CN114656435A

CN114656435A - Rockmilan alcohol hydroxyl derivative, preparation method and application thereof

Info

Publication number: CN114656435A
Application number: CN202210148323.XA
Authority: CN
Inventors: 苑春茂; 郝小江; 李亚男; 邱洁; 李艳梅; 杨珏; 胡占兴; 顾玮; 黄烈军
Original assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Current assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Priority date: 2022-02-17
Filing date: 2022-02-17
Publication date: 2022-06-24

Abstract

The invention discloses a loxagliflorin alcohol hydroxyl derivative, a preparation method and application thereof, wherein the loxagliflorin alcohol hydroxyl derivative has a structural formula (I), wherein: r is a benzoate group, a p-fluorobenzoate group, a p-methoxybenzoate group, a p-bromobenzoate group, a 4, 5-dimethoxy-2-nitro-benzoate group, a p-nitrocinnamate group, a 2-furoate group, a 2-picolinate group, a 6-chloronicotinate group, a butyrate group, a decanoate group, a dimethylamino group, a diethylamino group, a dibutylamino group, a dioctylamino group, a benzylamino group, a 3, 4-dimethoxyphenethylamino group, a 1-tetrahydropyrrolyl group, a 1-imidazolyl group, a 1-pyrazolyl group, a 1-morpholinyl group, a 1-piperidinyl group or a 4-methylpiperazin-1-yl group. The invention has nanomolar anti-colorectal cancerAnti-leukemia and anti-breast cancer activity.

Description

Rockmilan alcohol hydroxyl derivative, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a loxagliflorin alcohol hydroxyl derivative, a preparation method of the loxagliflorin alcohol hydroxyl derivative, and application of the loxagliflorin alcohol hydroxyl derivative in preparation of antitumor medicines.

Background

Cancer or tumor is a common and frequently occurring disease that seriously threatens human health, and the mortality rate has risen to the first according to the world health organization statistics. The treatment method of cancer or tumor mainly comprises three methods of operation, radiation and chemical drug treatment. But currently, chemotherapy is still mainly used clinically to a large extent. The existing chemotherapy drugs are one of the common methods for non-surgical treatment of cancer or tumor, and have the problems of very close effective dose and toxic dose, toxic and side effects and the like, wherein the reactions of impaired digestive function, inhibited bone marrow hematopoiesis function and the like are the most obvious, so that cancer or tumor patients are often difficult to receive chemotherapy or cannot insist on completing the whole course of treatment due to serious reactions. In addition, radiotherapy and chemotherapy are not selective, have serious side effects on normal tissues, and can induce cancer cells to generate drug resistance. The research of the high-efficiency and low-toxicity anticancer drugs is the hot spot and the key point of the current domestic and overseas research.

The Wnt signaling pathway is a complex network of protein action, the function of which is most common in embryonic development and cancer, but is also involved in the normal physiological processes in adult animals. In many malignancies, the Wnt signaling pathway is often in a highly activated state, especially prominent in colorectal, breast and liver cancers. Recent research shows that the classical Wnt/beta-catenin signal pathway plays a crucial role in regulating the self-renewal capacity of tumor cells and maintaining the dryness of the tumor cells. The discovery of drugs targeting Wnt signaling pathway anti-colorectal cancer drugs has been a hot spot.

Chinese patent publication No. CN113149942A discloses a rocagliflozin alcohol phenolic hydroxyl derivative and a preparation method thereof in 2021, 7.23.A structure of the rocagliflozin alcohol phenolic hydroxyl derivative is a rocagliflozin alcohol phenolic hydroxyl derivative, and a main action mechanism approach is a MAPK signal pathway inhibitor.

Disclosure of Invention

The object of the present invention is to overcome the above drawbacks and to provide a hydroxy derivative of loxaglucol having nanomolar activity against colorectal, leukemic and breast cancers.

The invention also aims to provide a preparation method of the rocagliflorin alcohol hydroxyl derivative.

The invention also aims to provide the application of the rocagliptin hydroxyl derivative in preparing medicaments for resisting colorectal cancer, leukemia, MAPK signal pathway and Wnt signal pathway inhibitors.

The purpose of the invention is realized by the following technical method:

the invention relates to a rocagliflorin alcohol hydroxyl derivative, which has the following structural formula (I):

wherein: r is methyl, ethyl, propyl, butyl, pentyl, hexyl, 5-bromopentyl, allyl, isopentenyl, propargyl, 2-butynyl, acetyl, propionyl, valeryl, benzoyl, p-nitrobenzoyl, ethylsulfonyl, propylsulfonyl or phenylsulfonyl.

The invention relates to a preparation method of a rocagliflorin alcohol hydroxyl derivative, which comprises the following steps:

(1) synthesis of Compound 3

20g of apigenin (compound 1, 0.0740mol) is weighed into a 500ml round bottom flask, 51g of anhydrous potassium carbonate (5.0eq) and 250ml of acetone are added into the flask as solvents, 24.5ml of dimethyl sulfate (3.5eq) is slowly added with stirring, and the system is put into an oil bath kettle and heated under reflux at 70 ℃ for 72 h. The reaction was followed by TLC, and after completion of the reaction, it was cooled to room temperature and adjusted to PH 11 with ammonia. Filtering to remove precipitate, washing the filtrate with saturated saline solution, drying with anhydrous sodium sulfate, spin-drying part of the solvent, adding silica gel, stirring, and purifying with flash silica gel column (chloroform: acetone ═ 8:2) to obtain light yellow solid compound;

700mg of Compound 2 was weighed, a mixed solvent of 80mL of dichloromethane and 60mL of acetone was added, and 140mL of an aqueous solution of potassium hydrogen monosulfate complex salt having a concentration of 11.6g/mL was slowly added to obtain Compound 3, according to the following reaction scheme:

(2) synthesis of mixtures of

Compounds

4 and 5

800mg of compound 3(2.44mmol) is weighed, 40ml of acetonitrile, 30ml of methanol and 12.6 equivalents of methyl trans-cinnamate 5.0g are added, and the mixture is irradiated by strong light with a xenon lamp and reacted for 17 hours to obtain a mixture of

product compounds

4 and 5, wherein the reaction route is as follows:

(3) synthesis of Compound 6

Weighing 1g of the mixture of the

compounds

4 and 5, adding 30mL of methanol and 10mL of 0.5M sodium methoxide methanol solution, and refluxing at 70 ℃ for 4h to obtain a compound 6, wherein the reaction route is as follows:

(4) synthesis of Compound 7

Weighing 2g of compound 6, adding 100mL of DMSO and 170mg of lithium chloride, and stirring at 100 ℃ for 8h to obtain compound 7, wherein the reaction route is as follows:

(5) synthesis of Compound 8

3.27g of sodium triacetoxyborohydride is weighed into 200mL of acetonitrile and 1.2mL of glacial acetic acid, then 700mg of Compound 7 is slowly added and stirred at 40 ℃ for 8h to obtain Compound 8, the reaction scheme is as follows:

(6) synthesis of Compound 9

30mg of Compound 8(0.069mmol) are weighed into a 25ml round-bottom flask, 2ml DCM are added as solvent, 28.7. mu.l Et are added₃N (0.207mmol), 11. mu.l chloroacetyl chloride (0.138mmol) and a catalytic amount of DMAP were stirred at room temperature for 10h to give compound 9, of the formula:

(7) synthesis of Compounds 10a to 10w

30mg of 1-chloroacetyl-4' -demethoxy-clotrimiol (9,0.059mmol) were weighed out, 5ml of DMF solution, 16.3mg of potassium carbonate (0.118mmol), 1.5 equivalents of acid (0.085mmol) were added, and the mixture was heated in a constant temperature oil bath at 70 ℃ for 12h to obtain the series of derivatives 10a to 10w, as follows:

the invention relates to an application of a rocomilanol hydroxyl derivative in preparing medicaments for resisting colorectal cancer, leukemia and breast cancer.

The invention relates to an application of a loxagliflorin hydroxyl derivative in preparation of MAPK signal channel and Wnt signal channel inhibitor drugs.

Compared with the prior art, the invention has obvious beneficial effects, and the technical scheme can show that: the invention takes apigenin as raw material, generates a compound 2 under the action of dimethyl sulfate, the compound is oxidized into a compound 3 under the action of potassium hydrogen peroxymonosulfate composite salt, the compound 3 and methyl cinnamate generate photocatalysis [3+2] reaction under the illumination of xenon lamp to obtain a tautomeric compound 4/5, the compound 4/5 generates similar pinacol rearrangement in a sodium methoxide solution of methanol to obtain a compound 6, the compound 7 removes methoxyl under the action of lithium chloride to obtain a compound 7, the compound 8 is reduced by sodium triacetoxyborohydride to obtain a compound 8, the compound 8 is subjected to chloroacetyl chloride action to obtain an active precursor 1-chloroacetyl clotrimiol (compound 9), and the 1-chloroacetyl clotrimiol (compound 9) is subjected to different acid or amine actions to obtain 1-acid group or amino group substituted clotrimiol derivatives with different structures. The compound has nanomolar activity of resisting colorectal cancer, leukemia and breast cancer, and further induces colorectal apoptosis and cell cycle arrest by inhibiting MAPK and Wnt signal pathways. When used as a medicament, it may be used as such or in the form of a pharmaceutical composition.

