Furoxan nitric oxide donor coupled chrysin derivatives: Synthesis and vasculoprotection

Article abstract of DOI:10.1016/j.bmcl.2010.12.077

A series of furoxan-based nitric oxide-releasing chrysin derivatives were synthesized. Pharmacological assays indicated that all chrysin derivatives exhibited in vitro inhibitory activities against aldose reductase and advanced glycation end-product formation. Some chrysin derivatives were also found to increase the glucose consumption of HepG2 cells. Furthermore, the compounds released a low amount of NO in the presence of l-cysteine (range from 0.20% to 1.89%). These hybrid furoxan-based NO donor chrysin derivatives offer a mutual prodrug design concept for the development of therapeutic or preventive agents for vascular complications due to diabetes.

Full text of DOI:10.1016/j.bmcl.2010.12.077

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1222–1226
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Furoxan nitric oxide donor coupled chrysin derivatives: Synthesis
and vasculoprotection
Xiao-Qing Zou a,b, Sheng-Ming Peng b, Chang-Ping Hu a, , Li-Feng Tan b, Han-Wu Deng a, Yuan-Jian Li a
⇑
a Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha 410078, China
b Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan 411105, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 May 2010
Revised 13 December 2010
Accepted 16 December 2010
Available online 24 December 2010
A series of furoxan-based nitric oxide-releasing chrysin derivatives were synthesized. Pharmacological
assays indicated that all chrysin derivatives exhibited in vitro inhibitory activities against aldose reduc-
tase and advanced glycation end-product formation. Some chrysin derivatives were also found to
increase the glucose consumption of HepG2 cells. Furthermore, the compounds released a low amount
of NO in the presence of L-cysteine (range from 0.20% to 1.89%). These hybrid furoxan-based NO donor
chrysin derivatives offer a mutual prodrug design concept for the development of therapeutic or preven-
tive agents for vascular complications due to diabetes.
Keywords:
Hybrid
Furoxan
Ó 2010 Elsevier Ltd. All rights reserved.
NO donor
Chrysin derivatives
Vasculoprotection
Diabetes is characterized by hyperglycemia and the develop-
ment of specific micro- and macro-vascular complications. Nitric
oxide (NO), endothelium-derived vasodilator, plays a crucial role
in the development of vascular complications.1 NO has diverse po-
tent physiological actions and helps maintain micro- and macro-
vascular homeostasis in the cardiovascular system through several
mechanisms, including vasodilation, inhibition of platelet aggrega-
tion, and modulation of platelet and leukocyte adhesion to the
endothelium.2 Decreased endothelial NO bioavailability and pro-
duction are critical contributors to endothelial dysfunction and
vascular complications in diabetes mellitus.3 Some NO donors are
currently being used to treat certain cardiovascular diseases.4
The most important NO donors are organic nitrates, such as glyc-
eryl trinitrate. However, tolerance for these agents during chronic
therapy is a cause for concern.5 Furoxan (1,2,5-oxadiazole-2-
oxides) derivatives are biologically active compounds that are
capable of releasing nitric oxide in the presence of thiols and lack
of tolerance.6,7 Hybrid NO donor furoxan-based drugs are a novel
type of drug that retains the pharmacological activity of the parent
compound but also has the biological actions of NO.
ing anti-diabetic effects.16–22 Based on the mutual prodrug con-
cept, this study aims to investigate a synthesis approach based
on grafting of an NO-releasing moiety furoxan to chrysin deriva-
tives via a variable spacer. We suppose that the new pharmacody-
namic hybrids may have combined advantages of hypoglycemic
effect with a slow release of NO. This may mimic the effects of
endogenous NO, which improve endothelial dysfunction and pre-
vent the progression of diabetic complications.
Here, we report the synthesis of several NO-donating furoxan
derivatives of chrysin. The NO-releasing capacities and glucose
consumption promotion, as well as aldose reductase (AR) and ad-
vanced glycation end-product (AGE) inhibitory effects of these
derivatives, were explored in vitro. The results will help in further
understanding the potential uses of chrysin NO-donating deriva-
tives for the development of therapeutic and preventive agents
for the treatment of vascular complications of diabetes.
