4.6- Dideoxynucleosides

DNA fragment formed under conditions of experiment terminates m indicated dideoxynucleoside... 

DNA polymerases normally use 3 -deoxynucleotide triphosphates as substrates for polymerization. Given an adequate concentration of substrate, DNA polymerase synthesizes a long strand of new DNA complementary to the substrate. The use of this reaction for sequencing DNA depends on the inclusion of a single 2/3 -dideoxynucleoside triphosphate (ddNTP) in each of four polymerization reactions. The dideoxynucleotides ate incorporated normally in the chain in response to a complementary residue in the template. Because no 3 -OH is available for further extension, polymerization is... 

Fig. 10. 2, 3 -Dideoxynucleosides active against human immunodeficiency vims (HIV).

AH 2/3 -dideoxynucleoside analogues are assumed to be intraceUularly phosphorylated to thek active form (5 -triphosphate), and then targeted at the vims-associated reverse transcriptase. The rate and extent of the 2 /3 -dideoxynucleosides phosphorylate to the 5 -triphosphates may be of equal or greater importance than the differences in the relative abiUties of these 5 -triphosphates to inhibit the vkal reverse transcriptase (171). At the level of vkal reverse transcriptase, the 5 -triphosphate of AZT and other dideoxynucleosides may either serve as a competitive inhibitor with respect to the natural substrates or may act as an alternate substrate, thus leading to chain termination (172). 

FIGURE 12.3 The chain termination or dideoxy method of DNA sequencing, (a) DNA polymerase reaction, (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate, (d) Electro-phoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. 

F. 3 Structural formulae of 2, 3 -dideoxynucleoside analogues (anti-HIV) agents... 

As has been demonstrated for tenofovir, when incorporated at the 3 -end of reverse transcriptase (RT)-driven DNA chain allows the PMPA residue to adopt multiple conformations [in contrast with the more rigid conformation of the 2, 3 -dideoxynucleosides (see infra) (Tuske et al. 2004). This greater flexibility in conformation may impede development of resistance to tenofovir. 

The 2, 3 -dideoxynucleoside (ddN) analogues (Fig. 3) encompass a vast group of compounds that have been found active against HIV and HBV, although they have been primarily pursued for the treatment of HIV infections (AIDS). They are targeted at the HIV-associated reverse transcriptase (RT) and therefore also referred to as nucleoside reverse transcriptase inhibitors (NRTIs). They have to be distinguished from the nucleotide reverse transcriptase inhibitors (NtRTIs) such as adefovir (PMEA) and tenofovir (PMPA) (see above) which, like the NRTIs, act as chain... 

All NRTIs, as exemplified for AZT (Fig. 7), act in a similar fashion following their uptake by the cells, they are phosphorylated successively to their 5 -monophosphate, 5 -diphosphate, and 5 -triphosphate form (De Clercq 2002). Unlike the first phosphorylation step in the metabolic pathway of the acyclic guanosine analogues (see above), which is carried out by a virus-encoded enzyme (thymidine kinase), the first as well as the subsequent phosphorylations of the 2, 3 -dideoxynucleosides are carried out by cellular enzymes, that is, a 2 -deoxynucleoside (e.g., dThd) kinase, a 2 -deoxynucleotide (e.g., dTMP) kinase, and a (2 -deoxy)nucleoside 5 -diphosphate (NDP) kinase. 

Kazi S, Cohen PR et al (1996) The diffuse infiltrative lymphocytosis syndrome. Clinical and immunogenetic features in 35 patients. Aids 10(4) 385-391 Keilbaugh SA, Prusoff WH et al (1991) The PC12 cell as a model for studies of the mechanism of induction of peripheral neuropathy by anti-HIV-1 dideoxynucleoside analogs. Biochem Pharmacol 42(1) R5-R8... 

In addition to the three anti-HIV agents [AZT (zidovudine), DDI (dida-nosine) and DDC (zalcitabine)] that have been formally approved by the U.S. Food and Drug Administration for the treatment of HIV infections, several other 2, 3 -dideoxynucleoside (ddN) analogues (Fig. 5), including 3 -fluoro-2, 3 -didcoxy-5-chlorouridine (FddClUrd) and 2, 3 -didehydro-... 

Figure 5 Dideoxynucleoside (ddN) analogues 2, 3 -dideoxycytidine (DDC), 3 -azido-2, 3 -dideoxythymidine (AZT), 3 -fluoro-2, 3 -dideoxythymidine (FLT), 2, 3 -didehydro-2, 3 -dideoxythymidine (D4T), 3 -thia-2, 3 -dideoxycytidine (3TC), 3 -thia-2, 3 -dideoxy-5-fluorocytidine (FTC), and 2, 3 -dideoxy-L-cytidine (l-DDC).

A modification of the azolide method for deoxygenation of alcohols is the conversion of the thiocarbonylimidazolide with methanol into the thionocarbonate, which is then treated with tri-w-butyl stannane/AIBN. Thus, 2,3-dideoxynucleosides (a), (b), and a secodeoxynucleoside (c) have been prepared as follows [52H56]... 

Such dideoxynucleosides as CNT (306) and the potent anti-HIV drug ddC (307) have been obtained (280), respectively, from the butenolide 248 and from its saturated analogue. Thus, conjugate addition of cyanide to 248, followed by reduction of the lactone group, acetylation of HO-1, and coupling with silylated thymine, afforded, after deprotection, compound 306. 

