Carbazole derivative and use thereof

Abstract

The present invention belongs to the technical field of medicine. Disclosed are a pyranocarbazole derivative and a preparation method therefor, and a pharmaceutical composition thereof and the use thereof. Specifically, disclosed in the present invention are pyranocarbazole derivatives as represented by general formulas I and II. The derivative is prepared by means of artificial synthesis. Further disclosed are a pharmaceutical composition containing the derivative, and the use thereof in resisting inflammation, resisting ischemic cerebral injury, relieving pain, resisting brain trauma, treating post-stroke depression, treating vascular dementia and cerebral small vessel disease, resisting Alzheimer's disease, and treating amyotrophic lateral sclerosis.

Description

Carbazole derivatives and uses thereof TECHNICAL FIELD

[0001] The present application relates to the field of natural medicine and medicinal chemistry, in particular to a class of pyrano-carbazole oxime and pyrano-carbazole nitro derivatives. The present application also discloses the preparation method, pharmaceutical composition and use in treating diseases of the derivatives. BACKGROUND

[0002] Pyrano-carbazole alkaloids have a wide range of biological activities. The present application introduces oxime and nitro on the pyrano-carbazole alkaloid mother nucleus to obtain a series of derivatives. Pharmacological experiments confirm that the derivatives have the following activities: (1) the derivatives can significantly inhibit the production of NO by LPS-stimulated Raw264.7 macrophages, suggesting that they have significant anti-inflammatory activity; (2) the derivatives can significantly reduce the volume of cerebral infarction and improve the neurological function, suggesting that they have significant anti-cerebral ischemia activity; (3) the derivatives have strong analgesic effect and can be developed into new analgesics with strong analgesic effect, no tolerance and addiction, and small side effects for chronic pain such as inflammatory pain, neuropathic pain and cancer pain; (4) the derivatives can significantly improve the neurological function of experimental animals, suggesting that they have obvious anti-brain injury activity; (5) the derivatives can significantly reduce the depressive behavior of animals and improve the neurological function, suggesting that they have significant post-stroke depression treatment activity; (6) the derivatives can significantly reduce the expression of various inflammatory factors in microglial cells after LPS stimulation, have significant anti-inflammatory ability, and can significantly increase the protein content of synaptophysin in the brain of chronic cerebral hypoperfusion model rats, suggesting that the derivatives can treat vascular dementia and cerebral small vessel disease by improving neuroinflammation and synaptic plasticity; (7) the derivatives can significantly improve the cognitive dysfunction of APP / PS1 mice and reduce the related pathological changes of Alzheimer's disease; (8) the derivatives can significantly improve the survival rate of SOD1 G93A and TDP43 M337V cells, suggesting that they can be used for preventing or treating amyotrophic lateral sclerosis. SUMMARY

[0003] The technical problem solved by the present application is to provide a class of carbazole derivatives and uses thereof.

[0004] The application discloses a kind of compounds of general formula I and general formula II, with the following pharmacological activity: (1) the compound of the present application has obvious improvement effect on the inflammatory response caused by lipopolysaccharide (LPS) stimulation macrophage Raw264.7 by pharmacological experiment.Therefore, the compound of the present application can be used for preventing and / or treating various inflammatory diseases, including but not limited to dermatitis, encephalitis, keratitis, conjunctivitis, rhinitis, otitis media, gingivitis, pharyngitis, tonsillitis, pneumonia, hepatitis, enteritis, dysentery, prostatitis, endometritis, cervicitis, pelvic inflammatory disease, paronychia, rheumatoid arthritis, asthma, psoriasis, etc.(2) The compound and its preparation of the present application have obvious improvement effect on the cerebral infarction volume and neurological behavior function of the rat acute cerebral ischemia model caused by middle cerebral artery occlusion.Therefore, the compound of the present application can be used for preventing and / or treating stroke.(3) The compound and its preparation of the present application have obvious improvement effect on the pain symptoms of mice.Therefore, the compound of the present application can be used for preparing pain prevention / treatment drugs.(4) The compound and its preparation of the present application have obvious improvement effect on the neurological behavior function of the rat traumatic brain injury (WDI) model caused by heavy object falling.Therefore, the compound of the present application can be used for preventing and / or treating brain injury.(5) The compound and its preparation of the present application have obvious improvement effect on the depression-like behavior of male rat bilateral common carotid artery ligation model.Therefore, the compound of the present application can be used for preventing and / or treating post-stroke depression.(6) The compound and its preparation of the present application can obviously improve neuroinflammation and regulate synaptic function, and have obvious improvement effect on the cognitive function defect caused by chronic cerebral hypoperfusion model constructed by bilateral common carotid artery occlusion operation.Therefore, the compound of the present application can be used for preventing and / or treating vascular dementia and cerebral small vessel disease.(7) The compound and its preparation of the present application have obvious improvement effect on the cognitive dysfunction and pathological characteristics of APP / PS1 mice.Therefore, the compound of the present application can be used for preventing and / or treating Alzheimer's disease.(8) The compound of the present application can significantly improve the survival rate of SOD1 G93A and TDP43 M337V cells.Therefore, the compound of the present application can be used for preventing or treating amyotrophic lateral sclerosis.

[0005] To solve the technical problems of the present application, the present application provides the following technical solutions:

[0006] The first aspect of the technical scheme of the present application is to provide a kind of pyrano-carbazole oxime derivatives or its pharmaceutically acceptable salt shown in general formula I:

[0007] R1, R2, independently of each other, can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, OCF3;

[0008] R3is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3.

[0009] The present application relates to a class of pyrano-carbazole nitro derivatives or its pharmaceutically acceptable salt represented by general formula II:

[0010] R1, R2, independently of each other, can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, OCF3;

[0011] R3is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3.

[0012] R4is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, C(CH3)3, Ph, Bn.

[0013] In the art, Bu t , t-Bu and C(CH3)3 all represent tert-butyl.

[0014] The structure and number of some compounds of the present application are as follows:

[0015] The second aspect of the technical scheme of the present application is to provide the compound represented by general formula I of the present application, which can be prepared by the following method:

[0016] Among them, the reaction condition (a) is hydroxylamine hydrochloride, 1 mol / L sodium hydroxide solution, the solvent is ethanol, and the reaction is carried out at room temperature.

[0017] The compound represented by general formula II of the present application can be prepared by the following method: The compound represented by general formula II of the present application can be prepared by the following method:

[0018] wherein the reaction condition (b) is R4NHOH, triethylamine, sodium sulfate, solvent ethanol, heating reaction at 90℃.

[0019] The preparation methods of intermediates 1 and 2 are described in “European Journal of Medicinal Chemistry. 2020, 190, 112079”.

[0020] A third aspect of the present application provides a pharmaceutical composition comprising the compound of the first aspect as an active ingredient. The pharmaceutical composition is prepared according to methods known in the art. The compound of the present application can be combined with one or more solid or liquid excipients and / or adjuvants that are pharmaceutically acceptable, and formulated into any dosage form suitable for human or animal use. The content of the compound of the present application in the pharmaceutical composition is usually 0.1-95% by weight.

[0021] The compound of the present application or the pharmaceutical composition containing it can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0022] The administration dosage form can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form can be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type and multiple emulsion), a suspension, an injection (including water injection, powder injection and infusion), eye drops, nose drops, lotion and liniment, etc.; the solid dosage form can be a tablet (including ordinary tablet, enteric-coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, oral disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric-coated capsule), a granule, a powder, a micro-pellet, a dripping pill, a suppository, a film, a patch, an aerosol (powder) mist, a spray, etc.; the semi-solid dosage form can be an ointment, a gel, a paste, etc.

[0023] The compound of the present application can be prepared into ordinary preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various micro-particle drug delivery systems.

[0024] For the preparation of tablets of the compounds of the present application, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropyl alcohol, etc.; the binders can be starch paste, dextrin, sugar syrup, honey, glucose solution, microcrystalline cellulose, acacia paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic acid resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfate, etc.; the lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0025] The tablets can also be further prepared into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0026] For the preparation of capsules of the administration units, the effective ingredient, the compounds of the present application, can be mixed with diluents and glidants, and the mixture can be directly placed in hard capsules or soft capsules. The effective ingredient, the compounds of the present application, can also be first mixed with diluents, binders and disintegrants to prepare granules or pellets, and then placed in hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants and glidants used for the preparation of tablets of the compounds of the present application can also be used for the preparation of capsules of the compounds of the present application.

[0027] For the preparation of injections of the compounds of the present application, water, ethanol, isopropyl alcohol, propylene glycol or their mixtures can be used as solvents, and appropriate amounts of solubilizers, co-solubilizers, pH adjustors and osmotic pressure adjustors commonly used in the art can be added. The solubilizers or co-solubilizers can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc. The pH adjustors can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure adjustors can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If lyophilized powder injections are prepared, mannitol, glucose, etc. can also be added as supporting agents.

[0028] In addition, if necessary, coloring agents, preservatives, flavors, taste correctors or other additives can also be added to the pharmaceutical preparations.

[0029] For the purpose of medication and enhancement of therapeutic effects, the drugs or pharmaceutical compositions of the present application can be administered by any known administration method.

[0030] The dosage of the pharmaceutical composition of the compound of the present application can vary greatly depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration and the dosage form, etc. Generally, the suitable dosage of the compound of the present application per day is in the range of 0.001-150 mg / kg body weight, preferably 0.1-100 mg / kg body weight, more preferably 1-60 mg / kg body weight, and most preferably 2-30 mg / kg body weight. The above dosage can be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the physician and the administration regimen including the use of other therapeutic agents.

[0031] The compound or composition of the present application can be used alone or in combination with other therapeutic agents or symptomatic agents. When the compound of the present application has a synergistic effect with other therapeutic agents, the dosage thereof should be adjusted according to the actual situation.

[0032] The fourth aspect of the technical solution of the present application is to provide the use of the compound or pharmaceutically acceptable salt of the first aspect.

[0033] Specifically, the use of the compound or pharmaceutically acceptable salt of the first aspect in the preparation of an anti-inflammatory, prophylactic or therapeutic drug for stroke, prophylactic or therapeutic drug for pain, prophylactic or therapeutic drug for brain trauma, prophylactic or therapeutic drug for post-stroke depression, prophylactic or therapeutic drug for vascular dementia and cerebral small vessel disease, prophylactic or therapeutic drug for Alzheimer's disease, and prophylactic or therapeutic drug for amyotrophic lateral sclerosis is provided. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1: Effect of the compound on the content of NO produced by LPS-stimulated macrophage Raw264.7 (means ± S.E.M, n = 3).

[0035] Figure 2: Effect of compound CXT-24 on cerebral infarction volume after pMCAO in SD rats

[0036] Figure 3: Effect of compound CXT-24 on behavioral impairment after pMCAO in SD rats (Bederson score)

[0037] Figure 4: Effect of compound CXT-24 on behavioral impairment after pMCAO in SD rats (mNSS score)

[0038] Figure 5: Digital photos of cerebral infarction volume of compound CXT-24 pMCAO model rats

[0039] Figure 6: Effect of compound CXT-24 injection on cerebral infarction volume after pMCAO in SD rats.

[0040] Figure 7: Effect of compound CXT-24 injection on behavioral impairment after pMCAO in SD rats (Bederson score)

[0041] Figure 8: Effect of compound CXT-24 injection on behavioral impairment after pMCAO in SD rats (mNSS score)

[0042] Figure 9: Digital photos of cerebral infarction volume of pMCAO model rats treated with compound CXT-24 injection

[0043] Figure 10: Effect of compound CXT-24 on pain threshold in mice hot plate test

[0044] Figure 11: Effect of compound CXT-24 on pain latency in mice acetic acid writhing test

[0045] Figure 12: Effect of compound CXT-24 on writhing times in mice acetic acid writhing test

[0046] Figure 13: Effect of compound CXT-24 on motor function of rats after TBI modeling

[0047] Figure 14: Effect of compound CXT-24 on behavioral impairment of rats after TBI modeling (mNSS score)

[0048] Figure 15: Effect of compound CXT-24 on cell survival rate of hydrogen peroxide induced neuronal injury (n = 9)

[0049] Figure 16: Effect of compound CXT-24 on forced swimming struggle time of bilateral common carotid artery ligation SD rats

[0050] Figure 17: Effect of compound CXT-24 on center time of open field test of bilateral common carotid artery ligation SD rats

[0051] Figure 18: Effect of compound CXT-24 on center times of open field test of bilateral common carotid artery ligation SD rats

[0052] Figure 19: Effect of compound CXT-24 on center distance of open field test of bilateral common carotid artery ligation SD rats

[0053] Figure 20: Body weight changes of male SD rats after three weeks of compound CXT-24 administration

[0054] Figure 21: Flow chart for verification of therapeutic effect of compound CXT-24 on chronic cerebral hypoperfusion model in rats

[0055] Figure 22: Effect of compound CXT-24 on swimming path and latency of rats in Morris water maze directional navigation experiment of chronic cerebral hypoperfusion model

[0056] Figure 23: Effect of compound CXT-24 on rats in Morris water maze spatial exploration experiment and visible platform experiment of chronic cerebral hypoperfusion model

[0057] Figure 24: Effect of compound CXT-24 on the novel object recognition index of rats in chronic cerebral hypoperfusion model

[0058] Figure 25: Effect of compound CXT-24 on the synaptophysin protein content in the brain tissue of rats in chronic cerebral hypoperfusion model

[0059] Figure 26: Effect of compound CXT-24 on the release of inflammatory factors IL-6 and TNF-a from microglial cells after LPS stimulation

[0060] Figure 27: Effect of compound CXT-24 on the transcriptional level of inflammatory factors IL-6, IL-1 β, TNF-a, iNOS and COX-2 from microglial cells after LPS stimulation

[0061] Figure 28: Compound CXT-24 can improve the cognitive dysfunction of APP / PS1 mice in Morris water maze test and step-down test. A: Effect of CXT-24 on the escape latency of APP / PS1 mice in the place navigation phase of Morris water maze test. B: Effect of CXT-24 on the latency to cross the platform of APP / PS1 mice in the spatial probe phase of Morris water maze test. C: Effect of CXT-24 on the number of crossing the platform of APP / PS1 mice in the spatial probe phase of Morris water maze test. D: Effect of CXT-24 on the latency to jump off the insulating platform of APP / PS1 mice in the step-down test. E: Effect of CXT-24 on the number of errors of APP / PS1 mice in the step-down test.

[0062] Figure 29: Compound CXT-24 can reduce the deposition of Aβ plaques and neuroinflammation in the cerebral cortex and hippocampus of APP / PS1 mice.

[0063] Figure 30: Compound CXT-24 can reduce the release of inflammatory factors from BV2 cells stimulated by LPS + Aβ1-42.

[0064] Figure 31: Compound CXT-24 and its derivatives can improve the survival of SKN cells under Aβ1-42 damage conditions.

