Class of magnolol / honokiol nitrone derivatives and use thereof

Abstract

The present invention belongs to the technical field of medicine. Disclosed are a class of magnolol / honokiol derivatives, a preparation method therefor, a pharmaceutical composition thereof and the use thereof. Specifically, disclosed in the present invention are magnolol / honokiol nitrone derivatives as represented by general formulas (I) and (II). Such derivatives are prepared by means of artificial synthesis. Further provided are a pharmaceutical composition containing same, and the use thereof against inflammation, against cerebral infarction, in the treatment of amyotrophic lateral sclerosis, and against cerebral trauma.

Description

Magnolol / norbuzlangaol nitrones derivatives and uses thereof

[0001] This application is based on and claims priority to Chinese Patent Application No. 202411239214.4, filed on September 5, 2024, Chinese Patent Application No. 202411557493.9, filed on November 4, 2024, Chinese Patent Application No. 202411557645.5, filed on November 4, 2024, and Chinese Patent Application No. 202411848703.X, filed on December 16, 2024, the contents of which are incorporated herein in their entirety. TECHNICAL FIELD

[0002] The present application relates to the field of natural medicine and medicinal chemistry, in particular to a class of magnolol / norbuzlangaol nitrones derivatives. The present application also discloses the preparation method, pharmaceutical composition and use in treating diseases of the derivatives. BACKGROUND

[0003] Magnolol / norbuzlangaol have a wide range of biological activities, and the present application introduces oxime and nitrones on the nucleus of magnolol / norbuzlangaol to obtain a series of derivatives. The 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 compounds and their preparations 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 compounds of the present application can be used for preventing and / or treating cerebral infarction. (3) The compounds of the present application can significantly improve the survival rate of SOD1 G93A and TDP43 M337V cells. Therefore, the compounds of the present application can be used for preventing or treating amyotrophic lateral sclerosis. (4) The compounds and their preparations of the present application show obvious therapeutic effect on the traumatic brain injury model (TBI) of SD rats. Therefore, the compounds of the present application can be used for preventing and / or treating brain trauma. SUMMARY

[0004] The technical problem solved by the present application is to provide a class of magnolol / norbuzlangaol derivatives and uses thereof.

[0005] The first aspect of the technical solution of the present application provides a magnolol nitrones derivative represented by general formula I or a pharmaceutically acceptable salt thereof:

[0006] R1 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3;

[0007] R2 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, t-Bu, Ph, Bn.

[0008] The present application relates to the naphtholactone derivatives shown in the general formula II or pharmaceutically acceptable salts thereof:

[0009] R3, R4 are independently selected from H or and R3, R4 are not H simultaneously; R5 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, t-Bu, Ph, Bn. Some compounds of the present application are structured and numbered as follows:

[0010] The second aspect of the present application is to provide a compound represented by the general formula I, which can be prepared by the following method:

[0011] The compound represented by the general formula II of the present application can be prepared by the following method:

[0012] Wherein, the reaction condition (a) is polyformaldehyde, magnesium chloride, triethylamine, solvent anhydrous tetrahydrofuran, heating at 60 degrees; reaction condition (b) is R2NHOH, triethylamine, sodium sulfate, solvent ethanol, heating at 90 degrees; reaction condition (c) is RI, potassium carbonate, solvent tetrahydrofuran, room temperature; reaction condition (d) is i: reactant urotropine, solvent glacial acetic acid, heating at 130 degrees; II: 33% sulfuric acid, heating at 100 degrees. Reaction condition (e) is 35% sodium hydroxide, solvent chloroform, heating at 60 degrees.

[0013] In the above compounds, the definition of R1, R2 is the same as that of R1, R2 in the general formula I.

[0014] I(a) includes compounds such as HK-1, HK-2, HK-3, HK-4, HK-5, etc.

[0015] I(b) includes compounds such as HK-6, HK-7, HK-8, HK-9, HK-10, etc.

[0016] II(a) includes compounds such as HL-1, HL-2, HL-3, HL-4, HL-5, etc.

[0017] II(b) includes compounds such as HL-6, HL-7, HL-8, HL-9, HL-10, etc.

[0018] II(c) includes compounds such as HL-11, HL-12, and the like.

[0019] A third aspect of the present application is to provide a pharmaceutical composition of the compound of the first aspect as an active ingredient. The pharmaceutical composition is prepared according to the methods known in the art. The compound of the present application can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants to form any dosage form suitable for human or animal use. The content of the compound of the present application in its pharmaceutical composition is usually 0.1-95% by weight.

[0020] 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.

[0021] 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 tablets (including ordinary tablets, enteric-coated tablets, chewing tablets, dispersible tablets, effervescent tablets, oral disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, pellets, dripping pills, suppositories, films, patches, gas (powder) mist, spray, etc.; the semi-solid dosage form can be ointment, gel, paste, etc.

[0022] The compound of the present application can be prepared into ordinary preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle drug delivery systems.

[0023] In order to prepare the compound of the present application into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrating agents, 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, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrating agents 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 dodecyl sulfonate, etc.; the lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0024] The tablets can be further coated with sugar, film, enteric, or double or triple layer tablets.

[0025] To prepare the administration unit into a capsule, the effective component, the compound of the present application, can be mixed with diluents, glidants, and the mixture can be directly put into a hard or soft capsule. The effective component, the compound of the present application, can also be mixed with diluents, binders, disintegrants to prepare granules or pellets, and then put into a hard or soft capsule. The various diluents, binders, wetting agents, disintegrants, glidants used for preparing the tablets of the compound of the present application can also be used for preparing the capsules of the compound of the present application.

[0026] To prepare the injection of the compound of the present application, water, ethanol, isopropyl alcohol, propylene glycol or their mixture can be used as solvent, and appropriate amount of solubilizers, co-solvents, pH adjustors, osmotic pressure adjustors commonly used in the art can be added. The solubilizers or co-solvents 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 a freeze-dried powder injection is prepared, mannitol, glucose, etc. can also be added as a supporting agent.

[0027] In addition, if necessary, colorants, preservatives, flavors, taste correctors or other additives can also be added to the pharmaceutical preparation.

[0028] To achieve the purpose of medication and enhance the therapeutic effect, the pharmaceutical or pharmaceutical composition of the present application can be administered by any known administration method.

[0029] The administration dose of the pharmaceutical composition of the compound of the present application can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the administration route and the dosage form, etc. Generally, the suitable dose 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 dose can be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the doctor and the administration regimen including the use of other therapeutic agents.

[0030] The compound or composition of the present application can be taken alone or in combination with other therapeutic or symptomatic drugs. When the compound of the present application has a synergistic effect with other therapeutic drugs, the dose thereof should be adjusted according to the actual situation.

[0031] 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.

[0032] In particular, the use of the compound or pharmaceutically acceptable salt of the first aspect in the manufacture of a medicament for anti-inflammatory, preventing or treating cerebral infarction, preventing or treating amyotrophic lateral sclerosis, preventing or treating brain trauma is provided. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1: Effect of some compounds on the production of NO content by LPS-stimulated macrophage Raw264.7 (means ± S.E.M, n = 3)

[0034] Figure 2: Effect of some compounds (different from Figure 1) on the production of NO content by LPS-stimulated macrophage Raw264.7 (means ± S.E.M, n = 3).

[0035] Figure 3: HL-3 improves the survival rate of SK-N-SH cells after oxygen-glucose deprivation / reoxygenation injury (OGDR). The difference in survival between non-injured control group SK-N-SH cells and OGDR-injured SK-N-SH cells, and the effect of 0.1-10 μM HL-3 on the survival of OGDR-injured SK-N-SH cells when administered to SK-N-SH cells at 4h of oxygen-glucose deprivation injury and 4h of reoxygenation were detected by MTT method. "#" indicates comparison with the empty load group, and "*" indicates comparison with the model group, wherein # P<0.05, ## P<0.01, "*" indicates comparison with the model group, wherein * P<0.05.

