A cyclopent-2-enecarbonyl group mimics proline at the P2 position of prolyl oligopeptidase inhibitors

Article abstract of DOI:10.1021/jm049503w

With the aim to replace the natural amino acid proline by a proline mimetic structure, a cyclopent-2-enecarbonyl moiety was studied at the P2 position of prolyl oligopeptidase (POP) inhibitors. The cyclopent-2-enecarbonyl moiety proved to be an excellent proline mimetic at the P2 position of POP inhibitors. The replacement is particularly useful when increased lipophilicity is needed.

Full text of DOI:10.1021/jm049503w

J. Med. Chem. 2004, 47, 5605-5607
5605
environment.13 It has also recently been reported that
the inhibiton of the POP of Trypanosoma cruzi could
be a therapeutic target.14-16
A Cyclopent-2-enecarbonyl Group Mimics
Proline at the P2 Position of Prolyl
Oligopeptidase Inhibitors
L-Proline is the preferred amino acid at the P2
position of POP inhibitors. Only a few successful re-
placements of this proline have been reported.17-20 The
L-prolyl residue has been replaced by groups that
contain a five-membered carbon ring, such as a cyclo-
pentanecarbonyl group and a cyclopent-1-enecarbonyl
group, but the resulting compounds had a decreased
inhibitory activity.21,22 In the present study, the pyrro-
lidine ring was successfully replaced by a cyclopent-2-
enecarbonyl group. The same replacement has previ-
ously been studied at the P2 position of thrombin
inhibitors with moderate results.23
Elina M. Jarho,*,† Jarkko I. Vena¨la¨inen,‡
Juhani Huuskonen,†,# Johannes A. M. Christiaans,†,§
J. Arturo Garcia-Horsman,‡ Markus M. Forsberg,‡
Tomi Ja¨rvinen,† Jukka Gynther,†
Pekka T. Ma¨nnisto¨,‡ and Erik A. A. Walle´n†
Department of Pharmaceutical Chemistry and
Department of Pharmacology and Toxicology, University of
Kuopio, P.O. Box 1627, FI-70211 Kuopio, Finland
Received June 23, 2004
The synthetic routes are presented in Scheme 1. (()-
2-Formyl-cyclopent-2-enecarboxylic acid (1) and (()-
cyclopent-2-ene-1,2-dicarboxylic acid 1-methyl ester (2)
were synthesized as described in the literature with
small modifications.23,24 DCC and HOBt were used to
couple benzylamine with 2, and the methyl ester of the
obtained product was hydrolyzed with LiOH to obtain
2-benzylcarbamoylcyclopent-2-enecarboxylic acid (3).
Reaction of 1 with (3-phenylpropyl)magnesium bromide
(prepared in situ) produced a secondary alcohol, which
was oxidized with oxalyl chloride/DMSO to obtain (()-
2-(4-phenylbutyryl)cyclopent-2-enecarboxylic acid (4).
Compounds 3 and 4 were activated with pivaloyl
chloride and reacted with pyrrolidine to yield end
products 9 and 8, respectively. The enantiomers of 9 and
8 were not separated. Compound 3 was activated with
pivaloyl chloride and reacted with 2(S)-(acetoxyacetyl)-
pyrrolidine. The acetyl group was hydrolyzed with
K2CO3 in water and methanol, yielding end product 5.
The diastereomers of 5 were separated on silica support.
The separated compounds were confirmed to be dia-
stereomers by NMR. Compound 4 was activated with
pivaloyl chloride and reacted with L-proline methyl
ester. The methyl ester group was hydrolyzed with
LiOH in water and methanol. Thus, the obtained free
carboxylic acid was activated with ethyl chloroformate
and treated with 25% aqueous NH3. The carboxylic acid
amide was then dehydrated with trifluoroacetic anhy-
dride to yield end product 7. The diastereomers of 7
were separated on silica support. The separated com-
pounds were confirmed to be diastereomers by NMR.
