Synthesis and evaluation of Glyψ(PO2R-N)pro-containing pseudopeptides as novel inhibitors of the human cyclophilin hCyp-18

Article abstract of DOI:10.1021/jm020865i

The human cyclophilin hCyp-18, an abundant peptidyl-prolyl cis-trans isomerase (PPIase) implicated in protein folding, controls the infection of CD4⁺ T-cells by HIV-1, the pathologic agent of AIDS. Therefore, hCyp-18 is an interesting target fo

Full text of DOI:10.1021/jm020865i

3928
J . Med. Chem. 2002, 45, 3928-3933
Syn th esis a n d Eva lu a tion of Glyψ(P O2R-N)P r o-Con ta in in g P seu d op ep tid es a s
Novel In h ibitor s of th e Hu m a n Cyclop h ilin h Cyp -18†
Luc Demange, Mireille Moutiez, and Christophe Dugave*
CEA/ Saclay, De´partement d’Inge´nierie et d’Etudes des Prote´ines, Baˆtiment 152, 91191 Gif-sur-Yvette, France
Received February 27, 2002
The human cyclophilin hCyp-18, an abundant peptidyl-prolyl cis-trans isomerase (PPIase)
implicated in protein folding, controls the infection of CD4+ T-cells by HIV-1, the pathologic
agent of AIDS. Therefore, hCyp-18 is an interesting target for the development of novel anti-
HIV-1 therapeutics. We focused on the design of transition-state analogue inhibitors of the
PPIase activity of cyclophilin. Most experimental results reported in the literature suggest
that hCyp-18 catalyzes the pyramidalization of the nitrogen of pyrrolidine via an H-bond
network which results in the deconjugation of the amino acyl-prolyl peptide bond. We proposed
the Glyψ(PO2R1-N)Pro motif (R ) alkyl or H) as a selective transition-state analogue inhibitor
of cyclophilin. This motif was inserted in Suc-Ala-Ala-Pro-Phe-pNA, a peptide substrate of hCyp-
18. The pseudopeptide Suc-Ala-Glyψ(PO2Et-N)Pro-Phe-pNA 1b bound to hCyp-18 (Kd ) 20 (
5 µM) and was able to selectively inhibit its PPIase activity (IC50 ) 15 ( 1 µM) but not hFKBP-
12, another important PPIase. Deprotection of the phosphonamidate moiety resulted in a
complete lack of inhibition. We previously demonstrated that reduction of the Phe-pNA moiety
caused a quantitative reduction of the affinity; however, Suc-Ala-Glyψ(PO2Et-N)Pro-Pheψ-
(CH2-NH)pNA 7b still bound and inhibited hCyp-18, suggesting that the Glyψ(PO2Et-N)Pro
motif plays the major role in the binding to cyclophilin. Consequently, we propose compound
1b as being a novel transition-state mimic inhibitor of hCyp-18.
In tr od u ction
tion state is a deconjugated amide (ketoamine form).17
The catalyzed isomerization is globally favored by the
hydrophobic environment of the active site.18 The struc-
ture of the complex of hCyp18 with tetrapeptide Suc-
Ala-Ala-Pro-Phe-pNA shows that Arg55 located at the
catalytic subsite strongly binds the carboxyl at the P1′
C-terminus. In addition, this arginine could drive the
pyramidalization of nitrogen (a prerequisite to the
amide hyperpolarization) by binding the pyrrolidine
nitrogen at P1′ via an H-bond.17 Arg55 might also assist
the rotation of the acyl moiety by a transient interaction
(Scheme 1, A and A′).19-23 This hypothesis is supported
by results from J anda and co-workers24 and Schultz and
collaborators25 who generated PPIase catalytic antibod-
ies using a ketoamide-containing hapten. In the latter
work, a ketoamide-containing transition-state analogue
inhibitor was developed. This compound, however, is not
selective and inhibits both hCyp-18 and hFKBP-12,
another important PPIase.25,26 Nevertheless, these data
indicate that by mimicking a rotating amide bond, novel
transition-state analogue inhibitors of hCyp-18 might
be able to be designed. For this pupose, it is important
to study the differences found in the various PPIase
subfamilies. The hypothesis of a tetrahedral covalent
intermediate has been ruled out for the cis-trans
isomerization process catalyzed by hCyp-18, while the
crystallographic structure of a complex between a
dipeptide and Pin1, a human parvulin implicated in the
control of mitosis27 and involved in neurodegeneration,28
suggests the existence of a tetrahedralized carbonyl
carbon at P1.29 Moreover, Pin1 possesses several nu-
cleophilic residues that might participate in the cataly-
sis.29 On the other hand, FKBP does not possess a polar
Immunophilins are abundant and ubiquitous pepti-
dyl-prolyl isomerases (PPIases) which specifically cata-
lyze the cis-trans interconversion of the amino acyl-
prolyl amide bond.1 As a consequence, they play a
critical role in protein folding and in several related
biological processes, such as cellular multiplication2 and
communication.3 hCyp-18, a cytosolic member of the
human cyclophilin subfamily, is also the main receptor
of the immunosuppressive undecapeptide cyclosporin A
and, hence, is implicated in immunosuppression.4 More-
over, hCyp-18 is involved in the infection of T-cells by
HIV-1, the pathological agent of AIDS, and, hence,
participates in the control of virus multiplication.5-7
Therefore, the design of specific inhibitors of hCyp-18
arouses considerable interest for the development of new
drugs, in particular, of anti-AIDS agents. Several
strategies have been proposed for the development of
cyclophilin inhibitors as novel therapeutics. These
include non-immunosuppressive analogues of cyclo-
sporin,8-11 short modified peptides derived from the
viral Gag polyprotein,12 nonisomerizable substrate ana-
logues,13,14 and ground-state analogue inhibitors.15 Here,
we present pseudopeptides mimicking the transition
state16 of the PPIase activity as selective inhibitors of
cyclophilin.
