The reactions of hexafluoropropene oxide (19), methyl trifluoropyruvate (21), and l,l,l-trifluorobutane-2,3-dione (45) with a series of ortho diamines were investigated as an approach to the synthesis of Muoromethyl-substituted quinoxalinones and lumazines. 6-(Trifluoromethyl)-7-oxo-8-(~-ribityl)lumazine (1 1) was synthesized by reaction of methyl trifluoropyruvate (21) with 5-amino-6-(~-ribitylamino)-2,4( lH,3H)-pyrimidinedione (3) hydrochloride and utilized as a 19F NMR probe of the light riboflavin synthase of Bacillus subtilis. The fluorolumazine 11 was found to be an inhibitor of the enzyme with an inhibition constant KI = 55 pM. Equilibrium dialysis experiments indicated the binding of six molecules of 11 per enzyme molecule, corresponding to one molecule bound at each of the three donor and three acceptor sites of the enzyme. The apparent dissociation constants KD were approximately 4 and 112 pM. The enzyme-bound ligand gave rise to several broad I9F NMR signals which were shifted to low field. The bound ligand 11 could be displaced from the enzyme by the enzyme product, riboflavin (2), and the product analog, 5-nitroso-6-(ribitylamino)-2,4(lH,3H)-pyrimidinedione (56). 6-(Trifluoromethyl)-7-methyl-8-(~-ribityl)lumazine (13) was synthesized by reaction of the hydrochloride salt of 3 with l,l,l-trifluorobutane-2,3-dione (45). Three molecules of 13 can be bound relatively tightly per mole of riboflavin synthase, i.e., one ligand molecule per protein subunit. The inhibition constant KI was determined to be 75 pM, while dissociation constants of 17 and 70 pM were determined by equilibrium dialysis and '9F NMR, respectively.The bound ligand 13 could also be displaced by riboflavin and product analog 56. A scheme for the catalytic cycle of riboflavin synthase is proposed.Riboflavin synthase (E.C. 2.5.1.9.) catalyzes an interesting dismutation reaction in which two molecules of 6,7-dimethyl-8-(~-ribityl)lumazine (1) form one molecule of riboflavin (2) and one molecule of the pyrimidinedione 3.1-3 During this reaction, substrate bound at the donor /OH /OH1 2 HQe,, OH R' H::q 3site of the enzyme donates a four-carbon unit to substrate bound at the acceptor site. The mechanism of the reaction is thought to proceed as outlined in Scheme I.4p5 In this process, the anion 5 formed from deprotonation of the C-7 methyl group of 1 at the acceptor site of the enzyme adds to the imine group of 4, which is formed by addition of an unidentified nucleophile to C-7 of 1 bound at the donor site of the enzyme. Various suggestions for the identity of the nucleophile have been offered, including a nucleophilic group from the enzyme: the 2'-hydroxyl or 3'-Purdue University.* Technische Universitiit Munchen.0022-3263/92/ l957-5630$03.00/0 hydroxyl of the ribityl side chain,'^^ and watereg A j3elimination involving cleavage of the N-5 to C-6 bond of the lumazine moiety of 6 at the donor site yields the alkene 7. A 1,g-elimination involving loss of a proton from the C-6 methyl group of the lumazine moiety bound at the acceptor site and "...

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Fiduxosin (1) has been under development at Abbott Laboratories for the treatment of benign prostatic hyperplasia. A convergent strategy required methodologies for preparation of an enantiomerically pure 3,4-cis-disubstituted pyrrolidine and a 2,3,5-trisubstituted thienopyrazine in a regiospecific manner. A [3+2] cycloaddition of an enantiopure azomethine ylide followed by a diastereoselective crystallization was employed to prepare the benzopyranopyrrolidine in high diastereomeric and enantiomeric purity. Conditions for reduction of an O-aryl lactone susceptible to epimerization were developed, and cyclization of the alcohol/phenol to the ether was accomplished in high yield. The thienopyrazine was prepared by condensation of methyl thioglycolate and a regiospecifically prepared 2-bromo-3-cyano-5-phenylpyrazine. Conditions for effective halogen substitutive deamination to prepare regiospecific trisubstituted pyrazines will be described.