Cauhinin type compound as well as preparation method and application thereof

Abstract

The invention discloses a Cauhinin type compound with structural formulas shown as formulas I and II. A C / D ring is constructed based on key reactions such as an asymmetric olefin insertion Mannich reaction, an N-alkylation reaction and a Mannich cyclization reaction, then a C15 site side chain is introduced through a Horner-Wadsworth-Emmons reaction to synthesize a general intermediate, finally oxidation and reduction are carried out at different stages through a chiral regulation unit, synthesis of C15-C20 site different chiral configurations of the C15-C20 site different chiral configurations of the C15-C20 site different chiral configurations of the C15-C20 site different chiral configurations of the C15-C20 site different chiral configurations of the C15-C20 site different chiral configurations is achieved, and the C15-C20 site different chiral configurations of the C15-C20 site different chiral configurations of the C15-C20 site different chiral configurations of the C15-C20 site different chiral configurations of the C15-C20 site different chiral configurations of The Cauhinin type compound provided by the invention has ferroptosis inhibitory activity and anti-neuroinflammation activity, and the compound provided by the invention has important medicinal value and wide application prospect in preparation of anti-ischemic stroke drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of drug synthesis, and specifically relates to a coranine-type compound and a preparation method thereof, and application of the compound in preparing a drug for treating ischemic stroke. Background Art

[0002] Natural products with complex and diverse structures and their analogs are the source of biological activity research and new drug development. The products launched can greatly improve and protect human health. Monoterpene indole alkaloids are the largest class of alkaloids. More than 15 families and more than 3,000 members have been discovered. They are the most diverse class of natural products in terms of stereochemistry and structure. Secondly, since most of these alkaloids are isolated from folk Chinese herbal medicine plants from the Rubiaceae, Loganaceae and Apocynaceae, they have a wide range of biological activities. Among them, there are dozens of clinical drugs, belonging to the classes of vinca alkaloids, camptothecins, quinines, strychnine, coryne and ellipticine.

[0003] The alkaloids of the konin family occupy an important position among the monoterpene indole alkaloids. Nearly 300 members have been reported so far, and most of the alkaloids in this family have a wide range of biological activities, mainly manifested in antiviral, antibacterial, anti-inflammatory, antimalarial, muscle relaxant, vascular relaxant, analgesic activity, and activity affecting the main nervous and cardiac systems. They also have NF-κB inhibition and Na + - Glucose co-transporter inhibitory properties. These alkaloids have very different biological activities due to their structural differences, and there is a small group of molecules in this family of natural products that are structurally similar, with only different chiral configurations at C15 and C20. We hope to find a universal intermediate to conduct synthetic studies on its analogs, so as to further explore the biological activity of this type of molecule through structure-activity analysis.

[0004] Ferroptosis is a type of programmed cell death discovered in 2012. It is iron-dependent and characterized by excessive lipid peroxidation. Under the action of ferrous iron or lipoxygenase, unsaturated fatty acids highly expressed on the cell membrane are catalyzed to cause lipid peroxidation, thereby inducing cell death. In addition, it is also manifested as a decrease in the regulatory core enzyme GPX4 of the antioxidant system (glutathione system). Therefore, three conditions must be met for cells to undergo ferroptosis: ① The substrate of lipid peroxidation reaction - phospholipids (PL) containing polyunsaturated fatty acids (PUFAs). Since the hydrogen atom of the methylene group on the non-conjugated diallyl group in the PUFAs structure (located between two double bonds) is easy to leave, PUFAs are prone to oxidation reactions; ② Free iron and iron embedded in specific enzymes such as lipoxygenase; ③ Impairment of lipid peroxidation repair function. Changing any of these conditions will affect the execution of ferroptosis.

[0005] At present, some ferroptosis inhibitors have been discovered: free radical scavenging antioxidants (RTAs), lipoxygenase inhibitors, acyl-CoA synthetase long-chain family member 4 (ACSL4) inhibitors, iron chelators and deuterated polyunsaturated fatty acids, as shown in Table 1. The existing ferroptosis inhibitors are not only of limited variety, but also have poor drugability, such as poor stability of Fer-1, low bioavailability of DFO, and difficulty of deferoxamine to penetrate the blood-brain barrier, which limits their application as drugs. Therefore, the development of new ferroptosis inhibitors with good drug-like properties and inhibitory activity has important research significance and application value.

[0006] Table 1 Types of action and applications of ferroptosis inhibitors

[0007]

[0008] Ferroptosis is closely related to the occurrence and development of neurological diseases such as neurodegenerative diseases and stroke, and inhibiting the ferroptosis process has become a new strategy for the treatment of the above diseases. Ischemic stroke (IS) is the main type of stroke. The cause is ischemia and hypoxia caused by obstruction of blood supply to the brain, resulting in damage or death of neurons in some brain tissues and dysfunction. After cerebrovascular blockage, excessive activation of N-methyl-D-aspartate receptors promotes neuronal iron uptake and produces ferroptosis. The dysregulated homeostasis of glutamatergic neurotransmission increases extracellular glutamate levels, thereby inhibiting cystine uptake and limiting glutathione biosynthesis. The reduction of soluble tau protein after cerebral ischemia prevents the dissociation of immature amyloid precursor protein from the endoplasmic reticulum, eliminates the transport of amyloid precursor protein to the surface of neurons, and amyloid precursor protein interacts with FPN protein to allow iron to be exported from neurons. The lack of this interaction leads to toxic accumulation of iron and ultimately leads to neuronal ferroptosis damage after ischemic stroke. In addition, the commonly used treatment for ischemic stroke is to restore blood circulation in brain tissue vessels, which can save nerve cells and promote the recovery of brain tissue function on the one hand; but on the other hand, it will cause a rapid cascade of damage in nerve cells, further aggravating brain damage. Therefore, ferroptosis is involved in both acute ischemic brain damage and secondary brain damage caused by stroke. Inhibiting ferroptosis and exerting a neuroprotective effect, that is, targeting the brain parenchyma during the ischemic period and restoring neuronal function in the late stage of stroke, is a promising strategy for the treatment of ischemic stroke. Summary of the invention

[0009] The present invention provides a class of coryne compounds, the structural formula of which is as follows:

[0010]

[0011] In the formula: R is selected from H, p-methoxybenzyl; R1 is selected from ethyl, vinyl, aldehyde, hydroxymethyl;

[0012] In formula I, the connection mode between C15 and C16, and between C15 and C20 is C=C or CC. When the connection mode between C15 and C16 is C=C, the conformation of hydrogen at C20 is α-configuration. In formula II, the conformation of hydrogen at C15 is α-configuration or β-configuration, and the conformation of hydrogen at C20 is α-configuration or β-configuration.

[0013] The preparation method of the coronin type compound of the present invention is as follows:

[0014] 1. Synthesis of general intermediate 9

[0015] (1) Compound 1 is subjected to Vilsmeier-Haack reaction to obtain compound 2, and compound 2 is condensed with tert-butylsulfenamide to obtain compound 3;

[0016]

[0017] wherein R is selected from p-methoxybenzyl (PMB), benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, trifluorosulfonyl, and R 2 are halogen (Cl, Br, I), OH, p-toluenesulfonyl (OTs), methylsulfonyl (OMs);

[0018] (2) Compound 3 was subjected to a vinylogous Mannich addition reaction to obtain compound 5 and its diastereoisomer 4;

[0019]

[0020] wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, trifluorosulfonyl, and R 2 is halogen, OH, p-toluenesulfonyl, methylsulfonyl;

[0021] (3) Compound 5 was deprotected from the sulfinyl group and then a base was added to construct the C ring through N-alkylation, and then compound 6 was obtained through Mannich cyclization reaction;

[0022]

[0023] wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, trifluorosulfonyl, and R 2 is halogen, OH, p-toluenesulfonyl, methylsulfonyl;

[0024] The removal of the sulfinyl protection is achieved by the following three methods: (1) the substrate is dissolved in a solvent and hydrochloric acid is added for removal; (2) the substrate is dissolved in a solvent and iodine is added for removal; (3) the substrate is dissolved in methanol and acetyl chloride is added for removal;

[0025] The base in the N-alkylation process is selected from one of NaHCO3, NaH, Na2CO3, K2CO3, and Cs2CO3. The carbon unit in the Mannich cyclization reaction for the construction of the D ring is selected from formaldehyde gas, formaldehyde aqueous solution, or paraformaldehyde. The reaction temperature is 50-100°C. (4) Compound 6 is freed from acetone protection to obtain compound 7. Compound 7 is reduced to an ester group to obtain compound 8.

[0026]

[0027] wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, and trifluorosulfonyl;

[0028] The acetone protection is opened by the following four methods: (1) the substrate is dissolved in methanol and sodium metal is added; (2) the substrate is dissolved in a mixed solvent of tetrahydrofuran and methanol (volume ratio 10:1) and sodium hydride is added; (3) the substrate is dissolved in tetrahydrofuran and sodium methoxide is added; (4) the substrate is dissolved in methanol and lithium hydroxide is added.

