1, 2-dioxetane derivative as well as preparation method and application thereof

Abstract

The invention provides a 1, 2-dioxetane derivative as well as a preparation method and application thereof, and belongs to the technical field of chemiluminescent substrates. The 1, 2-dioxetane derivative provided by the invention has high luminous efficiency and good stability. The result of the embodiment shows that the time required by the 1, 2-dioxetane derivative provided by the invention to reach the maximum luminescence peak value at 25 DEG C is only 5-30 minutes, and the continuous luminescence time can reach 70 minutes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemiluminescent substrate, in particular to a 1,2-dioxetane derivative and a preparation method and application thereof. BACKGROUND

[0002] Chemiluminescence technology is widely used in the fields of infectious diseases, heart diseases, tumors, pregnancy detection, etc. In the immune diagnosis market, chemiluminescence accounts for a large part of the market, especially in the medium and high throughput market. The AP platform (alkaline phosphatase platform) is currently the most widely used chemiluminescent detection platform, accounting for about 80%, and the main chemiluminescent substrates used in the market are AMPPD, APS-5 and CDP-Star, which have the following structures: However, these are early market products and all have certain defects. For example, APS-5 has poor stability, AMPPD has lower luminescence performance than CDP-Star, and the enhancer in CDP-Star is difficult to obtain, so the price is relatively high.

[0003]

[0004] If a new type of chemiluminescent substrate can be provided, it will be of great significance to promote the development of chemiluminescence detection. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a 1,2-dioxetane derivative and a preparation method and application thereof. The 1,2-dioxetane derivative provided by the present application has high luminescent efficiency and good stability.

[0006] The present application provides a 1,2-dioxetane derivative having the structure shown in formula I. The present application adds electron-withdrawing groups to the aromatic ring to enhance its stability while accelerating the alkaline phosphatase hydrolysis process. By optimizing the 1,2-dioxetane ortho (R position), introducing groups such as trifluoroethyl, hydroxymethyl cyclopropane, etc., the 1,2-dioxetane structure can be made more stable. By modifying the 5-position of adamantane ketone, the hydrophilic group can make the structure have better compatibility with other reagents in solution. The 1,2-dioxetane derivative provided by the present application has high luminescent efficiency and good stability. The results of the examples show that the 1,2-dioxetane derivative provided by the present application reaches the maximum luminescence peak value in 5-30 min at 25℃, and the continuous luminescence time can reach 70 min. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 The synthesis route of the 1,2-dioxetane derivative is shown in the following scheme:

[0008] Figure 2Results of the luminescent performance test for CL-1 to CL-6;

[0009] Figure 3 Results of the luminescent performance test for CL-7 to CL-12;

[0010] Figure 4 Results of the luminescent performance test for CL-13 to CL-18;

[0011] Figure 5 Results of the luminescent performance test for CL-19 to CL-24;

[0012] Figure 6 Results of the luminescent performance test for CL-25 to CL-30;

[0013] Figure 7 Results of the luminescent performance test for CL-31 to CL-36;

[0014] Figure 8 Results of the luminescent performance test for CL-37 to CL-39. DETAILED DESCRIPTION

[0015] The present application provides a 1,2-dioxetane derivative, having the structure shown in Formula I:

[0016]

[0017] In Formula I, R is C1-6 alkyl, In the present application, the C1-6 alkyl can be C1, C2, C3, C4, C5 or C6 alkyl, and is specifically preferably methyl or ethyl; m = 1-6, and is preferably 1, 2, 3, 4, 5 or 6; n = 1-6, and is preferably 1, 2, 3, 4, 5 or 6; x = 1-6, and is preferably 1, 2, 3, 4, 5 or 6; y = 1-6, and is preferably 1, 2, 3, 4, 5 or 6.

[0018] R1 is H, C1-6 alkyl, halogen, In the present application, the C1-6 alkyl can be C1, C2, C3, C4, C5 or C6 alkyl, and is specifically preferably methyl or ethyl; n = 1-6, and is preferably 1, 2, 3, 4, 5 or 6; x = 1-6, and is preferably 1, 2, 3, 4, 5 or 6; y = 1-6, and is preferably 1, 2, 3, 4, 5 or 6.

[0019] R2 is C1-6 alkyl, halogen, one of the following: in the present application, the C1-6 alkyl group can be C1, C2, C3, C4, C5 or C6 alkyl group, and is particularly preferably methyl or ethyl; m = 1-6, preferably 1, 2, 3, 4, 5 or 6; n = 1-6, preferably 1, 2, 3, 4, 5 or 6; x = 1-6, preferably 1, 2, 3, 4, 5 or 6; and y = 1-6, preferably 1, 2, 3, 4, 5 or 6.

[0020] in the present application, R is methyl, ethyl, one of the following:

[0021] R1 is H, Cl, one of the following:

[0022] R2 is methyl, ethyl, Cl, one of the following.

[0023] in the present application, R1 and R2 are not simultaneously halogen.

[0024] in the present application, the 1,2-dioxetane derivative preferably has a structure represented by any one of formulas CL-1 to CL-39:

[0025]

[0026]

[0027]

[0028] The present application provides a preparation method of the above-mentioned 1,2-dioxetane derivative, comprising the following steps:

[0029] a compound having a structure represented by formula a is subjected to nucleophilic substitution reaction with 3-hydroxypropionitrile and phosphorus oxychloride to obtain a compound having a structure represented by formula b;

[0030]

[0031] the compound having a structure represented by formula b is subjected to hydroxylation reaction with methanol and sodium methoxide to obtain a compound having a structure represented by formula c;

[0032]

[0033] the compound having a structure represented by formula c is mixed with sodium molybdate, H2O2 and an alcohol solvent to perform ring formation reaction to obtain a compound having a structure represented by formula I.

[0034] In the present application, the compound with the structure shown in formula a is subjected to nucleophilic substitution reaction with 3-hydroxypropionitrile and phosphorus oxychloride to obtain the compound with the structure shown in formula b. In the present application, the preparation method of the compound with the structure shown in formula a preferably comprises the following steps:

[0035] In the presence of boron trifluoride etherate, the compound with the structure shown in formula d is subjected to phosphorylation reaction with triethyl phosphite to obtain the compound with the structure shown in formula e.

[0036]

[0037] In the presence of n-butyllithium, the compound with the structure shown in formula e is subjected to olefination reaction with the compound with the structure shown in formula f to obtain the compound with the structure shown in formula g.

[0038]

[0039] The compound with the structure shown in formula g is subjected to hydroxylation reaction with sodium hydride and ethanethiol to obtain the compound with the structure shown in formula a.

[0040] In the present application, in the presence of boron trifluoride etherate, the compound with the structure shown in formula d is subjected to phosphorylation reaction with triethyl phosphite to obtain the compound with the structure shown in formula e. In the present application, the molar ratio of the compound with the structure shown in formula d to triethyl phosphite is preferably 1:1-1.5, more preferably 1:1-1.2; the molar ratio of triethyl phosphite to boron trifluoride etherate is preferably 1:1-1.5, more preferably 1:1-1.2. In the present application, the reaction solvent of the phosphorylation reaction is preferably dichloromethane; in the present application, the temperature of the phosphorylation reaction is preferably 0°C, and the time is preferably 2h.

[0041] After the phosphorylation reaction, the present application preferably performs post-treatment on the obtained phosphorylation reaction liquid, and the post-treatment preferably comprises: mixing the phosphorylation reaction liquid with saturated sodium bicarbonate solution, separating the organic phase, extracting the aqueous phase, and combining the organic phases; drying, filtering and concentrating the combined organic phase to obtain the pure compound with the structure shown in formula e. In the present application, the extractant used in the extraction is preferably dichloromethane; the drying is preferably anhydrous sodium sulfate drying, and the concentration is preferably rotary evaporation.

[0042] In the present application, the compound having the structure shown in formula e is subjected to olefination reaction with the compound having the structure shown in formula f in the presence of n-butyllithium to obtain the compound having the structure shown in formula g. In the present application, the molar ratio of the compound having the structure shown in formula e to the compound having the structure shown in formula f is 1:1-1.5, more preferably 1:1-1.2. In the present application, the reaction solvent of the olefination reaction is preferably tetrahydrofuran; the temperature of the olefination reaction is preferably -78℃- -60℃, and the time is preferably 1-3h, more preferably 2h.

[0043] After the olefination reaction, the present application preferably subjects the obtained olefination reaction liquid to post-treatment, which preferably comprises: mixing the olefination reaction liquid with saturated ammonium chloride solution, water and ethyl acetate, separating the organic phase, extracting the aqueous phase, and combining the organic phases; drying, filtering and concentrating the combined organic phase to obtain the pure compound having the structure shown in formula f. In the present application, the extractant used in the extraction is preferably ethyl acetate; the drying is preferably anhydrous sodium sulfate drying, and the concentration is preferably rotary evaporation.

[0044] In the present application, the compound having the structure shown in formula g is subjected to hydroxylation reaction with sodium hydride and ethanethiol to obtain the compound having the structure shown in formula a. In the present application, the molar ratio of the compound having the structure shown in formula g to sodium hydride and ethanethiol is preferably 1:(1-1.5):(1-4), more preferably 1:1:1. In the present application, the reaction solvent of the hydroxylation reaction is preferably tetrahydrofuran; the temperature of the hydroxylation reaction is 80℃, and the time is preferably 10h.

