Sulfide prodrug based on butylphthalide as carrier

Abstract

The invention discloses a sulfide prodrug composition based on taking butylphthalide as a carrier, which has a structure as shown in the following formula I. The butylphthalide prodrug can improve the curative effect of the drug and reduce the toxic and side effects of the drug. # imgabs0 #

Description

Technical Field

[0001] The invention relates to a prodrug design which uses butylphthalide as a carrier and releases hydrogen sulfide or sulfide substances by hydrolysis in vivo, and its application in increasing the efficacy of drugs and reducing the toxic and side effects of drugs. Background Art

[0002] In previous studies, hydrogen sulfide was considered a toxic gas with a rotten egg smell, but recent studies have shown that hydrogen sulfide is a third gas signal molecule, which has important physiological effects in many diseases, such as cardiovascular disease, neurodegenerative disease, metabolic disease, etc. It has a good protective effect on cardiovascular and cerebrovascular, gastrointestinal tract, liver, etc. Butylphthalide is a class of compounds found in celery seeds, which has been widely used in cerebral ischemia, but its efficacy is poor in clinical applications, and it often needs to be used in combination with Danshen oral liquid. Summary of the invention

[0003] The purpose of the invention is to provide a class of butylphthalide prodrugs to address the problems of existing butylphthalide, which can increase the efficacy of the drug and reduce its toxic side effects.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] A butylphthalide prodrug or a pharmaceutically acceptable salt thereof has a structure shown in the following formula I:

[0006]

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

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

[0009] The butylphthalide prodrug or the pharmaceutically acceptable salt thereof of the present invention can release hydrogen sulfide or sulfide and the original drug under the action of enzymes in the body, thereby exerting the physiological function of hydrogen sulfide or sulfide, increasing the efficacy of the drug and reducing its toxic side effects.

[0010] The enzyme described in the present invention may include any enzyme that can directly hydrolyze ester bonds or thiocarboxylic acids and directly release H2S or release hydrogen sulfide through chemical conformational changes. The inventors have 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 of the present invention releasing hydrogen sulfide or sulfide and the original drug is as follows:

[0012]

[0013] There are two metabolic processes for prodrugs, mainly through pathway 1 in vitro and pathway 2 in vivo. Pathway 1 is the enzymatic hydrolysis of thiocarboxylic acid to generate carboxylic acid, followed by hydrolysis of ester bonds to generate common lactones; in vivo, the ester bonds are mainly hydrolyzed first to form cyclic thiolactones, which are then hydrolyzed to generate common lactones.

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

[0015]

[0016] The present invention also provides a method for preparing the butylphthalide prodrug shown in formula I:

[0017]

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

[0019] The present invention also provides a pharmaceutical composition, which comprises the butylphthalide prodrug or a pharmaceutically acceptable salt thereof of the present invention, and a pharmaceutically acceptable carrier.

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

[0021] The present invention also provides the use of the hydrogen sulfide prodrug or a pharmaceutically acceptable salt thereof in preparing a drug for treating cerebral stroke, especially ischemic cerebral stroke.

[0022] The compounds of the present invention or their pharmaceutically acceptable salts can be used alone as a single therapeutic drug or in combination with other active drugs.

[0023] Beneficial effects of the present invention:

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

[0025] Figure 1 The hydrogen sulfide release curve of the compound of the present invention is measured by the methylene blue method;

[0026] Figure 2 The persulfide of the present invention releases the persulfide under the action of PLE, the persulfide is captured by DNFB, and the HPLC test result is obtained;

[0027] Figure 3Compound activity data: A is the MTT colorimetric detection of the cytotoxicity (cell survival rate) of the relevant compounds on PC12 cells, and B is the relevant compounds in the cell ischemia-reperfusion injury model, where the drug was administered 24 hours after modeling and the cell survival rate was detected after further culture for 24 hours.

[0028] Figure 4 Compound activity data: A is the detection of cell apoptosis by Houst33258 nuclear staining after compound ZYSH09 modeled PC12 cells, and B is the effect of related compounds on apoptosis caused by ischemia-reperfusion injury at the cellular level (AnnexinV-FITC / PI) measured by flow cytometry 24 hours after drug administration after PC12 modeling.

