The discovery of small molecule carbamates as potent dual α4β1/α4β7 integrin antagonists

Article abstract of DOI:10.1016/S0960-894X(01)00710-7

The α₄β₁ and α₄β₇ integrins are implicated in several inflammatory disease states. Systematic SAR studies of an α₄β₁-specific arylsulfonyl-Pro-Tyr lead led to the identification of a new α₄β₇ binding site, best captured by O-carbamates of Tyr for this structural class. Several compounds showed a 200- to 400-fold improvement in α₄β₇ binding affinity while maintaining subnanomolar α₄β₁ activity, for example 2l, VCAM-Ig α₄β₁ IC₅₀=0.13 nM, VCAM-Ig α₄β₇ IC₅₀=1.92 nM.

Full text of DOI:10.1016/S0960-894X(01)00710-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 159–163
The Discovery of Small Molecule Carbamates as Potent Dual
ꢀ4ꢁ1/ꢀ4ꢁ7 Integrin Antagonists
Linda L. Chang,a,* Quang Truong,a Richard A. Mumford,b Linda A. Egger,b
Usha Kidambi,b Kathryn Lyons,c Ermengilda McCauley,b Gail Van Riper,b
Stella Vincent,c John A. Schmidt,b Malcolm MacCossa and William K. Hagmanna
aDepartment of Basic Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Immunology and Rheumatology, Merck Research Laboratories, Rahway, NJ 07065, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 20 August 2001; accepted 16 October 2001
Abstract—The a4b1 and a4b7 integrins are implicated in several inflammatory disease states. Systematic SAR studies of an a4b1-
specific arylsulfonyl-Pro-Tyr lead led to the identification of a new a4b7 binding site, best captured by O-carbamates of Tyr for this
structural class. Several compounds showed a 200- to 400-fold improvement in a4b7 binding affinity while maintaining sub-
nanomolar a4b1 activity, for example 2l, VCAM-Ig a4b1 IC50=0.13 nM, VCAM-Ig a4b7 IC50=1.92 nM. # 2002 Elsevier Science
Ltd. All rights reserved.
Introduction
there is a substantial interest in developing small-mole-
cule therapeutics for these disorders.5
The integrins a4b1 (very late antigen-4; VLA-4) and a4b7
are heterodimeric cell surface glycoprotein transmem-
brane receptors that are divalent cation-dependent.1
a4b1 is expressed on all leukocytes except platelets and is
a key mediator in cell–cell and cell–matrix interactions.
As such, it mediates cell adhesion to vascular cell adhe-
sion molecule-1 (VCAM-1), which is expressed on acti-
vated endothelial cells at sites of inflammation, and CS-
1, an alternatively spliced form of fibronectin (Fn).2
a4b7 is expressed on leukocytes and is central to lym-
phocyte homing in the gastrointestinal tract via its
interaction with the mucosal addressing cell adhesion
molecule-1 (MAdCAM-1) expressed on endothelial cells
of gut-associated mucosal tissues. a4b7 also binds to
VCAM-1 and Fn.3
Screening of a combinatorial library identified a capped
dipeptide lead 1 (a4b1 IC50=58 nM, a4b7 IC50=8330
nM).6 Initial derivatization efforts led to 2a (a4b1
IC50=0.34 nM, a4b7 IC50=820 nM), a much more
potent a4b1 antagonist which also showed a somewhat
increased activity versus a4b7.7,8 The three major bind-
ing elements, as determined by the SAR for this class of
compounds, are designated P1, an arylsulfonyl moiety,
P2, a prolyl residue, and P3, a substituted phenylalanine,
as shown in 2a.9a We were intrigued by the possibility of
locating additional binding sites to these receptors by
elaboration of the tyrosine hydroxyl in 2a. This investi-
gation led to the identification of low nanomolar
dual antagonists of a4b1 and a4b7 which are reported
herein.9b
Therapeutic efficacy in several animal models of inflam-
mation and autoimmune diseases such as asthma,
multiple sclerosis, and inflammatory bowel disease via
the inhibition of the interaction between a4b1 and/or
a4b7 and their ligands has been demonstrated using
both a4-antibodies and peptidyl antagonists.4 Therefore,
*Corresponding author. Tel.: +1-732-594-6465; fax: +1-732-594-
5350; e-mail: linda_chang@merck.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00710-7

160
L. L. Chang, et al. / Bioorg. Med. Chem. Lett. 12 (2002) 159–163
Chemistry
in Scheme 1 rather than (O-t-butyl)-tyrosine methyl
ester. Subsequently, the target compounds were
obtained using reaction sequences analogous to those
shown for the preparation of 2i–j,l.
