Isocoumarin-based inhibitors of pancreatic cholesterol esterase

Article abstract of DOI:10.1016/j.bmc.2008.03.016

Pancreatic cholesterol esterase (CEase), which is secreted from the exocrine pancreas, is a serine hydrolase that aids in the bile salt-dependent hydrolysis of dietary cholesteryl esters and contributes to the hydrolysis of triglycerides and phospholipids. Additional roles for CEase in intestinal micelle formation and in transport of free cholesterol to the enterocyte have been suggested. There also are studies that point to a pathological role(s) for CEase in the circulation where CEase accumulates in atherosclerotic lesions and triggers proliferation of smooth muscle cells. Thus, there is interest in CEase as a potential drug target. 4-Chloro-3-alkoxyisocoumarins are a class of haloenol lactones that inhibit serine hydrolases and serine proteases and have the potential to be suicide inhibitors. In the present study, we have developed 3-alkoxychloroisocoumarins that are potent inhibitors of CEase. These inhibitors were designed to have a saturated cycloalkane ring incorporated into a 3-alkoxy substituent. The size of the ring as well as the length of the tether holding the ring was found to be important contributors to binding to CEase. 4-Chloro-3-(4-cyclohexylbutoxy)isocoumarin and 4-chloro-3-(3-cyclopentylpropoxy)isocoumarin were demonstrated to be potent reversible inhibitors of CEase, with dissociation constants of 11 nM and 19 nM, respectively. The kinetic results are consistent with predictions from molecular modeling.

Full text of DOI:10.1016/j.bmc.2008.03.016

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5285–5294
Isocoumarin-based inhibitors of pancreatic cholesterol esterase
Justin J. Heynekamp,a Lucy A. Hunsaker,b Thomas A. Vander Jagt,b Robert E. Royer,b
Lorraine M. Decka,* and David L. Vander Jagtb,*
aDepartment of Chemistry, University of New Mexico, Albuquerque, NM 87131, USA
bDepartment of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA
Received 10 July 2007; revised 26 February 2008; accepted 3 March 2008
Available online 6 March 2008
Abstract—Pancreatic cholesterol esterase (CEase), which is secreted from the exocrine pancreas, is a serine hydrolase that aids in the
bile salt-dependent hydrolysis of dietary cholesteryl esters and contributes to the hydrolysis of triglycerides and phospholipids. Addi-
tional roles for CEase in intestinal micelle formation and in transport of free cholesterol to the enterocyte have been suggested.
There also are studies that point to a pathological role(s) for CEase in the circulation where CEase accumulates in atherosclerotic
lesions and triggers proliferation of smooth muscle cells. Thus, there is interest in CEase as a potential drug target. 4-Chloro-3-
alkoxyisocoumarins are a class of haloenol lactones that inhibit serine hydrolases and serine proteases and have the potential to
be suicide inhibitors. In the present study, we have developed 3-alkoxychloroisocoumarins that are potent inhibitors of CEase.
These inhibitors were designed to have a saturated cycloalkane ring incorporated into a 3-alkoxy substituent. The size of the ring
as well as the length of the tether holding the ring was found to be important contributors to binding to CEase. 4-Chloro-3-(4-cyclo-
hexylbutoxy)isocoumarin and 4-chloro-3-(3-cyclopentylpropoxy)isocoumarin were demonstrated to be potent reversible inhibitors
of CEase, with dissociation constants of 11 nM and 19 nM, respectively. The kinetic results are consistent with predictions from
molecular modeling.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
blocking the Nieman-Pick C-1 like-1 protein
(NPC1L1).4 Not all hypercholesterolemic individuals
can tolerate statins.5 In addition, responsiveness of plas-
ma cholesterol levels to dietary changes in cholesterol is
highly variable.6 Therefore, there continues to be inter-
est in other targets for control of plasma cholesterol.
The well-established link between plasma cholesterol
levels and coronary artery disease and the contribution
of elevated plasma cholesterol, specifically LDL-choles-
terol, to other diseases including cancer, obesity, and
diabetes have made control of plasma cholesterol a ma-
jor health aim. Because cholesterol is both the product
of biosynthesis and of dietary intake, multiple ap-
proaches to control of plasma cholesterol have been ini-
tiated. The statins, inhibitors of the rate-limiting
biosynthetic enzyme HMGCoA reductase in the path-
way of cholesterol synthesis, have been highly successful
in controlling plasma cholesterol levels.1 In addition, po-
tent inhibitors of intestinal uptake of dietary cholesterol
have been introduced.2 Ezetimibe, which is a member of
this new class of drugs that limit the absorption of free
cholesterol,3 appears to act directly at the enterocyte
membrane to limit cholesterol uptake, possibly by
Pancreatic cholesterol esterase (CEase; EC 3.1.1.13),
also known as bile salt-stimulated lipase, bile salt-depen-
dent lipase, carboxyl ester lipase, pancreatic lysophos-
pholipase, and nonspecific lipase, has been studied
extensively as a potential target to prevent the absorp-
tion of dietary cholesterol. CEase, along with triglycer-
ide lipase and pancreatic phospholipase A2, is secreted
from the exocrine pancreas in response to a fat-contain-
ing meal where CEase aids in the bile salt-dependent
hydrolysis of dietary cholesteryl esters, as well as con-
tributes to the hydrolysis of triglycerides and phospho-
lipids, consistent with the known broad substrate
specificity of CEase.7 The major physiological role of
CEase in determining the bioavailability of cholesterol
that is derived from cholesteryl esters is supported by
CEase gene knockout studies.8 A second role for CEase
in transport of free cholesterol to the enterocyte has
been suggested, although there are conflicting reports
Keywords: Inhibitors; Cholesterol esterase; Haloenolactones.
*
Corresponding authors. Tel.: +1 505 277 5438 (L.M.D.); tel.: +1 505
272 5788; fax: +1 505 272 3518 (D.L.V.J.); e-mail addresses:
ldeck@unm.edu; dlvanderjagt@salud.unm.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.016

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J. J. Heynekamp et al. / Bioorg. Med. Chem. 16 (2008) 5285–5294
concerning this role for CEase.9,10 In addition, a role for
CEase in intestinal micelle formation has been sug-
gested, where CEase participates with phospholipase
A2 in hydrolysis of lecithin to lysolecithin which is re-
quired for the formation of intestinal micelles that effi-
ciently deliver free cholesterol.11 In the absence of
hydrolysis of lecithin to lysolecithin, the intestinal mi-
celles that form do not deliver cholesterol efficiently.
Figure 2. Structure of 3-alkoxy-4-chloroisocoumarin inhibitors of
CEase.
