TWI295994B - Derivatives of 20-o-β-d-glucopyranosyl-protopanaxadiol, preparation and use thereof - Google Patents

Description

1295994 IX. INSTRUCTIONS OF THE INVENTION: FIELD OF THE INVENTION The present invention relates to derivatives of 20-Ο_β·ϋ-σ-pyranosyl 2〇(S)·pro-human decanediol (compcnmdK, CK for short), especially A series of new CK succinate and glutarate derivatives and preparation methods thereof, and the application of the bamboo organism as a music, cosmetics and health food. Prior Art Ginsenoside CK is a unique component of red ginseng, and its content is only a few hundred thousandth. It has been confirmed by many studies that it has excellent clinical effects in the treatment and prevention of malignant tumors. U.S. Patent No. 5,919,770 and its parallel Chinese Patent No. CN1 182 433 A and International Patent No. WO-A No. 97-31013 disclose a human intestinal bacterial metabolite of human ginseng glycosides and an anticancer preparation thereof, one of which is CK, and one of the main products of the metabolite is prepared. The agent is a potential anticancer agent with immunopotentiating effect, which can inhibit tumor angiogenesis and extravasation of cancer cells. Chinese patent CN1417345A and parallel international patent W003-043 83 also disclose a method for preparing CK by hydrolyzing ginsenoside with an enzyme to provide a demand for preparing an anti-tumor therapeutic drug. CK has low polarity and poor water solubility. When using or developing anti-cancer injections, it is often necessary to add some micro-toxic co-solvents, such as Crem0ph0r0r0r0r0r emulsifier (dimethylsulfoxide), dimethylsulfoxide (DMSO), Ethanol and the like. Therefore, the present invention intends to chemically modify CK ginsenoside to solve its water solubility problem. SUMMARY OF THE INVENTION A primary object of the present invention is to provide a derivative of 2〇_〇-p_D_glucopyranosyl 2〇(S)-former quinonediol (CK) and a method for the wound thereof, And the use of this derivative as a drug, a cosmetic and a health food. Another object of the present invention is to provide a series of novel CK succinate and glutarate derivatives and processes for their preparation, and the use of such derivatives as pharmaceuticals, cosmetics and health foods. In order to achieve the above object, the present invention carries out a ring-opening reaction of dianhydride with KOH and a dianhydride in the presence of a suitable solvent and a base as a catalyst, thereby forming a CK diester derivative which can be represented by the following reaction formula.

1295994 where Glc is

OH A is -(CR4R9)n-; R1 = H or -C(0)(CR4R9)nC00H ; R2 = H or a C(0)(CR4R9)nC00H ;

Wherein R5, R6, R7 and R8 are independently Η, or -C(0)(CR4R9)nC00H; wherein η is an integer from 2 to 6, and R4 and R9 are independently Η or Cl-C4. Preferably, R4 and R9 are hydrogen and η is 2 or 3. Preferably, R2 is Η. Preferably, R1 is Η. Preferably, R5 is -C(0)(CH2)nC00H, and R6, R7 and R8 are both Η, wherein η is 2 or 3. Preferably, R7 is a C(0)(CH2)nC00H, and R5, R6 and R8 are both H, wherein η is 2 or 3. Preferably, R5 and R7 are both -C(0)(CH2)nC00H, and R6 and R8 1295994 are both H, wherein η is 2 or 3. Preferably, R5, R6 and R7 are both -C(0)(CH2)nC〇〇H, and R8 is H, wherein η is 2 or 3. The present invention also provides a pharmaceutical preparation comprising an aqueous solution and the aforementioned CK diester derivative of the present invention or a pharmaceutically acceptable salt thereof dissolved in the aqueous solution. Preferably, the derivative or a pharmaceutically acceptable salt thereof is present in the aqueous solution at a concentration greater than 10 mg/ml. Embodiments The present invention discloses a 20-Ο-β·ϋ·glucopyranosyl-20(S)-protoxindiol (CK) derivative having the formula: or a pharmaceutically acceptable salt thereof:

Wherein R1, R2 and R3 are as defined above. In a preferred embodiment of the invention, succinate and giutarnate derivatives of CK are synthesized and compared to the anticancer activity of CK. The synthesis comprises mixing CK with Succinic Anhydride or Glutaric Anhydride at a ratio of 1:0·1 to 100, preferably 1:1 to 1 molar, in a suitable organic solvent and as a The base of the catalyst was 1295994, and the ring opening reaction of the dianhydride was carried out. This organic solvent can be used! Ianes (such as Dichloromethane, Trichlorocarbazone, Dioxane, etc.); ethers (such as dialkyl ether or cycloaliphatic ether (Alicyclic ether), tetrahydroanthracene) Tetrahydrofuran, Dioxane; tertiary amines include tertiary amines (such as Triethylamine, Pyridine), or trialkyldiamines (such as Ν,Ν,Ν',Ν,-tetramethylethylenediamine (N,N,N',N'_tetramethyl ethylenediamine), N,N,N',N'-tetramethyldiamine (N, N, N', N'-tetramethyldiaminomethane)); low-burning quinones (such as Dimethyl sulfoxide (DMF)); low-burning bases. This test can be used with liquids such as tertiary amines including, but not limited to, tertiary alkylamines, 4-(Dimethylamino)pyridine, mouth-biting, N, N, N ',N'_Tetramethylethylidene diamine, N,N,N,N'-tetradecyldiamine decane, etc.; or solid phase base, such as Si-dimethylamine (Si-Dimethylamine) ), Si_Morpholine, Si-hexahydro^b^(Si_Piperidine), and the like. Preferably, the organic solvent and the base are the same as the tertiary amine. The ring opening reaction is at 0. 〜2 00 ° C (preferably 10 to 120 ° C) is shaken or turbulent and refluxed for 3 〇 minutes to 1 〇 day (preferably 30 minutes to 48 hours). If a reaction is carried out using a water-soluble solvent, such as a tertiary amine or a low-burning kiln, the solvent is first volatilized to remove the concentrate, and then partitioned with water and an acid ester or ether to obtain an organic layer. Concentrated and dried to obtain a CK succinate or glutarate product mixture. If a solvent that is immiscible with water is used, the reaction is stopped by directly adding ice water. The organic layer is taken, and partitioned with water and acid or ether. The organic layer is concentrated and dried to obtain CK succinate. Or pent! 295994 diester product mixture. The product mixture can also be further purified using a silica gel chromatography column or a high performance liquid chromatography column. The product of CK succinate or glutarate is dissolved in acetone, and then dropped into a methanol or ethanol solution containing NaOH or KOH, and the precipitate is completely precipitated. The concentrate is too concentrated to obtain the potassium or sodium salt of CK succinate. Or CK glutaric acid sulphate or sodium salt product, other pharmaceutically acceptable salts such as ammonium salts can also be prepared in a similar manner. The CK succinate or glutarate product can also be directly dissolved in a phosphate buffer (Phosphate buffer saline), sodium bicarbonate or potassium aqueous solution having a pH of 6 to 9 (preferably ΡΗ6·8 to 7.8) to become a ck. Aromatic acid vinegar or glutaric acid I salt solution. EXAMPLES 1. Synthesis of CK succinate 401 mg of 20-Ο_β_ϋ-glucopyranosyl-20(S)_protol decanediol (CK), 257 mg (4 equivalents) of succinic anhydride (Succinic Anhydride) And 393 mg (5 equivalents) of 4-(Dimethylamino)pyridine in a 50 ml two-necked round bottom flask. The middle of the two-necked round bottom bottle is provided with a serpentine countercurrent condensation tube; the right side of the bottle is pumped with a hydraulic pressure to remove the water from the reactants. The reaction was carried out by introducing a milk gas which was dehydrated with sulfuric acid and then injecting 35 ml of tetrahydroanthracene (THF) which was first dried by CaH2. (After 48 hours of the reaction, the reaction mixture was concentrated under reduced pressure, and the obtained concentrate was added to ice water and ethyl acetate for separation, the aqueous layer was discarded, and the organic layer was extracted twice with water, so that the excess was unreacted. The anhydride and base are extracted into the water layer. The extracted 1129994 layer is added with an appropriate amount of anhydrous sulfuric acid to remove water. After the transition of the Buchner funnel, the filtrate is concentrated and dried to obtain 369.7 mg of CK succinate derivative. The product obtained from product 0 was analyzed by a high performance liquid chromatography column (Analytic HPLC; GL Sciences Inc., Olertsil ODS_3V C-18; 3 〇mm χ 15 〇 mm). The mobile phase was 52% acrylonitrile (Acetonitrile)/H20. (containing 0.02% dish acid), the flow rate is 611.61111/111丨11, detected by the absorption value of ultraviolet light 203 11111, the time when the main products are flushed out are: lle0 min (yield: 21.1%), 19.9 Min (yield: 44.2%), 24.7 min (yield·· 20.7%), 31.7 min (yield: 1〇_4%) using a semi-preparative HPLC column (Preparative HPLC; GL Sciences) Inc., Inertsil ODS-3 C-18; 10 mm x 25 0 mm) for purification and separation 4i · 8 mg of 19.9 min product (GS_8), identified by mass spectrometry (Mass Spectrum) as a CK monosuccinate derivative with a succinate ester, molecular weight 723.44, 13C-NMR, i-NMR , HH COSY, CH COSY identification, acryl ester was attached to the 3rd position of the CK 20th position on the glucose group, the -OH functional group, and 14.8 mg of the 24.7 min product (GS-9), mass spectrometer It was identified as a CK disuccinate derivative with 2 succinates attached, and the molecular weight was 823.44. It was identified by I3c-NMR, 1H-NMR and HH COSY, and the succinate was attached to the glucosyl group at position 20 of CK. And 6, the position of the OH functional group, and obtained 11 2 mg of 3 1.7 min product (GS -11), identified by mass spectrometry as a succinate derivative with 3 succinates, the molecular weight is 923.46, identified by 13C-NMR, h-NMR and HH COSY, the succinate was attached to the _H functional group at the 3, 4, and 1299994 to 6' positions on the glucosyl group at position 20 of CK. % Example 2 Synthesis of CK succinate: Take 41 mg of CK and 40 mg (6 equivalents) of succinic anhydride, place in a 50 ml two-necked round bottom bottle, first remove the reactants. After the step, the water-removed nitrogen gas was introduced and 40 ml of triethylamine which was firstly dried by CaH2 was used, and after reacting at room temperature for 25 hours at 25 ° C, the reaction mixture was concentrated to remove the solvent. The mixture was separated into ice water and ethyl acetate at least three times. The organic layer was added with anhydrous magnesium sulfate to remove water. After the transition of the Buchner funnel, the filtrate was concentrated and dried to obtain a mixed product of 65_3 mg of CK succinate derivative. The product was analyzed by HPLC, and the time of the main product was 11.11 (11:11), 13.5 111111 (yield: 41.2%), 19.9 min (yield: 14.4%). ), 24.7 min (yield: 14.4%) and 31.7 min (yield: 2.4%). After separation and purification by semi-preparative HPLC, 175 mg of 13.5 min product (GS-6) was obtained, which was identified by mass spectrometry as a CK monosuccinate derivative with i-am φ vinegar, and the molecular weight was 723.44. Identification by 13C-NMR, i-NMR and H_HC〇SY, the succinate was attached to the 6th position of the glucosyl group at position 20 of cK. Example 3· Synthesis of CK glutarate Take 100 mg of CK, 1 84 mg (10 equivalents) of glutaric anhydride (Glutaric i58 mg (8 equivalents) of 4-(dimethylamino)pyridine, placed at 50 In the ml two-neck round bottom bottle, after the reactant water removal step, the nitrogen gas which has been dehydrated is introduced, and then 30 ml of THF which has been dried by CaH2 dry distillation is injected.