Drawings

FIG. 1 is a graph of the effect of Compound 10r on the cycle of HCT116 tumor cell treatment for 48 hours;

FIG. 2 is a graph of the percentage of compound 10r versus the different stages of the cycle of HCT116 tumor cells;

FIG. 3 is a graph of the induction of apoptosis of HCT116 tumor cells by Compound 10 r;

FIG. 4 is a histogram of Compound 10r induced apoptosis in tumor cells;

FIG. 5 Effect of Compound 10r on HCT116 clonal sphere formation

FIG. 6 statistical plot of the number of balls formed by compound 10r versus HCT116 clones

FIG. 7 is a graph of the effect of Compound 10r on related apoptotic proteins;

FIG. 8 is a graph of the effect of Compound 10r on related cyclins;

FIG. 9 is a graph of the effect of Compound 10r on MAPK signaling pathway-associated proteins;

figure 10 is a graph of the effect of compound 10r on Wnt signaling pathway related proteins;

the specific implementation mode is as follows:

the following examples are provided to further illustrate the essence of the present invention, but are not intended to limit the present invention.

Example 1: a process for the preparation of a target compound 10a comprising the steps of:

(1) synthesis of Compound 3

20g of apigenin (compound 1, 0.0740mol) is weighed into a 500ml round bottom flask, 51g of anhydrous potassium carbonate (5.0eq) and 250ml of acetone are added into the flask as solvents, 24.5ml of dimethyl sulfate (3.5eq) is slowly added with stirring, and the system is put into an oil bath kettle and heated under reflux at 70 ℃ for 72 h. The reaction was followed by TLC, and after completion of the reaction, it was cooled to room temperature and adjusted to PH 11 with ammonia. The precipitate was removed by filtration, and the filtrate was washed with a saturated aqueous solution of sodium chloride, dried over anhydrous sodium sulfate, and then a part of the solvent was dried, and a silica gel was added to stir the sample, followed by purification using a flash silica gel column (chloroform: acetone ═ 8:2) to obtain compound 2(12g) as a pale yellow solid in 52.3% yield.

700mg of Compound 2(2.24mmol) was weighed, and the mixture was charged into a 1000mL round-bottomed flask, 80mL of methylene chloride and 60mL of acetone were added, respectively, and the mixture was stirred and dissolved in a room-temperature stirrer, followed by preparing 200mL of a buffer (16g of sodium carbonate, 7.6g of sodium bicarbonate, and 200mL of water), adding the prepared buffer to the round-bottomed flask, and stirring for 30 minutes. 12g of potassium monopersulfate complex salt was weighed and dissolved by ultrasonic wave in 140ml of water. A dropping funnel was placed on the round bottom flask, and 140mL of saline solution was added and slowly added dropwise, about 5-7 seconds per drop. After the salt solution is added dropwise, detecting the reaction solution by using a pH test paper to ensure that the pH of the reaction system is 9. Stirring was carried out overnight and monitored by thin layer silica gel chromatography. And continuously preparing 140mL of potassium monopersulfate composite salt solution, repeatedly dropwise adding, detecting the reaction solution by using pH test paper after the dropwise adding of the salt solution is finished, and adjusting the pH to 9 by using saturated sodium carbonate aqueous solution. Stirring was again carried out overnight and monitored by thin layer silica gel chromatography. The previous experimental steps were repeated. After that, the room temperature stirrer was turned off, and the mixture was left to stand for about 30 min. The dichloromethane layer was separated and the organic layer was concentrated to give a small portion of concentrated solution, which was added p-toluenesulfonic acid monohydrate to adjust pH to 3 and stirred at room temperature for 2 h. Monitoring by thin-layer silica gel chromatography, and then carrying out sample mixing. Using chloroform: methanol 50: 1, flash normal phase silica gel column purification gave compound 3(350mg) as a yellow solid in 50% yield.

(2) Synthesis of mixtures of

Compounds

4 and 5

Synthesis of a mixture of compounds 4 and 5: 800mg of Compound 3(2.44mmol) was weighed into a 250mL round-bottom flask, and dissolved by adding 40mL of acetonitrile and 30mL of methanol with stirring. Then, 5.0g of trans-methyl cinnamate was added thereto, and the mixture was dissolved with stirring at room temperature. The flask was degassed with argon (Ar) using a diaphragm pump and purged several times to ensure that the round-bottom flask was completely filled with argon. The round-bottom flask was placed in a low temperature constant temperature stirrer at 0 ℃ for stirring. Then, the reaction was performed by intense light irradiation using a xenon lamp. Stirring at 0 deg.C for 17-20h, detecting with thin layer silica gel chromatography, and reacting to obtain the final product. The solvent was concentrated to dryness using a rotary evaporator under water pump reduced pressure to give a mixture of

compounds

4 and 5, which was used directly in the next step.

(3) Synthesis of Compound 6

Weighing 1g of the mixture of the compound 4 and the compound 5 after the reaction in the previous step, adding 30mL of methanol for dissolving, stirring at room temperature, slowly adding 10mL of 0.5M sodium methoxide methanol solution, placing the system at 70 ℃, and carrying out oil bath reflux for 4 h. The reaction was monitored by thin layer silica gel chromatography, after completion of the reaction, the system was cooled to room temperature, 20mL of saturated ammonium chloride solution was added to quench the reaction, and 100mL of water was added. Extracted 3 times with ethyl acetate, washed with saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate. Then, the product is purified by flash column chromatography. Using chloroform: elution with a mixed solvent of acetone 9:1 gave compound 6 as a brown oil in 920mg weight 76.9% yield.

(4) Synthesis of Compound 7

2g of Compound 6(0.0035mol) was weighed into a 250mL round-bottomed flask and dissolved in 100mL of DMSO. 170mg of lithium chloride (1.1 eq.) was then added, followed by 2mL of water and stirring at room temperature for 30min, after which the round bottom flask was placed in a constant temperature oil bath at 100 ℃ and heated overnight. And monitoring the reaction by using a thin-layer silica gel chromatography until the reaction is finished. The reaction system was cooled to room temperature, 100mL of water was added, extraction was performed with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated to obtain an organic layer. Using petroleum ether: ethyl acetate 7: 3 was subjected to flash normal phase column chromatography to give compound 7(820mg) as a yellow solid in 46.1% yield.

(5) Synthesis of Compound 8

3.27g of sodium triacetoxyborohydride (9.0 equiv.) are weighed into a 250mL round-bottom flask, 200mL of acetonitrile and 1.2mL of glacial acetic acid are added, and the mixture is stirred at room temperature for 20 min. 700mg of Compound 7(1.38mmol) was dissolved in 90mL of acetonitrile, and then slowly added dropwise to the reaction system, and after stirring for 30min, the round-bottom flask was placed in a constant-temperature oil bath at 40 ℃ and heated. Monitoring by thin-layer silica gel chromatography, and finishing the reaction. Adding 30mL of saturated ammonium chloride solution into a reaction system to quench the reaction, adding 100mL of water, extracting with ethyl acetate, washing with saturated sodium chloride solution, drying with anhydrous sodium sulfate, and concentrating to obtain a reaction product. Using petroleum ether: ethyl acetate ═ 1: normal phase column chromatography was performed on the mixed solvent of 0.5 to obtain compound 8(420mg) as a white solid with a yield of 69.2%.

Loxagliflonol (8). white solid; melting point 136- & ltSUB & gt 137- & lt/SUB & gt¹H NMR(400MHz,CDCl₃)δ: 7.13–7.07(5H,m),6.99(2H,d,J＝7.1Hz),6.69–6.66(2H,m),6.29(1H,d,J＝1.9 Hz),6.14(1H,d,J＝1.9Hz),4.81(1H,d,J＝6.3Hz),4.00(1H,dd,J＝14.1,6.5Hz), 3.90(3H,s),3.84(3H,s),3.71(3H,s),3.30(1H,s),2.74(1H,td,J＝14.0,6.5Hz), 2.20(1H,dd,J＝14.0,6.9Hz),1.72(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:163.9, 161.0,158.6,157.0,138.7,128.9,128.9,128.1,128.1,127.6,127.6,126.8,126.2, 112.7,107.7,103.5,94.8,92.4,89.4,79.0,55.8,55.6,55.0,53.2,36.4,30.9；ESIMS m/z 457.0[M+Na]⁺；HRESIMS m/z 457.1613[M+Na]⁺(Calcd.for C₂₆H₂₆O₆Na, 457.1622).

(6) Synthesis of Compound 9

Synthesis of compound 9: 30mg of Compound 8(0.065mmol) was weighed into a 25ml round-bottom flask, 2ml of DCM was added as a solvent, and the sample was dissolved with stirring at room temperature. Add 28.7. mu.l Et₃N (0.207mmol), 11. mu.l chloroacetyl chloride (0.138mmol) and a catalytic amount of DMAP were stirred at room temperature for 10h and the reaction was followed by TLC plates until the starting material spot disappeared. Then, 5ml of water was added to quench the reaction, the DCM layer was separated, the aqueous layer was extracted twice more with 5ml of DCM, the DCM layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was dried by spin-drying, and flash column chromatography was performed using silica gel (petroleum ether: ethyl acetate ═ 6:4) to give compound 9(23.5mg) as a white solid with a yield of 69.5%.

