CN115572250A

CN115572250A - Diaryl-beta-lactam compound, preparation method and application in pharmacy

Info

Publication number: CN115572250A
Application number: CN202110685894.2A
Authority: CN
Inventors: 王洋; 唐海荣; 梁玉茹; 丁奎岭
Original assignee: Shanghai Institute of Organic Chemistry of CAS; Fudan University
Current assignee: Shanghai Institute of Organic Chemistry of CAS; Fudan University
Priority date: 2021-06-21
Filing date: 2021-06-21
Publication date: 2023-01-06

Abstract

The invention belongs to the technical field of synthetic pharmaceutical chemistry, and relates to a novel chiral diaryl-beta-lactam HDAC/Tubulin bifunctional inhibitor with a following general formula structure and obvious antitumor activity and application thereof in research and development of antitumor drugs. The invention also discloses the application of the compound, the pharmaceutical salt thereof and the compound medicine thereof in preparing medicines for preventing or treating diseases related to tumors. The compound or the pharmaceutically acceptable salt thereof can inhibit the growth of tumor cells in vitro and in vivo effectively by inhibiting the aggregation of histone deacetylase and tubulin and inhibiting the regulation mechanism of tumor cell proliferation, and can be applied to the preparation of medicaments for preventing or treating tumor-related diseases. The tumor-related diseases include benign and malignant tumors and other diseases caused by tumors.

Description

Diaryl-beta-lactam compound, preparation method and application in pharmacy

Technical Field

The invention belongs to the field of synthetic pharmaceutical chemistry in the research and development of new drugs, relates to diaryl-beta-lactam compounds, and particularly relates to a novel chiral diaryl-beta-lactam HDAC/Tubulin bifunctional inhibitor with remarkable anti-tumor activity, a preparation method, in vivo and in vitro anti-tumor activity, and application of the compounds and acceptable pharmaceutical salts thereof or compound drugs taking the compounds as one of the components in preparation of drugs for preventing and treating tumor-related diseases.

Background

Microtubules (microtubule) are essential for mitosis of cells, and play an essential role in maintaining cell morphology, cell motility, cell division, and proliferation. The mitosis and metastasis of malignant tumor cells are extremely vigorous, so that the tumor cells can be selectively inhibited from division and proliferation by targeting microtubules and breaking the dynamic balance of polymerization and depolymerization of tubulin (Science 2013,339, 587-590). Although there have been some major advances in the research on tubulin aggregation inhibitors acting on colchicine, particularly in the research on structural modification of Combretastatin (Combretastatin a-4), such as the disodium phosphate salt of Combretastatin (CA-4P) and the disodium phosphate salt of BNC105 (BNC 105P) in phase I and phase II clinical trials, respectively, the research shows that these drugs have poor clinical effects, certain toxic and side effects, such as nausea, vomiting, visual disturbances and headache, and also have disadvantages in prolonging the lifetime of patients, and so on, and until now none of these drugs has been approved for marketing (j.med.chem.2016, 59, 8685-8711). Therefore, the development of tubulin aggregation inhibitors with better antitumor activity, higher selectivity and less toxic and side effects is the main direction for the development of such drugs in the future.

Histone Deacetylases (HDACs), which are key enzymes for regulating cellular epigenetics, are highly expressed in a number of tumor cell lines, are highly correlated with growth, proliferation and invasion of tumor cells, and are considered as one of the most promising targets for tumor therapy (nat. Rev. Drug Discovery 2012,11,384-400). The pan-HDAC inhibitors are mainly used for the treatment of hematological tumors and have poor therapeutic effect on solid tumors, so that HDAC inhibitors are often used in combination with other antitumor drugs, and although the combination mode can produce synergistic antitumor effect, the combination mode can cause complicated and unpredictable pharmacokinetics, poor patient compliance and even drug-drug interaction. The drug mode of 'one drug and multiple targets' can effectively overcome the defects and is an ideal alternative treatment mode of traditional 'single target and single drug' and combined drug treatment (J.Med. chem.2005,48, 6523-6543). A large number of research reports have proved that the design of the HDAC based dual-target medicine is reasonable and feasible, and is also obvious for improving the tumor treatment effect.

Preclinical studies have shown that tubulin aggregation inhibitor (vincristine) and HDAC inhibitor SAHA can produce synergistic antitumor effects when used in combination to treat leukemia. In a MOLT-4 nude mouse transplantation tumor model, the tumor inhibition rate of a vincristine and SAHA combined administration group is obviously higher than that of two single medicine groups, and the survival period of mice is greatly prolonged by the combined administration group (J.Hematol.Oncol.2017, 8,82). In addition, it is also shown by physical research that tubulin and HDAC are associated and cooperated with each other in the process of generating and developing tumors. In early studies, HDACs catalyzed primarily deacetylation of histone lysine residues located on the nuclear endosomal chromosome, but in fact, the catalytic substrates of HDACs were also found extensively in the cytoplasm, such as α -tubulin and HSP90. The over-expressed HDAC in tumor cells can over-deacetylate α -tubulin in cytoplasm, thereby accelerating the aggregation and disaggregation of microtubules and promoting the migration and invasion of tumor cells. The microtubules also have the dynamic characteristics of aggregation and depolymerization, and the synergistic interaction process of the microtubules and HDAC is very obvious in malignant tumor cells with vigorous division (J.Med.chem.2020, 63,23-39). The data from these mechanistic studies, as well as preclinical studies, have provided a rational basis for the design of HDAC/Tubulin dual-target inhibitors.

Based on the current state of the art, the inventors of the present application intend to provide diaryl- β -lactam compounds, as well as methods for their preparation and use in pharmaceutical applications.

Disclosure of Invention

The invention aims to provide diaryl-beta-lactam compounds, a preparation method and application thereof in pharmacy based on the current situation of the prior art. In particular to a novel chiral diaryl-beta-lactam HDAC/Tubulin bifunctional inhibitor, a preparation method thereof and application of the compound and pharmaceutical salts thereof or compound medicines taking the compound as a component in preparing medicines for preventing and treating tumor-related diseases.

According to the application, the structural characteristics of the Tubulin aggregation inhibitor and the HDAC inhibitor are combined, the design principle of a fused pharmacophore is applied, a chiral diaryl-beta-lactam HDAC/Tubulin double-target molecular compound library is designed and constructed, after the anti-tumor activity test at the molecular level and the cell level, a lead compound with better activity is screened out, and finally, a candidate compound with a novel structure and potential drug development prospect is provided for further research.

The invention provides a novel chiral diaryl-beta-lactam HDAC/Tubulin bifunctional inhibitor with a structure shown in a general formula I or a pharmaceutical salt thereof,

wherein R is ¹ And R ² Is selected from hydrogen atom, alkyl, substituted alkyl, alkoxy, halogen atom, amino, hydroxyl, acyloxy, methoxy formyl, allyloxy, propargyloxy, sulfonyloxy, alkylamino, acylamino, sulfonylamino or combination of 2-3 of the same or different groups; x is selected from carbon, nitrogen, oxygen, ester group, amide group; l is taken from

Wherein n is selected from 0, 1, 2, 3,4,5, 6, 7; l can also be taken from

Alicyclic and aromatic heterocycles; r is ³ Is taken from

R ⁴ Is selected from hydrogen atom, alkyl, substituted alkyl, alkoxy, acyloxy, hydroxyl, phenyl, substituted phenyl, pyridyl, cyclopropyl, vinyl, amino, alkylamino, amido, sulfonyloxy, and sulfonamido; preferred compounds are:

the invention also provides a novel chiral diaryl-beta-lactam HDAC/Tubulin bifunctional inhibitor with the structure of the general formula II or pharmaceutical salt thereof,

wherein R is ¹ And R ² Is selected from hydrogen atom, alkyl, substituted alkyl, alkoxy, halogen atom, amino, hydroxyl, acyloxy, methoxy formyl, allyloxy, propargyloxy, sulfonyloxy, alkylamino, acylamino, sulfonamido or combination of 2-3 same or different groups; x is selected from carbon, nitrogen, oxygen, ester group, amide group; l is taken from

Wherein n is selected from 1, 2, 3,4,5, 6, 7; l may also be derived from

Alicyclic and aromatic heterocycles; r ³ Is taken from

Preferred compounds are:

the "pharmaceutically acceptable salt" in the present invention includes, specifically, salts with organic acids such as malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid, and oxalic acid, and inorganic acids such as phosphoric acid, hydrohalic acid, sulfuric acid, and nitric acid.

