CN115403511B

CN115403511B - Piperlonguminide analogue, pharmaceutical composition, preparation method and application thereof

Info

Publication number: CN115403511B
Application number: CN202110894985.7A
Authority: CN
Inventors: 段勇涛; 李红良; 王旋; 姚永芳; 孙默然; 杨华
Original assignee: Henan Childrens Hospital Zhengzhou Childrens Hospital
Current assignee: Henan Childrens Hospital Zhengzhou Childrens Hospital
Priority date: 2021-08-03
Filing date: 2021-08-03
Publication date: 2024-01-26
Anticipated expiration: 2041-08-03
Also published as: CN115403511A

Abstract

The invention provides a piperlonguminine analogue, a preparation method thereof and application thereof in resisting tumors. The compound of the invention has obvious inhibition effect on various tumors including but not limited to U937 (human leukemia cells), MCF7 (human breast cancer cells), hepG2 (human liver cancer cells), A549 (human lung cancer cells), MGC-803 (human stomach cancer cells) and HeLa (human cervical cancer cells). The compound disclosed by the invention has better biological activity, higher selectivity and lower toxicity, and can be used for preparing antitumor drugs.

Description

Piperlonguminide analogue, pharmaceutical composition, preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and in particular relates to a piperlonguminine analogue, a pharmaceutical composition, a preparation method thereof and application thereof in preparing antitumor drugs.

Background

Malignant tumors seriously threaten the life and health of human beings, and are one of the main causes of human death worldwide. At present, the main treatment methods of malignant tumors are as follows: surgical excision, radiation therapy, drug therapy, biological therapy, and the like. However, even with the above treatments, the probability of distant metastasis and recurrence of tumors remains high, making malignant tumor treatment one of the most challenging problems worldwide. Currently, drug therapy is still the primary method of tumor treatment. The common problems of the existing clinically applied antitumor drugs are that: the medicine has poor selectivity, great toxic and side effects and easy drug resistance. Therefore, new antitumor drugs are being studied to solve these problems.

Natural products are an important source for the development of clinical therapeutic drugs. The structure of natural products is highly chemically diverse and biochemically specific, and these characteristics make it well suited as a lead structure for drug discovery. However, they generally have poor selectivity to tumor cells and often cause serious toxic and side effects, such as taxol, vinblastine, camptothecine, and the like, which are anti-malignant tumor drugs. Therefore, there is an urgent need to find a drug with better selectivity.

Piperlonguminine (PL), also known as piperlonguminine, is an alkaloid compound isolated from the plant Piper longum. Its molecular formula is C ₁₇ H ₁₉ NO ₅ The molecular weight is 317.34, the piperlongumin has two isomers of cis (cis) and trans (trans), the piperlongumin existing in natural plants is mainly trans, the trans piperlongumin is also an active main form, and the cis piperlongumin does not show obvious cytotoxicity. Unless otherwise indicated, piperlonguminine is referred to herein as the trans-form. Piperlongum amide is a natural product with broad-spectrum biological activity, such as anti-tumor, anti-inflammatory, anti-platelet coagulation, anti-diabetes, neuroprotection, etc. The anti-tumor activity of piperlonguminine becomes a hot spot for research by researchers, and has excellent selective inhibition effect on various tumor cells, but has no obvious toxic or side effect on normal cells. The excellent characteristic makes the piperlongumin an important lead compound, and provides an important structural basis for developing high-efficiency and low-toxicity antitumor new drugs. Therefore, the invention aims to explore the piperlonguminine analogues with better anti-tumor activity.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a compound represented by formula I, which is a racemate, a stereoisomer, a tautomer, an isotopic label, a solvate, a polymorph, a prodrug or a pharmaceutically acceptable salt thereof:

wherein R is ₁ Selected from aryl groups unsubstituted or optionally substituted by one, two or more Ra,Heteroaryl, heterocyclyl;

R ₂ absent or selected from the group consisting of alkenyl, arylene, heteroarylene optionally substituted with one, two or more Rb;

R ₃ selected from H, unsubstituted or optionally substituted alkyl, alkoxy, haloalkyl with one, two or more Rc;

R ₄ selected from C (O), S (O) ₂ ；

R ₅ Selected from H, halogen, alkyl, alkoxy, haloalkyl;

each Ra, rb, rc is the same or different and is independently selected from H, NO ₂ CN, OH, COOH, halogen, oxo (=o), alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, heterocyclyl.

According to an embodiment of the invention, R ₁ May be selected from C which is unsubstituted or optionally substituted by one, two or more Ra _6-14 Aryl, 5-14 membered heteroaryl, 3-14 membered heterocyclyl;

R ₂ may be absent or selected from unsubstituted or optionally substituted sub-C with one, two or more Rb _2-12 Alkenyl, sub-C _6-14 Aryl, 5-14 membered heteroaryl;

R ₃ may be selected from H, unsubstituted or optionally substituted C with one, two or more Rc _1-12 Alkyl, C _1-12 Alkoxy, halo C _1-12 An alkyl group;

R ₄ can be selected from C (O), S (O) ₂ ；

R ₅ Can be selected from H, halogen, C _1-12 Alkyl, C _1-12 Alkoxy, halo C _1-12 An alkyl group;

each Ra, rb, rc is the same or different and is independently selected from H, NO ₂ CN, OH, COOH, halogen, oxo (=o), C _1-12 Alkyl, C _1-12 Alkoxy, C _3-12 Cycloalkyl, C _6-14 Aryl, 5-14 membered heteroaryl, 3-14 membered heterocyclyl.

According to an embodiment of the invention, R ₁ Can be selected from unsubstituted or optionally substituted by one, two or moreMore Ra-substituted C _6-14 Aryl, 5-14 membered heteroaryl, 3-14 membered heterocyclyl;

R ₂ may be absent or selected from unsubstituted or optionally substituted sub-C with one, two or more Rb _2-6 Alkenyl, sub-C _6-14 Aryl, 5-14 membered heteroaryl;

R ₃ may be selected from H, unsubstituted or optionally substituted C with one, two or more Rc _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 An alkyl group;

R ₄ can be selected from C (O), S (O) ₂ ；

R ₅ Can be selected from H, halogen, C _1-6 An alkyl group;

each Ra, rb, rc is the same or different and is independently selected from H, NO ₂ CN, halogen, oxo (=o), C _1-6 Alkyl, C _1-6 An alkoxy group.

According to an embodiment of the invention, R ₁ May be selected from the following groups which are unsubstituted or optionally substituted with one, two or more Ra: phenyl, naphthyl, benzopyrazinyl, quinolinyl, 4H-chromen-4-one, carbazolyl, benzofuranyl, dibenzofuranyl, 2, 3-dihydrobenzofuranyl, 1, 3-benzodioxanyl and benzo [ b ]]Thienyl, dibenzothienyl;

R ₂ may be absent or selected from the group consisting of unsubstituted or optionally substituted phenylene, imidazolylene, pyridylene, ethenylene, propenylene, optionally substituted with one, two or more Rb;

R ₃ may be selected from H, methyl;

R ₄ can be selected from C (O), S (O) ₂ ；

R ₅ May be selected from H, F, cl, br, I;

ra is selected from H, NO ₂ F, cl, br, I, oxo (=o), methyl, methoxy;

rb is selected from H and methyl.

Preferably, R ₁ Is quinolinyl, R ₂ Is trans-vinylidene, R ₃ Is H, R ₄ Is C (O), R ₅ Is Cl.

According to an exemplary embodiment of the invention, the compound of formula I is selected from the following structures:

the invention also provides a preparation method of the compound shown in the formula I, which comprises the following steps:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ And R is ₅ Independently having the definition set forth above; x is selected from halogen or OC (O) -C _1-6 Alkyl radicals, e.g. Cl or OC (O) C (CH) ₃ ) ₃ ；

Reacting the compound a with the compound b to obtain a compound shown in a formula I;

according to an embodiment of the invention, the reaction is carried out under the action of a base, which may be an organometallic reagent, such as n-butyllithium, t-butyllithium, methyllithium, lithium diisopropylamide;

according to an embodiment of the present invention, the reaction may be performed in the presence of an organic solvent, which may be, for example, at least one of dioxane, tetrahydrofuran, 2-methyltetrahydrofuran.

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of at least one of a compound of formula I, racemate, stereoisomer, tautomer, isotopic label, solvate, polymorph, prodrug or a pharmaceutically acceptable salt thereof.

According to an embodiment of the invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

According to embodiments of the present invention, the pharmaceutical composition may further comprise one or more additional therapeutic agents.

The present invention also provides a method for treating or preventing a neoplastic disease, comprising administering to a patient a prophylactically or therapeutically effective amount of at least one of a compound of formula I, racemate, stereoisomer, tautomer, isotopic label, solvate, polymorph, prodrug, or a pharmaceutically acceptable salt thereof.

The invention also provides a method of treating or preventing a neoplastic disease comprising administering to a patient a prophylactically or therapeutically effective amount of the above pharmaceutical composition.

The tumor can be leukemia cells, breast cancer cells, liver cancer cells, lung cancer cells, stomach cancer cells and cervical cancer cells; preferably U937 leukemia cells, MCF7 breast cancer cells, hepG2 liver cancer cells, A549 lung cancer cells, MGC-803 stomach cancer cells and HeLa cervical cancer cells.

In some embodiments, the patient mammal, preferably a human.

The invention also provides the use of at least one of a compound shown in formula I, a racemate, a stereoisomer, a tautomer, an isotopic label, a solvate, a polymorph, a prodrug or pharmaceutically acceptable salts thereof for preparing a medicament.

According to an embodiment of the invention, the medicament is an anti-tumor medicament, such as a broad spectrum anti-tumor medicament.

According to an embodiment of the present invention, the tumor may be leukemia cells, breast cancer cells, liver cancer cells, lung cancer cells, stomach cancer cells, and cervical cancer cells; preferably U937 leukemia cells, MCF7 breast cancer cells, hepG2 liver cancer cells, A549 lung cancer cells, MGC-803 stomach cancer cells and HeLa cervical cancer cells.

Advantageous effects

The invention provides a piperlonguminine analogue, a preparation method thereof and application thereof in resisting tumors. The compound of the invention has obvious inhibition effect on various tumors including but not limited to U937 (human leukemia cells), MCF7 (human breast cancer cells), hepG2 (human liver cancer cells), A549 (human lung cancer cells), MGC-803 (human stomach cancer cells) and HeLa (human cervical cancer cells). Can be used for preparing antitumor drugs.

Definition and description of terms

Unless otherwise indicated, the radical and term definitions recited in the specification and claims of this application, including as examples, exemplary definitions, preferred definitions, definitions recited in tables, definitions of specific compounds in the examples, and the like, may be arbitrarily combined and coupled with each other. Such combinations and combined group definitions and structures of compounds should fall within the scope of the protection of the present application.

The numerical ranges recited in the specification and claims are equivalent to at least each specific integer number recited therein unless otherwise stated. For example, the numerical range "1-40" corresponds to the numerical values of each integer in the numerical range "1-12", i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and the numerical values of each integer in the numerical range "13-40", i.e., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40. Furthermore, when certain numerical ranges are defined as "numbers," it is to be understood that both endpoints of the range, each integer within the range, and each fraction within the range are delineated. For example, a "number of 0 to 10" should be understood to describe not only each integer of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, but also at least the sum of each integer with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, respectively.

It should be understood that in describing one, two or more herein, "more" shall mean an integer greater than 2, such as greater than or equal to 3, such as 3, 4, 5, 6, 7, 8, 9 or 10.

"halogen" as used herein refers to fluorine, chlorine, bromine or iodine.