1-chloroacetyl-locomitol (9), white solid; melting point 173-174 ℃;¹H NMR(400MHz, CDCl₃)δ:7.15–7.12(2H,m),7.10–7.08(5H,m),6.64(2H,d,J＝8.9Hz),6.23(1H, d,J＝1.9Hz),6.05(1H,d,J＝1.9Hz),5.94(1H,dd,J＝5.5,1.5Hz),4.06(1H,dd,J ＝13.7,6.2Hz),3.89(1H,d,J＝15.1Hz),3.84(3H,s),3.78(3H,s),3.74(1H,d,J＝ 15.1Hz),3.68(3H,s),2.88(1H,td,J＝13.8,5.4Hz),2.33(1H,ddd,J＝13.8,6.2, 1.6Hz),2.15(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:166.0,163.8,160.9,158.6, 157.9,138.2,128.7,128.7,128.0,128.0,127.9,127.9,126.9,126.4,112.7,106.4, 103.0,93.3,91.8,88.4,80.9,55.6,55.4,55.0,53.7,40.9,35.3,30.9；ESIMS m/z 533.0[M+Na]⁺；HRESIMS m/z 533.1330[M+Na]⁺(Calcd.for C₂₈H₂₇O₇ClNa, 533.1338).

(7) synthesis of Compound 10a

30mg of compound 9(0.0557mmol) are weighed into a 25ml round-bottomed flask, 2ml of (N, N-dimethylformamide) DMF is added as solvent, then 10.18mg of benzoic acid (NaHCO 1.5eq), NaHCO are added₃7mg (1.5eq), proper amount of CsF, then placing the system into a 75 ℃ oil bath and stirring for reaction for 2 hours, detecting the reaction by a TLC point plate, and stopping the reaction until the raw material point disappears. Then, 5ml of water was added to quench the reaction, DCM layer was separated, the aqueous layer was extracted twice with 5ml of ethyl acetate, the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, the solvent was dried by spin-drying, and flash column chromatography was performed using silica gel (petroleum ether: ethyl acetate: 6:4) to give 19.3mg of a white solid as the objective compound 10a, 48.7% yield.

1- (benzoyl-2-hydroxyacetyl) -rocomiranol (10a) as a white solid; melting point 137-138 ℃;¹H NMR(400MHz,CDCl₃)δ:7.99(2H,dd,J＝8.6,1.1Hz),7.58(1H,t,J＝7.4 Hz),7.44(2H,t,J＝7.8Hz),7.15-7.06(7H,m),6.64(2H,d,J＝9.0Hz),6.14(1H,d, J＝1.9Hz),6.05(1H,d,J＝1.9Hz),5.95(1H,dd,J＝5.3,1.2Hz),4.70(1H,d,J＝ 16.0Hz),4.61(1H,d,J＝16.0Hz),4.02(1H,dd,J＝13.8,6.1Hz),3.82(3H,s),3.79 (3H,s),3.68(3H,s),2.88(1H,td,J＝13.8,5.3Hz),2.36(1H,ddd,J＝13.8,6.2,1.4 Hz),2.12(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:166.6,165.7,163.7,160.7,158.6, 157.9,138.3,133.3,129.9,129.1,128.7,128.7,128.4,128.4,128.0,128.0,127.8, 127.8,127.0,126.3,112.7,106.5,102.9,93.3,91.8,88.3,80.1,60.9,55.5,55.5,55.5, 55.0,53.8,35.5,35.5；ESIMS m/z 619.2[M+Na]⁺；HRESIMS m/z 631.1740[M+Cl]^- (Calcd.for C₃₅H₃₂O₉Cl,631.1729).

example 2: preparation of target Compound 10b

The procedure is as in example 1 except that p-fluorobenzoic acid is substituted for benzoic acid to give the desired compound 10b in 58.9% yield.

1- (p-fluorobenzoyl-2-hydroxyacetyl) -rocomilanol (10b) as a white solid; melting point 159-160 ℃;¹H NMR(400MHz,CDCl₃)δ:8.03–7.96(2H,m),7.16–7.05(9H,m),6.64(2H, d,J＝9.0Hz),6.13(1H,d,J＝1.9Hz),6.04(1H,d,J＝1.9Hz),5.94(1H,d,J＝4.0 Hz),4.69(1H,d,J＝16.0Hz),4.60(1H,d,J＝16.0Hz),4.00(1H,dd,J＝13.9,6.1 Hz),3.82(3H,s),3.81(3H,s),3.68(3H,s),2.88(1H,td,J＝13.9,5.3Hz),2.35(1H, ddd,J＝13.8,6.1,1.3Hz),2.10(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:166.5,164.7, 163.7,160.7,158.6,157.9,138.2,132.5,132.4,128.7,128.7,128.0,128.0,127.8, 127.8,127.0,126.3,125.4,115.6,115.5,112.7,106.5,103.0,93.2,91.9,88.2,80.2, 60.9,55.5,55.5,55.4,55.0,53.7,35.5,35.5；ESIMS m/z 637.3[M+Na]⁺；HRESIMS m/z 649.1646[M+Cl]^-(Calcd.for C₃₅H₃₁O₉ClF,649.1635).

example 3: preparation of target Compound 10c

The procedure is as in example 1 except that p-methoxybenzoic acid is used instead of benzoic acid to obtain the desired compound 10c in 55.9% yield.

1- (p-methoxybenzoyl-2-hydroxyacetyl) -loklonol (10c) as a white solid; the melting point is 71-72 ℃;¹H NMR(400MHz,CDCl₃)δ:7.94(2H,d,J＝8.6Hz),7.14–7.06(7H,m), 6.90(2H,d,J＝8.6Hz),6.64(2H,d,J＝8.6Hz),6.15(1H,d,J＝1.9Hz),6.05(1H, d,J＝1.9Hz),5.94(1H,dd,J＝5.1,1.4Hz),4.67(1H,d,J＝16.0Hz),4.57(1H,d,J ＝16.0Hz),4.01(1H,dd,J＝13.7,6.1Hz),3.87(3H,s),3.82(3H,s),3.80(3H,s), 3.68(3H,s),2.87(1H,td,J＝13.5,5.3Hz),2.36(1H,ddd,13.8,6.4,1.7Hz),2.12 (1H,s)；¹³C NMR(100MHz,CDCl₃)δ:166.8,165.4,163.7,163.6,160.7,158.6, 157.9,138.3,131.9,128.7,128.7,128.0,128.0,127.8,127.8,127.0,127.0,126.3, 121.5,113.6,112.7,106.6,103.0,93.2,91.8,88.3,80.1,60.7,55.5,55.5,55.5,55.4, 55.0,53.7,35.5,35.5；ESIMS m/z 649.3[M+Na]⁺；HRESIMS m/z 661.1845[M +Cl]^-(Calcd.for C₃₆H₃₄O₁₀Cl,661.1835).

example 4: preparation of target Compound 10d

The procedure is as in example 1 except that the benzoic acid is replaced with p-bromobenzoic acid to give the title compound 10d in 58.5% yield.

1- (p-bromobenzoyl-2-hydroxyacetyl) -rocomilanol (10d), white solid; melting point 134-;¹H NMR(400MHz,CDCl₃)δ:7.82(2H,d,J＝8.2Hz),7.52(2H,d,J＝8.2 Hz),7.15(2H,t,J＝7.4Hz),7.10-7.05(5H,m),6.65(2H,d,J＝8.7Hz),6.11(1H,d, J＝1.9Hz),6.02(1H,d,J＝1.9Hz),5.93(1H,dd,J＝5.1,1.2Hz),4.69(1H,d,J＝ 16.0Hz),4.60(1H,d,J＝16.0Hz),3.98(1H,dd,J＝13.9,6.1Hz),3.81(3H,s),3.79 (3H,s),3.68(3H,s),2.88(1H,td,J＝13.7,5.3Hz),2.35(1H,ddd,J＝13.6,6.2,1.7 Hz),2.11(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:166.3,164.9,163.7,160.7,158.6, 157.9,138.2,131.7,131.3,128.7,128.7,128.5,128.5,128.0,128.0,128.0,128.0, 127.9,127.9,127.0,127.0,126.4,112.7,106.5,102.9,93.2,91.9,88.1,80.2,61.0, 55.5,55.5,55.4,55.0,53.7,35.5；ESIMS m/z 697.2[M+Na]⁺；HRESIMS m/z 697.1050[M+Na]⁺(Calcd.for C₃₅H₃₁O₉BrNa,697.1044).

example 5: preparation of target Compound 10e

The procedure is as in example 1 except that benzoic acid is replaced with 4, 5-dimethoxy-2-nitro-benzoic acid to give the title compound 10e in 58.8% yield.