[0029] (5) Compound 8 was subjected to Horner-Wadsworth-Emmons reaction to obtain common intermediate 9;

[0030]

[0031] wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, and trifluorosulfonyl;

[0032] The Witting reagent used in the Horner-Wadsworth-Emmons reaction is selected from H1, H2, H3, and H4; the solvent is selected from tetrahydrofuran, dichloromethane, 1,4-dioxane, 1,2-dichloroethane, toluene, and acetonitrile, and the reaction temperature is 50 to 100°C;

[0033]

[0034] 2. Synthesis of Conanine-type compounds

[0035] (1) Intermediate 9 is subjected to oxidation reaction to obtain compound 10, compound 10 is subjected to Witting reaction to obtain compound 11, the exocyclic double bond in compound 11 is reduced to obtain compound 12, the indole protecting group is removed to obtain compound 13, and finally the endocyclic double bond in compound 13 is reduced to obtain compound 14 and its diastereomer 15;

[0036]

[0037] wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, and trifluorosulfonyl;

[0038] The oxidant used in the reaction of preparing compound 10 by oxidation reaction of intermediate 9 is selected from Dess-Martin oxidant, 2-iodoacylbenzoic acid, pyridinium chlorochromate, pyridinium dichromate, pyridine sulfur trioxide; the catalyst used in reducing compound 13 is palladium carbon, the molar ratio of compound 13 to the catalyst is 1:0.05-0.5, the reaction solvent is selected from one of methanol, tetrahydrofuran, and ethanol; the hydrogen pressure is 0.1-5MPa;

[0039] (2) The α,β-unsaturated double bond of intermediate 9 is reduced to obtain compound 16. Compound 16 is oxidized to an aldehyde and subjected to a Witting reaction to obtain compound 17 and its diastereomer 18. The indole protecting group in compound 17 is removed to obtain compound 19. Finally, the double bond is reduced to obtain compound 20.

[0040] The catalyst used in the reduction reaction of the intermediate 9 to prepare the compound 16 is selected from palladium carbon, palladium hydroxide carbon, nickel chloride hexahydrate-sodium borohydride, and Raney nickel; the solvent is selected from methanol, tetrahydrofuran, and ethanol;

[0041]

[0042] wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, and trifluorosulfonyl;

[0043] (3) After removing the indole protecting group from intermediate 9, compound 21 is obtained, and the double bond is reduced to obtain compound 22, which is oxidized to aldehyde to obtain compound 23, and finally subjected to Witting reaction to obtain compound 24;

[0044]

[0045] wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, and trifluorosulfonyl;

[0046] The catalyst for the catalytic reduction reaction of compound 21 to prepare compound 22 is one of palladium carbon, rhodium carbon, and palladium hydroxide carbon; the molar ratio of compound 21 to the catalyst is 1:0.05-0.5; the reaction solvent is selected from methanol, tetrahydrofuran, and ethanol; and the hydrogen pressure is 0.1-5MPa.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The cluster synthesis route of the coryne-type compounds provided by the present invention can complete the synthesis of multiple natural products of this family through common intermediates, and is suitable for industrial production and market promotion and application;

[0049] The corynein-type compounds prepared by the present invention have ferroptosis inhibitory activity, anti-neuroinflammatory activity, cytoprotective activity and blood-brain barrier permeability. It can be seen that the compounds of the present invention have important medicinal value and broad application prospects in the application of preparing ferroptosis inhibitors for the treatment of ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a schematic diagram of the evaluation results of the cytotoxicity of compound 9-24 on HT22 cells;

[0051] Figure 2 Schematic diagram of the effect of compound 9-24 on STS-induced cell apoptosis;

[0052] Figure 3 This is a schematic diagram showing the effect of compound 9-24 on rapamycin-induced cell autophagy;

[0053] Figure 4 This is a schematic diagram of the effect of compound 9-24 on programmed cell necrosis induced by the combined use of TNF-α, compound SM-164 and zVAD-fmk;

[0054] Figure 5 Schematic diagram of the results of lipid peroxidation inhibition by compounds 21 and 24;

[0055] Figure 6 Schematic diagram of the results of compounds 21 and 24 inhibiting erastin-induced intracellular ROS;

[0056] Figure 7 The inhibition results of compounds 21 and 24 on MDA;

[0057] Figure 8 The results are the effects of compounds 21 and 24 on intracellular GSH levels;

[0058] Fig. 9The results of the NO inhibition activity test of compounds 21 and 24;

[0059] Fig.10 The results of the inhibition of TNF-α by compounds 21 and 24;

[0060] Fig.11 The results of the inhibition of IL-1β by compounds 21 and 24;

[0061] Fig.12 The results show the inhibition of IL-6 by compounds 21 and 24. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with the accompanying drawings and examples, but the contents should not be regarded as limiting the present invention. The methods in the present embodiments are operated according to conventional methods unless otherwise specified, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified.

[0063] Example 1: Synthesis of Conanine-type compound 9 (R is PMB, R2 is Cl)

[0064] 1. Synthesis of compound 3

[0065]

[0066] 150 mL of DMF was measured and POCl3 (22.7 mL, 249.1 mmol) was added at 0°C under nitrogen atmosphere. After reacting for 30 minutes, compound 1 (10.00 g, 24.91 mmol) dissolved in 50 mL of DMF was added, and the reaction was continued for 30 minutes and then the temperature was raised to 70°C. The reaction was continued until the reaction of the raw material was complete as monitored by TLC (about 15 hours). The reaction solution was placed in an ice bath, and 2 mol / L NaOH solution was slowly added to adjust the pH>8. The solution was extracted with ethyl acetate (EA) (3×500 mL), the organic phases were combined, and the organic phases were washed with saturated NaCl (3×500 mL). After drying over anhydrous Na2SO4, the solvent was evaporated under reduced pressure and purified on a silica gel column (petroleum ether PE: dichloromethane DCM=5:1) to obtain white solid compound 2 (5.71 g, 70%). 1 H NMR (400MHz, CDCl3) δ10.17(s,1H),7.75(d,J=8.4Hz,1H),7.44-7.40(m,2H),7.25-7.20(m,1H),7.08(d,J =8.8Hz,2H),6.81(d,J=8.8Hz,2H),5.75(s,2H),3.82(t,J=7.2Hz,2H),3.74(s,3H),3.57(t,J=7.2Hz,2H). 13CNMR(100MHz, CDCl3)δ181.4,158.9,139.4,131.3,129.9,127.9,127.6,126.3,126.2,121.0,114.0,111.2,55.2,47.3,44.8,27.2.HRMS(ESI):Calcd for C 19 H 19 ClNO2[M+H]+:328.1099,found:328.1098.

[0067] Weigh compound 2 (20 g, 61.01 mmol) and dissolve it in 200 mL THF. Add (s)-tert-butylsulfenamide (11.09 g, 91.52 mmol) and Ti(OEt)4 (31.4 mL, 152.53 mmol) in sequence. React at 50 °C in a nitrogen atmosphere until the reaction of the raw materials is complete as monitored by TLC (about 12 hours). Add saturated Na2SO4 with stirring until loose solids appear. Continue stirring for 10 minutes, filter through a sand core funnel, wash with ethyl acetate until no product appears as monitored by TLC, evaporate the solvent under reduced pressure, and purify on a silica gel column (PE:EA=5:1) to obtain compound 3 (26.1 g, 99%) as a yellow oil. 1 H NMR (400MHz, CDCl3) δ8.80(s,1H),7.72(d,J=8.0Hz,1H),7.36-7.31(m,2H),7.20-7.16(m,1H),6.88(d,J=8.4Hz,2H),6.76(d,J =8.8Hz,2H),5.94(d,J=16.4Hz,1H),5.68(d,J=16.4Hz,1H),3.78(t,J=7.2Hz,2H),3.73(s,3H),3.62-3.49(m,2H),1.10(s,9H). 13 C NMR (100MHz, CDCl3) δ158.9,151.8,139.5,130.2,129.9,127.2,127.0,126.4,122. 3,120.9,120.5,114.2,110.7,57.7,55.4,47.5,44.6,28.1,22.4.HRMS(ESI):Calcd for C 23 H 28 ClN2O2S[M+H] + :431.1555,found:431.1558.

[0068] 2. Synthesis of compound 5 and diastereomer 4

[0069]

[0070] Compound 3 (10.0 g, 23.2 mmol) was weighed into a two-necked flask, THF (100 mL) was added to dissolve it, and then placed at -78 °C. Then, BF3·OEt2 (7.2 mL, 58.0 mmol) was added under nitrogen atmosphere and reacted for 1 hour. At the same time, compound 1,3-dioxenone (9.89 g, 69.6 mmol) was dissolved in 100 mL In THF, LiHMDS (69.6 mL, 69.6 mmol) was added at -78 ° C in a nitrogen atmosphere. After reacting for 1 hour, the lithium reagent of 1,3-dioxenone was transferred to a two-necked bottle containing compound 3 at -78 ° C and reacted for 30 minutes. After TLC monitoring, 100 mL of saturated NH4Cl was added to quench the reaction after the reaction of the raw material was completed. EA was added for extraction (2×500 mL), and the organic phases were collected and combined. The organic phases were washed with saturated NaCl (3×200 mL), dried over anhydrous Na2SO4, and then the solvent was evaporated under reduced pressure. The compound 5 (10.21 g, 77%) and diastereomer 4 (1.99 g, 15%) were obtained as a light yellow foam solid by column chromatography purification (PE:EA=2:1 to 1:1). Compound 4: 1 H NMR (600MHz, CD3OD) δ7.60(d,J=7.8Hz,1H),7.15–7.07(m,3H),6.86–6.80(m,4H),5.67–5.62(m,1H),5.50–5.45(m,1H),5.10(br s,1H),4.92(br s,1H),3.92–3.88(m,1H),3.77–3.71(m,4H),3.40–3.27(m,2H),3.02(dd,J=15.0,7.2Hz,1H),2.77(br s,1H),1.56(s,3H),1.54(s,3H),1.15(s,9H). 13 C NMR (150MHz, CD3OD) δ169.2,162.7,160.5,139.2,133.3,131.1,128.4,123.9,121.0,119.8,115.2,11 4.0,111.4,108.3,96.1,57.0,55.7,49.7,48.1,45.0,40.3,29.8,25.4,24.7,23.0.HRMS(ESI):Calcd for C 30 H 38 ClN2O5S[M+H]+:573.2184,found:573.2184.[α]20D=+42.7(c 0.79,CHCl3).

