Andrographolide derivative and medical application

Abstract

The invention discloses an andrographolide derivative and medical application thereof. Andrographolide is a bicyclic diterpene lactone compound extracted from a natural plant andrographis paniculata, has wide pharmacological activity, but has the limitation of insufficient activity in the aspect of anti-AD activity. According to the invention, andrographolide is subjected to structural modification to obtain the andrographolide derivative. Compared with andrographolide, the derivative has significantly improved treatment activity on Alzheimer's disease, and has a prospect of being developed into drugs for preventing and treating Alzheimer's disease.

Description

Technical Field

[0001] The invention relates to the field of natural drug chemistry, and in particular to an andrographolide derivative and its medical use. Background Art

[0002] Alzheimer's Disease (AD) is a serious neurodegenerative disease, which is mainly manifested by symptoms such as memory loss, cognitive dysfunction and abnormal behavior, which seriously affects the quality of life of patients and brings a heavy burden to their families and society. At present, although there are some drugs for the treatment of AD on the market, they have limited effects in delaying disease progression and improving the quality of life of patients, and are often accompanied by obvious side effects. Therefore, it is particularly important to develop new, efficient and safe anti-AD drugs.

[0003] The pathogenesis of AD is complex. The most recognized pathological feature is the neurotoxicity caused by excessive deposition of β-amyloid protein (Aβ) in the brain, which leads to the death and loss of cholinergic neurons in the central nervous system. Therefore, inhibiting Aβ deposition or reducing the damage of Aβ to neuronal cells is one of the main directions for the development of anti-AD drugs. Andrographolide is a bicyclic diterpene lactone compound extracted from the natural plant Andrographis paniculata, which has a wide range of pharmacological activities, including anti-inflammatory, anti-tumor, antiviral, and immunomodulatory. In recent years, with the continuous deepening of research on andrographolide, its application prospects in neurodegenerative diseases have also received increasing attention. However, andrographolide still has limitations in anti-AD activity, such as weak activity.

[0004] In order to solve the deficiencies in the prior art, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an andrographolide derivative and its medical use.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] An andrographolide derivative, the chemical structure of which is shown in formula (I):

[0008]

[0009] The andrographolide derivatives are used for preparing medicines for preventing and treating Alzheimer's disease.

[0010] Furthermore, the drug uses the andrographolide derivative as an active ingredient and is prepared into a pharmaceutically acceptable dosage form through a pharmaceutically acceptable carrier or excipient.

[0011] Furthermore, the dosage forms include tablets, capsules and injections.

[0012] Beneficial effects:

[0013] Andrographolide is a bicyclic diterpene lactone compound extracted from the natural plant Andrographis paniculata, which has a wide range of pharmacological activities, but has the limitation of insufficient activity in anti-AD activity. The present invention modifies the structure of andrographolide to obtain an andrographolide derivative. Compared with andrographolide, the derivative has significantly improved therapeutic activity against Alzheimer's disease and has the prospect of being developed into a drug for preventing and treating Alzheimer's disease. DETAILED DESCRIPTION

[0014] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0015] Example 1: Preparation of compound (I)

[0016] The synthetic route is as follows:

[0017]

[0018] Step S1: 3-methyl-[1,1'-biphenyl]-2-carbonitrile (0.5mmol), N-bromosuccinimide (NBS, 0.6mmol) and dibenzoyl peroxide (0.05mmol) were added to a dry round-bottom flask, and then 5mL of carbon tetrachloride was added. Under nitrogen protection, the mixture was heated to reflux for 12 hours. After the reaction solution was cooled to room temperature, carbon tetrachloride was removed by distillation under reduced pressure, and then 5mL of ethyl acetate was added. The organic phase was washed twice with a saturated sodium thiosulfate solution, and then washed three times with water. After drying, it was concentrated to obtain a crude product of 3-(bromomethyl)-[1,1'-biphenyl]-2-carbonitrile, which was directly carried out to the next step.

[0019] Step S2: Dissolve the 3-(bromomethyl)-[1,1'-biphenyl]-2-carbonitrile and andrographolide (0.5mmol) obtained in the previous step into 5mL of anhydrous dichloromethane, then add silver trifluoromethanesulfonate (AgOTf, 0.5mmol), and stir at room temperature for 24 hours under nitrogen protection. Then add 5mL of ethyl acetate, concentrate the ethyl acetate solution and load it on a normal phase silica gel column chromatography, isocratically elute with a mixed solvent of petroleum ether / ethyl acetate in a volume ratio of 1:1, detect by TLC, collect the eluate corresponding to the target compound, concentrate and dry, and obtain the compound of formula (Ⅰ). The spectral data are as follows: 1H NMR(500MHz,Chloroform-d)δ7.84(m,1H),7.67(m,1H),7.50(m,6H),6.96(m ,1H),6.19(s,1H),5.05(m,1H),4.93(s,1H),4.64(s,1H),4.46(m,1H),4.35 (d,J=11.4Hz,1H),4.26(m,1H),3.72(m,1H),3.66(d,J=11.4Hz,1H),2.60(m ,4H),2.38(m,1H),2.00(m,1H),1.90(m,4H),1.33–1.26(m,6H),0.88(s,3H). 13 C NMR (126MHz, CDCl3) δ169.94,148.74,146.40,146.03,142.64,138.24,132.73,130.44,128.91,128.71,128.66,128.03,125.43,116. 38,110.03,109.38,93.00,81.36,74.29,69.94,66.19,55.77,54.85,38.93,37.58,36.99,36.02,26.08,24.83,22.79,21.49,15.41.