1- (4, 5-dimethoxy-2-nitro-benzoic acid-2-hydroxyacetyl) -rocomilanol (10e).A white solid; melting point 145-146 deg.C;¹H NMR(400MHz,CDCl₃)δ:7.45(1H,s),7.15–7.07(8H,m), 6.64(2H,d,J＝8.8Hz),6.16(1H,d,J＝1.9Hz),6.01(1H,d,J＝1.9Hz),5.96(1H, d,J＝4.0Hz),4.66(1H,d,J＝16.0Hz),4.58(1H,d,J＝16.0Hz),4.04(1H,dd,J＝ 13.6,6.2Hz),3.98(3H,s),3.97(3H,s),3.80(3H,s),3.78(3H,s),3.68(3H,s),2.89 (1H,td,J＝13.4,5.8Hz),2.37(1H,ddd,J＝14.1,6.1,1.7Hz),2.13(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:166.1,164.7,163.7,160.6,158.5,157.9,152.4,150.6, 141.0,138.1,128.7,128.7,127.9,127.9,127.8,127.8,126.9,126.3,120.5,112.7, 111.0,106.9,106.4,102.9,93.2,91.6,88.3,80.3,61.6,56.6,56.5,56.5,55.5,55.4, 55.0,53.7,35.4；ESIMS m/z 724.2[M+Na]⁺；HRESIMS m/z 724.2010[M+Na]⁺ (Calcd.for C₃₇H₃₅O₁₃NNa,724.2000).

example 6: preparation of target Compound 10f

The procedure is as in example 1, where benzoic acid is replaced by p-nitro cinnamic acid to prepare the target compound 10f with a yield of 54.8%.

1- (p-nitrocinnamoyl-2-hydroxyacetyl) -rocomiranol (10f) as a white solid; melting point 110-;¹H NMR(400MHz,CDCl₃)δ:8.26(2H,d,J＝8.8Hz),7.73–7.65(3H,m), 7.15–7.06(7H,m),6.65(2H,t,J＝6.0Hz),6.53(1H,d,J＝16.1Hz),6.17(1H,d,J ＝1.9Hz),6.08(1H,d,J＝1.9Hz),5.93(1H,d,J＝4.0Hz),4.60(1H,d,J＝16.0Hz), 4.42(1H,d,J＝16.0Hz),4.03(1H,dd,J＝13.9,6.1Hz),3.83(3H,s),3.77(3H,s), 3.68(3H,s),2.89(1H,td,J＝14.0,5.3Hz),2.37(1H,ddd,J＝14.1,6.1,1.7Hz), 2.11(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:166.3,165.0,163.7,160.8,158.6,158.0, 148.6,142.9,140.2,138.2,128.8,128.8,128.7,128.7,127.9,127.9,127.9,126.9, 126.4,124.1,121.0,112.7,106.6,103.0,93.2,91.8,88.2,77.2,77.0,76.7,60.8,55.5, 55.4,55.0,55.0,53.7,35.5；ESIMS m/z 690.2[M+Na]⁺；HRESIMS m/z 690.1954 [M+Na]⁺(Calcd.for C₃₇H₃₃O₁₁NNa,690.1946)。

example 7: preparation of target Compound 10g

The procedure is as in example 1 except that benzoic acid is replaced with 2-furancarboxylic acid to prepare 10g of the target compound with a yield of 45.7%.

1- (2-furoyl-2-hydroxyacetyl) -rocomilanol (10g) as a white solid; melting point 157-;¹H NMR(400MHz,CDCl₃)δ:7.60(1H,d,J＝0.8Hz),7.20(1H,dd,J＝3.6,0.8 Hz),7.15–7.05(7H,m),6.64(2H,d,J＝9.0Hz),6.52(1H,dd,J＝3.5,1.7Hz),6.17 (1H,d,J＝1.9Hz),6.07(1H,d,J＝1.9Hz),5.93(1H,dd,J＝5.3,1.Hz),4.64(1H,d, J＝16.0Hz),4.55(1H,d,J＝16.0Hz),4.03(1H,dd,J＝13.8,6.1Hz),3.83(3H,s), 3.82(3H,s),3.68(3H,s),2.88(1H,td,J＝13.9,8.6Hz),2.36(1H,ddd,J＝13.8,6.2, 1.4Hz),2.12(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:166.2,163.8,160.7,158.6, 157.9,157.5,146.9,143.6,138.3,128.7,128.0,128.0,127.8,127.0,127.0,126.3, 126.3,119.0,112.7,111.9,106.5,103.0,93.2,91.8,88.2,80.2,60.5,55.5,55.5,55.0, 53.7,35.5,30.9；ESIMS m/z 609.2[M+Na]⁺；HRESIMS m/z 587.1883[M+H]⁺ (Calcd.for C₃₃H₃₁O₁₀,587.1912).

example 8: preparation of target Compound 10h

The procedure is as in example 1, substituting benzoic acid for p-2-picolinic acid to give the title compound in a yield of 59.8% over 10 h.

1- (2-picolinoyl-2-hydroxyacetyl) -rocomilanol (10 h.) as a white solid; the melting point is 79-80 ℃;¹H NMR(400MHz,CDCl₃)δ:8.76(1H,dd,J＝4.7,0.7Hz),8.09(1H,d,J＝7.8Hz), 7.85(1H,t,J＝6.92Hz),7.50(1H,ddd,J＝8.16,4.87,1.36Hz),7.14–7.07(7H,m), 6.64(2H,d,J＝6.1Hz),6.14(1H,d,J＝1.9Hz),6.05(1H,d,J＝1.9Hz),5.94(1H, d,J＝4.0Hz),4.71(1H,d,J＝15.9Hz),4.66(1H,d,J＝15.9Hz),4.05(1H,dd,J＝ 13.9,6.1Hz),3.83(3H,s),3.80(3H,s),3.67(3H,s),2.92(1H,td,J＝14.3,5.3),2.38 (1H,ddd,J＝14.1,6.1,1.7Hz),2.12(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:166.1, 164.1,163.7,160.7,158.5,157.9,149.9,147.0,138.2,136.9,128.7,127.9,127.9, 127.8,127.8,127.2,127.0,126.3,125.5,112.7,106.4,102.9,93.2,91.7,88.2,80.2, 61.4,55.5,55.4,55.00,55.0,53.7,35.5；ESIMS m/z 620.2[M+Na]⁺；HRESIMS m/z 620.1901[M+Na]⁺(Calcd.for C₃₄H₃₁O₉NNa,620.1891).

example 9: preparation of target Compound 10i

The procedure is as in example 1 except that benzoic acid is replaced with 6-chloronicotinic acid to prepare the target compound 10i with a yield of 50.6%.

1- (6-chloronicotinyl-2-hydroxyacetyl) -rocomilanol (10i) as a white solid; melting point is 79-80 ℃;¹H NMR(400MHz,CDCl₃)δ:8.89(1H,dd,J＝2.4,0.6Hz),8.15(1H,dd,J＝8.3, 2.4Hz),7.41(1H,dd,J＝8.3,0.5Hz),7.17–7.05(7H,m),6.63(2H,d,J＝9.0Hz), 6.10(1H,d,J＝1.9Hz),6.00(1H,d,J＝1.9Hz),5.91(1H,d,J＝4.2Hz),4.72(1H, d,J＝16.0Hz),4.63(1H,d,J＝16.0Hz),3.98(1H,dd,J＝14.0,6.1Hz),3.80(3H,s), 3.79(3H,s),3.67(3H,s),2.89(1H,td,J＝14.0,5.2Hz),2.37(1H,dd,J＝14.0,6.1, 1.2Hz),2.08(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:165.9,163.7,163.5,160.7, 158.6,157.8,156.0,151.2,139.6,138.1,128.6,128.6,127.9,127.9,127.8,126.8, 126.8,126.3,124.1,124.0,112.7,106.3,102.9,93.2,91.9,88.0,80.3,61.1,55.5,55.5, 55.4,55.0,53.7,35.5；ESIMS m/z 654.2[M+Na]⁺；HRESIMS m/z 654.1510[M+ Na]⁺(Calcd.for C₃₄H₃₀O₉NClNa,654.1501).

example 10: preparation of target Compound 10j

The procedure was as in example 1 except that benzoic acid was replaced with butyric acid to give the title compound 10j in 52.3% yield.

1- (butyryl-2-hydroxyacetyl) -loklonol (10j) white solid; melting point 114-;¹H NMR(400MHz,CDCl₃)δ:7.15–7.08(7H,m),6.64(2H,d,J＝8.9Hz),6.23(1H,d, J＝1.9Hz),6.07(1H,d,J＝1.9Hz),5.91(1H,dd,J＝5.2,1.3Hz),4.45(1H,d,J＝ 16.0Hz),4.35(1H,d,J＝16.0Hz),4.02(1H,dd,J＝13.7,6.2Hz),3.84(3H,s),3.79 (3H,s),3.68(3H,s),2.86(1H,td,J＝14.7,5.4Hz),2.33(1H,ddd,J＝13.7,7.7,1.3 Hz),2.29(2H,td,J＝7.5,3.0Hz),2.12(1H,s),1.64(2H,dd,J＝14.8,7.4Hz),0.96 (3H,t,J＝7.4Hz)；¹³C NMR(100MHz,CDCl₃)δ:172.7,166.7,163.8,160.8,158.6, 158.0,138.3,128.7,128.7,128.0,128.0,127.8,127.8,127.0,127.0,126.3,112.7, 106.6,103.0,93.2,91.8,88.2,80.1,60.2,55.6,55.4,55.0,53.7,35.5,35.5,18.2,13.6； ESIMS m/z 585.3[M+Na]⁺；HRESIMS m/z 585.2104[M+Na]⁺(Calcd.for C₃₂H₃₄O₉Na,585.2095).

example 11: preparation of target Compound 10k

The procedure is as in example 1 except that benzoic acid is replaced by decanoic acid to afford the title compound 10k in 55.1% yield.

