CN113304129B - Application of mono-ketene monocarbonyl curcumin analogue in preparing antioxidant drugs - Google Patents

Application of mono-ketene monocarbonyl curcumin analogue in preparing antioxidant drugs Download PDF

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CN113304129B
CN113304129B CN202110665962.9A CN202110665962A CN113304129B CN 113304129 B CN113304129 B CN 113304129B CN 202110665962 A CN202110665962 A CN 202110665962A CN 113304129 B CN113304129 B CN 113304129B
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吴建章
何文斐
金琪玲
王靖松
张佳枫
沈梦雅
胡越
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Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
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Abstract

The invention belongs to the field of pharmaceutical chemistry, and particularly provides application of a mono-ketene monocarbonyl curcumin analogue with stable structure and good safety in medicines, in particular application in treating diseases caused by oxidative stress as an antioxidant medicine, aiming at the defects of instability of curcumin, high toxicity of a bis-ketene monocarbonyl curcumin analogue and high stability of the analogue to be prepared into medicines to be improved. The compounds have good in vitro and in vivo antioxidation effects: in vitro, the compounds have excellent antioxidant protection effect in a cell oxidative damage model induced by hydrogen peroxide; in a mouse body, the compounds have good antioxidant protection effect in cerebral ischemia-reperfusion injury related to oxidative stress.

Description

Application of mono-ketene monocarbonyl curcumin analogue in preparing antioxidant drugs
Technical Field
The invention belongs to the field of medicinal application, and particularly relates to application of a mono-ketene monocarbonyl curcumin analogue in preparation of an antioxidant medicament.
Background
Curcumin (figure 1. Cur.) is an active ingredient widely existing in Zingiberaceae plants, and has wide pharmacological activities of anti-inflammation, antioxidation, anti-tumor, etc. Due to its good pharmacological activity, over 260 clinical trials have been conducted: (https://clinicaltrials.gov) However, most of them have failed so far, and have not been approved for clinical use for the time being. The defect of pharmacokinetics due to instability is one of the main reasons for clinical failure of curcumin, and therefore, the research for maintaining the pharmacological activity of curcumin by improving the stability of curcumin is a research hotspot in the field.
The beta-diketone structure of the molecular skeleton of curcumin is the most main reason for causing the instability of curcumin, and the diketone structure is transformed into the diketene monocarbonyl curcumin analogue (the molecular skeleton is shown in figure 1. Ac) with a monoketone structure, so that the stability of curcumin can be effectively improved, and the good pharmacological activity of curcumin can be well kept (Current pharmaceutical design 2013,19 (11): 2114 CN, ZL200710066787.1 and the like). However, through literature research (ACS chem. Neurosci.2019,10,4545) and experimental comparison research, the compounds are actually higher in toxicity, and the stability of the molecular skeleton of the compounds is to be improved (fig. 1). The monoalkenone monocarbonyl curcumin analogue (a compound shown in a general formula (I)) is mainly used as a chemical raw material for synthesizing other curcumin analogues at present, and no related literature reports the pharmacological activity of the analogue.
Figure RE-GDA0003152666800000011
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the application of the monocarbonyl curcumin analogue in preparing the antioxidant drugs.
The technical scheme adopted by the invention is as follows:
the application of the compound with the general formula (I) in preparing antioxidant drugs,
Figure RE-GDA0003152666800000021
ar is a benzene ring or a naphthalene ring or an aromatic heterocyclic ring which is mono-substituted, poly-substituted or unsubstituted by a substituent R;
the substituent R is one or more of alkyl, alkenyl, alkynyl, alkoxy, halogen, halogenated alkyl, halogenated alkenyl, halogenated alkoxy, cycloalkyl alkyl, carboxyl and amino;
wherein, the substituent R in the polysubstituted Ar is the same substituent or different substituents.
Preferably, the medicament is for use in one or more of the following diseases: sepsis due to oxidative stress, ischemia and ischemia reperfusion injury of organs of brain, heart, kidney, and liver, acute mild and moderate pain (such as postoperative, traumatic, strain, primary dysmenorrhea, toothache, and headache); parkinson's disease, alzheimer's disease, atherosclerosis, diabetic complications caused by oxidative stress.
A pharmaceutical composition for the treatment of oxidative stress injury comprising a therapeutically effective amount of a compound of formula (I) or a salt or solvate thereof;
Figure RE-GDA0003152666800000022
ar is a benzene ring or a naphthalene ring or an aromatic heterocyclic ring which is mono-substituted, poly-substituted or unsubstituted by a substituent R;
the substituent R is one or more of alkyl, alkenyl, alkynyl, alkoxy, halogen, halogenated alkyl, halogenated alkenyl, halogenated alkoxy, cycloalkyl alkyl, carboxyl and amino;
wherein, the substituent R in the polysubstituted Ar is the same substituent or different substituents.
Preferably, the compound of formula (I) or a salt or solvate thereof is used as the sole active ingredient.
The preparation form of the pharmaceutical composition is selected from one of injection, tablet, suppository, membrane, capsule, ointment, aerosol, dripping pill, controlled release or sustained release agent and nano preparation.
The invention has the following beneficial effects: the invention finds the new application of the compound in the general formula (I) in preparing the medicines for treating oxidative stress injury, and compared with the existing molecular skeleton of curcumin and diketene analog curcumin with the effect of treating oxidative stress injury, the compound in the general formula (I) has the advantages of improved stability and reduced toxicity, so the compound has good application prospect in treating diseases caused by oxidative stress as an antioxidant medicine.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.
