Phosphaisocoumarins as a new class of potent inhibitors for pancreatic cholesterol esterase

Article abstract of DOI:10.1016/j.ejmech.2010.01.038

Due to the importance of pancreatic cholesterol esterase (CEase) as a potential target in atherosclerosis and for the development of hypocholesterolemic agents, there are increasing interests in designing and synthesizing CEase inhibitors. In the present study, we prepared forty-five isocoumarin phosphorus analogues (i.e., phosphaisocoumarins) and investigated the inhibition of these compounds on the CEase. The results showed that some phosphaisocoumarins could act as potent inhibitors of CEase. The most potent inhibitors, compounds 9d, 10a and 12e give IC₅₀ values of 4.8?μM, 2.3?μM and 1.9?μM, respectively. The inhibition mechanism and kinetic characterization studies indicate that they are reversible competitive inhibitors.

Full text of DOI:10.1016/j.ejmech.2010.01.038

European Journal of Medicinal Chemistry 45 (2010) 1955–1963
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Phosphaisocoumarins as a new class of potent inhibitors for pancreatic
cholesterol esterase
*
Baojian Li, Binhua Zhou, Hailiang Lu, Lin Ma, Ai-Yun Peng
School of Chemistry & Chemical Engineering, Sun Yat-sen University, 135 Xingangxi Lu, Guangzhou 510275, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Due to the importance of pancreatic cholesterol esterase (CEase) as a potential target in atherosclerosis
and for the development of hypocholesterolemic agents, there are increasing interests in designing and
synthesizing CEase inhibitors. In the present study, we prepared forty-five isocoumarin phosphorus
analogues (i.e., phosphaisocoumarins) and investigated the inhibition of these compounds on the CEase.
The results showed that some phosphaisocoumarins could act as potent inhibitors of CEase. The most
Received 19 October 2009
Received in revised form
15 January 2010
Accepted 18 January 2010
Available online 28 January 2010
potent inhibitors, compounds 9d, 10a and 12e give IC50 values of 4.8 mM, 2.3 mM and 1.9 mM, respectively.
The inhibition mechanism and kinetic characterization studies indicate that they are reversible
competitive inhibitors.
Keywords:
Cholesterol esterase
Inhibitor
Ó 2010 Published by Elsevier Masson SAS.
Isocoumarin
Phosphaisocoumarin
1. Introduction
lipoprotein particles to smaller and more atherogenic lipoprotein
subspecies [11–13]. A recent investigation also disclosed that
Since plasma cholesterol level is linked to many diseases such as
coronary artery disease, cancer, obesity, and diabetes, the control of
the cholesterol level has gained much attention. In the past decades,
pancreatic cholesterol esterase (CEase; EC 3.1.1.13) has been studied
extensively as a potential target to control the cholesterol level. It
has been suggested that CEase plays important roles in the hydro-
lysis of dietary cholesterol esters [1–3] and the transport of free
cholesterol to enterocytes [4,5], although there are some conflict
reports about the roles of CEase in the absorption and transport of
free cholesterol [6,7]. The gene knockout studies demonstrated that
the lack of CEase reduced the intestinal absorption of dietary cho-
lesteryl esters but had no influence on intestinal absorption of free
cholesterol [2,3]. These results were also supported by the fact that
some inhibitors of CEase were shown to inhibit the absorption of
cholesterol esters in animal studies [8]. All the available data thus far
shows that CEase has no direct role in absorption of unesterified
cholesterol. It is believed that this enzyme plays an important
compensatory role in providing sufficient lipolytic enzyme activities
for complete triglyceride and phospholipid hydrolysis, which are
required for optimal absorption of cholesterol [9,10].
plaque-associated CEase had exhibited proangiogenic properties by
promoting proliferation, migration, and capillary network formation
by endothelial cells [14]. These findings further stimulated research
interests in the inhibition of CEase as a potential drug target in
atherosclerosis and for the development of hypocholesterolemic
agents.
CEase and serine proteases have a similar Ser-His-Asp (Glu)
catalytic triad [15,16] and they might be inhibited by the similar
classes of compounds. Isocoumarins are a class of well-established
mechanism-based inhibitors of a variety of serine proteases such as
porcine pancreatic elastase (PPE), human leukocyte elastase (HLE),
chymotrypsin, trypsin and the trypsin-like enzymes that initiate
blood coagulation and the complement cascade [17–19]. Recently,
Deck and coworkers [20] found that some 3-alkoxy-4-chlor-
oisocoumarins (Fig. 1) were potent reversible inhibitors of CEase.
Taking into account that phosphonic group can be regarded as
bioisostere of carboxylic group and some phosphonic acid
analogues of naturally occurring carboxylic acids have exhibited
similar bioactivities with the carboxylic acids [21–25], we reasoned
that isocoumarin phosphorus analogues (i.e., phosphaisocoumar-
ins) (Fig. 2) [26–31] would have homologous bioactivities with
isocoumarins. Furthermore, some phosphonic acid derivatives have
been developed as potent irreversible inhibitors of CEase [32,33].
Herein, we decided to investigate the inhibition of CEase by phos-
phaisocoumarins in the present study and the results showed that
some of them are potent reversible competitive inhibitors of CEase.
