Synthesis and biological evaluation of novel flavonols as potential anti-prostate cancer agents

Article abstract of DOI:10.1016/j.ejmech.2012.06.031

A library of flavonol analogues was synthesised and evaluated as potential anticancer agents against a human prostate cancer cell line, 22rν1. Compounds 3, 8 and 11 (IC₅₀ 2.6, 3.3 and 4.0 μM respectively) showed potent cancer cell growth inhibi

Full text of DOI:10.1016/j.ejmech.2012.06.031

European Journal of Medicinal Chemistry 54 (2012) 952e958
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and biological evaluation of novel flavonols as potential anti-prostate
cancer agentsq
Robert G. Britton a, Emma Horner-Glister b, Odette A. Pomenya b, Ewan E. Smith c, Roanne Denton c,
,
Paul R. Jenkins c, William P. Steward b, Karen Brown b, Andreas Gescher b, Stewart Sale b
*
a Department of Cancer Studies and Molecular Medicine, George Porter Building, University of Leicester, Leicester LE1 7RH, UK
b Department of Cancer Studies and Molecular Medicine, RKCSB, Leicester Royal Infirmary, University of Leicester, Level 5, Leicester LE2 7LX, UK
c Department of Chemistry, George Porter Building, University of Leicester, Leicester LE1 7RH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of flavonol analogues was synthesised and evaluated as potential anticancer agents against
Received 29 March 2012
Received in revised form
7 June 2012
Accepted 14 June 2012
Available online 22 June 2012
a human prostate cancer cell line, 22r
showed potent cancer cell growth inhibition, comparable to the lead compound 30,40,50-trimethoxy-
flavonol (1) (IC50 3.1 M) and superior to the naturally occurring flavonols quercetin (16) and fisetin (22)
(both >15
M). Results indicate that the 30,40,50- arrangement of either hydroxy (OH) or methoxy (OMe)
n1. Compounds 3, 8 and 11 (IC50 2.6, 3.3 and 4.0 mM respectively)
m
m
residues is important for growth arrest of these cells and that the OMe analogues may be superior to
their OH counterparts. Compounds 1, 3, 8 and 11 warrant further investigation as potential agents for the
management of prostate cancer.
Keywords:
Flavonols
AR signalling pathway
Prostate cancer management
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
cancer properties [2e8]. Fisetin caused apoptosis in LNCaP pros-
tate cancer cells [2], and quercetin has been shown to attenuate
Prostate cancer is the most commonly diagnosed male cancer in
the developed world with the incidence in Western Europe being
93 per 100,000 men compared to the global incidence of 28 cases
per 100,000 and mortality being 12 and 7 per 100,000, respectively
[1]. Other than surgical intervention and/or anti-androgen
chemotherapy there are few alternatives for the treatment or
prevention of prostate cancer.
Flavonoids are produced ubiquitously in plants, and many of
them can be found in the diet or in traditional herbal medicines. A
sub-class of these are the flavonols, which are characterized by
a hydroxy group on C-3 of the flavone scaffold (Table 1). The
naturally occurring flavonols fisetin (30,40,7-trihydroxyflavonol, 22),
and quercetin (30,40,5,7-tetrahydroxyflavonol, 16), are produced
ubiquitously in the plant kingdom and may be found in high
concentrations in certain food plants, most notably grape, onion
and cucumber, and tea, apple and onion, respectively. Both
compounds have been demonstrated to possess anti-prostate
proliferation and decrease androgen receptor (AR) expression [3] in
22rn
1 cells. We have recently synthesized 30,40,50-trimethoxy-
flavonol (TMFol (1)) and found it to possess colorectal cancer che-
mopreventive activity [9] in preclinical systems. In a mouse
prostate tumour model TMFol displayed anti-prostate cancer
activity which was comparable, if not superior, to that of the
structurally related molecules quercetin (16) and fisetin (22) [10].
The normal development and maintenance of the prostate is
dependent on the hormone androgen acting through the AR.
Activation of the AR drives development of prostate cancer.
Androgen ablation is an important therapeutic option for patients
with primary androgen-dependent prostate cancer [11]. Androgen
ablation is a type of hormone therapy that specifically removes
testosterone from the body with the aim of controlling the
progression of the prostate cancer. Two common types of androgen
ablation are surgical castration or medical castration. However,
almost all patients in this group become refractory to androgen
ablation therapy [12]. In spite of this insensitivity, the AR and the
AR target gene Prostate Specific Antigen (PSA) continues to be
expressed in these patients, indicating that the AR signalling
pathway remains active [13]. It has been suggested that agents
which down-regulate AR can inhibit the growth of prostate cancer
cells [12], and TMFol decreased AR and PSA expression in cells
in vitro [11] significantly. We investigated a library of 27 flavonols
Abbreviations: AR, androgen receptor; PSA, Prostate Specific Antigen; TMFol,
30,40,50-trimethoxyflavonol.
q
Grant Support: Cancer Research UK (A325/A13101) and Prostate Action
(G2009/25).
* Corresponding author. Tel.: þ44 1162231859; fax: þ44 1162231855.
E-mail address: sls13@le.ac.uk (S. Sale).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.031

R.G. Britton et al. / European Journal of Medicinal Chemistry 54 (2012) 952e958
953
Table 1
Cytotoxicity of flavonol analogues against 22R
n
1 human prostate carcinoma cells and corresponding AR and PSA protein expression levels.
