A stigmasterol triazole derivative and its preparation method and application

Abstract

The present invention provides a stigmasterol triazole derivative represented by formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a preparation method thereof, and use thereof in preparing a drug for treating diseases related to anti-tumor activity.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a stigmasterol triazole derivative, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and a preparation method thereof, and use thereof in preparing a drug for treating diseases related to anti-tumor activity. Background Art

[0002] Cancer is a major public health problem and has become one of the most common causes of death in humans. With the continuous advancement of medicine, chemical prevention of tumors is one of the most direct ways to reduce the incidence of cancer. People are also constantly understanding and discovering natural products in order to find new therapeutic drugs from plants. In recent years, the anti-cancer effect of phytosterols has gradually been confirmed, especially for high-incidence cancers such as gastric cancer, breast cancer, lung cancer and ovarian cancer. Plant sterols have achieved good anti-cancer effects. Related studies have shown that compounds obtained by using a steroid structure as a parent and an aromatic heterocyclic structure as an external structure will have different physiological activities from the steroid parent itself, and they often show high-efficiency anti-tumor, antibacterial or antiviral activity.

[0003]

[0004] Stigmasterol, also known as stigmaster-5,22-diene-3β-ol, is a type of plant sterol widely distributed in nature. As one of the typical representatives of plant sterols, stigmasterol is widely present in various plants and deodorized distillates of vegetable oil refining, accounting for about 22%. The molecular structure is shown in the figure. As a natural active substance, stigmasterol has the characteristics of high nutritional value and strong physiological activity. Previous studies have found that its main physiological functions include anti-tumor effect, lowering human blood lipid concentration, anti-oxidation, analgesia, anti-diabetes and other pharmacological functions.

[0005] In recent years, with the isolation of stigmasterol as a single component, a large number of stigmasterol derivatives have been designed and synthesized. By modifying their structures to improve their solubility, their application range has become wider, and some new properties have also been developed. It is well known that aromatic rings with conjugated systems and negative electron characteristics are widely used in the structural modification of lead compounds, which can interact with target proteins in π-π ways and increase affinity with the target. Studies have found that different types of substituents in aromatic rings are closely related to charge distribution, the properties of compounds, and anti-tumor efficacy, especially halogen groups, which significantly enhance biological activity and improve the metabolic stability of the parent.

[0006] Triazole is a five-membered heterocyclic ring containing three nitrogen atoms, which has antibacterial, anti-allergic, anti-HIV, anti-tumor, anti-tuberculosis and other biological activities. In recent years, it is common to use natural active substances as lead compounds, introduce different types of triazole groups on the skeleton, and explore their structure-activity relationship. It can be seen that the introduction of triazole into the parent molecule can change the pharmacokinetic properties and solubility of the parent molecule and enhance the biological activity of the parent molecule. However, the existing literature reports have not disclosed the modification of stigmasterol with a heterocyclic ring containing triazole and the provision of a lead compound with better anticancer activity. Based on the existing public inhibition structure, the present invention screens and obtains a series of novel and highly active structural compounds. Summary of the invention

[0007] The object of the present invention is to provide a stigmasterol triazole derivative as shown in the structure of formula (I), specifically a compound as shown in formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0008]

[0009] Wherein, A, B, and C are independently selected from C or N, and at least one of A, B, and C is selected from N;

[0010] R is selected from any one of hydrogen, halogen, -OH, substituted or unsubstituted -C1-6 alkyl, substituted or unsubstituted -C2-6 alkenyl, substituted or unsubstituted -C2-6 alkynyl, substituted or unsubstituted -C1-6 alkoxy, substituted or unsubstituted -C6-C10 aryl, substituted or unsubstituted -C6-C10 heteroaryl;

[0011] Wherein, the substituted -C1-6 alkyl, substituted -C2-6 alkenyl, substituted -C2-6 alkynyl, substituted -C1-6 alkoxy, substituted -C6-C10 aryl, substituted -C6-C10 aryl, substituted -C6-C10 heteroaryl is R 1 Substitute, the R 1 Any one selected from hydrogen, -C1-C6 alkyl, halogen, -C1-C6 alkoxy.

[0012] Preferably, the R is selected from substituted or unsubstituted -C1-6 alkyl, substituted or unsubstituted -C6-C10 aryl, wherein the substituted -C1-6 alkyl, substituted -C6-C10 aryl is R 1 Substitute, the R 1 Any one selected from hydrogen, -C1-C6 alkyl, halogen, -C1-C6 alkoxy.

[0013] More preferably, the -C6-C10 aryl group is phenyl, the -C1-C6 alkoxy group is methoxy or ethoxy, and the halogen group is fluorine, chlorine or bromine.

[0014] Preferably, A, B, and C are independently selected from N; R is selected from More preferably, the compound described in formula I specifically comprises any one of the following:

[0015]

[0016] The present invention also provides a method for preparing the compound of formula (I), comprising the following preparation method:

[0017]

[0018] Step a: In an organic solvent, stigmasterol 1 reacts with an azide compound in the presence of triphenylphosphine and azodicarbonate, after which a dilute alkali solution is added for quenching, and then an organic solvent is added for extraction and separation to obtain a compound 2 in which the hydroxyl group is replaced by an azide;

[0019] Step b: Compound 2 reacts with a copper salt and an acetylene compound in a polar alkaline solvent. After the reaction is completed, an organic solvent is added for extraction and separation to obtain a compound of formula (I), wherein A, B, C and the substituent R in formula (I) are as defined in any one of claims 1 to 6.

