Use of proline bioisosteres in potential HIV protease inhibitors: Phenylalanine-2-thiophenoxy-3-pyrrolidinone: Synthesis and anti-HIV evaluation

Article abstract of DOI:10.1016/S0960-894X(00)00393-0

The synthesis of new phenylalanine-2-thiophenoxy-3-pyrrolidinones is described. Anti-HIV recombinant protease assays and HIV infected cell culture assays (observation of syncytia) demonstrated the potent anti-HIV activity of this new class of pseudopeptides. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00393-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2023±2026
Use of Proline Bioisosteres in Potential HIV Protease Inhibitors:
Phenylalanine-2-thiophenoxy-3-pyrrolidinone:
Synthesis and Anti-HIV Evaluation
J. L. Kraus,a,b,* M. Bouygues,a,b J. Courcambeck,a,b and J. C. Chermannb
a
    Â
Laboratoire de Chimie Biomoleculaire, Faculte des Sciences de Luminy, Case 901, Universite de la Mediterranee,
70 Route L. Lachamp, 13288 Marseille Cedex 9, France
b
Â
Â
Â
INSERM Unite 322 - Unite des retrovirus et maladies associees, Campus de Luminy, BP33, 13273 Marseille Cedex 9, France
Â
Received 11 April 2000; revised 5 July 2000; accepted 5 July 2000
AbstractÐThe synthesis of new phenylalanine-2-thiophenoxy-3-pyrrolidinones is described. Anti-HIV recombinant protease
assays and HIV infected cell culture assays (observation of syncytia) demonstrated the potent anti-HIV activity of this new class of
pseudopeptides. # 2000 Published by Elsevier Science Ltd.
Introduction
We have recently reported the synthesis of new 3-pyr-
rolidinone type inhibitors of HIV-1 replication.1,2 The
design of these inhibitors was based on the observation
that HIV protease displays an unusual preference for
the Tyr-Pro or Phe-Pro primary cleavage site.3,4 This
observation led us to replace the 1-carboxy-pyrrole ring
of the proline residue by a 3-pyrrolidinone moiety. In
this feature these new proline mimicking moieties were
coupled to various amino acids, including aromatic
residue (Phe, Tyr). We have evaluated the anti-HIV
potencies of this new class of analogues, through syn-
cytia formation inhibition assay as ®rst screening.5,6
Indeed, many peptidomimetic inhibitors possessing
e€ective activity on puri®ed protease inhibition assays
turned out to be sometimes inactive on HIV infected
cells, because of lack of cell membrane permeation.
Figure 1.
a chemical rearrangement allowing the release of thio-
phenol as shown in Scheme 1. This observation was
already reported by Kingsbury et al.7 in the case of Ala-
(S)-thiophenoxyglycine. Our research approach was ®rst
to use these reactive modi®ed dipeptides as new sca€olds
for the design of new potential anti-HIV agents.
We describe herein the synthesis of new Phe-Pro dipeptide
analogues in which the proline residue was replaced by a
2-thiophenoxy-3-pyrrolidinone (Fig. 1).
Chemistry
Introduction of a thiophenoxy group in position 2 of
the pyrrolidinone ring could be of interest since enzy-
matic cleavage of the amide bond leads to an unstable
phenylthioaminal intermediate, which rapidly undergoes
Since 2-thiophenoxy-3-pyrrolidinone is an unstable
species which cannot be isolated, the synthesis of the
target compounds through the direct coupling between
(l)-phenylalanine residue with 2-thiophenoxy-3-pyr-
rolidinone derivative cannot be considered. Therefore
the synthesis of the new class of compounds (Fig. 1)
required a speci®c synthetic route shown in Scheme 2.
*Corresponding author. Tel.: +33-0-4-91-82-91-41; fax: +33-0-4-91-
82-94-16; e-mail: kraus@luminy.univ-mrs.fr
0960-894X/00/$ - see front matter # 2000 Published by Elsevier Science Ltd.
PII: S0960-894X(00)00393-0

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J. L. Kraus et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2023±2026
Compounds 3a±d were obtained through standard
coupling reactions between 3-pyrrolidinol 2 and the cor-
responding N-protected amino acid residues 1a±d using
BOP8 as coupling reagent. Direct oxidation of deriva-
tives 3a±d under Swern9 conditions led quantitatively to
the corresponding 3-pyrrolidinone derivatives 4a±d. In
contrast, sulfenylation at the 2 position of the pyr-
rolidinone ring was rather dicult to perform and the
obtained yields were quite low. The best results were
obtained using the following conditions: 1 equiv of
sec-butyllithium, 1.5 equiv of diphenyl thiosulfonate
(Ph2S2O3) and 1.5 equiv of 3-pyrrolidinone derivatives
4a±d in toluene at 78 ꢀC. In the same reaction condi-
tions, analogues 5a±d were isolated in 25, 17, 10 and
10% yield respectively. Similarly compounds 8 and 9
were obtained in respectively 10 and 22% yields from
the N-deprotected precursors 6 and 7. The low yields
obtained in the desired mono 2-sulfenylated analogues
Scheme 1.
Scheme 2.