The first series of compounds studied are shown in
Table 1. They were prepared from key intermediates
3a,b (Scheme 1). Thus, sulfonylation of proline t-butyl
ester followed by deprotection provided N-(3,5-dichloro-
benzenesulfonyl)-proline.10 This was coupled with the t-
butyl ester or the methyl ester of tyrosine t-butyl ether.
Liberation of the phenol via trifluoroacetic acid pro-
vided compounds 3a,b.11 Alkylation of 3a with a variety
of halides followed by removal of the t-butyl ester pro-
vided 2b,d–g. For 2h, tetrazole formation from 4, where
R=CH2CN, preceded ester hydrolysis. Compounds 2i–j
and l were prepared from 3b in an analogous manner.
The esters 2k,m–n were obtained by coupling of 3a with
the appropriate acids followed by ester hydrolysis.
A slightly altered order of steps was used to prepare
6a,b (Scheme 2). Here, the fully elaborated Pro-Tyr
moiety (P2–P3) was assembled before modifications were
made to obtain the desired P1 unit.
Biological Evaluations
The a4b1 integrin binding affinity of the compounds
reported was assessed by measuring the reduction in
binding of 125I-VCAM-Ig to a suspension of Jurkat cells
(a human T Cell line a4+b1+b7À) in the presence of the
test compound, as previously described.7 The assays are
run in the presence of 1 mM of MnCl2 and all com-
pounds were tested at least in duplicate.
The a4b7 integrin binding affinity of these compounds
was determined in duplicate by a radioligand binding
assay using 125I-VCAM-Ig and a suspension of RPMI-
8866 cells (a human B cell line a4+b1Àb7+) in the pre-
sence of 1 mM of MnCl2, as described previously.7
Table 1. In vitro binding potencies of (N-arylsulfonyl)-prolyltyrosyl
derivatives 2 versus 125I-VCAM-Ig
Compd
R
a4b1
IC50 (nM)
a4b7
IC50 (nM)
Scheme 1. (a) (3,5-Cl2)C6H3SO2Cl, NEt(iPr)2, DMAP, DCM, 94%;
.
(b) 1/1 TFA/DCM, rt, 40–98%; (c) H-Tyr(t-Bu)-O(t-Bu or Me) HCl,
EDC, HOBt, NMM, DCM, rt, 80–85%; (d) excess 1/1 TFA/DCM,
0 ꢀC, 2 h, 56–70%; (e) K2CO3, RX, DMF, 40 ꢀC, 85–95%; (f)
Me3SnN3, toluene, reflux, 20%; (g) EDC, DMAP, RCO2H, DCM, rt,
90–97%; (h) 0.2 N NaOH/EtOH, rt, 40–70%.
