Selection of a 2-azabicyclo[2.2.2]octane-based α4β1 integrin antagonist as an inhaled anti-asthmatic agent

Article abstract of DOI:10.1016/j.bmc.2006.01.067

The α₄β₁ integrin, expressed on eosinophils and neutrophils, induces inflammation in the lung by facilitating cellular infiltration and activation. From a number of potent α₄β₁ antagonists that we evaluated for

Full text of DOI:10.1016/j.bmc.2006.01.067

Bioorganic & Medicinal Chemistry 14 (2006) 4208–4216
Selection of a 2-azabicyclo[2.2.2]octane-based a4b1
integrin antagonist as an inhaled anti-asthmatic agent
a,
a
a
Edward C. Lawson,a,* Rosemary J. Santulli, Alexey B. Dyatkin, Scott A. Ballentine,
William M. Abraham,b Sandra Rudman,c Clive P. Page,c Lawrence de Garavilla,a
Bruce P. Damiano,a William A. Kinneya and Bruce E. Maryanoffa
*
aResearch & Early Development, Johnson & Johnson Pharmaceutical Research & Development, Welsh and McKean Roads,
Spring House, PA 19477-0776, USA
bDivision of Pulmonary Disease, University of Miami at Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140, USA
cSackler Institute of Pulmonary Pharmacology, GKT School of Biomedical Sciences, King’s College London, London SE1 9RT, UK
Received 16 December 2005; revised 25 January 2006; accepted 26 January 2006
Available online 21 February 2006
Abstract—The a4b1 integrin, expressed on eosinophils and neutrophils, induces inflammation in the lung by facilitating cellular infil-
tration and activation. From a number of potent a4b1 antagonists that we evaluated for safety and efficacy, 1 was selected as a lead
candidate for anti-asthma therapy by the inhalation route. We devised an optimized stereoselective synthesis to facilitate the prep-
aration of a sufficiently large quantity of 1 for assessment in vivo. Administration of 1 to allergen-sensitive sheep by inhalation
blocked the late-phase response of asthma and abolished airway hyper-responsiveness at 24 h following the antigen challenge. Addi-
tionally, the recruitment of inflammatory cells into the lungs was inhibited. Administration of 1 to ovalbumin-sensitized guinea pigs
intraperitoneally blocked airway resistance and inhibited the recruitment of inflammatory cells.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
ed. The a4b1 integrin mediates cell adhesion by binding
to either of two ligands, VCAM-1 or the alternatively
spliced CS1-containing fibronectin variant, Fn-CS1.2
Additionally, a4b1 interacts with the matrix ligand oste-
opontin,3 which may be important in that osteopontin is
strongly up-regulated in inflammatory settings.4
Integrins are heterodimeric glycoproteins, composed of
an a and a b subunit, which are expressed on cell surfac-
es and involved in cell–cell and cell–extracellular matrix
interactions. Two integrins containing the a4 subunit
(CD49D) have been described: a4b1 and a4b7. The inte-
grin a4b1 (very late antigen-4, VLA-4, CD49d/CD29) is
expressed on eosinophils, mononuclear leukocytes, mast
cells, macrophages, basophils, and neutrophils.1 In con-
The interaction of a4b1 with VCAM-1 is responsible for
mononuclear leukocyte and eosinophil adhesion to the
endothelium and subsequent transendothelial migration.
Integrin a4b1 is believed to mediate airway inflammation
by facilitating infiltration of lymphocytes and eosino-
phils into the lung and inducing expression of inflamma-
tory mediators. Airway eosinophilia is associated with
pulmonary inflammation. Moreover, the interaction of
a4b1 with its ligands leads to the activation of eosino-
phils, mast cells, and T-cells, as well as inhibition of
apoptosis to increase cell survival.5 While eosinophil
recruitment appears to be involved in the early- and
late-phase responses of asthma, conflicting evidence
exists as to whether eosinophil recruitment plays
a role in airway hyper-responsiveness. For example, in
allergen-sensitized sheep and rats,6,7 inhibition of airway
hyper-responsiveness does not correlate to eosinophil
trast to the prototypical integrins, such as a5b1, aIIbbIIIa
,
and avb3, that recognize the Arg-Gly-Asp (RGD) pep-
tide sequence in their respective protein ligands, a4b1
binds to other primary protein sequences: Gln-Ile-Asp-
Ser (QIDS) in vascular cell adhesion molecule-1
(VCAM-1) and Ile-Leu-Asp-Val (ILDV) in fibronectin.
Although these a4b1 recognition motifs share a common
Asp (D) residue with RGD, they are otherwise unrelat-
Keywords: Integrin; Antagonist; VLA-4.
*
Corresponding authors. Tel.: +1 215 628 5644 (E.C.L.), +1 215 628
7804 (R.J.S.); fax: +1 215 628 4985; e-mail addresses:
elawson@prdus.jnj.com; rsantull@prdus.jnj.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.067

E. C. Lawson et al. / Bioorg. Med. Chem. 14 (2006) 4208–4216
4209
O
OH
accumulation in broncho-alveolar lavage (BAL) fluid.
The identification of a4b1 on neutrophils, particularly
those in the lungs, suggests that neutrophils may be a
key effector cell in airway hyper-responsiveness.1d,8 This
viewpoint is further supported by the ability of intrana-
sally delivered a4b1 monoclonal antibodies to bind pul-
monary neutrophils and block inflammatory responses,
such as cytokine release and mucus secretion. Taken
together, there is ample evidence that a4b1 is a major
player in inflammatory processes in the allergen-sensi-
tized lung via its capability to invoke key cellular and
biochemical signals by interacting with its ligands.
O
ii, iii
i
O
Ph
Ph
O
CHO
Ph
O
OH
O
3
2
O
O
O
O
O
iv, v
N
N
N
OH
Ph
Ph
Me
Me
Boc
Ph
Ph
5 (desired isomer)
6
4 (desired isomer)
Scheme 1. Synthesis of 6. Reagents and conditions: (i) HIO6, Et2O;
˚
(ii) (R)-(+)-a-methylbenzylamine, 4 A MS, CH2Cl2,
3 to 23 ꢁC;
(iii) CF3CO2H, BF3-OEt2, 1,3-cyclohexadiene, À25 ꢁC, 74% 4 + 5;
(iv) 10% Pd/C, H2O, MeOH, 50 psig H2, 85%; (v) Boc2O, 3 N NaOH,
1,4-dioxane, 0–23 ꢁC, 99%.
