Compounds for the treatment of ischemic brain injury related disorders

Abstract

The application discloses a compound for treating diseases related to ischemic brain injury, and the compound has the structure shown in the following formula (II). The compound has higher bioavailability, higher distribution concentration in rat plasma and brain tissue, and good effect on ischemic brain injury.

Description

TECHNICAL FIELD

[0001] The present application relates to a compound having anti-cerebral ischemia, anti-post-ischemic inflammation and anti-convulsant effects, capable of improving neurological impairment of ischemic stroke patients, a pharmaceutical composition and a method for treating ischemic cerebral neuron injury and necrosis related diseases. TECHNICAL BACKGROUND

[0002] The brain white matter is an important part of the central nervous system, and is a place where nerve fibers gather. White matter lesions (WML) are usually caused by reduced blood flow, insufficient oxygen supply of blood vessels. Cerebral blood supply disorder (insufficiency) that is difficult to meet the metabolic needs of brain tissue, thus producing a series of symptoms. Clinically, it can be manifested as dizziness, headache, limb numbness or transient loss of consciousness, and severe cases can cause irreversible damage to brain function and even death. Cerebral ischemia related diseases include transient ischemic attack (TIA), ischemic stroke (cerebral infarction), moyamoya disease, chronic cerebral hypoperfusion, etc., and is one of the causes of cognitive decline and vascular dementia in patients.

[0003] At present, there are many drugs for treating diseases caused by cerebral ischemia, but only a few are truly effective. The existing drug nimodipine has a preventive effect on cerebral ischemia, but the therapeutic effect is not certain. 3-n-butylphthalide (NBP) extracted from celery seed volatile oil was approved in China in 2005 for the treatment of mild to moderate acute ischemic stroke, but the mechanism is still unclear, and adverse reactions such as abnormal liver function and digestive tract reactions can occur during clinical use.

[0004] Cerebral ischemia can cause varying degrees of brain nerve injury and necrosis, leading to dysfunction of the corresponding system of the human body, greatly reducing the quality of life of patients, and there is still a need to provide other compounds with better efficacy, having anti-cerebral ischemia, anti-post-ischemic inflammation and anti-convulsant effects, capable of improving neurological impairment of ischemic stroke patients, and can improve memory impairment, protect nerve cells and blood-brain barrier, etc. SUMMARY

[0005] The present application provides a novel compound having anti-cerebral ischemia, anti-post-ischemic inflammation and anti-convulsant effects, capable of improving neurological impairment of ischemic stroke patients, for treating ischemic cerebral neuron injury and necrosis related diseases.

[0006] The present application provides a compound of formula (II) having the following structure, or a pharmaceutically acceptable salt thereof:

[0007]

[0008] The present application further provides a compound of formula (I) having the following structure, or a pharmaceutically acceptable salt thereof:

[0009]

[0010] The present application also provides a pharmaceutical composition comprising the compound of formula (II) or a pharmaceutically acceptable salt thereof according to the present application and one or more pharmaceutically acceptable carriers.

[0011] The present application also provides a pharmaceutical composition comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present application and one or more pharmaceutically acceptable carriers.

[0012] The present application also provides the use of the compound of formula (II) or a pharmaceutically acceptable salt thereof according to the present application in the manufacture of a medicament for treating and / or preventing a disease related to ischemic brain injury.

[0013] The present application also provides the use of the compound of formula (I) or a pharmaceutically acceptable salt thereof according to the present application in the manufacture of a medicament for treating and / or preventing a disease related to ischemic brain injury.

[0014] The present application also provides the use of the composition according to the present application in the manufacture of a medicament for treating and / or preventing a disease related to ischemic brain injury.

[0015] The disease related to ischemic brain injury according to the present application includes but is not limited to ischemic stroke, vascular dementia, inflammation after cerebral ischemia, convulsion, ischemic brain neuronal injury or necrosis, etc.

[0016] The present application also provides a method for synthesizing the compound of formula (II) and the compound of formula (I) according to the present application.

[0017] In a specific example, the present application also provides a method for preparing the compound of formula (I),

[0018]

[0019] The present application also provides a single crystal of the compound of formula (I) according to the present application, having the following cell parameters:

[0020]

[0021] In a specific example, the asymmetric unit of the single crystal of the compound of formula (I) is as shown in the following figure: Figure 2

[0022] The present application also provides a method for preparing the single crystal, wherein the compound of formula (I) according to the present application is dissolved in petroleum ether, filtered, the filtrate is covered with a pinhole membrane, and the solvent is slowly evaporated at room temperature in a ventilated environment.