In addition, CEase has been reported to be proatherogenic since it
facilitated the conversion of the larger and less atherogenic
* Corresponding author. Tel.: þ86 020 84110918; fax: þ86 020 84112245.
E-mail address: cespay@mail.sysu.edu.cn (A.-Y. Peng).
0223-5234/$ – see front matter Ó 2010 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2010.01.038

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B. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1955–1963
phenyl group at the 3-position (6a, 8b–9b, 6c–9c, 6d) exhibit rela-
tively weak inhibitory activities (IC50 > 25 M). To our delight,
m
p-ethylphenyl group at the 3-position and halogen group, phenyl-
ethynyl or benzoylmethyl at the 4-position could work together to
improve the inhibitory effect by approximately 4–10 fold
(compounds 7d–9d, 10b, 11 vs compounds 7c–9c). Among them,
compound 9d, with iodine at the 4-position and a p-ethylphenyl
group at the 3-position, displays the strongest activity
Fig. 1. Structure of 3-alkoxy-4-chloroisocoumarins.
(IC50 ¼ 4.8
mM). However, the replacement of the aryl groups with
small alkyl groups (such as cyclopropyl and n-butyl) at the 3-posi-
tion results in inactive or poor active compounds (6e–9e, 6f–8f).
Similarly, in cases where there is no substituent at the 7-posi-
tion (compounds 6g–9g, 6h–9h), the introduction of halogen at the
4-position can also lower the IC50 value. For example, the
replacement of hydrogen with chlorine will improve the bioactivity
by approximately 5 fold (6g vs 7g). Compounds with small alkyl
group at the 3-position (6h–9h) show no or low inhibitory activi-
ties. To our surprise, the increase in the size of the group at the 4-
position going from hydrogen to phenylethynyl group is very
2. Chemistry
Most of the compounds tested in this study were synthesized as
previously described by our group [26–31]. The new compounds 8b,
9b, 10b, 11 and 6k were prepared by similar methods. Scheme 1
outlines the synthesis of 4-unsubstituted and 4-halophos-
phaisocoumarin esters. A variety of 2-(1-alkynyl)phenylphosphonic
diethyl esters 4 could be easily synthesized from phenol derivatives 1
via phosphorylation, rearrangment, sulfonation, and Sonogashira
coupling reactions in sequence. It was worth mentioning that aryl
productive by comparison of compound 10a (IC50 ¼ 2.3
mM) with
compound 6g (IC50 ¼ 63.6 M).
m
nonafluorobutanesulfonates (Rf
¼
n-C4F9) were appropriate
Compounds 6i–9i with a methoxy group at the 7-position show
weakened bioactivities compared with their counterparts with no
substituent at the 7-position (6g–9g), indicating that the methoxy
group may negatively affect the inhibitory activity. Compounds 7j,
8j and 6k with a methoxy group at the 7-position and small alkyl
group at the 3-position are also poor inhibitors.
substrates just like aryl perfluoroalkanesulfonates (Rf ¼ (CF2)2
O(CF2)2H) described previously in the Sonogashira reaction [29]. 4-
Unsubstituted phosphaisocoumarin esters 6 could be obtained by
CuI-catalyzed cyclization of 2-(1-alkynyl)phenylphosphonic mono-
ethyl esters 5 [26]. 4-Halophosphaisocoumarin esters 7, 8 and 9 were
prepared by halocyclization of the diethyl esters 4 with I2 [27], CuBr2
or CuCl2 [30], or by halocyclization of the monoethyl esters 5 with
NBS or NCS [28].
The reaction of 4-iodo-3-phenylphosphaisocoumarin ester 9g
with phenylacetylene afforded the corresponding coupling product
10a in 83% yield [27]. While under the similar conditions, the
reaction of 7-chloro-4-iodo-3-(4-ethylphenyl)phosphaisocoumarin
ester 9d and phenylacetylene gave a mixture of the coupling
product 10b in 45% yield and its acetylenic hydration derivative 11
in 26% yield (Scheme 2).
Besides, we investigated the inhibition effect of phosphaiso-
coumarin acids (compounds 12a–12i) on the CEase. In general,
these compounds follow similar rules with the corresponding esters
mentioned above. The importance of halogen at the 4-position is
also observed by comparison of 12a with 12b, and 12d with 12e. It is
worthwhile to point out that compound 12e with chlorine at the 4-
position gives the lowest IC50 value (1.9
compounds tested in this paper.
mM) among all the
Compared with their ester counterparts, compounds 12a, 12b,
12e, 12f and 12g show better inhibitory activities while compounds
12c, 12d, 12h and 12i are weaker inhibitors. Herein, it may indicate
that the hydrophilicity is not the main factor in deciding the
inhibitory effects. Additionally, the effects of different halogens are
complex by comparing all the compounds with halogen at the
4-position.
Based on the above results, the following structure–activity
relationships could be deduced thus far: (1) halogen or other larger
(for example, phenylethynyl, benzoylmethyl) group at the 4-posi-
tion, (2) an aryl group at the 3-position, and (3) no methoxy group
at the 7-position are favorable for CEase inhibition. Deck et al. [20]
also observed the importance of chlorine at the 4-position of iso-
coumarins on their inhibition of CEase, which are consistent with
our results in this point.
