Beta-carboline alkaloid PFT217 in gastrodia elata as well as preparation method and application thereof

Abstract

The invention provides alkaloid 1-(4-hydroxyphenyl)-beta-carboline (PFT217) in Gastrodia elata Blume, a preparation method of the alkaloid 1-(4-hydroxyphenyl)-beta-carboline (PFT217) and application of the alkaloid 1-(4-hydroxyphenyl)-beta-carboline (PFT217) in the field of medicine. Belongs to the technical field of medicine. The PFT217 has remarkable inhibitory activity on acetylcholin esterase and butyrylcholin esterase, and the IC50 (half maximal inhibitory concentration) values of the PFT217 are 24.70 mu M and 0.84 mu M respectively. The alkaloid can be used for preparing medicines for resisting Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the field of botanical medicine technology, and more specifically, to a gastrodia elata β-carboline alkaloid PFT217, a pharmaceutical composition containing the same, a preparation method thereof, and an application thereof in preparing medicines. Background Art

[0002] Acetylcholine (ACh) is the first neurotransmitter discovered. It is a neurotransmitter used by all cholinergic neurons and plays a very important role in the peripheral and central nervous systems. Almost every area in the brain is controlled by cholinergic neurons. Alzheimer's disease (AD) is a progressive central nervous system degenerative disease. The main clinical manifestations of patients with AD are the gradual loss of memory and the gradual emergence of cognitive dysfunction. The poor cognitive ability of AD is related to the decrease in central acetylcholinergic activity, and the increase in ACh content in synapses can improve people's cognitive ability and memory. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are specific enzymes that hydrolyze acetylcholine. In the late stage of AD patients' brains, acetylcholinesterase will decrease, while the concentration of butyrylcholinesterase will increase compensatorily, playing a leading role in the hydrolysis of acetylcholine. Therefore, selective inhibition of butyrylcholinesterase is expected to become a new option for the treatment of advanced AD [Dong Chang'e, Li Rui, Xue Xuanyi, Zhang Zhaoyuan, Shi Dahua. Design and synthesis of quinoline-triazine derivatives and in vitro butyrylcholinesterase inhibitory activity. Journal of Hebei University (Natural Science Edition), 2024, 44(4), 382-389].

[0003] The traditional Chinese medicine Gastrodia elata is the dried tuber of the orchid plant Gastrodia elata Blume. It is a precious Chinese medicinal material in my country. It has the effects of calming wind and relieving spasms, calming liver yang, dispelling wind and unblocking meridians, etc. It is used to treat infantile convulsions, epilepsy, tetanus, headache and dizziness, limb paralysis, limb numbness, rheumatism and pain, etc. [China Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China (Part 1). Beijing: China Medical Science and Technology Press. 2020, 59-60]. However, there are no relevant reports on the Gastrodia elata alkaloid PFT217 and its inhibitory activity on cholinesterase. Summary of the invention

[0004] The object of the present invention is to provide a Gastrodia elata alkaloid PFT217, a pharmaceutical composition containing the same, a preparation method thereof, and their use in the preparation of acetylcholinesterase inhibitors and butyrylcholinesterase inhibitors, as well as their use in the preparation of anti-Alzheimer's disease drugs in view of the above-mentioned deficiencies in the prior art.

[0005] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0006] The compound PFT217 shown in the following structural formula,

[0007]

[0008] The preparation method of the compound PFT217 shown in the structural formula comprises the following steps:

