Diaryl-β-lactam compounds, preparation methods and uses in pharmaceuticals

Abstract

The present invention belongs to the technical field of synthetic pharmaceutical chemistry, and relates to novel chiral diaryl-β-lactam HDAC / Tubulin bifunctional inhibitors with significant anti-tumor activity having the following general formula structure and their applications in the research and development of anti-tumor drugs. The present invention also includes the applications of the compounds, their pharmaceutically acceptable salts and their compound drugs in the preparation of drugs for preventing or treating diseases related to tumors. The compounds of the present invention or their pharmaceutically acceptable salts can inhibit the regulation mechanism of tumor cell proliferation by inhibiting histone deacetylase and microtubulin aggregation, effectively inhibit the growth of tumor cells in vitro and in vivo, and can be applied to the preparation of drugs for preventing or treating diseases related to tumors. The diseases related to tumors include benign and malignant tumors and other diseases caused by tumors.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic medicinal chemistry in new drug research and development, relates to diaryl-β-lactam compounds, and particularly relates to novel chiral diaryl-β-lactam HDAC / Tubulin bifunctional inhibitors with significant anti-tumor activity, preparation methods, anti-tumor activities in vitro and in vivo, and the use of such compounds, their acceptable pharmaceutical salts, or compound drugs containing them as one of the components in the preparation of drugs for preventing and treating tumor-related diseases. Background Art

[0002] Microtubules are the main components of the cytoskeleton, are essential for cell mitosis, and also play an indispensable role in maintaining cell morphology, cell movement, cell division and proliferation. Malignant tumor cells have extremely active mitosis and metastasis. Therefore, targeting microtubules and disrupting the dynamic balance between tubulin polymerization and depolymerization can selectively inhibit the division and proliferation of tumor cells (Science 2013, 339, 587-590). Although some significant progress has been made in the research on tubulin aggregation inhibitors acting on colchicine, especially the structural modification research on Combretastatin A-4, such as the disodium phosphate of Combretastatin (CA-4P) and the disodium phosphate of BNC105 (BNC105P) have entered phase I and phase II clinical trials respectively, studies have shown that the clinical effects of these drugs are poor and they have certain toxic side effects, such as nausea, vomiting, visual impairment and headache. In addition, there are also deficiencies in prolonging the survival of patients. So far, no such drug has been approved for marketing (J. Med. Chem. 2016, 59, 8685-8711). Therefore, developing tubulin aggregation inhibitors with better anti-tumor activity, higher selectivity and lower toxic side effects is the main direction for the future research and development of such drugs.

[0003] As a key enzyme regulating cell epigenetics, histone deacetylases (HDACs) are highly expressed in multiple tumor cell lines. It is highly correlated with the growth, proliferation, and invasion of tumor cells and is considered one of the most promising targets for cancer treatment (Nat. Rev. Drug Discovery 2012, 11, 384 - 400). The marketed pan-HDAC inhibitors are mainly used for the treatment of hematological malignancies and have poor efficacy in solid tumors. Therefore, HDAC inhibition is often combined with other anti-tumor drugs. Although the combination drug mode can produce a synergistic anti-tumor effect, it will also cause complex and unpredictable pharmacokinetics, poor patient compliance, and even drug-drug interactions. The "one drug, multiple targets" drug mode can effectively overcome these disadvantages and is an ideal alternative treatment mode to traditional "single target, single drug" and combination drug therapy (J. Med. Chem. 2005, 48, 6523 - 6543). A large number of research reports have proven that the design of HDAC dual-target drugs is reasonable and feasible and is also obvious for improving the efficacy of cancer treatment.

[0004] Preclinical studies have shown that the microtubule polymerization inhibitor (vincristine) and the HDAC inhibitor SAHA can produce a synergistic anti-tumor effect in the combined treatment of leukemia. In the MOLT-4 nude mouse xenograft tumor model, the tumor inhibition rate of the vincristine and SAHA combination administration group was significantly higher than that of the two single-drug groups, and the combination administration group also greatly prolonged the survival time of the mice (J. Hematol. Oncol. 2017, 8, 82). In addition, mechanism studies have also shown that microtubulin and HDAC are interrelated and synergistic in the occurrence and development of tumors. In early studies, HDAC mainly catalyzed the deacetylation of histone lysine residues on chromosomes in the nucleus. However, in fact, the catalytic substrates of HDAC are also widely present in the cytoplasm, such as α-tubulin and HSP90. Overexpressed HDAC in tumor cells can cause excessive deacetylation of α-tubulin in the cytoplasm, thereby accelerating the aggregation and disassembly of microtubules and promoting the migration and invasion of tumor cells. Microtubules themselves also have the kinetic characteristics of aggregation and disassembly, and this synergistic process between microtubules and HDAC is very obvious in highly proliferating malignant tumor cells (J. Med. Chem. 2020, 63, 23 - 39). These mechanism studies and the data results of preclinical studies provide a reasonable basis for the design of HDAC / Tubulin dual-target inhibitors.

[0005] Based on the current situation of the existing technology, the inventors of the present application intend to provide diaryl-β-lactam compounds, preparation methods, and uses in pharmaceuticals. Summary of the Invention

[0006] Based on the current situation of the prior art, the object of the present invention is to provide diaryl-β-lactam compounds, their preparation methods and uses in pharmaceuticals. In particular, it relates to novel chiral diaryl-β-lactam HDAC / Tubulin bifunctional inhibitors, their preparation methods, and the use of such compounds, their pharmaceutically acceptable salts or pharmaceutical compositions containing them in the preparation of drugs for preventing and treating tumor-related diseases.

[0007] Combining the structural characteristics of Tubulin aggregation inhibitors and HDAC inhibitors, and applying the design principle of fused pharmacophores, the present application designed and constructed a library of chiral diaryl-β-lactam HDAC / Tubulin dual-target molecular compounds. After anti-tumor activity tests at the molecular and cellular levels, lead compounds with good activity were screened out, and finally candidate compounds with novel structures and potential for drug development were provided for further research.