[0071] Compound 5: 1 H NMR (600MHz, CD3OD) δ7.59 (d, J=7.8Hz, 1H), 7.23 (br s,1H),7.14(t,J=7.2Hz,1H),7.08(t,J=7.2Hz,1H),6.88-6.81(m,4H),5.56-5.48(m,2H)5.14(br s,2H),3.83(br s,2H),3.72(s,3H),3.41-3.33(m,2H),3.08(q,J=7.8Hz,1H),2.75(br s,1H),1.53(s,6H),1.10(s,9H). 13 C NMR (150MHz, CD3OD) δ169.5,162.9,160.6,138.9,136.0,131.4,128.5,128.3,123.9,121.0,119.8,11 5.4,112.2,111.3,108.2,96.2,57.6,55.7,49.7,47.9,45.2,40.6,29.7,25.1,22.9.HRMS(ESI):Calcd for C 30 H 38 ClN2O5S[M+H] + :573.2184,found:573.2183.

[0072] 3. Synthesis of Compound 6

[0073]

[0074] Compound 5 (5.73 g, 10.00 mmol) was dissolved in 50 mL of THF, and a tetrahydrofuran solution (12.5 mL) containing 4 mol / L HCl was added. The reaction of the raw material was monitored by TLC (about 2 hours). DMF (50 mL) and K2CO3 (13.82 g, 100.0 mmol) were added. The reaction of the raw material was monitored by TLC (about 12 hours). EA (500 mL) and H2O (20 mL) were used for extraction. The organic phase was collected, washed with saturated NaCl (3×300 mL), dried over anhydrous Na2SO4, and then the solvent was evaporated under reduced pressure to obtain a yellow foamy solid, which was directly used for the next step without purification.

[0075] The crude product obtained above was dissolved in 100 mL of THF, and 1.6 mL of 37% formaldehyde aqueous solution and KHSO4 (2.72 g, 20.0 mmol) were added in sequence. The reaction was carried out at 50°C for 1 hour. After the reaction of the raw materials was completed as monitored by TLC, saturated NaHCO3 was added to neutralize to weak alkalinity, and EA was extracted (2×600 mL). The organic phases were collected and combined, and the organic phases were washed with saturated NaCl (3×300 mL). After drying over anhydrous Na2SO4, the solvent was evaporated under reduced pressure and purified by column chromatography (PE:EA=2:1) ​​to obtain compound 6 (3.38 g, 76%) as a yellow foamy solid. 1 H NMR (600MHz, CDCl3) δ7.57-7.55(m,1H),7.15-7.12(m,3H),6.91(d,J=8.4Hz,2H),6.83(d ,J=8.4Hz,2H),5.28(d,J=17.4Hz,1H),5.19(d,J=16.8Hz,1H),3.75-7.78(m,5H),3.41(d t,J=15.0,3.0Hz,1H),3.22-3.19(m,1H),3.04-2.99(m,1H),2.87(dt,J=14.8,4.2Hz,1H) ,2.77(ddd,J=11.4,10.2,4.2Hz,1H),2.54-2.50(m,1H),2.44-2.38(m,1H),1.67(s,6H). 13 C NMR (150MHz, CDCl3) δ162.4,160.6,159.0,138.0,134.8,129.3,127.1,126.9,122.1,119.8,118.6,114. 4,109.96,109.94,106.6,101.1,55.4,54.2,50.5,50.2,47.3,33.5,26.9,23.8,22.1.HRMS(ESI):Calcd for C 27 H 29 N2O4[M+H] + :445.2122,found:445.2120.

[0076]

[0077] 4. Synthesis of Compound 8

[0078]

[0079] Weigh compound 6 (5.6 g, 12.6 mmol), add THF (100 mL) to dissolve, then directly add NaOMe (6.81 g, 126 mmol) and react for 30 minutes. After the reaction of the raw material is completed, 10 mL of saturated NH4Cl is added to quench the reaction, and EA is extracted (2×100 mL). The organic phases are combined, washed with saturated NaCl (3×50 mL), dried over anhydrous Na2SO4, and then the solvent is evaporated under reduced pressure. After simple filtration by column chromatography, a yellow foamy solid compound 7 (4.2 g, 85%) is obtained, which is directly used in the next step.

[0080] Compound 7 (4.2 g, 10.04 mmol) was dissolved in 150 mL of DCM, triethylamine (NEt3) (14.0 mL, 100.04 mmol) was added at 0°C and reacted for 30 minutes, TBSOTf (9.2 mL, 40.16 mmol) was added, and the mixture was warmed to room temperature. TLC monitored the complete reaction of the raw material (about 1 hour), and 100 mL of saturated NaHCO3 was added for quenching. The mixture was extracted with DCM (3×400 mL), and the organic phases were collected and combined. The organic phases were washed with saturated NaCl (3×200 mL), dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure to obtain a yellow foamy solid, which was directly used for the next step without purification.

[0081] The obtained intermediate enol silyl ether was dissolved in 150 mL of DCM, and diisobutylaluminum hydride (DIBAL-H) ​​(40.2 mL, 40.16 mmol) was slowly added dropwise at -40 ° C. under a nitrogen atmosphere. The reaction of the raw material was completed by TLC monitoring (about 1 hour). 100 mL of saturated potassium sodium tartrate solution was added to quench the reaction. The mixture was heated to room temperature and stirred for 1 hour. The mixture was extracted with dichloromethane (3×400 mL), and the organic phases were collected and combined. The organic phases were washed with saturated NaCl (3×300 mL), dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure to obtain a yellow foamy solid, which was directly used for the next step without purification.

[0082] The alcohol obtained by the reduction in the previous step was dissolved in THF (100 mL), and AcOH (1.72 mL, 30.12 mmol) and TBAF (6.34 g, 20.08 mmol) were added in sequence. After TLC monitoring of the reaction of the raw material was complete (about 2 hours), 20 mL of saturated NaHCO3 was added to quench the reaction, and EA was added for extraction (2×400 mL). The organic phases were collected and combined, and the organic phases were washed with saturated NaCl (3×200 mL). After drying over anhydrous Na2SO4, the solvent was evaporated under reduced pressure and purified by column chromatography (PE:EA=1:1to 1:2) to obtain a light yellow solid compound 8 (2.86 g, 73%). 1H NMR (600MHz, CDCl3) δ7.57-7.55(m,1H),7.16-7.11(m,3H),6.85(d,J=8.4Hz,2H),6.79(d, J=8.4Hz,2H),5.24(d,J=16.8Hz,1H),5.14(d,J=16.8Hz,1H),4.05(dd,J=11.4,3.0Hz,1H) ,3.75(s,5H),3.40(q,J=6.0Hz,1H),3.37-3.34(m,1H),3.05-3.00(m,3H),2.98-2.94(m,1 H),2.91-2.87(m,1H),2.66(dd,J=11.4,2.4Hz,1H),2.58(dd,J=13.8,3.0Hz,1H),2.48(br s,1H). 13 C NMR (150MHz, CDCl3) δ210.5,159.0,137.6,134.9,129.2,127.1,126.9,122.1,119.7,118.5, 114.4,110.0,108.6,60.4,57.3,57.1,55.4,49.2,47.1,46.7,45.2,22.5.HRMS(ESI):Calcd for C 24 H 27 N2O3[M+H] + :391.2016,found:391.2020.

[0083] 5. Synthesis of Compound 9

[0084]

[0085] Compound 8 (1.82 g, 4.66 mmol) and Witting reagent (3.12 g, 9.32 mmol) were added to a 100 mL sealed tube, and then 30 mL of DCM was added to dissolve it. The mixture was reacted at 55 °C for 24 hours, and then an equal amount of Witting reagent was added, once every 24 hours, for a total of four times. After TLC monitoring of the reaction of the raw material was complete (about 4 days), the solvent was evaporated under reduced pressure and purified by column chromatography (PE:EA:DCM=1:1:0.1) to obtain a light yellow solid compound 9 (2.0 g, 96%). 1H NMR (600MHz, CDCl3) δ7.53(d,J=7.2Hz,1H),7.21(d,J=7.8Hz,1H),7.14-7.09(m,2H),6.89(d,J=8.4Hz,2H),6.74(d,J=9.0Hz, 2H),5.59(s,1H),5.46(d,J=16.8Hz,1H),5.38(d,J=16.8Hz,1H),4.32(dd,J=13.2,2.4Hz,1H),3.85(q,J=5.4Hz,1H),3.81(d, J=10.2Hz,1H),3.72(s,3H),3.68(s,3H),3.60(dd,J=10.8,6.6Hz,1H),3.45(dd,J=12.6,4.2Hz,1H),3.31(dt,J=10.8,6.6Hz, 1H), 2.94 (t, J = 5.4Hz, 2H), 2.91-2.87 (m, 1H), 2.72 (q, J = 11.4Hz, 1H), 2.70-2.65 (m, 1H), 2.18 (t, J = 12.6Hz, 1H), 1.86 (s, 1H). 13 C NMR (150MHz, CDCl3) δ167.2,159.9,158.8,137.6,135.8,130.1,127.4,127.0,121.7,119.3,118.4,11 4.1,111.6,110.0,108.3,61.8,59.9,58.5,55.3,51.3,47.1,46.3,42.7,32.4,22.4.HRMS(ESI):Calcd for C 27 H 31 N2O4[M+H] + :447.2278,found:447.2278.