Compound 10 was prepared from cyclopent-1-ene-1,2-
dicarboxylic anhydride and benzylamine. Compound 10
was then activated with pivaloyl chloride and reacted
with pyrrolidine to yield reference compound 11. Refer-
ence compounds 6, SUAM-1221,25-27 and JTP-481928
were prepared as described in the literature.
Abstract: With the aim to replace the natural amino acid
proline by a proline mimetic structure, a cyclopent-2-enecar-
bonyl moiety was studied at the P2 position of prolyl oligopep-
tidase (POP) inhibitors. The cyclopent-2-enecarbonyl moiety
proved to be an excellent proline mimetic at the P2 position
of POP inhibitors. The replacement is particularly useful when
increased lipophilicity is needed.
Among the 20 common amino acids, proline has a
unique role because of its cyclic structure. It causes
constraints on a polypeptide backbone and can protect
a peptide from nonspecific hydrolysis by peptidases. It
may also serve as a regulatory structure in maturation
and degradation of peptides and in protein folding.
Thus, it is not surprising that proline is often highly
conserved in peptides and specific peptidases have
evolved to hydrolyze bonds before and after proline
residues.1-3
Several protease inhibitors are recently under inves-
tigation as potential drugs. Many of them have a
peptide-like structure, and they suffer from poor bio-
availability and susceptibility to nonspecific degrada-
tion. These problems have led to the development of
amino acid mimetics that could be used to replace the
natural amino acids. The unique structure of proline
makes it challenging to replace.4,5
Prolyl oligopeptidase (POP, EC 3.4.21.26) is a proline-
specific serine peptidase that preferentially hydrolyzes
peptides at the carboxyl side of proline. POP has been
related to cognitive disorders because several cognition-
enhancing neuropeptides are substrates of POP.6-8 The
inhibitors of POP have been shown to increase the levels
of these peptides in the brain9 and to prevent scopola-
mine-induced amnesia in rats.10 They have also been
shown to enhance cognition in a model of early Parkin-
sonism in monkeys.11 Furthermore, expression of the
POP gene is increased in the hypothalamus and the
cortex of old rats12 and decreased in an enriched
The partition coefficients (P) were determined in a
1-octanol-phosphate buffer system with the traditional
shake-flask method. The log P values are shown in
Table 1.
The inhibitory activities of the novel and reference
compounds are shown in Table 1. The tight-binding
inhibitors are highly potent compounds with the IC50
values in the same range as the enzyme concentration
* To whom correspondence should be addressed. Phone: +358-17-
162460. Fax: +358-17-162456. E-mail: Elina.Jarho@uku.fi.
† Department of Pharmaceutical Chemistry.
‡ Department of Pharmacology and Toxicology.
# Present address: Department of Chemistry, University of
Jyva¨skyla¨, P.O. Box 35, FI-40014 Jyva¨skyla¨, Finland.
§ Present address: Altana Pharma bv, P.O. Box 31, 2130 AA
Hoofddorp, The Netherlands.
10.1021/jm049503w CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/01/2004

5606 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Letters
Scheme 1a
a Reagents and conditions: (i) CH(OCH3)3, p-TsOH; (ii) resorcinol, NaH2PO4‚H2O, NaClO2/t-BuOH, H2O; (iii) benzylamine, HOBt, Et3N,
DCC/CH3CN, 0-20 °C; (iv) LiOH/MeOH, H2O; (v) (1) Et3N, (CH3)3CCOCl/DCM, 0 °C, (2) Et3N, an amine or a salt of an amine;
(vi) K2CO3/MeOH, H2O, 0-20 °C; (vii) (1) Mg, I2, 1-bromo-3-phenylpropane/Et2O, (2) compound 1; (viii) (1) oxalyl chloride, DMSO/DCM,
-78 °C, (2) Et3N; (ix) (1) Et3N, C2H5OCOCl/THF, -10 °C, (2) 25% aqueous NH3/THF, room temperature; (x) Et3N, trifluoroacetic anhydride/
THF, 0-20 °C; (xi) benzylamine/THF, 0-20 °C.
