Pyrrosia glabra alkaloid compound as well as preparation, application and medicine thereof

Abstract

The invention relates to a pyrrosia lingua alkaloid-containing compound as well as preparation, application and a medicine thereof. The novel compounds (Calvatine A and B) provided by the invention are obtained by extracting and separating from a water orthopedics (Polypodiaceae) pyrrosia plant (Pyrrosia calva (Bak.) Chig], and are alkaloids which are novel in structure and are connected with polyphenol through pyrrolidone. The invention further provides a preparation method of the novel compounds (Calvatine A and B) and a preparation method of the novel compounds (Calvatine A and B) of the novel compounds (Calvatine A and B) of the novel compounds (Calvatine B). A biological activity experiment shows that the alkaloid compound has muscarinic receptor M3 antagonistic activity and can be applied to preparation of drugs for treating pain, chronic obstructive pulmonary diseases, irritable bowel syndromes and the like related to muscarinic receptors.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicine chemistry and relates to a new skeleton alkaloid compound of Pyrrosia calvata. More specifically, it relates to the preparation and resolution methods of a pair of chiral alkaloids Calvatine A and B and their use as active ingredients of muscarinic receptor M3 for treating related diseases such as pain, chronic obstructive pulmonary disease, and irritable bowel syndrome. In vitro activity shows that the above compounds have significant muscarinic receptor M3 antagonistic activity and can be used as lead compounds for developing new drugs for treating or relieving pain and spasm-related diseases. Background Art

[0002] Pyrrosia calvata (Bak.) Ching is a plant of the family Polypodiaceae. It was included as a local conventional medicinal material in the "Guangxi Chinese Medicinal Materials Standard" in 1990 edition [1]. It is a commonly used herb in the Zhuang ethnic group in Guangxi. Zhuang medicine is often used for clearing heat toxins, regulating water channels, stopping bleeding, treating stranguria, and relieving cough, etc. [2]. The Chinese patent medicine compound Jinqiancao Granules is made by extracting and processing four traditional Chinese medicines, namely Desmodium styracifolium, Plantago asiatica, Pyrrosia calvata, and Cornus officinalis, further affirming that Pyrrosia calvata is a potential ethnic medicinal plant [3]. However, current domestic and foreign research on plants of the genus Pyrrosia mainly focuses on Pyrrosia petiolosa, Pyrrosia sheareri, and Pyrrosia lingua included in the "Chinese Pharmacopoeia" 2010 edition [4], and less research has been carried out on Pyrrosia calvata.

[0003] Currently, no more than 30 compounds have been isolated from Pyrrosia calvata, mainly including steroids, terpenoids, phenolic acids, and flavonoid compounds [5 - 8]. Mangiferin and isomangiferin are the main active ingredients of Pyrrosia calvata [9], accounting for more than 60%. This indicates that other potential natural molecules exist in trace or even trace amounts, increasing the difficulty of separation and thus limiting the discovery of new drugs from Pyrrosia calvata.

[0004] Muscarinic receptors (M receptors) include five pharmacological subtypes, M1 - M5. These subtypes have different distributions and functions. For example, M1 receptors have the function of regulating higher cognitive functions such as memory and learning, M2 receptors can regulate myocardial contractility, and M3 receptors have the function of regulating glandular and smooth muscle contractions. Pyrrosia alkaloids have good selective activity on M3 receptors. Therefore, it is of great research value to discover compounds with M3 receptor activity from Pyrrosia calvata.

[0005] In the present invention, a multidimensional liquid chromatography separation method for Pyrrosia calvata alkaloids is described. A pair of chiral alkaloid compounds are isolated for the first time in the genus Pyrrosia; and such alkaloid skeletons are also identified for the first time in nature. Pharmacological activity tests show that the new skeleton alkaloid compounds have significant muscarinic receptor M3 antagonistic activity and can be applied in drugs for treating diseases such as pain, chronic obstructive pulmonary disease, and irritable bowel syndrome.

[0006] References

[0007] [1] Guangxi Zhuang Autonomous Region Health Department. Guangxi Traditional Chinese Medicine Standards[S]. Nanning: Guangxi Science and Technology Press, 1990:184.

[0008] [2] Guangxi Zhuang Autonomous Region Food and Drug Administration. Quality Standards of Zhuang Medicine in Guangxi Zhuang Autonomous Region [M]. Volume 2. Nanning: Guangxi Science and Technology Press, 2011: 124-125.

[0009] [3] Ma Junhua, Qiu Hongcong, Chen Mingsheng, Wen Youmin, Chen Lu, Liu Buming. Study on the quality control of compound Qianqiancao granules[J]. Modern Drugs and Clinics, 2014, 29(04): 381-384.

