Use of bisamide curcumin compounds in the preparation of oxidative stress damage protective drugs

Abstract

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

Description

Technical fields:

[0001] This invention belongs to the field of medicinal chemistry. Specifically, this invention relates to the application of specific diamide curcumin compounds in the preparation of antioxidant drugs and in the treatment of oxidative stress damage. These compounds can effectively antagonize oxidative stress damage, thereby playing a good role in in vitro and in vivo antioxidant protection. Background technology:

[0002] Curcumin is a compound with excellent antioxidant and anti-inflammatory effects, but its instability and low bioavailability limit its clinical application. To address this issue, various structural modifications have been made to curcumin, but existing techniques significantly alter the curcumin skeleton, which compromises its intrinsic activity. This invention proposes designing and synthesizing nitrogen-containing dicarbonyl curcumin analogs by introducing stable amide bonds to improve stability and maintain pharmacological activity. Through long-term research and practice, the inventors have obtained specific compounds with good antioxidant activity from a series of diamide curcumin analogs without structure-function guidance. Summary of the Invention:

[0003] The purpose of this invention is to provide the application of 13 diamide 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 injury, comprising a therapeutically effective amount of any one or more of the diamide curcumin compounds of claim 1 or their pharmaceutically acceptable salts and excipients as active ingredients.

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

[0006]

[0007]

[0008] Effective 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, F₄ molecular formula is C₂O₄. 22 H 22O5, chemical name is (E)-3-(2,5-dimethoxyphenyl)-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, and F7 molecular formula is C 24 H 28 N₂O₈, chemically named (E)-3-(3,4,5-trimethoxyphenyl)-N'-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)acrylohydrazide, has the molecular formula C₂O₈. 22 H 24 N₂O₄, chemical name is (E)-3-(4-ethoxyphenyl)-N'-((E)-3-(4-ethoxyphenyl)acryloyl)acrylohydrazide, F₁ has the molecular formula C₂. 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, and the 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, and the 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, and the 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, chemically named ((1E,1'E)-hydrazine-1,2-diylbis(3-oxoprop-1-ene-3,1-diyl))bis(3,1-phenylene)diacetate, has the molecular formula C20. 25 H 21 BrN2O4, chemically named 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, and F3 has the molecular formula C. 22 H 22 O5, chemical name is (E)-3-(2,4-dime thoxyphenyl)-N'-((E)-3-(2,4-dimethoxyphenyl)acryloyl)acrylohydrazide, and F6 has the molecular formula C 25 H 28 O7, chemical name is (E)-3-(3,5-dimethoxyphenyl)-N'-((E)-3-(3,5-dimethoxyphenyl)acryloyl)acrylohydrazide, F11 molecular formula is C 18 H 12F4N2O2, chemical name is (E)-3-(2,5-difluorophenyl)-N'-((E)-3-(2,5-difluorophenyl)acryloyl)acrylohydrazide, and the molecular formula of F15 is C 18 H 14 Cl2N2O2, chemical name is (E)-3-(3-chlorophenyl)-N'-((E)-3-(3-chlorophenyl)acryloyl)acrylohydrazide, F16 molecular formula 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. Curcumin's molecular formula is C₂O₂. 21 H 20 O6, chemically named (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione.

[0010] Since the β-diketone structure in the curcumin chemical structure is a significant factor leading to its poor structural stability, this invention first chemically modifies the β-diketone in the curcumin skeleton by replacing it with an amide bond to obtain a class of diamide curcumin analogs. These diamide curcumin compounds are synthesized using classic synthetic methods such as the Knoevenagel reaction (see Example 1 for details). The stability of the obtained diamide curcumin skeleton is significantly improved (see Example 2 for details). Subsequently, based on this skeleton, a series of derivatives were synthesized and screened, yielding the following technical effects:

[0011] (1) Hydrogen peroxide (H₂O₂) 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., ultimately leading to cell death. Therefore, we selected the H₂O₂-induced PC12 cell oxidative damage model to evaluate the in vitro antioxidant activity of novel diamide curcumin analogs F1-F20 (see Example 3 for details). Among them, compound F19 showed the best cell protection effect and its antioxidant activity was also superior to its lead compound curcumin. Therefore, F19 can be regarded as a cell protectant against oxidative damage in 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 drastically increase the level of MDA in PC12 cells, while the level of MDA in cells decreased sharply after 18 hours of pretreatment with F19, indicating that F19 can resist the oxidative stress damage caused by excessive ROS to cells. In addition, compound F19 has an inhibitory effect on hydrogen peroxide-induced intracellular ROS generation. ROS levels were measured using DCFH-DA. Compared with the DMSO group, the ROS level in PC12 cells stimulated by hydrogen peroxide was significantly increased, while pretreatment with F19 for 24 hours could significantly clear the intracellular ROS generated by hydrogen peroxide stimulation (see Example 4 for details).

[0013] (3) Curcumin is an activator of Nrf2, exerting 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 antioxidant activity research. 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, activating 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 HO-1 expression in a dose-dependent manner. Therefore, compound F19 may resist hydrogen peroxide-induced oxidative damage in PC12 cells by promoting the entry of nuclear factor Nrf2 and the expression of the 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 clinical ischemic stroke. Compared with the sham-operated group, the infarct area in mice after modeling was significantly increased. Compared with the solvent group, the infarct area in the drug F19 group was significantly smaller, and the neurological function score was significantly improved. Therefore, the active compound F19 alleviates cerebral ischemia-reperfusion injury in mice and has the potential to treat ischemic stroke.

[0015] The diamide curcumin compounds described in this invention can be used in the preparation of antioxidant drugs and therapeutic drugs for diseases related to oxidative stress injury. These diseases include, but are not limited to, the following: acute oxidative stress-induced sepsis; ischemia and ischemia-reperfusion injury of organs such as the eyes, brain, heart, kidneys, and liver; acute mild to moderate pain (e.g., postoperative, post-traumatic, post-exhaustion, primary dysmenorrhea, toothache, headache); chronic oxidative stress-induced Parkinson's disease, Alzheimer's disease, atherosclerosis, diabetic complications; rheumatoid arthritis, osteoarthritis, spondyloarthritis, gouty arthritis; and non-articular soft tissue rheumatic pain (e.g., shoulder pain, tenosynovitis, bursitis, myalgia, or post-exercise injury pain).

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

[0017] The term "pharmaceutical excipients" as used in this article refers to conventional drug carriers in the pharmaceutical field, such as: diluents, excipients like water, fillers like starch and sucrose; binders like cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; humectants like glycerin; disintegrants like agar, calcium carbonate, and sodium bicarbonate; absorption enhancers like quaternary ammonium compounds; surfactants like hexadecyl alcohol; adsorbents like kaolin and soap clay; and lubricants like talc, calcium / magnesium stearate, and polyethylene glycol. Other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0018] Various dosage forms of the pharmaceutical compositions of this invention can be prepared according to conventional pharmaceutical manufacturing methods. For example, the active ingredient is mixed with one or more carriers and then formulated into the desired dosage form. The formulations of the drug include injections, tablets, capsules, aerosols, suppositories, films, pellets, ointments, controlled-release or sustained-release formulations, or nanoformulations. This invention can be administered to patients requiring this treatment via oral, nasal, rectal, or parenteral administration in the form of a composition. For oral administration, it can be formulated as conventional solid dosage forms such as tablets, powders, granules, capsules, etc., or as liquid dosage forms such as aqueous or oil suspensions or other liquid dosage forms such as syrups, elixirs, etc. For parenteral administration, it can be formulated as solutions for injection, aqueous or oil suspensions, etc. Attached image description:

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

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

[0021] Appendix Figure 2 The protective effect of the compound against hydrogen peroxide-induced PC12 cell injury model. PC12 cells were pre-incubated with the compound (1 μM) for 18 hours, followed by treatment with hydrogen peroxide (480 μM) for 24 hours. Finally, the viability of PC12 cells was assessed using the MTT assay. Data are expressed as mean ± standard deviation, n = 3. #### P<0.0001 vs DMSO; ****P<0.0001,***P<0.001,**P<0.01,*P<0.05 vs hydrogen peroxide.