% Controlb
20
PSA
10
% Controlb
20
a
Compound
C
A
B
22r
n
1 IC50
AR
10
3
5
6
7
30
40
50
(
mM)
m
M
m
M
m
M
mM
1 (TMFol)
2
3
4
5
6
7
8
OH
OH
OMe
OH
OH
OH
OH
OH
F
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
OH
F
F
OH
F
F
OH
F
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
OH
F
F
OH
F
F
OH
F
F
OH
F
F
OH
F
F
OH
F
F
OMe
OH
OMe
OMe
H
OMe
OH
OMe
H
OMe
OMe
H
OMe
OMe
OH
OMe
OH
OH
OMe
OH
OH
OMe
OH
OH
OMe
OH
OH
OMe
OH
OH
OMe
OH
H
OMe
OH
OMe
H
H
H
OMe
OMe
OMe
OH
OMe
OH
OH
OMe
OH
H
H
H
H
H
H
H
H
H
3.1 ꢀ 0.3
7.7 ꢀ 3.0
2.6 ꢀ 0.5
5.3 ꢀ 0.4
30.5 ꢀ 17.8
12.3 ꢀ 3.5
12.4 ꢀ 1.7
3.3 ꢀ 0.8
>40
7.1 ꢀ 2.9
4.0 ꢀ 0.7
11.6 ꢀ 6.9
13.1 ꢀ 4.7
5.9 ꢀ 0.7
6.3 ꢀ 0.8
15.5 ꢀ 6.2
7.4 ꢀ 1.3
16.3 ꢀ 11.5
>40
15.8 ꢀ 6.6
28.7 ꢀ 18.4
25.9 ꢀ 10.8
>40
9.2 ꢀ 1.5
30.2 ꢀ 4.7
7.1 ꢀ 0.9
8.4 ꢀ 1.3
14.9 ꢀ 8.3
96(18)
94(26)
94(47)
64(36)
71(27)
63(46)
74(20)
91(2)
110(4)
62(23)c
89(47)
80(30)
23(35)c
61(22)c
81(26)
72(23)
75(9)c
125(9)
102(68)
75(43)
94(41)
114(69)
103(55)
90(32)
120(36)
94(35)
86(14)
131(24)
111(16)
96(53)
101(39)
120(18)
59(43)
125(31)
104(15)
64(29)
92(60)
21(15)c
110(41)
167(99)
27(17)c
127(37)
61(21)c
133(48)
14(19)c
63(13)c
69(42)
23(15)c
109(40)
149(96)
13(23)c
115(39)
56(22)c
109(58)
5(6)c
OMe
OMe
OMe
OMe
OH
OMe
OH
OH
OMe
OH
OH
OMe
OH
H
H
H
OH
OMe
OH
H
9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
63(35)
111(48)
22(19)c
106(59)
121(40)
68(25)
132(72)
31(23)c
88(18)
22(19)c
37(15)c
66(16)c
33(41)c
44(20)c
70(18)c
18(16)c
39(10)c
62(2)c
10 (Robinetin)
11
12
13 (Myricetin)
14
15
16 (Quercetin)
17
18
19 (Kaempferol)
20
21
22 (Fisetin)
23
24
25
26
27
Flavonol
84(39)
112(62)
100(38)
119(72)
100(37)
118(52)
114(52)
110(42)
116(14)
128(30)
115(14)
136(36)
109(17)
129(31)
108(33)
141(36)
112(8)
37(16)c
111(58)
110(53)
95(47)
134(71)
40(23)c
78(14)
61(12)c
33(13)c
85(13)
F
77(27)
H
H
H
H
H
H
H
51(6)c
78(23)
21(19)c
47(9)c
H
H
H
H
H
H
H
70(15)c
32(14)c
124(38)
129(45)
100(38)
3(6)c
80(46)
H
a
IC50 calculated as the mean ꢀ standard deviation of 3e6 independent experiments.
b
c
Protein expression levels measured by densitometry and is the mean of 3e5 replicates. SD in brackets.
Indicates data point is significant from vehicle control (p < 0.05).
(Table 1) including quercetin (16) and fisetin (22), two of the most
widely studied flavonols for prostate cancer, to determine which
flavonol structural features may contribute to their abilities to
compromise prostate cancer cell growth and to modulate the
expression of the AR and PSA using the human-derived prostate
2. Chemistry
The library (Table 1) consisted of 27 flavonols, of which 7 occur in
plants and can be purchased, whereas 20 have been synthesised,
including 15 novel flavonols. The synthesis was performed using well
established chemistry [9] with a maximum of 4 consecutive steps
(Schemes 1, 3 and 4) and crystallisation purification. All starting
materials could be purchased, apart from 40,60-difluoro-20-hydrox-
yacetophenone (28), which was synthesised in high yield (89%) from
3,5-difluorophenol via O-acetylation followed by an AlCl3 catalysed
Fries rearrangement (Scheme 2). Methoxyflavonols were produced
by the ClaiseneSchmidt condensation of a 20-hydroxyacetophenone
with a benzaldehyde to afford the corresponding 20-hydrox-
ychalcones, which were then treated under AlgareFlynneOyamada
conditions (H2O2, NaOH) to produce the desired flavonols withyields
of 11%e78% over 2 steps following recrystallisation. Methoxy-
flavonols were then demethylated using BBr3 to produce the corre-
sponding hydroxyflavonols in good yields (76%e99%). 3,30,40,50-
Tetramethoxyflavone (3) was obtained by methylation of TMFol (1)
using iodomethane (Scheme 3). 3-Fluoro-30,40,50-trimethoxyflavone
(9) was synthesised in 4 steps (Scheme 4). Initially, a condensation
between 20-hydroxyacetophenone and 3,4,5-trimethoxybenzoyl
chloride produced 2-acetylphenyl 3,4,5-trimethoxybenzoate,
which was then treated with base (KOH) to afford 3-hydroxy-1-(20-
carcinoma cells, 22rn1 [14,15]. These cells mimic the transition
between androgen-dependent and androgen-independent cancer
types as they are androgen-independent but androgen-responsive,
they also express PSA. Androgen-independence means that the
cells are not reliant on androgens for growth however these cells
will respond to them if present. The work aimed to identify agents
which may warrant further development as potential anti-prostate
cancer chemotherapeutic or chemopreventive drugs.
The synthetic strategy employed involved placement of OH,
OMe or F in different positions on the flavonol scaffold generating
a library of natural product analogues (Table 1). Replacement of OH
groups with OMe moieties has been suggested to reduce the degree
of metabolic removal of flavones whilst retaining anti-proliferative
potency [16]. The use of fluorine as a bioisostere for CeH or CeOH
has afforded many useful compounds, and about 20% of all phar-
maceuticals and 30% of agrochemicals are now fluorinated [17].
Fluorine substituents can impart greater metabolic stability,
increase lipophilicity, thus increasing bioavailability and they can
also increase potency [18].

954
R.G. Britton et al. / European Journal of Medicinal Chemistry 54 (2012) 952e958
Scheme 3. Synthesis of 3,30,40,50-tetramethoxyflavone (3).
30,40,50 OH/OMe substitution pattern. For compound 9 a concen-
tration of 0.01 M inhibited cell growth by 18% and 0.1 M inhibited
by 46%. A high concentration, 50 M, caused cell growth inhibition
of 48%. Therefore an accurate IC50 value could not be established,
hence we state it is >40 M. Compounds 8 and 11 both contain
m
m
m
m
a fluorine on the A ring, 11 has a fluorine on C-7, which harbours OH
or OMe in natural compounds such as fisetin (22) and robinetin
(10), whereas the fluorine on compound 8 is on C-6, which is not
a site substituted in naturally occurring compounds.
Scheme 1. Synthesis of methoxy and hydroxyflavonols.
Several of the compounds tested altered expression levels of the
AR or PSA proteins (Tables 1 and S63 of Supporting Information
which shows western blot data for the expression of AR and PSA
hydroxyphenyl)-3-(300,400,500-trimethoxyphenyl)prop-2-en-1-one
(29) (47%, 2 steps) via a BakereVenkataraman rearrangement.
Compound 29 was fluorinated at the 2-position using N-fluo-
robenzenesulfonimide to give 30 (37%), and then cyclised and
dehydrated under acidic conditions to yield 9 (96%).
proteins in human 22r
library of compounds). TMFol (1) and compounds 4, 5 and 8 at
20 M were able to significantly inhibit AR expression. None of
n1 prostate carcinoma cells against the full
m
these compounds contain OH or OMe on the A ring, whilst
compound 8 has a fluorine at C-6.
