Synthesis and promotion angiogenesis effect of chrysin derivatives coupled to NO donors

Article abstract of DOI:10.1016/j.bmcl.2008.12.116

Two types of new chrysin derivatives were prepared by coupling NO donors of alkyl nitrate and furazan derivatives and were fully characterized by ¹H NMR and other techniques. These compounds were tested in human umbilical vein endothelial cells

Full text of DOI:10.1016/j.bmcl.2008.12.116

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1264–1266
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and promotion angiogenesis effect of chrysin derivatives coupled
to NO donors
*
Sheng-Ming Peng, Xiao-Qing Zou, Hua-Lan Ding, Yong-Lan Ding, Yuan-Bin Lin
Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, Hunan 411105, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two types of new chrysin derivatives were prepared by coupling NO donors of alkyl nitrate and furazan
derivatives and were fully characterized by 1H NMR and other techniques. These compounds were tested
in human umbilical vein endothelial cells (HUVECs-12) and all the compounds exhibited cell prolifera-
tion. Notable effects of promoting angiogenesis were observed for all the modified compounds using
chick chorioallantoic membrane (CAM) assay.
Received 2 August 2008
Revised 28 October 2008
Accepted 6 December 2008
Available online 10 January 2009
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Angiogenesis
Chrysin derivatives
Furazan derivatives
Alkyl nitrate
Couple
Angiogenesis, the formation of new blood vessels from pre-
existing vasculature, is a crucial step in the management of ische-
mic disease such as coronary heart disease, ischemic cerebral
embolism and fracture healing, due to its ability of supply oxygen
and nutrients to the ischemia target. Brownlee proposed that the
diabetic patients with vascular injuries in diabetic vascular compli-
cations is one of the main reasons.1 One of the key mediators of
blood vessel formation during development is vascular endothelial
growth factor (VEGF),2 which can stimulate the proliferation and
migration in endothelial cells. Endothelial cells proliferation plays
an essential role in the regulation of various vascular biological
function and diseases.
Recently, considerable fundamental researches and clinical re-
searches indicate that oxidative stress plays a key role in the
pathogenesis of diabetic vascular complications. An early step
of such damage is considered to be the development of an endo-
thelial dysfunction.3 Oxidative stress and diabetic vascular com-
plications are closely related to the development of diabetes,
suggesting that anti-oxidant treatment is expected to become
the prevention and treatment of vascular complications of diabe-
tes new ways.4
dant effects.14 However, research on the role of chrysin deriva-
tives in cardiovascular is rare.15 In order to investigate the
effect of chrysin derivatives in promoting angiogenesis, in this
paper, we made attempts to report that chrysin derivatives were
coupled to different NO donors and their effect on promoting
angiogenesis was evaluated by HUVEC-12 and CAM assay.
To begin our study, chrysin derivatives were coupled to ni-
trate following the synthetic route illustrated in Scheme 1. An
alkyl chain with bromide was introduced to the 7-position of
chrysin (1) selectively by reacting with the corresponding dibro-
mo alkane and yielded the 7-bromo alkane derivatives (2a–2c),
which were subsequently converted to the final nitro ester
(3a–3c) by treatment with AgNO3 in anhydrous acetonitrile.
The synthesis of furazan derivatives is shown in Scheme 2.16 Sat-
urated aqueous sodium nitrite was added to a solution of cin-
namyl alcohol (4) in glacial acetic acid. The resulting reaction
mixture was diluted with water and extracted with diethyl
ether. The combined organic layer was washed with brine and
dried over MgSO4. The volatiles were removed in vacuo and
the residue was recrystallized from dichloromethane–petroleum
ether to give 5. Chrysin derivatives were successfully coupled
to furazan as shown in Scheme 3. To a stirring solution of chry-
sin 1 in acetone, K2CO3 was added and then a mixture of ethyl
bromoacetate and acetone was added dropwise to give com-
pound 6. Compound 6 was treated in KOH solution (methanol)
and after that most of the volatiles were evaporated. The residue
was dissolved in water and the solution was adjusted to pH 2 by
using HCl solution to give 7. Chrysin derivatives 7 was sus-
pended in anhydrous dichloromethane with stirring at room
Chrysin(5,7-dihydroxyflavone) is a naturally wide distributed
flavonoid, which has been reported to have many different bio-
logical activities such as anti-viral,5 anti-cancer,6 anti-bacterici-
dal,7
anti-inflammatory,8
anti-allergic,9
DNA
cleavage,10
vasodilator,11 anti-mutagenic,12 anti-anxiolytic13 and anti-oxi-
* Corresponding author. Fax: +86 732 8292251.
E-mail address: pengshengming@21cn.com (Y.-B. Lin).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.116