[0065] Figure 32: 0.1-10 mM CXT-24 and CXT-5 improve survival of SOD1 G93A stable cells and TDP43 M337V stable cells. A: MTT assay to detect the difference in proliferation between empty vector control pEGFP stable cells and SOD1 G93A stable cells, and the effect of 0.1-10 mM CXT-24 and CXT-5 on the survival of SOD1 G93A stable cells after being given to SOD1 G93A stable cells for 24 hours. B: MTT assay to detect the difference in proliferation between empty vector control pCI stable cells and TDP43 M337V stable cells, and the effect of 0.1-10 mM CXT-24 and CXT-5 on the survival of TDP43 M337V stable cells after being given to TDP43 M337V stable cells for 24 hours. "#" indicates compared with the empty load group, wherein #P<0.05, ##P<0.01, "*" indicates compared with the model group, wherein *P<0.05, **P<0.01. DETAILED DESCRIPTION

[0066] Synthetic experiment:

[0067] Example 1 Preparation of CXT-1

[0068] Compound 1 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then tert-butyl hydroxylamine (53.5 mg, 2 eq), triethylamine (60.7 mg, 2 eq), anhydrous sodium sulfate (128 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), and the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 82 mg at a yield of 67.8%. 1 H NMR (800 MHz, Acetone) δ 10.58 (s, 1H), 10.15 (s, 1H), 8.17 (s, 1H), 8.04 (d, J = 1.9 Hz, 1H), 7.41 (dd, J = 8.4, 1.9 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 6.96 (d, J = 9.8 Hz, 1H), 5.83 (d, J = 9.8 Hz, 1H), 1.62 (s, 9H), 1.51 (s, 6H), 1.41 (s, 9H). 13C NMR (200 MHz, Acetone) δ 150.22, 143.42, 139.51, 138.86, 129.94, 124.76, 123.97, 123.46, 120.30, 118.34, 117.79, 116.61, 114.10, 111.21, 105.07, 77.58, 70.74, 35.25, 32.37, 28.54, 27.76. ESI-MS (m / z): 404.2 [M+H] + .

[0069] Example 2 Preparation of CXT-2

[0070] Compound 2 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then methyl hydroxylamine (28.2 mg, 2 eq), triethylamine (60.7 mg, 2 eq), anhydrous sodium sulfate (128 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 73 mg at a yield of 67%. 1 H NMR (800 MHz, Acetone) δ 10.05 (s, 1H), 8.02 (d, J = 1.4 Hz, 3H), 7.42 (dd, J = 8.4, 1.1 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 6.95 (dd, J = 9.8, 2.0 Hz, 1H), 5.82 (d, J = 9.7 Hz, 1H), 3.88 (s, 3H), 1.49 (s, 6H), 1.41 (s, 9H). 13 C NMR (200 MHz, Acetone) δ 149.82, 143.46, 139.40, 138.94, 130.11, 129.37, 124.65, 123.57, 120.05, 118.24, 117.82, 116.52, 113.88, 111.19, 105.07, 77.64, 54.75, 35.24, 32.34, 27.68. ESI-MS (m / z): 362.2 [M+H] + .

[0071] Example 3 Preparation of CXT-3

[0072] Compound 3 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then benzyl hydroxylamine (74 mg, 2 eq), triethylamine (60.7 mg, 2 eq), anhydrous sodium sulfate (128 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 92 mg at a yield of 70%. 1 H NMR (800 MHz, Acetone) δ 10.58 (s, 1H), 10.04 (s, 1H), 8.23 (s, 1H), 8.00 (s, 1H), 7.59 (d, J = 7.1 Hz, 1H), 7.40 (t, J = 7.4 Hz, 1H), 7.38 - 7.32 (m, 3H), 6.94 (d, J = 9.8 Hz, 1H), 5.80 (d, J = 9.8 Hz, 1H), 5.17 (s, 3H), 1.47 (s, 6H), 1.39 (s, 9H). 13 C NMR (200 MHz, Acetone) δ 150.07, 143.46, 139.50, 139.04, 136.27, 130.08, 129.95, 129.26, 129.09, 129.03, 124.61, 123.54, 120.18, 118.20, 117.87, 116.59, 113.67, 111.21, 105.13, 77.61, 71.45, 35.21, 32.32, 27.66. ESI-MS (m / z): 438.2 [M+H] + .

[0073] Example 4 Preparation of CXT-4

[0074] Compound 4 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then hydroxylamine hydrochloride (50.4 mg, 2 eq), 1M NaOH solution (0.6 ml, 2 eq) were added thereto, and stirring was performed at room temperature for 5 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 90 mg at a yield of 86%. 1HNMR (800 MHz, Acetone) δ 10.35 (s, 1H), 10.01 (s, 1H), 8.55 (s, 1H), 8.36 (s, 1H), 8.10 (d, J = 1.9 Hz, 1H), 7.42 (dd, J = 8.4, 1.9 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 9.8 Hz, 1H), 5.83 (d, J = 9.7 Hz, 1H), 1.50 (s, 6H), 1.42 (s, 9H). 13 C NMR (200 MHz, Acetone) δ 150.22, 145.66, 143.26, 139.66, 138.97, 130.35, 124.27, 123.74, 118.98, 118.17, 117.24, 116.77, 114.26, 111.10, 105.50, 77.29, 35.20, 32.31, 27.75. ESI-MS (m / z): 348.1 [M+H] + .

[0075] Example 5 Preparation of CXT-5

[0076] Compound 5 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then phenylhydroxylamine (64.5 mg, 2 eq), triethylamine (60.7 mg, 2 eq), anhydrous sodium sulfate (128 mg, 3 eq) were added, heated and stirred at 90 degrees for 2 hours, and the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) three times, combined the ethyl acetate layer, dried with anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to obtain a crude product. The crude product was column chromatographed on silica gel to obtain a yellow powder 86 mg, with a yield of 67.7%. ESI-MS (m / z): 424.2 [M+H] + .

[0077] Example 6 Preparation of CXT-6

[0078] Compound 6 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then tert-butylhydroxylamine (64.3 mg, 2 eq), triethylamine (73 mg, 2 eq), anhydrous sodium sulfate (154 mg, 3 eq) were added, heated and stirred at 90 degrees for 2 hours, and the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) three times, combined the ethyl acetate layer, dried with anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to obtain a crude product. The crude product was column chromatographed on silica gel to obtain a yellow powder 76 mg, with a yield of 60.3%. 1H NMR (800 MHz, Acetone) δ 10.66 (s, 1H), 10.15 (s, 1H), 8.17 (s, 1H), 7.98 (d, J = 7.7 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.30 (t, J = 7.0 Hz, 1H), 7.16 (t, J = 7.4 Hz, 1H), 6.97 (d, J = 9.8 Hz, 1H), 5.84 (d, J = 9.8 Hz, 1H), 1.61 (s, 9H), 1.51 (s, 6H). 13 C NMR (200 MHz, Acetone) δ 150.36, 141.39, 138.41, 130.09, 125.61, 124.96, 123.78, 120.31, 118.25, 117.46, 114.40, 111.71, 105.10, 77.64, 70.79, 28.53, 27.74. ESI-MS (m / z): 348.1 [M+H] + .

[0079] Example 7 Preparation of CXT-7

[0080] Compound 7 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then methyl hydroxylamine (34 mg, 2 eq), triethylamine (73 mg, 2 eq), anhydrous sodium sulfate (154 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 68 mg at a yield of 61.9%. 1 H NMR (800 MHz, MeOD) δ 9.70 (s, 1H), 8.10 (s, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 7.16 (t, J = 7.4 Hz, 1H), 6.82 (d, J = 9.7 Hz, 1H), 5.79 (dd, J = 9.8, 3.8 Hz, 1H), 3.89 (s, 4H), 1.50 (s, 6H). 13C NMR (200 MHz, MeOD) δ 151.65, 142.16, 140.27, 135.72, 130.59, 126.34, 124.96, 121.74, 120.99, 120.43, 118.36, 118.12, 112.09, 111.94, 105.53, 78.58, 53.82, 27.76. ESI-MS (m / z): 306.1 [M+H] + .

[0081] Example 8 Preparation of CXT-8

[0082] Compound 8 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then benzyl hydroxylamine (88.9 mg, 2 eq), triethylamine (73 mg, 2 eq), anhydrous sodium sulfate (154 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 93 mg at a yield of 67.9%. 1 H NMR (800 MHz, Acetone) δ 10.63 (s, 1H), 10.03 (s, 1H), 8.20 (s, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.59 (d, J = 6.9 Hz, 1H), 7.44 - 7.39 (m, 2H), 7.37 (t, J = 7.4 Hz, 1H), 7.31 (td, J = 7.6, 1.2 Hz, 1H), 7.17 (td, J = 7.6, 1.0 Hz, 1H), 6.94 (d, J = 9.7 Hz, 1H), 5.83 (d, J = 9.8 Hz, 1H), 5.15 (s, 2H), 1.48 (s, 6H). 13 C NMR (200 MHz, Acetone) δ 150.19, 141.42, 138.56, 136.30, 130.26, 130.00, 129.28, 128.72, 125.72, 124.86, 120.61, 120.33, 118.11, 117.53, 114.07, 111.74, 105.16, 77.69, 71.52, 27.66. ESI-MS (m / z): 382.2 [M+H] + .

[0083] Example 9 Preparation of CXT-9

[0084] Compound 9 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then hydroxylamine hydrochloride (60.6 mg, 2 eq), 1 M NaOH solution (0.72 ml, 2 eq) were added, stirred at room temperature for 5 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) for three times, combined the ethyl acetate layer, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to get the crude product. The crude product was purified by silica gel column chromatography to get yellow powder 76 mg, yield 72%. ESI-MS (m / z): 292.1 [M+H] + .

[0085] Example 10 Preparation of CXT-10

[0086] Compound 10 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then phenyl hydroxylamine (78.7 mg, 2 eq), triethylamine (73 mg, 2 eq), anhydrous sodium sulfate (154 mg, 3 eq) were added, stirred at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) for three times, combined the ethyl acetate layer, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to get the crude product. The crude product was purified by silica gel column chromatography to get yellow powder 90 mg, yield 68%. ESI-MS (m / z): 368.1 [M+H] + .

[0087] Example 11 Preparation of CXT-11

[0088] Compound 11 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then tert-butyl hydroxylamine (51.6 mg, 2 eq), triethylamine (58.6 mg, 2 eq), anhydrous sodium sulfate (123 mg, 3 eq) were added, stirred at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) for three times, combined the ethyl acetate layer, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to get the crude product. The crude product was purified by silica gel column chromatography to get yellow powder 89 mg, yield 73%. ESI-MS (m / z): 416.2 [M+H] + .

[0089] Example 12 Preparation of CXT-12

[0090] Compound 12 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then benzyl hydroxylamine (71.3 mg, 2 eq), triethylamine (58.6 mg, 2 eq), anhydrous sodium sulfate (123 mg, 3 eq) were added to it, heated and stirred at 90 degrees for 2 hours, and the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) three times, combined the ethyl acetate layer, dried with anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 72 mg with a yield of 55%. ESI-MS (m / z): 450.2 [M+H] 1 H NMR (800 MHz, Acetone) δ 11.11 (s, 1H), 10.12 (s, 1H), 8.35 (s, 1H), 8.05 (s, 1H), 7.62 (s, 2H), 6.96 (d, J = 9.8 Hz, 1H), 5.87 (d, J = 9.8 Hz, 1H), 3.90 (s, 3H), 1.51 (s, 6H). 13 C NMR (200 MHz, Acetone) δ 150.66, 143.21, 139.13, 130.71, 129.06, 127.15, 125.80, 124.64, 122.37, 120.37, 117.78, 116.84, 115.10, 112.18, 105.53, 78.05, 54.86, 27.70. ESI-MS (m / z): 374.1 [M+H] + .

[0091] Example 13 Preparation of CXT-13

[0092] Compound 12 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then benzyl hydroxylamine (71.3 mg, 2 eq), triethylamine (58.6 mg, 2 eq), anhydrous sodium sulfate (123 mg, 3 eq) were added to it, heated and stirred at 90 degrees for 2 hours, and the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) three times, combined the ethyl acetate layer, dried with anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 72 mg with a yield of 55%. ESI-MS (m / z): 450.2 [M+H] + .

[0093] Example 14 Preparation of CXT-14

[0094] Compound 14 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then hydroxylamine hydrochloride (49 mg, 2 eq) was added thereto, 1 M NaOH solution (0.58 ml, 2 eq) was added, and stirring was performed at room temperature for 5 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), the ethyl acetate layers were combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to column chromatography on silica gel to obtain a yellow powder of 62 mg at a yield of 59.6%. 1 H NMR (800 MHz, Acetone) δ 10.14 (s, 1H), 8.54 (s, 1H), 8.46 (d, J = 8.3 Hz, 1H), 7.65-7.59 (m, 2H), 6.94 (d, J = 9.8 Hz, 1H), 5.89 (d, J = 9.8 Hz, 1H), 1.53 (s, 6H). 13 C NMR (200 MHz, Acetone) δ 151.18, 145.36, 143.43, 139.23, 131.08, 127.26, 125.92, 124.44, 122.68, 122.20, 118.19, 118.06, 117.83, 115.79, 112.12, 106.06, 77.82, 27.89. ESI-MS (m / z): 360.1 [M+H] + .

[0095] Example 15 Preparation of CXT-15

[0096] Compound 15 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then phenylhydroxylamine (63.2 mg, 2 eq) was added thereto, triethylamine (58.6 mg, 2 eq) was added, anhydrous sodium sulfate (123 mg, 3 eq) was added, and stirring was performed at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), the ethyl acetate layers were combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to column chromatography on silica gel to obtain a yellow powder of mg at a yield of %. ESI-MS (m / z): 436.1 [M+H] + .

[0097] Example 16 Preparation of CXT-16

[0098] Compound 16 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then tert-butyl hydroxylamine (49.3 mg, 2 eq), triethylamine (56 mg, 2 eq), anhydrous sodium sulfate (118 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 76 mg at a yield of 63.9%. 1 H NMR (800 MHz, Acetone) δ 10.17 (s, 1H), 8.17 (s, 1H), 8.08 (d, J = 1.9 Hz, 1H), 7.50 (dd, J = 8.5, 1.9 Hz, 1H), 7.43 (d, J = 8.5 Hz, 1H), 7.12 (d, J = 9.9 Hz, 1H), 5.88 (d, J = 9.9 Hz, 1H), 4.53 (q, J = 7.2 Hz, 2H), 1.61 (s, 9H), 1.51 (s, 6H), 1.46 (t, J = 7.2 Hz, 3H), 1.45 (s, 9H). 13 C NMR (200 MHz, Acetone) δ 150.87, 143.54, 140.18, 137.69, 129.82, 124.12, 123.43, 123.41, 120.10, 118.93, 118.19, 116.25, 114.30, 109.07, 105.88, 76.30, 70.60, 40.29, 35.07, 32.16, 28.35, 26.93, 15.04. ESI-MS (m / z): 432.3 [M+H] + .

[0099] Example 17 Preparation of CXT-17

[0100] Compound 16 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then tert-butyl hydroxylamine (49.3 mg, 2 eq), triethylamine (56 mg, 2 eq), anhydrous sodium sulfate (118 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 76 mg at a yield of 63.9%. 1H NMR (800 MHz, Acetone) δ 10.02 (s, 1H), 8.07 (s, 1H), 8.04 (d, J = 1.9 Hz, 1H), 7.49 (dd, J = 8.5, 2.0 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.09 (d, J = 9.9 Hz, 1H), 5.86 (d, J = 9.9 Hz, 1H), 4.50 (q, J = 7.2 Hz, 2H), 3.88 (s, 2H), 3.31 (s, 3H), 1.49 (s, 6H), 1.44 (t, J = 7.2 Hz, 3H), 1.41 (s, 9H). 13 C NMR (200 MHz, Acetone) δ 150.90, 143.80, 140.37, 138.13, 130.24, 124.08, 123.77, 120.39, 118.89, 118.44, 116.35, 113.72, 109.31, 106.09, 76.62, 54.54, 49.71, 40.43, 35.18, 32.27, 26.97, 15.19. ESI-MS (m / z): 390.2 [M+H] + .

