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

Abstract

The invention relates to the fields of medicinal chemistry and pharmacotherapeutics, in particular to a ROCK inhibitor-dichloroacetic acid double salt or pharmaceutically acceptable salt thereof, a preparation method thereof, a medicinal composition containing the compounds and medicinal application thereof, especially application in preparing medicaments for preventing and/or treating cardiovascular and cerebrovascular diseases such as pulmonary hypertension, ischemic stroke, subarachnoid hemorrhage and the like.

Description

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

Technical Field

The invention relates to a ROCK inhibitor-dichloroacetic acid complex salt, in particular to a ROCK inhibitor-dichloroacetic acid complex salt, a preparation method thereof, a medicinal composition containing the compounds and medicinal application thereof, belonging to the technical field of pharmacy.

Background

Rho kinase (ROCK) is an important enzyme involved in a series of cell life phenomena such as cell mitosis adhesion, cytoskeleton regulation, muscle cell contraction, tumor cell infiltration, etc. since 1996, ROCK has been found to be classified into ROCK I (ROCK β) which is mainly present in cells of non-neural tissues such as heart, lung, skeletal muscle, etc., and ROCK II (ROCK α) which is mainly present in central nervous systems such as hippocampal pyramidal neurons, cerebral cortex, cerebellar purkinje cells, etc.

Fasudil [ hexahydro-1- (5-sulfonylisoquinoline) -1(H) -1, 4-diazepine, Fasudil, also known as HA1077], is a novel isoquinoline sulfonamide derivative developed by Asahi Kasei corporation and the pharmacological research laboratory of the university of ancient houses in Japan. As a novel and efficient vasodilator, fasudil can effectively relieve cerebral vasospasm and improve prognosis of patients with subarachnoid interstitial hemorrhage (SAH), since fasudil is marketed in Japan in 1996, the effect of fasudil on pulmonary blood vessels is widely concerned by researchers, and a large number of animal experiments and clinical studies show that fasudil can: 1) activating endogenous neural stem cells to promote brain tissue repair; 2) increasing astrocyte stimulating factor; 3) inhibiting the release of intracellular calcium ions; 4) relaxing cerebral blood vessels; 5) protecting nerve cells and improving the function of extending; 6) promoting axon regeneration. Therefore, fasudil is also used for treating cerebral ischemic stroke. In addition, fasudil can also treat pulmonary hypertension safely and effectively. The ROCK inhibitor fasudil can penetrate into vascular smooth muscle cells and compete with ATP for ATP binding sites in the Rho kinase catalytic region under normal or pathological conditions to specifically block Rho kinase activity. At present, the anti-PAH effect of fasudil hydrochloride is in the phase II clinical research stage.

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

Disclosure of Invention

The purpose is as follows: the invention provides a ROCK inhibitor-dichloroacetic acid complex salt, a preparation method and medical application thereof.

The technical scheme is as follows: in order to solve the technical problems, the technical scheme adopted by the invention is as follows:

one kind of compound is ROCK inhibitor-dichloroacetic acid double salt.

Further, the ROCK inhibitor is selected from fasudil, Netarsudil and Ripasudil.

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

the preparation method of the fasudil dichloroacetate comprises the following steps:

putting a proper amount of fasudil into a reaction container, adding a proper amount of reaction solvent, and mixing to obtain a mixed solution of fasudil and the reaction solvent;

adding a proper amount of dichloroacetic acid into the mixed solution while stirring at the reaction temperature of 0-100 ℃, and continuously stirring for a period of time after the dripping is finished to obtain a reaction solution;

and then decompressing and concentrating the reaction liquid to remove the solvent, washing and recrystallizing to obtain the fasudil dichloroacetate.

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

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

the preparation method comprises the following steps: dissolving the Netarsudil in tetrahydrofuran, dropwise adding dichloroacetic acid into a reaction system, stirring for a period of time at normal temperature, and spin-drying to obtain the Netarsudil dichloroacetate.