The term "alkyl" is understood to mean a straight-chain or branched saturated hydrocarbon radical having from 1 to 40 carbon atoms, for example from 1 to 20 carbon atoms. "C _1-12 Alkyl "is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having from 1 to 12 carbon atoms. For example, "C _1-10 Alkyl "means straight-chain and branched alkyl having 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms," C _1-8 Alkyl "means straight and branched alkyl having 1,2, 3, 4, 5, 6, 7, or 8 carbon atoms," C _1-6 Alkyl "means straight and branched alkyl groups having 1,2, 3, 4, 5 or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-ethylpropyl, 1, 2-dimethylpropyl, neopentyl, 1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 2, 3-dimethylbutyl, 1, 3-dimethylbutyl, or 1, 2-dimethylbutyl, or the like, or an isomer thereof.

The term "cycloalkyl" is understood to mean a saturated monocyclic, bicyclic (e.g. fused, bridged, spiro) hydrocarbon ring or tricyclic hydrocarbon ring having 3 to 20 carbon atoms, preferably "C _3-12 Cycloalkyl ", more preferably" C _3-8 Cycloalkyl groups). "C _3-12 Cycloalkyl "is understood to mean a saturated monovalent monocyclic, bicyclic (e.g. fused, bridged, spiro) hydrocarbon ring or tricyclic hydrocarbon ring having 3 to 12 carbon atoms, preferably" C _3-10 Cycloalkyl ", more preferably" C _3-8 Cycloalkyl groups). The term "C _3-10 Cycloalkyl "is understood to mean a saturated monovalent monocyclic, bicyclic (e.g. bridged, spiro) hydrocarbon ring or tricycloalkane having 3,4, 5, 6, 7, 8, 9 or 10 carbon atoms. The C is _3-10 Cycloalkyl may be a monocyclic hydrocarbon group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl, or a bicyclic hydrocarbon group such as campholyl, indolyl, hexahydroindolyl, tetrahydronaphthyl, decahydronaphthyl,Bicyclo [2.1.1]Hexyl, bicyclo [2.2.1]Heptyl, bicyclo [2.2.1]Heptenyl, 6-dimethylbicyclo [3.1.1]Heptyl, 2, 6-trimethylbicyclo [3.1.1]Heptyl, bicyclo [2.2.2]Octyl, 2, 7-diazaspiro [3,5 ]]Nonylalkyl, 2, 6-diazaspiro [3,4 ] ]Octyl, or tricyclic hydrocarbon groups such as adamantyl.

The term "aryl" is understood to mean preferably an aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring having 6 to 20 carbon atoms (e.g. 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 carbon atoms). "C _6-14 Aryl "is understood to mean preferably a mono-, bi-or tricyclic hydrocarbon ring (" C ") having a monovalent aromatic or partially aromatic character of 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms _6-14 Aryl), in particular a ring having 6 carbon atoms ("C) ₆ Aryl "), such as phenyl; or biphenyl, or a ring having 9 carbon atoms ("C ₉ Aryl "), e.g. indanyl or indenyl, or a ring having 10 carbon atoms (" C ₁₀ Aryl "), such as tetralin, dihydronaphthyl or naphthyl, or a ring having 13 carbon atoms (" C " ₁₃ Aryl "), e.g. fluorenyl, or a ring having 14 carbon atoms (" C) ₁₄ Aryl "), such as anthracenyl. When said C _6-14 When aryl is substituted, it may be mono-substituted or poly-substituted. The substitution site is not limited, and may be, for example, ortho, para or meta substitution. The term "aryl" also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjacent rings (the rings being "fused rings"), wherein at least one of the rings is aromatic and the other rings may be, for example, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and/or heterocyclyl. Examples of polycyclic rings include, but are not limited to, 2, 3-dihydro-1, 4-benzodioxadiene and 2, 3-dihydro-1-benzofuran.

The term "heteroaryl" is understood to include such monocyclic, bicyclic (e.g., fused, bridged, spiro) or tricyclic aromatic ring systems: having 5 to 20 ring atoms and containing one or more (e.g., 1 to 5) heteroatoms independently selected from N, O and S, such as "5-14 membered heteroaryl". "5-14 membered heteroaryl" is understood to include such monovalent monocyclic, bicyclic or tricyclic aromatic ring systems: it has 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, and it contains 1 to 5, preferably 1 to 3 heteroatoms each independently selected from N, O and S and, in addition, can be benzo-fused in each case. "heteroaryl" also refers to groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, wherein the attached radical or point is on the heteroaromatic ring. When the 5-14 membered heteroaryl is attached to other groups to form the compounds of the invention, the carbon atom on the 5-14 membered heteroaryl ring may be attached to other groups, or the heteroatom on the 5-14 membered heteroaryl ring may be attached to other groups. When the 5-14 membered heteroaryl is substituted, it may be mono-substituted or poly-substituted. And, the substitution site thereof is not limited, and for example, hydrogen attached to a carbon atom on a heteroaryl ring may be substituted, or hydrogen attached to a heteroatom on a heteroaryl ring may be substituted. Examples of heteroaryl groups include, but are not limited to, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furanyl, quinolinyl, isoquinolinyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrrolyl, oxazolyl, benzofuranyl, benzothienyl, benzothiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2, 4-thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, benzoxazolyl, azabenzoxazolyl, imidazothiazolyl, benzo [1,4] dioxanyl, benzo [1,3] dioxolyl, and the like. In some embodiments, heteroaryl groups have 3 to 40 carbon atoms and in other embodiments 3 to 20 carbon atoms. In some embodiments, heteroaryl groups comprise 3 to 14, 4 to 14, 3 to 7, or 5 to 6 ring forming atoms. In some embodiments, heteroaryl groups have 1 to 4, 1 to 3, or 1 to 2 heteroatoms. In some embodiments, the heteroaryl has 1 heteroatom.

The term "heterocyclyl" refers to a saturated or unsaturated, non-aromatic ring or ring system containing one or more heteroatoms independently selected from N, O and S and having 3 to 20 ring atoms (e.g., atoms)The numbers are 3, 4, 5, 6, 7, 8, 9, 10, etc.). "3-12 membered heterocyclyl" means a saturated or unsaturated, non-aromatic ring or ring system, e.g., which is a 4-, 5-, 6-, or 7-membered monocyclic, 7-, 8-, 9-, 10-, 11-, or 12-membered bicyclic (e.g., fused, bridged, spiro) or tricyclic ring system, and which contains at least one, e.g., 1, 2, 3, 4, 5 or more heteroatoms selected from O, S and N, wherein N and S may also optionally be oxidized to various oxidation states to form nitrogen oxides, -S (O) -or-S (O) ₂ -a state of the device. Preferably, the heterocyclic group may be selected from "3-10 membered heterocyclic groups". The term "3-10 membered heterocyclyl" means a saturated or unsaturated, non-aromatic ring or ring system and contains at least one heteroatom selected from O, S and N. The heterocyclic group may be attached to the remainder of the molecule through any of the carbon atoms or a nitrogen atom, if present. The heterocyclic group may include fused or bridged rings as well as spiro rings. In particular, the heterocyclic groups may include, but are not limited to: 4-membered rings such as azetidinyl, oxetanyl; a 5-membered ring such as tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl; or a 6 membered ring such as tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl or trithianyl; or a 7-membered ring such as diazepanyl. Optionally, the heterocyclyl may be benzo-fused. The heterocyclic group may be bicyclic, such as, but not limited to, a 5,5 membered ring, such as hexahydrocyclopenta [ c ] ]Pyrrol-2 (1H) -yl ring, or 5,6 membered bicyclic ring, e.g. hexahydropyrrolo [1,2-a ]]Pyrazin-2 (1H) -yl ring. The heterocyclic group may be partially unsaturated, i.e., it may contain one or more double bonds, such as, but not limited to, dihydrofuranyl, dihydropyranyl, 2, 5-dihydro-1H-pyrrolyl, 4H- [1,3,4]Thiadiazinyl, 1,2,3, 5-tetrahydrooxazolyl or 4H- [1,4]Thiazinyl, or it may be benzo-fused, such as, but not limited to, dihydroisoquinolinyl. When the 3-12 membered heterocyclic group is linked to other groups to form the compound of the present invention, the carbon atom on the 3-12 membered heterocyclic group may be linked to other groups, or the heterocyclic atom on the 3-12 membered heterocyclic ring may be linked to other groups. Unless otherwise indicated, it may be made by carbon orNitrogen to connect, wherein-CH ₂ -the group is optionally replaced by-C (O) -; and wherein, unless otherwise indicated to the contrary, the ring nitrogen or ring sulfur atom is optionally oxidized to form an N-oxide or S-oxide or the ring nitrogen atom is optionally quaternized; wherein the-NH in the ring is optionally substituted with acetyl, formyl, methyl or methanesulfonyl; and the ring is optionally substituted with one or more halogens. It will be appreciated that when the total number of S and O atoms in the heterocyclyl exceeds 1, these heteroatoms are not adjacent to one another. If the heterocyclyl is bicyclic or tricyclic, at least one ring may optionally be heteroaromatic or aromatic, provided that at least one ring is non-heteroaromatic. If the heterocyclyl is a single ring, it must not be aromatic. Examples of heterocyclyl groups include, but are not limited to, piperidinyl, N-acetylpiperidinyl, N-methylpiperidinyl, N-formylpiperazinyl, N-methylsulfonylpiperazinyl, homopiperazinyl, piperazinyl, azetidinyl, oxetanyl, morpholinyl, tetrahydroisoquinolyl, tetrahydroquinolinyl, indolinyl, tetrahydropyranyl, dihydro-2H-pyranyl, tetrahydrofuranyl, tetrahydrothiopyranyl, tetrahydrothiopyran-1-oxide, tetrahydrothiopyran-1, 1-dioxide, 1H-pyridin-2-one, and 2, 5-dioxoimidazolidinyl.

The term "spiro" refers to a ring system in which two rings share 1 ring-forming atom.

The term "fused ring" refers to a ring system in which two rings share 2 ring atoms.

The term "bridged ring" refers to a ring system in which two rings share more than 3 ring members.

Unless otherwise indicated, heterocyclyl, heteroaryl or heteroarylene include all possible isomeric forms thereof, e.g. positional isomers thereof. Thus, for some illustrative non-limiting examples, forms that may include substitution at 1, 2, or more of its 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-positions, etc. (if present) or bonding to other groups include pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, and pyridin-4-yl; thienyl or thienylene include thiophen-2-yl, thienylene-2-yl, thiophen-3-yl and thienylene-3-yl; pyrazol-1-yl, pyrazol-3-yl, pyrazol-4-yl, and pyrazol-5-yl.

The term "subunit" denotes a divalent group having two bonds substituted.

The compounds of formula I also include all of their respective possible stereoisomers, either in the form of a single stereoisomer or any mixture of any of the stereoisomers (e.g. R-isomer or S-isomer, or E-isomer or Z-isomer) in any proportion. Separation of individual stereoisomers (e.g., individual enantiomers or individual diastereomers) of the compounds of the invention may be accomplished by any suitable prior art method (e.g., chromatography, particularly, e.g., chiral chromatography). In addition, the compounds may also exist in tautomeric forms. The compounds of formula I of the present invention include all possible tautomers, either in the form of a single tautomer or any mixture of said tautomers in any proportions. All such isomers and mixtures thereof are included in the present invention.

The compounds of formula I according to the invention, their isomers, prodrugs, pharmaceutically acceptable salts and complexes thereof, respectively, may be present in the form of solvates, such as hydrates, wherein the compounds according to the invention comprise a polar solvent as a structural element of the compound lattice, in particular, for example, water, methanol or ethanol. The polar solvent, in particular water, may be present in stoichiometric or non-stoichiometric amounts. In the case of stoichiometric solvates (e.g., hydrates), there may be a half-solvate, a mono-solvate, a sesquisolvate, a di-solvate, a tri-solvate, a tetra-solvate, a penta-solvate, a semi-penta-solvate, etc., respectively. The present invention includes all such solvates, particularly hydrates.

