Compounds for treating ischemic brain injury-related diseases

Abstract

The present invention discloses a compound for treating ischemic brain injury related diseases, the structure of said compound is shown in the following formula (II), the compound described in the present invention has higher bioavailability, higher distribution concentration in rat plasma and brain tissue, and has good effect on ischemic brain injury. [Formula 1] TIFF2024526912000019.tif36135

Description

[Technical field]

[0001] The present invention relates to a compound, a pharmaceutical composition, and a method for treating ischemic cranial nerve damage and necrosis-related diseases, which have anti-cerebral ischemia, anti-post-ischemic inflammation, and anticonvulsant effects and can improve neurological dysfunction in ischemic stroke patients. [Background technology]

[0002] White matter is an important component of the central nervous system and is the site of nerve fiber accumulation. White matter lesions (WMLs) are usually caused by reduced blood flow or insufficient blood oxygen supply. Impaired (insufficient) cerebral blood supply makes it difficult to meet the metabolic demands of brain tissue, thus resulting in a series of symptoms. Clinically, dizziness, headache, numbness in the limbs, or transient loss of consciousness may be observed, and in severe cases, irreversible damage to brain function may occur, leading to death. Cerebral ischemia-related diseases include transient ischemic attack (TIA), ischemic stroke (cerebral infarction), moyamoya disease, and chronic cerebral insufficiency of blood supply, which are also 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 not many of them are truly effective. The conventional drug nimodipine has a preventive effect on cerebral ischemia, but its therapeutic effect is not certain. 3-n-butylphthalide (NBP), extracted from celery seed volatile oil, was approved in China in 2005 for use in treating mild to moderate acute ischemic stroke, but its current mechanism of action is still unclear, and it may cause side effects such as liver dysfunction and gastrointestinal reaction during clinical use.

[0004] Cerebral ischemia will cause different degrees of brain nerve damage and necrosis, leading to dysfunction of the corresponding systems in the human body, and greatly reducing the patient's quality of life. At present, there is still a need to provide other compounds that have better therapeutic effects, have anti-cerebral ischemia, anti-ischemic post-inflammation and anticonvulsant effects, can improve neurological dysfunction in ischemic stroke patients, improve memory impairment, protect nerve cells and the blood-brain barrier, etc. Summary of the Invention

[0005] The present invention provides a novel compound having anti-cerebral ischemia, anti-post-ischemic inflammation and anticonvulsant effects for treating ischemic cranial nerve damage and necrosis-related diseases, and capable of improving neurological dysfunction in ischemic stroke patients.

[0006] The present invention provides a compound of formula (II) having the structure: or a pharma- ceutically acceptable salt thereof. [ka]

[0007] The present invention also provides a compound of formula (I) having the structure: or a pharma- ceutically acceptable salt thereof. [ka]

[0008] The present invention further provides a pharmaceutical composition comprising a compound of formula (II) of the present invention or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable carriers.

[0009] The present invention further provides a pharmaceutical composition comprising a compound of formula (I) of this invention, or a pharma- ceutically acceptable salt thereof, and one or more pharma- ceutically acceptable carriers.

[0010] The present invention further provides use of the compound of formula (II) of the present invention or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for treating and / or preventing an ischemic brain injury-related disease.

[0011] The present invention further provides the use of the compound of formula (I) of the present invention or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for treating and / or preventing an ischemic brain injury-associated disease.

[0012] The present invention further provides the use of a composition according to the present invention in the manufacture of a medicament for treating and / or preventing an ischemic brain injury-related disease.

[0013] The ischemic brain damage-related diseases according to the present invention include, but are not limited to, ischemic stroke, vascular dementia, post-ischemic cerebral inflammation, convulsion, ischemic cranial nerve damage or necrosis, and the like.

[0014] The present invention further provides methods for synthesizing the compounds of formula (II) and compounds of formula (I) of the present invention.

[0015] In one specific embodiment, the present invention further provides a method for preparing a compound of formula (I).

