Comparison of the inhibition of human and Trypanosoma cruzi prolyl endopeptidases

Article abstract of DOI:10.1016/S0968-0896(02)00035-4

Prolyl endopeptidases (PEPs) have been found in numerous species. Inhibitors of human enzyme could correct cognitive deficits in Alzheimer patients while inhibition of Trypanosoma cruzi PEP could prevent invasion phase in Chagas disease. A structure-activity relationship study carried out in a tetrahydroisoquinoline series allowed to obtain potent competitive inhibitors superior to SUAM-1221. Besides, inhibitors expected to act according to an irreversible mechanism revealed to be superior to JPT-4819, for applications linked to human enzyme inhibition.

Full text of DOI:10.1016/S0968-0896(02)00035-4

Bioorganic & Medicinal Chemistry 10 (2002) 1719–1729
Comparison of the Inhibition of Human and Trypanosoma cruzi
Prolyl Endopeptidases
Sandrine Vendeville,a,y Filip Goossens,c Marie-Ange Debreu-Fontaine,a Valerie Landry,a
Elisabeth Davioud-Charvet,a Philippe Grellier,b Simon Scharpec
and Christian Sergheraerta,*
a
´
´
Institut de Biologie de Lille - Institut Pasteur de Lille, UMR CNRS 8525, Faculte de Pharmacie, Universite de Lille II,
1 rue du rofesseur Calmette, BP447, 59021 Lille cedex, France
bLaboratoire de Biologie Parasitaire, MuseumNational d’Histoire Naturelle, IFR CNRS 63, 61 rue Buffon, 75231 Paris, France
cLaboratory for Medicinal Biochemistry, University of Antwerp, Universiteitplein 1, B-2610 Antwerp, Belgium
Received 23 August 2001; accepted 15 January 2002
Abstract—Prolyl endopeptidases (PEPs) have been found in numerous species. Inhibitors of human enzyme could correct cognitive
deficits in Alzheimer patients while inhibition of Trypanosoma cruzi PEP could prevent invasion phase in Chagas disease. A struc-
ture–activity relationship study carried out in a tetrahydroisoquinoline series allowed to obtain potent competitive inhibitors
superior to SUAM-1221. Besides, inhibitors expected to act according to an irreversible mechanism revealed to be superior to JPT-
4819, for applications linked to human enzyme inhibition. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
Trypanosoma cruzi, the causative agent of Chagas dis-
ease, also contains a prolyl endopeptidase which exhi-
bits the unusual property of cleaving, in addition to the
small peptides, collagens of the extracellular matrix.10
These data suggest that the inhibition of this proline-
specific enzyme may represent a therapeutic approach to
the treatment of Chagas disease, by preventing the
invasion of mammalian cells by trypomastigotes.
Prolyl oligopeptidase (prolyl endopeptidase, post-pro-
line cleaving enzyme, PEP, EC 6.4.21.26) is a proline-
specific endopeptidase with a serine-type mechanism,
cleaving peptide bonds on the carboxylic side of prolyl
residues.1 This enzyme was first isolated in the human
uterus,2 and has later been identified and purified from
various mammalian tissues.3ꢀ6 In the central nervous
system, PEP degrades proline-containing neuropeptides
involved in the processes of learning and memory, such
as vasopressin, substance Pand thyrotropin-releasing
hormone (TRH).7 Therefore, as cognitive deficits in
Alzheimer patients are reported to show improvement
with TRH, one can postulate that PEP inhibitors could
prevent memory loss in patients suffering of senile
dementia. Interestingly, the enzyme was found to be
exclusively active on small peptides, which further
stresses the specificity of this particular enzyme.
While most of the PEP inhibitors described in the lit-
erature are structurally related to Z-prolyl-prolinal,11
the collagenase-like substrate Gly-Pro-Leu-Gly-Pro was
also identified as a lead molecule.10 To obtain new leads
against both human and T. cruzi PEPs, two orthogonal
peptide combinatorial libraries were screened and led to
a 1,2,3,4-tetrahydroisoquinoline carboxylic acid (Tic)
derivative, active in the low micromolar range against
the T. cruzi enzyme.12,13 Subsequently, the automated
parallel synthesis and the screening of a focused Tic-
based library allowed us to select lead 1 (Fig. 1), which
displayed an IC50 of 7 nM against the parasitic PEP
while its activity towards human PEP was found to be
less potent (IC50=550 nM).14 Here, we report our
efforts to establish structure–activity relationships based
upon lead 1 and to obtain more potent inhibitors against
human and T. cruzi PEPs. For the latter, obtaining a
good selectivity index was an additional aim.
PEPs have also been found in other species such as
fungi, bacteria and plants.8,9 We have reported that
*Corresponding author. Tel.:+33-3-2087-1211; fax; +33-3-2087-
1233; e-mail: christian.sergheraert@pasteur-lille.fr
yPresent address: TIBOTEC, L11 B3 General de Wittelaan,
B-2800 Mechelen, Belgium.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00035-4

1720
S. Vendeville et al. / Bioorg. Med. Chem. 10 (2002) 1719–1729
Chemistry
to the aldehyde 27 as a racemic mixture by treatment
with DIBAL-H (Scheme 2).
The starting materials Boc-l-Tic-pyrrolidine, Boc-l-Tic-
thiazolidine, Boc-l-Tic(7-OH)-pyrrolidine and Boc-l-
Tic-l-Pro-OCH3 were obtained by reacting Boc-l-Tic
or 7-OH-Boc-l-Tic(7-OH) with pyrrolidine, thiazolidine
or l-Pro-OCH3, using DCC/HOBt as coupling reagents
in dichloromethane (Scheme 1). Boc-l-Tic(7-OH) was
prepared from l-di-iodo-Tyr according to Verschueren
et al.15 All inhibitors were then prepared in solution
phase from Boc-l-Tic-pyrrolidine (1 and 6–26), Boc-l-
Tic-thiazolidine (2–3) Boc-l-Tic(7-OH)pyrrolidine (4–5)
or Boc-l-Tic-l-Pro-OCH3 (27–31). After TFA depro-
tection, each terminal group was substituted by reaction
with the appropriate carboxylic acid (1-–26) using the
conditions described previously (Scheme 1).
In the case of the nitrile derivative 31, 4-phenyl-butyryl-
l-Tic was coupled to Pro-NH2 before dehydratation
according to Pozdnev et al. using trifluoroacetic anhy-
dride in THF (Scheme 3).17
Assays of PEPs inhibition
T. cruzi PEP was purified following two steps of chro-
matography (DEAE and phenylsepharose) as pre-
viously described.10 Enzyme inhibition was evaluated by
using the fluorogenic substrate Suc-Gly-Pro-Leu-Gly-
Pro-AMC (Km=12.5 mM). The test compound was dis-
solved at various concentrations in Tris–HCl buffer (25
mM, pH 7.5), containing 10% DMSO. 20 mL of the
solution was preincubated for 15 min with 20 mL of
enzyme in the same buffer at 37 ꢁC. The enzymic reac-
tion was then initiated by adding 20 mL of the substrate
solution (33 mM). After 15 min, the reaction was stop-
ped by adding 100 mL of EtOH. The fluorescence of free
released AMC was measured at 440 nm upon excitation
at 380 nm in a Hitachi 2000 spectrofluorimeter. Inhibi-
tory potency was evaluated by the IC50 value, which
was defined as the concentration of the test compound
that resulted in 50% inhibition of the fluorescence with
respect to the DMSO control.
In the case of compounds 27–30, the methyl ester
obtained from Boc-l-Tic-l-Pro-OCH3 was transformed
to the chloromethylketone 28 according to Kowalski et
al. using LDA/CH2ICl as reagents (Scheme 2).16 Treat-
ment with potassium acetate in DMF led to the corre-
sponding acetyloxymethylketone 29, which was
hydrolysed into the hydroxymethylketone 30 with
Na2CO3 in MeOH (Scheme 2). The methyl ester also led
Human PEP was purified from human platelets follow-
ing two steps of chromatography (DEAE and phe-
nylsepharose) and enzyme activity was measured as
previously reported.18,19 At 100 mL of K-phosphate
buffer (100 mM, pH 7.5) containing 1 mM EDTA,
NaN3 and DTT, was added 5 mL of inhibitor in DMSO
at various concentrations and 20 mL of enzyme. After 20
min at 37 ꢁC, the reaction was started by addition of 5
mL of substrate (Z-Gly-Pro-AMC, Km=120 mM, 4.3
mM dissolved in 40% DMSO). After 15 min, the reac-
tion was stopped by adding 500 mL of acetic acid and
the fluorescence was measured in the same conditions as
for the T. cruzi assay.
Figure 1. Structure of compound 1.
Results
Modifications were successively introduced at the three
binding subsites named P1, P2 and P3 according to the
Scheme 1. Synthesis of compounds 1–26. Reagents: (a) DCC/HOBt,
CH2Cl2; (b) TFA/CH2Cl2 50/50; (c) RCOOH, DCC/HOBt, CH2Cl2.
Scheme 2. Synthesis of compounds 27–30. Reagents: (a) LDA/CH2ICl, THF, ꢀ78 ꢁC; (b) CH3COOK, DMF, 60 ꢁC; (c) Na2CO3, MeOH;
(d) DIBALH, THF, ꢀ78 ꢁC.

S. Vendeville et al. / Bioorg. Med. Chem. 10 (2002) 1719–1729
1721
Schechter–Berger nomenclature.20 Activity upon T.
cruzi and human PEPs are given in Tables 1–3, respec-
tively, for compounds 1–9, 10–26 and 27–31.
subsite P2. Reverse changes in the electronic density of
the phenyl ring (10–11) or replacement of the phenyl
ring with an isostere such as thiophene (12) led to an
increase of activity against human PEP with, however, a
decrease of selectivity towards T. cruzi PEP. Steric hin-
drance decreased activity against parasitic enzyme (13).
Whatever the distance to subsite P2, the presence of a
nitrogen atom in a phenyl ring was unfavourable for
both PEPs (14–15 versus 6–7).
While introduction of a sulphur atom in the pyrrolidine
ring (subsite P1) was reported to be very favourable
towards bovine brain PEP inhibition,21,22 only a slight
increase of activity was observed towards parasitic and
human PEPs compared to lead 1 (IC50=5 and 120 nM
respectively for compound 2) which was however asso-
ciated with a decrease of selectivity. The corresponding
sulphone derivative 3 was found to be less active
(IC50=90 and 2800 nM). Addition of a hydroxyl group
in the C-7 position of the Tic ring (subsite P2, com-
pound 4), was favourable towards human enzyme
(IC50=70 nM) and slightly unfavourable towards para-
sitic enzyme (IC50=19 nM). Ethyl ether derivative (5)
showed a similar potency upon T. cruzi enzyme com-
pared to the hydroxyl derivative 4, while showing a
better selectivity.
The potent inhibition found for compound 16 against
T. cruzi PEP within the Tic-based library (IC50=9 nM;
7 and 12 nM for its separated isomers),14 led us to syn-
thetize and evaluate activities of its analogues 17–26.