2a
2b
2c
2d
2e
2f
OH
OCH3
0.34
0.37
0.21
1.11
1.94
0.28
0.26
820
362
116
299
849
62.5
246
O-t-(C4H9)
O(CH2)3CH3
OCH2C6H5
O(CH2)2OCH3
OCH2CN
Compounds 5a–i (Table 2) were prepared via inter-
mediate 3c (obtained from a-methyl proline t-butyl ester
instead of proline t-butyl ester in Scheme 1) or its (3-
hydroxy)-Phe analogue, obtained by using (3-t-butoxy)-
Phe methyl ester in the amide bond formation reaction
2g
2h
0.21
166
2i
2j
OCH2COCH3
OCH2CO2-t-(C4H9)
0.18
0.13
51.4
211
2k
2l
0.45
0.13
0.22
0.26
43.2
1.92
58.6
13
2m
2n
Scheme 2. (a) 1/1 TFA/DCM, rt, 70%; (b) K2CO3, ClC(O)N(CH2)4,
DMF, 40 ꢀC, 90%; (c) H2, 10% Pd/C, MeOH, rt, quant; (d) 0.2 N
NaOH/EtOH, rt 70–90%; (e) RSO2Cl, NEt(iPr)2, DMAP, DCM, 80–
90%.

L. L. Chang, et al. / Bioorg. Med. Chem. Lett. 12 (2002) 159–163
161
Results and Discussion
Table 2. In vitro binding potencies of (N-arylsulfonyl)-prolyltyrosine
derivatives 5 versus 125I-VCAM-Ig
To begin, a series of simple alkyl ethers of the parent
compound 2a was evaluated (Table 1, 2a–e).12 The data
suggest that these compounds generally bind well to
a4b1, although as the length and lipophilicity of the
ether increased, the binding potency decreased (2d–e vs
2b–c). The binding to the a4b7 integrin was enhanced by
steric bulk proximal to the phenolic oxygen (2a vs 2b–c),
although, in general, they remained modest.
Compd
R
a4b1
IC50 (nM)
a4b7
IC50 (nM)
Data from 2d and 2f show that isosteric replacement of
an oxygen for a carbon atom provided a ꢁ4-fold
increase in potency towards both integrins, suggesting
that an appropriately placed heteroatom may bring
about substantial improvements to both the a4b1 and
a4b7 binding. Placing this heteroatom in a more direc-
tional manner (2g) or the addition of multiple hetero-
atoms (2h) had little effect in the a4b1 activity, but
decreased the a4b7 binding. Data from an alkoxy ketone
and an alkoxy ester (2i–j) suggest that the requirements
for good a4b7 binding in this vicinity are much more
limiting than those for the a4b1 site and binding to the
a4b7 site appear to be sensitive to the spatial positioning
of the hetero atom(s) incorporated.
5a
5b
4-O-t-(C4H9)
0.27
0.17
647
5.26
5c
5d
5e
4-OC(O)N(CH2CH3)2
4-OC(O)N(i-Pr)2
4-OC(O)N(CH3)(C6H5)
0.19
0.22
0.25
29.8
62.4
12.3
5f
0.13
3.51
5g
5h
3-O-t-(C4H9)
3-OCH2CO2-t-(C4H9)
1.79
0.41
Inactive @ 100 mM
Inactive @ 100 nM
5i
3.65
Inactive @ 100 nM
The effect of binding affinity induced by heteroatoms
closer to the phenolic oxygen was studied. Data from 2k
and 2e suggest that the presence of the carbonyl oxygen
in the ester 2k not only increases the a4b1 binding by
4-fold but more importantly, it produces a 20-fold
increase in the a4b7 binding. The carbamate 2l was pre-
pared to study the effect of heteroatoms in 1,3-relation-
ships with respect to the phenolic oxygen. This resulted
in subnanomolar potency for a4b1 and a 20- to 30-fold
increase in potency for a4b7 relative to the esters 2k and
2m. The data from 2k,m–n versus 2l show that the
nitrogen of the carbamate plays a pivotal role in
enhancing the a4b7 activity by conferring a 20- to 30-
fold increase in binding affinity. It also contributes a
ꢁ2-fold increase to the a4b1 activity. Moving it to a
neighboring position in the ring as in 2n decreased the
a4b7 activity. Thus, the addition of appropriately
arrayed oxygen and nitrogen proximal to the phenolic
oxygen of 2a, as in a carbamate (2l), can bring about a
>400-fold increase in a4b7 binding affinity and a 2-fold
increase in a4b1 activity.