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

the preparation method comprises the following steps: at room temperature, taking a proper amount of Ripasudil, placing the Ripasudil into a reaction container, adding water, slowly dripping dichloroacetic acid into Ripasudil suspension while stirring, and continuously stirring at room temperature for a period of time after dripping to obtain a reaction solution; then decompressing and concentrating the reaction liquid, washing, filtering and drying to obtain Ripasudil dichloroacetate.

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

On the other hand, the invention also provides application of the compound in preparing medicaments for preventing and/or treating cardiovascular and cerebrovascular diseases such as pulmonary hypertension, subarachnoid hemorrhage, ischemic stroke and the like.

In the present invention, the above-mentioned compounds and pharmaceutically acceptable salts thereof, and solvates of these compounds (collectively referred to herein as "therapeutic agents") can be administered to mammals either alone or, preferably, in combination with pharmaceutically acceptable carriers or diluents in accordance with standard pharmaceutical practice. The mode of administration can be by various routes, including oral, parenteral, or topical administration. Parenteral administration as used herein includes, but is not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and transdermal administration.

The invention firstly discloses fasudil dichloroacetate and a preparation method thereof, and the preparation method comprises the following steps: mixing free fasudil with water, slowly dripping dichloroacetic acid, continuously stirring for 5min, concentrating to remove purified water, adding diethyl ether into residues, washing for 3 times, and recrystallizing with isopropanol or other solvents to obtain high-purity fasudil dichloroacetate. And the structure of the compound is confirmed by hydrogen spectrum, carbon spectrum and mass spectrum. The method has the advantages of simple operation, low production cost, high product yield, small environmental pollution and contribution to industrial mass production.

Meanwhile, the invention discloses the inhibition activity of fasudil dichloroacetate, Netarsudil dichloroacetate and Ripasudil dichloroacetate on ROCK I and ROCK II. As a result, the salt formation of dichloroacetate and ROCK inhibitor is found to improve the ROCK inhibition activity.

The invention discloses a treatment effect of Fasudil Dichloroacetate (FDCA) on pulmonary hypertension, firstly, Fasudil Dichloroacetate (FDCA) in cell experiments can obviously inhibit the expression of inflammatory factors tumor necrosis factor- α (TNF- α) and interleukin-6 (I L-6) in platelet-derived growth factor BB (PDGF-BB) and hypoxia-induced Pulmonary Artery Smooth Muscle Cells (PASMCs) and Pulmonary Artery Endothelial Cells (PAECs), further animal experiments are carried out, in a treatment model of rat pulmonary hypertension induced by monocrotaline, FDCA (43.3mg/kg) is perfused to obviously reduce the average pulmonary artery pressure of a rat with pulmonary hypertension, the right ventricular systolic pressure and the right ventricular hypertrophy index, but has no obvious influence on the systemic circulatory pressure, and by pathological examination of rat and heart tissue, FDCA obviously reduces the ratio of the pulmonary arteriolar vascular wall thickness to the pulmonary arteriolar diameter (PAMT) and the pulmonary arteriolar fibrosis degree, and the FDCA is a candidate drug for obviously reducing the pulmonary hypertension and the pulmonary fibrosis, namely the molar reduction of FDCA and the pulmonary hypertension resistance of FDCA.

Meanwhile, the invention discloses an effect of Fasudil Dichloroacetate (FDCA) in preventing and/or treating subarachnoid hemorrhage. In a rat subarachnoid hemorrhage model (subarachnoid hemorrhage model perforation, administration for 0.5h and 6h after model creation, and evaluation for each index of a rat after 24 h), FDCA obviously reduces cerebral vasospasm injury after rat subarachnoid hemorrhage, improves cerebral edema and animal neurological score, obviously improves basilar artery diameter, lumen area, tube wall thickness and local cerebral blood flow (rCBF) of a top cortex, and is superior to F, DCA and combined administration of the F and DCA. The results suggest that FDCA is an effective candidate drug for combating subarachnoid hemorrhage and is worthy of further study.

