Synthesis and biological evaluation of novel pyridazinone-based α4 integrin receptor antagonists

Article abstract of DOI:10.1021/jm060031q

A novel series of pyridazinone-functionalized phenylalanine analogues was prepared and evaluated for inhibition of cellular adhesion mediated by α₄β₁/VCAM-1 and α₄β ₇MAdCAM-1 interactions. Concise syntheses were

Full text of DOI:10.1021/jm060031q

3402
J. Med. Chem. 2006, 49, 3402-3411
Synthesis and Biological Evaluation of Novel Pyridazinone-Based r4 Integrin Receptor
Antagonists
Yong Gong,* J. Kent Barbay,* Alexey B. Dyatkin, Tamara A. Miskowski, Edward S. Kimball, Stephen M. Prouty,
M. Carolyn Fisher, Rosemary J. Santulli, Craig R. Schneider, Nathaniel H. Wallace, Scott A. Ballentine, William E. Hageman,
John A. Masucci, Bruce E. Maryanoff, Bruce P. Damiano, Patricia Andrade-Gordon, Dennis J. Hlasta, Pamela J. Hornby, and
Wei He*
Drug DiscoVery, Johnson & Johnson Pharmaceutical Research and DeVelopment, L.L.C., Welsh & McKean Roads, P.O. Box 776,
Spring House, PennsylVania 19477
ReceiVed January 11, 2006
A novel series of pyridazinone-functionalized phenylalanine analogues was prepared and evaluated for
inhibition of cellular adhesion mediated by R4â1/VCAM-1 and R4â7/MAdCAM-1 interactions. Concise
syntheses were developed and applied for exploration of structure-activity relationships pertaining to the
pyridazinone ring as well as the N-acyl phenylalanine scaffold. Potent dual antagonists of R4â1 and R4â7
were generated from an amide subseries; antagonists selective for R4â7 were identified from urea and
carbamate-based subseries. The pharmacokinetic properties of selected members of the series have been
determined in rats and demonstrate that the use of ester prodrugs and alterations to the amide linkage can
lead to improved oral bioavailability in this series. An R4â7-selective member of the carbamate subseries
(36c), upon oral admininstration, demonstrated in vivo efficacy in the mouse DSS colitis model.
Introduction
Integrins are heterodimeric transmembrane receptors that
mediate cellular adhesion processes. The R4 integrin family is
composed of R4â1 (also known as VLA-4) and R4â7 integrins,
which are expressed on the surface of leukocytes and are
involved in their adhesion to the extracellular matrix, a key step
in recruitment to sites of inflammation.1,2 Vascular cell adhesion
molecule-1 (VCAM-1) is expressed on endothelial cell surfaces
Figure 1. Structures of (a) a representative antagonist of the N-acyl
phenylalanine class (1) and (b) a generalized member of the pyridazi-
none series.
and binds to R4â1, facilitating extravasation of leukocytes
through the endothelial cell layer. Integrin R4â7 mediates
leukocyte recruitment to the intestinal mucosa by binding
therapeutic intervention for the treatment of Crohn’s disease in
light of recent developments with the dual antibody Tysabri.
Anti-tumor necrosis factor-R (TNF-R) therapies, such as Remi-
cade, have significantly improved the quality of life for many
patients with Crohn’s disease, but there are still patients who
do not respond to the anti-TNF-R therapies. A combination of
an anti-TNF-R antibody and an R4â7 integrin antagonist may
offer an improved modality for treating such unmet medical
needs.
The discovery and development of orally active small-
molecule R4 integrin antagonists have been ongoing for some
time. Although many small molecules have been identified to
inhibit R4 integrins with nanomolar potency and selectivity, there
is still room for improvement in the discovery and development
of antagonists with improved pharmacokinetic properties.9 In
addition, most of the known compounds are either dual
antagonists against both R4â1 and R4â7 or are selective for R4â1
over R4â7; few compounds are selective for R4â7.10 N-Acyl-4-
arylphenylalanines constitute a major class of known R4 integrin
antagonists (e.g. 111 (SB683698); Figure 1a). As a potential
replacement for the substituted biphenyl group found in the
majority of antagonists in this class, we became interested in
4-heteroaryl functionalized N-acyl phenylalanines.12 We envi-
sioned that a pyridazinone might serve as a bioisostere for the
2,6-dimethoxy-substituted phenyl ring that may be prone to
metabolic demethylation (general structure of target antagonists;
Figure 1b). In addition, the pyridazinone group can be easily
mucosal addressin cell adhesion molecule-1 (MAdCAM-1)
on epithelial cell surfaces. The therapeutic potential of R4
integrin antagonists has recently been demonstrated by Tysabri
(natalizumab), a humanized monoclonal antibody that binds the
integrin R4 subunit, in the treatment of multiple sclerosis.3 In
November 2004, Tysabri received accelerated approval from
the FDA,4 but shortly thereafter it was voluntarily withdrawn
from the United States market due to reports of rare but serious
adverse events (progressive multifocal leukoencephalopathy, a
demyelinating disease of the central nervous system).5,6
In addition to its therapeutic utility for the treatment of
multiple sclerosis, Tysabri7 (and MLN02, an R4â7 integrin
antibody from Millennium Pharmaceuticals8) has shown promise
for treatment of Crohn’s disease in clinical trials. A nonselective
dual antibody such as Tysabri, active against both R4â1 and R4â7
integrins, may have broader therapeutic utility but may also have
more potential for side effects. Whereas selectively targeting
either R4â1 or R4â7 may provide better safety profiles, one would
expect more limited therapeutic utility. Presently, it is debatable
which integrin (R4â1 or R4â7) is a better target for producing
new safe and effective treatments. Targeting the tissue-specific
or -selective, gut-homing integrin R4â7 may represent a good
* To whom correspondence should be addressed. (Y.G.) Phone: (215)
628-5177. Fax: (215) 628-5047. E-mail: ygong@prdus.jnj.com. (J.K.B.)
Phone: (215) 628-5033. Fax (215) 628-3297. E-mail: kbarbay@prdus.jnj.com.
(W.H.) Phone: (215) 628-5525. Fax: (215) 628-5047. E-mail: whe@
prdus.jnj.com.
10.1021/jm060031q CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/09/2006

Pyridazinone-Based R4 Integrin Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3403
Table 1. Cellular Adhesion Assay Results for Benzamide Analoguesa,b
R4â1/VCAM-1
IC50 (µM)
R4â7/MAdCAM-1
IC50 (µM)
cmpd
R1
R2
5c
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OEt
OCHF2
NH2
pyrrolidin-1-yl
morpholin-4-yl
OCH2CH2OH
CH3
H
CH2Ph
Ph
cyclohexyl
t-Bu
0.031 ( 0.008 (4)
1.0 ( 0.25 (2)
0.52
3.9
>5
0.79
0.13 ( 0.026 (2)
0.36
0.48 ( 0.061 (3)
0.17
0.28 ( 0.066 (2)
0.37
0.11 ( 0.039 (3)
0.48 ( 0.14 (3)
0.30 ( 0.12 (3)
0.066 ( 0.013 (2)
0.15
0.003 ( 0.001 (6)
0.041 ( 0.013 (2)
0.10 ( 0.004 (2)
0.018 ( 0.010 (2)
0.33 ( 0.14 (2)
0.042 ( 0.005 (2)
0.002 ( 0.001 (5)
0.002 ( 0.002 (3)
0.049 ( 0.010 (7)
0.002 ( 0.001 (3)
0.008 ( 0.002 (4)
0.013 ( 0.009 (2)
0.016 ( 0.013 (3)
0.003 ( 0.003 (2)
0.002 ( 0.001 (3)
0.003 ( 0.001 (2)
0.011 ( 0.006 (3)
CH2CH2OH
2-morpholin-4-yl-ethyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
2-morpholin-4-yl-ethoxy
cyclohexyloxy
a Each IC50 is the mean of a duplicated run at six different concentrations. For repetitions (n), the IC50 is the mean ( SE with n in parentheses. b The IC50
of reference compound 111 was 0.20 ( 0.046 µM in the R4â1/VCAM-1 assay and 0.058 ( 0.010 µM in the R4â7/MAdCAM-1 assay. c The enantiomer of
compound 5 (prepared from 4-borono-D-phenylalanine) displayed reduced activity: IC50 (R4â1) > 1 µM, IC50 (R4â7) ) 0.17 µM.
functionalized to provide additional receptor interactions for
potency and selectivity improvements. Functionalization of the
pyridazinone will affect the chemical and physical properties
and may also change the pharmacokinetic profiles of the target
molecules. We also considered that functionalization of the
pyridazinone ring alone might not be sufficient to solve the poor
pharmacokinetic properties observed with N-acyl phenylalanines
since other parts of the target molecules may also be subject to
metabolism. Our strategy was to modify the N-acyl region of
the targeted molecules after the best pyridazinone was identified.
Finally, the corresponding ester prodrugs would also be prepared
for comparison with the parent acids. Our previously reported
microwave-assisted Suzuki coupling with unprotected free
amino acids13 enabled us to synthesize a variety of function-
alized, aryl-substituted pyridazinones. Since the initial member
of this series (compound 5, Table 1) proved to be a potent
antagonist of both R4â1 and R4â7 integrins, a limited set of
pyridazinone-functionalized phenylalanine analogues was ex-
plored further. In this report we describe in vitro cellular
adhesion activity, selectivity profiles, pharmacokinetic proper-
ties, and in vivo activity of selected members of this new series.