12 1295994 * After 60 hours of reaction at 60 ° C, the reaction mixture was concentrated to remove the solvent, and the mixture was separated into ice water and ethyl acetate at least three times. The organic layer was added to anhydrous k sulfuric acid to remove water and filtered by a Buchner funnel. The filtrate was concentrated and dried to obtain a mixed product of '13 5·6 mg CK glutarate derivative. The obtained product was analyzed by HPLC, and the main products were flushed for 13.5 min (yield··3.4%), 20.6 min (yield: 16.0%), 25.3 min (yield: 37.1%), and 31.3 min. Rate: 3.7%), 36.4 min (production rate: 12.5%) and 51.4111丨11 (yield: 25.8%). After separation and purification by semi-preparative 11?1^, 9.6 mg of 20·6 min product (GS-13) was obtained, which was identified by mass spectrometry as a CK monoglutarate derivative with 1 glutarate. The molecular weight was 737.55, identified by 13C-NMR, W-NMR, HH COSY and CH COSY. The glutarate was attached to the -OH functional group at the 3rd position on the glucosyl group at position 20 of CK. And obtained 1 8.1 mg of 25.3 min product (GS-14), which was identified by mass spectrometry as a CK diglutarate derivative with 2 glutarate, molecular weight of 85 1.59, 13C_N]V [R, h-NNIR ❿ and HH c〇SY were identified, and the glutarate was attached to the -OH functional group at the 3' and 6' positions on the glucosyl group at position 20 of CK. And obtained 7.5 mg of 36.4 min product (GS-15), which was identified by mass spectrometry as a kt-glutarate derivative with 3 glutarate, molecular weight of 965.64, by "C-NMR^H- NMR and HH COS Y identified that the glutarate was attached to the -0H functional group at the 3', 4' and 6' positions on the glucosyl group at position 20 of CK. And obtaining 51.4 min of product (GS-16) of ι 5·5 mg, which was identified by mass spectrometry as a CK pentaglutarate derivative with 5 glutaric acid vinegar, having a molecular weight of 1193.74, 13C-NMR, iH- NMR and Η·Η COSY identification, glutaric acid ester attached to the ck acutely bitter body 13 1295994 3rd position, and the 20th position of the glucose group 2, 3, 4, and 6, the position of the -OH functional group on. Example 4. Synthesis of CK bismuth vinegar 194 mg of CK and 178 mg (5 equivalents) of glutaric anhydride were placed in a 50 ml two-necked round bottom bottle, and the reaction was followed by a water removal step. After passing through the nitrogen gas, 150 ml of triethylamine which was dried by CaH2 dry distillation was introduced at room temperature 25. (6 hours after the reaction, the reaction mixture was concentrated to remove the solvent, and separated into ice water and ethyl acetate at least three times. The organic layer was added to anhydrous sulfuric acid to remove water, filtered by a Buchner funnel, and then taken. After concentration and drying, a mixture of 198 mg of CK glutarate derivative was obtained. The obtained product was analyzed by HPLC, and the main products were washed out for 10.8 min (yield: 31.0%) and 13.8 min (yield: 24.6%). ), 19.4 min (yield: 22.1%), 24.3 min (yield: 14.6%) and 31.0 min (yield ·5.2%). After separation and purification by semi-preparative HPLC, hydrazine can be obtained. · 5 of 1 〇.8 1^11 product ((^((^_12)))) and 3 〇91^ 138 瓜4 product (GS-17), identified by mass spectrometry as glutaric acid The ester of CK monoglutarate has a molecular weight of 737.55, which is identified by "c_NMr, 1h_nmr, HH COSY and c_H COSY. The glutarate is attached to the glucosyl group at the 2nd position of CK. On the h-functional group, and the 19.4 min product (GS_13) of 23·7, the 24 3 min product of 23 2 1^ (GS·14), and the 31.0 min product of 14·4 mg (GS-15). One And Table 2 lists the above examples to 4 in the HpLc pure

14 1295994 Results of 1H-NMR and 13C_NMR of CK and CK succinate and glutarate derivatives. CK: iH-NMR (500MHz, CD3OD) δ: 5.101 (t, 1H, J = 5.0Hz, H-24), 4.600 (d? 1H? J = 8.2? Η-Γ of 20-Glc), 3.766 (dd 2H? J = 12.5? 2.0Hz? H-6, a of 20_Glc), 3.672 (td, 1H, J = 9.5, 5.0Hz, H-12), 3.625 (dd,

2H, J=12.5, 5.0 Hz, H-6丨b of 20-Glc), 3.346 (td, 1H, J = 8.2, 2·0Ηζ, H-3' of 20-Glc), 3.210 (m, 1H, H-5, of 20-Glc), 3.29 (m, lH, H-4' of 20-Glc), 3.138 (dd, 1H, J = 12.5, 4.5 Hz, H-3), 3.072 (t, 1H, J = 8.2Hz, H-2' of 20-Glc), 2.283 (td, 1H, J=11.0, 8.5Hz, H-17), 2.06 (m, 2H, H-23a, b), 1.822 ( Dd,2H,J = 13.0, 5·0Ηζ,H-lla), 1.814 (dt,2H,J= 18.5, 7.5Hz, H-22a), 1.743 (t,1H,J=11.0Hz, H-13) , 1.676 (s, 3H, H-26), 1.638 (s, 3H, H-27), 1.39 (m, 2H, H-22b), 1.338 (s, 3H, H face 21), 1.220 (dt, 2H) , J=13.0, 9·5Ηζ, Η-11β), 1·017 (s, 3H, H-29), 0.960 (s, 3H, H-28), 0·924 (s, 3H, H-30) , 0.914 (s, 3H, H-18), 0.775 (s, 3H, H-19). 13C-NMR (125MHz, CD3OD) δ: aglycone moiety: Cl, 40.24; C-2, 27.99; C-3, 79.52; C-4, 40.03; C-5, 57.25; C-6, 19.41; C-7, 35.84; C-8, 40.96; C-9, 51.05; C_10, 38.17; C-ll, 30.99; 12, 71.93; C-13, 49.76; C_14, 52.49; C-15, 31.64; C-16, 27.19; C-17, 53.14, C-18, 16.23; C-19? 16.72; C-20, 84.94; C-21, 22.85; C-22, 36.65; C-23, 24.26; C-24, 125.84; C_25, 132.30; C-26, 25.89; 27, 17.95; C-28, 28.61; C-29, 16.13; C-30, 17.17; 20-Glc sugar moiety: C-l', 98.30; C-2,, 75.40; C-3,, 78.19; -4',71.17; C-5·,77.95; C-6,,62.50. 15 1295994 CK-Suc (GS-6): h-NMR (500MHz, CD3OD) δ: 5.118 (t,1H, J = 6.5 Hz, H-24), 4.582 (d, 1H, J = 8.3, Η-Γ of 20-Glc), 4.438 (d, 2H, J = 11.4Hz5 H-6fa of 20-Glc)? 4.104 (dd5 2H? J=11.4? 7.4Hz, H-6fb of 20-Glc), 3.704 (td, 1H, J=10.5, 4.5Hz, H-12), 3.427 (t, lH, J = 8.3Hz, H-4* of 20-Glc), 3.344 (dd, 1H, J = 8.3, 7.4Hz, H-5' of 20-Glc), 3.234 (t, 1H, J = 8.3Hz, H-3' of 20-Glc), 3.133 (dd, 1H, J = 9.5, 4.5Hz, H-3), 3.112 (t, 1H, J = 8.3Hz, H-2* of 20-Glc), 2.279

