Patent Application: US-21319298-A

Abstract:
an efficient and scalable method is reported for the isolation of costatolide , an hiv - 1 - specific nonnucleoside reverse transcriptase inhibitor , from the latex of calophyllum plants such as c . teysmannii var . inophylloide . an overall yield of 10 . 6 % of costatolide , with a purity of 96 %, was obtained by repetitive recrystallization of the latex from a single organic solvent after the oily material was removed by treatment with hexane . a second major component of the latex , soulattrolide , another hiv - 1 nnrti , was also isolated . both compounds were characterized by spectroscopic and chromatographic analyses and their in vitro anti - hiv activities were also confirmed . the results suggest that sufficient supplies of costatolide can be obtained in a relatively low - cost manner from natural sources .

Description:
all references , patents and published patent applications cited herein are incorporated by reference in their entirety . the latex starting material may be obtained from calophyllum plants such as c . teysmannii miq . and c . teysmannii miq . var . inophylloide ( ing ) p . f . stevens and c . lanigerum var . austrocoriaceum ( t . c . whitmore ) p . f . stevens , which are found typically in southeast asia , sri lanka , and northern australia . c . teysmannii latex , provided by the forest research centre of sarawak , malaysia , was isolated according to described procedures 19 and was analyzed by quantitative high performance liquid chromatography ( hplc ) analysis . the results indicated that the latex consisted of costatolide (˜ 37 %) and soulattrolide (˜ 23 %), along with other unidentified components . a preliminary solubility study revealed that the latex was soluble in almost all organic solvents at room temperature with the following order : ch 2 cl 2 ( 400 mg / ml )& gt ; ch 2 cl 2 / meoh ( 1 : 1 )≅ acoet (˜ 200 mg / ml )& gt ; ch 2 cl 2 / acoet ( 1 : 1 ) ( 50 mg / ml )& gt ; acetone ≅ meoh ( 40 mg / ml )& gt ; etoh ( 30 mg / ml )& gt ; hexane ( 20 mg / ml ). hence , for a preliminary extraction of mostly non - costatolide components from the latex , it is preferred that the latex be treated with any suitable solvent that least solubilizes the latex such as a non - polar solvent , e . g ., hexane and heptane . the amount of solvent used relative to latex ranges between about 5 l / kg latex and about 13 l / kg latex , preferably about 8l / kg latex . the preliminary extraction of the latex starting material may be conducted under solvent reflux conditions or preferably at room temperature . thus , treatment of the latex with hexane dissolved approximately 50 % of the latex by weight , resulting in an off - white solid residue which contained approximately 50 % costatolide ( 2 ) as determined by quantitative hplc analysis . additional amounts of crude costatolide may be obtained by allowing the hexane solution to stand at room temperature for several days . the resulting crude costatolide was then collected and pooled with the residue . the solid residue is then dissolved in a suitable solvent , e . g ., ch 2 cl 2 or acetone , to remove any insoluble bark debris by filtration of the resulting solution through a cotton cloth , glass cotton or other suitable filtering material . in practicing the invention , the solvent acetone is preferred because the resulting acetone solution was more convenient to filter than the ch 2 cl 2 solution . after removal of solvent by any suitable method such as reduced pressure evaporation