[0008] The present invention provides novel chiral diaryl-β-lactam HDAC / Tubulin bifunctional inhibitors of the following general formula I or their pharmaceutically acceptable salts,

[0009]

[0010] wherein, R 1 and R 2 are independently selected from a hydrogen atom, an alkyl group, a substituted alkyl group, an alkoxy group, a halogen atom, an amino group, a hydroxyl group, an acyloxy group, a methoxycarbonyl group, an allyloxy group, a propargyloxy group, a sulfonyloxy group, an alkylamino group, an acylamino group, a sulfonylamino group, or a combination of two or three of the above same or different groups; X is selected from carbon, nitrogen, oxygen, an ester group, an amide group; L is selected from where n is selected from 0, 1, 2, 3, 4, 5, 6, 7; L can also be selected from and other alicyclic and heteroaromatic rings; R 3 is selected from R 4 is selected from a hydrogen atom, an alkyl group, a substituted alkyl group, an alkoxy group, an acyloxy group, a hydroxyl group, a phenyl group, a substituted phenyl group, a pyridyl group, a cyclopropyl group, a vinyl group, an amino group, an alkylamino group, an acylamino group, a sulfonyloxy group, a sulfonylamino group; Preferred compounds are:

[0011]

[0012] The present invention also provides novel chiral diaryl-β-lactam HDAC / Tubulin bifunctional inhibitors of the following general formula II or their pharmaceutically acceptable salts,

[0013]

[0014] Among them, R 1 With R 2 is selected from hydrogen, alkyl, substituted alkyl, alkoxy, halogen, amino, hydroxy, acyloxy, methoxycarbonyl, allyloxy, propargyloxy, sulfonyloxy, alkylamino, acylamino, sulfonylamino, or a combination of 2-3 of the same or different groups; X is selected from carbon, nitrogen, oxygen, ester, or amide; L is selected from Where n is selected from 1, 2, 3, 4, 5, 6, 7; L can also be selected from Alicyclic and aromatic heterocyclic rings; R 3 Taken from Preferred compounds are:

[0015]

[0016] The "pharmaceutically acceptable salts" mentioned in the present invention include, specifically, salts formed 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.

[0017] A further object of the present invention is to provide the use of the above-mentioned compound or pharmaceutically acceptable salts of these compounds and compositions comprising the compound or its salt for preparing drugs for preventing or treating tumor-related diseases.

[0018] The tumor-related diseases can be specifically listed as neuroblastoma, thyroid cancer, lymphoma, prostate cancer, kidney cancer, bladder cancer, glioma, nasopharyngeal cancer, neuroendocrine cancer, head and neck squamous cell carcinoma, cervical cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, stromal sarcoma, choriocarcinoma, malignant hydatidiform mole, malignant teratoma, gastric cancer, lung cancer, liver cancer, melanoma, undifferentiated cancer and benign tumors, but are not limited thereto.

[0019] The present invention provides and demonstrates a novel diaryl-β-lactam HDAC / Tubulin dual-function inhibitor or a pharmaceutically acceptable salt thereof with significant anti-tumor activity, a regulatory mechanism by which tumor cell growth is inhibited by inhibiting the aggregation of HDAC and tubulin, and a significant inhibitory effect on tumor cell proliferation and angiogenesis in in vitro and in vivo anti-tumor experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 .In vitro inhibition experiment of microtubule self-assembly by compound 5a—absorbance-time curve.

[0021] Figure 2 .Effect of compound 5a on the expression of HDAC-related proteins.

[0022] Figure 3 . Effect of Compound 5a on the tumor cell cycle.

[0023] Figure 4 . Effect of Compound 5a on the expression of tumor cell cycle-related proteins.

[0024] Figure 5 . Test for apoptosis induction by Compound 5a.

[0025] Figure 6 . Effect of Compound 5a on the expression of apoptosis-related proteins.

[0026] Figure 7 . Inhibition of tumor cell colony formation by Compound 5a. Detailed implementation mode

[0027] The present invention will be further described below in conjunction with embodiments. These embodiments are only used to further illustrate the present invention and do not change the protection scope of the present invention. The preparation method of the target compound of the present invention can be further reflected by the preparation process of representative compounds as follows:

[0028] Example 1 Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-3-methyl-4-(3-hydroxy-4-methoxyphenyl)azetidin-2-one (1 g)

[0029] Referring to the method in the literature (J. Med. Chem. 2016, 59, 10329 - 10334), the intermediate compound 1g was synthesized according to the following route in the present invention:

[0030]

[0031] Reagents and conditions: (a) Pd2(dba)3 (0.1%), (R,R,R)-Ph-SKP (0.25%), K2CO3, CH2Cl2, 25 °C, 3 h; (b) i) Sn[N(TMS)2]2, toluene, reflux, 3 h; ii) TBAF, THF, 0 °C, 1 h; (c) Pd / C, H2, EtOH, 25 °C, 12 h; (d) i) BnCl, K2CO3, MeCN, reflux, 12 h; ii) B2(pin)2 (1.3 equiv), CuCl (5%), MeOH (1.5 equiv), PPh3 (15%), t-BuOLi (10%), THF, 25 °C, 12 h; iii) NaBO3·4H2O, H2O, THF, 25 °C, 2 h; (e) i) CBr4, PPh3, THF, 25 °C, 4 h; ii) Pd / C, AcONa, H2, EtOH, 25 °C, 12 h.

[0032] 1.1 Synthesis of Ethyl (S)-2-[1-(3-tert-butyldimethylsilyloxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenylamino)methyl]acrylate (1c)

[0033] In a 50 mL Schlenk tube, add starting material 1a (0.65 g, 1.59 mmol), 3,4,5-trimethoxyaniline (0.43 g, 2.07 mmol), potassium carbonate (0.66 g, 4.77 mmol), Pd2(dba)3 (1.5 mg, 0.0016 mmol) and (R,R,R)-Ph-SKP (2.6 mg, 0.004 mmol). After evacuating and filling with dry nitrogen three times, inject anhydrous dichloromethane (7 mL) into the system. Continue to react at room temperature for 3 hours under nitrogen protection. Add water, extract three times with dichloromethane. Combine the organic phases, wash once with saturated brine, and dry over Na2SO4. Concentrate by rotary evaporation and separate by column chromatography (PE / EA 3:1) to obtain 0.73 g of a colorless oily liquid (1c) with a yield of 85%. [α] D 20 +86.2 (c 0.41, CHCl3), 98% ee [determined by HPLC analysis using a Chiralcel AS-3 column; n-Hex / i-PrOH = 95:5, 1.0 mL / min, 254 nm; t R (major) = 7.47 min; t R (minor) = 9.72 min]. 1 1H NMR (400 MHz, CDCl3): δ 6.92 (dd, J = 8.3, 2.1 Hz, 1H), 6.82 (d, J = 2.1 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.34 (s, 1H), 5.88 (s, 1H), 5.81 (s, 2H), 5.28 (s, 1H), 4.15 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 3.77 (s, 6H), 3.74 (s, 3H), 1.22 (t, J = 7.1 Hz, 3H), 0.96 (s, 9H), 0.11 (s, 6H). ESI-MS (m / z): 532.1 (M+H + ).

