The design and synthesis of potent cyclic peptide VCAM-VLA-4 antagonists incorporating an achiral Asp-Pro mimetic

Article abstract of DOI:10.1016/S0960-894X(00)00174-8

The Asp-Pro sequence of the cyclic peptide Ac-HN-Tyr-Cys*-Asp-Pro-Cys*-OH (1) could be replaced with the achiral dipeptide mimetic 1-(2-aminoethyl)cyclpentylcarboxylic acid with retention of potent inhibition of the VCAM-VLA-4 interaction. (C) Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00174-8

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1171±1173
The Design and Synthesis of Potent Cyclic Peptide VCAM±VLA-4
Antagonists Incorporating an Achiral Asp-Pro Mimetic
Nader Fotouhi,* Pramod Joshi, David Fry, Charles Cook, Je€erson W. Tilley,
Gerry Kaplan, Angela Hanglow, Karen Rowan, Virginia Schwinge and Barry Wolitzky
Roche Research Center, Ho€mann-La Roche Inc, Nutley, NJ 07110, USA
Received 17 November 1999; accepted 9 March 2000
AbstractÐThe Asp-Pro sequence of the cyclic peptide Ac-HN-Tyr-Cys*-Asp-Pro-Cys*-OH (1) could be replaced with the achiral
dipeptide mimetic l-(2-aminoethyl)cyclpentylcarboxylic acid with retention of potent inhibition of the VCAM±VLA-4 interaction.
# 2000 Elsevier Science Ltd. All rights reserved.
Vascular cell adhesion molecule-l (VCAM-l), a member
of the immunoglobulin (Ig) supergene family, is expres-
sed on activated, but not resting, endothelium. The
principal receptor for VCAM-1, the integrin very late
antigen 4 (VLA-4, a4b1), is expressed on many lympho-
cytes including circulating eosinophils, basophils, and
monocytes, but not neutrophils. Antibodies to either
protein are e€ective at inhibiting leukocyte in®ltration
and preventing tissue damage in several animal models
of in¯ammation.1 Peptides derived from the connecting
segment 1 (CS1) sequence of ®bronectin have also been
shown to block VCAM±VLA-4 interactions and to
block allergen induced airway responses in a sheep model
of asthma.2,3 Thus, we are interested in discovering orally
active VCAM±VLA-4 antagonists that might be useful
for the treatment of asthma or rheumatoid arthritis.
incorporate both a hydrophobic portion capable of
mimicking the proline interaction as well as a suitable
spacer to maintain the appropriate geometry and con-
formational constraints of the cyclic peptide ring system.
As a starting point for this e€ort, we employed a NMR
based conformational model of the ring system of 1 using
the closely related peptide 1-[(9-oxo-¯uorenone)carbonyl]-
Arg-Cys*-Asp-thiaPro-Cys*-OH.5 While the position of
the N-terminal residue was indeterminent in these
structures, the ring is well de®ned, forming essentially
two families of conformers, which di€er in the direc-
tionality of the disul®de linkage. Since the ring con-
formation seems well conserved, we proceeded by
displaying a single member of the family and manually
building in potential spacers using the molecular mod-
eling package Sybyl. Each new structure was minimized,
subjected to a short molecular dynamics run and remi-
nimized using the maximin function within Sybyl. The
suitability of individual mimetics was evaluated visually
by inspecting their overall e€ect on ring conformation.
Recent work described structure±activity work on a
series of cyclic peptide VCAM±VLA-4 antagonists
which led to the synthesis of the potent lead pentapep-
tide 1.4 This e€ort highlighted the importance of the C-
terminal carboxyl group, demonstrated that the aspartic
acid was not important for activity, and showed that the
Asp-Pro dipeptide could be replaced by linear spacers
with only a modest decrease in potency. Unpublished
results from our laboratories additionally suggested that
the proline residue contributed to binding through a
hydrophobic interaction with VLA-4. Thus, we were
intrigued by the possibility of combining these results in
the design of Asp-Pro dipeptide replacements which
As a result of this e€ort, a series of l-(aminoalk-
yl)cyclopentane carboxylic acids was designed as easily
*Corresponding author. Tel.: +1-973-235-4660; fax: +1-973-235-
7122; e-mail: nader.fotouhi@roche.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00174-8

