Preparation method and application of fasudil dichloroacetate

Abstract

The invention discloses a preparation method and application of fasudil dichloroacetate, which comprises the following steps: reacting 5-isoquinoline sulfonic acid with thionyl chloride in a solvent to obtain an intermediate 2, and carrying out condensation reaction on the intermediate 2 and 5-Boc homopiperazine to obtain an intermediate 3; and directly reacting the intermediate 3 with dichloroacetic acid to remove the t-butyloxycarboryl protecting group and form dichloroacetate to obtain the target compound. According to the method, waste caused by preferential preparation of fasudil hydrochloride in the prior art is avoided, the yield is increased, and the cost is saved. The fasudil dichloroacetate is used for preparing the medicine for preventing or treating the vascular dementia, has the medicine effect of remarkably reducing cognitive impairment caused by chronic hypoperfusion of cerebral blood flow, and can become a novel medicine or composition for preventing or treating the vascular dementia.

Description

Technical Field

[0001] The present application relates to a method for preparing fasudil dichloroacetate (FDCA) and its application, and specifically to a method for preparing fasudil dichloroacetate and its application in preparing a drug for preventing and / or treating vascular dementia, belonging to the field of medical technology. Background Art

[0002] Vascular dementia (VaD) refers to a syndrome of severe cognitive dysfunction caused by ischemic stroke, hemorrhagic stroke, and cerebrovascular disease that causes low perfusion of brain areas such as memory, cognition, and behavior. According to epidemiological surveys, vascular dementia is the second most common cause of dementia after Alzheimer's disease, accounting for about 15% of cases. The incidence rate increases with age, and the risk of vascular dementia doubles approximately every 5.3 years. In addition, approximately 15% to 30% of subjects develop dementia 3 months after a stroke. VaD not only reduces the patient's own quality of life, but also brings a heavy burden to the patient's family and society. However, unlike Alzheimer's disease, there is no licensed treatment for vascular dementia. Therefore, there is an urgent need to find a low-risk, effective drug to treat vascular dementia.

[0003] The pathogenesis of vascular dementia is mainly related to the continuous decrease in cerebral blood flow. Chronic hypoperfusion and thromboembolism lead to a continuous decrease in cerebral blood flow, hypoxia, oxidative stress and inflammatory response. The periventricular white matter area, basal ganglia and hippocampus are extremely susceptible to lesions caused by hypoperfusion, and the interruption of the prefrontal lobe-basal ganglia circuit leads to cognitive deficits. The white matter of the brain is extremely susceptible to hypoxia-induced damage, causing demyelination, which delays neural signal transmission and leads to cognitive loss. Inflammatory factors further aggravate white matter damage (demyelination, axonal loss, oligodendrocyte degeneration), impair neurogenesis, neuronal progenitor cell proliferation, synaptic plasticity and dendritic spine density, causing neurodegeneration and cell death.

[0004] At present, cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonist memantine are the most popular methods in the treatment of vascular dementia, but their effects on improving patients' cognitive function are limited. Recent studies have shown that the Rho / Rho-associated coiled-coil containing protein kinase (ROCK) signaling pathway may be a potential therapeutic target. ROCK is the main downstream target of Rho and is a widely expressed serine-threonine protein kinase, including two isozymes, ROCK1 and ROCK2. In the nervous system, the Rho / ROCK signaling pathway is involved in regulating neuronal morphology, axon growth, synaptic plasticity and neural regeneration. Its abnormalities are associated with a variety of nervous system diseases, such as neurodegenerative diseases, ischemic brain injury and cognitive dysfunction. In addition, the pyruvate dehydrogenase kinase (PDK) signaling pathway has received increasing attention in the study of neurodegenerative diseases.

[0005] Dichloroacetate (DCA) is a PDK inhibitor that activates the pyruvate dehydrogenase complex (PDC) by inhibiting the activity of PDK, promoting the conversion of glucose to the tricarboxylic acid cycle and enhancing oxidative phosphorylation. Studies have shown that DCA can improve energy metabolism and exert a neuroprotective effect by promoting pyruvate to enter mitochondria for oxidative phosphorylation.