[0010] [4] Chinese Pharmacopoeia Committee. Chinese Pharmacopoeia [S]. Volume 1. Beijing: China Medical Science and Technology Press, 2010: 834.

[0011] [5] Zheng Xing, Yu Lin, Liao Duanfang, et al. Study on the chemical components of Psoralea corylifolia[J]. Chinese Herbal Medicine, 1999, 30(4):253.

[0012] [6] Bao Wenfang, Xi Xiaohong, Li Bin. Two xanthones of Psoralea corylifolia[J]. Northwest Journal of Pharmaceutical Sciences, 1989, 4(1):16.

[0013] [7] Zhang Qilong, Xu Hong, He Kang. Study on the chemical components of Psoralea corylifolia[J]. Chinese Journal of Experimental Traditional Chinese Medicine, 2014, 20(03): 49-51.

[0014] [8]Chen YJ,

[0015] [9] Huang Yan, Wen Youmin, Ma Junhua, et al. Study on HPLC fingerprint of Psoralea corylifolia[J]. Chinese Journal of Experimental Traditional Chinese Medicine, 2013, 19:152-155. Summary of the Invention

[0016] The present invention provides a novel alkaloid compound, or a compound in different crystal forms, or a chiral isomer thereof, or a glycoside thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a prodrug thereof, or a metabolite thereof, wherein the general structural formula (I) is as follows:

[0017]

[0018] In the two compounds of the general formula (I), R1 - R5 are each independently hydrogen, chlorine, hydroxyl, methoxy, ethoxy, O - glycosyl or OAr; Ar is a substituted or unsubstituted phenyl group, and the substituents on the substituted phenyl group are preferably one or more of hydroxyl, methoxy or acetyl; the glycosyl group (preferably O is connected to the anomeric carbon atom of the sugar) is one or more of a monosaccharide group or various disaccharide groups and polysaccharide groups formed by two or more monosaccharides: the monosaccharide is preferably one or more of glucose, fructose, galactose, xylose and apiose; the disaccharide with glucose is preferably glucose connected to the skeleton of compound 1 or 2, and another molecule of monosaccharide is connected to glucose, and the connection position of the another molecule is preferably one or more of the 2, 4, 6 positions of glucose; the polysaccharide is preferably one or more of random polymers of 3 - 10 molecules of monosaccharides.

[0019] Furthermore, the stereoconfiguration of the above stereocompound (I) is selected from one or both of (7S) and (7R).

[0020] Furthermore, the stereoconfigurations of compounds 1 - 2 are preferably 7S and 7R respectively; R1, R3 - R5 are selected from hydroxyl, and R2 is selected from hydrogen. The compounds are as shown in formula (II) and are named (S)-Calvatine A and (R)-Calvatine B respectively.

[0021]

[0022] The present invention also provides a method for preparing the above compounds (1 - 2), comprising the following steps:

[0023] (1) Medicinal material extraction: Take 30 - 50 kg of dry whole grass of Pyrrosia calvata, crush it into coarse powder with a particle size of 10 - 100 mm, add 40 - 80 L of ethanol with a volume concentration of 50% - 90%, heat and extract at a low temperature of 30 - 50 °C for 12 - 48 hours. After filtering the extract with a 70 - 80 - mesh filter, rotate and evaporate the solvent of the filtrate at 30 - 50 °C to obtain an extract paste. Dissolve the obtained paste in 1 - 3 L of distilled water, then add 1 - 5 L of petroleum ether for extraction, and remove the impurities in the petroleum ether layer; add 1 - 5 L of ethyl acetate to the remaining aqueous layer for extraction, and concentrate to obtain the ethyl acetate layer extract;

[0024] (2) Dissolve the ethyl acetate layer extract in step (1) with methanol having a volume concentration of 10% - 30%, pass it through an MCI column of a methanol - water system, and elute successively with 2 - 5 times the column volume of methanol with a volume concentration of 10% and methanol with a volume concentration of 30% to obtain two fractions GSWY - A and GSWY - B respectively;

[0025] (3) Combine the GSWY-A and GSWY-B fractions in step (2), perform dry loading and pass through a silica gel column with a particle size of 200 - 300 mesh, and elute with 3 - 5 column volumes of chloroform - methanol with a volume ratio of 50:1 - 10:1 to obtain the GSWY-AB-1 sub-fraction;

