3-Alkyl-6-chloro-2-pyrones: Selective inhibitors of pancreatic cholesterol esterase

Article abstract of DOI:10.1021/jm990309x

A series of 3-alkyl-6-chloro-2-pyrones with cyclohexane rings tethered to the 3-position was synthesized. The tether ranged from 0 to 4 methylene units. Inhibition of pancreatic cholesterol esterase by this series of pyrones was markedly dependent upon the length of the tether. Dissociation constants as low as 25 nM were observed for 6-chloro-3-(1-ethyl-2- cyclohexyl)-2-pyranone. This class of cholesterol esterase inhibitors functioned as simple competitive inhibitors of substrate binding rather than as suicide substrates or active site inactivators. Trypsin and chymotrypsin were not strongly inhibited by this class of pyrones. Selectivities for cholesterol esterase were greater than 10³. This is in contrast to 3-aryl-6- chloro-2-pyrones which are nonselective, irreversible inactivators of serine hydrolases. Thus, replacement of the 3-aryl group by an appropriately tethered 3-alkyl ring can produce highly selective inhibitors of cholesterol esterase. A second series of halogen-containing esters was prepared in which cholesterol was esterified with α-haloacyl halides. These haloesters were simple substrates of cholesterol esterase with no evidence of irreversible inactivation.

Full text of DOI:10.1021/jm990309x

4250
J . Med. Chem. 1999, 42, 4250-4256
3-Alk yl-6-Ch lor o-2-p yr on es: Selective In h ibitor s of P a n cr ea tic Ch olester ol
Ester a se
Lorraine M. Deck,*,† Miranda L. Baca,† Stephanie L. Salas,† Lucy A. Hunsaker,‡ and David L. Vander J agt*,‡
Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131, and Department of Biochemistry and
Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131
Received J une 15, 1999
A series of 3-alkyl-6-chloro-2-pyrones with cyclohexane rings tethered to the 3-position was
synthesized. The tether ranged from 0 to 4 methylene units. Inhibition of pancreatic cholesterol
esterase by this series of pyrones was markedly dependent upon the length of the tether.
Dissociation constants as low as 25 nM were observed for 6-chloro-3-(1-ethyl-2-cyclohexyl)-2-
pyranone. This class of cholesterol esterase inhibitors functioned as simple competitive inhibitors
of substrate binding rather than as suicide substrates or active site inactivators. Trypsin and
chymotrypsin were not strongly inhibited by this class of pyrones. Selectivities for cholesterol
esterase were greater than 103. This is in contrast to 3-aryl-6-chloro-2-pyrones which are
nonselective, irreversible inactivators of serine hydrolases. Thus, replacement of the 3-aryl
group by an appropriately tethered 3-alkyl ring can produce highly selective inhibitors of
cholesterol esterase. A second series of halogen-containing esters was prepared in which
cholesterol was esterified with R-haloacyl halides. These haloesters were simple substrates of
cholesterol esterase with no evidence of irreversible inactivation.
Bile salt-stimulated cholesterol esterase (CEase), also
known as bile salt-stimulated lipase, pancreatic carboxyl
ester lipase, pancreatic lysophospholipase, nonspecific
lipase, and pancreatic CEase, functions in the hydrolysis
of dietary cholesterol esters as well as other dietary
esters. CEase has been suggested to have a critical role
in determining the bioavailability of dietary cholesterol
by catalyzing both the hydrolysis of cholesterol esters
and the transport of free cholesterol across the brush
border of the intestinal villi.1,2 Recent gene knockout
studies, however, suggest that CEase primarily is
involved in hydrolysis of cholesterol esters. Knockout
mice exhibited a dramatically impaired ability to absorb
cholesterol derived from cholesterol esters but exhibited
normal absorption of free cholesterol.3
inactivators of serine proteinases.5 Their use as inacti-
vators of CEase is limited due to lack of selectivity.
In the present study, we have addressed the question
of development of selective inhibitors of CEase based
upon the 6-chloro-2-pyrone backbone. A series of 3-alkyl-
6-chloro-2-pyrones of general structure 2 was synthe-
sized in which the 3-substituent included an aliphatic
rather than aromatic ring. This approach was designed
to test the hypothesis that replacement of aromatic
groups in the 3-position of 1 with aliphatic groups would
lead to the development of selective inhibitors of CEase
compared to chymotrypsin. A second series of haloge-
nated esters of general structure 3 was synthesized in
CEase is a classical serine hydrolase in terms of its
catalytic triad of S194, H435, and D320 (numbered for
rat CEase) and the presence of an R/â-hydrolase fold.
Inhibitors of serine hydrolases, such as 3-benzyl-6-
chloro-2-pyrone (1), inactivate CEase and prevent ab-
sorption of cholesterol esters in animal studies,4 which
is in agreement with results from gene knockout studies,
which cholesterol was esterified with short or long chain
fatty acids containing chloro or bromo groups at the
2-position. Both series 2 and 3 have the potential to
function as (1) conventional substates of CEase where
both acylation and deacylation of the active site serine
are facile reactions; (2) pseudosubstrates where acyla-
tion is followed by slow deacylation; (3) suicide inhibitors
where formation of the acyl enzyme is followed by
nucleophilic attack by some enzyme reactive group at
the halogenated carbon to form an irreversible modifica-
tion and inactivation of CEase; (4) conventional com-
petitive inhibitors; or (5) irreversible inactivators where
binding to the active site is followed by covalent
modification of CEase in a reaction that is not part of
the catalytic cycle.
both of which argue for an important role of CEase in
digestion of cholesterol esters. 3-Aryl-6-chloro-2-pyrones
such as 1 were developed as potential mechanism-based
* Address correspondence to either Dr. Lorraine M. Deck, Depart-
ment of Chemistry, University of New Mexico, Albuquerque, NM
87131 (phone 505-277-5438, e-mail ldeck@unm.edu), or Dr. David L.