or distillation , the solid product obtained is recrystallized repeatedly from a suitable solvent , e . g . a polar aprotic solvent such as acetone , t - butylmethyl ether , ethyl acetate or solvent mixtures of acetone / ethyl acetate , ethyl acetate - t - butylmethyl ether , or acetone / t - butylmethyl ether , to produce a final product of costatolide with purity of greater than 96 %. in practicing this invention , acetone is the preferred recrystallization solvent for costatolide . mother liquors containing similar purity levels of costatolide ( 2 ) were combined , solvent was removed and the resulting residue was recrystallized from acetone to yield an additional quantity of costatolide ( 2 ). it has been observed that each iteration of the recrystallization step resulted in an increase in costatolide ( 2 ) purity by an average of 10 %, with recovery yield of 40 to 70 %. generally , the purity of the resulting crystalline product was inversely proportional to the recrystallization yield . in practicing the invention , the appropriate volume ( in milliliters ) of solvent needed for each recrystallization step ranged between about 5 ml to about 12 ml per grain of solid to be recystallized , preferably about 10 ml of solvent , e . g ., acetone , per gram of solid product . a typical example described in example no . 1 , starting with 1 . 96 kilograms of latex , is presented in scheme i . another batch of 2 . 64 kilograms of latex yielded similar results . from these two batches , representing a total of 4 . 60 kilograms of latex , a combined 490 grams of costatolide ( 2 ) was obtained with a purity greater than 96 % in an overall yield of 10 . 6 %, along with other less pure fractions ( table 1 ). a higher overall yield could be expected if the less pure fractions were combined and upgraded by silica gel column chromatography , followed by the recrystallization processes described above . during this study , organic solvents employed were hexane ( 95 l ), ch 2 cl 2 ( 25 l ), and acetone ( 45 l ). the fraction containing a mixture of costatolide ( 2 ) and soulattrolide ( 3 ) in a ratio of 1 : 1 was further purified either by repetitive recrystallization from ethanol ( ca . 3 - 5 times ) or by flash silica gel column chromatography followed by recrystallization from ethanol to afford soulattrolide ( 3 ) with a purity greater than 96 %. the structures of purified costatolide ( 2 ) and soulattrolide ( 3 ) were elucidated by ms and 2d - nmr and their physical ( mp , [ α ] d ) and spectral data ( uv , ir , 1 h nmr , and 13 c nmr ) were identical with those reported in the literature . 1 , 6 , 16 , 17 in addition , the anti - hiv activities of both compounds were also confirmed in the in vitro xtt assay . the isolated costatolide ( 2 ) and soulattrolide ( 3 ) had ec 50 values of 0 . 18 μm and 0 . 62 μm , respectively , in cem - ss cells against the hiv - 1 rf strain . table 1______________________________________summary of results obtained from 4 . 60 kilograms of latex fractions purity of costatolide______________________________________490 g & gt ; 96 % 75 g 90 %. sup . a 129 g 80 %. sup . a 80 g 60 %. sup . a 60 g 50 %. sup . a 917 g 30 - 40 % 2450 g oily material 4201 g ( total ). sup . b______________________________________ . sup . a the remaining major constituent was soulattrolide ( 3 ). . sup . b the bark debris and volatiles accounted for the loss . ## str2 ## in summary , an efficient and scalable method has been demonstrated for the isolation of costatolide ( 2 ), an anti - hiv agent , from