WO2023103733A1 - 一种大麻二酚衍生物及其制备方法和应用 - Google Patents

一种大麻二酚衍生物及其制备方法和应用 Download PDF

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WO2023103733A1
WO2023103733A1 PCT/CN2022/132572 CN2022132572W WO2023103733A1 WO 2023103733 A1 WO2023103733 A1 WO 2023103733A1 CN 2022132572 W CN2022132572 W CN 2022132572W WO 2023103733 A1 WO2023103733 A1 WO 2023103733A1
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cancer
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alkyl
substituted
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French (fr)
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王曙宾
杜业松
张平平
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德义制药有限公司
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Definitions

  • the invention relates to the technical field of chemistry and medicine, in particular to a cannabidiol derivative and its preparation method and application, especially the application in the prevention and treatment of nervous system diseases (such as epilepsy and Parkinson's disease).
  • nervous system diseases such as epilepsy and Parkinson's disease.
  • Cannabinoids are a class of secondary metabolites unique to cannabis plants containing alkyl and monoterpene groups. At present, more than one hundred cannabinoids have been isolated from cannabis plants, mainly including delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), cannabigerol (CBG), Cannabinol (CBN), etc., among which THC and CBD have the highest content. Studies have shown that cannabinoids have a wide range of pharmacological effects.
  • THC delta-9-tetrahydrocannabinol
  • CBD cannabidiol
  • CBD cannabichromene
  • CBD cannabigerol
  • CBD Cannabinol
  • Cannabidiol (CBD), chemical name 2-[(1R,6R)-3-methyl-6-(1-methylvinyl)-2-cyclohexen-1-yl]-5-pentyl -1,3-benzenediol, CAS registration number: 13956-29-1, the chemical structure is as follows:
  • CBD has pharmacological effects such as anti-spasmodic, anti-anxiety, anti-inflammatory, and anti-oxidative, and can be used for drug development.
  • high-purity CBD is a white or light yellow crystal with a melting point of 66-67°C, almost insoluble in water or 10% sodium hydroxide solution, and easily soluble in ethanol, methanol, ether, benzene, chloroform and petroleum ether and other organic solvents.
  • CBD has poor water solubility and low bioavailability, which greatly limits the application of CBD in medicine and other fields.
  • the present invention provides a cannabidiol derivative and its preparation method and application, especially the application in the prevention and treatment of nervous system diseases (such as epilepsy and Parkinson's disease).
  • nervous system diseases such as epilepsy and Parkinson's disease.
  • X 3 selected from:
  • R is selected from: H, alkyl, cycloalkyl
  • R 2 is selected from: OH, alkoxy
  • n is an integer of 1-5 (eg 1, 2, 3, 4, 5);
  • R is selected from: H,
  • R is selected from: H, alkyl, cycloalkyl
  • R is selected from: OH, alkoxy
  • n is an integer of 1-5 (eg, 1, 2, 3, 4, 5).
  • X 1 is selected from: single bond, C1-6 straight chain or branched chain alkylene, especially C1-3 straight chain alkyl; in one embodiment of the present invention, X 1 is methylene.
  • R can be selected from: H, methyl, isopropyl, sec-butyl, isobutyl, In one embodiment of the invention, R 7 is
  • X 3 is
  • n 1
  • R 1 is H.
  • R 2 is selected from: OH, C1-6 alkoxy; in some embodiments of the present invention, R 2 is OH, methoxy or ethoxy.
  • the above compound has the following structure:
  • R3 is H.
  • R 3 is
  • X 4 is selected from: single bond, C1-6 straight chain or branched chain alkylene, especially C1-3 straight chain alkyl; in one embodiment of the present invention, X 4 is methylene.
  • R can be selected from: H, methyl, isopropyl, sec-butyl, isobutyl, In one embodiment of the invention, R 8 is
  • X 6 is
  • n 1
  • R4 is H.
  • R 5 is selected from: OH, C1-6 alkoxy; in some embodiments of the present invention, R 5 is OH, methoxy or ethoxy.
  • the above compound has the following structure:
  • the above compound has the following structure:
  • the above compound has the following structure:
  • X 1 , X 2 , X 3 , R 1 , R 2 , R 3 , and n have the corresponding definitions as described in the first aspect of the present invention.
  • the above stereoisomers have the following structures:
  • the salt of the compound of the first aspect or the stereoisomer of the second aspect especially a pharmaceutically acceptable salt.
  • the salts are base addition salts, which may be formed with metals or amines and compounds, especially alkali metal salts, alkaline earth metal salts, eg sodium, potassium, magnesium, calcium salts.
  • the salt is N-(2-aminoethyl)-2-aminoethyl
  • esters, prodrugs, and solvates of the compounds described in the first aspect are provided.
  • R 3 is H
  • the preparation method may comprise the following reaction steps:
  • R 2 ' is an alkoxy group, such as a C1-6 alkoxy group, such as a methoxy group.
  • the preparation method may also include the following reaction steps:
  • the alkali in the above reaction step is an alkali metal hydroxide, such as lithium hydroxide, sodium hydroxide, potassium hydroxide.
  • the acid in the above reaction step is an inorganic acid, such as hydrochloric acid.
  • the preparation method may also include the following reaction steps:
  • R 9 is an alkyl group, such as a C1-6 alkyl group, such as a methyl group;
  • R 10 is a leaving group, such as -F, -Cl, -Br, -I, (-OTs), (-OMs), (-OBs), (-OTf).
  • the alkali in the above reaction step is an alkali metal hydroxide, such as lithium hydroxide, sodium hydroxide, potassium hydroxide.
  • the acid in the above reaction step is an inorganic acid, such as hydrochloric acid.
  • R 3 is The method of preparation may include separately or simultaneously (at this time and represent the same substance) as the steps of the reaction;
  • R 2 ′ and R 5 ′ are alkoxy groups, such as C1-6 alkoxy groups, such as methoxy groups.
  • a pharmaceutical composition comprising the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate thereof, and a One or more pharmaceutically acceptable excipients.
  • auxiliary materials can be selected from: fillers, binders, lubricants, disintegrants, antioxidants, buffers, suspending agents, solubilizers, thickeners, stabilizers and preservatives, etc. one or more of.
  • the pharmaceutical composition can be administered gastrointestinally or parenterally, such as intravenously, intramuscularly, intradermally and subcutaneously.
  • the pharmaceutical composition can be prepared into the following dosage forms: tablets, pills, powders, granules, capsules, lozenges, syrups, gels, emulsions, suspensions, controlled release preparations, aerosols, Film preparations, injections, intravenous drips, transdermal preparations, ointments, lotions, adhesive preparations, suppositories, nasal preparations, lung preparations, etc.
  • various dosage forms of the above pharmaceutical composition can be prepared according to conventional production methods in the field of pharmacy.
  • the pharmaceutical composition may also contain one or more other active ingredients for the same or different indications.
  • the compound described in the first aspect of the present invention or its pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate, and the pharmaceutical composition described in the sixth aspect.
  • the above diseases are selected from one or more of nervous system diseases, cancer, autoimmune diseases, cardiovascular diseases, pain, inflammation, liver damage, especially nervous system diseases.
  • neurological diseases include, but are not limited to, epilepsy, multiple sclerosis, Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, Huntington's disease, anxiety disorders, depression, and the like.
  • epilepsy may be acute epilepsy, chronic epilepsy, especially chronic epilepsy.
  • cancers include, but are not limited to, breast cancer, lung cancer, colorectal cancer, liver cancer, pancreatic cancer, gastric cancer, gastroesophageal adenocarcinoma, esophageal cancer, small intestine cancer, cardia cancer, endometrial cancer, ovarian cancer, fallopian tube cancer , vulvar cancer, testicular cancer, prostate cancer, leukemia, glioma, etc.
  • autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, autoimmune hepatitis, and the like.
  • a method for preventing and/or treating diseases which comprises administering the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt, stereo Isomer, ester, prodrug, solvate, or the step of the pharmaceutical composition described in the sixth aspect.
  • the above diseases are selected from one or more of nervous system diseases, cancer, autoimmune diseases, cardiovascular diseases, pain, inflammation, liver damage, especially nervous system diseases.
  • neurological diseases include, but are not limited to, epilepsy, multiple sclerosis, Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies, Huntington's disease, anxiety disorders, depression, and the like.
  • epilepsy may be acute epilepsy, chronic epilepsy, especially chronic epilepsy.
  • cancers include, but are not limited to, breast cancer, lung cancer, colorectal cancer, liver cancer, pancreatic cancer, gastric cancer, gastroesophageal adenocarcinoma, esophageal cancer, small intestine cancer, cardia cancer, endometrial cancer, ovarian cancer, fallopian tube cancer , vulvar cancer, testicular cancer, prostate cancer, leukemia, glioma, etc.
  • autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, ulcerative colitis, Crohn's disease, autoimmune hepatitis, and the like.
  • the subject may be a mammal, for example, a human, an orangutan, a monkey, a dog, a rabbit, a mouse, etc., especially a human.
  • the inventors of the present invention carried out structural modification on the basis of cannabidiol, and screened the compounds of the present invention in a series of synthetic derivatives. The results of animal experiments showed that these compounds can effectively shorten the duration of epileptic seizures in experimental animals and improve the experimental results.
  • Epilepsy symptoms in animals reduce the balance beam score of Parkinson's model animals, increase the dopamine level and the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN), and can be used for epilepsy, Parkinson's disease and other diseases.
  • Drug development and research have better application value.
  • Figure 1 shows the detection results of seizure duration in PTZ-ignited epileptic rats before and after treatment.
  • Figure 2 shows the Ono's grading of the behavioral seizure index of PTZ-ignited epileptic rats before and after treatment.
  • Figure 3 shows the detection results of seizure latency in PTZ-ignited epileptic rats before and after treatment.
  • Figure 4 shows the PAGE gel images of apoptosis-related proteins Bax and Bcl-2 in rat hippocampal tissue.
  • Figure 5 shows the changes of apoptosis-related proteins Bax and Bcl-2 in rat hippocampus.
  • Figure 6 shows the detection results of ⁇ -aminobutyric acid content in rat brain tissue homogenate.
  • Figure 7 shows the detection results of glycine content in rat brain tissue homogenate.
  • Figure 8 shows the detection results of glutamic acid content in rat brain tissue homogenate.
  • Figure 9 shows the detection results of aspartic acid content in rat brain tissue homogenate.
  • Figure 10 shows the behavioral balance beam scoring results of the Parkinson's model.
  • Figure 11 shows the test results of striatal dopamine content after Parkinson's model treatment.
  • Figure 12 shows the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN).
  • Figure 13 shows microscopic images of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN).
  • alkyl refers to a hydrocarbon group derived from an alkane molecule by the loss of one hydrogen atom, which may be straight or branched and which is attached to the rest of the molecule by a single bond.
  • Alkyl groups as used herein generally contain 1 to 12 (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, preferably 1 to 6 carbon atoms atom (ie, C 1 -C 6 alkyl).