Vander J agt, Department of Biochemistry and Molecular Biology,
University of New Mexico School of Medicine, Albuquerque, NM 87131
(phone 505-272-5788, e-mail dlvanderjagt@salud.unm.edu).
† University of New Mexico.
‡ University of New Mexico School of Medicine.
10.1021/jm990309x CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/18/1999

Selective Inhibitors of Pancreatic Cholesterol Esterase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4251
Sch em e 1
Sch em e 2
Ta ble 1. Inhibition of Cholesterol Esterase by
6-Chloro-2-pyrones
inhibitor
Ki (µM)
Ch em ica l Syn th eses
The synthesis of 3-alkyl-6-chloro-2-pyrones (2) was
accomplished as described in Scheme 1. The appropri-
ately substituted diethyl malonate compounds were
synthesized by published procedures. The resulting
diesters were reacted with sodium hydride to form the
sodium salts of compounds 4a -f and then heated with
methyl cis-2-chloroacrylate (5) in tetrahydrofuran. The
resulting triesters 6a -f were saponified, acidified, and
decarboxylated to give substituted pentenedioic acids
(glutaconic acids) 7a -f as mixtures of synthetically
equivalent isomers. Reflux of the mixture of isomers
with acetyl chloride formed the 3-alkyl-6-chloro-2-py-
rones 2a -f with minor amounts of the 5-alkyl-6-chloro-
2-pyrones 8a -f; the isomers are thermally interchange-
able with the equilibrium generally favoring the
3-substituted pyrones.
In h ibition of P a n cr ea tic CEa se, Ch ym otr yp sin ,
a n d Tr yp sin by 3-Alk yl-6-ch lor op yr on es a n d
Ch olester yl-r-h a loester s
Inhibition of CEase by 6-chloropyrones containing a
tethered cyclohexane ring at the 3-position is highly
dependent upon the length of the tether. For 3-cyclo-
hexyl-6-chloropyrone (2a ) where the cyclohexane ring
is attached to the pyrone ring without a spacer, Ki )
2.3 µM (Table 1) and is not altered by introduction of
a single methylene spacer (2b, Ki ) 2.2 µM). However,
introduction of a 2-methylene spacer improves binding
100-fold; for 2c, Ki ) 0.025 µM. Lengthening the spacer
to three or four methylenes (2d and 2e) increases the
Ki values. Thus, in the tethered cyclohexane series, the
2-methylene spacer produces the most potent inhibitor.
In all cases, inhibition is competitive as shown by the
double reciprocal plots in Figure 1 for 2c. The inhibition
of CEase by 2c was also analyzed by Dixon plots and
by Straus-Goldstein plots for high-affinity inhibitors; the
results were the same.
The synthesis of halogenated cholesterol esters was
carried out as described in Scheme 2. Cholesterol (9)
was reacted with an R-haloacyl halide (10a -g) in
methylene chloride containing triethylamine to form
esters 11a -g. Halides 10a -g are either commercially
available or were prepared from the corresponding
R-haloacid by reaction with thionyl chloride, except for
R-chloropalmitoyl chloride (10d ) and R-chlorohexanoyl
chloride (10c) which were synthesized by treatment of
the corresponding carboxylic acids with thionyl chloride
and N-chlorosuccinimide.

4252 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Deck et al.
problem of hypercholesterolemia has a long history.
Plant sterols such as campesterol and sitosterol have
long been known to reduce plasma cholesterol by
inhibiting cholesterol absorption.6 This appears to result
from competition with cholesterol for incorporation into
micelles, although other absorption steps may also be
involved. However, large doses of sitosterol are required
for modest reductions in plasma cholesterol. Although
interest in plant sterols decreased somewhat as new
approaches to treatment of hypercholesterolemia were
developed, such as introduction of the statins, the use
of agents that reduce cholesterol absorption has reached
commercial success. Sitostanol, a 5-R-saturated deriva-
tive of sitosterol, and its esters have been reported to
be more effective hypocholesterolemic agents than si-
tosterol.7 Sitostanol-ester margarine for treatment of
hypercholesterolemia8 is now available commercially.
Recently, it has been shown that margarines enriched
with oils containing either sitosterol-esters or sitostanol-
esters were equally effective in lowering LDL-choles-
terol,9 which suggests that margarines containing a
wide range of plant sterol-esters will likely be developed.
F igu r e 1. Double reciprocal plot of the inhibition of CEase
by 2c (0.1 µM, open circles) with p-nitrophenylbutyrate as
substrate demonstrates that inhibition is competitive, Ki )
0.025 µM.
The structural features of alkyl-6-chloropyrones that
are potent inhibitors of CEase are more complicated
than suggested by the tethered cyclohexane series.
Compound 2f in which a cyclopentane ring is attached
directly to the 3-position of the pyrone ring is almost
as strong an inhibitor as 2c; (for 2f, Ki ) 0.040 µM).
Likewise, 8c, the positional isomer of 2c in which the
tethered cyclohexane ring is attached to the 5-position,
is a good inhibitor, Ki ) 0.058 µM. It is apparent,
therefore, that inhibition of CEase by alkyl-6-chloropy-
rones is amenable to wide variation in the nature and
location of the alkyl groups.