The invention further aims to provide application of the compounds or the pharmaceutically acceptable salts of the compounds and compositions containing the compounds or the salts of the compounds in preparing medicines for preventing or treating diseases related to tumors.

The tumor-related diseases include, but are not limited to, neuroblastoma, thyroid cancer, lymphoma, prostate cancer, kidney cancer, bladder cancer, brain glioma, nasopharyngeal carcinoma, neuroendocrine cancer, head and neck squamous cell carcinoma, cervical cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, interstitial sarcoma, choriocarcinoma, malignant hydatidiform mole, malignant teratoma, gastric cancer, lung cancer, liver cancer, melanoma, undifferentiated carcinoma, and benign tumor.

The invention provides and proves that the novel diaryl-beta-lactam HDAC/Tubulin bifunctional inhibitor with remarkable anti-tumor activity or pharmaceutically acceptable salt thereof can inhibit the regulation mechanism of tumor cell growth by inhibiting the aggregation of HDAC and Tubulin and has remarkable proliferation inhibition and angiogenesis inhibition on tumor cells in-vitro and in-vivo anti-tumor experiments.

Drawings

FIG. 1. Compound 5a inhibits microtubule self-assembly assay in vitro-absorbance-time curves.

Figure 2. Effect of compound 5a on HDAC related protein expression.

FIG. 3. Effect of Compound 5a on tumor cell cycle.

FIG. 4. Effect of Compound 5a on tumor cell cycle-associated protein expression.

Figure 5. Compound 5a induces apoptosis assay.

FIG. 6. Effect of Compound 5a on apoptosis-related protein expression.

FIG. 7 inhibition of tumor cell colony formation by Compound 5 a.

Detailed Description

The present invention is further illustrated by the following examples. These examples are intended only to further illustrate the invention and do not alter the scope of protection of the invention. The process for the preparation of the object compounds of the present invention can be further embodied by the following representative compound preparation processes:

example 1 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -3-methyl-4- (3-hydroxy-4-methoxyphenyl) azetidin-2-one (1 g)

Referring to the literature (j.med.chem.2016, 59, 10329-10334) procedure, the present invention synthesizes 1g of intermediate compound according to the following route:

the reagent and the condition (a) Pd ₂ (dba) ₃ (0.1％),(R,R,R)-Ph-SKP(0.25％),K ₂ CO ₃ ,CH ₂ Cl ₂ ,25℃, 3h；(b)i)Sn[N(TMS) ₂ ] ₂ ,toluene,reflux,3h；ii)TBAF,THF,0℃,1h；(c)Pd/C,H ₂ , EtOH,25℃,12h；(d)i)BnCl,K ₂ CO ₃ ,MeCN,reflux,12h；ii)B ₂ (pin) ₂ (1.3equiv), CuCl(5％),MeOH(1.5equiv),PPh ₃ (15％),t-BuOLi(10％),THF,25℃,12h；iii) NaBO ₃ ·4H ₂ O,H ₂ O,THF,25℃,2h；(e)i)CBr ₄ ,PPh ₃ ,THF,25℃,4h；ii)Pd/C, AcONa,H ₂ ,EtOH,25℃,12h.

1.1 Synthesis of ethyl (S) -2- [1- (3-tert-butyldimethylsilyloxy-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenylamino) meth ] acrylate (1 c)

A50 mL Schlenk tube was charged with starting material 1a (0.65g, 1.59mmol), 3,4,5-trimethoxyaniline (0.43g, 2.07mmol), potassium carbonate (0.66g, 4.77mmol), pd ₂ (dba) ₃ (1.5mg, 0.0016 mmol) and (R, R, R) -Ph-SKP (2.6 mg, 0.004mmol), the system was filled with dry nitrogen after three times of air-purging, and oxygen-free dichloromethane (7 mL) was injected into the system. The reaction was continued for 3 hours at room temperature under nitrogen. Adding water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated salt water, and adding Na ₂ SO ₄ And (5) drying. After concentration by rotary evaporation, column chromatography separation (PE/EA 3:1) was carried out to obtain 0.73g of colorless oily liquid (1 c) with a yield of 85%. [ alpha ] of] _D ²⁰ +86.2(c 0.41,CHCl ₃ ),98％ee[determined by HPLC analysis using a Chiralcel AS-3column；n-Hex/i-PrOH＝95:5,1.0mL/min,254nm；t _R (major)＝ 7.47min；t _R (minor)＝9.72min]. ¹ H NMR(400MHz,CDCl ₃ ):δ6.92(dd,J＝8.3, 2.1Hz,1H),6.82(d,J＝2.1Hz,1H),6.80(d,J＝8.3Hz,1H),6.34(s,1H),5.88(s, 1H),5.81(s,2H),5.28(s,1H),4.15(q,J＝7.1Hz,2H),3.77(s,3H),3.77(s,6H), 3.74(s,3H),1.22(t,J＝7.1Hz,3H),0.96(s,9H),0.11(s,6H).ESI-MS(m/z):532.1 (M+H ⁺ ).

1.2 Synthesis of (S) -1- (3,4,5-trimethoxyphenyl) -4- (3-hydroxy-4-methoxyphenyl) -3-methyleneazetidin-2-one (1 d)

A50 mL Schlenk tube was charged with starting material 1c (0.44g, 0.83mmol), sn [ N (TMS) ₂ ] ₂ (0.4 mL, 1mmol) and 10mL of anhydrous toluene. The mixture is heated under reflux for 3 hours under the protection of nitrogen, and then is directly separated by flash column chromatography (PE/EA 4:1) to obtain colorless oily liquid, and then TBAF (0.39g, 15mmol) and tetrahydrofuran (8 mL) are added under the condition of ice bath. After further ice-bath reaction for 20 min, water was added, extraction was carried out three times with ethyl acetate, the organic phases were combined and washed once with saturated brine, na ₂ SO ₄ And (5) drying. Separating by column chromatography (PE/EA 2:1) to obtain light yellow oil0.19g of the liquid (1 d) was obtained, showing a yield of 62%. [ alpha ] to] _D ²⁰ +33.6 (c 1.00,CHCl ₃ ),99％ee[determined by HPLC analysis using a Chiralcel AD-H column；n-Hex/i-PrOH＝80:20,1.0mL/min,254nm；t _R (major)＝13.76min；t _R (minor)＝17.72min]. ¹ H NMR(400MHz,CDCl ₃ ):δ6.95(d,J＝2.1Hz,1H),6.90 (dd,J＝8.2,2.0Hz,2H),6.83(d,J＝8.2Hz,1H),6.59(s,2H),5.81(t,J＝1.6Hz, 1H),5.71(s,1H),5.27(s,1H),5.14(s,1H),3.88(s,3H),3.75(s,3H),3.73(s,6H). ESI-MS(m/z):372.1(M+H ⁺ ).