Most importantly, antibodies to a4b1 and small-mole-
cule a4b1 antagonists have been reported to inhibit cell
recruitment to the lungs and allergic airway responses
in animal models of asthma.6,9–12
room temperature over 18 h formed the corresponding
imine in situ. The formation of the imine was achieved
more effectively by this method versus using a 30-min
time period at 0 ꢁC, as reported in the literature. The
molecular sieves were removed and the solution was
cooled to À25 ꢁC. Trifluoroacetic acid, boron trifluoride
etherate, and 1,3-cyclohexadiene were added sequential-
ly followed by stirring at À25 ꢁC for 16 h to provide de-
sired diastereomers 4 and 5 in 74% overall yield after
chromatography. When the Diels–Alder reaction was
performed according to the literature18 without remov-
ing the molecular sieves and at À78 ꢁC for 5 h followed
by warming to room temperature, diastereomers 4 and 5
were isolated in only 38% overall yield after chromatog-
raphy. Hydrogenation of 4 and 5 reduced the alkene, re-
moved the benzyl ester, and cleaved the a-methylbenzyl
group to give the corresponding 2,2,2-bicyclic amino
acid in good yield as a single isomer. Treatment of this
amino acid with t-butoxycarbonylanhydride gave Boc-
protected amino acid 6.
Since a4b1 plays a key role in the migration of leukocytes
into tissues during inflammatory responses,13 an antag-
onist would be expected to have a therapeutic benefit
in a number of inflammatory and autoimmune diseas-
es.14 Natalizumab, a humanized monoclonal antibody
to a4b1, was approved by the FDA for treatment of
the autoimmune disease multiple sclerosis and was in
clinical trials for treatment of Crohn’s disease. However,
its marketing and clinical trials have been suspended due
to cases of progressive multifocal leukoencephalopathy
(PML). The extent of risk is unclear for this highly effi-
cacious drug and is currently under extensive debate and
review.15
We have been interested in developing a suitable a4b1
antagonist for inhaled anti-asthma treatment, especially
to avoid extensive systemic exposure. Previously, we de-
scribed 2-azabicyclo[2.2.2]octane and 1,2,4-triazolo[2,3-
a]pyrrole derivatives as novel antagonists of the inte-
grins a4b1 and a4b7.16,17 From the former series, we
selected a lead compound that was advanced into in vivo
evaluation. In this paper, we present details relating to
the synthesis, biological profile, and selection criteria
for this advanced lead, 2-azabicyclo[2.2.2]octane deriva-
tive 1. In particular, we report the in vivo evaluation of 1
in sheep and guinea pig models of asthma.
We synthesized dimethylcarbamate derivative 1 starting
with amino ester 8, which was obtained in good yield by
deprotonation of L-tyrosine methyl ester with NaH fol-
lowed by addition of dimethylcarbamoyl chloride at
À15 ꢁC (Scheme 2). Carboxylic acid 6 was coupled with
amine 8 by using bis(2-oxo-3-oxazolidinyl)phosphinic
chloride (BOP-Cl) to yield the N-Boc-protected 2,2,2-
azabicyclic amide. Removal of the Boc-protecting group
with trifluoroacetic acid, followed by sulfonamide for-
O
HN
O
N
S
O
NMe2
OH
O
O
O
OH
O
i
NMe2
H2N
H2N
OMe
O
OMe
1
O
O
8
7
O
2. Chemistry
ii, iii, iv
v
O
O
6
1 (Na salt)
N
S
O
HN
NMe2
The optimized stereoselective synthesis of the 2,2,2-aza-
bicyclic core is described in this report (Scheme 1). The
synthesis of 4 and 5 was realized by optimizing the liter-
ature procedure for reaction scale-up.18 Oxidative cleav-
age of diol 2 by using periodic acid provided
benzylglyoxylate 3. Treatment of benzylglyoxylate 3
O
OMe
Ph
O
9
Scheme 2. Synthesis of target compound 1. Reagents and conditions:
(i) NaH, dimethylcarbamoyl chloride, DMSO, THF, À15 to 0 ꢁC,
56%; (ii) 8, BOP-Cl, Et3N, CH2Cl2, 60%; (iii) CF3CO2H, CH2Cl2, 97%;
(iv) PhSO2Cl, Et3N, CH2Cl2, 0 to 23 ꢁC, 98%; (v) NaOH, H2O, THF,
99%.
˚
with (R)-a-methylbenzyl amine and 4 A molecular sieves
in CH2Cl2 at 3 ꢁC followed by gradually warming to

4210
E. C. Lawson et al. / Bioorg. Med. Chem. 14 (2006) 4208–4216
Table 1. Inhibition binding of a4b1 (Ramos) or a4b7 (K562) positive
cells to hVCAM-1 by N-phenylsulfonyl[2.2.2]bicyclooctane derivatives
increase in specific lung resistance (SRL), known as the
early-phase response. Between 4 and 6 h after antigen
challenge, SRL returns to baseline, but then 6–8 h fol-
lowing antigen challenge, SRL is elevated a second time,
resulting in a late-phase broncho-constrictive response.
Airway hyper-responsiveness is observed at 24 h follow-
ing antigen challenge. These three responses in the aller-
gic sheep model parallel the airway responses that are
seen in humans with asthma. In a preliminary study
(Fig. 1), a single aerosolized dose of 1 (0.25 mg/kg)
was administered, 30 min prior to antigen challenge. A
modest effect was seen on airway resistance in the ear-
ly-phase and the late-phase was completely blocked,
similar to what was observed with 12.16a All four com-
pounds blocked airway hyper-responsiveness, but 10
and 11 had no effect on the early phase of airway resis-
tance. Although 1 and 12 exhibited the best efficacy in
the initial asthma model, 12 possessed some cardiovas-
cular liabilities.20 Thus, 1 was selected for further
evaluation.
