Focused library approach for identification of new N-acylphenylalanines as VCAM/VLA-4 antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00201-9

A structure-based focused library approach was employed in an effort to identify more lipophilic replacements for the N-benzylpyroglutamyl group of the VCAM/VLA-4 antagonist 2. This effort led to the discovery of two new classes of potent antagonists char

Full text of DOI:10.1016/S0960-894X(02)00201-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1679–1682
Focused Library Approach for Identification of New
N-Acylphenylalanines as VCAM/VLA-4 Antagonists
Li Chen,* Richard Trilles, Dorota Miklowski, Tai-Nan Huang, David Fry,
Robert Campbell, Karen Rowan, Virginia Schwinge and Jefferson W. Tilley
Roche Research Center, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ07110, USA
Received 12 December 2001; accepted 26 February 2002
Abstract—A structure-based focused library approach was employed in an effort to identify more lipophilic replacements for the
N-benzylpyroglutamyl group of the VCAM/VLA-4 antagonist 2. This effort led to the discovery of two new classes of potent
antagonists characterized by the N-(a-phenylcyclopentanoyl- and the N-(2,6-dimethylbenzoyl)-derivatives 60 and 64. # 2002
Elsevier Science Ltd. All rights reserved.
The very late antigen 4 (VLA-4, a4b1) is an integrin
receptor expressed on many lymphocytes that mediates
cell adhesion, infiltration and survival of these cell
types, when it interacts with its counter ligands, such as
VCAM-1 and fibronectin. VLA-4 antagonists that inhi-
bit the interaction of VCAM-1/VLA-4 have been inten-
sively sought for the treatment of inflammatory diseases
such as asthma.1,2
on a class of antagonists characterized by 2 and devel-
oped the hypothesis that they mimic the cyclic peptide’s
interaction with the VLA-4 integrin.4d
Compounds related to 2 are rapidly cleared when
administered iv to mice and these findings prompted us
to employ a structure-based library design in an effort
to identify a more lipophilic replacement of the N-benzyl-
pyroglutamyl moiety. Herein, we report the implementa-
tion of such an approach that led to the identification of
the new lead series characterized by 3 and 4.
Recent reports on CH–p interactions suggested that
both phenyl–phenyl and alkyl–phenyl interactions are
dominated by dispersion forces where the surface con-
tact between the CH and the aromatic ring is the major
driving force.5 Thus CH–p interactions are postulated
to play an important role in protein folding and mole-
cular recognition, as well as conformational pre-organi-
zation of small molecules. We designed compounds 3b
and 4 based on the hypothesis that an intramolecular
CH-p interaction may facilitate the N-acylphenylala-
nines to adopt a gauche (À) conformation which mimics
the core structure of potent peptide VCAM/VLA-4
inhibitors such as 1. The published X-ray crystal struc-
ture of N-phenylacetyl-l-phenylalanine (3a) features a
gauche (À) conformation of the phenylalanine and an
edge-to-face interaction between the two benzene rings
as shown in Figure 1.6 The methylene hydrogens of 3a
were morphed into a cyclopentane ring and the resulting
compound 3b was minimized using the Sybyl molecular
modeling program.7
In previous papers,3 we have described a conforma-
tional analysis and the potent VCAM/VLA-4 antago-
nist activity of cyclic peptide 1. We have also reported
*Corresponding author. Fax: +1-973-235-6084; e-mail: li.chen@
roche.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00201-9

1680
L. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1679–1682
Figure 1. (left) X-ray structure of 3a showing the CH–p interaction and
(right) an overlay of NMR structures of 1 (in green and cyan), X-ray of
3a and computer models of 3b (in orange) and 4 (in magenta).
Scheme 1. (a) Wang resin, BOP, NMP; (b) Pd(Ph3P)2Cl2. N-Bu3SnH,
HOAc, DCM, rt; (c) acid 7, BOP, DIEA, NMP, rt; (d) 25% piper-
idine, NMP, rt; (e) 90% TFA, DCM, rt.
Chart 1. VCAM/VLA-4 inhibition screening at 10 nM in ELISA
assay.
The N-acylphenylalanine focused library was designed
to quickly explore the two new classes of N-acyl groups.
As a reference set, we have also incorporated N-acyl-
prolinyl building blocks (class A). Selection of the aroyl
(class B) and the alkanoyl (class C) are based on the
desire for rapid preliminary SAR development and
availability of these building blocks fromour in house
collection (Fig. 2).