[0101] Example 18 Preparation of CXT-18

[0102] Compound 18 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then benzyl hydroxylamine (68 mg, 2 eq), triethylamine (56 mg, 2 eq), anhydrous sodium sulfate (118 mg, 3 eq) were added, heated and stirred at 90 degrees for 2 hours, and the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) for three times, combined the ethyl acetate layer, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to get the crude product. The crude product was purified by silica gel column chromatography to get 86 mg of yellow powder, with a yield of 66.7%. 1 H NMR (800 MHz, Acetone) δ 9.80 (s, 1H), 8.02 (s, 1H), 7.83 (d, J = 1.9 Hz, 1H), 7.40 - 7.36 (m, 2H), 7.28 (dd, J = 8.5, 2.0 Hz, 1H), 7.23 - 7.18 (m, 3H), 7.18 - 7.14 (m, 1H), 6.89 (d, J = 9.9 Hz, 1H), 5.66 (d, J = 9.9 Hz, 1H), 4.95 (s, 2H), 4.30 (q, J = 7.2 Hz, 2H), 1.26 (s, 6H), 1.23 (t, J = 7.2 Hz, 3H), 1.21 (s, 9H). 13CNMR (200 MHz, Acetone) δ 151.00, 143.82, 140.36, 138.11, 136.16, 130.24, 130.02, 129.31, 129.28, 129.07, 124.11, 123.73, 120.32, 118.91, 118.47, 116.44, 113.81, 109.29, 106.11, 76.57, 71.37, 40.45, 35.20, 32.28, 26.99, 15.18. ESI-MS (m / z): 466.3 [M+H] + .

[0103] Example 19 Preparation of CXT-19

[0104] Compound 19 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then hydroxylamine hydrochloride (46.6 mg, 2 eq), 1 M NaOH solution (0.55 ml, 2 eq) were added, stirred at room temperature for 5 h, the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) for three times, the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to give the crude product. The crude product was purified by silica gel column chromatography to give a yellow powder 86 mg, yield 83%. 1 HNMR (800 MHz, Acetone) δ 10.17 (s, 1H), 8.17 (s, 1H), 8.08 (d, J = 1.8 Hz, 1H), 7.49 (dd, J = 8.5, 1.9 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.26 (d, J = 9.9 Hz, 1H), 5.81 (d, J = 9.9 Hz, 1H), 4.02 (s, 3H), 3.33 (d, J = 4.7 Hz, 2H), 1.62 (s, 9H), 1.51 (s, 6H), 1.44 (s, 9H). 13 C NMR (200 MHz, Acetone) δ 151.11, 143.59, 141.25, 139.04, 129.30, 124.02, 123.53, 120.22, 119.29, 118.11, 116.31, 114.33, 109.29, 106.43, 76.58, 70.77, 49.80, 35.23, 33.23, 32.35, 28.52, 27.19. ESI-MS (m / z): 376.2 [M+H] + .

[0105] Example 20 Preparation of CXT-20

[0106] Compound 20 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then benzyl hydroxylamine (68 mg, 2 eq), triethylamine (56 mg, 2 eq), anhydrous sodium sulfate (118 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 hours, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), and the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to column chromatography on silica gel to obtain 86 mg of a yellow powder at a yield of 69%. ESI-MS (m / z): 452.3 [M+H] + .

[0107] Example 21 Preparation of CXT-21

[0108] Compound 21 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then tert-butyl hydroxylamine (51.3 mg, 2 eq), triethylamine (58 mg, 2 eq), anhydrous sodium sulfate (123 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 hours, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), and the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to column chromatography on silica gel to obtain 85 mg of a yellow powder at a yield of 71%. ESI-MS (m / z): 418.3 [M+H] + .

[0109] Example 22 Preparation of CXT-22

[0110] Compound 22 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then methyl hydroxylamine (27 mg, 2 eq), triethylamine (58 mg, 2 eq), anhydrous sodium sulfate (123 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 hours, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), and the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to column chromatography on silica gel to obtain 76 mg of a yellow powder at a yield of 70%. 1 H NMR (800 MHz, Acetone) δ 9.22 (s, 1H), 7.21-7.18 (m, 1H), 6.66 (dd, J = 8.5, 2.0 Hz, 1H), 6.55 (d, J = 8.5 Hz, 1H), 6.42 (d, J = 9.9 Hz, 1H), 4.97 (d, J = 9.9 Hz, 1H), 3.19 (s, 3H), 3.03 (s, 1H), 2.49 (d, J = 3.9 Hz, 3H), 0.66 (s, 6H), 0.59 (s, 9H). 13C NMR (200 MHz, Acetone) δ 150.73, 143.63, 141.31, 139.15, 129.46, 123.92, 123.64, 119.98, 119.22, 118.15, 116.21, 114.08, 109.36, 106.48, 76.65, 54.77, 49.79, 35.22, 33.25, 32.32, 27.10. ESI-MS (m / z): 376.2 [M+H] + .

[0111] Example 23 Preparation of CXT-23

[0112] Compound 23 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then benzyl hydroxylamine (70.9 mg, 2 eq), triethylamine (58 mg, 2 eq), anhydrous sodium sulfate (123 mg, 3 eq) were added thereto, and stirring was performed at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, and extraction was performed three times with ethyl acetate (5 ml x 3), the ethyl acetate layer was combined, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain 86 mg of a yellow powder at a yield of 66%. 1 H NMR (800 MHz, Acetone) δ 8.21 (s, 1H), 8.03 (d, J = 1.9 Hz, 1H), 7.59 (d, J = 6.9 Hz, 1H), 7.49 (dd, J = 8.5, 1.9 Hz, 1H), 7.43 - 7.35 (m, 4H), 7.27 (d, J = 9.9 Hz, 1H), 5.82 (d, J = 10.0 Hz, 1H), 5.15 (s, 2H), 4.04 (s, 2H), 3.31 (d, J = 4.3 Hz, 3H), 1.48 (s, 6H), 1.42 (s, 9H). 13 C NMR (200 MHz, Acetone) δ 150.97, 143.70, 141.33, 139.24, 136.30, 130.03, 129.52, 129.28, 128.79, 123.94, 123.64, 120.10, 119.21, 118.22, 116.34, 114.00, 109.35, 106.53, 76.66, 71.53, 49.80, 35.23, 33.27, 32.32, 27.11. ESI-MS (m / z): 452.3 [M+H] + .

[0113] Example 24 Preparation of CXT-24

[0114] Compound 19 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then hydroxylamine hydrochloride (48 mg, 2 eq), 1 M NaOH solution (0.58 ml, 2 eq) were added thereto, stirred at room temperature for 5 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) three times, combined the ethyl acetate layer, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 86 mg with a yield of 83%. 1 HNMR (800 MHz, Acetone) δ 10.01 (s, 1H), 8.55 (s, 1H), 8.37 (s, 1H), 8.10 (d, J = 1.9 Hz, 1H), 7.49 (dd, J = 8.5, 1.9 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 9.9 Hz, 1H), 5.82 (d, J = 9.9 Hz, 1H), 4.03 (s, 3H), 1.51 (s, 6H), 1.42 (s, 9H). 13 C NMR (200 MHz, Acetone) δ 151.16, 145.73, 143.43, 141.50, 139.34, 129.75, 123.83, 123.58, 119.34, 119.20, 117.34, 116.52, 114.53, 109.22, 106.92, 76.37, 35.20, 33.27, 32.29, 27.23. ESI-MS (m / z): 362.2 [M+H] + .

[0115] Example 25 Preparation of CXT-25

[0116] Compound 23 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then benzyl hydroxylamine (71 mg, 2 eq), triethylamine (58 mg, 2 eq), anhydrous sodium sulfate (123 mg, 3 eq) were added thereto, and stirred at 90 degrees for 2 h, and the reaction was stopped. 10 ml of water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) three times, combined the ethyl acetate layer, dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was rotary evaporated to obtain a crude product. The crude product was subjected to silica gel column chromatography to obtain a yellow powder of 78 mg with a yield of 62%. ESI-MS (m / z): 438.2 [M+H] + .

[0117] Pharmacological experiments:

[0118] Example 1: Protective effect of compound CXT 1-25 on inflammatory response of LPS-stimulated macrophage Raw264.7

[0119] Method: The frozen cells were taken out from liquid nitrogen and placed in a 37°C water bath immediately. The cells were thawed by gently shaking and 3 mL of pre-warmed complete medium (DMEM + 10% fetal bovine serum + 1% penicillin-streptomycin mixture) was added. After centrifugation at 800g for 3 min, the supernatant was discarded and the cells were resuspended and transferred to a fresh culture dish containing fresh complete medium. The cells were incubated in a 37°C CO2 incubator. In this experiment, the resuscitated cells were considered stable after being subcultured for 2 passages and were used for experiments. Raw264.7 cells are semi-adherent cells, and in the resting state, they are round and several cells aggregate in a "grape-like" shape. When the cells grow to about 90% confluence, they are subcultured. The medium is aspirated and replaced with fresh complete medium. The cells are detached by blowing and collected in a centrifuge tube. After centrifugation at 800g for 3 min, the supernatant is discarded and the cell pellet is resuspended in 1 mL of medium. The cell pellet is gently blown to disperse the cells into single cells, and the cells are inoculated in a culture dish at a ratio of 1:3 and incubated in a 37°C CO2 incubator.

[0120] The cells were uniformly plated in a 96-well plate at a concentration of 5 x 10 5 After 17-24 hours of incubation in a 37°C CO2 incubator, the cells were treated with 1% fetal bovine serum starvation medium for 4 hours. After 4 hours, the medium was replaced with 8% fetal bovine serum maintenance medium, and 10 μL / well of drug was added for 2 hours. Then, 1 μg / mL of lipopolysaccharide (LPS) was added for 12 hours. The cell culture medium was collected for subsequent experiments.

[0121] NO content was measured by the Griess reagent method: 1% sulfanilamide solution and 0.1% NED solution were taken and restored to room temperature. 0.1 M sodium nitrite was diluted with complete medium to 100 μM, and then gradient diluted at a ratio of 1:2 to obtain 8 concentration standards. 50 μL / well of sample and standard was added to a 96-well plate, 50 μL / well of 1% sulfanilamide solution was added to the above wells, and incubated at room temperature for 5 min in the dark. 50 μL / well of 0.1% NED solution was added to the above wells, and the color reaction appeared immediately. Incubate at room temperature for 5 min in the dark. Read the absorbance value of the above wells at 525 nm. Linear fitting of the absorbance values of the standards gives the linear equation C=a*A525+b between concentration C and absorbance A525. The NO concentration in each sample was calculated by substituting the sample absorbance value into the above equation.

[0122] The results of the anti-inflammatory effect screening of compound CXT 1-25 on LPS-stimulated macrophages Raw264.7 are shown in Table 1. LPS stimulation can significantly increase the secretion of NO, and 10 μmol / L CXT 1-25 compounds can significantly reduce the secretion of NO, showing obvious anti-inflammatory effect.

[0123] Table 1 Effect of CXT 1-25 on NO release from macrophage Raw264.7 after LPS stimulation (means ± S.E.M, n=3)

[0124] Example 2: Effect of CXT-24 at 3 mg / kg, 10 mg / kg, 30 mg / kg on infarct volume in pMCAO rats

[0125] Materials and methods:

[0126] CXT-24 was provided by the Institute of Materia Medica, Peking Union Medical College, with a chemical purity of >99.5%. When prepared, DMSO and 0.5% CMC-Na were added and ultrasonicated for 30 min.

[0127] TTC (2,3,5-triphenyltetrazolium chloride): A 4% solution was prepared in TTC buffer solution and was produced by Sigma-Aldrich.

[0128] Permanent focal cerebral ischemia model (pMCAO) preparation:

[0129] Male SD rats, weighing 260-280 g, were housed 5 per cage in a room maintained at 23 °C with free access to food and water. The rats were anesthetized with a Zoletil small animal anesthetic machine. The right common carotid artery was exposed through a midline incision in the neck, and the internal and external carotid arteries (ICA, ECA) were carefully separated. The ECA was ligated and cut, and a 0.26 ± 0.01 mm diameter nylon thread with a 0.36 ± 0.02 mm diameter head was inserted into the ICA from the ECA for about 16 mm, reaching the origin of the middle cerebral artery. The thread was then fixed and the excess was cut off, and the ECA opening was ligated and 5 drops of gentamicin were added. The incision was sutured and the rats were returned to the cage. The room temperature was maintained at 23-25 °C throughout the procedure. During the entire operation, the rats were placed on a 37 °C heating plate to maintain body temperature. Drug administration was performed 10 minutes after ischemia.

[0130] Grouping and drug administration:

[0131] The rats were divided into 5 groups of 10 each, namely the solvent control group, the CXT-24 3 mg / kg group, the CXT-24 10 mg / kg group, the CXT-24 30 mg / kg group, and the edaravone group at 30 mg / kg of dextromethorphan; the edaravone group was administered by tail vein injection, and the others were administered by oral gavage.

[0132] Neurobehavioral scoring:

[0133] Behavioral observation was performed 24 hours after ischemia, and the evaluation methods included Bederson and mNSS scoring.

[0134] Measurement of infarct volume:

[0135] The determination of cerebral infarction volume is described in detail in Pharmacological Experimental Methods (Xu Shuyun, 3rd edition, p1066-1067). The rats were decapitated and their brains were removed immediately after 24 hours of ischemia. The olfactory tract, cerebellum and low brain stem were removed, and the brain was cut into 6 pieces in the coronal direction. The brain slices were quickly placed in 5 ml of 1% TTC solution (37°C, dark) for 30 minutes, with the slices being turned over every 5 minutes. After TTC staining, normal tissues were deeply stained red, while infarcted tissues were white. The brain slices of each group were arranged in order and photographed for preservation. The image analysis system software Photoshop was used for processing and statistics to calculate the infarct area of each slice, and finally the infarct volume was calculated.

[0136] The percentage of infarct volume was calculated by the following formula:

[0137] Cerebral infarction volume (%) = (unoperated half-brain volume - unimpaired volume of the operated half-brain) / unoperated half-brain volume x 100%

[0138] Inclusion and exclusion criteria for experimental animals:

[0139] In this experiment, the permanent focal cerebral ischemia model (pMCAO) was used in SD rats. The entire operation (anesthesia) process took about 20 minutes for one rat. All postoperative SD rats could drink water and eat freely after waking up from anesthesia. The main causes of death were bleeding caused by wire insertion and intestinal distension during anesthesia. The exclusion criteria included:

[0140] (1) SD rats died of brain hemorrhage caused by wire insertion within 24 hours after operation;

[0141] (2) SD rats died of intestinal distension caused by anesthesia within 24 hours after operation;

[0142] (3) SD rats whose body weight did not meet the experimental standards.