[0016] [ka]

[0017] The present invention further provides a single crystal of the compound of formula (I) according to the present invention, having the following unit cell parameters: [Table 1]

[0018] In one specific example, the asymmetric unit of a single crystal of the compound of formula (I) is shown in FIG.

[0019] The present invention further provides a method for preparing said single crystals by dissolving the compound of formula (I) according to the present invention in petroleum ether, filtering the solution, and then covering the filtrate with a pinhole membrane and placing it in a ventilated environment at room temperature to slowly evaporate the solvent.

[0020] The compounds described in the present invention have higher bioavailability, higher distribution concentration in rat plasma and brain tissue, and good effect on ischemic brain injury. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 is a micrograph of a (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one single crystal. [Diagram 2]This is the asymmetric unit of (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one single crystal. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention will be further described below in accordance with the general technical knowledge and conventional means in the art by combining specific examples. The following examples are only a part of the preferred embodiments of the present invention, and should not be construed as limiting the present invention. Those skilled in the art can make some modifications without departing from the scope of the present invention, and they should also be construed as being within the scope of protection of the present invention.

[0023] Example 1 Synthesis of (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one [ka]

[0024] 3-Benzofuranon (5.0g, 37.3mmol, 1.0eq.) was dissolved in dichloromethane (50mL), cooled to 5°C, potassium tert-butoxide (6g, 53.5mmol, 1.4eq.) was slowly added, reacted for 0.5h, n-butyryl chloride (8g, 75.0mmol, 2.0eq.) was slowly added, reacted for 1h, water (50mL) was added for extraction, the organic phase was taken and washed with 0.1N hydrochloric acid to acidify (pH<2), the organic phase was dried with anhydrous sodium sulfate (5g) for 0.5h, filtered and concentrated to obtain an oily substance, which was then purified using high pressure preparative chromatography, and the purified solution was freeze-dried to obtain the compound of formula (I) ((Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one (2.5g, 32.9%) as a white solid. 1 H NMR (CDCl 3 ,400MHz): δ12.02(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). 1H NMR (CDCl 3 +D 2 O,400MHz): δ7.34-7.32(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).

[0025] Example 2 Preparation of (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one single crystals 20 mg (0.098 mmol) of (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one prepared in Example 3 was dissolved in petroleum ether (0.4 mL) and filtered. The filtrate was covered with a pinhole membrane and placed in a fume hood to slowly evaporate the solvent at room temperature. After 24 h, (Z)-3-(1-hydroxybutenyl)benzofuran-2(3H)-one single crystals (Figure 1) were obtained.

[0026] 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 radiation (λ=0.71073 Å). The crystal was maintained at 296 K during the data collection process. The structure of the single crystal was solved in Olex2 software, the initial structure was calculated using the intrinsic phasing method of SHELXT program, and the structure was refined by the least square method of 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(3H)-one single crystal is shown in Figure 2.

[0027] [Table 2]

[0028] Biological measurements: The following measurements prove that compound I of the present invention has the effect of improving ischemic neurological dysfunction. The measurement results also show that compound I of the present invention has good bioavailability and efficacy after oral administration.

[0029] Study 1: Pharmacokinetic study in rats Compound I was administered to SD male rats (body weight 180-260g) via tail vein injection at a dose of 1.0mg / kg and orally at a dose of 10.0mg / kg, with 3 animals in each group. The administration solvent was a saline solution of 5% DMSO + 5% polyoxyethylene castor oil (Cremophor EL). The animals were fasted for about 12 hours before administration, and allowed to eat freely for 4 hours after administration, with no water allowed. Approximately 0.2mL of blood was collected from the orbit before administration and 5, 15, 30min, 1, 2, 4, 6, 8, and 24h after administration, and placed in an EDTA-K2 anticoagulant EP tube in an ice bath, and centrifuged at 3500 rpm at 4℃ for 10 minutes at low speed to separate plasma, which was then stored at -20℃ until analysis. Quantitative analysis of the concentration of Compound I in plasma was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The sample analysis results were used to calculate pharmacokinetic parameters using WinNonlin software.