[0020] Preferably, the organic solvent in step a is selected from one or two of ether, tetrahydrofuran, 1,4-dioxane, methyl tert-butyl ether, acetonitrile, toluene or xylene; the azodicarbonate is diethyl azodicarbonate (DEAD) or diisopropyl azodicarbonate (DIAD); the azide compound is bis(P-nitrophenyl) azidophosphate, 15-azido-4,7,10,13-tetraoxypentadecanoic acid-N-succinimidyl ester, diphenyl phosphate azide or azide-heptaethylene glycol-pentafluorobenzene ester; in the step b: the acetylene compound is hexyne, p-methoxyphenylacetylene, 2-fluorophenylacetylene, 4-fluorophenylacetylene, 3-chlorophenylacetylene, 4-chlorophenylacetylene, 2-bromophenylacetylene or 3-bromophenylacetylene; the polar solvent is selected from one of water-methanol, water-ethanol, water-isopropanol, water-n-butanol or water-tert-butanol; the copper salt is selected from one of copper sulfate, copper acetate, copper chloride, copper nitrate or copper carbonate; the base is selected from one of sodium carbonate, sodium bicarbonate, sodium sulfite, sodium bisulfite or sodium ascorbate.

[0021] The present invention also provides a pharmaceutical composition, characterized in that: the preparation is prepared by using the compound, or its stereoisomer, or its pharmaceutically acceptable salt as a pharmaceutical active ingredient and adding pharmaceutically acceptable excipients.

[0022] The present invention also provides the use of the compound represented by formula (I), or its stereoisomer, or its pharmaceutically acceptable salt, or the pharmaceutical composition in the preparation of drugs for treating anti-tumor and / or anti-cancer diseases.

[0023] As further preferred, the anticancer is selected from human cervical cancer cells, human gastric cancer cells or human renal epithelial cells.

[0024] The following is an explanation and description of the terms of the present invention:

[0025] "Cancer" or "malignancy" refers to any of a variety of diseases characterized by uncontrolled abnormal proliferation of cells, the ability of affected cells to spread locally or through the bloodstream and lymphatic system to other parts of the body (i.e., metastasis), and any of a number of characteristic structural and / or molecular features. "Cancer cells" refer to cells in the early, intermediate, or advanced stages of multi-step tumor progression. Cancers include sarcomas, breast cancer, lung cancer, brain cancer, bone cancer, liver cancer, kidney cancer, colon cancer, and prostate cancer. In some embodiments, the compounds of Formula I are used to treat cancers selected from colon cancer, brain cancer, breast cancer, fibrosarcoma, human cervical cancer cells, human gastric cancer cells, and human renal epithelial cells and squamous cell carcinoma. In some embodiments, cancer is selected from human cervical cancer cells, human gastric cancer cells, and human renal epithelial cells.

[0026] The compounds and derivatives provided in the present invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature system.

[0027] Definitions of terms used in the present invention: Unless otherwise stated, the initial definitions provided for groups or terms in this document apply to the groups or terms throughout the specification; for terms that are not specifically defined in this document, the meaning that a person skilled in the art can give them should be given based on the disclosure and context.

[0028] "Substitution" refers to the replacement of a hydrogen atom in a molecule by another different atom or molecule.

[0029] The minimum and maximum carbon atom content of a hydrocarbon group is indicated by a prefix, for example, the prefix C1-b alkyl indicates any alkyl group containing from "a" to "b" carbon atoms. Thus, for example, "C1-4 alkyl" refers to an alkyl group containing 1 to 4 carbon atoms.

[0030] "Alkyl" refers to a saturated hydrocarbon chain having a specified number of member atoms. For example, C1-6 alkyl refers to an alkyl group having 1 to 6 member atoms, such as 1 to 4 member atoms. The alkyl group can be straight or branched. Representative branched alkyl groups have one, two or three branches. The alkyl group may be optionally substituted with one or more substituents as defined herein. Alkyl includes methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl and tert-butyl), pentyl (n-pentyl, isopentyl and neopentyl) and hexyl. The alkyl group may also be part of other groups, such as C1-C6 alkoxy.

[0031] "Cycloalkyl", "cycloalkane" refers to a saturated or partially saturated cyclic group having carbon atoms and no ring heteroatoms and having a single ring or multiple rings (including fused, combined and). For polycyclic systems with aromatic and non-aromatic rings without ring heteroatoms, when the point of attachment is located at a non-aromatic carbon atom, the term "cycloalkyl" (e.g., 5,6,7,8,-tetrahydronaphthalene-5-yl) is applicable. The term "cycloalkyl" includes cycloalkenyl groups, such as cyclohexenyl. Examples of cycloalkyl groups include, for example, adamantyl, cyclopropyl, cyclobutyl, cyclohexyl, cyclopentyl, cyclooctyl, cyclopentenyl and cyclohexenyl. Examples of cycloalkyl groups including polybicycloalkyl ring systems are bicyclohexyl, bicyclopentyl, bicyclooctyl, etc.

[0032] "Alkenyl" refers to a straight or branched chain hydrocarbon group having 2 to 10 carbon atoms, and in some embodiments 2 to 6 carbon atoms or 2 to 4 carbon atoms, and having at least 1 site of vinyl unsaturation (>C=C<). For example, (Ca-Cb)alkenyl refers to an alkenyl group having a to b carbon atoms and is meant to include, for example, ethenyl, propenyl, isopropenyl, 1,3-butadienyl, and the like.

