CN110642918B

CN110642918B - Diosgenin derivative and preparation method and application thereof

Info

Publication number: CN110642918B
Application number: CN201910969223.1A
Authority: CN
Inventors: 刘玉法; 王目旋; 彭立增; 王继庆; 井晓琳; 王磊
Original assignee: Shandong Normal University
Current assignee: Shandong Normal University
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2021-01-08
Anticipated expiration: 2039-10-12
Also published as: CN110642918A

Abstract

The invention provides diosgenin derivativesThe diosgenin derivative has a structure shown in formula (I):

Description

Diosgenin derivative and preparation method and application thereof

Technical Field

The invention relates to the field of biological medicines, in particular to a diosgenin derivative and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Pancreatic cancer is a common pancreatic tumor, is a digestive tract malignant tumor with high malignancy degree and difficult diagnosis and treatment, and has 5-year survival rate of less than 1 percent. The early diagnosis rate of pancreatic cancer is low, the operative mortality rate is high, and the early diagnosis rate is one of the worst malignant tumors. At present, the main therapeutic drugs comprise fluorouracil, doxorubicin, ifosfamide, semustine, paclitaxel, gemcitabine, cisplatin and the like, but the clinical cure rate is very low, so that research and development of anti-pancreatic cancer drugs with better curative effect are very necessary.

Cholangiocarcinoma is a common biliary tract malignant tumor, with high malignancy, very poor prognosis, and increasing morbidity and mortality year by year (wangxian, et al, the clinical treatment progress of hepatoportal cholangiocarcinoma [ J ], electronic journal of Chinese liver surgery, 2018, 7(4): 253-containing 257). The common chemotherapy drugs in the clinic of bile duct cancer are 5-fluorouracil and cisplatin. When the 5-fluorouracil reaches 20ug/mL, the traditional Chinese medicine composition has a good effect of inhibiting the growth of human bile duct cancer QBC939 cells, and has side effects of bone marrow inhibition such as leukopenia and platelet drop, and gastrointestinal reactions such as inappetence, nausea, vomiting, stomatitis, gastritis, abdominal pain and diarrhea. When the cisplatin reaches 3.0 mu g/mL, the cisplatin has a good effect of inhibiting cell growth of human bile duct cancer QBC939, and mainly has severe digestive tract reactions such as nausea and vomiting, renal toxicity such as renal tubular injury, and more common neurotoxicity such as tinnitus and hearing loss caused by auditory nerve damage. Because biliary duct cancer has hidden onset and difficult early diagnosis and has late clinical symptoms, epidemiological studies show that only 10-15% of patients are likely to be cured by operation, and many patients find that the patients are in late stage, but the postoperative recurrence rate is still 50-60%, and the five-year survival rate is only 30%. At present, the treatment principle of the bile duct cancer still adopts a comprehensive treatment scheme mainly based on surgical resection, but only patients with deficiency of 1/3 can be surgically resected when the diagnosis is confirmed, the surgical prognosis is poor, and the combined chemotherapy has no ideal effect on locally advanced or metastatic unresectable bile duct cancer. Therefore, the development of new therapeutic drugs is urgently required.

Diosgenin (CAS number: 512-06-1) is a steroid compound obtained by hydrolyzing and deglycosylating dioscin contained in rhizome of Dioscoreaceae plants such as Dioscorea zingiberensis, Dioscorea nipponica, Dioscorea panthaica and the like, and is an important raw material for synthesizing steroid hormone drugs and steroid contraceptives. Dioscin has certain anticancer activity (application of protodioscin in preparing medicine for resisting drug-resistant osteosarcoma CN201810091212.3), and has effects in treating lung cancer cell A549(Wei Y, et al. Anti-cancer effects of Dioscin on third peptides of human lung cells through inducing DNA and activating pathological cancer, 2013, 59), gastric cancer cell MGC-803 (Zhuao X, et al. patent effects of Dioscin induced cancer in vitro and human physiology, human epidermis 2016, 23), hepatocarcinoma cell Huh7(Hsieh M J, et al. liver infection treatment and cancer cell III) and cancer cell of human liver, cancer cell III, 2016, 21) has anticancer activity, and diosgenin also has activity on sarcoma-180, ascites due to hepatic tumor, cervical cancer in mouse, and Ehrlich carcinoma (new application of diosgenin in preparing medicine CN 01129317.9).

Disclosure of Invention

The present invention is directed to overcoming the above-mentioned disadvantages and providing a diosgenin derivative, a method for preparing the same, and use thereof. The diosgenin derivative has good inhibitory activity on proliferation of bile duct cancer cells and human pancreatic cancer cells, the inhibitory rate of the diosgenin derivative on proliferation of bile duct cancer cells and human pancreatic cancer cells is generally higher than 80% at 0.2ug/mL, and the activity of the diosgenin derivative is far higher than that of 5-fluorouracil for clinical application.

Specifically, the invention has the following technical scheme:

in a first aspect of the invention, the invention provides a compound having the structure shown in formula (I):

wherein R is phenyl substituted with a substituent selected from the group consisting of alkyl, alkoxy, and halogen;

also, the present invention provides pharmaceutically acceptable salts of the compounds of formula (I).

Examples of the "pharmaceutically acceptable salt" may include, for example, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, phosphate and nitrate; organic acid salts such as acetate, propionate, oxalate, succinate, lactate, malate, tartrate, citrate, maleate, fumarate, methanesulfonate, benzenesulfonate, p-toluenesulfonate and ascorbate; inorganic base salts such as sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt and aluminum salt; and organic base salts such as arginine salt, benzathine salt, choline salt, diethylamine salt, dialcohol amine salt, glycinate salt, lysine salt, meglumine salt, ethanolamine salt, and tromethamine salt.

In an embodiment of the invention, R is selected from the following groups:

wherein, in the above optional groups, R₁Represents a single or multiple substitution at any position of a benzene ring, R₁Selected from halogens; r₂Is C_1-5Alkyl groups of (a); the polysubstitution is disubstituted, trisubstituted, tetrasubstituted or pentasubstituted.

In an embodiment of the invention, R₁One or more selected from the group consisting of F, Cl, Br and I, which may be the same or different; r₂Is C_1-3An alkyl group.

In an embodiment of the invention, R is

When R is₁Is mono-or polysubstituted, the polysubstitution being di-, tri-or penta-substituted, R₁Is F.

In an embodiment of the invention, R is selected from

When R is₁Is monosubstituted, selected from halogen, R₂Is methyl or ethyl; the halogen is selected from F, Cl, Br and I.

