Application of bisamide curcumin compound in preparation of oxidative stress injury protection medicine

Abstract

The invention relates to the field of medicinal chemistry, in particular to application of a specific bisamide curcumin compound in preparation of antioxidant drugs and treatment of oxidative stress injury related diseases. The bisamide curcumin compounds can effectively inhibit oxidative damage of hydrogen peroxide to PC12 cells, inhibit generation of active oxygen in the cells, activate an Nrf2 / HO-1 antioxidant signal channel and effectively relieve cerebral ischemia reperfusion injury of mice.

Description

Technical field:

[0001] The present invention belongs to the field of pharmaceutical chemistry. Specifically, the present invention relates to the use of specific diamide curcumin compounds in the preparation of antioxidant drugs and therapeutic drugs for oxidative stress damage. These compounds can effectively antagonize oxidative stress damage, thereby playing a good in vitro and in vivo antioxidant protection role. Background technology:

[0002] Curcumin is a compound with good antioxidant and anti-inflammatory effects, but its instability and low bioavailability limit its clinical application. To improve this problem, curcumin has been subjected to a variety of structural modifications, but the prior art has greatly changed the curcumin skeleton, which destroys the active characteristics of curcumin itself. The present invention proposes to design and synthesize nitrogen-containing dicarbonyl curcumin analogs by introducing stable amide bonds to improve stability and maintain pharmacological activity. After long-term research and practice, the inventors have obtained specific compounds from a series of diamide curcumin analogs without structure-function directional enlightenment, which have good antioxidant activity. Summary of the invention:

[0003] The present invention aims to provide the use of 13 bisamide curcumin compounds in the preparation of antioxidant drugs and therapeutic drugs for diseases related to oxidative stress damage.

[0004] Another object of the present invention is to provide a pharmaceutical composition for treating oxidative stress damage diseases, which contains a therapeutically effective amount of any one or more of the bisamide curcumin compounds described in claim 1 or their pharmaceutically acceptable salts and pharmaceutical excipients as active ingredients.

[0005] Specifically, the 13 active bisamide curcumin active compounds of the present invention have the following structures:

[0006]

[0007]

[0008] Active compound: The molecular formula of F1 is C 20 H 20 N2O4, chemical name is (E)-3-(2-methoxyphenyl)-N'-((E)-3-(2-methoxyphenyl)acryloyl)acrylohydrazide, molecular formula of F4 is C 22 H 22O5, chemical name is (E)-3-(2,5-dim ethoxyphenyl)-N'-((E)-3-(2,5-dimethoxyphenyl)acryloyl)acrylohydrazide, F5 molecular formula is C 25 H 28 O7, chemical name is (E)-3-(3,4-dimethoxyphenyl)-N'-((E)-3-(3,4-dimethoxyphenyl)acryloyl)acrylohydr azide, molecular formula of F7 is C 24 H 28 N2O8, chemical name is (E)-3-(3,4,5-trimethoxyphenyl)-N'-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)acrylohydrazide, molecular formula of F8 is C 22 H 24 N2O4, chemical name is (E)-3-(4-ethoxyphenyl)-N'-((E)-3-(4-ethoxyphenyl)acryloyl)acrylohydrazide, molecular formula of F9 is C 18 H 14 F2N2O2, chemical name is (E)-3-(2-fluorophenyl)-N'-((E)-3-(2-fluorophenyl)acryloyl)acrylohydrazide, molecular formula of F10 is C 18 H 14 F2N2O2, chemical name is (E)-3-(4-fluorophenyl)-N'-((E)-3-(4-fluorophenyl)acryloyl)acrylohydrazide, molecular formula of F12 is C 18 H 12 F4N2O2, chemical name is (E)-3-(2,6-difluorophenyl)-N'-((E)-3-(2,6-difluorophenyl)acryloyl)acrylohydrazide, molecular formula of F13 is C 18 H 12 F4N2O2, chemical name is (E)-3-(3,5-difluorophenyl)-N'-((E)-3-(3,5-difluorophenyl)acryloyl)acrylohydrazide, molecular formula of F14 is C 18H 14 Cl2N2O2, chemical name is (E)-3-(2-chlorophenyl)-N'-((E)-3-(2-chlorophenyl)acryloyl)acrylohydrazide, molecular formula of F18 is C 18 H 14 Br2N2O2, chemical name is (E)-3-(3-bromophenyl)-N'-((E)-3-(3-bromophenyl)acryloyl)acrylohydrazide, molecular formula of F19 is C 22 H 20 N2O6, chemical name is ((1E,1'E)-hydrazine-1,2-diylbis(3-oxoprop-1-ene-3,1-diyl))bis(3,1-phenylene)diacetate, molecular formula of F20 is C 25 H 21 BrN2O4, chemical name is 3'-(3-bromobenzoyl)-4'-(3,4-dihydroxyphenyl)-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one.

[0009] Comparative compound: The molecular formula of F2 is C 23 H 24 O6, chemical name is (E)-3-(3-methoxyphenyl)-N'-((E)-3-(3-methoxyphenyl)acryloyl)acrylohydrazide, molecular formula of F3 is C 22 H 22 O5, chemical name is (E)-3-(2,4-dimethoxyphenyl)-N'-((E)-3-(2,4-dimethoxyphenyl)acryloyl)acrylohydrazide, F6 molecular formula is C 25 H 28 O7, the chemical name is (E)-3-(3,5-dimethoxyphenyl)-N'-((E)-3-(3,5-dimethoxyphenyl)acryloyl)acrylohydrazide, and the molecular formula of F11 is C 18 H 12F4N2O2, chemical name is (E)-3-(2,5-difluorophenyl)-N'-((E)-3-(2,5-difluorophenyl)acryloyl)acrylohydrazide, molecular formula of F15 is C 18 H 14 Cl2N2O2, chemical name is (E)-3-(3-chlorophenyl)-N'-((E)-3-(3-chlorophenyl)acryloyl)acrylohydrazide, molecular formula of F16 is C 18 H 12 C l4 N2O2, chemical name is (E)-3-(2,6-dichlorophenyl)-N'-((E)-3-(2,6-dichlorophenyl)acryloyl)acrylohydrazide, molecular formula of F17 is C 18 H 12 C l4 N2O2, chemical name is (E)-3-(3,5-dichlorophenyl)-N'-((E)-3-(3,5-dichlorophenyl)acryloyl)acrylohydrazide. The molecular formula of curcumin is C 21 H 20 O6, chemical name is (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione.

