various embodiments and aspects of the invention will now be described by way of example only , with reference to the accompanying examples and drawings . activity of 2 ′, 3 ′- dideoxy - 3 - c - hydroxymethyl - cytosine against tam primer rescue - related resistant hiv in the phenosense hiv assay the susceptibility of 2 ′, 3 ′- dideoxy - 3 ′- c - hydroxymethyl - cytosine on hiv - 1 isolates from patient plasma samples that bear typical tam primer rescue mutant resistant genotypes is determined by the commercially available phenosense hiv assay ( described in petropoulos , c j et al ., ( 2000 ) antimicrob . agents chemother . 44 : 920 - 928 and performed by virologics , inc ). the assay is performed by amplifying the protease ( pr )- rt segment of the hiv pol gene from patient plasma and inserting the amplification products into a modified hiv - 1 vector derived from an nl4 - 3 molecular clone . viral stocks are prepared by co - transfecting 293 cell cultures with recombinant viral dna vector and an expression vector that produces the amphotropic murine leukemia virus envelope proteins . pseudotyped virus particles are harvested from the transfected cell cultures and are used to infect fresh 293 cell cultures . the recombinant viral dna contains a luciferase gene cassette within the hiv env gene region and the production of luciferase in target cells is dependent on the completion of one round of virus replication . drug susceptibility is measured by adding serial concentrations of the compound of the invention and the reference compounds to the cells . drugs that inhibit virus replication reduce luciferase signal in a dose - dependent manner , providing a quantitative measure of drug susceptibility . table 1 summarizes a main cluster of primer - rescue - related tam mutants used in the experiment are resistant to hiv and bear the characteristic tam genotype that typically emerges during azt - involved antiretroviral therapy . results are depicted in fig5 . wild - type hiv virus is used as the reference . here , the inhibition of the patient isolate 20 and 21 strains is expressed as the fold change in reduction of susceptibility to the treatment drug as compared to parallel runs of the reference . the following antiviral drugs were tested : azt , 3tc , tnf , abc , d4t , ftc and the compound of the invention . it is clearly apparent that the invention &# 39 ; s 2 ′, 3 ′- dideoxy - 3 ′- c - hydroxymethylcytosine retained activity against the tam bearing strains . the results show only a 1 . 0 fold reduction in susceptibility for the isolate 20 strain and less than a 1 . 0 fold reduction in susceptibility for the isolate 21 strain . this means that 2 ′, 3 ′- dideoxy - 3 ′- c - hydroxymethylcytosine retained activity against the patient &# 39 ; s primer rescue - related mutant hiv rt at a level of potency similar to its potency against wild type hiv rt . in contrast , other drugs , notably azt ( 451 fold reduction in susceptability ), but also to 3tc , tfn , abc , d4t and ftc , lost potency against the virus from these patients as compared to wildtype . in other words , the virus from these patients exhibited resistance , that is large reductions in susceptibility , to these drugs as shown in fig5 . it is important to note that the two patient isolates harbor different amino acid transitions at codon 215 ; t to f in isolate 20 and t to y in isolate 21 . this is a representative hallmark of primer rescue - related tam resistance mutants . table 2 outlines a primer rescue - related mutant hiv with the genetic background m184v ( a discriminative mutant ), which is typically selected by the very commonly employed antiretroviral therapy azt + 3tc ( combivir ). as shown in fig6 , 2 ′, 3 ′- dideoxy - 3 ′- c - hydroxymethylcytosine once again retained activity against this resistant virus , showing only a 1 . 