Patent Application: US-8874098-A

Abstract:
the present invention provides certain 6 - hetarylalkyloxy pyrimidine derivatives of formula ii ## str1 ## wherein r is a cyclic group having at least one 5 - or 6 - membered heterocyclic ring , optionally with a carbocyclic or heterocyclic ring fused thereto , the or each heterocyclic ring having at least one hetero atom chosen from o , n , or s , or a substituted derivative thereof ; or phenyl or a substituted derivative thereof , r 2 is selected from h , c 1 - c 5 alkyl , halogen or nh 2 , r 4 and r 5 which are the same or different are selected from h , nh 2 or no n where n = 1 or 2 , or r 4 and r 5 together with the pyrimidine ring form a 5 - or 6 - membered ring structure containing one or more heterocyclic atoms , and pharmaceutically acceptable salts thereof , exhibit the ability to deplete o 6 - alkylguanine - dna alkyltransferase activity .

Description:
examples of compounds of the invention are shown in tables 1a and 1b . they were synthesized by the procedures presented below , adapted as appropriate . a . o 6 - substituted hypoxanthines were made by the action of alkoxide rch 2 ona on the quaternary salt n , n , n - trimethyl - 1h - purin - 6aminium chloride . 1 b . o 6 - substituted 2 - methylhypoxanthines were made similarly , from the quaternary salt from diazabicyclooctane ( dabco ) and 6 - chloro - 2 - methylpurine . 2 c . o 6 - substituted 2 - fluorohypoxanthines were made by diazotisation of the corresponding guanines using sodium nitrite and concentrated fluoboric acid at - 25 ° c . 3 d . o 6 - substituted 9 -( 2 - hydroxyethoxymethyl ) guanines were made by condensing the corresponding guanines after silylation with 2 - acetoxyethoxymethyl bromide in the presence of mercuric cyanide followed by saponification of the o - acetyl group . 4 e . o 6 - substituted 8 - hydroxyguanines were made from 6 - hetarylmethyl - 2 , 4 , 5 - triaminopyrimidines and 1 , 1 - carbonyldiimidazole in dmf . 5 reaction of 6 - chloro - 2 , 4 - diaminopyrimidine with alkoxide in dmso , followed by nitrosation with sodium nitrite in aqueous acetic acid and reduction using sodium hydrosulphite in aqueous dmf , gave the 2 , 4 , 5 - triamines . a . o 6 - substituted 8 - azaguanines were made from the above triamines and sodium nitrite in aqueous acetic acid . 6 b . o 6 - substituted 8 - aza - 7 - deazaguanines were made from the alkoxide rch 2 ona and 2 - amino - 6 - chloro - 8 - aza - 7deazapurine 7 in sulfolane or from the dabco quaternary salt ( in dmso solvent ) derived from it . a . o 6 - substituted 8 - oxaguanines were made by lead tetraacetate oxidation 8 of 6 - hetarylmethyl - 2 , 4 - diamino - 5 - nitrosopyrimidines obtained as under type ie . b . o 6 - substituted 8 - thiaguanines were made from the triamine intermedites under type ie and n - tosylthionylimine in pyridine . 9 c . o 4 - substituted pterins were made from these triamines and glyoxal with sodium metabisulphite . 10 c . o 6 - substituted 2 , 4 - diamino - 5 - nitropyrimidines were made by the action of alkoxide rch 2 ona in dmso on 6 - chloro - 2 , 4 - diamino - 5 - nitropyrimidine . 11 s 6 - substituted 6 - thioguanines were prepared from the thiolate rch 2 sna and the quaternary salt 2 - amino - n , n , n - trimethyl - 1h - purin - 6 - aminium chloride ( wo 94 / 29312 ). o 6 - substituted guanines as listed in tables 6a and 6b were made by the standard preparation as described in wo 94 / 29312 , usually with 3 mmol alcohol rch 2 oh per mmol quaternary salt . the alcohols were made as described in u . s . patent application ser . no . 08 / 568 , 576 , filed dec . 7 , 1995 by sodium borohydride reduction of the corresponding aldehydes , with two exceptions . for 4 - bromothenyl alcohol 12 required for b . 4280 the aldehyde is commercially available . 5 - chlorothiophen - 2 - aldehyde 13 and 5 - methylthiothiophen - 2 - aldehyde 14 were prepared by vilsmeier reaction on 2 - chlorothiophen and 2 - methylthiothiophen respectively . sodium borohydride reduction of the methylthioaldehyde followed by sodium periodate oxidation 15 of the resulting methylthioalcohol yielded the methylsulphinylalcohol required for b . 4294 . reduction of the chloroaldehyde gave 5 - chlorothenyl alcohol 16 for b . 4281 . several other aldehydes were obtained by halogenation of the appropriate thiophen aldehyde or furfural . thus , direct bromination gave 5 - bromofurfural 17 and thence the alcohol 18 for b . 4336 . halogen in presence of aluminum chloride on thiophen - 2 - aldehyde yielded 4 - chlorothiophen - 2 - aldehyde 19 ( for the alcohol for b . 4298 ), on thiophen - 3 - aldehyde yielded 2 - bromothiophen - 4 - aldehyde 20 ( and eventually b . 4313 ), and on 5 - chlorothiophen - 2 - aldehyde yielded 4 , 5 - dichlorothiophen - 2 - aldehyde 21 ( for the alcohol 22 for b . 4318 ). cyanoaldehydes were obtained from copper cyanide and the corresponding bromoaldehydes in refluxing dimethylformamide . 5 - cyanothiophen - 2 - aldehyde 23 and its 4 - cyano isomer 24 then gave the 5 - cyano and 4 - cyano 25 alcohols , for b . 4283 and b . 4317 respectively . 4 - methoxythenyl alcohol 26 ( for b . 4300 ) was prepared as described from 2 , 3 - dibromosuccinic acid and methyl thioglycollate , and ultimate reduction of the methyl ester ( not aldehyde in this case ) by lithium aluminium hydride and 2 - chloro - 4 - picolyl alcohol 27 ( for b . 4321 ) by sodium borohydride reduction 28 of the corresponding acid chloride , made in turn from reaction 29 of phosphorus oxychloride / pentachloride on isonicotinic acid n - oxide . for b . 4282 , 3 - pyridinemethanol n - oxide is commercially available . 5 - methylsulphonylthenyl alcohol ( for b . 4309 ) was obtained by m - chloroperbenzoic acid ( mcpba ) oxidation of the alcohol resulting from reduction of 5 - methylthio - 2 - thiophenecarboxaldehyde 30 . 6 - chloro - 3 - pyridinemethanol ( for b . 4319 ) and 5 - bromo - 3 - pyridinemethanol ( for b . 4320 ) were made by treatment of 6 - chloro and 5 - bromonicotinic acids respectively with phosphorus oxychloride / pentachloride and reduction of the resulting acid chlorides with sodium borohydride 28 . isothiazole - 4 - methanol ( for b . 4354 ) was obtained by reduction of the corresponding methyl ester ( a . adams , and r . slack , j . chem . soc . 1959 , 3061 ) with lithium aluminium hydride ( m . hatanaka and t . ishimaru , j . med . chem . 16 , 1973 , 978 ). 4 - bromo - 2 - thiophenecarboxaldehyde was converted into the 4 - lithio derivative ( a . l . johnson , j . org . chem . 41 , 1976 , 1320 ) of its ethylene acetal and reaction of this organometallic with dimethyl disulphide followed by acid hydrolysis gave 4 - methylthio - 2 - thiophenecarboxaldehyde ( r . noto , l . lamartina , c . arnone and d . spinelli , j . chem . soc ., perkin trans . 2 , 1987 , 689 ). sodium borohydride reduced this aldehyde to the 4 - methylthio alcohol ( for b . 4356 ), which in turn with one of two equivalents of mcpba yielded the 4 - methylsulphinyl and 4 - methylsulphonyl alcohols ( for b . 4377 and b . 4361 respectively ). reaction of the above organometallic with naphthalene - 2 - sulphonyl azide ( a . b . khare and c . e . mckenna , synthesis , 1991 , 405 ) and sodium pyrophosphate followed by hydrolysis by the method ( p . spagnolo and p . zamirato , j . org . chem ., 43 , 1978 , 3539 ) for the preparation of other azidothiophene aldehydes gave 4 - azido - 2 - thiophenecarboxaldehyde leading to the alcohol for b . 4373 . 5 - iodo - 3 - thiophenemethanol ( for b . 4357 ) came from the aldehyde obtained by treatment of 3 - thiophenecarboxaldehyde with iodine - iodic acid - sulphuric acid ( r . guilard , p . fournari and m . person , bull . soc . chim . france , 1967 , 4121 ). 2 - naphtho [ 2 , 1 - b ] thienylmethanol ( for b . 4366 ) was prepared by lithium aluminium hydride reduction of the corresponding carboxylic acid ( m . l . tedjamulia ,, y . tominaga , r . n . castle and m . l . lee , j . heterocycl . chem ., 20 , 1983 , 1143 ). 5 - phenylthenyl alcohol ( m . p . 91 . 5 ° c . for b . 4378 ) resulted from sodium borohydride reduction of the aldehyde ( p . demerseman , n . p . buu - hoi and r . royer , j . chem . soc ., 1954 , 4193 ) obtained by vilsmeier reaction of 2 - phenylthiophene ( from gomberg - bachmann reaction ( n . p . buu - hoi and n . hoan , rec . trav . chim ., 69 , 1950 , 1455 ) of benzenediazonium chloride and alkali with thiophene ). by way of specific example , the preparation of o 6 -( 4 - bromothenyl ) guanine ( b . 4280 ) will now be described . a solution of 4 - bromothenyl alcohol 12 [ 4 . 63 g , 24 mmol ; r f 0 . 38 in tlc ( phme - meoh , 4 : 1 )] in dmso ( 4 ml ) was treated cautiously with sodium hydride ( 60 % in oil ; 0 . 64 g , 16 mmol ). after 1 hour &# 39 ; s stirring , 2 - amino - n , n , n - trimethyl - 1h - purin - 6 - aminium chloride ( 1 . 83 g , 8 mmol ) was added . after 1 hour &# 39 ; s further stirring , acetic acid ( 1 . 3 ml ) followed by ether ( 240 ml ) was added and the solid filtered off after 1 - 2 h . removal of solvents and excess of alcohol ( b . p . 85 - 90 ° c ./ 0 . 4 mm ) from the filtrate yielded a negligible second fraction ( 17 mg ). the main crop was triturated with water ( 10 ml ), affording substantially pure product ( 1 . 89 g , 73 %) with r f 0 . 22 in tlc ( phme - meoh , 4 : 1 ). it was recrystallized by dissolving in hot methanol ( 100 ml ) and then concentrating . analytical data are given in tables 6a and 6b , together with data for other compounds . other typical synthetic procedures are described by way of example in a special section later in this text . compounds of formula ii or xiii in which y &# 39 ; is r &# 34 ; xchr &# 34 ;&# 39 ; and r &# 34 ;&# 39 ; is alkyl ( seco - nucleosides ) may be prepared by an analogous preparation to the reaction of o 6 - benzylguanine with - chloro - ethers ( maccoss et al ., tetrahedron lett . ; european patent application no . 184 , 473 , loc . cit .) or with alkyl bromides ( e . g . kjellberg , liljenberg and johansson , tetrahedron lett ., 1986 , 27 , 877 ; moschel , mcdougall , dolan , stine , and pegg , j . med . chem ., 1992 , 35 , 4486 ). typical &# 34 ; sugar &# 34 ; components corresponding to r &# 34 ; xchr &# 34 ;&# 39 ;, leading to seco - nucleosides , are made by methods described in e . g . mccormick and mcelhinney , j . chem . soc ., perkin trans . 