Synthesis and biological evaluation of phosphorylated flavonoids as potent and selective inhibitors of cholesterol esterase

Article abstract of DOI:10.1016/j.ejmech.2013.03.025

A series of phosphorylated flavonoids were synthesized and investigated in vitro as inhibitors of pancreatic cholesterol esterase (CEase) and acetylcholinesterase (AChE). The results showed that most of the synthesized compounds exhibited nanomolar potenc

Full text of DOI:10.1016/j.ejmech.2013.03.025

European Journal of Medicinal Chemistry xxx (2013) 1e8
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of phosphorylated flavonoids as
potent and selective inhibitors of cholesterol esterase
,
a
a
b
a
Yingling Wei a, Ai-Yun Peng a , Bo Wang , Lin Ma , Guoping Peng , Yidan Du ,
*
Jingming Tang a
a School of Chemistry & Chemical Engineering, Sun Yat-sen University, 135 Xingangxi Lu, Guangzhou 510275, China
b College of Bioscience and Biotechnology, Hunan Agricultural University, Furong District, Changsha 410428, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of phosphorylated flavonoids were synthesized and investigated in vitro as inhibitors of
pancreatic cholesterol esterase (CEase) and acetylcholinesterase (AChE). The results showed that most of
the synthesized compounds exhibited nanomolar potency against CEase, much better than the parent
flavonoids. Furthermore, these phosphorylated flavonoids demonstrated good to high selectivity for
CEase over AChE, which only showed micromolar potency inhibition of AChE. The most selective and
potent inhibitor of CEase (3e) had IC50 value of 0.72 nM and 11800-fold selectivity for CEase over AChE.
The structureeactivity relationships revealed that the free hydroxyl group at position 5 and phosphate
group at position 7 of the phosphorylated flavonoids are favorable to the inhibition of CEase. The inhi-
bition mechanism and kinetic characterization studies indicated that they are irreversible competitive
inhibitors of CEase.
Received 17 September 2012
Received in revised form
9 March 2013
Accepted 13 March 2013
Available online xxx
Keywords:
Cholesterol esterase
Acetylcholinesterase
Inhibitor
Crown Copyright Ó 2013 Published by Elsevier Masson SAS. All rights reserved.
Phosphorylated flavonoids
Organophosphate
Selectivity
1. Introduction
usually can be inhibited by the same class of compounds and it is
challenging to improve the selectivity when developing inhibitors
Pancreatic cholesterol esterase (CEase; EC 3.1.1.13) is an impor-
tant serine hydrolase that plays significant roles in the absorption
of dietary cholesterol. Inhibition of CEase has attracted much
attention in the last decades as a potential approach to treat
hypocholesterolemia and atherosclerosis by limiting the bioavail-
ability of dietary cholesterol [1]. Several classes of potent CEase
inhibitors have been developed [2], including 6-chloro-2-pyrones
[3], thieno [1,3]-oxazin-4-ones [1,4], carbamates [5], aryl phos-
phates and phosphonates [6], chloroisocoumarins [7], phosphai-
socoumarins [8], and thiazolidinediones [9] (Fig. 1). However, most
of these inhibitors are not highly selective and they could also
inhibit other serine hydrolases, such as acetylcholinesterase (AChE;
EC 3.1.1.7), butyrylcholinesterase (BChE, EC 3.1.1.8), Pseudomonas
species lipase (PSL, EC 3.1.1.3), chymotrpsin (CT, EC 3.4.21.1) and
trypsin (EC 3.4.21.4) [1,3,5d,5f,5h]. One main reason for the poor
selectivity is that all serine hydrolases as well as serine proteases
share the similar catalytic triad of Ser-His-Asp (Glu) and mecha-
nism of acylationedeacylation [10]. Therefore, these enzymes
of them.
Nevertheless, someselective inhibitorsofcertainserinehydrolase
have been designed and developed. For example, acetylcholines-
terase (AChE) is one of the most studied serine hydrolases as a viable
target to treat Alzheimer’s disease (AD) and many high selective in-
hibitors of AChE with nanomolar to picomolar potency have been
identified, three of which have been used clinically to treat AD [11]. In
contrast, only a few selective inhibitors of CEase were reported. In
1999, Deck’s group [3a] found that 6-chloro-3-(1-ethyl-2-
cyclohexyl)-2-pyranone could effectively inhibit CEase with a Ki
value of 25 nM and its selectivity for CEase over chymotrypsin was
greater than 1000-fold. Recently, Gütschow and Pietsch developed
two CEase inhibitors with micromolar potency and medium selec-
tivityforCEaseoverAChE;oneisa1,3-oxazin-4-one[1], andtheother
is a thiazolidinedione [9]. Apparently, it is still desirable to develop
novel inhibitors of CEase with high potency and high selectivity.
Flavonoids, including flavones, flavonols, isoflavones and flava-
nones, are a large class of polyphenolic compounds widely
distributed in herbs and foods of plant origin, and they exhibit
diversified biological activities, such as antioxidant, antiviral, anti-
cancer and enzyme inhibition [12]. Recently, we prepared five fully
phosphorylated flavones and found that they displayed excellent
* Corresponding author. Tel.: þ86 020 84110918; fax: þ86 020 84112245.
E-mail address: cespay@mail.sysu.edu.cn (A.-Y. Peng).
0223-5234/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.025
Please cite this article in press as: Y. Wei, et al., Synthesis and biological evaluation of phosphorylated flavonoids as potent and selective
inhibitors of cholesterol esterase, European Journal of Medicinal Chemistry (2013), http://dx.doi.org/10.1016/j.ejmech.2013.03.025

2
Y. Wei et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8
Fig. 1. Structures of some reported inhibitors of CEase.
CEase inhibitory activities with IC50 values in the nanomolar range
[13]. This work indicated that we may develop inhibitors of CEase
with better activities by investigating more kinds of phosphory-
lated flavonoids. Furthermore, aryl phosphates, especially those
with good leaving groups, such as diethyl 4-nitrophenyl phosphate
(paraoxon), are well-known irreversible inhibitors of many serine
hydrolases and proteases and widely used as insecticides [6a,14].
Taking into account that many phosphate insecticides are toxic and
the neurotoxicity of them are mainly associated with their AChE
inhibition [14], we think it necessary to examine whether these
flavonoid phosphate derivatives can selectively inhibit CEase over
AChE so as to preliminarily know about their application prospect.
