Discovery of pyridazinopyridinones as potent and selective p38 mitogen-activated protein kinase inhibitors

Article abstract of DOI:10.1021/jm100567y

The p38 mitogen-activated protein kinase (MAPK) plays an important role in the production of proinflammatory cytokines, making it an attractive target for the treatment of various inflammatory diseases. A series of pyridazinopyridinone compounds were designed as novel p38 kinase inhibitors. A Structure-activity investigation identified several compounds possessing excellent potency in both enzyme and human whole blood assays. Among them, compound 31 exhibited good pharmacokinetic properties and showed excellent selectivity against other related kinases. In addition, 31 demonstrated efficacy in a collagen-induced arthritis disease model in rats.

Full text of DOI:10.1021/jm100567y

6398 J. Med. Chem. 2010, 53, 6398–6411
DOI: 10.1021/jm100567y
Discovery of Pyridazinopyridinones as Potent and Selective p38 Mitogen-Activated Protein
Kinase Inhibitors
Bin Wu,*,† Hui-Ling Wang,† Liping Pettus,† Ryan P. Wurz,† Elizabeth M. Doherty,† Bradley Henkle,‡ Helen J. McBride,‡
Christiaan J. M. Saris,‡ Lu Min Wong,‡ Matthew H. Plant, Lisa Sherman,‡ Matthew R. Lee,§ Faye Hsieh,# and
Andrew S. Tasker†
†Department of Chemistry Research and Discovery, ‡Inflammation, HTS Molecular Pharmacology, §Molecular Structure, and
#Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320
Received May 10, 2010
The p38 mitogen-activated protein kinase (MAPK) plays an important role in the production of
proinflammatory cytokines, making it an attractive target for the treatment of various inflammatory
diseases. A series of pyridazinopyridinone compounds were designed as novel p38 kinase inhibitors.
A structure-activity investigation identified several compounds possessing excellent potency in both
enzyme and human whole blood assays. Among them, compound 31 exhibited good pharmacokinetic
properties and showed excellent selectivity against other related kinases. In addition, 31 demonstrated
efficacy in a collagen-induced arthritis disease model in rats.
Introduction
The p38 mitogen-activated protein (MAP)a kinases are
major components of the MAP kinase pathway that trans-
duces extracellular signals to intracellular responses.1 There
are four isoforms in the p38 family, referred to as R, β, γ, and
δ, with differential expression and activation patterns.2 Of the
four known isoforms, p38R is considered to be the most relevant
kinase involved in the inflammatory response. Upon activa-
tion by extracellular stress stimuli, p38 activates several down-
stream kinases (i.e., MK2, MK3) and transcription factors
(i.e., Stat1, ATF-2) and plays an essential role in the produc-
tion of proinflammatory cytokines such as IL-1β, TNFR, and
IL-6.3 It is known that therapeutics that block the production
of these cytokines provide relief of the symptoms of a variety
of inflammatory diseases.4 Thus, p38 is an attractive target for
the treatment of rheumatoid arthritis (RA), chronic obstruc-
tive pulmonary disease (COPD), and psoriasis, and as such
has been the subject of intense research within the pharma-
ceutical industry since its discovery in the early 1990s.5
Figure 1. Phthalazine and pyrazolpyridinone as p38 inhibitors.
a hydrophobic pocket defined by the residues Thr106,
Leu104, and Lys53. The cyclopropyl amide of those inhibitors
forms polar interactions with Asp168 and Glu71. The phthal-
azine of 1 engages the strand that connects the N- and
C-terminal domains, referred to as the “linker strand”. Both
N-2 and N-3 of the phthalazine function as hydrogen bond
acceptors to the linker strand, one through a classical inter-
action with Met109, seen in all ATP-competitive kinase
inhibitors, and the second with Gly110, which is flipped when
compared to classical kinase inhibitors.9 The tolyl ring at C-1
forms a series of van der Waals contact with the backbone of
the linker strand, while the tolyl methyl group tightly fills a
small hydrophobic cavity on the floor of the binding site,
defined by Ala157 and Leu104. According to the multiple
sequence alignment used in the widely recognized analysis of
the human kinome in 2002 by Manning et al.,10 an alanine
residue in this floor region of the active site is common to only
three other protein kinases (TTBK1, TTBK2, and TSSK3)
outside of the p38 family (with no kinases having the smaller
glycine), so it is reasoned that the interaction with this residue
is the basis for the high selectivity of these agents. Finally, the
hydrophobic benzenoid ring of the phthalazine in 1 engages in
an edge-to-face interaction with Tyr35 from the tip of the
P-loop. In compound 2, N-2 of the pyrazolopyridinone forms
a hydrogen bond with the backbone NH of Met109, while the
We recently reported the discovery of a series of potent
ATP-competitive p38 inhibitors typified by the phthalazine 1
(p38R IC50 = 0.8 nM) and pyrazolopyridinone 2 (p38R IC50
=
3.2 nM) thatexploit features of the ATP binding site unique to
p38R (Figure 1).6 X-ray cocrystal structures revealed that
both 1 and 2 bind to p38R in the so-called DFG-in mode, in
which Phe169 of the DFG-motif is buried in its highly con-
served cognate hydrophobic pocket.7,8 As shown in Figure 2,
the methyl groups on the toluamide rings of 1 and 2 occupy
*To whom correspondence should be addressed: Tel 805-313-5661;
fax 805-480-3016; e-mail: binw@amgen.com.
a Abbreviations: MAP, mitogen-activated protein; RA, rheumatoid
arthritis; COPD, chronic obstructive pulmonary disease; DFG, Asp-
Phe-Gly sequence in ATP binding site; hWB, human whole blood;
THP1, human acute monocytic leukemia cell line; PMB, para-methoxy
benzyl; DIBAL, diisobutyl aluminum hydride; LAH, lithium aluminum
hydride; AUC, area under curve.
r
pubs.acs.org/jmc
Published on Web 08/16/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6399
Figure 2. Co-crystal structures of compounds 1 and 2 with unphosphorylated p38R.
Scheme 1. Selective Amination of 4,5-Dibromopyridazinonea
a Reagents and conditions: (a) PMBCl, K2CO3, Bu4NBr, MeCN, 60 °C, 60%; (b) MeNH2, toluene, microwave, 100 °C, 81%.
Scheme 2. Synthesis of Aldehyde Intermediate 10a
Figure 3. Pyridazinopyridinones as p38 inhibitors.
a Reagents and conditions: (a) CuCN, DMF, microwave, 200 °C,
ortho fluoro substituent is directed downward in a low energy
94%; (b) DIBAL, THF, 0 °C, 59%; (c) CO (30 psi), Pd(OAc)2,
XantPhos, MeNH(OMe), toluene, 110 °C, 64%; (d) LAH, THF, 100%.
conformation, orthogonal to the pyrazolopyridinone ring, to
form a well-defined hydrophobic contact with Ala157. In
contrast to compound 1, the P-loop maintains an extended
Chemistry
˚
conformation, in which the Tyr35 residue (not shown) lies 7 A
from the polar pyridinone carbonyl, separated by two crystallo-
graphically resolved water molecules, which also form a net-
work of hydrogen bonds involving the carbonyl of compound 2
and the charged side chain termini of Asp168 and Lys53.
While 1 and 2 demonstrated excellent potency in enzyme
assays and selectivity against other kinases, they suffered
from unfavorable pharmacokinetic properties such as mode-
rate clearance (1: Cl 1.7 L/h/kg) and low bioavailability (2:
F 12%). Consequently, as part of our continuing efforts to
pursue selective p38 inhibitors for the treatment of inflamma-
tory diseases, we designed pyridazinopyridinone analogues 3
and 4, by merging key elements of compounds 1 and 2 to
further exploit the unique features of the ATP binding site of
p38R (Figure 3). The goal of these novel inhibitors was to
improve the pharmacokinetic liabilities while retaining the
favorable pharmacological properties of the previous series.
Herein, we describe the synthesis, structure-activity relation-
ships (SAR), pharmacokinetics, and in vivo pharmacology of
these new classes of compounds.
The synthesis of the pyridazinopyridinone series com-
menced with 4,5-dibromo-pyridazinone (5, Scheme 1). N-
Protection of 5 with a p-methoxybenzyl group followed by
treatment with methylamine in p-dioxane led to a mixture of
regioisomers with a 2:1 ratio, favoring the desired 5-bromo-
4-(methylamino)pyridazin-3(2H)-one 7. It is well documented
that, in nucleophilic substitutions of 4,5-dihalo-pyridazin-
3(2H)-one, the solvent plays an important role in the product
distribution.11 By switching to toluene as the solvent, the ratio
of regioisomers 7 to 8 was improved to >5:1. The sub-
sequent cyanation of 7 with copper cyanide under micro-
wave irradiation led to 9, which was reduced to aldehyde 10
with diisobutyl aluminum hydride (DIBAL) (Scheme 2).
