Synthesis, characterization, anti-inflammatory and anti-proliferative activity against MCF-7 cells of O-alkyl and O-acyl flavonoid derivatives

Article abstract of DOI:10.1016/j.bioorg.2015.09.005

A series of O-alkyl and O-acyl flavonoid derivatives was synthesized in high efficiency. Alkylation and acylation of 5-hydroxyflavonoids showed that the low reactivity hydroxyl group, 5-OH, well reacted with strong reagents whereas with weaker reagents, the different products were obtained dependently on structural characteristic of ring C of respective flavonoid. In order to evaluate anti-inflammatory activity, all compounds were tested for in vitro inhibition of bovine serum albumin denaturation and in vivo inhibition of carrageenan-induced mouse paw edema. Among them, the compounds 3, 3b, 4b and 4c demonstrated more effective anti-inflammatory activity than standard drugs (diclofenac sodium and ketoprofen) in both tests. Meanwhile, the flavonoids 2, 2c, 3a and 4b displayed anti-proliferative activity against MCF-7 cell lines. Triacetyl derivative of hesperetin 4b inducing degradation of DNA in MCF-7 cells was observed.

Full text of DOI:10.1016/j.bioorg.2015.09.005

Accepted Manuscript
Synthesis, characterization, anti-inflammatory and anti-proliferative activity
against MCF-7 cells of O-alkyl and O-acyl flavonoid derivatives
T. Kim-Dung Hoang, T. Kim-Chi Huynh, Thanh-Danh Nguyen
PII:
S0045-2068(15)30020-1
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.09.005
YBIOO 1843
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
18 July 2015
17 September 2015
23 September 2015
Please cite this article as: T. Kim-Dung Hoang, T. Kim-Chi Huynh, T-D. Nguyen, Synthesis, characterization, anti-
inflammatory and anti-proliferative activity against MCF-7 cells of O-alkyl and O-acyl flavonoid derivatives,
Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bioorg.2015.09.005
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Title :
Synthesis, characterization, anti-inflammatory and anti-proliferative activity against MCF-7 cells of O-
alkyl and O-acyl flavonoid derivatives
Authors :
T. Kim-Dung Hoang*, T. Kim-Chi Huynh and Thanh-Danh Nguyen*
Institute of Chemical Technology, Vietnam Academy of Science and Technology. 1 Mac Dinh Chi Street,
District 1, Ho Chi Minh City, Vietnam.
* Email: htkdung@ict.vast.vn (T.K.D. Hoang), danh5463bd@yahoo.com (T. D. Nguyen)
Abstract
A series of O-alkyl and O-acyl flavonoid derivatives was synthesized in high efficiency. Alkylation
and acylation of 5-hydroxyflavonoids showed that the low reactivity hydroxyl group, 5-OH, well reacted
with strong reagents whereas with weaker reagents, the different products were obtained dependently on
structural characteristic of ring C of respective flavonoid. In order to evaluate anti-inflammatory activity,
all compounds were tested for in vitro inhibition of bovine serum albumin denaturation and in vivo
inhibition of carrageenan-induced mouse paw edema. Among them, the compounds 3, 3b, 4b and 4c
demonstrated more effective anti-inflammatory activity than standard drugs (diclofenac sodium and
ketoprofen) in both tests. Meanwhile, the flavonoids 2, 2c, 3a and 4b displayed anti-proliferative activity
against MCF-7 cell lines. Triacetyl derivative of hesperetin 4b inducing degradation of DNA in MCF-7
cells was observed.
Keywords :
Flavonoid, Synthesis, Anti-inflammatory, Anti-proliferative, MCF-7.
1

Introduction
Flavonoids consisting of a large group of polyphenolic compounds present in almost plants where
they are synthesized through the phenylpropanoid pathway. [1] The flavonoids possess a wide range of
biological activities such as antioxidant, [2-4] antifungal,[5] antiviral,[6-8] anticancer[9-11] and anti-
inflammatory agent.[12-14] In recent years, carrageenan-induced edema process is commonly used in
study on in vivo of anti-inflammatory drugs. This edema formation is a biphasic event. The first phase of
edema is attributed to the release of histamine and serotonin while the second phase is due to the release
of prostaglandins. The drugs possessed anti-inflammatory activity may inhibit one of the two phases.[15]
In addition, a common study method on in vitro is the interaction of drugs with normal proteins, which
strongly inhibit heat coagulation of serum, and may cause the biological activity of anti-inflammatory
drugs.[16, 17] These drugs have exerted an inhibitory activity on immune haemolysis and also have
suppressive effect on vascular reactivity. Denaturation as one of the causes of inflammation is well
documented. Anti-inflammatory drugs interact in some way with proteins. To cause the interaction
between the drug and the proteins, the stability of proteins against heat coagulation can be measured. [18]
Thus, the coagulated inhibitors originating flavonoids have recently been recognized as efficient anti-
inflammatory drugs. [19-21]
Recently, flavonoid compounds which occur naturally and are widely distributed in plants have
been reported as cancer chemopreventive agents.[22, 23] Many molecular mechanisms of inhibition of
cancer cells have been proposed for flavonoids. For example, quercetin may inhibit the expression of
mutant p53 to lead to arrest human breast cancer cells.[24] It has been experimentally proved that
quercetin may also interact with nuclear type II estrogen binding sites in breast cancer cells.[25] Since
MCF-7 cell line has become a prominent model system for the study of breast cancer, many flavonoid
derivatives against MCF-7 cells and inducing apoptosis in the cells have been found as a potent source of
new therapeutic anticancer drugs.[26, 27]
2

Flavonoid structures are based upon a fifteen-carbon skeleton consisting of two benzene rings linked
via a heterocyclic pyrane ring. They can be divided into a variety of classes such as flavones (e.g., flavone,
chrysin, diosmetin and luteolin), flavonols (e. g., quercetin, kaempferol and galangin), flavanones (e.g.
flavanone, hesperetin and naringenin) and others. The various classes of flavonoids differ in the level of
oxidation and pattern of substitution of pyrane ring, while individual compounds within a class differ in the
pattern of substitution of the benzene rings.[28] The biological activities depend remarkably on their
structural characteristics such as conjugations, degree of hydroxylation and substitutions. A small
alternation in the chemical structure of flavonoids may lead to significant changes in biological activities.
As a result, numerous flavonoid derivatives have recently been designed and synthesized with convenient
procedures.[29-31] Due to specifically biochemical properties, methyl-O and acetyl-O flavonoid
derivatives particularly have attracted significant attention of synthesis chemists.[32-35] In 5-
hydroxyflavonoids including quercetin (1), luteolin (2), diosmetin (3) and hesperetin (4) (Fig. 1), the low
reactivity of hydroxyl group at 5-position is specifically considered because of forming hydrogen bond
with carbonyl.[36] The acylation or alkylation of such a proton often requires more drastic conditions.
Some O-methyl and O- acetyl flavonoid derivatives have been previously reported in the literature. [37-39]
However, it is not revealed influence of flavonoid structures and reagent on acylation and alkylation of
these 5-hydroxyflavonoids. Our previous researches [40, 41] showed that acylation and alkylation of the
natural products including flavonoid derivatives were carried out in high efficiency and they possessed
significantly biological activities. In this paper, we describe the synthesis, NMR data, anti-inflammatory
activity and anti-proliferative activity against breast cancer cells MCF-7 of O-alkyl and O-acyl flavonoid
derivatives.
3