(td, 1H, J=11.0, 8.5Hz, H-17), 2.132 (dq, 2H, J=19, 6·5Ηζ, H-23a), 2.013 (dq5 2H, J=1 95 6.5Hz, H- 23b)? 1.891 (dt, 2H? J= 18, 6.5Hz? H-22a), 1.783 (dd, 2H, J=13.3, 4·5Ηζ, H-llcx), 1.738 (t, 1H, J=11.0Hz) , H-13), 1.671 (s, 3H, H-26), 1.611 (s, 3H, H-27), 1.345 (s, 3H, H-21), 1.243 (dt, 2H, J = 13.3, 10 ·5Ηζ,Η·11β), 1.004 (s,3H, H-29), 0.956 (s,3H,H-28), 0.915 (s,3H,H-30),0.907 (s,3H, H-18 ), 0.770 (s, 3H, H-19)·

CK-Suc (GS-8): h-NMR (500MHz, CD3OD) δ: 5.109 (t, 1H, J = 7.0Hz, H-24), 4.960 (t, 1H, J = 9.5Hz, H-3, Of 20-Glc), 4.71 (d, 1H, J = 8.3, Η-Γ of 20-Glc), 3.776 (dd, 2H, J = 12.5, 1.8 Hz, H-6'a of 20-Glc), 3.673 (td,1H,J= 1 1.05 5·0Ηζ, H-12), 3.649 (dd, 2H, J=12.5, 5.0Hz, H-6, b of 20-Glc), 3·484 (t, lH, J = 9.5 Hz, H-41 of 20-Glc), 3.29 (m, 1H, H-5, of 20-Glc), 3·215 (dd, 1H, J = 9.5, 7.8 Hz, H-2, of 20-Glc), 3.136 (dd, 1H, J=11.5, 5·0Ηζ, H-3), 2.272 (td, 1H, J=11.0, 8.0Hz, H-17), 2.08 (m, 2H, H-) 23ab), 1.913 (ddd, 2H, J= 19, 16 1295994 13.5, 9.5 Hz, H-22a), 1.828 (dd, 2H, J = 13.0, 5.0 Ηζ, Η-11α), 1.737 (t, 1H, J=11.0Hz, H-13), 1.679 (s, 3H, H-26), 1.622 (s, 3H, H-27), 1.345 (s, 3H, H-21), 1.217 (dt, 2H, J =13.5, 11.0 Hz, Η-11β), 1.012 (s, 3H, H-29), 0.958 (s5 3H, Η·28), 0.916 (s, 3H, H-30), 0.910 (s5 3H, H- 18), 0.771 (s, 3H, H-19) · CK-Sue (GS-9) : i-NMR (500MHz, CD3OD) δ: 5.120 (t 1H,

J = 7.3 Hz, H-24), 4.960 (t, 1H, J = 9.5 Hz, H-3' of 20-Glc), 4.685 (d, 1H, J = 8.5, Η-Γ of 20-Glc), 4.437 (dd, 2H, J=11.5, 2.0Hz, H-6'a of 20-Glc), 4.139 (dd, 2H, J=11.5, 6.5Hz, H-6, b of 20-Glc), 3.706 ( Td5 1H, J = 11.0, 5·5Ηζ, H-12), 3.537 (ddd, 1H, J = 9:5, 6.5, 2·0Ηζ, H-5' of 20-Glc), 3.418 (t, lH, J = 9.5 Hz, H-4' of 20-Glc), 3.260 (dd, 1H, J = 9.5, 8.5 Hz, H-2' of 20-Glc), 3.129 (dd, 1H, J = 10.3, 4.8 Hz , H-3), 2.274 (td? 1H5 J= 1 0.55 8.5Hz, H-17)? 2.132 (dq? 2H5 J=19.5? 6.8Hz? H-23a), 2.007 (dq, 2H, J=19.5, 6.8 Hz, H-23b), 1.89 (m5 2H, H-22a), 1.787 (dd, 2H, J = 12.8, 5.5 Hz, H-lla), 1.727 (t5 1H, J = 11.0 Hz, H-13) , 1.673 (s, 3H, H-26), 1.614 (s, 3H, H-27), 1.352 (s, 3H, H-21), 1.211 (dt, 2H, J = 12.8, 11·0Ηζ, Η· 11β), 0.999 (s, 3H, H-29), 0.954 (s, 3H, H-28), 0.906 (s, 3H, H-30), 0.906 (s, 3H, H-18), 0.769 (s , 3H, H-19)· CK-Suc (GS-11) : h-NMR (500MHz, CD3OD) δ: 5.162 (t,1H, J = 9.5 Hz, H-3' of 20-Glc), 4.914 (t, lH, J = 9.5 Hz, H-4' of 20-Glc), 4.769 (d, 1H, J = 7.5, Hl* of 20-Glc ), 4.176 (dd, 2H, J = 12.0, 2.0Hz, 17 1295994)