[0010] Since the β-diketone structure in the chemical structure of curcumin is an important factor leading to the poor structural stability of curcumin, the present invention first chemically transforms the β-diketone of the curcumin skeleton, that is, replaces the β-diketone with an amide bond to obtain a class of bisamide curcumin analogs, and the synthesis of such bisamide curcumin compounds adopts classical synthesis methods such as Knoevenagel reaction (see Example 1 for details). The stability of the obtained bisamide curcumin skeleton is significantly improved (see Example 2 for details). Subsequently, based on the skeleton, a series of derivatives are synthesized and screened to obtain the following technical effects:

[0011] (1) Hydrogen peroxide (hydrogen peroxide) is one of the most abundant reactive oxygen species (ROS) in cells. It can rapidly release exogenous oxygen free radicals, causing oxidative damage to proteins, lipids, DNA, etc., and ultimately leading to cell death. Therefore, we selected the PC12 cell oxidative damage model induced by hydrogen peroxide to evaluate the in vitro antioxidant activity of the novel bisamide curcumin analogs F1-F20 (see Example 3 for details). Among them, compound F19 has the best cell protection effect and its antioxidant activity is also better than its lead compound curcumin. Therefore, F19 can be regarded as a cell protectant against oxidative damage of PC12 cells and can be used for further antioxidant research.

[0012] (2) Compound F19 can resist the damage caused by hydrogen peroxide to PC12 cells and promote colony formation. Malondialdehyde (MDA) is an important biomarker of oxidative stress. ROS can lead to excessive accumulation of MDA. Hydrogen peroxide stimulation can sharply increase the level of MDA in PC12 cells, while after pretreatment with F19 for 18 hours, the level of MDA in the cells dropped sharply, indicating that F19 can resist the oxidative stress damage caused by excessive ROS to cells. In addition, compound F19 has an inhibitory effect on the generation of intracellular ROS induced by hydrogen peroxide. The ROS level was determined using DCFH-DA. Compared with the DMSO group, the ROS level in PC12 cells stimulated by hydrogen peroxide was significantly increased, and pretreatment with F19 for 24 hours can significantly eliminate the intracellular ROS generated by hydrogen peroxide stimulation (see Example 4 for details).

[0013] (3) Curcumin is an activator of Nrf2 and exerts its antioxidant effect by activating the Nrf2 signaling pathway. The Nrf2 signaling pathway is a classic antioxidant signaling pathway and plays an important role in the study of antioxidant activity. The effect of F19 nuclear translocation was detected by Western blot. The results showed that F19 promoted the expression of Nrf2 protein in the nucleus of PC12 cells and activated the Nrf2 antioxidant signaling pathway. Heme oxygenase-1 (HO-1) is an antioxidant protein downstream of the Nrf2 signaling pathway. The results showed that in cells pre-incubated with F19, F19 promoted the expression of HO-1 in a dose-dependent manner. Therefore, compound F19 may resist hydrogen peroxide-induced oxidative damage to PC12 cells by promoting the entry of nuclear factor Nrf2 and the expression of antioxidant protein HO-1.

[0014] (4) The middle cerebral artery occlusion (MCAO) model is a classic animal model for studying ischemic stroke and can simulate the animal model of clinical ischemic stroke. Compared with the sham operation group, the cerebral infarction area of ​​mice after modeling was significantly increased. Compared with the solvent group, the cerebral infarction area of ​​the drug F19 group was significantly smaller and the neurological function score was significantly improved. Therefore, the active compound F19 alleviated cerebral ischemia-reperfusion injury in mice and has the potential to treat ischemic stroke.

[0015] The bisamide curcumin compounds of the present invention can be used in the preparation of antioxidant drugs and therapeutic drugs for diseases related to oxidative stress damage, including but not limited to the following diseases: sepsis caused by acute oxidative stress, organ ischemia and ischemia-reperfusion injury of the eyes, brain, heart, kidneys, and liver, acute mild and moderate pain (such as post-operative, post-traumatic, post-strain, primary dysmenorrhea, toothache, headache); Parkinson's disease, Alzheimer's disease, atherosclerosis, diabetic complications caused by chronic oxidative stress, rheumatoid arthritis, osteoarthritis, spondyloarthropathy, gouty arthritis, non-articular soft tissue rheumatic pain (such as shoulder pain, tenosynovitis, bursitis, myalgia or pain caused by post-exercise injury).

[0016] A pharmaceutical composition for treating oxidative stress injury diseases, comprising a therapeutically effective amount of any one or more of the 13 bisamide curcumin compounds or their pharmaceutically acceptable salts and pharmaceutical excipients as active ingredients. "Pharmaceutical composition" refers to a composition of drugs for preventing and treating oxidative stress injury diseases prepared by combining any one or more of the 13 bisamide curcumin compounds or their pharmaceutically acceptable salts with an antioxidant drug currently on the market.

[0017] As used herein, "pharmaceutical excipients" refer to conventional drug carriers in the pharmaceutical field, such as: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium / magnesium stearate, polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents, sweeteners, etc. may be added to the composition.

