Aza-bicyclic amino acid sulfonamides as α4β 1/α4β7 integrin antagonists

Article abstract of DOI:10.1016/j.bmcl.2003.11.063

The design, synthesis, and biological activity of novel α ₄β₁ and α₄β₇ integrin antagonists, containing a bridged azabicyclic nucleus, are reported. Conformational analysis of targets containing an azabicyclo[2.2.2]octane carboxylic acid and known integrin antagonists indicated that this azabicycle would be a suitable molecular scaffold. Variation of substituents on the pendant arylsulfonamide and phenylalanine groups resulted in potent α ₄β₁-selective and dual α₄β ₁/α₄β₇ antagonists. Potent compounds 11i, 11h, and 14 were effective in the antigen-sensitized sheep model of asthma. 2003 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/j.bmcl.2003.11.063

Bioorganic & Medicinal Chemistry Letters 14 (2004) 591–596
Aza-bicyclic amino acid sulfonamides as ꢀ4ꢁ1/ꢀ4ꢁ7 integrin
antagonists
Alexey B. Dyatkin,a,* William J. Hoekstra,a William A. Kinney,a Maria Kontoyianni,a
Rosemary J. Santulli,a Edward S. Kimball,a M. Carolyn Fisher,a Stephen M. Prouty,a
William M. Abraham,b Patricia Andrade-Gordon,a Dennis J. Hlasta,a Wei He,a
Pamela J. Hornby,a Bruce P. Damianoa and Bruce E. Maryanoff a,*
aDrug Discovery, Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Spring House, PA 19477-0776, USA
bMount Sinai Medical Center, Miami Beach, FL 33140, USA
Received 5 September 2003; accepted 25 November 2003
Abstract—The design, synthesis, and biological activity of novel a4b1 and a4b7 integrin antagonists, containing a bridged azabi-
cyclic nucleus, are reported. Conformational analysis of targets containing an azabicyclo[2.2.2]octane carboxylic acid and known
integrin antagonists indicated that this azabicycle would be a suitable molecular scaffold. Variation of substituents on the pendant
arylsulfonamide and phenylalanine groups resulted in potent a4b1-selective and dual a4b1/a4b7 antagonists. Potent compounds 11i,
11h, and 14 were effective in the antigen-sensitized sheep model of asthma. #2003 Elsevier Science Ltd. All rights reserved.
# 2003 Elsevier Ltd. All rights reserved.
Integrins are heterodimeric receptors, with a and b
components, that are involved in cell adhesion. Two
representatives of the a4 integrin family, a4b1 and a4b7,
are important in the adhesion of lymphocytes to the
extracellular matrix.1 Integrin a4b1 (VLA-4) binds to
vascular cell adhesion molecule-1 (VCAM-1), which is
expressed on endothelial cell surfaces. The successful
blockade of leukocyte infiltration by antibodies to VLA-4
indicates the potential therapeutic utility of VLA-4
antagonists in treating inflammatory disorders, such as
asthma, multiple sclerosis, and rheumatoid arthritis.
Integrin a4b7 plays a key role in lymphocyte homing in
the intestinal mucosa via interaction with mucosal
addressin cell adhesion molecule-1 (MAdCAM-1), which
is expressed on epithelial cell surfaces. The blockade of
this interaction was found to be beneficial in the treat-
ment of inflammatory bowel and Crohn’s diseases.1 In
addition to binding to MAdCAM-1, a4b7 integrins inter-
act with VCAM-1 and fibronectin, albeit to a lesser extent.
major structural class is the N-acylphenylalanines, with
the N-acyl group frequently resembling proline. Repre-
sentative compounds 1,3 2,4 3,5 4,6 and 57 (Fig. 1) have
exhibited good in vitro activity in cell adhesion and
ELISA assays. We sought to investigate a novel
molecular scaffold for the N-acyl core in the form of
2-azabicyclo[2.2.2]octane (6, Fig. 2). Initially, we were
concerned that the 2-azabicyclic unit might adversely
affect the bioactive conformation of the a4b1 target
molecule. Thus, it was important to study the preferred
conformations of known a4b1 integrin antagonists, such
as 2–4, to determine whether the bicyclic targets would
be suitable. Extensive conformational analysis was per-
formed on condensed8 versions of compounds in Figure
1 by using the Monte Carlo Multiple Minimum
(MCMM)9 search algorithm, followed by energy mini-
mization. The OPLS-AA force field10 was used in all
calculations, with 25,000 steps for the searches and 2000
iterations for minimizations. With regard to protona-
tion of the ionizable groups, we assumed full ionization
at physiological pH. To assure that the Monte Carlo
simulations converged to a global minimum for each
compound, we carried out additional independent
Monte Carlo simulations or increased the number of
steps in those cases where one of the five low-energy
conformers had not converged. Because the low-lying
There are many reports in the scientific and patent lit-
erature of small-molecule a4 integrin antagonists.2
A
* Corresponding author. Tel.: +1-215-628-5008 (A.B.D.); +1-215-
628-5530 (B.E.M.); fax: +1-215-628-4985; e-mail: adyatkin@prdus.
jnj.com; bmaryano@prdus.jnj.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.063