1 , 1985 , 93 ; lucey , mccormick and mcelhinney , j . chem . soc . perkin trans . 1 , 1990 , 795 . compounds of formula ii or xiii in which y is ribosyl or deoxyribosyl ( nucleosides ) may be prepared by methods analogous to the syntheses of o 6 - benzylguanine riboside and 2 - deoxyriboside ( moschel et al . 1992 ; cf . gao , fathi , gaffney et al ., j . org . chem ., 1992 , 57 , 6954 ; moschel , hudgins and dipple , j . amer . chem . soc ., 1981 , 103 , 5489 ) ( see preparation of ribosides above ). the amount of the compound of the present invention to be used varies according to the effective amount required for treating tumour cells . a suitable dosage is that which will result in a concentration of the compound of the invention in the tumor cells to be treated which results in the depletion of atase activity , e . g . about 1 - 2000 mg / kg body weight , and preferably 1 - 800 mg / kg body weight , particularly 1 - 120 mg / kg body weight , prior to chemotherapy with an appropriate alkylating agent . the pharmaceutical composition of the invention may be formulated in conventional forms with conventional excipients , as described for example in and u . s . pat . nos . 5 , 525 , 606 and 5 , 091 , 430 and 5 , 352 , 669 , the contents of which are incorporated herein by reference in their entirety . the composition may contain the inactivator according to the invention together with an appropriate alkylating agent ; or the composition may comprise two parts , one containing the inactivator and the other containing the alkylating agent . the method of administering the compounds of the invention to a host may also be a conventional method , as described in wo 91 / 13898 for example . for administration of an inactivator according to the invention to patients , the pharmaceutical composition may suitably contain the inactivator in a suitable vehicle such as 40 % polyethyleneglycol 400 in saline solution , or in saline or 3 % ethanol ( in saline ), for intravenous injection , or in a powder form in suitable capsules for oral administration . alkylating agents may be administered in accordance with known techniques and in conventional forms of administration , as described in wo 91 / 13898 for example or preferably as a single dose immediately after or up to 24 hours after but preferably around 2 hours after administration of the atase inactivating agents and also at doses lower than those used in standard treatment regimen . a reduction in dose may be necessary because the inactivators would generally be anticipated to increase the toxicity of the alkylating agents . examples of chloroethylating agents include 1 , 3 bis ( 2 - chloroethyl )- 1 - nitrosourea ( bcnu ), 1 -( 2 - chloroethyl )- 3 - cyclohexyl - 1 - nitrosourea ( ccnu ), fotemustine , mitozolomide and clomesone and those described in mccormick , mcelhinney , mcmurry and maxwell j . chem . soc . perkin trans . 1 , 1991 , 877 and bibby , double , mccormick , mcelhinney , radacic , pratesi and dumont anti - cancer drug design , 1993 , 8 , 115 . examples of methylating agents include temozolomide and u . s . pat . no . 5 , 260 , 291 the contents of which are incorporated herein in their entirety ) and dacarbazine , procarbazine , and streptozocin . varying amounts of recombinant atase or cell / tissue extracts were incubated with [ 3 h ]- methylnitrosourea - methylated calf thymus dna ( specific activity , 17 ci / mmol ) at 37 ° c . for 1 hour in a total volume of 300 μl buffer 1 /[ 50 mm tris / hcl ( ph 8 . 3 ), 3 mm dithiothreitol ( dtt ), 1 mm edta ] containing 1 mg / ml bovine serum albumin ( ibsa ) for recombinant atases and tissue extracts , or 1 . 1 ml buffer 1 for cell extracts . after incubation , bovine serum albumin ( 100 μl of 10 mg / ml in buffer 1 ) and perchloric acid ( 100 μl of 4m perchloric acid for 300 μl volumes and 400 μl for 1 . 1 ml volumes ) and 2 ml of 1m perchloric acid were added . samples were then heated at 75 ° c . for 50 minutes to hydrolyze the dna . samples were then centrifuged at 3 , 000 rpm for 10 minutes and the precipitate washed once with 4 ml of 1m perchloric acid , before being resuspended in 300 μl of 0 . 01m sodium hydroxide and dissolved in 3 ml of aqueous scintillation fluid ( ecoscint a , national diagnostics ). counting efficiency was approximately 30 %. atase specific activity was calculated from the region where the activity was proportional to the amount of extract added , since with higher amounts of extracts the reaction becomes substrate limiting . atase activity is expressed as fmol methyl transferred to protein per mg of total protein in the extract . the cdna cloning and overexpression of the human atase has been reported previously 30 . purification of the recombinant proteins was achieved either by affinity chromatography through a dna - cellulose column as described by wilkinson et al ., 31 , 32 , or by deae - cellulose ion - exchange chromatography . for the latter , the atase protein was partially purified by ammonium sulphate precipitation ( 30 - 60 %) and dialyzed against 10 mm tris - hcl ph 7 . 5 , 1 mm dtt , 2 mm edta , 10 % glycerol , before loading on a deae - cellulose column . the atase was then eluted with a 0 - 0 . 1 m nacl gradient . the purified human atase protein retained activity for more than one year when stored at high concentration at - 20 ° c . in buffer 1 [ 50 mm - tris / hcl ( ph 8 . 3 )/ 3 mm - dithiothreitol / 1mm - edta ] and could be thawed and refrozen several times without substantial loss of activity . compounds to be tested were dissolved in dmso to a final concentration of 10 mm and diluted just before use in buffer 1 . recombinant atase was diluted in buffer 1 containing 1 mg / ml bovine serum albumin ( ibsa ) and tilrated as described above in order that the reaction be conducted under atase , and not substrate , limiting conditions . in each assay , fixed amounts of atase ( 60 - 75 fmol ) were incubated with varying amounts of o 6 - benzylguanine , or test compound in a total volume of 200 μl of ibsa containing 10 μg of calf thymus dna at 37 ° c . for 1 hour . the [ 3 h ]- methylated - dna substrate ( 100 μl ) containing 4 μg of dna and 100 fmol of o 6 - methylguanine ) was then added and incubation continued at 37 ° c . for 1 hour , until the reaction was complete . following acid hydrolysis of the dna as described above the [ 3 h ]- methylated protein was recovered and quantitated by liquid scintillation counting . i 50 is the concentration of inactivator required to produce a 50 % reduction in atase activity under the above conditions . mammalian cells including raji cells ( a human lymphoblastoid cell line from a burkitt &# 39 ; s lymphoma ), a375m cells ( human melanoma cells ), mcf - 7 cells ( human breast cancer cells ) and pc3 and du145 ( both human prostrate cancer cells ) were cultured under standard conditions . for example , raji cells were grown in suspension culture in rpmi medium supplemented with 10 % horse serum . cell pellets were resuspended in cold ( 4 ° c .) buffer i containing 2 μg / ml leupeptin and sonicated for 10 seconds at 12 μm peak to peak distance . after cooling in ice , the cells were sonicated for a further 10 seconds at 18 μm . immediately after sonification , 10 μl / ml of phenylmethanesulphonylfluoride ( pmsf 8 . 7 mg / ml in 100 % ethanol ) was added and the sonicates centrifuged at 15000cpm for 10 minutes at 4 ° c . to pellet cell debris . the supernatant was transferred to a tube on ice and kept for determination of atase activity ( see above ). inactivators ( 10mm in dmso ) were diluted to 0 . 1mm in prewarmed degassed pbs ( ph 7 - 7 . 2 ). pbs ( phosphate buffered saline ) is 0 . 8 % nacl , 0 . 02 % kcl , 0 . 15 % na 2 h 2 po 4 , 0 . 02 % kh 2 po 4 . samples were immediately transferred to a cary13 spectrophotometer ( cuvette block held at 37 ° c .) and scanned at an appropriate wavelength ( according to the spectral properties of the compound ) at 5 - 10 minute intervals for up to 80 hours . the results were expressed as percentage absorbance change versus time and t1 / 2 values ( half life ) extrapolated from this . in the tables the results of these tests are identified by &# 34 ; in pbs &# 34 ; or &# 34 ; by spec &# 34 ;. inactivators ( 10 μm in dmso ) were diluted to the appropriate concentration ( i 90 calculated from previous i 50 determination data ) in buffer i without dtt and incubated for varying times at 37 ° c . samples were then taken for use in the competition assay to assess the compound &# 39 ; s ability to inactivate human atase . the results were expressed as reduction in activating activity versus time and t 1 / 2 values extrapolated from this . raji cells were diluted to between 5 × 10 5 / ml and 10 6 / ml in medium containing either the appropriate concentration of inactivator or an equivalent volume of vehicle ( dmso ). following incubation at 37 ° c . for 2 hours the cells were harvested by centrifugation , washed twice with pbs and the resulting cell pellets ( between 5 × 10 6 and 10 7 cells per pellet ) stored at - 20 ° c . atase activity was determined as described above , in duplicate cell extracts and expressed as the percentage activity remaining , based on that present in the untreated controls ( 350 - 450 fm / mg depending on the assay ). i 50 ( i . e concentration of inactivator required to reduce atase activity by 50 %) values were extrapolated from this data . sensitization of mammalian cells to the cytotoxic effects of bcnu , temozolomide and other cytotoxic agents following a 2 hour pretreatment with inactivator was analysed using an xtt - based growth inhibition assay 22 . cells were plated in 96 well plates ( for example in the case of raji cells at 500 cells / well ) and incubated at 37 ° c . for 30 minutes prior to the addition of medium containing either the appropriate concentration of inactivator or an equivalent volume of vehicle . following a 2 hour incubation at 37 ° c ., medium containing either increasing doses of cytotoxic agent or equivalent vehicle was added and the cells allowed to grow for 6 days . at this time xtt solution was added and the cells incubated for a further 4 hours at 37 ° c . the resulting red / orange formazan reaction product was quantified by measuring adsorption at 450 nm on a microtitre platereader . from this data the percentage growth of cells relative to that in control wells was determined for a range of bcnu , temozolomide or other cytotoxic