Results underline the good recognition by the parasitic
enzyme of the cyclohexyl ring, substituted or not (16
Table 2. Activity of compounds 10–23 towards T. cruzi and human
PEPs
Optimization of the subsite P3 was approached accord-
ing to two types of modifications: (i) replacement of the
phenyl ring by heterocyclic, polyaromatic, alicyclic or
isosteric structures and (ii) variation of the distance to
Compd
R
n
T. cruzi
PEP
Human
PEP
IC50 (nM) IC50 (nM)
Selectivity
index
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4-Nitrophenyl
4-Methoxyphenyl
Thiophen-2-yl
Pyren-1-yl
Pyridin-3-yl
Pyridin-3-yl
4-Methylcyclohexyl
4-Methylcyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclopentyl
Norborn-2-yl
Adamant-1-yl
Dicyclohexylmethyl
3
3
3
3
1
0
1
0
0
1
2
3
4
1
1
1
0
52
54
10
100
1200
3500
9
370
395
12
105
150
>500
67
370
115
6500
220
150
55
4.2
2.7
5.5
0.5
0.5
9.7
1.7
2.8
1.5
14.6
1.4
0.5
0.5
5.9
4.7
2.6
0.2
50
600
34,000
16
1030
600
175
150
70
370
400
1750
300
1500
Scheme 3. Synthesis of compound 31. Reagents: (a) DCC/HOBt,
CH2Cl2; (b) (CF3CO)2O, THF.
Table 1. Activity of compounds 1–9 towards T. cruzi and human
PEPs
Table 3. Activity of compounds 27–31 towards T. cruzi and human
PEPs
Compd
X
Y
n
T. cruzi
PEP
Human Selectivity index
PEP
IC50 (nM) IC50 (nM)
IC50 human
PEP/IC50
T. cruzi PEP
Compd
X
T. cruzi
PEP IC50 (nM)
Human
PEP IC50 (nM)
1
2
3
4
5
6
7
8
9
CH2
S
SO2
CH2
H
H
H
3
3
3
3
3
0
1
2
4
7
5
90
19
550
120
2800
70
125
4000
400
850
100
78
24
31
3.7
7.3
3.1
3.8
1.6
6.6
27
28
29
30
31
CHO
12
Nd
1.5
3.5
1.5
40
OH
COCH2Cl
COCH2OCOCH3
COCH2OH
CN
2.6
3.9
4.5
11
CH2 OC2H5
17
CH2
CH2
CH2
CH2
H
H
H
H
1300
105
525
15
JTP-4819
16
3.6

1722
S. Vendeville et al. / Bioorg. Med. Chem. 10 (2002) 1719–1729
and 19) and the influence of the length of the spacer: the
best activities were obtained for one methylene group.
However, except for compound 19, no or a weak selec-
tivity between both enzymes was noted in this series.
Influence of the length of the spacer was also observed
in the phenyl series (6–9): the best inhibition activity
towards T. cruzi PEP associated to the best selectivity
was obtained for a spacer of three methylene groups.
ability of inhibitors by addition of hydrophilic or
hydrophobic side chains, maintains or enhances activ-
ity, compared to compound 1, respectively against
parasitic and human PEP. This result increases the
interest of the Tic residue as a P2 subsite.
In accordance with previous reports, good activities are
found with the alicyclic moieties as well as with the
aromatic ring chosen as subsite P3.25,26 These results
can be explained by the interaction with the hydro-
phobic environment of the subsite S3, due to the pres-
ence of residues Phe173, Met235, Cys255, Ile591 and
Ala594.23 For the same reason, it is not surprising that a
variation of electronic density in the ring does not lead
to significant difference of inhibitory potency (10–11).
However, length of the spacer has a different influence
according to each series. Recognition in the phenyl ser-
ies is optimal when aromatic ring and Tic residue are
linked by three methylene groups while the cyclohexyl
ring has to be closer, result reinforcing those obtained
with an open chain.27
Derivatives corresponding to additional reactive group
in compound 1 (27–31) were found to be very active
towards both enzymes with IC50 values in the low
nanomolar range (Table 3).
Discussion
In the aim of discovering new potent inhibitors against
T. cruzi and human PEPs, the screening of two ortho-
gonal d-tripeptide combinatorial libraries and of a
focused Tic-based library were carried out. They led to
the selection of compound 1 displaying IC50 of 7 and
550 nM against parasitic and human enzymes, respec-
tively. The three parts of the molecule P1, P2 and P3 as
well as the spacer between P2 and P3 were successively
modified to analyse their influence in the recognition of
the corresponding enzyme subsites S1, S2 and S3. These
latters have been recently well defined in the case of
porcine PEP co-crystallised with the Z-prolyl-prolinal
inhibitor.23 They were assumed identical in T. cruzi and
human PEPs because of the high sequence similarity
between PEPs of different organisms, particularly in the
region containing the catalytic triad.24 All reported
results so far, and those deduced from our focused Tic-
based library (32 amines tested), converge to point out
pyrrolidine ring as the most appropriate subsite P1 for
PEPs recognition, whatever their origin.
Except for the slight increase of activity observed by
introduction of a sulphur atom in the pyrrolidine ring,
compound 1 remained the most potent inhibitor and
was therefore modified by reactive groups likely to lead
from a competitive inhibition to a tight-binding or an
irreversible mode of action. The first mechanism could
be involved by addition of an aldehyde group capable of
generating a tetrahedric complex with the catalytic ser-
ine, which could mimic the transition state proved from
the co-crystallisation porcine PEP/Z-prolyl-prolinal.23
The aldehyde derivative 27 obtained under the racemic
form, was only tested upon the parasitic enzyme and
gave IC50 value in the low nanomolar range (12 nM).
The possibility of an irreversible mode of action was
also considered via the preparation of methylketones
substituted by good leaving groups. The groups chosen,
chloromethylketone, acetyloxymethyl ketone and
hydroxymethylketone, led to IC50 values below 4.5 nM
towards both enzymes (28–30). Compounds were found
to effectively act on T. cruzi PEP as irreversible inhibi-
tors or very tight-binding inhibitors since less than 1%
of enzymic activity was recovered following extensive
dialysis. Therefore, it can be expected that they act on
the human PEP in a similar way. It can be noted that
the hydroxymethylketone 30 displays a 2-fold decrease
of the IC50 value (1.5 nM) compared to JPT-4819
(Table 3 and Fig. 2), previously reported as capable to
improve memory impairment in rat by activating cere-
bral peptidergic and cholinergic neurons via inhibition of
PEP.28 Besides, nitrile derivative 31, likely to behave as a
non-competitive reversible inhibitor,29,30 yields to a more
moderate increase of activity upon human enzyme.
Pyrrolidine ring presents the optimal size to fit into the
hydrophobic pocket S1, defined by residues Trp595,
Phe476, Val644, Tyr599 and Asn555, in the case of
porcine PEP, the only crystallised PEP described so
far,23 and its replacement with other close rings such as
piperidine or azetidine, strongly decreases inhibition.21
Its replacement for a thiazolidine ring (2) improves
activity towards human PEP but at a lesser extent com-
pared to the bovine brain PEP where a 20-fold increase
was observed for similar replacements in a proline series.22
Conversely, the intermediate subsite P2 allows a widest
diversity and replacement of proline and thiaproline for
unnatural analogues such as perhydroindole, azabicy-
clo[2.2.2]- and azabicyclo[2.2.1]-heptane led to 10/20-
fold increases of activity against rat brain PEP.21 In the
same way, replacement of proline by the Tic analogue
as in compound 1, increases the activity against T. cruzi
PEP (data not shown). In all cases, stereochemistry of
the central moiety was important for recognition,21,22
for example, IC50 value for the d-Tic isomer of lead
compound 1 against T. cruzi PEP, was 200 nM instead
of 7 nM for the L-Tic isomer. Introduction of an
hydroxyl group or an alkoxy group in the C-7 position
of the Tic ring (4–5) in the aim of modulating bioavail-
Figure 2. Structure of SUAM-1221 and JPT-4819.

S. Vendeville et al. / Bioorg. Med. Chem. 10 (2002) 1719–1729
1723
In conclusion, the structure–activity relationship study
carried out in this 1,2,3,4-tetrahydroisoquinoline series
allowed us to obtain competitive inhibitors superior to
phenylpropylcarbonyl-Pro-pyrrolidine known as SUAM-
1221 (Fig. 2), a PEP inhibitor used as control in the lit-
erature against PEPs.20 Besides, inhibitors expected to
act according to an irreversible mechanism revealed
themselves to be superior to JPT-4819 for applications
linked to human enzyme inhibition. For the latter
applications, compared to proline analogues, presence
of the hydrophobic Tic residue at the subsite P2, con-
stitutes a major advantage for the blood–brain barrier
penetrability.
the amine (1.4 mmol, 1 equiv). After stirring the mixture
at room temperature for 15 h, the solvent was evapo-
rated and the residue taken up in 50 mL of AcOEt,
cooled at ꢀ20 ꢁC, filtered then washed with 40 mL of
Na2CO3 1 M, 40 mL of aqueous citric acid 20% and 30
mL of brine. The organic layer was separated, dried
over MgSO4, the solvent evaporated and the residue
purified by TLC (CH2Cl2/MeOH, 95:5 to 90:10) to yield
the desired product.
Method C. General procedure for deprotection of the
Boc group followed by a coupling reaction. The Boc-
aminoacid (1.4 mmol, 1 equiv) was diluted in 10 mL of
a TFA/CH2Cl2 1:1 mixture. After stirring the mixture at
room temperature for 1 h, the solvent was evaporated
and the residue taken up in 10 mL of CH2Cl2 with an
excess of DIEA (7.0 mmol, 5 equiv). To this solution
were added HOBt (1.4 mmol, 1 equiv) in 2 mL of DMF
and DCC (1.4 mmol, 1 equiv) in 2 mL of CH2Cl2, then,
after 10 min, the amine (1.4 mmol, 1 equiv). After stir-
ring the mixture at room temperature for 15 h, the sol-
vent was evaporated with the residue being taken up in
50 mL of AcOEt, cooled at ꢀ20 ꢁC, filtered then washed
with 40 mL of Na2CO3 1 M, 40 mL of aqueous citric
acid 20% and 30 mL of brine. The organic layer was
separated, dried over MgSO4, the solvent evaporated
and the residue purified by TLC (CH2Cl2/MeOH, 97:3
to 90:10) to yield the desired product.
Experimental
Chemistry
All reactions were monitored by thin-layer chromato-
graphy carried out on 0.2 mm E. Merck silica gel plates
(60F-254) using UV light as a visualising agent. Chro-
matography was undertaken using silica gel 60 (230–400
mesh ASTM) from Macherey-Nagel. Thick-layer chro-
matography (TLC) was performed using silica gel from
Merck, from which the compounds were extracted by
1
the following solvent system: CH2Cl2/MeOH, 85:15. H
NMR spectra were obtained using a Bruker 300 MHz
spectrometer, chemical shifts (d) were expressed in ppm
relative to TMS used as an internal standard. Mass
spectra were recorded on a time-of-flight (TOF) plasma
desorption spectrometer using a Californium source.
