Part 2: Structure-activity relationship (SAR) investigations of fused pyrazoles as potent, selective and orally available inhibitors of p38α mitogen-activated protein kinase

Article abstract of DOI:10.1016/j.bmcl.2010.01.059

A novel class of pyrazolopyridazine p38α mitogen-activated protein kinase (MAPK) inhibitors is disclosed. A structure activity relationship (SAR) investigation was conducted driven by the ability of these compounds to inhibit the p38α enzyme, the secretion of TNFα in a LPS-challenged THP1 cell line and TNFα-induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted in the discovery of several inhibitors with IC₅₀ values in the single-digit nanomolar range in hWB. Further investigation of the pharmacokinetic profiles of these lead compounds led to the identification of three potent and orally bioavailable p38α inhibitors 2h, 2m, and 13h. Inhibitor 2m was found to be highly selective for p38α/β over a panel of 402 other kinases in Ambit screening, and was highly efficacious in vivo in the inhibition of TNFα production in LPS-stimulated Lewis rats with an ED₅₀ of ca. 0.08 mg/kg.

Full text of DOI:10.1016/j.bmcl.2010.01.059

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1680–1684
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Part 2: Structure–activity relationship (SAR) investigations of fused pyrazoles
as potent, selective and orally available inhibitors of p38
a
mitogen-activated protein kinase
a
b
b
b
b
Ryan P. Wurz a, , Liping H. Pettus , Bradley Henkle , Lisa Sherman , Matthew Plant , Kent Miner ,
Helen J. McBride b, Lu Min Wong b, Christiaan J. M. Saris b, Matthew R. Lee c, Samer Chmait c,
Christopher Mohr c, Faye Hsieh d, Andrew S. Tasker a
*
a Department of Chemistry Research & Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b Department of Inflammation, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
c Department of Molecular Structure, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
d Department of Pharmacokinetics & Drug Metabolism, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of pyrazolopyridazine p38
a mitogen-activated protein kinase (MAPK) inhibitors is dis-
Received 22 November 2009
Revised 6 January 2010
Accepted 11 January 2010
Available online 21 January 2010
closed. A structure activity relationship (SAR) investigation was conducted driven by the ability of these
compounds to inhibit the p38 enzyme, the secretion of TNF in a LPS-challenged THP1 cell line and
TNF -induced production of IL-8 in the presence of 50% human whole blood (hWB). This study resulted
a
a
a
in the discovery of several inhibitors with IC50 values in the single-digit nanomolar range in hWB. Further
investigation of the pharmacokinetic profiles of these lead compounds led to the identification of three
Keywords:
p38
MAP kinase
TNF
potent and orally bioavailable p38
selective for p38 /b over a panel of 402 other kinases in Ambit screening, and was highly efficacious
in vivo in the inhibition of TNF
a inhibitors 2h, 2m, and 13h. Inhibitor 2m was found to be highly
a
a
production in LPS-stimulated Lewis rats with an ED50 of ca. 0.08 mg/kg.
Ó 2010 Elsevier Ltd. All rights reserved.
Pyrazolopyridazine
Orally active small molecules that modify the pro-inflammatory
cytokine release associated with rheumatoid arthritis (RA) have
generated considerable interest in the pharmaceutical industry.1
Small molecules potentially offer a cost-effective and convenient
alternative to biologics for patients and healthcare providers alike.2
In particular, the intracellular inhibition of a number of serine/
threonine and tyrosine kinases is the subject of multiple ongoing
phase II clinical trials.1
goal of our p38 program was to improve further upon kinase selec-
tivity and this led to the exploration of structurally related7 chem-
otypes to the pyrazolopyridinone class of inhibitors (1). Herein we
report significant progress made towards this goal following the
discovery of the novel pyrazolopyridazine class of p38a inhibitors,
exemplified by compound 2m.
