Synthesis and immunomodulation of human lymphocyte proliferation and cytokine (interferon-gamma) production of four novel malonitrilamides.

Leflunomide is an immunomodulator drug with applications in the management of arthritis rheumatoid. In this study, four novel analogs (4a-d) of A771726, the active metabolite of leflunomide were synthesized and examined in vitro for their immunomodulation activity by examining human lymphocyte proliferation and determination of the cytokine interferon-gamma concentrations in human lymphocyte cell culture. For this purpose, 5 x 10(4) human lymphocyte cells were incubated at 37 degrees C in 5% CO(2) with phytohemagglutinin and one of the analogs (concentrations 1-100 mM), negative controls or cyclosporine (0.1 mM). Effects of the compounds on lymphocyte proliferation and interferon-gamma production were determined by MTT assay and enzyme-linked immunosorbent assay, respectively. Our results showed that all four compounds dose-dependently suppressed lymphocyte proliferation. Moreover, these compounds at some concentrations reduced interferon-gamma production which is an indicator of the immune response. Generally, the most potent analog was 4b with an amide linkage (X=NH) and the weakest analog was 4a with an ester linkage (X=O). Compound 4a has little similarity with the leflunomide active metabolite which has an amide linkage. In this study, four novel compounds were synthesized that showed considerable immunosuppressive effects that deserve further investigations.