[0026] (4) Pass the GSWY-AB-1 component in step (3) through a 200 - 300 mesh silica gel column, and perform stepwise isocratic elution successively with 2 - 5 times the column volume of petroleum ether - acetone with volume ratios of 6:1, 5:1, 4:1, 3:1, 2:1, 1:1 at a flow rate of 0.8 - 2 ml / min, and collect 37 sub-fractions from GSWY-AB-1-1 to GSWY-AB-1-37 according to the ultraviolet absorption peaks;

[0027] (5) Use a C18HD reversed-phase chromatographic column for the first-dimensional separation and purification of the fraction GSWY-AB-1-32 in step (4), with a flow rate of 60 - 80 ml / min. Mobile phase A is methanol, and B is formic acid - water with a volume concentration of 0.1% - 0.3%. The gradient elution conditions are: 0 - 50 min: 15 - 65% A (volume ratio, the same below), 50 - 60 min: 65 - 95% A, 60 - 70 min: 95% A, and collect the crude fraction F2 with a retention time of 30 - 50 min according to the ultraviolet absorption chromatogram;

[0028] (6) Use a C18HD reversed-phase chromatographic column for the second-dimensional separation and purification of the sub-fraction F2 obtained in step (5), with a flow rate of 60 - 80 ml / min. Mobile phase A is formic acid - methanol with a volume concentration of 0.1% - 0.5%, and B is formic acid - water with a volume concentration of 0.1% - 0.3%. The gradient elution conditions are: 0 - 60 min: 25 - 65% A, 60 - 61 min: 65 - 95% A, 61 - 75 min: 95% A, and collect 6 sub-fractions F2-1 - F2-6 according to the ultraviolet absorption chromatographic peaks. The retention time of F2-2 is tR = 30 - 40 min;

[0029] (7) Use a QUIS reversed-phase chromatographic column for the third-dimensional separation and purification of the sub-fraction F2-2 obtained in step (6), with a flow rate of 3 - 5 ml / min. Mobile phase A is methanol, and B is formic acid - water with a volume concentration of 0.1% - 0.3%. The gradient elution conditions are: 0 - 30 min, 50 - 70% A; 30 - 32 min, 70 - 90% A; 32 - 45 min, 90% A, and collect 5 sub-fractions as F2-2-1 - F2-2-5. The retention time of F2-2-1 is tR = 10 - 15 min;

[0030] (8) The subfraction F2-2-1 obtained in step (7) was subjected to fourth-dimensional separation and purification using an AH chiral chromatographic column at a flow rate of 10-20 ml / min, mobile phase A was formic acid-ethanol with a volume concentration of 0.1-0.5%, B was supercritical fluid CO2, and the gradient elution conditions were: 0-13 min: 22% A; compound 1 was collected according to the chromatographic peak, with a retention time of tR = 4.80-6.90 min, named Calvatine A; compound 2, with a retention time of tR = 7.60-10.90 min, named Calvatine B.

[0031] The method comprises the following steps: pulverizing the medicinal material of Psoralea corylifolia into a coarse powder, extracting it multiple times with 75% ethanol at room temperature, and concentrating the extract under reduced pressure to obtain an extract. The extract is dissolved in 10% methanol-water and extracted with petroleum ether, ethyl acetate, and n-butanol in a ratio of 1:1–1:2. The ethyl acetate layer is subjected to multi-step high-performance preparative liquid chromatography to obtain a mixture of 1 and 2. This mixture is then separated using a chiral column to obtain calvatine A and calvatine B.

[0032] The present invention conducted an activity test on the muscarinic receptor M3 on the obtained pair of alkaloid compounds. The activity test was performed using human embryonic kidney cells HEK293 transfected with the M3 receptor. The results showed that the compounds showed low micromolar antagonistic activity on the muscarinic receptor M3 and could become potential lead compounds for the treatment of pain and spasm.

[0033] The compounds of the present invention can be obtained by isolation and purification from plants; they can also be synthesized by chemical methods well known to those skilled in the art. The compounds, their crystal forms, their isomers, their glycosides, their pharmaceutically acceptable salts, their solvates, their prodrugs, or their metabolites can be used in the preparation of one or more medicaments for preventing and / or treating muscarinic receptor-related diseases such as pain, chronic obstructive pulmonary disease, and irritable bowel syndrome.

[0034] A pharmaceutical composition comprising one or more of the compound or its crystal form, or its isomer, or its glycoside, or its pharmaceutically acceptable salt, or its solvate, or its prodrug, or its metabolites, and any other pharmaceutically acceptable excipients, carriers, diluents or other active ingredients.