Compounds necessary for the structure–activity relationship
(SAR) study could be accessed in an expedient fashion using the
two general routes illustrated in Schemes 1 and 2. Thus, eth-
oxy(methylene) compound (4) was prepared upon treatment of 38
with ethyl orthoformate (69–89%, Scheme 1).9 Condensation of 4
with the desired aryl hydrazine furnished pyrazole 5 in modest
p38a MAP kinase belongs to the serine/threonine family of ki-
nases and is intimately involved in the signaling cascade responsi-
ble for the development of inflammation.3 Increased activity of the
p38 enzyme results in cytokine overproduction (especially IL-1b
and TNF
as RA, inflammatory bowel disease and psoriasis. p38 exists in four
isoforms ( , b, d, and ) of which p38 is believed to be the pre-
dominant isoform involved in inflammation.4 Since p38
is an
integral component in the regulation of these processes, it has
emerged as an attractive target for small molecule drug discovery.5
Recent efforts in Amgen’s p38 program have been focused on a
novel pyrazolopyridinone scaffold (1, Fig. 1).6 The longstanding
a, which is a hallmark of inflammatory disorders, such
Cl
a
c
a
Cl
H
a
N
H
HN
N
HN
O
O
N
N
N
N
N
N
O
F
N
1
2m
F
p38α IC50 = 1.0 nM
p38α IC50 = 1.4 nM
F
F
* Corresponding author. Tel.: +1 805 313 5400; fax: +1 805 480 1337.
E-mail address: rwurz@amgen.com (R.P. Wurz).
Figure 1. Fused pyrazole-derived p38 inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.059

R. P. Wurz et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1680–1684
1681
(7, 8–42%)9 necessary for either the Buchwald–Hartwig amination
(15–87%)11 or etherification (20–60%)12 furnishing p38 inhibitors 2.
Building on the SAR established in the pyrazolopyridinone ser-
ies6 preliminary efforts were focused on optimization of the aryl
group of the N-1 position of the pyrazolopyridazine ring, the linker
atom of the benzamide top ring X, and R.1 Our SAR investigation
CO2Et
OEt
OEt
OEt
i
ii
OEt
OEt
N
EtO
CO2Et
EtO
CO2Et
N
O
O
Ar
3
4
5
R1
O
Cl
H
N
X
iii
iv
v
was driven using the inhibition of p38a, the potency in lipopoly-
NH
N
N
N
N
O
saccharide (LPS) challenged TNF production in a THP1 cell line
a
N
N
N
N
N
N
and in TNF
a stimulated IL-8 secretion in the presence of 50% hu-
Ar
Ar
N
6
7
2
man whole blood (hWB, Table 1) assays results.13
Ar
In a similar manner to the SAR observed in the pyrazolopyridi-
none class of p38 inhibitors,6 the substitution pattern at the N-1
pyrazolopyridazine aryl group had only a small influence on the
Scheme 1. General synthetic route used to access aniline NH- and O-linked
pyrazolopyridazine benzamides 2. Reagents and conditions: (i) Ac2O, (EtO)3CH,
130 °C; (ii) ArNHNH2, EtOH, 0 °C to rt; (iii) NH2NH2, AcOH, 90 °C; (iv) POCl3, 100 °C;
(v) X = NH: R–NH2, Pd2(dba)3, Dave-Phos, LiHMDS, THF, 60 °C; X = O: R–OH,
potency of the inhibitor in the p38a enzyme assay (Table 1, e.g.,
Pd2(dba)3, t-Bu2X-Phos, K3PO4, PhMe/THF 5:1,
lwave, 120 °C.
entries 2a, 2c, 2d, 2h, 2k, 2m). This enzyme potency generally
translated well into hWB potency with the most potent inhibitors
favoring 2,4-Di-F-Ph (2e–j) or 2,6-Di-F-Ph (2l–n) as N-1 aryl sub-
stituents. The slight dependence on potency of the N-1 aryl substi-
tution pattern may be related to the preference for a twisted
spatial orientation of this ring with respect to the pyrazolopyrid-
azine scaffold, favoring ortho-substitutions on the N-1 aryl group
that allow for enhanced dispersion interactions with the hydro-
phobic Ala157 residue on the floor of the binding pocket (for more
details see Fig. 2).