[0022] Appendix Figure 3 In a hydrogen peroxide-induced oxidative damage model of PC12 cells, 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 h. Cells were incubated in the dark with a dichlorofluorescein diacetate (DCFH-DA) assay and finally detected by inverted fluorescence microscopy or flow cytometry. Fluorescence intensity represents ROS levels. 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] Appendix Figure 4F19 activates the Nrf2 antioxidant signaling pathway. (A) F19 activates the Nrf2 antioxidant signaling pathway and promotes Nrf2 entry into the cell nucleus. PC12 cells were pretreated with F19 (1 μM) for 18 hours, nuclear proteins were extracted, and Nrf2 protein expression was measured by Western blotting. (B) F19 activates the Nrf2 antioxidant signaling pathway and promotes the expression of the antioxidant protein HO-1. PC12 cells were pre-incubated with F19 (0.5, 1, 2, 5 μM) for 18 hours, and HO-1 protein expression levels were measured by Western blotting. Data are expressed as mean SD compared to DMSO, n = 3, *P < 0.01, *P < 0.05

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

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

[0026] Example 1: Synthesis of diamide curcumin derivative:

[0027] The synthetic route for this compound is shown in Scheme 1. The substituted cinnamic acid was obtained by reacting the corresponding substituted benzaldehyde with malonic acid and piperazine in pyridine using Knoevenagel. Under nitrogen protection, the substituted cinnamic acid reacted with oxalyl chloride to generate the intermediate cinnamoyl chloride. After removing the solvent and excess oxalyl chloride under vacuum, the crude product was dissolved in dry THF, followed by the addition of hydrazine hydrate and triethylamine to obtain the target product. All compounds were purified by recrystallization from a dimethyl sulfoxide / anhydrous ethanol system. The target compounds were confirmed and characterized by NMR and LC-MS. The synthetic chemical reactions and compound data for all synthesized bisamide curcumin derivatives are as follows:

[0028]

[0029] Option 1: Reagents and conditions: (a) Malonic acid, piperazine, pyridine, 90℃, 3h; (b) Oxaloyl 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 °C. 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 °C. 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] Compare 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: Stability comparison between diamide curcumin analogue and curcumin:

[0071] The stability of curcumin and its analogues was determined using a UV-Vis spectrophotometer. A specific mass of the compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 μM solution, which was then diluted to 40 μM using Duchenne modified Eagle medium (DMEM, pH 7.0–7.4). The absorbance of the compound was measured using a UV-Vis spectrophotometer, scanning the entire wavelength range from 200 nm to 800 nm at 5 nm intervals over 30 min. Absorption curves were obtained through data analysis. Each experiment was repeated three times.

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

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

[0074] Rat adrenal pheochromocytoma cells (PC12) were purchased from the cell bank of Wuhan University. 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 cell culture incubator containing 5% CO2, with cells in the logarithmic growth phase used in the experiments. PC12 cells were seeded at a density of 5000 cells per well in 96-well plates and incubated overnight in 100 LMEM high-glucose medium. The next day, each well was incubated with a 1M compound for 18 hours, followed by stimulation with a specific concentration of hydrogen peroxide (i.e., a concentration that ensures 60% cell viability) for 24 hours. Finally, absorbance was measured using a microplate reader (Bio-Rad, USA). Each test was repeated at least three times.

[0075] Compared to 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 compounds, the survival rate could be increased to 80% in most cases, with compound F19 showing the best cell protective effect and better antioxidant activity than its lead compound curcumin (see appendix). Figure 2 Therefore, F19 can be considered a cytoprotective agent against oxidative damage in PC12 cells.

