Synthesis of lipophilic aldehydes and study of their inhibition effect on human digestive lipases.

Article abstract of DOI:10.1021/ol026039l

[reaction: see text] Novel inhibitors of human digestive lipases, aldehyde dialkyl and alkyl-acyl glycerol analogues, were developed. The inhibitors were prepared starting from 3-(benzyloxy)-1,2-propanediol. The inhibition of human pancreatic and gastric lipases by the aldehyde derivatives was studied using the monolayer technique. (1R)-1-[(Dodecyloxy)methyl]-4-oxobutyl decanoate caused a 50% decrease in HPL and HGL activities at 0.100 and 0.053 molar fractions, respectively.

Full text of DOI:10.1021/ol026039l

ORGANIC
LETTERS
2002
Vol. 4, No. 16
2625-2628
Synthesis of Lipophilic Aldehydes and
Study of Their Inhibition Effect on
Human Digestive Lipases
,†
Stavroula Kotsovolou,† Robert Verger,‡ and George Kokotos*
Department of Chemistry, UniVersity of Athens, Panepistimiopolis,
Athens 15771, Greece, and Laboratoire de Lipolyse Enzymatique CNRS-IFR1,
UPR 9025, 31 Chemin Joseph-Aiguier, 13402 Marseille, Cedex 20, France
gkokotos@cc.uoa.gr
Received April 18, 2002
ABSTRACT
Novel inhibitors of human digestive lipases, aldehyde dialkyl and alkyl-acyl glycerol analogues, were developed. The inhibitors were prepared
starting from 3-(benzyloxy)-1,2-propanediol. The inhibition of human pancreatic and gastric lipases by the aldehyde derivatives was studied
using the monolayer technique. (1R)-1-[(Dodecyloxy)methyl]-4-oxobutyl decanoate caused a 50% decrease in HPL and HGL activities at 0.100
and 0.053 molar fractions, respectively.
In humans and most mammals the digestion of dietary
triacylglycerols (TAGs) requires the successive intervention
of two lipases: gastric lipase (HGL), which is secreted and
is active in stomach, followed by the classical pancreatic
lipase (HPL) secreted into the duodenum. The hydrolysis of
TAGs by these lipases to monoacylglycerols and free fatty
acids is a necessary step for efficient fat digestion and
absorption by the enterocytes.1 Therefore, inhibitors of
digestive lipases are of special interest because they may
find applications as anti-obesity agents.2 A gastrointestinal
lipase inhibitor of microbial origin is now a registered drug
for weight reduction.3
the various classes of inhibitors reported, inhibitors of the
phosphonate type have been successfully used to solve the
3D structure of various lipases.5
The active site of pancreatic lipase contains Ser-His-Asp
residues, a triad resembling the catalytic triad of serine
proteases, as has been proven by site-directed mutagenesis6
and crystallographic data.7 HGL possesses the same catalytic
triad.8 We have recently proposed a strategy for the rational
design of lipase inhibitors. In general a lipase inhibitor should
consist of two components: a chemically reactive moiety,
(3) (a) Hauptman, J. B.; Jeunet, F. S.; Hartmann, D. Am. J. Clin. Nutr.
1992, 55, 309. (b) Drent, M. L.; Larsson, I.; William-Olson, T.; Quaade,
F.; Czubayko, F.; von Bergmann, K.; Strobel, W.; Sjo¨stro¨m, L.; van der
Veen, E. A. Int. J. Obes. 1995, 19, 221.
(4) For a recent review, see: Cavalier, J.-F.; Buono, G. Verger, R. Acc.
Chem. Res. 2000, 33, 579.
(5) (a) Egloff, M.-P.; Marguet, F.; Buono, G.; Verger, R.; Cambillau,
C.; van Tilbeurgh, H. Biochemistry 1995, 34, 2751. (b) Roussel, A.; Miled,
N.; Berti-Dupuis, L.; Riviere, M.; Spinelli, S.; Berna, P.; Gruber, V.; Verger,
R.; Cambillau, C. J. Biol. Chem. 2002, 277, 2266.
Synthetic inhibitors of lipases have been used in the study
of structural and mechanistic properties of lipases.4 Among
† University of Athens.
‡ CNRS Marseille.
(1) (a) Carriere, F.; Barrowman, J. A.; Verger, R.; Laugier, R. Gastro-
enterology 1993, 105, 876. (b) Lowe, M. E Gastroenterology 1994, 107,
1524.
(2) Thomson, A. B. R.; de Pover, A.; Keelan, M.; Jarocka-Kyrta, E.;
Clandinin, M. T. Methods Enzymol. 1997, 286, 3.
(6) (a) Lowe, M. E. J. Biol. Chem. 1992, 267, 17069. (b) Lowe, M. E.
Biochim. Biophys. Acta 1996, 1302, 177.
10.1021/ol026039l CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/13/2002