[0045] After the hydroxylation reaction, the present application preferably subjects the obtained hydroxylation reaction liquid to post-treatment, which preferably comprises: mixing the hydroxylation reaction liquid with water and ethyl acetate, separating the organic phase, extracting the aqueous phase, and combining the organic phases; drying, filtering and concentrating the combined organic phase to obtain the pure compound having the structure shown in formula a. In the present application, the extractant used in the extraction is preferably ethyl acetate; the drying is preferably anhydrous sodium sulfate drying, and the concentration is preferably rotary evaporation.

[0046] In the present application, the compound having the structure shown in formula a is subjected to nucleophilic substitution reaction with 3-hydroxypropionitrile and phosphorus oxychloride to obtain the compound having the structure shown in formula b. In the present application, the molar ratio of the compound having the structure shown in formula a to phosphorus oxychloride and 3-hydroxypropionitrile is preferably 1:1:(3-5), more preferably 1:1:5. In the present application, the nucleophilic substitution reaction is preferably carried out in the presence of pyridine; in the present application, the reaction solvent of the nucleophilic substitution reaction is preferably tetrahydrofuran; the temperature of the nucleophilic substitution reaction is preferably room temperature, and the time is preferably 24h.

[0047] After the nucleophilic substitution reaction, the present application preferably performs post-treatment on the obtained nucleophilic substitution reaction solution, and the post-treatment preferably includes: evaporating the nucleophilic substitution reaction solution to dryness, and performing column chromatography for purification.

[0048] In the present application, the compound having the structure shown in formula b is subjected to a hydroxylation reaction with methanol and sodium methoxide to obtain a compound having the structure shown in formula c. In the present application, the molar ratio of the compound having the structure shown in formula b to sodium methoxide is preferably 1:2-3. In the present application, the temperature of the hydroxylation reaction is preferably room temperature, and the time is preferably 1-3 h, more preferably 1-2 h.

[0049] After the hydroxylation reaction, the present application preferably performs post-treatment on the obtained hydroxylation reaction solution, and the post-treatment preferably includes: evaporating the hydroxylation reaction solution to dryness, adding ethyl acetate, and performing filtration to obtain a pure compound having the structure shown in formula c.

[0050] In the present application, the compound having the structure shown in formula c is mixed with sodium molybdate, H2O2, and an alcohol solvent to perform a ring formation reaction to obtain a compound having the structure shown in formula I. In the present application, the molar ratio of the compound having the structure shown in formula c to sodium molybdate is preferably 1:2. In the present application, the alcohol solvent is preferably methanol. In the present application, the volume ratio of H2O2 to the alcohol solvent is preferably 1:5-10. In the present application, the temperature of the ring formation reaction is preferably room temperature, and the time is preferably 12-24 h, more preferably 12-18 h.

[0051] After the ring formation reaction, the present application preferably performs post-treatment on the obtained ring formation reaction solution, and the post-treatment preferably includes: cooling the ring formation reaction solution to 0°C, adding a 10% sodium thiosulfate solution, performing first filtration, spinning dry the filtrate, adding methanol to reflux, performing second filtration, and drying the obtained solid to obtain a pure compound having the structure shown in formula I.

[0052] As a specific embodiment of the present application, the synthesis route of the 1,2-dioxetane derivative is shown in Figure 1 .

[0053] The present application provides the use of the above-mentioned 1,2-dioxetane derivative as a chemiluminescent substrate.

[0054] The 1,2-dioxetane derivative, the preparation method and the use thereof provided by the present application will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0055] Example 1 Synthesis of CL-2

[0056]

[0057] (1) Vanillin (15.2 g 0.1 mol) was added to a 500 mL reaction flask, 2-methoxyethyl p-toluenesulfonate (23.0 g 0.1 mol), cesium carbonate (48.8 g 0.15 mol), DMF 200 mL, the system was heated to 50 °C for 6 hours, TLC monitoring, the reaction was completed, the system was cooled to room temperature, added to 300 mL water, the mixture was extracted with ethyl acetate 3 times 100 mL each time, the combined organic phase was washed with saturated sodium chloride solution 3 times 100 mL each time, the organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to give a crude product, which was purified by column chromatography to give intermediate 1 (11.3 g, 54%). ESI-MS m / z: 211 [M+H].

[0058] (2) Intermediate 1 (11 g, 0.052 mol) was added to a reaction flask, 20 mL of methanol, trimethyl orthoformate (11.1 g, 0.14 mol), p-toluenesulfonic acid (0.9 g, 5.2 mmol), the system was reacted at room temperature for 24 hours, TLC monitoring, the reaction was completed, 2 mL of triethylamine was added to the system, the system was rotary evaporated, and after drying, 50 mL of saturated sodium bicarbonate solution, 100 mL of water, 100 mL of ethyl acetate were added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate 2 times 100 mL each time, the combined organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to give a yellow oil intermediate 2 (13.2 g, used directly in the next step).

[0059] (3) Intermediate 2 (13 g, 0.05 mol) was added to a reaction flask, 150 mL of dichloromethane, the system was cooled to 0 °C, then triethyl phosphite (7.45 g, 0.06 mol) was slowly added, and then boron trifluoride ether (8.46 g, 0.06 mol) was added dropwise, after the dropwise addition was completed, the system was kept at 0 °C for 2 hours, TLC monitoring, the reaction was completed, the system was warmed to room temperature, 100 mL of saturated sodium bicarbonate solution was added, the organic phase was separated, the aqueous phase was extracted with dichloromethane 2 times 100 mL each time, the combined organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to give a yellow oil intermediate 3 (14.2 g, used directly in the next step).

[0060] (4) Intermediate 3 (14 g, 0.04 mol) was added to a reaction flask, 100 mL of anhydrous tetrahydrofuran was added, the system was cooled to -78°C under nitrogen protection, then n-butyllithium (18 mL, 2.5 M / L) was added dropwise, after the dropwise addition was completed, the system was kept at -78°C for 1 hour, a solution of 5-chloro-2-adamantanone (7.36 g, 0.04 mol) in 50 mL of tetrahydrofuran was added dropwise, after the dropwise addition was completed, the system was kept at -78°C for 1 hour, TLC was used for monitoring, after the reaction was completed, the system was warmed to 0°C, 50 mL of saturated ammonium chloride solution was added, after stirring for 5 minutes, it was added to 100 mL of water, 100 mL of ethyl acetate was added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate twice, 100 mL each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product 15.7 g, which was purified by column chromatography to obtain intermediate 4 (12.3 g, 75%) ESI-MS m / z: 393 [M+H].

[0061] (5) Anhydrous DMF was added to a reaction flask, the temperature was reduced to 0°C under nitrogen protection, sodium hydride (1.44 g, 0.037 mol) was slowly added, after the addition was completed, the system was reacted at room temperature for 30 minutes, the system was warmed to 60°C, ethanethiol (1.94 g, 0.031 mol) was slowly added dropwise, after the dropwise addition was completed, the system was kept at 60°C for 30 minutes, a solution of intermediate 4 (12.3 g, 0.031 mol) in DMF was added, the system was warmed to 80°C and reacted for 10 hours, TLC was used for monitoring, after the reaction was completed, the system was cooled to room temperature, added to 100 mL of water, 100 mL of ethyl acetate was added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate twice, 100 mL each time, the organic phases were combined, the organic phase was washed with saturated sodium chloride solution three times, 100 mL each time, the organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product, which was purified by column chromatography to obtain intermediate 5 (5.9 g, 50%) ESI-MS m / z: 379 [M+H].

[0062] (6) Intermediate 5 (5.5 g, 0.015 mol) was added to a reaction flask, 100 mL of tetrahydrofuran and 20 mL of pyridine were added, the system was cooled to 0°C, phosphorus oxychloride (2.23 g, 0.015 mol) was added dropwise, the system was reacted at room temperature for 1 hour, 3-hydroxypropionitrile (5.33 g, 0.075 mol) was added, after the addition was completed, the system was kept at room temperature and reacted for 24 hours, TLC was used for monitoring, after the reaction was completed, the system was evaporated, and column chromatography was used for purification to obtain intermediate 6 (5.2 g, 63%) ESI-MS m / z: 379 [M+H].

[0063] (7) Intermediate 6 (5.0 g, 8.85 mmol) was added to a reaction flask, 100 mL of methanol was added, sodium methoxide (1 g, 17.7 mmol) was added, and the system was reacted at room temperature for 1 hour. TLC monitoring showed that the reaction was complete. The system was evaporated to dryness, 50 mL of ethyl acetate was added, the system was filtered, and light yellow solid intermediate 7 (4.0 g, 84%) was obtained. ESI-MS m / z: 459 [M+H].

[0064] (8) Intermediate 7 (2 g, 4.36 mmol) was added to a reaction flask, sodium molybdate (1.79 g, 8.72 mmol) H2O2 (10 mL), 50 mL of methanol was added, the system was reacted at room temperature for 24 hours, TLC monitoring showed that the reaction was complete. The system was cooled to 0°C, 10% sodium thiosulfate solution (5 mL) was added, the system was stirred at 0°C for 5 hours, filtered, the filtrate was evaporated to dryness, 20 mL of methanol was added, refluxed for 1 hour, hot filtered, and the solid was dried to obtain CL-2 (0.7 g, 33%). ESI-MS m / z: 491 [M+H], 489 [M-H].

[0065] Synthesis of CL-4

[0066]

[0067] The difference from Example 1 is that 2-methoxyethyl-p-toluenesulfonic acid is used instead of 2-methoxyethyl-p-toluenesulfonic acid in step (1) to obtain CL-4. Yield: 37%, ESI-MS m / z: 535 [M+H], 533 [M-H].

