Patent Application: US-52910603-A

Abstract:
the present invention is a method for preparing 2 - halo - 6 - aminopurines , and more specifically for preparing the clinical agent cladribine , a drug of choice against hairy - cell leukemia and other neoplasms , from 2 - amino - 6 - oxopurines , which are readily obtained from the naturally occurring compound 2 ′- deoxyguanosine . according to the methods of the present invention , the 6 - oxo group of a protected 2 ′- deoxyguanosine is converted to a 6 - leaving group , or alternatively to a 6 - chloro leaving group , the 2 - amino group is replaced with a 2 - chloro group , the 6 - leaving group , or alternatively the 6 - chloro leaving group , is replaced with a 6 - amino group or , alternatively , a 2 , 6 - dichloro substituted compound is selectively replaced with a 6 - amino group , and the protecting groups are removed .

Description:
in accordance with the present invention , synthesis of regio - and stereochemically pure 2 - chloro - 2 ′- deoxyadenosine ( cladrabine , or cldado ), which avoids separation of mixtures with fusion and sodium salt glycosylation procedures , is accomplished by transformation of the naturally occurring nucleoside 2 ′- deoxyguanosine ( dguo ) as the starting compound . 24 methods for producing cldado from 2 ′- deoxyguanosine ( dguo ) according to the present invention begin with protected forms of dguo including , but not restricted to , acyl , silyl , amide , and other derivatives useful in the field of nucleoside / nucleotide / nucleic acid chemistry and protection strategies . methods for obtaining protected forms of dguo are well - known in the art . 25 , 26 , 27 , 28 , 29 , 30 the preferred starting products for efficient synthesis of cldado are defined by the following chemical structure : where r is any suitable protecting group , and preferably r is ac or bz . in order to obtain the desired cldado compounds , protected dguo derivatives are treated with combinations of chemicals that effect functionalization at the c6 position to give groups that can be replaced , followed by transformation of the 2 - amino function to a 2 - chloro group , followed by replacement of the 6 - functional group to give a 6 - amino group ( or a 6 - substituent that can be converted into a 6 - amino group , followed by conversion to the 6 - amino group ), and concomitant or subsequent deprotection of the resulting 6 - amino - 2 - chloropurine derivative to give cldado . for example , from dguo , cldado ( 4 ) ( fig1 ) is synthesized by converting the 6 - oxo function into appropriate 6 -( substituted oxy ) leaving groups that can be replaced without protection of the 2 - amino moiety , transformation of the 2 - amino function to a 2 - chloro functional group by diazotization / chloro - dediazoniation of the 2 - amino function , and selective c6 ammonolysis of the 2 - chloro - 6 -( substituted ) purine derivatives , with accompanying sugar deprotection . this route advantageously provides retention of both β - anomeric stereochemistry and n9 isomeric purity . functionalization of the 6 - oxo group is accomplished by converting , without protection of the 2 - amino moiety , the 6 - oxo group to a 6 -( substituted oxy ) leaving group having greater reactivity than the 2 - chloro group in a s n ar displacement reaction . two preferred methods for functionalization at the c6 - oxy group include alkyl - or arylsulfonylation of the c6 - oxy group and chlorodeoxygenation at c6 . alkyl - or arylsulfonylation of the c6 - oxy group can be accomplished by treating protected dguo derivatives with ( alkyl or any substituted alkyl or cycloalkyl ) sulfonyl , phosphoryl or phosphonyl reagents or ( aryl or any substituted aryl ) sulfonyl , phosphoryl or phosphonyl reagents , which include , for example , sulfonyl compounds having the formula r ′ so 2 — x , where x is a halogen ( such as chloride ), imidazolide , triazolide or tetrazolide and r ′ is alkyl , substituted alkyl ( including but not limited to fluoroalkyl ), cycloalkyl , aryl , or substituted aryl , to convert the 6 - oxo group to a 6 - o -( alkyl , substituted alkyl , cycloalkyl , aryl or substituted aryl ) sulfonyl group . in preferred embodiments , 3 ′, 5 ′- di - o -( acetyl or benzoyl )- 2 ′- deoxyguanosine ( 1 ) is treated with ( 2 , 4 , 6 - triisopropyl or 4 - methyl ) benzenesulfonyl chloride , to give high yields of the 6 - o - arylsulfonyl derivatives 2 or 2 ′ b . in other preferred embodiments , 1 is treated with tipbs - cl , which gives the 6 - o - tipbs derivative 2a or 2b , or tscl , which gives the 6 - o - ts derivative 2 ′ b . several acyl - protected 6 - o - sulfonyl derivatives of dguo that can be utilized are known in the art . 28 , 29 , 30 treatment of 3 ′, 5 ′- di - o - acetyl - 2 ′- deoxyguanosine 31 ( 1a ) or its 3 ′, 5 ′- di - o - benzoyl analogue 1b 31 with tipbs - cl / et 3 n / dmap / chcl 3 by the general method of hata et al . 29 gave the 6 - o - tipbs derivatives 2a 32 ( 91 %) or 2b ( 86 %), respectively . similar treatment of 1b with tscl / et 3 n / dmap / chcl 3 gave the 6 - o - ts derivative 2 ′ b ( 89 %). efficient displacement of sulfonate from c6 in following steps requires a sterically hindered arylsulfonyl derivative . a more economical 6 - o - tosyl derivative gave lower yields at the final stage owing to attack of ammonia at both sulfonyl sulfur and c6 ( 3 ′ b gave 4 in 43 % yield ). by contrast , ammonolysis of the 6 - o - tipbs derivatives proceeded efficiently at c6 with minimal attack at the hindered sulfur atom . both types of such arylsulfonate derivatives , and especially the 6 - o - ts , underwent increased nucleophilic attack at sulfur with lower temperatures (− 20 to 0 ° c .) to give 6 - oxopurine derivatives , resulting in a lower yield . however , treatment of the 6 - o - tipbs compounds with nh 3 / meoh / ch 2 cl 2 in a pressure tube at 80 ° c . strongly favored nucleophilic attack at c6 to give good yields of cldado ( 4 ). alternatively , the 6 - oxo group can be replaced by a 6 - chloro leaving group by chlorodeoxygenation at c6 . chlorine is the most frequently used leaving group at c6 of purine nucleosides . methods for producing cldado from dguo begin with protected forms of dguo described above and involve treatment of such derivatives with combinations of chemicals that convert the 6 - oxo function into a 6 - chloro group that can be replaced on the resulting 2 - amino - 6 - chloropurine derivative . deoxychlorination at c6 of 1 results in excellent yields of the 2 - amino - 6 - chloropurine derivatives 5 . in preferred embodiments , protected dguo derivatives are treated with phosphoryl chloride , a source of soluble chloride , an organic base , and acetonitrile or other compatible solvent , to convert the 6 - oxo function into a 6 - chloro group . original studies on deoxychlorination of guanosine 33 ( guo ) and dguo 34 derivatives with pocl 3 gave moderate ( guo ) to poor ( dguo ) yields of 2 - amino - 6 - chloropurine products , and improved procedures have been reported . 26a , 35 , 36 the acid - labile 2 ′- deoxy derivatives 1 were deoxychlorinated with pocl 3 / n , n - dimethylaniline / btea - cl / mecn / δ , 26a , 36 and 6 - chloro derivatives 5a ( 90 %) and 5b ( 85 %) were obtained in high yields under carefully controlled conditions . following the step of converting the 6 - oxo group to a 6 -( substituted oxy ) leaving group , the 2 - amino group is replaced with a 2 - chloro group by a diazotization / halo - dediazoniation reaction . improved methods are disclosed for replacement of an amino group on purine nucleoside derivatives with chlorine , bromine , or iodine under non - aqueous conditions by diazotization / halo - dediazoniation methods . 