78 - fold difference in susceptibility compared to wild type hiv . both 3tc and azt lost activity and showed reduced potency ( i . e . a pronounced reduction in viral susceptibility ) to the resistance virus ( fig6 ). continuous challenge of patients with antiretroviral agents results in the emergence of mdr . a t69s mutation with a 6 - bp insertion between amino acids 68 and 70 in the finger region of rt is often seen in combination with various forms of tams and contributes to an enhanced primer rescue activity . a cluster of mdr ( with different forms of amino acid insertion ( s )) in combination with tam was chosen , as outlined in table 3 . as shown in fig7 , the compound of the invention inhibited these patient isolates , giving the smallest change in drug susceptibility compared with six reference antivirals currently used in conventional antiretroviral therapy . note that a pronounced ( 500 to 1000 - fold ) reduction in susceptibility to azt was observed for patient isolates 32 and 35 whereas the compound of the invention showed changes of 2 . 79 and 4 . 29 - fold respectively . this is consistent with the compound of the invention displaying a different mechanism of inhibition compared to the obligatory dna chain terminators represented by conventional nrtis . isolate 4 represents a further discriminative mutant bearing the k65r + m184v genotype in a non - essential tam background consisting of mutations at r211s and k219e . this isolate causes a typical cross - resistance to abacavir , 3tc and the newly approved nucleoside ftc , but retains its susceptibility to thymidine analogues , such as azt and d4t . this isolate does not bear typical primer rescue mutations , yet the compound of the invention still inhibits this viral phenotype as indicated by an fc value of 3 . 88 . this value is comparable to the thymidine analogues , azt ( fc = 1 . 11 ) and d4t ( fc = 0 . 71 ), whereas significant resistance was found for 3tc ( fc & gt ; 200 ), ftc ( fc & gt ; 40 ) and to some extent to abc ( fc & gt ; 9 . 0 ). this experimental data demonstrates that the compound of the invention not only bears unique properties against “ primer rescue ” mutants but is also able to inhibit hiv mutants from the discriminative family . this , therefore , contrasts with the inhibitory mechanism employed by 3tc and ftc as well as the likely mechanism of the kodama 4 ′- c - ethynyl compounds described above in which m184v together with one additional amino acid change in codon t165r in the catalytic region contributes to cross - resistance to 4 - c - ethynyl nucleoside ( kodama 2002 ). the antiviral performance of the compound of the invention against additional tam primer rescue - related resistant hiv isolates was assayed in a pbmc culture . isolates of hiv - 1 were generated and expanded to high titer by co - cultivation of infected patient pbmc with pha - stimulated donor pbmc ( virology manual for actg hiv laboratories ). the cell - free supernatants were harvested , sequenced , and stored in aliquots at − 70 ° c . for drug susceptibility assays . in vitro drug susceptibility assays were performed using a modified actg / dod consensus method ( virology manual for actg hiv laboratories ). pbmcs were pre - infected with viral stocks for 4 hrs at 37 ° c . in a humidified atmosphere of 5 % co 2 following 4 hr incubation . infected cells were washed twice in media and pipetted into a microtiter plate with eight serial drug dilutions . each well contained 100 , 000 pre - infected pbmc and all drug dilutions were made with cell culture medium . the drug dilutions were chosen to span the 50 % inhibitory concentration ( ic 50 ) for each single drug . control wells containing cells and virus were co - incubated on each plate . after a 7 - day incubation at 37 ° c . in a mummified atmosphere of 5 % co 2 , viral growth was determined using a p24 antigen assay on supernatants ( abbott laboratories , chicago , usa ). the percent inhibition of viral growth compared to the control well , which contained no drug , was calculated and expressed as fold changes ( reductions in compounds susceptibility ) compared to the control well . the reference compound azt was run in parallel with the compound of the invention . a cluster of representative of primer rescue - related mutant virus was selected that harbors the essential feature of primer rescue - related tam resistant rt mutations . strains with mutations at position m41l , d67n , k70r , l210w , t215y / f and k219q / e in various combinations with or without discriminative mutant m184v were used as indicated in table 4 . most of these selected primer rescue mutants conferred a pronounced resistance to azt susceptibility , dropping a couple of hundred folds in fc value . the exception was isolate 7086 ( fc = 3 . 