Macrocyclic GPX4 inhibitor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drug synthesis, and particularly relates to a preparation method and application of a macrocyclic GPX4 inhibitor. The structure of the macrocyclic GPX4 inhibitor disclosed by the invention is shown as a formula (I). Experiments prove that the compound has strong GPX4 inhibitory activity and ferroptosis-inducing activity, can efficiently induce ferroptosis of ferroptosis-sensitive tumor cell strains, and has activity obviously superior to that of the existing inhibitor ML162. The method has scientific research tool value and clinical application prospect, and can be used for mechanism research of ferroptosis-related diseases and preparation of medicines for treating diseases such as tumors.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis technology, specifically relating to a macrocyclic GPX4 inhibitor, its preparation method, and its application. Background Technology

[0002] In recent years, a large amount of research has focused on various non-classical pathways regulating cell death, primarily concentrating on oxidative cell damage. Ferroprelation (FRT) is a ferrous ion-dependent regulated cell death mechanism, first proposed by Brent R. Stockwell in 2012. Compared to apoptosis, autophagy, and necrosis, ferroptosis possesses unique ultrastructural features, metabolic regulatory networks, and molecular effector mechanisms. This redox-active process plays a crucial role in various pathological conditions, including malignant tumors, acute kidney injury (AKI), cardiovascular disease, neurodegenerative diseases, and the progression of liver disease. Notably, ferroptosis is also a key regulatory node in the tumor drug resistance microenvironment. In the initial years following the discovery of ferroptosis, the mechanisms controlling it were elucidated primarily around cysteine ​​and glutathione metabolism, and the phospholipid peroxidase GPX4's prevention of lipid peroxidation accumulation. Ferroprelation inducers act directly or indirectly on glutathione peroxidases (GPXs) through different pathways, leading to decreased cellular antioxidant capacity, ROS accumulation, and ultimately oxidative cell death. The complex interactions between lipid, iron, and cysteine ​​metabolism have become important regulators of this cell death pathway. Recently, designing novel ferroptosis inducers by targeting GPX4 has emerged as an attractive strategy for the treatment of drug-resistant tumors.

[0003] GPX4 is a key regulator of ferroptosis and a potential target for inducing ferroptosis in cancer therapy. Current research indicates that directly targeting GPX4 is more effective in inducing ferroptosis for cancer treatment. ML162, a representative GPX4 inhibitor, covalently binds to the Sec46 molecule of GPX4 via its chloroacetone fragment, inactivating GPX4 and exhibiting anti-tumor efficacy at the cellular level. However, because ML162 activity only moderately inhibits GPX4 protein activity, its efficacy on the ferroptosis-sensitive cell line HT1080 is limited, restricting its clinical application.

[0004] Therefore, the search for potent GPX4 inhibitors to induce ferroptosis in tumor cells has significant practical and economic value. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a macrocyclic GPX4 inhibitor, its preparation method, and its application. The compound designed in this invention has the structure shown in formula (I). Compounds with this structure possess excellent GPX4 inhibitory activity and ferroptosis-inducing activity, and can potently induce ferroptosis in ferroptosis-sensitive tumor cell lines, demonstrating significant scientific research value and clinical application prospects.

[0006] The technical solution of this invention is: This invention provides a macrocyclic GPX4 inhibitor, the structure of which is shown in formula (I): Or, an enantiomer of formula (I), a mixture of enantiomers, a diastereomer, a mixture of diastereomers, a tautomer, a mixture of tautomers, or an isotopic variant; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof; Among them, R 1 Selected from or –CH2 R 2b or or , where R 2a It is hydrogen or C1 C6 alkyl; and R 2b It is a halogen; R 2 Selected from C5-C 10 Aromatic heterocyclic or ferrocene-based; R 3 Selected from benzene rings or C1-C6 alkyl groups; R 4 Selected from C1-C8 alkyl, C4-C6 heterocyclic alkyl, and alkyl in which the carbon atom is replaced by one or more heteroatoms; R 5 Selected from amide or olefin groups and R 3 and R 4 Fragment concatenation; Each R 3 and R 4 All were independently selected from the above fragments and associated with R. 5 connect.

[0007] Preferably, the specific structural formulas of the macrocyclic GPX4 inhibitor compounds A1-A26 and B1-B14 are as follows: .

[0008] Furthermore, the present invention provides a method for preparing a macrocyclic GPX4 inhibitor of formula (I), wherein R 5 Amide group and R 3 and R 4 During fragment ligation, the compound of formula (I) obtained at this time is IA, and the synthetic route is as follows: The preparation method of compound IA includes the following steps: Compound I undergoes a nucleophilic elimination reaction with methyl formate to give compound II; compound II undergoes an oxidation reaction with phosphorus oxychloride to give compound III; compound IV undergoes a nucleophilic elimination reaction with di-tert-butyl dicarbonate ((BOC)₂O) to give compound V; compound V undergoes a nucleophilic substitution reaction with a brominated derivative (containing a BOC protecting group) to give compound VI; compound VI undergoes BOC removal under acidic conditions (TFA:DCM = 1:3), followed by a selective nucleophilic elimination reaction with (BOC)₂O to give compound VII; compound III undergoes a four-component ugi reaction with compounds VIII, VII, and IX to give compound X; compound X undergoes the removal of tert-butyl and tert-butyloxycarbonyl (BOC) groups under acidic conditions (TFA:DCM = 1:3) to give compound XI; compound XI undergoes an intramolecular condensation reaction to give the final compound IA.

[0009] Furthermore, the specific preparation method of compound IA includes the following steps: (1) Compound I and methyl formate were dissolved in methanol and reacted at 60°C for 12 hours. After the reaction was completed, the mixture was separated and purified to obtain a colorless oil or a white solid, namely compound II. (2) Compound II and N,N-diisopropylethylamine (DIPEA) were added to dry dichloromethane, and phosphorus oxychloride was added at -80°C. The reaction was continued at this temperature for 1 hour. After the reaction was completed, the compound III was obtained by separation and purification. (3) Compound IV and (BOC)2O were added to dry tetrahydrofuran and refluxed for 12 hours. After the reaction was completed, the mixture was separated and purified to obtain white solid compound V. (4) Compound V, the bromine-substituted product (containing the BOC protecting group) and potassium carbonate were added to NMP and reacted at 55°C for 3 hours. After the reaction was completed, the compound VI was obtained by separation and purification. (5) Compound VI was added to a mixed solution (TFA:DCM = 1:3), reacted for 1 hour, the solvent was removed by rotary evaporation, (BOC)2O and DIPEA were added, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the white solid compound VII was obtained by separation and purification. (6) Compound VIII and Compound VII were added to methanol and reacted at 55°C for 1 hour. Then the mixture was moved to room temperature and added to Compound III and Compound IX. The mixture was reacted at room temperature for 10 hours. After the reaction was completed, the mixture was separated and purified to obtain solid compound X. (7) Compound X was added to a mixed solution (TFA:DCM = 1:3), reacted for 2 hours, and the solvent was removed by rotation to obtain compound XI; (8) Compounds XI, BOP and DIPEA were dissolved in dichloromethane and reacted at room temperature for 24 hours. After the reaction was completed, the compounds were separated and purified to obtain a white solid compound IA.

[0010] Preferably, the specific method for separation and purification in step (2) is as follows: after the reaction is completed, a small amount of water is added to quench the residual phosphorus oxychloride, the mixture is moved to room temperature, water is removed by rapid filtration, the mixture is concentrated by dichloromethane layer, wet sample loading is performed, and rapid column chromatography is performed to obtain colorless oily substance III.

[0011] Preferably, the specific method for separation and purification in step (5) is as follows: after the reaction is completed, the mixed reaction solution is poured into a saturated ammonium chloride aqueous solution, extracted with dichloromethane, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain intermediate compound VII.

[0012] Preferably, the specific method for separation and purification in step (6) is as follows: after the reaction is completed, the solvent is evaporated under vacuum, ethyl acetate is added to dissolve it, the organic layer is washed with saturated ammonium chloride aqueous solution, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain intermediate compound X.

[0013] Preferably, the specific method for separation and purification in step (8) is as follows: after the reaction is completed, the organic layer is washed with water, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain a white solid compound IA.

[0014] Furthermore, the present invention provides a method for preparing a macrocyclic GPX4 inhibitor of formula (I), wherein R 5 For olefinic group and R 3 and R 4 When the fragments are joined, the compound of formula (I) obtained at this time is IB, and the synthetic route is as follows: The preparation method of the compound IB includes the following steps: Compound XII undergoes a nucleophilic addition-elimination reaction with (BOC)₂O to give compound XIII; compound XIII undergoes a nucleophilic substitution reaction with a brominated derivative (without the BOC protecting group) to give compound XIV; compound XIV undergoes BOC removal under acidic conditions (TFA:DCM = 1:3), followed by a nucleophilic addition-elimination reaction with methyl formate to give compound XV; compound XV undergoes oxidation with phosphorus oxychloride to give compound XVI; compound V undergoes a nucleophilic substitution reaction with a brominated derivative to give compound XVIII; compound XVIII undergoes BOC removal under acidic conditions (TFA:DCM = 1:3) to give compound XIX; compound IV undergoes a nucleophilic addition-elimination reaction with (BOC)₂O to give compound V; compound XVI undergoes a four-component reaction with compounds VIII, XIX, and IX to give compound XVII; compound XVII undergoes an olefin metathesis reaction to give the final compound IB; Preferably, R 3 Selected from C1-C6 alkyl groups; R 4 Selected from C1-C8 alkyl groups; n is selected from 0-3.