In an embodiment of the invention, said R is selected from the following groups: o-fluorophenyl group, m-fluorophenyl group, p-fluorophenyl group, (3, 4-difluoro) phenyl group, (2, 3-difluoro) phenyl group, (2, 4-difluoro) phenyl group, (2, 5-difluoro) phenyl group, (2, 6-difluoro) phenyl group, (3, 5-difluoro) phenyl group, (2,4, 6-trifluoro) phenyl group, (3,4, 5-trifluoro) phenyl group, pentafluorophenyl group, 2-methoxy-3-chlorophenyl group, 2-methoxy-4-chlorophenyl group, 2-methoxy-5-chlorophenyl group, 2-methoxy-6-chlorophenyl group, 2-methoxy-3-fluorophenyl group, 2-methoxy-4-fluorophenyl group, 2-methoxy-5-fluorophenyl group, 2-methoxy-6-fluorophenyl group, m-fluorophenyl group, p-fluorophenyl group, 3, 4-difluoro) phenyl group, 2, 3-difluoro) phenyl group, 2, 2-methoxy-3-bromophenyl, 2-methoxy-4-bromophenyl, 2-methoxy-5-bromophenyl, 2-methoxy-3-iodophenyl, 2-methoxy-4-iodophenyl, 2-methoxy-5-iodophenyl, 4-methoxy-2-chlorophenyl, 4-methoxy-3-chlorophenyl, 4-methoxy-2-fluorophenyl, 4-methoxy-3-fluorophenyl, 4-methoxy-2-bromophenyl, 4-methoxy-3-bromophenyl, 4-methoxy-2-iodophenyl, 4-methoxy-3-iodophenyl, 2-methyl-3-chlorophenyl, methyl-3-pyridyl, methyl-2, 2-methyl-4-chlorophenyl group, 2-methyl-5-chlorophenyl group, 2-methyl-6-chlorophenyl group, 2-methyl-3-fluorophenyl group, 2-methyl-4-fluorophenyl group, 2-methyl-5-fluorophenyl group, 2-methyl-6-fluorophenyl group, 2-methyl-3-bromophenyl group, 2-methyl-4-bromophenyl group, 2-methyl-5-bromophenyl group, 2-methyl-6-bromophenyl group, 2-methyl-3-iodophenyl group, 2-methyl-4-iodophenyl group, 2-methyl-5-iodophenyl group, 3-methyl-2-chlorophenyl group, 3-methyl-4-chlorophenyl group, 2-methyl-6-fluorophenyl group, 2-methyl-3-iodophenyl group, 2-methyl-5-iodo, 3-methyl-5-chlorophenyl group, 3-methyl-4-fluorophenyl group, 3-methyl-5-fluorophenyl group, 3-methyl-4-bromophenyl group, 3-methyl-5-bromophenyl group, 3-methyl-4-iodophenyl group, 3-methyl-5-iodophenyl group, 4-methyl-2-chlorophenyl group, 4-methyl-3-chlorophenyl group, 4-methyl-2-fluorophenyl group, 4-methyl-3-fluorophenyl group, 4-methyl-2-bromophenyl group, 4-methyl-3-bromophenyl group, 4-methyl-2-iodophenyl group, 4-methyl-3-iodophenyl group, 4-chloro-3-methoxyphenyl group, 3-fluorophenyl group, 3-methyl-4-fluorophenyl group, 3-iodophenyl group, 3-methyl-2-iodophenyl group, 3-methyl-3-, 4-bromo-3-methoxyphenyl, 4-fluoro-3-methoxyphenyl, 4-iodo-3-methoxyphenyl, 2-chloro-3-methoxyphenyl, 2-bromo-3-methoxyphenyl, 2-fluoro-3-methoxyphenyl, 2-iodo-3-methoxyphenyl, 5-chloro-3-methoxyphenyl, 5-bromo-3-methoxyphenyl, 5-fluoro-3-methoxyphenyl, 5-iodo-3-methoxyphenyl, 5-chloro-3-ethoxyphenyl, 5-bromo-3-ethoxyphenyl, 5-fluoro-3-ethoxyphenyl and 5-iodo-3-ethoxyphenyl.

Further, in some embodiments of the invention, the compounds of formula (I) of the present invention include the compounds shown in table 1:

TABLE 1 Structure of the compound of formula (I) and HR-MS structural analysis thereof

In a second aspect of the invention, there is also provided a process for the preparation of a compound of formula (I), which process comprises reacting diosgenin as an initial reactant with RCH₂X reacts to obtain a compound shown in a formula (I); wherein R is as defined above for the first aspect of the invention and X is a halogen selected from F, Cl, Br and I, preferably Cl or Br.

In an embodiment of the invention, the method comprises dissolving diosgenin in a solvent, adding RCH₂And reacting X with a basic substance, and keeping the solution to be basic (pH 7-14) in the reaction process to prepare the compound shown in the formula (I).

In the embodiment of the invention, the reaction temperature is-20-300 ℃, and the reaction time is 0.1-72 h; preferably, the reaction temperature is-20 to 150 ℃, more preferably-10 to 120 ℃, at which the reaction proceeds more easily.

In an embodiment of the present invention, the solvent is selected from one or more of water, methanol, ethanol (anhydrous ethanol is preferred), propanol, butanol, pentanol, glycerol, dichloromethane, trichloromethane, carbon tetrachloride, benzene, toluene, di-10 toluene, 1, 4-dioxane, 1, 2-dichloroethane, tetrahydrofuran, N-Dimethylformamide (DMF), and ethyl acetate and dimethyl sulfoxide (DMSO); any one of n-propanol, isopropanol, n-butanol, ethyl acetate, butyl acetate, absolute ethanol, methanol, dichloromethane, and tetrahydrofuran is preferable, and among these solvents, the reaction proceeds more easily.

In an embodiment of the present invention, the solvent is used in an amount of 5-100L per mole of diosgenin dissolved in the solvent.

In an embodiment of the invention, the alkaline substance is selected from γ -Al₂O₃-Na、γ-Al₂O₃-K、γ-Al₂O₃-NaOH-Na、γ-Al₂O₃-NaOH-K、γ-Al₂O₃-KOH-Na、γ-Al₂O₃-one or more of KOH-K, sodium methoxide, sodium ethoxide, sodium propoxide, sodium butoxide, sodium amide, sodium metal, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium oxide, potassium oxide, calcium oxide, strontium carbonate, potassium carbonate, sodium carbonate, potassium phosphate, ammonia, ethylamine, diethylamine, triethylamine, methylamine, dimethylamine, trimethylamine, pyridine, and piperidine.

In some preferred embodiments, the basic substance is trimethylamine, triethylamine, γ -Al₂O₃-NaOH-K、γ-Al₂O₃KOH-Na, sodium hydride, potassium hydride, sodium ethoxide, strontium carbonate, potassium carbonate, diethylamine, triethylamine or sodium tert-butoxide, in the presence of these basic substances, the reaction proceeds more readily.

In an embodiment of the invention, the diosgenin, RCH₂X and the molar ratio of the alkaline substances is 1: (0.05-15): (0.01-15), preferably 1: (0.1-10): (0.01-10), more preferably 1: (1.0-1.5): (0.6-1.5).

In an embodiment of the present invention, the preparation method of the compound of formula (I) according to the present invention further comprises detecting the end point of the reaction by chromatography, standing to room temperature after the reaction is finished, and separating and purifying to prepare the compound of formula (I).

In an embodiment of the present invention, the separation and purification method is selected from a combination of one or more of filtration, resin treatment, water washing, distillation, crystallization, extraction, activated carbon treatment, molecular sieve treatment, and chromatography.

The separation and purification method of the invention can be as follows: for example, in some further embodiments, the separation and purification method may be: detecting the total reaction of diosgenin by HPLC, reducing pressure to remove solvent, performing chromatography, such as chromatography with Agilent Zorbax SB-C18(4.6mm × 150mm,5um), eluting with acetonitrile-water (such as v/v ═ 90:10), performing HPLC tracing to separate and purify the reaction product, and drying the solid to obtain the target product. As another example, in some embodiments, the separation and purification process can be: detecting all reactions of diosgenin by HPLC, distilling off solvent until liquid volume is reduced to 1/4, cooling and crystallizing overnight, such as cooling to 5 deg.C and crystallizing overnight, filtering, tracking reaction and product separation and purification process by HPLC, and drying solid, such as drying at 60 deg.C for 4 hr, to obtain target product. And, for example, in some embodiments, TLC detects the total reaction of diosgenin, after warming to room temperature, water is added, dichloromethane, chloroform, acetone or ethyl acetate is used for extraction for 1 to more times, such as 3 times (such as 50-300mL × 3), TLC tracks the reaction and separation and purification process of the product, extract is recovered, and the obtained solid is dried, such as at 60 ℃, for 4 hours, to obtain the target product.

In a third aspect of the invention, the present invention also provides a composition comprising a compound of formula (I) as described in the above first aspect of the invention or a pharmaceutically acceptable salt thereof.

