Synthesis and study of a lipophilic alpha-keto amide inhibitor of pancreatic lipase.

Article abstract of DOI:10.1021/ol991295s

[reaction: see text] A lipophilic alpha-keto amide, inhibitor of pancreatic lipase, was synthesized using a lipidic 2-amino alcohol as backbone. The chiral key intermediate 2-(tert-butyloxycarbonylamino)-D-undecen-5-ol was synthesized starting from D-glutamic acid. The inhibitor formed a stable monomolecular film at the air/water interface as shown by a force/area curve. Inhibition studies using the monomolecular film technique with mixed films of 1,2-dicaprin containing variable proportions of the inhibitor showed a 50% decrease in lipase activity at a 0.14 molar fraction.

Full text of DOI:10.1021/ol991295s

ORGANIC
LETTERS
2000
Vol. 2, No. 3
347-350
Synthesis and Study of a Lipophilic
r-Keto Amide Inhibitor of Pancreatic
Lipase
Antonia Chiou,† Theodoros Markidis,† Violetta Constantinou-Kokotou,‡
,†
Robert Verger,§ and George Kokotos*
Department of Chemistry, UniVersity of Athens, Panepistimiopolis,
Athens 15771, Greece, Chemical Laboratories, Agricultural UniVersity of Athens,
Athens 11855, Greece, and Laboratoire de Lipolyse Enzymatique CNRS, UPR 9025,
31 Chemin Joseph-Aiguier, 13402 Marseile, Cedex 20, France
gkokotos@cc.uoa.gr
Received December 1, 1999
ABSTRACT
A lipophilic r-keto amide, inhibitor of pancreatic lipase, was synthesized using a lipidic 2-amino alcohol as backbone. The chiral key intermediate
2-(tert-butyloxycarbonylamino)-D-undecen-5-ol was synthesized starting from D-glutamic acid. The inhibitor formed a stable monomolecular
film at the air/water interface as shown by a force/area curve. Inhibition studies using the monomolecular film technique with mixed films of
1,2-dicaprin containing variable proportions of the inhibitor showed a 50% decrease in lipase activity at a 0.14 molar fraction.
Lipases are versatile tools for biotechnology and have been
employed by organic chemists for a long time to catalyze
chemo-, regio-, and stereoselective transformations.1,2 In
humans, pancreatic and gastric lipases are essential enzymes
for efficient fat digestion.3 The hydrolysis of dietary tri-
acylglycerols by these enzymes is a necessary step for fat
absorption by the enterocytes. Potent and specific inhibitors
of these enzymes are of interest because they may find
application as anti-obesity agents.4 Furthermore, inhibitors
of various lipases of animal and microbial origin may
contribute to a better understanding the mechanisms of lipase
action.5
The active site of pancreatic lipase contains Ser-His-Asp,
a triad resembling the catalytic triad of serine proteases, as
it has been proven by chemical modification,6 site-directed
mutagenesis,7 and crystallographic data.8
Many inhibitors of serine proteases consist of a substrate-
(5) For reviews, see: (a) Gargouri, Y.; Ransac, S.; Verger, R. Biochim.
Biophys. Acta 1997, 1344, 6. (b) Ransac, S.; Gargouri, Y.; Marguet, F.;
Buono, G.; Beglinger, C.; Hildebrand, P.; Lengsfeld, H.; Hadvary, P.;
Verger, R. Methods Enzymol. 1997, 286, 191.
(6) Guidoni, A.; Benkouka, F.; de Caro, J.; Rovery, M. Biochim. Biophys.
Acta 1981, 660, 148.
† University of Athens.
‡ Agricultural University of Athens.
§ CNRS Marseille.
(1) Schmid, R. D.; Verger, R. Angew. Chem., Int. Ed. 1998, 37, 1608.
(2) Reetz, M. T.; Jaeger, K.-E. Top. Curr. Chem. 1999, 200, 31.
(3) (a) Carrie`re, F.; Barrowman, J. A.; Verger, R.; Laugier, R. Gastro-
enterology 1993, 105, 876. (b) Lowe, M. E. Gastroenterology 1994, 107,
1524.
(4) (a) Drent, M. L.; Vanderveen, E. A. Int. J. Obesity 1993, 17, 241.
(b) Thomson, A. B. R.; De Pover, A.; Keelan, M.; Jarocka-Kyrta, E.;
Clandinin, M. T. Methods Enzymol. 1997, 286, 3.
(7) Lowe, M. E. J. Biol. Chem. 1992, 267, 17069.
(8) (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.
10.1021/ol991295s CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/06/2000

