Flavonoids as vasorelaxant agents: Synthesis, biological evaluation and quantitative structure activities relationship (QSAR) studies

Article abstract of DOI:10.3390/molecules16108257

A series of 2-(2-diethylamino)-ethoxychalcone and 6-prenyl(or its isomers)-flavanones 10a,b and 11a-g were synthesized and evaluated for their vasorelaxant activities against rat aorta rings pretreated with 1 μM phenylephrine (PE). Several compounds showe

Full text of DOI:10.3390/molecules16108257

Molecules 2011, 16, 8257-8272; doi:10.3390/molecules16108257
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Flavonoids as Vasorelaxant Agents: Synthesis, Biological
Evaluation and Quantitative Structure Activities Relationship
(QSAR) Studies
Xiaowu Dong 1, Yanming Wang 1, Tao Liu 1, Peng Wu 1, Jiadi Gao 1, Jianchao Xu 1, Bo Yang 2
and Yongzhou Hu 1,*
1
ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou 310058, Zhejiang, China
2
Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, Zhejiang, China
* Author to whom correspondence should be addressed; E-Mail: huyz@zju.edu.cn;
Tel./Fax: +86-571-88208460.
Received: 29 July 2011; in revised form: 14 September 2011 / Accepted: 16 September 2011 /
Published: 28 September 2011
Abstract: A series of 2-(2-diethylamino)-ethoxychalcone and 6-prenyl(or its
isomers)-flavanones 10a,b and 11a–g were synthesized and evaluated for their
vasorelaxant activities against rat aorta rings pretreated with 1 μM phenylephrine (PE).
Several compounds showed potent vasorelaxant activities. Compound 10a (EC50 = 7.6 μM,
Emax = 93.1%), the most potent one, would be a promising structural template for
development of novel and more efficient vasodilators. Further, 2D-QSAR analysis of
compounds 10a,b and 11c-e as well as thirty previously synthesized flavonoids 1-3 and
12-38 using Enhanced Replacement Method-Multiple Linear Regression (ERM-MLR) was
further performed based on an optimal set of molecular descriptors (H5m, SIC2, DISPe,
Mor03u and L3m), leading to a reliable model with good predictive ability (Rtrain2 = 0.839,
2
2
Qloo = 0.733 and Rtest = 0.804). The results provide good insights into the structure-
activity relationships of the target compounds.
Keywords: flavonoids; molecular descriptors; vasorelaxant agents; qsar; erm-mlr

Molecules 2011, 16
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1. Introduction
Flavonoids, a class of plant secondary metabolites, are polyphenols based around a
phenylbenzopyrone structure [1,2]. According to their different skeletons, they are categorized into
flavones, flavanones, chalcones, flavonols, isoflavones and aurones, etc. [3]. Associations of dietary
flavonoid intake with lower incidence of cardiovascular disease have been reported in several
epidemiological studies [4-7]. Since one of the main causes of cardiovascular diseases is the
involvement of increasing tonicity or loss of relaxation capacity of vascular tissues, vasodilators are a
beneficial treatment for cardiovascular diseases. Until now, a large number of flavonoids such as
quercetin, luteolin and apegenin (Figure 1A) have been found to show vasorelaxant activities [8-10]. In
addition, flavonoids can reduce the superoxide levels of vascular endothelium under oxidative stress
conditions and improve endothelial cell disfunction, which is also crucial for treatment of
cardiovascular diseases [11,12].
Previously, some quercetin analogues were synthesized by our group and evaluated for their
vasorelaxant activities, the results of which indicated that the LogP values of the synthesized
flavonoids were correlated with their vasorelaxant activities [13]. In order to further investigate the
effect of lipophilic change on vasorelaxant activity, the prenyl (or allyl) group was introduced into
various flavonoid scaffolds (e.g., chalcones, flavanones, flavones and aurones). Some of them exhibited
potent vasorelaxant activity, such as 8-prenyl (or allyl)-flavanone derivatives 1, 2 and chalcone
derivative 3 (Figure 1B; the EC50 values of 1, 2 and 3 are 9.3, 4.6 and 24.0 μM, respectively) [14-16].
These results prompted us to discover more potent lipophilic flavonoid derivatives and investigate the
comprehensive structure-activity relationship of these compounds.
Figure 1. The 2D structure of the flavonoids with potent vasorelaxant activities.
In this study, 6-prenyl(or its isomers)-flavanones and 2-(2-diethylamino)-ethoxychalcone
derivatives 10a,b and 11a–g (Figure 2) were prepared, considering the effect of prenyl(or its isomers)
in the C-6 position of flavanones as well as the introduction of 2-(diethylamino)ethyl groups in
chalcones. The vasorelaxant activities of the synthesized flavonoids were assayed on rat-aorta rings
pretreated with 1 μM phenylephrine (PE). Furthermore, Enhanced Replacement Method-Multiple
Linear Regression (ERM-MLR) was applied to select the most optimal set of molecular descriptors
and set up a linear model to probe the quantitative structure-activity relationships (QSAR) of the
target compounds.

Molecules 2011, 16
Figure 2. The structures of the target flavonoids synthesized in this study.
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2. Results and Discussion
2.1. Chemistry
The synthetic pathway to the nine prenylflavonoids 10a,b and 11a–g is outlined in Scheme 1.
Acetophenone 4 was allylated with prenyl bromide and successively heated at 220 °C to afford Claisen
rearrangement products 5. Condensation of 5 with the corresponding benzaldehydes in aqueous
alcoholic alkali at room temperature afforded chalcones 6. Cyclization of 6 in a solution of sodium
acetate in ethanol under reflux conditions gave flavanone 7. Compound 9 was obtained by the
treatment of chalcone 8 with 2-chloro-N,N-diethylethanamine hydrochloride in the presence of
potassium carbonate in acetone. Deprotection of the methoxymethyl groups of 7a-c, 7f, 7g and 9a,b
were carried out in 3N HCl/MeOH/THF (2:5:5, v/v) to give the expected products 11a-c, 11f, 11g and
10a,b, while demethoxymethylation of compounds 7d and 7e in 3N HCl/MeOH (1/1, v/v) was
observed to afford dihydropyranoflavones 11d and 11e bearing cyclic prenyl groups. The structures of
all the synthesized compounds were elucidated by 1H-NMR and ESI-MS.
2.2. Vasorelaxant Activity and SAR
Vasorelaxant activities of compounds 10a,b and 11a-g were evaluated against aortic rings with
endothelium pre-contracted with 1 μM phenylephrine. The promoted relaxation of all the compounds
was dose-dependent manner, with the maximal effect observed at 300 μM. Several compounds showed
more potent vasorelaxation than the positive control quercetin based on the result of either Emax or
EC50. As shown in Table 1, flavonoids 10a,b and 11c-e inducing 50% relaxation at small concentration
(EC50 < 100 μM) with good efficacy (Emax > 90%) were considered to be good relaxing agents, while the
remaining compounds 11a,b,f and 11g were regarded as weak vasodilators (EC50 > 100 μM; Emax < 70%).
Concentration–relaxation curves of the flavonoids in two categories are shown in Figure 3. The effects
on vasorelaxant activities of prenyl (or its isomer) on C-6 of flavonoids were investigated, showing
that the introduction of a cyclic prenyl group resulted in good vasorelaxant activity, as exemplified in
dihydropyranoflavones 11d and 11e (11d: EC50 = 78.7 μM, Emax = 93.5%; 11e: EC50 = 53.5 μM,
Emax = 93.6%). The introduction of a 6-prenyl or 8-(1,1-dimethyl)allyl group on A ring of flavanone
(e.g., 11a,b,f and 11g) led to the weak to moderate activity, except for compound 11c. The