27 these mild diazotization / halo - dediazoniation methods are applicable at c6 of dado derivatives as well as at c2 of 2 - amino - 6 - chloropurine nucleosides . in accordance with the present invention , cldado is produced from dguo by treatment of protected forms of dguo that contain a respective 6 - o -( alkyl , cycloalkyl , or aryl ) sulfonyl group with reagents that effect diazotization / chloro - dediazoniation at c2 to give a 2 - chloro group . cldado is also produced by treating protected 2 - amino - 6 - chloropurine derivatives with reagents that effect diazotization / chloro - dediazoniation at c2 to give respective 2 , 6 - dichloropurine derivatives . reagents that effect diazotization / chloro - dediazoniation at c2 to give a 2 - chloro group include a halide source ( such as metal chlorides , metal chloride salts , acyl chlorides , sulfonyl chlorides , and silyl chlorides , alkyl and aryl substituted ammonium chloride salts , including but not limited to tetraalkyl and aryl ammonium chloride salts ) and a nitrite source ( such as metal nitrites , metal nitrite salts , organic nitrites , such as tert - butyl nitrite , pentyl nitrite , and isoamyl nitrite , and complex quaternary ammonium nitrites , such as benzyltriethylammonium nitrite ). in preferred embodiments of the present invention , cldado is synthesized by employing acetyl chloride and benzyltriethylammonium nitrite ( btea - no 2 )- mediated diazotization / chloro - dediazoniation of 6 - o -( 2 , 4 , 6 - triisopropylbenzenesulfonyl ) ( tipbs ) or 6 - chloro derivatives that are readily obtained from dguo . non - aqueous diazotization / chloro - dediazoniation ( acetyl chloride / benzyltriethylammonium nitrite ) of 2 , 2 ′ b , or 5 gave the 2 - chloropurine derivatives 3 , 3 ′ b , or 6 , respectively . this new procedure for non - aqueous diazotization / chloro - dediazoniation 27 ( accl / btea - no 2 / ch 2 cl 2 /− 5 to 0 ° c .) worked well for replacement of the 2 - amino group of 2 , 2 ′ b , and 5 with chlorine to give 3a ( 89 %), 3b ( 90 %), 3 ′ b ( 87 %), 6a ( 95 %), and 6b ( 91 %). efficient diazotization / chloro - dediazoniation of 9 -( 2 , 3 , 5 - tri - o - acetyl - β - d - ribofuranosyl )- 2 - amino - 6 - chloropurine 26 , 33 ( 7 ) ( fig2 ) was effected with tms - cl ( 9 equivalents ) and benzyltriethylammonium nitrite ( beta - no 2 ) ( 3 equivalents ) in ch 2 cl 2 at ambient temperature . the process was rapid (& lt ; 30 min ), and the desired 9 -( 2 , 3 , 5 - tri - o - acetyl - β - d - ribofuranosyl )- 2 , 6 - dichloropurine 27 , 37 ( 8 ) ( 83 %, without chromatography ) was obtained as a white crystalline solid . comparable yields were obtained at 0 ° c . tms - cl ( 3 . 5 equivalents ) and btea - no 2 ( 1 . 5 equivalents ) with powdered nano 2 ( 5 equivalents ) gave 8 ( 86 %) within 1 hr . by contrast , an alternative method for non - aqueous diazotization / chloro - dediazoniation of 7 employed cl 2 / tbn / cucl in a strongly exothermic reaction , and removal of colloidal material by filtration was required prior to crystallization of 7 . 35 compound 7 underwent efficient diazotization / bromo - dediazoniation with tms - br and tert - butyl nitrite ( tbn ). competing redox interactions between nitrite anion and tms - br precluded the use of nano 2 . the 2 - bromo - 6 - chloropurine nucleoside 3 27 , 37 ( 85 %, without chromatography ) was obtained as a crystalline solid with tms - br ( 9 equivalents )/ tbn ( 20 equivalents )/ ch 2 br 2 / ambient temperature within 1 h . nocl / ch 2 cl 2 or nobr / ch 2 br 2 is presumed to be generated from ( me 3 six or accl ) and ( tbn or btea - no 2 ). these procedures provide efficient diazotization / halo - dediazoniation of protected ( 2 or 6 )- aminopurine nucleosides as well as the acid - sensitive 2 ′- deoxynucleosides . the reactions are cost - effective and proceed at or below ambient temperature with convenient reagents and standard laboratory equipment and conditions . following replacement of the 2 - amino group with a 2 - chloro group , the protected forms of dguo that contain a respective 6 - o -( alkyl , cycloalkyl , or aryl ) sulfonyl or phosphoryl group and a 2 - chloro group is reacted with chemicals that cause replacement of the 6 - o -( alkyl , cycloalkyl , or aryl ) sulfonyl or phorphoryl group to give a 6 - amino group ( or a substituent that can be converted into a 6 - amino group , resulting in overall conversion of the substituent into a 6 - amino group ) of a resulting 6 - amino - 2 - chloropurine derivative . in preferred embodiments , the 6 - leaving group is replaced with a 6 - amino group by reacting the product of step ( b ) with a nitrogen source capable of being converted to an amino group in a solvent compatible with the nitrogen source to replace the 6 - leaving group with a 6 - amino group by selective ammonolysis of the 6 - leaving group . the nitrogen source is selected from the group consisting of ammonia , azides , hydrazines , benzylic amines , or compatible ammonium salts . the solvent may be any solvent that is compatible with the nitrogen source , such as methanol , ethanol , higher alcohols , or aprotic solvents , such as 1 , 2 - dimethoxyethane , tetrahydrofuran , or dmf . in other preferred embodiments , the respective 6 - o -( alkyl , cycloalkyl , or aryl ) sulfonyl leaving group is reacted with ammonia in a compatible aprotic solvent to give a 6 - amino - 2 - chloropurine derivative , followed by deprotection ( if necessary ) to give cldado . in a more preferred embodiment , a 9 -( 3 , 5 - di - o - acyl - β - d - erythro - pentofuranosyl - 6 - o -( alkyl , cycloalkyl , or aryl ) sulfonyl - 2 - chloropurine is treated with ammonia in methanol or other compatible solvent to give cldado . further , protected derivatives of 2 , 6 - dichloropurine are treated with reagents that cause selective replacement of the 6 - chloro group to give a 6 - amino group ( or a substituent that can be converted into a 6 - amino group , followed by conversion of the substituent into a 6 - amino group ) of a resulting 6 - amino - 2 - chloropurine derivative . in a more preferred embodiment , 9 -( 3 , 5 - di - o - acyl - β - d - erythro - pentofuranosyl )- 2 , 6 - dichloropurine is treated with ammonia in methanol or other compatible solvent to give cldado . selective ammonolysis at c6 ( arylsulfonate with 3 or chloride with 6 ) and accompanying deprotection of the sugar moiety gave cldado ( 4 ) ( 64 - 75 % overall from 1 ). specifically , displacements of the hindered arylsulfonate ( from 3 ) or chloride ( from 6 ) at c6 with accompanying cleavage of the sugar esters were effected at 80 ° c . with nh 3 / meoh / ch 2 cl 2 . cladribine ( 4 ) was obtained in high yields from 3a ( 81 %), 3b ( 83 %), 6a ( 87 %), and 6b ( 94 %), but only in moderate yield from the 6 - o - tosyl derivative 3 ′ b ( 43 %). the r protecting groups are removed by deacylation using a basic reagent well known in the art in a solvent compatible with the basic reagent , to remove the r protecting groups and produce 2 - chloro - 2 ′- deoxyadenosine . the basic reagent is selected from the group consisting of ammonia , metal alkoxides , metal hydroxides , and metal carbonates . the solvent may be any solvent that is compatible with the basic reagent , such as methanol , ethanol , 1 , 2 - dimethoxyethane / h 2 o , or tetrahydrofuran / h 2 o . it is to be noted that removal of the r protecting groups may occur concomitantly with or subsequent to the