Spiro [acridine-1, 5 '-isoxazole] compound as well as preparation method and application thereof

Abstract

The invention relates to novel acridine spiroisoxazole compounds, a preparation method thereof and application of the novel acridine spiroisoxazole compounds in antifungal drugs. The novel acridine spiroisoxazole compounds have the antibacterial activity of 36 compounds, and the 36 compounds have the inhibiting effect on botrytis cinerea, parasite scalaria, rhizoctonia solani, fusarium graminearum and candida albicans. The antifungal biological activity result shows that the compound series all show obvious antibacterial activity. The compound is determined as a broad-spectrum antibacterial candidate due to the comprehensive antibacterial characteristics of the compound, and the EC50 value (5.0 + / -0.4 mu g / mL) of H35 shows 1.5 times, 2.2 times and 3.2 times of titer advantages compared with positive control drugs famoxadone (7.6 mu g / mL), chlorothalonil (11.1 mu g / mL) and carbendazim (15.9 mu g / mL) respectively. The spiro structure of the compound can significantly improve the antibacterial activity of the compound, the preliminary mechanism research target shows that the compound is related to fungal membrane damage, and in addition, the compound H35 has protection and treatment effects on botrytis cinerea infection in corn, which shows that the compound H35 has agricultural application potential.

Description

Technical Field

[0001] This invention relates to the technical field of medicinal chemistry, specifically to an acridine spiroisoxazole compound and its preparation method. Background Technology

[0002] Plant pathogenic fungi pose an extremely serious threat to crops. Statistics from the Food and Agriculture Organization of the United Nations (FAO) show that global crop yields are reduced by 20%–40% annually due to plant pathogenic fungi, with particularly severe losses in areas with high disease incidence. In my country, fungal diseases cause tens of millions of tons of grain losses annually, posing a significant threat to food security and the agricultural economy. Although existing fungicides have suppressed the spread of diseases to some extent, the long-term use of monocyclic planar fungicides has led to increasingly serious problems of pathogen resistance, significantly reducing their control effectiveness, necessitating the development of new fungicides.

[0003] Heterocyclic compounds have attracted significant attention in the field of pesticide chemistry due to their remarkable biological activity. Among them, nitrogen-containing heterocyclic compounds, with their unique chemical structures, possess high efficiency, low toxicity, and environmental friendliness, making them a key focus of novel pesticide development. Isoxazole compounds, with their unique chemical structures and broad-spectrum biological activity, have shown application potential in antifungal and antiviral applications. The spirocyclic structure enhances the compound's membrane penetration and achieves precise targeting through its rigid three-dimensional configuration, while the conjugated large π-bond system of the acridine ring can interfere with the energy metabolism and biosynthetic processes of pathogens. The synergistic effect of these three elements not only endows the compound with unique physicochemical properties but also significantly enhances its antifungal activity, effectively inhibiting pathogen hyphal growth, spore germination, and biofilm formation. As novel nitrogen-containing heterocyclic derivatives, acridine spiroisoxazole compounds hold promise for providing innovative solutions for the control of plant fungal diseases. Therefore, in-depth research into the synthetic methods and antifungal mechanisms of acridine spiroisoxazole compounds is of great significance for promoting pesticide innovation and crop disease control. Summary of the Invention

[0004] The purpose of this invention is to provide a novel acridine-spiroisoxazole compound and its uses. This invention relates to the generation of 3,4-dihydroacrylidine-1(2H)-ones with different substituents from o-nitrobenzaldehyde and 1,3-cyclohexanedione as starting materials under the action of iron powder and citric acid. Furthermore, acetophenone with different substituents undergoes a nucleophilic addition reaction with hydroxylamine hydrochloride to obtain acetophenone oximes, which are further deprotonated under the action of diisopropylaminolithium and undergo nucleophilic addition with 3,4-dihydroacrylidine-1(2H)-one to obtain 36 acridine-acetophenone oxime compounds. Finally, intramolecular cyclization is performed under the action of concentrated sulfuric acid to prepare 36 acridine-spiroisoxazole compounds. The obtained 36 acridine spiroisoxazole compounds all showed significant antibacterial activity against *Botrytis cinerea*, *Pseudomonas aeruginosa*, *Rhizoctonia solani*, *Fusarium graminearum*, and *Candida albicans*. Due to its comprehensive antibacterial properties, this compound was identified as a broad-spectrum antibacterial candidate. The spirocyclic structure of the compound significantly enhances its antibacterial activity. Preliminary mechanistic studies show that the target site is related to fungal membrane damage. Furthermore, compound H35 has protective and therapeutic effects against *Botrytis cinerea* infection in maize, indicating its potential for agricultural application.

[0005] To address the above problems, the present invention provides the following technical solution:

[0006] In a first aspect, this application provides an acridine spiroisoxazole compound, the structure of which is shown below:

[0007]

[0008] in:

[0009] R 1 Selected from any one of H, F, and Br;

[0010] R 2 Selected from either H or CH3;

[0011] R 3 Selected from any one of H, F, Br, Me, OMe, CF3

[0012] It has one of the following chemical formulas:

[0013]

[0014] Among them: Compound H1 is (S)-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H2 is (S)-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H3 is (S)-3'-(4-methoxyphenyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H4 is (S)-3'-(4-fluorophenyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H5 is (S)-3'-(4-bromophenyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H6 is (S)-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H7 is (S)-7-fluoro-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H8 is (S)-7-fluoro-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H9 is (S)-7-fluoro-3'-(4-methoxyphenyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H10 is (S)-7-fluoro-3'-(4-fluorophenyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H11 is (S)-3'-(4-bromophenyl)-7-fluoro-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H12 is (S)-7-fluoro-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H13 is (S)-7-bromo-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H14 is (S)-7-bromo-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H15 is (S)-7-bromo-3'-(4-methoxyphenyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H16 is (S)-7-bromo-3'-(4-fluorophenyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H17 is (S)-7-bromo-3'-(4-bromophenyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H18 is (S)-7-bromo-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole].Compound H19 is (S)-3,3-dimethyl-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H20 is (S)-3,3-dimethyl-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H21 is (S)-3'-(4-methoxyphenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H22 is (S)-3'-(4-fluorophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H23 is (S)-3'-(4-bromophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H24 is (S)-3,3-dimethyl-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H25 is (S)-7-fluoro-3,3-dimethyl-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H26 is (S)-7-fluoro-3,3-dimethyl-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H27 is (S)-7-fluoro-3'-(4-methoxyphenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H28 is (S)-7-fluoro-3'-(4-fluorophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H29 is (S)-3'-(4-bromophenyl)-7-fluoro-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H30 is (S)-7-fluoro-3,3-dimethyl-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H31 is (S)-7-bromo-3,3-dimethyl-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H32 is (S)-7-bromo-3,3-dimethyl-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H33 is (S)-7-bromo-3'-(4-methoxyphenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H34 is (S)-7-bromo-3'-(4-fluorophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole].Compound H35 is (S)-7-bromo-3'-(4-bromophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole]. Compound H36 is (S)-7-bromo-3,3-dimethyl-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole].

[0015] The synthetic route is shown below:

[0016]

[0017] Secondly, regarding the acridine spiroisoxazole compounds and their uses described in this invention, the preparation method of the acridine spiroisoxazole compounds is carried out according to the following steps:

[0018] (1) In a 1000 mL double-necked flask, add 81.2 mmol of o-nitrobenzaldehyde with different substituents, 99.2 mmol of 1,3-cyclohexanedione with different substituents, 14.82 g, 265.3 mmol of iron powder, 50.86 g, 264.7 mmol of citric acid, and 20.00 g of montmorillonite. Use water as solvent, attach a reflux tube and a mechanical stirrer, and place the flask on a heated magnetic stirrer. Heat the magnetic stirrer to reflux temperature, and then monitor the reaction progress with TLC every 1 h. After the o-nitrobenzaldehyde reacts completely, stop the reaction and cool to room temperature. Filter, extract the filter cake with CH2Cl2, extract the filtrate, combine the organic phases, dry with anhydrous Na2SO4, concentrate, and obtain a crude product of reddish-brown oily liquid. Perform silica gel column chromatography on the crude product. The silica gel is 200-300 mesh, and the eluent is PE:EA = 5:1 (v / v). Collect R. f The band with a ratio of 0.47 (developing solvent: PE:EA = 1:1) was concentrated under reduced pressure to obtain compounds E1-E6, respectively.

[0019] (2) In a 500 mL double-necked flask, add 41.6 mmol of acetophenone with different substituents, 8.67 g of hydroxylamine hydrochloride, 124.7 mmol of hydroxylamine hydrochloride, and 200 mL of water. Heat and stir until the substrate dissolves. Dissolve 17.23 g of potassium carbonate, 124.7 mmol of hydroxylamine hydrochloride in 50 mL of aqueous solution. Place the solution in a constant pressure dropping funnel and add it dropwise to the reaction system. Heat to reflux temperature with magnetic stirring. Then monitor the reaction progress with TLC every 1 h. After the acetophenone has reacted completely, cool to room temperature and wait for crystals to precipitate. Filter to obtain colorless needle-like crystals, wash three times with ice water (30 mL × 3), and obtain compounds F1-F6 respectively.