OH
OH
OR2
OCH3
O
O
O
O
HO
HO
R1
O
O
H
H
1: R1 = OH, R2 = H
2: R1 = R2 = H
4
3: R1 = H, R2 = CH3
Fig. 1. Structures of four important flavonoids: quercetin (1), luteolin (2), diosmetin (3), and hesperetin (4)
Results and Discussion
Chemistry
The methods used for synthesis of flavonoid derivatives are simple and highly effective because the
reagents are readily available and protocols are straightforward. The general synthetic routes are
illustrated in Scheme 1. We first investigated the direct alkylation of the flavonoids with excess of
alkylating reagent in order to get an insight into the reactivity of the 5-OH group. Dimethyl sulfate and
allyl bromide were chosen as alkylating reagents with reactivity difference. The reaction was carried out
in acetone with K2CO3 as a base. These derivatives were obtained in good yields. The methylation of
quercetin (1) and luteolin (2) revealed that all of the hydroxyl groups including 5-OH were replaced in
yield of 65.7 % and 90.7 %, respectively.
4

OR3
OH
OR2
OR2
O
O
R3O
O
O
HO
i or ii
R1
R1
OR4
OH
1a: R1 = OCH3, R2 = R3 = R4 = CH3
1: R1 = OH, R2 = H
2: R1 = R2 = H
3: R1 = H, R2 = CH3
1b: R1 = OCH2CH=CH2, R2 = R3 = CH2CH=CH2, R4 = H
1c: R1 = OCOCH3, R2 = R3 = R4 = COCH3
1d: R1 = OCOC2H5, R2 = R3 = R4 = COC2H5
1e: R1 = OCO(NH)(CH2)3CH3, R2 = R3 = CO(NH)(CH2)3CH3, R4 = H
2a: R1 = H, R2 = R3 = R4 = CH3
2b: R1 = H, R2 = R3 = R4 = CH2-CH=CH2
2c: R1 = H, R2 = R3 = R4 = COCH3
3a: R1 = H, R2 = CH3, R3 = R4 = CH2-CH=CH2
3b: R1 = H, R2 = CH3, R3 = R4 = COCH3
OR3
OH
O
OCH3
OCH3
OCH3
O
O
R3O
O
O
O
HO
O
ii
i
OR4
OH
OH
O
4b: R3 = R4 = COCH3
4c: R3 = CO(NH)(CH2)3CH3, R4 = H
4
4a
Scheme 1. Reagents. (i) Dimethyl sulfate or allyl bromide, K2CO3, acetone, reflux; (ii) CH3COCl or
C2H5COCl or CH3(CH2)3NCO, Py
In a similar fashion, one interesting result had come from reaction of all four flavonoids with allyl
bromide as a weak alkylating reagent. First, two flavones, luteolin (2) and diometin (3), were allylated at
all positions of hydroxyl group to afford the perallylated derivatives 2b and 3b, respectively. In next
consideration, allylation of quercetin (1) showed that the 5-OH group was not replaced to give 3,3’,4’,8-
tetra-O-allylquercetin (1b) in 82.5 % yield. It may be because the electron pair of oxygen at 3-position
stabilized the non-covalent six-membered ring (fig 1) which are formed from a hydrogen bond between 5-
OH group and adjacent carbonyl. Finally, to understand influence of double bond of ring C to the
flavonoid alkylation, hesperetin (4) was employed in comparison with allylation of 3. Outstandingly, in
the same conditions, allyl bromide opened the pyrane ring of hesperetin to afford a chalcone derivative
(4a) bearing one hydroxyl group. Although the detailed mechanism has been unknown, some flavanones
generally can be isomerized by ring opening into chalcones in alkaline media provided a hydroxyl
5

substituent at position 6' (or 2’) of the chalcone.[42, 43] These results indicated alkylation reaction of the
5-OH group depending not only on the alkylating reagent but also on structure of ring C of flavonoids.
The structure of 4a was unambiguously determined by NMR spectra as described in table 1. In the
1
13
HSQC spectrum, cross peaks between two H peaks at 7.73 and 7.64 ppm and two C peaks at 125.08
1
and 142.85 ppm, respectively, were observed. Moreover, these H peaks were long-range coupled to the
13C peak at 192.11 (C-1) in HMBC spectrum. Therefore, these shifts were assigned to H-2/C-2 and H-
3/C-3, respectively. The coupling constant between H-2 and H-3 (15.5 Hz) indicated that the
conformation of the bond between C-2 and C-3 was trans. The spectra demonstrated that compound 4a is
a derivative of tri-O-allylchalcone.
Employing a similar strategy for synthesis of O-acyl flavonoids, we used short fatty chloride acids as
strongly acylating reagent and n-butyl isocyanate as weak acylating reagent in pyridine at room
temperature. The reaction of all flavonoids with short fatty chloride acids, acetyl chloride and propyl
chloride, generated peracyl derivatives 1c, 1d, 2c, 3b and 4b whereas the 5-hydroxyl group of quercetin
(1) and hesperetin (4) was not acylated by n-butyl isocyanate in pyridine as deduced by 1H NMR (1e and
4c, table 3). All the synthesized compounds including three new derivatives 2b, 4a and 4c were identified
by ESI-MS, IR and NMR as shown in table 2 – 4.
6

13
Table 1 1H, C NMR, DEPT spectral data and HSQC and HMBC correlations in compound 4a*
c
b
OH
O
a
1
O
3
6'
3'
2''
5''
2
1''
6''
5'
4'
1'
2'
3''
4''
O
O
O
a
a
b
b
c
c
Position
1H NMR (ppm)
13C NMR
(ppm)
DEPT Correlations
HSQC HMBC
1
2
3
-
192.11
125.08
142.85
C
CH
CH
-
-
7.73 (d, 15.5 Hz)
7.64 (d, 15.5 Hz)
C-2
C-3
C-1, C-1’’, C-2’’, C-3,C-6’’
C-1, C-1’’, C-3
1’
2’
-
-
106.55
160.63
C
C
-
-
-
-
3’
4’
6.19 (d, 2.5 Hz)
-
92.39
CH
C
C-3’
C-1’, C-2’, C-4’, C-5’
165.39
-
-
5’
6’
6.13 (d, 2.5 Hz)
-
-
7.27 (s)
-
-
94.79
CH
C
C
CH
C
C
C-5’
-
-
C-2’’
-
-
C-5’’
C-6’’
C-1’, C-3’, C-4’, C-6’
164.16
127.45
112.01
147.77
151.45
112.01
123.48
1’’
2’’
3’’
4’’
5’’
6’’
C-1’’,C-3,C-3’’,C-4’’,C-6’’
-
-
C-1’’, C-3’’, C-4’’
C-1’’, C-3, C-4’’, C-5’’
7.04 (d, 8.5 Hz)
7.25 (d, 8.0 Hz)
CH
CH
OH
a
13.51 (s)
-
-
-
C-1’, C5’
4.68 (d, 5.5 Hz), 4.64 (d, 4.0 69.46, 68.98, 68.59
Hz), 4.60 (d, 5.5 Hz)
CH2
C-a
C-b, C-c, C-2’, C-3’’, C-4’
b
c
6.17 (m), 6.14-6.00 (m)
133.56, 132.96, 132.91
CH
C-b
C-c
-
5.47 (m), 5.44 (m), 5.41 (m), 118.30, 117.95, 117.71
5.30 (m), 5.29 (m), 5.27 (m)
CH2
C-a, C-b
4’’-OCH3
3.82 (s, 3H)
55.65
CH3
C-O
C-4’’
*1H-NMR and 13C- NMR experimental conditions: 500 MHz and 125 MHz, respectively, in DMSO-d6
7