[0015] Furthermore, the specific preparation method of the compound IB includes the following steps: (1) Compound XII, (BOC)2O and DIPEA were added to dichloromethane and reacted at room temperature for 1 hour. After the reaction was completed, the mixture was separated and purified to obtain an oily compound XIII. (2) Compound XIII, the bromine-substituted product (containing olefin fragments) and potassium carbonate were added to N-methylpyrrolidone (NMP) and reacted at 55°C for 3 hours. After the reaction was completed, the product was separated and purified to obtain colorless oily compound XIV. (3) Compound XIV was added to a mixed solution (TFA:DCM = 1:3), reacted for 1 hour, the solvent was removed by rotary evaporation, methyl formate and methanol were added, and the reaction was carried out at 60°C for 12 hours. After the reaction was completed, the white solid compound XV was obtained by separation and purification. (4) Compound XV and DIPEA were added to dry dichloromethane, and phosphorus oxychloride was added at -80°C. The reaction was continued at this temperature for 1 hour. After the reaction was completed, the compound XVI was obtained by separation and purification. (5) Compound V, the bromine-substituted compound and potassium carbonate were added to NMP and reacted at 55°C for 3 hours. After the reaction was completed, the compound was separated and purified to obtain a colorless oily compound XVIII. (6) Compound XVIII was added to a mixed solution (TFA:DCM = 1:3) and reacted for 1 hour. After the reaction was completed, the compound was separated and purified to obtain a white solid compound XIX. (7) Compound VIII and compound XIX were added to methanol and reacted at 55°C for 1 hour. Then the mixture was moved to room temperature and added to compound XVI and compound IX. The mixture was reacted at room temperature for 10 hours. After the reaction was completed, the mixture was separated and purified to obtain solid compound XVII. (8) Compound XVII and metal catalyst were added to dry dichloromethane and refluxed for 48 hours. After the reaction was completed, the mixture was separated and purified to obtain solid compound IB. Preferably, the specific method for separation and purification in step (2) is as follows: after the reaction is completed, the reaction solution is diluted with ethyl acetate, the organic layer is washed with saturated ammonium chloride aqueous solution, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain intermediate XIV.

[0016] Preferably, the specific method for separation and purification in step (8) is as follows: after the reaction is completed, the organic layer is washed with water, the organic layer is dried and concentrated, and the mixture is separated by column chromatography to obtain a white solid compound IB.

[0017] Furthermore, the present invention also provides a pharmaceutical composition comprising the above-mentioned macrocyclic GPX4 inhibitor, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0018] In addition, the present invention provides the use of the above-mentioned macrocyclic GPX4 inhibitor or the above-mentioned pharmaceutical composition in the preparation of ferroptosis inducers.

[0019] Furthermore, the ferroptosis inducer is used to prepare a drug for treating ferroptosis-related diseases.

[0020] Furthermore, the ferroptosis-related disease is a disorder, disease, or condition mediated by glutathione peroxidase 4.

[0021] Furthermore, the ferroptosis-related diseases are tumors, including lung cancer, colon cancer, neuroblastoma, melanoma, breast cancer, stomach cancer, leukemia, etc.

[0022] Furthermore, the present invention also provides the use of the above-mentioned macrocyclic GPX4 inhibitor or the above-mentioned pharmaceutical composition in the preparation of antitumor drugs.

[0023] Compared with existing technologies, the macrocyclic GPX4 inhibitor provided by this invention has the following advantages: This invention provides a macrocyclic GPX4 inhibitor. This compound exhibits excellent GPX4 inhibitory activity and ferroptosis-inducing activity, potently inducing ferroptosis in ferroptosis-sensitive tumor cell lines. Its activity is significantly superior to the existing inhibitor ML162, and it can precisely target diseased cells to exert its effects, providing a highly effective core active ingredient for the treatment of ferroptosis-related diseases. Furthermore, this compound possesses dual value as a research tool and for clinical applications. It can serve as a specific tool for studying the mechanisms of ferroptosis-related diseases, while also possessing excellent drug-like properties and significant potential for drug development. It can also be used to prepare drugs for treating tumors and other ferroptosis-related diseases, demonstrating significant research value and promising clinical application prospects. Attached Figure Description

[0024] Figure 1 This is a surface plasmon resonance sensing image of the interaction between compound B14 and GPX4. Detailed Implementation

[0025] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the protection scope of the present invention.

[0026] For all the following embodiments, standard processing and purification methods known to those skilled in the art can be used. Unless otherwise stated, all temperatures are expressed in degrees Celsius. Unless otherwise stated, all reactions are carried out at room temperature. The synthetic methods shown in this invention are intended to illustrate applicable chemical methods through specific examples and do not represent the scope of the invention.

[0027] In the following examples, unless otherwise specified, the reagents are conventional reagents and can be purchased from conventional reagent manufacturers and distributors.

[0028] The symbols and conventions used in the processes, protocols, and examples, whether or not specific abbreviations are specifically defined in this invention, are consistent with the symbols and conventions used in contemporary scientific literature, such as the Journal of the American Chemical Society, the Journal of Medicinal Chemistry, or the Journal of Biological Chemistry. Specifically, and not limited to, the following abbreviations may be used in the examples and throughout the specification: g (gram); mg (milligram); mL (milliliter); vL (microliter); mM (millimoles per liter); μM (micromoles per liter); mmol (millimoles); h (hours); min (minutes); EtOH (ethanol); MeOH (methanol); EtOAc (ethyl acetate); prep TLC (Preparative Thin-Layer Chromatography); LCMS (Liquid Chromatography) Mass spectrometry (MS / MS) and NMR (nuclear magnetic resonance).

[0029] This invention provides a macrocyclic GPX4 inhibitor, which is a compound with the structural formula shown in formula (I): (1) This invention provides a method for preparing a macrocyclic GPX4 inhibitor (I), wherein when the R 5 Amide group and R 3 and R 4 During fragment ligation, the compound of formula (I) obtained at this time is IA, and the synthetic route is as follows: The reagents and reaction conditions were as follows: (a) MF, 60℃, 12 h; (b) POCl3, DIPEA, dry-DCM, -80℃, 1 h; (c) (Boc)2O, reflux, 12 h; (d) K2CO3, NMP, 55℃, 3 h; (e) TFA:DCM = 3:1, 1 h, rt; (Boc)2O, DCM, DIPEA, 1 h, rt; (f) MeOH, rt, 10 h; (g) TFA:DCM = 3:1, 2 h, rt; (h) BOP, DIPEA,DCM, rt, 24 h. (2) This invention provides a method for preparing a macrocyclic GPX4 inhibitor (I), wherein R 5 Since it is an olefin linker, the compound of formula (I) becomes IB, and the synthetic route is as follows: Among them, (i) (Boc)2O, DCM, rt, 1 h; (d) K2CO3, NMP, 55℃, 3 h; (j) TFA:DCM = 3:1, 1 h, rt; MF, 60℃, 12 h; (b) POCl3, DIPEA, dry-DCM, -80℃, 1 h; (c) (Boc)2O, reflux, 12 h; (d) K2CO3, NMP, 55℃, 3 h; (m) TFA:DCM = 3:1, 1 h, rt; (f) MeOH, rt, 10 h; (k) Grubbs 2nd, dry-DCM, 40℃, 48 h. Example 1: Preparation of 10-chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-12,18-dioxa-4,7,15-triazatricyclo[17.2.2.28,11] tetavidin-1(22),8(25),9,11(24),19(23),20-hexaene-5,16-dione A1 Compound A1 was synthesized according to the route shown in Scheme 1.

[0030] Compound 1 (0.685 g, 5 mmol) and methyl formate (1.80 g, 30 mmol) were dissolved in 5 mL of methanol and reacted at 60 °C for 12 hours. The reaction mixture was then concentrated under vacuum and purified by column chromatography to give colorless oily intermediate 2 (570 mg, yield 69%).

[0031] Intermediate 2 (0.28 g, 1.73 mmol) and tert-butyl α-bromoacetate (0.5 g, 2.59 mmol) were dissolved in 3 mL of NMP solution, and potassium carbonate (0.36 g, 2.59 mmol) was added. After stirring at 55 °C for 3 hours, the reaction mixture was poured into a saturated ammonium chloride aqueous solution and extracted with ethyl acetate. The organic phases were combined, washed sequentially with saturated sodium chloride aqueous solution, dried with anhydrous Na2SO4, and then concentrated under vacuum to obtain a crude product. This crude product was purified by column chromatography to obtain an intermediate (0.38 g, 80% yield). The intermediate (0.38 g, 1.38 mmol) and DIPEA (1.25 g, 9.66 mmol) were then dissolved in 36 mL of dry dichloromethane. The reaction mixture was pre-cooled at -80°C for 10 minutes, followed by the dropwise addition of phosphorus oxychloride (0.64 g, 4.15 mmol) diluted in 1.5 mL of dry dichloromethane over 30 minutes. After stirring for 1 hour, 2 mL of water was added to the reaction mixture, and stirring was continued for 5 minutes. The mixture was filtered to remove ice residue, and the organic phase was concentrated under vacuum. Rapid separation by column chromatography (PE:EA = 10:1) yielded a colorless oily intermediate 3, which was directly used in the next reaction (90% yield).

[0032] Compound 4 (2.94 g, 22.52 mmol) and (BOC)₂O (4.93 g, 22.6 mmol) were dissolved in 15 mL of dry tetrahydrofuran and refluxed for 12 hours. The reaction mixture was then concentrated under vacuum and purified by column chromatography to give intermediate 5 (3.18 mg, 58% yield) as a white solid.

[0033] Intermediate 5 (0.36 g, 1.5 mmol) and N -BOC-bromoethylamine (0.32 g, 1.42 mmol) was dissolved in 2 mL of NMP solution, and potassium carbonate (0.31 g, 2.25 mmol) was added. After stirring at 55 °C for 3 hours, the reaction mixture was poured into a saturated ammonium chloride aqueous solution and extracted with ethyl acetate. The combined organic phases were washed successively with saturated sodium chloride aqueous solution, dried over anhydrous Na₂SO₄, and then concentrated under vacuum to give a crude product. The crude product was purified by column chromatography to give a colorless oily intermediate 6 (480 mg, yield 83%).

[0034] Intermediate 6 (0.248 g, 0.864 mmol) was added to 4 mL of a mixed solution (TFA:DCM = 1:3), and the reaction was carried out for 1 hour. After removing the solvent by rotary evaporation, (BOC)₂O (0.20 g, 0.950 mmol) and DIPEA (1.12 g, 8.64 mmol) were added. White fumes were produced. After dissolving the fumes in 3 mL of dichloromethane, the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the intermediate 7 (160 mg, yield 66%) was obtained by separation and purification.

[0035] Intermediates 7 (0.16 g, 0.576 mmol) and 8 (0.06 g, 0.576 mmol) were dissolved in 4 mL of methanol and reacted at 55 °C for 1 h with stirring. The mixture was then allowed to return to room temperature. Intermediates 3 (0.15 g, 0.576 mmol) and 9 (0.04 g, 0.576 mmol) were added with stirring, and the mixture was reacted at room temperature for 10 h with stirring. The reaction mixture was concentrated under vacuum, reconstituted with ethyl acetate, washed successively with aqueous solution and saturated sodium chloride solution, dried over anhydrous Na₂SO₄, and then concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain a colorless oily intermediate 10 (262 mg, 64% yield).

[0036] Intermediate 10 (0.184 g, 0.258 mmol) was added to 4 mL of mixed solution (TFA:DCM = 1:3), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was then concentrated under vacuum to obtain intermediate 11, which was used directly in the next step of the reaction without purification.