[0023] The compound according to the present application has higher bioavailability and higher distribution concentration in rat plasma and brain tissue, and has good effect on ischemic brain injury. BRIEF DESCRIPTION OF DRAWINGS ​

[0024] Figure 1 A single crystal microscope image of (Z)-3-(1-hydroxybutenyl)benzofuran-2-one;

[0025] Figure 2 An asymmetric unit of a single crystal of (Z)-3-(1-hydroxybutenyl)benzofuran-2-one. DETAILED DESCRIPTION

[0026] The application will be further described in conjunction with specific examples in light of ordinary knowledge and skill in the art. The following examples are only some of the preferred embodiments of the application and should not be deemed as limiting the application. Those skilled in the art can make several improvements without departing from the scope of the application, and these improvements should also be deemed as falling within the protection scope of the application.

[0027] Example 1

[0028] Synthesis of (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one

[0029]

[0030] A solution of 3-benzofuranone (5.0 g, 37.3 mmol, 1.0 eq.) in dichloromethane (50 mL) was cooled to 5 °C, and potassium tert-butoxide (6 g, 53.5 mmol, 1.4 eq.) was slowly added. After 0.5 h, n-butyryl chloride (8 g, 75.0 mmol, 2.0 eq.) was slowly added, and the reaction was allowed to proceed for 1 h. Water (50 mL) was added to extract the product, and the organic phase was washed with 0.1 N hydrochloric acid until the solution was acidic (pH < 2). The organic phase was dried with anhydrous sodium sulfate (5 g) for 0.5 h, filtered, and concentrated to obtain an oil. The oil was purified by high-pressure preparative chromatography, and the purified solution was freeze-dried to obtain (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one (2.5 g, 32.9%) as a white solid.

[0031] 1H NMR (CDCI3, 400 MHz): δ 12.02 (s, 1H), 7.35-7.33 (d, 1H), 7.28-7.17 (m, 3H), 2.76-2.73 (m, 2H), 1.90-1.80 (m, 2H), 1.12-1.08 (m, 3H).

[0032] 1H NMR (CDCI3+D2O, 400 MHz): δ 7.34-7.32 (d, 1H), 7.28-7.16 (m, 3H), 2.76-2.72 (m, 2H), 1.89-1.80 (m, 2H), 1.12-1.08 (m, 3H).

[0033] Preparation of (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one single crystal

[0034] The 20 mg (0.098 mmol) of (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one prepared in Example 1 was dissolved in petroleum ether (0.4 mL), after filtration, the filtrate was covered with a pinhole membrane, placed in a fume hood, and the solvent was slowly evaporated at room temperature, 24 h to obtain (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one single crystal Figure 1 ).

[0035] The X-ray data of (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one single crystal was collected on a Bruker D8 Venture diffractometer, with a Mo target Kα ray as the light source During the data collection process, the crystal was kept at 296 K. Single crystal structure analysis was performed in Olex2 software, the initial structure was calculated using the Intrinsic Phasing method of the SHELXT program, and the structure refinement was completed by the least squares method of the SHELXL program. The crystallographic data and structure refinement parameters of (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one are shown in Table 1, and the asymmetric unit of (Z)-3-(1-hydroxybutenyl)benzofuran-2-ketone single crystal is shown in Figure 2 .

[0036] Table 1. Crystallographic data and structure refinement parameters of (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one

[0037]

[0038] Biological assay:

[0039] The following assays demonstrate that the compound I of the present application has the effect of improving ischemic neurological dysfunction. The results of the assays also show that the compound I of the present application has good bioavailability and efficacy after oral administration.

[0040] Test 1: Rat pharmacokinetic test

[0041] SD male rats (body weight 180-260 g) were given compound I at a dose of 1.0 mg / kg by tail vein injection and at a dose of 10.0 mg / kg by oral administration, 3 animals in each group. The administration solvent was a 5% DMSO + 5% Cremophor EL solution in normal saline. The animals were fasted for about 12 hours before administration, and were allowed to eat freely 4 hours after administration; no water was restricted. About 0.2 mL of blood was collected from the orbit before administration and at 5, 15, 30 min, 1, 2, 4, 6, 8, 24 h after administration, and was placed in an EDTA-K2 anticoagulated EP tube in an ice bath, 4°C, 3500 rpm low-speed centrifugation for 10 minutes to separate the plasma, which was stored at -20°C until analysis. The concentration of compound I in the plasma was quantitatively analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method. The WinNonlin software was used to calculate the pharmacokinetic parameters from the sample analysis results.

