CN109734701B - ROCK inhibitor-dichloroacetic acid complex salt and preparation method and application thereof - Google Patents

Abstract

The present invention relates to the field of medicinal chemistry and pharmacotherapeutics, in particular to a ROCK inhibitor-dichloroacetic acid double salt or a pharmaceutically acceptable salt thereof, their preparation method, a pharmaceutical composition containing these compounds and their medicines Use, especially in the preparation of drugs for preventing and/or treating pulmonary hypertension, ischemic stroke, subarachnoid hemorrhage and other cardiovascular and cerebrovascular diseases.

Description

ROCK inhibitor-dichloroacetic acid double salt and preparation method and use thereof

technical field

The present invention relates to a kind of ROCK inhibitor-dichloroacetic acid double salt, in particular to a kind of ROCK inhibitor-dichloroacetic acid double salt, their preparation method, medicinal composition containing these compounds and their medicinal uses, belong to pharmacy technical field.

Background technique

Rho kinase (ROCK) is an important enzyme involved in a series of cell life phenomena such as cell mitotic adhesion, cytoskeleton adjustment, muscle cell contraction, and tumor cell infiltration. Since 1996, ROCKs that have been discovered are divided into ROCK I (ROCKβ) and ROCK II (ROCKα). The former mainly exists in non-neural tissues such as heart, lung, and skeletal muscle cells; the latter mainly exists in the central nervous system, such as hippocampal pyramidal neurons, cerebral cortex, and cerebellar Purkinje cells. Rho kinase (ROCK) plays an important intracellular signal transduction role in many cellular functions such as vascular smooth muscle cell contraction, cell migration, proliferation and apoptosis. Abnormal activation of Rho kinase has been found in a variety of cardiovascular diseases, such as atherosclerosis, restenosis, hypertension, pulmonary hypertension and cardiac hypertrophy. Studies have shown that chronic hypoxia and monocrotaline-induced rat pulmonary hypertension model and patients with severe pulmonary hypertension significantly increased Rho kinase activity in lung tissue and pulmonary arteries.

Fasudil [hexahydro-1-(5-sulfonylisoquinoline)-1(H)-1,4-diazepine, Fasudil, also known as HA1077], is a product of Asahi Kasei Co., Ltd. and Nagoya University Pharmacology A new type of isoquinoline sulfonamide derivative developed in cooperation with the scientific research laboratory. As a new and efficient vasodilator, fasudil can effectively relieve cerebral vasospasm and improve the prognosis of patients with subarachnoid hemorrhage (SAH). The role of pulmonary blood vessels has been widely concerned by researchers. A large number of animal experiments and clinical studies have shown that fasudil can: 1) activate endogenous neural stem cells to promote brain tissue repair; 2) increase the stimulation of astrocytes 3) Inhibit the release of intracellular calcium ions; 4) Dilate brain blood vessels; 5) Protect nerve cells and improve extension function; 6) Promote axon regeneration. Therefore, fasudil is also used in the treatment of ischemic stroke. In addition, fasudil is also safe and effective in the treatment of pulmonary hypertension. The ROCK inhibitor fasudil can penetrate into vascular smooth muscle cells, compete with ATP for the ATP binding site of the catalytic region of Rho kinase under normal or pathological conditions, and specifically block Rho kinase activity. Fasudil hydrochloride is currently in the phase II clinical research phase.

In addition, ROCK inhibitors include Ripasudil and Netarsudil, which are marketed for the treatment of glaucoma.

SUMMARY OF THE INVENTION

Objective: The present invention provides a ROCK inhibitor-dichloroacetic acid double salt, its preparation method and medical use.

Technical scheme: in order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is:

A class of compounds is a ROCK inhibitor-dichloroacetic acid double salt.

Further, the ROCK inhibitor is selected from Fasudil, Netarsudil, Ripasudil.

Specifically, the compound is fasudil dichloroacetate, and the structural formula is as follows:

The preparation method of fasudil dichloroacetate is:

Take an appropriate amount of fasudil and place it in a reaction vessel, add an appropriate amount of reaction solvent and mix to obtain a mixed solution of fasudil and reaction solvent;

Add an appropriate amount of dichloroacetic acid to the above-mentioned mixed solution while stirring at a reaction temperature of 0-100 degrees, and continue to stir for a period of time after dropping to obtain a reaction solution;

Then the reaction solution was concentrated under reduced pressure to remove the solvent, washed and recrystallized to obtain fasudil dichloroacetate.

