Identification of a novel antiangiogenic agent; 4-(N-imidazol-2-ylmethyl)amino benzopyran analogues

Article abstract of DOI:10.1016/S0960-894X(03)00248-8

A series of 4-(N-imidazol-2-ylmethyl)aminobenzopyran analogues, originally designed as K_ATP openers for ischemic diseases, showed antiangiogenic properties through the inhibition of HUVEC tube formation. Especially one of p-Cl substituted analo

Full text of DOI:10.1016/S0960-894X(03)00248-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1661–1663
Identification of a Novel Antiangiogenic Agent;
4-(N-Imidazol-2-ylmethyl)amino Benzopyran Analogues
Nakjeong Kim,b Sunkyung Lee,a Kyu Yang Yi,a,* Sung-eun Yoo,a Guncheol Kim,b
Chong Ock Lee,a Sung Hee Parka and Byung Ho Leea
aMedicinal Science Division, Korea Research Institute of Chemical Technology, 100 Jang-dong,
Yoosung-gu, Taejon 305-600, South Korea
bDepartment of Chemistry, Chungnam National University, Taejon 305-764, South Korea
Received 19 January 2003; accepted 11 March 2003
Abstract—A series of 4-(N-imidazol-2-ylmethyl)aminobenzopyran analogues, originally designed as KATP openers for ischemic
diseases, showed antiangiogenic properties through the inhibition of HUVEC tube formation. Especially one of p-Cl substituted
analogues (4c) completely inhibited HUVEC tube formation at 10 mM. The compound 4c significantly inhibited tumor growth by
52% on A549 (human non small cell lung carcinoma) in nude mice xenografts without any significant side effects.
# 2003 Elsevier Science Ltd. All rights reserved.
Angiogenesis plays critical roles in the pathophysiology
of ischemic and neoplastic disorders, the most common
causes of mortality, as wells as other chronic diseases
including age-related macular degeneration, chronic
lung diseases, neovascular retinopathies, and rheuma-
toid arthritis.1 While we have been working on the
identification of ATP sensitive potassium channel
(KATP) openers2 targeting ischemic diseases such as
myocardial infarction and stroke, some of the com-
pounds related to KATP showed the inhibitory effects on
HUVEC (human umbilical vein endothelial cell) tube
formation unexpectedly, indicating antiangiogenic
properties. We prepared that series of compounds, and
examined their inhibitory effects on HUVEC tube for-
mation, followed by in vivo antitumor activity.
reacted with an optically pure benzopyran epoxide,4 in
the presence of CoCl2 in CH3CN to provide the 4-(N-
imidazol-2-ylmethyl)amino-6-nitrobenzopyrans (3a–3j).
6-Aminobenzopyran derivatives (4a–4j) were obtained
by the catalytic hydrogenation of the 6-nitro derivatives
3a–3j (Scheme 1).
The imidazole analogue of 4-(N-aryl)-substituted ben-
zopyran (BMS-191095) has been reported as a cardio-
selective KATP opener.3 We prepared the same
analogues modified at the 2-position of benzopyran
from gem dimethyl to the acetal, and at the 6-position
from nitrile to the nitro or amino group. N-(1H-imida-
zol-2-ylmethyl)anilines (1) were prepared by the reduc-
tive amination of 2-imidazolecarboxaldehyde with
various 4-substituted anilines using NaBH4, which were
*Corresponding author. Tel.: +82-42-860-7143; fax: +82-42-861-
1291; e-mail: kyyi@krict.re.kr
Scheme 1. Preparation of 4-(N-imidazol-2-ylmethyl)aminobenzopyrans.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00248-8

