An indole derivative, a preparation method and application thereof

Abstract

The present application relates to a kind of indole derivatives and its preparation method and application, belong to the field of medicinal chemistry and medicine technology.The chemical structural formula of the indole derivative is as shown below.The present application uses L-tryptophan or its derivative as chiral starting material, realizes the stereoselective construction of the tetra-cyclic indole core skeleton by chiral source induction, and further realizes the synthesis of the indole derivative by efficient functional group transformation strategy.The indole derivative plays the role of treating and / or preventing anxiety or depression by improving / restoring hippocampal dentate gyrus neurogenesis, has important development value.

Description

Technical Field

[0001] This invention relates to an indole derivative, its preparation method, and its application, belonging to the fields of medicinal chemistry and pharmaceutical technology. Background Technology

[0002] Major Depressive Disorder (MDD) is a prevalent neuropsychiatric disorder characterized by depressed mood, anhedonia, and cognitive impairment. It is characterized by high incidence, high relapse rate, high disability rate, and high suicide rate. Compared to other physical illnesses, depression has an insidious onset and a prolonged course; early mild symptoms are easily overlooked, and most patients are diagnosed with persistent neurological damage and social decline, severely impacting individual mental and physical health, family stability, and social development. Social environmental pressures, chronic stress, sleep disorders, and neuroimmunological disorders contribute to the high incidence of depression, and the affected population is trending younger. More seriously, some patients with depression do not experience significant symptom improvement after routine interventions and gradually develop treatment-resistant depression, greatly increasing the difficulty of clinical treatment and the risk of death and disability. The pathogenesis of depression is complex, involving multiple pathological factors such as monoamine neurotransmitter imbalance, hippocampal neuroplasticity damage, chronic central inflammation, and adult hippocampal neurodevelopment disorders. Currently, first-line antidepressants in clinical practice are mainly selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs). These drugs mainly improve depressive symptoms by regulating the level of monoamine neurotransmitters. However, they generally have many clinical shortcomings, such as delayed onset of action, limited effective response rate, large individual differences, easy development of drug resistance with long-term use, and adverse reactions in the gastrointestinal and nervous systems. Moreover, they cannot fundamentally repair the pathological damage to the central nervous system, making it difficult to achieve radical cure and long-term prognosis improvement.

[0003] The indole heterocyclic skeleton is the dominant core structure for both natural alkaloids and synthetic centrally acting drugs. Its chemical structure is homologous to endogenous centrally active substances such as serotonin and tryptophan, making it an important lead structure for the development of antidepressant drugs. Existing research indicates that indole derivatives exert antidepressant effects through the regulation of the monoamine neurotransmitter system, antioxidant pathways, and anti-inflammatory mechanisms. For example, natural indole alkaloids (yohimbine, limonene) and gut microbiota metabolites (indoleacetic acid, indolepropionic acid) have shown certain neuroprotective activities. However, current research on the mechanisms of indole drugs largely focuses on the monoamine neurotransmitter system, receptors, and antioxidant and anti-inflammatory pathways. Whether indole derivatives can achieve antidepressant effects by regulating the proliferation and differentiation of hippocampal neural stem cells (NSCs) and repairing adult hippocampal neurogenesis has not yet been systematically studied, representing a key gap in the research on the antidepressant mechanisms of indole drugs.

[0004] Although some studies have reported that indole-based active substances can participate in the regulation of neural cells, it remains unclear whether they exert their antidepressant effects by targeting the proliferation and differentiation of neural stem cells (NSCs). Furthermore, the complete regulatory chain of "indole derivatives-neural stem cells-hippocampal neurogenesis" has not been elucidated, greatly limiting the structural optimization, mechanism refinement, and clinical translation of indole-based antidepressant lead compounds. Therefore, there is an urgent need to develop novel indole derivatives and elucidate their mechanism of action through regulating neural stem cell function to fill the gaps in existing technologies. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, this invention provides an indole derivative, its preparation method, and its applications.

[0006] In a first aspect, the present invention provides an indole derivative, the chemical structural formula of which is shown below: .

[0007] Secondly, the present invention provides a method for preparing the above-mentioned indole derivatives, comprising the following steps: (1) Starting from N-BOC-L-tryptophan or tryptophan, an amino-protected intermediate is prepared by nucleophilic substitution reaction with an amino-protecting reagent under alkaline conditions. The intermediate is then coupled with bromobutyne protected by TBS or TBDPS and the silyl protecting group is removed to obtain an alkynol intermediate. The alkynol intermediate is oxidized to carbonyl by an oxidant and then undergoes a tandem cyclization reaction under an acid catalyst to obtain product (-)-6 or product (±)-8. Product (-)-6 is hydrogenated under a metal catalyst to obtain product (-)-7. Product (-)-7 is reacted with a disulfide bond activating reagent and a phosphine reagent, and then a thiol reagent is added to carry out a desulfurization cyclization reaction to obtain product (-)-8. (2) The product (-)-8 or (±)-8 is deprotected under reducing deprotection conditions to obtain the product (-)-9 or (±)-9; (3) The product (-)-9 or (±)-9 undergoes a coupling reaction under the action of a palladium catalyst to obtain the product (-)-10 or (±)-10; (4) The product (-)-10 or (±)-10 undergoes an oxidation reaction under the action of an oxidizing agent, and then a reducing agent is added to carry out a reduction reaction to obtain the product (-)-11 or (±)-11; further protection with silicon-based materials yields the product (-)-12 or (±)-12. (5) The product (-)-12 or (±)-12 undergoes an alkylation reaction with a propargylating agent under alkaline conditions to give the product (-)-13 or (±)-13; (6) The product (-)-13 or (±)-13 undergoes intramolecular cyclization and imine reduction under the action of silver salt catalyst to obtain the product (+)-14 or (±)-14. (7) The product (+)-14 or (±)-14 reacts with a trifluoromethanesulfonating agent under alkaline conditions to give the product (+)-15 or the product (±)-15. (8) The product (+)-15 or (±)-15 undergoes a deprotection reaction in a hydrogen atmosphere under the action of a metal catalyst and an alkaline auxiliary agent, and is then treated with acid to obtain (+)-Limaspermidine or (±)-Limaspermidine.

[0008] Further, in step (1), when N-Boc-L-tryptophan is used as the starting material, it reacts with p-methoxybenzyl bromide under the action of potassium tert-butoxide to introduce a p-methoxybenzyl protecting group; then it reacts with benzyl bromide under the action of potassium carbonate to introduce a benzyl protecting group, thus obtaining an amino-protected intermediate; when tryptophan is used as the starting material, it reacts with p-methoxybenzyl bromide under the action of sodium hydride to introduce a p-methoxybenzyl protecting group; then it reacts with p-toluenesulfonyl chloride under the action of triethylamine to introduce a p-toluenesulfonyl protecting group, thus obtaining an amino-protected intermediate.

[0009] Further, in step (1), the oxidant is selected from one or more of manganese dioxide, Desmond-Martin oxidant, IBX or Swern oxidation system; the acid catalyst is selected from one or more of camphor sulfonic acid, p-toluene sulfonic acid, trifluoromethanesulfonic acid or trifluoroacetic acid; the metal catalyst is selected from one or more of palladium on carbon, platinum on carbon, Raney nickel or rhodium on carbon; the disulfide bond activator is selected from one or more of 2,2'-dithiopyridine-1,1'-dioxide, 2,2'-dithiobis(5-nitropyridine), dithiobenzoate compounds; the phosphine reagent is selected from one or more of tri-n-butylphosphine, triphenylphosphine, triethylphosphine; and the thiol reagent is selected from one or more of tert-dodecyl mercaptan, dodecyl mercaptan, tert-butyl mercaptan or benzyl mercaptan.

[0010] Furthermore, in step (2), the system in the reducing deprotection conditions is selected from one or more of sodium naphthalene, lithium naphthalene, sodium / naphthalene system or alkali metal aryl radical system.

[0011] Further, in step (3), the palladium catalyst is selected from one or more of tetra(triphenylphosphine)palladium, palladium acetate, palladium chloride, or palladium phosphine complexes.

[0012] Further, in step (4), the oxidant is selected from one or more of the following: potassium osmium tetroxide / sodium periodate system, potassium permanganate, osmium tetroxide / sodium periodate system, or ozone oxidation system; the reducing agent is selected from one or more of the following: sodium borohydride, lithium aluminum hydride, diisobutylaluminum hydride, or borane; and the silicon-based protecting agent is selected from one or more of the following: tert-butyldimethylchlorosilane, trimethylchlorosilane, tert-butyldiphenylchlorosilane, or triisopropylchlorosilane.

[0013] Further, in step (5), the propargylating agent is selected from one or more of propargyl bromide, propargyl chloride, propargyl iodide or propargyl sulfonate; the base is selected from one or more of n-butyllithium, sodium hydride, potassium tert-butoxide, potassium carbonate or hexamethyldisilamide lithium.

[0014] Further, in step (6), the silver salt is selected from one or more of silver trifluoromethanesulfonate, silver bis(trifluoromethanesulfonyl)imide, silver tetrafluoroborate, or silver hexafluorophosphate.

[0015] Further, in step (7), the trifluoromethanesulfonylating agent is selected from one or more of N,N-ditrifluoromethanesulfonyl-5-chloro-2-aminopyridine and aminosulfonyl reagents.

[0016] Further, in step (8), the metal catalyst is selected from one or more of palladium on carbon, platinum on carbon, Raney nickel or rhodium on carbon; the alkaline additive is selected from one or more of lithium carbonate, sodium carbonate, potassium carbonate or cesium carbonate; and the acid is selected from one or more of hydrochloric acid, sulfuric acid, trifluoroacetic acid or methanesulfonic acid.