The mechanism of cis-trans isomerization is still
unclear, but experimental data suggest that the transi-
† Part of this work was presented at the 26th European Peptide
Symposium (Montpellier, France, 2000); see: Peptides 2000; Martinez,
J ., Fehrentz, J .-A., Eds.; EDK: Paris, France, 2001; pp 809-810.
* To whom correspondence should be addressed. Tel: (33) 169 08
52 25. Fax: (33) 169 08 90 71. E-mail: christophe.dugave@cea.fr.
10.1021/jm020865i CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/25/2002

Glyψ(PO2R-N)Pro-Containing Pseudopeptides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3929
and coupling reagents were obtained from Novabiochem,
Bachem, or Sigma. Suc-Ala-Gly-Pro-Phe-pNA, Suc-Ala-Ala-
Pro-Phe-pNA, Suc-Ala-Ala-Pro-Arg-pNA, and Suc-Ala-Leu-
Pro-Phe-pNA were purchased from Bachem. Suc-Ala-Ala-Pro-
Phe-DFA was synthesized as previously reported.34 All other
solvents were of analytical grade and were used without
further purification. All intermediates were purified by flash
chromatography on 40-60 µm (230-400 mesh) Merck silica
gel. Peptides were purified by RP-HPLC. All compounds were
Sch em e 1. Proposed Mechanism of the
hCyp-18-Catalyzed Isomerization: (A) Arg55-Assisted
Deconjugation of the Amide via an H-Bond with the
Guanidinium Side Chain and Pyramidalization of the
Proline Nitrogen, (A′) Arg55-Assisted Rotation of the
Acyl Group via an H-Bond with the Guanidinium Side
Chain, (B) the Phosphonamide Transition-State
Analogue Which Might Mimic both Putative Transition
States A and A′, and (C) Ketoamide Transition-State
Analogue Which Is Supposed To Mimic the Rotating
Acyl Moiety
1
characterized by H, 13C, and 31P NMR (recorded on a Bruker
AVANCE 250 NMR spectrometer); δ values are given in parts
per million (ppm), and J values are in hertz. Electrospray mass
spectra (Atheris Laboratories, Geneva, Switzerland) were
recorded on a Micromass Platform II (Micromass, Altrincham,
U.K.). Fluorimetric determination of Kd was done using a J asco
FP-750 spectrofluorometer equipped with a 250 µL thermo-
stated cell. Kinetic assays were performed using a Kontron
Uvikon 930 spectrophotometer equipped with a 2 mL ther-
mostated cell. The recombinant hCyp-18 was obtained as
previously reported.12
P h tdGlyΨ(P O2Et-N)P r o-P h e-pNA (3a a n d 3b). Com-
pound 2 (2.97 g, 10 mmol) was refluxed with phosphorus
pentachloride (2.41 g, 1.1 equiv) in freshly dried benzene (15
mL) for 7 h under argon. After removal of the solvent under
reduced pressure, the crude product dissolved in freshly dried
THF (10 mL) was added dropwise to a solution of Pro-Phe-
pNA (0.5 equiv) and triethylamine (3.48 mL, 2.5 mmol) in
freshly dried THF. The mixture was stirred overnight under
argon at room temperature. After evaporation of the solvent,
the crude product was dissolved in dichloromethane and
washed twice with 10% citric acid, saturated sodium bicarbon-
ate, and brine. The organic layer was dried over sodium
sulfate, and the volume was reduced in vacuo. Purification by
silica gel flash chromatography (eluent, 65:35 ethyl acetate:
chexane ratio) gave 3a (939 mg, 1.48 mmol) and 3b (576 mg,
0.91 mmol) in 48% overall yield.
residue at the active site and interacts with its substrate
in a mode that is completely different from that of hCyp-
18 and Pin1.19
Phosphinic or phosphonamide amino acyl-proline
surrogates might mimic the rotating amide bond. We
recently reported the synthesis and biochemical evalu-
ation of the phosphinic pseudopeptides Ac-Alaψ(PO2R-
CH)Pro-pCMA (R ) H or CH3). Though the phosphinic
moiety is chemically stable and resistant to proteolysis,
these peptides are not able to inhibit the PPIase activity
of hCyp-18.30 We investigated the potential of phospho-
namide surrogates of Gly-Pro as novel transition-state
mimics of PPIase activity. Though these surrogates are
less chemically resistant than their phosphinic equiva-
lent,31 the phosphonamide nitrogen is slightly pyrami-
dalized,32 and therefore, its doublet is available for
establishing a strong H-bond with the guanidinium
moiety of Arg55 (Scheme 1, B).