[0018] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient is mixed with one or more carriers and then made into the desired dosage form. The preparation forms of the drug include injections, tablets, capsules, aerosols, suppositories, films, pills, ointments, controlled release or sustained release agents or nano preparations. The present invention can be administered to patients who need such treatment in the form of a composition by oral, nasal inhalation, rectal or parenteral administration. When used for oral administration, it can be made into conventional solid preparations such as tablets, powders, granules, capsules, etc., and into liquid preparations such as water or oil suspensions or other liquid preparations such as syrups, elixirs, etc.; when used for parenteral administration, it can be made into solutions for injection, water or oil suspensions, etc. Description of the drawings:

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0020] Attached Figure 1 :Design of bisamide curcumin analogs. (A) Design process from lead compound to target compound. (B) Structural stability of curcumin. (C) Structural stability of the skeleton of bisamide curcumin analogs.

[0021] Attached Figure 2 :Protective effect of the compound on the PC12 cell injury model induced by hydrogen peroxide. PC12 cells were pre-incubated with the compound (1 μM) for 18 hours and then treated with hydrogen peroxide (480 μM) for 24 hours. Finally, the survival rate of PC12 cells was detected by MTT assay. Data are expressed as mean ± SD, n = 3. #### P<0.0001 vs DMSO; ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05 vs hydrogen peroxide.

[0022] Attached Figure 3 : In the hydrogen peroxide-induced PC12 cell oxidative damage model, compound F19 promoted colony formation (A), inhibited MDA production (B), and reduced ROS production (C, D). PC12 cells were pretreated with 1 μM or 10 μM F19 and then stimulated with 1 mM hydrogen peroxide for 2 hours. Cells were incubated in the dark with dichlorodihydrofluorescein diacetate (DCFH-DA) test, and finally photographed with an inverted fluorescence microscope or detected on a flow cytometer. Fluorescence intensity represents ROS level. Data are expressed as mean ± standard deviation, n = 3.####P<0.0001,##P<0.001,##P<0.01,##P<0.05vs DMSO,****P<0.0001,***P<0.001,**P<0.01,*P<0.05vs hydrogen peroxide.

[0023] Attached Figure 4:F19 activates the Nrf2 antioxidant signaling pathway. (A) F19 activates the Nrf2 antioxidant signaling pathway and promotes the entry of Nrf2 into the cell nucleus. PC12 cells were pretreated with F19 (1 μM) for 18 hours, nuclear proteins were extracted, and the expression of Nrf2 protein was determined by WB test. (B) F19 activates the Nrf2 antioxidant signaling pathway and promotes the expression of antioxidant protein HO-1. PC12 cells were preincubated with F19 (0.5, 1, 2, 5 μM) for 18 hours, and the expression level of HO-1 protein was determined by WB test. Data are expressed as mean SD compared with DMSO, n=3, *P<0.01, *P<0.05

[0024] Attached Figure 5 :F19 alleviates brain damage caused by cerebral ischemia-reperfusion in mice. (A) TTC staining of the infarcted brain area. Normal saline (NS), solvent (DMSO castor oil: normal saline = 1: 19: 80) and F19 (10 mg / kg) were intraperitoneally injected into sham-operated mice or MCAO-reperfused mice for 1 hour in advance. (B) The infarct size of the mouse brain was quantified, and the ratio of infarct size to the whole brain size was calculated. (C) Behavioral neurological scores of mice. Data are expressed as mean ± SD, n = 6. ####P<0.0001 compared with the sham-operated group, *P<0.05 compared with the solvent group. Specific implementation method:

[0025] The present invention also provides methods for preparing these compounds and methods for evaluating their pharmacodynamics in cell models and animal models, as well as specific application details such as their dosage, administration method and administration cycle.

[0026] Example 1 Synthesis of bisamide curcumin derivatives:

[0027] The synthetic route of the compound is shown in Scheme 1. The substituted cinnamic acid is obtained by the Knoevenagel reaction of the corresponding substituted benzaldehyde with malonic acid and piperazine in pyridine. Under nitrogen protection, the substituted cinnamic acid reacts with oxalyl chloride to form the intermediate cinnamoyl chloride. After removing the solvent and excess oxalyl chloride in vacuo, the crude product is dissolved in dry THF, and then hydrazine hydrate and triethylamine are added to obtain the target product. All compounds are purified by recrystallization from a dimethyl sulfoxide / anhydrous ethanol system. The target compound is confirmed and characterized by nuclear magnetic resonance and liquid chromatography-mass spectrometry. The synthetic chemical reaction formulas and compound data of all synthesized bisamide curcumin derivatives are as follows:

[0028]

[0029] Scheme 1: Reagents and conditions: (a) malonic acid, piperazine, pyridine, 90°C, 3h; (b) oxalyl chloride, anhydrous dichloromethane (DCM), anhydrous N,N-dimethylformamide (DMF), N2, room temperature; (c) hydrazine hydrate, anhydrous tetrahydrofuran (THF), triethylamine, N2, room temperature.

[0030] Effective compound F1(E)-3-(2-methoxyphenyl)-N'-((E)-3-(2-methoxyphenyl)acryloyl)acrylohydrazide

[0031] White powder, 43.8% yield, mp 278.8-280.8℃. 1 H NMR (400MHz, DMSO) δ: 10.35 (s, 2H, NH × 2), 7.74 (d, J = 16.0Hz, 2H, Ar-CH = × 2), 7.54 (d, J = 7.6Hz, 2H, Ar-H 6 ×2),7.38(t,J=7.8Hz,2H,Ar-H 4 ×2),7.08(d,J=8.4Hz,2H,Ar-H 3 ×2),7.00(t,J=7.5Hz,2H,Ar-H 5 ×2),6.80(d,J=16.0Hz,2H,CO-CH=×2),3.86(s,6H,2-OCH3×2).LC-MS m / z:353.14(M+H) + ,calcd for C 20 H 20 N2O4:352.14.