Phosphaisocoumarin acids 12a–12i were synthesized via the
reaction of phosphaisocoumarin esters with Me3SiBr in chloroform
followed by treatment with methanol, or with Me3SiCl and NaI in
acetonitrile followed by hydrolysis (Scheme 3) [31].
3. Biological results and discussion
3.1. Structure–activity correlations
The inhibition of phosphaisocoumarins on the CEase was
investigated according to Hosie et al. [34] with some modifications
and the results are summarized in Table 1. As shown in Table 1, the
inhibitory activities were much dependent on the substituents at
the positions 3, 4 and 7 of phosphaisocoumarins. Compounds with
chlorine at the 7-position and p-nitrophenyl, p-methoxyphenyl or
3.2. Mechanism and kinetic characterization
The inhibition mechanism of the most potent inhibitors 9d, 10a
and 12e on the CEase for the hydrolysis of p-nitrophenylbutyrate
was examined. Fig. 3 shows the relationship of enzyme activity
with its concentration in the presence of different concentrations of
inhibitors. The plots give a family of straight lines with different
slopes, all of which pass through the origin. These results demon-
strate that the inhibitory manners of compounds 9d, 10a and 12e
on the CEase are reversible.
To further confirm the reversible inhibitory manner of phos-
phaisocoumarins, compounds 9d, 10a and 12e were incubated with
CEase separately in the absence of p-nitrophenylbutyrate according
to Deck et al. [35]. CEase and excess inhibitors were added to sodium
Fig. 2. Structure of phosphaisocoumarin esters and acids.

B. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1955–1963
1957
Scheme 1. Synthesis of 4-unsubstituted and 4-halophosphaisocoumarin esters 6–9.
phosphate buffer (pH 7.04) containing acetonitrile (5% by volume),
then they were incubated at 25 ꢀC for up to 6 h which were detected
periodically by TLC. In these processes, no new products were
observed and all the added inhibitors could be finally recycled.
These experimental results convinced that the phosphaisocou-
marins are not irreversible but reversible inhibitors of CEase.
The kinetic behavior of phosphaisocoumarins on the CEase
using p-nitrophenylbutyrate as substrate was then determined
from Lineweaver–Burk double reciprocal plots (Fig. 4) [35]. The
results indicated that they were competitive inhibitors of CEase.
The dissociation constants (Ki, calculated from the plots) for 9d, 10a
irreversible inactivators of serine proteases and CEase in early years
[36]. Later, Deck et al. [35] found that 3-alkyl-6-chloro-2-pyrones
were simple competitive inhibitors of CEase rather than suicide
inhibitors. Thieno[2,3-d][1,3]oxazin-4-ones and tricyclic 1,3-oxazin-
4-ones were characterized by Gu¨tschow’s group as irreversible
inhibitors of CEase based on the acylation–deacylation mechanism
[37,38]. Recently, Deck et al. [20] developed 3-alkoxychloroiso-
coumarins as potent reversible inhibitors of CEase. Hermetter and
colleagues have demonstrated that some phosphonic derivatives are
irreversible inhibitors of CEase [32,33]. Unlike phosphates and
phosphonates are mainly known as suicide inhibitors of serine
proteases and CEase, our phosphaisocoumarins in this work are
reversible competitive inhibitors of CEase. This finding is interesting
and may be useful for the design of new reversible phosphonates
inhibitors of CEase.
and 12e are 4.2 mM, 1.9 mM and 1.4 mM, repectively.
Before our work, there are several similar classes of inhibitors of
CEase with variable mechanistic and kinetic behaviors. For example,
3-aryl-6-chloro-2-pyrones have been developed as potential
Scheme 2. Synthesis of 4-(phenylethnyl)phosphaisocoumarin esters 10a, 10b and the derivative 11.

1958
B. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1955–1963
4. Conclusions
Forty-five phosphaisocoumarins were synthesized and evalu-
ated in vitro as inhibitors of porcine pancreatic CEase and some of
them with appropriate 3-aryl and 4-halogen, 4-phenylethynyl or
4-benzoylmethyl substituents were demonstrated to be potent
inhibitors of CEase. Compounds with methoxy at the 7-position or
small alkyl groups (for example, cyclopropyl, n-butyl) at the
3-position show no or weak inhibition activity. In contrast to
phosphates and phosphonates usually acting as potent irreversible
inhibitors of CEase, these class of cyclic phosphonates are reversible
competitive inhibitors. The present work suggested that phos-
phaisocoumarins might act as interesting lead compounds for
developing new CEase inhibitors.
Scheme 3. Synthesis of phosphaisocoumarin acids 12.
Table 1
Inhibitory effects on pancreatic Cholesterol Esterase of phosphaisocoumarin esters and acids.
