CN114573490B - Diaryl-2H-aziridine compounds, preparation method and application thereof - Google Patents
Diaryl-2H-aziridine compounds, preparation method and application thereof Download PDFInfo
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- CN114573490B CN114573490B CN202011386653.XA CN202011386653A CN114573490B CN 114573490 B CN114573490 B CN 114573490B CN 202011386653 A CN202011386653 A CN 202011386653A CN 114573490 B CN114573490 B CN 114573490B
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- YCWSUKQGVSGXJO-NTUHNPAUSA-N nifuroxazide Chemical group C1=CC(O)=CC=C1C(=O)N\N=C\C1=CC=C([N+]([O-])=O)O1 YCWSUKQGVSGXJO-NTUHNPAUSA-N 0.000 claims abstract description 10
- 150000001541 aziridines Chemical class 0.000 claims abstract description 3
- -1 Diaryl substituted-2H-azepine compounds Chemical class 0.000 claims description 118
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D203/00—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom
- C07D203/04—Heterocyclic compounds containing three-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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Abstract
The invention belongs to the field of chemical pharmacy in the research and development of new medicines, and relates to application of novel chiral diaryl substituted-2H-aziridine compounds and diaryl substituted aziridine compounds with remarkable antitumor activity, a preparation method, in-vitro and-in-vivo antitumor activity and acceptable pharmaceutical salts thereof, or compound medicines taking the same as one of components in preparation of medicines for preventing and treating tumor-related diseases. The compound or the pharmaceutically acceptable salt thereof provided by the invention can effectively inhibit the growth of nude mice transplanted tumor in vitro and in vivo by inhibiting the regulatory mechanism of inhibiting the proliferation of tumor cells through inhibiting the aggregation of tubulin, and can be applied to the preparation of medicaments for preventing or treating tumor-related diseases. The tumor-associated diseases include benign and malignant tumors and other diseases caused by the tumors.
Description
Technical Field
The invention belongs to the field of chemical pharmacy in the research and development of new medicines, and relates to application of novel chiral diaryl substituted-2H-aziridine compounds and diaryl substituted aziridine compounds with remarkable antitumor activity, a preparation method, in-vitro and-in-vivo antitumor activity and acceptable pharmaceutical salts thereof, or compound medicines taking the same as one of components in preparation of medicines for preventing and treating tumor-related diseases.
Background
Currently, there are nearly hundred antitumor drugs approved for marketing, mainly including the following types: (1) an antitumor drug acting on DNA: such as alkylating agents, metal platinum complexes, DNA topoisomerase inhibitors, antimetabolite antineoplastic agents, and the like; (2) an antitumor agent acting on a kinase: such as tyrosine kinase inhibitors and serine/threonine kinase inhibitors; (3) an antitumor agent acting on microtubules (Microtubule): including microtubule aggregation inhibitors (i.e., microtubule destabilizers, represented by vinca alkaloids, colchicines, podophyllotoxins, and Combretastatins) and microtubule aggregation promoters (i.e., microtubule stabilizers, represented by taxol and epothilones).
Because microtubule aggregation inhibitors have the capacity of inhibiting Tubulin (Tubulin) polymerization, have the effects of specifically targeting and destroying the generated tumor blood vessels and starving tumors, and most of the drugs do not have multi-drug resistance, the microtubule aggregation inhibitors become a type of anti-tumor drugs which are actively researched in recent years. The tubulin aggregation inhibitor Combretastatin A-4, CA-4 for short, targeted to the colchicine binding site is a series of cis-stilbene natural products separated from the bark or stem of African shrub dwarf willow (Combretum caffrum) in 1982, has strong activity of inhibiting tubulin aggregation and selectively inhibiting tumor vascular proliferation, and has been developed in a series of important progress in structural engineering research. For example, chiral β -lactam CA-4 analogues with significant antitumor activity (J.Med. Chem.2016,59,10329-10334; eur. J. Med. Chem.2018,144, 817-842).
DNA alkylating agents play an important role in clinical chemotherapy of tumors, and are nitrogen mustard compounds which are one of the most widely used DNA cross-linking agents at the same time. The pharmacophore N, N-dichloro ethylamine can be converted into reactive electrophilic aziridine, mitomycin C and other complex natural products through intramolecular conversion, and an aziridine structure also exists, and a cross-linking structure is formed between the aziridine structure and DNA, so that the antitumor activity is exerted.
Because of the complicated pathogenesis and regulation mechanism of tumors, single-target drugs often have unsatisfactory actions. The multi-target drugs can act on a plurality of sites of the regulation network at the same time, and compared with single-target drugs, the multi-target drugs can obtain better anti-tumor effect. Meanwhile, due to the synergistic effect, the multi-target medicine can use lower dosage, so that toxic and side effects are reduced. Therefore, the multi-target drug is favored in tumor treatment, such as imatinib, sorafenib, lapatinib and other molecular targeting drugs are all multi-target drugs.
According to the structural characteristics of compounds acting on colchicine loci and DNA, the structures of the compounds are fused, a novel diaryl substituted three-membered ring compound is designed and synthesized, and activity research shows that the novel diaryl substituted three-membered ring compound has double functions on Tubulin and DNA, has excellent multiple tumor cell proliferation inhibition activities and in-vivo antitumor activities, and is an antitumor candidate compound with novel structure and unique mechanism.
Disclosure of Invention
The invention aims to provide a novel tubulin aggregation inhibitor and an angiogenesis inhibitor, in particular to a novel diaryl substituted aza-ternary ring compound with remarkable anti-tumor activity, a preparation method thereof and application of the compound and pharmaceutical salt thereof or compound medicines taking the compound as components in preparing medicines for preventing and treating tumor-related diseases.
The invention provides diaryl substituted-2H-aziridine compounds with the following general structure or pharmaceutical salts thereof,
Wherein R 1 and R 2 are taken from the group consisting of a hydrogen atom, an alkyl group, a substituted alkyl group, an alkoxy group, an alkylthio group, an acyloxy group, a hydroxyl group, an amino group, an alkylamino group, an amido group, an aryl group, a heteroaryl group, a vinyl group, a halogen atom, a methoxyformyl group, an allyloxy group, a propargyloxy group, a sulfonyloxy group, a sulfonylamino group, or a combination of 2-3 of the same or different groups. R is selected from the group consisting of a hydrogen atom, an alkyl group, a substituted alkyl group, an alkoxy group, an alkylthio group, an acyloxy group, a hydroxyl group, an amino group, an aryl group, a heteroaryl group, a vinyl group, and an acyl group.
Preferred compounds of the invention are:
The "pharmaceutically acceptable salts" in the present invention include, specifically, salts with inorganic acids such as halogen acids, sulfuric acid, phosphoric acid, nitric acid, and organic acids such as citric acid, fumaric acid, maleic acid, oxalic acid, malic acid, lactic acid, and camphorsulfonic acid.
It is another object of the present invention to provide the use of the above-mentioned compounds or pharmaceutically acceptable salts of these compounds, and compositions comprising the compounds or salts thereof, for the preparation of a medicament for the prevention or treatment of tumor-associated diseases.
Specific examples of the tumor-associated diseases include thyroid cancer, squamous cell carcinoma of head and neck, cervical cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, renal cancer, gastric cancer, lung cancer, liver cancer, melanoma, lymphoma, prostate cancer, bladder cancer, brain glioma, nasopharyngeal cancer, neuroendocrine cancer, undifferentiated carcinoma, interstitial sarcoma, choriocarcinoma, malignant grape embryo, malignant teratoma, and benign tumors, but are not limited thereto.
The diaryl substituted aza-tricyclic compounds or pharmaceutically acceptable salts thereof with remarkable anti-tumor effect are provided and proved to have good inhibition effect on tumor growth in-vitro and in-vivo anti-tumor experiments through acting on regulatory mechanisms of Tubulin/DNA double-target inhibition on tumor cell growth.
Drawings
Figure 1, compound 8 in vitro inhibition microtubule self-assembly inhibition assay.
FIG. 2 immunofluorescence staining test compound 8 for effect on microtubule filament structure.
Figure 3, compound 8 inhibition of neovascularization in vitro.
Fig. 4, compound 8 comet assay.
FIG. 5, compound 8 upregulates expression of DNA damage inducing factors
FIG. 6, compound 8 inhibits HeLa cell colony formation assay
FIG. 7, effect of Compound 8 on cell cycle.
FIG. 8 effect of Compound 8 on expression of cycle-related proteins.
Figure 9, compound 8 induced apoptosis experiment.
FIG. 10 effect of Compound 8 on apoptosis-related protein expression.
Figure 11, liver microsomal stability of compound 8.
Fig. 12, nude mouse transplantation tumor experiment of compound 8.
Detailed Description
The invention is further illustrated below with reference to examples. These examples are provided only for further illustration of the invention and do not alter the scope of the invention. The preparation method of the target compound of the invention can be further represented by the following preparation process of the representative compound:
EXAMPLE 1 Synthesis of the Compound 3- (3-fluoro-4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (1)
Reference (J.org.chem.2017, 82:3631-3638.; org.biomol.chem.2018, 16:4333-4337.) the present invention synthesizes the target compound 1 as follows:
1.1 Synthesis of 1- (3-fluoro-4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one (1 c)
3,4, 5-Trimethoxybenzyl cyanide 1a (627mg, 3 mmol), 3-fluoro-4-methoxyboric acid 1b (1.02 g,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were further added to dissolve the substrate completely, and trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions with nitrogen replaced, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 850mg of white solid (1 c) in yield of 85%.mp 106.5-109.3℃.1HNMR(400MHz,CDCl3)δ7.81(d,J=8.4Hz,1H),7.76(d,J=11.9Hz,1H),7.00(t,J=8.3Hz,1H),6.46(s,2H),4.16(s,2H),3.96(s,3H),3.84(s,6H),3.83(s,3H).13C NMR(150MHz,CDCl3)δ195.32,153.37,152.81,152.05,151.98,151.16,136.94,130.03,129.75,129.72,126.00,125.98,116.27,116.14,112.37,106.35,60.83,56.30,56.11,45.47.ESI-HRMS(m/z):calcd for C18H19FO5Na(M+Na+),357.1108;found,357.1119.
1.Synthesis of 21- (3-fluoro-4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (1 d)
1- (3-Fluoro-4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one 1c (1.0 g,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared to stop the reaction. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) to give 899mg as a white solid (1 d) in yield 86%.mp 123.5-126.0℃.1HNMR(400MHz,CDCl3)δ7.43(d,J=12.6Hz,1H),7.34(d,J=7.6Hz,1H),6.91(t,J=8.6Hz,1H),6.46(s,2H),4.10(s,2H),3.89(s,3H),3.80(d,J=2.6Hz,9H).13C NMR(150MHz,CDCl3)δ156.16,153.34,152.99,151.36,148.68,148.61,136.58,131.90,128.65,128.61,122.71,114.16,114.03,112.90,105.40,60.85,56.20,56.08,31.97.ESI-HRMS(m/z):calcd for C18H20FNO5Na(M+Na+),372.1223;found,372.1228.