ArNHNH2.HCl
CO2Et
CO2Et
CO2Et
O
iii-iv
i
ii
CN
N
N
N
NH2
I
N
N
N
EtO
v
CO2Et
Ar
Ar
Ar
8
9
10
R2
R1
X
O
Cl
H
N
vi
vii
NH
N
N
N
N
N
N
The nature of the amide portion of the inhibitor also had a small
influence on the compound’s potency.6 A primary amide (2e)
proved to be fourfold less potent than its corresponding cyclopro-
O
N
N
N
N
Ar
Ar
N
11
12
13
Ar
pyl amide analogue (2f) in the p38a enzyme assay. The introduc-
tion of an ortho-chloro group (R1 = Cl) as a replacement for an
ortho-methyl group (R1 = Me) on the benzamide portion of the
inhibitor was generally well tolerated, resulting in equipotent
compounds (e.g., 2b vs 2c, 2f vs 2h, 2i vs 2j, and 2l vs 2m). How-
ever, introduction of a fluorine atom (R1 = F) in this position re-
sulted in a decline in both enzyme and cellular potency (2g),
which is consistent with previous findings.6 Replacement of the
benzamide tether atom from X = NH with X = O invariably led to
a decline in hWB potency which is also consistent with the trends
observed in the pyrazolopyridinone class of p38 inhibitors.6
Scheme 2. General synthetic route used to access the aniline NH- and O-linked
pyrazolo-7-methylpyridazine benzamides 13. Reagents and conditions: (i) Et3N,
EtOH, reflux; (ii) isopentylONO, I2, CHCl3, reflux; (iii) PdCl2[P(o-tol)3]2, 1-eth-
oxyvinylSnBu3, 120 °C; (iv) HCl, rt; (v) NH2NH2, EtOH, reflux; (vi) POCl3, PhCl, 90 °C;
(vii) X = NH: R–NH2, Pd2(dba)3, Dave-Phos, LiHMDS, THF, 60 °C; X = O: R–OH,
Pd2(dba)3, t-Bu2X-Phos, K3PO4, PhMe/THF 5:1, lwave, 120 °C.
yields (40–76%).8 Treatment with hydrazine in acetic acid which re-
sulted in deprotection of the acetal and cyclization to the desired
pyrazolopyridazinone 6 (36–72%).10 Chlorination of 6 with phos-
phorus oxychloride afforded the common chloride coupling partner
Table 1
SAR of modifications to Ar, X and R1a
R1
X
H
N
O
N
N
1
N
N
2
Ar
Compd
Ar
R1
X
p38a
IC50 (nM)
THP1 LPS/TNF
a
IC50 (nM)
hWB TNFa/IL-8 IC50 (nM)
2a
2b
2c
2d
2ec
2f
2g
2h
2i
2j
2k
2l
2m
2n
2-Cl-Ph
3-F-Ph
3-F-Ph
4-F-Ph
Cl
Me
Cl
NH
NH
NH
NH
NH
NH
NH
NH
O
1.6
4.6
5.0
8.5
12
3.0
43
3.3
10
4.9
18
19
25
14
2.8
64
3.7
8.7
52
8.8
2.1
1.7
7.9
26b
76b
103b
95b
30
Cl
2,4-Di-F-Ph
2,4-Di-F-Ph
2,4-Di-F-Ph
2,4-Di-F-Ph
2,4-Di-F-Ph
2,4-Di-F-Ph
2,5-Di-F-Ph
2,6-Di-F-Ph
2,6-Di-F-Ph
2,6-Di-F-Ph
Me
Me
F
Cl
Me
Cl
Cl
Me
Cl
7.8b
176
4.5b
56
O
36
371b
30b
2.6b
6.8
NH
NH
NH
O
2.1
1.7
1.4
3.0
Me
21b
a
b
c
The IC50 data are mean values derived from at least three independent dose–response curves. Variability around the mean value was <50%.
