2(S)-(Cycloalk-1-enecarbonyl)-1-(4-phenyl-butanoyl)pyrrolidines and 2(S)-(aroyl)-1-(4-phenylbutanoyl)pyrrolidines as prolyl oligopeptidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.12.036

In order to replace the P2-P1 amide group, different 1-cycloalkenyls and 2-aryls were studied in the place of the P1 pyrrolidine group of a 4-phenylbutanoyl-l-Pro-pyrrolidine structure, which is a well-known prolyl oligopeptidase inhibitor SUAM-1221. The

Full text of DOI:10.1016/j.bmc.2006.12.036

Bioorganic & Medicinal Chemistry 15 (2007) 2024–2031
2(S)-(Cycloalk-1-enecarbonyl)-1-(4-phenyl-butanoyl)pyrrolidines
and 2(S)-(aroyl)-1-(4-phenylbutanoyl)pyrrolidines as
prolyl oligopeptidase inhibitors
Elina M. Jarho,a,* Jarkko I. Venalainen,b Sami Poutiainen,a Harri Leskinen,a
¨ ¨
Jouko Vepsa¨la¨inen,c Johannes A. M. Christiaans,a,ꢀ Markus M. Forsberg,b
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 31 October 2006; revised 8 December 2006; accepted 22 December 2006
Available online 24 December 2006
Abstract—In order to replace the P2–P1 amide group, different 1-cycloalkenyls and 2-aryls were studied in the place of the P1
pyrrolidine group of a 4-phenylbutanoyl-L-Pro-pyrrolidine structure, which is a well-known prolyl oligopeptidase inhibitor
SUAM-1221. The 1-cyclopentenyl and the 2-thienyl groups gave novel compounds, which were equipotent with the corresponding
pyrrolidine-analog SUAM-1221. It was shown that the P2–P1 amide group of POP inhibitors can be replaced by an a,b-unsaturated
carbonyl group or the aryl conjugated carbonyl group.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
that POP has an important role in the neuropeptide
metabolism.9 The intracellular functions of POP started
to be revealed only in the late 1990s. Inhibition of POP
has been shown to elevate the intracellular IP3 level,
which was suggested to explain the memory-enhancing
properties of POP inhibitors.10,11 Recently, a specific
POP inhibitor Z-Pro-prolinal was shown to prevent the
translocation of glyceraldehyde-3-phosphate dehydroge-
nase from the cytosol to the nucleus and, to prevent the
formation of reactive oxygen species in monkey fibro-
blasts, which had been exposed to 6-hydroxydopamine.12
Furthermore, POP has been proposed to have a role in
protein trafficking and secretion.13
Human prolyl oligopeptidase (POP, EC 3.4.21.26) is an
80 kDa serine protease that was discovered in the begin-
ning of the 1970s.1 POP is a proline-specific peptidase
that hydrolyzes oligopeptides after prolyl residues and
has mainly been described as a cytosolic enzyme. POP
has gained pharmaceutical interest because specific
POP inhibitors have been shown to ameliorate age-
related and induced amnesia in laboratory animals.2–6
Furthermore, the expression of the POP gene is up-reg-
ulated in the hypothalamus7 and the hippocampus8 of
aged mice. Certain memory-enhancing neuropeptides,
like substance P, arginine-vasopressin, and thyroliberin,
are in vitro substrates of POP and it has been suggested
POP is a proline-specific peptidase and the S1 binding
site of POP has evolved to fit the pyrrolidine ring of
an L-prolyl residue.14 The pyrrolidine group is unques-
tionably the most utilized structure at the P1 position
of POP inhibitors and only few successful replacements
of the P1 pyrrolidine ring have been described in the
literature.15,16 An electrophilic substituent, like a 2(S)-
formyl or a 2(S)-cyano group, at the 2-position of the
pyrrolidine ring increases significantly the potency.17,18
However, these groups are chemically reactive, which
may limit their usability in drugs.
Keywords: Prolyl oligopeptidase; a,b-Unsaturated carbonyl group;
2-Thiophene; 1-Cyclopentene.
*
Corresponding author. Tel.: +358 17 162460; fax: +358 17 162456;
e-mail: Elina.Jarho@uku.fi
ꢀ
Present address: Altana Pharma bv, PO Box 31, 2130 AA Hoofd-
dorp, The Netherlands.
ꢁ
Present address: Division of Pharmacology and Toxicology, Faculty
of Pharmacy, University of Helsinki, PO Box 56, FI-00014 Helsinki,
Finland.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.036

E. M. Jarho et al. / Bioorg. Med. Chem. 15 (2007) 2024–2031
2025
In a peptide chain, a prolyl residue lacks the main chain
I
O
NNH2
amide hydrogen. Consequently, the P1 pyrrolidine
group of POP inhibitors (or the substrates of POP)
is not stabilized by hydrogen bonding.14,19 We have
previously replaced the P1 pyrrolidine group of the
well-known reference compound SUAM-122120 1a by
a series of alkyl groups and a phenyl group.21 To our
surprise the cyclopentyl and the phenyl groups gave
equipotent compounds 1b and 1c (Fig. 1). These
compounds were moderately potent but still one order
of magnitude less potent than the reference compound
1a. In the present study, the field between these two
replacements was further studied, focusing on mimick-
ing the amide bond geometry.
i
i i
n
n
n
n = 1 or 2
2a; n = 1
2b; n = 2
3a; n = 1
3b; n = 2
Scheme 1. Reagents and condition: (i) Hydrazine monohydrate/reflux;
(ii) 1—I2, tetramethylguanidine/Et2O; 2—compound 2a or 2b/Et2O.
ii for 5c
iii for 5a,b,d,e
i
N
R
N
H
N
R
Boc
Boc
Boc
O
O
OH
4a-e
5a-e
Boc-L-prolinal
iv
2. Chemistry
4a-6a;
4b-6b;
R = 1-cyclopentenyl
R = 1-cyclohexenyl
The synthetic routes are presented in Schemes 1–3.