1.3 Synthesis of 3- (3-fluoro-4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (1)
1- (3-Fluoro-4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (349 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice-bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 1e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) gave 261mg of yellow solid (1) in yield 78%.mp 121.1-123.8℃.1H NMR(400MHz,CDCl3)δ7.66(d,J=9.5Hz,2H),7.11(t,J=7.9Hz,1H),6.34(s,2H),3.98(s,3H),3.81(s,9H),3.24(s,1H).13C NMR(150MHz,CDCl3)δ162.43,153.34,153.27,151.98,151.91,151.62,137.32,136.46,127.31,116.91,116.79,116.65,116.60,113.40,102.78,60.88,56.38,56.06,34.93.ESI-HRMS(m/z):calcd for C18H18FNO4Na(M+Na+),354.1118;found,354.1113..
EXAMPLE 2 Synthesis of the Compound 3- (3-fluoro-4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (2)
The present invention synthesizes the target compound 2 according to the following route:
2.1 Synthesis of 1- (4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one (2 c)
3,4, 5-Trimethoxybenzyl cyanide 2a (6271 mg,3 mmol), 4-methoxyphenylboronic acid 2b (912 mg,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were further added to dissolve the substrate completely, nitrogen was replaced, and trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 805mg of yellow solid (2 c) in yield 85%.mp 84.9-87.5℃.1H NMR(400MHz,CDCl3)δ8.01(d,J=8.5Hz,2H),6.94(d,J=8.4Hz,2H),6.48(s,2H),4.18(s,2H),3.87(s,3H),3.83(s,9H).13C NMR(150MHz,CDCl3)δ196.20,163.62,153.29,136.80,130.92,130.52,129.57,113.83,106.40,60.83,56.09,55.50,45.46.ESI-HRMS(m/z):calcd for C18H20O5Na(M+Na+),339.1202;found,339.1199.
2.2 Synthesis of 1- (4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (2 d)
1- (4-Methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one 2c (948 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL of anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (823 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and the reaction was stopped by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) gave 814mg of yellow oil (2 d) in yield 82%.1HNMR(600MHz,CDCl3)δ7.57(d,J=8.9Hz,2H),6.87(d,J=8.9Hz,2H),6.47(s,2H),4.12(s,2H),3.81(s,3H),3.80(s,3H),3.79(s,6H).13C NMR(150MHz,CDCl3)δ160.55,157.10,153.28,136.50,132.27,128.14,127.87,113.94,105.50,60.83,56.06,55.30,32.22.ESI-HRMS(m/z):calcd for C18H21NO5Na(M+Na+),354.1317;found,354.1313.
2.3 Synthesis of 3- (4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (2)
1- (3-Fluoro-4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (331 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with dichloromethane, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 2e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) to yield 225mg of yellow solid (2) in yield 71%.mp 117.5-120.6℃.1HNMR(400MHz,CDCl3)δ7.86(d,J=8.2Hz,2H),7.06(d,J=8.2Hz,2H),6.35(s,2H),3.90(s,3H),3.81(s,3H),3.80(s,6H),3.21(s,1H).13C NMR(150MHz,CDCl3)δ163.58,162.38,153.29,137.15,137.02,131.91,116.28,114.81,102.75,60.87,56.05,55.58,34.41.ESI-HRMS(m/z):calcd for C18H19NO4Na(M+Na+),336.1212;found,336.1204..
EXAMPLE 3 Synthesis of the Compound 3- (benzo [ d ] [1,3] dioxin-5-yl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (3)
The present invention synthesizes the target compound 3 according to the following route:
3.1 Synthesis of 1- (benzo [ d ] [1,3] dioxin-5-yl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one (3 c)
3,4, 5-Trimethoxybenzyl cyanide 3a (627mg, 3 mmol), benzo [ d ] [1,3] dioxin-5-boronic acid 3b (996 mg,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were added to completely dissolve the substrate, nitrogen was replaced, trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice bath conditions, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 752mg of white solid (3 c) in the yield of 76%.mp 117.4-119.0℃.1H NMR(400MHz,CDCl3)δ7.64(d,J=8.1Hz,1H),7.48(s,1H),6.86(d,J=8.1Hz,1H),6.46(s,2H),6.05(s,2H),4.15(s,2H),3.84(s,6H),3.83(s,3H).13C NMR(151MHz,CDCl3)δ195.71,153.32,151.94,148.27,136.85,131.34,130.34,124.99,108.34,107.94,106.35,101.92,60.84,56.10,45.54.ESI-HRMS(m/z):calcd for C18H18O6Na(M+Na+),353.0995;found,353.0996.
3.2 Synthesis of 1- (benzo [ d ] [1,3] dioxin-5-yl) -2- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (3 d)
1- (Benzo [ d ] [1,3] dioxin-5-yl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one 3c (990 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (828 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrum until the reaction was stopped by disappearance of the starting material. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) gave 859mg of white oil (3 d) in yield 83%.mp 143.3-146.6℃.1HNMR(400MHz,CDCl3)δ7.17(s,1H),7.10(d,J=7.9Hz,1H),6.78(d,J=8.0Hz,1H),6.47(s,2H),5.97(s,2H),4.10(s,2H),3.80(s,9H).13C NMR(150MHz,CDCl3)δ157.04,153.30,148.68,147.96,136.54,132.12,129.81,120.91,108.15,106.62,105.45,101.34,60.84,56.09,32.30.ESI-HRMS(m/z):calcd for C18H19NO6Na(M+Na+),368.1104;found,368.1101.
3.3 Synthesis of 3- (benzo [ d ] [1,3] dioxin-5-yl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (3)
1- (Benzo [ d ] [1,3] dioxin-5-yl) -2- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (345 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with dichloromethane, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 3e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) gave 258mg of yellow solid (3) in yield 79%.mp 113.6-115.5℃.1H NMR(400MHz,CDCl3)δ7.40(s,2H),6.96(d,J=7.1Hz,1H),6.34(s,2H),6.10(s,2H),3.81(s,9H),3.22(s,1H),1.65(s,2H).13C NMR(150MHz,CDCl3)δ162.73,153.30,152.01,148.59,137.22,136.71,126.38,117.82,109.04,108.69,102.76,102.09,60.87,56.05,34.93.ESI-HRMS(m/z):calcd for C18H17NO5Na(M+Na+),350.1004;found,350.0990..
EXAMPLE 4 Synthesis of the Compound 3- (3, 4-dimethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (4)
The present invention synthesizes the target compound 4 according to the following route:
4.1 Synthesis of 1- (3, 4-dimethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one (4 c)
3,4, 5-Trimethoxybenzyl cyanide 4a (627mg, 3 mmol), 3, 4-dimethoxyphenylboronic acid 4b (1.09 g,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were further added to dissolve the substrate completely, and trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions with nitrogen replaced, and the temperature was raised to 80℃for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 820mg of white solid (4 c) in yield of 79%.mp 151.2-156.1℃.1H NMR(400MHz,CDCl3)δ7.68(d,J=8.3Hz,1H),7.57(s,1H),6.90(d,J=8.3Hz,1H),6.49(s,2H),4.19(s,2H),3.95(s,3H),3.93(s,3H),3.84(s,6H),3.83(s,3H).13C NMR(150MHz,CDCl3)δ196.25,153.44,153.32,149.11,136.85,130.58,129.72,123.44,110.61,110.00,106.36,60.84,56.10,55.97,45.37.ESI-HRMS(m/z):calcd for C19H22O6Na(M+Na+),369.1308;found,369.1302.
4.2 Synthesis of 1- (3, 4-dimethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (4 d)
1- (3, 4-Dimethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one 4c (1.03 g,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared to stop the reaction. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) gives 844mg of white solid (4 d) in yield 78%.mp 118.2-120.6℃.1HNMR(400MHz,CDCl3)δ7.88(s,1H),7.29(d,J=1.9Hz,1H),7.15(dd,J=8.4,2.0Hz,1H),6.83(d,J=8.4Hz,1H),6.49(s,2H),4.12(s,2H),3.89(s,3H),3.88(s,3H),3.80(s,3H),3.80(s,6H).13C NMR(150MHz,CDCl3)δ157.15,153.29,150.21,148.90,136.49,132.44,128.36,119.72,110.60,108.92,105.46,60.85,56.07,55.89,55.84,32.10,29.33.ESI-HRMS(m/z):calcd for C19H23NO6Na(M+Na+),384.1416;found,384.1417.
4.3 Synthesis of 3- (3, 4-dimethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (4)
1- (3, 4-Dimethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (361 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with dichloromethane, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 4e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) to give 257mg of yellow solid (4) in yield of 75%.mp 101.5-103.6℃.1HNMR(400MHz,CDCl3)δ7.48(d,J=8.6Hz,1H),7.43(dd,J=8.2,1.7Hz,1H),6.99(d,J=8.3Hz,1H),6.36(s,2H),3.97(s,6H),3.82(s,3H),3.81(s,6H),3.24(s,1H).13C NMR(150MHz,CDCl3)δ162.81,153.32,153.26,149.69,137.23,136.92,124.83,116.41,111.06,110.91,102.85,60.89,56.18,56.16,56.07,34.97.ESI-HRMS(m/z):calcd for C19H21NO5Na(M+Na+),366.1317;found,366.1312..
EXAMPLE 5 Synthesis of the Compound 3- (4-ethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-az-cine (5)
The present invention synthesizes the target compound 5 according to the following route:
5.1 Synthesis of 1- (3, 4-dimethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one (5 c)
3,4, 5-Trimethoxybenzyl cyanide 5a (6271 mg,3 mmol), 4-ethoxyphenyl boric acid 5b (996 mg,6 mmol), palladium acetate (11 mg,5 mol%), 2 '-bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to Schlenk's tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were further added to dissolve the substrate completely, nitrogen was replaced, trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice bath conditions, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 861mg of yellow solid (5 c) in the yield of 87%.mp 99.6-101.1℃.1H NMR(400MHz,CDCl3)δ7.99(d,J=8.4Hz,2H),6.93(d,J=8.4Hz,2H),6.48(s,2H),4.17(s,2H),4.10(q,J=6.6Hz,2H),3.83(s,6H),3.82(s,3H),1.44(t,J=6.8Hz,3H).13C NMR(150MHz,CDCl3)δ196.18,163.06,153.28,136.80,130.92,130.56,129.38,114.25,106.41,63.79,60.83,56.09,45.42,14.67.ESI-HRMS(m/z):calcd for C19H22O5Na(M+Na+),353.1359;found,353.1355.
