Constraining the amide bond in N-Sulfonylated dipeptide VLA-4 antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.01.045

The integrin VLA-4 is implicated in several inflammatory disease states. In search of non-peptidic antagonists of VLA-4, rotational constraints were imposed on the amide bond of prototypical N-sulfonylated dipeptide VLA-4 antagonists. By judicious structural modification of the side chains, trisubstituted imidazoles with moderate binding potencies were obtained, for example, 19, VLA-4 IC₅₀ = 237 nM.

Full text of DOI:10.1016/j.bmcl.2008.01.045

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1688–1691
Constraining the amide bond in N-Sulfonylated
dipeptide VLA-4 antagonists
Linda L. Chang,a,* Ginger X. Yang,a Ermengilda McCauley,b Richard A. Mumford,b
John A. Schmidtb and William K. Hagmanna
aDepartment of Medicinal Chemical Research, Merck Research Laboratories, Rahway, NJ, USA
bDepartment of Immunology and Rheumatology, Merck Research Laboratories, Rahway, NJ, USA
Received 9 November 2007; revised 9 January 2008; accepted 14 January 2008
Available online 18 January 2008
Abstract—The integrin VLA-4 is implicated in several inflammatory disease states. In search of non-peptidic antagonists of VLA-4,
rotational constraints were imposed on the amide bond of prototypical N-sulfonylated dipeptide VLA-4 antagonists. By judicious
structural modification of the side chains, trisubstituted imidazoles with moderate binding potencies were obtained, for example, 19,
VLA-4 IC50 = 237 nM.
Ó 2008 Elsevier Ltd. All rights reserved.
The integrin VLA-4 (very late antigen-4; a4b1, CD49d/
CD29) is a heterodimeric cell surface glycoprotein trans-
membrane receptor.1 Expressed on all leukocytes except
platelets and neutrophils, it is a key mediator in cell–cell
and cell–matrix interactions. It is involved in leukocyte
recruitment, activation, proliferation, and differentiation
during normal and/or pathophysiological processes.
Blockade of the interaction between VLA-4 and its li-
gands, vascular cell adhesion molecule-1 (VCAM-1)
and CS-1, an alternatively spliced form of fibronectin,
may reduce the vascular extravasation of inflammatory
cells into tissues during prolonged inflammation.2
Indeed, the equivalent of this amide bond is featured in
many of the VLA-4 antagonists that have been identi-
fied, for example, 2, 3.5a,c We were interested in trans-
forming compound 1 into active compounds where the
amide bond is incorporated into various cyclic struc-
tures as shown in Figure 1. The first route (I) connected
the amide nitrogen to the C-3 position of the proline
O
I
OH
MeO
MeO
Cl
Cl
Cl
Cl
HN
O
H
N
N
O
SO2
O
OH
2
P1
Recent Phase III clinical data of natalizumab, the
humanized anti-a4 antibody, in multiple sclerosis vali-
date the potential for a4 integrin antagonists in human
disease.3 In addition, peptidyl VLA-4 antagonists have
proven effective in several animal models of inflamma-
tion and autoimmune diseases.4 These results led to
much effort in the development of small molecule
VLA-4 antagonists.5
II
O
P3
1
R
S
H
N
OH
N
SO2
1a: R = H
1b: R = (2-OMe)Ph
O
O
N
N
O
N
1c:
=
-naphthyl
R
3
O
O
H
Initial efforts at Merck led to derivatives such as 1a
(IC50 = 1.4 nM),6b 1b (IC50 = 0.64 nM),6a and 1c
(IC50 = 0.56 nM),6a all containing a central amide bond.
OH
N
N
SO2
N
OH
N
Cl
N
SO2
Cl
O
Keywords: VLA-4; Peptide bond mimetic; Bicyclic VLA-4 antagonists;
Imidazole VLA-4 antagonists.
Cl
Cl
4
5
*
Corresponding author. Tel.: +1 732 594 6465; fax: +1 732 594
5350; e-mail: linda_chang@merck.com
Figure 1.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.045