Structure
IC50
(
m
M)
Structure
IC50
(m
M)
Structure
IC50 (mM)
Br
I
C6H4-p-NO2
C6H4-p-OMe
C6H4-p-OMe
O
6a
>100
56.9
47.9
6.4
47.9
26.2
O
O
Cl
P
9b
8b
Cl
P
Cl
P
OEt
O
OEt
OEt
O
O
Cl
Br
Ph
Ph
Ph
O
6c
7c
34.8
40.3
4.8
51.2
8.9
nia
ni
8c
O
O
Cl
P
Cl
P
Cl
P
OEt
O
O
OEt
OEt
O
O
I
Cl
C6H4-p-Et
Ph
C6H4-p-Et
O
6d
9c
O
Cl
P
O
7d
Cl
P
OEt
Cl
P
O
OEt
OEt
O
Br
I
C6H4-p-Et
C6H4-p-Et
O
6e
O
O
8d
9d
Cl
P
Cl
P
Cl
P
OEt
OEt
OEt
O
O
O
Cl
Br
I
9e
7e
8e
>100
ni
O
O
O
Cl
Cl
P
P
Cl
Cl
P
Cl
P
OEt
OEt
OEt
O
O
O
Cl
P
Br
P
n-Bu
n-Bu
n-Bu
O
6f
8f
43.1
ni
ni
7f
O
O
Cl
OEt
O
P
OEt
OEt
O
O
Cl
Br
Ph
Ph
Ph
O
6g
7g
63.6
12.7
18.9
O
O
8g
P
P
OEt
O
OEt
OEt
O
O

B. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1955–1963
1959
M)
Table 1 (continued)
Structure
IC50
(m
M)
Structure
n-Bu
IC50
(m
M)
Structure
IC50 (m
I
Cl
Ph
n-Bu
O
6h
30.3
ni
ni
9g
O
P
O
P
7h
P
OEt
O
OEt
OEt
O
O
O
Ph
Br
P
I
n-Bu
n-Bu
Ph
ni
ni
2.3
8h
O
O
9h
P
10a
OEt
OEt
O
O
O
P
OEt
Ph
Ph
Ph
O
C6H4-p-Et
C6H4-p-Et
O
6i
8.5
5.1
77.4
MeO
P
OEt
O
O
11
O
10b
Cl
P
Cl
P
OEt
Br
OEt
O
O
Cl
I
Ph
Ph
Ph
9i
7i
8i
89.6
34.4
54.5
79.4
58.4
14.1
57.4
O
O
O
MeO
P
MeO
P
MeO
MeO
P
OEt
OEt
OEt
O
O
O
Cl
P
Br
P
OH
O
7j
P
>150
38.7
96.3
62.6
8j
ni
6k
O
O
OEt
MeO
MeO
O
P
OEt
OEt
O
O
Cl
Ph
C6H4-p-Et
Ph
O
O
12a
12c
12b
14.6
Cl
P
O
Cl
OH
Cl
P
OH
O
P
O
OH
O
Cl
Br
P
Ph
Ph
Ph
O
12d
12e
1.9
12f
O
O
P
OH
O
O
OH
OH
O
O
Br
P
n-Bu
Ph
Ph
O
O
12h
12g
102.5
P
MeO
P
O
12i
OH
OH
MeO
O
OH
O
a
ni, no inhibition at 100 mM.

1960
B. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1955–1963
Fig. 3. Determination of the inhibition mechanism of CEase by phosphaisocoumarins with p-nitrophenylbutyrate as substrate. (A), (B) and (C) represent the results of compounds
9d, 10a and 12e, respectively.
5. Experimental section
NMR (300 MHz, CDCl3):
6.86–6.89 (m, 2H), 4.07–4.28 (m, 4H), 3.82 (s, 3H), 1.33 (t, J ¼ 7.2 Hz,
6H); 13C NMR (75.4 MHz, CDCl3):
d 7.99–7.94 (m, 1H), 7.41–7.53 (m, 4H),
5.1. Chemistry
d
159.87, 134.07 (d, J ¼ 10.4 Hz),
133.90 (d, J ¼ 7.2 Hz), 133.38 (d, J ¼ 10.5 Hz), 132.89, 131.90, 131.52
(d, J ¼ 184.7 Hz), 124.47 (d, J ¼ 6.5 Hz), 114.72, 113.96, 95.77, 85.71
(d, J ¼ 8.2 Hz), 62.49 (d, J ¼ 6.1 Hz), 55.23, 16.31 (d, J ¼ 7.7 Hz); 31P
Anhydrous solvents (e.g. THF, DMF) were purified and dried
according to standard procedures. Unless otherwise noted, all other
reagents were obtained from commercial sources and used without
further purification. NMR spectra were recorded on a Varian
Mercury-Plus 300 (1H 300 MHz; 13C 75.4 MHz; 31P 121 MHz). ESI-
mass spectra were recorded on an LCMS-2010A Liquid Chromato-
graph mass spectrometer. HRMS were determined by a Thermo
MAT95XP High Resolution mass spectrometer. IR spectra were
recorded as KBr pellets on a Bruker Equinox 55 FT/IR spectrometer.
Melting points were not corrected. Compounds 6a, 6c–6i [26], 7c–
7j, 8c–8j [28,30], 9c–9e, 9g–9i, 10a [27], and 12a–12i [31] were
prepared as described previously. Compounds 4b, 8b, 9b, 6k, 10b,
and 11 are new compounds, their synthetic procedures and spectral
data are as follows.
