A sulfide prodrug based on butylphthalide as a carrier

Abstract

The application discloses a sulfide prodrug combination based on butylphthalide as a carrier, which has the structure shown in the following formula I. The butylphthalide prodrug can increase the curative effect of drugs and reduce the toxic side effects.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of butylphthalide as carrier, in vivo hydrolysis release hydrogen sulfide or sulfide substance of prodrug design and its in increasing drug efficacy, reduce the application of drug side effects. BACKGROUND

[0002] In the past research, hydrogen sulfide is considered as a kind of toxic gas with rotten egg smell, but recent research shows that hydrogen sulfide is the third gas signal molecule, has important physiological role in a variety of diseases, such as cardiovascular disease, neurodegenerative disease, metabolic disease and so on, has good protection to cardiovascular, gastrointestinal, liver, etc. Butylphthalide is a kind of compound found in celery seed, has been widely used in cerebral ischemia, but in clinical application, it is found that its drug effect is poor, often need to be combined with Danshen oral liquid. SUMMARY

[0003] The purpose of the application is to solve the problems existing in the prior art butylphthalide, provide a kind of butylphthalide prodrug, which can increase the efficacy of the drug and reduce its side effects.

[0004] The purpose of the application is achieved by the following technical solutions:

[0005] A kind of butylphthalide prodrug or its pharmaceutically acceptable salt has the structure shown in the following formula I:

[0006]

[0007] Among them: R1 is H or R2 is C1-C 10 alkyl.

[0008] In some preferred examples, R2 is C1-C6 alkyl; more preferably methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, sec-butyl or isobutyl.

[0009] The butylphthalide prodrug or its pharmaceutically acceptable salt according to the application can release hydrogen sulfide or sulfide and the original drug under the action of enzyme in vivo, thereby exerting the physiological function of hydrogen sulfide or sulfide, increasing the efficacy of the drug and reducing its side effects.

[0010] The enzyme according to the application can include any enzyme capable of directly hydrolyzing ester bond or thiocarboxylic acid and directly releasing H2S or releasing hydrogen sulfide through chemical modification, the inventors found through various in vivo and in vitro experiments that various enzymes in vivo can achieve this function.

[0011] The mechanism of the butylphthalide prodrug according to the application releasing hydrogen sulfide or sulfide and the original drug is as follows:

[0012]

[0013] The prodrug has two metabolic processes, in vitro mainly through pathway 1, and in vivo mainly through pathway 2, pathway 1 is enzymatic hydrolysis of a thio carboxylic acid to generate a carboxylic acid, and then hydrolysis of an ester bond to finally generate a common lactone; in vivo, it is mainly hydrolyzed into an ester bond to form a cyclic thiolactone, and then hydrolyzed to generate a common lactone.

[0014] The application also provides the following specific prodrugs of butylphthalide or pharmaceutically acceptable salts thereof:

[0015]

[0016] The application also provides a preparation method of the prodrug of butylphthalide shown in formula I:

[0017]

[0018] wherein, the definition of R2 is as described above.

[0019] The application also provides a pharmaceutical composition comprising the prodrug of butylphthalide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0020] The hydrogen sulfide prodrug or the pharmaceutically acceptable salt thereof can be administered to a subject with the compound of the application or a pharmaceutical composition containing the composition of the application in an environment sufficient to form hydrogen sulfide or persulfide and the original drug.

[0021] The application also provides the use of the hydrogen sulfide prodrug or the pharmaceutically acceptable salt thereof in the preparation of a drug for treating stroke; in particular, ischemic stroke.

[0022] The compound of the application or the pharmaceutically acceptable salt thereof can be used alone as a single therapeutic drug, or can be used in combination with other active drugs.

