## 3 Anti-HIV Pronucleotides-Nucleotide Reverse-Transcriptase Inhibitors (NttTIs)
### Diphosphate Esters of 2\({}^{\prime}\),3\({}^{\prime}\)-Dideoxynucleosides
For Di_PP_ro pronucleotides **34**, it was indeed found that the desired ddNDPs were formed during incubation in cell extracts [106]. Although this route was dominant, some ddNMPs formation was also observed [108; 109]. Initially, the RC(O) acyl moieties of both benzyl-protecting groups at the P\({}_{\beta}\) center were identical, and the chain length of R, which correlated with the lipophilicity of R, was found to significantly affect the hydrolytic properties of Di_PP_ro pronucleotides **34**. The anti-HIV activity of the d4T derivatives of **34** was similar or slightly worse than that of d4T in CEM/0 cells; however, in TK' cells, in which d4T is completely inactive, diphosphates **34** (R = heptyl or phenyl) retained their activity. In contrast, Di_PP_ro with R = Me was probably too polar to penetrate the cell membrane, and the one with R = _t_Bu was speculated to be resistant to the second deprotection step (i.e., **36**\(\rightarrow\) d4 TDP) [106]. These results, along with the finding that the amount of undesirable monophosphate formed from pronucleotides **34** closely correlated with the increasing chain length of the acyl residue, prompted the [PERSON] group to design unsymmetrical Di_PP_ro derivatives **34a** (Figure 7), which contained two different masking groups at the P\({}_{\beta}\) phosphate, differing in the length of the carbon chains of the acyl residues [108; 109]. The R\({}^{1}\) group with a short aliphatic chain should undergo rapid hydrolysis in the presence of appropriate cellular enzymes, thus preventing cleavage of the P-O-P bond. In contrast, the acyl substituent in the second benzyl group, with a long carbon chain, should ensure the adequate lipophilicity of the molecule. When tested under various experimental conditions, the unsymmetrical compounds **34a** produced significantly less NMPs and more NDPs in all instances compared to the previous symmetrical Di_PP_ro compounds **34**. Figure 7: Structures of the developed pronucleotides of Di_PP_ro type [108]. The [PERSON] group also designed bis(benzylbenzyl) derivatives of type **34b** (Figure 7), containing in their structure electron-withdrawing and electron-donating substituents in the _para_ position of the benzoyl residues. Stability studies in cell extract revealed that derivatives of type **34b** with strong electron-withdrawing groups (e.g., CF\({}_{3}\), CN, NO\({}_{2}\)) released only or mostly the expected ddNDP [110]. Studies on the therapeutical parameters of d4T derivatives of type **34** have shown that that these compounds had similar antiviral activities to the parent nucleosides [106]. Most of these analogs were effective in inhibiting HIV replication, and in some cases improved biological activity, as compared to free nucleosides, was even apparent. Moreover, a high antiviral activity was also observe in CEM/TK' cells, which supported the pronucleotide mode of action for these compounds [108]. Among the investigated compounds, the most active were analogs of type **34b**[110]. For the synthesis of various types of Di_PP_ro pronucleotides, the [PERSON] group used a combination of P(III) and P(V) chemistries. The phosphitylating reagent **37** bearing two identical benzyl groups could be prepared relatively simply by reacting dichloro-_N_,_N_-disopropylaminophosphoramidine with 2 equiv. of an appropriate benzyl alcohol [106, 110, 111]. In the case of an unsymmetrical version of reagent **37**, this approach failed, and a more sophisticated protocol had to be developed. To this end, the first benzyl alcohol was reacted with PCI\({}_{3}\) to give benzyl phosphorodichloride intermediate, which was converted into the bis(_N_,_N_-diisopropylamidine) derivative, and subjected to the reaction with the second benzyl alcohol.