Compounds 1, 4, 6, 8, 11, 16, 18, 19, 22 and 24e27 significantly
3. Results and discussion
reduced PSA expression at 10 and 20
mM, and compounds 20, 21
and 23 at 20 M. The most potent inhibitors of PSA expression were
m
Twenty-eight flavonoids (1e27 plus flavonol) were tested for
compounds 1, 4, 8, 11 and 24, three of which (1, 8 and 11) were also
among the more potent inhibitors of cell growth. Of the twelve
compounds that significantly reduced PSA expression, eight (11, 16,
19, 22 and 24e27) contain hydroxy, methoxy or fluorine at C-7 on
the A ring, four (11, 24, 26 and 27) possess a fluorine at C-7 and
compound 8 bears a fluorine on C-6.
their anti-proliferative activity and their ability to modulate the AR
signalling pathway in the human prostate cancer cell line 22rn1.
The human-derived prostate cancer cell line 22rv1 was selected as
it has been used previously to explore the ability of the flavonol
fisetin to affect growth of cells in culture in vitro and in mice in vivo.
It was therefore selected to screen the chosen library of flavonols.
As 22rv1 cells express AR, produce PSA and are androgen respon-
sive, they were considered a suitable paradigm for our study. TMFol
(1) showed significant growth inhibition with an IC50 value of
Modulation of the AR signalling pathway may be one of the
mechanisms by which these compounds exert their anti-
proliferative activity. The most potent growth-inhibitory
compound (3) was clearly a less potent modulator of the expres-
sion of either AR or PSA than compounds 1 and 8. Assuming that
modulation of AR and PSA contributes to the growth inhibition
exerted by some of these flavonols, this finding hints at the possi-
bility that compound 3 may inhibit cell growth by engaging
mechanisms of action different from those operated by compounds
1 and 8. In addition the low growth inhibitory potential of
compound 5 may be related to its low ability to affect PSA
expression. Whilst the AR system is possibly a target for some of
these agents, we cannot make any inferences as to the ultimate role
it may play in mediating the compounds growth inhibition.
Replacement of the OH residue with F did not appear to
enhance, or interfere with, growth-inhibitory activity or the ability
to modulate AR signalling. In contrast, substitution of the hydroxy
3.1
comparable anti-proliferative activity, with IC50 values of 2.6, 3.3
and 4.0 M respectively. Compounds 1e4, 8, 10, 11, 14, 15, 17, 24, 26
M and are
mM (Table 1). Three of the 28 compounds tested, 3, 8 and 11, had
m
and 27 inhibited proliferation with IC50 values ꢁ10
m
more potent than fisetin (22) and quercetin (16), both of which
have been under investigation pre-clinically as anticancer agents
[2e8]. Of the thirteen compounds characterized by IC50 ꢁ 10
mM,
seven are novel, and of those that had an IC50 ꢁ 5 M (1, 3, 8 and 11),
m
only TMFol (1) has been reported before [9]. The IC50 value of TMFol
(1) was significantly lower (p < 0.05) than those of all the natural
flavonols (10, 13, 16, 19, 22 and 25). The rank order for anti-
proliferative activity of the natural products, as judged by IC50
value, was robinetin > myricetin > quercetin > fisetin > reso-
kaempferol (25) > kaempferol.
group on C-7 of the
A
ring of the natural product 40,7-
All compounds which inhibited cell growth with an IC50 ꢁ 5
mM
(1, 3, 8 and 11) have methoxy moieties at the 30,40,50 positions. Of
the natural products that were tested, only those with hydroxy
groups in positions 30,40,50 (10 and 13) showed an IC50 ꢁ 15
mM. The
results suggest that the 30,40,50 arrangement of OH or OMe on the B
ring of the flavonol scaffold is important for cell growth inhibition.
This notion is borne out by the findings that compounds 1e3, 8 and
10e15 were more potent than 4e7 and 16e27, which lack the
Scheme 2. Synthesis of 40,60-difluoro-20-hydroxyacetophenone (28).
Scheme 4. Synthesis of 3-fluoro-30,40,50-trimethoxyflavone (9).

R.G. Britton et al. / European Journal of Medicinal Chemistry 54 (2012) 952e958
955
dihydroxyflavonol (25) with fluorine yielding 27 increased growth-
5.2. Synthesis of methoxyflavonols
inhibitory potency and reduced PSA and AR expression when
compared to 25. A similar difference was observed for the pair
compound 24 versus fisetin (22), where replacement of the
hydroxyl on C-7 by fluorine imparted superior potency in terms of
5.2.1. General method
5.2.1.1. 5,7-Difluoro-30,40-dimethoxyflavonol. Sodium ethoxide (1.19
g,17.44 mmol) was dissolved in ethanol (20 mL) and cooled to room
temperature. To this solution was added 40,60-difluoro-20-hydrox-
yacetophenone (1.0 g, 5.81 mmol) and this was stirred at room
ability to affect PSA levels at 20 mM.
temperature for
1 h. 3,4-Dimethoxybenzaldehyde (0.96 g,
4. Conclusion
5.81 mmol) was then added and stirred at room temperature
overnight. The resulting yellow suspension was poured into H2O
(50 mL) and acidified to pH 1 with HCl (10% aq.). The yellow
precipitate was filtered, washed with H2O and dried. Conversion
and product verification was by TLC (SiO2, ethyl acetate/Pet. ether
(40e60 ꢄC) (1:2)) and ESI-MS. This chalcone (1.4 g, 4.37 mmol) was
suspended in methanol (50 mL), and NaOH (3 M aq.) (7.5 mL) was
added to produce a yellow solution, which was cooled to 0 ꢄC. To
this was added H2O2 (30% aq.) (1.90 mL, 16.76 mmol) and the
solution stirred at 0 ꢄC for 3 h then overnight at room temperature
if the reaction was not complete (by TLC). The resulting suspension
was poured into HCl (10% aq.) (50 mL), extracted with CHCl3 or
CH2Cl2 (3 ꢃ 30 mL), dried (MgSO4) and evaporated to yield a yellow
solid which was recrystallised from ethanol or ethanol/methanol to
yield yellow crystals of 5,7-difluoro-30,40-dimethoxyflavonol
(372 mg, 26% (from benzaldehyde)).
We synthesised a number of novel flavonol analogues and
investigated their potential ability to inhibit cell growth and
modulate components of the AR signalling pathway using the
human-derived prostate cancer cell line 22r
n1. TMFol(1), and novel
compounds 3, and 11 showed potent growth inhibition.
8
Compounds 1, 4 and 8 inhibited the protein expression of AR and
PSA proteins. The 30,40,50 arrangement of either OH or OMe groups
on the B ring of the flavonol structure may play an important role in
inhibition of cell growth. It seems the inclusion of fluorine in the A
ring failed to significantly alter the growth inhibition potency.
