Patent Application: US-84894704-A

Abstract:
compounds of formula , or pharmaceutically acceptable salts or esters thereof , are inhibitors of reverse transcriptase : r 4 is a nucleoside with q substituting a 3 ′ hydroxyl group , and q is a moiety of formulas -:

Description:
the present invention makes use of rational drug design and of the discovery that novel nucleoside analogs having structures with a branched 3 ′- group containing a carbon or sulfur central atom fit into the 3 ′- pocket of hiv rt , but do not bind to human dna polymerases . these novel molecules act as substrates for rt , and like other nucleoside drugs , terminate reverse transcription prematurely . unlike currently available nucleoside analogs , the molecules of the invention show greater specificity for rt compared to endogenous human dna polymerases . these molecules take advantage of the tertiary structural differences between human dna polymerases and hiv rt . a similar molecule ( i ) is ineffective in vivo ; in that case , a nitrogen at the 3 ′- branched group proved ineffective ( hossain et al ., 1993 ; huang et al ., 1995 ). furthermore , these inhibitors are expected to be effective against rt of other retroviruses . examining the crystal structure of ternary complexes of hiv rt covalently tethered via a disulfide bond to a dna template at 3 . 2 å ( huang et al ., 1998 ) revealed , for the first time , the details of the dntp binding site — where nucleoside analog inhibitors also bind ( fig1 ). like most other dna polymerases , hiv rt can be described as being a right hand , with a top 1 , thumb 2 , bottom ( palm ) 3 and fingers 4 . when hiv rt forms a complex with a dna template : primer and deoxythymidine triphosphate ( dttp ), the dttp occupies a cleft composed of part of the thumb 2 and fingers 4 , with the palm 3 as the base . the 3 ′- hydroxyl group of the dttp 5 , which is modified in nucleoside analog drugs , projects into a pocket . the triphosphate portion 6 is also shown for orientation . compared to that of human dna polymerases , this “ 3 ′- pocket ” is large , being able to accommodate an azido group like that of azt - triphosphate ( azttp ). the large 3 ′- pocket is the critical difference that allows for specificity , where nucleoside inhibitors bind to hiv rt but not human dna polymerases . the azido group of azttp can occupy the large 3 ′- pocket either at the top 1 or bottom 3 . a branched 3 ′- group with a sp2 central atom will fit into the entire 3 ′- pocket . further considerations of the size limitation of the 3 ′- pocket leads to a structure ( ii ): r 2 is h , f , ch 3 , oh , nh 2 or a moiety of structure eight members of this new class of nucleosides are of particular interest ( v )-( xii ): in another embodiment , the novel nucleoside rt inhibitors have the general structure ( xxiii ): r 2 is h , f , ch 3 , oh , nh 2 or a moiety of structure r 4 is a naturally - occurring nucleoside , wherein the 3 ′ hydroxyl group is substituted with the q moiety ( xiii )-( xvi ): in another embodiment , the r 4 is a universal ( non - natural or natural ) nucleoside , such as ( xvii )-( xxii ) these novel molecules inhibit hiv reverse transcriptase and represent new and important weapons in the molecular arsenal to combat the debilitating effects of hiv infection . nucleosides encompass bases conjugated to ribose and deoxyribose , yielding nucleosides and deoxynucleosides . natural nucleosides are those that are found in dna and rna , such as the nucleosides that contain adenine , thymine , guanine , cytosine and uracil . universal nucleosides refer to those that exhibit the ability to replace at least one of the natural nucleosides without significantly destabilizing neighboring base - pair interactions . examples of universal nucleosides include 3 - nitropyrrole 2 ′- deoxynucleoside , 5 - nitroindole 2 ′- deoxynucleoside , 6h , 8h - 3 , 4 - dihydropyrimido [ 4 , 5 - c ][ 1 , 2 ] oxazin - 7 - one - 2 ′- deoxynucleoside , 2 , 6 - diaminopurine - 2 ′- deoxynucleoside , inosine - 2 ′- deoxynucleoside and 2 - aminopurine - 2 ′- deoxynucleoside . the inhibitors ( v )-( xii ) may be synthesized using standard chemical synthesis ; reagents are generally available from sigma - aldrich ( st . louis , mo .). for the synthesis of ( v )-( xi ), the intermediate ( xxxvii ) is first made ( table 4 ). as an example describing the preparation of ( xxxvii ) ( table 4 ), 10 g of ( xxxiii ) ( t , thymidine ) is reacted with 10 g of 4 , 4 ′- dimethyoxytrityl chloride ( dmtrcl ) in the presence of 4 -( dimethylamino ) pyridine ( dmap ) as a catalyst and pyridine as a solvent . after purification of the reaction mixture by column chromatography , 21 g of ( xxxiv ) ( 93 % yield ) is typically obtained as pale yellow foam . reacting of 15 g of ( xxxiv ) with 7 . 