In a preferred preparation process, the reaction temperature is room temperature, the reaction solvent is water, the molar ratio of the added fasudil and dichloroacetic acid is 1:1.5, and the recrystallization solvent is isopropanol.

Specifically, the compound is Netarsudil dichloroacetate, and the structural formula is as follows:

The preparation method is as follows: Netarsudil is dissolved in tetrahydrofuran, dichloroacetic acid is added dropwise into the reaction system, stirred at normal temperature for a period of time, and spin-dried to obtain Netarsudil dichloroacetate.

Specifically, the compound is Ripasudil dichloroacetate; the structural formula is as follows:

The preparation method is as follows: at room temperature, taking an appropriate amount of Ripasudil and placing it in a reaction vessel, adding water, slowly adding dichloroacetic acid dropwise to the Ripasudil suspension while stirring, and continuing to stir at room temperature for a period of time after dropping to obtain a reaction solution; then The reaction solution was concentrated under reduced pressure, washed, filtered and dried to obtain Ripasudil dichloroacetate.

In another aspect, the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned compound or its optical isomer, enantiomer, diastereomer, racemate or racemic mixture, or A pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle.

On the other hand, the present invention also provides the application of the above-mentioned compounds in the preparation of medicines for preventing and/or treating pulmonary hypertension, subarachnoid hemorrhage, ischemic stroke and other cardiovascular and cerebrovascular diseases.

In the present invention, when administering the above-mentioned compounds and their pharmaceutically acceptable salts to mammals, as well as solvates of these compounds (herein collectively referred to as "therapeutic drugs"), they can be used alone, or preferably in accordance with standard pharmaceutical methods. It is used in combination with a pharmaceutically acceptable carrier or diluent. Administration can be by various routes, including oral, parenteral, or topical. Parenteral administration as referred to herein includes, but is not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and transdermal administration.

The invention first discloses fasudil dichloroacetate and a preparation method thereof, which comprises the following steps: firstly mixing free fasudil with water, slowly adding dichloroacetic acid dropwise, continuing to stir for 5 minutes, and then concentrating out pure water, the residue was washed with ether for 3 times, and then recrystallized with isopropanol or other solvents to obtain high-purity fasudil dichloroacetate. The structure was confirmed by hydrogen spectrum, carbon spectrum and mass spectrometry. The invention has the advantages of simple operation, low production cost, high product yield and little environmental pollution, and is favorable for industrialized large-scale production.

Meanwhile, the present invention discloses the inhibitory activities of fasudil dichloroacetate, Netarsudil dichloroacetate and Ripasudil dichloroacetate on ROCK I and II. It was found that salt formation of dichloroacetate and ROCK inhibitor enhanced its ROCK inhibitory activity.

The invention discloses the therapeutic effect of fasudil dichloroacetate (FDCA) on pulmonary hypertension. First, fasudil dichloroacetate (FDCA) significantly inhibited platelet-derived growth factor BB (PDGF-BB) and hypoxia-induced inflammation in pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs) in cell experiments. The expression of factors tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6); further animal experiments, in the treatment model of rat pulmonary hypertension induced by monocrotaline, FDCA (43.3mg/kg ) intragastric administration can significantly reduce the mean pulmonary artery pressure, right ventricular systolic pressure and right ventricular hypertrophy index in rats with pulmonary arterial hypertension, but have no significant effect on systemic pressure; FDCA significantly decreased the ratio of pulmonary arteriolar wall thickness to pulmonary arteriole diameter (PAMT) and the degree of pulmonary arteriolar fibrosis; FDCA significantly decreased the right ventricular cardiomyocyte area (CSA) and the degree of fibrosis. It is worth noting that the activity of FDCA against pulmonary hypertension is superior to that of equimolar doses of fasudil dihydrochloride (F), sodium dichloroacetate (DCA) and their combined administration, suggesting that FDCA is an effective drug. Candidate drugs against pulmonary arterial hypertension are worthy of further study.

Meanwhile, the invention discloses the effect of fasudil dichloroacetate (FDCA) in preventing and/or treating subarachnoid hemorrhage. In the rat subarachnoid hemorrhage model (subarachnoid intravascular perforation modeling, administration once 0.5h and 6h after modeling, and evaluating various indicators of rats after 24h), FDCA significantly reduced the rat spider web Cerebral vasospasm injury after submembranous hemorrhage, improved cerebral edema and animal neurological score, significantly improved basilar artery diameter, lumen area and wall thickness and regional cerebral blood flow (rCBF) in the parietal cortex, better than F, DCA and Combined administration of the two. The results suggest that FDCA is an effective anti-subarachnoid hemorrhage drug candidate and deserves further study.