FIG. 1 (A) design, (B) stability, (C) cytotoxicity and (D) antioxidant activity of monoalkenone monocarbonylcurcumin analogs. (A) The design process from parent compound curcumin (Cur.), curcumin skeleton (Cur-S), diketene monocarbonyl curcumin analogue skeleton (Ac) to monoalkenyl monocarbonyl curcumin analogue skeleton (sAc). (B) Cur., cur-S, ac and (sAc time-light absorption profile in phosphate buffer). (C) PC12 cells were subjected to 42h reaction with Cur-S, ac and sAc (10. Mu.M), and the cytotoxicity was detected by MTT method. (D)PC12 cells were preincubated with Cur, cur-S, ac and sAc (10. Mu.M) for 18H, followed by H 2 O 2 (640. Mu.M) for 24h (D) and the protective activity of the compounds was determined by the MTT method. The cell viability of the (C, D) solvent (DMSO) group was defined as 100%. Data are expressed as mean ± SD, n =3. #### p<0.0001， ## p<0.01vs DMSO， **** p<0.0001， ** p＜0.01vs H 2 O 2 ；
FIG. 2 cytotoxicity of compounds in normal human liver cell (MIHA) cells and rat kidney cell (NRK) cells. MIHA cells (A) and NRK cells (B) were seeded in 96-well plates, and cell survival was measured by MTT method after 42h treatment with different concentrations of compound (5, 10, 20. Mu.M). Results are expressed as mean ± standard deviation, n =3. * P<0.05, ** P<0.01, **** P<0.0001 compared to DMSO group;
FIG. 3 is a synthetic scheme for compounds;
FIG. 4 cytotoxicity screening of PC12 cells by Compounds (A) and (B) against H 2 O 2 Antioxidant protection of PC12 cells in a damage model. (A) Cells were incubated with sAc1-sAc, TBHQ, PL and curcumin (10 μ M) for 42h and tested for cytotoxicity by MTT method. (B) PC12 cells are preincubated with curcumin and derivatives thereof sAc1-sAc (10 mu M) for 18H, and then H is used 2 O 2 The cells were stimulated (640. Mu.M) for 24h and the cell activity was determined by the MTT method. The cell activity of the solvent (DMSO) group was defined as 100%. Data are expressed as mean ± SD, n =3. #### p<0.0001vs DMSO， **** p＜0.0001， *** p＜0.001， ** p<0.01， * p<0.05vs H 2 O 2 ；
FIG. 5sAc (or sAc; compound sAc or sAc in the present invention) vs. H 2 O 2 Protective effect of induced oxidative damage of PC12 cells. (A) sAc promotes H 2 O 2 Colony formation of PC12 cells in the oxidative damage model. PC12 cells were preincubated with compound (2.5, 5, 10. Mu.M) for 18h, followed by 300. Mu. M H 2 O 2 The treatment is carried out for 6 days. Pictures were taken after crystal violet staining. (B) sAc is able to dose-dependently increase the survival of oxidatively damaged cells. PC12 cells in different concentrationsAfter sAc pretreatment for 18h, it was exposed to 510 μ M H 2 O 2 In (1). The MTT method detects the cell survival rate. (C) sAc dose-dependently reduces intracellular ROS levels. PC12 cells were treated with 2.5, 5, 10. Mu.M sAc for 18H, respectively, followed by 1mM H 2 O 2 Stimulation was performed for 2h, and finally, 1. Mu.L of DCFH-DA (10. Mu.M) was used for 30min of dark incubation at 37 ℃. The fluorescence microscope takes images. Results are expressed as mean ± standard deviation, n =3. #### P<0.0001 compared with the DMSO group, * P<0.05, ** P<0.01 vs. H 2 O 2 Group (d);
FIG. 6sAc for H 2 O 2 Protective effect of induced oxidative stress injury of PC12 cells. (A) Compound sAc in H 2 O 2 The colony formation of PC12 cells was promoted under the injury. PC12 cells were preincubated with sAc (0.625, 1.25 and 2.5. Mu.M) and TBHQ (2.5. Mu.M) for 18H, followed by H 2 O 2 (200. Mu.M) for 8 days. (B) Compound sAc protects PC12 cells from H in a dose-dependent manner 2 O 2 Induced cell damage. PC12 cells were preincubated with different doses (0.625, 1.25, 2.5, 5, 10 and 20. Mu.M) of sAc and TBHQ (20. Mu.M) for 18H, followed by H 2 O 2 Cells were stimulated (650. Mu.M) for 24h. Cell viability was measured by the MTT method and the viability of the solvent (DMSO) group was defined as 100%. (C, D, E) PC12 cells were incubated with sAc (1, 5 and 10. Mu.M) and TBHQ (5. Mu.M) for 18H, followed by H 2 O 2 (1 mM) effect (C) 6h or (D, E) 2h, (C) MDA levels and (D, E) intracellular ROS levels were determined according to kit instructions. Data are expressed as mean ± SD, n =3. #### p<0.0001， ### p<0.001 vs DMSO， **** p<0.0001， *** p<0.001， ** p<0.01， * p<0.05vs H 2 O 2 ；