1.3 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -4- (3-benzyloxy-4-methoxyphenyl) -3-hydroxymethylazetidin-2-one (1 e) and (3R, 4R) -1- (3,4,5-trimethoxyphenyl) -4- (3-benzyloxy-4-methoxyphenyl) -3-hydroxymethylazetidin-2-one (1 f)

A50 mL eggplant-shaped bottle was charged with the starting materials 1d (0.22g, 0.59mmol), benzyl bromide (0.084mL, 0.71 mmol), potassium carbonate (0.1g, 0.71mmol) and acetonitrile (5 mL). Heating and refluxing for 8 hr, adding water, extracting with ethyl acetate for three times, mixing organic phases, washing with saturated salt water, and adding Na ₂ SO ₄ And (5) drying. Concentrating by rotary evaporation to obtain white solid. The white solid, pinacol diboron ester (0.2g, 0.8mmol), cuCl (3.0mg, 0.09mmol), triphenylphosphine (24mg, 0.09mmol), and lithium tert-butoxide (4.8mg, 0.06mmol) were charged into a 50mL Schlenk tube, and the system was filled with dry nitrogen by purging, followed by injecting methanol (15. Mu.L, 0.9 mmol) and anhydrous THF (10 mL). Reacting at room temperature for 12 hr under nitrogen protection, adding water, extracting with ethyl acetate for three times, mixing organic phases, washing with saturated NaCl aqueous solution, and adding Na ₂ SO ₄ And (5) drying. After the solvent was distilled off, sodium perborate tetrahydrate (0.46g, 1.5 mmol), THF (10 mL), and water (5 mL) were added and the mixture was reacted at room temperature for 2 hours. Adding water, extracting with ethyl acetate for three times, mixing the organic phases, washing with saturated aqueous NaCl solution, and adding Na ₂ SO ₄ And (5) drying. Column chromatography (PE/EA 1:2) afforded 138mg of (1 e) as a white solid in 48% yield. ¹ H NMR(400MHz,CDCl ₃ ):δ7.58-7.31(m,5H), 6.94-6.77(m,3H),6.55(s,2H),5.18(s,2H),5.10(d,J＝5.1Hz,1H),3.91(s,3H), 3.81(m,2H),3.76(s,3H),3.73(s,6H),3.65(m,1H).ESI-MS(m/z):480.1(M+H ⁺ ) 92mg of white solid (1 f) in 32% yield. ¹ H NMR(400MHz,CDCl ₃ ):δ7.48-7.32(m,5H), 6.97(d,J＝1.5Hz,1H),6.91(dd,J＝8.3,1.5Hz,1H),6.84(d,J＝8.3Hz,1H),6.56 (s,2H),5.16(s,2H),4.92(d,J＝1.8Hz,1H),4.00(dd,J＝11.9,3.3Hz,1H),3.90(s, 3H),3.76(s,3H),3.72(s,7H),3.28(d,J＝2.3Hz,1H).ESI-MS(m/z):480.1 (M+H ⁺ ).

1.5 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -4- (3-hydroxy-4-methoxyphenyl) -3-methylazetidin-2-one (1 g)

A25 mL Schlenk tube was charged with the starting materials 1f (20mg, 0.04mmol), carbon tetrabromide (69 mg,0.2 mmol) and triphenylphosphine (54mg, 0.2mmol), and after purging three times, the system was filled with dry nitrogen gas, and then anhydrous dichloromethane (3 mL) was injected into the system. The reaction was continued for 4 hours at room temperature under nitrogen. Adding water, extracting with dichloromethane for three times, mixing organic phases, washing with saturated aqueous NaCl solution, and adding Na ₂ SO ₄ And (5) drying. After concentration by rotary evaporation, 10% of Pd/C (3 mg), sodium acetate (6 mg, 0.08mmol) and ethanol (1.5 mL) were added and the reaction was carried out under hydrogen at room temperature under normal pressure for 12 hours. After removing Pd/C by filtration, the mixture is concentrated by rotary evaporation and purified by column chromatography on silica gel (300-400 mesh) (eluent: PE/EA 2:1). Spin-dry to give 10mg of white solid (1 g) in 66% yield. ¹ H NMR(400MHz,CDCl ₃ ):δ6.93(d,J＝1.5Hz,1H),6.87(dd, J＝8.3,1.5Hz,1H),6.83(d,J＝8.3Hz,1H),6.54(s,2H),5.69(s,1H),4.44(d,J＝ 2.2Hz,1H),3.89(s,3H),3.76(s,3H),3.72(s,6H),3.11(qd,J＝7.3,2.2Hz,1H), 1.45(d,J＝7.3Hz,3H).ESI-MS(m/z):374.0(M+H ⁺ ).。

EXAMPLE 2 Synthesis of intermediate Compound (2 a-d)

The synthetic method is reported in a reference document, and the specific operation is as follows: a25 mL eggplant-shaped flask was charged with 1g (45 mg, 0.12mmol) of the starting materials, potassium carbonate (33mg, 0.24mmol), bromoalkyl acid ester (0.24 mmol) and acetonitrile (2 mL), and the mixture was reacted under reflux overnight under heating. After the reaction, the mixture was cooled, water was added, extraction was carried out with ethyl acetate three times, the organic phases were combined, washed with saturated brine once, and dried over anhydrous sodium sulfate. Concentrating by rotary evaporation, separating by silica gel column chromatography (PE/EA 1:1), and rotary drying to obtain corresponding intermediate product.

2.1 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -3-methyl-4- (3-methoxyacylmethoxy-4-methoxyphenyl) azetidin-2-one (2 a)

Yellow-green oily liquid (2 a) in 90% yield. ¹ H NMR(400MHz,CDCl ₃ ):δ7.01(d,J＝ 7.5Hz,1H),6.91(dd,J＝7.5,1.5Hz,1H),6.81(s,1H),6.52(s,2H),4.67(s,2H), 4.47(s,1H),3.88(s,3H),3.76(s,3H),3.72(s,6H),3.69(s,3H),3.12-3.07(m,1H), 1.46(d,J＝6.3Hz,3H). ¹³ C NMR(150MHz,CDCl ₃ ):δ169.0,168.2,153.5,149.9, 147.8,134.4,134.0,130.3,120.3,112.5,112.1,94.7,66.5,62.8,60.9,56.1,55.2,52.1, 13.1.ESI-MS(m/z):446.0(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₃ H ₂₈ NO ₈ [M+H ⁺ ], 446.1809；found,446.1806.

2.2 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -3-methyl-4- (3-ethoxyacylpropoxy-4-methoxyphenyl) azetidin-2-one (2 b)

Yellow oily liquid (2 b) in 88% yield. ¹ H NMR(400MHz,CDCl ₃ ):δ6.95(d,J＝7.8 Hz,1H),6.88(m,2H),6.55(s,2H),4.47(s,1H),4.14(q,J＝6.9,6.3Hz,2H),4.01 (m,2H),3.86(s,3H),3.76(s,3H),3.72(s,6H),3.15(d,J＝7.9Hz,1H),2.52(t,J＝ 6.3Hz,3H),2.15(m,2H),1.47(d,J＝5.0Hz,3H),1.25(t,J＝6.9Hz,3H). ¹³ C NMR(150MHz,CDCl ₃ ):δ172.5,167.8,152.8,149.1,148.3,133.6,133.5,129.6, 118.4,111.3,109.9,93.9,67.4,62.5,60.3,59.8,55.4,54.4,30.0,23.8,13.6,12.5. ESI-MS(m/z):488.5(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₆ H ₃₄ NO ₈ [M+H ⁺ ], 488.2279；found,488.2279.

2.3 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -3-methyl-4- (3-ethoxycarbonyloxy-4-methoxyphenyl) azetidin-2-one (2 c)

Yellow oily liquid (2 c) in 86% yield. ¹ H NMR(400MHz,CDCl ₃ ):δ6.94(d,J＝7.6 Hz,1H),6.87(d,J＝7.6Hz,1H),6.83(s,1H),6.55(s,2H),4.47(s,1H),4.13(q,J＝ 6.8Hz,2H),3.97(s,2H),3.86(s,3H),3.76(s,3H),3.72(s,6H),3.15(d,J＝6.8Hz, 1H),1.83(s,4H),1.47(d,J＝6.6Hz,3H),1.26(t,J＝6.8Hz,3H). ¹³ C NMR(150 MHz,CDCl ₃ ):δ172.8,167.8,152.8,149.1,148.5,133.7,133.5,129.6,118.2,111.2, 109.6,93.9,68.0,62.5,60.3,59.7,55.4,54.4,33.3,27.9,20.9,13.6,12.5.ESI-MS (m/z):502.5(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₇ H ₃₆ NO ₈ [M+H ⁺ ],502.2435； found,502.2432.