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N. Kim et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1661–1663
As shown in Table 1, most p-Cl substituted analogues
(3a–3d, 4a–4c) demonstrated inhibitory effects on
HUVEC (primary cultured cells within passage 5 on
Matrigel) tube formation at 50 mM concentration,
respectively. The reduced 6-amino derivatives (4a–4c)
seemed to be more potent than the 6-nitro compounds
(3a–3d). Especially, the compound 4c completely inhib-
ited the tube formation at 50 mM, and even at 10 mM.
At those concentrations, the compound 4c didn’t show
any cytotoxicity on HUVECs. Other p-substituted ana-
logues, both 6-nitro and 6-amino, demonstrated weaker
activities than Cl-substituted compounds.
was injected intraperitoneally at 50 mg/kg once a day
from day 1 to day 20 after A549 implantation in
BALB/c-nu/nu.
No mice were died in both control and treated group
during experiments. As shown in Table 2, retardation or
loss of weight gain was not observed by the treatment of
4c. The compound 4c significantly inhibited
A549 NSCLC growth from 45 days to 61 days after
implantation by 49–52%. The fundamental goal of
antiangiogenic therapy is to return foci of proliferating
microvessels to their resting state, and to prevent their
re-growth.7 The inhibition by 4c was maintained until
61 days without any significant side effects, which gives
the prospective that the compound 4c may prevent the
re-growth of microvessels and tumor.
Although there are many therapeutic opportunities for
the use of antiangiogenic inhibitors in clinic, targeting
cancer has been most extensively investigated.5
A
growing tumor needs an extensive network of capil-
laries to provide nutrients and oxygen. In addition, the
intratumoral blood vessels provide a way for tumor
cells to enter the circulation and to metastasize to dis-
tant organs.6 Therefore, angiogenesis is a crucial step in
tumorigenesis. Approximately 75–80% of lung carci-
nomas are non-small cell lung carcinomas (NSCLC), of
which prognosis remains poor, especially in advanced
diseases.7 It is suggested that it will be difficult to
obtain better results in advanced NSCLC by chemo-
therapy alone, and novel treatment modalities are
urgently needed including antiangiogenesis therapy.
Then we investigated the effect of the compound 4c on
the growth of A549 (human NSCLC) in nude mice
xenograft experiments, primarily. The compound 4c
Further we examined the anti-ischemic effects of the
compounds to find out if those have KATP channel
opening properties or not, according to the published
procedures.4,9
As represented in Table 3, the compound 3b showed
good cardioprotective effects both in vitro and in vivo,
while its vasorelaxant effect was comparably weak (IC50
>30 mM). But the compound 4c neither showed any
cardiopretectve effect nor vasorelaxation. Then, the
antiangiogenic effects of this series of compounds may
not be related with the KATP opening properties. The
compound 4c showed antiangiogenic properties without
any KATP channel opening effects in this study.
Table 1. Inhibitory effects on HUVEC tube formation
X
Stereo (2, 3, 4)
Compd (50 mM)
Inhibitiona
Compd (50 mM)
Inhibition
Cl
SRS
SSR
RSR
RRS
SRS
RSR
RSR
RSR
SRS
RSR
3a
3b
3c
3d
3e
3f
3g
3h
3i
+
++
++
+
+
+
na
na
+
na
4a
4b
4c
4d
4e
4f
4g
4h
4i
++
++
+++
na
na
+
+
+
na
+
OCH3
CF3
Br
OCF3
3j
4j
a
À, control; +, inhibition; ++, significant inhibition; +++, Tubes were not formed; na, not assayed.
Table 2. Antitumor efficacy of the compound 4c on A549a human non-small cell lung carcinoma in nude mice xenografts
1 day
14 day
25 day
35 day
45 day
61 day
Body weightb (g)
Control
4c treatedc
22.50Æ0.34
22.30Æ0.29
25.16Æ0.26
25.42Æ0.30
25.43Æ0.45
26.17Æ0.38
25.88Æ0.48
27.10Æ0.42
Tumor volumed (mm3)
Control
4c treated
Inh. (%)
152.9Æ28.7
92.2Æ20.9
39.7
308.6Æ54.8
186.8Æ36.4
39.5
483.9Æ105.0
244.3*Æ47.0
49.5
1034.9Æ183.0
494.8*Æ66.7
52.2
aA549 was implanted sc into the right flanks of 8-week old nude mice (BALB/c-nu/nu).
bEach group consisted of 8 mice. Values represent meanÆS.E.
cNude mice were injected ip with 4c (50 mg/kg) or vehicle (phosphate buffered saline containing 0.5% tween80), once daily from day 1 to day 20
after implantation.
dSignificance was determined by student t test (p<0.05). Inhibition (%) was calculated as (1ÀT/C)Â100, where T and C were the mean tumor
volume of treated and control group, respectively.

