Dihydrotriticale flavones, methods for their preparation and uses thereof

Abstract

The present application relates to the technical field of medicine, and in particular to a dihydrotricin, a preparation method and use thereof, the preparation method comprising the following steps: taking compound A, diisopropylethylamine and bromomethyl methyl ether to prepare compound B; taking compound C, diisopropylethylamine and bromomethyl methyl ether to prepare compound D; taking compound B and compound D, adding sodium hydroxide to prepare compound E; taking compound E, adding hydrochloric acid to prepare dihydrotricin after washing, separation, and concentration. The preparation method realizes efficient and large-scale synthesis of dihydrotricin. It is first confirmed that dihydrotricin can significantly resist A 1‑42 Oligomer-induced neurotoxicity has broad prospects in preventing and treating middle and late AD and delaying the progression of AD.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a dihydrotrienol flavonoid, its preparation method, and its uses. Background Technology

[0002] Alzheimer's disease (AD) is an extremely common degenerative disease of the central nervous system, seriously endangering the physical and mental health of the elderly. Currently, the main drugs used clinically to treat AD are acetylcholinesterase inhibitors, which aim to increase the level of the neurotransmitter acetylcholine in the synaptic cleft of hippocampal neurons, thereby improving the learning and memory abilities of AD patients. Since the decline in acetylcholine levels mainly occurs in the early stages of AD, acetylcholinesterase inhibitors have achieved good efficacy in treating early-stage AD patients. However, as the disease progresses, a large number of functional neurons in the hippocampus are lost and die, and acetylcholinesterase inhibitors cannot protect these dying hippocampal neurons. Therefore, in the later stages of AD, the efficacy of these drugs is extremely limited. Developing drugs with hippocampal neuroprotective effects is of paramount importance for the prevention and treatment of AD.

[0003] Age spots are the core pathological product of Alzheimer's disease (AD), formed by abnormal formation of β-amyloid (Aβ) fibromas. Aβ is a polypeptide containing 39-43 amino acids, synthesized from amyloid precursor protein (APP) by cleavage with β- and γ-secretases. 1-40 and Aβ 1-42 Isotype. Aβ 1-40 and Aβ 1-42 After formation, its monomers gradually aggregate into oligomers and fibrils, and Aβ 1-42 They are more prone to aggregation. Traditionally, Aβ fibers were considered the main component of senile plaques and a core pathogenic factor in the development and progression of Alzheimer's disease (AD). Modern research has confirmed that Aβ oligomers possess extremely strong neurotoxicity; even at very low concentrations, they can induce synaptic damage in hippocampal neurons, inhibit synaptic plasticity, and ultimately lead to hippocampal neuronal death. They are currently widely recognized as the "culprit" driving the progression of AD. Therefore, it is crucial to explore effective methods to combat Aβ. 1-42 Oligomeric compounds are of great value for the development of drugs for the prevention and treatment of Alzheimer's disease.

[0004] Flavonoids are a class of natural products with a variety of beneficial biological activities. Dihydroflavonoids, a class of structurally similar compounds, also possess rich pharmacological activities and are a hot topic in small molecule drug research. Dihydrotrienylenic acid is a typical dihydroflavonoid, but there are few reports on its synthesis, and the limited availability of its sources further restricts in-depth pharmacological research.

[0005] Therefore, whether a method for preparing dihydrotrienol flavonoids that can be efficiently synthesized on a large scale is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] This invention utilizes the structural characteristics of dihydrotrienolone and designs an efficient chemical synthesis route, achieving the efficient and large-scale synthesis of dihydrotrienolone. Furthermore, regarding pharmacological activity, the internationally recognized Aβ... 1-42 In a mouse model of Alzheimer's disease (AD) cells induced by oligomer-induced damage to hippocampal neurons (HT22), dihydrotristylarinase was demonstrated for the first time to significantly antagonize Aβ. 1-42 Oligomeric neurotoxicity suggests that dihydrotrienolone may have broad prospects in preventing and treating mid-to-late-stage Alzheimer's disease and delaying its progression. The specific technical solution is as follows:

[0007] A compound or a pharmaceutically acceptable salt thereof, wherein the compound or a pharmaceutically acceptable salt thereof has the structure shown in Formula I:

[0008]

[0009] A pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable excipients.

