Parallel synthesis of new phenylalanino-2-(phenylthio)pyrrolidin-3-one scaffold-based analogues

Article abstract of DOI:10.1039/b201147f

Based on the specific PhePro proteolytic cleavage of the HIV-protease, we synthesized short pseudopeptides incorporating a 2-(phenylthio)pyrrolidin-3-one ring. We previously described various analogues based on this scaffold. We report herein a parallel m

Full text of DOI:10.1039/b201147f

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Parallel synthesis of new phenylalanino-2-(phenylthio)pyrrolidin-3-
one scaffold-based analogues
Gilles Quéléver, Martin Bouygues and Jean-Louis Kraus*
1
Laboratoire de Chimie Biomoléculaire, INSERM U 382, Faculté des Sciences de Luminy,
Case 901, Université de la Méditerranée, 70, route Léon Lachamp, F-13288 Marseille Cedex 9,
France. E-mail: kraus@luminy.univ-mrs.fr; Fax: (33) 491 82 94 16; Tel: (33) 491 82 91 41
Received (in Cambridge, UK) 1st February 2002, Accepted 5th March 2002
First published as an Advance Article on the web 8th April 2002
Based on the specific PhePro proteolytic cleavage of the HIV-protease, we synthesized short pseudopeptides
incorporating a 2-(phenylthio)pyrrolidin-3-one ring. We previously described various analogues based on this
scaffold. We report herein a parallel methodology with improved efficiency applied to the synthesis of new
derivatives. For this purpose, we selected an intermediate, i.e. 8, which can be used as a versatile synthon after
simple removal of the N-Boc protecting group of the starting material as the penultimate reaction step. This new
route improved the synthesis of these molecules in terms of numbers of reaction and in terms of both reagent and
reaction time, and allowed the preparation of a small library focused on the phenylalanino-2-(phenylthio)pyrrolidin-
3-one scaffold. A phenylthio group at the 2-position confers on the resulting derivative a high reactivity, which could
be unmasked upon hydrolysis, and which led to the release of toxophoric thiophenol as detected by Ellman’s reagent.
Biological activity of these compounds was analysed towards various representative proteolytic enzymes. Pure
diastereoisomers 7(R) and 7(S) displayed potent inhibitory activities against HIV-protease.
Introduction
Viral proteases are crucial in the replication cycle of many
viruses, including retroviruses such as Human Immuno-
deficiency Virus (HIV); herpes viruses; picorna viruses such as
rhinovirus; and flaviviruses such as Hepatitis C Virus (HCV).
Viral proteases have therefore been favoured as targets for anti-
viral agents.1 Proteases cleave newly expressed precursor poly-
proteins into smaller, mature viral proteins. For example, in
HIV replication, HIV protease is an aspartate protease which
cleaves gag and pol precursor proteins2 whereas herpes virus
proteases that have Ser-His-His as the catalytic triad3 or HCV
proteases encoded by the non-structural NS3 domain4 are
serine proteases. Rhinovirus 3C protease is a cysteine protease
involved in the proteolytic cleavage of the viral precursor poly-
protein to both capsid and functional proteins required
for RNA replication.5–7 Chiral 2-substituted pyrrolidin-3-ols
or pyrrolidin-3-ones have been widely used in the design
of pseudopeptides as aspartate,8–10 serine,11 and cysteine12,13
protease inhibitors or non-peptidic neuroamidase inhibitors.14
We have recently reported new HIV-aspartyl protease inhibi-
tors, which incorporate
a 2-(phenylthio)pyrrolidin-3-one
moiety as proline replacement.10 Introduction of a phenylthio
group at position 2 of a pyrrolidinone ring could be of interest
since hydrolysis of the amido bond leads to an unstable
phenylthioaminal intermediate which undergoes a chemical
rearrangement allowing the release of toxophoric thiophenol as
shown in Scheme 1. Such a mechanism has already been
reported by Kingsbury et al.15 during the enzymatic hydrolysis
of Ala-(S)-(phenylthio)glycine. Acylation of the intracyclic
amine provides stabilization of the resulting molecule by
delocalization of the nitrogen electrons into the amide bond. If
compounds such as 1 (Scheme 1), where R represents various
amino acid residues, are recognized as substrates by intra-
cellular peptidases, hydrolysis of the peptide bond should result
in the release of thiophenol. These observations prompted us to
investigate the synthesis of a focused library of new derivatives
containing a 2-(phenylthio)pyrrolidin-3-one moiety, to test
their activities (substrate or inhibitor) on representative families
Scheme 1 Structure of N-substituted 2-(phenylthio)pyrrolidin-3-ones
1 and their mode of breakdown after peptidase cleavage.15
of proteases: aspartate protease (HIV-aspartyl protease), serine
protease (α-chymotrypsin and trypsin) and cysteine protease
(caspase 3); and to confirm the ability of these compounds to
release the toxophoric thiophenol upon hydrolysis.
Results and discussion
Since 2-(phenylthio)pyrrolidin-3-one is an unstable species
which cannot be isolated (Scheme 1), the synthesis of the target
DOI: 10.1039/b201147f
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This journal is © The Royal Society of Chemistry 2002
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compounds through direct coupling between -amino acid
residues with 2-(phenylthio)pyrrolidin-3-one cannot be con-
sidered. Therefore the synthesis of the new derivatives required
a specific synthetic route as summarized in Scheme 2. Com-
phosphate (BOP) as coupling agent.16 Direct oxidation of this
compound under Swern’s conditions17 led to the correspond-
ing pyrrolidin-3-one derivative 3. As far as sulfenylation at the
2-position of the pyrrolidinone ring was the most crucial
step, we were able to use two different chemical pathways to
perform the synthesis of the target molecules. On the one hand,
intermediate 3 was first deprotected and then coupled to
various moieties. Resulting compounds could be submitted to a
sulfenylation reaction in order to obtain the desired derivatives.
We used and described previously this methodology to per-
form the synthesis of such nicotinoyl and quinolin-2-carbonyl
analogues 6a and 6b, respectively (Scheme 2, Route 1).10 On the
other hand, 3 could be first sulfenylated. The resulting N-Boc
2-phenylthio derivative 7 was then deprotected to lead to the
versatile TFA salt synthon 8. This intermediate was coupled to
various reagents to give the desired derivatives (Scheme 2,
Route 2).
As mentioned above and for different reasons, the most
crucial step of these synthetic pathways was the sulfenylation of
the pyrrolidin-3-one ring. Indeed, stereoselectivity and regio-
selectivity were both important parameters involved during this
reaction. Besides, it was the synthetic sequence’s yield-limiting
step.
Various attempts were made to optimize all these parameters
and, of course, the yield of the reaction. Compound 3, easily
obtained in a two-step reaction, was used to optimize the
limiting sulfenylation reaction.
The sulfenylation step was performed at Ϫ78 ЊC in toluene or
tetrahydrofuran (THF) using one equivalent of various bases
versus various stoichiometric amounts of substrate 3 and
sulfenylating agent; this latter could be phenyl disulfide or
S-phenyl benzenethiosulfonate.18 An α-keto carbanion in
equilibrium with its two possible corresponding enolates was
in-situ base-generated before an electrophilic addition of the
sulfenylating agent.19 Two α-keto positions were available on
the pyrrolidin-3-one ring during this α-sulfenylation. For this
reason, it was necessary to control the regioselectivity of the
monosubstitution reaction which must preferentially occur at
the 2-position of the ring and not at the 4-position.
The different bases used during this study were sec-butyl-
lithium, lithium bis(trimethylsilyl)amide (LiHMDS) and
lithium diisopropylamide (LDA). As shown in Table 1, the
best results were obtained using the following stoichiometric
conditions: base : sulfenylating agent : substrate 3 (1 : 1.5 : 1.5).
As expected, we observed the formation of 2- and 4-mono- and
polysubstituted pyrrolidin-3-one species, which were fully
characterized. The desired monosulfenylated derivative 7
was the main reaction product. The main by-product was the
4,4-disulfenylated analogue 7a and the minor one was the 2,4,4-
trisulfenylated derivative 7b (Scheme 3). We never observed the
presence of any analogues disubstituted at the 2-position or
monosubstituted at the 4-position of the pyrrolidin-3-one
ring. The presence of 4,4-disubstituted derivatives and the
absence of 4-monosubstituted analogues can be explained by
the acidic character of the geminal proton20 of the in-situ-
generated 4-monosubstituted intermediate. This proton was
then easily substituted by a second molecule of the electrophilic
agent.
At the 2-position of the pyrrolidin-3-one ring, the second
proton was less available for a second base abstraction. Indeed,
the steric hindrance due to the presence on this carbon of three
bulky groups, i.e. a trisubstituted nitrogen atom, a carbonyl
group and a phenylthio substituent, reduced drastically the
availability of the last proton towards the base. Even if a strong
base such as sec-butyllithium could extract this proton, the
steric hindrance was too great for electrophilic attack by
another bulky phenylthio group to succeed.