5.2 Synthesis of 1- (4-ethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (5 d)
1- (4-Ethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one 5c (990 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL of anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and the reaction was stopped by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) to give 890mg of red solid (5 d) in yield 86%.mp 112.1-115.3℃.1HNMR(400MHz,CDCl3)δ7.56(d,J=8.6Hz,2H),6.86(d,J=8.6Hz,2H),6.48(s,2H),4.13(s,2H),4.03(q,J=7.0Hz,2H),3.80(s,3H),3.78(s,6H),1.41(t,J=6.9Hz,3H).13C NMR(150MHz,CDCl3)δ159.94,157.04,153.25,136.44,132.28,127.91,127.84,114.45,105.47,63.50,60.83,56.05,32.26,14.75.ESI-HRMS(m/z):calcd for C19H23NO5Na(M+Na+),368.1468;found,368.1469.
5.3 Synthesis of 3- (4-ethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (5)
1- (4-Ethoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (345 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with dichloromethane, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 5e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) to give 245mg of yellow solid (5) in the yield of 75%.mp 99.8-102.1℃.1HNMR(400MHz,CDCl3)δ7.84(d,J=8.6Hz,2H),7.04(d,J=8.6Hz,2H),6.35(s,2H),4.12(q,J=6.9Hz,2H),3.81(s,3H),3.80(s,6H),3.20(s,1H),1.46(t,J=6.9Hz,3H).13C NMR(150MHz,CDCl3)δ163.00,162.34,153.28,137.13,137.07,131.91,116.03,115.22,102.74,63.90,60.87,56.04,34.37,14.66.ESI-HRMS(m/z):calcd for C19H21NO4Na(M+Na+),350.1368;found,350.1364..
EXAMPLE 6 Synthesis of the Compound 3- (4-methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-az-cine (6)
The present invention synthesizes the target compound 6 according to the following route:
6.1 Synthesis of 1- (4-methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one (6 c)
3,4, 5-Trimethoxybenzyl cyanide 6a (6271 mg,3 mmol), 4-methylphenylboronic acid 6b (816 mg,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to the Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were further added to dissolve the substrate completely, nitrogen was replaced, trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice bath conditions, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) gave a red solid (6 c) 756mg in yield 84%.mp 86.4-89.5℃.1H NMR(400MHz,CDCl3)δ7.92(d,J=8.2Hz,2H),7.27(d,J=8.2Hz,2H),6.48(s,2H),4.20(s,2H),3.83(s,6H),3.82(s,3H),2.41(s,3H).13C NMR(150MHz,CDCl3)δ197.28,153.29,144.16,136.83,134.06,130.30,129.38,128.73,106.45,60.83,56.09,45.62,21.68.ESI-HRMS(m/z):calcd for C18H20O4Na(M+Na+),323.1253;found,323.1251.
6.2 Synthesis of 1- (4-methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (6 d)
1- (4-Methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one 6c (900 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and the reaction was stopped by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) to give 746mg of yellow solid (6 d) in yield 79%.mp 124.1-126.9℃.1HNMR(400MHz,CDCl3)δ7.52(d,J=8.2Hz,2H),7.16(d,J=8.0Hz,2H),6.47(s,2H),4.14(s,2H),3.80(s,3H),3.78(s,6H),2.35(s,3H).13C NMR(150MHz,CDCl3)δ157.43,153.24,139.48,136.45,132.80,132.20,129.28,126.38,105.48,60.83,56.22,56.04,32.29,21.27.ESI-HRMS(m/z):calcd for C18H21NO4Na(M+Na+),338.1362;found,338.1360.
6.3 Synthesis of 3- (4-methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (6)
1- (4-Methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (315 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and dichloromethane (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 6e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) to give 216mg of yellow solid (6) in the yield of 73%.mp 79.8-82.6℃.1HNMR(400MHz,CDCl3)δ7.81(d,J=7.9Hz,2H),7.37(d,J=7.8Hz,2H),6.35(s,2H),3.81(s,3H),3.80(s,6H),3.23(s,1H),2.46(s,3H).13C NMR(150MHz,CDCl3)δ163.26,153.30,144.22,137.21,136.86,130.04,129.92,121.16,102.79,60.87,56.05,34.55,21.92.ESI-HRMS(m/z):calcd for C18H19NO3Na(M+Na+),320.1257;found,320.1250..
EXAMPLE 7 Synthesis of the Compound 3- (4-methoxy-3-methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (7)
The present invention synthesizes the target compound 7 as follows:
7.1 Synthesis of 1- (4-methoxy-3-methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one (7 c) 3,4, 5-trimethoxybenzyl acetonitrile 7a (6271 mg,3 mmol), 4-methoxy-3-methylbenzoboric acid 7b (996 mg,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were then added to dissolve the substrate completely, nitrogen was replaced, and trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 811mg of red solid (7 c) in yield of 82%.mp83.4-86.2℃.1H NMR(400MHz,CDCl3)δ7.89(dd,J=8.5,1.9Hz,1H),7.84(s,1H),6.85(d,J=8.6Hz,1H),6.48(s,2H),4.17(s,2H),3.89(s,3H),3.84(s,6H),3.82(s,3H),2.25(s,3H).13C NMR(150MHz,CDCl3)δ196.47,161.92,153.27,136.77,131.19,130.69,129.06,128.84,126.94,109.25,106.41,60.83,56.08,55.55,45.40,16.32.ESI-HRMS(m/z):calcd for C19H22O5Na(M+Na+),353.1359;found,353.1353.
7.2 Synthesis of 1- (4-methoxy-3-methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (7 d)
1- (4-Methoxy-3-methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one 7c (990 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared to stop the reaction. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) to give 879mg of yellow solid (7 d) in the yield of 85%.mp 117.1-120.4℃.1HNMR(400MHz,CDCl3)δ7.46(s,1H),7.42(dd,J=8.5,2.1Hz,1H),6.78(d,J=8.5Hz,1H),6.49(s,2H),4.13(s,2H),3.83(s,3H),3.80(s,3H),3.79(s,6H),2.20(s,3H).13C NMR(150MHz,CDCl3)δ158.82,157.22,153.23,136.42,132.41,128.63,127.57,126.80,125.43,109.63,105.49,60.83,56.04,55.35,32.26,16.37.ESI-HRMS(m/z):calcd for C19H23NO5Na(M+Na+),368.1468;found,368.1459.
7.3 Synthesis of 3- (4-methoxy-3-methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (7)
1- (4-Methoxy-3-methylphenyl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (345 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 7e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) to give 238mg of yellow solid (7) in the yield of 73%.mp 96.6-98.8℃.1H NMR(400MHz,CDCl3)δ7.73(d,J=8.7Hz,1H),7.70(s,1H),6.96(d,J=8.3Hz,1H),6.35(s,2H),3.92(s,3H),3.81(s,3H),3.81(s,6H),3.19(s,1H),2.27(s,3H).13C NMR(151MHz,CDCl3)δ162.36,161.83,153.28,137.22,137.10,131.98,129.91,128.08,115.64,110.21,102.75,60.87,56.05,55.61,34.37,16.18.ESI-HRMS(m/z):calcd for C19H21NO4Na(M+Na+),350.1362;found,350.1357..
EXAMPLE 8 Synthesis of Compound 2- (3-fluoro-4-methoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (8)
The present invention synthesizes the target compound 8 according to the following route:
8.1 Synthesis of 2- (3-fluoro-4-methoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one (8 c) 4-methoxy-3-methylbenzonitrile 8a (495mg, 3 mmol), 3,4, 5-trimethoxyphenylboronic acid 8b (1.27 g,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were then added to dissolve the substrate completely, and trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under the conditions of nitrogen substitution and ice bath, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 791mg of white solid (8 c) in yield of 79%.mp125.3-127.8℃.1H NMR(400MHz,DMSO)δ7.33(s,2H),7.16-7.07(m,2H),7.04-7.02(m,1H),4.35(s,2H),3.85(s,6H),3.81(s,3H),3.74(s,3H).13C NMR(150MHz,DMSO)δ196.30,152.94,152.68,151.87,150.26,145.66,145.59,141.82,131.45,128.03,127.98,125.74,117.08,116.96,113.58,105.90,104.47,60.06,59.96,56.01,55.95,55.81,43.32.ESI-HRMS(m/z):calcd for C18H19FO5Na(M+Na+),357.1108;found,357.1112.
8.2 Synthesis of 2- (3-fluoro-4-methoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (8 d)
2- (3-Fluoro-4-methoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one 8c (1.00 g,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared to stop the reaction. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) to give 890mg of white solid (8 d) in yield 85%.mp 96.9-99.1℃.1HNMR(400MHz,CDCl3)δ7.04(d,J=12.2Hz,1H),6.97(d,J=8.2Hz,1H),6.87(d,J=9.1Hz,1H),6.84(s,2H),4.10(s,2H),3.85(d,J=2.6Hz,6H),3.83(s,6H).13C NMR(150MHz,CDCl3)δ157.09,153.18,151.54,146.23,146.16,139.27,130.83,129.59,129.55,124.14,124.12,116.46,116.34,113.51,103.84,60.90,56.28,56.14,31.29.ESI-HRMS(m/z):calcd for C18H20FNO5Na(M+Na+),372.1217;found,372.1216.
8.3 Synthesis of 2- (3-fluoro-4-methoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (8) 2- (3-fluoro-4-methoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (349 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and solvent dichloromethane (15 mL) were added thereto, and acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise under ice-bath conditions and the mixture was warmed to room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with dichloromethane, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 8e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) gave 258mg of yellow solid (8) in yield 78%.mp 84.3-86.5℃.1HNMR(400MHz,CDCl3)δ7.12(s,1H),6.91(s,1H),6.90(d,J=11.9Hz,1H),6.84(d,J=11.9Hz,1H),3.95(s,3H),3.92(s,6H),3.87(s,3H),3.28(s,1H).13C NMR(150MHz,CDCl3)δ163.28,153.89,153.25,151.62,146.99,146.91,142.33,134.14,134.10,121.97,118.84,113.87,113.74,113.37,106.74,61.05,56.39,34.3.ESI-HRMS(m/z):calcd for C18H18FNO4Na(M+Na+),354.1112;found,354.1107..
EXAMPLE 9 Synthesis of Compound 2- (4-methoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-az-cine (9)
The present invention synthesizes the target compound 9 according to the following route:
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9.1 Synthesis of 2- (3-fluoro-4-methoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one (9 c) 4-methoxy-3-methylbenzonitrile 9a (495mg, 3 mmol), 3,4, 5-trimethoxyphenylboronic acid 9b (1.27 g,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were then added to dissolve the substrate completely, and trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under the conditions of nitrogen substitution and ice bath, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to yield 786mg of white solid (9 c) in yield 83%.mp87.2-89.0℃.1H NMR(400MHz,CDCl3)δ7.26(s,2H),7.19(d,J=8.2Hz,2H),6.87(d,J=8.1Hz,2H),4.19(s,2H),3.90(s,3H),3.89(s,6H),3.78(s,3H).13C NMR(150MHz,CDCl3)δ196.76,158.55,153.00,142.52,131.71,130.27,126.76,114.20,106.20,60.93,56.24,55.25,44.72.ESI-HRMS(m/z):calcd for C18H20O5Na(M+Na+),339.1202;found,339.1198.