The purity of final compounds was verified by high
pressure liquid chromatography (HPLC) with a C18
vydac column. Analytical HPLC was performed on a
Shimadzu system equiped with a UV detector set at
254 nm. Compounds were dissolved in EtOH and
injected through a 50 mL loop. The following eluent
systems were used: A (H2O/TFA, 100:0.05) and B
(CH3CN/H2O/TFA, 80:20:0.05). HPLC retention times
(HPLC tR) were obtained, at flow rates of 1 mL/min, using
the following conditions: a gradient run from 100% eluent
A during 5 min, then to 100% eluent B over the next 25
min. l-Tic was obtained from Lancaster, 3,5-diiodo-l-Tyr,
2H2O from Acros, coupling reagents from Novabiochem
and other reagents from Acros, Aldrich or Lancaster.
Boc-L-Tic. Boc-l-Tic was prepared from l-Tic (1 g, 5.6
mmol, 1 equiv) by method A and was obtained as a
white solid (1.36 g, 88% yield); Rf 0.60 (CH2Cl2/MeOH,
9:1); HPLC tR 20.27 min; 1H NMR (DMSO-d6) d
1.36+1.42 (s, 9H, C(CH3)3 cis/trans), 2.99–3.15 (m, 2H,
Tic–H), 4.32–4.62 (m, 2H, Tic–H), 4.82–4.85 (m, 1H,
Tic–H), 7.14–7.16 (m, 4H, Ar–H), 12.60 (s, 1H,
COOH); TOFMS m/z 177 (M+ꢀBoc).
Boc-L-Tic-pyrrolidine. Boc-l-Tic-pyrrolidine was pre-
pared from Boc-l-Tic (1 g, 3.6 mmol, 1 equiv) and pyr-
rolidine (300 mL, 3.6 mmol, 1 equiv) by method B and
was obtained after TLC (CH2Cl2/MeOH, 90:10) as a
white solid (1.18 g, 95% yield); Rf 0.85 (CH2Cl2/MeOH,
9:1); HPLC tR 21.93 min; 1H NMR (CDCl3) d
1.42+1.48 (s, 9H, C(CH3)3 cis/trans), 1.82–2.04 (m, 4H,
pyrrolidine), 3.01–3.09 (m, 2H, Tic–H), 3.38–3.65 (m,
4H, pyrrolidine), 4.34+4.52 (d, J=15.3+16.1 Hz, 1H,
Tic–H cis/trans), 4.53–4.57+5.02–5.05 (m, 1H, Tic–H
cis/trans), 4.82+4.96 (d, J=15.3+16.1 Hz, 1H, Tic–H
cis/trans), 7.03–7.33 (m, 4H, Ar–H); TOFMS m/z 330
(M+).
Method A. General procedure for the protection of
amines by a Boc group. To a solution of the amine (30
mmol, 1 equiv) in 120 mL of a H2O/dioxane/NaOH 1 M
1:2:1 mixture, was added, at 0 ꢁC, Boc2O (33 mmol, 1.1
equiv). After stirring the mixture at room temperature
for 5 h, the solvent was evaporated with the residue
being taken up in 30 mL of AcOEt and washed with 30
mL of aqueous citric acid 10% and 30 mL of brine. The
organic layer was separated, dried over MgSO4 and the
solvent evaporated to yield the desired product.
4-Phenylbutyryl-L-Tic-pyrrolidine (1). Compound 1 was
prepared from Boc-l-Tic-pyrrolidine (695 mg, 3.0
mmol, 1 equiv) and 4-phenylbutyric acid (405 mg, 3.0
mmol, 1 eqiv.) by method C and was obtained after
TLC (CH2Cl2/MeOH, 95:5) as a colourless oil (895 mg,
79% yield); Rf 0.55 (CH2Cl2/MeOH, 9.5:0.5); HPLC
tR 22.94 min; 1H NMR (CDCl3) d 1.82–2.04 (m,
6H, CH2CH2CH2C6H5 and pyrrolidine), 2.43–2.49 (m,
2H, CH2CH2CH2C6H5), 2.68 (t, J=7.3 Hz, 2H,
CH2CH2CH2C6H5), 3.07 (d, J=6.5 Hz, 2H, Tic–H),
3.35–3.53 (m, 4H, pyrrolidine), 3.74–3.80 (m, 1H, pyr-
Method B. General procedure for the coupling reaction.
At 0 ꢁC, to a solution of the carboxylic derivative (1.4
mmol, 1 equiv) in 10 mL of CH2Cl2, were added HOBt
(1.4 mmol, 1 equiv) in 2 mL of DMF and DCC (1.4
mmol, 1 equiv) in 2 mL of CH2Cl2, and, after 10 min,

1724
S. Vendeville et al. / Bioorg. Med. Chem. 10 (2002) 1719–1729
rolidine), 4.55 (d, J=15.3 Hz, 1H, Tic–H), 4.64 (d,
J=15.3 Hz, 1H, Tic–H), 5.16 (t, J=6.5 Hz, 1H, Tic–H),
7.05–7.30 (m, 9H, Ar–H); TOFMS m/z 376 (M+).
(DMSO-d6) d 3.02 (dd, J=10.9, 16.6 Hz, 1H, Tic–H),
3.16 (dd, J=4.8, 16.8 Hz, 1H, Tic–H), 3.95 (d, J=16.4
Hz, 1H, Tic–H), 4.04 (d, J=16.4 Hz, 1H, Tic–H), 4.26
(dd, J=4.8, 10.9 Hz, 1H, Tic–H), 7.65 (s, 1H, Ar–H),
9.63 (broad s, 1H, OH), 10.00 (broad s, 1H, COOH);
TOFMS m/z 445 (M+).
Boc-L-Tic-thiazolidine. Boc-l-Tic-thiazolidine was pre-
pared from Boc-l-Tic (255 mg, 0.93 mmol, 1 equiv)
and thiazolidine (73 mL, 0.93 mmol, 1 equiv) by
method B and was obtained after TLC (CH2Cl2/MeOH,
95:5) as a colourless oil (284 mg, 88% yield); Rf 0.90
7-OH-6,8-Diiodo-Boc-L-Tic. 7-OH-6,8-Diiodo-Boc-l-
Tic was prepared from 7-OH-6,8-diiodo-l-Tic (5.6 g,
12.6 mmol, 1 equiv) by method A and was obtained as a
yellow solid (5.8 g, 78% yield); Rf 0.90 (CH2Cl2/MeOH,
9:1); HPLC tR 23.07 min; 1H NMR (DMSO-d6) d
1.35+1.41 (s, 9H, C(CH3)3 cis/trans), 2.99 (m, 2H, Tic–
H), 4.14 (d, J=17.4 Hz, 1H, Tic–H), 4.36–4.42 (m, 1H,
Tic–H), 4.62–4.76 (m, 1H, Tic–H), 7.58 (s, 1H, Ar–H),
9.42 (broad s, 1H, OH), 12.72 (broad s, 1H, COOH);
TOFMS m/z 445 (M+-Boc).
1
(CH2Cl2/MeOH, 9:1); HPLC tR 21.99 min; H NMR
(CDCl3) d 1.28+1.34 (s, 9H, C(CH3)3 cis/trans), 2.69–
3.06 (m, 4H, Tic–H and thiazolidine), 3.83–3.85 (m, 2H,
thiazolidine), 4.19–5.00 (m, 5H, Tic–H and thiazolidine),
7.00–7.10 (m, 4H, Ar–H); TOFMS m/z 248 (M+ꢀBoc).
4-Phenylbutyryl-L-Tic-thiazolidine (2). Compound 2 was
prepared from Boc-l-Tic-thiazolidine (202 mg, 0.8
mmol, 1 equiv) and 4-phenylbutyric acid (135 mg, 0.8
mmol, 1 equiv) by method C and was obtained after
TLC (CH2Cl2/MeOH, 95:5) as a colourless oil (245
mg, 77% yield); Rf 0.75 (CH2Cl2/MeOH, 9.5:0.5);
HPLC tR 23.55 min; 1H NMR (CDCl3) d 1.97 (qt,
J=6.8 Hz, 2H, CH2CH2CH2C6H5), 2.41–2.47 (m, 2H,
CH2CH2CH2C6H5), 2.66 (t, J=7.4 Hz, 2H, CH2CH2
CH2C6H5), 2.86–3.07 (m, 4H, Tic–H and thiazolidine),
3.71–4.05 (m, 2H, thiazolidine), 4.46–4.71 (m, 4H, Tic–
H and thiazolidine), 5.15–5.19 (m, 1H, Tic–H), 7.03–
7.28 (m, 9H, Ar–H); TOFMS m/z 394 (M+).
7-OH-Boc-L-Tic. To a solution of 7-OH-6,8-diiodo-
Boc-l-Tic (10.4 g, 19.1 mmol, 1 equiv) in 385 mL of
distilled MeOH, were added distilled triethylamine (5.3
mL, 38.2 mmol, 2 equiv) and Pd/C 10% (1.18 g). After
stirring the mixture under hydrogene pressure (100 bars)
for 4 h, the solution was filtered on Celite, the filtrate
concentrated with the residue being taken up in 200 mL
of a H2O/AcOEt 1:1 mixture. The aqueous layer was
acidified with concentrated chlorhydric acid 35% until
pH 2–3 then extracted with 3ꢂ150 mL of AcOEt. The
organic layers were mixed, dried over MgSO4, the sol-
vent evaporated and the residue purified by column
chromatography (CH2Cl2/MeOH, 97:3) to yield 7-OH-
Boc-l-Tic as a yellow solid (3.0 mg, 54% yield); Rf 0.65
4-Phenylbutyryl-L-Tic-thiazolidin-1-sulfone (3). To
a
solution of compound 2 (120 mg, 0.3 mmol, 1 equiv) in
10 mL of CH2Cl2 dried on molecular sieves, was added
meta-chloroperbenzoic acid (265 mg, 1.5 mmol, 5
equiv). After stirring the mixture at room temperature
for 18 h, the solution was diluted in 20 mL of CH2Cl2,
washed with 20 mL of a saturated solution of NaHCO3
containing 10% of sodium sulfite. The organic layer was
dried over MgSO4, the solvent evaporated and the resi-
due purified by TLC (CH2Cl2/MeOH, 95:5) to yield
compound 3 as a colourless oil (90 mg, 70% yield); Rf
1
(CH2Cl2/MeOH, 9:1); HPLC tR 17.42 min; H NMR
(DMSO-d6) d 1.33+1.40 (s, 9H, C(CH3)3 cis/trans),
2.96 (m, 2H, Tic–H), 4.20–4.48 (m, 2H, Tic–H), 4.50–
4.74 (m, 1H, Tic–H), 6.49 (s, 1H, Ar–H), 6.62–6.65 (d,
J=7.7 Hz, 1H, Ar–H), 6.90 (d, J=7.9 Hz, 1H, Ar–H),
9.25 (broad s, 1H, OH); TOFMS m/z 193 (M+-Boc).