[0029] Figure 5 Effect of ZYSH09 on SOD activity of PC12 cells injured by ischemia-reperfusion at the cellular level;

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

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

[0032] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0033] Unless otherwise specified, the structures of the compounds were determined by nuclear magnetic resonance (1H-NMR) or high-resolution mass spectrometry (MS). NMR measurements were performed using a Bruker AV-300 / Bruker AV-500 NMR instrument, 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 synthetic routes of the intermediates involved in the following examples are as follows:

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

[0038]

[0039] At -10°C and N2 atmosphere, butylmagnesium bromide (3.2 g, 20 mmol) was added dropwise to a stirred ether solution of 2-formylbenzoic acid (1 g, 6.6 mmol). The reaction was complete after 5 minutes, and then the pH of the reaction solution was adjusted to 3-4, extracted with brine 3 times, and the organic phase was dried over anhydrous Na2SO4, filtered and concentrated to obtain a white solid (1.37 6.6 mmol, 100%), which was directly used for the next reaction.

[0040] (2) Compound 2: Synthesis of 2-(1-acetoxypentyl)benzoic acid

[0041]

[0042] 2-(1-Hydroxypentyl)benzoic acid (5.41 g, 26 mmol), triethylamine (10.98 mL, 79 mmol), DMAP (0.15 g, 1.3 mmol) were added to the DCM solution, and acetyl chloride (2.2 mL, 26 mmol) was added dropwise at -10°C, and stirred for 40 minutes, TLC PE / EA 4:1. After the reaction was completed, the pH of the mixture was adjusted to 2-3, extracted 3 times with DCM, and the combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated to obtain a yellow oil, which was directly put into the next reaction.

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

[0044]

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

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

[0047]

[0048] 2-(1-Hydroxypentyl)benzoic acid (5.41 g, 26 mmol), triethylamine (10.98 mL, 79 mmol), and DMAP (0.15 g, 1.3 mmol) were added to the DCM solution, and isovaleryl chloride (3.16 mL, 26 mmol) was added dropwise at -10°C. The mixture was stirred for 40 min and the reaction was completed after TLCPE:EA 4:1. The mixture was then adjusted to pH 2-3 and extracted 3 times with DCM. The combined organic phases were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated to obtain a yellow oil, which was directly used for the next step reaction.

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

[0050] Example 1 Synthesis of ZYSA09

[0051]

[0052] Synthesis of 2-(1-acetoxypentyl)phenylthio S-acid: Lawesson's reagent (1.75 g, 4.4 mmol) was added to 2-(1-acetoxypentyl)benzoic acid (2 g, 8 mmol) and refluxed in toluene at 130°C for 3 hours. After the reaction, the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography. TLC DCM / EA 4:1. Then, a methanol solution of NaOH (64 mg, 1.6 mmol) was added and ether was added to precipitate to obtain a white precipitate (425 mg, 1.6 mmol, 20%). 1 H NMR(DMSO-d6)δ7.55(dd,J=6.6,2.4Hz,1H),7.20(d,J=6.8Hz,1H),7.10(tt,J=7.8,5.6Hz,2H),6.32(dd,J =8.5,3.8Hz,1H),2.02(d,J=3.8Hz,3H),1.94–1.53(m,2H),1.42–1.11(m,4H),0.84(q,J=4.8,3.5Hz,3H). 13 C NMR(DMSO-d6)δ211.54,169.95,148.41,135.92,128.55,126.68,126.18,124.75,73.22,36.48,27.97,22.47,21.55,14.48.C 14 H 18 O3S(M+Na) + The calculated value is 289.08689, the measured value is 289.08666.

[0053] Example 2 Synthesis of ZYSA10

[0054]

[0055] Synthesis of 2-(1-(isobutyryloxy)pentyl)benzenethio S-acid: To 2-(1-(propionyloxy)pentyl)benzoic acid (2 g, 7.19 mmol) was added Lawesson's reagent (1.59, 0.39 mmol) and refluxed in toluene at 130 ° C for 3 hours. After the reaction, the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography. TLC DCM / EA 4:1. Then, a methanol solution of NaOH (52 mg, 1.3 mmol) was added and the solvent was removed by rotary evaporation to obtain a viscous yellow oil (382 mg, 1.3 mmol, 16%). 1 H NMR(CDCl3)δ7.79(d,J=7.5Hz,1H),7.53(d,J=13.2Hz,2H),7.42–7.31(m,1H),6.14(t,J=6.4Hz,1H), 2.60(p,J=7.0Hz,1H),2.06–1.73(m,2H),1.38(d,J=6.7Hz,4H),1.28(dd,6H),0.92(t,J=6.7Hz,3H). 13 C NMR(CDCl3)δ192.18,176.21,141.40,136.06,132.52,128.88,127.37,126.32,72.39,36.67,34.12,30.94,28.00,22.35,18.92,13.9.C 16 H 22 O3S(M+Na) + HRMS calculated value: 317.11819, found value: 317.11801.