[0020] Example 2: Compound (I) for Cu 2+ Induction of Aβ 25-35 The impact of aggregation

[0021] 1. Experimental Materials

[0022] Aβ 25-35 purchased from Beijing Bioson Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and thioflavin T (ThT) were purchased from Shanghai Yien Chemical Technology Co., Ltd. Compound (I) was prepared by Example 1. The experimental water was homemade ultrapure water.

[0023] 2. Experimental Methods

[0024] 1. Solution preparation

[0025] HEPES buffer: Weigh an appropriate amount of HEPES and dissolve it in ultrapure water to prepare a 10 mM buffer (pH 7.2).

[0026] Thioflavin T (ThT) solution: Weigh an appropriate amount of Thioflavin T and dissolve it in HEPES buffer to prepare an 80 μM solution.

[0027] Cu2+ Solution: Weigh an appropriate amount of CuCl2 and dissolve it in HEPES buffer to prepare a 200 μM solution.

[0028] Aβ 25-35 Solution: Weigh an appropriate amount of Aβ 25-35 Dissolved in HEPES buffer (containing 37.5% DMSO) to prepare a 500 μM solution.

[0029] Sample stock solution:

[0030] (1) Positive compound stock solution: Weigh an appropriate amount of curcumin and dissolve it in DMSO to prepare a 200 μM solution;

[0031] (2) Compound (I) stock solution: Weigh an appropriate amount of compound (I) and dissolve it in DMSO to prepare a 200 μM solution;

[0032] (3) Andrographolide stock solution: Weigh an appropriate amount of andrographolide and dissolve it in DMSO to prepare a 200 μM solution;

[0033] 2. Grouping, modeling and drug administration

[0034] Experimental group: 166 μL HEPES buffer, 10 μL Cu 2+ Solution, 4 μL Aβ 25-35 solution, 10 μL of the mother solution of the sample to be tested.

[0035] Blank group: 170 μL HEPES buffer, 10 μL Cu 2+ solution, 10 μL of the mother solution of the sample to be tested.

[0036] Control group: 166 μL HEPES buffer, 10 μL Cu 2+ Solution, 4 μL Aβ 25-35 solution, 10 μL DMSO solution.

[0037] Solvent group: 170 μL HEPES buffer, 10 μL Cu 2+ solution, 10 μL DMSO solution.

[0038] Add them to a black 96-well plate, with 3 replicates per group. Incubate at 37°C for 24 hours. After the incubation is complete, add 10 μL of ThT solution to each group and incubate at 37°C for 3 hours. Use a multifunctional microplate reader to measure the fluorescence value at an excitation wavelength of 440 nm and an emission wavelength of 480 nm. Calculate the Cu concentration of the sample to be tested according to the following formula 2+ Induction of Aβ 25-35 Percent inhibition of aggregation.

[0039] Inhibition percentage (%) = [1-(IF i -IF0) / (IFc -IF r )]×100%.

[0040] IF in the formula i represents the fluorescence value of the experimental group, IF0 represents the fluorescence value of the blank group, and IF c represents the fluorescence value of the control group, IF r Represents the fluorescence value of the solvent group.

[0041] 3. Statistical analysis

[0042] The data were expressed as mean ± SD, and the t test was used for comparison among the groups. P < 0.05 indicated a significant difference.

[0043] 3. Experimental Results

[0044] Each sample to be tested has Cu 2+ Induction of Aβ 25-35 The inhibition percentage of aggregation is shown in Table 1 (* indicates compared with the positive compound, **P < 0.01; & Compared with andrographolide, && P<0.01).

[0045] Table 1 The Cu of each sample to be tested 2+ Induction of Aβ 25-35 Percent inhibition of aggregation

[0046] Samples to be tested Inhibition percentage (%) Positive compounds 54.85±3.15 Compound (I) <![CDATA[71.35±2.33 **，&& ]]> Andrographolide 51.66±2.95

[0047] The above results show that compound (I) can effectively inhibit Cu 2+ Induction of Aβ 25-35 Aggregation, and the inhibitory effect was significantly stronger than that of positive compounds and andrographolide at the same concentration.