[0036] Figure 4: Therapeutic effect of 0.1-10 mg / kg HL-3 on pMCAO rats. A: Effect of HL-3 on the infarct volume of SD rats after pMCAO. B: Statistical results of the effect of HL-3 on the infarct volume of SD rats after pMCAO. C: Effect of compound HL-3 on the behavioral impairment of SD rats after pMCAO (Bederson score). D: Effect of compound HL-3 on the behavioral impairment of SD rats after pMCAO (mNSS score). "*" indicates comparison with the model group, wherein * P<0.05.

[0037] Figure 5: Therapeutic effect of 0.1-10 mg / kg HL-3 on tMCAO rats. A: Effect of HL-3 on the infarct volume of SD rats after tMCAO. B: Statistical results of the effect of HL-3 on the infarct volume of SD rats after tMCAO. C: Effect of compound HL-3 on the behavioral impairment of SD rats after tMCAO (Bederson score). D: Effect of compound HL-3 on the behavioral impairment of SD rats after tMCAO (mNSS score). "*" indicates comparison with the model group, wherein * P<0.05, ** P<0.01.

[0038] Figure 6: 0.1-10 μΜ HL-3 improves the 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 μΜ HL-3 on the survival of SOD1 G93A stable cells after 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 μΜ HL-3 on the survival of TDP43 M337V stable cells after 24 hours. "#" indicates comparison with the empty vector group, where #P<0.05, ##P<0.01, and "*" indicates comparison with the model group, where *P<0.05.

[0039] Figure 7: HL-3 significantly improves the neurological function score of brain injury rats. ###P<0.001 compared with the sham operation group; *P<0.05 compared with the brain injury group.

[0040] Figure 8: HL-3 significantly improves the fall latency of brain injury rats in the rotarod test. ###P<0.001 compared with the sham operation group; *P<0.05 compared with the brain injury group. DETAILED DESCRIPTION

[0041] Synthesis experiment:

[0042] Example 1 Preparation of 3-aldehyde-substituted magnolol

[0043] Magnolol (200 mg, 1 eq) was dissolved in tetrahydrofuran, and paraformaldehyde (90 mg, 4 eq), magnesium chloride (214 mg, 3 eq), and triethylamine (104 ul, 1 eq) were added. The mixture was heated to 60 degrees and stirred for 10 hours, and the reaction was detected by TLC. 10 ml of water was added to quench the reaction, and the mixture was extracted with ethyl acetate (5 ml x 3) three times. The combined ethyl acetate layer was 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 (petroleum ether: ethyl acetate = 20:3) to obtain 138 mg of yellow oil with a yield of 62%.

[0044] ESI-MS (m / z): 295.1 [M+H] + .

[0045] Example 2 Preparation of 3-methyl nitro-substituted magnolol (HK-1)

[0046] Intermediate 1 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then N-methylhydroxylamine hydrochloride (56.6 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 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), 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 (dichloromethane:methanol = 20:1) to obtain a yellow powder of 98 mg at a yield of 89%.

[0047] 1 H NMR (400 MHz, CDCl3) δ 14.39 (s, 1H), 7.59 (s, 1H), 7.35 (d, J = 2.2 Hz, 1H), 7.13 (dd, J = 8.2, 2.2 Hz, 1H), 7.07 (d, J = 1.9 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.92 (d, J = 2.1 Hz, 1H), 6.03 - 5.90 (m, 2H), 5.14 - 5.03 (m, 4H), 3.89 (s, 3H), 3.37 (t, J = 7.5 Hz, 4H).

[0048] 13 C NMR (100 MHz, CDCl3) δ 154.19, 153.14, 142.95, 138.07, 137.82, 137.07, 132.58, 131.75, 131.61, 131.25, 130.68, 129.64, 126.96, 118.94, 116.97, 116.50, 115.61, 51.96, 39.62, 39.05.

[0049] ESI-MS (m / z): 324.2 [M+H] + .

[0050] Example 3 Preparation of 3-isopropyl nitroketone-substituted magnolol (HK-2)

[0051] Intermediate 1 (100 mg, 1 eq), N-isopropylhydroxylamine hydrochloride (75.6 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) were used in accordance with the synthesis method of Example 2, and a yellow oil of 104 mg was obtained at a yield of 87%.

[0052] 1H NMR (400 MHz, CDC13) δ 14.58 (s, 1H), 7.76 - 7.69 (m, 1H), 7.66 (s, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.12 (dd, J = 8.2, 2.2 Hz, 1H), 7.07 (d, J = 2.2 Hz, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.94 (d, J = 2.2 Hz, 1H), 6.06 - 5.89 (m, 2H), 5.09 (ddt, J = 16.2, 3.6, 1.7 Hz, 4H), 4.29 (m, 1H), 3.37 (t, J = 7.3 Hz, 4H), 1.53 (d, J = 6.4 Hz, 6H).

[0053] 13 C NMR (100 MHz, CDC13) δ 154.36, 153.24, 139.60, 138.11, 137.43, 137.19, 132.42, 131.77, 131.39, 131.25, 130.61, 129.51, 127.10, 118.88, 117.13, 116.37, 115.54, 66.54, 39.61, 39.08, 21.06.

[0054] ESI-MS (m / z): 352.2 [M+H] + .

[0055] Example 4 Preparation of 3-tert-Butyl nitro substituted magnolol (HK-3)

[0056] Intermediate 1 (100 mg, 1 eq), N-tert-butylhydroxylamine (60.4 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) were used according to the synthetic procedure of Example 2 to give 107 mg of yellow oil in 86% yield.

[0057] 1 H NMR (400 MHz, CDC13) δ 14.56 (s, 1H), 7.79 (s, 1H), 7.35 (d, J = 2.1 Hz, 1H), 7.12 (dd, J = 8.2, 2.3 Hz, 1H), 7.09 - 7.06 (m, 1H), 7.02 - 6.96 (m, 2H), 6.04 - 5.89 (m, 2H), 5.13 - 5.03 (m, 4H), 3.38 (t, J = 5.5 Hz, 4H), 1.64 (s, 9H).

[0058] 13C NMR (100 MHz, CDC13) δ 154.63, 153.29, 138.55, 138.15, 137.28, 137.26, 132.32, 131.94, 131.27, 131.22, 130.75, 129.45, 127.16, 118.84, 117.38, 116.31, 115.50, 70.41, 39.61, 39.12, 28.38.

[0059] ESI-MS (m / z): 366.2 [M+H] + .

[0060] Example 5 Preparation of 3-phenyl nitroso substituted magnolol (HK-4)

[0061] Intermediate 1 (100 mg, 1 eq), phenyl hydroxylamine (74.0 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) were taken in reference to the synthetic procedure of Example 2 to get 86 mg of orange powder with 66% yield.

[0062] 1 H NMR (400 MHz, CDC13) δ 14.57 (s, 1H), 8.15 (s, 1H), 7.79 (dd, J = 6.9, 3.0 Hz, 2H), 7.56 - 7.50 (m, 3H), 7.41 (d, J = 2.1 Hz, 1H), 7.14 (dd, J = 8.2, 2.2 Hz, 1H), 7.11 - 7.06 (m, 2H), 7.02 (d, J = 8.2 Hz, 1H), 5.98 (tdt, J = 16.7, 9.9, 6.7 Hz, 2H), 5.10 (dt, J = 17.8, 7.7 Hz, 4H), 3.40 (d, J = 6.8 Hz, 4H).

[0063] 13 C NMR (100 MHz, CDC13) δ 154.60, 153.10, 145.56, 141.92, 138.36, 137.98, 136.91, 132.51, 132.48, 131.74, 131.16, 130.87, 130.83, 129.58, 129.54, 126.84, 121.89, 118.87, 117.26, 116.49, 115.52, 39.53, 38.97.

[0064] ESI-MS (m / z): 386.2 [M+H] + .

[0065] Example 6 Preparation of 3-benzyl nitroso substituted magnolol (HK-5)

[0066] Intermediate 1 (100 mg, 1 eq), N-benzylhydroxylamine (83.5 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) were added according to the synthetic procedure of Example 2 to give 102 mg of yellow powder in 75% yield.