3a : 1H NMR (CDCl3) δ 7.84 (d, J ) 5.1, 2H) + 7.63 (d, J )
7.1, 2H) + 7.42-7.17 (m, 12H), 5.05-4.94 (m, 1H), 4.33-4.02
(m, 5H), 3.81 (dd, J ) 14.1, J ′ ) 4.6, 1H), 3.53-3.45 (bm, 1H),
3.29-3.15 (m, 1H), 3.10 (dd, J ) 14.1, J ′ ) 12.3), 2.09-2.04 +
1.80-1.75 + 1.62-1.51 + 1.30-1.22 (4m, 4H), 1.42 (t, J ) 7.0,
3H); 13C NMR (CDCl3) δ 172.7 + 170.5 + 168.0, 144.1 + 142.8
+ 137.8 + 134.4 + 130.9 + 129.1 + 128.5 + 126.7 + 124.1 +
123.3 + 118.9, 62.7 (d, J 2
) 7.2), 61.4 (d, J 2
) 1.1), 53.5,
C-P
C-P
47.5 (d, J 2
) 2), 36.8, 33.7 (d, J 1
) 141.3), 31.3 (d, J 3
C-P C-P
C-P
) 7.5), 25.6 (d, J 3
) 7.2), 16.4 (d, J 3
) 6.0); 31P NMR
C-P
C-P
+
(CDCl3, decoupled) δ 25.8; DCI-MS (NH3) m/z 651 (MNH4
,
9%), 634 (MH+, 100%); combustion analysis (C31H32N5O8) C,
H, N.
3b: 1H NMR (CDCl3) δ 8.09 (d, J ) 9.25, 2H) + 7.96 (d, J
) 9.25, 2H), 7.88-7.85 + 7.78-7.75 (2m, 4H), 7.29-7.25 +
7.18-7.14 (5H), 5.01-4.92 (m, 1H), 4.21-4.07 (m, 5H), 3.83-
3.71 + 3.66-3.46 + 3.23-3.04 (3m, 4H), 2.41-2.20 + 2.09-
1.81 + 1.74-1.51 (3m + 4H), 1.19 (t, J ) 7, 3H); 13C NMR
(CDCl3) δ 172.4 + 170.4 + 167.2, 144.5 + 143.3 + 136.3 +
134.6 + 131.6 + 129.4 + 128.9 + 127.3 + 124.5 + 123.8 +
We report herein on the synthesis and biochemical
evaluation of phosphonamide-containing pseudopeptides
as transition-state analogue inhibitors of hCyp-18. The
corresponding ketoamide was prepared as a reference
for the test tube assays (Scheme 1, C).
119.9, 61.7 (d, J 2
) 6.2), 61.4 (d, J 2
) 6.5), 53.4, 48.6 (d,
C-P
C-P
J 2
) 2.6), 37.2, 32.5 (d, J 1
) 137.9), 31.7 (d, J 3
) 7.7),
C-P
C-P
C-P
25.2 (d, J 3
) 7.5), 16.3 (d, J 3
) 6.8); 31P NMR (CDCl3,
C-P
C-P
decoupled) δ 24.2; DCI-MS (NH3) m/z 651 (MNH4+, 14%), 634
Exp er im en ta l Section
(MH+, 100%); combustion analysis (C31H32N5O8) C, H, N.
Abbr evia tion s. Alaψ(COCO-N)Pro, ((S)-2-oxo-3-aminobu-
tanoyl)proline; DCC, dicyclohexylcarbodiimide; DIPEA, diiso-
propylethylamine; Glyψ(COCO-NH)Pro, (2-oxo-3-aminopro-
panoyl)proline; Glyψ(PO2R-NH)Pro, ethyl aminomethylphos-
phonylproline (R ) Et) or aminomethylphosphonylproline
sodium salt (R ) Na); HOBT, 1-hydroxybenzotriazole; pCMA,
4-carboxymethylaniline; Pheψ(CH2-NH)pNA, 1-(4-nitrophe-
nyl)amino-(R)-2-benzyl-2-aminoethane; pNA, 4-nitroaniline;
SD, standard deviation; Suc, succinyl; THF, tetrahydrofuran;
Valψ(COCO-NH)Pro, ((S)-2-oxo-3-amino-4-methylpentanoyl)-
proline.
F m oc-Ala -GlyΨ(P O2E t -N)P r o-P h e-p NA (4a a n d 4b ).
Compounds 3a (0.63 g, 1 mmol) and 3b (0.5 g, 0.79 mmol) were
treated with 1.0 M hydrazine in THF (4.0 equiv) overnight at
room temperature. The mixture was filtered, and the cake was
washed with THF (3 times). The crude product was dissolved
in dry dichloromethane (10 mL) and treated at 0 °C with Fmoc-
alanine (9 equiv), DCC (9.9 equiv), HOBT (9 equiv), and
DIPEA (18 equiv). The mixture was stirred overnight at room
temperature. The crude mixture was filtered and washed with
10% citric acid (2 times), saturated sodium bicarbonate, and
brine. The organic layer was dried over sodium sulfate.
Purification by silica gel flash chromatography (eluent, 65:35
All the reagents employed were of analytical grade and were
purchased from Aldrich Chemical Co. or Sigma. Amino acids

3930 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Demange et al.
ethyl acetate:chexane ratio) gave 4a (690 mg, 88%) and 4b
nitrile:acetic acid (0.25% in water) 25:75 to 75:25 in 30 min)
(580 mg, 92%).
to give 7a (121 mg, 77%) and 7b (128 mg, 82%).