[0068] Synthesis of CL-6

[0069]

[0070] The difference from Example 1 is that 3-(2-methoxy)ethyl-p-toluenesulfonic acid is used instead of 2-methoxyethyl-p-toluenesulfonic acid in step (1) to obtain CL-6. Yield: 37%, ESI-MS m / z: 579 [M+H], 577 [M-H].

[0071] Synthesis of CL-8

[0072]

[0073] The difference from Example 1 is that 4-(2-methoxy)ethyl-p-toluenesulfonic acid is used instead of 2-methoxyethyl-p-toluenesulfonic acid in step (1) to obtain CL-8. Yield: 41%, ESI-MS m / z: 623 [M+H], 621 [M-H].

[0074] Synthesis of CL-1

[0075] The difference from Example 1 is that 5-chloro-2-adamantanone in step (4) is replaced by 2-adamantanone to give CL-1. Yield: 33%, ESI-MS m / z: 457 [M+H], 455 [M-H].

[0076] Synthesis of Example 6 CL-3

[0077]

[0078] The difference from Example 1 is that 2-methoxyethyl-p-toluenesulfonate in step (1) is replaced by 3-(2-methoxy)ethyl-p-toluenesulfonic acid, and 5-chloro-2-adamantanone in step (4) is replaced by 2-adamantanone to give CL-5. Yield: 35%, ESI-MS m / z: 545 [M+H], 543 [M-H].

[0079] Synthesis of Example 7 CL-5

[0080]

[0081] The difference from Example 1 is that 2-methoxyethyl-p-toluenesulfonate in step (1) is replaced by 3-(2-methoxy)ethyl-p-toluenesulfonic acid, and 5-chloro-2-adamantanone in step (4) is replaced by 2-adamantanone to give CL-5. Yield: 35%, ESI-MS m / z: 545 [M+H], 543 [M-H].

[0082] Synthesis of Example 8 CL-7

[0083]

[0084] The difference from Example 1 is that 2-methoxyethyl-p-toluenesulfonate in step (1) is replaced by 3-(2-methoxy)ethyl-p-toluenesulfonic acid, and 5-chloro-2-adamantanone in step (4) is replaced by 2-adamantanone to give CL-5. Yield: 35%, ESI-MS m / z: 545 [M+H], 543 [M-H].

[0085] Synthesis of Example 9 CL-9

[0086]

[0087] (1) Vanillin (15.2 g 0.1 mol) was added to a 500 mL reaction flask, 2-methoxyethyl p-toluenesulfonate (23.0 g 0.1 mol) was added, cesium carbonate (48.8 g 0.15 mol), DMF 200 mL, the system was heated to 50 °C for 6 hours, TLC monitoring, the reaction was completed, the system was cooled to room temperature, added to 300 mL water, the mixture was extracted with ethyl acetate 3 times 100 mL each time, the combined organic phase was washed with saturated sodium chloride solution 3 times 100 mL each time, the organic phase was dried over anhydrous sodium sulfate, filtered, rotary evaporated to give a crude product, which was purified by column chromatography to give intermediate 1 (11.3 g, 54%), ESI-MS m / z: 211 [M+H].

[0088] (2) Intermediate 1 (10 g, 47.6 mmol) was added to a reaction flask, dichloromethane (100 mL) was added and cooled to 0 °C, phosphorus pentachloride (19.8 g, 95.2 mmol) was added, the system was kept at 0 °C for 3 hours, TLC detection, the reaction was completed, the system was poured into ice water, sodium hydroxide was added to adjust pH = 7, the organic phase was separated, the aqueous phase was extracted with dichloromethane 2 times 200 mL each time, the combined organic phase was dried over anhydrous sodium sulfate, filtered, evaporated to give a light yellow oil intermediate 2 (11.2 g, 89%).

[0089] (3) Intermediate 2 (11 g, 41.5 mmol) was added to a reaction flask, 2,2,2-trifluoroethanol (80 mL) was added and cooled to 0 °C, NaH (3.65 g, 91.3 mmol, 60% oil) was slowly added in portions, after addition, the system was kept at 0 °C for 30 minutes, silver oxide (19.2 g, 83 mmol) was added, after addition, the system was heated to 50 °C for 24 hours, TLC detection, the reaction was completed, the system was cooled to 0 °C, 5 mL ice water was added, the system was kept at 0 °C for 30 minutes, the system was poured into 200 mL ice water, the aqueous phase was extracted with ethyl acetate 3 times 100 mL each time, the combined organic phase was dried over anhydrous sodium sulfate, filtered, evaporated, and purified by column chromatography to give intermediate 3 (10.7 g) ESI-MS m / z: 393 [M+H].

[0090] (4) Intermediate 3 (10 g, 0.05 mol) was added to a reaction flask, dichloromethane 150 mL was added, the system was cooled to 0 °C, then triethyl phosphite (7.45 g, 0.06 mol) was slowly added, and boron trifluoride ether (8.46 g, 0.06 mol) was slowly added dropwise. After the dropwise addition was completed, the system was kept at 0 °C for 2 hours, TLC monitoring showed that the reaction was completed, the system was warmed to room temperature, 100 mL of saturated sodium bicarbonate solution was added, the organic phase was separated, the aqueous phase was extracted with dichloromethane twice, 100 mL each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain yellow oil intermediate 3 (14.2 g, which was directly used in the next step reaction).

[0091] (5) Intermediate 4 (14 g, 0.04 mol) was added to a reaction flask, anhydrous tetrahydrofuran 100 mL was added, the system was cooled to -78 °C under nitrogen protection, then n-butyllithium (18 mL, 2.5 M / L) was added dropwise. After the dropwise addition was completed, the system was kept at -78 °C for 1 hour, 5-chloro-2-adamantanone (7.36 g, 0.04 mol) in 50 mL of tetrahydrofuran was added dropwise, the system was kept at -78 °C for 1 hour after the dropwise addition was completed, TLC monitoring showed that the reaction was completed, the system was warmed to 0 °C, 50 mL of saturated ammonium chloride solution was added, stirred for 5 minutes, then added to 100 mL of water, 100 mL of ethyl acetate was added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate twice, 100 mL each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product 15.7 g, which was purified by column chromatography to obtain intermediate 5 (12.3 g, 75 %). ESI-MS m / z: 461 [M+H].

[0092] (6) Anhydrous DMF was added to a reaction flask, the temperature was reduced to 0 °C under nitrogen protection, sodium hydride (1.44 g, 0.037 mol) was slowly added, after the addition was completed, the system was kept at room temperature for 30 minutes, the temperature was increased to 60 °C, ethanethiol (1.94 g, 0.031 mol) was slowly added dropwise, after the dropwise addition was completed, the system was kept at 60 °C for 30 minutes, intermediate 5 (12.3 g, 0.031 mol) in DMF was added, the system was warmed to 80 °C for 10 hours, TLC monitoring showed that the reaction was completed, the system was cooled to room temperature, added to 100 mL of water, 100 mL of ethyl acetate was added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate twice, 100 mL each time, the organic phases were combined, the organic phase was washed with saturated sodium chloride solution three times, 100 mL each time, the organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product, which was purified by column chromatography to obtain intermediate 6 (5.9 g, 50 %). ESI-MS m / z: 447 [M+H].

[0093] (7) Intermediate 6 (5.5 g, 0.015 mol) was added to a reaction flask, 100 mL of tetrahydrofuran, 20 mL of pyridine, the system was cooled to 0 °C, and phosphorus oxychloride (2.23 g, 0.015 mol) was added dropwise. The system was allowed to react at room temperature for 1 h, and then 3-hydroxypropionitrile (5.33 g, 0.075 mol) was added. After the addition was completed, the system was allowed to react at room temperature for 24 h. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness, and column chromatography purification gave intermediate 7 (5.2 g, 63%). ESI-MS m / z: 634 [M+H].

[0094] (8) Intermediate 7 (5.0 g, 8.85 mmol) was added to a reaction flask, 100 mL of methanol, and sodium methoxide (1 g, 17.7 mmol) was added. The system was allowed to react at room temperature for 1 h. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness, 50 mL of ethyl acetate was added, and the system was filtered to give intermediate 8 (4.0 g, 84%) as a light yellow solid. ESI-MS m / z: 527 [M+H].

[0095] (9) Intermediate 8 (2 g, 4.36 mmol) was added to a reaction flask, 50 mL of methanol, sodium molybdate (1.79 g, 8.72 mmol), and H2O2 (10 mL) were added. The system was allowed to react at room temperature for 24 h. TLC monitoring showed that the reaction was completed. The system was cooled to 0 °C, 10% sodium thiosulfate solution (5 mL) was added, and the system was stirred at 0 °C for 5 h. Filtration, rotary evaporation of the filtrate, addition of 20 mL of methanol, reflux for 1 h, hot filtration, and drying of the solid gave CL-9 (0.7 g, 33%). ESI-MS m / z: 559 [M+H], 557 [M-H].

[0096] Example 10 Synthesis of CL-11

[0097]

[0098] The difference from Example 9 is that 2-methoxyethyl-p-toluenesulfonic acid was used instead of 2-methoxyethyl-p-toluenesulfonic acid in step (1) to obtain CL-11. Yield: 29%, ESI-MS m / z: 603 [M+H], 601 [M-H].

