Synthesis and SAR of pyridazinone-substituted phenylalanine amide α4 integrin antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.01.022

Structural modification and cellular adhesion inhibition activities of pyridazinone-substituted phenylalanine amide α₄ integrin antagonists are described. Functionality requirements for the arylamide moiety and the carboxylic acid group were de

Full text of DOI:10.1016/j.bmcl.2008.01.022

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1331–1335
Synthesis and SAR of pyridazinone-substituted phenylalanine amide
a4 integrin antagonists
Yong Gong,* J. Kent Barbay, Edward S. Kimball, Rosemary J. Santulli,
M. Carolyn Fisher, Alexey B. Dyatkin, Tamara A. Miskowski,
Pamela J. Hornby and Wei He
Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Welsh & McKean Roads, PO Box 776, Spring House,
PA 19477-0776, USA
Received 7 December 2007; accepted 7 January 2008
Available online 11 January 2008
Abstract—Structural modification and cellular adhesion inhibition activities of pyridazinone-substituted phenylalanine amide a4
integrin antagonists are described. Functionality requirements for the arylamide moiety and the carboxylic acid group were dem-
onstrated. The study also revealed novel structure–activity relationships (SAR) for arylated pyridazinones. A correlation between
bioavailability and permeability was also explored. A selected compound showed effectiveness in a mouse leukocytosis study.
Ó 2008 Elsevier Ltd. All rights reserved.
Integrins a4b1 and a4b7 are heterodimeric transmem-
brane receptors from the a4 integrin family, which are
expressed on the surface of leukocytes. They play an
essential role in leukocyte adhesion and trafficking.
Undesired interactions of integrin a4b1 with vascular cell
adhesion molecule-1 (VCAM-1), and integrin a4b7 with
mucosal addressin cell adhesion molecule-1 (MAd-
CAM-1), have been implicated in asthma, multiple scle-
rosis, ulcerative colitis, and Crohn’s disease. TysabriÒ, a
humanized monoclonal antibody that targets a4 inte-
grin, has been approved for the treatment of multiple
sclerosis.1 Many small molecules, especially N-acyl 4-
aryl phenylalanines, have been identified that inhibit
the interaction of a4 integrins with their receptors.2 Re-
cently, we discovered pyridazinone based a4 integrin
antagonists.3 Cellular adhesion activity, pharmacoki-
netic (PK) properties, and in vivo activity of additional
members of this series are presented herein.
Scheme 1. Suzuki coupling of 4-boronophenylalanine
(2) with 4-chloro-5-methoxy-2-methyl-2H-pyridazin-3-
one under our previously reported conditions3,4 yielded
4-pyridazinone-substituted phenylalanine 3. Arylamides
5–7 in Table 1 were prepared by direct acylation of
phenylalanine 3 with benzoyl chlorides. Arylamides 8–
12 were obtained through esterification (to ester 4),
EDC-mediated coupling with carboxylic acids, and
saponification. Thioamide analog 13 in Table 1 was pre-
pared by refluxing amide 5 with Lawesson’s reagent in
toluene.
R
N
N
CH3
O
O
O
Ar
N
H
OH
1
In this study, structural modifications to three groups
within the generalized lead antagonist 1 were targeted:
the arylamide, the carboxylic acid, and the 5-position
substituent on the pyridazinone. Variation of the
arylamide group in 1 was accomplished as depicted in
Compounds in Table 2 contain alternatives to the car-
boxylic acid within 5. Hydroxamic acid 14 and N-acyl-
methanesulfonamide 15 were prepared by acylation of
hydroxylamine hydrochloride and methanesulfonamide,
respectively, with parent acid 5 in the presence of EDC
under standard coupling conditions. Alcohol 16 was
Keywords: Pyridazinone; Phenylalanine; a4 Integrin antagonist.
*
Corresponding author. Tel.: +1 908 927 7923; fax: +1 908 526
6469; e-mail: ygong@prdus.jnj.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.022