Pharmaceutically acceptable salts of the compounds of formula I of the present invention may be acid addition salts of the compounds of the present invention having a nitrogen atom or other basic group in the chain or ring, with sufficient basicity, for example with inorganic acids such as: such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid, or acid addition salts with organic acids such as: for example formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4-hydroxybenzoyl) benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, pamoic acid, pectate acid, persulphuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptonic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, hemisulfuric acid, or thiocyanic acid.

In addition, another suitable pharmaceutically acceptable salt of a compound of the invention having sufficient acidity is an alkali metal salt (e.g., sodium or potassium salt), alkaline earth metal salt (e.g., calcium or magnesium salt), ammonium salt, or a salt with an organic base that provides a physiologically acceptable cation, such as a salt with: n-methylglucamine, dimethylglucamine, ethylglucamine, lysine, dicyclohexylamine, 1, 6-hexamethylenediamine, ethanolamine, glucamine, sarcosine, serinol, tris-hydroxymethyl aminomethane, aminopropanediol, 1-amino-2, 3, 4-butanetriol. Alternatively, the basic nitrogen-containing groups may be quaternized with the following agents: lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and dipentyl sulfate; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromides, and the like.

And, the pharmaceutically acceptable salts shall also include pharmaceutically acceptable salts of the compounds which are partly in the form of inner salts.

Detailed Description

The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.

The compounds of formula I of the present invention may be prepared by methods and process parameters known in the art. As an example, the present invention also provides a process for the preparation of a compound of formula I comprising one or more of the steps of examples 1 to 9 of the following route:

the english used in the reaction schemes of the following examples have the following meanings: dry represents dry; reflux represents reflux; overright indicates reaction overnight; grubbs 2nd-gen. Catalyst represents a Grubbs generation II catalyst; dimethyl succinate dimethyl succinate; pivaloyl chloride it represents pivaloyl chloride; dioxane represents 1, 4-Dioxane; bis (pinacolato) dibron represents bis (pinacolato) diboron; pyridine represents pyridine; carboxyacetic acid malonic acid, also known as abbreviated malic acid; tosMIC represents p-toluenesulfonylmethisonitrile; other english abbreviations refer to the common interpretation in the art.

Example 1

Preparation of compound 4 a: 3-butenamine hydrochloride (1.2 g,11.155 mmol) was added to a 50mL reaction flask, 16mL anhydrous dichloromethane was added, stirred to dissolve, placed in an ice-water bath, and Et was added sequentially under the protection of argon ₃ N (3.2 mL,22.309 mmol), DMAP (141 mg,1.116 mmol), 2-butenoyl chloride (1.3 mL,13.385 mmol), and then the system was reacted at room temperature for 2h. After TLC monitoring the reaction was completed, saturated NaHCO was added ₃ Extracting the solution with dichloromethane three times, collecting organic phase, adding appropriate amount of saturated saline water, washing once, drying with anhydrous magnesium sulfate, filtering, concentrating to obtain yellow oily substance, purifying with silica gel column chromatography, and eluting with petroleum as eluentEther: ethyl acetate (2:1) gave compound 3 as a clear oily liquid, 1.358g, 87.6% yield. ¹ H NMR(400MHz,CDCl ₃ )δ6.85–6.73(m,1H),6.01(s,1H),5.85–5.68(m,2H),5.06(dd,J＝15.4,7.8Hz,2H),3.36(q,J＝6.4Hz,2H),2.31–2.21(m,2H),1.82(dd,J＝6.9,1.0Hz,3H).

Compound 3 (1.356 g,9.769 mmol) was added to a 500mL reaction flask, 350mL of anhydrous dichloromethane was added, dissolved with stirring, oxygen in the solvent was removed, grubbs' II catalyst (207 mg,0.244 mmol) was added, oxygen in the solvent was removed once more, and the flask was transferred to a 40℃oil bath and refluxed under argon for 2h. After TLC monitoring reaction, the system is cooled to room temperature, stirred for 1h, concentrated under reduced pressure to obtain brown oily substance, purified by column chromatography, and the eluent is petroleum ether: ethyl acetate (1:1) gave compound 4a as a brown solid, 898mg, 94.7% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.04(s,1H),6.66(dt,J＝9.8,4.2Hz,1H),5.90(dd,J＝9.9,1.7Hz,1H),3.42(td,J＝7.2,2.4Hz,2H),2.41–2.26(m,2H).

Example 2

Preparation of compound 4 b: 2-piperidone (2 g,20.202 mmol) was added to a 50mL reaction flask, 25mL of anhydrous dichloromethane was added, stirred and dissolved, and placed in an ice-water bath, phosphorus pentachloride (8.4 g,40.404 mmol) was slowly added in portions under the protection of argon, and stirring was continued in the ice-water bath for 10min, then ZnI was added ₂ (194 mg,0.606 mmol) and the system was removed from the ice-water bath, warmed to room temperature and stirred for 1h, and Br was slowly added ₂ (2.1 mL,40.404 mmol) was stirred at room temperature for 12h. After TLC monitoring the reaction, the system was poured into ice water, stirred for 1h, extracted three times with dichloromethane, the organic phase was collected, then washed once with an appropriate amount of saturated brine, dried over anhydrous magnesium sulfate, and washed once with waterFiltering, concentrating, purifying by column chromatography, eluting with petroleum ether: ethyl acetate (2:1) gave compound 6b as a brown solid, 3.05g, 58.7% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.21(s,1H),3.51–3.44(m,2H),2.98(dd,J＝7.3,4.4Hz,2H),2.06–1.99(m,2H).

Compound 6b (3.05 g,11.87 mmol) was added to a 50mL reaction flask, 20mL anhydrous DMF was added, the mixture was stirred to dissolve, lithium carbonate (1.7 g,22.55 mmol) and lithium chloride (513mg, 12.1074 mmol) were added sequentially, the system was transferred to an oil bath at 130℃and reacted under argon for 8h. After the TLC monitoring reaction is finished, the system is cooled to room temperature, filtered, the solvent is removed under reduced pressure, the ethyl acetate is used for extraction three times, an organic phase is collected, then an appropriate amount of saturated saline water is used for washing once, anhydrous magnesium sulfate is used for drying, the filtration and the column chromatography purification are carried out after the concentration, and the eluent is petroleum ether: ethyl acetate (1:1) gave compound 4b as a brown solid, 1.56g, 74.7% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.45(s,1H),7.05(t,J＝4.6Hz,1H),3.49(td,J＝7.1,2.6Hz,2H),2.43(td,J＝7.1,4.7Hz,2H).

Preparation of compound 4 c: 2-piperidone (2 g,20.202 mmol) was added to a 100mL reaction flask, 60mL chloroform was added, the flask was put into an ice-water bath, phosphorus pentachloride (12.6 g,60.606 mmol) was added in portions under argon protection, the flask was transferred to room temperature and stirring was continued for 10min, the system was transferred to a 65℃oil bath, and the reaction was carried out under argon protection for 12h. After the TLC monitoring reaction was completed, the system was cooled to room temperature, poured into ice water, pH was adjusted to 8 to 11 with sodium hydroxide solution, extracted three times with methylene chloride, the organic phase was collected, then washed once with an appropriate amount of saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, and then a small amount of methylene chloride was added, and the mixture was filtered to give Compound 6c as a yellow solid in 2.3g in 65.7% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.51(s,1H),3.44(td,J＝6.2,2.4Hz,2H),2.81–2.74(m,2H),2.11–2.03(m,2H).

Compound 6c (2.3 g,13.691 mmol) was added to a 50mL reaction flask, 25mL anhydrous DMF was added, the mixture was stirred to dissolve, lithium carbonate (3.034 g,41.071 mmol) was added, the system was transferred to a 120℃oil bath and reacted under argon for 12h. After the TLC monitoring reaction is finished, the system is cooled to room temperature, filtered, the solvent is removed under reduced pressure, the ethyl acetate is used for extraction three times, an organic phase is collected, then an appropriate amount of saturated saline water is added for washing once, and then the reaction system is dried by anhydrous magnesium sulfate, filtered, concentrated and purified by column chromatography, and the eluent is petroleum ether: ethyl acetate (1:1) gave compound 4c as a brown solid, 1.1g, 61.1% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.39(s,1H),6.78(t,J＝4.6Hz,1H),3.48(td,J＝7.2,2.8Hz,2H),2.47(td,J＝7.1,4.6Hz,2H).

Example 3

Preparation of Compound I-1: 3,4, 5-trimethoxybenzaldehyde (2 g,10.204 mmol) and dimethyl succinate (1.6 mL, 12.248 mmol) were added to a 50mL reaction flask, 15mL anhydrous methanol was added for dissolution, a freshly prepared methanolic sodium methoxide solution (340mg 14.796mmol Na was added to 4mL anhydrous methanol) was added, the mixture was transferred to room temperature and stirring was continued for 10min, the system was transferred to 65℃oil bath and refluxed for 2h under argon. After TLC monitoring reaction, the system was cooled to room temperature, acidified, extracted three times with ethyl acetate, the organic phase was collected, then washed once with an appropriate amount of saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated and purified by column chromatography with stone as eluentOil ether: ethyl acetate (2:1) gave compound 8 as a yellow oily liquid, 2.1g, 65.6% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.87(s,1H),6.66(s,2H),3.90(s,3H),3.87(s,9H),3.64(s,2H).

Compound 8 (1, 1g, 3.258 mmol) was added to a 25mL reaction flask, 10mL acetic anhydride was added to dissolve, then anhydrous sodium acetate (349 mg,4.258 mmol) was added, the system was transferred to a 130℃oil bath and reacted under argon for 45min. After the TLC monitoring reaction was completed, the system was cooled to room temperature, the solvent was removed under reduced pressure, the saturated sodium bicarbonate solution was extracted three times with ethyl acetate, the organic phase was collected, then an appropriate amount of saturated brine was added for washing once, dried over anhydrous magnesium sulfate, filtered, concentrated and then dissolved in 15mL of methanol, transferred to a 50mL reaction flask, potassium carbonate (5.2 g,37.671 mmol) was added, the system was transferred to an oil bath at 65℃for 2 hours of reaction, after the TLC monitoring reaction was completed, the system was cooled to room temperature, the solvent was removed under reduced pressure, extracted three times with ethyl acetate, the organic phase was collected, then an appropriate amount of saturated brine was added for washing once, dried over anhydrous magnesium sulfate, filtered, concentrated and then purified by column chromatography, and the eluent was petroleum ether: ethyl acetate (5:1) afforded compound 10 as a white solid, 600mg, 60.8% yield in two steps. ¹ H NMR(400MHz,CDCl ₃ )δ9.50(s,1H),7.97(d,J＝1.4Hz,1H),7.34(d,J＝1.5Hz,1H),7.05(s,1H),4.18(s,3H),4.00(d,J＝3.2Hz,6H),3.97(s,3H),1.58(s,3H).

Compound 10 (1.1 g,3.957 mmol) was added to a 25mL reaction flask, 10mL DMF was added and dissolved with stirring, cesium carbonate (2.6 g, 7.284 mmol) and methyl iodide (370. Mu.L, 5.935 mmol) were added sequentially, and the reaction was carried out at room temperature for 12h. After the TLC monitoring was completed, the solvent was removed under reduced pressure, extracted three times with ethyl acetate, and the organic phase was collected, then washed once with an appropriate amount of saturated brine,drying with anhydrous magnesium sulfate, filtering, concentrating, purifying by column chromatography, and eluting with petroleum ether: ethyl acetate (7:1) gave compound 11a as a white solid, 1.1g, 91.6% yield. 1H NMR (400 MHz, CDCl) ₃ )δ8.05(d,J＝1.3Hz,1H),7.30(d,J＝1.4Hz,1H),7.04(s,1H),4.05(s,3H),4.00(s,3H),3.97(s,3H),3.96(s,3H),3.92(s,3H).