FIG. 7sAc (or sAc; compound sAc, sAc in the present patent) reduces PC12 cell oxidative damage by activating the Nrf2/HO-1 signaling pathway. (A) sAc increases the expression of HO-1 protein in a dose-dependent manner. PC12 cells were incubated with sAc (2.5, 5, 10. Mu.M) for 18h and the levels of HO-1 and GAPDH were measured by Western Blot. (B-C) silencing Nrf2 reversed the antioxidant protection of sAc. Inoculation of Nrf 2-silenced PC12 cells into 96 well plates, pretreated with 10. Mu.M sAc for 18h, and then exposed to 510. Mu. M H 2 O 2 In (1). The MTT method detects the survival rate of the cells. Results are expressed as mean ± standard deviation, n =3. ### P<0.001, #### P<0.0001 compared with the DMSO group, ** P<0.01 vs. H 2 O 2 Group (iv);
figure 8 compound sAc protects PC12 cells from oxidative stress damage by activating the Nrf2 signaling pathway. (A) Compound sAc induced Nrf2 nuclear translocation in PC12 cells. PC12 cells were incubated with sAc (10. Mu.M) and TBHQ (5. Mu.M) for 6h, then Nrf2 antibody was incubated and stained with DAPI. Images of nuclear translocation were taken under a fluorescent microscope. (B) Compound sAc upregulates HO-1 protein expression in PC12 cells. Cells were incubated with sAc (1, 5 and 10 μ M) and TBHQ (5 μ M) for 18h and the levels of HO-1 protein were measured by western blot. The cytoprotective effect of (C, D) sAc is attenuated following downregulation of Nrf2 levels by siRNA. PC12 cells were transfected with Nrf2 siRNA (si-Nrf 2) and control siRNA (NC), respectively, pretreated with sAc (0.625 μ M) for 18H, and then re-treated with H 2 O 2 (650. Mu.M) for 24h. The MTT method is used for determining the cell activity, and the western blot method is used for detecting the protein level. Data are expressed as mean ± SD, n =3. #### p<0.0001， ## p＜0.01， # p＜0.05vs DMSO， * p<0.05vs H 2 O 2 ， & p＜0.05vs NC；
Figure 9sAc (or sAc) protection in MCAO rats. (A) representative images of groups of brain sections after TTC staining. (B-C) cerebral infarct size and neurological score for each group. Representative images of Sham (Sham) rats and brain sections of MCAO rats injected with saline (NS), solvent (DMSO: NS = 1) and sAc (0.15 mg/kg) in the ventricles. Results are expressed as mean ± standard deviation, n =6. #### P<0.0001 compared to the Sham group, ** P<0.01, **** P<0.0001 compared to the Vehicle group;
figure 10 compound sAc shows pre-protection of mouse brain tissue following MCAO. (A) Representative images of TTC stained brain sections of different experimental groups. (B) Infarct area (%) level and neurological score (C) for each group. Sham (Sham) mice or MCAOThe perfused mice received saline (NS), solvent (DMSO: castor oil: saline =1 = 80), sAc (10 or 20 mg/kg) and BNP (10 mg/kg) by intraperitoneal injection, respectively. Data are expressed as mean + -SD, with n ≧ 6. #### p<0.0001 Representing the statistical difference between NS and sham, **** p＜0.0001， *** p<0.001， * p<0.05 represents the statistical difference between the solvent group and the drug treatment group;
Detailed Description
To make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.
Synthesis of the Compound of example 1
L-proline is used as a catalyst to synthesize the mono-ketene mono-carbonyl curcumin analogue in one step. All compounds were obtained by reacting acetone with the corresponding aldehyde at room temperature, and after column chromatography purification, higher yields (40% -71%) were obtained as shown in table 1.
Figure RE-GDA0003152666800000061
Synthesis and structure of mono-ketone mono-carbonyl curcumin analog. Reaction regulation (I) L-proline, DMSO, room temperature.
TABLE 1 Structure and yield of Monoketene Monocarbonyl curcumin analogs
Figure RE-GDA0003152666800000071
The compound is structurally identified by mass spectrum and H1-NMR, and the spectral and physicochemical data are shown in the following
.1. (E) -4-phenylbut-3-en-2-one (sAc or sAc)
Yellow powder,70％yield,mp 34.8～36.0℃. 1 H-NMR(600MHz,CDCl 3 ),δ: 7.558-7.542(m,2H,Ar-H 2 ,β-H),7.520(d,J＝16.2Hz,1H,Ar-H 6 ),7.409-7.398(m, 3H,Ar-H 3 ,Ar-H 4 ,Ar-H 5 ),6.725(d,J＝16.2Hz,1H,α-H),2.390(s,3H,CH 3 ). LC-MS m/z:147.08(M+H) + ,calcd for C 10 H 10 O:146.07.
2.(E)-4-(2-methoxyphenyl)but-3-en-2-one(sAc2)
Yellow powder,66％yield,mp 43.9～45.7℃. 1 H-NMR(600MHz,DMSO-d 6 ), δ:7.770(d,J＝16.2Hz,1H,β-H),7.682(t,J＝6.6Hz,1H,Ar-H 6 ),7.425-7.396(m, 1H,Ar-H 4 ),7.086(d,J＝8.4Hz,1H,Ar-H 3 ),6.983(t,J＝7.2Hz,1H,Ar-H 5 ),6.821 (d,J＝16.8Hz,1H,α-H),3.859(s,3H,2-OCH 3 ),2.293(s,3H,CH 3 ).LC-MS m/z: 177.09(M+H) + ,calcd for C 11 H 12 O 2 :176.08.
3.(E)-4-(3-methoxyphenyl)but-3-en-2-one(sAc3)
Orange oil,68％yield. 1 H-NMR(600MHz,DMSO-d 6 ),δ:7.578(d,J＝16.2Hz,
1H,β-H),7.330(t,J＝8.4 Hz,1H,Ar-H 5 ),7.264(d,J＝7.2 Hz,2H,Ar-H 2 ,Ar-H 6 ), 6.983(t,J＝7.8 Hz,1H,Ar-H 4 ),6.814(d,J＝16.8 Hz,1H,α-H),3.780(s,3H, 3-OCH 3 ),2.317(s,3H,CH 3 ).LC-MS m/z:177.09(M+H) + ,calcd for C 11 H 12 O 2 : 176.08.