Cyclopentylidene-hydrazine (2a) and cyclohexylidene-
hydrazine (2b) were prepared by the reaction of
hydrazine monohydrate with cyclopentanone and cyclo-
hexanone, respectively. While the reaction with cyclo-
pentanone gave N,N0-dicyclopentylidene-hydrazine as
a minor side-product (about 5 mol %), the reaction with
cyclohexanone produced a significant amount of N,N0-
dicyclohexylidene-hydrazine (about 20 mol %). All
attempts to distill 2b led to the decomposition of the
product. The unpurified compounds 2a and 2b were
reacted with iodine in the presence of tetramethylguani-
dine to yield 1-iodocyclopentene (3a) and 1-iodocyclo-
hexene (3b), respectively, which were distilled before
further use.22 Compounds 3a and 3b were treated with
tert-BuLi, and 2-bromopyridine and furan were treated
with n-BuLi to obtain the corresponding organolithium
reagents, which were reacted with Boc-L-prolinal to give
compounds 4a-c and 4e respectively. Compound 4d was
obtained from the reaction of the commercially avail-
able 2-thienyllithium and Boc-L-prolinal. Compounds
4a–e were oxidized with Dess–Martin periodinane or
SO3Æpyridine/dimethylsulfoxide and deprotected. The
resulting amines were coupled with 4-phenylbutyric acid
using EDC and HOBt to yield compounds 6a–e. Boc-L-
proline and Boc-D-proline were activated with ethyl
chloroformate and reacted with benzylamine or
N
R
4c-6c; R = 2-pyridyl
4d-6d; R = 2-thienyl
O
O
R = 2-furyl
4e-6e;
6a-e
Scheme 2. Reagents and conditions: (i) organolithium reagent (pre-
pared in situ or commercial reagent)/tetrahydrofuran, ꢀ80 ꢁC; (ii)
Et3N, SO3Æpyridine/dimethylsulfoxide; (iii) Dess–Martin periodinane/
CH2Cl2; (iv) 1—trifluoroacetic acid/ CH2Cl2; 2—4-phenylbutyric acid,
HOBtÆH2O, EDCÆHCl, Et3N/ CH2Cl2, 0–20 ꢁC.
i
H
7a; S, n = 1
HO
N
N
N
7b; R, n = 1
7c; S, n = 2
Boc
Boc
n
O
O
Boc-L-proline or
Boc-D-proline
ii, iii
N
8a; S, n = 1
8b; R, n = 1
8c; S, n = 2
H
N
n
O
O
Scheme 3. Reagents and conditions: (i) 1—Et3N, ethyl chloroformate/
tetrahydrofuran, ꢀ15 ꢁC, 2—benzylamine/tetrahydrofuran, ꢀ15 to
20 ꢁC; (ii) 1. trifluoroacetic acid/CH2Cl2, 0 ꢁC; (iii) 1. 1-cyclopentene-1-
carboxylic acid, Et3N, trimethylacetyl chloride/CH2Cl2, 0 ꢁC; 2—
Et3N, trifluoroacetic acid salt of the deprotected 6 from reaction (ii).
N
N
O
O
1a (SUAM-1221); IC50 = 2.2 nM
N
N
O
O
O
O
1b; IC50 = 30 nM
Figure 1. The reference compound 1a20 and the previously reported compounds 1b and 1c.21
1c; IC50 = 23 nM

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E. M. Jarho et al. / Bioorg. Med. Chem. 15 (2007) 2024–2031
phenethylamine to yield compounds 7a–c. 1-Cyclopen-
tene-1-carboxylic acid was activated with trimethylace-
tyl chloride and reacted with 7a–c after the removal of
the Boc-protection group to yield compounds 8a–c.
compared to compound 1c with the phenyl group at
the P1 site, compound 6c with the 2-pyridyl group had
strongly decreased inhibitory activity. In contrast, com-
pound 6d, with the 2-thienyl group at the P1 site, was
one order of magnitude more potent than compound
1c and equipotent with compound 6a. The difference be-
tween compounds 6d and 1c can be explained by the
smaller size of the 2-thienyl group. The 1-cyclopentenyl
and the 2-thienyl groups, instead, are almost equal in
size and compound 6d does not seem to have any bene-
ficial p-stacking over 6a. The potency of compound 6e
with the 2-furyl group at the P1 site was slightly
decreased but of the same order of magnitude as the
potency of compound 6d.
3. Results and discussion
Compounds 6a–e are presented in Table 1. The P1 pyr-
rolidine ring of the reference compound 1a (Fig. 1) has
been replaced by a 1-cyclopentene ring in compound
6a. A similar replacement at the P2 position has previ-
ously been shown to give potent POP inhibitors.23 In-
deed, compound 6a was equipotent with the reference
compound 1a. Compound 6a was one order of magni-
tude more potent than the previously reported com-
pound 1b with the cyclopentyl ring at the P1 site. The
difference in their potencies can be explained by differ-
ences in geometry and p-stacking. The P1 amide nitro-
gen is sp2 hybridized and, although the replacement by
an sp3 carbon was tolerated (IC50 of compound 1b
was 30 nM), an sp2 carbon provides a better mimetic
for the amide nitrogen. Furthermore, the double bond
may have beneficial p-stacking with the indole ring of
Trp595.14
The 2-thienyl group has previously been studied at the
P1 position of POP inhibitors but with opposite results.