agent doses in both the presence and absence of inactivator . sensitization factor ( sf ) based on d 50 ( d 50 . c / d 50 . i ) was determined by dividing the d 50 ( i . e . dose at which there was 50 % growth versus the controls untreated with alkylating agent ) calculated for the cytotoxic agent alone ( d 50 . c ) by that for the cytotoxic agent plus inactivator ( d 50 . i ). a value of one ( 1 ) thus indicates no sensitization by the inactivator . comparable sensitization factors were also determined in some cases based on d 60 and d 80 , i . e . the dose at which there was respectively 60 % or 80 % growth compared to the untreated controls . in table 3 the sensitization factor d 50 . c / d 50 . i is shown as d 50 control / d 50 ` b `, with the letter ` b ` referring to the inactivator compound . swiss mouse derived athymic male mice ( o / nu ) weighing between 20 - 30 g were obtained from zeneca pharmaceuticals , alderley park , macclesfield , cheshire , sk10 4t6 , england . animals were housed 4 - 5 / cage in filter top cages and had access to food and water ad libitum . all animals were maintained under a controlled 12h - light - 12h - dark cycle . these animals were used for all tests except those which are shown in fig1 and 17 and table 8 , as mentioned below . a375m ( human melanoma ) and du145 ( human prostrate cancer ) cells were grown in dmem containing 10 % foetal bovine serum ( fbs ), mcf - 7 ( human breast cancer cells ) were grown in dmem containing 10 % fbs supplemented with 100iu insulin . a375m , du145 and mcf - 7 cells ( 10 6 ) in 100 μl pbs were injected subcutaneously into the right - hand flank of 8 - 10 week old o / nu athymic mice . these cells were allowed to develop into a tumour for 3 - 4 weeks ( a375m and du145 cells ) and 4 - 6 weeks ( mcf - 7 cells ). once established , tumours were maintained by subcutaneous implantation of 2 mm 3 blocks into the right - hand flank of athymic o / nu mice . mcf - 7 tumours are oestrogen positive and require oestrogen for growth . this was supplied as a subcutaneous implant ( see below ) at the tail base simultaneously to the tumour implant and monthly thereafter . 468 mg β - oestradiol was added to 9 . 7 g silastic and mixed until evenly distributed . 1 . 1 g of curing agent was added and the whole mixture spread into 3 ( 26 mm × 12 mm × 1 mm ) glass fomers . these were then incubated at 42 ° c . overnight before being cut into 2 mm × 2 mm × 1 mm cubes , so that each pellet contained 2 mg estradiol . tumours were implanted as previously described and left 3 - 6 weeks to establish depending on tumour type . an inactivator was homogenized in corn oil at 5 mg / ml before administration by interperitoneal injection ( i . p .) or oral gavage ( p . o .). mice were sacrificed at various times up to 72 h and tumours and murine tissues taken for atase assay . samples were snap frozen and stored at - 20 ° c . until analysis . o / nu mice were treated with the appropriate dose of the inactivator as indicated ( 4 mg / ml in corn oil ) or the appropriate vehicle as a control 1 hour prior to administration of the appropriate dose of the cytotoxic agent ( e . g . temozolomide 6 mg / ml in pbs + 20 % dmso ) or fotemustine or bcnu ( 2 mg / ml in pbs + 3 % ethanol ) using the doses and schedules indicated . animals were weighed twice weekly and xenograft tumour measurements taken using digital calipers . tumour volume was calculated using the formula ( h × w × l ) π / 6 . measurements continued until the tumour reached the maximum allowable volume ( i . e . 1 cm 3 ). results were expressed as percentage tumour growth using day 1 tumour volumes as controls . in the tests on the compounds shown in table 6 and in fig9 to 17 , the methods used were as described in wo 94 / 29312 . the following items a ) to c ) are also to be noted : atase substrate dna was prepared by incubation of purified calf thymus dna with n --[ 3 h ]- methyl - n - nitrosourea ( 18 . 7 ci / mmole , amersham international ). cell or tissue extracts were incubated with [ 3 h ]- methylated - dna substrated ( 100 μl containing 6 . 7 μg of dna and 100fmol of o 6 -[ 3 h ] methylguanine ) at 37 ° c . for 60 mins . following acid hydrolysis of the dna as previously described 33 the [ 3 h ]- methylated protein was recovered and quantitated by liquid scintillation counting . mice were treated with the inactivator as a suspension in corn oil by intraperitoneal injection ( i . p .) or by oral gavage ( p . o .) 60 mins prior to temozolomide ( 100 mg / kg in 20 % dmso in phosphate - buffered saline ) which was always given in intraperitoneal injection : this schedule was repeated on days 1 to 5 inclusive . controls received vehicle alone , inactivator alone or temozolomide alone . the mice in the tests shown in fig1 and table 8 were balb - c derived athymic male mice ( nu / nu athymic ) from the in - house breeding colony of the paterson institute for cancer research as described in wo 94 / 29312 ( animal services - asu mice ). the mice in the tests shown in fig1 - 16 were swiss mouse derived athymic male mice ( o / nu athymic ) as described above . the mice in the tests shown in fig1 were dba 2 mice from the in - house breeding colony of the paterson institute for cancer research ( animal services unit ), originally from the jackson laboratory in 1970 . the results of the atase depletion assay on the compounds of table 1 are shown in table 2 or table 3 . many of the compounds tested were more efficient in inactivating atase than o 6 - benzylguanine . in accordance with the results in wo 94 / 29312 the parent application , compounds in which r is a heterocyclic group were more efficient than the comparable compounds having benzyloxy side chains . in general the compounds in which rch 2 is substituted or unsubstituted thenyl were the most efficient , the most preferred being halo - substituted thenyl having its halo substituent in a 1 , 3 - relationship with the methyleneoxy group attached to the pyrimidine residue . tables 3 , 4 and 5 summarize data for a number of parameters . table 3 includes depletion assay results for recombinant atase of the following types : ______________________________________ hat = human mat = mouse rat = rat chat = chinese hamster ogt = e . coli ogt gene ada = e . coli ada gene______________________________________ the combinations of properties for the various inactivators can be seen in the tables . the following surprising points are noted in particular : b . 4335 is a compound that is unexpectedly much more effective in the inactivation of atase in raji cells than of pure recombinant protein : generally , the i 50 for inactivation of recombinant atase in vitro is lower or similar to that in cultured cells . b . 4343 is a compound that has a very low i 50 for atase in vitro but is not as capable as agents with higher i 50 s ( e . g . b . 4335 ) in the sensitization of raji cells to the growth inhibitory effects of temozolomide . a similar example is b . 4351 versus b . 4349 . b . 4316 was twice as effective as b . 4280 but sensitization to temozolomide of raji cells was almost identical . thus different cell lines may respond surprisingly differently to these agents . fig1 to 3 show that temozolomide , bcnu and fotemustine inhibit the growth of raji cells in a dose - dependent manner but sensitivity is greatly increased by exposure to b . 4316 at 0 . 1 , 1 . 0 and 10 μm respectively . in contrast b . 4316 had no measurable effect on growth inhibition of raji cells by melphalan or cisplatin ( fig4 ). this indicates that the inactivators were specifically sensitizing cells to the o 6 - alkylating agents and not other classes of alkylating compound . fig5 and 6 respectively show the b . 4316 and b . 4349 sensitization factors for the above therapeutic agents in raji cells . fig7 shows that of the inactivators examined human melanoma xenograft atase depletion was complete only after administration of b . 4314 and b . 4351 under the experimental conditions used . the former was more effective in atase depletion in liver and kidney of host animals whilst the latter was more effective in the brain , suggesting its relative ease in passing the blood - brain barrier . noteworthy is the fact that whilst b . 4311 was one of the most effective agents in sensitizing raji cells to the toxic effects of temozolomide , it was surprisingly one of the least effective agents in depleting mouse tissue or tumour xenograft atase activity . fig8 shows that b . 4363 depletes atase more effectively in human melanoma xenografts than in murine host tissues under the conditions used : relatively little effect was seen in brain tissue , suggesting its poor ability to cross the blood brain barrier . the test results for the compounds of table 6 ( and some in table 1 ) are shown in table 7 and fig9 to 27 . b . 4280 , which is o 6 -( 4 - bromothenyl ) guanine and has its bromo substituent in a 1 , 3 - relationship with the methylene group attached to the guanine residue , was more efficient in inactivating atase in vitro than its 5 - bromo analogue b . 4269 , in which the bromo substituent is in a 1 , 4 - relationship with the methylene group . both b . 4280 and b . 4269 were more efficient than the unsubstituted thenyl derivative b . 4205 despite having considerably larger o 6 substituents . another preferred compound is b . 4317 which is o 6 -( 4 - cyanothenyl ) guanine . b . 4317 is a more efficient inactivator in vitro than its 5 - cyano analogue b . 4283 or the unsubstituted thenyl derivative b . 4205 . typical atase inactivation profiles for beg and b . 4205 and b . 4280 are shown in fig9 . the inactivation of atase resulted in the sensitization of raji cells to the growth inhibitory effects of temozolomide ( fig1 ). b . 4280 was considerably more effective than either b . 4205 or beg in this respect . atase in human melanoma xenografts was inactivated by beg , b . 4205 and b . 4280 ( fig1 ) with some indication that the rates of recovery of atase activity were different between the agents . b . 4280 was the most effective in vivo inactivator at the doses examined . b . 4280 was able to inactivate atase in most tissues as shown in table 8 . thus , activity in brain , testis and bone marrow was near to control levels by 24 hours whereas lung and spleen activity had not completely recovered by 48 hours . tumour activity was very low at 24 hours but had recovered completely by 48 hours . differential recovery rates might be an important factor in the toxicity of atase inactivators when used in combination with cnu or temozolomide . combination of b . 