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, t-pentyl base, n-hexyl, isohexyl, etc.
  • aralkyl e.g. C 7 -C 18 aralkyl, e.g.
  • alkylene refers to a hydrocarbon group (divalent alkyl group) formed by the loss of two hydrogen atoms from an alkane molecule, which may be straight or branched and which is connected to the rest of the molecule by a single bond.
  • Typical alkylene groups herein are 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, preferably containing 1 to 6 carbon atoms, Examples of alkylene groups are methylene ( -CH2- ), ethylene, propylene, butylene and the like.
  • alkoxy refers to a substituent formed by replacing hydrogen in a hydroxy group with an alkyl group.
  • Alkoxy groups as used herein typically contain 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, preferably 1 to 6 carbon atoms atom (ie, C 1 -C 6 alkoxy).
  • Examples of the alkoxy group include, but are not limited to, methoxy, ethoxy, propoxy, butoxy and the like.
  • cycloalkyl refers to an alicyclic hydrocarbon, such as containing 1 to 4 monocyclic and/or condensed rings, containing 3-18 carbon atoms, preferably 3-10 (eg 3, 4, 5, 6, 7 , 8, 9, 10) carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or adamantyl, etc.
  • aryl refers to any functional group or substituent derived from a simple aromatic ring, including monocyclic aryl groups and/or fused ring aryl groups, such as those containing 1-3 rings, single Cyclic or fused ring and having 6-18 (eg 6, 8, 10, 12, 14, 16, 18) carbon ring atoms.
  • the aryl group used herein is usually an aryl group containing 1-2 rings, monocyclic or condensed rings and having 6-12 carbon ring atoms (ie, C 6 -C 12 aryl group), wherein the carbon atom
  • the H on can be substituted, for example by alkyl, halogen and the like. Examples of the aryl group include, but are not limited to, phenyl, p-hydroxyphenyl, and the like.
  • heterocyclyl refers to a 3 to 18 membered non-aromatic ring group comprising 2 to 17 carbon atoms and 1 to 10 heteroatoms.
  • a heterocyclyl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can contain fused, spirocyclic, or bridged ring systems.
  • a heterocyclyl group can be partially saturated (heteroaryl) or fully saturated (heterocycloalkyl).
  • pharmaceutically acceptable salt includes acid addition salts and base addition salts.
  • stereoisomers includes the existence of enantiomers, diastereomers and geometric isomers.
  • solvate refers to a physical association of a compound of the invention with one or more solvent molecules. This physical association includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, solvates can be isolated, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. Solvates include solution-phase and isolatable solvates. Representative solvates include ethanolates, methanolates, and the like.
  • prodrug refers to a form of the compound of formula I which is suitable for administration to a patient without undue toxicity, irritation, allergic response, etc. and which is effective for the purpose for which it is used, including acetal, ester and zwitterionic forms. Prodrugs are transformed in vivo, eg, by hydrolysis in blood, to yield the parent compound.
  • subject refers to any animal or cell thereof, whether in vitro or in situ, that is subjected to the methods described herein.
  • the aforementioned animals include mammals, for example, rats, mice, guinea pigs, rabbits, dogs, monkeys, orangutans or humans, especially humans.
  • treating refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, arresting and/or stopping one or more clinical symptoms of a disease after onset of the disease.
  • prevention refers to avoiding, minimizing or making the onset or progression of a disease difficult by treatment, before the onset of the disease occurs.
  • Embodiment 1 the preparation of compound E1
  • CBD (20g, 64mmol, 1eq) was dissolved in 400ml butanone, cesium carbonate (31g, 96mmol, 1.5eq) was added, heated to 80°C for 2h. 12.6 g (0.082 mol, 1.3 eq) of methyl bromoacetate was added and reacted overnight.
  • methyl bromoacetate (5g) and cesium carbonate (15g) react for 2h, filter the reaction solution, add ethyl acetate (100ml) after concentration, wash with 2N hydrochloric acid, wash with saturated sodium chloride, collect the organic phase, anhydrous sulfuric acid Drying over sodium and concentration gave the crude product as a brown oil (30 g).
  • Column chromatography n-hexane/dichloromethane 20:1-1:1) gave compound 1 (11 g) and compound 5 (10.5 g) as yellow oil.
  • reaction solution Concentrate the reaction solution, add 100ml of ethyl acetate, adjust the pH to 2 with 2N hydrochloric acid, separate the layers, wash the organic phase with saturated sodium chloride, dry over anhydrous sodium sulfate, filter, and concentrate to obtain 5.5g of a yellow oily crude product.
  • the crude product was subjected to column chromatography (dichloro/methanol: 150/1-50/1) to obtain compound 4 (3.4 g) as a yellow oil.
  • Embodiment 2 the synthesis of compound E2
  • PTZ was purchased from Sigma; sodium valproate was purchased from Sigma; absolute ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.
  • the experimental animals were randomly divided into 4 groups: model group, positive drug group, compound 1 group, and compound 2 group, with 8 animals in each group.
  • the groups are randomized. Start the treatment, all Wistar rats are weighed, and the intragastric dosage is calculated.
  • the model group was given an equal volume of normal saline; the positive drug group (sodium valproate was given 100 mg/kg by stomach); the compound 1 group (compound E1 prepared in Example 1 was given by stomach, 100 mg/kg); the compound 2 group (the Compound E2 prepared in 2, intragastric administration, 100mg/kg).
  • the prepared PTZ solution was intraperitoneally injected 1 hour after intragastric administration, and the injection dose was 35 mg/kg.
  • the behavioral changes of the rats were observed within 30 min after the injection of PTZ.
  • the behavioral onset indicators before and after administration were recorded according to Ono's classification, and the onset latency, onset classification and onset duration were recorded.
  • the brain and hippocampus of the experimental animals were collected and frozen at -80°C.
  • the corresponding protein concentration ( ⁇ g/ ⁇ l) can be found on the standard curve, and multiplied by the sample dilution factor (10) is the actual concentration of the sample (unit: ⁇ g/ ⁇ l).
  • the loading amount of each well is about 25 ⁇ g protein, and the loading amount can be increased according to the experimental requirements.
  • the protein quantification results take the required protein and add an appropriate amount of loading buffer, centrifuge in a boiling water bath for 10 minutes, and take the supernatant for loading. Put the prepared PAGE gel into the electrophoresis tank, add an appropriate amount of electrophoresis buffer, remove the comb and gently blow the sample hole with a gun to avoid residual gel residue in the hole affecting sample loading. Slowly add the prepared sample into the corresponding hole with a sample gun, and be careful not to overflow the sample hole.
  • the concentrated gel is 80V for 20 minutes, and the separating gel is 120V for 60 minutes.
  • the voltage and time can be adjusted according to specific experimental requirements.
  • the membrane transfer is divided into wet transfer and semi-dry transfer, and semi-dry transfer was selected in this experiment.
  • the sandwich arrangement is: /filter paper/glue/membrane/filter paper, soaked with electrotransfer buffer, and placed directly between the positive and negative electrodes of the electrotransfer instrument.
  • the glue is placed on the negative electrode and the membrane is placed on the positive electrode.
  • the semi-dry electrotransfer buffer is different from the wet electrotransfer buffer, the recommended ones are: 48mM Tris, 39mM glycine, 0.04% SDS, 20% methanol. 25V transfer film for 30 minutes. Soak the PVDF membrane in methanol for 1-2 minutes before transferring the membrane (soak the NC membrane in the electrotransfer solution for 10-20 minutes), and then incubate in the ice-cold electrotransfer buffer for 5 minutes. The gel also needs to be in the ice-cold electrotransfer buffer Equilibrate for 3-5 minutes, otherwise the strips will be deformed when transferred to the membrane.
  • Staining method Wash the membrane once in TBST, then place it in Ponceau staining solution, shake and stain at room temperature for 5 minutes, wash the membrane with a large amount of water until the water becomes clear and the colorless protein bands are clear, (the membrane can also be used TBST or water re-washing before staining) PVDF membrane needs to be reactivated with methanol and then washed with TBST before blocking.
  • Blocking block with 5% skimmed milk powder (BSA for detection of phosphorylated protein) at room temperature for 1 hour or overnight at 4°C.
  • BSA skimmed milk powder
  • ECL chemiluminescent detection prepare ECL luminescent liquid, mix equal amounts of ECL luminescent liquid A and B according to the dosage, add it to the front of the membrane in a dark room for 5 minutes to avoid light. Pour off the chromogenic solution, carefully absorb the chromogenic solution with paper, and cover it with a flat layer of transparent paper. Put it in the imaging system and scan it.
  • the experimental operation and method refer to the kit instructions.
  • the duration of PTZ ignited epileptic seizures before and after treatment by comparing the data, it was found that the duration of each group before treatment did not change, and the duration of seizures was about 350s; as the treatment stage entered, there were significant differences among the groups, and the positive drug group was in The onset duration decreased on the 7th and 14th day of treatment, and it was statistically significant.
  • the other compound groups also showed a significant decrease in seizure duration on day 7 of treatment and before treatment, which was statistically significant; among them, compound 2 had a statistically significant decrease in seizure duration on day 14 of follow-up treatment and on day 7 of treatment. There is no change in the onset duration of compound 1 on the 14th day of treatment and the 7th day of treatment.
  • Ono s grading records the highest grade of epileptic seizures in rats.
  • epileptic seizures are a progressive process, and the highest seizure grades will have a duration during the epileptic seizures. Subsequent recording of the duration of each seizure grade is recommended.
  • the experimental results showed that the apoptosis-promoting Bax protein was highly expressed in the hippocampal tissue of the rats in the model group, while the apoptosis-promoting Bax protein was lower in the positive drug group and the model group, and the expression was statistically significant.
  • the expression of Bax protein was significantly down-regulated in both compound E1 and compound E2 with statistical significance.
  • Excitatory amino acids glutamic acid and aspartic acid
  • inhibitory neurotransmitters ⁇ -aminobutyric acid and glycine in brain tissue homogenate were detected by ELISA detection kit.
  • the results of ELISA experiments showed that the content of ⁇ -aminobutyric acid and glycine in the brain tissue homogenate of the model group was low, and the content of glutamic acid and aspartic acid was high.
  • the duration of seizures in the two compound groups on the 7th and 14th days of treatment were significantly reduced, and there were significant differences.
  • the duration of seizures was in the order of compound E2>compound E1; on the 14th day of treatment, the duration of seizures was in the order of compound E2>compound E1.
  • Embodiment 4 research on the treatment of Parkinson's
  • mice SPF grade, weighing 18-20 g, 6-8 weeks old, male, were purchased from Shanghai Slack Experimental Animal Co., Ltd.
  • the temperature of the feeding environment was kept at 22 ⁇ 1°C, the humidity was kept at 65-70%, the light/dark light cycle was 12 hours, and water was freely available.
  • MPTP was purchased from Sigma; amantadine was purchased from Sigma; absolute ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.