[0037] BOP (0.17 g, 0.387 mmol) and DIPEA (0.33 g, 2.58 mmol) were added sequentially to the concentrated intermediate 11 (0.258 mmol), producing white fumes. Then, 70 mL of dichloromethane was added to dissolve the fumes. After stirring at room temperature for 24 hours, the mixture was washed with saturated ammonium chloride aqueous solution. The organic phases were then combined and washed sequentially with saturated sodium chloride aqueous solution. The mixture was dried over anhydrous Na₂SO₄ and then concentrated under vacuum to obtain the crude product. The crude product was purified by preparative TLC to give a white solid compound A1 (30 mg, yield 22%). A1: 1 H NMR (400 MHz, Chloroform- d ) δ 7.2 (dd, J = 5.2, 1.1 Hz, 1H),7.1 (s, 1H), 7.0 (d, J = 8.3 Hz, 2H), 6.8 – 6.8 (m, 2H), 6.7 (dd, J= 5.2, 3.6Hz, 1H), 6.6 (d, J = 8.8 Hz, 1H), 6.5 (s, 1H), 6.4 – 6.3 (m, 1H), 6.2 (s, 1H), 5.7 (s, 2H), 4.2 (dt, J = 10.5, 5.3 Hz, 1H), 4.1 – 4.0 (m, 1H), 3.7 – 3.6 (m,2H), 3.6 – 3.5 (m, 1H), 3.4 (d, J = 13.0 Hz, 1H), 3.2 (q, J = 7.5 Hz, 2H), 3.0(s, 1H), 2.9 (m, 1H), 2.6 (m, 1H). 13 C NMR (101 MHz, Chloroform- d ) δ 169.0,166.8, 156.2, 154.4, 133.8, 132.3, 131.8, 130.8, 130.6, 130.2, 130.0, 128.0,126.9, 122.4, 114.8, 112.8, 82.1, 67.6, 67.5, 57.5, 55.5, 43.5, 39.1, 36.7,33.8, 26.9, 18.5, 17.0, 12.8. The synthesis processes of compounds A1 to A12 and A15 to A26 are similar to those of compound A1. In the synthesis routes of compounds A13 to A14, starting material 1 is an amino fragment containing a tert-butyl carboxylate structure, which can be directly used to prepare isonitriles. The remaining synthesis steps are similar to those of compound A1.