S.-M. Peng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1264–1266
1265
Brn(H2C)O
O
O
O
O
O2NOn(H2C)O
O
O
HO
AgNO3
Br(CH2)nBr
CH3CN, lucifugous
K2CO3, acetone
OH
OH
OH
1
2a-2c
3a-3c
2a
2b
2c
n = 2
n = 4
n = 6
3a n = 2
3b n = 4
3c n = 6
Scheme 1. Preparation methods for 3a–3c.
CAM assay was used to examine whether the compounds exhi-
bit an angiogenic activity. General CAM assay: neutral filter paper
was loaded with products (1, 3a, 3b, 3c, 8, ECGF, vehicle or PBS),
respectively, which, after air-drying, were then applied to the
CAM surface of 7-day-old chick embryos. Two days later, an appro-
priate volume of 10% formaldehyde solution was injected into the
9-day-old embryo chorioallantois and angiogenesis was observed
under a microscope (see Fig. 2). Whereas the control vehicle
(0.3% DMSO) and PBS both showed no angiogenic activity in the
HOH2C
NaNO2
Ph
C C CH2OH
H H
N
N
HAc
O
O
5
4
Scheme 2. Preparation methods for 6.
temperature, then furazan derivatives 5, dicyclohexylcarbodiim-
ide (DCC) and 4-dimethylaminopyridine (DMAP) were added.
The reaction solution was stirred for 12 h at room temperature
and dicyclohexylurea (DCU) was formed, which was removed
through filtering. The residue solution was evaporated to get a
pale yellow solid which was recrystallized from dichlorometh-
ane–petroleum ether and washed with K2CO3 solution and water
to give 8. All of the compounds were characterized by means of
1H NMR and MS (EI).17
1.2
**
1.0
0.8
0.6
0.4
0.2
0.0
MTT assay was performed to evaluate the effect of these com-
pounds on HUVECs-12 proliferation. Incubation with the com-
pounds at concentration of 10 lmol/L for 24 h showed promoted
proliferation of HUVECs-12. Generally, HUVECs were split at a den-
sity of 2.5 Â 105 cells/mL in 96-well plates and 200
lL suspension
per well cultured in DMEM containing 10% FCS for 24 h, then
starved by serum deprivation for 24 h, treated with the products
(10
MTT (5 mg/mL) was added and incubated for 4 h. Culture medium
was removed and 150 L DMSO was added to each well. The absor-
l
M) and vehicle (0.1% DMSO) for 24 h.18 Twenty microliter of
Control 0.1%
DMSO
1
3a
3c
8
3b
l
bance (OD) was measured at 490 nm using a microplate spectro-
photometer (Bio-Rad) (see Fig. 1). It was observed that all the
compounds exhibited cell proliferation. However, with a degree
of variation, cell proliferation of compound 3b is the most obvious.
Figure 1. Effects of products (10 lM) on the proliferation of HUVECs-12. The
proliferation of HUVECs-12 was determined by MTT assay. Each value is the
mean SEM of six determinations. **p < 0.05 versus 0.1% DMSO.
O
HO
O
O
C2H5OCH2CO
O
BrCH2COOC2H5
K2CO3, acetone
OH
OH
O
1
6
KOH/CH3OH
O
O
HOH2C
N
Ph
HOCH2CO
O
O
H2C
OCH2CO
Ph
N
O
O
N
O
O
N
O
DCC/DMAP,CH2Cl2
O
OH
OH
8
7
Scheme 3. Preparation methods for 8.