Compound 11a (1.1 g,3.767 mmol) was added to a 50mL reaction flask, 3mL of methanol, 3mL of tetrahydrofuran, 9mL of water were added, and the mixture was dissolved with stirring, followed by addition of sodium hydroxide (226 mg,5.651 mmol) and reacted at 65℃for 2 hours. After the completion of the reaction by TLC, the solvent was removed under reduced pressure, acidified, filtered and dried to give compound 12a as a white solid in 950mg with a yield of 86.4%.

Compound 12a (150 mg,0.539 mmol) was added to a 25mL reaction flask, 4mL anhydrous THF was added under argon, stirring was dissolved, the system was set at-20℃and then anhydrous triethylamine (51. Mu.L, 0.621 mmol) and pivaloyl chloride (73. Mu.L, 0.594 mmol) were sequentially added, and the system was reacted at-20℃for 1h. After the completion of the TLC monitoring reaction, 1mL of saturated sodium bicarbonate was added to quench the reaction, the reaction was extracted three times with methylene chloride, and the organic phase was collected, then washed once with an appropriate amount of saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated and dried by suction to give compound 13a. The key intermediate 4a (63 mg,0.648 mmol) was added to a 25mL two-port reaction flask, 4mL anhydrous THF was added under argon protection, stirring was performed to dissolve, the system was placed at-78deg.C, then 2.5M n-butyllithium in cyclohexane (26. Mu.L, 0.648 mmol) was added, the reaction was performed for 30min, and finally compound 13a (0.540 mmol) was added to the system, and the reaction was performed for 2h at-78deg.C. After TLC monitoring the reaction, adding saturated ammonium chloride solution to quench the reaction, heating the system to room temperature, removing the solvent under reduced pressure, extracting with ethyl acetate three times, collecting The organic phase is collected, then a proper amount of saturated saline is added for washing once, and then anhydrous magnesium sulfate is used for drying, filtration and column chromatography purification are carried out after concentration, and the eluent is petroleum ether: ethyl acetate (2:1) afforded compound I-1 as a yellow oil, 50mg, 25% yield in two steps. ¹ H NMR(400MHz,CDCl ₃ )δ7.51(d,J＝1.3Hz,1H),7.00(dt,J＝9.7,4.2Hz,1H),6.97–6.93(m,2H),6.02(dt,J＝9.8,1.8Hz,1H),4.05–4.00(m,5H),3.97(s,3H),3.95(s,3H),3.91(s,3H),2.64(tdd,J＝6.3,4.2,1.9Hz,2H). ¹³ C NMR(101MHz,CDCl3)δ174.11,165.69,156.04,153.19,149.47,145.49,144.04,132.92,131.97,125.35,121.13,117.98,104.63,102.78,62.16,61.41,56.14,55.80,43.66,24.97.

The preparation of reference I-1 of compounds I-2 to I-4 is carried out in combination with the above-described reaction scheme. Compound I-2 was a yellow oil in 26.2% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.57(dd,J＝7.5,4.3Hz,3H),7.41(dd,J＝10.0,4.6Hz,2H),7.35(d,J＝7.3Hz,1H),7.00(dt,J＝8.4,2.2Hz,3H),6.00(dt,J＝9.7,1.8Hz,1H),5.22(s,2H),4.02(t,J＝6.4Hz,2H),3.96(d,J＝6.2Hz,6H),3.78(s,3H),2.62(qd,J＝6.2,1.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ174.03,165.64,155.24,153.29,149.67,145.42,144.12,137.03,132.88,132.13,128.47,127.84,127.68,125.39,121.47,118.39,104.64,104.56,77.36,77.04,76.72,71.25,62.25,61.35,55.78,43.66,24.98.

Compound I-3 is a white solid with a melting point of 147.6-148.2℃and a yield of 28.5%. ¹ H NMR(400MHz,CDCl ₃ )δ7.50(d,J＝1.3Hz,1H),7.39(t,J＝4.6Hz,1H),6.97(s,1H),6.91(d,J＝1.5Hz,1H),4.05(t,J＝6.5Hz,2H),4.01(s,3H),3.98(s,3H),3.95(s,3H),3.91(s,3H),2.67(td,J＝6.4,4.7Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ173.76,161.00,156.15,153.26,149.47,145.85,144.18,132.41,131.96,121.10,118.60,118.08,104.71,102.49,77.44,77.12,76.80,62.18,61.43,56.19,55.84,43.88,26.88.

Compound I-4 is a yellow solid with a melting point of 114.4-115.0 ℃ and a yield of 30.1%. ¹ H NMR(400MHz,CDCl ₃ )δ7.51(d,J＝1.3Hz,1H),7.12(t,J＝4.6Hz,1H),6.97(s,1H),6.92(d,J＝1.5Hz,1H),4.05(t,J＝6.5Hz,2H),4.02(s,3H),3.98(s,3H),3.95(s,3H),3.91(s,3H),2.71(td,J＝6.4,4.7Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ ) Delta 173.65,161.24,156.19,153.30,149.50,144.23,140.91,132.36,131.97,127.90,121.19,118.15,104.74,102.57,77.39,77.07,76.75,62.16,61.40,56.20,55.83,43.84,25.50.HR-MS (ESI-TOF) m/z calculated C ₂₀ H ₂₀ ClNO ₆ Na[M+Na] ⁺ 428.0879, experimental 428.0871.

Example 4

Wherein, the terminal of R is linked to the left group in its structure and the terminal of R is linked to the right group in its structure;

preparation of Compound I-5: 3,4, 5-trimethoxybromobenzene (1 g,4.05 mmol) was added to a 50mL reaction flask, 30mL of 1, 4-dioxane was added, the mixture was stirred and dissolved, bis (pinacolato) diboron (1.233 g,4.856 mmol) and potassium acetate (1.747 g, 17.803 mmol) were then added in sequence, oxygen was removed from the system, and PdCl was added ₂ (dppf) (148 mg,0.202 mmol), and once more, the oxygen was removed, and the system was transferred to a 110℃oil bath and reacted under argon for 12h. After TLC monitoring the reaction, the system was cooled to room temperature, filtered, the solvent was removed under reduced pressure, extracted three times with ethyl acetate, and the organic phase was collected, thenAdding a proper amount of saturated saline water for washing once, drying by using anhydrous magnesium sulfate, filtering, concentrating, purifying by column chromatography, wherein the eluent is petroleum ether: ethyl acetate (16:1) afforded compound 15 as a white solid, 980mg, 81.7% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.05(s,2H),3.92(s,6H),3.89(s,3H),1.36(s,12H).

Compound 15 (1 g,3.401 mmol) was added to a 100mL reaction flask, 50mL of 1, 4-dioxane and 5mL of water were added, and the mixture was stirred and dissolved, followed by successively adding methyl o-bromobenzoate (731 mg,3.401 mmol) and cesium carbonate (4.4 g,3.401 mmol), removing oxygen from the system, and adding Pd (PPh) ₃ ) ₄ (196 mg,0.170 mmol) and once more oxygen, the system was transferred to a 100deg.C oil bath and reacted under argon for 12h. After the TLC monitoring reaction, the system was cooled to room temperature, filtered, the solvent was removed under reduced pressure, extracted three times with ethyl acetate, the organic phase was collected, then washed once with an appropriate amount of saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated, purified by column chromatography, and the eluent was petroleum ether: ethyl acetate (6:1) gave compound 16a as a yellow oil, 860mg, 84.1% yield. Compound 16a (633 mg,2.103 mmol) was transferred to a 50mL reaction flask, 3mL of methanol, 3mL of tetrahydrofuran, 9mL of water were added, and the mixture was dissolved with stirring, followed by addition of sodium hydroxide (168 mg,4.206 mmol) and reacted at 60℃for 2 hours. After the completion of the TLC monitoring, the solvent was removed under reduced pressure, acidified, filtered and dried to give compound 17a as a white solid, 550mg, in 91.2% yield.

The synthesis of compounds 17b and 17c was combined with the scheme and reference was made to the synthesis of 17a, and the operation of compounds 17 a-17 c for the synthesis of I-5-I-7 was combined with the scheme and reference was made to the synthesis of compounds 12a to I-1 in example 3.

Compound I-5 as a white solid, melting point 145.9-147.3 ℃ and the yield is 23.5 percent. ¹ H NMR(400MHz,CDCl ₃ )δ7.53–7.41(m,3H),7.30(dd,J＝7.0,1.5Hz,1H),6.66(dt,J＝9.6,4.2Hz,1H),6.48(s,2H),5.60(dt,J＝9.8,1.8Hz,1H),3.85(s,3H),3.81(s,6H). ¹³ C NMR(101MHz,CDCl ₃ ) Delta 172.97,164.07,152.91,145.53,138.71,137.26,137.10,136.51,129.77,129.06,127.68,127.46,124.73,105.90,77.35,77.03,76.71,60.87,56.20,41.87,24.31.HR-MS (ESI-TOF) m/z calculated C ₂₁ H ₂₁ NO ₅ Na[M+Na] ⁺ 390.1320, experimental 390.1312.

Compound I-6 was a yellow oil in 21.2% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.77(t,J＝1.6Hz,1H),7.65(ddd,J＝7.6,1.8,1.3Hz,1H),7.49(d,J＝1.6Hz,1H),7.46–7.41(m,1H),6.99(dt,J＝9.7,4.2Hz,1H),6.77(s,2H),5.99(dt,J＝9.8,1.8Hz,1H),4.04(t,J＝6.4Hz,2H),3.92(s,6H),3.89(s,3H),2.62(tdd,J＝6.3,4.2,1.9Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ173.74,165.49,153.47,145.71,141.48,137.89,136.63,136.44,130.24,128.29,127.02,127.00,125.21,104.71,77.41,77.09,76.78,60.95,56.28,43.35,24.94.

Compound I-7 is a yellow oil in 25.6% yield. ¹ H NMR(400MHz,CDCl ₃ )δ8.70(d,J＝5.0Hz,1H),8.59(s,1H),7.36(dd,J＝5.0,0.6Hz,1H),6.73(dt,J＝9.7,4.2Hz,1H),6.47(s,2H),5.64(dt,J＝9.8,1.8Hz,1H),3.86(s,3H),3.82(s,6H). ¹³ C NMR(101MHz,CDCl ₃ )δ170.39,163.85,153.18,149.53,148.98,146.45,144.41,137.79,132.88,132.45,124.28,120.68,105.98,77.38,77.06,76.74,60.93,56.26,41.50,24.24.

Example 5

Preparation of Compound I-8: 3,4, 5-trimethoxybenzaldehyde (1 g,5.097 mmol) was added to a 25mL reaction flask, 6mL DMF was added, stirring was performed to dissolve, malonic acid (2.1 g,20.387 mmol) and pyridine (1.66 mL,20.387 mmol) were added sequentially, the system was transferred to 90℃oil bath and reacted under argon for 12h. After the TLC monitoring reaction is finished, the system is cooled to room temperature, the solvent is removed under reduced pressure, acidification is carried out, extraction is carried out three times by using ethyl acetate, an organic phase is collected, then an appropriate amount of saturated saline water is added for washing once, and then anhydrous magnesium sulfate is used for drying, filtration, concentration and column chromatography purification are carried out, and the eluent is petroleum ether: ethyl acetate (3:1) gave compound 19 as a white solid, 1.4g, 93.3% yield.

Compound 19 (200 mg,0.794 mmol) was added to a 25mL reaction flask, 5mL of methanol was added, dissolved with stirring, placed in an ice-water bath, oxalyl chloride (169. Mu.L, 2 mmol) was slowly added dropwise, the ice-water bath was removed, and the reaction was carried out at room temperature under argon atmosphere for 12h. After the TLC monitoring reaction was completed, the solvent was removed under reduced pressure, then a saturated sodium bicarbonate solution was added, extraction was performed three times with ethyl acetate, the organic phase was collected, then an appropriate amount of saturated brine was added for washing once, and then dried over anhydrous magnesium sulfate, filtered, concentrated, purified by column chromatography, and the eluent was petroleum ether: ethyl acetate (5:1) afforded compound 20 as a white solid, 190mg, 90.1% yield.