[0032] Effective compound F4(E)-3-(2,5-dimethoxyphenyl)-N'-((E)-3-(2,5-dimethoxyphenyl)acryloyl)acrylohydrazide(F4)

[0033] Yellow powder,32.6%yield,mp 210.5-216.4℃. 1 H NMR (400MHz, DMSO) δ: 10.42 (s, 2H, NH × 2), 7.69 (d, J = 15.9Hz, 2H, Ar-CH = × 2), 7.08 (d, J = 2.9Hz, 2H, Ar-H 6 ×2),7.00(d,J=9.0Hz,2H,Ar-H 3×2), 6.95 (dd, J=9.0, 3.0 Hz, 2H, Ar-H 4 ×2), 6.81 (d, J=16.0 Hz, 2H, CO-CH=×2), 3.79 (d, J=4.8 Hz, 6H, 2-OCH3), 3.72 (d, J=4.3 Hz, 6H, 5-OCH3). LC-MS m / z: 413.16 (M+H) + , calcd for C 22 H 22 O5: 412.16. Active compound F5 (E)-3-(3,4-dimethoxyphenyl)-N'-((E)-3-(3,4-dimethoxyphenyl)acryloyl)acrylohydrazide (F5)

[0034] Light yellow powder, 36.9% yield, mp 245.4 - 246.6℃. 1 H NMR (400 MHz, DMSO) δ: 10.30 (s, 2H, NH×2), 7.48 (d, J=15.7 Hz, 2H, Ar-CH=×2), 7.21 - 7.15 (m, 4H, Ar-H 2 ×2, Ar-H 5 ×2), 7.00 (d, J=8.4 Hz, 2H, Ar-H 6 ×2), 6.65 (d, J=15.8 Hz, 2H, CO-CH=×2), 3.81 (s, 6H, 3-OCH3×2), 3.79 (s, 6H, 4-OCH3×2). LC-MS m / z: 413.16 (M+H) + , calcd for C 25 H 28 O7: 412.16.

[0035] Active compound F7 (E)-3-(3,4,5-trimethoxyphenyl)-N'-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)acrylohydrazide (F7)

[0036] Yellow powder, 40.6% yield, mp 277.5 - 279.5℃. 1 H NMR (400 MHz, DMSO) δ: 10.40 (s, 2H, NH×2), 7.49 (d, J=15.9 Hz, 2H, Ar-CH=×2), 6.94 (s, 4H, Ar-H2 ×2, Ar-H 6 ×2), 6.74 (d, J=15.8 Hz, 2H, CO-CH=×2), 3.82 (s, 12H, 3-OCH3×2, 5-OCH3×2), 3.69 (s, 6H, 4-OCH3×2). LC-MS m / z: 473.18 (M+H) + , calcd for C 24 H 28 N2O8: 472.18.

[0037] Active compound F8 (E)-3-(4-ethoxyphenyl)-N'-((E)-3-(4-ethoxyphenyl)acryloyl)acrylohydrazide (F8)

[0038] White powder, 32.8% yield, mp 285.6 - 286.9℃. 1 H NMR (400 MHz, DMSO) δ: 10.30 (s, 2H, NH×2), 7.53 (d, J=8.7 Hz, 4H, Ar-H 2 ×2, Ar-H 6 ×2), 7.47 (d, J=15.8 Hz, 2H, Ar-CH=×2), 6.96 (d, J=8.7 Hz, 4H, Ar-H 3 ×2, Ar-H 5 ×2), 6.59 (d, J=15.8 Hz, 2H, CO-CH=×2), 4.05 (q, J=7.0 Hz, 4H, 4-OCH2×2), 1.32 (t, J=7.0 Hz, 6H, 4-O-C-CH3×2). LC-MS m / z: 381.17 (M+H) + , calcd for C 22 H 24 N2O4: 380.17.

[0039] Active compound F9 (E)-3-(2-fluorophenyl)-N'-((E)-3-(2-fluorophenyl)acryloyl)acrylohydrazide (F9)

[0040] White powder, 26.9% yield, mp 276.5 - 278.5℃. 1 H NMR (400 MHz, DMSO) δ: 10.62 (s, 2H, NH×2), 7.72 - 7.67 (m, 2H, Ar-H 6×2), 7.60 (d, J = 16.0 Hz, 2H, Ar-CH=×2), 7.32 - 7.26 (m, 6H, Ar-H 3 ×2, Ar-H 4 ×2, Ar-H 5 ×2), 6.87 (d, J = 16.0 Hz, 2H, CO-CH=×2). LC-MS m / z: 329.10 (M+H) + , calcd for C 18 H 14 F2N2O2: 328.10.

[0041] Active compound F10 (E)-3-(4-fluorophenyl)-N'-((E)-3-(4-fluorophenyl)acryloyl)acrylohydrazide (F10)

[0042] Apricot powder, 27.8% yield, mp 276.8 - 278.7℃. 1 1H NMR (400 MHz, DMSO) δ: 10.05 (s, 2H, NH×2), 7.67 (dd, J = 8.6, 5.6 Hz, 4H, Ar-H 2 ×2, Ar-H 6 ×2), 7.54 (d, J = 15.8 Hz, 2H, Ar-CH=×2), 7.27 (t, J = 8.8 Hz, 4H, Ar-H 3 ×2, Ar-H 5 ×2), 6.71 (d, J = 15.9 Hz, 2H, CO-CH=×2). LC-MS m / z: 329.10 (M+H) + , calcd for C 18 H 14 F2N2O2: 328.10.