7 g of o -( p - tolyl ) chlorothionoformate ( toloc ( s ) cl ) in the presence of dmap , triethylamine , and methylene chloride , results in typically 16 . 6 g of ( xxxv ) ( 87 % yield ) after purification . compound ( xxxv ) is converted into ( xxxvi ) by reacting with β - tributylstannyl styrene using 2 , 2 ′- azobis ( 2 - methylpropionitrile ) ( aibn ) as a catalyst , affording typically 6 . 3 g of ( xxxvi ) ( 58 % yield ) after purification . the key intermediate ( xxxvii ) is obtained ( typically 2 . 6 g , 30 % yield ) by oxidative cleavage of the double bond in ( xxxvi ) with oso 4 / naio 4 . as an example of preparing ( v ), 100 mg of ( xxxvii ) is reacted with i 2 in a mixture of acetonitrole and water , affording typically 95 mg of ( xxxviii ) ( 93 % yield ) after purification . deprotection of the dmt group in ( xxxviii ) by treating with trichloroacetic acid ( ccl 3 cooh ) in methylene chloride ( ch 2 cl 2 ) typically results in 37 mg ( 98 % yield ) of ( v ). the scheme is depicted in table 5 . as an example of preparing ( vi ), 100 mg of ( xxxvii ) is first treated with aibn and n - bromosuccinimide ( nbs ) at 70 ° c . for two hours . the reaction mixture is cooled to 0 ° c ., and the ammonia gas is bubbled through the mixture . typically , 86 mg of ( xxxix ) ( 84 % yield ) is recovered after purification by column chromatography . the same deprotection procedure used in the synthesis of ( v ) is applied to obtain typically 33 mg of ( vi ) from ( xxxix ) ( 87 % yield ). the synthesis is shown in table 6 . as an example , 100 mg of ( xxxvii ) and cerium chloride ( cecl 3 ) are treated with methylmagnesium bromide ( memgbr ) at − 10 ° c . for 3 hours . after working up , the obtained alcohol intermediate was treated with dess - martin reagent to yield 87 mg of ( xli ) ( 85 %), which was then deprotected as for ( v ), giving 36 mg of ( vii ). as an example , 100 mg of ( xxxvii ) is treated with i 2 in methanol at 0 ° c . to convert ( xxxvii ) to ( xl ) ( typically 100 mg , 96 % yield ). deprotection of dmt group in ( xl ) typically results in 46 mg of ( viii ) ( 95 % yield ). the synthesis is shown in table 8 . pharmaceutically acceptable salts , such as alkali metal , alkaline earth or ammonium salts , and esters , such as methoxy , ethoxy , 5 ′- monophosphate , 5 ′- diphosphate and 5 ′- triphosphate , of any of the rt inhibitors herein described are contemplated . the compounds of the invention , such as ( v )-( xii ), can be incorporated into pharmaceutical compositions . such compositions typically comprise the compound and a pharmaceutically acceptable carrier . a “ pharmaceutically acceptable carrier ” includes solvents , dispersion media , coatings , antibacterial and antifungal agents , isotonic and absorption delaying agents , etc ., compatible with pharmaceutical administration . preferred examples of such carriers or diluents include water , saline , finger &# 39 ; s solutions , dextrose solution , and 5 % human serum albumin . liposomes and non - aqueous vehicles such as fixed oils may also be used . except when a conventional media or agent is incompatible with an active compound , use of these compositions is contemplated . supplementary active compounds can also be incorporated into the compositions . a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration , including intravenous , intradermal , subcutaneous , oral , inhalation , transdermal , transmucosal , and rectal administration . solutions or suspensions used for parenteral , intradermal or subcutaneous application include sterile diluents for injection , such as water , saline , fixed oils , polyethylene glycols , glycerine , propylene glycol or other synthetic solvents ; antibacterial agents such as benzyl alcohol or methyl parabens ; antioxidants such as ascorbic acid or sodium bisulfite ; chelating agents such as ethylenediaminetetraacetic acid ( edta ); buffers such as acetates , citrates or phosphates , and agents for the adjustment of tonicity such as sodium chloride or dextrose . the ph can be adjusted with acids or bases , such as hydrochloric acid or sodium hydroxide , if necessary . parenteral preparations can be enclosed in ampules , disposable syringes or multiple dose vials made of glass or plastic . pharmaceutical compositions suitable for injection include sterile aqueous solutions for water - soluble compounds , or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion . for intravenous administration , suitable carriers include physiological saline , bacteriostatic water , cremophor el ™ ( basf , parsippany , n . j .) or phosphate buffered saline ( pbs ). in all cases , the composition must be sterile and should be fluid for administration using a syringe . such compositions should be stable during manufacture and storage and must be preserved