2.4 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -3-methyl-4- (3-ethoxyacylpentyloxy-4-methoxyphenyl) azetidin-2-one (2 d)

Yellow oily liquid (2 d), yield 85%. ¹ H NMR(400MHz,CDCl ₃ ):δ6.94(d,J＝8.1 Hz,1H),6.86(d,J＝8.1Hz,1H),6.83(s,1H),6.55(s,2H),4.47(s,1H),4.12(q,J＝ 6.6Hz,2H),3.95(s,2H),3.86(s,3H),3.76(s,3H),3.71(s,6H),3.15(d,J＝7.0Hz, 2H),2.33(t,J＝6.5Hz,2H),1.83(m,2H),1.69(s,2H),1.47(d,J＝6.9Hz,4H), 1.25(t,J＝6.6Hz,3H). ¹³ C NMR(150MHz,CDCl ₃ ):δ172.9,167.8,152.8,149.0, 148.5,133.6,133.6,129.6,118.2,111.2,109.5,93.9,68.2,62.5,60.3,59.6,55.4,54.4, 33.6,28.2,24.9,24.1,13.6,12.5.ESI-MS(m/z):516.5(M+H ⁺ ).ESI-HRMS(m/z): calcd for C ₂₈ H ₃₈ NO ₈ [M+H ⁺ ],516.2592；found,516.2595.。

EXAMPLE 3 Synthesis of Compound (3 a-d)

A25 mL eggplant-shaped bottle was charged with the starting material (0.1 mmol) and sodium hydroxide (40mg, 1.0 mmol), dissolved in methylene chloride (0.5 mL) and methanol (1.0 mL), and then charged with an aqueous hydroxylamine solution (0.15 mL). Reacting in ice bath, adding water after the raw materials completely disappear, adjusting pH to 6 with acetic acid, extracting with ethyl acetate for three times, combining organic phases, washing with saturated salt water once, and drying with anhydrous sodium sulfate.Filtering, concentrating by rotary evaporation, separating by silica gel column Chromatography (CH) ₂ Cl ₂ /MeOH 20:1)。

3.1 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -3-methyl-4- [3- (N-hydroxyamidomethoxy) -4-methoxyphenyl ] azetidin-2-one (3 a)

Pale yellow solid (3 a), yield 79%. mp 86-88 deg.C] _D ²⁰ ＝+68.5(c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,CDCl ₃ ):δ9.72(s,1H),7.04(d,J＝7.9Hz,1H),6.90(d,J＝7.8Hz, 2H),6.50(s,2H),4.59(s,2H),4.47(s,1H),3.86(s,3H),3.73(s,3H),3.70(s,6H), 3.08(d,J＝6.3Hz,1H),1.44(d,J＝6.8Hz,3H). ¹³ C NMR(150MHz,CDCl ₃ ):δ 167.7,165.2,152.9,149.3,146.9,133.9,133.3,130.3,120.6,112.8,111.8,94.1,68.5, 61.9,60.3,55.5,54.5,12.4.ESI-MS(m/z):447.5(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₂ H ₂₇ N ₂ O ₈ [M+H ⁺ ],447.1762；found,447.1765.

3.2 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -3-methyl-4- [3- (N-hydroxyamidopropoxy) -4-methoxyphenyl ] azetidin-2-one (3 b)

Pale yellow solid (3 b), yield 65%. mp 82-83 deg.C] _D ²⁰ ＝+56.7(c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,CDCl ₃ ):δ6.97(d,J＝8.0Hz,1H),6.87(d,J＝8.0Hz,1H),6.83(s, 1H),6.53(s,2H),5.78(s,1H),4.47(s,1H),3.98(m,2H),3.89(s,3H),3.75(s,3H), 3.71(s,6H),3.13(m,1H),2.41(m,2H),2.10(m,2H),1.45(d,J＝7.2Hz,3H). ¹³ C NMR(150MHz,CDCl ₃ ):δ169.6,167.5,152.5,148.0,146.9,133.4,132.9,126.5, 119.3,110.5,93.9,67.3,59.9,57.5,55.1,54.9,48.3,28.7,23.5,8.7.ESI-MS(m/z): 475.5(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₄ H ₃₁ N ₂ O ₈ [M+H ⁺ ],475.2075；found, 475.2079.

3.3 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -3-methyl-4- [3- (N-hydroxyamidobutoxy) -4-methoxyphenyl ] azetidin-2-one (3 c)

Pale yellow solid (3 c), yield 45%. mp 66-68 deg.C] _D ²⁰ ＝+49.6(c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,CDCl ₃ ):δ6.94(d,J＝8.1Hz,1H),6.86(d,J＝8.1Hz,1H),6.82(s, 1H),6.54(s,2H),4.46(s,1H),3.98(m,2H),3.85(s,3H),3.76(s,3H),3.70(s,6H), 3.13(m,1H),2.42(m,2H),1.88-1.81(m,4H),1.46(d,J＝7.3Hz,3H). ¹³ C NMR (150MHz,CDCl ₃ ):δ167.9,152.8,149.0,148.4,133.7,133.5,129.6,118.4,111.2, 109.5,94.0,68.0,62.5,60.3,55.4,54.4,27.7,20.8,12.5.ESI-MS(m/z):489.5 (M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₅ H ₃₃ N ₂ O ₈ [M+H ⁺ ],489.2231；found, 489.2230.

3.4 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -3-methyl-4- [3- (N-hydroxyamidopentyloxy) -4-methoxyphenyl ] azetidin-2-one (3 d)

Pale yellow solid (3 d), yield 42%. mp 64-66 deg.C] _D ²⁰ ＝+42.3(c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,CDCl ₃ ):δ6.93(d,J＝8.1Hz,1H),6.86(d,J＝8.1Hz,1H),6.82(s, 1H),6.54(s,2H),4.46(d,J＝2.1Hz,1H),3.95(t,J＝6.6Hz,2H),3.86(s,3H),3.75 (s,3H),3.71(s,6H),3.17-3.12(m,1H),2.37(t,J＝7.2Hz,2H),1.82(q,J＝6.7Hz, 2H),1.68(q,J＝7.7Hz,2H),1.54-1.50(m,2H),1.46(d,J＝7.3Hz,2H). ¹³ C NMR (150MHz,CDCl ₃ ):δ178.4,168.6,153.4,149.7,149.1,134.3,134.1,130.2,118.9, 111.8,110.2,94.6,68.9,63.2,60.9,56.0,55.0,33.8,28.8,25.5,24.4,13.1.ESI-MS (m/z):503.5(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₆ H ₃₅ N ₂ O ₈ [M+H ⁺ ],503.2388； found,503.2388.。

EXAMPLE 4 Synthesis of the object Compound (5 a-b)

To a 25mL eggplant type bottle were added 4- (N-benzyloxycarboxamido) benzoic acid (81mg, 0.3mmol), HOBt (40mg, 0.3mmol) and EDCI (57mg, 0.3mmol), and DMF (1.5 mL) was added to dissolve. After stirring at room temperature for 6 hours, the starting material (0.1 mmol) and triethylamine (30mg, 0.3mmol) were added and the reaction was stirred at room temperature for a further 2 hours. After the raw materials completely disappear, adding water, extracting with ethyl acetate for three times, combining organic phases, washing with saturated salt once, and drying with anhydrous sodium sulfate. Filtering, concentrating by rotary evaporation, and separating by flash silica gel column chromatography (PE/EA 1:1). Then, 10% of Pd/C (3 mg) and ethanol (1 mL) were added, and the reaction was carried out under normal pressure and hydrogen at room temperature for 12 hours. Filtering to remove Pd/C, evaporating to dryness, and separating and purifying by silica gel (300-400 mesh) column chromatography.