[0143] Statistical methods:

[0144] All data were statistically analyzed on Graph Prism 8.0.1, and all results were expressed as mean ± SEM. The t-test was used for statistical analysis to compare the differences between groups, and P<0.05 was considered to be a significant difference.

[0145] Experimental results:

[0146] (1) Effect of compound CXT-24 on cerebral infarction volume in SD rats after acute ischemic stroke

[0147] The cerebral infarction volume of SD rats after acute cerebral ischemia for 24 h was (66.08 ± 2.021) %, while after administration of different doses of CXT-24 (3 mg / kg, 10 mg / kg, 30 mg / kg) and edaravone dextrose (30 mg / kg), the cerebral infarction volume after 24 h was (58.05 ± 6.774) %, (40.04 ± 5.823) %, (49.87 ± 6.463) % and (51.59 ± 6.225) %, respectively. The cerebral infarction volume of each group of compound CXT-24 orally administered was reduced compared with the control group, indicating that administration of compound CXT-24 had a certain therapeutic effect on acute ischemic stroke in SD rats.

[0148] Table 2. Effect of compound CXT-24 on cerebral infarction volume of SD rats after pMCAO. *P < 0.05 compared with the model group, **P < 0.01 compared with the model group, n = 8-11

[0149] (2) Effect of compound CXT-24 on behavioral deficits of SD rats after acute ischemic stroke

[0150] The Bederson score was used to determine the behavioral score of rats after acute cerebral ischemia. The results showed that the score of SD rats after cerebral ischemia for 24 h was 2.889 ± 0.2003. After administration of different doses of CXT-24 (3 mg / kg, 10 mg / kg, 30 mg / kg) and edaravone dextrose (30 mg / kg), the behavioral score after 24 h was 2.375 ± 0.1830, 2.182 ± 0.1220, 2.111 ± 0.1111 and 2.250 ± 0.1637, respectively. The results showed that the 10 mg / kg dose group and the 30 mg / kg dose group of compound CXT-24 had significant differences compared with the control group, indicating that administration of compound CXT-24 had a certain effect on improving the behavioral deficits of SD rats after acute ischemic stroke.

[0151] Table 3. Effect of compound CXT-24 on behavioral deficits of SD rats after pMCAO (Bederson score) *P < 0.05 compared with the model group, **P < 0.01 compared with the model group, n = 8-11

[0152] The mNSS score was used to determine the behavior score of rats after acute cerebral ischemia. The results showed that the score of SD rats was 11.44±0.6035 after 24h of cerebral ischemia. After administration of different doses of CXT-24 (3mg / kg, 10mg / kg, 30mg / kg) and edaravone dextrose (30mg / kg), the behavior scores after 24h were 9.500±0.5976, 9.091±0.3921, 8.556±0.8012, and 8.500±0.4226, respectively. This suggests that administration of compound CXT-24 has a certain effect on improving the behavior impairment of SD rats with acute ischemic stroke.

[0153] Table 4. Effect of compound CXT-24 on behavior impairment of SD rats after pMCAO (mNSS score) *P<0.05 compared with the model group, **P<0.01 compared with the model group, n=8-11

[0154] Example 3: Effect of 1mg / kg, 3mg / kg, 10mg / kg dose of CXT-24 injection on cerebral infarction volume of pMCAO rats

[0155] Experimental materials and methods:

[0156] Chloro triphenyl tetrazolium (TTC): a 4% solution was prepared with TTC buffer solution for use, produced by Source Leaf Biotech Co., Ltd.

[0157] Preparation of permanent focal cerebral ischemia model (pMCAO):

[0158] Male SD rats, weighing 260-280 grams, were placed in a cage of 5 rats each, and the room temperature was maintained at 23°C, with free access to food and water. The rats were anesthetized with a Ruiwode general-purpose small animal anesthetizing machine. The right common carotid artery was exposed through a midline incision in the neck, and the internal and external carotid arteries (ICA, ECA) were carefully separated. The ECA was ligated and cut, and a nylon thread with a body diameter of 0.26±0.01mm and a head diameter of 0.36±0.02mm was inserted into the ICA about 16mm from the ECA, reaching the middle cerebral artery. The thread was then fixed and the excess portion was cut off, and the ECA opening was ligated and 5 drops of gentamicin were added, and the incision was sutured and placed back in the cage. The room temperature was maintained at 23-25°C during the entire procedure. During the entire operation, the rats were placed on a 37°C heating plate to maintain body temperature. Drug administration was performed 1 minute after ischemia.

[0159] Grouping and administration:

[0160] The rats were divided into 5 groups, 10 rats in each group, namely control group, CXT-24 1 mg / kg group, CXT-24 3 mg / kg group, CXT-24 10 mg / kg group, and edaravone group (30 mg / kg). All the rats were given tail vein injection.

[0161] Neurobehavioral score:

[0162] Behavioral observation was performed 24 hours after ischemia, and the evaluation methods included Bederson and mNSS scores.

[0163] Measurement of cerebral infarction volume:

[0164] The measurement of cerebral infarction volume is described in detail in the pharmacological experimental method (Xu Shuyun, 3rd edition, p1066-1067). After 24 hours of ischemia, the rats were decapitated immediately, and the olfactory tract, cerebellum and low brain stem were removed. The brain was cut into 6 pieces in the coronal direction, and then quickly placed in 5 ml of 1% TTC solution for staining (37°C, dark) for 30 minutes, with turning every 5 minutes. After TTC staining, normal tissues were deeply stained red, and infarct tissues were white. The brain slices in each group were arranged in order, and photographed for preservation. Image analysis system software Photoshop was used for processing and statistics, and the infarction area of each slice was calculated, and finally converted into infarction volume.

[0165] The percentage of infarction volume was calculated by the following formula:

[0166] Cerebral infarction volume (%) = (unoperated half brain volume - unimpaired volume of the operated half brain) / unoperated half brain volume x 100%

[0167] Inclusion and exclusion criteria for experimental animals:

[0168] In this experiment, the permanent focal cerebral ischemia model (pMCAO) of SD rats was used. The whole operation (anesthesia) process took about 20 minutes for one rat. All postoperative SD rats could drink water and eat freely after waking up from anesthesia. The main causes of death were insertion of line causing brain hemorrhage or death due to intestinal distension during anesthesia. The exclusion criteria included:

[0169] (1) SD rats died of brain hemorrhage caused by line insertion within 24 hours after operation;

[0170] (2) SD rats died of intestinal distension caused by anesthesia within 24 hours after operation;

[0171] (3) SD rats whose body weight did not meet the experimental standard.

[0172] Statistical method:

[0173] All data were analyzed by Graph Prism 8.0.1, and all results were expressed as mean ± SEM. The differences between groups were compared by t test, and P < 0.05 was considered to be significantly different.

[0174] Experimental results

[0175] Effect of CXT-24 injection on cerebral infarction volume after acute ischemic stroke in SD rats:

[0176] The cerebral infarction volume of SD rats after 24 h of acute cerebral ischemia was (65.68 ± 3.582)%, while after administration of different doses of CXT-24 injection (1 mg / kg, 3 mg / kg, 10 mg / kg) and edaravone dextrose (30 mg / kg), the cerebral infarction volume after 24 h was (59.97 ± 2.347)%, (47.91 ± 4.583)%, (38.80 ± 6.849)%, and (50.22 ± 5.419)%, respectively. The cerebral infarction volume of each CXT-24 injection group was lower than that of the control group, indicating that CXT-24 injection had a certain therapeutic effect on acute ischemic stroke in SD rats.

[0177] Table 5. Effect of CXT-24 injection on cerebral infarction volume after acute ischemic stroke in SD rats. *P < 0.05 compared with the model group, **P < 0.01 compared with the model group, n = 8-13

[0178] Effect of CXT-24 injection on behavioral deficits after acute ischemic stroke in SD rats

[0179] The behavioral score of rats after acute cerebral ischemia was determined by Bederson score, and the results showed that the score of SD rats after 24 h of cerebral ischemia was 2.778 ± 0.1470. After administration of different doses of CXT-24 injection (1 mg / kg, 3 mg / kg, 10 mg / kg) and edaravone dextrose (30 mg / kg), the behavioral score after 24 h was 2.600 ± 0.2211, 2.231 ± 0.1216, 2.200 ± 0.1333, and 2.250 ± 0.1637, respectively. The results showed that the 3 mg / kg dose group and the 10 mg / kg dose group of CXT-24 injection had significant differences compared with the control group, indicating that CXT-24 injection had a certain effect on improving the behavioral deficits of SD rats after acute ischemic stroke.

[0180] Table 6. Effect of CXT-24 injection on behavioral deficits after pMCAO in SD rats (Bederson score) *P<0.05 vs model group, **P<0.01 vs model group, n=8-13

[0181] The mNSS score was used to determine the behavior score of rats after acute cerebral ischemia. The results showed that the score of SD rats was 10.44±0.4747 after 24h of cerebral ischemia. After administration of different doses of CXT-24 injection (1mg / kg, 3mg / kg, 10mg / kg) and edaravone (30mg / kg), the behavior scores after 24h were 9.300±0.5385, 8.692±0.3469, 8.200±0.3887, and 8.000±0.3780, respectively. This suggests that administration of CXT-24 injection has a certain effect on improving the behavior impairment of SD rats with acute ischemic stroke.

[0182] Table 7. Effect of CXT-24 injection on behavior impairment of SD rats after pMCAO (mNSS score) *P<0.05 vs model group, **P<0.01 vs model group, n=8-13

[0183] Example 4: Effect of compound CXT-24 on cell survival rate of neurons injured by oxygen-glucose deprivation / reoxygenation injury

[0184] Experimental grouping:

[0185] A blank control group (Control group), an oxygen-glucose deprivation / reoxygenation injury model group (Model group), and an oxygen-glucose deprivation / reoxygenation injury + test drug group were set up.

[0186] Experimental process:

[0187] (1) SK-N-SH cells were inoculated in a 96-well culture plate at a density of 6.5×10^4 cells / ml, 100μl / well;

[0188] (2) After the cells adhered, the medium in the 96-well plate was discarded (one row was discarded with a row gun, and one row was added), the Control group was added with complete culture medium, 100μl per well, the Model group was added with low-sugar culture medium, 100μl per well, and the neuroprotective agent screening group was added with low-sugar culture medium, 90μl per well. The neuroprotective agent screening group was further added with 10μl of the test compound at a concentration of 100μM. Three replicate wells were set up for each group, and the experiment was repeated 3 times;

[0189] (3) Put into the anoxic chamber and fill with 95% N2+5% CO2, cultivate at 37℃ for 4h;

[0190] (4) Aspirate the culture medium of the 96-well plate (use the gun to aspirate one row and add one row). Add 100 μl of complete culture medium to each well of the Control group. After opening the hypoxia chamber, add 100 μl of complete culture medium to each well of the Model group and add 90 μl of complete culture medium to each well of the neuroprotective agent screening group. Add 10 μl of the neuroprotective agent to be screened at a concentration of 100 μM to the neuroprotective agent screening group.

[0191] (5) Incubate the cells in a cell incubator at 37°C for 4 hours.

[0192] (6) Add 10 μl of MTT (5 mg / ml) to each well and continue the reaction at 37°C for 4 hours.

[0193] (7) Aspirate the culture medium in the 96-well plate and add 150 μl of DMSO to each well. Gently shake the plate to dissolve the crystal product. Measure the optical density (OD) value of each well at a wavelength of 570 nm using an enzyme marker. The OD value reflects the cell survival rate of each neuroprotective agent to be screened. Compounds that have a protective effect on the injury model are determined to be re-screened.

[0194] Calculate the relative cell survival rate of each group compared to the Model group:

[0195] (1) Calculate the cell survival rate of the Model group:

[0196] (Model group OD value / Control group OD value) x 100%

[0197] (2) Then calculate the cell survival rate of each drug compared to the Model group:

[0198] (Drug group cell survival rate - Model group cell survival rate) / Model group cell survival rate x 100%

[0199] Statistical method:

[0200] The statistical analysis method is T-test. P < 0.05 is considered to be significantly different.

[0201] Table 8 Effect of compound CXT-24 on the cell survival rate of neurons injured by oxygen-glucose deprivation / reoxygenation (n = 3)

[0202] Example 5: Effect of compound CXT-24 on the hot plate test of ICR mice at a dose of 3 mg / kg, 10 mg / kg and 30 mg / kg

[0203] Experimental method:

[0204] CXT-24 was provided by the Institute of Chemistry, Chinese Academy of Sciences, with a chemical purity of >99.5%. When preparing, 0.5% CMC-Na was added, ground, and then ultrasonicated for 30 min.

[0205] (1) Hot plate test of mice

[0206] Male ICR mice were randomly divided by weight the day before the test and acclimated to the equipment for 5 min. On the test day, the hot plate was set to 55.0°C. The animals were gently placed on the hot plate, and the instrument recorded the time at which the animals showed licking the hind foot or jumping behavior within 60 s as the pain threshold. The second test value was recorded 5 min apart, and the drug was given immediately. The animals were gently placed on the hot plate again 30 min after the positive control interval (60 min interval), and the post-dose pain threshold was tested. Animals with a pre-dose pain threshold <5 s or >30 s were excluded, and the post-dose pain threshold was calculated. After the animal experiment, the animals were euthanized using CO2 and disposed of.

[0207] (2) Grouping and drug administration

[0208] There were 5 groups, each with 20-22 animals, and the animals were divided into a solvent control group, a CXT-24 drug administration group (3 mg / kg, 10 mg / kg, 30 mg / kg), and a positive drug indomethacin group. The drug was administered at a volume of 0.1 mL / 10 g, all by oral gavage.

[0209] (3) Statistical method

[0210] All data were statistically analyzed using t-test to compare differences between groups, and P<0.05 was considered to be a significant difference.

[0211] Experimental results:

[0212] Effect of compound CXT-24 on hot plate test of ICR mice at doses of 3 mg / kg, 10 mg / kg, and 30 mg / kg

[0213] The pain threshold of male ICR mice on the hot plate was (8.66±1.07) s in the model group, while after administration of different doses of CXT-24 (3 mg / kg, 10 mg / kg, 30 mg / kg) and indomethacin (10 mg / kg), the pain threshold was (10.38±1.26) s, (12.59±1.59) s, (15.37±2.38) s, and (14.83±1.35) s, respectively. Compound CXT-24 at doses of 10 mg / kg and 30 mg / kg significantly increased the pain threshold of mice compared with the control group (p<0.05), suggesting that administration of compound CXT-24 had a certain analgesic effect.

[0214] Table 9. Effects of compound CXT-24 on the pain threshold of ICR mice in hot-plate test. *P<0.05 vs model group, ***P<0.001 vs model group n=20-22

[0215] Example 6: Effects of compound CXT-24 on the acetic acid writhing test of ICR mice at doses of 3 mg / kg, 10 mg / kg, and 30 mg / kg

[0216] Experimental method:

[0217] CXT-24 was provided by the Institute of Plant Chemistry, with a chemical purity of >99.5%. When preparing, 0.5% CMC-Na was added and ground before ultrasonic treatment for 30 min.