[0017] Preferably, the method for preparing the indole derivative includes the following steps: (1) Under nitrogen protection, N-BOC-L-tryptophan was dissolved in N,N-dimethylformamide, potassium tert-butoxide was added, and p-methoxybenzyl bromide was added dropwise at 0 °C to give crude product 1; crude product 1 was dissolved in N,N-dimethylformamide, potassium carbonate was added, and benzyl bromide was added dropwise at 0 °C to give crude product 2; crude product 2 was dissolved in dichloromethane, and 4 M 1,4-dioxane HCl solution was added dropwise at 0 °C to give crude product; crude product was dissolved in dichloromethane, triethylamine was added at 0 °C, and p-toluenesulfonyl chloride was added dropwise to give compound 3; compound 3 was coupled with TBS-protected bromobutyne in the presence of cuprous bromide, N,N′-dimethylethylenediamine and potassium carbonate to give crude product, which was then dissolved in tetrahydrofuran at 0 °C. A mixed solution of tetrabutylammonium fluoride and acetic acid was added at ℃, and the mixture was stirred at room temperature to obtain product 5. Product 5 was dissolved in dichloromethane with MnO2 added, and the mixture was stirred at room temperature to obtain a crude product. This crude product was then dissolved in anhydrous dichloromethane, and camphor sulfonic acid was added at 0 ℃, followed by stirring to obtain product (-)-6. 10% palladium on carbon was added to a methanol solution containing ammonium formate at 0 ℃, stirred, and then product (-)-6 was added. The mixture was stirred at room temperature to obtain product (-)-7. Product (-)-7 was reacted with 2,2'-dithiopyridine-1,1'-dioxide and tri-n-butylphosphine in dichloromethane at room temperature in the dark, and then tert-dodecyl mercaptan was added under red LED irradiation to obtain product (-)-8. Using tryptamine as a starting material, the mixture was dissolved in... N , N In dimethylformamide, NaH was added in portions at 0°C with stirring, followed by dropwise addition of p-methoxybenzyl bromide. The reaction was carried out at room temperature to give product 17. Product 17 was dissolved in dichloromethane, and triethylamine was added at 0°C, followed by p-toluenesulfonyl chloride. The reaction was carried out at room temperature to give product 18. Product 18 was coupled with TBDPS-protected bromobutyne in the presence of 1,10-phenanthroline, potassium phosphate, and copper sulfate pentahydrate to give product 20. Product 20 was dissolved in tetrahydrofuran, and tetrabutylammonium fluoride trihydrate was added in portions at 0°C. The reaction was carried out at room temperature to give product 21. Product 21 was dissolved in dichloromethane, and tetrabutylammonium fluoride trihydrate was added at 0°C. N -methyl- N -Oxymorpholine was stirred and then tetrapropylammonium perruthenate was added. The reaction was carried out to obtain product 22. Product 22 was dissolved in anhydrous dichloromethane, camphor sulfonic acid was added at 0 °C, and the reaction was stirred to obtain product (±)-8. (2) Dissolve product (-)-8 or (±)-8 in anhydrous tetrahydrofuran, and add sodium naphthalene tetrahydrofuran solution dropwise at -78 °C until the deep green color does not fade, to obtain product (-)-9 or (±)-9; (3) Dissolve product (-)-9 or (±)-9 in 1,4-dioxane, add tetra(triphenylphosphine)palladium and allyl methyl carbonate under nitrogen protection, and react at 40 °C to obtain product (-)-10 or (±)-10. (4) Dissolve product (-)-10 or (±)-10 in a mixed solvent of potassium tert-butoxide / tetrahydrofuran / water, add potassium osmium tetroxide, 1,4-diazabicyclo[2.2.2]octane, potassium ferricyanide and sodium periodate, react at room temperature and then cool to -20 °C and add sodium borohydride to obtain product (-)-11 or (±)-11; dissolve it in dichloromethane, add tert-butyldimethylchlorosilane and imidazole at 0 °C, react to obtain product (-)-12 or (±)-12; (5) Dissolve product (-)-12 or (±)-12 in tetrahydrofuran, add NaH and propargyl bromide at 0 °C, and react at 40 °C to obtain product (-)-13 or (±)-13; (6) Dissolve product (-)-13 or (±)-13 in dichloroethane, add silver bis(trifluoromethanesulfonyl)imide, react at room temperature, cool to -20 °C and add ethanol solution of sodium cyanoborohydride to obtain product (+)-14 or (±)-14. (7) Dissolve product (+)-14 or (±)-14 in anhydrous tetrahydrofuran, add bis(trimethylsilylaminolithium) at -78 °C, stir, add N,N-ditrifluoromethanesulfonyl-5-chloro-2-aminopyridine, react at room temperature to obtain product (+)-15 or product (±)-15. (8) Dissolve the product (+)-15 or (±)-15 in ethyl acetate, add 10% palladium on carbon and lithium carbonate, react under a hydrogen atmosphere, then add 2M HCl and continue stirring, and then add NaHCO3 at 0 °C to terminate the reaction to obtain (+)-Limaspermidine or (±)-Limaspermidine.

[0018] The preparation method of the present invention uses L-tryptophan or its derivatives as chiral starting materials, and achieves stereoselective construction of tetracyclic indole core skeleton through chiral source induction, and further achieves the synthesis of indole derivatives of the present invention through efficient functional group transformation strategy.

[0019] Thirdly, the present invention provides the use of the above-mentioned indole derivatives in the preparation of medicaments for treating and / or preventing anxiety.

[0020] Fourthly, the present invention provides the use of the above-mentioned indole derivatives in the preparation of medicaments for treating and / or preventing depression.

[0021] Furthermore, the anxiety or depression mentioned is chronic stress-induced anxiety or depression.

[0022] Furthermore, the indole derivatives exert therapeutic and / or preventive effects by restoring and improving the homeostasis of neurogenesis in the hippocampal dentate gyrus, wherein restoring and improving the homeostasis of neurogenesis in the hippocampal dentate gyrus includes: (a) Reduce the abnormal increase in GFAP-positive cells, MCM2-positive cells, and GFAP / MCM2 double-positive cells in the hippocampal dentate gyrus induced by a chronic depression model; (b) Increased abnormal reduction of DCX-positive cells in the hippocampal dentate gyrus induced by a chronic depression model; (c) Reduce the abnormal increase of DCX / MCM2 double-positive cells in the hippocampal dentate gyrus induced by a chronic depression model.

[0023] Fifthly, the present invention provides a pharmaceutical composition comprising the above-described indole derivatives.

[0024] The beneficial effects of this invention: The indole derivatives provided by this invention, after in vitro cell activity screening and in vivo animal experiments, can significantly promote the proliferation of primary neural stem cells in mice and reverse the typical depressive-like behavior of prolonged immobility time in tail suspension and forced swimming tests in mice with chronic restraint stress (CRS) depression models, confirming that the compound possesses in vivo antidepressant activity. Based on this, this invention further targets hippocampal neurogenesis, systematically investigating the regulatory effect of the compound on endogenous hippocampal neurogenesis by constructing a depression animal model. Experimental results show that the derivative can effectively improve hippocampal neurogenesis in depressed mice, and its mechanism of action differs from traditional monoamine neurotransmitter system targeted drugs. This invention reveals the antidepressant mechanism of the compound from the perspective of neurogenesis regulation, and provides new candidate drugs and experimental evidence for the treatment of treatment-resistant depression, possessing significant clinical application prospects and drug development value. Attached Figure Description

[0025] Figure 1 (A) Representation of primary neural stem cell spheres after treatment with (+)-Limaspermidine ((+)-lim) and (±)-Limaspermidine ((±)-lim) at 0.1, 1, and 10 μM, respectively. Primary neural stem cells were extracted from the subventricular zone (SVZ) of 14-day-old C57BL / 6J mice and seeded at 1000 cells / well in 96-well plates. The corresponding compounds were added 6 h after seeding, and observation and statistics were performed after 3 days of culture. Scale bar = 100 μm. Figure 1 (B) shows the statistical results of the number of neural stem cell spheres after treatment with (+)-lim and (±)-lim. Figure 1(C) shows the statistical results of neural stem cell sphere diameters after (+)-lim and (±)-lim treatments. Data are expressed as mean ± SEM (n = 10, P <0.05, P <0.01, P <0.001 vs. Veh group; one-way ANOVA with LSD test).

[0026] Figure 2 (A) is a schematic diagram of the process of establishing a chronic restraint stress (CRS) model, administering medication, conducting behavioral tests, and collecting samples. Male C57BL / 6J mice aged 9-10 weeks were treated with CRS for 2 h daily for 21 consecutive days; (+)-lim, (±)-lim, or Veh were administered via intraperitoneal injection starting from day 1 of CRS and continued for 4 weeks. Figure 2 (B) shows the statistical results of the total movement distance of mice in each group during the open field experiment (OFT). Figure 2 (C) shows the statistical results of the average movement speed of mice in each group in OFT. Figure 2 (D) shows the statistical results of the time spent in the central area of ​​mice in each group during OFT. Figure 2 (E) is a heatmap of the movement trajectories of mice in each group in OFT. Figure 2 (F) shows the statistical results of the open-arm dwell time of mice in each group during the elevated cross maze test (EPM). Data are expressed as mean ± SEM (n = 8-10, ...). P <0.05, P <0.01, P <0.001 vs. Control + Veh group; # P <0.05, ## P <0.01 vs. CRS + Veh group; one-way ANOVA with LSD test).

[0027] Figure 3 (A) shows the statistical results of the immobility time of mice in each group during the tail suspension test (TST). Figure 3 (B) Statistical results of immobility time for mice in each group during the forced swimming test (FST). Data are expressed as mean ± SEM (n = 9-10, ...). P <0.05, P <0.01, P <0.001 vs. Control+ Veh group; # P <0.05, ## P <0.01 vs. CRS+Veh group; one-way ANOVA with LSD test).

[0028] Figure 4 (A) Representative immunofluorescence images of GFAP / MCM2 / DAPI in the dentate gyrus (DG) region of the hippocampus of mice in each group. The magnified images show GFAP-positive cells, MCM2-positive cells, and GFAP / MCM2 double-positive cells. Scale bar = 20 μm. Figure 4 (BD) shows the statistical results for GFAP-positive cells (B), MCM2-positive cells (C), and GFAP / MCM2 double-positive cells (D) in the DG region. GFAP was used to label neural stem / progenitor-like cells, and MCM2 was used to label proliferation-related cells. Data are expressed as mean ± SEM (n=3, P <0.05, P <0.01, P <0.001 vs. Control+Veh group; # P <0.05, ## P <0.01 vs. CRS+Veh group; one-way ANOVA with LSD test).

[0029] Figure 5 (A) Representative immunofluorescence images of DCX / MCM2 / DAPI in the dentate gyrus (DG) region of the hippocampus of mice in each group. The magnified images show DCX-positive cells, MCM2-positive cells, and DCX / MCM2 double-positive cells. Scale bar = 20 μm. Figure 5 (BD) shows the statistical results for DCX-positive cells (B), MCM2-positive cells (C), and DCX / MCM2 double-positive cells (D) in the DG region. DCX was used to label neuroblasts / immature neurons, and MCM2 was used to label proliferation-related cells. Data are expressed as mean ± SEM (n = 3, P<0.05, P <0.01, P <0.001 vs. Control+Veh group; # P <0.05, ## P <0.01 vs. CRS+Veh group; one-way ANOVA with LSD test). Detailed Implementation

[0030] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0031] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0032] Example 1 Preparation of (+)-Limaspermidine:

[0033] (1) Under N2 protection, N- Boc protection L β-Tryptophan (2.0 g, 6.58 mmol, 1.0 equiv.) was dissolved in anhydrous DMF (0.2 M). Potassium tert-butoxide (1.5 g, 13.82 mmol, 2.1 equiv.) was then added, and the mixture was stirred until the solid was completely dissolved, yielding a pale yellow solution. The reaction solution was cooled to 0 °C, and PMBBr (1.5 g, 7.24 mmol, 1.1 equiv.) was added dropwise. After 5 min, the reaction was terminated by adding 1 M HCl, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic layers were collected and combined, treated with water (50 mL) and saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was used directly in subsequent reactions without further purification.

[0034] The crude product was dissolved in anhydrous DMF, and potassium carbonate (1.8 g, 13.16 mmol, 2 equiv.) was added at 0 °C. After 10 min, BnBr (1.2 g, 7.24 mmol, 1.1 equiv.) was added dropwise, and the reaction was continued with stirring at room temperature. After 1 h, the reaction was terminated by adding ammonium chloride aqueous solution (50 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). The organic layers were collected and combined, treated with water (50 mL) and saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Crude product 2 was used directly in subsequent reactions without further purification.

[0035] Crude product 2 was dissolved in DCM (50 mL), and 4 M HCl / 1,4-dioxane (8.2 mL, 32.88 mmol, 5 equiv.) was added dropwise at 0 °C. The reaction was then continued with stirring at room temperature. After 8 h, the solvent was removed under reduced pressure. The resulting crude product was used directly in subsequent reactions without further purification.

[0036] The crude product was dissolved in dichloromethane, and TEA (2.0 g, 19.74 mmol, 3 equiv.) was added at 0 °C, followed by p-toluenesulfonyl chloride (1.6 g, 7.24 mmol, 1.1 equiv.). The reaction mixture was brought to room temperature. After 2 h, the reaction was terminated by adding ammonium chloride aqueous solution (50 mL), and the mixture was extracted with dichloromethane (50 mL × 3). The organic layers were collected and combined, treated with water (50 mL) and saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate, 10:1–5:1) to give product 3 (2.6 g, 70%) as a white solid.

[0037] The structural identification data of compound 3 are as follows: = 33.4 (c = 0.5, CHCl3).

