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References and Notes
1. (a) For recent reviews, see: Lin, K.-C.; Castro, A. C. Curr.
Opin. Chem. Biol. 1998, 2, 453. (b) Elices, A. J. Curr. Anti-
inflam. Immun. Invest. Drugs 1999, 1, 14.
2. For a recent review, see: Panes, J.; Granger, D. N. Gastro-
enterology 1998, 114, 1066.
3. (a) For recent reports of small molecule a4b1 and/or a4b7
antagonists, see: Souers, A. J.; Virgilio, A. A.; Schurer, S. S.;
Ellman, J. A. Bioorg. Med. Chem. Lett. 1998, 8, 2297. (b)
Chen, L.; Tilley, J. W.; Huang, T.-N.; Miklowski, D.; Trilles,
R.; Guthrie, R. W.; Luk, K.; Hanglow, A.; Rowen, K.;
Schwinge, V.; Wolitzky, B. Bioorg. Med. Chem. Lett. 2000, 10,
725. (c) 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.; Rowen, K. Bioorg. Med.
Chem. Lett. 2000, 10, 729. (d) Archibald, S. C.; Head, J. C.;
Linsley, J. M.; Porter, J. R.; Robinson, M. K.; Shock, A.;
Warrellow, G. J. Bioorg. Med. Chem. Lett. 2000, 10, 993. (e)
Archibald, S. C.; Head, J. C.; Gozzard, N.; Howat, D. W.;
Parton, T. A. H.; Porter, J. R.; Robinson, M. K.; Shock, A.;
Warrellow, G. J.; Abraham, W. M. Bioorg. Med. Chem. Lett.
2000, 10, 997. (f) Tilley, J. W.; Kaplan, G.; Fotouhi, N.;
Wolitzky, B.; Rowan, K. Bioorg. Med. Chem. Lett. 2000, 10,
1163. (g) Fotouhi, N.; Joshi, P.; Tilley, J. W.; Rowan, K.;
Schwinge, V.; Wolitzky, B. Bioorg. Med. Chem. Lett. 2000, 10,
1167. (h) Fotouhi, N.; Joshi, P.; Fry, D.; Cook, C.; Tilley,
J. W.; Kaplan, G.; Hanglow, A.; Rowan, K.; Schwinge, V.;
Wolitzky, B. Bioorg. Med. Chem. Lett. 2000, 10, 1171.
4. Shieh, W.-C.; Carlson, J. A. J. Org. Chem. 1992, 57, 379.
5. Details of a competitive binding assay between human
Jurkat cells and a radiolabeled 125I-VCAM-immunoglobulin
fusion protein (125I-VCAM-Ig) have been disclosed (Durette,
P. L; Hagmann, W. K.; MacCoss, M.; Mills, S.; Mumford, R.
PCT Application WO98/53817A1, 1998; Chem. Abstr. 1998,
130, 52736s). Briefly, human Jurkat cells were suspended in
binding buffer (25 mM HEPES, 150 mM NaCl, 3 mM KCl, 2
mM glucose, 0.1% bovine serum albumin, pH 7.4) supple-
mented with MnCl2 (1 mM), placed in Millipore MHVB mul-
tiscreen platesÆcompounds in DMSO, incubated at room
temperature for 30 min, filtered on a vacuum box, and washed
with 100 mL of binding buffer containing 1 mM MnCl2. After
insertion of the plates into adapter plates, 100 mL of Micro-
scint-20 (Packard cat# 6013621) was added to each well. The
plates were then sealed, placed on a shaker for 30 s, and
counted on a Topcount microplate scintillation counter
(Packard). Control wells containing DMSO alone were used
to determine the level of VCAM-Ig binding corresponding to
0% inhibition. Control wells in which cells were omitted were
used to determine the level of binding corresponding to 100%
inhibition. Percent inhibition was then calculated for each test
well and the IC50 was determined from a 10-point titration
using a validated four parameter fit algorithm. All titrations
were run in duplicate.
Scheme 2. (a) 3,5-Cl2–PhSO2Cl, Na2CO3, H2O, rt; (b) PyBop,
iPr2NEt, CH2Cl2; (c) LiOH, MeOH/THF/H2O.
When studied in the binding assays, the 3-hydroxypro-
line derivatives (13b/13d) are significantly more potent
(5-fold) against a4b1 than the proline derivative (13a/
13c), which is in agreement with the hypothesis that an
S2 binding site exists for the a4b1 receptor. A 2-fold
increase in potency was also observed against a4b7,
which is consistent with additional contacts with the S2
site of the a4b7 integrin. The absolute binding affinities
for a4b7 are still relatively poor, presumably due to
unfavorable binding to the S1 site as described earlier.
Dappen and co-workers have reported a4b1 potency
enhancement of a 3,3-dimethyl but not the 3-(S)-phenyl
substituted proline analogue over the unsubstituted
proline derivative.9 The former may be explained by
additional interactions between the methyl groups and
the S2 site of a4b1. In the latter case, the phenyl group
may be too bulky to fit into the S2 site as also seen with
9b/10b.10 This binding site model was further explored
for the design of a series of extremely potent VLA-4
antagonists, which will be the subject of another report.
In summary, we have identified the N-3,5-dichloro-
phenylsulfonyl (R)-thioprolyl biarylalanine derivative
10a as a potent and specific antagonist of VLA-4.
Altering the configuration of thioproline from R to S
led to a series of dual antagonist of VLA-4 and a4b7,
and 8b was found to be the most potent dual antagonist.
Structure–activity relationship analysis led to a binding
site model, which served as a guide in the design of more
potent antagonists of VLA-4 and a4b7.
6. Details of a competitive binding assay between human
RPMI-8866 cells (a human B-cell line a4+b1Àb7 was a gift
from Professor John Wilkins, University of Manitoba,
Canada) and radiolabeled 125I-MAdCAM-immunoglobulin
fusion protein (125I-MAdCAM-Ig) have been disclosed5 and
are similar to the VLA-4 binding assay.
7. The N-substituents (H, MeCO and RSO2) also differ in
hydrogen bonding capability, hybridization of the nitrogen
and therefore the orientation the substituent, etc. Various
alkyl and aryl including heteroaryl sulfonyl groups were stud-
ied,8 and the SAR of these compounds are best correlated with
the size of the alkyl or aryl group.
Acknowledgements
8. (a) 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.;
We wish to thank Mr. Henry Murillo and Ms. Amy
Bernick for LC–MS measurements.