Dicarboxylic acid bis(L-prolyl-pyrrolidine) amides as prolyl oligopeptidase inhibitors

Article abstract of DOI:10.1021/jm020966g

New dicarboxylic acid bis(L-prolyl-pyrrolidine) amides were synthesized, and their inhibitory activity against prolyl oligopeptidase from pig brain was tested in vitro. As compared with earlier described prolyl oligopeptidase inhibitors, these new compounds have in common an L-prolyl-pyrrolidine moiety, but the typical lipophilic acyl end group is replaced by another L-prolyl-pyrrolidine moiety connected symmetrically with a short dicarboxylic acid linker. These compounds are a new type of peptidomimetic prolyl oligopeptidase inhibitor.

Full text of DOI:10.1021/jm020966g

J . Med. Chem. 2002, 45, 4581-4584
4581
Brief Articles
Dica r boxylic Acid bis(L-P r olyl-p yr r olid in e) Am id es a s P r olyl Oligop ep tid a se
In h ibitor s
Erik A. A. Walle´n,*,† J ohannes A. M. Christiaans,†,‡ Markus M. Forsberg,§ J arkko I. Vena¨la¨inen,§
Pekka T. Ma¨nnisto¨,§,| and J ukka Gynther†,|
Department of Pharmaceutical Chemistry and Department of Pharmacology and Toxicology, University of Kuopio,
P.O. Box 1627, FIN-70211 Kuopio, Finland, and Finncovery, Ltd., Kuopio, Finland
Received J une 18, 2002
New dicarboxylic acid bis(L-prolyl-pyrrolidine) amides were synthesized, and their inhibitory
activity against prolyl oligopeptidase from pig brain was tested in vitro. As compared with
earlier described prolyl oligopeptidase inhibitors, these new compounds have in common an
L-prolyl-pyrrolidine moiety, but the typical lipophilic acyl end group is replaced by another
L-prolyl-pyrrolidine moiety connected symmetrically with a short dicarboxylic acid linker. These
compounds are a new type of peptidomimetic prolyl oligopeptidase inhibitor.
Ch a r t 1
In tr od u ction
The serine protease prolyl oligopeptidase (POP, previ-
ously called prolyl endopeptidase or postproline cleaving
enzyme, EC 3.4.21.26) is a large intracellular enzyme
(80 kDa) that preferentially hydrolyses proline-contain-
ing oligopeptides at the carboxyl side of a prolyl residue.
It is presumably involved in the maturation and deg-
radation of peptide hormones and neuropeptides.1
A low level of substance P, which is a substrate of
POP, is characteristic in the brains of Alzheimer’s
patients. Supportingly, administration of â-amyloids
decreases substance P levels in the brain. Vice versa,
administration of substance P is able to alleviate amy-
loid peptide-induced toxicity.2
Ch a r t 2
There is no firm evidence of increased POP activity
in Alzheimer’s patients. In contrast, rather low enzyme
activities are correlated with the severity of Alzheimer’s
disease, which is thought to reflect the degree of
neuronal damage.3 However, in aged rats, POP gene
levels are manyfold increased in several brain areas.4
Correspondingly, in mice subjected to an enriched
environment, the POP gene levels are decreased.5 As a
whole, it seems that enhanced levels of neuroactive
peptides such as substance P, angiotensins, vasopressin,
oxytocin, and thyrotropine-releasing hormone, which are
all substrates of POP, may be beneficial in old age and
in patients with cognitive disturbances. Indeed, POP
inhibitors have been able to reverse scopolamine-
induced amnesia in rats.6-8 Centrally acting POP
inhibitors that increase neuropeptide concentrations9
may therefore be worthy of testing in Alzheimer’s
disease.
Most described low molecular weight POP inhibitors
have an acyl-L-prolyl-pyrrolidine backbone, wherein a
lipophilic acyl end group has been reported to be
important for high inhibitory activity.10 Four reference
compounds of this type with different lipophilic acyl end
groups are benzoyl-L-prolyl-pyrrolidine,11 caproyl-L-
prolyl-pyrrolidine,12 SUAM-1221,11-13 and Z-L-prolyl-L-
prolinal.14 SUAM-1221 and Z-L-prolyl-prolinal are the
two most potent ones, and the latter has the pyrrolidine
group replaced by an L-prolinal group, which increases
the activity further. The lipophilic acyl end group is the
P3 site, since it binds to the S3 binding site of the active
site of the enzyme (the P2 site is the L-prolyl residue in
the center of the molecule, and the P1 site is the
pyrrolidine and the L-prolinal end groups, respectively)
(Chart 1).