4205 or b . 4280 and temozolomide given over three days were more effective in atase inactivation in tumour xenografts than either agent alone ( fig1 ). decreasing the dose of b . 4205 had no major effect on the ability of the agent to inactivate atase , 10 mg / kg being as effective as 60 mg / kg . b . 4280 was more effective than b . 4205 at equivalent doses . as before ( fig1 ) there was some indication that atase recovery was less efficient in the tumour xenograft ( fig1 ) than in the liver ( fig1 ). b . 4205 ( fig1 a ) and b . 4280 ( fig1 a ) were effective in sensitizing human melanoma xenografts to the growth inhibitory effects of temozolomide . a comparison of the two sets of data indicates that b . 4280 was about twice as effective as b . 4205 in this respect . at equi - effective doses for tumour growth inhibition , b . 4280 seems to be less toxic than b . 4205 ( fig1 b and 15b ). in experiments using dba 2 mice in combination with bcnu , b . 4280 was considerably less acutely toxic than b . 4205 or beg as shown in table 9 . oral administration of b . 4280 was shown to be almost as effective as i . p . administration in sensitizing human melanoma xenografts to the growth inhibitory effects of temozolomide ( fig1 a ). furthermore the oral combination appeared to be marginally less toxic than the i . p . route ( fig1 b ). at a dose of 20 mg / kg of inactivator in combination with temozolomide in dba 2 mice , b . 4205 and b . 4280 were shown to be less acutely toxic than beg , with b . 4280 being less acutely toxic than b . 4205 ( fig1 ). fig1 and 19 show that b . 4280 ( patrin - 2 ) ( i . p . at 20 mg / kg and p . o . at 30 mg / kg respectively ) depletes atase in human melanoma xenografts more completely and for a more extensive period than it does in host tissues . fig2 show that despite the considerably higher initial level of atase activity in the human breast tumour , b . 4280 depletes atase therein more completely and for a longer period of time than in murine host tissues . in this study using 30 mg / kg b . 4280 i . p . extensive depletion was seen in brain tissue , indicating the ability to cross the blood - brain barrier . fig2 likewise shows that despite the considerably higher initial level of atase activity in the human prostrate tumour , b . 4280 depletes atase therein more completely and for a longer period of time than in murine host tissues . in this study using 20 mg / kg b . 4280 i . p . relatively little depletion was seen in brain tissue , indicating by reference to fig2 that the ability of b . 4280 to cross the blood - brain barrier may be dose - dependent . fig2 shows that b . 4280 ( 20 mg / kg i . p .) considerably increased the sensitivity of the human breast tumour xenograft to the growth inhibitory effects of temozolomide using a 5 day dosing schedule . this sensitization occurred despite the very high level of atase in this tumour . fig2 shows that a single dose of b . 4280 ( 30 mg / kg p . o .) considerably increased the sensitivity of the human melanoma tumour xenograft to the growth inhibitory effects of a single dose of the chloroethylating agent , fotemustine , without any substantial effect on toxicity . bromothenylaldehyde ( 0 . 79 mg , 66 . 8 umoles was reacted with nab [ 3 h ) 4 ( 0 . 0167 mmoles , 60 ci / mmole ) in isopropanol ( 350 μl ) for 1 h at room temperature . the resulting [ 3 h ]- 4 - bromothenylalcohol was extracted into pentane , dried , weighed and reacted with nah ( 5 . 44 mg ), and the quaternary ammonium salt of guanine ( 15 . 55 mg ) in dmso ( 250 μl ) for 1 hour at room temperature . the product was recovered by precipitation from acetic acid - ether ( 15 μl glacial acetic in 1 . 5 ml ether ), washed with ether , dried and triturated with h 2 o . after washing with water , the final product was dried to constant weight . fig2 shows the scheme for synthesis of the radio - labelled b . 4280 . an aliquot of the product was dissolved in buffer a ( 10mm kh 2 po 4 containing 7 . 5 % acetonitrile ) and subjected to high performance liquid chromatography on an ods - 5 column . elution at 1 ml / min was with a linear gradient over 20 minutes from 100 % a to 20 % a : 80 % b ( 10mm kh 2 po 4 containing 80 % acetonitrile ). the effluent was monitored for uv absorption at 254 nm and fractions ( 1 min ) were collected and assayed for radioactivity after addiition of 10 ml of ecoscint a . it was shown that 96 % of the radio activity co - chromatographed with authentic b . 4280 ( fig2 ). incubation of an aliquot of the product with known amounts of pure recombinant human atase resulted in the transfer of radioactivity to the protein ( fig3 ), strongly suggesting that the mechanism of atase inactivation involves the transfer of the thenyl group to the active site cysteine residue in the atase molecule . measurement of the amount of radioactivity transferred to protein indicated that the o 6 -([ 3 h ]- 4 - bromothenyl ) guanine had a radiochemical purity of & gt ; 96 % and a specific activity of 16 ci / mmole . o 6 -([ 3 h ]- 4 - bromothenyl ) guanine can be used as an alternative to the standard method , which presently uses [ 3 h ]- labelled substrate dna , to determine the amounts of atase , for example , in cell or tissue extracts . it may also be used to locate active atase molecules in tumour and other tissue sections by incubation with such sections on microscope slides followed by washing , autoradiography and histological staining . it may also be used to monitor the formation of the [ 3 h ]- labelled products of breakdown or metabolism of the agent after administration to mammals . it may also be used to determine the distribution of the b . 4280 or its breakdown products in animal tissues and tumours by means of whole body autoradiography . 4 - bromothenyl alcohol ( 1 . 16 g , 6 mmol ) was added to sodium hydride ( 60 % in oil ; 0 . 16 g , 2 mmol ) and dmso ( 1 ml ). the solution was stirred for 30 min . the trimethylammonium salt ( 0 . 427 g , 2 mmol ) was then added and stirring continued for 2 . 5 h at 20 ° c . the solution was cooled in an ice bath and poured into ether ( 60 ml ) containing acetic acid ( 0 . 32 ml ). a white precipitate was collected , triturated with water ( 4 ml ) and collected again to give b . 4292 ( 436 mg , 69 %) recrystallised from methanol . 6 - chloro - 2 - methylpurine ( 0 . 5 g , 3 mmol ) was dissolved in a mixture of dmf ( 5 ml ) and diglyme ( 25 ml ). dabco ( 0 . 66 g , 6 mmol ) was then added . the mixture was stirred for 1 h and the precipitate collected to give the quaternary salt ( 700 mg , 82 %). nmr ( 300 mhz , dmso - d 6 ): shift in ppm 2 . 65 ( s ), 3 . 27 ( t , j = 7 . 5 hz ), 3 . 78 ( s ), 4 . 14 ( t , j = 7 . 5 hz ), 8 . 21 ( s ). thenyl alcohol ( 684 mg , 6 mmol ) was added to sodium hybride ( 60 % in oil ; 80 mg , 2 mmol ) and dmso ( 0 . 5 ml ). the solution was stirred for 30 min . the dabco salt was then added and stirring continued for 5 h . the solution was then poured into ether ( 30 ml ) containing acetic acid ( 0 . 15 ml ). a precipitate was collected , triturated with water ( 4 ml ) and collected again to give o 6 - thenyl - 2 - methylhypoxanthine ( 96 mg , 35 %) recrystallised from acetonitrile . to 3 . 6 ml of 40 % fluoroboric acid precooled to - 25 ° c . in a bath was added o 6 -( 4 - bromothenyl ) guanine ( 326 mg , 1 mmol ) with vigorous stirring . a solution of sodium nitrite ( 0 . 116 g , 1 . 7 mmol ) in water ( 0 . 15 ml ) was added dropwise over a period of 10 min . after 20 min , the solution was poured into ice . the mixture was then allowed to stand at 0 ° c . for 15 h , then collected and dried to afford almost pure ( t . l . c .) b . 4353 ( 180 mg , 55 %). flash chromatography ( hexane - ethyl acetate decreasing polarity little by little ) afforded b . 4353 . a mixture of 5 - deazapterin 33 , 34 ( 2 . 0 g , 13 . 36 mmol ), 4 - dimethylaminopyridine ( 0 . 22 g , 1 . 8 mmol ) and pivalic anhydride ( 12 ml ) was heated to 165 ° c . excess pivalic anhydride was distilled off and the residue dissolved in dichloromethane and applied to a pad of silica gel , and eluted with 2 % methanol in dichloromethane . evaporation and recrystallisation of the product from ethanol gave shiny cream coloured crystals ( 2 . 25 g , 74 %) of the pivaloyl derivative , m . p . 258 - 259 ° c . ; λ max ( meoh ) 277 nm ; nmr ( 300 mhz , dmso - d 6 ) δ1 . 28 ( s ), 7 . 44 ( q ), 8 . 43 ( dd ), 8 . 88 ( dd ), 11 . 4 ( s ), 12 . 31 ( s ). a suspension of n 2 - pivaloyl - 5 - deazapterin ( 0 . 492 g , 2 mmol ) in tetrahydrofuran ( 8 ml ) was stirred for 10 min , and tri - n - butylphosphine ( 0 . 606 g , 3 mmol ), thenyl alcohol ( 0 . 432 g , 3 mmol ) and diisopropylazodicarboxylate ( 0 . 606 g , 3 mmol ) were added successively . the reaction was allowed to proceed for 2 h at room temperature and evaporation than gave an oil . hexane was added to induce crystallisation . filtration and recrystallisation from hexane gave bright yellow crystals of the thenyl derivative ( 0 . 32 g , 47 %) m . p . 107 - 108 ° c . ; λ max ( meoh ) 272 , 311 nm ; nmr ( 300 mhz , dmso - d 6 ) δ1 . 28 ( s ), 5 . 86 ( s ), 6 . 98 ( q ), 7 . 28 ( dd ), 7 . 43 ( dd ), 7 . 52 ( q ), 8 . 46 ( dd ), 8 . 89 ( dd ). n 2 - pivaloyl - o 4 - thenyl - 5 - deazapterin ( 0 . 28 g , 0 . 82 mmol ) was heated for 24 h under reflux with aqueous naoh ( 3m , 2 ml ) and ethanol ( 1 ml ). the solvent was removed by evaporation and the residual solid dissolved in water . acidification with acetic acid gave a white precipitate . filtration and recrystallisation of the solid from ethanol gave white crystals of o 4 - thenyl - 5 - deazapterin ( b . 4376 ), ( 0 . 107 g , 51 %). sodium hydride ( 60 % in oil ; 80 mg , 2 mmol ) was added to a stirred solution of 4 - bromothenyl alcohol ( 290 mg , 1 . 5 mmol ) in dry dmso ( 1 ml ). after 30 min , 4 - amino - 2 - chloro - 5 - nitropyrimidine 35 ( 174 mg , 1 mmol ) was added and the mixture heated at 50 ° c . for 2 h . the dmso was removed in vacuo and the ph adjusted to 7 with aqueous acetic acid . after extraction into ethyl acetate , the product b . 4380 was crystallised from methanol ( 51 mg , 15 %). sodium hydride ( 60 % in oil ; 44 mg , 1 . 1 mmol ) was added to a stirred solution of 4 - bromothenyl mercaptan ( 418 mg , 2 mmol ) in dry dmso ( 0 . 