NMR (121 MHz, CDCl3):
d
15.69; MS (ESI): m/z (%): 373 [(M þ Na)þ,
100]. Anal. Calcd for C19H20ClO4P: C, 60.25; H, 5.32. Found: C, 60.30;
H, 5.30. IR (KBr): 2982, 2215, 1606, 1461, 1289, 1250, 1025 cmꢁ1
.
5.1.2. 4-Bromo-1-ethoxy-3-(4-methoxyphenyl)benzo[c][1,2]-
oxaphosphinine 1-oxides (8b)
The mixture of 4b (140 mg, 0.50 mmol) with CuBr2 (447 mg,
2.0 mmol) and n-Bu4NBr (17 mg, 0.05 mmol), in DCE (10.0 mL) was
stirred at 85 ꢀC for 10 h. The reaction mixture was then diluted with
saturated brine and extracted with EtOAc. The combined organic
phase was dried over anhydrous Na2SO4. After removal of the
solvent in vacuo, the residue was purified by column chromatog-
raphy on silica gel with petroleum ether/EtOAc (7:1–2:1) as eluent
to afford 8b (192 mg, yield: 89%) as pale yellow solid, mp: 107–
5.1.1. 5-Chloro-2-(2-(4-methoxyphenyl)ethynyl)phenylphosphonic
acid diethyl esters (4b)
109 ꢀC. 1H NMR (300 MHz, CDCl3):
d 7.63–7.94 (m, 5H), 6.95–6.98
To a mixture of 3a (2734 mg, 5.0 mmol), PdCl2(PPh3)2 (175 mg,
0.25 mmol), Et3N (2.78 mL, 20.0 mmol), and DMF (15 mL) was
added dropwise the p-methoxyphenylethyne (991 mg, 7.5 mmol)
at room temperature. After stirring at 80 ꢀC for 5 h under nitrogen,
the reaction mixture was diluted with EtOAc and washed with
saturated NH4Cl until neutral and brine, dried (Na2SO4), and
evaporated in vacuo. The residue was chromatographed on silica
gel using petroleum ether/EtOAc (10:1–2:1) as eluent to give the
corresponding product 4b (1455 mg, yield: 77%) as yellow oil. 1H
(m, 2H), 4.25–4.35 (m, 2H), 3.89 (s, 3H), 1.39 (t, J ¼ 6.9 Hz, 3H); 13C
NMR (75.4 MHz, CDCl3):
d
160.67, 148.36 (d, J ¼ 12.5 Hz), 135.75 (d,
J ¼ 6.9 Hz), 134.37 (d, J ¼ 21.6 Hz), 133.30, 131.09, 129.99 (d,
J ¼ 12.4 Hz), 128.75 (d, J ¼ 11.1 Hz), 126.24 (d, J ¼ 4.7 Hz), 122.48 (d,
J ¼ 178.2 Hz), 113.25 (d, J ¼ 3.5 Hz), 102.76 (d, J ¼ 11.7 Hz), 63.80 (d,
J ¼ 5.8 Hz), 55.40, 16.54 (d, J ¼ 9.0 Hz); 31P NMR (121 MHz, CDCl3):
d
8.91; MS (ESI): m/z (%): 429 [(M þ 1)þ, 80], 431 [100]. Anal. Calcd
for C17H15ClBrO4P: C, 47.53; H, 3.52. Found: C, 47.65; H, 3.77. IR
(KBr): 2958, 1592, 1469, 1269, 1240, 1021 cmꢁ1
.

B. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1955–1963
1961
Fig. 4. Lineweaver–Burk plots for inhibition of CEase by phosphaisocoumarins with p-nitrophenylbutyrate as substrate. (A), (B) and (C) represent kinetics of compounds 9d, 10a and
12e, respectively.
5.1.3. 1-Ethoxy-4-iodo-3-(4-methoxyphenyl)benzo-
[c][1,2]oxaphosphinine 1-oxides (9b)
2.5 h. The mixture was evaporated in vacuo to remove the EtOH,
then diluted with H2O (20 L), cooed in an ice bath, neutralized with
concd HCl, and extracted with EtOAc. The combined extracts were
evaporated in vacuo to give the corresponding monoester quanti-
tatively, which was used without further purification. To a mixture
of the above monoester (130 mg, 0.48 mmol) in DMF (3 mL) was
added CuI (9 mg, 0.047 mmol). After heating at 90 ꢀC for 4 h, water
was added to the mixture and the resulting aqueous mixture was
extracted with EtOAc. The combined organic extract was washed
with saturated NH4Cl, brine, dried (Na2SO4), and evaporated in
vacuo. The residue was chromatographed on silica gel using
petroleum ether/EtOAc (2:1–1:1) as eluent to give 6k (100 mg,
A mixture of 4b (379 mg, 1.0 mmol) and I2 (508 mg, 2.0 mmol)
was dissolved in 10.0 mL CHCl3. After stirring at room temperature
for 18 h, the reaction mixture was then diluted with EtOAc and
washed with 5% aqueous Na2S2O3. The organic phase was washed
with brine, dried (Na2SO4), and evaporated in vacuo. The residue
was chromatographed on silica gel using petroleum ether/EtOAc
(7:1–2:1) as eluent to give 9b (308 mg, yield: 65%) as yellow solid,
mp: 115–117 ꢀC. 1H NMR (300 MHz, CDCl3):
6.95–6.98 (m, 2H), 4.23–4.36 (m, 2H), 3.89 (s, 3H), 1.40 (t, J ¼ 7.2 Hz,
3H); 13C NMR (75.4 MHz, CDCl3):
d 7.58–8.06 (m, 5H),
d
160.66, 150.86 (d, J ¼ 9.6 Hz),
137.40 (d, J ¼ 3.5 Hz), 134.74 (d, J ¼ 20.7 Hz), 133.42, 131.39, 128.83
(d, J ¼ 7.7 Hz), 128.57 (d, J ¼ 8.0 Hz), 126.48, 121.97 (d, J ¼ 181.9 Hz),
113.21, 63.79 (d, J ¼ 7.8 Hz), 55.38, 16.56 (d, J ¼ 6.2 Hz); 31P NMR
yield: 77%) as yellow oil. 1H NMR (300 MHz, CDCl3):
d 7.01–7.26 (m,
3H), 6.03 (s, 1H), 4.20 (d, J ¼ 3.6 Hz, 2H), 4.01–4.10 (m, 2H), 3.78 (s,
3H), 1.23 (t, J ¼ 6.9 Hz, 3H); 31P NMR (121 MHz, CDCl3):
d 12.96; MS
(121 MHz, CDCl3):
d
9.27; MS (ESI): m/z (%): 477 [(M þ 1)þ, 100].
