An introduction of a pyridine group into the structure of prolyl oligopeptidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.08.029

A series of ionizable prolyl oligopeptidase inhibitors were developed through the introduction of a pyridyl group to the P3 position of the prolyl oligopeptidase inhibitor structure. The study was performed on previously developed prolyl oligopeptidase in

Full text of DOI:10.1016/j.bmcl.2006.08.029

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5590–5593
An introduction of a pyridine group into the structure
of prolyl oligopeptidase inhibitors
Elina M. Jarho,a,* Jarkko I. Vena¨la¨inen,b Juha Juntunen,a A. Leena Yli-Kokko,a
Jouko Vepsa¨la¨inen,c Johannes A. M. Christiaans,a,ꢀ Markus M. Forsberg,b
a
Tomi Ja¨rvinen,a Pekka T. Ma¨nnisto¨b,ꢁ and Erik A. A. Wallen
´
aDepartment of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FI-70211 Kuopio, Finland
bDepartment of Pharmacology and Toxicology, University of Kuopio, PO Box 1627, FI-70211 Kuopio, Finland
cDepartment of Chemistry, University of Kuopio, PO Box 1627, FI-70211 Kuopio, Finland
Received 30 June 2006; revised 3 August 2006; accepted 3 August 2006
Available online 17 August 2006
Abstract—A series of ionizable prolyl oligopeptidase inhibitors were developed through the introduction of a pyridyl group to the
P3 position of the prolyl oligopeptidase inhibitor structure. The study was performed on previously developed prolyl oligopeptidase
inhibitors with proline mimetics at the P2 position. The 3-pyridyl group resulted in equipotent compounds as compared to the par-
ent compounds. It was shown that the pyridyl group improves water solubility and, in combination with a 5(R)-tert-butyl-L-prolyl
group at the P2 position, good lipophilicity can be achieved.
Ó 2006 Elsevier Ltd. All rights reserved.
Human prolyl oligopeptidase (POP) is an 80 kDa serine
protease that hydrolyzes oligopeptides after prolyl resi-
dues. Several studies have shown that specific POP
inhibitors can prevent memory impairments1–3 and have
neuroprotective effects4 in rats, improve cognition in a
model of early Parkinsonism in monkeys,5 and improve
performance in verbal memory tests in humans.6 In aged
mice, the expression of the POP gene has been shown to
be up-regulated in the hypothalamus7 and the hippo-
campus.8 All these results imply that POP is associated
with neurodegeneration and memory deficits but the
physiological role of POP is not still fully understood.
Initially, POP was suggested to have a function in the
maturation and the degradation of neuropeptides, which
are involved in memory and learning. This idea has been
called into question because POP is mainly described as
a cytosolic enzyme and neuropeptides are located in
transcellular space. During the last years, some light
has been shed on the intracellular functions of POP.
Inhibition of POP has been shown to elevate IP3 levels,
which was suggested to explain the memory-enhancing
properties of POP inhibitors.9,10 Recently, POP was also
proposed to have a role in protein trafficking and
secretion.11
Unlike many small-molecule drugs, typical POP inhibi-
tors are unionizable compounds. An ionizable group
may improve water solubility and it also allows salt for-
mation. In later stages of drug development, the proper
choice of salt can be used to modify stability, solubility,
and pharmaceutical processing properties.
Two unionizable POP inhibitors with different proline
mimetics at the P2 positions were earlier presented by
our group (Fig. 1).12,13 These compounds were further
investigated as to whether an ionizable pyridyl group
H
N
N
N
N
Keywords: Prolyl oligopeptidase; Inhibitor; Pyridine.
*
O
O
P2
O
O
Corresponding author. Tel.: +358 17 162460; fax: +358 17 162456;
e-mail: Elina.Jarho@uku.fi
P3
P2
P1
P3
P1
ꢀ
Present address: Altana Pharma bv, PO Box 31, 2130 AA Hoofd-
dorp, The Netherlands.