5 ml ). after 30 min , 2 - amino - n , n , n - trimethyl - 1h - purin - 6 - aminium chloride ( 228 mg , 1 mmol ) was added and stirring continued for 1 h . acetic acid ( 0 . 12 ml ) and ether ( 30 ml ) were added and after decantation and trituration with fresh ether , b . 4352 ( 38 mg , 11 %) was filtered off . o 6 -( 4 - bromothenyl ) guanine ( 652 mg , 2 mmol ) was dissolved in sodium ethoxide ( 1m , 2 ml , 2 mmol ). after 10 min , the ethanol was removed and the residue was dissolved in dry dmf . chloromethyl ethyl ether ( 189 mg , 2 mmol ) was added dropwise to the stirred solution under an atmosphere of argon . after 45 min , the solvent was removed . the oily product was crystallised from ethanol giving b . 4369 ( 158 mg ) as needles . a further 118 mg was obtained by flash chromatography of the mother liquor on silica gel with 5 % ethanol in ch 2 cl 2 . total yield , 39 %. a stirred mixture of o 6 -( 4 - bromothenyl ) guanine ( 294 mg , 1 mmol ), ( nh 4 ) 2 so 4 ( 47 mg ) and hexamethyldisilazane ( 5 ml ) was heated at reflux for 3 h . volatile material was then evaporated under vacuum . the residue was stirred with benzene ( 15 ml ) and hg ( cn ) 2 ( 344 mg , 1 . 3 mmol ) under reflux for 30 min . a solution of ( 2 - acetoxyethoxy ) methyl bromide { ref 4 p33 } ( 197 mg , 1 mmol ) in benzene ( 10 ml ) was added , reflux maintained for 2 h , and the cloudy diluted with chloroform ( 150 ml ). the organic phase was washed with saturated aqueous nahco 3 ( 30 ml ), followed by ki ( 1m ; 30 ml ), dried over mgso 4 and evaporated to give an oil ( 313 mg ). this oil was chromatographed on a silica gel column with chcl 3 -- meoh ( 12 : 1 ) as eluant , yielding almost pure ( t . l . c .) o - acetate ( 141 mg ) of b . 4335 . methanol ( 60 ml ) was saturated with dry ammonia and poured onto this o - acetate in a flash which was tightly stoppered . after dissolution , stirring was stopped and the flask left closed overnight . evaporation of methanol gave b . 4335 ( 135 mg , 46 %), recrystallised from isopropanol . a mixture of 2 &# 39 ;, 3 &# 39 ;, 5 &# 39 ;- tri -( o - acetyl ) guanosine 36 ( 409 mg , 1 mmol ), tri - n - butylphosphine ( 303 mg , 1 . 5 mmol ) and 4 - bromothenyl alcohol ( 290 mg , 1 . 5 mmol ) in dry tetrahydrofuran ( 16 ml ) was stirred at room temperature for 45 min . then diisopropyl azodicarboxylate ( 303 mg , 1 . 5 mmol ) was added dropwise and the mixture stirred for 2 h . the solution was evaporated leaving an oil which was dissolved in thf / meoh / 25 % aqueous ammonia ( 1 : 1 : 1 ; 5 ml ) and kept for 48 h at 4 ° c . adsorption on silica gel and column chromatography with chcl 3 / meoh ( 15 : 1 to 10 : 1 ) gave the riboside b . 4363 ( 205 mg , 44 %). a mixture of 3 &# 39 ;, 5 &# 39 ;- di -( o - acetyl )- 2 &# 39 ;- deoxyguanosine 37 ( 554 mg , 1 . 5 mmol ), tri - n - butylphosphine ( 666 . 6 mg , 3 . 3 mmol ) and 4 - bromothenyl alcohol ( 638 mg , 3 . 3 mmol ) in dry tetrahydrofuran ( 40 ml ) was stirred at 80 ° c . for 15 min . then diisopropyl azodicarboxylate ( 666 . 6 mg , 3 . 3 mmol ) was added dropwise and 15 min later , the reaction mixture was cooled and evaporated leaving an oil . this was dissolved in thf / meoh / 25 % aqueous ammonia ( 1 : 1 : 1 ; 5 ml ) and kept for 48 h at 4 ° c . adsorption on silica gel and column chromatography with chcl 3 / meoh ( 20 : 1 ) gave the 2 &# 39 ;- deoxyriboside b . 4379 ( 338 mg , 51 %). an alkoxide solution was made from sodium hydride ( 60 % in oil ; 60 mg , 1 . 5 mmol ) and 4 - bromothenyl alcohol ( 344 mg , 1 . 8 mmol ) in dry dmso ( 0 . 5 ml ) over 1 h . it was reacted with 2 - amino - 9 -( β - d - arabinofuranosyl )- 6 - chloropurine 38 ( 151 mg , 0 . 5 mmol ) and stirred for 5 min at room temperature , then 15 min at 60 - 65 ° c . cooling and trituration with ether ( 50 ml ) and filtration yielded a solid which was dissolved in water ( 5 ml ), neutralised with acetic acid and treated with silica gel . column chromatography with ethyl acetate / meoh ( 19 : 1 ) gave the arabinoside b . 4368 ( 87 mg , 38 %), pure on t . l . c . these were made by the standard procedure from the quaternary salt 2 - amino - n , n , n - trimethyl - 1h - purin - 6 - aminium chloride and the appropriate alkoxide derived from the alcohol and sodium hybride in dmso ( cf . pp . 16d , 17 , 18 , 47 of jul . 12 , 1995 ). table 1a__________________________________________________________________________ m . p . mole - o . sup . 6 - substituent yield solvent for ( decomp .) cular analysiscompound , test no . rch . sub . 3 % recrystn . (° c .) formula weight c h n__________________________________________________________________________type 1a . hypoxanthinesb . 4293 furfuryl 60 meoh 154 c . sub . 18 h . sub . 8 n . sub . 4 o . sub . 2 216b . 4291 thenyl 66 meoh 168 c . sub . 10 h . sub . 8 n . sub . 4 os 232 found 51 . 85 3 . 40 24 . 12 req . 51 . 71 3 . 47 24 . 12b . 4292 4 - bromothenyl 69 meoh 170 c . sub . 10 h . sub . 8 brn . sub . 4 os 311 found 38 . 33 2 . 18 17 . 66 req 38 . 6 2 . 26 18 . 001b . 2 - methylhypoxanthinesb . 4347 benzyl 43 mecn 191 - 193 . sup . a c . sub . 12 h . sub . 11 n . sub . 4 o 240 found 65 . 05 4 . 91 23 . 30 req 64 . 99 5 . 03 23 . 32b . 4350 thenyl 35 mecn 176 - 178 . sup . a c . sub . 12 h . sub . 11 n . sub . 4 os 325 found 53 . 63 3 . 90 22 . 67 req 53 . 64 4 . 09 22 . 751c . 2 - fluorohypoxanthinesb . 4353 4 - bromothenyl 55 column 142 c . sub . 10 h . sub . 4 brfn . sub . 4 3291d . 9 -( 2 - hydroxyethoxy - methylguaninesb . 4334 benzyl 46 i - proh 150 - 152 . sup . a c . sub . 15 h . sub . 12 n . sub . 5 o . sub . 4 315 found 57 . 19 5 . 59 21 . 93 req 57 . 13 5 . 43 22 . 21b . 4335 4 - bromothenyl 42 i - proh 156 - 158 . sup . a c . sub . 13 h . sub . 14 brn . sub . 3 o . sub . 2 s 400 found 39 . 16 3 . 68 17 . 20 req 39 . 01 3 . 53 17 . 531e . 8 - hydroxyguaninesb . 4349 4 - bromothenyl 56 aq . etoh & gt ; 230 c . sub . 10 h . sub . 8 brn . sub . 5 o . sub . 2 s . 351 found 34 . 53 2 . 48 19 . 50 1 / 2 h . sub . 2 o req . 34 . 20 2 . 58 19 . 94type 2a . 8 - azaguaninesb . 4270 4 - fluorobenzyl 40 aq . meoh & gt ; 280 c . sub . 10 h . sub . 9 fn . sub . 4 o 260 found 51 . 50 3 . 85 29 . 44 req . 50 . 77 3 . 49 32 . 30b . 4314 4 - chlorothenyl 26 aq . meoh & gt ; 200 c . sub . 2 h . sub . 5 cln . sub . 4 os 282 . 7 found 28 . 86 2 . 61 28 . 61 38 . 24 2 . 50 29 . 73b . 4289 4 - bromothenyl 12 mecn & gt ; 190 c . sub . 8 h . sub . 5 brn . sub . 6 os 327 found 35 . 91 2 . 78 24 . 60 req . 33 . 04 2 . 16 25 . 692b . 8 - aza - 7 - deazaguaninesb . 4310 benzyl meoh 160 c . sub . 18 h . sub . 11 n . sub . 5 o . sub . 1 h . sub . 2 o 259 found 55 . 53 4 . 9 26 . 41 req . 55 . 59 5 . 01 27 . 02b . 4340 4 - fluorobenzyl 65 etoh 188 c . sub . 12 h . sub . 12 fn . sub . 5 o . 263 . 7 found 53 . 82 3 . 76 25 . 97 1 / 4h . sub . 2 o req . 54 . 6 4 . 0 26 . 55b . 4339 4 - chlorobenzyl 92 etoh 242 - 244 * c . sub . 12 h . sub . 10 cln . sub . 5 296 found 51 . 15 3 . 89 23 . 43 1 / 4h . sub . 2 o 1 / 4etoh req . 50 . 7 4 . 25 23 . 64b . 4343 piperonyl 50 etoh 186b . 4348 furfuryl etoh 150 . sup . c c . sub . 13 h . sub . 11 n . sub . 5 c . sub . 3 28 . 5 found 54 . 52 3 . 82 24 . 50 req . 54 . 7 3 . 88 24 . 55b . 4338 thenyl 68 etoh 180 c . sub . 10 h . sub . 6 n . sub . 5 o . sub . 2 1 / 4 . sub . 2 o 236 . 7 found 50 . 96 3 . 87 29 . 54 req . 50 . 96 4 . 06 29 . 71b . 4337 4 - bromothenyl 79 etoh 180 c . sub . 10 h . sub . 6 n . sub . 5 os 247 found 47 . 58 3 . 54 27 . 41 req . 47 . 7 3 . 8 27 . 8 c . sub . 10 h . sub . 2 brnios 326 found 37 . 08 2 . 52 21 . 31 req . 36 . 8 2 . 5 21 . 5type 3a . 8 - ozaguaninesb . 4272 4 - fluorobenzyl 41 acetone 223 - 224 c . sub . 11 h . sub . 5 n . sub . 5 o . sub . 2 261 found 50 . 39 3 . 08 26 . 65 req . 50 . 58 3 . 09 26 . 81b . 4285 4 - chlorobenzyl 63 acetone 219 - 220 c . sub . 11 h . sub . 8 cln . sub . 5 o . sub . 2 277 . 7 found 47 . 59 2 . 88 25 . 25 req . 47 . 58 2 . 90 25 . 22b . 4299 4 - chlorothenyl 55 acetone 164 - 165 c . sub . 12 h . sub . 4 cln . sub . 5 o . sub . 2 s 283 . 7 found 37 . 68 2 . 15 24 . 43 req . 38 . 10 2 . 13 24 . 69b . 4287 4 - bromothenyl 61 acetone 170 - 172 c . sub . 2 h . sub . 6 brn . sub . 5 o . sub . 2 328 found 33 . 30 1 . 85 21 . 37 req . 32 . 94 1 . 84 21 . 343b . 8 - thioguaninesb . 4296 benzyl 39 etoh c . sub . 11 h . sub . 8 n . sub . 5 os 259b . 4286 4 - fluorobenzyl 11 plc 182 - 184 c . sub . 11 h . sub . 5 fn . sub . 2 os 277b . 4315 4 - chlorothenyl 13 meoh c . sub . 5 h . sub . 6 cln . sub . 5 os . sub . 2 299 . 8 found 36 . 27 2 . 04 23 . 07 req . 36 . 06 2 . 02 23 . 36b . 4351 4 - bromothenyl 41 meoh 156 - 160 c . sub . 9 h . sub . 6 brn . sub . 5 os . sub . 2 144 found 31 . 49 1 . 60 20 . 11 req . 31 . 41 1 . 76 20 . 353c . pterins ( o . sup . 2 - substituent ) b . 4290 4 - fluorobenzyl 55 meoh & gt ; 110 c . sub . 13 h . sub . 12 fn . sub . 5 o 271 found 57 . 87 3 . 88 25 . 65 req . 57 . 56 3 . 72 25 . 82b . 4316 4 - chlorobenzyl 41 meoh & gt ; 170 c . sub . 13 h . sub . 8 cln . sub . 5 os 293 . 7 found 44 . 93 2 . 84 23 . 72 req . 44 . 98 2 . 75 23 . 84b . 4288 4 - bromothenyl 63 meoh 178 - 179 c . sub . 11 h . sub . 8 brn . sub . 5 os 338 found 39 . 34 3 . 13 20 . 25 req . 39 . 07 2 . 38 20 . 71type 4a . 2 , 4 - diamino - 6 - hydroxypyrimidinesb . 4305 4 - fluorobenzyl 98 c . sub . 6 h . sub . 5 / petrol 133 - 134 c . sub . 11 h . sub . 10 fn . sub . 5 o 234 found 56 . 20 4 . 79 23 . 66 56 . 40 4 . 73 23 . 92b . 4304 4 - chlorobenzyl 31 c . sub . 6 h . sub . 5 122 - 123 c . sub . 12 h . sub . 11 cln . sub . 4 250 . 7 found 52 . 43 4 . 56 22 . 47 req . 52 . 70 4 . 42 22 . 35b . 4303 piperonyl 79 mecn 168 - 171 c . sub . 17 h . sub . 12 n . sub . 6 o . sub . 5 260 found 55 . 31 4 . 64 21 . 38 req . 55 . 38 4 . 65 21 . 52b . 4307 thenyl 97 meph 100 c . sub . 9 h . sub . 10 n . sub . 4 os 222 found 48 . 83 4 . 58 25 . 25 req . 48 . 63 4 . 54 25 . 21b . 4302 4 - chlorothenyl 45 meph 129 - 130 c . sub . 9 h . sub . 9 cln . sub . 4 os 256 . 7 found 42 . 40 3 . 68 22 . 00 req . 42 . 11 3 . 53 21 . 834b . 2 , 4 - diamino - 6 - hydroxy - 5 - autrosopyrimidinesb . 4301 4 - fluorobenzyl 76 meoh & gt ; 250 ch . sub . 10 fn . sub . 5 o . sub . 2 263 found 49 . 60 3 . 90 26 . 29 req . 50 . 19 3 . 83 26 . 61b . 4311 4 - chlorothenyl 84 acetone & gt ; 190 c . sub . 9 h . sub . 8 cln . sub . 5 o . sub . 2 285 . 7 found 37 . 54 2 . 79 24 . 22 req . 37 . 84 2 . 82 24 . 51b . 