Figure 2. Building blocks of N-acyl focused library.
The synthesis of the focused library started with alloc-
and Fmoc- orthogonally protected 4-aminophenylala-
nine (5) as shown in Scheme 1. Loading 5 to Wang resin
using BOP reagent followed by removal of the alloc
group gave compound 6, which was coupled with 4-
quinolinecarboxylic acid (7) to give 8. Removal of the
Fmoc followed by BOP mediated coupling of the three
classes of building blocks (9) gave the focused library after
cleavage fromWang resin using TFA in DCM. Library
products were analyzed by HPLC using UV detection at a
wavelength of 214 nM. Products with purity greater than
80% were screened in a VCAM/VLA-4 ELISA assay
using VCAM-1 coated plates allowing VLA-4 derived
from Ramos cells to compete with test compounds at 1
and 10nM. The % inhibition of library members at 10nM
is shown in Chart 1. Compounds that showed greater than
50% inhibition at 10nM were selected for purification and
were further evaluated in the VCAM/VLA4 ELISA
assay4c to determine the IC50 values as shown in Table 1.
A
preliminary study of N-aroyl-phenylalanines4a
prompted us to consider replacement of the N-benzyl-
pyroglutamyl moiety of 2 with a 2,6-dimethylbenzyl
group as well to give compound 4. Replacement of the
benzyl group in 3a with a 2,6-dimethylbenzoyl group
and minimization gave the conformer of 4 shown in
Figures 1 and 2. In this conformation one of the methyl
groups of 4 is in close proximity to the benzene ring of
l-phenylalanine and provides a CH–p interaction that
could facilitate pre-organization of the molecule into the
desired gauche (À) conformation. An overlay of the
modeled conformations of 3b and 4 with the NMR
structure of the peptide core of 1 is shown in Figure 1.
Compounds 3b and 4 present the 5-membered ring and
an ortho-methyl group to the putative receptor hydro-
phobic site occupied by the cyclopentyl group in
compound 1.

L. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1679–1682
1681
Results fromscreening at 10 nM showed a trend in the
SAR within each building block class. As expected,
compounds which gave >50% inhibition in the screen
showed IC50 values lower than 10 nM. For analogues
derived frombuilding blocks of class A, N-acetyl- or
substituted N-acetylprolinyl was better than N-benzoyl-
prolinyl. In the case of analogues derived frombuilding
blocks of class B, it appears that an ortho-substitution is
required for good activity and substitutions at both
ortho positions are preferred. This result is in agreement
with our model in which the aromatic ring of the N-
aroyl group is preferentially aligned orthogonal to the
plane of the amide bond in order to present an ortho-
substituent as a receptor recognition element in the
space occupied by the cyclopentyl group of cyclic pep-
tide VCAM/VLA-4 antagonist 1.
Scheme 2. (a) MeOH, HCl, rt, overnight; (b) HBTU, B11 or C19,
DIEA, DMF; (c) SnCl2, DMF; (d) RCOOH, HBTU, DIEA, DMF;
(e) 1 N NaOH, EtOH; (f) 2,6-dochlorobenzoyl chloride, DIEA.
ferred gauche (À) conformation through an aromatic-
aromatic interaction. The hydrophobic group at the N-
cap region seems essential for the potency and their
spatial orientation is governed, at least in part, by the a-
substituents of the class C analogues.