0 ), which bears the t215v amino acid mutation . a complete report of fc values is presented in fig8 . here , 2 ′, 3 ′- dideoxy - 3 ′- c - hydroxymethylcytosine inhibited all 8 - isolates , with the highest fc value being only 2 . 7 . the presence of a 3 ′- hydroxymethyl group in the compound of the invention should , in principle , support incorporation and elongation into the viral nucleic acid catalyzed by hiv - 1 rt . a rate - limiting amount of primer - template ( 16s and 23s ribosomal rna annealed with an oligo - dna primer with the sequence of 5 ′- taaccttgcggccgt - 3 ′ ( seq id no : 1 ), custom synthesized by innovagen ) was used . this was pre - incubated with 100 μm ( 55 times the ic 50 ) 2 ′- 3 ′- dideoxy - 3 ′- c - hydroxymethylcytosine - triphosphate , 6 . 0 μm ddc - triphosphate ( 54 times the ic 50 ddctp ), 20 μm deoxycytosine - triphosphate ( 20 times the km dctp ) or the control ( h 2 o ). at the time points indicated ( 0 , 10 , 30 , 60 , and 120 min ), the dna polymerization process was stopped by inactivation of the rt at 70 ° c . for 2 min during the first round of dna polymerization ( fig9 ). the residual amount of primer - template directly reflects the availability of free 3 ′- oh primer terminus present after the first round of reaction . in order to measure this , a new polymerization was initiated by addition of fresh rt in the presence of 150 μm ( 160 times the km ) dctp and tritium - labeled dctp which is sufficient to compete out any inhibitory effect from the residual 2 ′- 3 ′- dideoxy - 3 ′- c - hydroxymethylcytosine tp and ddctp that is left from the first round of dna polymerization . the availability of free 3 ′- oh at the primer terminus in the residual amount of primer - template that supported further dna polymerization was measured and expressed as a function of pre - incubation time ( fig1 ). with reference to fig9 & amp ; 10 , although ddctp and 2 ′ 3 ′- dideoxy - 3 ′- c - hydroxymethyl tp were set to provide an equal inhibitory level , there was a sharp contrast in their respective ability to support the second round of hiv rt - catalyzed dna polymerization . pre - incubation with an obligatory chain terminator such as ddctp causes chain termination and gives a significant reduction of free 3 - oh primer terminus compared to the triphosphate of the compound of the invention . at pre - incubation time points of 10 and 30 minutes , less than half the amount of residual 3 - oh primer terminus was left to support further dna prolongation when ddctp is present compared to the triphosphate of the compound of the invention , notwithstanding that comparable amounts of the tp compounds were used in the first round of dna polymerization . this clearly indicates that the incorporation of the tp of the compound of the invention into the nascent nucleic acid provides continued opportunity for some further dna synthesis . the dna polymerization must include binding of the enzyme to the template , complex with appropriate dntp , phospho - diester formation , liberation of pyrophosphate and translocation of the enzyme from the n - site into the p - site in order for the next round of synthesis to occur . it is therefore apparent that the compound of the invention is able to be incorporated and translocated by the enzyme into the next position , after which further elongation ceases . incorporation of 2 ′, 3 ′- dideoxy - 3 ′- c - hydroxymethylcytosine monophosphate leads to a different chain termination pattern compared to ddc two deoxycytosine analogues , the conventional nrti ddc lacking a 3 ′- hydroxyl group function and the compound of the invention , were subjected to a dna chain termination assay in which dna prolongation was conducted with m13 mp 18 single strand dna template pre - annealed to an oligo - dna primer ( the forward primer sequence of 5 ′- gttttcccagtcacgacgttgta - 3 ′ ( seq id no : 2 ) was purchased from amersham uk . m13 mp 18 single strand rna was annealed to this oligo - dna primer giving a final concentration of 1 mg / ml in a buffer containing 10 mm tris - hcl , ph 7 . 