Substituted N-(3,5-dichlorobenzenesulfonyl)-L-prolyl-phenylalanine analogues as potent VLA-4 antagonists

Article abstract of DOI:10.1016/S0960-894X(01)00821-6

A series of substituted N-(3,5-dichlorobenzenesulfonyl)-L-prolyl- and α-methyl-L-prolyl-phenylalanine derivatives was prepared as VLA-4/VCAM antagonists. The compounds showed excellent potency with a wide variety of neutral, polar, electron withdrawing or donating groups on the phenylalanine ring (IC₅₀～1 nM). Heteroaryl ring substitution for phenylalanine was also well tolerated. Pharmacokinetic studies in rat were performed on a representative set of compounds in both series.

Full text of DOI:10.1016/S0960-894X(01)00821-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 637–640
Substituted N-(3,5-Dichlorobenzenesulfonyl)-L-prolyl-
phenylalanine Analogues as Potent VLA-4Antagonists
Ihor E. Kopka,a,* David N. Young,a Linus S. Lin,a Richard A. Mumford,b Plato A. Magriotis,a
Malcolm MacCoss,a Sander G. Mills,a Gail Van Riper,b Ermengilda McCauley,b
Linda E. Egger,b Usha Kidambi,b John A. Schmidt,b Kathryn Lyons,c Ralph Stearns,c
Stella Vincent,c Adria Colletti,c Zhen Wang,c Sharon Tong,a Junying Wang,a
Song Zheng,a Karen Owens,a Dorothy Levorsea 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
cDepartment of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 10 September 2001; accepted 3 December 2001
Abstract—A series of substituted N-(3,5-dichlorobenzenesulfonyl)-l-prolyl- and a-methyl-l-prolyl-phenylalanine derivatives was
prepared as VLA-4/VCAM antagonists. The compounds showed excellent potency with a wide variety of neutral, polar, electron
withdrawing or donating groups on the phenylalanine ring (IC50ꢀ1 nM). Heteroaryl ring substitution for phenylalanine was also
well tolerated. Pharmacokinetic studies in rat were performed on a representative set of compounds in both series. # 2002 Elsevier
Science Ltd. All rights reserved.
The integrin VLA-4 (‘very late antigen-4’, a4b1) is
expressed on all leukocytes except platelets.1 Its ligands
include vascular cell adhesion molecule-1 (VCAM-1),
which is a member of the immunoglobulin (Ig) super
family, as well as peptides derived from the type III
connecting segment (CS-1) of fibronectin. Antibodies to
a4 are effective inhibitors of leukocyte infiltration and
prevent tissue damage in several animal models of
inflammation.2 Inhibition of VLA-4 may reduce the
migration and/or activation of cell types critical in sus-
taining a prolonged inflammatory response. VLA-4
inhibitors may be considered in the treatment of
asthma,3 multiple sclerosis4 and rheumatoid arthritis.5
The relatively poor pharmacokinetic characteristics of
this series of compounds, coupled with the appearance
of oxidized species attributed to the naphthyl moeity,
pointed to the need to replace this group with a meta-
bolically more stable entity. In this paper, we replace the
2-naphthylalanine in 1 with a variety of substituted
phenyl and heterocyclic alanine derivatives.8 Our goal
was to retain potency in the inhibition of VLA-4, reduce
serum protein binding that was observed with the
biphenyl series, and improve the pharmacokinetic char-
acteristics of these VLA-4 antagonists.
In Scheme 1, we outline the synthesis of substituted
phenylalanine VLA-4 inhibitors. In general, most of our
analogues were assembled from commercially available
amino acids. Treatment of the free amino acids with thio-
nyl chloride in cold methanol affords the corresponding
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.6,7 We
previously reported8 that sulfonylated dipeptides which
incorporate 2-(S)-a-naphthylalanine (1) and 4-biphenyl
replacement (2) provided substantial improvement over
initial screening leads in blocking binding of VLA-4 to
VCAM-1 (Fig. 1).
