N-(arylacetyl)-biphenylalanines as potent VLA-4 antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00366-9

A series of potent N-(aralkyl-, arylcycloalkyl-, and heteroaryl-acyl)-4-biphenylalanine VLA-4 antagonists was prepared by rapid analogue methods using solid-phase chemistry. Further optimization led to several highly potent compounds (IC(50) <1 nM). Evalu

Full text of DOI:10.1016/S0960-894X(02)00366-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2141–2144
N-(Arylacetyl)-biphenylalanines as Potent VLA-4 Antagonists
Bing Li,a,* Stephen E. de Laszlo,a Theodore M. Kamenecka,a Ihor E. Kopka,a
Philippe L. Durette,a Thomas Lanza, Jr.,a Malcolm MacCoss,a Sharon Tong,a
Richard A. Mumford,b Ermengilda D. McCauley,b Gail Van Riper,b John A. Schmidtb
and William K. Hagmanna
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Inflammation and Rheumatology Research, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 28 January 2002; accepted 6 May 2002
Abstract—A series of potent N-(aralkyl-, arylcycloalkyl-, and heteroaryl-acyl)-4-biphenylalanine VLA-4 antagonists was prepared
by rapid analogue methods using solid-phase chemistry. Further optimization led to several highly potent compounds (IC50 <1
nM). Evaluation of rat pharmacokinetic revealed generally high clearance. # 2002 Elsevier Science Ltd. All rights reserved.
VLA-4 (a4b1; CD49d/CD29) is a heterodimeric integrin
expressed on the surface of lymphocytes.1 Its ligands
include vascular cell adhesion molecule-1 (VCAM-1),
which is expressed on activated endothelial cells at sites
of inflammation and produced in response to inflammatory
cytokines, and an alternatively spliced (CS-1) domain of
fibronectin (FN). The adhesion of lymphocytes to
VCAM-1 and subsequent migration to extravascular
spaces appear to be critical steps in the inflammatory
response.2 Several reports have demonstrated that
blocking this interaction in animals with a4 antibodies
or VLA-4 antagonists can have therapeutic utility in a
variety of disease models, including models of airways
hyperresponsiveness, inflammatory bowel disease,
multiple sclerosis, and arthritis.3ꢀ6
related integrin, a4b7.12,14 Acyl components could be
relatively small and include N-benzylpyroglutamate,
substituted benzoyls, and tetrahydrofuroyl.
In our search for unique leads that retained potency and
had improved pharmacokinetic characteristics, a solid-
phase chemistry strategy for the synthesis of single
compounds was pursued. (l)-20-Methoxy-4-biphenyl-
alanine had been identified as a potency enhancing acid
component and was initially chosen to load on the resin.
For the acyl component, commercially available car-
boxylic acids were chosen from among substituted aryl
acetyls. The corresponding benzoyl derivatives have
previously been disclosed.15
The acids were incorporated via amide bond coupling to
the derivatized resin to afford the desired products after
cleavage from the resin (Scheme 1). (l)-4-Iodophenyl-
alanine t-butyl ester was reacted with FmocCl in the
presence of diisopropylethylamine to give N-Fmoc-(l)-
4-iodophenylalanine t-butyl ester which was reacted
with 2-methoxyphenylboronic acid under Suzuki aryl
coupling conditions to yield (l)-4-(20-methoxy)-
biphenylalanine t-butyl ester.16 The ester was removed
by treatment with trifluoroacetic acid to afford the free
acid which was subsequently loaded onto a Wang resin
using standard conditions.12 Approximately 3 g of the
free acid gave about 3 g of loaded resin which was
enough to prepare ꢁ100 compounds. The N-Fmoc
protecting group on the resin-loaded amino acid was
VLA-4 has been shown to bind to the sequences
-Ile-Asp-Ser- (-IDS-) in the C-D loop of VCAM-1 and
-Leu-Asp-Val- (-LDV-) in the CS-1 domain of FN.7,8 It
is believed that the center aspartic acid makes an essential
binding contact with VLA-4. In our search for unique
antagonists of VLA-4, we and others have reported
series of substituted acylated and sulfonylated phenyl-
alanine derivatives as potent VLA-4 antagonists.3,9ꢀ14
Substituted biphenylalanines emerged as one potency
enhancing pharmacophore for both VLA-4 and the
*Corresponding author. Fax: +1-732-594-6090; e-mail: bing_li@
merck.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00366-9