Meanwhile, the invention discloses an effect of Fasudil Dichloroacetate (FDCA) in preventing and/or treating cerebral arterial thrombosis. In a transient ischemia model of a rat (2 h of ischemia re-perfusion, 4h of ischemia once administration, 24h of administration, evaluation of various indexes of the rat after 48 h), FDCA effectively reduces the cerebral infarction area, is obviously superior to a marketed drug butylbenzene peptide (NBP) group, is obviously superior to a fasudil dihydrochloride group (F), a sodium Dichloroacetate (DCA) group and a combined administration group of F and DCA; in addition, FDCA also obviously improves the neurobehavioral dysfunction induced by ischemia, is obviously superior to F, DCA and the combined administration of the F and the DCA, and is slightly superior to NBP group. The result indicates that FDCA is an effective candidate drug for resisting cerebral arterial thrombosis and is worthy of further research.

As an inhibitor of ROCK, fasudil can relax blood vessels, reduce blood pressure, inhibit proliferation of vascular smooth muscle cells and inhibit vascular remodeling; the dichloroacetate is an inhibitor of pyruvate dehydrogenase kinase, can improve the activity of pyruvate dehydrogenase, promote the aerobic metabolism of glucose and reduce the generation of lactic acid; meanwhile, the expression of potassium ion channels, particularly Kv1.5, can be promoted, the proliferation of smooth muscle cells is inhibited, and the apoptosis of the smooth muscle cells is promoted. 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 the combined administration, Fasudil Dichloroacetate (FDCA) as a whole molecule can have differences in drug absorption, distribution, metabolism and the like and the combined administration, thereby showing better activity.

Drawings

FIG. 1 is a graph showing the effect of the compounds of example 5 on the expression of TNF- α and I L-6 in PDGF-BB and hypoxic culture conditions for PASMCs and PAECs, wherein PASMCs are pulmonary artery smooth muscle cells, PAECs are pulmonary artery endothelial cells, PDGF-BB is platelet-derived growth factor BB, I L-6, interleukin-6, CON is a blank control group, Hypoxia is Hypoxia, FDCA is fasudil dichloroacetate, F is fasudil dihydrochloride, DCA is dichloroacetic acid sodium salt, F + DCA is fasudil dihydrochloride and dichloroacetic acid sodium salt.

FIG. 2 is a graph showing the effect of the compound of example 5 on hemodynamics of MCT-induced PAH model rats, mPAP mean pulmonary artery pressure, RVSP right ventricular systolic pressure, mPAP mean systemic circulation pressure, RV/L V + S right heart hypertrophy index, Control, MCT monocrotaline, FDCA fasudil dichloroacetate, F fasudil dihydrochloride, DCA dichloroacetate sodium salt, F + DCA fasudil dihydrochloride, and dichloroacetate sodium salt.

FIG. 3 is a graph of the effect of each administration group on the ratio of pulmonary arteriole vascular wall thickness to pulmonary arteriole diameter (PAMT) and the degree of fibrosis in rats in example 5; PAMT: the ratio of the wall thickness of the pulmonary arteriole vessel to the diameter of the pulmonary arteriole of the rat; fibrosis: fiberizing; control: control, MCT: monocrotaline; FDCA: fasudil dichloroacetate; f: fasudil dihydrochloride; DCA: dichloroacetic acid sodium salt; f + DCA: and the fasudil dihydrochloride and the dichloroacetic acid sodium salt are jointly administered.

FIG. 4is a graph showing the effect of each administration group on the right ventricular cardiomyocyte area and the degree of fibrosis in example 5; CAS: myocardial cell cross-sectional area; fibrosis: fiberizing; control: control, MCT: monocrotaline; FDCA: fasudil dichloroacetate; f: fasudil dihydrochloride; DCA: dichloroacetic acid sodium salt; f + DCA: and the fasudil dihydrochloride and the dichloroacetic acid sodium salt are jointly administered.