[0056] Example 3 Synthesis of ZYSA11

[0057]

[0058] Synthesis of N-acetyl-S-((2-(1-(isobutyryloxy)pentyl)benzoyl)thio)cysteine: Add 2-(1-(isobutyryloxy)pentyl)phenylthio S-acid (300 mg, 1 mmol) and N-acetyl-S-(pyridin-2-ylthio)cysteine ​​(800 mg, 2 mmol) to DCM / MeOH 20:1 and stir at room temperature for 10 minutes. TLC DCM / EA: 4:1. After the solvent is removed by vortexing, a white foamy substance is obtained. Then a methanol solution of NaOH (52 mg, 1.3 mmol) is added and the solvent is removed by vortexing to obtain a viscous yellow oil (245 mg, 0.53 mmol, 53%). 1H NMR(CDCl3)δ7.92–7.63(m,1H),7.55(t,J=7.1Hz,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=8.2Hz,1H),2.23–2.06(m,3H),1.89(d,J=11.3Hz,2H),1.45–1.09(m,10H),0.92(d,J=8.3Hz,3H). 13 CNMR(CDCl3)δ193.08,176.37,171.87,141.08,134.21,132.85,128.89,12 7.65,126.54,72.44,53.50,41.41,36.72,27.85,22.45,18.89,14.01.HRMS C 21 H 29 NO6S2(M+Na) + Calculated value: 478.13285, actual value: 478.13192.

[0059] Example 4 Synthesis of ZYSO11NAC

[0060]

[0061] Synthesis of 2-(1-((3-methylbutyryl)oxy)pentyl)phenylthio S-acid: Lawesson's reagent (1.51 g, 3.74 mmol) was added to 2-(1-((3-methylbutyryl)oxy)pentyl)benzoic acid (2 g, 6.8 mmol) and refluxed in toluene at 130°C for 3 hours. After the reaction was completed, the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography. TLC DCM / EA 4:1. Then, a methanol solution of NaOH (62 mg, 1.56 mmol) was added and the solvent was removed by rotary evaporation to obtain an oily viscous product (408 mg, 1.56 mmol, 22%). 1 H NMR(CDCl3)δ.79(d,J=7.8Hz,1H),7.52(d,J=4.1Hz,2H),7.36(m,J=8.0,4.2Hz,1H),6.17(t,J=6.5Hz,1H),2.29 –2.20(m,2H),2.11(,J=13.5,6.6Hz,1H),1.81(t,J=6.8Hz,2H),1.45–1.29(m,4H),0.93(m,J=12.9,6.5Hz,9H). 13HRMS C 17 H 24 O3S(M+Na) + Calculated value: 331.13384, actual value: 331.13370.

[0062]

[0063] Example 5 Synthesis of ZYSO12NAC

[0064] Synthesis of S-((2-(1-acetoxypentyl)benzoyl)thio)-N-acetylcysteine: Add 1 equivalent of 2-(1-acetoxypentyl)phenylthio S-acid (300 mg, 1 mmol) and N-acetyl-S-(pyridin-2-ylthio)cysteine ​​(800 mg, 2 mmol) to DCM / MeOH 20:1 and stir at room temperature for 10 minutes. TLC DCM / EA: 4:1. After purification, add a methanol solution of NaOH and then spin off the solvent to obtain a white foam (223 mg, 0.5 mmol, 50%) 1 H NMR(CDCl3)δ7.80(dd,J=17.6,7.6Hz,1H),7.58(s,2H),7.40(s,1H),6.08(d,J=49.2Hz,1H),4.81(d,J=42.9Hz,1H ),3.91–3.29(m,2H),2.17(s,3H),2.09(d,J=8.7Hz,3H),1.89(s,2H),1.38(d,J=7.2Hz,4H),0.92(d,J=5.8Hz,3H). 13 HRMS C 19 H 25 NO6S2(M+Na) + Calculated value: 450.10155, actual value: 450.10097.

