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

Article abstract of DOI:10.1016/j.bmc.2005.07.022

A series of N-carboxy, N-alkyl, and N-carboxamido azabicyclo[2.2.2]octane carboxamides were prepared and assayed for inhibition of α ₄β₁-VCAM-1 and α₄β₇- MAdCAM-1 interactions. Potency and α₄β₁

Full text of DOI:10.1016/j.bmc.2005.07.022

Bioorganic & Medicinal Chemistry 13 (2005) 6693–6702
Aza-bicyclic amino acid carboxamides as
a4b1/a4b7 integrin receptor antagonists
Alexey B. Dyatkin,* Yong Gong, Tamara A. Miskowski, Edward S. Kimball,
Stephen M. Prouty, M. Carolyn Fisher, Rosemary J. Santulli, Craig R. Schneider,
Nathaniel H. Wallace, Pamela J. Hornby, Craig Diamond, William A. Kinney,
Bruce E. Maryanoff, Bruce P. Damiano and Wei He
Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, LLC., Spring House, PA 19477-0776, USA
Received 10 June 2005; revised 19 July 2005; accepted 19 July 2005
Available online 19 August 2005
Abstract—A series of N-carboxy, N-alkyl, and N-carboxamido azabicyclo[2.2.2]octane carboxamides were prepared and assayed for
inhibition of a4b1-VCAM-1 and a4b7-MAdCAM-1 interactions. Potency and a4b1/a4b7 selectivity were sensitive to the substituent
R1–R3 in the structures 6, 7, and 8. Several compounds demonstrated low nanomolar balanced a4b1/a4b7 in vitro activity. Two com-
pounds were selected for in vivo leukocytosis studies and demonstrated increases in circulating lymphocytes up to 250% over
control.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
gut mucosal endothelium. Inhibition of this interaction
was found to be beneficial in the treatment of inflammato-
ry bowel disease (CrohnÕs disease or ulcerative colitis).1
Integrins are members of a widely expressed group of
heterodimeric cell adhesion receptors, consisting of a
and b subunits. The a4 integrin family, in particular
a4b1 and a4b7 integrins, plays important roles in adhe-
sion of lymphocytes to extracellular matrix.1
The therapeutic potential of a4 integrin antagonists has
recently been highlighted by FDA approval of Tysabri
(natalizumab), a humanized monoclonal antibody that
binds the integrin a4 subunit, for treatment of multiple
sclerosis.2 However, serious complications resulted in
the voluntarily withdrawn of Tysabri shortly after its
introduction.3 In this paper, we describe the design, syn-
thesis, and in vivo properties of novel small-molecule
a4b1/a4b7 integrin antagonists.
The a4b1 integrins (very late antigen-4, VLA-4) bind to
their counter-receptor vascular cell adhesion molecule-
1 (VCAM-1), which is expressed on endothelial cell sur-
faces and mediates cell adhesion and infiltration. It was
shown that blockage of leukocyte infiltration may be
beneficial for therapeutic treatment of such inflammato-
ry diseases as asthma, multiple sclerosis, and rheuma-
toid arthritis.1
2. Chemistry
The a4b7 integrins are critical in lymphocyte homing to
the intestinal mucosa through interaction with its princi-
pal counter-receptor mucosal addressin cell adhesion
molecule-1 (MAdCAM-1), which is expressed on the
There are many reports in the scientific and patent liter-
ature of small-molecule a4 integrin antagonists.4
A major structural class of a-4 integrin antagonists is the
N-acylphenylalanines, with the N-acyl group frequently
resembling proline derivatives.5 Several representatives
of this class (1,6 2,7 3,8 and 49) that demonstrated good
in vitro activity in cell adhesion and ELISA assays are
shown in Figure 1. We recently discovered that sulfona-
mides of aza-bicyclic amino acid derivatives10 (e.g., 5)
are very potent a4b1 and a4b7 antagonists.
Keywords: a4b1 integrin; a4b7 integrin; VLA-4; VCAM-1; MAdCAM-
1; CrohnÕs disease; Inflammatory bowel disease; Asthma; Multiple
sclerosis; Rheumatoid arthritis; N-Acylphenylalanine; Azabicy-
clo[2.2.2]octane; Leukocytosis.
*
Corresponding author. Tel.: +1 215 628 5008; fax: +1 215 628
4985; e-mail: adyatkin@prdus.jnj.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.022

6694
A. B. Dyatkin et al. / Bioorg. Med. Chem. 13 (2005) 6693–6702
HO2C
O
HO2C
NH
O
Cl
Cl
N
H
N
NH
NH
O
NH
O
N
H
S O
O
N
O
Cl
Ph
2
1
Me
HO2C
HO2C
Ph
S
OMe
NH
O
N
N
O
Me
O
S O
O
Cl
MeO
O
Ph
4
3
HO2C
O
Cl
NH
N
H
N
O
Cl
S O
O
5
Ph
Figure 1. Nonpeptide a4b1/a4b7 integrin antagonists.
The sulfonamides of aza-bicyclic amino acids, such as 5,
have very high in vitro activity and in vivo efficacy, but
are not orally bioavailable. We had chosen to investigate
whether further manipulation of the structure, in particu-
lar replacement of the sulfonamide group, may lead
to orally active dual a4b1/a4b7 integrin antagonists. We
envisioned that by replacing the sulfonamide group with
amides, ureas or amines, we may achieve greater com-
pound diversity (prototype structures 6–8), which may
provide additional benefits in activity and selectivity (see
Fig. 2).
A large number of 4-substituted phenylalanines were
reported as components of the active a4 integrin antag-
onists. We opted to retain the 2,6-dichloroisonicotinoyl
amide derivatized analogs of phenylalanine13 since these
analogs demonstrated the highest activity with sulfona-
mides of aza-bicyclic amino acid derivatives.10
We used a convergent approach to the synthesis of the
target compounds (Schemes 1–3). The methodology
for preparation of the requisite bridged bicyclic amino
acids 9 is well developed.14 Acylation of 9 with ben-
zyloxycarbonyl chloride followed by hydrolysis result-
ed in acid 11 (Scheme 1). The acid was coupled with
amine 14, prepared with near quantitative yield from
Boc-4-nitrophenylalanine 12 (Scheme 2). Further
deprotection and modification of the nitrogen atom
of amine 15 followed by hydrolysis resulted in target
compounds 6, 7, and 8 (Scheme 3). Final products
Although the preparation of some amido derivatives of
proline analogs, along with their a4b1 activity, was de-
scribed in the scientific11 and patent12 literature, very
limited SAR data are available Our first goal was to
investigate the a4b1 and a4b7 activity of non-sulfon-
amide analogs of aza-bicyclic amino acids.