[0038] 10-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-12,20-dioxa-4,7,17-triazatricyclic[19.2.2.28,11]hexadecano-1(24),8(27),9,11(26),21(25),22-hexaen-18-one A2: 1 H NMR (400 MHz, Chloroform- d ) δ 7.2 (dd, J = 5.1, 1.2 Hz, 1H), 7.1 (d, J = 8.6 Hz, 2H), 6.9 – 6.8 (m, 3H), 6.7 (dd, J= 5.2, 3.6 Hz, 2H), 6.5 (d, J = 8.8 Hz, 1H), 6.5(s, 1H), 6.3 (dd, J = 8.7, 2.5 Hz, 2H), 6.0 (s, 1H), 4.2 (d, J = 14.1 Hz, 1H), 4.1 (td, J = 6.4, 5.7, 3.3 Hz, 3H), 3.7 (s, 2H), 3.6 (dt, J = 12.3, 6.4 Hz, 1H), 3.5 – 3.4 (m, 1H), 3.0 (s, 1H), 2.8 (t, J = 6.2 Hz, 2H), 1.8 (m, 4H). 13 C NMR (101 MHz, Chloroform- d ) δ 167.9, 167.2, 155.6, 154.1, 153.7, 133.6, 131.8,131.7, 130.5, 130.3, 130.3, 129.7, 128.2, 126.1, 122.7, 114.8, 112.7, 81.8,68.3, 66.9, 57.1, 39.2, 38.6, 34.6, 26.8, 25.0. 10-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-12,21-dioxa-4,7,18-triazatricyclo[20.2.2.28,11]octadecano-1(25),8(28),9,11(27),22(26),23-hexane-5,19-dione A3: 1 HNMR (400 MHz, Chloroform- d ) δ 7.1 (dt, J = 12.7, 4.2 Hz, 3H), 6.9 – 6.8 (m,2H), 6.7 (q, J = 4.4 Hz, 1H), 6.7 (d, J = 5.0 Hz, 1H), 6.5 (m, 3H), 6.4 (d, J = 4.1Hz, 1H), 6.4 (dq, J = 8.2, 2.6 Hz, 1H), 6.1 (d, J= 4.3 Hz, 1H), 4.4 (dd, J = 14.7, 3.9 Hz, 1H), 4.3 (dd, J = 14.6, 3.9 Hz, 1H), 4.1 – 4.0 (m, 2H), 3.7 (m, 2H), 3.3 (m, 2H), 3.0 (d, J = 4.2 Hz, 1H), 2.8 (dq, J = 9.2, 5.0 Hz, 2H), 1.8 (t, J =5.2 Hz, 2H), 1.6 (m, 4H). 13 C NMR (101 MHz, Chloroform- d ) δ 168.2, 167.2,155.8, 154.5, 153.7, 133.6, 132.0, 131.3, 130.3, 130.2, 130.1, 130.0, 128.3,126.1, 122.3, 115.0, 112.4, 81.8, 76.0, 68.8, 67.2, 57.1, 39.7, 38.4, 34.6,28.5, 27.2, 22.8. 28-Chloro-7-(1-oxoylidene-2-ynyl)-6-(thiophene-2-yl)-12,22-dioxa-4,7,19-triazatricyclic[21.2.2.28,11] 29-1(25),8(9),10,23(24),26,28-hexaden-5,20-dione A4: 1 H NMR (400 MHz, Chloroform- d ) δ 7.2 – 7.2 (m, 2H), 7.2 (dd, J = 5.1, 1.3 Hz, 1H), 6.9– 6.8 (m, 2H), 6.8 (dd, J = 5.1, 3.6 Hz, 1H), 6.7 (s, 1H), 6.6 (d, J = 3.1 Hz, 1H), 6.5 (d, J = 8.7 Hz, 1H), 6.5 (s, 1H), 6.3 (d, J = 2.6 Hz, 2H), 6.3 (dd, J =8.7, 2.6 Hz, 1H), 4.3 (d, J= 14.1 Hz, 1H), 4.1 (d, J = 14.1 Hz, 1H), 4.1 – 4.0(m, 1H), 4.0 (m, 1H), 3.9 (td, J = 8.6, 4.3 Hz, 1H), 3.6 (dt, J = 10.6, 3.6 Hz,1H), 3.5 – 3.5 (m, 1H), 3.3 – 3.2 (m, 1H), 3.0 (s, 1H), 2.9 – 2.9 (m, 1H), 2.7 (m, 1H), 1.9 (m, 2H), 1.6 – 1.5 (m, 6H). 10-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-4,7,18-triaza-12,15,21-trioxatricyclo[20.2.2.28,11]octadecano-1(25),8(28),9,11(27),22(26),23-hexane-5,19-dione A5: 1 HNMR (400 MHz, Chloroform- d ) δ 7.2 (dd, J = 5.2, 1.2 Hz, 1H), 7.1 – 7.0 (m, 3H), 6.8 – 6.8 (m, 2H), 6.7 (dd, J = 5.2, 3.6 Hz, 1H), 6.7 (d, J = 8.7 Hz, 1H), 6.6(d, J = 2.5 Hz, 1H), 6.5 (s, 1H), 6.3 (dd, J = 8.7, 2.6 Hz, 1H), 6.3 (d, J = 3.6Hz, 1H), 6.1 (t, J = 6.0 Hz, 1H), 4.4 – 4.2 (m, 2H), 4.2 (dt, J = 11.0, 4.0 Hz, 1H), 4.1 (dt, J = 11.0, 3.8 Hz, 1H), 3.8 (t, J = 4.2 Hz, 2H), 3.7 (ddt, J = 12.3,9.6, 6.6 Hz, 4H), 3.5 (m, 2H), 3.0 (s, 1H), 2.8 (dq,J = 8.7, 4.2, 3.1 Hz, 2H). 13 C NMR (101 MHz, Chloroform- d ) δ 168.2, 167.2, 155.8, 154.5, 153.7, 133.6,132.0, 131.3, 130.3, 130.2, 130.1, 130.0, 128.3, 126.1, 122.3, 115.0, 112.4,81.8, 76.0, 68.8, 67.2, 57.1, 39.7, 38.4, 34.6, 28.5, 27.2, 22.8. 29-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-12,21-dioxa-4,7,18-triazatetracyclo[20.2.2.28,11.215,18]tetra-1(24),8(9),10,22(23),25,29-hexane-5,14,19-trione A6: 1 H NMR (400 MHz, Chloroform- d ) δ 7.57 (d, J = 2.1 Hz, 1H), 7.41 (dd, J = 5.3,1.8 Hz, 1H), 7.36 – 7.29 (m, 2H), 7.17 – 7.09 (m, 4H), 6.99 (d, J = 8.5 Hz,1H), 6.86 – 6.80 (m, 2H), 6.04 (d, J = 0.7 Hz, 1H), 4.81 (s, 2H), 4.62 (s, 2H), 3.67 (ddd, J = 12.4, 8.7, 6.0 Hz, 2H), 3.63 – 3.50 (m, 5H), 2.91 – 2.83 (m,3H), 2.76 (p, J = 5.6 Hz, 1H), 2.18 (ddt, J = 12.7, 8.8, 5.9 Hz, 2H), 1.97 – 1.87(m, 2H). 28-Chloro-7-(1-oxoylidene-2-ynyl)-6-(thiophene-2-yl)-12,20-dioxa-4,7,17-triazatetracyclo[19.2.2.28,11.214,17] 29-1(23),8(9),10,21(22),24,28-hexaden-5,18-dione A7: 1 H NMR (400 MHz, Chloroform- d ) δ 7.57 (d, J = 2.2 Hz, 1H), 7.41 (dd, J = 5.3, 1.8Hz, 1H), 7.36 – 7.29 (m, 1H), 7.17 – 7.09 (m, 2H), 6.96 (d, J = 8.5 Hz, 1H), 6.86 – 6.80 (m, 1H), 6.04 (d, J = 0.7 Hz, 1H), 4.62 (s, 1H), 3.95 (d, J = 5.1 Hz,1H), 3.64 – 3.50 (m, 4H), 2.91 – 2.83 (m, 2H), 2.20 (dqd, J = 11.0, 5.9, 5.0Hz, 1H), 1.98 (ddt, J = 12.4, 8.4, 6.1 Hz, 1H), 1.80 – 1.70 (m, 1H). 10-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-12,20-dioxa-4,7,17-triazatetracyclo[19.2.2.28,11.114,17]octadecano-1(24),8(27),9,11(26),21(25),22-hexaden-5,18-dione A8: 1 H NMR (400 MHz, Chloroform- d ) δ 7.3 (s, 1H), 7.2 – 7.2 (m, 1H), 7.1 (dd, J = 15.9, 8.2 Hz, 2H), 6.9 (d, J = 8.5 Hz, 1H), 6.9 – 6.8 (m, 2H), 6.8 – 6.7 (m,1H), 6.6 (d, J = 12.5 Hz, 1H), 6.5 (d, J= 8.8 Hz, 1H), 6.0 (dd, J = 8.7, 2.5 Hz, 1H), 6.0 (d, J = 18.7 Hz, 1H), 4.5 – 4.4 (m, 2H), 4.2 (dd, J = 10.4, 4.1 Hz, 1H), 3.9 (d, J = 13.3 Hz, 2H), 3.7 (dt, J = 19.9, 9.6 Hz, 2H), 3.6 – 3.4 (m, 2H), 2.9(s, 1H), 2.8 – 2.7 (m, 2H), 2.1 – 2.0 (m, 1H), 1.7 – 1.7 (m, 1H), 1.6 (d, J =10.8 Hz, 1H). 25-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-12,19-dioxa-4,7,16-triazatetracyclo[18.2.2.28,11.114,16]hexadecano-1(22),8(9),10,20(21),23,25-hexaen-5,17-dione A9: 1 H NMR (400 MHz, Chloroform- d ) δ 7.57 (d, J = 2.2 Hz, 1H), 7.41 (dd, J = 5.3, 1.8Hz, 1H), 7.36 – 7.29 (m, 1H), 7.17 – 7.09 (m, 3H), 6.96 (d, J = 8.5 Hz, 1H), 6.86 – 6.80 (m, 2H), 6.04 (d, J = 0.7 Hz, 1H), 4.62 (s, 1H), 4.06 (d, J = 4.9 Hz, 2H), 3.82 (dd, J = 10.4, 4.6 Hz, 2H), 3.64 – 3.50 (m, 3H), 2.91 – 2.83 (m, 2H), 2.55 (hept, J = 4.8 Hz, 1H). 10-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-12,19-dioxa-4,7,16-triazatetracyclo[18.2.2.28,11.113,16]hexadecano-1(23),8(26),9,11(25),20(24),21-hexaen-5,17-dione A10: 1 H NMR (400 MHz, Chloroform- d ) δ 7.59 (d, J = 2.2 Hz, 1H), 7.41 (dd, J = 5.3,1.8 Hz, 1H), 7.35 – 7.27 (m, 2H), 7.17 – 7.09 (m, 4H), 6.97 (d, J = 8.8 Hz,1H), 6.86 – 6.80 (m, 2H), 6.04 (d, J = 0.7 Hz, 1H), 4.95 (tt, J = 3.7, 2.7 Hz,1H), 4.62 (s, 2H), 3.88 – 3.82 (m, 1H), 3.75 (ddd, J = 12.5, 7.2, 5.4 Hz, 1H), 3.65 – 3.50 (m, 4H), 2.91 – 2.83 (m, 3H), 2.41 – 2.32 (m, 1H), 2.12 (dddd, J =12.9, 7.2, 5.3, 3.4 Hz, 1H). 24-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-12,18-dioxa-4,7,15-triazatetracyclo[17.2.2.28,11.113,15]hexadecyl-1(21),8(9),10,19(20),22,24-hexaen-5,16-dione A11: 1 H NMR (400 MHz, Chloroform- d ) δ 7.59 (d, J = 2.2 Hz, 1H), 7.41 (dd, J = 5.3,1.8 Hz, 1H), 7.35 – 7.27 (m, 2H), 7.17 – 7.09 (m, 4H), 6.97 (d, J= 8.7 Hz,1H), 6.86 – 6.80 (m, 2H), 6.04 (d, J = 0.7 Hz, 1H), 4.82 (p, J = 3.1 Hz, 1H), 4.67 (s, 2H), 4.08 (dd, J = 10.8, 3.1 Hz, 2H), 3.83 (dd, J = 10.6, 3.1 Hz, 2H), 3.64 – 3.50 (m, 2H), 2.91 – 2.83 (m, 3H). 29-Chloro-7-(1-oxoylidene-2-ynyl)-6-(thiophene-2-yl)-12,21-dioxa-4,7,18-triazatetracyclo[20.2.2.28,11.215,18]tetra-1(24),8(9),10,22(23),25,29-hexaen-5,19-dione A12: 1 HNMR 400 MHz, Chloroform- d ) δ 7.57 (d, J = 2.2 Hz, 1H), 7.41 (dd, J = 5.3, 1.8 Hz,1H), 7.36 – 7.29 (m, 2H), 7.17 – 7.09 (m, 4H), 6.95 (d, J = 8.5 Hz, 1H), 6.86 –6.80 (m, 2H), 6.04 (d, J = 0.7 Hz, 1H), 4.62 (s, 2H), 4.07 (t, J = 5.8 Hz, 2H), 3.65 – 3.50 (m, 7H), 2.91 – 2.83 (m, 3H), 1.97 – 1.87 (m, 2H), 1.77 – 1.61(m, 5H). 22-Chloro-19-(1-oxoylideneprop-2-ynyl)-18-(thiophene-2-yl)-2-oxa-8,16,19-triazabicyclo[18.2.2]tetracosyl-1(23),20(24),21-triene-9,17-dione A13: 1 H NMR (400 MHz, Chloroform- d ) δ 7.57 (d, J = 2.2 Hz, 0H), 7.41 (dd, J= 5.3, 1.8 Hz, 0H), 7.36 – 7.29 (m,1H), 7.15 – 7.05 (m, 1H), 6.95 (d, J = 8.5 Hz, 0H), 6.04 (d, J = 0.7 Hz, 0H), 5.87 (t, J = 4.9 Hz, 0H), 4.03 (t, J = 6.1 Hz, 1H), 3.29 – 3.14 (m, 2H), 2.16 (t, J = 8.4 Hz, 1H), 1.80 (tt, J = 6.7, 5.9 Hz, 1H), 1.68 (ddd, J = 16.0, 8.3, 7.4 Hz,1H), 1.59 – 1.41 (m, 4H). 21-Chloro-18-(1-oxoylidene-2-ynyl)-17-(thiophene-2-yl)-2-oxa-7,15,18-triazabicyclo[17.2.2]tetraco-1(22),19(23),20-triene-8,16-dione A14: 1 H NMR (400 MHz, Chloroform- d ) δ 7.57 (d, J = 2.2 Hz, 1H), 7.41 (dd, J = 5.3, 1.8 Hz, 1H), 7.36 – 7.29 (m,1H), 7.15 – 7.05 (m, 2H), 6.95 (d, J = 8.5 Hz, 1H), 6.04 (d, J = 0.7 Hz, 1H), 5.75 (t, J = 4.9 Hz, 1H), 4.08 – 3.99 (m, 2H), 3.29 – 3.15 (m, 4H), 2.85 (s,1H), 2.16 (t, J = 8.4 Hz, 2H), 1.82 – 1.73 (m, 2H), 1.73 – 1.63 (m, 2H), 1.61 –1.52 (m, 2H), 1.54 – 1.47 (m, 2H), 1.50 – 1.45 (m, 1H), 1.47 – 1.41 (m, 1H). 