WO2020177291A1 - 法舒地尔复合盐及其制备方法和用途 - Google Patents

法舒地尔复合盐及其制备方法和用途 Download PDF

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WO2020177291A1
WO2020177291A1 PCT/CN2019/104910 CN2019104910W WO2020177291A1 WO 2020177291 A1 WO2020177291 A1 WO 2020177291A1 CN 2019104910 W CN2019104910 W CN 2019104910W WO 2020177291 A1 WO2020177291 A1 WO 2020177291A1
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fasudil
compound
dichloroacetate
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fdca
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French (fr)
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黄张建
张奕华
庞涛
孔辉
解卫平
王虹
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中国药科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/42—Separation; Purification; Stabilisation; Use of additives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C53/00—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen
    • C07C53/15—Saturated compounds having only one carboxyl group bound to an acyclic carbon atom or hydrogen containing halogen
    • C07C53/16—Halogenated acetic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to a fasudil compound salt, in particular to a fasudil compound salt, their preparation method, medicinal compositions containing these compounds and their medical uses, belonging to the technical field of pharmacy.
  • Rho kinase 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 (ROCK ⁇ ) and ROCK II (ROCK ⁇ ). The former mainly exists in non-neural tissues such as heart, lung, skeletal muscle and other 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 role in intracellular signal transduction in vascular smooth muscle cell contraction, cell migration, proliferation and apoptosis.
  • Rho kinase Abnormal activation of Rho kinase has been found in a variety of cardiovascular diseases, such as atherosclerosis, restenosis, hypertension, pulmonary hypertension, and myocardial hypertrophy. Studies have shown that the rat pulmonary hypertension model induced by chronic hypoxia and monocrotaline and the activity of Rho kinase in lung tissue and pulmonary artery of patients with severe pulmonary hypertension are significantly increased.
  • Fasudil hexahydro-1-(5-sulfonylisoquinoline)-1(H)-1,4-diazepine, Fasudil, also known as HA1077
  • Japan Asahi Kasei Corporation and Nagoya University
  • fasudil can effectively relieve cerebral vasospasm and improve the prognosis of patients with subarachnoid hemorrhage (SAH). Since fasudil was launched in Japan in 1996, it has The role of pulmonary blood vessels has been widely concerned by researchers.
  • Fasudil can: 1) activate endogenous neural stem cells to promote brain tissue repair; 2) increase astrocyte stimulation Factors; 3) inhibit the release of intracellular calcium ions; 4) relax 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 can safely and effectively treat pulmonary hypertension. ROCK inhibitor fasudil can penetrate into vascular smooth muscle cells, and under normal or pathological conditions, it can compete with ATP for the ATP binding site in the catalytic region of Rho kinase and specifically block the activity of Rho kinase. Fasudil hydrochloride anti-PAH is currently in phase II clinical research.
  • the present invention provides a fasudil compound salt, its preparation method and medical use.
  • a class of compounds the compound is selected from fasudil dichloroacetate, fasudil trifluoroacetate, fasudil trichloroacetate, fasudil chloroacetate, fasudil Dirchlorobromoacetate, fasudil tribromoacetate, fasudil dichloroacetic acid condensate.
  • reaction container Put an appropriate amount of fasudil in the reaction container, add an appropriate amount of reaction solvent and mix to obtain a mixed solution of fasudil and the reaction solvent;
  • reaction solution is concentrated under reduced pressure to remove the solvent, washed, and recrystallized to obtain fasudil dichloroacetate.
  • the reaction temperature is room temperature
  • the reaction solvent is water
  • the molar ratio of fasudil to dichloroacetic acid added is 1:1.5
  • the recrystallization solvent is isopropanol.
  • the present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of the above-mentioned compound or its optical isomers, enantiomers, diastereomers, racemates or racemic mixtures, or Its pharmaceutically acceptable salts and pharmaceutically acceptable carriers, adjuvants or vehicles.
  • the present invention also provides the application of the above-mentioned compounds in the preparation of drugs for preventing and/or treating pulmonary hypertension, subarachnoid hemorrhage, ischemic stroke and other cardiovascular and cerebrovascular diseases.
  • therapeutic drugs when administering the above-mentioned compounds and their pharmaceutically acceptable salts, and solvates of these compounds (herein collectively referred to as "therapeutic drugs") to mammals, they can be used alone, or preferably in accordance with standard pharmaceutical methods. It is used in combination with a carrier or diluent suitable for pharmaceutical use.
  • the mode of administration can be via various routes, including oral, parenteral or topical administration.
  • the parenteral administration referred to herein includes but is not limited to intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and transdermal administration.
  • the present invention first discloses fasudil dichloroacetate and a preparation method thereof, including the following steps: the free fasudil is first mixed with water, dichloroacetic acid is slowly added dropwise, and after stirring for 5 minutes, it is concentrated and purified. 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 spectrum.
  • the invention has simple operation, low production cost, high product yield, low environmental pollution, and is conducive to large-scale industrial production.
  • the present invention discloses the inhibitory activity of fasudil, fasudil dichloroacetate, other fasudil compound salts, and fasudil-dichloroacetic acid condensate on ROCK I and ROCK II . It was found that compared with fasudil, fasudil dichloroacetate increased its ROCK inhibitory activity.
  • FDCA fasudil dichloroacetate
  • PDGF-BB platelet-derived growth factor BB
  • PASMCs pulmonary artery smooth muscle cells
  • PAECs pulmonary artery endothelial cells
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-6 interleukin-6
  • Gavage can significantly reduce the average pulmonary artery pressure, right ventricular systolic pressure and right ventricular hypertrophy index in pulmonary hypertension rats, but has no significant effect on systemic circulation pressure; pathological examination of rat lung and heart tissue found that, FDCA significantly reduces the ratio of pulmonary artery wall thickness to pulmonary artery diameter (PAMT) and the degree of
  • FDCA fasudil dihydrochloride
  • DCA sodium dichloroacetate
  • the present invention discloses the effect of fasudil dichloroacetate (FDCA) in preventing and/or treating subarachnoid hemorrhage.
  • FDCA fasudil dichloroacetate
  • FDCA significantly reduced the rat arachnoid Cerebral vasospasm damage after submembrane hemorrhage, improved cerebral edema and animal neurological scores, significantly improved the diameter of the basilar artery, lumen area and wall thickness, and regional cerebral blood flow (rCBF) in the top cortex, better than F, DCA and The combined administration of the two.
  • FDCA is an effective anti-subarachnoid hemorrhage drug candidate, which is worthy of further study.
  • the present invention discloses the effect of fasudil dichloroacetate (FDCA) in preventing and/or treating ischemic stroke.
  • FDCA fasudil dichloroacetate
  • NBP drug bufenide
  • F fasudil dihydrochloride group
  • DCA sodium dichloroacetate
  • FDCA also significantly improved
  • the neurobehavioral dysfunction induced by ischemia is significantly better than F, DCA and the combined administration of the two, and slightly better than the NBP group.
  • fasudil can relax blood vessels, lower blood pressure, inhibit the proliferation of vascular smooth muscle cells, and inhibit vascular remodeling; and dichloroacetate is an inhibitor of pyruvate dehydrogenase kinase, which can increase pyruvate
  • the activity of dehydrogenase promotes the aerobic metabolism of glucose and reduces the production of lactic acid; it can also promote the expression of potassium 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 pulmonary hypertension, ischemic stroke and subarachnoid hemorrhage and other cardiovascular and cerebrovascular diseases from multiple mechanisms.