1- (decanoyl-2-hydroxyacetyl) -loklomanol (10 k.) white solid; melting point is 74-75 ℃;¹H NMR(400MHz,CDCl₃)δ:7.14–7.07(7H,m),6.65-6.63(2H,m),6.22(1H,d,J＝ 1.9Hz),6.07(1H,d,J＝1.9Hz),5.91(1H,dd,J＝5.2,1.4Hz),4.44(1H,d,J＝16.0 Hz),4.35(1H,d,J＝16.0Hz),4.02(1H,dd,J＝13.6,6.3Hz),3.84(3H,s),3.79(3H, s),3.68(3H,s),2.86(1H,td,J＝13.7,5.4Hz),2.34–2.28(3H,m),2.11(1H,s),1.66– 1.62(4H,m),1.28(10H,d,J＝14.4Hz),0.88(3H,t,J＝7.0Hz)；¹³C NMR(100 MHz,CDCl₃)δ:172.8,166.7,163.7,160.7,158.6,158.0,138.2,128.7,128.0,128.0, 127.85,127.85,127.02,126.33,112.73,106.61,103.00,93.24,91.84,88.25,80.07, 60.2,55.5,55.4,55.0,53.7,35.4,33.7,33.7,31.8,30.9,30.9,29.3,29.2,29.06,24.7, 22.6,14.1；ESIMS m/z 669.3[M+Na]⁺；HRESIMS m/z 647.3184[M+H]⁺(Calcd. for C₃₈.H₄₇.O₉,647.3215).

example 12: preparation of target Compound 10l

The procedure was as in example 1 except that dimethylamine was used instead of benzoic acid, to obtain 10l of the objective compound in 59.4% yield.

1- (2-methylaminoacetyl) -rocagliflonol (10 l.) as a white solid; melting point 149-150 deg.C;¹H NMR (400MHz,CDCl₃)δ:7.13-7.06(7H,m),6.63(2H,d,J＝8.9Hz),6.23(1H,d,J＝1.9 Hz),6.03(1H,d,J＝1.9Hz),5.86(1H,d,J＝3.7Hz),4.10(1H,dd,J＝13.8,6.1Hz), 3.84(3H,s),3.76(3H,s),3.67(3H,s),3.00(1H,d,J＝16.9Hz),2.86(1H,td,J＝ 13.9,4.9Hz),2.82(1H,d,J＝16.9Hz),2.33(1H,dd,J＝13.7,6.3Hz),2.25(6H,s), 1.77(1H,s)；¹³C NMR(100MHz,CDCl₃)δ169.3,163.7,161.0,158.5,157.9,138.5, 128.7,128.7,128.0,128.0,127.8,127.8,127.2,127.2,126.2,112.6,106.6,103.0,93.3, 91.7,88.2,79.1,59.8,55.6,55.4,55.0,53.8,44.9,44.9,35.7；ESIMS m/z 542.2[M+ Na]⁺；HRESIMS m/z 520.2338[M+H]⁺(Calcd.for C₃₀H₃₄O₇N,520.2329).

example 13: preparation of target Compound 10m

The procedure is as in example 1 except that the benzoic acid is replaced by diethylamine to produce the desired compound in a yield of 56.6% to 10 m.

1- (2 diethylaminoacetyl) -rocagliflonol (10 m.) as a white solid; the melting point is 50-51 ℃;¹H NMR (400MHz,CDCl₃)δ:7.10(7H,m),6.63(2H,d,J＝9.0Hz),6.23(1H,d,J＝1.9Hz), 6.03(1H,d,J＝1.9Hz),5.82(1H,d,J＝3.6Hz),4.14(1H,dd,J＝14.0,6.2Hz), 3.83(3H,s),3.76(3H,s),3.67(3H,s),3.15(1H,d,J＝17.1Hz),3.03(1H,d,J＝ 17.1Hz),2.88(1H,td,J＝13.9,4.9Hz),2.54–2.48(4H,m),2.37(1H,ddd,J＝ 13.4,6.1,1.5Hz),1.25(1H,s),0.96(6H,t,J＝7.2Hz)；¹³C NMR(100MHz,CDCl₃) δ:170.2,163.7,161.1,158.5,158.0,138.6,128.6,128.6,128.0,128.0,127.8,127.8, 127.3,127.3,126.2,126.2,112.6,103.1,93.3,91.7,88.2,79.0,55.6,55.4,55.0,53.8, 53.8,53.6,47.4,35.8,12.4,12.4；ESIMS m/z 548.3[M+H]⁺；HRESIMS m/z 548.2650[M+H]⁺(Calcd.for C₃₂H₃₈O₇N,548.2643).

example 14: preparation of target Compound 10n

The procedure is as in example 1 except that the benzoic acid is replaced by dibutylamine to produce the desired compound 10n in 48.2% yield.

1- (2-dibutylaminoacetyl) -rocagliflonol (10 n.) white solid; the melting point is 39-40 ℃;¹H NMR(400MHz,CDCl₃)δ:7.15-7.13(3H,m),7.10-7.07(4H,m),6.64(2H,dd,J＝ 6.8,2.18Hz),6.22(1H,dd,J＝6.7,2.0Hz),6.05(1H,dd,J＝17.7,1.9Hz),5.97(1H, dd,J＝5.1,1.6Hz),4.45(1H,d,J＝16.2Hz),4.35(1H,d,J＝16.2Hz),4.09(1H,dd, J＝13.8,6.1Hz),3.84(3H,s),3.78(3H,s),3.68(3H,s),3.14(1H,d,J＝16.8Hz), 2.91-2.82(1H,m),2.42(1H,td,J＝4.5,2.3Hz),2.37-2.33(1H,m),2.17(1H,s), 1.53–1.46(2H,m),1.35–1.21(7H,m),0.96–0.85(7H,m)；¹³C NMR(100MHz, CDCl₃)δ:167.6,163.7,163.7,161.1,160.7,158.6,158.5,158.0,128.7,128.6,128.0, 127.8,127.8,126.2,126.2,112.7,103.1,103.0,91.7,88.3,88.1,79.8,61.1,55.5,55.5, 55.4,55.3,55.0,55.0,53.8,53.8,53.7,29.9,20.4,14.0,13.8；ESIMS m/z 604.2[M+ H]⁺；HRESIMS m/z 604.3273[M+H]⁺(Calcd.for C₃₆H₄₆O₇N,604.3269).

example 15: preparation of the target Compound 10o

The procedure is as in example 1 except that the benzoic acid is replaced with dioctylamine to give the title compound 10o in 46.3% yield.

1- (2-dioctylaminoacetyl) -loc-milnacol (10 o.) as a white solid; melting point is 41-42 ℃;¹H NMR(400MHz,CDCl₃)δ:7.14–7.11(4H,m),7.08–7.05(3H,m),6.62(1H,d,J＝ 8.9Hz),6.22(1H,dd,J＝6.1,1.9Hz),6.02(1H,d,J＝8.9Hz),5.82(1H,d,J＝3.8 Hz),4.13(1H,dd,J＝14.0,6.1Hz),3.83(3H,s),3.75(3H,s),3.67(3H,s),3.14(1H, d,J＝17.0Hz),3.04(1H,d,J＝17.0Hz),2.87(1H,td,J＝13.1,6.5H,),2.40(4H,dd, J＝8.1,5.4Hz),2.35(1H,ddd,J＝13.5,6.1,1.0Hz),1.32(5H,dd,J＝14.5,7.4Hz), 1.28–1.22(18H,m),0.87(7H,t,J＝7.1Hz)；¹³C NMR(100MHz,CDCl₃)δ:170.4, 163.7,161.1,158.5,158.0,138.6,128.7,128.0,128.0,127.8,127.3,126.2,112.7, 106.6,103.1,93.4,91.8,88.2,78.9,55.5,55.4,55.0,54.7,54.1,53.8,35.8,35.8,35.8, 31.8,31.8,30.9,30.9,29.5,29.5,29.3,29.3,27.8,27.8,27.3,27.3,22.6,22.6,14.1, 14.1；ESIMS m/z 738.5[M+Na]⁺；HRESIMS m/z 716.4535[M+H]⁺(Calcd.for C₄₄H₆₂O₇N,716.4521).

example 16: preparation of target Compound 10p

The procedure is as in example 1 except that the benzoic acid is replaced with benzylamine to afford the title compound 10p with a yield of 55.6%.

1- (2-benzylaminoacetyl) -rocagliflonol(10p) a white solid; the melting point is 59-60 ℃;¹H NMR (400MHz,CDCl₃)δ:7.31-7.27(4H,m),7.26(1H,s),7.15–7.06(8H,m),6.67–6.62 (2H,m),6.21(1H,d,J＝1.9Hz),5.99(1H,d,J＝1.9Hz),5.88(1H,d,J＝3.6Hz), 4.08(1H,dd,J＝13.7,6.2Hz),3.80(3H,s),3.70(2H,s),3.69(3H,s),3.68(3H,s), 3.27(1H,d,J＝17.5Hz),3.12(1H,d,J＝17.5Hz),2.88(1H,td,J＝13.8,5.2Hz), 2.37–2.30(1H,m)；¹³C NMR(100MHz,CDCl₃)δ:170.9,163.7,160.9,158.5,157.9, 138.4,128.7,128.4,128.3,128.0,128.0,127.8,127.8,127.2,127.1,126.3,112.7, 106.5,103.0,93.3,91.8,88.3,79.6,55.5,55.3,55.3,55.0,53.8,53.1,49.8,49.8, 35.5,35.5,29.3,29.3；ESIMS m/z 604.2[M+Na]⁺；HRESIMS m/z 582.2484[M+ H]⁺(Calcd.for C₃₅H₃₆O₇N,582.2486).

example 17: preparation of target Compound 10q

The procedure is as in example 1 except that 3, 4-dimethoxyphenethylamine was used instead of benzoic acid to prepare the desired compound in an amount of 10q with a yield of 58.4%.