[0067] 1 H NMR (400 MHz, CDC13) δ 14.38 (s, 1H), 7.56 (s, 1H), 7.46 (s, 5H), 7.33 (d, J = 2.1 Hz, 1H), 7.12 (dd, J = 8.2, 2.2 Hz, 1H), 7.06 (d, J = 2.2 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 6.86 (d, J = 2.2 Hz, 1H), 6.06 - 5.86 (m, 2H), 5.11 - 5.03 (m, 6H), 3.35 (dd, J = 16.8, 6.7 Hz, 4H).

[0068] 13 C NMR (100 MHz, CDC13) δ 154.25, 153.15, 142.06, 138.07, 137.80, 137.09, 132.50, 132.08, 131.95, 131.54, 131.24, 130.64, 129.76, 129.59, 129.46, 129.38, 126.96, 118.90, 116.91, 116.42, 115.58, 68.63, 39.60, 39.02.

[0069] ESI-MS (m / z): 400.2 [M+H] + .

[0070] Example 7 Preparation of 4'-methoxymagnolol

[0071] Magnolol (200 mg, 1 eq) was dissolved in tetrahydrofuran, iodomethane (117 μl, 2.5 eq) and potassium carbonate (260 mg, 2.5 eq) were added, stirred at room temperature overnight. After the reaction was completed, 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, rotary evaporated the ethyl acetate to give the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:3) to give 196 mg of yellow oil in 93% yield.

[0072] ESI-MS (m / z): 281.2 [M+H] + .

[0073] Example 8 Preparation of 3- aldehyde-4'-methoxy substituted magnolol

[0074] Weighed hexamethylenetetramine (urotropin) (150 mg, 1.5 eq) into 2 ml glacial acetic acid, stirred the mixture to be uniform, then added the mixture into a round bottom flask containing intermediate 2 (200 mg, 1 eq), slowly warmed to 130 degrees, constant temperature stirring for 3 h. Cooled to 100 degrees, added 33% mass fraction of dilute sulfuric acid solution, continued to stir for 1 h, stopped the reaction. Added 10 ml water to quench the reaction, extracted with ethyl acetate (5 ml x 3) for three times, combined the ethyl acetate layer, dried with anhydrous magnesium sulfate, filtered, rotary evaporated the ethyl acetate to get the crude product. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:3) to get 193 mg of yellow oil, yield 88%.

[0075] ESI-MS (m / z): 309.1 [M+H] + .

[0076] Example 9 Preparation of 3-methyl nitro ketone-4'-methoxy substituted magnolol (HK-6)

[0077] Dissolved intermediate 3 (100 mg, 1 eq) in 2 ml ethanol, then added N-methyl hydroxylamine hydrochloride (54 mg, 2 eq), triethylamine (90 ul, 2 eq), anhydrous sodium sulfate (138 mg, 3 eq) to it, heated and stirred at 90 degrees for 2 h, stopped the reaction. Added 10 ml water to quench the reaction, extracted with ethyl acetate (5 ml x 3) for three times, combined the ethyl acetate layer, dried with anhydrous magnesium sulfate, filtered, rotary evaporated the ethyl acetate to get the crude product. The crude product was purified by silica gel column chromatography (dichloromethane: methanol = 20:1) to get 99 mg of yellow powder, yield 91%.

[0078] 1 H NMR (400 MHz, CDCl3) δ 7.55 (s, 1H), 7.21 (t, J = 1.5 Hz, 1H), 7.14 (dd, J = 8.4, 2.3 Hz, 1H), 7.08 (d, J = 2.2 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.90-6.88 (m, 1H), 6.02-5.91 (m, 2H), 5.13-5.02 (m, 4H), 3.86 (s, 3H), 3.78 (s, 3H), 3.35 (dd, J = 16.1, 6.7 Hz, 4H).

[0079] 13C NMR (100 MHz, CDC13) δ 155.70, 155.67, 142.07, 137.99, 137.43, 136.33, 132.04, 131.64, 130.82, 130.20, 130.11, 128.87, 127.69, 116.88, 116.15, 115.65, 111.55, 56.23, 52.24, 39.56, 39.11.

[0080] ESI-MS (m / z): 338.2 [M+H] + .

[0081] Example 10 Preparation of 3-isopropyl nitroketone-4'-methoxy substituted magnolol (HK-7)

[0082] Intermediate 3 (100 mg, 1 eq), N-isopropylhydroxylamine hydrochloride (72.2 mg, 2 eq), triethylamine (90 ul, 2 eq), anhydrous sodium sulfate (138 mg, 3 eq) were referred to the synthetic method of Example 9 to give 102 mg of yellow oil, yield 86%.

[0083] 1 H NMR (400 MHz, CDC13) δ 12.03 (s, 1H), 7.64 (s, 1H), 7.21 (d, J = 2.2 Hz, 1H), 7.14 (dd, J = 8.3, 2.3 Hz, 1H), 7.08 (d, J = 2.3 Hz, 1H), 6.94 - 6.89 (m, 2H), 5.97 (dtt, J = 16.5, 9.6, 6.6 Hz, 2H), 5.12 - 5.01 (m, 4H), 4.27 (p, J = 6.5 Hz, 1H), 3.77 (s, 3H), 3.35 (dd, J = 12.2, 6.7 Hz, 4H), 1.51 (d, J = 6.5 Hz, 6H).

[0084] 13 C NMR (100 MHz, CDC13) δ 155.74, 155.67, 138.67, 138.02, 137.55, 135.89, 131.90, 131.70, 130.73, 130.13, 129.92, 128.70, 127.81, 117.11, 116.00, 115.56, 111.50, 66.38, 56.20, 39.55, 39.14, 21.08.

[0085] ESI-MS (m / z): 366.2 [M+H] + .

[0086] Example 11 Preparation of 3-tert-butyl nitropane-4'-methoxy substituted magnolol (HK-8)

[0087] Intermediate 3 (100 mg, 1 eq), N-tert-butylhydroxylamine (57.7 mg, 2 eq), triethylamine (90 ul, 2 eq), anhydrous sodium sulfate (138 mg, 3 eq) following the synthetic procedure of Example 9, afforded 104 mg of yellow oil, yield 84.5%.

[0088] 1 H NMR (400 MHz, CDC13) δ 7.77 (s, 1H), 7.21 (d, J = 1.9 Hz, 1H), 7.13 (dd, J = 8.4, 2.3 Hz, 1H), 7.08 (d, J = 2.2 Hz, 1H), 6.94 - 6.89 (m, 2H), 6.02 - 5.90 (m, 2H), 5.11 - 5.01 (m, 4H), 3.76 (d, J = 1.2 Hz, 3H), 3.35 (t, J = 6.5 Hz, 4H), 1.61 (d, J = 1.2 Hz, 9H).

[0089] 13 C NMR (100 MHz, CDC13) δ 155.71, 138.09, 137.68, 137.46, 135.74, 131.89, 131.76, 130.91, 130.29, 129.87, 128.67, 127.91, 117.42, 115.96, 115.55, 111.51, 70.06, 56.23, 39.58, 39.21, 28.49.

[0090] ESI-MS (m / z): 380.2 [M+H] + .

[0091] Example 12 Preparation of 3-phenyl nitropane-4'-methoxy substituted magnolol (HK-9)

[0092] Intermediate 3 (100 mg, 1 eq), N-tert-butylhydroxylamine (57.7 mg, 2 eq), triethylamine (90 ul, 2 eq), anhydrous sodium sulfate (138 mg, 3 eq) following the synthetic procedure of Example 9, afforded 104 mg of yellow oil, yield 84.5%.

[0093] 1H NMR (400 MHz, CDC13) δ 8.10 (s, 1H), 7.77 (dd, J = 6.1, 2.8 Hz, 2H), 7.49 (dd, J = 4.6, 2.5 Hz, 3H), 7.28 (d, J = 1.7 Hz, 1H), 7.16 (dd, J = 8.3, 2.3 Hz, 1H), 7.11 (d, J = 2.1 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 5.98 (m, 2H), 5.13 - 5.05 (m, 4H), 3.80 (d, J = 1.3 Hz, 3H), 3.37 (t, J = 7.7 Hz, 4H).