Alternatively, intermediate 10 was also prepared through
Weinreb amide 11 in a two-step sequence which proved
more amenable to large-scale production. Palladium-cata-
lyzed aminocarbonylation generated the Weinreb amide
11,12 which was subsequently converted to 10 by lithium
aluminum hydride (LAH) reduction.

6400 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Wu et al.
Scheme 3. Preparation of Pyridazinopyridinone Analogues 17-25a
a Reagents and conditions: (a) piperidine, heat, 64%; (b) TFA, 125 °C; (c) POCl3, 110 °C, 86%; (d) 2,4-difluorophenyl boronic acid, Pd(PPh3)4,
Na2CO3, dioxane/H2O (4:1), microwave, 105 °C; then aq. NaOH, 72%; (e) LiOAc, NBS, THF/H2O, 55 °C, 81%; (f) ArNH2 or ArOH, Pd(OAc)2,
XantPhos, toluene, microwave, 125 °C; (g) ArCH2ZnCl, Pd(PPh3)4, THF, 75 °C.
Scheme 4. Synthesis of Pyridazinopyridinone Analoguesa
a Reagents and conditions: (a) 3-borono-4-methylbenzoic acid or 3-borono-4-chlorobenzoic acid, Pd(PPh3)4, Na2CO3, dioxane/H2O (4:1);
(b) MeNH2 or cyclopropylamine, HATU, DIPEA, DMSO; (c) ArB(OR)2, Pd(PPh3)4, Na2CO3, microwave, dioxane/H2O (4:1).
Asshownin Scheme3, cyclocondensationof10withdiethyl
malonate (12) in the presence of piperidine furnished pyrida-
zinonepyridinone 13.13 The p-methoxybenzyl (PMB) protect-
ing group was then removed by TFA, and treatment of the
resulting pyridazinone with POCl3 yielded aryl chloride 14, a
common intermediate for both series. Suzuki coupling reaction
of 14 with 2,4-difluorophenyl boronic acid, followed by saponi-
fication of the ester, yielded carboxylic acid 15. The subsequent
decarboxylative bromination, according to Chowdhury et al,14
afforded aryl bromide 16. Pyridazinopyridinones 17-23 were
obtained through Buchwald-Hartwig coupling with the
corresponding anilines or phenols, and analogues 24 and
25 were prepared by Negishi coupling with the alkyl zinc
reagents.15
Alternatively, Suzuki reaction of intermediate 16 with
2-methyl-5-carboxyphenylboronic acid or 2-chloro-5-carboxy-
phenylboronic acid followed by amide formation yielded
compound 28 and 29 (Scheme 4). Analogues 30-34 were
generated through Suzuki coupling reaction with the corres-
ponding boronic esters with the amide already installed.16

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6401
Table 1. SAR: Pyridazinopyridinones 17-25a
a Standard deviations are given when ng3. b Average of two measurements.
Results and Discussion
TNFR production in THP1 cells and TNF-induced IL-8
production in human whole blood was measured. The
results shown in Tables 1-3 are based on the average
of at least three independent experiments, unless noted
otherwise, and are reported as IC50 with standard devi-
ation.
In Vitro SAR. Compounds were evaluated for their
ability to inhibit the phosphorylation of substrate acti-
vating transcription factor 2 (ATF2) by recombinant
human p38R. In parallel, the inhibition of LPS-induced

6402 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Table 2. SAR: Toluamide Ringa
Wu et al.
a Standard deviations are given when n g 3. b Average of two measurements.
Analogues of pyridazinopyridinone represented by gen-
eric structure 3 (Figure 3), in which the aromatic ring
was attached to the pyridinone ring at the 3 position via
N, O, or CH2 linkage, were evaluated first (Table 1). The
4-fluoroaniline analogue 17 showed good potency, and
adding one more fluorine atom in the ortho position of
the aniline (18) further improved the potency by 5-fold in
the whole blood assay. In contrast, replacement of the
phenyl with a pyridine (i.e., 19 and 20) likely led to a
stabilization of the small molecule conformational prefer-
ence away from the binding mode and methylation of the
amine (21) likely led to an unfavorable interaction with the
protein; both resulted in substantial loss of activity.
modeling of compound 17 in the ATP binding site of un-
phosphorylated p38R suggested that, as in the case of the
cocrystal with compound 2, ligand binding was water
mediated, albeit through a single, highly ordered water
molecule rather than multiple water molecules. In the model
of 17, as illustrated in Figure 4, the coordinated water
molecule was completely shielded from bulk solvent with
both hydrogens held in place by hydrogen bonds with the
carbonyl on 17 and the carboxylate on Asp168 as well as
both oxygen lone pairs held in place by hydrogen bonds with
the anilinic NH of 17 and the primary amine of Lys53. With
O-linked analogues (22, 23), the coordinated water molecule
would experience a much less favorable environment. The
NH to O change presents one less hydrogen bond donor and
one additional hydrogen bond acceptor, leaving the bound
water molecule in the presence of three hydrogen bond
acceptors and one donor, a significant difference expected
Similarly, replacement of the linker -NH with either -O or
-CH2 also led to a loss in activity (22, 24, vs 17), although this
could be somewhat rescued by the introduction of a second
fluorine atom at the ortho position (23 and 25). Molecular

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6403
Table 3. SAR: C(8) Substitutiona
a Standard deviations are given when ng3.
to result in electrostatic repulsion for one of the water mole-
cule oxygen lone pairs. This likely accounts for the 10-40 fold
reduction in potency of these analogues. In the case of the CH2-
linked series (24, 25), the water molecule would be surrounded
by two hydrogen bond acceptors and one donor, leaving one of
the water molecule’s oxygen lone pair desolvated by the
nonpolar aliphatic carbon. In addition, the strong bias for a
tetrahedral geometry of the sp3 methylene spacer would impart
a significant penalty upon binding to the protein, creating
internal strain of the kinase inhibitor to adopt the same binding
mode. While the desolvation penalty of the coordinated water
molecule was likely not as significant as with the O-linkage, the
ligand’s internal strain penalty was greater; taken together,
these two factors likely accounted for the 7-25 fold reduction
in potency associated with the CH2-linkage that was compar-
able to the 10-40 fold loss imparted by the O-linkage. The
model of a tightly bound water molecule also is consistent with
the ∼100 fold loss of potency resulting from N-methylation of
the anilinic amine (21), as previously alluded to.
On the basis of its excellent potency in the whole blood
assay (IC50 = 12 nM), compound 18 was then profiled for its
pharmacokinetic parameters in male Sprague-Dawley rats.
18 displayed moderate plasma clearance and half-life (Cl =
1.42 L/h/kg and t1/2 = 2.79 h), but low oral bioavailability
F (5%). In light of this unfavorable pharmacokinetic profile
and the generally poor potency of the linker series in whole
blood assays, we turned our attention to the analogues
represented by the generic structure 4.
The preliminary SAR study of these analogues, in which
the aromatic ring is directly attached to the pyridinone at
the 3 position, is shown in Table 2. As anticipated based on
our previous SAR, carboxylic acid 26 was devoid of activ-
ity, presumably due to a repulsive electrostatic interac-
tion with Glu71 located on the C R-helix of the enzyme.
In contrast, the primary amide 30 was potent in all three
assays with IC50 values of 2.7, 19, and 21 nM in the enzyme,
THP1 cell, and whole blood assays, respectively. Further
extension of the amide from methyl, to cyclopropyl and

6404 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Wu et al.
Figure 5. Molecular model of compound 31 in the ATP binding site
of unphosphorylated p38R.
Figure 4. Molecular model of compound 17 in the ATP binding site
of unphosphorylated p38R.
1-methylcyclopropyl (compounds 28, 31, 32, respectively),
had little impact on enzyme activity, but afforded an
improvement in cell activity in the case of cyclopropyl
(31). We then looked at substitution of the aromatic ring.
Replacement of 4-methyl with 4-chloro (compare 29 to 31)
was well tolerated as was the introduction of the fluoro
atom at C5 (33). The pyridine analogue 34, however, was
less potent in all assays with over 30-fold reduction in
the whole blood assay compared with 31, again presum-
ably due to an unfavorable electrostatic interaction with
Glu71, the loss of a van der Waals interaction with Leu75,
or both.
Table 4. In Vitro Pharmacokinetic Profile of Compounds 31, 33, 35, 36,
and 38
iv (2.0 mg/kg in DMSO)a
po (2.0 mg/kg)a,b
CL
(L/h/kg)
Vdss
(L/kg)
AUC
(ng h/mL)
compound
t1/2 (h)
F (%)
3
31
33
35
36
38
0.23
0.30
4.91
0.79
1.84
1.04
1.19
2.09
1.65
1.73
3.06
4.66
2.15
2.73
3.35
7633
6737
543
86
55
132
57
1460
2410
116
a Values are the average of 3 runs. b Vehicle: 1% Pluronic F68,
1% HPMC, 15% hydroxypropyl β-cyclodextrin, 83% water.
