阅读说明：本技术 一种基于丁苯酞结构的前体药物或其药学上可接受的金属盐及其制备方法和应用 (Prodrug based on butylphthalide structure or pharmaceutically acceptable metal salt thereof, and preparation method and application thereof ) 是由 李建波 张金洁 刘志磊 蔡慧杰 高峰 王辰旭 于 2020-12-26 设计创作，主要内容包括：本发明公开了一种基于丁苯酞结构的前体药物或其药学上可接受的金属盐,结构通式I为该前体药物可稳定的靶向胃肠道单羧酸转运体,提高口服生物利用度和增加药物在脑部的聚集浓度。本发明还公开了合成该前体药物的方法,该类前体药物将丁苯酞开环,并对其α-羟基进行酯化修饰,形成具有一定稳定性的酯类前体药物,暴露出游离羧基,可在体内快速代谢为原药发挥治疗作用。本发明还公开了一种该前体药物及其药学上可接受的金属盐在制备预防和治疗心脑缺血性疾病、阻塞性疾病及中枢退行性疾病药物制剂中的应用。该类化合物稳定性优异且不易闭环,便于制备口服制剂。且口服后在体内可快速降解,显著高于原药丁苯酞的作用效果,具有明显的临床优势。(The invention discloses a prodrug based on a butylphthalide structure or a pharmaceutically acceptable metal salt thereof, and the structural general formula I is The prodrug can stably target gastrointestinal monocarboxylic acid transporters, improve oral bioavailability and increase the concentration of the drug accumulated in the brain. The invention also discloses a method for synthesizing the prodrug, the prodrug is prepared by opening the ring of butylphthalide and esterifying and modifying alpha-hydroxyl of butylphthalide to form an ester prodrug with certain stability, and free carboxyl is exposed, so that the butylphthalide prodrug can be rapidly metabolized into a raw drug in vivo to play a therapeutic role. The invention also discloses the application of the prodrug and the pharmaceutically acceptable metal salt thereof in preparing medicinal preparations for preventing and treating ischemic diseases of heart and brain, obstructive diseases and central degenerative diseases. The compound has excellent stability and is not easy to close rings, and is convenient for preparing oral preparations. And the oral liquid can be rapidly degraded in vivo after being taken, is obviously higher than the effect of the original drug butylphthalide, and has obvious clinical advantages.)

1. A prodrug based on butylphthalide structure or a pharmaceutically acceptable metal salt thereof, wherein the prodrug based on butylphthalide structure has the general structural formula I:

wherein R is selected from C₁-C₁₀One of alkyl or halogenated alkyl, aryl and heteroaryl; the chiral center is in R configuration, S configuration or R/S configuration.

2. The butylphthalide structure-based prodrug or a pharmaceutically acceptable metal salt thereof according to claim 1, wherein R is selected from one of methyl, ethyl and n-propyl.

3. The butylphthalide structure-based prodrug or a pharmaceutically acceptable metal salt thereof according to any one of claims 1 to 2, wherein the metal salt is one selected from potassium salt, calcium salt, sodium salt, magnesium salt, aluminum salt, zinc salt, iron salt, lithium salt and calcium salt.

4. The method for preparing the butylphthalide structure-based prodrug or the pharmaceutically acceptable metal salt thereof according to any one of claims 1 to 3, comprising the steps of:

dissolving butylphthalide in an organic solvent A, adding water and inorganic base, mixing, heating for reaction, and performing rotary evaporation to remove the organic solvent A and the water; then adding distilled water to dissolve the product, reacting at-20-10 ℃, dropwise adding dilute hydrochloric acid to adjust the pH value to 1-3, extracting with diethyl ether, and drying with anhydrous sodium sulfate to obtain diethyl ether solution of the product I-1;

continuously adding the organic solvent B into the ether solution of the product I-1 obtained in the previous step, stirring at the temperature of minus 20-10 ℃, then adding the organic base and the catalyst, uniformly mixing, and dropwise adding the acyl chloride solution or the anhydride solution to react to obtain the final product compound I.

5. The method for preparing the butylphthalide structure-based prodrug or the pharmaceutically acceptable metal salt thereof according to claim 4, wherein the organic solvent A is selected from one of methanol, ethanol, tetrahydrofuran, acetone, and acetonitrile;

the inorganic base is selected from one of sodium hydroxide, potassium hydroxide, sodium carbonate and potassium carbonate;

the organic solvent B is one or two selected from diethyl ether, acetonitrile, N-dimethylformamide, tetrahydrofuran, dichloromethane, chloroform and acetone.