In this paper, we presented a series of fully and partially phos-
phorylated flavonoids as inhibitors of CEase with low nanomolar
potency and high selectivity for porcine CEase over AChE from
Electrophorus electricus. The highest selectivity reached to over
10000-fold. Besides, we examined the inhibition mechanism and
kinetics, the results indicated that they are irreversible competitive
inhibitors of CEase.
2. Results and discussion
2.1. Chemistry
The synthesis of the phosphorylated flavonoids 2e4 was out-
lined in Scheme 1. According to our previous procedure [13],
the fully phosphorylated flavonoids 2ae2j could be obtained by
the treatment of 1ae1j with ClP(O)(OEt)2, Et3N and 4-
dimethylamiopryidine (DMAP) in THF at room temperature for
24 h. To accelerate the reaction process, we carried out these re-
actions under reflux conditions, and found that all the reactions
completed within 3 h (condition (1), Scheme 1). During these
Scheme 1. General synthetic procedure for the preparation of compounds 2, 3 and 4.a.
Please cite this article in press as: Y. Wei, et al., Synthesis and biological evaluation of phosphorylated flavonoids as potent and selective
inhibitors of cholesterol esterase, European Journal of Medicinal Chemistry (2013), http://dx.doi.org/10.1016/j.ejmech.2013.03.025

Y. Wei et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8
3
Table 1
courses, we noticed that for those parent flavonoids with a 5-
Inhibition of CEase and AChE by flavonoids and the phosphorylated derivatives.
hydroxy group, the fully phosphorylated products 2 were pro-
duced at first, but they would gradually dephosphorylate the 5-
phosphate to give the corresponding compounds 3. Compounds 3
became the main products when the reaction time was prolonged
to over 4 h in refluxing THF or when they were purified through
column chromatography using regular silica gel. This result can be
explained by the formation of the hydrogen bond between the 5-
hydroxy group and the nearby carbonyl group at position 4,
which makes compounds 3 more stable [15]. To get compounds 2
with high purity and avoid the undesired dephosphorylation re-
action, it is important to control the reaction time within 3 h under
reflux conditions and purify them through column chromatog-
raphy on deactivated silica gel [16]. Under these control conditions,
the transformation of compounds 2 into compounds 3 was negli-
gible, and compounds 2 could be obtained in pure and good yields
(see Experimental section). For example, the isolated yield of 2c
was 73% when using deactivated silica gel, while only a mixture of
2c and 3c were obtained when using the regular silica gel.
For the preparation of the phosphorylated flavonoids 3 with a 5-
hydroxy group, the above procedure is not convenient and effec-
tive. Compounds 3 could be synthesized readily by the reaction of
compounds 1 and HP(O)(OEt)2 using the classic AthertoneTodd
reaction [17] (condition (2), Scheme 1). Under these conditions, the
hydroxy group at position 5 is stable enough to resist the phos-
phorylation reaction.
Several methyl phosphorylated compounds 4 were prepared
under condition (3) (Scheme 1). As ClP(O)(OMe)2 is not easily
commercially available, we chose HP(O)(OMe)2 as the phosphory-
lation reagent. In this case, no matter using DMAP as catalyst or
increasing the equivalent of HP(O)(OMe)2, the 5-OH could not be
phosphorylated. Besides, when the starting material having more
than three hydroxyl groups, the reaction did not lead to the desired
product but to an unknown compound with high polarity (Rf ¼ 0,
CH2Cl2:MeOH ¼ 10:1) according to TLC. In the end, four methyl
phosphorylated compounds 4 were synthesized under typical
AthertoneTodd reaction conditions.
Compound
IC50 (nM)
CEase
Selectivity for CEase
over AChE (fold)
AChE
1a
2a
3a
1b
2b
1c
2c
3c
1d
2d
3d
4d
1e
2e
3e
4e
1f
4720 ꢀ 110
40.4 ꢀ 2.2
36.7 ꢀ 1.9
28100b
58200 ꢀ 500
3380 ꢀ 600
Nia
12.3
83.7
e
Ni
e
3.89b
170 ꢀ 40
62100 ꢀ 1000
Ni
Ni
Ni
280 ꢀ 60
970 ꢀ 20
6600 ꢀ 450
Nib
4350 ꢀ 120
8520 ꢀ 1010
6730 ꢀ 240
Ni
43.7
5.05
e
12300 ꢀ 800
1340 ꢀ 150
430 ꢀ 50
35700b
e
e
26.1b
10.7
232
24.4
Ni
1160
11800
39.6
4.18 ꢀ 0.15
270 ꢀ 60
Nib
3.76b
0.72 ꢀ 0.06
170 ꢀ 30
Nib
2f
4f
2.44b
2130 ꢀ 670
4190 ꢀ 240
Ni
873
6.07
e
690 ꢀ 120
1g
2g
4g
1h
2h
1i
2i
3i
1j
Nib
390b
12600 ꢀ 2900
32.3
e
Ni
Ni
Ni
Ni
e
20.6 ꢀ 0.4
20900 ꢀ 1600
4.62 ꢀ 0.56
1.85 ꢀ 0.01
Ni
2940 ꢀ 290
143
e
Ni
1260 ꢀ 20
3210 ꢀ 190
Ni
273
1735
e
2j
25.7 ꢀ 1.4
7650 ꢀ 240
298
a
Ni, no inhibition at 100
m
M.
b
Data taken from Ref. [13].
selectivity for CEase over AChE. Compounds 2e, 3e, 2f and 3i
were 1157-, 11800-, 873-, and 1735-fold more efficient for
inhibition of CEase than that of AChE (see Table 1). The
possible reasons for the superior inhibition of CEase are as
follows. On one hand, the catalytic triad of AChE is located at
the bottom of a deep and narrow cavity [23], while CEase has
a larger active site binding pocket than AChE [24]. The
phosphorylated flavonoids are relatively bulky that makes
them easier to enter the larger active site pocket of CEase. On
the other hand, these phosphate derivatives are not good
electrophiles with good leaving groups, and the nucleophi-
licity of AChE is less than that of CEase according to Lin and
coworkers’ research [5d].
2.2. Biology
2.2.1. Structureeactivity relationship (SAR) studies
The inhibition of CEase by compounds 1e4 was determined
spectrophotometrically by the Hosie assay [18] with some modifi-
cation as described before [6a,13]. The inhibition of AChE was
assayed according to the Ellman et al. method [19] and other lit-
eratures [6a,20]. The IC50 values of all compounds are summarized
in Table 1. Based on the data shown in Table 1, the preliminary
structureeactivity relationships could be summarized as follows.