Table 1. Structures, Inhibitory Activities (95% Confidence Intervals), and log P Values of the Reference and Novel Compounds
a The IC50 values were determined against POP of porcine brain homogenate. b The Ki values were determined against purified porcine
POP for those tight-binding inhibitors that had the IC50 value in the subnanomolar range. c The chiral center marked with the asterisk
was assumed to have R configuration. The absolute configuration was not verified. d The chiral center marked with the asterisk was
assumed to have S configuration. The absolute configuration was not verified.
in the in vitro assay. The Ki values were determined
for tight-binding inhibitors to obtain the accurate
inhibitory potencies.
oligopeptidase. Normally, these electrophilic groups
increase the inhibitory activity over 10-fold compared
to an unsubstituted pyrrolidine. Compound 8 has the
unsubstituted pyrrolidine at the P1 position. It was
tested as a mixture of enantiomers, and despite that,
the IC50 value was as low as 1.30 nM. On the basis of
the activity differences between 5a and 5b and between
7a and 7b, it can be assumed that the more active
enantiomer is even more potent than the reference
compound SUAM-1221.
POP inhibitors, which contain a cyclopent-1-enecar-
bonyl group at the P2 position, have been published in
the literature.22 One such inhibitor (11) and the corre-
sponding cyclopent-2-enecarbonyl analogue (9) were
synthesized. As expected, 11 was less active, with an
IC50 of 230 nM, than 9 with an IC50 of 9 nM. The
structure of the cyclopent-1-enecarbonyl group is not a
good proline mimetic and is therefore moderately active.
The cyclopent-2-enecarbonyl group mimics a proline
residue rather well, as observed by the inhibitory
activity.
The activities of diastereomers 5a and 5b differed
considerably from each other. The IC50 value of 5a was
0.3 nM, while the IC50 value of 5b was 52 nM. At the
P2 position L-amino acids have repeatedly caused better
activities than D-amino acids.25,29,30 Consequently, the
configurations of 5a and 5b were assigned assuming
that the more active diastereomer 5a has the L-proline
mimetic at the P2 position. The configurations were not
verified. Diastereomer 5a was almost equipotent with
reference compound JTP-4819.
Compound 7a was equipotent with reference com-
pound 6. It had a Ki value of 0.03 nM and was also
assumed to have the L-proline mimetic at the P2
position. The absolute configuration was not verified.
The other diastereomer 7b was less active with an IC50
of 5 nM.
The 2(S)-hydroxyacetyl derivative 5a and 2(S)-cyano
derivative 7a are tight-binding inhibitors of prolyl

Letters
To study the effect of the cyclopent-2-enecarbonyl
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5607
(6) Koida, M.; Walter, R. Post-proline cleaving enzyme. Purification
of this endopeptidase by affinity chromatography. J. Biol. Chem.
1976, 251, 7593-7599.
(7) Walter, R. Partial purification and characterization of post-
proline cleaving enzyme: enzymatic inactivation of neurohypo-
physeal hormones by kidney preparations of various species.
Biochim. Biophys. Acta 1976, 422, 138-158.
(8) Wilk, S. Prolyl endopeptidase. Life Sci. 1983, 33, 2149-2157.
(9) Toide, K.; Fujiwara, T.; Iwamoto, Y.; Shinoda, M.; Okamiya, K.;
et al. Effect of a novel prolyl endopeptidase inhibitor, JTP-4819,
on neuropeptide metabolism in the rat brain. Naunyn-Schmiede-
berg’s Arch. Pharmacol. 1996, 353, 355-362.
(10) Yoshimoto, T.; Kado, K.; Matsubara, F.; Koriyama, N.; Kaneto,
H.; et al. Specific inhibitors for prolyl endopeptidase and their
anti-amnesic effect. J. Pharmacobiodyn. 1987, 10, 730-735.
(11) Schneider, J. S.; Giardiniere, M.; Morain, P. Effects of the prolyl
endopeptidase inhibitor S 17092 on cognitive deficits in chronic
low dose MPTP-treated monkeys. Neuropsychopharmacology
2002, 26, 176-182.
(12) Jiang, C. H.; Tsien, J. Z.; Schultz, P. G.; Hu, Y. The effects of
aging on gene expression in the hypothalamus and cortex of
mice. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1930-1934.