[0010] Furthermore, the dosage form of the pharmaceutical composition is any one of tablets, capsules, drop pills, micro-pellets, oral liquids, lyophilized preparations, water injections, infusions, and suppositories.

[0011] A method for preparing a compound of Formula I or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0012]

[0013] Step 1: Compound A is reacted with diisopropylethylamine and bromomethyl methyl ether to prepare compound B;

[0014] Step 2: Compound C is reacted with diisopropylethylamine and bromomethyl methyl ether to prepare compound D;

[0015] Step 3: Take compound B and compound D, add sodium hydroxide and react to prepare compound E;

[0016] Step 4: Take compound E, add hydrochloric acid and react, then wash, separate and concentrate to prepare compound I, dihydrotrigranular wheat flavonoid.

[0017] Further, in step 1, compound 1 is dissolved in dichloromethane, and diisopropylethylamine and bromomethyl methyl ether are added to the system under ice-water bath conditions. After the reaction is completed, compound B is prepared by separation, drying, and impurity removal.

[0018] Further, in step 2, compound C is dissolved in dichloromethane, and diisopropylethylamine and bromomethyl methyl ether are added to the system under ice-water bath conditions. After the reaction is completed, compound D is prepared by separation, drying, and impurity removal.

[0019] Further, in step 3, compound B prepared in step 1 and compound D prepared in step 2 are taken, dissolved in ethanol, and sodium hydroxide is added. After the reaction is completed, dilute hydrochloric acid is added to adjust the pH value to precipitate the product. After filtration and drying, compound E is obtained.

[0020] Further, in step 4, the compound E prepared in step 3 is added to methanol, then hydrochloric acid is added. After the reaction is complete, the methanol is removed, and then water and ethyl acetate are added for washing. After separation and impurity removal, compound I, dihydrotrigranular wheat flavonoid, is obtained.

[0021] Furthermore, the impurity removal step includes concentration followed by silica gel column chromatography.

[0022] Use of a compound of Formula I or a pharmaceutically acceptable salt thereof or the above-described pharmaceutical composition in the preparation of a medicament for the prevention or treatment of Alzheimer's disease.

[0023] One or more technical solutions provided in the embodiments of the present invention have at least the following technical effects or advantages:

[0024] 1. This invention is the first to design and complete a highly efficient new synthetic route for dihydrotrigerminol flavonoids, realizing the efficient and large-scale synthesis of dihydrotrigerminol flavonoids, and providing a rich source of compounds for subsequent pharmacological activity studies.

[0025] 2. Current clinical treatments for Alzheimer's disease (AD) primarily involve acetylcholinesterase inhibitors, which aim to increase acetylcholine levels in the synaptic cleft of hippocampal neurons, but cannot salvage damaged hippocampal neurons. This invention is the first to discover that dihydrotrienoloflavones can significantly counteract Aβ, a key pathogenic factor in AD. 1-42 Oligomers induce neurotoxicity in mouse hippocampal neurons, protecting damaged hippocampal neurons. Compared to acetylcholinesterase inhibitors, dihydrotrienolone may exert neuroprotective effects, delaying the progression of Alzheimer's disease, and its efficacy may be more significant in the middle and late stages of Alzheimer's disease. Attached Figure Description

[0026] Figure 1 Example 2 of this invention demonstrates that dihydrotrienol flavonoids significantly antagonize Aβ. 1-42 Bar chart showing cell survival induced by oligomers in mouse hippocampal neurons;

[0027] Figure 2 Example 2 of this invention demonstrates that dihydrotrienol flavonoids significantly antagonize Aβ. 1-42Electron micrographs of cellular morphological changes induced by oligomers in mouse hippocampal neurons. Detailed Implementation

[0028] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially or prepared according to known teachings.

[0029] The structures of the compounds described in the examples were determined using conventional spectroscopic techniques (infrared, ultraviolet, nuclear magnetic resonance, or ESIMS mass spectrometry).