7a : tR 12.59 (>95%); H NMR (CDCl3) δ 8.02 (d, J ) 9.2,
1
4a : 1H NMR (CDCl3) δ 8.10 (d, J ) 7.5, 2H) + 7.88 (d, J )
9.0, 2H) + 7.75 (d, J ) 7.3, 2H) + 7.54 (d, J ) 7.2, 2H) + 7.42-
6.80 (bm, 9H), 5.48-5.45 (m, 1H), 4.97-4.93 (m, 1H), 4.41-
4.30 + 4.20-3.92 (2bm, 8H), 3.47-3.44 + 3.22-3.07 (2bm, 4H),
1.97-1.80 + 1.75-1.53 (2b, 4H), 1.38-1.20 (bm, 6H); 13C NMR
(CDCl3) δ 173.8 + 173.5 + 173.4 + 171.3, 156.9 + 156.2,
2H), 7.51 (bd, J ) 7.95, 1H), 7.33-7.13 (m, 5H), 6.50 (d, J )
9.2, 2H), 6.30 (bd, J ) 7.1, 1H), 6.19 (bt, J ) 5.6, 1H), 4.44 (t,
J ) 7.0, 1H), 4.40-4.31 (m, 1H), 4.02-3.88 (m, 5H), 3.41-
3.25 + 3.10-3.01 + 2.97-2.93 (3m, 6H), 1.99 (s, 3H), 2.04-
1.94 + 1.81-1.62 (2m, 4H), 1.36 (d, J ) 7.0, 3H), 1.26 (t, J )
7.0, 3H); 13C NMR (CDCl3) δ 173.8 + 172.7 + 172.6 + 170.5,
137.4 + 129.1 + 128.6 + 128.5 + 127.7 + 126.8 + 126.4 +
144.3-118.8 (complex), 67.3, 62.1, 61.4 (d, J 2
) 6.6), 54.9,
C-P
) 7.3), 18.0, 16.5 (d, J 3
C-P C-P
C-P
53.7 + 47.0, 50.4 + 49.1 + 46.7, 37.2, 36.3 (d, J 1
) 147.3),
126.3 + 110.9, 61.6 (d, J 2
) 4.15), 61.2 (d, J 2
) 6.8), 50.3
) 3.6), 46.2, 38.1, 34.8 (d, J 1
) 143.3),
C-P
31.4 (d, J 3
) 7.2), 25.8 (d, J 3
C-P
C-P
C-P
+ 49.1, 47.5 (d, J 2
) 5.9); 31P NMR (CDCl3, decoupled) δ 26.5 (minor) + 27.1
(major); DCI-MS (NH3) m/z 797 (MH+, 30%), 415 ([Pro-Phe-
pNA]H+, 35%), 383 ([Fmoc-Ala-CH2PO2Et]H+, 100%).
C-P
31.1 (d, J 3
C-P
) 6.5), 25.1 (d, J 3
) 6.9), 23.0, 17.9, 16.3 (d,
C-P
C-P
J 3
) 6.3); 31P NMR (CDCl3, decoupled) δ 27.1; ES-MS 602.4.
1
4b: 1H NMR (CDCl3) δ 8.08 (d, J ) 9.3), 7.91 (d, J ) 9.3),
7.78-7.64 + 7.57-7.50 + 7.46-7.11 (bm, 13H), 5.40-5.31 (m,
1H), 4.97-4.87 (m, 1H), 4.49-3.96 (2bm, 8H), 3.61-3.32 +
3.11-2.98 (2bm, 4H); 13C NMR (CDCl3) δ 173.4 + 172.9 +
170.3, 157.4 + 156.1 + 155.7, 144.2-110.8 (complex), 67.2, 63.8
7b: tR 11.90 (>95%); H NMR (CDCl3) δ 8.00 (d, J ) 10.0,
2H), 7.55-7.46 (b, 1H), 7.34-7.15 (m, 5H), 6.51 (d, J ) 10.0,
2H), 6.44 (d, J ) 7.5, 1H), 6.16-6.08 (b, 1H), 4.50-4.29 (m,
2H), 4.23-3.84 (m, 5H, Ha), 3.68-3.55 + 3.43-3.22 + 3.18-
2.97 (3m, 6H), 2.04 (minor) + 2.02 (major) (s, 3H), 2.11-1.99
+ 1.81-1.62 (2m, 4H), 1.34 (d, J ) 7.5, major) + 1.30 (d, J )
5.0, minor), 1.26 (t, J ) 5.0, major) + 1.25 (t, J ) 5.0, minor);
13C NMR (CDCl3) δ 174.5 + 173.0 + 172.8 + 170.4, 137.6 +
(d, J 2
) 6.2), 62.0 (d, J 2
) 4.5) + 61.0 (d, J 2
) 6.9),
C-P
C-P
C-P
53.9, 50.9 + 47.7 + 47.0 + 47.0 (d, J 2
) 9.2), 36.9, 34.1 (d,
C-P
J 1
) 141.8), 31.6 (d, J 3
) 6.9), 25.5 (d, J 3
) 8.2), 18.1,
C-P
C-P
C-P
137.4 + 129.1 + 128.5 + 126.7 + 110.9, 61.8 (d, J 2
) 6.8),
16.2 (d, J 3
) 6.8); 31P NMR (CDCl3, decoupled) δ 27.6; DCI-
C-P
C-P
60.9 (d, J 2
) 3.6), 50.2 + 49.0, 47.3 (d, J 2
) 3.4), 46.4,
C-P
MS (NH3) m/z 797 (MH+, 13%), 415 ([Pro-Phe-pNA]H+, 29%),
C-P
38.1, 36.2 (d, J 1
) 146.0), 31.5 (d, J 3
) 7.1), 25.7 (d, J 3
C-P C-P
383 ([Fmoc-Ala-CH2PO2Et]H+, 100%).
C-P
) 6.8), 23.1, 17.9, 16.3 (d, J 3
decoupled) δ 26.7; ES-MS m/z 602.3.