Data represents an average of two dose–response curves.
Compound corresponds to a primary amide instead of a cyclopropyl amide.

1682
R. P. Wurz et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1680–1684
Introduction of the 7-methyl group onto the pyrazolopyridazine
scaffold led to a modest increase in the potency of the inhibitor
resulting in several analogues with low single-digit nanomolar
IC50 values in the hWB TNFa/IL-8 assay (Table 2, e.g., 2d vs 13b
and 2m vs 13h). This improvement in potency could be attributed
to the addition of favorable van der Waals interactions with the
hydrophobic pocket, which may result from added stabilization
of an orthogonal orientation of the pyrazolopyridazine aryl group
in the aforementioned conformation that engages Ala157 with
ortho-substituents.
The excellent in vitro potencies of p38 inhibitors 2h, 2l, 2m, and
13h prompted further profiling of their pharmacokinetic (PK)
parameters in order to differentiate between these compounds
for further studies (Table 3). The compounds displayed moderate
to good oral bioavailability (%F) following oral dosing and excellent
exposure (AUC) with the exception of compound 2l. Moderate to
low clearance was also observed for all four compounds. Com-
pounds 13d and 13h containing a methyl group at C-7 were found
to have elevated levels of human pregnane receptor X (hPXR)
transactivation17 (11% and 31% @ 2
testing was halted. This compared unfavorably with compound
2m, which lacked this substitution (hPXR 7% @ 2 M).
The X-ray co-crystal structure of compound 2m bound to
unphosphorylated p38
was obtained (Fig. 2)18 and revealed several
lM, respectively) and further
l
Figure 2. X-ray co-crystal structure of compound 2m with unphosphorylated
p38a.
a
key binding interactions. The NH of the Met109 linker engages in a
hydrogen bonding interaction with N-2 of the pyrazole (3.3 Å), while
the N-1 aryl group is projected into the hydrophobic pocket near the
hinge region of the enzyme. The floor of this pocket contains an
Ala157 residue, which according to the kinome multiple sequence
alignment of Manning et al.19 is smaller than the corresponding res-
idue in over 98% of the catalytically active protein kinases. As dis-
cussed previously, this residue engages in a tight van der Waals
contact with one of the ortho-fluorophenyl substituents (3.2 Å).6
The ‘gate-keeper’ residue, Thr106, is internally hydrogen-bonded
to the carbonyl of His107, while the oxygen lone pair participates
in a hydrogen bond with the aniline NH (2.9 Å). The cyclopropyl
amide forms two key interactions with Asp168 and Glu71. The
amide carbonyl forms a hydrogen bond to the NH of Asp168 (2.9
Å), while the carboxylate of Glu71 forms a hydrogen bond with the
NH of the cyclopropyl amide (3.1 Å). The ‘DFG in’ configuration (res-
idues Asp168, Phe169, Gly170) is observed as expected.20
In an attempt to improve further upon the cellular potency of
compounds in this series, a methyl group was introduced at the 7-
position of the pyrazolopyridazine ring. These compounds were ac-
cessed using a seven step sequence depicted in Scheme 2. Condensa-
tion of ethyl (ethoxymethylene) cyanoacetate with the desired aryl
hydrazine in ethanol allowed access to 5-aminopyrazole carboxyl-
ate esters (8) in excellent yields (88–94%).14 Diazotization of the