In a fourth aspect of the invention, the invention also provides a pharmaceutical formulation comprising a compound of formula (I) as described in the above first aspect of the invention or a pharmaceutically acceptable salt thereof or a composition comprising said compound of formula (I) or a salt thereof, together with at least one pharmaceutical carrier or adjuvant.

The compounds of the present invention or pharmaceutical compositions containing the compounds of the present invention may be in a variety of pharmaceutical dosage forms and administered in unit dosage form. The pharmaceutical or administration form may be a liquid form, a solid form, a topical formulation, a spray, and the like. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or mixed suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.

The pharmaceutical composition or pharmaceutical preparation of the present invention may further comprise a conventional carrier, wherein the pharmaceutically acceptable carrier includes but is not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of 1% to 98% by weight, usually about 80% by weight.

Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may also be coated by methods known in the art of pharmacy.

The oral liquid can be made into suspension, solution, emulsion or syrup of water and oil, or made into dry product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoate, sorbic acid. Flavoring or coloring agents may be added if desired.

In a fifth aspect, the present invention also provides the use of a compound of formula (I) as described in the above first aspect of the present invention or a pharmaceutically acceptable salt thereof, or a composition comprising a compound of formula (I) or a salt thereof, or a pharmaceutical formulation comprising a compound of formula (I) or a salt thereof, in the manufacture of a medicament for the treatment of cancer; in an embodiment of the invention, the cancer is preferably pancreatic cancer and/or cholangiocarcinoma.

In some embodiments of the invention, the inhibition rate of 0.2ug/mL of the compound of formula (I) of the invention on the proliferation of bile duct cancer cells QBC-939 is higher than 80%, and can reach 95%; the inhibition rate of 0.2ug/mL of the compound shown in the formula (I) on the proliferation of human pancreatic cancer cells PANC-1 is higher than 80% and can reach 95%. The activity of the compound of the invention is far higher than that of 5-fluorouracil used clinically.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are exemplary only, and one skilled in the art can prepare the compounds of the present invention by simple modification based on the teachings of the present invention.

Example 1

Weighing 4.2g of diosgenin and 0.5g of sodium hydride, dissolving in 500mL of 100mL of absolute ethanol, adding 2.0g of o-fluorobenzyl chloride, stirring and freezing to 0 ℃, keeping the temperature and stirring for reaction for 72h, detecting the total reaction of the diosgenin by TLC (a developing agent: petroleum ether-ethyl acetate (7:3), a color developing agent: 5% phosphomolybdic acid ethanol solution), distilling out the ethanol, cooling to room temperature, adding 50mL of water, extracting for 3 times (60mL multiplied by 3) by using dichloromethane, tracking the reaction and the separation and purification process of the product by TLC, recovering the dichloromethane from an extract, and drying the solid at 60 ℃ for 4h to obtain 3.8g of a light yellow powdery product. Melting point of the target product: 198.2-199.5 deg.c,¹³C NMR(CDCl₃100MHz) delta 150.74,140.05,134.43,130.59,129.77,129.70,122.62,120.63,119.75,87.97,82.70,67.20,66.53,62.24,56.91,50.16,42.54,40.51,40.43,39.18,37.77,37.27,32.78,32.56,32.25,31.91,30.33,29.94,28.86,21.12,19.97,18.56,15.92, 15.28. TOF-HRMS: m/e (522.7330), formula: c₃₄H₄₇O₃F, compound 1 in table 1.

Example 2

Weighing 4.2g of diosgenin and 1.0g of potassium hydride, dissolving in 500mL of 100mL of methanol, adding 2.0g of m-fluorobenzyl chloride, stirring and heating to 65 ℃, keeping the temperature, stirring and reacting for 36h, detecting all reactions of the diosgenin by TLC (a developing agent: petroleum ether-ethyl acetate (7:3), a color developing agent: 5% phosphomolybdic acid ethanol solution), evaporating the methanol, cooling to room temperature, adding 50mL of water, extracting for 3 times (60mL multiplied by 3) by using dichloromethane, tracking the reactions and the separation and purification processes of products by TLC, recovering the dichloromethane from an extract, and drying the solid at 60 ℃ for 4h to obtain 3.5g of a light yellow powdery product. Melting point of the target product: 198.5-199.9 deg.C,¹³C NMR(CDCl₃,100MHz)δ152.21,140.01,131.40,130.79,129.55,128.77,123.54,120.65,119.74,87.95,82.64,67.15,66.53,62.19,56.86,50.11,42.50,40.46,40.38,39.13,37.72,37.22,32.73,32.51,32.20,31.86,30.28,29.89,28.81,21.07,19.92,18.50,15.87,15.23。TOF-HRMS：M/e(522.7328)，the molecular formula is as follows: c₃₄H₄₇O₃F, compound 2 in table 1.

Example 3

Weighing diosgenin 4.2g and sodium ethoxide 0.8g, dissolving in 100mL of n-propanol in a 500mL three-neck flask, adding p-fluorobenzyl bromide 2.2g, stirring and heating at 78.5 ℃, keeping the temperature and stirring for reaction for 36h, detecting the total reaction of the diosgenin by TLC (a developing agent: petroleum ether-ethyl acetate (7:3), a color developing agent: 5% phosphomolybdic acid ethanol solution), evaporating the n-propanol, cooling to room temperature, adding 80mL of water, extracting for 3 times (100mL multiplied by 3) by using trichloromethane, tracking the separation and purification process of the reaction and the product by TLC, recovering the trichloromethane from an extract, and drying the solid at 60 ℃ for 4h to obtain a light yellow powdery product 4.0 g. Melting point of the target product: 198.1-199.9 deg.C,¹³C NMR(CDCl₃100MHz) delta 151.79,140.11,135.35,131.65,131.65,122.76,122.76,120.81,119.74,87.97,82.70,67.20,66.53,62.24,56.91,50.16,42.54,40.51,40.43,39.18,37.77,37.27,32.78,32.56,32.25,31.91,30.33,29.94,28.86,21.12,19.97,18.56,15.92, 15.28. TOF-HRMS: m/e (522.7329), formula: c₃₄H₄₇O₃F, compound 3 in table 1.

Example 4

Weighing diosgenin 4.2g and strontium carbonate 0.05g, dissolving in 100mL n-propanol in 500mL three-neck flask, adding 3, 4-difluorobenzyl bromide 2.1g, heating and stirring at 95 deg.C, maintaining the temperature and stirring for reaction for 24h, detecting total reaction of diosgenin by TLC, distilling off solvent under reduced pressure, cooling to room temperature, adding water 50mL, extracting with acetone for 3 times (50mL × 3), tracking reaction and product separation and purification by TLC, extracting with anhydrous Na₂SO₄Drying for 8h, filtering, recovering acetone from the filtrate to obtain solid, and drying the solid at 60 deg.C for 4h to obtain light yellow powder 3.0 g. Melting point of the target product: 199.5-201.2 ℃ of water,¹³C NMR(101MHz,CDCl₃)δ150.20,140.04,137.43,126.79,125.55,124.24,120.81,119.54,115.77,87.98,82.67,67.00,66.56,62.22,56.89,50.14,42.53,40.49,40.41,39.16,37.75,37.25,32.76,32.54,32.23,31.89,30.31,29.92,28.84,21.10,20.01,18.53,15.90,15.26. TOF-HRMS: m/e (540.7234), formula: c₃₄H₄₆O₃F₂I.e. compound 4 in table 1.

