Conformationally rigid N-acyl-5-alkyl-L-prolyl-pyrrolidines as prolyl oligopeptidase inhibitors

Article abstract of DOI:10.1016/S0968-0896(03)00363-8

In the N-acyl-L-prolyl-pyrrolidine type of prolyl oligopeptidase inhibitors the L-prolyl group was replaced by different 5-alkyl-L-prolyl groups, resulting in a series of N-acyl-5-alkyl-L-prolyl-pyrrolidines. Since N-amides of 5-alkyl-L-prolines are conformationally more rigid than those of L-proline, the main objective was to make more rigid prolyl oligopeptidase inhibitors. In the series of compounds where the N-acyl group was a Boc group, the 5(R)-tert-butyl group increased the potency strongly. A similar effect was not observed for the 5(S)-tert-butyl group. In the series of compounds where the N-acyl group was a 4-phenylbutanoyl group, the 5(R)-tert-butyl, 5(R)-methyl and 5(S)-methyl groups did not have an effect on the potency [the 5(S)-tert-butyl group was not tested in this series]. As an additional effect, the 5-tert-butyl groups increased the log P of the compounds 1.5 log units, which might be beneficial when targeting the compounds to the brain.

Full text of DOI:10.1016/S0968-0896(03)00363-8

Bioorganic & Medicinal Chemistry 11 (2003) 3611–3619
Conformationally Rigid N-Acyl-5-alkyl-L-prolyl-pyrrolidines as
Prolyl Oligopeptidase Inhibitors
Erik A. A. Wallen,a,* Johannes A. M. Christiaans,a,y Taija J. Saarinen,a,{ Elina M. Jarho,a
Markus M. Forsberg,b Jarkko I. Venalainen,b Pekka T. Mannistob,c and Jukka Gynthera,c
aDepartment of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland
bDepartment of Pharmacology and Toxicology, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland
cFinncovery Ltd., Kuopio, Finland
Received 4 April 2003; accepted 28 May 2003
Abstract—In the N-acyl-l-prolyl-pyrrolidine type of prolyl oligopeptidase inhibitors the l-prolyl group was replaced by different
5-alkyl-l-prolyl groups, resulting in a series of N-acyl-5-alkyl-l-prolyl-pyrrolidines. Since N-amides of 5-alkyl-l-prolines are con-
formationally more rigid than those of l-proline, the main objective was to make more rigid prolyl oligopeptidase inhibitors. In the
series of compounds where the N-acyl group was a Boc group, the 5(R)-tert-butyl group increased the potency strongly. A similar
effect was not observed for the 5(S)-tert-butyl group. In the series of compounds where the N-acyl group was a 4-phenylbutanoyl
group, the 5(R)-tert-butyl, 5(R)-methyl and 5(S)-methyl groups did not have an effect on the potency [the 5(S)-tert-butyl group was
not tested in this series]. As an additional effect, the 5-tert-butyl groups increased the log Pof the compounds 1.5 log units, which
might be beneficial when targeting the compounds to the brain.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
thought to reflect the degree of neuronal damage.4
However, it was recently reported that the expression of
POP gene was increased many-fold in the hypothalamus
and the cortex in aged rats.5 Furthermore, the expres-
sion of POP gene was decreased in rats in an enriched
environment that enhances learning and memory.6 POP
inhibitors have been reported to increase the con-
centrations of substance P, vasopressin and thyroliberin
in the brain,7,8 which is suggested to be beneficial in
patients with cognitive disturbances. Indeed, POP inhi-
bitors have been shown to reverse scopolamine-induced
amnesia in rats and to improve cognition in MPTP-
treated monkeys.9À11
The serine protease prolyl oligopeptidase (POP, pre-
viously called prolyl endopeptidase or post-proline cleav-
ing enzyme, EC 3.4.21.26) is a large enzyme of the size of
80 kDa, that preferentially hydrolyses proline-containing
oligopeptides at the carboxyl side of a prolyl residue.1
Several substrates of POP, such as substance P, vaso-
pressin, neurotensin and thyroliberin, are implicated in
learning and memory.2 Furthermore, low levels of sub-
stance Pare characteristic in the brains of Alzheimer
patients and administration of substance Pis able to
block b amyloid-induced neurotoxicity.3 There is no
firm evidence of increased POP activity in Alzheimer
patients, since rather low POP activities have been cor-
related with the severity of Alzheimer’s disease, which is
In addition to human POP, a parasitic POP has recently
also been recognized as a therapeutic target.12 Trypano-
soma cruzi, which is the causative agent in Chagas dis-
ease, contains a POP which is linked to the invasion of
mammalian cells by trypomastigotes.
*Corresponding author at present address: Department of Pharmaco-
chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV
Amsterdam, The Netherlands.; E-mail: wallen@uku.fi
yPresent address: Altana Parma bv, PO Box 31, 2130 AA Hoofddorp,
The Netherlands.
{Present address: Orion Pharma, PO Box 1780, FIN-70701 Kuopio,
Finland.
Many low molecular weight POP inhibitors are based
on an N-acyl-L-prolyl-pyrrolidine structure.13 The
l-proline moiety at the P2 site has been very challenging
to replace. Only a few successful replacements that
increased the potency have been reported (Chart 1).
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00363-8

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3612
Synthesis
The (5R)- and (5S)-diastereomers of the N-Boc-5-alkyl-
l-proline esters 2a–d were synthesized according to dia-
stereoselective synthetic procedures (Scheme 1). The N-
Boc-5-alkyl-l-proline esters 2a–d were used as starting
materials for the synthesis of the compounds 3b, 3c, 3e–i,
3k, 3l and 3n.
Compound 2a was synthesized according to a reported
procedure.18 However, the amino group of the starting
material was protected with a trityl group instead of a
phenylfluorenyl group according to a general proce-
dure.22 This modification was made because the trityl
group is a less expensive protecting group. Compound
2b was also synthesized according to a reported proce-
dure.23 This procedure was modified slightly by per-
forming the last reductive amination step with the
methyl ester instead of the carboxylic acid. This modifi-
cation was made because the methyl esters were
assumed to be easier to purify by flash chromatography.
Synthesis of compounds 2c and 2d followed reported
procedures.24,25
Chart 1.
The well known compound SUAM-1221 1a has an
l-prolyl moiety at the P2 site. Compound 1b has a
l-thioproline moiety at the P2 site, which increased the
potency 2–3 times against bovine and canine POP as
compared to SUAM-1221 1a.14,15 Compounds 1c–e
have bicyclic l-proline analogues at the P2 site, which
increased the potency over 10 times against Flavo-
bacterium meningoseptum POP as compared to com-
pound 1b.16 Compound 1f was not so active against
human POP, but it had an increased activity against the
POP of T. cruzi.12 This compound also demonstrates
that a modified l-proline moiety at the P2 site can be
used to improve the selectivity between POP of different
species.