Chemistry
A series of 2,6-dichlorobenzoyl phenylalanines with varied
substituents on the pyridazinone was initially prepared. The 2,6-
dichlorophenylamide group, held constant in this portion of the
study, was selected on the basis of its presence in a number of
potent antagonists of the N-acyl phenylalanine class.9,11 Acyl-
ation of 4-borono-L-phenylalanine 2 with 2,6-dichlorobenzoyl
chloride and subsequent Suzuki coupling of the resulting amide
3 with 4-halopyridazinones 4 provided a convergent approach
(Scheme 1) to the target analogues 5-18.14 The commercial
Scheme 1a
a Reagents and conditions: (i) 2,6-dichlorobenzoyl chloride, Na2CO3, H2O, CH3CN, 50 °C, 71%; (ii) Pd(PPh3)2Cl2, Na2CO3, H2O, CH3CN, µW, 150 °C,
10 min or reflux in oil bath, 1 h, 10-62%; (iii) ROH, Na0, 80 °C, 30-42%.

3404 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Gong et al.
Scheme 2a
a Reagents and conditions: (i) Pd(PPh3)2Cl2, Na2CO3, H2O, CH3CN, µW, 150 °C, 10 min, 55%; (ii) MeOH, SOCl2, 66%; (iii) 1,1′-carbonyldiimidazole,
THF, CH2Cl2, 81%; (iv) R4R5NH, CH3CN, 23 °C (aliphatic amines) or µW, 130 °C, 10 min (anilines); (v) 2 N aq LiOH, 20-57% over two steps.
Table 2. Cellular Adhesion Assay Results for Urea and Carbamate Compoundsa
R4â1/VCAM-1
IC50 (µM)
R4â7/MAdCAM-1
IC50 (µM)
cmpd
R2
R4
R5
Y
25
26
27
28
29
30
CH3
CH3
CH3
CH3
CH3
CH3
Ph
Ph
Ph
CH3
CH2CH2OH
i-Bu
CH3
i-Pr
n-Pr
CH3
CH3
CH3
N
N
N
N
N
N
>5
>5
>5
>5
>5
1.8
0.063 ( 0.031 (5)
0.092 ( 0.015 (2)
0.35
1.5
0.094 ( 0.037 (5)
0.017 ( 0.009 (5)
31
CH3
1.8
0.029 ( 0.001 (2)
33
34
35
36
CH3
CH3
CH3
CH3
CH2Ph
t-Bu
-
-
-
-
O
O
O
O
>5
>5
>5
>5
0.71
0.57
0.81 ( 0.30 (2)
0.11 ( 0.025 (3)
CH2CH2OH
t-Bu
a As for Table 1.
Scheme 3a
availability and synthetic accessibility of substituted 4-halo-
pyridazinones enabled preparation of analogues differentially
substituted at the 3- and 5-positions. It was found that treatment
of 5-methoxy pyridazinone 5 with sodium alkoxide nucleophiles
resulted in substitution at the 5-position,15 facilitating efficient
preparation of additional analogues 19-21.
A second focus of targeted structural modifications was
replacement of the amide linkage with potential bioisosteres
(urea and carbamate) to explore the selectivity profile and
pharmacokinetic properties of the resulting compounds. The
synthesis of antagonists based on a urea linkage is presented in
Scheme 2. Unprotected amino acid 2 was coupled with
4-chloropyridazinone 22 under our previously reported micro-
wave-assisted Suzuki conditions.13 Following esterification, the
corresponding amino ester intermediate 23a was reacted with
1,1′-carbonyldiimidazole to afford carbamoyl imidazole 24. This
intermediate reacted smoothly with aliphatic amines at room
temperature, or with anilines under microwave irradiation, to
form ureas. Addition of aqueous base to the crude reaction
mixtures provided acids 25-31 (Table 2). Carbamate analogues
a Reagents and conditions: (i) R4OCOCl or Boc2O, Na2CO3, H2O,
CH3CN, 10-84%.
33-36 were readily prepared from amino acid intermediates
23 and 32 by reaction with chloroformates or dialkyl dicarbon-
ates (Scheme 3).
Ester prodrugs of several of the antagonists were prepared
in order to assess their pharmacokinetic properties. These
compounds were prepared from the corresponding carboxylic
acids either by thionyl chloride- or bis(2-oxo-3-oxazolidinyl)-
phosphinic chloride-mediated esterification, as detailed in the
Experimental Section.

Pyridazinone-Based R4 Integrin Receptor Antagonists
Table 3. Inhibition of Cellular Adhesion by Compound 5a
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3405
endothelial cell line
adhesion nominally
leukocyte type (cell line)
(method of stimulation)
mediated by
IC50, µM
human lymphocyte (Ramos)
human monocytes (U-937)
human granulocytes (HL-60)
HUVEC (TNF-R)
HUVEC (LPS)
HUVEC (TNF-R)
VCAM-1
ICAM-1/VCAM-1
ELAM-1
0.043
0.014
<0.005
a HUVEC: human umbilical vein endothelial cell. ICAM-1: intercellular adhesion molecule-1. ELAM-1: endothelial leukocyte adhesion molecule-1.
Table 4. Pharmacokinetic Properties of Selected Compounds in Ratsa
cmpd
ester form
AUCb (µM-h)
t1/2 (h)c
F (%)
5d
5a
5b
5c
30
30c
36
36c
none (carboxylic acid)
methyl ester
ethyl ester
2-hydroxyethyl ester
none (carboxylic acid)
2-hydroxyethyl ester
none (carboxylic acid)
2-hydroxyethyl ester
0.5 ( 0.3
1.7 ( 0.6
2.3 ( 0.7
3.3 ( 0.6
5.2 ( 2.9
47 ( 32
0.2 ( 0.05
0.7 ( 0.01
nd
1 ( 1
8 ( 3
4 ( 1
1.0
7 ( 1
3 ( 2
0.9 ( 0.5
0.4 ( 0.3
0.2 ( 0.03
1.1 ( 0.1
14 ( 10
1 ( 1
1.6 ( 0.9
51 ( 13
31 ( 11
a Averages from a group of four. Dosed at 30 mg/kg po (10% PEG 400 in PBS, pH 7.0-7.5), 3 mg/kg iv. In the case of ester prodrugs, data refer to
measured concentrations of the parent carboxylic acids (quantitated with authentic samples of the acids as calibration standards); esters were not detected.20
c
See Experimental Section for details. b Area under the plasma concentration-time curve following oral administration. t1/2 following iv administration; nd
) not determined. d Clearance (Clp) values for carboxylic acids 5, 30, and 36 were 22, 7.8, and 6.0 mL/min/kg, respectively; volume of distribution (Vdss)
values for 5, 30, and 36 were 0.40, 0.19, and 0.11 L/kg, respectively (iv administration).
Results and Discussion
2-hydroxyethyl group, identified as a potent ligand in the earlier
optimization of the amide series. The selectivity for R4â7
observed with the ureas and carbamates might be explained by
conformational changes when compared with the amides.
Since it has been shown that anti-R4 antibodies can block
synergistic effects between TNF-R and R4 integrins,16,17 we
turned our attention to whether our compounds can block the
adhesion of different types of cells induced by either TNF-R or
lipopolysaccharide (LPS) in addition to the primary cellular
assay that is activated by Mn2+. Consequently, we studied the
ability of compound 5 to block adhesion of human leukocytes
to stimulated human endothelial cells.18,19 Compound 5 was
found to block the adhesion of human lymphocytes, monocytes,
and granulocytes to stimulated human umbilical vein endothelial
(HUVEC) cells with IC50 values in the low nanomolar range
(Table 3). These results demonstrate that 5 blocks cellular
adhesion in different cell types activated by Mn2+, LPS, or the
proinflammatory cytokine TNF-R. Considering the role of both
TNF-R and R4 integrins in many diseases, and the therapeutic
effects of R4 integrin antagonists (whether potentially separated
from rare but fatal side effects) and anti-TNF-R antibodies, the
combination of these two approaches may offer an improved
treatment modality.
Pharmacokinetics. Pharmacokinetic (PK) data of selected
compounds in rats are presented in Table 4. Compound 5 was
found to have low bioavailability (F ) 1%) and a low area
under the plasma concentration-time curve (AUC ) 0.5 µM-
h) after oral dosing at 30 mg/kg. A few esters were selected as
potential prodrugs of these antagonists.21,22 Oral administration
of methyl, ethyl, and 2-hydroxyethyl esters (5a, 5b, and 5c,
respectively) of 5 resulted in enhanced plasma exposure of the
parent carboxylic acid, with the latter ester (5c) providing the
highest exposure (F ) 7%, AUC ) 3.3 µM-h). Alterations to
the amide functionality also proved to have the potential to
improve PK properties. Within the carbamate and urea subseries
substantial improvements in oral bioavailability and systemic
exposure level were achieved, provided that the compounds were
dosed as 2-hydroxyethyl esters (urea 30c: F ) 14%, AUC )
47 µM-h; carbamate 36c: F ) 31%, AUC ) 51 µM-h).
Dextran Sulfate Sodium (DSS) Colitis. With the discovery
that the prodrug 36c had considerably improved pharmaco-
kinetics, the in vivo activity of this R4â7-specific integrin
In Vitro Activity. Compounds were assayed for their ability
to inhibit the binding of human lymphocytes (Ramos) expressing
R4â1 to immobilized VCAM-1 and the binding of R4â7-
expressing K562 cells to immobilized MAdCAM-1. The results
of the cellular adhesion assays are presented in Tables 1 and 2.
Substitutions on the pyridazinone core were explored to
determine their effects on potency, selectivity, and pharmaco-
kinetic properties. 2-Methyl-5-methoxy pyridazinone 5 displayed
activity against both R4â1 (IC50 ) 31 nM) and R4â7 (IC50 ) 3
nM). The N-desmethyl analogue 6 and O-desmethyl analogue
13 each had reduced potency. Replacement of the methyl group
at the 2-position of the pyridazinone with larger hydrophobic
groups (benzyl, phenyl, cyclohexyl, and tert-butyl; compounds
7-10) generally decreased activity, particularly with respect to
R4â1, while certain polar groups (2-hydroxyethyl and 2-mor-
pholinoethyl; compounds 11 and 12) could be incorporated at
this position with retention of R4â7 activity. Substitution at the
5-position of the pyridazinone (R1) with a variety of primary
and secondary O-alkyl groups (compounds 14, 15, 19-21) were
well-tolerated in regard to inhibition of R4â7-mediated cellular
adhesion, but generally resulted in moderate decreases in R4â1
activity. Similarly, substitution at the 5-position with amino and
alkylamino groups resulted in analogues (16-18) that retained
activity versus R4â7, but displayed reduced inhibition of the
R4â1/VCAM-1 interaction.