Meanwhile, the invention discloses the effect of fasudil dichloroacetate (FDCA) in preventing and/or treating ischemic stroke. In the rat model of transient ischemia (reperfusion for 2 hours of ischemia, once for 4 hours of ischemia, once for 24 hours, and evaluating various indicators of rats after 48 hours), FDCA can effectively reduce the area of cerebral infarction, which is significantly better than that in the market. The drug butylbenzene peptide (NBP) group was significantly better than the fasudil dihydrochloride group (F) and sodium dichloroacetate (DCA) group, as well as the combined administration group of F and DCA; in addition, FDCA also significantly improved The neurobehavioral dysfunction induced by ischemia was significantly better than F, DCA and their combined administration, and slightly better than the NBP group. The results suggest that FDCA is an effective anti-ischemic stroke candidate drug and deserves further study.

As an inhibitor of ROCK, fasudil can relax blood vessels, lower blood pressure, inhibit the proliferation of vascular smooth muscle cells, and inhibit vascular remodeling; while dichloroacetate is an inhibitor of pyruvate dehydrogenase kinase, which can increase pyruvate The activity of dehydrogenase can promote the aerobic metabolism of glucose and reduce the production of lactic acid; at the same time, it can also promote the expression of potassium ion channels, especially Kv1.5, inhibit the proliferation of smooth muscle cells and promote their apoptosis. Therefore, the combined administration of fasudil and dichloroacetate can treat cardiovascular and cerebrovascular diseases such as pulmonary hypertension, ischemic stroke, and subarachnoid hemorrhage from multiple mechanisms. Compared with co-administration, fasudil dichloroacetate (FDCA) as a whole molecule may be different from co-administration in terms of drug absorption, distribution and metabolism, thus showing better activity .

Description of drawings

Figure 1 shows the effects of the compounds in Example 5 on the expressions of TNF-α and IL-6 in PASMCs and PAECs under PDGF-BB and hypoxic culture conditions; PASMs: pulmonary artery smooth muscle cells; PAECs: pulmonary artery endothelial cells; PDGF-BB: platelets Derived growth factor BB; IL-6; Interleukin-6; CON: blank control; Hypoxia: hypoxia; FDCA: fasudil dichloroacetate; F: fasudil dihydrochloride; DCA : dichloroacetic acid sodium salt; F+DCA: fasudil dihydrochloride and dichloroacetic acid sodium salt combined administration group.

Figure 2 is the effect of the compound in Example 5 on the hemodynamics of MCT-induced PAH model rats; mPAP: mean pulmonary artery pressure, RVSP: right ventricular systolic pressure; mSAP: mean systemic pressure; RV/LV+S: right heart Hypertrophy index; Control: control group, MCT: monocrotaline; FDCA: fasudil dichloroacetate; F: fasudil dihydrochloride; DCA: dichloroacetic acid sodium salt; F+DCA: method The combined administration group of sudil dihydrochloride and sodium dichloroacetate.

Figure 3 is the effect of each administration group on the ratio of pulmonary arteriole wall thickness to pulmonary arteriole diameter (PAMT) and the degree of fibrosis in Example 5; Ratio of arterial diameters; Fibrosis: fibrosis; Control: control group, MCT: monocrotaline; FDCA: fasudil dichloroacetate; F: fasudil dihydrochloride; DCA: sodium dichloroacetate Salt; F+DCA: fasudil dihydrochloride and dichloroacetic acid sodium salt combined administration group.

Figure 4 shows the effects of each administration group on the area of right ventricular cardiomyocytes and the degree of fibrosis in Example 5; CAS: cross-sectional area of cardiomyocytes; Fibrosis: fibrosis; Control: control group, MCT: monocrotaline; FDCA: Fasudil dichloroacetate; F: fasudil dihydrochloride; DCA: dichloroacetate sodium salt; F+DCA: fasudil dihydrochloride and dichloroacetate sodium salt combined administration Group.

Figure 5A is the effect of different compounds in Example 6 on brain edema in SAH rats; Figure 5B is the effect of different compounds in Example 6 on the spontaneous activity score of SAH rats.

FIG. 6 is a graph of TTC staining in the brain tissue of the tMCAO model rat in Example 7. FIG.

FIG. 7 is a statistical diagram of the cerebral infarction area of the tMCAO model rat in Example 7. FIG.

FIG. 8 is the neurological function score of the tMCAO model rat in Example 7. FIG.

FIG. 9 is a graph showing the area of cerebral infarction in the rat model of tMCAO and the neurological function score of the rat in Example 7. FIG.