[0033] "Alkynyl" refers to a straight chain monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical containing at least one triple bond. The term "alkynyl" is also intended to include those hydrocarbon groups having one triple bond and one double bond. For example, (C2-C6)alkynyl is intended to include ethynyl, propynyl, and the like.

[0034] "Halogen" is fluorine, chlorine, bromine or iodine.

[0035] "Halogen alkyl" refers to an alkyl group in which the hydrogen atoms may be replaced by one or more halogen atoms. For example, C1-4 halogen alkyl refers to an alkyl group containing 1 to 4 carbon atoms in which the hydrogen atoms are replaced by one or more halogen atoms.

[0036] "Heterocycle", "heterocycloalkyl" and "heterocycloalkane" refer to a saturated ring or a non-aromatic unsaturated ring containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, or a sulfur atom;

[0037] "Aromatic heterocycle" refers to an aromatic unsaturated ring containing at least one heteroatom; wherein the heteroatom refers to a nitrogen atom, an oxygen atom, or a sulfur atom;

[0038] The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist as different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including but not limited to, diastereomers, enantiomers, atropisomers, and mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane polarized light. When describing optically active compounds, the prefix D, L or R, S is used to indicate the absolute configuration of the chiral center of the molecule. The chemical structure of these stereoisomers is the same, but their stereostructure is different. Specific stereoisomers can be enantiomers, and mixtures of isomers are generally called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may result in no stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers that lacks optical activity.

[0039] The term "pharmaceutically acceptable" means that a carrier, vehicle, diluent, excipient, and / or formed salt is generally chemically or physically compatible with the other ingredients that constitute a pharmaceutical dosage form and physiologically compatible with the receptor.

[0040] The pharmaceutical composition of the present invention can be any reusable pharmaceutical preparation form, such as oral, injection, external use, etc., and the oral dosage form includes but is not limited to tablets, capsules, oral liquids, granules, pills, suspensions, injections selected from water injections and powder injections, and external preparations selected from patches and ointments. All preparations can be prepared according to conventional pharmaceutics techniques, such as using the compound of the present invention, or any one of its stereoisomers, or its pharmaceutically acceptable salt as a pharmaceutically active ingredient, adding a pharmaceutically acceptable carrier if necessary, to prepare the above-mentioned pharmaceutical dosage form suitable for taking, wherein the pharmaceutically active ingredient per unit dose can be 0.1 mg-1000 mg, such as each tablet containing 0.1 mg-1000 mg, preferably 5-500 mg, of the pharmaceutically active ingredient.

[0041] The terms "salt" and "pharmaceutically acceptable salt" refer to acidic and / or basic salts of the above-mentioned compounds or their stereoisomers, formed with inorganic and / or organic acids and bases, and also include zwitterionic salts (inner salts), and also include quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final separation and purification of the compound. It can also be obtained by mixing the above-mentioned compound, or its stereoisomer, with a certain amount of acid or base appropriately (e.g., equivalent). These salts may form a precipitate in the solution and be collected by filtering, or be recovered after solvent evaporation, or be obtained by freeze drying after reaction in an aqueous medium. The salt described in the present invention can be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

[0042] In certain embodiments, one or more compounds of the present invention may be used in combination with each other. It is also possible to select the compounds of the present invention and any other active agent for use in combination to prepare a drug or pharmaceutical composition for regulating cell function or treating a disease. If a group of compounds is used, these compounds may be administered to a subject simultaneously, separately or sequentially.

[0043] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0044] Beneficial technical effects of the present invention:

[0045] 1. The present invention modifies the hydroxyl group at position 3 of stigmasterol to provide a novel stigmasterol triazole derivative, thereby enriching the structural types of steroid compounds;

[0046] 2. The preparation method of the stigmasterol triazole derivative provided by the present invention is simple, the raw materials are cheap and readily available, the reaction conditions are mild, the post-treatment is simple, the yield is high, reaching more than 71%, and the solvent can be recycled, which is conducive to industrial production;

[0047] 3. The present invention introduces triazole active substructure units into the stigmasterol skeleton, greatly improving its anti-tumor activity, and obtains a plurality of stigmasterol triazole derivatives with high anti-tumor activity, providing a research basis and lead compounds for the development of new anti-cancer drugs, and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present invention. Moreover, the same reference numerals are used throughout the accompanying drawings to represent the same components. In the accompanying drawings:

[0049] Figure 1 The H NMR spectrum of compound 2 is schematically shown;

[0050] Figure 2 The carbon NMR spectrum of compound 2 is schematically shown;

[0051] Figure 3 The H NMR spectrum of compound 3a is schematically shown;

[0052] Figure 4 The carbon NMR spectrum of compound 3a is schematically shown;

[0053] Figure 5 The H NMR spectrum of compound 3b is schematically shown;

[0054] Figure 6 The carbon NMR spectrum of compound 3b is schematically shown;

[0055] Figure 7 The H NMR spectrum of compound 3c is schematically shown;

[0056] Figure 8 The carbon NMR spectrum of compound 3c is schematically shown;

[0057] Fig. 9 The H NMR spectrum of compound 3d is schematically shown;

[0058] Fig.10 The carbon NMR spectrum of compound 3d is schematically shown;

[0059] Fig.11 The H NMR spectrum of compound 3e is schematically shown;

[0060] Fig.12 The carbon NMR spectrum of compound 3e is schematically shown;

[0061] Fig.13 The H NMR spectrum of compound 3f is schematically shown;

[0062] Fig.14 The carbon NMR spectrum of compound 3f is schematically shown;

[0063] Fig.15The H NMR spectrum of compound 3g is schematically shown;

[0064] Fig.16 The carbon NMR spectrum of compound 3g is schematically shown;

[0065] Fig.17 The H NMR spectrum of compound 3h is schematically shown;

[0066] Fig.18 The carbon NMR spectrum of compound 3h is schematically shown. DETAILED DESCRIPTION

[0067] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art.