Following this screen of modifications to the pyridazinone
group, alternatives to the amide linkage were sought to explore
the improvement of pharmacokinetic properties and selectivity
for R4â7 versus R4â1. The majority of such analogues prepared
incorporated the 2-methyl-5-methoxy pyridazinone found in the
potent dual R4â1/R4â7 antagonist 5. Table 2 summarizes the
cellular inhibition results for urea and carbamate compounds,
which were found to display selective inhibition of R4â7/
MAdCAM-1 based cellular adhesion compared to that mediated
by R4â1/VCAM-1. Double-digit nanomolar activity versus R4â7
was achieved with a number of trisubstituted ureas, with the
most active (30, R4 ) i-Bu, R5 ) CH3) having an IC50 of 17
nM. It proved to be more difficult to optimize the carbamate
series to achieve low nanomolar activity, and the most potent
carbamate (36, R4 ) t-Bu, R4â7 IC50 ) 110 nM) required
concomitant substitution of the pyridazinone 2-position with a

3406 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Gong et al.
leucine enkephalin as an internal standard. Elemental analysis was
conducted by Robertson Microlit Laboratories, Inc. Madison, NJ.
Where noted, compounds were determined to be >95% pure by
analytical reverse-phase HPLC. The retention time (tr) was deter-
mined using two of the following three HPLC systems. HPLC-1:
Waters XTerra MS C18 column (2.5 µm, 2.1 × 30 mm), gradient
4-96% CH3CN-H2O, 0.05% HCO2H over 3.5 min, hold 96%
CH3CN for 2 min, flow rate 0.5 mL/min, 190-360 nm PDA
detection. HPLC-2: Supelco ABZ+PLUS column (3 µm, 2.1 ×
50 mm), gradient 10-90% CH3CN-H2O, 0.05% TFA over 4 min,
then to 100% CH3CN over 0.1 min, hold at 100% CH3CN for 1.9
min, flow rate 0.75 mL/min, detection at 220 and 254 nm; HPLC-
3: Supelco ABZ+PLUS column (3 µm, 4.6 × 33 mm), gradient
4-95% CH3CN-H2O, 0.1% TFA over 8.5 min, hold at 95%
CH3CN for 0.3 min, flow rate 1.2 mL/min, detection at 220 and
254 nm.
4-Halopyridazinone precursors were commercially available or
were prepared using published procedures.24,25 In cases where
preparation of the required halopyridazinone has not been reported,
experimental methods are provided preceding the synthesis in which
it was used.
Female Balb/c mice (Charles River Laboratories), 11-14 weeks
of age, were used for dextran sulfate sodium (DSS) colitis studies.
DSS (MW 36-44 kD) was purchased from ICN pharmaceuticals
(Newport, CA).
Figure 2. Prevention of DSS-induced colitis by compound 36c. Mice
were dosed with 60 mg/kg, bid, po, for 7 days. *p < 0.05 vs DSS +
vehicle-treated. Macroscopic score was the combined scores from
measurements of colon shrinkage, stool consistency, and colon damage.
antagonist in a mouse DSS colitis model was evaluated. After
oral dosing at 60 mg/kg, bid, for 7 days, the macroscopic
damage score was reduced by 54% (Figure 2). These data
demonstrate that an R4â7-specific integrin antagonist can ef-
fectively prevent the development of experimental DSS-induced
colitis, presumably by preventing the recruitment of R4â7
positive cells to the intestinal tract. Our results lend additional
support to the concept that inhibition of integrin-dependent
recruitment can have therapeutic value, consistent with data
reported for monoclonal antibodies to R4 integrins7a and for
small molecule integrin antagonists.23
(S)-2-(2,6-Dichloro-benzoylamino)-3-(4-boronophenyl)-propi-
onic Acid (3). 2,6-Dichlorobenzoyl chloride (1.47 mL, 10.2 mmol)
was added to a mixture of 4-borono-L-phenylalanine (2, 2.04 g,
9.76 mmol) and Na2CO3 (2.07 g, 19.5 mmol) in CH3CN-H2O (1:
1, 40 mL) at 50 °C. The resulting mixture was stirred at 50 °C for
1 h, then was cooled to 0 °C, and was acidified to pH 2 by addition
of concentrated HCl (aq). The suspension was stirred at 0 °C for
30 min, and the precipitated solid was collected by vacuum filtration
and was washed with water. The white solid was dried in a vacuum
Conclusion
A novel series of integrin antagonists based on a pyridazi-
none-substituted phenylalanine scaffold has been discovered.
Synthetic routes were developed that allow efficient preparation
of analogues with varied substitutions on the pyridazinone ring,
as well as compounds with an amide, urea, or carbamate linkage
to the phenylalanine nitrogen. These compounds are potent
antagonists of R4â1/VCAM-1 and/or R4â7/MAdCAM-1 medi-
ated cellular adhesion whether activated by metal ion, LPS, or
TNF-R. Dual antagonists were identified as well as selective
R4â7 antagonists that are very different from known R4
antagonists (most of which are R4â1 selective). The pharmaco-
kinetic properties of selected members of the series have been
determined in rats, leading to the finding that the use of ester
prodrugs and bioisosteric replacement of the amide functionality
has the potential to improve oral bioavailability and systemic
exposure level. Our findings in a mouse colitis model offer the
unique perspective that an integrin antagonist with specificity
for R4â7 integrin and with negligible activity against R4â1
integrin can prevent development of experimental colitis. Further
exploration of R4 integrin receptor antagonists may provide new
therapeutic interventions in diseases where the integrins play
critical roles.
1
oven at 50 °C, affording compound 3 (2.65 g, 71%). H NMR
(CD3OD, 300 MHz) δ 7.55 (d, 2H, J ) 7.6 Hz), 7.28-7.40 (m,
5H), 4.95 (dd, 1H, J ) 9.3, 4.7 Hz), 3.30 (dd, 1H, J ) 13.9, 5.3
Hz), 3.03 (dd, 1H, J ) 14.1, 9.4 Hz). MS m/z 382 (MH)+.
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(5-methoxy-2-methyl-
3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (5). A
10 mL vial containing a magnetic stir bar was charged with boronic
acid 3 (76 mg, 0.20 mmol), 4-chloro-5-methoxy-2-methyl-2H-
pyridazin-3-one (22, 35 mg, 0.20 mmol), Pd(PPh3)2Cl2 (7 mg, 0.01
mmol), 1.0 M sodium carbonate (0.50 mL, 0.50 mmol), and CH3CN
(0.50 mL). The vial was sealed, and the mixture was heated under
microwave irradiation at 150 °C for 10 min. Acidification (with
TFA) followed by reverse phase HPLC purification (20-40%
CH3CN-H2O, 0.1% TFA) gave compound 5 as a white solid (57
1
mg, 58%). H NMR (CD3OD, 300 MHz) δ 8.17 (s, 1H), 7.38-
7.33 (m, 7H), 4.99 (dd, 1H, J ) 5.0, 9.4 Hz), 3.94 (s, 3H), 3.78 (s,
3H), 3.33 (dd, 1H, J ) 5.1, 14.1 Hz), 3.08 (dd, 1H, J ) 9.4, 14.1
Hz). MS m/z 476 (MH)+. Anal. (C22H19Cl2N3O5‚H2O) C, H, N,
Cl.
Compounds 6-14 and 17-18 were prepared by the method
given for compound 5.
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(5-methoxy-3-oxo-2,3-
dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (6). Prepared
from coupling of 3 (96 mg, 0.25 mmol) and 4-chloro-5-methoxy-
2H-pyridazin-3-one. Yield: 29 mg (25%). 1H NMR (CD3OD, 300
MHz) δ 8.20 (s, 1H), 7.41-7.33 (m, 7H), 4.97 (dd, 1H, J ) 5.1,
9.3 Hz), 3.96 (s, 3H), 3.25 (dd, 1H, J ) 5.1, 14.0 Hz), 3.07 (dd,
1H, J ) 9.3, 14.0 Hz). MS m/z 462 (MH)+. TOF HRMS calcd for
C21H18Cl2N3O5 (MH)+ 462.0624; found 462.0605. HPLC-1, tr )
2.82 min; HPLC-3, tr ) 3.61 min.
Experimental Section
General. 1H NMR spectra were acquired on a Bruker 300-
Avance (300 MHz) or Avance DMX-400 (400 MHz) spectrometer,
with TMS as an internal standard; chemical shifts are expressed in
parts per million (ppm, δ scale). Reverse-phase preparative HPLC
purifications were performed using a Gilson system equipped with
a YMC-Pack ODS-A column (100 × 30 mm; 5 µM, 120 Å) eluting
at 32 mL/min with detection at 214 and 254 nm. Microwave-
accelerated reactions were performed using either a CEM Discover
or a Personal Chemistry Smith Synthesizer microwave instrument.
LC-MS was performed on an Agilent 1100 series LC/MSD.
Accurate mass determination was performed on a Micromass LCT
time-of-flight mass spectrometer using electrospray ionization, with
(S)-3-[4-(2-Benzyl-5-methoxy-3-oxo-2,3-dihydro-pyridazin-4-
yl)-phenyl]-2-(2,6-dichloro-benzoylamino)-propionic Acid (7).
