid as
solvent and catalyst. To optimize the best reaction condition, con-
densation of phthalic anhydride was carried out with 2-aminoben-
zothiazole in different ionic liquids at variable temperature and
time interval (Table 1). Full conversion was not observed without
using any solvent (Table 1, entry 1). Low to moderate yield was ob-
served in case of toluene and 1-butyl-3-methylimidazolium hydro-
gen sulfate ([bmim]HSO4) (Table 1, entry 2 and 6). Comparative
yield was observed in case of acetic acid, [bmim]BF4 and
⇑
Corresponding authors.
E-mail addresses: soovro@yahoo.ca (S. Chatterjee), bikram_npp@rediffmail.com
(B. Singh).
IHBT Communication No. 3419.
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.10.106

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S. Nagarajan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 287–290
Table 1
O
O
Condensation of phthalic anhydride with 2-aminobenzothiazole under different
O
conditionsa
O
O
O
O
O
NH
NH
N
O
N
O
O
O
NH
N
2
S
S
O
solvent
NH
2
H N
N
+
O
2
N
N
Thalidomide
Lenalidomide
CC-1069
O
O
Figure 1. Structure thalidomide, lenalidomide and CC-1069.
Entry
Solvent
Temperature (°C)
Time (h)
Yield (%)
1
2
3
4
5
6
7
8
9
8
9
10
11
Neat
Toluene
110
110
110
110
110
110
110
110
110
110
90
5
5
5
5
5
5
4
4
4
2
6
5
5
32
39
76
73
83
52
81
85b
72c
49
63
33
35
After optimizing the best reaction conditions the scope of reac-
tion was explored using different substrates (Table 2). Low to good
yield was obtained in most of the cases.
Acetic acid
[bmim]BF4
[bmim]PF6
[bmim]HSO4
[bmim]PF6
[bmim]PF6
[bmim]PF6
[bmim]PF6
[bmim]PF6
Silica-H3BO3
Silica-H2SO4
Numerous studies have revealed that many tissues and organs
like eyes and heart are affected by thalidomide (NKTA) during
embryonic development.16 In 1994, D’amato et al. demonstrated
that orally administered thalidomide (NKTA) was an inhibitor of
angiogenesis induced by basic fibroblast growth factor in a rabbit
cornea micropocket assay.8 Our previous study also demonstrated
that thalidomide (NKTA) exerts inhibitory effects on nitric oxide-
mediated angiogenesis by altering sub-cellular actin polymerization
pattern, which leads to inhibition of endothelial cell migration.17
Moreira et al. showed that thalidomide (NKTA) analogue CC–1069
(Fig. 1) significantly inhibited endothelial cell proliferation com-
pared to thalidomide (NKTA).18 There was a notable reduction in
the nuclear factor activity and a sensible inhibition of NF-rB activa-
tion in nuclear extracts of endothelial cells.
110
110
a
Reaction conditions: A mixture of phthalic anhydride (250 mg, 1.68 mmol),
2-aminobenzothiazole (253 mg, 1.68 mmol), 5 ml of solvent were heated in round
bottom 25 ml flask.
b
Under vacuum (0.4 bar).
Yield after third cycle.
c
[bmim]PF6 with maximum yield in [bmim]PF6 (Table 1, entry 3–5).
When the reaction was carried out under vacuum (0.4 bar) a slight
increase in yield was observed (Table 1, entry 8). In the presence of
silica supported boric acid and sulfuric acid low yield was recorded
(Table 1, entry 10 and 11) and yield was comparable to the reaction
where no solvent or catalyst were used (Table 1, entry 1).
The pharmacokinetics of thalidomide (NKTA) suggested that
thalidomide (NKTA) having two rings where the glutarimide moi-
ety was thought to be responsible for the teratogenic effects of the
molecule.11 These effects coupled with clinically effective thalido-
mide (NKTA) have led to the synthesis of more potent phthalimide
Table 2
Synthesis of different phthalimide derivativesa
Entry
Phthalic anhydride
Amine
Product
Yield (%)
85.1
O
O
N
S
NH2
S
O
N
1
N
(NK037)
O
O
O
O
N
S
O
N
H2N
NH2
2
3
N
83.9
84.5
O
O
S
O
O
(NK041)
S
N
N
N
N
S
O
O
N
(NK042)
O
O
O
N
S
H3C
NH2
NH2
S
O
N
4
5
76
N
(NK0139A)
(NK0148)
O
O
O
O
N
N
O
N
H
N
72.6
N
H
O
O
a
Conditions were not optimized separately for different substrates, reaction conditions: a mixture of phthalic anhydride (1.68 mmol), amine (1.68 mmol), 5 ml of
[bmim]PF6 were heated at 110 °C in round bottom 25 ml flask for 4 h under vacuum (0.4 bar).

S. Nagarajan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 287–290
289
Figure 2. Effect of thalidomide (NKTA) and phthalimide derivatives on CAM angiogenesis: CAM were treated with thalidomide analogs for 6 h and photographed. (A, B, C)
Data suggests that NK037 and NK042 inhibited angiogenesis significantly compared to DMSO on all the time points (⁄p = <0.001 vs DMSO). There was a statistically
significant inhibition of angiogenesis by NK041 compared to control (⁄p = <0.001 vs DMSO). (D) Representative images of chicken embryo chorioallantoic membrane treated
with phthalimide derivatives for 6 h. Images were analyzed using Angioquant image analysis software. The experiments were performed at least three times for each
condition and generated reproducible data. The error bars represent Standard error of the mean.
derivatives with decreased toxicity.11,19 Hence, structurally modi-
fied phthalimide derivatives were synthesized to test their inhibi-
tory effect on angiogenesis compared to control. Dredge et al. also
demonstrated in vivo anti-angiogenic effect of orally administered
lenalidomide (Fig. 1), inhibition of endothelial cell migration and
Akt phosphorylation in vitro.20
Results of the present study also elaborate the stronger anti-
angiogenesis effects of the phthalimide derivatives in comparison
to that of thalidomide (NKTA).
To evaluate the potency of the different phthalimide derivatives
and thalidomide (NKTA) itself on inhibiting angiogenesis, a more
complex egg yolk angiogenesis assay was performed. The potency
of phthalimide derivatives to inhibit angiogenesis in the developing
chick embryo (CAM assay) ex vivo was studied. Images depicted the
inhibition of angiogenesis with phthalimide derivatives. As shown
in Figure 2, treatment with NK037 and NK042 showed disorganiza-
tion and inhibition of growth of new vessels proximity to the area
covered by the discs containing the drug indicating a reduction in

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S. Nagarajan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 287–290
vascularization compared to other phthalimide derivatives and con-
trol. The number of length and junctions of tubule complexes was
quantified.
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In conclusion, benzimidazole, benzothiazole and benzothiadiaz-
ole containing phthalimide derivatives have been discovered as po-
tent inhibitors of angiogenesis in ex vivo egg yolk angiogenesis.
Further structural development studies based on the present com-
pounds may yield potent and non-teratogenic derivatives for anti-
cancer therapy.
Acknowledgments
Authors are grateful to the Director, CSIR-IHBT, Palampur for
providing necessary facilities during the course of the work.
Thanks are also due to CSIR for awarding Senior Research Fellow-
ship to U.S.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.10.
106.