(IC50
)
O
N
HN
O
S
Ph
O
O
R
OH
Compound R group
a4b1
(nM)
a4b7
(nM)
10
11
12
1
–NHC(O)-4-pyridyl 36
–NHC(O)-2,6-dichloro-4-pyridyl 12
9
120 40
4
3
9
46
110 40
9
Phthalimido
–OC(O)NMe2
26
39
440 150
mation with benzenesulfonyl chloride, gave the 2,2,2-
azabicyclic derivative 9. Hydrolysis of the methyl ester
with NaOH provided the corresponding sodium salt of
1. Derivatives 10–12 (Table 1) were prepared by similar
methods, as described elsewhere.16
Compound 1 was then evaluated in the sheep asthma
model at two doses, 0.075 and 0.25 mg/kg, administered
twice daily for three consecutive days and 0.5 h prior to
antigen challenge via inhalation. The early-phase
response was slightly affected only by the lower dose,
while the late-phase response was completely blocked
by either dose of 1. The airway hyper-responsiveness
was completely abolished with the 0.25 mg/kg dose,
while the 0.075 mg/kg dose was partially effective
(Fig. 2).
3. In vitro biological results
Previously, we described the SAR for the 2-azabicyclo-
[2.2.2]octane arylsulfonamide series of a4 integrin antag-
onists, along with some in vivo results at a single dose
for three of the best compounds (Table 1, 10–12).16a
Here, we present a more detailed pharmacological pro-
file of 1 and our rationale for designating 1 as a lead
compound. Compound 1 has good in vitro inhibition
against the a4b1 (IC50 = 39 nM) integrin with potency
similar to that of 10–12. Like 10 and 12, only modest
potency was observed in the a4b7 (IC50 = 440 nM) adhe-
sion assay. In contrast, the 2,6-dichloro-4-pyridyl deriv-
ative 11 showed good dual potency against the two a4
integrins. Only weak inhibition for 1 against the a5b1
integrin was observed (IC50 = 7200 nM). Compound 1
exhibits high aqueous solubility (>1 mg/mL) at pH 7.4,
and excellent stability in human liver microsomes
(t1/2 > 100 min) and in human and rat hepatic S9
cells (89% and 86% remaining at 90 min, respectively).
Human lung S9 fraction also had no effect on 1, with
>99% remaining at 90 min. By contrast, 11 was only
moderately stable in human liver microsomes
(t1/2 = 39 min). Compound 1 showed no inhibition of
any P450 enzymes. Reasonable plasma protein binding
was observed, 87% in plasma and 91% in serum albu-
min, and there was low binding to red blood cells (13%).
Broncho-alveolar lavage (BAL) fluid from antigen-treat-
ed sheep was also examined for changes in cell influx at
baseline (prior to antigen) and at 8 and 24 h after anti-
gen challenge at both doses of 1. A pronounced reduc-
tion of eosinophil recruitment was observed at 0.075
and 0.25 mg/kg, at both the 8 and 24 h time points
(Fig. 3). Neutrophil recruitment was also markedly re-
duced at 24 h for both doses relative to the vehicle
group. Compound 1 showed moderate bioavailability
by the inhalation route in guinea pigs (F = 9%, Cmax
of 1.5 lM, t1/2 = 0.8 h, 10 mg/kg) and was rapidly elim-
inated from the plasma. It was not orally bioavailable in
rats (F < 1%).
To demonstrate the efficacy of 1, a second species, we
used the passively sensitized guinea pig model,21 assess-
ing airway hyper-responsiveness and eosinophil recruit-
ment into the BAL fluid. Aerosol challenge of passively
sensitized guinea pigs with ovalbumin (OVA) for 1 h
produced a marked increase in the airway hyper-respon-
siveness at 24 h relative to a na¨ıve group. A large in-
crease of eosinophil recruitment was observed in the
BAL fluid at the 24 h time point. Compound 1 was
administered intraperitoneally for 7 days and at 24 h
prior to assessing lung function in the passively sensi-
tized guinea pigs at three different doses (0.1, 0.5, and
1 mg/kg). Examination of the BAL fluid showed a sig-
nificant reduction in eosinophil recruitment at the doses
of 0.5 and 1 mg/kg (Fig. 4). A reduction in eosinophil
recruitment was also observed at 0.1 mg/kg, but to a
lesser extent. Airway hyper-responsiveness at 24 h
4. In vivo biological studies
We proceeded to evaluate 1 in the well-characterized
preclinical sheep model of asthma19 via inhalation deliv-
ery and compare it to 10–12.16a The allergic sheep model
measures the efficacy of experimental compounds
against three distinct responses to allergen provoca-
tion.19 Aerosol challenge of naturally sensitive animals
with the antigen Ascaris suum results in an immediate

E. C. Lawson et al. / Bioorg. Med. Chem. 14 (2006) 4208–4216
4211
20
B
A
800
700
600
500
400
300
200
100
0
15
10
5
0
Baseline
24 hours
0
1
2
3
4
5
6
7
8
Hours
Figure 1. Effect of aerosol administration of 1 to conscious allergic sheep on airway mechanics. (A) Increase in SRL in control animals (d) and
animals treated with 1 (m), single dose of 0.25 mg/kg, 0.5 h prior to antigen challenge. (B) Change in airway responsiveness at baseline and 24 h post-
antigen challenge in animals treated with single 0.25 mg/kg dose of 1 (hatched bar) and control animals (solid bar).
A
B
40
30
20
10
0
600
500
400
300
200
100
0
0
2
4
6
8
Baseline
24 hours
Hours
D
C
40
30
20
10
0
600
500
400
300
200
100
0
Baseline
24 hours
0
2
4
6
8
Hours
Figure 2. Effect of aerosol administration of 1 to conscious allergic sheep by aerosol on airway mechanics. (A) Increase in SRL in control animals (d)
and animals treated with 1 (m) at 0.075 mg/kg. (B) Change in airway responsiveness at baseline and 24 h post-antigen challenge in animals treated
with 0.075 mg/kg of 1; solid bar, control animals; hatched bar animals treated with 1. (C) Increase in SRL in control animals (d) and animals treated
with 1 (m) at 0.25 mg/kg. (D) Change in airway responsiveness at baseline and 24 h post-antigen challenge in animals treated with 0.25 mg/kg of 1;
solid bar, control animals; hatched bar, treated animals.
showed a large reduction at the 0.5-mg/kg dose with re-
spect to the increase in airway resistance resulting from
ovalbumin treatment. In contrast, when 1 was adminis-
tered at 0.1 mg/kg, there was no effect on airway hyper-
responsiveness at 24 h.
reduction in eosinophil and neutrophil cell recruitment
to the lungs. In guinea pigs, 1 produced a large reduc-
tion in inflammatory cell recruitment, which is consis-
tent with antagonism of a4b1.