[0086] Example 2: Synthesis of Corinnamon-type compound 10 (R is PMB)

[0087]

[0088] Compound 9 (50 mg, 0.11 mmol) was weighed and dissolved in a mixture of 5 mL of DCM and 1 mL of DMSO. DIPEA (96 μL, 0.55 mmol) and SO3·Py (87.5 mg, 0.55 mmol) were added at 0°C. The reaction of the raw material was completed (about 30 minutes) after being monitored by TLC. 2 mL of saturated NH4Cl was added for quenching. The mixture was extracted with DCM (3×50 mL). The organic phases were collected and combined, washed with saturated NaCl (3×50 mL), dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The mixture was purified by column chromatography (PE:EA=1:1) to obtain compound 10 (31.8 mg, 65%) as a pink foamy solid. 1 H NMR (600MHz, CDCl3) δ10.04(s,1H),7.55(d,J=7.2Hz,1H),7.20-7.12(m,3H),6.92(d,J=8.4Hz,2H),6 .81(d,J=9.0Hz,2H),5.25(dd,J=17.4,7.2Hz,2H),3.82(d,J=16.8Hz,1H),3.77(s,3H),3.69(d,J=15 .6Hz,1H),3.68(s,3H),3.67-3.64(m,1H),3.30(dt,J=16.8,3.6Hz,1H),3.21(d,J=15.6Hz,1H),3.20 -3.16(m,1H),3.02-2.97(m,1H),2.85(dt,J=15.0,3.0Hz,1H),2.73-2.68(m,2H),2.45-2.40(m,1H). 13 C NMR (100MHz, CDCl3) δ188.9,169.8,159.0,147.5,138.1,135.3,134.9,129.7,127.2,126.9,121.9,119. 7,118.5,114.3,109.74,109.72,55.4,54.3,52.6,52.3,50.4,47.4,38.1,36.7,22.1.HRMS(ESI):Calcd for C 27 H 29 N2O4[M+H] + :445.2122,found:445.2119.

[0089] Example 3: Synthesis of Compound 11 (R is PMB)

[0090]

[0091] Weigh Ph3PCH3·Br (1.78 g, 5 mmol) and dissolve it in 7.5 mL of ultra-dry THF. Add NaHMDS (2.5 mL, 5 mmol) at 0°C in a nitrogen atmosphere, warm to room temperature and react for 1 hour. Let stand to allow the salt to precipitate completely to obtain methylphosphine ylide for later use.

[0092] Compound 10 (315.2 mg, 0.71 mmol) was dissolved in 15 mL of ultra-dry THF, and the prepared methylphosphine ylide (2.84 mL, 1.42 mmol) was added at 0°C in a nitrogen atmosphere, and the temperature was raised to room temperature for reaction. TLC monitored the complete reaction of the raw material (about 2 hours), and 5 mL of saturated NH4Cl was added for quenching. EA was extracted (3×100 mL), and the organic phases were collected and combined, washed with saturated NaCl (3×100 mL), dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure and purified by column chromatography (PE:EA=3:1) to obtain yellow oily compound 11 (254.5 mg, 81%). 1 H NMR (600MHz, CDCl3) δ7.54(d,J=7.2Hz,1H),7.18(d,J=7.2Hz,1H),7.15-7.10(m,2H),6.92(d,J=8.4Hz,2H),6.79(d,J=8.4Hz,2H),6.67(dd,J= 17.4,11.4Hz,1H),5.26(q,J=16.8Hz,2H),5.18(d,J=17.4Hz,1H),5.10 (d,J=10.8Hz,1H),3.76(s,3H),3.69-3.66(m,2H),3.63(s,3H),3.39(br d,J=15.6Hz,1H),3.28(d,J=15.0Hz,1H),3.22-3.19(m,1H),3.05-3.00(m,1H),2.93(d,J=15.6Hz,1H),2.86(dq,J =15.0,3.0Hz,1H),2.71(ddd,J=10.8,9.6,4.2Hz,1H),2.60(dt,J=18.0,3.0Hz,1H),2.33(dd,J=18.0,11.4Hz,1H). 13 C NMR (150MHz, CDCl3) δ171.4,158.9,138.0,136.3,131.9,130.7,129.9,127.2,127.1,127.0,121.6,119.5 ,118.4,114.2,113.3,109.7,109.3,55.4,54.8,54.6,52.0,50.5,47.3,37.7,36.6,22.2.HRMS(ESI)Calcd for C28 H 31 N2O3[M+H] + :443.2329,found:443.2325.

[0093] Example 4: Synthesis of Compound 13 (R is PMB)

[0094]

[0095] Compound 11 (254.5 mg, 0.58 mmol) was dissolved in 8 mL MeOH, and Pd / C catalyst (61.7 mg, 10%) was added. The mixture was reacted under a hydrogen atmosphere. The reaction of the raw material was completed (about 12 hours) after TLC monitoring. The mixture was filtered through a sand core funnel filled with diatomaceous earth, and the filter cake was washed with EA. The solvent was evaporated under reduced pressure and purified by column chromatography (PE:EA=3:1) to obtain a yellow oily compound 12. 1HNMR (600 MHz, Chloroform-d) δ7.56-7.51 (m, 1H), 7.17-7.14 (m, 1H), 7.14-7.08 (m, 2H), 6.91 (d, J=8.8 Hz, 2H), 6.79 (d, J=8.6 Hz, 2H), 5.29 (d, J=17.0 Hz, 1H), 5.22 (d, J=17.0 Hz, 1H) ,3.76(s,3H),3.65(dd,J=11.0,3.6Hz,1H),3.60(s,3H),3.30(q,J=16.2Hz,2H ),3.20-3.13(m,1H),3.10(d,J=15.2Hz,1H),3.04-2.99(m,1H),2.83(d,J=15.6 Hz,2H),2.65(ddd,J=10.8,9.4,4.2Hz,1H),2.46(dt,J=16.2,2.8Hz,1H),2.20 (t,J=13.9Hz,1H),2.15-2.10(m,1H),2.01-1.95(m,1H),1.01(t,J=7.6Hz,3H). 13 C NMR (151MHz, CDCl3) δ172.1,158.9,138.0,136.7,134.9,130.0,127.2,127.1,121.5,121.2,119.4,118.3 ,114.2,109.8,109.2,57.6,55.4,55.2,51.9,50.4,47.3,37.5,35.5,24.0,22.2,13.0.HRMS(ESI):Calcd for C 20 H 25N2O2[M+H] + :445.2486,found:445.2483.

[0096] p-MePhSH (720.4 mg, 5.8 mmol) was dissolved in a TFA-DCM mixture (TFA 5 mL, DCM 1 mL), and then added to the crude product obtained above. The reaction of the raw material was monitored by TLC (about 1 hour). Saturated NaHCO3 was added to quench the reaction until it was weakly alkaline. EA was extracted (2×100 mL), and the organic phases were collected and combined. The organic phases were washed with saturated NaCl (3×100 mL), dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The mixture was purified by column chromatography (3:1 to 1:1) to obtain a yellow oily compound 13 (133.4 mg, 71%). 1 HNMR(600MHz, CDCl3)7.82(s,1H),7.49(d,J=8.4Hz,1H),7.31(d,J=8.4Hz,1H),7.15(t,J=7.8Hz,1H),7.10(t,J=7.2Hz,1H),3.70(s,3H),3.55(br d,J=10.8Hz,1H),3.40(d,J=16.2Hz,1H),3.26(d,J=15.0Hz,1H),3.20(dd, J=11.4,4.2Hz,1H),3.09(dt,J=16.2,3.0Hz,1H),3.05-2.99(m,2H),2.78-2 .75(m,1H),2.62(td,J=11.4,4.2Hz,1H),2.57(dt,J=16.2,3.0Hz,1H),2.3 5-2.31(m,1H),2.22-2.16(m,1H),2.06-2.00(m,1H),1.05(t,J=7.8Hz,3H). 13 C NMR (150MHz, CDCl3) δ172.3,136.4,135.5,134.7,127.3,121.6,120.8,119.5,118.3,1 10.9,108.5,57.6,55.9,52.04,52.00,37.6,35.5,24.2,21.6,13.0.HRMS(ESI):Calcd for C 20 H 25 N2O2[M+H] + :325.1911,found:325.1916.

[0097] Example 5: Synthesis of Compound 14 and Compound 15

[0098]

[0099] Compound 13 (133.4 mg, 0.41 mmol) was dissolved in 8 mL of EtOH, and a Pd / C catalyst (223.5 mg, 10%) was added. The mixture was reacted in a hydrogen reactor (0.4 MPa). TLC monitored the complete reaction of the raw material (about 48 hours). The mixture was filtered using a sand core funnel filled with diatomaceous earth, and the filter cake was washed with EA. The solvent was evaporated under reduced pressure and purified by column chromatography to obtain yellow oily compounds 14 (100.6 mg, 75%, PE / EA=3:1) and 15 (6.5 mg, 5%, PE / EA=1:1). Compound 14: 1 H NMR (600MHz, CDCl3) δ7.82(br s,1H),7.47(d,J=7.8Hz,1H),7.29(d,J=7.8Hz,1H),7.13(t,J=7.8Hz,1H),7.08(t,J=7.2Hz,1H),3.74(s,3H),3.28(br s,1H),3.03-2.93(m,3H),2.71-2.67(m,1H),2.62-2.57(m,1H),2.42(dd,J=11.4,2.4Hz,2H),2.28(t,J=9.6Hz,1H),2.33- 2.27(m,2H),1.93(dq,J=12.6,3.0Hz,1H),1.70-1.62(m,1H),1.57-1.49(m,2H),1.29-1.22(m,1H),0.93(t,J=7.2Hz,3H). 13 C NMR (150MHz, CDCl3) δ173.8,136.0,135.2,127.5,121.3,119.5,118.2,110.9,108.3 ,60.1,57.7,53.5,51.8,40.0,38.1,36.8,31.8,21.8,18.2,12.6.HRMS(ESI):Calcd for C 20 H 27 N2O2[M+H] + :327.2067,found:327.2063. Compound 15: R f =0.23(PE:EA=2:1). 1H NMR (600MHz, CDCl3) δ7.80(s,1H),7.47(d,J=7.8Hz,1H),7.30(d,J=8.4Hz,1H),7.13(t,J=7.8Hz,1H),7.08(t,J=7.2Hz,1H),3.73(s,3H),3.26(br d,J=11.4Hz,1H),3.13(dd,J=11.4,4.2Hz,1H),3.08(q,J=6.0Hz,1H),3.04-2.9 8(m,1H),2.72(td,J=14.4,4.8Hz,2H),2.61(td,J=11.4,4.8Hz,1H),2.24(dt,J =12.6,3.6Hz,1H),2.16-2.09(m,2H),1.87-1.81(m,1H),1.66-1.59(m,1H),1.5 4-1.48(m,1H),1.38(q,J=12.0Hz,1H),1.22-1.14(m,1H),0.94(t,J=7.2Hz,3H). 13 C NMR (150MHz, CDCl3) δ173.9,136.1,134.7,127.5,121.5,119.5,118.3,110.9,108.3 ,60.3,59.7,53.3,51.8,41.8,38.1,37.5,36.2,23.7,21.9,11.1.HRMS(ESI):Calcd for C 20 H 27 N2O2[M+H] + :327.2067,found:327.2068.