(3) Ethyl phosphorylated flavonoids were more active than the
methyl derivatives in inhibiting CEase. For example, com-
pounds 2f, 3d and 3e have IC50 values of 2.44 nM, 3.18 nM and
0.72 nM against CEase, being 283-, 65-, and 236-fold more
potent than the methyl counterparts 4f, 4d and 4e. This is
probably due to the fact that the ethyl phosphates have better
lipophilicity than the methyl ones.
(4) Phosphorylated isoflavones inhibited CEase more effectively
than the phosphorylated flavones. For example, the only dif-
ference between 2d and 2i, 3d and 3i is the B benzene ring at
position 2 or 3. The isoflavone derivatives 3i and 2i were 2- to
6-fold more active than the flavone derivatives 3d and 2d (3i vs
3d, 2i vs 2d).
(5) The position of the phosphate group has much effect on the
inhibitory activities and the presence of the 7-phosphate and
5-OH is beneficial to the inhibition of CEase. From Table 1, we
can see that all potent inhibitors of CEase (IC50 < 5.0 nM) have a
7-phosphate group, indicating that the introduction of a
phosphate group at position 7 is important to the high inhi-
bition of CEase. Besides, compounds with a 5-OH were
(1) Phosphorylation of the phenolic groups of flavonoids signifi-
cantly improved their inhibitory potency against CEase. The
parent flavonoids 1 showed weak or no activities on CEase.
However, all tested derivatives (2e4) except 4g exhibited good
to excellent inhibitory activities against CEase, and seven of
them had IC50 values below 5.0 nM. For example, 3d showed an
IC50 value of 4.18 nM toward CEase that was 7223-fold better
than that of the parent compound 1d (IC50 ¼ 35.7
mM). Among
all compounds examined, 3e was the best CEase inhibitor
(IC50 ¼ 0.72 nM). The above results maybe attributed tothe facts
that these derivatives have better lipophilicity than the parent
compounds [1,21] and the tetrahedral phosphates can mimic
the transition-states of the hydrolysis of cholesterol esters [22].
(2) Comparing inhibition of CEase and AChE, all phosphate de-
rivatives examined only showed micromolar inhibition of
AChE and most of them demonstrated good to high
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4
Y. Wei et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8
The kinetic behavior of compounds 3d and 3e on the CEase
(Fig. 3) were then determined from LineweavereBurk double
reciprocal plots [25]. The results demonstrated that on a short time
scale, they were competitive inhibitors of CEase. The Michaelise
Menten constant Km value was calculated to be 130 ꢀ 10
mM from
the intercept of the X-axis when [I ¼ 0] of the plots in Fig. 3, which is
in agreement with the literature results [5b,5c]. The inhibition
constant Ki values (calculated from the plots) for the inhibition of
CEase by compounds 3d and 3e were 3.81 ꢀ 0.33 nM and
0.40 ꢀ 0.03 nM, respectively. These kinetic results are not sur-
prising. Tsou and Tian [27] have systematically investigated the
kinetics of irreversible inhibition of enzymes. They found that
competitive, noncompetitive, and uncompetitive inhibitions could
also be applied to irreversible inhibition and diisopropyl fluo-
rophosphate was as an irreversible competitive inhibitor of
chymotrypsin.
a-
Carbamates [5d], aryl phosphates and phosphonates [14c,22]
are well known irreversible inhibitors of many serine hydrolases
and proteases, their inhibition usually includes reversible binding
with the enzyme and irreversible carbamylation or phosphoryla-
tion of the active site. Similarly, the inhibition of CEase by the
phosphorylated flavonoids might involve reversible binding the
inhibitors with the enzyme followed by phosphorylation of the
active-site serine to give phosphorylated enzyme irreversibly
(Fig. 4).
Fig. 2. Time-dependent inhibition of CEase by compounds 3d, 3e, 2i and 3i. CEase was
preincubated with each inhibitor. At various incubation time intervals, the substrate
(3.0 mg/mL, 5 mL) was added to the incubation mixture, and the remain CEase activity
was immediately assayed. In the absence of inhibitor, no deactivation of enzyme was
observed during the time scale shown in the plot.
generally about 2- to 6-fold more active than their counterparts
with a 5-phosphate (2c vs 3c, 2d vs 3d, 2e vs 3e, 2i vs 3i). On the
contrary, the presence of a phosphate group at position 6 or 3
has little or adverse effect on the inhibition of CEase (2c vs 2e,
2a vs 2b, 3c vs 3e).
3. Conclusions
In conclusion, a series of phosphorylated flavonoids were syn-
thesized and identified as potent and selective inhibitors of CEase.
Most of the synthesized compounds showed nanomolar inhibitory
potency toward CEase and only micromolar inhibition of AChE,
indicating good to high selectivity between the two enzymes. The
inhibition mechanism and kinetic characterization studies sug-
gested that they inactivate CEase in an irreversible competitive
manner.
Among all compounds examined, 3e was the best inhibitor of
CEase with IC50 as low as 0.72 nM. The structureeactivity re-
lationships indicated that the 7-phosphate and 5-OH of the phos-
phorylated flavonoids are favorable to the inhibition of CEase.
Moreover, ethyl phosphorylated flavonoids were more active than
the methyl ones, and the phosphorylated isoflavones exhibited
better inhibition activity on CEase.
2.2.2. Mechanism and kinetic characterization
To determine whether phosphorylated flavonoids could pro-
duce irreversible inhibition of CEase or not, four most potent in-
hibitors were preincubated with the enzyme and the residual
activities of CEase were measured according to the literature pro-
tocol [25]. As indicated in Fig. 2, 3d, 3e, 2i and 3i (all at nanomolar
concentration) inactivated CEase progressively; the enzyme activ-
ity decreased as the preincubation time increased. The four com-
pounds were all characterized as active-site irreversible inhibitors
of CEase since they were time-dependent and followed the first-
order kinetics over the observed time period, which met some
criteria proposed by Abeles and Maycock [26]. In addition, no
enzyme activity was recovered after dialysis of the assay solution,
which further proved that they are irreversible inhibitors.
The present work suggested that the phosphorylated flavonoids
with excellent inhibitory activity and good selectivity for CEase
Fig. 3. LineweavereBurk plots for inhibition of CEase by 3d and 3e with p-nitrophenylbutyrate (pNPB) as the substrate. (A) and (B) represent kinetics of compounds 3d and 3e,
respectively. Ki ¼ [I]/[Km (app)/Km ꢁ 1]. Km (app) determined with inhibitor and substrate and Km with substrate alone.