against contamination from microorganisms , such as bacteria and fungi . the carrier can be a solvent or dispersion medium containing , for example , water , ethanol , polyol ( such as glycerol , propylene glycol , and liquid polyethylene glycol ), and suitable mixtures . proper fluidity can be maintained , for example , by using a coating such as lecithin , by maintaining the required particle size in the case of dispersion and by using surfactants . various antibacterial and antifungal agents , for example , parabens , chlorobutanol , phenol , ascorbic acid , and thimerosal , may be added . isotonic agents , for example , sugars , polyalcohols such as manitol , sorbitol , and sodium chloride can be included in the composition . compositions that can delay absorption include agents such as aluminum monostearate and gelatin . sterile injectable solutions can be prepared by incorporating the active compound ( e . g ., compounds ( v )-( xii )) in the required amount in an appropriate solvent with one or a combination of ingredients as required , followed by sterilization . generally , dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium , and the other required ingredients . sterile powders for the preparation of sterile injectable solutions , which methods of preparation include vacuum drying and freeze - drying to yield a powder containing the active compound and any desired ingredient from a sterile solution , may also be supplied . oral compositions generally include an inert diluent or an edible carrier . they can be enclosed in gelatin capsules or compressed into tablets . for the purpose of oral therapeutic administration , the active compound can be incorporated with excipients and used in the form of tablets , troches , or capsules . oral compositions can also be prepared using a fluid carrier for use as a mouthwash , wherein the compound in the fluid carrier is applied orally . pharmaceutically compatible binding agents , and / or adjuvant materials can be included . tablets , pills , capsules , troches , etc ., can contain any of the following ingredients , or compounds of a similar nature : a binder , such as microcrystalline cellulose , gum tragacanth or gelatin ; an excipient , such as starch or lactose ; a disintegrating agent , such as alginic acid , primogel , or corn starch ; a lubricant , such as magnesium stearate or sterotes ; a glidant , such as colloidal silicon dioxide ; a sweetening agent , such as sucrose or saccharin ; or a flavoring agent such as peppermint , methyl salicylate , or orange flavoring . for administration by inhalation , the compounds are delivered as an aerosol spray from a nebulizer or a pressurized container that contains a suitable propellant , e . g ., a gas such as carbon dioxide . administration can also be transmucosal or transdermal . for transmucosal or transdermal administration , penetrants that can permeate the target barrier ( s ) are selected . transmucosal penetrants include detergents , bile salts , and fusidic acid derivatives . nasal sprays or suppositories can be used for transmucosal administration . for transdermal administration , the active compounds are formulated into ointments , salves , gels , or creams . the compounds can also be prepared in the form of suppositories ( e . g ., with bases such as cocoa butter and other glycerides ) or retention enemas for rectal delivery . in one embodiment , compounds ( v )-( xii ) are prepared with carriers that protect them against rapid elimination from the body , such as a controlled release formulations , implants and micro - encapsulated delivery systems . biodegradable or biocompatible polymers can be used , such as ethylene vinyl acetate , polyanhydrides , polyglycolic acid , collagen , polyorthoesters and polylactic acid . such materials can be obtained commercially from alza corporation ( mountain view , calif .) and nova pharmaceuticals , inc . ( lake elsinore , calif .). liposomal suspensions can also be used as carriers . these can be prepared according to methods known to those skilled in the art , such as in ( eppstein et al ., u . s . pat . no . 4 , 522 , 811 , 1985 ). oral formulations or parenteral compositions in unit dosage form can be created to facilitate administration and dosage uniformity . unit dosage form refers to physically discrete units suited as single dosages for the subject to be treated , containing a therapeutically effective quantity of active compound , optionally in association with a pharmaceutical carrier . in the treatment of a condition involving a rt , such as hiv infection , an appropriate dosage level will generally be 0 . 