[0099] Example 11 Synthesis of CL-13

[0100]

[0101] The difference from Example 9 is that 3-(2-methoxy)ethyl-p-toluenesulfonic acid was used instead of 2-methoxyethyl-p-toluenesulfonic acid in step (1) to obtain CL-13. Yield: 28%, ESI-MS m / z: 647 [M+H], 645 [M-H].

[0102] Synthesis of Example 12 CL-15

[0103]

[0104] The difference from Example 9 is that 2-methoxyethyl-p-toluenesulfonic acid is replaced by 4-(2-methoxy)ethyl-p-toluenesulfonic acid in step (1) to obtain CL-15. Yield: 28%, ESI-MS m / z: 691 [M+H], 689 [M-H].

[0105] Synthesis of Example 13 CL-10

[0106]

[0107] (1) Vanillin (15.2 g 0.1 mol) was added to a 500 mL reaction bottle, 2-methoxyethyl-p-toluenesulfonic acid (23.0 g 0.1 mol), cesium carbonate (48.8 g 0.15 mol), DMF 200 mL, the system was heated to 50 °C for 6 hours, TLC monitoring, the reaction was completed, the system was cooled to room temperature, added to 300 mL water, the mixture was extracted with ethyl acetate 3 times 100 mL each time, the combined organic phase was washed with saturated sodium chloride solution three times 100 mL each time, the organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product, which was purified by column chromatography to obtain intermediate 1 (11.3 g, 54%). ESI-MS m / z: 211 [M+H].

[0108] (2) Intermediate 1 (10 g, 47.6 mmol) was added to a reaction bottle, dichloromethane (100 mL) was added and cooled to 0 °C, phosphorus pentachloride (19.8 g, 95.2 mmol) was added, the system was kept at 0 °C for 3 hours, TLC detection, the reaction was completed, the system was poured into ice water, sodium hydroxide was added to adjust pH = 7, the organic phase was separated, the aqueous phase was extracted with dichloromethane 2 times 200 mL each time, the combined organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to obtain a light yellow oil intermediate 2 (11.2 g, 89%).

[0109] (3) Intermediate 2 (11 g, 41.5 mmol) was added to a reaction flask, tetrahydrofuran (80 mL) was added, and the hydroxymethylcyclopropane (20 mL) was cooled to 0 °C. NaH (3.65 g, 91.3 mmol, 60% oil) was slowly added in portions, and after the addition was complete, the system was kept at 0 °C for 30 min. Silver oxide (19.2 g, 83 mmol) was added, and after the addition was complete, the system was warmed to 50 °C and reacted for 24 h. TLC detection showed that the reaction was complete. The system was cooled to 0 °C, 5 mL of ice water was added, the system was kept at 0 °C for 30 min, and then the system was poured into 200 mL of ice water. The aqueous phase was extracted with ethyl acetate (100 mL x 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Purification by column chromatography gave intermediate 3 (13.6 g). ESI-MS m / z: 337 [M+H].

[0110] (4) Intermediate 3 (10 g, 0.03 mol) was added to a reaction flask, dichloromethane (150 mL) was added, the system was cooled to 0 °C, and then triethyl phosphite (7.39 g, 0.05 mol) was slowly added dropwise. Boron trifluoride ether (6.28 g, 0.05 mol) was slowly added dropwise. After the dropwise addition was complete, the system was kept at 0 °C for 2 h. TLC detection showed that the reaction was complete. The system was warmed to room temperature, 100 mL of saturated sodium bicarbonate solution was added, the organic phase was separated, the aqueous phase was extracted with dichloromethane (100 mL x 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give yellow oil intermediate 3 (13.5 g, which was used directly in the next step).

[0111] (5) Intermediate 4 (13 g, 0.04 mol) was added to a reaction flask, anhydrous tetrahydrofuran (100 mL) was added, the system was cooled to -78 °C under nitrogen protection, and then n-butyllithium (18 mL, 2.5 M / L) was added dropwise. After the dropwise addition was complete, the system was kept at -78 °C for 1 h. A solution of 5-chloro-2-adamantanone (7.36 g, 0.04 mol) in tetrahydrofuran (50 mL) was added dropwise. After the dropwise addition was complete, the system was kept at -78 °C for 1 h. TLC detection showed that the reaction was complete. The system was warmed to 0 °C, 50 mL of saturated ammonium chloride solution was added, and after stirring for 5 min, the system was added to 100 mL of water. Ethyl acetate (100 mL) was added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate (100 mL x 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give a crude product (15.7 g). Purification by column chromatography gave intermediate 5 (10.6 g, 67%). ESI-MS m / z: 433 [M+H].

[0112] (6) Anhydrous DMF was added to the reaction flask, and the temperature was lowered to 0 °C under nitrogen protection. Sodium hydride (1.06 g, 0.028 mol) was slowly added. After the addition was completed, the reaction was carried out at room temperature for 30 min. The system was warmed to 60 °C, and ethanethiol (1.71 g, 0.028 mol) was slowly added dropwise. After the addition was completed, the system was kept at 60 °C for 30 min. The DMF solution of intermediate 4 (10.0 g, 0.023 mol) was added, and the system was warmed to 80 °C for 10 h. TLC monitoring showed that the reaction was completed. The system was cooled to room temperature, added to 100 mL of water, and added with 100 mL of ethyl acetate. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate twice, 100 mL each time. The combined organic phase was washed with saturated sodium chloride solution three times, 100 mL each time. The organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product. Column chromatography purification gave intermediate 6 (5.6 g, 60%). ESI-MS m / z: 419 [M+H].

[0113] (7) Intermediate 6 (5.5 g, 0.013 mol) was added to the reaction flask, and 100 mL of tetrahydrofuran and 20 mL of pyridine were added. The system was cooled to 0 °C, and phosphorus oxychloride (2.00 g, 0.013 mol) was added dropwise. The system was reacted at room temperature for 1 h. After the addition of 3-hydroxypropionitrile (4.70 g, 0.065 mol) was completed, the system was kept at room temperature for 24 h. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness, and column chromatography purification gave intermediate 7 (4.7 g, 59%). ESI-MS m / z: 607 [M+H].

[0114] (8) Intermediate 6 (4.0 g, 6.59 mmol) was added to the reaction flask, and 100 mL of methanol and sodium methoxide (0.78 g, 14.5 mmol) were added. The system was reacted at room temperature for 1 h. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness, and 50 mL of ethyl acetate was added. The system was filtered to obtain a light yellow solid intermediate 7 (2.1 g, 69%). ESI-MS m / z: 465 [M+H].

[0115] (9) Intermediate 7 (2 g, 4.30 mmol) was added to the reaction flask, and sodium molybdate (1.79 g, 8.60 mmol), H2O2 (10 mL), and 50 mL of methanol were added. The system was reacted at room temperature for 24 h. TLC monitoring showed that the reaction was completed. The system was cooled to 0 °C, and 10% sodium thiosulfate solution (5 mL) was added. The system was kept at 0 °C for 5 h, filtered, and the filtrate was rotary evaporated. 20 mL of methanol was added, refluxed for 1 h, and hot filtered. The solid was dried to obtain CL-10 (0.8 g, 35%). ESI-MS m / z: 531 [M+H], 529 [M-H].

[0116] Example 14 Synthesis of CL-12

[0117]

[0118] The difference between Example 13 is that 2-methoxyethyl-p-toluenesulfonate in step (1) is replaced by 2-(2-methoxy)ethyl-p-toluenesulfonic acid to give CL-12. Yield: 34%, ESI-MS m / z: 575 [M+H], 573 [M-H].

[0119] Example 15 Synthesis of CL-14

[0120]

[0121] The difference between Example 13 is that 2-methoxyethyl-p-toluenesulfonate in step (1) is replaced by 3-(2-methoxy)ethyl-p-toluenesulfonic acid to give CL-14. Yield: 35%, ESI-MS m / z: 619 [M+H], 617 [M-H].

[0122] Example 16 Synthesis of CL-16

[0123]

[0124] The difference between Example 13 is that 2-methoxyethyl-p-toluenesulfonate in step (1) is replaced by 4-(2-methoxy)ethyl-p-toluenesulfonic acid to give CL-16. Yield: 35%, ESI-MS m / z: 663 [M+H], 661 [M-H].

[0125] Example 17 Synthesis of CL-17

[0126]

[0127] The difference between Example 13 is that hydroxymethylcyclopropane in step (3) is replaced by trifluoroethane, and 5-chloro-2-adamantanone in step (5) is replaced by 2-adamantanone to give CL-17. Yield: 35%, ESI-MS m / z: 525 [M+H], 523 [M-H].

[0128] Example 18 Synthesis of CL-18

[0129]

[0130] The difference between Example 13 is that 5-chloro-2-adamantanone in step (5) is replaced by 2-adamantanone to give CL-17. Yield: 35%, ESI-MS m / z: 497 [M+H], 495 [M-H].

[0131] Example 19 Synthesis of CL-19

[0132]

[0133] (1) 4-ethyl-3-methoxybenzaldehyde (10 g, 0.061 mol) was added to a reaction flask, 20 mL of methanol was added, and trimethyl orthoformate (12.9 g, 0.122 mol) was added to the system with p-toluenesulfonic acid (1.1 g, 6.1 mmol) at room temperature. The reaction was stirred for 24 hours, and TLC was used to monitor the completion of the reaction. 2 mL of triethylamine was added to the system, which was then dried by rotary evaporation. After drying, 50 mL of saturated sodium bicarbonate solution, 100 mL of water, and 100 mL of ethyl acetate were added. The organic phase was separated, and the aqueous phase was extracted twice with 100 mL of ethyl acetate each time. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain yellow oil intermediate 1 (12.7 g, which was used directly in the next step).