[0035] The compounds of the present invention can be used alone or in combination, or combined with pharmaceutically suitable carriers or excipients to prepare oral or non-oral dosage forms according to conventional methods.

[0036] Obviously, based on the above content of the present invention, according to the common technical knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions or changes can be made.

[0037] The novel compounds (Calvatine A and Calvatine B) provided by the present invention are a class of alkaloids with a novel structure, which are isolated from the plant Pyrrosia calvata (Bak.) Ching of the genus Pyrrosia in the family Polypodiaceae and are alkaloids connecting polyphenols through pyrrolidone. Biological activity experiments show that the alkaloid compounds have muscarinic receptor M3 antagonistic activity and can be used in the preparation of drugs related to muscarinic receptors such as pain, chronic obstructive pulmonary disease and irritable bowel syndrome.

[0038] The present invention has the following advantages: the target compound is an alkaloid with a novel structure connecting polyphenols through pyrrolidone; the R-type structure of this compound has significant muscarinic receptor M3 antagonistic activity and has application prospects for the development of drugs for pain, spasm, chronic obstructive pulmonary disease and irritable bowel syndrome, etc., which are highly concerned now. Description of the Drawings

[0039] Figure 1 1H NMR (proton nuclear magnetic resonance) spectrum of Compound 1 / 2 1 1H NMR (proton nuclear magnetic resonance) spectrum

[0040] Figure 2 13C NMR (carbon nuclear magnetic resonance) spectrum of Compound 1 / 2 13 13C NMR (carbon nuclear magnetic resonance) spectrum

[0041] Figure 3 H,H-COSY (hydrogen-hydrogen correlation spectroscopy) and key HMBC (heteronuclear multiple bond correlation spectroscopy) of Compound 1 / 2

[0042] Figure 4 Experimental circular dichroism spectrum (ECD) and calculated ECD spectrum of Compound 1-2

[0043] Figure 5 Dose response of Compound 1 (left figure) - 2 (right figure) on muscarinic receptor M3;

[0044] Figure 6 Schematic diagram of the structure of Compound II in the preferred embodiment of the present invention.

[0045] The process of stereostructure analysis of the compound is as follows, involving Figures 1-4 and Table 1.

[0046] The quasi-molecular ion peak [M+H]+ was observed at m / z 448.1754 in the HRESIMS spectrum, from which the molecular formula was deduced to be C26H25NO6 (the calculated value of C26H26NO6+ is 448.1755). From 1 the 1H NMR spectrum ( Figure 1 ), ten proton signals on the aromatic ring were observed at δH 7.91 (2H, d, 8.5, H-2′ / 6′), 6.91 (2H, dt, 8.4, 2.0, H-2″ / 6″), 6.88 (2H, dt, 8.5, 2.0, H-3′ / 5′), 6.64 (2H, d, 8.4, 2.0, H-3″ / 5″), 6.24 (1H, d, 2.4, H-5), δH 6.14 (1H, d, 2.3, H-3); five pairs of methylene signals at δH 4.12 / 3.99 (H-8′), 3.53 / 2.79 (H-8″), 2.69 / 2.47 (H-7″), 2.54 / 2.34 (H-9), 2.17 / 2.10 (H-8); and an azomethine methylene signal at 4.57 (1H, dd, 5.5, 8.9, H-7). Combining with the carbon spectrum ( Figure 2 ), HSQC, and HMBC spectra ( Figure 3 ), two carbonyl carbon signals at δC 198.4 (C-7′), 178.3 (C-10) were observed; two para-substituted benzene rings with signals at δC 164.0 (C-4′), 157.0 (C-4″), 132.2 (C-2′ / 6′), 131.6 (C-1″), 130.8 (C-2″ / 6″), 129.7 (C-1′), 116.4 (C-3′ / 3″ / 5′ / 5″); a 3,5-substituted benzene ring at δC 159.2 (C-4), 158.8 (C-6), 137.7 (C-2), 117.6 (C-1), 110.6 (C-3), 103.5 (C-5); an azomethine methylene at δC 59.8 (C-7); and five methylene carbon signals at δC 44.2 (C-8′), 44.2 (C-8″), 33.7 (C-7″), 32.3 (C-9), 25.4 (C-8).