4312 4 - bromothenyl 62 acetone 200 - 201 c . sub . 9 h . sub . 8 brn . sub . 5 o . sub . 2 330 found 32 . 87 2 . 38 20 . 96 req . 32 . 74 2 . 44 21 . 214c . 2 , 4 - diamino - 6 - hydroxy - 5 - nitropyrtnidinesb . 4308 piperonyl 67 dmf & gt ; 175 c . sub . 12 h . sub . 11 n . sub . 5 o . sub . 5 305 found 47 . 44 4 . 07 22 . 83 req . 47 . 22 3 . 63 22 . 94b . 4306 thenyl 34 meoh 159 - 160 c . sub . 9 h . sub . 5 n . sub . 6 o . sub . 3 267 found 40 . 99 3 . 71 25 . 99 req . 40 . 44 3 . 39 26 . 21type 3d . 5 - deazapterins ( o . sup . 4 - substituent ) b . 4276 thenyl 51 etoh 215 - 216 c . sub . 12 n . sub . 10 n . sub . 4 os 2584d . 5 - nitrocytosines ( o . sup . 2 - substituent ) b . 4380 4 - bromothenyl 15 meoh 143 - 144 c . sub . 9 h . sub . 7 brn . sub . 4 o . sub . 3 3315 . 6 - thtoguanines ( s . sup . 6 - substituent ) b . 4228 piperonyl 69 ch . sub . 2 oh 204 - 212 c . sub . 13 h . sub . 11 n . sub . 5 o . sub . 2 301 found 50 . 25 3 . 60 23 . 66 req . 51 . 82 3 . 68 23 . 24b . 4352 4 - bromothenyl 11 ch . sub . 2 oh 180 - 184 c . sub . 14 h . sub . 8 brn . sub . 5 o . sub . 1 342 found 35 . 07 2 . 42 19 . 49 1 / 2ch . sub . 2 oh 35 . 16 2 . 66 19 . 84__________________________________________________________________________ m . p . molecular analysis . sup . acompound , test no . 9 - substituent yield % solvent for recrystn . (° c .) formula weight c h n__________________________________________________________________________b . 4369 ethoxymethyl 39 etoh 134 - 5 c . sub . 13 h . sub . 14 brn . sub . 5 o . sub . 2 s 384 40 . 58 3 . 71 17 . 97 ( 40 . 64 . sup . 3 . 67 . sup . 18 . 23 ) b . 4370 n - octyloxymethyl 39 etoh 90 c . sub . 10 h . sub . 20 brn . sub . 5 o . sub . 2 s 468 48 . 97 5 . 67 14 . 82 ( 48 . 72 . sup . 5 . 60 . sup . 14 . 95 ) b . 4334 . sup . b 2 - hydroxy - 46 i - proh 150 - 2 c . sub . 15 h . sub . 27 n . sub . 5 o . sub . 3 315 57 . 19 5 . 59 21 . 93 ethoxymethyl ( 57 . 13 . sup . 5 . 43 . sup . 22 . 21 ) b . 4335 2 - hydroxy - 42 i - proh 156 - 8 c . sub . 13 h . sub . 14 brn . sub . 5 o . sub . 3 s 400 39 . 16 3 . 68 17 . 20 ethoxymethyl ( 39 . 01 . sup . 3 . 53 . sup . 17 . 50 ) b . 4363 β - d - ribo - 44 c . sub . 15 h . sub . 16 brn . sub . 5 o . sub . 5 s 458 furanosylb . 4368 β - d - arabino - 38 c . sub . 15 h . sub . 16 brn . sub . 5 o . sub . 5 s 458 furanosylb . 4379 β - d - 2 - deoxyribo - 51 c . sub . 15 h . sub . 16 brn . sub . 5 o . sub . 4 s 442 furanosyl__________________________________________________________________________ . sup . a found , with required values in parenthesis . . sup . b o . sup . 6benzyl table 1b__________________________________________________________________________ o . sup . 6 - substituent . sub . max ( meoh ) compound type , test no . rch . sub . 2 ( nm ) δ . sub . h [ ppm from tms , ( cd . sub . 3 ). sub . 2 so . sub . 1 ]/( hz ) __________________________________________________________________________type 1a . hypoxanthinesb . 4293 furfuryl 252 5 . 60 ( s ), 6 . 53 ( dd , 3 . 1 , 1 . 9 ), 6 . 69 ( d , j ), 7 . 76 ( dd , 19 , 3 . 9 ) 8 . 39 ( s ), 8 . 55 ( s ) b . 4291 thenyl 240 5 . 83 ( s ), 7 . 08 ( dd , 5 . 1 , 3 . 4 ), 7 . 35 ( d , 3 . 4 ), 7 . 6 ( d , 5 . 1 ), 8 . 39 ( s ), 8 . 51 ( s ) b . 4292 4 - bromothenyl 251 5 . 80 ( s ), 7 . 38 ( d , 13 ), 7 . 73 ( d , 13 ), 8 . 42 ( s ), 8 . 58 ( s ) type 1b . 2 - methylhypoxanthinesb . 4347 benzyl 256 2 . 61 ( s ), 5 . 60 ( s ), 7 . 50 ( m ), 8 . 32 ( s ) b . 4350 thenyl 240 2 . 63 ( s ), 5 . 77 ( s ), 7 . 05 ( dd , 5 . 1 , 2 . 6 , 7 . 33 ( d ), 2 . 4 ), 7 . 58 ( dd , 5 . 1 , 1 . 0 ) 8 . 26 ( s ), 13 . 22 ( s ) type 1c . 2 - phorohypoxanthinesb . 4353 4 - bromothenyl 233 , 255 5 . 77 ( s ), 7 . 4 ( d , 1 . 5 ), 7 . 77 ( d , 1 . 5 ), 8 . 45 ( s ), 13 . 64 ( bs ) type 1d . 9 -( 2 - hydroxyethoxymethyl ) guaninesb . 4334 benzyl 247 , 283 3 . 48 ( m ), 4 . 70 ( s ), 5 . 45 ( s ), 6 . 59 ( s ), 7 . 45 ( m ), 8 . 03 ( s ), b . 4335 4 - bromothenyl 245 , 284 3 . 49 ( m ), 4 . 71 ( s ), 5 . 45 ( s ), 5 . 66 ( s ), 6 . 65 ( s ), 7 . 30 ( d , 1 . 5 ) 7 . 72 ( d , 1 . 5 ) 8 . 04 ( s ) type 1e . 8 - hydroxyguaninesb . 4349 4 - bromothenyl 239 , 293 5 . 54 ( s ), 6 . 24 ( s ), 7 . 33 ( d , 1 . 4 ) 7 . 70 ( d , 1 . 4 ), 10 . 49 ( s ) 11 . 12 ( s ) type 2a . δ - azaguaninesb . 4270 4 - fluorobenzyl 288 5 . 57 ( s ), 7 . 04 ( s ), 7 . 28 ( m ), 7 . 65 ( m ), 15 . 38 ( s ) b . 4314 4 - chlorothenyl 288 5 . 71 ( s ), 7 . 13 ( s ), 7 . 41 ( d , 1 . 5 ), 7 . 66 ( d , 1 . 5 ), 15 . 42 ( s ). b . 4289 4 - bromothenyl 287 5 . 73 ( s ), 7 . 12 ( s ), 7 . 43 ( d , 1 . 5 ), 7 . 76 ( d , 1 . 5 ), 15 . 39 ( s ). type 2b . 8 - aza - 7 - diazaguaninesb . 4310 benzyl 217 5 . 50 ( s ), 6 . 68 ( s ), 7 . 74 ( m ), 7 . 82 ( s ), 12 . 87 ( bs ) b . 4340 4 - fluorobenzyl 278 5 . 49 ( s ), 6 . 70 ( s ), 7 . 20 ( m ), 7 . 61 ( m ), 7 . 83 ( s ), 12 . 88 ( bs ) b . 4339 4 - chlorobenzyl 276 5 . 50 ( s ), 6 . 69 ( s ), 7 . 49 ( d , 8 . 4 ), 7 . 56 ( d , 8 . 4 ), 7 . 83 ( s ), 12 . 90 ( s ) b . 4343 piperonyl 282 5 . 39 ( s ), 6 . 95 ( s ), 6 . 69 ( s ), 6 . 94 ( d , 7 . 3 ), 7 . 04 ( dd , 7 . 9 , 1 . 5 ), 7 . 1 ( dd , 1 . 5 ), 7 . 50 ( s ) 12 . 86 ( bs ). b . 4348 furfuryl 277 5 . 46 ( s ), 6 . 52 ( s ), 0 . 70 ( s ), 6 . 71 ( s ), 7 . 73 ( s ), 7 . 79 ( s ), 12 . 85 ( bs ). b . 4338 thenyl 278 5 . 69 ( s ), 6 . 73 ( s ), 7 . 07 ( d , 3 . 5 ), 7 . 35 ( s ), 7 . 60 ( d , 1 . 1 ), 7 . 79 ( s ), 12 . 90 ( bs ) b . 4337 4 - bromothenyl 278 5 . 65 ( s ), 6 . 76 ( s ), 7 . 38 ( s ), 7 . 72 ( d , 1 . 3 ), 7 . 91 ( s ), 2 . 91 ( bs ) type 3a . n - oxaguaninesb . 4272 4 - chlorobenzyl 257 , 341 5 . 62 ( s ), 7 . 39 ( 1 , 9 . 1 ), 7 . 68 ( s ), 7 . 91 ( s ), 7 . 97 ( s ). b . 4285 4 - chlorobenzyl 256 , 340 5 . 63 ( s ), 7 . 53 ( d , 8 . 3 ), 7 . 65 ( d , 8 . 3 ), 7 . 90 ( s ), 7 . 97 ( s ) b . 4299 4 - chlorobenzyl 252 , 343 5 . 78 ( s ), 7 . 46 ( d , 1 . 6 ), 7 . 72 ( d , 1 . 6 ), 7 . 95 ( s ), 8 . 01 ( s ). b . 4287 4 - bromothenyl 253 , 343 5 . 79 ( s ), 7 . 49 ( d , 1 . 6 ), 7 . 8 ( d , 1 . 6 ), 7 . 95 ( s ), 8 . 01 ( s ) type 3b . 8 - thiaguaninesb . 4296 benzyl 227 , 361b . 4286 4 - fluorobenzyl 235 , 362 5 . 59 ( s ), 7 . 29 ( d , 8 . 9 ), 7 . 51 ( s ), 7 . 67 ( m ) b . 4315 4 - chlorobenzyl 228 , 360 5 . 75 ( s ), 7 . 44 ( d , 1 . 6 ), 7 . 55 ( bs ), 7 . 69 ( d , 1 . 6 ) b . 4351 4 - bromothenyl 228 , 361 5 . 78 ( s ), 7 . 45 ( d , 1 . 6 ), 7 . 46 ( bs ), 7 . 75 ( d , 1 . 6 ) type 3c . pterins ( o . sup . 6 - substituent ) b . 4290 4 - fluorobenzyl 232 , 264 ( sb , 5 . 56 ( s ), 7 . 29 ( 1 . 6 , s ), 7 . 4 ( bs ), 7 . 66 ( s ), 8 . 45 ( d , 1 . 8 ), 8 . 82 ( d , 1 . 8 ) 162b . 4316 4 - chlorothenyl 232 , 364 5 . 71 ( s ), 7 . 41 ( d , 1 . 6 ), 7 . 47 ( bs ), 7 . 67 ( d , 1 . 6 ), 8 . 40 ( d , 2 . 0 ), 8 . 83 ( d , 2 . 0 ) b . 4288 4 - bromothenyl 231 , 364 5 . 73 ( s , 7 . 44 ( d , 1 . 6 ), 7 . 50 ( bs ), 7 . 77 ( d , 1 . 6 ) 8 . 46 ( d , 2 ), 8 . 83 ( d , 2 ) type 4a . 2 , 4 - diamino - 6 - hydroxy - pyrimidinesb . 4305 4 - fluorobenzyl 238 , 267 5 . 18 ( s ), 5 . 19 ( s ), 5 . 96 ( s ), 6 . 10 ( s ), 7 . 19 ( t , 8 . 8 ), 7 . 44 ( dd , 8 . 8 , 5 . 8 ) b . 4304 4 - chlorobenzyl 238 , 268 5 . 11 ( s ), 5 . 22 ( s ), 5 . 96 ( s ), 6 . 10 ( s ), 7 . 44 ( s ) b . 4303 piperonyl 236 , 267 5 . 09 ( s ), 5 . 11 ( s ), 5 . 97 ( s ), 6 . 01 ( s ), 6 . 07 ( s ), 6 . 91 ( d , 1 . 1 ), 7 . 00 ( s ). b . 4307 thenyl 235 , 267 5 . 08 ( s ), 5 . 40 ( s ), 6 . 00 ( s ), 6 . 10 ( s ), 7 . 03 ( dd , 8 . 1 , 3 . 5 ) 7 . 20 ( dd , 8 . 1 , 1 . 1 ), 7 . 54 ( dd , 3 . 5 , 1 . 1 ) b . 4302 4 - chlorothenyl 236 , 265 5 . 08 ( s ), 5 . 35 ( s ), 6 . 03 ( s ), 6 . 13 ( s ), 7 . 19 ( s ), 7 . 55 ( d , 1 . 6 ). type 4b . 2 , 4 - diamino - 6 - hydroxy - 3 - nitroxopyrimidinesb . 4301 4 - fluorobenzyl 336 5 . 59 ( s ), 7 . 26 ( m ), 7 . 65 ( m ), 7 . 80 ( bs ), 7 . 85 ( bs ), 8 . 00 ( bs ), 10 . 05 ( bs ). b . 4311 4 - chlorothenyl 335 5 . 73 ( s ), 7 . 40 ( d , 1 . 6 ), 7 . 66 ( d , 1 . 6 ), 7 . 94 ( s ), 7 . 98 ( d , 2 . 7 ), 8 . 11 ( d , 4 . 2 ), 10 . 03 ( d , 4 . 2 ). b . 4312 4 - bromothenyl 335 5 . 75 ( s ), 7 . 42 ( d , 1 . 4 ), 7 . 75 ( d , 1 . 4 ), 7 . 93 ( s ), 7 . 98 ( s ), 8 . 12 ( d , 4 . 0 ), 10 . 04 ( d , 4 . 0 ) type 4c . 2 , 4 - diamino - 6 - hydroxy - 5 - nitropyrimidinesb . 4308 piperonyl 288 , 330 5 . 33 ( s ), 6 . 05 ( s ), 6 . 95 ( d , 8 . 0 ), 7 . 00 ( dd , 8 . 0 , 1 . 4 ), 7 . 10 ( d , 1 . 4 ); 7 . 26 ( bs ), 7 . 3 7 . 96 ( bs ). b . 4306 thenyl 234 , 329 5 . 59 ( s ), 7 . 03 ( dd , 5 . 1 , 3 . 5 ), 7 . 28 ( d , 3 . 5 ), 7 . 32 ( bs ), 7 . 56 ( d , 5 . 1 ), __________________________________________________________________________ 7 . 94 ( bs ) substituent . sub . max ( meoh ) compound type , test no . rch . sub . 2 ( nm ) δ . sub . h [ ppm from tms , ( cd . sub . 3 ). sub . 2 so . sub . 1 ]/( hz ) __________________________________________________________________________type 3d 5 - deazapterins ( o . sup . 4 - substituent ) b . 4376 thenyl 248 , 309 5 . 54 ( s ), 6 . 96 ( q ), 7 . 716 ( dd ), 7 . 38 ( dd ), 7 . 41 ( q ), 8 . 39 ( dd ), 8 . 79 ( dd ). type 4d 5 - nitrocytosines ( o . sup . 2 - substituent ) b . 4380 4 - bromothenyl 255 sh , 334 5 . 19 ( s ), 7 . 20 ( s ), 7 . 56 ( d ), 8 . 24 ( s ), 8 . 70 ( s ), 8 . 90 ( s ). type 5 6 - thioguanines ( s . sup . 6 - substituent ) b . 4228 piperonyl 245 , 311 4 . 56 ( s ), 6 . 06 ( s ), 6 . 55 ( s ), 7 . 03 ( d ), 7 . 06 ( d ), 7 . 14 ( s ), 8 . 08 ( s ), 12 . 67 ( bs ). b . 4352 4 - bromothenyl 241 , 314 4 . 77 ( s ), 6 . 52 ( s ), 7 . 18 ( d ), 7 . 51 ( d ), 7 . 93 ( s ), 12 . 61 ( bs ) __________________________________________________________________________ . sub . max ( meoh ) compound type , test no . 9 - substituent yield % ( nm ) δ . sub . h [ ppm from tms , ( cd . sub . 3 ). sub . 2 so . sub . 1 ]/( hz ) __________________________________________________________________________b . 4369 ethoxymethyl 245 , 284 3 . 35 ( s ), 5 . 41 ( s ), 5 . 66 ( s ), 6 . 66 ( s ), 7 . 38 ( d ), 7 . 73 ( d ), 8 . 04 ( s ). b . 4370 n - octyloxymethyl 245 , 284 0 . 09 ( t ), 1 . 17 ( m ), 3 . 36 ( t ), 5 . 41 ( s ), 5 . 66 ( s ), 6 . 66 ( s ), 7 . 38 ( d ), 7 . 72 ( d ), 8 . 03 ( s ). b . 4334 . sup . a 2 - hydroxy - 245 , 283 3 . 48 ( m ), 4 . 70 ( s ), 5 . 45 ( s ), 6 . 59 ( s ), 7 . 45 ( s ), 8 . 03 ( s ). ethoxymethylb . 4335 2 - hydroxy - 245 , 284 3 . 49 ( m ), 4 . 71 ( s ), 5 . 45 ( s ), 5 . 66 ( s ), 6 . 65 ( s ), 7 . 30 ( d , 1 . 5 ), ethoxymethyl 7 . 72 ( d , 1 . 5 ), 8 . 40 ( s ). b . 4363 β - d - ribo - -- 3 . 54 ( m ), 3 . 63 ( m ), 3 . 91 ( dd ), 4 . 