Among the compounds derived from building blocks in
class C, several trends were observed. First, analogues
derived fromunsubstituted cyclic alkanoyl groups
(building blocks: C1, C4, C7, and C10) were lower in
potency compared with the corresponding a-substituted
derivatives, for example, the a-phenyl species (building
blocks: C5, C18, and C19). Secondly, in both groups,
there was a tendency for increased potency associated
with increased ring size. Thus compounds with three-
and four-membered rings were relatively weak inhibi-
tors compared to those incorporating five- and six-
membered rings, which were essentially equipotent
(building blocks: C23, and C24). Third, there appears to
be a modest substitution effect in analogues derived
Following up the results fromthis focused library, we
decided to evaluate the effect of the substituent at the
4-position of N-acyl-l-phenylananines on the N-acyl
analogues derived from a-phenylcyclopentyl-carboxylic
acid (C19) and 2,6-dimethylbenzene carboxylic acid
(B11). Their synthesis is shown in Scheme 2. The more
potent compounds were further evaluated in a cell based
assay in which the ability of compounds to inhibit the
binding of fluorescently labeled Ramos cells to human
VCAM coated 96-well plates was determined.4c
fromthe
a-(4-substituted-phenyl)cyclopentane car-
boxylic acids (C15, C16, C19, and C23), indicating a
trend for increasing potency Cl, Me<H<MeO. We
infer fromthese results that the five- and six-member
cycloakanoyl rings mimic the hydrophobic cyclopentyl
group of cyclic peptide 1, while the a-substitutions
facilitate pre-organization of the molecule into the pre-
Table 2. VCAM/VLA4 inhibition activity of focused N-acyl library
and follow up analogues in ELISA and Ramos cell based assay
Table 1. VCAM/VLA4 inhibition activity of focused N-acyl library
in ELISA assay
Compd
R
ELISA (IC50, nM)
0.37
Ramos (IC50, nM)
12
Compd
BB
%
Inhiba
IC50
(nM)
Compd
BB
%
Inhiba
IC50
(nM)
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
A1
A2
A3
A4
A5
A6
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
C1
C2
C3
18
24
37
53
61
65
13
19
31
42
45
46
61
64
68
73
74
81
12
12
15
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
C4
C5
C6
C7
C8
C9
17
19
23
25
30
33
37
49
49
49
51
53
63
64
67
68
69
72
73
73
77
55
46
57
58
59
23
1.9
2
2.1
2.7
9.8
6.1
10
590
1300
2400
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
5.8
7
8.2
2.3
6.4
9.1
7.6
5.6
3.4
3
2.4
4.6
1.4
1.2
6.6
1.7
2.9
4.7
2.5
2.2
60
64
1.7
1.2
98
81
a% Inhibition at 10 nM of library compound in VCAM/VLA-4 ELISA
assay.

1682
L. Chen et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1679–1682
The results summarized in Table 2 is in agreement with
the trend observed previously among N-benzyl-
pyroglutamyl derivatives of compound 2.4b Excellent
activity was seen with the 4-[(2,6-dichlorobenzoyl)-
amino]- group in both series as evidenced by com-
pounds 60 and 64.
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. (b) Fotouhi, N.; Joshi, P.; Fry, D.; Cook, C.; Tilley,
J. W.; Kaplan, G.; Hanglow, A.; Rowan, K.; Schwinge, V.;
Wolitzy, B. Bioorg. Med. Chem. Lett. 2000, 10, 1171. (c)
Fotouhi, N.; Joshi, P.; Tilley, J. W.; Rowan, K.; Schwinge, V.;
Wolitzy, B. Bioorg. Med. Chem. Lett. 2000, 10, 1671.
4. (a) Chen, L.; Tilley, J. W.; Huang, T.-N.; Miklowski, D.;
Trilles, R. V.; Guthrie, R. W.; Luk, K.; Hanglow, A.; Rowan,
K.; Schwinge, V.; Wolitzky, B. Bioorg. Med. Chem. Lett. 2000,
10, 725. (b) Chen, L.; Tilley, J. W.; Guthrie, R. W.; Mennona,
F.; Huang, T.-N.; Kaplan, G.; Trilles, R.; Miklowski, D.;
Huby, N.; Schwinge, V.; Wolitzky, B.; Rowan, K. Bioorg.
Med. Chem. Lett. 2000, 10, 729. (c) Chen, L.; Guthrie, R. W.;
Huang, T.-N.; Hull, K. G.; Sidduri, A.; Tilley, J. W. WO
9910312 1999; Chem. Abstr. 1999, 130, 196,952. (d) Chen, L.;
Tilley, J. W.; Trilles, R. V.; Yun, W.; Fry, D.; Cook, R.;
Rowan, K.; Schwinge, V.; Campbell, R. Bioorg. Med. Chem.
Lett. 2002, 12, 137.
Recently, the Merck group reported a class of potent
VLA4 inhibitors that contain class A type of building
blocks in the N-acylphenylalanines. In that case, the
most potent compounds were derived from the N-(3,5-
dichlorobenzenesulfonyl)-l-Prolyl group.8 It has also
been reported that A3 and A5 analogues of 4-sub-
stituted-l-phenylalanine are potent VLA-4 inhibitors.9
Most of these compounds suffered fast clearance in
rodent PK studies as we have observed on the analogues
of 2.