[0094] 13 C NMR (100 MHz, CDC13) δ 155.61, 146.47, 141.20, 137.89, 137.27, 136.84, 131.94, 131.57, 130.36, 130.27, 129.36, 128.80, 127.56, 121.97, 117.15, 116.12, 115.55, 111.46, 56.16, 39.46, 39.02.

[0095] ESI-MS (m / z): 400.2 [M+H] + .

[0096] Example 13 Preparation of 3-benzyl nitroketone-4'-methoxy substituted magnolol (HK-10)

[0097] Intermediate 3 (100 mg, 1 eq), N-benzylhydroxylamine (79.7 mg, 2 eq), triethylamine (90 ul, 2 eq), anhydrous sodium sulfate (138 mg, 3 eq) were used according to the synthetic procedure of Example 9 to give yellow powder 99 mg, yield 74%.

[0098] 1 H NMR (400 MHz, CDC13) δ 7.54 (s, 1H), 7.47 - 7.40 (m, 5H), 7.20 (d, J = 2.2 Hz, 1H), 7.13 (dd, J = 8.2, 2.3 Hz, 1H), 7.07 (d, J = 2.2 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.85 (t, J = 1.5 Hz, 1H), 5.95 (dtd, J = 16.7, 10.1, 6.7 Hz, 2H), 5.11 - 5.01 (m, 6H), 3.76 (s, 3H), 3.33 (dd, J = 20.4, 6.7 Hz, 4H).

[0099] 13C NMR (100 MHz, CDC13) δ 155.73, 155.63, 141.25, 137.99, 137.46, 136.33, 132.64, 131.99, 131.67, 130.93, 130.16, 130.11, 129.44, 129.30, 128.81, 127.65, 116.87, 116.07, 115.62, 111.51, 68.92, 56.20, 39.55, 39.10.

[0100] ESI-MS (m / z): 414.2 [M+H] + .

[0101] Preparation of ortho-aldehyde substituted and magnolol and bis-aldehyde substituted and magnolol

[0102] The starting material and magnolol (200 mg, 1 eq) was dissolved in chloroform 5 ml solution, 35% mass fraction of sodium hydroxide solution 1 ml and phase transfer catalyst tetrabutylammonium fluoride (20 ul, 0.1 eq) was added, heated and stirred at 60 degrees for 3h, the reaction was stopped. 20 ml water was added to quench the reaction, extracted with dichloromethane (10 ml x 3) three times, combined dichloromethane layer, anhydrous magnesium sulfate was dried, filtered, rotary evaporation of dichloromethane to get the crude product. The crude product was subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) to get a mixture of intermediate 4 and intermediate 5, 6, the mixture was separated by medium pressure preparation. Finally, intermediate 4 73 mg, yield 33%, intermediate 5 55 mg, yield 25%, intermediate 6 27 mg, yield 11% were obtained. All of them were brown oil.

[0103] ESI-MS (m / z): intermediate 4, 5: 293.1 [M-H] + ; intermediate 6: 323.1 [M+H] + .

[0104] Preparation of 3'-methyl nitroketone substituted and magnolol (HL-1)

[0105] Intermediate 4 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then N-methyl hydroxylamine hydrochloride (56.6 mg, 2 eq) was added, followed by the addition of triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq), heated and stirred at 90 degrees for 2h, the reaction was stopped. 10 ml water was added to quench the reaction, extracted with ethyl acetate (5 ml x 3) three times, combined ethyl acetate layer, anhydrous magnesium sulfate was dried, filtered, rotary evaporation of ethyl acetate to get the crude product. The crude product was subjected to silica gel column chromatography (dichloromethane:methanol = 20:1) to get yellow powder 98 mg, yield 89%.

[0106] 1 H NMR (400 MHz, CDC13) δ 7.56 (s, 1H), 7.38 - 7.33 (m, 2H), 6.86 (d, J = 8.2 Hz, 1H), 6.82 (d, J = 2.2 Hz, 1H), 5.19 (dd, J = 20.9, 13.6 Hz, 2H), 5.09 (d, J = 5.7 Hz, 1H), 5.06 (s, 1H), 4.99 (s, 1H), 3.89 (s, 3H), 3.46 (d, J = 6.4 Hz, 2H), 3.33 (d, J = 6.6 Hz, 2H).

[0107] 13 C NMR (100 MHz, CDC13) δ 155.29, 153.51, 142.22, 137.29, 136.55, 135.39, 132.43, 131.63, 131.04, 130.44, 130.31, 129.13, 124.72, 117.18, 116.65, 116.08, 115.57, 52.10, 39.02, 35.48.

[0108] ESI-MS (m / z): 324.2 [M+H] + .

[0109] Example 16 Preparation of 3'-isopropyl nitroketone substituted and honokiol (HL-2)

[0110] Intermediate 4 (100 mg, 1 eq), N-isopropylhydroxylamine hydrochloride (75.6 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) were referred to the synthesis method of Example 15 to obtain 111 mg of yellow oil, yield 92.5%.

[0111] 1 H NMR (400 MHz, CDC13) δ 7.67 (s, 1H), 7.33 (d, J = 7.4 Hz, 2H), 7.26 (d, J = 2.5 Hz, 1H), 6.87 - 6.81 (m, 2H), 5.99 (dtt, J = 33.6, 13.1, 5.1 Hz, 2H), 5.55 (s, 1H), 5.21 - 5.04 (m, 4H), 4.29 (p, J = 6.5 Hz, 1H), 3.44 (d, J = 6.5 Hz, 2H), 3.33 (d, J = 6.6 Hz, 2H), 1.53 (d, J = 6.5 Hz, 6H).

[0112] 13C NMR (100 MHz, CDC13) δ 155.32, 153.58, 139.24, 137.50, 136.82, 135.16, 132.63, 131.53, 130.94, 130.52, 130.35, 129.11, 125.06, 117.46, 116.38, 116.07, 115.57, 66.44, 39.15, 35.34, 21.09.

[0113] ESI-MS (m / z): 352.2 [M+H] + .

[0114] Example 17 Preparation of 3'-tert-Butyl nitrore substituted and honokiol (HL-3)

[0115] Intermediate 4 (100 mg, 1 eq), N-tert-butylhydroxylamine (60.4 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) were used according to the synthetic procedure of Example 15 to give 110 mg of yellow oil, yield 89%.

[0116] 1 H NMR (400 MHz, CDC13) δ 12.17 (s, 1H), 7.80 (s, 1H), 7.34 (d, J = 6.5 Hz, 2H), 7.26 (d, J = 2.2 Hz, 1H), 6.88 (d, J = 2.3 Hz, 1H), 6.83 (d, J = 8.9 Hz, 1H), 6.09 - 5.90 (m, 2H), 5.36 - 5.33 (m, 1H), 5.21 - 5.08 (m, 3H), 5.06 (d, J = 1.6 Hz, 1H), 3.44 (d, J = 6.4 Hz, 2H), 3.34 (d, J = 6.6 Hz, 2H), 1.63 (s, 9H).

[0117] 13 C NMR (100 MHz, CDC13) δ 155.44, 153.54, 137.90, 137.60, 136.81, 134.94, 132.74, 131.62, 131.15, 130.53, 130.41, 129.17, 124.92, 117.80, 116.46, 116.03, 115.59, 70.19, 39.21, 35.45, 28.45.

[0118] ESI-MS (m / z): 366.2 [M+H] + .

[0119] Example 18 Preparation of 3'-phenyl nitrore substituted and honokiol (HL-4)

[0120] Intermediate 4 (100 mg, 1 eq), phenylhydroxylamine (74.0 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) following the synthetic procedure of example 15, afforded 89 mg of orange powder in 68% yield.