It gave a poor POP inhibitor against Flavobacterium
meningosepticum POP.15 On the other hand, an unusual
POP inhibitor Y-29794 possesses a 2-thienyl group
although it is not known where it binds. Y-29794 was
reported to have a Ki value of 0.95 nM against rat
POP.16 The excellent POP inhibitory activity of com-
pound 6d indicates that the 2-thienyl group of Y-
29794 may bind to the S1 pocket of POP. It also seems
that the S1 subsites of the mammalian and the bacterial
POP have differences.
The enlargement of the 1-cyclopentenyl group of com-
pound 6a resulted in compound 6b with a 1-cyclohexe-
nyl group at the P1 position. Compound 6b was 20·
less potent than compound 6a, which reflects the limited
space in the S1 pocket.14 However, we have previously
reported that a compound with the same skeleton as
6b but with a fully saturated cyclohexyl group at the
P1 site gives only week POP inhibitory activity
(IC50 = 1.1 lM). Compound 6b was 18· more potent
than this compound, while it was only 2.6· less potent
than the reference compound 1c, which has the phenyl
group at the P1 site. This small decrease in potency in
relation to compound 1c may be explained by the small-
er size of the phenyl ring or loss in p-stacking with
Trp595.
The use of the 1-cyclopentenyl group at the P1 position
allowed further modifications on the backbone. Com-
pounds 8a–c (Table 2) were prepared in order to study
whether it is possible to reverse the P2 prolyl residue;
the P1–P2 carbonyl group was kept in the original posi-
tion but the direction of the amide bond was changed.
Consequently, compounds 8a–c possess the a,b-unsatu-
rated amide group instead of the a,b-unsaturated ketone
group. a,b-Unsaturated ketones are usually avoided in
drug design because of their ability to undergo Michael
additions to the double bond. a,b-Unsaturated amides
may be less reactive due to the electron-donating effect
of the amide nitrogen. Compound 8a with a reversed
L-prolyl residue at the P2 position had an IC50 value
of 210 nM. The potency was significantly decreased as
compared to compound 6a. A D-prolyl residue was also
studied but the resulting compound 8b was over 200·
less active than compound 8a with the L-prolyl residue.
The P3 chain of compound 8a was elongated with one
methylene group resulting in compound 8c, which had
improved inhibitory activity. This reflects the known
In order to study different aryls at the P1 position, the 2-
pyridyl, 2-thienyl, and 2-furyl groups were selected. The
pyridyl group has previously been successfully employed
at the P10 and P3 positions of POP inhibitors.24,25 As
Table 1. Inhibitory activities with 95% confidence intervals of the
compounds with different 1-cycloalkenyl and 2-aryl groups at the P1
position
Table 2. Inhibitory activities with 95% confidence intervals of the
compounds with the reversed prolyl residue at the P2 position
N
R
H
O
O
*
N
N
n
a
IC50 (nM)
Compound
R
O
O
6a
6b
6c
6d
6e
1-Cyclopentenyl
1-Cyclohexenyl
2-Pyridyl
3.0 (2.7–3.4)
59 (52–69)
a
IC50 (nM)
Compound
n
Stereo center
510 (450–570)
3.0 (2.7–3.4)
5.2 (4.3–6.1)
8a
8b
8c
1
1
2
S
R
S
210 (180–240)
44,000 (28,000–67,000)
110 (91–130)
2-Thienyl
2-Furyl
a The IC50 values were determined against POP from porcine brain.26
a The IC50 values were determined against POP from porcine brain.26

E. M. Jarho et al. / Bioorg. Med. Chem. 15 (2007) 2024–2031
2027
structure–activity relationships of the P3 site suggesting
that the 1-cyclopentenyl group of compounds 8a–c also
binds to the S1 site of POP.
mixture was stirred for 2.5 h. Compound 2a (4.9 g,
about 50 mmol) in Et2O (50 mL) was added dropwise
during 2 h and the mixture was stirred overnight. The
mixture was refluxed for 2 h, cooled down to rt, and fil-
tered. The filtrate was concentrated from Et2O, heated
up to 90–95 ꢁC for 3 h, cooled to rt, and diluted with
Et2O. The organic phase was washed with 1 M HCl
aq, 20% Na2S2O3, satd NaHCO3 aq, and satd NaCl
aq, dried over Na2SO4, filtered, and evaporated (with
a rotary evaporator at 40 ꢁC and 100 mbar as the lowest
pressure point). The product was stored at ꢀ18 ꢁC un-
der argon and was distilled before further use at 39 ꢁC
4. Conclusions
The 1-cycloalkenyl groups at the P1 position gave better
POP inhibitory activities than the corresponding fully
saturated cycloalkyl groups. The 1-cyclopentenyl group
gave the most potent compound, which was equipotent
with the reference compound 1a. In the series of the aro-
matic groups, the 2-thienyl group gave the most potent
compound. This confirms again that the 5-membered
ring is preferred. The reversed P2 L-prolyl residue de-
creased potency significantly but the effect of the re-
versed D-prolyl residue was much more pronounced.
In conclusion, the P2–P1 amide group of POP inhibitors
can be replaced by the a,b-unsaturated carbonyl group
or the aryl conjugated carbonyl group.