6. The method for preparing a butylphthalide structure-based prodrug or a pharmaceutically acceptable metal salt thereof according to claim 4, wherein the organic base is triethylamine.

7. The method for preparing a butylphthalide structure-based prodrug, or a pharmaceutically acceptable metal salt thereof, according to claim 4, wherein the catalyst is dimethylaminopyridine or pyridine.

8. The use of the butylphthalide structure-based prodrug or the pharmaceutically acceptable metal salt thereof according to claim 1 for preparing a pharmaceutical preparation for preventing and treating ischemic cardiovascular and cerebrovascular diseases, obstructive diseases, and central degenerative diseases.

9. The use of the butylphthalide structure-based prodrug or the pharmaceutically acceptable metal salt thereof according to claim 8 for preparing a medicament for preventing and treating cardiac and cerebral arterial occlusive diseases, anti-platelet aggregation and senile dementia, and a medicinal preparation for improving cerebral microcirculation and improving memory.

10. The use according to any one of claims 8 to 9, wherein the formulation is one or more of a tablet, a soft capsule, a hard capsule, a solution, a suspension.

Technical Field

The invention relates to the field of medicines, and relates to a prodrug based on a butylphthalide structure and capable of targeting a gastrointestinal tract monocarboxylic acid transporter or a pharmaceutically acceptable metal salt thereof, and a preparation method and application thereof.

Background

Butylphthalide, chemical name is racemic 3-n-butyl-1 (3H) -isobenzofuranone (NBP), is an oily liquid extracted from southern cress, and has various biological activities of resisting platelet aggregation, inhibiting thrombosis, improving brain microcirculation, regulating brain energy metabolism and the like. Through years of clinical research, NBP tradename Enbipod is a new medicine with independent intellectual property rights in China. The racemate is marketed in 2004, and the oral preparation is soft capsule, is used for treating ischemic stroke diseases and recommends drugs for clinical treatment guidelines of stroke. Recent experimental studies have found that NBP has a positive effect on other central nervous system diseases such as neurodegenerative diseases, in addition to the ability to resist brain damage caused by ischemia.

Considering that NBP has poor water solubility, the NBP is an oily liquid at normal temperature, and compared with a solid bulk drug, a liquid bulk drug is more complex in preparation and higher in production cost, so that the current clinical oral dosage form is a soft capsule with oily contents. But its human oral absolute bioavailability is low [ cesarean. butylphthalide's human pharmacokinetic studies [ D ]. beijing: the result shows that the oral butylphthalide soft capsule has unsatisfactory human oral bioavailability according to the regulation of ' guiding principle of biological equivalence of highly variable drugs ', the variation coefficient is more than 30% and belongs to the highly variable drugs ', and the gelatin of the outer shell of the soft capsule is easy to age in the long-term storage process, so that the disintegration degree of the soft capsule in the stomach is seriously influenced, thereby influencing the action and effect of the drugs and hardly ensuring the curative effect of treating patients clinically. This also suggests the necessity of replacing and improving existing soft capsule delivery systems with oily contents if oral administration is continued clinically.

The open-loop prodrug racemic 2- (alpha-hydroxypentyl) benzoic acid of butylphthalide can automatically synthesize corresponding butylphthalide in vitro and in vivo, the conversion rate reaches over 90 percent, and the butylphthalide has biological activity and pharmacokinetic characteristics similar to those of butylphthalide. Chinese patent ZL01109795.7 discloses a method for salifying an NBP ring-opening compound, namely (R/S) -2- (1-hydroxy-n-pentyl) benzoic acid with potassium, sodium, calcium, magnesium, zinc, aniline, benzylamine, morpholine or diethylamine and application thereof, wherein a potassium salt ((R/S) -PHPB, referred to as PHPB) has higher water solubility and can be converted into NBP in vivo so as to exert anti-cerebral-ischemia activity, and the bioavailability of the NBP ring-opening compound is superior to that of NBP (Acta Pharmacol.sin.,2018,39,275-; chinese patent CN104086399B discloses a 2- (alpha-hydroxypentyl) benzoic acid halogenated derivative drug 5-bromo-2- (alpha-hydroxypentyl) benzoic acid sodium salt for treating cerebrovascular diseases, and the compound has a protective effect on cerebral ischemia reperfusion injury in a rat ischemic stroke animal model, can obviously reduce the cerebral infarction volume and relieve cerebral edema. And the compound is used as a prodrug of the butylphthalide derivative, so that the stability and the water solubility are greatly improved, and the wide application of the medicament is facilitated.