[0121] 1 H NMR (400 MHz, CDC13) δ 12.44 (s, 1H), 8.12 (s, 1H), 7.78 (dd, J = 6.7, 2.9 Hz, 2H), 7.54 - 7.47 (m, 3H), 7.39 (d, J = 7.7 Hz, 2H), 7.32 (d, J = 2.1 Hz, 1H), 6.97 (d, J = 2.2 Hz, 1H), 6.87 (d, J = 8.0 Hz, 1H), 6.12 - 5.91 (m, 2H), 5.26 - 5.07 (m, 5H), 3.47 (d, J = 6.4 Hz, 2H), 3.36 (d, J = 6.6 Hz, 2H).

[0122] 13 C NMR (100 MHz, CDC13) δ 155.90, 153.67, 146.33, 141.66, 137.34, 136.69, 136.14, 132.84, 131.72, 131.28, 131.02, 130.82, 130.58, 129.52, 129.23, 124.96, 122.04, 117.65, 116.68, 116.28, 115.70, 39.15, 35.53.

[0123] ESI-MS (m / z): 386.2 [M+H] + .

[0124] Example 19 Preparation of 3'-benzyl nitroketone substituted and Magnolol (HL-5)

[0125] Intermediate 4 (100 mg, 1 eq), N-benzylhydroxylamine (83.5 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) following the synthetic procedure of example 15, afforded 101 mg of yellow powder in 74% yield.

[0126] 1H NMR (400 MHz, CDC13) δ 7.56 (s, 1H), 7.48 - 7.41 (m, 5H), 7.33 (d, J = 8.0 Hz, 2H), 7.25 (s, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.77 (d, J = 2.1 Hz, 1H), 6.08 - 5.87 (m, 2H), 5.31 - 5.27 (m, 1H), 5.22 - 5.11 (m, 2H), 5.08 (d, J = 3.9 Hz, 3H), 5.04 (s, 1H), 3.44 (d, J = 6.5 Hz, 2H), 3.30 (d, J = 6.6 Hz, 2H).

[0127] 13 C NMR (100 MHz, CDC13) δ 155.43, 153.61, 141.70, 137.42, 136.73, 135.52, 132.60, 132.53, 131.63, 130.96, 130.57, 129.50, 129.35, 129.29, 129.16, 124.97, 117.24, 116.56, 116.13, 115.62, 68.86, 39.11, 35.45.

[0128] ESI-MS (m / z): 400.2 [M+H] + .

[0129] Example 20 Preparation of 3-methyl nitroketone substituted and honokiol (HL-6)

[0130] Intermediate 5 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then N-methyl hydroxylamine hydrochloride (56.6 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 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), 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 (dichloromethane:methanol = 20:1) to obtain a yellow powder of 96 mg at a yield of 88%.

[0131] 1H NMR (400 MHz, CDC13) δ 12.49 (s, 1H), 7.55 (s, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.05 (dd, J = 8.5, 2.2 Hz, 2H), 7.00 (d, J = 2.2 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.07 - 5.93 (m, 2H), 5.15 - 5.07 (m, 3H), 5.07 (s, 1H), 5.04 (t, J = 1.5 Hz, 1H), 3.88 (s, 3H), 3.49 (d, J = 6.7 Hz, 2H), 3.34 (d, J = 6.7 Hz, 2H).

[0132] 13 C NMR (100 MHz, CDC13) δ 157.45, 150.96, 142.14, 137.83, 136.48, 134.85, 132.53, 132.24, 130.58, 130.27, 129.14, 127.82, 127.15, 116.93, 116.21, 115.97, 115.79, 52.24, 39.51, 34.64.

[0133] ESI-MS (m / z): 324.2 [M+H] + .

[0134] Example 21 Preparation of 3-isopropyl nitroketone substituted and honokiol (HL-7)

[0135] Intermediate 5 (100 mg, 1 eq), N-isopropylhydroxylamine hydrochloride (75.6 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) were used according to the synthetic procedure of Example 20 to give 101 mg of yellow oil in 85% yield.

[0136] 1 H NMR (400 MHz, CDC13) δ 12.49 (s, 1H), 7.55 (s, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.05 (dd, J = 8.5, 2.2 Hz, 2H), 7.00 (d, J = 2.2 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.07 - 5.93 (m, 2H), 5.15 - 5.07 (m, 3H), 5.07 (s, 1H), 5.04 (t, J = 1.5 Hz, 1H), 3.88 (s, 3H), 3.49 (d, J = 6.7 Hz, 2H), 3.34 (d, J = 6.7 Hz, 2H).

[0137] 13C NMR (100 MHz, CDC13) δ 157.51, 150.96, 138.74, 137.86, 136.59, 134.51, 132.46, 132.27, 130.51, 130.25, 129.10, 127.64, 127.26, 117.19, 116.11, 115.88, 115.76, 66.62, 39.51, 34.70, 21.11.

[0138] ESI-MS (m / z): 352.2 [M+H] + .

[0139] Example 22 Preparation of 3-tert-butyl nitronate substituted and honokiol (HL-8)

[0140] Intermediate 5 (100 mg, 1 eq), N-tert-butylhydroxylamine (60.4 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) were referred to the synthetic method of Example 20 to give yellow oil 104 mg, yield 84%.

[0141] 1 H NMR (400 MHz, CDC13) δ 12.46 (s, 1H), 7.78 (s, 1H), 7.37 (d, J = 2.2 Hz, 1H), 7.09 (d, J = 2.2 Hz, 1H), 7.07 - 7.02 (m, 1H), 7.00 (d, J = 2.1 Hz, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.08 - 5.92 (m, 2H), 5.14 - 5.03 (m, 5H), 3.50 (d, J = 6.6 Hz, 2H), 3.34 (d, J = 6.6 Hz, 2H), 1.64 (s, 9H).

[0142] 13 C NMR (100 MHz, CDC13) δ 157.63, 150.96, 137.90, 137.47, 136.65, 134.32, 132.43, 132.42, 130.70, 130.25, 129.10, 127.51, 127.33, 117.54, 116.06, 115.83, 115.73, 70.44, 39.52, 34.72, 28.46.

[0143] ESI-MS (m / z): 366.2 [M+H] + .

[0144] Example 23 Preparation of 3-phenyl nitronate substituted and honokiol (HL-9)

[0145] Intermediate 5 (100 mg, 1 eq), phenylhydroxylamine (74.0 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) following the synthetic procedure of example 20, afforded 88 mg of orange powder in 67% yield.

[0146] 1 H NMR (400 MHz, CDC13) δ 8.11 (s, 1H), 7.78 (dd, J = 6.6, 3.1 Hz, 2H), 7.54 - 7.48 (m, 3H), 7.45 (d, J = 2.2 Hz, 1H), 7.19 (d, J = 2.2 Hz, 1H), 7.08 - 7.01 (m, 2H), 6.89 (d, J = 8.1 Hz, 1H), 6.11 - 5.91 (m, 2H), 5.18 - 5.02 (m, 5H), 3.53 (d, J = 6.6 Hz, 2H), 3.35 (d, J = 6.7 Hz, 2H).

[0147] 13 C NMR (100 MHz, CDC13) δ 157.88, 150.98, 146.20, 141.31, 137.83, 136.47, 135.43, 132.55, 132.50, 131.43, 130.74, 130.28, 129.58, 129.39, 129.18, 128.54, 128.10, 127.09, 121.99, 117.29, 116.27, 116.00, 115.80, 39.51, 34.70.

[0148] ESI-MS (m / z): 386.2 [M+H] + .

[0149] Example 24 Preparation of 3-benzyl nitroketone substituted and Magnolol (HL-10)

[0150] Intermediate 5 (100 mg, 1 eq), N-benzylhydroxylamine (83.5 mg, 2 eq), triethylamine (94 ul, 2 eq), anhydrous sodium sulfate (144.4 mg, 3 eq) following the synthetic procedure of example 20, afforded 103 mg of yellow powder in 76% yield.

[0151] 1H NMR (400 MHz, CDC13) δ 7.52 (s, 1H), 7.45 (m, 5H), 7.36 (d, J = 2.1 Hz, 1H), 7.06 - 7.02 (m, 1H), 6.97 (d, J = 2.5 Hz, 2H), 6.87 (d, J = 8.2 Hz, 1H), 5.99 (m, 2H), 5.14 - 5.01 (m, 7H), 3.47 (d, J = 6.7 Hz, 2H), 3.33 (d, J = 6.7 Hz, 2H).

