M. J. Laufersweiler et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4267–4272
4271
Table 4. Pharmacokinetic properties and in vivo data of selected
compounds
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W., Eds.; Elsevier: Amsterdam, 2001; Vol. 38, pp 1–60; (d)
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Pearson, G.; Xu, B.-e.; Wright, A.; Vanderbilt, C.; Cobb,
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TNF-a IC50
(nM)
Solubility t1=2 (h) %F
(mg/mL)
RIA (%
reduction)a
1
15
2
1.6
1.2
0.7
1.4
2.1
52.2
17.2
22.4
22.3
28
27
17
18
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2
0.04
0.7
12h
12j
36
9
0.81
a Percent reduction in joint damage as compared to vehicle control at a
dose of 25 mg/kg. Statistically significant at P < 0:05.
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pharmacokinetic properties of these molecules without
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Examination of the pharmacokinetics of selected com-
pounds prompted in vivo testing of three compounds
(Table 4). Compound 1 showed excellent solubility with
good bioavailability and an acceptable half-life in the
rat. Compound 2 was one of the most active compounds
tested, however it had very poor solubility, a fairly short
half-life, and only moderate bioavailablility. Compound
12h showed significant improvement in solubility, half-
life, and bioavailability however it was less active than
other compounds. Finally, compound 12j had excellent
whole cell activity (IC50 ¼ 9 nM), good solubility, the
longest half-life among the compounds tested, and
acceptable bioavailability. Compounds 1, 2, 12j, and 12h
were tested in the rat iodoacetate (RIA) model for
osteoarthritis15 and showed positive oral efficacy at a
dose of 25 mg/kg.
7. Clark, M. P.; Laughlin, S. K.; Laufersweiler, M. J.;
Bookland, R. G.; Brugel, T. A.; Golebiowski, A.; Sabat,
M. P.; Townes, J. A.; VanRens, J. C.; Djung, J. F.;
Natchus, M. G.; De, B.; Hsieh, L. C.; Xu, S. C.; Walter,
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8. Duplicate cultures of human monocytic cells (THP-1)9
cells (2.0 · 105/well) were incubated for 15 min in the
presence or absence of various concentrations of inhibitor
before the stimulation of cytokine release by the addition
of lipopolysaccharide (LPS, 2 lg/mL). The amount of
TNF-a released was measured 4 h later using an ELISA (R
& D Systems, Minneapolis, MN). The viability of the cells
after the 4 h incubation was measured using MTS assay10
(Promega Co., Madison, WI).
In summary, we have reported a novel series of substi-
tuted bicyclic pyrazolones that inhibit the release of
TNF-a in monocytic cells (THP-1). Efforts to eliminate
the chiral center in the substituted bicyclic pyrazolones
led to the development of spiroketal analogs. Excellent
potency was observed in both the dioxolane spiroketal
and the dioxane spiroketal with the dioxolane com-
pounds showing slightly higher potency. The potency
was preserved with benzyl amine substituents on the
pyrimidine ring even when no a-methyl chiral center was
present. We observed good oral bioavailability within
the series and described four compounds that displayed
oral efficacy (25 mg/kg) in the rat iodoacetate in vivo
model for osteoarthritis.
9. Mohler, K. M.; Sleath, P. R.; Fitzner, J. N.; Cerretti, D. P.;
Alderson, M.; Kerwar, S. S.; Torrance, D. S.; Otten-Evans,
C.; Greenstreet, T.; Weerawarna, K.; Kronhelm, S. R.;
Petersen, M.; Gerhart, M.; Kozlosky, C. J.; March, C. J.;
Black, R. A. Nature 1994, 370, 218–220.
Acknowledgements
10. Barltrop, J. A.; Owen, T. C.; Cory, A. H.; Cory, J. G.
Bioorg. Med. Chem. Lett. 1991, 1, 611–614.
We are grateful to: A. L. Roe, D. C. Ackley, A. M.
Walter, C. R. Dietsch, and D. M. Bornes for pharma-
cokinetic data; A. Greib for chiral HPLC separation;
and M. Buchalova for formulations and solubility data.
11. The mutated p38a herein described is a double mutant
(S180A, Y182F) of murine p38a.12 The mutant enzyme
cannot be phosphorylated and, therefore, it is not
competent for activation. Protein expression and purifica-
tion were carried out as previously described for the murine
enzyme.13 For crystallization, mutated p38a was incubated
overnight (12–16 h) with 1 mM compound. Co-crystals
were grown by hanging drop vapor diffusion using PEG as
a precipitating agent and overall protocols similar to those
previously described for the human enzyme.14 Crystals
References and notes
1. For recent reviews, see: (a) Palladino, M. A.; Bahjat, F.
R.; Theodorakis, E. A.; Moldawer, L. L. Nat. Rev. Drug
Discovery 2003, 2, 736–746; (b) Baugh, J. A.; Bucala, R.
Curr. Opin. Drug Discovery Dev. 2001, 4, 635–650; (c)
Adams, J. L.; Badger, A. M.; Kumar, S.; Lee, J. C. In
ꢀ
typically diffracted to 1.9 A resolution and were of the
ꢀ
previously reported space group: P212121; a ¼ 65:2 A,
0
ꢀ
ꢀ
b ¼ 74:6 A, c ¼ 78:1 A (4 ). X-ray data were collected at