[0068] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The NMR shift (δ) is given in units of 10-6 (ppm). The NMR measurements were performed using a (Bruker Avance III 400 and Bruker Avance 300) nuclear magnetic spectrometer, and the measurement solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl 3 ), deuterated methanol (CD3OD), and tetramethylsilane (TMS) as the internal standard. The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from commercial reagent platforms.

[0069] Preparation of intermediate compound 2

[0070]

[0071] Take a 250mL round-bottom flask, weigh triphenylphosphine (1.44g, 5.5mmol), dissolve it in 35mL of tetrahydrofuran and stir to dissolve, then add diisopropyl azodicarboxylate (1.18mL, 6mmol), stir at 0℃ for 10min, then add stigmasterol (2.06g, 5mmol), continue stirring for ten minutes, then slowly add diphenylphosphoryl azide (1.29mL, 6mmol), remove the ice bath, warm to room temperature, stir for 10h, and then detect by TLC. Prepare 0.1mol / L sodium hydroxide and add it for quenching, extract the reaction solution with dichloromethane, and extract the remaining aqueous phase with dichloromethane again, repeat the extraction three times, combine the obtained organic phases, wash with saturated brine, and then dry with anhydrous sodium sulfate to obtain crude product 2, and reduce the pressure and concentrate the obtained crude product 3, and separate it by column chromatography to obtain compound 2. Yield 66.5%; characterization spectrum as shown Figure 1 , Figure 2 As shown; 1 H NMR (400 MHz, CDCl 3 )δ5.40(d,J=2.3Hz,1H),5.16(dd,J=15.1,8.6Hz,1H),5.02(dd,J=15.1,8.6Hz,1H),3.88(s,1H),2.52(d, J=14.9Hz,1H),2.19(d,J=14.9Hz,1H),2.09–1.91(m,3H),1.74(dd,J=21.8,10.5Hz,3H),1.65(d,J=13.3H z,2H),1.53(d,J=9.2Hz,5H),1.48–1.35(m,4H),1.27(d,J=12.3Hz,2H),1.17(dd,J=12.7,5.9Hz,3H),1.0 3(s,2H),1.01(d,J=2.6Hz,5H),0.85(d,J=6.1Hz,3H),0.83(s,1H),0.81(s,3H),0.79(s,2H),0.70(s,3H); 13 C NMR (100 MHz, CDCl 3)δ138.30,138.02,129.25,123.14,58.23,56.78,55.89,51.22,49.91,42.17,40 .49,39.61,37.07,36.04,33.61,31.87,31.79,31.77,28.89,26.09,25.39,24.3 2,21.20,21.07,20.69,18.97,12.23,12.02; FT-IR(KBr)ν:3358.26,3127.57,30 63.38,2942.70,1731.28,1457.50,1236.09,1044.71,972.64,838.19,763.08cm -1 ;HR-MS(ESI-TOF)m / z:Calcd forC 29 H 48 N 3 {[M+H] +}438.3835, found438.3836.

[0072] Preparation of compounds 3a-3h

[0073]

[0074] Take two 100mL round-bottom flasks, weigh copper acetate (0.2mmol) and sodium ascorbate (0.4mmol) in the round-bottom flask, add 2mL of water and stir to dissolve, take compound 2 (1mmol) and acetylene compound (1mmol) in another flask, add 10mL of tert-butyl alcohol and stir to dissolve, combine the two systems, and react at room temperature. After the reaction is completed by TLC detection, the organic phase is extracted with ethyl acetate, and the remaining aqueous phase is extracted with ethyl acetate again, and the extraction is repeated three times, the organic phases are combined, washed with saturated brine, and dried with anhydrous sodium sulfate to obtain a crude product 3, and the crude product 3 is decompressed and concentrated, and compound 3a-3h is obtained by column chromatography.

[0075] Example 1: Preparation of Compound 3a

[0076]