By keeping the toluamide constant as cyclopropylbenza-
mide and we then turned our attention to substitution at C(8)
of the pyridazinopyridinone (Table 3). The strategy here was
to maintain, minimally, an ortho substitutent to ensure a 90°
torsion to engage Ala157 and hopefully clash with the more
common Leu/Met residues from other protein kinases, while
examining the impact of different functional groups on
potency and pharmacokinetics. Compounds 35-40 were
prepared in a manner similar to compound 31. As shown
in Table 3, replacement of the 2-fluoro with 2-methyl,
2-chloro, or 2-trifluoromethyl as well as replacement of the
4-fluoro substituent with 4-trifluoromethyl resulted in no
significant change in activity across enzyme, cell and whole
blood assays. In addition, a 5-fluoro substitution was well
tolerated.
Molecular modeling of compound 31 in the ATP binding
site of unphosphorylated p38R suggested that, similar to
compound 17, N-6 and N-7 of the pyridazinopyridinone
form hydrogen bonds with NH of Met109 and NH of the
flipped Gly110, respectively, while a water molecule forms a
hydrogen bond network with the carbonyl of the pyridazino-
pyridinone, Asp168, Ala34, and Lys53 (Figure 5). The
methyl group on the toluamide ring was placed in the
hydrophobic gatekeeper pocket and the cyclopropylamide
positioned to engage with Glu71 and Asp168 through two
polar interactions, analogous to the interactions observed in
the cocrystal of compound 1 and p38R (Figure 2). In addi-
tion, the model positioned the 2,4-difluorophenyl ring tightly
between Ala157 and Leu167 below the plane of pyridazino-
pyridinone.
Pharmacokinetic Profile and Kinase Selectivity
On the basis of their whole blood potency, selected com-
pounds were further evaluated in pharmacokinetic studies.
The results of pharmacokinetic profiling of 31, 33, 35, 36, and
38 in male Sprague-Dawley rats are described in Table 4.
While 35 and 38 displayed less favorable profiles such as
moderate to high clearance, 31, 33, and 36 exhibited both low
clearance and good bioavailability. Clearance was much high-
er for 35 presumably due in part to oxidative metabolism in
the ortho-methyl group, although hydroxylation in the para
position may also play a part as clearance was somewhat
improved when a para-F was introduced as in compound 38.
Improvement in clearance over 35 is further observed with
ortho-Cl analogue (36), but remained higher than the ortho,
para-di-F analogue (31), suggesting para-hydroxylation was a
site of oxidative metabolism.
In a kinase profiling study to probe potential off-target
liabilities, compound 31 exhibited activity against p38R and
p38β (IC50 = 4.8 nM) and was selective against the other
two isoforms, p38γ and p38δ (IC50 > 10 μM).17 In an Ambit
screen,18 31 was devoid of activity against over 300 protein
kinases with the exception of cKit, PDGFR, and ZAK,
where 31 displayed moderate activity (POC<35% at 1 μM).
In addition, 31 did not show any activity against a variety of
G-protein-coupled receptors and ion channels when tested
at 10 μM concentration. In a cardiovascular safety screen,
compound 31 displayed an IC50 of 19.5 μM for blockade of
the hERG cardiac channel determined by electrophysiology.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6405
protein crystallography, several compounds were identified as
potent inhibitors in enzyme, cell and whole blood assays.
Among them, compound 31 demonstrated favorable phar-
macokinetic properties with low clearance and good bioavail-
ability, and also exhibited excellent selectivity against a panel
of over 300 kinases. In addition, 31 displayed dose-dependent
effects in both acute biochemical challenge and chronic dis-
ease models with ED50 values of 0.06 and 0.03 mg/kg,
respectively.
Experimental Section
Chemistry. Unless otherwise noted, all materials were ob-
tained from commercial suppliers and used without further
purification. Anhydrous solvents were obtained from Aldrich
or EM Science and used directly. All reactions involving air- or
moisture-sensitive reagents were performed under a nitrogen or
argon atmosphere. All microwave-assisted reactions were con-
ducted with an Initiator microwave reactor from Personal
Chemistry, Biotage AB, Uppsala, Sweden. All final compounds
were purified to >95% purity as determined by LC/MS
obtained on Agilent 1100 spectrometers using one of the three
following methods: HPLC Method A: YMCODS-AM (100 ꢀ
2.1 mm, 5 μm, 40 °C); mobile phase: A = 0.1% HCOOH in
water, B = 0.1% HCOOH in acetonitrile; gradient: 0.0-0.5
min, 10% B; 0.5-7.0 min, 10-100% B; 7.0-9.5 min, 100% B;
9.5-10.0 min, 100-10% B; flow rate = 0.5 mL/min; λ = 254
nm; 1.5 min post time; 3.0 μL injection. HPLC Method B:
Phenomenex Synergi MAX-RP (50 ꢀ 2.0 mm, 4.0 μm, 40 °C);
mobile phase: A = 0.1% TFA in water, B = 0.1% TFA in
acetonitrile; gradient: 0.0-0.2 min, 10% B, 0.2-3.0 min,
10-100% B, 3.0-4.5 min, 100% B, 4.5-5.0 min, 100-10%
B; flow rate = 0.8 mL/min; λ = 254 nm; 1.5 min post time; 3.0
μL injection. HPLC Method C: Agilent SB-C18 column (50 ꢀ 3
mm, 3.5 μm, 40 °C); mobile phase: A = 0.1% TFA in Water,
B = 0.1% TFA in acetonitrile; gradient: 0.0-3.0 min, 5-95%
B; 3.0-3.5 min, 95% B; 3.5-3.6 min, 95-5% B; flow rate = 1.5
mL/min; λ = 254 nm; 0 min post time; 3.0 μL injection. Silica gel
chromatography was performed using either glass columns
packed with silica gel (200-400 mesh, Aldrich Chemical) or
prepacked silica gel cartridges (Biotage or RediSep). 1H NMR
spectra were determined with a DRX 400 MHz spectrometer.
Chemical shifts are reported in parts per million (ppm, δ units).
Low-resolution mass spectral (MS) data were determined on an
Agilent 1100 spectrometer using ES ionization modes (positive
or negative). High-resolution mass spectral (HRMS) data were
determined on an Agilent LC/MSD TOF 1200 series spectro-
meter. Combustion analyses were performed by Atlantic Micro-
lab, Inc., Norcross, GA, and were within (0.4% of calculated
values unless otherwise noted.
Figure 6. Effect of 31 on LPS-Induced TNFR in Lewis rats.
Figure 7. Effect of 31 on paw swelling in Lewis rats in collagen-
induced arthritis.
The excellent in vitro selectivity and safety profile demon-
strated by 31 warranted further development.
In Vivo Pharmacology. Compound 31 was further evalu-
ated in an acute biochemical challenge model-lipopolysac-
charides (LPS) induced TNFR production in female Lewis
rats. In these studies, vehicle or compound was given to the
rats 1 h prior to injection of LPS, and plasma TNFR levels
were determined after 90 min. As illustrated in Figure 6, 31
showed dose-dependent inhibition of TNFR production
with an ED50 about 0.06 mg/kg. In a collagen-induced
arthritis (CIA) model in Lewis rats, compound 31 showed
a dose-dependent reduction in paw swelling with an ED50 of
0.03 mg/kg (Figure 7).
2-(4-Methoxybenzyl)-4,5-dibromopyridazin-3(2H)-one (6). A
mixture of 4,5-dibromopyridazin-3(2H)-one (5) (10.0 g, 39.4
mmol), 4-methoxybenzyl chloride (5.61 mL, 41.4 mmol), tetra-
butylammonium bromide (0.635 g, 1.97 mmol), and potassium
carbonate (13.6 g, 98.5 mmol) in acetonitrile (80 mL) was heated
at 60 °C for 12 h. The mixture was cooled to room temperature
and then filtered through a plug of Celite. The filtrate was
concentrated to dryness and then sonicated in MeOH. The solid
was collected by filtration and washed with MeOH. The solid
was dried in vacuo to afford 6 (8.81 g, 60% yield) as a light
brown solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.78 (1 H, s),
7.40 (2 H, d, J = 8.6 Hz), 6.85 (2 H, d, J = 8.8 Hz), 5.25 (2 H, s),
3.78 (3 H, s); MS (ESI, pos. ion) m/z: 375 (M þ 1).
Conclusion
2-(4-Methoxybenzyl)-5-bromo-4-(methylamino)pyridazin-3(2H)-
one (7) and 2-(4-Methoxybenzyl)-4-bromo-5-(methylamino)pyri-
dazin-3(2H)-one (8). Method A. To a microwave vial containing
2-(4-methoxybenzyl)-4,5-dibromopyridazin-3(2H)-one 6 (0.475 g,
1.27 mmol) was added p-dioxane (5 mL) saturated with MeNH2.