The inhibition of CEase by 2a -f, 8c was analyzed
using p-nitrophenylbutyrate as substrate in an assay
system that included the bile salt taurocholate. In the
absence of bile salt, the Michaelis constant of p-
nitrophenylbutyrate increased from 64 to 175 µM.
Likewise, the Ki values of 2c increased from 0.025 µM
in the presence of bile salt to 0.090 µM in the absence
of bile salt.
The possibility that 2a -f, 8c are substrates of CEase
or pseudosubstrates was examined using 2c as a
representative inhibitor. CEase and excess 2c were
incubated at neutral pH for up to 6 h with samples
examined periodically by TLC for formation of 2-(2-
cyclohexylethyl)glutaconic acid (7c), the expected prod-
uct of the hydrolysis of 2c. No evidence for product
formation was obtained. Likewise, separation of CEase
from 2c after several hours of incubation provided active
enzyme, suggesting that 2c is neither a substrate nor a
pseudosubstrate where deacylation is slow. We con-
clude, therefore, that the substituted pyrones are simple
competitive inhibitors of CEase.
Inhibition of trypsin and chymotrypsin by 2a -f was
examined to determine the extent of selectivity for
CEase. This series of pyrones did not inhibit trypsin at
inhibitor concentrations up to 250 µM. Inhibition of
chymotrypsin was competitive. The most potent inhibi-
tor of chymotrypsin was 2c; Ki ) 50 µM compared to Ki
) 0.025 µM for inhibition of CEase by 2c. Thus,
selectivity for CEase is greater than 103.
Other approaches to interference with cholesterol
absorption with a long history include the use of the
nonabsorbable aminoglycoside antibiotic neomycin, which
appears to inhibit cholesterol absorption by forming
complexes with cholesterol that are excreted,10 and use
of the bile salt binder cholestyramine, an anion ex-
changer that indirectly alters cholesterol levels by
limiting the resorption of cholesterol-derived bile salts.
All of these approaches suffer to some extent from the
sizable quantities of therapeutic agents that must be
administered. The development of selective inhibitors
of cholesterol absorption that are targeted at specific
enzymes would appear to be a more attractive approach.
More recent studies of cholesterol absorption have
identified a scavenger receptor class B type I that is
present in the brush border membrane. This receptor
appears to function as a conduit for entry of a variety
of lipids into the mucosal cells. Apolipoprotein A-I
interacts with this receptor to inhibit lipid entry,
suggesting that this receptor may be a selective target
for therapeutic intervention.11
Digestion of dietary lipid prior to absorption primarily
involves gastric lipase and the three pancreatic enzymes
triglyceride lipase, phospholipase A2, and CEase. The
broad specificity of CEase suggests that this hydrolase
participates in the hydrolysis of triglycerides as well as
catalyzing the hydrolysis of cholesterol esters. However,
the activity of triglyceride lipase in pancreatic juice
appears to be much greater than CEase activity, at least
in rat, which suggests that selective inhibition of CEase
will not interfere with digestion of triglycerides. It is
noteworthy that in some species of fish CEase also
functions as triglyceride lipase.12
The second series of haloesters, compounds 11a -g,
was examined as potential suicide substrates or ir-
reversible active site inhibitors of CEase. These choles-
terol esters were incubated with CEase, and samples
were examined by TLC. These esters were substrates
of CEase with no evidence of inactivation of CEase.
The possible dual roles of CEase in hydrolysis of
cholesterol esters as well as in transport of cholesterol
into mucosal cells of the small intestine have been
studied extensively. Rat pancreatic CEase enhances
sterol transfer in small unilamellar vesicles that contain
negatively charged phospholipids, independent of es-
terase activity.13 CEase binds to membrane-associated
heparin on the intestinal brush border. This appears
to involve a proline-rich 11 amino acid repeat in the
Discu ssion
The concept that interference with the absorption of
cholesterol may represent a useful approach to the

Selective Inhibitors of Pancreatic Cholesterol Esterase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4253
C-terminal portion of CEase. The binding of human
CEase to membrane-bound heparin increases the en-
zyme activity, whereas soluble heparin is a potent
inhibitor.14 The results from gene knockout studies,
however, do not support a role for CEase in the absorp-
tion of cholesterol.3 This may reflect differences between
human and mouse CEase. Most species that have been
analyzed contain 2-4 repeat units while human CEase
contains 16 proline-rich repeats. The inhibitory proper-
ties of heparin depend on the size of the repeat, and it
has been suggested that this could regulate cholesterol
uptake.14 Thus the role of CEase in absorption of
cholesterol remains controversial. A recent report of
cholesterol uptake by human intestinal cells identified
pancreatic phospholipase A2 as the major cholesterol
transport protein.15 This was shown to be due to
phospholipase-induced modification of the micellar lipid
composition rather than to direct transport of choles-
terol. It was suggested that CEase may act similarly to
enhance cholesterol uptake, which would argue against
a direct transport function.
active site serine is followed by slow deacylation. Kinetic
studies of CEase with carbamates allowed SAR to be
developed that individually describe the binding, acyla-
tion, and deacylation steps of the CEase catalytic cycle
and suggest a role for bile salts in conformational
modulation of both the fatty acid and steroid binding
sites to increase the hydrophobicity of these sites, at
least in the case of porcine CEase.20
In the present study, we focused on the development
of 3-substituted 6-chloro-2-pyrones with a tethered
cyclohexane ring attached to the 3-position. The size of
the tether ranged from 0 to 4 methylene units (struc-
tures 2a -e). In addition, 6-chloro-2-pyrone with a
cyclopentane ring attached at the 3-position was syn-
thesized (2f) in order to compare a five-membered ring
substituent with a six-membered ring substituent.