derivatives.
Table 2. physical data, IR and HR-MS spectra of O-alkyl and O-acyl flavonoid
Sub. M.p. (oC)
Isolated
IR
HR-MS
Yield (%)
147-151
65.7
82.5
84.0
41.2
52.5
2921 (CH3), 1650 (C=O), 1160 (C-O)
2986 (CH2), 1664 (C=O), 1160 (C-O)
373.1 [M+H]+
1a
112-115
429.4, 463.4 [M+H]+
1b
194-197
2932 (CH3), 1773 (C=O, ester), 1641 (C=O,), 1189 (C-O) 428.9, 470.9, 512.9 [M+H]+
1c
118-122
2984 (CH3), 1770 (C=O, ester), 1644 (C=O), 1121 (C-O)
3335 (-CONH), 1725 (C=O, ester), 1657 (C=O), 1539 134.1, 175.1 (100), 481.1,
(N-H), 1251 (C-O)
3085 (CH3), 1647 (C=O), 1163 (C-O)
471.1, 527.1, 583.2 [M+H]+
1d
215-219
1e
675.3, 699.3 [M+H]+
343.1 [M+H]+
447.2 [M+H]+
90.7
67.6
2a
2b
192194
103107
2982 (CH2), 1634 (C=O), 1179 (C-O)
53.2
80.4
83.0
76.6
84.0
1772 (C=O, ester), 1639 (C=O), 1167 (C-O)
2983 (CH3), 1639 (C=O), 1178 (C-O)
413.06, 455.09 [M+H]+
2c
3a
3b
4a
4b
223225
100104
189191
98-100
421.2 [M+H]+
1770 (C=O, ester), 1645 (C=O), 1204 (C-O)
2916 (CH2), 1623 (C=O), 1136 (C-O)
427.1, 449.1 [M+H]+
233.1, 423.2 [M+H]+
135-139
2957 (CH3) 1771 (C=O, ester), 1619 (C=O), 1192 (C-O)
74.3, 88.2, 345.1, 387.1 (100),
429.1 [M+H]+
155-159
79.2
3332 (NH), 2916 (CH3), 1716 (C=O, ester), 1665 (C=O), 523.2 [M+Na]+
1136 (C-O)
4c
8

Table 3. Chemical shifts (ppm) of the protons in the 1H NMR spectra of O-alkyl and O-acyl flavonoid
derivatives.*
OR3
3'
4'
OR2
2'
1
8
1'
2
5'
9 O
R3O
7
6
6'
3
10
R1
4
5
OR4
O
Flavonoid moiety
O-alkyl or O-acyl groups
R1 (or H-3) R2
(dd, OMe: 3.89 (s, 3H), 3.85 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.75 (s 3H)
Sub.
H-2
-
H-6
H-8
H-2’
H-5’
H-6’
R3
R4
6.48 (d, 6.81 (d, 7.63 (d, 7.12 (d, 7.66
1a
2.5 Hz)
2.5 Hz)
2.0 Hz)
d, 7.21 d, 7.15 d, 7.70
2.0 Hz 8.5 Hz 2.0, 8.5 Hz
8,5 Hz)
2.0, 8.5)
-
6.38
(d,2.0
Hz
OCH2CH=CH2: 6.12 -6.03 (m, 3H), 6.02-5.94 (m, 12.58 (s, 1H)
 dd,
1b
6.75
2.0 Hz
1H), 5.46 -5.41 (m, 3H), 5.35-5.28 (m, 4H); 5.21-5.19
(dd, 11.0, 1.0Hz, 1H), 4.68- 4.56 (m, 8H)
-
-
6.87 (d, 7.33 (d, 7.73 (d, 7.37 (d, 7.69
2.0 Hz) 2.0 Hz) 2.0 Hz)
(dd, OCOCH3: 2.34 (s, 3H), 2.33 (s, 6H), 2.43 (s, 3H)
8.0 Hz ) 2.0, 8.0 Hz)
1c
7.17 (d, 7.65 (d, 7.86 (d, 7.55 (d, 7.83
2.0 Hz) 2.0 Hz) 2.0 Hz) 8.0 Hz) 2.0, 8.0 Hz)
(dd, OCOCH2CH3: 2.70-2.62 (m, 10H); 1.09-1.08 (m, 15H)
1d
-
6.64 (d, 7.10 (d, 7.75 (d, 7.40 (d, 7.83
(dd, OCONH(CH2)3CH3: 8.07 – 8.06 (m, 1H), 8.02-8.00 12.18 (s, 1H)
2.0 Hz) 2.0 Hz) 2.0 Hz) 8.5 Hz) 2.0, 8.5 Hz)
(m, 1H), 7.88-7.85 (m, 2H); 3.15-3.05 (m, 8H); 1.54-
1.44 (m, 8H); 1.37-1.29 (m, 8H); 0.92-0.89 (m, 12H)
1e
2a
-
-
6.49 (d, 6.86 (d, 7.52 (d, 7.09 (d, 7.63
(dd, 6.76 (s, 1H)
OCH3: 3.90 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.83
(s, 3H)
2.5Hz) 2.5 Hz) 2.0 Hz) 8.5 Hz) 2.0, 8.5 Hz)
6.53 (d, 6.87 (d, 7.57 (d, 7.13 (d, 7.61
(dd, 6.71 (s, 1H)
OCH2CH=CH2 6.12-6.02 (m, 4H), 5.69-5.65 (m, 1H),
5.59-5.48 (m, 1H), 5.46-5.44 (m, 1H), 5.43-5.42 (m,
1H), 5.34-5.27 (m,4H), 4.72-4.71 (m, 4H), 4.68-4.64
(m, 4H)
2b
2.5 Hz) 2.5 Hz) 2.0 Hz) 8.5 Hz) 2.0, 8.5 Hz)
-
-
7.09 (d, 7.64 (d, 8.04 (d, 7.50 (d, 8.05
2.5 Hz) 2.5Hz) 2.0 Hz) 9.0 Hz) 2.0, 9.0 Hz)
(dd, 6.95 (s, 1H)
COCH3: 2.34 (s, 3H), 2.34 (s, 6H), 2.32 (s, 3H)
2c
3a
6.53 (d, 6.87 (d, 7.55 (d, 7.13 (d, 7.65
2.0 Hz) 2.0 Hz) 2.0 Hz) 8.5 Hz) 2.0, 8.5 Hz)
(dd, 6.71 (s, 1H)
3.85
(s,
OCH2CH=CH2: 6.12-6.02 (m, 3H), 5.69-
5.27 (m, 1H), 5.48-5.43 (m, 2H), 5.33-5.27
(m, 3H), 4.72-4.68 (m, 4 H), 4.64-4.63 (m,
2H)
3H)
-
7.07 (d, 7.64 (d, 7.89 (d, 7.33 (d, 8.02
5.0 Hz) 5.0 Hz) 2.5Hz) 9.0 Hz) 2.5, 9.0 Hz)
(dd, 6.86 (s, 1H)
3.88
(s,
3H)
COCH3: 2.34 (s, 3H), 2.33 (s, 3H), 2.31 (s,
3H)
3b
4b
5.60
6.69 (d, 6.87 (d, 7.3 (d, 7.17 (d, 7.40
(dd, 3.24 (dd, 13.0, 3.79
2.30 (s, 3H), 2.27 (s, 3H), 2.26 (s, 3H)
(dd, 2.5, 2.0 Hz)
13 Hz)
2.0 Hz)
2.0 Hz)
8.5 Hz)
2.0, 8.5 Hz)
17.0 Hz, 1H); (s,
2.70 (dd. 2.5, 3H)
17.0 Hz, 1H)
5.63
6.29 (d, 6.31
(d, 7.26 (d, 7.14 (d, 7.35
2.0 Hz) 9.0 Hz)
(dd, 3.48 (dd, 13.5, 3.78
7.95 (t, 6.0 Hz, 1H), 7.72 (t, 11.94 (s,
6.5 Hz, 1H),3.07-3.02 (m, 1H)
4H), 1.46-1.41 (m, 4H), 1.36-
1.28 (m, 4H), 0.91-0.86 (m,
6H)
4c
(dd, 3.0, 2.5 Hz)
13.0 Hz)
2.0 Hz)
2.5, 9.0 Hz)
17.5 Hz, 1H); (s,
2.81 (dd, 3.0, 3H)
17.0 Hz, 1H)
*NMR experimental conditions: 500 MHz, DMSO-d6.
9