[0009] The dried tubers, peduncles and inflorescences of Gastrodia elata after collecting seeds were crushed together, extracted three times with 90% ethanol aqueous solution at 70°C, each time for 3 hours, the extracts were combined, filtered, and the filtrate was concentrated under reduced pressure to obtain an extract; the crude extract was added with distilled water to prepare a suspension, HCl was added to adjust the pH to 2-3, extracted 5 times with an equal volume of petroleum ether, and the organic phase was concentrated under reduced pressure at 60°C using a rotary evaporator to obtain a petroleum ether extraction part, and the remaining aqueous phase was extracted 5 times with an equal volume of ethyl acetate, and the organic phase was extracted 5 times with an equal volume of ethyl acetate, and the organic phase was concentrated using a rotary evaporator. After concentrated under reduced pressure at 60°C, an ethyl acetate extraction portion was obtained. After the ethyl acetate extraction, the remaining aqueous phase was adjusted to pH 9-10 with an aqueous NaOH solution, extracted 5 times with an equal volume of dichloromethane, and the organic phase was concentrated under reduced pressure at 60°C using a rotary evaporator to obtain a dichloromethane extraction portion; the dichloromethane extraction portion was subjected to 100-200 mesh silica gel column chromatography, and v / v acetone-petroleum ether 20:1→1:1 gradient elution was performed to obtain 10 components, namely, components A, B, C, D, E, F, G, H, I and J;

[0010] Component G passes through RP C 18 Silica gel column chromatography, v / v methanol-water 10%→100%, to obtain 50% methanol elution part G5; component G5 was chromatographed on a 200-300 mesh silica gel column, v / v petroleum ether-ethyl acetate 10:1→0:1 gradient elution to obtain G5-1; G5-1 was separated by Sephadex LH-20 column chromatography using methanol as the eluent to obtain G5-1-1; G5-1-1 was separated by HPLC to obtain compound PFT217.

[0011] The compound PFT217 represented by the structural formula is used in the preparation of acetylcholinesterase inhibitors and butyrylcholinesterase inhibitors.

[0012] The use of the compound PFT217 represented by the structural formula in the preparation of anti-Alzheimer's disease drugs.

[0013] A pharmaceutical composition comprising the compound PFT217 represented by the structural formula and a pharmaceutically acceptable carrier.

[0014] The pharmaceutical composition is used in the preparation of acetylcholinesterase inhibitors and butyrylcholinesterase inhibitors.

[0015] The application of the pharmaceutical composition in preparing drugs for treating Alzheimer's disease.

[0016] The method for preparing the pharmaceutical composition comprises first obtaining the compound PFT217 by the above-mentioned preparation method, and then adding a pharmaceutically acceptable carrier.

[0017] When the compound PFT217 is used to prepare a drug, the present invention preferably uses the compound PFT217 directly or in the form of a pharmaceutical composition.

[0018] The pharmaceutical composition provided by the present invention comprises the above-mentioned compound PFT217 and a pharmaceutically acceptable carrier. In the present invention, the pharmaceutically acceptable carrier is preferably a solid, semisolid or liquid diluent, a filler and a pharmaceutical product adjuvant. The present invention has no particular limitation on the pharmaceutically acceptable carrier, and any pharmaceutically acceptable carrier well known in the art, non-toxic and inert to humans and animals can be selected.

[0019] The present invention has no particular limitation on the method for preparing the pharmaceutical composition. The compound PFT217 can be directly mixed with a pharmaceutically acceptable carrier. The present invention has no particular limitation on the mixing process. The pharmaceutical composition can be obtained by selecting a process well known in the art.

[0020] The present invention provides the use of the pharmaceutical composition described in the above technical solution in the preparation of medicines. The present invention has no special limitation on the method of the application, and any method well known in the art can be selected.

[0021] In the present invention, when the pharmaceutical composition is used to prepare a drug, the content of the composition in the drug is preferably 0.1-99%; in the pharmaceutical composition, the content of the compound PFT217 in the pharmaceutical composition is preferably 0.5-90%. The pharmaceutical composition of the present invention is preferably used in the form of a dosage per unit body weight. In the present invention, the prepared drug can preferably be administered in two forms: injection (intravenous injection, intramuscular injection) and oral administration.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. PFT217 has significant inhibitory activity against butyrylcholinesterase, IC 50 The value is 0.84 μM.

[0024] 2. The inhibitory activity of PFT217 on butyrylcholinesterase is better than that on acetylcholinesterase, and the selectivity index (acetylcholinesterase IC50 / Butyrylcholinesterase IC 50 , SI) was 29.4, which was higher than tacrine (SI=5.3).

[0025] 3.PFT217 has a simple chemical structure and is easy to synthesize. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the chemical structure of compound PFT217.

