N-benzylpyroglutamyl-L-phenylalanine derivatives as VCAM/VLA-4 antagonists

Article abstract of DOI:10.1016/S0960-894X(00)00089-5

A series of N-(N-benzylpyroglutamyl)-4-substituted-L-phenylalanine derivatives was prepared as VLA-4/VCAM antagonists. Analogues substituted by electron deficient benzoylamino groups bearing bulky ortho substituents had low-nM potency in an ELISA assay an

Full text of DOI:10.1016/S0960-894X(00)00089-5

Bioorganic & Medicinal Chemistry Letters 10 (2000) 729±733
N-Benzylpyroglutamyl-L-phenylalanine Derivatives as
VCAM/VLA-4 Antagonists
Li Chen, Je€erson W. Tilley,* Robert W. Guthrie, Francis Mennona, Tai-Nan Huang,
Gerry Kaplan, Richard Trilles, Dorota Miklowski, Nicolas Huby, Virginia Schwinge,
Barry Wolitzky and Karen Rowan
Roche Research Center, Ho€mann-La Roche Inc., Nutley, NJ 07110, USA
Received 4 October 1999; accepted 1 February 2000
AbstractÐA series of N-(N-benzylpyroglutamyl)-4-substituted-l-phenylalanine derivatives was prepared as VLA-4/VCAM
antagonists. Analogues substituted by electron de®cient benzoylamino groups bearing bulky ortho substituents had low-nM
potency in an ELISA assay and low-mM activity in a cell based assay. # 2000 Published by Elsevier Science Ltd. All rights reserved.
Vascular cell adhesion molecule-1 (VCAM-1), a mem-
ber of the immunoglobulin (Ig) supergene family, is
expressed 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
lymphocytes including circulating eosinophils, baso-
phils, and monocytes, but not neutrophils. Antibodies
to either protein are e€ective at inhibiting leukocyte
in®ltration and preventing tissue damage in several ani-
mal 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 interac-
tions 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
which might be useful for the treatment of asthma or
rheumatoid arthritis.
exploiting promising candidate substitutions using high
throughput chemistry.
Derivatives 5±14 were prepared by acylation of the
appropriate amino acid methyl esters with N-benzyl-
pyroglutamic acid 3 and ester hydrolysis. The (2:1 cis:
trans) 4-methylcyclohexylalanine and trans-4-tert-butyl-
cyclohexylalanines required for 13 and 14 were available
from glycine imine alkylation of the appropriate cyclo-
hexylmethyl iodides.5 The 4-(phenylethyl)phenylalanine
15 was obtained from catalytic hydrogenation of the
acetylene 12. In order to investigate the potential role of
heteroatom linked substituents, we prepared the 4-
nitrobenzylether 17 via a Mitsunobu reaction (Scheme
2). Stannous chloride mediated reduction of the 4-nitro
derivative 18 gave the aniline 19 which was readily
acylated with carboxylic acids or sulfonyl chlorides
followed by ester hydrolysis to give the amides 20±23
and the sulfonamides 24 and 25 as shown in Scheme 3.
We previously reported our ®nding that certain N-acyl-
l-phenylalanine derivatives are e€ective inhibitors of
the VCAM/VLA-4 interaction.4 In this work, N-ben-
zylpyroglutamic acid emerged as a particularly favor-
able N-acyl group as demonstrated by the activities of 1
and 2 in an ELISA based VCAM/VLA-4 binding assay
(20 and 46 nM, respectively). In this paper, we focus
attention on the role of the phenylalanine ring and its
preferred substitution. The goal of this e€ort was to
scan a variety of substitution patterns with the intent of
To prepare homologous analogues, 4-(hydoxymethyl)-
S-phenylalanine 29 was synthesized by enantioselective
hydrogenation of the dehydrophenylalanine derivative
28 as shown in Scheme 4. The S-stereochemistry was
*Corresponding author. Tel.: +1-973-235-4660; fax: +1-973-235-
7122; e-mail: je€erson.tilley@roche.com
0960-894X/00/$ - see front matter # 2000 Published by Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00089-5