There is another developing literature that points to a
role(s) for CEase in the circulation. It has long been
noted that CEase is detectable in blood. This CEase ap-
pears to be derived from pancreatic CEase that initially
is secreted into the intestine. Part of this CEase, perhaps
as a complex with chaperone GRP94, is internalized by
enterocytes and is released into the circulation where it is
associated with apolipoprotein B-containing lipopro-
teins. This circulating CEase accumulates in atheroscle-
rotic lesions and triggers proliferation of smooth muscle
cells.12,13 Recently, plaque-associated CEase has also
been shown to exhibit pro-angiogenic properties by pro-
moting proliferation, migration, and capillary network
formation by endothelial cells.14 Thus, CEase may be in-
volved in pathophysiological angiogenesis, which adds
to interest in CEase as a potential drug target.
also tested to investigate the effect of hydrogen bond do-
nors and acceptors at the 7-position. We demonstrate
that potent inhibitors of CEase with low nanomolar dis-
sociation constants can be developed as lead compounds
using the chloroisocoumarin scaffold.
2. Chemistry
The synthesis of compounds 2q, 2r, 3q, 3r, 6a, 6b, and 7a
was reported by Heynekamp et al.19 The other com-
pounds used in this study were prepared by similar
methods. Scheme 1 outlines the synthesis of 3a–3r.
Homophthalic acid (1a) or 5-nitrohomophthalic acid
(1n) was esterified to give monoesters 2a–2r. Two equiv-
alents of alcohol was used in these esterifications in or-
der to minimize the amount of starting homophthalic
acid in the product. The amount of diester byproduct
is increased somewhat by using excess alcohol, but the
diester is much easier to separate from the monoester
product than is the starting diacid. Cyclization of esters
2a–2r with phosphorus pentachloride in toluene gave 3-
alkoxy-4-chloroisocoumarins 3a–3r.
CEase is a serine hydrolase utilizing serine 194, histidine
435 and aspartate 320 as the catalytic triad and is part of
the a/b hydrolase family of enzymes. Previously, we de-
scribed the synthesis and testing of a series of 3- and 5-
cycloalkyl-6-chloro-2-pyrones (Fig. 1) that reversibly
inhibited CEase and were effective at preventing the
absorption of dietary cholesteryl esters.15–17 The chloro-
pyrones are a class of haloenol lactones that inhibit ser-
ine hydrolases and serine proteases and have the
potential to be irreversible inhibitors.
Compound 4, (7-amino-4-chloro-3-methoxyisocouma-
rin), was prepared by catalytic hydrogenation of the ni-
tro group of 3n as shown in Scheme 2. Amides 5a and 5b
were prepared by treating 4 with cyclohexanecarbonyl
chloride and benzoyl chloride, respectively, in the pres-
ence of triethylamine (Scheme 2).
There is another class of haloenol lactones, the chloro-
isocoumarins, that also inhibit serine hydrolases and
serine proteases.18 Recently we have described 3-alk-
oxy-4-chloroisocoumarins that are potent inhibitors of
the serine protease urokinase plasminogen activator
(uPA).19 uPA functions through a catalytic mechanism
that is similar to CEase. In the present study, we have
found that some 3-alkoxy-4-chloroisocoumarins are po-
tent inhibitors of CEase. The general structure for these
compounds is shown in Figure 2. In the majority of the
compounds tested, the R group is an alkyl chain of zero
to five carbons terminating in a saturated cyclopentyl or
cyclohexyl ring or a bromine and X is hydrogen. In
addition, in order to investigate the importance of the
chlorine, compounds corresponding to one of the 3-alk-
oxy-4-chloroisocoumarins, but with a hydrogen or a tri-
fluoroacetyl group replacing the chlorine were tested.
Compounds where X is an amino or nitro group were
Scheme 3 shows the synthesis of compounds that lack
chlorine in the four position. These compounds were
prepared by cyclizing the corresponding monoesters
with trifluoroacetic anhydride. Cyclization of esters with
nitro groups on the ring gave the expected products
unsubstituted at the 4-position (6a, 6b, 6d, 6e). Under
the same conditions, esters without nitro groups on
the ring gave products with trifluoroacetyl groups at
the 4-position (6c, 6f, 6g).
Finally, reduction of the nitro group of compound 6b by
catalytic hydrogenation provided amino substituted
compound 7b.
3. Results and discussion
Molecular modeling studies were conducted to deter-
mine the inhibition potential of the designed compounds
by docking the inhibitors into the active site of CEase.
The results of the flexible ligand docking were quantita-
Figure 1. Structure of pyrone inhibitors of CEase.

J. J. Heynekamp et al. / Bioorg. Med. Chem. 16 (2008) 5285–5294
5287
Scheme 1.
Scheme 2.
Scheme 3.

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J. J. Heynekamp et al. / Bioorg. Med. Chem. 16 (2008) 5285–5294
tively and qualitatively analyzed to determine the pre-
dicted binding affinities. The results of the modeling
studies suggested that 4-chloro-isocoumarins substituted
at the 3-position with large lipophilic groups and longer
tethers would be the best inhibitors. The active site of
CEase contains a deep pocket to bind the long chain
fatty acid portion of the native cholesterol ester sub-
strates. The inhibitors were designed with the idea that
lipophilic cycloalkyl groups would occupy the fatty acid
site. Occupation of the fatty acid pocket by the lipo-
philic cycloalkyl groups would orient the reactive car-
bonyl of the isocoumarin backbone close to serine 194.
This in turn would provide the proper orientation for
potential irreversible binding. Structure–activity rela-
tionships were determined by comparing predicted ori-
entations and rankings with actual kinetic results. For
the chloroisocoumarins, it was predicted that 3-alkoxy
substituents with a terminal cyclopentyl group or cyclo-
hexyl group would be good inhibitors, and that the
length of the tether would be important. Figure 3 shows
a series of 4-chloro-3-cyclopentylalkoxyisocoumarins
docked in the active site of CEase. The interaction en-
ergy that the cyclopentane ring achieves with the fatty
acid pocket of cholesterol esterase dictates the location
of the chloroisocoumarin. Thus the ability of the chloro-
isocoumarin to adopt an orientation close to serine 194
that might allow the inhibitor to participate as a suicide
substrate appears to be critically dependent upon the
substituent in the 3-position.
ies, the length of the tether is also important, as shown
by comparison of the dissociation constants for 3h–3k.
Compound 3k shows a dissociation constant of
19 nM. Removal of the ring markedly affects binding,
as shown with compounds 3l and 3m.
A chlorine or other group in the 4-position makes a sig-
nificant but variable contribution. The increase in the
size of the group in the 4-position going from chlorine
to trifluoroacetyl is counterproductive. Compounds 6f,
6g, and 6c, all of which have trifluoroacetyl groups at
the 4-position, are poor inhibitors compared to 3b, 3c,
and 3l which have chlorine at the 4-position. The impor-
tance of chlorine to binding is shown by comparison of
3o and 6d, where the presence of the chlorine improved
binding by an order of magnitude, and by comparison
of 3p and 6e, where the presence of the chlorine im-
proved binding more than two orders of magnitude.