28-Chloro-7-(1-oxoylidene-2-enyl)-6-(thiophene-2-yl)-12,22-dioxa-4,7,19-triazatricyclic[21.2.2.28,11]nonadecano-1(25),8(9),10,23(24),26,28-hexaden-5,20-dione A15: 1 H NMR (400 MHz, Chloroform- d ) δ 7.1 (d, J = 7.8 Hz, 3H), 6.8 (d, J = 7.7 Hz, 2H), 6.8 –6.7 (m, 2H), 6.6 (d, J = 32.6 Hz, 2H), 6.4 (d, J = 46.0 Hz, 2H), 6.3 (dd, J = 8.7,2.1 Hz, 1H), 6.2 (s, 1H), 5.6 (t, J = 53.3 Hz, 1H), 4.4 – 4.2 (m, 2H), 4.1 –3.9 (m, 2H), 3.8 – 3.6 (m, 2H), 3.5 – 3.3 (m, 2H), 2.8 (dt, J = 9.2, 5.1 Hz,2H), 1.8 (s, 2H), 1.5 (d, J = 49.0 Hz, 6H). 28-Chloro-7-(2-chloroacetyl)-6-(thiophen-2-yl)-12,22-dioxa-4,7,19-triazatricyclic[21.2.2.28,11]nona-1(25),8(9),10,23(24),26,28-hexane-5,20-dione A16: 1 H NMR (400MHz, Chloroform- d ) δ 7.2 (dd, J = 18.1, 6.6 Hz, 3H), 6.9 (d, J = 8.2 Hz, 2H), 6.8(s, 2H), 6.5 (m, 3H), 6.4 (d, J = 2.7 Hz, 2H), 6.3 – 6.1 (m, 1H), 4.4 (d, J =14.2 Hz, 1H), 4.2 (d, J= 14.3 Hz, 1H), 4.1 (s, 1H), 3.9 (m 2H), 3.7 – 3.5 (m,3H), 3.3 (s, 1H), 3.1 (qd, J = 7.3, 3.7 Hz, 1H), 2.9 (dt, J = 14.8, 4.3 Hz, 1H), 2.8 – 2.7 (m, 1H), 1.9 (s, 2H), 1.7 – 1.4 (m, 6H). 28-Chloro-7-(1-oxoylidenebut-2-ynyl)-6-(thiophene-2-yl)-12,22-dioxa-4,7,19-triazatricyclic[21.2.2.28,11] 29-1(25),8(9),10,23(24),26,28-hexaden-5,20-dione A17: 1 H NMR (400 MHz, Chloroform- d ) δ 7.2 – 7.1 (m, 2H), 7.1 (d, J = 5.1 Hz, 1H), 6.8 – 6.8(m, 2H), 6.7 (dd, J = 5.2, 3.5 Hz, 2H), 6.6 (d, J = 3.6 Hz, 1H), 6.5 (d, J = 8.8Hz, 1H), 6.5 (s, 1H), 6.4 (dd, J = 8.1, 3.8 Hz, 1H), 6.3 (d, J = 2.6 Hz, 1H), 6.2(dd, J = 8.7, 2.5 Hz, 1H), 4.3 (d, J = 14.0 Hz, 1H), 4.1 – 4.0 (m, 2H), 3.9 (dtt, J = 11.1, 7.8, 3.8 Hz, 2H), 3.5 (m, 2H), 3.3 – 3.2 (m, 1H), 2.8 (m, 1H), 2.7(m, 1H), 1.9 – 1.8 (m, 2H), 1.8 (s, 3H), 1.7 – 1.4 (m, 6H). 28-Chloro-7-(2,2-difluoroacetyl)-6-(thiophen-2-yl)-12,22-dioxa-4,7,19-triazatricyclic[21.2.2.28,11] 26-hexaden-5,20-dione A18: 1 H NMR (400MHz, Chloroform- d ) δ 7.1 (d, J = 7.8 Hz, 3H), 6.8 (d, J = 7.7 Hz, 2H), 6.8 – 6.7(m, 2H), 6.6 (d, J = 32.6 Hz, 2H), 6.4 (d, J = 46.0 Hz, 2H), 6.3 (dd, J = 8.7, 2.1Hz, 1H), 6.2 (s, 1H), 5.6 (t, J = 53.3 Hz, 1H), 4.4 – 4.2 (m, 2H), 4.1 – 3.9(m, 2H), 3.8 – 3.6 (m, 2H), 3.5 – 3.3 (m, 2H), 2.8 (dt, J = 9.2, 5.1 Hz, 2H),1.8 (s, 2H), 1.5 (d, J = 49.0 Hz, 6H). 30-Chloro-7-(1-oxoylidene-2-ynyl)-6-(thiophene-2-yl)-12,24-dioxa-4,7,21-triazatricyclic[23.2.2.28,11]hexaeno-1(27),8(9),10,25(26),28,30-hexaen-5,22-dione A19: 1 H NMR (400 MHz, Chloroform- d ) δ 7.2 – 7.1 (m, 3H), 6.9 (d, J = 8.6 Hz, 2H), 6.8 (dd, J = 5.1, 3.6 Hz, 1H), 6.7 (d, J = 3.0 Hz, 1H), 6.7 (t, J = 5.5 Hz, 1H), 6.5 (d, J =8.7 Hz, 1H), 6.4 (t, J= 7.6 Hz, 3H), 6.3 (dd, J = 7.3, 3.8 Hz, 1H), 5.4 – 5.3(m, 2H), 4.4 – 4.3 (m, 2H), 4.1 – 4.0 (m, 1H), 3.9 (td, J = 8.8, 3.7 Hz, 2H),3.6 – 3.4 (m, 2H), 3.3 (dq, J = 12.2, 6.1 Hz, 1H), 3.0 (s, 1H), 2.9 – 2.8 (m,1H), 2.7 (ddd, J = 14.5, 10.7, 3.8 Hz, 1H), 2.0 (t, J = 6.0 Hz, 2H), 1.8 (dd, J =13.5, 5.4 Hz, 2H), 1.6 (ddd, J = 18.1, 13.2, 6.7 Hz, 6H). 6-(benzo[b]thiophen-2-yl)-28-chloro-7-(1-oxoylidene-2-ynyl)-12,22-dioxa-4,7,19-triazatricyclic[21.2.2.28,11]necto-1(25),8(9),10,23(24),26,28-hexaden-5,20-dione A20; 1 H NMR (400 MHz, Chloroform- d ) δ 7.1 (d, J = 7.8 Hz, 3H), 6.8 (d, J = 7.7 Hz,2H), 6.8 – 6.7 (m, 2H), 6.6 (d, J = 32.6 Hz, 2H), 6.4 (d, J = 46.0 Hz, 2H), 6.3(dd, J = 8.7, 2.1 Hz, 1H), 6.2 (s, 1H), 5.6 (t, J = 53.3 Hz, 1H), 4.4 – 4.2 (m,2H), 4.1 – 3.9 (m, 2H), 3.8 – 3.6 (m, 2H), 3.5 – 3.3 (m, 2H), 2.8 (dt, J = 9.2,5.1 Hz, 2H), 1.8 (s, 2H), 1.5 (d, J= 49.0 Hz, 6H). 2-[10-chloro-20-oxoylide-7-(1-oxoylide-prop-2-ynyl)-12,22-dioxa-4,7,19-triazatricyclo[21.2.2.28,11]necto-1(25),8(9),10,23(24),26,28-hexen-6-yl]cyclopentan-2,4-dien-1-yl anion cyclopentan-2,4-dien-1-yl anion iron(0) A21: 1 H NMR (400 MHz, Chloroform- d ) δ7.1 (d, J = 7.8 Hz, 3H), 6.8 (d, J = 7.7 Hz, 2H), 6.8 – 6.7 (m, 2H), 6.6 (d, J =32.6 Hz, 2H), 6.4 (d, J = 46.0 Hz, 2H), 6.3 (dd, J = 8.7, 2.1 Hz, 1H), 6.2 (s,1H), 5.6 (t, J = 53.3 Hz, 1H), 4.4 – 4.2 (m, 2H), 4.1 – 3.9 (m, 2H), 3.8 – 3.6(m, 2H), 3.5 – 3.3 (m, 2H), 2.8 (dt, J = 9.2, 5.1 Hz, 2H), 1.8 (s, 2H), 1.5 (d, J = 49.0 Hz, 6H). 28-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(1,3-thiazacyclopentanyl-5-yl)-12,22-dioxa-4,7,19-triazatricyclo[21.2.2.28,11] 26-hexaden-5,20-dione A22: 1 H NMR (400 MHz, CHLOROFORM- D ) δ 7.84 (d, J = 3.2 Hz, 1H), 7.57 (dd, J =6.9, 4.0 Hz, 1H), 7.39 (dd, J = 13.5, 2.9 Hz, 2H), 6.94 (d, J= 8.6 Hz, 2H), 6.73– 6.65 (m, 4H), 5.86 (d, J = 2.8 Hz, 1H), 5.48 (d, J = 2.9 Hz, 1H), 4.35 (d, J =2.6 Hz, 2H), 3.94 (td, J = 6.1, 2.5 Hz, 2H), 3.77 (tdd, J = 9.7, 8.1, 4.6 Hz,1H), 3.68 – 3.60 (m, 1H), 3.45 (qt, J = 13.7, 7.0 Hz, 2H), 2.87 – 2.79 (m, 2H), 1.88 (p, J = 6.2 Hz, 2H), 1.59 (m, 7H). 6-(benzo[b]thiophene-3-yl)-28-chloro-7-(1-oxoylidene-2-ynyl)-12,22-dioxa-4,7,19-triazatricyclic[21.2.2.28,11]necto-1(25),8(9),10,23(24),26,28-hexen-20-one A23: 1 HNMR (400 MHz, Chloroform- d ) δ 7.2 – 7.1 (m, 2H), 7.1 (d, J = 5.1 Hz, 1H), 6.8 –6.8 (m, 2H), 6.7 (dd, J = 5.2, 3.5 Hz, 2H), 6.6 (d, J = 3.6 Hz, 1H), 6.5 (d, J =8.8 Hz, 1H), 6.5 (s, 1H), 6.4 (dd, J = 8.1, 3.8 Hz, 1H), 6.3 (d, J = 2.6 Hz, 1H), 6.2 (dd, J = 8.7, 2.5 Hz, 1H), 4.3 (d, J = 14.0 Hz, 1H), 4.1 – 4.0 (m, 2H), 3.9(dtt, J= 11.1, 7.8, 3.8 Hz, 2H), 3.5 (m, 2H), 3.3 – 3.2 (m, 1H), 2.8 (m, 1H), 2.7 (m, 1H), 1.9 – 1.8 (m, 2H), 1.8 (s, 3H), 1.7 – 1.4 (m, 6H). 13 C NMR (101MHz, Chloroform- d ) δ 168.0, 167.6, 155.7, 154.6, 139.1, 138.1, 132.0, 130.4,130.1, 130.0, 129.4, 126.8, 124.9, 124.8, 122.9, 121.9, 120.8, 115.1, 111.7,81.7, 67.0, 54.8, 39.2, 38.7, 34.7. 30-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-12,15,18,24-tetraoxa-4,7,21-triazatricyclic[23.2.2.28,11]hexaeno-1(27),8(9),10,25(26),28,30-hexaen-5,22-dione A24: 1 H NMR (400 MHz, Chloroform- d ) δ 7.18 (dd, J = 5.3, 1.2 Hz, 1H), 7.13 (d, J = 8.6Hz, 2H), 6.84 – 6.81 (m, 2H), 6.78 – 6.75 (m, 1H), 6.61 – 6.57 (m, 2H), 6.54(s, 1H), 6.47 (d, J = 3.6 Hz, 1H), 6.34 (dd, J = 8.7, 2.6 Hz, 1H), 6.16 (t, J = 5.7Hz, 1H), 4.16 – 4.10 (m, 2H), 3.92 – 3.88 (m, 2H), 3.82 (ddd, J = 4.9, 3.3, 1.5Hz, 2H), 3.69 – 3.61 (m, 7H), 3.55 – 3.46 (m, 2H), 2.98 (s, 1H), 2.83 (d, J =5.4 Hz, 3H). 28-Chloro-7-[(2-ethynyl-1,3-thiazacyclopentanyl-4-yl)carbonyl]-6-(thiophen-2-yl)-12,22-dioxa-4,7,19-triazatricyclic[21.2.2.28,11]nona-1(25),8(9),10,23(24),26,28-hexaden-5,20-dione A25: 1 H NMR (400 MHz, Chloroform- d ) δ 7.6 – 7.4 (m, 1H), 7.2 (dd, J =5.1, 1.2 Hz, 1H), 7.1 – 7.1 (m, 2H), 6.8 (dd, J = 5.1, 3.6 Hz, 1H), 6.7 (d, J =3.4 Hz, 2H), 6.7 – 6.7 (m, 2H), 6.6 (s, 1H), 6.5 (dd, J = 9.6, 5.8 Hz, 3H), 6.0(d, J = 8.7 Hz, 1H), 4.3 – 4.2 (m, 2H), 4.0 (dt, J = 9.3, 5.6 Hz, 1H), 3.9 (ddd, J = 9.4, 6.7, 5.1 Hz, 2H), 3.6 (dq, J = 14.0, 4.7 Hz, 1H), 3.5 (dt, J = 11.5, 6.4Hz, 1H), 3.5 (s, 1H), 3.3 – 3.2 (m, 1H), 2.9 (dt, J = 14.7, 4.7 Hz, 1H), 2.8(ddd, J = 14.4, 10.0, 4.2 Hz, 1H), 1.8 (p, J = 6.2 Hz, 2H), 1.7 (d, J = 9.1 Hz, 2H), 1.6 (dd, J = 9.4, 4.3 Hz, 2H), 1.5 (q, J = 7.2 Hz, 2H). 28-Chloro-7-[(2-ethynyl-1,3-thiazacyclopentanyl-5-yl)carbonyl]-6-(thiophen-2-yl)-12,22-dioxa-4,7,19-triazatricyclo[21.2.2.28,11]nona-1(25),8(9),10,23(24),26,28-hexaden-5,20-dione A26: 1 H NMR (400 MHz, Chloroform- d ) δ 7.7 (dd, J = 12.0, 7.5 Hz, 1H),7.6 – 7.5 (m, 1H), 7.5 (dd, J = 8.2, 5.6 Hz, 1H), 7.2 – 7.1 (m, 1H), 7.1 – 7.0(m, 2H), 6.7 (d, J = 27.6 Hz, 3H), 6.5 (d, J = 8.6 Hz, 2H), 6.5 (s, 1H), 6.3 (s,1H), 6.2 (dd, J = 8.7, 2.6 Hz, 1H), 4.2 (s, 2H), 4.1 (s, 1H), 4.0 (dt, J = 10.1,5.6 Hz, 1H), 3.9 (s, 1H), 3.7 (d, J = 22.7 Hz, 1H), 3.6 (s, 1H), 3.4 (dd, J =14.7, 6.7 Hz, 2H), 2.8 (d, J = 15.1 Hz, 1H), 2.8 – 2.7 (m, 1H), 1.9 (s, 2H), 1.7 – 1.6 (m, 3H), 1.4 (d, J = 17.0 Hz, 3H). Example 2 Preparation of (4E)-9-chloro-12-(1-oxoylidenebut-2-ynyl)-13-(thiophene-2-yl)-12,15-diaza-2,7-dioxatricyclo[16.2.2.28,11]tetracosyl-1(21),4,8(24),9,11(23),18(22),19-heptaen-14-one B1 Compound B1 was synthesized according to the route shown in Scheme 2.