[0048] Example 3: Effect of Compound (I) on CL4176 Nematode Paralysis Model

[0049] 1. Experimental Materials

[0050] MgSO4 was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; agar powder, yeast powder, cholesterol, tryptone, NaCl, and CaCl2 were all purchased from Beijing Solebow Technology Co., Ltd.; Na2HPO4·12H2O and KH2PO4 were all purchased from Sinopharm Chemical Reagent Co., Ltd.; transgenic AD model Caenorhabditis elegans strain CL4176 [dvIs27 [myo-3p:: A-Beta (1-42):: let-8513'UTR) + rol-6 (su1006)]X] and uracil-deficient Escherichia coli OP50 (Escherichia Coli OP50) were purchased from the American Caenorhabditis elegans Genetics Center. Compound (I) was prepared by Example 1.

[0051] 2. Solution Preparation

[0052] 1. Solution preparation

[0053] M9 buffer: weigh 0.6 g disodium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g sodium chloride, and 0.025 g magnesium sulfate (dehydrated), dissolve in 100 mL deionized water, and sterilize at high temperature for later use.

[0054] NGM culture plate: weigh 3.0g sodium chloride, 2.5g tryptone, and 17g agar into a conical flask, add 975mL deionized water, and sterilize them by high pressure. When the temperature of the sterilization solution drops to about 55°C, add 0.5mL 1M calcium chloride solution, 1mL 5mg / mL cholesterol solution, 1mL 1M magnesium sulfate solution, and 25mL potassium phosphate buffer in sequence. Use a pipette to add the prepared culture medium liquid to the corresponding plate or well plate, and cool and solidify overnight.

[0055] Sample stock solution:

[0056] (1) Compound (I) stock solution: Weigh an appropriate amount of Compound (I) and dissolve it in DMSO to prepare a 10 mM solution;

[0057] (2) Andrographolide stock solution: Weigh an appropriate amount of andrographolide and dissolve it in DMSO to prepare a 10 mM solution;

[0058] 2. Grouping, modeling and drug administration

[0059] Take each group of mother liquor and dilute it 20 times with M9 buffer, then take 500 μL of each and evenly spread it on the freshly prepared NGM plate and dry it naturally at room temperature. Add 50 μL of OP50 bacterial solution to the center of the plate and dry it overnight. Inoculate the synchronized Caenorhabditis elegans into the drug-containing plate. At the same time, set up a group without drug as the model group.

[0060] The synchronized treated Caenorhabditis elegans were cultured at 16°C for 48 hours, ie, when the Caenorhabditis elegans grew to the L3 stage, the culture plate was transferred to 25°C to induce the transcriptional expression of the Aβ gene.

[0061] The culture plate was transferred to a 25°C culture condition, and the number of paralyzed C. elegans was recorded at 36 hours. The paralyzed C. elegans individuals showed partial rigidity of the trunk, resulting in movement disorders or even loss of movement function, and the head could be twisted.

[0062] 3. Detection indicators

[0063] In the paralysis rate test, the number of nematodes in each group was no less than 30. The paralysis rate of nematodes at 36 hours = the number of paralyzed nematodes / the total number of nematodes × 100%. Each group of experiments was repeated 3 times.

[0064] 4. Statistical analysis

[0065] The data were expressed as mean ± SD, and the t test was used for comparison among the groups. P < 0.05 indicated a significant difference.

[0066] 3. Experimental Results

[0067] The results of nematode paralysis rate detection in each group are shown in Table 2 (* indicates that compared with andrographolide, *P < 0.05; & Compared with the model group, & P<0.05).

[0068] Table 2 Paralysis rate of nematodes in each group

[0069] Samples to be tested Paralysis rate (%) Model Group 94.66±3.01 Compound (I) <![CDATA[69.53±4.01 *，& ]]> Andrographolide 89.39±2.18

[0070] The above results show that compound (I) can effectively inhibit the paralysis of AD model Caenorhabditis elegans, and the paralysis rate is significantly lower than that of the model group and the andrographolide group.

[0071] In summary, the andrographolide derivatives provided by the present invention have significantly stronger anti-Aβ aggregation activity than the positive compounds, and stronger than its precursor compound andrographolide. Further Caenorhabditis elegans AD model also showed that the andrographolide derivatives have significantly stronger anti-AD activity than andrographolide.

[0072] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. An andrographolide derivative, characterized in that: Its chemical structure is shown in formula (I):

2. The medical use of the andrographolide derivative according to claim 1 for preparing a drug for preventing and treating Alzheimer's disease.

3. The medical use according to claim 2, characterized in that: The andrographolide derivative is used as an active ingredient and is prepared into a pharmaceutically acceptable dosage form through a pharmaceutically acceptable carrier or auxiliary material.

4. The medical use according to claim 3, characterized in that: The dosage forms include tablets, capsules and injections.

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