[0023] The application has the following beneficial effects:

[0024] The sulfide prodrug system of butylphthalide as a carrier in the application releases hydrogen sulfide or sulfide and the original drug under the action of enzymes in vivo and in vitro, and can increase the efficacy of the drug. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a methylene blue method for determining the hydrogen sulfide release curve of the compound of the application;

[0026] Figure 2 is the release of persulfide under the action of PLE of the persulfide of the application, the capture of persulfide by DNFB, and the HPLC detection results;

[0027] Figure 3Compound activity data: A is the MTT colorimetric assay related to the cytotoxicity (cell survival rate) of the compound to PC12 cells, B is the cell survival rate detected after 24h of related compound administration after modeling in the cell ischemia-reperfusion injury model, and continued culture for 24h.

[0028] Figure 4 Compound activity data: A is the Houst33258 nuclear staining detection of cell apoptosis of compound ZYSH09 after modeling of PC12 cells, B is the effect of related compounds on cell level ischemia-reperfusion injury apoptosis (Annexin V-FITC / PI) detected by flow cytometry after 24h of administration after PC12 modeling.

[0029] Figure 5 Effect of ZYSH09 on SOD activity of PC12 cells at cell level ischemia-reperfusion injury;

[0030] Figure 6 Effect of related compounds on MCAO model: A is TTC staining of brain tissue 24h after MCAO ischemia-reperfusion injury; B is quantitative analysis of ZYSH09 on infarction area by TTC staining, C) serum LDH level of MCAO model mice, D) MDA level of brain tissue of MCAO model mice.

[0031] Figure 7 For the drug metabolism process of ZYSH09 / ZYSO01 in vivo, A) schematic diagram of in vivo metabolism of related compounds, B) metabolic pathway of ZYSH09 in vivo, C) metabolism of ZYSO01 in vivo. DETAILED DESCRIPTION

[0032] The following examples facilitate a better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the test materials used are commercially available from conventional biochemical reagent stores unless otherwise specified.

[0033] Unless otherwise specified, the structure of the compound is determined by nuclear magnetic resonance (1H-NMR) or high-resolution mass spectrometry (MS). NMR measurement is performed using a Bruker AV-300 / Bruker AV-500 nuclear magnetic resonance spectrometer, with deuterated dimethyl sulfoxide (DMSO-D6) or deuterated chloroform (CDCl3) as the solvent, and TMS as the internal standard.

[0034] In the following examples, the structures of NBP and NBP-2 are as follows:

[0035]

[0036] Unless otherwise specified, the synthesis route of the intermediates involved in the following examples is as follows:

[0037] (1) Synthesis of intermediate 1: 2-(1-hydroxypentyl)benzoic acid

[0038]

[0039] To a stirred solution of 2-formylbenzoic acid (1 g, 6.6 mmol) in ether was added dropwise butyl magnesium bromide (3.2 g, 20 mmol) at -10 °C under N2atmosphere. The reaction was complete after 5 min, then the pH of the reaction was adjusted to 3-4 and extracted with brine 3 times, the organic phase was dried over anhydrous Na2SO4, filtered and concentrated to give a white solid (1.37 6.6 mmol, 100%) which was directly used in the next reaction.

[0040] (2) Synthesis of compound 2: 2-(1-acetyloxy-pentyl)benzoic acid

[0041]

[0042] To a solution of 2-(1-hydroxypentyl)benzoic acid (5.41 g, 26 mmol), triethylamine (10.98 mL, 79 mmol), DMAP (0.15 g, 1.3 mmol) in DCM was added dropwise acetyl chloride (2.2 mL, 26 mmol) at -10 °C, stirred for 40 min, TLC PE:EA 4:1. The reaction was complete, then the pH of the mixture was adjusted to 2-3 and extracted with DCM 3 times, the combined organic phase was washed with brine, dried over anhydrous Na2SO4, filtered, concentrated to give a yellow oil which was directly used in the next reaction.

[0043] (3) Synthesis of compound 3: 2-(1-(isobutyryloxy)pentyl)benzoic acid

[0044]

[0045] To a solution of 2-(1-hydroxypentyl)benzoic acid (5.41 g, 26 mmol), triethylamine (10.98 mL, 79 mmol), DMAP (0.15 g, 1.3 mmol) in DCM was added dropwise isobutyryl chloride (2.74 mL, 26 mmol) at -10 °C, stirred for 40 min, TLC PE:EA 4:1. The reaction was complete, then the pH of the mixture was adjusted to 2-3 and extracted with DCM 3 times, the combined organic phase was washed with brine, dried over anhydrous Na2SO4, filtered, concentrated to give a yellow oil which was directly used in the next reaction.