[0100] Example 6: Synthesis of Compound 16

[0101]

[0102] Compound 9 (520 mg, 1.16 mmol) was weighed and dissolved in 20 mL of MeOH at 0°C. NiCl2·6H2O (110.3 mg, 0.46 mmol) and NaBH4 (263.4 mg, 6.96 mmol) were added in sequence. The reaction was continued for 30 minutes. After the reaction of the raw materials was completed, 10 mL of saturated NH4Cl was added to quench the reaction, and EA was added for extraction (2×200 mL). The organic phases were collected and combined, and the organic phases were washed with NaCl (3×100 mL). After drying over anhydrous Na2SO4, the solvent was evaporated under reduced pressure and purified by column chromatography (EA:MeOH=20:1) to obtain light yellow foamy solid compound 16 (338.2 mg, 65%). 1 H NMR (600MHz, CDCl3) δ7.52-7.50(m,1H),7.11-7.09(m,3H),6.91(d,J=8.4Hz,2H),6.80(d,J=9. 0Hz,2H),5.28(d,J=16.8Hz,1H),5.16(d,J=17.4Hz,1H),3.83-3.81(m,1H),3.76(s,3H),3.58(s ,3H),3.49-3.41(m,2H),3.21-3.17(m,1H),2.99-2.91(m,2H),2.83-2.74(m,2H),2.66(t,J=11. 4Hz,1H),2.50-2.43(m,2H),2.31(dd,J=14.4,7.8Hz,1H),2.25-2.21(m,1H),1.88-1.86(m,2H). 13 CNMR (150MHz, CDCl3) δ174.3,158.8,137.9,136.2,129.9,127.2,127.1,121.5,119.4,118.2,114.2,10 9.8,109.1,63.1,55.3,52.81,52.76,52.0,49.4,47.0,38.3,33.2,32.4,30.8,22.0.HRMS(ESI):Calcd for C 27 H 33 N2O4[M+H] + :449.2435,found:449.2430.

[0103] Example 7: Synthesis of Compounds 17 and 18

[0104]

[0105] Using compound 16 as raw material, the first step of the synthesis method refers to the synthesis method of compound 10 in Example 2; the second step of the synthesis method refers to the synthesis method of compound 11 in Example 3;

[0106] Compound 17: 1 H NMR (600MHz, CDCl3) δ7.53(d,J=7.2Hz,1H),7.24(d,J=7.8Hz,1H),7.16(t,J=7.2Hz,1H),7.12(t,J=7.2Hz,1H),6.88(d,J=8.4Hz,2H),6.79(d,J= 8.4Hz,2H),5.71-5.65(m,1H),5.41(d,J=17.4Hz,1H),5.35(d,J=17.4Hz,1H),5.08(dd,J=17.4,1.8Hz,1H),5.01(dd,J=10.2,1.8Hz,1H),3.99(br s,1H),3.76(s,3H),3.64(s,3H),3.16(dd,J=12.0,5.4Hz,1H),3.09-3.04(m,1H),2 .98(td,J=12.0,4.2Hz,1H),2.80(dd,J=11.4,8.4Hz,1H),2.71(dd,J=12.0,4.2Hz, 1H),2.66(dd,J=15.6,4.8Hz,1H),2.41(dd,J=15.6,4.8Hz,1H),2.32(td,J=14.4,3 .0Hz,1H),2.10-2.06(m,2H),1.84-1.78(m,1H),1.61(ddd,J=13.8,9.6,4.2Hz,1H). 13 C NMR (150MHz, CDCl3) δ173.5,158.8,140.1,138.1,134.7,130.3,127.1,121.7,119.4,118.1,116.4,11 4.3,109.6,109.5,55.4,54.9,52.5,51.8,51.6,47.2,46.0,38.1,33.3,32.9,19.2.HRMS(ESI):Calcd for C 28 H 33 N2O3[M+H] + :445.2486,found:445.2480. Compound 18: 1H NMR (600MHz, CDCl3) δ7.53-7.51(m,1H),7.12-7.09(m,3H),6.91(d,J=8.4Hz,2H),6.79(d,J=8.4 Hz,2H),5.73(ddd,J=16.8,10.8,6.0Hz,1H),5.29(d,J=16.8Hz,1H),5.15(d,J=17.4Hz,1H),5.10 (dt,J=10.8,1.2Hz,1H),5.03(dt,J=17.4,1.8Hz,1H),3.88-3.86(m,1H),3.76(s,3H),3.54(s,3H ),3.23-3.19(m,1H),2.99-2.94(m,2H),2.89-2.76(m,4H),2.45-2.41(m,3H),1.92-1.85(m,2H). 13 C NMR (150MHz, CDCl3) δ174.0,158.8,138.8,137.9,136.6,130.0,127.2,127.1,121.5,119.4,118.2,115.8 ,114.2,109.9,109.2,55.4,54.2,52.1,51.7,49.5,46.9,39.6,34.7,32.3,32.2,22.3.HRMS(ESI):Calcd for C 28 H 33 N2O3[M+H] + :445.2486,found:445.2489.

[0107] Example 8: Synthesis of Compound 19

[0108]

[0109] Compound 19 was prepared using compound 17 as raw material by referring to the synthetic method of compound 13 in Example 4. 1H NMR (600MHz, CDCl3) δ8.07(s,1H),7.49(d,J=7.8Hz,1H),7.41(d,J=7.8Hz,1H),7.17(t,J=7.2Hz,1H),7.1 2(t,J=7.8Hz,1H),5.43-5.37(m,1H),5.08(dd,J=16.8,1.8Hz,1H),5.02(dd,J=10.2,1.8Hz,1H),4.39(br s,1H),3.70(s,3H),3.30-3.22(m,2H),3.05-2.99(m,1H),2.64-2.56(m,4H),2.40(dt,J=1 3.2, 3.6Hz, 1H), 2.13-2.05 (m, 2H), 1.70 (ddd, J=16.2, 11.4, 4.8Hz, 1H), 1.65-1.58 (m, 1H). 13 C NMR (150MHz, CDCl3) δ173.9,139.0,136.0,133.0,127.8,121.6,119.5,118.1,117.4, 111.4,107.9,53.9,51.7,51.2,51.1,46.8,37.4,32.9,32.3,17.3.HRMS(ESI):Calcd for C 20 H 25 N2O2[M+H] + :325.1911,found:325.1909.

[0110] Example 9: Synthesis of Compound 20

[0111]

[0112] Use compound 19 as raw material and refer to the synthesis method of compound 12 in Example 4. 1H NMR (600MHz, CDCl3) δ7.99(s,1H),7.49(d,J=7.8Hz,1H),7.37(d,J=7.8Hz,1H),7.15(t,J=7.2Hz ,1H),7.10(t,J=7.2Hz,1H),4.06(s,1H),3.73(s,3H),3.21-3.16(m,1H),3.07-2.98(m,2H),2.7 1(dd,J=11.4,3.6Hz,1H),2.64-2.60(m,2H),2.52(dd,J=12.0,8.4Hz,1H),2.29-2.21(m,2H),1. 75-1.71(m,2H),1.55-1.48(m,1H),1.38-1.33(m,1H),1.24-1.18(m,1H),0.86(t,J=7.8Hz,3H). 13 C NMR (150MHz, CDCl3) δ174.0,136.0,133.9,127.8,121.4,119.4,118.1,111.2,108.1 ,54.3,52.1,51.8,51.6,41.5,37.2,33.0,32.5,24.3,18.9,11.7.HRMS(ESI):Calcd for C 20 H 27 N2O2[M+H] + :327.2067,found:327.2065.

[0113] Example 10: Synthesis of Compound 21

[0114]

[0115] Using compound 9 as raw material, refer to the synthesis method of compound 13. 1H NMR (600MHz, CDCl3) δ8.72(s,1H),7.47(d,J=7.8Hz,1H),7.34(d,J=7.8Hz,1H),7.15( t,J=7.8Hz,1H),7.08(t,J=7.8Hz,1H),5.71(s,1H),4.04(dd,J=10.8,5.4Hz,2H),3.8 3(dd,J=10.8,5.4Hz,1H),3.76(s,3H),3.69(dd,J=12.6,4.8Hz,1H),3.30-3.26(m,1H ),3.24-3.19(m,2H),3.03-2.95(m,2H),2.72-2.64(m,2H),2.52-2.49(m,1H),1.90(br s,1H). 13 C NMR (150MHz, CDCl3) δ168.0,159.1,136.2,133.1,127.5,121.6,119.5,118.2,114. 6,111.3,107.4,64.1,58.3,54.2,51.52,51.50,46.2,30.8,19.3.HRMS(ESI):Calcd for C 19 H 23 N2O3[M+H] + :327.1703,found:327.1702.