[0043] Active compound F12 (E)-3-(2,6-difluorophenyl)-N'-((E)-3-(2,6-difluorophenyl)acryloyl)acrylohydrazide (F12)

[0044] Apricot powder, 24.4% yield, mp 258.3 - 259.6℃. 11H NMR (400 MHz, DMSO) δ: 10.77 (s, 2H, NH×2), 7.53 (d, J = 16.2 Hz, 2H, Ar-CH=×2), 7.50 - 7.47 (m, Ar-H 4 ×2) 7.21 (t, J = 8.7 Hz, 4H, Ar-H 3 ×2, Ar-H 5 ×2), 7.00 (d, J = 16.2 Hz, 2H, CO-CH=×2). LC-MS m / z: 365.08 (M+H) + , calcd for C 18 H 12 F4N2O2: 364.08. Active compound F13 (E)-3-(3,5-difluorophenyl)-N'-((E)-3-(3,5-difluorophenyl)acryloyl)acrylohydrazide (F13)

[0045] White powder, 22.9% yield, mp 293.8 - 294.6 °C. 1 1H NMR (400 MHz, DMSO) δ: 10.61 (s, 2H, NH×2), 7.54 (d, J = 15.8 Hz, 2H, Ar-CH=×2), 7.36 (d, J = 6.5 Hz, 4H, Ar-H 2 ×2, Ar-H 6 ×2), 7.28 (tt, J = 9.2, 2.2 Hz, 2H, Ar-H 4 ×2), 6.83 (d, J = 15.9 Hz, 2H, CO-CH=×2). LC-MS m / z: 365.08 (M+H) + , calcd for C 18 H 12 F4N2O2: 364.08.

[0046] Active compound F14 (E)-3-(2-chlorophenyl)-N'-((E)-3-(2-chlorophenyl)acryloyl)acrylohydrazide (F14)

[0047] White powder, 26.1% yield, mp 292.6 - 294.3 °C. 11H NMR (400 MHz, DMSO) δ: 10.67 (s, 2H, NH×2), 7.82 (d, J = 15.8 Hz, 2H, Ar-CH=×2), 7.77 - 7.72 (m, 2H, Ar-H 3 ×2), 7.56 - 7.51 (m, 2H, Ar-H 4 ×2), 7.44 - 7.39 (m, 4H, Ar-H 5 ×2, Ar-H 6 ×2), 6.82 (d, J = 15.8 Hz, 2H, CO-CH=×2). LC-MS m / z: 361.04 (M+H) + , calcd for C 18 H 14 Cl2N2O2: 360.04.

[0048] Active compound F17 (E)-3-(3,5-dichlorophenyl)-N'-((E)-3-(3,5-dichlorophenyl)acryloyl)acrylohydrazide (F17)

[0049] White powder, 23.4% yield, mp > 300℃. 1 1H NMR (400 MHz, DMSO) δ: 10.59 (s, 2H, NH×2), 7.68 (d, J

[0050] =1.8 Hz, 4H, Ar-H 2 ×2, Ar-H 6 ×2), 7.63 (t, J = 1.8 Hz, 2H, Ar-H 4 ×2), 7.51 (d, J = 15.9 Hz, 2H, Ar-CH=×2), 6.87 (d, J = 15.9 Hz, 2H, CO-CH=×2). LC-MS m / z: 428.96 (M+H) + , calcd for C 18 H 12 C l4 N2O2: 427.96. Active compound F18 (E)-3-(3-bromophenyl)-N'-((E)-3-(3-bromophenyl)acryloyl)acrylohydrazide (F18)

[0051] Apricot powder, 22.6% yield, mp 262.0 - 264.5℃. 11H NMR (400 MHz, DMSO) δ: 10.51 (s, 2H, NH×2), 7.78 (s, 2H, Ar-H 2 ×2), 7.59 - 7.55 (m, 4H, Ar-H 4 ×2, Ar-H 6 ×2), 7.49 (d, J=15.8 Hz, 2H, Ar-CH=×2), 7.36 (t, J=7.8 Hz, 2H, Ar-H 5 ×2), 6.78 (d, J=15.9 Hz, 2H, CO-CH=×2). LC-MS m / z: 448.94 (M+H) + , calcd for C 18 H 14 Br2N2O2: 447.94.

[0052] Active compound F19 (1E,1'E)-hydrazine-1,2-diylbis(3-oxoprop-1-ene-3,1-diyl))bis(3,1-phenylene)diacetate (F19)

[0053] Yellow powder, 24.6% yield, mp 216.0 - 218.3℃. 1 1H NMR (400 MHz, DMSO) δ: 10.50 (s, 2H, NH×2), 7.53 (dd, J=15.8, 1.9 Hz, 2H, Ar-CH=×2), 7.49 (d, J=7.7 Hz, 2H, Ar-H 5 ×2), 7.45 (dd, J=7.6, 2.2 Hz, 2H, Ar-H 6 ×2), 7.37 (s, 2H, Ar-H 2 ×2), 7.16 (d, J=7.6 Hz, 2H, Ar-H 4 ×2), 6.77 (dd, J=15.8, 2.1 Hz, 2H, CO-CH=×2), 2.28 (d, J=2.3 Hz, 6H, 3-OCOCH3×2). 13 13C-NMR (400 MHz, DMSO-d6), δ169.66(2), 163.25(2), 151.41(2), 139.62(2), 136.71(2), 130.59(2), 125.71(2), 123.70(2), 121.21(2), 120.93(2), 21.37(2). LC-MS m / z: 409.13 (M+H) + , calcd for C22 H 20 N2O6: 408.13.