[0038] 1 H NMR (600 MHz, CDCl3) δ 7.61 (d, J = 8.3 Hz, 2H), 7.48 (dt, J = 8.0, 1.0Hz, 1H), 7.32 – 7.26 (m, 3H), 7.24 (dt, J= 8.3, 0.9 Hz, 1H), 7.19 – 7.11 (m,3H), 7.09 – 7.05 (m, 1H), 7.04 – 6.95 (m, 4H), 6.79 (d, J = 8.7 Hz, 2H), 6.75(s, 1H), 5.20 (d, J = 9.0 Hz, 1H), 5.15 – 5.06 (m, 2H), 4.80 (d, J = 12.1 Hz,1H), 4.75 (d, J = 12.1 Hz, 1H), 4.32 (dt, J = 9.0, 5.7 Hz, 1H), 3.75 (s, 3H),3.24 (t, J = 5.9 Hz, 2H), 2.37 (s, 3H)。 13 C NMR(150 MHz, CDCl3) δ 171.1, 159.2,143.5, 136.8, 136.5, 134.9, 129.6, 129.4, 128.6, 128.5, 128.4, 128.2, 128.2,127.4, 127.2, 122.0, 119.6, 119.0, 114.2, 109.9, 108.1, 67.4, 56.4, 55.4,49.5, 29.5, 21.6。

[0039]

[0040] (2) Compound 3 (5.00 mmol, 1.0 equiv.) and bromoalkyne 4 (3.28 g, 12.50 mmol, 2.5 equiv.) were dissolved in toluene (40 mL), and CuBr (0.36 g, 2.50 mmol, 0.5 equiv.), DMEDA (0.18 g, 2.00 mmol, 0.4 equiv.), and potassium carbonate (6.90 g, 50.00 mmol, 10.0 equiv.) were added. The reaction mixture was heated in an oil bath at 60 °C for 24 h. After the reaction was completed, the mixture was filtered through a silica gel pad and washed with EtOAc. The residue obtained after evaporating the filtrate to dryness was used directly in subsequent reactions without further purification. The crude residue was dissolved in THF (40 mL), and TBAF (1 M) / AcOH (20:1) (10 mL, 10.00 mmol, 2.0 equiv.) was added at 0 °C. The mixture was then stirred at room temperature for 2 h. After the starting material was completely eliminated by TLC, the reaction was terminated by adding ammonium chloride aqueous solution (50 mL), followed by extraction with ethyl acetate (300 mL × 3). The organic layers were collected and combined, treated with saturated brine (200 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate, 2:1) to obtain product 5, a pale yellow oil.

[0041] The structural identification data of compound 5 are as follows: = 75.3 (c = 0.5, CHCl3).

[0042] 1 H NMR (600 MHz, CDCl3) δ 7.56 – 7.54 (m, 1H), 7.40 (dd, J = 7.5, 2.9 Hz, 2H), 7.33 (dt, J = 2.8, 1.5 Hz, 2H), 7.24 – 7.21 (m, 3H), 7.19 – 7.16 (m, 1H), 7.12 – 7.08 (m, 1H), 6.99 – 6.96 (m, 4H), 6.90 (d, J = 4.8 Hz, 1H), 6.81 – 6.78(m, 2H), 5.14 – 5.01 (m, 3H), 4.98 (d, J = 12.4 Hz, 1H), 4.90 (dt, J= 9.7, 5.4Hz, 1H), 4.61 – 4.53 (m, 1H), 3.75 (s, 3H), 3.46 (dd, J = 14.9, 5.4 Hz, 1H), 3.24 (dd, J = 15.0, 10.0 Hz, 1H), 2.33 (d, J = 2.1 Hz, 3H), 1.71 (dd, J = 36.6, 5.3Hz, 1H), 1.40 – 1.36 (m, 3H). 13 C NMR (150 MHz, CDCl3) δ 169.3, 159.2, 144.3,136.5, 135.3, 134.7, 129.4, 129.3, 128.6, 128.5, 128.4, 128.2, 127.8, 127.73,127.69, 121.9, 119.5, 118.7, 114.2, 109.9, 108.6, 76.0, 75.0, 67.3, 61.4,58.7, 55.4, 49.5, 26.4, 24.5, 21.8.

[0043]

[0044] (3) Substrate 5 (5.00 mmol, 1.0 equiv.) was dissolved in DCM (50 mL), and MnO2 (13.1 g, 182.76 mmol, 36.6 equiv.) was added. The reaction mixture was stirred at room temperature for 2 h, then filtered through a Buchner funnel (washed with DCM). The filtrate was concentrated to obtain the crude ketone product, which was then dissolved in anhydrous DCM (50 mL) and cooled to 0 °C. Camphor sulfonic acid (0.1 g, 0.50 mmol, 0.1 equiv.) was added to the reaction mixture, and the mixture was stirred at 0 °C for 30 min. TEA was then added to terminate the reaction. After removing the solvent under reduced pressure, the residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate, 10:1-3:1) to obtain product (-)-6 (3.0 g, 95%), which was a yellow solid.

[0045] The structural data of compound (-)-6 are as follows: = -116.2 (c = 0.5, CHCl3).

[0046] 1H NMR(600 MHz, CDCl3) δ 8.09 – 8.06 (m, 2H), 7.29 (d, J = 7.9 Hz, 2H),7.24 – 7.21 (m, 2H), 7.21 – 7.19 (m, 1H), 7.18 (d, J = 3.3 Hz, 2H), 7.08 – 7.06(m, 2H), 7.03 (td, J = 7.7, 1.3 Hz, 1H), 6.96 (dd, J = 7.5, 1.2 Hz, 1H), 6.82 –6.77 (m, 2H), 6.56 (td, J = 7.6, 1.0 Hz, 1H), 6.43 (d, J = 7.8 Hz, 1H), 5.79 (s,1H), 5.13 (d, J = 12.1 Hz, 1H), 5.08 (dd, J = 10.8, 1.2 Hz, 1H), 5.01 (d, J = 12.1Hz, 1H), 4.28 (d, J = 14.0 Hz, 1H), 3.73 (s, 4H), 3.58 (dd, J = 10.1, 5.8 Hz,1H), 2.55 – 2.50 (m, 1H), 2.43 (dd, J = 13.2, 10.8 Hz, 1H), 2.38 (s, 4H), 1.99– 1.89 (m, 1H)。 13 C NMR(150 MHz, CDCl3) δ 196.0, 169.9, 159.5, 159.2, 148.5,145.9, 134.9, 134.5, 131.0, 130.2, 129.4, 129.3, 129.0, 128.8, 128.7, 128.6,128.4, 124.2, 119.1, 114.3, 109.2, 106.6, 69.5, 67.8, 61.7, 55.4, 53.1, 48.7,39.7, 35.1, 21.9。

[0047]

[0048] (7) Ammonium formate (19.9 g, 315.35 mmol, 20.0 equiv.) was dissolved in MeOH (50 mL) and cooled to 0 °C in an ice bath. 10% Pd / C (3.0 g, 1.58 mmol, 0.1 equiv.) was added, and after stirring for 5 min, (-)-6 (10.0 g, 15.77 mmol, 1.0 equiv.) was added. The reaction solution was then gradually raised to room temperature. After 2 h, the reaction was terminated by filtration through a silica gel pad and washed with ethyl acetate. After removing the solvent under reduced pressure, the residue was purified by rapid silica gel column chromatography (DCM / MeOH, 50:1-20:1) to obtain product (-)-7 (8.1 g, 95%), which was a pale yellow oil.

[0049] The structural data of compound (-)-7 are as follows: = -47.9 (c = 0.5, CHCl3).

[0050] 1 H NMR (600 MHz, DMSO-) d 6) δ 13.37 (s, 1H), 8.12 (d, J = 8.1 Hz, 2H), 7.50(d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 7.13 – 7.09 (m, 1H), 7.06 – 7.00(m, 1H), 6.92 (d, J = 8.3 Hz, 2H), 6.60 (t, J = 7.5 Hz, 1H), 6.46 (d, J = 7.9 Hz,1H), 5.51 (s, 1H), 5.30 (d, J = 10.8 Hz, 1H), 4.24 (d, J = 14.6 Hz, 1H), 3.99 (d, J = 14.6 Hz, 1H), 3.83 (dd, J = 9.7, 6.1 Hz, 1H), 3.73 (s, 3H), 2.74 – 2.66 (m,1H), 2.40 (s, 4H), 2.33 (d, J = 12.9 Hz, 1H), 1.77 (dd, J = 17.1, 9.7 Hz, 1H).13 CNMR (150 MHz, DMSO- d 6) δ 194.6, 171.3, 159.8, 158.4, 148.4, 145.5, 134.4,131.6, 130.2, 129.6, 128.8, 128.8, 128.3, 123.5, 118.1, 114.0, 108.9, 104.7,69.2, 62.2, 55.0, 52.6, 48.0, 39.1, 34.9, 21.1.

[0051]

[0052] (8) Add (-)-7 (10.0 g, 18.38 mmol, 1.0 equiv.), 2,2 ' -dithiopyridine-1,1 ' -dioxide (5.6 g, 22.06 mmol, 1.2 equiv.) and n -Bu3P (4.5 g, 22.06 mmol, 1.2 equiv.) was dissolved in dichloromethane and stirred at room temperature in the dark. After TLC monitoring showed that (-)-7 had completely disappeared, [the solution was added] t -DodSH (37.13 g, 183.82 mmol, 10.0 equiv.) was reacted under red LED (30 W) irradiation for 2 h. The reaction was then terminated by adding ammonium chloride aqueous solution (50 mL), followed by extraction with dichloromethane (50 mL × 3). The organic layers were collected and combined, treated with water (50 mL) and saturated brine (50 mL), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure. The residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate, 20:1–3:1) to give product (-)-8 (6.4 g, 70%) as a yellow solid.

[0053] The structural data of compound (-)-8 are as follows: = 106.5 (c = 0.5, CHCl3).

[0054] 1 H NMR (400 MHz, DMSO-) d 6) δ 7.98 – 7.90 (m, 2H), 7.56 (d, J= 8.1 Hz,2H), 7.34 – 7.26 (m, 2H), 7.02 (td, J = 7.7, 1.2 Hz, 1H), 6.93 – 6.87 (m, 2H),6.47 (d, J = 7.9 Hz, 1H), 6.39 (td, J = 7.5, 1.0 Hz, 1H), 5.92 (s, 1H), 5.86 (dd, J = 7.4, 1.2 Hz, 1H), 4.29 (d, J = 14.8 Hz, 1H), 4.12 – 4.00 (m, 2H), 3.93 (dd, J = 9.8, 6.0 Hz, 1H), 3.73 (s, 4H), 2.46 (s, 4H), 2.13 (td, J = 11.7, 8.3 Hz,1H), 1.95 (dd, J = 12.0, 5.4 Hz, 1H), 1.87 (dd, J = 16.7, 9.9 Hz, 1H)。 13 C NMR(100MHz, DMSO- d 6) δ 195.1, 159.4, 158.4, 148.1, 145.7, 134.1, 130.8, 130.5,129.6, 128.8, 128.7, 127.1, 121.3, 118.0, 113.9, 109.0, 105.4, 66.8, 55.0,53.3, 48.8, 47.7, 35.4, 34.5, 21.1。

[0055]

[0056] (9) Preparation of sodium naphthalene tetrahydrofuran solution: Freshly cut sodium (1.1 g, 48.00 mmol, 4.8 equiv.) was added to an anhydrous THF (120 mL) solution of naphthalene (3.1 g, 24.00 mmol, 2.4 equiv.). The mixture was sonicated at room temperature for 30 min to obtain a dark green sodium naphthalene tetrahydrofuran solution (0.2 M). This freshly prepared sodium naphthalene tetrahydrofuran solution was added dropwise to an anhydrous THF (200 mL) solution of (-)-8 (5 g, 10.00 mmol, 1.0 equiv.), and the reaction temperature was controlled at -78 °C with continuous stirring until the dark green color remained unchanged (50 mL, 20.00 mmol, 2.0 equiv.). After the dark green color remained unchanged for 30 min and the reaction was confirmed by TLC, MeOH (20 mL) was added to terminate the reaction, and the temperature was raised to room temperature within 30 min. After removing the solvent under reduced pressure, the residue was purified by silica gel rapid column chromatography (DCM / MeOH, 50:1-20:1) to obtain product (-)-9 (2.7 g, 81%), which was a yellow solid.

[0057] The structural data of compound (-)-9 are as follows: =-49.8 (c = 0.5, CHCl3).