the latex of c . teysmannii . it is therefore possible that sufficient supplies of 2 could be obtained in a relatively low - cost manner from natural sources . the purified 490 grams of 2 will be utilized for further preclinical development of this compound . another anti - hiv agent , soulattrolide ( 3 ), has also been isolated utilizing this process , using the mother liquor fractions that were enriched with soulattrolide , preferably the fractions containing a weight ratio of at least 1 : 1 ( w / w ) soulattrolide to costatolide . the soulattrolide , however , was preferably recrystallized from any suitable polar solvent such as ethanol , methanol , diethyl ether , or solvent mixtures such as diethylether with ethanol or methanol . in practicing this invention , the preferred recrystallization solvent for soulattrolide is ethanol . plant material . the latex of calophyllum teysmannii var . inophlylloide was collected 19 at kubah n . p . and bedaun ( pueh f . r . ), kuching division , sarawak , malaysia . general experimental procedure . optical rotations were measured on a perkin elmer model # 241 polarimeter serial # 4677 . nmr spectra were recorded with a varian gemini spectrometer ( 300 mhz for 1 h and 75 . 5 mhz for 13 c ) using cdcl 3 as solvent . uv spectra were recorded on a hitachi u - 2001 spectrometer . ir spectra were acquired on a perkin elmer model paragon 1000 ft - ir spectrometer , with samples being prepared as kbr pellets . ms were obtained with a finnigan mat 90 mass spectrometer . an apci or esi probe was used for apcims or eims spectra . hplc analyses were performed on a hitachi hplc system equipped with a l - 4000 h uv detector and l - 6000 pump . a normal phase column ( b17419 zorbax , 4 . 6 mm × 25 cm ) was purchased from rockland technologies , inc ., and the mobile phase was 30 % acoet in hexane with a flow rate of 1 ml / min . method a . at r . t . : latex ( 1 , 964 g ) containing approximately 37 % costatolide ( 2 ) and 23 % soulattrolide ( 3 ), as determined by quantitative hplc analysis , was stirred in 15 l of hexane at r . t . for 1 hr . the solid was allowed to settle , and then was filtered through a filter stick by vacuum transfer and washed with hexane ( 10 l × 2 ). the solid product thus obtained was dissolved in 15 l of ch 2 cl 2 and filtered through cotton cloth to remove bark debris . removal of solvent afforded 1 , 026 g of a light yellowish solid , with the purity of costatolide ( 2 ) being increased to approximately 50 %. the combined greenish yellow hexane solution and washes were concentrated under vacuum . when the volume was reduced to approximately 10 l , precipitates formed , which were collected by filtration to yield an additional 133 g of an off - white solid which contained 30 % of 2 , resulting in a total of 1 , 159 g of costatolide - containing solid in a yield of 59 % recovered from latex after the hexane treatment . the filtrate was concentrated to dryness to leave a 579 g of greenish oil with less than 3 % 2 , as determined by hplc . method b . refluxing : a quantity of 2 . 