replacement of the 6 -( substituted oxy ) leaving group with a 6 - amino group by ammonolysis . both steps of replacement of the 6 -( substituted oxy ) leaving group with a 6 - amino group and removal of the r protecting groups is accomplished by use of a nitrogen source in a solvent compatible with the nitrogen source . where the nitrogen source is ammonia in a protic solvent , such as methanol or ethanol , both steps proceed simultaneously . however , where the nitrogen source is anhydrous ammonia in a dry solvent , such as 1 , 2 - dimethoxyethane , or tetrahydrofuran , only the step of replacement of the 6 -( substituted oxy ) leaving group with a 6 - amino group proceeds . in this case , it is necessary and sometimes desirable to remove the r protecting groups in a separate step . in the most preferred embodiments of the present invention , synthesis of the clinical drug cladribine ( 2 - chloro - 2 ′- deoxyadenosine , cldado , 4 ) was accomplished in three steps from the readily available 3 ′, 5 ′- di - o - acetyl - 2 ′- deoxyguanosine ( 1a ) or its dibenzoyl analogue 1b . replacement of the 2 - amino group proceeded in high yields by diazotization / chloro - dediazoniation with accl / btea - no 2 . selective ammonolysis of 3a and 3b ( 6 - otipbs ) or 6 ( 6 - cl ), with accompanying deacylation , gave 4 ( 64 - 75 % overall ). ammonolysis of 3 ′ b ( 6 - ots ) was problematic and gave 4 in poor overall yield ( 33 %). routes that employed deoxychlorination of 1 were ˜ 10 % more efficient overall than those which involved 6 - o - tipbs intermediates . melting points for 4 were determined with a hot - stage apparatus . uv spectra were recorded with solutions in meoh . 1 h nmr spectra were recorded at 300 mhz with solutions in cdcl 3 unless otherwise indicated . “ apparent ” peak shapes are in quotation marks when first - order splitting should be more complex or when peaks were poorly resolved . mass spectra ( ms ) were determined with fab ( glycerol ) unless otherwise indicated . chemicals and solvents were of reagent quality . ch 2 cl 2 and mecn were dried by reflux over and distillation from cah 2 . chcl 3 was dried over p 2 o 5 and distilled . accl , pocl 3 , and n , n - dimethylaniline were freshly distilled before use . benzyltriethylammonium nitrite ( btea - no 2 ) was prepared from btea - cl by ion exchange [ dowex 1 × 2 ( no 2 − )]. column chromatography ( silica gel , 230 - 400 mesh ) was performed with ch 2 cl 2 / meoh . compounds 1a and 1b were prepared as described . 31 method 1 ( nucleoside / tipbs - cl / dmap / et 3 n / chcl 3 ) is described for 1a → 2a , method 2 ( nucleoside / accl / btea - no 2 / ch 2 cl 2 ) for 2a → 3a , method 3 ( nucleoside / n , n - dimethylaniline / pocl 3 / btea - cl / mecn ) for 1a → 5a , and method 4 ( nucleoside / nh 3 / meoh / δ ) for 3a → 4 . analogous reactions with equivalent molar proportions of other nucleosides gave the indicated products and quantities . method 1 . et 3 n ( 1 . 25 ml , 910 mg , 9 . 0 mmol ) was added to a stirred solution of 1a ( 1 . 67 g , 4 . 8 mmol ), tipbs - cl ( 2 . 73 g , 9 . 0 mmol ), and dmap ( 72 mg , 0 . 6 mmol ) in dried chcl 3 ( 70 ml ) under n 2 . stirring was continued for 24 h , and volatiles were evaporated . the orange residue was chromotographed ( ch 2 cl 2 / meoh ) give 2a 32 ( 2 . 67 g , 91 %) as a slightly yellow foam with uv max 238 , 291 nm , min 264 nm ; 1 h nmr ( 500 mhz ) δ 1 . 26 - 1 . 32 ( m , 18h ), 2 . 08 ( s , 3h ), 2 . 14 ( s , 3h ), 2 . 54 ( ddd , j = 4 . 7 , 9 . 0 , 14 . 0 hz , 1h ), 2 . 91 - 2 . 99 ( m , 2h ), 4 . 22 - 4 . 37 ( m , 3h ), 4 . 43 - 4 . 47 ( m , 2h ), 4 . 97 ( br s , 2h ), 5 . 41 - 5 . 42 (“ d ”, 1h ), 6 . 26 - 6 . 29 ( m , 1h ), 7 . 21 ( s , 2h ), 7 . 84 ( s , 1h ); lrms m / z 618 ( mh + [ c 29 h 40 n 5 o 8 s ]= 618 ); hrms m / z 640 . 