[0020] (3) Set the temperature of the low-temperature constant temperature reaction bath to 0℃, add 7.4 mmol of compound F1-F6 to a 50 mL round bottom flask, add ultra-dry tetrahydrofuran solution, add a flap stopper, perform inert gas replacement, and then place it in a low-temperature reactor. Add 7.4 mL of 2 M diisopropylaminolithium. After one hour, dissolve 1.78 mmol of compound E1-E6 in ultra-dry tetrahydrofuran and add it dropwise to the reaction system. Monitor the reaction progress with TLC. After the reaction is completed, add saturated ammonium chloride solution to quench the reaction, add ethyl acetate (5×30 mL) for extraction, separate the organic phase, wash the organic phase with saturated sodium chloride solution, combine the organic phases, concentrate to obtain crude product, and separate and purify the crude product by silica gel column chromatography. The silica gel for column chromatography is 200-300 mesh, and the eluent is PE:EA = 2:1 (v / v). Compounds G1-G36 are obtained by separation.

[0021] (4) Add 0.5 mmol of compound G1-G36 to a 50 mL round-bottom flask, add 1 mL of 98% concentrated sulfuric acid, stir at room temperature, monitor the reaction progress by TLC, and when the reaction is complete (about 20 min), slowly add saturated sodium bicarbonate until the reaction system is neutral, add ethyl acetate (5 × 30 mL) for extraction, combine the organic phases, wash the organic phases with saturated sodium chloride, dry with anhydrous Na2SO4, filter, concentrate, and obtain compounds H1-H36, which do not require purification.

[0022] Thirdly, this application also provides the use of the aforementioned acridine spiroisoxazole compounds in inhibiting the activity of Botrytis cinerea, Powdery mildew, Rhizoctonia solani, Fusarium graminearum, and Candida albicans.

[0023] Fourthly, this application also provides the use of the aforementioned acridine spiroisoxazole compounds in inhibiting Staphylococcus aureus infection.

[0024] Fifthly, this application also provides a pharmaceutical composition characterized in that it comprises a pharmaceutically acceptable excipient or carrier and an acridine spiroisoxazole compound as described in claim 1.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) Based on inexpensive and readily available raw materials, this application successfully prepared a series of acridine spiroisoxazole compounds through multi-step organic synthesis. Antifungal evaluation showed that the H1-H36 compound series all exhibited significant antibacterial activity, with H35 showing the most significant therapeutic effect. Due to its comprehensive antibacterial properties, this compound was identified as a broad-spectrum antibacterial candidate. The EC50 of H35... 50The concentration (5.0 ± 0.4 μg / mL) showed a potency advantage of 1.5 times, 2.2 times, and 3.2 times compared to the positive control drugs oxadiazon (7.6 μg / mL), chlorothalonil (11.1 μg / mL), and carbendazim (15.9 μg / mL), respectively. These results indicate that the spirocyclic structure significantly enhances its antibacterial activity. Preliminary mechanistic studies suggest a target site related to fungal membrane damage. Furthermore, compound H35 exhibits both protective and therapeutic effects against gray mold infection in maize, indicating its potential for agricultural application.

[0027] (2) Based on a comprehensive analysis of relevant domestic and international patents and literature, this invention conducts a systematic study on spiroisoxazole compounds. These compounds exhibit a variety of biological activities due to their unique spirocyclic structure, particularly showing potential applications in antifungal and antiviral activity. Their unique three-dimensional structure has attracted considerable attention in natural product and drug development. This study modifies and transforms acridine spiroisoxazole compounds by introducing different substituents, aiming to enhance their application potential in the control of fungal diseases in crops. Simultaneously, this study systematically investigates the inhibitory activity of these compounds against various pathogens, including *Botrytis cinerea*, *Pseudomonas aeruginosa*, *Rhizoctonia solani*, *Fusarium graminearum*, and *Candida albicans*, aiming to discover compounds with significant antibacterial activity and clearly defined targets, providing theoretical basis and practical guidance for the development of novel pesticides. Attached Figure Description

[0028] Figure 1 The diagram shows the synthetic route for the target compounds H1-H36.

[0029] Figure 2 The figure shows the inhibitory effects of H35 and carbendazim on the mycelial growth of Botrytis cinerea in vitro.

[0030] Figure 3 The image shown is a micro-electron microscope image.

[0031] Fluorescence microscopy analysis of Botrytis cinerea hyphae is shown as A1-E1; bright-field images of hyphal morphology are shown as A2-E2 and A3-E3; green fluorescence images highlighting structural details are shown as A1-A3; the untreated control group is shown as B1-C3; and hyphae treated with carbendazim are shown as D1-E3.

[0032] Figure 4 The protective and therapeutic activities of compound H35 on maize leaves are shown. Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Reagents used in this application: All reagents were commercially available analytical grade;

[0035] The fungi used in this application—Rhizoctonia solani, Botrytiscinerea, Gibberella zeae, and Ampelomyces humuli (Fautrey) Rudakov—were cultured on potato dextrose agar (PDA) at 28°C, and Candida albicans was cultured on yeast-mold agar (YM) at 28°C. All of the above strains are conventional strains and were purchased from Wuhan Gray Algae Biotechnology Co., Ltd., China. They can also be obtained by the general public through relevant biological companies.

[0036] Unless otherwise specified, the technical means used in the following implementation examples are conventional means well known to those skilled in the art.

[0037] Example 1: Preparation of the compound

[0038] For the synthetic route and principle of acridine spiroisoxazole compounds, please refer to the appendix. Figure 1 As shown, the specific steps are as follows:

[0039] (1) In a 1000 mL double-necked flask, add 81.2 mmol of o-nitrobenzaldehyde with different substituents, 99.2 mmol of 1,3-cyclohexanedione with different substituents, 14.82 g, 265.3 mmol of iron powder, 50.86 g, 264.7 mmol of citric acid, and 20.00 g of montmorillonite. Use water as solvent, attach a reflux tube and a mechanical stirrer, and place the flask on a heated magnetic stirrer. Heat the magnetic stirrer to reflux temperature, and then monitor the reaction progress by TLC every 1 h. After the o-nitrobenzaldehyde reacts completely, stop the reaction and cool to room temperature; filter, extract the filter cake with CH2Cl2, extract the filtrate, combine the organic phases, dry with anhydrous Na2SO4, concentrate, and obtain a crude product of reddish-brown oily liquid; perform silica gel column chromatography on the crude product, using 200-300 mesh silica gel and eluent with a volume ratio of PE:EA = 5:1, and collect R. f The band with a ratio of 0.47 (developing solvent: PE:EA = 1:1) was concentrated under reduced pressure to obtain compounds E1-E6, respectively.

[0040] (2) In a 500 mL double-necked flask, add 41.6 mmol of acetophenone with different substituents, 8.67 g of hydroxylamine hydrochloride, 124.7 mmol of hydroxylamine hydrochloride, and 200 mL of water. Heat and stir until the substrate dissolves. Dissolve 17.23 g of potassium carbonate, 124.7 mmol of hydroxylamine hydrochloride in 50 mL of aqueous solution. Place the solution in a constant pressure dropping funnel and add it dropwise to the reaction system. Heat to reflux temperature with magnetic stirring. Then monitor the reaction progress with TLC every 1 h. After the acetophenone has reacted completely, cool to room temperature and wait for crystals to precipitate. Filter to obtain colorless needle-like crystals, wash three times with ice water (30 mL × 3), and obtain compounds F1-F6 respectively.

[0041] (3) Set the temperature of the low-temperature constant temperature reaction bath to 0℃, add 7.4 mmol of compound F1-F6 to a 50 mL round bottom flask, add ultra-dry tetrahydrofuran solution, add a flap stopper, perform inert gas replacement, and then place it in a low-temperature reactor. Add 7.4 mL of 2 M diisopropylaminolithium. After one hour, dissolve 1.78 mmol of compound E1-E6 in ultra-dry tetrahydrofuran and add it dropwise to the reaction system. Monitor the reaction progress with TLC. After the reaction is completed, add saturated ammonium chloride solution to quench the reaction, add ethyl acetate (5×30 mL) for extraction, separate the organic phase, wash the organic phase with saturated sodium chloride solution, combine the organic phases, concentrate to obtain crude product, and separate and purify the crude product by silica gel column chromatography. The silica gel for column chromatography is 200-300 mesh, and the eluent is PE:EA = 2:1 (v / v). Compounds G1-G36 are obtained by separation.

[0042] (4) Add 0.5 mmol of compound G1-G36 to a 50 mL round-bottom flask, add 1 mL of 98% concentrated sulfuric acid, stir at room temperature, monitor the reaction progress by TLC, and when the reaction is complete (about 20 min), slowly add saturated sodium bicarbonate until the reaction system is neutral, add ethyl acetate (5 × 30 mL) for extraction, combine the organic phases, wash the organic phases with saturated sodium chloride, dry with anhydrous Na2SO4, filter, concentrate, and obtain compounds H1-H36, which do not require purification.

[0043] Example 2: Preparation of the compound

[0044] Preparation of compound H1: 0.5 mmol of compound G1 (S,E)-2-(1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-phenylethyl-1-one oxime was added to a 50 mL round-bottom flask. 1 mL of 98% concentrated sulfuric acid was added, and the mixture was stirred at room temperature. The reaction was monitored by TLC. After the reaction was complete (approximately 20 min), saturated sodium bicarbonate was slowly added until the reaction system was neutral. Ethyl acetate (5 × 30 mL) was added for extraction. The organic phases were combined, washed with saturated sodium chloride, dried over anhydrous Na2SO4, filtered, and concentrated to obtain compound H1 (S)-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole]. No purification was required. Yield: 94%. White solid.