7-OH-Boc-L-Tic-pyrrolidine. 7-OH-Boc-l-Tic-pyrroli-
dine was prepared from 7-OH-Boc-l-Tic (1 g, 3.4 mmol,
1 equiv) and pyrrolidine (285 mL, 3.4 mmol, 1 equiv) by
method B and was obtained after TLC (CH2Cl2/MeOH,
90:10) as a yellow solid (735 mg, 62% yield); Rf 0.75
1
0.75 (CH2Cl2/MeOH, 9.5:0.5); HPLC tR 22.00 min; H
NMR (CDCl3) d 1.91–2.01 (m, 2H, CH2CH2CH2C6H5),
2.41–2.47 (m, 2H, CH2CH2CH2C6H5), 2.66 (t, J=7.4
Hz, 2H, CH2CH2CH2C6H5), 3.06 (d, J=6.4 Hz, 2H,
Tic–H), 3.26–3.40 (m, 2H, thiazolidine), 4.00–4.05 (m,
2H, thiazolidine), 4.41–4.49 (m, 2H, thiazolidine), 4.50
(d, J=15.4 Hz, 1H, Tic–H), 4.57 (d, J=15.4 Hz, 1H,
Tic–H), 5.15–5.22 (m, 1H, Tic–H), 7.05–7.30 (m, 9H,
Ar–H); TOFMS m/z 426 (M+).
1
(CH2Cl2/MeOH, 9:1); HPLC tR 17.92 min; H NMR
(DMSO-d6) d 1.33+1.42 (s, 9H, C(CH3)3 cis/trans),
1.75–1.89 (m, 4H, pyrrolidine), 2.45–2.49+2.96–2.99 (m,
2H, Tic–H cis/trans), 3.29–3.50 (m, 4H, pyrrolidine),
4.14+4.59 (d, J=15.2 Hz, 2H, Tic–H), 4.42–4.45+4.81–
4.84 (m, 1H, Tic–H cis/trans), 6.51 (s, 1H, Ar–H), 6.59
(d, J=7.2 Hz, 1H, Ar–H), 6.90 (d, J=7.1 Hz, 1H, Ar–
H), 9.24 (broad s, 1H, OH); TOFMS m/z 346 (M+).
7-OH-6,8-Diiodo-L-Tic. To a solution of 3,5-diiodo-l-
Tyr, 2H2O (10 g, 22 mmol, 1 equiv) in 100 mL of
chlorhydric acid 35%, were added 1,2-dimethoxyethane
(7.3 mL) and formaldehyde (7 mL, 88.4 mmol, 4 equiv).
After stirring the mixture at 72 ꢁC for 30 min, were
added chlorhydric acid 35% (44.2 mL), 1,2-dimethoxy-
ethane (3.7 mL) and formaldehyde (3.5 mL, 44.2 mmol,
2 equiv). After further stirring of the mixture at 72 ꢁC
for 18 h, the solution was cooled in an ice bath and fil-
tered, the residue washed with 3ꢂ5 mL of 1,2-dime-
thoxyethane and dried under vacuum to yield 7-OH-6,8-
diiodo-l-Tic as a white solid (5.4 g, 55% yield); Rf 0.45
4-Phenylbutyryl-7-OH-L-Tic-pyrrolidine (4). Compound
4 was prepared from 7-OH-Boc-l-Tic-pyrrolidine (700
mg, 2.0 mmol, 1 equiv) and 4-phenylbutyric acid (335
mg, 2.0 mmol, 1 equiv) by method C and was obtained
after TLC (CH2Cl2/MeOH, 95:5) as a colourless oil (300
mg, 38% yield); Rf 0.70 (CH2Cl2/MeOH, 9:1); HPLC tR
1
19.57 min; H NMR (acetone d6) d 1.56–1.63 (m, 2H,
pyrrolidine), 1.74–1.78 (m, 4H, CH2CH2CH2C6H5 and
pyrrolidine), 2.32 (t, J=7.2 Hz, 2H, CH2CH2CH2C6H5),
2.46 (t, J=7.6 Hz, 2H, CH2CH2CH2C6H5), 2.74–2.86
1
(CH2Cl2/MeOH, 8:2); HPLC tR 11.57 min; H NMR

S. Vendeville et al. / Bioorg. Med. Chem. 10 (2002) 1719–1729
1725
(m, 2H, Tic–H), 3.08–3.25 (m, 2H, pyrrolidine), 3.35–
3.57 (m, 2H, pyrrolidine), 4.37 (d, J=15.3 Hz, 1H, Tic–
H), 4.47 (d, J=15.3 Hz, 1H, Tic–H), 4.61 (t, J=6.5 Hz,
1H, Tic–H), 6.52 (s, 1H, Ar–H), 6.53 (d, J=2.4 Hz, 1H,
Ar–H), 6.80–6.85 (m, 1H, Ar–H), 6.95–7.10 (m, 5H, Ar–
H); TOFMS m/z 392 (M+).
3.70–3.73 (m, 1H, pyrrolidine), 4.57 (d, J=15.3 Hz, 1H,
Tic–H), 4.64 (d, J=15.3 Hz, 1H, Tic–H), 5.12 (t, J=6.6
Hz, 1H, Tic–H), 7.00–7.27 (m, 9H, Ar–H); TOFMS m/z
362 (M+).
5-Phenylpentanoyl-L-Tic-pyrrolidine (9). Compound 9
was prepared from Boc-l-Tic-pyrrolidine (90 mg, 0.40
mmol, 1 equiv) and 5-phenylpentanoic acid (70 mg, 0.40
mmol, 1 equiv) by method C and was obtained after
TLC (CH2Cl2/MeOH, 96:4) as a colourless oil (100 mg,
63% yield); Rf 0.80 (CH2Cl2/MeOH, 9.5:0.5); HPLC tR
4-Phenylbutyryl-7-OEt-L-Tic-pyrrolidine (5). To a solu-
tion of compound 4 (100 mg, 0.26 mmol, 1 equiv) in 5
mL of DMF, were added ethylbromide (29 mL, 0.38
mmol, 1.5 equiv) and K2CO3 (70 mg, 0.51 mmol, 2
equiv). After stirring the mixture at 60 ꢁC for 18 h, the
solvent was evaporated and the residue taken up in 20
mL of AcOEt, washed by 2ꢂ20 mL of a solution of HCl
1 M and brine. The organic layer was then dried over
MgSO4 and the residue purified by TLC (CH2Cl2/
MeOH, 94:6) to yield compound 5 as a colourless oil (70
mg, 67% yield); Rf 0.65 (CH2Cl2/MeOH, 9.4:0.6); HPLC
tR 25.12 min; 1H NMR (CDCl3) d 1.39 (t, J=7.0 Hz, 3H,
OCH2CH3), 1.80–2.03 (m, 6H, CH2CH2CH2C6H5 and
pyrrolidine), 2.42–2.48 (m, 2H, CH2CH2CH2C6H5), 2.68
(t, J=7.3 Hz, 2H, CH2CH2CH2C6H5), 2.99 (d, J=6.6
Hz, 2H, Tic-H), 3.33–3.52 (m, 3H, pyrrolidine), 3.74–
3.80 (m, 1H, pyrrolidine), 3.98 (q, J=7.0 Hz, 2H,
OCH2CH3), 4.48 (d, J=15.3 Hz, 1H, Tic–H), 4.62 (d,
J=15.3 Hz, 1H, Tic–H), 5.11 (t, J=6.6 Hz, 1H, Tic–H),
6.60 (d, J=2.4 Hz, 1H, Ar–H), 6.74 (dd, J=2.4, 8.3 Hz,
1H, Ar–H), 7.04 (d, J=8.3 Hz, 1H, Ar–H), 7.14–7.30
(m, 5H, Ar–H); TOFMS m/z 420 (M+).
24.48 min; 1H NMR (CDCl3)
d 1.69 (m, 4H,
CH2CH2CH2CH2), 1.80–2.06 (m, 4H, pyrrolidine),
2.50–2.64 (m, 4H, CH2CH2CH2CH2), 3.07 (d, J=6.6
Hz, 2H, Tic–H), 3.30–3.52 (m, 3H, pyrrolidine), 3.75–
3.82 (m, 1H, pyrrolidine), 4.62 (d, J=15.3 Hz, 1H, Tic–
H), 4.69 (d, J=15.3 Hz, 1H, Tic–H), 5.14 (t, J=6.6 Hz,
1H, Tic–H), 7.07–7.29 (m, 9H, Ar–H); TOFMS m/z 390
(M+).
4-(4-Nitrophenyl)butyryl-L-Tic-pyrrolidine (10). Com-
pound 10 was prepared from Boc-l-Tic-pyrrolidine (110
mg, 0.48 mmol, 1 equiv) and 4-(4-nitrophenyl)butyric
acid (100 mg, 0.48 mmol, 1 equiv) by method C and was
obtained after TLC (CH2Cl2/MeOH, 95:5) as a colour-
less oil (115 mg, 58% yield); Rf 0.95 (CH2Cl2/MeOH,
9:1); HPLC tR 22.88 min; 1H NMR (acetone-d6) d 1.59–
1.86 (m, 6H, CH2CH2CH2C6H5 and pyrrolidine), 2.42
(t, J=7.4 Hz, 2H, CH2CH2CH2C6H4NO2), 2.69 (t,
J=7.7 Hz, 2H, CH2CH2CH2C6H5NO2), 2.84–2.99 (m,
2H, Tic–H), 3.05–3.58 (m, 4H, pyrrolidine), 4.47 (d,
J=15.8 Hz, 1H, Tic–H), 4.65 (d, J=15.8 Hz, 1H, Tic–
H), 5.03 (t, J=6.2 Hz, 1H, Tic–H), 7.00–7.09 (m, 4H,
Ar–H), 7.37 (d, J=8.7 Hz, 2H, Ar–H), 8.00 (d, J=8.7
Hz, 2H, Ar–H); TOFMS m/z 421 (M+).
4-Benzoyl-L-Tic-pyrrolidine (6). Compound 6 was pre-
pared from Boc-l-Tic-pyrrolidine (120 mg, 0.52 mmol,
1 equiv) and benzoic acid (65 mg, 0.52 mmol, 1 equiv)
by method C and was obtained after TLC (CH2Cl2/
MeOH, 95:5) as a colourless oil (105 mg, 69% yield); Rf
1
0.60 (CH2Cl2/MeOH, 9.5:0.5); HPLC tR 19.63 min; H
NMR (CDCl3) d 1.80–2.10 (m, 4H, pyrrolidine), 3.10–
3.25 (m, 2H, Tic–H), 3.41–3.92 (m, 4H, pyrrolidine),
4.60 (d, J=15.6 Hz, 1H, Tic–H), 4.68 (d, J=15.6 Hz,
1H, Tic–H), 5.18 (t, J=7.1 Hz, 1H, Tic–H), 6.90–7.61
(m, 9H, Ar–H); TOFMS m/z 334 (M+).