The reaction vessel was sealed and heated in a microwave at 100 °C
for 1 h, then cooled to room temperature. The solvent was then
On the basis of the insight provided by X-ray crystallo-
graphy of potent p38 inhibitors 1 and 2, we have designed
a novel pyridazinopyridinone scaffold by merging the key
pharmacophoric elements of the phathalazine and pyrazolo-
pyridinone series. Through focused SAR studies guided by

6406 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Wu et al.
removed in vacuo and the residue was purified by silica gel column
chromatography (10-40% EtOAc in hexanes) to give 7 (0.258 g,
62.7% yield) as a white solid and 8 (0.124 g, 30.1% yield) as a white
solid. 7:1H NMR (400 MHz, CDCl3) δppm 7.59 (1 H, s), 7.35 (2 H,
d, J= 8.6 Hz), 6.84 (2 H, d, J= 8.8 Hz), 5.95 (1 H, br. s.), 5.17 (2 H,
s), 3.78 (3 H, s), 3.30 (3 H, d, J = 5.7 Hz); MS (ESI, pos. ion) m/z:
324 (M þ 1). 8: 1H NMR (400 MHz, CDCl3) δ ppm 7.49 (1 H, s),
7.39 (2 H, d, J = 8.6 Hz), 6.84 (2 H, d, J = 8.6 Hz), 5.25 (2 H, s),
4.75 (1 H, br. s.), 3.77 (3 H, s), 3.03 (3 H, d, J = 5.1 Hz); MS (ESI,
pos. ion) m/z: 324 (M þ 1).
very slowly with MeOH (1.0 mL), water (1.0 mL) and 1 M HCl
aqueous solution (1.0 mL). The reaction mixture was then
filtered off and the filtrate was concentrated to give 10 (3.82 g,
100% yield), which was subjected to the next step without
purification.
Ethyl 7-(4-methoxybenzyl)-1-methyl-2,8-dioxo-1,2,7,8-tetra-
hydropyrido[2,3-d] pyridazine-3-carboxylate (13). A mixture
of 10 (3.78 g, 13.8 mmol), diethyl malonate (12) (4.20 mL, 27.7
mmol), piperidine (1.44 mL, 14.5 mmol), and EtOH (100 mL)
was stirred at reflux in an oil bath for 5 h. The reaction mixture
was allowed to stand at room temperature overnight to afford a
suspension. The solid was collected by suction filtration, washed
with EtOH (10 mL), and dried in vacuo to afford 13 (3.27 g,
63.9% yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ
ppm 8.38 (s, 1 H), 8.27 (s, 1 H), 7.31 (2 H, d, J = 8.6 Hz), 6.89
(2 H, d, J = 8.6 Hz), 5.21 (s, 2 H), 4.29 (2 H, q, J = 7.1 Hz), 4.01
(s, 3 H), 3.72 (s, 3 H), 1.29 (3 H, t, J = 7.0 Hz); MS (ESI, pos. ion)
m/z: 370 (M þ 1).
Method B. To a sealed tube was added 6 (18.0 g, 48.1 mmol) in
toluene (250 mL, saturated with MeNH2). The reaction was
heated in an oil bath at 90 °C for 18 h, and then the solvent was
removed in vacuo. The residue was purified by silica gel column
chromatography (10-40% EtOAc in hexanes) to give 7 (12.6 g,
80.8% yield) as a white solid.
1-(4-Methoxybenzyl)-5-(methylamino)-6-oxo-1,6-dihydropyr-
idazine-4-carbonitrile (9). To a microwave vial containing a
suspension of 7 (2.24 g, 6.91 mmol) in DMF (12 mL) was added
copper cyanide (0.743 g, 8.29 mmol). The reaction was heated in
a microwave at 200 °C for 1 h. The reaction mixture was then
diluted with a solution of FeCl3 (10.0 g), conc. HCl (3.0 mL) and
water (50 mL) and heated in an oil bath at 70 °C for 15 min, then
cooled to room temperature. The mixture was filtered and the
solid was washed with water to give 9 (1.75 g, 93.7% yield) as a
pale yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.56 (1 H,
s), 7.35 (2 H, d, J = 8.6 Hz), 6.85 (2 H, d, J = 8.6 Hz), 5.16 (2 H,
s), 3.78 (3 H, s), 3.37 (3 H, d, J = 5.9 Hz); MS (ESI, pos. ion) m/z:
271 (M þ 1).
Ethyl 8-Chloro-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyri-
dazine-3-carboxylate (14). A microwave vial was charged with
13 (2.00 g, 5.41 mmol) in TFA (12 mL). The reaction was heated
in a microwave at 125 °C for 2 h, cooled to room tempera-
ture, and then the solvent was removed in vacuo. The residue
was diluted with DCM (100 mL) and concentrated in vacuo.
The crude product was dissolved in phosphorus oxychloride
(8.00 mL, 85.8 mmol), and the solution was heated to reflux at
110 °C for 1 h in an oil bath. The mixture was cooled to room
temperature. The solvent was removed in vacuo, and the residue
was triturated with DCM. The suspension was filtered to give 14
(1.25 g, 86.2% yield) as a light green solid. 1H NMR (400 MHz,
CDCl3) δ ppm 9.11 (1 H, s), 8.29 (1 H, s), 4.45 (2 H, q, J =
7.0 Hz), 4.09 (3 H, s), 1.43 (3 H, t, J = 7.1 Hz); MS (ESI, pos.
ion) m/z: 268 (M þ 1).
1-(4-Methoxybenzyl)-N-methoxy-N-methyl-5-(methylamino)-
6-oxo-1,6-dihydropyridazine-4-carboxamide (11). A glass pres-
sure vessel equipped with CO regulator was charged with 7 (7.00 g,
21.5 mmol), n,o-dimethylhydroxylamine hydrochloride (3.20 g,
32.8 mmol), potassium phosphate (14 g, 65 mmol), and toluene
(60 mL). The mixture was purged with Ar then palladium (ii)
acetate (0.24 g, 1.1 mmol) and XantPhos (1.20 g, 2.2 mmol) were
added. The vessel was sealed and charged with CO (30 psi) and
the reaction mixture was stirred in a 110 °C oil bath behind a
blast shield for 44 h. After being cooled to room temperature,
the reaction mixture was partitioned between water and DCM.
The whole mixturewas filtered through a pad of Celite and washed
with DCM. The organic layer was separated, dried (Na2SO4),
filtered, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (0-3% MeOH in DCM)
8-(2,4-Difluorophenyl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]-
pyridazine-3-carboxylic acid (15). A microwave vial was charged
with 14 (0.75 g, 2.80 mmol), 2,4-difluorobenzeneboronic acid
(0.53 g, 3.36 mmol), and tetrakis(triphenylphosphine)-palladium
(0.26 g, 0.23 mmol) in p-dioxane/2 M aq. Na2CO3 (4:1, 12.5 mL).
The reaction was heated in a microwave at 105 °C for 90 min, and
then 1 M aq. NaOH (1.5 mL) was added. The reaction was heated
in a microwave at 105 °C for 1 h, and then cooled to room
temperature. The reaction was repeated once on a 0.75 g scale.
The two reaction mixtures were combined and extracted with
DCM. The aqueous layer was acidified with conc. HCl to pH ∼ 2
and extracted with CHCl3/iPrOH (4:1). The combined organic
layers were dried (MgSO4), filtered, and concentrated in vacuo to
give 15 (1.28 g, 72.0% yield) as an orange solid. 1H NMR (400
MHz, DMSO-d6) δ ppm 9.57 (1 H, s), 8.77 (1 H, s), 7.76-7.91
(1 H, m), 7.44-7.59 (1 H, m), 7.26-7.44 (1 H, m), 3.13 (3 H, s);
MS (ESI, pos. ion) m/z: 318 (M þ 1).
1
to give 11 (4.60 g, 64.2% yield) as a yellow foam. H NMR
(400 MHz, DMSO-d6) δ ppm 7.52 (1 H, s), 7.19-7.31 (3 H, m),
6.81-6.94 (2 H, m), 5.12 (2 H, s), 3.72 (3 H, s), 3.57 (3 H, s), 3.23
(3 H, s), 2.70 (3 H, d, J = 5.5 Hz); MS (ESI, pos. ion) m/z: 333
(M þ 1).