Scheme 2 Synthesis of 2-(phenylthio)pyrrolidin-3-one analogues 6a–b
and synthon 8. Reagents and conditions: (i) BOP, Et3N, CH2Cl2,
RT (91%); (ii) TFAA, DMSO, DIEA, CH2Cl2, Ϫ60 ЊC (80%); Route 1:
(iii) TFA, CH2Cl2 (10 : 90), RT; (iv) 4a or 4b, DMF, HOBt, DCC,
Et3N (5a: 70%, 5b: 38%); (v) (a) sec-BuLi, toluene, Ϫ78 ЊC;
(b) PhSO2SPh, toluene, Ϫ78 ЊC (6a: 10%; 6b: 22%); Route 2: (vi) (a) sec-
BuLi, THF, Ϫ78 ЊC; (b) PhSO2SPh, THF, Ϫ78 ЊC (39%); (vii) TFA–
CH2Cl2 (10 : 90), RT.
pound 2 was obtained through a standard coupling reaction
between Boc--PheOH and (R)-pyrrolidin-3-ol using (benzo-
triazol-1-yloxy) tris(dimethylamino)phosphonium hexafluoro-
The ratio between the sulfenylated isolated compounds was
dependent of the various reaction parameters mentioned above.
The 2,4,4-trisubstituted analogue 7b was always present in the
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Table 1 Sulfenylation study of 3 (Scheme 3)
Base (1.0 equiv.)
3 (mol equiv.)
PhSO2SPh (mol equiv.)
Molar ratio 7 : 7a
Molar ratio 7(R) : 7(S)
Yield of 7 (%)
sec-BuLia
sec-BuLia
sec-BuLib
sec-BuLia
sec-BuLia
LiHMDSa
LDAa
1.5
1.0
1.0
1.5
0.5
1.0
1.0
1.0
1.5
2.0
2.0
2.0
2.0
2.0
2.0
2.0
5.0 : 1.0
1.8 : 1.0
1.2 : 1.0
4.7 : 1.0
0.8 : 1.0
1.0 : 1.0
2.3 : 1.0
1.5 : 1.0
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
1 : 1
39
13
17
25
10
12
15
8
LDAb
Reaction time to generate the enolate of substrate 3:a 1 hour; b 2 hours.
Scheme 3 Synthesis and separation of the diastereoisomeric mixture 7. Reagents and conditions: (i) sec-BuLi, THF, Ϫ78 ЊC; (ii) PhSO2SPh, THF,
Ϫ78 ЊC; (iii) HPLC, C18 Waters Spherisorb column, eluent CH3CN–H2O (60 : 40) [7(R) : 7(S) 1 : 1].
crude reaction mixture but only in a small amount, less than
5%. As mentioned above, the two major compounds were the 2-
monosulfenylated and 4,4-disulfenylated derivatives, respect-
ively 7 and 7a.
The ratio of the two diastereoisomers was determined by
analytical HPLC. In each case, we observed the same ratio,
i.e. 7(R) : 7(S) 1 : 1 (Scheme 3).
During this sulfenylation reaction, we were able to control
the regioselectivity of the electrophilic addition. We always
obtained the desired monosulfenylated derivative 7 as a mixture
of its two diastereoisomers. The following steps were performed
on this mixture of diastereoisomers.
The best molar ratio between 7 and 7a was observed under
the following conditions: 1 equivalent of sec-butyllithium, 1.5
equivalent of S-phenyl benzenethiosulfonate and 1.5 equivalent
of pyrrolidin-3-one derivative 3 in toluene or THF at Ϫ78 ЊC
(Table 1). Under these experimental conditions, the yield in
compound 7 was 39%. Despite the real improvement in the
yield of this step, this reaction remained the yield-limiting step
of this synthetic route. We observed the same phenomenon
when the sulfenylation reaction was the final step involved in
the synthesis of the nicotinoyl (6a) and quinolin-2-ylcarbonyl
(6b) analogues with yields of 10 and 22%, respectively.10 It was
obvious that it was easier to prepare a large amount of the N-
Boc-protected synthon 3 via a two-step reaction than to prepare
each N-substituted phenylalanine analogue before the final
reaction which should be the sulfenylation reaction. In this case,
it was required to obtain a large amount of the penultimate
intermediate. For this reason, we performed the synthetic
sequence according to the procedure involving the sulfenylation
of the N-Boc-protected synthon 3 (Scheme 2, Route 2).
Synthesis of a small focused library from synthon 8 (Scheme 4).
The removal of the N-Boc protecting group led to the corre-
sponding TFA salt 8 which represents a versatile and very use-
ful synthon in a parallel synthetic procedure.
In order to prepare different analogues, we investigated the
parallel coupling reaction of this synthon 8 with several func-
tionalized reagents selected from the following families of
reagents: sulfonyl chlorides leading to the corresponding
sulfonamides; chloroformates to give carbamates; carbamoyl
chlorides and isocyanates to give ureas; amino acids and
peptides to give amides.
As representative sulfonyl chlorides, we selected 4-nitro-
benzenesulfonyl chloride 9a and 4-methoxybenzenesulfonyl
chloride 9b for the synthesis of the corresponding sulfonamides
10a and 10b. 4-Nitrophenyl chloroformate 9c was used to pre-
pare the carbamate analogue 10c. Morpholine-4-carbonyl
chloride 9d and isocyanates 9e and 9f gave the corresponding
ureas 10d, 10e and 10f.
The 2-monosubstituted pyrrolidin-3-one derivative 7 was
isolated as a mixture of the two corresponding diastereoisomers
7(R) and 7(S). Indeed, we did not observe any isomerization of
the phenylalanine residue, which conserved its -configuration.
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Scheme 4 Synthesis of 2-(phenylthio)pyrrolidin-3-one analogues 10a–j. Reagents and conditions: (i) 9a or 9b, Et3N, CH3CN (9a) or CH2Cl2 (9b), RT
[from 7 : 10a, 42% (R2 = NO2); 10b, 53% (R2 = OMe)]; (ii) 9c, Et3N, CH2Cl2, RT (from 7 : 10c, 34%); (iii) 9d, 9e or 9f, Et3N, CH2Cl2, RT [from 7 : 10d,
54%; 10e, 71% (R3 = 3-CF3); 10f, 67% (R3 = 4-MeO)]; (iv) 9g, EDC, DIEA, HOBt, DMF, RT [from 7 : 10g, 55% (R4 = Boc--Asn–)]; (v) BOP, DIEA,
9h, CH2Cl2, RT [from 7 : 10h, 64% (R4 = Boc--Leu--Tyr–)]; (vi) TFA, CH2Cl2 (10 : 90), RT [10i, quantitative (R5 = TFAؒH2N--Asn–); 10j, 93%
(R5 = TFAؒH2N--Leu--Tyr–)].
Boc--AsnOH 9g and dipeptide Boc--Leu--TyrOH 9h led,
respectively, to derivatives 10g and 10h. It should be pointed out
that activation of the carboxy group of asparagine derivatives is
known to be complicated by the formation of cyanoalanine
one analogues thanks to the versatility of 8. This procedure
could be extended to a larger library.
Diastereoisomeric separation. As mentioned previously, the
N-Boc-protected 2-sulfenylated pyrrolidin-3-one 7 and the final
compounds 10a–j were obtained as diastereoisomeric mixtures.
As we observed no isomerization of the C1 stereogenic center
when sec-butyllithium was used as base in the sulfenylation step
of 3, the derivatives were mixtures of only two diastereoisomers
in which asymmetric carbon C1 of the phenylalanine residue
was in its -configuration. Separation of the diastereoisomeric
mixtures could be achieved by a semipreparative method using
a reversed-phase C18 Waters Spherisorb column and CH3CN–
H2O (60 : 40) as eluent. This procedure, when applied to
diastereoisomeric mixture 7, led to both pure stereoisomers
7(R) and 7(S), which were fully characterized. It should be
underlined that R and S assignments of the asymmetric carbon
C2 had not then been determined.
analogues.21,22
1-[3-(dimethylamino)propyl]-3-ethylcarbodi-
imide (EDC)–HOBt was proven to been an efficient coupling
system for the activation of the carboxylic function of Boc--
AsnOH.22 Application of this procedure led to a moderate
yield (55%) for the two-step synthesis of the N-Boc--asparagyl
analogue 10g. In the case of the dipeptide 9h, BOP reagent was
successfully used to give the corresponding pyrrolidinone
derivative 10h in moderate yield (64%). Products 10g and 10h
were deprotected and the corresponding TFA salts 10i and 10j,
respectively, were isolated in good yields.