2142
B. Li et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2141–2144
Table 1. Inhibition of VLA-4 by substituted arylacetyl biphenylala-
nine derivatives (IC50, nM)
No.
R
H
HPLC VLA-4 No.
Purity%
R
HPLC VLA-4
Purity%
1
92
262
12
95
607
2
3
85
92
264
315
13
14
95
84
44.9
4.8
Scheme 1. (a) FmocCl, DIEA, CH2Cl2; (b) 2-OCH3Ph–B(OH)2,
Pd(PPh3)4, KBr, Na2CO3, toluene; (c) TFA, CH2Cl2; (d) Wang resin,
2.0 equiv EDC, 1.0 equiv DMAP; (e) 4.0 equiv AcOH, 4.0 equiv EDC,
2.0 equiv DMAP; (f) 20% piperdine, DMF; (g) 4.0 equiv acid, 4.0
equiv HBTU, 4.0 equiv HOBt, 5.0 equiv DIEA; (h) 95% TFA/5%
water.
4
84
328
15
81
64.9
5
6
7
8
86
92
89
84
36.8 16
8.0 17
84
87
89
93
246
removed by treatment with 20% piperidine in DMF
followed by coupling with the acid component. After
washing, the desired product was removed from the
resin with trifluoroacetic acid/water (95:5). The solvents
were removed in vacuo and the products characterized
by HPLC and mass spectrometry. The inhibition of
VLA-4 binding to a soluble radiolabeled VCAM-1-Ig
fusion protein12 by these compounds was listed in Table 1.
129
801
16.6 18
145
19
15.8
The addition of a phenyl ring 2 onto the acetamide 1 had
little effect on potency. Only when the conformation
about the a-carbon was changed by ortho substitution
(5, 6, 7, and 14) did potency improve. Potency was also
greatly enhanced by replacement of phenyl with a hetero-
aryl. Interestingly, all three positional isomers of pyridine
(10, 19, and 20) were equipotent. Tetrazolyl- (11) and
dichloro-imidazolyl- (21) acetyl derivatives were also
more potent than the phenyl derivative (2).
9
87
87
81
30.9 20
12.9 21
14.4
92
85
12.2
10
11
6.15
Conformational constraints offered by a-substitution on
the phenylacetyl group also significantly improved
VLA-4 potency (Table 2). The binding appeared to be
sensitive to the bond angle between the phenyl carbon
and carbonyl carbon at a-position noted in Table 2. The
methoxy compound (22) may have another specific
binding interaction between VLA-4 and the methoxyl.
products. The nature of substitution on the pyridyl ring
as well as other heteroaryls was explored and results
were listed in Table 3. Activity of this series against the
related a4b7 integrin was also included.
It appeared from the results in Table 3 that the unsub-
stituted 3-pyridyl 27 or equivalent 4-pyridazinyl 35
derivatives were the most potent VLA-4 antagonists of
this series of heteroaryls. Interestingly, the nature of the
specific binding interaction of the 3-pyridyl was not
entirely clear. Whereas the pyridyl nitrogen might be
expected to be a hydrogen bond acceptor, the pyridones
29 and 30 were also very potent. Likewise, with the
possible exception of isoquinolinyl 37, most substitu-
tions and positional isomers had comparable potencies.
If there was a specific polar interaction of VLA-4 with
The obvious hybrid molecules incorporating the
potency enhancing effects of phenyl to 3-pyridyl as well
as methylene to cyclopentyl were prepared. The desired
3-pyridyl cycloalkane and heterocycle derivatives were
prepared as illustrated in Scheme 2. Ethyl 3-pyridyl
acetate was treated with excess potassium hydride fol-
lowed by alkylation with a dihaloalkane to afford the
cyclized acetate. Saponification of the ester followed by
standard coupling conditions with (l)-4-(20-methoxy)
biphenylalanine, t-butyl ester afforded the desired