1
and 6 mbar. Yield 5.6 g, 42% for two steps. H NMR
d 1.93 (m, 2H), 2.32 (m, 2H), 2.60 (m, 2H), 6.10 (m, 1H).
5.2.2. 1-Iodo-cyclohexene (3b). Prepared according to
method A from cyclohexanone (10.4 mL, 100 mmol).
Step 1 (3a). Crude product 10.8 g. Step 2 (3b). Tetra-
methylguanidine (1.16 mol), I2 (212 mmol). Compound
3b was distilled at 50 ꢁC and 1 mbar. Yield 9.9 g, 47%
1
for two steps. H NMR d 1.63–1.72 (m, 4H), 2.09 (m,
2H), 2.50 (m, 2H), 6.34 (m, 1H).
5. Experimental
5.3. Method B
5.1. General methods
5.3.1. ((S)-1-Boc-pyrrolidin-2-yl)-cyclopent-1-enyl-metha-
nol (4a). 1.7 M tert-BuLi (17 mL, 29 mmol) was added
dropwise to 3a (5.6 g, 29 mmol) in THF (30 mL) and
the mixture was stirred for 1 h at ꢀ80 ꢁC. Boc-L-prolinal
(1.9 g, 9.5 mmol) in THF (10 mL) was added dropwise
and the mixture was stirred for 3 h at ꢀ80 ꢁC. The reac-
tion was quenched with satd NH4Cl aq at ꢀ80 ꢁC. The
mixture was let to warm up to rt, diluted with H2O, and
extracted with DCM. The organic phase was washed
with satd NaCl aq, dried over Na2SO4, filtered, and
evaporated. The product was purified by flash-chroma-
tography (15–50% EtOAc in hexane). Yield 1.9 g, 75%.
1H and 13C NMR spectra were recorded on a Bruker
Avance 500 spectrometer in CDCl3. Chemical shifts
(d) are given in ppm downfield from the internal stan-
dard tetramethylsilane (d 0.00). The N-amide bond of
a proline residue has energetically similar cis and trans
isomers (rotamers). These rotamers have slightly differ-
ent shifts. Minor rotamers that are less than 20% of
the major rotamers are not reported for 13C spectra.
Electrospray ionization mass spectra (ESI-MS) were ob-
tained on a LCQ ion trap mass spectrometer equipped
with an electrospray ionization source (Finnigan
MAT, San Jose, CA). Elemental analyses (CHN) were
carried out with a Thermo Quest CE Instruments EA
1110 CHNS-O elemental analyzer. Flash chromatogra-
phy was performed on J. T. Bakers silica gel for chroma-
5.3.2. ((S)-1-Boc-pyrrolidin-2-yl)-cyclohex-1-enyl-metha-
nol (4b). Prepared according to method B from 3b (3.1 g,
15 mmol) and Boc-L-prolinal (1.0 g, 5 mmol). The prod-
uct was purified twice by flash-chromatography. One
diastereomer was purified with 15% EtOAc in petroleum
ether (PE) but the other diastereomer co-eluted with the
unreacted Boc-prolinal. The other diastereomer was
purified with 5% acetonitrile in DCM. The diastereo-
mers were combined giving the total yield of 0.54 g,
38%.
˚
tography (pore size 60 A, particle size 50 lm). The silica
plate for the chromatotron was made of Merck silica gel
60 PF254 containing gypsum (particle size 5–40 lm).
Diethyl ether, furan, and tetrahydrofuran (THF) were
distilled from sodium and organolithium reactions were
performed under argon.
5.2. Method A
5.4. ((S)-1-Boc-pyrrolidin-2-yl)-pyridin-2-yl-methanol
(4c)
5.2.1. 1-Iodo-cyclopentene (3a). Step 1 (2a). Cyclopenta-
none (8.8 mL, 100 mmol) was added dropwise to hydra-
zine monohydrate (30 mL, 620 mmol) under vigorous
stirring. The mixture was refluxed for 2 h and cooled
down to room temperature (rt). Dichloromethane
(DCM) was cautiously added at cold water bath and
the phases were separated. The organic phase was
washed with saturated (satd) NaCl aq, dried over
Na2SO4, filtered, and evaporated. The product was used
without further purification. Crude product 7.1 g. Step 2
(3a). Tetramethylguanidine (75 mL, 600 mmol) in Et2O
(110 mL) was added dropwise to iodine (28 g,
110 mmol) in Et2O (160 mL) during 1.5 h and the
To a solution of 2-bromopyridine (3.2 g, 20 mmol) in
THF (40 mL) was added dropwise 1.6 M n-BuLi
(12.5 mL, 20 mmol) at ꢀ70 ꢁC and the mixture was stir-
red for 1 h. Boc-L-prolinal (3.6 g, 18 mmol) in THF
(40 mL) was added and the mixture was stirred for 2 h
at ꢀ70 ꢁC. The mixture was poured in water and THF
was evaporated. The water phase was extracted with
DCM and the organic phase was extracted with 0.1 M
HCl aq The water phase was made basic with 2 M
NaOH aq, extracted with DCM, dried over Na2SO4,
filtered, and evaporated. Yield 2.8 g, 56%.