Example 5

Weighing diosgenin 4.2g and potassium carbonate 1.5g, dissolving in 100mL n-propanol in a 500mL three-neck flask, adding 2, 3-difluorobenzyl bromide 2.1g, stirring and heating to boil (97 ℃), keeping the temperature and stirring for reaction for 16h, detecting all reactions of diosgenin by TLC, distilling off the solvent under reduced pressure, cooling to room temperature, adding 50mL of water, extracting with acetone for 3 times (50mL multiplied by 3), tracking the TLC reaction and the separation and purification process of the product, and using anhydrous Na for extraction liquid₂SO₄Drying for 8h, filtering, recovering acetone from the filtrate to obtain solid, and drying the solid at 60 deg.C for 4h to obtain light yellow powder 2.9 g. Melting point of the target product: 199.4-201.3 ℃ of the total weight of the mixture,¹³C NMR(101MHz,CDCl₃) δ 149.45,140.02,137.41,126.77,125.53,124.21,121.82,119.75,116.58,87.96,82.65,66.54,66.46,62.20,56.87,50.12,42.51,40.47,40.39,39.14,37.73,37.23,32.74,32.52,32.21,31.87,30.29,29.90,28.82,21.08,19.99,18.51,15.88, 15.24. TOF-HRMS: m/e (540.7227), formula: c₃₄H₄₆O₃F₂I.e. compound 5 in table 1.

Example 6

Weighing diosgenin 4.2g and potassium carbonate 1.5g, dissolving in 100mL isopropanol in 500mL three-neck flask, adding 2, 4-difluorobenzyl bromide 2.1g, heating and stirring at 98 deg.C, maintaining the temperature and stirring for reaction for 24h, detecting all reactions of diosgenin by TLC (developing agent: petroleum ether-ethyl acetate (7:3), developer: 5% phosphomolybdic acid ethanol solution), distilling off solvent under reduced pressure, cooling to room temperature, adding water 50mL, extracting with acetone for 3 times (50mL × 3), tracking reaction and separation and purification process of product by TLC, extracting with anhydrous Na₂SO₄Drying for 8h, filtering, recovering acetone from the filtrate to obtain solid, and drying the solid at 60 deg.C for 4h to obtain light yellow powder 3.2 g. Melting point of the target product: 199.0-200.7 ℃ of the total weight of the mixture,¹³C NMR(101MHz,CDCl₃)δ161.44,160.12,140.07,130.75,120.94,120.80,119.24,112.52,106.42,87.98,82.67,66.56,66.18,62.22,56.89,50.14,42.53,40.49,40.41,39.16,37.75,37.25,32.76,32.54,32.23,31.89,30.31,29.92,28.84,21.10,20.01,18.53,15.90,15.26. TOF-HRMS: m/e (540.7235), formula: c₃₄H₄₆O₃F₂I.e. compound 6 in table 1.

Compound 7 (substituent R is (2, 5-difluoro) phenyl) and compound 8 (substituent R is (2, 6-difluoro) phenyl) can be prepared according to a similar procedure to example 6, using the appropriate substituted benzyl halide (halogen can be Cl or Br).

Example 7

Weighing diosgenin 4.2g and gamma-Al₂O₃Dispersing 1.5g of-Na into 100mL of n-butanol in a 500mL three-neck flask, adding 2.1g of 3, 5-difluorobenzyl bromide, heating and stirring at 100 ℃, keeping the temperature and stirring for reaction for 12h, detecting all diosgenin by TLC (developing agent: petroleum ether-ethyl acetate (7:3), developer: 5% phosphomolybdic acid ethanol solution), distilling off the solvent under reduced pressure, cooling to room temperature, adding 50mL of water, extracting for 3 times (50mL × 3) by using acetone, tracking the reaction and the separation and purification process of the product by TLC, and using anhydrous Na for extraction liquid₂SO₄Drying for 8h, filtering, recovering acetone from the filtrate to obtain solid, and drying the solid at 60 deg.C for 4h to obtain light yellow powder 3.9 g. Melting point of the target product: 199.3-201.5 ℃ of the total weight of the mixture,¹³C NMR(101MHz,CDCl₃) δ 161.56,160.11,140.05,131.55,120.87,120.81,119.25,113.57,103.34,87.97,82.65,66.54,66.16,62.20,56.87,50.12,42.51,40.47,40.39,39.14,37.73,37.23,32.74,32.52,32.21,31.87,30.29,29.90,28.82,21.07,19.98,18.51,15.89, 15.24. TOF-HRMS: m/e (540.7232), formula: c₃₄H₄₆O₃F₂Compound 9 in table 1.

Example 8

Weighing diosgenin 4.2g and gamma-Al₂O₃Dispersing 1.2g of-NaOH-K in 500mL of 100mL of n-butanol in a three-necked flask, adding 2.5g of 3,4, 5-trifluorobenzyl bromide, heating and stirring at 90 ℃, keeping the temperature and stirring for reaction for 12h, and detecting the dioscorea by TLC (developing agent: petroleum ether-ethyl acetate (7:3), developer: 5% phosphomolybdic acid ethanol solution)Allowing sapogenin to react completely, evaporating solvent under reduced pressure, cooling to room temperature, adding 50mL of water, extracting with acetone for 3 times (50mL × 3), tracking reaction and product separation and purification process by TLC, and extracting with anhydrous Na₂SO₄Drying for 8h, filtering, recovering acetone from the filtrate to obtain solid, and drying the solid at 60 deg.C for 4h to obtain light yellow powder 4.1 g. Melting point of the target product: at the temperature of 197.5-199.3 ℃,¹³C NMR(101MHz,CDCl₃) δ 165.03,162.55,162.55,140.11,120.92,119.36,108.87,102.57,102.57,88.08,82.76,66.65,66.27,62.31,56.98,50.23,42.62,40.58,40.50,39.25,37.73,37.34,32.85,32.63,32.32,31.98,30.40,30.01,28.93,21.18,20.09,18.62,16.00, 15.35. TOF-HRMS: m/e (558.7140), formula: c₃₄H₄₅O₃F₃I.e. compound 11 in table 1.

Compound 10 (substituent R is (2,4, 6-trifluoro) phenyl) can be prepared in a similar manner to example 8, using the appropriate substituted benzyl halide (halogen can be Cl or Br).

Example 9

Weighing diosgenin 4.2g and potassium carbonate 1.5g, dissolving in 100mL n-butanol in 500mL three-neck flask, adding pentafluorobenzyl bromide 2.9g, heating and stirring at 50 deg.C, maintaining the temperature and stirring for reaction for 12h, detecting diosgenin by TLC (developing agent: petroleum ether-ethyl acetate (7:3), developer: 5% phosphomolybdic acid ethanol solution), distilling off solvent under reduced pressure, cooling to room temperature, adding water 50mL, extracting with acetone for 3 times (50mL × 3), tracking reaction and product separation and purification process by TLC, extracting with anhydrous Na₂SO₄Drying for 8h, filtering, recovering acetone from the filtrate to obtain solid, and drying the solid at 60 deg.C for 4h to obtain light yellow powder 4.2 g. Melting point of the target product: the temperature of the mixture is 195.4-197.2 ℃,¹³C NMR(101MHz,CDCl₃) δ 162.46,149.51,149.51,140.12,135.87,135.87,122.54,120.93,119.37,88.09,82.77,66.66,66.28,62.32,56.99,50.24,42.63,40.59,40.51,39.26,37.74,37.35,32.86,32.64,32.33,31.99,30.41,30.02,28.94,21.19,20.10,18.63,16.01, 15.36. TOF-HRMS: m/e (594.6947), formula: c₃₄H₄₃O₃F₅Instant watchCompound 12 of 1.

