Patent Application: US-201314040738-A

Abstract:
the present invention relates to a novel compound derived from ginsenoside rh2 , the preparation methods and the use thereof in treating cancers .

Description:
as used herein and in the claims , “ comprising ” means including the following elements but not excluding others . the following examples are given to enable a skilled person in the art to clearly understand and practice the present invention . accordingly , a new ginsenoside compound (“ the novel compound ”) is provided that is chemically modified from its parental ginsenoside 20 ( r )- rh2 , which exhibits specific anti - cancer efficacy in both in vitro and in vivo models . in the in vitro cell culture studies , the novel compound exhibited significant cytotoxicity in a panel of cancer cells , but no cytotoxic effect in normal human lung fibroblasts by using the same range of drug concentrations in cancer 175 cells was observed . furthermore , the novel compound was found to exhibit potent suppressive effect on the carcinoma growth in llc - 1 bearing c57 mice via oral administration without observable adverse impacts on the major organs and body weight of animals . these results provide solid evidences that the novel compound has potential to be developed as a novel chemotherapeutic agent . in one embodiment of the present invention , example 1 describes three methodologies for the organic synthesis of the novel compound from natural ginsenoside 20 ( r )- rh2 . the chemical synthesis and chemical structure thereof are shown in fig1 . there are three synthetic routes in obtaining the novel compound as discussed below : a mixture of oxone ® mono - persulfate compound ( 494 . 2 mg ) and nahco 3 ( 210 . 4 mg ) was added slowly to a solution of 20 ( r )- ginsenoside rh2 ( 100 mg ) in 60 ml of a 1 : 1 mixture of acetonitrile and na 2 ( edta ) ( 4 × 10 − 4 m in water ); in one embodiment , acetonitrile can be replaced by methanol or dmso . shi epoxidation diketal catalyst ( ketone , 124 . 5 mg ) in 15 ml of acetonitrile was then added dropwise during a period of 10 - 20 minutes . the reaction mixture was allowed to stand 8 - 24 hours at room temperature with magnetic stirring . after filtration and removal of organic solvents in vacuum , the reaction solution was directly loaded to an ods column and eluted with 50 % to 90 % methanol to obtain the pure novel compound ( 60 - 90 mg ) of formula ( i ). the aforesaid synthesis is illustrated in fig1 . alternatively , the products can also be purified by a silica gel column chromatography eluted with chloroform - methanol - water ( from 9 : 1 : 0 . 1 to 7 : 3 : 0 . 5 ) to obtain the pure novel compound . the total yield by this synthetic route - 1 was 60 - 90 %. zr ( otbu ) 4 ( 8 . 0 μl 0 . 0200 mmol ) or hf ( otbu ) 4 ( 8 . 0 μl 0 . 0198 mmol ) was added to a mixture of bishydroxamic acid ( bha , 0 . 0202 mmol ), dmpu ( 5 . 2 mg , 0 . 0406 mmol ) in thf , dmso or dioxane ( 1 ml ) to prepare a catalyst solution . the catalyst solution was cooled to 0 ° c . after 1 hour of stirring at room temperature . 20 ( r )- ginsenoside rh2 ( 100 mg , 0 . 16 mmol ) in organic solvents ( thf or dmso , or dioxane ) was added to the catalyst solution , followed by 80 % cumene hydroperoxide ( chp ) ( 0 . 26 ml , 1 . 5 mmol ). the reaction mixture was kept stirred at 0 ° c . for 4 - 12 hours , warmed to room temperature , and stirred for another 40 - 60 hours . afterwards , the solution was purified by ods or silica gel column chromatography as above mentioned in the synthetic route - 1 to obtain the pure novel compound of formula ( i ). the total yield by this synthetic route - 2 was between 20 - 50 %. vo ( o - i - pr ) 3 ( 0 . 005 ml , 0 . 0208 mmol ) was added to a solution of bha ( 0 . 0202 mmol ) in thf , dmso or dioxane ( 0 . 5 ml ), and then stirred for 8 hour at room temperature . 20 ( r )- ginsenoside rh2 ( 100 mg , 0 . 