2028
E. M. Jarho et al. / Bioorg. Med. Chem. 15 (2007) 2024–2031
5.5. ((S)-1-Boc-pyrrolidin-2-yl)-thiophen-2-yl-methanol
(4d)
fied with a chromatotron (1% EtOH in CHCl3). Yield
0.37 g, 61%. H NMR d 1.24 (s, 5.4H), 1.46 (s, 3.6H),
1
1.88–1.98 (m, 3H), 2.38–2.45 (m, 1 H), 3.48–3.70 (m,
2H), 5.69 (dd, 0.6H), 5.78 (dd, 0.4H), 7.44 (ddd,
J = 1.0, 4.8, 7.6 Hz, 0.4H), 7.48 (ddd, J = 1.1, 4.8,
7.7 Hz, 0.6H), 7.82 (td, J = 1.6, 7.7 Hz, 0.4H), 7.86 (td,
J = 1.7, 7.8 Hz, 0.6H), 8.08 (dt, J = 1.0, 7.8 Hz, 1H),
8.66 (d, 0.4H), 8.68 (d, J = 4.8 Hz, 0.6H).
To a solution of Boc-L-prolinal (0.31 g, 1.6 mmol) in
THF was added 1 M 2-thienyllithium solution in THF
(1.75 mL, 1.75 mmol) at ꢀ70 ꢁC. The mixture was stir-
red for 3 h and quenched with satd NH4Cl aq at
ꢀ70 ꢁC. The mixture was let to warm up to rt, diluted
with H2O, and extracted with DCM. The organic phase
was washed with satd NaCl aq, dried over Na2SO4, fil-
tered, and evaporated. The product was purified by
flash-chromatography (1% MeOH in DCM). Yield
0.32 g, 71%.
5.8. 1-Boc-2(S)-(furan-2-carbonyl)pyrrolidine (5e)
1.6 M n-BuLi (4.7 mL, 7.5 mmol) was added dropwise to
furan (10 mL) at 0 ꢁC and the mixture was stirred for 1 h.
Boc-L-prolinal (0.30 g, 1.5 mmol) in THF (10 mL) was
added dropwise at ꢀ80 ꢁC and the mixture was stirred
for 3 h. Satd NH4Cl aq was added and the mixture was
let to warm up to rt and diluted with DCM. The organic
phase was washed with satd NaCl aq, dried over Na2SO4,
and evaporated to yield 4e. Without further purification,
the residue was dissolved in DCM, added to a solution of
DMP (0.95 g, 2.2 mmol) in DCM (4 mL), and then con-
tinued according to method C. The product was purified
by flash-chromatography (10% EtOAc in PE). Yield
5.6. Method C
5.6.1.
(5a). To
1-Boc-2(S)-(cyclopent-1-enecarbonyl)pyrrolidine
solution of Dess–Martin periodinane
a
(DMP) (1.4 g, 3.3 mmol) in DCM (16 mL) was added
4a (0.80 g, 3.0 mmol) in DCM (10 mL) and the mixture
was stirred for 1 h at rt 30% Na2S2O3 aq was added and
the mixture was stirred for 5 min. The phases were sep-
arated and the organic phase was washed with 30%
Na2S2O3 aq and satd NaHCO3 aq, dried over Na2SO4,
filtered, and evaporated. The product was purified by
flash-chromatography (20% EtOAc in PE). Yield
0.58 g, 73%. 1H NMR d 1.34 (s, 5H), 1.45 (s, 4H),
1.80–1.96 (m, 5H), 2.13–2.25 (m, 1H), 2.50–2.69 (m,
4H), 3.38–3.61 (m, 2H), 4.78 (dd, J = 4.5, 8.9 Hz,
0.5H), 4.96 (dd, J = 3.5, 9.0 Hz, 0.5H), 6.77 (m, 0.5H),
6.83 (m, 0.5H).
1
110 mg, 28% (two steps). H NMR d 1.26 (s, 5.4H),
1.46 (s, 3.6H), 1.87–2.01 (m, 3H), 2.23–2.36 (m, 1H),
3.43–3.67 (m, 2H), 4.90 (dd, J = 4.6, 8.6 Hz, 0.6H), 5.09
(dd, 0.4H), 6.53 (dd, J = 1.5, 3.3 Hz, 0.4H), 6.56 (dd,
J = 1.6, 3.5 Hz, 0.6H), 7.23 (d, J = 3.5 Hz, 0.6H), 7.26
(m, 0.4H), 7.58 (d, 0.4H), 7.61 (d, 0.6).
5.9. Method D
5.6.2. 1-Boc-2(S)-(cyclohex-1-enecarbonyl)pyrrolidine
(5b). Prepared according to method C from 4b (0.54 g,
1.9 mmol) using 1.3 equiv of DMP. No chromatograph-
5.9.1. 2(S)-(Cyclopent-1-enecarbonyl)-1-(4-phenylbuta-
noyl)pyrrolidine (6a). To a solution of 5a (110 mg,
0.35 mmol) in DCM (2.5 mL) was added trifluoroacetic
acid (TFA) (0.7 mL, 9.4 mmol) at 0 ꢁC. The mixture was
stirred for 2.5 h at 0 ꢁC and evaporated. TFA salt of the
amine, 4-phenylbutyric acid (58 mg, 0.35 mmol), HOB-
tÆH2O (81 mg, 0.53 mmol), and EDCÆHCl (102 mg,
0.53 mmol) were dissolved/suspended in DCM. Et3N
(240 lL, 1.75 mmol) was added at 0 ꢁC and the mixture
was stirred for 0.5 h at 0 ꢁC and for 5 h at rt. The reac-
tion mixture was washed with 30% citric acid, satd NaCl
aq, and satd NaHCO3, dried over Na2SO4, filtered, and
evaporated. The product was purified with a chromato-
tron (40–50% EtOAc in PE; Rf = 0.12 in 30% EtOAc in
1
ic purification was performed. Yield 0.54 g, 100%. H
NMR d 1.34 (s, 4.7H), 1.45 (s, 4.3 H), 1.57–1.69 (m,
4H), 1.75–1.94 (m, 3H), 2.12–2.39 (m, 5H), 3.41 (m,
0.5H), 3.46–3.62 (m, 1.5H), 4.90 (dd, J = 4.1, 8.8 Hz,
0.5H), 5.05 (dd, J = 3.4, 9.1 Hz, 0.5H), 6.90 (m, 0.5H),
6.96 (m, 0.5H).