[0047] The COSY spectrum ( Figure 3 ) showed two para-substituted benzene rings H2′ / H3′ / H5′, H2″ / H3″ / H5″ and a 3,5-substituted benzene ring H3 / H5, and two pairs of coupled methylenes H7″ / H8″, H8 / H9. According to HMBC ( Figure 3)The relevant signals H2′ / H6′ to C7′ in it prove that one of the para-phenyl rings is connected to the carbonyl group; the HMBC signal H2″ / H6″ to C7″ indicates that the other para-substituted phenyl ring is connected to the aliphatic chain; H7 to C1 / C2 / C6 confirms that the 3,5-substituted phenyl ring is connected to the nitrogen-containing five-membered ring; the weaker signal of the methylene H8″ to C7 / C10 indicates the connection between the aliphatic chain and the five-membered ring, and combined with the fact that there is one nitrogen atom in the molecular formula, it shows that it is directly connected to N.

[0048] The experimental value of specific rotation is 0, indicating that the compound F2-2-1 is a racemate and contains a pair of chiral compounds. After separation by an AH chiral column, two enantiomers were obtained. Through the calculation results, it was found that the experimental values of the ECD curves of F2-2-1A and F2-2-1B corresponded to the curves of the calculated values (S) and (R) respectively. Thus, the absolute configuration of F2-2-1A was determined to be S; the absolute configuration of F2-2-1B was determined to be R( Figure 4 ).

[0049] Figure 5 Note: The drug addition process for activity screening is divided into two steps. In the first step (S1), the sample is added to HEK293-M3 cells, and the DMR response signal of the compound at different concentrations on the cells is detected (corresponding to the blue line). In the second step (S2), the probe molecule acetylcholine is added to the cells after 1 h of pretreatment, and the DMR response signal of the probe molecule on the cells is detected by the desensitization analysis method (red curve). Generally speaking, if there is no obvious signal in S1 and the signal in S2 decreases, it means that the test sample has antagonistic activity. If there is a signal in S1 and the signal in S2 decreases, it means that the test sample may have receptor agonist activity. The lower the signal in S2, the stronger the effect of the sample on the receptor. Specific Embodiments

[0050] The following examples are intended to illustrate the present invention rather than further limit the present invention. The present invention can be implemented in any manner described in the invention content.

[0051] Preparation Example of the Compound of Formula (II) of the Present Invention:

[0052] In the following Preparation Examples, the preparation system included Waters Alliance, including an e2695 separation unit, a 2998 PDA detector, and data processing was performed by Empower 3; Waters AutoP automatic purification system, including a 2545 separation unit, a 2767 sample manager, and a 2489 dual-wavelength detector. Reagents included chromatographic-grade methanol and acetonitrile purchased from Fisher Scientific (Loughborough, UK), chromatographic-grade formic acid purchased from J&K Scientific (Hebei, China), laboratory water from a Milli-Q ultrapure water purification system (Billerica, MA, USA), and preparative-grade methanol purchased from Shanghai Xingke High-Purity Solvent Co., Ltd. (Shanghai, China). Preparation columns and semi-preparation columns: MCI column (gel column, 50 mm × 400 mm), silica gel column (200 - 300 mesh, 75 - 150 μm, 35 mm × 200 mm), C18HD (high bond density C18, 10 μm, 50 mm × 253 mm), QUIS (quinine stationary phase, 3.5 μm, 10 mm × 150 mm), and AH column (polysaccharide derivative, 5 μm, 10 mm × 250 mm) (Dalian Sipu Precision Technology Co., Ltd.).

[0053] The nuclear magnetic resonance spectra used for structural identification were measured with a Bruker AVIII-600 nuclear magnetic resonance spectrometer (Bruker, German), and the compounds were all dissolved in deuterated methanol (MeOD). Mass spectrometry was performed using an Agilent 1290 Infinity LC / 6540 Q-TOF MS liquid chromatography-mass spectrometry system for sample separation and analysis.

[0054] The steps for preparation and compound identification were as follows:

[0055] (1) Medicinal material extraction: 41.8 kg of dry whole Pyrola saxatilis roots and herbs were crushed into coarse powder (particle size 10 - 100 mm), added with 50 L of 70% ethanol by volume, heated at 40 °C for 48 hours under low temperature, the extract was filtered through an 80-mesh sieve for solid-liquid separation, and the filtrate was rotary evaporated at 45 °C to remove the solvent to obtain 7 kg of extract. After the extract was dissolved in 2 L of distilled water, 1 L of petroleum ether was added for extraction, and the impurities in the petroleum ether layer were removed. 1 L of ethyl acetate was added to the remaining aqueous layer for liquid-liquid extraction, and the ethyl acetate layer was separated and concentrated to obtain 143 g of medium-polarity organic layer extract (GSWY fraction).

