2,3-Diphenylpropionic acids as potent VLA-4 antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.09.089

2,3-Diphenylpropionic acid derivatives as highly potent VLA-4 antagonists are described. One representative compound, 9cc has inhibited intercellular adhesion by a VCAM-1/VLA-4 interaction with an IC₅₀ of 1.7 nM, and has good pharmacokinetics a

Full text of DOI:10.1016/j.bmcl.2004.09.089

Bioorganic & Medicinal Chemistry Letters 15 (2005) 217–220
2,3-Diphenylpropionic acids as potent VLA-4 antagonists
Yoichiro Hoshina,a Satoru Ikegami,a,* Akihiko Okuyama,a Hideto Fukui,a
Kiyoshi Inoguchi,a Tatsuya Maruyama,a Kyoko Fujimoto,a Yuzuru Matsumura,a
Akinori Aoyama,a Tatsuhiro Harada,a Hiroshi Tanakab and Tsutomu Nakamuraa
aCentral Research Laboratories, Kaken Pharmaceutical Co., Ltd, 14 Shinomiya-minamikawara, Yamashina, Kyoto 607-8042, Japan
bDepartment of Applied Chemistry, Graduate School of Science and Engineering, Tokyo Institute of Technology,
2-12-1 Ookayama, Meguro, Tokyo 152-8552, Japan
Received 19 August 2004; accepted 30 September 2004
Available online 22 October 2004
Abstract—The discovery and SAR of 2,3-diphenylpropionic acid derivatives as highly potent VLA-4 antagonists are described. One
representative compound, 9cc has inhibited intercellular adhesion by a VCAM-1/VLA-4 interaction with an IC50 of 1.7nM, and has
good pharmacokinetics and oral bioavailability.
Ó 2004 Elsevier Ltd. All rights reserved.
The integrin very late antigen-4 (VLA-4) is a hetero di-
meric adhesion molecule (a4b1), expressed on the surface
of leukocytes, including T-lymphocytes and eosinoph-
ils.1 These leukocytes play key roles in inflammation
and autoimmune diseases, and VLA-4 is involved in
the cell adhesion, migration and activation of these cell
types.2 One of the ligands for VLA-4 is vascular cell
adhesion molecule-1 (VCAM-1), which is expressed on
activated endothelial cells at sites of inflammation, and
the other is the connecting segment 1 (CS-1) domain
of the extracellular matrix protein fibronectin.3 Interac-
tion between VLA-4 and VCAM-1 initiates firm adhe-
sion of the leukocytes to the vascular endothelium
followed by extravasation into inflamed tissues.4 Anti-
bodies to the a4 subunit of VLA-4 are reported to be effi-
cacious in inhibiting leukocyte infiltration and then
preventing tissue damage in several animal models of
inflammation. These experimental results jointly indi-
cate that a small molecule capable of antagonizing the
action of VLA-4 can be useful in the treatment of
chronic inflammatory diseases such as asthma,5 multiple
sclerosis,6 and rheumatoid arthritis.7 Therefore, we
launched a program to create a small molecule VLA-4
antagonist aimed at the development of a new medicine
for the previously mentioned diseases.8
Our anti-VLA-4 project initially started by working on
peptidemimetic-based drug discovery, as we could have
successfully utilized that technology in a previous study
of growth hormone secretagogues.9 We could generate
several lead compounds10 through the 3D-pharmaco-
phore development and the 3D-database searching tech-
nique using various peptide-like VLA-4 antagonists11 as
initial informative sources, but most of which showed
low oral bioavailabilities and unsuitable pharmacoki-
netic properties. Since our objective was to obtain orally
active VLA-4 antagonists, we redirected our focus to
phenylalanine derivatives such as 1 (TR-14035)12 and
28g shown in Figure 1.
In order to reduce the disadvantage intrinsically-linked
to the peptidyl features of these compounds, we de-
signed 2-(3-carbonylamino)phenyl-3-phenylpropionic
acids 3 as a new template. We postulated that the ben-
zene ring A would adequately serve as the peptide back-
bone of either 1 or 2, and that the tert-amide group13 at
the 30-position would be able to work as the N-terminal
variations. In addition to that, suitable substituent(s)
could also be introduced as R1 and/or R2 to this benzene
ring A for further manipulation. Taken together, we ex-
pected that the 2,3-diphenylpropionic acid derivatives
would have a potent VLA-4 antagonistic activity and
that a better oral bioavailability and metabolic stability
as a result of its nonpeptidyl structure would be
achieved.
Keywords: VLA-4; VCAM-1; 2,3-Diphenylpropionic acid.
*
Corresponding author. Tel.: +81 75 594 0787; fax: +81 75 594
0790; e-mail: ikegami_satoru@kaken.co.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.089