[0045] (S)-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H1): White solid, yield 94%, mp180.1~183.2℃; 1 H NMR (400MHz, DMSO-d6) δ8.38(s,1H),7.91(d,J=1.0Hz,1H),7.75(d,J=6.1Hz,1H) ,7.54-7.45(m,3H),3.76(d,J=5.9Hz,2H),3.12(s,1H),2.13(s,1H),1.23(s,3H); 13 C NMR(101MHz,DMSO-d6)δ158.09,156.41,147.37,134.61,133.89,130.52,130.22,130.09,12 9.28,128.60,128.24,127.36,127.16,126.32,87.10,49.05,35.41,33.17,19.86; MS:calcd for C 21 H 18 N₂O[M+H] + :315.14,found:315.14.

[0046] Example 3: Preparation of the compound

[0047] Based on the description in Example 2 above, compound H2 was prepared as follows: 0.5 mmol of compound G2, which is (S,E)-2-(1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-(p-tolyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same to obtain compound H2, which is (S)-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], with a yield of 92%.

[0048] (S)-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H2): White solid, yield 92%, mp171.4~173.9℃; 1 H NMR (400MHz, Chloroform-d) δ8.24(s,1H),7.99(d,J=8.5Hz,1H),7.74(d,J=8.1Hz,1H),7.67(ddd,J=8.4,6.9,1.4Hz,1H),7.61(d,J=8 .0Hz,2H),7.49-7.41(m,1H),7.24(d,J=7.9Hz,2H),3.55(s,2H),3.32-3.13(m,2H),2.40(s,3H),2.35-2.16(m,3H),2.11-1.95(m,1H); 13 CNMR(101MHz,Chloroform-d)δ157.52,155.22,147.67,140.66,134.28,134.09,130.04,129.66,12 8.40,127.97,127.46,126.88,126.73,126.13,87.14,50.07,35.63,33.14,21.60,20.18; MS:calcd for C 22 H 20 N₂O[M+H] + :329.16,found:329.16.

[0049] Example 4: Preparation of the compound

[0050] Compound H3 was prepared based on the description in Example 2 above: 0.5 mmol of compound G3, (S,E)-2-(1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-(4-methoxyphenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, and compound H3 was obtained as (S)-3'-(4-methoxyphenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 84%.

[0051] (S)-3'-(4-methoxyphenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H3): White solid, yield 84%, mp174.3~176.5℃; 1 H NMR (400MHz, Chloroform-d) δ8.26 (s, 1H), 8.01 (d, J = 8.5Hz, 1H), 7.74 (dd, J = 8.2, 1.4Hz, 1H), 7.70-7.62 (m, 3H), 7.51-7. 41(m,1H),6.98-6.92(m,2H),3.85(s,3H),3.54(s,2H),3.24(dd,J=5.8,2.5Hz,1H),2.35-2.14(m,1H),2.09-1.96(m,1H); 13 CNMR(101MHz,Chloroform-d)δ161.29,157.48,154.84,147.44,134.43,134.16,130.11,128.31,12 8.22,127.96,127.45,126.18,122.19,114.36,86.93,55.52,50.18,35.57,33.01,20.15; MS:calcd forC 22 H 20 N₂O₂[M+H] + :344.15,found:344.15.

[0052] Example 5: Preparation of the compound

[0053] Preparation of compound H4: 0.5 mmol of compound G4, (S,E)-1-(4-fluorophenyl)-2-(1'-hydroxy-1',2',3',4'-tetrahydroacridine)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, and compound H4 was obtained as (S)-3'-(4-fluorophenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 94%.

[0054] (S)-3'-(4-fluorophenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H4): White solid, yield 94%, mp205.0~206.4℃; 1 H NMR(400MHz,Chloroform-d)δ8.27(s,1H),8.06(d,J=8.4Hz,1H),7.76(d,J=8.2Hz,1H),7.73-7.68(m,3H),7.48(ddd,J=8.1, 6.8,1.2Hz,1H),7.17-7.08(m,2H),3.55(s,2H),3.36-3.17(m,2H),2.36-2.25(m,2H),2.24-2.15(m,1H),2.11-1.96(m,1H); 13 CNMR(101MHz,Chloroform-d)δ165.25,162.75,157.26,154.32,135.16,133.99,130.68,128.76(J C-F =8.7Hz),128.00,127.70,127.45,126.70(J C-F =21.7Hz), 125.88 (J) C-F =3.3Hz), 116.16(J) C-F =22.1Hz),87.35,49.97,35.49,32.57,19.98..;MS:calcd for C 21 H 17 FN2O[M+H] + :333.13,found:333.13.

[0055] Example 6: Preparation of Compounds

[0056] Preparation of compound H5: 0.5 mmol of compound G5, (S,E)-1-(4-bromophenyl)-2-(1'-hydroxy-1',2',3',4'-tetrahydroacridine)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H5 as (S)-3'-(4-bromophenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 96%.

[0057] (S)-3'-(4-bromophenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H5): White solid, yield 96%, mp166.5~169.3℃; 1 H NMR (400MHz, Chloroform-d) δ8.21(s,1H),8.00(d,J=8.5Hz,1H),7.75(dd,J=8.2,1.4Hz,1H),7.68(ddd,J=8.4,6.8,1.5Hz,1H),7.58(d, J=1.9Hz,4H),7.46(td,J=7.3,6.7,1.1Hz,1H),3.54(s,2H),3.32-3.15(m,2H),2.37-2.23(m,2H),2.23-2.15(m,1H),2.11-1.96(m,1H); 13 CNMR(101MHz,Chloroform-d)δ157.39,154.46,147.44,134.53,133.75,132.22,130.34,12 8.64,128.22,127.97,127.42,126.35,124.67,87.75,49.67,35.59,32.94,20.08; MS:calcd for C 21 H 17 BrN2O[M+H] + :393.05,found:393.05.

[0058] Example 7: Preparation of the compound

[0059] Preparation of compound H6: 0.5 mmol of compound G6, (S,E)-2-(1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-[4-(trifluoromethyl)phenyl]ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H6 as (S)-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 94%.

[0060] (S)-3'-(4-(trifluoromethyl)phenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-iso-xazole](H6): White solid, yield 94%, mp172.8~175.6℃; 1H NMR(400MHz,Chloroform-d)δ8.21(s,1H),8.00(dt,J=8.6,1.0Hz,1H),7.88-7.81(m,2H),7.75(dd,J=8.3,1.4Hz,1H),7.69(ddd,J=8.3,6.9,1 .4Hz,4H),7.47(ddd,J=8.1,6.9,1.2Hz,1H),3.58(d,J=1.1Hz,2H),3.2 9-3.20(m,2H),2.39-2.25(m,2H),2.26-2.17(m,1H),2.12-1.97(m,1H); 13 C NMR(101MHz,Chloroform-d)δ157.42,154.24,147.79,134.27,133.35(J C-F =33.2Hz),132.19,131.87,131.54,130.26,128.47,127.96,127.40,127.04,126.30,125.96(J C-F =3.7Hz),125.29,122.58,88.21,49.53,35.66,20.10.;MS:calcd for C 22 H 17 F3N2O[M+H] + :383.13,found:383.13.

[0061] Example 8: Preparation of Compounds

[0062] Preparation of compound H7: 0.5 mmol of compound G7, (S,E)-2-(7-fluoro-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-phenylethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, and compound H7 was obtained as (S)-7-fluoro-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 82%.

[0063] (S)-7-fluoro-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H7): White solid, yield 82%, mp168.4~170.3℃; 1H NMR(400MHz,Chloroform-d)δ8.21(s,1H),8.03(s,1H),7.73(dd,J=6.8,3.0Hz,2H),7.46(tt,J=7.6,3.3Hz,4H ),7.37(dd,J=8.8,2.8Hz,1H),3.57(s,2H),3.23(dt,J=9.2,5.6Hz,2H),2.39-2.17(m,3H),2.12-1.97(m,1H); 13 C NMR(101MHz,Chloroform-d)δ159.06,156.78,155.26,134.98,133.97(J C-F =2.6Hz),130.50,129.57,129.02,128.00(J C-F =10.2Hz), 126.82, 120.65 (J) C-F =25.3Hz), 110.80(J) C-F =21.8Hz),87.19,50.00,35.47,29.84,20.0; MS:calcdfor C 22 H 17 FN2O[M+H] + :333.13,found:333.13.

[0064] Example 9: Preparation of the compound

[0065] Preparation of compound H8: 0.5 mmol of compound G8, (S,E)-2-(7-fluoro-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-(p-tolyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H8 as (S)-7-fluoro-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 95%.

[0066] (S)-7-fluoro-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H8): White solid, yield 95%, mp105.6~107.2℃; 1H NMR(400MHz,Chloroform-d)δ8.17(s,1H),7.98(dd,J=9.3,5.2Hz,1H),7.64-7.57(m,2H),7.43(td,J=8.8,2.8Hz,1H),7.34(d d,J=8.8,2.8Hz,1H),7.24(d,J=7.9Hz,2H),3.54(s,2H),3.29-3.11(m,2H),2.39(s,3H),2.35-2.14(m,3H),2.10-1.94(m,1H); 13 C NMR(101MHz,Chloroform-d)δ161.41,158.95,156.85(J C-F =2.6Hz),155.20,144.79,140.73,134.98,133.57(J C-F =5.4Hz), 130.87(J) C-F =9.1Hz), 129.66, 127.94 (J) C-F =10.2Hz), 126.73, 120.34 (J) C-F =25.9Hz), 110.72(J) C-F =21.5Hz),87.00,50.08,35.48,32.92,21.60,20.11; MS:calcd for C 22 H 19 FN2O[M+H] + :347.15,found:347.15.

[0067] Example 10: Preparation of the compound

[0068] Preparation of compound H9: 0.5 mmol of compound G9, (S,E)-2-(7-fluoro-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-(4-methoxyphenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H9 as (S)-7-fluoro-3'-(4-methoxyphenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 91%.