FIG. 5A is the effect of different compounds of example 6 on cerebral edema in SAH rats; FIG. 5B is the effect of different compounds of example 6 on the score of spontaneous activity in SAH rats.

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

FIG. 7 is a statistical plot of cerebral infarct size of rats in the tMCAO model of example 7.

Figure 8 is the tMCAO model rat neurological function score of example 7.

FIG. 9 is a graph of the cerebral infarct area and the neurological function score of rats in the tMCAO model of example 7.

Detailed Description

To further illustrate the present invention, a series of examples are given below, which are purely illustrative and are intended to be a detailed description of the invention only and should not be understood as limiting the invention.

Example 1

Fasudil dichloroacetate

Compound synthesis and characterization data:

at room temperature, 10g of fasudil is placed in a 100m L eggplant-shaped bottle, 20m L of tap water or purified water is added for stirring, then 4.87g of dichloroacetic acid is weighed and slowly added into the suspension in a dropwise manner, fasudil is found to be gradually dissolved in the dropwise adding process, the dropwise adding is completed, the stirring is continued for 10min at room temperature, the reaction liquid is decompressed and concentrated, the remainder is added with ether for washing for 3 times, the ether is removed, organic solvent is used for recrystallization, white solid is obtained by filtering, the white solid is placed in a vacuum drying box for drying, 13.95g of target compound is obtained, the yield is 96.9%, and Mp is 143 ℃.¹H NMR(300MHz，D₂O，TMS)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).¹³C NMR(75MHz，D₂O)170.25，152.36，142.97，134.17，133.01，131.0，130.25，128.10，126.03，116.69，68.07，46.76，46.22，44.41，43.46，24.99.ESI-MS(70eV)m/z：292.2[M+H]⁺。

Example 2

Netarsuidil Dichloroacetate salt

Compound synthesis and characterization data:

4- (3-amino-1- (isoquinolin-6-ylamino) -1-oxoprop-2-yl) benzyl 2, 4-dimethylbenzoate (100mg, 0.22mmol) was dissolved in tetrahydrofuran, dichloroacetic acid (28mg, 0.22mmol) was added dropwise to the reaction system, stirred at room temperature for 10min, and spun dry to give a pale yellow solid, netarsuil dichloroacetate.¹H NMR(300MHz，DMSO)：11.07(s，1H)，9.22(s，1H)，8.43(s，2H)，8.18(s，1H)，8.11(d，J＝8.70Hz，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(70eV)m/z：454.2[M+H]⁺。

Example 3

Ripasudil dichloroacetate salt

Compound synthesis and characterization data:

at room temperature, 100mg of Ripasudil is placed in a 50m L eggplant-shaped bottle, 3m L of tap water or purified water is added for stirring, then 48mg of dichloroacetic acid is weighed and slowly dripped into the suspension, the reaction liquid is gradually dissolved in the dripping process, dripping is completed, the stirring is continued for 10min at room temperature, then the reaction liquid is decompressed and concentrated, the residue is added with diethyl ether for washing 3 times, the diethyl ether is discarded, a light yellow solid is obtained by filtering, and then the light yellow solid is placed in a vacuum drying oven for drying to obtain 113.1mg of a target compound, wherein the yield is 81%.¹H-NMR(300MHz，D₂O，)：1.11(3H，d，J＝6.6Hz)，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(70eV)m/z：324.2[M+H]⁺。

Example 4

Inhibitory Activity of Compounds against 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 inhibition activity of the fasudil dichloroacetate to ROCK-I and ROCK-II is stronger than that of the fasudil to ROCK-I and ROCK-II;

the inhibition activity of the Netarsudil dichloroacetate on ROCK-I and ROCK-II is stronger than that of the Netarsudil on ROCK-I and ROCK-II;

ripasudil dichloroacetate has stronger inhibitory activity on ROCK-I and ROCK-II than Ripasudil.