H-6, a of 20-Glc), 4.124 (dd, 2H, J = 12.0, 6.0 Hz, H-6'b of 20-Glc), 3.793 (ddd, 1H, J = 9.5, 6.0, 2.0 Hz, H-5· of 20-Glc), 3.716 (td, 1H, J=11.0, 5·5Ηζ, H-12), 3.366 (dd, 1H, J = 9.5, 7.5Hz, H-2f of 20-Glc) , 3.128 (dd, 1H, J=11.5, 4.5Hz, H-3), 2.282 (td, 1H, J=10.5, 8.5Hz, H-17), 2.14 (m, 2H, 6·8Ηζ, H-23a ), 2.018 (dq, 2H, J = 20.5, 6.5 Hz, H-23b), 2.018 (dq, 2H, J = 19.0, 6.5 Hz, H-22a), 1.734 (t, 1H, J = 11.0 Hz, H -13),1·673 (s,3H,H-26),1.616 (s,3H,H-27), 1.359 (s,3H,H-21),1.216 (dt,2H,J = 13.0,11.0 Hz, Η_11β), 1.179 (dd, 2H, J=13.0, 5·5Ηζ, H-lla), 1.002 (s, 3H, H_29), 0.954 (s, 3H, H-28), 0.909 (s, 3H, H-30), 0.905 (s, 3H, H-18), 0.769 (s, 3H, CK-Glutarate (GS-17): W-NMR (500MHz, CD3OD) δ: 5.11 (t5 1H, J = 6.5Hz , H-24), 4.58 (d, 1H, J = 8, Η-Γ of 20-Glc), 4.42 (dd, 2H, J = 11.5, 1Hz, H-6, a of 20-Glc), 4.11 ( Dd, 2H, J=12, 7Hz, H-6, b of 20-Glc), 3·71 (td, 1H, J= 15.5, 5Hz, H-12), 3.43 (broad, 1H, H-5f of 20-Glc), 3.35 (t, 1H, J = 9.5Hz, H-3, of 20-Glc), 3.23 (t, lH , J = 9Hz, H-4f of 20-Glc), 3.14 (dd, 1H, J=11.5, 5Hz, H-3), 3.11 (t, 1H, J = 8.5Hz, H-2, of 20-Glc ), 1.67 (s, 3H, H-26), 1.61 (s, 3H, H-27), 1.34 (s, 3H, H-21), 1.00 (s, 3H, H-29), 0.96 ( s, 3H, H-28), 0.91 (s5 3H, H-30), 0.91 (s, 3H, H-18), 0.77 (s, 3H, H-19) · CK-Glutarate (GS-13): iH-NMR (500MHz, CD3OD) δ: 5.11 (t, 1H, J = 7Hz, H-24), 4.96 (t, 1H, J = 9Hz, H-3' of 20-Glc), 4.70 (d, 1H) , 18 1295994 J=7.5, Η-Γ of 20-Glc), 3.77 (dd, 2H, J = 12, 2Hz, H-6, a of 20-Glc), 3.66 (td,1H,J=15.5, 5Hz , H-12), 3.64 (dd, 2H, J=12, 5Hz, H-6, b of 20-Glc), 3.47 (t, lH, J = 9.5Hz, H-4, of 20-Glc), 3.29 (broad, 1H, H-5· of 20-Glc), 3.20 (dd, 1H, J = 9, 8Hz, H-2, of 20-Glc), 3.14 (dd, 1H, J=11.5, 5Hz, H-3), 1.68 (s, 3H, H-26), 1.62 (s, 3H, H-27), 1.35 (s, 3H, H-21), 1.01 (s, 3H, H-29), 0 .96 (s, 3H, H-28), 0·92 (s, 3H, H-30), 0.91 (s, 3H, H-18), 0.77 (s, 3H, Η · 19).

CK-Glutarate (GS-14) : ^-NMR (500MHz, CD3〇D) δ: 5.12 (t, 1H? J = 7Hz, H-24), 4.97 (t, 1H, J = 9Hz, H- 3, of 20-Glc), 4.69 (d, 1H, J = 7.5, Hl· of 20-Glc), 4.43 (dd, 2H, J=12, 2Hz, H-6, a of 20-Glc), 3.71 (td, 1H, J = 15.5, 5Hz, H-12), 4.14 (dd, 2H, J = 11.5, 6.5Hz, H-6, b of 20-Glc), 3·55 (broad, 1H, H- 5, of 20-Glc), 3.41 (t, 1H, J=10Hz, H-4, of 20-Glc), 3.25 (dd, 1H, J = 9.5, 8Hz, H-2, of 20-Glc), 3.14 (dd, 1H, J=ll.5, 5Hz, H-3), 1.68 (s, 3H, H-26), 1.62 (s, 3H, H-27), 1.36 (s, 3H, H-21 ), 1.01 (s, 3H, H-29), 0.96 (s5 3H, H-28), 0.92 (s, 3H, H-30), 0.91 (s, 3H, H-18), 0.78 (s, 3H) , H-19)· CK-Glutarate (GS_15) : W-NMR (500MHz, CD3OD) δ: 5.35 (t,1H, J = 9.5Hz, H-3, of 20-Glc), 5.12 (t,1H, J = 7 Hz, H-24), 4.90 (t, 1H, J = 10 Hz, H-4, of 20-Glc), 4.77 (d, 1H, J = 8, H-l' of 20-Glc), 4.17 (dd, 2H, J=12, 5.5Hz, H-6丨b of 20-Glc), 4.12 (dd, 2H, J=12, 2.5Hz, H-6*a of 20-Glc), 3.81 (ddd , 1H, J=10, 5 .5, 2.5, H-5, of 20-Glc), 3·72 (td, 1H, J=15.5, 5Hz, H-12), 3.22 (dd, 1H, J = 9.5, 8Hz, H-2’