[0069] (S)-7-fluoro-3'-(4-methoxyphenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-iso-xazole](H9): White solid, yield 91%, mp150.7~153.4℃; 1H NMR(400MHz,Chloroform-d)δ8.19(s,1H),7.99(dd,J=9.3,5.3Hz,1H),7.69-7.61(m,2H),7.44(dd,J=2.8,1.0Hz,1H),7.35(dd,J =8.8,2.8Hz,1H),6.99-6.90(m,2H),3.85(s,3H),3.53(d,J=1.0Hz,2H),3.29-3.12(m,2H),2.33-2.16(m,3H),2.07-1.97(m,1H); 13 C NMR(101MHz,Chloroform-d)δ161.44,161.36,158.97,156.86(J C-F =2.7Hz),154.84,144.69,135.07,133.67(J C-F =5.6Hz), 130.80(J) C-F =9.1Hz), 128.34, 127.97 (J) C-F =10.0Hz), 122.10, 120.39 (J) C-F =25.9Hz), 114.40, 110.74 (J) C-F =21.6Hz),86.82,55.54,50.23,35.46,32.88,20.12; MS:calcd for C 22 H 19 FN2O2[M+H] + :363.14,found:363.14.

[0070] Example 11: Preparation of the compound

[0071] Preparation of compound H10: 0.5 mmol of compound G10, (S,E)-2-(7-fluoro-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-(4-fluorophenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H10 as (S)-7-fluoro-3'-(4-fluorophenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 93%.

[0072] (S)-7-fluoro-3'-(4-fluorophenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxa-zole](H10): White solid, yield 93%, mp160.4~162.1℃;1 H NMR(400MHz,Chloroform-d)δ8.18(s,1H),8.01(dd,J=9.4,5.2Hz,1H),7.75-7.67(m,2H),7.50-7.41(m,1H),7.36(dd,J=8.8,2.8 Hz,1H),7.13(t,J=8.6Hz,2H),3.54(s,2H),3.22(dt,J=9.0,5.1Hz,2H),2.35-2.24(m,2H),2.25-2.15(m,1H),2.09-1.97(m,1H); 13 C NMR(101MHz,Chloroform-d)δ165.27,162.77,161.52,159.06,156.76(J C-F =2.7Hz), 154.29(J) C-F =1.4Hz), 134.78, 133.79 (J) C-F =2.9Hz), 130.76(J) C-F =9.2Hz), 128.77(J) C-F =8.5Hz), 127.95 (J) C-F =10.0Hz), 125.86 (J) C-F =3.5Hz), 120.62(J) C-F =25.9Hz), 116.18(J) C-F =22.1Hz), 110.76(J) C-F =21.7Hz),87.38,50.01,35.46,32.77,20.05; MS:calcd for C 21 H 16 F2N2O[M+H] + :351.12,found:351.12.

[0073] Example 12: Preparation of the compound

[0074] Preparation of compound H11: 0.5 mmol of compound G11, (S,E)-1-(4-bromophenyl)-2-(7-fluoro-1'-hydroxy-1',2',3',4'-tetrahydroacridine)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H11 as (S)-3'-(4-bromophenyl)-7-fluoro-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], which did not require purification. Yield: 86%.

[0075] (S)-3'-(4-bromophenyl)-7-fluoro-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isox-azole](H11): White solid, yield 86%, mp132.6~134.3℃; 1 H NMR (400MHz, Chloroform-d) δ8.14(s,1H),7.98(dd,J=9.3,5.3Hz,1H),7.56(s,4H),7.44(ddd,J=9.3,8.3,2.8Hz,1H),7.34(dd,J=8. 8,2.8Hz,1H),3.53(s,2H),3.27-3.13(m,2H),2.35-2.23(m,2H),2.19(ddd,J=9.6,5.8,2.4Hz,1H),2.02(ddt,J=8.8,3.9,2.2Hz,1H); 13 C NMR(101MHz,Chloroform-d)δ161.42,158.96,156.75(J C-F =2.7Hz),154.41,144.83,134.55,133.53(J C-F =5.5Hz), 132.18, 130.90 (J) C-F =9.1Hz),128.52,128.19,127.86(J C-F =10.1Hz), 124.68, 120.46 (J) C-F =25.9Hz), 110.70(J) C-F =21.7Hz),87.63,49.65,35.45,32.86,20.02; MS:calcd for C 21 H 16 BrFN2O[M+H] + :411.04,found:411.04.

[0076] Example 13: Preparation of the compound

[0077] Preparation of compound H12: 0.5 mmol of compound G12, (S,E)-2-(7-fluoro-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-[4-(trifluoromethyl)phenyl]ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H12 as (S)-7-fluoro-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 89%.

[0078] (S)-7-fluoro-3'-(4-(trifluoromethyl)phenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H12): White solid, yield 89%, mp147.3~148.8℃; 1 HNMR(400MHz,Chloroform-d)δ8.17(s,1H),8.04(d,J=8.8Hz,1H),7.87-7.80(m,2H),7.70(d,J=8.2Hz,2H),7.52-7.42(m,1H),7.37(dd,J= 8.8,2.8Hz,1H),3.58(s,2H),3.31-3.15(m,2H),2.37-2.26(m,2H),2.22(ddd,J=13.3,6.5,2.9Hz,1H),2.05(dtt,J=12.4,9.6,6.7Hz,1H); 13 C NMR(101MHz,Chloroform-d)δ161.58,159.11,156.69(J C-F =2.6Hz),154.23,134.49,133.03,131.96(J C-F =3.0Hz), 127.93 (J) C-F =9.9Hz), 126.00(J) C-F =3.8Hz),125.27,122.56,120.77(J C-F =26.8Hz), 110.78 (J) C-F =21.7Hz),88.02,49.57,35.49,32.73,20.00; MS:calcd for C 22 H 16 F4N2O[M+H] + :401.12,found:401.12.

[0079] Example 14: Preparation of the compound

[0080] Preparation of compound H13: 0.5 mmol of compound G13, (S,E)-2-(7-bromo-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-phenylethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H13 as (S)-7-bromo-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 88%.

[0081] (S)-7-bromo-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H13): White solid, yield 88%, mp210.7~213.3℃; 1 H NMR(400MHz,Chloroform-d)δ8.14(s,1H),7.96-7.82(m,2H),7.76-7.67(m,3H),7.44(dd,J=5 .0,1.9Hz,3H),3.56(d,J=1.9Hz,2H),3.28-3.12(m,2H),2.37-2.15(m,3H),2.10-1.97(m,1H); 13 C NMR(101MHz,Chloroform-d)δ158.01,155.22,146.11,135.00,133.50,133.23,130.45,130.10 ,129.88,129.52,128.98,128.52,126.77,119.92,87.50,49.92,35.41,33.00,20.42; MS:calcd for C 21 H 17 BrN2O[M+H] + :393.05,found:393.05.

[0082] Example 15: Preparation of the compound

[0083] Preparation of compound H14: 0.5 mmol of compound G14, (S,E)-2-(7-bromo-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-(p-tolyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H14 as (S)-7-bromo-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 95%.

[0084] (S)-7-bromo-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H14): White solid, yield 95%, mp179.9~187.0℃; 1 H NMR(400MHz,Chloroform-d)δ8.14(s,1H),7.90(d,J=2.2Hz,1H),7.85(d,J=9.0Hz,1H),7.72(dd,J=9.0,2.2Hz,1H),7.63-7. 57(m,2H),7.24(d,J=8.0Hz,2H),3.54(d,J=2.5Hz,2H),3.25-3.15(m,2H),2.40(s,3H),2.36-2.15(m,3H),2.08-1.96(m,1H); 13 C NMR(101MHz,Chloroform-d)δ158.07,155.22,146.21,140.78,135.13,133.46,133.18,130.18,12 9.91,129.69,128.57,126.75,126.72,119.89,86.95,50.09,35.44,33.08,21.62,20.09; MS:calcd for C 22 H 19 BrN2O[M+H] + :407.07,found:407.07.

[0085] Example 16: Preparation of the compound

[0086] Preparation of compound H15: 0.5 mmol of compound G15, (S,E)-2-(7-bromo-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-(4-methoxyphenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H15 as (S)-7-bromo-3'-(4-methoxyphenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 93%.

[0087] (S)-7-bromo-3'-(4-methoxyphenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-iso-xazole](H15): White solid, yield 93%, mp211.6~214.0℃; 1H NMR(400MHz,Chloroform-d)δ8.14(s,1H),7.89(d,J=2.2Hz,1H),7.85(d,J=9.0Hz,1H),7.71(dd,J=9.0,2.2Hz,1H),7.67-7. 60(m,2H),6.98-6.90(m,2H),3.85(s,3H),3.52(d,J=2.7Hz,2H),3.26-3.15(m,2H),2.34-2.15(m,3H),2.01(d,J=2.5Hz,1H); 13 C NMR(101MHz,Chloroform-d)δ161.38,158.09,154.84,146.18,135.20,133.46,133.19,130.16,12 9.91,128.58,128.34,122.07,119.89,114.41,86.80,55.54,50.22,35.42,33.06,20.10; MS:calcd forC 22 H 19 BrN2O2[M+H] + :423.06,found:423.06.

[0088] Example 17: Preparation of Compounds

[0089] Preparation of compound H16: 0.5 mmol of compound G16, (S,E)-2-(7-bromo-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-(4-fluorophenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H16 as (S)-7-bromo-3'-(4-fluorophenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], which did not require purification. Yield: 87%.