1-(4-Methoxybenzyl)-5-(methylamino)-6-oxo-1,6-dihydropyr-
idazine-4 carbaldehyde (10). Method A. To a suspension of 9
(5.50 g, 20.3 mmol) in toluene at 0 °C was added diisobutylalu-
minum hydride (44.8 mL, 44.8 mmol, 1 M solution in hexanes)
dropwise. The reaction was stirred at 0 °C for 20 min, then
EtOAc (50 mL) was added and the mixture was warmed to room
temperature. After 20 min, 1 M HCl (120 mL) was added and the
mixture was heated at 50 °C for 4 h, then filtered through a plug
of Celite and washed with EtOAc. The aqueous layer was
extracted with EtOAc and the combined organic layers were
dried (MgSO4), filtered, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (20-35%
EtOAc in hexanes) to give 10 (3.27 g, 58.8% yield) as a light
3-Bromo-8-(2,4-difluorophenyl)-1-methylpyrido[3,2-d]pyridazin-
2(1H)-one (16). To a suspension of 15 (4.51 g, 14.2 mmol) in
THF/H2O (10:1, 55 mL) was added lithium acetate dihydrate
(0.218 g, 2.13 mmol) followed by n-bromosuccinimide (3.16 g,
17.8 mmol). The reaction was heated at 55 °C in an oil bath for
30 min, and then another 1.25 eq of n-bromosuccinimide was
added. The reaction was stirred an additional 30 min before
being cooled to room temperature. The mixture was diluted
with water and extracted with DCM. The combined organic
layers were dried (MgSO4), filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(5-15% EtOAc in DCM with 1% MeOH) to give 16 (4.05 g,
80.9% yield) as a light yellow solid. 1H NMR (400 MHz,
CDCl3) δ ppm 9.17 (1 H, s), 8.25 (1 H, s), 7.72-7.85 (1 H,
m), 7.09-7.20 (1 H, m), 6.92-7.05 (1 H, m), 3.36 (3 H, s); MS
(ESI, pos. ion) m/z: 352 (M þ 1).
1
yellow solid. H NMR (400 MHz, CDCl3) δ ppm 9.52-10.19
(1 H, m), 7.72 (1 H, br. s.), 7.38 (2 H, d, J = 8.6 Hz), 6.80-6.96
(2 H, m), 5.15 (2 H, s), 3.78 (3 H, s), 3.57 (3 H, br. s.); MS (ESI,
pos. ion) m/z: 274 (M þ 1).
Method B. To a solution of 11 (4.60 g, 13.8 mmol) in THF
(50.0 mL) was added lithium aluminum hydride (1 M solution in
THF) (15.23 mL, 15.23 mmol) at -78 °C. The mixture was then
stirred at 0 °C for 1 h. The reaction mixture was quenched at 0 °C
8-(2,4-Difluorophenyl)-3-(4-fluorophenylamino)-1-methylpyrido-
[3,2-d]pyridazin-2(1H)-one (17). A microwave vial was charged
with 16 (0.10 g, 0.284 mmol) in toluene (1 mL), 4-fluoroaniline
(0.034 mL, 0.355 mmol), palladium acetate (3.19 mg, 0.014 mmol),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6407
cesium carbonate (0.139 g, 0.426 mmol), and 4,5-bis(diphenyl-
phosphino)-9,9-dimethylxanthene (0.016 mg, 0.028 mmol). The
reaction was heated in a microwave at 125 °C for 30 min. The
mixture was diluted with water, and filtered, and the resulting solid
was washed with water, toluene, then dried to give 17 (0.078 g,
71.8% yield) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ
ppm 9.26 (1 H, s), 8.85 (1 H, s), 7.74-7.86 (1 H, m), 7.42-7.55
(3 H, m), 7.21-7.38 (4 H, m), 3.21 (3 H, s); HRMS(TOF-MS)
calcd: 383.1114 for C20H14F3N4O [M þ H]þ. Found: 383.1111;
HPLC (Method A); retention time 6.45 min.
3-(4-Fluorobenzyl)-8-(2,4-difluorophenyl)-1-methylpyrido[3,2-d]-
pyridazin-2(1H)-one (24). A microwave vial was charged with 16
(0.050 g, 0.14 mmol) in THF (0.5 mL), tetrakis(triphenylphos-
phine)palladium (0.010 g, 0.0091 mmol), and 4-fluorobenzylzinc
chloride (0.5 M solution in THF) (0.5 mL, 0.25 mmol). The
reaction was heated in an oil bath at 75 °C for 1 h, and then cooled
to room temperature. The reaction was quenched with sat. NH4Cl
and the mixture was extracted with EtOAc. The combined organic
layers were dried (MgSO4), filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
1
8-(2,4-Difluorophenyl)-3-(2,4-difluorophenylamino)-1-methyl-
pyrido[3,2-d]pyridazin-2(1H)-one (18). The title compound
(30 mg, 26%) was prepared according to the procedure de-
scribed for compound 17 from 16 (100 mg, 0.284 mmol) and 2,4-
difluorobenzenamine (45.8 mg, 0.355 mmol). 18 (1H NMR (400
MHz, DMSO-d6) δ ppm 9.25 (1 H, s), 8.64 (1 H, s), 7.72-7.88
(1 H, m), 7.41-7.61 (3 H, m), 7.32 (1 H, td, J = 8.5, 1.9 Hz), 7.21
(1 H, t, J = 9.4 Hz), 6.76 (1 H, d, J = 2.3 Hz), 3.21 (3 H, s);
HRMS(TOF-MS) calcd: 401.1020 for C20H13F4N4O [M þ H]þ.
Found: 401.1022; HPLC (method A); retention time 6.24 min.
8-(2,4-Difluorophenyl)-3-(5-fluoropyridin-2-ylamino)-1-methyl-
pyrido[3,2-d]pyridazin-2(1H)-one (19). The title compound (31
mg, 48%) was prepared according to the procedure described
for compound 17 from 16 (60 mg, 0.17 mmol) and 2-amino-5-
(35-85% EtOAc in hexanes) to give 24 (16 mg, 30% yield). H
NMR (400 MHz, DMSO-d6) δppm 9.37 (1 H, s), 7.87 (1 H, s), 7.79
(1 H, td, J = 8.5, 6.7 Hz), 7.42-7.51 (1 H, m), 7.28-7.42 (3 H, m),
7.16 (2 H, t, J = 8.9 Hz), 3.94 (2 H, s), 3.10 (3 H, s); HRMS(TOF-
MS) calcd: 382.1162 for C21H15F3N3O [M þ H]þ. Found:
382.1168; HPLC (Method C); retention time 2.05 min.
3-(2,4-Difluorobenzyl)-8-(2,4-difluorophenyl)-1-methylpyrido-
[3,2-d]pyridazin-2(1H)-one (25). The title compound (34 mg,
60%) was prepared according to the procedure described for
compound 24 from 16 (50 mg, 0.14 mmol) and (2,4-difluoro-
benzyl)zinc(II) bromide (0.4 mL, 0.5 M). 25: 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.40 (1 H, s), 7.74-7.86 (2 H, m), 7.39-7.52
(2 H, m), 7.22-7.38 (2 H, m), 7.09 (1 H, td, J = 8.6, 1.9 Hz), 3.95
(2 H, s), 3.11 (3 H, s); HRMS(TOF-MS) calcd: 400.1068 for
C21H14F4N3O [M þ H]þ. Found: 400.1074; HPLC (Method C);
retention time 2.10 min.
3-(8-(2,4-Difluorophenyl)-1-methyl-2-oxo-1,2-dihydropyrido-
[3,2-d]pyridazin-3-yl)-4-methylbenzoic acid (26). To a sealed
vessel was added 16 (2.00 g, 5.68 mmol) in p-dioxane/H2O
(4:1, 50 mL), 3-borono-4-methylbenzoic acid (1.124 g, 6.25
mmol), sodium carbonate monohydrate (1.761 g, 14.20 mmol),
and tetrakis(triphenylphosphine) palladium (0.328 g, 0.284
mmol). The reaction was heated in oil bath at 105 °C for 8 h,
and then at 55 °C overnight. The reaction mixture was diluted
with 1 N NaOH and washed with DCM twice. The aqueous
layer was acidified with 5 N HCl to pH 2, and the resulting
suspension was filtered to give 26 (1.98 g, 85.4% yield) as an off-
white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.45 (1 H,
s), 8.21 (1 H, s), 7.92 (1 H, dd, J = 7.8, 1.8 Hz), 7.80-7.89 (2 H,
m), 7.43-7.55 (2 H, m), 7.36 (1 H, td, J = 8.6, 2.4 Hz), 3.15 (3 H,
s), 2.25 (3 H, s); MS (ESI, pos. ion) m/z: 408 (M þ 1); HPLC
(Method B); retention time 1.71 min.
1
fluoropyridine (23 mg, 0.21 mmol). 19: H NMR (400 MHz,
DMSO-d6) δ ppm 9.59 (1 H, s), 9.38 (1 H, s), 8.88 (1 H, s), 8.37
(1 H, d, J = 2.9 Hz), 7.68-7.87 (2 H, m), 7.60 (1 H, dd, J = 9.3,
3.8 Hz), 7.41-7.53 (1 H, m), 7.28-7.39 (1 H, m), 3.22 (3 H, s);
HRMS(TOF-MS) calcd: 384.1067 for C19H13F3N5O [M þ H]þ.