The parallel synthesis of this small library using the versatile
TFA salt synthon 8 as intermediate involved only a small
amount of the N-Boc-protected 2-sulfenylated pyrrolidin-3-one
starting material 7, in contrast to the other synthetic sequence
(Scheme 2, Route 1) which required a larger amount of the
penultimate compound to perform the ultimate sulfenylation
reaction with a very low yield.
Thiophenol release evidenced by chemical hydrolysis. The con-
cept of the release of the toxophoric thiophenol (Scheme 1)
according to Kingsbury et al.15 was validated upon chemical
hydrolysis. Indeed, when the mixture 7 was submitted to basic
As demonstrated, the purpose of this study was to perform
the synthesis of a small library of 2-(phenylthio)pyrrolidin-3-
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hydrolysis, i.e. 0.1 M NaOH in H2O–THF 1 : 1 (v : v), the
release of thiophenol was identified by using Ellman’s reagent
5,5Ј-dithiobis(2-nitrobenzoic acid).23 Thiophenol release was
spectrophotometrically monitored (λmax = 412 nm), thereby
confirming the initial hypothesis summarized in Scheme 1.
Semi-preparative HPLC was performed by using a reversed-
phase C18 Waters Spherisorb column and CH3CN–H2O
(60 : 40) as eluent. Microanalyses were carried out by Service
Central d’Analyses du CNRS (Venaison, France) and were
within 0.4% of the theoretical values. UV spectra for chemical
analysis and biological assays were obtained with an Uvikon
930 spectrophotometer (Kontron Instruments). Analytical
thin-layer chromatography (TLC) was performed using silica
gel plates 0.2 mm thick (60F254 Merck). Preparative flash-
column chromatography was carried out on silica gel (230–240
mesh, G60 Merck).
Protease-inactivation studies. The activities of these new
2-(phenylthio)pyrrolidin-3-one analogues were evaluated on
representative classes of proteases including: aspartyl protease
(HIV-protease), serine protease (α-chymotrypsin and tryp-
sin) and cysteine protease (caspase 3). Enzymatic-activity
evaluation was first performed directly on diastereoisomeric
mixtures; only pure diastereoisomers which derived from
mixtures showing substantial inhibitory properties were separ-
ately assayed. We found that none of the different mixtures
10a–j showed any significant antiprotease activity on serine and
cysteine proteases. Only pure diastereoisomers 7(R) and 7(S),
which mixture was active on HIV-aspartyl protease (IC50 = 0.1
µM, the concentration required to inhibit 50% of the activity of
the enzyme), were tested separately. R and S assignment of the
asymmetric carbon C2 had not then been determined but one of
the two diastereoisomers was found to be more potent than its
homologue: IC50-values of 0.008 µM and 1 µM.10
In summary, we report herein the parallel synthesis of a small
focused library based on the phenylalanine-2-(phenylthio)-
pyrrolidin-3-one scaffold. The versatile synthon 8 was coupled
to different reagents as various as sulfonyl chlorides; chloro-
formates; carbamoyl chlorides and isocyanates; amino acids
and peptides. The synthetic results were promising and allowed
the preparation of a larger library. Our interest in the phenyl-
thio group at the 2-position of the pyrrolidinone ring was based
on the fact that hydrolysis (chemically or enzymatically) of the
exocyclic amide bond of these compounds led to the release of
toxophoric thiophenol as spectrophotometrically demonstrated
by using Ellman’s reagent. The small parallel library was
biologically tested towards various representative proteolytic
enzymes, i.e. HIV-aspartyl protease, α-chymotrypsin and tryp-
sin as serine protease, and caspase 3 as cysteine protease. Only
diastereoisomeric mixture 7 and both purified diastereoisomers
7(R) and 7(S) diplayed an inhibitory activity versus HIV-
aspartyl protease. No crucial activity was pointed out towards
the serine and cysteine proteases.
(R)-N-[N-(tert-Butoxycarbonyl)-L-phenylalanyl]pyrrolidin-3-ol
210,16
Boc--PheOH (4.44 g, 1.0 equiv., 16.75 mmol) was dissolved in
100 mL of anhydrous CH2Cl2. 1.2 Equiv. of BOP (8.89 g, 20.10
mmol) was then added. The resulting solution was cooled to
0 ЊC and 1.0 equiv. of Et3N (2.3 mL, 16.75 mmol) was added
dropwise. The reaction mixture was allowed to warm to room
temperature and was stirred for 30 min. The solution was then
cooled to 0 ЊC and a solution of (R)-pyrrolidin-3-ol (1.46 g, 1.0
equiv., 16.75 mmol) and Et3N (3.5 mL, 1.5 equiv., 25.13 mmol)
in 20 mL of anhydrous CH2Cl2 was added dropwise. The reac-
tion mixture was allowed to warm to room temperature and
was stirred for 2 hours. The reaction was monitored by TLC
(EtOAc–hexane 9 : 1). The solution was concentrated under
reduced pressure. The residue was diluted in ethyl acetate
(120 mL) and washed successively with H2O (50 mL), 5% aq.
NaHCO3 (50 mL), H2O (50 mL), 5% aq. citric acid (2 × 50 mL)
and brine (50 mL). The organic layer was dried over anhydrous
MgSO4, filtered and concentrated. The residue was purified by
flash chromatography (EtOAc–hexane 9 : 1) to give 2 as a white
hygroscopic powder (5.07 g, 91%), Rf 0.19 (EtOAc–hexane
9 : 1); mp 56–58 ЊC; 1H NMR (250 MHz; CDCl3) δ 1.21 (s, 9 H,
CH3 Boc), 1.40–1.80 (m, 2 H, –NCH2CH2CH(OH)–), 2.30–2.80
(m, 3 H, –NCH2CH2CH(OH)– ϩ CHaHb β Phe), 3.21–3.37 (m,
3 H, –NCH2CH(OH)– ϩ CHaHb β Phe), 3.90 (br s, 1 H, OH ),
4.05–4.20 (m, 1 H, –CHOH), 4.25–4.50 (m, 1 H, CH α Phe),
5.15–5.32 (m, 1 H, NH ), 7.01–7.07 (m, 5 H, C6H5); 13C NMR
(62.9 MHz; CDCl3) δC 27.1, 35.1, 39.4, 40.4, 50.8, 52.5, 73.1,
79.9, 126.8, 128.4, 129.5, 135.9, 155.2, 171.1; νmax (KBr)/cmϪ1
3335, 2952, 1750, 1680; MS (FAB > 0, GT) m/z 357 (M ϩ Na)ϩ.
Calc. for C18H26N2O4: 334.41 g molϪ1. Found: C, 64.28; H, 8.02;
N, 8.52. C18H26N2O4 requires C, 64.65; H, 7.84; N, 8.38%.
Experimental
Unless otherwise noticed, starting materials and reagents were
obtained from commercial suppliers and were used without
purification. All the protected amino acids and peptide-
coupling reagents were purchased from Bachem, Neosystem
and Aldrich. Tetrahydrofuran (THF) was distilled over sodium
benzophenone ketyl immediately prior to use. Methylene
dichloride (CH2Cl2) was distilled over P2O5 just prior to use.
Dimethylformamide (DMF), toluene and dimethylsulfoxide
(DMSO) were of anhydrous quality from commercial suppliers
(Aldrich). Nuclear magnetic resonance spectra were recorded at
N-[N-(tert-Butoxycarbonyl)-L-phenylalanyl]pyrrolidin-3-one
310,17
Anhydrous DMSO (2.54 mL, 2.0 equiv., 35.88 mmol) was dis-
solved in 32 mL of anhydrous CH2Cl2. The solution was cooled
to Ϫ60 ЊC and 1.5 equiv. of trifluoroacetic anhydride (TFAA,
3.80 mL, 26.91 mmol) was added dropwise. The reaction mix-
ture was stirred at Ϫ60 ЊC for 15 min and a solution of 1.0
equiv. of alcohol 2 (6.00 g, 17.94 mmol) in 32 mL of anhydrous
CH2Cl2 was then added. The solution was stirred at Ϫ60 ЊC for
1 hour. The reaction was monitored by TLC (EtOAc–hexane
9 : 1). Diisopropylethylamine (DIEA) (13.75 mL, 4.4 equiv.,
78.94 mmol) was then added dropwise to the reaction mixture,
which was then stirred for 15 min at Ϫ60 ЊC. The solution was
allowed to warm to room temperature and 100 mL of H2O
was added. The aqueous layer was extracted with CH2Cl2
(3 × 50 mL) and the combined organic layers were dried over
anhydrous MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by flash chromatography
(EtOAc–hexane 4 : 6) to give the desired ketone 3 as a pale
yellow hygroscopic solid (4.70 g, 80%), Rf 0.15 (EtOAc–hexane
4 : 6); mp 106–108 ЊC; 1H NMR (300 MHz; CDCl3) δ 1.25 (br s,
9 H, CH3 Boc), 2.17–2.28 (m, 2 H, –NCH2CH2C(O)–), 2.71–
2.88 (m, 4 H, CH2 β Phe ϩ –NCH2CH2C(O)–), 3.43–3.71 (m,
2 H, –NCH2C(O)–), 4.22–4.50 (m, 1 H, CH α Phe), 5.15 (br t,
1
250 MHz for H and 62.9 MHz for 13C on a Brüker AC-250
spectrometer and at 300 MHz for 1H on a Brüker Avance
300. Chemical shifts are expressed as δ-units (ppm) downfield
from TMS (tetramethylsilane). Fast-atom bombardment
(FABϩ) mass spectral analyses were obtained by Dr Astier
(Laboratoire de Mesures Physiques-RMN, USTL, Montpellier,
France) on a JEOL DX-100 using a caesium ion source and
glycerol/thioglycerol (1 : 1) or m-nitrobenzyl alcohol (NOBA)
as matrix. Mass calibration was performed using caesium
iodide. IR spectra were recorded on a Perkin-Elmer FTIR 1605
spectrophotometer. Analytical HPLC was performed on a
Waters 600E instrument with a Waters 991 detector using a
Waters Spherisorb S5 ODS2 column (4.6 × 150 mm; 5 µM)
and a two-mobile phase system (0.1 % formic acid in water/0.1
% formic acid in acetonitrile: 40 : 60; flow rate 1 mL minϪ1).