X
X
X
O
O
O
N
H
CO H
N
H
CO H
2
N
H
CO H
2
2
N
N
N
2
R
1
3
O
N
H
O
R
R
6
7
8
Figure 2. Prototype non-sulfonamide targets 6, 7, and 8.
O
O
O
a
b
OMe
OMe
OH
N
H
N
N
O
O
O
O
11
9
10
Ph
Ph
Scheme 1. Reagents and conditions: (a) CBZ-Cl, CH2Cl2, Et3N, 23 ꢁC (85%); (b) LiOH or NaOH, MeOH, water, 60 ꢁC (70%).

A. B. Dyatkin et al. / Bioorg. Med. Chem. 13 (2005) 6693–6702
6695
N
H
N
NO
NH
2
2
O
Cl
a, b
c, d
Boc
OH
Boc
OMe
OMe
N
H
N
H
H N
2
O
O
O
13
14
12
Scheme 2. Reagents and conditions: (a) Me3SiCHN2, MeOH, CHCl3, 0 ꢁC; (b) H2, Pd/C 10%, MeOH, 23 ꢁC; (c) 2,6-dichloroisonicotinoyl chloride,
CH2Cl2, Et3N, 23 ꢁC; (d) CF3CO2H, CH2Cl2, 23 ꢁC (90% total yield).
Cl
Cl
N
N
H
N
H
N
O
O
Cl
O
Cl
OH
O
+
a, b
N
OMe
OMe
e, f
H N
2
N
H
14
O
O
11
N
H
O
O
15
Ph
c, f
d, f
Cl
Cl
N
N
H
N
H
N
Cl
O
Cl
O
Cl
N
H
N
O
O
OH
OH
N
H
N
H
O
Cl
N
N
O
O
O
3
OH
1
O
R
R
N
H
8
6
N
O
2
R
N
H
O
7
Scheme 3. Reagents and conditions: (a) bis(2-oxo-3-oxazolidinyl)phosphinic chloride, i-Pr2NEt/CH2Cl2, 23 ꢁC; (b) HBr in AcOH, 23 ꢁC; (c)
RCO2H, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride/1-hydroxybenzotriazole hydrate, i-Pr2NEt/CH2Cl2, 23 ꢁC; (d) R2N@C@O/
CH2Cl2, 23 ꢁC; (e) RCHO, Na(OAc)3BH/ClCH2CH2Cl; (f) LiOH or NaOH, MeOH, water, 60 ꢁC (85%).
were isolated by HPLC and characterized by spectro-
scopic methods.
bonding of polar functional groups with substrate. The
length of the linker is very important, methylene and
ethylene were found to be optimal (18 is more potent
than 16). Further elongation of the linker diminished
the activity, especially for a4b7 (the activity of 20 was
lower than 19, for a4b7). Carbon atoms in the linker
may be replaced by O or S atoms with retention of activ-
ity (41–43 vs 28). The aromatic or heteroaromatic group
attached to the linker may be substituted with acceptor
groups without decreasing activity (38, 39). Electron-
donating groups slightly increased a4b1 as well as a4b7
activity (40 vs 38 and 39).
3. In vitro biological results
The aza-bicyclic target compounds were tested for inhib-
iting cell adhesions mediated by a4b1 and a4b7 integrins,
and the results are presented in the Table 1. All com-
pounds were prepared and tested as mixtures of (R,S)
and (S,S) diastereomers.17 We found that the size and
the type of R group have decisive effect on a4b1 and
a4b7 activity with several trends being observed. The
best substituents have a more or less bulky group (i.e.,
t-Bu, i-Pr, cycloalkyl or aryl) attached to the C0–C3 link-
er. The alternative to the bulky groups are smaller polar
functional groups such as substituted or non-substituted
amino (22–24), hydroxy (25) or alkoxy (26). These polar
groups attached to C2–C3 linker also yielded potent inte-
grin antagonists with dual activity. Retention of activity
in this case may be explained by additional hydrogen
Ureas were less potent in vitro than the amides and
sulfonamides. Compound 46 is the only example with
a4b1 activity below 100 nM. The activity against a4b7
integrins of all synthesized ureas was modest.
N-Alkylated analogs (47–50) were found to be less
potent than the N-acylated derivatives. The best
compound, 47, demonstrated balanced nanomolar

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A. B. Dyatkin et al. / Bioorg. Med. Chem. 13 (2005) 6693–6702
Table 1. Inhibition of cell adhesion mediated via a4b1 and a4b7 integrins by azabicyclo[2.2.2]octane derivatives (IC50
)
Compound
R
a4b1/VCAM-1 (nM)
a4b7/MAdCAM-1 (nM)
a4b1/a4b7 selectivity ratio
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
5
t-BuCO
t-BuCH2CO
i-Pr(CH2)2CO
390
830
0.5
0.6
0.3
1.0
0.2
0.3
0.5
0.1
6.7
0.3
0.1
2.9
0.1
0.4
0.1
0.3
0.1
0.2
0.9
0.8
0.3
0.1
0.1
1.3
0.3
0.4
0.9
1.0
0.9
4.7
0.2
0.5
0.2
2.0
1.8
2.0
0.2
89
5
1
1
5
150
15
6
1
9
2-Cyclopentyl(CH2)2CO
3-Cyclohexyl(CH2)3CO
BocNH(CH2)2CO
NH2(CH2)2CO
Me2N(CH2)2CO
HO(CH2)2CO
28
27
41 20
9
250 50
30 14
60 21
180 16
0.6 0.3
47 10
110 11
150
8
28 12
24 13
4
14
8
1
4
1
HOC(Me)2CH2CO
MeO(CH2)2CO
2-F–C6H4CO
440
1
Ph(CH2)2CO
0.5
4
7
30
2
2
2-ThienylCH2CO
3-ThienylCH2CO
4-MorpholinoCH2CO
4-(MeO)C6H4CH2CO
3,5-(MeO)2C6H3CH2CO
3,6-(MeO)2C6H3CH2CO
2-Pyridyl-CH2CO
3-Pyridyl-CH2CO
4-Pyridyl-CH2CO
2-F–C6H4 (CH2)2CO
4-Cl–C6H4 (CH2)2CO
4-(MeO)C6H4 (CH2)2CO
PhCH2OCO
12
4
1
41 14
30 10
110 50
140 70
130
10
8
6
3
23
120
25
20
3
5
8
7
1
1
4
1
1
1
2
32
62
4
8
32 15
46 20
24 14
4
31
2
7
24
10
6
1
4
4
2
9
2-Pyridyl-SCH2CO
4-Pyridyl-SCH2CO
PhNHCO
9
6
260
580
290
120 20
38
4-MeC6H4NHCO
PhCH2NHCO
t-Bu(CH2)2
72 16
5
47
62
5
6
94 21
260 90
260
Ph(CH2)3
4-MeC6H4(CH2)2
PhCH2
500
540
310
H
45 12
19
23 13
94 25
3
Data represent inhibition of binding of a4bþ1 cells to immobilized VCAM-1 or a4bþ7 cells to immobilized MAdCAM-1. Confidence intervals were
usually calculated with N = 3. Assay conditions were developed based on Ref. 15 and 16.