L. L. Chang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1688–1691
1689
O
a
b
c
O
N
a
OMe
b
OH
NHBn
N
Bn
COOMe
HCl.H2N
OMe
6
7
11
N3
OMe
I
d
e
NH
O
OMe
N
H
N
Bn
N
O
O
OH
Bn
O
O
HCl.H2N
MeO
9
c
8
N
SO2
+
O
Cl
O
NH
O
12
13
TfO
OMe
N
g, h
+
f
Cl
Cl
4
SO2
O
H
N
f, g
OMe
N
SO2
10
Cl
Cl
O
O
O
Scheme 1. Reagents and conditions: (a) i—MsCl, NEt3, DCM; ii—
BnNH2, 80%; (b) BrCH2CO2Me, NEt3, DMSO, rt, 12 h, 69%; (c) i—
LDA, ꢀ78 °C; ii—ZnBr2, ꢀ90 °C; iii—I2, 80%; (d) NaN3, DMF, rt,
16 h, 71%; (e) H2, Pd/C, EtOAc, 98%. H2, Pd/C, formic acid, MeOH,
99%; (f) (3,5-Cl2)C6H3SO2Cl, NEt(iPr)2, DMAP, DCM/THF (1:1),
41%; (g) NaH, THF/DMF, 51%; (h) NaOH/MeOH, 94%.
N
OH
N
SO2
Cl
14
Cl
S
d, e
5
15
Cl
Scheme 2. Reagents and conditions: (a) (C6H5)2C@NH, DCM, rt,
77%; (b) KO-t-Bu, THF, ꢀ70 °C, b-naphthyl-COCl; 2 N HCl, 92%; (c)
NMM, THF; t-Bu-OCOCl, NMM, ꢀ20 °C, 74%; (d) NH4OAc,
HOAc, 3A sieves, xylene, reflux, 16 h, 55%; (e) excess TMSI, 100 °C,
1–3 h, 40%; (f) Lawesson’s Reagent, THF, 3A sieves, reflux, 14 h. 69%;
(g) 2 equiv NaOH/MeOH, rt, 50%.
(P2) via a carbon bridge, as in 4.7 The second approach
(II) incorporated the amide bond and the b-carbon of
the P3 side chain into a 5-membered heteroaryl ring,
as in 5.8 Both approaches introduced a considerable
amount of conformational restriction to the resulting
non-peptidic entities. Herein, we describe the effects of
such constraints on receptor-ligand binding in this class
of VLA-4 antagonists.
coupling of 13 (prepared from proline t-butyl ester by
N-sulfonylation and ester hydrolysis) with 12 provided
the key intermediate 14.14 The imidazole ring formation
was achieved by treatment of 14 with acidic ammonium
acetate in refluxing xylene. Removal of the methyl ester
was best accomplished by treatment with iodotrimeth-
ylsilane, providing the imidazole 5.
Compound 4, the target from route I, contains a novel
diaza-5,5-fused ring system which provided considerable
synthetic challenges. The successful route began with the
conversion of homoallylic alcohol to the corresponding
homoallylic benzylamine 6,9 which was reacted with
methyl bromoacetate to yield the key intermediate 7
(Scheme 1). Deprotonation of 7 was followed by the
putative complexation of the resulting enolate oxygen,
amine nitrogen, and the homoallyl double bond with
zinc bromide.10 Subsequent treatment with iodine pro-
vided the substituted proline methyl ester 8, possessing
an iodomethyl group. The iodo functionality was con-
verted to an azide11 which was hydrogenated to com-
pound 9, containing the desired restricted amide bond.
Thus, the transformation from 8 to the strained, unsub-
stituted 5,5-fused ring system 9 was achieved in one pot
via reductive cyclization followed by N-debenzylation.12
The corresponding oxazole methyl ester was prepared
by treating a solution of 14 and DBU in a mixture of
carbon tetrachloride, pyridine, and acetonitrile with tri-
phenylphosphine.15 Unfortunately this compound could
not be hydrolyzed to the corresponding acid cleanly un-
der a variety of conditions. In contrast, treatment of 14
with Lawesson’s reagent followed by base hydrolysis
readily provided the thiazole 15.
Compounds 16–20 were prepared from iminoglycine 11
using sequences analogous to Scheme 2 by the appropri-
ate choice of acylating agent (in step b) and N-sulfony-
lated amino acid (in step c) for amide coupling.
Subsequently, the 3,5-dichlorobenzenesulfonyl moiety
(P1) was attached to the pyrrolidine nitrogen. Alkyl-
ation of the lactam nitrogen with the triflate of methyl
(L)-3-phenyllactate 10 (obtained from methylation of
(L)-3-phenyllactic acid and sulfonylation of the hydro-
xyl group) followed by base hydrolysis of the methyl es-
ter provided the racemate 4.
The a4b1 integrin binding affinity of the reported com-
pounds was assessed by measuring the reduction in
binding of 125I-VCAM-Ig to a suspension of Jurkat cells
in the presence of the test compound as described previ-
ously.6c All test compounds were assayed at least in
duplicate.
The imidazole 5 was prepared as shown in Scheme 2.
Glycine methyl ester was converted to its benzophenone
Schiff base 11.13 Addition of the corresponding potas-
sium enolate to a solution of b-naphthoyl chloride at
ꢀ70 °C provided the a-imino-b-keto compound which
was hydrolyzed to intermediate 12. Mixed anhydride
The data in Table 1 show that a significant amount of
binding was lost by the introduction of conformation
constraints via route I or route II (Fig. 1) relative to
the parent dipeptides (1a vs 4, and 1c vs 5, 15). Also,
N-methylation of the amide bond NAH in 1b resulted
in a compound with IC50 of 2400 nM, equivalent to a