like structure incorporating a reactive carbonyl group at the
site of the scissile amide bond, capable of reacting with the
active site of the enzyme. A number of reactive carbonyl
groups, such as fluorinated ketones,9 R-keto esters,10 R-keto
amides,11 and 1,2-diketones,12 have been successfully used
in the design of protease inhibitors. In such cases the
mechanism of action involves most likely a nucleophilic
attack by the hydroxyl group of the active site serine onto
the electrophilic carbonyl group of the inhibitors, followed
by the formation of a hemiacetal adduct, which mimics the
tetrahedral intermediate involved in the enzymatic cleavage.
Furthermore, fatty alkyl trifluoromethyl ketones have been
reported to inhibit phospholipases A2.13 Most recently R-keto
amide triglyceride analogues as inhibitors of Staphylococcus
hyicus lipase have been reported.14
Boc-glutamate (1) was reduced using DIBAL under con-
trolled conditions,15 and the resulting aldehyde was submitted
to Wittig reaction with the suitable ylide to produce the fully
protected unsaturated lipidic amino acid 2. The Boc-protected
amino acid 3, obtained by the appropriate deprotection-
protection procedure,15 was converted into the corresponding
fluoride and reduced in situ by treatment with sodium
borohydride and dropwise addition of methanol16 to produce
the amino alcohol 4.17
The etherification procedure took place under phase
transfer conditions. The hydroxy component 4 was treated
with n-decyl bromide in a biphasic system of benzene/
aqueous sodium hydroxide in the presence of a catalytic
amount of Bu4NHSO4 and afforded the ether derivative 5
(Scheme 2) in satisfactory yield. Removal of the Boc group
To develop novel inhibitors of lipases we incorporated the
R-keto amide function into a lipidic amino alcohol backbone,
ensuring the lipophilicity of the final product. In this paper
we thus report the synthesis of such a typical molecule, the
study of its surface properties, and its inhibitory effects on
pancreatic lipase activity studied by the monomolecular film
technique.
Scheme 2
The lipidic amino alcohol 4, used as backbone for the
R-keto amide inhibitor, was synthesized starting from D-
glutamic acid, as described in Scheme 1. Dimethyl N,N-di-
Scheme 1
using HCl/THF led to the corresponding free amino com-
pound 6, which was coupled with 2-hydroxyhexadecanoic
acid using 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide
(9) (a) Imperiali, B.; Abeles, R. H. Biochemistry 1986, 25, 3760. (b)
Beugue, J.-P.; Bonnet-Delpon, D. Tetrahedron 1991, 47, 3207.
(10) Li, Z.; Patil, G. S.; Colubski, Z. E.; Hori, H.; Tehrani, K.; Foreman,
J. E.; Eveleth, D. D.; Bartus, R. T.; Powers, J. C. J. Med. Chem. 1993, 36,
3472.
(11) Li, Z.; Ortega-Vilain, A.-C.; Patil, G. S.; Chu, D.-L.; Foreman, J.
E.; Eveleth, D. D.; Powers, J. C. J. Med. Chem. 1996, 39, 4089.
(12) Mehdi, S.; Angelastro, M. R.; Burkhart, J. P.; Kehl, J. R.; Peet, N.
P.; Bey, P. Biochem. Biophys. Res. Commun. 1990, 166, 595.
348
Org. Lett., Vol. 2, No. 3, 2000

