Synthesis and anti-angiogenesis activity of coumarin derivatives

Article abstract of DOI:10.1016/j.bmcl.2006.06.007

A series of 7-diethylaminocoumarin compounds were synthesized and the cytotoxicities were tested against human umbilical vein endothelial cell (HUVEC) and some cancer cells. We found that the introduction of cyano groups at the 4-position will promote the bioactivity. In particular, compounds 9 and 10 strongly inhibited the proliferation of various cancer cell lines, and 12 and 15 showed a high selectivity for HUVEC. Therefore, these coumarin molecules can be utilized as lead compounds to develop potential nontoxic angiogenesis inhibitors and small molecular ligands to target HUVEC.

Full text of DOI:10.1016/j.bmcl.2006.06.007

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4596–4599
Synthesis and anti-angiogenesis activity of coumarin derivatives
a,b,
a
c
*
Seokjoon Lee,
Krishnamoorthy Sivakumar, Woon-Seob Shin,
Fang Xie and Qian Wang
a
a,
*
a
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29203, USA
Department of Basic Science, Kwandong University College of Medicine, Gangneung 210-701, South Korea
Department of Microbiology, Kwandong University College of Medicine, Gangneung 210-701, South Korea
b
c
Received 29 March 2006; revised 24 May 2006; accepted 5 June 2006
Available online 21 June 2006
Abstract—A series of 7-diethylaminocoumarin compounds were synthesized and the cytotoxicities were tested against human umbil-
ical vein endothelial cell (HUVEC) and some cancer cells. We found that the introduction of cyano groups at the 4-position will
promote the bioactivity. In particular, compounds 9 and 10 strongly inhibited the proliferation of various cancer cell lines, and
12 and 15 showed a high selectivity for HUVEC. Therefore, these coumarin molecules can be utilized as lead compounds to develop
potential nontoxic angiogenesis inhibitors and small molecular ligands to target HUVEC.
Ó 2006 Elsevier Ltd. All rights reserved.
Angiogenesis, the formation of new blood vessels from
pre-existing host vasculatures stimulated by biochemical
early cancer cells and to discriminate between normal
and neoplastic lesions has recently attracted consider-
able attention due to its simplicity and efficiency. In
1
2
10
stimulators, is involved in wound healing, embryonic
3
4
development, and the female reproductive cycle, which
is under elaborate regulations in normal vascular sys-
tem. However, malignant angiogenesis plays a critical
role in several fatal diseases including cancer, vascular
insufficiency, diabetic retinopathy, and rheumatoid
arthritis by a normal delivering mechanism of oxygen
particular, fluorescent probes for detection and imaging
of angiogenesis are important and efficient to clinical
treatment against cancer because tumor angiogenesis oc-
curs drastically in the initial stage of the growing and
1
1
metastasis of cancer cell. Furthermore, if fluorescent
molecules have the antiangiogenic activity, we can
achieve both the imaging and the inhibition against
angiogenesis at the same time.
5
and nutrients to cell and tissue. Because tumor angio-
genesis caused by angiogenic inducers is the most critical
6
factor in the growth of solid tumors, as well as their
7
invasion and metastasis, early control of angiogenesis
may be a promising therapeutic strategy for the related
Coumarins are one of the most important classes of
fluorescent molecules, and they are found to possess
8
12
diseases.
versatile biological activities. In addition, 6,7-dimeth-
ylcoumarin-based novel angiogenesis inhibitors were
1
3
On the other hand, detection and imaging of specific
marker for each disease at the initial stage are the emerg-
ing research areas in medicinal chemistry. For example,
various fluorescent probes are useful in detecting the
reactive oxygen species such as super oxide radical,
hydrogen peroxide, singlet oxygen, and hydroxyl radi-
cal, which are involved in several biological malfunc-
reported recently. As an extension of our study in
the combinatorial synthesis of coumarin derivatives
1
4,15
for bioimaging,
we started to systemically screen
the antiangiogenic activity of coumarins. In this com-
munication, we report the synthesis of a series of 7-
dialkylamino-3,4-substituted coumarin derivatives and
the cell proliferation inhibitory activity test of these
compounds against cell lines such as U87 MG, B16
9
tions. The use of fluorescence microscopy to detect
1
6
BL6, HeLa, DLD-1, SiHa, NIH 3T3, and HUVEC.
In particular, by comparison of growth inhibition
Keywords: Coumarin derivaties; Angiogenesis; Anti-angigenesis; Fluo-
rescence; Synthesis; Small molecule; Imaging.
17
activity against HUVEC and other cell lines, we will
prove that the chemical library produced from a cou-
marin scaffold can produce novel angiogenesis inhibi-
tors with high selectivity over tumor cells, and it is
*
Corresponding authors. Tel.: +1 803 777 8436; fax: +1 803 777 9521
Q.W), tel.: +82 33 649 7454; fax: +82 33 641 1074 (S.L); e-mail
addresses: sjlee@kwandong.ac.kr; wang@mail.chem.sc.edu
(
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.007

S. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4596–4599
4597
˚
Scheme 1. Reagents and conditions: (a) ethyl nitroacetate (1 equiv), n-BuOH, morpholine (cat.), acetic acid (cat.), molecular sieve 3 A, 0 °C,
overnight, 79%; (b) NaCN (2 equiv), I (1 equiv), DMSO, rt, for 4: 83%, for 6: 95%, for 8: 57%; (c) SnCl Æ2H O (8 equiv), concd HCl, rt, 4 h, 67%; (d)
ethyl acetoacetate (1 equiv), EtOH, morpholine (cat.), reflux, overnight, 30%; (e) i—2-cyanomethylbenzimidazole (1 equiv), MeOH, DMF, piperidine
cat.), rt, overnight, ii—17% HCl, rt, overnight, 70% for 2 steps; (f) (3-methoxypropyl)benzooxazol-2-yl-acetate (1 equiv), iso-propanol, reflux, 12 h,
3%; (g) NaCN (2 equiv), lead tetraacetate (1 equiv), DMSO, 40 °C, 2 h to rt, 10 h, for 10: 88%, for 12: 79%; (h) ethyl 2-(2-benzothiazolyl)acetate (1
2
2
2
(
3
˚
equiv), EtOH, piperidine (cat.), 50 °C, 12 h, 82%; (i) Ethyl nitroacetate (1 equiv), n-BuOH, DMF, molecular sieve 3 A, reflux, 12 h, 74%; (j) i—2-
cyanomethylbenzimidazole (2 equiv), EtOH, piperidine (cat.), reflux, overnight, ii—concd HCl, reflux, 3 h, 70% for 2 steps.
potentially possible to inhibit and detect tumor angio-
genesis simultaneously.
For structural diversity, commercially available yellow
coumarin fluorescent dyes containing electron accepting
heteroaromatic groups at the 3-position were synthe-
1
9
In order to establish chemical library with various func-
tionalities and structural diversity, we began with the
synthesis of simple coumarin derivatives from 4-(N,N-
diethylamino)salicylaldehyde (1) and 8-hydroxy-9-form-
yljulolidine (13) initially. As shown in Scheme 1, 3-ami-
no-7-diethylaminocoumarin (3) was synthesized from
reaction of 1 with ethyl nitroacetate in butanol and suc-
cessive reduction of nitro group of 2 with SnCl Æ2H O in
sized by the known method. This was accomplished
through base-catalyzed condensation of 1 with 2-
0
cyanomethylbenzimidazole to give a 3-(2 -benzimidazol-
yl)coumarin known as coumarin-7 via hydrolysis of imi-
1
9
nolactone intermediate, which was transformed to 4-
2
0
cyanocoumarin derivative 8 by cyanation with NaCN.
0
4-Cyano-3-(2 -benzoxazolyl)coumarin (10) and 4-cyano-
0
3-(2 -benzothiazolyl)coumarin (12) were synthesized by
a similar pathway and the reaction conditions are shown
2
2
a concentrated HCl solution. The cyano group was
introduced at the 4-position in the coumarin moiety by
treating 3 with NaCN and iodine in DMSO regioselec-
tively to afford 3-amino-4-cyano-7-diethylamino couma-
2
1
in Scheme 1. Additionally, two benzoquinolizine cou-
marin structures 14 and 15 with similar electronic prop-
erty but different structural features at 7-position were
also synthesized.
1
8
rin (4).
synthesized from a similar condensation reaction of 4
3-Acetyl-7-diethylamino coumarin (5)
and ethyl acetoacetate in ethanol was transformed to
4
As listed in Table 1, simple coumarin derivatives (2–6)
did not show any cytotoxicity against both tested cancer
-cyanocoumarin (6) in 95% yield.