1332
Y. Gong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1331–1335
OH
B
CH3O
N
N
OH
CH3O
Cl
CH3
N
N
a
O
+
CH3
O
H2N
O
OH
O
H2N
OH
2
3
c
b
to 5-7
CH3O
CH3O
N
N
N
N
CH3
CH3
d, e
O
O
O
to 8-12
O
O
OH
H2N
Ar
N
H
5-12, Table 1
4
OCH3
Scheme 1. (a) Pd(PPh3)2Cl2, Na2CO3, H2O/CH3CN, reflux 2 h or lW 150 °C, 10 min; (b) CH3OH, SOCl2; (c) ArCOCl, Na2CO3, H2O/CH3CN; (d)
ArCO2H, EDC, HOBt, Et3N, CH2Cl2; (e) LiOH, CH3OH, H2O.
Table 1. Cellular adhesion assay resultsa for arylamides 5–13
CH3O
N
N
CH3
O
X
O
Ar
N
H
OH
Compound
Ar
X
IC50 (lM)
a4b1
a4b7
*
*
Cl
Cl
5
o
o
0.031 0.008 (4)
0.003 0.001 (6)
Cl
Cl
6
0.31 0.005 (2)
0.004 0.002 (2)
SO2CH3
*
7
8
o
o
1.2
0.019 0.007 (2)
0.018 0.013 (5)
F
*
Cl
Cl
Cl
Cl
0.005 0.001 (2)
N
*
9
o
o
o
o
0.73 0.043 (2)
0.035 0.013 (6)
N
*
10
11
12
>5
1.4
S
*
H3C
H3C
>5
0.080 0.024 (3)
0.25
N
*
CH3
0.79
N
*
O
Cl
Cl
13
s
0.35
0.23
a IC50’s were determined from a six point titration, with each individual point being an average of two measurements at a particular antagonist
concentration. IC50’s are reported as value SEM, with n in parentheses.