[0076] Example 4: Detection of the protective effect of the diamide curcumin analog F19 against hydrogen peroxide-induced oxidative stress in PC12 cells:

[0077] Cell colony formation assay: PC12 cells were seeded at a density of 2000 cells / well in 6-well plates. After pretreatment with F19 (1 μmol) for 18 hours, the cells were incubated with hydrogen peroxide of a certain concentration for 8 days. The culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 20 min, followed by fixation with crystal violet dye solution for 20 min. The cells were washed with PBS until the dye-free solution was dissolved, dried, and photographed. MDA assay: PC12 cells were seeded at a density of 3 × 10⁶ cells / well. 5 Cells were seeded at a density of 3 × 10⁶ cells / well in 6-well plates and allowed to adhere. Control, model, and drug-treated groups were established. The drug-treated groups were treated with F19 (1 μM) and incubated for 18 h, while the control group received an equal volume of DMSO solution to eliminate the influence of the solvent DMSO. Subsequently, cells in both the drug-treated and model groups were stimulated with 1 mM hydrogen peroxide for 6 h, proteins were collected, and MDA levels were measured according to the instructions of the MDA detection kit. This experiment was repeated three times. Reactive oxygen species (ROS) assay: ROS production in PC12 cells was detected using the dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. PC12 cells were seeded at a density of 3 × 10⁶ cells / well.5 Cells were seeded per well in 6-well plates and cultured for 24 h. The drug-treated groups were treated with F19 (1 μmol or 10 μmol). Cells were pretreated for 24 h, while the control group received an equal volume of DMSO solution. Cells were then exposed to hydrogen peroxide for 2 h, the culture medium was discarded, and the cells were washed with PBS. In the dark at 37°C, 1 mL of starved medium containing the DCFH-DA probe (1:1000) was added. After incubation for another 30 minutes, images were taken using an inverted fluorescence microscope (Nikon) in the dark. Cells were washed three times with PBS and then analyzed using a flow analyzer; fluorescence intensity correlated with ROS levels.

[0078] The antioxidant activity of F19 against hydrogen peroxide-induced oxidative damage was investigated. Compound F19 was able to resist hydrogen peroxide-induced damage to PC12 cells and promote colony formation, further confirming the cytoprotective effect of F19 (see appendix). Figure 3 A). Malondialdehyde (MDA) is an important biomarker of oxidative stress. ROS leads to excessive accumulation of MDA. The level of intracellular ROS can be evaluated by the intracellular MDA content. Hydrogen peroxide stimulation can drastically increase the MDA level in PC12 cells, while after 18 hours of pretreatment with F19, the MDA level in the cells decreased sharply, indicating that F19 can resist the oxidative stress damage caused by excessive ROS to cells (see appendix). Figure 3 B). Furthermore, compound F19 inhibited hydrogen peroxide-induced intracellular ROS production. ROS levels were measured using DCFH-DA, where green fluorescence intensity and peak shift were proportional to the amount of ROS. Compared to the DMSO group, hydrogen peroxide-stimulated PC12 cells showed a significant increase in ROS levels, while 24 hours of F19 pretreatment significantly cleared intracellular ROS generated by hydrogen peroxide stimulation (see attached image). Figure 3 C, D).

[0079] Example 5: Potential mechanism of antioxidant activity of diamide curcumin analog F19:

[0080] Western blot assay: PC12 cells were blotted at 3 × 10⁻⁶ m³ / s. 5Cells were seeded at a density in 6-well plates and incubated for 24 hours in a constant temperature incubator. Afterward, incubation was continued for 18 hours with DMSO and different concentrations of F19 (0.5, 1, 2, and 5 μM). The 6-well plates were then removed from the incubator, the liquid was carefully discarded, the wells were washed with 4°C PBS, and lysed on ice with 60 μL of pre-chilled RIPA lysate for 10 min. Adhering cells were scraped off with a protein scraper and collected into the corresponding 1.5 mL EP tubes (for nucleoproteins, extraction was performed according to kit instructions). The supernatant was separated by centrifugation at 12000 rpm for 15 min. After sample preparation, the samples were denatured in 5-fold loading buffer at 100°C for 10 min, cooled, and stored at -20°C. Protein samples were separated by electrophoresis on a 10% polyacrylamide gel and then transferred to a PVDF membrane in an ice-water bath at 300 mA for 90 min. The sample was then blocked with 5% skim milk powder for 1 hour at room temperature, washed three times with TBST buffer for 7 minutes each time, and the primary antibody was added and incubated overnight in a shaker at 4°C. The next day, the sample was washed with TBST and incubated with the secondary antibody in a shaker at room temperature for 1 hour. It was washed again with TBST and developed using a gel imaging system. Quantification was performed using Quantity One gel imaging analysis software to detect the optical density ratio of the target protein to its internal reference. Primary antibodies: HO-1 (1:1000), GADPH (1:1000), Nrf2 (1:500), PCNA (1:1000); secondary antibodies were rabbit or mouse antibodies (1:5000).