Molecules 2011, 16
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2-(2-diethylamino)ethoxychalcone derivatives 10a,b showed better vasorelaxant activities (EC50 of
10a and 10b were 7.6 and 13.7 μM, respectively), indicating that replacement of prenyl with a
2-(diethylamino)ethyl group on chalcone skeletons retained the potent activity.
Scheme 1. Synthesis of prenylflavonoids 10a,b and 11a–g.
Reagents and conditions: (a) prenyl bromide, K2CO3, acetone; (b) N,N-dimethylaniline, N2, 190 °C;
(c) appropriate benzaldehyde, 10% KOH (H2O/EtOH); (d): NaOAc, EtOH, reflux; (e)
2-chloro-N,N-diethylethanamine hydrochlororide, potassium carbonate, acetone; (f) 3N HCl,
MeOH, reflux; (g): 3N HCl, MeOH, THF, 50 °C.
Table 1. The vasorelaxant activities of flavonoids 10a,b and 11a-g, in rat aorta rings
pre-contracted with PE.
Compd.
Compd. EC50(μM) Emax(%)
EC50(μM) Emax(%)
Quercetin 244
91.3
93.1
99.5
66.2
50.6
11c
11d
11e
11f
11g
19.9
78.7
53.5
N.D.
N.D.
90.3
93.5
93.6
52.9
49.1
10a
10b
11a
11b
7.6
13.7
N.D.
N.D.

Molecules 2011, 16
Figure 3. Effects of flavonoids on relaxation in aortic rings with endothelium
8261
pre-contracted with 1 μM phenylephrine. Flavonoids were added cumulatively to achieve
the appropriate concentrations. Results are expressed as means ± standard error of mean in
terms of percentage relaxation of the contraction to PE (n = 3 ~ 4). (A) Flavonoids with
highly vasorelaxation effect; (B) Flavonoids with weakly vasorelaxation effect.
2.3. Development of QSAR Model Using ERM-MLR
For a quantitative understanding of the structure-vasorelaxant activities relationship of the
flavonoids, ERM-MLR was applied to set up a linear QSAR model to explore the EC50 of the newly
synthesized five flavonoids 10a,b and 11c-e with efficacious EC50 and the previously reported thirty
flavonoids 1-3 and 12-38 [14-16,19]. At first, the calculated molecular descriptors that stayed constant
for all molecules were eliminated and one of each correlated pair was deleted according to the results
of correlation analysis, resulting in a pool of 299 descriptors for further QSAR model development.
Then, ERM-MLR was applied to select an optimal set of molecular descriptors and four QSAR models
with different number of variables (n = 2~5) were obtained considering that the number of variables
selected should be kept lower than 20 percent of the number of coupounds in training set in avoidance
of overlay problem. Commonly, the higher values of correlation coefficient (R2) for both training and
test set, leave-one-out (loo) cross correlation coefficient (Qloo2) and the lower standard deviation value
(SD), the better the results. Thus, five molecular descriptors in ERM-MLR model has an optimal
influence on improving correlation (Table 2). The physical-chemical meanings of the molecular
descriptors in the best model are listed in Table 3, forming the equation as given in Equation (1):
−Log(EC50) = 0.442*H5m − 0.465*SIC2 + 0.287*DISPe − 0.518*Mor03u − 0.574*L3m + 4.376 (1)
Table 2. The statistical parameters of obtained ERM-MLR models.
ERM-MLR
No.
n a
2
2
2
Rtrain
Qloo
Rtest
1
2
3
4
0.603
0.734
0.786
0.839
0.483
0.648
0.705
0.733
0.366
0.336
0.527
0.804
n = 2
n = 3
n = 4
n = 5
a The number of molecular descriptors selected in ERM-MLR QSAR models.

Molecules 2011, 16
Table 3. The molecular descriptors involved in the ERM-MLR models and their
8262
corresponding definition.
Symbol Class
Definition
H5m
GETAWAY descriptors H autocorrelation of lag 5/weighted by atomic masses
structural information content (neighborhood symmetry of
2-order) information indices
SIC2
information indices
d COMMA2 value/weighted by atomic Sanderson
DISPe
geometrical descriptors
electronegativities
Mor03u 3D-MoRSE descriptors Mor03u 3D-MoRSE - signal 03/unweighted
3rd component size directional WHIM index/weighted by
L3m
WHIM descriptors
atomic masses
The quality of the fit for the resulting model [Equation (1)] can be judged by the determination
2
coefficient (Rtrain = 0.839), the standard deviation (SD = 0.263) and the Fishers estimate of statistical
significance value (F = 21.9). Its predictive ability is characterized by leave-one cross validation
2
2
(Qloo = 0.733, SD = 0.303) and test set validation (Rtest = 0.804, SD = 0.145). The predictions
provided by Equation (1) are shown in Table 4. In Figure 4A the predictions in function of the
experimental coefficients are plotted, suggesting that the thirty-five data points (overall data set)
follow a straight line trend. In addition, Figure 4B shows the graph between the residuals vs.
experimental vasorelaxant activity values. The lower residuals obtained in both the training and test set
of compounds in the model indicated that the developed model are valuable and has the capability to
establish the relationship between the structure and vasorelaxant activity for flavonoids used in this
study. For example, the vasorelaxant activities of the highly potent compound 10a and 11d were
accurately predicted using the established QSAR model. The values of H5m, SIC2, DISPe, Mor03u
and L3m are optimal for Equation(1), contributing to the fact that the prediction activity is as good as
its actual activity (predictive –log(EC50) of 10a: 5.245; actual −log(EC50) of 10a: 5.119), while the
unfavorable values of H5m, SIC2, DISPe, Mor03u and L3m led to the lower prediction activity
compared with compound 10a but close to its actual activity (predictive –log(EC50) of 11d: 4.048;
actual −log(EC50) of 11d: 4.104). However, inaccurate prediction of several compounds was also
observed. As exemplified by compound 38, the weak prediction [predictive –log(EC50) of 38: 3.399]
may due to the weak activities exhibited by flavone skeleton compounds [actual −log(EC50) of
compounds 33~37: <4.088], but compound 38 actually exhibited moderate activity [actual −log(EC50) of
38: 4.290], resulting in the worst fit of 38 (residual: 0.891). In fact, this problem would be fixed in further
study, accompanied that more vasorelaxant flavonoids were identified and applied in improving the model.
Table 4. The experimental and prediction vasorelaxant activities of flavonoids 1-3, 10a,b,
11c-e and 12-38 used in the established QSAR model.