The N-Boc-5-alkyl-l-proline esters 2a–d were then used
as starting materials for the latter part of the synthetic
route for the synthesis of compounds 3b, 3c, 3e–i, 3k, 3l
and 3n. The ester protecting group was hydrolyzed with
LiOH in water-methanol or alternatively with KOH in
water–ethanol. The pyrrolidinyl and the 2(S)-(acetoxy-
acetyl)pyrrolidinyl groups were coupled to the car-
boxylic acid group by activation with pivaloyl chloride
in the presence of triethyl amine in dichloromethane [in
the case of 2(S)-(acetoxyacetyl)pyrrolidinyl the acetyl
group was removed subsequently with K2CO3 in water–
methanol in order to obtain compounds 3k and 3l]. The
Boc protecting group was removed with trifluoroacetic
acid in dichloromethane and the resulting amine was
reacted with an acid chloride, an anhydride, an isocya-
nate or alternatively by activating the carboxylic acid
with pivaloyl chloride in the presence of triethyl amine.
The order of these reactions was not always the same. A
general observation from these reactions was that com-
pound 2c was less reactive than compounds 2a, 2b and 2d.
Due to the cyclic structure of proline, N-amide bonds of
proline have energetically similar trans and cis con-
formations. The ratio can be shifted towards the cis
conformation by adding a bulky 5-alkyl substituent to
the proline ring.17À21 A bulky 5-alkyl substituent affects
the N-amide bond of proline by making it
conformationally more rigid.
A series of N-acyl-5-alkyl-l-prolyl-pyrrolidines 3 were
synthesized in order to study the effect of the 5-sub-
stituted l-proline moiety at the P2 site of a POP inhi-
bitor. The tert-butyl and methyl groups were selected as
the 5-substituents, since they are the two opposite
extremes in bulkiness of alkyl groups. The 5-substituent
introduces a new stereocentre, which has to be con-
trolled in the synthesis. In order to establish the effect of
the 5-substituent on the log Pvalue, it was determined
for selected compounds.
Since the reactions were not completely diastereo-
selective, the major diastereomer was purified by flash
chromatography at some stage of the synthetic route.
The diastereomeric purity was verified by NMR.
The reference compounds 3a, 3d, 3j and 3m were syn-
thesized according to reported procedures (not included
in Scheme 1).14,15,26,27
In Vitro Assay for POP Activity
The inhibitory effect of the novel compounds on pig
brain POP activity was determined according to an
earlier described method.28 Suc-Gly-Pro-7-amino-4-
methylcoumarin was used as substrate and the forma-
tion of 7-amino-4-methylcoumarin was determined
fluorometrically with a microplate fluorescence reader.

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3613
Results and Discussion
indication which stereoisomer at the 5-position was
favoured. The inhibitory activities of compounds Boc-l-
prolyl-pyrrolidine 3a, Boc-5(R)-tert-butyl-l-prolyl-pyr-
rolidine 3b and Boc-5(S)-tert-butyl-L-prolyl-pyrrolidine
3c were compared. Interestingly, 5(R)-tert-butyl-l-pro-
lyl-pyrrolidine 3b, with an IC50 value of 2.2 nM, was 13
times more active than Boc-l-prolyl-pyrrolidine 3a,
The in vitro activities of the compounds of series 3
against pig brain POP were determined (Table 1). The
combined effect of a bulky Boc group at the P3 site and
a bulky tert-butyl substituent at the 5-position of the l-
proline group at the P2 site was expected to give an
Scheme 1. (a) (1) Me3SiCl/acetonitrile, DCM; (2) Et3N, TrCl; (3) MeOH; (b) (1) LiN(SiMe3)2/THF; (2) pivaloyl chloride; (c) NaOH, water/EtOH;
(d) CH2N2/THF; (e) H2, Pd/C, Boc2O/MeOH; (f) paraformaldehyde, p-TsOH/benzene; (g) oxalyl chloride, DMF/DCM; (h) CH2N2/diethylether,
benzene; (i) 48% HI; (j) K2CO3, MeOH, water; (k) EtI, K2CO3/DMF; (l) Boc2O, DMAP, Et3N/DCM; (m) LiBEt3H/THF; (n) p-TsOH, MeOH;
0
0
.
.
.
.
(o) R =t-Bu: (t-BuLi, CuBr Me2S, BF3 OEt2)/THF; R =Me: (MeMgBr, CuBr Me2S, BF3 OEt2)/diethyl ether; (p) LiOH, water/MeOH (or alter-
natively: KOH, water/EtOH); (q) (1) pivaloyl chloride, Et3N/DCM; (2) pyrrolidine, Et3N; (r) TFA/DCM; (s) Ph(CH2)3COCl or Ac2O, Et3N/DCM;
(t) PhCH2NCO, Et3N/DMF; (u) (3-(2(S)-benzoyl-pyrrolidine-1-carbonyl)-benzoic acid, pivaloyl chloride, Et3N/ DCM), Et3N/ DCM); (v) (1) piva-
loyl chloride, Et3N/DCM; (2) 2(S)-(acetoxyacetyl)pyrrolidine, Et3N; (w) K2CO3, water/MeOH.
Table 1. The in vitro activities (with 95% confidence intervals given in parentheses) against POP from pig brain and log P values for compounds of
series 3
Compd
RCO
R0
R00
IC50 (nM)
(22–38)
Ki (nM)
Log P
3a
3b
Boc
Boc
Boc
H
H
H
H
H
H
H
H
H
29
2.2
5(R)-t-Bu
5(S)-t-Bu
H
5(R)-t-Bu
5(R)-Me
5(S)-Me
5(R)-t-Bu
5(R)-t-Bu
H
(1.9–2.5)
(7.0–12)
(1.9–2.5)
3c
3d (also 1a)
3e
3f
3g
3h
3i
3j
3k
3l
9.2
2.2
1.2
0.71
1.4
2.0
4-Phenylbutanoyl
4-Phenylbutanoyl
4-Phenylbutanoyl
4-Phenylbutanoyl
Benzylcarbamoyl
Acetyl
1.8
3.3
(1.0–1.4)
(0.36–1.4)
(0.94–2.3)
(0.87–4.4)
(3800–13,000)
(0.14–0.38)
(0.17–0.41)
(0.08–0.29)
(15–22)
H
7100
4-Phenylbutanoyl
4-Phenylbutanoyl
4-Phenylbutanoyl
3-(2(S)-Benzoyl-pyrrolidine-1-
carbonyl)-benzoyl
3-(2(S)-Benzoyl-pyrrolidine-1-
carbonyl)-benzoyl
COCH2OH
COCH2OH
COCH2OH
H
0.24
0.26
0.15
0.018
0.015
0.026
(0.013–0.023)
(0.0006–0.029)
(0.015–0.038)
5(R)-t-Bu
5(R)-Me
H
2.3
3m
18
3n
5(R)-t-Bu
H
460
(370–570)

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3614
with an IC50 value of 29 nM, and 3 times more active
than Boc-5(S)-tert-butyl-l-prolyl-pyrrolidine 3c, with
an IC50 value of 9.2 nM. This result indicated that the
5(R)-tert-butyl-l-prolyl group was preferred over 5(S)-
tert-butyl-l-prolyl group and that the 5(R)-tert-butyl
group increased the potency of this type of compounds.