4-(4-Methoxyphenyl)butyryl-L-Tic-pyrrolidine (11). Com-
pound 11 was prepared from Boc-l-Tic-pyrrolidine
(120 mg, 0.52 mmol, 1 equiv) and 4-(4-methoxy-
phenyl)butyric acid (100 mg, 0.52 mmol, 1 equiv) by
method C and was obtained after TLC (CH2Cl2/MeOH,
95:5) as a colourless oil (145 mg, 70% yield); Rf
0.75 (CH2Cl2/MeOH, 9:1); HPLC tR 22.75 min;
Phenylacetyl-L-Tic-pyrrolidine (7). Compound 7 was
prepared from Boc-l-Tic-pyrrolidine (120 mg, 0.52
mmol, 1 equiv) and benzylic acid (70 mg, 0.52 mmol, 1
equiv) by method C and was obtained after TLC
(CH2Cl2/MeOH, 95:5) as a colourless oil (120 mg, 67%
yield); Rf 0.75 (CH2Cl2/MeOH, 9.5:0.5); HPLC tR 20.48
1H NMR (acetone-d6)
d
1.53–1.77 (m, 6H,
CH2CH2CH2C6H5 and pyrrolidine), 2.33 (t, J=7.3 Hz,
2H, CH2CH2CH2C6H4OMe), 2.44 (t, J=7.6 Hz, 2H,
CH2CH2CH2C6H5OMe), 2.92 (d, J=5.8 Hz, 2H, Tic–
H), 3.00–3.62 (m, 4H, pyrrolidine), 3.59 (s, 3H, OCH3),
4.45 (d, J=15.8 Hz, 1H, Tic–H), 4.59 (d, J=15.8 Hz,
1H, Tic–H), 5.04 (t, J=6.1 Hz, 1H, Tic–H), 6.68 (d,
J=8.5 Hz, 2H, Ar–H), 6.93 (d, J=8.5 Hz, 2H, Ar–H),
6.99–7.06 (m, 4H, Ar–H); TOFMS m/z 406 (M+).
1
min; H NMR (CDCl3) d 1.80–2.00 (m, 4H, pyrroli-
dine), 3.07 (d, J=6.8 Hz, 2H, Tic–H), 3.30–3.56 (m, 3H,
pyrrolidine), 3.72–3.75 (m, 1H, pyrrolidine), 3.85 (s, 2H,
CH2), 4.68 (s, 2H, Tic–H), 5.10 (t, J=6.8 Hz, 1H, Tic–
H), 6.90–7.30 (m, 9H, Ar–H); TOFMS m/z 348 (M+).
4-(Thiophen-2-yl)butyryl-L-Tic-pyrrolidine (12). Com-
pound 12 was prepared from Boc-l-Tic-pyrrolidine (110
mg, 0.48 mmol, 1 equiv) and 4-(thiophen-2-yl)butyric
acid (80 mg, 0.48 mmol, 1 equiv) by method C and was
obtained after TLC (CH2Cl2/MeOH, 98:2) as a yellow
oil (75 mg, 40% yield); Rf 0.85 (CH2Cl2/MeOH, 9:1);
3-Phenylpropanoyl-L-Tic-pyrrolidine (8). Compound 8
was prepared from Boc-l-Tic-pyrrolidine (120 mg, 0.52
mmol, 1 equiv) and 3-phenylpropanoic acid (80 mg,
0.52 mmol, 1 equiv) by method C and was obtained
after TLC (CH2Cl2/MeOH, 95:5) as a colourless oil (145
mg, 76% yield); Rf 0.55 (CH2Cl2/MeOH, 9.5:0.5);
HPLC tR 22.19 min; H NMR (CDCl3) d 1.70–1.90 (m,
4H, pyrrolidine), 2.60–3.00 (m, 4H, CH2CH2), 3.04 (d,
J=6.6 Hz, 2H, Tic–H), 3.30–3.51 (m, 3H, pyrrolidine),
1
HPLC tR 22.66 min; H NMR (acetone-d6) d 1.57–1.92
1
(m, 6H, COCH2CH2CH2 and pyrrolidine), 2.38 (t,
J=7.3 Hz, 2H, COCH2CH2CH2), 2.72 (t, J=6.2 Hz,
2H, COCH2CH2CH2), 2.88–2.98 (m, 2H, Tic–H), 3.03–

1726
S. Vendeville et al. / Bioorg. Med. Chem. 10 (2002) 1719–1729
3.75 (m, 4H, pyrrolidine), 4.46 (d, J=15.5 Hz, 1H, Tic–
H), 4.62 (d, J=15.5 Hz, 1H, Tic–H), 5.04 (t, J=6.2 Hz,
1H, Tic–H), 6.65–6.72 (m, 1H, Ar–H), 6.72–6.79 (m,
1H, Ar–H), 6.98–7.08 (m, 5H, Ar–H); TOFMS m/z 382
(M+).
2H, Tic–H), 3.27–3.47 (m, 3H, pyrrolidine), 3.68–3.72
(m, 1H, pyrrolidine), 4.62–4.65 (m, 2H, Tic–H), 5.06 (t,
J=6.6 Hz, 1H, Tic–H), 7.03–7.20 (m, 4H, Ar–H);
TOFMS m/z 368 (M+).
4-Methylcyclohexylcarbonyl-L-Tic-pyrrolidine (17). Com-
pound 17 was prepared from Boc-l-Tic-pyrrolidine (210
mg, 0.91 mmol, 1 equiv) and 4-methylcyclohexyl-
carboxylic acid (140 mg, 1 mmol, 1.1 equiv) by method
C and was obtained after TLC (CH2Cl2/MeOH, 93:7) as
a yellow oil (295 mg, 92% yield); Rf 0.45 (CH2Cl2/
MeOH, 9.5:0.5); HPLC tR 23.38 min; 1H NMR
(CDCl3) d 0.90+0.97 (d, J=7.0 Hz, 3H, CH3 cis/trans),
1.50–1.99 (m, 13H, cyclohexyle and pyrrolidine), 2.50–
2.69 (m, 1H, cyclohexyle), 3.03–3.11 (m, 2H, Tic–H),
3.37–3.53 (m, 3H, pyrrolidine), 3.69–3.75 (m, 1H, pyr-
rolidine), 4.65–4.72 (m, 2H, Tic–H), 5.11 (dd, J=6.7,
15.2 Hz, 1H, Tic–H), 7.13–7.24 (m, 4H, Ar–H);
TOFMS m/z 354 (M+).
4-(Pyren-1-yl)butyryl-L-Tic-pyrrolidine (13). Compound
13 was prepared from Boc-l-Tic-pyrrolidine (140 mg,
0.61 mmol, 1 equiv) and 4-(pyren-1-yl)butyric acid (175
mg, 0.61 mmol, 1 equiv) by method C and was obtained
after TLC (CH2Cl2/MeOH, 96:4) as a yellow oil (200
mg, 66% yield); Rf 0.60 (CH2Cl2/MeOH, 9.6:0.4);
1
HPLC tR 32.19 min; H NMR (CDCl3) d 1.82–2.03 (m,
4H, pyrrolidine), 2.17–2.30 (m, 2H, COCH2CH2CH2),
2.54–2.61 (m, 2H, COCH2CH2CH2), 3.07 (d, J=6.6 Hz,
2H, Tic–H), 3.38–3.55 (m, 5H, COCH2CH2CH2 and
pyrrolidine), 3.75–3.80 (m, 1H, pyrrolidine), 4.48 (d,
J=15.3 Hz, 1H, Tic–H), 4.62 (d, J=15.3 Hz, 1H, Tic–
H), 5.16 (t, J=6.6 Hz, 1H, Tic–H), 6.90 (d, J=7.4 Hz,
1H, Ar–H), 7.10–7.20 (m, 3H, Ar–H), 7.87 (d, J=7.8
Hz, 1H, Ar–H), 7.98–8.10 (m, 5H, Ar–H), 8.16 (d,
J=7.5 Hz, 2H, Ar–H), 8.31 (d, J=9.3 Hz, 1H, Ar–H);
TOFMS m/z 500 (M+).
Cyclohexylcarbonyl-L-Tic-pyrrolidine (18). Compound
18 was prepared from Boc-l-Tic-pyrrolidine (210 mg,
0.91 mmol, 1 equiv) and cyclohexylcarboxylic acid (130
mg, 1 mmol, 1.1 equiv) by method C and was obtained
after TLC (CH2Cl2/MeOH, 93:7) as a colourless oil (135
mg, 44% yield); Rf 0.40 (CH2Cl2/MeOH, 9.5:0.5);
Pyrid-1-ylacetyl-L-Tic-pyrrolidine (14). Compound 14
was prepared from Boc-l-Tic-pyrrolidine (210 mg, 0.91
mmol, 1 equiv) and 4-(pyrid-3-yl)acetic acid, HCl (175
mg, 1 mmol, 1.1 equiv) by method C and was obtained
after TLC (CH2Cl2/MeOH, 90:10) as a yellow solid (115
mg, 37% yield); Rf 0.20 (CH2Cl2/MeOH, 9.5:0.5);
1
HPLC tR 21.63 min; H NMR (CDCl3) d 1.25–2.03 (m,
13H, cyclohexyle and pyrrolidine), 2.59–2.67 (m, 1H,
cyclohexyle), 3.03 (dd, J=6.6, 15.4 Hz, 1H, Tic–H),
3.12 (dd, J=6.6, 15.4 Hz, 1H, Tic–H), 3.37–3.53 (m,
3H, pyrrolidine), 3.70–3.75 (m, 1H, pyrrolidine), 4.68
(d, J=15.3 Hz, 1H, Tic–H), 4.74 (d, J=15.3 Hz, 1H,
Tic–H), 5.13 (t, J=6.6 Hz, 1H, Tic–H), 7.13–7.24 (m,
4H, Ar–H); TOFMS m/z 340 (M+).
1
HPLC tR 13.27 min; H NMR (CDCl3) d 1.83–2.01 (m,
4H, pyrrolidine), 3.08–3.12 (m, 2H, Tic–H), 3.40–3.52
(m, 3H, pyrrolidine), 3.78–3.86 (m, 1H, pyrrolidine),
3.87 (s, 2H, CH2), 4.62 (d, J=14.9 Hz, 1H, Tic–H), 4.75 (d,
J=14.9 Hz, 1H, Tic–H), 5.06 (t, J=7.0 Hz, 1H, Tic–H),
6.99 (d, J=6.9 Hz, 1H, Ar–H), 7.16–7.24 (m, 5H, Ar–H),
8.53 (d, J=4.3 Hz, 2H, Ar–H); TOFMS m/z 349 (M+).
Cyclohexylacetyl-L-Tic-pyrrolidine (19). Compound 19
was prepared from Boc-l-Tic-pyrrolidine (210 mg, 0.91
mmol, 1 equiv) and cyclohexylacetic acid (140 mg, 1
mmol, 1.1 equiv) by method C and was obtained after
TLC (CH2Cl2/MeOH, 93:7) as a colourless oil (120 mg,
37% yield); Rf 0.40 (CH2Cl2/MeOH, 9.5:0.5); HPLC tR
23.51 min; 1H NMR (CDCl3) d 0.82–2.07 (m, 15H,
cyclohexyle and pyrrolidine), 2.38 (d, J=6.6 Hz, 2H,
COCH2), 3.04 (dd, J=6.7, 15.3 Hz, 1H, Tic–H), 3.12
(dd, J=6.8, 15.4 Hz, 1H, Tic–H), 3.37–3.51 (m, 3H,
pyrrolidine), 3.70–3.72 (m, 1H, pyrrolidine), 4.67 (d,
J=15.3 Hz, 1H, Tic–H), 4.74 (d, J=15.3 Hz, 1H, Tic–
H), 5.13 (t, J=6.5 Hz, 1H, Tic–H), 7.15–7.26 (m, 4H,
Ar–H); TOFMS m/z 354 (M+).