Inhibition of CEase was compared with inhibition of
trypsin and chymotrypsin to gain some insight into
selectivity. The results (Table 1) suggest that replace-
ment of 3-aryl substituents as in 1 with 3-alkyl sub-
stituents as in 2a -f results in the formation of com-
pounds that are highly selective for CEase. The results
also indicate that the compounds in Table 1 are simple
competitive inhibitors of substrate binding. There was
no indication that CEase was inactivated through
covalent modification. This is surprising in view of the
reported rapid inactivation of CEase by 3-benzyl-6-
chloro-2-pyrone.4 It is anticipated that further modifica-
tion of the structures of 2 will lead to the development
of potent inhibitors that will irreversibly modify and
inactivate CEase.
6-Chloro-2-pyrones and the related 3-chloroisocou-
marins were initially developed as irreversible inacti-
vators of serine proteases.5,16 Although it was suggested
that this class of pyrones operated as mechanism-based
(suicide) inactivators, subsequent studies suggested that
inactivation is due to slow turnover of the acylated
enzymes.17 The most extensive structure-activity stud-
ies of 6-chloro-2-pyrones with alkyl or aryl substituents
at positions 3, 4, or 5 were carried out by Boulanger
and Katzenellenbogen who demonstrated that both
binding and inactivation of chymotrypsin are markedly
dependent on the nature and location of the substitu-
ent.18 Chymotrypsin is inactivated by 3-benzyl-6-chloro-
2-pyrone through formation of a fairly stable acyl
enzyme intermediate.17 This same pyrone inactivates
CEase even more rapidly than chymotrypsin,4 suggest-
ing that 3-aryl-substituted 6-chloro-2-pyrones are poor
choices for development of selective inhibitors of CEase.
By comparison, 3-ethyl- or 3-butyl-6-chloro-2-pyrone
inhibit chymotrypsin poorly but reversibly (Ki ) 785 and
1980 µM, respectively).18 These results with chymo-
trypsin are in agreement with the present study where
the 3-alkyl-6-chloro-2-pyrones in Table 1 were generally
poor, reversible inhibitors of chymotrypsin. Thus the
selectivity that was attained in the development of
3-alkyl-6-chloro-2-pyrones as inhibitors of CEase re-
sulted from a combination of weak inhibition of chy-
motrypsin and potent inhibition of CEase. Weak inhi-
bition of chymotrypsin by 3-alkyl-6-chloro-2-pyrones is
markedly different from the inhibition of chymotrypsin
by 5-alkyl-6-chloro-2-pyrones. For example, 5-ethyl- or
5-butyl-6-chloro-2-pyrone are strong inhibitors of chy-
motrypsin (Ki ) 5.6 and 3.9 µM, respectively).18 This
suggests that 3-alkyl-6-chloro-2-pyrones are more prom-
ising than 5-alkyl-6-chloro-2-pyrones as selective inhibi-
tors of CEase.
Molecular modeling studies of CEase predicted that
CEase would have a relatively open active site for
substrate binding, unlike the situation with a number
of lipases where access to the active site requires the
movement of a hinge-like surface loop.21 Two recent
crystal structure studies of bovine CEase support the
conclusion that movement of a hinge-like surface loop
is not required for activity.22,23 Interestingly, bovine
CEase crystallized in the absence of detergent or bile
salts adopts a conformation in which the highly con-
served C-terminal hexapeptide is lodged in the active
site, with distortion of the oxyanion hole away from a
productive binding mode.23 In the presence of bile salts,
CEase adopts a conformation with an open active site
and a productive orientation of the oxyanion hole; one
molecule of bile salt is located near the active site while
a second is complexed at a remote site.22 These results
suggest that part of the mechanism of bile salt activa-
tion of CEase involves displacement of the C-terminal
peptide and conformational change leading to formation
of a competent active site. The fact that CEase is active
with small water soluble substrates such as p-nitrophe-
nylbutyrate in the absence of bile salts may indicate
that some substrates are capable of displacing the
C-terminal peptide from the active site. Inhibitor 2c
inhibits CEase both in the presence and absence of bile
salts; however, Ki increases from 0.025 µM in the
presence of bile salts to 0.090 µM in the absence of bile
salts which suggests that some energy is being expended
to displace the C-terminal peptide from the active site
when bile salts are absent.
The development of mechanism-based inhibitors of
CEase includes boronic and borinic acids, arylhaloke-
tones, aryl phosphates and phosphonates, and aryl and
cholesteryl carbamates.19 The most extensive molecular
recognition studies have been carried out with carbam-
ates,20 which are transient inhibitors, or pseudosub-
strates, wherein rapid acylation (carbamylation) of the
In summary, the present study has identified 3-alkyl-
6-chloro-2-pyrones as selective inhibitors of CEase.

4254 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Deck et al.