Molecules 2011, 16
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Ref.
Table 4. Cont.
Exp. activities
EC50(μM) p(EC50)
Pred.
pEC50(μM)
Cmpd.
R1
R2
Res.
1 a
2
5,7-diOH-8-prenyl
2',4'-diOH
9.3
4.6
5.032
5.337
4.620
5.119
4.863
4.762
4.104
4.272
4.373
4.697
4.991
4.432
4.493
5.027
4.570
3.848
4.879
4.606
3.996
4.807
4.138
4.672
4.648
4.050
4.376
3.991
3.910
4.971
4.434
4.088
3.018
3.192
3.307
3.754
4.290
4.977
4.976
4.841
0.055 [16]
0.361 [14]
−0.221 [15]
6-OH-8-allyl
4'-Cl
3
3-allyl-4,6-diOH
4'-OH
24.0
7.6
−0.126
0.603
/
/
/
/
/
10a
10b
11c
11d
11e
12
/
/
/
5.245
4.260
4.796
4.048
4.592
4.372
4.688
5.024
4.219
4.632
4.558
4.295
3.858
4.837
4.636
4.221
4.680
4.389
4.789
4.919
3.937
4.184
4.116
3.943
4.937
4.389
4.065
3.266
3.175
3.399
3.744
3.399
/
13.7
19.9
78.7
53.5
42.4
20.1
34.5
37.0
32.1
9.4
−0.034
0.056
/
/
/
/
−0.32
/
/
0.001 [14]
0.009 [14]
−0.033 [14]
0.213 [14]
−0.139 [14]
0.469 [14]
0.275 [14]
−0.01 [14]
0.042 [19]
−0.03 [19]
−0.225 [19]
0.459 [19]
−0.251 [16]
−0.117 [14]
−0.271 [14]
0.113 [14]
0.192 [19]
−0.125 [15]
−0.033 [15]
0.034 [15]
0.045 [14]
0.023 [14]
−0.248 [19]
0.017 [19]
−0.092 [19]
5,7-diOH
5,7-diOH
6-OH-8-allyl
5,7-diOH-8-allyl
5,7-diOH-8-allyl
7-OH-8-allyl
7-OH-8-allyl
7-OH-8-allyl
5,7-diOH-8-prenyl
3'-Br
13
3',4'-OCH2O-
4'-OH
14
15a
3'-OH
16
4'-OH
17
3',4'-OCH2O-
4'-OH
18
26.9
142
19
20a
4'-Cl
3',4'-OCH2O-
13.2
24.8
101
21
5,7-diOH-8-prenyl 3',4',5'-triMeO-
5,7-diOH-8-prenyl 3'-MeO-4'-OH
22
23a
5,7-diOH-8-prenyl
5,7-diOH-8-prenyl
4,6-diOH
3'-Br
4'-OH
15.6
72.7
21.3
22.5
89.1
42.1
102
24
25a
26a
27
3'-Br
4,6-diOH
3',4'-OCH2O-
4'-OH
3-allyl-4-OH
3-prenyl-4,6-diOH
3-prenyl-4-OH
4-OH-5-prenyl
4-OH-5-prenyl
5,7-diOH
28
29a
3',4'-OCH2O-
4'-OH
30
3'-OH
123
31a
32
2', 4'-diOH
3'-Br
10.7
36.8
81.7
959
33
7-OH
3',4'-OCH2O-
3'-Br
34
5,7-diOH-8-prenyl
35
5,7-diOH-8-prenyl 3',4',5'-triMeO-
5,7-diOH-8-prenyl 3'-MeO-4'-OH
643
493
36
0.01
[16]
37
5,7-diOH-8-prenyl
6-OH-8-allyl
2',4'-diOH
4'-OH
176
51.3
0.891 [14]
38
a The compounds were used as test set.

Molecules 2011, 16
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Figure 4. Predicted vs. experimental vasorelaxant activities (−LogEC50) of the QSAR
model developed by EMR-MLR.
3. Conclusions
In this study, some synthesized flavonoid derivatives were characterized as agents with remarkable
vasorelaxant activities. The preliminary structure-activity relationships studies revealed that cyclic
prenyl and 2-(diethylamino)ethyl groups are beneficial for vasorelaxant activity. 2D-QSAR analysis
using ERM-MLR was performed to set up a statistically reliable model with good predictive ability
(Rtrain2 = 0.839, Qloo2 = 0.733 and Rtest2 = 0.804), and five descriptors (H5m, SIC2, DISPe, Mor03u and
L3m) were found to be closely correlated with the observed vasorelaxant activities of the target
compounds.
4. Experimental
4.1. General
1
Melting points were measured on a Büchi B-540 apparatus and are uncorrected. All H-NMR
spectra were recorded on a 400 MHz spectrometer (Brüker AM). Chemical shifts were expressed as δ
values in parts in million (ppm) relative to tetramethylsilane (TMS). Mass spectral data were obtained
on an Esquire-LC-00075 spectrometer. Reagents and solvents were purchased from common
commercial suppliers and were used without further purification. Compounds 4a-c, 6c, 7c, 8a,b and
11c were prepared according to the procedures reported in previous references [14,17].
4.2. General Method for Synthesis of Prenylated Acetophenones 5
A solution of compound 4, prenyl bromide and potassium carbonate in DMF (100 mL) was heated
at 100 °C under a N2 atmosphere for 6 h, and then the reaction mixture was poured into cold water and
extracted with ethyl acetate. The organic phase was washed with brine and dried over anhydrous
sodium sulfate. After removal of the solvent, the residue was dissolved in N,N-dimethylaniline (50 mL)
and then heated at 190 °C for 3~5 h. The mixture was concentrated under vacuum, and the residue was
purified by column chromatography on silica gel using (30/1, v/v) as eluent to give compounds 5.