[0042] As can be seen from the data in Table 2, after oral administration, the elimination time of compound I in rats is longer, and the bioavailability is higher (the bioavailability is more than 100% which is speculated to be caused by non-linear pharmacokinetics) compared with the literature (Wang Ningning, Li Yue, Li Xiaohong, Jiang Mingyan, RP-HPLC method for determining the content of 3-n-butylphthalide in rat plasma and its pharmacokinetics, Chinese New Drugs and Clinical Journal, December 2012, Vol. 31, No. 12, pp. 743-747) reported value.

[0043] Table 2. Pharmacokinetic parameters

[0044]

[0045] * According to the AUC listed in the table 0-inf Literature value calculation

[0046] Test 2: Rat brain tissue distribution test

[0047] Male SD rats (body weight 200-270 g) were orally administered with compound I and butylphthalide (NBP) at a dose of 20 mg / kg, respectively. Plasma and brain tissue samples were collected from the animals at 0.5, 1, 4, 24 h after administration. Plasma collection: whole blood 0.2 mL was collected in an EP tube containing EDTA-K2, and after centrifugation at 3500 g for 10 minutes, the upper plasma was collected and stored at -20°C. Brain tissue collection: after the animals were euthanized, an appropriate amount of brain tissue was weighed, and homogenized according to brain tissue: 80% methanol water (w / v) = 1:4. The concentration of the compound in the plasma and brain tissue samples was quantitatively analyzed using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method.

[0048] As can be seen from the data in Table 3, after oral administration, the distribution concentration of compound I of the application in the plasma and brain tissue of rats is higher.

[0049] Table 3 Concentration of compounds in rat plasma and brain tissue

[0050]

[0051] NA: below the lower limit of quantification

[0052] Test 3: Pharmacodynamics study of compounds in rat stroke model: single treatment administration

[0053] The middle cerebral artery occlusion (MCAO) model of SD rats was constructed by using a thread plug method to evaluate the neuroprotective effect of the compounds on rat cerebral ischemia-reperfusion. After SD rats (240-270 g) were induced for anesthesia with 3.0% isoflurane, the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were surgically exposed. The ECA was ligated, the ICA was temporarily clamped, and a thread was passed through the CCA near the heart and the distal end, which was tied tightly. A small incision was made between the two threads, and a 4-0 thread plug was inserted through the incision of the CCA, then gently pushed into the internal carotid artery, and stopped at the position of the ICA clip. The pre-ligature was further tightened, and the ICA clip that blocked the blood flow was removed. The thread plug was immediately pushed into the ICA until it entered the intracranial cavity. When the thread plug was inserted to a depth of about 18 mm from the bifurcation of the common carotid artery, there was a slight resistance, indicating that the head of the thread plug had entered the anterior cerebral artery (ACA), and the thread plug had blocked the opening of the middle cerebral artery. At this time, the insertion was stopped, and the time was recorded. The ICA clip was removed, and the incision was closed after observing no active bleeding. The ischemic rats were placed at room temperature to maintain a body temperature of 37°C, and after 120 min, the rats were induced for anesthesia. Under the maintenance of anesthesia, the thread plug was gently pulled back to the external carotid artery, which achieved reperfusion of the middle cerebral artery. The animals were administered once immediately after reperfusion (within 10 min), and three groups were set up, namely the model control group, the compound I intravenous group (30 mg / kg), and the compound I oral group (60 mg / kg). The animals were euthanized 24 h after ischemia-reperfusion, and the brains were quickly removed and frozen for sectioning and TTC staining. Normal tissue was stained pink, and infarct tissue was white. The percentage of infarct tissue weight to whole brain weight was used as the infarction area (%), which was used to evaluate the degree of ischemic injury in rats.

[0054] One day after surgery, TTC staining analysis showed that the cerebral infarction range in the model control group was 21.63±5.66%. The cerebral infarction ranges in the intravenous and oral groups of Compound I were 13.61±3.66% and 14.88±5.11%, respectively. This indicates that both the intravenous and oral groups of Compound I significantly reduced the cerebral infarction range (P=0.0025 and P=0.0389). Furthermore, the cerebral infarction inhibition rates in the intravenous and oral groups of Compound I were 37.1% and 31.2%, respectively. These results suggest that Compound I effectively improves cerebral infarction in rats, as detailed in Table 4.