IC50 = 9.0 nM (compound 4)
IC50 = 1.2 nM (compound 12)
ꢁ
Figure 1. Previously published POP inhibitors, which possess proline
mimetics at the P2 site.
Present address: Division of Pharmacology and Toxicology, Faculty
of Pharmacy, PO Box 56, University of Helsinki, FI 00014, Finland.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.029

E. M. Jarho et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5590–5593
5591
could be introduced into the structure. The pKa value of
pyridine is 5.2 and thus, it is mostly in the unionized
form at the physiological pH 7.4. However, a significant
fraction is in the ionized form in the slightly acidic envi-
ronment of the duodenum,14 which allows dissolution
that is a prerequisite for the absorption.
respectively. EDC and HOBt were used to couple L-pro-
lyl-pyrrolidine with (pyridin-3-yl)acetic acid to obtain
compound 14b.
Compound 412 possesses a cyclopentenecarbonyl moiety
at the P2 position. The IC50 value of compound 4 is
9 nM for the racemic mixture. It was chosen as the par-
ent compound for the first series of compounds present-
ed in Table 1. The replacement of the phenyl group of
compound 4 with a 2- or 4-pyridyl group gave com-
pounds 5a and 5c, respectively. These substitutions
resulted in over 2-fold lower potency. However, the
replacement with a 3-pyridyl group gave compound
5b, which was equipotent with the parent compound 4.
In order to further optimize the position of the pyridyl
group, the chain length of compounds 4 and 5a–5c
was extended by one methylene group, which resulted
in compounds 6 and 7a–c, respectively. The extension
of the chain of compound 4 caused a 6-fold increase in
potency; the IC50 value of compound 6 was 1.5 nM.
Again, the replacement of the P3 phenyl group by the
3-pyridyl moiety gave the most potent compound 7b
A literature search revealed that a pyridyl moiety has
occasionally been included in some series of POP inhib-
itors, but no systematic study regarding the position and
the substitution of the pyridine group has been per-
formed.15–21 One potent peptide-like POP inhibitor,
which possessed a pyridine-2-carboxyl amide moiety at
the P3 position, was reported by Tsuda et al.18 In the
present study, the phenyl group at the P3 site was suc-
cessfully replaced by a pyridyl group in two series of
POP inhibitors. The chain length and the substitution
pattern were optimized. The effect of the pyridyl moiety
on lipophilicity and water solubility was also studied.
The synthetic routes for the novel compounds are pre-
sented in Scheme 1. Compounds 1, 4,12 and 1213 were
synthesized as described earlier. Compound 1 was
activated with pivaloyl chloride and reacted with pyrrol-
idine at 0 °C to obtain ( )-5-(pyrrolidine-1-carbonyl)-
cyclopent-1-enecarbaldehyde (2). The aldehyde group
of compound 2 was oxidized with NaClO2 to obtain
( )-5-(pyrrolidine-1-carbonyl)-cyclopent-1-enecarboxy-
lic acid (3). Compound 3 was reacted with DCC, HOBt,
and an appropriate amine to yield compounds 5a–c, 6,
and 7a–c. Boc-5(R)-tert-butyl-L-proline (8) was pre-
pared according to published procedures22,23 with slight
modifications as described earlier.13 Boc-5(R)-tert-butyl-
L-proline (8) and Boc-L-proline (9) were activated with
pivaloyl chloride and reacted with pyrrolidine to yield
compounds 10 and 11, respectively. Compounds 10
and 11 were deprotected in HCl-saturated ethyl acetate
to yield 5(R)-tert-butyl-L-prolyl-pyrrolidine and L-pro-
lyl-pyrrolidine. 4-Pyridin-3-yl-butyric acid and 3-pyri-
din-3-yl-propionic acid were reacted with EDC, HOBt,
and 5(R)-tert-butyl-L-prolyl-pyrrolidine to yield com-
pounds 13a and 13b, respectively. 4-Pyridin-3-yl-butyric
acid was prepared according to a published procedure24
with small modifications.25 L-Prolyl-pyrrolidine was
reacted with 3-pyridin-3-yl-propionyl chloride and
nicotinoyl chloride to yield compounds 14a and 14c,
Table 1. Inhibitory activities with 95% confidence intervals and logP
values of the ( )-5-(pyrrolidine-1-carbonyl)-cyclopent-1-enecarboxylic
acid amides
N
N
n
X
O
O
a
Compound
X
Subst
n
IC50 (nM)
9.0 (5.5–15)
24
9.7
19
1.5
logPb
4
C
—
2
1
1
1
1
1.6c
0.3d
0.4d
0.4d
5a
5b
5c
N
N
N
(22–26)
(8.5–11)
(15–26)
3
4
6
C
—
2
2
2
2
2
(1.3–1.9)
(4.4–6.2)
(1.9–2.6)
(3.8–6.1)
1.9c
0.6d
0.6d
0.5d
7a
7b
7c
N
N
N
5.2
2.2
4.8
3
4
a The IC50 values were determined against POP from porcine brain.28
b The reported values are for the unionized species.