amine with isopentyl nitrite and iodine in refluxing chloroform fur-
nished the corresponding 5-iodopyrazole esters (9) in moderate
yields (40–78%).15 Subsequent Stille coupling of the iodide (9) with
tributyl(1-ethoxyvinyl)tin followed by acid hydrolysis afforded the
desired 5-acetyl-1H-pyrazole-4-carboxylic acid ethyl ester (10,
61–64% over 2 steps).16 Cyclization of 10 with hydrazine furnished
pyrazolopyridazinone 11 (59–82%) which was chlorinated as in
Scheme 1 furnishing the common chloride coupling partners (12,
8–83%) for the Buchwald–Hartwig amination with the desired ani-
line (45–66%)10 or etherification with the desired phenol (30–
33%).11
Given the superior pharmacokinetic properties to other analogs,
compound 2m was chosen for further profiling. When screened at
10
l
M concentration, it was clean against a broad panel of GPCRs
Table 2
SAR of modifications to X, R1, R2 and Ara
R2
R1
X
H
N
O
N
N
1
7
N
13
N
Ar
Compd
Ar
R1
R2
X
p38
a
IC50 (nM)
THP1 LPS/TNF
a
IC50 (nM)
hWB TNFa
/IL-B IC50 (nM)b
13a
13b
13c
13d
13e
13f
4-F-Ph
4-F-Ph
4-F-Ph
2,4-Di-F-Ph
2,4-Di-F-Ph
2,4-Di-F-Ph
2,4-Di-F-Ph
2,6-Di-F-Ph
Me
Cl
Me
Me
Me
Cl
H
H
H
H
F
H
H
H
NH
NH
O
NH
NH
NH
O
4.1
2.3
7.7
2.7
10
1.6
6.3
1.7
5.0
4.9
8.2
1.7
9.4
2.1
8.4
3.2
21
13
46
2.6
11
3.8
23
1.8
13g
13h
Me
Cl
NH
a
The IC50 data are mean values derived from at least three independent dose–response curves. Variability around the mean value was <50%.
Data represents an average of two dose–response curves.
b

R. P. Wurz et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1680–1684
1683
Table 3
Acknowledgments
Mean pharmacokinetic parameters for a series of compounds in male Sprague–
Dawley ratsa
We acknowledge Randall Hungate and Terry Rosen for their
support.
Compd
iv (2.0 mg/kg in DMSO)
po (2.0 mg/kg)b
CL ((L/h)/kg) Vdss (L/kg) t1/2 (h) AUC(0-inf.) (ngÁh/ml) F (%)
References and notes
2h
2l
2m
13h
0.16
0.58
0.22
0.47
0.89
1.6
1.1
3.5
2.8
3.6
2.2
6550
671
6827
3883
53
19
75
91
1. Stanczyk, J.; Ospelt, C.; Gay, S. Curr. Opin. Rheum. 2008, 20, 257.
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1.4
a
3. Schett, G.; Zwerina, J.; Firestein, G. Ann. Rheum. Dis. 2008, 67, 909.
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Values are for an average of three rats.
Vehicle: 1% Pluronic F68, 1% HPMC, 15% hydroxypropyl b-cyclodextrin, 83%
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5. For recent reviews on the design of small molecule inhibitors for p38 MAP
kinase, see: (a) Pettus, L. H.; Wurz, R. P. Curr. Top. Med. Chem. 2008, 8, 1452; (b)
Wagner, G.; Laufer, S. Med. Res. Rev. 2006, 26, 1; (c) Westra, J.; Limburg, P. C.
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Curr. Med. Chem. 2005, 12, 763.
6. Wurz, R. P.; Pettus, L. H.; Xu, S.; Henkle, B.; Sherman, L.; Plant, M.; Miner, K.;
McBride, H.; Wong, L. M.; Saris, C. J. M.; Lee, M. R.; Chmait, S.; Mohr, C.;
McBride, H.; Hsieh, F.; Tasker, A. S. Bioorg. Med. Chem. Lett. 2009, 19, 4724.
and ion channels.21 With regard to kinase selectivity, in an Ambit
screen22 with a panel of 402 kinases, compound 2m (@ 1
lM) hit
only two kinases (POC <30%), p38a and p38b (IC50 = 4 nM, deter-
mined by Amgen). This exquisite kinome selectivity of 2m is con-
sistent with the tight van der Waals contact formed between its
ortho-F and the Ala157 floor residue, which is smaller than 98%
of the kinome.