13
derivatives. *
Table 4. Chemical shifts (ppm) of the carbon atoms in the C NMR spectra of O-alkyl and O-acyl flavonoid
Sub.
flavonoid moiety
O-alkyl or O-acyl
R1 R2
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-1’
C-2’
C-3’
C-4’
C-5’
C-6’
R3
R4
-
1a
1b
155.57
155.46
140.32
136.77
172.11
178.01
160.25
160.89
95.71
98.17
163.95
163.98
92.90
93.10
158.07
156.14
108.44
105.14
122.52
122.24
111.04
113.11
148.40
150.48
122.52
147.33
111.41
113.73
121.30
122.23
OMe: 59.18, 55.96, 55.87, 55.57, 55.54
72.48, 69.21, 68.92, 68.79 (OCH2); 133.63,
133.52, 133.27, 132.72 (CH=); 118.14,
118.05, 117.65, 117.51 (=CH2)
1c
150.16
149.69
133.84
133.22
169.89
169.13
154.12
154.46
113.76
114.74
156.64
156.37
108.89
109.97
153.54
153.31
114.53
114.12
127.49
126.64
123.65
123.46
142.03
142.18
144.24
144.47
123.79
123.64
126.25
127.11
OCOCH3: 169.10, 167.71, 167.64 (CO); 20.95, 20.86,
20.46, 20.33 (OCH3)
OCOCH2CH3: 172.04, 171.75, 171.45, 171.28, 171.09
(CO); 26.96, 26.83, 26.66, 26.50 (CH2); 8.82, 8.69, 8.63,
8.60 (CH3)
1d
1e
155.55
131.60
176.89
155.61
104.56
160.30
101.05
157.15
107.42
126.44
123.86
146.22
143.56
124.35
125.81
153.22, 152.99, 152.88, 152.67 (CO); 39.50,
39.33, 39.17, 39.00 (CH2NH); 32.55, 31.67,
31.29, 31.25 (CH2); 19.44, 19.41, 19.26, 19.16
(CH2); 13.63, 13.60, 13.54, 13.20 (CH3).
-
2a
2b
160.20
159.48
107.03
107.09
175.64
175.53
159.65
158.97
96.16
97.59
163.59
162.28
93.38
94.31
159.11
158.94
108.26
108.54
123.11
123.31
109.11
111.19
148.99
147.97
151.55
150.67
111.64
113.46
119.27
119.48
-
-
OCH3: 55.99, 55.92, 55.83, 55.65 (CH3)
OCH2CH=CH2: 133.68, 133.33, 132.89, 132.81
(CH=);118.09, 117.55, 117.47, 116.94 (=CH2);
69.19, 68.92, 68.84 (CH2)
2c
3a
156.99
159.60
114.23
107.05
175.33
175.59
153.91
159.00
109.79
97.63
160.15
162.32
108.30
94.34
149.37
158.98
114.36
108.56
129.09
123.07
121.87
111.98
142.50
147.76
144.77
151.86
124.48
110.80
124.89
119.60
-
-
COCH3: 168.72, 168.33, 168.09, 167.90 (CO);
20.81, 20.78, 20.33, 20.25 (CH3)
55.74
OCH2CH=CH2:
133.66,
132.91,
132.84 (=CH); 118.11, 117.76, 116.98
(=CH2); 69.24, 68.96, 68.87 (OCH2)
3b
4b
4c
156.94
78.36
78.09
113.20
43.79
42.06
175.22
189.35
197.62
153.92
155.81
158.96
109.73
110.91
101.76
160.86
162.82
152.73
106.84
109.35
100.71
153.75
151.35
151.97
114.07
111.62
105.02
125.64
130.72
130.38
121.07
112.87
112.60
139.61
139.34
139.85
149.33
150.82
153.92
114.31
121.64
121.99
122.80
125.79
124.75
-
-
-
56.23
55.07
55.82
COCH3: 168.72, 168.35 (CO); 20.80,
20.30 (CH3)
COCH3: 168.83, 168.69, 168.48 (CO);
20.94, 20.88, 20.45(CH3)
CONH(CH2)3CH3: 162.17, 162.01
(CO); 40.12, 40.10 31.32, 31.08,
19.33,19.29(CH2); 13.59, 13.55(CH3)
*NMR experimental conditions: 125 MHz, DMSO-d6
10