1- (2- (3, 4-dimethoxyphenethylamine) -acetyl) -rocagliflonol (10 q.) white solid; melting point is 54-55 ℃;¹H NMR(400MHz,CDCl₃)δ:7.16-7.15(7H,m),6.77(1H,d,J＝8.2Hz), 6.73–6.71(2H,m),6.65–6.62(2H,m),6.22(1H,d,J＝1.9Hz),6.00(1H,d,J＝1.9 Hz),5.85(1H,dd,J＝4.9,1.4Hz),4.08(1H,dd,J＝13.7,6.2Hz),3.86(3H,s),3.83 (3H,s),3.82(3H,s),3.70(3H,s),3.68(3H,s),3.25(1H,d,J＝17.6Hz),3.09(1H,d, J＝17.6Hz),2.86(1H,td,J＝13.8,5.1Hz),2.80–2.71(2H,m),2.73-2.66(2H,m), 2.33(1H,ddd,J＝13.6,6.2,1.5Hz)；¹³C NMR(100MHz,CDCl₃)δ:171.1,163.7, 160.9,158.6,157.9,148.9,147.4,138.4,132.2,128.7,128.7,128.0,128.0,127.8, 127.8,127.1,126.3,120.4,112.7,111.9,111.3,106.6,103.0,93.3,91.8,88.2,79.5, 55.9,55.8,55.6,55.4,55.4,55.0,53.8,50.8,50.8,36.0,35.6；ESIMS m/z 678.3[M+ Na]⁺；HRESIMS m/z 656.2857[M+H]⁺(Calcd.for C₃₈H₄₂O₉N,656.2854).

example 18: preparation of target Compound 10r

The procedure is as in example 1 except that the benzoic acid is replaced with tetrahydropyrrole to prepare the desired compound 10r in 57.2% yield.

1- (2- (1-tetrahydropyrrolyl) -acetyl) -rocagliflonol (10 r.) a yellow solid; melting point 121-122 ℃;¹H NMR(400MHz,CDCl₃)δ:7.16–7.05(7H,m),6.63(2H,d,J＝8.9Hz),6.23(1H, d,J＝1.9Hz),6.03(1H,d,J＝1.9Hz),5.86(1H,d,J＝3.7Hz),4.12–4.05(1H,m), 3.83(3H,s),3.76(3H,s),3.67(3H,s),3.19(1H,d,J＝17.0Hz),2.99(1H,d,J＝ 17.0Hz),2.86(1H,td,J＝13.8,5.1Hz),2.59–2.47(4H,m),2.33(1H,ddd,J＝13.7, 6.2,1.4Hz),1.80-1.75(3H,m)；¹³C NMR(100MHz,CDCl₃)δ:169.5,163.6,160.9, 158.5,157.9,138.5,128.7,128.7,128.0,128.0,127.8,127.8,127.2,126.2,112.6, 106.7,103.0,93.2,91.7,88.1,79.2,56.2,56.2,55.5,55.5,55.4,55.4,54.9,53.7,53.6, 35.6,35.6,23.7,23.7；ESIMS m/z 546.3[M+H]⁺；HRESIMS m/z 546.2485[M+H]⁺ (Calcd.for C₃₂H₃₆O₇N,546.2486).

example 19: preparation of target Compound 10s

The procedure is as in example 1 except that the benzoic acid is replaced by imidazole to afford the title compound in 10s, 49.3% yield.

1- (2- (1-imidazolyl) -acetyl) -rocagliflonol (10 s.) white solid; melting point 100-101 ℃;¹H NMR(400MHz,CDCl₃)δ:7.63(1H,s),7.14–7.01(8H,m),6.78(1H,s),6.62(2H, d,J＝8.9Hz),6.27(1H,d,J＝1.8Hz),6.08(1H,d,J＝1.8Hz),5.89(1H,d,J＝3.8 Hz),4.50(1H,d,J＝17.6Hz),4.28(1H,d,J＝17.6Hz),4.13-4.06(1H,m),3.87(3H, s),3.71(3H,s),3.67(3H,s),2.90(1H,td,J＝13.6,4.5Hz),2.37(1H,dd,J＝13.7, 6.0Hz),2.04(1H,s)；¹³C NMR(100MHz,CDCl₃)δ:164.0,161.2,158.6,157.9, 138.0,133.3,128.6,128.6,127.9,127.9,127.9,126.8,126.3,112.7,105.9,102.9, 93.3,91.8,88.6,80.8,80.0,65.1,55.8,55.6,55.3,55.0,53.7,53.4,52.0,48.2,45.6；ESIMS m/z 533.2[M+H]⁺；HRESIMS m/z 543.2124[M+H]⁺(Calcd.for C₃₁H₃₁O₇N₂,543.2125).

example 20: preparation of target Compound 10t

The procedure is as in example 1 except that the benzoic acid is replaced with pyrazole to give the title compound in 10t, 58.7% yield.

1- (2- (1-pyrazolyl) acetyl) -rocomitol (10t) white solid; the melting point is 95-96 ℃;¹H NMR (400MHz,CDCl₃)δ:7.48(1H,d,J＝1.6Hz),7.25(1H,d,J＝2.2Hz),7.17–7.06 (7H,m),6.63(2H,d,J＝8.9Hz),6.26(1H,t,J＝2.1Hz),6.24(1H,d,J＝1.9Hz), 6.08(1H,d,J＝1.8Hz),5.90(1H,d,J＝3.6Hz),4.71(1H,d,J＝17.5Hz),4.60(1H, d,J＝17.5Hz),4.01(1H,dd,J＝13.7,6.1Hz),3.86(3H,s),3.79(3H,s),3.68(3H, s),2.86(1H,td,J＝13.8,5.1Hz),2.35(1H,ddd,J＝13.7,6.2,1.5Hz),2.14(1H,s)； ¹³C NMR(100MHz,CDCl₃)δ:166.5,163.9,161.0,158.6,158.0,139.9,138.3,130.4, 128.9,128.7,128.1,128.0,127.8,127.0,126.3,112.7,106.5,106.4,103.0,94.8,93.3, 91.9,88.4,80.3,55.6,55.6,55.0,55.0,53.7,52.9,35.4；ESIMS m/z 565.2[M+Na]⁺； HRESIMS m/z 565.1953[M+Na]⁺(Calcd.for C₃₁H₃₀O₇N₂Na,565.1945).

example 21: preparation of target Compound 10u

The procedure is as in example 1 except that morpholine was substituted for benzoic acid to give the title compound 10u in 59.2% yield.

1- (2- (1-morpholinyl) acetyl) -rocagliflonol (10 u.) white solid; melting point is 97-98 ℃;¹H NMR(400MHz,CDCl₃)δ:7.16–7.05(7H,m),6.64(2H,t,J＝6.0Hz),6.24(1H,d, J＝1.9Hz),6.03(1H,d,J＝1.9Hz),5.85(1H,d,J＝3.7Hz),4.11(1H,dd,J＝13.9, 6.1Hz),3.84(3H,s),3.76(3H,s),3.67(3H,s),3.66(4H,m),3.05(1H,d,J＝16.8 Hz),2.93(1H,d,J＝16.8Hz),2.89–2.85(1H,m),2.50–2.31(5H,m),2.20(1H,s)； ¹³C NMR(100MHz,CDCl₃)δ:168.8,163.7,161.0,158.5,157.9,138.4,128.6,128.0, 128.0,127.8,127.8,127.1,127.1,126.2,126.2,112.6,106.5,103.0,93.2,91.7,88.1, 79.3,66.7,59.3,55.5,55.4,54.9,53.7,52.9,52.9,35.6,35.6；ESIMS m/z 562.2[M+ H]⁺；HRESIMS m/z 562.2424[M+H]⁺(Calcd.for C₃₂H₃₆O₈N,562.2435).

example 22: preparation of target Compound 10v

The procedure was as in example 1 except that piperidine was substituted for benzoic acid to afford the title compound 10v in 54.6% yield.

1- (2- (1-piperidinyl) dioctylaminoacetyl) -rocagliflonol (10 v.) as a white solid; the melting point is 85-86 ℃;¹H NMR(400MHz,CDCl₃)δ:7.14–7.06(7H,m),6.64(2H,m),6.23(1H,d,J＝1.9 Hz),6.03(1H,d,J＝1.9Hz),5.84(1H,dd,J＝4.8,1.3Hz),4.11(1H,dd,J＝13.8, 6.1Hz),3.84(3H,s),3.75(3H,s),3.67(3H,s),3.03(1H,dd,J＝16.7Hz),2.88(1H, dd,J＝16.7Hz),2.84(1H,td,J＝14.0,5.2Hz),2.43-2.29(5H,m),1.72–1.40(6H, m)；¹³C NMR(100MHz,CDCl₃)δ:169.4,163.6,161.0,158.5,158.0,138.5,128.7, 128.7,128.0,128.0,127.8,127.8,127.2,127.2,126.2,112.6,106.7,103.0,93.3,91.7, 88.2,79.1,59.8,55.5,55.5,55.4,55.0,53.8,53.7,35.7,25.8,25.8,23.8；ESIMS m/z 560.2[M+H]⁺；HRESIMS m/z 560.2650[M+H]⁺(Calcd.for C₃₃H₃₈O₇N,560.2643).

example 23: preparation of target Compound 10w

The procedure is as in example 1 except that N-methylpiperazine was substituted from benzoic acid to give the target compound 10w with a yield of 55.2%.