Found: 384.1064; HPLC (Method B); retention time 1.86 min.
8-(2,4-Difluorophenyl)-3-(3,5-difluoropyridin-2-ylamino)-1-
methylpyrido[3,2-d]pyridazin-2(1H)-one (20). The title com-
pound (27 mg, 47%) was prepared according to the procedure
described for compound 17 from 16 (50 mg, 0.14 mmol) and 3,5-
difluoropyridin-2-amine (28 mg, 0.21 mmol). 20: 1H NMR (400
MHz, CDCl3) δ ppm 9.29 (1 H, s), 8.90 (1 H, s), 8.57 (1 H, br. s.),
8.18 (1 H, d, J = 2.5 Hz), 7.78 (1 H, td, J = 8.4, 6.3 Hz), 7.32 (1 H,
ddd, J = 10.0, 7.6, 2.5 Hz), 7.14 (1 H, td, J = 8.2, 2.2 Hz),
6.92-7.04 (1 H, m), 3.42 (3 H, s); HRMS(TOF-MS) calcd:
402.0973 for C19H12F4N5O [M þ H]þ. Found: 402.0971; HPLC
(Method B); retention time 2.38 min.
8-(2,4-Difluorophenyl)-3-((4-fluorophenyl)(methyl)amino)-1-
methylpyrido[3,2-d]pyridazin-2(1H)-one (21). The title com-
pound (22 mg, 40%) was prepared according to the procedure
described for compound 17 from 16 (50 mg, 0.14 mmol) and
4-fluoro-N-methylaniline (21 mg, 0.17 mmol). 21: 1H NMR
(400 MHz, DMSO-d6) δ ppm 9.36 (1 H, s), 7.72-7.84 (1 H, m),
7.51 (1 H, s), 7.41-7.50 (1 H, m), 7.28-7.36 (1 H, m),
7.05-7.15 (4 H, m), 3.35 (3 H, s), 3.02 (3 H, s); HRMS(TOF-
MS) calcd: 397.1271 for C21H16F3N4O [M þ H]þ. Found:
397.1269; HPLC (Method B); retention time 1.87 min.
8-(2,4-Difluorophenyl)-3-(4-fluorophenoxy)-1-methylpyrido-
[3,2-d]pyridazin-2(1H)-one (22). The title compound (23 mg,
36%) was prepared according to the procedure described for
compound 17 from 16 (60 mg, 0.14 mmol) and 4-fluorophenol
(29 mg, 0.26 mmol). 22: 1H NMR (400 MHz, DMSO-d6) δ ppm
9.36 (1 H, s), 7.79 (1 H, q, J = 7.8 Hz), 7.48 (1 H, t, J = 9.1 Hz),
7.23-7.41 (6 H, m), 3.16 (3 H, s); HRMS(TOF-MS) calcd:
384.0954 for C20H13F3N3O2 [M þ H]þ. Found: 384.0953;
HPLC (Method A); retention time 6.05 min.
3-(2,4-Difluorophenoxy)-8-(2,4-difluorophenyl)-1-methylpyrido-
[3,2-d]pyridazin-2(1H)-one (23). The title compound (18 mg, 32%)
was prepared according to the procedure described for compound
17 from 16 (50 mg, 0.14 mmol) and 2,4-difluorophenol (23 mg,
0.18 mmol). 23: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.36 (1 H,
s), 7.79 (1 H, td, J = 8.6, 6.6 Hz), 7.61 (1 H, ddd, J = 11.2, 8.7, 3.0
Hz), 7.45-7.56 (2 H, m), 7.41 (1 H, s), 7.34 (1 H, td, J = 8.4, 2.0
Hz), 7.21-7.29 (1 H, m), 3.16 (3 H, s); HRMS(TOF-MS) calcd:
402.0860 for C20H12F4N3O2 [M þ H]þ. Found: 402.0858; HPLC
(Method A); retention time 6.25 min.
4-Chloro-3-(8-(2,4-difluorophenyl)-1-methyl-2-oxo-1,2-dihydro-
pyrido[3,2-d]pyridazin-3-yl)benzoic acid (27). The title compound
(15 mg, 10%) was prepared according to the procedure described
for compound 26 from 16 (120 mg, 0.34 mmol) and 4-chloro-
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (116
mg, 0.41 mmol). 27: 1H NMR (400 MHz, DMSO-d6) δ ppm 13.32
(1 H, br. s.), 9.46 (1 H, s), 8.32 (1 H, s), 7.98 - 8.05 (2 H, m), 7.87
(1 H, td, J = 8.6, 6.7 Hz), 7.70-7.78 (1 H, m), 7.50 (1 H, td, J =
9.7, 2.4 Hz), 7.37 (1 H, td, J = 8.5, 2.2 Hz), 3.16 (3 H, s); MS (ESI,
pos. ion) m/z: 428 (M þ 1).
3-(8-(2,4-Difluorophenyl)-1-methyl-2-oxo-1,2-dihydropyrido-
[3,2-d]pyridazin-3-yl)-N,4-dimethylbenzamide (28). To a solu-
tion of 26 (0.100 g, 0.245 mmol) in DMSO (1.5 mL) was added
triethylamine (85 μL, 0.614 mmol), 2-(1H-7-azabenzotriazol-1-
yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methan-
aminium (0.140 g, 0.368 mmol), followed by MeNH2 in THF
(2 M, 0.6 mL, 1.2 mmol). The reaction was stirred at room
temperature for 30 min, and then was diluted with CHCl3/
iPrOH (4:1). The mixture was washed with water four times,
dried (MgSO4), filtered, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (40-50%
EtOAc in DCM with 1% MeOH) to give 28 (64.0 mg, 62.0%
yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm
9.45 (1 H, s), 8.37-8.47 (1 H, m), 8.19 (1 H, s), 7.79-7.92 (2 H,
m), 7.76 (1 H, s), 7.45-7.55 (1 H, m), 7.31-7.44 (2 H, m), 3.16 (3
H, s), 2.78 (3 H, d, J = 4.3 Hz), 2.21 (3 H, s); HRMS(TOF-MS)
calcd: 421.1471 for C23H19F2N4O2 [M þ H]þ. Found: 421.1473;
HPLC (Method A); retention time 5.43 min.

6408 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Wu et al.
4-Chloro-N-cyclopropyl-3-(8-(2,4-difluorophenyl)-1-methyl-2-
oxo-1,2-dihydropyrido[3,2-d]pyridazin-3-yl)benzamide (29). The
title compound (15 mg, 9%) was prepared according to the
procedure described for compound 28 from 27 (150 mg, 0.35
mmol) and cyclopropylamine (100 mg, 1.75 mmol). 29: 1H
NMR (400 MHz, DMSO-d6) δ ppm 9.46 (1 H, s), 8.56 (1 H, d,
J = 3.7 Hz), 8.29 (1 H, s), 7.82-7.97 (3 H, m), 7.69 (1 H, d, J =
9.2 Hz), 7.43-7.57 (1 H, m), 7.37 (1 H, td, J = 8.9, 2.7 Hz),
3.16 (3 H, s), 2.81-2.92 (1 H, m), 0.66-0.75 (2 H, m),
0.54-0.61 (2 H, m); HRMS(TOF-MS) calcd: 467.1081 for
64.08; H, 4.06; N, 11.90; F, 12.38; HPLC (Method A); retention
time 6.00 min.
N-Cyclopropyl-4-(8-(2,4-difluorophenyl)-1-methyl-2-oxo-1,2-
dihydropyrido[3,2-d]pyridazin-3-yl)-5-methylpicolinamide (34).
The title compound (8 mg, 10%) was prepared according to
the procedure described for compound 30 from 16 (60 mg, 0.17
mmol) and 2-(cyclopropylcarbamoyl)-5-methylpyridin-4-yl-
boronic acid (45 mg, 0.21 mmol). 34: 1H NMR (400 MHz,
DMSO-d6) δ ppm 9.46 (1 H, s), 8.74 (1 H, d, J = 4.9 Hz), 8.58
(1 H, s), 8.30 (1 H, s), 7.94 (1 H, s), 7.77-7.90 (1 H, m), 7.51 (1 H,
td, J = 9.9, 2.2 Hz), 7.30-7.42 (1 H, m), 3.15 (3 H, s), 2.86-
2.98 (1 H, m), 2.27 (3 H, s), 0.66-0.74 (4 H, m); HRMS(TOF-
MS) calcd: 448.1580 for C24H20F2N5O2 [M þ H]þ. Found:
448.1570; HPLC (Method B); retention time 1.79 min.