Please cite this article in press as: Y. Wei, et al., Synthesis and biological evaluation of phosphorylated flavonoids as potent and selective
inhibitors of cholesterol esterase, European Journal of Medicinal Chemistry (2013), http://dx.doi.org/10.1016/j.ejmech.2013.03.025

Y. Wei et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8
5
Fig. 4. Proposed mechanism of inhibition of CEase by compound 3e.
over AChE (such as 3e) might act as lead compounds for developing
new, efficient and selective CEase inhibitors.
scanning spectrophotometer. Origin 7.5 was used to analyze the
enzyme assay data.
4.1. Chemistry
4. Experimental section
4.1.1. General procedure for the synthesis of compound 2
THF was dried according to standard procedures. Unless other-
wise noted, all other reagents were obtained from commercial
sources and used without further purification. NMR spectra was
recorded on a Varian Mercury-Plus 300 (1H 300 MHz; 13C
75.4 MHz; 31P 121 MHz). ESI-mass spectra was recorded on an
LCMS-2010A Liquid Chromatograph mass spectrometer. IR spectra
was recorded as KBr pellets on a Bruker Equinox 55FT/IR spec-
trometer. HRMS was determined by a Thermo MAT95XP High
Resolution mass spectrometer. Melting point was measured on a
SGWX-4 Micro Melting Point Apparatus and not corrected. Thin
layer chromatography was conducted on Kieselgel 60 F254. The
column chromatography was performed on deactivated 200e300
mesh silica gel, which was pretreated with a 0.025 M solution of
KH2PO4 in 1:l H2O/MeOH, filtered and oven-dried at 120 ꢂC for 12 h
[16]. Elemental analysis was determined at Vario EL Elemental
Analyzer, and the results were within 0.4% of the theoretical values.
The compounds without analytical purity were confirmed >95%
pure via HPLC methods that was performed on Shimadzu LC-10AT.
The synthesis and CEase inhibition of compounds 2b, 2de2g had
been reported previously by our group [13], and the characteriza-
tion data were in agreement with the published data. Compounds
2a and 2j was reported in a patent by one of our authors [28] and
compounds 2c, 2h, 2i, 3a, 3c, 3d, 3i, 4de4g are new compounds;
their synthetic procedures and spectral data are as follows. CEase
(porcine) was from Worthington. AChE (Electrophorus electricus)
and pNPB were from Sigma. S-acetylthiocholine iodine (ATCh) was
from Alfa. Enzymatic assays were done on a Shimadzu UV-2450
Compound 1 (1.0 mmol) was added to a solution of 20 mL THF,
DMAP (3.0 mmol per OH group) and Et3N (3.0 mmol per OH group),
and the mixture was stirred until dissolved.
A solution of
ClP(O)(OEt)2 (4.0 mmol per OH group) with 5 mL THF was then
added dropwise with vigorous stirring in an ice-water bath over
30 min. After stirring at 70 ꢂC for 3 h under nitrogen, the reaction
mixture was concentrated, diluted with EtOAc (3 ꢃ 30 mL), washed
with 0.5 M HCl (3 ꢃ 15 mL), 5% (w/v) NaOH (3 ꢃ 15 mL) and brine,
and dried over anhydrous Na2SO4. After removal of the solvent in
vacuo, the residue was purified by column chromatography on
deactivated silica gel with petroleum ether/EtOAc (1:1e1:4),
CH2Cl2/MeOH (100:1e60:1) as eluant to give the corresponding
product 2.
4.1.1.1. 2-[3,4-Bis[(diethoxyphosphinyl)oxy]phenyl]-4-oxo-4H-1-
benzopyran-3,5,7-triyl phosphoric acid hexaethyl ester (2a).
Yellow oil. Yield: 56%. 1H NMR (300 MHz, CDCl3):
d 7.84 (s, 1H), 7.69
(d, J ¼ 9.5 Hz, 1H), 7.50 (d, J ¼ 8.7 Hz, 1H), 7.20 (d, J ¼ 10.9 Hz, 2H),
4.36e4.02 (m, 20H), 1.25 (dt, J ¼ 39.2, 7.1 Hz, 30H); 13C NMR
(75 MHz, CDCl3):
d
169.8, 156.6, 153.8 (d, J ¼ 5.4 Hz), 152.0 (d,
J ¼ 6.2 Hz), 150.2 (d, J ¼ 6.0 Hz), 143.6 (t, J ¼ 5.7 Hz), 141.0 (t,
J ¼ 7.1 Hz), 134.4 (d, J ¼ 7.1 Hz), 126.3, 126.2, 122.0, 120.9, 113.2 (d,
J ¼ 6.1 Hz), 109.6, 105.2, 65.4e64.4 (m), 16.0 (d, J ¼ 6.4 Hz); 31P NMR
(121 MHz, CDCl3):
d
ꢁ4.02, ꢁ5.31, ꢁ5.80, ꢁ6.37, ꢁ6.77; MS (ESI): m/
z: 1005 (M þ Na)þ, 983 (M þ H)þ. Anal. Calcd for C35H55O22P5: C,
42.78; H, 5.64. Found C, 42.50; H, 5.54; IR (film, cmꢁ1): 2986, 2924,
1661, 1478, 1386, 1282, 1160, 1029.
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Y. Wei et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8
4.1.1.2. 4-Oxo-2-phenyl-4H-1-benzopyran-5,6,7-triyl phosphoric acid
column chromatography on deactivated silica gel with petroleum
ether/EtOAc (1:1e1:4), CH2Cl2/MeOH (100:1e60:1) as eluant to
give the corresponding product 3.
hexaethyl ester (2c). Yellow oil. Yield: 73%. 1H NMR (300 MHz,
CDCl3): d 7.89e7.82 (m, 2H), 7.69 (s, 1H), 7.55e7.48 (m, 3H), 6.70 (s,
1H), 4.48e4.29 (m, 12H), 1.41 (t, J ¼ 7.0 Hz, 18H); 13C NMR (75 MHz,
CDCl3):
d
175.5, 161.6, 153.1, 146.7, 141.2, 131.9, 131.3, 130.4, 128.6,
4.1.2.1. 2-[3,4-Bis[(diethoxyphosphinyl)oxy]phenyl]-5-hydroxy-4-
125.7, 114.8, 107.9 (d, J ¼ 9.1 Hz), 106.0, 65.7e64.3 (m), 15.9 (d,
oxo-4H-1-benzopyran-3,7-diyl phosphoric acid tetraethyl ester (3a).