[0065] Example 6 Synthesis of ZYSO13NAC

[0066]

[0067] Synthesis of N-acetyl-S-((2-(1-((3-methylbutyryl)oxy)pentyl)benzoyl)thio)cysteine: Add 1 equivalent of 2-(1-((3-methylbutyryl)oxy)pentyl)phenylthio S-acid (320 mg, 1 mmol) and N-acetyl-S-(pyridin-2-ylthio)cysteine ​​(800 mg, 2 mmol) to DCM / MeOH 20:1, and stir the solution at room temperature for 10 minutes. TLC DCM / EA: 4:1. Then add a methanol solution of NaOH, and then spin off the solvent to obtain a viscous yellow oil, and then spin off the solvent to obtain a white foam (231 mg, 0.49 mmol, 49%) 1 H NMR(CDCl3)δ7.89–7.71(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(CDCl3)δ193.26,140.85,134.21,133.14(d,J=22.2Hz),128.60,127.81,12 6.97,51.70,43.74,39.31,36.73,29.72,27.92,25.84,23.33,21.96,13.99.HRMS C 22 H 31 NO6S2(M+Na)+ calculated value: 492.1485, actual value: 492.14803.

[0068] Example 7 Synthesis of ZYSO01

[0069]

[0070] Synthesis of ZYSO01: Butylphthalide was dissolved in toluene and 0.55 equivalents of Lawesson's reagent were added, followed by reflux reaction at 130°C for 12 hours. After the reaction, the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography TLC DCM / EA 4:1. 1H NMR(300MHz, CDCl3)δ8.04(d,J=7.8Hz,1H),7.69(td,J=7.5,1.0Hz,1H),7.54–7.38(m,2H), 5.76(dd,J=7.6,4.3Hz,1H),2.20–1.80(m,2H),1.61–1.33(m,4H),0.92(t,J=7.1Hz,3H).13C NMR(75MHz, CDCl3)δ210.55,147.98,136.86,133.77,129.30,126.88,121.20,90.28,34.02,26.81,22.48,13.89.HRMS calcd for C 12 H 13 OS(MH) - Calculated value: 205.06926, actual value: 205.16005.

[0071] Example 8 Synthesis of ZYSO10NAC

[0072]

[0073] Synthesis of ZYSO10NAC: 1.1 eq of oxalyl chloride and a catalytic amount of DMF were added to 2-(1-acetoxypentyl)benzoic acid (2 g, 8 mmol). After the reaction, the solvent was removed by rotary evaporation and the residue was purified by silica gel chromatography. 1 H NMR(300MHz,DMSO-d6)δ8.34(dd,J=14.7,8.2Hz,1H),7.80(dt,J=13.2,7.4 Hz,1H),7.58(dq,J=15.0,7.4,7.0Hz,2H),7.50–7.31(m,1H),6.02(q,J=6.1 Hz,1H),4.49(dt,J=13.1,6.4Hz,1H),2.04(t,J=3.0Hz,3H),1.87(d,J=7.9H z,3H),1.75(d,J=6.6Hz,2H),1.28(d,J=11.2Hz,4H),0.86(d,J=6.0Hz,3H).

[0074] Example 9 In vitro kinetic studies

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

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

[0077] Table 1

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

[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 cells were plated at 10 cells per well. 4 Cells were inoculated into 96-well plates at a density of 100 cells. After pretreatment for 24 hours, they were incubated with different concentrations of drugs (NBP and Na2S as control drugs) for another 24 hours. 10 μL (5 mg / mL) MTT was added to each well. After incubation at 37°C for 4 hours, the solution was discarded and 100 μL DMSO was added to each well. After gentle shaking for 10 minutes, the plate was read at 570 or 490 nm using a microplate reader. Figure 3 The compounds had no effect on cell growth and proliferation at the indicated low doses.

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

[0083] Preparation of ischemic solution: modified Krebs-Henseleit buffer (137 mM NaCl, 3.8 mM KCl, 0.49 mM MgCl2, 0.9 mM CaCl2, and 4.0 mM HEPES) was used, and 20 mM 2-DOG, 20 mM Sodiumlactate, and 2.5 mM Na2S2O4 were added.

[0084] PC12 cells in the logarithmic growth phase with a cell confluence of 70% were inoculated into DMEM high-glucose medium to a confluence of 30%, cultured for 24 hours, and the old medium was discarded. The cells were washed once with PBS and divided into a blank control group, an ischemic group, and a post-ischemic drug group. The blank control group was added with DMEM medium, and the other groups were added with an equal volume of ischemic solution. The cells were placed in an incubator for 30 minutes to model, the ischemic solution was discarded, the drug was added, and the cells were incubated for 24 / 48 hours for subsequent experiments.

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

[0086] After 24 hours of modeling, the drug-containing culture medium was discarded, and DMEM medium containing 10% FBS was added to continue culturing for 24 hours. MTT was used to measure cell viability. The study found that ZYSA09 at 20 μM and ZYSO13NAC at 10 μM could significantly increase cell survival rate *p<0.05 Figure 3 .