[0218] (1) Acetic acid writhing test in mice

[0219] Seventy-five male ICR mice, weighing 18-22 g, were randomly divided by weight the day before the test. Fifteen minutes after oral administration of CXT-24 at 0.1 mL / 10 g (positive drug at 60 min), 0.6% acetic acid solution was injected intraperitoneally at 0.1 mL / 10 g BW, and the writhing response of the mice was observed within 15 min, including abdominal concave, stretching of the trunk and hind limbs, high buttocks, etc. The latency of the first writhing and the number of writhings were recorded. The experiment was scored by a single-blind experimenter. After the experiment, the mice were euthanized with CO2.

[0220] (2) Grouping and administration

[0221] The mice were divided into 5 groups, 15 mice in each group, and the animals were divided into a solvent control group, a CXT-24 administration group (3 mg / kg, 10 mg / kg, and 30 mg / kg), and a positive drug indomethacin group. The drug was administered at a volume of 0.1 mL / 10 g, all by oral gavage.

[0222] (3) Statistical method

[0223] All data were statistically analyzed using t-test to compare the differences between groups, and P<0.05 was considered to be a significant difference.

[0224] Experimental results:

[0225] Effects of compound CXT-24 on the acetic acid writhing test of ICR mice at doses of 3 mg / kg, 10 mg / kg, and 30 mg / kg

[0226] The pain latency of male ICR mice after injection of 0.6% acetic acid solution was: model group (346.7±44.23) s, and after administration of different doses of CXT-24 (3 mg / kg, 10 mg / kg, 30 mg / kg) and indomethacin (10 mg / kg), the latency was (450.3±73.45) s, (517.2±68.29) s, (506.7±69.66) s, (747.5±51.92) s, respectively. The pain latency of mice after injection of acetic acid in the 10 mg / kg group of compound CXT-24 administered orally was significantly increased compared with the control group (p<0.05), and the pain latency of mice after injection of acetic acid in the 30 mg / kg group had a trend of increasing compared with the control group (p=0.06), suggesting that administration of compound CXT-24 had certain analgesic effect.

[0227] Table 10. Effect of compound CXT-24 on the latency of acetic acid writhing test in ICR mice. *P<0.05 vs model group, ***P<0.001 vs model group n=15

[0228] The writhing times of male ICR mice after injection of 0.6% acetic acid solution were: model group (25.57±4.34) s, and after administration of different doses of CXT-24 (3 mg / kg, 10 mg / kg, 30 mg / kg) and indomethacin (10 mg / kg), the writhing times were (17.20±4.84) s, (9.93±2.37) s, (8.67±2.90) s, (2.67±1.09) s, respectively. The writhing times of mice after injection of acetic acid in the 10 mg / kg and 30 mg / kg groups of compound CXT-24 administered orally were significantly reduced compared with the control group (p<0.01), suggesting that administration of compound CXT-24 had certain analgesic effect.

[0229] Table 11. Effect of compound CXT-24 on the writhing times of acetic acid writhing test in ICR mice. **P<0.01 vs model group, ***P<0.001 vs model group n=15

[0230] Example 7: Effect of 10 mg / kg dose of CXT-24 on the neurological function of TBI rats

[0231] Experimental materials and methods:

[0232] CXT-24 was provided by the Institute of Plant Protection, with a chemical purity of >99.5%. When prepared, 0.5% CMC-Na was added and ground uniformly in a mortar.

[0233] Preparation of rat traumatic brain injury model (TBI):

[0234] After isoflurane anesthesia of rats, the scalp was incised 1.5 mm behind the right coronal suture and 2.5 mm lateral to the midline, and a 5-mm-diameter bone hole was drilled. Using a modified Feeney free-fall injury device, a 40-g hammer was dropped from 25 cm to impact the striker, with a 6-mm depth of impact, and the scalp was sutured. The control group of rats was sutured after the scalp was incised and drilled, and the hammer was not struck. Subsequently, the rats were administered drugs according to the grouping at the predetermined time, and the control group and the model group were administered the same amount of 0.5% CMC-Na solution.

[0235] Grouping and administration:

[0236] The rats were divided into 4 groups, 10 rats in each group, namely the control group, the model group, the CXT-24 10 mg / kg double-dose administration group, and the butylphthalide 200 mg / kg group. The administration method was oral gavage. The administration time of the control group, the model group, and the butylphthalide 200 mg / kg group was 0, 24, and 48 h after surgery, and the administration time of the CXT-24 10 mg / kg double-dose administration group was 0, 6, 24, 30, 48, and 54 h after surgery.

[0237] Neurobehavioral score:

[0238] Behavioral observation was performed at 24, 48, and 72 h after modeling, and the evaluation method was mNSS score.

[0239] Rotarod test:

[0240] Rotarod test was performed at 0 h before modeling, 24 h, 48 h, and 72 h after modeling to evaluate the motor ability of rats. The rats were placed on the rotarod apparatus, the initial speed was 5 rpm, and the speed was uniformly accelerated to 25 rpm within 180 s. The longest latency to fall was recorded, and each experiment was repeated 3 times and the average value was taken.

[0241] Inclusion and exclusion criteria for experimental animals

[0242] SD rats were used in this experiment to establish a traumatic brain injury model (TBI). The entire surgical (anesthesia) process took about 5 minutes per rat. All postoperative SD rats could drink water and eat freely after waking up from anesthesia. The main cause of death was intestinal distension during anesthesia, and the exclusion criteria included:

[0243] (1) SD rats died within 24 h after surgery due to intestinal distension during anesthesia;

[0244] (2) SD rats did not meet the experimental standards.

[0245] Statistical method

[0246] All data were analyzed by Graph Prism 8.0.1, and all results were expressed as mean ± SD. The differences between groups were compared by t test, and P < 0.05 was considered to be significant.

[0247] Results:

[0248] (1) Effect of compound CXT-24 on motor function of SD rats after traumatic brain injury

[0249] The model rats showed a significant decrease in the falling time of the rotating rod (62.46 ± 11.58 s) 24 h after brain injury, which was significantly different from the control group (P < 0.001), indicating that the TBI model in this experiment caused a significant impairment of motor function in rats. After administration of CXT-24 10 mg / kg, the motor function of rats was significantly improved, and the falling time was 85.70 ± 21.30 s, which was significantly increased compared with the model group (P < 0.01); the falling time of the butylphthalide 200 mg / kg group was 69.26 ± 27.43, which showed no significant difference compared with the model group.

[0250] At 72 h after brain injury, the falling time of the rotating rod of the model rats was 114.95 ± 15.89 s, which was significantly lower than that of the control group at 72 h after brain injury (P < 0.001), indicating that the TBI model in this experiment caused impairment of motor function in rats. After administration of CXT-24 (10 mg / kg), the falling time of the rotating rod of rats was increased compared with the model group, but there was no statistical difference, and the falling time was 133.21 ± 24.72 s (P = 0.06); the falling time of the butylphthalide 200 mg / kg group was 118.47 ± 37.93 s, which showed no significant difference compared with the model group. The above results suggest that the CXT-24 10 mg / kg group can protect the motor function injury caused by TBI at 24 h after operation.

[0251] Table 12. Effect of compound CXT-24 on motor function of rats after TBI modeling **P < 0.01 vs model.

[0252] (2) Effect of compound CXT-24 on behavioral deficits of SD rats after brain injury

[0253] The mNSS score was used to determine the behavior score of rats after brain injury. The results showed that the neurological impairment score of model rats increased significantly (7.30±1.06) 24 hours after brain injury, and there was a significant difference compared with the control group (P<0.001), indicating that the TBI model in this experiment caused significant damage to the neurological function of rats. After administration of CXT-24 10 mg / kg, the score was 7.70±0.95; the score of the butylphthalate 200 mg / kg group was 7.80±1.03; compared with the neurological impairment score of model rats, no significant difference was found.

[0254] After 72 hours of brain injury, the neurological impairment score of model rats was 7.50±1.27. Compared with the control group, the neurological impairment score of model rats at 72 hours after brain injury was still significantly increased, and there was a significant difference (P<0.001), indicating that the TBI model in this experiment caused damage to the neurological function of rats. After administration of CXT-24 10 mg / kg, the neurological impairment score of rats was significantly reduced, with a score of 6.40±0.70, which had a significant difference compared with the model group (P<0.05); the butylphthalate 200 mg / kg group was slightly lower than the model group, but did not reach statistical difference. It is suggested that the CXT-24 10 mg / kg group can protect the neurological impairment caused by TBI at 72 hours after operation.

[0255] Table 13. Effect of compound CXT-24 on behavioral impairment of rats after TBI modeling (mNSS score) *P<0.05 vs model.

[0256] Example 8 Effect of compound on survival rate of SK-N-SH cells induced by hydrogen peroxide damage

[0257] Experimental grouping

[0258] A blank control group, a hydrogen peroxide damage model group, and a hydrogen peroxide damage + test drug group (neuroprotective agent screening group) were set up.

[0259] Experimental process

[0260] (1) SK-N-SH cells were inoculated in 96-well culture plates at a density of 6.5×10 4

[0261] ​(2) After the cells adhere to the wall, change the liquid medium, add 100 μl of complete culture medium to the control group, add 90 μl of complete culture medium to the model group, and add 80 μl of complete culture medium to the neuroprotective agent screening group. Add 10 μl of 100 μM of the compound to be screened to the neuroprotective agent screening group. Then add 10 μl of 15 mM of hydrogen peroxide to the model group and the neuroprotective agent screening group. Set 6 replicates for each group, and repeat the experiment 9 times;

[0262] (3) Incubate the cells in a cell incubator at 37°C for 4 h;

[0263] (4) Add MTT (5 mg / ml) at 10 μl / well, and continue to react at 37°C for 4 h;

[0264] (5) Discard the culture medium in the 96-well plate, and add 150 μl of DMSO to each well. After gentle blowing, make the crystalline product fully dissolved. Detect the optical density (OD) value of each well on an enzyme marker at a wavelength of 570 nm. The size of the detected OD value can reflect the cell survival rate of each drug to be screened. Determine the compound having a certain protective effect on the damage model to enter the re-screening.

[0265] Calculate the relative cell survival rate value of each group

[0266] (1) First, calculate the cell survival rate of the model group:

[0267] (Model group OD value / control group OD value) x 100%

[0268] (2) Then, calculate the cell survival rate of each drug relative to the model group:

[0269] (Screening group cell survival rate-model group cell survival rate) / model group cell survival rate x 100%

[0270] Statistical method

[0271] The statistical processing adopts the T-test analysis method. P<0.05 is a significant difference.

[0272] Example 9: Effect of 3 mg / kg, 10 mg / kg, and 30 mg / kg doses of CXT-24 on depressive-like behavior of rats with bilateral common carotid artery ligation in forced swimming test

[0273] Experimental method:

[0274] (1) Preparation of bilateral common carotid artery ligation model:

[0275] Methods (Xu Shuyun, editor in chief, the third edition p1066-1067). Briefly, male SD rats were anesthetized with a gas anesthetic machine (1.5-2.5% isoflurane), the body temperature was maintained at 37±0.5℃, and the rats were fixed on the operating table in a supine position. The skin was incised along the median line of the neck, and the right common carotid artery (CCA) was carefully separated and ligated. The skin was incised along the median line of the neck, and the left CCA was carefully separated and ligated. The blood supply of the middle cerebral artery was blocked. The ECA opening was ligated and the surgical incision was sutured. The room temperature was maintained at 24-25℃ during the whole process, and the rats were fed for one week after the operation. The rats were randomly divided according to the body weight after the operation.

[0276] (2) Grouping and administration

[0277] Divided into 5 groups, 8-11 rats in each group, respectively, solvent control group, CXT-24 3mg / kg group, CXT-24 10mg / kg group, CXT-24 30mg / kg group, fumarizine group 200mg / kg; all were oral gavage administration, continuous administration for 3 weeks.

[0278] (3) Forced swimming test of rats

[0279] Pre-swimming stage (adaptation stage): the rats were placed in the water tank and swam for 15min. In the first 2-3min, the rats swam or climbed constantly, trying to escape from the water, then the activity decreased, and the state of inactivity or floating appeared intermittently, which was called despair behavior, and the duration of this behavior increased, and the duration of this behavior reached the peak at 5-6min after entering the tank, and the inactivity time accounted for about 80% in the remaining time. After the pre-test, the rats were taken out, wiped with a dry towel, and placed in a drying cage. After the pre-swimming of the next group of animals was completed, the previous group was returned to the feeding cage; all rats were pre-swimming in turn. Test stage: 24h after the pre-test, the formal experiment was carried out, and the rats were placed in the tank again. The cumulative inactivity time of the rats within 5min was observed and recorded; the drug was given 30min before the experiment.

[0280] (4) Statistical method

[0281] All data were statistically analyzed on Graph Prism 9.2.0, and all results were expressed as mean±SEM. T test was used for statistical analysis to compare the differences between groups, and P<0.05 was considered to be significantly different.

[0282] Experimental results:

[0283] Effect of compound CXT-24 on depressive-like behavior of rats with bilateral common carotid artery ligation in forced swimming test

[0284] The struggle time in forced swimming of SD rats after bilateral common carotid artery ligation was (27.7±5.986)%, while after administration of different doses of CXT-24 (3mg / kg, 10mg / kg, 30mg / kg) and butylphthalide (200mg / kg), the struggle time was (50.57±5.957)s, (46.50±5.290)s, (39.44±1.914)s, (46.55±5.096)s, respectively. The struggle time in forced swimming of each group of compound CXT-24 orally administered was improved compared with the control group, suggesting that administration of compound CXT-24 has a certain therapeutic effect on post-stroke depression in SD rats.

[0285] Table 14. Effect of compound CXT-24 on forced swimming struggle time of bilateral common carotid artery ligation SD rats. *P<0.05 vs control group, n=8-10

[0286] Example 10: Effect of 3mg / kg, 10mg / kg, 30mg / kg dose of CXT-24 on depressive-like behavior of bilateral common carotid artery ligation rats in open field test

[0287] Experimental method:

[0288] (1) Preparation of bilateral common carotid artery ligation model:

[0289] Method (Xu Shuyun, 3rd edition, p1066-1067). Briefly, male SD rats were anesthetized with a gas anesthetic machine (1.5-2.5% isoflurane), the body temperature was maintained at 37±0.5℃, and the rats were fixed on the operating table in supine position. The skin was incised along the midline of the neck, and the right common carotid artery (CCA) was carefully separated and ligated. The skin was incised along the midline of the neck, and the left common carotid artery (CCA) was carefully separated and ligated. The blood supply of the middle cerebral artery was blocked. The ECA opening was ligated and the surgical incision was sutured. The room temperature was maintained at 24-25℃ during the whole process, and the rats were fed for one week after operation. The postoperative rats were randomly divided according to body weight.

[0290] (2) Grouping and administration

[0291] Divided into 5 groups, 8-10 rats in each group, respectively, solvent control group, CXT-24 3mg / kg group, CXT-24 10mg / kg group, CXT-24 30mg / kg group, butylphthalide group 200mg / kg; all were oral gavage administration.

[0292] (3) Open field test of rats

[0293] Rats were administered 30 minutes in advance, and adapted to the open field for 5 minutes. At the beginning of the experiment, the animals were placed in the center of the open field device with their heads facing the camera direction. The video recorded the movement trajectory for 5 minutes. The time spent in the center and corners of the open field, the number of entries, and the distance were recorded. After the experiment, the open field was wiped with 75% alcohol gauze.