However, the 2- (alpha-hydroxypentyl) benzoic acid and salts thereof have the stability problems of easy ring closure and the like, the requirements on conditions such as pH value, temperature and the like in the preparation process are higher, the production cost is greatly increased, and the oral bioavailability is still lower, so that the distribution of the original drug butylphthalide in the brain is less.

It is well known that absorption of oral drugs in the intestinal tract is not only achieved by simple passive transport but also by drug transporters in the epithelial cells of the intestinal tract, which present a specific transporter protein, namely, monocarboxylate transporter (MCT), which is involved in absorption of monocarboxylate compounds in the intestinal tract. It is now found that there are 18 family members (MCT1-18), and MCT1 transporter plays an important role in promoting nutrient absorption, regulating intracellular pH, and regulating body metabolic balance based on its broad substrate specificity.

Disclosure of Invention

In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a prodrug based on a butylphthalide structure or a pharmaceutically acceptable metal salt thereof, which can target gastrointestinal monocarboxylic acid transporters, improve oral bioavailability and increase brain aggregation concentration.

The invention also aims to provide a method for preparing a prodrug based on the butylphthalide structure or a pharmaceutically acceptable metal salt thereof.

The invention also aims to provide an application of a pro-drug based on a butylphthalide structure or a pharmaceutically acceptable metal salt thereof in preparing a medicinal preparation for preventing and treating heart cerebral ischemic diseases, obstructive diseases and central degenerative diseases.

One of the purposes of the invention is realized by adopting the following technical scheme:

a prodrug based on butylphthalide structure or pharmaceutically acceptable metal salt thereof, wherein the prodrug based on butylphthalide structure has the general structural formula I:

wherein R is selected from C₁-C₁₀One of alkyl, haloalkyl, aryl and heteroaryl of (a); the chiral center is in R configuration, S configuration or R/S configuration.

Further, R is selected from one of methyl, ethyl and n-propyl.

Further, the metal salt is selected from one of potassium salt, calcium salt, sodium salt, magnesium salt, aluminum salt, zinc salt, iron salt, lithium salt and calcium salt.

The second purpose of the invention is realized by adopting the following technical scheme:

a preparation method of a prodrug based on a butylphthalide structure or a pharmaceutically acceptable metal salt thereof comprises the following steps:

dissolving butylphthalide in an organic solvent A, adding water and inorganic base, mixing, heating for reaction, and performing rotary evaporation to remove the organic solvent A and the water; then adding distilled water to dissolve the product, reacting at-20-10 ℃, dropwise adding dilute hydrochloric acid to adjust the pH to 1-3, extracting with diethyl ether, and drying with anhydrous sodium sulfate to obtain diethyl ether solution of the product I-1;

continuously adding the organic solvent B into the ether solution of the product I-1 obtained in the previous step, stirring at the temperature of minus 20-10 ℃, then adding the organic base and the catalyst, uniformly mixing, and dropwise adding the acyl chloride solution or the anhydride solution to react to obtain the final product compound I.

Further, the organic solvent A is selected from one of methanol, ethanol, tetrahydrofuran, acetone and acetonitrile.

Further, the inorganic base is selected from one of sodium hydroxide, potassium hydroxide, sodium carbonate and potassium carbonate.

Further, the organic solvent B is one or two selected from diethyl ether, acetonitrile, N-dimethylformamide, tetrahydrofuran, dichloromethane, chloroform and acetone.

Further, the organic base is triethylamine.

Further, the catalyst is dimethylaminopyridine or pyridine.

The invention also aims to provide an application of a pro-drug based on a butylphthalide structure or a pharmaceutically acceptable metal salt thereof in preparing a medicinal preparation for preventing and treating heart cerebral ischemic diseases, obstructive diseases and central degenerative diseases.

Further, an application of a pro-drug based on a butylphthalide structure or a pharmaceutically acceptable metal salt thereof in preparing a medicament for preventing and treating cardiac and cerebral arterial occlusive diseases, resisting platelet aggregation and senile dementia and a medicinal preparation for improving cerebral microcirculation and improving memory.