capable of reacting with the active site serine of the enzyme,
and a part that contains chemical motifs, necessary for
specific interactions and a proper orientation in the enzyme
binding pocket. Up to now we have shown that lipophilic
2-oxo amide9,10 and 2-oxo amide11 and bis-2-oxo amide12
triacylglycerol analogues are effective inhibitors of human
pancreatic and gastric lipases.
Scheme 1a
a (a) C12H25Br, Bu4NHSO4, 50% NaOH/C6H6, 45 °C; (b)
C9H19COOH, DCC, DMAP, CH2Cl2.
The removal of the benzyl group from compounds 3 and
4 was carried out by catalytic hydrogenation (Scheme 2).
According to the above strategy we decided to use the
aldehyde group as the reactive functionality. Peptide alde-
hydes have been reported to inhibit serine and cysteine
proteases.13 It should be noted that two tripeptide aldehyde
inhibitors of thrombin have entered clinical trials.14 The novel
lipase inhibitors were designed taking into consideration the
structure of triacylgycerols, which are the natural substrate
of lipases. The carbonyl of the ester bond at the sn-1 position
of the substrate was replaced by the carbonyl of the aldehyde
functionality. The ester bond at the sn-3 position was replaced
by an ether bond to avoid hydrolysis at this position. The
ester bond at the sn-2 position was either maintained or
replaced by a non-hydrolyzable ether bond. Given the
preference of HPL and HGL to hydrolyze ester bonds at the
sn-1 and sn-3 positions of triacylglycerol, the ester bond of
the inhibitor corresponding to that of the sn-2 position is
not anticipated to undergo enzymatic hydrolysis.
Scheme 2a
Etherification of 3-(benzyloxy)-1,2-propanediol (1) with
1-bromododecane took place under phase transfer conditions
and produced a mixture of compounds 2 (40%) and 3 (26%),
which were separated (Scheme 1). Compound 2 was then
coupled with decanoic acid using 1,3-dicyclohexylcarbodi-
imide (DCC) as a condensing agent in the presence of
4-(dimethylamino)pyridine (DMAP)15 to produce compound
4.
(7) (a) Winkler, F. K.; D’Arcy, A.; Hunziker, W. Nature 1990, 343, 771.
(b) Brady, L.; Brzozowski, A. M.; Derewenda, Z. S.; Dodson, E.; Dodson,
G.; Tolley, S.; Turkenburg, J. P.; Christiansen, L.; Hughe-Jensen, B.;
Norskov, L.; Thim, L.; Menge, U. Nature 1990, 343, 767. (c) van Tilbeurgh,
H.; Egloff, M.-P.; Martinez, C.; Rugani, N.; Verger, R.; Cambillau, C.
Nature 1993, 362, 814.
(8) Roussel, A.; Canaan, S.; Egloff, M.-P.; Riviere, M.; Dupuis, L.;
Verger, R.; Cambillau, C. J. Biol. Chem. 1999, 274, 16995.
(9) Chiou, A.; Markidis, T.; Constantinou-Kokotou, V.; Verger, R.;
Kokotos, G. Org. Lett. 2000, 2, 347.
(10) Chiou, A.; Verger, R.; Kokotos, G. Lipids 2001, 36, 535.
(11) Kokotos, G.; Verger, R.; Chiou, A. Chem. Eur. J. 2000, 6, 4211.
(12) Kotsovolou, S.; Chiou, A.; Verger, R.; Kokotos, G. J. Org. Chem.
2001, 66, 962.
(13) (a) Westerik, J. O.; Wolfenden, R. J. Biol. Chem. 1971, 247, 8195.
(b) Thompson, R. C. Biochemistry 1973, 12, 47.
a (a) H2, 10% Pd/C; (b) AcNH-TEMPO, NaOCl, NaBr, PhCH3/
EtOAc/H2O, -10 °C; (c) Ph3PdCHCOOBut, THF, 65 °C; (d) 50%
CF3COOH/CH2Cl2; (e) NMM, ClCOOEt, THF, -10 °C; (f) NaBH4,
MeOH.
Compounds 5, 6 were then oxidized to the corresponding
aldehydes by NaOCl in the presence of 4-acetamido-2,2,6,6-
tetramethyl-1-piperidinyloxy free radical (AcNH-TEMPO).16
(14) (a) Jackson, C. V.; Satterwhite, J.; Roberts, E. Clin. Appl. Thromb./
Hemostasis 1996, 2, 258. (b) Rebello, S. S.; Miller, B. V.; Basler, G. C.;
Lucchesi, B. R. J. CardioVasc. Pharmacol. 1997, 29, 240.
(15) Neises, B.; Steglich, W. Angew. Chem., Int. Ed. Engl. 1978, 17,
522.
(16) (a) Ma, Z.; Bobbit, J. M. J. Org. Chem. 1991, 56, 6110. (b) Leanna,
M. B.; Sowin, T. J.; Morton, H, E. Tetrahedron Lett. 1992, 33, 5029.
2626
Org. Lett., Vol. 4, No. 16, 2002

