Cell adhesion antagonists: Synthesis and evaluation of a novel series of phenylalanine based inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00300-0

Several phenylalanine based inhibitors were synthesized as antagonists of the leukocyte cell adhesion process that is mediated through the interactions of the mucosal addressin cell adhesion molecule (MAdCAM) and the intgerin α4β7. Analogues 20, 21, 22 and 24 displayed inhibition of adhesion in a cell based assay in the low micromolar range. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00300-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1497±1499
Cell Adhesion Antagonists: Synthesis and Evaluation of a
Novel Series of Phenylalanine Based Inhibitors
Geraldine C. B. Harriman,* Charles F. Schwender, Debra Gallant,
Nancy A. Cochran and Michael J. Briskin
Millennium Pharmaceutical, 75 Sidney St, Cambridge, MA 02139, USA
Received 19 July 1999; accepted 17 December 1999
AbstractÐSeveral phenylalanine based inhibitors were synthesized as antagonists of the leukocyte cell adhesion process that is
mediated through the interactions of the mucosal addressin cell adhesion molecule (MAdCAM) and the integrin a4b7. Analogues
20, 21, 22 and 24 displayed inhibition of adhesion in a cell based assay in the low micromolar range. # 2000 Published by Elsevier
Science Ltd.
Circulating leukocytes home to speci®c tissues through a
complex series of receptor±ligand interactions.1,2 The
multistep process that leads to the in®ltration of leukocytes
into target tissues involves multiple interactions, which
include the association of carbohydrates with selectins,
chemokine receptors with chemokines, and integrins with
immunoglobulin-like (Ig-like) cell adhesion molecules.
Over expression of the tissue speci®c cell adhesion
molecule MAdCAM (mucosal addressin cell adhesion
molecule) results in the increased in®ltration of lympho-
cytes bearing the a4b7 integrin.3 6 This phenomenon is
associated with tissue damage in both murine and non-
human primate models of in¯ammatory bowel disease
(IBD) where anti-a4b7 mAbs have shown ecacy.7,8 In
our e€orts to develop small molecule antagonists of
leukocyte tracking,9 11 we identi®ed 1 as an inhibitor
of cell adhesion mediated by the leukocyte cell surface
integrin a4b7 and the cell adhesion molecule MAd-
CAM. Using a cell based adhesion assay, 1 was found
to inhibit adhesion at an IC50 of 54 mM. Previously, a
series of phenylalanines has been shown to e€ectively act
as adhesion antagonists of ®brinogen, where their utility
as integrin recognition motif mimics were described.12
We herein report on the chemistry involved in our opti-
mization e€orts in this series of novel inhibitors.
aldehyde a€orded benzyl amines 2±13. The aldehydes
chosen were a group of hydrogen bond acceptor±donor
substituted benzaldehydes. The relative pKa of the phe-
nolic proton was decreased by the addition of a nitro
group to the aromatic ring, which resulted in a 2-fold
increase in potency, (Table 1, 9 vs 11).
Optimization of the ester functionality was then investi-
gated keeping the N-2-hydroxy-5-nitrobenzyl moiety on
the molecule constant. The synthesis of these compounds
began with the esteri®cation of N-BOC-phenylalanine
(14) with a variety of alcohols in the presence of HBTU
and DIEA (Scheme 2).14,15 This was subsequently followed
by the facile removal of the t-butoxycarbonyl group in 4
M HC1 in dioxane a€orded esters 15±19.
Optimization of inhibitory activity in the series began with
the synthesis of a series of t-butyl N-benzylphenylalanines
(Scheme 1). Reductive amination utilizing commercially
available t-butyl phenylalanate using standard Borch
alkylation conditions13 in the presence of the appropriate
Reductive amination using Borch conditions13 in the pre-
sence of 2-hydroxy-5-nitrobenzaldehyde a€orded the
products 20±23. In order to achieve a more stable mole-
cule for future in vivo lymphocyte recruitment studies, the
isopentyl amide 24 was synthesized using the aforemen-
tioned protocol. The amide proved to be equiactive to
the corresponding ester 20, Table 1.
*Corresponding author. Tel.: +1-617-551-3638; fax: +1-617-551-8911.
0960-894X/00/$ - see front matter # 2000 Published by Elsevier Science Ltd.
PII: S0960-894X(00)00300-0