[0134] (2) Intermediate 1 (10 g, 0.05 mol) was added to a reaction flask, 150 mL of dichloromethane was added, and the system was cooled to 0°C. Then triethyl phosphite (7.39 g, 0.05 mol) was slowly added dropwise, followed by the addition of boron trifluoride etherate (6.28 g, 0.05 mol). After the dropwise addition was completed, the system was kept at 0°C for 2 hours, and TLC was used to monitor the completion of the reaction. The system was warmed to room temperature, 100 mL of saturated sodium bicarbonate solution was added, and the organic phase was separated. The aqueous phase was extracted twice with 100 mL of dichloromethane each time, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain yellow oil intermediate 2 (13.2 g, which was used directly in the next step).

[0135] (3) Intermediate 2 (13 g, 0.05 mol) was added to a reaction flask, 100 mL of anhydrous tetrahydrofuran was added, and the system was cooled to -78°C under nitrogen protection. Then n-butyllithium (19 mL, 2.5 M / L) was added dropwise. After the dropwise addition was completed, the system was kept at -78°C for 1 hour, and a solution of 5-chloro-2-adamantanone (9.20 g, 0.05 mol) in 50 mL of tetrahydrofuran was added dropwise. After the dropwise addition was completed, the system was kept at -78°C for 1 hour, and TLC was used to monitor the completion of the reaction. The system was warmed to 0°C, 50 mL of saturated ammonium chloride solution was added, and after stirring for 5 minutes, it was added to 100 mL of water. 100 mL of ethyl acetate was added, the organic phase was separated, and the aqueous phase was extracted twice with 100 mL of ethyl acetate each time. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain 15.3 g of crude product. Column chromatography purification yielded intermediate 3 (10.2 g, 65%). ESI-MS m / z: 347 [M+H].

[0136] (4) Anhydrous DMF was added to the reaction flask, and the temperature was lowered to 0 °C under nitrogen protection. Sodium hydride (1.10 g, 0.029 mol) was slowly added. After the addition was completed, the reaction was carried out at room temperature for 30 minutes. The system was warmed to 60 °C. Ethyl mercaptan (1.64 g, 0.029 mol) was slowly added dropwise. After the addition was completed, the system was kept at 60 °C for 30 minutes. The DMF solution of intermediate 3 (10.0 g, 0.029 mol) was added. The system was warmed to 80 °C and reacted for 10 hours. TLC monitoring showed that the reaction was completed. The system was cooled to room temperature and added to 100 mL of water. 100 mL of ethyl acetate was added. The organic phase was separated. The aqueous phase was extracted with ethyl acetate twice, 100 mL each time. The organic phases were combined. The organic phase was washed with saturated sodium chloride solution three times, 100 mL each time. The organic phase was dried over anhydrous sodium sulfate. Filtration was performed. Rotary evaporation was performed to obtain a crude product. Column chromatography was used for purification to obtain intermediate 4 (6.1 g, 64%). ESI-MS m / z: 332 [M+H].

[0137] (5) Intermediate 4 (5.0 g, 0.015 mol) was added to the reaction flask. 100 mL of tetrahydrofuran and 20 mL of pyridine were added. The system was cooled to 0 °C. Phosphorus oxychloride (2.30 g, 0.015 mol) was added dropwise. The system was reacted at room temperature for 1 hour. 3-Hydroxypropionitrile (5.33 g, 0.075 mol) was added. After the addition was completed, the system was kept at room temperature and reacted for 24 hours. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness. Column chromatography was used for purification to obtain intermediate 5 (4.90 g, 63%). ESI-MS m / z: 519 [M+H].

[0138] (6) Dimethylpropanolamine (0.8 g, 7.71 mmol) was added to the reaction flask. 50 mL of tetrahydrofuran and silver oxide (1.78 g, 7.71 mmol) were added. The system was stirred at room temperature for 30 minutes. Intermediate 5 (4 g, 7.71 mmol) was added. The system was warmed to 50 °C and reacted overnight. The next day, TLC monitoring showed that the reaction was completed. Diatomite (10 g) was added. The system was stirred for 30 minutes. The system was cooled to room temperature. Filtration was performed. The filtrate was evaporated to dryness. 100 mL of ethyl acetate and 100 mL of water were added. The system was stirred for 10 minutes. The organic phase was separated. The aqueous phase was extracted with ethyl acetate twice, 50 mL each time. The organic phases were combined. The organic phase was dried over anhydrous sodium sulfate. Filtration was performed. Rotary evaporation was performed. Column chromatography was used for purification to obtain intermediate 6 (2.80 g, 62%). ESI-MS m / z: 586 [M+H].

[0139] (7) Intermediate 6 (2.0 g, 3.42 mmol) was added to the reaction flask. 100 mL of methanol and sodium methoxide (0.37 g, 6.84 mmol) were added. The system was reacted at room temperature for 1 hour. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness. 50 mL of ethyl acetate was added. The system was filtered to obtain a light yellow solid intermediate 7 (1.1 g, 68%). ESI-MS m / z: 479 [M+H].

[0140] (8) Intermediate 7 (1 g, 2.09 mmol) was added to a reaction flask, sodium molybdate (0.86 g, 4.18 mmol) H202(5 mL), methanol 50 mL, the system was reacted at room temperature for 24 hours, TLC monitoring, the reaction was completed, the system was cooled to 0 °C, 10% sodium thiosulfate solution (5 mL) was added, the system was stirred at 0 °C for 5 hours, filtered, the filtrate was rotary dried, 20 mL of methanol was added, refluxed for 1 hour, hot filtered, the solid was dried to obtain CL-19 (0.3 g, 28%). ESI-MS m / z: 512 [M+H], 510 [M-H].

[0141] Example 20 Synthesis of CL-21

[0142]

[0143] (1) 4-ethyl-3-methoxybenzaldehyde (10 g, 0.061 mol) was added to a reaction flask, dichloromethane (100 mL) was added and cooled to 0 °C, phosphorus pentachloride (24.8 g, 0.122 mol) was added, the system was kept at 0 °C for 3 hours, TLC detection, the reaction was completed, the system was poured into ice water, sodium hydroxide was added to adjust pH = 7, the organic phase was separated, the aqueous phase was extracted with dichloromethane twice, 200 mL each time, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and evaporated to obtain a light yellow oil intermediate 1 (12.1 g, 91%).

[0144] (2) Intermediate 1 (12 g, 0.06 mol) was added to a reaction flask, 2,2,2-trifluoroethanol (80 mL) was added and cooled to 0 °C, NaH (4.60 g, 0.12 mol, 60% oil) was slowly added in batches, after addition, the system was kept at 0 °C for 30 minutes, silver oxide (20.8 g, 0.09 mol) was added, after addition, the system was warmed to 50 °C and reacted for 24 hours, TLC detection, the reaction was completed, the system was cooled to 0 °C, 5 mL of ice water was added, the system was kept at 0 °C and stirred for 30 minutes, the system was poured into 200 mL of ice water, the aqueous phase was extracted with ethyl acetate three times, 100 mL each time, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, evaporated, and purified by column chromatography to obtain intermediate 2 (10.1 g, 53%). ESI-MS m / z: 347 [M+H].

[0145] (3) Intermediate 2 (10 g, 0.03 mol) was added to a reaction flask, dichloromethane 150 mL was added, the system was cooled to 0 °C, then triethyl phosphite (4.78 g, 0.03 mol) was slowly added, and then boron trifluoride ether (4.23 g, 0.03 mol) was slowly added dropwise. After the dropwise addition was completed, the system was kept at 0 °C for 2 hours, TLC monitoring showed that the reaction was completed, the system was warmed to room temperature, 100 mL of saturated sodium bicarbonate solution was added, the organic phase was separated, the aqueous phase was extracted with dichloromethane twice, 100 mL each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain yellow oil intermediate 3 (12.2 g, which was directly used in the next step reaction).

[0146] (4) Intermediate 3 (12 g, 0.03 mol) was added to a reaction flask, anhydrous tetrahydrofuran 100 mL was added, the system was cooled to -78 °C under nitrogen protection, then n-butyllithium (14 mL, 2.5 M / L) was added dropwise, after the dropwise addition was completed, the system was kept at -78 °C for 1 hour, 5-chloro-2-adamantanone (6.20 g, 0.03 mol) in tetrahydrofuran 50 mL was added dropwise, after the dropwise addition was completed, the system was kept at -78 °C for 1 hour, TLC monitoring showed that the reaction was completed, the system was warmed to 0 °C, 50 mL of saturated ammonium chloride solution was added, after stirring for 5 minutes, it was added to 100 mL of water, 100 mL of ethyl acetate was added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate twice, 100 mL each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product 14.3 g, which was purified by column chromatography to obtain intermediate 4 (10.7 g, 65 %). ESI-MS m / z: 415 [M+H].

