CN116102608A

CN116102608A - Stigmasterol triazole derivative and preparation method and application thereof

Info

Publication number: CN116102608A
Application number: CN202211632443.3A
Authority: CN
Inventors: 王勇; 刘从军; 王炜; 段冰潮; 熊泳格; 李靖靖; 李维实; 高天增; 李佳灿
Original assignee: Zhengzhou Institute of Technology
Current assignee: Zhengzhou Institute of Technology
Priority date: 2022-12-19
Filing date: 2022-12-19
Publication date: 2023-05-12

Abstract

The invention provides a stigmasterol triazole derivative shown in a formula (I), or a stereoisomer or a pharmaceutically acceptable salt thereof, a preparation method thereof and application thereof in preparing medicines for treating diseases related to antitumor activity.

Description

Stigmasterol triazole derivative and preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and in particular relates to a stigmasterol triazole derivative, or a stereoisomer or a pharmaceutically acceptable salt thereof, a preparation method thereof and application thereof in preparing medicaments for treating related diseases with anti-tumor activity.

Background

Cancer is a major public health problem and has become one of the most common causes of death in humans. With the continued advancement of medicine, chemoprevention of tumors is one of the most direct methods of reducing the incidence of cancer. Natural products are also continually being recognized and discovered in an effort to find new therapeutic agents from plants. In recent years, the anticancer effect of phytosterol is gradually confirmed by people, and especially, the phytosterol has good anticancer effect on high-incidence cancers such as gastric cancer, breast cancer, lung cancer, ovarian cancer and the like. Related researches show that the compound obtained by taking a steroid structure as a matrix and externally connecting an aromatic heterocyclic structure has different physiological activities from the steroid matrix, and often has high-efficiency anti-tumor, antibacterial or antiviral activities.

Stigmasterol, also known as Stigmasterol-5, 22-diene-3 beta-alcohol, is a type of plant sterol widely distributed in nature, and as one of typical representatives of plant sterols, stigmasterol widely exists in deodorized distillates of various plants and vegetable oil refinements, the content of Stigmasterol is about 22%, and the molecular structure is shown in the figure. Stigmasterol is used as a natural active substance and has the characteristics of high nutritive value, strong physiological activity and the like. Early studies found that: its main physiological functions include anti-tumor effect, reducing blood fat concentration, antioxidation, analgesia and antidiabetic.

In recent years, as stigmasterol is separated as a single component, a large number of stigmasterol derivatives have been designed and synthesized, and the solubility of stigmasterol derivatives has been improved by modifying the structure of stigmasterol derivatives, so that the stigmasterol derivatives have a wider application range and some new properties have been developed. It is well known that aromatic rings with conjugated systems and negative electron properties are widely used for structural modification of lead compounds, which can pi-pi interact with target proteins and increase affinity to targets. The different types of substituents in the aromatic ring have been found to be closely related to charge distribution, nature of the compound and anti-tumour efficacy, especially halogen groups, which significantly enhance bioactivity and improve metabolic stability of the parent.

Triazole is a five-membered heterocyclic ring containing 3 nitrogen atoms and has the biological activity effects of bacteriostasis, antiallergic, anti-HIV virus, anti-tumor, anti-tuberculosis and the like. In recent years, studies have been made on the structure-activity relationship of a natural active material as a lead compound, in which various types of triazole groups are introduced into a skeleton. It follows that the introduction of triazole into the parent molecule can alter the pharmacokinetic properties and solubility of the parent molecule, enhancing the biological activity of the parent molecule. However, prior literature reports have not disclosed lead compounds that modify stigmasterols with triazole-containing heterocycles and provide superior anticancer activity. Based on the existing disclosed inhibition structure, the invention screens and obtains a series of novel structural compounds with higher activity.

Disclosure of Invention

The invention aims to provide a stigmasterol triazole derivative shown in the structure of a formula (I), in particular to a compound shown in the formula (I), or a stereoisomer or a pharmaceutically acceptable salt thereof:

wherein A, B, C is independently selected from C or N, and at least one of said A, B, C is selected from N;

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 and substituted or unsubstituted-C6-C10 heteroaryl;

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 heteroaryl is substituted with R ¹ Substitution, said R ¹ Selected from any one of hydrogen, -C1-C6 alkyl, halogen and, -C1-C6 alkoxy.