6-Ch lor o-3-m eth ylcycloh exyl-2-p yr a n on e (2b). In the
same manner as for 2c diethyl 2-methylcyclohexylmalonate
(8.50 g, 33.2 mmol) was added to sodium hydride in tetrahy-
drofuran followed by methyl cis-2-chloroacrylate. Saponifica-
tion and decarboxylation of the resulting triester (10.5 g, 93%)
gave a mixture of 2-methylcyclohexyl glutaconic acids (>90%
E by NMR) as an oil which solidified to a buff solid (6.90 g,
92%). Trituration with ethyl acetate followed by recrystalli-
zation from ethyl acetate gave a white solid of E-2-(2-
cyclohexylmethyl)-2-pentenedioic acid (7b): mp 140-142 °C;
1H NMR (DMSO-d6) d 0.86-1.60 (m, 11H), 2.10 (d, 2H), 3.19
(d, 2H), 6.81 (t, 1H), 12.4 (s, 2H). Assignment of the isomer
was based upon previous reports of NMR data.29 Reaction with
acetyl chloride and chromatography of the resulting oil gave
These inhibitors represent promising lead compounds
for development of irreversible inhibitors of CEase as
potential drugs for treatment of hypercholesterolemia.
Exp er im en ta l Section
Ch em ica l Syn th esis. Reagent quality solvents were used
without further purification. Melting points were determined
on a VWR Scientific Electrothermal capillary melting point
apparatus and are uncorrected. Infrared spectra were obtained
on a FT-IR Bomem MB-100 spectrophotometer. NMR spectra
were recorded on a Bruker AC250 NMR spectrometer in
CDCl3, unless otherwise stated. Chemical shifts are in ppm
(δ) relative to TMS. Solvents and other chemicals are reagent
grade. The R-bromoacyl chlorides and R-chloroacyl chlorides
that were not readily available were synthesized by reported
procedures.24 cis-3-Chloroacrylic acid was made from propiolic
acid25 and then was esterified to form the methyl ester (5).26
The diethyl 2-alkylmalonates (4a -f) were prepared according
to published procedures.27,28
1
1.90 g (25%) of 2b as white crystals: mp 58-60 °C; H NMR
δ 0.81-1.80 (m, 11H), 2.45 (d, 2H), 6.16 (d, 1H, J ) 6.95 Hz),
7.05 (d, 1H, J ) 7.00 Hz). Anal. (C12H15ClO2) C, H.
6-Ch lor o-3-(1-p r op yl-3-cycloh exyl)-2-p yr a n on e (2d ). In
the same manner as for 2c diethyl 2-(3-propylcyclohexyl)-
malonate (9.50 g, 33.4 mmol) was added to sodium hydride in
tetrahydrofuran followed by methyl cis-2-chloroacrylate. Sa-
ponification and decarboxylation of the resulting triester (11.5
g, 93%) gave a mixture of 2-propylcyclohexyl glutaconic acids
(>90% E by NMR) as a semisolid (9.79 g, 93%): 1H NMR
(DMSO-d6) δ 0.75-1.70 (m, 15H), 2.15 (t, 2H), 3.22 (d, 2H),
6.73 (t, 1H). Reaction with acetyl chloride and chromatography
of the resulting oil gave 1.50 g (18%) of 2d as white waxy
6-Ch lor o-3-(1-eth yl-2-cycloh exyl)-2-p yr a n on e (2c) a n d
6-Ch lor o-5-(1-eth yl-2-cycloh exyl)-2-p yr a n on e (8c). Diethyl
2-(2-cyclohexylethyl)malonate (9.02 g, 33.4 mmol) was added
dropwise to a solution containing pentane-washed sodium
hydride (0.80 g, 33.4 mmol) in 100 mL of tetrahydrofuran.
After the mixture was stirred for 10 min, methyl cis-2-
chloroacrylate (4.03 g, 33.4 mmol) was added, and the resulting
mixture was heated under reflux for 14 h. After cooling, water
was added and the mixture was extracted with ether. The
ether extracts were combined, washed with saturated sodium
chloride, dried over magnesium sulfate, filtered, and evapo-
rated to afford the triester as an oil (10.5 g, 89%). The oil was
dissolved in 20 mL of ethanol, and 150 mL of water containing
sodium hydroxide (10 g, 250 mmol) was added. The mixture
was refluxed for 8 h, cooled, and adjusted to pH 1 with
concentrated hydrochloric acid. After foaming ceased, the
mixture was refluxed for 1 h, cooled, and extracted with ether.
The ether extracts were washed with saturated sodium
chloride, dried over magnesium sulfate, filtered, and evapo-
rated to give a mixture of 2-ethylcyclohexyl glutaconic acids,
7.92 g (99%), as an oily solid. Trituration of the mixture with
ethyl acetate afforded a buff solid which was recrystallized
from ethyl acetate to give E-2-(2-cyclohexylethyl)-2-pentene-
dioic acid as white crystals: mp 117-118 °C; 1H NMR (DMSO-
d6) δ 0.87-1.75 (m, 13 H), 2.20 (t, 2H), 3.15 (d, 2H), 6.71 (t,
1H), 12.34 (s, 2H). Evaporation of the filtrate and recrystal-
lization from chloroform gave Z-2-(2-cyclohexylethyl)-2-pen-
tenedioic acid as a white solid: mp 166-167 °C; 1H NMR
(DMSO-d6) δ 0.87-1.75 (m, 13H), 2.20 (t, 2H), 3.39 (d, 2H),
6.00 (t, 1H), 12.34 (s, 2H). Assignment of E and Z isomers was
based upon previous reports of NMR data.29 The mixture of
glutaconic acids (7.92 g, 33.0 mmol) and acetyl chloride (80
mL) was refluxed for 3 days. The cooled solution was concen-
trated to give a dark oil (7.92 g, 85%) that was flash chro-
matographed on silica gel with ethyl acetate/hexane to give
1
crystals: mp 31-32 °C; H NMR δ 0.80-1.71 (m, 15H), 2.40
(t, 2H), 6.15 (d, 1H, J ) 7.00 Hz), 7.05 (d, 1H, J ) 7.03 Hz).