[0027] Figure 2 Key COSY (bold) and HMBC (arrow) correlations for compound PFT217. DETAILED DESCRIPTION

[0028] The substantial content of the present invention is further described below with reference to the accompanying drawings using embodiments of the present invention, but the present invention is not limited thereto.

[0029] Example 1

[0030] Preparation of β-carboline alkaloid PFT217 from Gastrodia elata:

[0031] The dried tubers, peduncles and inflorescences (21.4 kg) of Gastrodia elata after collecting seeds were crushed together, extracted three times (3 hours each time) with 80 L of 90% ethanol aqueous solution at 70°C, the extracts were combined, filtered, and the filtrate was concentrated under reduced pressure to obtain 6.6 kg of extract; the crude extract was added with distilled water (6.8 L) to prepare a suspension, 0.1% HCl (8 L) was added to adjust the pH to 2-3, extracted with an equal volume of petroleum ether for 5 times, and the organic phase was concentrated under reduced pressure at 60°C using a rotary evaporator to obtain a petroleum ether extraction part (142.2 g). The remaining aqueous phase was extracted with an equal volume of ethyl acetate for 5 times, and the organic phase was concentrated under reduced pressure at 60°C using a rotary evaporator to obtain an ethyl acetate extraction part (104.4 g). After ethyl acetate extraction, the remaining aqueous phase was adjusted to pH 9-10 with 5% NaOH aqueous solution (4 L), extracted 5 times with an equal volume of dichloromethane, and the organic phase was concentrated under reduced pressure at 60° C. using a rotary evaporator to obtain a dichloromethane extraction portion (25.6 g). The dichloromethane extraction portion was subjected to 100-200 mesh silica gel column chromatography (acetone-petroleum ether, 20:1→1:1, v / v) gradient elution to obtain 10 components including component A (449.2 mg), B (103.0 mg), C (336.1 mg), D (57.5 mg), E (962.8 mg), F (422.7 mg), G (3.9 g), H (1.6 g), I (1.4 g) and J (10.2 g).

[0032] Component G passes through RP C 18Silica gel column chromatography (methanol-water, 10%→100%, v / v) obtained a major component, namely, 50% methanol elution portion G5 (130.0 mg); component G5 was subjected to 200-300 mesh silica gel column chromatography (petroleum ether-ethyl acetate, 10:1→0:1, v / v) gradient elution to obtain G5-1 (30.0 mg); G5-1 was separated by Sephadex LH-20 column chromatography using methanol as the eluent to obtain G5-1-1 (14.0 mg); G5-1-1 was separated by HPLC (Welch Ulimate AQ-C 18 Column; φ7.8×250mm; 5μm MeOH-H2O, 60:40, 2mL / min) to separate compound PFT217 (9.0mg, t R =18.1min).

[0033] Embodiment 2:

[0034] Alkaloid PFT217( Figure 1 ) Spectral data and structural identification:

[0035] PFT217, Chinese name 1-(4-Hydroxyphenyl)-β-carboline, English name 1-(4-Hydroxyphenyl)-β-carboline, yellow powder, molecular formula C 17 H 12 N2O; 1 H NMR (500 MHz, DMSO-d6)δ H 12.53(1H,s,H-9),10.51(1H,s,4′-OH),8.66(1H,d,J=6.1Hz,H-4),8.52(1H,br d,J=8.1Hz,H-5),8.51(1H,d,J=6.1Hz,H-3),7.90(2H,br d,J=8.8Hz,H-2′,6′),7.78(1H,m,H-8),7.76(1H,m,H-7),7.45(1H,m,H-6),7.15(2H,br d,J=8.8Hz,H-3′,5′); 13 C NMR (126 MHz, DMSO-d6)δ C160.6(C,C-4′),143.8(C,C-8a),138.5(C,C-1),133.0(C,C-4a),132.2(C,C-9a),131.4(CH,C-2′,6′),131.2(CH,C-7),130.1(C H,C-3),123.3(CH,C-5),121.4(CH,C-6),120.5(C,C-1′),120.0(C,C-4b),116.4(CH,C-3′,5′),115.3(CH,C-4),113.2(CH,C-8); 1 H NMR (500MHz, methanol-d4)δ H 8.56(1H,d,J=6.2Hz,H-4),8.42(1H,br d,J=8.1Hz,H-5),8.38(1H,d,J=6.2Hz,H-3),7.88(1H,br d,J=8.6Hz,H-2′,6′),7.78(1H,m,H-7),7.78(1H,m,H-8),7.47(1H,m,H-6),7.17(1H,br d,J=8.6Hz,H-3′,5′); 13 C NMR (126MHz,methanol-d4)δ C 162.6(C,C-4′),145.8(C,C-8a),140.4(C,C-1),135.5(C,C-4a),134.0(C,C-9a),132.9(CH,C-7),132.0(CH,C-2′,6′),130.4(CH, ESIMS m / z261[M+H] + ; HRESIMS m / z 261.1027[M+H] + (C 17 H 13 N2O, calcd 261.1028). The nuclear magnetic resonance (NMR) data of this compound were obtained by two-dimensional NMR correlation ( Figure 2 ) for identification.