*Corresponding author. Tel.: +1-732-594-5004; fax: +1-732-594-
5966; e-mail: ihor_kopka@merck.com
Figure 1.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00821-6

638
I. E. Kopka et al. / Bioorg. Med. Chem. Lett. 12 (2002) 637–640
4-biphenyl derivative 2. Sterically hindered derivative 8e
is essentially equipotent with the methyl ether 6e or the
tyrosine derivative 5e. Receptor binding for electron
withdrawing VLA-4 antagonists 12e, 13e, and 15e is
similar with either neutral or electron-rich derivatives
like 4e, 5e, and 6e. The 3,4- and 3,5-difluoro derivatives
12b and 12c, respectively, are nearly equipotent with the
other analogues.
Analogues of 3–14 were prepared using 2-(S)-methyl-
proline in place of (S)-proline in order to compare
potency, selectivity in vitro and resistance to central
amide bond hydrolysis in vivo. As seen in Table 2, the
impact of this 2-(S)-methyl-proline substitution ranged
from a negligible to modest loss of potency within each
paired set. The most potent analogues across both
paired groups were the 4-oxo derivatives 5e, 6e, 8e and
18, 19, 21, respectively. Binding data for the related a4b7
integrin are included in Table 2 in order to assess speci-
ficity. The compounds in Table 2 are several orders of
magnitude more specific for VLA-4 with respect to a4b7.
Scheme 1. (a) SO2Cl, MeOH, 0ꢁC; (b) N-(3,5-Cl2PhSO2)-l-Pro,
PyBOP, DCM; (c) LiOH, EtOH/H2O; (d) H+
.
methyl esters. The amino acid ester hydrochlorides are
then coupled to N-(3,5-dichlorobenzenesulfonyl)-l-pro-
line. Hydrolysis of the ester with lithium hydroxide in
water/ethanol and subsequent acidification gave the free
acids which are listed in Table 1.
Analogues with a 2-(S)-methyl-l-proline were prepared
to block potential cleavage of the central amide bond in
vivo. Table 2 consists of a cognate set of substituted
phenylalanine derivatives coupled to N-(3,5-dichloro-
benzenesulfonyl)-2-(S)-methyl-proline. Binding data for
two literature compounds described as a potent, selec-
tive VLA-4 antagonist (BIO-1211)9 and a dual a4b7/
VLA-4 antagonist (TR-14035)10 are included in Table 2
for comparison to the data reported herein.
A series of heteroaryl analogues was also prepared to
assess the effect heteroaryl substitution for phenylala-
nine has on the inhibition of VLA-4 (Table 3). The
results obtained for VLA-4 binding and a4b1/a4b7
selectivity from this limited set closely parallel those
seen in Tables 1 and 2. All are approximately equipo-
tent against VLA-4 (ꢀ1 nM) with the same modest loss
in VLA-4 binding potency among 2-(S)-proline and 2-
(S)-methyl-proline pairs 27a, 27b and 31a, 31b, respec-
tively. Only analogue 29 showed a marked drop in
potency.
An examination of the binding data in Table 1 suggests
that 4-substituted phenylalanine analogues of 2 are
relatively insensitive to both the steric and electronic
character of the substituent pattern, consistent with the
Table 2. Inhibition of VLA-4a and a4b7 by substituted N-sulfonyl-
b
ated 2-(S)-methyl-prolyl-phenylalanine derivatives (IC50, nM, n=3)
Table 1. Inhibition of VLA-4a by substituted N-(3,5-dichloro-
benzene-sulfonylated prolyl-phenylalanine derivatives (IC50
n=3)
,
nM,
Compd
R
VLA-4
a4b7
16a
17
18
19
20
21
22
23a
23b
23c
24a
24b
24c
25
26
BIO-1211
TR-14035
H
C2H5
OH
OCH3
OCF3
O-tC4H9
I
o-Cl
m-Cl
p-Cl
o-F
m-F
p-F
1.0
3546
2%c
1.88
0.86
0.59
3.8
0.27
4.77
1.8
1.7
2.3
1.7
0.9
1.2
1.6
3.9
0.13
0.11
—
Compd
R
2 (a)
3,4 (b) 3,5 (c)
3 (d)
4 (e)
16%c
—
1
2
3a
4e
5e
6e
7e
À0.5
À0.65
—
—
—
—
—
—
—
—
—
0.48
1.1
—
646
58%d
<16%c
<16%c
<16%c
<5%c
<5%c
<5%c
29%c
25%c
862b
H
C2H5
OH
OCH3
OCF3
O-tC4H9
I
Br
Cl
F
F2
CN
CF3
NO2
1.4
—
—
—
—
—
—
—
0.99
1.3
—
—
—
—
—
—
—
—
—
—
—
—
—
0.44
0.34
0.37
0.86
0.22
0.33
0.92
0.85
0.48
—
8e
9e
CN
CF3
10e
11a,d,e
12a,b,d,e
12c
13e
14d
15e
3.6
0.67
—
0.75b
—
3.2
—
0.79
aVCAM-Ig.