[0030] Example 1: A compound or a pharmaceutically acceptable salt thereof, wherein the compound or a pharmaceutically acceptable salt thereof has the structure shown in Formula I:

[0031]

[0032] A pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable excipients. The excipients may be one or more of fillers, binders, disintegrants, lubricants, flow aids, wetting agents, effervescent agents, colorants, sweeteners, flavorings, preservatives, dispersants, film-forming agents, plasticizers, pore-forming agents, light-blocking agents, and retardants. The dosage form of the pharmaceutical composition is any one of tablets, capsules, pellets, microgranules, oral liquids, lyophilized preparations, injections, infusions, and suppositories.

[0033] A method for preparing dihydrotrigeratin, a compound of formula I, includes the following steps:

[0034]

[0035] Step 1: Compound A (5 g, 30 mmol) was dissolved in 60 mL of dichloromethane. Under ice-water bath conditions, diisopropylethylamine (DIPEA, 90 mmol) and bromomethyl methyl ether (MOMBr, 66 mmol) were added to the system. The reaction was allowed to proceed naturally to room temperature for 12 hours. The reaction was quenched with 60 mL of water and the mixture was transferred to a separatory funnel for separation. The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain product B 7.38 g, yield 96%.

[0036] R f =0.8(petroleum ether / EtOAc,2:1); 1 H NMR (400MHz, Chloroform-d)δ

[0037] 13.72(s,1H),6.27(d,J=2.5Hz,1H),6.24(d,J=2.3Hz,1H),5.26(s,2H),5.17(s,2H),3.52(s,3H),3.47(s,3H),2.66(s,3H); 13 C NMR(101MHz,Chloroform-d)δ203.10,167.30,163.30,160.50,107.30,97.30,94.61,94.50,56.35,56.05,32.96; HRMS(ESI):m / zcalcd for C 12 H 16 NaO6 + [M+Na] + :279.0839,found:279.0841.

[0038] Step 2: Compound C (4.2 g, 27.1 mmol) was dissolved in 50 mL of dichloromethane. Under ice-water bath conditions, diisopropylethylamine (DIPEA, 108 mmol) and bromomethyl methyl ether (MOMBr, 103 mmol) were added, and the reaction was allowed to proceed naturally to room temperature for 12 hours. The reaction was quenched with 50 mL of water, and the mixture was transferred to a separatory funnel for separation. The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain product D 8.5 g, yield 99%.

[0039] R f =0.6(petroleum ether / EtOAc,3:1); 1 H NMR (400MHz, Chloroform-d)

[0040] δ9.87(s,1H,),7.40(s,2H),5.29(s,6H),3.66(s,3H),3.54(s,6H).; 13 C NMR (100MHz, CDCl3) δ191.9,151.2,140.3,132.2,106.2,98.0,94.9,55.9; HRMS (ESI): m / zcalcd for C 13 H 18 NaO7 + [M+Na] + :309.0945,found:309.0949.

[0041] Step 3: Compounds B (2 g, 7.8 mmol) and D (2.2 g, 7.8 mmol) were dissolved in 50 mL of ethanol, and sodium hydroxide (39 mmol, dissolved in 10 mL of water) was added. The reaction was continued at room temperature. After 12 hours, the reaction was stopped. Dilute hydrochloric acid was added to adjust the pH to 5, and the product precipitated. The product was filtered, placed in a 65°C oven for 6 hours, and dried to obtain product E 3.8 g, yield 93%.

[0042] R f =0.4(petroleum ether / EtOAc,2:1); 1 H NMR (400MHz, Chloroform-d)

[0043] δ7.86(d,1H,J=15.5Hz),7.69(d,2H,J=15.5Hz),7.18(s,2H),6.31(d,1H,J=2.5Hz),6.28(d,1H,J=2.5Hz) ,5.30(s,2H),5.22(s,4H),5.20(s,2H),5.19(s,2H),3.62(s,3H),3.54(s,3H),3.51(s,6H),3.48(s,3H); 13 C NMR (100MHz, CDCl3) δ193.02,165.60,161.30,159.46,151.00,145.30,139.61,132.47,126.58, 112.62,108.97,98.00,97.90,95.23,94.90,94.55,94.50,57.15,56.35,56.05; HRMS(ESI):m / z calcd for C 25 H 32 NaO 12 + [M+Na] + :547.1786,found:547.1789.