6. Experimental
5. Conclusion
6.1. General methods
Compound 1 was selected as our lead candidate for an
inhaled anti-asthma therapeutic, because of its good
potency in the a4b1 adhesion assay (IC50 = 39 nM), its
clean in vitro and in vivo safety profile, and its excellent
efficacy in the sheep model of asthma. When dosed by
inhalation to allergen-sensitive sheep, 1 blocked the
late-phase asthmatic response, abolished airway hyper-
responsiveness at 24 h post-dosing, and caused a marked
1H NMR spectra were acquired at 300.14 MHz on a
Bruker Avance-300 spectrometer in CDCl3 unless indi-
cated otherwise, using Me4Si as an internal standard.
NMR abbreviations used: s, singlet; d, doublet; dd, dou-
blet of doublets; t, triplet; m, multiplet; br, broad; ov,
overlapping. HPLC analyses were performed on a Hew-
lett Packard Series 1100 HPLC instrument eluting with

4212
E. C. Lawson et al. / Bioorg. Med. Chem. 14 (2006) 4208–4216
45
150
125
100
75
A
A
40
35
30
*
*
25
*
20
15
10
5
50
25
0
NI
Veh
0.1
0.5
1. 0
0
Baseline
8 h
24 h
compound 1 (mg/kg)
B
120
100
80
60
40
20
0
B
50
40
30
20
10
0
*
*
*
Baseline
8 h
24 h
NI
Veh
0.1
0.5
1.0
Figure 3. Effect of aerosol administration of 1 to conscious allergic
sheep by aerosol on cell influx to the BAL. (A) Number of eosinophils
in BAL at baseline, 8 and 24 h after antigen challenge. Solid bar
represents BAL from control animals; hatched bar represents BAL
from animals treated with 0.075 mg/kg of 1; gray bar represents
animals treated with 0.25 mg/kg of 1 in both graphs. (B) Number of
neutrophils in BAL at baseline, 8 and 24 h after antigen challenge.
compound 1 (mg/kg)
200
150
100
50
C
a gradient of water/MeCN/CF3CO2H (10:90:0.2 to
90:10:0.2) over 4 min with a flow rate of 0.75 mL/min
on a Kromasil C18 column (50 · 2.0 mm; 3.5 lm parti-
*
cle size) or on
a Supelcosil ABZ+Plus column
0
(50 · 2.0 mm; 3.5 lm particle size) at 32 ꢁC. Signals were
recorded simultaneously at 220 and 254 with a diode ar-
ray detector. Normal-phase preparative chromatogra-
phy was performed on an Isco Combiflash Separation
System Sg 100c equipped with a Biotage FLASH Si
Veh
0.1
0.5
compound 1 (mg/kg)
Figure 4. Effect of ip administration of 1 to passively sensitized guinea
pigs. (A) Total cell number in BAL collected at 24 h following antigen
challenge. Solid bars represent non-immunized animals (NI); gray bars
represent immunized vehicle (Veh)-treated animals; hatched bars
represent animals treated with 0.1, 0.5, and 1 mg/kg of 1. (B) Percent
eosinophils of total cells recovered from BAL collected at 24 h
following antigen challenge. Bars have the same meaning as in
previous graph. (C) Percent increase in RL following challenge with
OVA (100 lg/kg) at 24 h post-initial exposure to OVA. *Significance
of P < 0.05, ANOVA; N = 6.
˚
40M silica gel cartridge (KP-Sil Silica, 32–63 l, 60 A;
4 · 15 cm) eluting at 35 mL/min with detection at
254 nm. Reversed-phase preparative chromatography
was performed on a Gilson HPLC with a Kromasil col-
˚
umn (10 l, 100 A C18, column length 250 · 50 mm).
Optical rotations were measured on a Perkin-Elmer
241 polarimeter. Electrospray (ES) mass spectra were
obtained on a Micromass Platform LC single quadru-
pole mass spectrometer in the positive mode. Accurate
mass spectra were run on a Micromass Autospec-OA-
TOF double focusing mass spectrometer using fast atom
bombardment ionization with thioglycerol as the sample
matrix. Elemental analysis and Karl Fischer water anal-
ysis were determined by Quantitative Technologies Inc.,
Whitehouse, NJ.
(2, 86.5 g, 0.262 mol) and 2 L of anhydrous ethyl
ether. To the resulting solution was added periodic
acid dihydrate (59.7 g, 0.262 mol). The slurry was stir-
red at 23 ꢁC for 1.5 h during which a white precipitate
had formed. The cloudy solution was filtered through
Celite (washing with 200 mL ethyl ether) and the fil-
trate was concentrated in vacuo (not to dryness) to
give benzyl glyoxylate (3), which was used without
any further purification. 1H NMR (C6D6) d 8.87
(s, 1H), 7.28–7.43 (m, 5H), 5.12 (s, 2H). The crude
benzyl glyoxylate (85.9 g, 0.524 mol) was dissolved
in dichloromethane (2 L) and charged into a 3-L,
6.2. 2-(1-(S)-Phenethyl)-2-(S)-azabicyclo[2.2.2]oct-5-ene-
3-carboxylic acid benzyl ester (4 and 5)
A 3-L, one-necked round-bottomed flask (equipped
with nitrogen inlet) was charged with dibenzyl tartrate

E. C. Lawson et al. / Bioorg. Med. Chem. 14 (2006) 4208–4216
4213
four-necked flask (equipped with mechanical stirrer,
6.3. (S)-2-Azabicyclo[2.2.2]octane-3-carboxylic acid18
nitrogen inlet, thermocouple, and a glass stopper).