[0055] Table 4. Infarct extent and infarct inhibition rate in experimental animals

[0056]

[0057] Note: All data in the table are expressed as mean ± standard deviation (Mean ± SD); “N” represents the number of animals in each group for statistical analysis, “—” indicates that there is no data for that item, * indicates P < 0.05 compared with the model control group, and ** indicates P < 0.01 compared with the model control group.

[0058] Experiment 4: Pharmacodynamic study of the compound in a medium- to long-term rat model of stroke

[0059] A middle cerebral artery occlusion (MCAO) model was established in SD rats using the suture occlusion method. The test drug was administered for 28 consecutive days. The pharmacodynamics of the test drug in the treatment of stroke was evaluated through general observation and neurobehavioral scoring.

[0060] SD rats (240-280g) were anesthetized with 3.0% isoflurane before surgery to expose the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The ECA was ligated, and the ICA was temporarily clamped. Sutures were threaded through the proximal and distal ends of the CCA, with the distal end tied tightly and the proximal end loosely knotted for later use. A small incision was made between the two sutures, and a 4-0 suture plug was inserted through the CCA incision. It was then slowly and gently pushed into the internal carotid artery, pausing at the arterial clamp of the ICA. The pre-ligated suture was further tightened, and the arterial clamp blocking blood flow to the ICA was removed. The suture plug was immediately pushed into the ICA until it entered the intracranial cavity. When the suture plug was inserted to a depth of approximately 18mm from the bifurcation of the common carotid artery, a slight resistance was felt, indicating that the tip of the suture plug had entered the anterior cerebral artery (ACA), and the lateral wall of the suture plug had blocked the opening of the middle cerebral artery. At this point, insertion was stopped, and the time was recorded. After removing the arterial clamp on the CCA and observing no active bleeding, the incision was closed. Ischemic rats were kept at room temperature to maintain a body temperature of 37°C. Anesthesia was induced after 120 minutes. While maintaining anesthesia, the suture plug was slowly and gently pulled back into the external carotid artery, thus achieving reperfusion of the middle cerebral artery. The animals were administered the drug immediately after reperfusion (within 10 minutes), once daily for 28 consecutive days. The day of surgery was defined as Day 0. Five groups were established: a sham-operated group, a model control group, a butylphthalide (NBP) administration group (60 mg / kg, po, qd), a low-dose oral compound I group (6 mg / kg, po, qd), and a high-dose oral compound I group (20 mg / kg, po, qd). During the administration period, all animals underwent grid test (D7, D14, D21 and D28, a total of 4 times) and new object recognition test (new object recognition adaptation on D26 and detection on D27). After the administration was completed on D28, all surviving animals were euthanized and their brains were quickly harvested for pathological analysis.

[0061] (1) The grid test results showed that one week after surgery, the number of missteps in the model control group was 7.24±3.59 times, while the numbers in the NBP treatment group, the low-dose compound I group, and the high-dose compound I group were 7.37±3.03 times, 5.33±2.33 times, and 4.23±1.44 times, respectively. The number of missteps in the high-dose compound I group was significantly lower than that in the model control group (P=0.0172). Two to three weeks after surgery, as the motor function of the model group gradually recovered, the number of missteps gradually decreased. Although there was no significant difference in the number of missteps in the high-dose compound I group compared with the model control group, there was still a decreasing trend. Four weeks after modeling, the number of missteps in the model animals decreased to the level of the sham-operated group. The above results suggest that compound I has the effect of improving walking dysfunction in stroke animals (see Table 5 for details).

[0062] Table 5. Grid Test Results

[0063]

[0064]

[0065] Note: All data in the table are expressed as mean ± standard deviation (Mean ± SD); "N" represents the number of animals in each group for statistical analysis; "1W, 2W, 3W, 4W" represent 1, 2, 3, and 4 weeks after surgery, respectively; * indicates P < 0.05 compared with the model control group.

[0066] (2) On the last day of treatment, a new object recognition test was conducted, and the new object recognition index (NRI) of each group of animals was calculated. The new object recognition index of the model control group was 54.81±21.94%, which was not significantly different from the old object recognition index (FRI). The NRI of the NBP treatment group, the low-dose group and the high-dose group of compound I were 70.97±22.57%, 70.98±22.60% and 71.66±17.06%, respectively. Among them, there were significant differences in the new / old object recognition index of the NBP treatment group, the low-dose group and the high-dose group of compound I (P=0.0074, P=0.0212 and P=0.0009). At the same time, the new object recognition index of the low-dose group and the high-dose group of compound I was comparable to that of the sham surgery group (66.51±10.80%). The above results suggest that compound I has a significant effect on improving cognitive impairment in animals with stroke (see Table 6 for details).