7. For
a structurally related pyrazole-derived class of p38 inhibitors see:
Dyckman, A. J.; Das, J.; Leftheris, K.; Liu, C.; Moquin, R. V.; Wrobleski, S. T. US
Patent 20080004278 A1, 2008.
The in vivo efficacy of compound 2m was demonstrated in an
animal disease model of LPS induced TNF
a production in female
8. Tago, K.; Arai, M.; Kogen, H. J. Chem. Soc., Perkin Trans. 1 2000, 2073.
9. (a) Bisagni, E.; Bourzat, J.-D.; Marquet, J.-P.; André-Louisfert, J. Tetrahedron
1973, 29, 429; (b) Elbe, H.-L.; Gebauer, O.; Greul, J. N.; Hartmann, B.;
Wachendorff-Neumann, U.; Dahmen, P.; Kuck, K.-H. WO Patent 2005042493
A1, 2005.
10. (a) Michaely, W. J.; Curtis, J. K.; Knudsen, C. G. US Patent 5300478; (b) Marquet,
J.-P.; Bourzat, J.-D.; André-Louisfert, J.; Bisagni, E. Tetrahedron 1973, 29, 435.
11. Charles, M. D.; Schultz, P.; Buchwald, S. L. Org. Lett. 2005, 7, 3965.
12. Burgos, C. H.; Barder, T. E.; Huang, X.; Buchwald, S. L. Angew. Chem., Int. Ed.
2006, 45, 4321.
13. For a description of the biological assays, see: (a) 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; (b) Pettus, L. H.; Xu, S.;
Cao, G.-Q.; Chakrabarti, P. P.; Rzasa, R. M.; Sham, K.; Wurz, R. P.; Zhang, D.;
Middleton, S.; Henkle, B.; Plant, M. H.; Saris, C. J. M.; Sherman, L.; Wong, L. M.;
Powers, D. A.; Tudor, Y.; Yu, V.; Lee, M. R.; Syed, R.; Hsieh, F.; Tasker, A. S. J. Med.
Chem. 2008, 51, 6280.
14. Goldstein, D. M.; Alfredson, T.; Bertrand, J.; Browner, M. F.; Clifford, K.;
Dalrymple, S. A.; Dunn, J.; Freire-Moar, J.; Harris, S.; Labadie, S. S.; La Fargue, J.;
Lapierre, J. M.; Larrabee, S.; Li, F.; Papp, E.; McWeeney, D.; Ramesha, C.; Roberts,
R.; Rotstein, D.; Pablo, B. S.; Sjogren, E. B.; So, O.-Y.; Talamas, F. X.; Tao, W.;
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Lewis rats.23 Compound 2m was administered at 0.01, 0.03, 0.1,
and 0.3 mg/kg and resulted in an ED50 of ca. 0.08 mg/kg in a dose
dependent manner (Fig. 3). The plasma exposure at the ED50 level
is estimated to be between 5.5 and 5.9 ng/mL.
In conclusion, through an SAR investigation guided by previous
experience with the pyrazolopyridinone class (1) of p38
tors, a novel class of aniline NH- and O-linked pyrazolopyridazine
benzamide-derived p38 inhibitors has been identified. Several
compounds in this series were potent in the p38 enzyme, THP1
cell line and in TNF challenged IL-8 secretion assays in 50%
a inhibi-
a
a
a
hWB. Pharmacokinetic profiling revealed that inhibitors 2h, 2m
and 13h were orally bioavailable and displayed low clearance.