The reaction was cooled in an ice water bath to an
internal temperature of ꢀ3 ꢁC and was treated sequen-
A 2-L Parr bottle was charged (under nitrogen) with
10% palladium on carbon, ꢀ50% water (18 g, Degussa
type E101 NE/W), and methanol (250 mL). The 2-(1-
(S)-phenethyl)-2-(S)-azabicyclo[2.2.2]oct-5-ene-3-carbox-
ylic acid benzyl ester (4 and 5, 88.4 g, 0.254 mol) was
dissolved in methanol (250 mL), added the Parr bottle,
and diluted with methanol (500 mL). The reaction mix-
ture was agitated on a shaker at 23 ꢁC under 40 psig of
hydrogen for 2 h. The catalyst was removed by filtration
and the filtrate was concentrated in vacuo to give a semi-
solid product, which was dissolved in ethyl acetate
(200 mL), with a minimal amount of methanol, and
treated with 1 N HCl in ethyl ether (275 mL). The
mixture was concentrated until a slurry of white solid
formed, diluted with ethyl acetate (100 mL), and
concentrated again. This process was repeated two more
times to remove methanol, and the resulting off-white
solid was collected by filtration/washing with ethyl
acetate (200 mL) and ethyl ether (100 mL). After drying,
41.2 g (85%) of the desired amino acid hydrochloride
salt was obtained as an off-white solid.
˚
tially with 104 g of freshly crushed 4 A molecular sieves
and (R)-(+)-a-methyl benzylamine (62.2 g, 0.514 mol).
The reaction mixture was allowed to slowly warm to
room temperature over an 18 h period (overnight).
The molecular sieves were removed by filtration
through Celite (washing with dichloromethane) and
the filtrate of the desired imine was used directly with-
out any purification. The imine solution (ꢀ0.524 mol)
was charged into a 5-L, three-necked flask (equipped
with a mechanical stirrer, nitrogen inlet, and a thermo-
couple), diluted with dichloromethane (500 mL), and
chilled in a dry ice/acetonitrile bath to an internal tem-
perature of À25 ꢁC. To this solution were added
sequentially trifluoroacetic acid (59.7 g, 0.524 mol),
boron trifluoride etherate (74.4 g, 0.524 mol), and 1,3-
cyclohexadiene (46.2 g, 0.576 mol). The reaction
mixture was stirred for 5 h keeping the internal temper-
ature between À25 and À15 ꢁC by occasional addition
of additional dry ice. The reaction appeared to be
mostly complete by NMR (disappearance of imine
C-H) but the reaction mixture was stored in freezer
(À25 ꢁC) for 16 h (overnight). The reaction mixture
was split into two equal portions and each portion
was quenched into 1.5 L of saturated sodium bicarbon-
ate and stirred vigorously for 1.5 h. The layers were
separated and the aqueous phase was extracted with
dichloromethane (2 · 250 mL). The organic extracts
were each washed with saturated sodium bicarbonate
(250 mL) then combined, dried (MgSO4), and concen-
trated in vacuo to give 208 g of crude product. The
red oil was dissolved in ethyl acetate and passed
through a Biotage-activated carbon cartridge (75M,
ꢀ400 g), and eluted off with ethyl acetate (3 L). This
material was dissolved in heptane and dichloromethane
(1 L: 50 mL), and loaded onto a Biotage 150 L (5 kg
silica gel) and eluted with heptane (4 L), and then ethyl
acetate–heptane 3:97 (36 L), 1:19 (20 L), 1:9 (10 L), and
1:4 (20 L) to yield 101.7 g (57%) of 4 and 30.3 g (17%)
of 5.
[a]D +14.2ꢁ (c 2.15, MeOH; 23 ꢁC), literature [a]D +15.0ꢁ
(c 0.85, EtOH);18 1H NMR (CD3OD) d 4.13–4.16 (m,
1H), 3.49–3.53 (m, 1H), 2.33–2.37 (m, 1H), 1.74–1.90
(m, 9H); MS (ES) m/z = 156 (M + H)+. Anal. Calcd
for C8H13NO2ÆHCl: C, 50.14; H, 7.36; N, 7.31; Cl,
18.50. Found: C, 50.37; H, 7.63; N, 7.13; Cl, 18.35.
6.4. (S)-2-N-(t-Butyloxycarbonyl)azabicyclo[2.2.2]-
octane-3-carboxylic acid (6)
A 1-L round-bottomed flask containing (S)-2-azabicy-
clo-[2.2.2]octane-3-carboxylic acid (11.34 g, 59.4 mmol)
and 1,4-dioxane (300 mL) was cooled in an ice/water
bath. 3 N NaOH (60 mL, 180 mmol) was added, fol-
lowed by di-t-butyl dicarbonate (13.02 g, 59.7 mmol),
and the mixture was stirred at 0 ꢁC for 1 h, and then
at 23 ꢁC for 4 h. After quenching with citric acid
(34.7 g, 181 mmol), the mixture was diluted with water
(200 mL)
and
extracted
with
ethyl
acetate
(2 · 600 mL). The combined organic layers were dried
(MgSO4), filtered through Celiteꢂ, and concentrated in
vacuo to give 15.0 g (99%) of 6 as a white solid.
Compounnd 4: [a]D À64.6ꢁ (c 1.23, CHCl3, 23 ꢁC); one
isomer by 1H NMR (CDCl3) d7.16–7.40 (m, 10H),
6.37–6.41 (m, 1H), 6.25–6.30 (m, 1H), 4.94 (s, 2H),
3.60–3.66 (m, 1H), 3.40–3.47 (m, 1H), 2.92–2.98 (m,
1H), 2.73–2.76 (m, 1H), 2.01–2.06 (m, 2H), 1.52–1.63
(m, 3H), 1.21–1.33 (m, 5H), 1.01–1.06 (m, 1H), 0.86–
0.90 (m, 1H); MS (ES) m/z = 348 (M+H)+; HRMS
[a]D À29.2ꢁ (c 1.11, MeOH; 23 ꢁC); 1H NMR (CD3OD)
d 4.12–4.14 (m, 1H), 3.95–4.02 (m, 1H), 2.12–2.19 (m,
1H), 1.89–1.98 (m, 1H), 1.47–1.77 (m, 7H), 1.42 (s,
9H); HRMS (FAB) m/z 256.1550 (256.1549 calcd for
C13H21NO4 + H+).
(FAB)
m/z
348.1979
(348.1964
calcd
for
C13H21NO4 + H+).