[0152] 13 C NMR (100 MHz, CDC13) δ 157.40, 150.82, 141.10, 137.74, 136.38, 134.76, 132.37, 132.19, 130.62, 130.13, 129.56, 129.34, 129.32, 129.05, 127.63, 127.04, 116.81, 116.09, 115.79, 115.66, 68.87, 39.39, 34.54.

[0153] ESI-MS (m / z): 400.2 [M+H] + .

[0154] Example 25 Preparation of 3,3'-dimethyl nitroketone substituted and honokiol (HL-11)

[0155] Intermediate 6 (100 mg, 1 eq) was dissolved in 2 ml of ethanol, then N-methyl hydroxylamine hydrochloride (103 mg, 4 eq), triethylamine (172 ul, 4 eq), anhydrous sodium sulfate (264 mg, 6 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 (dichloromethane:methanol = 20:1) to obtain 103 mg of a yellow powder at a yield of 88%.

[0156] 1H NMR (400 MHz, CDC13) δ 12.59 (s, 1H), 12.31 (s, 1H), 7.56 (d, J = 3.3 Hz, 2H), 7.48 (d, J = 2.2 Hz, 1H), 7.27 - 7.22 (m, 3H), 6.82 (d, J = 2.2 Hz, 1H), 6.06 (tt, J = 13.2, 5.0 Hz, 1H), 5.98 - 5.89 (m, 1H), 5.14 - 5.04 (m, 5H), 3.87 (d, J = 8.4 Hz, 6H), 3.49 (d, J = 6.6 Hz, 2H), 3.32 (d, J = 6.7 Hz, 2H).

[0157] 13 C NMR (100 MHz, CDC13) δ 157.02, 155.39, 142.61, 142.40, 137.31, 136.96, 135.44, 135.21, 131.55, 131.41, 130.68, 130.65, 128.90, 117.41, 116.28, 116.14, 115.76, 52.20, 52.13, 39.09, 34.70.

[0158] ESI-MS (m / z): 381.2 [M+H] + .

[0159] Example 26 Preparation of 3,3'-diisopropyl nitroketone substituted and honokiol (HL-12)

[0160] Intermediate 6 (100 mg, 1 eq), N-isopropylhydroxylamine hydrochloride (137 mg, 4 eq), triethylamine (172 ul, 4 eq), anhydrous sodium sulfate (264 mg, 6 eq) were used according to the synthetic procedure of Example 25 to give 119 mg of yellow powder in 87.6% yield.

[0161] 1H NMR (400 MHz, CDC13) δ 12.66 (s, 1H), 12.31 (s, 1H), 7.64 (d, J = 12.9 Hz, 2H), 7.46 (s, 1H), 7.28 (d, J = 2.1 Hz, 1H), 7.22 (s, 1H), 6.85 (d, J = 2.2 Hz, 1H), 6.06 (ddt, J = 16.8, 10.2, 6.5 Hz, 1H), 5.94 (ddt, J = 16.5, 9.6, 6.7 Hz, 1H), 5.14 - 5.01 (m, 4H), 4.27 (dt, J = 13.6, 6.07 Hz, 2H), 3.48 (d, J = 6.5 Hz, 2H), 3.32 (d, J = 6.6 Hz, 2H), 1.51 (d, J = 6.6 Hz, 12H).

[0162] 13 C NMR (100 MHz, CDC13) δ 156.94, 155.40, 139.15, 139.02, 137.45, 137.11, 135.06, 134.77, 131.59, 131.42, 130.54, 130.51, 130.46, 128.93, 117.60, 116.29, 116.11, 115.55, 66.44, 66.33, 39.12, 34.76, 21.08.

[0163] ESI-MS (m / z): 437.2 [M+H] + .

[0164] Pharmacological experiments:

[0165] Example 1 Protective effect of compounds HK 1-10, HL 1-12 on inflammatory response of LPS-stimulated macrophage Raw264.7

[0166] Method: The frozen cells were taken out from liquid nitrogen and placed in 37°C water bath immediately, and thawed by shaking gently. 3 mL 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. The cells were resuspended and transferred to a fresh culture dish containing fresh complete medium, and incubated in a 37°C CO2 incubator. In this experiment, the resuscitated cells were considered stable after being subcultured for 2 passages. 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 culture medium is aspirated, and fresh complete medium is added. The cells are detached by blowing, and the cell suspension is collected in a centrifuge tube and centrifuged at 800g for 3 min. The supernatant is discarded, and the cell pellet is resuspended in 1 mL medium. The cell pellet is dispersed into single cells by gentle blowing, and inoculated in a culture dish at a ratio of 1:3, and incubated in a 37°C CO2 incubator.

[0167] 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 cell culture 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 lipopolysaccharide (LPS) was added for 12 hours. The cell culture medium was collected for subsequent experiments.

[0168] NO content was measured by 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, and 50 μL / well of 1% sulfanilamide solution was added to the above wells. The mixture was incubated at room temperature for 5 min in the dark. Then, 50 μL / well of 0.1% NED solution was added to the above wells, and the color reaction appeared immediately. The mixture was incubated at room temperature for 5 min in the dark. The absorbance of the above wells at 525 nm was read, and the absorbance of the standard was linearly fitted to obtain the linear equation C = a*A525 + b between the concentration C and the absorbance A525. The NO concentration in each sample was calculated by substituting the sample absorbance into the above equation.

[0169] The results are shown in Table 1. LPS stimulation can significantly increase the secretion of NO, and 10 μmol / L HK 1-10 and HL 1-12 compounds can significantly reduce the secretion of NO, showing obvious anti-inflammatory effect.

[0170] Table 1 Effect of compounds HK 1-10, HL 1-12 on the release of NO content from macrophage Raw264.7 after LPS stimulation (means ± S.E.M, n=3)

[0171] Experimental Example 2 HL-3 improves the survival rate of SK-N-SH cells after oxygen-glucose deprivation / reoxygenation injury (OGDR).

[0172] HL-3 can effectively reduce the OGDR injury of SK-N-SH cells at a concentration of 10 micromoles (μM), and significantly improve the cell survival rate. Specifically, 10 μM HL-3 has a significant improvement effect on the survival rate of the cells.

[0173] 1. Cells

[0174] Cells: human neuroblastoma cells SK-N-SH

[0175] 2. Methods

[0176] 2.1 Oxygen-glucose deprivation / reoxygenation injury

[0177] SK-N-SH cells were seeded in a 96-well culture plate at a density of 6.5 x 10 4 / ml, and 100 μl of cell suspension was added to each well. After the cells were completely attached, 10 μl of drug solution was added to each well to give a final concentration of 0.1 μM, 1 μM, and 10 μM of HL-3, respectively, and incubated for 12 hours. After incubation, the culture medium was aspirated, 100 μl of complete medium was added to the control group, 100 μl of low-sugar medium was added to the model group, and 90 μl of low-sugar medium was added to the experimental group and supplemented with 10 μl of HL-3 solution of the corresponding concentration prepared in low-sugar medium. The culture plate was placed in an anoxic chamber filled with 95% N2 and 5% CO2, and incubated at 37°C for 4 hours. After treatment, the culture medium was aspirated, 100 μl of complete medium was added to each group, and 10 μl of HL-3 solution of the corresponding concentration prepared in complete medium was added to the experimental group, and the plate was placed in a 37°C incubator for reoxygenation for 4 hours.

[0178] 2.2 MTT method for determining cell survival rate

[0179] After reoxygenation, 10 μl of MTT solution (5 mg / ml) was added to each well, and incubated at 37°C for 4 hours. Then the culture medium was aspirated, and 150 μl of DMSO was added to dissolve the formed crystals. The 96-well plate was gently shaken to ensure complete dissolution of the crystals, and the optical density (OD) value was measured on an enzyme marker at a wavelength of 570 nm to evaluate the effect of different concentrations of HL-3 on the survival rate of SK-N-SH cells. According to the test results, the cell survival rate of each experimental group relative to the model group was calculated.