1- (2- (4-methylpiperazin-1-yl) acetyl) -rocagliflonol (10 w.) white solid; the melting point is 88-89 ℃;¹H NMR(400MHz,CDCl₃)δ:7.15–7.04(7H,m),6.63(2H,d,J＝8.9Hz),6.22(1H, d,J＝1.9Hz),6.02(1H,d,J＝1.9Hz),5.84(1H,d,J＝3.8Hz),4.11–4.04(1H,m), 3.83(3H,s),3.75(3H,s),3.67(3H,s),3.03(1H,d,J＝16.8Hz),2.85(1H,d,J＝ 16.8Hz),2.84(1H,td,J＝3.8Hz),2.43-2.41(8H,m),2.30(1H,ddd,J＝13.1,6.5, 1.2Hz),2.29(3H,s)；¹³C NMR(100MHz,CDCl₃)δ:168.9,163.7,160.9,158.5, 158.0,138.4,128.7,128.7,128.0,128.0,127.8,127.8,127.2,126.2,112.6,112.6, 106.7,103.0,93.2,91.7,88.2,79.3,59.0,55.5,55.4,55.4,55.0,54.7,53.7,53.4,52.7, 45.9,35.6；ESIMS m/z 574.2[M+H]⁺；HRESIMS m/z 575.2751[M+H]⁺(Calcd. for C₃₃H₃₉O₇N₂,575.2752).

test example 1: screening for antitumor Activity in vitro

Cell lines: human red blood cell leukemia cell line (HEL), breast cancer cell (MDA-MB-231), and human colon cancer cell (HCT 116).

The experimental principle is as follows: and (3) detecting by adopting an MTT colorimetric method. MTT is known as 3- (4,5) -dimethylthiohiahiazo (-z-y1) -3, 5-di-phenylyttrazolimide, a yellow dye. The succinate dehydrogenase in the mitochondria of the living cells can reduce MTT, and can generate blue (or blue-violet) water-insoluble Formazan (Formazan) under the action of cytochrome C, and the content of the Formazan is determined at 490nm by using an enzyme-labeling instrument. In general, the generation amount of formazan is positively correlated with the number of living cells, so that the number of living cells can be estimated from the optical density OD value.

The experimental method comprises the following steps: cells in logarithmic growth phase were grown at 6X 10³The cells/well were seeded in 96-well plates at 37 ℃ in 5% CO₂Culturing in a cell culture box for 16h, adding compounds with different concentration gradients to continue culturing when the cell state is observed to reach logarithmic growth phase, and taking adriamycin as a positive control. 37 ℃ and 5% CO₂After 72h of culture, 20 mu L/well MTT (5mg/mL) is added, the culture is continued for 4h, a 96-well plate is centrifuged for 15min at 2500 r/min, the supernatant is discarded, 160 mu L/well DMSO is added, and the mixture is placed in a shaker at 37 ℃ and is kept away from light and shaken for 15min at a low speed to ensure that the precipitate is fully dissolved.

Color comparison: OD was measured using a multifunctional microplate reader. The absorption wavelength was adjusted to 490nm and the light absorption value of each well of the 96 well plate was measured and recorded. Then, the inhibition ratio of each well was calculated, and IC was performed₅₀And (4) measuring the value.

The experimental results are as follows: in vitro antitumor activity screening evaluation of three cell lines of HEL, MDA-MB-231 and HCT116 by using 20 synthesized compounds and using adriamycin as a positive control group, and IC (integrated Circuit) as a result₅₀Values are shown (see table 1). In HCT116 tumor cell screening, the results showed that 13 compounds (compounds 10g, 10h, 10j, 10k, 10l, 10m, 10p, 10q, 10r, 10t, 10u, 10u and 10w) had better antitumor activity than the positive control doxorubicin.

TABLE 124 data for the screening of the Activity of derivatives

Test example 2: research on anti-tumor action mechanism of high-activity representative compound 10r

1. Study of Effect of Compound 10r on cell cycle

HCT116 tumor cells were treated with varying concentrations of compound 10r (0.05,0.1 and 0.2. mu.M) following the following experimental procedure, and the results showed that this compound was able to dose-dependently block HCT116 tumor cells at stage G1.

The specific experimental procedure for the cycle effect of compound 10r on HCT116 tumor cells was as follows:

(1) sample processing and cell collection: taking cells in logarithmic growth phase, and culturing at 3.5 × 10⁴The culture medium was inoculated into 60 mm-diameter culture dishes at a concentration of 3 ml/ml, and placed at 37 ℃ in 5% CO₂After 24 hours in the cell incubator, the cells were divided into different time groups, each time group was a control group and a sample preparation group with different concentrations, and the control group was 0.1% DMSO. The cells of each group were placed at 37 ℃ in 5% CO₂Collecting cells after 48 hours in a cell incubator;

(2) fixing: collecting the cell culture medium, washing the cells with precooled PBS, adding 1.5mL pancreatin without EDTA, digesting for about 3min, observing the cells starting to loosen, blowing the cells with a micro pipetting gun, collecting the cell suspension, washing the culture dish with 1.5mL PBS, and collecting. Centrifuging at 1000r/min for 3min with a centrifuge, discarding supernatant, washing cells with PBS, centrifuging at 1000r/min for 3min, discarding supernatant, adding 500 μ L of precooled 75% ethanol, and fixing at-20 deg.C overnight;

(3) dyeing: before staining, washing with PBS for 2 times, discarding supernatant, adding 500 μ ML staining solution (0.05% TRITON, 0.5% RNase and 5% PI) prepared with PBS, and incubating at 37 deg.C in dark for 30 min;

(4) and (3) loading on a machine: the stained cells were centrifuged at 1000 rpm for 3min by a centrifuge, the supernatant was discarded, and then resuspended in 300. mu.l PBS, filtered and loaded onto a machine.

The experimental results are shown in fig. 1 and fig. 2: FIG. 1 is a graph of the cyclic effect of compound 10r on HCT116 tumor cell treatment for 48 hours; 10r prevent HCT116 cell cycle and cells were treated with different concentrations of compound (0.05. mu.M, 0.1. mu.M and 0.2. mu.M) for 48 hours. FIG. 2 is a graph of the percentage of compound 10r versus different stages of HCT116 tumor cells; at least three replicates per experiment, P <0.05, P <0.01, showed that this compound was able to dose-dependently block HCT116 tumor cells in the G1 phase.

2. Study of Effect of Compound 10r on apoptosis

HCT116 tumor cells were treated with varying concentrations of compound 10r (0.05. mu.M, 0.1. mu.M and 0.2. mu.M) following the following experimental procedure, and the results showed that this compound could induce HCT116 tumor cell apoptosis dose-dependently.

Apoptosis inducing Compounds

(1) Sample processing and cell collection: taking cells in logarithmic growth phase at 3.0 × 10⁵The culture medium is inoculated into 60mm culture dishes at a concentration of 3ml per dish, placed at 37 ℃ and 5% CO₂Dividing the cells into a control group and a 10r sample group after 24 hours in a cell incubator, wherein the control group is a 0.1% DMSO group, the sample groups are respectively three groups of 0.05 mu M,0.1 mu M and 0.2 mu M, and each group of cells are placed in the cell incubator to be incubated for 48 hours and then collected;

(2) the cell culture medium was collected, the cells were washed with PBS, 1.5mL of pancreatin without EDTA was added, digested for 3min, the cells were observed to start loosening, the cells were gently blown, the cell suspension was collected, and the dishes were washed with 1.5mL PBS and collected. Centrifuging at 1000r/min for 3min, discarding the supernatant, washing cells with PBS, and repeating twice;

(3) dyeing: adding 50 μ l Binding Buffer to resuspend the cells, transferring to a flow tube, adding 2.5 μ l/tube Propidium Iodide (PI) and 2.5 μ l/tube phospholipid Binding protein V (Annexin V-FITC) into each tube of the medicine adding set, gently mixing the cells, and incubating at room temperature in the dark for 20 min;

(4) and (3) loading: centrifuging the stained cells at 1000r/min for 3min, discarding the supernatant, adding 50 μ l of 1 × Binding Buffer into each tube, and analyzing by flow cytometry as soon as possible;

the experimental results are shown in fig. 3 and 4: FIG. 3 is a graph of the induction of apoptosis in HCT116 tumor cells by Compound 10 r; flow cytometry analysis of different concentrations of compound 10r (0.05. mu.M, 0.1. mu.M and 0.2M); FIG. 4 is a bar graph of Compound 10r induced apoptosis in tumor cells; each experiment was repeated at least three times, P <0.05, P <0.01 compared to the control group. The results show that the compound can induce HCT116 tumor cell apoptosis in a dose-dependent manner.