Detailed ways

In order to further illustrate the present invention, a series of examples are given below, which are purely illustrative, and are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

Example 1

Fasudil Dichloroacetate

Compound synthesis and characterization data:

At room temperature, 10g of fasudil was placed in a 100mL eggplant-shaped bottle, 20mL of tap water or purified water was added to stir, then 4.87g of dichloroacetic acid was weighed and slowly added dropwise to the above suspension. Fasudil or gradually dissolved, drip completed. Continue stirring at room temperature for 10 min. Then the reaction solution was concentrated under reduced pressure, the residue was washed with ether for 3 times, the ether was discarded, and then recrystallized with an organic solvent, filtered to obtain a white solid, which was then dried in a vacuum drying oven to obtain 13.95 g of the target compound. The yield was 13.95 g. was 96.9%. Mp: 141-143°C. ¹ H NMR (300 MHz, D ₂ O, TMS) δ 9.20 (s, 1H), 8.49 (d, J=6.2, 1H), 8.21-8.29 (m, 3H), 7.72 ( t, J=7.7, 1H), 6.03 (s, 1H), 3.72 (t, J=5.0, 2H), 3.53 (t, J=6.1, 2H), 3.35-3.41 (m, 4H), 2.09-2.17 (m, 2H). ¹³ C NMR (75MHz, D ₂ O) δ 170.25, 152.36, 142.97, 134.17, 133.01, 131.0, 130.25, 128.10, 126.03, 116.69, 68.07, 46.76, 46.22, 44.41, 43.46, 24.9 ESI-MS (70 eV) m/z: 292.2 [M+H] ⁺ .

Example 2

Netarsudil Dichloroacetate

Compound synthesis and characterization data:

4-(3-Amino-1-(isoquinolin-6-ylamino)-1-oxoprop-2-yl)benzyl 2,4-dimethylbenzoate (100 mg, 0.22 mmol) dissolved in In tetrahydrofuran, dichloroacetic acid (28 mg, 0.22 mmol) was added dropwise to the reaction system, stirred at room temperature for 10 min, and spin-dried to obtain a pale yellow solid, which was netarsudil dichloroacetate. ¹ H NMR (300 MHz, DMSO) δ: 11.07 (s, 1H), 9.22 (s, 1H), 8.43 (s, 2H), 8.18 (s, 1H), 8.11 (d, J=8.70 Hz, 2H), 7.82(t, J=15.60Hz, 3H), 7.51(s, 4H), 7.14(s, 2H), 6.52(s, 1H), 5.28(s, 2H), 4.32(m, 1H), 3.56(m , 2H), 2.51 (s, 3H), 2.31 (s, 3H). ESI-MS (70 eV) m/z: 454.2 [M+H] ⁺ .

Example 3

Ripasudil dichloroacetate

Compound synthesis and characterization data:

At room temperature, place 100 mg of Ripasudil in a 50 mL eggplant-shaped bottle, add 3 mL of tap water or purified water to stir, then weigh 48 mg of dichloroacetic acid, and slowly add it dropwise to the above suspension. During the dropwise addition, it is found that the reaction solution gradually dissolves. , dripping. Continue stirring at room temperature for 10 min. Then the reaction solution was concentrated under reduced pressure, the residue was washed with ether for 3 times, the ether was discarded, and the light yellow solid was obtained by filtration, which was then dried in a vacuum drying oven to obtain 113.1 mg of the target compound with a yield of 81%. ¹ H-NMR (300 MHz, D ₂ O, δ): 1.11 (3H, d, J=6.6 Hz), 1.96-2.24 (2H, m), 3.13-3.39 (2H, m), 3.44-3.72 (4H, m), 3.76 (1H, m), 5.99 (1H, s), 7.69 (1H, m), 8.16-8.30 (2H, m), 8.31-8.42 (1H, s), 8.93 (1H, s); ESI - MS (70 eV) m/z: 324.2 [M+H] ⁺ .

Example 4

Inhibitory activity of compounds on ROCK-I and II:

Table 1. Inhibitory activity (nM) of compounds on ROCK-I and ROCK-II

As shown in Table 1, it can be seen that the inhibitory activity of fasudil dichloroacetate on ROCK-I and ROCK-II is stronger than that of fasudil on ROCK-I and ROCK-II;

The inhibitory activity of Netarsudil dichloroacetate on ROCK-I and ROCK-II was stronger than that of Netarsudil on ROCK-I and ROCK-II;

The inhibitory activity of Ripasudil dichloroacetate on ROCK-I and ROCK-II was stronger than that of Ripasudil on ROCK-I and ROCK-II.