  • the experimental animals were randomly divided into 5 groups: blank group, model group, positive drug group, compound 1 group, and compound 2 group, with 8 animals in each group.
  • a wooden bar with a length of 80 cm and a width of 2.5 cm is fixed horizontally at a height of 10 cm from the table top, and then the animals are allowed to walk on the wooden bar.
  • mice After the mice were sacrificed quickly, the brains of the mice were removed, and the cerebral cortex (CP) of the mice was isolated. After exposing the striatum of the mouse brain, take the striatum and weigh it, add PBS according to the proportion, and grind and homogenate. Then the supernatant was separated to detect the content of dopamine in the striatum.
  • CP cerebral cortex
  • the corresponding protein concentration ( ⁇ g/ ⁇ l) can be found on the standard curve, and multiplied by the sample dilution factor (10) is the actual concentration of the sample (unit: ⁇ g/ ⁇ l);
  • Citric acid C 6 H 8 O 7 ⁇ H 2 O molecular weight 210.14; 0.1 mol/L solution is 21.01 g/L.
  • tissue block When cutting the tissue block, pull the cut tissue backwards from the root of the knife.
  • the thickness of the tissue block is about 0.2-0.3 cm, and the size is preferably 1.5 cm ⁇ 1.5 cm ⁇ 0.3 cm.
  • the tissue blocks were fixed in 10% formalin for 48 hours.
  • Ethanol with different concentrations is dehydrated step by step, 50%, 70%, 85%, 95% until pure alcohol (absolute ethanol), each stage for 2 hours. Dehydration must be carried out in a bottle with a cover to prevent high-concentration ethanol from absorbing moisture in the air, resulting in a decrease in concentration and incomplete dehydration. Materials that need to be preserved can be dehydrated to 70% ethanol and stay there. For long-term preservation, an equal amount of glycerin can be added.
  • tissue block Place the tissue block in an equal-volume mixture of pure ethanol and xylene for 2 hours, then into pure xylene for 2 hours, and again in pure xylene for 2 hours; the material will be transparent to show the effect of the previous step of dehydration, if dehydration Thoroughly, the tissue will show a transparent state, such as white clouds in the tissue, indicating that the dehydration is not clean, and rework is required, but the effect of rework is often not good.
  • xylene for transparency it should avoid volatilization and absorption of moisture in the air, and keep it in an anhydrous state.
  • Dip wax must be carried out in a constant temperature box. First place the tissue material block in an equal mixture of molten paraffin and xylene to immerse for 1-2 hours, then move it into two melted paraffin liquids and immerse for about 3 hours each.
  • Embedding is the encapsulation of a wax-soaked tissue block in paraffin.
  • the specific method is: first prepare the carton, pour the melted wax into the box, quickly use pre-warmed tweezers to pick up the tissue block and place it flat on the bottom of the carton, with the cut side facing down, then gently lift the carton and place it flat on the bottom of the carton. In cold water, after the surface paraffin solidifies, immediately press the carton into the water to make it cool and solidify rapidly, and take it out after 30 minutes.
  • the wax blocks were placed in a -20°C refrigerator for at least 30 minutes to increase the hardness.
  • the dewaxed sections were soaked in 100% alcohol, 95% alcohol, 85% alcohol, and 75% alcohol for 5 minutes respectively, and rinsed with tap water for 10 minutes.
  • the slides were placed in 3% H2O2 and incubated in a wet box for 10 min to eliminate the activity of endogenous peroxidase. Wash with 0.02M PBS for 3 min ⁇ 3 times.
  • DAB staining when the color change of the section is observed, immediately wash away the staining solution with tap water.
  • mice were prepared by intraperitoneal injection of MPTP for Parkinson's model, and the behavioral evaluation was carried out by the balance beam score. Seven days after the intraperitoneal injection of MPTP, the balance beam score was performed. All scores are 5-6 points, each group compared with the blank group P ⁇ 0.05, which is statistically significant and proves that the model is successful.
  • the balance beam score of the model group was 4-5 points, and the balance beam score of the positive drug was between 2-3 points.
  • the beam score for the other compound treatment groups was 3-4.
  • the positive drug group and each group of compounds showed a significant downward trend, P ⁇ 0.05, which was statistically significant.
  • Parkinson's model As a neurotransmitter, dopamine regulates various physiological functions of the central nervous system, and the content of dopamine in the striatum of Parkinson's model in treated mice was detected. To judge the effect of drug treatment on Parkinson's model.
  • the degree of dopamine decline in the model was judged by comparing the dopamine content of each group and the blank group. Except for the positive drug group, the dopamine content of the other groups all decreased significantly, P ⁇ 0.05, which was statistically significant.
  • the content of dopamine in striatum was blank group>positive drug group>compound 1 group>compound 2 group>model group.
  • Tyrosine hydroxylase is a monooxygenase, which is the rate-limiting enzyme that catalyzes the first step in the series of reactions of the organism's own synthesis of L-Dopamine (DA), and is only expressed in the cytoplasm .
  • DA L-Dopamine
  • the content of TH in neurons is abundant, because the midbrain lacks dopamine- ⁇ -hydroxylase, so the neurons positive for TH immune response in the midbrain are DA neurons, which can be used as a sign of dopamine neurons in the brain.
  • the body uses L-tyrosine to survive levodopa under the catalysis of tyrosine hydroxylase, and levodopa is decarboxylated under the catalysis of aromatic decarboxylase to finally generate L-dopamine. Due to the important position of tyrosine hydroxylase in the synthesis of dopamine, the loss or insufficient expression of its function directly affects the synthesis and secretion of dopamine. Dopamine is an important neurotransmitter, and the failure of dopaminergic neurons to synthesize or insufficient secretion of dopamine will lead to Parkinson's disease.