19 1295994 of 20-Glc), 3.13 (dd, 1H, J=11.5, 5Ηζ, Η·3), 1.67 (s, 3H, H-26), 1.61 (s5 3H, H-27), 1.36 (s, 3H, H-21), 1.00 (s, 3H, H-29), 0.95 (s, 3H, H-28), 0.91 (s, 3H, H_30), 0.90 (s, 3H, H-18), 0.77 (s,3H, H-19)· CK-Glutarate (GS-16) : iH-NMR (500MHz, CD3OD) δ: 5.35 (t,1H, J = 9.5Hz, H-3, of 20-Glc), 5.12 (t, 1H, J = 8Hz, H-2· of 20-Glc), 5.10 (t, 1H, J = 7Hz, H_24), 5_02 (t, 1H, J=10Hz, H_4' of 20- Glc) , 4.90 (d,1H,J = 8, H-l' of 20-Glc), 4.49 (dd,1H,J=ll,5·5Ηζ,H-3), 3.92 (broad,1H,H-5丨Of 20-Glc), 3.55 (td, 1H, J=15, 5Hz, H_12), 1.67 (s, 3H, H-26), 1.62 (s, 3H, H-27), 1.34 (s, 3H, H -21), 1.00 (s, 3H, Η·29), 0.94 (s, 3H, H-28), 0.91 (s, 3H, H-30), 0.89 (s, 3H, H-18), 0.86 ( s, 3H, H-19)·

Table 1. CK and CK succinate 13c-nmr c atom CK succinate derivative CK GS-6 (mono-) GS-8 (mono-) GS-9 (di-) GS-11 (tri-) 1 (Level 2) 40.24 40.25 40.26 40.26 40.25 2 (Level 2) 27.99 28.01 28.00 28.00 28.00 3 (Level 3) 79.52 79.57 79.49 79.57 76.83 4 (Level 4) 40.03 40.03 40.03 40.02 40.02 5 (Level 3) 57.25 57.29 57.25 57.28 57.28 6 (Level 2) 19.41 19.42 19.43 19.41 19.41 7 (Level 2) 35.84 35.85 35.85 35.85 35.85 8 (Level 4) 40.96 40.95 40.96 40.95 40.96 9 (Level 3) 51.05 51.11 51.06 51.11 51.11 10 (Level 4) 38.17 38.18 38.17 38.17 38.17 11 (Level 2) 30.99 30.83 30.94 30.75 30.75 12 (Level 3) 71.93 71.71 71.82 71.65 71.65 13 (Level 3) 49.76 49.69 49.76 49.71 49.74 14 (Level 4) 52.49 52.45 52.45 52.41 52.41 15(2 m 31.64 31.52 31.61 31.47 31.46 20 1295994

16 (Level 2) 27.19 27.29 27.20 27.26 27.28 17(3 m) 53.14 53.00 52.98 52.86 52.75 18(1 m) 16.23 16.29 16.29 16.31 16.31 19 (Level 1) 16.72 16.67 16.74 16.66 16.64 20 (Level 4) 84.94 85.00 85.20 85.35 85.63 21 (1 m 22.85 22.29 22.74 22.16 22.20 22 (level 2) 36.65 36.77 36.65 36.66 36.64 23 (level 2) 24.26 23.82 24.11 23.67 23.66 24 (level 3) 125.84 126.04 125.78 125.99 125.90 25 (4 m 132.30 132.21 132.25 132.26 132.33 26 ( Level 1) 25.89 25.91 25.91 25.91 25.90 27 (Level 1) 17.95 17.89 17.97 17.89 17.88 28 (Level 1) 28.61 28.63 28.64 28.63 28.63 29 (Level 1) 16.13 16.12 16.16 16.12 16.12 30 (Level 1) 17.17 17.32 17.25 17.35 17.36 20- Q-Glc _&_3' 3,&6, 3'&4'&6' Γ (Level 3) 98.30 97.99 98.01 97.74 97.72 2丨(Level 3) 75.40 75.17 73.54 73.46 72.69 3丨 (Level 3) 78.19 78.46 79.66 79.76 79.57 4丨 (Level 3) 71.17 71.71 69.21 69.86 70.10 5, (Level 3) 77.95 75.31 77.56 74.79 73.39 6丨 (Level 2) 62.50 65.11 62.20 64.78 63.89 Domain ^ Acid · _ _CK_ GS-6 ( Mono-) GS-8 (mono-) GS-9 (di-) GS-11 (tri_) 3'-Sl 3,-S2 3,-S3 3'-S4 173.90 29.94 30.29 176.28 173.91 29.79 30.05 175.87 173.24 29.55 29.98 175.84 4'-Sl 173.71 4 丨-S2 29.60 4' -S3 30.02 4'-S4 175.89 6'-Sl 174.05 173.98 173.73 6,-S2 29.97 29.89 29.79 6'-S3 30.16 30.24 30.05 6'-S4 176.06 176.41 176.10 Table II, 13c-nmr c atom of CK glutarate CK glutarate derivative GS-17 (mono-) GS-13 (mono-) GS-14 (di-) GS-15 (tri-) GS-16 (penta-) 1 (Level 2) 40.24 40.27 40.24 40.25 39.63 2(2 m) 28.00 28.00 28.02 28.02 27.33 3 (Level 3) 79.56 79.53 79.22 79.53 82.28 4 (Level 4) 40.02 40.03 40.03 40.03 38.99 21 1295994