5.6.3. 1-Boc-2(S)-(thiophene-2-carbonyl)pyrrolidine (5d).
Prepared according to method C from 4d (0.32 g,
1.1 mmol) using 1.3 equiv of DMP. The product was
purified by flash-chromatography (0.5% MeOH in
1
1
DCM). Yield 0.27 g, 87%. H NMR d 1.25 (s, 5.4 H),
PE). Yield 50 mg, 46%. H NMR d 1.81–2.06 (m, 7H),
1.46 (s, 3.6H), 1.87–2.05 (m, 3H), 2.25–2.38 (m, 1H),
3.47 (m, 0.4H), 3.56–3.69 (m, 1.6H), 4.88 (dd, J = 5.0,
8.6 Hz, 0.6H), 5.10 (dd, J = 3.2, 8.9 Hz, 0.4H), 7.13
(m, 0.4H), 7.15 (m, 0.6 H), 7.78 (d, J = 3.8 Hz, 0.6H),
7.81 (d, J = 3.5 Hz, 0.4H).
2.14 (m, 1H), 2.23–2.38 (m, 2H), 2.50–2.69 (m, 6H),
3.43 (m, 0.9H), 3.56-3.62 (m, 1H), 3.67 (m, 0.1H), 4.78
(dd, J = 3.0, 9.2 Hz, 0.1H), 5.16 (dd, J = 3.8, 8.8 Hz,
0.9H), 6.65 (m, 0.1H), 6.87 (m, 0.9H), 7.12–7.29 (m,
5H). 13C NMR d 22.5, 24.6, 26.0, 29.4, 30.9, 33.3,
34.2, 35.1, 47.2, 61.2, 125.8, 128.3, 128.6, 141.9, 143.4,
144.2, 171.1, 196.4. Anal. Calcd for C20H25NO2Æ0.1
H2O: C, 76.69; H, 8.11; N, 4.47. Found C, 76.72; H,
8.19; N, 4.52. ESI-MS m/z 312.1 [M+H]+.
5.7. 1-Boc-2(S)-(pyridine-2-carbonyl)pyrrolidine (5c)
To a solution of 4c (0.60 g, 2.2 mmol) and Et3N
(0.9 mL, 6.5 mmol) in 2.2 mL of dimethylsulfoxide
(DMSO) was added SO3Æpyridine (1.03 g, 6.5 mmol) in
DMSO (2.2 mL). The mixture was stirred for 1 h and
then poured in ice-cold water (25 mL). The water phase
was extracted with CHCl3 and the organic phase was
washed with water and satd NaHCO3 aq, dried over
Na2SO4, filtered, and evaporated. The product was puri-
5.9.2.
2(S)-(Cyclohex-1-enecarbonyl)-1-(4-phenylbuta-
noyl)pyrrolidine (6b). Prepared according to method D
from 5b (325 mg, 1.0 mmol) having a 20-h reaction time.
The product was purified by flash chromatography (50%
1
EtOAc in PE; Rf = 0.31). Yield 160 mg, 49%. H NMR
d 1.56–1.67 (m, 4H), 1.80 (m, 1H), 1.87–2.05 (m, 4H),

E. M. Jarho et al. / Bioorg. Med. Chem. 15 (2007) 2024–2031
2029
2.09-2.38 (m, 7H), 2.61 (m, 0.3H), 2.68 (t, J = 7.5 Hz,
1.7H), 3.43 (m, 0.9H), 3.56–3.62 (m, 1H), 3.67 (m,
0.1H), 4.88 (dd, J = 2.9, 9.2 Hz, 0.1H), 5.24 (dd,
J = 3.7, 8.9 Hz, 0.9H), 6.76 (m, 0.1H), 7.00 (m, 0.9H),
7.13–7.28 (m, 5H). 13C NMR d 21.5, 21.8, 23.2, 24.6,
26.0, 26.1, 29.5, 33.4, 35.1, 47.1, 59.6, 125.8, 128.3,
128.6, 137.4, 140.5, 141.9, 171.0, 198.6. Anal. Calcd
for C21H27NO2Æ0.1 H2O: C, 77.08; H, 8.38; N, 4.28.
Found C, 76.97; H, 8.50; N, 4.21. ESI-MS m/z 326.1
[M+H]+.
26.0, 29.1, 33.3, 35.1, 47.2, 60.9, 112.3, 117.9, 125.7,
128.2, 128.6, 141.9, 146.5, 151.4, 171.3, 187.1. Anal.
Calcd for C19H21NO3Æ0.1 H2O: C, 72.87; H, 6.82; N,
4.47. Found C, 72.74; H, 7.10; N, 4.31. ESI-MS m/z
312.1 [M+H]+.
5.10. Method E
5.10.1. 1-Boc-L-proline benzylamide (7a). To a solution
of Boc-L-proline (0.43 g, 2.0 mmol) and Et3N (310 lL,
2.2 mmol) in THF (4 mL) was added ethyl chlorofor-
mate (190 lL, 2.0 mmol) in THF (3 mL) dropwise at
ꢀ15 ꢁC, and the mixture was stirred for 30 min. Benzyl-
amine (440 lL, 4.0 mmol) in THF (1 mL) was added at
ꢀ15 ꢁC and the mixture was stirred overnight at rt. The
mixture was diluted with DCM, washed with 30% citric
acid, satd NaCl aq, and satd NaHCO3, dried over
Na2SO4, filtered, and evaporated. Yield 0.55 g, 90%.