(EI): m/z: 270 (Mþ, 43), 288 (93), 241 (23), 213 (58), 195 (21), 182
(44), 161 (51), 136 (23), 105 (10), 77 (25), 43 (100); Anal. Calcd for
C12H15O5P: C, 53.34; H, 5.60. Found: C, 52.98; H, 5.76; IR (film): 3381,
Anal. Calcd for C17H15ClIO4P: C, 42.84; H, 3.17. Found: C, 43.12; H,
3.41. IR (KBr): 2960, 1581, 1466, 1267, 1257, 1019 cmꢁ1
.
2928, 1605, 1495, 1242, 1033 cmꢁ1
.
5.1.4. (1-Ethoxy-7-methoxy-1-oxo-1H-1l
5-benzo[c][1,2]
oxaphosphinin-3-yl)-methanol (6k)
5.1.5. Synthesis of 10b and 11
The diethyl ester 4k [29] (500 mg,1.68 mmol) and aq 1.0 M NaOH
(10 mL, 10 mmol) were combined in EtOH (10 mL) and refluxed for
To a mixture of 9d (210 mg, 0.44 mmol), PdCl2(PPh3)2 (15 mg,
0.0214 mmol), CuI (15 mg, 0.079 mmol) and Et3N (3.0 mL), was

1962
B. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1955–1963
added dropwise phenylacetylene (0.10 mol) at room temperature.
After stirring at room temperature for 11 h under nitrogen, the
reaction mixture was diluted with EtOAc and washed with satu-
rated NH4Cl until neutral and brine, dried (Na2SO4), and evaporated
in vacuo. The residue was chromatographed on silica gel using
petroleum ether/EtOAc (10:1–4:1) as eluent to give the corre-
sponding product 10b (89, yield: 45%) as yellow solid and 11 (5 g,
yield: 26%) as yellow oil.
PNPB (0.2 mM) was added to the reaction mixture and the enzyme
reaction was monitored for 1 min by measuring the change in
absorbanceat 405 nm. Themeasurement was performed in triplicate
for each concentration and averaged before further calculation.
5.4. Kinetic characterization of CEase inhibition
The general procedure of the kinetic characterization of CEase is
also similar to the above inhibition assays. First, 0.0125 mU of
5.1.5.1. 7-Chloro-1-ethoxy-3-phenyl-4-phenylethynylbenzo[c][1,2]
CEase (0.53
mg/mL) were pre-incubated with the inhibitor
oxaphosphinine 1-oxide (10b). Pale yellow solid, mp 81–83 ꢀC; 1H
(concentration shown in Fig. 3) in sodium phosphate buffer for
1 min. Then, different concentrations of PNPB (0.07–0.3 mM) were
added to the reaction mixture and the enzyme reaction was
monitored for 1 min by measuring the change in absorbance at
405 nm. Triplicate sets of data were collected for each inhibitor
concentration. Kinetics of CEase inhibition was generally analyzed
by Lineweaver–Burk plots [35].