9 and 100 mm nacl , and was stored in aliquots at − 20 ° c . dna polymerization was conducted using this annealed template / primer , hiv - 1 rt and natural dntps in a reaction incubated at 37 ° c . for 25 min . the reaction was stopped by the addition of stop solution containing 95 % formamide , 20 mm edta , 0 . 05 % bromophenol blue and 0 . 05 % xylene cyanol ff ( purchased from usb , united states biochemical via amersham uk ). after denaturing the dna products , the elongated dna fragment was electrophoresed on an 8 . 0 % polyacrylamide gel and visualized using autoradiography . the assay included a negative control ( the native dntp ), dual positive ddctp controls ( ddctp , 8 um from sigma chemical , st . louis , miss ., usa and ddctp obtained from a usb sequence kit united states biochemical via amersham uk ). various molecule ratios of the triphosphate of the compound of the invention and the natural dntp were used . after the reaction was conducted as described above , the denatured dna fragments from each individual reaction was loaded onto an 8 . 0 % polyacrylamide gel in the following order : 1 — negative control 2 — 1 st positive control 8 μm ddctp ( purchased from sigma ) 3 — 20 μm invention tp in 200 μm dntp 4 — 30 μm invention tp in 300 μm dntp 5 — 40 μm invention tp in 400 μm dntp 6 — 50 μm invention tp in 500 μm dntp 7 — 20 μm invention tp in 80 μm dntp 8 — 40 μm invention tp in 80 μm dntp 9 — empty space ( no loading ) 10 — 2 nd positive control ddctp ( from usb sequence kit ) in order to avoid any other factor which may influence interpretation of assay outcome , such as the edge effect associated with polyacrylamide gels , a duplicate set of samples were loaded in the middle of the gel . fig1 shows a digital photo from depicting autoradiology results obtained from the central part of the gel : 1 . negative control ( dntp ): no pause in dna polymerization was found . 2 . 1 st positive control 8 μm ddc - tp : led to chain termination at the anticipated 2 ′ 3 ′- deoxydeoxycytosine sites . 3 . 20μ invention tp / 200 μmdntp : led to chain termination at the site after / behind 2 ′ 3 ′- deoxydeoxycytosine site . 4 . 30 μm invention tp / 300 μmdntp : led to chain termination at the site after 2 ′ 3 ′- deoxydeoxycytosine site compared to ddc - tp . 5 . 40 μm invention tp / 400 μmdntp : lead to chain termination at the site after 2 ′ 3 ′- deoxydeoxycytosine site compared to ddc - tp . 6 . 50 μm invention tp / 500 μmdntp : no specific pause ( chain termination pattern ) was found , but is considered to be within experimental error . 7 . 20 μm invention tp / 80 μmdntp : led to a more pronounced chain termination effect at the site after the 2 ′ 3 ′- deoxydeoxycytosine site compared to ddc - tp and the invention experimental sample intermediately above . 8 . 40 μm invention tp / 80 μmdntp : led to a more pronounced chain termination effect at the site after the 2 ′ 3 ′- deoxydeoxycytosine site compared to ddc - tp and the invention experimental sample intermediately above . 9 . empty space ( no sample loaded ) 10 . 2 nd positive control ddc - tp : led to chain termination at anticipated 2 ′ 3 ′- deoxydeoxycytosine sites . the compound of the invention has induced dna chain termination in all samples , with the exception of the sample no . 6 which contains 50 μm of the invention &# 39 ; s tp in 500 μm dntp . the reason for this exception is unknown , but is likely within experimental error . interestingly , dna fragments resulting from incorporation of 2 ′, 3 ′- dideoxy - 3 ′- c - hydroxymethylcytosine monophosphate migrate more slowly than those resulting from the two positive control ddctp terminated dna fragments ( fig1 ). the duplicated reactions show a consistent pattern which implies that the compound of the invention is incorporated into the newly synthesized dna strand and allows the formation of a further 3 ′, 5 ′- phosphodiester bond in the next round of nucleotide