Example 5

Prevention and/or treatment of pulmonary hypertension

First, the Effect of FDCA on the expression of inflammatory factors TNF- α and I L-6 in PASMCs and PAECs cells in PDGF-BB and hypoxic culture models

The effects of Fasudil Dichloroacetate (FDCA) on hypoxia culture conditions and platelet-derived growth factor BB (PDGF-BB) -induced Pulmonary Artery Smooth Muscle Cells (PASMCs) and Pulmonary Artery Endothelial Cells (PAECs) on TNF- α and I α 2-6 expression are examined through cell experiments firstly, the effects of Fasudil Dichloroacetate (FDCA) on hypoxia culture conditions and on platelet-derived growth factor BB (PDGF-BB) induced Pulmonary Artery Smooth Muscle Cells (PASMCs) and I α 2-6 expression in PAECs are shown by the fact that the effects of the Fasudil Dichloroacetate (FDCA) in a group of normal cells (Control), a group of platelet-derived growth factor BB (PDGF-BB) or a cultured model group of PAECs, a group of α model group + Fasudil Dichloroacetate (FDCA), a group of 638 model group + fasudil hydrochloride (F) therapy group, a group of 6869 model group + dichloroacetate sodium salt (DCA) therapy group, a group of 6 model group of model group and a group of models of hypoxia-3648, a α and 24-6-24-hours after the administration of the platelet-derived growth factor BB, and the platelet-derived growth factor BB, after the administration of platelet-BB, the platelet-derived growth factor BB, the proliferation of the PDGF, the PDGF-derived growth factor BB, the factor is shown by the experiments, the factor is considered as the statistics of the experiments, the effects of the factor, the factor of the factor, the factor of the factor, the factor of inhibiting the factor of the factor.

Second, Effect of FDCA Compounds on MCT-induced PAH model rat hemodynamics

The animal group is a ① normal control group, a ② normal control group + FDCA, a ③ MCT model group, a ④ fasudil dihydrochloride (F) treatment group, a ⑤ DCA treatment group, a L F + DCA combination treatment group, a L1 FDCA administration group, the establishment of the rat model is that the animal model group and the treatment group are subjected to one-time intraperitoneal injection of Monocrotaline (MCT)60mg/kg, the normal control group is subjected to injection of physiological saline, the experimental treatment is that on the 14 th day of monocrotaline injection, the administration groups are started at equal molar doses, the administration mode is intragastric administration, the F group is once a day, the F group is 37.5mg/kg, the A group is 15.5mg/kg, the F + DCA combination treatment group comprises F (37.5mg/kg) and DCRVA 15.5mg/kg, the normal control group is subjected to one-time daily administration of monocrotaline injection, the monocrotaline injection of MCT injection of monocrotaline injection, the monocrotaline injection of the compound and the compound of.

Third, the Effect of FDCA Compounds on MCT-induced PAH model rat pulmonary artery

As shown in fig. 3, the effect of different administration groups on the ratio of pulmonary arteriolar vascular wall thickness to pulmonary arteriolar diameter (PAMT) and the degree of fibrosis in rats can be found that FDCA can effectively reduce the degree of PAMT and pulmonary arteriolar fibrosis, which is slightly better than that of F, DCA and the combination of F and DCA.

Fourth, Effect of FDCA Compounds on right ventricle of MCT-induced PAH model rats

As shown in fig. 4, the effect of each administration group on the right ventricular cardiomyocyte area and the degree of fibrosis suggests that the MCT model group significantly increased the right ventricular cardiomyocyte area and the degree of fibrosis in rats compared to the blank control. The administration group, especially FDCA, can obviously reduce the area and fibrosis degree of the myocardial cells in the right ventricle, is superior to F, DCA and the combined administration group of the F and the DCA, and results indicate that FDCA can effectively inhibit the proliferation and the reconstruction of the myocardial cells in the right ventricle.