taken into account when measuring the inhibition
parameters. When tight binding inhibition was mea-
sured utilizing the Morrison equation (see Experimental
part), compounds 3j, 3k and 3l were found to be equi-
potent POP inhibitors with Ki values of 0.018, 0.015 and
0.026 nM, respectively.
This result is likely to reflect that the conformation of a
bulky acyl group at the P3 site, such as the Boc group, is
significantly altered by the bulky 5-tert-butyl substituent
of the l-prolyl group at the P2 site.
Recently, a new type of POP inhibitors based on a iso-
phthalic acid bis(l-prolyl-pyrrolidine) amide structure
was presented.29 These compounds were developed fur-
ther, resulting in compound 3m with an IC50 value of 18
nM.27 Incorporating the 5(R)-tert-butyl-l-proline group
into this structure resulted in compound 3n with an IC50
value of 460 nM. It was evident that a bulky 5-sub-
stituent of the l-prolyl group was not beneficial in this
type of inhibitors.
The combination of the 4-phenylbutanoyl group at the
P3 site and the 5-substituent of the l-prolyl group at the
P2 site was studied next. 4-Phenylbutanoyl-5(R)-tert-
butyl-l-prolyl-pyrrolidine 3e, with an IC50 value of 1.2
nM, did not show a noticeable increase of the inhibitory
activity as compared to SUAM-1221 (4-phenylbuta-
noyl-l-prolyl-pyrrolidine) 3d, with an IC50 value of 2.2
nM. However, extending this series of compounds to
the 5-methyl substituted l-prolyl groups at the P2 site,
showed that 4-phenylbutanoyl-5(R)-methyl-l-prolyl-
pyrrolidine 3f, with an IC50 value of 0.71 nM, was
slightly more active, while 4-phenylbutanoyl-5(S)-
methyl-l-prolyl-pyrrolidine 3g, with an IC50 value of 1.4
nM, was equipotent as compared to SUAM-1221 3d
and 4-phenylbutanoyl-5(R)-tert-butyl-l-prolyl-pyrroli-
dine 3e.
The log P values were determined for selected com-
pounds (Table 1). SUAM-1221 3d had a log P value of
1.8 and 4-phenylbutanoyl-5(R)-tert-butyl-l-prolyl-pyr-
rolidine 3e had a log P value of 3.3. The 5-tert-butyl
group increased the log P value by 1.5 log units. The
very potent 4-phenylbutanoyl-5(R)-tert-butyl-l-prolyl-
2(S)-(hydroxyacetyl)pyrrolidine 3k had a log P value of
2.3, which is very good for a compound that is targeted
to the brain.
Conclusions
The conformation of a flexible acyl group at the P3 site,
such as the 4-phenylbutanoyl group, is hardly affected
by the 5-substituent of the l-prolyl group at the P2 site,
and it can adopt the optimal conformation for binding
to the active site in all cases. The 5(S)-tert-butyl-l-prolyl
moiety was not tested in this group of compounds, since
it was expected to be less active based on the results with
a Boc group at the P3 site.
Introducing a bulky Boc group at the P3 site showed
that the 5(R)-tert-butyl-l-prolyl group is preferred over
the 5(S)-tert-butyl-l-prolyl group at the P2 site. The
inhibitory activity was improved noticeably by a 5(R)-
tert-butyl-l-prolyl group at the P2 site only for com-
pounds with a bulky Boc group at the P3 site. The con-
formation of a bulky Boc group at the P3 site was most
probably significantly altered by the bulky 5-tert-butyl
substituent of the l-prolyl moiety at the P2 site. A more
flexible 4-phenylbutanoyl group at the P3 site does not
seem to be affected by a 5(R)-tert-butyl substituent on
the adjacent l-prolyl group. The lipophilic acyl group at
the P3 site could not be mimicked by the 5-tert-butyl
substituent of the l-prolyl moiety at the P2 site. The 5-
tert-butyl substituent of the l-prolyl group at the P2 site
increases the log P by 1.5 log units. This is a significant
increase in lipophilicity and it may be necessary in order
to target the compounds better to the brain. Therefore,
these compounds are good candidates for further phar-
macological studies.
The acyl group at the P3 site was further modified.
Benzylcarbamoyl-5(R)-tert-butyl-l-prolyl-pyrrolidine
3h, with an IC50 value of 2.0 nM, was equipotent as
compared to 4-phenylbutanoyl-5(R)-tert-butyl-l-prolyl-
pyrrolidine 3e. Since the 5(R)-tert-butyl substituent of
the l-prolyl group at the P2 site is a rather large lipo-
philic group, it was thought to mimic the lipophilic acyl
group at the P3 site. However, this was not the case,
since acetyl-5(R)-tert-butyl-l-prolyl-pyrrolidine 3i, with
an IC50 value of 7100 nM, was almost inactive.
Replacing the pyrrolidinyl group at the P1 site by a 2(S)-
(hydroxyacetyl)pyrrolidinyl group increased the potency.
4-Phenylbutanoyl - 5(R) - tert -butyl- l -prolyl- 2(S) - (hy-
droxyacetyl)pyrrolidine 3k and 4-phenylbutanoyl-5(R)-
methyl-l-prolyl-2(S)-(hydroxyacetyl)pyrrolidine 3l, with
IC50 values of 0.26 and 0.15 nM, respectively, were
equipotent as compared to 4-phenylbutanoyl-l-prolyl-
2(S)-(hydroxyacetyl)pyrrolidine 3j, with an IC50 value of
0.24 nM. However, these values do not give reliable
information of the potencies of compounds 3j–l, since
they were found to be tight binding inhibitors of POP
(their IC50 values are of the same order of magnitude as
the enzyme concentration in the reaction mixture). In
this case the depletion of the free inhibitor has to be
Experimental
NMR spectra were recorded on a Bruker Avance 500
spectrometer (500.1 MHz for 1H and 125.8 MHz for
13C) or a Bruker AM 400 spectrometer (400.1 MHz for
1H and 100.6 MHz for 13C), CDCl3 was used as solvent
if not otherwise noted and chemical shifts are expressed
in ppm relative to tetramethylsilane as internal stan-
dard. Amides N-terminal to prolyl groups or proline
analogue groups have energetically similar cis and trans
isomers (also sometimes referred to as rotamers), which

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E. A. A. Wallen et al. / Bioorg. Med. Chem. 11 (2003) 3611–3619
3615
complicates the NMR spectra. Changing the solvent
from CDCl3 to acetone-d6 simplified the NMR spectra
for some compounds, for compound 3e the two isomers
had a ratio of 6:4 in CDCl3 and a ratio 8:2 in acetone-
d6. When the isomer ratio was 8:2 or larger, the minor
isomer could usually not be detected in the 13C NMR
spectrum.