01 to 1000 mg per day that can be administered in single or multiple doses . more preferable , dosage will be between 60 mg to 500 mg , 100 mg to 400 mg and 200 mg to 300 mg / day . the total dosage per day may be administered once every 24 hours , or divided over 24 hours , such as into fourths ( a quarter dose every 6 hours ), thirds ( ⅓ dose every 8 hours ) or halves ( ½ dose every 12 hours ). an appropriate dosage level will generally be about 0 . 01 to 100 mg per kg patient body weight per day which can be administered in single or multiple doses . preferably , the dosage level can be about 0 . 1 to about 75 mg / kg per day ; more preferably about 0 . 5 to about 50 mg / kg per day . combinations of the novel compounds , or with any of those listed in tables 1 - 3 , may be administered to maximize rt inhibitory effects . for example , trizivir ® is a composite of azt , 3tc and ziagen ®, and so too , may the compounds of the invention , such as compounds ( v )-( xii ) be administered in any combination with themselves , or other rt inhibitors . additionally , these compounds may also be administered with anti - hiv proteases and non - nucleoside inhibitors of rt . some side effects may be diminished by adjusting dosing regimes , or having a subject take the pharmaceutical compositions during a meal or with certain foods , or not with a meal . time of day of administration may also be adjusted to lessen side effects , such as administering a compound that induces insomnia early in the day . side effects of nucleoside rt inhibitors may include nausea , vomiting , stomach discomfort , loss of appetite , diarrhea , insomnia , muscle wasting , anemia , fatigue , peripheral neuropathy , rash , pancreatitis , lactic acidosis , mouth ulcers , and flatulence . other longer - term side effects may include mitochondrial damage , which may cause low red and white blood cell counts , muscle pain and wasting , fatigue , peripheral neuropathy , lactic acidosis and pancreas problems . also , some nucleoside inhibitors may not be combined , such as zerit ® and azt . the pharmaceutical compositions can be included in a kit , container , pack , or dispenser together with instructions for administration . when the invention is supplied as a kit , the different components of the composition may be packaged in separate containers and admixed immediately before use . such packaging of the components separately may permit long - term storage without losing the active components &# 39 ; functions . such kits are exceptionally useful when mixing various compounds into a single composition would result in interaction and diminished potency . the reagents included in kits can be supplied in containers of any sort such that the life of the different components are preserved , and are not adsorbed or altered by the materials of the container . for example , sealed glass ampules may contain lyophilized compounds ( v )-( xii ) or buffers that have been packaged under a neutral , non - reacting gas , such as nitrogen . ampules may consist of any suitable material , such as glass , organic polymers , such as polycarbonate , polystyrene , etc ., ceramic , metal or any other material . other examples of suitable containers include simple bottles that may be fabricated from similar substances as ampules , and envelopes having foil - lined interiors , such as aluminum or an alloy . other containers include test tubes , vials , flasks , bottles , syringes , etc . containers may have a sterile access port , such as a bottle having a stopper that can be pierced by a hypodermic injection needle . other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix . removable membranes may be glass , plastic , rubber , etc . kits may also be supplied with instructional materials . instructions may be printed on paper or other substrate , and / or may be supplied as an electronic - readable medium , such as a floppy disc , mini - cd - rom , cd - rom , dvd - rom , zip disc , videotape , laserdisc , audio tape , etc . detailed instructions may not be physically associated with the kit ; instead , a user may be directed to an internet web site specified by the manufacturer or distributor of the kit , or supplied as electronic mail . the following examples are provided to illustrate the invention . those skilled in the art can readily make insignificant variations in the compositions and methods of this invention . the examples are not meant to limit the invention in any way . the inhibitors ( v )-( viii ) were synthesized using standard chemical synthesis ; reagents are generally available from sigma - aldrich ( st . louis , mo .). for the synthesis of ( v )-( viii ), the intermediate ( xxxvii ) is first made ( table 4 ). ten grams of ( xxxiii ) was reacted with 10 g of 4 , 4 ′- dimethyoxytrityl chloride ( dmtrcl ) in the presence of 4 -( dimethylamino ) pyridine ( dmap ) as a catalyst and pyridine as a solvent . after purification of the reaction mixture by column chromatography , 21 g of ( xxxiv ) ( 93 % yield ) was obtained as pale yellow foam . reacting of 15 g of ( xxxiv ) with 7 . 