The aldehydes, without further purification, reacted with
Ph3PdCHCOOBut to afford compounds 7 and 8. The
corresponding carboxylic acids 9 and 10 were obtained after
catalytic hydrogenation and acidic removal of the But group.
Reduction of the mixed carbonic anhydrides of 9 and 10 by
To determine the film stability and the interfacial properties
at the air/water interface of the aldehyde derivatives syn-
thesized, we recorded their force/area curves. Figure 1 shows
17
NaBH4 and oxidation of the products produced the target
compounds 13 and 14.
To study the stereoselectivity of the inhibition, the
R-enantiomer of compound 14, (1R)-1-(dodecyloxymethyl)-
4-oxobutyl decanoate (21), was prepared by similar reactions
starting from compound 15, as outlined in Scheme 3. All
the intermediates and final products gave satisfactory analyti-
cal and spectroscopic data.18
Scheme 3a
Figure 1. Force/area curves for compounds 13 (×) and 14 (9).
The aqueous subphase was composed of Tris/HCl 10 mM, pH 8,
NaCl 150 mM, CaCl2 21 mM, EDTA 1 mM. The continuous
compression experiment was performed in the rectangular reservoir
of the “zero order” trough.22
the molecular area dependency as a function of the surface
pressure for compounds 13 and 14 spread over a buffered
subphase at pH 8.0. The inhibition of HPL and HGL was
studied by means of the monomolecular film technique22,23
with mixed films of 1,2-dicaprin containing variable propor-
tions of each inhibitor. The inhibition studies were performed
at a constant surface pressure of 25 mN m-1 for HPL and
27 mN m-1 for HGL.
Inhibitors of lipolytic enzymes are best reported in terms
of molar fraction of the inhibitor in the interface. Remaining
lipase activity was plotted as a function of the inhibitor molar
fraction (R). The data obtained for HPL and HGL are
presented in Figures 2 and 3, respectively. Lipase hydrolysis
rates of 1,2-dicaprin decreased sharply as the molar fraction
of inhibitors increased. The dotted line corresponds to the
a (a) DIBALH, Et2O, 0 °C; (b) C6H5CH2Br, Bu4NHSO4, 50%
NaOH/C6H6, 45 °C; (c) 4 N HCl/MeOH; (d) C12H25Br, Bu4NHSO4,
50% NaOH/C6H6, 45 °C; (e) C9H19COOH, DCC, DMAP, CH2Cl2;
(f) H2, 10% Pd/C; (g) AcNH-TEMPO, NaOCl, NaBr, PhCH3/
AcOEt/H2O, -10 °C; (h) Ph3PdCHCOOBn, THF, 65 °C; (i)
NMM, ClCOOEt, THF, -10 °C; (j) NaBH4, MeOH.
The study of inhibition of lipolytic enzymes is a difficult
task, because of nonmutually exclusive processes such as
interfacial denaturation, changes in interfacial quality19 and
surface dilution phenomena.20 The use of the monolayer
technique, which is based upon surface pressure decrease
owing to lipid-film hydrolysis, is advantageous for the study
of lipases inhibitors since with conventional emulsified
systems it is not possible to control the interfacial quality.
The kinetic studies of the lipase hydrolysis reactions require
that the lipids used form a stable monomolecular film at the
air/water interface.21
Figure 2. Effect of increasing concentrations of 13 (×), 14 (9),
and 21 (0) on the remaining activity of HPL on 1,2-dicaprin
(17) Kokotos, G. Synthesis 1990, 299.
(18) See Supporting Information.
monolayer maintained at a constant surface pressure of 25 mN m-1
.
(19) Verger, R.; de Haas G. H. Annu. ReV. Biophys. Bioeng. 1976, 5,
77.
The aqueous subphase was composed of Tris/HCl 10 mM, pH 8,
NaCl 100 mM, CaCl2 21 mM, EDTA 1 mM. The kinetics of
hydrolysis were recorded during 15-20 min.
(20) Dennis, E. A. Drug DeV. Res. 1987, 10, 205.
(21) Ransac, S.; Ivanova, M. G.; Verger, R.; Panaiotov, I. Methods
Enzymol. 1997, 286, 263.
Org. Lett., Vol. 4, No. 16, 2002
2627