a4b1/a4b7 activity. A free amine 51 (R1 = H) was very ac-
tive, probably due to additional hydrogen bonding.
bioavailability, we prepared the Me ester of 30—pro-
drug 52. Results of PK studies are presented in the Table
2.
4. In vivo PK studies
5. In vivo leukocytosis studies
The pharmacokinetic profiles in Sprague–Dawley rats of
several N-amido and N-alkyl bicyclic derivatives were
determined. Unfortunately, oral bioavailability of all
tested compounds, (30, 47, and 51) was below 5%. These
compounds also showed t1/2 values below 30 min upon
intravenous administration while being stable in vitro
in the presence of human or rat liver microsomes. This
is an evidence of primarily non-first pass in vivo metab-
olism for this class of compounds. To overcome low oral
Two compounds, 5 and 28, representing the sulfona-
mides and amides, were selected for evaluation for ef-
fects on in vivo leukocytosis. Leukocytosis is the
increase in circulating white blood cells (leukocytes) that
can be brought about by preventing leukocyte binding
to leukocyte adhesion molecule counter-receptors ex-
pressed on endothelium. This cell–cell adhesion occurs
between immunoglobulin superfamily molecules and

A. B. Dyatkin et al. / Bioorg. Med. Chem. 13 (2005) 6693–6702
6697
Table 2. Oral PK data of 52 in Sprague–Dawley rats
circulation was prevented. Similar emigration of cells
out of the circulation into inflamed tissues is responsible
for the progression and maintenance of the inflammato-
ry state. Leukocytosis is an indication that lymphocyte
and leukocyte extravasation is prevented, and may be
predictive of general anti-inflammatory activity. In vivo
administration of the two compounds produced signifi-
cant elevations in circulating lymphocytes and total leu-
kocytes. Among the latter, circulating counts for cells of
various granulocytic lineages remained unchanged.
Cmax, lM t1/2, h (i.v.) AUC Clearance (dose/AUC), F%
lM-h mg/kg/lM-h
Dose–responsive increases in lymphocyte counts were
observed 1 h after subcutaneous administration of Com-
pound 5 and Compound 28 to na¨ıve animals. The lym-
phocytosis responses are shown in Figure 3. Compound
5 induced an increase in lymphocyte counts to 177% of
vehicle control, and compound 28 induced lymphocyte
counts to rise to 253% of vehicle control. Nearly, identi-
cal increases in total leukocyte counts were found to oc-
cur. Granulocyte counts remained unchanged (data not
shown).
0.533
0.238
1.047 28.7
22
Mature male rats (250–300 g) were used. Each compound was admin-
istered at a dose of 30 mg/kg p.o. (N = 3) and 3 mg/kg i.v. (N = 3). The
plasma levels for the compounds were determined by LC–MS. All
parameters were measured for the acid 30. Pro-drug 52 was not detected
in plasma.
integrins. Relevant examples of these paired interactions
include vascular cell adhesion molecule-1 with a4b1 inte-
grin, and mucosal addressin cell adhesion molecule-1
with a4b7 integrin, respectively.
The increased numbers of circulating leukocytes were
dose-dependent and indicate that blockade of integrin
receptor-mediated binding to endothelial counter-recep-
tors (a4b1 to VCAM-1 and a4b7 to MAdCAM-1) results
in an inability of the cells to extravasate as part of a nor-
mal trafficking function. This further supports the utility
of this method as a possible clinical surrogate and for
evaluating dosing regimens, as discussed previously in
the same context in studies with natalizumab, an anti-
body to a4 integrin,18 and with the synthetic integrin
antagonist BIO5192.19
In this model, a compound that antagonizes these leuko-
cyte–endothelial interactions will cause an increase in
circulating leukocytes, defined as leukocytosis, as mea-
sured at 1 h post-administration, a time when drug plas-
ma levels were allowed to achieve a maximum and have
had sufficient time to manifest a sufficient biological ef-
fect. This leukocytosis is indicative that normal lympho-
cyte or leukocyte emigration from the peripheral
Compound 28
Compound 5
**
** *
16
14
12
10
10
8
**
6
8
6
4
2
0
4
2
0
Veh
3mg/kg
30mg/kg
Veh
3mg/kg
30mg/kg
Treatment
Treatment
Leukocytosis
Compound 28
Compound 5
18
16
14
12
10
8
**
***
14
12
10
**
8
6
4
2
0
6
4
2
0
Veh
3 mg/kg
30 mg/kg
Veh
3mg/kg
30 mg/kg
Treatment
Treatment
Figure 3. In vivo leukocytosis studies. Dose–responsive lymphocytosis occurs following subcutaneous administration of 5 and 28. Female, Balb/c
mice, n = 8, were given vehicle, 3 mg/kg or 30 mg/kg of Compound 5 or Compound 28 in 0.5% methyl cellulose. One hour later, blood samples were
drawn and were analyzed for lymphocyte numbers. Numbers represent means and standard error of counts · 10ꢀ9/L: ** = p < 0.01, *** = p < 0.001
versus vehicle-treated group; ANOVA.