Further, the preparation is one or more of tablets, soft capsules, hard capsules, solutions and suspensions.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a prodrug based on a butylphthalide structure or a pharmaceutically acceptable metal salt thereof, which can stably target a gastrointestinal tract monocarboxylic acid transporter, improve oral bioavailability and increase the concentration of the drug in the brain. The invention also provides a method for synthesizing the prodrug or the pharmaceutically acceptable metal salt thereof, the prodrug is prepared by ring-opening butylphthalide, and esterifying and modifying alpha-hydroxyl to form an ester prodrug with certain stability, and free carboxyl is exposed, so that the prodrug can be rapidly metabolized into a raw drug in vivo to play a therapeutic role. The invention also provides an application of the prodrug or the pharmaceutically acceptable metal salt thereof in preparing a medicinal preparation for preventing and treating heart and cerebral ischemic diseases, obstructive diseases and central degenerative diseases. The prodrug or the pharmaceutically acceptable metal salt thereof has good appearance and white solid in physical form, excellent stability, difficult ring closure and convenient preparation of oral preparations. And can be rapidly degraded in vivo after being orally taken, has the oral bioavailability, the brain aggregation and the better drug action effect which are obviously higher than the original drug butylphthalide, and has obvious clinical advantages.

Drawings

FIG. 1 shows the stability of the prodrug based on butylphthalide structure in water in experimental example 1 of the present invention: the residual rate of the compound in water changes at different time points;

FIG. 2 is a graph showing the effect of different external factors on the amount of drug taken into cells in Experimental example 3 of the present invention: comparing the uptake of NBP, compound 1, compound 2 and compound 3 by Caco-2 (human colon adenocarcinoma) cells under the intervention of low temperature and energy inhibitors;

FIG. 3 is a graph showing the effect of different external factors on the amount of drug taken into cells in Experimental example 3 of the present invention: comparing the uptake of NBP, compound 1, compound 2 and compound 3 by Caco-2 (human colon adenocarcinoma) cells under the intervention of a monocarboxylic acid transporter inhibitor and acetylsalicylic acid;

FIG. 4 shows the results of the pharmacokinetic study of the prodrug of the invention 4 based on butylphthalide structure in rats: time-concentration curves of drug in vivo following pharmacokinetic administration of oral NBP, oral compounds 1, 2 and 3 in SD rats;

FIG. 5 is a study of in vivo distribution in mice of the prodrug oral formulation based on butylphthalide structure of Experimental example 5 of the present invention: comparing the drug accumulation amount in different organs of the mice after oral administration of NBP and oral administration of compounds 1, 2 and 3;

FIG. 6 shows the results of the anti-ischemic activity of Experimental example 6 of the present invention: the in vivo SOD concentration of rats after oral administration of NBP, oral compounds 1, 2 and 3 via cerebral ischemia reperfusion;

FIG. 7 shows the results of the anti-ischemic activity of Experimental example 6 of the present invention: in vivo GSH-Px concentration in rats after oral administration of NBP, compounds 1, 2 and 3 via cerebral ischemia-reperfusion;

FIG. 8 shows the results of the anti-ischemic activity of Experimental example 6 of the present invention: in vivo MDA concentration following oral administration of NBP, compounds 1, 2 and 3 orally to rats via cerebral ischemia reperfusion;

in the figure: p < 0.01; b represents p <0.05 compared to saline group; c represents p <0.01 compared to the saline group.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.

Example 1

Dissolving butylphthalide (0.5g,2.6mmol) in 20mL methanol, adding 10mL water and 0.2g sodium hydroxide (5mmol), mixing, heating at 55 deg.C for 0.5h, and rotary evaporating to remove methanol and water; then adding 20mL of distilled water to dissolve the reaction product, stirring for 0.5h at-5 ℃, dropwise adding dilute hydrochloric acid to adjust the pH of the solution to 1-3, extracting with diethyl ether, and drying with anhydrous sodium sulfate to obtain an diethyl ether solution of a product I;

adding 20mL of chloroform into the ether solution of the product I obtained in the step, stirring for 15min at 0 ℃, then adding 0.6mL of triethylamine (5mmol), 0.1g of Dimethylaminopyridine (DMAP), 0.21mL of acetyl chloride (3mmol) and 10mL of chloroform solution, uniformly mixing, then slowly dripping the mixture by using a constant-pressure dropping funnel, stirring for reacting for 12h, detecting by a spot plate, extracting for three times by using water after 5min of dripping is finished, separating an organic layer, drying by using anhydrous sodium sulfate, filtering to obtain concentrated oily liquid, and purifying by using a dichloromethane/methanol column to obtain the compound 1. The yield thereof was found to be 67.4%.

¹H NMR(400MHz,Chloroform-d)δ8.10–8.03(m,3H),7.63–7.50(m,6H),7.38(ddd,J＝8.2,6.0,2.6Hz,3H),6.62(dd,J＝8.4,4.3Hz,3H),2.13(s,7H),1.95–1.85(m,5H),1.85–1.77(m,2H),1.51–1.38(m,9H),1.38(d,J＝2.5Hz,1H),1.38–1.25(m,3H),0.93(t,J＝7.0Hz,9H).

