Sulphonamide-based small molecule VLA-4 antagonists

Article abstract of DOI:10.1016/S0960-894X(03)00760-1

The discovery of a sulphonamide by-product with VLA-4 antagonistic activity led to a series of potent, small molecule VLA-4 antagonists. Synthesis, SAR and in vivo evaluation of the selected compound will be presented.

Full text of DOI:10.1016/S0960-894X(03)00760-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3875–3878
Sulphonamide-Based Small Molecule VLA-4 Antagonists
Marcin Stasiak,y Christopher Mehlin,{ Erica Boni,{ Tomas Vaisar,{ Thomas Little,x
Hwa-Ok Kim{ and Maher Qabar{*
Molecumetics, 2023 120th Ave. N E., Bellevue, WA 98005, USA
Received 14 March 2003; accepted 3 June 2003
Abstract—The discovery of a sulphonamide by-product with VLA-4 antagonistic activity led to a series of potent, small molecule
VLA-4 antagonists. Synthesis, SAR and in vivo evaluation of the selected compound will be presented.
# 2003 Elsevier Ltd. All rights reserved.
Integrins comprise a large family of cell-surface recep-
tors that bind to the extracellular matrix proteins, such
as fibronectin (FN), vitronectin, fibrinogen, collagen,
laminin and invasin, and to the cell-surface ligands such
as vascular cell adhesion molecule-1 (VCAM-1) or
intercellular adhesion molecules (ICAMs) to mediate
cell-matrix and cell-cell interactions. These interactions
are important for the regulation of cell migration.
Integrins are large membrane glycoproteins consisting
of two subunits, a and b. VLA-4 (CD49d/CD29), which
consists of a4 and b1 subunits, is a member of b1 or
VLA subfamily. It is expressed on monocytes, T- and B-
lymphocytes, basophils and eosinophils.1 VLA-4 med-
iates both the initial tethering as well as firm adhesion of
cells to the walls of the inflamed vessels through the
interaction with cytokine-inducible vascular cell adhe-
sion molecule VCAM-1.
VLA-4 has received considerable attention as a target
for the design of small molecule anti-inflammatory
therapeutics.7 Our initial effort to identify a potent
VLA-4 antagonist was based on the belief that we could
mimic the LDV motif in the CS-1 segment of FN8 or the
IDS motif in the C-D loop of VCAM-19 using our pep-
tide secondary structure mimetic scaffolds.10
However, during the course of our work we discovered
that the 2-naphthylsulphonyl amide 1, a minor byproduct
in a tested crude material, was a potent VLA-4 antagonist
VLA-4 antagonists inhibit leukocyte migration into
lung tissues during inflammatory responses and lym-
phocyte trafficking. Hence, these agents may reduce tis-
sue damage caused by inflammation and could
potentially be effective therapies for asthma. Animal
studies also indicate that antagonists of a4-containing
integrins may have utility in the treatment of other
inflammatory diseases, including multiple sclerosis,
rheumatoid arthritis, heart failure and inflammatory
bowel disease.2ꢀ6
*Corresponding author. Tel.: +1-781-541-6727x108; fax: +1-452-
941-8989; e-mail: maher_q@aurigene.com
yCurrent address: Corus Pharma, Seattle, WA, USA.
{Current address: University of Washington, Seattle, WA, USA.
xCurrent address: Ceptyr, Bothell, WA, USA.
{Current address: Aurigene Discovery Technologies, Lexington, MA,
USA.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00760-1

3876
M. Stasiaket al. / Bioorg. Med. Chem. Lett. 13 (2003) 3875–3878
with an IC50 of 180 nM in the RAMOS/CS-1 cell-adhesion
assay.11 This discovery prompted us to further explore a
series of sulphonylated dipeptides. Herein, we disclose the
SAR and the identification of the potent VLA-4 antago-
nist 6 that is orally available in cynomologous monkey and
efficacious in the murine model of asthma.
In the attempt to reduce the number of hydrogen-bond
donors and to restrict some of the flexibility of the
molecule, we synthesized compound 3 which is the N-
methyl sulfonamide analogue of compound 2. This
compound displayed a 2-fold increase in activity over
compound 2. Importantly, the replacement of the
naphthyl moiety in 3 by a smaller, phenyl substituent
produced compound 4 that was 10-fold more potent
(IC50 0.1nM) than 3. To ascertain the stereoselectivity
of the interaction the compound with d-isomer at the
tyrosine moiety was also synthesized and was found to
be 50-fold less active than the l-isomer (4) at that posi-
tion. The synthesis of compound 4 (Scheme 2) exem-
plifies the methodology we had used in the synthesis of
analogues (Table 1).
One of the early modifications involved the introduction
of a para-substituent at the phenylalanine aromatic ring.
Several groups have reported12 a dramatic potency
enhancement achieved by linking an aryl group to that
position. The obvious choice was to utilize tyrosine to
introduce diversity at that site. This selection was moti-
vated by the ease of synthesis of analogues, for example,
by alkylation (Scheme 1) or Mitsunobu reactions. We
favored using an ether linkage at that position (vs amide
or ester linkage) due to the benign nature (i.e., resistance
to esterases or amidases) and that could be advantageous
in terms of ‘drug-likeness’. To our satisfaction, the
introduction of the 2,6-dichlorobenzyloxy moiety led to
compound 2 which was 90-fold more potent than 1.
In further optimization we investigated the influence of
the proximity of the N-terminal carboxyl group to the
phenyl moiety on the activity. The replacement of the
glutamic acid with the aspartyl moiety led to compound
5 and a significant decrease in potency.
Scheme 1.
Scheme 2.