Example 5

Prevention and/or treatment of pulmonary hypertension

1. The effect of FDCA on the expressions of inflammatory factors TNF-α and IL-6 in PASMCs and PAECs cells in PDGF-BB and hypoxia culture models

Pulmonary hypertension, especially pulmonary hypertension related to connective tissue diseases, is often accompanied by inflammation. The inflammatory factor tumor necrosis factor-α (TNF-α) can activate the inflammatory factor interleukin-6 (IL-6), promote the proliferation of smooth muscle cells, Vascular fibrosis and remodeling of pulmonary arterioles. Firstly, we investigated the effect of fasudil dichloroacetate (FDCA) on hypoxic culture conditions and platelet-derived growth factor BB (PDGF-BB)-induced pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs) by cell experiments. Effects of TNF-α and IL-6 expression. Cell grouping: ① normal cell group (Control); ② PDGF-BB or hypoxia cultured model group; ③ model group + fasudil dichloroacetate (FDCA); ④ model group + fasudil hydrochloride (F) treatment group; ⑤ model group + sodium dichloroacetic acid (DCA) treatment group; ⑥ model group + F and DCA combined administration group. The experimental method is as follows. PGDFBB model group: firstly the cells were passaged to 3-6 passages, after 24 hours of culture, PDGFBB (5 μl to 10 ml) was added for 24 hours, then starved for 48 hours, and the drug was added. The expressions of TNF-α and IL-6 in the cells of each group were counted; hypoxia model group: cells were passaged to 3-6 passages, then starved for 24 hours, and then cultured in hypoxia for 24 hours before administration. The concentration of each administration group was 50 μM, the expression of TNF-α and IL-6 in each group of cells was counted by ELISA after 72 h of culture). As shown in Figure 1, both hypoxic culture conditions and PDGF-BB significantly increased the expression of TNF-α and IL-6 in PASMCs and PAECs, suggesting that both hypoxic culture conditions and PDGF-BB could significantly increase inflammation; The administration group inhibited the expression of TNF-α and IL-6 to different degrees in the two cell lines and reduced inflammation. Among them, the FDCA group had the best inhibitory effect on inflammation, which was better than that of F, DCA and the combined administration group of the two. It is suggested that F and DCA have a certain synergistic effect in anti-inflammatory. The possible reason is that FDCA as a whole molecule has better ability to cross the cell membrane than F and DCA.

2. Effects of FDCA compounds on hemodynamics in MCT-induced PAH model rats

To further study the therapeutic effects of FDCA and related compounds in the rat model of PAH induced by monocrotaline (MCT). Animal groups: ① normal control group; ② normal control group + FDCA; ③ MCT model group; ④ fasudil dihydrochloride (F) treatment group; ⑤ DCA treatment group; ⑥ F + DCA combined treatment group; ⑦ FDCA administration group. Establishment of rat model: The animal model group and the treatment group were injected intraperitoneally with monocrotaline (MCT) 60 mg/kg, and the normal control group was injected with the same amount of normal saline. Experimental treatment: On the 14th day of injection of monocrotaline, each administration group started administration with an equimolar dose, and the administration method was intragastric administration, once a day, 37.5 mg/kg in group F, and 15.5 mg/kg in group F, 15.5 mg/kg in DCA group , F+DCA combined treatment group included F (37.5mg/kg) and DCA 15.5mg/kg, normal control group+FDCA group 43.3mg/kg; FDCA group 43.3mg/kg. The normal group and the model group were fed with the same amount of normal saline. On the 28th day, the mean pulmonary artery pressure (mPAP), right ventricular systolic pressure (RVSP) and mean systemic pressure (mSAP) were measured in each group, and then the rats were sacrificed to collect lung tissue and heart for right ventricular hypertrophy index (RV/LV+ S), PCNA detection, immunohistochemical staining, hematoxylin-eosin staining, Masson staining, etc., to evaluate the hemodynamics, average thickness of pulmonary artery, degree of pulmonary fibrosis, right heart function, etc. active. The results are shown in Figure 2. Compared with the normal control group, the direct administration of FDCA had little effect on the mPAP, RVSP and RV/LV+S of normal rats, indicating that FDCA has higher safety. The MCT model group could significantly increase mPAP, RVSP and RV/LV+S. Each administration group could effectively reduce mPAP, RVSP and RV/LV+S, among which the FDCA group had the strongest activity in reducing mPAP, RVSP and RV/LV+S, which was better than F, DCA and their combined administration. In addition, each administration group had little effect on mSAP.