6.5. Methyl 2-(S)-amino-3-(4-dimethylcarbamoyloxy-
phenyl)propanoate (8)
Compound 5: [a]D À13.5ꢁ (c 1.26, CHCl3, 23 ꢁC); one
isomer by 1H NMR (CDCl3) d 7.14–7.36 (m, 8H),
7.04–7.07 (m, 2H), 6.65–6.71 (m, 1H), 6.08–6.13 (m,
1H), 4.49–4.69 (m, 2H), 3.93–3.96 (m, 1H), 3.59–3.63
(m, 1H), 3.07–3.11 (m, 1H), 2.66–2.68 (m, 1H), 2.08–
2.12 (m, 2H), 1.61–1.65 (m, 3H), 1.32–1.59 (m, 5H),
1.11–1.26 (m, 1H), 0.85–0.88 (m, 1H); MS (ES)
m/z = 348 (M+H)+; HRMS (FAB) m/z 348.1964
(348.1964 calcd for C13H21NO4 + H+).
A 1-L round-bottomed flask containing L-tyrosine
methyl ester (21.0 g, 0.11 mol), tetrahydrofuran
(540 mL), and dimethylsulfoxide (42 mL, 0.59 mol)
was cooled to 0 ꢁC with a dry ice/acetone bath. Sodium
hydride (3.0 g, 0.13 mol, 95%) was added in 4 equal por-
tions over 15 min. The mixture was stirred in the dry ice/
acetone bath at 0 ꢁC until all hydrogen evolution ceases,

4214
E. C. Lawson et al. / Bioorg. Med. Chem. 14 (2006) 4208–4216
cooled to À20 ꢁC, and treated with dimethylcarbamoyl
chloride (9.9 mL, 0.11 mol), added gradually over
5 min. The mixture was stirred at À15 ꢁC for 30 min
and at 0 ꢁC for 3 h. The mixture was quenched with
1 N NaOH (350 mL), transferred to a separatory funnel,
and extracted with dichloromethane (2· 500 mL). The
organic layers were combined, dried (MgSO4), filtered
through Celiteꢂ, and concentrated in vacuo to give
16.0 g (56%) of 8 as a white solid. This compound is
not stable at 23 ꢁC over time and was used without fur-
ther purification.
(s, 3H), 2.23–2.27 (m, 1H), 1.66–1.83 (m, 8H). Anal.
Calcd for C21H29N3O5Æ1.4C2HF3O2Æ0.4H2O: C, 50.12;
H, 5.51; N, 7.37; F, 13.99; H2O, 1.26. Found: C,
50.47; H, 5.21; N, 7.20; F, 14.09; H2O, 1.30.
6.8. Methyl 2-(S)-[(2-benzenesulfonyl-2-(S)-azabicyclo-
[2.2.2]octane-3-carbonyl)amino]-3-(4-dimethyl-carba-
moyloxyphenyl)propanoate (9)
A 200-mL round-bottomed flask containing methyl 2-
[(2-(S)-azabicyclo[2.2.2]octane-3-carbonyl)amino]-3-(S)-
(4-dimethylcarbamoyloxyphenyl)propanoate trifluoro-
acetic acid salt (11.0 g, 21.3 mmol) and dichloromethane
(110 mL) was cooled in an ice/water bath. Triethylamine
(7.4 mL, 53.1 mmol) was added followed by benze-
nesulfonyl chloride (3.0 mL, 23.5 mmol). The mixture
was stirred in the bath for 30 min, and at 23 ꢁC for
2 h, then placed in a separatory funnel, and diluted with
dichloromethane (400 mL). The organic phase was
washed with 1 N HCl (200 mL) and 1 N NaOH
(200 mL). The organic layer was dried (MgSO4), filtered
through Celiteꢂ, and concentrated in vacuo. The prod-
uct was purified via flash chromatography (230–400
mesh silica gel 60, 99:1 dichloromethane/MeOH) to give
11.4 g (98%) of 9 as a white solid.
1H NMR (CD3OD) d 7.30 (d, J = 8.4 Hz, 2H), 7.12 (d,
J = 8.4 Hz, 2H), 4.31 (t, J = 6.6 Hz, 1H), 3.84 (s, 3H),
3.27–3.36 (m, 2H), 3.13 (s, 3H), 3.01 (s, 3H); MS (ES)
m/z = 267 (M+H)+.
6.6. Methyl 2-(S)-[(2-(S)-N-(t-butyloxycarbonyl)aza-bi-
cyclo[2.2.2]octane-3-carbonyl)amino]-3-(4-dimethyl-
carbamoyloxyphenyl)propanoate
A 500-mL round-bottomed flask containing 6 (15.2 g,
59.6 mmol), dichloromethane (200 mL), and triethyl-
amine (17.0 mL, 122 mmol) was cooled with an ice/
water bath. Bis(2-oxo-3-oxazolidinyl)phosphinic chlo-
ride (23.4 g, 91.9 mmol) was added and the mixture
was stirred in the ice/water bath for 15 min. Compound
8 (16.0 g, 60.2 mmol) was added via cannulation in
dichloromethane (100 mL). The mixture was stirred in
the ice/water bath for 1 h and then at 23 ꢁC for 16 h.
The mixture was concentrated in vacuo and purified
by flash chromatography (230–400 mesh silica gel
60, 98:2 dichloromethane/MeOH) to give 17.9 g (60%)
of methyl 2-(S)-[(2-(S)-N-Boc-azabicyclo[2.2.2]octane-
3-carbonyl)amino]-3-(4-dimethylcarbamoyloxyphenyl)-
propanoate as a white solid.
[a]D À82.9ꢁ (c 1.04, CHCl3; 23 ꢁC); 1H NMR (CDCl3) d
7.91 (d, J = 7.2 Hz, 2H), 7.60–7.62 (m, 1H), 7.48–7.58
(m, 2H), 7.15 (d, J = 6.4 Hz, 2H), 7.02 (d, J = 8.6 Hz,
2H), 4.88–4.91 (m, 1H), 4.00–4.03 (m, 1H), 3.76 (s,
3H), 3.19–3.22 (m, 2H), 3.08 (s, 3H), 2.99 (s, 3H),
2.20–2.25 (m, 1H), 1.93–1.98 (m, 1H), 1.42–1.59 (m,
4H), 1.13–1.28 (m, 4H). Anal. Calcd for C27H33N3O7-
SÆ0.45H2O: C, 58.78; H, 6.19; N, 7.62; H2O, 1.47.