[0116] Example 11: Synthesis of Compound 22

[0117]

[0118] Using compound 21 as raw material, refer to the synthesis method of compound 12. 1H NMR (600MHz, CDCl3) δ7.89(s,1H),7.47(d,J=7.8Hz,1H),7.29(d,J=7.8Hz,1H),7.13(t,J=7.2Hz,1H),7.09(t,J=7 .2Hz,1H),3.73(s,3H),3.64(dd,J=11.4,3.6Hz,1H),3.58(dd,J=11.4,4.8Hz,1H),3.27(dd,J=11.4,2.4Hz,1H),3. 09(dd,J=11.4,4.2Hz,2H),3.02-2.96(m,1H),2.75-2.72(m,1H),2.64-2.60(m,2H),2.42(t,J=11.4Hz,1H),2.24( dd,J=16.2,7.2Hz,1H),2.19(dt,J=12.0,4.2Hz,1H),2.10-2.04(m,1H),1.71-1.65(m,1H),1.43(q,J=12.0Hz,1H). 13 C NMR (150MHz, CDCl3) δ174.3,136.2,134.4,127.5,121.6,119.6,118.3,110.9,10 8.4,63.2,59.5,58.6,53.1,52.1,43.8,38.5,36.2,33.7,21.8.HRMS(ESI):Calcd for C 19 H 25 N2O3[M+H] + :329.1860,found:329.1855.

[0119] Example 12: Synthesis of Compound 23

[0120]

[0121] Using compound 22 as raw material, refer to the synthesis method of compound 10. 1H NMR (600MHz, CDCl3) δ9.71(d,J=2.4Hz,1H),7.82(s,1H),7.48(d,J=7.8Hz,1H),7.30(d,J=7.8Hz,1H), 7.15(t,J=7.8Hz,1H),7.10(t,J=7.2Hz,1H),3.72(s,3H),3.34(dd,J=11.4,2.4Hz,1H),3.26(dd,J=11 .4,4.2Hz,1H),3.12(dd,J=11.4,4.2Hz,1H),3.02-2.96(m,1H),2.78-2.74(m,1H),2.71-2.63(m,3H), 2.47-2.38(m,2H),2.35(dd,J=12.6,3.0Hz,1H),2.25(dd,J=15.6,9.0Hz,1H),1.41(q,J=12.0Hz,1H). 13 C NMR (150MHz, CDCl3) δ202.1,172.6,136.2,133.8,127.4,121.8,119.7,118.3,111 .0,108.5,59.0,54.5,53.7,53.0,52.0,38.3,35.0,32.5,21.8.HRMS(ESI):Calcd for C 19 H 23 N2O3[M+H] + :327.1703,found:327.1705.

[0122] Example 13: Synthesis of Compound 24

[0123]

[0124] Using compound 23 as raw material, refer to the synthesis method of compound 11. 1H NMR (600MHz, CDCl3) δ7.82(s,1H),7.47(d,J=7.8Hz,1H),7.30(d,J=7.8Hz,1H),7.13(t,J=7.8Hz,1H ),7.08(t,J=7.2Hz,1H),5.62-5.56(m,1H),5.17-5.12(m,2H),3.71(s,3H),3.32(d,J=11.4Hz,1H), 3.08(dd,J=11.4,6.0Hz,1H),3.02-2.97(m,2H),2.74-2.60(m,3H),2.33(q,J=11.4Hz,2H),2.23(dd d,J=13.8,10.8,3.6Hz,1H),2.08(dd,J=15.6,9.6Hz,1H),1.98-1.91(m,1H),1.37(q,J=12.0Hz,1H). 13 C NMR (150MHz, CDCl3) δ173.7,138.8,136.2,134.5,127.5,121.6,119.6,118.3,117.9, 110.9,108.4,61.0,59.5,53.0,51.8,47.1,38.5,37.0,35.5,21.9.HRMS(ESI):Calcd for C 20 H 25 N2O5[M+H] + :325.1911,found:325.1916.

[0125] Example 14: HT22 cytotoxicity evaluation experiment

[0126] 1. HT22 cells (mouse hippocampal neuronal cells) were cultured in vitro using DMEM medium containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin at 37°C and 5% CO2 for routine culture and passage;

[0127] 2. HT22 cells were seeded in a 96-well plate at 5000 cells / well. After culturing for 24 hours, the old culture medium was removed, and DMEM culture medium (containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin) containing 50 μmol / L coronin-type compounds was added to the sample group. At the same time, a control group (DMEM culture medium containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin) was set up. After continuing to culture for 48 hours, the culture medium was removed, and the survival rate of the cells was detected by the MTS method (a culture medium containing 20% ​​MTS was added). The cells were incubated for 1 hour at 37°C and 5% CO2. The blank group was a culture medium containing 20% ​​MTS, and the OD was detected using an enzyme reader. 490 , calculation formula: cell survival rate (%) = (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100%;

[0128] Results Figure 1 As can be seen from the figure, compared with the control group, the survival rate of the test compound group at a concentration of 50 μmol / L was higher than 80%, indicating that the coronin-type compound had no effect on the survival or proliferation of HT22 cells at this concentration.

[0129] Example 15: Experimental study on the inhibitory activity of coryneform compounds on Erastin-induced ferroptosis in HT22 cells

[0130] 1. HT22 cells were cultured in vitro using DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 units / mL penicillin at 37°C and 5% carbon dioxide for routine culture and passage;

[0131] 2. HT22 cells were seeded in 96-well plates at 5000 cells / well and cultured for 24 hours. The original culture medium was removed and the control group was added with normal DMEM medium (containing 10% FBS, 100 μg / mL streptomycin, 100 units / mL penicillin) without Erastin and the test compound; the Erastin-treated group was added with DMEM medium (containing 10% FBS, 100 μg / mL streptomycin, 100 units / mL penicillin) containing 10 μmol / L Erastin but not containing the test compound; the sample group was added with DMEM medium (containing 10% FBS, 100 μg / mL streptomycin, 100 units / mL penicillin) containing 10 μmol / L Erastin and different concentrations of coronin-type compounds; the positive control group: containing 10 μmol / L Erastin and Fer-1 were cultured in DMEM medium (containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin); after 24 hours of continuous culture, the drug-containing medium was removed, and the cell survival rate was detected by MTS method (addition of medium containing 20% ​​MTS), and the blank group was incubated with 20% MTS medium at 37°C and 5% CO2 for 1 hour, and the OD was detected using an enzyme reader. 490 , calculate the cell survival rate of adding different concentrations of coryneform alkaloid natural products or natural product-like products, the calculation formula is: cell survival rate (%) = (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100%.

[0132] The experimental results are shown in Table 2. It can be seen from the table that Ferrostatin-1 (Fer-1) is a known ferroptosis inhibitor and is used as a positive drug in this experiment. All 16 compounds have inhibitory activity against Erastin-induced ferroptosis in HT22 cells, among which compounds 10, 18, 21, and 24 all show better inhibitory activity than the positive drug;

[0133] Table 2 Inhibitory activity of compounds on Erastin-induced ferroptosis in HT22 cells

[0134]

[0135]

[0136] a The values ​​are expressed as the mean±SD of three independent experiments. b The positive control. Example 16: Experiment on the inhibitory activity of coryneform compounds on RSL3-induced ferroptosis in HT22 cells

[0137] 1. HT22 cells were cultured in vitro using DMEM medium containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin at 37°C and 5% CO2 for routine maintenance and passage;

[0138] 2. HT22 cells were seeded in 96-well plates at 5000 cells / well. After culturing for 24 hours, the original culture medium was removed. The control group was added with normal DMEM culture medium (containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin) without RSL3 and the test compound; the RSL3-treated group was added with DMEM culture medium (containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin) containing 1 μmol / L RSL3 but not containing the test compound; the sample group was added with DMEM culture medium (containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin) containing 1 μmol / L RSL3 and different concentrations of coronin-type compounds; the positive control group: containing 1 μmol / L RSL3 and different concentrations of Fer-1 in DMEM medium (containing 10% FBS, 100 μg / mL streptomycin, 100 units / mL penicillin); after 24 hours of continuous culture, the drug-containing medium was removed, and the cell survival rate was detected by MTS method (addition of medium containing 20% ​​MTS), and the blank group was added with medium containing 20% ​​MTS, incubated at 37°C, 5% CO2 for 1 hour, and the OD was detected by microplate reader 490 , calculate the cell survival rate of adding different concentrations of corydaline-type alkaloid natural products and natural product-like products, the calculation formula is: cell survival rate (%) = (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100%, with cell survival rate as the ordinate and compound concentration as the abscissa, to draw the cell survival curve of each compound under RSL3-induced ferroptosis conditions, and calculate the EC of each compound 50 The values ​​were used to evaluate the inhibitory activity of each compound on RSL3-induced ferroptosis;

[0139] The results are shown in Table 3. The experimental results show that the results of this example are consistent with the Erastin induction trend in Example 2. The 16 compounds have different degrees of inhibitory activity on RSL3-induced HT22 cell ferroptosis, among which compounds 21 and 24 show better inhibitory activity than the positive drug;

[0140] Inhibitory activity of the three compounds on RSL3-induced ferroptosis in HT22 cells

[0141]

[0142]

[0143] a represents the mean ± SD of three independent experiments, and b represents the positive control.

[0144] Example 17: Inhibitory effects of coryneform compounds 9-24 on other modes of death

[0145] In order to evaluate the specificity of the compounds, we tested the inhibitory effects of the compounds on common cell death modes such as apoptosis, autophagy, and programmed necrosis.

[0146] 1. HT22 cells were cultured in vitro using DMEM medium containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin at 37°C and 5% carbon dioxide for routine maintenance and passage;

[0147] 2. Inoculate HT22 cells into a 96-well plate, culture the cells in an incubator for 24 hours until they adhere to the plate, and then discard the culture medium.