[0077] According to the same preparation method as in Example 1, hexyne was added to prepare compound 3a. Compound 3a: yield 92.5%; characterization spectrum as shown Figure 3 , Figure 4 As shown; 1 HNMR (400MHz, CDCl 3)δ7.42(s,1H),5.38(s,1H),5.07(dd,J=15.1,8.6Hz,1H),4.94(dd,J=15.1,8.6Hz,1H),4.77(d,J=1.6Hz,1H),2.84(d,J=14.6H z,1H),2.59(t,J=7.6Hz,2H),2.41(d,J=15.7Hz,1H),2.10(d,J=14.0Hz,1H),2.03(d,J=14.0Hz,1H),2.00–1.90(m,3H),1.89(s, 1H),1.70–1.51(m,5H),1.51–1.35(m,6H),1.35–1.29(m,1H),1.25(dd,J=6.6,3.3Hz,5H),1.17(s,1H),1.14–1.03(m,3H),1.01 (s,3H),0.97(s,2H),0.93(d,J=6.5Hz,4H),0.81(t,J=6.7Hz,3H),0.76(d,J=6.2Hz,3H),0.72(s,3H),0.70(s,2H),0.62(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ146.84,138.02,137.97,129.07,123.74,119.90,56.50,55.98,55.69,50.98,49.89,41. 90,40.22,39.26,36.79,35.32,32.62,31.71,31.61,31.45,31.15,28.90,28.62,26.92,25 .40,25.14,24.04,22.17,20.97,20.82,20.39,19.02,18.75,13.83,11.98,11.78; FT-IR(K Br)ν:3415.11,2958.35,2868.49,1466.89,1367.12,1261.11,1080.50,1025.63,771.62cm -1 ;HR-MS(ESI-TOF)m / z:Calcd for C 36 H 60 N 3 {[M+H] +}534.4782, found 534.4788.

[0078] Example 2: Preparation of Compound 3b

[0079]

[0080] According to the same preparation method as in Example 1, p-methoxyphenylacetylene was added to prepare compound 3b. Compound 3b: yield 86.5%; characterization spectrum as shown Figure 5 , Figure 6 As shown; 1 HNMR (400MHz, CDCl 3 )δ7.85(s,1H),7.65(d,J=8.7Hz,2H),6.88(d,J=8.7Hz,2H),5.50–5.44(m,1H),5.08(dd,J=15.1,8.5Hz,1H),4.94(dd,J=15.1,8.6Hz,1H),4.86( s,1H),3.76(s,3H),2.91(d,J=14.7Hz,1H),2.50(d,J=15.7Hz,1H),2.16 (d,J=14.0Hz,1H),2.12–2.06(m,1H),1.97(d,J=7.0Hz,1H),1.94–1.87( m,1H),1.62(dd,J=14.0,6.6Hz,2H),1.54(d,J=18.3Hz,1H),1.43(ddd,J=11.8,9.2,7.5Hz,6H),1.34(dd,J=8.6,4.7Hz,1H),1.19(d,J=11.5Hz,2 H),1.07(d,J=9.3Hz,3H),1.04(s,3H),0.98(d,J=6.4Hz,2H),0.94(d,J= 6.6Hz,5H),0.77(d,J=6.2Hz,3H),0.73(s,3H),0.71(s,2H),0.63(s,3H). 13 C NMR (100 MHz, CDCl 3 )δ159.40,146.57,138.25,138.20,129.33,126.92,124.27,123.82,118.47,114.18,56.69,56 .53,55.89,55.28,51.20,50.10,42.15,40.43,39.44,37.10,35.56,32.76,32.01,31.84,31.70 ,30.17,29.66,28.84,27.16,25.36,24.27,21.17,21.04,20.61,19.28,18.96,12.20,12.02; FT -IR(KBr)ν:3126.14,2933.10,2869.28,1458.60,1380.55,1261.30,1045.64,971.70,800.19cm-1 ;HR-MS(ESI-TOF)m / z:Calcd for C 38 H 56 N 3 O{[M+H] +}570.4418, found 570.4428.

[0081] Example 3: Preparation of Compound 3c

[0082]

[0083] According to the same preparation method as in Example 1, 2-fluorophenylacetylene was added to prepare compound 3c. Compound 3c: yield 72.7%; characterization spectrum as shown Figure 7 , Figure 8 As shown; 1 H NMR (400 MHz, CDCl 3 )δ8.21(dd,J=10.6,4.5Hz,1H),8.16(d,J=3.5Hz,1H),7.23–7.11(m,2H),7.07–6.99(m,1H),5.47(s,1H),5.12–5 .01(m,1H),4.93(dd,J=15.5,8.9Hz,2H),2.91(d,J=15.3Hz,1H),2.51(d,J=15.6Hz,1H),2.15(t,J=12.7Hz,2H),1 .94(dd,J=36.3,15.1Hz,4H),1.62(d,J=14.1Hz,3H),1.55–1.41(m,5H),1.36(dd,J=17.3,4.1Hz,4H),1.25(d,J=5 .2Hz,1H),1.08(s,3H),1.03(s,5H),0.93(d,J=6.5Hz,4H),0.79–0.74(m,5H),0.72(s,3H),0.62(d,J=7.0Hz,3H). 13 CNMR (100MHz, CDCl 3)δ160.04,157.58,139.79,139.76,137.90,137.52,128.94,128.58,128.50,127.35,127.32,124.36,124.18,124. 15,122.48,122.34,118.73,118.60,115.35,115.13,56.31,56.28,55.52,50.87,49.75,41.79,40.13,39.09,36.7 3,35.26,32.38,31.64,31.52,31.34,29.35,28.53,26.89,25.04,23.94,20.85,20.73,20.27,18.98,18.63,11.89 ,11.69; FT-IR(KBr)ν:3454.49,3179.33,2943.14,1737.87,1467.54,1219.48,1079.39,1047.81,800.02,770.91cm -1 ;HR-MS(ESI-TOF)m / z:Calcd for C 37 H 53 FN 3 {[M+H] +}558.4218, found 558.4218.