Prepared by coupling of 3 (76 mg, 0.20 mmol) and 2-benzyl-4-
1
chloro-5-methoxy-2H-pyridazin-3-one. Yield: 52 mg (46%). H
NMR (CD3OD, 300 MHz) δ 8.20 (s, 1H), 7.35-7.27 (m, 12H),
5.34 (s, 2H), 4.97 (dd, 1H, J ) 5.1, 9.2 Hz), 3.93 (s, 3H), 3.35 (dd,
1H, J ) 5.0, 13.9 Hz), 3.08 (dd, 1H, J ) 9.4, 13.9 Hz). MS m/z

Pyridazinone-Based R4 Integrin Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3407
552 (MH)+. TOF HRMS calcd for C28H24Cl2N3O5 (MH)+ 552.1093;
found 552.1094. Anal. (C28H23Cl2N3O5‚0.5H2O) C, H, N, Cl.
4-Bromo-5-difluoromethoxy-2-methyl-2H-pyridazin-3-one. A
pressure tube was charged sequentially with 4-bromo-5-hydroxy-
2-methyl-2H-pyridazin-3-one24 (1.29 g, 6.28 mmol), chlorodi-
fluoroacetic acid sodium salt (1.15 g, 7.54 mmol), and NaOH (314
mg, 7.85 mmol). The vessel was purged with nitrogen, and DMF
(3.0 mL) was added. The mixture was heated to 130 °C for 1 h,
then was allowed to cool to 23 °C. The mixture was diluted with
EtOAc (50 mL), and the resulting solution was washed with a
saturated solution of NaCl (aq) (2 × 50 mL). The organic phase
was dried (Na2SO4), filtered, and concentrated, to yield a tan solid
which was purified by column chromatography (SiO2, 50-70%
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(5-methoxy-3-oxo-2-
phenyl-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (8).
Prepared by coupling of 3 (76 mg, 0.20 mmol) and 4-chloro-5-
1
methoxy-2-phenyl-2H-pyridazin-3-one. Yield: 46 mg (42%). H
NMR (CD3OD, 300 MHz) δ 8.34 (s, 1H), 7.53-7.35 (m, 12H),
4.97 (dd, 1H, J ) 5.1, 9.4 Hz), 4.01 (s, 3H), 3.35 (dd, 1H, J ) 5.1,
14.0 Hz), 3.06 (dd, 1H, J ) 9.3, 14.0 Hz). MS m/z 538 (MH)+.
TOF HRMS calcd for C27H22Cl2N3O5 (MH)+ 538.0937; found
538.0935. Anal. (C27H21Cl2N3O5‚0.5H2O) C, H, N, Cl.
(S)-3-[4-(2-Cyclohexyl-5-methoxy-3-oxo-2,3-dihydro-pyridazin-
4-yl)-phenyl]-2-(2,6-dichloro-benzoylamino)-propionic Acid (9).
Prepared from coupling of 3 (40 mg, 0.10 mmol) and 4-chloro-2-
cyclohexyl-5-methoxy-2H-pyridazin-3-one. Yield: 15 mg (27%).
1H NMR (CD3OD, 300 MHz) δ 8.22 (s, 1H), 7.36-7.33 (m, 7H),
4.99 (dd, 1H, J ) 5.0, 9.3 Hz), 4.91 (m, 1H), 4.00 (s, 3H), 3.35
(dd, 1H, J ) 5.0, 14.1 Hz), 3.07 (dd, 1H, J ) 9.3, 14.1 Hz), 1.92-
1.24 (m, 10H). MS m/z 544 (MH)+. TOF HRMS calcd for
C27H28Cl2N3O5 (MH)+ 544.1406; found 544.1391. HPLC-1, tr )
3.63 min; HPLC-3, tr ) 5.46 min.
(S)-3-[4-(2-tert-Butyl-5-methoxy-3-oxo-2,3-dihydro-pyridazin-
4-yl)-phenyl]-2-(2,6-dichloro-benzoylamino)-propionic Acid (10).
Prepared from coupling of 3 (76 mg, 0.20 mmol) and 2-tert-butyl-
4-chloro-5-methoxy-2H-pyridazin-3-one. Yield: 16 mg (15%). 1H
NMR (CD3OD, 300 MHz) δ 8.11 (s, 1H), 7.39-7.29 (m, 7H), 4.99
(dd, 1H, J ) 5.0, 9.0 Hz), 3.92 (s, 3H), 3.31 (dd, 1H, J ) 5.0, 14.0
Hz), 3.08 (dd, 1H, J ) 9.1, 14.0 Hz), 1.66 (s, 9H). MS m/z 518
(MH)+. TOF HRMS calcd for C25H24Cl2N3O5 (MH)+ 518.1250;
found 518.1235. HPLC-1, tr ) 3.53 min; HPLC-3, tr ) 5.44 min.
1
EtOAc-hexanes), yielding a white solid (1.09 g, 68%). H NMR
(CDCl3, 300 MHz) δ 7.79 (s, 1H), 6.70 (t, 1H, JHF ) 71.0 Hz),
3.86 (s, 3H). TOF HRMS calcd for C6H6N2O2BrF2 (MH)+
254.9581; found 254.9582.
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(5-difluoromethoxy-
2-methyl-3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid
(15). To a mixture of 3 (370 mg, 0.968 mmol), 4-bromo-5-
difluoromethoxy-2-methyl-2H-pyridazin-3-one (218 mg, 1.06 mmol)
and Pd(PPh3)2Cl2 (34 mg, 0.048 mmol) were sequentially added
an aqueous solution of Na2CO3 (2 M, 2 mL, 4 mmol) and CH3CN
(2 mL). The resulting suspension was heated at reflux under N2
for 1 h, then was allowed to cool to 23 °C. The mixture was partially
concentrated to remove volatile solvent. The resulting mixture was
diluted with half-saturated NaHCO3 (aq) (20 mL) and was washed
with Et2O (20 mL). The combined aqueous extracts were cooled
to 0 °C and were acidified to pH 2 by addition of 2 N aqueous
HCl. The precipitated solid was collected by vacuum filtration and
was purified by reverse-phase HPLC (35-55% CH3CN-H2O, 0.1%
TFA), affording the title compound as a white powder (305 mg,
1
62%). H NMR (CD3OD, 300 MHz) δ 8.09 (s, 1H), 7.38-7.44
(S)-2-(2,6-Dichloro-benzoylamino)-3-{4-[2-(2-hydroxy-ethyl)-
5-methoxy-3-oxo-2,3-dihydro-pyridazin-4-yl]-phenyl}-propion-
ic Acid (11). Prepared from coupling of 3 (153 mg, 0.40 mmol)
and 4-chloro-2-(2-hydroxy-ethyl)-5-methoxy-2H-pyridazin-3-one.
Yield: 74 mg (35%). 1H NMR (CD3OD, 300 MHz) δ 8.20 (s, 1H),
7.36-7.33 (m, 7H), 4.99 (dd, 1H, J ) 5.0, 9.3 Hz), 4.32 (t, 2H, J
) 5.7 Hz), 3.94 (s, 3H), 3.92 (t, 2H, J ) 5.7 Hz), 3.32 (dd, 1H, J
) 5.0, 14.1 Hz), 3.07 (dd, 1H, J ) 9.3, 14.1 Hz). MS m/z 506
(MH)+. TOF HRMS calcd for C23H22Cl2N3O6 (MH)+ 506.0886;
found 506.0882. Anal. (C23H21Cl2N3O6‚0.25F3CCO2H) C, H, N.
(m, 4H), 7.27-7.37 (m, 3H), 6.97 (t, 1H, JHF ) 72.2 Hz), 4.98
(dd, 1H, J ) 9.3, 5.0 Hz), 3.81 (s, 3H), 3.33 (dd, 1H, J ) 14.2, 5.1
Hz), 3.07 (dd, 1H, J ) 14.1, 9.4 Hz). TOF HRMS calcd for
C22H18N3O5Cl2F2 (MH)+ 512.0592; found 512.0604. Anal.
(C22H17N3O5Cl2F2‚0.1F3CCO2H) C, H, N.
(S)-3-[4-(5-Amino-2-methyl-3-oxo-2,3-dihydro-pyridazin-4-
yl)-phenyl]-2-(2,6-dichloro-benzoylamino)-propionic Acid (16).
Prepared by the method given above for compound 15 using 370
mg (0.968 mmol) of 3 and 5-amino-4-bromo-2-methyl-2H-
pyridazin-3-one25 (326 mg, 1.60 mmol). The crude product was a
tan solid (599 mg, 90%). A purified sample was obtained by
reverse-phase HPLC (20-40% CH3CN-H2O, 0.1% TFA). 1H
NMR (CD3OD, 300 MHz) δ 7.66 (s, 1H), 7.27-7.45 (m, 7 H),
4.98 (dd, 1H, J ) 8.4, 5.6 Hz), 3.68 (s, 3H), 3.30 (dd, 1H, J )
13.9, 5.5 Hz), 3.13 (dd, 1H, J ) 14.0, 8.4 Hz). TOF HRMS calcd
for C21H19N4O4Cl2 (MH)+ 461.0783; found 461.0794. HPLC-1, tr
) 2.78 min; HPLC-2, tr ) 2.72 min.
(S)-2-(2,6-Dichloro-benzoylamino)-3-{4-[5-methoxy-2-(2-mor-
pholin-4-yl-ethyl)-3-oxo-2,3-dihydro-pyridazin-4-yl]-phenyl}-
propionic Acid (12). Prepared from coupling of 3 (153 mg, 0.40
mmol) and 4-chloro-5-methoxy-2-(2-morpholin-4-yl-ethyl)-2H-
1
pyridazin-3-one. Yield: 116 mg (42%). H NMR (CD3OD, 300
MHz) δ 8.28 (s, 1H), 7.42-7.33 (m, 7H), 4.98 (dd, 1H, J ) 4.9,
9.6 Hz), 4.61 (t, 2H, J ) 5.5 Hz), 3.98 (s, 3H), 3.90 (br, 4H), 3.64
(t, 2H, J ) 5.5 Hz), 3.43 (br, 4H), 3.34 (dd, 1H, J ) 4.9, 14.1 Hz),
3.06 (dd, 1H, J ) 9.6, 14.1 Hz). MS m/z 575 (MH)+. TOF HRMS
calcd for C27H29Cl2N4O6 (MH)+ 575.1464; found 575.1464. HPLC-
1, tr ) 2.52 min; HPLC-3, tr ) 3.18 min.
