Synthesis and biological activity of pyridopyridazin-6-one p38 MAP kinase inhibitors. Part 1

Article abstract of DOI:10.1016/j.bmcl.2010.10.128

The development and synthesis of potent p38α MAP kinase inhibitors containing a pyridazinone platform is described. Evolution of the p38α selective pyridopyridazin-6-one series from the p38α/β dual inhibitor 2H-quinolizin-2-one series will be discussed in

Full text of DOI:10.1016/j.bmcl.2010.10.128

Bioorganic & Medicinal Chemistry Letters 21 (2011) 411–416
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of pyridopyridazin-6-one p38 MAP kinase
inhibitors. Part 1
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:
The development and synthesis of potent p38
is described. Evolution of the p38a selective pyridopyridazin-6-one series from the p38a/b dual inhibitor
2H-quinolizin-2-one series will be discussed in full detail.
a
MAP kinase inhibitors containing a pyridazinone platform
Received 15 September 2010
Revised 21 October 2010
Accepted 25 October 2010
Available online 30 October 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
p38 inhibitors
Selective
Pyridopyridazin-6-one
Rheumatoid arthritis
Autoimmune driven diseases such as rheumatoid arthritis (RA),
psoriatic arthritis, and inflammatory bowel disease can be treated
by suppressing the activity of the proinflammatory cytokine tumor
p38a inhibitor by modifying the previously disclosed quinolizin-2-
one core (1)7 (Fig. 1).
In 2000, the discovery of VX-745 (2) introduced a new class of
heterobiaryl p38 inhibitors that possessed a high level of selectiv-
ity against a large spectrum of kinases.8 This enhanced selectivity
was a result of a novel structure-induced conformation or ‘flip’ of
necrosis factor (TNF-a
).1 The monoclonal antibodies Infliximab
(RemicadeÒ), Adalimumab (HumiraÒ), and the fusion protein Eta-
nercept (EnbrelÒ) are currently used as effective treatment op-
tions.2 An alternative therapeutic approach would be suppressing
p38
a/b Gly-110 and was critical in producing several structurally
the production of TNFa with small molecule inhibitors designed
diverse second generation inhibitors.9 VX-745 was also unique
to block the p38 mitogen-activated protein (MAP) kinase signal
transduction cascade.3 Although in vivo models indicate p38 is a
viable therapeutic target to reduce symptoms of RA, no small mol-
ecule therapy has successfully reached the market.2 This may be
due to the fact that the majority of small molecule p38 inhibitors
are only semi-selective, possessing minimal activity against p38d
due to a moderate 18-fold selectivity for the p38
a over the p38b
isoform.10
Merck developed a novel class of p38a selective cyclic ureas
represented by compound 3.11 Cyclic urea derivatives and 2 bind
by using a carbonyl based B-ring to induce a Gly-110 ‘flip’, occupy-
ing hydrophobic pocket 1 with a 2,6-dihalophenyl substituted
A-ring, and occupying hydrophobic pocket 2 with a 2,4-dihaloben-
zenethiol C-ring (Fig. 2). Quinolizin-2-one 1 lacks the thio ether
and p38
c
but binding equally well to p38
and p38b inhibitory activity is a
a
and p38b isoforms.4
The difficulty in separating p38
a
result of these isoforms having an ATP-inhibitor binding site that
is 97% similar in sequence.5 Recently it was reported that a dual
p38
duced TNF
p38
.6 These results demonstrated p38b has no role in modulating
TNF levels and suggest that inhibition of this isoform would offer
a/b inhibitor could not suppress lipopolysaccharide (LPS) in-
a
production in mice expressing T106 M modified
a
a
no positive therapeutic advantage in treating inflammation based
diseases. Our research therefore focused on developing a selective
⇑
Corresponding author.
E-mail address: robert_tynebor@merck.com (R.M. Tynebor).
Figure 1. Isoform selectivity of quinolizin-2-one 1, VX-745 (2), and urea 3.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.128

412
R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 21 (2011) 411–416
of compounds the 2,4 difluoro aryl group occupy the same hydro-
phobic pocket and therefore selectivity may be a result of the dif-
ferent B–C ring connectivity.