Preferably, the R is selected from the group consisting of substituted or unsubstituted-C1-6 alkyl, substituted or unsubstituted-C6-C10 aryl, the substituted-C1-6 alkyl, substituted-C6-C10 aryl being substituted by R ¹ Substitution, said R ¹ Selected from any one of hydrogen, -C1-C6 alkyl, halogen and, -C1-C6 alkoxy.

Still more preferably, the-C6-C10 aryl is phenyl, the-C1-C6 alkoxy is methoxy or ethoxy, and the halogen is fluorine, chlorine or bromine.

Preferably, A, B, C is independently selected from N; r is selected from

Any one of the following. More preferably, the compound of formula I specifically comprises any one of the following: />

The invention also provides a preparation method of the compound shown in the formula (I), which comprises the following preparation methods:

step a: in an organic solvent, stigmasterol 1 and an azide compound react under the action of triphenylphosphine and azodicarbonate, dilute alkali solution is added for quenching after the reaction is finished, then the organic solvent is added for extraction, and the compound 2 with hydroxyl replaced by azide is obtained after separation;

step b: and reacting the compound 2 with copper salt in a polar solvent of alkali and alkyne compound, adding an organic solvent for extraction after the reaction is finished, and separating to obtain the compound shown in the formula (I), wherein A, B, C and substituent R in the formula (I) are defined as in any one of claims 1-6.

Preferably, in the step a, the organic solvent is selected from one or two of diethyl ether, tetrahydrofuran, 1, 4-dioxane, methyl tertiary butyl ether, acetonitrile, toluene and xylene; the azodicarbonate adopts diethyl azodicarbonate (DEAD) or diisopropyl azodicarbonate (DIAD); the azide compound adopts one of bis (P-nitrophenyl) azido phosphate, 15-azido-4, 7,10, 13-tetraoxypentadecanoic acid-N-succinimidyl ester, diphenyl azide phosphate or azido-heptapolyethylene glycol-pentafluorophenyl ester; in the step b: the alkyne compound is hexyne, p-methoxyphenylacetylene, 2-fluorophenylacetylene, 4-fluorophenylacetylene, 3-chlorophenylacetylene, 4-chlorophenylacetylene, 2-bromophenylacetylene or 3-bromophenylacetylene, and the polar solvent is 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 alkali is selected from one of sodium carbonate, sodium bicarbonate, sodium sulfite, sodium bisulphite or sodium ascorbate.

The invention also provides a pharmaceutical composition, which is characterized in that: the preparation is prepared by taking the compound, or a stereoisomer or a pharmaceutically acceptable salt thereof as a pharmaceutically active ingredient and adding pharmaceutically acceptable auxiliary materials.

The invention also provides application of the compound shown in the formula (I), or a stereoisomer, or a pharmaceutically acceptable salt or the pharmaceutical composition thereof in preparing medicines for treating tumor and/or anticancer diseases.

Further preferably, the anticancer agent is selected from the group consisting of human cervical cancer cells, human gastric cancer cells and human kidney epithelial cells.

The following is an explanation and explanation of the noun terminology of the invention:

"cancer" or "malignancy" refers to any of a variety of diseases characterized by uncontrolled cellular abnormal proliferation, the ability of affected cells to spread locally or through the blood stream and lymphatic system to other sites, the body (i.e., metastasis), and any of a number of characteristic structures and/or molecular features. "cancer cells" refers to cells that undergo early, mid, or late stages of multistep 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 useful for treating 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, the cancer is selected from the group consisting of human cervical cancer cells, human gastric cancer cells, and human kidney epithelial cells.

The compounds and derivatives provided in the present invention may be named according to IUPAC (international union of pure and applied chemistry) or CAS (chemical abstract service, columbus, OH) naming system.

Definition of terms used in connection with the present invention: unless otherwise indicated, the initial definitions provided for groups or terms herein apply to the groups or terms throughout the specification; for terms not specifically defined herein, the meanings that one skilled in the art can impart based on the disclosure and the context.

"substituted" means that a hydrogen atom in a molecule is replaced by a different atom or molecule.

The minimum and maximum values of the carbon atom content of the hydrocarbon groups are indicated by a prefix, for example, the prefix Ca-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.

"alkyl" refers to a saturated hydrocarbon chain having the indicated 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 may be linear 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 groups include 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.