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(2-methyl-3-oxo-5-
pyrrolidin-1-yl-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid
(17). Prepared from coupling of 3 (153 mg, 0.40 mmol) and
4-bromo-2-methyl-5-pyrrolidin-1-yl-2H-pyridazin-3-one. Yield: 31
1
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(5-hydroxy-2-methyl-
3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (13).
Prepared from coupling of 3 (96 mg, 0.25 mmol) and 4-bromo-5-
mg (14%). H NMR (CD3OD, 300 MHz) δ 7.85 (s, 1H), 7.41-
7.31 (m, 5H), 7.19 (d, 2H, J ) 7.7 Hz), 4.92 (dd, 1H, J ) 5.2, 8.4
Hz), 3.67 (s, 3H), 3.27 (dd, 1H, J ) 5.2, 14.1 Hz), 3.13 (dd, 1H,
J ) 8.4, 14.1 Hz), 3.06 (br, 4H), 1.75 (br, 4H). MS m/z 515 (MH)+.
TOF HRMS calcd for C25H25Cl2N4O4 (MH)+ 515.1253; found
515.1252. HPLC-1, tr ) 3.15 min; HPLC-3, tr ) 4.56 min.
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(2-methyl-5-morpho-
lin-4-yl-3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid
(18). Prepared from coupling of 3 (153 mg, 0.40 mmol) and
4-chloro-2-methyl-5-morpholin-4-yl-2H-pyridazin-3-one. Yield: 117
1
hydroxy-2-methyl-2H-pyridazin-3-one. Yield: 11 mg (10%). H
NMR (CD3OD, 300 MHz) δ 7.77 (s, 1H), 7.35-7.44 (m, 7H), 4.98
(dd, 1H, J ) 5.0, 8.6), 3.73 (s, 3H), 3.30 (obscured by CD3OD
signal), 3.12 (dd, 1H, J ) 8.6, 14.0 Hz). MS m/z 462 (MH)+. TOF
HRMS calcd for C21H18Cl2N3O5 (MH)+ 462.0624; found 462.0613.
HPLC-1, tr ) 2.75 min; HPLC-3, tr ) 3.90 min.
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(5-ethoxy-2-methyl-3-
oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (14).
Prepared from coupling of 3 (76 mg, 0.20 mmol) and 4-chloro-5-
ethoxy-2-methyl-2H-pyridazin-3-one. Yield: 44 mg (44%). 1H
NMR (CD3OD, 300 MHz) δ 8.15 (s, 1H), 7.32-7.44 (m, 7H), 5.01
(dd, 1H, J ) 9.3, 5.0 Hz), 4.27 (q, 2H, J ) 7.0 Hz), 3.79 (s, 3H),
3.35 (dd, 1H, J ) 14.4, 4.8 Hz), 3.10 (dd, 1H, J ) 14.1, 9.4 Hz),
1.33 (t, 3H, J ) 7.0 Hz). MS m/z 490 (MH)+. TOF HRMS calcd
for C23H22Cl2N3O5 (MH)+ 490.0937; found 490.0937. Anal.
(C23H21Cl2N3O5‚0.5H2O) C, H, N, Cl.
1
mg (52%). H NMR (CD3OD, 300 MHz) δ 7.92 (s, 1H), 7.42-
7.32 (m, 7H), 5.06 (dd, 1H, J ) 5.5, 8.2 Hz), 3.71 (s, 3H), 3.68 (s,
3H), 3.52 (t, 4H, J ) 4.6 Hz), 3.26 (dd, 1H, J ) 5.5, 14.0 Hz),
3.14 (dd, 1H, J ) 8.2, 14.0 Hz), 3.01 (t, 4H, J ) 4.6 Hz). MS m/z
531 (MH)+. TOF HRMS calcd for C25H25Cl2N4O5 (MH)+ 531.1202;
found 531.1199. Anal. (C25H24Cl2N4O5‚0.3F3CCO2H) C, H, N, Cl.
General Procedure for the Synthesis of Analogues 19-21 by
Michael Addition/Elimination. A solution of an alkoxide nucleo-
phile was generated by addition of sodium metal (25 mg, 1.08

3408 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Gong et al.
mmol) to an alcohol (1 mL) at 23 °C. The mixture was stirred
until solid metal was consumed. Compound 5 (100 mg, 0.20 mmol)
was added, and the mixture was heated in an 85 °C oil bath for 2
h. The mixture was concentrated, and the residue was suspended
in CH3CN, acidified by addition of TFA, filtered, and purified by
reverse-phase HPLC (CH3CN-H2O, 0.1% TFA).
(S)-2-(2,6-Dichloro-benzoylamino)-3-{4-[5-(2-hydroxy-ethoxy)-
2-methyl-3-oxo-2,3-dihydro-pyridazin-4-yl]-phenyl}-propionic
Acid (19). Prepared from ethylene glycol. Yield: 32 mg (30%).
1H NMR (CD3OD, 300 MHz) δ 8.17 (s, 1H), 7.33-7.46 (m, 7H),
4.99 (dd, 1H, J ) 9.0, 5.1 Hz), 4.24 (t, 2H, J ) 4.7 Hz), 3.73-
3.78 (m, 5H), 3.30-3.34 (m, obscured by CD3OD signal), 3.09
(dd, 1H, J ) 14.1, 9.0 Hz). TOF HRMS calcd for C23H22N3O6Cl2
(MH)+ 506.0886; found 506.0908. HPLC-1, tr ) 2.77 min; HPLC-
2, tr ) 2.73 min.
(S)-2-(2,6-Dichloro-benzoylamino)-3-{4-[2-methyl-5-(2-mor-
pholin-4-yl-ethoxy)-3-oxo-2,3-dihydro-pyridazin-4-yl]-phenyl}-
propionic Acid (20). Prepared from 4-(2-hydroxyethyl)morpholine.
Yield: 64 mg (42%). 1H NMR (CD3OD, 300 MHz) δ 8.16 (s, 1H),
7.33-7.44 (m, 7H), 4.95 (dd, 1H, J ) 8.7, 5.4 Hz), 4.52-4.58 (m,
2H), 3.80 (s, 3H), 3.72-3.84 (br m, 4H), 3.42-3.52 (m, 2H), 3.33
(m, obscured by CD3OD signal), 3.08-3.20 (m, 4 H), 3.09 (dd,
1H, J ) 13.9, 8.8 Hz). TOF HRMS calcd for C27H29N4O6Cl2 (MH)+
575.1464; found 575.1472. HPLC-1, tr ) 2.41 min; HPLC-2, tr )
2.08 min.
(S)-3-[4-(5-Cyclohexyloxy-2-methyl-3-oxo-2,3-dihydro-pyrid-
azin-4-yl)-phenyl]-2-(2,6-dichloro-benzoylamino)-propionic Acid
(21). Prepared from cyclohexanol. Yield: 53 mg (46%). 1H NMR
(CD3OD, 300 MHz) δ 8.12 (s, 1H), 7.34-7.43 (m, 7H), 4.99 (dd,
1H, J ) 9.0, 5.1 Hz), 4.55-4.61 (m, 1H), 3.77 (s, 3H), 3.33 (m,
obscured by CD3OD signal), 3.10 (dd, 1H, J ) 14.2, 9.2 Hz), 1.22-
1.94 (m, 10 H). TOF HRMS calcd for C27H28N3O5Cl2 (MH)+
544.1406; found 544.1417. Anal. (C27H27N3O5Cl2‚0.3 F3CCO2H)
C, H, N.
(CDCl3, 300 MHz) δ 8.05 (s, 1H), 7.89 (s, 1H), 7.48 (d, 2H, J )
8.2 Hz), 7.17 (d, 2H, J ) 8.2 Hz), 7.03 (s, 1H), 6.55 (d, 1H, J )
7.5 Hz), 4.91 (q, 1H, J ) 6.3 Hz), 3.90 (s, 3H), 3.81 (s, 3H), 3.80
(s, 3H), 3.29 (d, 2H, J ) 6.0 Hz). TOF HRMS calcd for C20H22N5O5
(MH)+ 412.1621; found 412.1630.
General Procedure for Preparation of Ureas 25-27 from
Anilines. To a solution of carbamoyl imidazole 24 (50 mg, 0.122
mmol) in acetonitrile (200 µL) was added the aniline (0.146 mmol).
The mixture was heated by microwave irradiation (130 °C, 10 min).
A 2 N aqueous solution of lithium hydroxide (200 µL) was added,
and the resulting mixture was stirred at 23 °C for 9 h. The mixture
was acidified by addition of TFA (100 µL), and the product was
isolated by reverse-phase HPLC (CH3CN-H2O, 0.1% TFA).
(S)-3-[4-(5-Methoxy-2-methyl-3-oxo-2,3-dihydro-pyridazin-4-
yl)-phenyl]-2-(3-methyl-3-phenyl-ureido)-propionic Acid (25).
Prepared from 20 mg of 24 (0.049 mmol) and 6.3 µL of
1
N-methylaniline (0.058 mmol). Yield: 5.5 mg (26%). H NMR
(CD3OD, 300 MHz) δ 8.21 (s, 1H), 7.31-7.44 (m, 5H), 7.12-
7.18 (m, 4H), 4.58 (dd, 1H, J ) 7.9, 5.1 Hz), 3.97 (s, 3H), 3.81 (s,
3H), 3.17-3.21 (m, 4H), 3.00 (dd, 1H, J ) 13.7, 7.9 Hz). TOF
HRMS calcd for C23H25N4O5 (MH)+ 437.1825; found 437.1839.