Notably though, compounds 6 and 23 were exceptions, in that their
growth-inhibitory ability was significantly lower than that of their
non-fluorinated counterparts. We have identified novel flavonols
which may possess anti-prostate cancer properties superior to
those of fisetin (22) and quercetin (16).
5.2.1.2. 30,40,50-Trimethoxyflavonol (1). 38% Yield; ESI-MS m/z 329
(M þ H)þ, 361 (M þ Na)þ; dH (400 MHz; CDCl3) 3.92 (3H, s, OCH3),
3.93 (6H, s, 2 ꢃ OCH3), 7.37 (1H, ddd, J 8.0, 7.1, 0.7, C(6)H), 7.50 (2H,
s, C(20,60)H), 7.54 (1H, dd, J 8.6, 0.7, C(8)H), 7.66 (1H, ddd, J 8.6, 7.1,
1.6, C(7)H), 8.20 (1H, dd, J 8.0, 1.6, C(5)H); dc (75 MHz; CDCl3) 56.13,
60.84, 105.26, 118.03, 120.45, 124.39, 125.26, 126.07, 133.41, 138.08,
139.80, 144.61, 153.00, 155.03, 173.10; HRMS (EI) 328.09420 (Mþ
C18H16O6 requires 328.09429).
5. Experimental section
5.1. General
NMR spectra were obtained on a Bruker DPX 300, Bruker DRX
400 or Bruker AV500 spectrometer. Chemical shifts (d) are reported
in ppm for a solution of the compound in specified deuterated
solvent internally referenced to either TMS or residual protonated
solvent and coupling constants (J) are quoted in hertz. Electrospray
mass spectra were recorded on a Micromass Quattro. Dry solvents
were obtained from an Innovative Technology, PureSolv solvent
purification system, except pyridine, which was purchased from
SigmaAldrich. All other chemicals were purchased from SigmaAl-
drich except compounds 10 and 25, which were from Indofine
Chemical Company. All compounds submitted for biological tests
were confirmed to be ꢂ95% pure by HPLC analysis.
5.2.1.3. 30-Methoxyflavonol (4). 21% Yield; ESI-MS m/z 269 (M þ H)þ,
267 (M ꢅ H)ꢅ; dH (300 MHz, CDCl3) 3.89 (3H, s, OCH3), 7.02 (1H, s,
OH), 7.05 (1H, dd, J 2.6, 0.8, C(40)H), 7.39-7.49 (2H, m, C(50,6)H), 7.60
(1H, dd, J 8.5, 0.5, C(8)H), 7.72 (1H, ddd, J 8.5, 7.0,1.6, C(7)H), 7.82-7.89
(2H, m, C(20,60)H), 8.26 (1H, dd, J 8.0,1.6, C(5)H); dc (100 MHz; CDCl3)
55.42, 113.25, 115.97, 118.30, 120.26, 120.63, 124.52, 125.47, 129.62,
132.33, 133.67, 138.59, 144.66, 155.41, 159.68, 173.50.
5.2.1.4. 40-Methoxyflavonol (5). 11% Yield; ESI-MS m/z 269
(M þ H)þ, 267 (M ꢅ H)ꢅ; dH (300 MHz, CDCl3) 3.81 (3H, s, OCH3),
6.87 (1H, s, OH), 6.99 (2H, d, J 9.0, C(20,60)H), 7.34 (1H, ddd, J 8.1, 7.0,
1.7, C(7)H), 7.52 (1H, d, J 8.1, C(8)H), 7.63 (1H, ddd, J 7.8, 7.0, 1.0, C(6)
H), 8.15e8.20 (3H, m, C(30,50,5)H); dc (100 MHz; d6-DMSO) 55.35,
114.02, 118.29, 121.33, 123.56, 124.45, 124.70, 129.38, 133.46, 138.13,
145.57, 154.42, 160.42, 172.61.
5.1.1. 40,60-Difluoro-20-hydroxyacetophenone (28)
To a solution of 3,5-difluorophenol (10 g, 76.9 mmol) and dry
pyridine (9.32 mL, 115 mmol) in dry CH2Cl2 (100 mL) was slowly
added acetyl chloride (7.11 mL, 100 mmol) and the reaction was
stirred at room temperature for 30 min NaHCO3 (sat. aq.) (50 mL)
was added and the product extracted with CH2Cl2, washed with
H2O (2 ꢃ 50 mL), dried (MgSO4) and evaporated to yield 3,5-
difluorophenyl acetate which was used without further purifica-
tion. To 3,5-difluorophenyl acetate (13.2 g, 76.9 mmol) was added
AlCl3 (13.35 g, 100 mmol) and the mixture was heated (neat) to
150 ꢄC with stirring for 10 min. The reaction mixture was cooled to
room temperature and dissolved in ethyl acetate (100 mL) before
careful quenching with H2O (100 mL). The product was extracted
with ethyl acetate, washed with H2O, dried (MgSO4) and evapo-
rated to afford an oil that crystallised on standing (may be recrys-
tallised from ethanol) (11.2 g, 85% (from phenol)). ESI-MS m/z 171
(M ꢅ H)ꢅ; dH (300 MHz, CDCl3) 2.59 (3H, d, J 7.3, CH3), 6.30 (1H, ddd,
J 11.8, 9.1, 2.6, C(50)H), 6.41 (1H, ddd, J 10.1, 1.7, 2.6, C(30)H); dC
(75 MHz, CDCl3) 32.10 (d, J 11.2), 95.9 (dd, J 28.2, 27.1), 101.33 (dd, J
23.6, 3.7),107.35 (dd, J 14.4, 2.9),164.11 (dd, J 169.8, 17.1),165.91 (dd,
J 16.6, 7.2), 167.51 (dd, J 168.5, 17.2), 202.06 (d, J 3.6); dF{H} (282 MHz,
CDCl3) ꢅ97.5 (d, J 13.6), ꢅ101.1 (d, J 13.6).
5.2.1.5. 30,40-Dimethoxyflavonol (6). 22% Yield; ESI-MS m/z 299
(M þ H)þ, 321 (M þ Na)þ; dH (300 MHz, CDCl3) 3.95 (3H, s, OCH3),
3.99 (3H, s, OCH3), 6.98 (1H, br s, OH), 7.05 (1H, dd, J 8.2, 2.0, C(60)
H), 7.42 (1H, ddd, J 7.5, 7.0, 1.6, C(6)H), 7.61 (1H, d, J 8.2, C(50)H),
7.71 (1H, ddd, J 9.1, 7.0, 1.5, C(7)H), 7.86 (1H, d, J 2.0, C(20)H), 7.91
(1H, dd, J 9.1, 1.6, C(8)H), 8.25 (1H, dd, J 7.5, 1.5, C(5)H); dc
(100 MHz; d6-DMSO) 55.57, 55.63, 111.01, 111.51, 118.36, 121.27,
121.50, 123.63, 124.43, 124.68, 133.41, 138.26, 145.45, 148.39,
150.32, 154.38, 172.58.