The effects of reduction of the 7-nitro group to an amino
group are complex. Reduction of the 7-nitro group to an
amino group had little effect when a small group was at
the 3-position, as shown by comparison of 3n and 4.
Addition of a large hydrophobic group to the amine at
the 7-position (5a and 5b) did not improve binding, in
marked distinction from our earlier studies of uPA.
The effect of reduction of the 7-nitro group to an amino
group was more pronounced when a larger group was in
the 3-position, as shown by comparison of 3q and 7a,
where the reduction markedly increased the dissociation
constant.
Table 1 shows the results from kinetic analysis of the
isocoumarin-based compounds as inhibitors of CEase.
As predicted, the length of the tether is important in
determining the binding constants. For the cyclohexyl
series, there is a marked improvement in binding going
from a 3-methylene spacer to a 4-methylene spacer, after
which further increase in the length of the tether mark-
edly decreases binding. Compound 3e with a 4-methy-
lene spacer shows a dissociation constant of 11 nM.
Placing the 4-methylene spacer into a cyclohexane ring
(compound 3g) is less effective. By comparison, increas-
ing the spacer to 5-methylenes (compound 3f) raises the
dissociation constant to 62 lM. For the cyclopentyl ser-
Replacement of the ring at the end of the 3-alkoxy sub-
stituent with a bromine demonstrated that the ring can
be replaced. The importance of the chlorine in the 4-po-
sition to binding is also observed in the compounds
where bromine is present in the 3-alkoxy group, as ob-
served by comparison of 7a with 7b, 3q with 6a, and
3r with 6b. In all cases, the inhibition of CEase by the
compounds in Table 1 was reversible, with no evidence
of inactivation of the enzyme.
4. Conclusions
The isocoumarin scaffold with appropriate 3-alkoxy
substituents can provide inhibitors of CEase with low
nanomolar dissociation constants. The molecular mod-
eling results suggest that the 3-alkoxy substituent an-
chors the inhibitors into the fatty acid binding pocket
of CEase such that the lactone moiety of the isocouma-
rin ring is not optimally located for mechanism-based
attack by serine 194. Consequently, these compounds
are reversible active site inhibitors.
5. Experimental
5.1. Modeling
The X-ray crystal structure of CEase (pdb code 1F6W)
was downloaded from the protein data bank. All com-
pounds shown in Table 1 were docked to the enzyme
Figure 3. Molecular modeling of CEase with a series of 4-chloro-3-
cyclopentylalkoxyisocoumarins where the cyclopentyl groups are
attached with tethers of different lengths.

J. J. Heynekamp et al. / Bioorg. Med. Chem. 16 (2008) 5285–5294
5289
Table 1. Dissociation constants for the reversible inhibition of CEase by 3-alkoxy isocoumarins
Structure
Ki (lM)
Structure
Ki (lM)
O
O
O
O
O
O
O
O
O
1.2
1.8
0.18
62
O
O
O
O
O
Cl
O
Cl
O
3a
3b
1.2
Cl
O
Cl
O
3c
3e
3d
3f
0.011
O
Cl
O
Cl
O
O
O
0.19
0.36
0.019
6.5
0.95
0.26
3.6
O
O
O
O
Cl
O
3h
3j
Cl
O
3g
3i
O
O
Cl
O
Cl
O
O
CH3
O
O
O
O
Cl
O
Cl
O
3k
3l
O
O
>100
CH3
Cl
O
3m
CF3
6f
(continued on next page)

5290
Table 1 (continued)
J. J. Heynekamp et al. / Bioorg. Med. Chem. 16 (2008) 5285–5294
Structure
Ki (lM)
Structure
Ki (lM)
O
O
O
O
73
>25
CH3
O
O
O
CF3
O
CF3
6g
6c
O
O
H2N
O2N
O
O
O
19
17
CH3
CH3
O
O
O
3n
4
Cl
O
Cl
O
O2N
O2N
O
5.7
54
O
6d
3o
Cl
O
O
O2N
O
O2N
0.7
>25
26
>100
>250
>100
50
O
O
O
6e
3p
Cl
O
O
H
N
H
N
O
O
CH3
CH3
O
O
O
O
5b
Cl
5a
Cl
O
O
H2N
H2N
O
O
Br
O
Br
Br
7b
O
7a
Cl
O
O
O2N
O2N
O
0.26
O
Br
O
6a
O
3q
Cl
O
O
O2N
O
O2N
1.6
33
O
O
Br
O
Br
6b
3r
Cl

J. J. Heynekamp et al. / Bioorg. Med. Chem. 16 (2008) 5285–5294
5291
using Autodock 3.0 on a cluster of Silicon Graphics
workstations consisting of Octanes and O2s. The com-
pounds were prepared using Sybyl 7.0 (Tripos Inc., St.
Louis, MO). The molecules were drawn in, assigned par-
Compound 4 was synthesized by a previously published
procedure.19 Compound 7b was synthesized by the same
published procedure as used for 7a.19 Spectral data are
reported for new compounds and for compounds whose
spectra are not previously reported in the literature.
tial charges using the included Gasteiger–Huckel meth-
¨
od, and energy minimized using the BFGS method.
Energy minimizations were run for 10,000 iterations
and the rotatable bonds defined to allow flexible ligand
docking. The protein was prepared using Sybyl before
docking by removing non-native substrates and water
molecules. Polar hydrogens and Kollman Uni charges
were added to the protein as well. The molecules were
docked in an area around the active site serine defined
5.3.1. 2-[2-(Cyclohexyloxy)-2-oxoethyl]benzoic acid (2a).
A solution of homophthalic acid (1a, 4.0 g, 20 mmol),
cyclohexanol (4.2 mL, 40 mmol), and 5 drops of concen-
trated sulfuric acid was refluxed in benzene (100 mL) for
six hours with a water trap. The solution was cooled,
washed with water (2 · 50 mL), brine (1 · 50 mL), and
dried over magnesium sulfate. Filtration and evapora-
tion of the solvent gave a pale yellow oil. Addition of
hexane (125 mL) and chilling in an ice bath produced
3.45 g (63%) of compound 2a as white crystals: mp
˚
by a cube of 60 · 60 · 60 A using a Lamarckian genetic
algorithm.
1
5.2. Kinetics
115–116 ꢁC; H NMR: d 1.54 (m, 10H), 4.03 (s, 2H),
4.80 (m, 1H), 7.28 (d, 1H, J = 9.14 Hz), 7.39 (t, 1H,
J = 7.55 Hz), 7.53 (t, 1H, J = 7.54 Hz), 8.12 (d, 1H,
J = 7.74 Hz); 13C NMR: d 23.67, 25.46, 31.53, 41.18,
73.03, 127.32, 128.65, 131.73, 132.26, 133.09, 137.07,
170.75, 172.46.