"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, and). For polycyclic systems having aromatic and non-aromatic rings that do not contain ring heteroatoms, the term "cycloalkyl" (e.g., 5,6,7,8, -tetrahydronaphthalen-5-yl) applies when the point of attachment is at a non-aromatic carbon atom. 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 comprising a multicycloalkyl ring system are dicyclohexyl, dicyclopentyl, bicyclooctyl, and the like.

"alkenyl" refers to a straight or branched 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 ethylenic unsaturation (> c=c <). For example, (Ca-Cb) alkenyl refers to an alkenyl group having a to b carbon atoms and is intended to include, for example, ethenyl, propenyl, isopropenyl, 1, 3-butadienyl, and the like.

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

"halogen" is fluorine, chlorine, bromine or iodine.

"haloalkyl" means that a hydrogen atom in an alkyl group may be substituted with one or more halogen atoms. For example, a C1-4 haloalkyl group refers to an alkyl group containing 1-4 carbon atoms wherein a hydrogen atom is replaced with one or more halogen atoms.

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

"aromatic heterocycle" refers to an aromatic unsaturated ring comprising at least one heteroatom; wherein the heteroatom means a nitrogen atom, an oxygen atom, and a sulfur atom;

the compounds of the invention may contain asymmetric or chiral centers and thus exist as different stereoisomers. All stereoisomeric forms of the compounds of the invention, including, but in no way 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 form, i.e. they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefix D, L or R, S is used to denote the absolute configuration of the chiral center of the molecule. The chemical structures of these stereoisomers are identical, but their stereoisomers are different. The particular stereoisomer may be an enantiomer, and the mixture of isomers is commonly referred to as an enantiomeric mixture. The 50:50 enantiomeric mixture is known as a racemic mixture or racemate, which may result in the absence of stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to a mixture of two enantiomers in equimolar amounts, lacking optical activity.

The term "pharmaceutically acceptable" means that the carrier, cargo, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients comprising the pharmaceutical dosage form, and physiologically compatible with the recipient.

The pharmaceutical composition of the invention can be in any of the forms of reusable pharmaceutical preparations, such as: oral, injectable, topical, etc. forms, oral dosage forms include, but are not limited to: the tablet, capsule, oral liquid, granule, pill, suspension, injection is selected from water injection, powder injection, and topical preparation is selected from patch and paste. All formulations can be prepared according to conventional techniques of pharmacy, for example, by taking any one of the compounds of the invention, or stereoisomers thereof, or pharmaceutically acceptable salts thereof, as a pharmaceutically active ingredient, and adding pharmaceutically acceptable carriers if necessary, to prepare the above pharmaceutical dosage forms suitable for administration, wherein the unit dose of the pharmaceutically active ingredient can be 0.1mg to 1000mg, for example, each tablet contains 0.1mg to 1000mg, preferably 5mg to 500mg, of the pharmaceutically active ingredient.

The terms "salts" and "pharmaceutically acceptable salts" refer to the acid and/or base salts of the above compounds or stereoisomers thereof, 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 may be obtained directly in the final isolation and purification of the compounds. The compound may be obtained by mixing the above compound or a stereoisomer thereof with a predetermined amount of an acid or a base as appropriate (for example, equivalent). These salts may be obtained by precipitation in solution and collected by filtration, or recovered after evaporation of the solvent, or by lyophilization after reaction in an aqueous medium. The salts of the present invention may be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoric, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate salts of the compounds.

In certain embodiments, one or more compounds of the present invention may be used in combination with one another. The compounds of the invention may alternatively be used in combination with any other active agent for the preparation of a medicament or pharmaceutical composition for modulating cellular function or treating a disease. If a group of compounds is used, the compounds may be administered to a subject simultaneously, separately or sequentially.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The beneficial technical effects of the invention are as follows:

1. the invention carries out structural modification on the 3-hydroxyl of stigmasterol, provides a stigmasterol triazole derivative with novel structure, and enriches the structural types of steroid compounds;

2. the stigmasterol triazole derivative provided by the invention has the advantages of simple preparation method, low-cost and easily obtained raw materials, mild reaction conditions, simple post-treatment, high yield which is more than 71%, and recyclable solvent, and is beneficial to industrial production;