3. Study of Effect of Compound 10r on the formation of HCT116 clonal balls

HCT116 cells in logarithmic growth phase were seeded at 500/well in 6-well plates and placed at 37 ℃ in 5% CO₂The cell incubator is used overnight, the fresh culture medium is replaced, and a control group and a dry pre-treatment group with different concentrations of 10r (0.00625, 0.0125, 0.025,0.05 and 0.1 mu M) are arranged, wherein the control group is a 0.1% DMSO group. The cells were incubated at 37 ℃ with 5% CO₂Culturing under the condition, changing the culture medium once every three days, and continuously culturing for 15 days. Washing with PBS for three times, adding 500 μ L methanol, fixing for 30min, washing with PBS for two times, dyeing with crystal violet for 20min, washing with PBS for three times, air drying, and taking picture.

The experimental results are shown in fig. 5 and 6: FIG. 5 is a graph of the effect of Compound 10r on the formation of spheres of HCT116 clones; FIG. 6 is a bar graph of the number of clones formed after HCT116 was treated with compound 10r (0.05. mu.M, 0.1. mu.M and 0.2. mu.M) for 15 days; each experiment was repeated at least three times, P <0.05, P <0.01 compared to the control group. The results show that the compound can inhibit the formation of HCT116 clone balls in a dose-dependent manner.

Previous experiments showed that 10r has the effect of inhibiting HCT116 cell proliferation and induces apoptosis in a dose-dependent manner and blocks cells at G1. To confirm the effect of 10r on the inhibition of cell growth, HCT116 was used as a subject to investigate the effect of 10r on colony sphere formation. Results as shown in 5 and 6, 10r significantly inhibited HCT116 clonogenic sphere formation and was dose dependent. The 10r (0.025. mu.M) induced clonality was almost completely inhibited compared to the control group.

4. Study of Effect of Compound 10r on expression of Key proteins in related apoptotic proteins, cyclins, MAPK signaling pathway and Wnt signaling pathway

HCT116 cells at 1X 10⁶The culture dish was inoculated in a 100mm culture dish and cultured overnight. Cells were treated at various concentrations of 10r (0.05,0.1 and 0.2. mu.M) and 0.1% DMSO was added to the control. Cells were collected and lysed. The total protein was quantified by BCA and the protein was separated by 10% SDS-PAGE and transferred to PVDF membrane. 5% skim milk was blocked and incubated overnight at 4 deg.C with primary antibody (c-Myc, G1/S-specific cyclin-E, G1/S-specific cyclin-D, cell cycle dependent kinase 4, cell cycle dependent kinase 6, full-length-PARP 1, cleaved-PARP 1, procaspase-3, cleaved procaspase-3, procaspase-9, cleaved procaspase-9, Bcl-2, P-ERK, P-JNK, P-P38, P38, beta-catenin, Axin-2, GSK-3 beta, TCF-4, beta-actin and glyceraldehyde phosphate dehydrogenase) and after incubation for 2h at room temperature with secondary antibody. Detection was performed using the Odyssey Platform.

The experimental results are shown in fig. 7 and 8: FIG. 7 is a graph of the effect of Compound 10r on expression of related apoptotic proteins; . FIG. 8 is a graph of the effect of Compound 10r on the expression of the relevant cyclin; .

Based on the previous experimental results, the influence of 10r on apoptosis and cell cycle related proteins is analyzed by Western bolt, and the molecular mechanism of inducing HCT116 apoptosis and cell cycle arrest by 10r is further discussed. As shown in FIG. 7, 10r induced specific cleavage of the apoptotic proteins PARP, procaspase-3 and procaspase-9 in HCT116 and was dose dependent. Furthermore, as 10r concentration increased, expression of the apoptosis-inhibiting protein Bcl-2 was down-regulated, suggesting that 10r may induce apoptosis via the mitochondrial pathway. 10r induces HCT116 to arrest in the G1 phase of the cell cycle, and the effect of 10r on the expression of cyclin and related protein kinases in the G1 phase was analyzed. As shown in FIG. 8, 10r (0.1. mu.M) significantly reduced the expression of c-Myc, G1/S-specific cyclin-D, G1/S-specific cyclin-E, cyclin-dependent kinase 4 and cyclin-dependent kinase 6, and it was speculated that 10r might be due to the failure of the cyclin-CDK complex to form normally by degrading c-Myc, G1/S-specific cyclin-D, G1/S-specific cyclin-E, cyclin-dependent kinase 4 and cyclin-dependent kinase 6, resulting in the arrest of HCT116 in G1.

The experimental results are shown in fig. 9 and 10: FIG. 9 is a graph showing the effect of Compound 10r on protein expression associated with the mitogen-activated protein kinase (MAPK) signaling pathway; . Figure 10 is a graph of the effect of compound 10r on the expression of key proteins of the Wnt signaling pathway.

Mitogen-activated protein kinase (MAPK) and Wnt families are used as key signal molecules for regulating cell proliferation, homeostasis and development, and participate in a series of cell physiological activities such as cell growth, differentiation, apoptosis and the like. The effect of 10r on MAPK and wnt signaling pathways was examined using Western felt. As shown in FIG. 9, 10r significantly up-regulated the expression levels of P-P38 and P-JNK, down-regulated the expression level of P-ERK, and both were dose-dependent. As shown in FIG. 10, the expression of beta-catenin, Axin-2, GSK-3 beta and TCF-4 was significantly reduced in a dose-dependent manner after 10r treatment.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the present invention without departing from the technical spirit of the present invention.

Claims

1. A hydroxy derivative of loxagliflorin having the following structural formula (I):

wherein: r is a benzoate group, a p-fluorobenzoate group, a p-methoxybenzoate group, a p-bromobenzoate group, a 4, 5-dimethoxy-2-nitro-benzoate group, a p-nitrocinnamate group, a 2-furoate group, a 2-picolinate group, a 6-chloronicotinate group, a butyrate group, a decanoate group, a dimethylamino group, a diethylamino group, a dibutylamino group, a dioctylamino group, a benzylamino group, a 3, 4-dimethoxyphenethylamino group, a 1-tetrahydropyrrolyl group, a 1-imidazolyl group, a 1-pyrazolyl group, a 1-morpholinyl group, a 1-piperidinyl group or a 4-methylpiperazin-1-yl group.

2. A preparation method of a rocomimol hydroxyl derivative comprises the following steps:

(1)

synthesis of (2)

Weighing 20g

Putting the mixture into a 500ml round bottom flask, adding 51g of anhydrous potassium carbonate and 250ml of acetone into the flask as solvents, slowly adding 24.5ml of dimethyl sulfate while stirring, and putting the system into an oil bath kettle for heating and refluxing at 70 ℃ for 72 hours. TLC tracing reaction, after the reaction is finished, cooling to room temperature, adjusting pH to 11 with ammonia water, filtering to remove precipitate, washing filtrate with saturated saline solution, drying with anhydrous sodium sulfate, spin-drying partial solvent, adding silica gel for sample mixing, purifying with flash silica gel column, and obtaining light yellow solid with acetone of 8:2

Weighing 700mg

Adding a mixed solvent of 80mL of dichloromethane and 60mL of acetone, and slowly adding 140mL of potassium hydrogen monosulfate complex salt aqueous solution with the concentration of 11.6g/mL to obtain

(2)

And with

Synthesis of the mixture

Weighing 800mg of

Adding 40ml of acetonitrile, 30ml of methanol and 12.6 equivalents of trans-methyl cinnamate 5.0g, irradiating by using a xenon lamp for strong light, and reacting for 17 hours to obtain a product

And

a mixture of (a);

(3)

synthesis of (2)

Weighing 1g of

And

30mL of methanol and 10mL of a 0.5M sodium methoxide solution in methanol were added to the mixture, and the mixture was refluxed at 70 ℃ for 4 hours to obtain

(4)

Synthesis of (2)

Weighing 2g

100mL of DMSO and 170mg of lithium chloride were added thereto, and the mixture was stirred at 100 ℃ for 8 hours to obtain

(5)

Synthesis of (2)

3.27g of sodium triacetoxyborohydride are weighed into 200mL of acetonitrile and 1.2mL of glacial acetic acid, and 700mg of sodium triacetoxyborohydride are slowly added

Stirring at 40 deg.C for 8h to obtain

(6)

Synthesis of (2)

Weighing 30mg

Placed in a 25ml round bottom flask, 2ml DCM was added as solvent and 28.7. mu.l Et was added₃Stirring N, 11 mu l of chloroacetyl chloride and a catalytic amount of DMAP at room temperature for 10h to obtain

Namely 1-chloroacetyl-4' -demethoxy-clomipraminol;

(7)

synthesis of series of derivatives

Weighing 30mg of

5ml of DMF solution, 16.3mg of potassium carbonate (0.118mmol) and 1.5 equivalents of acid (0.085mmol) are added and the mixture is heated in a thermostatic oil bath at 70 ℃ for 12 hours to give the series of derivatives

3. Application of a loxagliflorol hydroxyl derivative in preparation of medicaments for resisting colorectal cancer, leukemia and breast cancer.

4. An application of a loxagliflorin hydroxyl derivative in the preparation of MAPK signal channel and Wnt signal channel inhibitor drugs.