J ¼ 4.5 Hz); 31P NMR (121 MHz, CDCl3):
d
ꢁ4.77 (d, J ¼ 3.6 Hz), ꢁ5.81
Yellow oil. Yield: 40%. 1H NMR (300 MHz, CDCl3):
d 12.22 (s, 1H),
(d, J ¼ 3.6 Hz), ꢁ6.59; MS (ESI): m/z: 701 (M þ Na)þ, 679 (M þ H)þ.
HRMS m/z calcd for C27H37O14P3 (Mþ), 678.1391; found, 678.1391. IR
(film, cmꢁ1): 2986, 2926, 0.1654, 1452, 1358, 1287, 1107, 1027.
7.95 (d, J ¼ 1.0 Hz,1H), 7.79 (d, J ¼ 8.7 Hz,1H), 7.60 (d, J ¼ 8.7 Hz,1H),
6.93e6.90 (m,1H), 6.68 (d, J ¼ 2.1 Hz,1H), 4.36e4.10 (m,16H),1.64e
1.13 (m, 24H); 13C NMR (75 MHz, CDCl3):
d 176.2, 161.6, 156.0 (d,
J ¼ 5.8 Hz), 155.6, 154.6 (d, J ¼ 5.7 Hz), 143.9, 141.2 (t, J ¼ 6.7 Hz),
4.1.1.3. 2-[3-(Diethoxyphosphinyl)oxy]phenyl]-4-oxo-4H-1-benzop-
132.4 (d, J ¼ 7.2 Hz),126.5,126.2,122.2,121.1,108.0,103.5, 98.8, 65.0,
yran-5,7-diyl phosphoric acid tetraethyl ester (2h). Yellow oil. Yield:
16.0; 31P NMR (121 MHz, CDCl3):
d
ꢁ3.92, ꢁ5.27, ꢁ5.79, ꢁ6.27; MS
80%. 1H NMR (300 MHz, CDCl3):
d
7.35 (d, J ¼ 1.9 Hz, 1H), 7.17 (d,
(ESI): m/z: 845 (M ꢁ H)e. Anal. Calcd for C31H46O19P4: C, 43.98; H,
5.48. Found C, 43.90; H, 5.71; IR (film, cmꢁ1): 3077, 2986, 2913,
1655, 1509, 1446, 1346, 1279, 1158, 1030.
J ¼ 9.0 Hz, 1H), 6.95 (d, J ¼ 8.5 Hz, 1H), 6.90 (d, J ¼ 1.0 Hz, 1H), 6.77 (d,
J ¼ 2.3 Hz,1H), 5.36 (dd, J ¼ 13.2, 2.8 Hz, 1H), 4.40e4.14 (m,12H), 3.87
(s, 3H), 3.02 (dd, J ¼ 16.5, 13.2 Hz, 1H), 2.77 (dd, J ¼ 16.5, 2.9 Hz, 1H),
1.42e1.32 (m, 18H); 13C NMR (75 MHz, CDCl3):
d
188.1, 163.2, 155.5 (d,
4.1.2.2. 5-Hydroxy-4-oxo-2-phenyl-4H-1-benzopyran-5,7-diyl phos-
J ¼ 6.1 Hz), 151.2 (d, J ¼ 6.0 Hz), 150.9 (d, J ¼ 4.5 Hz), 139.8 (d,
J ¼ 6.8 Hz), 130.4,123.4,119.4, 112.6, 110.6 (d, J ¼ 6.5 Hz), 106.77, 105.7
(d, J ¼ 16.9 Hz), 78.7 (d, J ¼ 7.2 Hz), 64.8 (dd, J ¼ 33.5, 6.1 Hz), 56.0,
phoric acid tetraethyl ester (3c). Yellow solid. Yield: 75%. M.p.: 119e
120 ꢂC. 1H NMR (300 MHz, CDCl3):
d
13.03 (s, 1H) 7.86 (dd, J ¼ 7.5,
2.1 Hz, 2H), 7.69 (s, 1H), 7.557.48 (m, 3H), 6.70 (s, 1H), 4.47e4.28 (m,
45.3, 16.5e15.8
(m);
31P
NMR
(121
MHz,
CDCl3):
8H), 1.41 (t, J ¼ 7.0 Hz, 12H); 13C NMR (75 MHz, CDCl3):
d 182.6,
d
ꢁ5.00, ꢁ6.38, ꢁ6.58; MS (ESI): m/z: 733 (M þ Na)þ, 749 (M þ K)þ,
164.8, 152.9, 152.6, 148.4, 132.4, 130.6, 129.0, 126.3, 125.7 (d,
J ¼ 8.6 Hz), 108.3e108.1 (m), 105.4 (d, J ¼ 11.3 Hz), 98.6 (d,
J ¼ 5.3 Hz), 65.1 (dd, J ¼ 35.3, 6.4 Hz), 16.8e15.8 (m); 31P NMR
711 (M þ H)þ. Anal. Calcd for C28H41O15P3: C, 47.33; H, 5.82. Found C,
47.12; H, 5.67; IR (film, cmꢁ1): 2986, 2924, 1696, 1612, 1517, 1434,
1380, 1276, 1148, 1027.
(121 MHz, CDCl3):
d
ꢁ4.28, ꢁ6.33; MS (ESI): m/z: 541 (M ꢁ H)ꢁ.
Anal. Calcd for C23H28O11P2: C, 50.93; H, 5.20. Found C, 51.22; H,
4.1.1.4. 3-[4-(Diethoxyphosphinyl)oxy]phenyl]-4-oxo-4H-1-benzop-
4.92; IR (KBr, cmꢁ1): 3081, 2989, 1620, 1454, 1360, 1290, 1168, 1053.
yran-5,7-diyl phosphoric acid tetraethyl ester (2i). Yellow oil. Yield:
73%. 1H NMR (300 MHz, CDCl3):
d
7.83 (s, 1H), 7.47 (d, J ¼ 8.4 Hz, 2H),
7.27 (d, J ¼ 1.3 Hz, 3H), 7.24 (d, J ¼ 1.0 Hz, 1H), 4.41e4.19 (m, 12H),
1.43e1.34 (m,18H); 13C NMR (75 MHz, CDCl3):
173.2,157.5,153.1 (d,
4.1.2.3. 2-[4-(Diethoxyphosphinyl)oxy]phenyl]-5-hydroxy-4-oxo-4H-
1-benzopyran-7-yl phosphoric acid diethyl ester (3d). Yellow oil.