N-Cyclopropyl-4-methyl-3-(1-methyl-2-oxo-8-o-tolyl-1,2-dihydro-
pyrido[3,2-d]pyridazin-3-yl)benzamide (35). The title compound
(43.5 mg, 23%) was prepared according to the procedure described
for compound 30 from 14 and o-tolylboronic acid. 35: 1H NMR
(400 MHz, DMSO-d6) δ ppm 9.42 (1 H, s), 8.41 (1 H, d, J = 3.5
Hz), 8.17(1H, s), 7.81(1H, d, J=7.0Hz), 7.74(1H, s), 7.29-7.54
(5 H, m), 3.00 (3 H, s), 2.76-2.92 (1 H, m), 2.21 (3 H, s), 2.12 (3 H,
s), 0.64-0.74 (2 H, m), 0.50-0.60 (2 H, m); HRMS(TOF-MS)
calcd: 425.1972 for C26H25N4O2 [M þ H]þ. Found: 425.1968;
HPLC (Method A); retention time 5.67 min.
C
24H18ClF2N4O2 [M þ H]þ. Found: 467.1085; HPLC
(Method A); retention time 5.88 min.
3-(8-(2,4-Difluorophenyl)-1-methyl-2-oxo-1,2-dihydropyrido-
[3,2-d]pyridazin-3-yl)-4-methylbenzamide (30). A microwave
vial was charged with 16 (0.022 g, 0.062 mmol) in p-dioxane/
1 M Na2CO3 (3:1, 1.6 mL), 4-methyl-3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzamide (0.018 mg, 0.069 mmol)16d
and tetrakis(triphenylphosphine) palladium (0.0058 g, 0.005
mmol). The reaction was heated in a microwave at 100 °C for
50 min. The mixture was diluted with water and extracted with
CHCl3/iPrOH (4:1). The combined organic layers were dried
(MgSO4), filtered, and concentrated in vacuo. The residue was
purified with reverse phase HPLC to give 30 (0.008 g, 32%
yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm
9.45 (1 H, s), 8.20 (1 H, s), 7.96 (1 H, br. s.), 7.83-7.91 (2 H, m),
7.81 (1 H, d, J = 1.6 Hz), 7.46-7.55 (1 H, m), 7.29-7.44 (3 H,
m), 3.16 (3 H, s), 2.22 (3 H, s); HRMS(TOF-MS) calcd:
407.1314 for C22H17F2N4O2 [M þ H]þ. Found: 407.1317;
HPLC (Method B); retention time 1.56 min.
3-(8-(2-Chlorophenyl)-1-methyl-2-oxo-1,2-dihydropyrido[3,2-d]-
pyridazin-3-yl)-N-cyclopropyl-4-methylbenzamide (36). The title
compound (31 mg, 15%) was prepared according to the proce-
dure described for compound 30 from 14 and 2-chlorophenyl-
1
boronic acid. 36: H NMR (400 MHz, DMSO-d6) δ ppm 9.46
(1 H, s), 8.41 (1 H, d, J = 3.9 Hz), 8.20 (1 H, s), 7.77-7.84 (2 H,
m), 7.75 (1 H, d, J = 1.8 Hz), 7.66-7.70 (1 H, m), 7.56-7.65(2 H,
m), 7.39 (1 H, d, J = 7.8 Hz), 3.11 (3 H, s), 2.80-2.89 (1 H, m),
2.20 (3 H, s), 0.65-0.72 (2 H, m), 0.52-0.59 (2 H, m); HRMS-
(TOF-MS) calcd: 445.1426 for C25H22ClN4O2 [M þ H]þ. Found:
445.1419; HPLC (Method A); retention time 5.74 min.
N-Cyclopropyl-4-methyl-3-(1-methyl-2-oxo-8-(2-(trifluoromethyl)-
phenyl)-1,2-dihydropyrido[3,2-d]pyridazin-3-yl)benzamide (37).
The title compound (31 mg, 14%) was prepared according
to the procedure described for compound 30 from 14 and
2-(trifluoromethyl)phenylboronic acid. 37: 1H NMR (400
MHz, DMSO-d6) δ ppm 9.47 (1 H, s), 8.41 (1 H, d, J = 3.9
Hz), 8.19 (1 H, s), 7.99 (1 H, d, J = 7.6 Hz), 7.78-7.92 (4 H, m),
7.74 (1 H, d, J = 1.6 Hz), 7.39 (1 H, d, J = 8.0 Hz), 3.03 (3 H, s),
2.80-2.90 (1 H, m), 2.20 (3 H, s), 0.65-0.73 (2 H, m),
0.51-0.61 (2 H, m); HRMS(TOF-MS) calcd: 479.1689 for
C26H22F3N4O2 [M þ H]þ. Found: 479.1689; HPLC (Method
B); retention time 1.85 min.
N-Cyclopropyl-3-(8-(2,4-difluorophenyl)-1-methyl-2-oxo-1,2-
dihydropyrido[3,2-d]pyridazin-3-yl)-4-methylbenzamide (31). The
title compound (1.89 g, 60%) was prepared according to the
procedure described for compound 30 from 16 (2.5 g, 7.10 mmol)
and N-cyclopropyl-4-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)benzamide (2.46 g, 8.17 mmol). 31: 1H NMR (400
MHz, DMSO-d6) δ ppm 9.45 (1 H, s), 8.40 (1 H, d, J = 3.9 Hz),
8.19 (1 H, s), 7.78-7.92 (2 H, m), 7.74 (1 H, s), 7.50 (1 H, td, J =
9.6, 2.2 Hz), 7.32-7.42(2 H, m), 3.16 (3 H, s), 2.80-2.90 (1 H, m),
2.21 (3 H, s), 0.64-0.74 (2 H, m), 0.52-0.61 (2 H, m); HRMS-
(TOF-MS) calcd: 447.1627 for C25H21F2N4O2 [M þ H]þ. Found:
447.1619. Anal. Calcd. for C25H20F2N4O2: C, 67.26; H, 4.52; N,
12.55; F, 8.51. Found: C, 67.09; H, 4.48; N, 12.33; F, 8.52; HPLC
(Method A); retention time 5.71 min.
3-(8-(2,4-Difluorophenyl)-1-methyl-2-oxo-1,2-dihydropyrido-
[3,2-d]pyridazin-3-yl)-4-methyl-N-(1-methylcyclopropyl)benza-
mide (32). The title compound (41 mg, 63%) was prepared
according to the procedure described for compound 30 from 16
(50 mg, 0.14 mmol) and 4-methyl-N-(1-methylcyclopropyl)-
3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (52 mg,
0.16 mmol).16d 32: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.45
(1 H, s), 8.63 (1H, s), 8.19 (1H, s), 7.78-7.93 (2 H, m), 7.74 (1 H, d,
J = 1.8 Hz), 7.50 (1 H, td, J = 9.9, 2.4 Hz), 7.32-7.42 (2 H, m),
3.16 (3 H, s), 2.20 (3 H, s), 1.36 (3 H, s), 0.69-0.77 (2 H, m),
0.56-0.64 (2 H, m); HRMS(TOF-MS) calcd: 461.1784 for
C26H23F2N4O2 [M þ H]þ. Found: 461.1779; HPLC (Method B);
retention time 1.84 min.
N-Cyclopropyl-3-(8-(2,4-difluorophenyl)-1-methyl-2-oxo-1,2-
dihydropyrido[3,2-d]pyridazin-3-yl)-5-fluoro-4-methylbenzamide
(33). The title compound (57 mg, 72%) was prepared according
to the procedure described for compound 30 from 16 (60 mg,
0.17 mmol) and N-cyclopropyl-3-fluoro-4-methyl-5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (65 mg, 0.20
mmol).16e 33: 1H NMR (400 MHz, DMSO-d6) δ ppm 9.46
(1 H, s), 8.50 (1 H, d, J = 4.1 Hz), 8.24 (1 H, s), 7.85 (1 H, td, J =
8.5, 6.7 Hz), 7.70 (1 H, d, J = 10.6 Hz), 7.64 (1 H, s), 7.50 (1 H,
td, J = 9.7, 2.4 Hz), 7.36 (1 H, td, J = 8.4, 2.3 Hz), 3.15 (3 H, s),
2.80-2.91 (1 H, m), 2.10 (3 H, d, J = 1.8 Hz), 0.66-0.75 (2 H,
m), 0.51-0.61 (2 H, m); HRMS(TOF-MS) calcd: 465.1533 for
C25H20F3N4O2 [M þ H]þ. Found: 465.1534; Anal. Calcd. For
C25H19F3N4O2: C, 64.15; H, 4.12; N, 12.06; F, 12.27. Found: C,
N-Cyclopropyl-3-(8-(4-fluoro-2-methylphenyl)-1-methyl-2-
oxo-1,2-dihydropyrido[3,2-d]pyridazin-3-yl)-4-methylbenzamide
(38). The title compound (53 mg, 43%) was prepared according
to the procedure described for compound 30 from 14 and
1
4-floro-2-methylphenylboronic acid. 38: H NMR (400 MHz,
DMSO-d6) δ ppm 9.42 (1 H, s), 8.41 (1 H, d, J = 4.1 Hz), 8.17
(1 H, s), 7.81 (1 H, dd, J = 8.0, 1.8 Hz), 7.73 (1 H, d, J = 1.6 Hz),
7.49 (1 H, dd, J = 8.5, 6.0 Hz), 7.38 (1 H, d, J = 8.0 Hz), 7.30
(1 H, dd, J = 9.9, 2.4 Hz), 7.22 (1 H, td, J = 8.5, 2.6 Hz), 3.02 (3
H, s), 2.79-2.90 (1 H, m), 2.21 (3 H, s), 2.14 (3 H, s), 0.64-0.73
(2 H, m), 0.50-0.61 (2 H, m); HRMS(TOF-MS) calcd: 443.1878
for C26H24FN4O2 [M þ H]þ. Found: 443.1871; HPLC (Method
B); retention time 1.75 min.