  • the results of this experiment showed that in the mouse Parkinson's model induced by intraperitoneal injection of MPTP, the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the mouse decreased, P ⁇ 0.05, which was statistically significant. significance.
  • the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the positive drug group was significantly higher than that of the model group, indicating that the modeling was successful.
  • the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the brain substantia nigra (SubN) of each compound group was compared, and all of them increased.
  • the positive rate of tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the compound 1 group was compared with that of the model group, P ⁇ 0.01, and it was statistically significant.
  • the mouse Parkinson's model was established by intraperitoneal injection of MPTP (25mg/kg) once a day for 7 consecutive days, and the mouse Parkinson's model was screened by the balance beam score.
  • the model mice were treated by intragastric administration of positive drugs and various compounds.
  • the balance beam scores of each group after treatment were analyzed, the dopamine content in the striatum was detected by ELISA, and the substantia nigra (SubN) in tyrosine hydroxylase (TH) cell positive rate and other detection methods, identify each compound and blank group, model group difference, then carry out statistical analysis, the effectiveness of each compound is analyzed.
  • SubN substantia nigra
  • TH tyrosine hydroxylase
  • the dopamine content of the model group was compared with other compound groups, and the dopamine content of each group was higher than that of the model group, P ⁇ 0.01, which was statistically significant.
  • the positive rate of the positive drug group and each compound comparison model group wherein the tyrosine hydroxylase (TH) cells in the substantia nigra (SubN) of the compound 1 group
  • the positive rate was compared with the model group, P ⁇ 0.01, and it was statistically significant.
  • each compound group has a certain therapeutic effect compared with the model group. Compared with each detection index, compound 1 has a better therapeutic effect.

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Abstract

本发明公开了一种大麻二酚衍生物及其制备方法和应用,特别是神经系统疾病(例如癫痫、帕金森病)的预防和治疗中的应用。上述大麻二酚衍生物是从一系列合成衍生物中筛选得到的,动物试验结果显示,这些化合物可有效缩短实验动物癫痫发作持续时间,改善实验动物癫痫发作症状,降低帕金森模型动物的平衡木评分,提高多巴胺水平和脑黑质(SubN)中酪氨酸羟化酶(TH)细胞阳性率,可用于癫痫、帕金森病等多种疾病的药物开发和研究,具有较佳的应用价值。

Description

一种大麻二酚衍生物及其制备方法和应用 技术领域
本发明涉及化学医药技术领域,具体涉及一种大麻二酚衍生物及其制备方法和应用,特别是神经系统疾病(例如癫痫、帕金森病)的预防和治疗中的应用。
背景技术
大麻素(cannabinoids)是大麻植物中特有的含有烷基和单萜基团结构的一类次生代谢产物。目前已从大麻植物中分离出百余种大麻素,主要包括δ-9-四氢大麻酚(THC)、大麻二酚(CBD)、大麻环萜酚(CBC)、大麻萜酚(CBG)、大麻酚(CBN)等,其中以THC和CBD的含量最高。研究显示,大麻素具有广泛的药理作用。
大麻二酚(CBD),化学名称为2-[(1R,6R)-3-甲基-6-(1-甲基乙烯基)-2-环己烯-1-基]-5-戊基-1,3-苯二酚,CAS登记号:13956-29-1,化学结构如下所示:
Figure PCTCN2022132572-appb-000001
研究发现CBD具有抗痉挛、抗焦虑、抗炎、抗氧化等药理作用,可用于药物开发。但是,高纯度的CBD是一种白色或淡黄色结晶,熔点为66-67℃,几乎不溶于水或者10%的氢氧化钠溶液,易溶于乙醇、甲醇、乙醚、苯、氯仿及石油醚等有机溶剂。CBD水溶性不佳,生物利用度低,大大限制了CBD在医药等领域的应用。
发明内容
为克服现有技术的不足,本发明提供一种大麻二酚衍生物及其制备方法和应用,特别是神经系统疾病(例如癫痫、帕金森病)的预防和治疗中的应用。
在本发明第一方面,提供一种化合物,其具有如下结构:
Figure PCTCN2022132572-appb-000002
其中,
X 1和X 2独立地选自:单键、取代或未取代的亚烷基、被一个或多个-C(=O)O-间隔的亚烷基、被一个或多个-OC(=O)-间隔的亚烷基、被一个或多个-C(=O)NH-间隔的亚烷基;
X 3选自:
Figure PCTCN2022132572-appb-000003
R 1选自:H、烷基、环烷基;
R 2选自:OH、烷氧基;
n为1-5的整数(例如1、2、3、4、5);
R 3选自:H、
Figure PCTCN2022132572-appb-000004
其中,X 4和X 5独立地选自:单键、取代或未取代的亚烷基、被一个或多个-C(=O)O-间隔的亚烷基、被一个或多个-OC(=O)-间隔的亚烷基、被一个或多个-C(=O)NH-间隔的亚烷基;
X 6选自:
Figure PCTCN2022132572-appb-000005
R 4选自:H、烷基、环烷基;
R 5选自:OH、烷氧基;
m为1-5的整数(例如1、2、3、4、5)。
具体地,X 1选自:单键、C1-6直链或支链亚烷基,特别是C1-3直链烷基;在本发明的一个实施例中,X 1为亚甲基。
在本发明的一个实施方式中,X 2具有如下结构:
Figure PCTCN2022132572-appb-000006
其中R 7选自:H、烷基、被烷基巯基取代的烷基、被羟基取代的烷基、被巯基取代的烷基、被-C(=O)NH 2取代的烷基、被羧基取代的烷基、被氨基取代的烷基、被
Figure PCTCN2022132572-appb-000007
取代的烷基、杂环基烷基、芳烷基。
具体地,R 7可以选自:H、甲基、异丙基、仲丁基、异丁基、
Figure PCTCN2022132572-appb-000008
Figure PCTCN2022132572-appb-000009
Figure PCTCN2022132572-appb-000010
在本发明的一个实施例中,R 7
Figure PCTCN2022132572-appb-000011
在本发明的一些实施例中,X 3
Figure PCTCN2022132572-appb-000012
在本发明的一些实施例中,n为1。
在本发明的一些实施例中,R 1为H。
具体地,R 2选自:OH、C1-6烷氧基;在本发明的一些实施例中,R 2为OH、甲氧基或乙氧基。
在本发明的一个实施方式中,上述化合物具有如下结构:
Figure PCTCN2022132572-appb-000013
更具体地,上述化合物具有如下结构:
Figure PCTCN2022132572-appb-000014
在本发明的一个实施方式中,R 3为H。
在本发明另一个实施方式中,R 3
Figure PCTCN2022132572-appb-000015
具体地,X 4选自:单键、C1-6直链或支链亚烷基,特别是C1-3直链烷基;在本发明的一个实施例中,X 4为亚甲基。
在本发明的一个实施方式中,X 5具有如下结构:
Figure PCTCN2022132572-appb-000016
其中R 8选自:H、烷基、被烷基巯基取代的烷基、被羟基取代的烷基、被巯基取代的烷基、被-C(=O)NH 2取代的烷基、被羧基取代的烷基、被氨基取代的烷基、被
Figure PCTCN2022132572-appb-000017
取代的烷基、杂环基烷基、芳烷基。
具体地,R 8可以选自:H、甲基、异丙基、仲丁基、异丁基、
Figure PCTCN2022132572-appb-000018
Figure PCTCN2022132572-appb-000019
Figure PCTCN2022132572-appb-000020
在本发明的一个实施例中,R 8
Figure PCTCN2022132572-appb-000021
在本发明的一些实施例中,X 6
Figure PCTCN2022132572-appb-000022
在本发明的一些实施例中,m为1。
在本发明的一些实施例中,R 4为H。
具体地,R 5选自:OH、C1-6烷氧基;在本发明的一些实施例中,R 5为OH、甲氧基或乙氧基。
在本发明的一个实施方式中,上述化合物具有如下结构:
Figure PCTCN2022132572-appb-000023
在本发明另一个实施方式中,上述化合物具有如下结构:
Figure PCTCN2022132572-appb-000024
在本发明的一些实施例中,上述化合物具有如下结构:
Figure PCTCN2022132572-appb-000025
在本发明第二方面,提供第一方面所述化合物的立体异构体,其具有如下结构:
Figure PCTCN2022132572-appb-000026
其中,X 1、X 2、X 3、R 1、R 2、R 3、n具有本发明第一方面所述相应定义。
在本发明的一些实施例中,上述立体异构体具有如下结构:
Figure PCTCN2022132572-appb-000027
在本发明第三方面,提供第一方面所述化合物或第二方面所述立体异构体的盐,特别是药学上可接受的盐。
具体地,该盐为碱加成盐,其可以由金属或胺与化合物形成,特别是碱金属盐、碱土金属盐,例如钠盐、钾盐、镁盐、钙盐。
在本发明的一些实施例中,该盐为
Figure PCTCN2022132572-appb-000028
Figure PCTCN2022132572-appb-000029
在本发明第四方面,提供第一方面所述化合物的酯、前药、溶剂化物。
在本发明第五方面,提供第一方面所述化合物的制备方法。
在本发明的一个实施方式中,R 3为H,该制备方法可以包括如下反应步骤:
Figure PCTCN2022132572-appb-000030
其中,R 2'为烷氧基,例如C1-6烷氧基,例如甲氧基。
在本发明的一些实施例中,上述反应步骤中的
Figure PCTCN2022132572-appb-000031
Figure PCTCN2022132572-appb-000032
具体地,该制备方法还可以包括如下反应步骤:
Figure PCTCN2022132572-appb-000033
具体地,上述反应步骤中的碱为碱金属的氢氧化物,例如氢氧化锂、氢氧化钠、氢氧化钾。