[0039] Compound 1 (1.37 g, 10.0 mmol) and (Boc)2O (2.4 g, 11.0 mmol) were dissolved in 10 mL of dichloromethane solution. After stirring at room temperature for 1 hour, the mixture was concentrated under vacuum and separated by column chromatography to obtain a colorless oily intermediate 12 (1.85 g, yield 78%).

[0040] Intermediate 12 (7.11 g, 3.0 mmol) and allyl bromo (0.4 g, 3.3 mmol) were dissolved in 2 mL of NMP solution, and potassium carbonate (0.622 g, 4.5 mmol) was added. After stirring at 55 °C for 3 hours, the reaction mixture was poured into a saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The combined organic phases were washed successively with saturated aqueous sodium chloride solution, dried over anhydrous Na₂SO₄, and then concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain a colorless oily intermediate 13 (580 mg, 85% yield).

[0041] Intermediate 13 (0.416 g, 1.50 mmol) was added to 4 mL of a mixed solution (TFA:DCM = 1:3) and reacted for 1 hour. After removing the solvent by rotary evaporation, DIPEA (1.938 g, 15 mmol) free amino groups were added, and white fumes were emitted. Methyl formate (0.54 mmol, 9 mmol) was then added and dissolved in 2 mL of methanol. The mixture was stirred at 60 °C for 12 hours. The reaction mixture was then concentrated under vacuum and purified by column chromatography to obtain a colorless oily intermediate 14 (228 mg, yield 74%).

[0042] Intermediate 14 (0.164 g, 0.800 mmol) and DIPEA (0.723 g, 5.6 mmol) were dissolved in 21 mL of dry dichloromethane. The reaction solution was pre-cooled at -80°C for 10 minutes, and then phosphorus oxychloride (0.368 g, 2.40 mmol) diluted in 1 mL of dry dichloromethane was added dropwise at a controlled rate (completed over 30 minutes). After stirring for 1 hour, 2 mL of water was added to the reaction solution, and stirring was continued for 5 minutes. Ice residue was removed by filtration, and the organic phase was concentrated under vacuum. The organic phase was rapidly separated by column chromatography (PE:EA = 10:1) and concentrated to obtain a colorless oily intermediate 15, which was not weighed and was used directly in the next reaction (estimated yield of 90%).

[0043] Intermediate 5 (0.29 g, 1.20 mmol) and allyl bromo (0.17 g, 1.44 mmol) were dissolved in 2 mL of NMP solution, and potassium carbonate (0.25 g, 1.80 mmol) was added. After stirring at 55 °C for 3 hours, the reaction mixture was poured into a saturated aqueous ammonium chloride solution and extracted with ethyl acetate. The combined organic phases were washed successively with saturated aqueous sodium chloride solution, dried over anhydrous Na₂SO₄, and then concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain a colorless oily intermediate 16 (255 mg, 75% yield).

[0044] Intermediate 16 (0.23 g, 0.800 mmol) was added to 4 mL of a mixed solution (TFA:DCM = 1:3), and the mixture was stirred at room temperature for 1 hour. The reaction mixture was then poured into a saturated aqueous sodium bicarbonate solution and extracted with dichloromethane. The combined organic phases were washed successively with a saturated aqueous sodium chloride solution, dried over anhydrous Na₂SO₄, and then concentrated under vacuum to obtain the crude product. The crude product was purified by column chromatography to obtain a colorless oily intermediate 17 (117 mg, 80% yield).

[0045] Intermediates 17 (0.1 g, 0.546 mmol) and 8 (0.06 g, 0.546 mmol) were dissolved in 3 mL of methanol and reacted at 55 °C for 1 h with stirring. The mixture was then allowed to return to room temperature. Intermediates 15 (0.1 g, 0.546 mmol) and 18 (0.046 g, 0.546 mmol) were added with stirring, and the mixture was reacted at room temperature for 10 h with stirring. The reaction mixture was concentrated under vacuum, reconstituted with ethyl acetate, washed successively with aqueous solution and saturated sodium chloride solution, dried over anhydrous Na₂SO₄, and then concentrated under vacuum to give the crude product. The crude product was purified by column chromatography to give a white solid intermediate 19 (200 mg, 66% yield).

[0046] Intermediate 19 (0.08 g, 0.145 mmol) was dissolved in 360 mL of anhydrous dichloromethane solution, and Grubbs-2nd catalyst (0.025 g, 0.029 mmol) was added. The mixture was stirred at 40 °C for 48 hours under a nitrogen atmosphere. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography to give a white solid compound B1 (15 mg, 20% yield). 1 H NMR (400 MHz, Chloroform- d ) δ 7.2 (d, J = 5.1 Hz, 1H), 7.0 (d, J = 8.5 Hz, 2H), 6.8 (d, J= 9.9Hz, 2H), 6.7 – 6.7 (m, 4H), 6.6 (dd, J = 5.2, 3.6 Hz, 1H), 6.0 (dt, J = 16.6, 5.4Hz, 1H), 5.8 – 5.7 (m, 1H), 5.5 – 5.4 (m, 2H), 4.8 – 4.7 (m, 3H), 4.6 (dd, J =14.8, 6.9 Hz, 1H), 4.1 – 3.9 (m, 1H), 3.3 – 3.2 (m, 1H), 3.0 (dt, J = 14.5,5.7 Hz, 1H), 2.6 (ddd, J = 14.5, 9.1, 4.6 Hz, 1H), 1.7 (s, 3H). 13 C NMR (101MHz, Chloroform- d ) δ 167.3, 156.9, 153.2, 134.6, 132.5, 132.3, 131.6, 130.5,130.2, 129.6, 129.3, 127.8, 127.6, 126.9, 123.1, 116.0, 114.9, 73.7, 67.5,57.2, 39.5, 33.2, 29.7, 4.0. The synthesis processes of compounds B2 to B14 are similar to those of compound B1.