3. Effects of FDCA compounds on the pulmonary artery of MCT-induced PAH model rats

As shown in Figure 3, the effects of different administration groups on the ratio of pulmonary arteriolar wall thickness to pulmonary arteriolar diameter (PAMT) and the degree of fibrosis in rats can be found that FDCA can effectively reduce PAMT and the degree of pulmonary arteriolar fibrosis , slightly better than F, DCA and their combined administration.

4. Effects of FDCA compounds on the right ventricle of MCT-induced PAH model rats

As shown in Figure 4, the effect of each administration group on the area of right ventricular cardiomyocytes and the degree of fibrosis indicated that the MCT model group significantly increased the area of right ventricular cardiomyocytes and the degree of fibrosis in rats compared with the blank control. The administration group, especially FDCA, can significantly reduce the area and degree of fibrosis of right ventricular cardiomyocytes, which is better than that of F, DCA and their combined administration groups. The results suggest that FDCA can effectively inhibit the proliferation and remodeling of right ventricular cardiomyocytes.

Example 6

Prevention and/or treatment of subarachnoid hemorrhage

laboratory animal

SPF grade SD rats, weighing 260-340g, half male and female, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., were raised in SPF grade rearing environment, the indoor temperature was controlled at 23±2°C, and the food and water were ad libitum. . The total number of animals was 32. Sham operation group: equal volume of normal saline containing 1% DMSO (n=8);

experimental method

Trial grouping and drug concentration selection:

SAH model group: equal volume of normal saline containing 1% DMSO (n=8); fasudil dihydrochloride (F) group: (26.0 mg/kg) (n=8); sodium dichloroacetate (DCA) ) group: (10.7 mg/kg) (n=8): fasudil dihydrochloride combined with sodium dichloroacetate (F+DDCA) group: (F: 26.0 mg/kg; DCA: 10.7 mg/kg) (n=8); FDCA group: (30 mg/kg) (n=8); all drugs were formulated into physiological saline solution containing 1% DMSO, and the administration method was tail vein injection. Administered once at 0.5h and 6h after SAH (rat model of subarachnoid hemorrhage), respectively, the sham operation group and the model group were replaced with equal volume of normal saline containing 1% DMSO.

Model and method of administration

An endovascular perforation model for SAH was performed as described in reference (Stroke, 1995, 26, 1086-1092). Rats were anesthetized, intubated and maintained artificially ventilated with 3% isoflurane in 70%/30% medical air/oxygen during surgery. Body temperature was monitored with a rectal probe and normal heat was maintained with a heat lamp. A sharpened 4-0 nylon suture was introduced into the left internal carotid artery (ICA) until resistance was felt (approximately 18 mm from the common carotid bifurcation). The suture is then advanced further to puncture the bifurcation of the anterior and middle cerebral arteries until resistance is overcome and withdrawn immediately after perforation. In sham-operated animals, sutures were inserted into the left ICA, but no perforation was made. After the sutures were removed, the incisions were closed and the rats were individually housed in heated cages until recovery.

Spontaneous activity scoring: The rats were placed in a spacious cage with free movement and four walls accessible for scoring of spontaneous activity. Twenty-four hours after SAH modeling, the rats were evaluated and recorded by two experimental personnel in a double-blind manner, and the average of the two groups was taken as the final score. The spontaneous activity score is divided into 4 grades according to the animal's mental state and movement conditions: grade 1, the rat has normal activities, no movement disorder, actively explores the surrounding environment, and touches at least the upper edge of the three cage walls; grade 2, mild activity disorder, namely The rat is mentally poor, lethargic, has a certain delay in action, and does not reach all the cage walls, but he touches the upper edge of at least one cage wall; Grade 3, moderate mobility impairment, that is, the rat can barely stand, and can barely stand in the cage. No activity; grade 4, severe dyskinesia, i.e. the mouse does not move and shows paralysis of the limbs. The results are shown in Figure 5A.

Determination of brain water content: The rats were sacrificed 24 hours after SAH modeling, and the brain and cerebellum were quickly removed, and the surface blood was removed with filter paper. The mass (wet weight) of the brain and cerebellum was respectively weighed with an electronic balance, then the brain tissue was placed in an oven, baked at 105°C to constant weight, and the mass (dry weight) of the brain and cerebellum was weighed again. The calculation formula of brain tissue water content is: brain tissue water content (%)=(wet weight-dry weight)/wet weight×100%. The tissue water content of the cerebellum was used as a normal control. The results are shown in Figure 5B.