6698
A. B. Dyatkin et al. / Bioorg. Med. Chem. 13 (2005) 6693–6702
6. Conclusion
and evaporated. The residue was dissolved in 100 mL
H2O, acidified by 1 N HCl to pH 2 and extracted by
EtOAc (3· 25 mL). Organic fractions were combined,
dried over MgSO4, filtered, and evaporated, providing
white solid material (6.34 g, 90%).
In summary, we have identified a novel series of potent
non-sulfonamide a4 integrin antagonists. While amine
and urea substitutions provided moderately active com-
pounds, amides were equipotent or even more potent
than the corresponding sulfonamides. Several com-
pounds had low-nanomolar or even sub-nanomolar
potency; the ratio of a4b1/a4b7 activity varied from 0.1
to 6.7. In vivo administration of amide 28 as well as
sulfonamide 5 produced a significant elevation in
circulating lymphocytes and total white cells. Further
investigation of such compounds may lead to new treat-
ments of diseases mediated by a4 integrins.
1H NMR (CDCl3): d 7.34–7.26 (5H, m), 5.29-5-.10 (2H,
m), 4.70–4.13 (2H, m), 2.29–2.23 (1H, m), 2.09–2.00
(1H, m), 2.00–1.40 (8H, m); MS (ESꢀ) 288.
7.3. 4-Amino-N-Boc phenylalanine methyl ester (13)
N-Boc-4-nitro-L-phenylalanine, 12, (5 g) was dissolved
in 100 ml MeOH/chloroform 1:1 mixture, the solution
was cooled in the ice bath. Trimethylsilyl diazomethane
(1 M solution in hexane) was added dropwise until the
solution remained yellow. The reaction mixture was
evaporated in vacuum, the residue was dissolved in
50 ml MeOH/ethyl acetate 1:1 mixture and was hydroge-
nated at 30 psi overnight over Pd/C 10% (100 mg). After
filtration the solvent was evaporated, providing 5.1 g of
13 as white solid.
7. Experimental
All commercially available chemicals were used as pur-
chased. Melting points were obtained with a Mel-Temp
capillary melting point apparatus and are uncorrected.
1H NMR spectra were recorded with a Bruker AC-400
spectrometer with TMS as an internal standard. Electro-
spray ionization mass spectra (ESI) were obtained using
a Fisons spectrometer (Hewlett-Packard HPLC driven
electrospray MS instrument). Compounds were purified
using ISCO CombiFlashꢂ Sq16x system with silica
RediSep columns or Gilson HPLC system with YMC
ODS-H80 column. The purities of each compound were
determined using a Hewlett-Packard LC 1100 system
(YMC JÕSphere H80 S4 column, 4.0 · 50 mm, 4 mm
C18; mobile phase of 90% H2O (0.1% TFA) to 10%
H2O (0.1% TFA) with a flow rate of 1 mL/min; detec-
tion at 220 and 254 nM). Elemental analyses were con-
ducted by Robertson Microlit Laboratories.
1H NMR (CDCl3): d 6.89 (d, J = 8.2 Hz, 2H), 6.60 (d,
J = 8.3 Hz, 2H), 5.05–4.95 (m, 1H), 4.59–4.50 (m, 1H),
3.01–2.95 (m, 2H), 1.41 (s, 9H); MS (ESI+) m/z 295
(M+H)+. Anal. Calcd for C15H22N2O4: C, 61.21; H,
7.53; N, 9.52. Found: C, 61.24; H, 7.80; N, 9.46.
7.4. 4-Dichloroisonicotinamido phenylalanine methyl ester
(14)
Compound 13 (5.0 g, 0.017 mol) was dissolved in 50 ml
CH2Cl2 containing 3 ml of Et3N followed by 5.31 g
(0.025 mol) of 3,5-dichloroisonocotinoyl chloride,11 The
reaction mixture was kept overnight at room tempera-
ture, washed with 0.1 N HCl, 10% NaHCO3, dried over
MgSO4, filtered, and evaporated. The product was
purified by crystallization from hexane/ethyl acetate, pro-
viding 6.22 g (78% yield) of N-BOC-4-dichloroisonicoti-
namido phenylalanine methyl ester as white solid, mp
124–126 ꢁC.
7.1. N-Benzyloxycarbonyl-[2.2.2]azabicyclooctane-1 ethyl
carboxylate (10)
Aminoester 9 (5.90 g, 0.0322 mol)was dissolved in 100 ml
of dry DCM containing 9.43 mL (0.067 mol) of Et3N and
solution was cooled in the ice bath. Benzyl chloroformate
(5.77 g, 4.83 mL, 0.0338 mol) was added dropwise by
syringe. The reaction was stirred 2 h at 0 ꢁC, then warmed
up to room temperature and stirred overnight. The reac-
tion mixture was washed with 0.1 N HCl, 5% NaHCO3,
and water, dried over MgSO4 and concentrate, resulting
10 as viscous oil. Product was analyzed by TLC (hex-
ane/EtOAc 1:1, Rf 0.75). The crude material was purified
by column chromatography (silica, heptane/EtOAc 2:1)
resulting 7.73 g (76%) of yellow solid.
1H NMR (DMSO-d6): d 8.79 (s, 2H), 7.56 (d,
J = 8.4 Hz, 2H), 7.30 (d, J = 8.1 Hz, 1H), 7.24 (d,
J = 8.4 Hz, 2H), 4.17–4.13 (m, 1H), 2.97 (dd, J = 13.7
and 5.0, 1H), 2.83 (dd, J = 13.6 and 9.9, 1H); 1.33 (s,
9H); MS (ESI+Å ) m/z 469 (M+H)+. Anal. Calcd for
C21H23Cl2N3O5 0.8 Et2O: C, 55.09; H, 5.92; N, 7.96.
Found: C, 54.94; H, 5.86; N, 8.00.
N-BOC-4-dichloroisonicotinamido phenylalanine meth-
yl ester (4.68 g, 0.01 mol) was dissolved in 30 ml of
CH2Cl2 followed by 1 ml of TFA. Reaction was kept
overnight at room temperature, evaporated in vacuum,
and the viscous residue was recrystallized from
CH2Cl2/ether, providing 14 as white solid (5.1 g, 80%
yield); mp 257–259 ꢁC.