The preparation of furoxan derivatives 8a,b and 9a,b follows
the synthetic routes illustrated in Scheme 1. For our study,
hydroxybenzaldehyde was reduced with potassium borohydride
to obtain hydroxybenzyl alcohols 4a and 4b. Sodium nitrate was
added to cinnamyl alcohol under acidic conditions and thionyl
chloride was used for chlorination to obtain 6. Compounds 7a
and 7b were synthesized through a reaction between 4a,b and 6
in acetonitrile. Compounds 8a and 8b were then obtained through
esterification of 7-carboxymethyl chrysin 2. Finally, acetone was
used as a solvent and potassium carbonate as a base to obtain 9a
and 9b through a reaction with acetic anhydride.
Chrysin (5,7-dihydroxyflavone), a natural, widely distributed
flavonoid, has diverse biologically active properties, including anti-
oxidative,8,9 anticancer,10 anxiolytic,11 anti-inflammatory,12,13
anti-diabetic,14 and anti-glucosidase characteristics.15 Recently,
several researchers attempted to modify chrysin properties and
found that some chrysin derivatives have diverse activities, includ-
⇑
The synthesis of compounds 10 and 11 was accomplished
according to the general pathway illustrated in Scheme 2.
Corresponding author. Tel.: +86 731 8235 5079; fax: +86 731 8235 5078.
E-mail address: huchangping@yahoo.com (C.-P. Hu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.077

X.-Q. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1222–1226
1223
HOOCCH2O
O
O
HO
O
O
(1)
,K2CO3
BrCH2COOC2H5
(2)
/KOH
CH3OH
OH
OH
2
1
DCC/DMAP
CH2Cl2
KBH4
C2H5OH
CHO
CH2OH
HO
HO
4a-4b
m OH
3a-3b
CH2OH
H2C
N
O
Ph
K2CO3
KI
3a
3b
4a
4b
m OH
p
p
OH
OH
N
O
O
7a-7b
CH2Cl
O
(1)
NaNO2
/AcOH
Ph
C
H
CH
CH2OH
7a
7b
m OR
OR
SOCl
(2)
2
N
N
p
O
5
6
O
C
O
C
O
O
O
O
O
O
H2C
H2C
(CH3CO)2O
H2C
N
O
CH2O
H2C
N
O
CH2O
Ph
Ph
K2CO3 CH3COCH3
N
N
H3CCOO
OH
O
O
O
O
9a-9b
8a-8b
9a
9b
8a
8b
m OR
OR
m OR
p
p
OR
Scheme 1. Synthesis of target chemical compounds (8a,b and 9a,b).
HOH2C
Ph
O
C
HOOCCH2O
O
O
CH2O
N
H2C
O
O
O
Ph
N
N
O
O
N
DCC/DMAP,CH2Cl2
O
O
OH
OH
2
10
O
C
CH2O
Ph
O
H2C
O
O
(CH3CO)2O
K2CO3 CH3COCH3
N
N
O
O
H3CCOO
11
Scheme 2. Synthesis of target chemical compounds 10 and 11.
7-Carboxymethyl chrysin 2 and furoxan were used to obtain the
chemical compound 10 of chrysin coupled with furoxan through
dehydration under DCC/DMAP action. With the use of potassium
carbonate as base and acetone as solvent, 11 was obtained by
reacting with acetic anhydride.