218
Y. Hoshina et al. / Bioorg. Med. Chem. Lett. 15 (2005) 217–220
Based on the aforementioned procedure, we were able to
synthesize many compounds with a great diversity of
structures.
H3CO
CO2H
Cl
H
N
OCH3
O
Cl
Cl
O
Br
O
Compounds were assayed for their ability to inhibit the
binding of VLA-4-expressing human leukemia cells
(HL-60) to human VCAM-1 expressed on Chinese ham-
ster ovary (CHO) cells.15 The IC50 values of the rela-
tively simple compounds with no substituents at
the R1 and R2 positions are summarized in Table 1.
N
H
N
H
CO2H
Cl
CH3
1 (TR-14035)
2
R5
B
R4
O
The comparison among 9a, 9b, and 9c clearly showed
that the R3 substituent was crucial for the VLA-4 antag-
onistic activities and that the isobutyl group, the bulkier
one, was much better than the methyl group. The same
tendency was also observed when the R4 group
was changed from benzyl to cyclohexyl (9d and 9e).
Based on this significant achievement in which the 2,3-
diphenylpropionic acid core unit without a peptide bond
in it could definitely bestow the VLA-4 antagonist activi-
ties on the small molecules, a variety of functional
groups were examined in order to optimize the R4
group. These results are shown in Table 2. It clearly
N 3'
R1
CO2H
R3
A
R2
3
Figure 1.
The general method for the synthesis of the 2,3-diphen-
ylpropionic acid derivatives is outlined in Scheme 1.
Ethyl 3-nitrophenylacetate derivatives 4 were hydrogen-
ated to the corresponding aniline derivatives 5. Benzal-
dehyde imines 6, prepared in an ordinary manner from
5, were deprotonated at the a-position to the ethyl ester
group by lithium diisopropylamide and then quenched
with 4-nitrobenzyl bromide to give the benzylated imine
intermediates. Subsequent hydrolysis of the imine moi-
ety with aqueous hydrochloric acid provided the ethyl
2-(3-aminophenyl)-3-(4-nitrophenyl)acetate derivatives
7 in good yields. The free amino group in 7 was then
converted to various kinds of tert-amide groups to af-
ford 8 by a two-step sequence (reductive alkylation
and acylation). Another nitro group located at the upper
end of 8 was also converted to two aroyl amide groups,
i.e., 2,6-dichlorobenzamide and 3,5-dichloroisonicotin-
amide.14 Finally, hydrolysis of the ethyl esters with aque-
ous sodium hydroxide provided the target compounds 9.
Table 1. Inhibitory activities of VLA-4 antagonists (1)
Cl
H
N
O
R4
O
Cl
N
R3
CO2H
R4
Compd
R3
IC50 (nM)
9a
9b
9c
9d
9e
H
CH3
CH2Ph
CH2Ph
CH2Ph
>100,000
5000
620
CH2CH(CH3)2
H
CH2CH(CH3)2
Cyclohexyl
Cyclohexyl
>100,000
930
NO2
Table 2. Inhibitory activities of VLA-4 antagonists (2)
Cl
X
CO2Et
H
N
c
d, e
H2N
R1
R1
CO2Et
O
R4
R2
O
Cl
R2
N
R3
CO2H
X = NO2
4:
5:
6:
7
a
b
X = NH2
Compd
R3
R4
IC50 (nM)
X = NCHPh
Cl
9f
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2(CH2)CH3
CH2Ph
CH3
3300
940
Z
H
NO2
9g
9h
9i
CH2CH3
CH(CH3)2
C(CH3)2
N
54
85
R4
R4
O
Cl
O
O
f, g, h
N
N
9j
CH(CH2CH3)2
CH2C(CH3)3
Ph
48
650
CO2Et
R3
R3
CO2H
Z = CH, N
9k
9l
R1
R1
1700
1700
1100
7000
5300
7100
1700
580
R2
R2
8
9
9m
9n
9o
9p
9q
9r
9s
2-Thienyl
C6H4(4-OCH3)
2-Furyl
Scheme 1. (a) H2, 10% Pd–C, MeOH; (b) PhCHO, toluene, reflux; (c)
lithium diisopropylamide, 4-nitrobenzylbromide, THF, ꢀ78°C ꢁ rt
then HCl (aq); (d) aldehydes, NaBH(OAc)3, MeOH; (e) R4COCl,
Et3N, CHCl3; (f) H2, 10% Pd–C, MeOH, AcOEt; (g) 2,6-di-
chlorobenzoylchloride or 3,5-dichloroisonicotinoyl chloride, Et3N,
CHCl3; (h) NaOH (aq), THF, MeOH.
trans-CH=CHPh
OC(CH3)3
C(CH3)3
C(CH3)3