[0090] (S)-7-bromo-3'-(4-fluorophenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isox-azole](H16): White solid, yield 87%, mp136.7~138.5℃; 1H NMR(400MHz,Chloroform-d)δ8.15(s,1H),7.95-7.85(m,2H),7.78-7.66(m,3H),7.18-7.09(m,2H) ,3.53(d,J=3.0Hz,2H),3.30-3.13(m,2H),2.36-2.23(m,2H),2.25-2.15(m,1H),2.11-1.95(m,1H); 13 C NMR(101MHz,Chloroform-d)δ165.29,162.79,157.95,154.28,134.95,133.58(J C-F =30.6Hz), 129.92, 128.77 (J) C-F =8.5Hz), 128.55, 125.81 (J) C-F =3.5Hz), 116.20(J) C-F =22.1Hz),87.31,50.00,35.40,32.89,20.01.;MS:calcd for C 21 H 16 BrFN2O[M+H] + :411.04,found:411.04.

[0091] Example 18: Preparation of the compound

[0092] Preparation of compound H17: 0.5 mmol of compound G17, (S,E)-2-(7-bromo-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-(4-bromophenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H17 as (S)-7-bromo-3'-(4-bromophenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 83%.

[0093] (S)-7-bromo-3'-(4-bromophenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isox-azole](H17): White solid, yield 83%, mp182.3~185.1℃; 1H NMR(400MHz,Chloroform-d)δ8.11(s,1H),7.91(d,J=2.2Hz,1H),7.85(s,1H),7.73(dd,J=9.0,2.2Hz,1H),7. 57(s,4H),3.52(d,J=2.1Hz,2H),3.28-3.11(m,2H),2.37-2.24(m,2H),2.23-2.16(m,1H),2.07-1.97(m,1H); 13 C NMR(101MHz,Chloroform-d)δ157.97,154.43,146.16,134.76,133.65,133.25,132.24,130.12 ,129.91,128.51,128.49,128.22,124.77,120.05,87.59,49.68,35.42,32.96,20.00; MS:calcd for C 21 H 16 B r2 N₂O[M+H] + :470.96,found:470.96.

[0094] Example 19: Preparation of the compound

[0095] Preparation of compound H18: 0.5 mmol of compound G18, (S,E)-2-(7-bromo-1'-hydroxy-1',2',3',4'-tetrahydroacridine)-1-[4-(trifluoromethyl)phenyl]ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H18 as (S)-7-bromo-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 94%.

[0096] (S)-7-bromo-3'-(4-(trifluoromethyl)phenyl)-3,4-dihydro-2H,4'H-spiro[acridin-e-1,5'-isoxazole](H18): White solid, yield 94%, mp191.9~293.7℃; 1HNMR(400MHz,Chloroform-d)δ8.12(s,1H),7.94-7.80(m,4H),7.78-7.67(m,3H),3.57(d,J=1.9Hz,2H),3.28- 3.16(m,2H),2.39-2.24(m,2H),2.22(ddd,J=13.6,6.4,2.9Hz,1H),2.05(dddd,J=15.1,12.3,10.1,6.8Hz,1H); 13 C NMR(101MHz,Chloroform-d)δ157.93,154.22,134.58,133.73,133.13(J C-F =21.9Hz),132.30,131.97,130.18,129.91,128.50,127.06,126.00(J=3.8Hz),120.12,87.99,49.56,35.46,32.98,19.99; MS:calcd forC 22 H 16 BrF3N2O[M+H] + :461.04,found:461.04.

[0097] Example 20: Preparation of the compound

[0098] Preparation of compound H19: 0.5 mmol of compound G19, (S,E)-2-(1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridine)-1-phenylethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, and compound H19 was obtained as (S)-3,3-dimethyl-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], which did not require purification. Yield: 86%, white solid.

[0099] (S)-3,3-dimethyl-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H19): White solid, yield 86%, mp172.6~174.4℃; 1H NMR(400MHz,Chloroform-d)δ8.20(s,1H),8.02(d,J=8.5Hz,1H),7.76-7.65(m,5H),7.50-7.40(m,4H),3.72 -3.59(m,2H),3.03(d,J=5.9Hz,2H),2.36(dd,J=14.1,0.9Hz,1H),2.09(dd,J=14.0,1.0Hz,1H),1.17(s,6H); 13 C NMR(101MHz,Chloroform-d)δ157.18,155.41,147.77,134.90,133.49,130.35,130.19,129.69,128.9 9,128.42,127.97,127.71,126.79,126.23,86.24,51.62,49.70,47.78,31.10,29.29,29.20; MS:calcd for C 23 H 22 N₂O[M+H] + :343.17,found:343.17.

[0100] Example 21: Preparation of the compound

[0101] Preparation of compound H20: 0.5 mmol of compound G20, (S,E)-2-(1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridin)-1-(p-tolyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, and compound H20 was obtained as (S)-3,3-dimethyl-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole], which did not require purification. Yield: 93%, white solid.

[0102] (S)-3,3-dimethyl-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H20): White solid, yield 93%, mp173.4~175.8℃; 1H NMR(400MHz,Chloroform-d)δ8.21(s,1H),8.04(d,J=8.5Hz,1H),7.77-7.60(m,4H),7.47(t,J=7.8Hz,1H),7.29-7.22(m ,2H),3.64(d,J=3.2Hz,2H),3.12-2.96(m,2H),2.41(s,3H),2.36(d,J=14.0Hz,1H),2.09(d,J=14.0Hz,1H),1.18(s,6H); 13 C NMR(101MHz,Chloroform-d)δ157.04,155.42,140.72,135.53,133.76,130.54,129.71,128.0 0,127.75,126.79,126.76,126.49,86.32,51.77,49.62,31.09,29.29,29.17,21.63;MS:calcd for C 24 H 24 N₂O[M+H] + :357.19,found:357.19.

[0103] Example 22: Preparation of the compound

[0104] Preparation of compound H21: 0.5 mmol of compound G21, (S,E)-2-(1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridine)-1-(4-methoxyphenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H21 as (S)-3'-(4-methoxyphenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 87%.

[0105] (S)-3'-(4-methoxyphenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H21): White solid, yield 87%, mp171.2~173.0℃; 1HNMR(400MHz,Chloroform-d)δ8.21(s,1H),8.02(d,J=8.5Hz,1H),7.74(dd,J=8.2,1.4Hz,1H),7.72-7.64(m,3H),7.46(ddd,J=8.1,6.9,1.2Hz, 1H),7.01-6.92(m,2H),3.86(s,3H),3.63(d,J=3.6Hz,2H),3.10-2.95(m ,2H),2.35(d,J=14.0Hz,1H),2.09(dd,J=14.0,1.0Hz,1H),1.17(s,6H); 13 C NMR(101MHz,Chloroform-d)δ161.31,157.19,155.04,135.02,133.70,130.23,128.34,127.9 9,127.74,126.26,122.21,114.41,86.29,55.56,51.90,49.68,31.10,29.32,29.22; MS:calcd for C 24 H 24 N₂O₂[M+H] + :373.18,found:373.18.

[0106] Example 23: Preparation of the compound

[0107] Preparation of compound H22: 0.5 mmol of compound G22, (S,E)-1-(4-fluorophenyl)-2-(1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridine)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H22 as (S)-3'-(4-fluorophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 94%.

[0108] (S)-3'-(4-fluorophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H22): White solid, yield 94%, mp162.4~165.0℃; 1HNMR(400MHz,Chloroform-d)δ8.19(s,1H),8.03(d,J=8.5Hz,1H),7.78-7.64(m,4H),7.47(ddd,J=8.1,6.9,1.2Hz,1H),7.19 -7.07(m,2H),3.72-3.55(m,2H),3.11-2.95(m,2H),2.36(dd,J=14.1,0.8Hz,1H),2.09(dd,J=14.0,1.0Hz,1H),1.18(s,6H); 13 CNMR(101MHz,Chloroform-d)δ165.19,162.70,157.16,154.46,147.79,134.86,133.32,130.25,128.73(J C-F =8.5Hz),128.45,127.95,127.68,126.27,125.96(J C-F =3.5Hz), 116.16(J) C-F =22.0Hz),86.89,51.64,49.68,47.76,31.10,29.28,29.21; MS:calcd for C 23 H 21 FN2O[M+H] + :361.16,found:361.16.

[0109] Example 24: Preparation of the compound

[0110] Preparation of compound H23: 0.5 mmol of compound G23, (S,E)-1-(4-bromophenyl)-2-(1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridine)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H23 as (S)-3'-(4-bromophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 91%.

[0111] (S)-3'-(4-bromophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H23): White solid, yield 91%, mp183.6~186.2℃; 1HNMR(400MHz,Chloroform-d)δ8.17(s,1H),8.01(d,J=8.5Hz,1H),7.72(s,0H),7.71-7.64(m,1H),7.63-7.54(m,4H),7.46(d dd,J=8.1,6.8,1.2Hz,1H),3.70-3.54(m,2H),3.11-2.94(m,2H),2.35(d,J=14.0Hz,1H),2.08(d,J=14.0Hz,1H),1.17(s,6H); 13 C NMR(101MHz,Chloroform-d)δ157.14,154.57,147.93,134.68,133.16,131.78,130.17,128.66,12 8.54,128.20,127.93,127.64,126.22,124.57,87.16,51.29,49.68,47.82,31.07,29.24; MS:calcd for C 23 H 21 BrN2O[M+H] + :421.08,found:421.08.

[0112] Example 25: Preparation of the compound

[0113] Preparation of compound H24: 0.5 mmol of compound G24, (S,E)-2-(1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridin)-1-[4-(trifluoromethyl)phenyl]ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H24 as (S)-3,3-dimethyl-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole], yield: 96%.