(13) Rampon, C.; Jiang, C. H.; Dong, H.; Tang, Y. P.; Lockhart, D.
J.; et al. Effects of environmental enrichment on gene expression
in the brain. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12880-
12884.
(14) Grellier, P.; Vendeville, S.; Joyeau, R.; Bastos, I. M.; Drobecq,
H.; et al. Trypanosoma cruzi prolyl oligopeptidase Tc80 is
involved in nonphagocytic mammalian cell invasion by trypo-
mastigotes. J. Biol. Chem. 2001, 276, 47078-47086.
(15) Bal, G.; Van der Veken, P.; Antonov, D.; Lambeir, A. M.; Grellier,
P.; et al. Prolylisoxazoles: potent inhibitors of prolyloligopepti-
dase with antitrypanosomal activity. Bioorg. Med. Chem. Lett.
2003, 13, 2875-2878.
(16) Vendeville, S.; Goossens, F.; Debreu-Fontaine, M. A.; Landry,
V.; Davioud-Charvet, E.; et al. Comparison of the inhibition of
human and Trypanosoma cruzi prolyl endopeptidases. Bioorg.
Med. Chem. 2002, 10, 1719-1729.
(17) Tsuru, D.; Yoshimoto, T.; Koriyama, N.; Furukawa, S. Thiazo-
lidine derivatives as potent inhibitors specific for prolyl endopep-
tidase. J. Biochem. (Tokyo) 1988, 104, 580-586.
moiety on the lipophilicity, the log P values were
determined for a selection of compounds (Table 1). The
replacement of the prolyl moiety by the cyclopent-2-
enecarbonyl moiety changed the urea group of JTP-4819
to the amide group of 5a. This change increased the
log P value by 0.4 units. The change of the amide group
of 6 to the ketone group of 7a increased the log P value
by 0.8 units. On the basis of these results, it seems that
the replacement of a proline with a cyclopent-2-enecar-
bonyl group increases the log P value but the magnitude
depends on the substituents.
The replacement of the L-prolyl residue by the cyclo-
pent-2-enecarbonyl group introduced an R,â-unsatur-
ated ketone or amide to the novel compounds. The R,â-
unsaturated ketones gave valuable information about
the structure-activity relationships, but their stability
needs further evaluation because of the activated double
bond. However, the R,â-unsaturated amides are not as
reactive because of the electron-donating effect of the
nitrogen atom.
The L-prolyl moiety was replaced by the cyclopent-2-
enecarbonyl structure at the P2 position of prolyl
oligopeptidase inhibitors. In all cases, the cyclopent-2-
enecarbonyl analogue proved to be equipotent to the
reference compound. Furthermore, this replacement
increased the lipophilicity of the compounds. This study
shows that a cyclopent-2-enecarbonyl structure can be
used to replace a prolyl residue at the P2 position of
POP inhibitors.
(18) Barelli, H.; Petit, A.; Hirsch, E.; Wilk, S.; De Nanteuil, G.; et al.
S 17092-1, a highly potent, specific and cell permeant inhibitor
of human proline endopeptidase. Biochem. Biophys. Res. Com-
mun. 1999, 257, 657-661.
Acknowledgment. We thank Mrs. Tiina Koivunen,
Mrs. Jaana Leskinen, and Mrs. Pa¨ivi Sutinen for their
excellent technical assistance and Dr. Seppo Auriola for
performing the ESI-MS analyses. We also thank Profes-
sor Antti Poso for discussions on the 3D structure of
the POP enzyme and Professor Jouko Vepsa¨la¨inen for
his valuable remarks on the manuscript. We also
acknowledge National Technology Agency in Finland,
Orion Pharma Oy, Finnish Cultural Foundationof North-
ern Savo, and the Academy of Finland for financial
support.
(19) De Nanteuil, G. U.S. Patent 5506237, 1996.