1266
S.-M. Peng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1264–1266
In conclusion, we have prepared two types of chrysin deriva-
tives coupled with NO donors of alkyl nitrate and furazan deriva-
tives, and have tested in HUVEC-12 and CAM assay. The results
indicated that all of the compounds exhibited cell proliferation
in vitro and angiogenic activity in vivo. Our study on the hypogly-
cemic effect of these compounds is ongoing. Further results will be
released in subsequent papers.
Acknowledgment
We are grateful to the Hunan Province Office of Education
Funds (05C079) for financial support.
References and notes
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Granger, H. J. Am. J. Physiol. 1998, 274, H1054.
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A.; Goldfine, A. B.; Gordon, M. B. Circulation 2001, 103, 1618.
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Moreno, L.; López-López, G.; Tamargo, J. J. Pharmacol. Exp. Ther. 2002, 301, 66.
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Behav. 1994, 47, 1.
Figure 2. Effect of products (10 lL/egg) on angiogenesis in the CAM assay.
14. (a) Grassmann, J.; Hippeli, S.; Elstner, E. F. Plant Physiol. Biochem. 2002, 40, 471;
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eggs, the products (3a, 3b, 3c, 8, 10 lL/egg) and the positive control
ECGF showed strong angiogenic activities (D, E, F, G, H). These re-
sults indicated that these compounds showed excellent angiogenic
effects in vivo.
From the cell proliferation and CAM assay experimental results,
it can be seen that angiogenesis was caused partly due to a vascu-
lar endothelial cell proliferation.
Angiogenesis is impaired in conditions where NO activity is
attenuated. Exogenously applied NO donors have been shown to
stimulate endothelial cells (ECs) growth and migration in vitro
and promote angiogenesis. The fact indicates that these com-
pounds merit further investigation as potential pharmaceutical
agents in processes in which angiogenesis plays an important role,
for example, wound healing, fracture repair, peptic ulcers and the
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17. Compound 3a: Mp 135–136 °C; 1H NMR (400 MHz, DMSO-d6, ppm) d 12.79 (s,
1H), 8.09–8.08 (d, J = 8.4 Hz, 2H), 7.60–7.56 (m, 3H), 7.03 (s, 1H), 6.81–6.80 (d,
J = 2.0 Hz, 1H), 6.39–6.38 (d, J = 2.0 Hz, 1H), 4.59 (s, 2H), 4.14 (s, 2H); MS (EI) m/
z: 343.Compound 3b: Mp 119–120 °C; 1H NMR (400 MHz,CDCl3, ppm) d 12.73
(s, 1H), 7.90–7.88 (dd, J = 1.2 Hz, 2H), 7.54–7.52 (m, 3H), 6.67 (s, 1H), 6.50–6.49
(d, J = 2.0 Hz, 1H), 6.36–6.35 (d, J = 2.0 Hz, 1H), 4.56 (s, 2H), 4.09 (s, 2H), 1.96 (s,
4H); MS (EI) m/z: 371.Compound 3c: Mp 105–106 °C; 1H NMR (400 MHz,CDCl3,
ppm) d 12.71 (s, 1H), 7.90–7.88 (m, 2H), 7.57–7.50 (m, 3H), 6.67 (s, 1H), 6.50–
6.49 (d, J = 2.0 Hz, 1H), 6.37–6.36 (d, J = 2.0, 1H), 4.49–4.46 (t, J = 7.0 Hz, 2H),
4.06–4.03 (t, J = 7.0 Hz, 2H), 1.88–1.75 (m, 4H), 1.53–1.50 (m, 4H); MS (EI) m/z:
399.Compound 8: Mp 174–175 °C; 1H NMR (400 MHz, CDCl3, ppm) d 12.77 (s,
1H), 7.89–7.87 (m, 2H), 7.66–7.64 (m, 2H), 7.57–7.49 (m, 6H), 6.69 (s, 1H),
6.48–6.47 (d, J = 2.0 Hz, 1H), 6.34–6.33 (d, J = 2.0 Hz, 1H), 5.32 (s, 2H), 4.77 (s,
2H); MS (EI) m/z: 486.
18. Jiang, D. J.; Jia, S. J.; Dai, Z.; Li, Y. J. J. Mol. Cell. Cardiol. 2006, 40, 529.