[0030] The data in Table 2 show that after oral administration, compound I had a longer elimination time and higher bioavailability in rats compared with the values ​​reported in the butylphthalide literature (Wang Ningning, Li Yue, Li Xiaohong, Jiang Mingyan, Determination of the content of 3-n-butylphthalide in rat plasma and its pharmacokinetics by RP-HPLC method, Chinese Journal of New Drugs and Clinical Practice, December 2012, Vol. 31, No. 12, pp. 743-747). (The reason for the bioavailability exceeding 100% was presumed to be nonlinear pharmacokinetics.)

[0031] [Table 3]

[0032] Test 2: Rat brain tissue distribution test Compound I and butylphthalide (NBP) were orally administered at a dose of 20 mg / kg to male SD rats (body weight 200-270 g). Plasma and brain tissue samples were collected from the animals at 0.5, 1, 4, and 24 h after administration. Plasma collection: 0.2 mL of whole blood was collected in an EDTA-K2-containing EP tube, and centrifuged at 3500 g for 10 min, after which the upper layer plasma was collected and stored at -20°C. Brain tissue collection: After euthanasia of the animals, an appropriate amount of brain tissue was weighed and homogenized according to brain tissue: 80% methanol water (w / v) = 1:4. Quantitative analysis of the concentration of the compound in plasma and brain tissue samples was performed using a combined liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0033] The data in Table 3 show that compound I of the present invention has higher distribution concentrations in rat plasma and brain tissue after oral administration.

[0034] [Table 4]

[0035] Study 3: Pharmacodynamic study of compounds in a rat stroke model: Single treatment dose To evaluate the neuroprotective effect of compounds on cerebral ischemia-reperfusion in rats, a middle cerebral artery occlusion (MCAO) model was established in SD rats using the thread embolization method. SD rats (240-270 g) were anesthetized with 3.0% isoflurane, and then surgery was performed. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated and exposed. The ECA was ligated, the ICA was clamped, and threads were threaded into the proximal and distal ends of the CCA, respectively, and the distal end was tightened, and a loose knot was made at the proximal end, and a small incision was made between the two threads as necessary. A 4-0 thread embolization was inserted through the incision of the CCA, and then gently pushed into the internal carotid artery. When it was applied to the arterial clip of the ICA, it stopped once. After further tightening the ligature, the arterial clip that had been blocking the blood flow of the ICA was removed, and immediately after that, the thread embolization was pushed into the ICA and inserted into the skull. When the thread plug was inserted to a depth of about 18 mm from the common carotid artery bifurcation, there was a slight resistance, i.e., the tip of the thread plug had already entered the anterior cerebral artery (ACA) and the side wall of the thread plug had already occluded the opening of the middle cerebral artery. The insertion was stopped and the time was recorded. The arterial clip on the CCA was removed, and after observing that there was no active bleeding, the incision was closed. The ischemic rat was placed at room temperature and the body temperature was maintained at 37°C. After 120 min, anesthesia was induced, and the thread plug was gently pulled while maintaining anesthesia, and its tip was returned to the external carotid artery to achieve middle cerebral artery reperfusion. The animals were administered once immediately after reperfusion (within 10 min), and were divided into a total of three groups: a model control group, a compound I intravenous group (30 mg / kg), and a compound I oral group (60 mg / kg). The animals were euthanized 24 h after ischemia-reperfusion, and the brains were quickly removed, frozen, sectioned, and TTC stained. After staining, normal tissue turned rose color, and infarcted tissue turned white color. To evaluate the degree of cerebral ischemic injury in rats, the percentage of infarcted tissue to the total brain weight was defined as the infarction area (Infarction Area%).

[0036] According to the TTC staining analysis on the first day after the operation, the cerebral infarction range of the animals in the model control group was 21.63±5.66%. The cerebral infarction range of the animals in the compound I intravenous group and the compound I oral administration group was 13.61±3.66% and 14.88±5.11%, respectively, which shows that the compound I intravenous group and the compound I oral administration group described in the present invention can significantly reduce the cerebral infarction range of the animals (P=0.0025 and P=0.0389), and at the same time, the cerebral infarction inhibition rate of the animals in the compound I intravenous group and the compound I oral administration group was 37.1% and 31.2%, respectively. From the above results, it was shown that compound I has a high improving effect on cerebral infarction in rats, which is specifically shown in Table 4.