9.2 Synthesis of 2- (4-methoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (9 d)
2- (4-Methoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one 9c (948 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL of anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (823 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and the reaction was stopped by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) gave 715mg of a white solid (9 d) in yield 72%.mp 123.4-125.3℃.1HNMR(400MHz,CDCl3)δ8.97(s,1H),7.19(d,J=7.7Hz,2H),6.85(s,2H),6.81(d,J=7.5Hz,2H),4.11(s,2H),3.84(s,3H),3.81(s,6H),3.76(s,3H).13C NMR(150MHz,CDCl3)δ158.16,157.62,153.10,139.08,131.14,129.53,128.65,114.08,103.90,60.88,56.10,55.24,31.41.ESI-HRMS(m/z):calcd for C18H21NO5Na(M+Na+),354.1311;found,354.1305.
9.3 Synthesis of 2- (4-methoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (9)
2- (4-Methoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (331 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 9e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) to yield 225mg of yellow solid (9) in yield 72%.mp 88.8-91.5℃.1HNMR(400MHz,CDCl3)δ7.14(s,2H),7.09(d,J=7.8Hz,2H),6.84(d,J=7.8Hz,2H),3.94(s,3H),3.91(s,6H),3.79(s,3H),3.32(s,1H).13C NMR(150MHz,CDCl3)δ163.80,159.04,153.84,142.11,132.89,127.38,119.38,113.87,106.64,61.04,56.38,55.33,34.84.ESI-HRMS(m/z):calcd for C18H19NO4Na(M+Na+),336.1206;found,336.1208..
EXAMPLE 10 Synthesis of the Compound 2- (benzo [ d ] [1,3] dioxin-5-yl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (10)
The present invention synthesizes the target compound 10 according to the following route:
10.1 Synthesis of 2- (benzo [ d ] [1,3] dioxin-5-yl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one (10 c)
Benzo [ d ] [1,3] dioxin-5-ylacetonitrile 10a (483 mg,3 mmol), 3,4, 5-trimethoxyphenylboronic acid 10b (1.27 g,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were added to completely dissolve the substrate, nitrogen was replaced, trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 742mg of white solid (10 c) in yield of 75%.mp 98.9-101.1℃.1H NMR(400MHz,CDCl3)δ7.26(s,2H),6.82–6.67(m,3H),5.94(s,2H),4.16(s,2H),3.91(s,3H),3.90(s,6H).13C NMR(150MHz,CDCl3)δ196.50,153.03,147.93,146.60,142.62,131.62,128.32,122.37,109.69,108.49,106.18,101.05,60.95,56.28,45.17.ESI-HRMS(m/z):calcd for C18H18O6Na(M+Na+),353.0995;found,353.0989.
10.2 Synthesis of 2- (benzo [ d ] [1,3] dioxin-5-yl) -1- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (10 d)
2- (Benzo [ d ] [1,3] dioxin-5-yl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one 10c (990 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (828 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrum until the reaction was stopped by disappearance of the starting material. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) to yield 786mg of white solid (10 d) in yield 76%.mp 96.3-98.7℃.1HNMR(400MHz,CDCl3)δ8.17(s,1H),6.86(s,2H),6.78(s,1H),6.73(s,2H),5.91(s,2H),4.09(s,2H),3.85(s,3H),3.83(s,6H).13C NMR(150MHz,CDCl3)δ157.44,153.12,147.91,146.14,139.16,130.97,130.36,121.46,109.07,108.34,103.86,100.94,60.89,56.13,31.73.ESI-HRMS(m/z):calcd for C18H19NO6Na(M+Na+),368.1104;found,368.1109.
10.3 Synthesis of 2- (benzo [ d ] [1,3] dioxin-5-yl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (10)
2- (Benzo [ d ] [1,3] dioxin-5-yl) -1- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (345 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 10e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) to give 222mg of yellow solid (10) in the yield of 68%.mp 89.2-91.5℃.1H NMR(400MHz,CDCl3)δ7.13(s,2H),6.73(m,2H),6.56(s,1H),5.93(s,2H),3.94(s,3H),3.92(s,6H),3.29(s,1H).13C NMR(150MHz,CDCl3)δ163.69,153.86,147.86,147.00,142.21,134.96,119.84,119.11,108.22,106.69,106.39,101.05,61.04,56.39,35.17.ESI-HRMS(m/z):calcd for C18H17NO5Na(M+Na+),350.0998;found,350.0991..
EXAMPLE 11 Synthesis of Compound 2- (3, 4-dimethoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (11)
The present invention synthesizes the target compound 11 according to the following route:
11.1 Synthesis of 2- (3, 4-dimethoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one (11 c) 3, 4-dimethoxyphenyl acetonitrile 11a (531 mg,3 mmol), 3,4, 5-trimethoxyphenyl boric acid 11b (1.27 g,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were then added to dissolve the substrate completely, nitrogen was replaced, and trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions, and the temperature was raised to 80℃for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 840mg of white solid (11 c) in yield 81%.mp92.5-93.5℃.1H NMR(400MHz,CDCl3)δ7.27(s,2H),6.83(s,2H),6.79(s,1H),4.20(s,2H),3.91(s,3H),3.90(s,6H),3.86(s,6H).13C NMR(150MHz,CDCl3)δ196.71,153.02,149.11,148.04,142.60,131.70,127.21,121.43,112.32,111.35,106.23,60.95,56.27,55.89,55.86,45.20.ESI-HRMS(m/z):calcd for C19H22O6Na(M+Na+),369.1308;found,369.1311.
11.2 Synthesis of 2- (3, 4-dimethoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (11 d)
2- (3, 4-Dimethoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one 11c (1.03 g,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared to stop the reaction. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) to give 898mg as a white solid (11 d) in yield 83%.mp 137.4-139.9℃.1HNMR(400MHz,CDCl3)δ8.73(s,1H),6.86(s,2H),6.83(s,1H),6.79(d,J=5.6Hz,2H),4.12(s,2H),3.84(s,6H),3.81(s,9H).13C NMR(150MHz,CDCl3)δ157.58,153.11,149.06,147.65,139.13,131.22,129.16,120.49,111.86,111.28,103.93,60.89,56.12,55.88,55.81,31.86.ESI-HRMS(m/z):calcd for C19H23NO6Na(M+Na+),384.1417;found,384.1421.
11.3 Synthesis of 2- (3, 4-dimethoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (11)
2- (3, 4-Dimethoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (361 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 11e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) gave 264mg of yellow solid (11) in yield 77%.mp 121.5-124.7℃.1H NMR(400MHz,CDCl3)δ7.14(s,2H),6.82(d,J=8.0Hz,1H),6.74(d,J=7.7Hz,1H),6.66(s,1H),3.94(s,3H),3.92(s,6H),3.87(s,3H),3.84(s,3H),3.31(s,1H).13C NMR(150MHz,CDCl3)δ163.78,153.85,149.01,148.49,142.17,133.44,119.26,118.56,111.18,109.18,106.67,61.04,56.39,55.99,55.89,35.16.ESI-HRMS(m/z):calcd for C19H21NO5Na(M+Na+),366.1311;found,366.1312..
EXAMPLE 12 Synthesis of Compound 2- (4-ethoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-az-cine (12)
The present invention synthesizes the target compound 12 as follows:
12.1 Synthesis of 2- (4-ethoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one (12 c)
4-Ethoxyphenylacetonitrile 12a (483 mg,3 mmol), 3,4, 5-trimethoxyphenylboronic acid 12b (1.27 g,6 mmol), palladium acetate (11 mg,5 mol%), 2 '-bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to Schlenk's tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were added to dissolve the substrate completely, nitrogen was replaced, trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice bath conditions, and the temperature was raised to 80℃for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 831mg of white solid (12 c) in yield of 84%.mp 62.8-64.9℃.1H NMR(400MHz,CDCl3)δ7.26(s,2H),7.17(d,J=7.6Hz,2H),6.86(d,J=7.4Hz,2H),4.18(s,2H),4.01(q,J=6.4Hz,2H),3.90(s,3H),3.89(s,6H),1.40(t,J=6.9Hz,3H).13C NMR(150MHz,CDCl3)δ196.82,157.94,153.00,142.51,131.73,130.24,126.62,114.78,106.23,63.43,60.94,56.25,44.79,14.84.ESI-HRMS(m/z):calcd for C19H22O5Na(M+Na+),353.1359found,353.1357.
12.2 Synthesis of 2- (4-ethoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (12 d)
2- (4-Ethoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one 12c (990 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL of anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and the reaction was stopped by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) to give 859mg of white solid (12 d) in yield 85%.mp 148.0-150.3℃.1HNMR(400MHz,CDCl3)δ9.01(s,1H),7.18(d,J=8.6Hz,2H),6.85(s,2H),6.80(d,J=8.7Hz,2H),4.11(s,2H),3.98(q,J=7.0Hz,2H),3.84(s,3H),3.80(s,6H),1.38(t,J=7.0Hz,3H).13C NMR(150MHz,CDCl3)δ157.63,157.53,153.08,139.06,131.17,129.51,128.51,114.65,103.90,63.40,60.88,56.09,31.45,14.84.ESI-HRMS(m/z):calcd for C19H23NO5Na(M+Na+),368.1468;found,368.1469.
12.3 Synthesis of 2- (4-ethoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (12) 2- (4-ethoxyphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (345 mg,1 mmol) was added to a 100mL eggplant-type bottle, triethylamine (0.28 mL,2 mmol) and solvent dichloromethane (15 mL) were added, and acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise under ice-bath conditions and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 12e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) to give 242mg of yellow solid (12) in the yield of 74%.mp 103.1-105.7℃.1HNMR(400MHz,CDCl3)δ7.14(s,2H),7.08(d,J=8.6Hz,2H),6.83(d,J=8.6Hz,2H),4.01(q,J=7.0Hz,2H),3.94(s,3H),3.91(s,6H),3.31(s,1H),1.40(t,J=7.0Hz,3H).13C NMR(150MHz,CDCl3)δ163.83,158.41,153.84,142.09,132.72,127.38,119.43,114.44,106.63,63.50,61.04,56.39,34.89,14.83.ESI-HRMS(m/z):calcd for C19H21NO4Na(M+Na+),350.1362;found,350.1354..