be accessed from the 3-position (5g–i). The data from
these carbamates show that for both integrins, particu-
larly a4b7, the more favorable receptor–ligand interac-
tion projects from the phenolic position of Tyr rather
than from the aromatic 3-position of Phe. Although in
the case of 5h, extending the length of the 3-substituent
provided significant improvement in a4b1 binding, sug-
gesting that the longer chain was able to access partially
the a4b1 binding site.
A limited study was undertaken to probe the role of the
N-arylsulfonyl substituent in a4b1 and a4b7 binding. As
shown in Table 3, removal of the two chlorines in 2l made
Table 3. In vitro binding potencies of prolyltyrosyl carbamates 6
versus 125I-VCAM-Ig
The effect of a-alkylation of the prolyl moiety was
examined (Table 2). Data from two pairs of com-
pounds, 2c/5a, and 2l/5b show that the addition of the
a-methyl group had little effect on the a4b1 binding
while the a4b7 binding decreased by 3- to 6-fold. The
scope and limitation of N-substituents on the carbamate
are demonstrated by analogues 5b–f. Data from the
acyclic carbamates (5c–e) suggest that the a4b7 binding
is quite sensitive to steric congestion around the carba-
mate nitrogen while the a4b1 binding is much less
affected.
Compd
R
a4b1
IC50 (nM)
a4b7
IC50 (nM)
21
0.13
1.92
6a
6b
0.14
0.5
2.83
4.4
Several compounds with substituted (3-hydroxy)-Phe’s
at P3 were prepared in an attempt to probe if either the
a4b1 or the a4b7 potency-enhancing binding sites could
SO2CH3

162
L. L. Chang, et al. / Bioorg. Med. Chem. Lett. 12 (2002) 159–163
little difference in binding towards either integrin (6a vs
2l). Replacing the aryl moiety by a methyl group pro-
duced a compound with a more balanced profile, mostly
at the expense of increased a4b1 binding (6b vs 6a).
2. (a) Bevilacqua, M. P. Annu. Rev. Immunol. 1993, 11, 767.
(b) Postigo, A. A.; Teixido, J.; Sanchez-Madrid, F. Res.
Immunol. 1993, 144, 723.
3. (a) Berlin, C.; Berg, E. L.; Briskin, M. J.; Andrew, D. P.;
Kilshaw, P. J.; Holzmann, I. L.; Weissman, A.; Hamann, A.;
Butcher, E. C. Cell 1993, 74, 185. (b) Briskin, J. J.; McEvoy,
L. M.; Butcher, E. C. Nature 1993, 363, 461. (c) Hamann, A.;
Andrew, D. P.; Jablonski-Westrich, D.; Holzmann, B.;
Butcher, E. C. J. Immunol. 1994, 152, 3282. (d) Bargatze, R. F.;
Jutila, M. A.; Butcher, E. C. Immunity 1995, 3, 99.
4. (a) Lobb, R. R.; Hemler, M. E. J. Clin. Invest. 1994, 94,
1722. (b) Lin, K.-C.; Castro, A. C. Curr. Opin. Chem. Bio.
1998, 2, 453. (c) Molossi, S.; Elices, M.; Arrhenius, T.; Diaz,
R.; Coulber, C.; Rabinovitch, M. J. Clin. Invest. 1995, 95,
2601. (d) Foster, C. A. J. Allergy. Clin. Immunol. 1996, 98,
S270. (e) Elices, M. Curr. Opin. Anti-Inflam. Immunomod.
Invest. Drugs 2000, 2, 228.
5. (a) Lobb, R. R.; Adams, S. P. Exp. Opin. Invest. Drugs
1999, 8, 935. (b) Adams, S. P.; Lobb, R. R. Annu. Rep. Med.