[0037] [Table 5]

[0038] Study 4: Pharmacodynamic study of compounds in mid- to long-term stroke models in rats A middle cerebral artery occlusion (MCAO) model was established in SD rats using the thread embolization method, and the test drugs were administered for 28 consecutive days. The pharmacological effects of the test drugs on stroke were evaluated based on general observations and neurobehavioral scores.

[0039] Sprague-Dawley rats (240-280g) were anesthetized with 3.0% isoflurane, and then the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated and exposed. The ECA was ligated, the ICA was clamped, and the proximal and distal ends of the CCA were threaded, respectively, and the distal end was tightened, and a loose knot was made at the proximal end, and a small incision was made between the two threads in preparation for need. A No. 4-0 thread obturator was inserted through the incision of the CCA, and gently pushed into the internal carotid artery. It stopped once it hit the arterial clip of the ICA. The ligature was further tightened, and the arterial clip that had been blocking the blood flow of the ICA was removed. Immediately after that, the thread obturator was pushed into the ICA and inserted into the skull. When the thread plug was inserted to a depth of about 18 mm from the common carotid artery bifurcation, there was a slight resistance, i.e., the tip of the thread plug had already entered the anterior cerebral artery (ACA) and the side wall of the thread plug had already occluded the opening of the middle cerebral artery. The insertion was stopped and the time was recorded. The arterial clip on the CCA was removed, and after observing that there was no active bleeding, the incision was closed. The ischemic rat was placed at room temperature and the body temperature was maintained at 37°C. After 120 min, anesthesia was induced, and the thread plug was gently pulled while maintaining anesthesia, and its tip was returned to the external carotid artery to achieve middle cerebral artery reperfusion. The animals were administered the drug immediately after reperfusion (within 10 min), and then once daily for 28 consecutive days. The day of surgery was defined as D1 (Day 0). A total of five groups were established: a sham operation group, a model control group, a butylphthalide (NBP) administration group (60 mg / kg, po, qd), a low-dose oral administration group of compound I (6 mg / kg, po, qd), and a high-dose oral administration group of compound I (20 mg / kg, po, qd). During the administration, all animals were subjected to grid tests (4 times in total on D7, D14, D21, and D28) and novel object recognition tests (D26 novel object recognition adaptation, D27 detection). After the administration was completed on D28, all surviving animals in the groups were euthanized, and their brains were quickly removed and subjected to pathological analysis.

[0040] (1) According to the results of the grid test, one week after the operation, the stepping frequency of the animals in the model control group was 7.24 ± 3.59 times, and the stepping frequency of the NBP treatment group, the compound I low dose group, and the high dose group were 7.37 ± 3.03 times, 5.33 ± 2.33 times, and 4.23 ± 1.44 times, respectively. Among them, the stepping frequency of the animals in the compound I high dose group was significantly lower than that of the model control group (P = 0.0172). 2 to 3 weeks after the operation, the animals in the model group gradually recovered their motor function, so the stepping frequency gradually decreased. The stepping frequency of the compound I high dose group was not significantly different from that of the model control group, but still tended to decrease. 4 weeks after the model construction, the stepping frequency of the model animals was reduced to the level of the animals in the sham operation group. From the above results, it was shown that compound I has an improving effect on the behavioral dysfunction of stroke animals (see Table 5 for details).

[0041] [Table 6]

[0042] (2) On the final day of treatment, a novel object recognition test was conducted and the novel object recognition index (NRI) for animals in each group was calculated. The novel object recognition index of the animals in 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 animals in the NBP treatment group, the low-dose group and the high-dose group of compound I was 70.97±22.57%, 70.98±22.60% and 71.66±17.06%, respectively, among which, the novel / old object recognition index of the NBP treatment group, the low-dose group and the high-dose group of compound I were all significantly different (P=0.0074, P=0.0212 and P=0.0009); at the same time, the novel object recognition index of the low-dose group and the high-dose group of compound I was comparable to that of the animals in the sham-operated group (66.51±10.80%). These results showed that compound I significantly improved the cognitive impairment of stroke animals (see Table 6 for details).

