Synthesis and biological activity of pyridopyridazin-6-one p38α MAP kinase inhibitors. Part 2

Article abstract of DOI:10.1016/j.bmcl.2012.07.035

This manuscript concludes the Structure Activity Relationship (SAR) on the pyridazinone scaffold and identifies a compound with subnanomolar p38α activity and 24 h coverage in the rat arthritis efficacy model.

Full text of DOI:10.1016/j.bmcl.2012.07.035

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5979–5983
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of pyridopyridazin-6-one p38
inhibitors. Part 2
a MAP kinase
Robert M. Tynebor a, , Meng-Hsin Chen a, Swaminathan R. Natarajan a, Edward A. O’Neill b,
⇑
James E. Thompson b, Catherine E. Fitzgerald b, Stephen J. O’Keefe b, James B. Doherty a
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
b Department of Inflammation Research, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 March 2012
Revised 3 July 2012
Accepted 6 July 2012
Available online 2 August 2012
This manuscript concludes the Structure Activity Relationship (SAR) on the pyridazinone scaffold and
identifies a compound with subnanomolar p38
model.
a
activity and 24 h coverage in the rat arthritis efficacy
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
p38a MAP kinase inhibitors
Pyridopyridazin-6-one
Heterocycles
Inflammation
Rheumatoid arthritis
Regulating over expression of pro-inflammatory cytokine TNF-
a
The initial step in optimizing the A-ring was remove the 6 fluo-
rine substituent of compound 1 to serve as base line activity for
assessing future modifications. Eliminating the fluorine at the 6 po-
(tumor necrosis factor) to treat such diseases as rheumatoid arthri-
tis, psoriatic arthritis, and inflammatory bowel disease has been a
well studied topic for two decades.1 Continuing our efforts to de-
sition led to a fourfold decrease in p38
a potency and hWB func-
velop a small molecule p38
a
inhibitor to reduce TNF-
a
levels2
tional activity shift for compound 5 (Table 1). As seen in the
we focused on expanding the Structure Activity Relationship
(SAR) of the previously published pyridopyridazin-6-one scaffold
quinolizin series, methyl ester intermediate 6 yielded potent single
nanomolar p38
tency.4 Transforming the ester (6) into 5-methyl-1,3,4-oxadiazol-
2-yl (7) led to equipotent p38 activity and similar THP and
a activity, but possessed a 20-fold shift in hWB po-
1 (Fig. 1).3 Our primary goal was to improve p38
a human whole
blood (hWB) potency and pharmacokinetic properties to drive effi-
a
cacy in the rat arthritis model while maintaining moderate levels
of p38a vs p38b isoform selectivity.
hWB results. Previously when a 5-methyl-1,3,4-oxadiazol-2-yl
was converted to a 5-methyl ester only a fourfold shift of hWB
activity was observed rather than 20-fold.4 Expanding the SAR at
During the process of optimizing the A-ring of 2H-quinolizin-2-
one 2 it was discovered that two main structural features were
necessary to yield an active compound in the THP-1 and hWB func-
tional assays with minimal off target activity.4 The first critical ele-
ment was a 5-methyl-1,3,4-oxadiazol-2-yl (3 and 4) that served to
reduce pregnane X receptor (PXR) activation while maintaining
p38a potency. The second important feature was a 2,5 substitution
pattern (4) on the A-ring that led to a major improvement in func-
tional activity over 2,4,6 substituted analog 3 (Fig. 2). Due to the
success of the A-ring optimization in the quinolizin-2-one series
a similar strategy was implemented to optimize the 2,6 substituted
A-ring of compound 1 while maintaining the p38a selective qual-
ities of the pyridopyridazin-6-one core.3
⇑
Corresponding author.
E-mail address: robert_tynebor@merck.com (R.M. Tynebor).
Figure 1. Pyridopyridazin-6-one scaffold.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.035

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R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5979–5983
Figure 2. A-ring evolution of 2H-quinolizin-2-ones 2–4.
Table 1
p38a Inhibitory activity of 2-F, 5-modified A-ring derivatives
Compound
R
p38a5 IC50 (nM)
p38b5 IC50 (nM)
a
/b ratio
THP-1/TNFa6 IC50 (nM)
hWB/TNFa5 IC50 (nM)
1
5
6
4.7
17
1.3
86
300
27
18
17
20
29.0
45
8.7
26
130
24
H
COOMe
7
8
9
2.3
32
9
66
400
200
28
12
22
19
120
43
45
330
290
10
99
3000
30
2000
n/a
the 5 position by synthesizing smaller primary and secondary
amides 8–10 only resulted in a significant loss in THP-1 and WB
activity.
Due to the large hWB activity shift of 2 fluoro A-ring pyridopy-
ridazin-6-ones 5–10, SAR at the 2 position was explored to
improve activity. Replacing the 2 fluoro with an ethenyl group
synthesized in either the quinolizin-2-one or pyridopyridazin-6-
one series (Table 2). Unfortunately, ester 11 still possessed a large
hWB activity shift and this trend was also observed for both amide
analogs 12–13. Ethyl derivatives 14–15 appear to maintain a lower
functional activity shift when compared to their ethenyl counter-
parts 12–13, but hWB activity was still too weak to warrant further
evaluation. Introducing a 2 methoxy group (17–19) further im-
(11) improved p38
a enzyme potency with respect to similar fluoro
analog 6 and resulted in the first sub nanomolar compound
proved p38
a and hWB potency with respect to similar analogs

