Synthesis of phosphinic alanyl-proline surrogates AlaΨ(PO2R-CH)Pro as potential inhibitors of the human cyclophilin hCyp-18

Article abstract of DOI:10.1016/S0040-4039(01)01255-2

Pseudopeptides containing the phosphinic analogue of the alanyl-proline motif AlaΨ(PO₂R-CH)Pro, were synthesized via three- (R = H) and four-step (R = CH₃) procedures. The mixtures of diastereomers were evaluated as inhibitors of the

Full text of DOI:10.1016/S0040-4039(01)01255-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 6295–6297
Synthesis of phosphinic alanyl-proline surrogates
Alac(PO2R-CH)Pro as potential inhibitors of the human
cyclophilin hCyp-18
Luc Demange and Christophe Dugave*
CEA/Saclay, De´partement d’Inge´nierie et d’Etudes des Prote´ines (DIEP), Baˆtiment 152, Gif-sur-Yvette, France
Received 21 June 2001; accepted 6 July 2001
Abstract—Pseudopeptides containing the phosphinic analogue of the alanyl-proline motif Alac(PO2R-CH)Pro, were synthesized
via three- (R=H) and four-step (R=CH3) procedures. The mixtures of diastereomers were evaluated as inhibitors of the human
cyclophilin hCyp-18, an important peptidyl-prolyl isomerase. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
N
N
O
In the past decade, an impressive number of novel
pseudopeptides has been developed as enzyme
inhibitors, in particular protease inhibitors. Some of
them have been used as novel therapeutic agents.1
Among other mimetics, phosphinic pseudopeptides are
potent inhibitors of aspartyl proteases2 and zinc-
metalloproteases2,3 such as angiotensin-converting
enzyme (ACE)4 or matrix metalloproteases (MMPs).5
For this purpose, many different phosphinic motifs
have been designed and synthesized. We investigated
the synthesis of pseudopeptides 1 and 2 containing,
respectively, a charged and a non-charged Ala-Pro
phosphinic isostere, in order to test them as potential
inhibitors of the human cyclophilin hCyp-18. HCyp-18
is an important peptidyl-prolyl cis–trans isomerase
(PPIase) implicated in protein folding,6 cellular
multiplication7 and communication,8 as well as
immunosuppression.9 Moreover, hCyp18 is involved in
the infection of T-cells by HIV-1 and in the multiplica-
tion of the virus.10 The design of transition-state mimics
of the PPIase activity might lead to the development of
non-immunosuppressive and potent inhibitors of hCyp-
18.11
O
R
R
O
O
NH
NH
cis-proline
trans-proline
H
H
N
N+
H
O
N
O
H
Arg55
H2N
P
OR
O
O
A
B
hydrophobic
pocket
H
H
N
N+
H
O
O
H
O
N
Arg55
H2N
O
P
OR
B'
A'
Though the mechanism of the reaction is not clearly
established, recent results suggest an hyperpolarization
of the amide, a quaternarization of the nitrogen12
(Scheme 1, A) and an enzyme-assisted rotation of the
hydrophobic
pocket
Scheme 1. Amino acyl-proline cis–trans isomerization: pro-
posed transition-states12 for the hCyp-18-catalyzed reaction
and corresponding phosphinic transition-state isostere.
* Corresponding author. Tel.: (33) 169 08 52 25; fax: (33) 169 08 90
71; e-mail: christophe.dugave@cea.fr
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01255-2