Example 6

Prevention and/or treatment of subarachnoid hemorrhage

Laboratory animal

SPF grade SD rat with weight of 260-. The total number of animals was 32. The sham operation group: an equal volume of saline containing 1% DMSO (n ═ 8);

experimental methods

Test grouping and drug concentration selection:

SAH model group: an equal volume of saline containing 1% DMSO (n ═ 8); fasudil dihydrochloride (group F): (26.0mg/kg) (n ═ 8); sodium Dichloroacetate (DCA) group: (10.7mg/kg) (n ═ 8): fasudil dihydrochloride in combination with sodium dichloroacetate (F + DDCA) group: (F: 26.0 mg/kg; DCA: 10.7mg/kg) (n ═ 8); FDCA group: (30mg/kg) (n-8); all the medicines are prepared into a normal saline solution containing 1% DMSO, and the administration mode is tail vein injection. The drug was administered once each 0.5h and once after 6h after SAH (rat subarachnoid hemorrhage modeling), and the sham and model groups were replaced with an equal volume of physiological saline containing 1% DMSO.

Model and method of administration

The reference (Stroke,1995,26, 1086-. I.e. rats were anesthetized, intubated and kept artificially ventilated with 3% isoflurane in 70%/30% medical air/oxygen during surgery. Body temperature was monitored by a rectal probe and maintained normothermia by a heating lamp. A sharpened 4-0 nylon suture was introduced into the left Internal Carotid Artery (ICA) until resistance was felt (about 18mm from the common carotid bifurcation). The suture is then pushed further to pierce the bifurcation of the anterior and middle cerebral arteries until the resistance is overcome and withdrawn immediately after perforation. In sham animals, sutures were inserted into the left ICA, but no perforation was performed. After removal of the sutures, the incision was closed and the rats were individually housed in heated cages until recovery.

Spontaneous activity scoring: rats were scored for spontaneous activity in a spacious, freely movable, accessible cage on all four walls. The experimental rats were evaluated and recorded by 2 experimenters 24h after SAH modeling by a double-blind method, 2 groups of mean values were taken as final scores, and the rats were sacrificed immediately after observation of spontaneous activity. Spontaneous activity scores were classified into 4 grades according to animal mental status and movement: level 1, the rat moves normally without movement obstacle, actively explores the surrounding environment and at least touches the upper edges of three cage walls; grade 2, mild dyskinesia, i.e. poor spirit, lethargy, with a certain delay in locomotion, without reaching all cage walls, but he touched at least the upper edge of one cage wall; grade 3, moderate dyskinesia, i.e. rats were almost unable to stand and moved little in the cages; grade 4, severe motility disorder, i.e. mice did not move and showed paralysis with limbs. The results are shown in FIG. 5A.

And (3) measuring the water content of the brain, namely, killing the rats 24h after SAH molding, quickly taking out the brain and the cerebellum, sucking surface blood by using filter paper, weighing the masses (wet weights) of the brain and the cerebellum by using an electronic balance, placing the brain tissue in an oven, baking the brain tissue at 105 ℃ to constant weight, and weighing the masses (dry weights) of the brain and the cerebellum again, wherein the water content of the brain tissue is calculated according to the formula of (wet weight-dry weight)/wet weight × 100%, the water content of the tissue of the cerebellum is used as a normal control, and the result is shown in a figure 5B.

Measuring the diameter, area and wall thickness of basilar artery by HE staining tissue slices of the basilar artery, observing and photographing under an optical microscope, and measuring the diameter, area and wall thickness of the basilar artery by using an image pro-plus6.0 image analysis system, wherein the circumference (L) of the lumen of the basilar artery is measured along the inner surface of the basilar artery, the diameter (d) L/pi of the lumen is calculated according to a formula, the radius (r) L/2 pi of the lumen is calculated, and the area (S) of the lumen is calculated according to a formula, S pi r²And (6) obtaining. The method for measuring the thickness of the pipe wall comprises the following steps: the distance from the inner surface of the basilar artery to the outer edge of the media was measured, excluding the adventitia. And 4 different detection points are selected for each blood vessel to measure the thickness of the tube wall, and the average value of the thicknesses is taken as the measurement value of the blood vessel. The results are shown in Table 1.