CEase (porcine) was from Sigma. CEase was assayed in
0.1 M Hepes, pH 7, containing 6 mM taurocholate and
1 mM p-nitrophenylbutyrate, at 405 nm. Inhibitors of
CEase were generally analyzed by double reciprocal
and Dixon plots and, for high-affinity inhibitors, by
Straus–Goldstein plots. Kinetic analysis of Michaelis
constants and kcat values was carried out by nonlinear
regression analysis. Dissociation constants of inhibitors
were determined by linear regression analysis of the dou-
ble reciprocal, Dixon, and Straus–Goldstein plots.
5.3.2. 2-[2-(Cyclohexylmethoxy)-2-oxoethyl]benzoic acid
1
(2b). White crystals (60%): mp 107–109 ꢁC; H NMR: d
1.31 (m, 11H), 3.90 (d, 2H, J = 6.36 Hz), 4.05 (s, 2H),
7.27 (d, 1H, J = 7.75 Hz), 7.38 (t, 1H, 7.65 Hz), 7.52
(dd, 1H, J = 1.29, 7.45 Hz), 8.11 (d, 1H, J = 7.75 Hz);
13C NMR: d 25.27, 26.44, 29.67, 37.14, 40.74, 70.02,
127.40, 128.58, 131.80, 132.31, 133.16, 136.96, 171.43,
172.32.
5.3. Synthesis
Unless otherwise noted, all other reagents were obtained
from commercial sources and used without further puri-
fication. Reagent quality solvents were used without
purification with the exception of THF which was dis-
tilled from calcium hydride before use. Benzoyl chloride
and cyclohexane carbonyl chloride were distilled before
use. Alcohols that were not commercially available were
prepared by the lithium aluminum hydride reduction of
the corresponding carboxylic acids. Melting points were
determined on a Thomas Hoover capillary melting point
apparatus and are uncorrected. NMR spectra were re-
corded on a Bruker AC250 (250 MHz) NMR spectrom-
eter in CDCl3 unless otherwise noted. Chemical shifts
are reported in ppm (d) relative to CHCl3 at 7.24 ppm
5.3.3. 2-[2-(2-Cyclohexylethoxy)-2-oxoethyl]benzoic acid
(2c). White crystals (61%): mp 75–76 ꢁC; H NMR: d
1
1.27 (m, 13H), 4.06 (s, 2H), 4.14 (t, 2H, J = 6.85 Hz),
7.28 (d, 1H, J = 7.75 Hz), 7.39 (t, 1H, J = 7.65 Hz),
7.54 (td, 1H, J = 1.39 Hz, 7.58 Hz), 8.13 (dd, 1 H,
J = 1.19 Hz, 7.75 Hz); 13C NMR: d 26.20 26.49, 33.12,
34.56, 35.90, 40.80, 63.16, 127.32, 128.52, 131.75,
132.25, 133.10, 136.91, 171.33, 172.33.
5.3.4. 2-[2-(3-Cyclohexylpropoxy)-2-oxoethyl]benzoic acid
(2d). White crystals (75%): mp 88–90 ꢁC; 1H NMR: d 0.83
(m, 15H), 4.07 (m, 4H), 7.27 (d, 1H, J = 7.94 Hz), 7.37 (t,
1H, J = 7.55 Hz), 7.52 (t, 1H, J = 7.35 Hz), 8.13 (d, 1H,
J = 7.74 Hz); 13C NMR: d 25.99, 26.33, 26.65, 33.26,
33.44, 37.30, 40.77, 65.30, 127.33, 128.52, 131.76,
132.26, 133.11, 136.92, 171.33, 172.43.
1
for H NMR and 77.0 for 13C NMR. High resolution
mass spectra were performed at the UNM Mass Spec-
trometry Facility, University of New Mexico, Albuquer-
que, NM. Analytical data were obtained from Galbraith
Laboratories, Knoxville TN. Homophthalic acids were
esterified to provide monoesters 2b–2r by the same gen-
eral procedure described below for 2-[2-(cyclohexyloxy)-
2-oxoethyl]benzoic acid (2a). The esters were cyclized
with phosphorous pentachloride to form 4-chloro-3-
alkyloxyisocoumarins 3b–3r by the general procedure
described below for 4-chloro-3-cyclohexyloxyisocouma-
rin (3a). Amide 5b was synthesized by the same proce-
dure described for 5a, substituting benzoyl chloride for
cyclohexanecarbonyl chloride. Compounds 6a–6g were
all synthesized by the same general procedure involving
cyclization with trifluoroacetic anhydride, which is de-
scribed in detail for 6c. Compounds 2q, 2r, 3q, 3r, 6a,
6b, and 7a were prepared as previously reported.19–21
5.3.5. 2-[2-(4-Cyclohexylbutoxy)-2-oxoethyl]benzoic acid
1
(2e). White crystals (42%): mp 94–95 ꢁC; H NMR: d
1.14 (m, 17H), 4.04 (m, 4H), 7.27 (d, 1H, J = 7.54 Hz),
7.38 (t, 1H, J = 7.75 Hz), 7.52 (t, 1H, J = 7.15 Hz),
8.12 (d, 1H, J = 7.75 Hz), 13C NMR:
d 23.17,
26.44, 26.77, 28.94, 33.37, 37.06, 37.56, 40.81, 65.04,
127.38, 128.58, 131.79, 132.29, 133.13, 136.92, 171.40,
172.19.
5.3.6. 2-[2-(5-Cyclohexylpentoxy)-2-oxoethyl]benzoic acid
1
(2f). Buff solid (75%): mp 110–111 ꢁC; H NMR: d 1.26
(m, 19H), 4.05 (s, 2H), 4.09 (t, 2H, J = 6.76 Hz), 7.28 (d,
1H, J = 7.55 Hz), 7.39 (t, 1H, J = 7.76 Hz), 7.52 (t, 1H,
J = 7.16 Hz), 8.12 (d, 1H, J = 7.76 Hz).

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J. J. Heynekamp et al. / Bioorg. Med. Chem. 16 (2008) 5285–5294
5.3.7. 2-[2-(4-Cyclohexylcyclohexyloxy)-2-oxoethyl]ben-
zoic acid (2g). Cis/trans product (60%): Recrystallization
from ethyl acetate/hexane provided (21%) of the cis iso-
mer. White crystals: mp 147–148 ꢁC; H NMR: d 1.37
3H), 4.15 (s, 2H), 7.65 (d, 1H, J = 8.54 Hz), 8.33 (dd,
1H, J = 2.59, 8.35 Hz), 8.60 (d, 1H, J = 2.39 Hz); 13C
NMR: (DMSO-d6) d 51.65, 95.63, 124.92, 126.14,
131.71, 133.98, 143.08, 146.42, 166.22, 170.44.
1
(m, 20H), 4.04 (s, 2H), 4.99 (s, 1H), 7.27 (d, 1H,
J = 7.55 Hz), 7.37 (t, 1H, J = 7.65 Hz), 7.52 (t, 1H,
J = 7.35 Hz), 8.12 (d, 1H, J = 7.54 Hz); 13C NMR: d
24.21, 26.80, 30.06, 30.17, 41.23, 42.20, 42.63, 70.49,
127.20, 128.45, 131.72, 132.23, 133.03, 137.21, 170.61,
171.54.