具体地,上述反应步骤中的酸为无机酸,例如盐酸。
具体地,该制备方法还可以包括如下反应步骤:
Figure PCTCN2022132572-appb-000034
其中,R 9为烷基,例如C1-6烷基,例如甲基;
R 10为离去基团,例如-F、-Cl、-Br、-I、
Figure PCTCN2022132572-appb-000035
(-OTs)、
Figure PCTCN2022132572-appb-000036
(-OMs)、
Figure PCTCN2022132572-appb-000037
(-OBs)、
Figure PCTCN2022132572-appb-000038
(-OTf)。
在本发明的一些实施例中,上述反应步骤中的
Figure PCTCN2022132572-appb-000039
Figure PCTCN2022132572-appb-000040
具体地,上述反应步骤中的碱为碱金属的氢氧化物,例如氢氧化锂、氢氧化钠、氢氧化钾。
具体地,上述反应步骤中的酸为无机酸,例如盐酸。
在本发明另一个实施方式中,R 3
Figure PCTCN2022132572-appb-000041
该制备方法可以包括通过
Figure PCTCN2022132572-appb-000042
分别或同时(此时
Figure PCTCN2022132572-appb-000043
Figure PCTCN2022132572-appb-000044
代表相同的物质)与
Figure PCTCN2022132572-appb-000045
反应的步骤;
其中,R 2'、R 5'为烷氧基,例如C1-6烷氧基,例如甲氧基。
在本发明的一个实施例中,
Figure PCTCN2022132572-appb-000046
Figure PCTCN2022132572-appb-000047
代表相同的物质,例如
Figure PCTCN2022132572-appb-000048
在本发明第六方面,提供一种药物组合物,其包含本发明第一方面所述的化合物,或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物,以及一种或多种药学上可接受的辅料。
具体地,药学上可接受的辅料可以选自:填充剂、粘合剂、润滑剂、崩解剂、抗氧化剂、缓冲剂、助悬剂、增溶剂、增稠剂、稳定剂和防腐剂等中的一种或多种。
具体地,该药物组合物可通过胃肠给药或非胃肠给药,如通过静脉内、肌内、皮内和皮下途径给药。
具体地,该药物组合物可以制备成以下剂型:片剂、丸剂、粉剂、颗粒剂、胶囊剂、锭剂、糖浆剂、凝胶剂、乳剂、混悬剂、控制释放制剂、气雾剂、膜剂、注射剂、静脉滴注剂、透皮吸收制剂、软膏剂、洗剂、粘附制剂、栓剂、鼻制剂、肺制剂等。
具体地,上述药物组合物的各种剂型可以按照药学领域的常规生产方法制备。
具体地,该药物组合物还可以包含一种或多种其他针对相同或不同适应症的活性成分。
在本发明第七方面,提供本发明第一方面所述的化合物,或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物、第六方面所述的药物组合物在制备预防和/或治疗疾病的药物中的应用。
具体地,上述疾病选自:神经系统疾病、癌症、自身免疫性疾病、心血管疾病、疼痛、炎症、肝损伤中的一种或多种,特别是神经系统疾病。
具体地,神经系统疾病包括,但不限于,癫痫、多发性硬化症、帕金森氏病、阿尔茨海默症、路易体痴呆、亨廷顿病、焦虑症、抑郁症等。
具体地,癫痫可以为急性癫痫、慢性癫痫,特别是慢性癫痫。
具体地,癌症包括,但不限于,乳腺癌、肺癌、结直肠癌、肝癌、胰腺癌、胃癌、胃食管腺癌、食管癌、小肠癌、贲门癌、子宫内膜癌、卵巢癌、输卵管癌、外阴癌、睾丸癌、前列腺癌、白血病、神经胶质瘤等。
具体地,自身免疫性疾病包括,但不限于,系统性红斑狼疮、类风湿关节炎、多发性硬化症、溃疡性结肠炎、克罗恩氏病、自身免疫性肝炎等。
在本发明第八方面,提供一种预防和/或治疗疾病的方法,其包括向有此需求的受试者施用本发明第一方面所述的化合物,或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物,或第六方面所述的药物组合物的步骤。
具体地,上述疾病选自:神经系统疾病、癌症、自身免疫性疾病、心血管疾病、疼痛、炎症、肝损伤中的一种或多种,特别是神经系统疾病。
具体地,神经系统疾病包括,但不限于,癫痫、多发性硬化症、帕金森氏病、阿尔茨海默症、路易体痴呆、亨廷顿病、焦虑症、抑郁症等。
具体地,癫痫可以为急性癫痫、慢性癫痫,特别是慢性癫痫。
具体地,癌症包括,但不限于,乳腺癌、肺癌、结直肠癌、肝癌、胰腺癌、胃癌、胃食管腺癌、食管癌、小肠癌、贲门癌、子宫内膜癌、卵巢癌、输卵管癌、外阴癌、睾丸癌、前列腺癌、白血病、神经胶质瘤等。
具体地,自身免疫性疾病包括,但不限于,系统性红斑狼疮、类风湿关节炎、多发性硬化症、溃疡性结肠炎、克罗恩氏病、自身免疫性肝炎等。
具体地,受试者可以为哺乳动物,例如,人类、猩猩、猴、犬、兔、鼠等,特别是人类。
本发明的发明人在大麻二酚的基础上进行结构修饰改造,在一系列合成衍生物中筛选得到本发明的化合物,动物试验结果显示,这些化合物可有效缩短实验动物癫痫发作持续时间,改善实验动物癫痫发作症状,降低帕金森模型动物的平衡木评分,提高多巴胺水平和脑黑质(SubN)中酪氨酸羟化酶(TH)细胞阳性率,可用于癫痫、帕金森病等多种疾病的药物开发和研究,具有较佳的应用价值。
附图说明
图1所示为治疗前后PTZ点燃癫痫大鼠的癫痫发作持续时间检测结果。
图2所示为治疗前后PTZ点燃癫痫大鼠的行为发作指标Ono’s分级。
图3所示为治疗前后PTZ点燃癫痫大鼠的癫痫发作潜伏期检测结果。
图4所示为大鼠海马组织中凋亡相关蛋白Bax和Bcl-2的PAGE胶图。
图5所示为大鼠海马组织中凋亡相关蛋白Bax和Bcl-2变化。
图6所示为大鼠脑组织匀浆液中γ-氨基丁酸含量检测结果。
图7所示为大鼠脑组织匀浆液中甘氨酸含量检测结果。
图8所示为大鼠脑组织匀浆液中谷氨酸含量检测结果。
图9所示为大鼠脑组织匀浆液中天冬氨酸含量检测结果。
图10所示为帕金森模型行为学平衡木评分结果。
图11所示为帕金森模型治疗后纹状体多巴胺含量检测结果。
图12所示为脑黑质(SubN)中酪氨酸羟化酶(TH)细胞阳性率。
图13所示为脑黑质(SubN)中酪氨酸羟化酶(TH)细胞的显微镜图像。
具体实施方式
除非另有定义,本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义,例如:
术语“烷基”指的是烷烃分子失去一个氢原子而成的烃基,其可以为直链或支链且其以单键与分子其它部分连接。。在本文中所使用的烷基通常含有1至12(例如1、2、3、4、5、6、7、8、9、10、11、12)个碳原子,优选含有1至6个碳原子(即,C 1-C 6烷基)。所述烷基的实例包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、叔戊基、正己基、异己基等。如果烷基被芳基取代,那么其相应为“芳烷基”(例如C 7-C 18芳烷基,例如C 7-C 15芳烷基,C 7-C 12芳烷基;例如,(C 1-C 6亚烷基)-(C 6-C 12芳基),(C 1-C 3亚烷基)-苯基),如苄基、二苯甲基或苯乙基。如果烷基被杂环基取代,那么其相应为“杂环基烷基”。
术语“亚烷基”指的是烷烃分子失去两个氢原子而成的烃基(二价烷基),其可以是直链或支链且其以单键与分子其它部分连接。在本文中典型的亚烷基1至12(例如1、2、3、4、5、6、7、8、9、10、11、12)个碳原子,优选含有1至6个碳原子,亚烷基的实例如亚甲基(-CH 2-)、亚乙基、亚丙基、亚丁基等。
术语“烷氧基”指的是羟基中的氢被烷基取代后形成的取代基。本文中所使用的烷氧基通常含有1至12(例如1、2、3、4、5、6、7、8、9、10、11、12)个碳原子,优选含有1至6个碳原子(即,C 1-C 6烷氧基)。所述烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基、丁氧基等。
术语“环烷基”指的是脂环烃,如含1至4个单环和/或稠环、含3-18个碳原子,优选3-10(例如3、4、5、6、7、8、9、10)个碳原子,如环丙基、环丁基、环戊基、环己基或金刚烷基等。
术语“芳基”指的是任何从简单芳香环衍生出的官能团或取代基,包括单环的芳基基团和/或稠环的芳基基团,如包含1-3个环的、单环或稠环的且具有6-18(例如6、8、10、12、14、16、18)个碳环原子。本文中所使用的芳基通常为包含1-2个环的、单环或稠环的且具有6-12个碳环原子的芳基(即,C 6-C 12芳基),其中碳原子上的H可以被取代,例如被烷基、卤素等基团取代。所述芳基的实例包括但不限于苯基、对羟基苯基等。
术语“杂环基”是指3至18元非芳香环基团,其包含2至17个碳原子以及1至10个杂原子。杂环基可以为单环、双环、三环、或四环的环系统,其可包含稠合的、螺环的或桥接的环系统。杂环基可以是部分饱和的(杂芳基)或完全饱和的(杂环烷基)。
术语“药学上可接受的盐”包括酸加成盐和碱加成盐。
术语“立体异构体”包括对映体、非对映体和几何异构体的形式存在。
术语“溶剂化物”是指本发明的化合物与一种或多种溶剂分子的物理结合。该物理结合包括各种程度的离子和共价键合,包括氢键合。在某些情况下,溶剂化物可被分离出来,例如当一个或多个溶剂分子掺入到结晶固体的晶格中。溶剂化物包括溶液相和可分离的溶剂化物。代表性的溶剂化物包括乙醇化物、甲醇化物等。
术语“前药”指适于对患者给药的无过分毒性、刺激性和变态反应等的并且对其应用目的有效的式Ⅰ化合物形式,包括缩醛、酯和两性离子形式。前药在体内转化,如通过在血液中水解,得到母体化合物。
术语“受试者”是指接受本文所述的方法的任何动物或其细胞,不论是体外或原位。具体地,前述动物包括哺乳动物,例如,大鼠、小鼠、豚鼠、兔、犬、猴、猩猩或人类,特别是人类。
术语“治疗”是指在疾病发作之后预防、治愈、逆转、减弱、减轻、最小化、抑制、制止和/或停止疾病的一种或多种临床症状。
术语“预防”指在疾病发作之前,通过治疗以避免、最小化或令疾病难于发作或发展。
本文所引用的各种出版物、专利和公开的专利说明书,其公开内容通过引用整体并入本文。
下面将结合本发明实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:化合物E1的制备
Figure PCTCN2022132572-appb-000049
1、合成路线
Figure PCTCN2022132572-appb-000050
2、合成方法
(1)化合物2的合成
Figure PCTCN2022132572-appb-000051
实验过程:
CBD(20g,64mmol,1eq)溶于400ml丁酮中,加入碳酸铯(31g,96mmol,1.5eq),加热到80℃反应2h。加入溴乙酸甲酯12.6g(0.082mol,1.3eq),反应过夜。补加溴乙酸甲酯(5g)和碳酸铯(15g),反应2h,过滤反应液,浓缩后加入乙酸乙酯(100ml),2N盐酸洗涤,饱和氯化钠洗涤,收集有机相,无水硫酸钠干燥,浓缩得到棕色油状粗品(30g)。柱层析(正己烷/二氯甲烷20:1-1:1),得到黄色油状化合物1(11g)和化合物5(10.5g)。
化合物1(11g,28.5mmol,1eq)溶于THF(100ml)中,氢氧化锂(3.6g,85mmol,3eq)溶于15ml 水中,混合搅拌过夜。
用2N稀盐酸调pH为3-4,加入乙酸乙酯(300ml),分液,有机相用饱和氯化钠洗涤,无水硫酸钠干燥,过滤,浓缩后得黄色油状化合物2(9.5g),直接用于下步。
(2)化合物3的合成
Figure PCTCN2022132572-appb-000052
实验过程:
将化合物2(9.5g,24.6mmol,1eq)、蛋氨酸甲酯盐酸盐(6.4g,32mol,1.3eq)、DMAP(4.5g,37mol,1.5eq)溶于二氯甲烷(60ml)中,加入DCC(7.6g,37mmol,1.5eq),室温搅拌过夜。
过滤反应液,滤饼用50ml二氯甲烷洗涤,滤液用2N盐酸洗涤、饱和氯化钠洗涤、无水硫酸钠干燥、过滤、浓缩,粗品经柱层析(正己烷/二氯甲烷:5/1-1/1,正己烷/二氯甲烷/乙酸乙酯:1/1/0.1)得到黄色油状化合物3(6.6g)。
(3)化合物4(游离酸)的合成
Figure PCTCN2022132572-appb-000053
实验过程:
6.6g(0.0128mol,1eq)的化合物3溶于80ml THF中,1.6g(0.0383mol,3eq)氢氧化锂溶于10ml水中加入到反应液中,室温反应过夜。
浓缩反应液,加入乙酸乙酯100ml,用2N盐酸调pH=2,分液,有机相用饱和氯化钠洗涤、无水硫酸钠干燥、过滤、浓缩后得到黄色油状粗品5.5g。粗品经柱层析(二氯/甲醇:150/1-50/1)得到黄色油状化合物4(3.4g)。
(4)化合物E1(钠盐)的合成
Figure PCTCN2022132572-appb-000054
实验过程:
将化合物4(3.4g,6.8mmol,1eq)溶于20ml乙醇中,碳酸氢钠(0.625g,7.4mmol,1.1eq)溶解于10ml水中,混合搅拌,加热到50℃反应1h。
浓缩反应液,加入30ml乙醇并蒸干,残余物用60ml正己烷和20ml二氯甲烷混合溶剂洗涤并干燥后得到粉色固体E1(2.4g),HPLC>98%。
MS(ESI,positive):504.1[M+H]+.
1H-NMR(400MHz,CD 3OD):6.30(d,J=0.8Hz,1H),6.20(s,1H),5.29(s,1H),4.55-4.36(m,5H),4.03-4.05(m,1H),2.97-2.91(m,1H),2.46(t,J=7.6Hz,2H),2.42-2.36(m,2H),2.26-2.16(m,2H),2.05(s,3H),2.04-1.91(m,2H),1.80-1.74(m,2H),1.63(s,3H),1.561(s,3H),1.61-1.53(m,2H),1.37-1.32(m,4H),0.90(t,J=6.8Hz,3H).