M. Stasiaket al. / Bioorg. Med. Chem. Lett. 13 (2003) 3875–3878
3877
Scheme 3.
Compound 6 displayed sufficiently good in vitro
ADME properties (pH, plasma and microsomal stabi-
lity, as well as no inhibition of several CYP450 isoforms
up to 50 mM) to warrant in vivo efficacy study in murine
asthma model and pharmacokinetic study in primate.
The murine asthma model (with sensitization by oval-
bumin and dexamethasone as a control) data is shown
in Table 2. Compound 6 reduced eosinophil influx by
50% at 10 mpk (in) and by 69% at 10 mpk (po). It also
reduced leukocyte infiltration into BALF by 39 and
59%, respectively (data not shown). The pharmacoki-
netic study in cynomologous monkey (Table 3) revealed
that although 6 had acceptable oral availability, it suf-
fered from high clearance and a very short elimination-
phase half life, both of which were detrimental to
further in vivo study in monkey.
Table 1. IC50 data for the selected compounds in the cell adhesion
assay11 utilizing VLA-4-expressing Ramos cells and immobilized, bio-
tinylated CS-1 fragment
Compd
Ramos/CS-1
IC50 (nM)
Ramos/CS-1 with
10%FBS IC50 (nM)
1
2
3
4
5
6
7
8
9
180
2
1
0.1
2
0.6
0.7
1.5
0.8
ND
ND
ND
2
ND
8
10
10
10
FBS, fetal bovine serum; ND, not determined.
In summary, we have developed SAR around the sul-
phonamide chemotype of VLA-4 antagonists and
devised an expedient synthesis of the most promising
compound (6) in the series. This compound has shown
good in vivo efficacy as indicated by inhibition of eosi-
nophil and leukocyte lung infiltration in the murine
asthma model.
Table 2. Compound 6 inhibition of eosinophil infiltration in the
murine asthma model13
Saline OVA in-5 in-10 po-10 po-25 DEX-ip-1
EOS count (1.0ꢁE5) 0.01 8.73 6.26 4.36 2.67 2.16
EOS inhibition (%) 28 50 69 75
3.79
57
—
—
Table 3. PK data of 6 in cynomologous monkey
References and Notes
Cmax
(ng/mL)
t1/2
(min)
CL
(mL/min/kg)
Vdss
(L/kg)
F
(%)
1. Hemler, M. Annu. Rev. Immunol. 1990, 8, 365.
2. Lobb, R. R.; Abraham, W. M.; Burkly, L. C.; Ma, W.;
Knight, J. A.; Leone, D. R.; Antognetti, G.; Pepinsky, R. B.
Ann. N.Y. Acad. Sci. 1996, 796, 113.
2
528 (iv) 101 (po)
6.6
58.8
0.303
26.9
3. Yusf-Makagianasar, H.; Andersen, M. E.; Song, X.; Mur-
ray, J. S.; Siahaan, T. J. Curr. Top. Biochem. Res. 2000, 2, 33.
4. Gonzalez-Amaro, R.; Portales-Perez, D.; Baranda, L.;
Redondo, J. M.; Martinez-Martinez, S.; Yanez-Mo, M.; Gar-
cia-Vicuna, R.; Cabanas, C.; Sanchez-Mardrid, F. J. Immunol.
1998, 161, 65.
5. Furuzawa-Carballeda, J.; Alcocer-Varela, J. Scand. J.
Immunol. 1999, 50, 215.
6. Allison, M. C.; Poulter, L. W. Clin. Exp. Immunol. 1991,
85, 504.
Interestingly, transposition of the aspartic acid side
chain to the sulphonamide nitrogen produced com-
pound 6 with the potency almost in the same range as
for compound 4. Additional advantages of compound 6
are the elimination of one center of chirality and sim-
plification of the synthesis (see Scheme 3). Several other
analogues (i.e., 7, 8, and 9) in which the sulphonamide
aryl group was varied, were also prepared and were
found to be almost equipotent to compound 6. This was
a pleasant discovery since one can potentially utilize this
part of the molecule to improve the PK profile without
compromising the potency of the compound.
7. For a review, see: (a) Adams, S. P.; Lobb, R. R. In Annual
Reports in Medicinal Chemistry; Doherty, A. M., Ed., Academic:
New York, 1999; Vol. 34, p 179. (b) Lobb, R. R.; Adams, S. P.
Exp. Opin. Invest. Drugs 1999, 8, 935. (c) Holland, G. W.;