2413 ( mna + [ c 29 h 39 n 5 o 8 sna ]= 640 . 2417 ). treatment of 1b ( 950 mg , 2 . 0 mmol ) by method 1 gave 2b ( 1 . 27 g , 86 %) as a white solid foam with uv max 289 nm , min 264 nm ; 1 h nmr δ 1 . 29 - 1 . 32 ( m , 18h ), 2 . 76 ( ddd , j = 2 . 1 , 6 . 0 , 14 . 3 hz , 1h ), 2 . 96 (“ quint ”, j = 6 . 8 hz , 1h ), 3 . 15 - 3 . 25 ( m , 1h ), 4 . 34 (“ quint ”, j = 6 . 8 hz , 2h ), 4 . 65 - 4 . 74 ( m , 2h ), 4 . 85 - 4 . 90 ( m , 1 h ), 5 . 00 ( br s , 2h ), 5 . 84 - 5 . 86 (“ d ”, 1h ), 6 . 38 - 6 . 43 ( m , 1h ), 7 . 30 ( s , 2h ), 7 . 44 - 7 . 55 ( m , 4h ), 7 . 58 - 7 . 69 ( m , 2h ), 7 . 85 ( s , 1h ), 8 . 04 ( d , j = 7 . 1 hz , 2h ), 8 . 11 ( d , j = 7 . 1 hz , 2h ); lrms m / z 742 ( mh + [ c 39 h 44 n 5 o 8 s ]= 742 ), 764 ( mna + [ c 39 h 43 n 5 o 8 sna ]= 764 ); hrms m / z 764 . 2730 ( mna + [ c 39 h 43 n 5 o 8 sna ]= 764 . 2730 ). et 3 n ( 700 μl , 506 mg , 5 . 0 mmol ) was added to a stirred solution of 1b ( 1 . 43 g , 3 . 0 mmol ), tscl ( 858 mg , 4 . 5 mmol ), and dmap ( 36 mg , 0 . 3 mmol ) in dried chcl 3 ( 45 ml ) under n 2 . stirring was continued for 15 h , and volatiles were evaporated . the slightly yellow residue was chromotographed ( ch 2 cl 2 / meoh ) to give 2 ′ b ( 1 . 68 g , 89 %) as a white solid foam with uv max 300 nm ; 1 h nmr ( dmso - d 6 ) δ 2 . 43 ( s , 3h ), 2 . 73 ( ddd , j = 2 . 1 , 8 . 4 , 14 . 4 hz , 1h ), 3 . 17 - 3 . 27 (“ quint ”, j = 7 . 2 hz , 1h ), 4 . 52 - 4 . 65 ( m , 3h ), 5 . 76 - 5 . 78 (“ d ”, 1h ), 6 . 37 - 6 . 41 ( m , 1h ), 6 . 95 ( br s , 2h ), 7 . 47 - 7 . 73 ( m , 8h ), 7 . 95 ( d , j = 7 . 8 hz , 2h ), 8 . 03 - 8 . 11 ( m , 4h ), 8 . 30 ( s , 1h ); lrms m / z 630 ( mh + [ c 31 h 28 n 5 o 8 s ]= 630 ), m / z 652 ( mna + [ c 31 h 27 n 5 o 8 sna ]= 652 ); lrms m / z 652 . 1467 ( mna + [ c 31 h 27 n 5 o 8 sna ]= 652 . 1478 ). method 2 . a solution of accl ( 200 μl , 220 mg , 2 . 8 mmol ) in dried ch 2 cl 2 ( 12 ml ) under n 2 was chilled in a nacl / ice / h 2 o bath (− 5 to 0 ° c .) for 15 min . btea - no 2 ( 520 mg , 2 . 2 mmol ) was dissolved in dried ch 2 cl 2 ( 8 ml ) and this solution was immediately added dropwise to the cold , stirred solution of accl / ch 2 cl 2 . a solution of 2a ( 288 mg , 0 . 5 mmol ) in dried ch 2 cl 2 ( 5 ml ) was then added dropwise to the cold solution , and stirring was continued for 5 min ( tlc , ch 2 cl 2 / meoh , 95 : 5 , showed complete conversion of 2a into a single product ). the reaction mixture was added dropwise at a rapid rate to a cold ( ice / h 2 o bath ), vigorously stirred mixture of saturated nahco 3 / h 2 o ( 100 ml )// ch 2 cl 2 ( 100 ml ). the layers were separated , and the organic phase was washed with cold ( 0 ° c .) h 2 o ( 2 × 100 ml ) and dried ( mgso 4 ) for 1 h . volatiles were evaporated , and the residue was chromatographed ( ch 2 cl 2 / meoh ) to give 3a ( 267 mg , 89 %) as a white solid foam with uv max 240 , 266 nm , min 255 nm ; 1 h nmr ( 500 mhz ) δ 1 . 24 - 1 . 31 ( m , 18h ), 2 . 08 ( s , 3h ), 2 . 13 ( s , 3h ), 2 . 67 ( ddd , j = 2 . 5 , 7 . 0 , 15 . 5 hz , 1h ), 2 . 76 - 2 . 81 ( m , 1h ), 2 . 90 - 2 . 96 (“ quint ”, j = 7 . 0 hz , 1h ), 4 . 28 - 4 . 33 (“ quint ”, j = 7 . 0 hz , 2h ), 4 . 35 - 4 . 39 ( m , 3h ), 5 . 38 - 5 . 39 (“ d ”, 1h ), 6 . 42 - 6 . 45 ( m , 1h ), 7 . 22 ( s , 2h ), 8 . 23 ( s , 1h ); lrms m / z 637 ( mh + [ c 29 h 38 cln 4 o 8 s ]= 637 ); hrms m / z 659 . 1902 ( mna + [ c 29 h 37 cln 4 o 8 sna ]= 659 . 1918 ). treatment 2b ( 1 . 20 g , 1 . 6 mmol ) by method 2 gave 3b ( 1 . 10 g , 90 %) as a yellow solid foam with uv max 230 , 266 nm , min 255 nm ; 1 h nmr δ 1 . 22 - 1 . 34 ( m , 18h ), 2 . 92 - 3 . 00 ( m , 3h ), 4 . 30 - 4 . 34 ( m , 2h ), 4 . 68 - 4 . 77 ( m , 3h ), 5 . 80 - 5 . 82 (“ d ”, 1h ), 6 . 54 - 6 . 57 ( m , 1h ), 7 . 42 - 7 . 64 ( m , 8h ), 8 . 00 ( d , j = 8 . 4 hz , 2h ), 8 . 