The LPS induced TNFa production in female Lewis rats was used
to evaluate compound 2m in vivo, which was found to be highly
efficacious with an ED50 of 0.08 mg/kg. In addition, compound
2m shows excellent selectivity across a 402 member panel of
kinases.
15. Beck, J. R.; Gajewski, R. P.; Lynch, M. P.; Wright, F. L. J. Heterocycl. Chem. 1987,
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16. Atkinson, R. N.; Drizin, I.; Gregg, R. J.; Gross, M. F.; Kort, M. E.; Shi, L. US Patent
20050020564 A1, 2005.
17. The compounds were tested in the PXR luciferase reporter gene assay using
HepG2 cells transfected with a luciferase reporter construct driven by human
CYP3A4 gene and human PXR cDNA. The incubation duration was 24 h.
18. The X-ray coordinates have been deposited in the RCSB Protein Data Bank
database (RCSB ID code: RCSB054861 and PDB ID code: 3ITZ). We also thank
the Advanced Light Source staff at beamline 5.0.2 for their support. The
Advanced Light Source is supported by the Director, Office of Science, Office of
Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of
Energy under Contract DE-AC03-76SF00098 at Lawrence Berkeley National
Laboratory.
19. Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science
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20. For leading references and examples see: (a) Bukhtiyarova, M.; Karpusas, M.;
Northrop, K.; Namboodiri, H. V. M.; Springman, E. B. Biochemistry 2007, 46,
5687; (b) Angell, R. M.; Angell, T. D.; Bamborough, P.; Bamford, M. J.; Chung, C.-
w.; Cockerill, S. G.; Flack, S. S.; Jones, K. L.; Laine, D. I.; Longstaff, T.; Ludbrook,
S.; Pearson, R.; Smith, K. J.; Smee, P. A.; Somers, D. O.; Walker, A. L. Bioorg. Med.
Chem. Lett. 2008, 18, 4433.
21. Compound 2m showed weak inhibition against most of the CYP 450 isoforms,
with IC50 >27
l
M for 3A4, 2D6, 2C9, 2C19, and 2D6; and moderate inhibition
against 1A2 (IC50 = 5.4
l
M). The plasma protein binding (determined by
ultrafiltration methods) was measured in four species: rat, human,
cynomolgus monkey, and mouse and found to be 97.1%, 97.3%, 97.6% and
96.7% protein bound, respectively.
Figure 3. Effect of 2m on LPS induced TNF
Vehicle or compound was dosed p.o. 60 min prior to injection with LPS IV (100
rat). Blood was collected 90 min later. Serum TNF levels were determined by ELISA
(Biosource). Data points represent mean STE (n = 6 rats/group): (*) p < 0.01 versus
vehicle control.
a
production in female Lewis rats.
22. Assays were performed by Ambit Biosciences (San Diego, CA: http://
lg/
www.ambitbio.com/) utilizing KINOMEscan. Activity is recorded via
a
a
competition binding assay of selected kinases that are fused to a proprietary
tag. Measurements of the amount of kinase bound to an immobilized, active-

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R. P. Wurz et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1680–1684
site directed ligand in the presence and absence of the test compound provide
M.; Patel, H. K.; Zarrinkar P. P.; Lockhart, D. J. Nat. Biotechnol. 2005, 23, 329.
Compound 2m was screened at 1 M and considered active if <30% of binding
to immobilized probes remained compared to DMSO control.
23. Hegen, M.; Keith, J. C., Jr.; Collins, M.; Nickerson-Nutter, C. L. Ann. Rheum. Dis.
2008, 67, 1505.
a% of DMSO control for binding of ligand. For more information on this method,
see: Fabian, M. A.; Biggs III, W. H.; Treiber, D. K.; Atteridge, C. E.; Azimioara, M.
D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen, P. T.; Floyd, M.; Ford, J. M.;
Galvin, M.; Gerlach, J. L.; Grotzfeld, R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.;
Lai, A. G.; Lélias, J.-M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L.
l