[0054] Active compound F20

[0055] (E)-3-(2-(trifluoromethyl)phenyl)-N'-((E)-3-(2-(trifluoromethyl)phenyl)acryloyl)acrylohydrazide (F20)

[0056] White powder, 22.1% yield, mp 290.6 - 292.1 °C. 1 H NMR (400 MHz, DMSO) δ: 10.71 (s, 2H, NH×2), 7.89 (d, J = 5.2 Hz, 2H, Ar-H 3 ×2), 7.84 - 7.75 (m, 6H, Ar-CH=×2, Ar-H 5 ×2, Ar-H 6 ×2), 7.66 - 7.61 (m, 2H, Ar-H 4 ×2), 6.84 (dd, J = 15.5, 3.4 Hz, 2H, CO-CH=×2). LC-MS m / z: 429.10 (M + H) + , calcd for C 20 H 14 F6N2O2: 428.10.

[0057] Comparative compound F2 (E)-3-(3-methoxyphenyl)-N'-((E)-3-(3-methoxyphenyl)acryloyl)acrylohydrazide (F2)

[0058] Light yellow powder, 40.9% yield, mp 183.6 - 185.3 °C. 1 H NMR (400 MHz, DMSO) δ: 10.46 (s, 2H, NH×2), 7.48 (d, J = 15.8 Hz, 2H, Ar-CH=×2), 7.31 (t, J = 7.9 Hz, 2H, Ar-H 5 ×2), 7.17 - 7.11 (m, 4H, Ar-H 2 ×2, Ar-H 6 ×2), 6.94 (dd, J = 8.2, 2.4 Hz, 2H, Ar-H 4×2), 6.74 (d, J = 15.8 Hz, 2H, CO-CH=×2), 3.75 (s, 6H, 3-OCH3×2). LC-MS m / z: 353.14 (M+H) + , calcd for C 23 H 24 O6: 352.14.

[0059] Comparative compound F3 (E)-3-(2,4-dimethoxyphenyl)-N'-((E)-3-(2,4-dimethoxyphenyl)acryloyl)acrylohydrazide (F3)

[0060] White powder, 35.8% yield, mp 284.8 - 287.4 °C. 1 H NMR (400 MHz, DMSO) δ: 10.22 (s, 2H, NH×2), 7.65 (d, J = 15.9 Hz, 2H, Ar-CH=×2), 7.47 (d, J = 8.5 Hz, 2H, Ar-H 6 ×2), 6.66 (d, J = 15.9 Hz, 2H, CO-CH=×2), 6.62 - 6.57 (m, 4H, Ar-H 3 ×2, Ar-H 5 ×2), 3.86 (s, 6H, 2-OCH3), 3.80 (s, 6H, 4-OCH3). LC-MS m / z: 413.16 (M+H) + , calcd for C 22 H 22 O5: 412.16.

[0061] Comparative compound F6 (E)-3-(3,5-dimethoxyphenyl)-N'-((E)-3-(3,5-dimethoxyphenyl)acryloyl)acrylohydrazide (F6)

[0062] Yellow powder, 35.2% yield, mp 268.8 - 269.9 °C. 1 H NMR (400 MHz, DMSO) δ: 10.47 (s, 2H, NH×2), 7.45 (d, J = 15.8 Hz, 2H, Ar-CH=×2), 6.78 - 6.74 (m, 6H, Ar-H 2 ×2, Ar-H 6 ×2, CO-CH=×2), 6.53 (t, J = 2.1 Hz, 2H, Ar-H4 ×2), 3.76 (s, 12H, 3 - OCH3×2, 5 - OCH3×2). LC - MS m / z: 413.16 (M + H) + , calcd for C 25 H 28 O7: 412.16.

[0063] Comparative compound F11 (E)-3-(2,5 - difluorophenyl)-N'-((E)-3-(2,5 - difluorophenyl)acryloyl)acrylohydrazide (F11)

[0064] White powder, 24.4% yield, mp 258.3 - 259.6℃. 1 H NMR (400 MHz, DMSO) δ: 10.64 (s, 2H, NH×2), 7.57 - 7.54 (m, Ar - H 3 ×2) 7.52 (d, J=16.0 Hz, 2H, Ar - CH=×2), 7.37 - 7.25 (m, 4H, Ar - H 4 ×2, Ar - H 6 ×2), 6.88 (d, J=16.0 Hz, 2H, CO - CH=×2). LC - MS m / z: 365.08 (M + H) + , calcd for C 18 H 12 F4N2O2: 364.08.

[0065] Comparative compound F15 (E)-3-(3 - chlorophenyl)-N'-((E)-3-(3 - chlorophenyl)acryloyl)acrylohydrazide (F15)

[0066] Apricot powder, 25.9% yield, mp 251.4 - 252.8℃. 1 H NMR (400 MHz, DMSO) δ: 10.44 (s, 2H, NH×2), 7.58 (s, 2H, Ar - H 6 ×2), 7.50 - 7.47 (m, 2H, Ar - H 5 ×2), 7.43 (d, J=15.9 Hz, 2H, Ar - CH=×2), 7.36 (dd, J=3.6, 1.6 Hz, 4H, Ar - H 2 ×2, Ar - H 4×2),6.72(d,J=15.9Hz,2H,CO-CH=×2).LC-MS m / z:361.04(M+H) + ,calcd for C 18 H 14 Cl2N2O2:360.04.

[0067] Comparative compound F16 (E)-3-(2,6-dichlorophenyl)-N'-((E)-3-(2,6-dichlorophenyl)acryloyl)acrylohydrazide (F16)

[0068] White powder,20.1%yield,mp>300℃. 1 H-NMR(400MHz,DMSO),δ:10.14(s,2H,NH×2),7.52(d,J

[0069] =8.4Hz,4H,Ar-CH=×2,Ar-H 4 ×2),6.82(d,J=8.4Hz,4H,Ar-H 3 ×2,Ar-H 5 ×2),6.19(d,J=16.8Hz,2H,CO-CH=×2).LC-MS m / z:428.96(M+H) + ,calcd for C 18 H 12 C l4 N2O2:427.96.