Anal. (C23H24N4O5) C, H, N.
(S)-2-(3-Isopropyl-3-phenyl-ureido)-3-[4-(5-methoxy-2-meth-
yl-3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (26).
Prepared from N-isopropylaniline. Yield: 23 mg (34%). 1H NMR
(CD3OD, 300 MHz) δ 8.19 (s, 1H), 7.40-7.43 (m, 3H), 7.32 (d,
2H, J ) 8.0 Hz), 7.04 (d, 2H, J ) 5.9 Hz), 7.01 (d, 2H, J ) 8.1
Hz), 4.70 (sept, 1H, J ) 6.7 Hz), 4.53 (t, 1H, J ) 6.2 Hz), 3.95 (s,
3H), 3.80 (s, 3H), 3.14 (dd, 1H, J ) 13.8, 5.1 Hz), 2.94 (dd, 1H,
J ) 13.8, 7.4 Hz), 1.03 (d, 3H, J ) 6.7 Hz), 1.02 (d, 3H, J ) 6.6
Hz). TOF HRMS calcd for C25H29N4O5 (MH)+ 465.2138; found
465.2122. Anal. (C25H28N4O5‚0.8 F3CCO2H) C, H, N.
(S)-3-[4-(5-Methoxy-2-methyl-3-oxo-2,3-dihydro-pyridazin-4-
yl)-phenyl]-2-(3-phenyl-3-propyl-ureido)-propionic Acid (27).
1
Prepared from N-n-propylaniline. Yield: 24 mg (35%). H NMR
(S)-2-Amino-3-[4-(5-methoxy-2-methyl-3-oxo-2,3-dihydro-pyrid-
azin-4-yl)-phenyl]-propionic Acid (23). A mixture of 4-borono-
L-phenylalanine (2, 105 mg, 0.50 mmol), 4-chloro-5-methoxy-2-
methyl-2H-pyridazin-3-one (22, 87 mg, 0.50 mmol) and Pd(PPh3)2Cl2
(18 mg, 0.025 mmol) in 1.0 M sodium carbonate (1.0 mL, 1.0
mmol) and acetonitrile (1.0 mL) was heated under microwave
irradiation at 150 °C for 10 min. The crude mixture, upon
acidification with TFA, was purified by reverse phase HPLC (0-
20% CH3CN-H2O, 0.1% TFA) to yield compound 23 as a white
(CD3OD, 300 MHz) δ 8.19 (s, 1H), 7.29-7.43 (m, 5H), 7.06-
7.12 (m, 4H), 4.55 (dd, 1H, J ) 7.7, 5.2 Hz), 3.95 (s, 3H), 3.79 (s,
3H), 3.62 (dd, 1H, J ) 14.0, 7.7 Hz), 3.53 (dd, 1H, J ) 13.8, 7.6
Hz), 3.17 (dd, 1H, J ) 13.8, 5.1 Hz), 2.97 (dd, 1H, J ) 13.9, 7.7
Hz), 1.46 (sext, 2H, J ) 7.4 Hz), 0.85 (t, 3H, J ) 7.4 Hz). TOF
HRMS calcd for C25H29N4O5 (MH)+ 465.2138; found 465.2118.
Anal. (C25H28N4O5‚0.8F3CCO2H) C, H, N.
General Procedure for Preparation of Ureas 28-31 from
Aliphatic Amines. To a solution of carbamoyl imidazole 24 (40
mg, 0.097 mmol) in acetonitrile (200 µL) was added the amine
(0.117 mmol). The resulting mixture was stirred at 23 °C for 19 h.
A 2 N aqueous solution of lithium hydroxide (200 µL) was added,
and the resulting mixture was stirred at 23 °C for 9 h. The mixture
was diluted with H2O (400 µL) and acidified by addition of TFA
(100 µL), and the product was isolated by reverse-phase HPLC
(CH3CN-H2O, 0.1% TFA).
(S)-2-(3,3-Dimethyl-ureido)-3-[4-(5-methoxy-2-methyl-3-oxo-
2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (28). Pre-
pared from Me2NH (2M in THF). Yield: 23.5 mg (51%). 1H NMR
(CD3OD, 300 MHz) δ 8.17 (s, 1H), 7.38 (d, 2H, J ) 8.1 Hz), 7.29
(d, 2H, J ) 7.9 Hz), 4.53 (dd, 1H, J ) 9.0, 4.8 Hz), 3.93 (s, 3H),
3.78 (s, 3H), 3.22 (dd, 1H, J ) 13.8, 4.8 Hz), 3.06 (dd, 1H, J )
13.9, 8.9 Hz), 2.86 (s, 6H). TOF HRMS calcd for C18H23N4O5
(MH)+ 375.1668; found 375.1676. Anal. (C18H22N4O5‚0.9F3CCO2H)
C, H, N.
1
solid (TFA salt, 125 mg, 55%). H NMR (CD3OD, 300 MHz) δ
8.20 (s, 1H), 7.42 (d, 2H, J ) 8.3 Hz), 7.35 (d, 2H, J ) 8.3 Hz),
4.24, (dd, 1H, J ) 5.0, 8.3 Hz), 3.92 (s, 3H), 3.80 (s, 3H), 3.37
(dd, 1H, J ) 5.0, 14.5 Hz), 3.14 (dd, 1H, J ) 8.3, 14.5 Hz). MS
m/z 304 (MH)+. TOF HRMS calcd for C15H18N3O4 (MH)+
304.1297; found 304.1302. Anal. (C15H17N3O4‚1.3F3CCO2H) C, H,
N.
(S)-2-Amino-3-[4-(5-methoxy-2-methyl-3-oxo-2,3-dihydro-
pyridazin-4-yl)-phenyl]-propionic Acid Methyl Ester (23a).
Compound 23 (TFA salt, 0.20 g, 0.48 mmol) was dissolved in
MeOH (8 mL) and heated at reflux in the presence of SOCl2 (0.2
mL) for 2 h. The solution was concentrated, and the resulting solid
was treated with saturated NaHCO3 (aq) and extracted with CH2Cl2
(3 × 2 mL). The organic phase was dried (MgSO4), filtered, and
1
concentrated, yielding ester 23a as a clear gum (0.10 g, 66%). H
NMR (CDCl3) δ 7.87 (s, 1H), 7.47 (d, 2H, J ) 8.2 Hz), 7.24 (d,
2H, J ) 8.2 Hz), 3.90 (s, 3H), 3.80 (s, 3H), 3.77 (dd, 1H, J ) 5.0,
8.3 Hz), 3.73 (s, 3H), 3.15 (dd, 1H, J ) 5.0, 13.6 Hz), 2.86 (dd,
1H, J ) 8.3, 13.6 Hz). MS m/z 318 (MH)+. TOF HRMS calcd for
C16H20N3O4 (MH)+ 318.1454; found 318.1454.
(S)-2-[(Imidazole-1-carbonyl)-amino]-3-[4-(5-methoxy-2-methyl-
3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid Meth-
yl Ester (24). To a solution of 23a (336 mg, 1.06 mmol) in
CH2Cl2-THF (5:1, 6 mL) was added 1,1′-carbonyldiimidazole (259
mg, 1.59 mmol). The resulting solution was stirred at 23 °C for 1
h. The mixture was concentrated, and the residue was purified by
column chromatography (SiO2, 2-10% MeOH-CH2Cl2). The title
compound was obtained as a white solid (355 mg, 81%). 1H NMR
(S)-2-[3-(2-Hydroxy-ethyl)-3-methyl-ureido]-3-[4-(5-methoxy-
2-methyl-3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid
(29). Prepared from N-methylethanolamine. HPLC gradient: 15-
1
35%, CH3CN-H2O, 0.05% HCO2H. Yield: 8.6 mg (20%). H
NMR (CD3OD, 300 MHz) δ 8.18 (s, 1H), 7.38 (d, 2H, J ) 8.3
Hz), 7.29 (d, 2H, J ) 8.3 Hz), 4.53 (dd, 1H, J ) 8.8, 4.9 Hz), 3.94
(s, 3H), 3.78 (s, 3H), 3.60 (t, 2H, J ) 5.5 Hz), 3.33 (t, 2H, partially
obscured by CD3OD signal), 3.22 (dd, 1H, J ) 14.0, 5.0 Hz), 3.05
(dd, 1H, J ) 13.8, 8.8 Hz), 2.91 (s, 3H). TOF HRMS calcd for
C19H25N4O6 (MH)+ 405.1774; found 405.1790. Anal. (C19H24N4O6‚
0.8HCO2H) C, H, N.

Pyridazinone-Based R4 Integrin Receptor Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3409
(S)-2-(3-Isobutyl-3-methyl-ureido)-3-[4-(5-methoxy-2-methyl-
3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (30).
Prepared from N-methylisobutylamine. Yield: 26.5 mg (55%). 1H
NMR (CD3OD, 300 MHz) δ 8.17 (s, 1H), 7.38 (d, 2H, J ) 8.3
Hz), 7.28 (d, 2H, J ) 8.2 Hz), 4.56 (dd, 1H, J ) 8.7, 5.0 Hz), 3.93
(s, 3H), 3.78 (s, 3H), 2.94-3.26 (m, 4H), 2.85 (s, 3H), 1.86-1.93
(m, 1H), 0.84 (d, 3H, J ) 6.6 Hz), 0.83 (d, 3H, J ) 6.7 Hz). TOF
HRMS calcd for C21H29N4O5 (MH)+ 417.2138; found 417.2128.
Anal. (C21H28N4O5‚0.7F3CCO2H) C, H, N.