[0147] (5) Anhydrous DMF was added to a reaction flask, the temperature was reduced to 0 °C under nitrogen protection, sodium hydride (0.93 g, 0.024 mol) was slowly added, after the addition was completed, the system was reacted at room temperature for 30 minutes, the temperature was increased to 60 °C, ethanethiol (1.50 g, 0.024 mol) was slowly added dropwise, after the dropwise addition was completed, the system was kept at 60 °C for 30 minutes, intermediate 4 (10.0 g, 0.024 mol) in DMF was added, the system was warmed to 80 °C and reacted for 10 hours, TLC monitoring showed that the reaction was completed, the system was cooled to room temperature, added to 100 mL of water, 100 mL of ethyl acetate was added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate twice, 100 mL each time, the organic phases were combined, the organic phase was washed with saturated sodium chloride solution three times, 100 mL each time, the organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product, which was purified by column chromatography to obtain intermediate 5 (6.5 g, 67 %). ESI-MS m / z: 401 [M+H].

[0148] (6) Intermediate 5 (5.0 g, 0.013 mol) was added to a reaction flask, 100 mL of tetrahydrofuran, 20 mL of pyridine, the system was cooled to 0 °C, and phosphorus oxychloride (1.92 g, 0.013 mol) was added dropwise. The system was allowed to react at room temperature for 1 h, and 3-hydroxypropionitrile (4.62 g, 0.065 mol) was added. After the addition was completed, the system was allowed to react at room temperature for 24 h. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness, and column chromatography purification gave intermediate 6 (4.74 g, 61%). ESI-MS m / z: 587 [M+H].

[0149] (7) Dimethylpropanolamine (0.7 g, 6.83 mmol) was added to a reaction flask, 50 mL of tetrahydrofuran, and silver oxide (1.58 g, 6.83 mmol) was added. After the system was stirred at room temperature for 30 min, intermediate 6 (4 g, 6.83 mmol) was added, and the system was allowed to react at 50 °C overnight. The next day, TLC monitoring showed that the reaction was completed. Diatomite (10 g) was added, and the system was allowed to stir for 30 min. The system was cooled to room temperature, filtered, and the filtrate was evaporated to dryness. Then 100 mL of ethyl acetate and 100 mL of water were added, and the system was stirred for 10 min. The organic phase was separated, and the aqueous phase was extracted twice with 50 mL of ethyl acetate. The combined organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Column chromatography purification gave intermediate 7 (2.66 g, 60%). ESI-MS m / z: 654 [M+H].

[0150] (8) Intermediate 7 (2.0 g, 3.06 mmol) was added to a reaction flask, 100 mL of methanol, and sodium methoxide (0.33 g, 6.12 mmol) was added. The system was allowed to react at room temperature for 1 h. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness, 50 mL of ethyl acetate was added, and the system was filtered to give intermediate 8 (0.9 g, 54%) as a light yellow solid. ESI-MS m / z: 548 [M+H].

[0151] (9) Intermediate 8 (0.7 g, 1.28 mmol) was added to a reaction flask, 50 mL of methanol, sodium molybdate (0.53 g, 3.56 mmol), and H2O2 (5 mL) were added. The system was allowed to react at room temperature for 24 h. TLC monitoring showed that the reaction was completed. The system was cooled to 0 °C, 10% sodium thiosulfate solution (5 mL) was added, and the system was allowed to stir at 0 °C for 5 h. The system was filtered, the filtrate was evaporated to dryness, 20 mL of methanol was added, and the system was refluxed for 1 h. The hot filtrate was evaporated to dryness, and the solid was dried to give CL-21 product (0.2 g, 27%). ESI-MS m / z: 580 [M+H], 578 [M-H]. Yield: 27%.

[0152] Example 21 Synthesis of CL-23

[0153]

[0154] The difference between Example 20 is that 4-ethyl-3-methoxybenzaldehyde in the starting material is replaced by 4-methyl-3-methoxybenzaldehyde to obtain CL-23. Yield: 27%, ESI-MS m / z: 552 [M+H], 550 [M-H].

[0155] Example 22 Synthesis of CL-20

[0156]

[0157] The difference between Example 20 is that 4-ethyl-3-methoxybenzaldehyde in the starting material is replaced by 4-methyl-3-methoxybenzaldehyde, and 2,2,2-trifluoroethanol in step (2) is replaced by hydroxymethylcyclopropane to obtain CL-20. Yield: 28%, ESI-MS m / z: 538 [M+H], 536 [M-H].

[0158] Example 23 Synthesis of CL-22

[0159]

[0160] The difference between Example 19 is that 4-ethyl-3-methoxybenzaldehyde in the starting material is replaced by 4-methyl-3-methoxybenzaldehyde to obtain CL-22. Yield: 27%, ESI-MS m / z: 484 [M+H], 482 [M-H].

[0161] Example 24 Synthesis of CL-24

[0162]

[0163] The difference between Example 20 is that 4-ethyl-3-methoxybenzaldehyde in the starting material is replaced by 4-methyl-3-methoxybenzaldehyde, and 2,2,2-trifluoroethanol in step (2) is replaced by hydroxymethylcyclopropane to obtain CL-24. Yield: 27%, ESI-MS m / z: 524 [M+H], 521 [M-H].

[0164] Example 25 Synthesis of CL-31

[0165]

[0166] The difference between Example 19 is that 4-ethyl-3-methoxybenzaldehyde in the starting material is replaced by 4-methyl-3-methoxybenzaldehyde, and dimethylpropanolamine in step (6) is replaced by ethylene glycol methyl ether to obtain CL-31. Yield: 27%, ESI-MS m / z: 471 [M+H], 469 [M-H].

[0167] Example 26 Synthesis of CL-32

[0168]

[0169] The difference from Example 20 is that the starting material 4-ethyl-3-methoxybenzaldehyde is replaced by 4-methyl-3-methoxybenzaldehyde, 2,2,2-trifluoroethanol in step (2) is replaced by hydroxymethylcyclopropane, and dimethylaminopropanol in step (7) is replaced by ethylene glycol monomethyl ether to obtain CL-33. Yield: 29%, ESI-MS m / z: 511 [M+H], 509 [M-H].

[0170] Example 27 Synthesis of CL-33

[0171]

[0172] The difference from Example 20 is that the starting material 4-ethyl-3-methoxybenzaldehyde is replaced by 4-methyl-3-methoxybenzaldehyde, 2,2,2-trifluoroethanol in step (2) is replaced by hydroxymethylcyclopropane, and dimethylaminopropanol in step (7) is replaced by ethylene glycol monomethyl ether to obtain CL-33. Yield: 29%, ESI-MS m / z: 511 [M+H], 509 [M-H].

[0173] Example 28 Synthesis of CL-26

[0174]

[0175] (1) Vanillin (10 g, 0.066 mol) was added to a reaction flask, DMF (70 mL) was added to cool to 0°C, K2CO3 (18.2 g, 0.132 mol) was added, the system was stirred at 0°C for 30 minutes, then trifluoroiodoethane (13.8 g, 0.066) was added, the system was warmed to 50°C and reacted for 3 hours, TLC detection showed that the reaction was completed, the system was slowly poured into water, a solid was precipitated, after stirring for 30 minutes, it was filtered, the filter cake was dried and recrystallized with ethanol to obtain a yellow solid, intermediate 1 (11.2 g, 73%). ESI-MS m / z: 235 [M+H].

[0176] (2) Intermediate 1 (11 g, 0.047 mol) was added to a reaction flask, dichloromethane (150 mL) was added to cool to 0°C, phosphorus pentachloride (19.6 g, 0.094 mol) was added, the system was kept at 0°C for 3 hours, TLC detection showed that the reaction was completed, the system was poured into ice water, sodium hydroxide was added to adjust pH = 7, the organic phase was separated, the aqueous phase was extracted with dichloromethane twice, 200 mL each time, the combined organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to obtain a light yellow oil, intermediate 2 (10.2 g, 75%).

[0177] (3) Intermediate 2 (10 g, 0.04 mol) was added to a reaction flask, 2,2,2-trifluoroethanol (80 mL) was added, and the temperature was lowered to 0 °C. NaH (2.65 g, 0.08 mol, 60% oil) was slowly added in portions. After the addition was completed, the system was kept at 0 °C for 30 min. Silver oxide (13.9 g, 0.06 mol) was added, and after the addition was completed, the system was warmed to 50 °C and reacted for 24 h. TLC detection showed that the reaction was completed. The system was cooled to 0 °C, and 5 mL of ice water was added. The system was kept at 0 °C for 30 min, and then poured into 200 mL of ice water. The aqueous phase was extracted with ethyl acetate (100 mL) three times. The combined organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Column chromatography purification gave intermediate 3 (8.1 g, 58%). ESI-MS m / z: 417 [M+H].

[0178] (4) Intermediate 3 (8.0 g, 0.02 mol) was added to a reaction flask, dichloromethane (150 mL) was added, and the system was cooled to 0 °C. Triethyl phosphite (3.18 g, 0.02 mol) was slowly added dropwise, and then boron trifluoride ether (4.23 g, 0.03 mol) was slowly added dropwise. After the addition was completed, the system was kept at 0 °C for 2 h. TLC detection showed that the reaction was completed. The system was warmed to room temperature, and 100 mL of saturated sodium bicarbonate solution was added. The organic phase was separated, and the aqueous phase was extracted with dichloromethane (100 mL) twice. The combined organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give yellow oil intermediate 4 (10.2 g, which was used directly in the next step).