[0006] The ROCK inhibitor fasudil (F) and the PDK inhibitor DCA have shown neuroprotective effects in a variety of neurological diseases and may have synergistic effects. To date, there is no relevant literature on the use of fasudil dichloroacetate in the treatment of vascular dementia.

[0007] The currently reported preparation routes of fasudil dichloroacetate (such as CN109734701A and CN109761958A) all directly use commercial fasudil hydrochloride as a raw material for synthesis, while the traditional preparation routes of fasudil hydrochloride (such as US4678783, CN101092413A and CN101863880A) use isoquinoline to react with a sulfonating agent to prepare 5-isoquinoline sulfonate, and 5-isoquinoline sulfonate reacts with a chlorinating agent to prepare 5-isoquinolinesulfonyl chloride hydrochloride solid, the latter is freed with a sodium bicarbonate aqueous solution, and then extracted with dichloromethane to obtain a dichloromethane solution of 5-isoquinolinesulfonyl chloride, the latter is then reacted with homopiperazine to prepare fasudil, and then the hydrochloride is formed to complete the synthesis of fasudil hydrochloride. This process is relatively mature, but in production, since homopiperazine has no reaction selectivity at all, both primary amines at both ends of the reaction may participate in the reaction, which easily generates impurities. The expensive raw material homopiperazine must be excessive in the reaction, which cannot be fully utilized, resulting in waste, and the cost is relatively high. The reaction yield is low, with a maximum total yield of 62.7%, and it needs to be freed before it can be salified with dichloroacetic acid. Therefore, the current synthesis model of using commercial fasudil hydrochloride as a raw material, or first synthesizing fasudil hydrochloride, and then reacting with dichloroacetic acid to obtain fasudil dichloroacetate after being freed is relatively costly. Summary of the invention

[0008] Purpose of the invention: In view of the shortcomings of the prior art, one of the purposes of the present invention is to provide a method for preparing fasudil dichloroacetate, another purpose of the present invention is to provide the use of the fasudil dichloroacetate in the preparation of a medicament for preventing and / or treating vascular dementia, and the third purpose of the present invention is to provide a pharmaceutical composition containing the fasudil dichloroacetate.

[0009] Technical solution: The preparation method of fasudil dichloroacetate of the present invention is synthesized according to the following route:

[0010]

[0011] Furthermore, the preparation method comprises the following steps:

[0012] (1) 5-isoquinolinesulfonic acid reacts with thionyl chloride in a solvent to obtain intermediate 2;

[0013] (2) Intermediate 2 was precooled and stirred with anhydrous dichloromethane, chloroform and anhydrous triethylamine in an ice bath, and then DCM and 5-Boc homopiperazine were added dropwise to carry out a condensation reaction at room temperature to obtain Intermediate 3;

[0014] (3) Intermediate 3 reacts with dichloroacetic acid to remove the tert-butyloxycarbonyl protecting group and form dichloroacetate to obtain the target compound FDCA.

[0015] Furthermore, in step (1), the solvent is one or more of anhydrous dichloromethane, chloroform, ethyl acetate, redistilled acetone, anhydrous tetrahydrofuran, anhydrous N,N-dimethylformamide, dimethyl sulfoxide or dioxane.

[0016] Furthermore, in step (1), the reaction temperature is 0 to 100° C., and the reaction time is 1 to 12 h.

[0017] Furthermore, in step (2), the solvent is selected from one or more of anhydrous acetonitrile, anhydrous dichloromethane, chloroform, ethyl acetate, redistilled acetone, anhydrous tetrahydrofuran, anhydrous N,N-dimethylformamide, dimethyl sulfoxide or dioxane.

[0018] Furthermore, in step (2), the temperature of the condensation reaction is -20 to 25°C, and the time of the condensation reaction is 1 to 12 hours.

[0019] Furthermore, in step (3), the solvent is selected from one or more of dichloroacetic acid, anhydrous piperidine, anhydrous acetonitrile, anhydrous dichloromethane, chloroform, redistilled acetone or anhydrous tetrahydrofuran.

[0020] Furthermore, in step (3), the reaction temperature is -20 to 25°C, and the reaction time is 1 to 12 hours.