[0070] Example 2 Comparison of stability between bisamide curcumin analogs and curcumin:

[0071] The stability of curcumin and its analogs was determined by UV-visible spectrophotometer. A certain mass of the compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a solution with a concentration of 1 μM, and the solution was diluted to 40 μM with Dulbecco's modified Eagle medium (DMEM medium, pH = 7.0-7.4). The light absorbance of the compound was determined by UV-visible spectrophotometer, scanning the full wavelength range from 200 nm to 800 nm at intervals of 5 nm within 30 min, and measuring every 5 min. The absorption curve was obtained by data analysis. Each experiment was repeated 3 times.

[0072] Taking advantage of the instability of the β-diketone structure of curcumin and the stability of the amide bond, a bisamide curcumin skeleton was designed and synthesized by constructing two amide groups between the two carbonyl groups. Compared with the curcumin skeleton, the stability of the bisamide curcumin skeleton is significantly improved (see Appendix). Figure 1 ).

[0073] Example 3 Detection of the protective effect of bisamide curcumin analogs on PC12 cells in vitro:

[0074] Rat adrenal pheochromocytoma cells (PC12) were purchased from the Wuhan University Cell Bank. The cells were cultured in DMEM high glucose complete medium containing 10% FBS and 0.2% double antibody. PC12 cells were cultured at 37°C in a constant temperature cell culture incubator containing 5% CO2, and cells in the logarithmic growth phase were selected for the experiment. PC12 cells were seeded in a 96-well plate at a density of 5000 cells per well, and 100LDMEM high glucose medium was placed in a constant temperature incubator for overnight culture. The next day, each well was incubated with 1M compound for 18h, then stimulated with a certain concentration of hydrogen peroxide (i.e., a hydrogen peroxide concentration with a cell survival rate of 60%) for 24h, and finally the absorbance was measured with a microplate reader (Bio-Rad, USA). Each test was repeated at least three times.

[0075] Compared with the blank control group (DMSO group), the survival rate of PC12 cells in the hydrogen peroxide treatment group was only about 60%. However, after pretreatment with the compounds, most of the survival rates could be increased to 80%, among which compound F19 showed the best cell protection effect and had better antioxidant activity than its lead compound curcumin (Appendix Figure 2 ). Therefore, F19 can be considered as a cytoprotective agent against oxidative damage in PC12 cells.

[0076] Example 4 Protection of bisamide curcumin analog F19 against hydrogen peroxide-induced oxidative stress in PC12 cells:

[0077] Cell colony formation test: PC12 cells were seeded in a 6-well plate at a density of 2000 cells / well. After pretreatment with F19 (1 μM) for 18 hours, the cells were incubated with a certain concentration of hydrogen peroxide for 8 days. Finally, the culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 20 minutes, and then fixed with crystal violet dye solution for 20 minutes. PBS was washed until no dye solution was dissolved, and the cells were dried and photographed. MDA determination: PC12 cells were 3×10 5 The cells were seeded in a 6-well plate at a density of 1 μM / well and adhered to the wall. A control group, a model group, and a drug-treated group were set up. The drug-treated group was given F19 (1 μM) and incubated for 18 h, and an equal volume of DMSO solution was added to the control group to eliminate the effect of the solvent DMSO. Afterwards, the cells were stimulated with 1 mM hydrogen peroxide for 6 h in the drug-treated group and the model group, proteins were collected, and the MDA level was determined according to the instructions of the MDA detection kit. This experiment was repeated three times. Reactive oxygen species determination: The production of ROS in PC12 cells was detected using dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. PC12 cells were cultured at 3×105 Cells were inoculated in 6-well plates and cultured for 24 hours. The drug-treated group was treated with F19 (1 μM or 10 μM). The cells were pretreated for 24 hours, and an equal volume of DMSO solution was added to the control group at the same time. They were exposed to hydrogen peroxide for another 2 hours, the culture medium was discarded, washed with PBS, and 1 mL of starvation medium containing the DCFH-DA probe (1:1000) was added in the dark at 37°C. After incubation for another 30 minutes, the cells were photographed with a light-proof inverted fluorescence microscope (Nikon), and finally washed three times with PBS and then detected on a flow analyzer. The fluorescence intensity reacted to the ROS level.

[0078] The antioxidant activity of F19 against hydrogen peroxide-induced oxidative damage was tested. Compound F19 was able to resist the damage caused by hydrogen peroxide to PC12 cells and promote colony formation, which further confirmed the cytoprotective effect of F19 (Appendix Figure 3 A). Malondialdehyde (MDA) is an important biomarker of oxidative stress. ROS can lead to excessive accumulation of MDA. The extent of intracellular ROS can be evaluated by the intracellular MDA content. Hydrogen peroxide stimulation can sharply increase the MDA level in PC12 cells. After pretreatment with F19 for 18 hours, the MDA level in the cells dropped sharply, indicating that F19 can resist the oxidative stress damage caused by excessive ROS to cells (Appendix Figure 3 B). In addition, compound F19 has an inhibitory effect on the production of intracellular ROS induced by hydrogen peroxide. The ROS level was measured by DCFH-DA, where the green fluorescence intensity and peak shift are proportional to the amount of ROS. Compared with the DMSO group, the ROS level in PC12 cells stimulated by hydrogen peroxide increased significantly, while F19 pretreatment for 24 hours significantly eliminated the intracellular ROS generated by hydrogen peroxide stimulation (attached Figure 3 C, D).