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A. B. Dyatkin et al. / Bioorg. Med. Chem. Lett. 14(2004) 591–596
Figure 1. Nonpeptide a4b1/a4b7 integrin antagonists.
minima were identified several hundred times, it is likely
that the sampling was exhaustive.
overlap of 6 and 2, which suggests that the bicyclic
moiety does not prevent 6 from achieving the preferred
conformation. It should also be noted that the ring
nitrogen atoms are positioned on top of each other,
which implies that the electron distribution has not been
changed.
The global minima of the modeled compounds had the
key functional groups, carboxylate, amide nitrogen, and
phenyl ring (phenylalanine, Phe), oriented in a similar
fashion. This was particularly gratifying, given that 3
(Fig. 1) has a different stereochemistry in the acyl
group. This can be appreciated from Figure 3, where a
root-means-square fitting approach was used for the
overlay.
We have described the successful replacement of proline
by azabicyclic amino acids in novel vasopressin receptor
antagonists.11 The synthetic methodology for preparing
the requisite bridged bicyclic amino acid 7 in enantio-
merically enriched form is well developed.12 Arylsulfo-
nylation, followed by hydrolysis,13 then provided key
intermediate 8, and the coupling of 8 with substituted
Phe esters provided 9 (Scheme 1), which was used to
obtain the targets.
Next, the conformation of target molecule 6 was com-
pared to pyrrolidine 2. Figure 4 shows a representative
Examples of subsequent transformation of 9 are pre-
sented in Schemes 2–4. In Scheme 2, reduction of the
nitro group in 9a, X=NO2, with Zn powder, followed
by acylation of the aniline and hydrolysis of the ester
group afforded target 11 in 75–90% yield. The prepara-
tion of phthalimide 14 is presented in Scheme 3. Because
the phthalimide was opened to 13 on basic hydrolysis,
treatment with acetic acid was required to regenerate
Figure 2. Prototype azabicyclic target 6.
Figure 3. Superposition of the low-energy conformations of
(orange), 3 (magenta), and 4 (green).
2
Figure 4. Overlay of 2 (magenta) and 6 (green).