In order to introduce selectivity into the quinolizin-2-one series
a linker was installed at the 7 position and various attributes, such
as length and composition were explored (Table 1). Single atom
linkers 4–6 possessed moderate p38a activity and introduced an
average of 10-fold selectivity. Elongation of the linker region to a
methyl amine (7), ethyl (8), or ethylene (9) resulted in a substantial
loss in p38a potency and offered no selectivity advantage over sin-
gle atom linkers 4–6.
Although our initial screening led to compounds with moderate
p38a activity and enhanced selectivity, further changes to the mol-
ecule were necessary to increase potency and selectivity. Substi-
tuting the quinolizin-2-one core with a pyridopyridazin-6-one
core reduces the calculated Log D and therefore may improve
physical properties and functional activity.14 Another advantage
the pyridopyridazin-6-one core possessed is that 16 can serve as
a valuable intermediate for future synthetic manipulations.
Synthesis of quinolizin-2-one derivatives 4–9 began with a
Negishi coupling between 2,5-dibromopyridine and 2,6-difluorob-
enzylzinc bromide to yield diaryl methylene 11 (Scheme 1).
LiHMDS generated the dibenzylic anion of 11 at ꢀ78 °C and was
quenched with TMS propenyl ethyl ester to afford the protected
diarylbutynone 12 in moderate yield. The TMS protecting group
was removed and thermal cyclization generated the 2H-quinoli-
zin-2-one platform 14.15 Compounds 4, 5, and 7 were synthesized
using standard Ullmann procedures. Negishi coupling between 14
and 2,4-difluorobenzylzinc bromide yielded methylene linker 6.
Sonogashira coupling between bromide 14 and 1-ethynyl-2,4-
difluorobenzene and subsequent alkyne hydrogenation using Lind-
lar’s catalyst afforded alkene derivative 8, while alkyne hydrogena-
tion using 10% Pd/C yielded ethyl derivative 9.
Figure 2. Molecular modeling image of compound 3 binding to the p38
a active
site.13
Table 1
p38a/b Activity of 7-substituted quinolizin-2-one derivatives
Compound
X
p38a12 IC50 (nM)
p38b12 IC50 (nM)
a/b ratio
Pyridopyridazin-6-one analogs were synthesized using a similar
synthetic protocol described in Scheme 1 (Scheme 2). After the
2-chloro pyridopyridazin-6-one 16 core was synthesized the chlo-
ride was readily displaced with various nucleophiles or coupled
using synthetic protocols previously mentioned in Scheme 1 to
yield analogs 17–61 (Tables 2–4).
1
4
5
6
7
8
9
—
S
O
CH2
NHCH2
C2H2
C2H4
7
320
190
93
870
890
490
9
3100
1530
1300
10,100
5900
6610
1.3
9.7
8.1
13.9
11.6
6.6
13.4
Evaluating the B–C linker length of analogs 17–31 revealed a
similar trend observed in the quinolizin-2-one series (Table 1)
where potency dramatically decreased with an increase in linker
length (Table 2). This trend is clearly observed when the B–C linker
region was successively elongated from phenoxy 22 to phenyleth-
oxy 23. For each additional atom introduced into the linker region
linker and as a result the 2,4 difluorophenyl C-ring is shifted over
to the 6 position and therefore directly attached to the heterobiaryl
core. This different B–C ring connectivity produces quinolizin-2-
ones that only behave as dual p38a/b inhibitors. In both classes
Scheme 1. Synthesis of quinolizin-2-ones 4–9. Reagents and conditions: (a) Pd(PPh3)4, THF, reflux, 1 h, 46%; (b) LiHMDS, THF, ꢀ78 °C, 1 h, 51%; (c) TBAF, THF, 0 °C, 15 min,
60%; (d) TMEDA, 90 °C, 0.5 h, 50%; (e) 2,4-difluorophenol, 2,2,6,6-tetramethylheptane-3,5-dione, CuCl, Cs2CO3, NMP, 120 °C, 2 h, 62%; (f) 2,4-difluorobenzenethiol,
2,2,6,6-tetramethylheptane-3,5-dione, CuCl, Cs2CO3, NMP, 120 °C, 2 h, 68%; (g) bromo(2,4-difluorobenzyl)zinc, Pd(PPh3)4, THF, reflux, 1 h, 67%; (h) 1-(2,4-difluoro-
phenyl)methanamine, 2,2,6,6-tetramethylheptane-3,5-dione, CuCl, Cs2CO3, NMP, 120 °C, 2 h, 34%; (i) 1-ethynyl-2,4-difluorobenzene, TEA, CuI, Pd(PPh3)2Cl2, DMF, 100 °C, 4 h,
80%; (j) Lindlar catalyst, H2, MeOH, 1 h, 55%; (k) Pd/C, H2, MeOH, 1 h, 85%.