Anti-inflammatory activity
In vitro biochemical screening
The in vitro anti-inflammatory activity was carried out using inhibition of bovine serum albumin
denaturation method using diclofenac sodium as a reference standard. All the compounds were screened
for in vitro anti-inflammatory activity depicted in table 5. Eight compounds 1b, 1c, 1d, 2c, 3, 3b, 4b and
4c showed good anti-inflammatory activity. Among them, the flavonoids 3, 3b and 4c revealed excellent
activity over standard diclofenac sodium for all tested concentrations. However, all the other compounds
which were not selected to study in vivo anti-inflammatory activity, showed weak inhibitory activity of
bovine serum albumin denaturation.
In vivo biological evaluation
Compounds showing significant inhibition of bovine serum albumin denaturation were selected for
further anti-inflammatory evaluation by carrageenan-induced mouse paw edema method using ketoprofen
as a reference standard. The results of all the tested compounds are showed in table 6. The results
revealed that among the tested flavonoids, derivatives 3, 3b, 4b and 4c inhibited the edema formation
significantly (p < 0.01). In particular, the derivative 4b (Fig. 2) showed highest inhibition of paw edema
formation after only 1 day while the other compounds exhibited greater anti-inflammatory activity than
ketoprofen on fourth day after the carrageenan injection.
Antiproliferative effect of flavonoids
The in vitro anti-proliferative activity of all flavonoid derivatives was tested in the breast cancer cell
line MCF-7 according to the sulphorhodamine (SRB) assay method. [44] The pharmacological results are
summarized in table 7. Although the IC50 values of the flavonoids are higher than that of the standard
(campthotecin), compounds 1e, 2c, 3a and 4b displayed anti-proliferative activity against MCF-7 cells.
Among them, derivative 4b reveals the highest anti-proliferative activity (fig. 3). Therefore, 4b has been
chosen for the study on inducing degradation of DNA in MCF-7 cells.
Induction of apoptosis by 4b treatment.
11

Biochemically, apoptosis is characterized by fragmentation of chromosomal DNA. Due to significant
inhibition of proliferation, compound 4b is used for further study on degradation of DNA in MCF-7 cells.
As expected, Agarose gel electrophoresis of 4b treated chromosomal DNA revealed a ladder-like pattern
of DNA fragments consisting of multiples of approximately 180-200 bp (fig. 4). This suggests that 4b is
as a potential agent for the further studies and promotion of the clinical use.
Fig. 2. Photographs of mouse paw at 6th day after the carrageenan injection: Control (A) and 4b (B).
A
B
Fig. 3. Control (A) and MCF-7 cells treated with 4b for 48h (B)
Fig. 4. Agarose gel electrophoresis pattern of DNA isolated from 4b treated MCF-7 cells. Lane 1, marker.
Lane 2, treated MCF-7 in 36 h. Lane 3, in 48h. Lane 4, campthotecin-treated MCF-7 in 48h. Lane 5,
untreated MCF-7.
12

Table 5. In vitro serum albumin inhibitory activity of flavonoids at various concentrations.
Inhibition (%)
25 50
(μg/mL) (μg/mL)
Sub.
100
(μg/mL)
0.67
7.33
23.33
9.33
10.00
7.33
14.67
8.00
20.67
38.67
92.00
0
0.67
2.00
1
1a
1b
1c
1d
1e
2
2a
2b
2c
3
3a
10.00
25.33
39.33
35.33
12.00
15.33
14.00
21.33
38.67
93.33
0
10.00
43.33
36.67
30.00
12.67
14.67
14.67
24.67
34.67
94.00
0
79.33
0
98.67
0
97.33
0
3b
4
23.33
72.67
96.67
73.33
26.67
83.33
96.67
90.00
26.00
84.67
98.67
94.67
4a
4b
4c
Diclofenac
sodium
Table 6. Anti-inflammatory activity of flavonoids against carrageenan-induced mouse paw edema with
different time intervals.
Sub.
% Edema
3h
1 day
2 days
79.7 ± 4.45
3 days
68.2 ± 4.90
4 days
62.23 ± 4.07
5 days
50. ± 6.83
6 days
45.9 ± 6.06
Control
79.84 ± 3.33 90.0 ± 5.50
Standard# 76.28 ± 2.75 77.99 ± 2.88
65.4 ± 3.33 * 56.68 ± 3.47* 46.1 ± 4.42*
22.3 ± 4.77**
43.82 ± 6.31
46.18 ± 6.48
38.96 ± 8.85
51.67 ± 9.07
19.00 ±4.21**
30.81 ± 6.13
34.68 ± 6.72
27.96 ± 9.17
41.19 ± 9.58
0.854 ± 0.85**
77.54 ± 6.96 72.26 ± 9.21
77.65 ± 4.88 77.8 ± 13.3
77.84 ± 4.60 75.6 ± 5.3
75.58 ± 5.36 85.53 ± 9.94
85.12 ± 4.49 87.94 ± 6.18
86.28 ± 3.83 88.32 ± 5.00
67.6 ± 10.9
75.8 ± 11.3
67.2 ± 15.2
85.8 ± 13.7
70.02 ± 6.14
70.78 ± 2.23
59.07 ± 7.38
54.7 ± 10.5
50.3 ± 10.5
69.9 ± 11.2
58.90 ± 4.50
51.0 ± 5.90
63.47 ± 7.48
46.24 ± 7.67
54.4 ± 10.0
1b
1c
1d
2c
3
47.22 ± 6.70* 6.11 ± 2.73**
46.53 ± 3.96* 33.52 ±6.56** 16.41 ± 5.72** 5.15 ± 2.23**
48.15 ± 5.36* 36.41 ± 9.59* 17.64 ± 6.35** 19.67 ± 4.46**
3b
4b
4c
68.25 ± 5.68 54.2 ± 10.5** 46.34±7.21**
85.37 ± 4.91 77.04 ± 4.54 66.46 ± 4.67
56.76 ± 4.93
41.71 ± 8.63* 20.43 ± 5.24*
8.90 ± 3.77**
#Standard: Ketoprofen cream (2.5 %), each value is the mean ± SEM for six mice.
*p<0.05, **p<0.01 when compared with control group
13

Table 7. Anti-proliferative activity of flavonoids against MCF-7 Cells.
IC50 (μmol/mL)
Sub.
0.128 ± 0.016
0.208 ± 0.014
-
1
1a
1b*
0.084 ± 0.004
0.049 ± 0.003
0.036 ± 0.011
0.060 ± 0.002
-
1c
1d
1e
2
2a*
-
2b*
0.036 ± 0.006
-
2c
3*
0.035 ± 0.002
0.190 ± 0.007
0.125 ± 0.007
-
3a
3b
4
4a
0.020 ± 0.001
0.067 ± 0.001
0.001± 0.00002
4b
4c
Camptothecin
*compounds get low solubility
Experimental
Chemistry
All of the materials were purchased from Merck (Germany) or Aldrich. The other solvents were
purchased from Fluka and used without further purification. Column chromatography was performed with
Merck Kieselgel 60. Melting points were measured with an Electrothermal Model 9200 (England). IR
spectra were recorded on a BRUKER EQUINOX 55 IR spectrophotometer. The ESI-MS were performed
1
on a VG Zab Spec (70 eV) instrument. H (500 MHz) and 13C (125 MHz) NMR were recorded on a
BRUKER AVANCE 500 NMR spectrometer using DMSO-d6 as solvent and tetramethylsilane (TMS) as
internal standard. Chemical shifts are reported in δ relative to TMS.
General procedure for Alkylation of flavonoids
To a stirred solution of flavonoid (1 mmol), K2CO3 (552 mg, 4 mmol) in acetone (50 mL) was added
dimethyl sulfate (5.5 mmol) or allyl bromide (5.5 mmol). The mixture was stirred and refluxed for 10 h,
monitoring by TLC. After removing potassium carbonate, the solvent was evaporated in reduced pressure.
The residue was purified by column chromatography on silicagel.
14