Molecules 2011, 16
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2-Hydroxy-5-(3,3-dimethyl)allyl-4,6-dimethoxymethoxyacetophenone (5a): Reagents: compound 4a
(10.0 g, 39.1 mmol), prenyl bromide (8.73 g, 58.6 mmol) and potassium carbonate (10.8 g, 78.2 mmol).
1
Product: yellow oil (4.93 g, 39%); H-NMR (CDCl3, δ): 1.68 (s, 3H), 1.76 (s, 3H), 2.69 (s, 3H), 3.30
(d, 1H, J = 6.4 Hz), 3.45 (s, 3H), 3.51 (s, 3H), 4.95 (s, 2H), 5.14 (m, 1H), 5.21 (s, 2H), 6.46 (s, 1H),
12.93 (s, 1H, OH). ESI-MS: m/z [M-H]− 323.
2-Hydroxy-5-(3,3-dimethyl)allyl-4-dimethoxymethoxyacetophenone (5b): Reagents: compound 4b
(10.0 g, 51.0 mmol), prenyl bromide (11.4 g, 76.5 mmol) and potassium carbonate (14.1 g, 102.0 mmol).
Product: yellow oil (7.4 g, 55%). 1H-NMR (CDCl3, δ): 1.72 (s, 3H), 1.75 (s,3H), 2.55 (s, 3H), 3.25 (d,
2H, J = 7.2Hz), 3.47 (s, 3H), 5.23 (s, 2H), 5.26 (m, 1H), 6.60(s,1H), 7.43(s,1H), 12.54(s,1H). ESI-MS:
m/z [M-H]− 263.
2-Hydroxy-3-(1,1-dimethyl)allyl-5-dimethoxymethoxyacetophenone (5c): Reagents: compound 4c
(10.0 g, 51.0 mmol), prenyl bromide (11.4 g, 76.5 mmol) and potassium carbonate (14.1 g, 102.0
mmol). Product: yellow oil (6.19 g, 46%). 1H-NMR (CDCl3, δ): 1.44 (s, 3H), 1.47 (s, 3H), 2.55 (s, 3H),
3.48 (s, 3H), 4.85 (d, 1H, J = 17.2 Hz), 4.93 (d, 1H, J = 10.8 Hz), 5.24 (s, 2H), 6.05 (dd, 1H, J = 10.8,
17.2 Hz), 7.06 (d, 1H, J = 2.0 Hz), 7.33 (d, 1H, J = 2.0 Hz), 12.87 (s, 1H, OH). ESI-MS: m/z [M-H]− 263.
4.3. General Method for Synthesis of Prenylated Chalcones 6
To a cold solution of the acetophenone 5 and appropriate benzaldehyde in H2O-EtOH (1/4, v/v, 3 mL),
20% KOH in H2O-EtOH (1/4, v/v, 3 mL) was added with stirring. The resulting mixture was stirred
under nitrogen at room temperature for 36 h, and then poured into ice-water (50 mL). The solution was
acidified to pH ~ 2 with 1 N HCl, and extracted with ethyl acetate (20 mL × 3 times). The organic
phase was washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel using petroleum ether-ethyl acetate as
eluant to give the desired compound 6.
2-Hydoxy-2'-chloro-4,6-dimethoxymethoxy-5-(3,3-dimethyl)allylchalcone (6a): Reagents: compound
5a (500.2 mg, 1.54 mmol), 2-chlorobenzaldehyde (227.8 mg, 1.62 mmol). Eluent: petroleum ether-
ethyl acetate (20:1, v/v). Product: yellow oil (468.7 mg, 68%). 1H-NMR (CDCl3, δ): 1.68 (s, 3H,), 1.78
(s, 3H), 3.32 (d, 2H, J = 6.8 Hz), 3.43 (s, 3H), 3.49 (s, 3H), 5.21 (m, 1H), 5.25 (s, 2H), 5.26 (s, 2H),
6.39 (s, 1H,), 7.30 (m, 2H), 7.43 (m, 1H), 7.67 (m, 1H), 7.88 (d, 1H, J = 16.0 Hz), 8.12 (d, 1H, J = 16.0 Hz),
13.86 (s, 1H, OH). ESI-MS: m/z [M+H]+ 447.
2-Hydoxy-3',4'-dioxymethylene-4,6-dimethoxymethoxy-5-(3,3-dimethyl)allylchalcone (6b): Reagents:
compound 5a (500.0 mg, 1.54 mmol), 3,4-dioxymethylenebenzaldehyde (243.1 mg, 1.62 mmol).
1
Eluent: petroleum ether-ethyl acetate (18:1, v/v). Product: yellow oil (443.3 mg, 63%). H-NMR
(CDCl3, δ): 1.70 (s, 3H,), 1.74 (s, 3H), 3.31 (d, 2H, J = 6.8 Hz), 3.50 (s, 3H), 3.54 (s, 3H), 5.21 (m, 1H),
5.24 (s, 2H), 5.28 (s, 2H), 6.01 (s, 2H), 6.43 (s, 1H), 6.83 (d, 1H, J = 7.6 Hz), 7.06 (dd, 1H, J = 7.6, 2.0 Hz),
7.08 (d, 1H, J = 2.0 Hz), 7.71 (d, 1H, J = 16.0 Hz), 7.78 (d, 1H, J = 16.0 Hz), 13.90 (s, 1H). ESI-MS:
m/z [M+H]+ 457.

Molecules 2011, 16
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2-Hydoxy-4,4',6-trimethoxymethoxy-5-(3,3-dimethyl)allylchalcone (6d): Reagents: compound 5a
(500.2 mg, 1.54 mmol), 3,4-dimethoxymethoxybenzaldehyde (269.1 mg, 1.62 mmol). Eluent:
petroleum ether-ethyl acetate (18:1, v/v). Product: yellow oil (437.2 mg, 60%) . 1H-NMR (CDCl3, δ):
1.68 (s, 3H), 1.79 (s, 3H), 3.32 (d, 2H, J = 6.8 Hz), 3.47 (s, 3H), 3.52 (s, 3H), 3.53 (s, 3H), 5.21 (m, 1H),
5.22 (s, 2H), 5.23 (s, 2H), 5.25 (s, 2H), 6.42 (s, 1H,), 7.06 (d, 2H, J = 8.8 Hz), 7.55 (d, 2H, J = 8.8 Hz),
7.76 (d, 1H, J = 15.6 Hz), 7.85 (d, 1H, J = 15.6 Hz), 13.82 (s, 1H). ESI-MS: m/z [M+H]+ 473.
2-Hydoxy-3',4'-dioxymethylene-4-methoxymethoxy-5-(3,3-dimethyl)allylchalcone (6e): Reagents:
compound 5b (500.3 mg, 1.90 mmol), 3,4-dimethoxymethoxybenzaldehyde (298.5 mg, 1.99 mmol).
1
Eluent: petroleum ether-ethyl acetate (20:1, v/v). Product: yellow oil (540.3 mg, 72%). H-NMR
(CDCl3, δ): 1.77 (s, 3H,), 1.78 (s, 3H), 3.31 (d, 2H, J = 6.8 Hz), 3.50 (s, 3H), 5.26 (s, 2H), 5.30 (m, 1H),
6.06 (s, 2H), 6.66 (s, 1H), 6.88 (d, 1H, J = 8.4 Hz), 7.15 (d, 1H, J = 8.4 Hz), 7.18 (d, 1H, J = 1.2 Hz), 7.40
(d, 1H, J = 16.0 Hz), 7.62 (s, 1H), 7.81 (d, 1H, J = 16.0 Hz), 13.30 (s, 1H, OH). ESI-MS: m/z [M+H]+ 397.
2-Hydoxy-3-(1,1-methyl)allyl-4'-methoxy-5-methoxymethoxychalcone (6f): Reagents: compound 5c
(500.1 mg, 1.89 mmol), 4-methoxybenzaldehyde (270.5 mg, 1.99 mmol). Eluent: petroleum ether-
ethyl acetate (25:1, v/v). Product: yellow oil (180.9 mg, 25%). 1H-NMR (CDCl3, δ): 1.52 (s, 3H), 1.57
(s, 3H), 3.52 (s, 3H), 3.86 (s, 3H), 5.01 (d, 1H, J = 18.0 Hz), 5.02 (d, 1H, J = 9.6 Hz), 5.15 (s, 2H),
6.26 (dd, J = 18.0, 9.6 Hz), 6.94 (d, 2H, J = 8.4 Hz), 7.25 (d, 1H, J = 2.0 Hz), 7.46 (d, 1H, J = 2.0 Hz),
7.47 (d, 1H, J = 15.6 Hz), 7.61 (d, 2H, J = 8.4 Hz), 7.88 (d, 1H, J = 15.6 Hz), 13.40 (s, 1H, OH).
ESI-MS: m/z [M+H]+ 383.
2-Hydoxy-3-(1,1-methyl)allyl-4'-chloro-5-methoxymethoxychalcone (6g): Reagents: compound 5c (500
mg, 1.89 mmol), 4-chlorobenzaldehyde (279.4 mg, 1.99 mmol). Eluent: petroleum ether-ethyl acetate
(25:1, v/v). Product: yellow oil (219.6 mg, 30%). 1H-NMR (CDCl3, δ): 1.52 (s, 3H), 1.55 (s, 3H), 3.51
(s, 3H), 5.01 (d, 1H, J = 17.2 Hz), 5.03 (d, 1H, J = 9.2 Hz), 5.15 (s, 2H), 6.26 (dd, J = 17.2, 9.2 Hz),
7.28 (d, 1H, J = 2.0 Hz), 7.40 (d, 2H, J = 8.4 Hz), 7.45 (d, 1H, J = 2.0 Hz), 7.55 (d, 1H, J = 15.6 Hz),
7.58 (d, 2H, J = 8.4 Hz), 7.83 (d, 1H, J = 15.6 Hz), 13.6 (s, 1H, OH). ESI-MS: m/z [M+H]+ 387.
4.4. General Method for Synthesis of Prenylated Flavanones 7
A solution of 6 and sodium acetate (500 mg, 6.10 mmol) in ethanol (5 mL) containing 3 drops of
water was refluxed for 24 h. The mixture was poured into cold water (30 mL) and extracted with ethyl
acetate (10 mL × 3 times). The organic phase was washed with brine, dried over sodium sulfate. After
removing the solvent, the residue was purified by column chromatography on silica gel using
petroleum ether-ethyl acetate as eluent to give 7.
2'-Chloro-5,7-dimethoxymethoxy-6-(3,3-dimethyl)allylflavanone (7a): Reagents: compound 6a (300 mg,
0.67 mmol); Eluent: petroleum ether-ethyl acetate (15:1, v/v). Product: pale yellow syrup (162 mg,
54%). 1H-NMR (CDCl3, δ): 1.66 (s, 3H), 1.69 (s, 3H), 2.75 (dd, 1H, J = 16.4 Hz, 2.8 Hz), 2.93 (dd, 1H,
J = 13.4, 16.4 Hz), 3.36 (d, 2H, J = 6.8 Hz), 3.48 (s, 3H), 3.52 (s, 3H), 5.17 (m, 1H), 5.20 (s, 2H), 5.23
(s, 2H), 5.68 (dd, 1H, J = 2.8, 13.4 Hz), 6.65 (s, 1H), 7.30 (td, 1H, J = 2.0, 7.6 Hz), 7.36 (td, 1H, J = 2.0,
7.6 Hz), 7.39 (dd, 1H, J =2.0, 7.6 Hz), 7.20 ( dd, 1H, J =2.0, 7.6 Hz). ESI-MS: m/z [M+H]+ 447.