4.1 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -4- (3-hydroxy-4-methoxyphenyl) -3- [4- (N-hydroxycarbamido) benzoyloxy ] methylazetidin-2-one (5 a)

Red solid (5 a), yield 68%. mp 136-140 deg.C] _D ²⁰ ＝+13.6(c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,DMSO-d ₆ ):δ11.40(s,1H),9.19(s,1H),9.12(s,1H),7.95(d,J＝ 7.9Hz,2H),7.82(d,J＝7.9Hz,2H),6.92(m,2H),6.86(m,1H),6.55(s,2H),5.13 (d,J＝2.2Hz,1H),4.68(m,2H),3.73(s,3H),3.62(s,7H),3.56(s,3H).[α] _D ²⁰ ＝ +11.3(c 1.0,CHCl ₃ ). ¹³ C NMR(150MHz,DMSO-d ₆ ):δ164.7,163.8,162.9,152.9, 147.7,146.7,136.9,133.7,133.1,131.3,129.6,129.1,127.1,117.7,112.9,112.1, 94.7,61.2,59.9,57.7,57.1,55.6,55.4.ESI-MS(m/z):553.5(M+H ⁺ ).ESI-HRMS (m/z):calcd for C ₂₈ H ₂₈ N ₂ NaO ₁₀ [M+Na ⁺ ],575.1636；found,575.1631.

4.2 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -4- (3-hydroxy-4-methoxyphenyl) -3- [4- (N-hydroxycarbamido) benzamido ] methylazetidin-2-one (5 b)

Red solid (5 b), yield 72%. mp 154-156 deg.C] _D ²⁰ ＝+14.6(c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,DMSO-d ₆ ):δ11.29(s,1H),9.09(s,1H),9.05(s,1H),8.86(s,1H), 7.83-7.77(m,4H),6.84(d,J＝8.3Hz,1H),6.75(d,J＝8.3Hz,2H),6.47(s,2H), 4.92(d,J＝2.1Hz,1H),3.78-3.68(m,3H),3.68(s,3H),3.58(s,6H),3.52(s,3H). ¹³ C NMR(150MHz,DMSO-d ₆ ):δ165.9,164.9,153.0,147.6,146.8,133.6,133.4, 130.0,127.1,117.2,112.7,112.2,94.4,59.9,59.1,58.6,55.6,55.4,37.7.ESI-MS (m/z):552.5(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₈ H ₂₉ N ₃ NaO ₉ [M+Na ⁺ ],574.1796； found,574.1816.。

EXAMPLE 5 Synthesis of the object Compound (6 a-b)

The synthesis method is similar to that of 5 a-b.

5.1 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -4- (3-hydroxy-4-methoxyphenyl) -3- [4- (N-hydroxycarbamido) propionyloxy ] methylazetidin-2-one (6 a)

Pale green oily liquid (6 a), yield 40%. [ alpha ] to] _D ²⁰ ＝+34.2(c 1.0,CHCl ₃ ). ¹ H NMR(400 MHz,DMSO-d ₆ ):δ6.87(d,J＝8.5Hz,1H),6.82(d,J＝8.5Hz,1H),6.77(s,1H), 6.48(s,2H),4.90(s,1H),4.31(m,1H),3.69(s,5H),3.58(s,6H),3.51(s,3H),2.45 (s,4H). ¹³ C NMR(150MHz,DMSO-d ₆ ):δ172.7,165.6,152.9,147.5,146.7,133.5, 133.4,130.3,117.6,112.9,112.2,94.7,94.3,61.6,59.9,56.9,56.5,55.5,25.0. ESI-MS(m/z):505.5(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₄ H ₂₈ N ₂ NaO ₁₀ [M+Na ⁺ ], 527.1636；found,527.1642.

5.2 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -4- (3-hydroxy-4-methoxyphenyl) -3- [4- (N-hydroxycarbamido) butyryloxy ] methylazetidin-2-one (6 b)

Red oily liquid (6 b) in 45% yield. [ alpha ] to] _D ²⁰ ＝+29.7(c 1.0,CHCl ₃ ). ¹ H NMR(400 MHz,DMSO-d ₆ ):δ6.87(d,J＝8.3Hz,1H),6.83(d,J＝8.3Hz,1H),6.77(s,1H), 6.48(s,2H),4.90(s,1H),4.35(s,2H),3.69(s,3H),3.58(s,6H),3.51(s,3H),3.41 (m,1H),2.25(m,2H),1.92(m,2H),1.68(m,2H). ¹³ C NMR(150MHz,DMSO-d ₆ ): δ172.2,165.6,163.8,152.9,147.7,146.7,133.7,133.7,129.5,117.6,112.9,112.2, 94.6,94.3,61.6,60.0,57.7,57.1,56.5,55.6,55.4,32.5,20.4.ESI-MS(m/z):519.5 (M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₅ H ₃₀ N ₂ NaO ₁₀ [M+Na ⁺ ],540.1714；found, 540.1718.

5.3 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -4- (3-hydroxy-4-methoxyphenyl) -3- [4- (N-hydroxycarbamoylamino) propionylamino ] methylazetidin-2-one (6 c)

Black solid (6 c), yield 43%. mp 118-120 deg.C] _D ²⁰ ＝+32.7(c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,DMSO-d ₆ ):δ6.85-6.81(m,2H),6.59(s,1H),6.48(s,2H),4.85(d, J＝2.2Hz,1H),4.01(m,1H),3.68(s,3H),3.60(s,7H),3.55(s,3H),3.12(m,1H), 2.03(m,2H),1.65(m,2H). ¹³ C NMR(150MHz,DMSO-d ₆ ):δ172.5,170.2,164.9, 152.9,147.5,133.5,130.0,113.1,112.3,94.4,59.9,59.3,58.4,55.5,36.9,34.6,20.6, 13.9.ESI-MS(m/z):504.5(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₄ H ₂₉ N ₃ NaO ₉ [M+Na ⁺ ],526.1796；found,526.1793.

5.4 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -4- (3-hydroxy-4-methoxyphenyl) -3- [4- (N-hydroxycarbamido) butanamido ] methylazetidin-2-one (6 d)

Pale yellow oily liquid (6 d), yield 50%. [ alpha ] to] _D ²⁰ ＝+34.8(c 1.0,CHCl ₃ ). ¹ H NMR(400 MHz,DMSO-d ₆ ):δ8.84(s,2H),7.44(s,1H),7.26(d,J＝8.2Hz,1H),7.06(s,2H), 6.92(d,J＝7.4Hz,1H),5.45(d,J＝2.2Hz,1H),4.54(q,J＝7.1Hz,1H),4.22(s, 2H),4.19(s,3H),4.16(s,6H),4.11(s,3H),3.01(m,4H),1.68(t,J＝7.1Hz,2H). ¹³ C NMR(150MHz,DMSO-d ₆ ):δ175.4,173.2,170.2,164.7,152.9,152.3,148.3, 147.3,133.7,130.0,114.0,111.9,97.1,94.5,59.9,58.9,55.6,55.3,37.3,32.9,32.5, 20.6,13.9.ESI-MS(m/z):518.6(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₅ H ₃₁ N ₃ NaO ₉ [M+Na ⁺ ],540.1953；found,540.1949.。

EXAMPLE 6 Synthesis of the object Compound (7 g-h)

6.1 Synthesis of ethyl (S) -2- [1- (3-chloro-4-methoxyphenyl) -1- (3,4,5-trimethoxyphenylamino) methyl ] acrylate (7 c)

Synthetic procedure reference 1c, yellow oily liquid (7 c), yield 65%. [ alpha ] to] _D ²⁰ ＝+70.5(c 0.17, CHCl ₃ ). ¹ H NMR(400MHz,CDCl ₃ ):δ7.37(d,J＝2.1Hz,1H),7.24(dd,J＝8.8, 2.1Hz,1H),6.89(d,J＝8.8Hz,1H),6.39(s,1H),5.94(s,1H),5.81(s,2H),5.30(s, 1H),4.21-4.13(m,2H),3.89(s,3H),3.77(s,6H),3.75(s,3H),1.24(t,J＝7.1Hz, 3H). ¹³ C NMR(150MHz,CDCl ₃ ):δ165.5,153.9,153.2,142.7,139.6,133.2,129.8, 128.5,126.3,125.6,122.0,111.4,90.5,60.5,60.4,57.8,55.6,55.3,13.5.ESI-MS (m/z):436.1(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₂ H ₂₇ ClNO ₆ [M+H ⁺ ],436.1521； found 436.1523.

