## 3 Anti-HIV Pronucleotides-Nucleotide Reverse-Transcriptase Inhibitors (NttTIs)
### Diphosphate Esters of 2\({}^{\prime}\),3\({}^{\prime}\)-Dideoxynucleosides
The biological evaluation of the activity of the AZT analogs of **31** showed that their EC\({}_{50}\) values were of the same order as for the parent AZT nucleoside, and for the d4T derivatives, even being 100 times lower than that for d4T nucleoside. The strategy, based on the introduction of long aliphatic chains into the nucleotides, was extended to alkoxylkyl derivatives of acyclic nucleoside phosphonates (ANPs) by [PERSON] et al., who developed derivatives of type **32** as antiviral agents (Figure 6) [105]. In the  
Figure 4: Lipophilic nucleoside diphosphate analogs designed by [PERSON] et al. [102]. Figure 5: Examples of nucleoside mono-, di-, and triphosphate pronucleotides with a structural motif of the mixed carboxylic-phosphoric anhydrides [103,104]. synthesis of this class of compounds, alkoxyalkyl phosphoromorpholidates were used as intermediates that were coupled with the appropriate C-phosphonates of antiviral acyclic nucleoside analogs, adefovir and cidofovir. In vitro biological assays showed that analog **32a-HDP** inhibited HIV replication much better (EC\({}_{50}\) = 0.003 uM) than adefovir alone (EC\({}_{50}\) = 1.3 uM); unfortunately, its cytotoxicity in MT-2 cells was clearly higher (CC\({}_{50}\) = 0.018 uM vs. CC\({}_{50}\) = 157 uM), which resulted in the SI\({}_{50}\) selectivity index for this compound being only 6 (SI\({}_{50}\) = 121 for adefovir) [105]. Interestingly, adefovir esterified with the HDP group, having similar cytotoxicity, showed extremely high activity against HIV, EC\({}_{50}\) = 0.02 nM, which gave SI\({}_{50}\) = 3000. Cidofovir derivatives were tested against HCMV, HSV-1, vaccinia, and cowpso infections. Of the five compounds tested in this series, the pyrophosphate derivative was found to be the superior in one case. Derivative **32b-ODE** showed the strongest inhibition of HSV (EC\({}_{50}\) = 0.2 nM), while being moderately cytotoxic (EC\({}_{50}\) = 9.5 uM), which resulted in SI\({}_{50}\) \(\approx\) 500,000. No information on the possible mechanism of action of this type of pronucleotides was provided by the authors. The most comprehensive and extensive research on di- and triphosphate pronucleotides was carried out by [PERSON] and co-workers. In 2008, the first attempts were made to adapt the _cycloS_al approach of pronucleotides to diphosphate derivatives [106]. This choice seemed justified considering the promising biological results of the _cycloS_al-NMP derivatives [21]. Unfortunately, under hydrolytic conditions, compounds of type **33** (Scheme 18) hydrolyzed with cleavage of the P-O-P bond (path **B**), resulting mainly in the release of ddNMP rather than the expected ddNDP (path **A**) [106]. Thus, _cycloS_al-NDP derivatives appeared to be precursors of the same nucleoside monophosphates as the simpler _cycloS_al-NMP constructs. Due to the unfavorable course of the hydrolysis of _cycloS_al-NDP **33**, [PERSON] et al. developed another pronucleotide strategy, called DiPPro. In this approach, the terminal phosphate group of pronucleotide **34** was protected with stable benzyl-type groups, which required the action of cellular enzymes to release unmasked phosphate. This allowed them to avoid the unfavorable nucleophilic attack on the phosphorus center (Scheme 19) [106]. The protecting groups contained a carboxysester moiety that was prone to cleavage by cellular carboxysesterases, releasing the labile 4-hydroxybenzyl ester in intermediate **35**, which spontaneously collapsed to diphosphate **36**. The second benzyl group could be removed by the same mechanism or could be cleaved directly by phosphoesterases. This mode of action was analogous to the bis(4-acyloxybenzyl)-pronucleotide strategy proposed  
Figure 6: Lipophilic acyclic nucleoside phosphonates (ANP) analogs **32** designed by [PERSON] et al. [105]. by [PERSON] et al. in 1993 to achieve the in-cell formation of ddMPs, which did not receive much attention at that time [107].