Example 2

The procedure of example 1 was repeated except for replacing 0.21mL of acetyl chloride (3mmol) in the step of example 1 with 0.25mL of propionyl chloride (3 mmol). The yield thereof was found to be 62.1%.

¹H NMR(400MHz,Chloroform-d)δ8.07–8.00(m,1H),7.61–7.49(m,2H),7.36(ddd,J＝8.4,6.4,2.2Hz,1H),6.60(dd,J＝8.3,4.5Hz,1H),2.50–2.30(m,2H),1.86(dtd,J＝15.5,8.5,7.5,4.7Hz,2H),1.47–1.30(m,4H),1.15(t,J＝7.6Hz,3H),0.91(t,J＝7.0Hz,3H).

Example 3

The same procedures used in example 1 were repeated except for changing 0.21mL of acetyl chloride (3mmol) in step 1) of example 1 to 0.32mL of butyryl chloride (3 mmol). The yield thereof was found to be 68.7%.

¹H NMR(400MHz,Chloroform-d)δ8.03(dd,J＝7.6,1.1Hz,1H),7.64–7.49(m,2H),7.36(ddd,J＝8.3,6.2,2.4Hz,1H),6.58(dd,J＝8.2,4.5Hz,1H),2.36(dtd,J＝11.1,7.4,3.5Hz,2H),1.93–1.74(m,2H),1.67(h,J＝7.5Hz,2H),1.38(dddd,J＝17.6,15.0,10.1,4.2Hz,4H),1.07–0.86(m,6H).

Experimental example 1

Stability of compound 1, compound 2, compound 3 in water:

respectively adding 10mL of ultrapure water into three groups of test tubes with plug scales, respectively weighing an appropriate amount of compound, placing in a water bath at 37 ℃, oscillating, sampling at 0h, 0.5h, 1h, 2h, 4h, 8h and 12h, respectively, and centrifuging to determine the content of each compound. The stability of the compound in water was characterized by the residual rate (measured content/initial content) 100%.

The results are shown in fig. 1, and the stability of the compound 1, the compound 2 and the compound 3 in water after 12 hours is examined, the residual rate of the three compounds in water within 12 hours is close to 100%, and the compound prepared by the invention has good stability in water.

Experimental example 2

Preparation and hygroscopicity examination of calcium salts of compound 1, compound 2, compound 3:

preparation of calcium salt of compound 1: NaOH (2.0g, 5) was added to the reaction flask0mmol), 30mL of methanol and a compound 1(5.0g, 17mmol), stirring at room temperature for 2h, adding 1000mL of diethyl ether in batches, immediately precipitating a white solid, continuing stirring for 2h, filtering, and drying in vacuum to obtain 5.1g of a white solid; dissolving the obtained white solid in 200mL of water, and taking CaCl₂(1.1g, 10mmol) was dissolved in 150mL of water to prepare a solution, after the reaction was completed, the solution was cooled to room temperature, and then CaCl was added₂The aqueous solution is slowly dripped into the aqueous solution of the compound 1 under stirring, after dripping for 1h, the stirring is continued for 2h, and after filtration and pumping, vacuum drying is carried out to obtain 3.5g of white solid with the yield of 80.1 percent.

Preparation of calcium salt of compound 2: the preparation process is carried out in 85.7% yield with compound 1 calcium salt.

Preparation of calcium salt of compound 3: the preparation process was carried out in 84.1% yield with the compound 1 calcium salt.

Three calcium salt samples of compounds 1, 2 and 3, 0.2g each, were precisely weighed into a weighing bottle, and the total mass of each sample and the weighing bottle was weighed and recorded. Then placing the mixture in a constant humidity closed environment with the temperature of 25 ℃ and the relative humidity of 75 percent, weighing the total mass in 0, 1, 2, 3, 4 and 5 days respectively, and calculating the moisture absorption weight gain percentage. Percent (%) weight gain upon moisture absorption (mass of sample after moisture absorption-mass of sample before moisture absorption)/mass of sample before moisture absorption × 100%. The results of percentage increase in moisture absorption (%) of compound 1, compound 2 and compound 3 are shown in table 1:

TABLE 1

Time(d)	1	2	3	4	5
						Compound 1	0.53±0.4	0.71±0.29	0.80±0.16	0.92±0.36	1.19±0.08
Compound 2	0.41±0.71	0.65±0.31	0.81±0.54	0.93±0.66	1.21±0.11
						Compound 3	0.48±0.55	0.51±0.39	0.62±0.66	0.74±0.16	1.02±0.14