2.97 (dd, 1H, J ) 14.0, 9.3 Hz). TOF HRMS calcd for C17H20N3O6
(MH)+ 362.1352; found 362.1336. Anal. (C17H19N3O6‚0.8F3CCO2H)
C, H, N.
(S)-2-Benzyloxycarbonylamino-3-[4-(5-methoxy-2-methyl-3-
oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (34).
1
Prepared from benzyl chloroformate. Yield: 3.1 mg (10%). H
NMR (CD3OD, 300 MHz) δ 8.19 (s, 1H), 7.27-7.40 (m, 9H), 5.07
(s, 2H), 4.47 (dd, 1H, J ) 9.1, 4.7 Hz), 3.93 (s, 3H), 3.80 (s, 3H),
3.25 (dd, 1H, J ) 13.9, 4.7 Hz), 3.00 (dd, 1H, J ) 13.9, 9.2 Hz).
TOF HRMS calcd for C23H24N3O6 (MH)+ 438.1665; found
438.1656. Anal. (C23H23N3O6‚0.3F3CCO2H) C, H, N.
(S)-3-[4-(5-Methoxy-2-methyl-3-oxo-2,3-dihydro-pyridazin-4-
yl)-phenyl]-2-[(2-methyl-piperidine-1-carbonyl)-amino]-propi-
onic Acid (31). Prepared from 2-methylpiperidine. Yield: 29 mg
(S)-2-tert-Butoxycarbonylamino-3-[4-(5-methoxy-2-methyl-3-
oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (35).
Prepared from di-tert-butyl dicarbonate. HPLC gradient: 25-45%
1
(56%). H NMR (CD3OD, 300 MHz, diastereomer ratio 1.1:1) δ
8.17 (s, 2H), 7.37 (d, 4H, J ) 8.4 Hz), 7.28 (d, 4H, J ) 7.0 Hz),
4.48-4.57 (m, 2H), 4.16-4.27 (br m, 2H), 3.93 (s, 3H), 3.92 (s,
3H), 3.77 (s, 6H), 3.67-3.78 (m, 2H), 3.13-3.26 (m, 2H), 2.98-
3.10 (m, 2H), 2.80-2.90 (m, 2H), 1.24-1.68 (m, 12H), 1.16 (d,
3H, J ) 6.9 Hz), 1.08 (d, 3H, J ) 6.9 Hz). TOF HRMS calcd for
C22H29N4O5 (MH)+ 429.2138; found 429.2144. Anal. (C22H28N4O5‚
0.9F3CCO2H) C, H, N.
1
CH3CN-H2O, 0.05% HCO2H. Yield: 11.5 mg (47%). H NMR
(CD3OD, 400 MHz) δ 8.18 (s, 1H), 7.38 (d, 2H, J ) 8.2 Hz), 7.28
(d, 2H, J ) 8.2 Hz), 4.37 (dd, 1H, J ) 9.0, 4.9 Hz), 3.93 (s, 3H),
3.78 (s, 3H), 3.19 (dd, 1H, J ) 14.1, 4.9 Hz), 2.96 (dd, 1H, J )
14.0, 9.1 Hz), 1.40 (s, 9 H). TOF HRMS calcd for C20H26N3O6
(MH)+ 404.1822; found 404.1819. Anal. (C20H25N3O6‚0.4HCO2H)
C, H, N.
4,5-Dibromo-2-(2-hydroxy-ethyl)-2H-pyridazin-3-one. To a
solution of mucobromic acid (38.68 g, 150 mmol) in EtOH (128
mL) at 5 °C was added 2-hydroxyethyl hydrazine (13.2 mL, 195
mmol) dropwise, maintaining an internal temperature below 10 °C
during the addition. The resulting mixture was stirred at 5 °C for
1 h, then was allowed to warm to 23 °C, and finally was heated at
reflux for 2 h. The mixture was concentrated, and the residue was
purified by flash column chromatography (SiO2, 50-75% EtOAc-
hexanes), affording the title compound as a tan solid (22.93 g, 51%).
1H NMR (CDCl3, 300 MHz) δ 7.85 (s, 1H), 4.38 (t, 2H, J ) 4.7
Hz), 4.04 (t, 2H, J ) 5.1 Hz), 2.41 (br s, 1H). TOF HRMS calcd
for C6H7N2O2Br2 (MH)+ 296.8874; found 296.8887.
(S)-2-tert-Butoxycarbonylamino-3-{4-[2-(2-hydroxy-ethyl)-5-
methoxy-3-oxo-2,3-dihydro-pyridazin-4-yl]-phenyl}-propionic
Acid (36). Prepared from amino acid 32 (72 mg, 0.16 mmol) and
di-tert-butyl dicarbonate. HPLC gradient: 25-45% CH3CN-H2O,
0.05% HCO2H. Yield: 58 mg (84%). 1H NMR (CD3OD, 300 MHz)
δ 8.22 (s, 1H), 7.40 (d, 2H, J ) 8.1 Hz), 7.30 (d, 2H, J ) 8.1 Hz),
4.39 (dd, 1H, J ) 8.9, 4.7 Hz), 4.34 (t, 2H, J ) 5.7 Hz), 3.96 (s,
3H), 3.94 (t, 2H, J ) 5.8 Hz), 3.21 (dd, 1H, J ) 13.9, 4.9 Hz),
2.97 (dd, 1H, J ) 14.0, 9.2 Hz), 1.42 (s, 9H). TOF HRMS calcd
for C21H28N3O7 (MH)+ 434.1927; found 434.1935. Anal.
(C21H27N3O7) C, H, N.
4-Bromo-2-(2-hydroxy-ethyl)-5-methoxy-2H-pyridazin-3-
one. Sodium methoxide (30 wt % in MeOH, 4.85 mL, 25.8 mmol)
was added to an ice-cold solution of 4,5-dibromo-2-(2-hydroxy-
ethyl)-2H-pyridazin-3-one (7.00 g, 23.5 mmol) in MeOH (40 mL).
The mixture was allowed to warm to 23 °C with stirring overnight
and was concentrated. The residual white solid was partitioned
between CH2Cl2 (100 mL) and a saturated aqueous solution of NaCl
(100 mL). A white solid precipated and was collected by vacuum
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(5-methoxy-2-methyl-
3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid Meth-
yl Ester (5a). 2,6-Dichlorobenzoyl chloride (0.29 mL, 2.0 mmol)
was added to a solution of 23a (0.50 g, 1.6 mmol) and triethyamine
(0.35 mL, 2.5 mmol) in dichloromethane (10 mL). The resulting
mixture was stirred at 23 °C for 1 h, then was washed with NaHCO3
(aq). The dichloromethane layer was concentrated, and the residue
was purified by reverse-phase HPLC, affording compound 5a (0.40
1
1
filtration, affording the title compound (4.73 g, 81%). H NMR
g, 51%) as a white solid. H NMR (CDCl3) δ: 7.86 (s, 1H), 7.46
(CDCl3, 300 MHz) δ 7.77 (s, 1H), 4.43 (t, 2H, J ) 4.9 Hz), 4.09
(s, 3H), 4.02 (app q, 2H, J ) 5.2 Hz), 2.87 (t, 1H, J ) 5.7 Hz).
TOF HRMS calcd for C7H10N2O3Br (MH)+ 248.9875; found
248.9882.
(d, 2H, J ) 8.2 Hz), 7.33-7.25 (m, 5H), 6.30 (d, 1H, J ) 8.6 Hz),
5.25 (m, 1H), 3.89 (s, 3H), 3.80 (s, 3H), 3.77 (s, 3H), 3.30 (m,
2H). MS m/z 490 (MH)+. Anal. (C23H21Cl2N3O5) C, H, N, Cl.
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(5-methoxy-2-methyl-
3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid Ethyl
Ester (5b). Compound 5 (0.50 g, 1.0 mmol) was dissolved in EtOH
(25 mL) and heated at reflux in the presence of SOCl2 (2 mL) for
2 h. The solution was concentrated. The residue was dissolved in
EtOAc and washed with saturated NaHCO3 (aq) and H2O. The
organic phase was dried (MgSO4) and concentrated. The ester 5b
was purified by reverse-phase HPLC, yielding a white solid (0.32
g, 63%). 1H NMR (CDCl3, 300 MHz) δ 7.87 (s, 1H), 7.44 (d, 2H,
J ) 8.2 Hz), 7.32-7.23 (m, 5H), 6.36 (d, 1H, J ) 8.2 Hz), 5.22
(m, 1H), 4.24 (m, 2H), 3.89 (s, 3H), 3.80 (s, 3H), 3.30 (m, 2H),
1.27 (t, 3H, J ) 7.1 Hz). MS m/z 504 (MH)+. TOF HRMS calcd
for C24H24Cl2N3O5 (MH)+ 504.1093; found 504.1071. HPLC-1, tr
) 3.30 min; HPLC-3, tr ) 4.70 min.