In the present study, we performed AR and AGE assays as de-
scribed previously.16 NO-releasing capacities were also determined
for all the target compounds in vitro.16 Structure–activity relation-
ships acquired from the assays indicated that the 5-OH group was
the key group responsible for the high inhibitory effects on AR
tubular cells23 and ameliorates AGE-induced dysfunction of endo-
thelial progenitor cells.24 In our study, SNP was verified to inhibit
the AGE formation effectively but the intermediate compounds
4-phenyl-3-methanol-furoxan showed no effect on in vitro AGE
formation, the possible reason is that there is no sufficient sulfhy-
dryl in the AGE preparation system which affects the release of NO
from furazan. These results indicate that NO donors should play a
protective role on chronic vascular complications induced by AGE
in type 2 diabetes. NO was released from all the chrysin derivatives
upon incubation with phosphate-buffered saline solution (PBS at
(5-OH group: IC50 = 0.22 0.01 to 0.75 0.06
lM
vs 5-acetyl
pH 7.4) in the presence of L-cysteine. The percentage of released
group: IC50 = 0.90 0.02 to 1.93 0.07 M). The potency of AR
l
NO varied from 0.20 0.03% to 1.89 0.03%, indicating a slow
release. Correspondingly, the NO releasing from 4-phenyl-3-meth-
anol-furoxan was also lower (0.41 0.02%). In contrast, the amount
of NO released from SNP was substantially higher (10.42 1.80%).
These results indicate that the release of adequate amounts of NO
to provide protective effects was balanced with the concentration
range required for the sufficient activity of chrysin derivatives.
Turnbull et al. found that NO release from furoxan–aspirin
hybrids was undetectable in buffer alone, but was accelerated in
the presence of either plasma or plasma components, albumin,
glutathione and ascorbate.25 They therefore conclude that the
inhibition was over 10 times higher than the positive reference
compound quercetin, the parent compound chrysin, the intermedi-
ate compounds 4-phenyl-3-methanol-furoxan and the NO releas-
ing positive reference sodium nitroprusside (SNP). The potency of
the AGE formation inhibition was 22.3–33 times higher than that
of the positive reference compound aminoguanidine and was
greater than nitrate-based chrysin derivatives reported recently,16
meanwhile, 5-substituent did not affect the inhibitory effects on
AGE formation. It has been found that SNP treatments significantly
attenuates AGE-induced hypertrophic growth in human renal

1224
X.-Q. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1222–1226
decomposition of furoxan–aspirin hybrids to generate biologically
active NO is catalyzed by endogenous agents which may instil a
potential for primarily intracellular delivery of NO. In our settings,
the furoxan-based nitric oxide-releasing chrysin derivatives
showed better inhibitory effect on AR and AGE in spite of their low-
er NO release rate, which is reasonable because we also found that
chrysin itself had inhibitory activities on AR and AGE. Whether the
decomposition of these furoxan–chrysin hybrids to generate bio-
logically active NO also need to be catalyzed by endogenous agents
deserves to be further investigated (Table 1).
Hyperglycemia increases the risk of both micro- and macro-vas-
cular diseases in correspondingly diabetic patients.26 The metabo-
lism of excess glucose drives several damage pathways, such as
oxidative stress, generation of AGE, increased AR-related polyol
pathway flux, activation of protein kinase C, and increased
hexosamine pathway flux, which leads to endothelial dysfunction.
This indicates that hyperglycemia is a major cause of vascular com-
plications in diabetes. To evaluate the hypoglycemic effects of the
target compounds, we determined the glucose consumption of
HepG2 cells treated with chrysin, chrysin derivatives, 4-phenyl-
3-methanol-furoxan, SNP and a positive reference rosiglitazone.
The results showed that 8a, 8b, 10, 4-phenyl-3-methanol-furoxan,
SNP and rosiglitazone significantly promoted glucose consumption
of HepG2 cells compared to the blank vehicle 0.1% DMSO (P <0.05).