3878
M. Stasiaket al. / Bioorg. Med. Chem. Lett. 13 (2003) 3875–3878
Biediger, R. J.; Vanderslice, P. In Annual Reports in Medicinal
Chemistry; Doherty, A. M., Ed.; Academic: New York, 2002;
Vol. 37, p 65.
8. Komoriya, A.; Green, L. J.; Mervic, M.; Yamada, S. S.;
Yamada, K. M.; Humphries, M. J. J. Biol. Chem. 1991, 266,
15075.
9. Wang, J. H.; Pepinsky, R. B.; Stehle, T.; Liu, J.-H.; Kar-
pusas, M.; Browning, B.; Osborn, L. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 5714.
10. (a) For a review, see: Kim, H.-O.; Kahn, M. Res. Adv. In
Organic Chem. 2000, 1, 43. (b) Kim, H.-O.; Kahn, M. Comb.
Chem. High Throughput Screening 2000, 3, 167.
11. Assay conditions were developed based on the publication
by Vanderslice, P.; Ren, K.; Revelle, J. K.; Kim, D. C.; Scott,
D.; Bjercke, R. J.; Yeh, E. T. H.; Beck, P. J.; Kogan, T. P. J.
Immunol. 1997, 158, 1710. BiotinGPEILDVPST-amide was
allowed to bind to black NeutrAvidin-coated, 96-well plates
(Pierce #15117) for 1 h at rt at the concentration of 10 mg/mL,
100 mL/well, in the following assay buffer: 50 mM Tris (pH
7.5), 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM
MnCl2. The plate was washed three times with water and
blocked overnight with 200 mL of the same buffer plus 3%
BSA. The plate was washed the same way just prior to use.
RAMOS cells were incubated with Calcein-AM (Molecular
Probes) in the dark in DPBS for 1 h prior to use at 10 million
cells/mL, then spun down and resuspended in assay buffer at
500,000 cells/mL with 5 mM ethidium homodimer. Cells were
incubated at 37 ꢂC in the presence or absence of drugs for 30
min at 37 ꢂC and cell lysis was assessed by fluorescence at 535/
617 nM. Unbound cells were rinsed free with three gentle
additions of assay buffer followed by quick inversion. Cells
were then lysed with 50 mL/well of lysis solution: 1% Nonidet
P-40, 50 mM Tris (pH 7.5), 5 mM EDTA, and remaining cells
were quantified by fluorescence at 485/535 nM. Cyclic hexa-
peptide (with disulfide bond) CWLDVC was used as a control,
with a reproducible IC50 of 100 nM.
Huang, T.-N.; Kaplan, G.; Trilles, R.; Miklowski, D.; Huby,
N.; Schwinge, V.; Wolitzky, B.; Rowan, K. Bioorg. Med.
Chem. Lett. 2000, 10, 729. (b) 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.; 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.; Vincent,
S.; Steans, R.; Colletti, A.; Teffera, J.; Tong, S.; Fenyk-Mel-
ody, J.; Owens, K.; Levorse, D.; Kim, P.; Schmidt, J. A.;
Mumford, R. A. Bioorg. Med. Chem. Lett. 2001, 11, 2709. (c)
Porter, J. R.; Archibald, S. C.; Childs, K.; Critchley, D.; Head,
J. C.; Linsley, J. M.; Parton, T. A. H.; Robinson, M. K.;
Shock, A.; Taylor, R. J.; Warrellow, G. J.; Alexander, R. P.;
Langham, B. Bioorg. Med. Chem. Lett. 2002, 12, 1051. (d)
Kopka, I. E.; Lin, S. L.; Mumford, R. A.; Lanza, T.;
Magriotis, P. A.; Young, D.; de Laszlo, S. E.; MacCoss,
M.; Mills, S. G.; Van Riper, G.; McCauley, E.; Lyons, K.;
Vincent, S.; Egger, L. A.; Kidambi, U.; Steans, R.; Colletti,
A.; Teffera, J.; Tong, S.; Owens, K.; Levorse, D.; Schmidt,
J. A.; Hagmann, W. K. Bioorg. Med. Chem. Lett. 2002, 12,
2415.
13. The measurement of eosinophil influx into broncoalveolar
lavage (BAL) was done as follows: BALB/C female mice
received compound 6 on day 15, 18 and 21 at 15 min before
OVA challenge. Twenty-four hours after final OVA challenge,
the mice underwent exsanguinations by posterior vena cava
puncture and then BAL (0.4 mL of saline three times) was
performed on the right lung after tying of the left lung at the
mainstem bronchus. Total BAL fluid cells were counted from
a 0.05 mL aliquot and the remaining fluid was centrifuged at
1200 rpm (Microcentrifuge, 5415 C, Eppendorf) for 5 min.
The cell pellets were resuspended in saline containing 10%
BSA (Calbiochem, San Diego, CA, USA) and smears were
made on glass slides. Eosinophiles were stained for 5–8 min
with Discombe’s diluting fluid containing (0.05% aqueous
eosin and 5% (v/v) acetone in distilled water, and then coun-
terstained with 0.07% methylene blue.
12. (a) Chen, L.; Tilley, J. W.; Guthrie, R. W.; Mennona, F.;