6296
L. Demange, C. Duga6e / Tetrahedron Letters 42 (2001) 6295–6297
‘keto-amine’ intermediate13 (Scheme 1, A%). The phos-
phinic motif displays some similarities with the postulated
PPIase transition state (Scheme 1, B and B%). We report
herein the synthesis of an Ala-Pro phosphinic isostere
which might be able to mimic the catalytic intermediates
of the reaction. This motif was inserted inside a tripeptide
Ac-Ala-Pro-Phe-pCMA14 derived from the standard
model substrates of hCyp-18 Suc-Ala-Ala-Pro-Phe-pNA.
O
1- HMDS
2- Ad-Br
Ag2O
Z-HN
PH
OH
O
P
3
Z-HN
+
AdO
46%
CO2Me
4
CO2Me
(mixture of diastereomers)
1- 4N NaOH/MeOH
2- Phe-pCMA,
DCC, HOBT
2. Synthesis of phosphinic alanyl-proline surrogates
O
R1-HN
P
A general method of preparation of aminoalkyl phos-
phinic dipeptides equivalents has been previously
reported. It implies a conjugate addition of the trivalent
form of a protected aminoalkylphophinic acid on to a
C2-substituted acrylate.15 This strategy is particularly
attractive since it employs easily available starting mate-
rials, in particular substituted acrylates.16 The versatile
method of preparation of aminoalkylphosphinic acids
described by Baylis and co-workers also provides a large
molecular diversity.17 Moreover, most of the
diastereomers can be separated by reverse phase HPLC
(RP-HPLC) after deprotection of the phosphinyl moi-
ety.18 As a consequence, this strategy has been successfully
employed for generating large librairies of phosphinic
inhibitors with wide varieties of substituents and absolute
configurations.17–19 However, to our knowledge, no
preparation of C2 and C3 bis-substituted aminoalkyl
phosphinate Xaac(PO2H-CH)Pro has been formally
reported in the literature. We investigated the conjugate
addition of a bis-silyl derivative of benzyloxycarbonyl-1-
aminoethyl-1-phosphinate3ontomethyl1-cyclopentene-
1-carboxylate as a straightforward method for generating
an orthogonally protected Alac(PO2H-CH)Pro tem-
plates.
R2O
37%
CO-Phe-pCMA
1- HCO2- NH4+ Pd-C
2- AcCl
5 (R1 = Z; R2 = Ad)
3- TFA
1 (R1 = Ac; R2 = H) 19%
2 (R1 = Ac; R2 = Me) quant.
4- RP-HPLC purification
CH2N2
quant.
Scheme 2. Synthesis of Alac(PO2H-CH)Pro-containing pep-
tides 1 and 2.
semi-preparative RP-HPLC, the mixture was used in
preliminary biological assays.
3. Biological evaluation of phosphinic pseudopeptides as
inhibitors of hCyp-18
Phosphinic tripeptide isosteres were tested both as ligands
of hCyp-18 and inhibitors of its PPIase activity. Fluori-
metric titration of Trp12111 showed that compounds 1
and 2 bind to hCyp-18 (1: Kd=107 6 mM; 2: Kd=74 4
mM)withanaffinityequivalenttothereferencetripeptides
Ac-Ala-Pro-Phe-pNA (Kd=140 40 mM) and Suc-Ala-
Pro-Phe-pCMA (Kd=55 6 mM). Unfortunately, neither
1 nor 2 were able to inhibit the cyclophilin-catalyzed
isomerization of the model subtrate Suc-Ala-Ala-Pro-
Arg-pNA. This indicates that these compounds interact
close to hCyp-18 Trp121. In turn, the Alac(PO2R-
CH)Pro (R is H or CH3) moieties do not fit inside the
proline recognition pocket which catalyzes the cis–trans
isomerization.6,24
Compound 3 was prepared as a racemic mixture as
previously described.17–19 The conjugate addition of 3 on
to methyl 1-cyclopentene-1-carboxylate was carried out
using hexamethyldisilazane for the generation of the
nucleophilic trivalent form of the phosphinic acid. The
adduct was protected in situ using 1-bromoadamantane
and silver oxide (Scheme 2).20 The dipeptide isostere 4 was
isolated as a complex mixture of diastereomers which can
be separated by RP-HPLC. Saponification of the methyl
ester and standard peptide coupling to phenylalanyl(4-
carboxymethyl)aniline gave tripeptide 5 in low yield
(37%). This might be the result of a concurrent saponifi-
cation of the adamantane as previously observed.21
Deprotection of the benzyloxycarbonyl moiety was car-
ried out using ammonium formate and 10% Pd–C.20
Indeed, previous attempts for hydrogenolyzing the benzyl
carbamate under an hydrogen atmosphere caused a
significative deprotection of the adamantyl ester. Acetyl-
ation followed by acidolysis of adamantyl ester 6 yielded
pure Ac-Alac(PO2H-CH)Pro-Phe-pCMA 1.22 Only three
of the eight possible diastereomers of 1, generated by the
creation of three stereogenic centers, were observed by
RP-HPLC. The uncharged derivative of acid 1, methyl
ester 2,23 was obtained in quantitative yield by treatment
of compound 1 with diazomethane in THF. Even though
some diastereomers of 1 and 2 could be separated by
In summary, we reported herein an expeditious synthesis
of the orthogonally protected phosphinic Ala-Pro isostere
Alac(PO2Ad-CH)Pro and its use in solution peptide
synthesis for the preparation of Alac(PO2H-CH)Pro and
Alac(PO2CH3-CH)Pro-containing peptides.
Acknowledgements
This work was supported by SIDACTION (grant
c70000016/2), the Agence Nationale de Recherches sur
le Sida (ANRS) (grant c 99014) and the Atomic Energy
Commission (CEA). We gratefully acknowledge Dr. A.
Lecoq for helpful advise and discussion and Dr. M.
Moutiez for providing recombinant hCyp-18.

L. Demange, C. Duga6e / Tetrahedron Letters 42 (2001) 6295–6297
6297
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7.82–7.67+7.60–7.55 (3m, 4H, H Ar. pCMA), 7.27–7.17
(m, 5H, H Ar. Phe), 4.41–4.26 (bm, 1H, NCH
6 (CH3)P),
3.87 (s, 3H, CO2CH3 pCMA), 3.37–3.21+3.14–2.87+2.72–
6
2.48 (3m, 4H, P-CH+CH-CO cpentane+2Hb Phe), 1.99–
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172.4+168.2+168.1+144.2+144.1+138.7+138.3+138.2+131.4
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