[0180] 2.3 Statistical analysis of data

[0181] Data are expressed as mean ± SD. Student's t-test was used for statistical analysis. "#" indicates the comparison between model group and control group, where #P<0.05, ##P<0.01. "*" indicates the comparison between each drug group and model group, where *P<0.05.

[0182] 3. Results

[0183] 3.1 HL-3 improves the survival of SK-N-SH cells after OGDR injury

[0184] The results of this example show that, compared with the non-injured control cell group, OGDR injury significantly reduces cell survival rate, while the administration of 10 μM HL-3 can significantly improve cell survival rate (Figure 3).

[0185] Example 3 Therapeutic effect of 0.1-10 mg / kg HL-3 on pMCAO rats

[0186] HL-3 showed obvious therapeutic effect on the permanent middle cerebral artery occlusion (pMCAO) model of SD rats at the dose of 1 mg / kg and 10 mg / kg. Specifically, it reduced the infarct volume and improved the neurological function score.

[0187] 1. Animals

[0188] Animals: SPF male SD rats, 250-280 g.

[0189] 2. Methods

[0190] 2.1 Test drug and preparation method

[0191] Name: HL-3

[0192] Dosing concentration: 0.1 mg / kg; 1 mg / kg; 10 mg / kg

[0193] Dosing route: oral administration

[0194] 2.2 MCAO operation

[0195] 44 SD rats were divided into model group, HL-3 treatment group (0.1 mg / kg; 1 mg / kg; 10 mg / kg) each group of 11, in accordance with the principle of parallelism, four groups of alternating middle cerebral artery occlusion (MCAO) operation. Rats with isoflurane 2% induction, 1% isoflurane anesthesia after supine position on the thermostat, the body temperature was maintained at 37 ± 0.5 ℃, fixed on the operating table. Along the median line of the neck incision, carefully separate the right common carotid artery (CCA), external carotid artery (ECA), internal carotid artery (ICA). The ECA ligation cut, straight and ICA in a straight line. On the ECA cut a small mouth, a long 4.0 cm, 0.26 mm diameter of the round head of the silicon plug line from the opening inserted into the ICA 1.6 cm, to the rat cerebral artery origin, block the middle cerebral artery blood supply. Ligation of the ECA opening and suture the surgical incision. After the operation, the rats were incubated at 37 ℃ for 1 h. The experimenters after 24 h of postoperative behavior evaluation and animal sampling.

[0196] 2.3 Measurement of cerebral infarction volume

[0197] The measurement of cerebral infarction volume is described in detail in pharmacological experimental methods (Xu Shuyun, third edition, p1066-1067).

[0198] 2.4 Behavioral testing

[0199] SD rats were used Bederson and mNSS neurological deficit score method

[0200] 2.5 Statistical analysis of data

[0201] The data were expressed as mean ± SD, and the statistical analysis was performed by Student's t-test. "*" indicates that compared with the model group, wherein *P<0.05, **P<0.01.

[0202] 3. Results

[0203] The results of the test are shown in Figures 4A and B, SD rats after acute cerebral ischemia 24h cerebral infarction volume was (56.35 ± 8.30)%, after giving 10 mg / kg and 1 mg / kg HL-3, 24h after cerebral infarction volume was (44.03 ± 13.39)% and (44.34 ± 11.82)%, compared with the model group, there was a significant difference (P = 0.0260 and P = 0.0153).

[0204] The Bederson score was used to determine the behavior score of rats after acute cerebral ischemia. The results showed that the scores of each group were decreased compared with the model group 24 h after SD rat cerebral ischemia, but there was no significant difference. It is suggested that compound HL-3 may have the effect of improving the neurological behavior impairment of rats after acute cerebral ischemia (Figure 4C).

[0205] The mNSS score was used to determine the behavior score of rats after acute cerebral ischemia. The results showed that the score was 11.30±1.25 24 h after SD rat cerebral ischemia. After administration of 10 mg / kg and 1 mg / kg HL-3, the behavior scores were 9.44±2.35 and 9.73±2.05 respectively 24 h later. There was a significant difference in the scores compared with the model group (p=0.0436 and 0.0369). It is suggested that compound HL-3 has the effect of improving the neurological behavior impairment of rats after acute cerebral ischemia (Figure 4D).

[0206] Experimental Example 4: Therapeutic effect of 0.1-10 mg / kg HL-3 on tMCAO rats

[0207] HL-3 showed obvious therapeutic effect on the tMCAO model of SD rats at a dose of 1 mg / kg and 10 mg / kg. It specifically showed the reduction of cerebral infarction volume and improvement of neurological function score.

[0208] 1. Animals

[0209] Animals: SPF male SD rats, 250-280 g.

[0210] 2. Methods

[0211] 2.1 Test drugs and preparation methods

[0212] Name: HL-3

[0213] Dosing concentration: 0.1 mg / kg; 1 mg / kg; 10 mg / kg

[0214] Dosing route: oral administration

[0215] 2.2 MCAO operation

[0216] 44 SD rats were divided into model group, HL-3 treatment group (0.1 mg / kg; 1 mg / kg; 10 mg / kg) and honokiol group (50 μg / kg), each group of 11, in accordance with the principle of parallelism, four groups of MCAO operation. Rats with isoflurane 2% induction, 1% isoflurane anesthesia after supine position on the thermostat, the temperature was maintained at 37 ± 0.5 ℃, fixed on the operating table. Along the median line of the neck incision, carefully separate the right common carotid artery (CCA), external carotid artery (ECA), internal carotid artery (ICA). The ECA ligation cut, straight and ICA in a straight line. On the ECA cut a small mouth, a long 4.0 cm, 0.26 mm diameter of a round of siliconized thread from the opening inserted into the ICA 1.6 cm, to the rat cerebral artery origin, block the blood supply of the middle cerebral artery. Ligation of the ECA opening and suture incision. After the operation, the rats were kept at 37 ℃ for 1 h. The experiment personnel after 2 h after pulling out the line, 24 h after the behavioral assessment and animal material.

[0217] 2.3 Measurement of cerebral infarction volume

[0218] The measurement of cerebral infarction volume is described in detail in pharmacological experimental methods (Xu Shuyun, third edition, p1066-1067).

[0219] 2.4 Behavioral testing

[0220] SD rats after 24 h of operation using Bederson and mNSS neurological deficit score method

[0221] 2.5 Statistical analysis of data

[0222] The data were expressed as mean ± SD, and the statistical analysis was performed using Student's t-test. "*" indicates that compared with the model group, wherein *P<0.05, **P<0.01.

[0223] 3. Results

[0224] The test results are shown in Figure 3, SD rats acute cerebral ischemia 2h, reperfusion 24h after cerebral infarction volume was (48.36 ± 10.81)%, after 10 mg / kg and 1 mg / kg HL-3 acute ischemia 2h, reperfusion 24h after cerebral infarction volume was (38.29 ± 5.3)% and (34.02 ± 12.47)%, compared with the model group, there was a significant difference (P = 0.0202 and P = 0.0130) (Figure 5A-B).

[0225] The behavior score of rats after acute cerebral ischemia was determined by Bederson score. The results showed that the score of SD rats after 2h of cerebral ischemia and 24h of reperfusion was 2.818±0.375. After 2h of acute ischemia and 24h of reperfusion, the behavior score of rats treated with 10mg / kg and 1mg / kg HL-3 was (1.889±0.60)% and (1.889±0.333)%, respectively, which was significantly different from the model group (P=0.0076 and P=0.0030) (Figure 5C).

[0226] The behavior score of rats after acute cerebral ischemia was determined by mNSS score. The results showed that the score of SD rats after 2h of cerebral ischemia and 24h of reperfusion was 11.09±2.427. After 24h of reperfusion, the behavior score of rats treated with 1mg / kg HL-3 was 8.111±1.764, which was significantly different from the model group (p=0.0066) (Figure 5D).