[0036] Example 3

[0037] Acetylcholinesterase inhibitory activity assay:

[0038] 1. Experimental Principle

[0039] Acetylcholinesterase can catalyze the degradation of its substrate analog iodinated thioacetylcholine to generate thiocholine and acetic acid. The reaction product reacts with the color developer 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) to generate a yellow substance with specific light absorption at 405nm. The compound and acetylcholinesterase mixture react at 37℃. If the compound has an inhibitory effect on acetylcholinesterase, the amount of iodinated thioacetylcholine degradation catalyzed by acetylcholinesterase will decrease, and the corresponding yellow compound generated by the reaction with DTNB will decrease, that is, the light absorption value at 405nm will decrease, so as to screen compounds with inhibitory activity.

[0040] 2. Reagents and Instruments

[0041] Reagents: Na2HPO4, NaH2PO4, acetylcholinesterase, thioacetylcholine iodide, DTNB and tacrine were purchased from Sigma.

[0042] Instrument: Thermo Multiskan FC.

[0043] 3. Experimental Methods

[0044] 1. Use phosphate buffer (each 100 mL of phosphate buffer contains 94.7 mL of 0.1 M Na2HPO4 solution; 5.3 mL of 0.1 M NaH2PO4 solution, and the pH is adjusted to 8.0) to dilute AChE to a 0.1 U / mL working solution;

[0045] 2. Prepare a 6.25 mM solution (working solution) of iodinated thioacetylcholine and DTNB in ​​phosphate buffer;

[0046] 3. The compound was diluted with DMSO to form a concentration gradient. The positive control was tacrine, which was diluted with DMSO to form a concentration gradient; the negative control group (NC group) was a 2% DMSO solvent control.

[0047] 4. The reaction was carried out in a 96-well plate, with 200 μL / well plate, and 3 replicates for each sample;

[0048] (1) The concentration gradient of compound PFT217 and tacrine (diluted with 1% DMSO) is shown in Table 1.

[0049] Table 1. Compound PFT217 and tacrine concentration gradient settings

[0050]

[0051] (2) Plating (Table 2): 200 μL / well, the final concentration of DMSO in each well was 0.1%, and 3 replicate wells were prepared for each sample.

[0052] Table 2. Order of sample addition for plating

[0053]

[0054]

[0055] 5. Within 1 hour after adding the color developer and substrate, measure the absorbance at 405 nm every 30 seconds.

[0056] 6. Select the sample absorbance value when the average absorbance value of the NC group is about 1, calculate the average absorbance value of the compound (compound measurement value-background value), and calculate the compound AChE inhibition rate according to (NC-compound absorbance average value) / NC×100%.

[0057] IV. Experimental Results

[0058] As shown in Table 3, compound PFT217 has a certain inhibitory activity on acetylcholinesterase and has a concentration-dependent effect.