c Determined with the shake-flask method.
d Determined with pH metric titration using a Sirius PCA200 auto-
matic titrator.
n = 2
6: X=C
7a: X=N, 2-subst.
7b: X=N, 3-subst.
7c: X=N, 4-subst.
n = 1, X = N
5a: 2-subst.
5b: 3-subst.
5c: 4-subst.
i
ii
iii
H
H
OH
H
N
HO
N
N
N
n
X
O
O
R
O
O
O
O
O
O
1
2
3
vi for 13a-b, 14b
vii for 14a
R
R
n
viii for 14c
iv
v
R = H
R = t-Bu
13a: n=3
13b: n=2
O
N
OH
O
N
N
N
N
14a: n=2
14b: n=1
14c: n=0
O
O
O
O
O
O
N
8: R=t-Bu
9: R=H
10: R=t-Bu
11: R=H
Scheme 1. Synthesis of the novel compounds. Reagents and conditions: (i) 1—Et3N, (CH3)3CCOCl/CH2Cl2, 0 °C, 2—Et3N, pyrrolidine/CH2Cl2,
0 °C; (ii) resorcinol, NaH2PO4ÆH2O, NaClO2/t-BuOH, H2O; (iii) an appropriate amine, Et3N, HOBt, DCC/CH3CN, 0–20 °C; (iv) 1—Et3N,
(CH3)3CCOCl/CH2Cl2, 0 °C, 2—Et3N, pyrrolidine/CH2Cl2, room temperature; (v) HCl/EtOAc; (vi) an appropriate carboxylic acid, Et3N, HOBt,
EDCÆHCl/CH2Cl2, 0–20 °C; (vii) 3-pyridin-3-yl-propionyl chloride HCl, Et3N/CH2Cl2, 0–20 °C; (viii) DMAP, nicotinoyl chloride HCl/pyridine.

5592
E. M. Jarho et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5590–5593
(2.2 nM), while the 2- and 4-pyridyl analogs, 7a and 7c,
were less active. Compound 7b was almost equipotent
with compound 6 and over 4-fold more potent than its
shorter counterpart 5b. These results confirmed that
the 3-pyridyl group can be used to replace the phenyl
group at the P3 position of POP inhibitors and the opti-
mal chain length is the same as for the phenyl group, de-
spite the difference in their polarities.
an IC50 value of 2.1 nM. It was only slightly less potent
than compound 12 and equipotent with compound 7b,
which was the most potent pyridyl derivative in the first
series. However, it has to be kept in mind that 13a pos-
sesses the more active configuration of L-proline, while
7b was tested as a racemic mixture. To study whether
the change of the skeleton affects the optimal position
of the pyridyl group, the P3 chain of compound 13a
was shortened by one methylene group resulting in com-
pound 13b. The results were consistent with the first ser-
ies; a decrease in the chain length decreased the potency
and the IC50 value of compound 13b was 13 nM. The ef-
fect of even shorter chain lengths was studied with com-
pounds 14a–c, which have an L-prolyl residue at the P2
site. The change to an L-prolyl group at the P2 site was
made to avoid steric hindrance that the bulky 5(R)-tert-
butyl group causes when the P3 ring is brought closer to
the P2 ring. The removal of the 5(R)-tert-butyl group of
compound 13b resulted in compound 14a and in over
2-fold lower potency; the IC50 value of 14a was
30 nM. The P3 chain length of compound 14a was
shortened by one and two methylene groups to obtain
compounds 14b and 14c, respectively. Both compounds
were less potent than compound 14a, confirming that
the optimal position for the pyridyl moiety is three car-
bon atoms away from the carbonyl group.