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1 H, NH ), 6.95–7.15 (m, 5 H, C6H5); 13C NMR (62.9 MHz;
CDCl3) δC 28.4, 36.1, 39.5, 42.3, 52.0, 53.9, 79.5, 126.9, 128.4,
129.3, 136.4, 155.1, 170.9, 209.1; νmax (KBr)/cmϪ1 2980, 1762,
1760; MS (FAB > 0, GT) m/z 333 (M ϩ H)ϩ. Calc. for
C18H24N2O4: 332.39 g molϪ1. Found: C, 64.90; H, 7.14; N, 8.72.
C18H24N2O4 requires C, 65.04; H, 7.28; N, 8.43%.
(toluene–MeOH 9 : 1). After 30 min at Ϫ78 ЊC, the solution was
allowed to warm to room temperature and the reaction was
quenched by addition of 5 mL of saturated aq. NH4Cl. The
aqueous layer was extracted with EtOAc (3 × 10 mL). The
combined organic layers were dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The residue
was purified by flash chromatography to give the desired com-
pound 6a as a pale yellow oil (40 mg, 10%), Rf 0.20 (toluene–
N-(N-Nicotinoyl-L-phenylalanyl)pyrrolidin-3-one 5a10
1
MeOH 9 : 1); H NMR (250 MHz; CD3OD) δ 2.15–2.25 (m,
The previous ketone 3 (560 mg, 1.0 equiv., 1.68 mmol) was
dissolved in 5 mL of CH2Cl2. 10.0 Equiv. of TFA (1.29 mL,
16.80 mmol) were added dropwise. The reaction mixture was
stirred for 2 hours at room temperature. The reaction was moni-
tored by TLC (EtOAc–hexane 4 : 6). The solvent and excess of
TFA were removed under reduced pressure. The desired TFA
salt was used immediately without any further purification for
the following step. The TFA salt was diluted in 15 mL of
anhydrous DMF with 1.1 equiv. of nicotinic acid 4a (228 mg,
1.85 mmol) and 2.0 equiv. of HOBt (454 mg, 3.36 mmol). DCC
(519 mg, 1.5 equiv., 2.52 mmol) was added as a solid followed
by Et3N (700 µL, 3.0 equiv., 5.04 mmol). The reaction mixture
was stirred overnight at room temperature. The reaction was
monitored by TLC (CH2Cl2–MeOH 9 : 1). The solution was
diluted with 10 mL of diethyl ether and filtered to remove the
solid 1,3-dicyclohexylurea (DCU). The solid was washed with
diethyl ether (3 × 10 mL) and the filtrate was concentrated
under reduced pressure. The crude material was purified by
flash chromatography (CH2Cl2–MeOH 9 : 1) to give the desired
compound 5a (389 mg, 70%) as a colourless oil, Rf 0.36
2 H, –NCH2CH2C(O)–), 2.70–3.00 (m, 4 H, CH2 β Phe ϩ
–NCH2CH2C(O)–), 4.75–4.85 (m, 1 H, CH α Phe), 5.13–5.30
(m, 1 H, CHSPh), 7.00–7.15 (m, 10 H, C6H5), 7.40–8.75 (m, 4H,
C5H4N); 13C NMR (62.9 MHz; CD3OD) δC 36.1, 39.2, 42.6,
52.1, 70.4, 123.1, 126.9, 127.3, 128.3, 129.3, 129.8, 130.1, 134.0,
134.4, 136.0, 136.4, 151.7, 158.4, 171.0, 174.0, 210.2; νmax (KBr)/
cmϪ1 2980, 1763, 1760, 1680, 1640; MS (FAB > 0, GT) m/z 446
(M ϩ H)ϩ. Calc. for C25H23N3O3S: 445.53 g molϪ1. Found: C,
67.03; H, 4.88; N, 9.36. C25H23N3O3S requires C, 67.39; H, 5.20;
N, 9.43%.
N-[N-(Quinolin-2-ylcarbonyl)-L-phenylalanyl]-2-(phenylthio)-
pyrrolidin-3-one 6b10
The title compound 6b was obtained according to the pro-
cedure described above from the corresponding ketone 5b
(90 mg, 1.5 equiv., 0.23 mmol) and after purification by flash
chromatography (EtOAc–hexane 2 : 3) as a pale yellow oil (25
mg, 22%), Rf 0.29 (EtOAc–hexane 2 : 3); 1H NMR (250 MHz;
CD3OD) δ 2.30–2.45 (m, 2 H, –NCH2CH2C(O)–), 2.75–3.50
(m, 4 H, CH2 β Phe ϩ –NCH2CH2C(O)–), 4.80–4.95 (m, 1 H,
CH α Phe), 5.15–5.25 (m, 1 H, CHSPh), 7.00–7.15 (m, 10 H,
C6H5), 7.40–8.15 (m, 6H, C9H6N); 13C NMR (62.9 MHz;