64 kg of latex and 15 l of hexane was heated to reflux for one hour . the solid was allowed to settle , and then was filtered through a filter stick by vacuum transfer and washed with hexane ( 15 l × 2 ) to afford 844 g of an off - white solid containing approximately 50 % of costatolide ( 2 ). an additional 242 g of a light yellow solid , with 39 % of 2 , was obtained from the combined hexane solutions after standing at r . t . for one week . the filtrate was concentrated to 1 / 3 volume and allowed to stand at r . t . for one week to yield 107 g of a pale yellow solid with a purity of 57 % of costatolide ( 2 ). a total of 1 , 080 g of oily material was obtained after concentration from all the filtrates , which had less than 3 % 2 as determined by hplc . the recovery yield of costatolide - containing solid from this batch was 45 % after the hexane treatment . step 1 recrystallization to increase the purity of 2 from 50 % to 60 %: an amount of 1 , 026 g ( 50 % pure ) of the off - white solid was refluxed in 10 l of acetone until a clear brown solution was obtained , which was filtered hot through a glass fritted funnel under vacuum . the resulting solution was allowed to stand at r . t . for 2 days . the resulting solid was collected and washed with hexane to yield 678 g ( 66 % yield ) of a crystalline product , with the purity of 2 being increased to 60 %. the filtrate was concentrated to 1 / 2 the volume and 50 g of precipitate , with a purity of 32 % of 2 were collected through filtration . the filtrate was evaporated to dryness to afford 250 g of oil without detectable amounts of 2 . step 2 recrystallization to increase the purity of 2 from 60 % to 80 %: an amount of 678 g ( 60 % pure ) of the solid obtained above was recrystallized in the same manner described above from 5 . 5 l of acetone to yield 184 g ( 27 % yield ) of crystals with a purity of 82 % of 2 and 492 g ( 50 % pure ) of material from the mother liquor . step 3 recrystallization to increase the purity of 2 from 80 % to 90 %: an amount of 184 g ( 82 % pure ) of the solid obtained above was recrystallized in the same manner described above from 1 . 9 l of acetone to yield 119 g ( 65 % yield ) of crystals with a purity of 93 % of 2 and 65 g ( 50 % pure ) of material from the mother liquor . step 4 recrystallization to increase the purity of 2 from 90 % to 96 %: an amount of 393 g ( 93 % pure ) of the solid was recrystallized in the same manner described above from 2 . 9 l of acetone to yield 277 g ( 71 %) of crystalline costatolide ( 2 ) with purity of 96 % and 125 g ( 82 % pure ) of material from the mother liquor . costatolide ( 2 ): 1 , 6 , 16 mp . 174 - 175 ° c . ( lit . 16 181 - 182 ° c . ); [ α ] d - 22 . 2 ( c 0 . 71 , chcl 3 ) { lit . [ α ] d - 19 . 9 ( c 0 . 42 , chcl 3 )}; uv ( chcl 3 ) λ max ( log ε ) 222 ( 4 . 67 ), 285 ( 4 . 43 ), 327 ( 4 . 10 ) nm ; ir ( kbr ) ν max 3477 , 2961 , 2931 , 1703 , 1638 , 1584 , 1569 cm - 1 ; apci - ms : m / z 371 ( m + h ) + ; 1 h nmr : 1 . 03 ( 3h , t , j = 7 . 2 hz , ch 3 ), 1 . 14 ( 3h , d , j = 6 . 9 hz , ch 3 ), 1 . 43 ( 3h , d , j = 6 . 3 hz , ch 3 ), 1 . 48 ( 3h , s , ch 3 ), 1 . 49 ( 3h , s , ch 3 ), 1 . 65 ( 2h , sextet , j = 7 . 2 hz , ch 2 ), 1 . 74 ( 1h , m , h - 11 ), 2 . 67 ( 1h , d , j = 3 . 9 hz , oh ), 2 . 88 ( 2h , m , ch 2 ), 4 . 27 ( 1h , dq , j = 6 . 6 , 6 . 9 hz , h - 10 ), 4 . 97 ( 1h , broad , h - 12 ), 5 . 53 ( 1h , d , j = 9 . 9 hz , h - 7 ), 5 . 94 ( 1h , s , h - 3 ), 6 . 63 ( 1h , d , j = 9 . 9 13 c nmr : 12 . 5 ( ch 3 ), 14 . 0 ( ch 3 ), 18 . 8 ( ch 3 ), 23 . 2 ( ch 2 ), 27 . 6 ( ch 3 ), 27 . 8 ( ch 3 ), 38 . 2 ( c - 11 ), 38 . 5 ( ch 2 ), 61 . 8 ( c - 12 ), 73 . 0 ( c - 6 ), 77 . 6 ( c - 10 ), 103 . 5 ( c - 4a ), 106 . 1 ( c - 8a or c - 12a ), 106 . 3 ( c - 12a or c - 8a ), 110 . 3 ( c - 3 ), 116 . 6 ( c - 8 ), 126 . 7 ( c - 7 ), 151 . 4 ( c - 4b ), 153 . 1 ( c - 8b or c - 12b ), 153 . 9 ( c - 12b or c - 8b ), 158 . 7 ( c - 4 ), 161 . 0 ( c = 0 ); elem . anal . : c 71 . 40 %, h 7 . 20 %; calcd . for c 22 h 26 o 5 : c 71 . 