4.(E)-4-(4-methoxyphenyl)but-3-en-2-one(sAc4)
Yellow powder,71％yield,mp 67.4～68.2℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.505-7.463(m,3H,Ar-H 2 ,Ar-H 6 ,β-H),6.917(d,J＝8.4 Hz,2H,Ar-H 3 ,Ar-H 5 ), 6.608(t,J＝7.2 Hz,1H,α-H),3.838(d,J＝7.2 Hz,3H,4-OCH 3 ),2.354(d,J＝7.2 Hz,3H,CH 3 ).LC-MS m/z:177.09(M+H) + ,calcd for C 11 H 12 O 2 :176.08.
5.(E)-4-(2,3-dimethoxyphenyl)but-3-en-2-one(sAc5)
Yellow oil,65％yield. 1 H-NMR(600 MHz,DMSO-d 6 ),δ:7.724(d,J＝16.2 Hz, 1H,β-H),7.315(dd,J＝1.8 Hz,J＝4.8 Hz,1H,Ar-H 6 ),7.114(d,J＝2.4 Hz,2H, Ar-H 4 ,Ar-H 5 ),6.815(d,J＝16.8 Hz,1H,α-H),3.814(s,3H,2-OCH 3 ),3.762(s,3H, 3-OCH 3 ),2.317(s,3H,CH 3 ).LC-MS m/z:207.10(M+H) + ,calcd for C 12 H 14 O 3 : 206.09.
6.(E)-4-(2,4-dimethoxyphenyl)but-3-en-2-one(sAc6)
White powder,67％yield,mp 56.1～57.2℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.806(d,J＝16.2 Hz,1H,Ar-H 6 ),7.487(d,J＝9.0 Hz,1H,β-H),6.675(d,J＝16.2 Hz,1H,Ar-H 5 ),6.517(dd,J＝2.4 Hz,J＝6.6 Hz,1H,Ar-H 3 ),6.455(d,J＝2.4 Hz, 1H,α-H),3.877(s,3H,2-OCH 3 ),3.844(s,3H,4-OCH 3 ),2.360(s,3H,CH 3 ).LC-MS m/z:207.10(M+H) + ,calcd for C 12 H 14 O 3 :206.09.
7.(E)-4-(2,5-dimethoxyphenyl)but-3-en-2-one(sAc7)
Yellow powder,64％yield,mp 41.2～42.1℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.859(d,J＝16.2 Hz,1H,β-H),7.076(d,J＝3.0 Hz,1H,Ar-H 3 ),6.931(dd,J＝3.0 Hz,J＝6.0 Hz,1H,Ar-H 6 ),6.859(d,J＝9.0 Hz,1H,Ar-H 4 ),6.713(d,J＝16.2 Hz, 1H,α-H),3.855(s,3H,2-OCH 3 ),3.791(s,3H,5-OCH 3 ),2.391(s,3H,CH 3 ).LC-MS m/z:207.10(M+H) + ,calcd for C 12 H 14 O 3 :206.09.
8.(E)-4-(3,4-dimethoxyphenyl)but-3-en-2-one(sAc8)
Yellow powder,61％yield,mp 78.1～79.9℃. 1 H-NMR(600 MHz,DMSO-d 6 ), δ:7.541(d,J＝16.2 Hz,1H,β-H),7.305(d,J＝1.2 Hz,1H,Ar-H 2 ),7.236(dd,J＝ 1.2 Hz,J＝7.2 Hz,1H,Ar-H 5 ),6.986(d,J＝8.4 Hz,1H,Ar-H 6 ),6.720(d,J＝16.2 Hz,1H,α-H),3.785(d,J＝6.0 Hz,6H,3-OCH 3 ,4-OCH 3 ),2.287(s,3H,CH 3 ). LC-MS m/z:207.10(M+H) + ,calcd for C 12 H 14 O 3 :206.09.
9.(E)-4-(3,5-dimethoxyphenyl)but-3-en-2-one(sAc9)
White powder,67％yield,mp 68.5～69.4℃. 1 H-NMR(600 MHz,DMSO-d 6 ),δ: 7.529(d,J＝16.2 Hz,1H,β-H),6.873(d,J＝1.8 Hz,2H,Ar-H 2 ,Ar-H 6 ),6.820(d,J ＝16.8 Hz,1H,α-H),6.542(s,1H,Ar-H 4 ),3.761(s,6H,3-OCH 3 ,5-OCH 3 ),2.310(s, 3H,CH 3 ).LC-MS m/z:207.10(M+H) + ,calcd for C 12 H 14 O 3 :206.09.
10.(E)-4-(2,4,5-trimethoxyphenyl)but-3-en-2-one(sAc10)
White powder,59％yield,mp 94.5～95.0℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.732(d,J＝16.2 Hz,1H,β-H),7.225(s,1H,α-H),6.734(t,J＝16.2 Hz,2H,Ar-H 3 , Ar-H 5 ),3.839(d,J＝11.4 Hz,6H,2-OCH 3 ,5-OCH 3 ),3.733(s,3H,4-OCH 3 ),2.251 (s,3H,CH 3 ).LC-MS m/z:237.11(M+H) + ,calcd for C 13 H 16 O 4 :236.10.
11.(E)-4-(2,4,6-trimethoxyphenyl)but-3-en-2-one(sAc11)
Yellow powder,57％yield,mp 116.8～117.6℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.937(d,J＝16.8 Hz,1H,β-H),7.063(d,J＝16.8 Hz,1H,α-H),6.115(s,2H,Ar-H 3 , Ar-H 5 ),3.858(d,J＝15.6 Hz,9H,2-OCH 3 ,4-OCH 3 ,6-OCH 3 ),2.343(s,3H,CH 3 ). LC-MS m/z:237.11(M+H) + ,calcd for C 13 H 16 O 4 :236.10.