[0179] (5) Intermediate 4 (10 g, 0.03 mol) was added to a reaction flask, anhydrous tetrahydrofuran (100 mL) was added, and the system was cooled to -78 °C under nitrogen protection. n-Butyllithium (9.5 mL, 2.5 M / L) was added dropwise. After the addition was completed, the system was kept at -78 °C for 1 h. A solution of 5-chloro-2-adamantanone (6.20 g, 0.03 mol) in tetrahydrofuran (50 mL) was added dropwise. After the addition was completed, the system was kept at -78 °C for 1 h. TLC detection showed that the reaction was completed. The system was warmed to 0 °C, and 50 mL of saturated ammonium chloride solution was added. After stirring for 5 min, it was added to 100 mL of water. 100 mL of ethyl acetate was added, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (100 mL) twice. The combined organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give a crude product (13.5 g). Column chromatography purification gave intermediate 5 (7.7 g, 68%). ESI-MS m / z: 485 [M+H].

[0180] (6) Anhydrous DMF was added to the reaction flask, and the temperature was lowered to 0 °C under nitrogen protection. Sodium hydride (0.60 g, 0.016 mol) was slowly added. After the addition was completed, the system was reacted at room temperature for 30 minutes. The temperature was raised to 60 °C. Ethyl mercaptan (1.0 g, 0.016 mol) was slowly added dropwise. After the addition was completed, the system was kept at 60 °C for 30 minutes. The DMF solution of intermediate 5 (7.5 g, 0.016 mol) was added. The temperature of the system was raised to 80 °C. The reaction was carried out for 10 hours. TLC monitoring showed that the reaction was completed. The system was cooled to room temperature. It was added to 100 mL of water. 100 mL of ethyl acetate was added. The organic phase was separated. The aqueous phase was extracted with ethyl acetate twice, 100 mL each time. The organic phases were combined. The organic phase was washed with saturated sodium chloride solution three times, 100 mL each time. The organic phase was dried over anhydrous sodium sulfate. Filtration was performed. The solvent was removed by rotary evaporation. The crude product was obtained. Column chromatography was used for purification. Intermediate 6 (5.5 g, 76%) was obtained. ESI-MS m / z: 471 [M+H].

[0181] (7) Intermediate 6 (5.0 g, 0.011 mol) was added to the reaction flask. 100 mL of tetrahydrofuran and 20 mL of pyridine were added. The temperature of the system was lowered to 0 °C. Phosphorus oxychloride (1.63 g, 0.011 mol) was added dropwise. The system was reacted at room temperature for 1 hour. 3-Hydroxypropionitrile (3.91 g, 0.055 mol) was added. After the addition was completed, the system was kept at room temperature for 24 hours. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness. Column chromatography was used for purification. Intermediate 7 (4.83 g, 69%) was obtained. ESI-MS m / z: 657 [M+H].

[0182] (8) Dimethylpropanolamine (0.7 g, 6.85 mmol) was added to the reaction flask. 50 mL of tetrahydrofuran and silver oxide (1.58 g, 6.85 mmol) were added. The system was stirred at room temperature for 30 minutes. Intermediate 7 (4.5 g, 6.85 mmol) was added. The temperature of the system was raised to 50 °C. The reaction was carried out overnight. The next day, TLC monitoring showed that the reaction was completed. Diatomite (10 g) was added. The system was stirred for 30 minutes. The system was cooled to room temperature. Filtration was performed. The solvent was removed by rotary evaporation. 100 mL of ethyl acetate and 100 mL of water were added. The system was stirred for 10 minutes. The organic phase was separated. The aqueous phase was extracted with ethyl acetate twice, 50 mL each time. The organic phases were combined. The organic phase was dried over anhydrous sodium sulfate. Filtration was performed. The solvent was evaporated to dryness. Column chromatography was used for purification. Intermediate 8 (2.66 g, 60%) was obtained. ESI-MS m / z: 654 [M+H].

[0183] (9) Intermediate 8 (2.5 g, 3.82 mmol) was added to the reaction flask. 100 mL of methanol and sodium methoxide (0.42 g, 7.64 mmol) were added. The system was reacted at room temperature for 1 hour. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness. The system was filtered. Intermediate 9 (1.1 g, 47%) was obtained as a light yellow solid. ESI-MS m / z: 618 [M+H].

[0184] (10) Intermediate 9 (1.0 g, 1.62 mmol) was added to the reaction flask, sodium molybdate (0.66 g, 3.24 mmol) H2O2(5 mL), methanol 50 mL, the system was reacted at room temperature for 24 hours, TLC monitoring, the reaction was completed, the system was cooled to 0 °C, 10% sodium thiosulfate solution (5 mL) was added, the system was stirred at 0 °C for 5 hours, filtered, the filtrate was rotary dried, 20 mL of methanol was added, refluxed for 1 hour, hot filtered, the solid was dried to obtain the product (0.3 g, 29%). ESI-MS m / z: 650 [M+H], 648 [M-H].

[0185] Example 29 Synthesis of CL-25

[0186]

[0187] The difference from Example 28 is that 2,2,2-trifluoroethanol in step (2) is replaced by methoxy to obtain CL-25. Yield: 23%, ESI-MS m / z: 568 [M+H], 565 [M-H].

[0188] Example 30 Synthesis of CL-27

[0189]

[0190] The difference from Example 28 is that 2,2,2-trifluoroethanol in step (2) is replaced by hydroxymethylcyclopropane to obtain CL-27. Yield: 22%, ESI-MS m / z: 608 [M+H], 606 [M-H].

[0191] Example 31 Synthesis of CL-28

[0192]

[0193] The difference from Example 28 is that 2,2,2-trifluoroethanol in step (2) is replaced by methoxy, and dimethylpropanolamine in step 8 is replaced by (2-methoxy)ethyl to obtain CL-28. Yield: 18%, ESI-MS m / z: 555 [M+H], 553 [M-H].

[0194] Example 32 Synthesis of CL-29

[0195]

[0196] The difference from Example 28 is that dimethylaminopropanol group in step 8 is replaced by (2-methoxy)ethyl to obtain CL-29. Yield: 15%, ESI-MS m / z: 623 [M+H], 621 [M-H].

[0197] Synthesis of Example 33 CL-30

[0198]

[0199] The difference from Example 28 is that 2,2,2-trifluoroethanol in step (2) is replaced by hydroxymethylcyclopropane, dimethylaminopropanol group in step 8 is replaced by (2-methoxy)ethyl to obtain CL-30. Yield: 15%, ESI-MS m / z: 595 [M+H], 593 [M-H].

[0200] Synthesis of Example 34 CL-38

[0201]

[0202] (1) 4-Chloro-3-methoxybenzaldehyde (10 g, 0.059 mol) was added to a reaction flask, dichloromethane (150 mL) was added and the system was cooled to 0 °C, phosphorus pentachloride (24.5 g, 0.118 mol) was added and the system was kept at 0 °C for 3 hours, TLC detection, the reaction was completed, the system was poured into ice water, sodium hydroxide was added to adjust pH = 7, the organic phase was separated, the aqueous phase was extracted with dichloromethane twice, 200 mL each time, the organic phase was combined, dried over anhydrous sodium sulfate, filtered and evaporated to give a light yellow oily intermediate 1 (10.1 g, 77%).

[0203] (2) Intermediate 1 (10 g, 0.045 mol) was added to a reaction flask, 2,2,2-trifluoroethanol (80 mL) was added, and the system was cooled to 0 °C, NaH (3.44 g, 0.090 mol, 60% oil) was slowly added in portions, after addition, the system was kept at 0 °C for 30 minutes, silver oxide (20.8 g, 0.090 mol) was added, after addition, the system was warmed to 50 °C and reacted for 24 hours, TLC detection, the reaction was completed, the system was cooled to 0 °C, 5 mL of ice water was added, the system was kept at 0 °C and stirred for 30 minutes, the system was poured into 200 mL of ice water, the aqueous phase was extracted with ethyl acetate three times, 100 mL each time, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, evaporated and purified by column chromatography to give intermediate 2 (9.7 g). ESI-MS m / z: 353 [M+H].

[0204] (3) Intermediate 2 (9.0 g, 25.5 mmol) was added to a reaction flask, dichloromethane 150 mL was added, the system was cooled to 0 °C, then triethyl phosphite (4.24 g, 25.5 mmol) was slowly added, and then boron trifluoride ether (3.60 g, 25.5 mmol) was slowly added dropwise. After the dropwise addition was completed, the system was kept at 0 °C for 2 hours. TLC monitoring showed that the reaction was completed. The system was warmed to room temperature, 100 mL of saturated sodium bicarbonate solution was added, the organic phase was separated, the aqueous phase was extracted with dichloromethane twice, 100 mL each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain intermediate 3 (10.1 g, which was used directly in the next step) as a yellow oil.

[0205] (4) Intermediate 3 (10 g, 27.6 mol) was added to a reaction flask, anhydrous tetrahydrofuran 100 mL was added, and the system was cooled to -78 °C under nitrogen protection. Then n-butyllithium (12 mL, 2.5 M / L) was added dropwise. After the dropwise addition was completed, the system was kept at -78 °C for 1 hour. A solution of 5-p-toluenesulfonyl-2-adamantanone (8.8 g, 27.6 mol) in tetrahydrofuran 50 mL was added dropwise. After the dropwise addition was completed, the system was kept at -78 °C for 1 hour. TLC monitoring showed that the reaction was completed. The system was warmed to 0 °C, 50 mL of saturated ammonium chloride solution was added, stirred for 5 minutes, then added to 100 mL of water, 100 mL of ethyl acetate was added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate twice, 100 mL each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain 13.5 g of a crude product. Column chromatography purification gave intermediate 4 (7.3 g, 47%). ESI-MS m / z: 557 [M+H].