NMR (300 MHz, CDCl3):
d
8.01–8.13 (m, 3H), 7.89 (dd, J1 ¼ 15.3 Hz,
J2 ¼ 2.1 Hz, 1H), 7.67 (dd, J1 ¼ 8.7 Hz, J2 ¼ 2.1 Hz, 1H), 7.31–7.52 (m,
7H), 4.25–4.37 (m, 2H), 2.75 (q, J ¼ 7.5 Hz, 2H), 1.38 (t, J ¼ 7.2 Hz,
3H), 1.32 (t, J ¼ 7.5 Hz, 3H); 13C NMR (75.4 MHz, CDCl3):
d 154.68 (d,
J ¼ 11.7 Hz), 146.96, 136.18 (d, J ¼ 5.6 Hz), 134.04, 133.75, 133.27,
131.13,130.68 (d, J ¼ 6.7 Hz),128.88 (d, J ¼ 9.2 Hz),128.62,128.60 (d,
J ¼ 9.3 Hz), 128.39, 127.44, 122.77, 121.68 (d, J ¼ 180.8 Hz), 101.18,
96.73, 84.44, 63.62 (d, J ¼ 7.5 Hz), 28.91, 16.54 (d, J ¼ 6.4 Hz), 15.31;
Acknowledgements
31P NMR (121 MHz, CDCl3):
d
9.12; MS (ESI): m/z (%): 449 [(M þ 1)þ,
100]. Anal. Calcd for C26H22ClO3P: C, 69.57; H, 4.94. Found: C, 69.35;
This work was supported by the research grants from the
National Natural Science Foundation of China (Grant No. 20602043)
and Guangdong Natural Science Foundation (Grant No. 5300530).
H, 4.97. IR (KBr): 2981, 2207, 1651, 1471, 1279, 1248, 1077, 1020 cmꢁ1
.
5.1.5.2. 7-Chloro-1-ethoxy-3-phenyl-4-(benzoylmethyl)benzo[c][1,2]
oxaphosphinine 1-oxide (11). Yellow oil. 1H NMR (300 MHz, CDCl3):
References
d
7.84–7.93 (m, 3H), 7.28–7.44 (m, 4H), 7.19–7.23 (m, 3H), 6.94–6.99
(m, 1H), 3.97–4.03 (m, 2H), 3.53–3.70 (m, 3H), 2.77 (q, J ¼ 7.8 Hz,
2H), 1.31 (t, J ¼ 7.8 Hz, 3H), 1.11 (t, J ¼ 6.9 Hz, 3H); 13C NMR
[1] D.Y. Hui, Biochim. Biophys. Acta 1303 (1996) 1–14.
[2] P.N. Howles, C.P. Carter, D.Y. Hui, J. Biol. Chem. 271 (1996) 7196–7202.
[3] W. Weng, L. Li, A.M. van Bennekum, S.H. Potter, E.H. Harrison, W.S. Blaner,
J.L. Breslow, E.A. Fisher, Biochemistry 38 (1999) 4143–4149.
[4] A. Lopez-Candales, M.S. Bosner, C.A. Spilburg, L.G. Lange, Biochemistry 32
(1993) 12085–12089.
[5] S.C. Myers-Payne, D.Y. Hui, H.L. Brockman, F. Schroeder, Biochemistry 34
(1995) 3942–3947.
[6] R. Shamir, W.J. Johnson, R. Zolfaghari, H.S. Lee, E.A. Fisher, Biochemistry 34
(1995) 6351–6358.
[7] R.J. Kirby, S. Zheng, P. Tso, P.N. Howles, D.Y. Hui, J. Biol. Chem. 277 (2002)
4104–4109.
[8] J.E. Heidrich, L.M. Contos, L.A. Hunsaker, L.M. Deck, D.L. Vander Jagt, BMC
Pharmacol. 4 (2004) 5.
[9] K. Mackay, J.R. Starr, R.M. Lawn, J.L. Ellsworth, J. Biol. Chem. 272 (1997)
13380–13389.
(75.4 MHz, CDCl3):
d
194.01, 151.81, 151.18 (d, J ¼ 11.1 Hz), 135.22,
134.65, 134.58, 133.90 (d, J ¼ 20.5 Hz), 133.21, 130.13, 129.46 (d,
J ¼ 11.0 Hz), 129.26, 128.57, 128.43, 127.15, 127.00 (d, J ¼ 5.1 Hz),
121.95 (d, J ¼ 179.8 Hz), 118.02 (d, J ¼ 11.2 Hz), 62.95 (d, J ¼ 7.1 Hz),
39.35 (d, J ¼ 4.8 Hz), 29.15, 16.18 (d, J ¼ 6.7 Hz), 15.06; 31P NMR
(121 MHz, CDCl3):
d
9.42; MS (ESI): m/z (%): 373 [(M þ Na)þ, 100].
Anal. Calcd for C17H16ClO4P: C, 58.22; H, 4.60. Found: C, 58.07; H,
4.60. IR (KBr): 2966, 1667, 1603, 1474, 1288, 1264, 1027 cmꢁ1
.
5.2. Inhibition of CEase
[10] S.C. Young, D.Y. Hui, Biochem. J. 339 (1999) 615–620.
[11] O. Zscho¨rnig, M. Pietsch, R. Su¨ß, J. Schiller, M. Gu¨tschow, J. Lipid Res. 46 (2005)
803–811.
[12] D.Y. Hui, P.N. Howles, J. Lipid Res. 43 (2002) 2017–2030.
[13] J. Brodt-Eppley, P. White, S. Jenkins, D.Y. Hui, Biochim. Biophys. Acta 1272
(1995) 69–72.
CEase (porcine) and p-nitrophenylbutyrate (PNPB) were from
Sigma. The CEase inhibition was assayed according to Hosie et al.
[34] with some modifications. CEase activity was measured by
following the hydrolysis of the colorimetric substrate PNPB.