R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5979–5983
5981
Table 2
p38 Inhibitory activity of 2-modified A-ring derivatives
a
Compound
R1
R2
p38a5 IC50 (nM)
p38b5 IC50 (nM)
a
/b ratio
THP-1/TNFa6 IC50 (nM)
hWB/TNFa5 IC50 (nM)
11
COOMe
0.58
7.7
13
5.2
40
12
13
14
15
3.3
2.3
5.8
9.9
53.5
36
16
80
18
41
47
290
16
31
23
150
100
44
180
230
16
17
OMe
OMe
H
8.8
0.58
140
9.3
16
16
120
4.4
430
17
COOMe
18
19
20
OMe
OMe
OMe
1.6
110
64
68
40
25
88
46
4.6
150
48
1.6
0.33
8.5
5.0
Table 3
11–15, while 5-methyl-1,3,4-oxadiazol-2-yl 20 produced subn-
anomolar p38 potency and improved hWB activity to 5 nM.
Although the p38 enzyme/hWB potency shift is 15-fold, 5 nM
hWB was a fivefold increase in potency over lead compound 1
and potent enough to warrant further studies.
Compounds 7 and 20 possessed the most desirable potency pro-
file and were further evaluated to assess pharmacokinetic proper-
ties and efficacy in the LPS induced arthritic model.4 When
Pharmacokinetic properties of compounds 7 and 20
a
Compounda,b Species
t1/2 (h) Cl (mL/min/kg) AUC (
lM h) F (%)
a
1
7
Ratc
1.1
4.4
4.5
11.6
2.0
2.8
2.0
4.9
4.1
3.4
3.6
20.0
2.1
96
93
15
31
87
87
60
Ratd
Doge,f
Monkeye,f 6.9
5.6
20
Ratc
1.4
3.0
3.8
6.4
7.6
6.3
Dogf
Monkeyf
compared to lead compound 1, fluoro derivative
7 had an
improved rodent PK profile with a half life to 4.4 h, clearance of
2.0 mL/min/kg, AUC of 20 uM⁄h, and 93% oral bioavailability (Table
3). However, analog 7 had poor dog and monkey oral bioavailabil-
ity (15% and 31%, respectively). The rat pharmokinetic profile of
methoxy derivative 20 resembled the profile of 1 but with a
slightly longer half life and better exposure, but more importantly
possessed similar oral bioavailability across all 3 species (60–87%).
Compounds 7 and 20 were tested in LPS induced rats to mea-
a
b
c
d
e
f
IV dose = 1.0 mg/kg.
PO dose = 2.0 mg/kg.
PO vehicle = PEG-200:water (70:30) (v:v).
PO vehicle = EtOH:PEG-200:water (20:30:50) (v:v).
IV dose = 0.25 mg/kg.
PO dose = 0.5% methylcellulose.
Table 4
sure their ability to reduce high levels of TNF-
4). As a reference, lead compound 1 failed to reduce TNF-
after administered and only displayed a marginal effect (30%) on
TNF- levels after 4 h with a 3 mg/kg dose. Optimizing 1 signifi-
cantly improved efficacy of the pyridopyridazin-6-one series by
reducing TNF levels 52% and 85% at the 24 h time point for com-
pounds 20 and 7, respectively. At the 4 h time point 20 reduced
TNF- by 80% compared to a 30% reduction by 1.
a
at 4 and 24 h (Table
Reduction of TNFa levels in the rat LPS induced arthritic
model at 4 and 24 h
a
24 h
Compounda
4 hb (%)
24 hb (%)
a
1
7
20
30
n/a
80
0
85
52
a
a
3 mg/kg PO dose.
% reduction with respect to vehicle.
b
a