6.2 Synthesis of (S) -1- (3,4,5-trimethoxyphenyl) -4- (3-chloro-4-methoxyphenyl) -3-methyleneazetidin-2-one (7 d)

Synthetic procedure reference 1d gave white solid (7 d) in 68% yield. mp 119-120 deg.C] _D ²⁰ ＝+35.1 (c 0.29,CHCl ₃ ),97％ee[determined by HPLC analysis using a Chiralcel AD-H column；n-Hex/i-PrOH＝80:20,1.0mL/min,254nm；t _R (minor)＝10.62min；t _R (major)＝13.97min]. ¹ H NMR(400MHz,CDCl ₃ ):δ7.41(d,J＝2.0Hz,1H),7.25 (dd,J＝8.5,2.0Hz,1H),6.92(d,J＝8.5Hz,1H),6.56(s,2H),5.83(s,1H),5.28(s, 1H),5.16(s,1H),3.88(s,3H),3.75(s,3H),3.73(s,6H)； ¹³ C NMR(150MHz, CDCl ₃ ):δ160.1,154.8,153.0,148.8,134.2,133.0,128.9,128.2,125.7,122.6,111.8, 110.5,94.2,62.3,60.3,55.6,55.5.ESI-MS(m/z):390.1[M+H ⁺ ].ESI-HRMS(m/z): calcd for C ₂₀ H ₂₁ ClNO ₅ [M+H ⁺ ].390.1103；found 390.1103.

6.3 Synthesis of (S, E) -1- (3,4,5-trimethoxyphenyl) -4- (3-ethoxycarbonyl-4-methoxyphenyl acrylate) -3-methyleneazetidin-2-one (7E)

The synthetic method is reported in a reference document, and the specific operation is as follows: a15 mL stoppered tube was charged with the starting material (0.15 mmol), ethyl acrylate (1.17 mmol), triethylamine (0.2mL, 1.46mmol) and N-methylpyrrolidinone (1 mL), purged with nitrogen to remove oxygen sufficiently, and then added with palladium acetate (4.9mg, 0.02mmol) and tris (o-methylphenyl) phosphine (13.3mg, 0.04mmol), strictly sealed and heated at 128 ℃ for 12 hours. After the reaction, the mixture was cooled, ethyl acetate was added thereto, and the mixture was washed with water three times, and the organic phase was washed with saturated brine once and dried over anhydrous sodium sulfate. Concentrating by rotary evaporation, separating by silica gel column chromatography (PE/EA 2:1), and rotary drying to obtain corresponding intermediate. Pale yellow oily liquid (7 e), yield 46%. ¹ H NMR(400MHz,CDCl ₃ ): δ7.93(d,J＝16.2Hz,1H),7.55(s,1H),7.38(d,J＝8.4Hz,1H),6.94(d,J＝8.6Hz, 1H),6.59(s,2H),6.55(d,J＝16.2Hz,1H),5.86(s,1H),5.34(s,1H),5.18(s,1H), 4.26(q,J＝6.7Hz,2H),3.90(s,3H),3.77(s,3H),3.74(s,6H),1.34(t,J＝6.7Hz, 3H). ¹³ C NMR(150MHz,CDCl ₃ ):δ167.2,160.8,158.7,153.6,149.8,139.2,134.9, 133.7,129.7,128.7,127.6,124.2,119.9,111.9,110.9,94.9,63.4,60.9,60.5,56.1, 55.7,14.4.ESI-MS(m/z):454.4[M+H ⁺ ].ESI-HRMS(m/z):calcd for C ₂₅ H ₂₈ NO ₇ [M+H ⁺ ].454.4912；found 454.4918.

6.4 Synthesis of (S, E) -1- (3,4,5-trimethoxyphenyl) -4- (3-butenoic acid tert-butyl ester group-4-methoxyphenyl) -3-methylene azetidin-2-one (7 f)

Synthetic procedure reference 7e, yellow oily liquid (7 f), yield 48%. ¹ H NMR(400MHz,CDCl ₃ ): δ7.48(s,1H),7.32(m,2H),6.86(d,J＝7.9Hz,1H),6.73(d,J＝16.2Hz,1H),6.60 (s,2H),6.34-6.27(m,1H),5.85(s,1H),5.32(s,1H),5.17(s,1H),3.84(s,3H),3.76 (s,3H),3.73(s,6H),3.18(d,J＝6.2Hz,1H),1.47(s,9H). ¹³ C NMR(150MHz, CDCl ₃ ):δ170.4,160.4,156.3,152.9,149.2,133.9,133.2,127.7,126.5,126.2,124.9, 123.4,110.7,110.2,94.2,80.3,63.1,60.3,55.4,54.9,39.2,27.6,27.5.ESI-MS(m/z): 496.5[M+H ⁺ ].ESI-HRMS(m/z):calcd for C ₂₈ H ₃₄ NO ₇ [M+H ⁺ ].496.2330；found 496.2333.

6.5 Synthesis of (S, E) -1- (3,4,5-trimethoxyphenyl) -4- (3- (N-hydroxyacrylamido) -4-methoxyphenyl) -3-methyleneazetidin-2-one (7 g)

A25 mL eggplant-shaped bottle was charged with the starting material 7e (0.1 mmol), sodium hydroxide (40mg, 1.0 mmol) and an aqueous hydroxylamine solution (0.15 mL), and the mixture was dissolved in methylene chloride (0.5 mL) and methanol (1.0 mL). Reacting in ice bath, adding water after the raw materials completely disappear, adjusting pH to 6 with acetic acid, extracting with ethyl acetate for three times, combining organic phases, washing with saturated salt water once, and drying with anhydrous sodium sulfate. Filtering, concentrating by rotary evaporation, separating by silica gel column Chromatography (CH) ₂ Cl ₂ MeOH 20. Pale yellow solid (7 g), yield 70%. mp 126-128 deg.C] _D ²⁰ ＝+51.7(c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,CDCl ₃ ):δ7.86(d, J＝15.7Hz,1H),7.52(s,1H),7.33(d,J＝8.3Hz,1H),6.86(d,J＝8.3Hz,1H),6.56 (s,2H),5.81(s,1H),5.33(s,1H),5.15(s,1H),3.79(s,3H),3.74(s,3H),3.69(s,6H). ¹³ C NMR(150MHz,CDCl ₃ ):δ165.2,161.1,158.6,153.5,149.4,136.3,134.8,133.6, 129.5,128.5,127.5,124.2,117.6,111.7,111.3,94.9,63.4,60.9,56.1,55.6.ESI-MS (m/z):441.4(M+H ⁺ ).ESI-HRMS(m/z):calcd for C ₂₃ H ₂₅ N ₂ NaO ₇ [M+Na ⁺ ],463.1476； found,463.1473.