Pyrid-1-ylcarbonyl-L-Tic-pyrrolidine (15). Compound 15
was prepared from Boc-l-Tic-pyrrolidine (210 mg, 0.91
mmol, 1 equiv) and isonicotinic acid (125 mg, 1 mmol,
1.1 equiv) by method C and was obtained after TLC
(CH2Cl2/MeOH, 93:7) as a yellow solid (185 mg, 60%
yield); Rf 0.20 (CH2Cl2/MeOH, 9.5:0.5); HPLC tR 13.64
1
min; H NMR (CDCl3) d 1.88–2.05 (m, 4H, pyrroli-
dine), 3.19 (d, J=7.3 Hz, 2H, Tic–H), 3.42–3.63 (m, 3H,
pyrrolidine), 3.86–3.93 (m, 1H, pyrrolidine), 4.43 (d,
J=15.1 Hz, 1H, Tic–H), 4.71 (d, J=15.1 Hz, 1H, Tic–
H), 5.09 (t, J=7.3 Hz, 1H, Tic–H), 6.95 (d, J=7.3 Hz,
1H, Ar–H), 7.19–7.27 (m, 3H, Ar–H), 7.35 (dd, J=1.6,
4.4 Hz, 2H, Ar–H), 8.73 (dd, J=1.6, 4.4 Hz, 2H, Ar–
H); TOFMS m/z 335 (M+).
3-Cyclohexylpropanoyl-L-Tic-pyrrolidine (20). Com-
pound 20 was prepared from Boc-l-Tic-pyrrolidine (210
mg, 0.91 mmol, 1 equiv) and 3-cyclohexylpropanoic
acid (155 mg, 1 mmol, 1.1 equiv) by method C and was
obtained after TLC (CH2Cl2/MeOH, 93:7) as a colour-
less oil (95 mg, 29% yield); Rf 0.40 (CH2Cl2/MeOH,
9.5:0.5); HPLC tR 25.32 min; 1H NMR (CDCl3) d 0.90–
1.00 (m, 2H, cyclohexyle), 1.18–1.28 (m, 5H, cyclohex-
yle), 1.50–1.55 (m, 2H, COCH2CH2), 1.66–1.74 (m, 4H,
cyclohexyle), 1.82–2.04 (m, 4H, pyrrolidine), 2.43–2.49
(m, 2H, COCH2CH2), 3.07 (d, J=6.6 Hz, 2H, Tic–H),
3.35–3.53 (m, 3H, pyrrolidine), 3.75–3.80 (m, 1H, pyr-
rolidine), 4.66 (d, J=15.3 Hz, 1H, Tic–H), 4.72 (d,
4-Methylcyclohexylacetyl-L-Tic-pyrrolidine (16). Com-
pound 16 was prepared from Boc-l-Tic-pyrrolidine (90
mg, 0.40 mmol, 1 equiv) and 4-methylcyclohexylacetic
acid (60 mg, 0.40 mmol, 1 equiv) by method C and was
obtained after TLC (CH2Cl2/MeOH, 97:3) as a colour-
less oil (90 mg, 61% yield); Rf 0.70 (CH2Cl2/MeOH,
9.5:0.5); HPLC tR 25.29 min; 1H NMR (CDCl3) d
0.77+0.84 (d, J=6.8 Hz, 3H, CH3 cis/trans), 1.18–1.96
(m, 14H, cyclohexyle and pyrrolidine), 2.26+2.36 (d,
J=7.0 Hz, 2H, COCH2 cis/trans), 3.00 (d, J=6.6 Hz,

S. Vendeville et al. / Bioorg. Med. Chem. 10 (2002) 1719–1729
1727
J=15.3 Hz, 1H, Tic–H), 5.15 (t, J=6.6 Hz, 1H, Tic–H),
7.12–7.23 (m, 4H, Ar–H); TOFMS m/z 368 (M+).
Adamant-1-ylacetyl-L-Tic-pyrrolidine (25). Compound
25 was prepared from Boc-l-Tic-pyrrolidine (210 mg,
0.91 mmol, 1 equiv) and adamant-1-ylacetic acid (195
mg, 1 mmol, 1.1 equiv) by method C and was obtained
after TLC (CH2Cl2/MeOH, 93:7) as a colourless oil (145
mg, 39% yield); Rf 0.35 (CH2Cl2/MeOH, 9.5:0.5);
4-Cyclohexylbutyryl-L-Tic-pyrrolidine (21). Compound
21 was prepared from Boc-l-Tic-pyrrolidine (210 mg,
0.91 mmol, 1 equiv) and 4-cyclohexylbutyric acid (170
mg, 1 mmol, 1.1 equiv) by method C and was obtained
after TLC (CH2Cl2/MeOH, 93:7) as a yellow oil (280
mg, 81% yield); Rf 0.45 (CH2Cl2/MeOH, 9.5:0.5);
1
HPLC tR 27.58 min; H NMR (CDCl3) d 1.61–1.99 (m,
19H, adamantyl and pyrrolidine), 2.30 (d, J=7.1 Hz,
2H, COCH2), 3.07 (d, J=6.7 Hz, 2H, Tic–H), 3.36–3.53
(m, 3H, pyrrolidine), 3.75–3.81 (m, 1H, pyrrolidine),
4.68 (d, J=15.2 Hz, 1H, Tic–H), 4.80 (d, J=15.2 Hz,
1H, Tic–H), 5.15 (t, J=6.7 Hz, 1H, Tic–H), 7.11–7.22
(m, 4H, Ar–H); TOFMS m/z 406 (M+).
1
HPLC tR 27.93 min; H NMR (CDCl3) d 0.78–0.96 (m,
2H, cyclohexyle), 1.18–1.26 (m, 6H, COCH2CH2CH2
and cyclohexyle), 1.65–2.04 (m, 11H, COCH2CH2CH2
and cyclohexyle and pyrrolidine), 2.42–2.46 (m, 2H,
COCH2CH2CH2), 3.07 (d, J=6.6 Hz, 2H, Tic–H),
3.36–3.54 (m, 3H, pyrrolidine), 3.75–3.83 (m, 1H, pyr-
rolidine), 4.66 (d, J=15.3 Hz, 1H, Tic–H), 4.72 (d,
J=15.3 Hz, 1H, Tic–H), 5.15 (t, J=6.6 Hz, 1H, Tic–H),
7.12–7.23 (m, 4H, Ar–H); TOFMS m/z 382 (M+).
Dicyclohexylacetyl-L-Tic-pyrrolidine (26). Compound 26
was prepared from Boc-l-Tic-pyrrolidine (210 mg, 0.91
mmol, 1 equiv) and dicyclohexylacetic acid (225 mg, 1
mmol, 1.1 equiv) by method C and was obtained after
TLC (CH2Cl2/MeOH, 93:7) as a colourless oil (185 mg,
46% yield); Rf 0.55 (CH2Cl2/MeOH, 9.5:0.5); HPLC tR
20.05 min; 1H NMR (CDCl3) d 0.98–1.27 (m, 10H,
cyclohexyle), 1.66–1.74 (m, 12H, cyclohexyle), 1.86–1.94
(m, 2H, pyrrolidine), 1.99–2.09 (m, 3H, COCH and
pyrrolidine), 3.12–3.20 (m, 2H, Tic–H), 3.41–3.60 (m,
3H, pyrrolidine), 3.76–3.84(m, 1H, pyrrolidine), 4.74
(d, J=15.2 Hz, 1H, Tic–H), 4.82–4.89 (m, 2H, Tic-
H), 7.17–7.30 (m, 4H, Ar–H); TOFMS m/z 436
(M+).
5-Cyclohexylpentanoyl-L-Tic-pyrrolidine (22). Com-
pound 22 was prepared from Boc-l-Tic-pyrrolidine (210
mg, 0.91 mmol, 1 equiv) and 5-cyclohexylpentanoic acid
(185 mg, 1 mmol, 1.1 equiv) by method C and was
obtained after TLC (CH2Cl2/MeOH, 93:7) as a yellow
oil (180 mg, 50% yield); Rf 0.40 (CH2Cl2/MeOH,
9.5:0.5); HPLC tR 29.90 min; 1H NMR (CDCl3) d 0.79–
0.88 (m, 2H, cyclohexyle), 1.16–1.37 (m, 6H,
COCH2CH2CH2CH2 and cyclohexyle), 1.56–1.69 (m,
8H, COCH2CH2CH2CH2 and cyclohexyle), 1.83–2.02
(m, 5H, cyclohexyle and pyrrolidine), 2.42–2.46 (m, 2H,
COCH2CH2CH2), 3.08 (d, J=6.6 Hz, 2H, Tic–H),
3.35–3.54 (m, 3H, pyrrolidine), 3.73–3.83 (m, 1H, pyr-
rolidine), 4.66 (d, J=15.6 Hz, 1H, Tic–H), 4.72 (d,
J=15.6 Hz, 1H, Tic–H), 5.17 (t, J=6.6 Hz, 1H, Tic–H),
7.12–7.26 (m, 4H, Ar–H); TOFMS m/z 396 (M+).
Boc-L-Pro-OMe. To a solution of Boc-l-Pro (1 g, 4.7
mmol, 1 equiv) in 20 mL of MeOH, was added 1.3 mL
of a solution of Cs2CO3 1M (2.3 mmol, 0.5 equiv). The
solvent was evaporated and the residue taken up in
3ꢂ30 mL of a toluene/EtOH 1:1 mixture and con-
centrated. To a solution of the cesium salt in 10 mL of
DMF was added CH3I (725 mg, 5.1 mmol, 1.1 equiv).
After stirring the mixture at room temperature for 2 h,
the solvent was evaporated with the residue being taken
up in 20 mL of AcOEt and washed with 20 mL of brine.
The organic layer was separated, dried over MgSO4 and
the solvent evaporated to yield Boc-l-Pro-OMe as a
colourless oil (985 mg, 93% yield); Rf 0.75 (CH2Cl2/
MeOH, 9.4:0.6); HPLC tR 18.15 min; 1H NMR
(CDCl3) d 1.41+1.46 (s, 9H, C(CH3)3 cis/trans), 1.83–
2.27 (m, 4H, CH2), 3.37–3.60 (m, 2H, CH2), 3.72 (s, 3H,
OCH3), 4.20–4.24+4.30–4.34 (m, 1H, CH cis/trans);
TOFMS m/z 229 (M+).