incorporation . although a fragment longer by one base was observed , it cannot be excluded that the sequence of the template employed may play a role . example 3 clearly shows that the 3 ′- oh primer terminus which was pre - terminated by the compound of the invention supports further nucleotide incorporation more than a ddctp pre - terminated 3 ′- oh primer terminus , when a ribosomal rna template is used . this feature causes the slow electrophoresis mobility and implies that the rt has undergone translocation to begin the next round of polymerization . although not wishing to be bound by this mechanism , it is believed that the compound of the invention therefore represents a new strategy in inhibiting primer excision mutants . that is the compound is incorporated into the growing viral genome while simultaneously retaining the ability to allow the rt molecule to undergo the necessary transformational change in order to prepare for the next round of dna synthesis . examples 3 and 4 have clearly demonstrated that the compound of the invention bears such properties and thus it is able to defeat / counteract the primer rescue resistant mechanism as demonstrated in examples 1 and 2 . sarafinano et al . ( 2002 and 2003 ) provide compelling experimental data supporting the conclusion that the primer rescue reaction can only occur before rt translocates into the next position . that is , the pre - translocation complex is a prerequisite condition for a primer rescue mutant to be effective . the evidence presented in the examples suggests that is not the case for the compound and methods of the current invention . the 3 ′- mmtr / 5 ′- tmbds differentially protected compound 1 is prepared as the corresponding uridine as described by sanghvi et al : synthesis ( 1994 ) p 1163 & amp ; tetrahedron lett v 35 ( 1994 ) p 4697 & amp ; nucleosides & amp ; nucleotides v 15 ( 1996 ) 1383 . the u to c conversion is shown in koziov et al nucleosides & amp ; nucleotides , v17 ( 1998 ) 2249 . compound 1 ( 5 . 0 g , 6 . 7 mmol ) was dissolved in 80 % acetic acid ( 30 ml ) and stirred for 24 h at room temperature . the mixture was evaporated and the product was purified by flash chromatography 5 to 10 % meoh in ch 2 cl 2 as eluent . the yield 2 . 1 g ( 64 %). compound 1 ( 2 . 3 g , 3 . 06 mmol ) in thf ( 150 ml ) was treated with tetrabutylammonium fluoride ( 1m in thf , 3 . 0 ml ) for 1 h at room temperature . sodium bicarbonate ( sat , 100 ml ) was added and the mixture was extracted with dichloromethane ( 3 × 50 ml ). the organic layer was dried and evaporated . the residue was purified by flash chromatography to give 1 . 3 g ( 82 %) of compound 3 . triethylamine ( 0 . 455 g , 4 . 5 mmol ) and ethyl chloroformate ( 0 . 26 g , 2 . 4 mmol ) were added to the solution of boc - valine ( 0 . 49 g , 2 . 25 mmol ) in thf ( 15 ml ) at 0 ° c . the reaction mixture was stirred for 3 h at same temperature and then filtrated to the solution of compound 2 ( 0 . 72 g , 1 . 5 mmol ) and dmap ( 0 . 55 g , 4 . 5 mmol ) in thf ( 15 ml ). the reaction mixture was stirred overnight at room temperature . etoac was added to the mixture and it was washed three times with 2 % citric acid and once with sat . nahco 3 . the organic layer was dried over na 2 so 4 , filtrated and evaporated . the residue was purified by flash chromatography 2 to 5 % meoh in ch 2 cl 2 as eluent to give 0 . 35 g ( 34 %) of compound 4 . compound 4 ( 30 mg , 0 . 066 mmol ) was dissolved in conc . hcl ( 1 ml ) at room temperature and stirred for 5 min . acetone was added to the solution and it was evaporated . acetone was added again and the solution was evaporated and dried under vacuum to give 18 mg ( 69 %) of compound 5 . compound 4 ( 0 . 35 g , 0 . 5 mmol ) was dissolved in thf ( 20 ml ) and 1 . 0 m tetrabutylammonium fluoride in thf ( 0 . 5 ml , 0 . 5 mmol ) was added . the reaction mixture was stirred for 2 days at room temperature . the mixture was evaporated and the product was purified by flash chromatography 5 to 10 % meoh in ch 2 cl 2 as eluent to give 0 . 225 g ( 98 %) of compound 6 . the synthesis was made in the same manner as for the compound 4 using compound 6 as starting material . compound 7 ( 75 mg , 0 . 