5 (Level 3) 57.27 57.26 57.26 57.26 57.11 6 (Level 2) 19.41 19.41 19.43 19.43 19.26 7 (Level 2) 35.84 35.85 35.85 35.85 35.66 8 (Level 4) 40.94 40.96 40.95 40.95 40.94 9 (Level 3) 51.09 51.06 51.08 51.10 50.77 10 (Level 4) 38.17 38.17 38.17 38.17 38.14 11 (Level 2) 30.81 30.96 30.78 30.77 30.83 12 (Level 3) 71.68 71.88 71.62 71.62 71.90 13 (Level 3) 49.68 49.77 49.69 49.72 49.63 14 (Level 4) 52.43 52.47 52.40 52.40 52.48 15 (Level 2) 31.50 31.61 31.48 31.45 31.41 16 (Level 2) 27.28 27.19 27.27 27.29 25.93 17 (Level 3) 52.97 53.06 52.90 52.78 53.82 18 (Level 1) 16.29 16.27 16.32 16.33 16.66 19 (Level 1) 16.66 16.72 16.67 16.67 17.07 20 (Level 4) 84.99 85.25 85.31 85.61 86.45 21 (Level 1) 22.23 22.79 22.18 22.21 22.51 22 (Level 2) 36.72 36.63 36.66 36.64 36.80 23 (Level 2) 23.74 24.16 23.68 23.66 23.96 24 (Level 3) 126.07 125.82 125.94 125.85 125.67 25 (Level 4) 132.17 132.33 132.15 132.23 132.44 26 (Level 1) 25.94 25.90 25.94 25.94 24.72 27 (Level 1) 17.91 17.96 17.93 17.93 17.98 28 (1 28.63 28.63 28.64 28.64 28.55 29 (Level 1) 16.13 16.13 16.14 16.14 16.24 30 (Level 1) 17.33 17.22 17.36 17.38 17.30 20-O-Glc 6, 3' 3,&6, 3',4丨,6· 3 , 2',3·,4',6, Γ (Level 3) 97.95 98.15 97.78 97.72 95.65 2· (Level 3) 75.28 73.64 73.50 72.65 72.85 3, (Level 3) 78.50 79.08 79.53 76.79 74.25 4, (Level 3) 71.75 69.25 69.91 70.09 69.83 5丨 (Level 3) 75.08 77.71 74.85 73.35 73.05 6, (Level 2) 64.96 62.21 64.60 63.53 63.37 Glycolate GS-17 (mono-) GS-13 (mono-) GS-14 (di -) GS-15 (tri-) GS-16 (penta-) 3-G1 172.90 3-G2 33.76 3-G3 21.11 3-G4 33.87 3-G5 176.31 2'-Gl 173.21 2'-G2 33.76 2,-G3 21.11 2,-G4 33.93 2'-G5 176.45

22 1295994 3,-Gl 174.56 174.35 173.38 173.61 3,-G2 33.88 34.04 33.84 33.81 3,-G3 21.36 21.36 21.13 21.16 3'-G4 34.31 34.04 33.96 33.93 3,-G5 176.98 176.69 176.60 176.51 4,-Gl 174.05 174.13 4. -G2 33.92 33.83 4,-G3 21.23 21.18 4,-G4 33.96 33.99 4'-G5 176.67 176.63 6,-Gl 174.53 174.45 174.12 174.58 6,-G2 33.97 34.04 33.92 33.87 6,-G3 21.30 21.39 21.23 21.52 6,-G4 34.05 34.28 34.05 34.57 6,-G5 176.70 177.09 176.77 176.63

Control test: CK anticancer activity analysis used human ovarian cancer cell line OVCAR-3 (Adenocarcinoma, ovary, human), human lung cancer cell line A549 (Carcinoma, lung, human), human rectal cancer cell line HT-29 (Adenocarcinoma, Colon, moderately well-differentiated grade II, human), and human breast cancer cell line MCF-7 (Breast adenocarcinoma, pleural effusion, human). Cell line sources were obtained from the American Type Culture Collection (ATCC) organization and were numbered ATCC HTB-161, ATCC CCL-185, ATC HTB-38, and ATCC HTB-22, respectively. Ovarian cancer cell lines were cultured in RPMI 1640 medium containing 20% fetal bovine serum, and supplemented with 1 mg bovine insulin and 1% Antimycotic antibiotic per ml. The lung cancer cell line was cultured in a F-12K nutrient mixture (Kaighn's Modification) containing 10% fetal bovine serum. The rectal cancer cell line was cultured in MeCo/s 5A medium containing 10% fetal bovine 23 1295994 serum. The breast cancer cell line was cultured in Minimum Essential Medium containing 10% fetal bovine serum. CK is first mixed with ethanol and Cremophor RH40 (ethanol:

Dissolve Cremophor RH40 = 1:1), dilute with steam to CK concentration of 10000, 1000, 100, 10, 1 pg/m and reduce the concentration of ethanol and Cremophor RH40 to 5% 〇

A 100 μΐ cell suspension (containing approximately 1·0 to 3.0 X 1〇3 cells) was added to a 96-well microtiter plate at 37. <: Incubate in air containing 5% CO 2 , after 24 hours, add 100 μΐ growth medium and 2 μΐ different concentrations of CK solution, or 2 μΐ carrier (ie 5% ethanol and 5% Cremophor RH40), so the final ethanol The concentration of Cremophor RH40 was 0.05%, and the concentration of CK was 100, 10, 1, 0.1, 0·01 pg/ml. Perform 2 repetitions and continue to culture for 72 hours. At the end of the culture, 20 μL of 90% alamarBlue reagent was added to each well for another 6 hours, and then the fluorescence intensity (detection at 53 0 nm and absorption at 590 nm) was detected to know the survival of the cells. φ ability. Test Example 2: Anticancer activity analysis of CK succinate and CK glutarate derivative The test was carried out in the same manner as the cancer cell culture solution of Comparative Test Example 1. CK shotate acid ester and CK glutarate derivative are directly dissolved in pH 7.4 Phosphate Buffered Saline (PBS), and then diluted with distilled water to a concentration of CK derivative of 2000, 200, 20, 2 , 0.2 pg/ml 〇24 1295994 Take a 100 μΐ cell suspension (about 5·0 x 1 〇3 cells) and add it to a 96-well microtiter plate and incubate in 37°c 5% C〇2 air. After 24 hours, add 90μ1 growth medium and different concentrations of ck succinate or CK glutarate derivative solution, or 10 μl carrier (ie PBS with pH 7-4), so the final CK succinic acid The ester or CK-glutaric acid vinegar derivative was tested at a concentration of 100, 10, 1, 0.1, 〇·〇1 pg/ml; 2 replicates were carried out and incubation was continued for 72 hours. At the end of the culture, 2〇μ1 of 9〇% alamarBlue reagent was added to each well for another 6 hours, and then the fluorescence intensity (detection at 530 nm and 590 nm absorption light detection) was detected to know the cells. Survival ability. Percent Growth (PG) is calculated according to the following formula: PG(%) = 100% X (Mean Ftest - Mean Ftime〇) / (Mean Fctrl - Mean Ftime〇) If (Mean Ftest - Mean Ftime0) &lt ; 〇, 贝!] PG(%) = 100〇/〇x (Mean Ftest - Mean Ftime0) / (Mean Ftime〇- Mean Fblank)

