Isoquinoline alkaloid compound and its preparation, application and composition

Abstract

The present invention relates to a novel isoquinoline alkaloid compound and a preparation method and use thereof. The novel compound (Corybungine A‑L) provided by the present invention is a novel isoquinoline alkaloid extracted and separated from Corydalis bungeana Turcz. in the genus Corydalis of the family Papaveraceae. Biological activity experiments show that the alkaloid compound has dopamine D2 receptor antagonist activity and can be used in the preparation of drugs for dopamine D2 receptor-related pain, schizophrenia, Lesch‑Nyhan syndrome and other nervous system diseases.

Description

Technical Field

[0001] The present invention belongs to the field of natural drug chemistry and relates to a new isoquinoline alkaloid compound. More specifically, it relates to a new isoquinoline compound (CorybungineA-L) and a preparation method thereof and its use as a dopamine D2 receptor active ingredient for the treatment of diseases related to the nervous system, such as pain, schizophrenia, Lesch-Nyhan syndrome and the like. In vitro activity shows that the above-mentioned compound has significant dopamine D2 receptor antagonist activity and can be used to develop new drugs or lead compounds for the treatment or inhibition of nervous system diseases. Background Art

[0002] Screening pharmacologically active lead compounds from natural products is an important approach to drug development. Natural products are characterized by structural complexity and diversity, and their toxic side effects are often small, making them a class of compounds with good drugability. According to statistics, about 50% of the 1,881 new drugs launched between 1981 and 2019 were directly or indirectly derived from natural products. According to literature reports, alkaloids account for about 50% of pharmacologically active natural products. Systematic separation and purification of this type of alkaloid-rich medicinal plants is conducive to the discovery of more potential highly active alkaloid compounds and provides guidance for the discovery of lead compounds and the development of new drugs.

[0003] Corydalis bungeana Turcz. is a commonly used medicinal material in traditional Chinese medicine and Mongolian medicine. It is the dried whole herb with roots of Corydalis bungeana, a plant of the genus Corydalis in the family Papaveraceae. Corydalis bungeana is bitter and cold in nature. It is often used to clear away heat and detoxify, dissipate stagnation and reduce swelling, and treat a variety of diseases such as colds and coughs. It has a good effect on rheumatism and myocarditis. According to literature reports, Corydalis bungeana contains flavonoids and alkaloid components. Alkaloids are the most diverse and abundant chemical components in Corydalis bungeana. So far, more than 30 alkaloids have been found in Corydalis bungeana, including corydalis, acetyl corydalis, protopine, dihydrosanguinarine and other components. Modern pharmacological studies have shown that Corydalis bungeana has a variety of pharmacological effects such as analgesia and anti-inflammatory, sedative and hypnotic, antiviral, antibacterial, immune suppression, and cytotoxicity.

[0004] Dopamine receptors are a type of G protein-coupled receptors. The dopamine receptors discovered so far can be divided into two major categories: D1 receptors, mainly including D1 and D5 receptors; D2 receptors, mainly including D2, D3 and D4 receptors. G protein-coupled dopamine receptors (D1, D2, D3, D4 and D5) mediate all physiological functions of the catecholamine neurotransmitter dopamine, ranging from voluntary movement and motivation to hormone regulation and hypertension. Dopamine D2 receptor antagonist activity has been reported to be associated with many neurological diseases, such as pain, schizophrenia, Lesch-Nyhan syndrome and other diseases. Modern pharmacological studies have shown that bitter herb has analgesic, anti-inflammatory, sedative and hypnotic effects. Therefore, it is of great research value to discover compounds with dopamine D2 receptor activity from bitter herb.

[0005] The six aporphenanthrene isoquinoline compounds (Corybungine AF), one protoberberine isoquinoline compound (Corybungine G) and five open-ring protoberberine isoquinoline compounds (Corybungine H-L) in the present invention are new compounds extracted and separated from Corybungine for the first time, which have not been reported in other natural sources, and there are no reports on the compounds and their pharmacological activities in existing literature. Pharmacological activity tests show that the compounds have significant dopamine D2 receptor antagonist activity and can be used in the preparation of drugs for treating pain, schizophrenia, Lesch-Nyhan syndrome and other diseases. Summary of the invention

[0006] The present invention provides a novel isoquinoline compound, or the compound in different crystal forms, or its chiral isomers, or its glycosides, or its pharmaceutically acceptable salts, or its solvates, or its prodrugs, or its metabolites, wherein the general structural formula (I) is as follows:

[0007]

[0008] Where R 1 -R 14 Each is independently hydrogen, chlorine, hydroxyl, methoxy, ethoxy, O-glycosyl or OAr; Ar is substituted or unsubstituted phenyl; and the glycosyl is a monosaccharide or various disaccharides and polysaccharides formed from monosaccharides.

