Silvano Cruz-Gregorio

Six-Membered Ring Phosphates and Phosphonates As Model Compounds for Cyclic Phosphate Prodrugs: Is the Anomeric Effect Involved in the Selective and Spontaneous Cleavage of Cyclic Phosphate Prodrugs?

In recent years, several six-membered ring phosph(on)ates and phosphonamides have been reported as potent prodrugs against liver diseases such as hepatitis B and C and also as antitumor agents. Apparently, the success for their biological activity depends on the selective cleavage of the C4-O3 bond within the respective P-heterocyclic ring. Empirical observations have suggested that the group attached to the C4 position (aryl or pyridyl group) is responsible for the selective cleavage. In this regard, we show in the present work that the configuration at the P-atom, the conformation of the P-heterocyclic ring, and particularly, the anomeric effect are involved in the spontaneous and selective cleavage of the C4-O3 bond in cyclic phosph(on)ates. We arrived at this assumption based on the conformational and configurational study of simple model phosphates and phosphonates, where it was observed that the spontaneous conversion of unstable six-membered ring phosphates to their most stable six-membered ring phosphate (4d, 6d and 7d to 5d), by a selective C4-O3 bond cleavage, depends on both: the stereochemistry of the aryl group at C4 and the electronic nature of the substituent attached to the P-atom. Thus, we postulated that the anomeric effect weakens the C4-O3 bond within the 1,3,2-dioxaphosphorinane ring, favoring thus their selective cleavage and spontaneous conversion, similarly to the proposed mechanistic mode of action of six-membered ring P-heterocyclic prodrugs.

On the Existence of the Chair Conformation in Six-Membered Ring Phosphates Bearing an Aryl Group Axially Oriented at the C4 Position: Cyclic Nucleotides As Model Compounds for Cyclic Phosph(on)ate and Phosphoramide Prodrugs

A series of cyclic nucleotide analogues to HepDirect prodrugs were prepared by a three-component reaction of protected thymine, phosphoryl chloride, and 5-aryl-alpha-D-xylofuranoses derivatives. One of the cyclic nucleotides showed NMR data that suggest a predominant twisted conformation; however, in spite of having an aryl group at the C4 position within the crystal lattice, the cyclic nucleotide had a chair conformation with the aryl group axially oriented. By analyzing the unprecedented X-ray structure, it was observed that the oxygen atom from the phoshoryl group (P=O) is found in close proximity to the o-hydrogen atom of the aryl group (2.51 A), suggesting thus an attractive nonbonding electrostatic interaction, which might be the driving force that overcomes the steric diaxial interactions imposed by the aryl group. Theoretical studies (NBO) for two model compounds showed that there are indeed interactions between filled (donor) Lewis-type NBOs and empty (acceptor) non-Lewis NBOs corresponding to the nO-->sigma*(C-H) interaction. Additionally, conversion of a diastereomeric mixture of cyclic nucleotides into the more stable diastereomeric cyclic nucleotide was observed and explained by spontaneous isomerization in the phosphorinane ring. This finding supports the recently established hypothesis for the mode of action of prodrug cleavage, for which the anomeric effect plays an important role.

Further evidence on the favorable role of the anomeric effect on the cleavage of HepDirect and cyclophosphamide prodrugs.

On the basis of previous conformational and configurational studies of 4-aryl-substituted cyclophosph(on)ates derived from d-xylofuranose derivatives, wherein it was proposed that the anomeric effect is involved in the spontaneous isomerization of the P atom and the C4 carbon, and consequently, this unusual behavior was associated with the cleavage of the HepDirect prodrugs. We synthesized an analogous series of 2-amino-2-oxo-1,3,2-dioxaphosphorinanes and performed a conformational and configurational analysis in solution and the solid state followed by an examination of their mutagenic activity. The results showed that the 2-amino-2-oxo-1,3,2-dioxaphosphorinanes with the largest mutagenic activity contain either a 4-methoxyphenyl or 4-fluorophenyl group at C4 carbon and presented a major chair conformation, which is prone to weaken the C4-O3 bond via the anomeric effect and facilitates the cleavage for the release of the biologically active metabolite.