[0034] 1.2 Synthesis of (S)-N-(3,4,5-trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-3-methylenepyrrolidin-2-one (1d)

[0035] Add starting material 1c (0.44 g, 0.83 mmol), Sn[N(TMS)₂]₂ (0.4 mL, 1 mmol) and 10 mL of anhydrous toluene into a 50 mL Schlenk tube. Heat under reflux for 3 hours under nitrogen protection, and then directly perform flash column chromatography separation (PE / EA 4:1) to obtain a colorless oily liquid. Then, under ice bath conditions, add TBAF (0.39 g, 15 mmol) and tetrahydrofuran (8 mL). Continue the reaction in the ice bath for 20 minutes, add water, extract three times with ethyl acetate, combine the organic phases, wash once with saturated brine, and dry over Na₂SO₄. Perform column chromatography separation (PE / EA 2:1) to obtain 0.19 g of a pale yellow oily liquid (1d) with a yield of 62%. [α] D 20 +33.6 (c 1.00, CHCl₃), 99% ee [determined by HPLC analysis using a Chiralcel AD-H column; n-Hex / i-PrOH = 80:20, 1.0 mL / min, 254 nm; t R (major) = 13.76 min; t R (minor) = 17.72 min]. 1 ¹H NMR (400 MHz, CDCl₃): δ 6.95 (d, J = 2.1 Hz, 1H), 6.90 (dd, J = 8.2, 2.0 Hz, 2H), 6.83 (d, J = 8.2 Hz, 1H), 6.59 (s, 2H), 5.81 (t, J = 1.6 Hz, 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 + ).

[0036] Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-4-(3-benzyloxy-4-methoxyphenyl)-3-hydroxymethylazetidin-2-one (1e) and (3R,4R)-1-(3,4,5-trimethoxyphenyl)-4-(3-benzyloxy-4-methoxyphenyl)-3-hydroxymethylazetidin-2-one (1f)

[0037] Add raw material 1d (0.22 g, 0.59 mmol), benzyl bromide (0.084 mL, 0.71 mmol), potassium carbonate (0.1 g, 0.71 mmol) and acetonitrile (5 mL) into a 50 mL eggplant-shaped flask. After heating under reflux for 8 hours, add water, extract three times with ethyl acetate, combine the organic phases, wash once with saturated brine, and dry over Na2SO4. Concentrate by rotary evaporation to obtain a white solid. Add this white solid, bis(pinacolato)diboron (0.2 g, 0.8 mmol), CuCl (3.0 mg, 0.09 mmol), triphenylphosphine (24 mg, 0.09 mmol), lithium tert-butoxide (4.8 mg, 0.06 mmol) into a 50 mL Schlenk tube, evacuate and refill with dry nitrogen, then inject methanol (15 μL, 0.9 mmol) and anhydrous THF (10 mL). Continue to react at room temperature for 12 hours under nitrogen protection, add water, extract three times with ethyl acetate, combine the organic phases, wash once with saturated NaCl aqueous solution, and dry over Na2SO4. After evaporating the solvent, add sodium perborate tetrahydrate (0.46 g, 1.5 mmol), THF (10 mL), and water (5 mL), and react at room temperature for 2 hours. Add water, extract three times with ethyl acetate, combine the organic phases, wash once with saturated NaCl aqueous solution, and dry over Na2SO4. Separate by column chromatography (PE / EA 1:2) to obtain 138 mg of white solid (1e), with a yield of 48%. 1 H NMR (400 MHz, CDCl3): δ 7.58 - 7.31 (m, 5H), 6.94 - 6.77 (m, 3H), 6.55 (s, 2H), 5.18 (s, 2H), 5.10 (d, J = 5.1 Hz, 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 + ). 92 mg of white solid (1f), with a yield of 32%. 1 H NMR (400 MHz, CDCl3): δ 7.48 - 7.32 (m, 5H), 6.97 (d, J = 1.5 Hz, 1H), 6.91 (dd, J = 8.3, 1.5 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.56 (s, 2H), 5.16 (s, 2H), 4.92 (d, J = 1.8 Hz, 1H), 4.00 (dd, J = 11.9, 3.3 Hz, 1H), 3.90 (s, 3H), 3.76 (s, 3H), 3.72 (s, 7H), 3.28 (d, J = 2.3 Hz, 1H). ESI-MS (m / z): 480.1 (M + H + ).

[0038] Synthesis of 1.5(3S,4R)-1-(3,4,5-trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-3-methylazetidin-2-one (1g)

[0039] Add starting material 1f (20 mg, 0.04 mmol), carbon tetrabromide (69 mg, 0.2 mmol), triphenylphosphine (54 mg, 0.2 mmol) into a 25 mL Schlenk tube. After evacuating and filling with dry nitrogen three times, inject anhydrous dichloromethane (3 mL) into the system. React at room temperature for 4 hours under nitrogen protection. Add water, extract three times with dichloromethane. Combine the organic phases, wash once with saturated NaCl aqueous solution, and dry over Na2SO4. After rotary evaporation and concentration, add 10% Pd / C (3 mg), sodium acetate (6 mg, 0.08 mmol), and ethanol (1.5 mL). React at room temperature for 12 hours under normal pressure hydrogen. Filter off Pd / C and then rotary evaporate and concentrate. Purify by silica gel (300 - 400 mesh) column chromatography (eluent: PE / EA 2:1). Rotary dry to obtain a white solid (1g) 10 mg, yield 66%. 1 H NMR (400 MHz, CDCl3): δ 6.93 (d, J = 1.5 Hz, 1H), 6.87 (dd, J = 8.3, 1.5 Hz, 1H), 6.83 (d, J = 8.3 Hz, 1H), 6.54 (s, 2H), 5.69 (s, 1H), 4.44 (d, J = 2.2 Hz, 1H), 3.89 (s, 3H), 3.76 (s, 3H), 3.72 (s, 6H), 3.11 (qd, J = 7.3, 2.2 Hz, 1H), 1.45 (d, J = 7.3 Hz, 3H). ESI-MS (m / z): 374.0 (M + H + ).

[0040] Synthesis of Intermediate Compounds (2a - d) in Example 2

[0041]

[0042] The synthesis method refers to the literature report. The specific operation is as follows: Add starting material 1g (45 mg, 0.12 mmol), potassium carbonate (33 mg, 0.24 mmol), bromoalkyl acid ester (0.24 mmol) and acetonitrile (2 mL) into a 25 mL eggplant-shaped flask. React overnight under heating and reflux conditions. After the reaction is completed, cool, add water, extract three times with ethyl acetate. Combine the organic phases, wash once with saturated brine, and dry over anhydrous sodium sulfate. After rotary evaporation and concentration, separate by silica gel column chromatography (PE / EA 1:1), and rotary dry to obtain the corresponding intermediate product.

[0043] 2.1 Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-3-methyl-4-(3-methoxycarbonylmethoxy-4-methoxyphenyl)azetidin-2-one (2a)

[0044] Yellowish green oily liquid (2a), yield 90%. 1 H NMR (400 MHz, CDCl3): δ 7.01 (d, J = 7.5 Hz, 1H), 6.91 (dd, J = 7.5, 1.5 Hz, 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 = 6.3 Hz, 3H). 13 C NMR (150 MHz, CDCl3): δ 169.0, 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 NO8[M + H + , 446.1809; found, 446.1806.