[0058] 1 H NMR (400 MHz, DMSO-) d 6) δ 8.24 (s, 1H), 7.39 – 7.31 (m, 2H), 7.07(td, J = 7.7, 1.3 Hz, 1H), 6.97 – 6.89 (m, 3H), 6.67 (td, J = 7.4, 0.9 Hz, 1H), 6.51 (d, J = 7.8 Hz, 1H), 4.97 (s, 1H), 4.33 (d, J = 14.6 Hz, 1H), 4.06 (d, J =14.6 Hz, 1H), 3.81 (dd, J = 9.4, 6.3 Hz, 1H), 3.74 (s, 3H), 3.51 – 3.41 (m,2H), 2.29 (dd, J = 16.5, 6.3 Hz, 1H), 2.08 (dt, J= 11.5, 9.6 Hz, 1H), 1.95 –1.79 (m, 2H). 13 CNMR (100 MHz, DMSO- d 6) δ 191.6, 169.4, 158.4, 148.4, 131.9,129.9, 128.8, 128.5, 121.6, 118.1, 113.9, 108.7, 93.0, 66.9, 55.0, 52.8,47.9, 43.4, 37.4, 35.7.

[0059]

[0060] (10) Compound (-)-9 (10.0 g, 28.86 mmol, 1.0 equiv.) was dissolved in 1,4-dioxane (200 mL). Under N2 protection, Pd(PPh3)4 (1.7 g, 1.44 mmol, 0.05 equiv.) and allyl methyl carbonate (3.7 g, 31.77 mmol, 1.1 equiv.) were added. The reaction mixture was then heated to 40 °C. After 3 h, the reaction was terminated by adding an aqueous solution of ammonium chloride (200 mL) and extracted with ethyl acetate (200 mL × 3). The organic layers were collected and combined, treated with water (50 mL) and saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate, 2:1-1:1) to give product (-)-10 (7.8 g, 70%), which was a yellow solid.

[0061] The structural data of compound (-)-10 are as follows: =-86.4 (c = 0.5, CHCl3).

[0062] 1 H NMR (600 MHz, CDCl3) δ 7.34 (d, J = 8.2 Hz, 2H), 7.18 – 7.12 (m, 1H), 7.00 (d, J = 7.2 Hz, 1H), 6.91 – 6.87 (m, 2H), 6.74 (t, J = 7.4 Hz, 1H), 6.55 (d, J = 7.8 Hz, 1H), 5.75 (dt, J= 16.4, 11.7, 6.0 Hz, 1H), 5.52 (s, 1H), 5.07 –4.96 (m, 2H), 4.41 (d, J = 14.1 Hz, 1H), 3.96 (d, J = 14.1 Hz, 1H), 3.81 (s, 3H), 3.76 (dd, J = 9.7, 6.2 Hz, 1H), 3.64 (td, J = 10.5, 5.5 Hz, 1H), 3.57 – 3.51 (m,1H), 3.23 (dd, J = 16.4, 5.5 Hz, 1H), 2.90 (dd, J = 16.3, 6.3 Hz, 1H), 2.54 (dd, J = 16.5, 6.2 Hz, 1H), 2.24 (dd, J = 16.5, 9.8 Hz, 1H), 2.19 – 2.07 (m, 2H). 13 CNMR(150 MHz, CDCl3) δ 192.3, 165.7, 159.1, 148.7, 136.7, 131.9, 129.5, 129.2,129.0, 122.1, 118.8, 114.5, 114.1, 109.1, 103.3, 67.0, 55.4, 53.1, 48.6,44.3, 38.1, 35.8, 27.6. 3446, 3138, 1699, 1483, 1058, 953, 862, 544.

[0063]

[0064] (11) Dissolve compound (-)-10 (5.0 g, 12.94 mmol, 1.0 equiv.) in... tIn a 2:2:1 mixture of BuOH / THF / water (50 mL), K₂O₄·2H₂O (0.9 g, 2.59 mmol, 0.2 equiv.), DABCO (1.5 g, 12.94 mmol, 1.0 equiv.), and K₃Fe(CN)₆ (12.8 g, 38.84 mmol, 3 equiv.) were added, followed by NaIO₄ (4.2 g, 19.42 mmol, 1.5 equiv.). The mixture was stirred at room temperature for 8 h. After the starting material was completely eliminated by TLC, the reaction mixture was cooled to -20 °C, and NaBH₄ (1.5 g, 38.84 mmol, 3 equiv.) was added, followed by stirring for 10 min. The reaction was then terminated by adding an aqueous solution of sodium thiosulfate (50 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). After collecting and combining the organic layers, the mixture was treated with water (50 mL) and saturated brine (50 mL). The organic phase was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The resulting crude residue (-)-11 was used directly in subsequent reactions without further separation or purification.

[0065] The crude residue (-)-11 was dissolved in DCM (50 mL) and cooled to 0 °C in an ice bath. TBSCl (3.9 g, 25.89 mmol, 2 equiv.) and imidazole (1.3 g, 19.42 mmol, 1.5 equiv.) were added. After 30 min, the reaction was terminated by adding ammonium chloride aqueous solution (50 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). The organic layers were collected and combined, treated with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate, 5:1) to give product (-)-12 (4.4 g, 68%), a pale yellow oil.

[0066] The structural data of compound (-)-12 are as follows: =-152.5 (c = 0.5, CHCl3).

[0067] 1 H NMR (600 MHz, CDCl3) δ 7.33 (d, J = 8.3 Hz, 2H), 7.14 (td, J = 7.7, 1.3Hz, 1H), 7.03 – 6.98 (m, 1H), 6.92 – 6.86 (m, 2H), 6.73 (t, J= 7.4 Hz, 1H),6.68 (s, 1H), 6.54 (d, J = 7.8 Hz, 1H), 4.40 (d, J = 14.1 Hz, 1H), 3.97 (d, J =14.1 Hz, 1H), 3.81 (s, 3H), 3.77 – 3.68 (m, 2H), 3.68 – 3.58 (m, 2H), 3.49(dd, J = 10.6, 7.9, 2.7 Hz, 1H), 2.67 – 2.60 (m, 1H), 2.52 (dd, J = 16.4, 6.2 Hz,1H), 2.45 – 2.38 (m, 1H), 2.22 (dd, J = 16.4, 9.7 Hz, 1H), 2.19 – 2.08 (m, 2H),0.92 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H)。 13 C NMR(150 MHz, CDCl3) δ 192.5,167.4, 159.1, 148.8, 132.1, 129.6, 129.2, 128.9, 122.3, 118.7, 114.1, 109.0,105.1, 67.1, 65.1, 55.4, 53.1, 48.6, 43.9, 38.5, 35.9, 27.1, 26.1, 18.4, -5.2, -5.3。

[0068]

[0069] (12) The substrate (-)-12 (5 g, 9.92 mmol, 1.0 equiv.) was dissolved in THF (50 mL) and cooled to 0 °C in an ice bath. Sodium hydride (0.5 g, 60% dispersion in mineral oil, 11.90 mmol, 1.2 equiv.) and propargyl bromide (1.3 g, 10.91 mmol, 1.1 equiv.) were then added to the reaction solution. The reaction solution was then heated to 40 °C in an oil bath. After 6 h, the reaction was terminated by adding an aqueous solution of ammonium chloride (50 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). The organic layers were collected and combined, treated with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate, 10:1-5:1) to give product (-)-13 (4.7 g, 85%), which is a pale yellow oil.

[0070] The structural data of compound (-)-13 are as follows: = 75.4 (c = 0.5, CHCl3).

[0071] 1 H NMR (600 MHz, CDCl3) δ 7.32 (d, J = 8.2 Hz, 2H), 7.12 (t, J = 7.7 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H), 6.91 – 6.85 (m, 2H), 6.71 (t, J = 7.4 Hz, 1H), 6.51 (d, J = 7.8 Hz, 1H), 4.90 (dd, J = 18.1, 2.5 Hz, 1H), 4.57 (dd, J = 18.2, 2.4Hz, 1H), 4.38 (d, J = 14.2 Hz, 1H), 3.96 (d, J = 14.2 Hz, 1H), 3.83 – 3.80 (m,3H), 3.77 – 3.61 (m, 4H), 3.49 (t, J = 9.1 Hz, 1H), 2.78 – 2.68 (m, 2H), 2.52(dd, J= 16.1, 5.9 Hz, 1H), 2.41 (td, J = 2.5, 1.0 Hz, 1H), 2.19 (dd, J = 16.2,10.2 Hz, 1H), 2.12 (dd, J = 11.6, 5.5 Hz, 1H), 2.04 (td, J = 11.2, 8.2 Hz, 1H), 0.85 (d, J = 1.0 Hz, 9H), -0.04 – -0.07 (m, 6H). 13 C NMR (150 MHz, CDCl3) δ 162.1,159.0, 148.5, 132.1, 129.7, 129.1, 128.8, 122.5, 118.6, 114.1, 109.0, 105.9,79.4, 73.3, 66.6, 62.8, 55.4, 55.3, 50.8, 48.7, 39.1, 35.8, 35.8, 26.7, 26.2,18.5, -5.2, -5.5.

[0072]

[0073] (13) Compound (-)-13 (4.0 g, 7.38 mmol, 1 equiv.) was dissolved in DCE (80 mL), and AgNTf2 (5.7 g, 14.76 mmol, 2 equiv.) was added at room temperature, and the reaction was carried out for 5 h. After the starting material was completely eliminated by TLC monitoring, the reaction solution was cooled to -20 °C, and then NaBH3CN (0.8 g, 12.55 mmol, 1.7 equiv.) in ethanol (10 mL) solution was added and stirred for 1 h. Subsequently, the mixture was filtered through an alkaline alumina pad, and the filter cake was thoroughly washed with ethyl acetate. After removing the solvent under reduced pressure, the residue was purified by alkaline alumina rapid column chromatography (petroleum ether / ethyl acetate, 10:1) to obtain product (+)-14 (2.6 g, 65%), which was a pale yellow oil.

[0074] The structural data of compound (+)-14 are as follows: = +229.2 (c = 0.5, CHCl3).

[0075] 1 H NMR (600 MHz, CDCl3) δ 7.17 (d, J= 8.3 Hz, 2H), 7.14 – 7.10 (m, 1H),7.04 (td, J = 7.7, 1.2 Hz, 1H), 6.86 – 6.80 (m, 2H), 6.74 (t, J = 7.4 Hz, 1H),6.47 (d, J = 7.9 Hz, 1H), 5.79 – 5.75 (m, 1H), 5.70 – 5.66 (m, 1H), 4.42 (d, J =15.5 Hz, 1H), 4.02 (d, J = 15.6 Hz, 1H), 3.78 (s, 3H), 3.60 (t, J = 3.5 Hz, 1H),3.46 – 3.39 (m, 2H), 3.30 (td, J = 9.7, 4.5 Hz, 2H), 3.19 (dd, J = 10.0, 8.6, 6.7Hz, 1H), 2.71 – 2.60 (m, 2H), 2.44 – 2.36 (m, 3H), 2.01 – 1.91 (m, 1H), 1.78– 1.65 (m, 2H), 0.76 (s, 9H), -0.18 (d, J = 13.5 Hz, 6H)。 13 C NMR(150 MHz, CDCl3)δ 212.1, 158.9, 151.7, 136.0, 130.2, 129.7, 129.2, 128.6, 125.5, 123.2,119.2, 114.1, 108.5, 72.8, 71.6, 59.3, 55.4, 54.2, 52.3, 51.6, 50.9, 50.0,41.2, 37.6, 37.0, 26.0, 18.2, -5.2, -5.3。

[0076]

[0077] (14) The substrate (+)-14 (2.0 g, 3.68 mmol, 1 equiv.) was dissolved in anhydrous THF (50 mL), and LiHMDS (1.0 M in THF, 7.4 mL, 7.35 mmol, 2 equiv.) was added at -78 °C. After stirring the mixture at -78 °C for 1 h, Comins reagent (2.6 g, 7.35 mmol, 2 equiv.) was added. The reaction mixture was then brought to room temperature. After 10 min, the reaction was terminated by adding an aqueous solution of ammonium chloride (50 mL), and the mixture was extracted with ethyl acetate (50 mL × 3). The organic layers were collected and combined, treated with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel rapid column chromatography (petroleum ether / ethyl acetate, 20:1) to give product (+)-15 (2.2 g, 90%), which is a pale yellow oil.