B. Li et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2141–2144
2143
Table 2. Inhibition of VLA-4 by substituted phenylacetyl biphenyl-
alanine derivatives (IC50, nM)
Table 3. Inhibition of VLA-4 and a4b7 by heteroaryl cyclopentyl
acetyl biphenylalanine derivatives (IC50, nM)
No.
R
VLA-4
3.2
a4b7
No.
X
VLA-4
264
a*
No.
X
VLA-4
90.7
a*
25
110
2
111.4
24
115.5
27
28
29
30
31
32
33
34
35
36
37
38
39
40
0.5
3.2
1.4
2.1
1.5
1.2
1.3
1.6
0.5
3.0
9.8
3.9
0.7
0.8
9.9
180
26
22
23
7.1
110.3
108.9
25
26
3.2
6.5
106.6
106.6
14.1
*a represents degree of phentl carbon–carbon–carbonyl carbon angle.
18
44
22
36.3
165
33
Scheme 2. (a) 3 equiv KH; Br(CH2)4Br, THF; (b) NaOH, CH3OH; (c)
(l)-4-(20-methoxy)biphenylalanine, t-butyl ester, HBTU, HOBt,
DIEA, CH2Cl2; (d) TFA, CH2Cl2.
this portion of the molecule, it appeared to be fairly
nonspecific or was spread over an area that was not
unidirectional.
215
120
98
Most of the analogues in Table 3 were not very specific
for VLA-4 over a4b7. The most specific compound was
the 5-bromo analogue 34 (ꢁ100ꢂ). In an attempt to
improve specificity while retaining potency, the nature
of the spirocyclic ring was explored with substituents
and heterocyclic replacements. These derivatives were
prepared in an analogous fashion to 27 (Scheme 2).
Inhibition data was reported in Table 4.
49
As can be seen in Table 4, modification of the spiro-
cyclopentyl portion of 27 had little effect on VLA-4
potency or specificity with respect to a4b7. There was
ꢁ10-fold increase in specificity with the pyranyl analogue
44. The major contribution of the spirocyclic portion of
the molecule might be largely conformational.
15
spirocycle resulted in high plasma clearance rates and
generally poor oral bioavailability. It was hoped that if
the pyridyl nitrogen in 27 was a target for metabolic
oxidation that the ortho methyl substitution in 31 and
33 might reduce this metabolism. The data clearly
showed that this was not the case.
The pharmacokinetic (PK) properties of several VLA-4
antagonists from this study were measured in rats
(Table 5). Whereas the unsubstituted phenyl (23 and 25)
and pyridyl (27) derivatives had very promising PK
characteristics, any change to the heterocycle or the

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B. Li et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2141–2144
Table 4. Inhibition of VLA-4 and a4b7 by substituted 3-pyridylacetyl
biphenylalanine derivatives (IC50, nM)
Acknowledgements
The authors are grateful to Song Zheng and Junying
Wang for formulation and mass spectral analysis, to
Marcie Donnelly and Judy Fenyk-Melody for dosing of
animals for pharmacokinetic evaluation and to Laurie
Castonguay for modeling work.
No.
R1/R2
VLA-4
0.5
a4b7
References and Notes
27
9.9
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47
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aMixture of four isomers.
Table 5. Pharmacokinetic parameters of selected compoundsa
No.
Fb (%)
Clp (mL/kg/min)
t1/2c (h)
23
25
27
29
31
32
33
36
39
43
44
94
55
35
1
11
1
12
<1
17
6
7.5
21.5
24
2.0
1.0
1.1
NDd
0.6
0.2
>100
>100
>100
>100
>100
>100
82
0.3
ND
2.5
0.4
<1
>100
ND
aSprague–Dawley rats.
bDose: 1 mg/kg iv; 2 mg/kg po.
ct1/2=plasma half-life(0ꢀ8 h)
.
dND, not determined.
In summary, a series of potent N-(aralkyl-, arylcyclo-
alkyl-, and heteroaryl-acyl)-4-biphenylalanine VLA-4
antagonists was prepared by rapid analogue methods
using solid phase chemistry. Further optimization led to
several highly potent compounds (IC50 <1 nM). Eva-
luation of rat pharmacokinetics revealed generally high
clearance.
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Nomura, S.; Jayakumar, H.; Teegarden, B.; Nowlin, D. M.;
Caradelli, P. M.; Mah, J. R.; Connell, S.; Griffith, R. C.;
Lazarides, E. A. Bioorg. Med. Chem. 2002, 12, 2051.
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