[0079] Example 5 Potential mechanism of antioxidant effect of bisamide curcumin analog F19:

[0080] Western blot assay: PC12 cells were cultured at 3×10 5The density of cells / well was seeded in a 6-well plate and incubated in a constant temperature incubator for 24 hours. After that, incubation continued for 18 hours with DMSO and different concentrations of F19 (0.5, 1, 2 and 5 μM). Then remove the 6-well plate from the incubator, carefully discard the liquid, wash the wells with 4 ° C PBS, and lyse with pre-cooled 60 μL of RIPA lysate on ice for 10 minutes. Scrape off the adhered cells with a protein scraper and collect them into the corresponding 1.5mL EP tube (for nuclear protein, extract according to the kit instructions). The supernatant was separated by centrifugation at 12000rpm for 15 minutes. After sample preparation, denature 10min in 100 ° C boiling water with 5 times loading buffer, cool and store in a -20 ° C refrigerator. Protein samples were separated by electrophoresis in 10% polyacrylamide gel and then transferred to PVDF membrane in an ice water bath, with transfer conditions of 300mA for 90 minutes. The samples were then blocked with 5% skim milk powder for 1 hour at room temperature, washed 3 times with TBST buffer for 7 minutes each time, added with the primary antibody and incubated overnight in a shaker at 4°C, washed with TBST the next day and incubated with the secondary antibody in a shaker for 1 hour at room temperature, washed again with TBST and developed with a gel imaging system, and quantified by Quantity One gel imaging analysis software to detect the optical density ratio of the target protein to its internal reference. Primary antibody: HO-1 (1:1000), GADPH (1:1000), Nrf2 (1:500), PCNA (1:1000), secondary antibody is rabbit or mouse antibody (1:5000).

[0081] F19 promoted the expression of Nrf2 protein in the nucleus of PC12 cells and activated the Nrf2 antioxidant signaling pathway (Appendix Figure 4 A). Heme oxygenase-1 (HO-1) is an antioxidant protein downstream of the Nrf2 signaling pathway. Next, the effect of F19 on HO-1 protein expression in PC12 cells was evaluated. The results showed that in cells pre-incubated with F19, F19 promoted HO-1 expression in a dose-dependent manner (see Appendix Figure 4 B). Therefore, compound F19 can protect PC12 cells from hydrogen peroxide-induced oxidative damage by promoting the entry of nuclear factor Nrf2 and the expression of antioxidant protein HO-1.

[0082] Example 6 Bisamide curcumin analog F19 alleviates brain damage after ischemia-reperfusion in mice:

[0083] MCAO-induced cerebral ischemia / reperfusion injury model: All animals were divided into 4 groups and injected with saline, solvent (DMSO: castor oil: saline = 1:19:80) and F19 1 hour before modeling. First, mice were anesthetized with 1% sodium pentobarbital (50 mg / kg) and then fixed in supine position. Secondly, the inner side of the neck skin was incised, the anterior neck muscle group was separated, the common carotid artery was exposed, and the internal carotid artery and external carotid artery were extended upward to separate. Again, the external carotid artery and its small blood vessels were ligated with fine surgical thread, and the internal carotid artery and common carotid artery were temporarily closed with an artery clamp. Finally, a small incision was made in the external carotid artery about 2 mm from the branch of the common carotid artery, and a wire was inserted to block the middle cerebral artery. After 1 hour, the wire was slowly removed and the mice were placed on a heating pad until they woke up. The sham group underwent the same procedure, but no wire was inserted. Behavioral testing and calculation of infarct area: The neurological deficit scores of mice were evaluated 48 hours after reperfusion. Scoring criteria are: 0, no signs of nerve damage; 1. Inability to fully extend the contralateral forepaw; 2. Entanglement to the paralyzed side; 3. Tilt to the contralateral side; 4. No spontaneous motor activity and loss of consciousness. For 2,3,5-triphenyltetrazolium chloride (TTC) staining, first precool the six-well plate containing PBS and brain bath on ice for 5 minutes, and place the culture dish containing TTC in a 37°C oven. Place the removed brain in precooled PBS, and after it hardens slightly, place it in a brain mold, cut 5 1mm thick brain slices in succession, and gently place the brain slices in 2% TTC staining solution with tweezers to avoid light staining, flip for about 5 minutes, until the normal brain tissue is red, the infarcted tissue is white, and the color on both sides is uniform and beautiful, add paraformaldehyde for fixation, and take pictures after 6 hours, and measure the infarct area with Image J software.

[0084] Given the good cell protection effect of compound F19 in vitro, the neuroprotective effect of F19 in mice was further studied. The middle cerebral artery occlusion (MCAO) model is a classic animal model for studying ischemic stroke and can simulate the animal model of clinical ischemic stroke. Therefore, the mouse MCAO model was used to further evaluate the in vivo antioxidant activity of compound F19 (see Appendix Figure 5 A). Compared with the sham operation group, the cerebral infarction area of ​​the mice after modeling was significantly increased (see Appendix Figure 5 B). Compared with the vehicle group, the drug group had significantly smaller infarct volume and significantly improved neurological scores (Appendix Figure 5 C). Therefore, active compound F19 alleviated cerebral ischemia-reperfusion injury in mice and has the potential to treat ischemic stroke.

[0085] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of any one of the compounds shown below in the preparation of antioxidant drugs.

2. The use according to claim 1, characterized in that: The antioxidant is able to inhibit various diseases caused by oxidative stress damage caused by free radicals.

3. The use according to claims 1 and 2, wherein the oxidative stress injury includes but is not limited to the following: cerebral ischemia-reperfusion oxidative stress injury.

4. The use according to claim 1, wherein the compound is preferably selected from the following compounds:

5. A pharmaceutical composition for treating oxidative stress damage, comprising a therapeutically effective amount of the compound according to claim 1 or a pharmaceutical excipient thereof.

6. The pharmaceutical composition according to claim 5, wherein the compound according to claim 1 or 4 or a pharmaceutically acceptable salt thereof is the sole active ingredient.

7. The pharmaceutical composition according to claim 6, characterized in that: The pharmaceutical composition is in the form of an injection, tablet, suppository, film, capsule, ointment, aerosol, pellet, controlled-release or sustained-release preparation and nano preparation.

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