[0056] (2) The GSWY fraction in step (1) was dissolved in 100 ml of 10% methanol (by volume, the same below), passed through an MCI column (methanol-water system), and eluted successively with 4 column volumes of 10% methanol and 30% methanol, and the corresponding fractions were collected and concentrated under pressure to obtain an extract, yielding two fractions GSWY-A and GSWY-B.

[0057] (3) Combine the GSWY-A fraction and the GSWY-B fraction in step (2), perform dry loading and pass through a silica gel column (200 - 300 mesh), and elute with 5 column volumes of chloroform - methanol (volume ratio 30:1) to obtain GSWY-AB-1.

[0058] (4) Pass the GSWY-AB-1 component in step (3) through a silica gel column (200 - 300 mesh), and perform stepwise isocratic elution successively with 3 column volumes of petroleum ether - acetone at volume ratios of 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1. The flow rate is 1 ml / min, and collect 37 fractions in total, namely GSWY-AB-1-1 to GSWY-AB-1-37, according to the ultraviolet absorption peaks.

[0059] (5) Perform the first - dimension separation and purification of the fraction GSWY-AB-1-32 (900 mg) in step (4) using C18HD (reversed - phase chromatographic column). The flow rate is 80 ml / min, mobile phase A is methanol, and B is formic acid - water (volume concentration 0.1%). The gradient elution conditions are: 0 - 50 min: 15 - 65% A (volume ratio, the same below), 50 - 60 min: 65 - 95% A, 60 - 70 min: 95% A. Collect the crude fraction F2 with a retention time of 30 - 40 min according to the ultraviolet absorption chromatographic peaks.

[0060] (6) Perform the second - dimension separation and purification of the sub - fraction F2 (520 mg) obtained in step (5) using C18HD (reversed - phase chromatographic column). The flow rate is 80 ml / min, mobile phase A is formic acid - methanol with a volume concentration of 0.1%, and B is formic acid - water with a volume concentration of 0.1%. The gradient elution conditions are: 0 - 60 min: 25 - 65% A, 60 - 61 min: 65 - 95% A, 61 - 75 min: 95% A. Collect 6 sub - fractions, namely F2-1 to F2-6, according to the ultraviolet absorption chromatographic peaks. The retention time of F2-2 is tR = 32 - 38 min.

[0061] (7) Perform the third - dimension separation and purification of the sub - fraction F2-2 (70.6 mg) obtained in step (6) using QUIS (reversed - phase chromatographic column). The flow rate is 3.3 ml / min, mobile phase A is methanol, and B is formic acid - water (volume concentration 0.1%). The gradient elution conditions are: 0 - 30 min, 50 - 70% A; 30 - 32 min, 70 - 90% A; 32 - 45 min, 90% A. Collect 5 sub - fractions, namely F2-2-1 to F2-2-5, according to the chromatographic peaks. The retention time of F2-2-1 is tR = 10.5 - 14 min.

[0062] (8) The sub-fraction F2-2-1 (10.1 mg) obtained in step (7) was separated and purified in the fourth dimension using AH (chiral chromatographic column) at a flow rate of 10 ml / min. Mobile phase A was formic acid-ethanol (volume concentration 0.1%), and B was supercritical fluid CO2. The gradient elution conditions were as follows: 0 - 13 min: 22% A. Compound 1 (tR = 4.80 - 6.90 min, 1.13 mg) was collected according to the chromatographic peak and named Calvatine A; Compound 2 (tR = 7.60 - 10.90 min, 1.37 mg) was named Calvatine B.

[0063] (9) The above compounds have the following physicochemical properties and spectroscopic characteristics:

[0064] Calvatine A: White powder; specific rotation Ultraviolet absorption UV (MeOH) λmax = 279 nm; Circular dichroism ECD (MeOH) λmax (Δε) = λ 214 (+14.4), λ 235 (-19.8), λ 270 (+5.20); High-resolution mass spectrometry HRESIMS m / z 448.1754 [M + H] + (calcd.for C 26 H 25 NO6,448.1755).

[0065] Calvatine B: White powder; specific rotation Ultraviolet absorption UV (MeOH) λ max = 279 nm; Circular dichroism ECD (MeOH) λ max (Δε) = λ 210 (-6.15), λ 235 (+10.4), λ 281 (-3.94); High-resolution mass spectrometry HRESIMS m / z 448.1754 [M + H] + (calcd.for C 26 H 25 NO6,448.1755).

[0066] The 1H-NMR and 13C-NMR nuclear magnetic data of Compound 1 are shown in Table 1, and its 1H-NMR and 13C-NMR nuclear magnetic spectra are as Figures 1-2 shown, and the key two-dimensional nuclear magnetic information of the compound is as Figure 3As shown (Note: Since Compounds 1 and 2 are enantiomers, their planar NMR data are exactly the same), the absolute configurations of Compounds 1 and 2 were determined by comparing their circular dichroism (ECD) spectra and quantum chemical calculated ECD, such as Figure 4 .