3. according to the invention, the triazole active subunit is introduced into the stigmasterol skeleton, so that the anti-tumor activity of the stigmasterol is greatly improved, a plurality of stigmasterol triazole derivatives with higher anti-tumor activity are obtained, a research basis and a lead compound are provided for the development of new anticancer drugs, and the stigmasterol triazole derivative has a good application prospect.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to designate like parts throughout the figures. In the drawings:

fig. 1 schematically shows a nuclear magnetic resonance hydrogen spectrum of a compound 2;

FIG. 2 schematically shows a nuclear magnetic resonance carbon spectrum of Compound 2;

FIG. 3 schematically shows a nuclear magnetic resonance hydrogen spectrum of compound 3 a;

FIG. 4 schematically shows a nuclear magnetic resonance carbon spectrum of compound 3 a;

fig. 5 schematically shows a nuclear magnetic resonance hydrogen spectrum of the compound 3 b;

FIG. 6 schematically shows a nuclear magnetic resonance carbon spectrum of compound 3 b;

fig. 7 schematically shows a nuclear magnetic resonance hydrogen spectrum of the compound 3 c;

FIG. 8 schematically shows a nuclear magnetic resonance carbon spectrum of compound 3 c;

FIG. 9 schematically shows a nuclear magnetic resonance hydrogen spectrum of compound 3 d;

FIG. 10 schematically shows a nuclear magnetic resonance carbon spectrum of compound 3 d;

FIG. 11 schematically shows a nuclear magnetic resonance hydrogen spectrum of compound 3 e;

FIG. 12 schematically shows a nuclear magnetic resonance carbon spectrum of compound 3 e;

FIG. 13 schematically shows a nuclear magnetic resonance hydrogen spectrum of compound 3 f;

FIG. 14 schematically shows a nuclear magnetic resonance carbon spectrum of compound 3 f;

FIG. 15 schematically shows a nuclear magnetic resonance hydrogen spectrum of 3g of the compound;

FIG. 16 schematically shows a nuclear magnetic resonance carbon spectrum of 3g of the compound;

FIG. 17 schematically shows a nuclear magnetic resonance hydrogen spectrum of compound 3 h;

fig. 18 schematically shows a nuclear magnetic resonance carbon spectrum of compound 3h.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

The structure of the compounds of the present invention is determined by Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS). NMR shift (. Delta.) is given in units of 10-6 (ppm). NMR was performed using a Bruker Avance III 400 and Bruker Avance300 magnetonucleuses in which the solvent was deuterated dimethyl sulfoxide (DMSO-d 6), deuterated chloroform (CDCl) ₃ ) Deuterated methanol (CD 3 OD), internal standard was Tetramethylsilane (TMS). The known starting materials of the present invention may be synthesized using or according to methods known in the art or may be purchased from commercial reagent platforms.

Preparation of intermediate compound 2

A250 mL round bottom flask was taken, triphenylphosphine (1.44 g,5.5 mmol) was weighed and dissolved in 35mL tetrahydrofuran with stirring, diisopropyl azodicarboxylate (1.18 mL,6 mmol) was added, stimulative 10min at 0deg.C, stigmasterol (2.06 g,5 mmol) was added, stirring was continued for ten minutes, diphenyl azide phosphate (1.29 mL,6 mmol) was slowly added, the ice bath was removed and stirred for 10h at room temperature, and TLC detection was performed. Preparing 0.1mol/L sodium hydroxide, adding the sodium hydroxide for quenching, extracting a reaction liquid with dichloromethane, extracting the remaining water phase with dichloromethane, repeating the extraction for three times, combining the obtained organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate to obtain a crude product 2, decompressing and concentrating the obtained crude product 3, and separating by column chromatography to obtain the compound 2. The yield was 66.5%; the characterization map is shown in fig. 1 and 2; ¹ H NMR(400MHz,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＝13.3Hz,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),1.03(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(100MHz,CDCl ₃ )δ138.30,138.02,129.25,123.14,58.23,56.78,55.89,51.22,49.91,42.17,40.49,39.61,37.07,36.04,33.61,31.87,31.79,31.77,28.89,26.09,25.39,24.32,21.20,21.07,20.69,18.97,12.23,12.02；FT-IR(KBr)ν:3358.26,3127.57,3063.38,2942.70,1731.28,1457.50,1236.09,1044.71,972.64,838.19,763.08cm ^-1 ；HR-MS(ESI-TOF)m/z:Calcd for C ₂₉ H ₄₈ N ₃ {[M+H] ⁺ }438.3835，found438.3836.