[0059] Table 3. Inhibition rate of compound PFT217 on acetylcholinesterase

[0060]

[0061] Example 4

[0062] Butyrylcholinesterase inhibitory activity assay:

[0063] 1. Experimental Principle

[0064] Butyrylcholinesterase can catalyze the degradation of its substrate analog iodinated thiobutyrylcholine to generate thiocholine and butyric acid. The reaction product reacts with the color developer DTNB to generate a yellow substance with specific light absorption at 405nm. The compound and butyrylcholinesterase mixture react at 30℃. If the compound has an inhibitory effect on butyrylcholinesterase, the amount of butyrylcholinesterase-catalyzed degradation of the substrate analog iodinated thiobutyrylcholine will decrease, and the corresponding yellow compound generated by the reaction with DTNB will decrease, that is, the light absorption value at 405nm will decrease, so as to screen compounds with inhibitory activity.

[0065] 2. Reagents and Instruments

[0066] Reagents: Na2HPO4, NaH2PO4, butyrylcholinesterase, iodinated thiobutyrylcholine, DTNB, and tacrine were purchased from Sigma;

[0067] Instrument: Thermo Multiskan FC.

[0068] 3. Experimental Methods

[0069] 1. Dilute BuChE to 0.04U / mL with phosphate buffer (94.7mL of 0.1M Na2HPO4 solution and 5.3mL of 0.1M NaH2PO4 solution per 100mL of phosphate buffer, pH adjusted to 8.0);

[0070] 2. Prepare a 6.25 mM solution (working solution) of iodinated thiobutyrylcholine and DTNB in ​​phosphate buffer;

[0071] 3. The compound was diluted with DMSO to form a concentration gradient. The positive control was tacrine, which was diluted with DMSO to form a concentration gradient; the negative control group (NC group) was a 2% DMSO solvent control.

[0072] 4. The reaction was carried out in a 96-well plate, with 200 μL / well plate, and 3 replicates for each sample;

[0073] (1) See Table 4 for the setting of compound and tacrine concentration gradient (diluted with 1% DMSO).

[0074] Table 4. Compound and tacrine concentration gradient settings

[0075]

[0076] (2) Plating (Table 5): 200 μL / well, the final concentration of DMSO in each well was 0.1%, and 3 replicate wells were prepared for each sample.

[0077] Table 5. Order of sample addition for plating

[0078]

[0079]

[0080] 5. Within 1 hour after adding the color developer and substrate, measure the absorbance at 405 nm every 30 seconds.

[0081] 6. Select the sample absorbance value when the average absorbance value of the NC group is about 1, calculate the average absorbance value of the compound (compound measurement value - background value), and calculate according to (NC - average absorbance value of the compound) / NC ×

[0082] The BuChE inhibition rate of the compound was calculated as 100%.

[0083] IV. Experimental Results

[0084] As shown in Table 6, compound PFT217 has significant inhibitory activity on butyrylcholinesterase in a concentration-dependent manner.

[0085] Table 6. Inhibition rate of compound PFT217 on butyrylcholinesterase

[0086]

[0087] Example 5

[0088] Origin8 software was used to fit the curve and calculate the half inhibitory concentration (IC 50 )value.

[0089] According to the inhibition rates in Table 3 and Table 6, IC 50 The IC values ​​of PFT217 for inhibition of acetylcholinesterase and butyrylcholinesterase were calculated and the results are shown in Table 7. 50 The values ​​were 24.70 μM and 0.84 μM, respectively. The inhibitory activity of PFT217 on butyrylcholinesterase was better than that on acetylcholinesterase, and the selectivity index (acetylcholinesterase IC 50 / Butyrylcholinesterase IC 50 , SI) was 29.4, which was higher than tacrine (SI=5.3).

[0090] Table 7. IC of PFT217 for inhibition of acetylcholinesterase and butyrylcholinesterase 50 Value and selection index

[0091]

[0092] Preparation Example:

[0093] In the following formulation examples, conventional reagents are selected and the formulations are prepared according to conventional methods. This formulation example only reflects that the compound PFT217 of the present invention can be prepared into different formulations, and no specific restrictions are imposed on the specific reagents and operations.