Molecules 2011, 16
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3',4'-Dioxymethylene-5,7-dimethoxymethoxy-6-(3,3-dimethyl)allylflavanone (7b): Reagents: compound
6b (300.2 mg, 0.66 mmol). Eluent: petroleum ether-ethyl acetate (15:1, v/v). Product: pale yellow
syrup (204.1 mg, 68%). 1H-NMR (CDCl3, δ): 1.71 (s, 3H), 1.73 (s, 3H), 2.76 (dd, 1H, J = 16.8 Hz, 2.8 Hz),
2.98 (dd, 1H, J = 12.8, 16.4 Hz), 3.27 (d, 2H, J = 7.2 Hz), 3.42 (s, 3H), 3.47 (s, 3H), 5.22 (s, 2H), 5.26
(m, 1H), 5.27 (s, 2H), 5.34 (dd, J = 2.8, 12.8Hz), 5.99 (s, 2H), 6.69 (s, 1H), 6.83 (d, 1H, J = 8.0 Hz),
6.90 (dd, 1H, J = 2.0, 8.0), 6.98 (d, 1H, J = 2.0 Hz), 7.69 (s, 1H). ESI-MS: m/z [M+H]+ 457.
4',5,7-Trimethoxymethoxy-6-(3,3-dimethyl)allylflavanone (7d): Reagent: compound 6d (300.5 mg,
0.64 mmol). Eluent: petroleum ether-ethyl acetate (15:1, v/v). Product: pale yellow syrup (156.3 mg,
52%). 1H-NMR (CDCl3, δ): 1.68 (s, 3H), 1.70 (s, 3H), 2.82 (dd, 1H, J = 16.4 Hz, 2.8 Hz), 2.98 (dd, 1H,
J =13.4, 16.4 Hz), 3.33 (d, 2H, J = 7.2 Hz), 3.48 (s, 3H), 3.50 (s, 3H), 3.54 (s, 3H), 5.20 (m, 1H), 5.22
(s, 2H), 5.23 (s, 2H), 5.26 (s, 2H), 5.37 (dd, 1H, J = 2.8 Hz, 13.4 Hz), 6.68 (s, 1H), 7.10 (d, 2H,
J = 8.0 Hz), 7.39 (d, 2H, J = 8.0 Hz). ESI-MS: m/z [M+H]+ 473.
3',4'-Dioxymethylene-5-methoxymethoxy-6-(3,3-dimethyl)allylflavanone (7e): Reagents: compound 6e
(300.2 mg, 0.76 mmol). Elent: petroleum ether-ethyl acetate (12:1, v/v). Product: pale yellow syrup
(180.1 mg, 60%). 1H-NMR (CDCl3, δ): 1.71 (s, 3H), 1.73 (s, 3H), 2.76 (dd, 1H, J = 16.8 Hz, 2.8 Hz),
2.98 (dd, 1H, J =12.8, 16.4 Hz), 3.27 (d, 2H, J = 7.2 Hz), 3.47 (s, 3H), 5.22 (s, 2H), 5.26 (m, 1H), 5.34
(dd, J = 2.8, 12.8Hz), 5.99 (s, 2H), 6.69 (s, 1H), 6.83 (d, 1H, J = 8.0 Hz), 6.90 (dd, 1H, J = 2.0, 8.0),
6.98 (d, 1H, J = 2.0 Hz), 7.69 (s, 1H). ESI-MS: m/z [M+H]+ 397.
4'-Methoxy-6-methoxymethoxy-8-(1,1-dimethyl)allyl-flavanone (7f): Reagent: compound 6f (300.0 mg,
0.79 mmol). Elu-nt: petroleum ether-ethyl acetate (15:1, v/v). Product: pale yellow syrup (150 mg,
50%). 1H-NMR (CDCl3, δ): 1.41 (s, 3H), 1.45 (s, 3H), 2.85 (dd, J = 2.0, 16.8 Hz, 1H), 3.02 (dd, J = 13.2,
16.8 Hz, 1H), 3.48 (s, 3H), 3.92 (s, 3H), 4.94 (d, 1H, J = 17.2 Hz), 4.95 (d, 1H, J = 10.8 Hz), 5.21
(s, 2H), 5.24 (dd, J = 2.0, 13.2 Hz), 6.09 (dd, 1H, J = 10.8, 17.2 Hz), 7.01 (d, 2H, J = 8.4 Hz), 7.12 (d,
1H, J = 2.4 Hz), 7.38 (d, 1H, J = 2.4 Hz), 7.40 (d, 2H, J = 8.4 Hz). ESI-MS: m/z [M+H]+ 383.
4'-Chloro-6-methoxymethoxy-8-(1,1-dimethyl)allyl-flavanone (7g): Reagents: compound 6g (300.3 mg,
0.78 mmol). Eluent: petroleum ether-ethyl acetate (15:1, v/v). Product: pale yellow syrup (129.1 mg,
43%). 1H-NMR (CDCl3 δ): 1.42 (s, 3H), 1.48 (s, 3H), 2.83 (dd, 1H, J = 2.0, 16.8 Hz), 3.95 (dd, 1H, J = 13.2,
16.8 Hz), 3.44 (s, 3H), 4.90 (d, 1H, J = 17.2 Hz), 4.98 (d, 1H, J = 10.8 Hz), 5.25 (s, 2H), 5.32 (dd, 1H,
J = 2.0, 13.2 Hz), 6.03 (dd, 1H, J = 10.8, 17.2 Hz), 7.13 (d, 1H, J = 2.4 Hz), 7.39 (d, 1H, J = 2.4 Hz),
7.44 (brd, 4H). ESI-MS: m/z [M+H]+ 387.
4.5. General Method for Synthesis of Compounds 10
A solution of compound 8, 2-chloro-diethylethanamine hydrochloride and potassium carbonate in
acetone (5 mL) was refluxed for 10 h. After cooling to room temperature, the mixture was filtered and
the solution was concentrated in vacuo. The residue was purified by column chromatography on silica
gel using petroleum ether-ethyl acetate as eluant (3:1, v/v) to give 9, which was further dissolved in 5 mL
3N HCl/methanol (1/4, v/v) and refluxed for 1 h. Then, the reaction mixture was evaporated in vacuo
to give yellow solid and washed with ether to afford 10.