Hygroscopicity is a very important property during the preparation and storage of a drug, and directly affects the stability of the drug and even the action effect of the drug. The results of the determination of the moisture absorption weight gain percentage of the compound 1, the compound 2 and the compound 3 are shown in table 1, and the moisture absorption weight gains of the three compounds after being placed in a constant humidity closed environment with the temperature of 25 ℃ and the relative humidity of 75 percent for 5 days are respectively 1.19 +/-0.08 percent, 1.21 +/-0.11 percent and 1.02 +/-0.14 percent. According to the rules of pharmacopoeia, the compounds 1 to 3 have slight hygroscopicity, and reasonable hygroscopicity, thereby laying a foundation for subsequent research and providing more possibility for the pharmaceutical property of the compounds.

Experimental example 3

Influence on the amount of drug taken up by cells under different external factor intervention conditions:

(1) caco-2 cells (human colon adenocarcinoma cells) were cultured at 4 x 10⁵Each 1.5mL of the suspension was inoculated into a 6-well plate and cultured in Solebao 1640. When the cells grow to about 80%, the medium is discarded.

(2) Adding 1.5mL of culture medium containing compounds 1 to 3, respectively, culturing for 1h, immediately discarding the culture medium, gently washing the cells with 1mL of PBS three times, sucking up PBS with a pipette, adding 200. mu.L of PBS, and mixing. And (3) repeatedly freezing and thawing at 37-80 ℃ for three times, taking 10 mu L of the protein, measuring the protein content after 5 times of dilution by using a BCA method, and operating according to the instruction. And adding 360 mu L of methanol into 120 mu L of the residual sample to precipitate the protein, vortexing for 5min and centrifuging at 12000rpm for 10min, and measuring the concentrations of intracellular butylphthalide (NBP) and compounds 1-3 by using 20 mu L of the mixture through a high performance liquid phase.

(3) Effect of Low temperature, energy inhibitor intervention on the amount of drug taken up by cells

The incubation concentration of the drug was determined to be 400. mu.M for 1 hour, and sodium azide (NaN) at 4 ℃ was examined₃) And effect on cellular uptake at 37 ℃. Wherein sodium azide (NaN)₃) For energy inhibitor, the cells cultured in 37 ℃ incubator without any addition are used as a control group, the transport function of the cells is normal under the condition of 37 ℃, and exogenous substances can enter the cells through passive diffusion or mediated by corresponding transporters. Prior to the experiment, the cells were incubated at 4 deg.C (on ice or in a refrigerator) with 1mg/mL NaN₃The medium and the 37 ℃ cell culture box are respectively balanced for 60min, then 400 mu M of the medicine is added into each hole, and the incubation is carried out for 60 min. The subsequent operation is the same as the step (2).

Results are shown in fig. 2, which shows the comparison of the uptake of NBP, compound 1, compound 2, compound 3 by Caco-2 (human colon adenocarcinoma) cells under low temperature and energy inhibitor intervention: compound 1, compound 2, and compound 3 all showed significant differences compared to NBP. Compared with the content of the medicine in the cells cultured in the medium at 37 ℃, the content of the compound 1, the compound 2 and the compound 3 in the cells cultured in the medium containing the energy inhibitor sodium azide at the low temperature of 4 ℃ is obviously reduced. Temperature and energy have a large influence on the transport of compounds 1 to 3 into cells.

(4) Effect of monocarboxylic acid transporter inhibitors, acetylsalicylic acid on the amount of drug taken up by cells

To the culture medium of Caco-2 cells, 100 μ M acetylsalicylic acid (Aspirin, prodrug competitive inhibitor), CHC (monocarboxylic acid transporter inhibitor) and Control (Control) were added, respectively, to compare the difference in the uptake of butylphthalide and its prodrug.

As shown in fig. 3, the uptake of NBP, compound 1, compound 2, compound 3 by Caco-2 (human colon adenocarcinoma) cells under the intervention of monocarboxylate transporter inhibitors, acetylsalicylic acid, was compared: upon addition of the monocarboxylic acid transporter inhibitor CHC, uptake of NBP was almost unchanged, but uptake of compounds 1 to 3 was significantly reduced. And upon addition of acetylsalicylic acid, which competes with the prodrug for inhibition, the uptake of compounds 1 to 3 was also significantly reduced, indicating that the prodrug is dependent on the transport of the monocarboxylic acid transporter into the cell in an active transport manner.