12.(E)-4-(3,4,5-trimethoxyphenyl)but-3-en-2-one(sAc12)
White powder,57％yield,mp 87.0～88.1℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.433(d,J＝16.2 Hz,1H,β-H),6.775(s,2H,Ar-H 2 ,Ar-H 6 ),6.631(d,J＝16.2 Hz, 1H,α-H),3.893(d,J＝4.2 Hz,9H,3-OCH 3 ,4-OCH 3 ,5-OCH 3 ),2.384(s,3H,CH 3 ). LC-MS m/z:237.11(M+H) + ,calcd for C 13 H 16 O 4 :236.10.
13.(E)-4-(3-hydroxyphenyl)but-3-en-2-one(sAc13)
White powder,50％yield,mp 91.3～92.1℃. 1 H-NMR(600 MHz,DMSO-d 6 ),δ: 9.654(s,1H,3-OH),7.508(d,J＝16.2 Hz,1H,β-H),7.214(t,J＝7.8 Hz,1H,Ar-H 5 ), 7.110(d,J＝7.8 Hz,1H,Ar-H 6 ),7.025(s,1H,Ar-H 4 ),6.819(dd,J＝1.2 Hz,J＝6.6 Hz,1H,Ar-H 2 ),6.665(d,J＝16.2 Hz,1H,α-H),2.304(s,3H,CH 3 ).LC-MS m/z: 163.07(M+H) + ,calcd for C 10 H 10 O 2 :162.07.
14.(E)-4-(4-hydroxyphenyl)but-3-en-2-one(sAc14)
Yellow powder,48％yield,mp 83.2～85.5℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.495(d,J＝16.2 Hz,1H,β-H),7.453(dt,J＝8.4 Hz,J＝9.6 Hz,2H,Ar-H 2 ,Ar-H 6 ), 6.889(dd,J＝1.8 Hz,J＝4.8 Hz,2H,Ar-H 3 ,Ar-H 5 ),6.609(d,J＝16.2 Hz,1H,α-H), 2.383(s,3H,CH 3 ).LC-MS m/z:163.07(M+H) + ,calcd for C 10 H 10 O 2 :162.07.
15.(E)-4-(3,4-dihydroxyphenyl)but-3-en-2-one(sAc15)
Brown powder,45％yield,mp 162.8～164.0℃. 1 H-NMR(600 MHz, DMSO-d 6 ),δ:9.587(s,1H,3-OH),9.159(s,1H,4-OH),7.432(d,J＝16.2 Hz,1H, β-H),7.042(d,J＝1.8 Hz,1H,Ar-H 2 ),6.990(dd,J＝1.8 Hz,J＝6.6 Hz,1H,Ar-H 5 ), 6.758(d,J＝8.4 Hz,1H,Ar-H 6 ),6.462(d,J＝16.2 Hz,1H,α-H),2.258(s,3H,CH 3 ). LC-MS m/z:179.07(M+H) + ,calcd for C 10 H 10 O 3 :178.06.
16.(E)-4-(3-hydroxy-4-methoxyphenyl)but-3-en-2-one(sAc16)
Beige powder,52％yield,mp 84.0～85.5℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.428(d,J＝16.2 Hz,1H,β-H),7.155(d,J＝2.4 Hz,1H,Ar-H 2 ),7.059(dd,J＝2.4 Hz,J＝6.0 Hz,1H,Ar-H 5 ),6.861(d,J＝7.8 Hz,1H,Ar-H 6 ),6.588(d,J＝16.2 Hz, 1H,α-H),3.936(s,3H,4-OCH 3 ),2.358(s,3H,CH 3 ).LC-MS m/z:193.08(M+H) + , calcd for C 11 H 12 O 3 :192.08.
17.(E)-4-(4-hydroxy-3-methoxyphenyl)but-3-en-2-one(sAc17)
Beige powder,56％yield,mp 115.9～117.8℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.451(d,J＝16.2 Hz,1H,β-H),7.093(dd,J＝1.8 Hz,J＝6.6 Hz,1H,Ar-H 2 ),7.060 (d,J＝1.8 Hz,1H,Ar-H 5 ),6.935(d,J＝8.4 Hz,1H,α-H),6.589(d,J＝16.2 Hz,1H, Ar-H 6 ),3.937(s,3H,3-OCH 3 ),2.368(s,3H,CH 3 ).LC-MS m/z:193.08(M+H) + , calcd for C 11 H 12 O 3 :192.08.
18.(E)-4-(4-hydroxy-3,5-dimethoxyphenyl)but-3-en-2-one(sAc18)
Beige powder,44％yield,mp 131.2～132.5℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.429(d,J＝16.2 Hz,1H,β-H),6.794(s,2H,Ar-H 2 ,Ar-H 6 ),6.597(d,J＝16.2 Hz, 1H,α-H),3.926(s,6H,3-OCH 3 ,5-OCH 3 ),2.369(s,3H,α-CH 3 ).LC-MS m/z:223.09 (M+H) + ,calcd for C 12 H 14 O 4 :222.09.
19.(E)-3-(3-oxobut-1-en-1-yl)benzaldehyde(sAc19)
White powder,40％yield,mp 47.9～48.8℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 10.052(s,1H,3-CHO),8.048(t,J＝1.8 Hz,1H,Ar-H 6 ),7.905(dt,J＝7.8 Hz,J＝ 7.2 Hz,1H,Ar-H 4 ),7.798(dd,J＝1.2 Hz,J＝6.6 Hz,1H,β-H),7.601-7.547(m,2H, Ar-H 2 ,Ar-H 5 ),6.809(d,J＝16.2 Hz,1H,α-H),2.406(s,3H,CH 3 ).LC-MS m/z: 175.07(M+H) + ,calcd for C 11 H 10 O 2 :174.07.