EXAMPLE 13 Synthesis of Compound 2- (4-methylphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-az-cine (13)
The present invention synthesizes the target compound 13 as follows:
13.1 Synthesis of 2- (4-methylphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one (13 c)
4-Methylphenylacetonitrile 13a (393 mg,3 mmol), 3,4, 5-trimethoxyphenylboronic acid 13b (1.27 g,6 mmol), palladium acetate (11 mg,5 mol%), 2 '-bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to Schlenk's tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were added to dissolve the substrate completely, nitrogen was replaced, trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice bath conditions, and the temperature was raised to 80℃for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to yield 783mg of white solid (13 c) in yield 87%.mp 75.1-76.3℃.1H NMR(400MHz,CDCl3)δ7.26(s,2H),7.15(dd,J=11.6,8.4Hz,4H),4.21(s,2H),3.90(s,3H),3.88(s,6H),2.32(s,3H).13C NMR(151MHz,CDCl3)δ196.66,152.99,142.51,136.55,131.71,129.48,129.11,106.24,60.93,56.24,45.28,21.07.ESI-HRMS(m/z):calcd for C18H20O4Na(M+Na+),323.1253;found,323.1247.
13.2 Synthesis of 2- (4-methylphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (13 d)
2- (4-Methylphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one 13c (900 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and the reaction was stopped by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) gave 812mg of a clear colorless oil (13 d) in the yield of 86%.1HNMR(400MHz,CDCl3)δ7.16(d,J=8.0Hz,2H),7.08(d,J=7.9Hz,2H),6.86(s,2H),4.14(s,2H),3.84(s,3H),3.81(s,6H),2.30(s,3H).13C NMR(150MHz,CDCl3)δ157.50,153.09,139.08,135.98,133.59,131.14,129.36,128.38,103.88,60.87,60.44,56.09,31.82,21.07,21.02,14.20.ESI-HRMS(m/z):calcd for C18H21NO4Na(M+Na+),338.1362;found,338.1360.
13.3 Synthesis of 2- (4-methylphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (13) 2- (4-methylphenyl) -1- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (315 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and solvent dichloromethane (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice-bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 13e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) gave 237mg of yellow solid (13) in yield 80%.mp 89.6-92.4℃.1HNMR(400MHz,CDCl3)δ7.14(s,2H),7.11(d,J=8.0Hz,1H),7.06(d,J=8.1Hz,1H),3.94(s,3H),3.91(s,6H),3.32(s,1H),2.33(s,3H).13C NMR(150MHz,CDCl3)δ163.44,153.83,142.13,137.84,136.92,129.05,126.18,119.28,106.70,61.04,56.38,35.08,21.14.ESI-HRMS(m/z):calcd for C18H19NO3Na(M+Na+),320.1257;found,320.1254..
EXAMPLE 14 Synthesis of Compound 2- (4-methylthiophenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (14)
The present invention synthesizes the target compound 14 as follows:
14.1 Synthesis of 2- (4-methylthiophenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one (14 c)
4-Methylthiophenylacetonitrile 14a (4819 mg,3 mmol), 3,4, 5-trimethoxyphenylboronic acid 14b (1.27 g,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were further added to dissolve the substrate completely, and trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions in place of nitrogen, and the reaction was carried out at 80℃for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 866mg of red solid (14 c) in the yield of 87%.mp 104.0-106.8℃.1HNMR(400MHz,CDCl3)δ7.25(s,2H),7.21(q,J=8.3Hz,4H),4.21(s,2H),3.91(s,3H),3.89(s,6H),2.47(s,3H).13C NMR(151MHz,CDCl3)δ196.33,153.03,142.64,137.04,131.61,131.54,129.77,127.01,106.19,60.95,56.27,44.99,15.92.ESI-HRMS(m/z):calcd for C18H20O4SNa(M+Na+),355.0974;found,355.0971.
14.2 Synthesis of 2- (4-methylthiophenyl) -1- (3, 4, 5-trimethoxyphenyl) ethane-1-ketoxime (14 d)
2- (4-Methylsulfanyl-phenyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one 14c (996 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared to stop the reaction. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) to give 822mg of white solid (14 d) in yield 79%.mp 95.6-98.9℃.1HNMR(400MHz,CDCl3)δ7.19(m,4H),6.84(s,2H),4.13(s,2H),3.84(s,3H),3.81(s,6H),2.45(s,3H).13C NMR(150MHz,CDCl3)δ157.24,153.13,139.19,136.31,133.59,130.94,129.02,127.10,103.87,60.89,56.12,31.68,16.02.ESI-HRMS(m/z):calcd for C18H21NO4SNa(M+Na+),370.1083;found,370.1081.
14.3 Synthesis of 2- (4-methylthiophenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (14)
2- (4-Methylsulfanyl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one oxime (277 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise under ice-bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 14e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) gave 233mg of yellow solid (14) in yield 71%.mp 84.7-86.0℃.1HNMR(400MHz,CDCl3)δ7.20(d,J=8.3Hz,2H),7.13(s,2H),7.09(d,J=8.3Hz,2H),3.94(s,3H),3.91(s,6H),3.31(s,1H),2.47(s,3H).13C NMR(150MHz,CDCl3)δ163.28,153.86,142.25,137.92,137.22,126.72,119.01,106.73,61.05,56.40,34.84,16.05.ESI-HRMS(m/z):calcd for C18H19NO3SNa(M+Na+),352.0977;found,352.0981..
EXAMPLE 15 Synthesis of the Compound 3- (naphthalen-2-yl) -2- (3, 4, 5-trimethoxyphenyl) -2H-az-cine (15)
The present invention synthesizes the target compound 15 as follows:
15.1 Synthesis of 1- (naphthalen-1-yl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one (15 c)
3,4, 5-Trimethoxybenzyl cyanide 15a (627mg, 3 mmol), 2-naphthylboric acid 15b (1.03 g,6 mmol), palladium acetate (11 mg,5 mol%), 2 '-bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to Schlenk's tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were further added to dissolve the substrate completely, nitrogen was replaced, trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) gave 849mg of yellow oil (15 c) in yield 84%.1HNMR(400MHz,CDCl3)δ8.55(s,1H),8.06(d,J=8.6Hz,1H),7.96(d,J=8.0Hz,1H),7.88(t,J=8.9Hz,2H),7.58(m,2H),6.53(s,2H),4.35(s,2H),3.83(d,J=3.7Hz,9H).13C NMR(150MHz,CDCl3)δ197.54,153.35,136.91,135.62,133.89,132.48,130.35,130.19,129.59,128.64,128.57,127.80,126.88,124.19,106.52,60.83,56.10,45.76.ESI-HRMS(m/z):calcdfor C21H21O4(M+H+),337.1434;found,337.1432.
15.2 Synthesis of 1- (naphthalen-1-yl) -2- (3, 4, 5-trimethoxyphenyl) ethane-1-oxime (15 d)
1- (Naphthalen-1-yl) -2- (3, 4, 5-trimethoxyphenyl) ethan-1-one 15c (1.01 g,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared to stop the reaction. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) gave 770mg of colorless oil (15 d) in yield 73%.1HNMR(400MHz,CDCl3)δ8.04(s,1H),7.84(m,4H),7.55–7.44(m,2H),6.53(s,2H),4.27(s,2H),3.80(s,3H),3.78(s,6H).13C NMR(150MHz,CDCl3)δ157.54,153.32,136.52,133.70,133.05,132.98,132.19,128.50,128.27,127.65,126.82,126.49,126.45,123.65,105.48,105.00,60.83,56.22,56.06,32.11.ESI-HRMS(m/z):calcdfor C21H22NO4(M+H+),352.1543;found,352.1537.
15.3 Synthesis of 3- (naphthalen-2-yl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (15)
1- (Naphthalen-1-yl) -2- (3, 4, 5-trimethoxyphenyl) ethane-1-oxime (352 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 15e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) gave 223mg of yellow oil (15) in yield 67%.1HNMR(400MHz,CDCl3)δ8.32(s,1H),8.03(q,J=8.5Hz,2H),7.93(t,J=6.9Hz,2H),7.60(m,2H),6.41(s,2H),3.82(s,3H),3.80(s,6H),3.35(s,1H).13C NMR(150MHz,CDCl3)δ191.09,163.79,153.64,153.36,137.32,136.67,135.62,132.84,132.05,129.39,129.08,128.75,128.11,127.23,124.56,121.23,106.70,102.87,60.87,56.05,35.05.ESI-HRMS(m/z):calcd for C21H20NO3(M+H+),334.1438;found,334.1435..
EXAMPLE 16 Synthesis of Compound 3-phenyl-2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (16)
The present invention synthesizes the target compound 16 as follows:
16.1 Synthesis of 1-phenyl-2- (3, 4, 5-trimethoxyphenyl) ethan-1-one (16 c)
3,4, 5-Trimethoxybenzyl cyanide 16a (627mg, 3 mmol), phenylboronic acid 16b (732 mg,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were added to dissolve the substrate completely, nitrogen was replaced, and trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions, and the temperature was raised to 80℃for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) gave a yellow oil in the yield of yellow oil 79%.1H NMR(400MHz,CDCl3)δ8.07–7.95(m,2H),7.56(d,J=7.3Hz,1H),7.47(t,J=7.5Hz,2H),6.48(s,2H),4.22(s,2H),3.83(s,6H),3.83(s,3H).13C NMR(150MHz,CDCl3)δ197.59,153.33,136.90,136.57,133.29,130.04,128.69,128.57,106.47,60.83,56.09,45.71.ESI-HRMS(m/z):calcd for C17H19O4(M+H+),287.1278;found,287.1275.
16.2 Synthesis of 1-phenyl-2- (3, 4, 5-trimethoxyphenyl) ethane-1-oxime (16 d)
1-Phenyl-2- (3, 4, 5-trimethoxyphenyl) ethan-1-one 16c (861 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared to stop the reaction. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) gave 697mg of yellow oil (16 d) in yield 77%.1H NMR(400MHz,CDCl3)δ7.67–7.57(m,2H),7.36(s,3H),6.47(s,2H),4.15(s,2H),3.80(s,3H),3.78(s,6H).13C NMR(150MHz,CDCl3)δ157.50,153.25,136.49,135.68,132.03,129.39,128.56,126.51,105.49,60.83,56.04,32.39.ESI-HRMS(m/z):calcd for C17H20NO4(M+H+),302.1387;found,302.1385.
16.3 Synthesis of 3-phenyl-2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (16)
1-Phenyl-2- (3, 4, 5-trimethoxyphenyl) ethane-1-oxime (302 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 16e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) gave 181mg of yellow oil (16) in yield 64%.1HNMR(400MHz,CDCl3)δ7.92(d,J=7.0Hz,2H),7.66–7.48(m,3H),6.36(s,2H),3.82(s,3H),3.80(s,6H),3.26(s,1H).13C NMR(150MHz,CDCl3)δ163.80,153.32,137.29,136.61,133.29,129.88,129.29,124.00,102.81,60.86,56.05,34.79.ESI-HRMS(m/z):calcd for C17H18NO3(M+H+),284.1281;found,284.1279..
