## 3 Anti-HIV Pronucleotides-Nucleotide Reverse-Transcriptase Inhibitors (NttTIs)
### Diphosphate Esters of 2\({}^{\prime}\),3\({}^{\prime}\)-Dideoxynucleosides  
At the onset, the strategy of di- and triphosphate prodrugs was developed mainly for AZT derivatives, since for this particular 2\({}^{\prime}\),3\({}^{\prime}\)-dideoxynucleoside, the second stage of phosphorylation (conversion of AZTMP to AZTMP) is the most problematic and is a source of the acute toxicity of the AZT therapy [7,8]. Studies on diphosphate pronucleotides date back to early 1990s and the works of [PERSON] and collaborators, who introduced lipidic moieties at the terminal phosphate residue of AZ TDP and ddCDP, and evaluated the anti-HIV potential of such phospholipid conjugates (compounds of type **28**, Scheme 17) [98-100]. It should be noted, however, that these compounds were designed as precursors of ddN monophosphates, not their diphosphates. In order to obtain these derivatives, suitable phosphoromorpholidate derivatives (originally introduced by [PERSON] et al.) [101] were coupled with glycerophospholipids to give diphosphates of type **28**. It was argued that glyceride nucleoside diphosphates, due to the presence of highly lipophilic acyl groups, should easily penetrate the cell membrane. [PERSON] et al. predicted an intracellular metabolism of these compounds to be, by analogy to natural glycerolipids,initiated by phospholipase A or lysophospholipase. This should lead to the formation of a derivative of type **29**, which then can be degraded by another enzyme (phosphodiesterase) to the corresponding ddNMP (path **A**, Scheme 17). Alternatively, compound **28** with the P-O-P anhydride bond could be directly hydrolyzed by cellular pyrophosphatases to generate nucleoside monophosphate and phosphatidic acid (path **B**, Scheme 17) [100]. Studies on the stability of the pyrophosphate anhydride bond in pyrophosphate **28** showed that path **B** of ddNMP formation was preferred [99]. The potential therapeutic value of the obtained diphosphates was rather disappointing, as the antiviral activity of the AZTDP derivative of type **28** (R = C\({}_{15}\)H\({}_{31}\)) in CEM cells was lower than that of AZT alone (EC\({}_{50}\) = 7 uM vs. 0.2 uM, respectively) [100]. These studies were followed a few years later by [PERSON] and collaborators. They reported on the synthesis of alkyl and thioalkyl glyceride ether derivatives of type **30** (Figure 4) [102]. These compounds were synthesized via nucleoside 5'-phosphooromorpholidates that were subjected to condensation with the corresponding glycerophospholipids. All the investigated compounds had a low cytotoxicity (CC\({}_{50}\) > 200 uM), and the thioether derivative **30a** showed greater anti-HIV activity (EC\({}_{50}\) < 0.58 uM) than the ether analog **30b** (EC\({}_{50}\) = 57 uM) [102]. At the same time, [PERSON] and collaborators designed mixed carboxylic-phosphoric anhydrides of type **31** as potential pronucleotides (Figure 5). These derivatives contain acyl groups with long alkyl chains at the terminal phosphate moiety, which should provide appropriate lipophilicity, similarly to the above glycerolipid analogs [103,104]. Nucleotide derivatives of type **31** were synthetized from the corresponding carboxylic acids and nucleoside diphosphates using the carbodimide approach for the formation of an anhydride bond. Derivatives of type **31** were assumed to penetrate cell membranes and release ddNMP, ddNDP or ddNTP for \(n\) = 1, 2 or 3, respectively, as a result of the expected preferential hydrolysis of the mixed carboxylic-phosphoric acid anhydride bond (C-O-P) over the pyrophosphate bond (P-O-P). Indeed, this cleavage order was confirmed in both triethylammonium acetate buffer at physiological pH and in RPMI; however, in the last medium the reaction was so rapid (t\({}_{1/2}\) < 2 h) that the lipophilic compounds **31** apparently did not have enough time to enter the cells [104].