br Conclusion br Competing interests br Authors contribution
Acknowledgements The work was supported by Aurigene Discovery Technologies (M) Sdn. Bhd., Industrial Doctoral Program sponsorship by Ministry of Higher Education, Malaysia and research grants by the University of Malaya, grant number PG038-2014A and UMRGRP027-15AFR.
Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that catalyzes the fourth step in the pyrimidine biosynthetic pathway, namely the conversion of dihydroorotate to orotate. Inhibitors of DHODH exhibit anti-inflammatory and immunosuppressant properties and have demonstrated to be efficacious in the treatment of autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Leflunomide and brequinar () are two examples of low-molecular weight inhibitors of DHODH that have been in clinical development., , , Leflunomide (Arava®, Sanofi-Aventis) is the only member of its class in the market. It is approved for the treatment of rheumatoid arthritis and psoriatic arthritis. Teriflunomide is the active metabolite of leflunomide and is currently in Phase III clinical trials for multiple sclerosis. However, teriflunomide has a low potency against human DHODH and a long half-life of approximately 2weeks in plasma, which could represent a serious obstacle for patients who need to withdraw the treatment in case of toxicity or pregnancy. Herein, we report our efforts to increase inhibitory potency against human DHODH enzyme and to adjust pharmacokinetic properties by reducing half-life. Our strategy to develop DHODH inhibitors was based on the recently published co-crystal structure of the human enzyme with teriflunomide. Analyses of this crystal () provided a good starting point for structure-based design of more potent inhibitors. The crystal revealed that trifluoromethyl phenyl group present in only partially filled the binding cavity that is mainly lined by hydrophobic amino GSK481 synthesis residues (). Initially, methyl group of teriflunomide was changed by ethyl and a two-fold increase in potency was observed. Then, in compound (), where a bulkier hydrophobic group was introduced to better fill the cavity, an improvement in in vitro hDHODH activity by an order of magnitude was obtained (). In addition, the iv half-life of was significantly reduced compared to , but was still perceived as too long having in mind the different behaviour observed with teriflunomide between rat (14h) and man (2weeks) (). The use of soft metabolic centres was then investigated as a mean to modulate and reduce . The idea of introducing a methyl ester led to compound . This molecule proved to be 15-fold more active than in in vitro hDHODH activity and a greatly modified iv PK profile in rat (). A concern with compound , however, was that it contained a potentially displaceable triflate in the β-position of an α,β-unsaturated carboxylate which could be prone to promote covalent binding to proteins. The triflate group in was put to good synthetic use, however, as a cross-coupling partner, which allowed a rapid assessment of SAR. Thus, introduction of a phenyl ring was effected using a Suzuki reaction and variation of the substituents on this ring were explored with respect to both enzymatic and cellular immunosuppressive activity (). A flexible synthetic route () was employed in the synthesis of these series of compounds. Thus, synthesis of intermediate was carried out by methylation of 2-hydroxy-5-nitrobenzoic acid , subsequent reduction of the nitro group to give the corresponding aniline and coupling of this aniline with cyanoacetic acid. Intermediate was treated with a triflating agent to give the cyanoacetamide , which was then acylated to afford the β-hydroxyenamide using sodium hydride and propionic anhydride. Finally, compounds – were synthesized by standard Suzuki coupling of compound and the corresponding boronic acid or boronic acid pinacol ester employing tetrakis(triphenylphosphine)palladium(0) as a catalyst. Most boronic acids were either commercially available or prepared by standard literature methods.