General procedure for Acylation of flavonoids
To a stirred solution of flavonoid (1 mmol) in pyridine (2 mL) was added acyl chloride (6 mmol) or
n-butyl isocyanate (6 mmol) in nitrogen atmosphere. The mixture was stirred for 24 h at room
temperature. The reaction solution was poured into 100 mL of water with ice. Solid was filtered, washed
with water and dried. The residue was purified by column chromatography or crystallization from mixture
of methanol and acetone.
In vitro Anti-inflammatory activity
All compounds were screened for in-vitro anti-inflammatory activity by inhibition of bovine serum
albumin denaturation method according to Saso et al.[17] The test compounds were dissolved in DMSO
and diluted with acetate buffer (pH 5.5). The test solution (2 mL) containing different concentrations of
drug was mixed with 3 mL albumin solution (0.2 %, w/v) in acetate buffer and incubated at 37o ±1o C for
30 min. Denaturation was induced by keeping the reaction mixture at 67o ± 1o C in a water bath for 3 min.
After cooling the turbidity was measured at 660 nm. Percentage inhibition of denaturation was calculated
from control where no drug was added. Each experiment was done in triplicate and the average was taken.
The standard solution was also prepared as similar to that of the test solution. Diclofenac sodium was
used as a standard. The percentage of inhibition was calculated from the following formula.
Where, Vt = drug absorbance of triplicate average, Vc = control absorbance of triplicate average.
In vivo Anti-inflammatory activity
Mice weighing 22  2g of either sex were kept in appropriate cages at a temperature controlled room
(25  2oC) under a 12h light/dark cycle. The animals were assigned into several groups randomly, each
group consisting of six mice. Before the experiment, all the animals were acclimatized at least two days
with food and water ad libitum in accordance with the World Health Organization’s International Guiding
Principles for Animal Research. [45] The methods used for test compounds were Carrageenan-induced
mice paw edema. [46] The flavonoids were prepared as cream at 5 % of the dose and ketoprofen cream
15

(2.5 %) was used as a reference standard. The mice were divided into various groups (n = 5). Different
groups administered with various test compounds and ketoprofen (2.5 %) cream. One group of mice
served as a control. A solution of carrageenan (0.025 mL, 1%) was injected subcutaneously into the
subplanter region of hind paw. The thickness of the paw was measured before and 3 h after carrageenan
injection using a plethysmometer (Ugo Basile 7140, Italy). The mice having the paw edema volume over
50 % of normal paw volume were chosen into the experiments. The mice were creamed twice each day, in
the morning after measurement the paw edema and in the afternoon, during six days. The average paw
edema was calculated according to the following formula
Where V0 = the paw volume before induction of inflammation and Vt = the inflamed paw volume.
Cell Culture
MCF-7 cell line was purchased from National Cancer Institute (Frederick, MD, U.S.A). Cells were
cultured in E’MEM medium supplemented with L-glutamine (200 mM), 2-[4-(2-hydroxyethyl)-1-
piperazinyl]ethanesulfonic acid (HEPES) (1M), amphotericin B 0.1 % (v/v), 100 U/mL of penicillin, 100
mg/L of streptomycin and 10 % (v/v) foetal bovine serum (FBS) at 37oC in 5% (v/v) CO2 incubator. For
synchronization, cells at confluence of 70-80% were incubated for 32 h in E’MEM medium without FBS.
Antiproliferative Activity Assay
Cell viability was determined using SRB assay.[44, 47] In brief, cells were harvested and plated in a
96-well plate. After a 24 h culture, cells were incubated for 48 h with varying concentrations of test
specimens. A solution of TCA 50% and SRB 0.2% was added into each well. The level of coloured was
then analyzed with a Multiskan Ascent reader (Thermo Electron Corporation) at 492 nm. Concentration
inducing a 50% inhibition of cell growth (IC50) was determined graphically using the curve fitting
algorithm. Values were means ± STD from three independent experiments.
Analysis of DNA Fragmentation
Briefly, 5 x 106 cells were lysed in a 600 μl buffer containing 10 mM Tris-HCl pH 8.0, 5 mM EDTA,
16

100 mM NaCl, 0.2 % Triton X-100 for 10 min in ice. The lysate was then centrifuged at 14,000 rpm for
10 min. The supernatant was incubated with 1 mg/mL RNase A at 37oC for 2h. Samples were extracted
twice with phenol: chloroform: isoamyl alcohol (25:24:1, v/v/v) and once with chloroform: isoamyl
alcohol (24:1, v/v). After ethanol precipitation, DNA was analyzed by 2 % agarose gel electrophoresis at
50 V for 1h.
Acknowledgements
The authors gratefully acknowledge Prof. Dr Phung-Nguyen Vo (Faculty of pharmacy, University of
Medicine and Pharmacy, Ho Chi Minh City) and Dr. Quoc-Tuan Tran (University of Natural Sciences, Ho
Chi Minh City National University) for bioactivity assay.
Conclusions
We herein report synthesis, spectral studies and biological evaluation of a series of O-alkyl and O-
acyl flavonoid derivatives including novel compounds 2b, 4a and 4c. Alkylation of 5-OH group depends
on the alkylated reagent and structure of ring C of flavonoids. All compounds were evaluated for in vitro
inhibition of bovine serum albumin denaturation and in vivo Anti-inflammatory activity. Some
compounds which showed significant inhibition of albumin denaturation were subjected to anti-
inflammatory study. The present study revealed that the flavonoids 3, 3b, 4b and 4c were effective
against the carrageenan-induced paw edema. It is clear that acyl derivatives of diosmetin and hesperetin
constitute an interesting template for the evaluation of new anti-inflammatory agents and may be helpful
for design of new therapeutic tools against inflammation. In the other hand, the flavonoids 2, 2c, 3a and
4b displayed anti-proliferative activity against breast cancer cells MCF-7. Particularly, triacetyl derivative
of hesperetin (4b) inducing degradation of DNA in MCF-7 cells was observed. This suggests that 4b is as
a potential agent for the further studies on a new anticancer drug.
References
[1] M. L. F. Ferreyra, S. P. Rius, P. Casati, Flavonoids: biosynthesis, biological functions, and
biotechnological applications, Front. Plant Sci., 3 (2012), 222.
17