114 mmol ) was dissolved in 3 ml of meoh and conc . hcl ( 0 . 5 ml ) was added at 0 ° c . the mixture was stirred for 5 min at 0 ° c . and for 3 min at room temperature and after that evaporated . acetone was added to the residue and it was evaporated . ch 2 cl 2 was added and the residue was evaporated and dried under vacuum to give 58 mg ( 96 %) of compound 8 . ethyl chloroformate ( 110 mg , 1 . 0 mmol ) was added to the solution of boc - valine ( 220 mg , 1 . 0 mmol ) and trithylamine ( 200 mg , 2 . 0 mmol ) in thf ( 30 ml ) at 0 ° c . and the mixture was stirred for 3 h . the temperature was allowed to reach room temperature and the mixture was filtered . the filtrate was added to the solution of compound 3 ( 350 mg , 0 . 68 mmol ) and dmap ( 244 mg , 2 . 0 mmol ) in thf ( 20 ml ). the reaction mixture was stirred overnight at room temperature , ethyl acetate ( 100 ml ) was added to the mixture and it was washed with citric acid ( 10 %, 2 × 30 ml ) and sodium bicarbonate ( sat , 30 ml ). solvent was removed and the product was separated on silica gel column to give compound 9 ( 220 mg , 45 %). compound 9 ( 200 mg , 0 . 28 mmol ) was dissolved in 3 ml of conc . hcl and stirred for 3 min at room temperature . the mixture was evaporated , washed with acetone , acetonitrile and diethyl ether and dried under vacuum to give 85 mg ( 77 %) of compound 10 . ethyl chloroformate ( 110 mg , 1 . 0 mmol ) was added to the solution of boc - valyl - lactic acid ( 290 mg , 1 . 0 mmol ) and triethylamine ( 200 mg , 2 . 0 mmol ) in thf ( 30 ml ) at 0 ° c . and the mixture was stirred for 3 h at 0 ° c . the temperature was allowed to reach room temperature . the mixture was filtered and the filtrate was added to the solution of compound 3 ( 300 mg , 0 . 58 mmol ) and dmap ( 244 mg , 2 . 0 mmol ) in thf ( 20 ml ). the reaction mixture was stirred overnight at room temperature , ethyl acetate ( 100 ml ) was added to the mixture and it was washed with citric acid ( 10 %, 2 × 30 ml ) and sodium bicarbonate ( sat , 30 ml ). solvent was removed and the product was separated on silica gel column to give compound 11 ( 250 mg , 38 %). the compound was prepared from compound 11 in the same manner as compound 8 . the compound was prepared in the same manner from compound 2 as compound 4 by using boc - valyl - lactic acid as starting material instead of boc - valine . compound 1 ( 1 g , 1 . 33 mmol ) was dissolved in 14 ml of conc . hcl and the mixture was stirred for 8 min at room temperature and then evaporated . the residue was washed with acetone and filtrated to give 334 mg ( 90 %) of compound 15 . triethylamine ( 0 . 487 g , 4 . 82 mmol ) and ethyl chloroformate ( 0 . 30 g , 2 . 77 mmol ) were added to the solution of boc - valine - lactic acid ( 0 . 77 g , 2 . 65 mmol ) in thf ( 30 ml ) at 0 ° c . the reaction mixture was stirred for 3 h at same temperature and then filtrated to the solution of compound 15 ( 0 . 334 g , 1 . 20 mmol ) and dmap ( 0 . 74 g , 6 . 0 mmol ) in thf ( 30 ml ) and dmf ( 30 ml ). the mixture was stirred overnight at room temperature . etoac was added to the mixture and it was washed three times with 2 % citric acid and once with sat . nahco 3 . the organic layer was dried over na 2 so 4 , filtrated and evaporated . the crude product was purified by flash chromatography 2 to 5 % meoh in ch 2 cl 2 as eluent to give only 65 mg ( 8 %) of compound 16 . valeryl chloride ( 460 mg , 3 . 8 mmol ) was added to the solution of valeric acid ( 390 mg , 3 . 8 mmol ) and triethylamine ( 770 mg , 7 . 6 mmol ) in thf ( 50 ml ) at 0 ° c . and the mixture was stirred for 3 h , and then filtered . the filtrate was added to the solution of compound 3 ( 1 . 3 g , 2 . 53 mmol ) and dmap ( 930 mg , 7 . 6 mmol ) in thf ( 50 ml ). the reaction mixture was stirred overnight at room temperature . citric acid ( 10 %, 50 ml ) was added to the mixture and it was extracted with ethyl acetate ( 2 × 50 ml ). the combined organic layer was washed with citric acid ( 10 %, 30 ml ) and then with sodium carbonate ( 50 ml ) and brine ( 50 ml ). after drying , the organic solvent was removed and the residue was separated on silica gel column to give compound 18 ( 850 mg , 56 %). compound 18 ( 850 mg , 1 . 