* To whom correspondence should be addressed. E-mail: wallen@
uku.fi.
† Department of Pharmaceutical Chemistry, University of Kuopio.
‡ Present address: Altana Pharma, P.O. Box 61, NL-1160 AB
Zwanenburg, The Netherlands.
In a new series of POP inhibitors 1a -g, two L-prolyl-
pyrrolidine groups were connected with a short dicar-
boxylic acid linker. These compounds do not have the
typical lipophilic acyl end group found in the most
§ Department of Pharmacology and Toxicology, University of Kuopio.
| Finncovery, Ltd.
10.1021/jm020966g CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/27/2002

4582 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Brief Articles
Sch em e 1a
a
Reagents: (a) L-Proline, NaOH/H2O, diethyl ether. (b) (1) Pivaloyl chloride, Et3N/DCM; (2) pyrrolidine, Et3N/DCM. (c) L-Proline methyl
ester HCl salt, Et3N/DCM. (d) (1) Pivaloyl chloride, Et3N/DCM; (2) L-proline methyl ester HCl salt, Et3N/DCM. (e) LiOH/MeOH, H2O. (f)
(1) Pivaloyl chloride, Et3N/DCM; (2) L-Prolyl-pyrrolidine TFA salt, Et3N/DCM. (g) (1) Pivaloyl chloride, Et3N/DCM; (2) L-Prolinol, Et3N/
DCM. (h) SO3 pyridine complex, Et3N, DMSO. (i) (1) Pivaloyl chloride, Et3N/DCM; (2) 2(S)-Cyanopyrrolidine TFA salt, Et3N/DCM. (j) (1)
Pivaloyl chloride, Et3N/DCM; (2) 2(S)-(acetoxyacetyl)pyrrolidine TFA salt, Et3N/DCM. (k) K2CO3/MeOH, H2O.
Ta ble 1. Inhibitory Activity (IC50) of the Compounds 1 (R ) H)
potent POP inhibitors. The linking dicarboxylic acid in
against POP from Pig Brain (95% Confidence Intervals Given
in Parentheses)
the center of the molecule might act as the lipophilic
P3 moiety, although the lipophilic chain is not very long.
The second L-prolyl-pyrrolidine group will reach the S4
and most probably also the S5 binding sites of the
enzyme. As compared with an oligopeptide substrate,
which is also bound to the same active site, the amide
bond direction in the new compounds is reversed in the
center of the molecule. The new compounds are thereby
a new type of peptidomimetic POP inhibitors (Chart 2).
A series of substituted derivatives of compound 1f
were also synthesized, where the P1 pyrrolidine group
was replaced by either L-prolinal, 2(S)-cyanopyrrolidine,
or 2(S)-(hydroxyacetyl)pyrrolidine, resulting in com-
pounds 2a , 2b, and 2c, respectively. These P1 groups
have earlier been reported to increase the inhibitory
activity.14-18
compd
Q
IC50 (nM)
1a
1b
1c
1d
1e
1f
-(CH2)2-
-(CH2)3-
-(CH2)4-
-CH2C(CH3)2CH2-
para-phenylene
meta-phenylene
ortho-phenylene
77 (59-100)
48 (38-62)
68 (44-110)
13 (12-14)
81 (72-92)
26 (21-32)
1g
21 000 (7800-54 000)
ing compound was activated with pivaloyl chloride and
then reacted with L-prolinol, 2(S)-cyanopyrrolidine, or
2(S)-(acetoxyacetyl)pyrrolidine, respectively. Compound
2a was obtained after the product from the reaction with
L-prolinol was oxidized with SO3 pyridine complex in
dimethyl sulfoxide. Compound 2b was obtained directly
from the reaction with 2(S)-cyanopyrrolidine. Compound
2c was obtained after the product from the reaction with
2(S)-(acetoxyacetyl)pyrrolidine was hydrolyzed with
K2CO3 in water-methanol.