And (3) measuring the local cerebral blood flow (rCBF) of the top cortex, namely, cutting a bone window on the top of mail by using a small trephine with the diameter of 5mm, positioning the center of the bone window 1mm behind the Bergma point, and positioning the bone window 3mm behind and outside, fixing an L DF3 type laser Doppler blood flow instrument probe on a directional instrument micro-propeller, and observing the rCBF in time before SAH preparation and 1, 4, 12 and 24h after SAH preparation respectively, wherein the results are shown in a table 2.

The statistical method comprises the following steps: the spontaneous activity scoring data is represented by a scatter plot, and the rest data are represented by means +/-SD; statistical differences between groups were determined by Kruskal-Wallis test and Mann-Whitney U test, and between the remaining groups by one-way ANOVA and Tukey's test, and significant differences were considered for P values less than 0.05.

2.3 results of the experiment

As shown in fig. 5A-5B, administration of different test compounds F, F + DCA and FDCA significantly improved animal neurological scores and significantly reduced brain water content in rats induced by SAH, compared to the SAH model control group, where FDCA showed the strongest activity, significantly better than F and DCA, slightly better than F + DCA. In addition, the FDCA group significantly improved the basilar artery caliber, luminal area and wall thickness (table 1) and the apical cortical local cerebral blood flow (rCBF) (table 2), significantly better than the F, DCA and F + DCA groups. The results show that FDCA has remarkable activity for resisting subarachnoid hemorrhage, and is superior to the marketed drug fasudil dihydrochloride and the combined administration of fasudil dihydrochloride and sodium dichloroacetate.

TABLE 1 measurement of the lumen diameter, lumen area and lumen thickness of the basilar artery of rats in each administration group

Note: p <0.05 for model, # P <0.01 for F + DCA group, # P <0.05 for # P, # P <0.01 for model.

TABLE 2 Effect of Compounds on regional cerebral blood flow changes in SAH rats

Note: p <0.05, P <0.01 compared to model, # P <0.05, # P <0.01 compared to F + DCA group

Example 7

Prevention and/or treatment of ischemic stroke

To investigate whether FDCA has neuroprotective effects in vivo, transient rat cerebral ischemia model (tMCAO) was chosen for the experiments.

Model and administration method: anesthetizing a rat by injecting 10% chloral hydrate (350mg/kg) into the abdominal cavity, fixing the rat in a supine position on a laboratory bench, performing a median cervical incision, incising the skin by using a scalpel, separating tissues in a blunt manner, separating a left Common Carotid Artery (CCA) under a stereomicroscope according to a neck blood vessel anatomical diagram of the rat, placing a suture for standby upward separation of a left External Carotid Artery (ECA) and an internal carotid artery, performing double ligation, cutting two branches of the external carotid artery and an upper thyroid artery, performing double ligation on the ECA at a position about 5mm-8mm away from the bifurcation of the CCA, respectively retaining a arteriolar clamp at the near-core ends of the ICA and the CCA, beating a single-knot but not tightening the suture at the near-bifurcation of the ECA, performing a V-shaped micro incision with the diameter of about 0.2mm between the proximal ligation position of the ECA and the branch of the common carotid artery, slightly inserting a nylon thread from the incision, slightly tightening the knot, and cutting the internal carotid artery between the two ligation threads, the direction of the artificial artery is consistent with that of the internal carotid artery, the artery clamp is loosened, the nylon thread is sent into the cranium along the ECA through the ICA, the insertion depth is stopped when meeting resistance slightly at about 18 mm-20 mm, the head end of the nylon thread is positioned at the starting position of the MCA, the blood flow of the MCA is blocked, the silk thread is tightened, the incision is sutured, and the tail end of the nylon thread is kept out of the body.