[0094] 1. The compound PFT217 of the present invention is dissolved in DMSO, and then water for injection is added according to a conventional method, finely filtered, and sterilized by filling to prepare an injection solution, wherein the concentration of the injection solution is 0.5-5 mg / mL.

[0095] 2. Dissolve the compound PFT217 of the present invention in DMSO and then dissolve it in sterile water for injection. Stir to dissolve it, filter it with a sterile suction funnel, filter it with sterile fine filtration, dispense it into ampoules, freeze-dry it at low temperature, and seal it with sterile sealing to obtain a powder injection.

[0096] 3. The compound PFT217 of the present invention was added to the excipient at a mass ratio of 9:1 to the excipient to prepare a powder.

[0097] 4. The compound PFT217 of the present invention was added to the excipients at a mass ratio of 5:1, and the mixture was granulated and tableted.

[0098] 5. The compound PFT217 of the present invention is prepared into an oral liquid according to a conventional oral liquid preparation method.

[0099] 6. The compound PFT217 of the present invention was added to the excipients at a mass ratio of 5:1 to the excipients to prepare capsules.

[0100] 7. The compound PFT217 of the present invention was added to the excipient at a mass ratio of 5:1 to the excipient to prepare granules.

[0101] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Compound PFT217 shown in the following structural formula, 2. A method for preparing the compound PFT217 represented by the structural formula according to claim 1, characterized in that: The method comprises the following steps: Grind the dried tubers, peduncles and inflorescences of Gastrodia elata after collecting seeds together, extract them three times with 90% ethanol aqueous solution at 70°C, each time for 3 hours, combine the extracts, filter, and concentrate the filtrate under reduced pressure to obtain an extract; The crude extract was added with distilled water to prepare a suspension, HCl was added to adjust the pH to 2-3, and the extract was extracted with an equal volume of petroleum ether for 5 times. The organic phase was concentrated under reduced pressure at 60°C using a rotary evaporator to obtain a petroleum ether extraction part, and the remaining aqueous phase was extracted with an equal volume of ethyl acetate for 5 times. The organic phase was concentrated under reduced pressure at 60°C using a rotary evaporator to obtain an ethyl acetate extraction part. After the ethyl acetate extraction, the remaining aqueous phase was adjusted to pH 9-10 with a NaOH aqueous solution, extracted with an equal volume of dichloromethane for 5 times, and the organic phase was concentrated under reduced pressure at 60°C using a rotary evaporator to obtain a dichloromethane extraction part; the dichloromethane extraction part was subjected to 100-200 mesh silica gel column chromatography, and v / v acetone-petroleum ether 20:1→1:1 gradient elution was performed to obtain 10 components A, B, C, D, E, F, G, H, I and J; Component G passes through RP C 18 Silica gel column chromatography, v / v methanol-water 10%→100%, to obtain 50% methanol elution part G5; component G5 was chromatographed on a 200-300 mesh silica gel column, v / v petroleum ether-ethyl acetate 10:1→0:1 gradient elution to obtain G5-1; G5-1 was separated by Sephadex LH-20 column chromatography using methanol as the eluent to obtain G5-1-1; G5-1-1 was separated by HPLC to obtain compound PFT217.

3. Use of the compound PFT217 represented by the structural formula according to claim 1 in the preparation of acetylcholinesterase inhibitors and butyrylcholinesterase inhibitors.

4. Use of the compound PFT217 represented by the structural formula according to claim 1 in the preparation of anti-Alzheimer's disease drugs.

5. A pharmaceutical composition comprising the compound PFT217 represented by the structural formula of claim 1 and a pharmaceutically acceptable carrier.

6. Use of the pharmaceutical composition according to claim 5 in the preparation of acetylcholinesterase inhibitors and butyrylcholinesterase inhibitors.

7. Use of the pharmaceutical composition according to claim 5 in the preparation of anti-Alzheimer's disease drugs.

8. The method for preparing the pharmaceutical composition according to claim 5, characterized in that: The method comprises first obtaining the compound PFT217 by the preparation method described in claim 2, and then adding a pharmaceutically acceptable carrier.

Images