[0046] (4) Compound 4: 2-(1-((3-methylbutanoyl)oxy)pentyl)benzoic acid

[0047]

[0048] To a solution of DCM was added 2-(1-hydroxypentyl)benzoic acid (5.41 g, 26 mmol), triethylamine (10.98 mL, 79 mmol), DMAP (0.15 g, 1.3 mmol), isovaleryl chloride (3.16 mL, 26 mmol) was added dropwise at -10 °C, stirred for 40 min, TLC PE:EA 4:1 reaction was complete, then the mixture was adjusted to pH 2-3 and extracted with DCM 3 times, the combined organic phase was washed with brine, dried over anhydrous Na2S04, filtered, concentrated to give a yellow oil, which was directly used in the next step.

[0049] The synthesis of the relevant end product is as follows:

[0050] Example 1 Synthesis of ZYSA09

[0051]

[0052] Synthesis of 2-(1-acetyloxy-pentyl)-benzenethioic acid S-acid: To 2-(1- acetyloxy-pentyl)-benzoic acid (2 g, 8 mmol) was added Lawesson's reagent (1.75 g, 4.4 mmol) in toluene and refluxed at 130 °C for 3 h. After the reaction the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography. TLC DCM / EA 4:1. NaOH (64 mg, 1.6 mmol) in methanol was added afterwards and the precipitate was filtered to give a white solid (425 mg, 1.6 mmol, 20%) 1 H NMR (DMSO-d6) δ 7.55 (dd, J = 6.6, 2.4 Hz, 1H), 7.20 (d, J = 6.8 Hz, 1H), 7.10 (tt, J = 7.8, 5.6 Hz, 2H), 6.32 (dd, J = 8.5, 3.8 Hz, 1H), 2.02 (d, J = 3.8 Hz, 3H), 1.94 - 1.53 (m, 2H), 1.42 - 1.11 (m, 4H), 0.84 (q, J = 4.8, 3.5 Hz, 3H). 13 C NMR (DMSO-d6) δ 211.54, 169.95, 148.41, 135.92, 128.55, 126.68, 126.18, 124.75, 73.22, 36.48, 27.97, 22.47, 21.55, 14.48.C 14 H 18 O3S (M+Na) + Calculated 289.08689, found: 289.08666.

[0053] Example 2 Synthesis of ZYSA10

[0054]

[0055] 2-(1-(isobutyryloxy)pentyl)benzenethioic acid S-acid: To 2-(1-(propionyloxy)pentyl)benzoic acid (2 g, 7.19 mmol) was added Lawesson's reagent (1.59, 0.39 mmol) in toluene and refluxed at 130 °C for 3 h. After the reaction, the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography. TLC DCM / EA 4:1. After the addition of NaOH (52 mg, 1.3 mmol) in methanol, the solvent was removed to give a sticky yellow oil (382 mg, 1.3 mmol, 16%) 1 H NMR (CDCI3) δ 7.79 (d, J = 7.5 Hz, 1 H), 7.53 (d, J = 13.2 Hz, 2 H), 7.42 - 7.31 (m, 1 H), 6.14 (t, J = 6.4 Hz, 1 H), 2.60 (p, J = 7.0 Hz, 1 H), 2.06 - 1.73 (m, 2 H), 1.38 (d, J = 6.7 Hz, 4 H), 1.28 (dd, 6 H), 0.92 (t, J = 6.7 Hz, 3 H). 13 C NMR (CDCI3) δ 192.18, 176.21, 141.40, 136.06, 132.52, 128.88, 127.37, 126.32, 72.39, 36.67, 34.12, 30.94, 28.00, 22.35, 18.92, 13.9.C 16 H 22 O3S(M+Na) + HRMS calcd for C20H28NO3S 317.11819, found: 317.11801.