[0084] Example 4: Preparation of Compound 3d

[0085]

[0086] According to the same preparation method as in Example 1, 4-fluorophenylacetylene was added to prepare compound 3d. Compound 3d: yield 71.2%; characterization spectrum as shown Fig. 9 , Fig.10 As shown; 1 HNMR (400MHz, CDCl 3)δ7.96(s,1H),7.77(dd,J=8.0,5.7Hz,2H),7.11(t,J=8.5Hz,2H),5.55(s,1H) ,5.15(dd,J=15.1,8.5Hz,1H),5.08–4.89(m,2H),3.00(d,J=14.9Hz,1H),2.58( d,J=15.7Hz,1H),2.13(ddd,J=50.3,28.6,13.4Hz,5H),1.58(tdd,J=36.4,28.3 ,15.6Hz,11H),1.08(dd,J=50.9,10.8Hz,13H),0.88–0.76(m,9H),0.71(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ163.73,161.28,145.90,138.19,129.36,127.38,127.30,124.37,118.98,115.81,115.60 ,56.70,56.66,55.91,51.21,50.13,42.17,40.44,39.44,37.13,35.55,32.75,32.02,31.85 ,31.70,28.84,27.14,25.36,24.28,21.18,21.05,20.62,19.28,18.97,12.21,12.03; FT-IR (KBr)ν:3416.23,3154.02,2816.45,1740.00,1432.51,1262.52,1024.89,868.99,767.41cm -1 ;HR-MS(ESI-TOF)m / z:Calcd for C 37 H 53 FN 3 {[M+H] +}558.4218, found 558.4212.

[0087] Example 5: Preparation of Compound 3e

[0088]

[0089] According to the same preparation method as in Example 1, 3-chlorophenylacetylene was added to prepare compound 3e. Compound 3e: yield 74.4%; characterization spectrum as shown Fig.11 , Fig.12 As shown; 1 HNMR (400MHz, CDCl 3)δ8.00(s,1H),7.80(s,1H),7.69(d,J=7.6Hz,1H),7.35(t,J=7.8Hz,1H),7 .31–7.24(m,1H),5.60–5.52(m,1H),5.15(dd,J=15.2,8.5Hz,1H),5.06–4. 90(m,2H),3.01(d,J=14.2Hz,1H),2.59(d,J=15.7Hz,1H),2.34–1.93(m,5H ),1.80–1.35(m,11H),1.22–0.96(m,14H),0.89–0.75(m,9H),0.71(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ145.45,138.18,138.04,134.68,132.83,130.00,129.32,127.79,125.65,124.43,123.71 ,119.53,56.72,56.61,,55.86,51.18,50.04,42.13,40.42,39.39,37.10,35.49,32.72,31. 97,31.83,31.67,28.82,27.05,25.35,24.25,21.17,21.03,20.59,19.24,18.95,12.20,12. 01; FT-IR(KBr)ν:3148.32,2957.40,2868.32,1458.30,1286.83,1105.53,1077.35,815.37cm -1 ;HR-MS(ESI-TOF)m / z:Calcd forC 37 H 53 C1N 3 {[M+H] +}574.3923, found 574.3916.

[0090] Example 6: Preparation of Compound 3f

[0091]

[0092] According to the same preparation method as in Example 1, 4-chlorophenylacetylene was added to prepare compound 3f. Compound 3f: yield 79.6%; characterization spectrum as shown Fig.13 , Fig.14 As shown; 1 HNMR (400MHz, CDCl 3)δ7.91(s,1H),7.65(d,J=8.4Hz,2H),7.29(d,J=8.4Hz,2H),5.45(d,J=1.8Hz,1H),5.07(dd,J=15.2,8.5Hz,1H),4.94(dd,J=15. 1,8.6Hz,1H),4.84(s,1H),2.90(d,J=14.8Hz,1H),2.49(d,J=15.7Hz,1H),2.14(dd,J=17.4,9.0Hz,1H),2.04(d,J=29.2Hz,1H),1 .94(dt,J=18.1,10.7Hz,3H),1.62(d,J=14.1Hz,2H),1.51(s,2H),1.49–1.42(m,3H),1.26(s,1H),1.20(s,2H),1.17(s,2H),1.0 6(d,J=8.5Hz,3H),1.03(s,4H),0.93(d,J=6.5Hz,4H),0.77(d,J=6.2Hz,4H),0.74(s,1H),0.72(s,3H),0.71(s,2H),0.62(s,3H); 13 C NMR (100 MHz, CDCl 3 )δ145.63,138.14,138.08,133.50,129.53,129.31,128.89,126.82,124.35,119.25,56.6 6,56.63,55.85,51.16,50.07,42.11,40.39,39.39,37.07,35.48,32.71,31.96,31.81,31 .64,29.64,28.80,27.05,25.32,24.23,21.15,21.02,20.58,19.22,18.94,12.18,11.98; FT-IR(KBr)ν:3105.61,1956.76,2867.68,1498.62,1249.27,1177.53,1035.19,836.61cm -1 ;HR-MS(ESI-TOF)m / z:Calcd for C 37 H 53 C1N 3 {[M+H] +}574.3923, found574.3925.