  • FDCA fasudil dichloroacetate
  • Figure 1 shows the effect of the compound in Example 9 on the expression of TNF- ⁇ 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 group;
  • Hypoxia hypoxia;
  • FDCA fasudil dichloroacetate;
  • F fasudil dihydrochloride;
  • DCA Dichloroacetate sodium salt;
  • F+DCA Fasudil dihydrochloride and dichloroacetate sodium salt combined administration group.
  • Figure 2 shows the effect of the compound in Example 9 on the hemodynamics of MCT-induced PAH model rats; mPAP: mean pulmonary artery pressure, RVSP: right ventricular systolic pressure; mSAP: mean systemic circulation pressure; RV/LV+S: right heart Hypertrophy index; Control: control group, MCT: monocrotaline; FDCA: fasudil dichloroacetate; F: fasudil dihydrochloride; DCA: dichloroacetate sodium; F+DCA: method Sudil dihydrochloride and sodium dichloroacetate combined administration group.
  • mPAP mean pulmonary artery pressure
  • RVSP right ventricular systolic pressure
  • mSAP mean systemic circulation pressure
  • RV/LV+S right heart Hypertrophy index
  • Control control group, MCT: monocrotaline; FDCA: fasudil dichloroacetate; F: fasudil dihydrochloride; DCA: dichlor
  • Figure 3 shows the effect of each administration group in Example 9 on the ratio of pulmonary artery wall thickness to pulmonary artery diameter (PAMT) and the degree of fibrosis;
  • PAMT rat pulmonary artery wall thickness and lung size Ratio of artery diameter;
  • Fibrosis fibrosis;
  • Control control group, MCT: monocrotaline;
  • FDCA fasudil dichloroacetate;
  • F fasudil dihydrochloride;
  • DCA sodium dichloroacetate Salt;
  • F+DCA Fasudil dihydrochloride and sodium dichloroacetate combined administration group.
  • Figure 4 shows the effect of each administration group on the area of right ventricular myocardial cells and the degree of fibrosis in Example 9; 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 shows the effect of different compounds in Example 10 on brain edema in SAH rats
  • Figure 5B shows the effect of different compounds in Example 10 on the spontaneous activity score of SAH rats.
  • Figure 6 is a TTC staining image of the brain tissue of the tMCAO model rat in Example 11.
  • Fig. 7 is a statistical diagram of the cerebral infarct area of tMCAO model rats in Example 11.
  • Figure 8 shows the neurological function scores of tMCAO model rats in Example 11.
  • Fig. 9 is a graph showing the cerebral infarct area of the tMCAO model rat and the score of the rat’s neurological function in Example 11.
  • Pulmonary hypertension is often accompanied by inflammation.
  • the inflammatory factor tumor necrosis factor- ⁇ can activate the inflammatory factor interleukin-6 (IL-6) to promote the proliferation of smooth muscle cells. Fibrosis of blood vessels and remodeling of pulmonary arterioles.
  • FDCA fasudil dichloroacetate
  • PDGF-BB platelet-derived growth factor BB
  • PASMCs pulmonary artery smooth muscle cells
  • PAECs pulmonary artery endothelial cells
  • Cell grouping 1 Normal cell group (Control); 2 PDGF-BB or hypoxic cultured model group; 3 Model group + Fasudil dichloroacetate (FDCA); 4 Model group + Fasudil hydrochloride (F) Treatment group; 5Model group + sodium dichloroacetate (DCA) treatment group; 6Model group + F and DCA combined administration group.
  • the experimental method is as follows.
  • PGDFBB model group Cells were passaged to 3-6 generations, 24h after culture, PDGFBB (5 ⁇ l for 10 ml) was added for 24h, and then starved for 48h, and the drug was added. The concentration of each administration group was 50 ⁇ M.
  • both hypoxic culture conditions and PDGF-BB can significantly increase the expression of TNF- ⁇ and IL-6 in PASMCs and PAECs, suggesting that both hypoxic culture conditions and PDGF-BB can significantly increase inflammation;
  • the administration group inhibited the expression of TNF- ⁇ and IL-6 to varying degrees in the two cell strains and reduced inflammation.
  • the FDCA group has the best anti-inflammatory effect, which is better than 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 a better ability to cross the cell membrane than F and DCA.
  • the administration method was intragastric administration, once a day, 37.5mg/kg in F group and 15.5mg/kg in DCA group , F+DCA combination treatment group includes 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 the same amount of normal saline.
  • the mean pulmonary artery pressure (mPAP), right ventricular systolic pressure (RVSP) and mean systemic circulation pressure (mSAP) were measured on the 28th day in each group.
  • RV/LV+ S right heart hypertrophy index
  • PCNA detection immunohistochemical staining
  • hematoxylin-eosin staining Masson staining and other treatments to evaluate the hemodynamics, average pulmonary artery thickness, pulmonary fibrosis degree, right heart function and other aspects of each administration group active.
  • FIG 2. Compared with the normal control group, direct administration of FDCA has little effect on the mPAP, RVSP and RV/LV+S of normal rats, indicating that FDCA is safer.
  • the MCT model group can significantly increase mPAP, RVSP and RV/LV+S.
  • Each administration group can effectively reduce mPAP, RVSP and RV/LV+S, and the FDCA group has the strongest activity in reducing mPAP, RVSP and RV/LV+S, which is better than F, DCA and the combined administration of the two. In addition, each administration group had less influence on mSAP.
  • the effects of each administration group on the area of right ventricular cardiomyocytes and the degree of fibrosis showed that compared with the blank control, the MCT model group significantly increased the area of right ventricular cardiomyocytes and the degree of fibrosis.
  • the administration group, especially FDCA can significantly reduce the area and fibrosis of right ventricle myocardial cells, which is better than F, DCA and the combined administration group.
  • the results suggest that FDCA can effectively inhibit the proliferation and remodeling of right ventricular myocardial cells.
  • Scoring of spontaneous activity Place the rat in a spacious cage that can move freely and all four walls can reach to score the spontaneous activity. 24 hours after the SAH model was established, two experimenters evaluated and recorded the experimental rats in a double-blind manner. The average of the two groups was taken as the final score. The rats were killed immediately after the spontaneous activity was observed.
  • the spontaneous activity score is divided into 4 levels according to the animal’s mental state and movement: level 1, normal activity of the rat, no activity disorder, actively explore the surrounding environment, at least touch the upper edge of the three cage walls; level 2, mild activity disorder, namely The rat is mentally poor, lethargic, and has a certain delay in action.
  • brain tissue water content Rats were sacrificed 24 hours after SAH modeling, the brain and cerebellum were quickly taken out, and the surface blood was sucked off with filter paper. Weigh the masses of the brain and cerebellum (wet weight) with an electronic balance, then put the brain tissue in an oven and bake at 105°C to a constant weight, and weigh the masses of the brain and cerebellum (dry weight) again.
  • Measurement of basilar artery diameter, lumen area and wall thickness HE stain the tissue sections of the above groups of basilar arteries, observe and take pictures under an optical microscope, and use image pro-plus6.0 image analysis system to measure the basilar artery Diameter, lumen area and wall thickness.
  • the measurement method of wall thickness is as follows: measure the distance from the inner surface of the basilar artery to the outer edge of the media, excluding the adventitia. Each vessel selects 4 different detection points to measure the thickness of the vessel wall, and takes the average value as the measured value of the vessel. The results are shown in Table 1.
  • rCBF regional cerebral blood flow
  • Spontaneous activity score data is represented by a scatter plot, and the rest of the data are represented by means ⁇ SD; the spontaneous activity score uses Kruskal-Wallis test and Mann-Whitney U test according to statistical differences between groups, and the remaining data groups have statistical differences Using one-way ANOVA and Tukey's test, a P value less than 0.05 is considered a significant difference.