1172
N. Fotouhi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1171±1173
Tyr, and then cyclized. Final deprotection then yielded
compound 8.
Compounds were assayed for VLA-4 antagonist activity
using a solid-phase, dual antibody ELISA in which VLA-4
derived from Ramos cells was allowed to compete for
bound recombinant human VCAM in the presence of
serial dilutions of test compound. VLA-4 bound to
VCAM- 1 was detected by a complex of anti-b1 anti-
body and HRP-conjugated anti-mouse IgG: chromo-
genic substrate (K-Blue).6 A secondary, cell based assay
was also run in which ¯uorescently labeled Ramos cells
were allowed to compete for immobilized VCAM. As the
data summarized in Table 1 indicate, 9 in which amino-
ethylcyclopentyl moiety replaces the Asp-Pro of 1 was 2-
and 15-fold more potent than the parent compound 1 in
the solid phase and cell based assays, respectively.
Figure 1. Overlay of the NMR structure of core ring system of 1-
FCA-Arg-CysÃ-Asp-thiaPro-CysÃ-OH9 (grey) and two spirocyclic sys-
tems where the Asp-Gly is substituted with 1-(aminomethyl)cyclo-
pentane carboxylic acid (light grey) or 1-(2-aminoethyl)cyclopentane
carboxylic acid (dark grey).
The similar potencies of N-acetyl derivative 9 and the
unsubstituted N-terminal amine 11 indicate that activity
among compounds in this series is relatively insensitive to
accessible, achiral dipeptide mimetics. As illustrated in
Figure 1, modeling experiments suggested that a deri-
vative incorporating 1-(2-aminoethyl)cyclopentane car-
boxylic acid linker 11 o€ered the best opportunity to
maintain the ring conformation. Shorter spacers caused
a considerable shift in the vectors and thus changed the
relationship between the critical C-terminal carboxyl
group and the N-terminal tyrosine. It is interesting to
note that while 1 has a 14-membered ring, the analogue
11 is one atom smaller.
Table 1.
The required cyclopentane carboxylic acids 2±6 were syn-
thesized as outlined in Scheme 1. Incorporation into the
cyclic peptides shown in Table 1 involved standard HBTU
mediated peptide coupling of these acids with 1-tritylcys-
teine derivatives to give the intermediates 7 (P represents
Boc or Fmoc protection) which could be cyclized by
treatment with I2 in methylene chloride-methanol. The
remaining steps required routine deprotection, coupling
with the appropriate tyrosine derivatives and ®nal
deprotection as outlined in Scheme 2. Alternatively the
linear peptide could be fully elaborated to incorporate
Solid phase assay Ramos cell assay
No.
R
X
IC50 (nM)
IC50 (nM)
1
9
Ac
Ac
Asp-Pro
1.8
0.70
200
12
10
11
12
H
H
H
5.0
0.50
19
24
480
13
14
H
H
112
3.9
Scheme 1. a. NaH, R-I, THF, 0 ꢀC. b. Br(CH2)4Br, K2CO3, CH3CN,
heat, 4 d. c. H2, 5% Rh on Alumina. d. Boc2O. e. NaOH, dioxane. f.
nBuLi, THF, 65 ꢀC, then Br(CH2)2Br. g. NaN3, DMF, 50 ꢀC, 5 h. h.
nBuLi, THF, 65 ꢀC, then Br(CH2)3Br.

N. Fotouhi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1171±1173
1173
could be the in¯uence of increased hydrophobicity or con-
formational control as a consequence of the gem-dialkyl
e€ect.
In conclusion, aminoethylcyclopentane carboxylic acid
was incorporated into 1 as an Asp-Pro mimetic. It not
only provided the proper hydrophobic contacts while
preserving the general conformation of the macrocycle,
but actually provided an enhancement in potency while
markedly simplifying the structure of the macrocyclic
system.
References and Notes
1. Elices, M. In Cell Adhesion Molecules and Matrix Proteins:
Role in Health and Diseases: Mousa, S. A., Ed.; Springer-
Verlag: 1998; pp 133±147.
Scheme 2.
2. Lin, K.-c.; Ateeq, H. S.; Hsiung, S. H.; Chong, L. T.; Zim-
merman, C. N.; Castro, A.; Lee, W.-c.; Hammond, C. E.;
Kalkunte, S.; Chen, L.-L.; Pepinsky, R. B.; Leone, D. R.;
Sprague, A. G.; Abraham, W. M.; Gill, A.; Lobb, R. A.;
Adams, S. P. J. Med. Chem. 1999, 42, 920. Lin, K.-C., Castro,
A. C. Curr. Opin. Chem. Biol. 1998, 2, 453.
substitution on the N-terminus. Comparison of a series of
analogues possessing free N-terminal nitrogen atoms, in
which the ring size is varied is in accord with our modeling
predictions. Thus, 11, which possesses a 13-membered ring,
was 10-fold more potent than the 12-member ring analo-
gue 10, which incorporates 1-(aminomethyl)cyclopentane
carboxylic acid and 20-times more potent than the homo-
logue 12, which incorporates l-(3-aminopropyl)cyclo-
pentane carboxylic acid. In order to determine whether the
cyclopentane ring was required, we prepared the analogues
13 and 14 which were constructed from 4-amino-2,2-dime-
thyl- (2) and 4-amino-2,2-diethylbutanoic acid (3), respec-
tively. Both compounds were active, although somewhat
less potent than the corresponding cyclopentyl derivative
11. The higher potency of the bulkier diethyl analogue
3. Elices, M. Curr. Opin. in Antiin¯ammat. and Immunomodu-
lat. Investigat. Drugs 1999, 1, 14.
4. Jackson, D.; Quan, C.; Artis, D. R.; Rawson, T.; Black-
burn, B.; Struble, M.; Fitzgerald, G.; Chan, K.; Mullins, S.;
Burnier, J. P.; Fairbrother, W. J.; Clark, K.; Berisini, M.;
Chui, H.; Renz, M.; Jones, S.; Fong, S. J. Med. Chem. 1997,
40, 3359.
5. Lobl, T. J.; Chiang, S.-L.; Cardarelli, P. M. WO 9200995,
1992.
6. Chen, I.; Guthrie, R.; Huang, T.-N.; Hull, K.; Sidduri, A.;
Tilley, J. W. WO 9910312, 1999.