42 mmol ) was dissolved in methanol ( 10 ml ) and conc . hcl ( 1 . 5 ml ) was added to the solution at 0 ° c . the reaction mixture was stirred for 0 . 5 h . sodium carbonate ( 50 ml ) was added to the mixture and it was extracted with dichloromethane ( 3 × 50 ml ). solvent was removed and the product was separated on silica gel column to give compound 19 ( 230 mg , 50 %). compound 2 ( 1 . 02 g , 2 . 1 mmol ) and dmap ( 1 . 05 g , 8 . 61 mmol ) were dissolved in thf ( 35 ml ) and cooled to − 78 ° c . valeryl chloride ( 6 × 42 μl , 2 . 14 mmol ) was added during 15 min . the mixture was stirred in cold temperature for 2 h and then 1 h without cooling bath . the reaction mixture was poured to 5 % citric acid and then extracted with etoac . the combined organic layers were washed with brine , dried over na 2 so 4 and evaporated to give a white solid which was purified on silica gel column with 0 to 6 % meoh in ch 2 cl 2 as eluent to give 0 . 31 g ( 25 %) of compound 23 . compound 23 ( 0 . 35 g , 0 . 58 mmol ) was dissolved in thf ( 20 ml ), 1 m tbaf in thf ( 0 . 58 ml , 0 . 58 mmol ) was added and the mixture was stirred for 90 min at room temperature . the solvent was evaporated and the residue was purified by flash chromatography with 0 to 10 % meoh in ch 2 cl 2 as eluent to give 166 mg ( 92 %) of compound 24 . confirmation that the prodrugs of the invention convert completely to active 2 ′ 3 ′- dideoxy - 3 ′- c - hydromethylcytosine parent compound can be assessed by monitoring the appearance of the parent compound in pooled human plasma , 37c , spiked with 5 um of the prodrug : prodrugs are typically blended in mq grade water , 3 mg / ml and orally administered by intubation to duplicate rats . a suitable dose is 5 mg / kg . plasma samples are taken at suitable timepoints , such as t0 , 15 & amp ; 30 minutes , 1 , 2 , 4 and 6 hours . recovery ( as the metabolite 2 ′, 3 ′- dideoxy - 3 ′- c - hydroxymethyl - β - d - erythropentofuranosylcytosine ) in the plasma is measured with mass spectrometry , detected as the sodium adduct m / z 264 ( m + na )+. results are plotted as plasma concentration against time and generally show a cmax of the order of 3 to 5 um . absolute bioavailability % f is calculated in the conventional manner , ie by reference to clearance of an in vitro dose of the parent , as shown in wo97 / 30051 . as rats cannot be infected with hiv , the antiretroviral activity of such oral formulations cannot be directly measured , but it is noted that the ed 50 for the metabolite 2 ′, 3 ′- dideoxy , 3 ′- c - hydroxymethyl - β - d - erythropento - furanosylcytosine is typically around 0 . 01 um in human h9 cells . this in turn means that peak plasma concentrations of the order of magnituted seen with these prodrugs is several hundredfold over the ed 50 . other pharmaceutical parameters such as auc and clearance are typically consistent with achieving a 24 hour trough level well over the ed 50 with qd or bid dosing . each of the patent and scientific references cited in the text are listed below and are hereby incorporated by reference in their entirety . brigitte montes and michel segondy ( 2002 ) prevalence of the mutational pattern e44d / a and / or v118l in the reverse transcriptase ( rt ) gene of hiv - 1 in relation to treatment with nucleoside analogue rt inhibitors , j med virol . 66 ( 3 ): 299 - 303 . boyer p l , imamichi t , sarafianos s g , arnold e , hughes s h ( 2004 ) effects of the delta67 complex of mutations in human immunodeficiency virus type 1 reverse transcriptase on nucleoside analog excision . j virol . 78 ( 18 ): 9987 - 9997 . boyer p l , sarafianos s g , arnold e , hughes s h ( 2002 ) nucleoside analog resistance caused by insertions in the fingers of human immunodeficiency virus type 1 reverse transcriptase involves atp - mediated excision . 