(WSC‚HCl)18 as a condensing agent in the presence of
1-hydroxybenzotriazole (HOBt).
the kinetic studies a surface pressure of 20 mN‚m-1 was
selected. At this value of surface pressure PPL is highly
active and characterized by linear kinetics.
Remaining lipase activity was measured as a function of
the inhibitor molar fraction (R) (Figure 2). Lipase hydrolysis
The racemic 2-hydroxy fatty acid was prepared by
deamination of the corresponding 2-aminohexadecanoic
acid19 using NaNO2 under acidic conditions. The R-hydroxy
amide 7 was converted to the corresponding R-keto amide
820 using pyridinium dichromate (PDC) in acetic acid, which
proved to be an effective agent for the oxidation affording
the desired product in good yield.
The use of the monolayer technique, which is based upon
surface pressure decrease due to the film hydrolysis, is
advantageous for the study of lipase inhibition since with
conventional emulsified systems it is not possible to control
their “interfacial quality”.21 The kinetic studies of the lipase
hydrolysis reactions requires that the lipids used as substrates
form a rather stable monomolecular film at the air/water
interface.22
To determine the film stability and the interfacial properties
of the R-keto amide derivative 8, we have recorded its force/
area curve at the air/water interface. The experiment was
performed in the reservoir compartment of a “zero-order”
trough. Figure 1 gives the surface pressure dependency as a
Figure 2. Effect of increasing concentrations of 8 on the hydrolysis
rate by PPL of 1,2-dicaprin monolayer maintained at a constant
surface pressure of 20 mN‚m-1. 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.
rates of 1,2-dicaprin decreased as the molar fraction of the
inhibitor increased. The dotted line corresponds to the surface
dilution phenomena, which reflects the decrease in lipase
activity that would be observed if a nonsubstrate, noninhibitor
compound, i.e., so-called “surface dilutor”, were present in
the monomolecular film. A 50% decrease in lipase activity
was observed when 14.1 ( 3.5% of 1,2-dicaprin had been
substituted by the inhibitor compound 8. Although this value
corresponds to a rather weak inhibition of pancreatic lipase,
Figure 1. Force/area curve of compound 8. The aqueous subphase
was composed of Tris/HCl 10 mM, pH 8, NaCl 100 mM, CaCl2
21 mM, EDTA 1 mM. The continuous compression experiment
was performed in the rectangular reservoir of the “zero order”
trough.23
(15) Kokotos, G.; Padron, J.-M.; Martin, T.; Gibbons, W. A.; Martin,
V. S. J. Org. Chem. 1998, 63, 3741.
(16) Kokotos, G.; Noula, C. J. Org. Chem. 1996, 61, 6994.
(17) Yield 84%; oil; [R]D +3.6 (c 0.5, CHCl3); 1H NMR (200 MHz,
CDCl3) δ 5.4 (m, 2H), 4.6 (br, 1H), 3.7-3.5 (m, 3H), 2.2-1.9 (m, 4H),
1.6-1.4 (m, 11H), 1.3 (m, 6H), 0.9 (t, J ) 7 Hz, 3H); MS (FAB) m/z (%)
308 (12) [M + Na+], 286 (9) [M + H+], 230 (68), 186 (100). Anal. Calcd
for C16H31NO3: C, 67.33; H, 10.95; N, 4.91. Found: C, 67.09; H, 11.24;
N, 4.82. Enantiomeric excess >95% was indicated by 1H NMR and 19F
NMR analysis of the corresponding Mosher ester.