[0148] STS induced cell apoptosis: the sample group was added with DMEM medium containing 25 μmol / L test compound and 0.5 μmol / L STS (same as above); the positive control group was added with DMEM medium containing 25 μmol / L Z-VAD-FMK and 0.5 μmol / L STS (same as above);

[0149] Rapamycin induced cell autophagy: DMEM medium containing 25 μmol / L test compound and 50 μmol / L rapamycin was added to the sample group (the components are the same as above); the positive control group was DMEM medium containing 25 μmol / L 3-MA and 50 μmol / L rapamycin (the same as above);

[0150] Combined use of tumor necrosis factor-α (TNF-α), compound SM-164 and zVAD-fmk (T / S / Z) to induce programmed cell necrosis: the sample group was added with DMEM medium containing 25 μmol / L of the test compound and 20 μmol / LT / S / Z (same as above); the positive control group was cultured in DMEM medium containing 25 μmol / L Nec-1 and 20 μmol / LT / S / Z for 24 hours, and the cell survival rate was tested using the MTS method. Cell survival rate (%) = (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100%, calculate the cell survival rate of each compound at different concentrations to evaluate the inhibitory activity of each compound on various types of cell death. The blank group and control group are the same as those in Example 14.

[0151] The experimental results are shown in Figure 2-4 ,As can be seen from the figure, the coryne-type compounds have no inhibitory activity on the above three types of cell death.

[0152] Example 18: Inhibitory effects of compounds 21 and 24 on lipid oxidation

[0153] 1. Detection of lipid ROS

[0154] HT22 cells were cultured at 3×10 4 Cells were inoculated into 6-well plates per well. After culturing for 24 hours, normal DMEM medium (same components as above) was added to the blank group. DMEM medium (same components as above) containing 10 μmol / L Erastin but not containing the test compound was added to the Erastin-treated group. DMEM medium (same components as above) containing 10 μmol / L Erastin and 25 μmol / L test compound was added to the sample group. The cells were incubated for 12 hours and then incubated with 5 μmol / L BODIPY-C11 probe at 37°C for half an hour. The cells were washed once with PBS, digested with trypsin, centrifuged and collected in 1 mL PBS buffer, and then the fluorescence intensity of the FL1 channel was detected at 488 nm using a flow cytometer. At least 10,000 cells were detected for each sample.

[0155] The experimental results are shown in Figure 5 As can be seen from the figure, 10 μmol / L Erastin significantly induced lipid peroxidation in HT22 cells after 12 hours of treatment; in the sample group, when the cells were co-incubated with compounds 21 and 24, the fluorescence intensity of the probe decreased significantly, indicating that compounds 21 and 24 could inhibit the generation of ROS in lipids;

[0156] 2. Detection of intracellular ROS

[0157] HT22 cells were cultured at 3×10 4 After 24 hours of inoculation in a 6-well plate, the sample group was added with Erastin±25μmol / L test compound for 12 hours (same as above), and then incubated with 10μmol / L carboxy-H2DCFDA probe at 37°C for half an hour. The cells were washed once with PBS, digested with trypsin, centrifuged and collected in 1mL PBS buffer, and then the fluorescence intensity of FL1 channel was detected at 488nm by flow cytometry. At least 10,000 cells were detected for each sample. The settings of the blank group and the Erastin treatment group were the same as step 1;

[0158] The fluorescent probe carboxy-H2DCFDA was used to detect the level of intracellular reactive oxygen species (ROS). The probe itself has no fluorescence. After entering living cells, it is hydrolyzed by esterases in the cells to generate DCFH. The non-fluorescent DCFH can be oxidized by intracellular ROS to generate fluorescent DCF. Therefore, the fluorescence intensity of DCF reflects the level of intracellular ROS. The experimental results are shown in Figure 6 The results showed that Erastin significantly induced an increase in ROS in HT22 cells after 12 hours of treatment, while compounds 21 and 24 concentration-dependently inhibited the increase in intracellular ROS induced by Erastin. This data result is consistent with the data results of lipid reactive oxygen species.

[0159] 3. Detection of the inhibitory effect of compounds on MDA

[0160] Malondialdehyde (MDA) is also an important indicator for evaluating ferroptosis. The lipid hydroperoxides generated during lipid peroxidation not only react with Fe 2+ The reaction generates lipid alkoxy free radicals, which attack the surrounding polyunsaturated fatty acids and promote lipid peroxidation. It also decomposes to generate toxic and active aldehyde metabolites such as malondialdehyde. These aldehydes can cross-link to proteins and DNA, inactivating these biological macromolecules and affecting the normal activities of cells.

[0161] HT22 cells were cultured at 3×10 4 After inoculating cells / well in a 6-well plate and culturing for 24 hours, the sample group was added with Erastin±compound for 12 hours (compound concentrations of 25μmol / L, 12.5μmol / L, and 6.25μmol / L), and lysed with lysis buffer on ice, centrifuged at 4°C and 13000rpm for 10min, and the supernatant was transferred to a new EP tube and placed on ice for later use. The blank group and Erastin-treated group were set up in the same way as in step 1, and the positive control group Fer-1 was added with DMEM medium containing 10μmol / L Erastin and the test compound at concentrations of 25μmol / L, 12.5μmol / L, and 6.25μmol / L;

[0162] MDA was detected using a lipid oxidation MDA detection kit. The MDA detection working solution was prepared according to the instructions, and then 100 μL of supernatant and 200 μL of MDA detection working solution were added to the EP tube, mixed, and heated at 100°C in a metal bath for 15 min; cooled to room temperature, centrifuged at 10,000 rpm for 10 min, the supernatant was added to a 96-well plate, and the absorbance at 532 nm was detected using an enzyme reader.

[0163] The experimental results are shown in Figure 7 The MDA level in the model group was significantly increased, indicating that Erastin induced cellular lipid peroxidation; while compounds 21 and 24 significantly reduced the MDA level, indicating that compounds 21 and 24 inhibited lipid peroxidation.

[0164] 4. Detection of intracellular GSH

[0165] GPX4 uses glutathione GSH as a cofactor to catalyze the reduction of lipid hydroperoxides and inhibit lipid peroxidation and ferroptosis. We used a commercial GSH content kit for detection;

[0166] HT22 cells were cultured at 3×10 4 After culturing for 24 hours, the sample group was added with Erastin±compound for 12 hours (compound concentrations of 25μmol / L, 12.5μmol / L, and 6.25μmol / L), and the blank group and Erastin treatment group were set up in the same way as in step 1; the positive control group Fer-1 was added with DMEM medium containing 10μmol / L Erastin and the test compound at concentrations of 25μmol / L, 12.5μmol / L, and 6.25μmol / L;

[0167] The experimental results are shown in Figure 8 , the GSH level in the Erastin-treated group was significantly decreased, indicating that Erastin induced GSH depletion; while compounds 21 and 24 inhibited lipid peroxidation by increasing GSH levels.

[0168] Example 19: Test of the protective activity of coryza-type compounds against glutamate-induced HT22 cell death

[0169] 1. HT22 cells were cultured in vitro using DMEM medium containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin at 37°C and 5% CO2 for routine maintenance and passage;

[0170] 2. HT22 cells were seeded in 96-well plates at 5000 cells / well. After culturing for 24 hours, the old culture medium was removed, and the control group was added with DMEM culture medium (same components as above); the glutamate treatment group was added with DMEM culture medium containing 10mmol / L glutamate but not containing the test compound (same components as above); the sample group was added with DMEM culture medium containing 10mmol / L glutamate and 25μmol / L test compound (same components as above); the positive control group: DMEM culture medium containing 10mmol / L glutamate and 25μmol / L Edaravone (same components as above); after further culturing for 24 hours, the drug-containing culture medium was removed, and the cell survival rate was detected by MTS method (addition of culture medium containing 20% ​​MTS), and the blank group was added with culture medium containing 20% ​​MTS, incubated at 37°C, 5% CO2 for 1 hour, and the OD was detected using an enzyme reader. 490 , calculation formula: cell survival rate (%) = (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100%.

[0171] The experimental results are shown in Table 4. The survival rate of cells in the glutamate treatment group was only about 30-38.9%, and the cell survival rate of the compound group was higher than that of the glutamate treatment group, indicating that the 16 compounds had a certain protective effect on HT22 cells at 25 μmol / L, among which compounds 21 and 24 showed significant neuroprotective activity;

[0172] Table 4 Inhibitory activity of compounds on Glu-induced HT22 cell death

[0173]

[0174] a represents the mean ± SD of three independent experiments, and b represents the positive control.

[0175] Example 20: Corinnamon-type compounds inhibit LPS-induced inflammation in BV2 cells

[0176] 1. NO inhibition activity test

[0177] (1) BV2 cells (mouse microglia) were cultured in vitro using DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 units / mL penicillin at 37°C and 5% CO2 for routine maintenance and passage;

[0178] (2) Take cells in the logarithmic growth phase, digest, centrifuge, and resuspend them in 100 μL per well. The number of cells per well is 4×10 4The cells were inoculated in 96-well plates, and the original culture medium was removed after culturing for 24 hours; the blank group was added with DMEM medium (containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 units / mL penicillin); the LPS-treated group was added with DMEM medium (containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 units / mL penicillin) containing 10 μg / mL LPS but not containing the test compound; the sample group was added with DMEM medium (containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 units / mL penicillin) containing 10 μg / mL LPS and different concentrations of the test compound (25 μmol / L, 12.5 μmol / L, and 6.25 μmol / L); the positive control group: containing 10 μg / mL The cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 units / mL penicillin) containing LPS and 25 μmol / L edaravone for 24 hours, and then the NO level of each group was detected using a commercially available NO kit.

[0179] (3) Griess method to detect the NO inhibition experiment of the target compound. The NO level in the culture medium was determined according to the kit instructions. The main steps are as follows:

[0180] ① Add standards and samples to a 96-well plate at 50 μL / well;

[0181] ② Add room temperature Griess reagent I to each well at 50 μL / well;

[0182] ③ Add room temperature Griess reagent II to each well at 50 μL / well;

[0183] ④Measure the absorbance at 540nm.