Found: C, 58.46; H, 6.16; N, 7.66; H2O, 1.35.
6.9. 2-(S)-[(2-Benzenesulfonyl-2-(S)-azabicyclo[2.2.2]-oc-
tane-3-carbonyl)amino]-3-(4-dimethylcarbamoyl-oxyphe-
nyl)propanoic acid, sodium salt (1-Na)
[a]D À28.5ꢁ (c 1.11, MeOH, 23 ꢁC); 1H NMR (CD3OD)
d 7.26 (d, J = 7.8 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H),
4.75–4.81 (m, 1H), 4.02–4.06 (m, 1H), 3.95–4.00 (m,
1H), 3.71 (s, 3H), 3.17–3.23 (m, 2H), 3.10 (s, 3H), 2.98
(s, 3H), 2.05–2.09 (m, 1H), 1.46–1.77 (m, 8H), 1.32 (s,
9H); HRMS (FAB) m/z 504.2716 (504.2710 calcd for
C26H37N3O7+H+).
A 500-mL round-bottomed flask containing 9 (11.79 g,
21.9 mmol) and tetrahydrofuran (110 mL) was treated
with a solution of NaOH (0.90 g, 22.5 mmol) in water
(210 mL), added by addition funnel. The mixture was
stirred at 23 ꢁC for 30 min and concentrated in vacuo.
The product was precipitated from 2-propanol/diethyl
ether to give 11.8 g (99%) of 1 as a white solid.
6.7. Methyl 2-(S)-[(2-(S)-azabicyclo[2.2.2]octane-3-car-
bonyl)amino]-3-(4-dimethylcarbamoyloxyphenyl)-propan-
oate, trifluoroacetic acid salt
1
[a]D À42.7ꢁ (c 1.53, CH3OH; 23 ꢁC); H NMR (CDCl3)
A 500-mL round-bottomed flask containing methyl 2-
[(2-(S)-N-Boc-azabicyclo[2.2.2]octane-3-carbonyl)ami-
no]-3-(S)-(4-dimethylcarbamoyloxyphenyl)propanoate
(11.0 g, 21.9 mmol) and dichloromethane (110 mL) was
treated with trifluoroacetic acid (20 mL) and then stirred
at 23 ꢁC for 1 h. The mixture was concentrated in vacuo
to give 11.0 g (97%) of methyl 2-(S)-[(2-(S)-azabicy-
clo[2.2.2]octane-3-carbonyl)amino]-3-(4-dimethylcarba-
moyloxyphenyl)-propanoate trifluoroacetic acid salt as a
white solid.
d 7.97 (d, J = 7.2 Hz, 2H), 7.58–7.68 (m, 3H), 7.29 (d,
J = 8.4 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 4.41–4.44
(m, 1H), 4.02–4.06 (m, 1H), 3.68–3.72 (m, 1H), 3.16–
3.33 (m, 2H), 3.10 (s, 3H), 2.99 (s, 3H), 2.04–2.08 (m,
1H), 1.72–1.80 (m, 1H), 1.51–1.55 (m, 4H), 1.20–1.29
(m, 4H); MS (ES) m/z = 551 (M+Na)+; Anal. Calcd
for C26H30N3O7SÆNaÆ2.2H2O: C, 52.82; H, 5.86; N,
7.11; H2O, 6.70. Found: C, 52.61; H, 5.62; N, 7.03;
H2O, 6.88.
6.10. Adhesion of ramos cells (a4b1 specific) to VCAM-1
1H NMR (CD3OD) d 7.23 (d, J = 8.5 Hz, 2H), 7.02 (d,
J = 8.6 Hz, 2H), 4.76–4.80 (m, 1H), 3.88–3.98 (m, 1H),
3.73 (s, 3H), 3.31–3.45 (m, 2H), 3.10 (s, 3H), 2.98
This adhesion assay was modified from that reported by
Jackson et al.22 Ultrahigh binding 96-well plates (Dynex

E. C. Lawson et al. / Bioorg. Med. Chem. 14 (2006) 4208–4216
4215
Technologies, Chantilly, VA) were coated with 100 lL
recombinant hVCAM-1 at 4.0 lg/mL in 0.05 M NaCO3
buffer, pH 9.0, overnight at 4 ꢁC(R&D Systems). Plates
were washed twice in calcium- and magnesium-free PBS
with 1% BSA and blocked for 1 h at 23 ꢁC in this buffer.
PBS was removed and compounds (50 lL) were added
at 2· concentration. Dose–responses were then deter-
mined in duplicate. Ramos cells (ATCC, Manassas,
VA), 50 lL at 2 · 106 cells/mL, labeled with 5 lM Calce-
in AM (Molecular Probes) for 30 min at 37 ꢁC, were
added to each well and allowed to adhere for 1 h at
23 ꢁC. Plates were washed four times in PBS containing
1% BSA and cells were lysed for 15 min in 100 lL of 1
M Tris–HCl buffer at pH 8.0 with 1% SDS. Plates were
read at 485-nm excitation and 530-nm emission on a
CytoFluor 4000 fluorescent plate reader (Applied Bio-
systems, Foster City, CA).
(SRL = RL · Vtg). Post-drug measurements of SRL were
obtained immediately prior to challenge with A. suum
antigen. Measurements of SRL were obtained immedi-
ately after challenge, hourly from 1 to 6 h after challenge
and on the half-hour from 6.5, 5–8 h after challenge.
Measurements of SRL were obtained 24 h after chal-
lenge, followed by the 24 h post-challenge dose–re-
sponse curve for carbachol. The results for these
studies are compared to each sheep’s historical control.
To determine the effect of 1 on the hyper-reactivity
stage, measurements of SRL were repeated immediately
after inhalation of buffer and after each administration
of 10 breaths of increasing concentrations of carbachol
solution (0.25%, 0.5%, 1.0%, 2.0%, and 4.0% wt/vol).
To assess airway responsiveness, the cumulative
carbachol dose in breath units (BU) that increases
SRL 400% over the post-buffer value (i.e., PC400) was
calculated from the dose-response curve. One breath
unit is defined as one breath of a 1% wt/vol carbachol
solution.
6.11. Adhesion of K562 cells that express a4b7 to VCAM-1
K562 cells expressing human a4b7 were licensed from
Dr. David Erle (UCSF).23 Compounds were evaluated
for their ability to inhibit adhesion of a4b7-expressing
K562 cells to hVCAM-1 by using identical methods de-
scribed for Ramos cell adhesion to hVCAM-1.