Molecules 2011, 16
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2-(2-(Diethylamino)ethoxy-3'-bromo-4,6-dimethoxymethoxychalcone (10a): Reagents: compound 8a
(200 mg, 0.47 mmol), 2-chloro-N,N-diethylethanamine hydrochlororide (122 mg, 0.71 mmol) and
potassium carbonate (194.6 mg, 1.41 mmol). Product: yellow solid (167.3 mg, 72%), m.p. 180–182 °C.
1H-NMR (MeOH-d4, δ): 1.08 (d, 1H, J = 6.8 Hz), 3.10 (m, 4H), 3.48 (m, 2H), 4.38 (brs, 2H), 6.03
(s, 1H), 6.07 (s, 1H), 7.40 (d, 1H, J = 8.0 Hz), 7.51 (d, 1H, J = 16.0 Hz), 7.63 (d, 1H, J = 8.0 Hz ), 7.65
(d, 1H, J = 16.0 Hz), 7.78 (d, 1H, J = 8.0 Hz), 8.01 (s, 1H), 10.06 (s, 1H, OH), 10.37 (s, 1H, OH),
12.48 (s, 1H, OH). ESI-MS: m/z [M+H]+ 434.
2-(2-(Diethylamino)ethoxy-3',4'-dioxymethylene-4,6-dimethoxymethoxy-chalcone (10b): Reagents:
compound 8b (201 mg, 0.52 mmol), 2-chloro-N,N-diethylethanamine hydrochlororide (133.7 mg, 0.78
mmol) and potassium carbonate (215.3 mg, 1.56 mmol). Product: yellow solid (161.8 mg, 78%), m.p.
227–230 °C. 1H-NMR (MeOH-d4, δ): 1.10 (d, 1H, J = 6.8 Hz), 3.10 (m, 4H), 3.49 (m, 2H), 4.39 (brs,
2H), 6.02 (s, 1H), 6.06 (s, 1H), 6.10 (s, 2H), 6.97 (d, 1H, J = 8.0 Hz), 7.25 (d, 1H, J = 8.0 Hz), 7.46
(s, 1H), 7.45 (d, 1H, J = 16.0 Hz), 7.51 (d, 1H, J = 16.0 Hz), 10.07 (s, 1H, OH), 10.55 (s, 1H, OH),
12.55 (s, 1H, OH). ESI-MS: m/z [M+H]+ 400.
4.6. General Method for Synthesis of Compounds 11
A solution of 7 in 3N HCl/methanol (1/4, v/v, 3 mL) was refluxed for 2 h, then poured into cold
water (15 mL) and extracted with ethyl acetate (5 mL × 3 times). The organic phase was washed with
brine and then dried over anhydrous sodium sulfate. After removal of the solvent, the residue was
purified by column chromatography on silica gel using petroleum ether-ethyl acetate as eluant to give 11.
2'-Chloro-5,7-dihydroxy-6-(3,3-dimethyallyl)flavanone (11a): Reagents: compound 11a (150 mg, 0.34
mmol). Eluent: petroleum ether-ethyl acetate (6:1, v/v). Product: white solid (90.3 mg, 75%), m.p.
130–133 °C. 1H-NMR (acetone-d6, δ): 1.59 (s, 3H), 1.69 (s, 3H), 2.78 (dd, 1H, J = 2.8, 17.2 Hz), 3.07
(dd, 1H, J = 12.8, 17.2 Hz), 3.12 (d, 2H, J = 6.8 Hz), 5.17 (m, 1H), 5.79 (dd, 1H, J = 2.8, 12.8 Hz),
5.99 (s, 1H), 7.39–7.48 (m, 3H), 7.77 (dd, 1H, J =1.6, 7.6 Hz), 9.66 (s, 1H), 12.35 (s, 1H). ESI-MS:
m/z [M+H]+ 359.
3',4'-Dioxymethylene-5,7-dihydroxy-6-(3,3-dimethyl)allylflavanone (11b): Reagents: compound 11b
(150 mg, 0.33 mmol). Eluent: petroleum ether-ethyl acetate (6:1, v/v). Product: white solid (79.9 mg,
68%), m.p. 156–158 °C. 1H-NMR (acetone-d6, δ): 1.59 (s, 3H), 1.68 (s, 3H), 2.69 (dd, J =3.2, 16.8 Hz,
1H), 3.10 (dd, 1H, J = 12.8, 16.8 Hz), 3.20 (d, 2H, J = 7.2 Hz), 5.18 (m, 1H), 5.39 (dd, 1H, J = 12.8 Hz,
3.2 Hz), 5.98 (s, 2H), 6.00 (s, 1H), 6.83 (d, 1H, J = 8.0 Hz), 6.97 (d, J = 8.0 Hz), 7.04 (s, 1H), 9.53
(s, 1H), 12.39 (s, 1H). ESI-MS: m/z [M+H]+ 369.
2-(4'-Hydroxyphenyl)-5-hydroxy-6,7-dihydro-8,8-dimethyl-4H,8H-benzo[1,2-b;5,4-b']dipyran-4-one
(11d): Reagents: compound 11d (150 mg, 0.32 mmol). Eluent: petroleum ether-ethyl acetate (6:1, v/v).
Product: white solid (41.1 mg, 38%), m.p. 122–125 °C. 1H-NMR (DMSO-d6, δ): 1.33 (s, 6H), 1.82 (t,
2H, J = 7.2 Hz), 2.58 (t, 2H, J = 7.2 Hz), 2.73 (dd, 1H, J = 2.8, 17.2 Hz), 3.16 (dd, 1H, J = 12.8, 17.2 Hz),
5.42 (dd, 1H, J = 2.8, 12.8 Hz), 5.84 (s, 1H), 6.89 (d, 1H, J = 8.0 Hz), 7.38 (dd, 1H, J = 8.0 Hz), 8.51
(1H, OH), 12.57 (1H, OH). ESI-MS: m/z [M+H]+ 341.