According to Pietsch et al. [38], the PNPB is susceptible to nonen-
zymatic hydrolysis, which should be considered when the testing is
more than 5 min. The testing in the present study only last for
1 min, so the effect of nonenzymatic hydrolysis was not considered
here. The temperature was maintained at 25.0 ꢂ 0.2 ꢀC. All the
compounds and PNPB were dissolved in acetonitrile. The final
concentration of acetonitrile in the test solution was 2.0%. First,
[14] O. Rebai, J. Le Petet-Thevenin, N. Bruneau, D. Lombardo, A. Verine, Arterioscl.
Thromb. Vasc. Biol. 25 (2005) 359–364.
[15] W. Boland, C. Froessl, M. Lorenz, Synthesis 12 (1991) 1049–1072.
[16] A. Svendsen, in: P. Woolley, S.B. Petersen (Eds.), Lipases, Their Structure
Biochemistry and Application, Cambridge University Press, Cambridge, 1994,
pp. 1–21.
¨
[17] J.C. Powers, J.L. Asgian, O.D. Ekici, K.E. James, Chem. Rev. 102 (2002) 4639–
4750 (and references therein).
´ ´
´
´
[18] F. Bihel, G. Quelever, H. Lelouard, A. Petit, C. Alves da Costa, O. Pourquie,
F. Checler, A. Thellend, P. Pierre, J.-L. Kraus, Bioorg. Med. Chem. 11 (2003)
3141–3152.
0.0125 mU of CEase (0.53 mg/mL) were pre-incubated with different
concentrations of inhibitor in 0.1 M sodium phosphate buffer (pH
7.04, containing 0.1 M NaCl) for 1 min. Then, the PNPB (0.1 mM) was
added to the reaction mixture and the enzyme reaction was
monitored for 1 min by measuring the change in absorbance at
405 nm. The measurement was performed in triplicate for each
concentration and averaged before further calculation. The
computer program used for the analysis of data was Origin 7.5.
[19] L. Pochet, R. Fre´de´rick, B. Masereel, Curr. Pharm. Des. 10 (2004) 3781–3796.
[20] J.J. Heynekamp, L.A. Hunsaker, T.A. Vander Jagt, R.E. Royer, L.M. Deck,
D.L. Vander Jagt, Bioorg. Med. Chem. 16 (2008) 5285–5294.
[21] K.B. Dillon, F. Mathey, J.F. Nixon FRS (Eds.), Phosphorus: The Carbon Copy, John
Wiley & Sons, Chichester, 1998.
[22] R.G. Almquist, W.-R. Chao, C. Jennings-White, J. Med. Chem. 28 (1985)
1067–1071.
[23] R. Engel (Ed.), Handbook of Orgnophosphorus Chemistry, Marcel Dekker, New
York, 1992.
[24] F.R. Hartley (Ed.), The Chemistry of Organophosphorus Compounds, Vol. 4,
John Wiley & Sons, New York, 1996.
5.3. Mechanism of CEase inhibition
[25] L.D. Quin (Ed.), A Guide to Organophosphorus Chemistry, John Wiley & Sons,
New York, 2000.
[26] A.-Y. Peng, Y.-X. Ding, J. Am. Chem. Soc. 125 (2003) 15006–15007.
[27] A.-Y. Peng, Y.-X. Ding, Org. Lett. 6 (2004) 1119–1121.
[28] A.-Y. Peng, Y.-X. Ding, Tetrahedron 61 (2005) 10303–10308.
[29] A.-Y. Peng, X.-Y. Zhang, Y.-X. Ding, Heteroat. Chem. 16 (2005) 529–534.
The general procedure of the mechanism study is similar to the
above inhibition assays. First, different concentrations of CEase
(0.2–1.0
mg/mL) were pre-incubated with the inhibitor (concentra-
tion shown in Fig. 3) in sodium phosphate buffer for 1 min. Then, the

B. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1955–1963
1963
[30] A.-Y. Peng, F. Hao, B. Li, Z. Wang, Y. Du, J. Org. Chem. 73 (2008) 9012–9015.
[31] A.-Y. Peng, B. Li, X. Yang, J. Lin, Synthesis 15 (2008) 2412–2416.
[32] H. Schmidinger, R. Birner-Gruenberger, G. Riesenhuber, R. Saf, H. Susani-
Etzerodt, A. Hermetter, ChemBioChem 6 (2005) 1776–1781.
[33] G.B. Quistad, S.N. Liang, K.J. Fisher, D.K. Nomura, J.E. Casida, Toxicol. Sci. 91
(2006) 166–172.
[34] L. Hosie, L.D. Sutton, D.M. Quinn, J. Biol. Chem. 262 (1987) 260–264.
[35] L.M. Deck, M.L. Baca, S.L. Salas, L.A. Hunsaker, D.L. Vander Jagt, J. Med. Chem.
42 (1999) 4250–4256.
[36] R.B. Westkaemper, R.H. Abeles, Biochemistry 22 (1983) 3256–3264.
[37] M. Pietsch, M. Gu¨tschow, J. Biol. Chem. 277 (2002) 24006–24013.
[38] M. Pietsch, M. Gu¨ tschow, J. Med. Chem. 48 (2005) 8270–8288.