7 g of o -( p - tolyl ) chlorothionoformate ( toloc ( s ) cl ) in the presence of dmap , triehylamine , and methylene chloride , gave 16 . 6 g of ( xxxv ) ( 87 % yield ) after purification . compound ( xxxv ) was converted into ( xxxvi ) by reacting with β - tributylstannyl styrene using 2 , 2 ′- azobis ( 2 - methylpropionitrile ) ( aibn ) as a catalyst , affording 6 . 3 g of ( xxxvi ) ( 58 % yield ) after purification . the key intermediate ( xxxvii ) was obtained ( 2 . 6 g , 30 % yield ) by oxidative cleavage of the double bond in ( xxxvi ) with oso 4 / naio 4 . one hundred mg of ( xxxvii ) is reacted with i 2 in a mixture of water and acetonitrile , yielding 95 mg of ( xxxviii ) ( 93 % yield ) after purification . deprotection of the dmt group in ( xxxviii ) by treating with trichloroacetic acid ( ccl 3 cooh ) in methylene chloride ( ch 2 cl 2 ) typically gave 37 mg ( 98 % yield ) of ( v ). synthesis of ( vi ) ( see also table 6 ) p one hundred milligrams of ( xxxvii ) was first treated with aibn and n - bromosuccinimide ( nbs ) at 70 ° c . for two hours . the reaction mixture was cooled to 0 ° c ., and the ammonia gas was bubbled through the mixture . eighty - six milligrams of ( xxxix ) ( 84 % yield ) was recovered after purification by column chromatography . ( xxxix ) was deprotected as for ( v ), obtaining 33 mg of ( vi ) from ( xxxix ) ( 87 % yield ). one hundred mg of ( xxxvii ) and cerium chloride ( cecl 3 ) was treated with methylmagnesium bromide ( memgbr ) at − 10 ° c . for 3 hours . after working up , the obtained alcohol intermediate was treated with dess - martin reagent to yield 87 mg of ( xli ) ( 85 %), which was then deprotected as for ( v ), giving 36 mg of ( vii ). one hundred mg of ( xxxvii ) was treated with i 2 in methanol at 0 ° c . to convert ( xxxvii ) to ( xl ), yielding 100 mg ( 96 % yield ). deprotection of dmt group in ( xl ) gave 46 mg of ( viii ) ( 95 % yield ). for primer extension experiments ( example 3 ), some of the novel nucleoside analog inhibitors were converted to triphosphate form in vitro , such as occurs in mammalian cells by endogenous enzymes . the corresponding triphosphate derivatives of ( v )-( vii ) are shown in table 12 . in vitro demonstration for the incorporation of nucleoside rt inhibitors ( v )-( vii ) into dna by rt the suggested mechanism by which nucleoside rt inhibitors exert their effects requires their incorporation into growing dna chains as they are reverse - transcribed from rna . once incorporated into the growing dna strand , transcription is terminated because the nucleosides lack the necessary 3 ′ hydroxyl group . this experiment demonstrates that the nucleoside analogs in their triphosphate forms ( v )-( vii ) are incorporated into growing dna chains by rt . the method is analogous to the sanger sequencing . specifically , in the presence of a template and four natural nucleotide triphosphates ( datp , dgtp , dctp and dttp ), a radiolabeled primer is extended to full length by rt , as indicated by a single band on the sequencing gel . addition of ddttp , which competes with dttp , results in the termination of primer extension at the positions of nucleotides opposite adenosine in the template strand . azttp , which is also a chain terminator , gives a similar result as ddttp . if a compounds , e . g ., ( v ) to ( vii ), in triphosphate form has a branched 3 ′- group that cannot be accommodated in the 3 ′- pocket of rt , the compound is not a substrate for rt . therefore , it will be unable to compete with dttp , resulting in a completed , extended product . however , if an added compound can productively occupy the active site and serve as a substrate of rt , like ddttp and azttp , primer extension is blocked by the compound at positions opposite adenosine in the template . if a chain terminator competes well , it is more probable that it is incorporated early during dna synthesis , resulting in more lower molecular weight products , represented by relatively higher intensity of bands when the products are resolved using polyacrylamide or agarose gel electrophoresis . if a chain terminator competes poorly , more products with higher molecular weight are produced , resulting in relatively higher intensity of bands in the upper part of the gel . by varying the ratio of the natural nucleotides to the inhibitors , the relative kinetics of incorporation of new inhibitors , as compared to the current drugs , into dna by rt and human dna polymerase can be studied . the results of such an experiment are shown in fig2 . template cagatagtcttcacgaggcaggtcgtcttgtcctggtactcgtttgcgttccg ( seq id no : 1 ) and primer gtctatcagaagtgctccgtcc ( seq