经检测,化合物E1在水中溶解度达到2.7g/100ml水。
实施例2:化合物E2的合成
Figure PCTCN2022132572-appb-000055
1、合成路线
Figure PCTCN2022132572-appb-000056
2、合成方法
(1)化合物6的合成
将化合物5(10.5g,0.0229mol,实施例1制备)溶于100ml甲醇中,加入氢氧化钾(3.8g,0.0688mol),搅拌,加热回流1h。浓缩反应液,残余物用100ml乙酸乙酯洗涤,过滤。滤饼再加入200ml乙酸乙酯,用2N稀盐酸调pH=2,分液。有机相用饱和氯化钠洗涤,无水硫酸钠干燥,过滤,浓缩后得黄色油状化合物6(9g)。MS(ESI);431[M+H] +
(2)化合物7的合成
Figure PCTCN2022132572-appb-000057
化合物6(6.3g,0.0145mol)、蛋氨酸甲酯盐酸盐(11.7g,0.0586mol)、DMAP(7.15g,0.0586mol)溶于200ml二氯甲烷中,加入DCC(12g,0.0586mmol),室温反应过夜。过滤反应液,滤饼用50ml二氯甲烷洗涤,滤液先后用2N盐酸洗涤、饱和氯化钠洗涤、无水硫酸钠干燥,浓缩后柱层析(正己烷:二氯甲烷=5:1-1:1,正己烷:二氯甲烷:乙酸乙酯=1;1:0.1)得到黄色油状化合物7(6g)。MS(ESI):721[M+H] +
(3)化合物8的合成
Figure PCTCN2022132572-appb-000058
化合物7(10g,0.014mol,1eq)溶于80mlTHF中,加入氢氧化锂(1.7g,0.042mol,3eq)8ml溶液,室温下反应过夜。浓缩反应液,加入100ml乙醇带干一次,残余物用正己烷洗涤,浓缩后加入乙酸乙酯100ml,2N盐酸调pH=2,分液,饱和氯化钠洗涤,无水硫酸钠干燥,浓缩后得到黄色固体化合物8(10g)。MS(ESI):693[M+H] +
(4)化合物E2的合成
Figure PCTCN2022132572-appb-000059
将化合物8(10g,0.0144mol)溶于80ml乙醇中,碳酸氢钠(2.5g,0.0304mol,2.1mol)溶于20ml水中,混合搅拌,50℃反应1h。浓缩反应液,加入100ml乙醇并蒸干,残余物用20ml二氯甲烷洗涤并干燥得到类白色固体E2(8.1g)。MS(ESI):693[M+H] +1H-NMR(CD 3OD):6.48(s,2H),5.32(s,1H),4.62-4.55(m,4H),4.44-4.39(m,4H),4.13-4.18(m,1H),4.15((d,1H),3.01-2.95(m,1H),2.58-2.54(t,2H),2.42-2.38(t.4H),2.17-2.19(m,2H),2.03(s,6H),1.99-1.95(m,3H),1.84-1.81(t,2H),1.72(s,3H),1.66(s,3H),1.64-1.60(m,2H),1.39-1.33(m,4H),0.94-0.91(t,3H)
经检测,化合物E2在水中溶解度达到20g/100ml水。
实施例3:抗癫痫活性研究
1、实验动物和部分试剂
Wistar大鼠,SPF级别,体重240-260g,周龄6-8,雌性,购自上海斯莱克实验动物有限责任 公司。饲养环境温度保持在22±1℃,湿度保持在65-70%,12小时明/暗光照周期,可自由饮水。
PTZ,购自Sigma公司;丙戊酸钠,购自Sigma公司;无水乙醇,购自国药集团化学试剂有限公司。
2、实验分组
将实验动物随机分为4组:模型组、阳性药物组、化合物1组、化合物2组,每组8只。
3、实验方法
(1)腹腔给药造模
所有大鼠适应性饲养1周后,开始造模。用生理盐水新鲜配置1.75%的PTZ溶液,腹腔注射配制好的PTZ溶液,注射剂量为35mg/kg。隔日注射1次PTZ,注射PTZ后30min内观察大鼠行为学变化,记录发作分级。
实验过程中连续注射11次PTZ后,记录发作分级,所有大鼠已经连续5次出现2级或2级以上的记录的大鼠,大鼠连续注射11次PZT后所有大鼠模型成功。
(2)灌胃给药治疗
模型成功后,随机分组。开始治疗,所有Wistar大鼠称重,计算灌胃剂量。模型组灌胃等体积生理盐水;阳性药物组(丙戊酸钠灌胃100mg/kg);化合物1组(实施例1制备的化合物E1,灌胃,100mg/kg);化合物2组(实施例2制备的化合物E2,灌胃,100mg/kg)。
(3)Ono’s分级
在给药前、给药后第7天、14天,灌胃给药1h后腹腔注射配制好的PTZ溶液,注射剂量为35mg/kg。注射PTZ后30min内观察大鼠行为学变化。
记录给药前后的行为发作指标按照Ono’s分级,记录发作潜伏期,发作分级和发作持续时间。
表1 Ono’s分级
Ono’s分级 发作表现
0 无惊厥
1 点头或头部抽搐
2 全身肌肉阵挛
3 头部抽搐加前肢阵挛
4 阵挛惊厥加后肢站立
5 跌倒
6 全身强直-阵挛性惊厥
实验结束后,采集实验动物的脑、海马,-80℃冻存。
4、WB检测大鼠海马组织中凋亡相关蛋白Bax和Bcl-2的表达:
4.1试剂、抗体和仪器
实验所用部分试剂、抗体和仪器分别如表2-4所示。
表2 试剂
Figure PCTCN2022132572-appb-000060
Figure PCTCN2022132572-appb-000061
表3 抗体
Figure PCTCN2022132572-appb-000062
表4 仪器
Figure PCTCN2022132572-appb-000063
4.2样品准备
将海马组织剪成细小的碎片,按每20mg组织加入150-250μl裂解液的比例加入裂解液(裂解液中加入蛋白酶和磷酸酶抑制剂),匀浆器匀浆直至完全裂解。裂解后的样品4℃12000g离心15分钟,取上清,进行蛋白质定量后贮存于-80℃冰箱。
4.3蛋白定量
(1)标准曲线的绘制:取一块酶标板,按照下表加入试剂;
表5 试剂
孔号 1 2 3 4 5 6 7 8
蛋白标准液(μl) 0 2 4 6 8 12 16 20
去离子水(μl) 20 18 16 14 12 8 4 0
蛋白浓度(μg/μl) 0 0.05 0.1 0.15 0.2 0.3 0.4 0.5
(2)根据样品数量,将BCA试剂A与试剂B按体积比为50:1配制适量BCA工作液,充分混匀;
(3)各孔加入160μl BCA工作液;
(4)把酶标板放在振荡器上振荡30sec,37℃放置30分钟,然后在562nm下测定吸光度;以吸光值为横坐标,蛋白浓度(μg/μl)为纵坐标,绘出标准曲线;
(5)在酶标板中加入2μl待测蛋白和18μl PBS(稀释10倍),加入160μl BCA工作液,把酶标板放在振荡器上振荡30sec,37℃放置30分钟,然后在562nm下测定吸光度;
(6)根据所测样品的吸光值,在标准曲线上即可查得相应的蛋白浓度(μg/μl),乘以样品稀释倍数(10)即为样品实际浓度(单位:μg/μl)。
4.4上样及电泳
(1)上样
每孔上样量约为25μg蛋白,可以根据实验要求加大上样量。根据蛋白定量结果取所需蛋白加入适量上样缓冲液,沸水浴10min后离心取上清上样。将配制好的PAGE胶放入电泳槽中,加入适量电泳缓冲液,取下梳子用枪轻轻吹打加样孔,避免孔内有余胶残留影响上样。将准备好的样品用加样枪慢慢加到对应的孔内,注意勿溢出加样孔。
(2)电泳
一般浓缩胶80V 20分钟,分离胶120V 60分钟,可以根据具体实验要求调整电压和时间。当染料到 达胶底部时切断电源,停止电泳,进行下一步转膜。
转膜
转膜分为湿转和半干转,本实验选用半干转。
半干式转膜中,三明治的排列为:/滤纸/胶/膜/滤纸,用电转缓冲液浸湿后,直接置于电转仪的正负极之间。胶于负极而膜置于正极。半干式的电转缓冲液不同于湿式的电转缓冲液,推荐为:48mM Tris,39mM glycine,0.04%SDS,20%甲醇。25V转膜30分钟即可。转膜前将PVDF膜在甲醇中浸泡1-2分钟(NC膜在电转液中浸泡10-20分钟),再孵育于冰冷的电转缓冲液中5分钟,胶也需在冰冷的电转缓冲液中平衡3-5分钟,否则转膜时会导致条带变形。
膜上蛋白的检测
为检测转膜是否成功,可用丽春红染色,丽春红染色工作液:2%的丽春红贮备液1:10稀释,即加9倍的ddH 2O。
染色方法:将膜放入TBST洗一次,再置于丽春红染色工作液中,在室温下摇动染色5分钟,大量的水洗膜直至水变清无色蛋白条带清晰,(膜也可以用TBST或水重新洗后再进行染色)PVDF膜需用甲醇再活化后用TBST洗后进行封闭。
4.5膜的封闭及抗体孵育
(1)封闭:5%脱脂奶粉(检测磷酸化蛋白用BSA)室温封闭1小时或4℃过夜。
(2)一抗:根据说明书 Bcl-2 1:500 Bax 1:5000 GAPDH 1:30000稀释抗体,抗体加入封闭液中稀释到所需浓度,和膜室温孵育2小时或4℃孵育过夜。
(3)二抗:孵育一抗的膜用TBST洗涤3次,每次5min。随后根据用量,按照1:1000稀释HRP标记的二抗,与膜37℃孵育1h。用TBST洗涤3次,每次5min。
4.6显色
ECL化学发光检测:配制ECL发光液,根据用量,取ECL发光液A和B等量混匀,加在膜的正面暗室避光5分钟。倒掉显色液,用纸小心吸取显色液,在上面盖上一层平整的透明纸。将其放入成像系统中进行扫描。
5、试剂盒法检测大鼠大脑组织中γ-氨基丁酸和谷氨酸、甘氨酸、天冬氨酸的含量
5.1实验材料
实验所用试剂盒、仪器如下表所示。
表6 试剂盒信息
Figure PCTCN2022132572-appb-000064
表7 主要仪器及耗材
Figure PCTCN2022132572-appb-000065
5.2实验方法
实验操作及方法参考试剂盒说明书。
6、实验结果
(1)治疗前后PTZ点燃癫痫大鼠的癫痫发作持续时间如下表及图1所示。
表8 治疗前后PTZ点燃癫痫大鼠的癫痫发作持续时间
分组 治疗前持续时间/s 治疗7天持续时间/s 治疗14天持续时间/s
模型组 338.33±27.16 327.50±12.01 311.83±29.02
阳性药物组 347.25±44.10 200.29±18.43 178.71±16.96
化合物1组 359.83±14.41 290.50±66.93 299.67±39.25
化合物2组 358.57±40.33 280.17±65.01 250.33±48.64
结果显示:治疗前后PTZ点燃癫痫发作持续时间,通过对比数据发现治疗前的各组的持续时间没有变化,持续发作时间在350s左右;随着进入治疗阶段,各组出现明显差异,阳性药物组在治疗7天和14天出现了发作持续时间出现下降,且具有统计学意义。其他化合物组也在治疗7天出现和治疗前明显发作持续时间下降,具有统计学意义;其中化合物2在后续治疗第14天的发作持续时间和治疗第7天对比均出现发作持续时间下降具有统计学意义;化合物1在治疗第14天的发作持续时间和治疗第7天对比出现发作持续时间没有出现变化。
(2)治疗前后PTZ点燃癫痫大鼠的行为发作指标Ono’s分级如下表及图2所示。