5.2.1.6. 30,50-Dimethoxyflavonol (7). 21% Yield; ESI-MS m/z 299
(M þ H)þ, 321 (M þ Na)þ; dH (300 MHz, CDCl3) 3.81 (6H, s, 2 ꢃ OCH3),
6.52 (1H, t, J 2.3, C(40)H), 6.98 (1H, br s, OH), 7.37 (1H, ddd, J 8.4, 7.1,
1.5, C(7)H), 7.40 (2H, d, J 2.3, C(20,60)H), 7.52 (1H, dd, J 8.4,1.6, C(8)H),
7.65 (1H, ddd, J 8.4, 7.1, 1.6, C(6)H), 8.19 (1H, dd, J 8.4, 1.5, C(5)H); dc

956
R.G. Britton et al. / European Journal of Medicinal Chemistry 54 (2012) 952e958
(100 MHz; d6-DMSO) 56.37, 102.50, 106.99, 119.49, 122.15, 125.54,
125.72, 133.91, 134.72, 140.28, 145.65, 155.47, 161.36, 173.98.
104.82 (d, J 25.6), 113.35 (d, J 23.5),113.99, 118.55, 123.32, 127.51 (d, J
11.0), 129.28, 138.05, 145.95, 155.37 (d, J 14.3), 160.44, 164.66 (d, J
251.1), 172.00; dF{H} (376 MHz, d6-DMSO) ꢅ104.66 (s).
5.2.1.7. 6-Fluoro-30,40,50-trimethoxyflavonol (8). 29% Yield; ESI-MS
m/z 347 (M þ H)þ, 346 (M ꢅ H)ꢅ; dH (300 MHz, CDCl3) 3.88 (3H,
s, OCH3), 3.90 (6H, s, 2 ꢃ OCH3), 6.90 (1H, s, OH), 7.39 (1H, ddd, J 9.2,
7.7, 3.1, C(7)H), 7.46 (2H, s, C(20,60)H), 7.57 (1H, dd, J 9.2, 4.1, C(8)H),
7.82 (1H, dd, J 8.0, 3.1, C(5)H); dc (100 MHz; d6-DMSO) 56.31, 60.99,
105.53, 109.89 (d, J 23.8),120.36 (d, J 8.3),121.55 (d, J 8.1),122.10 (d, J
25.8), 125.90, 137.94, 140.22, 145.22, 151.51, 153.23, 159.06 (d, J
246.6), 172.59 (d, J 2.4); þdF{H} (282 MHz, CDCl3) ꢅ116.60 (s); HRMS
(FAB) 347.09274 (M þ H C18H16O6F requires 347.09269).
5.2.1.14. 3,30,40,50-Tetramethoxyflavone
(3). 30,40,50-Trimethoxy-
flavonol (0.21 g, 0.642 mmol) was dissolved in acetone (20 ml), this
was followed by addition of K2CO3 (1.97 g, 14.3 mmol), and
subsequent addition of iodomethane (1.8 ml, 29 mmol). This was
refluxed at 60 ꢄC for 2 days. The reaction was cooled to room
temperature, excess K2CO3 was filtered out, washed with acetone
(15 ml), and the acetone was evaporated. The residue was dissolved
in CH2Cl2 (15 ml), washed with H2O (20 ml), dried (MgSO4) and the
solvent was removed under reduced pressure. The product was
recrystallised from hot ethanol and water and dried under reduced
pressure over P2O5 to give 3 as a white solid (0.137 g, 62%). ESI-MS
m/z 343 (M þ H)þ; dH (400 MHz, CDCl3) 3.83 (3H, s, OCH3), 3.89 (9H,
s, 3 ꢃ OCH3), 7.34 (1H, ddd, J 8.0, 7.0, 0.9, C(6)H), 7.35 (2H, s, C(20,60)
H), 7.48 (1H, dd, J 8.4, 0.9, C(8)H), 7.62 (1H, ddd, J 8.4, 7.0, 1.5, C(7)H),
8.21 (1H, dd, J 8.0, 1.5, C(5)H); dc (100 MHz; CDCl3) 56.34, 60.10,
60.99, 106.21, 117.97, 124.16, 124.73, 125.80, 125.96, 133.46, 140.46,
141.32, 153.10, 155.13, 155.26, 174.95; HRMS (FAB) 343.11775
(M þ Hþ C19H19O6 requires 343.11769).
5.2.1.8. 7-Fluoro-30,40,50-trimethoxyflavonol (11). 78% Yield; ESI-
MS m/z 347 (M þ H)þ; dH (400 MHz, d6-DMSO/CDCl3) 3.79 (3H, s,
OCH3), 3.89 (6H, s, 2 ꢃ OCH3), 7.29 (1H, td, J 8.8, 2.4, C(6)H), 7.55
(2H, s, C(20,60)H), 7.66 (1H, dd, J 9.7, 2.4, C(8)H), 8.16 (1H, dd, J 8.8,
6.4, C(5)H), 9.52 (1H, s, C(3)OH); dc (100 MHz; d6-DMSO) 55.98,
60.11, 104.92 (d, J 25.7), 105.45, 113.34 (d, J 23.5), 118.33, 127.44 (d, J
11.0), 138.70, 139.13, 145.15, 152.63, 155.30 (d, J 14.3), 164.71 (d, J
251.2), 172.10; dF{H} (376 MHz, d6-DMSO) ꢅ104.53 (s); HRMS (FAB)
347.09259 (M þ Hþ C18H16O6F requires 347.09269).
5.2.1.9. 5,7-Difluoro-30,40,50-trimethoxyflavonol (14). 25% Yield; ESI-
MS m/z 365 (M þ H)þ; dH (400 MHz, d6-DMSO/CDCl3) 3.78 (3H, s,
OCH3), 3.87 (6H, s, 2 ꢃ OCH3), 7.25 (1H, ddd, J 11.5, 9.5, 2.4, C(6)H),
7.52 (2H, s, C(20,60)H), 7.60 (1H, br d, J 9.5, C(8)H), 9.60 (1H, s, C(3)
OH); dc (100 MHz; d6-DMSO) 56.00, 60.13, 101.03 (t, J 25.9), 101.63
(dd, J 25.9, 4.1), 105.32, 109.07 (d, J 12.9), 125.79, 138.96, 139.18,
143.93, 152.67, 156.02 (dd, J 16.8, 6.3), 160.47 (dd, J 263.3, 15.8),
5.3. Demethylation
5.3.1. Method A
5, 7-Difluoro-30,40-dimethoxyflavonol (200 mg, 0.60 mmol) was
suspended in dry CH2Cl2 (5 mL) and cooled to ꢅ78 ꢄC before BBr3
(1.0 M in CH2Cl2) (1.20 mL, 1.20 mmol) was added dropwise. The
reaction was allowed to warm to 0 ꢄC and stirred for a further 3 h.