EXAMPLE 17 Synthesis of Compound 2- (naphthalen-2-yl) -3- (3, 4, 5-trimethoxyphenyl) -2H-az-cine (17)
The present invention synthesizes the target compound 17 according to the following route:
17.1 Synthesis of 2- (naphthalen-2-yl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one (17 c)
2-Naphthylacetonitrile 17a (501 mg,3 mmol), 3,4, 5-trimethoxyphenylboronic acid 17b (1.27 g,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were further added to dissolve the substrate completely, nitrogen was replaced, trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions, and the temperature was raised to 80℃for reaction for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) gave 836mg of yellow oil (17 c) in yield 83%.1HNMR(400MHz,CDCl3)δ7.80(m,3H),7.72(s,1H),7.50–7.42(m,2H),7.40(d,J=8.4Hz,1H),7.30(s,1H),4.41(s,2H),3.89(s,3H),3.88(s,6H).13C NMR(150MHz,CDCl3)δ196.44,153.03,142.62,133.57,132.36,131.67,128.42,127.94,127.68,127.57,127.40,126.21,125.80,106.27,60.93,56.25,45.81.ESI-HRMS(m/z):calcd for C21H21O4(M+H+),337.1434;found,337.1432.
17.2 Synthesis of 2- (naphthalen-2-yl) -1- (3, 4, 5-trimethoxyphenyl) ethane-1-oxime (17 d)
2- (Naphthalen-2-yl) -1- (3, 4, 5-trimethoxyphenyl) ethan-1-one 17c (1.01 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared to stop the reaction. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) gave 834mg of yellow oil (17 d) in yield 79%.1HNMR(400MHz,CDCl3)δ7.74(m,4H),7.43(s,3H),6.90(s,2H),4.34(s,2H),3.82(s,3H),3.77(s,6H).13C NMR(150MHz,CDCl3)δ157.26,153.13,139.17,134.22,133.61,132.17,131.05,128.36,127.62,127.49,126.98,126.85,126.12,125.55,103.92,60.86,56.08,32.47.ESI-HRMS(m/z):calcd for C21H22NO4(M+H+),352.1543;found,352.1540.
17.3 Synthesis of 2- (naphthalen-2-yl) -3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (17)
2- (Naphthalen-2-yl) -1- (3, 4, 5-trimethoxyphenyl) ethane-1-oxime (352 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 17e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) gave 253mg of yellow oil (17) in yield 76%.1HNMR(400MHz,CDCl3)δ7.86–7.72(m,3H),7.67(s,1H),7.45(d,J=6.1Hz,2H),7.23(d,J=8.5Hz,1H),7.17(s,2H),3.94(s,3H),3.90(s,6H),3.50(s,1H).13C NMR(150MHz,CDCl3)δ163.21,153.88,142.28,138.44,133.25,132.79,128.08,127.73,127.53,126.30,125.62,125.05,124.08,119.05,106.82,61.04,56.37,35.41.ESI-HRMS(m/z):calcd for C21H20NO3(M+H+),334.1438;found,334.1435..
EXAMPLE 18 Synthesis of Compound 2-phenyl-3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (18)
The present invention synthesizes the target compound 18 as follows:
18.1 Synthesis of 2-phenyl-1- (3, 4, 5-trimethoxyphenyl) ethan-1-one (18 c)
Phenyl acetonitrile 18a (351 mg,3 mmol), 3,4, 5-trimethoxyphenylboronic acid 18b (1.27 g,6 mmol), palladium acetate (11 mg,5 mol%), 2' -bipyridine (15 mg,10 mol%) and potassium fluoride (348 mg,6 mmol) were added to a Schlenk tube (100 mL), tetrahydrofuran (40 mL) and water (10 mL) were added to dissolve the substrate completely, nitrogen was replaced, and trifluoroacetic acid (2.23 mL,30 mmol) was slowly added dropwise under ice-bath conditions, and the temperature was raised to 80℃for 2 to 3 hours. The TLC plate detects the reaction, when the raw materials completely disappear, the reaction is quenched by adding saturated sodium bicarbonate solution (30 mL), the aqueous phase (3X 15 mL) is extracted by ethyl acetate, the organic phases are combined, the organic phases are washed by saturated saline water, dried by anhydrous sodium sulfate and filtered, the obtained filtrate is concentrated, and then the filtrate is separated and purified by wet-process loading silica gel column chromatography (200-300 meshes), and the eluent: petroleum ether-ethyl acetate (5:1) to give 720mg of white solid (18 c) in the yield of 84%.1HNMR(400MHz,CDCl3)δ7.33(t,J=7.2Hz,2H),7.29–7.22(m,5H),4.25(s,2H),3.90(s,3H),3.88(s,6H).13C NMR(150MHz,CDCl3)δ196.45,153.01,142.57,134.84,131.69,129.27,128.76,126.94,106.24,60.93,56.24,45.65.ESI-HRMS(m/z):calcd for C17H19O4(M+H+),287.1278;found,287.1279.
18.2 Synthesis of 2-phenyl-1- (3, 4, 5-trimethoxyphenyl) ethane-1-oxime (18 d)
2-Phenyl-1- (3, 4, 5-trimethoxyphenyl) ethan-1-one 18c (861 mg,3 mmol) was added to a 100mL eggplant-shaped bottle, 35mL anhydrous methanol was added, hydroxylamine hydrochloride (310 mg,4.5 mmol) and potassium carbonate (8238 mg,6 mmol) were added respectively, the temperature was raised to 50℃for reaction, and detection was performed by TLC plate or LC/MS low resolution mass spectrometry until the starting material disappeared to stop the reaction. Concentrating the reaction solution, separating and purifying by wet-process sample loading silica gel column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (10:3) gave 679mg of yellow oil (18 d) in the yield of 75%.1HNMR(400MHz,CDCl3)δ7.27(t,J=4.6Hz,4H),7.20(dd,J=8.2,4.0Hz,1H),6.85(s,2H),4.18(s,2H),3.84(s,3H),3.80(s,6H).13C NMR(150MHz,CDCl3)δ157.30,153.10,139.12,136.73,131.07,128.68,128.52,126.46,103.89,60.88,56.08,32.31.ESI-HRMS(m/z):calcd for C17H20NO4(M+H+),302.1387;found,302.1386.
18.3 Synthesis of 2-phenyl-3- (3, 4, 5-trimethoxyphenyl) -2H-azepine (18)
2-Phenyl-1- (3, 4, 5-trimethoxyphenyl) ethane-1-oxime (302 mg,1 mmol) was added to a 100mL eggplant-shaped bottle, triethylamine (0.28 mL,2 mmol) and methylene chloride (15 mL) were added thereto, acetic anhydride (0.15 mL,1.5 mmol) was slowly added dropwise thereto under ice bath conditions, and the mixture was allowed to react at room temperature. The reaction was quenched by adding water (15 mL) to the reaction system, extracting the aqueous phase (3X 10 mL) with methylene chloride, combining the organic phases, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating the filtrate to give crude 18e, which was then directly put into the next reaction without further isolation and purification. The crude product was dissolved in DMF (10 mL), cesium carbonate (276 mg,2 mmol) was added, stirring was performed at 80℃for 1 hour under nitrogen protection, TLC plates were used to detect the disappearance of the starting material, water (15 mL) was added to the reaction system, the aqueous phase (3X 10 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, the obtained filtrate was concentrated, and wet-loaded onto silica gel column chromatography (200-300 mesh) for separation and purification, eluent: petroleum ether-ethyl acetate (5:1) gave 207mg of yellow oil (18) in yield 73%.1HNMR(400MHz,CDCl3)δ7.34–7.21(m,3H),7.20–7.15(m,2H),7.14(s,2H),3.94(d,J=1.6Hz,3H),3.90(d,J=1.6Hz,6H),3.33(s,1H).13C NMR(150MHz,CDCl3)δ163.12,153.85,142.21,140.89,128.33,127.15,126.23,119.08,106.76,61.03,56.39,35.17.ESI-HRMS(m/z):calcd for C17H18NO3(M+H+),284.1281;found,284.1279..
EXAMPLE 19 Synthesis of Compound 2- (3-fluoro-4-methoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -aziridine (19)
The present invention synthesizes the target compound 19 according to the following scheme:
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2- (3-fluoro-4-methoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -2H-az-cause (115 mg,0.345 mmol) and sodium borohydride (50 mg,1.39 mmol) were added to a 25mL eggplant-shaped bottle, tetrahydrofuran (5 mL) was added, and the mixture was stirred at room temperature for two hours, and the TLC plate was used to detect the completion of the reaction. The reaction solution was concentrated to dryness, water (5 mL) was added to the eggplant-shaped flask, the aqueous phase (3×5 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the obtained filtrate was concentrated, and then purified by column chromatography (200 to 300 mesh), eluent: petroleum ether-ethyl acetate (10:3) to give 105mg of yellow solid (19), yield 91%.mp 117.4-120.3℃.1H NMR(400MHz,CDCl3)δ6.99(dd,J=12.4,1.8Hz,1H),6.91(d,J=8.4Hz,1H),6.75(t,J=8.5Hz,1H),6.39(s,2H),3.81(s,3H),3.76(s,3H),3.74(s,6H),3.50(q,J=6.5Hz,2H).13C NMR(150MHz,CDCl3)δ152.65,151.06,146.36,146.29,136.67,132.06,129.81,129.77,123.58,123.56,115.86,115.74,112.73,104.73,60.80,56.21,55.96,39.90,39.17.ESI-HRMS(m/z):calcd for C18H20FNO4(M+H+),334.1449;found,334.1445..
EXAMPLE 20 Synthesis of the Compound 2- (3-fluoro-4-methoxyphenyl) -1-methyl-3- (3, 4, 5-trimethoxyphenyl) aziridine (20)
The present invention synthesizes the target compound 20 as follows:
2- (3-fluoro-4-methoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) -aziridine (100 mg,0.298 mmol) was added to a 25mL eggplant-shaped bottle, methyl iodide (5 mL) was added, and the mixture was stirred at room temperature for 3 hours, and the completion of the reaction was detected by TLC plate. Concentrating the reaction solution, separating and purifying by column chromatography (200-300 meshes), eluting with an eluent: petroleum ether-ethyl acetate (2:1) to give a white solid (20) 84mg, yield 81%.mp 110.6-112.8℃.1H NMR(400MHz,CDCl3)δ6.97(d,J=12.4Hz,1H),6.87(d,J=8.4Hz,1H),6.75(t,J=8.5Hz,1H),6.35(s,2H),3.81(s,3H),3.75(s,3H),3.73(s,6H),2.74(q,J=6.5Hz,2H),2.68(s,3H).13C NMR(150MHz,CDCl3)δ152.67,151.08,146.30,146.22,136.60,131.97,129.68,129.64,123.47,115.82,115.69,112.79,104.63,60.80,56.25,55.95,50.54,49.70,47.77.ESI-HRMS(m/z):calcd for C19H22FNO4Na(M+Na+),370.1403;found,370.1425..