A. B. Dyatkin et al. / Bioorg. Med. Chem. Lett. 14(2004) 591–596
593
the phthalimide. The Suzuki transformation of 9b
(X=Br) to the aryl-substituted Phe derivative 15 is pre-
sented in Scheme 4.
the cellular adhesion assays are presented in Tables 1
and 2. The SAR in this series are similar to other N-acyl
derivatives of 4-substituted phenylalanine.14 High
potency of the benzamide analogues against the a4b1
integrin was dependent on the presence of 2,6-dis-
ubstitution (e.g., 11b) in the phenyl ring. Mono-
substituted analogues demonstrated moderate potency,
whereas the unsubstituted compound 11a has low
activity. Substitution of phenyl group of Phe in the
2-position or 3-position, leaving the 4-position unsub-
stituted, or inversion of the chirality of Phe resulted in a
larger than 100-fold decrease in potency.
The azabicyclic target compounds were tested for
antagonism of a4b1 and a4b7 integrins. The results of
The configuration of the chiral center of the bicyclic
amino acid strongly affects in vitro activity as well. The
(S)-enantiomers are significantly more active than the
R-enantiomers (11f vs 11g). Additional substitution of
the 4-position of the benzamide (11c) does not affect the
a4b1 and a4b7 activities, while introduction of the basic
nitrogen atom improves potency, compensating for
the lack of 2,6-disubstitution (11h). Linkers between the
aromatic ring carbonyl and R2, such as CH2 (11d) or
NH (11e), dramatically decreased in vitro potency.
Various arylsulfonamide groups could be introduced to
provide compounds with better dual activity, with
phenyl (e.g., 11i) and 2-thienyl (e.g., 11k) derivatives
being the best. Phthalimide15 14 was a strong a4b1
antagonist, with the carbonyl groups probably playing a
similar role to the 2,6-disubstitution of other analogues.
The disubstituted biphenyl 15 has good potency,
Scheme 1. (i) Et3N, CH2Cl2, 23 ꢀC; (ii) LiOH or NaOH, MeOH,
water, 60 ꢀC; (iii) substituted Phe methyl ester, EDAC/HOBt,
i-Pr2NEt/CH2Cl2, 23 ꢀC.
Scheme 2. (i) Zn, NH4Cl, MeOH, reflux, 3 h; (ii) RCOCl, Et3N,
CH2Cl2; (iii) LiOH, MeOH, water, 23 ꢀC.
Scheme 4. (i) ArB(OH)2, Pd(PPh3)4, 50–65%; dimethoxyethane; (ii)
LiOH, MeOH, water, 75–90%.
Scheme 3. (i) phthalic anhydride, AcOH, 120 ꢀC; (ii) NaOH, MeOH/water, rt; (iii) AcOH, 120 ꢀC.