R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 21 (2011) 411–416
413
Scheme 2. Synthesis of pyridopyridazin-6-ones 17–61. Reagents and conditions: (a) X = O; Cs2CO3, phenol, NMP, 90 °C, 2 h; (b) X = S; NaH, benzenethiol, THF, rt, 15 min; (c)
X = CH2; Pd(PPh3)4, benzyl zinc bromide, THF, 80 °C, 30 min; (d) X = N; tris(dibenzylideneacetone)dipalladium, 1,10-bis(diphenylphosphino)ferrocene, sodium t-butoxide,
aniline, toluene, 80 °C, 3 h.
Table 2
p38a Inhibitory activity of 2 substituted pyridopyridazin-6-one derivatives
Compound
X
p38a12 IC50 (nM)
p38b12 IC50 (nM)
a/b
THP-1/TNFa16 IC50 (nM)
hWB/TNFa12 IC50 (nM)
17
3.2
33
10.3
—
17.9
29
18
19
720
4.7
>10,000
86
N/A
N/A
26
18.3
29.0
20
21
22
23
>10,000
1300
>10,000
>10,000
1800
—
N/A
N/A
505
N/A
N/A
N/A
N/A
N/A
—
160
11.3
—
>10,000
>10,000
24
25
1300
719
>10,000
>10,000
—
—
N/A
N/A
N/A
N/A
26
3.5
45
12.8
16.3
64
27
28
20
230
11.5
—
150
N/A
100
N/A
650
>10,000
29
30
31
480
1430
7.6
5500
>10,000
86
11.4
—
N/A
N/A
23.2
N/A
N/A
72
11.3

414
R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 21 (2011) 411–416
the potency decreased approximately 10-fold. The most selective
compound in the two atom linker series was benzylamine 27
(11.5-fold) but failed to improve selectivity with respect to the
quinolizin-2-one series.
of compound 5 positions the C-ring approximately 90° away from
hydrophobic pocket 2, while compound 19 it positions the C-ring
in a conformation more suitable to interact with hydrophobic
pocket 2 (Fig. 3). This difference in low energy conformations
may explain why the quinolizin-2-one series was significantly
more potent than the pyridopyridazin-6-one series.
Due to the enhanced potency and selectivity of ether derivative
19, the C-ring was further explored to determine the correlation
between substitution and potency/selectivity in the pyridopyrida-
zin-6-one series (Table 3). Fluoro and chloro substituted analogs
drastically favored ortho or para substitution over the meta posi-
tion. For example, 2-fluorophenyl 32 (12 nM) and 4-fluorophenyl
42 (48 nM) were 10–40 times more potent than 3-fluorophenyl
37 (420 nM), while 2-chlorophenyl 33 (44 nM), and 4-chloro-
phenyl 43 (79 nM) were 3-5 times more potent than 3-chloro-
phenyl 38 (240 nM). Methyl substituted derivatives followed a
different trend in which the 2-methylphenyl (34, 92 nM) and
Oxygen linker 19 possessed p38
greater than similar quinolizin-2-one derivative 5 and improved
p38 /b selectivity to 18-fold. Derivatives containing a thioether
a inhibitory activity 40-fold
a
(17) or a methylene B–C linker (31) also dramatically increased po-
tency with respect to quinolizin-2-ones 4 and 6, but failed to im-
prove selectivity. Since a dramatic shift in enzyme potency was
observed between quinolizin-2-one 5 and pyridopyridazin-6-one
19, both compounds were further investigated to understand the
role of the additional nitrogen present in 19.