[0294] (4) Statistical methods

[0295] All data were statistically analyzed using Graph Prism 9.2.0, and all results were expressed as mean ± SEM. The t-test was used for statistical analysis to compare the differences between groups. P < 0.05 was considered to be a significant difference.

[0296] Experimental results

[0297] Experimental results:

[0298] Effect of compound CXT-24 on depressive-like behavior in bilateral common carotid artery ligation rats in the open field experiment

[0299] After bilateral common carotid artery ligation in SD rats, the time spent in the center of the open field was (21.35 ± 3.940) s, while after administration of different doses of CXT-24 (3 mg / kg, 10 mg / kg, 30 mg / kg) and butylphthalide (200 mg / kg), the struggling time was (42.91 ± 8.730) s, (30.78 ± 4.042) s, (47.68 ± 10.30) s, and (37.44 ± 8.898) s, respectively. The time spent in the center of the open field experiment in the compound CXT-24 oral administration groups was improved compared with the control group, indicating that administration of compound CXT-24 had a certain therapeutic effect on post-stroke depression in SD rats.

[0300] Table 15. Effect of compound CXT-24 on the center time of bilateral common carotid artery ligation SD rats in the open field experiment. *P < 0.05 vs control group, n = 8-10

[0301] After bilateral common carotid artery ligation in SD rats, the number of entries into the center of the open field experiment was (9.50 ± 1.258) times, while after administration of different doses of CXT-24 (3 mg / kg, 10 mg / kg, 30 mg / kg) and butylphthalide (200 mg / kg), the number of entries into the center was (18.33 ± 4.048) times, (14.75 ± 1.449) times, (17.44 ± 2.274) times, and (14.22 ± 1.597) times, respectively. The number of entries into the center of the open field experiment in the compound CXT-24 oral administration groups was improved compared with the control group, indicating that administration of compound CXT-24 had a certain therapeutic effect on post-stroke depression in SD rats.

[0302] Table 16. Effect of compound CXT-24 on the number of times in the center of the open field test in bilateral common carotid artery ligation SD rats. *P<0.05 vs control group, n=8-10

[0303] The distance of movement in the center area of the open field test in SD rats after bilateral common carotid artery ligation was (1748±259.1) mm, while after administration of different doses of CXT-24 (3 mg / kg, 10 mg / kg, 30 mg / kg) and butylphthalide (200 mg / kg), the center area distances were (4101±1101) mm, (2968±361.2) mm, (3726±603.1) mm, and (3357±477.9) mm, respectively. The distance of movement in the center area of the open field test in each group of orally administered compound CXT-24 was improved compared with the control group, indicating that administration of compound CXT-24 has a certain therapeutic effect on post-stroke depression in SD rats.

[0304] Table 17. Effect of compound CXT-24 on the distance in the center of the open field test in bilateral common carotid artery ligation SD rats. *P<0.05 vs control group, n=8-10.

[0305] Example 11: Effect of 3 mg / kg, 10 mg / kg, 30 mg / kg dose of CXT-24 on the Morris water maze behavior test in rats with chronic cerebral hypoperfusion model caused by bilateral common carotid artery occlusion (BCCAO).

[0306] Experimental method:

[0307] Preparation of bilateral common carotid artery occlusion model (BCCAO) in rats:

[0308] Male SD rats weighing 230-250 grams were placed in a cage of 5 rats each, and the room temperature was maintained at 23°C with free access to food and water. The rats were anesthetized with a Ruiweide general-purpose small animal anesthetic machine (1.5%-2.5% isoflurane), the body temperature was maintained at 37±0.5°C, and the rats were fixed in a supine position on the operating table. The left and right common carotid arteries were exposed through a midline incision in the neck and ligated. The blood supply of the middle cerebral artery was blocked. At the same time, the middle cerebral artery opening was ligated and 5 drops of gentamicin were added, and the incision was sutured and placed back in the cage. The room temperature was maintained at 23-25°C throughout the procedure. During the entire operation, the rats were placed on a heating plate at 37°C to maintain body temperature. On the day after the operation, 5 mL of sugar saline was supplemented per rat, and 5 mL of sugar saline was supplemented per rat in the morning and evening on the second and third days.

[0309] Experimental grouping and administration

[0310] The rats were randomly grouped by weight 7 days after surgery, and divided into 5 groups, 14 rats in each group, namely, sham operation group (control group), solvent CMC-Na group (model group), CXT-24 3 mg / kg group, CXT-24 10 mg / kg group, CXT-24 30 mg / kg group and butylphthalate group 200 mg / kg group, all of which were orally administered by gavage.

[0311] Morris water maze experiment

[0312] Morris water maze experiment: The device is composed of a circular pool, a hidden platform, a camera system and a trajectory analysis system. The diameter of the mouse pool is usually 90-120 cm, the pool is about 0.5 m high, and the inner surface is made of non-reflective material. The pool is divided into four quadrants by four equidistant points on the pool wall, and the hidden platform is placed in the center of one of the quadrants, with a diameter of 10-12 cm, located 1-2 cm below the liquid surface. The liquid surface is turned white by pouring 100 g of titanium dioxide, and the camera system and trajectory analysis system use Shanghai Xinsoft Supermaze+ and its attached image acquisition system.

[0313] Morris water maze experiment includes two stages of positioning navigation and spatial exploration. The positioning navigation experiment lasts for 5 days, and each day is trained twice. Each training puts the animals into the water from an arbitrary quadrant facing the pool wall and starts timing, and lasts for 60 s. If the animal successfully finds the platform within 60 s, it will stay on the platform for 30 s. Otherwise, the experimenter needs to guide it to find the platform and stay for the same time. The spatial exploration experiment is usually carried out 24 hours after the last training. Remove the hidden platform, and put the animals into the pool from the opposite side of the original platform quadrant. Record the number of times the animals cross the original platform location within 60 s, and the time spent in the original platform quadrant, etc. for analysis. After the spatial exploration experiment, put in the platform and mark the platform for visible platform experiment. Put the animals into the four quadrants in turn, and record the time and path of the animals to find the platform, to exclude the influence of vision and swimming speed on finding the platform.

[0314] Statistical method:

[0315] The data were processed by SPSS statistical analysis software and expressed as mean ± standard error (mean ± SEM). Two-way ANOVA and one-way ANOVA analysis were used, and P<0.05 was considered to be significantly different.

[0316] Experimental results:

[0317] 1. Positioning navigation experiment

[0318] (1) Escape latency: With the increase of training days, the escape latency of rats in each group gradually shortened, indicating that rats could learn to find the hidden platform. Compared with the model group, the escape latency of rats in CXT-24 10 mg / kg and 30 mg / kg treatment groups and batroxobin 200 mg / kg group was significantly shortened, among which the effect of 30 mg / kg treatment group was the most significant.

[0319] (2) Swimming path: The swimming path of rats in the sham operation group gradually tended to be straight, indicating that their spatial learning and memory ability was good. Compared with the model group, the swimming path of rats in CXT-24 10 mg / kg and 30 mg / kg treatment groups and batroxobin 200 mg / kg group tended to be straight, among which the effect of 30 mg / kg treatment group was the most significant, indicating that their spatial learning and memory ability was enhanced.

[0320] 2. Spatial exploration experiment

[0321] (1) Latency to find platform: The results of spatial exploration experiment showed that the latency to find platform of rats in the sham operation group was 22.14±5.45, the number of platform crossing of rats in the solvent control group was 45.84±5.51, and the number of platform crossing of rats in different dose CXT-24 (3 mg / kg, 10 mg / kg and 30 mg / kg) and batroxobin (200 mg / kg) treatment groups was 35.33±5.14, 31.90±6.20, 24.73±5.84 and 22.49±4.08, respectively. The results showed that there was a significant difference between the 30 mg / kg dose group of compound CXT-24 and the solvent control group (p<0.05). It is suggested that giving compound CXT-24 has a certain improvement effect on cognitive impairment of SD rats after chronic cerebral hypoperfusion.

[0322] Table 18. Effect of compound CXT-24 on spatial exploration ability of chronic cerebral hypoperfusion model rats in water maze (latency) # P<0.05 vs sham group, * P<0.05 vs model group, n=12-14

[0323] (2)Cross-platform times: Spatial exploration experiment results showed that the number of times of crossing the platform for the sham operation group was 2.0 ± 0.37, and the number of times of crossing the platform for the solvent control group was 0.57 ± 0.23. The number of times of crossing the platform for the rats in the treatment groups given different doses of CXT-24 (3 mg / kg, 10 mg / kg and 30 mg / kg) and butylphthalide (200 mg / kg) was 1.00 ± 0.23, 1.43 ± 0.43, 2.00 ± 0.41, and 2.08 ± 0.35, respectively. The results showed that the 30 mg / kg dose group of compound CXT-24 had a significant difference compared with the solvent control group (p < 0.05). It is suggested that giving compound CXT-24 has a certain improvement effect on cognitive impairment caused by chronic cerebral hypoperfusion in SD rats.

[0324] Table 19. Effect of compound CXT-24 on spatial exploration ability of chronic cerebral hypoperfusion model in water maze (cross-platform times) # P < 0.05 vs sham group, * P < 0.05 vs model group, n = 12-14

[0325] (3) Target quadrant residence time: Spatial exploration experiment results showed that the target quadrant residence time for the sham operation group was 22.90 ± 2.77, and the target quadrant residence time for the solvent control group was 13.97 ± 1.77. The target quadrant residence time for the rats in the treatment groups given different doses of CXT-24 (3 mg / kg, 10 mg / kg and 30 mg / kg) and butylphthalide (200 mg / kg) was 14.32 ± 1.85, 20.95 ± 2.03, 21.19 ± 1.76, and 15.22 ± 1.74, respectively. The results showed that the target quadrant residence time of the 10 mg / kg and 30 mg / kg dose groups of compound CXT-24 increased significantly compared with the solvent control group, and the effect of the 30 mg / kg dose group was more significant (p = 0.078). It is suggested that giving compound CXT-24 has a certain improvement effect on cognitive impairment caused by chronic cerebral hypoperfusion in SD rats. Table 20. Effect of compound CXT-24 on spatial exploration ability of chronic cerebral hypoperfusion model in water maze (target quadrant residence time) # P < 0.05 vs sham group, * P < 0.05 vs model group, n = 12-14

[0326] 3. Visible platform experiment

[0327] In the visible platform experiment, the swimming speed of the sham operation group was (157.5 ± 5.35) mm / s, the swimming speed of the solvent control group was (198.5 ± 16.12) mm / s, and the swimming speed of the CXT-24 treatment groups with different doses (3 mg / kg, 10 mg / kg and 30 mg / kg) and the treatment group with 200 mg / kg of dl-3-butylphthalide was (178.8 ± 9.11) mm / s, (196.5 ± 10.96) mm / s, (175.7 ± 9.70) mm / s, (2204.0 ± 12.5) mm / s, respectively. The results showed that there was no significant difference in swimming speed between the animals in each group.

[0328] Table 21. Effect of compound CXT-24 on swimming speed of rats in the water maze in a chronic cerebral hypoperfusion model n = 12-14

[0329] Example 12: Effect of 3 mg / kg, 10 mg / kg, 30 mg / kg doses of CXT-24 on the novel object recognition behavior of rats in a chronic cerebral hypoperfusion model.

[0330] Experimental materials and methods:

[0331] The novel object recognition test (NOR) is a classic behavioral experiment for evaluating the recognition memory ability of rodents. Based on the natural tendency of rodents to explore novel objects, the experiment compares the exploration time of familiar objects and novel objects to evaluate the recognition memory ability of animals.

[0332] Experimental device: a square open box (60 cm x 60 cm x 40 cm) made of opaque material, the bottom is divided into 16 equal-sized squares. Two new and old objects are placed inside, and a camera system and trajectory analysis system are used, Shanghai Xinsoft Supermaze+ and its attached image acquisition system.

[0333] Experimental procedure: One hour before the experiment, rats were moved to the experimental room to acclimate to the environment. The experimental apparatus and objects were wiped with 75% alcohol to remove odor interference. On the first day, rats were placed in the open field box for 5 minutes of free exploration. After the acclimation phase, rats were returned to their home cages. On the second day, two identical objects (Al and A2) were placed symmetrically in the open field box, 10 cm from the walls of the box. Rats were placed in the open field box for 10 minutes of free exploration. The time spent exploring each object (nose touching the object or less than 2 cm from the object) was recorded. After the familiarization phase, rats were returned to their home cages. On the third day, one of the familiar objects (Al) was replaced with a novel object (B), and the position remained unchanged. Rats were placed in the open field box for 10 minutes of free exploration. The time spent exploring each object was recorded. After the test phase, rats were returned to their home cages. The time spent exploring the novel object (B) and the familiar object (A2) during the test phase was calculated. The recognition index (RI) was calculated as follows: RI = (TB-TA2) / (TB+TA2) x 100%, where TB is the time spent exploring the novel object, and TA2 is the time spent exploring the familiar object.

[0334] Statistical methods:

[0335] All data were analyzed using Graph Prism 8.0.1, and all results were expressed as mean ± standard error (mean ± SEM). Statistical analysis was performed using t-test to compare differences between groups. P<0.05 was considered to be a significant difference.

[0336] Experimental results:

[0337] Effect of CXT-24 at doses of 3 mg / kg, 10 mg / kg, and 30 mg / kg on the novel object recognition behavior of rats with chronic cerebral hypoperfusion model

[0338] The novel object recognition index of the sham-operated group of rats with chronic cerebral hypoperfusion model was (59.97 ± 3.56)%, and the novel object recognition index of the solvent control group of rats was (54.68 ± 2.45)%. After treatment with different doses of CXT-24 (3 mg / kg, 10 mg / kg, and 30 mg / kg) and dl-3-butylphthalide (200 mg / kg), the novel object recognition indexes were (63.28 ± 2.85)%, (63.31 ± 3.21)%, (50.78 ± 4.03)%, and (64.93 ± 3.78)%, respectively. The novel object recognition indexes of the CXT-24 3 mg / kg and 10 mg / kg treatment groups increased, with the most significant increase in the 3 mg / kg group (p<0.05), indicating that the compound CXT-24 has a certain effect on improving the cognitive ability of rats with chronic cerebral hypoperfusion model

[0339] Table 22. Effect of CXT-24 on the new object recognition index of rats in a chronic cerebral hypoperfusion model * P<0.05 vs vehicle group, n=9-13

[0340] Example 13: Effect of CXT-24 on the content of synaptic protein (Synaptophysin) in the brain tissue of rats in a chronic cerebral hypoperfusion model

[0341] Experimental grouping

[0342] A blank control group (Control group), a BCCAO model group (Model group), and a CXT-24 30 mg / kg treatment group were set up

[0343] Experimental process

[0344] Liquid nitrogen grinding of tissue protein: The half brain tissue of the rat was ground in liquid nitrogen, and the mortar, spatula, and EP tube were pre-cooled. The rat brain tissue stored at -80°C was placed in the mortar, and one spoonful of liquid nitrogen was added for immediate grinding. The process was repeated three times. After the rat brain tissue was completely ground into powder and mixed evenly, it was loaded into a pre-cooled EP tube and stored at -80°C.