preparation of Compounds 3a-3h

Two round bottom flasks of 100mL size were taken, copper acetate (0.2 mmol) and sodium ascorbate (0.4 mmol) were weighed into the round bottom flasks, 2mL of water was added and stirred to dissolve, compound 2 (1 mmol) and acetylenic compound (1 mmol) were taken into the other flask, 10mL of t-butanol was added and stirred to dissolve, the two systems were combined and reacted at room temperature. After the TLC detection reaction was completed, the organic phase was extracted with ethyl acetate, the remaining aqueous phase was extracted with ethyl acetate, extraction was repeated three times in total, the obtained organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate to obtain crude product 3, and the obtained crude product 3 was subjected to reduced pressure, concentration and column chromatography to obtain compounds 3a to 3h.

Example 1: preparation of Compound 3a

Compound 3a was prepared by following the same procedure as in example 1, except that hexyne was added. Compound 3a, 92.5% yield; the characterization map is shown in fig. 3 and 4; 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＝14.6Hz,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(100MHz,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(KBr)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.

example 2: preparation of Compound 3b

According to the same manner as that of example 1, p-methoxyphenylacetylene was added to prepare a compound 3b. Compound 3b, 86.5% yield; the characterization map is shown in fig. 5 and 6; 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,2H),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(100MHz,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.

example 3: preparation of Compound 3c

Compound 3c was prepared by following the same procedure as in example 1, adding 2-fluorophenylacetylene. Compound 3c in 72.7% yield; the characterization map is shown in fig. 7 and 8; ¹ H NMR(400MHz,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 )δ160.04,157.58,139.79,139.76,137.90,137.52,128.94,128.58,128.50,127.35,127.32,124.36,124.18,124.15,122.48,122.34,118.73,118.60,115.35,115.13,56.31,56.28,55.52,50.87,49.75,41.79,40.13,39.09,36.73,35.26,32.38,31.64,31.52,31.34,29.35,28.53,26.89,25.04,23.94,20.85,20.73,20.27,18.98,18.63,11.89,11.69；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.

example 4: preparation of Compound 3d

Following the same procedure as in example 1, 4-fluorophenylacetylene was added to prepare compound 3d. Compound 3d, 71.2% yield; the characterization map is shown in fig. 9 and 10; 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(100MHz,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.

example 5: preparation of Compound 3e

According to the same manner as that of example 1, 3-chloroacetylene was added to prepare compound 3e. Compound 3e in 74.4% yield; the characterization map is shown in fig. 11 and 12; 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(100MHz,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 for C ₃₇ H ₅₃ ClN ₃ {[M+H] ⁺ }574.3923，found 574.3916.

example 6: preparation of Compound 3f

According to the same manner as that of example 1, 4-chloroacetylene was added to prepare compound 3f. Compound 3f, 79.6% yield; the characterization map is shown in fig. 13 and 14; 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),1.06(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(100MHz,CDCl ₃ )δ145.63,138.14,138.08,133.50,129.53,129.31,128.89,126.82,124.35,119.25,56.66,56.63,55.85,51.16,50.07,42.11,40.39,39.39,37.07,35.48,32.71,31.96,31.81,31.64,29.64,28.80,27.05,25.32,24.23,21.15,21.02,20.58,19.22,18.94,12.18,11.98；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 ClN ₃ {[M+H] ⁺ }574.3923，found 574.3925.

example 7: preparation of Compound 3g

According to the same manner as that of example 1, 2-bromophenylacetylene was added to prepare 3g of a compound. 3g of compound with the yield of 83.0 percent; the characterization map is shown in fig. 15 and 16; 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,1H),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,4H),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(100MHz,CDCl ₃ )δ144.05,138.17,138.08,133.49,131.57,130.31,129.28,128.91,127.63,124.70,123.27,121.01,56.81,56.54,55.82,51.17,50.18,42.12,40.40,39.44,37.06,35.60,32.55,32.07,31.81,31.65,30.14,29.64,28.80,27.42,25.34,24.24,21.15,21.03,20.58,19.28,18.94；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 H ₅₃ BrN ₃ {[M+H] ⁺ }618.3417，found 618.3419.