) 6.2); 31P NMR (CDCl3,
C-P
Su c-Ala -GlyΨ(P O2Et-N)P r o-P h e-pNA (1a a n d 1b). Com-
pounds 4a (500 mg, 0.79 mmol) and 4b (450 mg, 0.58 mmol)
were treated with 20% diisopropylamine in DMF for 3 h at
room temperature. After evaporation of the solvent under
reduced pressure, methanol was chased over the product (3
times). The crude product was treated with succinic anhydride
(2.0 equiv) and trietylamine (3.0 equiv) overnight at room
temperature. After evaporation of the solvent under reduced
pressure, the crude product was purified by RP-HPLC (semi-
preparative column Vydac C18; rate, 4 mL min-1; linear
gradient, acetonitrile:acetic acid (0.25% in water) 25:75 to 75:
25 in 30 min).
Ac-Ala -GlyΨ(P O2Bn -N)P r o-P h e-pCMA (11). Compound
10 (85 mg, 87 µmol) was treated with 20% diisopropylamine
in DMF for 3 h at room temperature. After evaporation of the
solvent under reduced pressure, methanol was chased over the
product (3 times). The crude product was treated with acetyl
chloride (13 mL, 2.0 equiv) and DIPEA (45 mL, 3.0 equiv)
overnight at room temperature. After evaporation of the
solvent under reduced pressure, the crude product was purified
by preparative thin-layer chromatography (eluent, 94:6 chlo-
roform:methanol ratio) to give 11 (31 mg, 52%): 1H NMR
(CDCl3) δ 7.98 (d, J ) 10.0, 2H) + 7.84 (d, J ) 10.0, 2H), 7.68
(d, J ) 7.5, 1H) + 7.37-7.18 (m, 9H), 5.96 (d, J ) 7.3, 1H),
1
1a (50 mg, 12%): tR 11.09 min (95%); H NMR (CD3OD) δ
8.19 (d, J ) 9.0) + 7.84 (d, J ) 9.2), 7.36-7.16 (bm, 5H), 4.90-
4.82 (m, 1H), 4.39 (q, J ) 7.2, 1H), 4.28-4.17 (m, 1H), 4.06-
3.90 (m, 2H), 3.83-3.69 (m, 2H), 3.30-3.10 (bm, 4H), 2.68-
2.55 + 2.55-2.41 (bm, 4H), 2.19-2.03 + 1.84-1.52 (2bm, 4H),
1.35 (d, J ) 7.2, 3H), 1.26 (t, J ) 7.0, 3H); 13C NMR (CD3OD)
δ 176.4 + 175.3 + 175.2 + 174.6 + 172.4, 145.7 + 144.7 +
5.00 (AB, δA ) 5.07, δB ) 4.94, J AB ) 11.8) + 5.00 (AB, δA
)
5.03, δB ) 4.97, J AB ) 11.7) (m, 2H), 4.85-4.96 + 4.42-4.34
+ 4.08-4.05 + 3.94-3.84 (4m, 5H), 3.87 (s, 3H), 3.51-2.95
(m, 4H), 1.79 (s, 3H), 1.80-1.73 + 1.54-1.48 (2m, 4H), 1.26
(d, J ) 7.1, 3H); 13C NMR (CDCl3) δ 173.7 + 173.2 + 171.0 +
170.7 + 166.6, 142.6 + 137.7 + 136.0 + 135.9 + 130.6 + 129.3
+ 128.8 + 128.4 + 128.0 + 126.7 + 125.4 + 119.3, 66.7 (d,
138.5 + 130.5 + 129.5 + 127.9 + 125.7 + 120.7, 62.7 (d, J 2
C-P
) 3.6), 62.3 (d, J 2
142.7), 32.7 (d, J 3
) 6.8), 56.7, 50.5, 38.5, 37.3 (d, J 1
)
C-P
J 2
) 7.0), 61.3 (d, J 2
) 2.3), 54.9 + 52.0 + 48.6 + 47.0,
C-P
C-P
C-P
) 6.9), 31.4 + 30.2, 26.6 (d, J 3
) 6.5),
C-P
36.6, 36.6 (d, J 1
) 146.0), 31.1 (d, J 3
) 7.0), 25.2 (d, J 3
C-P C-P
C-P
C-P
17.7, 16.6 (d, J 3
) 6.4); 31P NMR (CD3OD, decoupled) δ 25.7;
) 7.0), 22.8, 16.9; 31P NMR (CDCl3, decoupled) δ 28.1; ES-MS
m/z 691.5.
C-P
ES-MS (negative ionization) m/z 674.19.