Chem. 1999, 34, 179. (c) Engleman, V. W.; Kellogg, M. S.;
Rogers, T. E. Annu. Rep. Med. Chem. 1996, 31, 191. (d)
Shroff, H. N.; Schwender, C. F.; Dottavio, D.; Yang, L.-L.;
Briskin, M. J. Bioorg. Med. Chem. Lett. 1996, 6, 2495. (e)
Harriman, G. C. B.; Schwender, C. F.; Gallant, D.; Cochran,
N. A.; Briskin, M. J. Bioorg. Med. Chem. Lett. 2000, 10, 1497.
6. Durette, P. L.; Hagmann, W. K.; MacCoss, M.; Mills, S.
G.; Mumford, R. A.; Van Riper, G. M.; Schmidt, J. A.;
Kevin, N. J. WO98/53814 A1, 1998; Chem. Abstr. 1999, 130,
52724m.
Thus, systematic SAR studies of the binding of a4b1
and a4b7 integrins to (N-arylsulfonyl)-prolyl-(O-sub-
stituted)-tyrosines revealed that an O-tyrosylcarbamate
could confer a 300- to 400-fold increase to the a4b7
binding affinity over that obtained from the parent
tyrosyl derivative, yielding low nanomolar a4b7 antago-
nists 2l, 5f, and 6a. In this study, this a4b7 binding site is
reached most effectively by a carbamate moiety derived
from the phenolic hydroxyl of P3-Tyr. This binding site
is averse to steric congestion of the carbamate sub-
stituents. The a4b1 integrin also accommodates this
carbamate but the receptor–ligand interaction here is
much less stringent in its spatial requirement of and
tolerance for the positioning of various heteroatoms,
enabling several different structural motifs to achieve a
modest gain in potency.
The pharmacokinetic (PK) profile of several of the
foregoing compounds were evaluated in rats (Table 4).
Generally, these compounds exhibited moderate to high
plasma clearance, low plasma half-life and poor oral
absorption. The metabolic potential of the acidic moiety
and the amide bond could both contribute to this type
of PK profile, in addition to possible metabolic lability
associated with carbamates of phenols.13
7. (a) Durette, P. L.; Hagmann, W. K.; MacCoss, M.; Mills,
S. G.; Mumford, R. A. WO98/53818 A1, 1998; Chem. Abstr.
1999, 130, 33016r. (b) Durette, P. L.; Hagmann, W. K.;
MacCoss, M.; Mills, S. G.; Mumford, R. A. WO98/53817 A1,
1998; Chem. Abstr. 1999, 130, 52736s.
Table 4. Pharmacokinetic parametersa of selected compounds
8. (a) Several structurally similar compounds were described
recently: Dappen, M. S.; Dressen, D. B.; Grant, F. S.; Konradi,
A. W.; Pleiss, M. A.; Semko, C. M.; Thorsett, E. D.; Freedman,
S. B.; Holsztynska, E. J.; Quinn, K. P.; Ashwell, S.; Banker, A.
L.; Baudy, R. B.; Bicksler, J. J.; Giverson, J.; Leeson, P. D.;
Lombardo, L. J.; Sarantakis, D. Abstract of Papers, Part X,
220th National Meeting of the American Chemical Society,
Washington, DC, Aug. 20–24, 2000; American Chemical
Society: Washington, DC, 2000; MEDI #135. (b) Thorsett, E.
D.; Dappen, M. S.; Dressen, D. B.; Ellingboe, J. W.; Grant, F.
S.; Jacobson, M.; Kincaid, S. L.; Konradi, A. W.; Kreft, A.;
Lombardo, L. J.; Mann, W.; Nunn, D.; Pleiss, M. A.;
Sabalski, J. E.; Sacchi, K.; Sarantakis, D.; Semko, C. M.