[0021] Furthermore, in step (3), after the reaction is completed, the mixture is washed with anhydrous ether, and then acetone is added to dissolve the yellow oil, and then ether is added to precipitate the solid, sonicated, filtered, and the solid is washed with pre-cooled acetone until it turns light yellow and dried to obtain FDCA.

[0022] Application of fasudil dichloroacetate obtained by the preparation method of the present invention in preparing medicine for preventing or treating vascular dementia.

[0023] Furthermore, when preparing the drug, fasudil dichloroacetate is added in the form of fasudil dichloroacetate or its optical isomer, enantiomer, diastereomer, racemate or racemic mixture.

[0024] Furthermore, the drug also includes a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant or medium.

[0025] Furthermore, fasudil dichloroacetate exerts neuroprotective effects against vascular dementia by improving cerebral blood flow after chronic hypoperfusion.

[0026] A pharmaceutical composition comprises fasudil dichloroacetate prepared by the method of the invention.

[0027] The present invention confirms through rat experiments that fasudil dichloroacetate can improve the cerebral blood flow of rats after hypoperfusion and reduce damage to nerve cells. Moreover, fasudil dichloroacetate can significantly shorten the escape latency of Morris water maze rats, increase the number of platform position crossings, platform quadrant swimming distance and time, and increase the number and time of exploring new objects. Fasudil dichloroacetate can significantly improve the neuroimmune inflammatory microenvironment in the rat brain and play a role in treating vascular dementia. Fasudil dichloroacetate is safe and non-toxic, and has no effect on body weight and organ coefficients.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0029] The invention discloses a preparation method and application of fasudil dichloroacetate. In the invention, 5-isoquinolinesulfonic acid and thionyl chloride are reacted in a solvent to obtain an intermediate 2, which is then subjected to a condensation reaction with 5-Boc homopiperazine to obtain an intermediate 3. Intermediate 3 is directly reacted with dichloroacetic acid to remove the tert-butyloxycarbonyl protecting group while forming dichloroacetate to obtain a target compound FDCA. The method avoids the waste caused by the need to prepare fasudil hydrochloride first in the reported synthetic route, improves the yield and saves the cost. The invention discloses the mechanism of action of fasudil dichloroacetate complex administration in treating a rat vascular dementia model, and finds that the fasudil dichloroacetate complex administration has better curative effect than fasudil administration alone or fasudil and dichloroacetate combined administration; the escape latency of Morris water maze rats can be significantly shortened, the number of platform position crossings, the platform quadrant swimming distance and time can be increased, and the number and time of exploring new objects can be increased; in addition, the fasudil dichloroacetate complex administration can more significantly improve the rat cerebral blood flow than fasudil administration alone or fasudil and dichloroacetate combined administration, and can become a novel drug or composition for preventing or treating vascular dementia. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The figure is the establishment and experimental design diagram of the BCCAO rat model in Example 2;

[0031] Figure 2 Graph showing the representative swimming path and escape latency of the water maze navigation experiment in Example 2, wherein A is the representative swimming path of the navigation experiment in the water maze, and B is the escape latency on different training days;

[0032] Figure 3 : is a representative swimming trajectory and water maze parameter diagram of the water maze space exploration experiment in Example 2; wherein A is a representative swimming trajectory in the water maze space exploration experiment; B is a water maze parameter, including swimming speed, percentage time in the target quadrant, total distance in the target quadrant, and number of platform crossings;

[0033] Figure 4 The figure is a representative swimming trajectory and escape latency diagram of rats in the water maze navigation experiment after BCCAO modeling and drug administration in Example 2; wherein A is a representative swimming trajectory of rats in the water maze navigation experiment after BCCAO modeling and drug administration, and B is the escape latency in the water maze test;

[0034] Figure 5 The following are representative swimming trajectories and water maze parameter diagrams of rats in the water maze space exploration experiment after BCCAO modeling and drug administration in Example 2; wherein A is a representative swimming trajectory of rats in the water maze space exploration experiment after BCCAO modeling and drug administration, and B is a water maze parameter of the water maze space exploration experiment after BCCAO modeling and drug administration, including the average swimming speed of rats, the total time and total distance percentage in the target quadrant, and the number of platform crossings;

[0035] Figure 6 is a discrimination index graph in the new object recognition test in Example 2;

[0036] Figure 7 3 is a diagram showing the detection results of the laser speckle blood flow imaging system in Example 3; wherein A is a representative image of cerebral blood flow imaging of each group of rats detected by the laser speckle blood flow imaging system, and B is the percentage change of cerebral blood flow.