12 ( dd ), 4 . 48 ( ddd ), 5 . 12 ( dd ), furanosyl 5 . 18 ( d ), 5 . 45 ( d ), 5 . 66 ( s ), 5 . 80 ( dd ), 6 . 61 ( s ), 7 . 38 ( d ), 7 . 71 ( d ), 8 . 15 ( s ). b . 4368 β - d - arabino - 245 , 284 3 . 64 ( m ), 3 . 76 ( dd ), 4 . 07 ( m ), 5 . 09 ( dd ), 5 . 51 ( d ), 5 . 53 ( m ), furanosyl 6 . 13 ( d ), 6 . 60 ( d ), 7 . 37 ( d ), 7 . 71 ( d ), 7 . 95 ( s ) b . 4379 β - d - 2 - deoxyribo - 2 . 39 ( ddd ), 2 . 72 ( ddd ), 3 . 65 ( ddd ), 3 . 98 ( dd ), 4 . 40 ( dd ), 5 . 11 ( s ) furanosyl 5 . 41 ( d ), 5 . 80 ( s ), 6 . 38 ( dd ), 6 . 67 ( s ), 7 . 49 ( d ), 7 . 83 ( d ), 8 . 25 ( s ). __________________________________________________________________________ . sup . a o . sup . 6benzyl table 2______________________________________ i . sub . 50 ( μm ) t 1 / 2 ( h ) inactivat0r type hat in pbs______________________________________1ab . 4291o . sup . 6 -( thenyl )- hypoxanthine & gt ; 201 . 9b . 4293o . sup . 6 -( furfuryl )- hypoxanthine & gt ; 168b . 4292o . sup . 6 -( 4 - bromothenyl )- hypoxanthine & gt ; 16o . sup . 6 -( benzyl )- hypoxanthine . sup . b 851bb . 4347o . sup . 6 -( benzyl )- 2 - methylhypoxanthine 75b . 4350o . sup . 6 -( thenyl )- 2 - methylhypoxanthine 141cb . 4353o . sup . 6 -( 4 - bromothenyl )- 2 - fluorohypoxanthine 1 . 4o . sup . 6 -( benzyl )- 2 - fluorohypoxanthine . sup . a 481db . 4334o . sup . 6 -( benzyl )- 9 -( 2 - hydroxyethoxymethyl ) 8 & gt ; 20guanineb . 4335o . sup . 6 -( 4 - bromothenyl )- 9 -( 2 - hydroxy see table 3ethoxymethyl ) guanine1eb . 4349o . sup . 6 -( 4 - bromothenyl )- 8 - hydroxyguanine see table 3o . sup . 6 -( benzyl )- 8 - hydroxyguanine . sup . a 0 . 32ab . 4270o . sup . 6 -( 4 - fluorobenzyl )- 8 - azaguanine 0 . 08b . 4314o . sup . 6 -( 4 - chlorothenyl )- 8 - azaguanine see table 3b . 4289o . sup . 6 -( 4 - bromothenyl )- 8 - azaguanine & gt ; 10o . sup . 6 -( benzyl )- 8 - azaguanine . sup . a 0 . 072bb . 4310o . sup . 6 -( benzyl )- 7 - deaza - 8 - azaguanine & gt ; 16b . 4340o . sup . 6 -( 4 - fluorobenzyl )- 8 - aza - 7 - deazaguanine 0 . 018 & gt ; 16b . 4339o . sup . 6 -( 4 - chlorobenzyl )- 8 - aza - 7 - deazaguanine 0 . 02 1 . 5b . 4343o . sup . 6 -( piperonyl )- 8 - aza - 7 - deazaguanine see table 3b . 4348o . sup . 6 -( furfuryl )- 8 - aza - 7 - deazaguanine 0 . 27b . 4338o . sup . 6 -( thenyl )- 8 - aza - 7 - deazaguanine 0 . 01b . 4337o . sup . 6 -( 4 - bromothenyl )- 8 - aza - 7 - deazaguanine 0 . 007 & gt ; 203ab . 4272o . sup . 6 -( 4 - fluorobenzyl )- 8 - oxaguanine see table 3b . 4285o . sup . 6 -( 4 - chlorobenzyl )- 8 - oxaguanine 4 . 6b . 4299o . sup . 6 ( 4 - chlorothenyl )- 8 - oxaguanine 9 . 23b . 4287o . sup . 6 -(- 4 - bromothenyl )- 8 - oxaguanine 2 . 6b . 4232o . sup . 6 -( benzyl )- 8 - oxaguanine 0 . 253bb . 4296o . sup . 6 -( benzyl )- 8 - thiaguanine & gt ; 17 0 . 02b . 4286o . sup . 6 -( 4 - fluorobenzyl )- 8 - thiaguanine & gt ; 17b . 4315o . sup . 6 -( 4 - chlorothenyl )- 8 - thiaguanine . sup . c 0 . 006b . 4351o . sup . 6 -( 4 - bromothenyl )- 8 - thiaguanine see table 33cb . 4290o . sup . 4 -( 4 - fluorobenzyl )- pterin & gt ; 10 0 . 088b . 4316o . sup . 4 -( 4 - chlorothenyl )- pterin see table 3b . 4288o . sup . 4 -( 4 - bromothenyl )- pterin & gt ; 10 0 . 0254ab . 43052 , 4 - diamino - 6 -( 4 - fluorobenzyloxy ) pyrimidine 4 . 0 & gt ; 16b . 43042 , 4 - diamino - 6 -( 4 - chlorobenzyloxy ) pyrimidine 5 . 0 & gt ; 16b . 43032 , 4 - diamino - 6 -( 3 , 4 - piperonyloxy ) pyrimidine 0 . 8 12 . 5b . 43072 , 4 - diamino - 6 -( thenyloxy ) pyrimidine 4 . 2b . 43022 , 4 - diamino - 6 -( 4 - chlorothenyloxy ) pyrimidine 0 . 17 & gt ; 162 , 4 - diamino - 6 -( benzyloxy ) pyrimidine . sup . a 154bb . 43012 , 4 - diamino - 6 -( 4 - fluorobenzyloxy )- 5 - 0 . 0175 & gt ; 16nitrosopyrimidineb . 43112 , 4 - diamino -( 4 - chlorothenyloxy )- 5 - see table 3nitrosopyrimidineb . 43122 , 4 - diamino - 6 -( 4 - bromothenyloxy )- 5 - 0 . 045 4nitrosopyrimidine2 , 4 - diamino - 6 -( benzyloxy )- 5 - 0 . 06nitrosopyrimidine . sup . a4cb . 43062 , 4 - diamino - 6 -( thenyloxy )- 5 - 2 . 3 & gt ; 16nitropyrimidineb . 43082 , 4 - diamino - 6 - piperonyloxy - 5 - nitropyrimidine 0 . 5 9 . 22 , 4 - diamino - 6 - benzyloxy - 5 - nitropyrimidine . sup . a 0 . 064db . 4380o . sup . 2 ( 4 - bromothenyl )- 5 - nitrocytosine 50b . 4228s . sup . 6 -( piperonyl )- 6 - thioguanine 50b . 4352s . sup . 6 -( 4 - bromothenyl )- 6 - thioguanine 8comparativeb . 4376o . sup . 6 - thenyl - 5 - deazapterin 1 , 600______________________________________ results for some 9substituted o . sup . 6 ( 4bromothenyl ) guanines are included in table 7 . . sup . a data taken from chae et al , j . med . chem . 1995 , 38 , 359 - 365 . sup . b data taken from moschel et al ., j . med . chem . 1992 , 35 , 4486 - 4491 . . sup . c b . 4315 raji i . sub . 50 ( um ) 0 . 002 blank space = not done . table 3__________________________________________________________________________ sol - ubili - t . sup . 1 / 2 raji cell sensitisation ty raji cell ( h ) ( d . sub . 50 control / d . sub . 50 wa -) toxicity at i . sub . 50 i . sub . 50 i . sub . 50 i . sub . 50 i . sub . 50 i . sub . 50 i . sub . 50 t . sup . 1 / 2 by bcnu temozolomide ter 10 μm ` b ` mol hat raji mat rat chat ogt ada ( h ) as - inactivator concentration ( mg /. m ) aloneinactivatorwgt ( μm ) ( μm ) ( μm ) ( μm ) ( μm ) ( μm ) ( μm ) pbs say 10 10 1 . 0 0 . 5 0 . 1 ml ) (% __________________________________________________________________________ growth ) b4272261 0 . 05 0 . 023 0 . 125 0 . 075 0 . 04 & gt ; 1000 & gt ; 1000 5 . 7 2 . 6 1 . 88 1 . 41 -- -- -- 0 . 002 111 . 21 + 23 . 3b4311286 0 . 009 0 . 009 0 . 008 0 . 016 0 . 02 1 . 8 & gt ; 1000 10 12 . 5 8 . 0 73 . 3 8 . 25 -- 1 . 4 0 113 . 0 + 31 . 0b4314283 0 . 011 0 . 012 0 . 073 0 . 037 0 . 03 2 & gt ; 1000 & gt ; 19 & gt ; 48 7 . 62 84 4 . 61 -- 3 . 46 not 55 . 5 + 7 . 3 done ( d . sub . 50 16 μm ) b4316294 0 . 025 0 . 011 0 . 068 0 . 03 0 . 04 3 . 8 & gt ; 1000 & gt ; 19 32 6 . 4 66 13 . 2 -- 1 . 4 0 . 3 85 . 5 + 20 . 0b4335400 0 . 33 0 . 07 15 . 63 6 . 5 1 . 8 156 & gt ; 1000 & gt ; 19 & gt ; 48 5 . 33 38 3 . 5 -- 1 . 0 0 . 009 98 . 4 + 12 . 2b4343285 0 . 007 0 . 0085 0 . 31 0 . 045 0 . 02 30 & gt ; 1000 7 . 5 3 3 . 81 9 . 5 2 . 12 -- 1 . 6 0 . 01 97 . 0 + 10 . 0b4349342 0 . 018 0 . 007 0 . 043 0 . 074 0 . 02 0 . 08 & gt ; 1000 7 . 3 & gt ; 48 4 . 8 50 . 8 33 -- 2 . 4 0 . 002 90 . 0 + 13 . 0b4351344 0 . 003 0 . 005 0 . 071 0 . 027 0 . 03 5 . 8 & gt ; 1000 & gt ; 16 12 4 . 8 18 . 1 1 . 32 -- 1 . 2 not 117 . 5 + 29 . 1 donebeg 241 0 . 04 0 . 1 0 . 2 0 . 076 0 . 01 17 & gt ; 1000 & gt ; 64 & gt ; 75 4 . 33 27 . 5 1 . 89 -- 1 . 03 0 . 023 82 . 2 + 11 . 0patrin - 2326 0 . 003 0 . 003 0 . 05 0 . 019 0 . 03 0 . 85 & gt ; 1000 & gt ; 16 & gt ; 48 6 . 0 60 33 8 5 . 5 not 69 . 8 + 10 . 3 doneb . 4280 ( d50 44__________________________________________________________________________ μm ) -- = not done table 4______________________________________effect of inactivator pretreatment on sensitisationof varous human cancer cell lines to temozolomide sensitisation factor ( d . sub . 50 control / l ). sub . 50 ` b `) mcf - 7 pc3 du145 ** raji inactivator dose inactivator dose ( μm ) inactivator ( 10 μm ) 10 1 . 0 0 . 5 0 . 1______________________________________b4311 -- 5 . 56 3 . 75 73 . 3 8 . 25 1 . 4b4314 * 2 . 0 1 . 71 84 . 0 4 . 61 3 . 46 -- b4316 7 . 6 3 . 53 66 13 . 2 1 . 4 -- b4349 3 . 6 4 . 0 50 . 8 33 . 0 2 . 4 -- bcg 2 . 88 . 94 5 . 45 27 . 5 1 . 89 1 . 03 -- patrin - 2 3 . 13 4 . 6 4 . 14 60 33 . 0 8 . 0 5 . 5______________________________________ * toxic to raji cells at 10 μm ** sensitisation factor = d . sub . 60 control / d . sub . 60 ` b -- not done table 5______________________________________effect of inactivator pretreatment on sensitisationof various human cancer cell lines to bcnu sensitisation factor ( d . sub . 50 control / d . sub . 50 ` b `) rajiinactivator inactivator dose ( μm )( 10 μm ) mcf - 7 pc3 du145 ** 10 1 . 0 0 . 1______________________________________b4311 -- 1 . 47 1 . 56 8 . 0 -- -- b4314 * -- 1 . 25 7 . 62 7 . 6 3 . 45b4316 1 . 37 1 . 35 3 . 57 6 . 4 -- -- b4349 1 . 85 1 . 63 2 . 78 4 . 8 -- -- bcg 1 . 94 1 . 41 1 . 79 4 . 33 -- -- patrin - 2 1 . 61 2 . 11 2 . 08 6 . 0 -- -- ______________________________________ * toxic to raji cells at 10 μm ** sensitisation factor = d . sub . 60 control / d . sub . 60 ` b -- not done table 6a__________________________________________________________________________ yield % ( based solvent for m . p . ( decomp .) analysistest no . o . sup . 6 - substiutent on solvate ) recrystn . (° c .) formula c h n__________________________________________________________________________b . 42804 - bromothenyl 73 meoh 204 - 205 c . sub . 10 h . sub . 8 brn . sub . 5 os found 36 . 7 2 . 45 21 . 46 req . 36 . 82 2 . 47 21 . 47b 42815 - chlorothenyl . sup . a 39 mecn 155 - 158 c . sub . 10 h . sub . 8 cln . sub . 5 os found 41 . 81 2 . 86 24 . 10 req . 42 . 63 2 . 86 24 . 86b . 42835 - cyanothenyl . sup . b 10 meoh 200 upwards c . sub . 11 h . sub . 8 n . sub . 6 os found 47 . 01 2 . 94 28 . 24 0 . 5 h . sub . 2 o req . 46 . 97 3 . 23 29 . 88b . 42945 - methylsulph - 32 meoh 200 upwards c . sub . 11 h . sub . 11 n . sub . 5 o . sub . 2 s . sub . 2 found 42 . 58 3 . 62 22 . 27inylthenyl req 42 . 71 3 . 58 22 . 64b . 42984 - chlorothenyl 34 mecn 194 - 198 c . sub . 12 h . sub . 8 cln . sub . 5 os found 42 . 70 2 . 94 24 . 84 req 42 . 63 2 . 86 24 . 86b . 43004 - methoxythenyl 44 meoh 189 - 190 c . sub . 11 h . sub . 11 n . sub . 5 o . sub . 2 found 47 . 73 4 . 15 25 . 05 req 47 . 64 4 . 00 25 . 26b . 43135 - bromo - 3 - 7 . 6 mecn 190 upwards c . sub . 10 h . sub . 2 brn . sub . 5 os found 37 . 02 2 . 43 20 . 95thienylmethyl req 36 . 82 2 . 47 21 . 47b . 43174 - cyanothenyl 32 meoh 213 - 216 c . sub . 11 h . sub . 8 n . sub . 6 os found 48 . 50 2 . 84 30 . 66 req 48 . 52 2 . 96 30 . 87b . 43184 , 5 - dichlorothenyl 38 meoh 210 upwards c . sub . 10 h . sub . 7 cl . sub . 2 n . sub . 5 found 35 . 94 2 . 67 20 . 96 1h . sub . 2 o req 35 . 94 2 . 71 20 . 96b . 43212 - chloro - 4 - picolyl 10 meoh 234 upwards c . sub . 11 h . sub . 9 cln . sub . 6 o found 47 . 15 3 . 52 29 . 32 req 47 . 75 3 . 29 30 . 37b . 43365 - bromofurfuryl 39 meoh 180 upwards c . sub . 10 h . sub . 8 brn . sub . 5 o . sub . 2 . found 38 . 22 2 . 71 21 . 93 0 . 25 h . sub . 2 o req 38 . 18 2 . 72 22 . 26__________________________________________________________________________o . sup . 