Compound 20 (650 mg,2.444 mmol) and p-toluenesulfonyl methyl isonitrile (525 mg,2.688 mmol) were added to a 25mL reaction flask, 8mL anhydrous THF was added, stirred for dissolution, placed in an ice-water bath, sodium hydride (88 mg,3.665 mmol) was slowly added, the ice-water bath was removed, the system was transferred to an 80℃oil bath, and reacted for 2h under argon. After the TLC monitoring reaction was completed, the system was cooled toQuenching with ice water at room temperature, extracting with ethyl acetate three times, collecting organic phase, washing with appropriate amount of saturated saline water once, drying with anhydrous magnesium sulfate, filtering, concentrating, purifying with column chromatography, and eluting with petroleum ether: ethyl acetate (2:1) afforded compound 21 as a white solid, 600mg, 88.4% yield. ¹ H NMR(400MHz,CDCl ₃ )δ9.15(s,1H),7.48(d,J＝2.0Hz,1H),6.79(s,3H),3.89(d,J＝1.9Hz,9H),3.78(s,3H).

Manipulation of the methyl protecting group on Compound 21 reference was made to the procedure for the preparation of Compound 11a in example 3 to give Compound 22. Compound 22 (600 mg,2.069 mmol) was transferred to a 50mL reaction flask, 3mL of methanol, 3mL of tetrahydrofuran, 9mL of water were added, and the mixture was dissolved with stirring, followed by addition of lithium hydroxide (396 mg,16.553 mmol) and reacted at 60℃for 2h. After the completion of the reaction by TLC, the solvent was removed under reduced pressure, acidified, filtered and dried to give compound 23 as a white solid, 533mg, in 93.5% yield.

Synthesis of I-8 from Compound 23 the synthesis of I-1 was carried out with reference to Compound 12a in example 3. Compound I-8 is a yellow oil in 29% yield. ¹ H NMR(400MHz,CDCl ₃ )δ7.15(d,J＝2.4Hz,1H),6.62–6.55(m,2H),6.48(s,2H),5.56(dt,J＝9.7,1.9Hz,1H),3.86–3.81(m,11H),3.70(s,3H),2.25(tdd,J＝6.3,4.2,1.9Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.81,165.06,152.70,143.97,136.89,130.89,126.84,125.90,125.24,121.00,119.31,106.70,77.40,77.08,76.76,56.29,43.02,36.59,24.37.

Example 6

The synthesis of the compounds I-9 to I-13 was carried out using readily available aromatic acids as starting materials, and the synthetic procedure was combined with the synthesis of the compounds 12a to I-1 of reference example 3.

Compound I-9 is a yellow solid with a melting point of 102.1-103.3 ℃ and a yield of 26.2%. ¹ H NMR(400MHz,CDCl ₃ )δ9.01(s,1H),8.17–8.12(m,1H),8.10–8.04(m,1H),7.86–7.75(m,2H),7.05(dt,J＝9.7,4.2Hz,1H),5.97(dt,J＝9.8,1.9Hz,1H),4.17(t,J＝6.5Hz,2H),2.70(tdd,J＝6.4,4.2,1.9Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ170.33,164.89,149.03,146.83,143.29,142.57,140.83,130.93,130.52,129.79,129.36,124.58,42.23,24.63.

Compound I-10 is a yellow oil in 24.6% yield. ¹ H NMR(400MHz,CDCl ₃ )δ8.89(s,2H),8.31(dd,J＝1.9,0.5Hz,1H),8.13(dd,J＝8.7,0.5Hz,1H),7.91(dd,J＝8.7,1.9Hz,1H),7.04(dt,J＝9.7,4.2Hz,1H),5.99(dt,J＝9.8,1.9Hz,1H),4.10(t,J＝6.5Hz,2H),2.66(tdd,J＝6.3,4.2,1.9Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ172.60,165.38,146.21,146.08,145.70,144.12,142.32,137.80,129.54,129.43,128.93,125.03,43.23,24.95.

Compound I-11 is green solid with a melting point of 82.3-84.5 ℃ and a yield of 27.4%. ¹ H NMR(400MHz,CDCl ₃ )δ8.25(d,J＝8.4Hz,1H),8.06(d,J＝8.5Hz,1H),7.84(dd,J＝8.2,1.0Hz,1H),7.71(ddd,J＝8.4,6.9,1.4Hz,1H),7.64–7.60(m,1H),7.57(ddd,J＝8.1,6.9,1.2Hz,1H),7.00(dt,J＝9.8,4.2Hz,1H),5.94(dt,J＝9.8,1.9Hz,1H),4.15(t,J＝6.5Hz,2H),2.68(tdd,J＝6.4,4.2,1.9Hz,2H).

Compound I-12 is a yellow solid with a melting point of 221.4-222.1℃and a yield of 29.5%. ¹ H NMR(400MHz,CDCl ₃ )δ8.03(d,J＝7.8Hz,1H),8.01(d,J＝1.4Hz,1H),7.49–7.45(m,1H),7.39(d,J＝8.2Hz,1H),7.28–7.21(m,2H),7.00–6.94(m,1H),6.02(dt,J＝9.7,1.8Hz,1H),4.16(s,3H),4.03–3.98(m,5H),2.68–2.59(m,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ174.70,165.94,146.49,144.97,141.86,132.97,126.54,126.04,125.63,123.69,123.22,120.46,119.61,115.76,109.05,107.63,55.78,44.15,32.19,25.05.

Compound I-13 is a white solid with a melting point of 145.8-147.9 ℃ and a yield of 30%. ¹ H NMR(400MHz,CDCl ₃ )δ8.26(s,1H),8.19(dd,J＝8.0,1.5Hz,1H),7.69(ddd,J＝8.7,5.4,1.9Hz,1H),7.50–7.46(m,1H),7.43–7.39(m,1H),7.02(dt,J＝9.7,4.2Hz,1H),6.01(dt,J＝9.8,1.9Hz,1H),4.08(t,J＝6.4Hz,2H),2.62(tdd,J＝6.3,4.2,1.9Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ173.53,165.95,165.20,157.22,155.99,146.48,134.18,126.49,125.80,124.98,124.36,124.09,118.20,42.47,24.95.

Example 7

Preparation of Compound I-14: the key intermediate 4a (63 mg,0.648 mmol) was added to a 25mL two-port reaction flask, 4mL anhydrous THF was added under argon protection, stirring was performed to dissolve, the system was placed at-78deg.C, then 2.5M n-butyllithium in cyclohexane (26. Mu.L, 0.648 mmol) was added, the reaction was performed for 30min, and finally 2-naphthalenesulfonyl chloride (0.5396 mmol) was added to the system, and the reaction was performed for 2h at-78deg.C. After the TLC monitoring has been completed, saturated ammonium chloride is addedThe solution is quenched, the system is warmed to room temperature, the solvent is removed under reduced pressure, the ethyl acetate is used for extraction for three times, the organic phase is collected, then an appropriate amount of saturated saline water is added for washing once, and then the solution is dried by anhydrous magnesium sulfate, filtered, concentrated and purified by column chromatography, and the eluent is petroleum ether: ethyl acetate (2:1) afforded compound I-14 as a white solid with a melting point of 97.5-98.7 ℃ and a yield of 26%. ¹ H NMR(400MHz,CDCl ₃ )δ8.67(s,1H),8.02(d,J＝7.9Hz,1H),7.98–7.93(m,2H),7.90(d,J＝8.1Hz,1H),7.68–7.58(m,2H),6.81(dt,J＝9.7,4.2Hz,1H),5.84(dt,J＝9.8,1.8Hz,1H),4.14(t,J＝6.5Hz,2H),2.58(tdd,J＝6.4,4.3,1.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ ) Delta 162.97,144.74,135.61,135.35,131.89,130.68,129.62,129.29,128.98,127.89,127.54,125.06,122.94,44.23,25.31.HR-MS (ESI-TOF) m/z calculated C ₁₅ H ₁₃ NO ₃ S Na[M+Na] ⁺ 310.0516, experimental 310.0508.

The procedure for compounds I-15 and I-16 is described in example 7 with reference to the synthesis of I-14.

Compound I-15 is a white solid with a melting point of 136.9-137.5 ℃ and a yield of 27.5%. ¹ H NMR(400MHz,CDCl ₃ )δ8.68(d,J＝0.9Hz,1H),8.02(d,J＝7.6Hz,1H),7.97(d,J＝1.3Hz,2H),7.93–7.89(m,1H),7.65(dddd,J＝19.5,8.2,6.9,1.4Hz,2H),7.19(t,J＝4.6Hz,1H),4.20(t,J＝6.5Hz,2H),2.62(td,J＝6.5,4.6Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ158.42,145.00,135.48,134.98,131.89,131.16,129.74,129.50,129.16,127.91,127.64,122.94,117.93,44.34,27.28.

Compound I-16 is a white solid with a melting point of 144.7-146.2 ℃ and a yield of 26.3%. ¹ H NMR(400MHz,CDCl ₃ )δ8.68(d,J＝0.9Hz,1H),8.01(d,J＝7.8Hz,1H),7.96(d,J＝1.3Hz,2H),7.94–7.87(m,1H),7.64(dddd,J＝19.7,8.2,6.9,1.4Hz,2H),6.92(t,J＝4.6Hz,1H),4.18(t,J＝6.5Hz,2H),2.65(td,J＝6.5,4.7Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ158.76,140.33,135.48,134.93,131.87,131.16,129.72,129.53,129.18,127.92,127.67,127.32,122.90,44.28,25.84.

Example 8

Preparation of Compound II-1: benzofuran-2-carbaldehyde (185 mg,1.267 mmol) was added to a 25mL reaction flask, 3mL DMF was added, the mixture was stirred and dissolved, malonic acid (227 g,5.069 mmol) and pyridine (409. Mu.L, 5.069 mmol) were then added in sequence, the system was transferred to a 90℃oil bath and reacted under argon for 12h. After the completion of the TLC monitoring reaction, the system was cooled to room temperature, the solvent was removed under reduced pressure, acidification was performed, extraction was performed three times with ethyl acetate, the organic phase was collected, then an appropriate amount of saturated brine was added for washing once, drying was performed with anhydrous magnesium sulfate, filtration was performed, a small amount of dichloromethane was added after concentration, and filtration was performed, to obtain compound 29a as a yellow solid, 125mg, and a yield of 52.5%.

Compound 29a (100 mg,0.532 mmol) was added to a 25mL reaction flask, 4mL anhydrous THF was added under argon, the system was dissolved with stirring, and the system was left at-20℃and then anhydrous triethylamine (85. Mu.L, 0.612 mmol) and pivaloyl chloride (73. Mu.L, 0.585 mmol) were added in sequence, and the system was reacted at-20℃for 1h. After the TLC monitoring was completed, the reaction was quenched by adding 1mL of saturated sodium bicarbonate, extracted three times with methylene chloride, and the organic phase was collected, then washed once with an appropriate amount of saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated and dried by suction to give compound 30a. Key intermediate 4a (64 mg,0.665mm ol) was added to a 25mL two-port flask, 4mL anhydrous THF was added under argon protection, stirring was performed to dissolve, the system was placed at-78deg.C, then 2.5M n-butyllithium in cyclohexane (27. Mu.L, 0.665 mmol) was added, the reaction was performed for 30min, and finally compound 30a (0.532 mmol) was added to the system, and the reaction was performed for 2h at-78deg.C. After TLC monitoring reaction, adding saturated ammonium chloride solution to quench reaction, heating the system to room temperature, removing solvent under reduced pressure, extracting with ethyl acetate three times, collecting organic phase, then adding appropriate amount of saturated saline water for washing once, drying with anhydrous magnesium sulfate, filtering, concentrating, purifying by column chromatography, eluting with petroleum ether: ethyl acetate (3:1) afforded compound II-1 as a yellow solid with a melting point of 119.7-120.9 ℃ and a two-step yield of 28.2%. ¹ H NMR(400MHz,CDCl ₃ )δ7.61(s,2H),7.57(d,J＝7.8Hz,1H),7.47(d,J＝8.2Hz,1H),7.36–7.30(m,1H),7.22(t,J＝7.5Hz,1H),6.96–6.91(m,2H),6.06(d,J＝9.7Hz,1H),4.04(t,J＝6.5Hz,2H),2.47(tdd,J＝6.3,4.2,1.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.52,165.71,155.52,153.08,145.67,130.19,128.50,126.31,125.69,123.21,122.34,121.65,111.49,111.12,77.43,77.11,76.80,41.71,24.79.