1
1b (50 mg, 12%): tR 12.45 min (92%); H NMR (CD3OD) δ
8.19 (d, J ) 9.15, 2H) + 7.87 (d, J ) 9.15, 2H), 7.33-7.17 (bm,
5H), 4.81-4.73 (m, 2H), 4.34 (q, J ) 7.2, 1H), 4.15-3.96 (m,
4H), 3.70-3.56 + 3.26-3.13 (2m, 4H), 2.64-2.47 (bm, 4H),
2.21-2.02 + 1.91-1.69 (2bm, 4H), 1.35 (d, J ) 7.5, 3H), 1.24
(t, J ) 7.05, 3H); 13C NMR (CD3OD) δ 176.3 + 175.2 + 175.1
+ 174.7 + 172.5, 145.7 + 144.8 + 138.1 + 130.4 + 129.6 +
128.0 + 125.7 + 125.6 + 120.8 (complex), 62.6-62.3 (m), 56.5,
Ac-Ala -GlyΨ(P O2H-N)P r o-P h e-pCMA (12). Compound
11 (23 mg, 33 µmol) dissolved in a 50:50 methanol/water
mixture (7 mL) was treated with sodium bicarbonate (8 mg,
2.8 equiv) and 10% Pd-C (11 mg) under 4 bar of hydrogen for
2 h. The catalyst was removed by filtration, and the solvent
was evaporated under reduced pressure. The crude product
was dissolved in a 50:50 water/acetonitrile mixture and freeze-
dried. Compound 12 was used without further purification:
1H NMR (CD3OD) δ 8.01-7.92 (m, 4H), 7.32-7.19 (m, 5H),
4.90-4.78 (m), 4.32 (q, J ) 7.0, 1H), 4.00-3.93 (m, 1H), 3.88
(s, 3H), 3.64 (dd, J ) 15.2, J ′ ) 11.8, 1H) + 3.52 (dd, J ) 13.9,
J ′ ) 4.0, 1H) + 3.30-3.19 (m, 1H) + 3.01-2.91 (m, 1H), 1.96
(s, 3H), 1.90-1.75 + 1.65-1.50 (2m, 4H), 1.29 (d, J ) 5.0, 3H);
13C NMR (CD3OD) δ 178.5 + 174.4 + 174.3 + 173.1 + 173.0 +
168.3, 144.3 + 139.1 + 131.1 + 130.2 + 129.5 + 127.7 + 126.6
50.2 (d, J 2
) 7.3), 38.4, 35.6 (d, J 1
) 144.9), 32.8 (d, J 3
C-P
C-P
C-P
) 7.0), 31.4 + 30.3, 26.3 (d, J 3
) 7.5), 17.7, 16.6 (d, J 3
)
C-P
C-P
5.8); 31P NMR (CD3OD, decoupled) δ 25.8; ES-MS (negative
ionization) m/z 674.30.
Ac-Ala -GlyΨ(P O2Et-N)P r o-P h eY(CH2-NH)pNA (7a a n d
7b). Compounds 6a (210 mg, 0.26 mmol) and 6b (208 mg, 0.26
mmol) were treated with 20% diisopropylamine in DMF for 3
h at room temperature. After evaporation of the solvent under
reduced pressure, methanol was chased over the product (3
times). The crude product was treated with acetyl chloride (2.0
equiv) and DIPEA (3.0 equiv) overnight at room temperature.
After evaporation of the solvent under reduced pressure, the
crude product was purified by preparative thin-layer chroma-
tography (eluent, chloroform:methanol with 98:2, then 97:3,
and finally 95:5 ratios) or by RP-HPLC (semipreparative
column Vydac C18; rate, 4 mL min-1; linear gradient, aceto-
+ 121.7, 63.6, 56.3 + 52.5 + 50.5, 38.3, 37.3 (d, J 1
) 128.9),
C-P
32.6, 26.3 (d, J 3
) 5.5), 22.5, 17.9; 31P NMR (CD3OD,
C-P
decoupled) δ 16.4; ES-MS (negative ionization) m/z 601.4.
Bioch em ica l Eva lu a tion . (1) Deter m in a tion of Kd .
Fluorimetric titration was carried out as previously re-
ported.12,33
(2) En zym a tic Assa ys. The PPIase uncoupled assay was
carried out in a 35 mM Hepes buffer (pH 7.8) at 10 ( 0.5 °C
using Suc-Ala-Ala-Pro-Phe-DFA as a substrate.34 The PPIase

Glyψ(PO2R-N)Pro-Containing Pseudopeptides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3931
trypsin-hCyp-18-coupled assay was carried out in a 35 mM
Hepes buffer (pH 8.6) at 10 ( 0.5 °C using Suc-Ala-Ala-Pro-
Arg-pNA,3,35 and the R-chymotrypsin-FKBP-coupled assay was
carried out in a 35 mM Hepes buffer (pH 7.8) at 10 ( 0.5 °C
using Suc-Ala-Leu-Pro-Phe-pNA as a substrate.21,36 Data were
processed according to the literature.12
Sch em e 2. Synthesis of
Suc-Ala-Glyψ(PO2Et-N)Pro-Phe-pNA 1a /b,
Ac-Ala-Glyψ(COCO-N)Pro-Pheψ(CH2-NH)pNA 7a /b, and
Ac-Ala-Glyψ(PO2Na-N)Pro-Phe-pCMA 12
Resu lts a n d Discu ssion
Design Ra tion a le. Transition-state analogues were
designed on the basis of information obtained from
crystallographic data and structure-activity relation-
ship studies. We chose a substrate of immunophilins,
the tetrapeptide Suc-Ala-Ala-Pro-Phe-pNA,36 as a tem-
plate for the development of novel hCyp-18 inhibitors.