[0081] F19 promoted the expression of Nrf2 protein in the nucleus of PC12 cells and activated the Nrf2 antioxidant signaling pathway (see appendix). Figure 4 A). Heme oxygenase-1 (HO-1) is an antioxidant protein downstream of the Nrf2 signaling pathway. Next, we evaluated the effect of F19 on HO-1 protein expression in PC12 cells. 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 counteract hydrogen peroxide-induced oxidative damage in PC12 cells by promoting the entry of nuclear factor Nrf2 and the expression of the antioxidant protein HO-1.

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

[0083] MCAO-induced cerebral ischemia / reperfusion injury model: All animals were equally divided into four groups, and injected with saline, solvent (DMSO:castor oil:saline = 1:19:80), and F19, respectively, one hour before modeling. First, mice were anesthetized with 1% sodium pentobarbital (50 mg / kg) and then fixed in a supine position. Second, an incision was made on the medial side of the neck, the anterior neck muscles were separated, the common carotid artery was exposed, and the internal and external carotid arteries were separated upwards. Third, the external carotid artery and its small vessels were ligated with fine surgical sutures, and the internal and common carotid arteries were temporarily blocked with arterial clamps. Finally, a small incision was made on the external carotid artery approximately 2 mm from a branch of the common carotid artery, and a wire was inserted to block the middle cerebral artery. One hour later, the wire was slowly removed, and the mice were placed on a heating pad until they woke up. The sham-operated group underwent the same procedure but without the insertion of a wire. Behavioral testing and infarct area calculation: Neurological deficit scores were assessed in mice 48 hours after reperfusion. The scoring criteria were: 0, no signs of neurological damage; 1, inability to fully extend the contralateral forepaw; 2, entanglement to the paralyzed side; 3, tilting to the contralateral side; 4, no spontaneous motor activity and loss of consciousness. For 2,3,5-triphenyltetrazolium chloride (TTC) staining, six-well plates containing PBS and a brain bath were first pre-cooled on ice for 5 minutes, and culture dishes containing TTC were placed in a 37°C oven. The removed brain tissue was placed in pre-cooled PBS, and after slightly hardening, it was placed in a brain mold. Five 1mm thick brain slices were cut consecutively. The brain slices were gently placed into 2% TTC staining solution with forceps, avoiding light staining. The slices were inverted for about 5 minutes until normal brain tissue appeared red and infarcted tissue appeared white, with uniform and aesthetically pleasing color on both sides. Paraformaldehyde was added for fixation. After 6 hours, photographs were taken, and the infarct area was measured using ImageJ software.

[0084] Given the favorable cytoprotective effects of compound F19 in vitro, its neuroprotective effects in mice were further investigated. The middle cerebral artery occlusion (MCAO) model is a classic animal model for studying ischemic stroke, simulating 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-operated group, the infarct area in mice after modeling was significantly increased (see appendix). Figure 5 B). Compared with the solvent group, the drug group showed significantly smaller infarct volume and significantly improved neurological scores (see appendix). Figure 5 C). Therefore, the active compound F19 alleviated cerebral ischemia-reperfusion injury in mice and has the potential to treat ischemic stroke.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the following compounds in the preparation of drugs for oxidative stress injury caused by cerebral ischemia-reperfusion injury. 。 2. A pharmaceutical composition for treating oxidative stress injury caused by cerebral ischemia-reperfusion, comprising a therapeutically effective amount of the compound of claim 1 or its pharmaceutically acceptable salts and excipients.

3. The pharmaceutical composition according to claim 2, wherein the compound of claim 1 or a pharmaceutically acceptable salt thereof is the sole active ingredient.

4. The pharmaceutical composition according to claim 3, characterized in that: The pharmaceutical composition is formulated in the form of injections, tablets, suppositories, films, capsules, ointments, aerosols, pellets, controlled-release or sustained-release formulations, and nanoformulations.

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