[0067] Table 6. Results of the New Object Recognition Experiment

[0068]

[0069] Note: All data in the table are expressed as mean ± standard deviation (Mean ± SD); "N" represents the number of animals in each group for statistical analysis; * indicates P < 0.05 compared with the model control group, ** indicates P < 0.01 compared with the model control group, and *** indicates P < 0.001 compared with the model control group.

[0070] (3) Pathological examination: 1) Repair range: Samples with a repair area >30% were considered to have good recovery, while samples with ≤30% were considered to have poor recovery. The proportion of animals with good recovery in the model control group was 17.6%. The proportions of animals with a repair area >30% in the NBP treatment group, the low-dose group and the high-dose group of compound I were 21.4%, 33.3% and 75.0%, respectively. The high-dose group of compound I had the best recovery, which was significantly different from the model control group (Chi-square, P=0.0080). The above results suggest that compound I has the effect of promoting the repair of the infarct area (see Table 7 for details); 2) Number of vessels filling the infarct repair area: Two criteria were set: ≤10 and >10 red blood cell vessels in the infarct repair area. In the sham surgery group, all animals had more than 10 engorged blood vessels, accounting for 100%. In the model control group, 10 animals had ≤10 erythrocyte vessels and 7 animals had >10 erythrocyte vessels in the infarct repair area, with 41.2% having more than 10 erythrocyte vessels. In the NBP treatment group, 1 animal had ≤10 erythrocyte vessels and 14 animals had >10 erythrocyte vessels in the infarct repair area, with 93.3% having more than 10 erythrocyte vessels. This was statistically significant compared to the model control group (Chi-square, P = 0.0028). In the low-dose and high-dose groups of compound I, 0 / 12 animals had ≤10 erythrocyte vessels and 1 / 12 animals had >10 erythrocyte vessels in the infarct repair area, with the number of animals having more than 10 erythrocyte vessels significantly higher than that in the model control group (Chi-square, P = 0.0012, P = 0.0076). Secondly, the proportions of samples containing more than 10 erythrocyte vessels in the repair area of ​​animals in the low-dose group and the high-dose group of compound I were 100% and 92.3%, respectively, and all treatment groups were above 90%. These results suggest that compound I can significantly improve the vascular filling of the cerebral infarction repair area of ​​stroke animals after 28 days of continuous treatment (see Table 8 for details).

[0071] Table 7. Statistical table of sample recovery status for each group

[0072]

[0073] Note: ":. represents the number of animals in each group for statistical analysis; ** represents P<0.01 compared with the model control group Chi-square."

[0074] Table 8. Abundance of filled blood vessels in each group of samples.

[0075]

[0076] Note: ":. represents the number of animals in each group for statistical analysis; ** represents P<0.01 compared with the model control group Chi-square."

Claims

1. Compounds with the structure of formula (I) or pharmaceutically acceptable salts thereof: .

2. A single crystal of the compound of formula (I), characterized in that, It has the following unit cell parameters: 。 3. A pharmaceutical composition, characterized in that... A compound comprising the structure of formula (I) as described in claim 1 or a pharmaceutically acceptable salt thereof or a single crystal as described in claim 2, with one or more pharmaceutically acceptable carriers.

4. The use of the compound of formula (I) of claim 1 or a pharmaceutically acceptable salt thereof, the single crystal of claim 2, or the composition of claim 3 in the preparation of a medicament for the treatment and / or prevention of ischemic brain injury-related diseases.

5. The application according to claim 4, characterized in that, The ischemic brain injury-related diseases include ischemic stroke, vascular dementia, post-ischemic cerebral inflammation, seizures, and ischemic brain neuronal damage or necrosis.

6. A method for synthesizing compound (I), wherein the method for preparing compound (I) is as follows: .

7. A method for preparing the single crystal according to claim 2, wherein the compound of formula (I) according to claim 1 is dissolved in petroleum ether, filtered, and the filtrate is covered with a pinhole membrane and placed in a ventilated environment at room temperature to slowly evaporate the solvent to obtain the crystal.

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