Tetsuji Kawamoto

Synthesis and reaction of novel 5-deazaflavins with axial chirality at pyrimidine ring moiety

Abstract A series of novel 5-deazaflavin derivatives possessing axial chirality at pyrimidine ring moiety have been prepared to investigate effects of the pyrimidine site on the stereoselective reactions between flavins and substrates. Successful optical resolution of the racemic compounds has been achieved by HPLC method on a chiral stationary phase and a diastereomer formation method. The chiral recognition ability of the 5-deazaflavin enantiomers was investigated in a model reaction of asymmetric intercoenzyme “(net) hydride transfer” reactions.

Synthesis and antitumor activities of novel 5-deazaflavin-sialic acid conjugate molecules.

6-Nitro-5-deazaflavin derivatives bearing O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha- and beta-D-galacto-non-2-ulopyranosylonate)alkyl group (sialosylalkyl group) at N(3) or N(10) and 8-amino-5-deazaflavin substituted with the sialosylalkyl group at the amino group were synthesized and their physicochemical properties as well as antitumor effects on KB and L1210 cells have been investigated. The configurations of the glycosides were determined by 1H NMR and rate of hydrolysis of the glycosidic bond. It has been found that these conjugate molecules show significant antitumor activities. Combination of an 8-amino-5-deazaflavin with the sialosylalkyl group have been found to give rise to significant increase in antitumor activities of the compound. Antitumor effects of 6-nitro-5-deazaflavin-sialic acid conjugate molecules were similar or rather weak in comparison with those of the 6-nitro-5-deazaflavin derivatives without sialosylalkyl group.

Synthesis and evaluation of nitro 5-deazaflavins as novel bioreductive antitumor agents

Abstract A series of nitro 5-deazaflavins, 5-deazaflavins possessing a nitro group at C(6)–C(9) position, has been designed and synthesized as a novel class of bioreductive nitrohetero-aromatic compounds and their cytotoxicities towards L1210 and KB cells were evaluated. It has been found that the nitro 5-deazaflavins undergo one electron reduction on the nitro group and undergo two electrons or “(net) hydride” reduction on the C(5)-C(4a)-C(10a)-N(1) redox system. They showed much more potent antitumor activities than the other 5-deazaflavins bearing no nitro group. These results suggest that an activation of nitro group by biological one electron reduction is crucial for an expression of cytotoxicity.

Diastereoface differentiating “(net) hydride transfer” in novel 5-deazaflavins modified at pyrimidine ring

Abstract In novel 5-deazaflavin models ( 1 )–( 5 ) where one face of the pyrimidine ring moiety is flanked, “(net) hydride transfer” from BNAH ocurred at mainly C(5) on the face which aligns with the open side of the pyrimidine ring. The degree of diastereoface differentiation depends on the bulkiness of the substituent on phenyl group at N(3). The results revealed that the pyrimidine ring moiety of flavin ring system interacts with the carbamoyl group of BNAH in the transition of the “(net) hydride transfer” reaction. Diastereoface differentiating “(net) hydride abstraction” from the reduced 5-deazaflavin was also investigated.

Synthesis and evaluation of nitro 5-deazaflavin-pyrrolecarboxamide(s) hybrid molecules as novel DNA targeted bioreductive antitumor agents.

A series of 6-nitro-5-deazaflavins bearing at N(3) or N(10) position the pyrrolecarboxamide(s) group as DNA minor groove binder has been synthesized. These hybrid molecules show similar redox properties to those of 6-nitro-5-deazaflavins with no pyrrolecarboxamide(s) group, suggesting that they generate stable one- and two-electron reduction product(s). Electrolytic reductions of the hybrid molecules were carried out at a controlled potential under anaerobic conditions in the presence of plasmid pBR322 DNA. Significant conversions of the supercoiled circular pBR322 DNA (form I) to the open circular DNA (form II) have been found by treatment with the reductively activated 6-nitro-5-deazaflavin derivatives. Their DNA damaging effects have been found to be enhanced as the number of pyrrolecarboxamide group as the DNA binder increases. Antitumor activities of the hybrid molecules towards KB and L1210 cells were evaluated in vitro. It has been found that the antitumor effects of the compounds on KB cells slightly change and those on L1210 cells decrease as the number of the pyrrolecarboxamide group increases. These results reveal that the combination of 6-nitro-5-deazaflavin molecule with the pyrrolecarboxamide(s) group increase the DNA binding properties of the compounds, giving rise to promoted DNA damaging effects, and also suggest that the combination would affect the capacity of the compounds to act as the substrate for intracellular reductases and/or the cellular uptake of the compounds.