16 mmol ) in organic solvents ( thf or dmso , or dioxane ) was added to the aforesaid solution , subsequently followed by 80 % chp ( 0 . 5 ml , 3 mmol ). the reaction mixture was stirred at room temperature for 20 - 30 hours until the substrate of 20 ( r )- ginsenoside rh2 cannot be detected by tlc . the products were then purified by column chromatography on silica gel or ods also as mentioned in the synthetic route - 1 above to obtain the pure novel compound of formula ( i ). the total yield by this synthetic route - 3 was around 40 - 60 %. the novel compound obtained by either of the synthetic routes 1 - 3 was identified as below : 3 - o - β - d - glucopyranosyl 20 ( r ), 24 - epoxydammarane - 3β , 12 , β - triol ( i . e . the novel compound ): white amorphous powder , optical rotation [ α ] d 20 + 5 . 29 ( c = 0 . 32 , meoh ); high resolution - esi - ms ( positive ion mode ): m / z 639 . 4480 [ m + h ] + ( calculated for c 36 h 63 o 9 : 639 . 4467 ); 1 h - nmr ( 400 mhz , c 5 d 5 n ) δ : 5 . 04 ( 2h , d , j = 7 . 8 hz , h - 1 ′), 4 . 61 ( 2h , d , j = 11 . 4 hz , h - 6 ′ a ), 4 . 42 ( 2h , dd , j = 11 . 4 , 5 . 5 hz , h - 6 ′ b ), 4 . 39 ( 2h , m , h - 3 ′), 4 . 23 ( 2h , m , h - 4 ′), 4 . 20 ( 2h , m h - 2 ′), 4 . 15 ( 2h , m , h - 5 ′), 4 . 12 ( 2h , m , h - 24 ), 3 . 88 ( 2h , m , h - 12 ), 3 . 48 ( 2h , dd , j = 11 . 7 , 4 . 3 , h - 3 ), 1 . 61 , 1 . 60 ( 3h each , s , h - 27 ), 1 . 58 , 1 . 56 ( 3h each , s , h - 21 ), 1 . 41 , 1 . 40 ( 3h each , s , h - 26 ), 1 . 40 , 1 . 39 ( 3h each , s , h - 18 ), 1 . 10 ( 6h , s , h - 28 ), 1 . 08 , 1 . 07 ( 3h each , s , h - 30 ), 1 . 06 , 1 . 00 ( 3h each , s , h - 19 ), 0 . 90 ( 6h , s , h - 29 ); 13 c - nmr ( 100 mhz , c 5 d 5 n ) δ : 39 . 7 ( c - 1 ), 27 . 2 ( c - 2 ), 89 . 3 ( c - 3 ), 40 . 2 ( c - 4 ), 56 . 9 ( c - 5 ), 19 . 0 ( c - 6 ), 35 . 8 ( c - 7 ), 40 . 6 ( c - 8 ), 50 . 8 and 50 . 7 ( c - 9 ), 37 . 6 ( c - 10 ), 32 . 0 and 31 . 9 ( c - 11 ), 71 . 3 ( c - 12 ), 50 . 2 and 50 . 1 ( c - 13 ), 52 . 2 and 52 . 1 ( c - 14 ), 32 . 1 ( c - 15 , 24s - epimer ), 31 . 7 ( c - 15 , 24r - epimer ), 27 . 3 ( c - 16 , 24s - epimer ), 27 . 0 ( c - 16 , 24r - epimer ), 51 . 4 and 51 . 3 ( c - 17 ), 17 . 0 and 16 . 9 ( c - 18 ), 16 . 3 and 16 . 2 ( c - 19 ), 86 . 8 and 86 . 7 ( c - 20 ), 21 . 9 ( c - 21 , 24r - epimer ), 19 . 6 ( c - 21 , 24s - epimer ), 39 . 8 ( c - 22 , 24r - epimer ), 38 . 7 ( c - 22 , 24s - epimer ), 27 . 5 and 27 . 4 ( c - 23 ), 87 . 6 ( c - 24 , 24s - epimer ) 86 . 4 ( c - 24 , 24r - epimer ), 71 . 1 ( c - 25 , 24s - epimer ), 70 . 7 ( c - 25 , 24r - epimer ), 26 . 6 and 26 . 5 ( c - 26 ), 28 . 0 ( c - 27 , 24r - epimer ), 27 . 6 ( c - 27 , 24s - epimer ), 28 . 7 ( c - 28 ), 17 . 4 ( c - 29 ), 17 . 7 and 17 . 6 ( c - 30 ), 107 . 4 ( c - 1 ′), 76 . 3 ( c - 2 ′), 79 . 3 ( c - 3 ′), 72 . 4 ( c - 4 ′), 78 . 9 ( c - 5 ′), 63 . 6 ( c - 6 ′). the oxidation of double bond on the side chain of 20 ( r )- ginsenoside rh2 with oxone / nahco 3 catalyzed by ketone in the synthetic route - 1 ; or , by zirconium or hafnium in the synthetic route - 2 ; or by vanacium in the synthetic route - 3 with bha ligands , first led to its 24 , 25 - epoxy compounds , as shown in fig1 . the hydroxyl group located at c - 20 of these epoxides immediately attacked the electron - demand c - 24 to form 20 , 24 - epoxides in an 1 : 1 mixture of 24 - epimers [ 5 ] ( checked by uplc - ms analysis ) which was purified by ods column chromatography ( 90 % methanol ). structure of the novel compound was characterized on the basis of spectroscopic evidence . its molecular formula c 36 h 62 o 9 was determined by positive high - resolution esi - ms . 1 h - nmr spectrum showed an anomeric proton at δ 5 . 04 ( d , j = 7 . 8 hz ), three oxygenated methine proton signals at δ 4 . 12 ( m ), 3 . 88 ( m ) and 3 . 48 ( dd , j = 11 . 7 , 4 . 3 ), and sixteen quaternary methyl groups at δ 1 . 61 , 1 . 60 , 1 . 58 , 1 . 56 , 1 . 41 , 1 . 40 ( 6h ), 1 . 39 , 1 . 10 ( 6h ), 1 . 08 , 1 . 07 , 1 . 06 , 1 . 00 , 0 . 90 ( 6h ). 