[0078] The structural data of compound (+)-15 are as follows: = +4.6 (c = 0.5, CHCl3).

[0079] 1 H NMR (600 MHz, CDCl3) δ 7.17 (d, J = 8.1 Hz, 2H), 7.15 – 7.10 (m, 1H), 7.04 (td, J = 7.7, 1.2 Hz, 1H), 6.85 – 6.80 (m, 2H), 6.74 (t, J = 7.4 Hz, 1H), 6.47 (d, J = 7.9 Hz, 1H), 5.77 (t, J = 9.8, 5.0, 1.6 Hz, 1H), 5.68 (dt, J = 9.9, 2.0 Hz, 1H), 4.42 (d, J = 15.5 Hz, 1H), 4.02 (d, J = 15.6 Hz, 1H), 3.78 (s, 3H), 3.60 (t, J = 3.5 Hz, 1H), 3.42 (dd, J = 17.3, 13.1, 4.1 Hz, 2H), 3.30 (td, J = 9.7, 4.5 Hz, 2H), 3.19 (dd,J = 10.0, 8.5, 6.7 Hz, 1H), 2.73 – 2.65 (m, 1H), 2.64(dd, J = 15.4, 3.7 Hz, 1H), 2.44 – 2.35 (m, 3H), 2.01 – 1.89 (m, 1H), 1.80 –1.65 (m, 2H), 0.76 (s, 9H), -0.18 (d, J = 13.5 Hz, 6H). 13 C NMR(150 MHz, CDCl3) δ212.1, 158.9, 151.7, 136.0, 130.2, 129.7, 129.2, 128.6, 125.5, 123.2, 119.2,114.1, 108.5, 72.8, 71.6, 59.3, 55.4, 54.2, 52.3, 51.6, 50.9, 50.0, 41.2,37.6, 37.0, 26.0, 18.2, -5.2, -5.3.

[0080]

[0081] (15) The substrate (+)-15 (200 mg, 0.30 mmol, 1 equiv.) was dissolved in ethyl acetate (5 mL), and 10% Pd / C (285 mg, 0.15 mmol, 0.5 equiv.) and Li2CO3 (44 mg, 0.60 mmol, 2 equiv.) were added. The mixture was stirred for 8 h under a hydrogen atmosphere (H2balloon). After the starting material was completely eliminated by TLC, 2 M HCl (2 mL) was added, and stirring was continued for 30 min under a hydrogen atmosphere. The reaction was then terminated by adding sodium bicarbonate aqueous solution (10 mL) at 0 °C, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic layers were collected and combined, treated with water (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel rapid column chromatography (DCM / MeOH, 50:1-20:1) to obtain (+)-limaspermidine (56 mg, 63%) as a white solid.

[0082] The structural data of compound (+)-limaspermidine were identified as follows: = +9.2 (c = 0.5, CHCl3).

[0083] 1 H NMR (600 MHz, CDCl3) δ 7.08 (d, J = 7.3 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.73 (t, J = 7.4 Hz, 1H), 6.63 (d, J = 7.7 Hz, 1H), 3.62 (td, J = 10.1, 5.4 Hz, 1H), 3.57 – 3.48 (m, 2H), 3.15 – 3.09 (m, 1H), 3.04 (d, J = 11.0 Hz, 1H), 2.33 – 2.21 (m, 3H), 2.05 (td, J = 13.9, 3.5 Hz, 1H), 2.01 – 1.95 (m, 1H), 1.84 – 1.58 (m, 4H), 1.49 (td, J = 14.6, 6.8 Hz, 3H), 1.30 – 1.22 (m, 1H), 1.18 (ddd, J = 14.3, 9.3, 5.4 Hz, 1H), 1.03 (d, J = 13.7 Hz, 1H). 13 C NMR (150 MHz, CDCl3) δ 149.6, 135.4, 127.5, 122.9, 119.3, 110.6, 70.8, 65.5, 58.7, 53.9, 53.6, 53.0, 40.6, 38.7, 35.6, 35.5, 28.4, 24.4, 21.8.

[0084] Preparation of (±)-Limaspermidine:

[0085] (1) Tryptamine (20.0 g, 0.125 mol, 1.0 equiv.) was dissolved in N,N-dimethylformamide (100 mL) and cooled to 0 °C under ice bath conditions. Sodium hydride (6.0 g, 60% dispersion in mineral oil, 0.150 mol, 1.2 equiv.) was added dropwise to the reaction solution, and stirring was continued for 30 min. Then PMBBr (27.6 g, 0.137 mol, 1.1 equiv.) was added dropwise to the reaction solution. The ice bath was removed and the temperature of the reaction solution was allowed to gradually rise to room temperature. Stirring was continued for 2 h. The reaction was quenched by adding saturated ammonium chloride aqueous solution (500 mL), extracted with ethyl acetate (200 mL x 3), the organic layers were combined, washed once with saturated sodium chloride aqueous solution (200 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and directly proceeded to the next reaction without purification.

[0086] The crude product 17 obtained from the previous reaction was dissolved in dichloromethane (200 mL), and triethylamine (26.0 mL, 0.188 mol, 1.5 equiv.) was added under ice bath conditions, followed by a single addition of TsCl (26.2 g, 0.137 mol, 1.1 equiv.). The ice bath was removed, and the reaction mixture was allowed to reach room temperature. Stirring was continued for 2 h, and the reaction was quenched by adding saturated ammonium chloride aqueous solution (200 mL). The mixture was extracted with dichloromethane (200 mL x 3), and the organic layers were combined. The mixture was washed once with saturated sodium chloride aqueous solution (200 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by rapid silica gel column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain a pale yellow oily compound 18 (45.0 g, 83%).

[0087] The structural data of compound 18 are as follows: 1 H NMR (600 MHz, CDCl3) δ 7.67 – 7.60 (m, 2H), 7.42 (dt, J = 7.9, 1.0 Hz, 1H), 7.28 (dt, J = 8.3, 0.9 Hz, 1H), 7.23 – 7.15 (m, 3H), 7.10 – 7.04 (m, 3H), 6.89 – 6.82 (m, 3H), 5.17 (s, 2H), 4.54 (t, J = 6.1 Hz, 1H), 3.78 (s, 3H), 3.27(q, J= 6.6 Hz, 2H), 2.94 – 2.88 (m, 2H), 2.40 (s, 3H); 13 C NMR(150 MHz, CDCl3) δ159.2, 143.4, 137.0, 136.8, 129.7, 129.4, 128.4, 127.7, 127.1, 126.5, 122.1,119.3, 118.8, 114.3, 110.7, 110.0, 55.4, 49.5, 43.3, 25.6, 21.6.

[0088]

[0089] (2) Tryptophan derivative 18 (45.0 g, 0.104 mol, 1.0 equiv.) and bromoalkyne 19 (60.4 g, 0.156 mol, 1.5 equiv.) were dissolved in toluene (300 mL). 1,10-phenanthroline (7.5 g, 0.042 mol, 0.4 equiv.), potassium phosphate (44.1 g, 0.208 mol, 2.0 equiv.), and copper sulfate pentahydrate (5.2 g, 0.021 mol, 0.2 equiv.) were added to the reaction solution in sequence. The mixture was wrapped in aluminum foil to protect it from light and heated and stirred at 90 °C for 48 h. The reaction solution was filtered through silica gel (100~200 mesh), washed with ethyl acetate, and the filtrate was concentrated. The crude product was directly used for the next reaction without purification.

[0090] The crude product 20 obtained from the previous reaction was dissolved in tetrahydrofuran (300 mL). Under ice bath conditions, TBAF•3H2O (49.3 g, 0.156 mol, 1.5 equiv.) was added in portions to the reaction solution. The ice bath was removed, and the reaction solution was allowed to reach room temperature. Stirring was continued for 2 h. TLC monitoring showed that no raw material remained. The reaction was quenched by adding saturated ammonium chloride aqueous solution (100 mL). The mixture was extracted with ethyl acetate (300 mL x 3), and the organic layers were combined. The mixture was washed once with saturated sodium chloride aqueous solution (200 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by rapid silica gel column chromatography (petroleum ether / ethyl acetate = 2:1) to obtain a white solid compound 21 (37.5 g, 72%).

[0091] The structural data of compound 21 are as follows: 1 H NMR (600 MHz, DMSO) -d 6) δ 7.70 (d, J= 8.2 Hz, 2H), 7.46 (d, J = 7.9 Hz,1H), 7.42 (s, 1H), 7.40 (d, J = 8.2 Hz, 2H), 7.27 (s, 1H), 7.15 (d, J = 8.7 Hz, 2H), 7.09 (t, J = 7.4 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 5.31 (d, J = 5.3 Hz, 1H), 5.23 (s, 2H), 4.52 – 4.45 (m, 1H), 3.69 (s, 3H), 3.57– 3.49 (m, 2H), 2.96 (t, J = 7.5 Hz, 2H), 2.40 (s, 3H), 1.29 (d, J = 6.6 Hz, 3H); 13 C NMR (150 MHz, DMSO) -d 6) δ 158.6, 144.7, 135.9, 133.8, 130.1, 130.0, 128.6,127.5, 127.3, 127.1, 121.3, 118.8, 118.3, 113.9, 110.2, 109.5, 75.5, 74.3,56.4, 55.1, 51.4, 48.5, 24.8, 23.3, 21.1.

[0092]

[0093] (3) Add to a solution of substrate 21 (37.5 g, 0.075 mol, 1.0 equiv.) in dichloromethane (300 mL) N -methyl- N -Oxymorpholine (13.1 g, 0.112 mol, 1.5 equiv.) was added, and the reaction mixture was stirred in an ice bath for 20 min. TPAP (5.3 g, 0.015 mol, 0.2 equiv.) was then added. The reaction mixture was stirred at 0 °C for 2 h. The reaction was monitored by TLC until complete. The mixture was filtered through silica gel (100–200 mesh) (washed with ethyl acetate), and the filtrate was concentrated to obtain the crude acetylene compound 22.

[0094] The crude product 22 obtained from the previous step was dissolved in anhydrous dichloromethane (250 mL), and camphor sulfonic acid (1.9 g, 0.008 mol, 0.1 equiv.) was added under ice bath conditions. The reaction solution was stirred for 30 min under ice bath conditions. The reaction was monitored by TLC until it was complete. The reaction was quenched by adding triethylamine, concentrated, and purified by recrystallization (petroleum ether / dichloromethane = 10:1) to obtain a white solid tetracyclic indole skeleton (±)-8 (35.7 g, 95%).

[0095] The structural data of compound (±)-8 are as follows: 1 H NMR (600 MHz, DMSO -d 6) δ 7.95 (d, J = 8.1 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.03 (t, J = 7.7 Hz, 1H), 6.92 (d, J = 8.4 Hz, 2H), 6.49 (d, J = 7.7 Hz, 1H), 6.40 (t, J = 7.3 Hz, 1H), 5.94 (s, 1H), 5.86 (d, J = 7.3Hz, 1H), 4.30 (d, J = 14.8 Hz, 1H), 4.08–4.04 (m, 2H), 3.94 (dd, J = 9.9, 6.0 Hz,1H), 3.74 (s, 4H), 2.49–2.43 (m, 4H), 2.17–2.12 (m, 1H), 1.96 (dd, J = 11.9, 5.3 Hz, 1H), 1.88(dd, J = 16.7, 9.9 Hz, 1H); 13 C NMR (150 MHz, DMSO -d6) δ 195.1,159.5, 158.4, 148.2, 145.7, 134.1, 130.8, 130.6, 129.6, 128.8, 128.7, 127.2,121.3, 118.0, 113.9, 109. 0, 105.4, 66.8, 55.0, 53.4, 48.8, 47.7, 35.4, 34.5,21.1.

[0096] The subsequent preparation process followed steps (9) to (15) of (+)-Limaspermidine to obtain compound (±)-Limaspermidine.