33 %, h 7 . 08 %. soulattrolide ( 3 ): an amount of 20 g of solid obtained from the above acetone recrystallization , which contained approximately 1 : 1 ratio of costatolide ( 2 ) and soulattrolide ( 3 ), was subjected to flash silica gel column chromatography ( biotage flash column ), eluting with a stepwise gradient of acoet in hexane : 10 % ( 5l ), 15 % ( 10 l ), 20 % ( 15 l ), and 100 % acoet ( 5 l ). a volume of 500 ml was collected for each fraction . fractions 28 and 29 contained soulattrolide ( 3 ) (˜ 70 % by hplc ). crystalline needles ( 300 mg , 89 % purity of 3 ) were formed in fraction 29 and collected . the crystals were recrystallized from etoh twice to afford 170 mg of soulattrolide ( 3 ) with a purity of 97 % by hplc . soulattrolide ( 3 ): 17 mp 200 - 201 ° c . ( lit . 201 - 202 ° c . ); [ α ] d - 34 . 2 ( c 0 . 24 , chcl 3 ) { lit . 17 [ α ] d - 29 . 6 ° ( chcl 3 )}; uv ( chcl 3 ) λ max ( log ε ) 216 ( 4 . 74 ), 232 ( 4 . 53 ), 286 ( 4 . 56 ), 335 ( 4 . 29 ) nm ; ir ( kbr ) ν max 3457 , 2862 , 1698 , 1584 , 1366 , 1139 , 837 , 762 cm - 1 ; apcims m / z 405 [ m + h + ]; 1 h nmr : 0 . 93 ( 6h , s , 2 ch 3 ), 1 . 16 ( 3h , d , j = 7 . 2 hz , ch 3 ), 1 . 44 ( 3h , d , j = 7 . 2 hz , ch 3 ), 1 . 76 ( 1h , m , h - 11 ), 2 . 79 ( 1h , d , j = 3 . 6 hz , oh ), 4 . 26 ( 1h , dq , j = 6 . 4 , 10 . 7 hz , h - 10 ), 4 . 96 ( 1h , d , j = 3 . 2 hz h - 12 ), 5 . 34 ( 1h , d , j = 9 . 8 hz , h - 7 ), 5 . 94 ( 1h , s , h - 3 ), 6 . 51 ( 1h , d , j = 9 . 8 hz , h - 8 ), 7 . 3 ( 5h , m , c 6 h 5 ); 13 c nmr : 12 . 4 ( ch 3 ), 18 . 7 ( ch 3 ), 26 . 7 ( ch 3 ), 26 . 8 ( ch 3 ), 38 . 7 ( c - 11 ), 61 . 7 ( c - 12 ), 73 . 0 ( c - 10 ), 76 . 6 ( c - 6 ), 103 . 6 ( c - 4a ), 106 . 1 ( c - 8a or c - 12a ), 106 . 2 ( c - 12a or c - 8a ), 111 . 7 ( c - 3 ), 116 . 0 ( c - 8 ), 127 . 3 ( para - and meta - c 6 h 5 ), 127 . 4 ( ortho - c6h 5 ), 127 . 6 ( c - 7 ), 140 . 1 ( ipso - c 6 h5 ), 151 . 2 ( c - 4b ), 153 . 6 ( c - 8b or c - 12b ), 153 . 9 ( c - 12b or c - 8b ), 156 . 4 ( c - 4 ), 160 . 8 ( c = 0 ); elem . anal . : c 73 . 94 %, h 6 . 10 %, calcd . for c 25 h 24 o 5 , c 74 . 24 %, h 5 . 98 %. solid material obtained from mother liquor of costatolide recrystallization as described in experiment no . 1 , which contained soulattrolide and costatolide in the weight ratio of about 1 : 1 , was purified via recrystallization ( 3 ×) from ethanol to produce solatrrolide ( 3 ) of 95 . 5 % purity in a total yield of 41 . 5 % starting from latex . step no . 1 : recrystallization to purity of ( 3 ) from 49 % to 63 %: a quantity of 13 g of solid ( soulattrolide 49 %, costatolide 49 %) was dissolved in 260 ml of hot ethanol by refluxing , and filtered . the clear filtrate was let stand at room temperature overnight . the precipatate shown was filtered to produce 9 . 28 g of off - white crystals with a purity of 63 . 4 % of 3 ( yield 71 . 5 %). step no . 2 . recrystallization to increase purity of ( 3 ) from 63 % to 93 %: an amount of 9 . 28 g of solid ( soulattrolide 63 %, costatolide 33 %) was recrystallized in the same manner described above from 250 ml of ethanol to produce 7 . 04 g of white crystals with a purity of 93 % of 3 ( yield 75 . 9 %). step no . 3 . recrystallization to increase purity of ( 3 ) from 93 % to 95 . 5 %: an amount of 7 . 04 g of solid ( soulattrolide 93 %, costatolide 5 . 1 %) was recrystallized in the same manner described above from 250 ml of ethanol to produce 5 . 38 g of white crystals with a purity of 95 . 5 % of 3 ( yield 76 . 4 %). 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