Experimental example 4

Preparation of capsules containing compounds 1, 2 and 3:

weighing 5g of calcium salt of the compound 1, adding 132g of microcrystalline cellulose, 70g of mannitol, 90g of sodium carboxymethyl starch and 3g of magnesium stearate, mixing, sieving for three times by a 60-mesh sieve, performing dry granulation, and filling into 1000 capsules.

Weighing 5g of compound 2 calcium salt, adding 132g of microcrystalline cellulose, 70g of mannitol, 90g of sodium carboxymethyl starch and 3g of magnesium stearate, mixing, sieving for three times by a 60-mesh sieve, performing dry granulation, and filling into 1000 capsules.

Weighing 5g of compound 3 calcium salt, adding 132g of microcrystalline cellulose, 70g of mannitol, 90g of sodium carboxymethyl starch and 3g of magnesium stearate, mixing, sieving for three times by a 60-mesh sieve, performing dry granulation, and filling into 1000 capsules.

Pharmacokinetic studies of the prodrug based on butylphthalide structure in rats:

30 SD mice (male, 200 +/-5 g) were taken, fasted for 12h before the experiment, and freely drunk water, and the experiment was randomly divided into 5 groups, namely oral administration groups of butylphthalide, compounds 1, 2 and 3. Compound 1, compound 2 and compound 3 are administered in equimolar amounts per 30mg/kg butylphthalide. All groups were bled at the angle at the indicated time points and plasma was measured by centrifugation. 0.1mL of plasma was collected and placed in a 0.5mL EP tube, and 0.3mL of methanol was added to precipitate the protein. Vortex and shake for 5min, centrifuge at 10000rpm for 10min, sample 50 μ L of supernatant, determine drug concentration at each time point by HPLC, and calculate AUC.

Experimental results as shown in fig. 4, time-concentration curves of drug in vivo after pharmacokinetic administration of oral NBP, oral compounds 1, 2 and 3 in SD rats: the AUC of the oral compound 1, the oral compound 2 and the oral compound 3 of the SD rat respectively reach 89.45, 73.97 and 10.71 mu g/L.min, while the AUC of the NBP group is only 1.405 mu g/L.min, the AUC of the compounds 1 to 3 are respectively 63, 52 and 8 times higher than that of the original drug, and the oral bioavailability is greatly improved. And compound 1, compound 2, compound 3C orally administered to rats_max(maximum in vivo drug concentration) 28.91, 29.27 and 12.83. mu.g/mL, respectively, whereas NBP has a C_maxOnly 0.75. mu.g/mL, C_maxThe improvement is 39 times, 39 times and 17 times respectively.

Experimental example 5

Study of in vivo distribution in mice of prodrug oral formulations based on butylphthalide structure and its derivatives:

125 Kunming mice (male, 20 +/-2 g) are taken, fasting is carried out for 12h before the experiment, water can be freely drunk in the fasting period, the experiment is randomly divided into 4 groups, butylphthalide is administered at 30mg/kg, and the compounds 1, 2 and 3 are orally administered, and the administration amount of the oral administration group is equal to that of butylphthalide. All groups were sacrificed after blood was collected from the mouse eyeballs 15min after administration, and the mouse Heart (Heart), Liver (Liver), Spleen (Spleen), Lung (Lung), Kidney (Kidney) and Brain (Brain) were immediately separated, washed with physiological saline and dried by blotting with filter paper, weighed and homogenized with 2 times the mass of physiological saline. 0.1mL of each homogenate was placed in a 0.5mL EP tube and precipitated by adding 0.3mL of methanol protein. Vortex and shake for 5min, centrifuge at 10000rpm for 10min, sample 50 μ L of supernatant, and determine the concentration of each test sample by HPLC.

The results are shown in fig. 5, comparing the accumulation of NBP orally administered and compounds 1, 2 and 3 orally administered in different organs of mice: the results show that compared with oral NBP, the oral compounds 1, 2 and 3 have higher accumulation concentration in heart and brain, increase the accumulation concentration of the heart and brain drugs and are more beneficial to the drug effect.

Each pharmacokinetic parameter is calculated by DAS3.2.5 software, wherein the targeting evaluation indexes are relative uptake rate Re and peak concentration ratio Ce for evaluating whether the medicament has brain targeting, the target evaluation index is greater than 1, the medicament has brain targeting, the larger the value is, the better the brain targeting effect is, and the calculation formula is as follows:

Re_brain＝(AUC_{0-t, brain})_{Compound (I)}/(AUC_0-t,brain)_{Butylphthalide}

Ce_Brain＝(C_max,brain)_{Compound (I)}/(C_max,brain)_{Butylphthalide}

TABLE 2

Target evaluation index	NBP	Compound 1	Compound 2	Compound 3
					Re	/	2.94	2.11	1.35
Ce	/	1.61	1.24	1.06

The evaluation results of the brain targeting of the mice after oral administration of the drug are shown in table 2, and compared with the original drug, the compound 1, the compound 2 and the compound 3 show better brain targeting, the peak concentration ratio Ce and the relative uptake Re are all greater than 1, the brain aggregation is obvious, and the compound 1 shows better targeting.