CD3OD) δC 37.0, 39.4, 42.4, 52.1, 72.4, 126.9, 128.3, 128.7,
129.3, 129.6, 129.8, 130.1, 130.2, 133.2, 134.0, 136.4, 138.3,
149.3, 170.9, 172.0, 209.1; νmax (KBr)/cmϪ1 2980, 1760, 1758,
1680, 1640; MS (FAB > 0, GT) m/z 496 (M ϩ H)ϩ. Calc. for
C29H25N3O3S: 495.59 g molϪ1. Found: C, 70.07; H, 4.81; N,
8.26. C29H25N3O3S requires C, 70.28; H, 5.08; N, 8.48%.
1
(CH2Cl2–MeOH 9 : 1); H NMR (250 MHz; CD3OD) δ 2.15–
2.27 (m, 2 H, –NCH2CH2C(O)–), 2.70–3.05 (m, 4 H, CH2 β Phe
ϩ –NCH2CH2C(O)–), 3.45–3.75 (m, 2 H, –NCH2C(O)–), 4.70–
4.85 (m, 1 H, CH α Phe), 6.95–7.15 (m, 5 H, C6H5), 7.40–7.75
(m, 4H, C5H4N); 13C NMR (62.9 MHz; CD3OD) δC 36.2, 39.6,
42.5, 52.1, 53.9, 123.1, 126.9, 127.4, 128.4, 136.1, 136.4, 151.7,
158.4, 171.0, 173.1, 210.2; νmax (KBr)/cmϪ1 2979, 1763, 1762,
1759, 1640; MS (FAB > 0, GT) m/z 338 (M ϩ H)ϩ. Calc. for
C19H19N3O3: 337.37 g molϪ1. Found: C, 67.89; H, 5.32; N,
12.75. C19H19N3O3 requires C, 67.54; H, 5.68; N, 12.46%.
N-[N-(tert-Butoxycarbonyl)-L-phenylalanyl]-2-(phenylthio)-
pyrrolidin-3-one 710
N-[N-(Quinolin-2-ylcarbonyl)-L-phenylalanyl]pyrrolidin-3-one
5b10
The ketone 3 (1.00 g, 1.5 equiv., 2.99 mmol) was dissolved in
12.5 mL of freshly distilled THF. The solution was cooled to
Ϫ78 ЊC. sec-BuLi [1.3 M in cyclohexane (1.53 mL, 1.0 equiv.,
1.99 mmol)] was then added dropwise. The reaction mixture
was stirred for 1 hour at Ϫ78 ЊC. A solution of 1.5 equiv. of
S-phenyl benzenethiosulfonate (0.75 g, 2.99 mmol) in 3 mL of
freshly distilled THF was then added dropwise. The resulting
mixture was stirred for 1 hour at Ϫ78 ЊC. The reaction was
monitored by TLC (EtOAc–hexane 3 : 7). The reaction mixture
was then allowed to warm to room temperature and 5 mL
of saturated aq. NH4Cl was added. The aqueous layer was
extracted with EtOAc (3 × 10 mL). The combined organic
layers were dried over anhydrous MgSO4, filtered, and concen-
trated under reduced pressure. The residue was purified by flash
chromatography to give the desired compound 7 as a pale
yellow solid (514 mg, 39%), Rf 0.20 (EtOAc–hexane 3 : 7); mp
The title compound was prepared according to the two-
step procedure described above from the protected ketone 3
(294 mg, 1.0 equiv., 0.75 mmol) which was deprotected with
TFA before the coupling reaction with 2-quinaldic acid 4b
(144 mg, 1.1 equiv., 0.83 mmol). The crude material was puri-
fied by flash chromatography (CH2Cl2–MeOH 9 : 1) to give the
desired compound 5b (110 mg, 38%) as a colourless oil, Rf
1
0.71 (CH2Cl2–MeOH 9 : 1); H NMR (250 MHz; CD3OD) δ
2.31–2.53 (m, 2 H, –NCH2CH2C(O)–), 3.00–3.90 (m, 6 H, CH2
β Phe ϩ –NCH2CH2C(O)– ϩ –NCH2C(O)–), 4.80–4.88 (m, 1
H, CH α Phe), 7.05–7.20 (m, 5 H, C6H5), 7.40–8.15 (m, 6H,
C9H6N); 13C NMR (62.9 MHz; CD3OD) δC 37.0, 39.4, 42.3,
52.2, 53.8, 126.9, 128.3, 128.4, 129.2, 129.3, 129.7, 129.7, 130.2,
133.1, 136.3, 138.2, 149.3, 171.0, 172.2, 210.0; νmax (KBr)/cmϪ1
2981, 1762, 1760, 1757, 1680, 1641; MS (FAB > 0, GT) m/z 388
(M ϩ H)ϩ. Calc. for C23H21N3O3: 387.43 g molϪ1. Found: C,
70.15; H, 5.74; N, 11.10. C23H21N3O3 requires C, 71.30; H, 5.46;
N, 10.85%.
1
56–58 ЊC; H NMR (300 MHz; CDCl3) δ 1.45 (br s, 9 H, CH3
Boc), 1.75–2.45 (m, 2 H, –NCH2CH2C(O)–), 2.70–3.05 (m, 4 H,
CH2 β Phe ϩ –NCH2CH2C(O)–), 4.40–4.90 (m, 1 H, CH
α Phe), 5.00–5.30 (m, 1 H, CHSPh), 5.50 (br s, 1 H, NH ), 6.90–
7.40 (m, 10 H, ArH ); 13C NMR (62.9 MHz; CDCl3) δC 28.4,
39.7, 39.9, 42.7, 53.2, 65.6, 78.0, 126.0–138.0, 155.0, 170.0,
206.1; νmax (KBr)/cmϪ1 2980, 1760, 1757, 1680; MS (FAB > 0,
N-(N-Nicotinoyl-L-phenylalanyl)-2-(phenylthio)pyrrolidin-3-one
6a10
The N-substituted ketone 5a (300 mg, 1.5 equiv., 0.89 mmol)
was dissolved in 5 mL of anhydrous toluene and the solution
was cooled to Ϫ78 ЊC. 1.0 Equiv. of sec-butyllithium [1.3 M in
cyclohexane (460 µL)] was then added dropwise. The reaction
mixture was stirred at Ϫ78 ЊC for 1 hour before addition of a
toluene solution of 1.5 equiv. of S-phenyl benzenethiosulfonate
(223 mg, 0.89 mmol). The reaction was monitored by TLC
GT) m/z 441 (M ϩ H)ϩ. Calc. for C24H28N2O4S: 440.54 g molϪ1
.
Found: C, 65.38; H, 6.65; N, 6.27. C24H28N2O4S requires C,
65.43; H, 6.41; N, 6.36%.
The pure diastereoisomers were separated by semi-
preparative HPLC using
a reversed-phase C18 Waters
Spherisorb column and CH3CN–H2O (60 : 40) as eluent.
1186
J. Chem. Soc., Perkin Trans. 1, 2002, 1181–1189

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First stereoisomer: tR: 11.9 min; 1H NMR (250 MHz; CDCl3)
δ 1.50 (br s, 9 H, CH3 Boc), 1.75–2.45 (m, 2 H, –NCH2-
CH2C(O)–), 2.72–3.05 (m, 4 H, CH2 β Phe ϩ –NCH2CH2-
C(O)–), 4.85–5.00 (m, 1 H, CH α Phe), 5.25–5.45 (m, 1 H,
CHSPh), 5.65 (br s, 1 H, NH ), 7.08–7.50 (m, 10 H, ArH ); MS
(FAB > 0, GT) m/z 441 (M ϩ H)ϩ.
tography (EtOAc–hexane 1 : 1) to give the desired compound
10b (61 mg, 53%) as a yellow solid, Rf 0.37 (EtOAc–hexane
1 : 1); mp 112–114 ЊC; 1H NMR (300 MHz; CDCl3) δ 1.60–2.20
(m, 2 H, –NCH2CH2C(O)–), 2.60–2.90 (m, 4 H, CH2 β Phe ϩ
–NCH2CH2C(O)–), 3.60 (br s, 3 H, OCH3), 4.25–4.55 (m, 1 H,
CH α Phe), 4.90–5.10 (m, 1 H, CHSPh), 5.50 (br s, 1 H, NH ),
6.55–7.70 (m, 14 H, ArH ); 13C NMR (62.9 MHz; CDCl3)
δC 38.7, 39.7, 42.5, 53.1, 56.0, 71.8, 114.4, 125.0, 125.1, 126.5,
126.6, 127.5, 127.8, 128.0, 131.6, 134.6, 139.3, 165.2, 171.0,
209.8; νmax (KBr)/cmϪ1 1765, 1760, 1757, 1155; MS (FAB > 0,
GT) m/z 511 (M ϩ H)ϩ. Calc. for C26H26N2O5S2: 510.61 g
molϪ1. Found: C, 60.95; H, 5.02; N, 5.86. C26H26N2O5S2
requires C, 61.16; H, 5.13; N, 5.49%.
Second stereoisomer: tR: 12.6 min; 1H NMR (250 MHz;
CDCl3) δ 1.41 (br s, 9 H, CH3 Boc), 1.75–2.45 (m, 2 H, –NCH2-
CH2C(O)–), 2.69–3.07 (m, 4 H, CH2 β Phe ϩ –NCH2CH2-
C(O)–), 4.40–4.75 (m, 1 H, CH α Phe), 5.10–5.30 (m, 1 H,
CHSPh), 5.55 (br s, 1 H, NH ), 7.05–7.50 (m, 10 H, ArH ); MS
(FAB > 0, GT) m/z 441 (M ϩ H)ϩ.
N-[L-Phenylalanyl-2-(phenylthio)pyrrolidinone trifluoroacetic
acid salt 8
N-[N-(4-Nitrophenyloxycarbonyl)-L-phenylalanyl]-2-
(phenylthio)pyrrolidin-3-one 10c
General procedure. The N-Boc-protected ketone 7 (1.0 equiv.)
was dissolved at 0 ЊC in 2 mL of a 10% solution of TFA in
CH2Cl2. The reaction mixture was allowed to warm to room
temperature and was stirred for 2 hours. The reaction was
monitored by TLC (EtOAc–hexane 3 : 7 and EtOAc). The
solvent was removed under reduced pressure and the excess of
TFA was co-evaporated successively with CH2Cl2 and toluene
to give the desired TFA salt 8 as a black velvet solid which was
used immediately without any further purification for the
following step, Rf 0.13 (EtOAc); MS (FAB > 0, GT) m/z 341
The title compound 10c was prepared according to a similar
procedure as described as previously, starting from 90 mg (0.20
mmol) of N-Boc-protected 7. The resulting TFA salt 8 was
treated with 4-nitrophenyl chloroformate 9c (49 mg, 1.2 equiv.,
0.25 mmol) and Et3N (65 µL, 2.3 equiv., 0.47 mmol). The
residue of the reaction after work-up was purified by flash
chromatography (EtOAc–hexane 3 : 7) to give the desired com-
pound 10c (35 mg, 34%) as an yellow oil, Rf 0.22 (EtOAc–
hexane 3 : 7); 1H NMR (300 MHz; CDCl3) δ 2.00–2.50 (m, 2 H,
–NCH2CH2C(O)–), 2.65–3.10 (m, 4 H, –NCH2CH2C(O)– ϩ
CH2 β Phe), 4.70–4.95 (m, 1 H, CH α Phe), 5.05–5.40 (m, 1 H,
CHSPh), 6.45 (br s, 1 H, NH ), 6.70 (d, 2 H, ArH, 3J = 9.0 Hz),
(M ϩ H)ϩ. Calc. for C19H20N2O2S: 340.43 g molϪ1
.