[0114] (S)-3,3-dimethyl-3'-(4-(trifluoromethyl)phenyl)-3,4-dihydro-2H,4'H-spiro[acri-dine-1,5'-isoxazole](H24): White solid, yield 96%, mp183.6~185.5℃; 1H NMR(400MHz,Chloroform-d)δ8.18(s,1H),8.05(d,J=8.5Hz,1H),7.85(d,J=8.1Hz,2H),7.78-7.66(m,4H),7.48(t,J =7.0Hz,1H),3.74-3.58(m,2H),3.14-2.98(m,2H),2.38(d,J=14.0Hz,1H),2.11(dd,J=14.0,1.0Hz,1H),1.19(s,6H); 13 C NMR(101MHz,Chloroform-d)δ157.08,154.39,134.99,133.10(J C-F =8.5Hz),132.18,131.85,130.45,128.40,127.96,127.66,127.04,126.42,125.99(J C-F =3.8Hz),125.30,87.54,51.20,49.71,31.13,29.23,29.21; MS:calcd for C 24 H 21 F3N2O[M+H] + :411.16,found:411.16.

[0115] Example 26: Preparation of the compound

[0116] Preparation of compound H25: 0.5 mmol of compound G25, (S,E)-2-(7-fluoro-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridin)-1-phenylethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H25 as (S)-7-fluoro-3,3-dimethyl-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole], yield: 93%.

[0117] (S)-7-fluoro-3,3-dimethyl-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H25): White solid, yield 93%, mp147.2~149.6℃; 1HNMR(400MHz,Chloroform-d)δ8.17(s,1H),8.05(s,1H),7.78-7.69(m,2H),7.51-7.42(m,4H),7.36( dd,J=8.8,2.8Hz,1H),3.66(s,2H),3.11-2.95(m,2H),2.36(d,J=14.0Hz,1H),1.18(d,J=2.2Hz,6H); 13 C NMR(101MHz,Chloroform-d)δ161.55,159.08,156.50,155.44,134.48,130.47,129.56,129.04,128.27(J C-F =10.1Hz), 126.83, 110.81 (J) C-F =21.7Hz),86.50,51.64,49.59,31.08,29.32,29.20; MS:calcd for C 23 H 21 FN2O[M+H] + :361.16,found:361.16.

[0118] Example 27: Preparation of the compound

[0119] Preparation of compound H26: 0.5 mmol of compound G26, (S,E)-2-(7-fluoro-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridin)-1-(p-tolyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H26 as (S)-7-fluoro-3,3-dimethyl-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole], yield: 87%.

[0120] (S)-7-fluoro-3,3-dimethyl-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H26): White solid, yield 87%, mp152.4~154.8℃; 1HNMR(400MHz,Chloroform-d)δ8.14(s,1H),8.00(dd,J=9.3,5.3Hz,1H),7.62(d,J=8.2Hz,2H),7.44(ddd,J=9.3,8.3,2.8Hz,1H),7.34(dd,J=8.8,2 .8Hz,1H),7.26(d,2H),3.63(s,2H),3.00(d,J=5.6Hz,2H),2.41(s,3H),2 .35(d,J=14.0Hz,1H),2.09(dd,J=14.1,1.0Hz,1H),1.17(d,J=1.8Hz,6H); 13 C NMR(101MHz,Chloroform-d)δ161.85,159.26,156.32(J C-F =3.1Hz),155.46,140.89,134.89,129.75,128.39(J C-F =10.2Hz),126.79,126.58,110.93(J C-F =22.0Hz),86.01,51.77,49.45,31.06,29.29,29.15,21.64; MS:calcdfor C 24 H 23 FN2O[M+H] + :375.18,found:375.18.

[0121] Example 28: Preparation of the compound

[0122] Preparation of compound H27: 0.5 mmol of compound G27, (S,E)-2-(7-fluoro-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridine)-1-(4-methoxyphenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H27 as (S)-7-fluoro-3'-(4-methoxyphenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 94%.

[0123] (S)-7-fluoro-3'-(4-methoxyphenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acr-idine-1,5'-isoxazole](H27): White solid, yield 94%, mp146.9~148.5℃; 1H NMR(400MHz,Chloroform-d)δ8.18(s,1H),8.05(s,1H),7.71-7.62(m,2H),7.47(t,J=8.3Hz,1H),7.37(d,J=8.7Hz,1H),7.0 1-6.93(m,2H),3.87(s,3H),3.63(s,2H),3.04(s,2H),2.35(d,J=14.0Hz,1H),2.10(d,J=14.0Hz,1H),1.18(d,J=2.7Hz,6H); 13 C NMR(101MHz,Chloroform-d)δ161.50,159.41,156.15,155.12,135.18(J C-F =2.0Hz),128.42,121.80,114.49,114.27,111.05(J C-F =21.7Hz),85.63,55.58,51.94,49.35,31.03,29.84,29.23,29.17; MS:calcd for C 24 H 23 FN2O2[M+H] + :391.17,found:391.17.

[0124] Example 29: Preparation of the compound

[0125] Preparation of compound H28: 0.5 mmol of compound G28, (S,E)-2-(7-fluoro-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridin)-1-(4-fluorophenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H28 as (S)-7-fluoro-3'-(4-fluorophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole], yield: 96%.

[0126] (S)-7-fluoro-3'-(4-fluorophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acrid-ine-1,5'-isoxazole](H28): White solid, yield 96%, mp197.8~199.5℃; 1H NMR(400MHz, DMSO-d6)δ8.35(s,1H),7.99(dd,J=9.3,5.4Hz,1H),7.85-7.74(m,3H),7.63(td,J=8.9,2.9Hz,1H),7.39- 7.30(m,2H),3.85(d,J=17.8Hz,1H),3.76(d,J=17.8Hz,1H),2.92(s,2H),2.14(d,J=2.1Hz,2H),1.09(d,J=14.8Hz,6H); 13 C NMR(101MHz,DMSO-d6)δ164.27,161.81,160.44,158.01,156.61(J C-F =2.6Hz),154.84,144.33,134.28(J C-F =5.2Hz), 133.93, 130.70 (J) C-F =9.3Hz), 129.04(J) C-F =8.6Hz), 127.75 (J) C-F =10.6Hz), 126.11(J) C-F =3.3Hz), 119.98 (J) C-F =26.1Hz), 115.86(J) C-F =21.9Hz), 111.01(J C-F =21.8Hz),86.23,50.51,48.39,46.88,30.49,28.68,28.58; MS:calcd for C 23 H 20 F2N2O[M+H] + :379.15,found:379.15.

[0127] Example 30: Preparation of the compound

[0128] Preparation of compound H29: 0.5 mmol of compound G29, (S,E)-1-(4-bromophenyl)-2-(7-fluoro-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridine) ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H29 as (S)-3'-(4-bromophenyl)-7-fluoro-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 95%.

[0129] (S)-3'-(4-bromophenyl)-7-fluoro-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acrid-ine-1,5'-isoxazole](H29): White solid, yield 95%, mp191.2~193.8℃; 1 HNMR(400MHz,Chloroform-d)δ8.15(s,1H),8.08(s,1H),7.59(s,4H),7.48(td,J=8.6,2.7Hz,1H),7.37(dd,J=8.9,2 .7Hz,1H),3.62(s,2H),3.04(dd,J=5.8Hz,2H),2.35(d,J=14.0Hz,1H),2.09(d,J=14.1Hz,1H),1.17(d,J=1.1Hz,6H); 13 C NMR(101MHz,Chloroform-d)δ161.77,159.28,156.25,154.66,134.43,132.29,128.31(J C-F =12.0Hz), 124.83, 110.92 (J) C-F =21.7Hz),86.69,51.35,49.45,31.05,29.23,29.16; MS:calcd forC 23 H 20 BrFN2O[M+H] + :439.07,found:439.07.

[0130] Example 31: Preparation of the compound

[0131] Preparation of compound H30: 0.5 mmol of compound G30, (S,E)-2-(7-fluoro-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridin)-1-[4-(trifluoromethyl)phenyl]ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H30 as (S)-7-fluoro-3,3-dimethyl-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole], yield: 86%.

[0132] (S)-7-fluoro-3,3-dimethyl-3'-(4-(trifluoromethyl)phenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H30): White solid, yield 86%, mp167.3~169.7℃; 1 HNMR(400MHz,Chloroform-d)δ8.14(s,1H),8.07(s,1H),7.85(d,J=8.1Hz,2H),7.72(d,J=8.2Hz,2H),7.49(t,J=8.7Hz, 1H),7.41-7.33(m,1H),3.66(s,2H),3.05(s,2H),2.37(d,J=14.0Hz,1H),2.11(d,J=14.1Hz,1H),1.19(d,J=2.3Hz,6H); 13 C NMR(101MHz,Chloroform-d)δ157.67,154.38,146.53,134.04,133.64(J C-F =7.0Hz),132.99,132.55,132.23,131.90,131.58,130.33,129.88,128.74,127.03,125.99(J C-F =3.8Hz),125.27,122.56,120.06,87.35,51.17,47.76,31.07,29.25,29.20; MS:calcd forC 24 H 20 F4N2O[M+H] + :429.15,found:429.15.

[0133] Example 32: Preparation of the compound

[0134] Preparation of compound H31: 0.5 mmol of compound G31, (S,E)-2-(7-bromo-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridine)-1-phenylethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H31 as (S)-7-bromo-3,3-dimethyl-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 94%.