[0056] Example 3 Synthesis of ZYSA11

[0057]

[0058] Synthesis of N-acetyl-S-((2-(1-(isobutyryloxy)pentyl)benzoyl)thio)cysteine: To 2-(1-(isobutyryloxy)pentyl)benzenethioic acid S-acid (300 mg, 1 mmol) and N-acetyl-S-(pyridin-2-ylthio)cysteine (800 mg, 2 mmol) was added DCM / MeOH 20:1 and stirred at room temperature for 10 min. TLC DCM / EA: 4:1. After the addition of NaOH (52 mg, 1.3 mmol) in methanol, the solvent was removed to give a white foamy material. After the addition of NaOH (52 mg, 1.3 mmol) in methanol, the solvent was removed to give a sticky yellow oil (245 mg, 0.53 mmol, 53%) 1H NMR (CDCI3) δ 7.92 - 7.63 (m, 1H), 7.55 (t, J = 7.1 Hz, 2H), 7.40 - 7.33 (m, 1H), 6.20 - 5.77 (m, 1H), 5.07 - 4.64 (m, 1H), 3.67 (m, 2H), 2.58 (d, J = 8.2 Hz, 1H), 2.23 - 2.06 (m, 3H), 1.89 (d, J = 11.3 Hz, 2H), 1.45 - 1.09 (m, 10H), 0.92 (d, J = 8.3 Hz, 3H). 13 CNMR (CDCI3) δ 193.08, 176.37, 171.87, 141.08, 134.21, 132.85, 128.89, 127.65, 126.54, 72.44, 53.50, 41.41, 36.72, 27.85, 22.45, 18.89, 14.01. HRMS C 21 H 29 NO6S2 (M + Na) + Calculated 478.13285, Actual: 478.13192.

[0059] Example 4 Synthesis of ZYSO11NAC

[0060]

[0061] Synthesis of 2-(l-((3-methylbutanoyl)oxy)pentyl)benzenethio S-acid: To 2-(l-((3-methylbutanoyl)oxy)pentyl)benzoic acid (2 g, 6.8 mmol) was added Lawesson's reagent (1.51 g, 3.74 mmol) and refluxed in toluene at 130 °C for 3 h. After completion of the reaction, the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography. TLC DCM / EA 4: 1. To this was added NaOH (62 mg, 1.56 mmol) in methanol and the solvent was removed to get the product as an oily sticky mass (408 mg, 1.56 mmol, 22%). 1 H NMR (CDCI3) δ 7.92 - 7.63 (m, 1H), 7.55 (t, J = 7.1 Hz, 2H), 7.40 - 7.33 (m, 1H), 6.20 - 5.77 (m, 1H), 5.07 - 4.64 (m, 1H), 3.67 (m, 2H), 2.58 (d, J = 8.2 Hz, 1H), 2.23 - 2.06 (m, 3H), 1.89 (d, J = 11.3 Hz, 2H), 1.45 - 1.09 (m, 10H), 0.92 (d, J = 8.3 Hz, 3H). 13C NMR (CDC13) δ 192.21, 172.36, 141.25, 136.12, 132.47, 127.40, 126.57, 72.40, 43.67, 36.57, 30.93, 27.99, 25.77, 22.49, 13.98. HRMS C 17 H 24 O3S(M+Na) + Calcd 331.13384, Found: 331.13370.