derivatives 14 and 21 were better inhibitors than the ether
derivative 13 both for HPL and HGL. No stereoselective
discrimination was observed during the inhibition of HPL
or HGL. The racemic compound 14 and its R-enantiomer
21 caused almost the same inhibitory effect on both digestive
lipases. These findings may be explained on the basis of the
three-dimensional model proposed for the inhibited forms
of HPL5a and HGL.5b As shown by crystallographic data,
both enantiomers of alkyl phosphonate inhibitors may be
accommodated into the catalytic crevice. Alcohol 12 was
also tested, but no inhibition of either HPL or HGL was
observed, indicating that the presence of the aldehyde group
is critical for the inhibitory activity.
The R50 values reported for a series of chiral organophos-
phorus acylglycerol analogues,24 in which one carbonyl was
replaced by a phosphonate group, varied from 0.13 to 0.20
for HPL and 0.05 to 0.22 for HGL. Up to now the best
synthetic inhibitor of HPL reported is O-hexadecyl-O-(p-
nitrophenyl) n-undecyl phosphonate, with an R50 value of
0.003. For HGL the highest inhibition was obtained with
O-undecyl-O-(p-nitrophenyl) n-decyl phosphonate, which
exhibits an R50 value of 0.008.25 The most active 1,3-bis-2-
oxo amide triacylglycerol analogue12 exhibited R50 values
of 0.076 and 0.020 for HPL and HGL, respectively.
In conclusion, we have demonstrated that aldehyde dialkyl
and alkyl-acyl glycerol analogues inhibit both digestive
lipases. To the best of our knowledge this is the first time
that the inhibition of a lipolytic enzyme by a synthetic
aldehyde derivative was observed.
Figure 3. Effect of increasing concentrations of 13 (×), 14 (9),
and 21 (0) on the remaining activity of HGL on 1,2-dicaprin
monolayer maintained at a constant surface pressure 27 mN m-1
.
The aqueous subphase was composed of CH3COONa 10 mM, pH
5, NaCl 150 mM, CaCl2 21 mM, EDTA 1 mM. The kinetics of
hydrolysis were recorded during 15-20 min.
surface dilution phenomena, which reflects the decrease in
lipase activity that would be observed if a nonsubstrate,
noninhibitor compound were present in the monomolecular
film. The molar fractions, R50, obtained for all the inhibitors
tested are summarized in Table 1. The R50 is defined as the
molar fraction of inhibitor that reduces by 50% the initial
rate of lipolysis.
Acknowledgment. This work was supported by the
GSRT and the French Foreign Ministry.
Table 1. Inhibition Constants (R50) of Inhibitors Tested on
HPL and HGL with the Monolayer Technique
R50
Supporting Information Available: Experimental pro-
cedures and characterization data. This material is available
free of charge via the Internet at http://pubs.acs.org.
compound
HPLa
HGLb
13
14
21
0.180 ( 0.019
0.143 ( 0.012
0.100 ( 0.007
0.086 ( 0.009
0.052 ( 0.003
0.053 ( 0.004
OL026039L
(22) Verger, R.; de Haas G. H. Chem. Phys. Lipids 1973, 10, 127.
(23) The reasons for using lipid monolayers as substrates for lipolytic
enzymes are summarized in Supporting Information.
(24) Marguet, F.; Douchet, I.; Cavalier, J.-F.; Buono, G.; Verger, R.
Colloids Surf., B 1999, 13, 37.
(25) Cavalier, J.-F.; Ransac, S.; Verger, R.; Buono, G. Chem. Phys. Lipids
1999, 100, 3.
a Surface pressure 25 mN m-1 b Surface pressure 27 mN m-1
. .
As shown from these data, all inhibitors tested were 2-3
times more powerful on HGL as compared to HPL. The ester
2628
Org. Lett., Vol. 4, No. 16, 2002