—
—
bMadCAM-Ig.
0.3
c% inhibition @ 0.1 mM.
d% inhibition @ 20 mM.
aVCAM-Ig.

I. E. Kopka et al. / Bioorg. Med. Chem. Lett. 12 (2002) 637–640
639
Since there was little difference observed in the binding
of most of our antagonists against the VLA-4 receptor,
we sought to differentiate these derivatives based on
their susceptibility to metabolism in vitro as well as their
in vivo pharmacokinetic properties. A preliminary
examination of the metabolism of compounds in this
class suggests that there are oxidation products asso-
ciated with the central proline ring as well as hydro-
xylation of the aromatic phenyl ring as determined by
preincubation of 16a in rat hepatocytes or rat liver
microsomes (10 mm/30 min).8 The pharmacokinetic
properties of a representative subset of VLA-4 deriva-
tives from the previous tables were measured in rats and
are reported in Table 4.
macokinetic parameters of all four compounds are
described in Table 5.
The compounds described generally had low to moder-
ate oral bioavailability and moderate to fast plasma
clearance rates. Several compounds (26 and 29) had
clearance rates approaching rat hepatic blood flow
(ꢀ60 mL/min). The plasma concentration versus time
curves (AUC) for these compounds (3a, 16a, and 24c)
exhibited a very rapid drop in plasma concentration
(data not shown) followed by low sustained circulating
concentrations (ꢀ1 nM).
The data from Table 5 show that only 16a with the (S,S)
configuration has reasonable binding potency against
VLA-4. The other three isomers show moderate to good
oral bioavailability, but suffer from exceptionally high
clearance rates with the previously observed rapid drop
in plasma concentration versus time (AUC) followed by
very low but sustained circulating concentrations. The
low but sustained serum levels may be due to high
serum protein binding (ꢀ99.5% in human and rat
plasma). Compound 16a was less potent in a plasma
shift assay (9.2 nM @ 10% rat plasma). Most of the
compounds are rapidly cleared by the liver and excreted
intact in the bile. The pharmacokinetic properties of 16a
were studied in beagle dogs, rhesus monkeys and sheep.
Table 6 summarizes these results.
The attractive pharmacokinetic profile of 16a encour-
aged us to explore the structure–function relationship of
all its stereoisomers.11 By introducing the unnatural (R)
configuration for either phenylalanine or a-methyl-pro-
line, we hoped to increase the circulating plasma levels
of drug without unduly affecting VLA-4 binding affi-
nity. The stereoisomers of 16a were synthesized by the
route described in Scheme 1. Binding data and phar-
We observed modest to good oral bioavailability in all
four species. The clearance rates across all species were
moderate, with acceptably long half-lives. The major
b
Table 3. Inhibition of VLA-4 and a4b7 by substituted sulfonylated
proline heterocyclic amino acid derivatives (IC50, nM, n=3)
Table 4. Pharmacokinetic parametersa of selected compounds
c
Compd
F
b (%) Clp
(mL/kg/min)
Vds (L/kg)
t1=2 (h)
3a
16a
19
46
59
9
34
32
53
55
5
32
30
47
36
22
44
53
58
0.56
1.25
1.4
1.1
1.0
0.6
0.9
0.5
1.2
0.9
0.3
20
1.4
23b
24c
26
0.36
1.2
NDd
0.48
a
Compd
R
Heterocycle
VLA-4a
a4b7
29
aSprague–Dawley rats.