Cyclopentylacetyl-L-Tic-pyrrolidine (23). Compound 23
was prepared from Boc-l-Tic-pyrrolidine (210 mg, 0.91
mmol, 1 equiv) and cyclopentylacetic acid (130 mg, 1
mmol, 1.1 equiv) by method C and was obtained after
TLC (CH2Cl2/MeOH, 93:7) as a colourless oil (200 mg,
65% yield); Rf 0.50 (CH2Cl2/MeOH, 9.5:0.5); HPLC tR
22.09 min; 1H NMR (CDCl3) d 1.06–1.62 (m, 8H,
cyclopentyle), 1.82–2.02 (m, 4H, pyrrolidine), 2.25–2.36
(m, 1H, cyclopentyle), 2.44 (d, J=7.1 Hz, 2H, COCH2),
3.07 (d, J=6.4 Hz, 2H, Tic–H), 3.33–3.52 (m, 3H, pyr-
rolidine), 3.73–3.82 (m, 1H, pyrrolidine), 4.70 (s, 2H,
Tic–H), 5.19 (t, J=6.4 Hz, 1H, Tic–H), 7.12–7.26 (m,
4H, Ar–H); TOFMS m/z 340 (M+).
Boc-L-Tic-L-Pro-OMe. Boc-l-Tic-l-Pro-OMe was pre-
pared from Boc-l-Pro-OMe (1 g, 4.4 mmol, 1 equiv)
and Boc-l-Tic (1.21 g, 4.4 mmol, 1 equiv) by method C
and was obtained after column chromatography
(CH2Cl2/MeOH, 95:5) as a colourless oil (1.29 g, 75%
yield); Rf 0.50 (CH2Cl2/MeOH, 9.5:0.5); HPLC tR 21.39
Norborn-2-ylacetyl-L-Tic-pyrrolidine (24). Compound 24
was prepared from Boc-l-Tic-pyrrolidine (210 mg, 0.91
mmol, 1 equiv) and norborn-2-ylacetic acid (155 mg, 1
mmol, 1.1 equiv) by method C and was obtained after
TLC (CH2Cl2/MeOH, 93:7) as a colourless oil (100 mg,
30% yield); Rf 0.40 (CH2Cl2/MeOH, 9.5:0.5); HPLC tR
24.25 min; 1H NMR (CDCl3) d 1.10–2.43 (m, 17H,
COCH2 and norbornyl and pyrrolidine), 3.07 (d, J=6.3
Hz, 2H, Tic–H), 3.35–3.54 (m, 3H, pyrrolidine), 3.73–
3.79 (m, 1H, pyrrolidine), 4.69 (s, 2H, Tic–H), 5.22 (t,
J=6.3 Hz, 1H, Tic–H), 7.12–7.23 (m, 4H, Ar–H);
TOFMS m/z 366 (M+).
1
min; H NMR (CDCl3) d 1.42+1.48 (s, 9H, C(CH3)3
cis/trans), 1.94–2.18 (m, 4H, pyrrolidine), 3.04–3.14 (m,
2H, Tic–H), 3.60+3.69 (s, 3H, OCH3 cis/trans), 3.65–
3.77
(m,
2H,
pyrrolidine),
4.31+4.48
(d,
J=15.5+15.9 Hz, 1H, Tic–H cis/trans), 4.51–4.55 (m,
1H, pyrrolidine), 4.61+5.02 (t, J=7.0+5.9 Hz, 1H,
Tic–H), 4.80+4.96 (d, J=15.5+15.9 Hz, 1H, Tic–H
cis/trans), 7.11–7.26 (m, 4H, Ar–H); TOFMS m/z 388
(M+).

1728
S. Vendeville et al. / Bioorg. Med. Chem. 10 (2002) 1719–1729
4-Phenylbutyryl-L-Tic-L-Pro-OMe. 4-phenylbutyryl-l-
Tic-l-Pro-OMe was prepared from Boc-l-Tic-l-Pro-
OMe (425 mg, 1.1 mmol, 1 equiv) and 4-phenylbutyric
acid (180 mg, 1.1 mmol, 1 equiv) by method C and was
obtained after TLC (CH2Cl2/MeOH, 94:6) as a colour-
less oil (252 mg, 54% yield); Rf 0.40 (CH2Cl2/MeOH,
9.5:0.5); HPLC tR 22.97 min; 1H NMR (CDCl3) d 1.93–
2.20 (m, 6H, COCH2CH2CH2 and pyrrolidine), 2.42 (t,
J=7.4 Hz, 2H, COCH2CH2CH2), 2.65 (t, J=7.4 Hz,
2H, COCH2CH2CH2), 3.07–3.11 (m, 2H, Tic–H), 3.62
(s, 3H, OCH3), 3.64–3.71 (m, 2H, pyrrolidine), 4.47–4.60
(m, 3H, Tic–H and pyrrolidine), 4.99 (t, J=7.3 Hz, 1H,
Tic–H), 7.02–7.26 (m, 9H, Ar–H); TOFMS m/z 434 (M+).
4-Phenylbutyryl-L-Tic-L-Pro-OCH2OCOMe (29). To a
solution of compound 28 (100 mg, 0.22 mmol, 1 equiv)
in 5 mL of DMF, was added potassium acetate (21 mg,
0.22 mmol, 1 equiv). After stirring the mixture at 60 ꢁC
for 3 h, the solvent was evaporated with the residue
being taken up in 20 mL of a AcOEt/brine 1:1 mixture.
The organic layer was dried over MgSO4 and the sol-
vent evaporated to yield compound 29 as a colourless
oil (45 mg, 43% yield); Rf 0.25 (CH2Cl2/MeOH,
9.5:0.5); HPLC tR 22.92 min; 1H NMR (CDCl3) d 1.90–
2.25 (m, 9H, COCH2CH2CH2 and pyrrolidine and
COCH3), 2.53 (t, J=7.4 Hz, 2H, COCH2CH2CH2),
2.72 (t, J=7.4 Hz, 2H, COCH2CH2CH2), 3.00–3.15 (m,
2H, Tic–H), 3.63–3.87 (m, 2H, pyrrolidine), 4.42–4.62
(m, 3H, Tic–H and pyrrolidine), 4.76–4.91 (m, 3H, Tic–
4-Phenylbutyryl-L-Tic-Pro-CHO (27). To a solution of
4-phenylbutyryl-l-Tic-l-Pro-OMe (200 mg, 0.46 mmol,
1 equiv) in 5 mL of CH2Cl2 dried over molecular sieves,
was added at ꢀ70 ꢁC, a solution of DIBAL 1 M in
CH2Cl2 (1.38 mL, 1.38 mmol, 3 equiv). After stirring
the mixture at ꢀ70 ꢁC for 2 h, 2 mL of isopropanol was
added, the solution stirred for a further 10 min, warmed
to room temperature, then washed with 2ꢂ10 mL of
water. The organic layer was dried over MgSO4, the
solvent evaporated and the residue purified by column
chromatography (CH2Cl2/MeOH/pyridine, 90:10:0.1)
to yield 27 as a colourless oil under the racemic form (30
mg, 16% yield); Rf 0.50 (AcOEt/cyclohexane, 5:5);
H
and COCH2OAc), 7.02–7.31 (m, 9H, Ar–H);
TOFMS m/z 476 (M+).
4-Phenylbutyryl-L-Tic-L-Pro-OCH2OH (30). To a solu-
tion of compound 29 (95 mg, 0.20 mmol, 1 equiv) in 5
mL of MeOH, was added Na2CO3 (23 mg, 0.22 mmol,
1.1 equiv). After stirring the mixture at room tempera-
ture for 30 min, the solvent was evaporated with the
residue being taken up in 15 mL of CH2Cl2, washed
with 2ꢂ15 mL of brine. The organic layer was dried
over MgSO4, the solvent evaporated and the residue
purified by TLC (CH2Cl2/MeOH, 92:8) to yield com-
pound 30 as a colourless oil (25 mg, 31% yield); Rf 0.20
(CH2Cl2/MeOH, 9.5:0.5); HPLC tR 20.40 min; 1H NMR
(CDCl3) d 1.87–2.23 (m, 6H, COCH2CH2CH2 and pyr-
rolidine), 2.55 (t, J=7.4 Hz, 2H, COCH2CH2CH2), 2.70
(t, J=7.4 Hz, 2H, COCH2CH2CH2), 3.01–3.16 (m, H,
Tic-H), 3.25 (broad s, 1H, OH), 3.63–3.91 (m, H, pyr-
rolidine), 4.25+439 (d, J=18.9 Hz, 2H, CH2OH cis/
trans), 4.46–4.63 (m, 3H, Tic–H and pyrrolidine), 4.74–
4.80 (m, 1H, Tic–H), 7.06–7.31 (m, 9H, Ar–H); TOFMS
m/z 434 (M+).
1
HPLC tR 21.17 min (broad); H NMR (CDCl3) d 1.84–
2.06 (m, 6H, COCH2CH2CH2 and pyrrolidine), 2.41–
2.48 (m, 2H, COCH2CH2CH2), 2.63–2.70 (m, 2H,
COCH2CH2CH2), 3.00–3.20 (m, 2H, Tic–H), 3.71–3.87
(m, 2H, pyrrolidine), 4.47–4.59 (m, 3H, Tic–H and pyr-
rolidine), 5.10 (t, J=7.0 Hz, 1H, Tic–H), 7.03–7.31 (m,
9H, Ar–H), 9.42+9.44 (d+s, 0.7H, J=2.0 Hz, CHO),
9.74+9.75 (d+s, 0.3H, J=2.0 Hz, CHO); TOFMS m/z
404 (M+).
4-Phenylbutyryl-L-Tic-L-Pro-OCH2Cl (28). A solution
of LDA (5 mmol) was prepared from a solution of di-
isopropylamine (770 mL, 5.5 mmol, 5.5 equiv) in 5 mL
of THF and a solution of n-BuLi 2.5 M (2 mL, 5 mmol,
5 equiv) in hexane. To a solution of 4-phenylbutyryl-l-
Tic-l-Pro-OMe (435 mg, 1 mmol, 1 equiv) and chloro-
iodoethane (291 mL, 4 mmol, 4 equiv) in 5 mL of THF
was added, at ꢀ70 ꢁC, the solution of LDA (5 mmol)
prepared previously. After stirring the mixture at
ꢀ70 ꢁC for 10 min, 10 mL of a solution of acetic acid
10% in THF was added dropwise within 10 min. The
solution was diluted at room temperature in 75 mL of
AcOEt, washed with 75 mL of brine, 75 mL of an aqu-
eous solution of NaHCO3 1 M then 75 mL of brine. The
organic layer was dried over MgSO4, the solvent evap-
orated and the residue purified by column chromato-
graphy (AcOEt/cyclohexane, 8:2) to yield compound 28
as a colourless oil (215 mg, 48% yield); Rf 0.40 (AcOEt/
cyclohexane, 8:2); HPLC tR 23.72 min; 1H NMR
(CDCl3) d 1.91–2.27 (m, 6H, COCH2CH2CH2 and pyr-
rolidine), 2.50 (t, J=7.4 Hz, 2H, COCH2CH2CH2), 2.68
(t, J=7.4 Hz, 2H, COCH2CH2CH2), 3.02–3.16 (m, 2H,
Tic–H), 3.63–3.81 (m, 2H, pyrrolidine), 4.15 (s, 2H,
COCH2Cl), 4.46–4.63 (m, 3H, Tic–H and pyrrolidine),
4.76–4.82 (m, 1H, Tic–H), 7.06–7.31 (m, 9H, Ar–H);
TOFMS m/z 452.5 (M+).