J. L. Kraus et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2023±2026
2025
are due to the low regioselectivity of the enolate forma-
tion, which could lead to the formation of mono and
polysulfenylated derivatives in positions 2 and 4 of the
pyrrolidinone ring. Final compounds 5a±d were obtained
as diastereoisomeric mixtures. We observed no epimeri-
sation of the C1 stereogenic center, when sec-butyl-
lithium is used in the following conditions (1 equiv sec-
BuLi for 1,5 equiv of ketone), the a proton of the ketone
is predominantly abstracted. Separation of these dia-
stereoisomeric mixtures was achieved by semipreparative
HPLC method using a reverse C18 waters spherisorb
column and CH3CN:H2O (60:40) as eluent. Four pairs of
diastereoisomers were obtained in which the asymmetric
carbon C1 coming from the (l)-phenylalanine residue
is in the S con®guration and asymmetric carbon C2
bearing the thiophenoxy group at the 2 position of
the pyrrolidinone ring is either R or S. It should be
underlined that R, S assignment of the asymmetric
carbon bearing the thiophenoxy group (compounds 5a1,
5a2, 5b1, 5b2, 5c1, 5c2, 5d1, 5d2) is not yet available
(Table 1). Compounds 8 and 9 were tested as diastereo-
isomeric mixtures since HPLC separation was not
satisfactory.
phenylalanine residue using the same procedure shown
in Scheme 2.
Antiviral Activity
The whole new synthesized analogues were evaluated
for their anti-HIV activity on both screening assays:
syncytia formation observation and recombinant pro-
tease assay. Syncytia formation: The fusogenic e€ect of
HIV in MT4 cells line10 was determined as already
described by Rey et al.5,6 The appearance of syncytia
was measured ®ve days after infection, the inhibitory
dose was expressed as the concentration of the tested
compound which causes 50% inhibition of syncytia for-
mation (EC50). Results are presented in Table 1. Anti-
protease assay: Evaluation of the synthesized compounds
as HIV protease inhibitors was carried out according to a
classical HIV recombinant protease assay.11 Inhibitory
activity of the compounds was expressed as IC50 values
which correspond to the concentration required to
inhibit 50% of the substrate hydrolysis by the recombi-
nant HIV-1 protease. IC50 values are given in Table 1.
The structures of all these new analogues were deter-
1
From the obtained results, it can be seen that some
of the new analogues (5a1, 5b1 and 5c1), which are
described in Table 1, elicited remarkable anti-HIV
activities on both, infected MT4 cells culture assay and
recombinant HIV protease assay. It can be also
observed that the con®guration of the asymmetric
mined by H and 13C NMR, mass spectrometry and
elemental analyses. The synthesis of compound 5a3
mixture of diastereoisomers (Table 1) which asymmetric
carbon C1 is in the d con®guration and C2 mixture of R
and S con®gurations, was achieved starting from (d)-
Table 1. Anti-HIV potencies of new phenylalanine-2-thiophenoxy-3-pyrrolidinone derivatives
SIc
IC50
a
b
d
Compound
Con®guration
C2
EC50
CC50
R1
R2
C1
mM
mM
mM
5a1
5a2
5a3
5b1
5b2
5c1
5c2
5d1
5d2
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
H
H
H
OBn
OBn
OH
OH
OCH3
OCH3
l
l
d
l
l
l
l
l
l
R
S
(R,S)
R
S
R
S
R
S
1
50
NAe
0.1
NA
0.1
50
NA
NA
>100
100
>100
50
10
50
>100
50
>100
>100
2
Ð
500
Ð
500
>2
Ð
0.008
1
NA
0.020
0.020
0.026
0.025
10
10
Ð
8
9
H
H
l
l
(R,S)
(R,S)
1
50
50
50
Ð
0.1
NA
5
aEC50: concentration in mM required to inhibit syncytia formation by 50% on MT4 cells.
bCC50: concentration in mM required to cause 50% death of uninfected MT4 cells.
cSI: selective index=CC50/EC50
.
dIC50: enzyme inhibition values average of two separate runs.
eNA: nonactive at nontoxic concentrations.

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J. L. Kraus et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2023±2026
carbons C1 and C2 appears to be crucial for anti-HIV
activity particularly in the cell culture assay. Compound
5a3 (Table 1) which asymmetric carbon C1 is in d con-
®guration and C2 is a mixture of R, S con®guration is
denied of any anti-HIV activity in both assays. In con-
trast the most active compounds have the asymmetric
carbon C1 in the l con®guration. Substitution on the
aromatic ring of the phenylalanine residue also in¯uenced
the antiviral activity, the presence of a methoxy group
(compound 5d) abolished the antiviral activity while the
presence of hydroxy or benzyloxy groups seems to
improve the activity (compounds 5c1 and 5b1) compared
to that of compound 5a1. Concerning the recombinant
HIV protease assay, the results are more dicult to
explain since if as expected IC50 values found for
diastereoisomers 5a1 and 5a2 (Table 1) are quite di€er-
ent, respectively 8 nm and 1 mM, in the case of the
diastereisomers (5b1, 5b2) or (5c1, 5c2) (Table 1), the
IC50 values for both isomers are similar, respectively
20 nm and 26 nm. More enzymatic experiments are
required in order to clearly elucidate this point.
Acknowledgements
We wish to thank ANRS (Agence National de
Recherche contre le SIDA) for ®nancial support and the
INSERM U-322 for virological testing.
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Conclusion
Nevertheless, this new series of 2-thiophenoxy-3-pyrro-
lidinone analogues appear to be a promising model for
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