[0345] Protein quantification: 50 mg of ground brain tissue was weighed and added to 200 μL / tube RIPA after cell sonication. The BCA protein concentration determination kit was used for protein quantification. According to the kit instructions, different concentrations of standard were added to the standard curve well at 25 μL / well. The sample was diluted 25 times with PBS, and 25 μL / well was added to the detection well. After 200 μL / well of detection solution A:B was added at a ratio of 1:50 and incubation at 37°C for 30 min, the absorbance value of each well at 562 nm was immediately detected by a microplate reader. The protein concentration was calculated according to the standard curve.

[0346] Western Blot: The extracted protein supernatant was mixed with 5X protein loading buffer and placed in boiling water for 10 min of boiling water bath. After denaturation, it was cooled to room temperature. Prepare 15% separating gel and 5% concentrated gel. The prepared gel was fixed to the electrophoresis tank, and the electrophoresis solution was poured into the reservoir. The prepared protein sample and marker were added to the loading well. After loading, constant voltage electrophoresis was performed at 80V for 30 min, and then constant voltage electrophoresis was performed at 120V to the bottom of the gel, which took about 1.5h. The gel was removed and the target band was cut according to the marker. The same size PVDF membrane and filter paper as the gel were cut. The PVDF membrane was soaked in methanol for a few seconds and then soaked in the transmembrane buffer together with the filter paper. According to the black plate-fiber pad-filter paper-gel-PVDF membrane-filter paper-fiber pad-white plate, they were placed in turn, clamped and placed in the transmembrane instrument. Add enough transmembrane liquid to the transmembrane tank and start transmembrane. After transmembrane, remove the transmembrane clamp and soak the PVDF membrane in TBST containing 5% skim milk powder for 2h at room temperature. Dilute the corresponding primary antibody in blocking solution and incubate the PVDF membrane in the primary antibody incubation solution. Incubate overnight at 4°C. Wash the PVDF membrane with TBST for 3 times, 8 min each time. Dilute the corresponding HRP-labeled secondary antibody in blocking solution and incubate the PVDF membrane in the secondary antibody incubation solution. Incubate for 2h at 37°C on a shaker. Wash the PVDF membrane with TBST for 3 times, 8 min each time. Mix A and B liquids of ECL reagent at a ratio of 1:1, and drop the working solution onto the PVDF membrane. Place it in the developing instrument for development.

[0347] Statistical methods

[0348] All data statistics were performed on Graph Prism 8.0.1, and all results were expressed as mean ± standard error (mean ± SEM). Statistical analysis was performed using t-test to compare the differences between groups, and P<0.05 was considered to be a significant difference.

[0349] Experimental results

[0350] The effect of CXT-24 on the change of synaptophysin protein content in the brain tissue of chronic cerebral hypoperfusion model rats, Western Blot results showed that the gray value statistics of the control group was 1.00±0.67, and the gray value statistics of the model group was 0.66±0.03. Compared with the control group, the synaptophysin protein level of the model group was significantly decreased (p<0.001). The gray value statistics of the 30mg / kg CXT-24 treatment group was 0.80±0.06, which was significantly increased compared with the protein content of the model group (p<0.05). It can be seen that CXT-24 treatment can significantly increase the synaptophysin protein content in the brain of chronic cerebral hypoperfusion model rats, and play a therapeutic role by improving synaptic function.

[0351] Example 14: Effect of CXT-24 on the release of inflammatory factors at the transcriptional and protein levels by BV2 cells after lipopolysaccharide (LPS) stimulation.

[0352] Experimental grouping

[0353] The blank control group (Control group), LPS stimulation model group (Model group), and CXT-24 0.01 μM, 0.1 μM and 1 μM treatment groups were set up.

[0354] Experimental process

[0355] Establishment of LPS-stimulated BV2 inflammatory model: After the BV2 cells were subcultured to the logarithmic growth phase for three generations, the cells were plated in 96-well plates or 6-well plates for modeling. After the cells were cultured in the well plates for 24 hours, the control group was replaced with complete culture medium (i.e., DMEM culture medium containing 10% FBS and 1% PS), and the model and CXT-24 administration groups were replaced with starvation medium (i.e., DMEM culture medium containing 1% FBS and 1% PS), while pre-incubating CXT-24 at different concentrations (0.01 μM, 0.1 μM and 1 μM). After 6 hours, the cells were stimulated with 1 μg / mL LPS, and after 6 hours of stimulation, the supernatant or cell samples were collected and stored at -20°C.

[0356] Determination of IL-6 and TNF-α release from BV2 cells: The supernatant of the cells after LPS stimulation was collected, and the determination was performed according to the kit instructions. The corresponding capture antibody was diluted with coating buffer at a ratio of 1:250, the 96-well plate was coated, and incubated at 4°C overnight. The coating solution was discarded, and 300 μL / well of 1X wash buffer was added for washing 3 times. 200 μL / well of 1X diluent was added for blocking, and incubated at room temperature for 1 hour. The blocking solution was discarded, 300 μL / well of 1X wash buffer was added for washing 3 times, 100 μL / well of sample and gradient-diluted standard was added, and incubated at room temperature for 2 hours. 300 μL / well of 1X wash buffer was added for washing 3 times, the working detector was configured, the detection antibody and HRP were diluted with 1X diluent, 100 μL / well was added to the well plate, and incubated at room temperature for 1 hour. 100 μL / well of TMB was added, and incubated at room temperature for several minutes until the color in the plate stopped turning blue. 100 μL / well of stop solution was added to terminate the reaction, and the absorbance values of each well at 450 nm and 570 nm were immediately detected by a microplate reader. The release level of the corresponding cytokine was calculated according to the standard curve.

[0357] BV2 cell inflammatory factor transcription level change determination: (1) RNA extraction and reverse transcription: collect LPS stimulated cell precipitate, prepare TransZol UP kit, add 1 mL TransZol to each tube of cells, blow and suck evenly to turbidity, lyse the cells on ice for 10 minutes, add 200 μL RNA extraction agent, vortex for 5 minutes, 10000xg, 4°C centrifuge for 15 minutes, suck about 500 μL supernatant into a new EP tube, add equal volume of isopropanol, upside down and mix well. After standing for 10 minutes, centrifuge at 10000xg for 10 minutes. Discard the supernatant, add 1 mL, add 1 mL 75% ethanol, tap the tube bottom to suspend the precipitate, centrifuge at 7500xg for 5 minutes. Discard the supernatant, dry the precipitate until the RNA is transparent and milky white, add 20 μL RNase-free water, blow and suck 30 times, and stand at room temperature for 10 minutes. Measure the concentration of the RNA sample and perform reverse transcription. Add RNase-free water, RNA sample and DNA scavenger in turn, and use the DNA scavenging program to scavenge. After the program is completed, add reverse transcriptase for reverse transcription, and store the sample at -80°C. (2) qPCR: First, dissolve the primer dry powder, centrifuge to make the primer sink to the bottom, and add the corresponding 10 times ddH2O according to the number on the primer tube. Configure the enzyme and primer mixture, add 10 μL enzyme, 1 μL forward and reverse primer mixture and 9 μL cDNA template to each well. Vortex and check for air bubbles. According to the instructions, perform PCR detection.

[0358] Statistical method

[0359] All data statistics were performed on Graph Prism 8.0.1, and all results were expressed as mean ± standard error (mean ± SEM). Statistical analysis was performed using t-test to compare the differences between groups, and P<0.05 was considered to be a significant difference.

[0360] Experimental results

[0361] (1) The effect of CXT-24 on the release of inflammatory factors IL-6 and TNF-α at the protein level in BV2 cells after LPS stimulation.

[0362] The results showed that for IL-6, the IL-6 content of the control group cells was 12.38 ± 6.06 pg / mL, and the IL-6 content of the model group stimulated by LPS was significantly increased to 1027 ± 64.55 pg / mL (P < 0.001). The IL-6 content of the CXT-24 treatment groups with different concentrations (3 nM, 10 nM, 30 nM and 100 nM) was 774.4 ± 105.3 pg / mL, 597.9 ± 84.06 pg / mL and 594.5 ± 36.57 pg / mL and 664.0 ± 19.44 pg / mL, respectively, among which the IL-6 release amount of the CXT-24 treatment groups was significantly decreased compared with the LPS-stimulated model group, and the effect of 30 nM and 100 nM doses was the most obvious (P < 0.001).

[0363] For TNF-α, the results showed that the TNF-α content of the control group cells was 0 pg / mL, and the TNF-α content of the model group stimulated by LPS was significantly increased to 819.1 ± 71.71 pg / mL (P < 0.001). The TNF-α content of the CXT-24 treatment groups with different concentrations (3 nM, 10 nM, 30 nM and 100 nM) was 698.4 ± 60.01 pg / mL, 585.3 ± 42.45 pg / mL and 589.9 ± 29.58 pg / mL and 571.4 ± 22.98 pg / mL, respectively, among which the TNF-α release amount of the CXT-24 treatment groups was significantly decreased compared with the LPS-stimulated model group, and the effect of 30 nM and 100 nM doses was the most obvious (P < 0.01).

[0364] Table 23. Effect of different concentrations of CXT-24 on the levels of inflammatory factors in LPS-stimulated BV2 cells ### P < 0.001 compared to the Control group; *** P < 0.001, ** P < 0.01 compared to the Model group. n = 6.

[0365] (2) Effect of CXT-24 on the transcriptional level of inflammatory factors IL-6, IL-1β, TNF-α, iNOS and COX-2 released by BV2 cells after LPS stimulation.

[0366] The results showed that for IL-6, the relative level of IL-6 in the control group was 1.00 ± 0.29, and the relative level of IL-6 in the model group stimulated by LPS was significantly increased to 13.47 ± 1.54 (P < 0.01). The relative levels of IL-6 in the CXT-24 treatment groups with different concentrations (0.01 μM, 0.1 μM, and 1 μM) were 9.05 ± 1.45, 8.51 ± 1.16, and 8.21 ± 0.98, respectively. Compared with the LPS-stimulated model group, the relative levels of IL-6 in the CXT-24 treatment groups were decreased, and the effect of the 1 μM dose group was the most obvious (P < 0.05).

[0367] For IL-1β, the results showed that the relative level of IL-1β in the control group was 1.00 ± 0.33, and the relative level of IL-1β in the model group stimulated by LPS was significantly increased to 7.62 ± 1.05 (P < 0.01). The relative levels of IL-1β in the CXT-24 treatment groups with different concentrations (0.01 μM, 0.1 μM, and 1 μM) were 4.42 ± 0.85, 4.58 ± 0.30, and 4.45 ± 0.02, respectively. Compared with the LPS-stimulated model group, the relative levels of IL-1β in the CXT-24 treatment groups were decreased, and the effects of the 0.1 μM and 1 μM dose groups were the most obvious (P < 0.05).

[0368] For TNF-α, the results showed that the relative level of TNF-α in the control group was 1.00 ± 0.23, and the relative level of TNF-α in the model group stimulated by LPS was significantly increased to 3.61 ± 0.31 (P < 0.01). The relative levels of TNF-α in the CXT-24 treatment groups with different concentrations (0.01 μM, 0.1 μM, and 1 μM) were 2.56 ± 0.12, 2.10 ± 0.21, and 2.39 ± 0.30, respectively. Compared with the LPS-stimulated model group, the relative levels of TNF-α in the CXT-24 treatment groups were significantly decreased (P < 0.05).

[0369] For iNOS, the results showed that the relative level of iNOS in the control group was 1.00 ± 0.12, and the relative level of iNOS in the model group stimulated by LPS was significantly increased to 1.85 ± 0.02 (P < 0.01). The relative levels of iNOS in the CXT-24 treatment groups with different concentrations (0.01 μM, 0.1 μM, and 1 μM) were 1.08 ± 0.27, 0.62 ± 0.21, and 0.69 ± 0.07, respectively. Compared with the LPS-stimulated model group, the relative levels of iNOS in the CXT-24 treatment groups were significantly decreased, and the effects of the 0.1 μM and 1 μM dose groups were the most obvious (P < 0.001).

[0370] For COX-2, the results showed that the relative level of COX-2 in the control group was 1.00 ± 0.25, and the relative level of COX-2 in the model group stimulated by LPS was significantly increased to 4.91 ± 0.26 (P < 0.001). The relative levels of COX-2 in the CXT-24 treatment groups with different concentrations (0.01 μM, 0.1 μM, 1 μM) were 22.57 ± 0.35, 3.14 ± 0.50, and 2.14 ± 0.31, respectively. The relative levels of COX-2 in the CXT-24 treatment groups were significantly decreased compared with the LPS-stimulated model group (P < 0.05). The above results suggest that the compound CXT-24 has a significant anti-inflammatory effect.

[0371] Table 24. Effect of different concentrations of CXT-24 on the relative mRNA levels of inflammatory factors in LPS-stimulated BV2 cells ## P < 0.01 compared with the Control group; * P < 0.05, * P < 0.05 compared with the Model group. n = 3.

[0372] Table 25. Effect of different concentrations of CXT-24 on the relative mRNA levels of inflammatory factors in LPS-stimulated BV2 cells ## P < 0.01 compared with the Control group; * P < 0.05, ** P < 0.01 compared with the Model group. n = 3.

[0373] Example 15: Improvement of cognitive dysfunction in APP / PS1-AD transgenic mice by compound CXT-24 in Morris water maze test and step-down test

[0374] 1. Animal grouping and administration

[0375] Animals: APP / PS1-AD transgenic mice and age-matched control WT mice were bred in our laboratory.

[0376] Grouping and administration: divided into 4 groups, WT solvent control group, n = 8, administered with solvent CMC-Na; APP / PS1 solvent control group, n = 12, administered with solvent CMC-Na; APP / PS1 CXT-24 10mg / kg group, n = 14, administered with CXT-24 10mg / kg; APP / PS1 CXT-24 20mg / kg group, n = 13, administered with CXT-24 20mg / kg. Each group was administered by gavage, the administration volume was 0.1mL / 10g, the administration started from 7 months old, 6 times a week, and the Morris water maze experiment and step-down test were performed at the 19th week of administration.

[0377] 2. Method

[0378] (1) Morris water maze experiment

[0379] The device of the Morris water maze experiment consists of a circular pool, a hidden platform, a camera system and a trajectory analysis system. The mouse pool is 120cm in diameter and is divided into 4 quadrants. The hidden platform is located in the center of one of the quadrants and is 12cm in diameter, located 1cm below the liquid surface. The water temperature in the pool is maintained at 23±1℃ during the experiment. Warm water dissolving titanium dioxide is poured into the pool to make the liquid surface white, which can form a sharp contrast with the black mouse. Adjust the light to make the camera positioning system accurately capture the mouse swimming path. The pool is covered with curtains around the pool, and a certain number of markers are set to provide reference for the mouse's own spatial exploration.

[0380] The Morris water maze experiment is divided into two stages: positioning navigation and spatial exploration. The positioning navigation experiment lasts for 7 days. The mouse is trained 4 times a day, and the mouse is put into the water from an arbitrary quadrant facing the pool wall and the timing starts. If the mouse successfully finds the hidden platform within 60s, the time is recorded as the escape latency, otherwise the latency is calculated as 60s. Whether the mouse finds the platform within 60s or not, it is allowed to stay on the platform for 30s at the end to deepen the memory of the platform position. The spatial exploration experiment is performed on the 8th day. At this time, the platform is removed, and the mouse is put into the water from the opposite quadrant of the previous platform position, and freely explores for 60s. The time spent in the original platform position and the number of times the mouse crosses the platform are recorded.