[0045] 2.2 Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-3-methyl-4-(3-ethoxycarbonylpropoxy-4-methoxyphenyl)azetidin-2-one (2b)

[0046] Yellow oily liquid (2b), yield 88%. 1 H NMR (400 MHz, CDCl3): δ 6.95 (d, J = 7.8 Hz, 1H), 6.88 (m, 2H), 6.55 (s, 2H), 4.47 (s, 1H), 4.14 (q, J = 6.9, 6.3 Hz, 2H), 4.01 (m, 2H), 3.86 (s, 3H), 3.76 (s, 3H), 3.72 (s, 6H), 3.15 (d, J = 7.9 Hz, 1H), 2.52 (t, J = 6.3 Hz, 3H), 2.15 (m, 2H), 1.47 (d, J = 5.0 Hz, 3H), 1.25 (t, J = 6.9 Hz, 3H). 1313C NMR (150 MHz, CDCl3): δ 172.5, 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 NO8 [M+H + , 488.2279; found, 488.2279.

[0047] 2.3 Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-3-methyl-4-(3-ethoxycarbonylbutoxy-4-methoxyphenyl)azetidin-2-one (2c)

[0048] Yellow oily liquid (2c), yield 86%. 1 1H NMR (400 MHz, CDCl3): δ 6.94 (d, J = 7.6 Hz, 1H), 6.87 (d, J = 7.6 Hz, 1H), 6.83 (s, 1H), 6.55 (s, 2H), 4.47 (s, 1H), 4.13 (q, J = 6.8 Hz, 2H), 3.97 (s, 2H), 3.86 (s, 3H), 3.76 (s, 3H), 3.72 (s, 6H), 3.15 (d, J = 6.8 Hz, 1H), 1.83 (s, 4H), 1.47 (d, J = 6.6 Hz, 3H), 1.26 (t, J = 6.8 Hz, 3H). 13 13C NMR (150 MHz, CDCl3): δ 172.8, 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 NO8 [M+H + , 502.2435; found, 502.2432.

[0049] 2.4 Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-3-methyl-4-(3-ethoxycarbonylpentyloxy-4-methoxyphenyl)azetidin-2-one (2d)

[0050] Yellow oily liquid (2d), yield 85%. 1 H NMR (400 MHz, CDCl3): δ 6.94 (d, J = 8.1 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.83 (s, 1H), 6.55 (s, 2H), 4.47 (s, 1H), 4.12 (q, J = 6.6 Hz, 2H), 3.95 (s, 2H), 3.86 (s, 3H), 3.76 (s, 3H), 3.71 (s, 6H), 3.15 (d, J = 7.0 Hz, 2H), 2.33 (t, J = 6.5 Hz, 2H), 1.83 (m, 2H), 1.69 (s, 2H), 1.47 (d, J = 6.9 Hz, 4H), 1.25 (t, J = 6.6 Hz, 3H). 13 C NMR (150 MHz, CDCl3): δ 172.9, 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 NO8 [M + H + , 516.2592; found, 516.2595.

[0051] Synthesis of Compounds (3a-d) in Example 3

[0052]

[0053] Add the raw material (0.1 mmol) and sodium hydroxide (40 mg, 1.0 mmol) into a 25 mL eggplant-shaped flask. After dissolving with dichloromethane (0.5 mL) and methanol (1.0 mL), add aqueous hydroxylamine solution (0.15 mL). React under ice bath conditions. After the raw material completely disappears, add water, adjust the pH to 6 with acetic acid, extract three times with ethyl acetate, combine the organic phases, wash once with saturated brine, and dry over anhydrous sodium sulfate. Filter, concentrate by rotary evaporation, and separate by silica gel column chromatography (CH2Cl2 / MeOH 20:1).

[0054] 3.1 Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-3-methyl-4-[3-(N-hydroxycarbamoylmethoxy)-4-methoxyphenyl]azetidin-2-one (3a)

[0055] Pale yellow solid (3a), yield 79%. mp 86 - 88 °C. [α] D 20 = +68.5 (c 1.0, CHCl3). 1 1H NMR (400 MHz, CDCl3): δ 9.72 (s, 1H), 7.04 (d, J = 7.9 Hz, 1H), 6.90 (d, J = 7.8 Hz, 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 = 6.3 Hz, 1H), 1.44 (d, J = 6.8 Hz, 3H). 13 13C NMR (150 MHz, CDCl3): 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 N2O8 [M + H + , 447.1762; found, 447.1765.

[0056] 3.2 Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-3-methyl-4-[3-(N-hydroxycarbamoylpropoxy)-4-methoxyphenyl]azetidin-2-one (3b)

[0057] Pale yellow solid (3b), yield 65%. mp 82 - 83 °C. [α] D 20 = +56.7 (c 1.0, CHCl3). 11H NMR (400 MHz, CDCl3): δ 6.97 (d, J = 8.0 Hz, 1H), 6.87 (d, J = 8.0 Hz, 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 = 7.2 Hz, 3H). 13 13C NMR (150 MHz, CDCl3): δ 169.6, 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 + [[ID=**4**]]). ESI-HRMS (m / z): calcd for C 24 H 31 N2O8 [M + H + , 475.2075; found, 475.2079.

[0058] Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-3-methyl-4-[3-(N-hydroxyamidobutoxy)-4-methoxyphenyl]azetidin-2-one (3c)

[0059] Pale yellow solid (3c), yield 45%. mp 66 - 68 °C. [α] D 20 = +49.6 (c 1.0, CHCl3). 1 1H NMR (400 MHz, CDCl3): δ 6.94 (d, J = 8.1 Hz, 1H), 6.86 (d, J = 8.1 Hz, 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 = 7.3 Hz, 3H). 13 Note: There seems to be a formatting issue in the original text where the "C NMR" part has some text split across lines in an unusual way. I've tried to make sense of it in the translation. Also, the "calcd forC H N2O8" in the original seems to be missing some characters between "C" and "H", which I've left as is in the translation for the sake of following the rules. If this is a critical error in the original, it might need to be corrected in the source before translation.13C NMR (150 MHz, CDCl3): δ 167.9, 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 N2O8 [M+H + , 489.2231; found, 489.2230.