[0062]

[0063] Example 5 Synthesis of ZYSO12NAC

[0064] Synthesis of S-((2-(l-acetyloxy-pentyl)benzoyl)thio)-N-acetyl cysteine: 1 equivalent of 2-(l-acetyloxy-pentyl)benzene thio S-acid (300 mg, 1 mmol) and N-acetyl-S-(pyridin-2-ylthio) cysteine (800 mg, 2 mmol) were added to DCM / MeOH 20:1, stirred at room temperature for 10 minutes. TLC DCM / EA: 4:1. After purification, NaOH in methanol was added, and the solvent was spun off to give a white foamy material (223 mg, 0.5 mmol, 50%) 1 H NMR (CDC13) δ 7.80 (dd, J = 17.6, 7.6 Hz, 1H), 7.58 (s, 2H), 7.40 (s, 1H), 6.08 (d, J = 49.2 Hz, 1H), 4.81 (d, J = 42.9 Hz, 1H), 3.91 - 3.29 (m, 2H), 2.17 (s, 3H), 2.09 (d, J = 8.7 Hz, 3H), 1.89 (s, 2H), 1.38 (d, J = 7.2 Hz, 4H), 0.92 (d, J = 5.8 Hz, 3H). 13 C NMR (CDC13) δ 193.25, 170.73, 140.63, 134.54, 132.79, 128.82, 127.72, 126.89, 72.73, 54.22, 41.75, 36.53, 27.81, 22.50, 21.18, 14.00. HRMS C 19 H 25 NO6S2(M+Na) + Calcd 450.10155, Found: 450.10097.

[0065] Example 6 Synthesis of ZYSO13NAC

[0066]

[0067] Synthesis of N-acetyl-S-((2-(l-((3-methylbutanoyl)oxy)pentyl)benzoyl)thio)cysteine: 1 equivalent of 2-(l-((3-methylbutanoyl)oxy)pentyl)benzenethioate (320 mg, 1 mmol) and N-acetyl-S-(pyridin-2-ylthio)cysteine (800 mg, 2 mmol) were added to DCM / MeOH 20:1 and the solution was stirred at room temperature for 10 minutes. TLC DCM / EA: 4:1. A solution of NaOH in methanol was added and the solvent was removed to give a sticky yellow oil which was removed to give a white foamy material (231 mg, 0.49 mmol, 49%) 1 H NMR (CDCI3) δ 7.89 - 7.71 (m, 1H), 7.67 - 7.51 (m, 2H), 7.44 - 7.39 (m, 1H), 6.18 - 5.91 (m, 1H), 5.03 - 4.77 (m, 1H), 3.89 - 3.18 (m, 2H), 2.27 - 2.12 (m, 5H), 2.00 - 1.80 (m, 2H), 1.51 - 1.31 (m, 4H), 0.98 - 0.87 (m, 9H). 13 CNMR (CDCI3) δ 193.26, 140.85, 134.21, 133.14 (d, J = 22.2 Hz), 128.60, 127.81, 126.97, 51.70, 43.74, 39.31, 36.73, 29.72, 27.92, 25.84, 23.33, 21.96, 13.99. HRMS C 22 H 31 NO6S2 (M+Na)+calcd 492.1485, found: 492.14803.

[0068] Example 7 Synthesis of ZYSO01

[0069]

[0070] Synthesis of ZYSO01 : Butyl orthophthalide was dissolved in toluene and 0.55 equivalents of Lawesson’s reagent were added and the reaction was refluxed at 130 °C for 12 hours. After the reaction was complete, the solvent was removed by rotary evaporation and the residue was purified using silica gel chromatography TLC DCM / EA 4:1. 1H NMR (300 MHz, CDC13) δ 8.04 (d, J = 7.8 Hz, 1H), 7.69 (td, J = 7.5, 1.0 Hz, 1H), 7.54 - 7.38 (m, 2H), 5.76 (dd, J = 7.6, 4.3 Hz, 1H), 2.20 - 1.80 (m, 2H), 1.61 - 1.33 (m, 4H), 0.92 (t, J = 7.1 Hz, 3H).13C NMR (75 MHz, CDC13) δ 210.55, 147.98, 136.86, 133.77, 129.30, 126.88, 121.20, 90.28, 34.02, 26.81, 22.48, 13.89. HRMS calcd for C 12 H 13 OS(M-H) - Calcd 205.06926, Found: 205.16005.