After 2h of ischemia, the rats were re-anesthetized with 10% chloral hydrate, and the nylon wire was gently pulled to return the tip to the micro-incision (slightly resistive), thereby restoring blood supply to the middle cerebral artery and performing reperfusion. The rats in the sham operation group are only subjected to anesthesia and blood vessel separation, blood vessels and lead-in thread plugs are not ligated, and the animals are kept warm after the operation. The administration mode comprises the following steps: rats were administered by tail vein injection 4h and 24h after ischemia. After 48h of ischemia, neurological function was scored and the rats were sacrificed.

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

TTC dyeing: making four coronary cutting knives at the positions 2mm before and after the intersection of the whole brain vision, cutting into five slices, quickly placing the slices into 5ml of phosphoric acid buffer solution containing 2 percent TTC, incubating for 10min in a dark place at 37 ℃, turning over every 7-8 min in the incubating process, taking out the slices after incubating for 10min, taking a picture by using a digital camera (Olympus C-4000, Japan), separating a pale area (infarct area) and a non-pale area (normal area) by using an ophthalmic forceps, and calculating the infarct percentage by using Image pro-plus6.0 as follows:

percentage of infarct (%) < weight of pale area/(weight of pale area + weight of non-pale area) × 100%

Neurological ratings animals were graded for neurological deficits following 48h ischemia according to the L onga's method, as follows:

0 minute: no neurological symptoms were observed;

1 minute: when the tail is lifted and suspended, the operation of the animal shows that the contralateral forelimb shows that the wrist and elbow are bent, the shoulder is rotated inwards, the elbow is expanded outwards and is tightly attached to the chest wall;

and 2, dividing: the animal is placed on a smooth plane, and when the side shoulder of the operation is pushed to move towards the opposite side, the resistance is reduced;

and 3, dividing: when the animal walks freely, the animal rotates towards the opposite side of the operation or rotates around;

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

The statistical method comprises the following steps: the grading scoring data of the neurological deficit is expressed by median, and the rest data are expressed by means +/-SD; statistical differences between groups of neurological deficit grade scoring data were determined by Kruskal-Wallis test and Mann-Whitney U test, and statistical differences between the remaining groups were determined by one-way ANOVA and Tukey's test, and significant differences were considered for P values less than 0.05.

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

The activity of FDCA with equimolar doses of fasudil dihydrochloride (F), dichloroacetate sodium salt (DCA) and equimolar combinations of the two was further investigated in the rat tMCAO model. The molding method and the administration time point were the same as above. The results showed that FDCA (30mg/kg) administered to rats was effective in reducing the cerebral infarct size (percent infarct size: 6.78%), significantly better than F (26.0mg/kg, percent infarct size: 22.8%), DCA (10.7mg/kg, percent infarct size: 23.4%) and, in combination with the two (percent infarct size: 15.2%), after 4h of ischemia (FIG. 9). In addition, FDCA also significantly improved ischemia-induced neurobehavioral dysfunction over F, DCA and its combination (fig. 9).

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Claims (5)

1. A compound is characterized in that the compound is ROCK inhibitor-dichloroacetic acid double salt; the ROCK inhibitor is selected from Ripasudil; the compound is selected from:

ripasudil dichloroacetate; the structural formula is as follows:

2. a pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, or an enantiomer, diastereomer, racemate, or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

3. Use of a compound according to claim 1 for the preparation of a medicament for the prophylaxis and/or treatment of pulmonary hypertension diseases.

4. Use of a compound according to claim 1 for the preparation of a medicament for the prophylaxis and/or treatment of subarachnoid hemorrhage disorders.

5. Use of the compound of claim 1 for the preparation of a medicament for the prevention and/or treatment of ischemic stroke diseases.

Images