[0060] 3.4 Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-3-methyl-4-[3-(N-hydroxyamidopentoxy)-4-methoxyphenyl]azetidin-2-one (3d)

[0061] Pale yellow solid (3d), yield 42%. mp 64 - 66 °C. [α] D 20 = +42.3 (c 1.0, CHCl3). 1 1H NMR (400 MHz, CDCl3): δ 6.93 (d, J = 8.1 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 6.82 (s, 1H), 6.54 (s, 2H), 4.46 (d, J = 2.1 Hz, 1H), 3.95 (t, J = 6.6 Hz, 2H), 3.86 (s, 3H), 3.75 (s, 3H), 3.71 (s, 6H), 3.17 - 3.12 (m, 1H), 2.37 (t, J = 7.2 Hz, 2H), 1.82 (q, J = 6.7 Hz, 2H), 1.68 (q, J = 7.7 Hz, 2H), 1.54 - 1.50 (m, 2H), 1.46 (d, J = 7.3 Hz, 2H). 13 13C NMR (150 MHz, CDCl3): δ 178.4, 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 N2O8 [M+H+ ,503.2388; found,503.2388..

[0062] Synthesis of the target compound (5a - b) in Example 4

[0063]

[0064] Add 4-(N-benzyloxycarbonylamino)benzoic acid (81 mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol) and EDCI (57 mg, 0.3 mmol) into a 25 mL eggplant-shaped flask, and dissolve them with DMF (1.5 mL). Stir at room temperature for 6 hours, then add the raw material (0.1 mmol) and triethylamine (30 mg, 0.3 mmol), and continue to stir the reaction at room temperature for 2 hours. After the raw material completely disappears, add water, extract three times with ethyl acetate, combine the organic phases, wash once with saturated brine, and dry over anhydrous sodium sulfate. Filter, concentrate by rotary evaporation, and then rapidly separate by silica gel column chromatography (PE / EA 1:1). Then add 10% Pd / C (3 mg) and ethanol (1 mL), and react at room temperature under normal pressure hydrogen for 12 hours. Filter to remove Pd / C and evaporate to dryness, and purify by silica gel (300 - 400 mesh) column chromatography.

[0065] 4.1 Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-3-[4-(N-hydroxycarbonylamino)benzoyloxymethyl]azetidin-2-one (5a)

[0066] Red solid (5a), yield 68%. mp 136 - 140 °C. [α] D 20 = +13.6 (c 1.0, CHCl3). 1 H NMR (400 MHz, DMSO-d6): δ 11.40 (s, 1H), 9.19 (s, 1H), 9.12 (s, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.82 (d, J = 7.9 Hz, 2H), 6.92 (m, 2H), 6.86 (m, 1H), 6.55 (s, 2H), 5.13 (d, J = 2.2 Hz, 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, CHCl3). 1313C NMR (150 MHz, DMSO-d6): δ 164.7, 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 N2NaO 10 [M + Na + , 575.1636; found, 575.1631.

[0067] Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-3-[4-(N-hydroxyformamido)benzamido]methylazetidin-2-one (5b)

[0068] Red solid (5b), yield 72%. mp 154 - 156 °C. [α] D 20 = +14.6 (c 1.0, CHCl3). 1 1H NMR (400 MHz, DMSO-d6): δ 11.29 (s, 1H), 9.09 (s, 1H), 9.05 (s, 1H), 8.86 (s, 1H), 7.83 - 7.77 (m, 4H), 6.84 (d, J = 8.3 Hz, 1H), 6.75 (d, J = 8.3 Hz, 2H), 6.47 (s, 2H), 4.92 (d, J = 2.1 Hz, 1H), 3.78 - 3.68 (m, 3H), 3.68 (s, 3H), 3.58 (s, 6H), 3.52 (s, 3H). 13 13C NMR (150 MHz, DMSO-d6): δ 165.9, 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 N3NaO9 [M + Na +,574.1796; found,574.1816.

[0069] Synthesis of Target Compound (6a - b) in Example 5

[0070]

[0071] The synthesis method is similar to that of 5a - b.

[0072] 5.1 Synthesis of (3S,4R)-1-(3,4,5 - trimethoxyphenyl)-4-(3 - hydroxy - 4 - methoxyphenyl)-3 - [4-(N - hydroxymethanamido)propanoyloxy]methylazetidin - 2 - one (6a)

[0073] Pale green oily liquid (6a), yield 40%. [α] D 20 = +34.2 (c 1.0, CHCl3). 1 1H NMR (400 MHz, DMSO - d6): 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 13C NMR (150MHz, DMSO - d6): 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 N2NaO 10 [M + Na + , 527.1636; found, 527.1642.

[0074] 5.2 Synthesis of (3S,4R)-1-(3,4,5 - trimethoxyphenyl)-4-(3 - hydroxy - 4 - methoxyphenyl)-3 - [4-(N - hydroxymethanamido)butanoyloxy]methylazetidin - 2 - one (6b)

[0075] Red oily liquid (6b), yield 45%. [α] D 20= +29.7 (c 1.0, CHCl3). 1 1H NMR (400 MHz, DMSO-d6): δ 6.87 (d, J = 8.3 Hz, 1H), 6.83 (d, J = 8.3 Hz, 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 13C NMR (150 MHz, DMSO-d6): δ 172.2, 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 N2NaO 10 [M+Na + , 540.1714; found, 540.1718.

[0076] Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-3-[4-(N-hydroxyformamido)propanamido]methylazetidin-2-one (6c)

[0077] Black solid (6c), yield 43%. mp 118 - 120 °C. [α] D 20 = +32.7 (c 1.0, CHCl3). 1 1H NMR (400 MHz, DMSO-d6): δ 6.85 - 6.81 (m, 2H), 6.59 (s, 1H), 6.48 (s, 2H), 4.85 (d, J = 2.2 Hz, 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). 1313C NMR (150 MHz, DMSO-d6): δ 172.5, 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 N3NaO9 [M + Na + , 526.1796; found, 526.1793.

[0078] Synthesis of (3S,4R)-1-(3,4,5-trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-3-[4-(N-hydroxyformamido)butanamido]methylazetidin-2-one (6d)

[0079] Pale yellow oily liquid (6d), yield 50%. [α] D 20 = +34.8 (c 1.0, CHCl3). 1 1H NMR (400 MHz, DMSO-d6): δ 8.84 (s, 2H), 7.44 (s, 1H), 7.26 (d, J = 8.2 Hz, 1H), 7.06 (s, 2H), 6.92 (d, J = 7.4 Hz, 1H), 5.45 (d, J = 2.2 Hz, 1H), 4.54 (q, J = 7.1 Hz, 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 = 7.1 Hz, 2H). 13 13C NMR (150 MHz, DMSO-d6): δ 175.4, 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 N3NaO9 [M + Na + , 540.1953; found, 540.1949.