The preparation of the compounds II-2 to II-14, II-1a to II-10a and II-1b to II-10b was carried out in combination with the reaction scheme and reference was made to the synthesis of the compound II-1 in example 8.

Compound II-2 is a yellow solid with a melting point of 93.2-94.4 ℃ and a yield of 26.4%. ¹ H NMR(400MHz,CDCl ₃ )δ7.98–7.94(m,1H),7.89(d,J＝15.8Hz,2H),7.78(d,J＝16.2Hz,1H),7.52(ddd,J＝4.8,2.3,0.4Hz,1H),7.39–7.32(m,2H),6.96(dt,J＝9.6,4.2Hz,1H),6.08(dt,J＝9.7,1.8Hz,1H),4.07(t,J＝6.5Hz,2H),2.49(tdd,J＝6.3,4.2,1.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.82,165.91,156.08,147.99,145.58,133.70,125.92,125.30,124.91,123.86,121.93,121.39,118.65,111.83,41.57,24.81.

Compound II-3 is a white solid with a melting point of 80.9-82.1 ℃ and a yield of 25.5%. ¹ H NMR(400MHz,CDCl ₃ )δ7.74(d,J＝15.2Hz,1H),7.56(d,J＝15.2Hz,1H),7.51(d,J＝7.5Hz,1H),7.43(d,J＝8.2Hz,1H),7.37–7.31(m,1H),7.25–7.20(m,1H),6.94(dt,J＝9.6,4.2Hz,1H),6.07(dt,J＝9.7,1.8Hz,1H),4.05(t,J＝6.5Hz,2H),2.48(tdd,J＝6.3,4.2,1.8Hz,2H),2.38(s,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.89,165.71,154.82,149.11,145.42,129.92,128.39,126.56,125.84,122.68,120.69,120.01,111.34,41.73,24.82,8.43.

Compound II-4 is a white solid with a melting point of 101.2-103.2 ℃ and a yield of 27.5%. ¹ H NMR(400MHz,CDCl ₃ )δ7.87(d,J＝15.6Hz,1H),7.83(d,J＝1.5Hz,1H),7.63(d,J＝2.2Hz,1H),7.55(dd,J＝16.0,8.7Hz,2H),7.48(d,J＝8.6Hz,1H),6.97–6.90(m,1H),6.80–6.76(m,1H),6.05(dt,J＝9.7,1.8Hz,1H),4.05(t,J＝6.5Hz,2H),2.48(tdd,J＝6.3,4.2,1.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ169.06,165.88,155.99,145.93,145.47,144.21,130.21,127.97,125.89,124.74,121.81,120.70,111.80,106.83,41.64,24.83.

Compound II-5 is a white solid with a melting point of 141.3-143.2 ℃ and a yield of 24.3%. ¹ H NMR(400MHz,CDCl ₃ )δ8.18(d,J＝1.5Hz,1H),7.98(d,J＝7.6Hz,1H),7.93(d,J＝15.6Hz,1H),7.70(dd,J＝8.6,1.7Hz,1H),7.57(dd,J＝18.6,11.9Hz,3H),7.48(d,J＝1.0Hz,1H),7.37(dd,J＝10.9,4.0Hz,1H),6.99–6.92(m,1H),6.08(dt,J＝9.7,1.7Hz,1H),4.07(t,J＝6.5Hz,2H),2.50(tdd,J＝6.3,4.2,1.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.98,165.92,157.25,156.67,145.55,143.81,130.15Calculated C.s.m/z.127.93, 127.65,125.86,124.87,123.74,123.10,120.91,120.90,120.63,112.00,111.81,41.67,24.84.HR-MS (ESI-TOF) ₂₀ H ₁₅ NO ₃ Na[M+Na] ⁺ 340.0952; experimental value 340.0944.

Compound II-6 is a yellow solid with a melting point of 117.0-119.7deg.C and a yield of 23.6%. ¹ H NMR(400MHz,CDCl ₃ )δ7.73(d,J＝15.6Hz,1H),7.50–7.47(m,1H),7.39(d,J＝15.6Hz,1H),7.36–7.32(m,1H),6.93(dt,J＝9.7,4.2Hz,1H),6.77(d,J＝8.3Hz,1H),6.04(dt,J＝9.7,1.8Hz,1H),4.61(t,J＝8.7Hz,2H),4.03(t,J＝6.5Hz,2H),3.22(t,J＝8.7Hz,2H),2.46(tdd,J＝6.3,4.2,1.9Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ169.20,165.89,162.17,145.33,144.14,130.08,127.98,127.96,125.95,124.57,118.70,109.53,71.85,41.61,29.25,24.83.

Compound II-7 is a white solid with a melting point of 91.2-93.3℃and a yield of 29.5%. ¹ H NMR(400MHz,CDCl ₃ )δ7.67(d,J＝1.0Hz,2H),6.99–6.90(m,2H),6.83–6.80(m,2H),6.07–6.02(m,3H),4.03(t,J＝6.5Hz,2H),2.51–2.44(m,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ169.15,165.73,147.93,146.57,145.46,138.05,125.81,124.30,122.82,121.67,118.10,109.57,101.46,41.66,24.80.

Compound II-8 is a yellow solid with a melting point of 169.8-171.3℃and a yield of 26.7%. ¹ H NMR(400MHz,CDCl ₃ )δ8.04(d,J＝15.5Hz,1H),7.32(d,J＝15.4Hz,1H),7.17(s,1H),7.04(s,1H),6.95(dt,J＝9.7,4.2Hz,1H),6.06–5.99(m,3H),4.04(t,J＝6.5Hz,2H),2.48(tdd,J＝6.3,4.2,1.9Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.53,165.81,149.87,147.84,145.60,141.65,128.38,125.78,122.60,117.95,113.03,106.89,102.19,41.61,24.79.

Compound II-9 is a yellow solid with a melting point of 169.7-170.6℃and a yield of 30%. ¹ H NMR(400MHz,CDCl ₃ )δ8.12(d,J＝15.5Hz,1H),7.54(s,1H),7.32(d,J＝15.4Hz,1H),7.11(s,1H),7.00–6.93(m,1H),6.15(s,2H),6.04(dt,J＝9.7,1.8Hz,1H),4.05(t,J＝6.5Hz,2H),2.50(tdd,J＝6.3,4.2,1.9Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ167.98,165.81,151.97,148.77,145.94,138.60,128.09,125.78,125.60,107.78,105.58,103.31,41.61,39.64,24.75.

Compound II-10 is a yellow solid with a melting point of 122.9-125.7deg.C and a yield of 24.2%. ¹ H NMR(400MHz,CDCl ₃ )δ7.95(dd,J＝15.3,0.5Hz,1H),7.80–7.73(m,2H),7.49(s,1H),7.40(d,J＝15.1Hz,1H),7.37–7.32(m,2H),6.95(dt,J＝9.6,4.2Hz,1H),6.06(dt,J＝9.7,1.8Hz,1H),4.04(t,J＝6.5Hz,2H),2.48(tdd,J＝6.3,4.2,1.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.36,165.68,145.52,140.49,140.34,139.67,136.67,128.46,126.03,125.79,124.73,124.32,123.07,122.47,41.65,24.79.

Compound II-11 is a yellow solid with a melting point of 110.4-112.5℃and a yield of 23.3%. ¹ H NMR(400MHz,CDCl ₃ )δ8.13(d,J＝15.1Hz,1H),7.78–7.74(m,1H),7.72–7.68(m,1H),6.94(dt,J＝9.6,4.2Hz,1H),6.06(dt,J＝9.7,1.8Hz,1H),4.05(t,J＝6.5Hz,2H),2.54(s,3H),2.52–2.45(m,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.63,165.70,145.45,140.65,139.36,136.24,134.84,134.71,126.36,125.84,124.38,122.63,122.44,122.30,41.69,24.81,12.21.

Compound II-12 is a white solid with a melting point of 131.2-133.4 ℃ and a yield of 18.4%. ¹ H NMR(400MHz,CDCl ₃ )δ8.30(d,J＝1.5Hz,1H),8.20–8.16(m,1H),7.93(d,J＝15.6Hz,1H),7.85–7.81(m,2H),7.69(dd,J＝8.4,1.6Hz,1H),7.64(d,J＝15.6Hz,1H),7.49–7.44(m,2H),6.94(dt,J＝9.6,4.2Hz,1H),6.07(dt,J＝9.7,1.8Hz,1H),4.06(t,J＝6.5Hz,2H),2.48(tdd,J＝6.3,4.2,1.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.99,165.92,145.58,143.75,141.37,139.76,136.01,135.13,131.64,127.15,126.35,125.87,124.68,123.07,122.91,121.81,121.73,121.44,41.69,24.84.

Compound II-13 is a yellow solid with a melting point of 123.4-125.2 ℃ and a yield of 21.2%. ¹ H NMR(400MHz,CDCl ₃ )δ9.06(s,1H),8.14–8.05(m,3H),7.89(d,J＝15.6Hz,1H),7.81–7.75(m,2H),6.99(dt,J＝9.6,4.2Hz,1H),6.09(dt,J＝9.7,1.8Hz,1H),4.09(t,J＝6.5Hz,2H),2.53(tdd,J＝6.3,4.2,1.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ ) Delta 168.24,165.71,148.75,145.98,144.88,142.50,142.37,138.56,130.53,130.50,129.87,129.21,128.90,125.55,41.72,24.79.HR-MS (ESI-TOF) m/z calculated C ₁₆ H ₁₃ N ₃ O ₂ [M+Na] ⁺ 302.1008, experimental 302.0900.

Compound II-14 is a white solid with a melting point of 171.8-173.7 ℃ and a yield of 31%. ¹ H NMR(400MHz,CDCl ₃ )δ8.92(dd,J＝4.2,1.7Hz,1H),8.18(d,J＝8.3Hz,1H),8.11–8.06(m,1H),7.97(dt,J＝3.8,1.8Hz,2H),7.90(d,J＝15.6Hz,1H),7.66(d,J＝15.6Hz,1H),7.43(dd,J＝8.3,4.2Hz,1H),7.00–6.94(m,1H),6.08(dt,J＝9.7,1.8Hz,1H),4.08(t,J＝6.5Hz,2H),2.51(tdd,J＝6.3,4.2,1.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.77,165.87,151.18,148.99,145.71,142.47,136.49,133.39,130.08,129.07,128.27,127.99,125.78,123.21,121.76,41.67,24.82.

Compound II-1a is a white solid with a melting point of 147.9-149.5℃and a yield of 27.5%. ¹ H NMR(400MHz,CDCl ₃ )δ7.62(d,J＝2.7Hz,2H),7.58(d,J＝7.7Hz,1H),7.47(d,J＝8.3Hz,1H),7.38–7.33(m,2H),7.23(t,J＝7.5Hz,1H),6.97(s,1H),4.10(t,J＝6.4Hz,2H),2.59–2.49(m,2H). ¹³ C NMR(101MHz,CDCl ₃ ) Delta 168.25,161.06,155.62,152.88,146.00,131.12,128.45,126.54,123.28,121.72,121.59,119.03,111.59,111.56,41.90,26.74.HR-MS (ESI-TOF) m/z calculated C ₁₆ H ₁₂ BrNO ₃ Na[M+Na] ⁺ 369.9880, experimental 369.9872.