20.(E)-4-(3-fluorophenyl)but-3-en-2-one(sAc20)
Yellow oil,57％yield. 1 H-NMR(600 MHz,DMSO-d 6 ),δ:7.604(t,J＝4.2 Hz, 2H,β-H,Ar-H 6 ),7.537(d,J＝7.2 Hz,1H,Ar-H 5 ),7.481-7.444(m,1H,Ar-H 4 ), 7.266-7.235(m,1H,Ar-H 2 ),6.863(d,J＝16.2 Hz,1H,α-H),2.322(s,3H,CH 3 ). LC-MS m/z:165.07(M+H) + ,calcd for C 10 H 9 FO:164.06.
21.(E)-4-(4-fluorophenyl)but-3-en-2-one(sAc21)
Yellow oil,60％yield. 1 H-NMR(600 MHz,DMSO-d 6 ),δ:7.788-7.764(m,2H, Ar-H 3 ,Ar-H 5 ),7.613(d,J＝16.8 Hz,1H,β-H),7.261(t,J＝9.0 Hz,2H,Ar-H 2 , Ar-H 6 ),6.762(d,J＝16.2 Hz,1H,α-H),2.312(s,3H,CH 3 ).LC-MS m/z:165.07 (M+H) + ,calcd for C 10 H 9 FO:164.06.
22.(E)-4-(3,4-difluorophenyl)but-3-en-2-one(sAc22)
Yellow oil,51％yield. 1 H-NMR(600 MHz,DMSO-d 6 ),δ:7.893-7.860(m,1H, β-H),7.578(d,J＝16.8 Hz,2H,Ar-H 5 ,Ar-H 6 ),7.491(d,J＝10.2 Hz,1H,Ar-H 2 ), 6.829(d,J＝16.2 Hz,1H,α-H),2.310(s,3H,CH 3 ).LC-MS m/z:183.06(M+H) + , calcd for C 10 H 8 F 2 O:182.05.
23.(E)-4-(3,5-difluorophenyl)but-3-en-2-one(sAc23)
Yellow oil,55％yield. 1 H-NMR(600 MHz,DMSO-d 6 ),δ:7.572(d,J＝16.2 Hz, 1H,β-H),7.501(d,J＝6.6 Hz,2H,Ar-H 2 ,Ar-H 6 ),7.312-7.282(m,1H,α-H),6.928 (d,J＝16.2 Hz,1H,Ar-H 4 ),2.319(s,3H,CH 3 ).LC-MS m/z:183.06(M+H) + ,calcd for C 10 H 8 F 2 O:182.05.
24.(E)-4-(3-chlorophenyl)but-3-en-2-one(sAc24)
Yellow oil,56％yield. 1 H-NMR(600 MHz,DMSO-d 6 ),δ:7.812(s,1H,β-H), 7.671(d,J＝7.2 Hz,1H,Ar-H 6 ),7.587(d,J＝16.2 Hz,1H,Ar-H 5 ),7.477-7.430(m, 2H,Ar-H 2 ,Ar-H 4 ),6.880(d,J＝16.2 Hz,1H,α-H),2.319(s,3H,CH 3 ).LC-MS m/z: 181.04(M+H) + ,calcd for C 10 H 9 ClO:180.03.
25.(E)-4-(4-chlorophenyl)but-3-en-2-one(sAc25)
White powder,61％yield,mp 51.6～52.2℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.485-7.448(m,3H,Ar-H 2 ,Ar-H 6 ,β-H),7.387-7.365(m,2H,Ar-H 3 ,Ar-H 5 ),6.689(d, J＝16.2 Hz,1H,α-H),2.382(s,3H,CH 3 ).LC-MS m/z:181.04(M+H) + ,calcd for C 10 H 9 ClO:180.03.
26.(E)-4-(3,5-dichlorophenyl)but-3-en-2-one(sAc26)
White powder,57％yield,mp 72.0～72.5℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.407(d,J＝1.8 Hz,2H,β-H,Ar-H 4 ),7.367(t,J＝1.8 Hz,2H,Ar-H 2 ,Ar-H 6 ),6.700 (d,J＝16.2 Hz,1H,α-H),2.379(s,3H,CH 3 ).LC-MS m/z:215.99(M+H) + ,calcd for C 10 H 8 Cl 2 O:214.00.
27.(E)-4-(4-bromophenyl)but-3-en-2-one(sAc27)
White powder,62％yield,mp 74.9～76.4℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.532(d,J＝8.4 Hz,2H,Ar-H 3 ,Ar-H 5 ),7.442(d,J＝16.2 Hz,1H,β-H),7.405(d,J ＝8.4 Hz,2H,Ar-H 2 ,Ar-H 6 ),6.700(d,J＝16.2 Hz,1H,α-H),2.376(s,3H,CH 3 ). LC-MS m/z:224.99(M+H) + ,calcd for C 10 H 9 BrO:223.98.
28.(E)-4-(4-(dimethylamino)phenyl)but-3-en-2-one(sAc28)
Yellow powder,61％yield,mp 129.7～130.7℃. 1 H-NMR(600 MHz,CDCl 3 ),δ: 7.453(t,J＝16.8 Hz,3H,Ar-H 2 ,Ar-H 6 ,β-H),6.678(d,J＝8.4 Hz,2H,Ar-H 3 ,Ar-H 5 ), 6.546(d,J＝16.8 Hz,1H,α-H),3.031(s,6H,4-N(CH 3 ) 2 ),2.340(s,3H,CH 3 ). LC-MS m/z:190.12(M+H) + ,calcd for C 12 H 15 NO:189.12.