BAL was sampled before antigen challenge, and at 8 and
24 h following antigen challenge, to evaluate cell influx
into the lungs. Lung lavage was performed as previously
described via infusion and aspiration of 30 mL aliquots
of PBS (pH 7.4) at 39 ꢁC.9
6.12. Adhesion of K562 cells (a5b1 specific) to human
fibronectin
6.14. Antigen-induced airway resistance and eosinophilia
in passively sensitized guinea pigs
K562 cells (ATCC) possess a single a chain (a5) and spe-
cifically bind to human fibronectin via a5b1. Adhesion
assays were carried out similar to the above methods
with the exception that human fibronectin (Sigma,
St. Louis, MO) at 10 lg/mL was plated onto microtiter
plates in 0.05 M Na2CO3 buffer, pH 9.0, overnight at
4 ꢁC.24
Methods were performed as described in previous pub-
lications with slight modifications.21b,25 Briefly, male
Dunkin Hartley guinea pigs were immunized ip with a
1.0 mL solution of ovalbumin (OVA) in Al(OH)3
(10 lg OVA per animal), while control animals received
Al(OH)3 alone (N = 5 per group). This procedure was
repeated on day 14. On day 21, blood was collected, cen-
trifuged, and plasma collected. This anti-OVA plasma
was injected into na¨ıve guinea pigs (1 mL/animal). In
the initial study, seven days later and 24 h prior to
assessing lung function, passively sensitized guinea pigs
were injected with 1 (dissolved in DMSO; 0.1, 0.5, and
1 mg/kg; ip) or vehicle. Thirty minutes later, animals
were exposed for 1 h to aerosolized ovalbumin (dis-
solved in sterile 0.9% physiological saline, 100 lg/ml)
in an exposure chamber in which the guinea pigs could
move freely. Twenty-four hours following OVA expo-
sure, animals were anesthetized and the trachea was
cannulated and attached to a ventilator (60 breaths/
min). The carotid artery was cannulated for the mea-
surement of blood pressure. Changes in airway resis-
tance in response to histamine (1, 2, and 4 lg/kg) and
OVA (100 lg/kg) were measured. Data was collected
for animals treated with either vehicle or 0.1 and
0.5 mg/kg of 1. BAL fluid was also collected at 24 h.
6.13. Antigen-induced airway response in sheep
The validated model of A. suum antigen-induced asth-
matic response in conscious sheep was used to evaluate
the effectiveness of compound 1 in inhibiting antigen-in-
duced early and late airway bronchoconstriction.6 Ani-
mals used in these studies (N = 4 per treatment)
exhibited both early and late airway responses as well
as airway hyper-responsiveness to inhalation challenge
with A. suum antigen. Briefly, aerosol of A. suum extract
was reproducibly generated using a disposable medical
nebulizer (Raindropꢂ, Puritan Bennett). The aerosol
was delivered at a tidal volume of 500 mL and a rate
of 20 breaths/min for 20 min. Compound 1 was admin-
istered by aerosol, BID, for 3 consecutive days, and a
single dose on day 4, 30 min prior to antigen challenge.
Two doses, 3 and 10 mg (ca. 0.075 and 0.25 mg/kg based
on an average body weight of 40 kg), were evaluated
using this protocol (N = 3 sheep at each dose). Analysis
of 5–10 breaths, by previously described methods, was
used for the determination of RL, which is calculated
by dividing the change in transpulmonary pressure by
the change in flow at mid-tidal volume. Immediately
after the measurement of RL, thoracic gas volume
(Vtg) was determined in a constant volume body plethys-
mograph to obtain specific lung resistance
To obtain BAL fluid, 5 mL of sterile saline was slowly
instilled into the lungs via the trachea cannula and the
fluid immediately aspirated. This process was repeated
three times. This procedure resulted in 40–50% recovery
of BAL fluid from the lungs of each guinea pig. Total
cell counts in each BAL sample were determined by

4216
E. C. Lawson et al. / Bioorg. Med. Chem. 14 (2006) 4208–4216
12. Archibald, S. H.; 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. A. Bioorg. Med.
Chem. Lett. 2000, 10, 997–999.
13. Springer, T. A. Cell 1994, 76, 301–314.
14. Sharar, S. R.; Winn, R. K.; Harlan, J. M. Springer Semin.
Immunopathol. 1995, 16, 359–378.
counting. To differentiate the different cell types in the
BAL, cytospin preparations were made and the resulting
slides were fixed and stained for differential cell analysis.
On each slide, in an area selected at random, 200 cells
were counted under light microscope, with cells being
classified as neutrophils, eosinophils and mononuclear
cells according to standard morphologic criteria.
15. Steinman, L. Nat. Rev. Drug Discov. 2005, 4, 510–518.
16. (a) Dyatkin, A. B.; Hoekstra, W. J.; Kinney, W. A.;
Kontoyianni, M.; Santulli, R. J.; Kimball, E. S.; Fisher, C.
M.; Prouty, S. M.; Abraham, W. M.; Andrade-Gordon,
P.; Hlasta, D. J.; He, W.; Hornby, P. J.; Damiano, B. P.;
Maryanoff, B. E. Bioorg. Med. Chem. Lett. 2004, 14, 591–
596; (b) Dyatkin, A. B.; Gong, Y.; Miskowski, T. A.;
Kimball, E. S.; Prouty, S. M.; Fisher, C. M.; Santulli, R.
J.; Schneider, C. R.; Wallace, N. H.; Hornby, P. J.;
Diamond, C.; Kinney, W. A.; Maryanoff, B. E.; Damiano,
B. P.; He, W. Bioorg. Med. Chem. 2005, 13, 6693–6702.
17. Lawson, E. C.; Kinney, W. A.; Santulli, R. J.; Fisher, C.
M.; Damiano, B. P.; Maryanoff, B. E. Lett. Drug Des.
Discov 2005, 2, 563–566.
Acknowledgments
We thank Grace Wells, William Hageman, Jeffery Hall,
Bryan Raferty, Wensheng Lang, John Masucci, and
Gary Caldwell for pharmacokinetic and pharmacody-
namic studies.
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