10 ( d , j = 8 . 4 hz , 2h ), 8 . 26 ( s , 1h ); hrms m / z 783 . 2224 ( mna + [ c 39 h 41 cln 4 o 8 sna ]= 783 . 2231 ). treatment of 2 ′ b ( 1 . 45 g , 2 . 3 mmol ) by method 2 gave 3 ′ b ( 1 . 30 g , 87 %) as a slightly yellow foam with uv max 267 nm , min 255 nm ; 1 h nmr ( dmso - d 6 ) δ 2 . 45 ( s , 3h ), 2 . 86 ( ddd , j = 3 . 4 , 6 . 2 , 14 . 0 hz , 1h ), 3 . 21 - 3 . 30 (“ quint ”, j = 7 . 0 hz , 1h ), 4 . 55 - 4 . 67 ( m , 3h ), 5 . 82 - 5 . 84 ( m , 1h ), 6 . 56 - 6 . 61 ( m , 1h ), 7 . 42 - 7 . 72 ( m , 8h ), 7 . 88 ( d , j = 7 . 8 hz , 2h ), 8 . 04 - 8 . 07 ( m , 4h ), 8 . 88 ( s , 1h ); hrms m / z 671 . 0983 ( mna + [ c 31 h 25 cln 4 o 8 sna ]= 671 . 0979 . a mixture of 1a ( 540 mg , 1 . 54 mmol ), btea - cl ( 710 mg , 3 . 1 mmol ), n , n - dimethylaniline ( 215 μl , 206 mg , 1 . 7 mmol ), and pocl 3 ( 720 μl , 1 . 2 g , 7 . 7 mmol ) in mecn ( 6 ml ) was stirred in a preheated oil bath ( 85 ° c .) for 10 min . volatiles were flash evaporated immediately ( in vacuo ), and the yellow foam was dissolved ( chcl 3 , 15 ml ) and stirred vigorously with crushed ice for 15 min . the layers were separated , and the aqueous phase was extracted ( chcl 3 , 3 × 5 ml ). crushed ice was frequently added to the combined organic phase , which was washed [( ice - h 2 o ( 3 × 5 ml ), 5 % nahco 3 / h 2 o ( to ph ˜ 7 )] and dried ( mgso 4 ). volatiles were evaporated , and the residue was chromatographed ( ch 2 cl 2 / meoh ) to give 5a 36 , 38 ( 517 mg , 90 %) as a white solid foam with uv max 248 , 310 nm , min 268 nm ; 1 h nmr ( 500 mhz , dmso - d 6 ) δ 2 . 02 ( s , 3h ), 2 . 08 ( s , 3h ), 2 . 49 - 2 . 52 ( m , 1h ), 3 . 04 - 3 . 06 ( m , 1h ), 4 . 20 - 4 . 29 ( m , 3h ), 5 . 32 - 5 . 34 (“ d ”, 1h ), 6 . 23 - 6 . 26 ( m , 1h ); 7 . 03 ( br s , 2h ), 8 . 35 ( s , 1h ); 1 h nmr δ 2 . 03 ( s , 3h ), 2 . 08 ( s , 3h ), 2 . 51 ( ddd , j = 2 . 7 , 5 . 9 , 14 . 2 hz , 1h ), 2 . 85 - 2 . 94 (“ quint ”, j = 7 . 1 hz , 1h ), 4 . 28 - 4 . 42 ( m , 3h ), 5 . 35 - 5 . 37 ( m , 1h ), 6 . 21 - 6 . 26 ( m , 1h ), 7 . 89 ( s , 1h ); hrms ( ei ) m / z 369 . 0844 ( m + [ c 14 h 16 cln 5 o 5 ]= 369 . 0840 ). treatment of 1b ( 2 . 38 g , 5 mmol ) by method 3 gave 5b ( 2 . 10 g , 85 %) as a slightly yellow solid foam with uv max 310 nm ; 1 h nmr ( dmso - d 6 ) δ 2 . 69 - 2 . 78 (“ ddd ”, 1h ), 3 . 20 - 3 . 24 (“ quint ”, j = 7 . 2 hz , 1h ), 4 . 56 - 4 . 67 ( m , 3h ), 5 . 77 - 5 . 79 (“ d ”, 1h ), 6 . 39 - 6 . 44 ( m , 1h ), 7 . 02 ( br s , 2h ), 7 . 48 - 7 . 71 ( m , 6h ), 7 . 96 ( d , j = 8 . 4 hz , 2h ), 8 . 06 ( d , j = 8 . 7 hz , 2h ), 8 . 37 ( s , 1h ); lrms m / z 494 ( mh + [ c 24 h 21 cln 5 o 5 ]= 494 ); hrms m / z 516 . 1042 ( mna + [ c 24 h 20 cln 5 o 5 na ]= 516 . 1051 ). treatment of 5a ( 265 mg , 0 . 7 mmol ) by method 2 gave 6a 39 ( 266 mg , 95 %) as a white solid foam with uv max 274 nm , min 232 nm ; 1 h nmr ( 500 mhz ) δ 2 . 12 ( s , 3h ), 2 . 15 ( s , 3h ), 2 . 73 ( dddd , j = 2 . 4 , 5 . 9 , 14 . 2 hz , 1h ), 2 . 83 - 2 . 86 ( m , 1h ), 4 . 38 - 4 . 39 ( m , 3h ), 5 . 41 - 5 . 42 (“ d ”, 1h ), 6 . 45 - 6 . 50 ( m , 1h ), 8 . 33 ( s , 1h ); hrms m / z 411 . 0230 ( mna + [ c 14 h 14 cl 2 n 4 o 5 na ]= 411 . 0239 ). treatment of 5b ( 407 mg , 0 . 8 mmol ) by method 2 gave 6b ( 386 mg , 91 %) as a slightly yellow solid foam with uv max 274 nm , min 257 nm ; 1 h nmr ( 500 mhz , dmso - d 6 ) δ 2 . 87 ( ddd , j = 3 . 5 , 8 . 0 , 17 . 5 hz , i h ), 3 . 25 - 3 . 31 ( m , 1h ), 4 . 57 - 4 . 71 ( m , 3h ), 5 . 83 - 5 . 85 (“ d ”, 1h ), 6 . 59 - 6 . 62 ( m , 1h ), 7 . 46 - 7 . 72 ( m , 6h ), 7 . 91 ( d , j = 7 . 5 hz , 2h ), 8 . 09 ( d , j = 7 . 5 hz , 2h ), 8 . 96 ( s , 1h ); hrms m / z 535 . 0562 ( mna + [ c 24 h 18 cl 2 n 4 o 5 na ]= 535 . 0552 ). nh 3 / meoh ( 12 ml , saturated at 0 ° c .) was added to a solution of 3a ( 159 mg , 0 . 