[0097]

[0098] Example 2 Test of antidepressant activity of indole derivatives obtained in Example 1 1. Experimental Methods 1.1. Laboratory animals This animal experiment was approved by the Laboratory Animal Ethics Committee of Shenyang Pharmaceutical University and complied with the internationally recognized ethical standards of the "Guidelines for the Care and Use of Laboratory Animals". The adaptation, reproduction, husbandry, and subsequent experimental procedures of the laboratory animals were all conducted in an SPF-grade animal laboratory. The SPF-grade C57BL / 6J mice used in the experiment were purchased from Liaoning Changsheng Biotechnology Co., Ltd. The SPF-grade animal room temperature was maintained at (22 ± 2℃), and the animals were kept in alternating light and dark environments (light: 8:00 ~ 20:00, dark: 20:00 ~ 8:00). Except for periods requiring specific behavioral experiments, the subjects had free access to food and water. All experiments in this study were approved by the Laboratory Animal Ethics Committee of Shenyang Pharmaceutical University.

[0099] 1.2 Culture of Neural Stem Cells (1) Extraction and culture of neural stem cells 1) After euthanizing 4-week-old C57BL / 6J mice by cervical dislocation, the brains were immediately removed and placed in a culture dish containing pre-cooled dissection solution.

[0100] 2) Under a dissecting microscope, the SVZ brain region tissue of the mouse was dissected, and the tissue block was cut into 1 mm pieces using microforceps. 3 Transfer the small pieces to a pre-cooled 15 mL EP tube containing dissection fluid.

[0101] 3) Aspirate the supernatant, add 200 μL of Accutase, and digest at 37 °C for 7 min.

[0102] 4) Carefully aspirate the Accutase, add 200 μL of preheated Sorting buffer, and pipette until the tissue block is completely dispersed. Then add 800 μL of preheated SFM medium and pipette until well mixed.

[0103] 5) Filter using a 5 mL polystyrene round-bottom tube (Falcon, 352235) with a 70 μm cell filter and transfer to a 15 mL EP tube.

[0104] 6) Centrifuge at 900 rpm for 6 minutes. 7) Aspirate the supernatant, add 1 mL of SFM medium and mix thoroughly. Seed the cell suspension into a 6-well plate containing 2 mL of SFM medium. Shake in a cross shape and observe the cells under a microscope.

[0105] 8) Culture in a 37 ℃, 5% CO2 constant temperature cell culture incubator for 7 days.

[0106] (2) Subculture of neurospheres 1) Transfer the culture medium containing neurospheres to a 15 mL EP tube and flush the bottom of the well plate with SFM.

[0107] 2) Centrifuge at 900 rpm for 6 min.

[0108] 3) Remove the supernatant, add 200 μL of Accutase, digest at 37 ℃ for 7 min, then add 800 μL of SFM medium and mix well.

[0109] 4) Centrifuge at 900 rpm for 6 min.

[0110] 5) Remove the supernatant, add SFM medium and mix thoroughly. Pass the cells at a ratio of 1:2 to 1:3 according to the cell density, and seed them into 6-well plates. 6) After culturing at 37 ℃ and 5% CO2 for 3 days, new neurospheres are formed and can be passaged again.

[0111] (3) Cryopreservation and thawing of neural stem cells Cryopreservation: Prepare cryopreservation buffer by mixing SFM (excluding EGF and FGF) and DMSO at a ratio of 9:1. Collect cells in 15 mL centrifuge tubes. Centrifuge at 900 rpm for 6 min. Discard the supernatant, add the cryopreservation buffer, and gently pipette to resuspend the cells. Transfer to 1.5 mL cryovials and incubate overnight at -80 °C. For long-term storage, store in liquid nitrogen.

[0112] Thawing: Thaw cells in a 37 °C water bath, transfer to 15 mL EP tubes, and rinse the cryovials with SFM (without EGF, FGF) medium. Centrifuge at 900 rpm for 6 min. Discard the supernatant, add SFM complete medium and gently pipette to resuspend the cells, seed in 6-well plates, and incubate at 37 °C with 5% CO2. Change the medium on the second day, and passage after 3 days.

[0113] (4) Neural stem cell proliferation experiment In the in vitro experiment of neurosphere proliferation, cells were seeded at a density of 1000 cells / well in 96-well plates, and the drug was added 6 hours later. Neurospheres were counted after 3 days of culture.

[0114] 1.3 Behavioral Experiments (1) Open field test (OFT) OFT used a 50 cm × 50 cm × 50 cm open white open-field box for behavioral testing. Experimental animals were gently placed in the open-field box facing the box wall from the same position and allowed to move freely for 1 hour with dim lighting. Data was collected using Bandicam software. The total distance the mice moved in the open-field box within 5 minutes and the percentage of time spent in the central area (25 cm × 25 cm × 25 cm) were recorded to evaluate the mice's basic spontaneous activity ability and anxiety level.

[0115] (2) Elevated plus maze (EPM) The EPM (Extended Physical Model) used a 50 cm × 10 cm × 40 cm elevated maze for behavioral testing. It consisted of two open arms (unobstructed) and two closed arms (obstructed). During the experiment, mice were gently placed in the central area with their heads facing the closed arms, under dim lighting. Data was collected using Bandicam software, and a total of 5 minutes of video was recorded. After each test, feces and urine were removed by spraying with 75% ethanol solution. The experiment recorded the number of times mice entered the open and closed arms and the time spent in each arm during the recording period. By combining the mice's exploratory nature in new environments with their fear of the suspended open arms, a contrasting conflict was created to detect anxiety and depression-like behavioral indicators in the experimental animals.

[0116] (3) Tail suspension test (TST) The TST test method is as follows: The mouse's tail is fixed with tape 1 cm from the tip, and it is suspended upside down on a support 35 cm above the table. The mouse's behavior is recorded for 5 minutes, and the immobility time and percentage of immobility in the last 4 minutes are calculated to assess the mouse's anxiety and depression level.

[0117] (4) Forced swimming test (FST) FST used a cylindrical transparent acrylic water tank, 25 cm high and 12 cm in diameter, for behavioral testing. Water was added to the tank, with the water depth for mice at 10 cm and the water temperature controlled at (25±1) ℃. Each mouse entered the tank independently, and swimming behavior was recorded for 6 minutes. The immobility time within the last 4 minutes was calculated: animals floating on the surface, maintaining their heads above water only through minor limb movements, without active swimming, were considered immobile; active paddling, forelimb pawing, and diving were not included in the immobility time. This was used to assess the mice's level of depression.

[0118] 1.4 Brain tissue sampling (1) Irrigation After anesthetizing mice with isoflurane (4% for induction, 2% for maintenance), the skin was cut along both sides of the ribs from the lower xiphoid process of the sternum, and the muscles and fascia were separated to fully expose the diaphragm in the lower thoracic cavity. The right atrial appendage was cut to allow sufficient blood to flow out, and the needle was inserted into the left ventricle to the 1 / 3 of the heart and fixed. Physiological saline was injected until the liver turned grayish-white, and then 4% PFA solution was injected until the mouse tail rose up, ending the perfusion.

[0119] (2) Collection of materials The surgical scissors cut open the scalp along the midline of the skull, cut the skull along the sagittal suture, and cut the brow bone between the eyes. Excess meninges are removed, and the entire brain tissue is completely removed using a brain scoop. The brain tissue is placed in a centrifuge tube, fixed with 4% PFA solution for 24 hours, and then the solution is changed to 0.4% PFA solution for long-term preservation.

[0120] (3) Brain tissue slices 1) Remove the whole brain tissue, cut off the cerebellum and olfactory bulb tissue of the mouse in the vertical direction on the paper, apply strong glue evenly on the stage, and fix the brain tissue horizontally at the glue.

[0121] 2) Adjust the parameters of the oscillating microtome: amplitude 0.9 - 1.1, thickness 40 μm, continuous slicing; collect brain slices into 48-well plates, 3 brain slices per well, add 0.4% PFA solution to the well plate, and store in a refrigerator at 4℃ in the dark for a long time.

[0122] 1.5 Immunofluorescence staining (1) Pre-processing 1) Remove the coronal facial brain slices of mice (3 slices per mouse) from the 0.4% PFA preservation solution and rinse them twice with 0.01 M PBS for 10 min each time.

[0123] 2) Place the brain slices in an EP tube containing citrate buffer (0.1 M, pH = 6.80) and treat in a metal bath at 80 °C for 30 min. Alternatively, place the brain slices in a 2 M hydrochloric acid solution and acidify at 37 °C for 20 min, then rinse immediately after treatment.

[0124] 3) After the brain slices have cooled naturally at room temperature for 30 min, rinse them twice with 0.01 M PBST for 10 min each time.

[0125] 4) Block the brain slices with 5% serum prepared from 0.01 M PBST for 30 min.

[0126] 5) Add the primary antibody diluted with 5% serum to a certain ratio, incubate the brain slices at room temperature for 30 min, and then incubate at 4°C overnight.

[0127] 6) The next day, after the brain slices have returned to room temperature, continue incubation for 30 min, and rinse twice with 0.01 M PBST for 10 min each time.

[0128] 7) Add fluorescent secondary antibody diluted with 5% serum to a certain ratio and incubate the brain slices at room temperature for 60 min.

[0129] 8) Rinse twice with 0.01 M PBST for 10 min each time, and then rinse with 0.01 M PBS in the dark for 10 min.

[0130] 9) Attach the brain slices to a glass slide, add anti-fluorescence quenching solution, seal the slide, and store it in a dark box away from light.

[0131] (2) Laser confocal microscope imaging Select the target area and uniformly set the laser power and signal-to-noise ratio for each fluorescence channel. hv The image includes the background (offset). Rotate the fine focus knob to position the z-axis, and scan 10 layers continuously at a slice thickness of 1 μm (the diameter of a neuron is approximately 5-10 μm). For quantitative fluorescence experiments, all tissue sections must be stained in the same batch and imaged under fixed imaging parameters.

[0132] 1.6 Statistical Methods Experimental results are expressed as mean ± standard error (Mean ± SEM). Laser confocal microscopy images were processed and analyzed using ImageJ software, and sample experimental data were processed and analyzed using GraphPad 8.3.0. Graphs were created using Adobe Illustrator 2024. Results were compared using one-way ANOVA or two-way ANOVA combined with the LSD test. PA value <0.05 is considered statistically significant.

[0133] 2. Experimental Results 2.1 Study on the ability of indole derivatives obtained in Example 1 to promote the self-renewal of primary neural stem cells This study first extracted primary neural stem cells from the subventricular zone (SVZ) of 14-day-old C57BL / 6J mice, and then evaluated the effects of different compounds on the self-renewal and proliferation capacity of neural stem cells using a neural stem cell spheroid formation assay. Primary neural stem cells were seeded at a density of 1000 cells / well in 96-well plates. After 6 hours of seeding, different concentrations of candidate compounds were added, and the cells were cultured for another 3 days. The formation of neural stem cell spheroids was observed, and the number and diameter of spheroids were recorded. The results are shown in [Table data would be inserted here]. Figure 1 .

[0134] Figure 1 (A) Representation of primary neural stem cell spheres after treatment with (+)-Limaspermidine ((+)-lim) and (±)-Limaspermidine ((±)-lim) at 0.1, 1, and 10 μM, respectively. Primary neural stem cells were extracted from the subventricular zone (SVZ) of 14-day-old C57BL / 6J mice and seeded at 1000 cells / well in 96-well plates. The corresponding compounds were added 6 h after seeding, and observation and statistics were performed after 3 days of culture. Scale bar = 100 μm. Figure 1 (B) shows the statistical results of the number of neural stem cell spheres after treatment with (+)-lim and (±)-lim. Figure 1 (C) shows the statistical results of neural stem cell sphere diameters after (+)-lim and (±)-lim treatments. Data are expressed as mean ± SEM (n = 10, P <0.05, P <0.01, P <0.001 vs. Veh group; one-way ANOVA with LSD test).

[0135] In this study, each compound was prepared at three concentrations: 0.1 μM, 1 μM, and 10 μM. The results showed that, compared with the Veh group, the different compounds had significantly different effects on the ability of primary neural stem cells to form spheres. Among them, (+)-lim significantly increased the number of neural stem cell spheres at a concentration of 0.1 μM, and its promoting effect was the most significant, suggesting that (+)-lim has a strong ability to promote the self-renewal and proliferation of primary neural stem cells. In contrast, (±)-lim had a weaker or less significant promoting effect on the number of spheres, and some compounds even showed a trend of decreasing the number of spheres at specific concentrations.