Abstract of Papers, Part X, 220th National Meeting of the
American Chemical Society, Washington, DC, Aug. 20–24,
2000; American Chemical Society: Washington, DC, 2000;
MEDI #134.
9. (a) Hagmann, W. K.; Durette, P. L.; Lanza, T. J.; Kevin,
N. J.; De Laszlo, S. E.; Kopka, I. E.; Young, D. N.; Magriotis,
P. A.; Li, B.; Lin, L. S.; Yang, G. X.-Q.; Kamenecka, T.; Chang,
L. L.; Wilson, J.; MacCoss, M.; Mills, S. G.; Van Riper, G.;
McCauley, E. D.; Egger, L. A.; Kidambi, U.; Lyons, K. A.;
Vincent, S. H.; Stearns, R. A.; Colleti, A. E.; Teffera, Y.; Tong,
X.; Wang, J.; Zheng, S.; Owens, K. A.; Levorse, D. A.; Kim, P.;
Schmidt, J. A.; Mumford, R. A. Bioorg. Med. Chem. Lett. 2001,
11, 2709. (b) For a study on substituted Phe’s and related
compounds at P3, see: Kopka, I. E.; Young, D.; Lin, L.;
Mumford, R. A.; MacCoss, M.; Mills, S. G.; Van Riper, G.;
McCauley, E.; Egger, L.; Kidambi, U.; Schmidt, J. A.; Lyons,
K.; Stearns, R.; Vincent, S.; Colletti, A.; Wang, Z.; Tong, S.;
Wang, J.; Zheng, S.; Owens, K.; Levorse, D.; Hagmann, W.
K. Bioorg. Med. Chem. Lett. In press.
b
c
Compd
Clp
(mL/kg/min)
t1/2 (h)
F (%)
5b
5e
5f
27
38
50
176
NDd
0.2
ND
0.2
6
11
3
ND
6a
aSprague–Dawley rats.
bDose: 1 mg/kg iv; 2 mg/kg po (cassette dosing).
ct1=2=plasma half-life(0À8h)
.
dND, not determined.
This investigation resulted in the discovery of highly
potent small molecule dual a4b1/a4b7 integrin antago-
nists. Indeed, the dual antagonists identified from this
study, for example, 6a [a4b1 (VCAM-Ig) IC50=0.14 nM,
and a4b7 (VCAM-Ig) IC50=2.83 nM] have been instru-
mental as tools for understanding the biochemistry of
these integrins and their role in inflammatory processes.
Acknowledgement
We thank Ms. Amy Bernick for mass spectral analysis.
References and Notes
1. (a) Butcher, E. C. Cell 1991, 67, 1033. (b) Springer, T. A.
Cell 1994, 76, 301. (c) Cox, D.; Aoki, T.; Seki, J.; Motoyama,
Y.; Yoshida, K. Med. Res. Rev. 1994, 14, 195.
10. This and all other compounds prepared were character-
ized by mass spectrum (FAB or LC/MS), and 300, 400, or
500 MHz H NMR.
1

L. L. Chang, et al. / Bioorg. Med. Chem. Lett. 12 (2002) 159–163
163
11. A similar method has been described: Gibson, F. S.;
Bergmeier, S. C.; Rapoport, H. J. Org. Chem. 1994, 59,
3216.
12. (a) N-Benzylpyroglutamyl-l-phenylalanines have been
evaluated as VLA-4 receptor antagonists: Tilley, J. W.;
Kaplan, G.; Rowan, K.; Schwinge, V.; Wolitzky, B. Bioorg.
Med. Chem. Lett. 2001, 11, 1. (b) 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. Chem. Lett. 2000, 10, 729, and
references cited therein.
13. (a) Hansen, K. T.; Faarup, P.; Bundgaard, H. J. Pharm.
Sci. 1991, 80, 793. (b) Hansen, J.; Mørk, N.; Bundgaard, H.
Int. J. Pharm. 1992, 81, 253.