Measurement of basilar artery diameter, lumen area and wall thickness: The tissue sections of the basilar artery in the above groups were stained with HE, observed and photographed under an optical microscope, and the basilar artery was measured by the image pro-plus6.0 image analysis system. diameter, lumen area, and wall thickness. The measurement method of the lumen area is as follows: measure the lumen circumference (L) along the inner surface of the basilar artery, calculate the lumen diameter according to the formula: diameter (d)=L/π, and calculate the lumen diameter (r)=L/2π. The cavity radius and the lumen area (S) are obtained according to the formula: S=πr ² . Wall thickness was measured as follows: measure the distance from the inner surface of the basilar artery to the outer edge of the media, excluding the adventitia. Four different detection points were selected for each blood vessel to measure the wall thickness, and the average value was taken as the measured value of the blood vessel. The results are shown in Table 1.

Measurement of regional cerebral blood flow (rCBF) in the parietal cortex: a small trephine window with a diameter of 5 mm was used to open the bone window at the top of the post, the center was located 1 mm posterior to the Bergma point and 3 mm posterior to the outer side. The LDF3 laser Doppler flowmeter probe was fixed on the directional instrument micro-propeller, and the rCBF was observed in time before SAH preparation and 1, 4, 12, and 24 hours after SAH preparation. The results are shown in Table 2.

Statistical methods: Spontaneous activity score data is represented by scatter plot, and other data are represented by means ± SD; Kruskal-Wallis test and Mann-Whitney U test are used for statistical differences in spontaneous activity scores between groups, and the remaining data are statistically different between groups One-way ANOVA and Tukey's test were used, and P values less than 0.05 were considered significant differences.

2.3 Experimental results

As shown in Figure 5A-5B, compared with the SAH model control group, administration of different test compounds F, F+DCA and FDCA can significantly improve the neurological score of animals, and significantly reduce the brain water content of rats caused by SAH. Among them, FDCA showed the strongest activity, significantly better than F and DCA, and slightly better than F+DCA. In addition, the basilar artery diameter, lumen area, and wall thickness (Table 1) and regional cerebral blood flow (rCBF) in the parietal cortex (Table 2) were significantly improved in the FDCA group, which were significantly better than those in the F, DCA, and F+DCA groups. The above results show that FDCA has a significant anti-subarachnoid hemorrhage activity, which is superior to the marketed drug fasudil dihydrochloride and the combined administration of fasudil dihydrochloride and sodium dichloroacetate.

Table 1. Measurement of lumen diameter, lumen area and lumen thickness of rat basilar artery in each administration group

Note: *P<0.05, **P<0.01 compared with model, #P<0.05, ##P<0.01 compared with F+DCA group.

Table 2 Effects of compounds on regional cerebral blood flow in SAH rats

Note: *P<0.05, **P<0.01 compared with model, #P<0.05, ##P<0.01 compared with F+DCA group

Example 7

Prevention and/or treatment of ischemic stroke

In order to study whether FDCA has neuroprotective effect in vivo, the transient rat cerebral ischemia model (tMCAO) was selected for the experiment.

Model and administration method: After the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (350 mg/kg), they were fixed in the supine position on the experimental table, the mid-neck incision was made, the skin was incised with a scalpel, and each layer of tissue was bluntly separated. Anatomical diagram of the neck blood vessels of the rat, the left common carotid artery (CCA) was isolated under a stereo microscope, the left external carotid artery (ECA) and the left internal carotid artery were separated upwards, double ligated, and the superior thyroid artery and occipital artery were cut off. Two external carotid artery branches, double ligation of ECA at about 5mm-8mm near the CCA bifurcation, clipped with micro-arterial clips at the proximal end of the ICA and CCA respectively, and indwelling a dozen single knots near the bifurcation of the ECA but not. For the tightened silk thread, make a V-shaped micro-incision with a diameter of about 0.2 mm between the proximal ligation of the ECA and the bifurcation of the common carotid artery. Gently insert the nylon thread from the incision, and gently tighten the knot. The internal carotid artery is cut between the two ligatures to make it consistent with the direction of the internal carotid artery. Release the arterial clip. The nylon thread is sent into the skull along the ECA through the ICA. The insertion depth is about 18mm to 20mm. The end is located at the beginning of the MCA, the blood flow of the MCA is blocked, the silk thread is tightened, the incision is sutured, and the end of the nylon thread is indwelled in vitro.