Methanol (5 mL) was added dropwise and stirred at room
temperature for 30 min then the volatiles removed in vacuo. The
resulting yellow solid was crystallised from ethanol to yield 5, 7-
difluoro-30,40-dihydroxyflavonol (164 mg, 89%).
163.92 (dd,
J 250.8, 15.6), 170.44; dF{H} (376 MHz, d6-
DMSO) ꢅ109.06 (d, J 11.2), ꢅ102.03 (d, J 11.2).
5.2.1.10. 5,7-Difluoro-30,40,-dimethoxyflavonol (17). 26% Yield; ESI-
MS m/z 335 (M þ H)þ; dH (400 MHz, d6-DMSO/CDCl3) 3.86 (3H, s,
OCH3), 3.87 (3H, s, OCH3), 7.11 (1H, d, J 8.6, C(50)H), 7.22 (1H, ddd, J
11.5, 9.5, 2.3, C(6)H), 7.55 (1H, br d, J 9.5, C(8)H), 7.77 (1H, d, J 2.1,
C(20)H), 7.85 (1H, dd, J 8.6, 2.1, C(60)H), 9.44 (1H, s, C(3)OH); dC
(75 MHz, DMSO) 55.51, 55.63, 101.00 (t, J 25.8), 102.07 (dd, J 26.1,
4.1), 109.61 (dd, J 11.2, 1.8), 110.59, 111.40, 121.33, 122.88, 138.36,
144.54, 148.40, 150.31, 156.52 (dd, J 16.2, 6.8) 161.04 (dd, J 256.7,
13.3), 164.37 (dd, J 246.1, 14.7), 170.33; dF{H} (376 MHz, d6-
DMSO) ꢅ109.15 (d, J 11.0), ꢅ102.26 (d, J 11.0).
5.3.2. Method B
5, 7-Difluoro-40-methoxyflavonol (210 mg, 0.69 mmol) was
suspended in dry toluene (5 mL) and cooled to 0 ꢄC before BBr3
(1.0 M in CH2Cl2) (1.5 mL, 1.50 mmol) was added dropwise. The
reaction was warmed to room temperature and stirred for 1 h
before being heated to reflux (110 ꢄC) for 2 h. After cooling to 0 ꢄC,
methanol (5 mL) was added dropwise and stirred at room
temperature for 30 min then the volatiles removed in vacuo. The
resulting yellow solid was crystallised from ethanol to yield 5, 7-
difluoro-40-hydroxyflavonol (200 mg, 99%).
5.2.1.11. 5,7-Difluoro-40,-methoxyflavonol (20). 37%Yield; ESI-MSm/
z 305 (M þ H)þ; dH (400 MHz, d6-DMSO/CDCl3) 3.86 (3H, s, OCH3),
7.09 (2H, d, J 9.0, C(30,50)H), 7.22 (1H, ddd, J 11.5, 9.5, 2.3, C(6)H), 7.49
(1H, br d, J 9.5, C(8)H), 8.17 (2H, d, J 9.0, C(20,60)H), 9.42 (1H, s, C(3)
OH); dF{H} (376 MHz, d6-DMSO) ꢅ109.10 (d, J 10.8), ꢅ102.14 (d, J 10.8).
5.3.2.1. 30,40,50-Trihydroxyflavonol (2). Method A. 20% yield; ESI-MS
m/z 287 (M þ H)þ, 285 (M ꢅ H)ꢅ; dH (400 MHz, d6-DMSO) 7.31
(2H, s, C(20,60)H), 7.45 (1H, ddd, J 8.2, 7.0, 1.4, C(7)H), 7.67 (1H, dd, J
8.2,1.6, C(8)H), 7.29 (1H, ddd, J 8.3, 7.0,1.6, C(6)H), 8.10 (1H, dd, J 8.3,
1.4, C(5)H), 9.12e9.29 (3H, br s, 3 ꢃ OH); dc (100 MHz; d6-DMSO)
107.29, 118.00, 121.11, 121.22, 124.32, 124.68, 133.35, 135.76, 137.97,
145.72, 146.14, 154.27, 172.37.
5.2.1.12. 7-Fluoro-30,40-dimethoxyflavonol (23). 77% Yield; ESI-MS
m/z 317 (M þ H)þ; dH (400 MHz, d6-DMSO/CDCl3) 3.87 (6H, s,
2 ꢃ OCH3), 7.12 (1H, d, J 8.6, C(50)H), 7.30 (1H, td, J 8.8, 2.4, C(6)H),
7.66 (1H, dd, J 9.8, 2.4, C(8)H), 7.81 (1H, d, J 2.0, C(20)H), 7.88 (1H, dd,
J 8.6, 2.0, C(60)H), 8.16 (1H, dd, J 8.8, 6.4, C(5)H), 9.42 (1H, s, C(3)OH);
dC (75 MHz, DMSO) 55.49, 55.61, 105.37 (d, J 25.7), 110.80, 111.54,
113.32 (d, J 23.2), 118.52, 121.46, 123.44, 127.38 (d, J 10.7), 138.21,
145.79, 148.37, 150.33, 155.31 (d, J 14.1), 164.67 (d, J 250.7), 172.01;
dF{H} (376 MHz, d6-DMSO) ꢅ104.75 (s).
5.3.2.2. 7-Fluoro-30,40,50-trihydroxyflavonol (12). Method A. 99%
yield; ESI-MS m/z 303 (M ꢅ H)ꢅ; dH (400 MHz, d6-DMSO) 7.28 (2H,
s, C(20,60)H), 7.33 (1H, td, J 8.8, 2.4, C(6)H), 7.60 (1H, dd, J 9.8, 2.4,
C(8)H), 8.14 (1H, dd, J 8.8, 6.4, C(5)H); dc (100 MHz; d6-DMSO)
104.57 (d, J 25.5),107.29,113.23 (d, J 23.4), 118.45, 120.89,127.47 (d, J
11.0), 135.83, 137.87, 145.71, 146.63, 155.23 (d, J 14.0), 164.62 (d, J
251.0), 171.77; dF{H} (376 MHz, d6-DMSO) ꢅ104.77 (s).