High lipid solubility is a prerequisite for the passive
brain penetration of small-molecule drugs26 and the the-
oretical optimum for the logP value is near 2.27
Although lipophilicity is not the only factor that deter-
mines brain penetration and the optimal lipophilicity
can vary between compound classes, it should be taken
into account when novel CNS targeted compounds are
designed. The logP values were determined for com-
pounds 4, 5a–c, 6, and 7a–c (Table 1). The presented
logP values for compounds 5a–c and 7a–c are values
for the unionized species, which are mainly found at
the physiological pH 7.4 (the pKa values range between
4.4 and 5.5). The logP value of compound 4 was 1.6.
The replacement of the phenyl moiety by the more polar
pyridyl moiety dropped the logP values to 0.3–0.4 for
compounds 5a–5c. The extension of the chain increased
the logP value to 1.9 for compound 6, but again the
replacement of the phenyl moiety by the pyridyl moiety
dropped the logP values to 0.5–0.6 for compounds
7a–7c.
The logP values of the second series of compounds are
presented in Table 2. Again, the reported logP values
are for the unionized species. The replacement of the
phenyl moiety of compound 12 by the polar pyridyl
moiety dropped the logP value from 3.3 to the theoret-
ical optimum 2.0 for compound 13a. The drop was of
the same order of magnitude as in the first series of
the compounds; 20-fold drop in partition coefficient P.
Compound 13b with shorter chain length had a logP
value 1.8. A comparison between the logP values of
compounds 13b and 14a (logP = 0.3) shows that the
tert-butyl group greatly increases lipophilicity and con-
sequently, the logP values of compounds 14a–c were
low. The IC50 and logP values of compound 13a are
comparable to those of the unionizable reference com-
pound 15, SUAM-1221, which is a potent POP inhibitor
that can penetrate into the CNS (Table 2).29
Compound 1213 possesses a lipophilic 5(R)-tert-butyl-L-
prolyl moiety at the P2 position. The IC50 value of com-
pound 12 is 1.2 nM and the logP value is 3.3. It was
chosen as the parent compound for the second series
of compounds presented in Table 2. In this series, only
the 3-pyridyl group was studied because it had given
the most potent compounds in the first series. The
replacement of the P3 phenyl group of compound 12
by the 3-pyridyl group resulted in compound 13a having
Table 2. Inhibitory activities with 95% confidence intervals and logP
values of the N-alkanoyl 5(R)-tert-butyl-L-prolyl-pyrrolidines and
N-alkanoyl L-prolyl-pyrrolidines
R
The water solubility was determined for the parent com-
pounds of the two series, compounds 4 and 12, their
3-pyridyl analogs 5b and 13a, and for the reference com-
pound 15, SUAM-1221. The solubilities were deter-
mined after 3-day-shaking in 50 mM phosphate buffer
(pH 7.4, ionic strength 0.15 M) up to 6 mg/mL, which
can be considered adequate even for high-dose com-
pounds with poor permeability.30 While the solubility
of compound 15 was at least 6 mg/mL, compounds 4
and 12 with proline mimetics at the P2 sites had de-
creased values; 3.0 and 0.9 mg/mL, respectively. Howev-
er, the change of the phenyl moiety to the 3-pyridyl
moiety overcame this decrease and the solubilities of
compounds 5b and 13a were at least 6 mg/mL.