An analytical sample was obtained by sublimation at 80-90
°C (0.1 Torr). Anal. (C14H19ClO2) C, H.
6-Ch lor o-3-(1-bu tyl-4-cycloh exyl)-2-p yr a n on e (2e). In
the same manner as for 2c diethyl 2-(4-butylcyclohexyl)-
malonate (7.50 g, 25.1 mmol) was added to sodium hydride in
tetrahydrofuran followed by methyl cis-2-chloroacrylate. Sa-
ponification and decarboxylation of the resulting triester (8.80
g, 92%) gave a mixture of 2-butylcyclohexyl glutaconic acids
as an oily solid (5.50 g, 82%). Reaction with acetyl chloride
and chromatography of the resulting oil gave 1.20 g (18%) of
2e as a waxlike solid: 1H NMR δ 0.75-1.72 (m, 17H), 2.42 (t,
2H), 6.14 (d, 1H, J ) 6.96 Hz), 7.05 (d, 1H, J ) 6.97 Hz) Anal.
(C15H21ClO2) C, H.
6-Ch lor o-3-cyclop en tyl-2-p yr a n on e (2f). In the same
manner as for 2c diethyl 2-cyclopentylmalonate (11.0 g, 48.2
mmol) was added to sodium hydride in tetrahydrofuran
followed by methyl cis-2-chloroacrylate. Saponification and
decarboxylation of the resulting triester gave a 50/50 mixture
(indicated by NMR) of 2-cyclopentyl glutaconic acids (9.00 g,
94%). Reaction with acetyl chloride and chromatography of the
resulting oil gave 2.10 g (22%) of 2f as a clear oil: 1H NMR δ
1.32-2.05 (m, 8H), 2.95 (br p, 1H), 6.16 (d, 1H, J ) 7.08 Hz),
7.09 (d, 1H, J ) 7.04 Hz). Anal. (C10H11ClO2) C, H.
Ch olester yl 2-Ch lor op a lm ita te (10d ). Cholesterol (2.00
g, 5.20 mmol) was dissolved in methylene chloride, and
triethylamine (1.45 mL, 10.4 mmol) and 2-chloropalmitoyl
chloride (1.76 mL, 5.70 mmol) were added to the mixture. The
reaction was allowed to stir overnight at which time TLC
indicated complete disappearance of starting material. The
methylene chloride reaction mixture was first washed twice
with dilute HCl then twice with saturated sodium chloride and
dried over magnesium sulfate. The mixture was then filtered,
and the solvent was removed by rotary evaporation. The
resulting solid was purified on a silica gel column. Elution with
methylene chloride gave 2.73 g (80%) of 10d as a white solid:
1
2.00 g (25%) of 2c as white crystals: mp 33-34 °C; H NMR
δ 0.86-1.77 (m, 13H), 2.44 (t, 2H), 6.16 (d, 1H, J ) 6.97 Hz),
7.17 (d, 1H, J ) 6.99 Hz). An analytical sample was obtained
by sublimation at 80 °C (0.1 Torr). Anal. (C13H17ClO2) C, H.
Compound 8c (0.9 g, 12%) was eluted second from the column
and obtained as an oil: 1H NMR δ 1.16-1.66 (m, 13H), 2.41
(t, 2H), 6.23 (d, 1H, J ) 9.3 Hz), 7.29 (d, 1H, J ) 9.4 Hz).
Assignment of 3 and 5 isomers was based upon previous
reports of NMR data.30
6-Ch lor o-3-cycloh exyl-2-p yr a n on e (2a ). In the same
manner as for 2c diethyl 2-cyclohexylmalonate (10.5 g, 43.3
mmol) was added to sodium hydride in tetrahydrofuran
followed by methyl cis-2-chloroacrylate. Saponification and
decarboxylation of the resulting triester (13.5 g, 96%) gave a
mixture of 2-cyclohexyl glutaconic acids as an oily solid (8.00
g, 87%). Reaction with acetyl chloride and chromatography of
the resulting oil gave 2.00 g (22%) of 2a as white crystals: mp
69-70 °C; 1H NMR δ 1.14-1.92 (m, 10H), 2.60 (br t, 1H), 6.16
1
mp 52-53 °C; IR 1740 (carbonyl) cm-1; H NMR δ 0.65-2.39
(m, 72H), 4.20 (t, 1H), 4.62 (m, 1H), 5.37 (br d, 1H). Anal.
(C43H75BrO2) C, H.
Ch olester yl 2-Ch lor oh exa n oa te (10c). Cholesterol (2.00
g, 5.2 mmol), triethylamine (1.45 mL, 10.4 mmol), and 2-chlo-
rohexanoyl chloride (0.96 mL, 5.7 mmol) were combined in
methylene chloride. The same procedure as that used for 10d
was followed to afford 2.23 g (83%) of 10c as white needles:
(d, 1H, J ) 7.07 Hz), 7.02 (d, 1H, J ) 7.10 Hz). Anal. (C11H13
ClO2) C, H.