N. Kim et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1661–1663
Table 3. Vasorelaxant potencies and cardioprotective effects
1663
Vasoa IC50 (mM)
In vitro cadioprotection (10 mM)b
In vivo anti-infarctionc (0.3 mg/Kg)
(IZ/AAR%/AAR/LV%)
LVDPÂHR(%)
EDP (mmHg)
TTC (min)
LDH (U/g)
vehicle
3b
4c
23.0
55.7
19.3
43.4
14.0
62.3
20.3
28.0
23.5
29.9
10.7
nad
61/40
41/33
nad
>30
>30
aVasorelaxant potency was assessed by measurement of IC50 for inhibition of methoxamine-contracted rat aorta. IC50 value is represented as a mean
of 3 experiments.
bCardioprotective effects were evaluated by measuring the contractile function (LVDPÂHR), TTC, and LDH in the globally ischemic rat heart.
Each value is an average of 3 experiments.
cIn vivo anti-infarction effects were determined by measuring a ratio of myocardial infarct zone to area at risk (IZ/AAR) in ischemic myocardium
damage rat model (0.3 mg/kg), n=3. Each value given is an average and withinÆ10%.
dNot assayed.
References and Notes
From this study we identified a novel chemical
class8 of angiogenesis inhibitors originally designed
1. Semenza, G. L. Annu. Rev. Med. 2003, 54, 17.
2. Coghlan, M. J.; Carroll, W. A.; Gopalakrishnan, M. J.
Med. Chem. 2001, 44, 1627.
3. Rovnyak, G. C.; Ahmed, S. Z.; Ding, C. Z.; Dzwonczyk,
S.; Ferrara, F. N.; Humphreys, W. G.; Grove, G. J.; Santa-
fianos, D.; Atwal, K. S.; Baird, A. J.; McLaughlin, L. G.;
Normandin, D. E.; Sleph, P. G.; Traeger, S. C. J. Med. Chem.
1997, 40, 24.
4. Yoo, S.; Yi, K. Y.; Lee, S.; Suh, J. K. N.; Lee, B.; Seo,
H. W.; Kim, S.-O.; Lee, D.-H.; Lim, H.; Shin, H. S. J. Med.
Chem. 2001, 44, 4207.
5. Sepp-Lorenzino, L.; Thomas, K. Exp. Opin. Investig. Drugs
2002, 11, 1447.
as a KATP opener targeting ischemic diseases, and
confirmed its in vivo antitumor activity. We are
going to prepare more analogues of the compound
4c and investigate their antiangiogenic potencies to
study the structure–activity relationships of this
novel class of compounds, and to find more potent
compounds. In addition, we will continuously study
the antitumor activity of the compound 4c through
the optimization of dosing schedule, extension of
tumor cell lines, and the combination with chemo-
therapeutic agents as well as its mechanism on
antiangiogenic activity, pharmacokinetic profiles, and
toxicity.
6. Liekens, S.; De Clercq, E.; Neyts, J. Biochem. Pharmacol.
2001, 61, 253.
7. Ferreira, C. G.; Huisman, C.; Giaccone, G. Clinical
Reviews in Oncology and Hematology 2002, 41, 57.
8. Hamby, J. M.; Showalter, H. D. Pharmacol. Ther. 1999, 82,
169.
9. D’Alonzo, A. J.; Darbenzio, R. B.; Sewter, J. C.; Hess,
T. A.; Grover, G. J.; Sleph, P. G.; Normandin, D. E.; Lodge,
N. J. Eur. J. Pharmacol. 1995, 294, 271.
Acknowledgements
We are greatful to the Ministry of Science and
Technology of Korea for financial support of this
research.