  • the administration of different test compounds F and FDCA can significantly improve animal neurological scores and significantly reduce the brain water content of rats caused by SAH.
  • FDCA shows The strongest activity, significantly better than F and DCA, slightly better than F+DCA.
  • FDCA has significant anti-subarachnoid hemorrhage activity, which is superior to the marketed drug fasudil dihydrochloride, and the combined administration of fasudil dihydrochloride and sodium dichloroacetate.
  • tMCAO transient rat cerebral ischemia model
  • Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (350mg/kg), fixed on the experimental table in supine position, neck incision, skin incision with scalpel, blunt separation of various layers of tissue, according to Anatomy of the rat’s neck vessels.
  • the left common carotid artery (CCA) was separated under a stereo microscope, and the left external carotid artery (ECA) and internal carotid artery were separated upwards.
  • Double ligation, and the superior thyroid artery and occipital artery were cut off Two branches of external carotid arteries, double ligation of ECA at approximately 5mm-8mm near the bifurcation of CCA, clamp the proximal end of ICA and CCA with arteriole clamps respectively, indwelling a single knot at the proximal bifurcation of ECA but not Tighten the silk thread, make a 0.2mm diameter V-shaped micro incision between the proximal ECA ligature and the bifurcation of the common carotid artery, gently insert the nylon thread through the incision, and gently tighten the knot, Cut the internal carotid artery between the two ligatures to make it consistent with the direction of the internal carotid artery.
  • the insertion depth is about 18mm-20mm and stops when there is slight resistance. 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 tail end of the nylon thread is left outside the body.
  • Rats in the sham operation group only underwent anesthesia and vascular separation without ligating the blood vessels and introducing thread plugs, and keeping the animals warm after the operation.
  • Administration method rats were injected into the tail vein after 4h and 24h ischemia. After 48 hours of ischemia, the neurological function was scored and the rats were sacrificed.
  • sham operation group (Sham); blank solvent group (Vehicle); fasudil dihydrochloride group (F, 30mg/kg, tail vein injection); FDCA group (30mg/kg, tail vein injection); D Phthalide group (NBP, 5mg/kg, tail vein injection).
  • TTC staining make four coronal cuts at the optic chiasma and 2mm before and after the whole brain. After cutting into five slices, quickly place the brain slices in 5ml of phosphate buffer solution containing 2% TTC, and incubate at 37°C in the dark for 10 minutes. Turn over every 7 to 8 minutes during the incubation process, take out the brain slices after 10 minutes of incubation, take a photo with a digital camera (Olympus C-4000, Japan), and then separate the pale area (infarct area) and non-pale area with ophthalmic forceps (Normal area), calculate the percentage of infarction through Image pro-plus 6.0 as follows:
  • Neural function rating 48 hours after ischemia, the animal’s neurological deficits were graded and scored according to Longa’s method.
  • the standards are as follows:
  • the neurological deficit grading and scoring data are represented by the median, and the rest of the data are represented by means ⁇ SD; the statistical difference between the neurological deficit grading and scoring data groups uses the Kruskal-Wallis test and the Mann-Whitney U test, and the remaining data groups One-way ANOVA and Tukey's test were used for statistical differences between the two, and a P value less than 0.05 was considered to be significant.
  • Fasudil (F) intravenous injection (6.24mg/kg) 0.25 and 1h tissue distribution in rats (ng/g, n 3)

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Abstract

一种法舒地尔复合盐，选自法舒地尔二氯乙酸盐、法舒地尔三氟乙酸盐、法舒地尔三氯乙酸盐、法舒地尔氯乙酸盐、法舒地尔氯溴乙酸盐、法舒地尔三溴乙酸盐、法舒地尔二氯乙酸缩合物，或其药学上可接受的盐，它们的制备方法、含有这些化合物的药用组合物以及它们的医药用途，特别是在制备预防和/或治疗肺动脉高压、缺血性脑卒中、蛛网膜下腔出血等心脑血管疾病的药物中的应用。

Description

法舒地尔复合盐及其制备方法和用途 技术领域
本发明涉及一种法舒地尔复合盐，具体涉及一种法舒地尔复合盐，它们的制备方法、含有这些化合物的药用组合物以及它们的医药用途，属于药学技术领域。
背景技术
Rho激酶(Rho associa ted kinase，ROCK)，是参与细胞有丝分裂粘附、细胞骨架调整、肌肉细胞收缩、肿瘤细胞浸润等一系列细胞生命现象的重要酶。自1996年以来，已发现的ROCK分为ROCK I(ROCKβ)和ROCK II(ROCKα)。前者主要存在于非神经组织如心脏、肺、骨胳肌等细胞；后者主要存在于中枢神经系统，如海马锥体神经元、大脑皮质、小脑浦肯野细胞等。Rho激酶(ROCK)在血管平滑肌细胞收缩、细胞迁移、增殖以及凋亡等多项细胞功能中具有重要的细胞内信号转导作用。在多种心血管疾病中都发现了Rho激酶异常活化，如动脉粥样硬化、再狭窄、高血压、肺动脉高压和心肌肥厚等。研究表明，慢性缺氧和野百合碱所致大鼠肺动脉高压模型以及严重肺动脉高压患者肺组织和肺动脉中Rho激酶活性均显著增高。
法舒地尔[六氢-1-(5-磺酰基异喹啉)-1(H)-1，4–二氮杂卓，Fasudil，又名HA1077]，是日本旭化成株式会社和名古屋大学药理学研究室合作开发的一种新型异喹啉磺胺衍生物。做为一种新型、高效的血管扩张药，法舒地尔可以有效缓解脑血管痉挛，改善蛛网膜下隙出血(SAH)患者的预后，自1996年法舒地尔在日本上市以来，其对于肺血管的作用一直受到研究者的广泛关注，大量的动物实验和临床研究均表明法舒地尔可以：1)激活内源性的神经干细胞促进脑组织修复；2)增加星状胶质细胞刺激因子；3)抑制细胞内钙离子的释放；4)舒张脑部血管；5)保护神经细胞和改善伸进功能；6)促进轴突的再生。因此法舒地尔也用于缺血性脑卒中的治疗。此外，法舒地尔也能够安全有效地治疗肺动脉高压。ROCK抑制药法舒地尔可以渗透入血管平滑肌细胞，在正常或病理情况下都能与ATP竞争Rho激酶催化区的ATP结合位点，特异地阻断Rho激酶活性。目前盐酸法舒地尔抗PAH处于II期临床研究阶段。
发明内容
目的：本发明提供一种法舒地尔复合盐、其制备方法及医药用途。
技术方案：本发明采用的技术方案为：
一类化合物，所述化合物选自法舒地尔二氯乙酸盐、法舒地尔三氟乙酸盐、法舒地尔三氯乙酸盐、法舒地尔氯乙酸盐、法舒地尔氯溴乙酸盐、法舒地尔三溴乙酸盐、法舒地尔二氯乙酸缩合物。
化合物1：法舒地尔二氯乙酸盐，结构式如下：
Figure PCTCN2019104910-appb-000001
法舒地尔二氯乙酸盐的制备方法为：
取适量法舒地尔置于反应容器中，加入适量反应溶剂混合，得法舒地尔与反应溶剂的混合液；
在反应温度0-100度下边搅拌边加入适量的二氯乙酸到上述混合液中，滴完后继续搅拌一段时间，得反应液；
然后将反应液减压浓缩出去溶剂，洗涤、重结晶，即得法舒地尔二氯乙酸盐。
优选的制备过程中，反应温度为室温，反应溶剂为水，加入的法舒地尔与二氯乙酸的摩尔比为1:1.5，重结晶溶剂为异丙醇。
化合物2：法舒地尔三氟乙酸盐，结构式如下：
Figure PCTCN2019104910-appb-000002
化合物3：法舒地尔三氯乙酸盐，结构式如下：
Figure PCTCN2019104910-appb-000003
化合物4：法舒地尔氯乙酸盐，结构式如下：
Figure PCTCN2019104910-appb-000004
化合物5：法舒地尔氯溴乙酸盐，结构式如下：
Figure PCTCN2019104910-appb-000005
化合物6：法舒地尔三溴乙酸盐，结构式如下：
Figure PCTCN2019104910-appb-000006
化合物7：法舒地尔二氯乙酸缩合物(FD)，结构式如下：
Figure PCTCN2019104910-appb-000007
另一方面，本发明还提供一种药物组合物，其中含有治疗有效量的上述的化合物或其旋光异构体、对映体、非对映体、外消旋体或外消旋混合物，或其药学上可接受的盐及可药用的载体、佐剂或媒剂。
另一方面，本发明还提供上述的化合物在制备预防和/或治疗肺动脉高压、蛛网膜下腔出血、缺血性脑卒中等心脑血管疾病的药物中的应用。
本发明中，在给予哺乳动物上述化合物及其药学上可接受的盐，以及这些化合物的溶剂化物(这里统称为“治疗药物”)时，可以单独使用，或者最好是按照 规范的制药方法将其与适于药用的载体或稀释剂配合后使用。给药方式可以经各种途径，包括口服、非胃肠道给药或局部给药。这里所指的非胃肠道给药包括但并不限于静脉注射、肌肉注射、腹腔注射、皮下注射和透皮给药。
本发明首先公开了法舒地尔二氯乙酸盐及其制备方法，包括以下步骤：游离的法舒地尔首先与水混合，缓缓滴加入二氯乙酸，继续搅拌5min后，浓缩出去纯净水，剩余物加入乙醚洗涤3次后，再用异丙醇或其他溶剂进行重结晶后得到高纯度的法舒地尔二氯乙酸盐。并通过经氢谱，碳谱，质谱对其结构进行了确证。本发明操作简单，生产成本低，产品收率高，环境污染小，利于工业化大生产。
同时，本发明公开了法舒地尔、法舒地尔二氯乙酸盐、其他的法舒地尔复合盐，以及法舒地尔-二氯乙酸缩合物对ROCK I和ROCK II的抑制活性。结果发现相比于法舒地尔，法舒地尔二氯乙酸盐提高了其ROCK抑制活性。
本发明公开了法舒地尔二氯乙酸盐(FDCA)对肺动脉高压的治疗作用。首先，细胞实验中法舒地尔二氯乙酸盐(FDCA)可显著抑制血小板衍生生长因子BB(PDGF-BB)和低氧诱导的肺动脉平滑肌细胞(PASMCs)和肺动脉内皮细胞(PAECs)中炎症因子肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的表达；进一步进行动物实验，在野百合碱诱导的大鼠肺动脉高压的治疗模型中，FDCA(43.3mg/kg)灌胃给药可明显降低肺动脉高压大鼠的平均肺动脉压，右心室收缩压和右心室肥厚指数，而对体循环压无明显影响；通过将大鼠肺部和心脏组织进行病理学检查发现，FDCA显著降低大鼠肺小动脉血管壁厚度与肺小动脉直径的比值(PAMT)和肺小动脉纤维化程度；FDCA显著降低右心室心肌细胞面积(CSA)和纤维化程度。值得注意的是FDCA抗肺动脉高压的活性优于等摩尔剂量的法舒地尔二盐酸盐(F)，二氯乙酸钠(DCA)及二者的联合给药，提示FDCA是一种有效的抗肺动脉高压的候选药物，值得进一步研究。