Compound II-2a is a white solid with a melting point of 161.3-164.2 ℃ and a yield of 25.4%. ¹ H NMR(400MHz,CDCl ₃ )δ7.94(ddd,J＝16.9,9.9,6.6Hz,3H),7.74(d,J＝15.7Hz,1H),7.54–7.50(m,1H),7.40–7.33(m,3H),4.13(t,J＝6.5Hz,2H),2.53(td,J＝6.4,4.7Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.53,161.11,156.10,148.27,145.95,134.80,125.40,124.77,123.99,121.46,121.08,119.22,118.60,111.82,41.84,26.71.

Compound II-3a is yellow solid with melting point of 120.4-122.2 ℃ and yield25.2％。 ¹ H NMR(400MHz,CDCl ₃ )δ7.75(d,J＝15.1Hz,1H),7.56(d,J＝15.1Hz,1H),7.53–7.49(m,1H),7.44–7.39(m,1H),7.38–7.31(m,2H),7.26–7.20(m,1H),4.10(t,J＝6.5Hz,2H),2.52(td,J＝6.5,4.6Hz,2H),2.38(s,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.50,161.01,154.84,148.94,145.98,129.82,129.17,126.82,122.78,121.30,120.13,119.91,119.03,111.35,41.91,26.74,8.51.

Compound II-4a is a white solid with a melting point of 128.5-129.1℃and a yield of 26.5%. ¹ H NMR(400MHz,CDCl ₃ )δ7.93–7.80(m,2H),7.64(dd,J＝3.6,2.2Hz,1H),7.60–7.45(m,3H),7.39–7.30(m,1H),6.78(dd,J＝2.0,1.2Hz,1H),4.10(dd,J＝8.3,4.5Hz,2H),2.52(dd,J＝6.3,4.6Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.70,161.18,156.13,146.01,145.88,145.23,129.99,128.03,124.90,121.90,120.02,119.19,111.84,106.82,41.80,26.74.

Compound II-5a is a white solid with a melting point of 178.7-179.5℃and a yield of 28.3%. ¹ H NMR(400MHz,CDCl ₃ )δ8.18(s,1H),7.95(dd,J＝21.9,11.6Hz,2H),7.68(d,J＝8.6Hz,1H),7.57(ddd,J＝14.4,7.2,5.5Hz,3H),7.48(t,J＝7.8Hz,1H),7.41–7.32(m,2H),4.11(t,J＝6.4Hz,2H),2.57–2.48(m,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.65,161.23,157.43,156.72,145.94,144.90,129.92,128.17,127.71,124.96,123.71,123.17,120.98,120.66,120.16,119.17,112.05,111.83,41.85,26.75.

Compound II-6a is a yellow solid with a melting point of 149.3-151.2 ℃ and a yield of 29.4%. ¹ H NMR(400MHz,CDCl ₃ )δ7.74–7.68(m,1H),7.63(dd,J＝8.9,7.8Hz,1H),7.34(dd,J＝5.0,4.1Hz,1H),7.01–6.94(m,1H),6.84–6.80(m,2H),6.07(t,J＝0.9Hz,2H),4.09(t,J＝6.5Hz,2H),2.55–2.47(m,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.86,160.95,147.97,146.71,145.84,138.88,123.48,122.64,121.69,119.11,117.82,109.78,101.54,41.91,26.72.

Compound II-7a is a yellow solid with a melting point of 219.7-220.8 ℃ and a yield of 30.3%. ¹ H NMR(400MHz,CDCl ₃ )δ8.06(d,J＝15.5Hz,1H),7.36(t,J＝4.6Hz,1H),7.31(d,J＝15.4Hz,1H),7.19(s,1H),7.04(s,1H),6.02(s,2H),4.10(t,J＝6.5Hz,2H),2.53(td,J＝6.5,4.6Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.21,161.17,150.11,147.93,145.99,142.65,128.17,121.87,119.06,118.14,113.05,106.94,102.26,41.80,26.71.

Compound II-8a is a yellow solid with a melting point of 143.9-145.5℃and a yield of 25.4%. ¹ H NMR(400MHz,CDCl ₃ )δ8.15(d,J＝15.1Hz,1H),7.72(ddd,J＝9.0,5.7,2.3Hz,2H),7.41–7.30(m,4H),4.08(t,J＝6.3Hz,2H),2.56–2.45(m,5H). ¹³ C NMR(101MHz,CDCl ₃ ) Delta 168.29,160.95,145.85,140.58,139.51,136.74,135.86,134.53,126.53,124.43,122.70,122.48,121.48,119.12,41.90,26.71,12.20.HR-MS (ESI-TOF) m/z calculated C ₁₇ H ₁₄ BrNO ₂ S Na[M+Na] ⁺ 397.9829, experimental 397.9821.

Compound II-9a is a red solid with a melting point of 174.8-176.1℃and a yield of 25.5%. ¹ H NMR(400MHz,CDCl ₃ )δ9.08–9.04(m,1H),8.09(ddd,J＝21.1,10.0,9.0Hz,3H),7.92(s,1H),7.83–7.75(m,2H),7.40(s,1H),4.14(t,J＝6.4Hz,2H),2.58(dd,J＝4.7,1.6Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.01,161.11,148.43,146.34,144.90,142.48,142.45,139.47,130.63,130.59,129.89,129.23,128.17,118.72,41.96,26.72.

Compound II-10a is a white solid with a melting point of 176.7-178.3 ℃ and a yield of 28.7%. ¹ H NMR(400MHz,CDCl ₃ )δ8.97–8.88(m,1H),8.18(t,J＝7.9Hz,1H),8.08(d,J＝8.1Hz,1H),7.94(dt,J＝25.6,11.9Hz,3H),7.64(dd,J＝15.6,8.2Hz,1H),7.47–7.34(m,2H),4.12(t,J＝6.5Hz,2H),2.62–2.50(m,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.36,161.16,151.26,149.09,146.00,143.39,136.44,133.17,130.14,129.07,128.25,128.05,122.58,121.76,119.02,41.83,26.70.

Compound II-1b is a yellow solid with a melting point of 131.1-132.8℃and a yield of 29.6%. ¹ H NMR(400MHz,CDCl ₃ )δ7.62(d,J＝3.7Hz,2H),7.57(d,J＝6.7Hz,1H),7.47(dd,J＝8.3,0.7Hz,1H),7.38–7.32(m,1H),7.25–7.20(m,1H),7.09(t,J＝4.6Hz,1H),6.97(d,J＝0.4Hz,1H),4.09(t,J＝6.5Hz,2H),2.57(td,J＝6.4,4.8Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.14,161.33,155.59,152.86,141.24,131.10,131.09,128.44,128.13,126.55,123.29,121.73,121.56,111.61,111.53,41.82,25.33.

Compound II-2b is a yellow solid with a melting point of 149.3-150.5℃and a yield of 28.4%. ¹ H NMR(400MHz,CDCl ₃ )δ7.98–7.89(m,3H),7.76(d,J＝15.7Hz,1H),7.52(dt,J＝5.2,3.0Hz,1H),7.39–7.34(m,2H),7.10(t,J＝4.6Hz,1H),4.12(t,J＝6.5Hz,2H),2.56(td,J＝6.4,4.7Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.42,161.42,156.09,148.36,141.20,134.88,128.31,125.41,124.73,124.00,121.46,121.02,118.58,111.83,41.74,25.30.

Compound II-3b is a yellow solid with a melting point of 78.2-80.3℃and a yield of 26.8%. ¹ H NMR(400MHz,CDCl ₃ )δ7.76(d,J＝15.1Hz,1H),7.56(d,J＝15.2Hz,1H),7.54–7.49(m,1H),7.42(dd,J＝5.0,4.1Hz,1H),7.38–7.33(m,1H),7.26–7.20(m,1H),7.08(t,J＝4.6Hz,1H),4.09(t,J＝6.5Hz,2H),2.57(td,J＝6.5,4.6Hz,2H),2.38(s,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.47,161.31,154.87,148.94,141.08,129.83,129.26,128.22,126.82,122.77,121.33,120.12,119.87,111.38,41.84,25.35,8.51.

Compound II-4b is a white solid with a melting point of 114.5-115.1℃and a yield of 25.5%. ¹ H NMR(400MHz,CDCl ₃ )δ7.87(dd,J＝19.4,8.5Hz,2H),7.64(d,J＝2.2Hz,1H),7.54(ddd,J＝26.5,9.8,5.2Hz,3H),7.09(t,J＝4.6Hz,1H),6.80–6.77(m,1H),4.10(t,J＝6.5Hz,2H),2.57(dd,J＝11.1,6.4Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.63,161.48,156.13,146.02,145.26,141.08,129.97,128.28,128.03,124.87,121.92,119.97,111.85,106.83,41.73,25.34.

Compound II-5b is a yellow solid with a melting point of 154.6-155.9℃and a yield of 27.3%. ¹ H NMR(400MHz,CDCl ₃ )δ8.19(d,J＝1.8Hz,1H),7.98(ddd,J＝7.7,1.3,0.6Hz,1H),7.94(d,J＝15.5Hz,1H),7.70–7.67(m,1H),7.61–7.52(m,3H),7.48(ddd,J＝8.3,7.3,1.4Hz,1H),7.41–7.35(m,1H),7.10(t,J＝4.6Hz,1H),4.11(t,J＝6.5Hz,2H),2.58(td,J＝6.5,4.6Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.57,161.52,157.42,156.71,156.70,144.91,141.16,129.90,128.26,128.17,127.72,124.96,123.69,123.17,120.97,120.64,120.12,112.06,111.83,41.76,25.35.

Compound II-6b is a yellow solid with a melting point of 144.7-145.1℃and a yield of 29.4%. ¹ H NMR(400MHz,CDCl ₃ )δ7.74–7.69(m,1H),7.64(d,J＝15.7Hz,1H),7.08(t,J＝4.6Hz,1H),6.99–6.95(m,1H),6.84–6.80(m,2H),6.07(s,2H),4.08(t,J＝6.5Hz,2H),2.56(td,J＝6.5,4.6Hz, ¹³ C NMR(101MHz,CDCl ₃ )δ168.78,161.25,147.97,146.70,141.09,139.02,128.20,123.46,122.76,121.71,121.70,117.80,109.81,101.56,41.82,25.32.

Compound II-7b is a yellow solid with a melting point of 194.2-195.1 ℃ and a yield of 30%. ¹ H NMR(400MHz,CDCl ₃ )δ8.06(d,J＝15.5Hz,1H),7.31(d,J＝15.4Hz,1H),7.19(s,1H),7.09(t,J＝4.6Hz,1H),7.05(s,1H),6.02(s,2H),4.09(t,J＝6.5Hz,2H),2.64–2.50(m,2H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.13,161.46,150.12,147.93,142.63,141.24,128.15,128.11,121.81,118.16,113.04,106.89,102.28,41.73,25.31.

Compound II-8b is a yellow solid with a melting point of 151.7-152.4℃and a yield of 25.4%. ¹ H NMR(400MHz,CDCl ₃ )δ8.16(d,J＝15.1Hz,1H),7.77(ddd,J＝4.0,2.1,0.7Hz,1H),7.71(ddd,J＝3.3,2.2,0.6Hz,1H),7.40–7.33(m,3H),7.09(t,J＝4.6Hz,1H),4.10(dd,J＝12.0,5.5Hz,2H),2.60–2.55(m,2H),2.54(s,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ168.25,161.28,141.07,140.57,139.49,136.84,135.93,134.50,128.27,126.55,124.44,122.72,122.50,121.39,41.83,25.32,12.26.