example 8: preparation of Compound 3h

According to the same manner as that of example 1, 3-bromophenylacetylene was added to prepare a compound 3h. Compound 3h, 84.9% yield; the characterization map is shown in fig. 17 and 18; 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,2H),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(100MHz,CDCl ₃ )δ145.27,138.17,137.99,133.07,130.69,130.25,129.27,128.51,124.41,124.15,122.84,119.50,56.70,56.58,55.82,51.16,49.98,42.10,40.42,39.36,37.07,35.46,32.70,31.94,31.81,31.64,28.81,27.02,25.34,24.24,21.16,21.04,20.57,19.22,18.94,12.20,11.99；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 BrN ₃ {[M+H] ⁺ }618.3417，found 618.3423.

effect examples: in vitro antitumor Activity test

The MTT method is adopted to test the in vitro growth proliferation inhibition activity of the target compound on three cell strains of human cervical cancer cells (HeLa), human gastric cancer cells (HGC-27) and human kidney epithelial cells (HEK-293T). All cells were incubated with DMEM medium (10% bovine serum and 0.1G/L penicillin G+0.1G/L streptomycin sulfate) at 37℃in 5% CO ₂ Is cultured in a humid atmosphere. The target compound was dissolved in sterile DMSO (Sigma) at a concentration of 10mg/mLWhen in use, the culture solution is diluted according to the use concentration. Cell viability assessment: the cells in logarithmic growth phase were grown at about 3X 10, respectively ⁴ The density of each mL is inoculated in a 96-well plate, 200 mu L of each well is inoculated and placed in CO ₂ Culturing in an incubator for 24 hours. Test compounds at different concentrations were then added to the test wells, 6 parallel wells were set per concentration gradient, and an equivalent amount of DMSO was used as a blank. Culturing was continued in a carbon dioxide incubator for 48 hours, the cells were washed with sterile PBS (phosphate buffer saline), then 190. Mu.L of medium and 10. Mu.L of MTT (5 mg/mL) solution were added to each well, and incubation was continued in the carbon dioxide incubator for 4 hours; the supernatant was discarded, 200. Mu. LDMSO was added, and the formed purple precipitate was dissolved by shaking on a shaker for 10min, followed by measurement of OD at 490nm using an ELISA reader.

TABLE 1 in vitro antiproliferative Activity of stigmasterol derivatives [ IC ₅₀ /(μmol·L ^-1 )]

As can be seen from the data in Table 1, the crude drug stigmasterol has inhibitory activity IC on human cervical cancer cells (HeLa) ₅₀ The value was 55.6. Mu. Mol.L ^-1 Inhibitory Activity of human gastric cancer cell (HGC-27) IC ₅₀ The value was 72.6. Mu. Mol.L ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Inhibitory Activity of 5-fluorouracil (5-Fu) against human cervical cancer cells (HeLa) ₅₀ The value was 72.0. Mu. Mol.L ^-1 Inhibitory Activity (IC) without inhibitory Activity against human gastric cancer cells (HGC-27) ₅₀ The value is more than 100 mu mol.L ^-1 ) The method comprises the steps of carrying out a first treatment on the surface of the Among the synthesized stigmasterol derivatives, other derivatives showed no significant inhibitory activity on both the tumor cell line tested and human normal cell 293T, except for compounds 3c and 3d. While the compounds 3c and 3d show good inhibitory activity on the tested tumor cell lines, such as inhibitory activity IC on human cervical cancer cells (HeLa) ₅₀ The values were 52.3. Mu. Mol.L, respectively ^-1 And 31.4. Mu. Mol.L ^-1 Inhibitory Activity of human gastric cancer cell (HGC-27) IC ₅₀ The value is only 40.6 mu mol.L ^-1 And 44.6. Mu. Mol.L ^-1 But is provided withShows substantially no inhibitory activity (IC) on human normal kidney epithelial cells (HEK-293T) ₅₀ The value is more than 100 mu mol.L ^-1 ) The method comprises the steps of carrying out a first treatment on the surface of the The two compounds have certain activity of inhibiting the growth and proliferation of tumor cells, and have less damage to normal cells. And both compounds 3c and 3d showed better inhibitory activity against the tested tumor cell lines than the starting stigmasterol and 5-Fu.

It is to be understood that the terminology used herein is for the purpose of describing particular example embodiments only, and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The terms "comprises," "comprising," "includes," "including," and "having" are inclusive and therefore specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. The method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order described or illustrated, unless an order of performance is explicitly stated. It should also be appreciated that additional or alternative steps may be used.

