N-Tetrahydrofuroyl-(L)-phenylalanine derivatives as potent VLA-4 antagonists.

Article abstract of DOI:10.1016/S0960-894X(02)00210-X

Given the proposed involvement of VLA-4 in inflammatory processes, a program to identify orally active VLA-4 antagonists was initiated. Herein, we report the discovery of a N-tetrahydrofuroyl-(L)-phenylalanine derivative (17) and related analogues as potent VLA-4 antagonists with good oral bioavailability.

Full text of DOI:10.1016/S0960-894X(02)00210-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1497–1500
N-Tetrahydrofuroyl-(L)-Phenylalanine Derivatives as Potent
VLA-4 Antagonists
Ginger X. Yang,a,* Linda L. Chang,a Quang Truong,a George A. Doherty,a
Plato A. Magriotis,a Stephen E. de Laszlo,a Bing Li,a Malcolm MacCoss,a
Usha Kidambi,b Linda A. Egger,b Ermengilda McCauley,b Gail Van Riper,b
Richard A. Mumford,b John A. Schmidtb and William K. Hagmanna
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Immunology and Rheumatology Research, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 11 December 2001; accepted 8 March 2002
Abstract—Given the proposed involvement of VLA-4 in inflammatory processes, a program to identify orally active VLA-4
antagonists was initiated. Herein, we report the discovery of a N-tetrahydrofuroyl-(l)-phenylalanine derivative (17) and related
analogues as potent VLA-4 antagonists with good oral bioavailability. # 2002 Elsevier Science Ltd. All rights reserved.
VLA-4 (a4b1; ‘very late antigen-4’) is a heterodimeric
cell-surface protein expressed at high levels on lympho-
cytes. VLA-4 binds to an alternatively spliced segment
of fibronectin (CS-1) on extracellular matrix and to
vascular cell adhesion molecule-1 (VCAM-1) on endo-
thelium. Both of these ligand molecules are expressed in
inflamed tissues and actively participate in physiologic
and pathologic responses in inflammatory and
autoimmune diseases.
being potent VLA-4 antogonists, such dipeptides show
good bioavailability as well. Structure 1 consists of three
distinctive regions of pharmacophore depicted as P1,
P2, and P3 (Fig. 1). Presently, we wish to report our
efforts in the optimization of P1, P2, and P3.
That sulfonamides in the P1 region were not essential
for potency came as a surprise during a Suzuki-coupling
reaction (Fig. 2) of a sulfonylated pyrazolidine. Analysis
of the major product of this coupling showed that the
sulfonyl group had been eliminated giving rise to the
corresponding dehydropyrazolidine 3, which showed
interesting VLA-4 activity.5 The minor product was the
desired coupling product 4.
The binding of VLA-4 to VCAM-1 leads to lymphocyte
infiltration to extravascular tissues. Antibodies against
VLA-4 have been demonstrated to block lymphocyte
infiltration and prevent tissue damage in animal models
of inflammatory diseases such as asthma,1 multiple
sclerosis,2 rheumatoid arthritis,3 and inflammatory
bowel disease.4 Therefore, an orally active VLA-4
antagonist might be useful in the treatment of these
diseases.
To ensure that the observed activity was not due to
contamination from the potent sulfonylated pyr-
azolidine 4, the dehydropyrazolidine 3 was synthesized
via an alternative route (Scheme 1). Starting from the
In a previous communication,5 we described sulfony-
lated prolyl biphenylalanine derivatives 1 as potent
VLA-4 antagonists. In this paper, we wish to report our
discovery of a series of low molecular weight dipeptides
that emerged from the development of 1. Apart from
*Corresponding author. Tel.: +1-732-594-4734; fax: +1-732-594-
9556; e-mail: ginger_yang@merck.com
Figure 1. Binding domains of sulfonylated dipeptides.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00210-X