[0080] Synthesis of Target Compound (7g-h) in Example 6

[0081]

[0082] 6.1 Synthesis of Ethyl (S)-2-[1-(3-chloro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenylamino)methyl]acrylate (7c)

[0083] The synthesis procedure referred to 1c, and a yellow oily liquid (7c) was obtained with a yield of 65%. [α] D 20 = +70.5 (c 0.17, CHCl3). 1 H NMR (400 MHz, CDCl3): δ 7.37 (d, J = 2.1 Hz, 1H), 7.24 (dd, J = 8.8, 2.1 Hz, 1H), 6.89 (d, J = 8.8 Hz, 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 = 7.1 Hz, 3H). 13 C NMR (150 MHz, CDCl3): δ 165.5, 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 ClNO6 [M+H + , 436.1521; found 436.1523.

[0084] 6.2 Synthesis of (S)-1-(3,4,5-trimethoxyphenyl)-4-(3-chloro-4-methoxyphenyl)-3-methylideneazetidin-2-one (7d)

[0085] The synthesis procedure referred to 1d, and a white solid (7d) was obtained with a yield of 68%. mp 119 - 120 °C. [α] D 20= +35.1 (c 0.29, CHCl3), 97% ee [determined by HPLC analysis using a Chiralcel AD-H column; n-Hex / i-PrOH = 80:20, 1.0 mL / min, 254 nm; t R (minor) = 10.62 min; t R (major) = 13.97 min]. 1 1H NMR (400 MHz, CDCl3): δ 7.41 (d, J = 2.0 Hz, 1H), 7.25 (dd, J = 8.5, 2.0 Hz, 1H), 6.92 (d, J = 8.5 Hz, 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 13C NMR (150 MHz, CDCl3): δ 160.1, 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 ClNO5 [M+H + . 390.1103; found 390.1103.

[0086] Synthesis of (S,E)-1-(3,4,5-trimethoxyphenyl)-4-(3-(ethyl acryloyl)-4-methoxyphenyl)-3-methylideneazetidin-2-one (7e)

[0087] The synthetic method is reported in the reference literature, and the specific operation is as follows: Add the raw material (0.15 mmol), ethyl acrylate (1.17 mmol), triethylamine (0.2 mL, 1.46 mmol) and N-methylpyrrolidone (1 mL) into a 15 mL screw-capped tube with a stopper. After purging with nitrogen to remove oxygen thoroughly, add palladium acetate (4.9 mg, 0.02 mmol) and tris(ortho-methylphenyl)phosphine (13.3 mg, 0.04 mmol). After sealing tightly, heat at 128 °C for 12 hours. After the reaction is completed, cool, add ethyl acetate, wash three times with water, wash the organic phase once with saturated brine, and dry over anhydrous sodium sulfate. After rotary evaporation and concentration, separate by silica gel column chromatography (PE / EA 2:1), and rotary dry to obtain the corresponding intermediate. Pale yellow oily liquid (7e), yield 46%. 1 H NMR (400 MHz, CDCl3): δ 7.93 (d, J = 16.2 Hz, 1H), 7.55 (s, 1H), 7.38 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.6 Hz, 1H), 6.59 (s, 2H), 6.55 (d, J = 16.2 Hz, 1H), 5.86 (s, 1H), 5.34 (s, 1H), 5.18 (s, 1H), 4.26 (q, J = 6.7 Hz, 2H), 3.90 (s, 3H), 3.77 (s, 3H), 3.74 (s, 6H), 1.34 (t, J = 6.7 Hz, 3H). 13 C NMR (150 MHz, CDCl3): δ 167.2, 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 NO7 [M+H + . 454.4912; found 454.4918.

[0088] Synthesis of (S,E)-1-(3,4,5-trimethoxyphenyl)-4-(tert-butyl 3-butenoate-4-methoxyphenyl)-3-methylideneazetidin-2-one (7f)

[0089] The synthetic method refers to 7e, pale yellow oily liquid (7f), yield 48%. 11H NMR (400 MHz, CDCl3): δ 7.48 (s, 1H), 7.32 (m, 2H), 6.86 (d, J = 7.9 Hz, 1H), 6.73 (d, J = 16.2 Hz, 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 = 6.2 Hz, 1H), 1.47 (s, 9H). 13 13C NMR (150 MHz, CDCl3): δ 170.4, 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 NO7 [M + H + . 496.2330; found 496.2333.

[0090] Synthesis of (6.5(S,E))-1-(3,4,5-Trimethoxyphenyl)-4-(3-(N-hydroxyacrylamido)-4-methoxyphenyl)-3-methylidenetropane-2-one (7g)

[0091] Add the raw material 7e (0.1 mmol), sodium hydroxide (40 mg, 1.0 mmol), and hydroxylamine aqueous solution (0.15 mL) to a 25 mL eggplant-shaped flask, and dissolve them with dichloromethane (0.5 mL) and methanol (1.0 mL). React under an ice bath. After the raw material completely disappears, add water, adjust the pH to 6 with acetic acid, extract three times with ethyl acetate, combine the organic phases, wash once with saturated brine, and dry over anhydrous sodium sulfate. Filter, concentrate by rotary evaporation, and separate by silica gel column chromatography (CH2Cl2 / MeOH 20:1). Pale yellow solid (7g), yield 70%. mp 126 - 128 °C. [α] D 20 = +51.7 (c 1.0, CHCl3). 11H NMR (400 MHz, CDCl3): δ 7.86 (d, J = 15.7 Hz, 1H), 7.52 (s, 1H), 7.33 (d, J = 8.3 Hz, 1H), 6.86 (d, J = 8.3 Hz, 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 13C NMR (150 MHz, CDCl3): δ 165.2, 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 N2NaO7 [M + Na + , 463.1476; found, 463.1473.

[0092] Synthesis of (6.5(S,E))-1-(3,4,5-trimethoxyphenyl)-4-(3-(N-hydroxycrotonamido)-4-methoxyphenyl)-3-methylidenetropane-2-one (7h)

[0093] The synthesis method was referred to 7g. Pale red solid (7h), yield 56%. mp 110 - 113 °C. [α] D 20 = +41.8 (c 1.0, CHCl3). 1 1H NMR (400 MHz, DMSO-d6): δ 10.51 (s, 1H), 8.80 (s, 1H), 7.60 (s, 1H), 7.30 (d, J = 8.7 Hz, 1H), 7.00 (d, J = 8.7 Hz, 1H), 6.65 (d, J = 15.9 Hz, 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 = 6.2 Hz, 2H). 1313C NMR (150 MHz, DMSO-d6): 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 N2NaO7 [M+Na + . 477.1356; found 477.1352..