1498
G. C. B. Harriman et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1497±1499
Scheme 1.
Table 1. Inhibition of a4 b7/MAdCAM mediated cell adhesion16
synthesized and was inactive in the cell adhesion assay.
Stereochemical integrity was evaluated by creating cor-
responding chiral phenolic ester of 27.
Cell Adhesion Assay
A cell adhesion assay involving the B-cell lymphoma,
RPMI 8866 cells, and soluble human MAdCAM-IG
chimera was used in a 96-well plate format. RPMI 8866
cells have previously been shown to bind avidly to
MAdCAM.
Compound
R1
R2
IC50 (mM)
1
3
4
5
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
3-Hydroxyphenyl
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
2-Methoxyphenyl
54
117
176
121
50
6
RPMI 8866 cells were ¯uorescently labeled by pre-
incubation with BCECF stain (Molecular Probes),
washed, and resuspended in assay bu€er containing 2%
fetal calf serum and 2 mM Mg2+. Compounds were
tested in HBSS/2% FCS/25 mM HEPES bu€er at 2.5
106 cells per mL. The typical assay consisted of a ®nal
volume of 200 mL containing 50 mL of cells at 1.25 105
cells per well. Adhesion assays for MAdCAM were
washed on an automatic plate washer using a bu€er
consisting of 50 mM Tris/150 mM NaCl/2 mM MnCl2,
pH 7.2, in a wash volume of 500 mL for two wash cycles.
Assays were then read on an Idexx ¯uorescent plate
reader at 485/535 Zm. Inhibition was determined by the
number of cells adhering to the MAdCAM lined plates
in the presence and absence of an inhibitor and IC50
values were determined using Kaleidagraph (Adelbeck
Software) and are reported as an average of several
determinations.
7
8
9
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
Isopentyl
Benzyl
3-Methoxyphenyl
4-Methoxyphenyl
2-Hydroxyphenyl
4-Hydroxyphenyl
122
122
60
85
30
100
Inactive
7
10
11
12
13
20
21
22
23
24
27
2-Hydroxy-5-nitrophenyl
Phenyl
3-Hydroxy-4-nitrophenyl
2-Hydroxy-5-nitrophenyl
2-Hydroxy-5-nitrophenyl
2-Hydroxy-5-nitrophenyl
2-Hydroxy-5-nitrophenyl
7
9
82
Neopentyl
Isopentenyl
Isopentyl aniide 2-Hydroxy-5-nitrophenyl
Isopentyl ``D'' 2-Hydroxy-5-nitrophenyl
6
Inactive
The stereospeci®c interaction of the inhibitor with the
protein is illustrated by the synthesis and evaluation of
the unnatural `D' isomer 27, Scheme 3. Using the stan-
dard protocol described above the enantiomer of 24 was
Scheme 2.

G. C. B. Harriman et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1497±1499
1499
Scheme 3.
References and Notes
9. Carson, K. G.; Schwender, C. F.; Shro€, H. N.; Cochran,
N. A.; Gallant, D. L.; Briskin, M. J Bioorg. Med. Chem. Lett.
1997, 7, 711.
10. Schro€, H. N.; Schwender, C. F.; Baxter, A. D.; Brook-
®eld, F.; Payne, L. J.; Cochran, N. A.; Gallant, D. L.; Briskin,
M. J. Bioorg. Med. Chem. Lett. 1998, 8, 1601.
11. Shro€, H. N.; Schwender, C. F.; Dottavio, D.; Yang, L.;
Briskin, M. J. Bioorg. Med. Chem. Lett. 1996, 6, 2495.
12. Alig, L.; Edenhofer, A.; Hadvary, P.; Hurzeler, M.;
Knopp, D.; Muller, M.; Steiner, B.; Trzeciak, A.; Weller, T. J.
Med. Chem. 1992, 35, 4393.
1. Butcher, E. C. Cell 1991, 67, 1033.
2. Springer, T. A. Cell 1994, 65, 301.
3. Brezinschek, R. I.; Brezinschek, H.-P.; Lazarovits, A. I.;
Lipsky, P. E.; Oppenheimer-Marks, N. Am. J. Pathol. 1996, 149,
1651.
4. Berlin, C.; Berg, E. L.; Briskin, M. J.; Andrew, D.; Kil-
shaw, P. J.; Holzmann, B.; Weissman, I. L.; Hainann, A.;
Butcher, E. C. Cell 1993, 74.
5. Bargatze, R. F.; Jutila, M. A.; Butcher, E. C. Immunity
1995, 3, 99.
6. Briskin, M.; Winsor-Hines, D.; Shyan, A.; Cochran, N.;
Bloom, S.; Wilson, J.; Butcher, E. C.; Kassam, N.; Mackay, C.
R.; Newman, W.; Ringler, D. J. Am. J. Pathol. 1997, 151, 97.
7. Picarella, D.; Hurlbut, P.; Rottman, J.; Shi, X.; Butcher, E.;
Ringler, D. J. Immunol. 1997, 158, 2099.
8. Hesterberg, P. E.; Winsor-Hines, D.; Briskin, M. J.; Soler-
Ferran, D.; Merrill, C.; Mackay, C. R.; Newman, W.; Ringler,
D. J. Gastroenterology 1996, 111, 1373.
13. Borch, R.; Bernstein, M.; Durst, H. J. Am. Chem. Soc.
1971, 93, 2897.
14. Dourtoglou, V.; Gross, B. Synthesis 1984, 572.
15. Hudson, D. J. Org. Chem. 1988, 53, 617.
16. Compounds were analyzed via LC/MS (with diodearray
1
detection), H NMR, 13C NMR, and elemental analysis. All
compounds assayed were >95% pure.