EXAMPLE 21 test of in vitro Activity of target Compounds to inhibit proliferation of human tumor cells
Five human tumor cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM): the human ovarian cancer cell line (A2780), the human colon cancer cell line (HCT-116), the human lung adenocarcinoma cell line (A549), the human cervical cancer cell line (HeLa) and the human acute T-lymphoblastic leukemia cell line (Jurkat) were cultured at 37℃and with 5% CO 2, the medium containing 100 units/mL penicillin G, 100. Mu.g/mL streptomycin and 10% (V/V) fetal bovine serum. Compounds were tested for in vitro antiproliferative activity by MTT method and positive samples CA-4 and mitomycin C were used as controls. Briefly, cells were first seeded in 96-well plates (2.5X10 3 cells per well) containing 100. Mu.L of growth medium, after incubation for 24 hours, cells were treated with different concentrations of the test compound, after 48 hours of incubation, 20. Mu. LMTT solution (5 mg/mL) was added to each well, then incubated at 37℃for another 4 hours, the suspension was discarded and dimethylsulfoxide (150. Mu.L) was added to each well and lysed by shaking for 10 minutes. The absorbance of each well was measured at 570nm wavelength using a microplate reader (Biotech ELX 800) and the inhibition and IC 50 values were calculated using GRAPHPAD PRISM software (version 6.0). The results are shown in Table 1:
table 1.2,3-diaryl-2H-aziridines and 2, 3-diaryl-aziridines in vitro anti-tumor cell proliferation Activity (IC 50/. Mu.M)
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Example 22 inhibition of tubulin aggregation assay: in vitro tubulin self-assembly experiments
Purified porcine brain tubulin polymerization kit was purchased from Cytoskeleton, usa, and the test compounds were evaluated for inhibition of microtubule aggregation in vitro by nephelometry. Tubulin aggregation buffer contains 100mM PIPES (pH=6.7), 10mM magnesium chloride, 1mM ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA), 1mM GTP and 3.4M glycerol. And adding the compounds to be tested with different concentrations into the buffer solution, and taking colchicine as a positive control sample and dimethyl sulfoxide as a negative control sample. And then placing the treated tubulin sample in a 37 ℃ environment, detecting the absorbance of the system at 340nm by using a SpectraMax 190 spectrophotometer of a light absorption enzyme label instrument of Molecular Devices company in the United states, drawing an absorbance curve, and analyzing to obtain the aggregation inhibition activity of the compound on the tubulin. Plotting a graph (as shown in fig. 1) from absorbance; the result shows that the compound 8 can obviously inhibit the aggregation of microtubulin, the IC 50 is 3.3 mu M, and the inhibition effect is more obvious than that of the positive control colchicine (shown in table 2);
TABLE 2 microtubule aggregation inhibiting Activity of Compound 8
Example 23 inhibition of tubulin aggregation assay: immunofluorescence experiment for detecting tubulin morphology
Human cervical cancer cells (HeLa) were seeded in 6-well plates, cells were treated with different concentrations of the preferred compound (100 nm,200 nm) while 0.1% dimethyl sulfoxide was used as negative control and CA-4 (10 nm) as positive control, after incubation at 37℃for 24 hours under 5% CO 2, the medium was washed twice with PBS, the cells were fixed with methanol, and 4 minutes of permeation in PBS with 0.1% Triton X-100. Cells were incubated in PBS solution containing 1% Bovine Serum Albumin (BSA) for 1 hour to block non-specific antibody binding. The cells were then incubated with monoclonal antibody (anti- α -tubulin) for 4h at room temperature, stained with fluorescent antibody, washed three times with PBS, and then nuclei were labeled with 4', 6-diamidino-2-phenylindole (DAPI). Finally, the cells were washed three times with PBS and the effect of the test compound on the tubulin wire structure was observed under a fluorescence microscope (OLYMPUS), and the results of the experiment were recorded by photographing (see FIG. 2).
Example 24 in vitro inhibition of neovascularization experiments
Matrigel MatriGel was thawed at 4 ℃, human Umbilical Vein Endothelial Cells (HUVEC) suspended in Dulbecco's Modified Eagle Medium (DMEM) were incubated at 37 ℃ for 30min, after which they were inoculated into 96-well plates, and then the cells were treated with different concentrations of the preferred compound (75 nm,150 nm) while using 0.1% dimethyl sulfoxide as a negative control, and after incubation at 37 ℃ for 12h at 5% co 2, the capillary formation was observed and photographed under an inverted microscope (OLYMPUS) (as shown in fig. 3).
EXAMPLE 25 alkaline Single cell gel electrophoresis (comet assay)
The test kit is purchased from Trevigen company in the United states, and is used for detecting the damage effect of a preferred compound on DNA by adopting an alkaline comet assay, firstly, acute T lymphocyte leukemia cells (Jurkat) are amplified and cultured, and are washed by using RPMI-1640 complete culture medium, and when the cells grow to the logarithmic phase, the cell density is adjusted to be 1 multiplied by 10 5 cells/mL. Jurkat cells were seeded into six well plates with 3mL per well. Then, the cells were treated with different concentrations of the preferred compound (100 nm,200nm,400 nm) while using dimethyl sulfoxide as a negative control and mitomycin C (200 nm) as a positive control, respectively culturing at 37℃under 5% CO 2 for 48 hours, discarding the upper layer solution in the culture plate, washing the cells with 2mL of PBS for 2 times, adding pancreatin, adding 1mL of DMEM medium into the well plate after the cells were detached, gently beating to completely detach the cells, sucking the cells into a 5mL centrifuge tube after the cells were evenly beaten, centrifuging (1000 r/min,3 min), sucking the supernatant after centrifuging, washing the cells with PBS twice. The cell suspension was mixed with low melting point agarose at 37 ℃ and immediately smeared with a pipette onto a comet assay slide. The slide glass is solidified for 10 minutes at 4 ℃, immersed in the newly prepared lysate for 2 hours, taken out from the lysate, washed out of excessive salt in distilled water and dried. This was immersed in a newly prepared alkaline DNA solution (1mM EDTA,200mM NaOH) for 30 minutes, and subjected to electrophoresis separation (25V, 15 min) at 4 ℃. After electrophoresis, the slide was placed in a plate, rinsed twice with deionized water, rinsed once with 70% ethanol to render the slide neutral, dried at 37 ℃ for 15 minutes, stained with 50 μl to 100 μl acridine orange solution (EB) for 10 minutes, and rinsed twice with deionized water or more. After staining, the staining was performed as soon as possible using a fluorescence microscope (OLYMPOS BX a) (as shown in fig. 4), and too long a time would lead to fluorescent discoloration. The above procedure was all performed in the dark to avoid additional DNA damage.
Example 26 in vitro detection of expression of DNA damage inducible factors
HeLa cells in the logarithmic growth phase were digested completely with pancreatin and blown into single-cell solutions, which were counted and inoculated into culture dishes 60mm in diameter, each containing 1X 10 6 cells. The cells were incubated at 37℃under 5% carbon dioxide for 24 hours, respectively, and treated with different concentrations of the preferred compound 8 (0.8 μm,1.6 μm,3.2 μm) while the incubation was continued for 48 hours with dimethyl sulfoxide as a negative control. The culture broth was then discarded, the cells were added to a centrifuge tube and centrifuged at low speed for 10 minutes, the supernatant was discarded, the cells were collected, the protein samples were obtained by lysis with RIPA lysate, and the samples were quantitatively loaded on polyacrylamide gels, subjected to electrophoresis (SDS-PAGE) at 100V voltage, SDS-PAGE protein gel transfer (PVDF membrane), and membrane-blocked. Then, the primary antibody incubation, primary antibody washing, secondary antibody incubation and secondary antibody washing are sequentially carried out. At the end of incubation, development imaging was performed (as shown in fig. 5).
EXAMPLE 27 cell colony formation assay
HeLa cells in the logarithmic growth phase were digested completely with pancreatin and blown into single cell solutions, counted and seeded into six well plates with 1000 cells per well. After the cells were attached, the cells were treated with various concentrations of the preferred compound 8 (0.5 μm,1.0 μm,2.0 μm,4.0 μm,8.0 μm) while being cultured with dimethyl sulfoxide as a negative control at 37℃under 5% carbon dioxide for 48 hours, the medium was changed, and after the culture was continued under the same conditions for one week, the liquid was sucked off, and then a layer of 2 to 3mm ice-cold methanol was covered on the cells. Cells were fixed at-20℃for 15 min. The fixative was blotted three times with PBS, stained with Giemsa or with crystal violet, the stain washed off with PBS, dried well, and colony formation observed under a microscope and counted (as shown in FIG. 6).
EXAMPLE 28 in vitro cell G2/M phase blocking assay
HeLa cells in the logarithmic growth phase were digested completely with pancreatin and blown into single-cell solutions, which were counted and inoculated into culture dishes 60mm in diameter, each containing 1X 10 6 cells. The cells were treated with different concentrations of the preferred compound 8 (0.4 μm,0.8 μm,1.6 μm,3.2 μm) at 37℃under 5% carbon dioxide for 24 hours, respectively, while the culture was continued with dimethyl sulfoxide as a negative control for 12 hours, the liquid was sucked off, and then the cells were covered with a layer of 2 to 3mm ice-cold 75% ethanol. Cells were fixed at-20 ℃. The fixative was blotted, rinsed three times with PBS, added with RNaseA-containing PBS, then stained with Propidium Iodide (PI) for 30 minutes in the dark, detected with a flow cytometer, and the test results analyzed with software (as shown in fig. 7).
Example 29 in vitro cell cycle related regulatory protein detection
HeLa cells in the logarithmic growth phase were digested completely with pancreatin and blown into single-cell solutions, which were counted and inoculated into culture dishes 60mm in diameter, each containing 1X 10 6 cells. The cells were incubated at 37℃under 5% carbon dioxide for 24 hours, respectively, and treated with different concentrations of the preferred compound 8 (0.8 μm,1.6 μm,3.2 μm) while the incubation was continued for 48 hours with dimethyl sulfoxide as a negative control. The culture broth was then discarded, the cells were added to a centrifuge tube and centrifuged at low speed for 10 minutes, the supernatant was discarded, the cells were collected, the protein samples were obtained by lysis with RIPA lysate, and the samples were quantitatively loaded on polyacrylamide gels, subjected to electrophoresis (SDS-PAGE) at 100V voltage, SDS-PAGE protein gel transfer (PVDF membrane), and membrane-blocked. Then, the primary antibody incubation, primary antibody washing, secondary antibody incubation and secondary antibody washing are sequentially carried out. At the end of incubation, development imaging was performed (as shown in fig. 8).