[0067] Table 1. 1 1H-NMR data of Compound 1 / 2 (δ H , J in Hz, MeOD)

[0068]

[0069]

[0070] Examples of activity tests:

[0071] Experimental method:

[0072] The human embryonic kidney cell line HEK293-M3 that highly expresses the muscarinic acetylcholine receptor subtype M3 is derived from laboratory transfection (the transfection method is to transfect the M3 plasmid into HEK293 cells to obtain the cell line HEK293-M3 that highly expresses the M3 receptor, M3 plasmid: transfection reagent = 1:3; the M3 plasmid is CHRM3-pcDNA3.1+, and the transfection reagent is Lipofectamine2000), and it is cultured in DMEM medium with 10% fetal bovine serum (FBS) by volume concentration. The cells are seeded in an Epic 384-well plate at a density of approximately 25,000 cells / well. An EPIC instrument (Corning) is used for activity screening.

[0073] For the dose-activity determination of the compound (Compound 1 or Compound 2) against muscarinic M3, in the first step, 10 μL of the compound (Compound 1 or Compound 2) gradient-diluted with HBSS buffer (containing 20 mM HEPES, pH = 7.4) was added respectively, with the highest final concentration of 100 μM, diluted in a 2-fold gradient, and 14 concentration points (100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.563 μM, 0.782 μM, 0.391 μM, 0.196 μM, 0.098 μM, 0.049 μM, 0.024 μM, 0.012 μM) were used. The EPIC activity system was used for real-time detection for 1 h, and then a 2-min baseline was re-established. In the second step of adding the drug, a fixed amount of 1.25 μM ACh (acetylcholine) was added to each well, and real-time detection continued for 1 hour. The results showed that the compounds at each concentration did not have a response signal or had a weak response signal themselves, but could antagonize the response signal generated by the M3 receptor agonist acetylcholine on HEK293 cells in a dose-dependent manner, indicating that the compound has M3 receptor antagonistic activity and is an M3 receptor antagonist, such asFigure 4 as shown

[0074] The muscarinic receptor M3 activity information of Calvatine A and B is shown in Table 2. The test results show that the activities of a pair of chiral compounds are completely different. Calvatine A does not show M3 activity, but Calvatine B can significantly inhibit the Ca2+ current on HEK293 cells, indicating that it has obvious antagonistic activity against the M3 muscarinic receptor. Therefore, when the 7-position stereoconfiguration of this compound is R, that is, Calvatine B is a lead compound with activity for developing new drugs for treating or alleviating pain, spasm and other related diseases.

[0075] Table 2. Activity information of compounds

[0076]

[0077] The new compounds (Calvatine A and Calvatine B) provided by the present invention are a class of alkaloids with a novel structure that connect polyphenols through pyrrolidone, which are extracted and isolated from the plant Pyrrosia calvata (Bak.) Ching of the genus Pyrrosia in Polypodiaceae. Biological activity experiments show that this alkaloid compound has antagonistic activity against the muscarinic receptor M3 and can be used in the preparation of drugs for preventing and treating muscarinic receptor-related pain, chronic obstructive pulmonary disease, irritable bowel syndrome and the like.

Claims

1. A new skeleton alkaloid compound of Psoralea corylifolia, characterized in that: The alkaloid compound is one or two of the two structural compounds represented by general formula (I), or compounds of general formula (I) in different crystalline forms thereof, or chiral isomers thereof, or glycosides thereof, or pharmaceutically acceptable salts thereof, or solvates thereof, or prodrugs thereof, or metabolites thereof, or more than one of the following: In the two compounds of the following general formula (I), R1-R5 are each independently hydrogen, hydroxyl, methoxyl, ethoxyl, -O-glycosyl or -OAr; Ar is a substituted or unsubstituted phenyl group, and the substituent on the substituted phenyl group is preferably one or more of hydroxyl, methoxyl or acetyl; the glycosyl group is a monosaccharide group or one or more of a disaccharide group and a polysaccharide group formed by two or more monosaccharides; 2. The compound according to claim 1, characterized in that: R1, R3-R5 are selected from hydroxyl groups, R2 is selected from hydrogen, and the compound is one or two of the two structural compounds shown in formula (II):