Molecular modeling calculations minimized the rotational free-
dom associated with the ether linker and determined the energy
difference between the lowest energy conformation of 5 and 19
was 1.3 kcal/mol.13 As a result, the lowest energy conformation
Table 3
p38a Inhibitory activity for mono substituted C-ring derivatives 32–46
Compounda
a
b
c
p38a12 IC50 (nM)
p38b12 IC50 (nM)
b/
a
THP-1/TNFa16 IC50 (nM)
hWB/TNFa12 IC50 (nM)
22
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
—
F
—
—
—
—
—
—
F
Cl
Me
OMe
CF3
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
F
Cl
Me
OMe
CF3
160
12
44
92
82
2400
420
240
70
>10,000
>10,000
48
79
260
1800
180
360
520
110
11.3
15
8.2
5.6
1.3
505
67
N/A
171
352
N/A
475
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Cl
Me
OMe
CF3
—
—
—
—
—
—
—
—
—
102
386
172
N/A
977
351
376
N/A
N/A
160
170
N/A
N/A
N/A
N/A
3700
2067
660
N/A
N/A
1400
1900
1900
N/A
8.8
8.6
9.4
29
24
7.3
>10,000
>10,000
—
N/A
a
d = e = H for 22 and 32–46.
Table 4
p38 Activity for substituted C-ring derivatives
a
Compound
a
b
c
d
e
p38a12 IC50 (nM)
p38b12 IC50 (nM)
b/
a
THP-1/TNFa16 IC50 (nM)
hWB/TNFa12 IC50 (nM)
22
47
48
19
49
50
51
52
53
54
55
56
57
58
59
60
61
—
F
Cl
F
—
F
—
—
—
F
Cl
F
Cl
—
—
—
—
F
—
—
—
—
—
—
—
F
Cl
—
—
—
—
F
—
—
—
—
—
—
—
—
—
F
Cl
F
Cl
F
—
—
—
160
32
32
4.7
11
14
1800
300
220
86
180
11.3
9.4
6.8
18.3
16.3
9.3
7.3
13.1
5.0
12.6
6.8
12.0
4.6
505
97
61
29
27
52
57
92
>10,000
295
183
115
58
>10,000
>10,000
>10,000
>10,000
480
392
689
26
75
154
552
265
>10,000
>10,000
183
462
453
>10,000
>10,000
>10,000
>10,000
Cl
—
—
—
—
—
—
—
—
—
—
F
F
Cl
Cl
F
Cl
F
Cl
F
Cl
F
130
220
30
130
280
95
90
200
68
4300
270
140
260
1700
1400
1200
610
2400
310
>10,000
3300
1200
2700
F
F
F
F
—
12.3
8.6
—
—
—
F
Cl
Cl
—
—
—
Cl
10.4

R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 21 (2011) 411–416
415
Table 5
Pharmacokinetic properties of compounds 17, 19, and 31
Compounda,b
t1/2 (h)
Cl (mL/min/kg)
AUC (
lM h)
F (%)
17c
19d
31d
0.9
1.1
6.5
37.9
11.6
1.8
0.33
3.6
26.8
30
96
71
a
b
c
IV dose = 1.0 mg/kg.
PO dose = 2.0 mg/kg.
PO vehicle = PEG-200/DMSO/water 70:10:20 (v/v/v).
PO vehicle = PEG-200/water 70:30 (v/v).
d
Table 6
Reduction of TNF
a levels in rat LPS induced arthritic model at 4 and 24 h
Compounda
4 h (%)
24 h (%)
19
31
30
75
0
55
a
3 mg/kg PO dose.
Figure 3. Lowest energy conformation for compounds 5 and 19.
tional fluoro substituted derivatives such as trifluorophenyl 56 and
pentafluoro 58 derivatives failed to improve potency with respect
to the bis fluoro derivative 19.
Although disubstituted C-ring analogs yielded enhanced bind-
ing potency when compared to monosubstituted derivatives, selec-
tivity failed to improve. For example, 2,4-difluorophenyl analog 19
was 10-fold more potent than 42, but less selective. A similar trend
was also observed between 4-chlorophenyl analog (43) and 2,4-
dichlorophenyl analog (51).