1H NMR (CDCl3): d 7.37–7.26 (5H, m), 5.20–5.10 (2H,
m), 4.71–4.69 (1H, m), 4.26–4.00 (3H, m), 2.24–2.22
(1H, m), 2.16–2.15 and 2.13–2.04 (1H, m), 2.00–1.40
(m, 9H), 1.25 and 1.15 (3H, J = 7.3 Hz).
7.2. N-Benzyloxycarbonyl-[2.2.2]azabicyclooctane-1
carboxylic acid (11)
1H NMR (DMSO-d6): d 8.80 (s, 2H), 8.44 (br s, 3H),
7.63 (d, J = 8.4 Hz, 2H), 7.63 (d, J = 8.5 Hz, 2H), 7.24
(d, J = 8.5 Hz, 2H), 4.33 (t, J = 6.4 Hz, 1H), 3.09 (d,
J = 6.4, 2H), MS (ESI+) m/z 369 (M+H)+. Anal. Calcd
Ester 10 (7.73 g, 24.36 mmol) was dissolved in 100 ml
MeOH and 5 equiv of 1.0 N NaOH aq were added as
one portion. Reaction was warmed to 70 ꢁC for 5 h

A. B. Dyatkin et al. / Bioorg. Med. Chem. 13 (2005) 6693–6702
6699
Å
for C16H15Cl2N3O3 CF3CO2H: C, 44.83; H, 3.34; F,
11.82; N, 8.71. Found: C, 44.49; H, 3.23; F, 11.78; N,
8.61.
This amide was dissolved in MeOH/water (2 mL, 1:1)
and LiOH (4 mg, 0.1 mmol) was added in one portion.
The reaction was homogenized in an ultrasonic bath
and kept overnight at room temperature. The reaction
mixture was diluted with water (20 mL), extracted with
Et2O (10 mL), and the organic layer was discarded.
The aqueous layer was acidified with 1 N HCl to pH 2
and extracted with EtOAc (2· 10 mL). The organic lay-
ers were combined, dried (MgSO4), filtered, and evapo-
rated to give a white residue which was purified by
HPLC. The desired fractions were pooled and lyophi-
lized to yield target product 6, which was characterized
by NMR, MS, and elemental analysis.
7.5. 2-[(2-Aza-bicyclo[2.2.2]octane-3-carbonyl)-amino]-3-
(4-[(3,5-dichloro-pyridine-4-carbonyl)-amino]-phenyl)-
propionic acid methyl ester (15)
The acid 11 (2.50 g, 8.6 lmol) and di-TFA salt of amine 14
(5.14 g, 8.6 lmol) were dissolved in 15 mL CH2Cl2,
containing 500 lL of Et3N, followed by bis(2-oxo-3-oxa-
zolidinyl)phosphinic chloride (4.40 g, 2 equiv). The reac-
tion was stirred overnight at room temperature under
nitrogen and then washed H2O. The residue after evapo-
ration of solvent was subjected to column chromatogra-
phy on silica gel eluted with ethyl acetate to give 3-(2-(4-
[(3,5-dichloro-pyridine-4-carbonyl)-amino]-phenyl)-1-meth-
oxycarbonyl ethylcarbamoyl)-2-aza-bicyclo[2.2.2]octane-2-
carboxylic acid benzyl ester (4.66 g, 85%) as a white
solid.
7.7. Typical procedure for preparation of ureas (7)
Compound 15 (73 mg, 0.10 mmol), the isocyanate
(0.105 mmol) and 150 lL of Et3N were suspended in
DCM (1 mL). The reaction was kept at room tempera-
ture for 12 h and loaded into a silica column. Flash
chromatography (silica gel, EtOAc) provided target
amide with 50–85% yield.
1H NMR (300 MHz, CD3CN): d 8.90 (1H, s), 8.65 (2H,
s), 7.65–7.50 (2H, m), 7.45–7.15 (9H, m), 6.97 (1H, d,
J = 8.0 Hz), 5.12–4.99 (2H, m), 4.75–4.60 (1H, m),
4.09–4.01 (2H, m), 3.69–3.62 (3H, m), 3.20–2.90 (1H,
m); MS (ESI+) m/z 639 (M+H)+.
This urea was dissolved in MeOH/water (2 mL, 1:1) and
LiOH (4 mg, 0.1 mmol) was added in one portion. The
reaction was homogenized in an ultrasonic bath and
kept overnight at room temperature. The reaction mix-
ture was diluted with water (20 mL), extracted with
Et2O (10 mL), and the organic layer was discarded.
The aqueous layer was acidified with 1 N HCl to pH 2
and extracted with EtOAc (2· 10 mL). The organic lay-
ers were combined, dried (MgSO4), filtered, and evapo-
rated to give a white residue which was purified by
HPLC. The desired fractions were pooled and lyophi-
lized to yield target product 7, which was characterized
by NMR, MS, and elemental analysis.
This benzyloxycarbonyl derivative (3.65 g, 5.72 mmol)
was added to 33% HBr in AcOH (45 mL) under vigor-
ous stirring. The reaction was kept at room temperature
for 3 h (reaction became homogeneous after 45 min).
The viscous liquid was evaporated under vacuum, and
the resulting residue was dissolved in water (250 mL),
then extracted with Et2O. The organic layer was discard-
ed. The aqueous layer was basified to pH 7 with Na2CO3
and extracted with EtOAc (5· 20 mL). The organic lay-
ers were combined, dried (Na2SO4), and evaporated to
provide Compound 15 (2.85 g) as a pale yellow solid.
Compound 15 was purified by column chromatography
on silica gel eluted with 9:1 CHCl3/MeOH to give 2.5 g
(87%) of pure compound 15 a white solid. The analyti-
cally pure material was purified by HPLC, providing
after lyophilization a salt with 1.3 equivalents of TFA.