[0136] Further statistical analysis of the diameter of neural stem cell spheres revealed that different compounds also had a certain influence on the growth and size of neurospheres, but the changes in neurosphere diameter were not entirely consistent with the changes in the number of neurospheres. Since the number of neurospheres better reflects the ability of primary neural stem cells to form clones and self-renew, this study mainly used the number of neurospheres as the core in vitro indicator, and then conducted subsequent in vivo pharmacodynamic evaluations of (+)-lim and its corresponding racemic mixture (±)-lim.

[0137] 2.2 (+)-lim significantly improved anxiety-like behavior in CRS mice This study further established a mouse model of depression using chronic restraint stress (CRS) to evaluate whether (+)-lim and (±)-lim had any effect on improving stress-related emotional and behavioral abnormalities in vivo. Male C57BL / 6J mice aged 9-10 weeks were treated with CRS for 2 h daily for 21 consecutive days; the drugs were administered concurrently from day 1 of model establishment for 4 consecutive weeks. CRS mice were given 1, 3, and 9 mg / kg of (+)-lim or (±)-lim, respectively. A high-dose control group of normal mice was also included to observe the effects of the drugs on baseline behavior. The results are shown below. Figure 2 .

[0138] Figure 2 (A) is a schematic diagram of the process of establishing a chronic restraint stress (CRS) model, administering medication, conducting behavioral tests, and collecting samples. Male C57BL / 6J mice aged 9-10 weeks were treated with CRS for 2 h daily for 21 consecutive days; (+)-lim, (±)-lim, or Veh were administered via intraperitoneal injection starting from day 1 of CRS and continued for 4 weeks. Figure 2 (B) shows the statistical results of the total movement distance of mice in each group during the open field experiment (OFT). Figure 2 (C) shows the statistical results of the average movement speed of mice in each group in OFT. Figure 2 (D) shows the statistical results of the time spent in the central area of ​​mice in each group during OFT. Figure 2 (E) is a heatmap of the movement trajectories of mice in each group in OFT. Figure 2 (F) shows the statistical results of the open-arm dwell time of mice in each group during the elevated cross maze test (EPM). Data are expressed as mean ± SEM (n = 8-10, ...). P <0.05, P <0.01, P <0.001 vs. Control + Veh group; # P <0.05, ## P <0.01 vs. CRS + Veh group; one-way ANOVA with LSD test).

[0139] First, the open field test was used to evaluate the mice's spontaneous movement ability and anxiety-like behavior. The results showed that, compared with the Control+Veh group, the CRS+Veh group mice did not show significant changes in total movement distance and average movement speed in the open field, indicating that CRS treatment did not significantly affect the mice's basic motor ability. Administration of (+)-lim or (±)-lim also did not significantly change the mice's total movement distance and average movement speed, suggesting that the drug itself did not cause significant motor dysfunction.

[0140] Further analysis of the time mice spent in the central region of the open field revealed that, compared to the Control+Veh group, the CRS+Veh group exhibited a significantly reduced time spent in the central region, suggesting that CRS induces significant anxiety-like behavior in the mice. Compared to the CRS+Veh group, treatment with (+)-lim at 3 mg / kg and 9 mg / kg significantly increased the time CRS mice spent in the central region, while the (±)-lim groups did not show the same degree of improvement. Movement trajectory heatmaps also showed that the CRS+Veh group mice concentrated their activity more in the periphery of the open field, while (+)-lim treatment increased activity in the central region.

[0141] The elevated cruciate maze test was then used to further evaluate anxiety-like behavior in mice. The results showed that, compared with the Control+Veh group, the open-arm dwell time of mice in the CRS+Veh group was significantly reduced, further demonstrating that CRS can induce anxiety-like behavior in mice. Compared with the CRS+Veh group, treatment with (+)-lim at 3 mg / kg and 9 mg / kg significantly increased the open-arm dwell time in CRS-treated mice, while the (±)-lim treatment group showed no significant improvement. These results indicate that (+)-lim can alleviate CRS-induced anxiety-like behavior without affecting the spontaneous activity ability of mice, and its effect is superior to that of the corresponding racemic mixture (±)-lim.

[0142] 2.3 (+)-lim significantly improved depressive-like behavior in CRS mice After confirming that (+)-lim can improve anxiety-like behavior in CRS mice, this study further evaluated the effect of (+)-lim on depressive-like behavior in CRS mice using the tail suspension test and forced swimming test. In the tail suspension test and forced swimming test, the immobility time of mice is often used to reflect a hopeless state; increased immobility time suggests enhanced depressive-like behavior in mice. The results are shown in […]. Figure 3 .

[0143] Figure 3 (A) shows the statistical results of the immobility time of mice in each group during the tail suspension test (TST). Figure 3 (B) Statistical results of immobility time in each group of mice during the forced swimming test (FST). CRS treatment increased immobility time in both TST and FST, while (+)-lim treatment decreased immobility time in CRS mice. Data are expressed as mean ± SEM (n = 9-10, ...). P <0.05, P <0.01, P <0.001 vs. Control+Veh group; # P <0.05, ## P <0.01 vs. CRS+Veh group; one-way ANOVA with LSD test).

[0144] Tail suspension test results showed that, compared with the Control+Veh group, the immobility time of mice in the CRS+Veh group was significantly increased, indicating that CRS treatment can induce significant depressive-like behavior in mice. Compared with the CRS+Veh group, treatment with (+)-lim 3 mg / kg and 9 mg / kg significantly reduced the immobility time in the tail suspension test of CRS mice, while the (±)-lim dose groups did not show a stable improvement. In normal mice, administration of high doses of (+)-lim or (±)-lim did not show significant abnormal changes in immobility time, suggesting that the drug had no significant effect on the baseline depressive-like behavior in normal mice.

[0145] The results of the forced swimming test were largely consistent with those of the tail suspension test. Compared with the Control+Veh group, the immobility time in the CRS+Veh group mice during the forced swimming test was significantly increased. Compared with the CRS+Veh group, treatment with (+)-lim at 3 mg / kg and 9 mg / kg significantly reduced the immobility time in CRS mice, while treatment with (±)-lim did not produce the same degree of improvement. These results indicate that (+)-lim can effectively alleviate CRS-induced depressive-like behavior in mice, and its efficacy is superior to that of the corresponding racemic mixture (±)-lim.

[0146] 2.4 (+)-lim regulates abnormal changes in GFAP / MCM2-related neural stem cells in the DG region of CRS mice To further investigate whether the improvement of anxiety-like and depression-like behaviors in CRS mice by (+)-lim is related to hippocampal neurogenesis, brain tissue was collected from mice after the behavioral experiments, and immunofluorescence staining was performed on the dentate gyrus (DG) region of the hippocampus. First, GFAP / MCM2 / DAPI co-staining was used to evaluate changes in GFAP-positive neural stem / progenitor-like cells, MCM2-positive proliferating cells, and GFAP / MCM2 double-positive cells in the DG region. GFAP can be used to label neural stem / progenitor cell populations with radial glial-like features, MCM2 can reflect cell proliferation status, and GFAP / MCM2 double-positive cells can reflect GFAP-positive neural stem / progenitor-like cells in a proliferating or activated state. Results are shown in [Figure number missing]. Figure 4 .

[0147] Figure 4 (A) Representative immunofluorescence images of GFAP / MCM2 / DAPI in the dentate gyrus (DG) region of the hippocampus of mice in each group. The magnified images show GFAP-positive cells, MCM2-positive cells, and GFAP / MCM2 double-positive cells. Scale bar = 20 μm. Figure 4(BD) shows the statistical results for GFAP-positive cells (B), MCM2-positive cells (C), and GFAP / MCM2 double-positive cells (D) in the DG region. GFAP was used to label neural stem / progenitor-like cells, and MCM2 was used to label proliferation-related cells. Data are expressed as mean ± SEM (n=3, P <0.05, P <0.01, P <0.001 vs. Control+Veh group; # P <0.05, ## P <0.01 vs. CRS+Veh group; one-way ANOVA with LSD test).

[0148] Immunofluorescence results showed that, compared with the Control+Veh group, the CRS+Veh group mice had significantly increased numbers of GFAP-positive cells, MCM2-positive cells, and GFAP / MCM2 double-positive cells in the DG region. This result suggests that, under the experimental conditions, CRS did not simply reduce all neurogenesis-related cell populations, but rather induced an abnormal increase in GFAP / MCM2-related neural stem / progenitor-like cells and proliferation-related cells in the DG region, potentially reflecting an abnormal activation state of early neural stem / progenitor cells or glial-related cells after chronic stress.

[0149] Compared with the CRS+Veh group, treatment with (+)-lim 9 mg / kg reduced the abnormally elevated number of GFAP-positive cells, MCM2-positive cells, and GFAP / MCM2 double-positive cells in the DG region of CRS mice, bringing them closer to normal levels. (±)-lim 9 mg / kg also showed some regulatory effects on certain indicators, but the overall effect was not as stable as that of (+)-lim. These results indicate that (+)-lim can regulate the abnormal changes in GFAP / MCM2-related neural stem / progenitor-like cells in the DG region induced by CRS, suggesting that it may improve behavior by restoring homeostasis in the early neurogenesis process of the DG region under stress.

[0150] 2.5 (+)-lim improves the reduction of DCX-positive neuroblasts / immature neurons in the DG region of CRS mice. To further evaluate the effects of CRS and (+)-lim on neuroblasts and immature neurons in the DG region, this study used DCX / MCM2 / DAPI co-staining to detect changes in DCX-positive cells, MCM2-positive cells, and DCX / MCM2 double-positive cells in the DG region. DCX is a commonly used marker for neuroblasts and immature neurons, and DCX / MCM2 double-positive cells can be used to reflect a proliferating population of neuroblast-like cells. Results are shown in […]. Figure 5 .

[0151] Figure 5 (A) Representative immunofluorescence images of DCX / MCM2 / DAPI in the dentate gyrus (DG) region of the hippocampus of mice in each group. The magnified images show DCX-positive cells, MCM2-positive cells, and DCX / MCM2 double-positive cells. Scale bar = 20 μm. Figure 5 (BD) shows the statistical results for DCX-positive cells (B), MCM2-positive cells (C), and DCX / MCM2 double-positive cells (D) in the DG region. DCX was used to label neuroblasts / immature neurons, and MCM2 was used to label proliferation-related cells. Data are expressed as mean ± SEM (n = 3, P <0.05, P <0.01, P <0.001 vs. Control+Veh group; # P <0.05, ## P <0.01 vs. CRS+Veh group; one-way ANOVA with LSD test).

[0152] Immunofluorescence results showed that, compared with the Control+Veh group, the CRS+Veh group had a significantly reduced number of DCX-positive cells in the DG region, suggesting that CRS treatment inhibits the generation or maintenance of neuroblasts / immature neurons in the DG region. In contrast, the CRS+Veh group showed an increased number of MCM2-positive cells and DCX / MCM2 double-positive cells, suggesting that CRS may cause a phased imbalance in the neurogenesis process in the DG region, i.e., an abnormal increase in some proliferation-related cell populations, but ultimately a decrease in the overall output of DCX-positive neuroblasts / immature neurons.

[0153] Compared with the CRS+Veh group, treatment with (+)-lim 9 mg / kg increased the number of DCX-positive cells in the DG region of CRS mice, suggesting that it can improve the CRS-induced reduction in neuroblasts / immature neurons. Simultaneously, (+)-lim treatment reduced the abnormally elevated number of MCM2-positive cells and DCX / MCM2 double-positive cells in CRS mice, bringing them closer to normal levels. In contrast, (±)-lim 9 mg / kg had a certain regulatory effect on some indicators, but the overall improvement effect was not as significant as that of (+)-lim. These results indicate that CRS can lead to an imbalance in neurogenesis in the DG region, manifested as abnormal changes in early or proliferation-related cell populations and a reduction in DCX-positive neuroblasts / immature neurons; (+)-lim can improve this abnormal state and promote the restoration of homeostasis in neurogenesis in the DG region.