Y. Gong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1331–1335
1333
Table 2. Cellular adhesion assay results for carboxylic acid
alternatives
and a4b1. Compared to 5, 2-chloro-4-methanesulfonyl-
benzamide 6 was an equally potent a4b7 antagonist
and was more selective with regard to a4b1 inhibition.8
2-Chloro-5-fluorobenzamide 7 was fairly potent and
selective for a4b7. 3,5-Dichloro-isonicotinic amide 8
was a potent dual antagonist. Other heteroarylamides
9–12 were less potent. Thioamide analog 13 displayed
only moderate activity, with sub-micromolar antago-
nism of both a4b7 and a4b1.
CH3O
N
N
CH3
O
Cl
O
N
H
Y
Cl
Compound
Y
IC50 (lM)
a4b7
Efforts to replace the carboxylic acid group in 5 resulted,
to varying degrees, in a reduction of potency (Table 2).
Hydroxamic acid 14 and N-acyl-methanesulfonamide 15
produced only micromolar range activity against a4b7.
Alcohol 16, surprisingly, displayed sub-micromolar
activity against a4b7. Tetrazole 20 provided double digit
nM activity against a4b7 and micromolar range activity
against a4b1. Although carboxylic acid group replace-
ment did not generate the desired potency, it could
potentially alter PK properties of this class of
compounds.
a4b1
14
15
16
–CONHOH
–CONHSO2CH3
–CH2OH
>5
>5
>5
0.61
1.0
0.23
N
N
N
20
2.1
0.089
N
H
prepared by reduction of the corresponding methyl ester
with lithium borohydride. Tetrazole 20 was prepared by
conversion of carboxylic acid 173 to nitrile 18, followed
by conversion of 18 to unprotected tetrazole 19 and sub-
sequent acylation, as shown in Scheme 2.
Previously reported structure–activity relationships
within the N-acyl 4-aryl phenylalanine series suggested
that ortho substitution of the terminal aryl ring, espe-
cially with methoxy group(s), was important in main-
taining potency.3,9 The methoxy groups, however, are
potentially metabolically labile, which may contribute
to the less than favorable PK properties associated
with this series of compounds. When the methoxy
Synthesis of arylated pyridazinones 27–36 (Table 3) was
accomplished through double Suzuki couplings (Scheme
3). 4,5-Dichloro-2-methyl-2H-pyridazin-3-one 25 was
selectively mono-arylated to 5-aryl-4-chloro-2-methyl-
2H-pyridazin-3-ones 26 under our previously developed
conditions.5 The second Suzuki arylation of compounds
26 with borono phenylalanine amide 24 furnished the
targets. Azole substituted pyridazinones 37–40 were pre-
pared by substitution of the methoxy group in 5 with an
azole (sodium hydride, microwave heating).6
group in pyridazinone
5 was removed, activity
dropped significantly (compound 21). Introduction
of 5-methyl and -ethyl groups to the pyridazinone
(22 and 23) resulted in partial recovery of potency
versus a4b7. Further enhancement of potency was
achieved in arylated pyridazinones 27–40. Substituted
phenyls (27–35), as well as other heteroaryl groups
(36–40), were well tolerated. The arylated compounds
were more active in blocking a4b7 than a4b1-mediated
cell adhesion.10 Single to double digit nM potency
versus a4b7 and sub-lM to lM activity with respect
to a4b1 was generally observed. For example, 4-meth-
anesulfonyl-phenyl analog 28 displayed an IC50 of
2 nM against a4b7 and 150 nM against a4b1; imidaz-
ole 37 exhibited IC50’s of 7 and 790 nM, respectively.
The successful extension of SAR into arylated
pyridazinones provides a novel handle to poten-
tially modify the PK properties of this class of
compounds.
Table 1 lists in vitro activities of a selected set of aryla-
mides in assays of a4b7/MAdCAM-1 and a4b1/VCAM-1
mediated cellular adhesion.7 Compound 5, containing
the well-optimized 2,6-dichlorobenzamide moiety, dem-
onstrated potent antagonist activity versus both a4b7
CH3O
N
N
CH3O
N
N
CH3
a, b
CH3
O
O
O
BocHN
17
18
BocHN
CN
Pharmacokinetic data of selected compounds in rats are
presented in Table 4. Compounds 5, 6, 7, and 31, each
containing a carboxylic acid, showed almost no oral bio-
availability. Prodrug hydroxyethyl ester 41 provided en-
hanced plasma levels of the parent carboxylic acid 7
(Cmax = 19 lM, F = 5%).11 Tetrazole 20 failed to im-
prove PK properties. Alcohol 16, however, offered mod-
est improvements in systemic exposure level
(Cmax = 0.9 lM) and oral bioavailability (F = 8%). This
compound also displayed an increased rate of cellular
permeability (as indicated by the Caco-2 model), sug-
gesting that increasing intestinal absorption may be
important in improving oral bioavailability in the series.
OH
c
CH3O
CH3O
N
N
N
N
CH3
CH3
d
O
O
Cl
O
N
N
N
N
N
H
H2N
N
HN
20
HN
19
N
Cl
Scheme 2. (a) Boc2O, NH4HCO3, pyridine, CH3CN; (b) cyanuric
chloride, DMF; (c) NaN3, ZnBr2 (5 equiv), i-PrOH, H2O; (d) 2,6-
dichlorobenzoyl chloride, Et3N, CH2Cl2.