[0071] Example 8 Synthesis of ZYSO10NAC

[0072]

[0073] Synthesis of ZYSO10NAC: To 2-(l-acetyloxy-pentyl)-benzoic acid (2 g, 8 mmol) was added 1.1 eq of oxalyl chloride and catalytic amount of DMF. After reaction, the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography. 1 H NMR (300 MHz, DMSO-d6) δ 8.34 (dd, J = 14.7, 8.2 Hz, 1H), 7.80 (dt, J = 13.2, 7.4 Hz, 1H), 7.58 (dq, J = 15.0, 7.4, 7.0 Hz, 2H), 7.50 - 7.31 (m, 1H), 6.02 (q, J = 6.1 Hz, 1H), 4.49 (dt, J = 13.1, 6.4 Hz, 1H), 2.04 (t, J = 3.0 Hz, 3H), 1.87 (d, J = 7.9 Hz, 3H), 1.75 (d, J = 6.6 Hz, 2H), 1.28 (d, J = 11.2 Hz, 4H), 0.86 (d, J = 6.0 Hz, 3H).

[0074] Example 9 In vitro kinetic study

[0075] The compound was prepared as a 500 μΜ stock solution, and esterase (PLE) was added to a final concentration of 3 unit / mL, and stirred at 37 °C. The rate of drug release of hydrogen sulfide under the action of PLE was analyzed by methylene blue method.

[0076] The experimental results are shown in Table 1 Figure 1As shown, by HPLC analysis of the rate of compound hydrolysis by PLE, it was found that under the action of PLE, the compounds described in the examples can be hydrolyzed by PLE to carboxylic acid from thiocarboxylic acid, while releasing hydrogen sulfide; its t 1 / 2 As shown in Table 1. From the data of compounds ZYSH09-ZYSH11 in Table 1, it can be seen that changing the size of the protection of butylphthalide can change the release rate of hydrogen sulfide, and from the data of ZYSO11NAC-ZYSO13NAC in Table 1, it can be seen that PLE can hydrolyze the thioester bond, and under the same experimental conditions, the release rate of the compound with the thioester bond is faster than that of the compound with-SH. The released peroxide is difficult to detect by high-resolution mass spectrometry due to its high activity and high reactivity, so we use electrophilic reagents to capture the enzymatic products of ZYSO11NAC. We synthesized DNB-NAC as a reference, and as shown in Figure 2 We successfully captured peroxide by DNFB.

[0077] Table 1

[0078] Compound ZYSH11 ZYSH10 ZYSH09 <![CDATA[t 1 / 2 ]]> 415 ± 4.1 min 900 ± 60 min 1400 ± 200 min Compound ZYSO11NAC ZYSO12NAC ZYSO13NAC <![CDATA[t 1 / 2 ]]> 178.80075 ± 2 min 13.5 ± 0.5 min 13.2 ± 1.5 min

[0079] Example 10 Study of drug efficacy in an in vitro ischemia-reperfusion injury model

[0080] 1. Drug cytotoxicity study

[0081] PC12 cells were used in this study. PC12 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum (Gemni, A84F00i) and 1% penicillin-streptomycin (shenggong, shanghai) at 37°C and 5% CO2. The viability and proliferation of PC12 were measured by MTT colorimetric assay, PC12 were seeded into 96-well plates at a density of 10 4 cells per well, and after 24 hours of pre-treatment, different concentrations of drugs (NBP and Na2S as control drugs) were incubated for another 24 hours, 10 μL (5 mg / mL) MTT was added to each well, and after 4 h of incubation at 37°C, the solution was discarded, and 100 μL DMSO was added to each well. After gently shaking for 10 minutes, the plate was read at 570 or 490 nm using a microplate reader. As shown in Figure 3 The compounds had no effect on cell growth and proliferation at low doses.

[0082] 2. Effect of drugs on cell survival and apoptosis in an ischemia-reperfusion injury model at the cellular level

[0083] Ischemia solution configuration: using contains modified krebs-Henseleit buffer solution (137 mM NaCl, 3.8 mM KCl, 0.49 mM MgCl2, 0.9 mM CaCl2 and 4.0 mM HEPES), and add 20 mM 2-DOG, 20 mM Sodium lactate, 2.5 mM Na2S2O4.