id no : 2 ) were used . with this template and primer , there is only one adenosine in the template to be read into the growing chain as a thymidine ( capital and boldface in seq id no : 1 ). if the tested compound is a substrate for rt , it will be incorporated into the growing chain at this position and the chain will prematurely terminate , giving a product 38 base pairs long instead of the full - length 53 base pairs . compounds ( c1 ) and ( c2 ) were used as controls . compounds ( v ) ( lane 7 ), ( vi ) ( lane 8 ) and ( vii ) ( lane 9 ) were active , as indicated by the appearance of a band corresponding to the chain termination of the primer extension opposite the only a in the template . the efficiency of incorporation of these compounds by rt was roughly equivalent to ddttp ( lane 5 ) and azttp ( lane 6 ), both also chain terminators . the assay did not show the appearance of the terminated products with control compounds , which possess larger 3 ′- groups ( additional methylene groups ; lanes 10 ( c1 ) and 11 ( c2 )), indicating that they were not substrates for rt . in vitro demonstration for specificity : nucleoside rt inhibitors ( v )-( xii ) for rt compared to human dna polymerase ( prophetic example ) the experiment of example 4 is repeated but instead of rt , human dna polymerase is used . comparing these two experiments reveals the discrimination of inhibition of rt versus human dna polymerase for ddttp , azttp and xtp . a chain terminator , ( ddttp ), competes against natural nucleotide , a dttp in this case , for incorporation into dna . the effectiveness of its ability to compete is reflected in relative distribution of terminated products . the suggested mechanism by which nucleoside rt inhibitors exert their effects requires their incorporation into growing dna chains as they are reverse - transcribed from rna . once incorporated into the growing dna strand , transcription is terminated because the nucleosides lack the necessary 3 ′ hydroxyl group . this experiment demonstrates that the nucleoside analogs in their triphosphate forms , such as ( viii )-( xxii ), are incorporated into growing dna chains by rt . in the presence of a template and four natural nucleotide triphosphates ( datp , dgtp , dctp and dttp ), a radiolabeled primer is extended to full length by rt , as indicated by a single band on the sequencing gel . addition of a chain inhibitor , such as ddatp , ddgtp , dditp , ddctp as controls , which compete respectively with datp , dgtp , ditp and dctp , results in the termination of primer extension at the positions of nucleotides opposite the corresponding complement in the template strand . if the candidate compound , e . g ., ( viii )-( xxii ) in triphosphate form has a branched 3 ′- group that cannot be accommodated in the 3 ′- pocket of rt , the compound is not a substrate for rt . therefore , it will be unable to compete with the corresponding dntp , resulting in a completed , extended product . however , if an added compound can productively occupy the active site and serve as a substrate of rt , like ddntps , primer extension is blocked by the compound at positions opposite adenosine in the template . in vivo assay for anti - hiv activities of new nucleoside analogs using an hiv - infected cell line ( prophetic example ) anti - viral susceptibility assays of these inhibitors on clinical isolates and drug resistant laboratory mutants will be performed . in vitro test of the inhibition of hiv replication by these new nucleoside analogs using an hiv infected cell - line will be carried out according to the method of averett ( 1989 ). averett , d . r . 1989 . anti - hiv compound assessment by two novel high capacity assays . j . virol . meth . 23 : 263 - 276 . barre - sinoussi , f ., j . c . chermann , f . rey , m . t . nugeyre , s . chamaret , j . gruest , c . dauguet , c . axler - blin , f . vezinet - brun , c . rouzioux , w . rozenbaum , and l . montagnier . 1983 . isolation of a t - lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome ( aids ). science . 220 : 868 - 71 . beral , v ., and r . newton . 1998 . overview of the epidemiology of immunodeficiency - associated cancers . j natl cancer inst monogr : 1 - 6 . gallo , r . c ., s . z . salahuddin , m . popovic , g . m . shearer , m . kaplan , b . f . haynes , t . j . palker , r . redfield , j . oleske , b . safai , and et al . 1984 . frequent detection and isolation of cytopathic retroviruses ( htlv - iii ) from patients with aids and at risk for aids . science . 224 : 500 - 3 . hossain , n ., a . papchikhin , n . garg , i . fedoriv and j . chattopadhyaya . 1993 . 208 . solution structure of lariat rna by 500 mhz nmr spectroscopy and molecular dynamics studies in water . nucleosides & amp ; 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