[0043] [Table 7]

[0044] (3) Pathological examination: 1) Repair range: Samples with repair area range >30% were considered to have good recovery, and samples with ≦30% were considered to have bad recovery. The proportion of animals with good recovery in the model control group was 17.6%, and the proportions of repair area >30% in the NBP treatment group, compound I low dose group and high dose group were 21.4%, 33.3% and 75.0%, respectively, with the compound I high dose group showing the best recovery, with a significant difference compared to the model control group (Chi-square, P=0.0080). From the above results, it was shown that compound I has a promoting effect on the repair of the infarcted area (see Table 7 for details). 2) Number of filled blood vessels in the infarcted repair area: Two standards were set: the number of blood vessels containing red blood cells in the infarcted repair area was ≦10 and >10. In the sham-operated group, the number of filled blood vessels was more than 10 in all animals, accounting for 100%. In the model control group, the number of animals with ≦10 and >10 blood vessels containing red blood cells in the infarct repair area was 10 and 7, respectively, accounting for 41.2%. In the NBP-treated group, the number of animals with ≦10 and >10 blood vessels containing red blood cells in the infarct repair area was 1 and 14, respectively, accounting for 41.2%. The proportion of animals with blood vessels containing red blood cells >10 was 93.3%, which was statistically significant compared with the animals in the model control group (Chi-square, P=0.0028). The number of animals with blood vessels containing red blood cells ≦10 and >10 in the infarct repair area in the low-dose and high-dose compound I groups was 0 / 12 and 1 / 12, respectively, and the number of animals with blood vessels containing red blood cells >10 was significantly higher than that in the model control group (Chi-square, P=0.0012, P=0.0076). Next, the proportion of specimens with blood vessels containing red blood cells >10 in the repair area in the low-dose and high-dose compound I groups was 100% and 92.3%, respectively, and each treatment group was above 90%. The above results showed that compound I significantly increased the vascular fullness of the cerebral infarct repair area of ​​stroke animals during 28 consecutive days of treatment (see Table 8 for details).

[0045] [Table 8]

[0046]

Table 9

Claims

1. Formula (II): [Formula 1] or a pharma- ceutically acceptable salt thereof.

2. Formula (I): [Case 2] or a pharma- ceutically acceptable salt thereof.

3. The following unit cell parameters: [Table 1] A single crystal of the compound of formula (I) having the following structure:

4. A pharmaceutical composition comprising a compound having the structure of formula (II) according to claim 1 or a pharma- ceutically acceptable salt thereof, a compound having the structure of formula (I) according to claim 2 or a pharma- ceutically acceptable salt thereof, or the single crystal according to claim 3, and one or more pharma- ceutically acceptable carriers. A pharmaceutical composition comprising:

5. Use of a compound having the structure of formula (II) according to claim 1 or a pharma- ceutically acceptable salt thereof, a compound having the structure of formula (I) according to claim 2 or a pharma- ceutically acceptable salt thereof, a single crystal according to claim 3, or a composition according to claim 4, in the manufacture of a drug for treating and / or preventing an ischemic brain injury-related disease.

6. The ischemic brain damage-related disease includes ischemic stroke, vascular dementia, post-ischemic inflammation, convulsion, ischemic cranial nerve damage or necrosis; 6. Use according to claim 5.

7. A method for synthesizing a compound of formula (II) and a compound of formula (I), wherein the compound of formula (I) is preferably prepared as follows: [C3]

8. The method for producing the single crystal of claim 3, comprising dissolving the compound of formula (I) of claim 2 in petroleum ether, filtering the mixture, covering the filtrate with a pinhole membrane, and allowing the solvent to slowly evaporate at room temperature in a ventilated environment.

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