First example of “diastereotopic face activation” and chiral recognition without metal assistance. A novel 5-deazaflavin enzyme model.

Abstract A chiral 5-deazaflavin 1 with 2-hydroxynaphthyl group at N(3) position of the pyrimidine ring moiety was synthesized. 1 underwent, in the absence of magnesium ion, a “(net) hydride transfer” from BNAH to C(5) almost exclusively on the face where OH group is present. It was also the case with PNPH, and enantioselectivity for PNPH by 1 was higher in the absence of magnesium ion than in its presence. These results indicate that the “diastereotopic face activation” in 1 is operated as well as chiral recognition for PNPH by 1 .

Evaluation of differential hypoxic cytotoxicity and electrochemical studies of nitro 5-deazaflavins

Abstract Cytotoxicities of nitro 5-deazaflavins were evaluated in vitro towards hypoxic and oxic Chinese hamster cells (V79). 6-Nitro and 8-nitro derivatives were generally more toxic towards hypoxic cells than oxic cells, showing marked hypoxic selectivity. In contrast, 7-nitro and 9-nitro derivatives showed no significant hypoxic selectivity. Electrochemical study using cyclic voltammetry (CV) revealed that 6-nitro and 8-nitro derivatives generate a stable two electrons reduction product as well as a stable one electron reduction product and that 7-nitro and 9-nitro derivatives afford an unstable one electron reduction product. These results strongly support that not solely electron affinity but also stability of the one electron reduction products is crucial for the differential hypoxic cytotoxicities of nitro 5-deazaflavins.

Synthesis and reaction of a new type of 5-deazaflavin with axial and planar chirality

Abstract A new type of 5-deazaflavin derivative with axial and planar chirality was synthesized as a flavoenzyme model. A novel optical resolution gave an enantiomeric pair of the 5-deazaflavins 1a,b . Compounds 1a,b were optically stable and effectively discriminated PNPH enantiomers in a model reaction of intercoenzyme hydrogen transfer.

Stereochemistry of asymmetric “(net) hydride transfer” in an intercoenzyme model reaction system

Abstract X-ray crystal structure analysis revealed the absolute configuration of compound 2 and thus proved the absolute configuration of 5-deazaflavin (+)-1 possessing both axial and planar chirality to be (S). Model reactions of “(net) hydride transfer” between 1 and Me2PNPH enanatiomers revealed that (S)-(+)-1 oxidizes (4S)-Me2PNPH more rapidly than its (4R) isomer and that (R)-(−)-1 oxidizes (4R)-Me2PNPH more rapidly than its (4S) isomer. These results strongly suggest that an enatioselectivity on Me2PNPH by chiral 1 is not solely determined by the availability of C(4) hydrogen but by the “(net) hydride donor-acceptor” interactions involving the interaction between the pyrimidine site of 5-deazaflavin molecule and the carbamoyl group of the NAD(P)H model as the important contributor.

Variation of reaction channel in a flavoenzyme model with a modified pyrimidine ring

Abstract A chiral 5-deazaflavin derivative with 2-hydroxymethylphenyl group at N(3) position of the pyrimidine ring has been synthesized. Oxidized form of the 5-deazaflavin derivative is reduced stereo-specifically: the hydroxymethyl group exerts steric inhibition in the absence of Mg 2+ , whereas it facilitates the approach of a reducing agent in the presence of Mg 2+ .