[0135] (S)-7-bromo-3,3-dimethyl-3'-phenyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H31): White solid, yield 94%, mp174.2~176.0℃; 1 H NMR(400MHz,Chloroform-d)δ8.12(s,1H),7.91(d,J=2.2Hz,2H),7.74(ddd,J=7.1,5.3,2.8Hz,3H),7.46(p,J=3 .5Hz,3H),3.64(s,2H),3.10-2.94(m,2H),2.36(d,J=14.0Hz,1H),2.10(d,J=14.1Hz,1H),1.17(d,J=1.8Hz,6H); 13 C NMR(101MHz,Chloroform-d)δ157.53,155.47,134.77,134.05,130.53,129.96,12 9.44,129.05,128.84,126.83,86.31,51.65,49.46,31.06,29.29,29.19; MS:calcd for C 23 H 21 BrN2O[M+H] + :421.08,found:421.08.

[0136] Example 33: Preparation of the compound

[0137] Preparation of compound H32: 0.5 mmol of compound G32, (S,E)-2-(7-bromo-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridin)-1-(p-tolyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H32 as (S)-7-bromo-3,3-dimethyl-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole], yield: 96%.

[0138] (S)-7-bromo-3,3-dimethyl-3'-(p-tolyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H32): White solid, yield 96%, mp167.8~169.1℃; 1HNMR(400MHz,Chloroform-d)δ8.12(s,1H),7.90(d,J=2.3Hz,1H),7.75(s,0H),7.65-7.58(m,1H),7.25(d,J =6.5Hz,2H),3.62(s,1H),3.01(d,J=6.0Hz,1H),2.41(s,1H),2.09(d,J=14.0Hz,1H),1.17(d,J=1.4Hz,3H); 13 C NMR(101MHz,Chloroform-d)δ157.72,155.44,140.78,134.71,133.87,133.64,130.06,129.92,12 9.72,128.84,126.76,126.67,120.05,86.21,51.74,49.50,31.05,29.34,29.20,21.63; MS:calcd for C 24 H 23 BrN2O[M+H] + :435.10,found:435.10.

[0139] Example 34: Preparation of Compounds

[0140] Preparation of compound H33: 0.5 mmol of compound G33, (S,E)-2-(7-bromo-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridine)-1-(4-methoxyphenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H33 as (S)-7-bromo-3'-(4-methoxyphenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 88%.

[0141] (S)-7-bromo-3'-(4-methoxyphenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acr-idine-1,5'-isoxazole](H33): White solid, yield 88%, mp172.4~175.0℃; 1H NMR(400MHz,Chloroform-d)δ8.12(s,1H),7.89(t,J=2.7Hz,1H),7.73(dd,J=9.0,2.1Hz,1H),7.68-7.62(m,2H),6.99-6.92( m,2H),3.86(s,3H),3.60(s,2H),3.00(d,J=6.1Hz,2H),2.34(d,J=14.0Hz,1H),2.08(d,J=14.1Hz,1H),1.16(d,J=2.5Hz,6H); 13 CNMR(101MHz,Chloroform-d)δ161.38,157.72,155.07,134.78,133.93,133.65,130.01,129.92,1 28.85,128.35,122.02,120.07,114.43,86.50,55.56,51.86,49.49,31.01,29.33,29.23; MS:calcd for C 24 H 23 BrN2O2[M+H] + :451.09,found:451.09.

[0142] Example 35: Preparation of the compound

[0143] Preparation of compound H34: 0.5 mmol of compound G34, (S,E)-2-(7-bromo-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridine)-1-(4-fluorophenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H34 as (S)-7-bromo-3'-(4-fluorophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 89%.

[0144] (S)-7-bromo-3'-(4-fluorophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acrid-ine-1,5'-isoxazole]](H34): White solid, yield 89%, mp188.6~190.2℃; 1HNMR(400MHz, DMSO-d6)δ8.36(s,1H),8.27(d,J=2.2Hz,1H),7.87(d,J=9.0Hz,1H),7.84-7.76(m,3H),7.39-7.30( m,2H),3.84(d,J=17.7Hz,1H),3.75(d,J=17.8Hz,1H),2.92(s,2H),2.14(d,J=2.7Hz,2H),1.08(d,J=13.9Hz,6H); 13 CNMR(101MHz,DMSO-d6)δ164.26,161.80,157.86,154.85,145.64,134.09,133.97,132.86,130.12(J C-F =2.7Hz), 129.04(J) C-F =8.5Hz), 128.50, 126.11 (J) C-F =3.1Hz), 118.66, 115.84 (J) C-F =21.9Hz),86.18,50.51,48.32,46.99,30.47,28.66,28.61; MS:calcd for C 23 H 20 BrFN2O[M+H] + :439.07,found:439.07.

[0145] Example 36: Preparation of the compound

[0146] Preparation of compound H35: 0.5 mmol of compound G35, (S,E)-2-(7-bromo-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridine)-1-(4-bromophenyl)ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H35 as (S)-7-bromo-3'-(4-bromophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole], yield: 92%.

[0147] (S)-7-bromo-3'-(4-bromophenyl)-3,3-dimethyl-3,4-dihydro-2H,4'H-spiro[acrid-ine-1,5'-isoxazole](H35): White solid, yield 92%, mp139.8~142.1℃; 1HNMR (400MHz, DMSO-d6) δ8.36 (s, 1H), 8.27 (d, J = 2.2Hz, 1H), 7.90-7.78 (m, 2H), 7.70 (s, 4H), 3.83 (d ,J=17.8Hz,1H),3.75(d,J=17.8Hz,1H),2.92(s,2H),2.15(d,J=2.2Hz,2H),1.08(d,J=12.6Hz,6H); 13 C NMR (101MHz, DMSO-d6) δ157.84,155.06,145.66,133.99,132.88,131.78,130.14,130.11,128. 74,128.69,128.49,123.34,118.66,86.46,50.17,48.27,46.98,30.47,28.64,28.58; MS:calcd for C 23 H 20 Br2N2O[M+H] + :498.99,found:498.99.

[0148] Example 37: Preparation of the compound

[0149] Preparation of compound H36: 0.5 mmol of compound G36, (S,E)-2-(7-bromo-1'-hydroxy-3,3-dimethyl-1',2',3',4'-tetrahydroacridin)-1-[4-(trifluoromethyl)phenyl]ethyl-1-one oxime, was added to a 50 mL round-bottom flask. The rest of the preparation method was the same, yielding compound H36 as (S)-7-bromo-3,3-dimethyl-3'-[4-(trifluoromethyl)phenyl]-3,4-dihydro-2H,4'H-spiro[acridin-1,5'-isoxazole], yield: 93%.

[0150] (S)-7-bromo-3,3-dimethyl-3'-(4-(trifluoromethyl)phenyl)-3,4-dihydro-2H,4'H-spiro[acridine-1,5'-isoxazole](H36): White solid, yield 93%, mp147.2~149.1℃; 1HNMR(400MHz,Chloroform-d)δ8.06(s,1H),7.92-7.80(m,4H),7.77-7.66(m,3H),3.64(s,2 H),3.09-2.92(m,2H),2.36(d,J=14.1Hz,1H),2.10(d,J=14.0Hz,1H),1.17(d,J=2.0Hz,6H); 13 C NMR(101MHz,DMSO-d6)δ157.81,155.01,145.70,134.08,133.89,132.93,130.14(J C-F =2.7Hz),128.50,127.48,125.69(J C-F =3.5Hz),118.68,86.90,50.01,48.26,46.96,30.50,28.63,28.53; MS:calcd for C 24 H 20 BrF3N2O[M+H] + :489.07,found:489.07.

[0151] Example 38: Determination of Antibacterial Activity

[0152] Screening and determination of the antibacterial activity of the acridine spiroisoxazole compounds described in this invention:

[0153] Minimum inhibitory concentration (MIC) determination:

[0154] Experimental reagents, materials, and instruments: Pathogens: Botrytis cinerea (gray mold), Ampelomyces humuli (Fautrey) Rudakov (powdery mildew), Rhizoctonia solani (root rot), Gibberella zeae (grass spores), and Candida albicans (spore suspension, concentration 1×10⁻⁶). 5 CFU / mL). Culture medium: liquid culture medium (e.g., PDB, YM), compound solution: serial dilution (e.g., 100 μg / mL), solvent is DMSO (final concentration ≤0.1%) and sterile water, experimental equipment: 96-well plate, pipette, microplate reader, clean bench.

[0155] Experimental Procedure: Compound Dilution: In a 96-well plate, perform serial 2-fold dilutions of the compound using culture medium (columns 1 to 11, column 12 is the control without the compound). Example: Add 200 μL of 100 μg / mL compound to well 1, add 100 μL of culture medium + 100 μL of the solution from well 1 to well 2 (after mixing, the concentration is 50 μg / mL), and so on. Inoculation: Add 10 μL of bacterial suspension to each well (final concentration 5 × 10⁻⁶). 4 CFU / mL). Culture and observation: Static culture was performed at 28℃, with specific times varying depending on the strain: 24 hours for *Candida albicans*, 48 hours for *Botrytis cinerea* and *Rhizoctonia solani*, and 72 hours for other strains. Optical density (OD) was measured using a BioTek Synergy H1 microplate reader. 600 Turbidity method: Observe the turbidity inside the well with the naked eye; no turbidity indicates complete inhibition.

[0156] Formula 1 for calculating antibacterial rate:

[0157]

[0158] Where C and T represent the OD of the blank control and the test well, respectively. 600 The EC value was obtained through nonlinear regression analysis (GraphPadPrism). 50 The values ​​are expressed as mean ± SD (n = 3 independent replicates).