After 2 hours of ischemia, the rats were anesthetized again with 10% chloral hydrate, and the nylon thread was gently pulled back to the micro-incision (a slight resistance feeling) to restore blood supply to the middle cerebral artery for reperfusion. Rats in the sham operation group were only subjected to anesthesia and vascular separation, without ligation of blood vessels and introduction of sutures, and the animals were kept warm after operation. Mode of administration: rats were administered by tail vein injection after 4 h and 24 h of ischemia. After 48 hours of ischemia, the neurological function was scored, and then the rats were sacrificed.

Grouping: sham operation group (Sham); blank solvent group (Vehicle); fasudil dihydrochloride group (F, 30 mg/kg, tail vein injection); FDCA group (30 mg/kg, tail vein injection); D Phthalide group (NBP, 5mg/kg, tail vein injection)

TTC staining: Four coronal incisions were made at the optic chiasm of the whole brain and 2 mm before and after, and the brain slices were quickly placed in 5 ml of phosphate buffer solution containing 2% TTC, and incubated at 37 °C for 10 min in the dark. During the incubation process, it was turned every 7-8 minutes. After incubation for 10 minutes, the brain slices were taken out and photographed with a digital camera (Olympus C-4000, Japan), and then the pale area (infarct area) and the non-pale area were separated with ophthalmic forceps. (Normal area), the infarct percentage was calculated by Image pro-plus 6.0 as follows:

Infarct percentage (%) = pale area weight / (pallor area weight + non-pallor area weight) × 100%

Neurological function grading: After 48 hours of ischemia, animals were graded for neurological deficits according to Longa's method, with the following criteria:

0 points: no neurological symptoms were observed;

1 point: When the tail is lifted, the contralateral forelimb of the animal shows flexion of the wrist and elbow, internal rotation of the shoulder, abduction of the elbow, and close to the chest wall;

2 points: The resistance decreases when the animal is placed on a smooth surface and the operated shoulder is pushed to the opposite side;

3 points: The animal turns or turns in a circle to the contralateral side of the operation when the animal walks freely;

4 points; flaccid paralysis, no spontaneous movement of limbs.

Statistical methods: The neurological deficit grading score data is expressed as the median, and the rest of the data are expressed as means ± SD; the statistical difference between the neurological deficit grading scores between groups is using the Kruskal-Wallis test and the Mann-Whitney U test, and the rest of the data groups One-way ANOVA and Tukey's test were used for statistical differences between the two groups, and a P value less than 0.05 was considered to have a significant difference.

The results showed that after 4 hours of ischemia, administration of FDCA (30 mg/kg) to rats effectively reduced the area of cerebral infarction (infarction area percentage: 7.48%), which was significantly lower than that of the blank solvent group (31.4%) and the marketed drug NBP group (21.1%). %), was significantly better than the fasudil dihydrochloride (30mg/kg) group (13.6%) (as shown in Figure 6, Figure 7); in addition, FDCA also significantly improved ischemia-induced neurobehavior Dysfunction, significantly better than fasudil hydrochloride and slightly better than NBP (Figure 8).

The activities of FDCA and equimolar doses of fasudil dihydrochloride (F), sodium dichloroacetate (DCA) and equimolar combination of the two were further investigated in the rat tMCAO model. The modeling method and administration time point are the same as above. The results showed that FDCA (30 mg/kg) administered to rats after 4 hours of ischemia effectively reduced the cerebral infarct size (infarct size percentage: 6.78%), which was significantly better than F (26.0 mg/kg, infarct size percentage: 22.8%) DCA (10.7 mg/kg, percentage of infarct size: 23.4%) and, and the combined administration group of both (percentage of infarct size: 15.2%) ( FIG. 9 ). In addition, FDCA also significantly improved ischemia-induced neurobehavioral dysfunction better than F, DCA, and their combined administration (Figure 9).

The above is only the preferred embodiment of the present invention, it should be pointed out that: for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.

Claims (5)

1. a class of compound, is characterized in that, is ROCK inhibitor-dichloroacetic acid double salt; Described ROCK inhibitor is selected from Ripasudil; Described compound is selected from: Ripasudil dichloroacetate; the structural formula is as follows:

2. A pharmaceutical composition comprising a therapeutically effective amount of the compound of claim 1 or its enantiomer, diastereomer, racemate, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant or vehicle. 3. Use of the compound of claim 1 in the preparation of a medicament for preventing and/or treating pulmonary hypertension. 4. Use of the compound of claim 1 in the preparation of a medicament for preventing and/or treating subarachnoid hemorrhage. 5. Use of the compound of claim 1 in the preparation of a medicament for preventing and/or treating ischemic stroke.

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