A large number of cycloSal-nucleotide triesters 1-49 have been studied concerning their ability to inhibit cholinesterases of different origins as well as to inhibit HIV replication in cell culture. It was shown that none of the triesters showed inhibitory effects against human acetylcholinesterase (AChE; isolated enzyme) as well as against AChE from beef erythrocytes and calf serum. In contrast, inhibition of butyrylcholinesterase (BChE) has been observed for some triesters in human and mouse serum. cycloSal pronucleotides showed strong competitive inhibition with respect to the substrate acetylcholine chloride (K(i)/K(m): approximately 2 x 10(-5)) and acted by time-dependent irreversible inhibition of the human serum BChE. Detailed studies demonstrated that the inhibitory effect against BChE is dependent on the nucleoside analogue, the substitution pattern of the cycloSal-moiety, and particularly on the stereochemistry at the phosphorus atom. Structural requirements to avoid the inhibition of BChE by cycloSal-nucleotide triesters have been elucidated in the reported study.

Two new classes of lipophilic prodrugs of the antiviral active phosphonate 9-[2-phosphonomethoxyethyl]adenine (PMEA 1) have been prepared and were studied with regard to their hydrolysis properties and biological activity. A first series of compounds was prepared on the basis of the cycloSal nucleotide approach. Because of the surprisingly low hydrolysis stability of these cycloSal-PMEA derivatives, more stable derivatives have to be developed. Instead of using salicyl alcohol, in cycloAmb-PMEA derivatives 2-aminobenzyl alcohols were attached to PMEA 1. The latter compounds showed a considerably higher stability compared to the cycloSal counterparts. Stability studies revealed that all lipophilic prodrugs delivered PMEA selectively by chemical means. All compounds proved to be noninhibiting to acetyl- and butyrylcholinesterase, and some of the phosphonate diesters were found to be more active against HIV compared to the parent PMEA.

A new, considerably improved cycloSal masking group has been developed. This new group combines four desirable properties and has been attached to the anti‐HIV drug 2′,3′‐dideoxy‐2′,3′‐didehydrothymidine (d4T, 1) to give 3,5‐(di‐tert‐butyl)‐6‐fluoro‐cycloSal‐d4TMP (2i). This phosphate triester has a reasonable chemical half‐life, highly selectively released d4TMP, has poor − if any − inhibitory effect on butyrylcholinesterase (BChE), and achieved the TK‐bypass. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Bis-cycloSal-d4T-monophosphates have been synthesized as potentially anti-HIV active "dimeric" prodrugs of 2',3'-dideoxy-2',3'-didehydrothymidine monophosphate (d4TMP). These pronucleotides display a mask-drug ratio of 1:2, a novelty in the field of pronucleotides. Both bis-cycloSal-d4TMP 6 and bis-5-methyl-cycloSal-d4TMP 7 showed increased hydrolytic stability as compared to their "monomeric" counterparts and a completely selective hydrolytic release of d4TMP. The hydrolysis pathway was investigated via 31P NMR spectroscopy. Moreover, due to the steric bulkiness, compound 6 already displayed strongly reduced inhibitor potency toward human butyrylcholinesterase (BChE), while compound 7 turned out to be devoid of any inhibitory activity against BChE. Partial separation of the diastereomeric mixture of 6 revealed strong dependence of the pronucleotides' properties on the stereochemistry at the phosphorus centers. Both 6 and 7 showed good activity against HIV-1 and HIV-2 in wild-type CEM cells in vitro. These compounds were significantly more potent than the parent nucleoside d4T 1 in HIV-2-infected TK-deficient CEM cells, indicating an efficient TK-bypass.