[0094] Synthesis of Example 7 (2R,3R,E)-1-(3,4,5-trimethoxyphenyl)-4-(3-(N-hydroxyacrylamido)-4-methoxyphenyl)-3-methylazetidin-2-one (8a)

[0095]

[0096] The synthesis method refers to 7g, light red solid (8a), total yield 36%. mp 124 - 126 °C. [α] D 20 = +11.7 (c 1.0, CHCl3). 1 1H NMR (400 MHz, DMSO-d6): δ 7.83 (d, J = 15.6 Hz, 1H), 7.33 (s, 1H), 7.15 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.3 Hz, 1H), 6.56 (d, J = 15.6 Hz, 1H), 6.53 (s, 2H), 5.10 (d, J = 4.8 Hz, 1H), 3.79 (s, 3H), 3.74 (s, 3H), 3.67 (s, 6H), 3.64 - 3.61 (m, 1H), 0.83 (d, J = 7.9 Hz, 3H). 13 13C NMR (150 MHz, DMSO-d6): δ 168.7, 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 26N2NaO7[M+Na + .465.1632; found 465.1639.

[0097] Synthesis of Example 8 (3S,4R)-1-(3,4,5-trimethoxyphenyl)-4-(3-hydroxy-4-methoxyphenyl)-3-[4-carboxybenzoyloxy]methylazetidin-2-one (9a)

[0098]

[0099] The synthesis method refers to 5a, light yellow solid (9a), total yield 46%. mp 120 - 122 °C. [α] D 20 = +21.7 (c 1.0, CHCl3). 1 1H NMR (400 MHz, CDCl3): δ 8.10 - 8.05 (m, 4H), 6.96 (s, 1H), 6.87 (d, J = 8.1 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 6.56 (s, 2H), 4.88 (d, J = 2.1 Hz, 1H), 4.78 (m, 2H), 3.88 (s, 3H), 3.77 (s, 3H), 3.71 (s, 6H), 3.54 (m, 1H). 13 13C NMR (400 MHz, CDCl3): δ 164.8, 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.

[0100] Example 9 In vitro tumor cell proliferation inhibition activity detection experiment

[0101] Tumor cells in the logarithmic growth phase were treated and seeded into 96-well plates. They were cultured for 24 hours at 37°C and 5% CO2. Six gradient concentrations of the test compound were added, and CA-4 was used as a positive control. Culture was continued under the same conditions for 48 hours, and then MTT was added. After 4 hours of culture, the supernatant containing MTT was discarded, and DMSO was added to each well. The purple crystals were dissolved by shaking. The OD value was then measured at 490nm or 540nm on a microplate reader, and the inhibition rate was calculated. The half-maximal inhibitory concentration (IC50) of the compound was 100%. 50 The inhibitory values (values) were calculated based on the inhibition rates at six concentrations. Three replicate wells were set for each concentration gradient, and the assay was repeated three times. The activity results are shown in Tables 1 and 2.

[0102] Table 1 Inhibitory activity of compounds on tumor cell proliferation (IC 50 / μM)

[0103]

[0104]

[0105]

[0106] Notes: a The antitumor activity was determined by the MTT assay, and the data are the average of three measurements; b BE-(2)-C is a human neuroblastoma cell line; c A549 is a human lung cancer cell line; d U87MG is a human malignant glioblastoma cell line; e HCT-116 is a human colon cancer cell line.

[0107] Table 2 Inhibitory activity of compound 5a on tumor cell proliferation (IC 50 / μM)

[0108]

[0109] Notes: a The antitumor activity was determined by the MTT assay, and the data are the average of three measurements; b HeLa is a human cervical cancer cell line; c A2780 is a human ovarian cancer cell line; d MDA-MB-231 is a human breast cancer cell line; e HUH-7 is a human hepatocellular carcinoma 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.

[0110] Example 10 In Vitro Tubulin Self-Assembly Experiment

[0111] The inhibitory effect of the preferred compound 5a on microtubule aggregation in vitro was tested by turbidimetry. The detection kit was purchased from Cytoskeleton, Inc., USA. The operation steps were as follows: The microtubule aggregation system (0.1 M PIPES, 10 mM MgCl2, 1 mM GTP, 1 mM EGTA, and 3.4 M glycerol) with pH = 6.6 was pre-incubated on ice in advance. Different concentrations of the test compound 5a were added, and the Colchicine treatment group was set as the positive control and the DMSO (4%, v / v) treatment group was set as the negative control. After adding tubulin (10 mM) to the above system, it was immediately transferred and placed at 37 °C for the aggregation reaction while keeping the temperature constant. The absorbance was measured at 340 nm every 1 minute using a spectrophotometer (Synergy H4 Hybrid) for a total of 30 minutes, and then an absorbance curve was plotted (as Figure 1 shown). The results showed that compound 5a could significantly inhibit microtubule aggregation, and its IC 50 was 5.4 μM (as shown in Table 3).

[0112] Table 3. In Vitro Experiment on the Inhibition of Microtubule Self-Assembly by Compound 5a

[0113]

[0114] Example 11 In Vitro HDAC Inhibitory Activity Test

[0115] The inhibitory activity of the compound on HDACs was tested by enzyme-linked immunofluorescence assay. All enzymatic reactions were carried out at 37 °C for 30 minutes. The compound was diluted to different concentration ranges with 5% DMSO. 5 μL of the compound solution was added to 50 μL of the reaction mixture containing 25 mM Tris, 1 mM MgCl2, 0.1 mg / ml BSA, 137 mM NaCl, 2.7 mM KCl, HDAC, and its substrate at pH = 8.0. After the enzymatic reaction was completed, the fluorescence intensity was detected on a SpectraMax M5 microplate reader at an excitation wavelength of 350 - 360 nm and an emission wavelength of 450 - 460 nm. Finally, the IC 50 value of the test compound was calculated by non-linear regression fitting after normalization using Prism GraphPad software. The test results are shown in Table 4. Both 5a and 6a could significantly inhibit the activity of HDAC8, and their IC 50The values are 0.177 μM and 0.288 μM respectively. At the same time, both of them have similar inhibitory activities against HDAC1 as HDAC8, while the inhibitory activities against HDAC6 decrease by 6-fold and 8-fold respectively, indicating that these two compounds are class I HDAC inhibitors.

[0116] Table 4 Inhibitory activities of compounds against HDAC1 / 6 / 8 (IC 50 / μM)

[0117]

[0118]

[0119] Note: a IC 50 All are the averages of three measurements; b SAHA is a non-selective HDACs inhibitor; c PCI-34051 is a selective HDAC8 inhibitor; NT = not tested.

[0120] Example 12 Detection of HDAC-related protein expression

[0121] Take BE-(2)-C cells in the logarithmic growth phase. After treatment, inoculate them in 6-well plates at a density of 2×10 5 cells / well, and culture them in an incubator at 37°C, 5% CO2 and saturated humidity for 24 hours. After adding gradient concentrations of compound 5a to treat the tumor cells (set the DMSO treatment group as the negative control at the same time), collect the cells after 24 hours and lyse them with lysis buffer. After the protein samples are heated and denatured, load them onto a polyacrylamide gel, separate by SDS-PAGE electrophoresis, transfer by wet method, block, and then react with the primary antibody and the secondary antibody in turn, and then expose and develop the color. The results are as Figure 2 shown. Compound 5a can significantly promote the acetylation of the HDAC8 substrate protein SMC3 and the HDAC1 substrate protein histone H3, while having no obvious effect on the acetylation of the HDAC6 substrate protein α-tubulin, indicating that the preferred compound 5a can inhibit HDAC1 and HDAC8.