730
L. Chen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 729±733
assigned based on the literature precedent6 and by ana-
logy with the corresponding 3-hydroxymethyl series in
which the stereochemical outcome of the reduction was
veri®ed by crystallography. The 4-nitrophenyl ether 35
was prepared via a Mitsunobu reaction between the
alcohol 29 and 4-nitrophenol and the styrene derivative
36 was available from a 3-step sequence involving Swern
oxidation of 29 followed by a Wittig reaction and ester
hydrolysis as outlined in Scheme 5.
13 and 14, the benzene ring of the phenylalanines con-
tributes signi®cantly to potency. Among the phenyl-
alanine derivatives, comparison of 5±8 with 12, 15, 17
and 35, suggests that activity generally increases with
the size of the substituent in the 4-position. By far the
most interesting group of compounds to emerge from
this e€ort are the acylamino derivatives 20±23, each
of which have single digit nM potency in the VCAM/
VLA-4 ELISA assay. Furthermore the benzoylamino
analogues 20 and 21 are also the only compounds of this
series to block the interaction between ¯uorescently
labeled Ramos cells, which express VLA-4, with VCAM
coated microtiter plates at submicromolar concentra-
tions.
Results and Discussion
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 com-
pete 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
antibody and HRP-conjugated anti-mouse IgG: chro-
mogenic substrate (K-Blue).7 From the data summar-
ized in Table 1, it is evident from the lack of activity of
As a consequence of this initial e€ort, we chose to
expand the series of benzoylamino derivatives by the
Scheme 4. (a) TBDMS-C1, imidazole (92%), (b) LAH, Et20 (92%).
(c) Excess MnO2, 1:1 CH2C12:hexane, 6 h (86%). (d) Trimethyl Z-
phosphonogycine, tetramethylguanidine,
DuPhos, H2, MeOH, 50 psi, rt (97%), (f) HCO2NH4, MeOH, Pd(C),
(g) N-Bzl-pyroglutamic acid, HBTU, DIPEA, DMF (78%, 2 steps),
(h) 2 N HC1, MeOH (95%), (i) NaOH, EtOH, rt.
Scheme 1. (a) HBTU, DIPEA, DMF, 25 ꢀC, (b) NaOH, THF,
MeOH, 25 ꢀC.
40 ꢀC (85%), (e) Rh-
Scheme 2. (a) 4-Nitrobenzylether, DEAD, f3P, CH2C12, (b) NaOH,
EtOH.
Scheme 5. (a) DPPA, DBU, (b) NaOH, EtOH, (c) H2, Pd(C), EtOH,
(d) R2CO2H, HBTU, DIPEA, DMF, (e) DEAD, f3P, 4-nitrophenol,
(f) DMSO, oxalyl chloride, NEt3, (g) 4-nitrobenzylphosphonium bro-
mide, NEt3, DMF.
Scheme 3. (a) SnC12, EtOH, re¯ux, 50 min (75%), (b) R2CO2H,
HBTU, DIPEA, DMF, (c) R3SO2C1, pyridine, rt, d. NaOH, EtOH,
rt.

L. Chen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 729±733
731
Table 1. VCAM/VLA-4 binding inhibition of N-benzylpyroglutamyl-l-phenylalanine derivatives
Compound
R
ELISA
IC50 nM
Compound
R
ELISA
IC50 nM
Ramos cell
IC50 nM
5
460
20
6.3
310
6
7
160
560
21
22
2.2
8.2
560
2100
8
1000
23
5.3
1900
9
59
24
25
17
20
16,000
26.000
10
230
11
12
220
53
30
32
152
74
Ð
Ð
13
42,500
33
53
Ð
14
15
558% @ 250,000
34
35
20
32
Ð
Ð
38
17
18
36
90
Ð

732
L. Chen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 729±733
solid-phase synthesis of approximately 100 analogues.
These compounds were initially screened at concentra-
tions of 10 and 100 nM and only those combining
ꢁ60% inhibition of binding at 10 nM with ꢁ90% inhi-
bition at 100 nM were puri®ed or resynthesized for IC50
determination. Compounds thus characterized are
shown in Table 2. A number of these compounds have
low-nM IC50s in the ELISA assay and low nM activity
in the Ramos cell assay as well. Features common to the
most potent compounds include electron de®cient aro-
matic rings and at least one bulky ortho substituent.
The data suggest that the presence of these elements
is more important than their identity. For example the
2-nitro- (37), the 2-methoxy-5-nitro- (40) and the 2,6-
dichloro- (44) analogues all have similar pro®les.
One remarkable observation from this work is that vir-
tually all members of the N-benzylpyroglutamyl-4-sub-
stituted-l-phenylalanine class investigated e€ectively
inhibit the VCAM/VLA-4 interaction in our ELISA
assay with submicromolar IC50s. The preferred sub-
stitution is an electron de®cient, ortho-substituted ben-
zoylamino- moiety in the 4-position. We speculate that
the VLA-4 recognition elements present in these mole-
cules include the p-electron systems of both the benzoyl
and the phenylalanine aromatic rings as well as a
hydrogen bonding interaction with the carboxamido
group in the 4-position. The requirement for high
potency becomes evident when one considers the cell
binding results, in which it is necessary to block many
VCAM-VLA-4 interactions simultaneously along the
Table 2. Inhibition of VCAM/VLA-4 binding induced by N-benzylglutamyl-4-acylamino-l-phenylalanines
Compound
R
ELISA
IC50 nM
Ramos cell
IC50 nM
Compound
R
ELISA
IC50 nM
Ramos cell
IC50 nM
37
0.87
0.74
33
46
0.75
1.6
28
38
39
77
47
48
127
0.26
0.82
11
28
2.4
2.4
33
40
41
49
50
102
0.37
7
4.0
1850
42
43
4.6
3.5
260
51
52
3.6
1.4
260
80
1060
44
45
0.37
1.1
12
54
53
0.88
29

L. Chen et al. / Bioorg. Med. Chem. Lett. 10 (2000) 729±733
733
Ramos cell-microtiter plate boundary. We have employed
compounds from this work, particularly the 2,6-
dichlorobenzoyl analogue 44 to initiate studies of their
potential role as therapeutics in vivo.
Sprague, A. G.; Abraham, W. M.; Gill, A.; Lobb, R. A.;
Adams, S. P. J. Med. Chem. 1999, 42, 920.
3. Lin, K.-C.; Castro, A. C. Curr. Opin. Chem. Biol. 1998, 2,
453.
4. Chen, L.; Tilley, J. W.; Huang, T.-N.; Miklowski, D.;
Trilles, R.; Guthrie, R. W.; Luk, K.; Hanglow, A.; Rowan, K.;
Schwinge, V.; Woltizky, B. Bioorg. Med. Chem. Lett. 2000, 10,
725.
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