N-Tr-L-glutamic acid 5-methyl ester. Prepared according
to a reported general procedure.22
Boc-5(R)-tert-butyl-L-proline methyl ester. Prepared
starting from N-Tr-l-glutamic acid 5-methyl ester.
according to a reported procedure,18 with the modifi-
cation that the Tr group was used instead of the 9-(9-
phenylfluorenyl) group to protect the amine. This pro-
cedure yields the (5R)-diastereomer as the major
product, which was isolated by flash chromatography.
Positive ion mass spectra were acquired with ESI-MS,
using a Finnegan MAT LCQ quadropole ion trap mass
spectrometer equipped with an ESI source. Combustion
analyses for CHN were measured on an EA1110
ThermoQuest CE Instruments elemental analysator.
Boc-5(R)-tert-butyl-L-proline. The methyl ester group of
Boc-5(R)-tert-butyl-l-proline methyl ester (1.14 g, 4.0
mmol) was hydrolyzed according to procedure B with
LiOH in water–MeOH. Yield 0.88 g (3.2 mmol, 80%).
All chemicals and solvents were of commercial quality and
were purified if necessary following standard procedures.
Boc-5(R)-tert-butyl-L-prolyl-pyrrolidine 3b. Boc-5(R)-
tert-butyl-l-proline (0.88 g, 3.2 mmol) and pyrrolidine
(0.27 mL, 3.2 mmol) were coupled according to proce-
dure A. Purification by flash chromatography, yield
Procedure A: General procedure for coupling an amine
to a carboxylic acid with pivaloyl chloride
1
Pivaloyl chloride (1.0 mmol) was added to a solution of
the carboxylic acid (1.0 mmol) and triethyl amine (1.1
mmol) in dichloromethane at 0 ꢀC. After 1 h triethyl
amine (1.1 mmol, or if the amine is in the form of a HCl
or trifluoroacetic acid salt then 3.3 mmol) and the amine
(1.0–1.1 mmol) was added, where after the reaction
mixture was allowed to react 3–20 h at rt. The di-
chloromethane solution was washed with 30% citric
acid, saturated NaCl and saturated NaHCO3. The di-
chloromethane phase was dried and evaporated.
0.87 g (2.7 mmol, 84%). H NMR: d 1.01 (s, 9H), 1.42
(s, 9H), 1.80–2.11 (m, 8H), 3.36–3.70 (m, 4H), 3.79 (d,
1H, J=7.9 Hz), 4.39 (br s, 1H).13C NMR: d 24.09,
26.35, 27.08, 27.59, 28.38, 28.85, 36.36, 45.96, 45.99,
61.00, 66.69, 79.60, 156.21, 171.15. ESI-MS: m/z 325
(M+H)+. Anal. (C18H32N2O3) calcd C: 66.63, H: 9.94,
N: 8.63; found C: 66.28, H: 9.95, N: 8.57.
Boc-5(S)-tert-butyl-L-proline ethyl ester. Prepared
according to a reported procedure.25 This procedure
yields the (5S)-diastereomer as the as the major product.
The product was purified without separating the
diastereomers by flash chromatography.
Procedure B: Procedure for hydrolyzing a methyl or
ethyl ester group
LiOH (or KOH) (1.5–6.0 mmol) and carboxylic acid
ester (1.0 mmol) were dissolved in a small volume of
water–MeOH (or water–EtOH in the case of KOH).
After the reaction was complete the solvent was evapo-
rated and water was added. The aqueous phase was
washed with dichloromethane or diethyl ether. The
aqueous phase was then made acidic with hydrochloric
acid and the product was extracted with dichloro-
methane or diethyl ether. The organic phase was dried
and evaporated.
Boc-5(S)-tert-butyl-L-proline. The ethyl ester group of
Boc-5(S)-tert-butyl-l-proline ethyl ester (0.51 g, 1.7
mmol) was hydrolyzed according to procedure B with
LiOH in water–MeOH. Yield 0.46 g (1.7 mmol, 100%).
Boc-5(S)-tert-butyl-L-prolyl-pyrrolidine 3c. Boc-5(S)-
tert-butyl-l-proline (0.46 g, 1.7 mmol) and pyrrolidine
(0.16 mL, 2.1 mmol) were coupled according to proce-
dure A. Purification by flash chromatography, yield
1
0.050 g (0.15 mmol, 9%). H NMR: d 0.90 (s, 9H), 1.37
(s, 6.3H), 1.46 (s, 2.7H), 1.70–2.23 (m, 8H), 3.35–3.70
(m, 4H), 3.91 (br s, 0.3H), 4.04 (d, 0.7H, J=7.8 Hz),
4.42 (d, 0.7H, J=7.8 Hz), 4.50 (br s, 0.3H). 13C NMR: d
24.19, 25.03, 26.33, 27.52, 28.24, 29.66, 36.89, 45.91,
46.06, 60.18, 66.25, 79.01, 155.79, 172.02. ESI-MS: m/z
Procedure C: Deprotecting a Boc protected amine
The Boc protected amine (1.0 mmol) was dissolved in
dichloromethane (5–10 mL) and trifluoroacetic acid (2–
4 mL) was added at 0 ꢀC. The reaction was stirred at
0 ꢀC for 2 h. The solvent was evaporated, yielding the
trifluoroacetic acid salt of the amine.
+
.
325 (M+H) . Anal. (C18H32N2O3 0.3H2O) calcd C:
65.54, H: 9.96, N: 8.49; found C: 65.29, H: 9.87, N: 8.99.
4-Phenylbutanoyl-5(R)-tert-butyl-L-prolyl-pyrrolidine 3e.
