NF-B-induced gene expression contributes significantly to the pathogenesis of inflammatory diseases such as arthritis. I B kinase (IKK) is the converging point for the activation of NF-B by a broad spectrum of inflammatory agonists and is thus a novel target for therapeutic intervention. We describe a small molecule, selective inhibitor of IKK-2, SC-514, which does not inhibit other IKK isoforms or other serine-threonine and tyrosine kinases. SC-514 inhibits the native IKK complex or recombinant human IKK-1/IKK-2 heterodimer and IKK-2 homodimer similarly. IKK-2 inhibition by SC-514 is selective, reversible, and competitive with ATP. SC-514 inhibits transcription of NF-B-dependent genes in IL-1␤-induced rheumatoid arthritis-derived synovial fibroblasts in a dosedependent manner. When the mechanism of NF-B activation was evaluated in the presence of this inhibitor, several interesting observations were found. First, SC-514 did not inhibit the phosphorylation and activation of the IKK complex. Second, there was a delay but not a complete blockade in I B␣ phosphorylation and degradation; likewise there was a slightly slowed, decreased import of p65 into the nucleus and a faster export of p65 from the nucleus. Finally, both I B␣ and p65 were comparable substrates for IKK-2, with similar K m and K cat values, and SC-514 inhibited the phosphorylation of either substrate similarly. Thus, the effect of SC-514 on cytokine gene expression may be a combination of inhibiting I B␣ phosphorylation/degradation, affecting NF-B nuclear import/ export as well as the phosphorylation and transactivation of p65.

Adenosine has been suggested to induce bronchial hyperresponsiveness in asthmatics, which is believed to be an A(2B) adenosine receptor (AdoR) mediated pathway. We hypothesize that a selective, high-affinity A(2B) AdoR antagonist may provide therapeutic benefit in the treatment of asthma. In an attempt to identify a high-affinity, selective antagonist for the A(2B) AdoR, we synthesized 8-(C-4-pyrazolyl) xanthines. Compound 22, 8-(1H-pyrazol-4-yl)-1,3-dipropyl xanthine, is a N-1 unsubstituted pyrazole derivative that has favorable binding affinity (K(i) = 9 nM) for the A(2B) AdoR, but it is only 2-fold selective versus the A(1) AdoR. Introduction of a benzyl group at the N-1-pyrazole position of 22 resulted in 19, which had moderate selectivity. The initial focus of the SAR study was on the preparation of substituted benzyl derivatives of 19 because the corresponding phenyl, phenethyl, and phenpropyl derivatives showed a decrease in A(2B) AdoR affinity and selectivity relative to 19. The preferred substitution on the phenyl ring of 19 contains an electron-withdrawing group, specifically F or CF(3) at the m-position, as in 33 and 36 respectively, increases the selectivity while retaining the affinity for the A(2B) AdoR. Exploring disubstitutions on the phenyl ring of derivatives 33 and36 led to the 2-chloro-5-trifluoromethylphenyl derivative 50, which retained the A(2B) AdoR affinity but enhanced the selectivity relative to 36. After optimization of the substitution on the 8-pyrazole xanthine, 1,3-disubstitution of the xanthine core was explored with methyl, ethyl, butyl, and isobutyl groups. In comparison to the corresponding dipropyl analogues, the smaller 1,3-dialkyl groups (methyl and ethyl) increased the A(2B) AdoR binding selectivity of the xanthine derivatives while retaining the affinity. However, the larger 1,3-dialkyl groups (isobutyl and butyl) resulted in a decrease in both A(2B) AdoR affinity and selectivity. This final SAR optimization led to the discovery of 1,3-dimethyl derivative 60, 8-(1-(3-(trifluoromethyl) benzyl)-1H-pyrazol-4-yl)-1,3-dimethyl xanthine, a high-affinity (K(i) = 1 nM) A(2B) AdoR antagonist with high selectivity (990-, 690-, and 1,000-) for the human A(1), A(2A,) and A(3) AdoRs.

Recently, we have reported a series of new 1,3-symmetrically (R 1 = R 3) substituted xanthines ( 3 and 4) which have high affinity and selectivity for the human adenosine A 2B receptors (hA(2B)-AdoR). Unfortunately, this class of compounds had poor pharmacokinetic properties. This prompted us to investigate the effect of differential alkyl substitution at the N-1 and N-3 positions ( N 1-R not equal to N 3-R) on A(2B)-AdoR affinity and selectivity; we had the dual objectives of enhancing affinity and selectivity for the A(2B)-AdoR, as well as improving oral bioavailability. This effort has led to the discovery of compound 62, that displayed high affinity and selectivity for the hA(2B)-AdoR (K(i) = 22 nM). In addition, compound 62 showed high functional potency in inhibiting the accumulation of cyclic adenosine monophosphate induced by 5'- N-ethylcarboxamidoadenosine in HEK-A(2B)-AdoR and NIH3T3 cells with K(B) values of 6 and 2 nM, respectively. In a single ascending-dose phase I clinical study, compound 62 had no serious adverse events and was well tolerated.

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