6 - substituted guanines m . p . compound , yield solvent for ( decomp ) molecular analysistest no . o . sup . 6 - substituted rch . sub . 2 % recrystn . ( c . *) formula weight c h n__________________________________________________________________________b . 4282 3 - picolyl 54 meoh 244 - 254 c . sub . 11 h . sub . 10 n . sub . 6 o . sub . 2 258 n - oxideb . 4309 5 - methylsulphonyl - 12 etoh 206 - 209 c . sub . 11 h . sub . 11 n . sub . 5 o . sub . 3 s . sub . 2 . 348 found 41 . 46 3 . 83 20 . 13 thenyl 1 / 2 c . sub . 2 h . sub . 5 oh req . 41 . 37 4 . 05 20 . 10b . 4319 6 - chloro - 3 - picolyl 58 meoh & gt ; 215 c . sub . 11 h . sub . 9 cln . sub . 6 o . 285 . 7 found 46 . 01 3 . 49 29 . 05 1 / 2 h . sub . 2 o req . 46 . 25 3 . 53 29 . 42b . 4320 5 - bromo - 3 - picolyl 56 meoh & gt ; 220 c . sub . 11 h . sub . 9 brn . sub . 6 o . 330 found 40 . 02 3 . 05 25 . 28 1 / 2 h . sub . 2 o req . 39 . 87 3 . 01 25 . 28b . 4354 4 - isothiazolyl 28 meoh & gt ; 200 c . sub . 9 h . sub . 8 n . sub . 6 os . 261 . 8 found 41 . 59 3 . 64 31 . 53 3 / 4 h . sub . 2 o req . 41 . 32 3 . 59 32 . 16b . 4356 4 - methylthiothenyl 30 meoh c . sub . 11 h . sub . 11 n . sub . 5 os . sub . 2 293 . 4b . 4357 5 - iodo - 3 - thienyl - 23 meoh & gt ; 200 c . sub . 10 h . sub . 8 ln . sub . 5 os 373 methylb . 4361 4 - methyl - 95 meoh 170 - 172 c . sub . 11 h . sub . 11 n . sub . 5 o . sub . 3 s . sub . 2 325 found 40 . 2 3 . 39 21 . 01 sulphonylthenyl req . 40 . 61 3 . 41 21 . 53b . 4366 naphtho [ 2 , 1 - b ]- 81 meoh & gt ; 150 c . sub . 18 h . sub . 13 n . sub . 5 os 347 thiophen - 2 - yl - methylb . 4373 4 - azidothenyl 37 meoh & gt ; 195 . sup . a c . sub . 10 h . sub . 8 n . sub . 8 so 288b . 4377 4 - methyl - 55 meoh 204 - 206 c . sub . 11 h . sub . 11 n . sub . 5 o . sub . 2 s . sub . 2 309 sulphinylthenylb . 4378 5 - phenylthenyl 54 ch . sub . 3 cn & gt ; 170 c . sub . 16 h . sub . 13 n . sub . 5 os 323__________________________________________________________________________ . sup . a 5 6 mmol alcohol per mmol quaternary salt used in synthesis . . sup . b dimethylformamide reaction solvent . table 6b__________________________________________________________________________test no . o . sup . c - substituent . sub . max ( nm )( meoh ) δ . sub . h [ ppm from tms ; ( cd . sub . 3 ). sub . 2 so . sub . 1 ] j__________________________________________________________________________ ( hz ) b . 4280 4 - bromothenyl 238 , 284 5 . 65 ( s ), 6 . 40 ( s ), 7 . 37 ( d ), 7 . 71 ( d ), 7 . 85 ( s ), 12 . 49 ( s ). ( rch . sub . 2 oh : 233 ). b . 4281 5 - chlorothenyl 247 , 284 5 . 59 ( s ), 6 . 40 ( s ), 7 . 06 ( d ), 7 . 22 ( d ), 7 . 87 ( s ), 12 . 47 ( bs ). ( rch . sub . 2 oh : 245 ). b . 4283 5 - cyanothenyl 247 , 272 5 . 73 ( s ), 6 . 46 ( s ), 7 . 49 ( d ), 7 . 87 ( s ), 7 . 92 ( d ), 12 . 54 ( bs ). b . 4294 5 - methylsulphinylthenyl 243 , 284 ( sh ) 2 . 93 ( s ), 5 . 73 ( s ), 6 . 41 ( s ), 7 . 40 ( d ), 7 . 52 ( d ), 7 . 88 ( s ), 12 . 52 ( bs ). [ rch . sub . 2 oh : 240 , 274 ( sh )]. b . 4298 4 - chlorothenyl 238 , 284 5 . 64 ( s ), 6 . 42 ( s ), 7 . 34 ( d ), 7 . 62 ( d ), 7 . 86 ( s ), 12 . 51 ( s ) ( rch . sub . 2 oh : 240 ). b . 4300 4 - methoxythenyl 245 ( sh ), 282 3 . 75 ( s ), 5 . 57 ( s ), 6 . 37 ( s ), 6 . 60 ( d ), 7 . 01 ( d ), 7 . 85 ( s ), 12 . 48 ( s ). ( rch . sub . 2 oh : 258 ). b . 4313 5 - bromo - 3 - thienylmethyl 240 , 284 5 . 42 ( s ), 6 . 38 ( s ), 7 . 40 ( d ), 7 . 72 ( d ), 7 . 85 ( s ), 12 . 47 ( s ). ( rch . sub . 2 oh : 236 ). b . 4317 4 - cyanothenyl 244 , 284 5 . 68 ( s ), 6 . 44 ( s ), 7 . 74 ( d ), 7 . 86 ( s ), 8 . 60 ( d ), 12 . 50 ( s ). ( rch . sub . 2 oh : 244 ). b . 4318 4 , 5 - dichlorothenyl 243 , 285 5 . 58 ( s ), 6 . 45 ( s ), 7 . 41 ( s ), 7 . 87 ( s ), 12 . 52 ( s ) ( rch . sub . 2 oh : 243 ). b . 4321 2 - chloro - 4 - picolyl 241 , 272 ( sh ), 285 5 . 58 ( s ), 6 . 36 ( s ), 7 . 51 ( bs ), 7 . 61 ( bs ), 7 . 91 ( bs ), 8 . 44 ( bs ), 12 . 56 ( bs ). [ rch . sub . 2 oh : 262 , 268 ( sh )]. b . 4336 5 - bromofurfuryl 220 , 284 5 . 42 ( s ), 6 . 39 ( s ), 6 . 64 ( d ), 6 . 78 ( d ), 7 . 85 ( s ), 12 . 49 ( s ). ( rch . sub . 2 oh : 223 ) __________________________________________________________________________o . sup . 6 - substituted guanines o . sup . 6 - substituent . sub . max ( meoh ) compound type , test no . rch . sub . 2 ( nm ) δ . sub . h [ ppm from tms , ( cd . sub . 3 ). sub . 2 so ,] j__________________________________________________________________________ ( hz ) b . 4282 3 - picolyl n - oxide 271 5 . 48 ( s ), 6 . 41 ( s ), 7 . 47 ( m ), 7 . 87 ( s ), 8 . 22 ( m ), 8 . 42 ( s ), 12 . 52 ( s ) b . 4309 5 - methylsulphonyl - 242 , 284 5 . 75 ( s ), 6 . 43 ( s ), 7 . 47 ( d ), 7 . 74 ( d ), 7 . 87 ( s ), 12 . 52 ( s ). thenylb . 4319 6 - chloro - 3 - picolyl 242 , 276 5 . 53 ( s ), 6 . 38 ( s ), 7 . 59 ( d ), 7 . 87 ( s ), 8 . 05 ( dd ), 8 . 64 ( d ), 12 . 48 ( s ) b . 4320 5 - bromo - 3 - picolyl 242 , 281 5 . 53 ( s ), 6 . 41 ( s ), 7 . 86 ( s ), 7 . 86 ( s ), 8 . 26 ( dd ), 8 . 73 ( d ), 8 . 78 ( d ), 12 . 50 ( s ). b . 4354 4 - isothiazolyl 244 , 284 5 . 58 ( s ), 6 . 41 ( s ), 7 . 84 ( s ), 8 . 81 ( s ), 9 . 22 ( s ), 12 . 47 ( s ) b . 4356 4 - methylthio - thenyl 236 , 283 2 . 48 ( s ), 5 . 62 ( s ), 6 . 40 ( s ), 7 . 26 ( m ), 7 . 85 ( s ), 12 . 48 ( s ). b . 4357 5 - iodo - 3 - 240 , 283 5 . 43 ( s ), 6 . 38 ( s ), 7 . 48 ( d ), 7 . 77 ( s ), 7 . 84 ( s ), 12 . 47 ( s ). thienylmethylb . 4361 4 - methylsulphonyl - 240 , 285 3 . 26 ( s ), 5 . 70 ( s ), 6 . 40 ( s ), 7 . 72 ( s ), 7 . 85 ( s ), 8 . 38 ( d ), thenyl 12 . 49 ( s ). b . 4366 naphtho [ 2 , 1 - b ]- 244 , 286 sh 5 . 90 ( s ), 6 . 47 ( s ), 7 . 60 ( t ), 7 . 69 ( t ), 7 . 86 ( t ), 8 . 04 ( dd ), thiophen - 2 - ylmethyl 295 , 306 sh 8 . 44 ( s ), 8 . 51 ( d ), 12 . 51 ( s ). b . 4373 4 - azidothenyl 227 , 280 5 . 64 ( s ), 6 . 36 ( s ), 7 . 20 ( s ), 7 . 28 ( s ), 7 . 84 ( s ), 12 . 47 ( s ). b . 4377 4 - methylsulphinyl - 241 , 285 2 . 82 ( s ), 5 . 68 ( s ), 6 . 33 ( s ), 7 . 60 ( s ), 7 . 82 ( s ), 8 . 01 ( s ), thenyl 12 . 45 ( s ). b . 4378 5 - phenylthenyl 244 sh , 289 5 . 57 ( s ), 6 . 32 ( s ), 7 . 31 ( m ), 7 . 41 ( m ), 7 . 41 ( m ), 7 . 63 ( d ), 7 . 82 ( s ), 12 . 43 ( s ). __________________________________________________________________________ table 7__________________________________________________________________________ i . sub . 50 ( μm ) raji i . sub . 50 stability t 1 / 2 ( h ) inactivator hat by specm ) __________________________________________________________________________b . 4280o . sup . 6 -( 4 - bromothenyl ) guanine 0 . 0034 326b . 4281o . sup . 6 -( 5 - chlorothenyl ) guanine 0 . 004 281 . 7 & gt ; 10b . 4282o . sup . 6 -( oxido - 3 - picolyl ) guanine & gt ; 20b . 4283o . sup . 6 -( 5 - cyanothenyl ) guanine & gt ; 20 2b . 4294o . sup . 6 -( 5 - methylsulphinylthenyl ) guanine & gt ; 10b . 4298o . sup . 6 -( 4 - chlorothenyl ) guanine & gt ; 16 282b . 4300o . sup . 6 -( 4 - methoxythenyl ) guanine 0 . 83b . 4309o . sup . 6 -( 5 - methylsulphonylthenyl ) guanine 0 . 072 325 & gt ; 16b . 4313o . sup . 6 -( 5 - bromo - 3 - thienylmethyl ) guanine 326 0 . 0065 0 . 035b . 4317o . sup . 6 -( 4 - cyanothenyl ) guanine & gt ; 19 72b . 4318o . sup . 6 -( 4 , 5 - dichlorothenyl ) guanine 0 . 015 348 2 . 5b . 4319o . sup . 6 -( 6 - chloro - 3 - picolyl ) guanine 0 . 2 & gt ; 13b . 4320o . sup . 6 -( 5 - bromo - 3 - picolyl ) guanine & gt ; 13b . 4321o . sup . 6 -( 2 - chloro - 4 - picolyl ) guanine & gt ; 16b . 4336o . sup . 6 -( 5 - bromofurfuryl ) guanine 0 . 32 0b . 4354o . sup . 6 -( 4 - isothiazolylmethyl ) guanine 0 . 07 248b . 4356o . sup . 6 -( 4 - methylthiothenyl ) guanine 0 . 0095 293b . 4357o . sup . 6 -( 5 - iodo - 3 - thienylmethyl ) guanine 0 . 009 & gt ; 16b . 4361o . sup . 6 -( 4 - methylsulphonylthenyl ) guanine & gt ; 16b . 4366o . sup . 6 -( naphtho [ 2 , 1 - b ] thiophen - 2 - ylmethyl ) guanine 0 . 05 347b . 43689 -( b - d - arabinofuranosyl )- o . sup . 6 -( 4 - bromothenyl ) guanine 0 . 115b . 4369o . sup . 6 -( 4 - bromothenyl )- 9 -( ethoxymethyl ) guanine 0 . 28 384b . 4370o . sup . 6 -( 4 - bromothenyl )- 9 ( octyloxymethyl ) guanine 1 . 2 468b . 4373o . sup . 6 -( 4 - azidothenyl ) guanine 0 . 0063 288b . 4377o . sup . 6 -( 4 - methylsulphinylthenyl ) guanine 0 . 15 309b . 4378o . sup . 6 ( 5 - phenylthenyl ) guanine 0 . 75 323b . 4379o . sup . 6 -( 4 - bromothenyl )- 2 - deoxyguanosine 0 . 095 442__________________________________________________________________________ table 7b__________________________________________________________________________ stability t 1 / 2 ( h ) in vitro i . sub . 50 ( μm ) ( μm ) inactivator m . wt hat mat rat chat agt raji i . sub . 50 by spec by assay__________________________________________________________________________b . 4363o . sup . 6 -( 4 - bromothenyl ) guanosine 0 . 24 & gt ; 4816__________________________________________________________________________ blank space = not done table 8______________________________________atase activity in various tissues of nu / nu mice aftertreatment with 10 mg / kg ( ip ) b . 4280mean activity ( fm / mg ) tissue 24 h 48 h control * ______________________________________tumour 36 ± 7 . 79 140 ± 43 . 87 125liver 100 . 7 ± 8 . 73 . 14 110 ** lung 24 ± 2 . 83 . 2 . 05 43kidney 28 . 7 ± 4 . 11 . 4 . 03 33spleen 68 . 3 ± 9 . 53 . 35 81brain 16 . 3 ± 1 . 25 -. 2 . 05 14testis 44 ± 1 . 418 45bone marrow ( pooled ) 42 30______________________________________ * control values taken from a separate experiment ** mean of 2 control liver values table 8 . effect of b . 4280 on atase activity in several tissues of nude mice . animals were given a single dose of b . 4280 ( 10 mg / kg i . p .) and sacrificed 24 or 48 hours later . table 9______________________________________toxicity of inactivators in combination withbcnu in dba . sub . 2 mice % survival after 14 daysinactivator 12 mg / kg ( 60 mg / kg ) 20 mg / kg bcnu 16 mg / kg bcnu bcnu______________________________________o . sup . 6 - benzylguanine 33 ( 2 / 6 ) 0 ( 0 / 6 )* 50 ( 3 / 6 )** b . 4205 0 ( 0 / 6 ) 100 ( 6 / 6 )** b . 4280 93 ( 14 / 15 ) 100 ( 15 / 15 ) ______________________________________ * 15 mg / kg bcnu ** 10 mg / kg bcnu all agents were given as a single i . p . dose table 9 effect of atase inactivators on the acute toxicity of bischloroethylnitrosourea ( bcnu ) in dba . sub . 2 mice . 1 . kiburis j . and lister , j . h . j . chem . soc . 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