27a
27b
H
0.67
1.31
45% @ 20 mM
13% @ 0.1 mM
bDose: 1 mg/kg iv; 2 mg/kg po.
ct1=2=plasma half-life (0–8 h).
dND, not determined.
CH3
28
H
0.86
2100
Table 5. Pharmacokinetic parametersa of (R)- and (S)-a-methyl-pro-
line and phenylalanine VLA-4 inhibitors
29
30
H
H
26.5
0.73
ND
c
Compd
VCAM-Ig
(IC50, nM)
Fb
(%)
Clp
(mL/kg/min)
Vdss
(L/kg)
t1=2
(h)
16a
32
33
34
1.0
2055
28450
152
59
26
91
22
30
60
62
84
1.25
3.5
5.6
2.0
1.0
0.7
2.6
0.8
2350
31a
31b
H
CH3
0.92
1.61
2850
45% @ 20 mM
aSprague–Dawley rats.
bDose: 1 mg/kg iv; 2 mg/kg po.
aVCAM-Ig.
ct1=2 plasma half-life (0–8 h).

640
I. E. Kopka et al. / Bioorg. Med. Chem. Lett. 12 (2002) 637–640
Table 6. Pharmacokinetic parametersa of 16a in different animal
species
4. Tubridy, N.; Behan, P. O.; Capildeo, R.; Chaudhuri, A.;
Forbes, R.; Hawkins, C. P.; Hughes, R. A. C.; Mosely, I. F.;
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1999, 53, 466.
Species
F
b (%)
Clp (mL/kg/min)
Vdss (L/kg)
t1=2c (h)
Rata
59
39
23
42
30
1.25
2.6
2.7
4.2
1.0
6.7
7.9
6.9
5. Seiffge, D. J. Rheumatology 1996, 23, 2086.
Dogd
15.4
16.3
20
6. Wang, J. H.; Pepinsky, R. B.; Stehle, T.; Liu, J. H.; Kar-
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U.S.A. 1995, 92, 5714.
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Zimmerman, C. N.; Castro, A.; Lee, W.-C.; Hammond, C. E.;
Kalkunte, S.; Chen, L.-L.; Pepinsky, R. B.; Leone, D. R.;
Sprague, A. G.; Abraham, W. M.; Gill, A.; Lobb, R. R.;
Adams, S. P. J. Med. Chem. 1999, 42, 90.
9. 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.; Vin-
cent, S.; Stearns, R.; Colletti, A.; Teffera, J.; Tong, S.;
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aSprague–Dawley rats.
bDose: 1 mg/kg iv; 2 mg/kg po.
ct1=2=plasma half-life (0–8 h).
dBeagle (male) dose: 1 mg/kg iv; 2 mg/kg po.
eRhesus (male) dose: 1 mg/kg iv; 2 mg/kg po.
fAbraham Sheep (female) dose: 1 mg/kg iv; 2 mg/kg po.
metabolism product of 16a in all the species was the
acylglucuronide of the carboxylic acid.
In summary, we described a series of potent and selec-
tive VLA-4 inhibitors that have good oral bioavail-
ability in several species and low but sustained
circulating plasma drug levels in vivo. Further work on
related sulfonylated dipeptide derivatives will be repor-
ted in due course.
10. Sircar, I.; Gudmundsson, K. S.; Martin, R.; Nomura, S.;
Jayakumar, H.; Nowlin, D. M.; Caradelli, P. M.; Mah, J. R.;
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References and Notes
11. Elan reported on a related series of arylsulfonamide deri-
vatives. The PK profile of their compounds in the rats sug-
gested they were poorly absorbed and/or rapidly metabolized.
Semko, C. M.; Dressen, D. B.; Grant, F. S.; Konradi, A. W.;
Pleiss, M. A.; Thorsett, E. D.; Freedman, S. B.; Holsztynska,
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