Molecules 2011, 16
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2-(3,4-Dioxymethylenephenyl)-6,7-dihydro-8,8-dimethyl-4H,8H-benzo[1,2-b;5,4-b']dipyran-4-one (11e):
Reagents: compound 11e (150 mg, 0.38 mmol). Eluent: petroleum ether: ethyl acetate (6:1, v/v).
Product: white solid (60 mg, 45%), m.p. 95–98 °C .1H-NMR (DMSO-d6, δ): 1.34 (s, 3H), 1.35 (s, 3H),
1.81 (t, 1H, J = 6.8 Hz), 2.76 (t, 1H, J = 6.8 Hz), 2.77 (dd, 1H, J = 2.4, 17.2 Hz), 2.98 (dd, 1H, J = 12.8,
17.2 Hz), 5.32 (dd, 1H, J = 2.4, 12.8 Hz), 5.97 (s, 2H), 6.38 (s, 1H), 6.82 (d, 1H, J = 7.6 Hz), 6.90 (dd,
1H, J = 2.0, 7.6 Hz), 6.97 (d, 1H 1H, J = 2.0 Hz), 7.26 (s, 1H), 7.67 (s, 1H). ESI-MS: m/z [M+H]+ 353.
4'-Methoxy-6-hydroxy-8-(1,1-dimethyl)allyl-flavanone (11f): Reagents: compound 11f (150 mg,
0.39 mmol). Eluent: petroleum ether: ethyl acetate (10:1, v/v). Product: yellow syrup (55.7, 42%).
1H-NMR (CDCl3 δ): 1.43 (s, 3H), 1.44 (s, 3H), 2.82 (dd, J = 2.0, 16.8 Hz, 1H), 3.00 (dd, J = 13.2, 16.8
Hz, 1H), 3.84 (s, 3H), 4.89 (d, 1H, J = 17.2 Hz), 4.95 (d, 1H, J = 10.8 Hz), 4.97 (s, 1H, OH), 5.30 (dd,
J = 2.0, 13.2 Hz), 6.11 (dd, 1H, J = 10.8, 17.2 Hz), 6.95 (d, 2H, J = 8.4 Hz), 7.11 (d, 1H, J = 2.4 Hz),
7.27 (d, 1H, J = 2.4 Hz), 7.40 (d, 2H, J = 8.4 Hz). ESI-MS: m/z [M+H]+ 339.
4'-Chloro-6-hydroxy-8-(1,1-dimethyl)allyl-flavanone (11g): Reagents: compound 11g (100 mg,
0.26 mmol). Eluent: petroleum ether-ethyl acetate (10:1, v/v). Product: yellow solid (29.2 mg, 33%),
1
m.p. 90–92 °C. H-NMR (CDCl3 δ): 1.42 (s, 3H), 1.45 (s, 3H), 2.83 (dd, 1H, J = 2.0, 16.8 Hz), 3.92
(dd, 1H, J = 13.2, 16.8 Hz), 4.89 (d, 1H, J = 17.2 Hz), 4.95 (d, 1H, J = 10.8 Hz), 5.00 (s, 1H, OH),
5.30 (dd, 1H, J = 2.0, 13.2 Hz), 6.09 (dd, 1H, J = 10.8, 17.2 Hz), 7.13 (d, 1H, J = 2.4 Hz), 7.36 (d, 1H,
J = 2.4 H), 7.41 (brd, 4H). ESI-MS: m/z [M+H]+ 343.
4.7. Vasorelaxant Activities Assay
Vascular rings were prepared from the aorta of male Male Sprague-Dawley rats (four to six months
old and weighing on average 250 g), and contraction studies were performed following the general
procedure detailed in the literature [18]. After an equilibration period of at least 1 h, isometric
contractions induced by PE (1 μM) were obtained. When contraction of the tissue in response to this
vasoconstrictor agent had stabilized (after about 20 min), cumulatively increasing concentrations of the
tested compounds were added to the bath at 15~20 min intervals (the time needed to obtain
steady-state relaxation). Control tissues were simultaneously subjected to the same procedures, but
omitting the compounds and adding the vehicle. The flavonoids-induced maximal relaxation (Emax) in
aortic rings was calculated as a percentage of the contraction in response to PE (1 μM). The half
maximum effective concentration (EC50) was defined as the concentration of the flavonoids that
induced 50% of maximum relaxation from the contraction elicited by PE (1 μM) and was calculated
from the concentration response curve by nonlinear regression (curve fit) using GraphPad Prism
(Version 4.0).

Molecules 2011, 16
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4.8. Computational Methods
4.8.1. Data Preparation
For the development of 2D-QSAR model, five newly synthesized compounds and thirty previous
compounds [14-16,19] with available EC50 were selected. They were divided into training set (26
molecules) and test set (nine molecules) considering both structural diversity and wide coverage of
biological activity ranges. The structures of all compounds were optimized in Discovery Studio 2.0
software (Accelrys, Inc. San Diego, CA, USA). The resulted geometry of molecules were inputted into
Dragon software [20], which can calculate constitutional descriptors, topological descriptors, walk and
path counts, information indices, 2D autocorrelations, edge adjacency indices, Burden eigenvalue
descriptors, Burden eigenvalue descriptors, etc. After the calculation of the molecular descriptors,
those that stayed constant for all molecules were eliminated, and pairs of variables with a correlation
coefficient greater than 0.75 were classified as intercorrelated with one of each correlated pair was deleted.
4.8.2. Enhanced Replacement Method-Multiple Linear Regressions (ERM-MLR)
Enhanced Replacement Method (ERM) proposed by Mercader et al. [21] is a modified version of
Replacement Method (RM) [22,23]. The purpose of both algorithms is to choose an optimal subset of
d (d < D) descriptors from the pool of D descriptors with minimum standard deviation SD:
N
1
SD2 =
res 2
,
∑
i
N − d −1 i=1
where N is the number of molecules in the training set, and resi the residual for molecule i (difference
between the experimental and predicted property). Considering that SD(dn) is a distribution on a
discrete space of D!/d!(D–d) disordered points dn, ERM produces linear models that are quite similar
with the full search (FS) but with much less computational work. First, an initial set of descriptors dk
was selected at random. And one of the descriptors, say Xki, with all the remaining descriptors (D-d)
was replaced by other descriptor, one by one, and the set with the smallest value of SD was kept.
Second, from the resulting set the descriptor with the greatest SD in its coefficient is chosen and all the
remaining D-d descriptors, one by one, until the set remains unmodified. More detailed information
about these algorithms can be found in reference [21].
4.8.3. Leave-One-Out Cross Validation
Validation of the models was required to test the predictive ability and generalization of the
methods by cross validation as well as test set prediction. The leave-one-out cross validation, a special
case of the cross-validation technique [24] was employed to find the promising QSAR model. Given n
samples available in a data set and m candidate models, each model is trained with n − 1 samples and
then is tested on the sample that was left out. This process is repeated n times until every sample in the
data set have been used once as a cross-validation instance. Finally, cross validation correlation
coefficient (Qloo2) of LOO-CV, a measure of the model generalization capability, for all candidate
models can be obtained as below:

Molecules 2011, 16
8271
n
)
(y − y )2
∑
i
i
,
Ql2oo =1−
i=1
n
y − ( i=1 yi )/ n 2
]
n
[
∑
∑
i
i=1
)
yi
where yi is the desired output and
is the actual output of the model, and n is the number of
compounds in the analyzed set.
Acknowledgements
The authors are grateful for support from the Medical Scientific Research Foundation of Zhejiang
Province (NO. 2010KYA061), and the Administration of Traditional Chinese Medicine of Zhejiang
Province (NO. 2009CB033).
References
1. Grassi, D.; Desideri, G.; Croce, G.; Tiberti, S.; Aggio, A.; Ferri, C. Flavonoids, vascular function
and cardiovascular protection. Curr. Pharm. Design 2009, 10, 1072–1084.
2. Harborne, J.B.; Williams, C.A. Advances in flavonoid research since 1992. Phytochemistry 2000,
55, 481–504.
3. Bravo, L. Polyphenols: Chemistry, dietary sources, metabolism, and nutritional significance. Nutr.
Rev. 1986, 56, 317–333.
4. García-Lafuente, A.; Guillamón, E.; Villares, A.; Rostagno, M.A.; Martínez, J.A. Flavonoids as
anti-inflammatory agents: Implications in cancer and cardiovascular disease. Inflamm. Res. 2009,
58, 537–552.
5. Tripoli, E.; Guardia, M.L.; Giammanco, S.; Majo, D.D.; Giammanco, M. Citrus flavonoids:
Molecular structure, biological activity and nutritional properties: A review. Food Chem. 2007,
104, 466–479.
6. Stoclet, J.C.; Chataigneau, T.; Ndiaye, M.; Oak, M.H.; El Bedoui, J.; Chataigneau, M.;
Schini-Kerth, V.B. Vascular protection by dietary polyphenols. Eur. J. Pharmacol. 2004, 500,
299–313.
7. Kris-Etherton, P.M.; Keen, C.L. Evidence that the antioxidant flavonoids in tea and cocoa are
beneficial for cardiovascular health. Curr. Opin. Lipidol. 2002, 13, 41–49.
8. Lin, Y.L.; Dai, Z.K.; Lin, R.J.; Chu, K.S.; Chen, I.J.; Wu, J.R.; Wu, B.N. Baicalin, a flavonoid
from Scutellaria baicalensis Georgi, activates large-conductance Ca2+-activated K+ channels via
cyclic nucleotide-dependent protein kinases in mesenteric artery. Phytomedicine 2010, 17,
760–770.
9. Xu, Y.C.; Leung, S.W.S.; Yeung, D.K.Y.; Hu, L.H.; Chen, G.H.; Che, C.M.; Man, Y.K.R.
Structure–activity relationships of flavonoids for vascular relaxation in porcine coronary artery
Phytochemistry 2007, 68, 1179–1188.
10. Benavente-Garcia, O.; Castillo, J. Update on uses and properties of citrus flavonoids: New
findings in anticancer, cardiovascular, and anti-inflammatory activity. J. Agric. Food Chem. 2008,
56, 6185–6205.

Molecules 2011, 16
8272
11. Ma, X.; Li, Y.; Gao, Q.; Ye, Z.; Lu, X.; Wang, H.; Jiang, H.; Bruce, I.C.; Xia, Q. Inhibition of
superoxide anion-mediated impairment of endothelium by treatment with luteolin and apigenin in
rat mesenteric artery. Life Sci. 2008, 83, 110–117.
12. Woodman, O.L.; Meeker, W.F.; Boujaoude, M. Vasorelaxant and antioxidant activity of flavonols
and flavones: Structure-activity relationships. J. Cardiovasc. Pharmacol. 2005, 46, 302–309.
13. Chen, Z.; Hu, Y.; Wu, H.; Jiang, H. Synthesis and biological evaluation of flavonoids as
vasorelaxant agents. Bioorg. Med. Chem. Lett. 2004, 14, 3949–3952.
14. Dong, X. Liu, T.; Yan, J.; Wu, P.; Chen, J.; Hu, Y. Synthesis, biological evaluation and quantitative
structure-activities relationship of flavonoids as vasorelaxant agents. Bioorg. Med. Chem. 2009,
17, 716–726.
15. Dong, X.; Chen, J.; Jiang, C.; Liu, T.; Hu, Y. Design, synthesis, and biological evaluation of
prenylated chalcones as vasorelaxant agents. Arch. Pharm. 2009, 342, 428–432.
16. Dong, X.; Qi, L.; Jiang, C.; Chen, J.; Wei, E.; Hu, Y. Synthesis, biological evaluation of
prenylflavonoids as vasorelaxant and neuroprotective agents. Bioorg. Med. Chem. Lett. 2009, 19,
3196-3198.
17. Dong, X.; Fan, Y.; Yu, L.; Hu, Y. Synthesis of four natural prenylflavonoids and their
estrogen-like activities. Arch. Pharm. 2007, 340, 372–376.
18. Qian, L.; Wang, H.; Qiu, W.; Huang, H.; Bruce, I.C.; Xia, Q. Interleukin-2 protects against
endothelial dysfunction induced by high glucose levels in rats. Vascul. Pharmacol. 2006, 45,
374–382.
19. Dong, X.; Liu, Y.; Yan, J.; Jiang, C.; Chen, J.; Liu, T.; Hu, Y. Identification of SVM-based
classification model, synthesis and evaluation of prenylated flavonoids as vasorelaxant agents.
Bioorg. Med. Chem. 2008, 16, 8151–8160.
20. Talete srl. DRAGON for Windows software for molecular descriptor calculation. Available online:
http://talete.mi.it. (accessed on 14 September 2011).
21. Mercader, A.G.; Duchowicz, P.R.; Fernández, F.M.; Castro, E.A. Modified and enhanced
replacement method for the selection of molecular descriptors in QSAR and QSPR theories.
Chemom. Intell. Lab. Syst. 2008, 92, 138–144.
22 Duchowicz, P.R.; Castro, E.A.; Fernández, F.M. Alternative algorithm for the search of an optimal
set of descriptors in QSAR - QSPR studies. MATCH Commun. Math. Comput. Chem. 2006, 55,
179–192.
23 Duchowicz, P.R.; Fernández, M.; Caballero, J.; Castro, E.A.; Fernández, F.M. QSAR for
non-nucleoside inhibitors of HIV-1 reverse transcriptase. Bioorg. Med. Chem. 2006, 14,
5876–5889.
24 Gong, G. Cross-validation, the jackknife and the bootstrap excess error estimation in forward
regression logistic regression, J. Am. Stat. Assoc. 1986, 81, 108–113.
Sample Availability: Samples of the compounds 10a,b and 11a-g are available from the authors.
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