76 ( 18 ): 9143 - 9151 girouard m , diallo k , marchand b , mccormick s and gotte m ( 2003 ) mutations e44d and v1181 in the reverse transcriptase of hiv - 1 play distinct mechanistic roles in dual resistance to azt and 3tc . j biol chem : 5 ; 278 ( 36 ): 34403 - 34410 . harrigan , p . r ., c . stone , p . griffin , i . nájera , s . bloor , s . kemp , m . tisdale , b . larder , and the cna 2001 investigative group ( 2000 ) resistance profile of the human immunodeficiency virus type 1 reverse transcriptase inhibitor abacavir ( 1592u89 ) after monotherapy and combination therapy . j . infect . dis . 181 : 912 - 920 imamichi , t ., t . sinha , h . imamichi , y .- m . zhang , j . a . metcalf , j . falloon , and h . c . lane . 2000 . high - level resistance to 3 ′- azido - 3 ′- deoxythymidine due to a deletion in the reverse transcriptase gene of human immunodeficiency virus type 1 . j . virol . 74 : 1023 - 1028 . imamichi , t ., m . a . murphy , h . imamichi , and h . c . lane . 2001 . amino acid deletion at codon 67 and thr - to - gly change at codon 69 of human immunodeficiency virus type 1 reverse transcriptase confer novel drug resistant profiles . j . virol . 75 : 3988 - 3992 . jacobo - molina , a ., j . ding , r . g . nanni , a . d . clark , jr ., x . lu , c . tantillo , r . l . williams , g . kamer , a . l . ferris , p . clark , and e . arnold ( 1993 ) crystal structure of human immunodeficiency virus type 1 reverse transcriptase complexed with double - stranded dna at 3 . 0 å resolution shows bent dna . proc . natl . acad . sci . usa 90 : 6320 - 6324 kemp , s . d ., c . shi , s . bloor , p . r . harrigan , j . w . mellors , and b . a . larder . 1998 . a novel polymorphism at codon 333 of human immunodeficiency virus type 1 reverse transcriptase can facilitate dual resistance to zidovudine and l - 2 ′, 3 ′- dideoxy - 3 ′- thiacytidine . j . virol . 72 : 5093 - 5098 larder b a , kemp s d ( 1989 ) multiple mutations in hiv - 1 reverse transcriptase confer high - level resistance to zidovudine ( azt ). science . 1 ; 246 ( 4934 ): 1155 - 1158 larder , b . a ., s . bloor , s . d . kemp , k . hertogs , r . l . desmet , v . miller , m . sturmer , s . staszewski , j . ren , d . k . stammers , d . i . stuart , and r . pauwels . 1999 . a family of insertion mutations between codons 67 and 70 of human immunodeficiency virus type 1 reverse transcriptase confer multinucleoside analog resistance . antimicrob agents chemother . 43 : 1961 - 1967 miller , v ., a . phillips , c . rottmann , s . staszewski , r . pauwels , k . hertogs , m . p . de béthune , s . d . kemp , s . bloor , p . r . harrigan , and b . a . larder . 1998 . dual resistance to zidovudine ( zdv ) and lamivudine ( 3tc ) in patients treated with zdv / 3tc combination therapy : association with therapy failure . j . infect . dis . 177 : 1521 - 1532 . mas a ., m . parera , c . briones , v . soriano , m . a . martinez , e . domingo , and l . menéndez - arias . 2000 . role of a dipeptide insertion between codons 69 and 70 of hiv - 1 reverse transcriptase in the mechanism of azt resistance . embo j . 21 : 5752 - 5761 meyer p r , matsuura s e , tolun a a , pfeifer i , so a g , mellors j w , scott w a ( 2002 ) effects of specific zidovudine resistance mutations and substrate structure on nucleotide - dependent primer unblocking by human immunodeficiency virus type 1 reverse transcriptase antimicrob agents chemother . 46 ( 5 ): 1540 - 1545 . meyer p r , lennerstrand j , matsuura s e , larder b a , scott w a ( 2003 ) effects of dipeptide insertions between codons 69 and 70 of human immunodeficiency virus type 1 reverse transcriptase on primer unblocking , deoxynucleoside triphosphate inhibition , and dna chain elongation . j virol . 77 ( 6 ): 3871 - 3877 miller , v ., m . ait - khaled , c . stone , p . griffin , d . mesogiti , a . cutrell , r . harrigan , s . staszewski , c . katlama , g . pearce , and m . tisdale ( 2000 ) hiv - 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there is provided inter alia use of 2 ′, 3 ′- dideoxy - 3 ′- hydroxymethylcytosine or a prodrug or salt thereof in the manufacture of a medicament for the treatment of hiv infection wherein the reverse transcriptase of the hiv bears at least one mutation that allows an obligate chain terminating nucleoside - or nucleotide phosphate to be excised from the nascent dna strand by atp - or pyrophosphate - mediated excision .