The structure-activity relationships acquired for this assay indi-
cated that the 5-OH group is the key influential factor on glucose
consumption of HepG2 cells. In contrast, chrysin significantly
reduced glucose consumption (P <0.05 vs 0.1% DMSO, Fig. 1). Com-
pounds 9a, 9b, and 11 had no effect on the glucose consumption of
HepG2 cells. It has been shown that acute infusion of SNP increases
Table 1
Structure and in vitro AR and AGE inhibition and NO releasing properties of the target compounds
O
O
O
R2
O
R1
R1=H,CH3CO
H2C
Ph
O
X
R2 =
N
N
O
O
O
O
X=CCH2,
CH2COCH2Ph
Compound
R1
R2
IC50
(lmol/L)
%NO releasedc
AR inhibitiona
AGE inhibitionb
0.1% DMSO
/
/
/
Chrysin
8a
9a
8b
9b
H
H
OH
7.78 0.57
0.22 0.01
1.62 0.16
0.40 0.05
1.93 0.07
0.75 0.06
0.90 0.02
63.54 6.42
37.50 7.92
26.39 2.05
36.59 16.55
25.38 1.97
35.9 11.3
/
X@CH2COOCH2Ph(m)
X@CH2COOCH2Ph(m)
X@CH2COOCH2Ph(p)
X@CH2COOCH2Ph(p)
X@COCH2
0.76 0.13
1.89 0.03
0.75 0.08
0.85 0.01
0.24 0.01
0.20 0.03
CH3CO
H
CH3CO
H
10
11
CH3CO
X@COCH2
31.45 8.95
OH
H2C
Ph
N
4-Phenyl-3-methanol-furoxand
P10
P1000
0.41 0.02
N
O
O
SNPe
7.71 1.11
25.87 0.37
10.42 1.80
OH
OH
HO
O
O
Quercetinf
2.85 0.04
OH
OH
NH
Aminoguanidineg
826.22 9.26
H2N HN
NH2
Each value represents the mean SD (n = 3).
a
The concentration required for a 50% inhibition of the decrease in the optical density of NADPH at 340 nm relative to 0.1% DMSO. IC50 values were calculated from the
dose inhibition curve.
b
The concentration required for a 50% inhibition of the fluorescence intensity of AGE relative to 0.1% DMSO. IC50 values were calculated from the dose inhibition curve.
Percent of nitric oxide released based on a theoretical maximum release of 1 mol of NO/mol of the target compounds.
4-Phenyl-3-methanol-furoxan was the intermediate compounds for the synthesis of NO-releasing chrysin derivatives.
Sodium nitroprusside (SNP) was used as NO releasing positive reference.
Quercetin was used as positive control for AR inhibition test.
Aminoguanidine was used as positive control for AGE inhibition test.
c
d
e
f
g

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compounds (10µmol/L)
lmol/L chrysin, chrysin derivatives (8a,b, 9a,b, 10 and 11), 4-
phenyl-3-methanol-furoxan, SNP and rosiglitazone on HepG2 glucose consumption
(values are mean SD; n = 3; ⁄P < 0.05 vs 0.1% DMSO. Differences between
individual groups were analyzed by using ANOVA followed by Dunett’s test).
Figure 1. Effects of 10
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leg glucose uptake at rest in patients with type 2 diabetes.27 The
present study indicated that the NO donor 4-phenyl-3-methanol-
furoxan and SNP significantly promoted glucose consumption of
HepG2 cells. These findings provide a rationale that NO may there-
fore be an important mediator of peripheral glucose disposal and
the potential therapeutic role of NO donor on chronic vascular
complications in diabetic patients warrants more attention.
An O7-furoxan-based NO donor moiety was attached to chrysin.
The AR, AGE formation, and glucose consumption assays, as well as
NO-releasingcapacities, showedthat:(i) theparentcompoundchry-
sin and the NO releasing positive reference SNP are potent AGE
inhibitors; (ii) the intermediate compounds 4-phenyl-3-methanol-
furoxan and the NO releasing positive reference SNP are strong
promoter on glucose consumption of HepG2 cells; (iii) all the hybrid
prodrugs release NO slowly upon incubation with PBS in the pres-
Further reading
28. 3-(Methylol phenoxymethyl)-4-phenyl-1,2,5-oxadiazole-2-oxide acylation
coupling-carboxymethyl chrysin (8a, 8b): 8a (69.2% yield, mp: 134–135 °C).