[0084] PC12 cells in logarithmic growth phase with a confluence of 70% were seeded in DMEM high glucose medium to reach a confluence of 30%, cultured for 24 h, and the old culture medium was discarded. The cells were washed once with PBS and divided into blank control group, ischemia group and post-ischemia drug administration group. The blank control group was added with DMEM medium, and the rest groups were added with the same volume of ischemia solution. They were placed in the incubator for 30 minutes to model, the ischemia solution was discarded, the drugs were added, and the incubation was continued for 24 / 48 hours for subsequent experiments.

[0085] (1) Effect of drugs on cell survival.

[0086] The culture medium containing drugs was discarded after 24 hours of modeling, and DMEM medium containing 10% FBS was added for continuous culture for 24 hours, and MTT was used to determine cell viability. It was found that ZYSA09 at 20 μM and ZYSO13 NAC at 10 μM could significantly increase cell survival rate *p<0.05 as Figure 3 .

[0087] (2) Hoechst 33258 cell nucleus staining

[0088] When the confluence of cells reached 70%, they were seeded in 96-well plates to reach a confluence of 20%, and drug administration was performed after 24 h of culture. After 24 h of drug culture, the culture medium was discarded, and the cells were washed once with PBS. The cells were fixed with 4% paraformaldehyde at 4°C for 10 minutes, then 1 mL of 0.5 μg / mL Hoechst 33258 staining solution was added to each well in the dark, and stained at room temperature for 25 minutes. Washed with PBS for 3 times, observed and photographed under 400 times fluorescence microscope. Hoechst 33258 staining detects the morphological characteristics of apoptotic cell nucleus chromatin. As Figure 4 A, compared with the blank control group, the number of apoptotic cells in the ischemia group increased, with obvious nuclear condensation and rupture and apoptotic bodies, showing strong blue fluorescence. After treatment, the cell nucleus showed no obvious condensation and rupture, and showed light blue fluorescence, which was uniformly dispersed as Figure 4 A.

[0089] (3) Flow cytometry determination of cell apoptosis

[0090] After modeling, the administration was continued for 24 h, and the anti-apoptotic effect of ZYSA09 was further confirmed by flow cytometry Annexin V-FITC / PI, and 5 μM ZYSH09 showed better anti-apoptotic potential than 5 μM Na2S and BNP as Figure 4 B.

[0091] (4) Effect of drugs on superoxide dismutase (SOD) activity of cells after ischemia-reperfusion injury.

[0092] After modeling, the administration was continued for 24 h, and the anti-apoptotic effect of ZYSA09 was further confirmed by flow cytometry Annexin V-FITC / PI, and 5 μM ZYSH09 showed better anti-apoptotic potential than 5 μM Na2S and BNP as Figure 5 The experimental results showed that the SOD level was significantly reduced after modeling, and the SOD level was significantly increased when ZYSA09 was administered. Compared with the normal group: #P<0.05, compared with the model group: *P<0.05.

[0093] Example 11 Pharmacodynamic study in ischemia-reperfusion injury model

[0094] 1. Establishment of animal ischemia-reperfusion injury model

[0095] The 25-30 g C57 / 6J mice were randomly divided into 14 groups: sham operation group, model group, different administration groups, 8 mice in each group. We selected NBP and Na2S as positive drugs, and the remaining two groups were administration groups, which were divided into 3 dose groups (1 x 10 -4 mol / kg, 0.5 x 10 -4 mol / kg, 0.25 x 10 -4 mol / kg). The sham operation group was not inserted with the thread, and the rest of the groups were subjected to the middle cerebral artery occlusion model. Anesthesia was induced with 3% isoflurane, and anesthesia was maintained with 0.6% isoflurane after anesthesia. After disinfection, a midline incision was made in the neck. Blunt dissection was performed to expose the left common carotid artery (CCA), internal carotid artery (ICA) and external carotid artery (ECA). The proximal end of the CCA was separated from the distal end of the ECA and ligated. The CCA was ligated at the distal end, the suture thread (Beijing Xinnong Biological Technology Co., Ltd.) was inserted and passed through the ICA, and the middle cerebral artery (MCA) was blocked. The administration was injected through the tail vein during the occlusion, and 1 hour and 20 minutes after the occlusion, we pulled out the suture thread and reperfused. Other treatments were the same as above. After 24 hours, the blood was collected, the mice were sacrificed by cervical dislocation, the skull was opened, and the brain tissue was collected for subsequent experiments.