5.3.15. 2-(2-Cyclohexylmethoxy-2-oxoethyl)-5-nitrobenzoic
acid (2o). Buff crystals (80%): mp 135–136 ꢁC; 1H NMR: d
1.35 (m, 11H), 3.94 (d, 2H, J = 6.16 Hz), 4.19 (s, 2H), 7.51
(d, 1H, J = 8.54 Hz), 8.39 (dd, 1H, J = 8.34, 2.58 Hz), 8.97
(d, 1H, J = 2.38 Hz), 11.33 (s, 1 H).
5.3.8. 2-[2-(Cyclopentyloxy)-2-oxoethyl]benzoic acid (2h).
White crystals (72%): mp 131–133 ꢁC; 1H NMR: d 1.66 (m,
8H), 4.00 (s, 2H), 5.16 (m, 1H), 7.26 (d, 1H, J = 7.15 Hz),
7.38 (td, 1H, J = 1.20, 7.65 Hz), 7.51 (td, 1H, J = 1.46,
7.40 Hz), 8.11 (dd, 1H, J = 1.30, 7.85 Hz), 11.61 (br s,
1H); 13C NMR: d 23.67, 32.53, 41.04, 127.26, 128.57,
131.68, 132.21, 133.03, 136.97, 171.05, 172.45.
5.3.16. 2-(2-Cyclohexylethoxy-2-oxoethyl)-5-nitrobenzoic
acid (2p). Buff crystals (82%): mp 126–127 ꢁC; 1H NMR:
d 1.29 (m, 13H), 4.17 (t, 2H, J = 6.95 Hz), 4.19 (s, 2H),
7.52 (d, 1H, J = 8.54 Hz), 8.39 (dd, 1H, J = 8.35,
2.39 Hz), 8.98 (d, 1H, J = 2.39 Hz), 11.34 (s, 1H).
5.3.17. 4-Chloro-3-(cyclohexyloxy)isocoumarin (3a). A
solution of 2a (1.5 g, 5.4 mmol) in benzene (125 mL)
and phosphorous pentachloride (2.78 g, 13 mmol) was re-
fluxed for sixteen hours. The solution was cooled and
washed with water (2· 25 mL), saturated sodium bicar-
bonate solution (2· 50 mL) and brine (1· 25 mL). The
benzene layer was dried over magnesium sulfate, filtered,
and evaporated to give a dark red oil that was chromato-
graphed (hexane/ethyl acetate). The resulting yellow oil
was triturated with hexane (3· 20 mL) to give 1.0 g
5.3.9. 2-[2-(Cyclopentylmethoxy)-2-oxoethyl]benzoic acid
(2i). White crystals (65%): mp 94–95 ꢁC; 1H NMR d 1.46
(m, 8H), 2.17 (m, 1H), 3.98 (d, 2H, J = 7.15 Hz), 4.05 (s,
2H), 7.27 (d, 1H, J = 7.55 Hz), 7.38 (t, 1H, J = 7.65 Hz),
7.52 (t, 1H, J = 7.35 Hz), 8.12 (d, 1H, J = 7.75 Hz); 13C
NMR: d 25.33, 29.28, 38.50, 40.76, 68.77, 127.34,
128.55, 131.74, 132.26, 133.09, 136.91, 171.41, 172.36.
1
5.3.10. 2-[2-(2-Cyclopentylethoxy)-2-oxoethyl]benzoic acid
(2j). White crystals (47%): mp 71–73 ꢁC; 1H NMR: d 1.40
(m, 11H), 4.06 (s, 2H), 4.12 (t, 2H, J = 6.85 Hz), 7.28 (d,
1H, J = 7.55 Hz), 7.39 (t, 1H, J = 7.65 Hz), 7.53 (t, 1H,
J = 7.05 Hz), 8.13 (d, 1H, J = 7.75 Hz); 13C NMR: d
25.08, 32.56, 34.70, 36.86, 40.80, 64.55, 127.36, 128.55,
131.77, 132.30, 133.13, 136.91, 171.39, 172.47.
(62%) of 3a as a pale yellow solid: mp 67–69 ꢁC; H
NMR: d 1.60 (m, 10H), 4.79 (m, 1H), 7.35 (m, 1H), 7.68
(m, 2H), 8.16 (d, 1H, J = 7.95 Hz); 13C NMR: d 23.31,
25.31, 31.93, 92.97, 117.62, 122.32, 126.16, 129.98,
135.43, 137.79, 152.59, 159.88; Anal. calcd for
C15H15ClO3: C, 64.64; H, 5.42. Found: C, 64.33; H, 5.59.
5.3.18. 4-Chloro-3-(cyclohexylmethoxy)isocoumarin (3b).
1
5.3.11. 2-[2-(3-Cyclopentylpropoxy)-2-oxoethyl]benzoic acid
(2k). White crystals (75%): mp 72–73 ꢁC; H NMR: d
1.27 (m, 13H), 4.08 (m, 4H), 7.27 (d, 1H, J = 7.55 Hz),
7.37 (t, 1H, J = 7.75 Hz), 7.51 (t, 1H, J = 7.54 Hz),
8.12 (d, 1H, J = 7.74 Hz); 13C NMR: d 25.20, 27.88,
32.26, 32.67, 39.80, 40.80, 65.23, 127.37, 128.57,
131.80, 132.30, 133.15, 136.95, 171.37, 172.40.
Pale yellow solid (60%): mp 81–82 ꢁC; H NMR: d 1.60
1
(m, 11H), 4.17 (d, 2H, J = 6.16 Hz), 7.37 (m, 1H), 7.72
(m, 2H), 8.19 (d, 1H, J = 7.74 Hz); 13C NMR: d 25.62,
26.32, 29.36, 37.68, 75.65, 90.85, 117.15, 122.07,
125.90, 129.92, 135.42, 137.85, 153.23, 159.47. Anal.
calcd for C16H17ClO3: C, 65.64; H, 5.85. Found: C,
65.44; H, 5.96.
5.3.12. 2-(2-Oxo-2-propoxyethyl)benzoic acid (2l). White
crystals (68%): mp 103–104 ꢁC; 1H NMR: d 0.90 (t, 2H,
J = 7.35 Hz), 1.63 (m, 2H), 4.05 (m, 4H), 7.27 (d, 1H,
J = 7.54 Hz), 7.38 (t, 1H, J = 7.65 Hz), 7.52 (td, 1H,
J = 1.39, 7.45 Hz), 8.12 (dd, 1H, J = 1.20, 7.74 Hz);
13C NMR: d 10.39, 21.99, 40.78, 66.42, 127.40, 128.56,
131.78, 132.31, 133.15, 136.87, 171.42, 172.53.