1H NMR (CDCl3) d (ppm): 12.70 (s, 1H), 7.76–7.82 (m, 4H), 7.45–7.51 (m, 6H),
7.27–7.29 (d, 2H), 6.91–6.93(m, 2H), 6.62 (s, 1H), 6.43–6.44 (d, 1H), 6.29–6.30
(d, 1H), 5.15(s, 2H), 5.02(s, 2H), 4.67 (s, 2H). EI-MS (m/z): 592. Anal. Calcd for
C
33H24N2O9: C, 66.89; H, 4.08; N, 4.73. Found: C, 66.58; H, 4.42; N, 4.37.
Compound 8b (68% yield, mp: 155–156 °C). 1H NMR (CDCl3) d (ppm): 12.76 (s,
1H), 7.87 (s, 4H), 7.55 (s, 6H), 7.32 (s, 1H), 6.97–7.02 (d, 3H), 6.69 (s, 1H), 6.53 (s,
1H), 6.39 (d, 1H), 5.25 (s, 2H), 5.10 (s, 2H), 4.80 (s, 2H), EI-MS (m/z): 592. Anal.
Calcd for C33H24N2O9: C, 66.89; H, 4.08; N, 4.73; Found: C, 66.51; H, 4.45; N,
4.34. Characteristic of O5-acetyl-O7-Chrysin acetic acid[3-(4-phenyl-1,2,
5-oxadiazole-2-oxide-3-)methoxyl-]benzyl ester (9a, 9b): 9a (31.5% yield,
mp: 119–121 °C). 1H NMR (CDCl3) d (ppm): 7.83–7.84 (m, 4H), 7.50–7.57 (m,
6H), 7.30–7.32 (d, 2H), 6.95–6.97 (d, 2H), 6.81–6.82 (d, 1H), 6.65 (s, 2H), 5.21 (s,
2H), 5.05 (s, 2H), 4.75 (s, 2H), 2.43 (s, 3H). EI-MS (m/z): 634. Anal. Calcd for
ence of L-cysteine; (iv) all the furoxan based derivatives have high
inhibitory effects on AR and AGE formation; (v) the 5-OH group is
the key group that determines the inhibition of AR and promotion
of glucose consumption; (vi) O7-[3-(methylol phenoxymethyl)-
4-phenyl-1,2,5-oxadiazole-2-oxide carboxymethyl acylation] 8a,
8b and O7-[3-methylol-4-phenyl-1,2,5-oxadiazole-2-oxide car-
boxymethyl acylation] analog 10 significantly promote glucose
consumption of HepG2 compared to the blank vehicle 0.1% DMSO.
In conclusion, these hybrid furoxan-based NO donor derivatives
offer a mutual prodrug design concept for the development of ther-
apeutic or preventive agents for vascular complications due to
diabetes.
C
35H26N2O10: C, 66.24; H, 4.13; N, 4.41. Found: C, 66.62; H, 4.29; N, 4.05.
Compound 9b (47.3% yield, mp: 165–166 °C). 1H NMR (CDCl3) d (ppm): 7.83–
7.84 (m, 4H), 7.50–7.56 (m, 6H), 7.29–7.31(d, 1H), 7.00 (s, 2H), 6.93–6.95(d, 1H),
6.85 (s, 1H), 6.67 (s, 1H), 6.60 (s, 1H), 5.24 (s, 2H), 5.08 (s, 2H), 4.80 (s, 2H), 2.43
(s, 3H), EI-MS (m/z): 634. Anal. Calcd for C35H26N2O10: C, 66.24; H, 4.13; N, 4.41.