4-Phenylbutanoylchloride (prepared from 4-phenylbu-
tanoic acid (0.39 g, 2.4 mmol) and thionyl chloride (0.21
mL, 2.9 mmol)) was added to a solution of the 5(R)-
tert-butyl-l-prolyl-pyrrolidine trifluroacetic acid salt
(prepared from Boc-5(R)-tert-butyl-l-prolyl-pyrrolidine
(0.63 g, 1.9 mmol) according to procedure C) and tri-
ethyl amine (0.89 mL, 6.4 mmol) in dichloromethane at
0 ꢀC. The reaction mixture was stirred at rt for 3 h. The
dichloromethane phase was washed with 30% citric
acid, saturated NaCl and saturated NaHCO3. The
Procedure D: Hydrolysis of an O-acetyl group
K2CO3 (1.1 mmol) was added to a solution of O-acetyl
compound (1.0 mmol) in water–methanol (6 mL) at
0 ꢀC. The reaction was stirred 10 min at 0 ꢀC and then
50 min at rt. The solvent methanol was evaporated. Di-
chloromethane and saturated NaCl were added and the
phases were separated. The dichloromethane phase was
washed once with saturated NaCl. The dichloromethane
phase was dried and evaporated.

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E. A. A. Wallen et al. / Bioorg. Med. Chem. 11 (2003) 3611–3619
3616
dichloromethane phase was dried and evaporated. Puri-
fication by flash chromatography, yield 0.61 g (1.6
mmol, 84%) H NMR: d 1.00 (s, 9H), 1.65–2.47 (m,
ester (0.74 g, 2.6 mmol) was hydrolyzed according to
procedure B with KOH in water–EtOH. Yield 0.67 g
(2.4 mmol, 92%).
1
12H), 2.57–2.71 (m, 2H), 3.17–3.65 (m, 4H), 3.75–3.81
(m, 0.4H), 4.22 (t, 0.6H, J=9.0 Hz, 4.30 (d, 0.6H,
J=8.5 Hz), 4.59 (t, 0.4H, J=9.0 Hz), 7.15–7.28 (m,
5H). 13C NMR: d 23.90, 24.09, 25.92, 26.18, 26.34,
26.78, 27.41, 27.68, 27.93, 28.12, 29.60, 29.71, 33.07,
33.88, 35.12, 35.27, 36.44, 36.62, 45.76, 45.97, 46.00,
46.17, 60.82, 60.99, 65.72, 67.04, 125.74, 125.86, 128.25,
128.30, 128.51, 128.62, 141.75, 142.03, 170.34, 170.53,
4-Phenylbutanoyl-5(R)-methyl-L-prolyl-pyrrolidine 3f. 4-
Phenylbutanoyl-5(R)-methyl-l-proline (0.67 g, 2.4
mmol) and pyrrolidine (0.22 mL, 2.7 mmol) were cou-
pled according to procedure A. Purification by flash
chromatography, yield 0.53 g (1.6 mmol, 59%). 1H
NMR: d 1.32 (d, 2.6H, J=6.5 Hz), 1.37 (d, 0.4H, J=6.3
Hz), 1.65–2.20 (m, 10H), 2.30–2.34 (m, 2H), 2.67 (t, 2H,
J=7.6 Hz), 3.13–3.90 (m, 4H), 3.94–4.00 (m, 0.9H),
4.25–4.33 (m, 0.2H), 4.56 (t, 0.9H, J=8.1 Hz), 7.13–7.28
(m, 5H). 13C NMR: d 20.51, 24.16, 26.21, 26.22, 26.99,
32.85, 32.89, 35.21, 46.02, 46.35, 54.28, 58.87, 125.80,
1
173.99, 174.26. H NMR (acetone-d6): d 0.97 (s, 7.6H),
0.99 (s, 1.4H), 1.73–2.45 (m, 12H), 2.54–2.67 (m, 2H),
3.25–3.50 (m, 4H), 3.73 (br s, 0.2H), 4.23 (d, 0.8H,
J=8.7 Hz), 4.52 (t, 0.8H, J=8.8 Hz), 4.61 (m, 0.2H),
7.15–7.28 (m, 5H). 13C NMR (acetone-d6): d 24.49,
26.62, 26.91, 27.23, 28.10, 30.21, 33.64, 35.86, 37.14,
46.30, 46.67, 61.56, 66.09, 126.42, 128.99, 129.27,
143.09, 171.06, 173.94. ESI-MS: m/z 371 (M+H)+.
128.27, 128.52, 141.75, 170.69, 171.03. ESI-MS: m/z 329
+
.
(M+H) . Anal. (C20H28N2O2 0.3H2O) calcd C: 71.95,
H: 8.63, N: 8.39; found C: 72.14, H: 8.76, N: 8.34.
.
Anal. (C23H34N2O2 0.2H2O) calcd C: 73.84, H: 9.27, N:
7.49; found C: 73.91, H: 9.35, N: 7.17.
Boc-5(S)-methyl-L-proline ethyl ester. Prepared accord-
ing to a reported procedure.24 Purification without
separating the diastereomers by flash chromatography.
This procedure yielded the (5S)-diastereomer as the as
the major product.
(2S)-5-Oxo-2-[N-(benzyloxycarbonyl)-amino]hexanoic
acid. Prepared according to a reported procedure.23
(2S)-5-Oxo-2-[N-(benzyloxycarbonyl)-amino]hexanoic
(2S)-5-Oxo-2-[N-(benzyloxy-
4-Phenylbutanoyl-5(S)-methyl-L-proline ethyl ester. 4-
Phenylbutanoylchloride (prepared from 4-phenylbuta-
noic acid (1.42 g, 8.6 mmol) and thionyl chloride (0.93
mL, 13.0 mmol)) was added to a solution of the 5(S)-
methyl-l-proline ethyl ester trifluroacetic acid salt (pre-
pared from Boc-5(S)-methyl-l-proline ethyl ester (1.85
g, 7.2 mmol) according to procedure C) and triethyl
amine (4.0 mL, 28.7 mmol) in dichloromethane at 0 ꢀC.
The reaction was stirred 3 h in rt. The dichloromethane
phase was washed with 30% citric acid, saturated NaCl
and saturated NaHCO3. The dichloromethane phase
was dried and evaporated. Purification by flash
chromatography, yield 1.56 g (5.1 mmol, 71%).
acid
methyl
ester.
carbonyl)-amino]hexanoic acid (3.45 g, 12.3 mmol) was
methylated with an excess of diazomethane in anhy-
drous tetrahydrofuran at 0 ꢀC. The reaction mixture was
left at 4 ꢀC overnight. The solvent was evaporated and
the residue was dissolved in diethyl ether. The diethyl
ether phase was washed with water and saturated
NaHCO3. The diethyl ether phase was dried and eva-
porated. Purification by flash chromatography, yield 1.5
g (5.1 mmol, 41%).