[0096] 2. Measurement of infarct size

[0097] The mouse brain was frozen at -20°C for 20 minutes, cut into 5 pieces, each 1 mm thick. Each slice was immediately stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution at 37°C for 30 minutes and fixed in 4% paraformaldehyde (PFA) solution. The infarct area was not stained and the normal area was stained red. Then each pair of slices was photographed and the brain infarction area was calculated by ImageJ. The formula is as follows: infarction volume = sum of infarction area / total area. The results of the study showed that the positive drugs and prodrugs ZYSH09, ZYSO13NAC can significantly reduce the infarction area, and show a dose-dependent effect ZYSA09 group ZYSO13NAC, ZYSO10NAC drug efficacy is significantly better than the positive drug, among which ZYSO13NAC shows greater superiority in reducing infarction area Figure 6 A, 6B.

[0098] 3. Determination of serum LDH levels and brain tissue LDA levels

[0099] After blood collection, centrifuge at 3000g for 10 minutes at 4°C to collect serum. The determination method of LDH is described in the kit (solarbio, Beijing) instruction. The brain tissue is taken to determine the MDA level in the tissue, and the determination method is described in the MDA kit (solarbio, Beijing) instruction. LDH level reflects the damage to cell membrane caused by ischemia-reperfusion injury, and MDA level reflects the peroxidation of membrane lipids caused by ischemia-reperfusion injury. It is found that ZYSA09 has greater potential in MCAO model, and 0.25 x 10 - 4 mol / kg ZYSA09 can significantly reduce the LDH level and MDA level of the model group, and show significant difference with the NBP group**P<0.01 Figure 6 C, 6D.

[0100] Example 12 Release of original drug of ZYSH09 / ZYSO01 in vivo in rats

[0101] Take 5 SD rats, fast for 10h before administration, free water, give 8.13 x 10 -4 mol / kg ZYSH09 / ZYSO01, collect blood samples at 0, 1, 5, 10, 15, 20, 30, 45, 60 min after administration respectively. Add 200 μL methanol to the mixture to precipitate proteins. Mix the sample by vortex for 1 minute, centrifuge at 12000 r·min -1 for 5 minutes at 4°C, and finally determine the supernatant by triple quadrupole liquid chromatography-mass spectrometry Figure 7 A) Schematic diagram of in vivo drug metabolism of related compounds, ZYSH09 can be directly hydrolyzed to sulfocarboxylic acid to generate NBP in vivo, or can be hydrolyzed to generate ZYSO01 and then be enzymatically degraded to generate ZYSO01Figure 7 B), ZYSO01 hydrolyzes in vivo to generate NBP-2 Figure 7 C).

Claims

1. A class of butylphthalide prodrugs or pharmaceutically acceptable salts thereof, having the structure shown in Formula I: in: R1 is H or R2 is a C1-C6 alkyl group.

2. The butylphthalide prodrug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, R1 is H.

3. The butylphthalide prodrug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, R1 is 4. The butylphthalide prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, characterized in that, R2 is methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, or isobutyl.

5. A butylphthalide prodrug or a pharmaceutically acceptable salt thereof having the following structure:

6. The method for preparing the butylphthalide prodrug according to claim 1: in, R2 is defined as described in claim 1.

7. A pharmaceutical composition comprising the butylphthalide prodrug of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

8. The use of the butylphthalide prodrug of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 7, in the preparation of a medicament for treating stroke.

9. The application according to claim 8, characterized in that, The stroke mentioned is ischemic stroke.

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