同时，本发明公开了法舒地尔二氯乙酸盐(FDCA)预防和/或治疗蛛网膜下腔出血的作用。在大鼠蛛网膜下腔出血模型中(蛛网膜下腔血管内穿孔造模，造模后0.5h和6h各给药一次，评价24h后大鼠的各项指标)，FDCA显著降低大鼠蛛网膜下腔出血后脑血管痉挛损伤，改善脑水肿和动物神经学评分，明显改善了基底动脉管径、管腔面积及管壁厚度和顶部皮层局部脑血流量(rCBF)，优于 F，DCA以及二者的联合给药。结果提示FDCA是一种有效的抗蛛网膜下腔出血的候选药物，值得进一步研究。
同时，本发明公开了法舒地尔二氯乙酸盐(FDCA)预防和/或治疗缺血性脑卒中的作用。在大鼠短暂缺血模型中(缺血2h复灌，缺血4h给药一次，24h给药，评价48h后的大鼠的各项指标)，FDCA有效地降低脑梗死面积，显著优于上市药物丁苯肽(NBP)组，显著优于法舒地尔二盐酸盐组(F)和二氯乙酸钠(DCA)组，以及F和DCA的联合给药组；此外FDCA还显著地改善了缺血诱导的神经行为功能障碍，明显优于F，DCA以及二者的联合给药，略优于NBP组。结果提示FDCA是一种有效的抗缺血性脑卒中的候选药物，值得进一步研究。
作为ROCK的抑制剂，法舒地尔可以舒张血管、降低血压，抑制血管平滑肌细胞的增殖，抑制血管重构；而二氯乙酸盐是丙酮酸脱氢酶激酶的抑制剂，可以提高丙酮酸脱氢酶的活性，促进葡萄糖的有氧代谢，减少乳酸的生成；同时还可以促进钾离子通道尤其是Kv1.5的表达，抑制平滑肌细胞的增殖和促进其凋亡。因此，法舒地尔和二氯乙酸盐的联合给药可以从多个机制治疗肺动脉高压，缺血性脑卒中以及蛛网膜下腔出血等心脑血管疾病。和联合给药相比，法舒地尔二氯乙酸盐(FDCA)作为一个整体的分子，具有较法舒地尔盐酸盐更高的生物利用度和脑内分布浓度，因此表现出更好的活性。
附图说明
图1是实施例9中化合物对PASMCs和PAECs在PDGF-BB以及低氧培养条件下TNF-α和IL-6表达的影响；PASMs：肺动脉平滑肌细胞；PAECs：肺动脉内皮细胞；PDGF-BB：血小板衍生生长因子BB；IL-6；白细胞介素-6；CON：空白对照组；Hypoxia：低氧；FDCA：法舒地尔二氯乙酸盐；F：法舒地尔二盐酸盐；DCA：二氯乙酸钠盐；F+DCA：法舒地尔二盐酸盐和二氯乙酸钠盐联合给药组。
图2是实施例9中化合物对MCT诱导的PAH模型大鼠血流动力学的影响；mPAP：平均肺动脉压，RVSP：右心室收缩压；mSAP：平均体循环压；RV/LV+S：右心肥厚指数；Control：对照组，MCT：野百合碱；FDCA：法舒地尔二氯乙酸盐；F：法舒地尔二盐酸盐；DCA：二氯乙酸钠盐；F+DCA：法舒地尔二盐酸盐 和二氯乙酸钠盐联合给药组。
图3是实施例9中各给药组对大鼠肺小动脉血管壁厚度与肺小动脉直径的比值(PAMT)和纤维化程度的影响；PAMT：大鼠肺小动脉血管壁厚度与肺小动脉直径的比值；Fibrosis：纤维化；Control：对照组，MCT：野百合碱；FDCA：法舒地尔二氯乙酸盐；F：法舒地尔二盐酸盐；DCA：二氯乙酸钠盐；F+DCA：法舒地尔二盐酸盐和二氯乙酸钠盐联合给药组。
图4是实施例9中各给药组对右心室心肌细胞面积及纤维化程度的影响；CAS：心肌细胞横断面面积；Fibrosis：纤维化；Control：对照组，MCT：野百合碱；FDCA：法舒地尔二氯乙酸盐；F：法舒地尔二盐酸盐；DCA：二氯乙酸钠盐；F+DCA：法舒地尔二盐酸盐和二氯乙酸钠盐联合给药组。
图5A是实施例10中不同化合物对SAH大鼠脑水肿的影响；图5B是实施例10中不同化合物对SAH大鼠自发性活动评分的影响。
图6是实施例11中tMCAO模型大鼠脑组织TTC染色图。
图7是实施例11中tMCAO模型大鼠脑梗死面积统计图。
图8是实施例11中tMCAO模型大鼠神经功能评分。
图9是实施例11中tMCAO模型大鼠脑梗死面积和大鼠神经功能评分图。
具体实施方式
为了进一步阐明本发明，下面给出一系列实施例，这些实施例完全是例证性的，它们仅用来对本发明具体描述，不应当理解为对本发明的限制。
实施例1
法舒地尔二氯乙酸盐
Figure PCTCN2019104910-appb-000008
化合物合成及结构确证：
室温下，将10g法舒地尔置于100mL茄形瓶中，加入20mL自来水或纯净水进行搅拌，然后称取4.87g二氯乙酸，缓缓滴加入上述混悬液中，滴加过程中 发现法舒地尔或逐渐溶解，滴毕。继续室温搅拌10min。后将反应液减压浓缩，剩余物加入乙醚进行洗涤3次，弃去乙醚，再用有机溶剂进行重结晶，过滤得到白色固体，后置于真空干燥箱中干燥得到13.95g目标化合物，收率为96.9％。Mp：141-143℃. 1H NMR(300MHz，D 2O，TMS)δ9.20(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，D 2O)δ170.25，152.36，142.97，134.17，133.01，131.0，130.25，128.10，126.03，116.69，68.07，46.76，46.22，44.41，43.46，24.99.ESI-MS(70eV)m/z：292.2[M+H] +。
实施例2
法舒地尔三氟乙酸盐
Figure PCTCN2019104910-appb-000009
化合物合成及结构确证：
室温下，将100mg法舒地尔置于50mL茄形瓶中，加入10mL丙酮进行搅拌，然后称取40mg三氟乙酸，缓缓滴加入上述混悬液中，滴加过程中发现法舒地尔或逐渐溶解，滴毕。继续室温搅拌10min。后将反应液减压浓缩，剩余物加入乙醚搅拌后过滤，滤饼用乙醚进行洗涤3次，得到白色固体，后置于真空干燥箱中干燥得到130.8mg目标化合物，收率为94％。 1H NMR(500MHz，D 2O，TMS)δ9.72(s，1H)，8.84(d，J＝5.2，1H)，8.64-8.72(m，3H)，8.10(t，J＝7.7，1H)，3.84(t，J＝4.7，2H)，3.65(t，J＝5.9，2H)，3.44-3.52(m，4H)，2.23-2.26(m，2H).ESI-MS(70eV)m/z：292.2[M+H] +。
实施例3
法舒地尔三氯乙酸盐
Figure PCTCN2019104910-appb-000010
化合物合成及结构确证：
室温下，将100mg法舒地尔置于50mL茄形瓶中，加入10mL丙酮进行搅拌，然后称取66.76mg三氯乙酸，缓缓滴加入上述混悬液中，滴加过程中发现法舒地尔或逐渐溶解，滴毕。继续室温搅拌10min。后将反应液减压浓缩，剩余物加入乙醚搅拌后过滤，滤饼用乙醚进行洗涤3次，得到白色固体，后置于真空干燥箱中干燥得到135mg目标化合物，收率为87％。 1H NMR(300MHz，D 2O，TMS)δ9.89(s，1H)，9.08(d，J＝6.8，1H)，8.75-8.81(m，3H)，8.20(t，J＝7.1，1H)，3.85(t，J＝4.8，2H)，3.67(t，J＝5.8，2H)，3.48-3.62(m，4H)，2.22-2.26(m，2H).ESI-MS(70eV)m/z：292.2[M+H] +。
实施例4
法舒地尔氯乙酸盐
Figure PCTCN2019104910-appb-000011
化合物合成及结构确证：