6.5 Synthesis of (S, E) -1- (3,4,5-trimethoxyphenyl) -4- (3- (N-hydroxycrotonamido) -4-methoxyphenyl) -3-methyleneazetidin-2-one (7 h)

Synthetic methods refer to 7g, light red solid (7 h), yield 56%. mp 110-113 deg.C] _D ²⁰ ＝+41.8 (c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,DMSO-d ₆ ):δ10.51(s,1H),8.80(s,1H),7.60(s, 1H),7.30(d,J＝8.7Hz,1H),7.00(d,J＝8.7Hz,1H),6.65(d,J＝15.9Hz,1H),6.60 (s,2H),6.30-6.25(m,1H),5.82(s,1H),5.69(s,1H),5.31(s,1H),3.77(s,3H),3.64 (s,6H),3.55(s,3H),2.90(d,J＝6.2Hz,2H). ¹³ C NMR(150MHz,DMSO-d ₆ ):δ 166.7,160.1,156.0,152.9,149.4,133.8,132.9,128.2,127.2,126.0,125.4,125.1, 111.6,111.4,94.6,62.1,59.9,55.6,55.4,37.1.ESI-MS(m/z):455.4[M+H ⁺ ]. ESI-HRMS(m/z):calcd for C ₂₄ H ₂₆ N ₂ NaO ₇ [M+Na ⁺ ].477.1356；found 477.1352.。

Example 7 Synthesis of (2R, 3R, E) -1- (3,4,5-trimethoxyphenyl) -4- (3- (N-hydroxyacrylamido) -4-methoxyphenyl) -3-methylazetidin-2-one (8 a)

Synthetic methods refer to 7g, light red solid (8 a), total yield 36%. mp 124-126 deg.C] _D ²⁰ ＝ +11.7(c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,DMSO-d ₆ ):δ7.83(d,J＝15.6Hz,1H), 7.33(s,1H),7.15(d,J＝8.3Hz,1H),6.84(d,J＝8.3Hz,1H),6.56(d,J＝15.6Hz, 1H),6.53(s,2H),5.10(d,J＝4.8Hz,1H),3.79(s,3H),3.74(s,3H),3.67(s,6H), 3.64-3.61(m,1H),0.83(d,J＝7.9Hz,3H). ¹³ C NMR(150MHz,DMSO-d ₆ ):δ168.7, 165.4,158.1,153.5,136.5,134.4,133.7,129.5,127.7,126.8,123.8,117.7,111.3, 94.9,60.9,58.2,56.1,55.5,49.2,9.7.ESI-MS(m/z):443.4[M+H ⁺ ].ESI-HRMS(m/z): calcd for C ₂₃ H ₂₆ N ₂ NaO ₇ [M+Na ⁺ ].465.1632；found 465.1639.。

EXAMPLE 8 Synthesis of (3S, 4R) -1- (3,4,5-trimethoxyphenyl) -4- (3-hydroxy-4-methoxyphenyl) -3- [ 4-carboxybenzoyloxy ] methylazetidin-2-one (9 a)

Synthetic methods reference 5a, a light yellow solid (9 a), in 46% overall yield. mp 120-122 deg.C] _D ²⁰ ＝ +21.7(c 1.0,CHCl ₃ ). ¹ H NMR(400MHz,CDCl ₃ ):δ8.10-8.05(m,4H),6.96(s,1H), 6.87(d,J＝8.1Hz,1H),6.84(d,J＝8.0Hz,1H),6.56(s,2H),4.88(d,J＝2.1Hz, 1H),4.78(m,2H),3.88(s,3H),3.77(s,3H),3.71(s,6H),3.54(m,1H). ¹³ C NMR (400MHz,CDCl ₃ ):δ164.8,163.2,152.9,146.3,145.8,134.0,132.9,129.6,129.4, 129.1,117.2,111.4,110.4,94.3,61.1,60.3,58.6,58.4,55.4.ESI-MS(m/z):538.5 [M+H ⁺ ].ESI-HRMS(m/z):calcd for C ₂₈ H ₂₈ NO ₁₀ [M+H ⁺ ].560.1527；found 560.1532.。

Example 9 in vitro assay of tumor cell proliferation inhibitory Activity

Tumor cells in logarithmic growth phase were taken, treated, inoculated into 96-well plates, and subjected to CO-5% at 37 ℃% ₂ Was cultured for 24 hours under the conditions of (1). Test compounds were added at 6 gradient concentrations and CA-4 was used as a positive control. And (3) continuing to culture for 48 hours under the same condition, adding MTT, culturing for 4 hours, discarding supernatant containing MTT, adding DMSO into each hole, shaking to dissolve purple crystals, measuring an OD value at 490nm or 540nm on an enzyme labeling instrument, and calculating the inhibition rate. Median Inhibitory Concentration (IC) of Compounds ₅₀ Values) were calculated from the inhibition ratios of 6 concentrations. Three duplicate wells were set for each concentration gradient and the assay was repeated three times. The activity results are shown in tables 1 and 2.

TABLE 1 inhibitory Activity of Compounds on tumor cell proliferation (IC) ₅₀ /μM)

Note that: ^a the antitumor activity is determined by an MTT method, and the data are average values of three measurements; ^b BE- (2) -C is a human neuroblastoma cell strain; ^c a549 is a human lung cancer cell strain; ^d u87MG is human malignant glueA glioblastoma cell strain; ^e HCT-116 is a human colon cancer cell line.

TABLE 2 inhibitory Activity of Compound 5a on tumor cell proliferation (IC) ₅₀ /μM)

Note that: ^a the antitumor activity is determined by an MTT method, and the data are average values of three measurements; ^b HeLa is a human cervical cancer cell line; ^c a2780 is a human ovarian cancer cell strain; ^d MDA-MB-231 is a human breast cancer cell line; ^e HUH-7 is a human hepatoma cell line; ^f SKOV3 is a human ovarian cancer cell line; ^g MCF-7 is a human breast cancer cell line; ^h MDA-MB-468 is a human breast cancer cell line.

Example 10 in vitro tubulin self-Assembly assay

The inhibition of microtubule aggregation in vitro by the preferred compound 5a was tested by turbidity assay, with a test kit available from cytosketon, usa. The procedure was as follows, pH =6.6 microtubule aggregation system (0.1M PIPES, 10mM MgCl) ₂ 1mM GTP, 1mM EGTA and 3.4M glycerol) were pre-incubated on ice in advance, different concentrations of test compound 5a were added, and the Colchicine-treated group was set as a positive control and the DMSO (4%, v/v) -treated group was set as a negative control. After tubulin (10 mM) was added to the above system, it was immediately transferred, and the aggregation reaction was carried out at 37 ℃ with keeping the temperature constant, and absorbance was measured at 340nm with a spectrophotometer (Synergy H4 Hybrid) every 1 minute for 30 minutes, and then a graph of absorbance was plotted (as shown in FIG. 1). The results show that Compound 5a is capable of significantly inhibiting microtubule aggregation, its IC ₅₀ At 5.4. Mu.M (as shown in Table 3).

TABLE 3 Compound 5a in vitro inhibition of microtubule self-assembly assay

Example 11 in vitro HDAC inhibitory Activity assay

The inhibitory activity of the compounds on HDACs was tested by elisa, and all enzymatic reactions were performed at 37 ℃ for 30 min. Compounds were diluted to different concentration ranges with 5% DMSO, 5 microliters of compound solution was added to 50 microliters of pH =8.0 and contained 25mM Tris, 1mM MgCl ₂ 0.1mg/ml BSA, 137mM NaCl, 2.7mM KCl, HDAC and their substrates. After the enzymatic reaction is complete, the fluorescence intensity is measured at an excitation of 350-360nm and an emission wavelength of 450-460nm in a SpectraMax M5 microtiter plate reader. Finally, using Prism GraphPad software to perform non-linear regression calculation by fitting after normalization to obtain IC of the tested compound ₅₀ The value is obtained. The results are shown in Table 4, where 5a and 6a both significantly inhibited HDAC8 activity, IC ₅₀ The values were 0.177 μ M and 0.288 μ M, respectively, and both had similar inhibitory activity to HDAC8 on HDAC1, while the inhibitory activity to HDAC6 decreased 6-fold and 8-fold, respectively, indicating that these two compounds are class I HDAC inhibitors.