[0037] Figure 8 This is the organ coefficient diagram of the heart, liver, spleen, lung and kidney in each group after administration in Example 4. DETAILED DESCRIPTION

[0038] The technical problem of the present invention will be further described below in conjunction with the accompanying drawings.

[0039] A series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention, and should not be understood as limiting the present invention.

[0040] Drugs and reagents: Sodium dichloroacetate (DCA) and Fasudil used in the following experiments were purchased from Adamas.

[0041] Animals: Forty-five male Sprague-dawley rats (9-10 weeks old, 220-250 g) were used and purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., license number: SCXK(Su)2020-0009.

[0042] Instruments: Morris water maze (Shanghai Yuyan Scientific Instrument Co., Ltd.), new object recognition and analysis system (Beijing Zhongshi Technology Instrument Co., Ltd.), ANY-maze animal behavior analysis system (Stoeling, USA), laser speckle blood flow imaging system (Beijing Jiandeer Technology Co., Ltd.), gas isoflurane anesthetic, R500 universal small animal anesthesia machine (both from Shenzhen Ruiwode Life Science Technology Co., Ltd.)

[0043] Example 1 Synthesis and Identification Method of Fasudil Dichloroacetate

[0044] Synthesized according to the following route:

[0045]

[0046] The synthesis steps of intermediate 2 are: weigh 30g of compound 1 (5-isoquinolinesulfonic acid, 143.53mmol, 1.0eq) and add it to a reaction bottle, then add 30mL of thionyl chloride and 0.4mL of DMF, and react at 80°C for 5h after nitrogen replacement. A large amount of white solid precipitation is observed, and the reaction is monitored by TLC. After the raw materials react completely, the reaction solution is concentrated by a rotary evaporator to obtain a light yellow solid, which is washed 3 times with dichloromethane and dried to obtain a white solid intermediate 2 (about 32.5g, purity>95%, yield 86.1%).

[0047] The synthesis steps of intermediate 3 are as follows: 32.5 g of compound 2 (124.04 mmol, 1.0 eq) was added to a reaction bottle, and then 100 mL of anhydrous dichloromethane and 51.7 mL of anhydrous triethylamine (327.12 mmol, 3.0 eq) were added, and the mixture was stirred in an ice bath for about 5 minutes, and 50 mL of DCM and 24.8 g of 5-Boc homopiperazine (124.04 mmol, 1.0 eq) was added dropwise in about 30 minutes, the ice bath was removed, and the reaction solution was gradually restored to room temperature for 12 hours. The reaction was monitored by TLC. After the raw material reacted completely, the reaction solution was concentrated by rotary evaporator, 100 mL of water was added and extracted with ethyl acetate (100 mL) 3 times, the organic layer was washed with 10% citric acid (100 mL3) 3 times, saturated sodium bicarbonate (50 mL) 3 times, and saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a crude yellow viscous oily compound 3 (about 43.8 g, yield 90.3%).

[0048] The synthesis steps of the target compound FDCA are as follows: 43.8 g of compound 3 (111.97 mmol, 1.0 eq) is added to a reaction bottle, 50 mL of 0°C pre-cooled dichloroacetic acid is added under an ice bath, at this time the reaction solution is a yellow viscous liquid, the ice bath is removed, the reaction solution is gradually restored to room temperature and reacts for 5 hours, TLC monitors the completion of the reaction, a large amount of anhydrous ether is added to wash the yellow oil, excess dichloroacetic acid is removed, and then a small amount of acetone is added to dissolve the yellow oil, a large amount of ether is added, at this time a large amount of yellow solid is precipitated, ultrasonication is performed, insoluble matter is filtered out to obtain a yellow solid, the solid is washed with pre-cooled acetone until light yellow, and dried to obtain a light yellow solid FDCA (about 30.6 g, purity>98%, yield 62.8%).