Several structures of hCyp-18-peptide complexes have
been resolved and the main interactions delineated.17
Structure-activity relationship studies also provided
interesting information; we recently demonstrated37 the
existence of two functionally independent subsites: (i)
the S1′ subsite which contains the catalytic residue and
controls the selectivity for proline at P1′ and (ii) the S2′-
S3′ subsite which specifically recognizes the Xaa-pNA
moiety or Xaa-pNA surrogates.35,37 Therefore, the P1′
proline is dispensable for the binding of a ligand by
hCyp-18 provided the peptide possesses either an Xaa-
pNA or an Xaa-pNA analogue at P2′-P3′.37 However,
pNA-containing peptides without either a proline resi-
due or a proline mimic cannot inhibit hCyp-18.37,38 The
N-terminus of the tetrapeptide does not participate in
the binding because it protrudes outside the catalytic
site. The binding is mainly controlled by the carboxyl
at P2′ which might establish a strong H-bond with
Trp121.37 The residues at P1, P1′, and P2′ must be in
the L-configuration.35 Cyclophilins prefer Ala, Val, Leu,
Glu, Phe, or Gly at P1, whereas FKBPs recognize
preferentially Phe-Pro or Leu-Pro. In particular, the
selectivity factor for hCyp-18 versus hFKBP-12 is 3500
and 1000 for Glu and Gly, respectively, but only 60 for
Ala at P1′.21 Though Glu is preferred at P1, hCyp-18 is
indeed quite selective of the Gly-Pro moiety. The
introduction of the Glyψ(PO2Et-NH)Pro motif generates
only one novel stereogenic center and, hence, provides
only two diastereomers, whereas the Glu analogue
would give a mixture of four diastereomers. Therefore,
we designed the tetrapeptide analogue 1 (mixture of
both diastereomers 1a and 1b) which contains a phos-
phonamide isostere of Gly-Pro.32
Syn th esis of P seu d op ep tid es. Phosphonamidate-
containing pseudopeptides have been widely used in
medicinal chemistry. However, to our knowledge, there
is only one example of synthesis of phosphonamide
isosteres of the Xaa-Pro motif reported in the litera-
ture.39 Diethyl phthalimidomethylphosphonate 2 was
prepared according to Yamauchi et al.40 Activation with
phosphorus pentachloride in refluxing benzene, as
previously described,40 and coupling to Pro-Phe-pNA
yielded the diastereomeric mixture of 3a and 3b which
were separated by silica gel flash chromatography
(Scheme 2). Hydrazinolysis of the phthalimide and
subsequent coupling with Fmoc-alanine using DCC and
HOBT gave the corresponding pseudopeptides 4a and
4b in good yields. Removal of the Fmoc group and
succinylation led to the target compounds 1a and 1b
which were purified by RP-HPLC (Scheme 2). The
corresponding phosphonamidate could not be obtained
Ta ble 1. Synthesis of Suc-Ala-Glyψ(PO2Et-N)Pro-Phe-pNA
1a /b, Ac-Ala-Glyψ(COCO-N)Pro-Pheψ(CH2-NH)pNA 7a /b, and
Ac-Ala-Glyψ(PO2Na-N)Pro-Phe-pCMA 12
R1
R2
R3
compounds
Et
Et
Et
Et
Et
Et
Et
Bn
Bn
Bn
Bn
H
Phe-pNA
Suc
-
1a /1b
2
-
Phe-pNA
-
3a /3b
4a /4b
5a /5b
6a /6b
7a /7b
8
9
10
11
12
Phe-pNA
-
Pheψ(CH2-NH)pNA
Pheψ(CH2-NH)pNA
Pheψ(CH2-NH)pNA
-
Phe-pCMA
Phe-pCMA
Phe-pCMA
Phe-pCMA
-
-
Ac
-
-
-
Ac
Ac
by saponification of the ethyl esters 1a and 1b. This
might be due to the sensitivity of the Phe-pNA moiety
to the harsh basic conditions employed to hydrolyze
phosphonamidic esters. Therefore, compounds 7a and
7b were prepared by a similar route starting from Pheψ-
(CH2-NH)pNA, a reduced equivalent of Phe-pNA which
has an enhanced chemical stability.25 The peptides were
capped with an acetyl group in order to avoid a possible
intramolecular hydrolysis of the acid-sensitive phos-
phonamide bond39 as observed with compounds 1a and
1b. However, we did not succeed in deprotecting the
ethyl ester of pseudopeptides 7a and 7b. Finally, we
protected the phosphonamidate moiety with a benzyl
ester which can be removed by hydrogenolysis.41 This
strategy implied a replacement of the pNA C-terminus
with a pCMA equivalent which is resistant to hydro-
genolysis and does not affect the affinity for hCyp-18.37
The phosphonamidate 11 was obtained from dibenzyl
ester 8 as depicted in Scheme 2. Due to the low chemical
stability of protonated phosphonamidates, the benzyl
phosphonamidate was selectively deprotected by hydro-

3932 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Demange et al.
Ta ble 2. Biochemical Evaluation of Pseudopeptides 1a /b, 7a /b, 12, and 13
compounds
Suc-Ala-Gly-Pro-Phe-pNA
Kd ( SD (µM)a
IC50 ( SD (µM)b
140 ( 10
210 ( 100
20 ( 5
1300 ( 200
200 ( 15
79 ( 4
127 ( 7
135 ( 20
145 ( 15
1450 ( 60
5400 ( 400
15 ( 1
Suc-Ala-Glyψ(PO2Et-N)Pro-Phe-pNA 1a
Suc-Ala-Glyψ(PO2Et-N)Pro-Phe-pNA 1b
Ac-Ala-Glyψ(PO2Et-N)Pro-Pheψ(CH2-NH)pNA 7a
Ac-Ala-Glyψ(PO2Et-N)Pro-Pheψ(CH2-NH)pNA 7b
Ac-Ala-Glyψ(PO2--N)Pro-Phe-pCMA 12
Ac-Ala-Glyψ(COCO-N)Pro-Phe-pNA 13
Suc-Ala-Ala-Pro-Phe-pNA
no inhibition at 500 µMc
30% inhibition at 500 µMc
no inhibition at 100 µMd
215 ( 40
540 ( 70
640 ( 120
Ac-Ala-Ala-Pro-Phe-pNA
a
b
Kd values were deduced from the fluorescence titration curves of the hCyp-18 Trp121 upon addition of inhibitors12,33 (n ) 2). IC50
values were obtained using the standard trypsin-coupled PPIase assay13,35,36 at 10 °C (n ) 2). c Due to the significant absorbance at 390
nm of Pheψ(CH2-NH)-pNA-containing peptides 7a /b, the IC50 values were evaluated using the standard uncoupled PPIase assay34 at 10
d
°C; the maximum inhibitor concentration employed was 0.5 mM due to residual absorbance at 246 nm. Because of its low solubility, the
maximum inhibitor concentration employed was 0.1 mM.