5.9.3. 1-(4-Phenylbutanoyl)-2(S)-(pyridine-2-carbonyl)pyr-
rolidine (6c). Prepared according to method D from 5c
(280 mg, 1.0 mmol) having a 20-h reaction time. The
reaction mixture was not washed with 30% citric acid.
The product was purified by flash chromatography
1
(50% EtOAc in PE; Rf = 0.14). Yield 130 mg, 40%. H
NMR d 1.79–2.17 (m, 5.5H), 2.28–2.49 (m, 2.5H), 2.56
(m, 0.4H) 2.68 (t, J = 7.5 Hz, 1.6H), 3.52 (m, 0.8H),
3.64–3.71 (m, 1H), 3.76 (m, 0.2H), 5.85 (dd, J = 3.0,
9.4 Hz, 0.2H), 5.93 (dd, 0.8H), 7.07 (m, 0.5 H), 7.13–
7.21 (m, 2.5H), 7.26–7.29 (m, 2H), 7.46 (ddd, J = 1.2,
4.8, 7.6 Hz, 0.8H), 7.53 (ddd, J = 1.2, 4.7, 7.6 Hz, 0.2
H), 7.83 (td, J = 1.7, 7.7 Hz, 0.8H), 7.89 (td, J = 1.7,
7.7 Hz, 0.2H), 8.04–8.08 (m, 1H), 8.66–8.69 (m, 1H).
13C NMR d 25.0, 26.1, 29.3, 33.4, 35.1, 47.5, 60.4,
122.7, 125.8, 127.1, 128.3, 128.6, 136.9, 141.9, 148.8,
152.3, 171.0, 198.6. Anal. Calcd for C20H22N2O2: C,
74.15; H, 6.88; N, 8.69. Found C, 74.36; H, 7.09; N,
8.73. ESI-MS m/z 323.2 [M+H]+.
5.10.2. 1-Boc-D-proline benzylamide (7b). Prepared
according to method E from Boc-D-proline (0.90 g,
4.2 mmol). At the end of the reaction, the mixture was
diluted with EtOAc. Yield 1.08 g, 84%.
5.10.3. 1-Boc-L-proline phenethyl-amide (7c). Prepared
according to method E from Boc-L-proline (0.54 g,
2.5 mmol) and phenethylamine (0.63 mL, 5 mmol). At
the end of the reaction, the mixture was diluted with
EtOAc. Yield 0.80 g, 100%.
5.11. Method F
5.9.4. 1-(4-Phenylbutanoyl)-2(S)-(thiophene-2-carbonyl)pyr-
rolidine (6d). Prepared according to method D from 5d
(270 mg, 1.0 mmol) having a 20-h reaction time and
using 2.5 equiv of EDC and HOBt. The product was
purified by flash chromatography (15–50% EtOAc in
PE; Rf = 0.30 in 50% EtOAc in PE). Yield 155 mg,
47%. 1H NMR d 1.88–2.15 (m, 5.3H), 2.22–2.44 (m,
2.7H), 2.57 (m, 0.3H), 2.69 (t, J = 7.5 Hz, 1.7H), 3.48
(m, 0.85H), 3.63–3.69 (m, 1H), 3.75 (m, 0.15H), 4.93
(dd, J = 3.2, 9.1 Hz, 0.15H), 5.32 (dd, J = 3.6, 9.0 Hz,
0.85H), 7.07–7.21 (m, 4H), 7.26–7.29 (m, 2H), 7.64
(dd, J = 0.9, 5.0 Hz, 0.85H), 7.66 (dd, 0.15H), 7.72
(dd, J = 0.9, 5.0 Hz, 0.15H), 7.83 (dd, J = 0.9, 3.8 Hz,
0.85H). 13C NMR d 24.8, 26.0, 29.6, 33.3, 35.1, 47.2,
61.8, 125.8, 128.1, 128.3, 128.6, 132.3, 133.8, 141.7,
141.8, 171.4, 191.1. Anal. Calcd for C19H21NO2SÆ0.8
H2O: C, 66.75; H, 6.66; N, 4.10. Found C, 66.61; H,
6.34; N, 4.12. ESI-MS m/z 328.1 [M+H]+.
5.11.1. 1-(Cyclopent-1-enecarbonyl)-L-proline benzyla-
mide (8a). Step 1. To a solution of 7a (0.55 g, 1.8 mmol)
in DCM (4 mL) was added TFA (3.6 mL, 48 mmol) in
DCM (4 mL) dropwise at 0 ꢁC. The mixture was stirred
for 1 h at 0 ꢁC and evaporated. Step 2. To a solution of
1-cyclopentene-1-carboxylic acid (0.20 g, 1.8 mmol) and
Et3N (0.28 mL, 2.0 mmol) in DCM (4 mL) was added
trimethylacetyl chloride (0.22 mL, 1.8 mmol) in DCM
(4 mL) dropwise at 0 ꢁC. The mixture was stirred for
1 h at 0 ꢁC and the ice bath was removed. Et3N
(0.82 mL, 5.9 mmol) and the product of STEP 1 in
DCM (4 mL) were added in this order and the mixture
was stirred for 2 h. The mixture was washed with 30%
citric acid, satd NaCl aq, and satd NaHCO3, dried over
Na2SO4, filtered, and evaporated. The product was puri-
fied by flash-chromatography (40–80% EtOAc in PE;
Rf = 0.26 in EtOAc). Yield 380 mg, 71%. Crystallized
from EtOAc–hexane, mp 77.1–78.8 ꢁC. 1H NMR d
1.85–1.98 (m, 4H), 2.10 (m, 1H), 2.45–2.61 (m, 4H),
2.70 (m, 1H), 3.56–3.65 (m, 2H), 4.39 (dd, J = 5.0,
15.1 Hz, 1H), 4.48 (dd, J = 5.7, 15.1 Hz, 1H), 4.72 (m,
1H), 6.16 (m, 1H), 7.24–7.32 (m, 5H), 7.42 (m, 1H).