Boc-5(R)-methyl-L-proline methyl ester. Prepared by
reacting (2S)-5-oxo-2-[N-(benzyloxycarbonyl)-amino]-
hexanoic acid methyl ester 1.5 g (5.1 mmol) and di-tert-
butyl-dicarbonat (3.1 g, 14.0 mmol) with 10% Pd/C
(0.28 g) in methanol under 4 atm pressure of H2 over-
night. The solution was filtered through Celite and evap-
orated. This procedure yields the (5R)-diastereomer as
the major product, which was isolated by flash chroma-
tography, yield 0.90 g (3.7 mmol, 73%).
4-Phenylbutanoyl-5(S)-methyl-L-proline. The ethyl ester
group of 4-phenylbutanoyl-5(S)-methyl-l-proline ethyl
ester (1.54 g, 5.1 mmol) was hydrolyzed according to
procedure B with KOH in water–EtOH. Yield 1.36 g
(4.9 mmol, 96%).
4-Phenylbutanoyl-5(S)-methyl-L-prolyl-pyrrolidine 3g. 4-
Phenylbutanoyl-5(S)-methyl-l-proline (0.67 g, 2.4
mmol) and pyrrolidine (0.20 mL, 2.4 mmol) were cou-
pled according to procedure A. Purification by flash
chromatography, yield 0.64 g (2.0 mmol, 83%). 1H
NMR: d 1.12 (d, 2.5H, J=6.4 Hz), 1.21 (d, 0.5H, J=6.4
Hz) 1.47–2.54 (m, 12H), 2.65–2.70 (m, 2H), 3.33–3.43
(m, 2H), 3.54–3.59 (m, 1H), 3.75–3.80 (m, 1H), 4.08–
4.15 (m, 1H), 4.26 (d, 0.2H, J=8.6 Hz), 4.63 (d, 0.8H,
J=8.9 Hz), 7.14–7.28 (m, 5H). 13C NMR: d 21.72,
24.15, 26.25, 26.51, 26.54, 31.72, 32.99, 35.11, 45.87,
46.22, 53.72, 58.06, 125.76, 128.26, 128.64, 141.95,
170.53, 171.70. ESI-MS: m/z 329 (M+H)+. Anal.
4-Phenylbutanoyl-5(R)-methyl-L-proline ethyl ester. 4-
Phenylbutanoylchloride (prepared from 4-phenylbuta-
noic acid (0.73 g, 4.4 mmol) and thionyl chloride (0.64
mL, 8.9 mmol)) was added to a solution of the 5(R)-
methyl-l-proline ethyl ester trifluoroacetic acid salt
(prepared from Boc-5(R)-methyl-l-proline ethyl ester
(0.90 g, 3.7 mmol) according to procedure C) and tri-
ethyl amine (2.1 mL, 15.0 mmol) in dichloromethane at
0 ꢀC, where after it was stirred at rt for 3 h. The di-
chloromethane phase was washed with 30% citric acid,
saturated NaCl and saturated NaHCO3. The dichloro-
methane phase was dried and evaporated. Purification
by flash chromatography, yield 0.74 g (2.6 mmol, 70%).
.
(C20H28N2O2 0.2H2O) calcd C: 72.34, H: 8.62, N: 8.44;
found C: 72.08, H: 8.86, N: 8.55.
4-Phenylbutanoyl-5(R)-methyl-L-proline. The ethyl ester
group of 4-phenylbutanoyl-5(R)-methyl-l-proline ethyl
Benzylcarbamoyl-5(R)-tert-butyl-L-prolyl-pyrrolidine 3h.
Benzylisocyanate (0.55 mL, 4.5 mmol) was added to a

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E. A. A. Wallen et al. / Bioorg. Med. Chem. 11 (2003) 3611–3619
3617
solution of the 5(R)-tert-butyl-l-proline methyl ester
trifluroacetic acid salt [prepared from Boc-5(R)-tert-
butyl-l-proline methyl ester (1.46 g, 4.5 mmol) accord-
ing to procedure C] and triethyl amine (1.9 mL, 13.5
mmol) in dimethylformamide at 0 ꢀC. The reaction was
stirred 3 h in rt. The dimethylformamide solution was
poured into ice-water and the product was extracted
with dichloromethane. The dichloromethane phase was
washed with 30% citric acid, saturated NaCl and satu-
rated NaHCO3. The dichloromethane phase was dried
and evaporated. Purification by flash chromatography,
according to procedure B with LiOH in water–MeOH.
Yield 0.58 g (1.8 mmol, 86%).
Boc-2(S)-(acetoxyacetyl)pyrrolidine. Prepared according
to a reported procedure.26
4-Phenylbutanoyl-5(R)-tert-butyl-L-prolyl-2(S)-(acetoxy-
acetyl)pyrrolidine. 4-Phenylbutanoyl-5(R)-tert-butyl-l-
proline (0.58 g, 1.8 mmol) and 2(S)-(acetoxyacetyl)
pyrrolidine trifluoroacetic acid salt [prepared from Boc-
2(S)-(acetoxyacetyl)pyrrolidine (0.50 g, 1.8 mmol)
according to procedure C] were coupled accoroding to
procedure A. Purification by flash chromatography,
yield 0.30 g (0.64 mmol, 36%).
1
yield 1.24 g (3.5 mmol, 78%). H NMR: d 0.99 (s, 9H),
1.78–2.06 (m, 7H), 2.19–2.26 (m, 1H), 3.35–3.46 (m,
2H), 3.50–3.56 (m, 1H), 3.59–3.65 (m, 1H), 3.91 (d, 2H,
J=8.1 Hz), 4.31 (m, 2H), 4.48–4.53 (m, 1H), 4.98 (br t,
1H, J=5.3 Hz), 7.21–7.32 (m, 5H). 13C NMR: d 23.90,
26.34, 26.84, 27.54, 29.32, 36.46, 44.96, 46.16, 46.33,
62.56, 66.51, 127.07, 127.41, 128.54, 139.56, 160.29,
171.54. ESI-MS: m/z 358 (M+H)+. Anal.
(C21H31N3O2) calcd C: 70.55, H: 8.74, N: 11.75; found
C: 70.72, H: 8.85, N: 12.08.
4-Phenylbutanoyl-5(R)-tert-butyl-L-prolyl-2(S)-(hydroxy-
acetyl)pyrrolidine 3k. Prepared according to procedure
D using 4-phenylbutanoyl-5(R)-tert-butyl-l-prolyl-2(S)-
(acetoxyacetyl)pyrrolidine (0.30 g, 0.64 mmol) as start-
ing material. Purification by flash chromatography,
yield 0.26 g (0.61 mmol, 95%). 1H NMR: d 0.94 (s, 9H),
1.72 (m, 12H), 2.64 (t, 2H, J=7.3 Hz), 3.04 (br s, 0.4H),
3.10 (br s, 0.6H), 3.26–4.76 (m, 7H), 7.14–7.28 (m, 5H).