5.2.1.13. 7-Fluoro-40-methoxyflavonol (26). 75% Yield; ESI-MS m/z
287 (M þ H)þ; dH (400 MHz, d6-DMSO/CDCl3) 3.86 (3H, s, OCH3),
7.08 (2H, d, J 9.1, C(30,50)H), 7.27 (1H, td, J 8.8, 2.4, C(6)H), 7.55 (1H,
dd, J 9.7, 2.4, C(8)H), 8.16 (1H, dd, J 8.8, 6.4, C(5)H), 8.19 (2H, d, J 9.1,
C(20,60)H), 9.35 (1H, br s, C(3)OH); dC (100 MHz, DMSO) 55.33,
5.3.2.3. 5,7-Difluoro-30,40,50-trihydroxyflavonol (15). Method A. 76%
yield; ESI-MS m/z 321 (M ꢅ H)ꢅ; dH (400 MHz, d6-DMSO) 7.26 (2H,
s, C(20,60)H), 7.31 (1H, ddd, J 11.7, 9.7, 2.4, C(6)H), 7.48 (1H, dt, J 9.6,

R.G. Britton et al. / European Journal of Medicinal Chemistry 54 (2012) 952e958
957
1.6, C(8)H); dc (100 MHz; d6-DMSO) 100.92 (t, J 25.8), 101.22 (dd, J
25.2, 4.0), 107.18, 109.11 (dd, J 10.7, 2.1), 120.44, 135.88, 138.10,
145.88, 145.73, 155.97 (dd, J 16.6, 6.3), 160.51 (dd, J 263.1, 15.9),
163.78 (dd, J 251.0, 15.4), 170.14; dF{H} (376 MHz, d6-DMSO) ꢅ109.18
(d, J 11.0), ꢅ102.26 (d, J 11.0).
dd, J 8.2, 1.6, C(60)H), 11.97 (1H, C(3)OH); dC (75 MHz, CDCl3) 56.38,
61.03, 91.94, 104.32, 106.49, 118.83, 119.07, 128.42, 128.78, 135.80,
142.08, 153.32, 162.39, 177.52, 195.13.
5.4.2. 2-Fluoro-3-hydroxy-1-(20-hydroxyphenyl)-3-(300,400,500-trime
thoxyphenyl)prop-2-en-1-one (30)
5.3.2.4. 5,7-Difluoro-30,40,-dihydroxyflavonol (18). Method A. 89%
yield; ESI-MS m/z 305 (M ꢅ H)ꢅ; dH (400 MHz, d6-DMSO) 6.90 (1H,
d, J 8.5, C(50)H), 7.31 (1H, ddd, J 11.7, 9.6, 2.4, C(6)H), 7.53 (1H, m,
C(8)H), 7.55 (1H, dd, J 8.5, 2.2, C(60)H), 7.72 (1H, d, J 2.2, C(20)H), 9.27
(1H, br s, ArOH), 9.36 (1H, s, C(3)OH), 9.60 (1H, br s, ArOH); dC
(125 MHz, d6-DMSO) 100.88 (t, J 26.0),101.27 (dd, J 25.3, 2.8),109.10
(d, J 10.0), 115.22, 115.57, 119.75, 121.62, 137.98, 145.08, 145.26,
147.10, 155.98 (dd, J 16.3, 5.9), 160.50 (dd, J 263.5, 15.6), 163.77 (dd, J
251.2, 15.1), 170.16; dF{H} (376 MHz, d6-DMSO) ꢅ109.20 (d, J
11.0), ꢅ102.30 (d, J 11.0).
To a solution of 29 (1.57 g, 4.76 mmol) in dry CH2Cl2 (20 mL) and
dry CH3CN (70 mL) was added N-fluorobenzenesulfonimide (1.80 g,
5.71 mmol) and the reaction was stirred at room temperature for 7
days. The volatiles were removed in vacuo and the crude mixture was
purified by FC(SiO2, ethylacetate/hexanes (3:7))toafford 30asawhite
solid (0.62 g, 37%). dH (400 MHz, CDCl3) 3.95 (3H, s, OCH3), 3.96 (6H, s,
2 ꢃ OCH3), 7.27 (2H, s, C(200,600)H), 7.45 (1H, ddd, J 8.0, 7.1, 0.6, C(50)H),
7.60 (1H, dd, J 8.6, 0.6, C(30)H), 7.73 (1H, ddd, J 8.6, 7.1, 1.7, C(40)H), 8.30
(1H, dd, J 8.0, 1.7, C(60)H); dF{H} (282 MHz, CDCl3) ꢅ160.31 (s).
5.4.3. 3-Fluoro-30,40,50-trimethoxyflavone (9)
5.3.2.5. 5,7-Difluoro-40,-hydroxyflavonol (21). Method B. 99% yield;
ESI-MS m/z 289 (M ꢅ H)ꢅ; dH (400 MHz, d6-DMSO) 6.94 (2H, d, J
9.0, C(30,50)H), 7.31 (1H, ddd, J 11.7, 9.6, 2.4, C(6)H), 7.56 (1H, dt, J 9.6,
1.9, C(8)H), 8.07 (2H, d, J 9.0, C(20,60)H), 9.40 (1H, br s, OH), 10.10
(1H, br s, OH); dC (100 MHz, d6-DMSO) 100.91 (t, J 24.8), 101.35 (dd,
J 25.8, 4.4), 109.16 (dd, J 10.9, 2.1), 115.39, 121.24, 129.31, 137.89,
145.17, 156.00 (dd, J 16.6, 6.4), 159.17, 160.48 (dd, J 263.1, 15.6),
163.74 (dd, J 251.0, 15.3), 170.19; dF{H} (376 MHz, d6-DMSO) ꢅ109.18
(d, J 11.0), ꢅ102.30 (d, J 11.0).
30 (0.46 g, 1.32 mmol) was suspended in acetic acid (20 mL) and
H2SO4 (conc.) (0.2 mL) was added. The mixture was heated to reflux
(125 ꢄC) for 10 min then cooled to room temperature before cold
H2O (100 mL) was added. The precipitate was collected by filtration
and air dried to afford 9 as a white solid (0.42 g, 96%). ESI-MS m/z
331 (M þ H)þ; dH (400 MHz, CDCl3) 3.96 (3H, s, OCH3), 3.96 (6H, s,
2ꢃ OCH3), 7.28 (2H, s, C(20,60)H), 7.46 (1H, ddd, J 8.0, 7.1, 0.5, C(6)H),
7.60 (1H, dd, J 8.5, 0.5, C(8)H), 7.74 (1H, ddd, J 8.5, 7.1, 1.6, C(7)H),
8.31 (1H, dd, J 8.0, 1.6, C(5)H); dC (75 MHz, CDCl3) 56.20, 60.87,
105.48, 105.60, 117.98, 123.59 (d, J 5.1), 123.97 (d, J 7.1), 125.00,
125.65 (d, J 3.2), 133.84, 140.88, 144.19, 148.90 (d, J 213.7), 150.62,
153.21, 154.82, 170.56 (d, J 17.0); dF{H} (282 MHz, CDCl3) ꢅ160.33 (s).
5.3.2.6. 7-Fluoro-30,40-dihydroxyflavonol (24). Method A. 87% yield;
ESI-MS m/z 287 (M ꢅ H)ꢅ; dH (400 MHz, d6-DMSO) 6.90 (1H, d, J
8.5, C(50)H), 7.33 (1H, td, J 8.8, 2.3, C(6)H), 7.58 (1H, dd, J 8.5, 2.3,
C(60)H), 7.65 (1H, dd, J 9.8, 2.3, C(8)H), 7.74 (1H, d, J 2.3, C(20)H), 8.14
(1H, dd, J 8.8, 6.4, C(5)H), 9.39 (3H, br s, 3 ꢃ OH); dC (100 MHz, d6-
DMSO) 104.65 (d, J 25.4), 113.22 (d, J 23.4), 115.40 (d, J 25.6), 118.48,
119.84, 122.03, 127.44 (d, J 10.9), 137.72, 145.03, 146.50, 147.63,
155.26 (d, J 14.1), 164.59 (d, J 251.0), 171.81; dF{H} (376 MHz, d6-
DMSO) ꢅ104.81 (s).