A whole family of 2, 3 -dideoxynucleoside exists, and it is apparent that they may vary considerably in their antiviral and anticellular activities. Removal of the oxygen at the 3 carbon of the sugar of 2 -deoxyadenosine changes the molecule into a powerful antiviral compound, 2, 3 -dideoxyadenosine. 

Singhal D, Ho NF and Anderson BD (1998) Absorption and Intestinal Metabolism of Purine Dideoxynucleosides and an Adenosine Deaminase-Activated Prodrug of 2/,3/-Dideoxyinosine in the Mesenteric Vein Cannulated Rat Ileum. J Pharm Sci 87 pp 569-577. 

T. Tuntland, A. Odinecs, C.M. Pereira, C. Nosbisch, and J.D. Unadkat. In vitro models to predict the in vivo mechanism, rate, and extent of placental transfer of dideoxynucleoside drugs against human immunodeficiency virus. Am J Obstet Gynecol. 180 198-206 (1999). 

Schott H, et al. Synthesis and in vitro anti-HIV activities of amphiphilic heterodinucleoside phosphate derivatives containing the 2, 3,-dideoxynucleosides ddC, AZT and ddl. Eur J Med Chem 1999 34 343. 

As shown in Scheme 18, the reaction of 3-azido-2,3-dideoxynucleoside 36 with polystyryl diphenylphosphane resin (37) affords iminophosphorane 38, which gives amine 39 after hydrolysis. Dioxane or pyridine is used as a solvent, and the purity of the products obtained is identical with those obtained by the normal method (94S789). 

Didanosine (2 3 -dideoxyinosine or ddl) is a dideoxynucleoside purine analogue. Its mechanism of action is identical to that of zidovudine and resistance to didanosine is known to occur rapidly in patients who were already treated with zidovudine. Didanosine shows in vitro synergy with zidovudine while their toxicity profiles are different. Oral absorption is decreased by food and didanoside penetrates into the brain to a limited extend. Pancreatitis is the most serious complication. Other adverse reactions include peripheral neuropathy, diarrhoea and other gastrointestinal disturbances. 

Additional nucleoside analogues like the purine dideoxynucleosides lamivudine (3TC) and dideoxy-cytidine (ddC, zalcitabine) act in the same way as AZT. Resistance against these agents may show different patterns. They are generally less toxic than AZT. Adverse effects include diarrhoea and other gastrointestinal disturbances, headache, anxiety, restlessness and insomnia. Also hepatotoxicity can occur, probably because some of these drugs might have also some affinity for human DNA polymerases in the liver. 

Various isomers of 2,3-difluoro-2,3-dideoxynucleosides have been synthesized (Figure 6.7). The configuration of the fluorine atoms plays an essential role in the antiviral activity. ° In the case of the D-lyxo compound, coupling with thymine has failed, due to the participation of the protecting group. 

When the trifluoromethylation occurs on aketonic carbonyl (such as position 2 or 3 in furanose series), it must be followed by a Barton-McCombie deoxygenation of the hydroxyl. It thus leads to the 2- or 3-C-trifluoromethyl deoxyfuranoses. The transformation of these latter compounds into deoxynucleosides has been reported. Trifluoromethyl 2, 3 -dideoxynucleosides and A-2 3 -vinylic nucleosides have also been prepared according to this approach (Figure 6.34). The CF3 group protects the glycosyl base bound from proteolysis in acidic medium, especially in the case of A-2 3 compounds. ... 

One of the motive powers for the design of host molecules for nucleotides is their potential utility in chemotherapy. A number of nucleotide analogues exhibit antiviral activity in vitro [73] for example, 2, 3 -dideoxynucleosides are potent chain-terminating inhibitors of HIV reverse transcripase [74]. However, charged and hydrophilic as they are, they can hardly penetrate across cell... 

Shirasaka T, Kavlick MF, Ueno T, Gao W-Y, Kojima E, Alcaide ML, et al. Emergence of human immunodeficiency virus type 1 variants with resistance to multiple dideoxynucleosides in patients receiving therapy with dideoxynucleosides. Proc Natl Acad Sci USA 1995 92 2398-2402. 

Pauwels R, Andries K, Debyser Z, Kukla MJ, Schols D, Breslin HJ, et al. New TIBO derivatives are potent inhibitors of HIV-1 replication and are synergistic with 2, 3 -dideoxynucleoside analogues. Antimicrob Agents Chemother 1994 38 2863-2870. 

Phosphonoformate is a pyrophosphate analog and inhibits both DNA polymerases and reverse transcriptase. However, toxicity may prevent longterm treatment of AIDS patients. Amantadine has a narrow antiviral specificity. It specifically inhibits initiation of the replication of influenza virus RNA of type A (but not of type B). Active only against retroviruses, 3 -azidothymidine is a reverse transcriptase inhibitor, which acts by a chain termination mechanism. It was synthesized in the early 1960s but only recently has been used in treatment of AIDS victims. More recently a series of 2, 3 -dideoxynucleosides, such as dideoxyinosine, have also been used.d Acyclic phosphonates, such as phosphonylmethoxypropyladenine, avoid the need for metabolic phosphorylation of the drug.6... 

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