13C NMR: d 25.37, 25.42, 25.82, 26.06, 26.76, 27.15,
27.57, 27.82, 28.06, 28.07, 29.15, 29.43, 33.01, 33.79,
34.97, 35.24, 36.43, 36.53, 46.50, 46.79, 60.44, 60.63,
61.24, 61.30, 65.83, 66.90, 66.97, 67.08, 125.77, 125.91,
128.26, 128.33, 128.49, 128.65, 141.64, 141.97, 170.78,
Acetyl-5(R)-tert-butyl-L-prolyl-pyrrolidine 3i. Acetic
anhydride (0.15 mL, 1.5 mmol) was added to a solution
of the 5(R)-tert-butyl-l-prolyl-pyrrolidine trifluoro-
acetic acid salt [prepared from Boc-5(R)-tert-butyl-l-
prolyl-pyrrolidine (0.25 g, 0.77 mmol) according to
procedure C] and triethyl amine (0.40 mL, 3.1 mmol) in
dichloromethane at 0 ꢀC. The reaction was stirred at rt
for 3 h. The dichloromethane solutution was washed
with 30% citric acid, saturated NaCl and saturated
NaHCO3. The dichloromethane phase was dried and
evaporated. Purification by flash chromatography, yield
171.01, 173.74, 174.39, 208.42, 209.31. ESI-MS: m/z 429
+
.
(M+H) . Anal. (C25H36N2O4 0.1H2O) calcd C: 69.77,
H: 8.48, N: 6.51; found C: 69.62, H: 8.48, N: 6.73.
4-Phenylbutanoyl-5(R)-methyl-L-prolyl-2(S)-(acetoxy-
acetyl)pyrrolidine.
1
0.17 g (0.65 mmol, 84%). H NMR: d 1.00 (s, 4.5H),
4-Phenylbutanoyl-5(R)-methyl-l-
1.08 (s, 4.5H), 11.76–2.39 (m, 8H), 1.94 (s, 1.5H), 2.16
(s, 1.5H), 3.35–3.81 (m, 4H), 3.72 (d, 0.5H, J=8.1 Hz),
4.27 (d, 0.5H, J=8.7 Hz), 4.42 (t, 0.5H, J=9.2 Hz),
4.62 (t, 0.5H, J=9.2 Hz). 13C NMR: d 22.74, 23.17,
23.94, 24.08, 26.25, 26.29, 26.42, 27.61, 27.95, 28.12,
29.65, 36.62, 36.64, 45.97, 45.98, 46.01, 46.31, 60.78,
61.81, 65.64, 68.18, 170.30, 170.46, 172.00, 172.02
(all except one carbon give double peaks). ESI-MS:
m/z 267 (M+H)+. Anal. (C15H26N2O2) calcd C:
67.63, H: 9.84, N: 10.52; found C: 67.79, H: 10.16,
N: 10.68.
proline (0.23 g, 0.84 mmol) and 2(S)-(acetoxyacetyl)-
pyrrolidine trifluoroacetic acid salt (prepared from Boc-
2(S)-(acetoxyacetyl)pyrrolidine (0.23 g, 0.84 mmol)
according to procedure C) were coupled according to
procedure A. Purification by flash chromatography,
yield 0.23 g (0.54 mmol, 64%).
4-Phenylbutanoyl-5(R)-methyl-L-prolyl-2(S)-(hydroxy-
acetyl)pyrrolidine 3l. Prepared according to procedure
D using 4-phenylbutanoyl-5(R)-methyl-l-prolyl-2(S)-
(acetoxyacetyl)pyrrolidine (0.23 g, 0.54 mmol) as start-
ing material. Purification by flash chromatography,
yield 0.11 g (0.29 mmol, 54%). 1H NMR: d 1.28 (d, 3H,
J=6.6 Hz), 1.66–2.21 (m, 10H), 2.29–2.33 (m, 2H), 2.66
(t, 2H, J=7.4H), 3.05 (t, 1H, J=5.1 Hz), 3.58–3.63 (m,
1H), 3.93–4.02 (m, 2H), 4.30–4.47 (m, 2H), 4.55 (t, 1H,
J=8.1 Hz), 4.70 (dd, 1H, J=5.1 Hz, J=8.4 Hz). 13C
NMR: d 20.65, 25.34, 26.23, 26.82, 28.25, 32.84, 32.90,
35.23, 47.19, 54.30, 58.56, 61.27, 66.96, 125.88, 128.32,
4-Phenylbutanoyl-5(R)-tert-butyl-L-proline methyl ester.
4-Phenylbutanoylchloride [prepared from 4-phenylbu-
tanoic acid (0.76 g, 4.6 mmol) and thionyl chloride (0.50
mL, 6.9 mmol)] was added to a solution of the 5(R)-tert-
butyl-l-proline methyl ester trifluroacetic acid salt (pre-
pared from Boc-5(R)-tert-butyl-l-proline methyl ester
(1.1 g, 3.8 mmol) according to procedure C) and triethyl
amine (2.1 mL, 15.3 mmol) in dichloromethane at 0 ꢀC.
The reaction was stirred 4 h in rt. The dichloromethane
solution was washed with 30% citric acid, saturated
NaCl and saturated NaHCO3. The dichloromethane
phase was dried and evaporated. Purification by flash
chromatography, yield 0.73 g (2.2 mmol, 58%).
128.50, 141.66, 171.21, 171.33, 209.05. ESI-MS: m/z 387
+
.
(M+H) . Anal. (C22H30N2O4 0.5H2O) calcd C: 66.81,
H: 7.90, N: 7.08; found C: 66.82, H: 7.83, N: 6.83.
Boc-2(S)-benzoylpyrrolidine. Prepared according to
reported procedure.26
4-Phenylbutanoyl-5(R)-tert-butyl-L-proline. The methyl
ester group of 4-phenylbutanoyl-5(R)-tert-butyl-l-pro-
line methyl ester (0.68 g, 2.1 mmol) was hydrolyzed
3-(2(S)-Benzoylpyrrolidine-1-carbonyl)-benzoic acid methyl
ester. Isophthalic acid mono methyl ester (1.28 g, 7.1
mmol) and 2(S)-benzoylpyrrolidine trifluoroacetic acid

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E. A. A. Wallen et al. / Bioorg. Med. Chem. 11 (2003) 3611–3619
3618
salt [prepared from Boc-2(S)-benzoyl-pyrrolidine (1.96
g, 7.1 mmol) according to procedure C], were coupled
according to procedure A. Purification by flash
chromatography, yield 1.1 g (3.3 mmol, 46%).
inhibitory activities (percent of control) were plotted
against the log concentration of the compound, and the
IC50 value was determined by non-linear regression uti-
lizing GraphPad Prism 3.02 software.