Syn th etic Ch em istr y
The used synthesis routes are outlined in Scheme 1.
The synthesis of 1a was started from succinyl dichloride,
which was reacted with 2 equiv of L-proline. The
resulting compound was activated with 2 equiv of
pivaloyl chloride and then reacted with 2 equiv of
pyrrolidine. The compounds 1b-f were synthesized
using the same synthesis route, starting from glutaryl
dichloride, adipoyl dichloride, 3,3-dimethyl glutaryl
dichloride, terephthaloyl dichloride, and isophthaloyl
dichloride, respectively.
In Vitr o Assa y of P OP Activity
The inhibitory activity of the compounds against POP
from pig brain was determined by a method based on
that of Toide et al.19 Suc-Gly-Pro-7-amido-4-methyl-
coumarin was used as substrate, and the formation of
7-amido-4-methylcoumarin was determined fluoro-
metrically with a microplate fluorescence reader.
Compound 1g was synthesized starting from phthalic
anhydride, which was reacted with L-proline methyl
ester. The resulting compound was activated with
pivaloyl chloride and then reacted with L-proline methyl
ester. The methyl ester groups were hydrolyzed with
LiOH in water-methanol. The resulting compound was
activated with 2 equiv of pivaloyl chloride and then
reacted with 2 equiv of pyrrolidine.
The synthesis of compounds 2a -c was started from
isophthalic acid monomethyl ester, which was activated
with pivaloyl chloride and then reacted with L-prolyl-
pyrrolidine. The methyl ester group was hydrolyzed
with LiOH in water-methanol. The resulting compound
was activated with pivaloyl chloride and then reacted
with L-proline methyl ester. The methyl ester group was
hydrolyzed with LiOH in water-methanol. The result-
Resu lts a n d Discu ssion
In the series of compounds 1, the linking group Q was
varied (Table 1). A flexible alkylene linker gives very
potent compounds 1a -c, the glutaric acid derivative 1b
being the most active one. The optimum chain length
seems to be three carbon atoms. A branched alkylene
with the optimum chain length results in an even more
potent compound, as is indicated by the 3,3-dimethyl
glutaric acid derivative 1d . Compounds with rigid para-,
meta-, and ortho-phenylene linkers have a wider range
of inhibitory activities. The terephthalic acid derivative
1e and the isophthalic acid derivative 1f are very potent
compounds, the latter being the more active one. On the
other hand, the phthalic acid derivative 1g is almost
inactive. These rigid compounds seem to require a

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20 4583
Ta ble 2. Inhibitory Activity (IC50) of the Compounds 2 (Q )
m-Phenylene) against POP from Pig Brain (95% Confidence
Intervals Given in Parentheses)
pounds are structurally made of two L-prolyl-pyrrolidine
moieties connected symmetrically with a short dicar-
boxylic acid linker. The dicarboxylic acid linker in the
center of the molecule has a strong effect on the
inhibitory activity. The two best dicarboxylic acids are
3,3-dimethylglutamic acid and isophthalic acid. Replac-
ing one of the pyrrolidine ends of the symmetrical
molecule with L-prolinal, 2(S)-cyanopyrrolidine, or 2(S)-
(hydroxyacetyl)pyrrolidine increases the inhibitory ac-
tivity strongly. The most potent new inhibitor has an
IC50 value of 0.39 nM.
compd
R
IC50 (nM)
2a
2b
2c
CHO
CN
COCH2OH
1.3 (1.1-1.6)
1.5 (1.1-2.2)
0.39 (1.1-2.2)
Ta ble 3. Inhibitory Activity (IC50) of Reference Compounds
against POP from Pig Brain (95% Confidence Intervals Given
in Parentheses)
ref compd
IC50 (nM)
benzoyl-L-prolyl-pyrrolidine
SUAM-1221
Z-L-prolyl-L-prolinal
66 (38-110)
2.2 (1.9-2.5)
0.33 (0.24-0.47)
Exp er im en ta l Section
Cou p lin g
a Dica r boxylic Acid Dich lor id e w ith L-
P r olin e. A solution of 1.0 mmol dicarboxylic acid dichloride
in a suitable organic solvent was added to a solution of 2.0-
2.2 mmol L-proline and 4.0-6.0 mmol NaOH in water at 0 °C.