7.8. Typical procedure for preparation of amines (8)
A 10-mL vial (SmithProcess) containing a magnetic stir
bar was charged with 15 (73 mg, 0.10 mmol), aldehyde/
ketone (0.15 mmol), acetic acid (10 lL), sodium triacet-
oxyborohydride (32 mg 0.15 mmol) in ethylene dichlo-
ride (0.5 mL). The vial was sealed and the mixture was
heated under microwave (SmithSynthesizer) at 120 ꢁC
for 5 min. The reaction mixture was concentrated and
treated with 1 N LiOH (0.5 mL) in MeOH (0.5 mL) at
room temperature for 4 h. Acidification and purification
gave desired compound, which was purified by HPLC.
The desired fractions were pooled and lyophilized to
yield target product 8, which was characterized by
NMR, MS, and elemental analysis.
1H NMR (CD3OD): d 8.66 (s, 2H), 7.59 (d, J = 8.5 Hz,
2H), 7.24 (d, J = 8.5 Hz, 2H), 4.83 (m, 1H), 3.92 (s, 1H),
3.74 (s, 3H), 3. 42 (s, 1H), 3.25 (m, 1H), 2.97 (m, 1H),
2.28 (s, 1H), 2.01–1.54 (m, 8H), MS (ESI+Å) m/z 506
(M+H)+. Anal. Calcd for C24H26Cl2N4O4 1.3 CF3
COOH: C, 48.88; H, 4.21; Cl, 10.85; F, 11.34; N, 8.57.
Found: C, 48.53; H, 3.82; Cl, 10.90; F, 11.12; N, 8.35.
7.6. Typical procedure for preparation of amides (6)
After lyophilization compounds usually contained 0.2–
1.5 equivalent of TFA. Most compounds existed as rota-
mers, which complicated NMR spectra. Below are
presented examples of the target compounds character-
ization (S,S-isomers).
Compound 15 (73 mg, 0.10 mmol), the acid (0.105 mmol),
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride (21 mg, 0.11 mmol), and 1-hydroxybenzotriaz-
ole hydrate (14 mg, 0.105 mmol) were suspended in
DCM (1 mL) at room temperature and N-methyl-mor-
pholine (14 lL, 0.120 mmol) was added in one portion.
The reaction was kept at room temperature for 4 h and
loaded into a silica column. Flash chromatography (silica
gel, EtOAc) provided target amide with 65–90% yield.
16: 1H NMR (DMSO-d6): d 0.80 (s, 1H); 8.73 (s, 2H), 7.71
(d, J = 6.97 d, 1H), 7.49 (d, J = 8.5 Hz, 2H), 7.21 (d,
J = 8.5 Hz, 2H), 4.23–4.28 (m, 1H), 4.09–4.05 (m, 2H),
3.00–2.80 (m, 2H), 2.03 (s, 1H), 1.90–1.73 (m, 1H),

6700
A. B. Dyatkin et al. / Bioorg. Med. Chem. 13 (2005) 6693–6702
1.65–1.40 (m, 6H), 1.35–1.15 (m, 1H), 1.08 (s, 9H). MS
7.67 (m, 0.5H), 7.52–7.42 (m, 2H), 7.33–7.21 (m, 2H),
4.68–4.62 (m, 1H), 4.42–4.30 (m, 1H), 4.00–3.95 (m,
1H), 3.56–3.29 (m, 1H), 3.17–2.92 (m, 3H), 2.30–2.17
(m, 1H), 2.08–1.15 (m, 8H). MS (ESI+) m/z 610
(ESI+) m/z 575 (M+H)+. Anal. Calcd (C28H32Cl2N4O5
Å
0.25 TFA): C, 56.67; H, 5.38; F, 2.36; Cl, 11.74; N, 9.28.
Found: C, 56.22; H, 5.01; F, 2.30; Cl, 12.02; N, 9.02.
(M+H)+. Anal. Calcd (C30H29Cl2 N5O5 1.5 H2O, 1.3
Å
1
17: H NMR (CD3OD, rotamers): d 8.65 (s, 2H), 7.60–
TFA): C, 49.83; H, 4.27; Cl, 9.02; F, 9.43; N, 8.91.
Found: C, 49.92; H, 3.78; N, 8.87; Cl, 8.95; F, 9.68;
KF 3.30.
7.55 (m, 2H), 7.34–7.25 (m, 2H), 4.71 (m, 0.5H), 4.46
(m, 0.5H), 4.32 (m, 0.5H), 4.26 (m, 0.5H), 4.03 (m, 1H),
3.20–2.90 (m, 2H), 2.20–1.20 (m, 12H), 1.05 and 0.94 (s,
9H). MS (ESIÅ+) m/z 589 (M+H)+. Anal. Calcd
(C29H34Cl2N4O5 1.2 TFA): C, 51.92; H, 4.88; F, 9.42;
Cl, 9.76; N, 7.71. Found: C, 52.55; H, 4.80; F, 10.08; Cl,
10.14; N, 7.78.
1
37: H NMR (CD3OD, rotamers): d 8.78–8.26 (m, 5H),
8.00–7.93 (m, 1H), 7.56–7.48 (m, 2H), 7.35–7.29 (m,
2H), 4.70–4.67 (m, 1H), 4.41–4.35 (m, 1H), 4.21–4.17
(m, 1H), 4.05–3.95 (m, 1H), 3.70–3.34 (m, 1H), 3.20–
2.97 (m, 2H), 2.28–2.20 (m, 1H), 2.03–1.13 (m, 8H). MS
18: 1H NMR (DMSO-d6, rotamers): d 10.74 and 10.72 (s,
1H); 8.71 (s, 2H), 7.42–7.35 (m, 2.5H), 7.14–7.06 (m,
2.5H), 4.24 (m, 0.5H), 4.08–4.00 (m, 1H), 3.98–3.95 (m,
0.5H), 3.87–3.85 (m, 0.5H), 3.79 (m, 0.5H), 3.00–2.90
(m, 2H), 2.32–2.22 (m, 0.5H), 2.16–2.05 (m, 0.5H), 2.05
and 2.00 (m, 1H), 1.96–1.89 (m, 0.5H), 1.85–1.75 (m,
0.5H), 1.65–1.15 (m, 11H), 0.80 (d, J = 6.6 Hz, 3H), 0.72
(dd, J = 6.4 and 3.7 Hz, 3H). MS (ESI+) m/z 589
(ESI+) m/z 610 (M+H)+. Anal. Calcd (C30H29Cl2N5O5
Å
1 H2O, 1 TFA): C, 51.76; H, 4.34; F, 7.68; N, 9.43. Found:
C, 51.46; H, 4.69; F, 7.53; N, 9.69; KF 2.35.