[0381] After the spatial exploration experiment, the cueing experiment is performed. At this time, the hidden platform is put back into the pool, and a small flag is fixed on it. It is observed whether the animal can directly locate the position of the hidden platform under the prompt of the small flag. If the animal fails to complete this task more than twice, it may have problems in vision, motor ability and escape motivation, etc. The animal is excluded.

[0382] (2) Step-down test

[0383] The apparatus of mouse step-down test consists of test box, electric grid and insulating step-down. The test box is composed of several square compartments with the bottom area of 10x10 cm. Several electric grids are arranged side by side under each compartment and connected with electric stimulator. A 4x4x4 cm insulating step-down is placed in the corner of each compartment for the mouse to avoid electric shock. The step-down test is divided into three stages: adaptation, training and test. On the first day, the mouse is placed on the platform with the face to the corner and allowed to freely explore the compartment for 5 min to adapt to the environment. On the second day, the mouse is again placed on the insulating platform with the face to the corner, and the electric grid is turned on with 62 V and 0.8 mA current. When the mouse jumps off the insulating platform and touches the electric grid with the limbs, it will be shocked. The mouse must jump back to the platform to avoid injury. The training period lasts for 3 min, and the number of electric shocks received by each mouse is recorded. If the number is less than 3, the mouse is pushed off the step-down platform in the last minute to ensure that the number of electric shocks is at least 3 to ensure the training effect. On the third day, the mouse is again placed on the insulating platform, and the time for the mouse to jump off the platform for the first time is observed and recorded as the step-down latency. The number of times the mouse jumps off the platform during the test period is recorded as the error number. After each group of operations, 75% alcohol is sprayed to remove the odor.

[0384] (3) Statistical analysis of data

[0385] The data are expressed as mean ± SD, and the Student's t-test method is used for statistical analysis. "#" indicates comparison with the control group, where #p<0.05, ##p<0.01, and "*" indicates comparison with the model group, where *p<0.05, **p<0.01.

[0386] 3. Experimental results

[0387] (1) Compound CXT-24 can improve the cognitive dysfunction of APP / PS1 mice in the Morris water maze and step-down test

[0388] The results show that, in the Morris water maze experiment, compared with the WT Control group, the APP / PS1 Control group showed obvious spatial memory impairment, the escape latency in the seven-day positioning navigation training was significantly increased, the number of platform crossings in the spatial exploration stage was significantly reduced, and the platform crossing latency was significantly increased; and after administration, the spatial memory impairment of the APP / PS1 mice was improved to a certain extent, the escape latency in the seven-day positioning navigation training was significantly reduced, the number of platform crossings in the spatial exploration stage was significantly increased, and the platform crossing latency was significantly reduced. In the step-down test, compared with the WT Control group, the latency of the APP / PS1 Control group to jump off the insulating platform was significantly shortened, and the number of errors was significantly increased; after administration, the latency of the APP / PS1 mice to jump off the insulating platform was significantly increased, the number of errors was significantly reduced, and the learning and memory impairment was significantly improved.

[0389] In summary, the results of the present embodiment suggest that compound CXT-24 can improve the cognitive dysfunction of APP / PS1 mice in the Morris water maze and step-down test.

[0390] Example 16: Improvement of compound CXT-24 on the deposition of Aβ plaques and neuroinflammation in the cerebral cortex and hippocampus of APP / PS1 mice

[0391] 1. Preparation of mouse brain tissue frozen sections

[0392] After the animal behavior experiment in Example 1 was completed, the sample was taken. The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate, and the brain was taken by heart perfusion and decapitation. Half of the brain was fixed in 4% PFA solution for 24 hrs, and then dehydrated in 10%-20%-30% sucrose solution gradient. The soaking volume of PFA and sucrose solution should be at least 5 times the volume of the mouse half brain. After the brain tissue was settled in the 30% sucrose solution, the half brain was taken out and embedded with OCT embedding agent, and stored at -80℃. The embedded half brain was taken out from the -80℃ refrigerator and fixed on the sample holder of the freezing microtome with the help of OCT embedding agent. Adjust the temperature of the freezing microtome box, the temperature of the sample holder and the angle of the sample holder. The embedded brain tissue was cut into sagittal sections with a thickness of 8 μm. The brain slices with complete hippocampal development were collected and flattened on glass slides and stored at -80℃.

[0393] 2. Methods

[0394] (1) Immunofluorescence staining

[0395] The frozen sections were taken out from -80 °C, and after 15 min of equilibration at room temperature, they were rinsed with PBS for 3 times, 3 min each time. The sections were incubated with 10% blocked sheep serum (containing 0.5% Triton-X100 to increase membrane permeability) for 1 hrs to block non-specific binding sites. The appropriate dilution of the primary antibody (prepared in PBS) was added, and the sections were incubated at 4 °C overnight. The primary antibody was discarded, and the sections were rinsed in PBS for 3 times, 3 min each time. The fluorescent secondary antibody was added at a dilution of 1:200, and the sections were incubated at 37 °C for 1 h in the dark. The secondary antibody was discarded, and the Hoechst staining solution was added at a dilution of 1:10000, and the sections were incubated at 37 °C for 15 min in the dark. The sections were rinsed in PBS for 3 times, 3 min each time. The sections were passed through water once, and 90% glycerol was added to the tissue, and a cover glass was added, and nail polish was used to seal the cover glass.

[0396] (2) Fluorescence microscope observation

[0397] The Aβ plaques in the hippocampus and cortex regions of the brain tissue sections, as well as the activation of astrocytes and microglia cells, were observed under 4 and 10 times objectives. Five animals in each group were subjected to immunofluorescence staining of frozen sections, and five brain slices were taken for each animal. At least 4 fields of view were selected for each brain slice for imaging and counting. Image J software was used for image analysis and processing.

[0398] (3) Statistical analysis of data

[0399] The data are presented as mean ± SD, and the Student’s t-test was used for statistical analysis. "#" indicates comparison with the control group, in which ###P<0.001. "*" indicates comparison with the model group, in which *P<0.05, **P<0.01.

[0400] 3. Experimental results

[0401] (1) Compound CXT-24 can reduce Aβ plaque deposition and neuroinflammation in the cortex and hippocampus regions of APP / PS1 mice

[0402] The results of this example show that the Aβ deposition in the hippocampus and cortex regions of APP / PS1 mice was reduced after administration, and the expression of the astrocyte marker protein GFAP and the microglia marker protein Iba-1 was also significantly reduced.

[0403] In summary, the results of this example suggest that compound CXT-24 can significantly improve Aβ plaque deposition and neuroinflammation in the cortex and hippocampus regions of APP / PS1 mice.

[0404] Example 17: Compound CXT-24 can reduce the release of inflammatory factors in BV2 cells stimulated by LPS+Aβ 1-42 ​

[0405] 1. Cells: BV2 cells.

[0406] 2. Methods

[0407] (1) Preparation of cell supernatant samples

[0408] When the growth density of BV2 cells was 80-90%, the cells were inoculated in a 96-well plate at a density of 2*10 5 cells / mL and incubated for 24 hours. Subsequently, the original complete culture medium was discarded, low serum culture medium containing 2% FBS was added for starvation for 6 hours, and then the culture plate was divided into the following 8 groups: the first group was a negative control group, 90 μL of culture medium containing 2% FBS and 10 μL of blank medium were added; the second group was a model group, 90 μL of low serum culture medium and 10 μL of 1 μg / mL LPS+100 μM Aβ 1-42 mixed working solution were added; groups 3-8 were drug administration groups, 80 μL of low serum culture medium and 10 μL of 1 μg / mL LPS+100 μM Aβ 1-42 mixed working solution were added to each well, respectively, and 10 μL of CXT-24 working solution at different concentration gradients: 30 nM, 100 nM, 300 nM, 1 μM, 3 μM or 10 μM was added, and incubated for 6 hours. Subsequently, the cell culture supernatant was aspirated and used for detection of inflammatory factors IL-6 and TNF-α.

[0409] (2) ELISA detection of the secretion levels of IL-6 and TNF-α in the supernatant

[0410] The measurement process was performed according to the instructions of the ELISA kit.

[0411] (3) Statistical analysis of data

[0412] The data were expressed as mean ± standard deviation (mean ± SD), and Student's t-test was used for statistical analysis. "#" indicates comparison with the control group, in which ###P<0.001; "*" indicates comparison with the model group, in which *P<0.05, **P<0.01, ***P<0.05.

[0413] 3. Results

[0414] (1) Compound CXT-24 can reduce the release of inflammatory factors in BV2 cells stimulated by LPS+Aβ 1-42

[0415] The results of this example show that, compared with the control group, the secretion levels of IL-6 and TNF-α in the model group were significantly increased, and compared with the model group, the secretion levels of IL-6 and TNF-α were significantly reduced after administration of CXT-24 for 6 hours. ​

[0416] In summary, the results of the present example suggest that compound CXT-24 can reduce LPS + Abeta 1-42 stimulation of BV2 cells to release inflammatory factors.

[0417] Example 18: Compound CXT-24 and its derivatives can improve the survival of SKN cells under Abeta 1-42 damage conditions

[0418] 1. Cells: SK-N-SH cells.

[0419] 2. Methods

[0420] (1) MTT assay of cell survival rate

[0421] SK-N-SH cells were seeded in a 96-well plate at a density of 1*10 5 μM Abeta 1-42 and different concentrations of CXT-24 or 10 μM CXT-24 series derivatives were added for incubation for 24 h, then 10 μL MTT solution (5 mg / mL) was added to each well of the cultured cells, incubated for 4 h, the supernatant was discarded, 150 μL DMSO solution was added to each well, and the absorbance value was measured at 570 nm after dissolution.

[0422] (2) Statistical analysis of data

[0423] The data are expressed as mean ± SD, and the statistical analysis was performed using Student's t-test. "#" indicates comparison with the control group, and ###P<0.001, "*" indicates comparison with the model group, and ***P<0.001.

[0424] 3. Experimental results

[0425] (1) Compound CXT-24 and its derivatives can improve the survival of SKN cells under Abeta 1-42 damage conditions

[0426] The results show that compared with the control group, the cell survival rate of the model group was significantly reduced, and compared with the model group, the administration of CXT-24 10 μM can significantly improve the cell survival. And as shown in Table 26, many of its series derivatives can improve cell survival.

[0427] Table 26. Effect of CXT-24 and its derivatives on the cell survival rate of Abeta 1-42 induced neuronal damage (n=3)

[0428] Example 19: CXT-24 and CXT-5 have protective effects on SOD1 G93A and TDP43 M337V stably transfected cells

[0429] This example found that the survival of stably transfected cells was significantly reduced compared with the corresponding empty vector control cells, and that CXT-24 and CXT-5 had a significant protective effect on SOD1 G93A and TDP43 M337V stably transfected cells 24 hours after administration, specifically 10 μM CXT-24 and CXT-5 significantly improved the survival rate of the cells.

[0430] 1. Cells and plasmids

[0431] Cell lines: SOD1 G93A and TDP43 M337V

[0432] The NSC-34 cell line was purchased from Shanghai Hongshun Biotechnology Co., Ltd.

[0433] The SOD1 G93A stably transfected cell line and the pEGFP empty vector control cell line were constructed by the laboratory.

[0434] The TDP43 M337V stably transfected cell line and the pCI empty vector control cell line were constructed by the laboratory.

[0435] 2. Methods

[0436] (1) MTT method for determining cell survival rate

[0437] The pEGFP empty vector control cells and SOD1 G93A stably transfected cells were inoculated in a 96-well plate at a density of 5*10 4 μL of blank medium was added to each well. 10 μL of blank medium was added to the pEGFP empty vector control cell and SOD1 G93A model group cell wells, and 10 μL of medium containing CXT-24 and CXT-5 was added to the SOD1 G93A dosing group cell wells to make the final concentration in each dosing group well 0.1, 1 and 10 μM. After 4 hours and 24 hours in the incubator, 10 μL of MTT was added to each well, and after another 4 hours of incubation in the incubator, 100 μL of triplex solution was added to each well. After overnight dissolution at 37°C, the absorbance value was measured at 570 nm.

[0438] The pCI empty vector control cells and TDP43 M337V stably transfected cells were inoculated in a 96-well plate at a density of 5*10 4The cells were inoculated in 96-well plates at a density of 1 x 104 / mL, 100 μL per well, and placed in an incubator for culture. After 24 hours of culture, the medium was replaced, and 90 μL of blank medium was added to each well. 10 μL of blank medium was added to the wells of the pCI empty vector control cells and the TDP43 M337V model cells; 10 μL of medium containing CXT-24 and CXT-5 was added to the wells of the TDP43 M337V cells of each drug administration group, so that the final concentration in each well was 0.1, 1 and 10 μM. After 24 hours of incubation in the incubator, 10 μL of MTT was added to each well, and after 4 hours of incubation in the incubator, 100 μL of trihydrochloride was added to each well. After overnight dissolution at 37°C, the absorbance value was measured at 570 nm.

[0439] (2) Statistical analysis of data

[0440] The data are expressed as mean ± SD, and the Student's t-test was used for statistical analysis. "#" indicates that the model cells were compared with the empty vector control cells (pEGFP and pCI), and #P<0.05, ##P<0.01. "*" indicates that the cells of each drug administration group were compared with the model cells, and *P<0.05, **P<0.01.

[0441] 3. Results

[0442] The results of this example show that the survival rate of SOD1 G93A cells and TDP43 M337V cells was significantly lower than that of the empty vector control cell group, and that the administration of 10 μM CXT-24 and CXT-5 significantly improved the survival rate of SOD1 G93A cells and TDP43 M337V cells.

Claims

A class of pyrano-carbazole oxime derivatives represented by general formula I or a pharmaceutically acceptable salt thereof: R1, R2, independently of each other, can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, OCF3; R3is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3. a class of pyrano-carbazole nitrones represented by general formula II or a pharmaceutically acceptable salt thereof: R1, R2, independently of each other, can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, OCF3; R3is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3. R4is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, C(CH3)3, Ph, Bn. The compound according to any one of claims 1 and 2, or a pharmaceutically acceptable salt thereof, characterized in that, The compounds are selected from the group consisting of: A pharmaceutical composition comprising, as an effective ingredient, a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient. Pharmaceutical composition according to claim 4, characterized in that The pharmaceutically acceptable carrier is selected from microcapsules and microspheres, nanoparticles or liposomes. Use according to claim 5, characterized in that The pharmaceutical composition is selected from injections, tablets, capsules, pills, granules, oral liquids, suspensions, controlled-release preparations, subcutaneous implants, nanosuspensions. Use of a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of an anti-inflammatory drug. Use according to claim 7, characterized in that The inflammation is selected from respiratory system inflammation, bone and joint system inflammation, skin inflammation and autoimmune diseases. Use of a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating stroke. Use of a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating pain. Use of a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating brain trauma. Use of a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating post-stroke depression. Use of a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating vascular dementia and cerebral small vessel disease. Use of a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating Alzheimer's disease. Use of a compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the prevention or treatment of amyotrophic lateral sclerosis.

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