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G. X. Yang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1497–1500
10 were incorporated. N-Benzyl pyroglutamate deriva-
tives as potent VLA-4 antagonists have been reported.7
Increasing the ring size resulted in a loss of potency (8
versus 11). Shifting the oxygen in 8 to an adjacent posi-
tion in the ring (12) also decreased potency. These
results suggest a specific binding interaction between the
ring oxygen and VLA-4.
The corresponding a-methyl analogues (13 and 14) of
the two most potent compounds (8 and 10) were also
found to be quite potent. Compound 13 had an excel-
lent PK profile in rats (F=72%; CLp=5.3 mL/min/kg;
t1/2=1.4 h). The PK profile of the sulfone 14 was by far
less impressive in rats (F=6%; CLp=82 mL/min/kg; t1/2
=2.3 h).
Figure 2. Discovery of potent VLA-4 antagonists lacking a P1 sub-
stituent.
commercially available 4-iodo-phenylalanine, the car-
boxyl and amino groups were initially protected. A
subsequent Suzuki coupling with an arylboronic acid
gave the biaryl-alanine derivative. The BOC group was
then removed and the resulting amine salt was con-
verted to p-nitrophenylcarbamate and reacted with pyr-
azoline. Finally, deprotection of the t-butyl ester
furnished pure 3. The potency of this material was
approximately the same as that of the first batch
(IC50=26.8 nM) and it cannot be contaminated by 4.
Thus, we demonstrated that potent VLA-4 ligands may
be prepared lacking the P1 binding interaction.
Keeping the a-methyl-tetrahydrofuranoyl constant, fur-
ther optimization of substituted phenylalanine analo-
gues was explored. The disasteromers of 13 were
separated via preparative TLC and the more active iso-
mer 15 was assigned as R as proven by X-ray crystal-
lographic analysis of the corresponding resolved R-
tetrahydrofuroic acid.8 The less active S isomer has an
IC50 of 13.7 nM in VCAM binding assay. The pref-
erence of R configuration was a surprise because when
the P1 region is occupied by a large group (3,5-dichloro-
phenylsulfonyl) like 1 it was the S proline that gave the
more active diastereomer. This reversal of stereo pref-
erence may suggest that the oxygen atom in the tetra-
hydrofuran ring is interacting with the P2 binding site
instead of the P1 binding region as the oxygens of the
sulfonyl group. The existence of the P2 binding site was
also discussed in a previous report.9 The enantiomeri-
cally pure 2-(R)-tetrahydrofuryl acid was employed for
the preparation of compounds in Table 2 and was
obtained through resolution as has been described.8 The
Small cyclic P2 analogues devoid of the 3,5-dichlor-
ophenylfulfonyl5 group were prepared by methods
similar to those in Scheme 1. Compound 6 was prepared
via the p-nitrophenylcarbamate intermediate. Com-
pound 7 was synthesized from enantiomerically pure
and commercially available BOC-l-proline as a single
diastereomer demonstrating no racemization occurred
during the Suzuki coupling reaction. The requisite sul-
fones were prepared as shown in Scheme 2. Their
binding affinities for VLA-4 are shown in Table 1.6
Table 1. Inhibition of VLA-4 by compounds
Comparison of compounds 6 and 7 to 5 suggests that
the presence of the nitrogen atom improved potency.
Potency increased even further when other heterocycles
such as tetrahydrofuran 8, pyroglutamate 9, and sulfone
a
a
Compd
R
IC50
(nM)
Compd
R
IC50
(nM)
2.1
5
63
10
11
12
13
14
6
7
8
9
17
4.9
36
Scheme 1. (a) Isobutylene, diglyme, H2SO4 (60%); (b) BOC2O, DIEA
(100%); (c) (2-methoxyphenyl) boronic acid, Pd(PPh3)4, Na2CO3, tol-
uene/EtOH; (d) HCl/EtOAc; (e) p-nitrophenylchloroformate, DIEA,
pyrazoline or PyBOP, RCOOH, DIEA; (f) TFA/CH2Cl2.
11
1.7
2.8
3.9
1.3
Scheme 2. (a) ClCO2Bn, 2 equiv LiHMDS, (64%); (b) LiHMDS, MeI,
(78%); (c) H2, Pd/C, EtOAc, (75%).
a125I-VCAM-Ig was used as the ligand in a binding assay; see ref 5.6