[0227] Example 5: HL-3 has protective effect on SOD1 G93A and TDP43 M337V stable transfection cells

[0228] This example found that SOD1 G93A and TDP43 M337V stable transfection cells could significantly reduce cell survival compared with the corresponding empty vector control cells. HL-3 had a significant protective effect on SOD1 G93A and TDP43 M337V stable transfection cells after 24h, which was manifested as a significant improvement in the survival rate of 0.1μM HL-3 on the cells.

[0229] 1. Cells and plasmids

[0230] Cell lines:

[0231] The NSC-34 cell line was purchased from Shanghai Hongshun Biotechnology Co., Ltd.

[0232] The SOD1 G93A stable transfection cell line and the pEGFP empty vector control cell line were constructed by the laboratory.

[0233] The TDP43 M337V stable transfection cell line and the pCI empty vector control cell line were constructed by the laboratory.

[0234] 2. Methods

[0235] 2.1 MTT method for determining cell survival rate

[0236] The pEGFP empty vector control cells and SOD1 G93A stable transfection cells were seeded at 5*10 4Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated. After 24 hours of incubation, the medium was changed, and 90 μL of blank medium was added to each well. 10 μL of blank medium was added to the wells of the pEGFP empty vector control cells and the SOD1 G93A model group cells; 10 μL of medium containing HL-3 was added to the wells of each SOD1 G93A treatment group cells, bringing the final concentrations in each well to 0.1, 1, and 10 μM, respectively. After 4 hours and 24 hours of incubation, 10 μL of MTT was added to each well. After another 4 hours of incubation, 100 μL of triplet solution was added to each well, and the cells were dissolved overnight at 37°C. The absorbance was then measured at 570 nm.

[0237] pCI empty vector control cells and TDP43 M337V stable cells were divided into 5*10 cells. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated. After 24 hours of incubation, the medium was changed, 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 group cells; 10 μL of medium containing HL-3 was added to the wells of each TDP43 M337V treatment group cells, bringing the final concentrations in each well to 0.1, 1, and 10 μM, respectively. After incubation for 24 hours, 10 μL of MTT was added to each well, followed by another 4 hours of incubation. Then, 100 μL of triplet solution was added to each well, and the cells were dissolved overnight at 37°C. The absorbance was then measured at 570 nm.

[0238] 2.2 Data Statistical Analysis

[0239] Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using Student's t-test. "#" indicates that the model group cells were compared with their respective empty vector control groups (pEGFP and pCI). # P<0.05, ## P<0.01, "*" indicates that the cells in each drug-treated group were compared with their respective model group cells. * P<0.05.

[0240] 3. Results

[0241] 3.1 HL-3 enhances the survival of SOD1 G93A cells and TDP43 M337V cells

[0242] The results of this embodiment show that, compared with the empty vector control cell group, the survival rate of SOD1 G93A cells and TDP43M337V cells was significantly reduced, while administration of 0.1 μM HL-3 significantly improved the survival rate of SOD1 G93A cells and TDP43 M337V cells (Figures 6A and 6B).

[0243] Protective effect of HL-3 (1 mg / kg) on traumatic brain injury in SD rats

[0244] HL-3 at a dose of 1 mg / kg showed significant therapeutic effect on traumatic brain injury model in SD rats. Specifically, it can significantly improve the motor function of SD rats and improve the neurobehavioral score.

[0245] 1. Experimental animals

[0246] Male SD rats, SPF level, body weight 170-190g, purchased from Sperof Bio-technology Co., Ltd.

[0247] 2. Brain injury model

[0248] After isoflurane anesthesia, the scalp was incised at 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. A modified Feeney free fall injury device was used, and a 40g hammer was used to impact the striker from a height of 25cm, with a striking depth of 6mm. The scalp was sutured. The scalp of the sham-operated rats was sutured after incision and drilling, and the hammer was not hit.

[0249] 3. Animal grouping and administration:

[0250] The rats were divided into sham operation group, model group, HL-3 1 mg / kg group and butylphthalide 200 mg / kg group. Oral administration 5 minutes after operation, continuous administration for three days.

[0251] 4. Neurological function injury score

[0252] The mNSS neurological function deficit score was used at 24h, 48h and 72h after modeling, with a score of 0 to 18 (0 normal, 18 maximum neurological function deficit). It includes a comprehensive assessment of rat motor, sensory, reflex and balance functions. The higher the score, the more severe the injury. The detection method and scoring criteria are shown in the table below.

[0253] Rat neurological function score (mNSS)

[0254] 5. Rotarod test:

[0255] The rats were placed on the rotarod instrument, the initial speed was 5 rpm, and the speed was accelerated to 25 rpm within 180 seconds. The latency of falling was recorded, and 180 seconds was taken as the cutoff value. More than 180 seconds was recorded as 180 seconds, and less than 180 seconds was recorded as the actual recording time. Each experiment was repeated 3 times and the average value was taken.

[0256] 6. Data statistical analysis

[0257] Data are expressed as mean ± SEM. Statistical analysis was performed using Student's t-test. # indicates comparison between model and control groups, where ### P<0.001, * indicates comparison between HL-3 administration group and model group, where *P<0.05, **P<0.01, ***P<0.001.

[0258] 7. Experimental results

[0259] 7.1 Effect of HL-3 on behavioral impairment of rats after TBI modeling

[0260] After TBI modeling, the neurological impairment score (mNSS) of the model group rats was significantly increased, which was significantly different (P<0.001), indicating that the TBI model in this experiment caused significant damage to the neurological function of rats. Continuous three-day administration of HL-3 (1 mg / kg) drug treatment can significantly reduce the neurological impairment score of rats at 24 h, 48 h and 72 h, and has a significant difference (P<0.05) compared with the model group after 72 h of brain injury. It is suggested that the HL-3 (1 mg / kg) group can protect the neurological impairment caused by TBI at 72 h after operation. (Table 1, Figure 7)

[0261] 7.2 Effect of HL-3 on the rotarod falling time of rats after TBI modeling

[0262] The inventors detected the motor ability of brain injury rats by rotarod test. The motor time of rats in the brain injury model group on the rotarod was significantly reduced. Continuous three-day administration of HL-3 (1 mg / kg) drug treatment can improve the motor ability of rats on the rotarod at 24 h, 48 h and 72 h, and has a significant difference (P<0.05) compared with the model group after 24 h of brain injury, indicating that HL-3 drug treatment can reduce the damage of rotarod motor ability of brain injury rats. (Table 2, Figure 8)

[0263] Table 1. Effect of HL-3 on behavioral impairment of rats after TBI modeling ### P<0.001 compared with the sham operation group, *P<0.05 compared with the model group.

[0264] Table 2. Effect of HL-3 on motor function of rats after TBI modeling ### P<0.001 compared with the sham operation group, *P<0.05 compared with the model group.

[0265] The above examples are only for illustrating the technical concept features of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and implement it accordingly, and cannot limit the protection scope of the present application. Any equivalent changes or modifications made according to the spirit and principle of the present application shall be covered within the protection scope of the present application.

Claims

1. A class of magnolol nitroketone derivatives or pharmaceutically acceptable salts thereof, represented by general formula I: R1is selected from the group consisting of H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3; R2is selected from the group consisting of CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, t-Bu, Ph, Bn.

2. A class of honokiol nitrone derivatives represented by the general formula II: ###0002### or a pharmaceutically acceptable salt thereof. R3, R4, independently of each other, can be selected from H or and R3, R4are not simultaneously H; R5is selected from the group consisting of CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, t-Bu, Ph, Bn.

3. 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:

4. A pharmaceutical composition comprising, as an effective ingredient, a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 and a pharmaceutically acceptable carrier or excipient.

5. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the manufacture of an anti-inflammatory medicament.

6. Use according to claim 5, characterised in that, The inflammation is selected from the group consisting of respiratory system inflammation, bone and joint system inflammation, skin inflammation, and autoimmune diseases.

7. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the manufacture of a medicament for preventing or treating cerebral infarction.

8. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the manufacture of a medicament for preventing or treating amyotrophic lateral sclerosis.

9. Use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the manufacture of a medicament for preventing or treating traumatic brain injury.

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