-
1
mp 67-69 °C; IR 1740 (carbonyl) cm-1; H NMR δ 0.65-2.38

Selective Inhibitors of Pancreatic Cholesterol Esterase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4255
(4) Bailey, J . M.; Gallo, L. L.; Gillespie, J . Inhibition of Dietary
Cholesterol Ester Absorption by 3-BCP, a Suicide Inhibitor of
Cholesterol-esterase. Biochem. Soc. Trans. 1995, 23, 408S.
(5) Westkaemper, R. B.; Abeles, R. H. Inactivators of Serine
Proteases Based on 6-Chloro-2-pyrone. Biochemistry 1983, 22,
3256-3264.
(6) Kudchodkar, B. J .; Horlick, L.; Sodhi, H. S. Effects of Plant
Sterols on Cholesterol Metabolism in Man. Atherosclerosis 1976,
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(7) Becker, M.; Staab, D.; von Bergmann, K. Treatment of Severe
Familial Hypercholesterolemia in Childhood with Sitosterol and
Sitostanol. J . Pediatr. 1993, 122, 292-296.
(8) Miettinen, T. A.; Puska, P.; Gylling, H.; Vanhanen, H.; Vartiain-
en, E. Reduction of Serum Cholesterol with Sitostanol-ester
(m, 52H), 4.20 (t, 1H), 4.65 (m, 1H), 5.37 (br d, 1H). Anal.
(C33H55ClO2) C, H.
Ch olester yl 2-Ch lor op r op ion a te (10b). Cholesterol (1.00
g, 2.60 mmol), triethylamine (0.73 mL, 5.20 mmol), and
2-chloropropionyl chloride (0.36 mL, 2.90 mmol) were combined
in methylene chloride. The same procedure as that used for
10d was followed to afford 0.99 g (80%) of 10b as white
needles: mp 123-125 °C; IR 1740 (carbonyl) cm-1; 1H NMR δ
0.65-2.38 (m, 46H), 4.33 (q, 1H), 4.64 (m, 1H) 5.36 (br d, 1H).
Anal. (C30H49ClO2) C, H.
Ch olester yl 2-Ch lor oa ceta te (10a ). Cholesterol (2.00 g,
5.20 mmol), triethylamine (1.45 mL, 10.4 mmol), and chloro-
acetyl chloride (0.64 mL, 5.70 mmol) were combined in
methylene chloride. The same procedure as that used for 10d
was followed to afford 1.92 g (80%) of 10a as a white solid:
mp 161-162 °C (lit.31 mp 160-161 °C); IR 1740 (carbonyl)
Margarine in
a Mildly Hypercholesterolemic Population. N.
Engl. J . Med. 1995, 333, 1308-1312.
(9) Weststrate, J . A.; Meijer, G. W. Plant Sterol-enriched Margarines
and Reduction of Plasma Total- and LDL-cholesterol Concentra-
tions in Normocholesterolaemic and Mildly Hypercholestero-
laemic Subjects. Eur. J . Clin. Nutr. 1998, 52, 334-343.
(10) Sedaghat, A.; Samuel, P.; Crouse, J . R.; Ahrens, E. H., J r. Effects
of Neomycin on Absorption, Synthesis, and/or Flux of Cholesterol
in Man. J . Clin. Invest. 1975, 55, 12-21.
cm-1 1H NMR δ 0.65-2.48 (m, 43H), 4.02 (s, 2H), 4.68 (m,
;
1H), 5.37 (br d, 1H). Anal. (C29H47ClO2) C, H.
Ch olester yl 2-Br om op a lm ita te (10g). Cholesterol (2.00
g, 5.20 mmol), triethylamine (1.45 mL, 10.4 mmol), and
2-bromopalmitoyl chloride (2.00 mL, 5.70 mmol) were com-
bined in methylene chloride. The same procedure as that used
for 10d was followed to afford 3.10 g (85%) of 10g as a white
solid: mp 50-52 °C; IR 1740 (carbonyl) cm-1; 1H NMR δ 0.65-
2.38 (72H), 4.15 (t, 1H), 4.64 (m, 1H), 5.37 (br d, 1H). Anal.
(C43H75BrO2) C, H.
(11) Hauser, H,; Dyer, J . H.; Nandy, A.; Vega, M. A.; Werder, M.;
Bieliauskaite, E.; Weber, F. E.; Compassi, S.; Gemperli, A.;
Boffelli, D.; Wehrli, E.; Schulthess, G.; Phillips, M. C. Identifica-
tion of a Receptor Mediating Absorption of Dietary Cholesterol
in the Intestine. Biochemistry 1998, 37, 17843-17850.
(12) Wang, C.-S.; Hartsuck, J . A. Bile Salt-activated Lipase:
A
Multiple Function Lipolytic Enzyme. Biochim. Biophys. Acta
1993, 1166, 1-19.
(13) Myers-Payne, S. C.; Hui, D. Y.; Brockman, H. L.; Schroeder, F.
Cholesterol Esterase: A Cholesterol Transfer Protein. Biochem-
istry 1995, 34, 3942-3947.
(14) Spilburg, C. A.; Cox, D. G.; Wang, X.; Bernat, B. A.; Bosner, M.
S.; Lange, L. G. Identification of a Species Specific Regulatory
Site in Human Pancreatic Cholesterol Esterase. Biochemistry
1995, 34, 15532-15538.
(15) Mackay, K.; Starr, J . R.; Lawn, R. M.; Ellsworth, J . L. Phos-
phatidylcholine Hydrolysis Is Required for Pancreatic Choles-
terol Esterase- and Phospholipase A2-facilitated Cholesterol
Uptake into Intestinal Caco-2 Cells. J . Biol. Chem. 1997, 272,
13380-13389.