[2] C. A. Rice-Evans, N. J. Miller and G. Paganga, structure-antioxidant activity relationships of
flavonoids and phenolic acids, Free Radical Biology and Medicine, 20 (1996), 933-956.
[3] C. Rice-Evans, Flavonoid Antioxidants, Current Medicinal Chemistry, 8 (2001), 797-807.
[4] P. G. Pietta, Flavonoids as Antioxidants, J. Nat. Prod., 63 (2000), 1035-1042.
[5] T. C. Tempesti, M. G. Alvarez, M. F. D Araujo, F. E. A. C. Junior, M. G. d. Carvalho, E. N.
Durantini, Antifungal activity of a novel quercetin derivative bearing a trifluoromethyl group on
Candida albicans, Med. Chem. Res., 21 (2012), 2217-2222.
[6] T.B. Ng, B. Huang, W.P. Fong, H. W. Yeung, Anti-human immunodeficiency virus (anti-hiv)
natural products with special emphasis on hiv reverse transcriptase inhibitors, Life Sciences, 61
(1997), 933-949.
[7] D. D. Orhana, B. ozcelikb, S. Ozgen, F. Ergun, Antibacterial, antifungal, and antiviral activities
of some flavonoids, Microbiological Research, 165 (2010), 496-504.
[8] T. N. Kaul, J. M.D. E. Middleton, P. L. Ogra, Antiviral effect of flavonoids on human viruses,
Journal of Medical Virology, 15 (1985), 71-79.
[9] M. K. Chahar, N. Sharma, M. P. Dobhal, Y. C. Joshi, Flavonoids: A versatile source of
anticancer drugs, Pharmacogn Rev., 5 (2011), 1-12.
[10] C. Kanadaswami, L.T. Lee, P. P. Lee, J. J. Hwang, F. C. Ke, Y. T. Huang and M. T. Lee, The
Antitumor Activities of Flavonoids, in vivo, 19 (2005), 895-910.
[11] D. Ravishankar, A. K. Rajora, F. Greco, H. M. I. Osborn, Flavonoids as prospective
compounds for anti-cancer therapy, The International Journal of Biochemistry and Cell Biology, 45
(2013), 2821-2831.
[12] M. H. Pan, C. S. Lai, C. T. Ho, Anti-inflammatory activity of natural dietary flavonoids, Food
& Function, 1 (2010), 15-31.
[13] F. Cottigliaa, L. Casua, L. Bonsignorea, M. Casu, C. Florisb, S. Sosa, G. Altinier, R. D.
Loggia, Topical Anti-inflammatory Activity of Flavonoids and a New Xanthone from Santolina
18

insularis, Z. Naturforsch., 60c (2005), 63-66.
[14] M. Candiracci, E. Piatti, M. Dominguez-Barragan, D. Garcia-Antras, B. Morgado, D. Ruano, J.
F. Gutierrez, J. Parrado, A. Castano, Anti-inflammatory Activity of a Honey Flavonoid Extract on
Lipopolysaccharide-Activated N13 Microglial Cells, J. Agric. Food Chem., 60 (2012), 12304-
12311.
[15] H. V. Chavan, L. K. Adsul, A. S. Kotmale, V. D. Dhakane,V. N. Thakare, B. P. Bandgar,
Design, synthesis, characterization and in vitro and in vivo anti-inflammatory evaluation of novel
pyrazole-based chalcones, Journal of Enzyme inhibition and medicinal chemistry, 30 (2015), 22-31.
[16] Y. Mizushima, M. Kobayashi, Interaction of anti-inflammatory drugs with serum proteins,
especially with some biologically active proteins, J. Pharm. pharmac., 20 (1968), 169-173.
[17] L. A. D. Williams, E. A. Vasquez and W. Kraus, In vitro anti-inflammatory and anti-microbial
activities of phenylpropanoids from Piper betle L. (Piperaceae), Proceeding of the Phytochemical
Society of Europe: Natural products in the new millennium: Kluwer Academic Publisher,
Dordrecht. The Netherlands, 74 (2002), 221-227.
[18] S. Deka, S. Mohan, J. Saravanan, M. Kakati, A. Talukdar, B. J. Sahariah, B. K. Dey, R. K.
Sarma, Syntheses, Characterization and In-Vitro Anti-Inflammatory Activity of Some Novel
Thiophenes, Macedonian Journal of Medical Sciences, 5 (2012), 159-163.
[19] S. S. Sakat, A. R. Juvekar and M. N. Gambhire, In•vitro antioxidant and anti•inflammatory
activity of methanol extract of oxalis corniculata linn, International Journal of Pharmacy and
Pharmaceutical Sciences, 2 (2010), 146-155.
[20] K. F. Oyedapo, O. O. Akinwunmi, In vitro Anti-inflammatory Evaluation of African Nutmeg
(Monodora myristica) Seeds, European Journal of Medicinal Plants, 8 (2015), 167-174.
[21] P. Chatterjee, S. Chandra, P. Dey, S. Bhattacharya, Evaluation of anti-inflammatory effects of
green tea and black tea: A comparative in vitro study, J. Adv. Pharm. Technol. Res., 3 (2012), 136-
138.
19

[22] B. Koen, V. Ruth, V. Guido and V.S. Johannes, Induction of Cancer Cell Apoptosis by
Flavonoids Is Associated with Their Ability to Inhibit Fatty Acid Synthase Activity, J. Bio. Chem.,
280 (2005), 5636-5645.
[23] A. Mishra, A. K. Sharma, S. Kumar, A. K. Saxena, A. K. Pandey, Bauhinia variegata Leaf
Extracts Exhibit Considerable Antibacterial, Antioxidant, and Anticancer Activities, Biomed
research International, 2013, Article ID 915436.
[24] D. W. Lamson, and M. S. Brignall, Antioxidants and cancer III: quercetin, Altern. Med. Rev., 5
(2000), 196-208.
[25] R. L. Singhal, Y. A. Yeh, N. Prajda, E. Olah, G. W. Sledge and G. Weber, Quercetin Down-
Regulates Signal Transduction in Human Breast Carcinoma Cells, Biochemical and Biophysical
Research Communications, 208 (1995), 425-431.
[26] C. Pouget, F. Lauthier, A. Simon, C. Fagnere, J. P. Basly, C. Delage, A. J. Chulia, Flavonoids:
Structural Requirements for Antiproliferative Activity on Breast Cancer Cells, Bioorganic&
Medicinal Chemistry Letters, 11 (2001), 3095-3097.
[27] T. Y. Wong, S. M. Lin, C. H. Poon, L. K. Leung, Chemico-Biological Interactions, 2014, 221,
70.
[28] S. Kumar and A. K. Pandey, Chemistry and biological activities of flavonoids: an overview,
The scientific world journal, 2013, article ID 162750.
[29] R. S. Li, X. B. Wang, X. J. Hu and L.Y. Kong, Design, synthesis and evaluation of flavonoid
derivatives as potential multifunctional acetylcholinesterase inhibitors against Alzheimer’s disease,
Bioorganic & Medicinal Chemistry Letters, 23 (2013), 2636-2641.
[30] Y. Shen, J. Zhang, R Sheng, X. Dong, Q. He, B. Yang, Y. Hu, Synthesis and biological
evaluation of novel flavonoid derivatives as dual binding acetylcholinesterase inhibitors, Journal of
Enzyme Inhibition and Medicinal Chemistry, 24 (2009), 372-380.
[31] M. A. Selepe, F. R. V. Heerden, Application of the Suzuki-Miyaura Reaction in the Synthesis
20