25 mmol ) in ch 2 cl 2 ( 8 ml ) in a pressure tube . the tube was sealed and immediately immersed in an oil bath preheated to 80 ° c . heating ( 80 ° c .) was continued for 7 h , and volatiles were evaporated . the residue was dissolved ( h 2 o , 2 ml ), and the solution was applied to a column of dowex 1 × 2 ( oh − , 20 ml ), the flask was rinsed ( h 2 o , 5 ml ) and applied to the column . the column was washed ( h 2 o ) until the ph of the eluate was neutral , and then meoh / h 2 o ( 1 : 1 ) was applied . uv - absorbing fractions were pooled , and volatiles were evaporated . etoh ( 3 × 10 ml ) was added and evaporated , and the residue was dried ( in vacuo ) to give 4 ( 58 mg , 81 %). the white powder was recrystallized from meoh to give 4 ( 2 crops , 84 % recovery ) with mp & gt ; 300 ° c . ( crystals slowly became brown at ˜ 220 ° c . ), or from h 2 o ( 2 crops , 72 % recovery ) with mp & gt ; 300 ° c . ( lit . mp softening at 210 - 215 ° c ., 16 then dec . ; & gt ; 300 ° c . ; 18 , 21 232 ° c . ); uv , 1 h nmr , and ms data were in agreement with published values . 21 , 22 anal . calcd for c 10 h 12 cln 5 o 3 : c , 42 . 04 ; h , 4 . 23 ; n , 24 . 51 . found : c , 41 . 85 ; h , 4 . 40 ; n , 24 . 46 . treatment of 3b ( 83 %), 3 ′ b ( 43 %), 6a ( 87 %), or 6b ( 94 %) by method 4 gave 4 as white , tlc homogeneous powders with identical spectral data . method a . tms - cl ( 1 . 14 ml , 978 mg , 9 . 0 mmol ) was added dropwise to a stirred solution of 7 ( 428 mg , 1 . 0 mmol ) in ch 2 cl 2 ( 30 ml ) under n 2 . btea - no 2 ( 714 mg , 3 . 0 mmol ) in ch 2 cl 2 ( 10 ml ) was added dropwise ( 1 drop / 2 sec ) and the solution was stirred at ambient temperature for 30 min ( tlc ). the solution was diluted ( ch 2 cl 2 , 200 ml ) and washed ( 5 % nahco 3 / h 2 o , 5 × 100 ml ). the combined aqueous phase was extracted ( ch 2 cl 2 , 2 × 100 ml ), and the combined organic phase was dried ( mgso 4 ) and filtered . volatiles were evaporated , et 2 o was added to and evaporated ( 3 × 25 ml ) from the yellow oil , and the residue was recrystallized ( etoh ) to give 8 ( 373 mg , 83 %) as pale - yellow crystals with mp , uv , 1 h nmr , and ms data as reported . 27 method b . tms - cl ( 444 , ul , 280 mg , 3 . 50 mmol ) was added to dropwise to a stirred mixture of 7 ( 428 mg , 1 . 0 mmol ) and powdered nano 2 ( 345 mg , 5 . 0 mmol ) in ch 2 cl 2 ( 25 ml ) under n 2 . btea - no 2 ( 357 mg , 1 . 5 mmol ) in ch 2 cl 2 ( 25 ml ) was added dropwise ( 1 drop / sec ) and the solution was stirred at ambient temperature for 1 h ( tlc ). the mixture was cooled for 15 min and added dropwise to a cold , vigorously stirred mixture of saturated nahco 3 / h 2 o ( 200 ml )/ ch 2 cl 2 ( 200 ml ). the aqueous layer was extracted ( ch 2 cl 2 100 ml ), and the combined organic phase was washed ( h 2 o , 100 ml ) and dried mgso 4 ). volatiles were evaporated , and the yellow oil was recrystallized ( buoh ) to give 8 ( 384 mg , 85 %) as pale - yellow crystals . method c . a solution of accl / ch 2 cl 2 ( 1m , 2 . 5ml , 2 . 5 mmol ) was added to dried ch 2 cl 2 ( 10 ml ) under n 2 , and the solution was cooled for 15 min . btea - no 2 ( 535 . 5 mg , 2 . 25 mmol ) in ch 2 cl 2 ( 5 ml ) was then added dropwise ( 1 drop / 2 sec ) to the cold , stirred solution of accl / ch 2 cl 2 . a cold solution of 7 ( 214 mg ., 0 . 5 mmol ) in dried ch 2 cl 2 ( 5 ml ) was then added dropwise to the cooled , stirred accl / beta - no 2 cl 2 solution ( tlc , hexanes / etoac , 3 : 7 ). immediate workup and recrystallization ( as in method b ) gave 8 ( 187 mg , 84 %) as pale - yellow crystals . 1 ( a ) carson , d . a . ; wasson , d . b . ; kaye , j . ; ullman , b . ; martin , d . w ., jr . ; robins , r . k . ; montgomery , j . a . proc . natl . acad . sci . usa 1980 , 77 , 6865 - 6869 . 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