328
R. M. Angell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 324–328
modification of the Cheng–Prusoff equation was used
(Cheng, H. C. Pharmacol. Res. 2005, 50, 21–40).
Ki = IC50*(1+((n[E])/Kd)) where n is the fraction of enzyme
competent to bind fluoroligand. Ki values for 381 com-
pounds in the fluorescence polarisation assay correlate with
those from an activity assay (details to be published) with
r2 = 0.9.
synthesis and activity of pro-inflammatory cytokines.
The compound shows dose-dependent anti-inflamma-
tory activity with an ED50 of 15 mg/kg. Figure 4 shows
results for dosing 17 at 3.3, 10 and 30 mg/kg. Also
shown are results for the vehicle and prednisolone at
4 mg/kg. At 30 mg/kg, activity is comparable to that of
4 mg/kg prednisolone.
3. Gas-phase optimisation of the three phenyltriazole tautom-
ers in the model compound 3-methyl-5-phenyl-1H-1,2,4-
triazole was performed using B3LYP DFT with a 6-31g**+
basis set in JAGUAR v4.0; Schro}dinger Inc.: Portland, OR,
2000. Triazole tautomer N4-H would be capable of
donating the H-bond to Glu71, but its enthalpy of
formation is 7.0 kcal/mol less stable than tautomer N2-H.
Tautomer N1-H is only 0.4 kcal/mol less stable than N2-H.
4. Human peripheral blood mononuclear cells (PBMCs) were
prepared from heparinised human blood from normal
volunteers by centrifugation on hystopaque 1077 at 1000g
for 30 min. The cells were collected from the interface,
washed by centrifugation (1300g, 10 min) and re-suspended
in assay buffer (RPMI1640 containing 10% foetal calf
serum, 1% L-glutamine and 1% penicillin/streptomycin) at
1 · 106 cells/ml. Fifty microliter cells were added to micro-
titre wells containing 50 ll of an appropriately diluted
compound solution (prepared from 10 mM stock in DMSO
by serial dilution in assay buffer giving maximally 0.1%
DMSO final). After 10–15 min incubation, lipopolysaccha-
ride (s. typhosa, sigma) was added giving 1 ng/ml final
concentration. The assay plates were incubated at 37 ꢁC,
5% CO2 for 18–20 h and cell free supernatants collected
following centrifugation at 800g. The supernatant was then
assayed for TNFa using a commercially available ELISA
(Pharmingen).
The in vivo activity is very encouraging for this new tem-
plate, especially considering its modest enzyme potency.
Furthermore, given the selective kinase inhibition profile
of this compound, it is likely that the in vivo activity is
driven by inhibition of p38.
In summary, compound 17 shows significant progress
over compound 1. It is a potent, selective inhibitor of
p38a with cellular activity, oral bioavailability and
activity in an in vivo model of joint inflammation. Fu-
ture publications will describe the continuing develop-
ment of the biphenyl amide series.
Acknowledgment
The authors thank Rick Williamson for suggestions and
for contributing experimental details.
References and notes
1. Angell, R. M.; Bamborough, P.; Cleasby, A.; Cockerill, S.
G.; Jones, K. L.; Mooney, C. J.; Somers, D. O.; Walker, A.
L. Bioorg. Med. Chem. Lett. 2008, 18, 318.
5. Pharmacokinetic parameters in male Lewis rats were
determined following intravenous (iv) and oral (po) admin-
istration at 1 and 3 mg/kg, respectively. Compound was
administered as a solution in 20% DMSO: 80% PEG200
(iv) or 5% DMSO: 40% Vit E: 40% PEG400: 15% Mannitol
(po). Blood was collected over a 24-h time period. Plasma
was prepared following centrifugation and compound
extracted from 50 lL plasma using protein precipitation
with acetonitrile. Samples were then evaporated under
nitrogen and re-suspended in 100 ll of 10:90 acetoni-
trile:water. Analysis was performed using LC–MSMS on
the API365 with a 5 min fast gradient comprising 0.1%
formic acid in water and 0.1% formic acid in acetonitrile
(mobile phases), 20ll injection volume, flow rate 4 ml/min
and ODS3 Prodigy column (5 cm · 2.1 mm, 5 lm). Phar-
macokinetic data were generated using PKTools.
6. PG–PS (peptidoglycan–polysaccharide from Streptococcal
cell walls) was injected intra-articularly and followed two
weeks later by a systemic reactivation using the same
bacterial cell wall component. The reactivated joint
swelling was measured for 3 days after systemic challenge
while dosing orally, twice daily, with the target
compound.
2. (a) Fluorescence polarisation assays used GST-tagged p38a
(activated using MKK6 and re-purified) or GST-tagged
truncated JNK3 (residues 39–402) and an ATP-competitive
rhodamine-green labelled fluoroligand (2-(6-amino-3-imi-
no-3H-xanthen-9-yl)-5-{[({4-[4-(4-Cl-3-hydroxyphenyl)-
5-(4-pyridinyl)-1H-imidazol-2yl]phenyl}methyl)amino]car-
bonyl}benzoic acid). These components were dissolved in a
buffer of final composition 62.5 mM Hepes, pH 7.5,
1.25 mM CHAPS, 1 mM DTT, 12.5 mM MgCl2, with final
concentrations of 12 nM of p38a or 50 nM JNK3 and
5 nM fluoroligand. Thirty microliters of this mixture were
added to wells containing 1 ll of test compound (0.28 nM–
16.6 lM) and incubated for 30–60 min at room tempera-
ture. Fluorescence anisotropy was read in a Molecular
Devices Acquest (excitation 485 nm/emission 535 nm); (b)
The Cheng–Prusoff equation (Cheng, Y.-C.; Prusoff, W. H.
Biochem. Pharmacol. 1973, 22, 3099–3108), Ki =
IC50*(1+([S]/Km)), was used to calculate Ki from deter-
mined IC50 for activity assays. To calculate Ki from
determined IC50 for fluorescence polarisation assays a