d
Yield: 57%. 1H NMR (300 MHz, CDCl3):
d 12.72 (s, 1H), 7.88 (d,
J ¼ 5.8 Hz), 151.1, 150.4e150.1 (m), 150.0, 129.9, 127.6, 125.0, 119.3,
J ¼ 8.7 Hz, 2H), 7.38 (d, J ¼ 8.3 Hz, 2H), 6.99 (d, J ¼ 1.3 Hz, 1H), 6.67
113.9 (d, J ¼ 6.2 Hz), 109.5, 105.1, 65.1e63.9 (m), 15.7; 31P NMR
(s, 1H), 6.64 (d, J ¼ 1.8 Hz, 1H), 4.38e4.16 (m, 8H), 1.48e1.31 (m,
(121 MHz, CDCl3):
d
ꢁ5.26, ꢁ6.26, ꢁ6.45; MS (ESI): m/z: 701
12H); 13C NMR (75 MHz, CDCl3):
d 182.33, 163.5, 161.9, 156.7, 155.8
(M þ Na)þ, 717 (M þ K)þ, 679 (M þ H)þ. HRMS m/z calcd for
(d, J ¼ 5.7 Hz),153.5 (d, J ¼ 6.2 Hz),128.0,127.3,120.6,108.1,105.7 (d,
J ¼ 11.9 Hz), 103.8 (d, J ¼ 10.6 Hz), 98.9 (d, J ¼ 10.2 Hz), 65.0 (dd,
J ¼ 11.1, 6.0 Hz), 16.9e15.7 (m); 31P NMR (121 MHz, CDCl3):
C
27H37O14P3 ( Mþ), 678.1391; found, 678.1391. IR (film, cmꢁ1): 2986,
2923, 1654, 1433, 1371. 1282, 1170, 1029.
d
ꢁ5.65, ꢁ6.15; MS (ESI): m/z: 541 (M ꢁ H)ꢁ. HRMS m/z calcd for
4.1.1.5. 3-[4-(Diethoxyphosphinyl)oxy]phenyl]-4-oxo-4H-1-
C
23H28O11P2 (Mþ), 542.1101; found, 542.1100. IR (film, cmꢁ1): 3078,
benzopyran-7-yl phosphoric acid diethyl ester (2j). Yellow oil. Yield:
2986, 1655, 1496, 1343, 1281, 1156, 1030.
92%. 1H NMR (300 MHz, CDCl3):
d
8.28 (d, J ¼ 8.7 Hz, 1H), 7.98 (s,
1H), 7.54 (d, J ¼ 8.3 Hz, 2H), 7.41 (dd, J ¼ 2.1, 1.0 Hz, 1H), 7.32e7.26
4.1.2.4. 5-Hydroxy-4-oxo-2-phenyl-4H-1-benzopyran-7-yl phosphoric
(m, 3H), 4.34e4.18 (m, 8H), 1.39 (dd, J ¼ 7.1, 4.8, 1.1 Hz, 12H); 13C
acid diethyl ester (3e). Pale yellow solid. Yield: 66%. M.p.: 84e85 ꢂC
NMR (75 MHz, CDCl3):
d
175.1, 156.6, 154.4, 154.3, 152.9 (d,
(lit [29]., 87e89 ꢂC). 1H NMR (300 MHz, CDCl3):
d 12.74 (s, 1H), 7.89
J ¼ 5.6 Hz),150.5 (d, J ¼ 6.8 Hz),130.1, 128.1,124.4,121.3,119.9, 118.0,
(dd, J ¼ 7.7, 1.6 Hz, 2H), 7.55 (s, 2H), 7.53 (s, 1H), 7.00 (s, 1H), 6.72 (s,
108.4, 64.8 (dd, J ¼ 33.7, 6.0 Hz), 16.5e15.9 (m); 31P NMR (121 MHz,
1H), 6.66 (s, 1H), 4.34e4.22 (m, 4H), 1.41 (t, J ¼ 7.1 Hz, 6H); 13C NMR
CDCl3):
d
ꢁ5.28, ꢁ5.89; MS (ESI): m/z: 549 (M þ Na)þ, 565 (M þ K)þ,
(75 MHz, CDCl3): d 182.5, 164.5,162.0, 156.9, 155.9, 132.0, 130.9, 129.1,
527 (M þ H)þ. HRMS m/z calcd for C23H28O10P2 (Mþ), 526.1152;
found, 526.1154. IR (film, cmꢁ1): 2986, 2922, 1650, 1441, 1368, 1278,
1175, 1030.
126.3, 108.3, 106.0, 103.8 (d, J ¼ 5.3 Hz), 99.0 (d, J ¼ 4.3 Hz), 65.1 (d,
J ¼ 5.8 Hz), 16.2 (d, J ¼ 6.8 Hz); 31P NMR (121 MHz, CDCl3):
ꢁ6.16;
d
MS (ESI): m/z: 389 (M ꢁ H)ꢁ. IR (KBr, cmꢁ1): 3081, 2984, 1623, 1495,
1346, 1285, 1150, 1027.
4.1.2. General procedure for the synthesis of compound 3
Compound 1 (1.0 mmol) was added to a solution of 20 mL THF
and 5 mL Et3N (35.6 mmol), and the mixture was stirred until
dissolved. A solution of HP(O)(OEt)2 (1.1 equiv. per OH; the
numbers of OH were calculated one less than that of compound 1)
and 5 mL CCl4 (35.6 mmol) was added dropwise with vigorous
stirring in an ice-water bath over 30 min. The reaction proceeded
for 24 h at room temperature. The resulting salt of triethylamine
was filtered. The filtrate was evaporated in vacuo, and 10 mL of
water was added. The solution was extracted with EtOAc
(3 ꢃ 30 mL), washed with 1 M HCl (3 ꢃ 15 mL), saturated NaHCO3
solution (3 ꢃ 15 mL) and brine, and dried over anhydrous Na2SO4.
After removal of the solvent in vacuo, the residue was purified by
4.1.2.5. 3-[4-(Diethoxyphosphinyl)oxy]phenyl]-5-hydroxy-4-oxo-4H-
1-benzopyran-7-yl phosphoric acid diethyl ester (3i). Yellow oil.