1690
L. L. Chang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1688–1691
Table 1. VLA-4 binding affinities of prototype conformationally restrained compounds, 4, 5, 15 and reference compounds 1a, b
Compound
1a
1b
1c
4
5
15
VLA-4 IC50 (nM)
1.4
0.64
0.56
2100
8% inhibition at 100 lM
19% inhibition at 100 lM
3750-fold loss in binding affinity versus 1b. By compar-
ison, the data for 4 and 1a suggest that constraining the
P2 amide bond in a 5,5-fused system provides limited
advantage in binding. These data suggest that free
NAH is required for adequate binding with VLA-4.17
center and retains the NAH of the amide bond was cho-
sen as the platform for this study. We monitored the ef-
fects of relaxing the rigidity of the substituents around
the imidazole ring on binding affinity.
To begin, a spacer was introduced between the naph-
thyl group and the imidazole ring, as in 16 (Table 2).
This resulted in no improvement in binding affinity
(16 vs 5). The rigidity conferred by the pyrolidinyl
group of the prolyl moiety (P2) in 16 was then re-
laxed. Thus, the spatially diverse (N-methyl)leucyl
and (N-cyclohexyl)glycyl groups16 were chosen as pro-
lyl replacements while the b-naphthylmethyl group
was retained in P3 (17, 18). As shown in Table 2,
by opening up the proline ring, a compound that
was inactive at 100 lM (16) could be transformed to
a modest antagonist (18).
In compound 4, the methylene unit in P3 is freely rotat-
ing. In contrast, in compounds 5 and 15, the equivalent
P3 methylene group is incorporated into the heterocyclic
ring (cf. 1c) and the resulting structure is much more ri-
gid at that point. In fact, in these compounds, the P3
side chain extends out linearly from the heterocyclic
moiety. This rigidity contributed to the drastic loss of
potency in 5. Thus, the data from 4, 5, and 15 suggest
that in order to achieve reasonable binding with VLA-
4, the equivalent of the P2 amide bond in 1 should not
be quasi-coplanar with the P3 side chain.
We were intrigued by the possibility of transforming
inactive but non-peptide entities derived from peptides
such as 1 into moderately potent compounds. The tri-
substituted imidazole 5, which contains only one chiral
Retaining the (N-cyclohexyl)glycyl moiety in 18, we
examined 40-biphenyl groups with and without a meth-
ylene linker to the imidazole as a surrogate of the b-
naphthyl group (19, 20). The biphenylmethyl compound
19 was at least as active as the b-naphthyl compound
(18), suggesting a rather broad spatial tolerance for
hydrophobic groups beyond the methylene spacer.
Analogous to 5, compound 20 which has no spacer be-
tween the imidazole ring and the aryl substituent lost
all activity. This underlines the importance of the meth-
ylene spacer to provide the necessary flexibility to P3
and to confer activity thereby.
Table 2. VLA-4 binding affinities of conformationally restrained
imidazoles 16–20
O
H
N
N
SO2
OH
N
Cl
R
Taken together, these data show that reasonable flexibil-
ity built into both the P2 and the P3 moieties of 5 is re-
quired in order to have a reasonable affinity for the
VLA-4 receptor. This flexibility compensates for the
constraint inherent in the imidazole ring compared to
an amide bond. In this manner, these dipeptide-derived
imidazoles can be transformed from totally inactive enti-
ties to submicromolar VLA-4 antagonists. Our data
show that this can be achieved while retaining the struc-
tural characteristics of the side chains present in the ori-
ginal capped dipeptide lead.
Cl
Compound
R
VLA-4
IC50 (nM)
N
16
17
30% inhibition
at 100 lM
N
Me
Me
2535
Me
Many medicinal chemistry leads contain at least one
amide bond. As lead optimization progresses, non-pep-
tide entities are often sought. This investigation demon-
strates that, in a series of N-sulfonylated dipeptide
VLA-4 antagonists, an imidazole has the potential to
act as an amide bond surrogate when care is taken to
introduce adequate flexibility to its substituents and to
array them with appropriate binding motifs.
N
18
19
20
285
237
N
N
N
Acknowledgment
45% inhibition
at 100 lM
We thank Ms. Amy Bernick for mass spectral analysis.

L. L. Chang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1688–1691
1691
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