(16) Harper, J . W.; Hemmi, K.; Powers, J . C. New Mechanism-Based
Serine Protease Inhibitors: Inhibition of Human Leukocyte
Elastase, Porcine Pancreatic Elastase, Human Leukocyte Cathe-
psin G, and Chymotrypsin by 3-Chloroisocoumarin and 3,3-
Dichlorophthalide. J . Am. Chem. Soc. 1983, 105, 6518-6520.
(17) Ringe, D.; Mottonen, J . M.; Gelb, M. H.; Abeles, R. H. X-ray
Diffraction Analysis of the Inactivation of Chymotrypsin by
3-Benzyl-6-chloro-2-pyrone. Biochemistry 1986, 25, 5633-5638.
(18) Boulanger, W. A.; Katzenellenbogen, J . A. Structure-Activity
Study of 6-Substituted 2-Pyranones as Inactivators of R-Chy-
motrypsin. J . Med. Chem. 1986, 29, 1159-1163.
(19) Feaster, S. R.; Quinn, D. M. Mechanism-Based Inhibitors of
Mammalian Cholesterol Esterase. Methods Enzymol. 1997, 286,
231-252.
(20) Feaster, S. R.; Lee, K.; Baker, N.; Hui, D. Y.; Quinn, D. M.
Molecular Recognition by Cholesterol Esterase of Active Site
Ligands: Structure-Reactivity Effects for Inhibition by Aryl
Carbamates and Subsequent Carbamylenzyme Turnover. Bio-
chemistry 1996, 35, 16723-16734.
Ch olester yl 2-Br om oh exa n oa te (10f). Cholesterol (2.00
g, 5.20 mmol), triethylamine (1.45 mL, 10.4 mmol), and
2-bromohexanoyl bromide (1.47 mL, 5.7 mmol) were combined
in methylene chloride. The same procedure as that used for
10d was followed to afford 2.50 g (85%) of 10f as white
1
needles: mp 60-62 °C; IR 1740 (carbonyl) cm-1; H NMR δ
0.65-2.38 (m, 52H), 4.15 (t, 1H), 4.62 (m, 1H), 5.37 (br d, 1H).
Anal. (C33H55BrO2) C, H.
Ch olester yl 2-Br om op r op ion a te (10e). Cholesterol (2.00
g, 5.20 mmol), triethylamine (1.45 mL, 10.4 mmol), and
2-bromopropionyl bromide (1.23 mL, 5.7 mmol) were combined
in methylene chloride. The same procedure as that used for
10d was followed to afford 1.97 g (100%) of 10e as white
needles: mp 123-124 °C; IR 1740 (carbonyl) cm-1; 1H NMR δ
0.66-2.40 (m, 46H), 4.33 (q, 1H), 4.65 (m, 1H) 5.36 (br d, 1H).
Anal. (C30H49BrO2) C, H.
En zym e Assa ys a n d Kin etics. CEase (porcine), chymot-
rypsin (bovine), and trypsin (porcine) were from Sigma. CEase
was assayed in 0.1 M Hepes, pH 7, containing 6 mM tauro-
cholate 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 such as 2c, by
Straus-Goldstein plots. Kinetic analysis of Michaelis constants
and kcat values was carried out by nonlinear regression
analysis with the Enzfitter program. Dissociation constants
of inhibitors were determined by linear regression analysis of
the double reciprocal, Dixon, and Straus-Goldstein plots.
Chymotrypsin was assayed using the same substrate and
buffer as for CEase. Trypsin was assayed in 0.05 M Tris, pH
7.4, with 0.4 mM p-nitrophenyl-p′-guanidino benzoate at 405
nm.
(21) Feaster, S. R.; Quinn, D. M.; Barnett, B. L. Molecular Modeling
of the Structures of Human and Rat Pancreatic Cholesterol
Esterases. Protein Science 1997, 6, 73-79.
(22) Wang, X.; Wang, C. S.; Tang, J .; Dyda, F.; Zhang, X. C. The
Crystal Structure of Bovine Bile Salt Activated Lipase: Insights
into the Bile Salt Activation Mechanism. Structure 1997, 5,
1209-1218.
(23) Chen, J . C. H.; Miercke, L. J . W.; Krucinski, J .; Starr, J . R.;
Saenz, G.; Wang, X.; Spilburg, C. A.; Lange, L. G.; Ellsworth, J .
L.; Stroud, R. M. Structure of Bovine Pancreatic Cholesterol
Esterase at 1.6 A: Novel Structural Features Involved in Lipase
Activation. Biochemistry 1998, 37, 5107-5117.
Ack n ow led gm en t. This work was supported by a
grant from the American Heart Association (SW Affili-
ate), by Grant GM52576 from the Minority Biomedical
Sciences Support (MBRS) program, NIH, and by the
REU program, NSF.
(24) Harpp, D. N.; Bao, L. Q.; Black, C. J .; Gleason, J . G.; Smith, R.
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succinamide, N-Chlorosuccinamide and Molecular Iodine. J . Org.
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1965, 30, 3141-3146.
(26) House, H. O.; Roelofs, W. L.; Trost, B. M. The Chemistry of
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(27) Hiers, G. S.; Adams, R. Omega-Cyclohexyl Derivatives of Various
Normal Aliphatic Acids. IV. J . Am. Chem. Soc. 1926, 48, 2385-
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