Boc-L-Pro-NH2. To a solution of Boc-l-Pro (2.15 g, 10
mmol, 1 equiv), pyridine (500 mL) and Boc2O (3 g, 13
mmol, 1.3 equiv) in 50 mL of dioxane was added
amnonium carbonate (1 g, 13 mmol, 1.3 equiv). After
stirring the mixture at room temperature for 18 h, the
solvent was evaporated and the residue taken up in 50
mL of AcOEt, washed with 50 mL of an aqueous solu-
tion of citric acid 20% and 50 mL of brine. The aqueous
layers were mixed and extracted by 250 mL of AcOEt.
The organic layers were mixed then dried over MgSO4
and the solvent evaporated to yield Boc-l-Pro-NH2 as a
colourless oil (2.14 g, 99% yield); Rf 0.75 (CH2Cl2/
MeOH, 9.5:0.5); HPLC tR 11.89 min; 1H NMR
(CDCl3) d 1.44 (s, 9H, C(CH3)3), 1.84–2.21 (m, 4H,
pyrrolidine), 3.20–3.48 (m, 2H, pyrrolidine), 4.20–4.30
(m, 1H, pyrrolidine), 6.60+6.84 (broad s, 2H, NH2 cis/
trans 50:50); TOFMS m/z 214 (M+).
4-Phenylbutyryl-OSu. To a solution of 4-phenylbutyric
acid (2 g, 12.2 mmol, 1 equiv) in 100 mL of CH2Cl2,
were added N-OHSu (1.4 g, 12.2 mmol, 1 equiv) and
DCC (2.5 g, 12.2 mmol, 1 equiv). After stirring the
mixture at room temperature for 4 h, the solution was
filtered and the filtrate washed with 2ꢂ60 mL of an
aqueous solution of NaHCO3 5% and 2ꢂ60 mL of

S. Vendeville et al. / Bioorg. Med. Chem. 10 (2002) 1719–1729
1729
brine. The organic layer was then dried over MgSO4
and the solvent evaporated to yield 4-phenyl-
butyryl-Osu as a white solid (3 g, 95% yield); Rf 0.90
(CH2Cl2/MeOH, 9.5:0.5); HPLC tR 21.35 min; 1H NMR
(CDCl3) d 2.04 (qt, J=7.4 Hz, 2H, CH2CH2CH2C6H5),
2.58 (t, J=7.4 Hz, 2H, CH2CH2CH2C6H5), 2.71 (t,
J=7.4 Hz, 2H, CH2CH2CH2C6H5), 2.80 (s, 4H, succi-
nimide); TOFMS m/z 261 (M+).
We also thank Sandrine Delarue for her kind help for
manuscript writing. This work was supported by CNRS
(IFR CNRS 63, UMR CNRS 8525) and Universite de
Lille II. Sandrine Vendeville was a recipient of fellow-
ships from the CNRS-Region Nord-Pas de Calais.
References and Notes
4-Phenylbutyryl-L-Tic. To a solution of l-Tic (780 mg,
4.4 mmol, 1 equiv) in 10 mL of dioxane, were added 8.8
mL of NaOH 0.5 M and 4-phenylbutyryl-OSu (1.15 g,
4.4 mmol, 1 equiv). After stirring the mixture at room
temperature for 18 h, the solvent was evaporated and
the residue taken up in 40 mL of water, acidified by con-
centrated chlorhydric acid until pH 3. The solution was
extracted with 3ꢂ40 mL of AcOEt, the organic layer
dried over MgSO4, the solvent evaporated and the residue
purified by TLC (CH2Cl2/MeOH, 90:10) to yield 4-phe-
nylbutyryl-l-Tic as a colourless oil (755 mg, 53% yield);
1. Yoshimoto, T.; Orlowski, R.; Walter, R. Biochemistry
1977, 16, 2942.
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D. Biochim. Biophys. Acta 1979, 569, 184.
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1980, 32, 117.
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6. Goossens, F.; De Meester, I.; Vanhoof, G.; Scharpe, S. Eur.
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9. Yoshimoto, T.; Sattar, A. K. M. A.; Hirose, W.; Tsuru, D.
Biochim. Biophys. Acta 1987, 916, 29.
10. Santana, J. M.; Grellier, P.; Schrevel, J.; Teixeira, A. R. L.
Biochem. J. 1997, 324, 129.
1
Rf 0.70 (CH2Cl2/MeOH, 9:1); HPLC tR 21.26 min; H
NMR (CDCl3) d 1.78–2.07 (m, 2H, CH2CH2CH2C6H5),
2.30–2.43 (m, 2H, CH2CH2CH2C6H5), 2.58–2.66 (m,
2H, CH2CH2CH2C6H5), 2.95–3.24 (m, 2H, H–Tic), 4.50
(s, 2H, H–Tic), 5.30 (m, 1H, H–Tic), 7.02–7.29 (m, 4H,
Ar–H); TOFMS m/z 323 (M+).
11. Wilk, S.; Orlowski, M. J. Neurochem. 1983, 41, 69.
12. Deprez, B.; Williard, X.; Bourel, L.; Coste, H.; Hyafil, F.;
Tartar, A. J. Am. Chem. Soc. 1995, 117, 5405.
13. Vendeville, S.; Buisine, E.; Williard, X.; Schrevel, J.;
Grellier, P.; Santana, J.; Sergheraert, C. Chem. Pharm. Bull.
1999, 47, 194.
14. Vendeville, S.; Bourel, L.; Davioud-Charvet, E.; Grellier,
P.; Deprez, B.; Sergheraert, C. Bioorg. Med. Chem. Lett. 1999,
9, 437.
4-Phenylbutyryl-L-Tic-L-Pro-NH2. 4-phenylbutyryl-l-
Tic-l-Pro-NH2 was prepared from Boc-l-Pro-NH2 (275
mg, 1.3 mmol, 1 equiv) and 4-phenylbutyryl-l-Tic (415
mg, 3.9 mmol, 3 equiv) by method C and was obtained
after TLC (CH2Cl2/MeOH, 95:5) as a colourless oil (150
mg, 28% yield); Rf 0.75 (CH2Cl2/MeOH, 9:1); HPLC tR
18.00 min; 1H NMR (CDCl3) d 1.82–2.07 (m, 2H,
CH2CH2CH2C6H5), 2.18–2.44 (m, 6H, CH2CH2CH2C6H5
and pyrrolidine), 2.59–2.64 (m, 2H, CH2CH2CH2C6H5),
2.95–3.18 (m, 2H, H–Tic), 3.58–3.74 (m, 2H, pyrrolidine),
4.38–4.93 (m, 4H, H–Tic and pyrrolidine), 6.61+6.82
(broad s, 2H, NH2), 7.05–7.25 (m, 9H, Ar–H); TOFMS
m/z 373 (M+).
15. Verschueren, K.; Toth, G.; Tourwe, D.; Lebl, M.; Van
Binst, G.; Hruby, V. Synthesis 1992, 458.
16. Kowalski, C. J.; Reddy, R. E. J. Org. Chem. 1992, 57, 7194.
17. Pozdnev, V. F. Tetrahedron Lett. 1995, 36, 7115.
18. Goossens, F.; Vanhoof, G.; De Meester, I.; Augustyns,
K.; Borloo, M.; Tourwe, D.; Haemers, A.; Scharpe, S. Eur. J.
Biochem. 1997, 250, 177.
19. Goossens, F.; De Meester, I.; Vanhoof, G.; Hendricks, D.;
Vriend, J.; Scharpe, S. Eur. J. Biochem. 1995, 233, 432.
20. Schechter, I.; Berger, A. Biochem. Biophys. Res. Commun.
1967, 27, 157.
21. Portevin, B.; Benoist, A.; Remond, G.; Herve, Y.; Vincent,
M.; Lepagnol, J.; De Nanteuil, G. J. Med. Chem. 1996, 39, 2379.
22. Yoshimoto, T.; Tsuru, D.; Yamamoto, N.; Ikezawa, R.;
Furukawa, S. Agri. Biol. Chem. 1991, 55, 37.
23. Fulop, V.; Bocskei, Z.; Polgar, L. Cell 1998, 94, 161.
24. Kabashima, T.; Fujii, M.; Meng, Y.; Ito, K.; Yoshimoto,
T. Archiv. Biochem. Biophysics 1998, 358, 141.
25. Tsuru, D.; Yoshimoto, T.; Koriyama, N.; Furukawa, S. J.
Biochem. 1988, 104, 580.
26. Arai, H.; Nishioka, H.; Niwa, S.; Yamanaka, T.; Tanaka,
Y.; Yoshinaga, K.; Kobayashi, N.; Miura, N.; Ikade, Y.
Chem. Pharm. Bull. 1993, 41, 1583.
4-Phenylbutyryl-L-Tic-L-Pro-CN (31). To a solution of
4-phenylbutyryl-l-Tic-l-Pro-NH2 (100 mg, 0.24 mmol,
1 equiv) in 5 mL of THF was added trifluoroacetic
anhydride (67 mL, 0.48 mmol, 2 equiv). After stirring
the mixture at room temperature for 3 h, the solvent
was evaporated and the residue purified by TLC
(CH2Cl2/MeOH, 96:4) to yield 31 as a colourless oil (80
mg, 84% yield); Rf 0.80 (CH2Cl2/MeOH, 9:1); HPLC tR
22.51 min; 1H NMR (CDCl3) d 1.92–2.04 (m, 2H,
CH2CH2CH2C6H5), 2.18–2.25 (m, 4H, pyrrolidine),
2.44–2.51 (m, 2H, CH2CH2CH2C6H5), 2.64–2.69 (m,
2H, CH2CH2CH2C6H5), 3.12 (d, J=5.6 Hz, 2H, H-Tic),
3.67–3.88 (m, 2H, pyrrolidine), 4.48 (d, J=14.5 Hz, 1H,
H–Tic), 4.60 (d, J=14.5 Hz, 1H, H–Tic), 4.72 (m, 1H,
Tic–H), 4.85 (m, 1H, pyrrolidine), 7.08–7.32 (m, 9H,
Ar–H); TOFMS m/z 401 (M+).
27. Kanai, K.; Erdo, S.; Susan, E.; Feher, M.; Sipos, J.;
Podanyi, B.; Szappanos, A.; Hermecz, I. Bioorg. Med. Chem.
Lett. 1997, 7, 1701.
28. Toide, K.; Shinoda, M.; Iwamoto, Y.; Fujiwara, T.; Oka-
miya, K.; Uemura, A. Behavioural Brain Res. 1997, 83, 147.
29. Li, J.; Wilk, E.; Wilk, S. Neurochem. 1996, 66, 2105.
30. Tanaka, Y.; Niwa, S.; Nishioka, H.; Yamanaka, T.; Tor-
izuka, M.; Yoshinaga, K.; Kobayashi, N.; Ikeda, Y.; Arai, H.
J. Med. Chem. 1994, 37, 2071.
Acknowledgements
We express our thanks to Gerard Montagne for NMR
experiments and Dr. Steve Brooks for proof reading.