表9 治疗前后PTZ点燃癫痫大鼠的行为发作指标Ono’s分级
分组 治疗前Ono’s分级 治疗7天Ono’s分级 治疗14天Ono’s分级
模型组 3.67±0.52 3.50±0.55 3.67±0.52
阳性药物组 3.38±0.52 3.43±0.53 3.57±0.79
化合物1组 3.17±0.41 3.33±0.52 3.33±0.52
化合物2组 3.29±0.49 3.17±0.41 3.50±0.84
结果显示:治疗前后的行为发作指标按照Ono’s分级记录显示,在造模完成后和治疗期间,注射PTZ进行癫痫点燃模型,发作Ono’s分级都是在等级3-4之间,各组之间没有明显差异。在治疗14天后,也未出现明显的组间差异。
实验过程中Ono’s分级记录的是大鼠癫痫发作的最高分级,在实验过程中癫痫发作是进程性过程,最高发作等级在癫痫发作期间都会有持续期。建议后续记录每个发作分级的持续时间。
实验过程中发现出现Ono’s分级在5-6的分级状态下,不采取措施,大多都会出现死亡,且如果没有出现死亡的大鼠接下来也会出现PTZ点燃情况下死亡现象。
(3)治疗前后PTZ点燃癫痫大鼠的癫痫发作潜伏期如下表及图3所示。
表10 治疗前后PTZ点燃癫痫大鼠的癫痫发作潜伏期
分组 治疗前潜伏期/s 治疗7天潜伏期/s 治疗14天潜伏期/s
模型组 99.14±15.52 87.67±16.82 90.00±22.73
阳性药物组 97.38±15.86 87.00±13.90 84.43±14.77
化合物1组 85.75±21.45 82.83±20.90 87.00±20.96
化合物2组 99.00±1914 80.50±17.51 81.17±21.31
结果显示:治疗前后PTZ点燃癫痫发作潜伏期,在治疗前和治疗期间统计PTZ点燃癫痫发作潜伏期的均值80s-100s,发作潜伏期时间数据表明PTZ点燃癫痫的时间在60s-120s之间。且各组之间没有明显相关性,而且随着时间变化同组之间也没有看到发作潜伏期时间明显变化趋势。
(4)WB检测大鼠海马组织中凋亡相关蛋白Bax和Bcl-2变化的结果如图4和5所示。
实验结果表明模型组大鼠海马组织中促进凋亡相关Bax蛋白出现高表达,而阳性药物组和模型组对比促进凋亡相关Bax蛋白出现了低表达,且具有统计学意义。化合物E1、化合物E2都出现Bax蛋白表达明显下调且具有统计学意义。
相反检测各组大鼠海马组织中抗凋亡的Bcl-2蛋白,实验结果表明模型组的Bcl-2蛋白和其他各组相比出现明显低表达,阳性药物组中Bcl-2蛋白和其他各组相比均出现高表达,而化合物组中化合物E1、化合物E2和模型组对比都出现明显的Bcl-2蛋白的高表达,且具有统计学意义。
(5)大鼠脑组织匀浆液中γ-氨基丁酸、谷氨酸、甘氨酸、天冬氨酸含量测定结果如下表及图6-9所示。
表11 大鼠脑组织匀浆液中γ-氨基丁酸、谷氨酸、甘氨酸、天冬氨酸含量测定结果
Figure PCTCN2022132572-appb-000066
Figure PCTCN2022132572-appb-000067
通过ELISA检测试剂盒检测脑组织匀浆中,兴奋性氨基酸:谷氨酸和天冬氨酸,抑制性神经递质:γ-氨基丁酸和甘氨酸含量。
ELISA实验结果表现,模型组脑组织匀浆中γ-氨基丁酸和甘氨酸含量低,谷氨酸和天冬氨酸含量高。
阳性药物和化合物组对比模型组,兴奋性氨基酸均出现表达量降低,均具有统计学意义;抑制性神经递质的γ-氨基丁酸和甘氨酸表达量对比模型组出现含量升高,且具有统计学意义。
7、实验总结
PTZ点燃大鼠癫痫模型大鼠治疗过程中,阳性药物和各化合物对大鼠治疗效果在PTZ点燃大鼠癫痫的持续时间上具有显著性差异;在潜伏期和癫痫模型程度上具有作用趋势,但没有显著性差异。
与模型组相比,2个化合物组治疗第7、14天的癫痫发作持续时间均明显减少,均具有显著性差异。其中治疗第7天,癫痫发作持续时间依次为化合物E2>化合物E1;治疗第14天,癫痫发作持续时间依次为化合物E2>化合物E1。
通过对比治疗第14天的大鼠海马组织中凋亡相关蛋白Bax和Bcl-2,和脑组织匀浆中,兴奋性氨基酸:谷氨酸和天冬氨酸,抑制性神经递质:γ-氨基丁酸和甘氨酸含量。
实施例4:对帕金森的治疗研究
1、实验动物和部分试剂
C57/BL6小鼠,SPF级别,体重18-20g,周龄6-8,雄性,购自上海斯莱克实验动物有限责任公司。饲养环境温度保持在22±1℃,湿度保持在65-70%,12小时明/暗光照周期,可自由饮水。
MPTP,购自Sigma公司;金刚烷胺,购自Sigma公司;无水乙醇,购自国药集团化学试剂有限公司。
2、实验分组
将实验动物随机分为5组:空白组、模型组、阳性药物组、化合物1组、化合物2组,每组8只。
3、实验方法
(1)购买6-8周龄SPF级的C57小鼠,适应性饲养一周,开始造模。
(2)每天腹腔注射MPTP(25mg/kg)每天1次,连续7天。空白组不做处理。
(3)造模完成后,开始给药。阳性药物组灌胃金刚烷胺(40mg/kg),受试物组(100mg/kg)灌胃确定好的药物剂量,每天灌胃1次,连续14天。
(4)先进行行为学检测:平衡木检测评分
一根长为80cm、宽为2.5cm的木条,水平固定在离台面10cm高度的地方,然后让动物在木条上行走。
能跳上平衡木条,可以自由行走但不跌倒:0分;能跳上平衡木条,动物在上面行走会跌下,但跌下的机会小于50%:1分;能跳上平衡木条,动物在上面行走会跌下,但跌下的机会大于50%:2分;动物在正常侧身体的帮助下可以跳上平衡木条;但是瘫痪侧的后肢不能够帮助身体向前移动:3分;动物无法在平衡木条上行走,但是可以坐在木条上:4分;将动物放在木条上,动物很快会掉落下来:5分。
(5)评分结束后,开始取材
迅速处死小鼠后,取小鼠大脑,分离小鼠大脑皮质(CP)。暴露小鼠大脑纹状体后,取纹状体进行称量后,按照比例添加PBS后,进行研磨匀浆。然后分离上清检测脑纹状体中多巴胺含量。
4、试剂盒法检测检测脑纹状体中多巴胺含量
4.1试剂盒和部分仪器如下表所示。
表12 试剂盒信息
Figure PCTCN2022132572-appb-000068
表13 主要仪器及耗材
Figure PCTCN2022132572-appb-000069
4.2蛋白定量
(1)标准曲线的绘制:取一块酶标板,按照下表加入试剂;
表14 试剂
孔号 1 2 3 4 5 6 7 8
蛋白标准液(μl) 0 2 4 6 8 12 16 20
去离子水(μl) 20 18 16 14 12 8 4 0
蛋白浓度(μg/μl) 0 0.05 0.1 0.15 0.2 0.3 0.4 0.5
(2)根据样品数量,将BCA试剂A与试剂B按体积比为50:1配制适量BCA工作液,充分混匀;
(3)各孔加入160μl BCA工作液;
(4)把酶标板放在振荡器上振荡30sec,37℃放置30分钟,然后在562nm下测定吸光度。以吸光值为横坐标,蛋白浓度(μg/μl)为纵坐标,绘出标准曲线;
(5)在酶标板中加入2μl待测蛋白和18μl PBS(稀释10倍),加入160μl BCA工作液,把酶标板放在振荡器上振荡30sec,37℃放置30分钟,然后在562nm下测定吸光度;
(6)根据所测样品的吸光值,在标准曲线上即可查得相应的蛋白浓度(μg/μl),乘以样品稀释倍数(10)即为样品实际浓度(单位:μg/μl);
(7)小鼠多巴胺检测实验操作及方法参考试剂盒说明书。
5、免疫组化测定脑黑质(SubN)中酪氨酸羟化酶(TH)细胞
5.1实验材料
表15 主要试剂
Figure PCTCN2022132572-appb-000070
表16 主要仪器及耗材
Figure PCTCN2022132572-appb-000071
Figure PCTCN2022132572-appb-000072
溶液配制
(1)PBS溶液
600ml的ddH 2O中溶解8g NaCl、0.2g KCl、1.44g Na 2HPO 4和0.24g KH 2PO 4,用HCl调节溶液pH至7.4,定容至1L,滤器过滤后,高压灭菌,室温保存。
(2)0.1mol/L枸橼酸酸钠缓冲溶液
0.1mol/L柠檬酸3.8ml、0.1mol/L柠檬酸钠16.2ml。
柠檬酸C 6H 8O 7·H 2O:分子量210.14;0.1mol/L溶液为21.01克/升。
柠檬酸钠Na 3C 6H 5O 7·2H 2O:分子量294.12;0.1mol/L溶液为29.41克/毫升。
5.2实验方法
5.2.1样本包埋与固定
(1)取材和固定
切取组织时应使用锋利的刀、剪,切取组织块时,从刀的根部开始向后拉动切开组织。组织块的厚度约为0.2~0.3cm,大小为1.5cm×1.5cm×0.3cm为宜。取好的组织块置于10%福尔马林中固定48小时。
(2)洗涤和脱水
将固定后的组织用流水冲洗,去除残留的固定液和杂质。不同浓度的乙醇逐级脱水,50%、70%、85%、95%直至纯酒精(无水乙醇),每级2小时。脱水必须在有盖的瓶中进行,以防止高浓度乙醇吸收空气中水分导致浓度降低而使脱水不彻底。需要保存的材料可脱水至70%乙醇时停留其中,如需长期保存,可加入等量的甘油。
(3)透明
将组织块置入纯乙醇和二甲苯的等体积混合液中2小时,再进入纯二甲苯两小时,再一次纯二甲苯两小时;材料经过透明,会显示出前一步脱水的效果如何,若脱水彻底,组织则显现透明状态,如组织中有白色云雾状,说明脱水不净,须返工处理,但返工的效果往往不好。使用二甲苯透明时,应避免其挥发和吸收空气中的水分,并保持其无水状态。
(4)浸蜡
浸蜡须在恒温箱中进行。先把组织材料块放在熔化的石蜡和二甲苯的等量混合液浸渍1-2小时,再先后移入2个熔化的石蜡液中浸渍各3小时左右。
(5)包埋
包埋是使浸透蜡的组织块包裹在石蜡中。具体做法是:先准备好纸盒,将熔蜡倒入盒内,迅速用预温的镊子夹取组织块平放在纸盒底部,切面朝下,再轻轻提起纸盒,平放在冷水中,待表面石蜡凝固后立即将纸盒按入水中,使其迅速冷却凝固,30min后取出。
(6)切片
切片前将蜡块在-20℃冰箱中至少放置30min,以增加硬度。将切片刀装在刀片机的刀架上并固定紧,固定蜡块底座或蜡块,调整蜡块与刀至合适位置,刀刃与蜡块表面呈5度角。调整切片机上的切片厚度为4-7μm,然后切片。
5.2.2免疫组化染色
(1)烤片和脱蜡
将玻片置于65℃恒温烘箱中烤片30min;置于二甲苯I中浸泡15min,再置于二甲苯II中浸泡15min。
(2)水化
将脱蜡后的切片经100%酒精、95%酒精、85%酒精、75%酒精分别浸泡5min,自来水冲洗10min。
(3)抗原修复
采用0.01M柠檬酸钠缓冲溶液中高压修复15min,自然冷却后,0.02M PBS洗3min×3次。
(4)阻断
将玻片置于3%H2O2中,湿盒孵育10min,以消除内源性过氧化物酶的活性。0.02M PBS冲洗3min×3次。
(5)一抗孵育
滴加一抗(1:2000稀释),湿盒孵育,室温下放置1h(或者4℃孵育过夜)。0.02M PBS冲洗 3min×3次。