Claims

1. An indole derivative, characterized in that: The chemical structural formula of the indole derivative is shown below: 。 2. The method for preparing the indole derivative according to claim 1, characterized in that: Includes the following steps: (1) Starting from N-BOC-L-tryptophan or tryptophan, an amino-protected intermediate is prepared by nucleophilic substitution reaction with an amino-protecting reagent under alkaline conditions. The intermediate is then coupled with bromobutyne protected by TBS or TBDPS and the silyl protecting group is removed to obtain an alkynol intermediate. The alkynol intermediate is oxidized to carbonyl by an oxidant and then undergoes a tandem cyclization reaction under an acid catalyst to obtain product (-)-6 or product (±)-8. Product (-)-6 is hydrogenated under a metal catalyst to obtain product (-)-7. Product (-)-7 is reacted with a disulfide bond activating reagent and a phosphine reagent, and then a thiol reagent is added to carry out a desulfurization cyclization reaction to obtain product (-)-8. (2) The product (-)-8 or (±)-8 is deprotected under reducing deprotection conditions to obtain the product (-)-9 or (±)-9; (3) The product (-)-9 or (±)-9 undergoes a coupling reaction under the action of a palladium catalyst to obtain the product (-)-10 or (±)-10; (4) The product (-)-10 or (±)-10 undergoes an oxidation reaction under the action of an oxidizing agent, and then a reducing agent is added to carry out a reduction reaction to obtain the product (-)-11 or (±)-11; further protection with silicon-based materials yields the product (-)-12 or (±)-12. (5) The product (-)-12 or (±)-12 undergoes an alkylation reaction with a propargylating agent under alkaline conditions to give the product (-)-13 or (±)-13; (6) The product (-)-13 or (±)-13 undergoes intramolecular cyclization and imine reduction under the action of silver salt catalyst to obtain the product (+)-14 or (±)-14. (7) The product (+)-14 or (±)-14 reacts with a trifluoromethanesulfonating agent under alkaline conditions to give the product (+)-15 or the product (±)-15. (8) The product (+)-15 or (±)-15 undergoes a deprotection reaction in a hydrogen atmosphere under the action of a metal catalyst and an alkaline auxiliary agent, and is then treated with acid to obtain (+)-Limaspermidine or (±)-Limaspermidine.

3. The method for preparing indole derivatives according to claim 2, characterized in that: In step (1), when N-Boc-L-tryptophan is used as the starting material, it reacts with p-methoxybenzyl bromide under the action of potassium tert-butoxide to introduce a p-methoxybenzyl protecting group; then it reacts with benzyl bromide under the action of potassium carbonate to introduce a benzyl protecting group, thus obtaining an amino-protected intermediate; when tryptophan is used as the starting material, it reacts with p-methoxybenzyl bromide under the action of sodium hydride to introduce a p-methoxybenzyl protecting group; then it reacts with p-toluenesulfonyl chloride under the action of triethylamine to introduce a p-toluenesulfonyl protecting group, thus obtaining an amino-protected intermediate.

4. The method for preparing indole derivatives according to claim 2, characterized in that: In step (1), the oxidant is selected from one or more of manganese dioxide, Dys-Martin oxidant, IBX or Swern oxidation system; the acid catalyst is selected from one or more of camphor sulfonic acid, p-toluene sulfonic acid, trifluoromethanesulfonic acid or trifluoroacetic acid; the metal catalyst is selected from one or more of palladium on carbon, platinum on carbon, Raney nickel or rhodium on carbon; the disulfide bond activator is selected from one or more of 2,2'-dithiopyridine-1,1'-dioxide, 2,2'-dithiobis(5-nitropyridine), dithiobenzoate compounds; the phosphine reagent is selected from one or more of tri-n-butylphosphine, triphenylphosphine, triethylphosphine; and the thiol reagent is selected from one or more of tert-dodecyl mercaptan, dodecyl mercaptan, tert-butyl mercaptan or benzyl mercaptan. In step (2), the system in the reducing deprotection conditions is selected from one or more of sodium naphthalene, lithium naphthalene, sodium / naphthalene system or alkali metal aryl radical system; In step (3), the palladium catalyst is selected from one or more of tetra(triphenylphosphine)palladium, palladium acetate, palladium chloride, or palladium phosphine complexes; In step (4), the oxidant is selected from one or more of the following: potassium osmium tetroxide / sodium periodate system, potassium permanganate, osmium tetroxide / sodium periodate system, or ozone oxidation system; the reducing agent is selected from one or more of the following: sodium borohydride, lithium aluminum hydride, diisobutylaluminum hydride, or borane; and the silicon-based protecting agent is selected from one or more of the following: tert-butyldimethylchlorosilane, trimethylchlorosilane, tert-butyldiphenylchlorosilane, or triisopropylchlorosilane. In step (5), the propargylating agent is selected from one or more of propargyl bromide, propargyl chloride, propargyl iodide, or propargyl sulfonate; the base is selected from one or more of n-butyllithium, sodium hydride, potassium tert-butoxide, potassium carbonate, or hexamethyldisilamide lithium. In step (6), the silver salt is selected from one or more of silver trifluoromethanesulfonate, silver bis(trifluoromethanesulfonyl)imide, silver tetrafluoroborate, or silver hexafluorophosphate. In step (7), the trifluoromethanesulfonylating agent is selected from one or more of N,N-ditrifluoromethanesulfonyl-5-chloro-2-aminopyridine and aminosulfonyl reagents; In step (8), the metal catalyst is selected from one or more of palladium on carbon, platinum on carbon, Raney nickel or rhodium on carbon; the alkaline additive is selected from one or more of lithium carbonate, sodium carbonate, potassium carbonate or cesium carbonate; and the acid is selected from one or more of hydrochloric acid, sulfuric acid, trifluoroacetic acid or methanesulfonic acid.

5. The method for preparing indole derivatives according to claim 2, characterized in that: (1) Under nitrogen protection, N-BOC-L-tryptophan was dissolved in N,N-dimethylformamide, potassium tert-butoxide was added, and p-methoxybenzyl bromide was added dropwise at 0 °C to give crude product 1; crude product 1 was dissolved in N,N-dimethylformamide, potassium carbonate was added, and benzyl bromide was added dropwise at 0 °C to give crude product 2; crude product 2 was dissolved in dichloromethane, and 4 M 1,4-dioxane HCl solution was added dropwise at 0 °C to give crude product; crude product was dissolved in dichloromethane, triethylamine was added at 0 °C, and p-toluenesulfonyl chloride was added dropwise to give compound 3; compound 3 was reacted with TBS-protected bromobutyne in cuprous bromide, N,N′- A coupling reaction was carried out in the presence of dimethylethylenediamine and potassium carbonate to obtain a crude product. This crude product was dissolved in tetrahydrofuran, and a mixed solution of tetrabutylammonium fluoride and acetic acid was added at 0 °C. The mixture was stirred at room temperature to obtain product 5. Product 5 was dissolved in dichloromethane, and MnO2 was added. The mixture was stirred at room temperature to obtain a crude product. This crude product was then dissolved in anhydrous dichloromethane, and camphor sulfonic acid was added at 0 °C. The mixture was stirred to obtain product (-)-6. 10% palladium on carbon was added to a methanol solution containing ammonium formate at 0 °C, and product (-)-6 was added. The mixture was stirred at room temperature to obtain product (-)-7. Product (-)-7 was reacted with 2,2'-dithiopyridine-1,1'-dioxide and tri-n-butylphosphine in dichloromethane at room temperature in the dark. Then, tert-dodecyl mercaptan was added under red LED irradiation to obtain product (-)-8. Using tryptamine as a starting material, the mixture was dissolved in… N , N In dimethylformamide, NaH was added in portions at 0 °C with stirring, followed by dropwise addition of p-methoxybenzyl bromide. The reaction was carried out at room temperature to give product 17. Product 17 was dissolved in dichloromethane, and triethylamine was added at 0 °C, followed by p-toluenesulfonyl chloride. The reaction was carried out at room temperature to give product 18. Product 18 was coupled with TBDPS-protected bromobutyne in the presence of 1,10-phenanthroline, potassium phosphate, and copper sulfate pentahydrate to give product 20. Product 20 was dissolved in tetrahydrofuran, and tetrabutylammonium fluoride trihydrate was added in portions at 0 °C. The reaction was carried out at room temperature to give product 21. Product 21 was dissolved in dichloromethane, and tetrabutylammonium fluoride trihydrate was added at 0 °C. N -methyl- N -Oxymorpholine was stirred and then tetrapropylammonium perruthenate was added. The reaction was carried out to obtain product 22. Product 22 was dissolved in anhydrous dichloromethane, camphor sulfonic acid was added at 0 °C, and the reaction was stirred to obtain product (±)-8. (2) Dissolve product (-)-8 or (±)-8 in anhydrous tetrahydrofuran, and add sodium naphthalene tetrahydrofuran solution dropwise at -78 °C until the deep green color does not fade, to obtain product (-)-9 or (±)-9; (3) Dissolve product (-)-9 or (±)-9 in 1,4-dioxane, add tetra(triphenylphosphine)palladium and allyl methyl carbonate under nitrogen protection, and react at 40 °C to obtain product (-)-10 or (±)-10. (4) Dissolve product (-)-10 or (±)-10 in a mixed solvent of potassium tert-butoxide / tetrahydrofuran / water, add potassium osmium tetroxide, 1,4-diazabicyclo[2.2.2]octane, potassium ferricyanide and sodium periodate, react at room temperature and then cool to -20 °C and add sodium borohydride to obtain product (-)-11 or (±)-11; dissolve it in dichloromethane, add tert-butyldimethylchlorosilane and imidazole at 0 °C, react to obtain product (-)-12 or (±)-12; (5) Dissolve product (-)-12 or (±)-12 in tetrahydrofuran, add NaH and propargyl bromide at 0 °C, and react at 40 °C to obtain product (-)-13 or (±)-13; (6) Dissolve product (-)-13 or (±)-13 in dichloroethane, add silver bis(trifluoromethanesulfonyl)imide, react at room temperature, cool to -20 °C and add ethanol solution of sodium cyanoborohydride to obtain product (+)-14 or (±)-14. (7) Dissolve product (+)-14 or (±)-14 in anhydrous tetrahydrofuran, add bis(trimethylsilylaminolithium) at -78 °C, stir, add N,N-ditrifluoromethanesulfonyl-5-chloro-2-aminopyridine, react at room temperature to obtain product (+)-15 or product (±)-15. (8) Dissolve the product (+)-15 or (±)-15 in ethyl acetate, add 10% palladium on carbon and lithium carbonate, react under a hydrogen atmosphere, then add 2M HCl and continue stirring, and then add NaHCO3 at 0 °C to terminate the reaction to obtain (+)-Limaspermidine or (±)-Limaspermidine.

6. The use of the indole derivative of claim 1 in the preparation of a medicament for treating and / or preventing anxiety.

7. The use of the indole derivative of claim 1 in the preparation of a medicament for treating and / or preventing depression.

8. The application according to claim 6 or 7, characterized in that: The anxiety or depression mentioned refers to chronic stress-induced anxiety or depression.

9. The application according to claim 8, characterized in that: The indole derivatives exert therapeutic and / or preventive effects by restoring and improving the homeostasis of neurogenesis in the hippocampal dentate gyrus, wherein restoring and improving the homeostasis of neurogenesis in the hippocampal dentate gyrus includes: (a) Reduce the abnormal increase in GFAP-positive cells, MCM2-positive cells, and GFAP / MCM2 double-positive cells in the hippocampal dentate gyrus induced by a chronic depression model; (b) Increased abnormal reduction of DCX-positive cells in the hippocampal dentate gyrus induced by a chronic depression model; (c) Reduce the abnormal increase of DCX / MCM2 double-positive cells in the hippocampal dentate gyrus induced by a chronic depression model.

10. A pharmaceutical composition, characterized in that: It includes the indole derivatives as described in claim 1.

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