室温下，将100mg法舒地尔置于50mL茄形瓶中，加入10mL丙酮进行搅拌，然后称取38.76mg氯乙酸，缓缓滴加入上述混悬液中，滴加过程中发现法舒地尔或逐渐溶解，滴毕。继续室温搅拌10min。后将反应液减压浓缩，剩余物加入乙醚搅拌后过滤，滤饼用乙醚进行洗涤3次，得到白色固体，后置于真空干燥箱中干燥得到121mg目标化合物，收率为91.5％。 1H NMR(300MHz，D 2O，TMS)δ9.21(s，1H)，8.49(d，J＝6.1，1H)，8.24-8.30(m，3H)，7.74(t，J＝8.0，1H)，4.06(s，2H)，3.74(t，J＝3.9，2H)，3.54(t，J＝5.7，2H)， 3.41-3.43(m，4H)，2.15-2.18(m，2H).ESI-MS(70eV)m/z：292.2[M+H] +。
实施例5
法舒地尔氯溴乙酸盐
Figure PCTCN2019104910-appb-000012
化合物合成及结构确证：
室温下，将100mg法舒地尔置于50mL茄形瓶中，加入10mL丙酮进行搅拌，然后称取70.88mg氯溴乙酸，缓缓滴加入上述混悬液中，滴加过程中发现法舒地尔或逐渐溶解，滴毕。继续室温搅拌10min。后将反应液减压浓缩，剩余物加入乙醚搅拌后过滤，滤饼用乙醚进行洗涤3次，得到白色固体，后置于真空干燥箱中干燥得到140mg目标化合物，收率为88％。 1H NMR(300MHz，D 2O，TMS)δ9.63(s，1H)，8.74(d，J＝6.5，1H)，8.58-8.68(m，3H)，8.02(t，J＝7.5，1H)，6.07(s，1H)，3.81(t，J＝4.5，2H)，3.62(t，J＝6.0，2H)，3.43-3.47(m，4H)，2.17-2.21(m，2H).ESI-MS(70eV)m/z：292.2[M+H] +。
实施例6
法舒地尔三溴乙酸盐
Figure PCTCN2019104910-appb-000013
化合物合成及结构确证：
室温下，将100mg法舒地尔置于50mL茄形瓶中，加入10mL丙酮进行搅拌，然后称取121mg三溴乙酸，缓缓滴加入上述混悬液中，滴加过程中发现法舒地尔或逐渐溶解，滴毕。继续室温搅拌10min。后将反应液减压浓缩，剩余物加入乙醚搅拌后过滤，滤饼用乙醚进行洗涤3次，得到白色固体，后置于真空干 燥箱中干燥得到164.5mg目标化合物，收率为82％。 1H NMR(300MHz，D 2O，TMS)δ9.18(s，1H)，8.49(d，J＝5.8，1H)，8.22-8.26(m，3H)，7.71(t，J＝7.8，1H)，3.61(t，J＝3.9，2H)，3.50(t，J＝6.1，2H)，3.14-3.16(m，4H)，1.98-2.0(m，2H).ESI-MS(70eV)m/z：292.2[M+H] +。
实施例7
法舒地尔二氯乙酸缩合物(FD)
Figure PCTCN2019104910-appb-000014
化合物合成及结构确证：室温下，将200mg法舒地尔，227μL的DIEA，522mg的HBUT和68μL二氯乙酸置于50mL的茄形瓶中，加入20mL无水二氯甲烷搅拌反应过夜。后将反应液中加入10mL水洗涤，二氯甲烷层再浓缩，制砂，柱层析(石油醚/乙酸乙酯＝1:1)得到目标产物175mg，收率为61.4％。 1H-NMR(300MHz,CDCl 3,TMS)δ9.36(s,1H),8.69-8.72(m,1H),8.32-8.36(m,2H),8.22(d,J＝8.1,1H),7.67-7.74(m,1H),6.21(s,1H),3.91(s,1H),3.75-3.84(m,3H),3.61(s,1H),3.43-3.49(m,3H),2.05-2.11(m,2H).ESI-MS(70eV)m/z:416.1[M+H] +。
实施例8
化合物对ROCK-I和II抑制活性：
表1.化合物对ROCK-I和ROCK-II的抑制活性(nM)
Figure PCTCN2019104910-appb-000015
Figure PCTCN2019104910-appb-000016
实施例9
预防和/或治疗肺动脉高压
一、FDCA在PDGF-BB以及低氧培养模型中对PASMCs和PAECs细胞中炎症因子TNF-α和IL-6表达的影响
肺动脉高压特别是结缔组织疾病相关的肺动脉高压往往伴随炎症的产生，炎症因子肿瘤坏死因子-α(TNF-α)可激活炎症因子白细胞介素-6(IL-6)，促进平滑肌细胞的增殖、血管的纤维化以及肺小动脉的重构。首先通过细胞实验考察了法舒地尔二氯乙酸盐(FDCA)对低氧培养条件以及血小板衍生生长因子BB(PDGF-BB)诱导的肺动脉平滑肌细胞(PASMCs)和肺动脉内皮细胞(PAECs)中TNF-α和IL-6表达的影响。细胞分组：①正常细胞组(Control)；②PDGF-BB或者低氧培养的模型组；③模型组+法舒地尔二氯乙酸盐(FDCA)；④模型组+法舒地尔盐酸盐(F)治疗组；⑤模型组+二氯乙酸钠盐(DCA)治疗组；⑥模型组+F与DCA联合给药组。实验方法如下。PGDFBB模型组：先细胞传代至3-6代，培养后24h后加PDGFBB(5微升配10毫升)养24h，再饥饿48h，加药，各给药组浓度为50μM，培养72h后通过ELISA统计各组细胞中TNF-α和IL-6的表达情况；缺氧模型组：先细胞传代至3-6代，再饥饿24小时后缺氧培养24小时，给药，各给药组浓度为50μM，培养72h后通过ELISA统计各组细胞中TNF-α和IL-6的表达情况)。如图1所示，低氧培养条件和PDGF-BB均可显著提高PASMCs和PAECs中的TNF-α和IL-6的表达，提示低氧培养条件和PDGF-BB均可显著提高炎症；而各给药组在两株细胞中均不同程度地抑制TNF-α和IL-6的表达，减轻炎症。其中FDCA组的抑制炎症效果最佳，优于F、DCA以及二者的联合给药组。提示F和DCA在抗炎方面有一定的协同作用，其可能的原因是FDCA作为一个整体分子相比于F以及DCA具有更好的跨过细胞膜的能力。
二、FDCA化合物对MCT诱导的PAH模型大鼠血流动力学的影响
进一步研究野百合碱(MCT)引起的大鼠PAH模型中，FDCA及相关化合物的治疗作用。动物分组：①正常对照组；②正常对照组+FDCA；③MCT模型 组；④法舒地尔二盐酸盐(F)治疗组；⑤DCA治疗组；⑥F+DCA联合治疗组；⑦FDCA给药组。大鼠模型的建立：动物模型组及治疗组一次性腹腔注射野百合碱(MCT)60mg/kg，正常对照组注射等量生理盐水。实验处理：于注射野百合碱的第14天，各给药组以等摩尔剂量开始给药，给药方式为灌胃给药，每天一次，F组37.5mg/kg，DCA组15.5mg/kg，F+DCA联合治疗组包括F(37.5mg/kg)和DCA 15.5mg/kg，正常对照组+FDCA组43.3mg/kg；FDCA组43.3mg/kg。正常组和模型组予以等量的生理盐水喂养。各组在第28天进行平均肺动脉压力(mPAP)，右心室收缩压(RVSP)和平均体循环压(mSAP)的测量，随后处死大鼠取肺组织和心脏进行右心肥厚指数(RV/LV+S)，PCNA检测，免疫组织化学染色、苏木精-伊红染色，Masson染色等处理，评价各给药组在血流动力学、肺动脉平均厚度，肺纤维化程度，右心功能等方面的活性。结果如图2所示，和正常对照组相比，直接给予FDCA对正常大鼠的mPAP，RVSP和RV/LV+S的影响不大，说明FDCA的安全性较高。而MCT模型组可明显升高mPAP，RVSP和RV/LV+S。各给药组均可有效地降低mPAP，RVSP和RV/LV+S，其中FDCA组降低mPAP，RVSP和RV/LV+S的活性最强，优于F，DCA以及二者的联合给药。另外，各给药组对mSAP的影响较小。
三、FDCA化合物对MCT诱导的PAH模型大鼠肺动脉的影响
如图3所示，不同给药组对大鼠肺小动脉血管壁厚度与肺小动脉直径的比值(PAMT)和纤维化程度的影响，可以发现FDCA可有效降低PAMT和肺小动脉纤维化程度，略优于F，DCA及二者的联合给药。
四、FDCA化合物对MCT诱导的PAH模型大鼠右心室的影响