Example 10

Weighing diosgenin 4.2g, dissolving in ethyl acetate 80mL in a three-necked 500mL flask, adding triethylamine 10mL and 2-methoxy-3-chlorobenzyl chloride 1.8g, stirring, freezing to-10 ℃, carrying out heat preservation reaction for 72h, detecting the total reaction of the diosgenin by TLC (a developing agent: petroleum ether-ethyl acetate (7:3) and a color developing agent: 5% phosphomolybdic acid ethanol solution), heating to room temperature, carrying out heat preservation for 2h, carrying out reduced pressure evaporation until the liquid volume is 20mL, cooling to 5 ℃, crystallizing overnight, filtering, tracking the separation and purification process of the reaction and the product by TLC, and drying the solid at 60 ℃ for 4h to obtain a light yellow powdery product 3.2 g. Melting point of the target product: the temperature of the mixture is 194.2 to 196.0 ℃,¹³C NMR(101MHz,CDCl₃) δ 157.43,140.46,134.20,128.75,125.53,122.24,121.11,119.84,108.77,88.28,82.97,71.07,66.81,62.53,57.20,54.22,50.45,42.84,40.80,40.72,39.47,38.06,37.56,33.07,32.85,32.54,32.20,30.31,30.03,29.15,21.41,20.32,18.84,16.21, 15.57. TOF-HRMS: m/e (569.2131), formula: c₃₅H₄₉O₄Cl, compound 13 in table 1.

In a similar manner to example 10, compound 14 (substituent R is 2-methoxy-4-chlorophenyl), compound 15 (substituent R is 2-methoxy-5-chlorophenyl), compound 16 (substituent R is 2-methoxy-6-chlorophenyl), compound 17 (substituent R is 2-methoxy-3-fluorophenyl), and compound 18 (substituent R is 2-methoxy-4-fluorophenyl) can be prepared by using an appropriate substituted benzyl halide (halogen may be Cl or Br).

Example 11

Weighing diosgenin 4.2g, dissolving in ethyl acetate 80mL in a three-neck flask 500mL, adding triethylamine 10mL and 2-methoxy-5-fluorobenzyl chloride 2.0g, stirring, freezing to 0 deg.C, maintaining the temperature for reaction for 36h, detecting total reaction of diosgenin by TLC (developing agent: petroleum ether-ethyl acetate (7:3), developer: 5% phosphomolybdic acid ethanol solution), heating to room temperature, maintaining the temperature for 2h, evaporating under reduced pressure to liquid volume of 20mL, cooling to 5 deg.C, crystallizing overnight, filtering, tracking the separation and purification process of reaction and product by TLC, drying the solid at 60 deg.C for 4h to obtain light diosgenin3.4g of product in yellow powder form. Melting point of the target product: the temperature of the mixture is 194.5 to 196.3 ℃,¹³C NMR(101MHz,CDCl₃) δ 160.22,151.11,140.35,122.70,117.53,120.95,119.69,109.24,107.75,88.13,82.82,66.64,65.07,62.48,57.05,55.25,50.30,42.69,40.65,40.67,39.32,37.95,37.31,32.92,32.70,31.39,32.05,30.16,29.98,29.00,21.26,20.17,18.69,16.06, 15.42. TOF-HRMS: m/e (552.7592), formula: c₃₅H₄₉O₄F, compound 19 in table 1.

In a manner similar to that described in example 11, when an appropriate substituted benzyl halide (halogen may be Cl or Br) is selected, compound 20 (substituent R is 2-methoxy-6-fluorophenyl), compound 21 (substituent R is 2-methoxy-3-bromophenyl), compound 22 (substituent R is 2-methoxy-4-bromophenyl), compound 23 (substituent R is 2-methoxy-5-bromophenyl), compound 24 (substituent R is 2-methoxy-3-iodophenyl), compound 25 (substituent R is 2-methoxy-4-iodophenyl), compound 26 (substituent R is 2-methoxy-5-iodophenyl), compound 27 (substituent R is 4-methoxy-2-chlorophenyl) and pharmaceutically acceptable salts thereof can be prepared, Compound 28 (substituent R is 4-methoxy-3-chlorphenyl) and compound 29 (substituent R is 4-methoxy-2-fluorophenyl).

Example 12

Weighing diosgenin 4.2g, dissolving in butyl acetate 80mL in a three-necked 500mL flask, adding sodium ethoxide 1.0g and 4-methoxy-3-fluorobenzyl chloride 2.0g, stirring, reacting at room temperature for 24h, detecting all reactions of diosgenin by TLC (developing agent: petroleum ether-ethyl acetate (7:3), developer: 5% phosphomolybdic acid ethanol solution), evaporating under reduced pressure until the liquid volume is 20mL, cooling to 5 ℃, crystallizing overnight, filtering, tracking the reaction by TLC, separating and purifying the product, and drying the solid at 60 ℃ for 4h to obtain a light yellow powder product 3.5 g. Melting point of the target product: at a temperature of 193.7-195.3 ℃,¹³C NMR(101MHz,CDCl₃) δ 158.47,140.32,139.11,131.71,127.53,120.92,119.67,110.33,102.70,88.10,82.79,71.03,66.61,62.45,57.02,54.46,50.27,42.66,40.65,37.92,37.67,37.28,36.32,32.89,32.67,31.37,32.02,30.13,29.95,28.97,21.23,20.14,18.66,16.03, 15.39. TOF-HRMS: m/e (552.7592), formula: c₃₅H₄₉O₄F, compound 30 in table 1.

Compound 31 (substituent R is 4-methoxy-2-bromophenyl), compound 32 (substituent R is 4-methoxy-3-bromophenyl) can be prepared according to a similar manner to example 12 by using an appropriate substituted benzyl halide (halogen may be Cl or Br).

Example 13

Weighing diosgenin 4.2g, dissolving in 80mL dichloromethane in a 500mL three-neck flask, adding sodium tert-butoxide 1.8g and 4-methoxy-2-iodobenzyl bromide 3.5g, stirring, reacting at room temperature for 24h, detecting all reactions of diosgenin by TLC (developing agent: petroleum ether-ethyl acetate (7:3) and color developing agent: 5% phosphomolybdic acid ethanol solution), evaporating under reduced pressure until the liquid volume is 20mL, cooling to 5 ℃, crystallizing overnight, filtering, tracking the reaction by TLC, separating and purifying the reaction product, and drying the solid at 60 ℃ for 4h to obtain light yellow powder 4.3g of the product. Melting point of the target product: at a temperature of 193.5-195.3 ℃,¹³C NMR(101MHz,CDCl₃) δ 158.60,140.29,139.64,131.89,128.33,120.89,119.64,110.84,90.61,88.07,82.76,71.94,66.58,62.42,56.99,54.65,50.24,42.63,40.62,37.89,37.64,37.25,36.29,32.86,32.64,31.34,31.99,30.10,29.92,28.94,21.20,20.11,18.63,16.00, 15.36. TOF-HRMS: m/e (660.6648), formula: c₃₅H₄₉O₄I, compound 33 in table 1.

Compound 34 (substituent R is 4-methoxy-3-iodophenyl) can be prepared in a similar manner to example 13, using the appropriate substituted benzyl halide (halogen can be Cl or Br).

Example 14

Weighing diosgenin 4.2g and sodium ethoxide 0.8g, dissolving in 100mL of n-propanol in a 500mL three-neck flask, adding 2-methyl-5-chlorobenzyl chloride 2.1g, stirring and heating at 60 deg.C, maintaining the temperature, stirring and reacting for 36h, detecting by TLC (developing agent: petroleum ether-ethyl acetate (7:3), developer: 5% phosphomolybdic acid ethanol solution), distilling off n-propanol, cooling to room temperature, adding water 80mL, extracting with chloroform for 3 times (100mL × 3), tracking the reaction and separation and purification process of the product by TLC, recovering trisodium from the extractMethyl chloride, and drying the solid at 60 deg.C for 4 hr to obtain light yellow powder 3.5 g. Melting point of the target product: 197.1-199.7 ℃ of water,¹³C NMR(CDCl₃100MHz) delta 140.25,138.76,136.45,132.74,129.33,128.81,125.60,120.85,119.60,88.03,82.72,66.54,65.56,62.38,56.95,50.20,42.59,40.58,37.85,37.60,37.20,36.25,32.82,32.60,31.30,31.95,30.06,29.88,28.90,21.16,20.07,18.75,18.59,15.96, 15.32. TOF-HRMS: m/e (553.2149), formula: c₃₅H₄₉O₃Cl, compound 37 in table 1.