1334
Y. Gong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1331–1335
Table 3. Cellular adhesion assay results for pyridazinone
modifications
OH
B
OH
a
R
N
2
Cl
O
N
O
CH3
N
H
O
OH
Cl
Ar
Cl
24
Cl
O
+
O
N
H
Cl
Cl
N
N
N
N
OH
b
Cl
CH3
CH3
O
O
Compound R
IC50 (lM)
a4b7
25
26
a4b1
c
to 27-36
21
22
23
27
28
29
30
31
32
33
34
H
CH3
>1
3.2
>5
0.36
0.078 0.006 (2)
0.061
Ar
N
N
CH3CH2
Ph
CH3
0.57 0.075 (2) 0.021 0.008 (2)
0.15 0.039 (2) 0.002 0.001 (3)
0.094 0.019 (2) 0.003 0.002 (4)
d
O
5
4-CH3SO2-Ph
4-NH2CO-Ph
4-CN-Ph
Cl
O
to 37-40
O
N
H
0.74
0.023 0.005 (3)
OH
4-F-Ph
0.46 0.047 (2) 0.019 0.012 (2)
0.34 0.082 (2) 0.013 0.003 (2)
27-40, Table 3
Cl
4-CH3O-Ph
3-CF3O-Ph
3-CF3-Ph
Scheme 3. (a) 2,6-Cl2C6H3COCl, Na2CO3, H2O/CH3CN; (b)
Pd(PEt3)2Cl2, ArB(OH)2, Na2CO3, H2O/DMF; (c) Pd(PPh3)2Cl2,
Na2CO3, H2O/CH3CN, lW 150 °C, 10 min; (d) azole (5 equiv), NaH
(5 equiv), THF, lW 100 °C, 10 min.
1.54
0.52
0.025 0.009 (4)
0.021 0.009 (3)
O
35
36
37
38
0.40
0.005 0.002 (4)
O
*
S
Compound 5 was selected for testing in a mouse leuko-
cytosis study. The compound was dosed subcutaneously
rather than orally to achieve a higher systemic drug le-
vel.12 In vivo subcutaneous administration of 5 in na¨ıve
mice produced a dose-dependent elevation in circulating
total leukocytes (Fig. 1). Circulating leukocytes were
more than doubled over control at a 30 mg/kg dose,
which reflects that inhibition of binding of a4b1 to
VCAM-1 and a4b7 to MAdCAM-1 results in a blockage
of the cell’s extravasation.
0.83 0.026 (2) 0.069 0.033 (2)
0.79 0.019 (2) 0.007 0.001 (2)
*
*
N
CH3
N
CH3
Br
CH3
>5
2.7
0.024 0.009 (2)
0.039 0.008 (2)
N
N
*
N
39
40
N
*
N
In summary, convergent synthesis was applied to the
SAR exploration of a pyridazinone-substituted phenyl-
0.51 0.039 (2) 0.036 0.017 (3)
N
*
Table 4. Pharmacokinetic properties of selected compounds in ratsa
R
N
N
CH3
O
Cl
O
N
H
Y
Z
Compound
Z
Y
R
Caco-2
PappA ! B (10ꢀ6 cm/s)
0.13
Cmax (lM)
F (%)
5
6-Cl
–CO2H
–CO2H
–CO2H
OCH3
OCH3
OCH3
OCH3
4-F-Ph
OCH3
0.1
0
1
0
0
5
0
8
6
4-CH3SO2
5-F
5-F
—
—
7
0
41
31
16
–CO2(CH2)2OH
–CO2H
–CH2OH
—
19
0
6-Cl
6-Cl
0.16
2.5
0.9
N
N
N
20
6-Cl
OCH3
0.02
0
0
N
H
a Dosed at 30 mg/kg po, 3 mg/kg iv.

Y. Gong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1331–1335
1335
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Figure 1. Effect on total leukocyte counts by compound 5 (sc, 1 h
before sampling). *p < 0.05.
alanine amide class of a4 integrin antagonists. PK prop-
erties of this series of compounds were associated with
their permeabilities. A selected compound (5) demon-
strated dose-responsive effects on the elevation of circu-
lating leukocytes in a mouse leukocytosis study. The
results should serve as a guide for further investigation.
Acknowledgments
We are grateful to members of Chemical & Biological
Support Team, as well as Enterology Team at Johnson
& Johnson Pharmaceutical Research and Development,
Spring House, PA, for pharmacokinetic analyses and
biological support.
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pyridazinone to the corresponding phenol.
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assay were used to determine the inhibition of the
adhesion of a4b1+ cells to immobilized VCAM-1 and of
a4b7+ cells to immobilized MAdCAM-1. For details, see
Ref. 3.
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withdrawal of blood samples and continued hydrolysis
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8. The 2-chloro-4-methanesulfonyl-benzoyl moiety was also
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