[0087] (2) Hoechst 33258 nuclear staining

[0088] When the cell confluence reached 70%, they were seeded into a 96-well plate and the confluence reached 20%. The drug was administered after 24 hours of culture. After 24 hours 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, and then 1 mL of 0.5 μg / mL Hoechst 33258 staining solution was added to each well in the dark and stained for 25 minutes at room temperature. Washed with PBS three times, observed and photographed under a 400x fluorescence microscope. Hoechst33258 staining detects the morphological characteristics of apoptotic cell nuclear chromatin. Figure 4 As shown in A, compared with the blank control group, the number of apoptotic cells in the ischemic group increased, with obvious nuclear condensation, nuclear rupture and apoptotic bodies, showing strong blue fluorescence. After treatment, the cell nuclei showed no obvious condensation and rupture, showing light blue fluorescence, and were evenly dispersed. Figure 4 A.

[0089] (3) Flow cytometry to measure cell apoptosis

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

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

[0092] After modeling, continue to administer the drug for 24 hours, extract cell protein, and measure SOD activity. The SOD level reflects the ability of cells to cope with oxidative stress levels. The test method is shown in the instructions of the SOD detection kit (Biyuntian, S0109). Figure 5 The experimental results showed that the SOD level was significantly reduced after modeling, and the SOD level was significantly increased after ZYSA09 was given. Compared with the normal group: #P<0.05, compared with the model group: *P<0.05.

[0093] Example 11 Study on drug efficacy in ischemia-reperfusion injury model

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

[0095] Mouse middle cerebral artery occlusion model 25-30g C57 / 6J mice were randomly divided into 14 groups: sham operation group, model group, and different drug administration groups, with 8 mice in each group. We selected NBP and Na2S as positive drugs, and the other two groups were drug administration groups, which were divided into 3 dose groups (1×10 -4 mol / kg,0.5×10 -4 mol / kg,0.25×10 -4 mol / kg). The sham-operated group did not have sutures inserted, and the rest of the groups were subjected to a 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, and a suture (Beijing Xinong Biotechnology Co., Ltd.) was inserted and passed through the ICA until the cerebral artery (MCA) was blocked in the middle. The drug was injected into the tail vein during infarction, and we removed the suture and reperfused 1 hour and 20 minutes after infarction. Other treatments were the same as above. Blood was collected 24 hours later, and the mice were killed by cervical dislocation, the skull was opened, and brain tissue was collected for subsequent experiments.

[0096] 2. Determination of infarct area

[0097] The mouse brain was frozen at -20°C for 20 minutes and 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. Each pair of slices was then photographed, and the cerebral infarction area was calculated using ImageJ. The formula is as follows: Infarction volume = sum of infarct areas / total area. The results showed that both the positive drug and the prodrug ZYSH09 and ZYSO13NAC could significantly reduce the infarct area, and showed a dose-dependent effect. The ZYSA09 group and ZYSO13NAC, ZYSO10NAC were significantly better than the positive drug, among which ZYSO13NAC showed greater superiority in reducing the infarct area. Figure 6 A, 6B.

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

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

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

[0101] Five SD rats were fasted for 10 h before administration and allowed to drink water freely. -4 mol / kg ZYSH09 / ZYSO01, blood samples were collected at 0, 1, 5, 10, 15, 20, 30, 45, and 60 min after administration. 200 μL of methanol was added to the mixture to precipitate proteins. The samples were vortexed for 1 min and heated at 12000 r / min at 4 °C. -1 Centrifuge for 5 minutes, and finally use triple quadrupole liquid chromatography-mass spectrometry to measure the supernatant ( Figure 7 A) Schematic diagram of drug metabolism of related compounds in vivo. ZYSH09 can directly hydrolyze thiocarboxylic acid to generate NBP in vivo, or it can be hydrolyzed to generate ZYSO01 and then enzymatically hydrolyzed to generate ZYSO01( Figure 7 B), ZYSO01 is hydrolyzed in vivo to generate NBP-2 ( Figure 7 C).

Claims

1. A butylphthalide prodrug or a pharmaceutically acceptable salt thereof having a structure as shown in the following formula I: in: R1 is H or R2 is C1-C 10 alkyl.

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 a C1-C6 alkyl group.

5. 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.

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

7. The preparation method of the butylphthalide prodrug according to claim 1: in, R2 is as defined in claim 1.

8. A pharmaceutical composition comprising the butylphthalide prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier.

9. Use of the butylphthalide prodrug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 in the preparation of a drug for treating cerebral stroke.

10. The use according to claim 9, characterized in that: The cerebral stroke is ischemic cerebral stroke.

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