1
39: H NMR (DMSO-d6, rotamers): d 10.85 and 10.83
(s, 1H), 8.76 and 8.75 (s, 2H), 7.50–7.43 (m, 2H),
7.30–7.18 (m, 7H), 4.25 (m, 0.5H), 4.20 (dd, J = 12.7
and 7.2 Hz, 0.5H), 4.05 (m, 0.5H), 4.20 (dd, J = 7.2
and 5.2 Hz, 0.5H), 3.90 (m, 0.5H), 3.84 (m, 0.5H),
3.06–3.00 (m, 1H), 2.95–2.55 (m, 5H), 2.25–2.10 (m,
1H), 1.65–1.15 (m, 9H). MS (ESI+) m/z 671 (M+H)+.
Å
(M+H)+. Anal. Calcd (C29H34Cl2N4O5 2.5 TFAÅ 0.5
H2O): C, 46.22; H, 4.28; Cl, 8.02; F, 16.13; N, 6.34. Found:
C, 46.21; H, 4.34; Cl, 7.73; F, 15.69; N, 6.46; KF 1.04.
Å
Anal. Calcd (C32H31Cl3N5O5 3.5 H2O, 3.5 TFA): C,
41.30; H, 3.69; Cl, 9.38; F, 17.58; N, 6.18. Found: C,
41.26; H, 3.43; Cl, 9.07; F, 17.92; N, 6.36; KF 5.35.
21: 1H NMR (CD3OD, rotamers): d 8.65 (m, 2H), 7.60–
7.50 (m, 2H), 7.35–7.20 (m, 2H), 4.75–3.70 (m, 3H),
3.20–2.80 (m, 2H), 2.65–2.35 (m, 2H), 2.15 (m, 2H),
2.00–1.20 (m, 19H). MS (ESI+) m/z 662 (M+H)+. Anal.
Calcd (C31H37Cl2N5O7Å 1.1 TFA): C, 50.60; H, 4.87; Cl,
9.00; F, 7.96; N, 8.89. Found: C, 50.72; H, 4.80; Cl, 9.43;
F, 8.00; N, 9.27.
41:1H NMR (CD3OD, rotamers): d 8.63 (s, 2H), 7.60–
7.50 (m, 2H), 7.40–7.15 (m, 7H), 5.11 and 5.07 (m,
1H), 4.80–4.65 (m, 1H), 4.17 (m, 1H), 4.04 (m, 1H),
3.25–3.15 (m, 1H), 3.03–2.80 (m, 1H), 2.20–1.20 (m,
10H). MS (ESI+) m/z 625 (M+H)+. Anal. Calcd
(C31H30Cl2N4O6): C, 59.53; H, 4.83; Cl, 11.34; N, 8.96.
Found: C, 59.26; H, 4.56; Cl, 11.59; N, 8.70.
28: 1H NMR (DMSO-d6, rotamers): d 10.75 (s, 1H), 8.72
(s, 1H), 7.66 (m, 1H), 7.45–7.39 (m, 2H), 7.20–7.15 (m,
3H), 7.10–7.03 (m, 2H), 4.25 (m, 1H), 4.04 (m, 1H),
3.85–3.75 (m, 1H), 3.10–2.50 (m, 4H), 2.00–1.10 (m,
10H). MS (ESI+) m/z 623 (M+H)+. Anal. Calcd
(C32H32Cl2N4O5Å 1 TFA): C, 55.37; H, 4.51; Cl, 9.61; F,
7.73; N, 7.60. Found: C, 55.29; H, 4.46; Cl, 9.45; F,
7.55; N, 7.42.
48:1H NMR (CD3OD, rotamers): d 8.65–8.61 (m, 2H),
7.65–7.55 (m, 2H), 7.31–7.08 (m, 7H), 3.81–3.72 (m,
1H), 3.58–3.31 (m, 3H), 3.16–2.78 (m, 2H), 2.75–2.48
(m, 2H), 2.23 (br s, 1H), 2.05–1.11 (m, 11H). MS
Å
(ESI+) m/z 609 (M+H)+. Anal. Calcd (C32H34Cl2N4O4
0.5 H2O, 1.6 TFA): C, 52.78; H, 4.61; Cl, 8.85; F,
11.38; N, 6.99. Found: C, 52.72; H, 4.04; Cl, 9.00; F,
11.05; N, 6.97; KF 1.20.
30: 1H NMR (DMSO-d6, rotamers): d 0.79 (s, 1H), 8.78
(m, 2H), 7.52–7.38 (m, 4H), 7.22–6.94 (m, 4H), 4.30–
3.60 (m, 4H), 3.15–2.95 (m, 2H), 2.07 (m, 1H), 1.75–
1.20 (m, 9H). MS (ESI+) m/z 615 (M+H)+. Anal. Calcd
(C29H28Cl2N4O5SÅ 2 H2O, 1.5 TFA): C, 46.72; H, 4.10;
Cl 8.62; F, 10.39; N, 6.81; S 3.90. Found: C, 46.24;
H,3.96; Cl, 9.02; F, 10.07; N, 6.61; S, 3.84; KF 4.06.
1
49: H NMR (CD3OD, rotamers): d 8.63 (m, 2H), 7.57
(M, 2H), 7.34–7.24 (m, 7H), 3.97 (m, 1H), 3.16–3.04 (m,
3H), 2.52–2.29 (m, 4H), 2.29 (br s, 1H), 2.20–1.50 (m,
9H). MS (ESIÅ+) m/z 609 (M+H)+. Anal. Calcd
(C32H34Cl2N4O4 0.4 H2O, 1.4 TFA): C, 53.84; H,
4.70; Cl, 9.13; F, 10.28; N, 7.25. Found: C, 53.92; H,
4.91; Cl, 9.29; F, 10.35; KF 1.01.