The reaction was stirred vigorously for several hours at room
temperature. The organic phase was separated, and the
aqueous phase was made acidic. The aqueous phase was
evaporated, and the residue was dissolved in dichloromethane
or chloroform, or alternatively, the product was extracted from
the aqueous phase with ethanol-chloroform. The organic
phase was dried and evaporated.
Am id e F or m a tion Usin g P iva loyl Ch lor id e Activa tion .
A 1.0 mmol amount of pivaloyl chloride was added to a solution
of 1.0 mmol of carboxylic acid (or 0.5 mmol of dicarboxylic acid)
and 1.1 mmol of triethylamine in dichloromethane at 0 °C.
The reaction was stirred at 0 °C for 1 h. A solution of 1.1 mmol
of triethylamine and 1.0-1.1 mmol of amine in dichloro-
methane was added slowly at 0 °C, or if the amine was in the
form of a trifluoroacetic acid salt or HCl salt, then 2.2-3.3
mmol of triethylamine was used and the triethylamine was
added separately before the addition of the amine. The reaction
mixture was stirred for several hours at room temperature.
The dichloromethane solution was washed with 30% citric acid,
saturated NaCl, and saturated NaHCO3. The dichloromethane
phase was dried and evaporated.
Cou p lin g of P h th a lic An h yd r id e w ith L-P r olin e Meth -
yl Ester . A 1.5 g (10 mmol) amount of phthalic anhydride was
added to a solution of 1.5 g (10 mmol) of L-proline methyl ester
HCl salt and 3.1 mL (22 mmol) of triethylamine in di-
chloromethane at 0 °C. The reaction was stirred for 2 h at room
temperature. The organic phase was extracted with saturated
NaHCO3. The aqueous phase was made acidic, and the
aqueous phase was extracted with chloroform. The chloroform
phase was dried and evaporated.
Hyd r olysis of a Meth yl Ester . A 1.1-1.5 mmol amount
of LiOH was added to a solution of 1.0 mmol of the methyl
ester (or 3.0 mmol of LiOH in the case of a dicarboxylic acid
dimethyl ester) in water-methanol. The reaction was stirred
overnight or longer at room temperature. The methanol was
evaporated, and the residue was dissolved in water. The
aqueous phase was washed with dichloromethane. The aque-
ous phase was made acidic, and the product was extracted with
dichloromethane. The dichloromethane phase was dried and
evaporated.
correct geometry for high activity. In the series of
compounds 1, the 3,3-dimethylglutaric acid derivative
1d and the isophthalic acid derivative 1f are the most
potent inhibitors, with IC50 values of 13 and 26 nM,
respectively.
The second series of compounds 2 are derivatives of
compound 1f, wherein one of the pyrrolidine ends is
replaced by either L-prolinal, 2(S)-cyanopyrrolidine, or
2(S)-(hydroxyacetyl)pyrrolidine, resulting in compounds
2a , 2b, and 2c, respectively (Table 2). All three com-
pounds 2a -c are very potent inhibitors with IC50 values
ranging from 0.39 to 1.5 nM.
The increased potency of the compounds of series 2
results from the interaction of the formyl, cyano, and
hydroxyacetyl groups, respectively, with the active
serine residue (Ser554) of the POP enzyme. The formyl
group is known to form a hemiacetal adduct with the
active serine residue.20 The hemiacetal adduct mimics
the tetrahedral transition state of the enzyme-catalyzed
reaction. Therefore, it can be classified as a transition
state analogue inhibitor. It is very likely that the cyano
and hydroxyacetyl groups interact via a similar mech-
anism with the active serine residue, forming an imino
ether and a hemiketal adduct, respectively. The com-
pounds of series 1 mimic the substrate, and they do not
form a covalent adduct with the enzyme, which makes
these compounds substrate analogue inhibitors.