13 c - nmr spectrum revealed that some signals were in pair indicating the existence of a pair of epimers . the carbon signals at δ 107 . 4 ( c - 1 ′), 76 . 3 ( c - 2 ′), 79 . 3 ( c - 3 ′), 72 . 4 ( c - 4 ′), 78 . 9 ( c - 5 ′), 63 . 6 ( c - 6 ′) were assigned to the β - glucopyranosyl moiety of the novel compound [ 6 ]. the other carbon signals were almost identical to those of 20r , 24s - epoxy - dammarane - 3β - 12β , 25 - triol and its 24r - epimer by comparison with the literature data [ 7 - 9 ]. the downfield shift of c - 3 at δ 89 . 3 suggested that the glucosyl moiety was attached to c - 3 of the aglycone . the method of chemically synthesizing the novel compound from 20 ( r )- rh2 is described herein . the chemical structure thereof was characterized by spectroscopic evidence and uplc - ms analysis . this example describes the result of in vitro cytotoxicity study in a panel of cancer cells by the novel compound . the novel compound was dissolved in dmso at final concentration of 100 mmol / l and stored at − 20 ° c . cytotoxicity was assessed using the 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide assay as described previously [ 10 ]. several kinds of cells were seeded on 96 - well plates , namely mcf - 7 , 4000 cells ; hepg2 , 4000 cells ; hela , 3000 cells ; a549 , 4000 cells ; llc - 1 , 3000 cells ; h1299 , 3000 cells ; ccd19lu , 4000 cells per well . after overnight pre - incubation , the cells were exposed to different concentrations of the novel compound ( 0 . 039 - 100 μmol / l ) or control compounds 20 ( s )- rh2 and 20 ( r )- rh2 for 3 days . subsequently , 10 μl of mtt reagents was added to each well and incubated at 37 ° c . for 4 hours followed by the addition of 100 μl solubilization buffer ( 10 % sds in 0 . 01 mol / l hcl ) and overnight incubation . a 585 nm was determined from each well the next day . the percentage of viable cells was calculated using the following formula : cell viability (%)= a treated / a control × 100 . data was obtained from three independent experiments . as shown in fig2 a , the novel compound has a significant cytotoxic effect on lung cancer cells ( llc - 1 ) with ic 50 of 56 μm , but without marked impairment to human normal lung fibroblasts ccd19lu under the drug concentration of over 100 μm . the novel compound also displayed dose - dependent cytotoxic potency against other five cancer cell lines with different genetic backgrounds , as shown in fig2 b . for structure - activity comparison of the two subtypes of the natural rh2 ginsenoside , 20 ( s )- rh2 displayed its cytotoxicity in both llc - 1 and ccd19lu cells , with ic 50 of 46 . 5 μm and 80 . 5 μm , respectively , while 20 ( r )- rh2 exhibited no cytotoxicity in both llc - 1 and ccd19lu cells , as shown in fig2 c & amp ; d . structure modification on the non - effective natural ginsenoside 20 ( r )- rh2 successfully leads to production of a novel compound , which exhibits specific cell cytotoxicity toward a panel of cancer cells but non - toxic to normal human cells and tissues . this example describes an animal study to demonstrate that oral administration of the novel compound is useful in suppression of tumor growth in vivo . the llc - 1 cells harvested from in vitro culture were adjusted to a concentration of 1 . 5 × 10 7 / ml and 0 . 1 ml cell suspension was injected subcutaneously into the dorsal region of the male c57bl / 6j mice . subsequently , when 70 % of mice had developed tumor into a volume ( length × width 2 × 0 . 52 ) larger than 50 mm 3 , the animals were randomly divided into the vehicle control and several treatment groups . the novel compound powder was dissolved with peg400 : ethanol : distilled water in a ratio of 60 %: 10 %: 30 % ( v / v / v ), and orally administrated to mice by gavage feeding at doses of 10 , 20 , 40 and 80 mg / kg / day for 21 consecutive days . oral administration of 80 mg / kg / day 20 ( r )- rh2 , 20 ( s )- rh2 and 20 ( s )- rg3 , and 10 mg / kg / day 5 - fu were designed as control groups . in order to make sure all groups were appropriately blended , all the experimental procedures such as subcutaneous tumor cells injection , treatment and tumor size measurement were performed by three individual persons who were blended for animal group identity . oral administration of the novel compound at dosages of 10 , 20 , 40 and 80 mg / kg / day demonstrated significant and dose - dependent suppression on the tumor growth , up to 35 . 