5.5. HPLC
HPLC analyses for purity determination were performed using
a Varian Prostar HPLC system (Varian Ltd) consisting of a Varian
ProStar 230 solvent delivery system, a ProStar 325 UV detector, and
a 410 Varian autosampler. Separation was achieved on a Gemini C18
column (4.6 mm ꢃ 150 mm, 3
mm, Phenomenex Ltd) at a flow rate
5.3.2.7. 7-Fluoro-40-hydroxyflavonol (27). Method B. 84% yield; ESI-
MS m/z 271 (M ꢅ H)ꢅ; dH (400 MHz, d6-DMSO) 6.94 (2H, d, J 8.9,
C(30,50)H), 7.34 (1H, td, J 8.7, 2.4, C(6)H), 7.68 (1H, dd, J 9.8, 2.4, C(8)
H), 8.10 (2H, d, J 8.9, C(20,60)H), 8.15 (1H, dd, J 8.9, 6.4, C(5)H), 9.40
(1H, br s, OH), 10.10 (1H, br s, OH); dC (100 MHz, d6-DMSO) 104.72
(d, J 25.6), 113.24 (d, J 23.4), 115.39, 118.53, 121.71, 127.43 (d, J 11.0),
129.44, 137.67, 146.45, 155.28 (d, J 14.2), 159.14, 164.58 (d, J 250.9),
171.85; dF{H} (376 MHz, d6-DMSO) ꢅ104.81 (s).
of 0.75 mL/min, using an isocratic mobile phase of 69% methanol in
0.1 M ammonium acetate buffer (pH 5.1). UV absorbance at 352 nm
was used to detect compounds.
5.6. Cell proliferation assay
22rn1 cells were kindly provided by Dr. Imran Ahmad from the
Beatson Institute for Cancer Research (Glasgow, UK). Cells were
cultured in RPMI 1640 medium supplemented with 10% foetal
bovine serum (Gibco, Paisley, UK) and harvested by a 5 min treat-
ment with trypsineEDTA solution (Gibco, Paisley, UK). Cells were
seeded (5 ꢃ 103) onto 24 well plates and allowed to adhere over-
night, then agents dissolved in DMSO were added and cells incu-
bated for periods of up to 144 h to furnish a final DMSO
concentration of <0.1%. Cells were washed with phosphate buff-
ered saline (PBS), harvested by trypsinisation and resuspended in
cell culture media (1 ml). Aliquots (1 ml) were diluted 10-fold
5.4. Synthesis of 3-fluoro-30,40,50-trimethoxyflavone (9)
5.4.1. 3-Hydroxy-1-(20-hydroxyphenyl)-3-(300,400,500-trimethoxy
phenyl)prop-2-en-1-one (29)
20-Hydroxyacetophenone (2.68 g, 19.71 mmol) was dissolved in
dry pyridine (70 mL) and 3,4,5-trimethoxybenzoyl chloride (5.0 g,
21.68 mmol) was added. The reaction was heated to 70 ꢄC and
stirred overnight. The reaction mixture was then cooled to room
temperature and acidified to pH 1 by addition of HCl (10% aq.). The
precipitate was filtered, washed with H2O and dried. This white
solid was dissolved in dry pyridine (100 mL) and KOH powder
(1.6 g) was added. The reaction was heated to 65 ꢄC with stirring for
5 h then cooled in an ice bath before cold HCl was added to
neutralise to pH 7. The precipitate was filtered and recrystallised
from ethanol to yield 29 as a yellow solid (3.07 g, 47% from 20-
hydroxyacetophenone). ESI-MS m/z 329 (M ꢅ H)ꢅ; dH (400 MHz,
CDCl3) 3.94 (3H, s, OCH3), 3.96 (6H, s, 2 ꢃ OCH3), 6.75 (1H, s, C(2)H),
6.93 (1H, ddd, J 8.2, 7.3, 1.1, C(50)H), 7.01 (1H, dd, J 8.5, 1.1, C(30)H),
7.17 (2H, s, C(200,600)H), 7.47 (1H, ddd, J 8.5, 7.3, 1.6, C(40)H), 7.77 (1H,
(Isoton II buffer) and 500
ml was analysed using a Z2 Coulter
Particle Count and Size Analyser (Beckman Coulter, UK). IC50 values
were calculated from the plot of cell number as percentage of
DMSO control versus agent log concentration at 144 h, at which
time cells were still in linear growth phase. When the data points
were plotted a line of best fit was added and the equation of the
straight line was used to calculate the IC50 value. The number
generated from the equation was converted back from the log10
concentration to give the actual IC50 value. Values are the mean of
3e6 separate experiments.

958
R.G. Britton et al. / European Journal of Medicinal Chemistry 54 (2012) 952e958
5.7. Western blot analysis
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pended in lysis buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate,
0.1% SDS, with protease inhibitors PMSF 1 mM, aprotinin 2
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1 mM and sodium fluoride 1 mM). The homogenate was placed on
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concentration in cell lysate was determined using the Biorad
protein assay (Biorad, Hemel Hempstead, UK) with bovine serum
mg/ml,
m
albumin as standard. An aliquot of protein lysate (15
mg) was
separated by SDSePAGE and transferred for 1 h onto a nitrocellu-
lose membrane (Schleicher & Schuell, NH, USA). Blots were blocked
for 2 h with 5% skimmed milk in PBS/Tween 0.05% (PBS-T), and
probed with a specific primary antiserum (AR (1:1000) and Actin
(1:2000); Santa Cruz Biotechnology; PSA (1:1000), DAKO) in PBS
containing 0.05% PBS-T and 5% non-fat dry milk (4 ꢄC, overnight).
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5 min) in PBS-T. Proteins were detected by the enhanced chem-
iluminescence system (Geneflow, Staffordshire, UK). Protein
densitometry data were collected using a Syngene (Cambridge,
Cambs., UK) GeneGnome gel documentation system and protein
expression was normalised to actin levels.
5.8. Statistical analyses
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Values shown in the results are the mean ꢀ SD. Statistical
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of flavonols on AR and PSA expression were subjected to one-way
analysis of variance (ANOVA) with post-hoc Bonferroni correc-
tion. P values <0.05 were considered significant.
Acknowledgement
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This work was funded by a programme grant from Cancer
Research UK (C325/A13101) and a grant from Prostate Action
(G2009/25). Thanks to Dr A. Stuart for proof reading.
Appendix A. Supporting information
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compounds, Chembiochem 5 (2004) 622e627.
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.ejmech.2012.06.031.