Y. Hoshina et al. / Bioorg. Med. Chem. Lett. 15 (2005) 217–220
219
indicated that the a-branched aliphatic alkyl groups
(9h–9j) were perfectly used as the R4 substituent. The
aromatic ring with/without a hetero atom did not pro-
vide any advantage for on the VLA-4 antagonist activi-
ties. Carbamate instead of acylamide at the R4 position
represented by the comparison between 9k and 9q de-
creased the antagonistic activity. Along with the afore-
a limited way, it turned out that the existence of R2
group also favorably affected the VLA-4 antagonist
activities and that there would be great possibilities for
the R2 substituent with further activity enhancement.
Finally, we would like to briefly mention the R5 group.
3,5-Dichloroisonicotinamide in place of 2,6-dichloro-
benzamide generally gave a better result for the VLA-4
antagonistic activity in each case as shown in Table
4.8e,h,14
mentioned
structure–activity
relationships,
the
comparison of the VLA-4 antagonist activities between
9j in Table 2 and 9e in Table 1 strongly suggested that
the steric tolerance of the particular space occupied by
the R4 group was rather small as the electronic character
of the 1-ethylpropyl group and cyclohexyl group is al-
most the same.
The series of 2,3-diphenylpropionic acid derivatives we
have explained in this letter had fairly good pharmaco-
kinetics and oral bioavailabilities. The selected data of
the representative compounds 9t and 9cc are shown in
Table 5. Further optimization of the 2,3-diphenylpropi-
onic acid derivatives and preclinical investigations of a
group of selected candidates including 9cc are proceed-
ing and these results will be published in due course.
Now that we have obtained a suitable R4 group, we
attempted to further optimize the R3 group by using
the tert-butyl group as the fixed R4 group. The n-pentyl
group and benzyl group were examined as the R3 group
(9r and 9s), but neither of them gave a good result.
Though there might be a possibility of other kinds of
R3 groups working well, we fixed the iso-butyl
group as the R3 group for further exploration.
In conclusion, we launched the VLA-4 antagonist pro-
gram based on the structure of the reported phenylala-
nine derivatives and created the proprietary core
structure, 2,3-diphenylpropionic acid. After a process
of trial and error, we discovered several very potent
VLA-4 antagonists with subnanomolar activities. Since
all of the compounds explained above are racemics,
the optically pure forms would have the preferable
characteristics.
The introduction of substituent(s) at the R1 and/or R2
position(s) on the benzene ring depicted in Table 3 pro-
vided another important effect. The simple methoxy
group dramatically increased the VLA-4 antagonist
activity up to the IC50 value of 2.0nM which is 40-fold
more potent than the basic template 9i. Generally, any
substituents applied here at the R1 position constantly
increased the activities by more than ten times. Com-
pound 9w bearing an n-propoxy group revealed a subna-
nomolar activity for the first time in this series. These
results suggested that the interaction between R1 and
the binding site on VLA-4 was quite important and that
the particular space occupied by R4 was rather large or
enlarged by an induced-fit protein mobilization, other-
wise it worked just as a supervisory group for the direc-
tion of the vicinal tert-amide. Though the R2 group was
also varied from hydrogen to some functional groups in
Table 4. Inhibitory activities of VLA-4 antagonists (4)
Cl
N
H
N
O
O
Cl
N
CO2H
R1
R2
Compd
R1
OCH3
R2
IC50 (nM)
9cc
9dd
9ee
9ff
H
1.7
Table 3. Inhibitory activities of VLA-4 antagonists (3)
OCH2CH3
OCH2CH2CH3
OCH(CH3)2
CH2CH3
H
H
0.25
0.65
1.1
H
Cl
H
N
H
9gg
9hh
H
CF3
0.1
0.4
O
O
Cl
N
CO2H
R1
R2
Table 5. Pharmacological comparison among our compounds and
representative VLA-4 antagonists
Compd
R1
OCH3
R2
IC50 (nM)
1 (TR-14035)
2
9t
9cc
1.7
440
9t
9u
H
2.0
3.1
3.8
0.1
8.4
16
OCH2CH3
OCH2OCH3
OCH2CH2CH3
OCH(CH3)2
OCH3
H
IC50 (nM)
Solubility in water
(lg/mL)
61
219
17
330 107
2.0
9v
H
9w
9x
9y
H
Protein binding (%)a
CLint (mL/min/mg)a
F (%)b
—
—
—
—
—
—
95.17
0.013
34
92.53
0.030
33
H
OCH3
H
9z
OH
CH2CH3
H
11
9aa
9bb
H
CF3
4.9
23
a Rats.
b SD rats, 10mg/kg, po.

220
Y. Hoshina et al. / Bioorg. Med. Chem. Lett. 15 (2005) 217–220
2002, 12, 159–163; (d) Yang, G. X.; Chang, L. L.; Truong,
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We appreciate the constructive contributions of Profes-
sor Takashi Takahashi (TIT), Dr. Jun Nakano (Kaken),
and Dr. Masahiro Satsukawa (Kaken) to discussions
relevant to this study. We thank Mr. Atsushi Matsuo
and Mr. Naoya Nagata for supports of computational
drug design approach. We also thank Ms. Miyako
Ohara and Ms. Kazumi Sumita for measurement of
NMR and mass spectra.
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