The impact of the second L-prolyl-pyrrolidine moiety
is best seen by comparing the compounds 1e-g (Table
1) with benzoyl-L-prolyl-pyrrolidine, which has an IC50
value of 66 nM in our in vitro assay (Table 3). The only
difference between the new compounds and this refer-
ence compound is the second L-prolyl-pyrrolidine moiety
connected from a second carboxylic acid group. The
meta-substituted compound 1f has an IC50 value of 26
nM, and it is thereby more active than the reference
compound. On the other hand, the para-substituted
compound 1e is slightly less active and the ortho-
substituted compound 1g is almost inactive as compared
to the reference compound. The two very potent refer-
ence compounds SUAM-1221 and Z-L-prolyl-L-prolinal
have also been tested in our in vitro assay, and their
IC50 values are 2.2 and 0.33 nM, respectively (Table 3).
These reference compounds and our best new com-
pounds are equipotent.
Oxid a t ion w it h SO3 P yr id in e Com p lex in Dim et h yl
Su lfoxid e. A 370 mg (0.75 mmol) amount of isophthalic acid
L-prolyl-pyrrolidine L-prolyl-L-prolinol amide and 0.31 mL (2.3
mmol) of triethylamine were dissolved in 5 mL of anhydrous
dimethyl sulfoxide. A solution of 360 mg (2.3 mmol) of SO3
pyridine complex dissolved in 5 mL of anhydrous dimethyl
sulfoxide was added at room temperature, and the reaction
was stirred for 2 h. The reaction mixture was poured in 50
mL of ice-water. The product was extracted with chloroform.
The chloroform phase was washed with 30% citric acid,
saturated NaCl, and saturated NaHCO3. The chloroform phase
was dried and evaporated.
Con clu sion s
Hyd r olysis of a n Acet yl E st er . A 270 mg (1.94 mmol)
amount of K2CO3 was added to a solution of 1.0 g (1.76 mmol)
of isophthalic acid L-prolyl-2(S)-(acetoxyacetyl)pyrrolidine L-
New potent POP inhibitors with a symmetrical pep-
tidomimetic structure were synthesized. These com-

4584 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 20
Brief Articles
prolyl-pyrrolidine amide in 10 mL of 50% methanol in water
at 0 °C. The reaction was stirred for 1 h at room temperature.
The methanol was evaporated, and the residue was dissolved
in dichloromethane and saturated NaCl. The phases were
separated, and the dichloromethane phase was washed with
saturated NaCl. The dichloromethane phase was dried and
evaporated.
students Mr. J anne Lo¨tjo¨nen and Mr. Mikko Gynther
for helping us by performing some synthetic steps.
Su p p or tin g In for m a tion Ava ila ble: Complete experi-
mental details. This material is available free of charge via
the Internet at http://pubs.acs.org.
Su ccin ic Acid bis(L-P r olyl-p yr r olid in e) Am id e (1a ). MS
(ESI) m/z ) 419 (MH+). 13C NMR (CDCl3): δ 24.11, 24.67,
26.21, 28.80, 28.87, 45.90, 46.22, 47.15, 57.83, 170.69 (same
shift for both carbonyls). Anal. (C22H34N4O4‚1.4H2O) C, H, N.
Glu ta r ic Acid bis(L-P r olyl-p yr r olid in e) Am id e (1b). MS
(ESI) m/z ) 433 (MH+). 13C NMR (CDCl3): δ 19.80, 24.08,
24.81, 26.16, 28.86, 33.28, 45.86, 46.24, 47.32, 57.69, 170.65,
171.27. Anal. (C23H36N4O4‚0.7H2O) C, H, N.
Ad ip ic Acid bis(L-P r olyl-p yr r olid in e) Am id e (1c). MS
(ESI) m/z ) 447 (MH+). 13C NMR (CDCl3): δ 24.14, 24.29,
24.84, 26.21, 28.87, 34.26, 45.87, 46.28, 47.28, 57.63, 170.75,
171.38. Anal. (C24H38N4O4‚1.0H2O) C, H, N.
3,3-Dim eth yl Glu ta r ic Acid bis(L-P r olyl-p yr r olid in e)
Am id e (1d ). MS (ESI) m/z ) 461 (MH+). 13C NMR (CDCl3):
δ 24.13, 24.94, 26.22, 28.39, 28.86, 34.30, 44.32, 45.91, 46.20,
48.12, 57.72, 170.74, 170.78. Anal. (C25H40N4O4‚1.0H2O) C, H,
N.