[0049] The pale yellow solid FDCA was analyzed by nuclear magnetic hydrogen spectrum and carbon spectrum, and the results are as follows:

[0050] FDCA 1 H NMR (500MHz, DMSO-d6) δ9.67(s,1H),9.49(s,1H),8.71(d,J=6.1Hz,1H),8.48(d,J=8.2Hz,1H),8.37–8.33(m,2H),7. 85(t,J=7.8Hz,1H),5.98(s,1H),3.69(t,J=4.7Hz,2H),3.50(t,J=6.1Hz,2H),3.25–3.17(m,4H),2.07–1.99(m,2H).

[0051] 13 C NMR(126MHz,DMSO-d6)δ166.42,154.08,145.56,134.50,133.73,133.25,1 31.12,129.38,127.10,117.42,70.93,47.19,46.71,44.78,44.76,25.74.

[0052] From the above results, it can be seen that the structural formula of the light yellow solid FDCA prepared in this example is:

[0053]

[0054] Example 2: Effect of Fasudil dichloroacetate on the improvement of cognitive impairment

[0055] 1. Experimental methods

[0056] (1) Experimental grouping: 45 male SD rats were randomly divided into 5 groups (n=9 / group) after adaptive feeding: sham group, vehicle group, FDCA group, F group, and F+DCA group. Morris water maze training was performed one week before modeling, followed by surgery. The sham group only isolated the common carotid artery, and the other groups underwent BCCAO modeling. One week after surgery, intraperitoneal administration began once a day. The FDCA group was given 10 mg / Kg body weight of FDCA, the F group was given 7.875 mg / Kg body weight of fasudil, and the F+DCA group was given 7.875 mg / Kg body weight of fasudil and 3.6 mg / Kg body weight of DCA. The sham group and vehicle group were given an equal amount of saline (10 mg / Kg) for four weeks. Morris water maze test and new and old object recognition test were performed 28 days after surgery. The rats were killed at 35 days to remove the brain, and the organ coefficient was calculated.

[0057] (2) Establishment of BCCAO model: On the day of surgery, rats were anesthetized with an anesthesia mask (isoflurane partial pressure 2-3%) and placed in a supine position. A dry cotton ball was placed behind the rat's neck to make the neck straight, which was convenient for the subsequent separation of the common carotid artery. The neck was disinfected by wiping with alcohol cotton balls, and the skin was cut along the middle of the neck with a sterile instrument. After blunt separation of the subcutaneous tissue, the common carotid artery was bluntly separated between the anterior and lateral neck muscles on one side, and the common carotid artery was found and separated from the adjacent vagus nerve. It was ligated with 5-0 silk thread in different ways. After ligation, the skin was sutured and disinfected again with iodine tincture. In the sham operation group, only the common carotid artery was exposed, and no ligation was performed. No food or water was allowed within 12 hours after surgery, and then a normal diet was resumed.

[0058] (3) Morris water maze experiment: Morris water maze is an indispensable experimental method for studying learning and memory ability. It is used to test the spatial positioning ability of animals by forcing them to swim and learn to find a platform hidden in the water. Water maze training was performed on each group of mice one week before modeling, and water maze testing was performed on them one week before the end of drug administration. The experiment was divided into two parts. The first part was a 5-day positioning navigation experiment. Water was filled into a pool of r = 1.5m, h = 0.6m and the temperature control system was turned on to keep the water temperature at (23±3)℃. The water depth was about 0.3m. The platform height was slightly lower than the water height and hidden in the water. Triangular and square markers were posted on the opposite sides of the pool to facilitate spatial memory of rats. Black ink was added to the pool to eliminate the interference of the bottom of the pool and the platform seen by the rats. The analysis software of the computer was set up to divide the pool into four quadrants: east, west, south and north. A circular anti-slip platform was placed in the center of the second quadrant, slightly below the water surface. First, let the rats adapt to the environment and temperature of the pool, then randomly place them into the pool from the four quadrants by sticking to the wall. Use video tracking technology to record the movement trajectory of the rats in the water and the time it takes to climb onto the central platform. This time is the escape latency, with an upper limit of 90 seconds. Twice a day, 4 tests each time, for 5 days. The second part is a spatial exploration experiment. On the last day, the platform is removed, and the spatial learning and memory abilities of the rats are observed by recording the number of platform position crossings, platform quadrant swimming distance, and time.