24 % ( p & gt ; 0 . 05 ), 40 . 49 % ( p & gt ; 0 . 05 ), 44 . 9 % ( p & lt ; 0 . 05 ) and 52 . 04 % ( p & lt ; 0 . 01 ), respectively , after a 21 - day treatment course as compared to the mice treated by the vehicle ( as shown in fig3 & amp ; 4 ). the suppressive effect of the novel compound at the treatment groups of 40 and 80 mg / kg / day were significantly observed from day 12 to day 21 after oral administration ( as shown in fig5 ( a ) & amp ; ( b )). moreover , it was noted that there was no significant difference in body weights between the vehicle control and the novel compound - treated group after deduction of the solid tumors weight of each animal in all of the groups ( see fig6 ( a ) & amp ; ( b )). the novel compound at dosages of 40 and 80 mg / kg / day exhibit significant tumor suppressive effect in tumor growth in mice via oral administration without affecting the body weight . this example describes sub - chronic lethal dose study of the novel compound in which oral administration of the novel compound in high dosages is not harmful to mice 335 in 7 - day repeated dosing . 10 male c57bl / 6j mice were divided into two treatment groups , i . e . 160 mg / kg / day and 320 mg / kg / day . the novel compound powder was dissolved with peg400 : ethanol : distilled water in a ratio of 60 %: 10 %: 30 % ( v / v / v ), and orally administrated to mice by gavage feeding at doses of 160 and 320 mg / kg / day for 7 consecutive days . the health condition of the animals was monitored and their body weights were recorded during this 7 days treatment duration . as shown in fig7 ( a ) to ( b ), oral administration of the novel compound at dosages of 160 and 320 mg / kg / day were not harmful to the animal that 100 % of animal survived and no significant drop of body weight was encountered after a 7 - day treatment course . the novel compound derived from 20 ( r )- rh2 is non - toxic to mice up to 320 mg / kg / day for 7 days by oral administration . this example describes acute oral toxicity in 14 - day lethal dose study of the novel compound in which oral administration of the novel compound at 2000 mg / kg is not harmful to mice in 14 days after single dosing . the acute oral toxicity of the novel compound was evaluated in line with the organization for economic cooperation and development ( oecd ) guideline for testing chemicals — acute oral toxicity — acute toxic class method ( test guideline 423 , adopted on 17 dec . 2001 ). based on the result of example 4 , the test procedure with a starting dose of 2000 mg / kg ( annex 2d of oecd 423 ) was selected to evaluate the acute toxicity of the novel compound . in the test , 3 female and 3 male c57bl / 6j mice ( spf grade ) were fasted overnight and then orally administrated by gavage feeding with single dose of the novel compound solution at 2000 mg / kg . the mortality and health condition of the animals were monitored for 14 days and the result showed zero animal death in both gender . in addition , no significant drop of body weight and sign of toxicity were observed . based on the testing principle and evaluation criteria of oecd test guideline 423 , the novel compound can be classified as class 5 or unclassified under globally harmonized classification system ( ghs ) and the ld 50 of the novel compound is estimated larger than 5000 mg / kg . the novel compound is non - toxic to mice after single dosing at 2000 mg / kg oral administration and the ld 50 of the novel compound is classified as & gt ; 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