TABLE 4 inhibitory Activity of Compounds on HDAC1/6/8 (IC) ₅₀ /μM)

And (3) annotation: ^a IC ₅₀ are all averages of three measurements; ^b SAHA is a non-selective HDACs inhibitor; ^c PCI-34051 is a selective HDAC8 inhibitor; NT = not tested.

Example 12 HDAC-related protein expression assay

Taking BE- (2) -C cells in logarithmic growth phase, treating according to 2 × 10 ⁵ Number of cells/well was seeded in 6-well plates, at 37 ℃ C. And 5% CO ₂ And culturing in an incubator with saturated humidity for 24 hours, and adding the compound 5a with gradient concentration to treat the tumor cells (setting DMSO positions at the same time)Positive control), collected after 24 hours and lysed with lysis buffer. Heating and denaturing protein sample, loading to polyacrylamide gel, SDS-PAGE electrophoretic separation, wet filming, closing, primary antibody reaction and secondary antibody reaction, and exposure to develop color. As shown in fig. 2, the compound 5a can significantly promote the acetylation of the HDAC8 substrate protein SMC3 and the HDAC1 substrate protein histone H3, but has no significant effect on the acetylation of the HDAC6 substrate protein α -tubulin, which indicates that the compound 5a can preferably inhibit HDAC1 and HDAC8.

Example 13 in vitro cell cycle arrest assay

Taking BE- (2) -C cells in logarithmic growth phase, treating according to 2X 10 ⁵ Number of cells/well was inoculated in 6-well plates, at 37 ℃ and 5% ₂ And culturing for 12 hours in an incubator with saturated humidity, replacing fresh culture solution after the cells adhere to the wall, treating the cells for 24 hours by using the compound 5a with different concentrations, and setting a DMSO treatment group as negative control. Discarding supernatant, collecting adherent cells, washing twice with PBS, fixing with 75% ethanol at-20 deg.C overnight, staining with PI, and testing with flow cytometer, with the result that compound 5a can obviously block tumor cell cycle in G ₂ the/M phase (as shown in FIG. 3).

Example 14 in vitro cell cycle-related protein assay

Taking BE- (2) -C cells in logarithmic growth phase, treating according to 2 × 10 ⁵ Number of cells/well was seeded in 6-well plates, at 37 ℃ C. And 5% CO ₂ And culturing in an incubator with saturated humidity for 24 hours, adding a compound 5a with gradient concentration to treat the tumor cells (setting a DMSO treatment group as a negative control), and collecting and lysing the cells by using a lysate after 48 hours. Heating and denaturing protein sample, loading to polyacrylamide gel, SDS-PAGE electrophoretic separation, wet filming, closing, primary antibody reaction and secondary antibody reaction, and exposure to develop color. The results are shown in FIG. 4, and the compound 5a can obviously promote the expression of mitotic checkpoint protein Bubr-1, phosphorylated Histone P-Histone 3 and cyclin B1.

Example 15 in vitro apoptosis Induction assay

Taking BE- (2) -C cells in logarithmic growth phase, treating according to 2 × 10 ⁵ Number of cells/well was seeded in 6-well plates, at 37 ℃ C. And 5% CO ₂ And incubation in an incubator at saturated humidity for 24 hours, adding compound 5a at a gradient concentration, and setting the DMSO-treated group as a negative control. After culturing for 48 hours, collecting supernatant cells and adherent cells, carrying out double staining by PI and Annexin V, and detecting by using a flow cytometer. The results are shown in FIG. 5, where Compound 5a induces apoptosis in a concentration-dependent manner.

EXAMPLE 16 detection assay for apoptosis-related proteins in vitro

Taking BE- (2) -C cells in logarithmic growth phase, treating according to 2 × 10 ⁵ Number of cells/well was seeded in 6-well plates, at 37 ℃ C. And 5% CO ₂ And incubation in an incubator at saturated humidity for 24 hours, adding compound 5a at a gradient concentration, and setting the DMSO-treated group as a negative control. After 48 hours the cells were collected and lysed with lysis buffer. Loading on polyacrylamide gel, performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) separation, performing wet membrane transfer, sealing, sequentially performing primary antibody reaction and secondary antibody reaction, and exposing and developing. As shown in FIG. 6, the compound 5a can significantly promote the expression of the tumor suppressor gene p53, the pro-apoptotic protein Bax and the sheared DNA repair enzyme PAPR-1.

Example 17 colony formation inhibition experiment

Taking logarithmic growth phase BE- (2) -C cells, treating, inoculating into 6-well plate, inoculating 1500 cells per well, and making 5% CO at 37 ℃% ₂ And culturing for 24 hours under the condition of humidity saturation, adding compounds 5a with different concentrations to treat the cells for 48 hours after the cells adhere to the wall, and setting a DMSO treatment group as a negative control. After replacing the fresh culture medium and continuing to culture for two weeks, the supernatant was discarded, the cells were washed twice with PBS, fixed with anhydrous methanol for 30 minutes, stained with crystal violet dye for 1 hour, washed off the staining solution, and sufficiently dried, and then the number of colonies formed by more than 50 cells was counted under a microscope, and the result showed that compound 5a was able to significantly inhibit the formation of colonies of HeLa cells (as shown in FIG. 7).

Claims

1. Diaryl-beta-lactam compounds, characterized in that they have the structure of the general formula I,

wherein R is ¹ And R ² Is selected from hydrogen atom, alkyl, substituted alkyl, alkoxy, halogen atom, amino, hydroxyl, acyloxy, methoxy formyl, allyloxy, propargyloxy, sulfonyloxy, alkylamino, acylamino, sulfonylamino or combination of 2-3 of the same or different groups; x is selected from carbon, nitrogen, oxygen, sulfur, ester group, amide group; l is taken from

Wherein n is selected from 0, 1, 2, 3,4,5, 6, 7; l may also be derived from

Iso-alicyclic and aromatic (hetero) rings; r is ³ Is taken from

R ⁴ Is selected from hydrogen atom, alkyl, substituted alkyl, alkoxy, acyloxy, hydroxyl, phenyl, substituted phenyl, pyridyl, cyclopropyl, vinyl, amino, alkylamino, amido, sulfonyloxy and sulfonamido.

2. Diaryl- β -lactams according to claim 1, wherein said preferred compounds are:

3. diaryl-beta-lactam compounds, characterized in that they have the general formula II,

Wherein n is selected from 1, 2, 3,4,5, 6, 7; l can also be taken from

Iso-alicyclic and aromatic (hetero) rings; r ³ Is taken from

4. A diaryl- β -lactam compound according to claim 3, wherein said preferred compound is:

5. the use of a compound according to any one of claims 1 to 4, and pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the prevention and treatment of diseases associated with tumors,

the pharmaceutically acceptable salt is formed by organic acids such as malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid, oxalic acid and the like, and inorganic acids such as phosphoric acid, halogen acid, sulfuric acid and nitric acid;

the disease comprises neuroblastoma, interstitial sarcoma, choriocarcinoma, malignant hydatidiform mole, thyroid cancer, head and neck squamous cell carcinoma, cervical cancer, prostate cancer, renal cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, gastric cancer, lung cancer, liver cancer, melanoma, lymphoma, brain glioma, nasopharyngeal cancer, neuroendocrine cancer, undifferentiated cancer, malignant teratoma and benign tumor.

6. A combination drug for the prevention and treatment of tumor-related diseases comprising the compound according to any one of claims 1 to 4, wherein the tumor-related diseases are neuroblastoma, thyroid cancer, lung cancer, liver cancer, melanoma, lymphoma, prostate cancer, head and neck squamous cell carcinoma, cervical cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, renal cancer, undifferentiated carcinoma, interstitial sarcoma, choriocarcinoma, gastric cancer, bladder cancer, brain glioma, nasopharyngeal cancer, neuroendocrine cancer, malignant hydatidiform mole, malignant teratoma, and benign tumor.