[0122] Example 13 In vitro cell cycle arrest experiment

[0123] Take BE-(2)-C cells in the logarithmic growth phase. After treatment, inoculate them in 6-well plates at a density of 2×10 5The cells were seeded in 6-well plates at a density of 2×10 cells / well and cultured in an incubator at 37 °C, 5% CO2 and saturated humidity for 12 hours. After the cells adhered to the plate, the fresh culture medium was replaced, and the cells were treated with compound 5a at different concentrations for 24 hours. At the same time, the DMSO treatment group was set as the negative control. The supernatant was discarded, and the adherent cells were collected, rinsed twice with PBS, fixed with 75% ethanol, fixed overnight at -20 °C, stained with PI, and then tested by flow cytometry. The results showed that compound 5a could significantly arrest the tumor cell cycle at the G2 / M phase (as Figure 3 shown).

[0124] Example 14 Detection of cell cycle-related proteins in vitro

[0125] BE-(2)-C cells in the logarithmic growth phase were taken, and after treatment, they were seeded in 6-well plates at a density of 2×10 5 cells / well and cultured in an incubator at 37 °C, 5% CO2 and saturated humidity for 24 hours. After adding gradient concentrations of compound 5a to treat the tumor cells (while setting the DMSO treatment group as the negative control), the cells were collected and lysed with lysis buffer after 48 hours. After the protein samples were heated and denatured, they were loaded onto a polyacrylamide gel, separated by SDS-PAGE electrophoresis, transferred by wet transfer, blocked, and then reacted with the primary antibody and the secondary antibody in sequence, and then developed by exposure. The results were as Figure 4 shown. Compound 5a could significantly promote the expression of mitotic checkpoint protein Bubr-1, phosphorylated histone P-Histone 3, and cyclin B1.

[0126] Example 15 In vitro induction of apoptosis experiment

[0127] BE-(2)-C cells in the logarithmic growth phase were taken, and after treatment, they were seeded in 6-well plates at a density of 2×10 5 cells / well and cultured in an incubator at 37 °C, 5% CO2 and saturated humidity for 24 hours. Gradient concentrations of compound 5a were added, and the DMSO treatment group was set as the negative control. After continuous culture for 48 hours, the supernatant cells and adherent cells were collected, double-stained with PI and Annexin V, and detected by flow cytometry. The results were as Figure 5 shown. Compound 5a could induce apoptosis in a concentration-dependent manner.

[0128] Example 16 Detection experiment of apoptosis-related proteins in vitro

[0129] BE-(2)-C cells in the logarithmic growth phase were taken, and after treatment, they were seeded in 6-well plates at a density of 2×10 5The cells were seeded in a 6-well plate at a density of Figure 6 per well and cultured in an incubator at 37 °C, 5% CO2 and saturated humidity for 24 hours. Then, compounds 5a at gradient concentrations were added, and a DMSO-treated group was set as the negative control. After 48 hours, the cells were collected and lysed with lysis buffer. The samples were loaded onto a polyacrylamide gel, separated by SDS-PAGE electrophoresis, transferred by wet transfer, blocked, reacted with primary antibody and then secondary antibody in sequence, and developed by exposure. The results are shown as

[0130] Example 17 Colony Formation Inhibition Assay

[0131] BE-(2)-C cells in the logarithmic growth phase were harvested, seeded in a 6-well plate at a density of 1500 cells per well after treatment, and cultured at 37 °C, 5% CO2 and saturated humidity for 24 hours. After the cells adhered, different concentrations of compound 5a were added to treat the cells for 48 hours, and a DMSO-treated group was set as the negative control. The medium was replaced with fresh medium and the cells were cultured for another two weeks. Then, the supernatant was discarded, and the cells were washed twice with PBS, fixed with absolute methanol for 30 minutes, stained with crystal violet dye for 1 hour, the staining solution was washed off, and after being thoroughly dried, the number of colonies formed by more than 50 cells was counted under a microscope. The results showed that compound 5a could significantly inhibit the colony formation of HeLa cells (as shown in Figure 7 )

Claims

1. A novel chiral diaryl-β-lactam HDAC / Tubulin bifunctional inhibitor of general formula I structure or a pharmaceutically acceptable salt thereof, Among them, R 1 With R 2 Selected from a hydrogen atom, a methoxy group, a halogen atom, an amino group, a hydroxyl group, or a combination of 2 to 3 of the same or different groups above; L is Where n is selected from 1, 2, 3, 4, 5, 6, 7; m is selected from 0, 1; R 3 Is R 4 Selected from a hydrogen atom, a methyl group, a methylene group.

2. The novel chiral diaryl-β-lactam HDAC / Tubulin bifunctional inhibitor according to claim 1, wherein the compound is:

3. The novel chiral diaryl-β-lactam HDAC / Tubulin bifunctional inhibitor or its pharmaceutically acceptable salt according to claim 1, characterized in that, A novel chiral diaryl-β-lactam HDAC / Tubulin bifunctional inhibitor of the following general formula II structure, Among them, R 1 and R 2 are selected from a hydrogen atom, a methoxy group, a halogen atom, an amino group, a hydroxyl group, or a combination of 2 to 3 of the same or different groups above; X is selected from an ester group, an amide group; L is selected from where n is selected from 1, 2, 3, 4, 5, 6, 7; L can also be R 3 is 4. The novel chiral diaryl-β-lactam HDAC / Tubulin bifunctional inhibitor according to claim 3, wherein the preferred compound is:

5. Use of the compound according to any one of claims 1-4 and a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and treating diseases related to tumors, said diseases being neuroblastoma, interstitial sarcoma, choriocarcinoma, malignant mole, thyroid cancer, head and neck squamous cell carcinoma, cervical cancer, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, gastric cancer, lung cancer, liver cancer, melanoma, lymphoma, glioma, nasopharyngeal cancer, neuroendocrine cancer, undifferentiated cancer, malignant teratoma, and benign tumors; the "pharmaceutically acceptable salt" is a salt formed with malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid, oxalic acid, and phosphoric acid, hydrohalic acid, sulfuric acid, and nitric acid.

6. A compound drug for preventing and treating tumor-related diseases, characterized in that, It contains the compound according to any one of claims 1-4, and the diseases related to tumors 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, kidney cancer, undifferentiated cancer, interstitial sarcoma, choriocarcinoma, gastric cancer, bladder cancer, glioma, nasopharyngeal cancer, neuroendocrine cancer, malignant mole, malignant teratoma.

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