[0009] Furthermore, the stereo configurations of compounds 1 to 6 are selected from one or two of 6aR and 6aS, respectively, and the stereo configuration of compound 7 is selected from one or two of 14R and 14S, respectively; R 1 -R 14wherein Ar is a substituted or unsubstituted phenyl group; the substituted phenyl group refers to a phenyl group optionally substituted by one or more substituents, wherein each substituent group can independently be hydroxyl, hydroxymethyl, methoxy or OAr'; the Ar' is a substituted or unsubstituted phenyl group; the substituted phenyl group Ar' refers to a phenyl group optionally substituted by one or more substituents, wherein each substituent group can independently be hydroxyl, hydroxymethyl, methoxy or ethoxy. R 1 -R 14 In the above, the sugar group refers to, but is not limited to, glucose, glucuronic acid, mannosyl, galactosyl, allosyl, fructosyl, sorbosyl, fusosyl, rhamnosyl, chinonosyl, arabinosyl, lyxosyl, xylosyl, ribosyl, and various disaccharides and polysaccharides formed by the above monosaccharides.

[0010]

[0011] Furthermore, the stereo configurations of compounds 1 to 4 and 6 are preferably 6aR, the stereo configuration of compound 5 is preferably 6aS, and the stereo configuration of compound 7 is preferably 14S; compounds 1 to 12 have the structures of the following formula (II) and are named Corybungine AL respectively.

[0012] The present invention also provides a method for preparing the above-mentioned compound (II), the steps of which are as follows: grinding the dried whole herb with roots of Corybungine to obtain medicinal powder, heating and extracting with 50% to 90% ethanol at 60 to 70°C for 6 to 24 hours, filtering the extract with an 80-mesh filter, and concentrating the extract under reduced pressure to obtain an extract; the extract is dissolved in 0.01 to 0.2 mol / L sulfuric acid water, adjusting the pH to 2 to 3, adding petroleum ether at a volume ratio of 1:1 to 1:2 to extract, and taking a water layer. The obtained water layer is adjusted to a pH of 9-10 with 0.01 to 2 mol / L sodium hydroxide, and dichloromethane is added at a volume ratio of 1:1 to 1:2 to extract, and a dichloromethane layer is obtained. The dichloromethane layer is separated by multi-step high-efficiency preparative liquid chromatography to obtain the compound Corybungine AL.

[0013] The invention conducts an activity test on dopamine D2 receptors on the obtained isoquinoline alkaloid compound, and uses Chinese hamster ovary cells (CHO) transfected with dopamine D2 receptors for the activity test. The results show that the compound exhibits low micromolar antagonistic activity on dopamine D2 receptors, and can become a potential lead compound for treating pain, schizophrenia, Lesch-Nyhan syndrome and other nervous system diseases.

[0014] In the present invention, the sugar group refers to, but is not limited to, glucose, glucuronic acid, mannosyl, galactosyl, allosyl, fructosyl, sorbosyl, fusosyl, rhamnosyl, chinonosyl, arabinosyl, lyxosyl, xylosyl, ribosyl, and various disaccharides and polysaccharides formed by the above monosaccharides.

[0015] The compounds of the present invention can be obtained by separation and purification from plants; they can also be synthesized by chemical methods well known to those skilled in the art.

[0016] 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.

[0017] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes may be made.

[0018] The new compound (Corybungine AL) provided by the present invention is a novel isoquinoline alkaloid extracted and separated from Corydalis bungeana Turcz. of the genus Corydalis of the family Papaveraceae. Biological activity experiments show that the alkaloid compound has dopamine D2 receptor antagonist activity and can be used in the preparation of drugs for dopamine D2 receptor-related pain, schizophrenia, Lesch-Nyhan syndrome and other nervous system diseases.

[0019] The present invention has the following advantages: the target compound is a novel isoquinoline alkaloid compound; the compound has significant dopamine D2 receptor antagonist activity and has application prospects in the development of drugs for central nervous system diseases that are currently receiving much attention, such as pain, schizophrenia, Lesch-Nyhan syndrome, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Compounds 1-12 1 H NMR and 13 C NMR spectrum, where a is the 1 HNMR and 13 CNMR spectrum, b is compound 2 1 HNMR and 13 CNMR spectrum, c is compound 3 1 HNMR and 13 CNMR spectrum, d is compound 4 1 HNMR and 13 CNMR spectrum, e is compound 51 HNMR and 13 CNMR spectrum, f is compound 6 1 HNMR and 13 CNMR spectrum, g is compound 7 1 HNMR and 13 CNMR spectrum, h is compound 8 1 HNMR and 13 CNMR spectrum, i is compound 9 1 HNMR and 13 CNMR spectrum, j is compound 10 1 HNMR and 13 CNMR spectrum, k is the 1 HNMR and 13 CNMR spectrum, l is compound 12 1 HNMR and 13 CNMR spectrum;

[0021] Figure 2 Compounds 1-12 1 H, 1 H-COSY and key HMBC;

[0022] Figure 3 Characterization of the antagonistic activity of compounds 1, 3, and 8 at D2 receptors;

[0023] Figure 4 Schematic diagram of the structure of compound 1-12 according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0024] The following examples are intended to illustrate the present invention rather than to further limit the present invention. The present invention can be implemented in any manner described in the present invention.