genolysis in the presence of sodium bicarbonate, and
compound 12 was isolated as a sodium salt and used
without further purification.41 The ketoamide analogue
13 was synthesized starting from racemic 3-aminolac-
tate as previously reported.12
inhibition of the PPIase activity was observed at 100
µM. This is probably due to the introduction of a
negative charge because N-terminal modification does
not affect the biological properties of the peptides.35
Therefore, compound 12 is likely to interact with hCyp-
18 via the Phe-pCMA moiety.37
Bioch em ica l Eva lu a tion of Com p ou n d s 1a /b, 7a /
b, 12, a n d 13. We first checked that the Ala to Gly
change at P1 did not significantly modify the affinity.
For this purpose, Suc-Ala-Gly-Pro-Phe-pNA was tested
as a ligand of hCyp-18 by fluorescence titration.12,33,37
As expected, the Kd was similar to those observed for
other Phe-pNA-terminated peptides. We have previ-
ously demonstrated that efficient binding at the S2′-
S3′ subsite of cyclophilin does not imply that the
compound is able to inhibit the PPIase activity occurring
at the S1′ subsite.37 Consequently, we tested the ability
of the peptides to inhibit the PPIase activity of hCyp-
18 using the standard trypsin-coupled assay with Suc-
Ala-Ala-Pro-Arg-pNA as a substrate.13 The Ala to Gly
substitution caused a 3-fold increase in the IC50 value
(Table 2). This might be related to the particular
behavior of the Gly-Pro moiety.20
An important question to be addressed was the real
influence of the phosphonamide motif on the potency
of the inhibitor. Therefore, compounds 7a and 7b
bearing a reduced amide at P2′ were tested as well.
Reduction of the C-terminal amide has been shown to
dramatically affect both the affinity and inhibitory
potency of model peptides.37 Not surprisingly, we noticed
that the reduction of the important Phe-pNA moiety
caused a fall in affinity by 1 order of magnitude relative
to that of the corresponding amide. However, 7b, the
reduced equivalent of 1b, still inhibited hCyp-18. The
reduction of the amide resulted in a 50-fold increase in
the IC50 value for the most active compound 7b versus
1b; this probably reflects the lack of interaction of the
compound at the S2′-S3′ subsite.37 As observed with
compounds 1a and 1b, a strong difference in efficiency
was observed for both diastereomers. These data strongly
suggest that 7b mainly binds cyclophilin via an interac-
tion at the S1′ subsite. Therefore, the Glyψ(PO2Et-N)-
Pro moiety plays a major role in the interaction. Even
though the affinity of compound 1b is modest compared
to those of nanomolar inhibitors of proteases, the results
reported herein suggest that the phosphonamide-proline
pattern might be a transition-state mimic of the PPIase
activity of hCyp-18.
To compare between phosphonamide and ketoamide,
we tested peptide 13 which contains a glycine analogue
of the ketoamide motif Alaψ(COCO-N)Pro, a canonical
transition-state mimic of PPIases.25 We were surprised
to notice that compound 13 inhibits hCyp-18 with a
relatively low efficiency (Table 2). Several reasons may
be given for this lack of potency: (i) the conformation of
the Glyψ(COCO-N)Pro moiety, related to the particular
behavior of the Gly-Pro moiety compared to other Xaa-
Pro motifs,20 and (ii) a putative tendency of the ami-
nopyruvyl motif to enolize, facilitated by the absence of
electron-donating groups at C3 of the aminopyruvate.
In conclusion, we have reported the design, synthesis,
and biochemical evaluation of Glyψ(PO2Et-N)Pro-
containing peptides as novel transition-state analogues
of the PPIase activity of hCyp18. The biochemical assays
have shown that pseudopeptide 1b is a good and
selective inhibitor of cyclophilin, whereas the Glyψ-
Pseudopeptides 1a /b, 7a /b, 12, and 13 were tested as
ligands of hCyp-18. The results, summarized in Table
2, show that peptides 1a and 13 bind the enzyme with
an affinity equivalent to that of the model substrate.
Pseudopeptide 1b is a good ligand of hCyp-18 (Kd ) 20
µM). Evaluation of the IC50 value demonstrated that
compound 1b is a good inhibitor of cyclophilin (IC50
)
15 ( 1 µM): Replacement of the amide with an ethyl
phosphonamidate surrogate resulted in an improvement
in the IC50 value by 2 orders of magnitude. Time-
dependent inhibition experiments demonstrated that
compound 1b is not a slow-binding inhibitor of cyclo-
philin because the IC50 value does not significantly
change after 2 h of preincubation. In contrast, diaste-
reomer 1a is a poor inhibitor of the PPIase activity.
Absolute configuration of the phosphorus stereogenic
center seems to be critical for the interaction, suggesting
that the ethyl phosphonamidate moiety tightly interacts
at the S1′ subsite.
Compound 1b at concentrations up to 1 mM did not
inhibit the human FK506-binding protein hFKBP-12.
Although compound 1b is slightly less potent than the
ketoamide-containing peptide developed by Schultz and
co-workers,25 it selectively inhibits hCyp-18.
We have also investigated the influence of a charged
phosphonamidate isostere (peptide 12) on the biochemi-
cal activity. Though peptide 12 binds cyclophilin, no

Glyψ(PO2R-N)Pro-Containing Pseudopeptides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18 3933
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Ack n ow led gm en t. This work was supported by
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