[0059] (4) New and old object recognition experiment: The new and old object recognition experiment is a test of the experimental animals' memory ability for objects through three stages: adaptation, training, and recognition. Compared with the water maze experiment, it lasts for a shorter time and is closer to the conditions for studying human cognition. Because rodents have a natural preference for novel things, rodents that remember familiar objects will spend more time exploring new objects. We conducted behavioral tests for the new object recognition experiment 3 days before the end of drug administration. The first day was the adaptation stage. The rats were placed in a square plastic box (80cm×80cm×80cm) with an opening on the top. They were allowed to freely explore the adaptation environment for 15 minutes and then disinfected with alcohol. The second day was the training stage. Two objects (4cm×4cm×4cm) that were exactly the same (size, color, shape) were placed on the diagonal line of the box in advance. The rats were placed in the box and allowed to freely explore for 15 minutes. The exploration time of each object was recorded. The third day was the recognition stage. One of the objects was replaced with a new object of the same size but different color and shape. The rats were placed in the box to explore freely for 15 minutes, and the time they spent exploring different objects within 15 minutes was recorded. The discrimination index of each group of rats was calculated using the following formula: discrimination index = (new object time - old object time) / (new object time + old object time). The larger the discrimination index value, the better the rats' spatial learning and memory abilities.

[0060] 2. Experimental results

[0061] The results are shown in Tables 1, 2, 3 and Figure 1-6

[0062] Table 1 Water maze test indicators of different groups before BCCAO modeling

[0063]

[0064] From Table 1 and Figure 1-3 It can be seen that all groups of rats successfully formed spatial memory of the platform location, and all groups of rats had memory of the platform location. And after training, there were no significant differences in the swimming speed, platform quadrant swimming distance, time, and number of platform location crossings of the rats in each group, indicating that before modeling, the spatial memory ability of the rats in each group was similar.

[0065] Experimental design and dosage of each group Figure 1 As shown, Figure 1 Figure 1 shows the establishment of the BCCAO rat model and the experimental design. The experimental design schedule includes pre-training, BCCAO surgery, drug treatment, and behavioral testing. During the orientation navigation test training, the movement trajectory of each group of rats ( Figure 2 Middle A) shows that each group of rats entered the water from the designated starting point and located the platform through the path. During the training process, the escape latency of rats in the sham, vehicle, FDCA, F+DCA, and F groups was significantly shortened, and there was no significant difference between the groups ( Figure 2 This indicates that rats in each group successfully formed spatial memory of the platform location, providing a reliable basis for subsequent experimental comparisons.

[0066] In the spatial exploration experimental training, after the platform in the second quadrant was removed, it can be observed from the motion trajectory representation diagram that the activity trajectories of rats in each group were more in the quadrant where the original platform was located ( Figure 3 A), indicating that all groups of rats had memory of the platform position. Further analysis showed that after training, there were no significant differences in the swimming speed, platform quadrant swimming distance, time, and platform position crossing times of the rats in each group ( Figure 3 Middle B) shows that before modeling, the spatial memory abilities of rats in each group were similar.

[0067] Table 2: Drug administration can improve various indicators of water maze in rats after BCCAO modeling

[0068]

[0069]

[0070] As shown in Table 2 and Figure 4-5As shown, the Morris water maze test was used to evaluate the effect of FDCA on spatial learning and memory ability.

[0071] In the navigation test, the vehicle group had difficulty finding the platform, and the drug-treated groups showed some improvement ( Figure 4 Middle A); Movement trajectory data showed that the escape latency of rats in the BCCAO group was significantly longer than that in the sham group, confirming that BCCAO can lead to a decrease in the spatial learning and memory ability of rats. The escape latency of rats in the FDCA-administered group was significantly shorter than that in the vehicle group (P<0.001) ( Figure 4 Middle B), indicating that FDCA can effectively improve BCCAO-induced spatial learning and memory impairment.