N
N
n
O
O
X
Compound
X
n
R
logPb
a
IC50 (nM)
12
C
N
N
3
3
2
t-Bu
t-Bu
t-Bu
1.2
2.1
13
(1.0–1.4)
(1.9–2.4)
(8.4–20)
3.3c
2.0d
1.8d
13a
13b
14a
14b
14c
N
N
N
2
1
0
H
H
H
30
56
44
(24–39)
(46–70)
(29–67)
0.3d
À0.2d
À0.3d
15 SUAM-1221
C
3
H
2.2
(1.9–2.5)
1.8c
a The IC50 values were determined against POP from porcine brain.28
b The reported values are for the unionized species.
c Determined with the shake-flask method.
d Determined with pH metric titration using a Sirius PCA200 auto-
matic titrator.
The present study proved that a pyridyl group can be
introduced to the P3 position of the POP inhibitor struc-
ture. However, the inhibitory activity was dependent on

E. M. Jarho et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5590–5593
5593
11. Schulz, I.; Zeitschel, U.; Rudolph, T.; Ruiz-Carrillo, D.;
Rahfeld, J. U.; Gerhartz, B.; Bigl, V.; Demuth, H. U.;
Roßner, S. J. Neurochem. 2005, 94, 970.
12. Jarho, E. M.; Vena¨la¨inen, J. I.; Huuskonen, J.; Christia-
ans, J. A. M.; Garcia-Horsman, J. A.; Forsberg, M. M.;
the substitution of the pyridyl group and the P3 chain
length. The 3-pyridyl group gave the most potent com-
pounds and its optimal position was the same as for
the phenyl group; three atoms between the P3 carbonyl
group and the aromatic ring. The studied parent com-
pounds had decreased water solubility. The introduction
of an ionizable pyridyl group gave excellent water solu-
bility to the novel compounds. In conclusion, the intro-
duction of the 3-pyridyl group at the P3 position in
combination with a 5(R)-tert-butyl-L-prolyl moiety at
P2 position was used to optimize the physico-chemical
properties while maintaining an excellent inhibitory
activity.
´
Ja¨rvinen, T.; Gynther, J.; Ma¨nnisto¨, P. T.; Wallen, E. A.
A. J. Med. Chem. 2004, 47, 5605.
´
13. Wallen, E. A. A.; Christiaans, J. A. M.; Saarinen, T. J.;
Jarho, E. M.; Forsberg, M. M.; Vena¨la¨inen, J. I.;
Ma¨nnisto¨, P. T.; Gynther, J. Bioorg. Med. Chem. 2003,
11, 3611.
14. Dressman, J. B.; Berardi, R. R.; Dermentzoglou, L. C.;
Russell, T. L.; Schmaltz, S. P.; Barnett, J. L.; Jarvenpaa,
K. M. Pharm. Res. 1990, 7, 756.
15. Tsutsumi, S.; Okonogi, T.; Shibahara, S.; Ohuchi, S.;
Hatsushiba, E.; Patchett, A. A.; Christensen, B. G. J.
Med. Chem. 1994, 37, 3492.
Acknowledgments
16. Nakajima, T.; Ono, Y.; Kato, A.; Maeda, J.; Ohe, T.
Neurosci. Lett. 1992, 141, 156.
17. Saito, M.; Hashimoto, M.; Kawaguchi, N.; Shibata, H.;
Fukami, H.; Tanaka, T.; Higuchi, N. J. Enzyme Inhib.
1991, 5, 51.
18. Tsuda, M.; Muraoka, Y.; Nagai, M.; Aoyagi, T.; Takeu-
chi, T. J. Antibiot. 1996, 49, 909.
We thank Ms. Tiina Koivunen, Ms. Jaana Leskinen,
Ms. Miia Reponen, and Ms. Maritta Salminkoski for
their technical assistance and the Academy of Finland,
the Association of Finnish Pharmacies and Research
and Science Foundation of Farmos for their financial
support.
19. Tsuda, M.; Muraoka, Y.; Nagai, M.; Aoyagi, T.; Takeu-
chi, T. J. Antibiot. 1996, 49, 1022.