1
33: H NMR (CD3OD, rotamers): d 8.64–8.63 (m, 2H),
7.59–7.52 (m, 2H), 7.32–7.28 (m, 2H), 6.92–6.75 (m,
3H), 4.70–4.73 (m, 1H), 4.43–4.21 (m, 1H), 3.91 (s,
1H), 3.81–3.72 (m, 6H), 3.61–3.48 (m, 1H), 3.39–3.31
(m, 1H), 3.16–2.91 (m, 2H), 2.17 (s, 1H), 1.93–1.45 (m,
8H). MS (ESIÅ+) m/z 669 (M+H)+. Anal. Calcd
(C33H34Cl2N4O7 1 H2O, 1 TFA): C, 52.44; H, 4.65;
Cl, 8.85; F, 7.11; N, 6.99. Found: C, 52.20; H, 4.35;
Cl, 9.00; F, 6.76; N, 6.59; KF 2.20.
1
50: H NMR (CD3OD, rotamers): d 8.67 (s, 2H), 7.57
(m, 2H), 7.42 (m, 2H), 7.39–7.23 (m, 3H), 7.02 (m,
2H), 4.43–4.32 (m, 2H), 4.30–4.20 (m, 1H), 3.80 (s,
1H), 3.57 (m, 1H), 3.03–2.92 (m, 1H), 2.77–2.67 (m,
1H), 2.42–2.21 (m, H), 2.18–2.02 (m, 1H), 1.98–1.72
(m, 4H), 1.63–1.52 (m, 2H). (ESI+) m/z 581 (M+H)+.
Å
Anal. Calcd (C30H30Cl2N4O4 1.0 H2O, 1.6 TFA): C,
51.00; H, 4.33; Cl, 9.07; F, 11.66; N, 7.17. Found: C,
50.93; H, 3.75; Cl, 9.25; F, 11.45; N, 7.12; KF 2.15.
1
35: H NMR (CD3OD, rotamers): d 8.79–8.71 (m, 1H),
8.62 (m, 2H), 8.47–8.34 (m, 1H), 7.92–7.77 (m, 1.5H),

A. B. Dyatkin et al. / Bioorg. Med. Chem. 13 (2005) 6693–6702
6701
1
51: H NMR(DMSO-d6): d 10.91 (s, 1H); 9.06–9.00 (m,
standards were prepared by adding appropriate volumes
of stock solution directly into plasma and treated iden-
tically to collected plasma samples. Calibration stan-
dards were prepared in the range of 0.01–10 lM for
quantitation. LC–MS–MS analysis was performed uti-
lizing multiple reaction monitoring for detection of
characteristic ions for each tested compound, additional
related analytes, and internal standard. The low detec-
tion limit was 0.01 lM.
1H); 8.83 (d, J = 7.8 Hz, 1H); 8.80 (s, 2H), 8.00–7.92 (m,
1H), 7.57 (d, J = 8.5 Hz, 2H), 7.27 (d, J = 8.5 Hz, 2H),
4.57–4.52 (m, 1H), 3.92 (s, 1H), 3.37 (s, 1H), 3.13 (dd,
J = 14.0 and 4.6 Hz, 1H), 2.91 (dd, J = 14.0 and
9.6 Hz, 1H, 2.12 s, 1H), 1.85–1.48 (m, 8H). MS (ESI+)
m/z 492 (M+H)+. Anal. Calcd (C23H24Cl2N4O4 1.1
Å
TFA): C, 49.07; H, 4.10; Cl, 11.50; F, 10.16; N, 9.08.
Found: C, 48.85; H, 3.72; Cl, 11.49; F, 10.25; N, 8.85.
7.9. Ramos cell adhesion assay (a4b1 mediated adhesion/
VCAM-1)
7.12. Leukocytosis studies
Selected compounds were administered subcutaneously
in 0.5% methyl cellulose, at doses of 3 and 30 mg/kg,
to 8-week-old female Balb/C mice (Charles River Labo-
ratories, Kingston, NC). One hour later, 250 lL of
blood were removed and immediately added to micro-
tainer tubes containing lithium heparin (Becton–Dickin-
son, Franklin Lakes, NJ). Samples were analyzed for
total white blood cell counts and for lymphocyte counts
using an Advia 120 Hematology System (Bayer Diag-
nostics, Tarrytown, NY). ANOVA was used to evaluate
statistical significance.
Immulon 96-well plates (Dynex) 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 Sys-
tems). Plates were washed two times in PBS with 1%
BSA and blocked for 1 h at room temperature in this
buffer. PBS was removed and compounds to be tested
(50 lL) were added at 2 times concentration. Ramos
cells (50 lL at 2 · 106/mL), labeled with 5 lM Calcein
AM (Molecular Probes) for 1 h at 37 ꢁC, were added
to each well and allowed to adhere for 1 h at room tem-
perature. Plates were washed 4 times in PBS + 1% BSA
and cells were lysed for 15 min in 100 lL of 1 M Tris,
pH 8.0, with 1% SDS. The plate was read at 485 nm
excitation and 530 nm emission.
Acknowledgments
The authors thank Diane A. Gauthier, Dr. Gregory C.
Leo, Dr. John A. Masucci, and Dr. William E. Hag-
eman for assistance with spectroscopy and PK studies.
7.10. a4b7-K562 cell adhesion assay (a4b7 mediated
adhesion/MAdCAM-1)
M2 anti-FLAG antibody coated 96-well plates (Sigma)
were coated for 1 h at 4 ꢁC with 2–8 lL/well recombi-
nant FLAG-hMAdCAM-1 contained in 100 lL of Dul-
beccoÕs PBS, pH 7.4, with 1% BSA and 1 mM Mn2+
(PBS–BSA–Mn). Plates were washed once with PBS–
BSA–Mn. Buffer was removed and compounds to be
tested (50 lL) were added at 2 times concentration. Sta-
bly transfected K562 cells expressing human a4b7 inte-
grin, (50 lL at 2 · 106/mL) that had been labeled with
100 lg/mL carboxymethyl fluorescein diacetate succin-
imidyl ester (CFDA-SE; Molecular Probes) for 15 min
at 37 ꢁC were added to each well and allowed to adhere
for 1 h at room temperature. Plates were washed 4 times
in PBS–BSA–Mn and then cells were lysed for 2 min by
addition of 100 lL of PBS without Ca, Mg supplement-
ed with 0.1 M NaOH. The plate was read on a 96-well
fluorescent plate reader at 485 nm excitation and
530 nm emission.
References and notes
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6702
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