G. X. Yang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1497–1500
1499
Table 2. Inhibition of VLA-4 binding by compounds 15–22 and
pharmacokinetic parametersb
c
Compd
R
VLA-4a IC50 (nM)
2.7
F
b (%) CLp t1/2 (h)
15
57
17
0.6
16
17
18
11
NDd
42
0.3
3.9
58
14
3.4
0.6
76
19
20
2.4
0.2
NDd
3
11
0.4
Scheme 3. (a) CuI, Cs2CO3, DMF, 150 ꢀC, 66%; (b) TFA, anisole
DCM, 25 ꢀC, 70%; (c) SOCl2, MeOH, 75 ꢀC, 80%; (d) PyBOP, DIEA,
DCM, 75%; (e) NaOH, MeOH, rt, 95%.
21
22
4.1
NDd
27
12.9
66
0.5
profile in rats but its corresponding di-substituted
analogue 1915 did not exhibit the expected increase in
potency. Given the attractive PK of compound 17 in
rats, it was examined in rhesus monkey and it showed a
low plasma clearance (3.3 mL/min/kg) and high oral
bioavailability (F=51%).
a125I-VCAM-Ig was used as the ligand in a binding assay; see ref 5.
bSpraguen–Dawley rats, dose: 1 mg/kg iv; 2 mg/kg po.
cCLp, mL/kg/min.
dND, not determined.
To further improve potency and pharmacokinetic para-
meters, 4-amino phenylalanine derivatives such as com-
pounds 20–22 were prepared. The most potent of this
series of compounds, 20 was equipotent in VLA-4
binding to compound 17. However, it had a less favor-
able PK profile than that of the di-methoxy analogue
17.
biaryl phenylalanine derivatives (15–22) were prepared
according to the methodology outlined in Scheme 1.
The dichloro-isonicotinoyl group in 20 was prepared
according to literature procedures.10 The syntheses of
the imidazolone 21 and the pyridone 22 are illustrated
in Scheme 3. The successful execution of the Goldberg
variant11 of the Ullmann reaction12 en route to 21 and
22, which proceeded without concomitant epimerization
at the phenylalanine stereogenic center is noteworthy.13
The results of this P3 substituted optimization are
shown in Table 2 along with selected pharmacokinetic
(PK) parameters from rats.
In summary, the synthesis, biological properties, and
pharmacokinetic profile of several interesting VLA-4
antagonists containing a tetrahydrofuranoyl have been
described.
Acknowledgements
Comparison of 15 and 16 showed that replacing the
methoxy group with a cyano group decreased potency.
An 8-fold increase in potency was observed when a sec-
ond methoxy group was introduced (15–17) as it was
reported previously.5,14 Di-methoxy-biphenyl 17 dis-
played an increased plasma clearance rate compared to
that of 15. In order to address the metabolic stability of
the methoxy groups, more stable equivalents were
sought. Cyclopropyloxy 1815 indeed showed a better PK
The authors are grateful to Sharon Tong, Kathryn
Lyons, Stella Vincent, Ralph Stearns, Adria Colletti,
Marcie Donnelly, Ken Vakerich, and Chris Nunes for
the performance of the in vivo phase of the rodent
pharmacokinetic studies and sample preparation and
Susan Iliff, Bonnie Friscino, Cesaire Gai, and Alison
Kulick for conducting the in vivo phase of the non-
human primate pharmacokinetic studies and sample

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G. X. Yang et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1497–1500
preparation. We thank Linus Lin and Thomas Lanza,
Jr. for supplying the boronic acid intermediate used for
the preparation of compound 18.
7. 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.
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Chem.-Chimica Ther. 1976, 11, 159.
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5. VLA-4 activity was assayed following the same procedure
as described in: Hagmann, W. K.; Durette, P. L.; Lanza, T.;
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