20. Tsuru, D.; Yoshimoto, T.; Koriyama, N.; Furukawa, S. J.
Biochem. 1988, 104, 580.
References and notes
21. Vendeville, S.; Goossens, F.; Debreu-Fontaine, M. A.;
Landry, V.; Davioud-Charvet, E.; Grellier, P.; Scharpe, S.;
Sergheraert, C. Bioorg. Med. Chem. 2002, 10, 1719.
1. Toide, K.; Iwamoto, Y.; Fujiwara, T.; Abe, H. J.
Pharmacol. Exp. Ther. 1995, 274, 1370.
2. Shishido, Y.; Furushiro, M.; Tanabe, S.; Taniguchi, A.;
Hashimoto, S.; Yokokura, T.; Shibata, S.; Yamamoto, T.;
Watanabe, S. Pharm. Res. 1998, 15, 1907.
3. Shishido, Y.; Furushiro, M.; Tanabe, S.; Nishiyama, S.;
Hashimoto, S.; Ohno, M.; Yamamoto, T.; Watanabe, S.
Pharmacol. Biochem. Behav. 1996, 55, 333.
4. Shishido, Y.; Furushiro, M.; Tanabe, S.; Shibata, S.;
Hashimoto, S.; Yokokura, T. Eur. J. Pharmacol. 1999,
372, 135.
ˆ
´
22. Beausoleil, E.; L’Archeveque, B.; Belec, L.; Atfani, M.;
Lubell, W. D. J. Org. Chem. 1996, 61, 9447.
23. Sim, T. B.; Rapoport, H. J. Org. Chem. 1999, 64, 2532.
24. Menghin, S.; Pertz, H. H.; Kramer, K.; Seifert, R.;
Schunack, W.; Elz, S. J. Med. Chem. 2003, 46, 5458.
25. In the synthesis of 4-pyridin-3-yl-butyronitrile, NaCN was
used instead of KCN and no purification was needed after
evaporation of DMSO. In the work-up of 4-pyridin-3-yl-
butyric acid, the reaction mixture was washed with DCM
before the adjustment of pH and after continuous extrac-
tion of the product no further purification was needed.
26. Pardridge, W. NeuroRx 2005, 2, 3.
5. Schneider, J. S.; Giardiniere, M.; Morain, P. Neuropsy-
chopharmacology 2002, 26, 176.
27. Tute, M. S. Lipophilicity in Drug Action and Toxicology;
1st ed.; Pliska, V., Testa, B., van de Waterbeemd, H. Eds.;
VCH: Weinheim, 1996; p 7.
6. Morain, P.; Robin, J. L.; De Nanteuil, G.; Jochemsen, R.;
Heidet, V.; Guez, D. Br. J. Clin. Pharmacol. 2000, 50, 350.
7. Jiang, C. H.; Tsien, J. Z.; Schultz, P. G.; Hu, Y. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 1930.
8. Roßner, S.; Schulz, I.; Zeitschel, U.; Schliebs, R.; Bigl, V.;
Demuth, H. U. Neurochem. Res. 2005, 30, 695.
9. Schulz, I.; Gerhartz, B.; Neubauer, A.; Holloschi, A.;
Heiser, U.; Hafner, M.; Demuth, H. U. Eur. J. Biochem.
2002, 269, 5813.
28. Vena¨la¨inen, J. I.; Juvonen, R. O.; Forsberg, M. M.;
´
Garcia-Horsman, J. A.; Poso, A.; Wallen, E. A. A.;
Gynther, J.; Ma¨nnisto¨, P. T. Biochem. Pharmacol. 2002,
64, 463.
29. Atack, J. R.; Suman-Chauhan, N.; Dawson, G.; Kula-
gowski, J. J. Eur. J. Pharmacol. 1991, 205, 157.
30. Lipinski, C. A. J. Pharmacol. Toxicol. Methods 2000, 44,
235.
10. Williams, R. S.; Eames, M.; Ryves, W. J.; Viggars, J.;
Harwood, A. J. EMBO J. 1999, 18, 2734.