Specific and dual antagonists of α4β1 and α4β7 integrins

Article abstract of DOI:10.1016/S0960-894X(01)00705-3

N-(3,5-Dichlorophenylsulfonyl)-(R)-thioprolyl biarylalanine 10a has been identified as a potent and specific antagonist of the α₄β₁ integrin. Altering the configuration of thioproline from R to S led to a series of dual antagonists of α₄β₁ and α₄β₇, and the N-acetyl analogue 8b was found to be the most potent dual antagonist. A binding site model for α₄β₁ and α₄β₇ is proposed to explain the structure-activity relationship.

Full text of DOI:10.1016/S0960-894X(01)00705-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 133–136
Specific and Dual Antagonists of ꢀ4ꢁ1 and ꢀ4ꢁ7 Integrins
Linus S. Lin,a,* Thomas Lanza, Jr.,a Ermenegilda McCauley,b Gail Van Riper,b
Usha Kidambi,b Jin Cao,b Linda A. Egger,b Richard A. Mumford,b John A. Schmidt,b
Malcolm MacCossa and William K. Hagmanna
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
bDepartment of Immunology and Rheumatology Research, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
Received 5 September 2001; accepted 6 October 2001
Abstract—N-(3,5-Dichlorophenylsulfonyl)-(R)-thioprolyl biarylalanine 10a has been identified as a potent and specific antagonist
of the a4b1 integrin. Altering the configuration of thioproline from R to S led to a series of dual antagonists of a4b1 and a4b7, and the
N-acetyl analogue 8b was found to be the most potent dual antagonist. A binding site model for a4b1 and a4b7 is proposed to explain
the structure–activity relationship. # 2002 Elsevier Science Ltd. All rights reserved.
6
Integrins are a super family of structurally related het-
erodimeric glycoproteins, which are involved in cell
adhesion and cell trafficking. The a4b1 integrin (very late
antigen-4 or VLA-4) is expressed on many leukocytes
including eosinophils, basophils, and monocytes, but
not on platelets. VLA-4 antagonists may have potential
for the treatment of allergic diseases such as asthma,
and other chronic inflammatory diseases.1 The a4b7
integrin is found primarily on mucosal lymphocytes,
and blockade of a4b7 may be beneficial in the treatment
of inflammatory bowel disease.2 Although a specific
inhibitor of either a4b1 or a4b7 may be desirable, a dual
antagonist may be advantageous to achieve maximum
efficacy.3 In this paper, we wish to report a series
of N-substituted thioprolyl biarylalanine derivatives as
specific or dual antagonists of VLA-4 and a4b7, and the
specificity can be modulated by varying the N-sub-
stituent and the stereochemistry of thioproline. A bind-
ing site model was developed, and was used to design
more potent VLA-4 antagonists.
The inhibition of VLA-45 or a4b7 was determined for
each compound (Table 1).
In the 2(R)-thioproline series (4a and 8a–10a), the
potency against VLA-4 increased as the N-substituent
The synthesis of these molecules is illustrated in Scheme
1. Either 2(R)- or 2(S)-thioproline (1a or 1b) was reacted
with 24 to provide the respective diastereomers 3a/3b.
Deprotection and hydrolysis provided 4a/4b. Acylation or
sulfonylation afforded derivatives 8–10 after hydrolysis.
Scheme 1. (a) EDC, HOBt, N-methylmorpholine, CH2Cl2; (b) LiOH,
MeOH/THF/H2O; (c) TFA/CH2Cl2; (d) HCl/EtOAc; (e) Ac2O, iPr2-
NEt, CH2Cl2/THF, 0 ꢀC to rt.
*Corresponding author. Tel.: +1-732-594-8391; fax: +1-732-594-
5790; e-mail: linus_lin@merck.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00705-3