In a manner similar to that described in example 14, when an appropriately substituted benzyl halide (halogen may be Cl or Br) is selected, compound 35 (substituent R is 2-methyl-3-chlorophenyl), compound 36 (substituent R is 2-methyl-4-chlorophenyl), compound 38 (substituent R is 2-methyl-6-chlorophenyl), compound 39 (substituent R is 2-methyl-3-fluorophenyl), compound 40 (substituent R is 2-methyl-4-fluorophenyl), compound 41 (substituent R is 2-methyl-5-fluorophenyl), compound 42 (substituent R is 2-methyl-6-fluorophenyl), compound 43 (substituent R is 2-methyl-3-bromophenyl), and compound, Compound 44 (substituent R is 2-methyl-4-bromophenyl), compound 45 (substituent R is 2-methyl-5-bromophenyl), compound 46 (substituent R is 2-methyl-6-bromophenyl), compound 47 (substituent R is 2-methyl-3-iodophenyl), compound 48 (substituent R is 2-methyl-4-iodophenyl), compound 49 (substituent R is 2-methyl-5-iodophenyl), compound 50 (substituent R is 3-methyl-2-chlorophenyl), compound 51 (substituent R is 3-methyl-4-chlorophenyl), compound 52 (substituent R is 3-methyl-5-chlorophenyl).

Example 15

Weighing diosgenin 4.2g and potassium carbonate 1.5g, dissolving in 100mL isopropanol in 500mL three-neck flask, adding 3-methyl-4-fluorobenzyl bromide 2.6g, heating and stirring at 110 deg.C, maintaining the temperature and stirring for reaction for 12h, detecting total reaction of diosgenin by TLC (developing agent: petroleum ether-ethyl acetate (7:3), developer: 5% phosphomolybdic acid ethanol solution), distilling off solvent under reduced pressure, cooling to room temperature, adding water 50mL, extracting with acetone for 3 times (50mL × 3), tracking reaction and product separation and purification process by TLC, extracting with anhydrous Na₂SO₄Drying for 8h, filtering, recovering acetone from the filtrate to obtain solid, and drying at 60 deg.C for 4h to obtain light yellow powder 3.6 g. Melting point of the target product: 196.9-198.5 ℃ of water,¹³C NMR(101MHz,CDCl₃) δ 158.25,140.22,134.24,130.71,127.30,122.81,120.82,119.57,105.60,88.00,82.69,66.51,65.47,62.35,56.92,50.17,42.56,40.55,37.82,37.57,37.17,36.22,32.79,32.57,31.27,31.92,30.03,29.85,28.87,21.13,20.04,18.28,18.56,15.93, 15.29. TOF-HRMS: m/e (536.7608), formula: c₃₅H₄₉O₃F, compound 53 in table 1.

According to a method similar to example 15, using an appropriate substituted benzyl halide (halogen may be Cl or Br), compounds 54 (substituent R is 3-methyl-5-fluorophenyl), compounds 55 (substituent R is 3-methyl-4-bromophenyl), compounds 56 (substituent R is 3-methyl-5-bromophenyl), compounds 57 (substituent R is 3-methyl-4-iodophenyl), compounds 58 (substituent R is 3-methyl-5-iodophenyl), compounds 59 (substituent R is 4-methyl-2-chlorophenyl), compounds 60 (substituent R is 4-methyl-3-chlorophenyl), compounds 61 (substituent R is 4-methyl-2-fluorophenyl), Compound 62 (substituent R is 4-methyl-3-fluorophenyl), compound 63 (substituent R is 4-methyl-2-bromophenyl).

Example 16

Weighing diosgenin 4.2g and potassium carbonate 1.5g, dissolving in 100mL isopropanol in 500mL three-necked flask, adding 4-methyl-3-bromobenzyl bromide 2.8g, heating and stirring at 120 deg.C, stirring at constant temperature for 24 hr, and performing HPLC (chromatographic conditions: Agilent Zorbax SB-C18(4.6mm × 150mm,5um) on chromatographic column, acetonitrile-water (90: 10) as mobile phase, and flow rate of 1.0mL.min^-1Detecting total reaction of diosgenin at detection wavelength of 223nm and column temperature of 35 deg.C, distilling off solvent under reduced pressure, cooling to room temperature, adding 50mL of water, extracting with acetone for 3 times (50mL × 3), tracking reaction and product separation and purification process by HPLC, and extracting with anhydrous Na₂SO₄Drying for 8h, filtering, recovering acetone from the filtrate to obtain solid, and drying the solid at 60 deg.C for 4h to obtain light yellow powder 3.6 g. Melting point of the target product: 196.5-198.2 ℃ of water,¹³C NMR(101MHz,CDCl₃) δ 140.21,139.13,135.22,131.71,128.37,121.98,120.81,119.56,113.77,87.99,82.68,66.50,65.47,62.34,56.91,50.16,42.55,40.54,37.81,37.56,37.16,36.21,32.78,32.56,31.26,31.91,30.02,29.84,28.86,21.12,20.03,18.65,18.55,15.92, 15.28. TOF-HRMS: m/e (597.6654), formula: c₃₅H₄₉O₃Br, compound 64 in table 1.

In a manner similar to that described in example 16, compound 65 (substituent R is 4-methyl-2-iodophenyl), compound 66 (substituent R is 4-methyl-3-iodophenyl), and compound 67 (substituent R is 4-chloro-3-methoxyphenyl) were prepared by selecting an appropriate substituted benzyl halide (halogen may be Cl or Br).

Example 17

Weighing diosgenin 4.2g, dissolving in 80mL ethyl acetate in 500mL three-neck flask, adding triethylamine 10mL and 4-bromo-3-methoxybenzyl bromide 3.0g, stirring, freezing to-10 deg.C, reacting for 72h under constant temperature, and performing HPLC (chromatographic conditions: Agilent Zorbax SB-C18(4.6mm × 150mm,5um) on chromatographic column, acetonitrile-water (90: 10) as mobile phase, and flow rate of 1.0mL.min^-1Detecting the total reaction of diosgenin at a detection wavelength of 223nm and a column temperature of 35 ℃), heating to room temperature, keeping the temperature for 2h, steaming under reduced pressure until the liquid volume is 20mL, cooling to 5 ℃, crystallizing overnight, filtering, tracing the reaction and the separation and purification process of the product by HPLC, and drying the solid at 60 ℃ for 4h to obtain 5.0g of a light yellow powdery product. Melting point of the target product: the temperature of the mixture is 194.0 to 195.6 ℃,¹³C NMR(101MHz,CDCl₃) δ 157.02,140.27,136.65,130.56,124.38,120.87,119.62,110.44,104.69,88.05,82.74,72.54,66.56,62.40,56.97,55.46,50.22,42.61,40.60,37.87,37.62,37.23,36.27,32.84,32.62,31.32,31.97,30.08,29.90,28.92,21.18,20.09,18.61,15.98, 15.34. TOF-HRMS: m/e (613.6658), formula: c₃₅H₄₉O₄Br, compound 68 in table 1.

In a manner similar to that described in example 17, compound 69 (substituent R is 4-fluoro-3-methoxyphenyl), compound 70 (substituent R is 4-iodo-3-methoxyphenyl), compound 71 (substituent R is 2-chloro-3-methoxyphenyl), compound 72 (substituent R is 2-bromo-3-methoxyphenyl), compound 73 (substituent R is 2-fluoro-3-methoxyphenyl), and compound 74 (substituent R is 2-iodo-3-methoxyphenyl) can be prepared by using an appropriate substituted benzyl halide (halogen may be Cl or Br).