of Flavonoids, Molecules, 18 (2013), 4739-4765.
[32] V. A. Bandyukova and V. D. Ponomarev, Catalytic acetylation of flavonoids, Khimiya
Prirodnykh Soedinenii, 6 (1970), 418-421.
[33] M. Deodhar, K. Wood, D. S. Black and N. Kumar, Oxidative Dimerisation of Isoflavones:
Synthesis of Kudzu isoflavone A and Related Compounds, Aust. J. Chem., 65 (2012), 1377-1383.
[34] H. Matsuda, Y. Kogami, S. Nakamura, T. Sugiyama, T. Ueno and M. Yoshikawa, Structural
requirements of flavonoids for the adipogenesis of 3T3-L1 cells, Bioorganic & Medicinal
Chemistry, 19 (2011), 2835-2841.
[35] M. Freitas, D. Ribeiro, S. M. Tom, A. M.S. Silva, E. Fernandes, Synthesis of chlorinated
flavonoids with anti-inflammatory and proapoptotic activities in human neutrophils, European
Journal of Medicinal Chemistry, 86 (2014), 153-164.
[36] N. Deka, A. Mariotte, A. Boumendjel, Microwave mediated solvent-free acetylation of
deactivated and hindered phenols, Green Chemistry, 3 (2001), 263.
[37] M. Bouktaib, S. Lebrun, A. Atmani and C. Rolando, Hemisynthesis of all the O-
monomethylated analogues of quercetin including the major metabolites, through selective
protection of phenolic functions, Tetrahedron, 58 (2002), 10001-10009.
[38] M. Pica, A. F. Prigent, B. Chabannes and H. Pacheco, O-alkylation de la quegcetine et
synthese de la tetra O-ethyl-3,7,3',4' O-ethyl [3H]-5-quercetine, Tetrahedron letters, 25 (1984),
2227-2230.
[39] A. Mattarei 1, L. Biasutto, F. Rastrelli, S. Garbisa, E. Marotta, M Zoratti and C. Paradisi,
Regioselective O-Derivatization of Quercetin via Ester Intermediates. An Improved Synthesis of
Rhamnetin and Development of a New Mitochondriotropic Derivative, Molecules, 15 (2010), 4722-
4736.
[40] C. H. Dang, C. H. Nguyen, T. D. Nguyen and C. Im, Synthesis and characterization of N-acyl-
tetra-O-acyl glucosamine derivatives, RSC Adv., 4 (2014), 6239-6245.
21

[41] T. K. D. Hoang, C. K. Nguyen, N. H. Nguyen, T. V. Nguyen and H.T.D. Ho, Synthesis,
cytostatic and apoptotic activities of 3',5,7-triactyl-4'-methoxyflavanone on the MCF-7 tumor cell
line, Vietnam Journal of medicine and pharmacy, 49 (2009), 30-32.
[42] F. A. Tomas-Barberan, M. N. Clifford, Flavanones, chalcones and dihydrochalcones-nature,
occurrence and dietary burden, Journal of the Science of Food and Agriculture, 80 (2000), 1073-
1080.
[43] D. Ferreira, E. V. Brandt, F. D. R. Volsteedt and D. G. Roux, Parameters regulating the α- and
β-Cyclization of Chalcones, J. Chem. Soc. Perkin I, 1975 (1975), 1437-1446.
[44] K. T. Papazisis, G. D. Geromichalos, K.A. Dimitriadis, A.H. Kortsaris, Optimization of the
sulforhodamine B colorimetric assay, Journal of Immunological Methods, 208 (1997), 151-158.
[45] (CIOMS), Council for International Organizations of Medical Sciences, International Guiding
Principles for Bio-medical Research involving Animals, The WHO Chronicle, 39 (1985), 51-56.
[46] C. A. Winter, E. A. Risley, G. W. Nuss, Carrageenin-induced edema in hind paw of the rat as
an assay for antiiflammatory drugs, Proc. Soc. Exp. Biol. Med., 111 (1962), 544.
[47] P. Houghton, R. Fang, I. Techatanawat, G. Steventon, P. J. Hylands, C.C. Lee, The
sulphorhodamine (SRB) assay and other approaches to testing plant extracts and derived
compounds for activities related to reputed anticancer activity, Methods, 42 (2007), 377-387.
22

Scheme and Figure Captions
Fig. 1. Structures of four important flavonoids: quercetin (1), luteolin (2), diosmetin (3), and hesperetin (4)
Scheme 1. Reagents. (i) Dimethyl sulfate or allyl bromide, K2CO3, acetone, reflux; (ii) CH3COCl or
C2H5COCl or CH3(CH2)3NCO, Py
Fig. 2. Photographs of mouse paw at 6th day after the carrageenan injection: Control (A) and 4b (B).
Fig. 3. Control (A) and MCF-7 cells treated with 4b for 48h (B)
Fig. 4. Agarose gel electrophoresis pattern of DNA isolated from 4b treated MCF-7 cells. Lane 1, marker.
Lane 2, treated MCF-7 in 36 h. Lane 3, in 48h. Lane 4, campthotecin-treated MCF-7 in 48h. Lane 5,
untreated MCF-7.
23

Table Captions
13
Table 1 1H, C NMR, DEPT spectral data and HSQC and HMBC correlations in compound 4a
derivatives.
Table 2. physical data, IR and HR-MS spectra of O-alkyl and O-acyl flavonoid
1
Table 3. Chemical shifts (ppm) of the protons in the H NMR spectra of O-alkyl and O-acyl flavonoid
derivatives.
Table 4. Chemical shifts (ppm) of the carbon atoms in the 13C NMR spectra of O-alkyl and O-acyl
derivatives.
flavonoid
Table 5. In vitro serum albumin inhibitory activity of flavonoids at various concentrations.
Table 6. Anti-inflammatory activity of flavonoids against carrageenan-induced mouse paw edema with
different time intervals.
Table 7. Anti-proliferative activity of flavonoids against MCF-7 Cells.
24

Graphical abstract
Highlights
-
Six O-alkyl flavonoid derivatives including two novel compounds were synthesized with two
alkylation reagents.
-
Seven O-acyl flavonoid derivatives including a novel compound were synthesized with two
acylation reagents
-
-
-
Effect of structure of flavonoid on hydroxyl group reactivity at 5-position was showed.
Anti-inflammatory activity of all flavonoids was examined by in vitro and in vivo.
Anti-proliferative activity of all flavonoids was examined by SRB assay and apoptosis.
25