(18) Sheehan, J. C.; Cruickshank, P. A.; Boshart, G. L. J. Org. Chem.
1961, 26, 2525.
(19) Kokotos, G.; Martin, V. S.; Constantinou-Kokotou, V.; Gibbons,
W. A. Amino Acids 1996, 11, 329.
function of the molecular area of a film spread over a
buffered subphase at pH 8.0. The large molecular area of
the film formed by this compound may be attributed to the
presence of the double bond as well as the two alkyl chains.
The inhibition of pancreatic lipase was studied using the
monomolecular film technique22,23 with mixed films of 1,2-
dicaprin containing variable proportions of compound 8. For
(20) Yield 57%; oil; [R]D +3.1 (c 0.5, CHCl3); 1H NMR (200 MHz,
CDCl3) δ 7.1 (d, J ) 8 Hz, 1H), 5.4 (m, 2H), 4.0 (m, 1H), 3.4 (m, 4H), 2.9
(t, J ) 7 Hz, 2H), 2.1-1.9 (m, 4H), 1.8-1.5 (m, 6H), 1.4-1.1 (m, 42H),
0.9 (m, 9H); 13C NMR (200 MHz, CDCl3) δ 199.4, 159.7, 130.9, 128.2,
71.5, 49.0, 36.8, 31.9-22.3, 14.1-14.0; MS (FAB) m/z (%) 579 (24) [M
+ H+], 578 (100) [M+], 352 (12), 225 (18). Anal. Calcd for C37H71NO3:
C, 76.89; H, 12.38; N, 2.42. Found: C, 76.62; H, 12.56; N, 2.34.
(21) Verger, R.; de Haas, G. H. Annu. ReV. Biophys. Bioeng. 1976, 5,
77.
(13) (a) Street, I. P.; Lin, H.-K.; Laliberte, F.; Ghomashchi, F.; Wang,
Z.; Perrier, H.; Tremblay, N. M.; Huang, Z.; Weech, P. K.; Gelb, M. H.
Biochemistry 1993, 32, 5935. (b) Ackermann, E. J.; Conde-Frieboes, K.;
Dennis, E. A. J. Biol. Chem. 1995, 270, 445. (c) Ghomashchi, F.; Loo, R.;
Balsinde, J.; Bartoli, F.; Apitz-Castro, R.; Clark, J. D.; Dennis, E. A.; Gelb,
M. H. Biochim. Biophys. Acta 1999, 1420, 45.
(22) Ransac, S.; Ivanova, M. G.; Verger, R.; Panaiotov, I. Methods
Enzymol. 1997, 286, 263.
(23) Verger, R.; de Haas,G. H. Chem. Phys. Lipids 1973, 10, 127.
(14) Simons, J.-W. F. A.; Cox, R. C.; Egmond, M. R.; Verheij H. M.
Biochemistry 1999, 38, 6346.
Org. Lett., Vol. 2, No. 3, 2000
349

it is comparable to the R50 values recently reported for chiral
acylglycerol analogues belonging to the phosphonate type
inhibitors.24 However, more potent alkylphosphonate inhibi-
tors have been synthesized recently by Cavalier et al.25
amino alcohol backbone. The methology developed in this
paper allows for the synthesis of chiral inhibitors bearing
various saturated or unsaturated alkyl chains.
Acknowledgment. This work was supported by the
European Community (BIO2-CT943041), the Greek Secre-
tariat for Research and Technology, and the French Foreign
Minister (PLATON program 98054).
In conclusion, the R-keto amide group seems to be a
promising approach for the development of novel lipase
inhibitors after its incorporation into an optimized lipophilic
Supporting Information Available: Characterization
data for compounds 2-8. This material is available free of
charge via the Internet at http://pubs.acs.org.
(24) Marguet, F.; Douchet, I.; Cavalier, J.-F.; Buono, G.; Verger, R.
Colloids Surf., B 1999, 13, 37.
(25) (a) Cavalier, J.-F.; Ransac, S.; Verger, R.; Buono, G. Biochemistry
1995, 34, 2751. (b) Cavalier, J.-F.; Ransac, S.; Verger, R.; Buono, G. Chem.
Phys. Lipids 1999, 100, 3.
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