5.3.19. 4-Chloro-3-(2-cyclohexylethoxy)isocoumarin (3c).
Pale yellow solid (55%): mp 71–72 ꢁC; H NMR: d 1.28
1
(m, 13H), 4.41 (t, 2H, J = 6.75 Hz), 7.37 (m, 1H), 7.71
(m, 2H), 8.19 (d, 1H, J = 7.95 Hz); 13C NMR: d 26.21,
26.49, 33.16, 34.32. 36.49, 69.12, 91.20, 117.27, 122.17,
126.01, 129.98, 135.48, 137.85,153.17, 159.54; Anal.
calcd for C17H19ClO3: C, 66.56; H, 6.24. Found: C,
66.84; H, 6.42.
5.3.13. 2-(2-Butoxy-2-oxoethyl)benzoic acid (2m). White
1
crystals (70%): mp 84–86 ꢁC; H NMR: d 0.89 (t, 2H,
5.3.20. 4-Chloro-3-(3-cyclohexylpropoxy)isocoumarin (3d).
Pale yellow solid (78%): mp 55–56 ꢁC; H NMR: d 1.31
(m, 15H), 4.35 (t, 2H, J = 6.65 Hz), 7.37 (m, 1H), 7.71
(m, 2H), 8.18 (d, 1H, J = 7.74 Hz); 13C NMR: d 26.32,
26.63, 33.18, 33.26, 37.25, 71.23, 91.16, 117.25, 122.15,
125.98, 129.95, 135.44, 137.83, 153.12, 159.51, 172.91.
Anal. (C18H21ClO3).
1
J = 7.25 Hz), 1.34 (m, 2H), 1.59 (m, 2H), 4.04 (s, 2H),
4.09 (t, 2H, J = 6.66Hz), 7.27 (d, 1H, J = 7.55 Hz),
7.38 (t, 1H, J = 7.75 Hz), 7.52 (t, 1H, J = 7.35 Hz),
8.12 (d, 1H, J = 7.75 Hz); 13C NMR: d 13.71, 19.92,
30.65, 40.79, 64.69, 127.39, 128.55, 131.78, 132.31,
133.16, 136.89, 171.43, 172.57.
5.3.14. 2-(2-Methoxy-2-oxoethyl)-5-nitrobenzoic acid
(2n). Pale yellow crystals (99%): mp 164–166 ꢁC
5.3.21. 4-Chloro-3-(4-cyclohexylbutoxy)isocoumarin (3e).
Pale yellow solid (73%): mp 41–42 ꢁC; H NMR: d 1.31
(m, 17H), 4.36 (t, 2H, J = 6.56 Hz), 7.36 (m, 1H), 7.71
1
1
(lit.20,21 167–168 ꢁC); H NMR: (DMSO-d6) d 3.58 (s,

J. J. Heynekamp et al. / Bioorg. Med. Chem. 16 (2008) 5285–5294
5293
(m, 2H), 8.17 (d, 1H, J = 7.55 Hz); 13C NMR: d 22.94,
26.44, 26.75, 29.55, 33.38, 37.00, 37.57, 70.95, 91.26,
117.30, 122.20, 126.04, 130.01, 135.50, 137.89, 153.16,
159.60. Anal. (C19H23ClO3) C, H.
J = 6.46 Hz), 7.33 (t, 1H, J = 7.35 Hz), 7.67 (m, 2H),
8.14 (d, 1H, J = 7.55 Hz); 13C NMR: d 13.67, 18.91,
31.24, 70.55, 91.12, 117.21, 122.09, 125.95, 129.91,
135.40, 137.78, 153.11, 159.42.
5.3.22. 4-Chloro-3-(5-cyclohexylpentoxy)isocoumarin (3f).
Pale yellow solid (84%): mp 51–52 ꢁC; H NMR: d 1.39
(m, 19H), 4.36 (t, 2H, J = 6.56 Hz), 7.37 (m, 1H), 7.71
(m, 2H), 8.18 (d, 1H, J = 7.55 Hz); 13C NMR: d 25.98,
26.49, 26.81, 29.31, 33.48, 37.35, 37.62, 70.98, 91.27,
117.35, 122.23, 126.05, 130.04, 135.51, 137.92, 153.21,
159.59. Anal. (C20H25ClO3) C, H.
5.3.30. 4-Chloro-3-methoxy-7-nitroisocoumarin (3n). Yel-
low solid (73%): mp 129–132 ꢁC (lit.20,21 128–131 ꢁC);
1H NMR: d 4.14 (s, 3H), 7.78 (d, 1H, J = 8.93 Hz),
8.48 (dd, 1H, J = 2.38, 8.94 Hz), 8.97 (d, 1H,
J = 2.18 Hz); 13C NMR: d 57.21, 89.50, 116.44, 123.26,
126.19, 129.62, 142.93, 144.94, 155.87, 157.15.
1
5.3.31. 4-Chloro-3-cyclohexylmethoxy-7-nitroisocouma-
1
5.3.23. 4-Chloro-3-(4-cyclohexylcyclohexyloxy)isocoum-
arin (3g). Yellow solid (62%): mp 125–126 ꢁC; 1H
NMR: d 1.28 (m, 20H), 5.06 (s, 1H), 7.37 (m, 1H),
7.71 (m, 2H), 8.17 (d, 1H, J = 7.74 Hz); 13C NMR: d
23.88, 26.83, 30.18, 30.43, 42.34, 42.79, 77.66, 92.94,
117.55, 122.20, 126.06, 129.94, 135.35, 137.80, 152.58,
159.81. Anal. (C21H25ClO3) C, H.
rin (3o). Pale yellow solid (70%): mp 106–108 ꢁC; H
NMR: d 1.45 (m, 11H), 4.28 (d, 2H, J = 6.16 Hz), 7.83
(d, 1H, J = 8.93 Hz), 8.53 (dd, 1H, J = 2.19, 8.93 Hz),
9.04 (d, 1H, J = 2.19 Hz). Exact mass calcd for
C16H16ClNO5: 337.0717, observed (M+H) 338.0785.
5.3.32. 4-Chloro-3-(2-cyclohexylethoxy)-7-nitroisocoum-
1
arin (3p). Pale yellow solid (71%): mp 120–121 ꢁC; H
5.3.24. 4-Chloro-3-(cyclopentyloxy)isocoumarin (3h). Pale
yellow crystals (59%): mp 52–54 ꢁC; 1H NMR: d 1.76 (m,
8H), 5.27 (m, 1H), 7.36 (m, 1H), 7.67 (m, 2H), 8.17 (d, 1H,
J = 7.94 Hz); 13C NMR: d 23.49, 32.90, 84.52, 92.99,
117.53, 122.25, 126.13, 129.95, 135.38, 137.71, 152.65,
159.76. Anal. (C14H13ClO3) C, H.
NMR: d 1.38 (m, 13H), 4.52 (t, 2H, J = 6.56 Hz), 7.83
(d, 1H, J = 8.94 Hz), 8.52 (dd, 1H, J = 2.18, 8.94 Hz),
9.04 (d, 1H, J = 2.18 Hz). Exact mass calcd for
C17H18ClNO5: 351.0874, observed (M+H) 352.0942.