Ter ep h t h a lic Acid bis(L-P r olyl-p yr r olid in e) Am id e
(1e). MS (ESI) m/z ) 467 (MH+). 13C NMR (CDCl3): δ 24.19,
25.57, 26.24, 28.98, 46.03, 46.41, 50.24, 58.15, 127.29, 138.00,
168.71, 170.35. Anal. (C26H34N4O4‚0.4H2O) C, H, N.
Isop h th a lic Acid bis(L-P r olyl-p yr r olid in e) Am id e (1f).
MS (ESI) m/z ) 467 (MH+). 13C NMR (CDCl3): δ 24.18, 25.57,
26.25, 28.99, 46.04, 46.42, 50.30, 58.23, 126.06, 128.24, 129.12,
136.60, 168.61, 170.34. Anal. (C26H34N4O4‚0.2H2O) C, H, N.
P h th a lic Acid bis(L-P r olyl-p yr r olid in e) Am id e (1g). MS
(ESI) m/z ) 467 (MH+). 13C NMR (CDCl3): δ 23.30, 23.97,
24.13, 25.19, 25.89, 26.23, 29.09, 30.54, 45.64, 45.79, 45.99,
46.21, 46.86, 49.93, 57.51, 59.19, 126.89, 127.37, 128.26,
128.87, 134.78, 135.60, 168.75, 168.78, 170.45, 170.80. Anal.
(C26H34N4O4‚0.7H2O) C, H, N.
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Isop h th a lic Acid L-P r olyl-p yr r olid in e L-P r olyl-L-p r o-
lin a l Am id e (2a ). MS (ESI) m/z ) 495 (MH)+. 13C NMR
(CDCl3) δ 24.19, 25.04, 25.52, 25.55, 25.55, 26.24, 28.98, 29.26,
46.04, 46.40, 47.08, 50.27, 50.29, 58.05, 58.24, 64.89, 126.04,
128.30, 128.99, 129.19, 136.34, 136.69, 168.53, 168.73, 170.29,
171.38, 198.92. Anal. (C27H34N4O5‚2.2H2O) C, H, N.
Isop h th a lic Acid L-P r olyl-2(S)-cya n op yr r olid in e L-P r o-
lyl-p yr r olid in e Am id e (2b). MS (ESI) m/z ) 492 (MH)+. 13
C
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NMR (CDCl3) δ 23.01, 24.20, 25.43, 25.56, 26.25, 28.98, 29.80,
30.84, 46.05, 46.42, 46.50, 46.63, 50.27, 50.30, 58.03, 58.25,
118.63, 126.09, 128.36, 128.93, 129.29, 136.12, 136.77, 168.50,
168.83, 170.31, 171.13. Anal. (C27H33N5O4‚1.0H2O) C, H, N.
Isop h t h a lic Acid L-P r olyl-2(S)-(h yd r oxya cet yl)p yr -
r olid in e L-P r olyl-p yr r olid in e Am id e (2c). MS (ESI) m/z )
525 (MH)+. 13C NMR (CDCl3) δ 24.20, 25.39, 25.51, 25.55,
26.25, 28.25, 28.77, 28.99, 46.04, 46.41, 47.14, 47.20, 50.28,
58.04, 58.21, 61.16, 67.15, 126.08, 128.29, 128.96, 129.18,
136.32, 136.73, 168.52, 168.73, 170.30, 170.91, 209.03. Anal.
(C28H36N4O6‚0.4H2O) C, H, N.
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The yields for all synthesis routes mentioned above varied
between 7 and 55%.
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Ack n ow led gm en t. This research was supported by
Finncovery Ltd. and the National Technology Agency
in Finland (TEKES). We also thank Ms. Tiina Koivunen
and Ms. Pa¨ivi Sutinen for their outstanding technical
assistance, Prof. Dr. J ouko Vepsa¨la¨inen for his help in
assigning the NMR spectra, senior assistant Dr. Seppo
Auriola for performing the ESI-MS analysis, and the
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J M020966G