[0072] The spatial exploration test further verified the protective effect of FDCA on cognitive function. The vehicle group did not show a preference for the quadrant where the platform was located ( Figure 5 Middle A), compared with the sham group, the swimming time (P<0.001) and swimming distance (P<0.001) in the target quadrant of the vehicle group were significantly reduced, and the number of platform crossings was also significantly reduced (P<0.001). After drug treatment, the swimming distance in the target quadrant of the FDCA group (P<0.001), the F group (P<0.01) and the F+DCA group (P<0.001) was significantly higher than that of the vehicle group, and the swimming time in the target quadrant was significantly increased compared with the vehicle group (P<0.01). At the same time, the number of platform crossings in the FDCA group (P<0.001), the F group (P<0.05) and the F+DCA group (P<0.01) was also significantly higher than that of the vehicle group ( Figure 5 These results suggest that FDCA can improve cognitive impairment after chronic hypoperfusion in rats.

[0073] Table 3 Drug administration can improve the discrimination index of new and old objects in rats after BCCAO modeling

[0074]

[0075] As shown in Table 3 and Figure 6 As shown in the figure, in the new and old object recognition test, the discrimination index of the rats in the vehicle group was significantly lower than that in the sham group (P<0.001). After drug treatment, the discrimination index of the rats in the FDCA group (P<0.001), the F group (P<0.01) and the F+DCA group (P<0.01) were significantly higher than those in the vehicle group ( Figure 6 ), indicating that FDCA can improve not only spatial memory but also object recognition memory.

[0076] Example 3 Fasudil dichloroacetate improves cerebral blood flow in rats after modeling

[0077] 1. Experimental methods

[0078] Laser speckle contrast imaging: Laser speckle contrast imaging uses panoramic laser technology to provide real-time images of blood flow. When blood cells move in the area of ​​interest, the random speckle pattern generated by laser irradiation of the tissue will change. If the blood flow in a certain area is faster, the changing pattern will also speed up and become blurred, so the contrast will also decrease. Therefore, the higher the contrast, the lower the blood flow, and vice versa, the higher the contrast, the greater and faster the blood flow. We measured cerebral blood flow in different groups. On the day of measurement, the rats were placed in the equipment room in advance to adapt to the environment. Then the body weight was weighed, and 5% chloral hydrate was used for anesthesia according to the body weight of 0.7ml / 100g. The anesthesia time was 3-5min. Individual anesthesia was incomplete according to 25% of the first dose. After complete anesthesia, the rat was placed in a prone position on the operating table, and the head was wiped with an alcohol cotton ball for disinfection. The skin was cut along the midline with a sterile instrument to expose the skull on both sides, and the film and other tissues covering the skull were wiped with a cotton swab. During the measurement process, physiological saline should be wiped frequently to avoid drying of the skull and affecting the experimental results. Laser scanning technology is used for imaging, and finally the image is processed by moorFLPIReview software to generate a color-coded image representing the blood flow. Laser speckle contrast imaging detects blood flow by analyzing the changes in the speckle pattern produced by laser irradiation of tissue.

[0079] 2. Experimental results

[0080] The results are shown in Table 4 and Figure 7

[0081] Table 4FDCA administration has an improving effect on cerebral blood flow after BCCAO modeling

[0082]

[0083] 3. Results Analysis

[0084] Considering that chronic cerebral hypoperfusion is closely related to cerebral blood flow, this study used a laser speckle blood flow imaging system to detect the cerebral blood flow of each group of rats ( Figure 7 The results showed that compared with the sham group, the cerebral blood flow in the vehicle group decreased significantly to about 60% of the initial level (P<0.001), confirming that BCCAO successfully induced a chronic cerebral hypoperfusion state; while the cerebral blood flow of rats in the FDCA-administered group was significantly improved compared with that in the vehicle group (P<0.05) ( Figure 7 (middle B).