Mean Ftime() is the two average values of the fluorescence intensity detected by the alamarBlue reagent in the cell suspension before the test article is to be added;

Mean Ftest detected two average values of fluorescence intensity for the alamarBlue reagent 72 hours after the cell suspension was added to the test substance;

Mean Fetrl detected two average values of fluorescence intensity for the alamarBlue reagent after 72 hours of cell suspension without addition of the test substance;

The average of the fluorescence intensity was detected by Mean Fblank's alamarBlue reagent after 72 hours of cell suspension without cell.

25 1295994 50. /. Inhibitory concentration dcy is defined as the concentration of a test compound at which the increase in the number or quality of cells tested from the time of the test to the end of the test period is the blank test without the test compound. The ICw plots the concentration of the test compound against the viability of the test cells and is obtained via non-linear regression. Table 5 below lists the ic5 values of CK succinate and glutaric acid ester derivatives purified by HPLC from the above Examples 丨 to 3 for cancer cell lines. Table 2, IC5G (μΜ) of CK, CK alkanoate and CK glutarate GS-12 (CK) GS-6 GS-8 GS-9 GS-11 GS-13 GS-14 OVCAR-3 MCF -7 HT-29 A549 2.7μΜ 11·2μΜ 9.3 μΜ 17.7μΜ 22.1μΜ 19·3μΜ 53.9μΜ 71.9μΜ 31·8μΜ 89.9μΜ >100μΜ 69.1μΜ VJ kJ IT 79.8μΜ In addition, the inventors of the present invention will use the above examples 1 to 3. The HPLC-purified CK succinate and glutarate derivatives were dissolved in PBS for solubility testing and found to have a solubility of greater than 1 〇 mg/ml in PBS. While the invention has been described above, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention. 26

Claims (1)

1295994 X. Patent application scope: 1. A 20-Ο-β-ϋ-glucopyranosyl·20(8)_protoldiol derivative having the following formula, or a pharmaceutically acceptable salt thereof: Where R1 = Η or -C(0)(CR4R9)nC00H ; R2 = Η or -C(0)(CR4R9)nC00H ; R3 = Wherein R4 and R9 are independently Η or C1-C4 alkyl; η is an integer from 2 to 6; R5, R6, R7 and R8 are independently Η, or -C(0)(CR4R9)nC00H, wherein R4 and η The definition is the same as above; where R1, R2, R5, R6, R7 and R8 are not the same. 2. A derivative according to claim 1 wherein R4 and R9 are hydrogen and η is 2 or 3. 27 1295994 3. The derivative of claim 2, which is Η. 4. If the derivative of the third paragraph of the patent application is in the form of a derivative of the third paragraph of the patent application, wherein y is -C(0)(CH2)nC00H, and R6, R? R8 is either % or 3. 8:11 is 2 6. For the derivative of the third paragraph of the patent application, wherein -C(0)(CH2)nC00H, and R5, r6 and R8 are both & Tanjung η is 2 7·If applying for a patent The derivative of the third item, wherein Han 5 and y κ are -C(0)(CH2)nCOOH, and r^r8 are both H, and the average of them is 2 or 3. 8. A derivative according to item 3 of the patent application, wherein r5 r6 岣 is _C(0)(CH2)nC〇〇H, and R« is η, wherein n is 2 or 3 and 11 9. The derivative of the fourth item, wherein r5 is -C(0)(CH2)nC00H, and R6, r7 and r8 are homogeneous, and -N is: 10. A derivative according to claim 4, wherein Κ 28 1295994 〇 (dianhydride) (CK) where Glc is OH, R4 and R9 independently A is -(CR4R9)n_, wherein n is an integer of 2-6 which is Η or C1-C4 alkyl. 16. The method of claim 15, wherein R4 and R, rat, and η are 2 or 3. 17. The method of claim 15, wherein the organic solvent is a halogenated alkane, an ether, a tertiary amine, a low alkyl sulfonium, or a low alkyl ketone. 18. The method of claim 17, wherein the halogenated alkane is dichloromethane, trioxane or dichloroethene; the ether is dialkyl ether, cycloaliphatic Alicyclic ether, Tetrahydrofuran, Dioxane; the tertiary amine is 1299994 - Triethylamine, ° ratio (Pyridine), N, N, N' , N'_ N, N, N', N'-tetramethyl ethylenediamine, or 'N, N, N', N'-tetramethyldiamine decane • (N , N, N', N'-tetramethyldiaminomethane)); the low-burning kibbles are, for example, Dimethyl sulfoxide (DMF). 19. The method of claim 15, wherein the base is a liquid test or a solid phase test. 20. The method of claim 19, wherein the base is a liquid base and the liquid base is a tertiary amine. 2. The method of claim 20, wherein the organic solvent is a tertiary amine together with the base. Φ 22. The method of claim 21, wherein the tertiary amine is triethylamine, 4-dimethylaminopyridine, β, N, N, N', N'-tetradecylethylidene, or N,N,N,N*·tetradecyldiaminedecane. An anti-cancer pharmaceutical composition comprising the derivative according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, as an active ingredient. 31