[0159] Dose-response curve: EC50 is calculated by fitting a logistic curve with the logarithm of compound concentration on the x-axis and the inhibition rate on the y-axis. 50 (Concentration that inhibits 50% of bacterial growth).

[0160] The inhibitory activity data of compounds H1-H36 are shown in Table 1. Compounds with optimal activity against each fungus were screened from the H1-H36 series. After activity screening, three candidate compounds (H35, H17, H27, and H10) were finally selected for dose-response characterization, and their EC50 values ​​were calculated. 50 Value, EC 50 The values ​​are shown in Table 2. Through comprehensive evaluation of antifungal properties across taxa, H35 was identified as a broad-spectrum candidate compound. H35's EC50 values ​​are shown in Table 2. 50 The concentration (5.0 ± 0.4 μg / mL) showed 1.5-fold, 2.2-fold, and 3.2-fold antibacterial effects compared to the commercial control agents azoxystrobin (7.6 μg / mL), chlorothalonil (11.1 μg / mL), and carbendazim (15.9 μg / mL), respectively. This enhanced bioactivity is closely related to the spirocyclic structure of H35, which exhibits higher receptor binding affinity and a broader target spectrum. These mechanistic advantages indicate that the spirocyclic structure possesses significant antibacterial activity.

[0161] Table 1: In vitro antifungal activity of H1-H36 against five fungi at a concentration of 100 μg / ml

[0162]

[0163] Table 2: 50% effective concentration (EC50) of compounds against plant pathogenic fungi 50 )value

[0164]

[0165]

[0166] Example 39: Mycelial Growth Rate Method (Botrytis cinerea)

[0167] Materials and Reagents: Culture Medium: Potato Dextrose Agar (PDA) plates (sterilized and then inverted). Reagents and Solutions: Test Compound: H35 (dissolved in 0.1% DMSO, concentration preset to 25 μg / mL). Positive Control: Carbendazim (25 μg / mL, dissolved in sterile water), Solvent Control: 0.1% DMSO. Consumables and Tools: Inoculation Tool: 5 mm diameter punch (for preparing mycelial blocks). Petri Dishes: 90 mm diameter sterile petri dishes. Measuring Tools: Ruler or Vernier calipers (accurate to 0.1 mm). Incubator: 28℃ dark incubator.

[0168] Mycelial growth inhibition assays were performed on PDA plates inoculated with *Botrytis cinerea* mycelial blocks (5 mm in diameter). Compound H35 and carbendazim served as negative and positive controls, respectively. All plates were incubated at 28°C in the dark for 7 days, with colony diameter measured daily to calculate growth rate. The inhibition rate was determined by comparing the treatment group with the solvent control (0.1% DMSO), and the antibacterial effect is shown in [the table below]. Figure 1 The antifungal effect of compound H35 against *Botrytis cinerea* was evaluated using a potato dextrose agar (PDA) plate test after incubation at 28°C for 7 days, with carbendazim as a positive control. Figure 1 H35 exhibited concentration- and time-dependent inhibitory effects on fungal proliferation, with significantly higher activity than carbendazim. At 25 μg / mL, H35 achieved 93.41% ± 1.4% inhibition of mycelial growth, equivalent to twice the potency of carbendazim (46.90% ± 2.6%) at the same concentration.

[0169] Formula 2

[0170]

[0171] Wherein, C and T represent the average colony diameter (mm) of the control group and treatment group, respectively. All experiments were performed in triplicate (n=3) to ensure data reliability.

[0172] Example 40: Assessment of the damage of compound H35 to fungal cell membranes by SYTO-9 / PI staining

[0173] Biofilm culture system: The culture medium is PDB (used for biofilm formation and treatment). The bacterial suspension is Botrytis cinerea suspension (1×10⁻⁶). 6 (CFU / mL, prepared with 0.85% physiological saline). The staining reagent was the G1521 Viability Kit containing SYTO-9 (green fluorescence, for live cells) and PI (red fluorescence, for dead cells). The washing buffer was PBS buffer (pH 7.4, sterile). The diluent was 0.85% NaCl solution (used to prepare the working staining solution).

[0174] Consumables and Instruments: 96-well sterile microplates (for biofilm adhesion and treatment). Fluorescence Microscope: Inverted fluorescence microscope (with 488nm excitation filter for SYTO-9; 535nm excitation filter for PI). Pipettes and Tips: 200μL and 1000μL pipettes (sterile). Shaker: for gently washing biofilms (avoiding mechanical damage). ImageJ Software: for fluorescence image analysis (calculating the live / dead cell area ratio).

[0175] The in vitro antifungal activity and cell membrane damage of H35 and carbendazim against *Botrytis cinerea* were determined using fluorescence staining. The antifungal activity of compound H35 against *Botrytis cinerea* biofilms was evaluated using a 96-well microplate model.

[0176] fungal suspension (1×10) 6 Incubation with H35 (50 / 100 μg / mL) at 28°C for 2 hours to initiate biofilm adhesion, followed by washing with PBS to remove planktonic cells. Biofilms were treated in PDB with H35 (50 / 100 μg / mL) or carbendazim (positive control) for 48 hours. After PBS rinsing, double staining was performed using the G1521 Viability Kit (SYTO-9 / PI = 1:1, dissolved in 0.85% NaCl, incubated for 25 minutes), followed by imaging with a fluorescence microscope. Finally, samples were observed under a fluorescence inverted microscope. This experiment was conducted with three parallel samples and repeated three times to ensure data reliability and reproducibility. The degree of damage to the fungal cell membrane by compound H35 was determined by fluorescence staining. Figure 2 The effect of H35 on the induction rate of membrane damage by fluorescent staining is shown in Table 3, which further verifies its membrane destruction mechanism: H35 caused 83.0% and 48.5% membrane damage at concentrations of 100 μg / mL and 50 μg / mL, respectively, which was better than carbendazim (51.9% and 33.6% under the same conditions).

[0177] Formula 3

[0178]

[0179] Formula 4

[0180]

[0181] Among them, ImageJ was used to quantize the survival rate as SYTO-9. + / (SYTO-9 + +PI + The area ratio was determined by three biological and technical replicates per group to ensure statistical reliability (n = 9 for each group).

[0182] Table 3: Effects of H35 and carbendazim on the induction rate of film damage from gray mold fungus.

[0183]

[0184] Example 41: Antifungal activity of compound H35 against *Botrytis cinerea* in maize plants. Materials: Inoculated strain was *Botrytis cinerea*; plant was maize leaf with a wound diameter of 3 mm; inoculation amount was 10 μL of spore suspension (1×10⁻⁶). 5 (CFU / mL); the culture conditions were 28℃, 90% relative humidity, and 72 hours.

[0185] The protective and therapeutic activities of compound H35 on maize leaves, such as Figure 3 As shown, in vivo experiments demonstrated that H35 exhibited dual protective (80.00±1.7% at 100 μg / mL) and therapeutic (83.90±2.6% at 100 μg / mL) effects. At lower concentrations (e.g., at 50 μg / mL, the protective and therapeutic activities were 62.25±1.1% and 75.50±0.7%, respectively), it was superior to carbendazim. The data are shown in Table 4.

[0186] Antibacterial rate calculation:

[0187] Formula 5

[0188]

[0189] Among them, Disease Severity Treated Disease Severity: The severity of disease on the leaves of the treated plants. Control The severity of disease on the leaves of untreated (control group) plants.

[0190] Table 4: Protective and therapeutic effects of compound H35 in maize

[0191]

[0192] The above experimental results indicate that H35 exhibits significant broad-spectrum antifungal activity. Through microdilution tests, H35 showed effective EC50 activity against Botrytis cinerea. 50 The concentration of H35 was significantly better than that of carbendazim (15.9±1.2 μg / mL), at only 5.0±0.4 μg / mL. At a concentration of 25 μg / mL, H35 inhibited mycelial growth by 93.41±1.4%, far exceeding that of carbendazim (46.90±2.6%). Fluorescent staining further verified its membrane-damaging mechanism: H35 caused 83.0% and 48.5% membrane damage at 100 μg / mL and 50 μg / mL, respectively, which was superior to that of carbendazim (51.9% and 33.6% under the same conditions). In vivo experiments showed that H35 had a dual protective effect on maize plants (80.00±1.7% at 100 μg / mL) and a curative effect (83.90±2.6% at 100 μg / mL). At lower concentrations (e.g., 62.25±1.1% protection and 75.50±0.7% curative activity at 50 μg / mL), it was superior to carbendazim.

[0193] The above embodiments are only for illustrating the technical concept and features of the present invention in a specific scenario. Their purpose is to enable those who need this technology to understand the content of the present invention and implement it. They do not limit the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An acridine spiroisoxazole compound, characterized in that, The structure of the acridine spiroisoxazole compound is shown below: in: R1 is selected from any one of H, F, and Br; R2 is selected from either H or CH3; R3 is selected from any one of H, F, Br, Me, OMe, and CF3.

2. The acridine spiroisoxazole compound as described in claim 1, characterized in that, The acridine spiroisoxazole compound described herein has one of the following chemical formulas H1-H36: 。 3. The application of the acridine spiroxazole compound as described in claim 1 in inhibiting the activity of Botrytis cinerea, Powdery mildew, Rhizoctonia solani, Fusarium graminearum and Candida albicans.

4. The application of the acridine spiroisoxazole compound as described in claim 1 in inhibiting Staphylococcus aureus infection.

5. A pharmaceutical composition, characterized in that, It contains pharmaceutically acceptable excipients or carriers and acridine spiroisoxazole compounds as described in claim 1.

Images