Example 30 in vitro apoptosis experiments
HeLa cells in the logarithmic growth phase were digested completely with pancreatin and blown into single-cell solutions, which were counted and inoculated into culture dishes 60mm in diameter, each containing 1X 10 5 cells. The cells were incubated at 37℃under 5% carbon dioxide for 24 hours, respectively, and treated with different concentrations of the preferred compound 8 (1 μm,2 μm,4 μm,8 μm) while the incubation was continued for 48 hours with dimethyl sulfoxide as a negative control. The culture was then discarded, washed three times with ice-cold PBS, the cells were added to a centrifuge tube and centrifuged at low speed for 10 minutes, the supernatant was discarded, the cells were collected, and then the cells were covered with a layer of ice-cold 75% ethanol of 2 to 3 mm. Cells were fixed at-20 ℃. The fixative was blotted dry, rinsed three times with PBS, double stained with Annexin V-APC and 7-AAD in the dark, and analyzed for apoptosis by flow cytometry (as shown in FIG. 9).
Example 31 in vitro apoptosis-related protein detection
HeLa cells in the logarithmic growth phase were digested completely with pancreatin and blown into single-cell solutions, which were counted and inoculated into culture dishes 60mm in diameter, each containing 1X 10 6 cells. The cells were incubated at 37℃under 5% carbon dioxide for 24 hours, respectively, and treated with different concentrations of the preferred compound 8 (0.8 μm,1.6 μm,3.2 μm) while the incubation was continued for 48 hours with dimethyl sulfoxide as a negative control. The culture broth was then discarded, the cells were added to a centrifuge tube and centrifuged at low speed for 10 minutes, the supernatant was discarded, the cells were collected, the protein samples were obtained by lysis with RIPA lysate, and the samples were quantitatively loaded on polyacrylamide gels, subjected to electrophoresis (SDS-PAGE) at 100V voltage, SDS-PAGE protein gel transfer (PVDF membrane), and membrane-blocked. Then, the primary antibody incubation, primary antibody washing, secondary antibody incubation and secondary antibody washing are sequentially carried out. At the end of incubation, development imaging was performed (as shown in fig. 10).
EXAMPLE 32 in vitro liver microsomal metabolic stability study
The preferred compound 8 was dissolved in dimethyl sulfoxide to prepare a test solution, while using CA-4, testosterone (substrate of P4503A4 enzyme), diclofenac (substrate of P4502C9 enzyme), and propafenone (substrate of P4502D6 enzyme) as positive control groups, added to a 96-well plate at 10. Mu.l per well, and stored at 4 ℃. The mixture containing the test compound and liver microsomes was pre-incubated for 10 minutes at 37 ℃. To all reaction plates, 80 microliters per well, all reaction plates containing the compound and microsome mixture were incubated at 37 ℃ for 10 minutes, and then the reaction was stopped by adding acetonitrile (MeCN) at five time points 5, 10, 20, 30, 60min, respectively, 300 microliters per well. After quenching the reaction at the above five time points, the samples were sealed and shaken for 10 minutes, after which each sample was subjected to detection by liquid chromatography mass spectrometry/mass spectrometry at 4 ℃ for 20 minutes at 4000 rpm for 100 μl of supernatant. The peak value of all compounds tested at the initial time (t=0 min) was set to 100% and the percentage of compounds tested at different metabolic times via hepatic microsome metabolism was converted to the percentage of residual content. The in vitro half-life (t 1/2) and other relevant data (shown in fig. 11 and table 3) were calculated using the linear regression slope of the percent compound remaining versus incubation time measured.
TABLE 3 half-life in vitro of Compound 8
EXAMPLE 33 study of tumor therapeutic action at animal level
The animal experiment scheme is approved by the animal ethics committee of the university of double denier pharmaceutical college. We purchased 6 week old female Balb/C nude mice from Shanghai Laek laboratory animals Inc., and implanted A2780 cells suspended in PBS on both subcutaneous sides of the mice to establish a nude mice tumor metastasis model. When the tumor volume reached 100mm 3, mice were randomly divided into four groups of ten animals each, and given by intraperitoneal injection: different concentrations of 8 (25 mg/kg,50 mg/kg), colchicine (10 m/kg) and blank (10% castor oil and 10% dimethyl sulfoxide). The signs of poisoning and death were observed daily, the body weight of the mice was weighed each time and the tumor volume was measured with vernier calipers (volume (mm 3) = (pi/6) ×length×width×width). When the tumor volume reached 2000mm 3, mice were sacrificed, tumors were isolated and weighed, and tumor suppression rates were calculated (as shown in fig. 12).
EXAMPLE 34 preliminary stability study
The 8 (5 mg/mL methanol, 20. Mu.L) solution was mixed with phosphate buffer (480. Mu.L, pH 7.4) and the resulting solution was filtered through a 0.22 μm microfiltration membrane. The filtrate was analyzed by HPLC (Poroshell ec-C183.0X10 mm,2.7 μm; mobile phase methanol/water=60:40, 1 mL/min) at pH 7.4 after 4h, 8h, 12h, 24h and 48h, respectively. After 48h, the content of compound 8 was found to be 95.77% in the pH 7.4 medium.
Example 35 enantiomer resolution and specific optical rotation measurement
Chiral resolution of enantiomer 8 was performed on Themo Fisher Ultimate 3000,3000 high performance liquid chromatograph using DAICEL CHIRALPAK IC chiral column with mobile phase n-hexane/isopropanol (80/20), flow rate of 1.0mL/min, and uv response detected at 230nm wavelength at 25 ℃. Specific optical rotation data were measured using Rudolph Autopol IV polarimeter at 25℃using chloroform as the solvent and a sample concentration of 1.0mg/mL (as shown in Table 4);
TABLE 4 in vitro antitumor Activity of enantiomers (IC 50/. Mu.M)
Example 36 absolute configuration determination
Synthesis of Compound (2R, 3S) -2- (3-fluoro-4-methoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) aziridine (21)
The present invention synthesizes the target compound 21 according to the following scheme:
Compound 21a was obtained by chiral column resolution of Compound 8, and (S) -3- (3-fluoro-4-methoxyphenyl) -2- (3, 4, 5-trimethoxyphenyl) -2H-azepine (115 mg,0.345 mmol) and sodium borohydride (50 mg,1.39 mmol) were added to a 25mL eggplant-shaped bottle, tetrahydrofuran (5 mL) was added, and stirring was performed at room temperature for two hours, and the reaction was detected by TLC plate. The reaction solution was concentrated to dryness, water (5 mL) was added to the eggplant-shaped flask, the aqueous phase (3×5 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the obtained filtrate was concentrated, and then purified by column chromatography (200 to 300 mesh), eluent: petroleum ether-ethyl acetate (10:3) to give 105mg of yellow solid (21), yield 91%.mp 117.4-120.3℃.1H NMR(400MHz,CDCl3)δ6.99(dd,J=12.4,1.8Hz,1H),6.91(d,J=8.4Hz,1H),6.75(t,J=8.5Hz,1H),6.39(s,2H),3.81(s,3H),3.76(s,3H),3.74(s,6H),3.50(q,J=6.5Hz,2H).13C NMR(150MHz,CDCl3)δ152.65,151.06,146.36,146.29,136.67,132.06,129.81,129.77,123.58,123.56,115.86,115.74,112.73,104.73,60.80,56.21,55.96,39.90,39.17.ESI-HRMS(m/z):calcd for C18H20FNO4(M+H+),334.1449;found,334.1445.
Synthesis of Compound (2R, 3S) -2- (3-fluoro-4-methoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) aziridine (22)
The present invention synthesizes the target compound 22 as follows:
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(2R, 3S) -2- (3-fluoro-4-methoxyphenyl) -3- (3, 4, 5-trimethoxyphenyl) aziridine (20 mg,0.06 mmol), p-nitrobenzenesulfonyl chloride (20 mg,0.09 mmol), triethylamine (17. Mu.L, 0.12 mmol) and 4-dimethylaminopyridine (1 mg,0.01 mmol) were added to a 25mL eggplant-shaped bottle, methylene chloride (3 mL) was added, and stirred at room temperature for 1 hour, and the TLC plate was used to detect the completion of the reaction. The reaction solution was concentrated to dryness, water (5 mL) was added to the eggplant-shaped flask, the aqueous phase (3×5 mL) was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the obtained filtrate was concentrated, and then purified by column chromatography (200 to 300 mesh), eluent: petroleum ether-ethyl acetate (5:1) to give 27mg of pale yellow solid (22), yield 87%.mp 133.1-134.2℃.1HNMR(400MHz,CDCl3)δ8.42(d,J=8.9Hz,2H),8.27(d,J=9.0Hz,2H),6.85–6.72(m,3H),6.22(s,2H),4.28–4.20(m,2H),3.80(s,3H),3.75(s,3H),3.65(s,6H).13C NMR(150MHz,CDCl3)δ153.06,152.72,151.08,150.83,147.70,147.63,143.69,138.03,129.29,126.38,124.52,124.11,124.07,123.70,115.59,115.46,113.08,104.84,60.82,56.19,56.02,48.34,47.44.ESI-HRMS(m/z):calcd for C24H23FN2O8S(M+Na+),541.1051;found,541.1050.
After single crystals were grown in the compound 22, the configuration of the 2-position carbon atom of the target compound 22 was obtained as (R) -configuration and the configuration of the 3-position carbon atom was obtained as (S) -configuration by X-ray diffraction. It was further deduced that (+) -8 is in the (R) configuration.
Single crystals of22suitable for X-ray crystallographic analysis were obtained by slow evaporation ofa solution of22in petroleum ether/Dichloromethane.
Meter 5.Crystal data and structure refinement for 22
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Claims (4)
1. Diaryl substituted-2H-azepine compounds characterized by the following structural compounds:
。
2. diaryl substituted aziridine compounds are characterized by the following structural compounds:
。
3. The compound according to any one of claims 1-2 and the use of pharmaceutically acceptable salts thereof for the preparation of a medicament for the prophylaxis and treatment of diseases associated with tumors, such as human ovarian cancer, human colon cancer, human lung adenocarcinoma, human cervical cancer;
The pharmaceutically acceptable salt is a salt formed by malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid, oxalic acid, phosphoric acid, halogen acid, sulfuric acid and nitric acid.
4. A compound medicine for preventing and treating tumor-related diseases, which is characterized by comprising the compound of any one of claims 1-2, wherein the tumor-related diseases are human ovarian cancer, human colon cancer, human lung adenocarcinoma and human cervical cancer.
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