5.3.33. 7-Amino-4-chloro-3-methoxyisocoumarin (4). Yel-
low crystals (89%): mp 183–185 ꢁC (lit.20,21 175–176 ꢁC);
1H NMR: (DMSO-d6) d 3.93 (s, 3H), 5.74 (br s, 2H),
7.14 (dd, 1H, J = 1.99, 8.54 Hz), 7.25 (d, 1H,
J = 1.79 Hz), 7.40 (d, 1H, J = 8.54 Hz); 13C NMR:
(DMSO-d6) d 57.62, 91.12, 93.63, 110.79, 118.36,
122.86, 122.95, 125.07, 148.03, 150.22, 159.00.
5.3.25. 4-Chloro-3-(cyclopentylmethoxy)isocoumarin (3i).
1
Pale yellow solid (59%): mp 44–46 ꢁC; H NMR: d 1.60
(m, 8H), 2.36 (m, 1H), 4.24 (d, 2H, J = 7.15 Hz), 7.36
(m, 1H), 7.70 (m, 2H), 8.17 (d, 1H, J = 7.75 Hz); 13C
NMR: d 25.38, 29.18, 39.09, 74.61, 91.17, 117.27,
122.16, 125.97, 129.95, 135.44, 137.86, 153.20, 159.55.
Anal. (C15H15ClO3) C, H.
5.3.34. 4-Chloro-7-cyclohexanecarboxamide-3-methoxy-
4
isocoumarin (5a). To
a
solution of
(0.09 g,
5.3.26. 4-Chloro-3-(2-cyclopentylethoxy)isocoumarin (3j).
Pale yellow crystals (65%): mp 47–48 ꢁC; H NMR: d
1.42 (m, 11H), 4.38 (t, 2H, J = 6.75 Hz), 7.36 (m, 1H),
7.70 (m, 2H), 8.18 (d, 1H, J = 7.94 Hz); 13C NMR: d
25.13, 32.65, 35.37, 36.61, 70.44, 91.13, 117.28, 122.20,
126.02, 130.02, 135.51, 137.93, 153.20, 159.58. Anal.
(C16H17ClO3) C, H.
0.39 mmol) in dry tetrahydrofuran (10 mL) was added
cyclohexanecarbonyl chloride (65 lL, 0.47 mmol) and
triethylamine (60 lL, 0.39 mmol). The solution was
stirred at room temperature for six hours after which
time the triethylamine hydrochloride was filtered off
and washed with hot tetrahydrofuran (3 · 10 mL).
The mother liquor was evaporated to give a pale yel-
low semi solid that was crystallized from tetrahydro-
furan/hexane to afford 100 mg (76%) of a crude
yellow solid. Recrystallization from ethanol provided
75 mg of 5a as a pale yellow solid: mp 213–214 ꢁC;
1H NMR: (DMSO-d6) 1.33 (m, 6H), 1.78 (m, 4H),
2.31 (m, 1H), 4.00 (s, 3H), 7.58 (d, 1H,
J = 8.54 Hz), 7.98 (d, 1H, J = 8.34 Hz), 8.49 (s, 1H),
10.17 (s, 1H); 13C NMR: (DMSO-d6) d 25.16, 25.36,
29.01, 44.84, 57.27, 89.45, 117.17, 118.06, 122.23,
127.12, 131.80, 137.83, 152.35, 158.50, 174.49. Exact
mass calcd for C17H18ClNO4: 335.0924, observed
(M+H) 336.1003.
1
5.3.27. 4-Chloro-3-(3-cyclopentylpropoxy)isocoumarin (3k).
Pale yellow oil (87%): 1H NMR: d 1.42 (m, 13H), 4.30 (t,
2H, J = 6.66 Hz), 7.30 (m, 1H), 7.63 (m, 2H), 8.11 (d, 1H,
J = 7.34 Hz); 13C NMR: d 25.20, 28.49, 31.97, 32.66,
39.71, 71.09, 91.13, 117.24, 122.13, 125.98, 129.95,
135.44, 137.82, 153.13, 159.48. Anal. (C17H19ClO3) C, H.
5.3.28. 4-Chloro-3-propoxyisocoumarin (3l). Yellow solid
(68%): mp 48–50 ꢁC; 1H NMR: d 1.04 (t, 2H,
J = 7.45 Hz), 1.82 (m, 2H), 4.33 (t, 2H, J = 6.55 Hz),
7.39 (m, 1H), 7.70 (m, 2H), 8.17 (d, 1H, J = 7.74 Hz);
13C NMR: d 10.25, 22.73, 72.34, 91.26, 117.32, 122.22,
126.07, 130.03, 135.52, 137.90, 153.17, 159.60. Exact mass
calcd for C12H11ClO3: 238.0397, observed (M+H)
239.0475.
5.3.35. 7-Benzamido-4-chloro-3-methoxyisocoumarin (5b).
Pale yellow solid (33%): mp 220–221 ꢁC; 1H NMR:
(DMSO-d6) d 4.02 (s, 3H), 7.56 (m, 3H), 7.68 (d, 1H,
J = 8.74 Hz), 7.99 (m, 2H), 8.26 (dd, 1H, J = 2.19,
8.71 Hz), 8.68 (d, 1H, J = 2.19 Hz), 10.62 (s, 1H); 13C
NMR: (DMSO-d6) d 40.51, 89.34, 117.15, 119.35,
122.19, 127.55, 128.18, 128.30, 131.71, 132.44, 134.20,
5.3.29. 3-Butoxy-4-chloroisocoumarin (3m). Yellow solid
(75%): mp 55–56 ꢁC; 1H NMR: d 0.96 (t, 2H,
J = 7.35 Hz), 1.48 (m, 2H), 1.76 (m, 2H), 4.35 (t, 2H,

5294
J. J. Heynekamp et al. / Bioorg. Med. Chem. 16 (2008) 5285–5294
137.55, 152.61, 158.51, 165.53. Exact mass calcd for
C17H12ClNO4: 329.0455, observed (M+H) 330.0535.
Acknowledgment
This work was supported by a grant from the National
Institutes of Health, HL68598.
5.3.36. 3-Propoxy-4-(trifluoroacetyl)isocoumarin (6c). A
solution of 2l (0.5 g, 2 mmol) and trifluoroacetic anhy-
dride (0.38 mL, 2.7 mmol) in dichloromethane (20 mL)
was stirred for four hours at room temperature. The solu-
tion was evaporated and chromatographed (chloroform)
to give 0.11 g (59% yield based on recovered starting
material) of compound 6c as white crystals: mp 126–
References and notes
1. Grundy, S. M.; Cleeman, J. I.; Merz, C. N.; Brewer, H. B.,
Jr.; Clark, L. T.; Hunninghake, D. B.; Pasternak, R. C.;
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Biol. 2004, 24, e149–e161.