Example 18

Weighing diosgenin 4.2g, dissolving in dichloromethane 100mL, adding diethylamine 10mL and 5-chloro-3-methoxybenzyl chloride 2.9g, stirring, reacting at 20 deg.C for 18h, and performing HPLC (chromatography conditions: Agilent Zorbax SB-C18(4.6mm × 150mm,5um) with mobile phase of acetonitrile-water (90: 10) and flow rate of 1.0mL. min^-1Detecting the total reaction of diosgenin at a detection wavelength of 223nm and a column temperature of 35 ℃), steaming out dichloromethane until the volume of liquid is reduced to 1/4, cooling to 5 ℃, crystallizing overnight, filtering, tracing the separation and purification process of reaction and product by HPLC, and drying the solid at 60 ℃ for 4h to obtain 4.1g of yellow powdery product. Melting point of the target product: 193.9-195.4 ℃ of the total weight of the mixture,¹³C NMR(CDCl₃100MHz) delta 158.61,140.25,131.55,126.56,122.65,120.85,119.60,111.47,105.57,88.03,82.72,73.77,66.54,62.38,56.95,56.07,50.20,42.59,40.58,37.85,37.60,37.21,36.25,32.82,32.60,31.30,31.95,30.06,29.88,28.90,21.16,20.07,18.59,15.96, 15.32. TOF-HRMS: m/e (569.2141), formula: c₃₅H₄₉O₄Cl, compound 75 in table 1.

Compound 76 (substituent R is 5-bromo-3-methoxyphenyl) and compound 77 (substituent R is 5-fluoro-3-methoxyphenyl) can be prepared in a similar manner to example 18, using the appropriate substituted benzyl halide (halogen can be Cl or Br).

Example 19

Weighing diosgenin 4.2g, dissolving in dichloromethane 100mL, adding trimethylamine 10mL, piperidine mL, and 5-iodo-3-methoxybenzyl bromide 3.5g, stirring, reacting at 40 deg.C for 8h, and performing HPLC (under chromatographic conditions of Agilent Zorbax SB-C18(4.6mm × 150mm,5um) on chromatographic column, acetonitrile-water (90: 10) as mobile phase, and flow rate of 1.0mL.min^-1Detecting the total reaction of diosgenin at detection wavelength of 223nm and column temperature of 35 deg.C, and evaporating dichloromethane to liquidThe product was reduced to 1/4, cooled to 5 ℃ for crystallization overnight, filtered, HPLC followed the reaction and product separation and purification, and the solid was dried at 60 ℃ for 4h to give 4.1g of product as a yellow powder. Melting point of the target product: at the temperature of between 195.5 and 197.4 ℃,¹³C NMR(CDCl₃100MHz) delta 158.78,140.24,139.34,131.56,128.60,120.84,119.59,110.85,90.94,88.02,82.71,71.98,66.53,62.37,56.94,55.05,50.19,42.58,40.57,37.84,37.59,37.20,36.24,32.81,32.59,31.29,31.94,30.05,29.87,28.89,21.15,20.06,18.58,15.95, 15.31. TOF-HRMS: m/e (660.6662), formula: c₃₅H₄₉O₄I, compound 78 in table 1.

Example 20

Weighing diosgenin 4.2g, dissolving in tetrahydrofuran 100mL, adding diethylamine 10mL and 5-chloro-3-ethoxybenzyl chloride 3.0g, stirring, reacting at 66 deg.C (heating reflux) for 0.5h, and performing HPLC (chromatographic conditions: Agilent Zorbax SB-C18(4.6mm × 150mm,5um) with mobile phase of acetonitrile-water (90: 10) and flow rate of 1.0mL.min^-1Detecting the total reaction of diosgenin at a detection wavelength of 223nm and a column temperature of 35 ℃), steaming out dichloromethane until the volume of liquid is reduced to 1/4, cooling to 5 ℃, crystallizing overnight, filtering, tracing the separation and purification process of reaction and product by HPLC, and drying the solid at 60 ℃ for 4 hours to obtain 3.6g of yellow powdery product. Melting point of the target product: the temperature of the mixed solution is between 191.5 and 193.4 ℃,¹³C NMR(CDCl₃100MHz) delta 158.75,140.25,131.65,126.64,122.75,120.85,119.60,111.57,105.67,88.03,82.72,73.87,66.54,62.38,56.95,58.06,50.20,42.59,40.58,37.85,37.60,37.21,36.25,32.82,32.60,31.30,31.95,30.06,29.88,28.90,21.16,20.07,18.59,18.25,15.96, 15.32. TOF-HRMS: m/e (583.22389), formula: c₃₆H₅₁O₄Cl, compound 79 in table 1.

In a similar manner to example 20, compound 80 (substituent R is 5-bromo-3-ethoxyphenyl), compound 81 (substituent R is 5-fluoro-3-ethoxyphenyl), and compound 82 (substituent R is 5-iodo-3-ethoxyphenyl) were prepared by selecting the appropriate substituted benzyl halide (halogen may be Cl or Br).

Example 21

Weighing diosgenin 4.2g, dissolving in DMF 60mL in 500mL three-necked flask, adding triethylamine 10mL and benzyl chloride 3.0g, stirring, heating to 110 deg.C, reacting for 2h under constant temperature, and performing HPLC (chromatographic conditions: Agilent Zorbax SB-C18(4.6mm × 150mm,5um) with mobile phase of acetonitrile-water (90: 10) at flow rate of 1.0mL.min^-1Detecting the total reaction of diosgenin at a detection wavelength of 223nm and a column temperature of 35 ℃), steaming under reduced pressure until the volume of liquid is 20mL, cooling to 5 ℃, crystallizing overnight, filtering, tracing the separation and purification process of the reaction and the product by HPLC, and drying the solid at 60 ℃ for 4 hours in vacuum to obtain 4.4g of a light yellow powdery product. Melting point of the target product: the temperature of the mixture is 186.2-188.3 ℃,¹³C NMR(100MHz,CDCl₃) δ 140.85,137.53,128.62,128.62,127.80,127.42,127.42,121.85,119.63,87.17,82.92,72.83,66.92,62.84,56.64,50.81,42.04,40.81,40.03,39.58,37.73,37.50,32.81,32.11,32.05,31.31,30.33,29.54,28.86,21.12,19.33,17.06,15.52, 14.82. TOF-HRMS: m/e (504.7417), formula: c₃₄H₄₈O₃Compound 83 in table 1.

Example 22

Screening experiment for inhibitory activity on proliferation of bile duct cancer cell QBC-939 and human pancreatic cancer cell PANC-1

Collecting bile duct cancer cell QBC-939 and human pancreatic cancer cell PANC-1 with good growth state in logarithmic growth phase at 2 × 10³Per 0.1 mL/well, plated in 96-well plates, each set of 5 parallel wells, 5% CO at 37 ℃²Culturing in incubator, culturing in culture solution containing 10% calf serum for 24 hr, and respectively changing to compound 1-83[ concentration of 0.2ug/mL ] in Table 1]The culture medium is continuously cultured, the culture medium is added into a negative group, 0.2ug/mL 5-fluorouracil (national drug group chemical reagent Co., Ltd.) is added into a control group 1, and 0.2ug/mL diosgenin (Aladdin chemical reagent Co., Ltd.) is added into a control group 2. After 24h of culture, adding 20uL (5mg/mL) of MTT (methanol to ethanol) for further culture for 4h, discarding the supernatant, adding 100uL of dimethyl sulfoxide (DMSO), oscillating for 10min, after the precipitate is completely dissolved, adjusting the wavelength to 570nm on an enzyme-linked detection analyzer, adjusting the blank to zero, measuring the OD value of each well, and repeating the steps for 3 times.The inhibition rate is (1-drug-added OD value/negative group OD value) × 100%

The results of the experiments on the inhibitory activity of synthesized typical compounds 1 to 83 (prepared according to the examples of the present invention), 5-fluorouracil and diosgenin on the proliferation of bile duct cancer cells QBC-939 and human pancreatic cancer cells PANC-1 are shown in Table 2.