3. A method for preparing the compound according to claim 2, characterized in that: The following steps are involved: (1) Extraction of medicinal materials: 30-50 kg of dried whole herb of Pyrola glabra was crushed into a coarse powder with a particle size of 10-100 mm, and 40-80 L of 50%-90% ethanol was added. The mixture was heated at 30-50° C. for 12-48 hours. The extract was filtered through a 70-80 mesh filter, and the filtrate was rotary evaporated at 30-50° C. to remove the solvent to obtain an extract. The obtained extract was dissolved in 1-3 L of distilled water, and then 1-5 L of petroleum ether was added for extraction, and impurities in the petroleum ether layer were removed. The remaining water layer was extracted with 1-5 L of ethyl acetate, and concentrated to obtain an ethyl acetate layer extract. (2) dissolving the ethyl acetate extract in step (1) with 10-30% methanol by volume, passing it through an MCI column in a methanol-water system, and eluting it with 2-5 column volumes of 10% methanol and then 30% methanol by volume, respectively, to obtain two fractions, GSWY-A and GSWY-B; (3) The GSWY-A and GSWY-B fractions in step (2) were combined, dry-loaded onto a 200-300 mesh silica gel column, and eluted with chloroform-methanol in a volume ratio of 50:1-10:1 for 3-5 column volumes to obtain a GSWY-AB-1 subfraction; (4) The GSWY-AB-1 component in step (3) was passed through a 200-300 mesh silica gel column, and step elution was performed with 2-5 column volumes of petroleum ether-acetone volume ratios of 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1, respectively, at a flow rate of 0.8-2 ml / min, and 37 sub-fractions, GSWY-AB-1-1 to GSWY-AB-1-37, were collected according to the ultraviolet absorption peak; (5) The fraction GSWY-AB-1-32 in step (4) was separated and purified in the first dimension using a C18HD reverse phase chromatography column at a flow rate of 60-80 ml / min, the mobile phase A was methanol, and B was formic acid-water with a volume concentration of 0.1% to 0.3%. The gradient elution conditions were: 0-50 min: 15-65% A (volume ratio, the same below), 50-60 min: 65-95% A, 60-70 min: 95% A, and the crude fraction F2 with a retention time of 30-50 min was collected according to the UV absorption chromatogram; (6) The subfraction F2 obtained in step (5) was subjected to second-dimensional separation and purification using a C18HD reverse phase chromatography column at a flow rate of 60-80 ml / min, mobile phase A was 0.1%-0.5% by volume formic acid-methanol, mobile phase B was 0.1%-0.3% by volume formic acid-water, and gradient elution conditions were: 0-60 min: 25-65% A, 60-61 min: 65-95% A, 61-75 min: 95% A, and 6 subfractions F2-1 to F2-6 were collected according to the UV absorption chromatographic peaks. The retention time of F2-2 was tR = 30-40 min; (7) The subfraction F2-2 obtained in step (6) was subjected to third-dimensional separation and purification using a QUIS reverse phase chromatography column at a flow rate of 3-5 ml / min, mobile phase A was methanol, mobile phase B was 0.1%-0.3% by volume formic acid-water, and the gradient elution conditions were: 0-30 min, 50-70% A; 30-32 min, 70-90% A; 32-45 min, 90% A, and 5 subfractions were collected according to the chromatographic peaks, namely F2-2-1 to F2-2-5, with a retention time tR of F2-2-1 of 10-15 min; (8) The subfraction F2-2-1 obtained in step (7) was subjected to fourth-dimensional separation and purification using an AH chiral chromatographic column at a flow rate of 10-20 ml / min, mobile phase A was formic acid-ethanol with a volume concentration of 0.1-0.5%, B was supercritical fluid CO2, and the gradient elution conditions were: 0-13 min: 22% A; compound 1 was collected according to the chromatographic peak, with a retention time of tR = 4.80-6.90 min, named Calvatine A; compound 2, with a retention time of tR = 7.60-10.90 min, named Calvatine B.

4. Use of one or more of the compound according to any one of claims 1 to 2, or its crystal form, or its isomer, or its glycoside, or its pharmaceutically acceptable salt, or its solvate, or its prodrug, or its metabolite in the preparation of a medicament for preventing and / or treating one or more diseases associated with muscarinic receptors, such as pain, chronic obstructive pulmonary disease, and irritable bowel syndrome.

5. A drug comprising a pharmaceutical composition comprising one or more of the compound according to any one of claims 1 to 2, or its crystal form, or its isomer, or its glycoside, or its pharmaceutically acceptable salt, or its solvate, or its prodrug, or its metabolites, and any other pharmaceutically acceptable excipients, carriers, diluents or other active ingredients.

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