Synthesis and immunomodulation of human lymphocyte proliferation and cytokine (interferon-γ) production of four novel leflunomide analogues.

Leflunomide is an immunomodulating drug that has been used clinically for the treatment of rheumatoid arthritis and other immune system disorders. As a continuation of our previous work, four novel analogues of leflunomide (6a, 6b, 7a and 7b) were synthesized; here, an imidazolyl group has replaced the isoxazolyl moiety, while the 4-trifluoromethylphenyl group has been retained. These analogues were synthesized and investigated in vitro for their immunomodulating activity by examining human lymphocyte proliferation and determining the cytokine interferon-γ concentrations in human lymphocyte cells. For this purpose, 5 x 10(4) human lymphocyte cells were incubated at 37°C in 5% CO(2) with phytohemagglutinin and one of the analogues (concentrations ranging from 1 to 100 mM), negative controls or cyclosporine (0.1 mM). The compounds' effects on lymphocyte proliferation and interferon-γ (IFN-γ) production were determined using an MTT assay and an ELISA, respectively. All compounds were found to have significant effects on both lymphocyte proliferation and IFN-γ production in comparison to the negative control. However, the compounds' effects were weaker than those of the positive control. Some differences among compounds 6a, 6b, 7a and 7b were seen on lymphocyte proliferation and cytokine production. Compound 6a (R=CH(3) containing a trifluoromethylaniline moiety) suppressed lymphocyte proliferation and IFN-γ production with a potency comparable to the positive control. Therefore, further studies to evaluate the compound's effects in clinical conditions are suggested.