N-[N-(4-Nitrophenylsulfonyl)-L-phenylalanyl]-2-(phenylthio)-
pyrrolidin-3-one 10a
6.90–7.40 (m, 10 H, C6H5), 7.95 (d, 2 H, ArH, 3J = 9.0 Hz); 13
C
NMR (62.9 MHz; CDCl3) δC 37.8, 41.4, 42.0, 56.4, 70.2, 122.0,
124.3, 124.8, 126.6, 127.5, 127.9, 128.8, 128.9, 135.6, 141.2,
145.2, 157.5, 158.5, 171.0, 209.3; νmax (KBr)/cmϪ1 2975, 1763,
1757, 1753, 1342; MS (FAB > 0, GT) m/z 506 (M ϩ H)ϩ. Calc.
for C26H23N3O6S: 505.52 g molϪ1. Found: C, 61.50; H, 4.22; N,
8.12. C26H23N3O6S requires C, 61.77; H, 4.59; N, 8.31%.
The TFA salt 8 (obtained from 90 mg, 0.20 mmol of 7) was
dissolved in 1 mL of anhydrous acetonitrile and the resulting
solution was cooled to 0 ЊC. 4-Nitrobenzenesulfonyl chloride 9a
(58 mg, 1.3 equiv., 0.27 mmol) was then added as a solid
followed by K2CO3 (34 mg, 1.2 equiv., 0.25 mmol). The
reaction mixture was stirred overnight at room temperature.
The reaction was monitored by TLC (EtOAc and EtOAc–
hexane 3 : 7). The solvent was removed under reduced pressure.
The residue was dissolved in 10 mL of EtOAc and washed with
H2O (2 × 5 mL), dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified
by flash chromatography (EtOAc–hexane 3 : 7) to give the
desired compound 10a (45 mg, 42%) as a yellow solid, Rf 0.23
N-[N-(4-Morpholinylcarbonyl)-L-phenylalanyl]-2-(phenylthio)-
pyrrolidin-3-one 10d
The title compound 10d was prepared according to a similar
procedure as described as previously, starting from 52 mg (0.12
mmol) of N-Boc-protected 7. The resulting TFA salt 8 was
treated with morpholine-N-carbonyl chloride 9d (17 µL, 1.2
equiv., 0.15 mmol) and Et3N (38 µL, 2.3 equiv., 0.27 mmol).
The residue of the reaction after work-up was purified by flash
chromatography (EtOAc) to give the desired compound 10d (40
1
(EtOAc–hexane 3 : 7); mp 140–142 ЊC; H NMR (300 MHz;
CDCl3) δ 2.00–2.50 (m, 2 H, –NCH2CH2C(O)–), 2.80–3.30 (m,
4 H, CH2 β Phe ϩ –NCH2CH2C(O)–), 4.90–5.10 (m, 1 H, CH α
Phe), 5.25–5.40 (m, 1 H, CHSPh), 5.60 (br s, 1 H, NH ), 7.10–
7.55 (m, 14 H, ArH ); 13C NMR (62.9 MHz; CDCl3) δC 39.5,
39.6, 42.0, 52.5, 71.0, 123.9, 124.9, 125.7, 126.4, 126.6, 127.9,
128.4, 128.7, 135.6, 140.2, 145.4, 151.6, 173.9, 207.1; νmax (KBr)/
cmϪ1 1766, 1761, 1758, 1325, 1160; MS (FAB > 0, GT) m/z 526
(M ϩ H)ϩ. Calc. for C25H23N3O6S2: 525.57 g molϪ1. Found: C,
56.95; H, 4.18; N, 8.12. C25H23N3O6S2 requires C, 57.13; H,
4.41; N, 7.99%.
1
mg, 54%) as a brown solid, Rf 0.33 (EtOAc); mp 86–88 ЊC; H
NMR (300 MHz; CDCl3) δ 1.80–2.50 (m, 2 H, –NCH2-
CH2C(O)–), 2.60–3.55 (m, 12 H, 4 × CH2 morpholine ϩ CH2
β Phe ϩ –NCH2CH2C(O)–), 4.65–5.00 (m, 1 H, CH α Phe),
5.05–5.35 (m, 1 H, CHSPh), 5.72 (br s, 1 H, NH ), 6.95–7.40 (m,
10 H, ArH ); 13C NMR (62.9 MHz; CDCl3) δC 36.3, 39.1, 42.4,
52.5, 58.1, 70.2, 70.8, 125.0, 125.9, 126.6, 127.6, 128.4, 128.7,
137.1, 138.6, 164.2, 172.1, 208.0; νmax (KBr)/cmϪ1 2981, 1760,
1756, 1754; MS (FAB > 0, GT) m/z 454 (M ϩ H)ϩ. Calc. for
C24H27N3O4S: 453.54 g molϪ1. Found: C, 63.18; H, 6.25; N,
9.43. C24H27N3O4S requires C, 63.55; H, 6.00; N, 9.26%.
N-[N-(4-Methoxyphenylsulfonyl)-L-phenylalanyl]-2-
(phenylthio)pyrrolidin-3-one 10b
The TFA salt 8 (obtained from 100 mg, 0.23 mmol of 7) was
dissolved in 1 mL of anhydrous CH2Cl2 and the resulting solu-
tion was cooled to 0 ЊC. 4-Methoxybenzenesulfonyl chloride 9b
(61 mg, 1.3 equiv., 0.29 mmol) was then added as a solid fol-
lowed by Et3N (73 µL, 2.3 equiv., 0.52 mmol). The reaction
mixture was stirred for 2 hours at room temperature. The reac-
tion was monitored by TLC (EtOAc and EtOAc–hexane 1 : 1).
The solvent was removed under reduced pressure. The residue
was dissolved in 10 mL of EtOAc and successively washed with
H2O (5 mL), 5% aq. citric acid (5 mL), and H2O (5 mL), dried
over anhydrous MgSO4, filtered, and concentrated under
reduced pressure. The residue was purified by flash chroma-
N-[N-(ꢀ,ꢀ,ꢀ-Trifluoro-m-tolylcarbamoyl)-L-phenylalanyl]-2-
(phenylthio)pyrrolidin-3-one 10e
The title compound 10e was prepared according to a similar
procedure as described as previously, starting from 70 mg (0.16
mmol) of N-Boc-protected 7. The resulting TFA salt 8 was
treated with α,α,α-trifluoro-m-tolyl isocyanate 9e (26 µL, 1.2
equiv., 0.19 mmol) and Et3N (66 µL, 3.0 equiv., 0.43 mmol).
The residue of the reaction after work-up was purified by flash
chromatography (EtOAc–hexane 3 : 7) to give the desired com-
pound 10e (60 mg, 71%) as a brown oil, Rf 0.10 (EtOAc–hexane
3 : 7); 1H NMR (300 MHz; CDCl3) δ 1.80–2.50 (m, 2 H,
J. Chem. Soc., Perkin Trans. 1, 2002, 1181–1189
1187

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–NCH2CH2C(O)–), 2.75–3.20 (m, 4 H, –NCH2CH2C(O)– ϩ
CH2 β Phe), 4.80–5.20 (m, 4 H, CH α Phe ϩ CHSPh ϩ 2 NH ),
6.80–7.80 (m, 14 H, ArH ); 13C NMR (62.9 MHz; CDCl3) δC
37.4, 39.2, 41.4, 57.0, 79.7, 117.2, 119.3, 120.9, 123.7, 124.9,
125.7, 126.8, 127.9, 128.4, 128.7, 129.0, 131.2, 136.5, 138.5,
140.1, 158.0, 171.0, 209.5; νmax (KBr)/cmϪ1 2975, 1770, 1765,
1680; MS (FAB > 0, GT) m/z 528 (M ϩ H)ϩ. Calc. for
C27H24F3N3O3S: 527.55 g molϪ1. Found: C, 61.12; H, 4.42; N,
11.05. C27H24F3N3O3S requires C, 61.47; H, 4.59; N, 10.80%.
were removed under reduced pressure and co-evaporated suc-
cessively with CH2Cl2 and toluene to give quantitatively the
desired TFA salt (1.04 g) as an oil, 1H NMR (250 MHz;
CD3OD) δ 3.10 (d, 2 H, CH2 β Tyr), 4.27 (t, 2 H, CH α Tyr),
5.23 (AB, 2 H, CH2C6H5, Jgem = 12.0 Hz), 6.71 (d, 2 H, ArH, 3J
3
= 8.5 Hz), 6.97 (d, 2 H, ArH, J = 8.5 Hz), 7.32–7.43 (m, 5 H,
C6H5).
