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The prevention of xenograft rejection is substantially dependent on inhibiting antibodies (Ab) produced by B-cells independently of T-cell signals (TI-1). Due to their ubiquitous biochemical mechanisms of action, the immunosuppressants currently employed not only fail to discriminate between B- and T-cells but also have a narrow therapeutic window and, thus, their prolonged use in complex immunosuppressive regimens is problematic. By capitalizing on the target enzyme-bound (DHODH) structure 1b of one of these compounds, leflunomide, and modulating part of its multiple mechanisms of action to gain selectivity, the quinoline-8-carboxamide 3 was designed as a potentially weak enzyme inhibitor but effective immunosuppressant. Compound 3 fulfilled the mechanistic criteria set and had 10-fold B-cell over T-cell selectivity. Its pyridyl analogue 4 was found to be a highly potent and selective B-cell immunosuppressant with a 75-fold selectivity for B- over T-cells (as judged by the MLR data) and no general cytotoxicity at concentrations up to 160-fold higher than those required to inhibit B-cells. In the mouse, 4 effectively blocked TI-1 Ab production and suppressed Ab-mediated xenograft rejection in a xenotransplantation model under a once-daily dosing regimen, with efficacy down to 0.3 mg/kg/day po. These are the first data demonstrating the feasibility of the development of drugs specific for impeding Ab production.

T-cell immunosuppressant-based therapies efficiently control early graft rejection in allotransplantation settings. They fail, however, to prevent those rejection events which are mediated by transplant-induced antibody (Ab) responses such as those involved in xenograft and chronic allograft rejection. This is mainly due to their inability to block T-cell-independent Ab production against the transplanted organs. The bioactive metabolite 2(Z) of leflunomide (1) inhibits the formation of such Ab, but the drug has pharmacokinetic properties and a therapeutic window incompatible with transplantation indications. Pyrazole 3, a constrained analogue of 2(Z), was designed and shown to be conformationally and biologically similar to 2(Z). Further investigations with derivatives of 3 demonstrated that the pyrazoles had very tight structure-activity relationships, the only equipotent compound being 3o. However, in contrast to 2(Z), both 3 and 3o were inactive in vivo due to short half-life and drug concentrations lower than the in vitro obtained IC50 values. Compound 3o inhibits T-cell-independent Ab production by a different biochemical mechanism from that of 2(Z) and 3 and may therefore represent a valuable tool for the identification of new targets for B-cell inhibition.

Analysis of the contact surface of the cyclophilin A (CypA)/cyclosporin A (CsA, 1) crystal structure delineates a unique cavity between both molecules in the vicinity of the Abu-2 side chain atoms of 1 (Abu pocket). Therefore, (5-hydroxynorvaline)-2-cyclosporin (2) was designed and prepared as a CsA derivative which could mediate additional interactions within the pocket. The X-ray crystal structure of the CypA/2 complex at 1.76 A resolution shows that 1 and 2 have identical backbone conformations and that the introduced hydroxypropyl chain makes indeed the expected supplemental interactions with CypA. However, 2 has 8-9-fold lower binding affinity than 1 for CypA. This results from a presumed unfavorable free energy change associated with the displacement of one of the tightly bound water molecules within the pocket and a change in prebinding equilibria. The role of the later was assessed by comparing the conformation distribution of 1 and 2 to that of norvaline-2-cyclosporin (3) and norvaline-2-(D-MeSer)-3-cyclosporin (4).