5. (a) Kim, E.; Paliwal, S.; Wilcox, C. S. J. Am. Chem. Soc.
1998, 120, 11192. (b) Nishio, M.; Umezawa, Y.; Hirota, M.;
Takeuchi, Y. Tetrahedron 1995, 51, 8665.
6. (a) Burley, S. K.; Petsko, G. A. Science 1985, 229, 23. (b)
Burley, S. K.; Wang, A. H.-J. Acta Crystal. 1987, 43, 1316.
7. Modeling was conducted on an Indigo 2 silicon graphis
station using Sybyl 6 (Tripos, Inc., St. Louis, MO, USA).
Minimization was done under Sybyl standard force field over
100 iterations.
8. (a) Hagmann, W. K.; Durette, P. L.; Lanza, T.; Kevin,
N. J.; de Laszlo, S. E.; Kopka, I. E.; Young, D.; Magriotis,
P. A.; Li, B.; Lin, L. S.; Yang, G.; Kamenecka, T.; Chang,
L. L.; Wilson, J.; MacCoss, M.; Mills, S. G.; Van Riper, G.;
McCauley, E.; Egger, L. A.; Kidambi, U.; Lyons, K.; Vincent,
S.; Stearns, R.; Colletti, A.; Teffera, J.; Tong, S.; Fenyk-
Melody, J.; Owens, K.; Levorse, D.; Kim, P.; Schmidt, J. A.;
Mumford, R. A. Bioorg. Med. Chem. Lett. 2001, 11, 2709. (b)
Chang, L. L.; Truong, Q.; Mumford, R. A.; Egger, L. A.;
Kidambi, U.; McCauley, E.; Lyons, K.; Riper, G.; Vincent, S.;
Schmidt, J.; MacCoss, M.; Hagmann, W. K. Bioorg. Med.
Chem. Lett. 2002, 12, 159.
In summary, we have identified two new classes of
potent VCAM/VLA-4 antagonists, 3 and 4, through a
structure-based lead generation process. The results
suggest that increased potency of the N-acylphenyl-
alanine is associated with the capability of the N-acyl
moiety to facilitate the pre-organization of the molecule
and to present a receptor recognition element mimick-
ing the cyclopentyl or thioprolyl group of the cyclic
peptide VLA-4 antagonists.3 This focused library
approach provided a fast entry into new classes N-acyl-
phenylalanine series and provides a good starting point
for the further lead optimization of these classes of
VCAM/VLA-4 antagonists. The VCAM/VLA-4
antagonist activity and the X-ray structures of the deri-
vatives of 60 and 64 will be reported in due course to
further support the SAR of these classes.10
9. (a) Archibald, S. C.; Head, J. C.; Gozzard, N.; Howat,
D. W.; Parton, T. A. H.; Porter, J. R.; Robinson, M. K.;
Shock, A.; Warrellow, G. J.; Abraham, W. M. Bioorg. Med.
Chem. Lett. 2000, 10, 997. (b) Lin, L. S.; Lanza, T., Jr.; Mac-
Cauley, E.; Van Riper, G.; Kidambi, U.; Cao, J.; Egger, L. A.;
Mumford, R. A.; Schmidt, J. A.; MacCoss, M.; Hagmann,
W. K. Bioorg. Med. Chem. Lett. 2002, 12, 133.
10. Sidduri, A.; Tilley, J. W.; Lou, J. P.; Chen, L.; Kaplan, G.;
Campbell, R.; Guthrie, R.; Huang, T.-N.; Rowan, K.;
Schwinge, V.; Renzetti, L. M. Bioorg. Med. Chem. Lett. In
Press.
References and Notes
1. Elices, M. J. In Cell Adhes. Mol. Matrix Proteins; Mouse,
S. A., Ed.; Springer: Berlin, 1999; p 133.
2. (a) Tilley, J. W.; Sidduri, A. Drugs Future 2001, 24, 985. (b)
Porter, J. R. Idrugs 2000, 3, 788. (c) Adams, S. P.; Lobb, R. R.
Prog. Respir. Res. 2001, 31, 302. (d) Boer, J.; Gottschling, D.;
Schuster, A.; Semmrich, M.; Holzmann, B.; Kessler, H. J.
Med. Chem. 2001, 44, 2586.
3. (a) Jackson, D.; Quan, C.; Artis, D. R.; Rawson, T.;
Blackburn, B.; Struble, M.; Fitzgerald, G.; Chan, K.; Mullins,