2. Murdoch, D.; Scott, L. J. Am. J. Cardiovasc. Drugs 2004,
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Paolini, J. F.; Bergman, A. J.; Alton, K. B. Clin.
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1
127 ꢁC; H NMR: d 1.03 (t, 3H, J = 7.45 Hz), 1.87 (m,
2H), 4.49 (t, 2H, J = 6.76 Hz), 7.39 (t, 1H, J = 7.65 Hz),
7.71 (m, 1H), 8.11 (d, 1H, J = 8.35 Hz), 8.19 (d, 1H,
J = 7.94 Hz); 13C NMR: d 10.03, 22.02, 73.13, 77.20,
90.70, 115.91, 115.96, 123.38, 126.51, 130.23, 136.25,
158.07, 162.63, 180.26. Anal. (C14H11F3O4) C, H.
5.3.37. 3-Cyclohexylmethoxy-7-nitroisocoumarin (6d).
Yellow crystals (60%): mp 122–123 ꢁC; 1H NMR: d
1.04 (m, 11H), 4.00 (d, 2H, J = 5.96 Hz), 5.66 (s, 1H),
7.40 (d, 1H, J = 8.74 Hz), 8.40 (dd, 1H, J = 2.38,
8.74 Hz), 9.03 (d, 1H, J = 1.98 Hz). Exact mass calcd
for C16H17NO5: 303.1107, observed (M+H) 304.1185.
5. Pearson, T. A. Am. J. Cardiol. 2004, 94, 4F–8F.
6. Wilson, M. M.; Thomas, D. R. Curr. Atheroscler. Rep.
2003, 5, 324–330.
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1996, 271, 7196–7202.
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Lange, L. G. Biochemistry 1993, 32, 12085–12089.
10. Shamir, R.; Johnson, W. J.; Zolfaghari, R.; Lee, H. S.;
Fisher, E. A. Biochemistry 1995, 34, 6351–6358.
11. Ikeda, I.; Matsuoka, R.; Hamada, T.; Mitsui, K.;
Imabayashi, S.; Uchino, A.; Sato, M.; Kuwano, E.;
Itamura, T.; Yamada, K.; Tanaka, K.; Imaizumi, K.
Biochim. Biophys. Acta 2002, 1571, 34–44.
5.3.38. 3-(2-Cyclohexylethoxy)-7-nitroisocoumarin (6e).
1
Buff crystals (62%): mp 143–144 ꢁC; H NMR: d 1.33
(m, 13H), 4.25 (t, 2H, J = 6.55 Hz), 5.67 (s, 1H), 7.40
(d, 1H, J = 8.74 Hz), 8.39 (dd, 1H, J = 2.38, 8.74 Hz),
9.02 (d, 1H, J = 2.18 Hz). Exact mass calcd for
C17H19NO5: 317.1263, observed (M+H) 318.1349.
12. Bruneau, N.; Bendayan, M.; Gingras, D.; Ghitescu, L.;
Levy, E.; Lombardo, D. Gastroenterology 2003, 124, 470–
480.
13. Bruneau, N.; Richard, S.; Silvy, F.; Verine, A.; Lombardo,
D. Mol. Biol. Cell 2003, 14, 2861–2875.
14. Rebai, O.; Le Petet-Thevenin, J.; Bruneau, N.; Lombardo,
D.; Verine, A. Arterioscler. Thromb Vasc. Biol. 2005, 25,
359–364.
15. Deck, L. M.; Baca, M. L.; Salas, S. L.; Hunsaker, L. A.;
Vander Jagt, D. L. J. Med. Chem. 1999, 42, 4250–4256.
16. Stoddard-Hatch, M.; Brown, W. M.; Deck, J. A.; Hun-
saker, L. A.; Deck, L. M.; Vander Jagt, D. L. Biochim.
Biophys. Acta 2002, 1596, 381–391.
5.3.39. 3-Cyclohexylmethyl-4-(trifluoroacetyl)isocouma-
rin (6f). White crystals (60%): mp 112–113 ꢁC; 1H
NMR: d 1.45 (m, 11H), 4.36 (d, 2H, J = 6.36 Hz), 7.42
(t, 1H, J = 8.14 Hz), 7.74 (t, 1H, J = 7.15 Hz), 8.12 (d,
1H, J = 8.34 Hz), 8.23 (d, 1H, J = 7.95 Hz). Exact mass
calcd for C18H17F3O4: 354.1079, observed (M+H)
355.1145.
5.3.40. 3-(2-Cyclohexylethoxy)-4-trifluoroacetylisocoum-
arin (6g). White crystals (60%): mp 80–81 ꢁC. 1H NMR: d
1.37 (m, 13H), 4.59 (t, 2H, J = 6.95 Hz), 7.42 (t, 1H,
J = 8.14 Hz), 7.74 (t, 1H, J = 7.15 Hz), 8.15 (d, 1H,
J = 8.34 Hz), 8.24 (d, 1H, J = 8.15 Hz). Exact mass calcd
for C19H19F3O4: 368.1235, observed (M+H) 369.1245.
17. Heidrich, J. E.; Contos, L. M.; Hunsaker, L. A.; Deck, L.
M.; Vander Jagt, D. L. BMC Pharmacol. 2004, 4, 5.
18. Kam, C.; Kerrigan, J.; Plaskon, R.; Duffy, E.; Lollar, P.;
Suddath, F.; Powers, J. J. Med. Chem. 1994, 37, 1298–
1306.
5.3.41. 7-Amino-3-(2-bromoethoxy)isocoumarin (7b).
Yellow crystals (81%): mp >280 ꢁC; 1H NMR:
(DMSO-d6) d 3.79 (t, 2H, J = 5.17 Hz), 4.36 (t, 2H,
J = 4.97 Hz), 5.50 (s, 2H), 7.03 (d, 1H, J = 8.54 Hz),
7.19 (m, 2H); 13C NMR: (DMSO-d6) d 30.30, 68.71,
80.35, 110.20, 117.91, 123.17, 125.84, 128.03, 147.16,
154.71, 160.45. Exact mass calcd for C11H10BrNO3:
282.9844, observed (M+H) 283.9927.
19. Heynekamp, J. J.; Hunsaker, L. A.; Vander Jagt, T. A.;
Deck, L. M.; Vander Jagt, D. L. BMC Chem. Biol. 2006,
6, 1.
20. Bihel, F.; Quelever, G.; Lelouard, H.; Petit, A.; Alves de
Costa, C.; Pourquie, O.; Checler, F.; Thelland, A.; Pierre,
P.; Karaus, J. Bioorg. Med. Chem. 2003, 11, 3141–3152.
21. Choksey, I.; Usagaonkar, R. N. Indian J. Chem. Sect. B
1976, 14B, 596–598.