3-methylphenyl (39, 70 nM) derivatives possessed similar potency
and were 3–4-fold more potent than 4-methylphenyl 44 (260 nM).
Analogs bearing the larger methoxy or trifluoromethyl groups
exhibited a large drop in activity regardless of substituent position.
Selectivity was enhanced with respect to the unsubstituted phenyl
22 only when a 2-fluorophenyl (32), 4-fluorophenyl (42), or 4-
chlorophenyl (43) were present. The 4-fluorophenyl analog (42)
possessed the highest p38a/b selectivity ratio (29-fold) and was
approximately threefold more selective than 22.
SAR of the C-ring indicated that only small hydrophobic groups
were tolerated at the 2 and 4 positions. Molecular modeling of 3 in
Multi substituted C-ring analogs followed a potency and selec-
tivity trend similar to that observed in the mono substituted series
(Table 4). ortho and para Substituted analogs consistently yielded
the highest potency when compared with other substitution pat-
terns. The addition of a 2 fluoro substituent to a 3 or 4 substituted
phenyl group improved potency approximately 10-fold for analogs
19, 47, and 49. A similar increase in potency was also observed
when a chlorine was added to the 2 position, but the potency shift
was less dramatic than analogous 2 fluoro analogs. Fluorines were
the p38
a crystal structure shows the small size of hydrophobic
pocket 2 suggesting an explanation for the SAR potency trend we
observed (Fig. 4).13 However, the overlapping crystal structure of
3 in p38
tivity trend. The residues that comprise hydrophobic pocket 2 are
identical for p38 and p38b and very little movement is observed
a and p38b failed to suggest an explanation for the selec-
a
when 3 binds. The only residue which moved significantly was
D168, but C-ring substitution in this region failed to improve selec-
tivity. This would imply that modifications in the B–C linker and C-
ring region only affect selectivity by causing other parts of the mol-
ecule to shift in the binding site with respect to dual inhibitors.
This hypothesis will be communicated in a separate manuscript.
critical for p38a binding potency and significantly improved whole
blood data when compared to chlorines. This trend in functional
potency was due to the increased hydrophobicity of the chloro
derivatives and their effects on physical properties. However, addi-
Figure 4. Model of 3 occupying hydrophobic pocket 2 (a) and 3 binding to superimposed p38a and p38b enzymes (b).

416
R. M. Tynebor et al. / Bioorg. Med. Chem. Lett. 21 (2011) 411–416
Figure 5. Calculated EC50 of 31 at 24 h in the LPS induced TNFa rodent model.
4. (a) Hynes, J.; Leftheris, K. Curr. Top. Med. Chem. 2005, 5, 967; (b) Goldstein, D.
M.; Kuglstatter, A.; Lou, Y.; Soth, M. J. J. Med. Chem. 2010, 53, 2345.
5. Patel, S. P.; Cameron, P. M.; O’Keefe, S. J.; Frantz-Wattley, B.; Thompson, J. B.;
O’Neill, E. A.; Tennis, T.; Liu, L.; Becker, J. W.; Scapin, G. Acta Crystallogr., Sect. D
2009, 65, 777.
6. O’Keefe, S. J.; Mudgett, J. S.; Cupo, S.; Parsons, J. N.; Chartrain, N. A.; Fitzgerald,
C.; Chen, S.-L.; Lowitz, K.; Rasa, C.; Visco, D.; Luell, S.; Carballo-Jane, E.; Owens,
K.; Zaller, D. M. J. Biol. Chem. 2007, 282, 34663.
Compounds 17, 19, and 31 possessed the best balance between
functional activity and selectivity and were further evaluated to
determine pharmacokinetic properties (Table 5). Composition of
the linker region played a major role in determining the pharmaco-
kinetic profile of pyridopyridazin-6-one derivatives. Oxygen deriv-
ative 19 significantly increased the AUC and bioavailability when
compared to the historical thioether linker of 17. Methylene linker
31 further improved the pharmacokinetic profile of 19 by increas-
ing the AUC an additional 10-fold, lowering clearance to 1.8 mL/
min/kg, and increasing t1/2 to 6.5 h.