Found: C, 66.64; H, 4.26; N, 4.03. Characteristic of O7-chrysin acetic acid(4-
phenyl-1,2,5-oxadiazole-2-oxide-3-)methyl ester 10 (1.53 g, 63% yield, mp:
174–175 °C). 1H NMR (CDCl3) d (ppm): 12.76 (s, 1H), 7.87–7.89 (m, 2H), 7.63–
7.65 (m, 2H), 7.49–7.57 (m, 6H), 6.68 (s, 1H), 6.46–6.47 (d, 1H), 6.32–6.33
(d, 1H), 5.31 (s, 2H), 4.76 (s, 2H), EI-MS (m/z): 486; Anal. Calcd for C26H18N2O8: C,
64.20; H, 3.73; N, 5.76. Found: C, 64.07; H, 3.98; N, 5.38. Characteristic of O5-
acetyl-O7-Chrysin acetic acid(4-phenyl-1,2,5-oxadiazole-2-oxide-3-)methyl
ester 11 (0.23 g, 43.5% yield, mp: 179–180 °C). 1H NMR (CDCl3) d (ppm):
7.86–7.88 (m, 2H), 7.65–7.66 (d, 2H), 7.50–7.53 (m, 6H), 7.26 (s, 1H), 6.64–6.66
(d, 2H), 5.33 (s, 2H), 4.78 (s, 2H), 2.43 (s, 3H), EI-MS (m/z): 528. Anal. Calcd for
Acknowledgments
We are grateful to the Hunan Province office of Education Funds
(10 C1288) and Dr. Xiangtan University Start Funds (06KZ|
KZ08035) for financial support for this research.
C
28H20N2O9: C, 63.64; H, 3.81; N, 5.30. Found: C, 63.93; H, 3.97; N, 4.96.
29. Glucose consumption assays: HepG2 cells were plated into 96-well tissue
culture plates with some wells left blank. After the cells reached 80–90%
confluence, the medium was removed and the same culture medium
containing target compounds, chrysin or rosiglitazone (standard product)
was added to wells. After 24 h of treatment, glucose was assayed with
diagnostic kits, a MTT assay was used to monitor the cell number and to
adjust the glucose consumption values. [Zou, X. Q.; Peng, S. M.; Hu, C. P.; Tan,
L. F.; Yuan, Q.; Deng, H. W.; Li, Y. J. Bioorg. Med. Chem. 2010, 18, 3020.]
30. AR inhibitory activity: The inhibitory activity of the compounds on aldose
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l
0.24 mM NADPH, 0.4 M Li2SO4 and 2.5 mM of glyceraldehyde (substrate)
were added. The reaction was initiated by addition of glyceraldehyde and the

1226
X.-Q. Zou et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1222–1226
decrease in the optical density of NADPH at 340 nm was recorded for 3 min.
[Zou, X. Q.; Peng, S. M.; Hu, C. P.; Tan, L. F.; Yuan, Q.; Deng, H. W.; Li, Y. J.
Bioorg. Med. Chem. 2010, 18, 3020.]
32. Detection of nitrite: A solution of the appropriate compound was added to
2 mL of 1:1 v/v mixture of 50 mM PBS (pH 7.4) with MeOH, containing of
5 Â 10À4
M L-cysteine. The final concentration of target compounds was
31. Inhibitory activity of AGE formation: To prepare the AGE reaction solution,
10 mg/mL of bovine serum albumin in 50 mM PBS (pH 7.4) was added to
0.2 M glucose, and 0.02% sodium azide was added to prevent bacterial growth.
10À4 M. After 1 h at 37 °C, 1 mL of the reaction mixture was treated with
250 lL of Griess reagent. After 10 min at room temperature, the absorbance
was measured at 540 nm. Sodium nitrite standard solutions (10–80 nmol/mL)
were used to construct the calibration curve. The results were expressed as
the percentage of NO released (n = 3) relative to a theoretical maximum
release of 1 mol NO/mol of test compound. [Zou, X. Q.; Peng, S. M.; Hu, C. P.;
Tan, L. F.; Yuan, Q.; Deng, H. W.; Li, Y. J. Bioorg. Med. Chem. 2010, 18, 3020.]
The reaction mixture (950
the target compounds (1–300
fluorescence intensity of AGE was determined by
l
L) was then mixed with various concentrations of
mol/L). After incubating at 37 °C for 14 d, the
LS-55 fluorospec-
l
a
trophotometer (PE, USA) with excitation and emission wavelengths at
350 nm and 420 nm. [Zou, X. Q.; Peng, S. M.; Hu, C. P.; Tan, L. F.; Yuan, Q.;
Deng, H. W.; Li, Y. J. Bioorg. Med. Chem. 2010, 18, 3020.]