[0047] (4E)-9-chloro-12-[(2-ethynyl-1,3-thiazazetacyclopentanyl-4-yl)carbonyl]-13-(thiophen-2-yl)-12,15-diaza-2,7-dioxatricyclo[16.2.2.28,11]tetracosyl-1(21),4,8(24),9,11(23),18(22),19-heptaen-14-one B2: 1 H NMR (400 MHz, Chloroform- d ) δ 7.7 – 7.4 (m, 1H), 7.2(dd, J = 5.1, 1.2 Hz, 1H), 7.0 (d, J = 8.6 Hz, 2H), 6.9 (s, 1H), 6.8 – 6.7 (m,3H), 6.7 (t, J = 4.4 Hz, 1H), 6.6 (d, J = 8.8 Hz, 1H), 5.9 (dt,J = 16.3, 5.2 Hz,1H), 5.8 – 5.6 (m, 2H), 4.8 – 4.6 (m, 4H), 4.0 – 3.9 (m, 1H), 3.4 (s, 2H),3.0 (d, J = 15.4 Hz, 1H), 2.6 (ddd, J = 14.4, 8.8, 4.4 Hz, 1H), 2.0 (d, J = 17.0Hz, 1H). (4E)-20-chloro-17-[(5-nitrofuran-2-yl)carbonyl]-16-(thiophen-2-yl)-14,17-diaza-2,7-dioxatricyclo[16.2.2.28,11]tetracosyl-1(21),4,8(24),9,11(23),18(22),19-heptaen-15-one B3: 1 H NMR (400 MHz, Chloroform- d ) δ 7.2 (d, J = 5.1 Hz, 1H), 7.0 (d, J = 3.8 Hz,1H), 7.0 – 6.9 (m, 2H), 6.9 (s, 1H), 6.8 (d, J = 8.8 Hz, 1H), 6.8 – 6.7 (m,2H), 6.7 (s, 1H), 6.3 (d, J = 3.6 Hz, 1H), 6.0 (d, J = 16.2 Hz, 1H), 5.7 (d, J =16.4 Hz, 1H), 5.6 (dd, J = 7.8, 3.3 Hz, 1H), 5.1 – 4.7 (m, 3H), 4.6 (dd, J =14.6, 6.4 Hz, 1H), 4.2 – 4.0 (m, 1H), 3.3 – 3.2 (m, 1H), 3.0 (d, J = 44.4 Hz, 1H), 2.6 (s, 1H). 13 C NMR (101 MHz, Chloroform- d) δ 166.7, 157.2, 147.2, 133.9,132.2, 131.0, 130.1, 129.5, 128.0, 127.2, 118.6, 114.9, 111.0, 58.7. (4E)-20-chloro-17-(2-chloroacetyl)-16-(thiophen-2-yl)-14,17-diaza-2,7-dioxatricyclo[16.2.2.28,11]tetracosyl-1(21),4,8(24),9,11(23),18(22),19-heptaen-15-one B4: 1 H NMR (400 MHz, Chloroform- d ) δ 7.2 (d, J = 5.0 Hz, 1H), 7.0 (d, J = 8.1 Hz, 2H), 6.8 –6.6 (m, 5H), 6.4 (m, 1H), 5.9 (s, 1H), 5.7 (s, 1H), 5.5 (s, 2H), 4.9 – 4.6(m, 4H), 4.2 – 4.0 (m, 1H), 3.9 – 3.8 (m, 2H), 3.1 (m, 2H), 2.6 (d, J = 13.3Hz, 1H). (4E)-13-(benzo[b]thiophene-3-yl)-9-chloro-12-(1-oxoylideneprop-2-ynyl)-12,15-diaza-2,7-dioxatricyclo[16.2.2.28,11]tetracosyl-1(21),4,8(24),9,11(23),18(22),19-heptaen-14-one B5: 1 H NMR (400 MHz, Chloroform- d ) δ 7.8 – 7.8 (m, 1H), 7.7 (s, 1H), 7.4 –7.4 (m, 2H), 7.1 – 7.0 (m, 3H), 7.0 (s, 1H), 6.9 – 6.8 (m, 3H), 6.7 (d, J = 8.2Hz, 1H), 6.0 (d, J = 15.9 Hz, 1H), 5.7 – 5.7 (m, 1H), 5.5 (d, J = 6.9 Hz, 1H), 4.9 – 4.8 (m, 3H), 4.6 (dd, J= 15.0, 7.7 Hz, 2H), 4.2 (s, 1H), 3.2 (d, J = 9.7Hz, 2H), 2.8 (s, 1H), 2.7 (t, J = 9.2 Hz, 1H). (4E)-9-chloro-13-(naphth-1-yl)-12-(1-oxoylidene-2-ynyl)-12,15-diaza-2,7-dioxatricyclo[16.2.2.28,11]tetracosyl-1(21),4,8(24),9,11(23),18(22),19-heptaen-14-one B6: 1 H NMR (500 MHz, Chloroform- d ) δ 8.11 – 8.05 (m, 1H), 7.80 (ddt, J = 7.9, 3.2, 1.5 Hz,2H), 7.57 – 7.43 (m, 5H), 7.26 (dd, J = 8.4, 2.2 Hz, 1H), 7.15 (d, J = 9.7 Hz, 1H), 7.06 (dt, J = 8.6, 0.9 Hz, 2H), 6.98 (d, J = 8.5 Hz, 1H), 6.86 – 6.80 (m,2H), 5.82 (d, J = 0.7 Hz, 1H), 5.72 (p, J = 2.7 Hz, 2H), 4.63 (td, J = 2.5, 1.3 Hz, 2H), 4.55 (td, J = 2.5, 1.0 Hz, 2H), 3.65 – 3.51 (m, 2H), 2.91 – 2.83 (m, 3H). (4E)-13-(1H-indol-3-yl)-9-chloro-12-(1-oxoylidene-2-ynyl)-12,15-diaza-2,7-dioxatricyclo[16.2.2.28,11]tetracosyl-1(21),4,8(24),9,11(23),18(22),19-heptaen-14-one B7: 1 H NMR (400 MHz, DMSO- d6 ) δ 7.3 (d, J = 8.1 Hz, 2H), 7.1 (d, J= 7.5 Hz, 1H),7.0 – 7.0 (m, 3H), 6.9 – 6.8 (m, 4H), 6.8 – 6.8 (m, 2H), 6.6 (s, 1H), 5.9 (d, J = 17.0 Hz, 1H), 5.7 – 5.6 (m, 2H), 4.8 – 4.6 (m, 5H), 3.9 (d, J = 12.0 Hz, 1H), 3.2 (d, J = 6.2 Hz, 1H), 2.9 – 2.9 (m, 1H), 2.6 (dd, J = 10.0, 5.1 Hz, 1H), 2.0 (d, J = 7.6 Hz, 1H). 10-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-4,7-diaza-12,18-dioxatricyclo[17.2.2.28,11] tetavidin-1(22),8(25),9,11(24),15,19(23),20-heptaen-5-one B8: 1 H NMR (400 MHz, Chloroform- d ) δ 7.4 (s, 1H), 7.2 – 7.2 (m, 1H), 7.0 (d, J = 6.6 Hz,2H), 6.8 – 6.8 (m, 2H), 6.7 (s, 1H), 6.6 (t, J = 4.4 Hz, 1H), 6.6 (s, 1H), 6.2– 6.2 (m, 1H), 5.8 – 5.8 (m, 1H), 5.7 (s, 2H), 5.5 – 5.4 (m, 1H), 4.6 (d, J =5.2 Hz, 2H), 4.3 (dd, J = 6.0, 4.5 Hz, 2H), 3.9 (s, 1H), 3.3 (s, 1H), 3.1 (d, J =9.7 Hz, 1H), 2.9 (s, 1H), 2.7 – 2.7 (m, 1H), 2.5 (dd, J = 14.4, 6.4 Hz, 1H),2.4 (s, 1H). 10-Chloro-7-(1-oxoylideneprop-2-ynyl)-6-(thiophene-2-yl)-4,7-diaza-12,18-dioxatricyclo[17.2.2.28,11] tetavidin-1(22),8(25),9,11(24),14,19(23),20-heptaen-5-one B9: 1 H NMR (400 MHz, Chloroform- d ) δ 7.2 – 7.1 (m, 3H), 6.8 (dd, J = 5.2, 3.6 Hz, 1H), 6.8– 6.7 (m, 2H), 6.7 – 6.6 (m, 3H), 6.4 (s, 2H), 5.9 – 5.8 (m, 1H), 5.6 – 5.5(m, 2H), 4.8 (ddd, J = 14.5, 3.8, 1.7 Hz, 1H), 4.7 – 4.6 (m, 1H), 4.0 (ddd, J =8.7, 5.8, 3.1 Hz, 2H), 3.7 – 3.6 (m, 1H), 3.5 (d, J = 10.5 Hz, 1H), 3.0 (s,1H), 2.9 (dt, J = 14.5, 4.3 Hz, 1H), 2.7 (ddd, J = 14.8, 11.1, 4.0 Hz, 1H), 2.5 –2.5 (m, 1H), 2.4 (dt, J = 14.4, 6.1 Hz, 1H). (4E)-13-(1H-indol-3-yl)-9-chloro-12-(1-oxoylidenebut-2-ynyl)-12,15-diaza-2,7-dioxatricyclo[16.2.2.28,11]tetracosyl-1(21),4,8(24),9,11(23),18(22),19-heptaen-14-one B10: 1 H NMR (400 MHz, Chloroform- d ) δ 8.5 (m, 1H), 7.3 (d, J = 8.2 Hz, 1H), 7.2– 7.1 (m, 1H), 7.0 – 7.0 (m, 3H), 6.9 (d, J = 7.1 Hz, 3H), 6.8 – 6.7 (m, 3H), 6.7 (d, J= 8.7 Hz, 1H), 6.0 – 5.9 (m, 1H), 5.8 – 5.7 (m, 2H), 4.8 – 4.7 (m,3H), 4.7 – 4.6 (m, 1H), 4.1 – 4.1 (m, 1H), 3.2 – 3.0 (m, 2H), 2.6 – 2.5 (m,1H), 1.6 (s, 3H). (14E)-9-chloro-6-(1-oxoylideneprop-2-ynyl)-5-(thiophene-2-yl)-3,6-diaza-11,17-dioxatricyclo[16.2.2.27,10]tetracosyl-1(21),7(24),8,10(23),14,18(22),19-heptaen-4-one B11: 1 HNMR (400 MHz, Chloroform- d ) δ 7.5 (s, 1H), 7.4 (d, J = 5.2 Hz, 1H), 7.1 – 7.0(m, 1H), 7.0 (d, J = 3.6 Hz, 1H), 6.9 (d, J = 7.4 Hz, 1H), 6.8 (s, 1H), 6.7 (d, J =4.3 Hz, 4H), 6.6 (d, J = 8.8 Hz, 1H), 6.0 – 5.9 (m, 1H), 5.7 – 5.7 (m, 1H), 4.8– 4.7 (m, 1H), 4.7 (d, J = 4.2 Hz, 2H), 4.1 (s, 1H), 4.0 (s, 1H), 3.8 – 3.7 (m,1H), 2.9 (s, 1H), 2.5 (d, J = 9.6 Hz, 3H). (13Z)-9-chloro-6-(1-oxoylideneprop-2-ynyl)-5-(thiophene-2-yl)-3,6-diaza-11,16-dioxatricyclo[15.2.2.27,10]tetradecano-1(20),7(23),8,10(22),13,17(21),18-heptaen-4-one B12: 1 HNMR (400 MHz, Chloroform- d ) δ 7.4 (d, J = 5.1 Hz, 1H), 7.1 (d, J= 8.2 Hz, 2H), 7.0 (dd, J = 5.2, 3.6 Hz, 1H), 6.9 (s, 1H), 6.9 (d, J = 3.6 Hz, 1H), 6.8 – 6.8(m, 2H), 6.7 (dd, J = 8.9, 2.6 Hz, 1H), 6.6 (dd, J = 13.4, 5.0 Hz, 2H), 6.4 (d, J =8.9 Hz, 1H), 5.9 – 5.8 (m, 2H), 4.9 (d, J = 14.3 Hz, 1H), 4.7 (q, J = 2.4 Hz, 2H), 4.6 (dt, J = 14.6, 4.0 Hz, 1H), 4.4 (dd, J = 14.3, 6.9 Hz, 1H), 4.1 (dd, J =14.5, 5.9 Hz, 1H), 2.8 (s, 1H). (4E)-9-chloro-12-(1-oxoylideneprop-2-ynyl)-13-(thiophene-2-yl)-12,15-diaza-2,7-dioxatricyclo[16.2.2.28,11]tetracosyl-1(21),4,8(24),9,11(23),18(22),19-heptaen-14-one B13: 1 HNMR (400 MHz, Chloroform- d ) δ 7.2 – 7.2 (m, 1H), 7.0 (d, J = 8.5 Hz, 2H), 6.8(d, J = 18.8 Hz, 2H), 6.8 – 6.7 (m, 4H), 6.7 – 6.6 (m, 1H), 6.0 (dt, J = 16.5,5.2 Hz, 1H), 5.8 – 5.6 (m, 1H), 5.6 – 5.3 (m, 2H), 4.8 – 4.7 (m, 3H), 4.6(dd, J = 14.8, 7.0 Hz, 1H), 4.1 (dddd, J = 17.7, 13.8, 9.4, 5.8 Hz, 1H), 3.2 (dq, J= 10.5, 4.6 Hz, 1H), 3.0 (dt, J = 15.0, 5.6 Hz, 1H), 2.8 (s, 1H), 2.6 (ddd, J =14.7, 9.3, 4.7 Hz, 1H). 13 C NMR (101 MHz, Chloroform- d ) δ 166.8, 157.0, 153.6,134.1, 132.7, 131.6, 130.7, 130.2, 129.5, 129.5, 127.8, 127.7, 127.1, 114.9,81.2, 67.5, 57.3, 39.5, 33.2, 29.7. 6-(1H-indol-3-yl)-10-chloro-7-(1-oxoylideneprop-2-ynyl)-4,7-diaza-12,18-dioxatricyclo[17.2.2.28,11] tetavidin-1(22),8(25),9,11(24),15,19(23),20-heptaen-5-one B14: 1 HNMR (400 MHz, Chloroform-d) δ 7.72 – 7.65 (m, 1H), 7.60 – 7.53 (m, 2H), 7.39 – 7.30 (m, 2H), 7.22 – 7.10 (m, 2H), 7.06 (dt, J = 8.7, 0.8 Hz, 2H), 6.95 (d, J =8.5 Hz, 1H), 6.87 – 6.79 (m, 2H), 5.76 (d, J = 0.7 Hz, 1H), 5.74 – 5.57 (m,2H), 4.54 – 4.48 (m, 2H), 4.16 (td, J = 6.1, 0.8 Hz, 2H), 3.57 (tt, J = 5.7, 4.7Hz, 2H), 2.92 – 2.82 (m, 3H), 2.39 (tddt, J = 6.0, 5.1, 1.8, 1.0 Hz, 2H). Example 3: Assay of the compound's ferroptosis-inducing activity and affinity for GPX4 protein (1) Studies have shown that GPX4 inhibitors such as ML162 can induce ferroptosis in cells, which can be blocked by other small molecules, such as lipophilic antioxidants, such as Ferrostatin-1 (fer-1) and Liproxstatin. Therefore, whether a designed compound can be a ferroptosis inducer can be determined by whether its ability to kill tumor cells can be blocked by Fer-1.