[0206] (5) Anhydrous DMF was added to a reaction flask, and the temperature was reduced to 0 °C under nitrogen protection. Sodium hydride (0.52 g, 13.5 mmol) was slowly added. After the addition was completed, the system was kept at room temperature for 30 minutes. The system was warmed to 60 °C, and ethanethiol (0.84 g, 13.5 mol) was slowly added dropwise. After the dropwise addition was completed, the system was kept at 60 °C for 30 minutes. Intermediate 4 (7.5 g, 13.5 mmol) in DMF solution was added, the system was warmed to 80 °C, and the reaction was kept for 10 hours. TLC monitoring showed that the reaction was completed. The system was cooled to room temperature, added to 100 mL of water, 100 mL of ethyl acetate was added, the organic phase was separated, the aqueous phase was extracted with ethyl acetate twice, 100 mL each time, the organic phases were combined, and the organic phase was washed with saturated sodium chloride solution three times, 100 mL each time. The organic phase was dried over anhydrous sodium sulfate, filtered, and rotary evaporated to obtain a crude product. Column chromatography purification gave intermediate 5 (5.2 g, 72%). ESI-MS m / z: 543 [M+H].

[0207] (6) Intermediate 5 (5.0 g, 9.10 mmol) was added to a reaction flask, 100 mL of tetrahydrofuran, 20 mL of pyridine were added, the system was cooled to 0 °C, and phosphorus oxychloride (1.39 g, 9.10 mmol) was added dropwise. The system was allowed to react at room temperature for 1 h, and then 3-hydroxypropionitrile (3.23 g, 45.5 mol) was added. After the addition was completed, the system was allowed to react at room temperature for 24 h. TLC monitoring showed that the reaction was completed. The system was evaporated to dryness, and column chromatography purification gave intermediate 6 (4.66 g, 70%). ESI-MS m / z: 730 [M+H].

[0208] (7) Intermediate 6 (4.5 g, 6.17 mmol) was added to a reaction flask, 50 mL of tetrahydrofuran, and silver oxide (1.43 g, 6.17 mmol) were added. The system was stirred at room temperature for 30 min, and then trifluoroiodoethane (1.2 g, 6.17 mmol) was added. The system was allowed to react at 50 °C overnight. The next day, TLC monitoring showed that the reaction was completed. Diatomite (10 g) was added, and the system was stirred for 30 min. The system was cooled to room temperature, filtered, and the filtrate was evaporated to dryness. Then 100 mL of ethyl acetate and 100 mL of water were added, and the system was stirred for 10 min. The organic phase was separated, and the aqueous phase was extracted twice with 50 mL of ethyl acetate. The combined organic phase was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Column chromatography purification gave intermediate 7 (2.31 g, 56%). ESI-MS m / z: 657 [M+H].

[0209] (8) Intermediate 8 (0.9 g, 1.64 mmol) was added to a reaction flask, and sodium molybdate (0.67 g, 3.27 mmol), H2O2 (5 mL), and 50 mL of methanol were added. The system was allowed to react at room temperature for 24 h. TLC monitoring showed that the reaction was completed. The system was cooled to 0 °C, and 10% sodium thiosulfate solution (5 mL) was added. The system was stirred at 0 °C for 5 h, filtered, and the filtrate was evaporated to dryness. Then 20 mL of methanol was added, and the system was refluxed for 1 h. The hot filtrate was evaporated to dryness, and the solid was dried to give the product (0.1 g, 1%). ESI-MS m / z: 583 [M+H], 581 [M-H].

[0210] Synthesis of CL-34

[0211]

[0212] The difference from Example 34 is that 2,2,2-trifluoroethanol in step 2 is replaced by methoxy, and 4-chloro-3-methoxybenzaldehyde in the starting material is replaced by 4-methyl-3-methoxybenzaldehyde to give CL-34. Yield: 20%, ESI-MS m / z: 495 [M+H], 493 [M-H].

[0213] Synthesis of CL-35

[0214]

[0215] The difference between Example 34 is that 4-chloro-3-methoxybenzaldehyde in the starting material is replaced by 4-methyl-3-methoxybenzaldehyde, and 2,2,2-trifluoroethanol in step (2) is replaced by hydroxymethylcyclopropane to obtain CL-35. Yield: 14%, ESI-MS m / z: 563 [M+H], 561 [M-H].

[0216] Synthesis of Example 37 CL-36

[0217]

[0218] The difference between Example 34 is that 4-chloro-3-methoxybenzaldehyde in the starting material is replaced by 4-methyl-3-methoxybenzaldehyde, and 2,2,2-trifluoroethanol in step (2) is replaced by hydroxymethylcyclopropane to obtain CL-35. Yield: 14%, ESI-MS m / z: 563 [M+H], 561 [M-H].

[0219] Synthesis of Example 38 CL-37

[0220]

[0221] The difference between Example 34 is that 2,2,2-trifluoroethanol in step (2) is replaced by methoxymethyl to obtain CL-37. Yield: 12%, ESI-MS m / z: 515 [M+H], 513 [M-H].

[0222] Synthesis of Example 39 CL-39

[0223]

[0224] The difference between Example 34 is that 2,2,2-trifluoroethanol in step (2) is replaced by hydroxymethylcyclopropane to obtain CL-39. Yield: 11%, ESI-MS m / z: 555 [M+H], 553 [M-H].

[0225] Test Example

[0226] The RULS (Relative Light Units) of CL-1 to CL-39 were tested, and the test instrument was RLUs (62fg AP / Well). The test results of CL-1 to CL-6 are shown in Table 1, the test results of CL-7 to CL-12 are shown in Table 2, the test results of CL-13 to CL-18 are shown in Table 3, the test results of CL-19 to CL-24 are shown in Table 4, the test results of CL-25 to CL-30 are shown in Table 5, and the test results of CL-31 to CL-39 are shown in Table 6. Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 ​​​​The test results of CL-31 ~ CL-36 are shown in Table 1. Figure 7 The test results of CL-37 ~ CL-39 are shown in Table 1. Figure 8

[0227] The time required for CL-1 ~ CL-39 to reach maximum luminescence (peak) at 25℃ is shown in Table 1.

[0228] Table 1 The time required for CL-1 ~ CL-39 to reach maximum luminescence (peak) at 25℃

[0229]

[0230]

[0231] According to the literature and COA data provided by the manufacturers of AMPPD and CDP-Star, the time required for AMPPD and CDP-Star to reach maximum luminescence (peak) at 25℃ is 30 min ~ 60 min.

[0232] It can be seen that the 1,2-dioxetane derivative provided by the present application has high luminescence efficiency and good stability.

[0233] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A 1,2-dioxetane derivative, characterized in that, A compound having the structure of formula I: In formula I, R is C1-6alkyl, one of the following: R1is H, C1-6alkyl, halogen, one of the following: R2is one of C1-6alkyl, halogen, one of C1-6alkyl, halogen, In the R, R1 and R2, m = 1-6, n = 1-6, x = 1-6, y = 1-6; R1 and R2 are not simultaneously halogen.

2. The 1,2-dioxetane derivative according to claim 1, characterized in that, R is methyl, ethyl, or... One of them; R1is H, Cl, one of the following: R2is methyl, ethyl, Cl, one of methyl, ethyl, Cl, 3. The 1,2-dioxetane derivative according to claim 1 or 2, characterized in that A compound having the structure of any one of formula CL-1 to CL-39:

4. The method for producing the 1,2-dioxetane derivative according to any one of claims 1 to 3, characterized by, The method comprises the following steps: The compound having the structure of formula a is subjected to nucleophilic substitution reaction with 3-hydroxypropionitrile and phosphorus oxychloride to obtain a compound having the structure of formula b; The compound having the structure of formula b is subjected to hydroxylation reaction with methanol and sodium methoxide to obtain a compound having the structure of formula c; The compound having the structure of formula c is subjected to ring formation reaction with sodium molybdate, H2O2 and an alcohol solvent to obtain a compound having the structure of formula I.

5. The production method according to claim 4, characterized by, The molar ratio of the compound having the structure of formula a to phosphorus oxychloride and 3-hydroxypropionitrile is 1:1:(3-5); The molar ratio of the compound having the structure of formula b to sodium methoxide is 1:2-3.

6. The preparation method according to claim 4, characterized in that, The molar ratio of the compound having the structure of formula c to sodium molybdate is 1:

2.

7. The preparation method according to claim 4, characterized in that, The preparation method of the compound having the structure of formula a comprises the following steps: The compound having the structure of formula d is subjected to phosphonation reaction with triethyl phosphite in the presence of boron trifluoride ether to obtain a compound having the structure of formula e; The compound having the structure of formula e is subjected to olefination reaction with a compound having the structure of formula f in the presence of n-butyllithium to obtain a compound having the structure of formula g; The compound having the structure of formula g is subjected to hydroxylation reaction with sodium hydride and ethanethiol to obtain a compound having the structure of formula a.

8. The preparation method according to claim 7, characterized in that, The molar ratio of the compound having the structure of formula d to triethyl phosphite is 1:1-1.5; the molar ratio of triethyl phosphite to boron trifluoride ether is 1:1-1.5; The molar ratio of the compound having the structure of formula e to the compound having the structure of formula f is 1:1-1.

5.

9. The preparation method according to claim 7, characterized in that, The molar ratio of the compound having the structure of formula g to sodium hydride and ethanethiol is 1:(1-1.5):(1-4).

10. Application of the 1,2-dioxetane derivative of any one of claims 1-3 or the 1,2-dioxetane derivative prepared by the preparation method of any one of claims 4-9 as a chemiluminescent substrate.

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