如图4所示，各给药组对右心室心肌细胞面积及纤维化程度的影响，结果提示与空白对照相比，MCT模型组显著增加大鼠右心室心肌细胞面积和纤维化程度。而给药组，尤其是FDCA可显著降低右心室心肌细胞面积和纤维化程度，优于F，DCA及两者联合给药组，结果提示FDCA可有效抑制右心室心肌细胞的增殖与重构。
实施例10
预防和/或治疗蛛网膜下腔出血
实验动物
SPF级SD大鼠，体重260-340g，雌雄对半，购自北京维通利华实验动物技术有限公司，饲养于SPF级饲养环境中，室内温度控制在23±2℃，自由饮食和摄水。动物总数32只。假手术组：等体积生理盐水含1％的DMSO(n＝8)；
实验方法
试验分组情况及药物浓度选择：
SAH模型组：等体积生理盐水含1％的DMSO(n＝8)；法舒地尔二盐酸盐(F)组：(26.0mg/kg)(n＝8)；二氯乙酸钠(DCA)组：(10.7mg/kg)(n＝8)：法舒地尔二盐酸盐联合二氯乙酸钠(F+DDCA)组：(F：26.0mg/kg；DCA：10.7mg/kg)(n＝8)；FDCA组：(30mg/kg)(n＝8)；所有药物配成含1％DMSO的生理盐水溶液，给药方式为尾静脉注射给药。分别在SAH(大鼠蛛网膜下腔出血造模)后0.5h和6h后给药各一次，假手术组和模型组使用等体积生理盐水含1％DMSO代替。
模型及给药方法
参考文献(Stroke,1995,26,1086–1092)所述进行SAH的血管内穿孔模型。即将大鼠麻醉，插管并在手术期间用3％异氟烷在70％/30％医用空气/氧气中保持人工通气。通过直肠探针监测体温，并通过加热灯维持正常热。将锐化的4-0尼龙缝合线引入左颈内动脉(ICA)直至感觉到阻力(距颈总动脉分叉约18mm)。然后将缝合线进一步推入以刺穿前脑动脉和大脑中动脉的分叉，直到克服阻力并在穿孔后立即撤回。在假手术动物中，将缝线插入左ICA，但没有进行穿孔。缝合线移除后，关闭切口，将大鼠单独圈养在加热的笼子中直至恢复。
自发活动评分：将大鼠置于一宽敞、可以自由活动、四壁均可触及的笼中进行自发活动评分。SAH造模后24h由2位实验人员分别以双盲法对实验大鼠进行评价和记录，取2组均值为最后得分，自发活动观察后立即处死大鼠。自发活动评分根据动物精神状态及运动情况分为4级：1级，大鼠活动正常，无活动障碍，积极探索四周环境，至少触及三面笼壁的上缘；2级，轻度活动障碍，即大鼠精神差、嗜睡，行动有一定的延迟，没有到达所有的笼壁，但他至少触及一面笼壁的上缘；3级，中度活动障碍，即大鼠几乎不能站立，在笼中几乎不进行活动；4级，重度活动障碍，即老鼠没有动弹，并显示有肢体的瘫痪。结果见图5A。
脑含水量测定：分别于SAH造模后24h处死大鼠，迅速取出大脑和小脑，用滤纸吸去表面血液。用电子天平分别秤取大脑和小脑的质量(湿重)，然后， 将脑组织置于烤箱中，105℃烘烤至恒重，再次称取大脑和小脑的质量(干重)。脑组织含水量的计算公式为：脑组织含水量(％)＝(湿重-干重)/湿重×100％。其中小脑的组织含水量作为正常对照。结果见图5B。
基底动脉管径、管腔面积及管壁厚度的测量：将上述各组基底动脉的组织切片进行HE染色，光学显微镜下观察照相后，采用image pro-plus6.0图像分析系统测量基底动脉的管径、管腔面积和管壁厚度。管腔面积的测量方法如下：沿基底动脉内表面测定其管腔周长(L)，在根据公式：直径(d)＝L/π计算管腔直径，半径(r)＝L/2π计算管腔半径，管腔面积(S)根据公式：S＝πr 2求得。管壁厚度的测量方法如下：测量基底动脉内表面至中膜外缘之距，不包括外膜。每根血管选取4个不同的检测点测量管壁厚度，取其平均值作为该血管的测定值。结果见表1。
顶部皮层局部脑血流量(rCBF)测定：在邮顶部用直径5mm小型环钻开骨窗，中心位于Bergma点后1mm，后外方3mm。将LDF3型激光多普勒血流仪探头固定于定向仪微推进器上，分别于制备SAH前及SAH后1、4、12、24h及时观察rCBF。结果见表2。
统计方法：自发活动评分数据采用散点图表示，其余数据均以means±SD表示；自发活动评分据组间统计学差异采用Kruskal-Wallis检验和Mann-Whitney U检验，其余数据组间统计学差异采用one-way ANOVA和Tukey’s检验，P值小于0.05认为有显著性差异。
2.3实验结果
如图5A-图5B所示，与SAH模型对照组相比，给予不同的受试化合物F和FDCA均可明显改善动物神经学评分，明显降低SAH导致的大鼠脑含水量，其中FDCA表现出最强的活性，显著优于F和DCA，略优于F+DCA。
此外，FDCA组明显改善基底动脉管径、管腔面积及管壁厚度(表1)和顶部皮层局部脑血流量(rCBF)(表2)，显著优于F、DCA以及F+DCA组。以上结果显示FDCA具有显著的抗蛛网膜下腔出血的活性，优于上市药物法舒地尔二盐酸盐，以及法舒地尔二盐酸盐和二氯乙酸钠的联合给药。
表1.各给药组大鼠基底动脉管腔直径、管腔面积和管腔厚度的测量
组别 管腔直径(μm) 管腔面积(μm 2) 管腔厚度(μm)
Sham 180.66±20.64 25912.41±6017.4 5.94±0.20
Model 122.11±17.00 11903.30±3367.5 11.67±0.61
F 153.73±18.87 ** 18796.09±4730.3 ** 8.13±0.80 **
DCA 129.10±9.87 * 13092.20±1788.6 * 10.12±1.21 *
F+DCA 160.78±12.65 ** 20.7162±2987.76 ** 8.01±0.41 **
FDCA 175.91±25.70 **# 24745.73±7235.4 **## 6.62±0.80 **##
注：与model相比*P<0.05,**P<0.01，与F+DCA组相比#P<0.05,##P<0.01。
表2化合物对SAH大鼠局部脑血流变化的影响
Figure PCTCN2019104910-appb-000017
注：与model相比*P<0.05,**P<0.01，与F+DCA组相比#P<0.05,##P<0.01
实施例11
预防和/或治疗缺血性脑卒中
为研究FDCA在体内是否具有神经保护作用，选用短暂性大鼠脑缺血模型(tMCAO)进行实验。
模型及给药方法：将大鼠腹腔注射10％水合氯醛(350mg/kg)麻醉后，仰卧位固定在实验台上，颈正中切口，手术刀切开皮肤，钝性分离各层组织，按照大鼠颈部血管解剖图，于体视显微镜下分离左侧颈总动脉(CCA)，置线备用向上分离左颈外动脉(ECA)和颈内动脉，双重结扎，剪断甲状腺上动脉及枕动脉两条颈外动脉分支，在近CCA分叉约5mm-8mm处双重结扎ECA，于ICA及CCA近心端分别用微动脉夹夹闭，在ECA近分叉处留置一打好单结但不收紧的丝线，在ECA近端结扎处与颈总动脉分叉处之间做一直径约0.2mm的V型微切口，将尼龙线头自切口处轻轻插入，轻轻收紧线结，将颈内动脉于两结扎线间剪断，使之与颈内动脉方向一致松开动脉夹，将尼龙线顺ECA经ICA送入颅内，插入深度约18mm～20mm微遇阻力时停止，使尼龙线头端位于MCA起始处，阻断MCA的血流收紧丝线，缝合切口，留置尼龙线尾端于体外。
缺血2h后，用10％水合氯醛再次麻醉大鼠，轻轻拉动尼龙线使其头端回到微切口处(略有阻力感)，使大脑中动脉恢复血供，进行再灌注。假手术组大鼠只 进行麻醉和血管分离术，不结扎血管及导入线栓，术后动物保温。给药方式：缺血4h、24h后大鼠分别尾静脉注射给药。缺血48h后，神经功能评分，再处死大鼠。
分组：假手术组(Sham)；空白溶剂组(Vehicle)；法舒地尔二盐酸盐组(F，30mg/kg，尾静脉注射)；FDCA组(30mg/kg，尾静脉注射)；丁苯酞组(NBP，5mg/kg，尾静脉注射)。
TTC染色：在全脑视交叉及其前后各2mm处，做冠状切四刀，切成五片后迅速将脑片置5ml含有2％TTC的磷酸缓冲溶液中，37℃避光温孵10min，在温孵过程中每隔7～8分钟翻动一次，温孵10min后取出脑片，用数码相机(Olympus C-4000，Japan)拍照，之后用眼科镊分离苍白区(梗塞区)和非苍白区(正常区)，通过Image pro-plus 6.0计算梗塞百分比如下：