(R groups are shown in Table 2)

TABLE 2 inhibition of proliferation of bile duct cancer QBC-939 and human pancreatic cancer PANC-1 cells

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of diosgenin derivatives in preparing medicines for treating pancreatic cancer and cholangiocarcinoma is characterized in that the diosgenin derivatives have a structure shown as a formula (I):

or the diosgenin derivative is a pharmaceutically acceptable salt of the compound of formula (I);

r is selected from the following groups:

removing device

Outer, R₁Substituted on the benzene ring, mono-or disubstituted, trisubstituted, tetrasubstituted, R₁Selected from halogen, R₂Is C_1-5An alkyl group;

r is

When R is₁Is mono-or disubstituted, trisubstituted, tetrasubstituted, pentasubstituted, R₁Is F.

2. Use according to claim 1, characterised in that

Outer, R₁One or more selected from the group consisting of F, Cl, Br and I; r₂Is C_1-3An alkyl group.

3. Use according to claim 1 or 2, wherein R is selected from

When R is₁Is monosubstituted, R₁Selected from halogen, R₂Is methyl or ethyl.

4. Use according to claim 1 or 2, wherein R is selected from the following groups: o-fluorophenyl group, m-fluorophenyl group, p-fluorophenyl group, (3, 4-difluoro) phenyl group, (2, 3-difluoro) phenyl group, (2, 4-difluoro) phenyl group, (2, 5-difluoro) phenyl group, (2, 6-difluoro) phenyl group, (3, 5-difluoro) phenyl group, (2,4, 6-trifluoro) phenyl group, (3,4, 5-trifluoro) phenyl group, pentafluorophenyl group, 2-methoxy-3-chlorophenyl group, 2-methoxy-4-chlorophenyl group, 2-methoxy-5-chlorophenyl group, 2-methoxy-6-chlorophenyl group, 2-methoxy-3-fluorophenyl group, 2-methoxy-4-fluorophenyl group, 2-methoxy-5-fluorophenyl group, 2-methoxy-6-fluorophenyl group, m-fluorophenyl group, p-fluorophenyl group, 3, 4-difluoro) phenyl group, 2, 3-difluoro) phenyl group, 2, 2-methoxy-3-bromophenyl, 2-methoxy-4-bromophenyl, 2-methoxy-5-bromophenyl, 2-methoxy-3-iodophenyl, 2-methoxy-4-iodophenyl, 2-methoxy-5-iodophenyl, 4-methoxy-2-chlorophenyl, 4-methoxy-3-chlorophenyl, 4-methoxy-2-fluorophenyl, 4-methoxy-3-fluorophenyl, 4-methoxy-2-bromophenyl, 4-methoxy-3-bromophenyl, 4-methoxy-2-iodophenyl, 4-methoxy-3-iodophenyl, 2-methyl-3-chlorophenyl, methyl-3-pyridyl, methyl-2, 2-methyl-4-chlorophenyl group, 2-methyl-5-chlorophenyl group, 2-methyl-6-chlorophenyl group, 2-methyl-3-fluorophenyl group, 2-methyl-4-fluorophenyl group, 2-methyl-5-fluorophenyl group, 2-methyl-6-fluorophenyl group, 2-methyl-3-bromophenyl group, 2-methyl-4-bromophenyl group, 2-methyl-5-bromophenyl group, 2-methyl-6-bromophenyl group, 2-methyl-3-iodophenyl group, 2-methyl-4-iodophenyl group, 2-methyl-5-iodophenyl group, 3-methyl-2-chlorophenyl group, 3-methyl-4-chlorophenyl group, 2-methyl-6-fluorophenyl group, 2-methyl-3-iodophenyl group, 2-methyl-5-iodo, 3-methyl-5-chlorophenyl group, 3-methyl-4-fluorophenyl group, 3-methyl-5-fluorophenyl group, 3-methyl-4-bromophenyl group, 3-methyl-5-bromophenyl group, 3-methyl-4-iodophenyl group, 3-methyl-5-iodophenyl group, 4-methyl-2-chlorophenyl group, 4-methyl-3-chlorophenyl group, 4-methyl-2-fluorophenyl group, 4-methyl-3-fluorophenyl group, 4-methyl-2-bromophenyl group, 4-methyl-3-bromophenyl group, 4-methyl-2-iodophenyl group, 4-methyl-3-iodophenyl group, 4-chloro-3-methoxyphenyl group, 3-fluorophenyl group, 3-methyl-4-fluorophenyl group, 3-iodophenyl group, 3-methyl-2-iodophenyl group, 3-methyl-3-, 4-bromo-3-methoxyphenyl, 4-fluoro-3-methoxyphenyl, 4-iodo-3-methoxyphenyl, 2-chloro-3-methoxyphenyl, 2-bromo-3-methoxyphenyl, 2-fluoro-3-methoxyphenyl, 2-iodo-3-methoxyphenyl, 5-chloro-3-methoxyphenyl, 5-bromo-3-methoxyphenyl, 5-fluoro-3-methoxyphenyl, 5-iodo-3-methoxyphenyl, 5-chloro-3-ethoxyphenyl, 5-bromo-3-ethoxyphenyl, 5-fluoro-3-ethoxyphenyl and 5-iodo-3-ethoxyphenyl.

5. The preparation method of the diosgenin derivative shown in the formula (I) is characterized by comprising the steps of taking diosgenin as an initial reactant and reacting with RCH (Richardson fatty acid)₂X reacts to obtain a compound shown in a formula (I);

wherein R is as defined in any one of claims 1 to 4, X is halogen;

a compound of formula (I):

r is selected from the following groups:

removing device

r is

6. A process according to claim 5, which comprises dissolving diosgenin in a solvent and adding RCH₂X reacts with alkaline substances, and the solution is kept alkaline in the reaction process to prepare the compound shown in the formula (I);

the solvent is selected from any one of n-propanol, isopropanol, n-butanol, ethyl acetate, butyl acetate, anhydrous ethanol, methanol, dichloromethane and tetrahydrofuran.

7. The process according to claim 6, wherein the reaction temperature is-20 to 300 ℃ and the reaction time is 0.1 to 72 hours.

8. A process according to claim 6, wherein the solvent is used in an amount such that each mole of diosgenin is dissolved in 5-100L of solvent.

9. The method according to claim 6, wherein the alkaline substance is selected from γ -Al₂O₃-Na、γ-Al₂O₃-K、γ-Al₂O₃-NaOH-Na、γ-Al₂O₃-NaOH-K、γ-Al₂O₃-KOH-Na、γ-Al₂O₃-KOH-K, sodium methoxide, sodium ethoxideOne or more of sodium propoxide, sodium butoxide, sodium amide, sodium metal, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium oxide, potassium oxide, calcium oxide, strontium carbonate, potassium carbonate, sodium carbonate, potassium phosphate, ammonia, ethylamine, diethylamine, triethylamine, methylamine, dimethylamine, trimethylamine, pyridine, and piperidine.

10. The method according to claim 9, wherein the basic substance is selected from the group consisting of trimethylamine, triethylamine, and γ -Al₂O₃-NaOH-K、γ-Al₂O₃KOH-Na, sodium hydride, sodium ethoxide, strontium carbonate, potassium carbonate, diethylamine.

11. The method of claim 6, wherein the diosgenin and RCH are administered in the form of a solution₂X and the molar ratio of the alkaline substances is 1: (0.05-15): (0.01-15).

12. The process according to claim 6, further comprising detecting the end of the reaction by chromatography, allowing the reaction mixture to stand at room temperature after the reaction is completed, and separating and purifying the reaction mixture to obtain the compound of formula (I).

13. The method according to claim 12, wherein the separation and purification is performed by a method selected from the group consisting of filtration, resin treatment, water washing, distillation, crystallization, extraction, activated carbon treatment, molecular sieve treatment, and chromatography.