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L. S. Lin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 133–136
Table 1. Binding results of 4, 8, 9, and 10
a4b7
a4b1
a4b7
a4b1
Compd
R
a4b1
IC50 (nM)
a4b7
IC50 (nM)
Compd
R
a4b1
IC50 (nM)
a4b7
IC50 (nM)
4a
8a
9a
10a
H
Ac
13
1232
50
13
95
56
31
1020
4b
8b
9b
10b
H
Ac
8.2
197
2.1
8.9
160
24
10
23
27
0.90
0.42
0.30
0.20
0.39
6.0
MeSO2
3,5-Cl2PhSO2
MeSO2
3,5-Cl2PhSO2
306
changed from a hydrogen to the larger 3,5-dichloro-
phenylsulfonyl group, suggesting specific interactions
between the arenensulfonyl group and VLA-4. The
potency against the a4b7 integrin varied with 9a being
the most potent. In the 2(S)-thioproline series (4b and
8b–10b), potencies against both VLA-4 and a4b7 varied
as the size of the N-substituent increased.
modate the larger 3,5-dichlorophenylsulfonyl group. As
a result, 10a is also the more specific antagonist of VLA-
4. In the 2(S)-thioproline series, it is assumed that the
configuration alteration causes a conformation change,
and the S1 site is no longer accessible by the N-sub-
stituent. Instead, the N-substituent is better oriented to
fit into the S2 site. Since the S2 site may be comparable
in size for both a4b1 and a4b7, similar potency was
observed against both receptors for the series. Due to
moderate size of the S2 site, the N-acetyl analogue 8b is
likely to fit the best, and it is the most potent dual
antagonist. A related structure was reported by Archi-
bald and co-workers as a dual a4b1 and a4b7 antago-
nist,3e which is also consistent with this binding site
model.
To explain these results, a working hypothesis was for-
mulated as illustrated in Figure 1. It is proposed that
there are three major binding sites (S1, S2, and S3) for
both a4b1 and a4b7. The common biarylalanine segment
anchors the compounds into the S3 site as shown. The
two receptors may also share an S2 site, which are
comparable in size. The major difference between the
two integrins may be the size discrepancy of their
respective S1 site. Thus, in the 2(R) series, binding
To support this binding site model, two pairs of com-
pounds were prepared to probe the S2 site of a4b1 by
incorporating a hydroxy group at the 3-position of
proline (Scheme 2). It is reasonable to assume that the
conformation of (l)-proline is similar to that of (R)-
thioproline.8
7
interactions with a4b1 increased as the size of the
N-substituent changes from hydrogen to 3,5-dichloro-
phenylsulfonyl group due to extra contacts with the S1
site of a4b1, and 10a is the most potent in the series. The
S1 site of a4b7 may be smaller, and it cannot accom-
Figure 1. Proposed binding interactions of a4b1 and a4b7.

L. S. Lin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 133–136
135
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.

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L. S. Lin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 133–136
Wilson, J.; MacCoss, M.; Mills, S. G.; Van Riper, G.; McCau-
ley, E.; Egger, L. A.; Kidambi, U.; Lyons, K.; Vincent, S.;
Stearns, R.; Colletti, A.; Teffera, Y.; Tong, S.; Fenyk-Melody,
J.; Owens, K.; Levorse, D.; Kim, P.; Schmidt, J. A.; Mumford,
R. A. Bioorg. Med. Chem. Lett. 2001, 11, 2709. (b) Kopka, I.
E.; Young, D.; Lin, L. S.; Durette, P. L.; MacCoss, M.; Van
Riper, G.; McCauley, E.; Egger, L. A.; Kidambi, U.; Lyons,
K.; Vincent, S.; Schmidt, J. A.; Mumford, R. A.; Hagmann,
W. K. Bioorg. Med. Chem. Lett. Submitted for publication.
9. Dappen, M. S.; Dressen, D. B.; Ellingboe, J. W.; Grant, F.
S.; Konradi, A. W.; Pleiss, M. A.; Semko, C. M.; Thorsett, E.
D.; Freeman, S. B.; Holsztynska, E. J.; Quinn, K. P.; Ashwell,
S.; Banker, A. L.; Baudy, R. B.; Bicksler, J. J.; Giberson, J.;
Leeson, P. D.; Lombado, L. J.; Sarantakis, D. Abstracts of
Papers, 220th National Meeting of the American Chemical
Society, Washington, DC, August, 2000; American Chemical
Society: Washington, DC, 2000; MEDI 135.
10. The substituents that can favorably interact with the S2
site include small groups such as Ac, MeSO2, Me and OH, but
not groups that can ionize under physiological conditions such
as COOH and NH2. More detailed studies will be the subject
of another report.