N-Cyclopropyl-3-(8-(2-fluoro-4-(trifluoromethyl)phenyl)-1-
methyl-2-oxo-1,2-dihydropyrido[3,2-d]pyridazin-3-yl)-4-methyl-
benzamide (39). The title compound (35 mg, 14%) was prepared
according to the procedure described for compound 30 from
14 and 2-fluoro-4-(trifluoromethyl)phenylboronic acid. 39: 1H
NMR (400 MHz, DMSO-d6) δ ppm 9.49 (1 H, s), 8.42 (1 H, d,
J = 4.1 Hz), 8.21 (1 H, s), 8.07 (1 H, t, J = 7.5 Hz), 7.95 (1 H, d,
J = 9.6 Hz), 7.77 - 7.91 (2 H, m), 7.75 (1 H, d, J = 1.6 Hz), 7.39
(1 H, d, J = 8.0 Hz), 3.15 (3 H, s), 2.80-2.90 (1 H, m), 2.21 (3 H,
s), 0.63-0.75 (2 H, m), 0.50-0.61 (2 H, m); HRMS(TOF-MS)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6409
calcd: 497.1595 for C26H21F4N4O2 [M þ H]þ. Found: 497.1594;
HPLC (Method A); retention time 6.15 min.
at 37 °C with 5% CO2. TNFR with a final concentration of 1 nM
was then added to the blood and incubated overnight (16-18 h)
at 37 °C with 5% CO2. Plasma was harvested and cytokines
(IL8) were measured by MSD (Meso Scale Discovery) ECL
based antibody sandwich assay. All reagents were prepared in
RPMI 1640, 10% v/v human serum AB (Gemini Bio-Products),
1ꢀ Pen/Strep/Glu. Final concentration of human whole blood
was 50%. Data were analyzed using XLfit/Activity Base soft-
ware package (IDBS).
Lipopolysaccharide (LPS)-Induced TNF Study. Lipopoly-
saccharide (Sigma Chemical Co.) was diluted in phosphate
buffered saline (PBS, Life Technologies) to a concentration of
500 μg/mL. The vehicle or compound 31 as a solution in 15%
hydoxypropyl-cyclodextrin, 1% hydroxymethylcellulose, 1%
Pluronic F68 was administrated orally to female Lewis rats (n =
6) 1 h prior to injection with LPS (100 μg/rat, iv, tail vein).
Blood was harvested 90 min following the administration of
LPS. Serum TNFR levels were analyzed using rat TNFR CytoSet
kit from Biosource International. Data points represent the
mean ( SEM.
Collagen Induced Arthritis Model. Arthritis was induced by
intradermal injection of Porcine type II collagen emulsified
1:1 in incomplete Freunds adjuvant (IFA). Animals were
assigned to treatment groups at disease onset (study day 0),
which occurred 10-12 days following immunization. Com-
pound 31 or vehicle was administered orally once a day for
7 days. Paw diameter was measured daily from day 0 through
day 7.
Area under the paw swelling curve (AUC) was calculated and
used to determine percent inhibition of inflammation compared
with vehicle controls. Data points represent the mean ( SEM
(n=8 rats/group).
3-(8-(2-Chloro-5-fluorophenyl)-1-methyl-2-oxo-1,2-dihydro-
pyrido[2,3-d]pyridazin-3-yl)-N-cyclopropyl-4-methylbenzamide
(40). The title compound (396 mg, 23%) was prepared according
to the procedure described for compound 30 from 14 and
1
2-chloro-5-fluorophenylboronic acid. 40: H NMR (400 MHz,
DMSO-d6) δ ppm 9.48 (1 H, s), 8.41 (1 H, d, J = 3.9 Hz), 8.20
(1 H, s), 7.82 (1 H, dd, J = 7.9, 1.7 Hz), 7.69-7.77 (3 H, m), 7.47-
7.59 (1 H, m), 7.39 (1 H, d, J = 8.0 Hz), 3.15 (3 H, s), 2.80-
2.90 (1 H, m), 2.20 (3 H, s), 0.65-0.73 (2 H, m), 0.53-0.59
(2 H, m); HRMS(TOF-MS) calcd: 463.1332 for C25H21ClFN4O2
[M þ H]þ. Found: 463.1333; HPLC (Method B); retention time
1.91 min.
Biological Methods. p38r MAP Kinase in Vitro Assay. The
p38R kinase reaction was carried out in a polypropylene 96-well
black round bottomed assay plate in a total volume of 30 μL
of kinase reaction buffer (50 mM Tris-pH 7.5, 5 mM MgCl2,
0.1 mg/mL BSA, 100 μM Na3VO4, and 0.5 mM DTT). Recom-
binant activated human p38 enzyme (1 nM) was mixed with
50 μM ATP and 100 nM GST-ATF2-Avitag, in the presence
or absence of inhibitor. The reaction was allowed to incubate for
1 h at RT. The kinase reaction was terminated and phospho-
ATF2 was revealed by addition of 30 μL of HTRF detection
buffer (100 mM HEPES-pH 7.5, 100 mM NaCl, 0.1% BSA,
0.05% Tween-20, and 10 mM EDTA) supplemented with
0.1 nM Eu-anti-pTP and 4 nM SA-APC. After 1 h incubation
at RT, the assay plate was read in a Discovery Plate Reader
(Perkin-Elmer). The wells were excited with coherent 320 nm
light and the ratio of delayed (50 ms post excitation) emissions at
620 nm (native europium fluorescence) and 665 nm (europium
fluorescence transferred to allophycocyanin - an index of sub-
strate phosphorylation) was determined.19
LPS-Induced TNFr Production in THP-1 Cells. THP1 cells
were resuspended in fresh THP1 media (RPMI 1640, 10% heat-
inactivated FBS, 1ꢀ PGS, 1ꢀ NEAA, plus 30 μM βME) at a
concentration of 1.5 ꢀ 106 cells per mL. One hundred microliters
of cells per well were plated in a flat bottom polystyrene 96-well
tissue culture plate. Two micrograms per milliliter of bacterial
LPS (Sigma) was prepared in THP1 media and transferred to
the first 11 columns of a 96-well polypropylene plate. Column 12
contained only THP1 media for the LO control. Compounds
were dissolved in 100% DMSO and serially diluted 3-fold in a
polypropylene 96-well microtiter plate (drug plate). Columns 6
and 12 were reserved as controls (HI control and LO control,
respectively) and contained only DMSO. One microliter of
inhibitor compound from the drug plate followed by 10 μL of
LPS was transferred to the cell plate. The treated cells were
induced to synthesize and secrete TNFR in a 37 °C humidified
incubator with 5% CO2 for 3 h. TNFR production was
determined by transferring 50 μL of conditioned media to a
96-well small spot TNFR plate (MSD - Meso Scale Discovery)
containing 100 μL of 2ꢀ Read Buffer P supplemented with an
anti-TNFR polyclonal Ab labeled with ruthenium (MSD-
Sulfo-TAG - NHS ester). After overnight incubation at RT
with shaking, the reaction was read on the Sector Imager 6000
(MSD). A low voltage was applied to the ruthenylated TNFR
immune complexes, which in the presence of TPA (the active
component in the ECL reaction buffer, Read Buffer P),
resulted in a cyclical redox reaction generating light at 620
nm. The amount of secreted TNFR in the presence of com-
pound compared with that in the presence of DMSO vehicle
alone (HI control) was calculated using the formula: % con-
trol (POC) = (compd - average LO)/(average HI - average
LO)*100.
Acknowledgment. We thank Dr. Randy Jensen and Chris
Wilde for providing NMR analyses. We also acknowledge all
of our colleagues on the p38 MAP kinase research team.
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