Although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms may be only used to distinguish one element, component, region, layer or section from another region, layer or section. Terms such as "first," "second," and other numerical terms when used herein do not imply a sequence or order unless clearly indicated by the context. Thus, a first element, component, region, layer or section discussed below could be termed a second element, component, region, layer or section without departing from the teachings of the example embodiments.

For ease of description, spatially relative terms, such as "inner," "outer," "lower," "below," "upper," "above," and the like, may be used herein to describe one element or feature's relationship to another element or feature as illustrated in the figures. Such spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is turned over, elements described as "below" or "beneath" other elements or features would then be oriented "above" or "over" the other elements or features. Thus, the example term "below … …" may include both upper and lower orientations. The device may be otherwise oriented (rotated 90 degrees or in other directions) and the spatial relative relationship descriptors used herein interpreted accordingly.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the invention is subject to the protection scope of the claims.

Claims

1. A compound of formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

wherein the substitution-C1-6 alkyl, substituted-C2-6 alkenyl, substituted-C2-6 alkynyl, substituted-C1-6 alkoxy, substituted-C6-C10 aryl, substituted-C6-C10 heteroaryl is substituted by R ¹ Substitution, said R ¹ Selected from any one of hydrogen, -C1-C6 alkyl, halogen and, -C1-C6 alkoxy.

2. The compound of claim 1, or a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein: the R is selected from the group consisting of substituted or unsubstituted-C1-6 alkyl, substituted or unsubstituted-C6-C10 aryl, the substituted-C1-6 alkyl, substituted-C6-C10 aryl being substituted with R ¹ Substitution, said R ¹ Selected from any one of hydrogen, -C1-C6 alkyl, halogen and, -C1-C6 alkoxy.

3. The compound of claim 2, or a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein: the-C6-C10 aryl is phenyl, the-C1-C6 alkoxy is methoxy or ethoxy, and the halogen is fluorine, chlorine or bromine.

4. A compound according to any one of claims 1-3, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, A, B, C is independently selected from N; r is selected from

Any one of the following.

5. The compound of claim 1, or a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein: the compound shown in the formula I specifically comprises any one of the following components:

6. a process for the preparation of a compound of formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 5, comprising the process for the preparation of:

step b: the compound 2 reacts with copper salt in polar solvent of alkali and alkyne compound, after the reaction, organic solvent is added for extraction, and the compound shown in the formula (I) is obtained after separation, wherein A, B, C and substituent R in the formula (I) are defined as in any one of claims 1-5.

7. The method of manufacturing according to claim 6, wherein: in the step a, the organic solvent is selected from one or two or more of diethyl ether, tetrahydrofuran, 1, 4-dioxane, methyl tertiary butyl ether, acetonitrile, toluene or xylene; the azodicarbonate adopts diethyl azodicarbonate or diisopropyl azodicarbonate; the azide compound adopts one of bis (P-nitrophenyl) azido phosphate, 15-azido-4, 7,10, 13-tetraoxypentadecanoic acid-N-succinimidyl ester, diphenyl azide phosphate or azido-heptapolyethylene glycol-pentafluorophenyl ester;

in the step b, the alkyne compound is selected from any one of hexyne, p-methoxyphenylacetylene, 2-fluorophenylacetylene, 4-fluorophenylacetylene, 3-chlorophenylacetylene, 4-chlorophenylacetylene, 2-bromophenylacetylene or 3-bromophenylacetylene, and 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 alkali is selected from any one of sodium carbonate, sodium bicarbonate, sodium sulfite, sodium bisulphite or sodium ascorbate.

8. A pharmaceutical composition characterized by: a preparation prepared by taking the compound as defined in any one of claims 1 to 5, or a stereoisomer or a pharmaceutically acceptable salt thereof or the compound prepared by the method as defined in any one of claims 6 to 7, or a stereoisomer or a pharmaceutically acceptable salt thereof as a pharmaceutically active ingredient and adding pharmaceutically acceptable auxiliary materials.

9. Use of a compound according to any one of claims 1 to 5, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a compound prepared by a method according to any one of claims 6 to 7, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 8, for the preparation of a medicament for the treatment of an anti-tumour and/or anti-cancer disease.

10. The use according to claim 9, characterized in that: the anticancer is selected from human cervical cancer cells, human gastric cancer cells or human renal epithelial cells.