Boc--LeuOH (0.67 g, 1.0 equiv., 2.70 mmol) was dissolved
in 20 mL of anhydrous CH2Cl2. 1.0 Equiv. of BOP (1.19 g, 2.70
mmol) was added. The resulting solution was cooled to 0 ЊC
and 1.0 equiv. of DIEA (0.47 mmol, 2.70 mmol) was added
dropwise. The solution was allowed to warm up to room tem-
perature and was stirred for 30 min before being cooled to 0 ЊC.
A solution of 1.0 equiv. of the previous TFA salt (1.04 g, 2.70
mmol) and 3.0 equiv. of DIEA (1.41 mL, 8.10 mmol) in 5 mL
of anhydrous CH2Cl2 was added dropwise. The reaction mix-
ture was allowed to warm to room temperature and was stirred
for 2 hours. The reaction was monitored by TLC (EtOAc–
hexane 2 : 3). The solvent was removed under reduced pressure.
The residue was diluted in 30 mL of EtOAc, washed succes-
sively with H2O (20 mL), 1 M HCl (20 mL), brine (20 mL), and
5% aq. NaHCO3 (20 mL), dried over anhydrous MgSO4, fil-
tered, and concentrated under reduced pressure. The residue
was purified by flash chromatography (EtOAc–hexane 2 : 3)
to give the desired diprotected dipeptide (1.14 g, 87%), Rf
0.20 (EtOAc–hexane 3 : 7); 1H NMR (300 MHz; CDCl3) δ 0.69
(d, 3 H, CH3 δ Leu), 0.70 (d, 3 H, CH3 δ Leu), 1.20–1.32 (m,
11 H, CH3 Boc ϩ CH2 β Leu), 1.35–1.46 (m, 1 H, CH γ Leu),
2.81–2.88 (m, 2 H, CH2 β Tyr), 3.90 (br s, 1 H, CH α Leu),
4.63–4.78 (m, 2 H, CH α Tyr ϩ OH ), 4.95 (AB, 2 H, CH2C6H5,
Jgem = 12.1 Hz), 5.65 (br s, 1 H, NH ), 6.39–6.52 (m, 3 H, NH ϩ
ArH, 3J = 8.5 Hz), 6.65 (d, 2 H, ArH, 3J = 8.5 Hz), 7.10–7.25 (m,
5 H, C6H5); MS (FAB > 0, GT) m/z 485 (M ϩ H)ϩ. Calc. for
N-[N-(4-Methoxyphenylcarbamoyl)-L-phenylalanyl]-2-
(phenylthio)pyrrolidin-3-one 10f
The title compound 10f was prepared according to a similar
procedure as described previously, starting from 85 mg (0.19
mmol) of N-Boc-protected 7. The resulting TFA salt 8 was
treated with 4-methoxyphenyl isocyanate 9f (30 µL, 1.2 equiv.,
0.23 mmol) and Et3N (81 µL, 3.0 equiv., 0.59 mmol). The resi-
due of the reaction after work-up was purified by flash chroma-
tography (EtOAc–hexane 3 : 7 then 1 : 1) to give the desired
compound 10f (65 mg, 67%) as a clear brown solid, Rf 0.28
(EtOAc–hexane 1 : 1); mp 90–92 ЊC; 1H NMR (300 MHz;
CDCl3) δ 1.90–2.40 (m, 2 H, –NCH2CH2C(O)–), 2.75–3.10 (m,
4 H, –NCH2CH2C(O)–) ϩ CH2 β Phe), 3.56 (s, 3 H, OCH3),
4.25–5.15 (m, 2 H, CH α Phe ϩ CHSPh), 6.50–7.35 (m, 14 H,
ArH ); 13C NMR (62.9 MHz; CDCl3) δC 37.4, 39.2, 42.3, 58.0,
72.9, 114.3, 121.4, 124.9, 125.4, 125.7, 127.1, 128.7, 128.8,
130.5, 134.3, 138.7, 157.6, 161.0, 171.2, 174.7, 210.2; νmax (KBr)/
cmϪ1 1762, 1750, 1640; MS (FAB > 0, GT) m/z 490 (M ϩ H)ϩ.
Calc. for C27H27N3O4S: 489.57 g molϪ1. Found: C, 65.95; H,
5.72; N, 8.21. C27H27N3O4S requires C, 66.24; H, 5.56; N,
8.58%.
N-[N-(tert-Butoxycarbonyl)-L-asparagyl-L-phenylalanyl]-2-
(phenylthio)pyrrolidin-3-one 10g
C27H36N2O6: 484.58 g molϪ1
.
1.0 Equiv. of this N-Boc-protected dipeptide (1.02 g, 2.10
mmol) was dissolved in 20 mL of MeOH. A suspension of 100
mg of 10% Pd/C in 1 mL of MeOH was then added and the
resulting mixture was stirred for 3 hours at room temperature
under H2 at atmospheric pressure. The reaction was monitored
by TLC (EtOAc–hexane 3 : 7 and EtOAc). The suspension was
filtered on Celite and the solvent was removed under reduced
pressure to give the corresponding acid 9h as a white solid
Boc--AsnOH 9g (142 mg, 3.0 equiv., 0.16 mmol) was dissolved
in 2 mL of anhydrous DMF. HOBt (55 mg, 2.0 equiv., 0.41
mmol) was then added and the reaction mixture was cooled to 0
ЊC. EDC (78 mg, 2.0 equiv., 0.41 mmol) was added as a solid.
The TFA salt 8 (1.0 equiv., 0.20 mmol) obtained from 90 mg
(0.20 mmol) of 7 dissolved in 2 mL of anhydrous DMF was
then added to the reaction mixture. After a few minutes, 3.0
equiv. of DIEA (107 µL, 0.61 mmol) were added and the reac-
tion mixture was stirred at 0 ЊC for 1 hour, then overnight at
room temperature. The reaction was monitored by TLC
(EtOAc). The solvent was removed under reduced pressure and
the residue was dissolved in 10 mL of EtOAc. The organic layer
was successively washed with H2O (2 × 5 mL), 5% aq. NaHCO3
(2 × 5 mL), and brine (5 mL), dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The crude
material was purified by flash chromatography (EtOAc) to give
the desired compound 10g as a yellow solid (85 mg, 55%), Rf
1
(0.82 g, quantitative), Rf 0.15 (EtOAc); H NMR (300 MHz;
DMSO-d6) δ 0.62 (d, 3 H, CH3 δ Leu), 0.65 (d, 3 H, CH3 δ Leu),
1.12–1.22 (m, 11 H, CH3 Boc ϩ CH2 β Leu), 1.27–1.40 (m, 1 H,
CH γ Leu), 2.59 (dd, 1 H, CHaHb β Tyr, Jgem = 13.9 Hz,
3
3J = 8.2 Hz), 2.70 (dd, 1 H, CHaHb β Tyr, Jgem = 13.9 Hz, J =
5.2 Hz), 3.70–3.80 (m, 1 H, CH α Leu), 4.10–4.22 (m, 1 H, CH α
Tyr), 6.43 (d, 2 H, ArH, 3J = 8.4 Hz), 6.66 (d, 1 H, NH, 3J = 8.7
Hz), 6.77 (d, 2 H, ArH, 3J = 8.4 Hz), 7.57 (d, 1 H, NH, 3J = 7.7
Hz), 8.98 (s, 1 H, OH ), 12.53 (br s, 1 H, COOH ); MS (FAB > 0,
GT) m/z 395 (M ϩ H)ϩ. Calc. for C20H30N2O6: 394.46 g molϪ1
.
1
0.22 (EtOAc); mp 116–118 ЊC; H NMR (250 MHz; CDCl3) δ
1.37–1.42 (2 br s, 9 H, CH3 Boc), 1.90–2.40 (m, 2 H, –
NCH2CH2C(O)–), 2.60–3.10 (m, 6 H, –NCH2CH2C(O)– ϩ CH2
β Phe ϩ CH2 β Asn), 4.30–4.50 (m, 1 H, CH α Asn), 4.70–5.30
N-{N-[N-(tert-Butoxycarbonyl)-L-leucyl-L-tyrosyl]-L-phenyl-
alanyl}-2-(phenylthio)pyrrolidin-3-one 10h
Boc--Leu--TyrOH 9h (90 mg, 1.0 equiv., 0.23 mmol) was dis-
solved in 1 mL of anhydrous CH2Cl2. The solution was cooled
to 0 ЊC. BOP (100 mg, 1.0 equiv., 0.23 mmol) was then added
followed by 1.0 equiv. of DIEA (40 µL, 0.23 mmol). The reac-
tion mixture was allowed to warm to room temperature and
was stirred for 30 min. The solution was once again cooled to
0 ЊC. A solution of the TFA salt 8 (obtained from 100 mg,
0.23 mmol of 7) in 1 mL of anhydrous CH2Cl2 was then added
followed by 2.0 equiv. of DIEA (40 µL, 0.45 mmol). The
reaction mixture was stirred overnight at room temperature.