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R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5979–5983
Scheme 1. Generic synthetic scheme for compounds 18–20.
Synthesis of pyridopyridazin-6-one derivative 20 started with a
of the TMS group followed by thermal cyclization generated the
pyridopyridazin-6-one platform 25 with a 2 step yield of 58%.7
The final step in constructing the A–B–C ring scaffold was to intro-
duce the C-ring through a SnAr reaction between the chloride of 25
and 2,4 difluoro phenol using cesium carbonate in NMP at 80 °C for
1 h. The first step in modifying the A-ring was to hydrolyze ester 17
to quantitatively yield acid 26. The crude acid was treated with
ethyl chloroformate to generate the anhydride intermediate 27,
NBS and benzoyl peroxide bromination of methyl 4-methoxy-3-
methylbenzoate 21 to afford bromide 22 in quantitative yield
(Scheme 1). Negishi coupling between bromide 22 and 2,5-dichlo-
ropyridazine yielded bis aryl methylene intermediate 23. Treating
23 with LiHMDS generated the benzylic anion in situ at À78 °C
and was quenched with TMS propenyl ethyl ester to afford the
protected diarylbutynone 24 in 61% from bromide 22. Deprotection

R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5979–5983
5983
4. Tynebor, R. M.; Chen, M-H; Natarajan, S. R.; O’Neill, E. A.; Thompson, J. E.;
Fitzgerald, C. E.; O’Keefe, S. J.; Doherty, J. B. Bioorg. Med. Chem. Lett. 2010, 20, 2765.
5. (a) Liverton, N. J.; Butcher, J. W.; Claiborne, C. F.; Claremon, D. A.; Libby, B. E.;
Nguyen, K. T.; Pitzenberger, S. M.; Selnick, H. G.; Smith, G. R.; Tebben, A.; Vacca,
J. P.; Varga, S. L.; Agarwal, L.; Dancheck, K.; Forsyth, A. J.; Fletcher, D. S.; Frantz,
B.; Hanlon, W. A.; Harper, C. F.; Hofsess, S. J.; Kostura, M.; Lin, J.; Luell, S.; O’Neill,
E. A.; Orevillo, C. J.; Pang, M.; Parsons, J.; Rolando, A.; Sahly, Y.; Visco, D. M.;
O’Keefe, S. J. J. Med. Chem. 1999, 42, 2180
which was immediately reacted with either hydrazine to yield
hydrazone 28 or an amine to yield amides 18–19. Crude hydrazone
28 was then heated in trimethyl orthoacetate to generate oxadiaz-
ole 20 with a 3 step yield of 58% from acid 26.
In summary, our research efforts completed SAR in the pyrido-
pyridazin-6-one series and led to the discovery of 2-methoxy, 5(-5-
methyl-1,3,4-oxadiazol-2-yl) phenyl as the optimal A-ring for the
pyridopyridazin-6-one scaffold. As a result of our efforts, com-
(b) A SPA-bead based assay was carried out using mouse p38. Compounds were
serially diluted into a 96 well plate containing a MOPS based p38 assay buffer.
The assay was initiated by addition of cold ATP, 33P ATP (gamma) and biotin
pound 20 possessed subnanomolar p38
a activity with moderate
labeled GST-ATF2 substrate (4 lM). After incubation at 30 °C for 3 h, the
p38b isoform selectivity while significantly improving cellular/
functional potency and efficacy in the rat LPS model over lead 1.
reaction was stopped by addition of a PBS based quench buffer with 2Â moles of
SPA beads over the amount of substrate used. The extent of phosphorylation of
GST-ATF2 was measured using
a topcount reader and subtracted from
background. IC50s are means of two experiments.
References and notes
6. Anti human TNF-
a was coated on immulon 4 plates. THP-1 cells
(density = 2.5 Â 106/mL) were suspended into 96-well plates containing a PBS
based medium. Compound was added as solution in DMSO followed by addition
1. (a) Palladino, M. A.; Bahjat, F. R.; Theodorakis, E. A.; Moldawer, L. L. Nat. Rev.
Drug Disc. 2003, 2, 736; (b) Saklatvala, J. Curr. Opin. Pharmacol. 2004, 4, 372; (c)
Kumar, S.; Boehm, J.; Lee, J. C. Nat. Rev. Drug Disc. 2003, 2, 717; (d) Pettus, L. H.;
Wurz, R. P. Curr. Topics Med. Chem. 2008, 8, 1452.
of LPS. The reaction was incubated for 4 h at 37 °C under CO2. TNFa release was
measured in the supernatants by ELISA. Reported IC50s are means from three
measurements.
7. Natarajan, S. R.; Chen, M.-H.; Heller, S. T.; Tynebor, R. M.; Crawford, E. M.;
Minxiang, C.; Kaizheng, H.; Dong, J.; Hu, B.; Hao, W.; Chen, S.-H. Tetrahedron Lett.
2006, 47, 5063.
2. Stoll, J. G.; Yasothan, U. Nat. Rev. Drug Disc. 2009, 8, 693.
3. Tynebor, R. M.; Chen, M.-H.; Natarajan, S. R.; O’Neill, E. A.; Thompson, J. E.;
Fitzgerald, C. E.; O’Keefe, S. J.; Doherty, J. B. Bioorg. Med. Chem. Lett. 2011, 21, 411.