[0085] Example 4 Safety Assessment of Fasudil Dichloroacetate

[0086] 1. Experimental methods

[0087] Determination of organ coefficients of rats in different groups: On the 35th day of the experiment, the organs of the rats were taken and the organ coefficients were measured. The coefficients were calculated based on body weight and organ weight. The calculation formula was: organ coefficient = organ weight / rat body weight × 100%.

[0088] 2. Experimental results

[0089] The results are shown in Table 5 and Figure 8

[0090] Table 5 There was no significant difference in organ coefficients of rats in each group after modeling and drug administration

[0091]

[0092] 3. Results Analysis

[0093] While evaluating the therapeutic effect of FDCA, this study also focused on its safety. By comparing the organ coefficients of rats in each group 35 days after BCCAO modeling, we found that there was no significant difference among the five groups: sham, vehicle, FDCA, F, and F+DCA (Appendix Figure 8 ). This result shows that FDCA, F and F+DCA have no obvious toxicity at the doses used in this study and meet the clinical safety drug standards.

Claims

1. A method for preparing fasudil dichloroacetate, characterized in that: Synthesized according to the following route:

2. The method for preparing fasudil dichloroacetate according to claim 1, characterized in that: The following steps are involved: (1) 5-isoquinolinesulfonic acid reacts with thionyl chloride in a solvent to obtain intermediate 2; (2) Intermediate 2 was precooled and stirred with anhydrous dichloromethane, chloroform and anhydrous triethylamine in an ice bath, and then DCM and 5-Boc homopiperazine were added dropwise to carry out a condensation reaction at room temperature to obtain Intermediate 3; (3) Intermediate 3 reacts with dichloroacetic acid to remove the tert-butyloxycarbonyl protecting group and form dichloroacetate to obtain the target compound FDCA.

3. The method for preparing fasudil dichloroacetate according to claim 2, characterized in that: In step (1), the solvent is one or more of anhydrous dichloromethane, chloroform, ethyl acetate, redistilled acetone, anhydrous tetrahydrofuran, anhydrous N,N-dimethylformamide, dimethyl sulfoxide or dioxane, the reaction temperature is 0 to 100° C., and the reaction time is 1 to 12 hours.

4. The method for preparing fasudil dichloroacetate according to claim 2, characterized in that: In step (2), the solvent is selected from one or more of anhydrous acetonitrile, anhydrous dichloromethane, chloroform, ethyl acetate, redistilled acetone, anhydrous tetrahydrofuran, anhydrous N,N-dimethylformamide, dimethyl sulfoxide or dioxane, the condensation reaction temperature is -20 to 25°C, and the condensation reaction time is 1 to 12 hours.

5. The method for preparing fasudil dichloroacetate according to claim 2, characterized in that: In step (3), the solvent is selected from one or more of dichloroacetic acid, anhydrous piperidine, anhydrous acetonitrile, anhydrous dichloromethane, chloroform, redistilled acetone or anhydrous tetrahydrofuran, the reaction temperature is -20 to 25° C., and the reaction time is 1 to 12 h.

6. The method for preparing fasudil dichloroacetate according to claim 2, characterized in that: In step (3), after the reaction is completed, the reaction mixture is washed with anhydrous ether, and then acetone is added to dissolve the yellow oil. Then, ether is added to precipitate the solid, and the mixture is ultrasonicated and filtered. The solid is washed with pre-cooled acetone until it turns light yellow and dried to obtain FDCA.

7. Use of fasudil dichloroacetate prepared by the method according to any one of claims 1 to 6 in preparing a medicament for preventing or treating vascular dementia.

8. The use according to claim, characterized in that: When preparing the medicine, fasudil dichloroacetate is added in the form of fasudil dichloroacetate or its optical isomer, enantiomer, diastereomer, racemate or racemic mixture.

9. The use according to claim, characterized in that: The medicine also includes pharmaceutically acceptable salts thereof and pharmaceutically acceptable carriers, adjuvants or vehicles.

10. A pharmaceutical composition, characterized in that The invention relates to fasudil dichloroacetate prepared by the method according to any one of claims 1 to 6.

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