Experimental example 6

Anti-cerebral ischemia activity study:

establishing a middle cerebral artery occlusion model of a rat: model preparation A rat focal cerebral ischemia reperfusion model was prepared by a wire-plug method. The rat was fixed on the operating table in supine position, and 1% of pentobarbital sodium was intraperitoneally injected with anesthetized rat at a dose of 40mg/kg, the anterior cervical region was sterilized, the median longitudinal incision of the neck was used, the muscle and fascia were bluntly isolated, the right Common Carotid Artery (CCA) was fully exposed, and the vagus nerve was carefully isolated. The External Carotid Artery (ECA) and the Internal Carotid Artery (ICA) were then sequentially dissected. The ECA is ligated, the ICA distal end is closed by a arteriole clamp, the CCA is ligated again, a small opening is cut at about 10mm below the bifurcation of the CCA, a thread plug is inserted, the arteriole clamp for closing the ICA is released, and the thread plug is slowly fed into the ICA until slight resistance is met, at which time the insertion depth (18.0 +/-0.5) mm of the thread plug is reached. The external thread plug is painted black to facilitate the thread drawing and the incision suturing during the reperfusion. And (3) reperfusion is carried out after 1h, namely the wire plug is pumped into CCA, the blood supply of the middle cerebral artery is recovered, 20mg/kg of butylphthalide group is orally taken, and the compounds 1, 2 and 3 are orally taken according to the equal molar quantity of the dose of the butylphthalide group. The sham operation group is not inserted with a wire plug, and the neck operation and the blood vessel treatment are the same as the model group. The whole process is carried out at room temperature (24-25 ℃). The successful model mark is Horner's sign and hemiplegia mainly in contralateral forelimb after the model animal is anaesthetized and awake. Rat reperfusion for 24h for determining various pathological indexes.

The results of the measurement of the cerebral tissue infarct volume and the inhibition rate of the infarct volume by using Image J are shown in table 3, and compared with the model group, each administration group significantly reduced the infarct area of the cerebral tissue of rats, wherein the compound 1 had the strongest inhibitory effect in the oral group, and the infarct volume ratio was 23.17 ± 4.51%. The infarct volume after oral administration of the compound 1, the compound 2 and the compound 3 groups was significantly lower than that of the oral NBP group, and the inhibition rate of the infarct volume of the compound 1 was the greatest.

TABLE 3

SOD (superoxide dismutase), an antioxidant enzyme that scavenges free radicals and reduces reactive oxygen levels, is often used in conjunction with MDA (malondialdehyde), a lipid peroxide product that is cytotoxic, and GSH-Px (glutathione peroxidase), a superoxide dismutase, to assess cellular hyperoxia levels. In order to detect the antioxidant level of the compound obtained by the invention, the brain of an experimental rat is cut off 24 hours after reperfusion, ischemic lateral brain tissue is taken, residual blood is removed by flushing, and the compound is accurately weighed immediately after being sucked dry. Weight (mg) on ice bath: adding 0.9% physiological saline (1:9) into the mixture (uL) to prepare 10% homogenate at 3000 r.min^-1Centrifuging for 10min, collecting supernatant, and storing in 20 deg.C refrigerator. Adding samples and reagents according to the kit operation instructions, fully and uniformly mixing, taking 200 mu L of liquid from each tube, respectively adding the liquid into a disposable colorimetric plate with 96 holes, measuring the absorbance of the liquid in each hole by an enzyme-labeling instrument, and calculating the SOD activity, the MDA content and the GSH-Px activity according to a formula.

FIG. 6, FIG. 7, and FIG. 8 show the concentrations of SOD, GSH-Px, and MDA in mice after oral administration of NBP and oral administration of compounds 1, 2, and 3 after cerebral ischemia reperfusion, and after cerebral ischemia, the values of SOD and GSH-Px are significantly reduced, MDA is significantly increased, and reactive cell free radicals are increased. The compound 1, the compound 2 and the compound 3 have significant difference with the normal saline group, and have better antioxidant effect.

The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.