Hydrolytic Reactions of Thymidine 5′‐O‐Phenyl‐N‐Alkylphosphoramidates, Models of Nucleoside 5′‐Monophosphate Prodrugs

Abstract

To obtain detailed data on the kinetics of hydrolytic reactions of triester-like nucleoside 5'-O-aryl-N-alkylphosphoramidates, potential prodrugs of antiviral nucleoside monophosphates, the hydrolysis of diastereomeric (Rp/Sp) thymidine 5'-{O-phenyl-N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (3), a phosphoramidate derived from the methyl ester of L-alanine, has been followed by reversed-phase HPLC over the range from Ho=0 to pH 8 at 90 degrees C. According to the time-dependent product distributions, the hydrolysis of 3 proceeds at pH<4 by two parallel routes, namely by nucleophilic displacement of the alaninyl ester moiety by a water molecule and by hydrolysis of the carboxylic ester linkage that allows intramolecular attack of the carboxy group on the phosphorus atom, thereby resulting in the departure of either thymidine or phenol without marked accumulation of any intermediates. Both routes represent about half of the overall disappearance of 3. The departure of phenol eventually leads to the formation of thymidine 5'-phosphate. At pH>5, the predominant reaction is hydrolysis of the carboxylic ester linkage followed by intramolecular displacement of a phenoxide ion by the carboxylate ion and hydrolysis of the resulting cyclic mixed anhydride into an acyclic diester-like thymidine 5'-phosphoramidate. The latter product accumulated quantitatively without any indication of further decomposition. Hydroxide-ion-catalyzed P--OPh bond cleavage of the starting material 3 occurred as a side reaction. Comparative measurements with thymidine 5'-{N-[(1S)-2-oxo-2-methoxy-1-methylethyl]phosphoramidate} (4) revealed that, under acidic conditions, this diester-like compound is hydrolyzed by P--N bond cleavage three orders of magnitude more rapidly than the triester-like 3. At pH>5, the stability order is reversed, with 3 being hydrolyzed six times as rapidly as 4. Mechanisms of the partial reactions are discussed.

Influence of the Nucleobase and Anchimeric Assistance of the Carboxyl Acid Groups in the Hydrolysis of Amino Acid Nucleoside Phosphoramidates

“…The reactions are expected to follow pseudo-first-order kinetics with respect to phosphoramidate concentration, since D 2 O was present in large excess and there was no significant change in the pD of the solution after hydrolysis. The first-order kinetics (as reported previously for other phosphor-A C H T U N G T R E N N U N G amidates) [13,23,24] is also apparent from the exponential decay in the concentration of phosphoramidate (Figure 3, top left) and from the corresponding linear transformed data Scheme 1. a) The dicyclohexylcarbodiimide (DCC)-assisted coupling and methyl ester deprotection. b) The synthesis of 1-deazaadenine-containing phosphoramidate analogues (21)(22)(23).…”

“…In the titration of 18, the C2 carbon in the 1 H- 13 C HSQC spectrum showed upfield CSP, whereas the C8 carbon remains unchanged ( Figure S7 in the Supporting Information), clearly indicating that the protonation site is N3, with a pK a of 4.6. Similarly, the protonation site for 2'-deoxy-3-deazaadenosine was found to be N1, with a pK a of 7.5.…”

“…Temperature-and pH-dependent kinetics were observed for all of the aaNPs, that is, faster kinetics under acidic conditions (Figure S2 in the Supporting Information) and at higher temperatures ( Figure S3 in the Supporting Information). The acid-catalyzed hydrolysis demonstrates a first-order dependence on the hydronium ion concentration, [13,23,25] as shown by the exponential decay curve in the pD-rate profile ( Figure 3, bottom left) and from the corresponding linear relationship ( Figure 3, bottom right). The overall first-order rate constants for the hydrolysis of aaNPs are presented in Table 1.…”

“…It was shown that the a-COOH group is involved in the nucleophilic attack on phosphorus [26,27] in a neighboring-group participation (NGP) reaction mode during the hydrolysis, resulting in the P À O bond cleavage. [13] The presence of an additional b-COOH group was suggested to have an extra catalytic role through intramolecular hydrogen bonding. [26,28] These roles suggest that in aspartyl and glycinyl NPs the a-COOH group is involved in the P À O bond hydrolysis.…”

“…Recently, the kinetics of the hydrolysis of l-alanine methyl esters of NP prodrugs was reported and a mechanism for the hydrolysis reaction in both acidic and basic solutions was proposed. [13][14][15] However, the physicochemical properties and structural determinants that influence the chemical stability and hydrolysis mechanism of this class of compounds are not known in detail. We have synthesized a series of aaNPs, containing either natural or modified nucleobases (Figure 1).…”