[0044] Step 4: Compound E (3.8 g, 7.2 mmol) was added to 50 mL of methanol, followed by 10 mL of 4N hydrochloric acid, and the mixture was heated to reflux. The reaction was allowed to proceed for 8 hours, then heating was stopped, and the methanol was directly concentrated to remove it. The mixture was then washed with 50 mL each of water and ethyl acetate, and separated. The organic phase was concentrated and purified by silica gel column chromatography to obtain 1.8 g of a white solid product, dihydrotristalin, in 82% yield.

[0045] R f =0.7(EtOAc / MeOH, 10:1); 1H NMR(400MHz,DMSO-d6)δ12.14(s,

[0046] 1H),10.79(s,1H),8.96(s,2H),8.26(s,1H),6.38(s,2H),5.87(q,J=2.3Hz,2H),5.31 (dd,J=12.2,3.2Hz,1H),3.11(dd,J=17.1,12.2Hz,1H),2.66(dd,J=17.2,3.2Hz,1H); 13 C NMR(100MHz,DMSO-d6)δ194.16,166.94,164.36,163.45,146.60,133.83,133.31,104.20,102.26,96.65,95.62,79.27,43.35.HRMS(ESI):m / z calcd for C 15 H 12 NaO7 + [M+Na] + :327.0475,found:327.0479.

[0047] Example 2:

[0048] Use of a compound of Formula I or a pharmaceutically acceptable salt thereof or the above-described pharmaceutical composition in the preparation of a medicament for the prevention or treatment of Alzheimer's disease.

[0049] The dihydrotrigeratin prepared in Example 1 was used for verification experiments:

[0050] Experimental materials

[0051] HT22 hippocampal neurons, DMEM culture medium, fetal bovine serum, Aβ 1-42 Hexafluoroisopropanol, thiazolyl blue (MTT), fluorescein diacetate (FDA), propidium iodide (PI).

[0052] ② Experimental methods

[0053] 1)Aβ 1-42 Oligomer preparation

[0054] Take 5mg Aβ 1-42 [Jier Biochemical (Shanghai) Co., Ltd.] Dissolve in 1.1 ml hexafluoroisopropanol, incubate at room temperature for 60 minutes, aliquot into 1.5 ml centrifuge tubes (100 μl / tube), and store at -80℃. Take one tube, add 900 μl of sterile H2O, mix well, and blow dry the hexafluoroisopropanol. Centrifuge at 14,000 g for 15 minutes at 4℃, transfer the supernatant to a new centrifuge tube, incubate on a mixer for 48 hours, and store at 4℃. At this point, Aβ...1-42 100 μMAβ 1-42 Oligomer.

[0055] 2) Construction of AD cell model and determination of cell viability

[0056] HT22 hippocampal neurons were cultured in DMEM medium containing 10% fetal bovine serum at a concentration of 0.8 × 10⁻⁶ mg / L. 5 Cells were seeded at a density of 100 μl / mL in 96-well cell culture plates. After adhesion, control and model groups (Aβ) were set up. 1-42 Oligomers and treatment groups were used. The treatment groups were given final concentrations of dihydrotriterpenoids (0.1 μM, 1 μM, 10 μM, 30 μM, and 100 μM) and pre-incubated for 2 hours. The control and model groups were given the same volume of sterile H2O. Subsequently, both the model and treatment groups were given Aβ at a final concentration of 5 μM. 1-42 For oligomers, the control group was given the same volume of sterile H2O. After 24 hours, 10 μL of MTT solution with a final concentration of 0.5 mg / mL was added to each well, and incubation continued for another 4 hours. Then, the culture medium was aspirated, and 150 μL of dimethyl sulfoxide was added to each well. Both were measured at 570 nm using a ELISA reader (SpectraMax i3x, Molecular Devices) to calculate cell viability.