13C NMR d 22.7, 25.4, 26.8, 33.7, 33.9, 43.4, 49.2,
59.9, 127.2, 127.4, 128.6, 136.3, 138.5, 139.1, 168.7,
171.2. Anal. Calcd for C18H22N2O2: C, 72.46; H, 7.43;
N, 9.39. Found C, 72.57; H, 7.51; N, 9.46. ESI-MS
m/z 299.0 [M+H]+.
5.9.5. 2(S)-(Furan-2-carbonyl)-1-(4-phenylbutanoyl)pyr-
rolidine (6e). Prepared according to method D from 5e
(100 mg, 0.37 mmol) having a 20-h reaction time. The
product was purified by flash chromatography (27%
EtOAc in PE; Rf = 0.26 in 50% EtOAc in PE). Yield
42 mg, 36%. 1H NMR d 1.86–2.12 (m, 5.4H), 2.17–
2.40 (m, 2.6 H), 2.58 (m, 0.3H), 2.69 (t, J = 7.5 Hz,
1.7H), 3.47 (m, 0.85H), 3.61–3.67 (m, 1H), 3.73 (m,
0.15H), 4.97 (dd, J = 3.0, 9.2 Hz, 0.15H), 5.30 (dd,
J = 3.9, 8.8 Hz, 0.85H), 6.54 (dd, J = 1.6, 3.6 Hz,
0.85H), 6.59 (dd, J = 1.6, 3.5 Hz, 0.15H), 7.09–7.22 (m,
3.7H), 7.26–7.29 (m, 2.3H), 7.59 (d, J = 1.6 Hz,
0.85H), 7.63 (d, J = 1.6 Hz, 0.15H). 13C NMR d 24.8,
5.11.2. 1-(Cyclopent-1-enecarbonyl)-D-proline benzyla-
mide (8b). Prepared according to method F from 7b
(0.30 g, 1.0 mmol). The product was purified by

2030
E. M. Jarho et al. / Bioorg. Med. Chem. 15 (2007) 2024–2031
flash-chromatography (50–60% EtOAc in hexane;
Rf = 0.27 in EtOAc). Yield 180 mg, 60%. Crystallized
from EtOAc–hexane, mp 75.8–78.8 ꢁC. 1H NMR d
1.85–1.98 (m, 4H), 2.10 (m, 1H), 2.45–2.61 (m, 4H),
2.70 (m, 1H), 3.56–3.65 (m, 2H), 4.39 (dd, J = 5.2,
15.1 Hz, 1H), 4.48 (dd, J = 5.9, 15.1 Hz, 1H), 4.72 (m,
1H), 6.16 (m, 1H), 7.24–7.32 (m, 5H), 7.42 (m, 1H).
13C NMR d 22.7, 25.4, 26.8, 33.7, 33.9, 43.4, 49.2,
59.9, 127.2, 127.4, 128.6, 136.3, 138.5, 139.1, 168.7,
171.2. Anal. Calcd for C18H22N2O2: C, 72.46; H, 7.43;
N, 9.39. Found C, 72.30; H, 7.56; N, 9.34. ESI-MS m/
z 299.0 [M+H]+.
non-linear regression utilizing GraphPad Prism 3.0
software.
Acknowledgments
We thank Ms. Tiina Koivunen and Ms. Jaana Leskinen
for their technical assistance and the Academy of Fin-
land and The Finnish Cultural Foundation for their
financial support.
References and notes
5.11.3. 1-(Cyclopent-1-enecarbonyl)-L-proline phenethyl-
amide (8c). Prepared according to method F from 7c
(0.40 g, 1.25 mmol). The product was purified by flash-
chromatography (50% EtOAc in PE; Rf = 0.11 in 50%
EtOAc in PE). Yield 330 mg, 85%. Crystallized from
EtOAc–hexane, mp 95.0–96.2 ꢁC. 1H NMR d 1.82–
2.05 (m, 5H), 2.33–2.73 (m, 5H), 2.75–2.85 (m, 2H),
3.44–3.66 (m, 4H), 4.42 (m, 0.2H), 4.62 (m, 0.8H), 5.91
(m, 0.2H), 6.13 (m, 0.8H), 6.23 (m, 0.2H), 7.04 (m,
0.8H), 7.17–7.21 (m, 3H), 7.25–7.28 (m, 2H). 13C
NMR d 22.7, 25.3, 27.1, 33.7, 33.8, 35.7, 40.6, 49.2,
60.0, 126.3, 128.4, 128.8, 136.4, 139.1, 139.1, 168.3,
171.3. Anal. Calcd for C19H24N2O2: C, 73.05; H, 7.74;
N, 8.97. Found C, 72.88; H, 7.75; N, 8.95. ESI-MS
m/z 313.0 [M+H]+.
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