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A. B. Dyatkin et al. / Bioorg. Med. Chem. Lett. 14(2004) 591–596
Table 1. Inhibition of a4b1 and a4b7 integrins by N-arylsulfonyl[2.2.2] azabicyclooctane amide derivatives (IC50
)
Compd
R1
R2
Chirality*
a4b1/VCAM-1
a4b7/MAdCAM-1
(mM)
(mM)
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11r
3
4-MeC6H4
4-MeC6H4
4-MeC6H4
4-MeC6H4
4-MeC6H4
Ph
Ph
2,6-Cl2C6H3
2,4,6-Cl3C6H2
CH2-2,6-Cl2C6H3
NH-2,6-Cl2C6H3
2,6-Cl2C6H3
2,6-Cl2C6H3
4-pyridyl
2,6-Dichloro-4-pyridyl
2,6-Cl2C6H3
2,6-Dichloro-4-pyridyl
2,6-Dichloro-4-pyridyl
2,6-Dichloro-4-pyridyl
2,6-Dichloro-4-pyridyl
2,6-Dichloro-4-pyridyl
2,6-Dichloro-4-pyridyl
2,6-Dichloro-4-pyridyl
RS
RS
RS
RS
RS
S
R
S
S
S
0.90
30% @ 5
0.32
0.045Æ0.006
0.051Æ0.019
1.1
0.43Æ0.23
28% @ 5
NT
1.6
0.019Æ0.003
0.094Æ0.065
Ph
Ph
Ph
1.6
NT
0.036Æ0.009
0.012Æ0.004
0.030Æ0.011
0.005Æ0.001
0.011Æ0.002
0.069Æ0.011
0.105
0.081Æ0.015
0.012Æ0.002
0.15
0.175Æ0.107
0.013Æ0.008
0.062Æ0.029
0.032Æ0.019
0.064Æ0.001
0.014Æ0.002
0.011Æ0.003
0.102Æ0.045
0.022Æ0.011
0.151Æ0.009
0.090Æ0.031
2-Thienyl
2-Thienyl
5-Cl-2-thienyl
4,5-Cl2-2-thienyl
2,6-Cl2C6H3
2-CF3C6H4
8-Isoquinolinyl
8-Quinolinyl
S
RS
RS
RS
RS
S
S
0.41Æ0.14
Data represent inhibition of binding of a4b1+ cells to immobilized VCAM-1 or a4b7+ cells to immobilized MAdCAM-1. Confidence intervals were
usually calculated with N=3. All compounds were characterized by using NMR and MS data; they also gave satisfactory elemental analyses. For 3,
the reported literature value for a4b1: IC50=0.034 mM.5
Table 2. IC50 data for other azabicyclic a4b1 and a4b7 integrin
antagonists
carbachol required to provoke a 400% increase in SRL
(PC400). The test compounds were administered in a
single 10-mg aerosol dose 0.5 h prior to antigen chal-
lenge and our results are presented in Figure 5. In the
control animals, antigen challenge caused an increase in
airway resistance in the early phase (EP), and again
during the late phase (LP). Airway hyperreactivity
(AHHR) was increased at 24 h post challenge. Com-
pound 11h (N=3) and 11i (N=3) exhibited similar
degrees of efficacy and potency. Both compounds com-
pletely blocked the LP and the AHHR responses. Nei-
ther compound affected the EP response. In contrast, 14
completely blocked both the LP and AHHR response
and also demonstrated inhibition of the EP response.
Results are similar to those reported by Archibald et al.
for 3,5 which demonstrated efficacy of an a4b1 antago-
nist in the sheep model at 0.3 mg/kg administered iv 1 h
prior to antigen challenge.
Compd
X
a4b1/VCAM-1
(mM)
a4b7/MAdCAM-1
(mM)
14
15
5
Phthalimido
2,6-(MeO)2C6H3
0.026Æ0.003
0.081
0.030Æ0.004
0.312Æ0.056
0.090
0.119Æ0.013
For 5, the reported literature values for a4b1 and a4b7 are, respectively:
IC50=0.002 and 0.26 mM.7
whereas the unsubstituted analogue of 15 lost activity;
similar correlations were observed for Pro analogues.16
In summary, we have discovered novel, potent a4b1-
selective and a4b1/a4b7 dual integrin antagonists by
incorporating azabicyclic amino acid group as a proline-
mimetic scaffold. Selected compounds exhibited good in
vivo activity in the sheep model of asthma, which could
reflect their potential in the treatment of asthma and
other inflammatory diseases in humans.
On the basis of in vitro potency and chemical diversity,
we selected 11h, 11i and 14 for in vivo studies, and
evaluated them in the antigen-sensitized sheep model of
asthma.17 In untreated sheep, aerosol challenge with
Ascaris suum results in an immediate increase in specific
lung resistance (SRL), which persists for 2–4 h (early-
phase bronchoconstrictive response). Between 6 and 8 h
post challenge, SRL increases a second time in the late-
phase bronchoconstrictive response, similarly to that
observed in humans. Airway hyperreactivity is then
examined 24 h post antigen challenge by the amount of
Acknowledgements
We thank Dr. Lawrence de Garavilla for valuable
contributions.

A. B. Dyatkin et al. / Bioorg. Med. Chem. Lett. 14(2004) 591–596
595
Figure 5. Antigen-induced early-phase and late-phase responses (A, C, E), and airway hyperresponsiveness to carbachol (B, D, F) in the allergic
sheep: Solid circles represent control trial and open circles represent treatment trial in the same animals (A, C, E). Hatched bars represent control
trial and solid bars represent treatment trial in the same sheep (B, D, F). Results for 11i are shown in A and B; 14 in Cand D; 11h in E and F.
Antigen-induced LAR was completely inhibited by all three compounds (A, C, E) while 14 also inhibited the EAR (C). Each compound completely
reversed the airway hyperresponsiveness (indicated by the decrease in post-antigen PC400) to carbachol (B, D, F). A 10-mg dose in the sheep model
is equivalent to 0.25 mg/kg based on sheep weights.5
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