3-(2(S)-Benzoylpyrrolidine-1-carbonyl)-benzoic acid. The
methyl ester group of 3-(2(S)-benzoylpyrrolidine-1-car-
bonyl)-benzoic acid methyl ester (1.1 g, 3.3 mmol) was
hydrolyzed according to procedure B with LiOH in
water–MeOH. Yield 0.99 g (3.1 mmol, 94%).
The Ki values were determined for the tight binding
inhibitors 3j–l employing the following procedure. 10
mL of enzyme preparation was incubated with 465 mL of
0.1 M sodium–potassium phosphate buffer (pH 7.0) for
2 h in the presence of various concentrations of inhibi-
tors at room temperature. The reaction was started with
25 mL of 4 mM Suc-Gly-Pro-7-amino-4-methylcou-
marin and the reaction was monitored every 1 min for
30 min. Over that time scale, the product formations
were linear, indicating that the inhibitors did not dis-
sociate markedly from the enzyme. The Ki values were
calculated using the Morrison equation that takes the
tight binding inhibition into account:30
3-(2(S)-Benzoyl-pyrrolidine-1-carbonyl)-benzoyl-5(R)-tert-
butyl-L-prolyl-pyrrolidine 3n. 3-(2(S)-Benzoyl-pyrroli-
dine-1-carbonyl)-benzoic acid (0.35 g, 1.1 mmol) and
5(R)-tert-butyl-l-prolyl-pyrrolidine trifluoroacetic acid
salt (prepared from Boc-5(R)-tert-butyl-l-prolyl-pyrro-
lidine (0.36 g, 1.1 mmol) according to procedure C) were
coupled according to procedure A. Purification by flash
chromatography, yield 0.15 g (0.28 mmol, 25%). 1H
NMR: d 1.14 (s, 9H), 1.40–2.44 (m, 12H), 2.87–3.91 (m,
6H), 4.19 (br s, 1H), 4.46 (br s, 1H), 5.64–5.69 (m, 1H),
7.14–8.06 (m, 9H). 13C NMR: d 197.41, 173.01, 170.81,
168.34, 138.04, 136.48, 135.18, 133.37, 128.71, 128.60,
128.55, 128.29, 128.05, 124.69, 50.19, 46.03, 45.71,
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ðE þ I þ KiÞ À ðE þ I þ KiÞ À4E ꢀ I
2E
vi
¼ 1 À
v0
36.48, 29.60, 29.48, 27.66, 26.02, 25.21, 23.88. ESI-MS:
+
where v0 and vi are the reaction velocities in the absence
and presence of the inhibitor (I), respectively, Ki is the
inhibition constant of the inhibitor and E is the active
enzyme concentration in the reaction medium. Since the
inhibitors did not dissociate from the enzyme during the
measurements, competition of binding between sub-
strate and inhibitor did not occur and hence the calcu-
lated Ki values are the real dissociation constants of the
inhibitors.31
.
m/z 530 (M+H) . Anal. (C32H39N3O4 1.2H2O) calcd
C: 69.72, H: 7.57, N: 7.62; found C: 69.90, H: 7.51, N:
7.26.
In vitro assay for POP activity
The whole pig brains, excluding cerebellum and most of
the brain stem, of three pigs were frozen in liquid
nitrogen within 30 min from slaughtering and stored at
À80 ꢀC until homogenized. The brains were homo-
genized in 3 volumes (w/v) of ice-cold 0.1 M sodium–
potassium phosphate buffer (pH 7.0) and the homo-
genates were centrifuged for 20 min at 4 ꢀC at 10,000g.
The supernatants were pooled and stored in small ali-
quots at À80 ꢀC until used. The supernatant was thawed
in ice and diluted (1:2) with homogenization buffer. In
the microplate assay procedure, 10 mL of the enzyme
preparation (protein concentration 4.3 mg/mL) was
preincubated with 460 mL of 0.1 M sodium–potassium
phosphate buffer (pH 7.0) and 5 mL of a solution of the
compound dissolved in DMSO and diluted with 0.1 M
sodium–potassium phosphate buffer at 30 ꢀC for 30 min
(final DMSO concetration was less than 0.1%). The
controls contained 10 mL enzyme preparation and 465
mL of 0.1 M sodium–potassium phosphate buffer (pH
7.0). The reaction was initiated by adding 25 mL of 4
mM Suc-Gly-Pro-7-amino-4-methylcoumarin dissolved
in 0.1 M sodium–potassium phosphate buffer (pH 7.0),
and the mixture was incubated at 30 ꢀC for 60 min. The
reaction was terminated by adding 500 mL of 1 M
sodium acetate buffer (pH 4.2). Formation of 7-amino-
4-methylcoumarin was determined fluorometrically with
microplate fluorescence reader (excitation at 360 nm
and emission at 460 nm). 5–7 inhibitor concentrations
were used to determine the IC50 values and the final
concentrations of the compounds in the assay mixture
varied from 10À12 M up to 10À5 M. 2–4 independent
measurements were made for each inhibitor. The
Determination of the log P values
A known concentration of an inhibitor in phosphate
buffer (saturated with 1-octanol, pH 7.4) was shaken
with a suitable volume of 1-octanol for 60 min at room
temperature. The phases were separated by centrifuga-
tion for 5 min at 2000 rpm and the aqueous phase was
analysed. The partition coefficient was calculated in
relation to a control that was treated in the same way as
the samples but did not contain 1-octanol. Each parti-
tion coefficient was determined at least in triplicate. For
each HPLC method 20 mM KH2PO4 of pH 7 was used
as the aqueous phase and 90% acetonitrile was used as
the organic phase. The HPLC methods were tested for
linearity and repeatability. The Merck Hitachi HPLC
system consisted of an UV-detector (L-7400), an inter-
face module (D-7000), a pump (L-7100), an auto-
sampler (L-7250) and a Purospher RP-C18e column
(125ꢁ4 mm, 5 mm).
Acknowledgements
This research was supported by Finncovery Ltd. and
the National Technology Agency in Finland (TEKES).
We also thank Ms. Tiina Koivunen, Ms. Helly Rissanen
and Ms. Paivi Sutinen for their outstanding technical
assistance, Prof. Dr. Jouko Vepsalainen for his coop-
eration in the synthesis of the 5(S)-tert-butyl-l-proline

´
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3619
moiety, Dr. Antti Poso for the discussions regarding the
3D structure of the active site of the enzyme, Dr. Seppo
Auriola and Ms. Unni Tengvall, M.Sc., for performing
the ESI-MS analysis, and Ms. Piia Palonen, Mr. Pekka
Keski-Rahkonen, Mr. Rustam Safin Ms. Anne Riekki-
nen and Ms. Virpi Turunen for helping us by perform-
ing some synthetic steps.
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