Yield: 37%. 1H NMR (300 MHz, CDCl3):
d 12.75 (s, 1H), 7.95 (s, 1H),
7.51 (d, J ¼ 8.5 Hz, 2H), 7.31 (d, J ¼ 8.0 Hz, 2H), 6.90 (d, J ¼ 1.4 Hz,
1H), 6.69 (d, J ¼ 2.1 Hz, 1H), 4.33e4.20 (m, 8H), 1.45e1.35 (m, 12H);
13C NMR (75 MHz, CDCl3):
d
180.5, 162.3, 156.8, 155.7 (d, J ¼ 5.9 Hz),
153.6, 150.7 (d, J ¼ 6.6 Hz), 130.0, 126.8, 123.2, 120.0, 108.6, 103.6,
98.7, 64.8 (dd, J ¼ 29.9, 5.4 Hz), 16.1; 31P NMR (121 MHz, CDCl3):
d
ꢁ5.32, ꢁ6.19; MS (ESI): m/z: 565 (M þ Na)þ, 581 (M þ K)þ, 543
(M þ H)þ. HRMS m/z calcd for C23H28O11P2 (Mþ), 542.1101; found,
542.1102. IR (film, cmꢁ1): 3073, 2986, 2918, 1652, 1507, 1443, 1366,
1247, 1170, 1033.
Please cite this article in press as: Y. Wei, et al., Synthesis and biological evaluation of phosphorylated flavonoids as potent and selective
inhibitors of cholesterol esterase, European Journal of Medicinal Chemistry (2013), http://dx.doi.org/10.1016/j.ejmech.2013.03.025

Y. Wei et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8
7
4.1.3. General procedure for the synthesis of compound 4
4.2. Biology
Compound 1 (1.0 mmol) was added to a solution of 20 mL THF
and 5 mL Et3N (35.6 mmol), and the mixture was stirred until
dissolved. A solution of HP(O)(OMe)2 (1.1 equiv. per OH; the
numbers of OH were calculated one less than that of compound 1)
and 5 mL CCl4 (35.6 mmol) was added dropwise with vigorous
stirring in an ice-water bath. The reaction proceeded for 24 h at
room temperature. The resulting salt of triethylamine was filtered.
The filtrate was evaporated in vacuo, and 10 mL of water was
added. The solution was extracted with EtOAc (3 ꢃ 30 mL),
washed with 1 M HCl (3 ꢃ 15 mL) until neutral, saturated NaHCO3
solution (3 ꢃ 15 mL) and brine, and then dried over anhydrous
Na2SO4. After removal of the solvent in vacuo, the residue was
purified by column chromatography on deactivated silica gel
CH2Cl2/MeOH (60:1e30:1) with as eluant to give the corre-
sponding product 4.
4.2.1. CEase inhibition assay
CEase inhibition was assayed spectrophotometrically according
to Hoise et al. [18] with some modification as previously described
[6a,13]. CEase activity was measured by following the hydrolysis of
the colorimetric substrate pNPB. Assay buffer was 0.1 M sodium
phosphate buffer (pH 7.04, containing 0.1 M NaCl). The tempera-
ture was maintained at 25.0 ꢀ 0.2 ꢂC. The inhibitors and the sub-
strate were dissolved in acetonitrile. The final concentration of
acetonitrile was 1.5%. The inhibitors were pre-incubated with
CEase (3.25 U/mL, 5 mL) for 15 min. The substrate (3.0 mg/mL, 5 mL)
was then added to initiate the enzyme reaction (1.0 mL in a final
total volume), which was monitored immediately for 1 min by
measuring the absorbance at 405 nm. Each inhibitor was assayed
with six different concentrations around the IC50 values that were
roughly estimated in the first round of experiments. The mea-
surement was performed in triplicate for each concentration and
averaged before further calculation.
4.1.3.1. Dimethyl 5-hydroxy-4-oxo-2-phenyl-4H-chromen- 40, 7-yl
phosphate (4d). Pale yellow solid. Yield: 53%. M.p.: 117e119 ꢂC.
1H NMR (300 MHz, CDCl3):
d
12.68 (s, 1H), 7.86 (d, J ¼ 8.7 Hz,
2H), 7.36 (d, J ¼ 8.5 Hz, 2H), 6.94 (dd, J ¼ 2.1, 0.8 Hz, 1H), 6.64 (s,
4.2.2. AChE inhibition assay
1H), 6.62 (d, J ¼ 2.1 Hz, 1H), 3.89 (dd, J ¼ 11.4, 2.0 Hz, 12H); 13C
AChE inhibition was assayed spectrophotometrically at
37.0 ꢀ 0.2 ꢂC according to the method of Ellman et al. [19] and other
literature [6a,20]. AChE activity was measured by following the
hydrolysis of the colorimetric substrate ATCh. Assay buffer was
0.1 M sodium phosphate buffer (pH 7.04, containing 0.1 M NaCl).
The temperature was maintained at 37.0 ꢀ 0.2 ꢂC. The inhibitors
and the substrate were dissolved in acetonitrile. The final concen-
tration of acetonitrile was 1.5%. The inhibitors were pre-incubated
with AChE (1.0 U/mL, 10
acid) (DTNB) (3.0 mg/mL, 50
15 min. ATCh (3.0 mg/mL, 10
enzyme reaction (1.0 mL in a final total volume), which was
monitored immediately for 1 min by measuring the absorbance at
412 nm. Each inhibitor was assayed with six different concentra-
tions around the IC50 values that were roughly estimated in the first
round of experiments. The measurement was performed in tripli-
cate for each concentration and averaged before further calculation.
NMR (75 MHz, CDCl3):
d 182.3, 163.4, 162.0, 156.7, 155.6 (d,
J ¼ 6.0 Hz), 153.4 (d, J ¼ 6.2 Hz), 128.1, 127.5, 120.5 (d, J ¼ 4.4 Hz),
108.2, 105.8 (d, J ¼ 3.7 Hz), 103.7, 98.9, 55.2 (t, J ¼ 6.6 Hz); 31P
NMR (121 MHz, CDCl3):
d
ꢁ3.48, ꢁ3.95; MS (ESI): m/z: 485
(M ꢁ H)ꢁ. Anal. Calcd for C19H20O11P2: C, 46.93; H, 4.15. Found C,
47.06; H, 4.37; IR (KBr, cmꢁ1): 3080, 2958, 1658, 1499, 1346, 1289,
1159, 1058.