[0093] Example 7: Preparation of Compound 3g

[0094]

[0095] According to the same preparation method as in Example 1, 2-bromophenylacetylene was added to prepare compound 3g. Compound 3g: yield 83.0%; characterization spectrum as shown Fig.15 , Fig.16 As shown; 1 HNMR (400MHz, CDCl 3 )δ8.59(s,1H),8.09(dd,J=7.8,1.4Hz,1H),7.54(d,J=8.0Hz,1H),7.30(dd,J=11.7,4.3Hz,1H),7.08(td,J=8.0,1.6Hz,1H),5.48(s,1 H),5.07(dd,J=15.1,8.5Hz,1H),4.94(dd,J=14.8,8.5Hz,2H),2.91(d,J=14.8Hz,1H),2.47(d,J=15.7Hz,1H),2.10(s,2H),2.07(s,1H ),2.05–1.99(m,1H),2.00–1.93(m,2H),1.93–1.86(m,1H),1.61(d,J=14.2Hz,3H),1.57(s,1H),1.51(d,J=4.9Hz,1H),1.51–1.42(m,4 H),1.26(s,1H),1.19(d,J=11.4Hz,5H),1.03(s,4H),0.93(d,J=6.6Hz,5H),0.77(d,J=6.2Hz,4H),0.72(s,3H),0.62(d,J=6.1Hz,3H); 13 C NMR (100 MHz, CDCl 3 )δ144.05,138.17,138.08,133.49,131.57,130.31,129.28,128.91,127.63,124.70,123.2 7,121.01,56.81,56.54,55.82,51.17,50.18,42.12,40.40,39.44,37.06,35.60,32.55,32. 07,31.81,31.65,30.14,29.64,28.80,27.42,25.34,24.24,21.15,21.03,20.58,19.28,18. 94; FT-IR(KBr)ν:3137.86,2954.22,2866.43,1480.47,1261.53,1091.59,974.55,807.94cm -1 ;HR-MS(ESI-TOF)m / z:Calcd for C 37 H53 Bn 3 {[M+H] +}618.3417, found 618.3419.

[0096] Example 8: Preparation of Compound 3h

[0097]

[0098] According to the same preparation method as in Example 1, 3-bromophenylacetylene was added to prepare compound 3h. Compound 3h: yield 84.9%; characterization spectrum as shown Fig.17 , Fig.18 As shown; 1 HNMR (400MHz, CDCl 3 )δ8.00(s,1H),7.96(s,1H),7.73(d,J=7.7Hz,1H),7.45(d,J=8.0Hz,1H),7.28(dd,J=13.3,5.0Hz,1H),5.56(s,1H),5.15(dd,J =15.1,8.5Hz,1H),5.06–4.93(m,2H),3.01(d,J=15.6Hz,1H),2.59(d,J=15.8Hz,1H),2.23(dd,J=27.5,13.9Hz,2H),2.15–1.93 (m,3H),1.73(d,J=13.9Hz,3H),1.61(s,1H),1.59–1.44(m,5H),1.38(d,J=38.0Hz,1H),1.27(d,J=11.8Hz,2H),1.19–1.15(m,2 H),1.12(s,5H),1.07(s,2H),1.01(d,J=6.5Hz,4H),0.85(d,J=6.1Hz,3H),0.82(s,1H),0.80(s,3H),0.71(s,3H),0.07(s,1H). 13 C NMR (100 MHz, CDCl 3)δ145.27,138.17,137.99,133.07,130.69,130.25,129.27,128.51,124.41,124.15,122.84, 119.50,56.70,56.58,55.82,51.16,49.98,42.10,40.42,39.36,37.07,35.46,32.70,31.94,3 1.81,31.64,28.81,27.02,25.34,24.24,21.16,21.04,20.57,19.22,18.94,12.20,11.99; FT -IR(KBr)ν:3423.41,2952.86,2877.52,1485.44,1260.72,1230.30,817.60,754.67,702.25cm -1 ;HR-MS(ESI-TOF)m / z:Calcd for C 37 H 53 Bn 3 {[M+H] +}618.3417, found618.3423.

[0099] Effect Example: In vitro antitumor activity test

[0100] The MTT method was used to test the in vitro growth inhibition activity of the target compound on three cell lines: human cervical cancer cells (HeLa), human gastric cancer cells (HGC-27) and human kidney epithelial cells (HEK-293T). All cells were cultured in DMEM medium (added with 10% bovine serum and 0.1 g / L penicillin G + 0.1 g / L streptomycin sulfate) at 37°C and 5% CO 2 The target compound was dissolved in sterile DMSO (Sigma) at a concentration of 10 mg / mL and diluted with culture medium according to the concentration used. Cell viability assessment: Cells in the logarithmic growth phase were cultured at approximately 3×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, with 200 μL in each well. 2Incubate in the incubator for 24 hours. Then add different concentrations of the test compound to the test wells, set up 6 parallel wells for each concentration gradient, and use an equal amount of DMSO as a blank control. Continue to culture in the carbon dioxide incubator for 48 hours, wash the cells with sterile PBS (phosphate buffer saline), then add 190μL of culture medium and 10μL of MTT (5mg / mL) solution to each well, and continue to incubate in the carbon dioxide incubator for 4 hours; discard the supernatant, add 200μLDMSO, shake on a shaker for 10 minutes to dissolve the formed purple precipitate, and then use an enzyme marker to measure the OD value at a wavelength of 490nm.