4598
S. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4596–4599
Table 1. Biological activity of fluorescenic coumarin derivatives against various cancer cell lines, and HUVEC
a
Compound
Cytotoxic activity against cancer cell lines (IC50, lM)
HUVEC growth
a
inhibition (IC50, lM)
U87
B16
HeLa
DLD-1
SiHa
NIH 3T3
2
3
4
5
6
7
8
9
>70
>70
>70
>70
>70
15.0
43.0
0.21
1.8
>70
>70
>70
>70
>70
12.6
43.0
1.3
>70
>70
>70
>70
>70
>70
54.4
1.2
>70
>70
>70
>70
>70
>70
>70
0.21
1.8
42.3
>70
>70
>70
>70
62.1
>70
2.0
37.7
>70
31.2
>70
17.9
9.3
25.9
>70
>70
>70
>70
9.6
20.4
2.8
19.8
0.12
3.0
1
1
1
1
1
1
0
1
2
3
4
5
5.6
4.8
>70
>70
>70
>70
>70
14.6
3.5
4.8
>70
>70
>70
>70
33.4
0.08
>70
>70
>70
>70
8.9
>70
>70
>70
>70
11.6
0.47
>70
>70
>70
>70
35.2
0.09
>70
>70
>70
>70
41.4
1.29
>70
4.8
35.4
>70
3.3
Paclitaxel
0.08
0.06
a
IC50 was calculated from the nonlinear regression by Graphpad Prism software.
cell lines and HUVEC, except that compound 2 showed
weak inhibition activity against HUVEC. Because 2 has
selective activity against HUVEC over other cancer
cells, it could be interesting to perform further synthesis
and screening of analog coumarin molecules for devel-
opment of selective angiogenesis inhibitors.
and analogous groups at 3-position improved the over-
all inhibition activity and introducing a cyano group at
the fourth position afforded the selectivity towards HU-
VEC. In particular, the inhibition activity of 9 against
tested cancer cell lines was comparable to a positive con-
trol, paclitaxel, and compounds 12 and 15 showed a
high selectivity for HUVEC, which can be potentially
utilized as lead compounds to develop nontoxic angio-
genesis inhibitors and small molecular ligands to target
HUVEC. Moreover, it is potentially possible to inhibit
and detect tumor angiogenesis at the same time from
further screening in vivo.
In the next trial of synthesis, we introduced hydrophobic
functional groups at 3-position for enhancing van der
Waals interaction to the active site. Cytotoxicity of com-
pounds 7 and 9 designed from this postulation was rea-
sonably increased. In particular, when compared to the
control drug, paclitaxel, compound 9 with oxazolidine
substitution at the third position showed a higher inhib-
itory activity in submicromolar range, but, unfortunate-
ly, showed no selectivity for HUVEC over cancer cells.
The most interesting results were obtained by 4-cyana-
tion of 7, 9, and 11, which led to 3-aromatic-4-cya-
nocoumarins showing some impacts on the inhibition
activity or high selectivity, respectively. As shown in Ta-
ble 1, although the cyanide compounds, 8 and 10
showed weaker inhibition activity upon cancer cells
Acknowledgments
We are grateful for the financial support from the
University of South Carolina, USC Research and
Productive Scholarship, and DOD Breast Cancer
Research Program.
0
and HUVEC, 3-(2 -benzothiazole)-4-cyanocoumarin
(
References and notes
12) gave a high selectivity as well as antiproliferation
activity against HUVEC. Based on this synthetic trial,
we could find a possibility to improve the potency and
selectivity by adding a new functionality to mother scaf-
fold. Therefore, it can potentially be a selective angio-
genesis inhibitor from further elaboration.
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tain coumarin molecules showed growth inhibition
activity against both cancer cells and HUVEC. Further-
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1
1
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35.

S. Lee et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4596–4599
4599
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1
8
3
NMR (300 MHz, CDCl ): d 7.40 (d, 1H), 6.63 (d, 1H),
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J = 7.2 Hz, 6H); HRMS: calcd for (M+H) C H N O
+
1
4
15
3
2
1
258.1235, found 258.1242. For 6: H NMR (300 MHz,
CDCl ): d 7.79 (d, J = 9.0 Hz, 1H), 6.73 (dd, J = 9.3,
2.7 Hz, 1H), 6.47 (d, J = 2.4 Hz, 1H), 3.49 (q, J = 7.2 Hz,
3
4H), 4.99 (s, 2H), 1.27 (t, J = 7.2 Hz, 6H). For 8: mp 265–
267 °C; IR (KBr): 2210 (w), 1705 (s), 1620 (vs), 1528 (s),
1
1
1
1
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1420 (m). H NMR (300 MHz, CDCl
3
): d 11.3 (s, 1H),
7.89 (m, 1H), 7.32–7.51 (m, 4H), 6.60 (d, J = 6.7 Hz, 1H),
6.5 (s, 1H), 3.48 (q, J = 7.4 Hz, 4H), 1.27 (t, J = 7.4 Hz,
6H). For 10: d 7.88 (dt, J = 6.9, 2.4 Hz, 1H), 7.77 (d,
J = 9.0 Hz, 1H), 7.66 (dt, J = 6.9, 2.4 Hz, 1H), 7.43–7.40
(m, 2H), 6.75 (dd, J = 9.3, 2.4 Hz, 1H), 6.54 (d, J = 2.4 Hz,
1H), 3.51 (q, J = 7.2 Hz, 4H), 1.28 (t, J = 7.2 Hz, 6H). For
12: mp 237–239 °C; IR (KBr): 2221 (m), 1712 (m), 1620
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3
835.
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human cervical carcinoma; DLD-1: human colorectal
adenocarcinoma; SiHa: human cervical carcinoma; NIH
1
3T3: mouse embryonic fibroblast cell line; HUVEC:
human umbilical vein endothelial cell.
(m), 1558 (m), 1504 (s), 1420 (m). H NMR (300 MHz,
CDCl ): d 8.20 (d, J = 7.9 Hz, 1H), 7.87–7.98 (m, 2H),
3
1
1
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1
H NMR (300 MHz, CDCl ): d 8.60 (s, 1H), 6.99 (s, 1H),
3
1
980, 1, 3.
3.23 (m, 4H), 2.64–2.70 (m, 4H), 1.84–1.89 (m, 4H).