m
L) and 5,50-dithiobis(2-nitrobenzoic
L) as chromogenic agent for
L) was then added to initiate the
4.1.3.2. 5-Hydroxy-4-oxo-2-phenyl-4H-1-benzopyran-7-yl phosphoric
acid dimethyl ester (4e). Pale yellow solid. Yield: 58%. M.p.: 112e
m
m
113 ꢂC. 1H NMR (300 MHz, CDCl3):
d
12.76 (s, 1H), 7.90 (dd, J ¼ 7.9,
1.7 Hz, 2H), 7.60e7.50 (m, 3H), 7.00 (dd, J ¼ 2.2, 0.9 Hz, 1H), 6.74 (s,
1H), 6.66 (dd, J ¼ 2.2, 0.7 Hz, 1H), 3.93 (d, J ¼ 11.4 Hz, 6H); 13C NMR
(75 MHz, CDCl3):
d
182.5, 164.5, 162.1, 156.9, 155.6 (d, J ¼ 5.8 Hz),
132.1, 130.7, 129.0, 126.3, 108.3, 106.0, 103.7, 98.9, 55.2 (d,
J ¼ 6.1 Hz); 31P NMR (121 MHz, CDCl3):
ꢁ3.89; MS (ESI): m/z: 361
d
(M ꢁ H)e. Anal. Calcd for C17H15O7P: C, 56.36; H, 4.17. Found C,
56.08; H, 4.12; IR (KBr, cmꢁ1): 3077, 2962, 1621, 1493, 1345, 1293,
1144, 1026.
4.2.3. Mechanism of CEase inhibition
Four most potent inhibitors (3d, 3e, 2i or 3i) were chosen to
examine their inhibition mechanisms of CEase. CEase (3.25 U/mL,
4.1.3.3. 4-Oxo-2-phenyl-4H-1-benzopyran-7-yl phosphoric acid
dimethyl ester (4f). Pale yellow solid. Yield: 48%. M.p.: 100e
5 mL) was preincubated with each inhibitor at a certain concen-
tration near the IC50 value at 25.0 ꢀ 0.2 ꢂC. At various incubation
101 ꢂC. 1H NMR (300 MHz, CDCl3):
d
8.20 (d, J ¼ 8.5 Hz, 1H), 7.88
time intervals (0e60 min), the substrate (3.0 mg/mL, 5
mL) was
(s, 2H), 7.51 (s, 5H), 6.79 (s, 1H), 3.93 (d, J ¼ 11.4 Hz, 6H); 13C NMR
added to the above mixture (1.0 mL in a final total volume), and the
remain CEase activity was immediately monitored for 1 min by
measuring the absorbance at 405 nm. The measurement was per-
formed in triplicate for each time interval and averaged before
further calculation.
(75 MHz, CDCl3):
d
177.2, 163.4, 156.7, 154.2 (d, J ¼ 6.4 Hz), 131.5,
131.2, 128.9, 127.5, 126.1, 121.0, 117.7 (d, J ¼ 5.1 Hz), 108.8 (d,
J ¼ 4.0 Hz), 107.4, 55.2 (d, J ¼ 6.1 Hz); 31P NMR (121 MHz, CDCl3):
d
ꢁ3.67; MS (ESI): m/z: 347 (M þ 1)þ, 369 (M þ Na)þ, 385
(M þ K)þ. Anal. Calcd for C17H15O6P: C, 58.97; H, 4.37. Found C,
58.77; H, 4.66; IR (KBr, cmꢁ1): 2959, 1644, 1446, 1371, 1286, 1154,
1048.
4.2.4. Kinetic characterization of CEase inhibition
Compounds 3d and 3e were chosen to determine the kinetic
characterization of CEase according to the literature method [25].
CEase activity was measured by varying the concentration of the
4.1.3.4. 4-Oxo-2-phenyl-4H-1-benzopyran-6-yl phosphoric acid
dimethyl ester (4g). Pale yellow solid. Yield: 54%. M.p.: 85e87 ꢂC.
1H NMR (300 MHz, CDCl3):
d
7.95 (s, 1H), 7.92e7.85 (m, 2H), 7.64e
7.55 (m, 2H), 7.54e7.47 (m, 3H), 6.79 (s, 1H), 3.90 (d, J ¼ 11.3 Hz,
6H); 13C NMR (75 MHz, CDCl3):
177.3, 163.4, 153.0, 147.4 (d,
substrate. CEase (3.25 U/mL, 5
mL) was preincubated with each
inhibitor at two known concentrations or with no inhibitor at
25.0 ꢀ 0.2 ꢂC for 15 min. The enzyme reaction was initiated by
the addition of the substrate (1.0 mL in a final total volume), and
the enzyme activity was monitored for 1 min by measuring the
absorbance at 405 nm. Triplicate sets of data were collected for
each inhibitor concentration. The inhibition type, Km (without
inhibitor) and Ki (with inhibitor) were determined by Line-
weavereBurk double reciprocal plots.
d
J ¼ 6.8 Hz), 131.6, 131.3, 128.9, 126.3 (d, J ¼ 4.0 Hz), 126.1, 124.7,
119.8, 115.6 (d, J ¼ 4.9 Hz), 106.9, 55.1 (d, J ¼ 6.1 Hz); 31P NMR
(121 MHz, CDCl3):
d
ꢁ3.00; MS (ESI): m/z: 347 (M þ 1)þ, 369
(M þ Na)þ, 385 (M þ K)þ. Anal. Calcd for C17H15O6P: C, 58.97; H,
4.37. Found C, 59.04; H, 4.61; IR (KBr, cmꢁ1): 2923, 1634, 1467,
1361, 1270, 1178, 1034.
Please cite this article in press as: Y. Wei, et al., Synthesis and biological evaluation of phosphorylated flavonoids as potent and selective
inhibitors of cholesterol esterase, European Journal of Medicinal Chemistry (2013), http://dx.doi.org/10.1016/j.ejmech.2013.03.025

8
Y. Wei et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8
http://dx.doi.org/10.1155/2012/728983 (online article);
Acknowledgments
(c) M.I. Fernández-Bachiller, C. Pérez, L. Monjas, J. Rademann, M.I. Rodríguez-
Franco, New tacrineꢁ4-oxo-4H-chromene hybrids as multifunctional agents
for the treatment of Alzheimer’s disease, with cholinergic, antioxidant, and
We thank the National Natural Science Foundation of China
(Project No. 20602043) and the Fundamental Research Funds for
the Central Universities for financial support.
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candidates, Mini-Rev. Med. Chem. 8 (2008) 1429e1440.
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Appendix A. Supplementary material
Supplementary material related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.03.025.
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Please cite this article in press as: Y. Wei, et al., Synthesis and biological evaluation of phosphorylated flavonoids as potent and selective
inhibitors of cholesterol esterase, European Journal of Medicinal Chemistry (2013), http://dx.doi.org/10.1016/j.ejmech.2013.03.025