[0101] Table 1 In vitro antiproliferative activity of stigmasterol derivatives [IC 50 / (μmol·L -1 )]

[0102]

[0103] From the data in Table 1, we can see that the inhibitory activity of the raw material drug stigmasterol on human cervical cancer cells (HeLa) is IC 50 The value is 55.6 μmol·L -1 , inhibitory activity IC of human gastric cancer cells (HGC-27) 50 The value is 72.6 μmol·L -1 ; Inhibitory activity of 5-fluorouracil (5-Fu) on human cervical cancer cells (HeLa) IC 50 The value is 72.0 μmol·L -1 , had no inhibitory activity against human gastric cancer cells (HGC-27) (IC 50 Value>100μmol·L -1 ); Among the synthesized stigmasterol derivatives, except for compounds 3c and 3d, the other derivatives did not show obvious inhibitory activity against the tested tumor cell lines and human normal cells 293T. However, compounds 3c and 3d showed good inhibitory activity against the tested tumor cell lines, such as the inhibitory activity IC 50 The values ​​were 52.3 μmol·L -1 and 31.4 μmol·L -1 , inhibitory activity IC of human gastric cancer cells (HGC-27) 50 The value is only 40.6 μmol·L -1 and 44.6 μmol·L -1 However, it showed little inhibitory activity against normal human renal epithelial cells (HEK-293T) (IC 50 Value>100μmol·L -1); It shows that these two compounds have certain activity of inhibiting tumor cell growth and proliferation, but have less damage to normal cells. And compared with the raw materials stigmasterol and 5-Fu, compounds 3c and 3d show better inhibitory activity against the tested tumor cell lines.

[0104] It should be understood that the terms used herein are only for the purpose of describing specific example embodiments and are not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms "one", "an" and "said" as used herein may also be meant to include plural forms. The terms "include", "comprise", "contain", and "have" are inclusive, and therefore specify the existence of stated features, steps, operations, elements and / or parts, but do not exclude the existence or addition of one or more other features, steps, operations, elements, parts, and / or combinations thereof. The method steps, processes, and operations described herein are not interpreted as necessarily requiring them to be performed in the specific order described or illustrated, unless the execution order is clearly indicated. It should also be understood that additional or alternative steps may be used.

[0105] Although the terms first, second, third, etc. can be used in the text to describe multiple elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. These terms can only be used to distinguish an element, component, region, layer or section from another region, layer or section. Unless the context clearly indicates, terms such as "first", "second" and other numerical terms do not imply order or sequence when used in the text. Therefore, the first element, component, region, layer or section discussed below can be referred to as the second element, component, region, layer or section without departing from the teaching of the example embodiments.

[0106] For ease of description, spatial relative terms may be used herein to describe the relationship of one element or feature relative to another element or feature as shown in the figure, such as "inside", "outside", "inner side", "outer side", "below", "below", "above", "above", etc. Such spatial relative terms are intended to include different orientations of the device in use or operation in addition to the orientation depicted in the figure. For example, if the device in the figure is turned over, then the elements described as "below other elements or features" or "below other elements or features" will subsequently be oriented as "above other elements or features" or "above other elements or features". Therefore, the example term "below..." can include both upper and lower orientations. The device can be oriented otherwise (rotated 90 degrees or in other directions) and the spatial relative descriptors used in the text are interpreted accordingly.

[0107] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A compound represented by Formula 3c, Formula 3d and Formula 3h, or one or more of their stereoisomers, or their pharmaceutically acceptable salts:

2. A method for preparing a compound represented by formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, comprising the following preparation method: Step a: In an organic solvent, stigmasterol 1 reacts with an azide compound in the presence of triphenylphosphine and azodicarbonate, after which a dilute alkali solution is added for quenching, followed by extraction with an organic solvent and separation to obtain a compound 2 in which the hydroxyl group is replaced by an azide; Step b: The compound 2 reacts with a copper salt and an acetylene compound in a polar alkaline solvent. After the reaction is completed, an organic solvent is added for extraction to separate and obtain a compound represented by formula (I), wherein A, B, and C are nitrogen, and the substituent R is Any of .

3. The preparation method according to claim 2, characterized in that: In the step a, the organic solvent is selected from one or two or more of diethyl ether, tetrahydrofuran, 1,4-dioxane, methyl tert-butyl ether, acetonitrile, toluene or xylene; The azodicarbonate is diethyl azodicarbonate or diisopropyl azodicarbonate; The azide compound is one of bis(P-nitrophenyl) azidophosphate, 15-azido-4,7,10,13-tetraoxypentadecanoic acid-N-succinimidyl ester, diphenyl azidophosphate or azide-heptapolyethylene glycol-pentafluorophenyl ester.

4. The preparation method according to any one of claims 2 to 3, characterized in that: In the step b, the acetylene compound is selected from any one of 2-fluorophenylacetylene, 4-fluorophenylacetylene, and 3-bromophenylacetylene; The polar solvent is selected from any one of water-methanol, water-ethanol, water-isopropanol, water-n-butanol or water-tert-butanol; The copper salt is selected from any one of copper sulfate, copper acetate, copper chloride, copper nitrate or copper carbonate; The base is selected from any one of sodium carbonate, sodium bicarbonate, sodium sulfite, sodium bisulfite or sodium ascorbate.

5. A pharmaceutical composition, characterized in that: A preparation prepared by using the compound according to claim 1, or its stereoisomer, or its pharmaceutically acceptable salt as a pharmaceutical active ingredient and adding pharmaceutically acceptable excipients.

6. Use of the compound according to claim 1, or its stereoisomer, or its pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 5 in the preparation of drugs for treating human cervical cancer cells and / or human gastric cancer cells.

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