梗塞百分比(％)＝苍白区重量/(苍白区重量+非苍白区重量)×100％
神经功能评级：在缺血48h后，根据Longa’s方法对动物的神经功能缺陷进行分级评分，标准如下：
0分：未观察到神经症状；
1分：提尾悬空时，动物的手术对侧前肢表现为腕肘屈曲，肩内旋，肘外展，紧贴胸壁；
2分：将动物置于光滑平面上，推手术侧肩向对侧移动时，阻力降低；
3分：动物自由行走时向手术对侧环转或转圈；
4分；软瘫，肢体无自发活动。
统计方法：神经功能缺陷分级评分数据采用中位数表示，其余数据均以means±SD表示；神经功能缺陷分级评分数据组间统计学差异采用Kruskal-Wallis检验和Mann-Whitney U检验，其余数据组间统计学差异采用one-way ANOVA和Tukey’s检验，P值小于0.05认为有显著性差异。
结果表明，在缺血4h后，给予大鼠FDCA(30mg/kg)有效地降低脑梗死面积(梗死面积百分比：7.48％)，明显低于空白溶剂组(31.4％)和上市药物NBP组(21.1％)，有显著优于法舒地尔二盐酸盐(30mg/kg)组(13.6％)(如图6、图7所示)；此外，FDCA还显著地改善了缺血诱导的神经行为功能障碍，明显优于法舒地尔盐酸盐，略优于NBP(图8)。
进一步考察在大鼠tMCAO模型中，FDCA和等摩尔剂量的法舒地尔二盐酸 盐(F)、二氯乙酸钠盐(DCA)以及二者等摩尔联合给药的活性。造模方法和给药时间点同上。结果表明，在缺血4h后，给予大鼠FDCA(30mg/kg)有效地降低脑梗死面积(梗死面积百分比：6.78％)，显著优于F(26.0mg/kg，梗死面积百分比：22.8％)DCA(10.7mg/kg，梗死面积百分比：23.4％)和，以及二者的联合给药组(梗死面积百分比：15.2％)(图9)。此外，FDCA还显著地改善了缺血诱导的神经行为功能障碍，优于F、DCA及二者的联合给药(图9)。
实施例8
法舒地尔盐酸盐(F)法舒地尔二氯乙酸盐(FDCA)药代动力学和组织分布研究
考察了F和FDCA在静脉注射和灌胃两种给药途径下的药代动力学性质。如表3所示，静脉注射给药组，FDCA表现出相比于F略长的半衰期(0.440vs0.265小时)，而灌胃给药组，FDCA表现出比F更大的Cmax，AUC以及生物利用度，F的生物利用度为4.41±1.23％，FDCA的生物利用度为8.43±2.7％，提高了一倍左右。这些结果可以初步很解释在肺动脉高压大鼠模型试验中，灌胃给药的FDCA具有更高的生物利用度和体内浓度，从而产生相比于F更强的活性。
表3.F和FDCA药代动力学参数(n＝3)
参数 单位 F(静脉) FDCA(静脉) F(灌胃) FDCA(灌胃)
给药剂量 mg/kg 6.24 7.2 12.48 14.4
K el h -1 2.67±0.444 1.59±0.172 0.910±0.432 0.790±0.2581
t 1/2 h 0.265±0.05 0.440±0.05 0.867±0.34 0.943±0.30
t max h / / 0.25±0 0.25±0
C max ng·mL -1 1715±120 1511±330 84.2±46.5 113±46.5
C 0 ng·mL -1 2815±689 1950±475 / /
AUC 0-t h·ng·mL -1 669±26 649±183 55.4±16.5 109±35
AUC 0-inf h·ng·mL -1 672±26 651±182 59.8±18.4 117±43
AUMC 0-t h·h·ng·mL -1 182±5 211±75 52.8±26.6 119±51
AUMC 0-inf h·h·ng·mL -1 190±7 222±75 75.7±42.7 160±98
CL mL·min -1·kg -1 155±6 194±54 / /
MRT IV h 0.283±0.01 0.336±0.04 / /
Vd SS L·kg -1 2.63±0.1 3.86±0.8 / /
MRT PO h / / 1.20±0.52 1.29±0.3
F ％ / / 4.14±1.23 8.43±2.7
进一步考察了F和FDCA在静脉给药后在全身的组织分布情况。如表4和5所示，等摩尔剂量给药，FDCA在脑部的分布显著高于F组(463vs 193 0.25h，95.1vs 53.9 1h，ng/g)。这些结果初步解释了在蛛网膜下腔出血引起的血管痉挛以及脑缺血模型中，静脉注射给药FDCA要比F活性更佳的可能原因。
表4.法舒地尔(F)静脉注射给药(6.24mg/kg)0.25及1h后在大鼠体内的组织分布(ng/g，n＝3)
Figure PCTCN2019104910-appb-000018
表5.法舒地尔二氯乙酸盐(FDCA)静脉注射给药(7.20mg/kg)0.25及1h后在大鼠体内的组织分布(ng/g，n＝3)
Figure PCTCN2019104910-appb-000019
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一类化合物，其特征在于，所述化合物为法舒地尔复合盐，选自法舒地尔二氯乙酸盐、法舒地尔三氟乙酸盐、法舒地尔三氯乙酸盐、法舒地尔氯乙酸盐、法舒地尔氯溴乙酸盐、法舒地尔三溴乙酸盐。
  2. 根据权利要求1所述的化合物，其特征在于，所述化合物为法舒地尔二氯乙酸盐，结构式如下：
    Figure PCTCN2019104910-appb-100001
  3. 根据权利要求2所述的化合物，其特征在于，法舒地尔二氯乙酸盐的制备方法为：
    取适量法舒地尔置于反应容器中，加入适量反应溶剂混合，得法舒地尔与反应溶剂的混合液；
    在反应温度0-100度下边搅拌边加入适量的二氯乙酸到上述混合液中，滴完后继续搅拌一段时间，得反应液；
    然后将反应液减压浓缩出去溶剂，洗涤、重结晶，即得法舒地尔二氯乙酸盐。
  4. 根据权利要求3所述的化合物，其特征在于，制备过程中，反应温度为室温，反应溶剂为水，加入的法舒地尔与二氯乙酸的摩尔比为1:1.5，重结晶溶剂为异丙醇。
  5. 根据权利要求1所述的化合物，其特征在于，所述化合物为法舒地尔三氟乙酸盐，结构式如下：
    Figure PCTCN2019104910-appb-100002
    或，所述化合物为法舒地尔三氯乙酸盐，结构式如下：
    Figure PCTCN2019104910-appb-100003
  6. 根据权利要求1所述的化合物，其特征在于，所述化合物为法舒地尔氯乙酸盐，结构式如下：
    Figure PCTCN2019104910-appb-100004
    或，所述化合物为法舒地尔氯溴乙酸盐，结构式如下：
    Figure PCTCN2019104910-appb-100005
    或，所述化合物为法舒地尔三溴乙酸盐，结构式如下：
    Figure PCTCN2019104910-appb-100006
  7. 一种药物组合物，其中含有治疗有效量的权利要求1-6任一项所述的化合物或其旋光异构体、对映体、非对映体、外消旋体或外消旋混合物，或其药学上可接受的盐及可药用的载体、佐剂或媒剂。
  8. 权利要求1-6任一项所述的化合物在制备预防和/或治疗肺动脉高压疾病的药物中的应用。
  9. 权利要求1-6任一项所述的化合物在制备预防和/或治疗蛛网膜下腔出血 疾病的药物中的应用。
  10. 权利要求1-6任一项所述的化合物在制备预防和/或治疗缺血性脑卒中疾病的药物中的应用。
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