Design, Synthesis, and Evaluation of 9-d-Ribitylamino-1,3,7,9-tetrahydro-2,6,8-purinetriones Bearing Alkyl Phosphate and α,α-Difluorophosphonate Substituents as Inhibitors of Riboflavin Synthase and Lumazine Synthase

Abstract

Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport processes. To obtain structural probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a series of ribitylpurinetriones bearing alkyl phosphate and alpha,alpha-difluorophosphonate substituents were synthesized. Since the purinetrione ring system and the ribityl hydroxyl groups can be alkylated, the synthesis required the generation of these two moieties in protected form before the desired alkylation reaction could be carried out. These substances were designed as intermediate analogue inhibitors of lumazine synthase that would bind to its phosphate-binding site. All of the compounds were found to be effective inhibitors of both Bacillus subtilis lumazine synthase as well as Escherichia coli riboflavin synthase. Molecular modeling of the binding of 3-(1,3,7,9-tetrahydro-9-D-ribityl-2,6,8-trioxopurin-7-yl)propane 1-phosphate provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. Interestingly, the enzyme kinetics of these new compounds in comparison with the parent purinetrione demonstrated unexpectedly that the phosphate and phosphonate substituents contributed negatively to the binding. A possible explanation for these effects on lumazine synthase would be that the inorganic phosphate in the assay buffer competes with the substituted purinetriones for binding to the enzyme. This would be consistent with the observed increase in K(m) of the 3,4-dihydroxybutanone-4-phosphate substrate from 5.2 microM in Tris buffer or from 6.7 microM in MOPS buffer to 50 microM in phosphate buffer when tested on Bacillus subtilis lumazine synthase. However, when tested in Tris buffer vs Mycobacterium tuberculosis lumazine synthase, three of the phosphate inhibitors displayed inhibition constants in the 4-5 nM range, indicating that they are much more potent than the parent purinetrione. Under these conditions, the phosphate moieties of the inhibitors do contribute positively to their binding. The alpha,alpha-difluorophosphonate analogue, which is expected to have enhanced metabolic stability relative to the phosphates, was also found to be an inhibitor of Mycobacterium tuberculosis lumazine synthase with a K(i) of 60 nM.

Lumazine Synthase from Candida albicans as an Anti-fungal Target Enzyme

“…The design of those inhibitors has been based on the structures of both substrates of LS and the putative Schiff base intermediate of the enzymatic reaction (17,18). The synthesis of all compounds used in our experiments has been published earlier (20,21). Fig.…”

“…Because our experiments were performed in phosphate buffer, and the phosphate ion has been recognized as a component bound to the LS active site, we would like to emphasize that we in principle are dealing with a ternary binding reaction, involving a phosphate ion, an inhibitor molecule and free enzyme. It has been previously documented that with M. tuberculosis lumazine synthase, phosphate increases the apparent K m of the substrate 2 and decreases the overall reaction rate (20). This presumably occurs because inor- 1,3,7-trihydro-9-D-ribityl-2,4,8-purinetrione-7-yl (TS13) (a), 3-(1,3-dihydro-9-D-ribityl-2,4,8-purinetrione-7-yl)butane-1-phosphate (TS44) (b), 4-(6,7(5H,8H)-dioxo-8-D-ribityllumazine-5-yl)butane 1-phosphate (GJ43) (c), and [4-(6-chloro-2,4-dioxo-1,2,3,…”

“…At present the kinetic inhibition constants together with the experimentally determined complex structures with several substrate analogues, such as 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (11,18), 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (10,19), and 5-(6-D-ribitylamino-2,4(1H,3H)-pyrimidinedione-5-yl)1-pentylphosphonic acid (9,18), as well as the product analogues 6,7(5H,8H)-dioxo-8-ribityllumazine and 6-methyl-7(8H)-oxo-8-ribityllumazine (18), are available. Recently, a new series of compounds based on the aromatic purinetrione system and the ribityllumazinedione system bearing alkyl phosphate chains was designed by Cushman et al (20,21). In the present report we describe the threedimensional structure of lumazine synthase from the fungal pathogen C. albicans, the binding analysis of four inhibitors by isothermal titration calorimetry and the docking of those compounds into the CALS active site.…”

“…, and 4-(6-chloro-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-yl)-butyl (JC33) were prepared as described elsewhere (20,21).…”

Three-component process for the synthesis of 4-amino-5- pyrimidinecarbonitriles under thermal aqueous conditions or microwave irradiation

“…1,2 Structures containing such units often play an essential role due to of their biological activity, particularly in cancer and virus research. [3][4][5][6] Among these heterocycles, pyrimidine derivatives are an important class in pharmaceutical discovery. 7,8 Some such compounds are analgesics, 9 antihypertensives, 10 antipyretics, 11 and anti-inflammatory drugs.…”