29.(E)-4-(4-(diethylamino)phenyl)but-3-en-2-one(sAc29)
Yellow oil,58％yield. 1 H-NMR(600 MHz,DMSO-d 6 ),δ:7.480-7.451(m,3H, Ar-H 2 ,Ar-H 6 ,β-H),6.658(d,J＝9.0 Hz,2H,Ar-H 3 ,Ar-H 5 ),6.485(d,J＝16.2 Hz, 1H,α-H),3.388-3.353(m,4H,N(CH 2 ) 2 ),2.235(s,3H,CH 3 ),1.086(t,J＝6.6 Hz,6H, C(CH 3 ) 2 ).LC-MS m/z:218.15(M+H) + ,calcd for C 14 H 19 NO:217.15.
30.(E)-4-(4-(piperidin-1-yl)phenyl)but-3-en-2-one(sAc30)
Yellow powder,54％yield,mp 98.4～99.4℃. 1 H-NMR(600 MHz,DMSO-d 6 ), δ:7.490(t,J＝8.4 Hz,3H,Ar-H 2 ,Ar-H 6 ,β-H),6.913(d,J＝9.0 Hz,2H,Ar-H 3 , Ar-H 5 ),6.545(d,J＝16.2 Hz,1H,α-H),3.031(s,4H,N(CH 2 ) 2 ),2.250(s,3H,CH 3 ), 1.557(s,6H,CH 2 CH 2 CH 2 ).LC-MS m/z:230.15(M+H) + ,calcd for C 15 H 19 NO: 229.15.
31.(E)-4-(4-morpholinophenyl)but-3-en-2-one(sAc31)
Yellow powder,51％yield,mp 131.7～132.8℃. 1 H-NMR(600MHz,CDCl 3 ),δ: 7.466-7.415(m,3H,Ar-H 2 ,Ar-H 6 ,β-H),6.884(t,J＝7.8Hz,2H,Ar-H 3 ,Ar-H 5 ), 6.603-6.524(m,1H,α-H),3.841(d,J＝30.0Hz,4H,O(CH 2 ) 2 ),3.257-3.206(m,4H, N(CH 2 ) 2 ),2.351-2.298(m,3H,CH 3 ).LC-MS m/z:232.13(M+H) + ,calcd for C 14 H 17 NO 2 :231.13.
32.(E)-4-(naphthalen-2-yl)but-3-en-2-one(sAc32)
White powder,55％yield,mp 97.0～98.3℃. 1 H-NMR(600MHz,CDCl 3 ),δ: 7.965(s,1H,Ar-H 3 ),7.878-7.836(m,3H,Ar-H 2 ,Ar-H 6 ,Ar-H 7 ),7.698-7.672(m,2H, Ar-H 8 ,β-H),7.539-7.519(m,2H,Ar-H 4 ,Ar-H 5 ),6.842(d,J＝16.2Hz,1H,α-H), 2.431(s,3H,CH 3 ).LC-MS m/z:197.09(M+H) + ,calcd for C 14 H 12 O:196.09.
Example 2
The monocarbonyl curcumin analogue molecular skeleton (sAc or sAc) has good stability, low toxicity and good activity for protecting cells.
In phosphate buffer, the stability of curcumin (Cur.), curcumin skeleton (Cur-S), diketene monocarbonyl curcumin analogue molecular skeleton (Ac) and the monoene monocarbonyl curcumin analogue molecular skeleton (sAc) of the invention were determined (fig. 1B). The results show that the light absorption of Cur, cur-S, ac decreases greatly in a time-dependent manner within 30 minutes, while sAc remains unchanged. Thus, the cue sAc has a more stable structure than Cur, cur-S, ac.
The cytotoxicity of the molecular skeleton (Ac) of the diketene monocarbonyl curcumin analogue and the molecular skeleton (sAc) of the monoalkenone monocarbonyl curcumin analogue of the invention on normal liver cells (MIHA) and kidney cells (NRK) is determined by an MTT method (figure 2). The results show that, at the same concentration, the cell survival rate of the sAc group is much greater than that of the Ac group in both cells, i.e., ac has a greater growth inhibition rate on the cells. Thus sAc is relatively less cytotoxic than Ac.
The adrenal pheochromocytoma cell line (PC 12) from rats, which exhibits the general characteristics of neuroendocrine cells, is widely used in neurophysiological and neuropharmacological studies. Further cytotoxicity tests on PC12 cells found that sAc was also relatively less cytotoxic to PC12 cells compared to curcumin and Ac (fig. 1C).
Hydrogen peroxide (H2O 2) is an important oxygen free Radical (ROS) and is commonly used in the study of oxidative stress injury models for various diseases. Therefore, the H2O 2-induced oxidative damage model was used to evaluate the antioxidant protective activity of compounds on cells. The cell viability of PC12 after H2O2 oxidative damage was about 55% of the solvent [ dimethyl sulfoxide (DMSO) ] group (fig. 2B). After the mono-ketene monocarbonyl curcumin analogue molecular skeleton (sAc) is added, the cell survival rate is obviously increased, namely sAc shows obvious antioxidant protection activity on PC12 cells, and curcumin and Ac groups have no activity (figure 1D).
Example 3
The monocarbonyl curcumin analogs (sAc) have good antioxidant protection activity on PC12 cells and low cytotoxicity
The cytotoxicity of 32 sAc compounds on PC12 cells was determined by MTT method, and the results showed that all compounds showed significant cytotoxicity to PC12 cells at the determined concentrations compared to curcumin control group (fig. 4A).
In an H2O2 induced PC12 cell oxidative damage model, the antioxidant protection activity of 32 sAc compounds is measured. The results showed that cell viability was increased to 70-80% in most groups pre-incubated with compound (fig. 4B). Of these, 20 compounds (sAc (1-5), sAc, sAc, sAc, sAc (15-24), sAc and sAc) had protective activity even better than that of the antioxidant TBHQ. While the cell survival rate of the curcumin group was lower than that of the H2O2 group.