[0048] Cell line: Human fibrosarcoma cell line HT1080, purchased from Shanghai Cell Bank, Chinese Academy of Sciences.

[0049] The activity of the test compound was determined by proliferation assays using the human fibrosarcoma cell line HT1080. Cells were seeded in 96-well tissue culture plates (3500 cells per well). After overnight incubation, the predetermined concentration of the test compound was added, starting at 10 μM and then serially diluted 3-fold. Cell viability was determined using an MTT assay after 48 hours of cell culture. The IC50 of the test compound was determined. 50 Value. Ferrostatin, an inhibitor of ferroptosis. The antiproliferative activity of the compounds was tested in the presence of 1(Fer-1). The results are summarized in Table 1, where A represents values ​​not greater than 100 nM, B represents values ​​greater than 100 nM but not greater than 500 nM, C represents values ​​greater than 500 nM but not greater than 2 μM, D represents values ​​greater than 2 μM but not greater than 10 μM, and E represents values ​​greater than 10 μM. In this test, ML The IC50 of 162 is 77 nM.

[0050] Table 1: Results of the ferroptosis inhibitory activity test of the compounds As can be seen from Table 1, most of the macrocyclic GPX4 inhibitors prepared in this invention have better or equal activity and selectivity against ferroptosis-sensitive cell line HT1080 than ML162.

[0051] Cell lines: human clear cell carcinoma cell line 786-O, breast cancer cell line 4T1, human breast cancer cell line MDA-MB-231, human neuroblastoma cell line SH-SY5Y, human non-small cell lung cancer cell line A549, human cervical cancer cell line HeLa, and human liver cancer cell line HepG2 were all purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0052] The above-described tumor cells were used for proliferation assays to determine the antiproliferative activity of the test compound. Cells were seeded in 96-well tissue culture plates (4000-5000 cells per well). After overnight incubation, a predetermined concentration of the test compound was added, starting at 100 μM and then serially diluted 3-fold. After culturing the cells for 48 hours, cell viability was determined using an MTT assay. The IC50 of the test compound was determined. 50 Values. The results are summarized in Table 2, where A represents values ​​no greater than 100 nM, B represents values ​​greater than 100 nM but not greater than 500 nM, C represents values ​​greater than 500 nM but not greater than 2 μM, D represents values ​​greater than 2 μM but not greater than 10 μM, and E represents values ​​greater than 10 μM.

[0053] Table 2: Antiproliferative activity of some compounds As can be seen from Table 2, the selected macrocyclic GPX4 inhibitors showed significantly better activity against ferroptosis-sensitive cell lines such as 786-O, 4T1, and MDA-MB-231 than against ML162, and poorer activity against ferroptosis-insensitive tumor cell lines such as A549, HeLa, and HepG2. This fully demonstrates that the macrocyclic GPX4 inhibitors prepared in this invention have superior GPX4 inhibitory activity and selectivity.

[0054] (2) SPR (Surface Plasmon Resonance) measurement.

[0055] First, the CM5 chip was activated using an amino-coupling kit. Then, the all-cysteine-deficient (AllCys(-)) glutathione peroxidase 4 cysteine ​​mutant (GPX4U46C) in sodium acetate (pH 4.5) buffer was added at 10 μL / min. - ¹ The compound was injected into the CM5 chip surface at a flow rate of 15 min. The chip was then blocked with ethanolamine. Different concentrations (0-20 μM) of compound B14 were injected, allowing binding for 120 seconds, with a dissociation time set to 120 seconds. The dissociation constant (KD value) of the compound-protein complex was calculated using Biacore T200 evaluation software. The experimental results are shown below. Figure 1 As shown.

[0056] from Figure 1 As can be seen, B14, as a representative compound of macrocyclic GPX4 inhibitors, has a strong affinity for GPX4 protein.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A macrocyclic GPX4 inhibitor, characterized in that, It is a compound with the structural formula shown in formula (I): Or, an enantiomer of formula (I), a mixture of enantiomers, a diastereomer, a mixture of diastereomers, a tautomer, a mixture of tautomers, or an isotopic variant; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof; Among them, R 1 Selected from Or –CH2 R 2b or or , where R 2a It is hydrogen or C1 C6 alkyl, R 2b It is a halogen; R 2 Selected from C5-C 10 Aromatic heterocyclic or ferrocene-based; R 3 Selected from benzene rings or C1-C6 alkyl groups; R 4 Selected from C1-C8 alkyl, C4-C6 heterocyclic alkyl, and alkyl in which the carbon atom is replaced by one or more heteroatoms; R 5 Selected from amide or olefin groups, and R 3 and R 4 Fragment connection.

2. The macrocyclic GPX4 inhibitor according to claim 1, characterized in that, The specific structural formulas of the macrocyclic GPX4 inhibitor compounds A1-A26, B1-B14 are as follows: 。 3. A method for preparing a macrocyclic GPX4 inhibitor according to claim 1 or 2, characterized in that, When the R 5 Amide group and R 3 and R 4 During fragment ligation, the compound of formula (I) obtained at this time is IA, and the synthetic route is as follows: The preparation method of compound IA includes the following steps: Compound I undergoes a nucleophilic elimination reaction with methyl formate to give compound II; compound II undergoes an oxidation reaction with phosphorus oxychloride to give compound III; compound IV undergoes a nucleophilic elimination reaction with (BOC)₂O to give compound V; compound V undergoes a nucleophilic substitution reaction with a brominated derivative containing a BOC protecting group to give compound VI; compound VI undergoes BOC removal under acidic conditions, followed by a selective nucleophilic elimination reaction with (BOC)₂O to give compound VII; compound III undergoes a UGI four-component reaction with compounds VIII, VII, and IX to give compound X; compound X undergoes tert-butyl and BOC removal under acidic conditions to give compound XI; compound XI undergoes an intramolecular condensation reaction to give the final compound IA, where R 1 R 2 R 3 R 4 As described in claim 1.

4. A method for preparing a macrocyclic GPX4 inhibitor according to claim 1 or 2, characterized in that, When R 5 For olefinic group and R 3 and R 4 During fragment ligation, the compound of formula (I) obtained at this time is IB, and the synthetic route is as follows: The preparation method of the compound IB includes the following steps: Compound XII undergoes a nucleophilic elimination reaction with (BOC)₂O to give compound XIII; compound XIII undergoes a nucleophilic substitution reaction with a brominated derivative without a BOC protecting group to give compound XIV; compound XIV undergoes BOC removal under acidic conditions, followed by a nucleophilic elimination reaction with methyl formate to give compound XV; compound XV undergoes oxidation with phosphorus oxychloride to give compound XVI; compound V undergoes a nucleophilic substitution reaction with a brominated derivative to give compound XVIII; compound XVIII undergoes BOC removal under acidic conditions to give compound XIX; compound IV undergoes a nucleophilic elimination reaction with (BOC)₂O to give compound V; compound XVI undergoes a four-component reaction with compounds VIII, XIX, and IX to give compound XVII; compound XVII undergoes an olefin metathesis reaction to give the final compound IB; where R 1 R 2 As described in claim 1, R 3 Selected from C1-C6 alkyl groups; R 4 Selected from C1-C8 alkyl groups; n is selected from 0-3.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a macrocyclic GPX4 inhibitor as described in claim 1 or 2, and further comprises a pharmaceutically acceptable excipient.

6. The use of a macrocyclic GPX4 inhibitor according to claim 1 or 2, or the pharmaceutical composition according to claim 5, in the preparation of a ferroptosis inducer.

7. The application according to claim 6, characterized in that, The ferroptosis inducer is used to prepare drugs for treating ferroptosis-related diseases.

8. The application according to claim 6, characterized in that, The ferroptosis-related diseases include disorders, diseases, or conditions mediated by glutathione peroxidase 4.

9. The application according to claim 6, characterized in that, The ferroptosis-related diseases are tumors, including lung cancer, colon cancer, neuroblastoma, melanoma, breast cancer, stomach cancer, and leukemia.

10. The use of a macrocyclic GPX4 inhibitor according to claim 1 or 2 or the pharmaceutical composition according to claim 5 in the preparation of an antitumor drug.

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