The application of phosphoramidate protide technology to acyclovir confers anti-HIV inhibition

Abstract

Recently, it has been reported that phosphorylated acyclovir (ACV) inhibits human immunodeficiency virus type 1 (HIV-1) reverse transcriptase in a cell-free system. To deliver phosphorylated ACV inside cells, we designed ACV monophosphorylated derivatives using ProTide technology. We found that the L-alanine derived ProTides show anti-HIV activity at noncytotoxic concentrations; ester and aryl variation was tolerated. ACV ProTides with other amino acids, other than L-phenylalanine, showed no detectable activity against HIV in cell culture. The inhibitory activity of the prodrugs against herpes simplex virus (HSV) types -1 and -2 and thymidine kinase-deficient HSV-1 revealed different structure-activity relationships but was again consistent with successful nucleoside kinase bypass. Enzymatic and molecular modeling studies have been performed in order to better understand the antiviral behavior of these compounds. ProTides showing diminished carboxypeptidase lability translated to poor anti-HIV agents and vice versa, so the assay became predictive.

Fig. 1

Proposed activation pathway of the acyclovir ProTides.

Fig. 2

ACV and its ProTides.

Fig. 3

Carboxypeptidase-mediated cleavage of compound 9, monitored by ³¹P NMR

Fig. 4

Carboxypeptidase-mediated cleavage of compound 10, monitored by ³¹P NMR

Fig. 5

Carboxypeptidase-mediated cleavage of compound 6, monitored by ³¹P NMR

Fig. 6

Carboxypeptidase-mediated cleavage of compound 17, monitored by ³¹P NMR

Fig. 7

Carboxypeptidase-mediated cleavage of compound 19, monitored by ³¹P NMR

Fig. 8

Stability of ACV ProTides in crude CEM cell extracts as a function of incubation time.

Fig. 9

Stability of compound 9 in human serum, monitored by ³¹P NMR.

Fig. 10

a. Docking of compounds 9 within the catalytic site of carboxypeptidase Y enzyme. b. Docking of compounds 6 within the catalytic site of carboxypeptidase Y enzyme.

Fig. 11

a. interactions of 90 (L-Ala) with the active site of human Hint-1. b. Docking of compound 90 (L-Ala) ACV-MP phosphoramidate within the catalytic site of human HINT (I) enzyme

Fig. 11

a. interactions of 90 (L-Ala) with the active site of human Hint-1. b. Docking of compound 90 (L-Ala) ACV-MP phosphoramidate within the catalytic site of human HINT (I) enzyme

Scheme 1

Reagents and conditions: (i) POCl₃, anhydrous TEA, anhydrous Et₂O, -78 °C, 1 h then rt, overnight.

Scheme 2

Reagents and conditions: (i) anhydrous TEA, anhydrous DCM, -78 °C, 30 min - 1 h, then rt, 30 min - 4 h.

Scheme 3

Reagents and conditions: (i) dimethylformamide dimethyl acetal, anhydrous DMF, rt, 1 day; (ii) ^tBuMgCl, THF, rt, overnight or NMI, THF/pyridine = 3/2, rt, overnight; (iii) 1-propanol, reflux, for 18 h or 2-propanol, reflux, 24-96 h.