Example 4
sAc1 and sAc protection against H2O 2-induced oxidative damage to PC12 cells
The protective effect of sAc1 (sAc and sAc of the present invention are the same compound) and sAc15 on H2O 2-induced oxidative damage to PC12 cells was further investigated. MTT experiments show that sAc (FIG. 5B) and sAc (FIG. 6B) can improve the survival rate of PC12 cell oxidative damage induced by hydrogen peroxide in a concentration-dose-dependent manner. In the PC12 cell colony formation experiment, H2O2 obviously inhibits the cell colony formation (figure 4A), but the effect is reversed by sAc (figure 5A) and sAc (figure 6A), and the effect is far better than that of positive control drug TBHQ, so that both compounds have antioxidant protection effect on the cell colony formation.
The free radical of oxygen, ROS, is the major cause of oxidative damage, and therefore the effect of compounds on the production of ROS by hydrogen peroxide-induced PC12 cells was determined. ROS levels were measured by dichlorodihydrofluorescein diacetate (DCFH-DA) assay. ROS levels were significantly increased in PC12 cells exposed to H2O2 compared to the DMSO group. Compounds sAc (fig. 5C) and sAc (fig. 6 DE) were both able to eliminate intracellular ROS in a dose-dependent manner after 18 hours of pretreatment.
An imbalance in the production and clearance of ROS results in oxidative stress, destruction of cellular components such as proteins, enzymes, lipids, etc., and the formation of the lipid peroxidation product Malondialdehyde (MDA). Therefore, the effect of the compound on MDA production of PC12 cells induced by hydrogen peroxide is determined. The results show that the MDA content in the H2O2 damaged group is significantly higher than the MDA content in the solvent group. After pre-incubation with compound sAc (fig. 6C) for 18 hours, MDA content decreased significantly.
Example 5
Active compounds reduce oxidative damage to PC12 cells by activating the Nrf2/HO-1 signaling pathway
The compounds sAc and sAc exhibit excellent antioxidant properties, and their mechanisms of action are further explored. Nrf2 is a "primary" regulator of cellular oxidative damage. Therefore, immunofluorescence assays were used to determine whether sAc could promote accumulation of Nrf2 in the nucleus. Blue and red staining represent nuclei and Nrf2, respectively. Compared with the DMSO group, the sAc treated group showed red fluorescence in nuclei and was stronger than the positive control group (TBHQ) (fig. 9A). These results indicate that sAc can promote accumulation of Nrf2 in the nucleus. During oxidative stress, nrf2 is activated to induce downstream expression of heme oxygenase-1 (HO-1) and other antioxidant enzymes. The effect of sAc (fig. 8A) and sAc (fig. 9B) on HO-1 protein expression in PC12 cells was evaluated. Increased expression of antioxidant enzyme HO-1 was observed in cells incubated with both compounds; furthermore, the expression of HO-1 protein was higher with increasing concentration of the compound. To further demonstrate the protective effect of compounds on H2O 2-induced oxidative stress damaged cells, nrf2 protein expression was downregulated by transfecting Nrf2 small interference (si) RNA into PC12 cells. The protective effect of the compounds on oxidative stress injury was evaluated against cells in the Nrf2 siRNA and control siRNA groups. Downregulation of Nrf2 expression inhibited the protective effect of sAc (fig. 7 BC) and sAc (fig. 8 CD). Therefore, sAc and sAc can both protect PC12 cells from oxidative damage by targeting Nrf2 to enhance HO-1 expression.
Example 6
Protection effect of compound on oxidative stress injury after cerebral ischemia reperfusion of MCAO rat
Cerebral ischemia-reperfusion can bring irreversible damage to nerve cells, wherein oxidative stress is a core mechanism of the damage, so the cerebral ischemia-reperfusion damage of mice can be used as a classical model for evaluating the in vivo antioxidant activity of the compound. Middle Cerebral Artery Occlusion (MCAO) is a common focal cerebral ischemia model, and because the pathogenesis of the MCAO is similar to that of human ischemic stroke, the MCAO is widely used for researching the pathogenesis of cerebral ischemia and screening therapeutic drugs. Thus, sAc1 and sAc were investigated for their antioxidant protective activity in vivo using the mouse MCAO model. The brain sections of the sham-operated group had no infarcted tissue, while the infarct incidence of the brain tissue of the model group was-30%. The cerebral infarction incidence rate of the solvent group and the model group has no statistical difference, which indicates that the solvent has no neuroprotective effect. Compared with the model group or the solvent group, the administration groups sAc (FIG. 9) and sAc (FIG. 10) had significantly reduced infarct size and significantly improved neurological scores. Wherein sAc has the same degree of activity as clinical medicine butylphthalide. Therefore, the active compounds sAc and sAc have good protective effect on oxidative stress injury of cerebral ischemia-reperfusion.
Example 7
Effect of active Compounds on survival of mice dying by LPS
Male B6 mice were given a 15mg/kg dose intraperitoneally (sAc 1 and sAc), 15 minutes later by LPS (20 mg/kg). Survival was recorded every 12h for 7 consecutive days. The LPS group mice all die within 5 days, and the lethality of the mice in the drug-added group is obviously reduced. Therefore, sAc1 and sAc can obviously increase the survival rate of LPS to mice lethality, and has the prospect of being developed into antioxidant drugs.
The above embodiments are described in detail for the purpose of further illustrating the present invention and should not be construed as limiting the scope of the present invention, and the skilled engineer can make insubstantial modifications and variations of the present invention based on the above disclosure.

Claims (1)

1. The application of the compound with the general formula (I) in preparing the medicine for treating ischemic stroke,
Figure DEST_PATH_IMAGE002
I
ar is an unsubstituted benzene ring.
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