7. 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.
8. (a) Bemis, G. W.; Salituro, F. G.; Duffy, J. P.; Harrington, E. M. U.S. Patent
6147,080, 2000; (b) Salituro, F.; Bemis, G.; Cochran, J. WO 99/64,400.
9. (a) Natarajan, R. S.; Doherty, J. B. Curr. Top. Med. Chem. 2005, 5, 987; (b)
Herberich, B.; Cao, G.-Q.; Chakrabarti, P. P.; Falsey, J. R.; Pettus, L.; Rzasa, R. M.;
Reed, A. B.; Reichelt, A.; Sham, K.; Thaman, M.; Wurz, R. P.; Xu, S.; Zhang, D.;
Hsieh, F.; Lee, M. R.; Syed, R.; Li, V.; Grosfeld, D.; Plant, M. H.; Henkle, B.;
Sherman, L.; Middleton, S.; Wong, L. M.; Tasker, A. S. J. Med. Chem. 2008, 51,
6271.
Compounds 19 and 31 were evaluated in the LPS induced ar-
thritic rodent model to assess their ability to decrease high levels
of TNF
levels by 75% while 19 only reduced levels by 30% after 4 h. At the
24 h time point 19 showed no apparent reduction in TNF levels
a at 4 and 24 h (Table 6). A 3 mg/kg dose of 31 reduced TNFa
a
10. In house data.
while 31 reduced levels by 55% with respect to the vehicle. Addi-
tional doses of 31 were tested to determine that the effective con-
11. Liu, L.; Stelmach, J. E.; Natarajan, S. R.; Chen, M. H.; Singh, S. B.; Schwartz, C. D.;
Fitzgerald, C. E.; O’Keefe, S. J.; Zaller, D. M.; Schmatz, D. M.; Doherty, J. B. Bioorg.
Med. Chem. Lett. 2003, 13, 3979.
centration needed to reduce TNFa by 50% (EC50) at the 24 h time
12. (a) Liverton, N. J.; Butcher, J. W.; Claiborne, C. F.; Claremon, D. A.; Libby, B. E.;
Ngyuen, 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; (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 labeled GST-ATF2 substrate
point was 2.4 mg/kg (Fig. 5). Although both compounds have sim-
ilar functional activity, the superior pharmacokinetic properties of
31 resulted in a higher degree of efficacy in the LPS rodent model.
Our efforts led to the discovery of a novel class of pyridopyrida-
zin-6-one derived p38
selectivity, excellent functional activity, and efficacy in the rat LPs
model. The process of transforming a p38 /b dual inhibitor into a
p38 selective inhibitor identified several important structural
a inhibitors that possessed moderate p38a/b
a
(4 lM). After incubation at 30 °C for 3 h, the reaction was stopped by addition
a
of a PBS based quench buffer with 2ꢁ moles of SPA beads over the amount of
substrate used. The extent of phosphorylation of GST-ATF2 was measured
using a top count reader and subtracted from background. IC50s are means of
two experiments.
traits necessary to balance selectivity and potency. The end result
of this research was the development of a structurally unique ser-
ies that could serve as a template for further optimization.
13. Computed using the mixed torsional/low-mode sampling algorithm as
implemented in Maestro v 9.02 from the software company, Schrodinger.
Used the MMFFs force field in the gas phase with a distance dielectric constant
of 2.
Acknowledgment
14. Calculated Log D values: 5 = 6.08 0.88, 19 = 4.53 0.90. ACDLabs 11.0.
15. 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.
The authors would like to thanks Dr. Georgia B. McGaughey for
all molecular modeling images presented in this Letter.
16. Anti human TNF-
a was coated on immulon 4 plates. THP-1 cells
(density = 2.5 ꢁ 106/mL) were suspended into 96-well plates containing
a
References and notes
PBS based medium. Compound was added as solution in DMSO followed by
addition of LPS. The reaction was incubated for 4 h at 37 °C under CO2. TNF
release was measured in the supernatants by ELISA. Reported IC50s are means
from three measurements.
a
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