The reaction was monitored by TLC (EtOAc and EtOAc–
hexane 1 : 1). The organic layer was diluted with 10 mL of
CH2Cl2 and successively washed with H2O (5 mL), 5% aq. citric
acid (2 × 5 mL), H2O (5 mL), 5% aq. NaHCO3 (2 × 5 mL),
and brine (5 mL), dried over anhydrous MgSO4, filtered, and
(m, 2 H, CH α Phe ϩ CHSPh), 7.00–7.60 (m, 10 H, ArH ); 13
C
NMR (62.9 MHz; CDCl3) δC 28.7, 37.9, 38.2, 38.8, 41.4, 52.5,
54.9, 70.6, 79.7, 124.9, 125.7, 126.6, 127.9, 128.4, 128.7, 135.6,
140.2, 157.5, 173.9, 175.4, 177.2, 207.1; νmax (KBr)/cmϪ1 1762,
1757, 1750, 1680; MS (FAB > 0, GT) m/z 555 (M ϩ H)ϩ. Calc.
for C28H34N4O6S: 554.64 g molϪ1. Found: C, 60.86; H, 6.35; N,
9.98. C28H34N4O6S requires C, 60.63; H, 6.18; N, 10.10%.
N-[N-(tert-Butoxycarbonyl)-L-leucyl]-L-tyrosine 9h
Boc--Tyr-OBzl (1.00 g, 1.0 equiv., 2.70 mmol) was dissolved in
15 mL of CH2Cl2. At 0 ЊC, 10.0 equiv. of TFA (2.1 mL, 27.00
mmol) were added dropwise and the solution was stirred at
room temperature for 2 hours. The reaction was monitored by
TLC (EtOAc–hexane 1 : 1). The solvent and the excess of TFA
1188
J. Chem. Soc., Perkin Trans. 1, 2002, 1181–1189

View Article Online
concentrated under reduced pressure. The residue was purified
by flash chromatography (EtOAc–hexane 1 : 1 then 2 : 1) to give
the desired compound 10h (103 mg, 64%) as a white solid, Rf
References
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1
0.33 (EtOAc–hexane 2 : 1); mp 130–132 ЊC; H NMR (300
MHz; CDCl3) δ 0.70–0.90 (m, 6 H, 2 × CH3 δ Leu), 1.35 (br s,
11 H, CH3 Boc ϩ CH2 β Leu), 1.50 (br s, 1 H, CH γ Leu), 1.80–
2.40 (m, 2 H, –NCH2CH2C(O)–), 2.65–3.15 (m, 6 H, CH2 β Tyr
ϩ CH2 β Phe ϩ –NCH2CH2C(O)–), 3.60–3.80 (m, 1 H, CH α
Leu), 3.92–4.15 (m, 1 H, CH α Tyr), 4.60–5.40 (m, 3 H, CH α
Phe ϩ CHSPh ϩ OH Tyr), 6.50–7.50 (m, 14 H, ArH ); 13C
NMR (62.9 MHz; CDCl3) δC 21.6, 22.4, 28.4, 37.6, 37.9, 39.8,
41.1, 41.4, 54.3, 54.9, 56.7, 72.3, 77.1, 115.6, 124.9, 125.7, 126.6,
127.9, 128.4, 128.7, 129.3, 132.8, 135.6, 140.2, 154.5, 157.5,
173.9, 174.7, 175.4, 209.5; νmax (KBr)/cmϪ1 1762, 1759, 1755,
1750, 1680; MS (FAB > 0, GT) m/z 717 (M ϩ H)ϩ. Calc. for
C39H48N4O7S: 716.90 g molϪ1. Found: C, 64.98; H, 6.45; N,
7.61. C39H48N4O7S requires C, 65.34; H, 6.75; N, 7.82%.
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N-(N-L-Asparagyl-L-phenylalanyl)-2-(phenylthio)pyrrolidin-3-one
trifluoroacetic acid salt 10i
The N-Boc-protected derivative 10g (30 mg, 0.05 mmol)
was dissolved at 0 ЊC in 1 mL of a solution of TFA in CH2Cl2
(v/v 1 : 9). The reaction mixture was stirred at room temper-
ature for 2 hours. The reaction was monitored by TLC
(EtOAc). The solvents were evaporated off under reduced pres-
sure and the excess of TFA was co-evaporated with CH2Cl2.
The desired TFA salt was isolated as a brown solid (31 mg,
10 J. L. Kraus, M. Bouygues, J. Courcambeck and J. C. Chermann,
Bioorg. Med. Chem. Lett., 2000, 10(17), 2023–2026.
1
quantitative), H NMR (250 MHz; CD3OD) δ 1.80–2.40 (br s,
11 Y. M. Choi-Sledeski, D. G. McGarry, D. M. Green, H. J. Mason,
M. R. Becker, R. S. Davis, W. R. Ewing, W. P. Dankulich,
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E. Hansell and P. J. Rosenthal, Bioorg. Med. Chem., 1998, 6(12),
2477–2494.
13 N. P. Peet, H. O. Kim, A. L. Marquart, M. R. Angelastro,
T. R. Nieduzak, J. N. White, D. Friedrich, G. A. Flynn, M. E.
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2 H, –NCH2CH2C(O)–), 2.70–3.20 (m, 6 H, –NCH2CH2C(O)–
ϩ CH2 β Phe ϩ CH2 β Asn), 4.00–4.20 (m, 1 H, CH α Asn),
4.90–5.30 (m, 2 H, CH α Phe ϩ CHSPh), 7.05–7.55 (m, 10 H,
ArH ); 13C NMR (62.9 MHz; CD3OD) δC 37.5, 39.7, 41.0, 41.2,
53.9, 57.5, 68.3, 123.9, 125.4, 125.7, 126.6, 128.2, 128.6, 132.3,
138.5, 174.3, 175.4, 177.2, 208.0; MS (FAB > 0, GT) m/z 455
(M ϩ H)ϩ. Calc. for C23H26N4O4S: 454.53 g molϪ1. Calc. for
C23H26N4O4SؒCF3COOH: 568.56 g molϪ1
.
N-{N-[N-(L-Leucyl-L-tyrosyl)-L-phenylalanyl]-2-(phenylthio)-
pyrrolidin-3-one trifluoroacetic acid salt 10j
14 V. R. Atigadda, W. J. Brouillette, F. Duarte, S. M. Ali, Y. S. Babu,
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The title TFA salt 10j was prepared according to a similar pro-
cedure as described as previously, from the N-Boc-protected
derivative 10h (42 mg, 0.06 mmol). After evaporation and
co-evaporation of the solvent and excess of TFA, the salt was
1
isolated as a black solid (40 mg, 93%), H NMR (300 MHz;
DMSO-d6) δ 0.54–0.74 (m, 6 H, 2 × CH3 δ Leu), 0.95–1.05
(m, 3 H, CH2 β Leu ϩ CH γ Leu), 2.00–2.50 (m, 2 H,
–NCH2CH2C(O)–), 2.70–3.10 (m, 6 H, CH2 β Tyr ϩ CH2
β Phe ϩ –NCH2CH2C(O)–), 3.40–3.80 (m, 2 H, CH α Tyr ϩ
CH α Leu), 4.30–4.70 (m, 2 H, CH α Phe ϩ CHSPh), 6.25–7.50
(m, 14 H, ArH ); 13C NMR (62.9 MHz; DMSO-d6) δC 22.5,
23.9, 37.9, 38.3, 38.4, 42.4, 43.9, 54.8, 55.7, 57.7, 69.3, 116.5,
123.3, 126.6, 127.5, 127.9, 128.4, 128.6, 129.1, 131.2, 136.5,
139.3, 155.4, 173.9, 174.7, 174.8, 206.3; MS (FAB > 0, GT) m/z
617 (M ϩ H)ϩ. Calc. for C34H40N4O5S: 616.75 g molϪ1. Calc. for
C34H40N4O5SؒCF3COOH: m/z 730.78 g molϪ1
.
20 D. Seebach, Angew. Chem., Int. Ed. Engl., 1969, 8(9), 639–649.
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references therein; M. Bodanszky and J. Martinez, in The Peptides,
ed. E. Gross and J. Meienhofer, 1983, pp. 152–156.
Acknowledgements
We thank Mr Frédéric Bihel for his helpful comments. We
are indebted to Miss Delphine Palet and Dr Pascale Galéa
(Trophos, Faculté des Sciences de Luminy, Marseille, France)
for their biological technical assistance.
22 D. A. Evans and J. A. Ellman, J. Am. Chem. Soc., 1989, 111(3),
1063–1072.
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J. Chem. Soc., Perkin Trans. 1, 2002, 1181–1189
1189