[0057] 3) FDA / PI double staining to detect the number of live / dead neurons

[0058] HT22 hippocampal neurons were seeded in 35mm glass-bottom confocal culture dishes and treated according to the drug administration method for cell viability assays. After drug treatment, 10 μl / ml FDA and 5 μl / ml PI were added to each well, and the cells were observed and photographed under a confocal microscope.

[0059] ③ Experimental Results

[0060] 1) such as Figure 1 As shown, Aβ 1-42 Four hours after the oligomers were applied to mouse hippocampal neurons HT222, the cell viability decreased significantly from (100.00±0.48) in the control group to (58.67±1.78). Pre-incubation of HT22 cells with 10, 30, and 100 μM dihydrotrisaccharide for 2 hours significantly antagonized Aβ. 1-42 Oligomeric neurotoxicity was observed, with 30 μM dihydrotriamcinolone exhibiting the greatest efficacy, significantly increasing cell viability to (86.45 ± 2.15%).

[0061] 2) such as Figure 2 As shown, Aβ 1-42Four hours after oligomers were applied to mouse hippocampal neurons HT22, the number of HT22 neurons decreased, cell bodies shrank, and connections between neurons were disrupted. The number of FDA-labeled live cells was significantly reduced, while the number of PI-labeled dead cells was significantly increased. Pre-incubation with dihydrotrienol significantly inhibited Aβ. 1-42 Oligomeric neurotoxicity induced in HT22 neurons.

[0062] In summary, this invention provides a compound of formula I, dihydrotrienolone, which is structurally similar to known trienolone but has an unexpected use: its use in the preparation of medicaments for the prevention or treatment of Alzheimer's disease. This use differs from known uses of trienolone, such as antioxidant, anti-inflammatory, and anticancer effects. Furthermore, this use is not explicitly stated in common knowledge or cannot be deduced from common sense. Based on the above experiments, this invention is the first to discover that dihydrotrienolone can significantly counteract the key pathogenic factor Aβ in Alzheimer's disease. 1-42 Oligomers induce neurotoxicity in mouse hippocampal neurons, protecting damaged hippocampal neurons. Compared to acetylcholinesterase inhibitors, dihydrotrienolone may exert neuroprotective effects, delaying the progression of Alzheimer's disease, and its efficacy may be more significant in the middle and late stages of Alzheimer's disease.

[0063] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0064] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing dihydrotrigermina flavonoids, characterized in that: Includes the following steps: ； Step 1: Dissolve 30 mmol of compound A in 60 mL of dichloromethane. Under ice-water bath conditions, add 90 mmol of diisopropylethylamine and 66 mmol of bromomethyl methyl ether to the system. React for 12 hours, allowing the mixture to return to room temperature naturally. Quench the reaction with 60 mL of water and transfer the mixture to a separatory funnel for separation. Dry the organic phase with anhydrous sodium sulfate, concentrate it, and then perform silica gel column chromatography to obtain compound B. Step 2: Dissolve 27.1 mmol of compound C in 50 mL of dichloromethane. Under ice-water bath conditions, add 108 mmol of diisopropylethylamine and 103 mmol of bromomethyl methyl ether. React for 12 hours, allowing the mixture to return to room temperature naturally. Quench the reaction with 50 mL of water and transfer the mixture to a separatory funnel for separation. Dry the organic phase with anhydrous sodium sulfate, concentrate it, and then perform silica gel column chromatography to obtain compound D. Step 3: Take 7.8 mmol of compound B and 7.8 mmol of compound D, add 50 mL of ethanol to dissolve them, add 39 mmol of sodium hydroxide, and continue to react at room temperature. After 12 hours, stop the reaction, add dilute hydrochloric acid to adjust the pH value to 5, the product precipitates, filter, place the product in a 65℃ oven for 6 hours, and dry to obtain compound E; Step 4: Take 7.2 mmol of compound E and add 50 mL of methanol, then add 10 mL of 4N hydrochloric acid, heat to reflux, react for 8 hours, stop heating, directly concentrate to remove methanol, then add 50 mL each of water and ethyl acetate to wash, separate the liquid, concentrate the organic phase, and obtain dihydrotristal wheat flavonoids by silica gel column chromatography.

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