(20) Walle´n, E. A.; Christiaans, J. A.; Saarinen, T. J.; Jarho, E. M.;
Forsberg, M. M.; et al. Conformationally rigid N-acyl-5-alkyl-L-
prolyl-pyrrolidines as prolyl oligopeptidase inhibitors. Bioorg.
Med. Chem. 2003, 11, 3611-3619.
(21) Ohno, H. European Patent 0268190, 1988.
(22) Faraci, S. World Patent 9118891, 1991.
(23) No¨teberg, D.; Brånalt, J.; Kvarnstro¨m, I.; Linschoten, M.; Musil,
D.; et al. New proline mimetics: synthesis of thrombin inhibitors
incorporating cyclopentane- and cyclopentenedicarboxylic acid
templates in the P2 position. Binding conformation investigated
by X-ray crystallography. J. Med. Chem. 2000, 43, 1705-1713.
(24) Stevens, H. C.; Rinehart, J. K.; Lavanish, J. M.; Trenta, G. M.
The hydrolysis of 7,7-dichlorobicyclo[3.2.0]hept-2-en-6-one. J.
Org. Chem. 1971, 36, 2780-2784.
(25) Arai, H.; Nishioka, H.; Niwa, S.; Yamanaka, T.; Tanaka, Y.; et
al. Synthesis of prolyl endopeptidase inhibitors and evaluation
of their structure-activity relationships: in vitro inhibition of
prolyl endopeptidase from canine brain. Chem. Pharm. Bull.
(Tokyo) 1993, 41, 1583-1588.
Supporting Information Available: Experimental de-
tails and analytical data. This material is available free of
charge via the Internet at http://pubs.acs.org.
Note Added after ASAP Posting. This manuscript
was released ASAP on 10/1/2004 with an author missing
from the byline. The correct version was posted on
10/11/2004.
(26) Saito, M.; Hashimoto, M.; Kawaguchi, N.; Shibata, H.; Fukami,
H.; et al. Synthesis and inhibitory activity of acyl-peptidyl-
pyrrolidine derivatives toward post-proline cleaving enzyme; a
study of subsite specificity. J. Enzyme Inhib. 1991, 5, 51-75.
(27) Houssin, R.; Bernier, J.-L.; He´nichart, J.-P. A convenient and
general method for the preparation of tert-butoxycarbonylami-
noalkanenitriles and their conversion to mono-tert-butoxycar-
bonylalkanediamines. Synthesis 1988, 259-261.
References
(1) Brandts, J. F.; Halvorson, H. R.; Brennan, M. Consideration of
the possibility that the slow step in protein denaturation
reactions is due to cis-trans isomerism of proline residues.
Biochemistry 1975, 14, 4953-4963.
(28) Kobayashi, K. U.S. Patent 5536737, 1996.
(2) Vanhoof, G.; Goossens, F.; De Meester, I.; Hendriks, D.; Scharpe,
S. Proline motifs in peptides and their biological processing.
FASEB J. 1995, 9, 736-744.
(29) Portevin, B.; Benoist, A.; Remond, G.; Herve, Y.; Vincent, M.; et
al. New prolyl endopeptidase inhibitors: in vitro and in vivo
activities of azabicyclo[2.2.2]octane, azabicyclo[2.2.1]heptane,
and perhydroindole derivatives. J. Med. Chem. 1996, 39, 2379-
2391.
(3) Yaron, A.; Naider, F. Proline-dependent structural and biological
properties of peptides and proteins. Crit. Rev. Biochem. Mol. Biol.
1993, 28, 31-81.
(30) Yoshimoto, T.; Tsuru, D.; Yamamoto, N.; Ikezawa, R.; Furukawa,
S. Structure activity relationship of inhibitors specific for prolyl
endopeptidase. Agric. Biol. Chem. 1991, 55, 37-43.
(4) Leung, D.; Abbenante, G.; Fairlie, D. P. Protease inhibitors:
current status and future prospects. J. Med. Chem. 2000, 43,
305-341.
(5) De Nanteuil, G.; Portevin, B.; Lepagnol, J. Prolyl endopeptidase
inhibitors: a new class of memory enhancing drugs. Drugs
Future 1998, 23, 167-179.
JM049503W