[0184] The experimental results are shown in Fig. 9 The standard curve of NO generation of each compound was drawn with absorbance as the ordinate and compound concentration as the abscissa, and the half NO generation amount of each compound was calculated to evaluate the inhibitory activity of each compound on NO generation. Compounds 21 and 24 had significant inhibitory activity on LPS-induced BV2 cell inflammatory NO generation at a concentration of 25 μM, and we will further evaluate their inhibition of inflammatory factors.

[0185] 2. Detection experiment of the effect of coronin-type compounds on inflammatory factors (TNF-α, IL-1β, IL-6)

[0186] (1) BV2 cells (mouse microglia) were cultured in vitro using DMEM medium containing 10% fetal bovine serum, 100 μg / mL streptomycin, and 100 units / mL penicillin at 37°C and 5% CO2 for routine maintenance and passage;

[0187] (2) Take cells in the logarithmic growth phase, digest, centrifuge, and resuspend them in 100 μL per well. The number of cells per well is 4×10 4 The cells were inoculated in a 96-well plate, and the original culture medium was removed after culturing for 24 hours; the blank group was added with normal DMEM culture medium (containing 10% FBS, 100 μg / mL streptomycin, and 100 units / mL penicillin); the lipopolysaccharide (LPS)-treated group was added with DMEM culture medium containing 10 μg / mL LPS but not containing the test compound (the components are the same as above); the sample group was added with DMEM culture medium containing 10 μg / mL LPS and multiple concentrations of the test compound (25 μmol / L, 12.5 μmol / L, 6.25 μmol / L) (the components are the same as above); the positive control group: DMEM culture medium containing 10 μg / mL LPS and 25 μM Edaravone (the components are the same as above), and after continuing to culture for 24 hours, the target compound was used to detect the inhibition of inflammatory factor release experiment using a commercially available ELISA kit, and the level of inflammatory factors in the culture medium was determined according to the instructions of the kit.

[0188] The experimental results are shown in Figure 10-12 Compounds 21 and 24 had a significant inhibitory effect on LPS-induced BV2 cell inflammation at a concentration of 25 μmol / L.

[0189] Example 21: Brain-blood barrier permeation test of compounds (PAMPA-BBB)

[0190] 1. Prepare 2% porcine brain lipid (PBL): weigh 20 mg porcine brain extract and add 1 mL of n-dodecane to fully dissolve it. Prepare 50 mmol / L PBS: weigh 1.36 g K2HPO4 in 200 mL ultrapure water and adjust the pH to 7.4 with KOH;

[0191] 2. Carefully pipette 4 μL of 2% pig brain extract onto the hydrophobic membrane of the 96-well plate as the dosing pool, quickly pipette 200 μL of the sample solution to be tested into the 96-well plate as the dosing pool, add 300 μL of buffer (pH 7.4, PBS: ethanol: DMSO = 68: 30: 2) to the receiving pool, carefully place the dosing pool flat on the receiving pool, and make the membrane fully contact with the receiving liquid, let it stand at room temperature for 10 hours, and carefully remove the dosing pool; use a multifunctional microplate reader to test the OD value of the compound in the receiving pool at its maximum absorption peak; pipette 200 μL of the sample solution to be tested into 300 μL of buffer (pH 7.4, PBS: ethanol: DMSO = 68: 30: 2) and mix well, as a theoretical equilibrium solution, measure the OD value of the compound at the maximum absorption peak. Use a 2% DMSO buffer as a blank control. Calculate P according to the formula e Value, Pe =-V d ×V a / [(V d +V a )A×t]×ln(1-OD 接收池内待测样品 / OD 理论平衡溶液待测样品 ). a is the volume of the drug delivery pool, V d is the volume of the receiving pool, A is the permeation area, and t is the permeation time. e When >4.7, it means that the compound has a good ability to pass through the blood-brain barrier.

[0192] Table 5. Limit values ​​of blood-brain barrier

[0193]

[0194] The experimental results are shown in Table 6. The P values ​​of compounds 21 and 24 are e The values ​​are 6.7 and 5.7, respectively, indicating that the compound has good blood-brain barrier penetration ability;

[0195] Table 6. BBB results of PAMPA tests 21 and 24

[0196]

Claims

1. The structural formula of the coryne compounds shown in Formula I and II: In the formula: R is selected from H, p-methoxybenzyl; R1 is selected from ethyl, vinyl, aldehyde, hydroxymethyl; In formula I, the connection mode between C15 and C16, and between C15 and C20 is C=C or CC. When the connection mode between C15 and C16 is C=C, the conformation of hydrogen at C20 is α-configuration. In formula II, the conformation of hydrogen at C15 is α-configuration or β-configuration, and the conformation of hydrogen at C20 is α-configuration or β-configuration.

2. The method for preparing the coronin-type compound according to claim 1, characterized in that: Here are the steps: (1) Compound 1 is subjected to Vilsmeier-Haack reaction to obtain compound 2, and compound 2 is condensed with tert-butylsulfenamide to obtain compound 3; wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, trifluorosulfonyl, and R 2 is halogen, OH, p-toluenesulfonyl, methylsulfonyl; (2) Compound 3 was subjected to a vinylogous Mannich addition reaction to obtain compound 5 and diastereomer 4; wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, trifluorosulfonyl, and R 2 is halogen, OH, p-toluenesulfonyl, methylsulfonyl; (3) Compound 5 was deprotected from the sulfinyl group and then a base was added to construct the C ring through N-alkylation, and then compound 6 was obtained through Mannich cyclization reaction; wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, trifluorosulfonyl, and R 2 is halogen, OH, p-toluenesulfonyl, methylsulfonyl; (4) Compound 6 is unprotected by acetone to obtain compound 7, and compound 7 is reduced to the ester group to obtain compound 8; wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, and trifluorosulfonyl; (5) Compound 8 was subjected to Horner-Wadsworth-Emmons reaction to obtain intermediate 9; wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, and trifluorosulfonyl; (6) Intermediate 9 is subjected to oxidation reaction to obtain compound 10, compound 10 is subjected to Witting reaction to obtain compound 11, the exocyclic double bond in compound 11 is reduced to obtain compound 12, the indole protecting group is removed to obtain compound 13, and finally the endocyclic double bond in compound 13 is reduced to obtain compound 14 and its diastereomer 15; wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, and trifluorosulfonyl; (7) The α,β-unsaturated double bond of intermediate 9 is reduced to obtain compound 16. Compound 16 is oxidized to an aldehyde and subjected to a Witting reaction to obtain compound 17 and its diastereomer 18. The indole protecting group in compound 17 is removed to obtain compound 19. Finally, the double bond is reduced to obtain compound 20. wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, and trifluorosulfonyl; (8) After removing the indole protecting group from intermediate 9, compound 21 is obtained, and the double bond is reduced to obtain compound 22, which is oxidized to aldehyde to obtain compound 23, and finally subjected to Witting reaction to obtain compound 24; wherein R is selected from p-methoxybenzyl, benzyl, benzyloxycarbonyl, tert-butyloxycarbonyl, p-toluenesulfonyl, benzenesulfonyl, p-nitrobenzenesulfonyl, trimethylbenzenesulfonyl, methyl, methyl formate, and trifluorosulfonyl.

3. The method for preparing a coronin-type compound according to claim 2, characterized in that: In step (3), the removal of the sulfinyl group is achieved by adding hydrochloric acid, iodine or acetyl chloride-methanol; the base in the N-alkylation process is selected from one of NaHCO3, NaH, Na2CO3, K2CO3, and Cs2CO3; the carbon unit in the Mannich cyclization reaction for the construction of the D ring is selected from formaldehyde gas, formaldehyde aqueous solution or polyformaldehyde, and the reaction temperature is 50-100°C.

4. The method for preparing a coronin-type compound according to claim 2, characterized in that: The Witting reagent used in the Horner-Wadsworth-Emmons reaction of step (5) is selected from H1, H2, H3, and H4; the solvent is selected from tetrahydrofuran, dichloromethane, 1,4-dioxane, 1,2-dichloroethane, toluene, and acetonitrile, and the reaction temperature is 50 to 100° C.; 5. The method for preparing a coronin-type compound according to claim 2, characterized in that: In step (6), the oxidant used in the oxidation reaction of intermediate 9 to prepare compound 10 is selected from Dess-Martin periodinane, 2-iodoacylbenzoic acid, pyridinium chlorochromate, pyridinium dichromate, and pyridine sulfur trioxide; the catalyst used in the reduction of compound 13 is palladium carbon, the molar ratio of compound 13 to the catalyst is 1:0.05-0.5, and the hydrogen pressure is 0.1-5MPa.

6. The method for preparing a coronin-type compound according to claim 2, characterized in that: In step (7), the catalyst used in the hydrogenation reduction reaction of compound 9 to prepare compound 16 is selected from palladium carbon, palladium hydroxide on carbon, nickel chloride hexahydrate-sodium borohydride, and Raney nickel; and the solvent is selected from methanol, tetrahydrofuran, and ethanol.

7. The method for preparing a coronin-type compound according to claim 2, characterized in that: In step (8), the catalyst for the catalytic reduction reaction of compound 21 to prepare compound 22 is one of palladium carbon, rhodium carbon, and palladium hydroxide carbon; the molar ratio of compound 21 to the catalyst is 1:0.05-0.5; the reaction solvent is selected from methanol, tetrahydrofuran, and ethanol; and the hydrogen pressure is 0.1-5MPa.

8. Use of the coronin-type compound according to claim 1 in the preparation of anti-ischemic stroke drugs.

9. The use according to claim 8, characterized in that: Conanine-type compounds have ferroptosis inhibitory activity and anti-neuroinflammatory activity.

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