(6)二抗孵育
滴加HRP标记广谱二抗,湿盒孵育,室温下放置20min-30min。0.02M PBS冲洗3min×3次。
(7)显色
DAB染色,观察到切片有颜色改变时,立即用自来水洗去染色液。
(8)苏木素染色
苏木素复染3min,1%盐酸酒精分化,显微镜下观察,控制染色程度。自来水冲洗10min,放入65℃烘箱中烘干水分。
(9)透明和封片
将玻片置于二甲苯中透明3min×2次,中性树胶封片,放入65℃烘箱中15min。
5.2.3图像采集和分析
通过显微镜拍照,采集分析样本相关部位,计算阳性面积。
6、实验结果
(1)帕金森模型行为学平衡木评分结果如下表及图10所示。
表17 帕金森模型行为学平衡木评分结果
分组 造模后 治疗后
空白组 0 0
模型组 4.63±0.52*** 4.50±0.53
阳性药物组 4.75±0.46*** 2.13±0.83###
化合物1组 4.63±0.52*** 3.75±0.89#
化合物2组 4.13±0.35*** 3.50±0.93##
小鼠通过腹腔注射MPTP进行帕金森模型制备,通过平衡木评分进行行为学评价,MPTP腹腔注射7天后,进行平衡木评分,除空白组不进行造模,平衡木评分为0,其他各组小鼠的评分均在5-6分,各组对比空白组P≤0.05,具有统计学意义,证明模型成功。
后续通过阳性药物和各化合物组进行治疗后,模型组的平衡木评分4-5分,阳性药物平衡木评分在2-3分之间。其他化合物治疗组平衡木评分是3-4分。阳性药物组和各组化合物对比模型组出现明显下降趋势,P≤0.05,具有统计学意义。
治疗后各组评分为空白组<阳性药物组<化合物2组<化合物1组<模型组。
(2)帕金森模型治疗后纹状体多巴胺含量如下表及图11所示。
表18 帕金森模型治疗后纹状体多巴胺含量
分组 DA(ng/mg)
空白组 0.71±0.16
模型组 0.30±0.07***
阳性药物组 0.61±0.12###
化合物1组 0.45±0.09##
化合物2组 0.38±0.08#
多巴胺作为神经递质调控中枢神经系统的多种生理功能,通过检测治疗小鼠帕金森模型纹状体中多巴胺含量。判断药物治疗帕金森模型效果。
通过各组和空白组多巴胺含量进行对比判断模型中多巴胺下降程度,其中除阳性药物组外,其他各组的多巴胺含量均出现明显下降,P≤0.05,具有统计学意义。
再通过其他化合物组对比模型组多巴胺含量,各组的多巴胺含量对比模型组都出现增高,P≤0.01,具有统计学意义,表明各药物组具有升高脑内多巴胺含量的作用。
纹状体多巴胺含量是空白组>阳性药物组>化合物1组>化合物2组>模型组。
(3)免疫组化测定脑黑质(SubN)中酪氨酸羟化酶(TH)细胞的实验结果如下表及图12和13所示。
表19 免疫组化测定脑黑质(SubN)中酪氨酸羟化酶(TH)细胞的实验结果
分组 TH细胞阳性率(%)
空白组 34.27±5.08
模型组 12.74±1.71***
阳性药物组 20.82±5.77##
化合物1组 16.92±3.13##
化合物2组 15.18±3.37
酪氨酸羟化酶(TH)是一种单加氧酶,它是催化生物体自身合成左旋多巴胺(L-Dopamine,DA)系列反应的第一步反应的限速酶,仅在胞浆表达。神经元中TH含量丰富,因中脑缺乏多巴胺-β-羟化酶,故中脑TH免疫反应阳性神经元为DA神经元,可作为脑内多巴胺神经元的标志。机体利用左旋酪氨酸,在酪氨酸羟化酶的催化下生存左旋多巴,左旋多巴在芳香族脱羧酶的催化下脱去羧基最后生成左旋多巴胺。由于酪氨酸羟化酶在多巴胺合成中的重要地位,其功能的缺失或表达不足直接影响多巴胺的合成与分泌。多巴胺是一种重要的神经递质,多巴胺能神经元对多巴胺不能合成或分泌不足会导致帕金森氏病。
本实验结果显示,通过腹腔注射MPTP造成的小鼠帕金森模型中,小鼠的脑黑质(SubN)中酪氨酸羟化酶(TH)细胞阳性率出现降低,P≤0.05,具有统计学意义。阳性药物组对比模型组的脑黑质(SubN)中酪氨酸羟化酶(TH)细胞阳性率,出现明显升高现象,表明造模成功。
对比各化合物对小鼠帕金森模型治疗后,通过各化合物组对比模型组的脑黑质(SubN)中酪氨酸羟化酶(TH)细胞阳性率,都出现升高现象。其中化合物1组的脑黑质(SubN)中酪氨酸羟化酶(TH)细胞阳性率对比模型组,P≤0.01,且具有统计学意义。
通过对比各组的治疗后的脑黑质(SubN)中酪氨酸羟化酶(TH)细胞阳性率,空白组>阳性药物组>化合物1组>化合物2组>模型组。
7、实验总结
本次实验通过腹腔注射MPTP(25mg/kg)每天1次,连续7天,造模成小鼠帕金森模型,通过平衡木评分对小鼠帕金森模型进行筛选。
后续通过阳性药物和各化合物对模型小鼠进行灌胃治疗,连续灌胃治疗14天后,通过分析治疗后各组的平衡木评分,ELISA检测纹状体中多巴胺含量,免疫组化检测脑黑质(SubN)中酪氨酸羟化酶(TH)细胞阳性率等检测方法,鉴定各化合物和空白组,模型组差别,再进行统计学分析,对各化合物有效性进行分析。
通过治疗后平衡木评分鉴定,阳性药物组和各组化合物对比模型组出现明显下降趋势,P≤0.05,具有统计学意义。
再通过其他化合物组对比模型组多巴胺含量,各组的多巴胺含量对比模型组都出现增高,P≤0.01,具有统计学意义。
对比各化合物和阳性药物对小鼠帕金森模型治疗后,阳性药物组和各化合物对比模型组的阳性率,其中化合物1组的脑黑质(SubN)中酪氨酸羟化酶(TH)细胞阳性率对比模型组,P≤0.01,且具有统计学意义。
通过整体分析各化合物组对比模型组,均具有一定的治疗效果,对比各检测指标,其中化合物1具有较佳的治疗效果。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换等,均应包含在本发明的保护范围之内。
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。

Claims (10)

  1. 一种化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物,所述化合物具有如下结构:
    Figure PCTCN2022132572-appb-100001
    其中,
    X 1和X 2独立地选自:单键、取代或未取代的亚烷基、被一个或多个-C(=O)O-间隔的亚烷基、被一个或多个-OC(=O)-间隔的亚烷基、被一个或多个-C(=O)NH-间隔的亚烷基;
    X 3选自:
    Figure PCTCN2022132572-appb-100002
    R 1选自:H、烷基、环烷基;
    R 2选自:OH、烷氧基;
    n为1-5的整数;
    R 3选自:H、
    Figure PCTCN2022132572-appb-100003
    其中,X 4和X 5独立地选自:单键、取代或未取代的亚烷基、被一个或多个-C(=O)O-间隔的亚烷基、被一个或多个-OC(=O)-间隔的亚烷基、被一个或多个-C(=O)NH-间隔的亚烷基;
    X 6选自:
    Figure PCTCN2022132572-appb-100004
    R 4选自:H、烷基、环烷基;
    R 5选自:OH、烷氧基;
    m为1-5的整数。
  2. 如权利要求1所述的化合物,其特征在于,X 1和X 4独立地选自:单键、C1-6直链或支链亚烷基;
    优选地,X 1为亚甲基;
    优选地,X 4为亚甲基。
  3. 如权利要求1所述的化合物,其特征在于,X 2具有如下结构:
    Figure PCTCN2022132572-appb-100005
    其中R 7选自:H、烷基、被烷基巯基取代的烷基、被羟基取代的烷基、被巯基取代的烷基、被-C(=O)NH 2取代的烷基、被羧基取代的烷基、被氨基取代的烷基、被
    Figure PCTCN2022132572-appb-100006
    取代的烷基、杂环基烷基、芳烷基;
    优选地,R 7选自:H、甲基、异丙基、仲丁基、异丁基、
    Figure PCTCN2022132572-appb-100007
    Figure PCTCN2022132572-appb-100008
    Figure PCTCN2022132572-appb-100009
  4. 如权利要求1所述的化合物,其特征在于,X 5具有如下结构:
    Figure PCTCN2022132572-appb-100010
    其中R 8选自:H、烷基、被烷基巯基取代的烷基、被羟基取代的烷基、被巯基取代的烷基、被-C(=O)NH 2取代的烷基、被羧基取代的烷基、被氨基取代的烷基、被
    Figure PCTCN2022132572-appb-100011
    取代的烷基、杂环基烷基、芳烷基;
    优选地,R 8选自:H、甲基、异丙基、仲丁基、异丁基、
    Figure PCTCN2022132572-appb-100012
    Figure PCTCN2022132572-appb-100013
  5. 如权利要求1所述的化合物,其特征在于,R 2和R 5独立地选自:OH、C1-6烷氧基;
    优选地,R 2为OH、甲氧基或乙氧基;
    优选地,R 5为OH、甲氧基或乙氧基。
  6. 如权利要求1所述的化合物,其特征在于,所述化合物具有如下结构:
    Figure PCTCN2022132572-appb-100014
  7. 如权利要求1所述的化合物,其特征在于,所述立体异构体具有如下结构:
    Figure PCTCN2022132572-appb-100015
  8. 如权利要求7所述的化合物,其特征在于,所述立体异构体具有如下结构:
    Figure PCTCN2022132572-appb-100016
  9. 一种药物组合物,其包含权利要求1-8任一项所述的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物,以及一种或多种药学上可接受的辅料。
  10. 权利要求1-8任一项所述的化合物或其药学上可接受的盐、立体异构体、酯、前药、溶剂化物在制备预防和/或治疗疾病的药物中的应用,其中,所述疾病选自:神经系统疾病、癌症、自身免疫性疾病、心血管疾病、疼痛、炎症、肝损伤中的一种或多种;
    优选地,所述神经系统疾病选自:癫痫、多发性硬化症、帕金森氏病、阿尔茨海默症、路易体痴呆、亨廷顿病、焦虑症、抑郁症;
    优选地,所述癌症选自:乳腺癌、肺癌、结直肠癌、肝癌、胰腺癌、胃癌、胃食管腺癌、食管癌、小肠癌、贲门癌、子宫内膜癌、卵巢癌、输卵管癌、外阴癌、睾丸癌、前列腺癌、白血病、神经胶质瘤;
    优选地,所述自身免疫性疾病选自:系统性红斑狼疮、类风湿关节炎、多发性硬化症、溃疡性结肠炎、克罗恩氏病、自身免疫性肝炎。
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