Compound II-9b is a red solid with a melting point of 135.3-137.8deg.C and a yield of 26.6%. ¹ H NMR(400MHz,CDCl ₃ )δ9.06(s,1H),8.13–8.03(m,3H),7.91(d,J＝15.5Hz,1H),7.83–7.75(m,2H),7.14(t,J＝4.6Hz,1H),4.13(t,J＝6.5Hz,2H),2.62(td,J＝6.4,4.7Hz,2H). ¹³ C NMR(101MHz,CDCl ₃ ) Delta 167.89,161.38,148.38,144.89,142.44,142.42,141.59,139.48,130.65,130.60,129.86,129.21,128.10,127.93,41.87, 25.hr-MS (ESI-TOF) m/z calculated C ₁₆ H ₁₂ ClN ₃ O ₂ Na[M+Na] ⁺ 336.0518, experimental 336.0510.

Compound II-10b is a white solid with a melting point of 92.5-93.2℃and a yield of 28.7%. ¹ H NMR(400MHz,CDCl ₃ )δ8.95–8.90(m,1H),8.18(d,J＝8.3Hz,1H),8.09(d,J＝8.7Hz,1H),8.00–7.95(m,2H),7.92(d,J＝15.6Hz,1H),7.64(d,J＝15.6Hz,1H),7.44(dd,J＝8.3,4.2Hz,1H),7.12(t,J＝4.5Hz,1H),4.12(t,J＝6.4Hz,2H),2.60(dd,J＝11.7,5.7Hz,2H). ¹³ C NMR (101 MHz, CDCl 3) delta 168.33,161.50,151.30,149.04,143.43,141.31,136.51,133.11,130.12,129.17,128.24,128.14,128.03,122.50,121.81,41.77,25.32.HR-MS (ESI-TOF) m/z calculated C ₁₇ H ₁₃ ClN ₂ O ₂ Na[M+Na] ⁺ 335.0566, experimental 335.0558.

Example 9

Preparation of Compound II-1 c: a25 mL reaction flask was taken, 4mL of anhydrous THF was added thereto, the flask was placed in an ice-water bath, sodium hydride (30 g,0.740 mmol) and triethyl 2-phosphonopropionate (146. Mu.L, 0.678 mmol) were sequentially added thereto, and reacted under argon atmosphere for 10 minutes, then benzofuran-2-carbaldehyde (90 mg,0.616 mmol) was added, and the system was taken out of the ice-water bath and warmed to room temperature and stirred for 1 hour. After the completion of the TLC monitoring reaction, the reaction was quenched with ice water, the solvent was removed under reduced pressure, acidified, extracted three times with ethyl acetate, the organic phase was collected, then washed once with an appropriate amount of saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to give Compound 32a. Compound 32a (133 mg,0.616 mmol) was transferred to a 50mL reaction flask, 3mL ethanol, 3mL tetrahydrofuran, 9mL water were added, and the mixture was dissolved with stirring, followed by addition of sodium hydroxide (168 mg,4.206 mmol) and reacted at 60℃for 2h. After the completion of the TLC monitoring, the solvent was removed under reduced pressure, acidified, filtered and air-dried to give compound 33a as a white solid, 90mg, two-step yield 72%.

Compound 33a (90 mg, 0.4476 mmol) was added to a 25mL reaction flask, 4mL anhydrous THF was added under argon, the system was stirred to dissolve, the system was placed at-20deg.C, then anhydrous triethylamine (42 μL,0.512 mmol) and pivaloyl chloride (61 μL,0.490 mmol) were added sequentially, and the system was reacted at-20deg.C for 1h. After the completion of the TLC monitoring reaction, 1mL of saturated sodium bicarbonate was added to quench the reaction, the reaction was extracted three times with methylene chloride, and the organic phase was collected, then washed once with an appropriate amount of saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated and dried by suction to give compound 34a. The key intermediate 4c (59 mg,0.446 mmol) was added to a 25mL two-port reaction flask, 4mL anhydrous THF was added under argon protection, stirring was performed to dissolve, the system was placed at-78deg.C, then 2.5M n-butyllithium in cyclohexane (18. Mu.L, 0.446 mmol) was added, the reaction was performed for 30min, and finally compound 35a (0.446 mmol) was added to the system, and the reaction was performed for 2h at-78deg.C. After TLC monitoring reaction, adding saturated ammonium chloride solution to quench reaction, heating the system to room temperature,the solvent was removed under reduced pressure, extracted three times with ethyl acetate, the organic phase was collected, then washed once with an appropriate amount of saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated and purified by column chromatography with petroleum ether as eluent: ethyl acetate (3:1) afforded compound II-1c as a white solid with a melting point of 153.2-154.9 ℃, two-step yield 31.6%. ¹ H NMR(400MHz,CDCl ₃ )δ7.58(ddd,J＝7.7,1.3,0.7Hz,1H),7.47(dd,J＝8.2,0.8Hz,1H),7.31(ddd,J＝8.3,7.3,1.3Hz,1H),7.25–7.20(m,1H),7.08(t,J＝4.6Hz,1H),6.90–6.88(m,1H),6.87(s,1H),3.95(t,J＝6.5Hz,2H),2.63(td,J＝6.5,4.7Hz,2H),2.33(d,J＝1.1Hz,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ175.07,160.76,155.05,153.51,140.97,134.87,128.38,127.74,125.46,123.11,122.54,121.38,111.28,109.91,43.24,25.32,16.43.

Preparation of Compounds II-2c to II-4c reference was made to the synthesis of Compound II-1c in example 9.

Compound II-2c is a yellow solid with a melting point of 108.1-109.5℃and a yield of 28.5%. ¹ H NMR(400MHz,CDCl ₃ )δ7.07(t,J＝4.6Hz,1H),6.93(ddd,J＝7.9,1.2,0.5Hz,1H),6.86(d,J＝1.2Hz,1H),6.82(t,J＝7.8Hz,1H),6.79–6.75(m,1H),5.97(s,2H),3.94(t,J＝6.5Hz,2H),2.62(td,J＝6.5,4.7Hz,2H),2.11(d,J＝1.4Hz,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ175.42,160.80,147.37,145.71,140.94,135.83,127.75,126.60,122.07,121.28,118.11,108.28,100.92,43.05,25.33,15.75.

Compound II-3c is a red solid with a melting point of 141.9-142.5 ℃ and a yield of 23.6%. ¹ H NMR(400MHz,CDCl ₃ )δ8.85(s,1H),8.07(ddd,J＝5.0,3.0,1.2Hz,2H),7.79–7.71(m,2H),7.12(t,J＝4.6Hz,1H),6.92(dd,J＝2.8,1.3Hz,1H),4.03(t,J＝6.5Hz,2H),2.67(td,J＝6.5,4.7Hz,2H),2.55(d,J＝1.5Hz,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ174.75,160.72,150.66,146.60,143.20,142.22,141.36,140.74,130.24,130.00,129.61,129.03,127.63,126.41,42.62,25.28,16.69.

Compound II-4c was a yellow oil in 21.8% yield. ¹ H NMR(400MHz,CDCl ₃ )δ8.95(dd,J＝4.3,1.7Hz,1H),8.20(dd,J＝8.5,1.0Hz,1H),8.13(d,J＝8.8Hz,1H),7.86(d,J＝1.5Hz,1H),7.75(dd,J＝8.8,1.9Hz,1H),7.46(dd,J＝8.3,4.3Hz,1H),7.12(t,J＝4.6Hz,1H),7.05(s,1H),4.01(t,J＝6.5Hz,2H),2.67(td,J＝6.5,4.7Hz,2H),2.21(d,J＝1.4Hz,3H). ¹³ C NMR(101MHz,CDCl ₃ )δ175.37,160.85,150.34,146.95,141.12,136.80,136.08,134.33,132.26,131.04,128.73,128.49,127.77,121.60,42.96,25.33,15.67.

Test example: determination of anti-tumor cell proliferation Activity of Compounds

(1) Determination of adherent cell proliferation inhibitory Activity

Cells in the logarithmic growth phase were collected and counted, followed by 4X 10 ³ Number/hole, according to the formula: c (C) ₁ V ₁ ＝C ₂ V ₂ Calculating the cell suspension amount required by each 96-well plate, adding 100 mu L of cell suspension into each well, culturing overnight for adherence, discarding old culture medium, adding culture medium containing different drugs and different concentration gradients, culturing for 72h, and measuring IC by SRB method ₅₀ Values.

SRB method: collecting HepG-2, hela, MCF-7, MGC-803 and A549 cells in logarithmic growth phase, inoculating into 96-well plate, 3-4X10 ³ And/or holes. At 37 ℃, contain 5% CO ₂ Culturing overnight in incubator to adhere, discarding old culture medium, adding different concentration gradient medicated culture medium, culturing for 72 hr, discarding old culture medium, adding 100 μl 10% (w/v) trichloroacetic acid into each well, fixing at 4deg.C for 30min, discarding fixing solution, washing residual reagent with ultrapure water, oven drying at 65deg.C, adding 100 μl 0.4% SRB into each well, shake-staining at room temperature for 20min, The dye liquor was discarded, and the residual dye liquor was washed off with freshly prepared 1% glacial acetic acid and dried at 65 ℃. Finally, 150. Mu.L of 10mM unbuffered Trisbase was added to each well, the plate was oscillated for 5min to dissolve the dye, and the absorbance (OD) at 560nm wavelength was measured with a microplate reader. Inhibition (%) = (OD _{Control wells} －OD _{Medicine adding hole} )/OD _{Control wells} 100% and the concentration of the drug at 50% inhibition was calculated. Experiments were repeated three times and data are presented as mean and standard deviation.

(2) Measurement of proliferation inhibitory Activity of suspension cells

Cells in the logarithmic growth phase were collected and counted at 1X 10 ⁴ Number/hole, according to the formula: c (C) ₁ V ₁ ＝C ₂ V ₂ Calculating the cell suspension amount required by each 96-well plate, adding 50 mu L of cell suspension into each well, adding 50 mu L of drug-containing RPMI-1640 culture medium with different concentration gradients into each well, culturing for 72h, and measuring IC by adopting a CCK-8 method ₅₀ Values.

Cell Counting Kit (CCK-8) method: collecting U937 cells in logarithmic phase, inoculating into 96-well plate, and inoculating into 8×10 ³ Drug-containing medium is added to the wells along with the cell suspension. Three wells were taken and blank medium was added as a blank group. At 37 ℃, contain 5% CO ₂ Incubate in incubator for 72h. 10 mu L of CCK-8 dye solution is added into each hole, incubation is continued for 3-4 hours in an incubator, an enzyme-labeled instrument is used for measuring absorbance value at the wavelength of 450nm, and the OD value of a non-drug control group is preferably controlled to be 2.0. IC was measured according to the following formula ₅₀ Values. Calculation formula of proliferation inhibition rate: inhibition (%) = (OD _{Control wells} －OD _{Medicine adding hole} )/(OD _{Control wells} －OD _{Blank hole} )×100％。

Compound IC of the present invention ₅₀ The values are detailed in tables 1-5 below:

TABLE 1

TABLE 2

TABLE 3 Table 3

TABLE 4 Table 4

TABLE 5

From the above test, R is ₁ When the intermediate contains methoxy for substitution, the activity of the compound can be improved; r is R ₂ In the case of vinylidene, the compounds have higher activity.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound or pharmaceutically acceptable salt thereof as shown below:

。

2. a pharmaceutical composition comprising a therapeutically effective amount of at least one of the compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. Use of at least one of the compounds of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament;

the medicine is an anti-tumor medicine;

the tumor is selected from leukemia cells, breast cancer cells, liver cancer cells, lung cancer cells, stomach cancer cells and cervical cancer cells.