(S)-2-(2,6-Dichloro-benzoylamino)-3-[4-(5-methoxy-2-methyl-
3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid 2-Hy-
droxy-ethyl Ester (5c). Compound 5 (0.94 g, 1.9 mmol), bis(2-
oxo-3-oxazolidinyl)phosphinic chloride (0.59 g, 2.3 mmol), ethylene
glycol (200 µL, 3.6 mmol), and N,N-diisopropylethylamine (1.0
mL, 5.7 mmol) were added to dichloromethane (3 mL). The reaction
mixture was stirred overnight at 23 °C and then concentrated. The
residue was subjected to column chromatography (SiO2, EtOAc-
heptanes gradient), providing a clear viscous oil. The oil was
dissolved in MeOH-H2O (1:1), frozen and lyophilized, yielding
5c as a white powder (0.31 g, 31%). 1H NMR (CDCl3, 300 MHz)
δ 7.90 (s, 1H), 7.40-7.24 (m, 7H), 6.74 (d, 1H, J ) 8.1 Hz), 5.23
(dd, 1H, J ) 5.3, 6.8 Hz), 4.15 (m, 2H), 3.90 (s, 3H), 3.80 (s, 3H),
(S)-2-Amino-3-{4-[2-(2-hydroxy-ethyl)-5-methoxy-3-oxo-2,3-
dihydro-pyridazin-4-yl]-phenyl}-propionic Acid (32). Prepared
from 4-bromo-2-(2-hydroxy-ethyl)-5-methoxy-2H-pyridazin-3-one
by the method given above for compound 23. HPLC gradient:
5-15% CH3CN-H2O, 0.05% HCO2H. Yield: 107 mg (HCO2H
salt, 57%). 1H NMR (D2O, 300 MHz) δ 8.16 (s, 1H), 7.27 (d, 2H,
J ) 8.5 Hz), 7.24 (d, 2H, J ) 8.6 Hz), 4.22 (t, 2H, J ) 5.4 Hz),
3.89 (dd, 1H, J ) 7.9, 5.2 Hz), 3.83 (t, 2H, J ) 5.5 Hz), 3.81 (s,
3H), 3.21 (dd, 1H, J ) 14.5, 5.3 Hz), 3.03 (dd, 1H, J ) 14.6, 8.0
Hz). TOF HRMS calcd for C16H20N3O5 (MH)+ 334.1403; found
334.1415.
General Method for the Synthesis of Carbamates 33-36.
Sodium bicarbonate (14 mg, 0.17 mmol) and an alkylchloroformate
or dialkyl dicarbonate (0.17 mmol) were added in sequence to a
solution of the amino acid (23, 26 mg, 0.058 mmol except as noted
otherwise) in a mixture of acetonitrile (0.1 mL) and water (0.3 mL).
The resulting suspension was stirred at 23 °C for 18 h. The reaction
mixture was acidified to pH 2 by addition of trifluoroacetic acid
(10 µL), and the product was isolated by reverse-phase HPLC
(CH3CN-H2O, 0.1% TFA).
(S)-2-Methoxycarbonylamino-3-[4-(5-methoxy-2-methyl-3-
oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid (33).
1
Prepared from methyl chloroformate. Yield: 9.9 mg (38%). H
NMR (CD3OD, 300 MHz) δ 8.18 (s, 1H), 7.37 (d, 2H, J ) 8.1
Hz), 7.28 (d, 2H, J ) 8.1 Hz), 4.42 (dd, 1H, J ) 9.0, 4.8 Hz), 3.93
(s, 3H), 3.77 (s, 3H), 3.60 (s, 3H), 3.21 (dd, 1H, J ) 14.0, 4.7 Hz),

3410 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
Gong et al.
3.53 (t, 2H, J ) 4.4 Hz), 3.47 (dd, 1H, J ) 5.3, 13.6 Hz), 3.15 (dd,
1H, J ) 6.8, 13.6 Hz). MS m/z 520 (MH)+. TOF HRMS calcd for
C24H24Cl2N3O6 (MH)+ 520.1042; found 520.1040. HPLC-1, tr )
2.96 min; HPLC-3, tr ) 3.87 min.
removed for analysis by LC-MS-MS. Calibration standards were
prepared by adding appropriate volumes of stock solution directly
into plasma and treated identically to collected plasma samples.
Calibration standards were prepared in the range of 0.01 to 10 µM
for quantitation. LC-MS-MS analysis was performed utilizing
multiple reaction monitoring for detection of characteristic ions for
each tested compound, additional related analytes, and internal
standard. The limit of quantitation was 0.01 µM.
Dextran Sulfate Sodium Colitis Protocol. Details for this
method have previously been reported.26 In brief, mice (n ) 10/
treatment group) were provided with a solution of tap water
containing 5% DSS ad libitum over a 7 day period. The DSS
solution was changed daily and its consumption noted. During this
same time, selected groups of test animals were administered a
preparation of experimental antagonists. Mice were dosed by oral
gavage either with vehicle (10% poly(ethylene glycol) 400 (Sigma-
Aldrich; St. Louis, MO) in D5W, v/v) or with experimental
compound starting on the day of induction with DSS and twice
daily thereafter for 7 days. At the end of this period, the animals
were euthanized and their colons dissected for further analysis.
Among the parameters analyzed were length of the colon from the
aboral end of the cecum to the anus, signs of diarrhea, and
indications of gross inflammation. These data and observations were
assigned a score, and the sums of the individual macroscopic indices
were combined into a macroscopic score for each colon, where 0
) normal and 11 ) maximally affected, as previously described.26
(S)-2-(3-Isobutyl-3-methyl-ureido)-3-[4-(5-methoxy-2-methyl-
3-oxo-2,3-dihydro-pyridazin-4-yl)-phenyl]-propionic Acid 2-Hy-
droxy-ethyl Ester (30c). Ethylene glycol (434 µL, 7.78 mmol),
bis(2-oxo-3-oxazolidinyl)phosphinic chloride (515 mg, 2.02 mmol),
and triethylamine (564 µL, 4.05 mmol) were added in sequence to
a solution of 30 (648 mg, 1.56 mmol) in CH2Cl2 (7.8 mL). The
resulting mixture was stirred at 23 °C for 5 days. The mixture was
concentrated, and the residue was purified by reverse-phase HPLC
(20-40% CH3CN-H2O, 0.05% HCO2H), yielding ester 30c as a
white powder (450 mg, 63%). 1H NMR (CD3OD, 300 MHz) 8.17
(s, 1H), 7.38 (d, 2H, J ) 8.2 Hz), 7.28 (d, 2H, J ) 8.4 Hz), 6.21
(d, 1H, J ) 7.9 Hz), 4.52-4.60 (m, 1H), 4.11-4.24 (m, 2H), 3.93
(s, 3H), 3.77 (s, 3H), 3.68-3.72 (m, 2H), 2.96-3.25 (m, 4H), 2.86
(s, 3H), 1.80-1.94 (m, 1H), 0.84 (d, 3H, J ) 6.4 Hz), 0.83 (d, 3H,
J ) 6.7 Hz). TOF HRMS calcd for C23H33N4O6 (MH)+ 461.2400;
found 461.2390. Anal. (C23H32N4O6‚0.5H2O) C, H, N.
(S)-2-tert-Butoxycarbonylamino-3-{4-[2-(2-hydroxy-ethyl)-5-
methoxy-3-oxo-2,3-dihydro-pyridazin-4-yl]-phenyl}-propionic
Acid 2-Hydroxy-ethyl Ester (36c). Prepared from compound 36
(574 mg, 1.32 mmol) by the method given for ester 30c (reaction
time 23 h). HPLC gradient: 20-40% CH3CN-H2O, 0.05%
1
HCO2H. Yield: 171 mg (27%). H NMR (CD3OD, 300 MHz) δ
8.22 (s, 1H), 7.40 (d, 2H, J ) 8.0 Hz), 7.29 (d, 2H, J ) 8.1 Hz),
6.98 (d, 1H, J ) 8.2 Hz), 4.37-4.45 (br m, 1H), 4.34 (t, 2H, J )
5.7 Hz), 4.16-4.21 (br m, 2H), 3.96 (s, 3H), 3.94 (t, 2H, J ) 5.7
Hz), 3.69-3.74 (br m), 3.20 (dd, 1H, J ) 13.8, 5.4 Hz), 3.01 (dd,
1H, J ) 13.5, 8.8 Hz), 1.42 (s, 9H). TOF HRMS calcd for
C23H32N3O8 (MH)+ 478.2189; found 478.2188. Anal. (C23H31N3O8‚
0.3H2O) C, H, N.
Acknowledgment. We are grateful to members of Chemical
and Biological Support Team at Johnson & Johnson Pharma-
ceutical Research and Development, Spring House, PA, for
pharmacokinetic analyses.
Ramos Cell Adhesion Assay (R4â1 Mediated Adhesion/
VCAM-1). Immulon 96-well plates (Dynex) were coated with 100
µL recombinant hVCAM-1 at 4.0 µg/mL in 0.05 M Na2CO3 buffer
pH 9.0 overnight at 4 °C (R&D Systems). 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
µL) were added at 2× concentration. Ramos cells (50 µL at 2 ×
106/mL), labeled with 5 µM Calcein AM (Molecular Probes) for 1
h 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 + 1%
BSA, and cells were lysed for 15 min in 100 µL of 1 M Tris pH
8.0 with 1% SDS. The plate was read at 485 nm excitation and
530 nm emission.
Supporting Information Available: Table of elemental analysis
results. This material is available free of charge via the Internet at
http://pubs.acs.org.
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R4â7-K562 Cell Adhesion Assay (R4â7 Mediated Adhesion/
MAdCAM-1). M2 anti-FLAG antibody coated 96-well plates
(Sigma) were coated for 1 h at 4 °C with 2-8 µL/well recombinant
FLAG-hMAdCAM-1 contained in 100 µL of Dulbecco’s 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 µL) were added at 2× concentration.
Stably transfected K562 cells expressing human R4â7 integrin (50
µL at 2 × 106/mL) that had been labeled with 100 µg/mL
carboxymethyl fluorescein diacetate succinimidyl 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 µL of PBS without Ca2+ or Mg2+
,
supplemented with 0.1 M NaOH. The plate was read on a 96-well
fluorescent plate reader at 485 nm excitation and 530 nm emission.
Pharmacokinetic Assay. Rats were dosed intravenously (iv) at
3 mg/kg and by oral gavage at 30 mg/kg with tested compound.
Blood samples (0.5-1.0 mL) were collected post dose into
heparinized tubes and centrifuged for cell removal. Precisely 200
µL of plasma supernatant was then transferred to a clean vial, placed
on dry ice, and subsequently stored in a -70 °C freezer prior to
analysis. Plasma samples were prepared by adding 400 µL of
acetonitrile containing internal standard to 200 µL of plasma to
precipitate proteins. Samples were centrifuged, and supernatant was

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