[0025] Preparation Example of the compound of formula (II) of the present invention:

[0026] Compound preparation and structure identification:

[0027] In the following preparation examples, the preparation system includes Waters Alliance, including e2695 separation unit, 2998 PDA detector, data processing is performed by empower 3; Waters AutoP automatic purification system, including 2545 separation unit, 2767 sample manager, 2489 dual wavelength detector; Novase HPLC industrial grade high pressure liquid phase preparative chromatography and HIPERSEP software. Reagents including chromatographic grade methanol and acetonitrile were purchased from Fisher Scientific (Loughborough, UK), chromatographic grade formic acid, acetic acid, ammonia, triethylamine, ammonium formate, sodium monohydrogen phosphate, sodium dihydrogen phosphate were purchased from J&K Technology (Hebei, China), laboratory water was from Milli-Q ultrapure water purification system (Billerica, MA, USA), and preparation grade methanol was purchased from Shanghai Xingke High Purity Solvent Co., Ltd. (Shanghai, China). Preparative columns and semi-preparative columns: C18HCE (15 μm, 100 mm × 250 mm), C18HCE (10 μm, 100 mm × 325 mm), C18CE (7 μm, 50 mm × 250 mm), FC8HL (3.5 μm, 30 mm × 150 mm) and C18HCE (5 μm, 10 mm × 150 mm) (Dalian Sipu Precision Industry Co., Ltd.).

[0028] The nuclear magnetic resonance spectrum used for structural identification was determined by Bruker AVIII-600 nuclear magnetic resonance spectrometer (Bruker, Germany), and the compounds were dissolved in deuterated methanol (MeOD). The mass spectrometry was performed by Agilent 1290 Infinity LC / 6540 Q-TOF MS liquid chromatography-mass spectrometry system for sample separation and analysis.

[0029] The preparation and compound identification steps are as follows:

[0030] (1) Extraction of medicinal materials: Take the dried whole herb with roots of Corydalis eryngii, and crush it to obtain a total of 100 kg of medicinal material powder. Use 1000 liters of 70% ethanol by volume and heat it at 70°C for 6 hours. After filtering the extract through an 80-mesh filter, remove the solvent by rotary evaporation to obtain an extract. The extract is dissolved in 0.01 mol / L sulfuric acid water, the pH is adjusted to 2, and petroleum ether is added in a 1:1 volume ratio to extract the water layer. The obtained water layer is adjusted to pH 10 with 2 mol / L sodium hydroxide, and dichloromethane is added in a 1:1 volume ratio to extract the water layer. The obtained dichloromethane layer is the crude alkali.

[0031] (2) The crude alkali obtained in step (1) was separated and purified in the first dimension using C18HCE (15um, 100×250mm, reverse phase chromatography column) at a flow rate of 320mL / min. The mobile phase A was formic acid-methanol (volume concentration 0.1%), and the mobile phase B was formic acid-water (volume concentration 0.1%). The gradient elution conditions were: 0-8min: 25%A (volume ratio, the same below), 8-23min: 40%A, 23-38min: 80%A, 38-55min: 100%A. The sample was taken from 4.2 to 26.0min and concentrated as a total alkaloid sample of Corydalis bispinosa.

[0032] (3) The total alkaloids of bitter herbs obtained in step (2) were separated and purified in the second dimension using C18HCE (10um, 100×325mm, reverse phase chromatography column) at a flow rate of 320mL / min. The mobile phase A was formic acid-methanol (volume concentration 0.1%), and the mobile phase B was formic acid-water (volume concentration 0.1%). The gradient elution conditions were: 0-5min: 20%A, 5-50min: 20-45%A, 50-60min: 95%A, and 11 sub-fractions F1-F11 were collected according to the chromatographic peaks.

[0033] (4) The sub-fraction F2 obtained in step (3) was separated and purified in the third dimension using C8CE (reverse phase chromatography column), the mobile phase A was 25% ammonia-methanol (volume concentration 0.1%), B was 25% ammonia-water (volume concentration 0.1%), the gradient elution conditions were 0-5 min: 5% A, 5-50 min: 5-95% A, 50-60 min: 95% A, and 14 sub-fractions F2-1 to F2-14 were obtained by collecting according to the chromatographic peaks.

[0034] (5) The sub-fraction F2-10 obtained in step (4) was separated and purified in the fourth dimension using FC8HL (reverse phase chromatography column), the mobile phase A was (20 mM) ammonium formate-95% methanol / water (volume concentration), B was (20 mM) ammonium formate-water, and the gradient elution conditions were 0-2.38 min: 50% A, 2.38-13.88 min: 50%-100% A, 13.88-32 min: 100% A, and 5 sub-fractions were collected according to the chromatographic peaks to obtain F2-10-1 to F2-10-5.

[0035] (6) The subfraction F2-10-1 obtained in step (5) was separated and purified in the fifth dimension by C18HCE (reverse phase chromatography column), the mobile phase A was formic acid-methanol (volume concentration 0.1%), B was formic acid-water (volume concentration 0.1%), the gradient elution conditions were 0-8 min: 40% A, 8-20 min: 55% A, and compound 4 (t R =8.0min), named Corybungine D.

[0036] (7) The sub-fraction F3 obtained in step (3) was separated and purified in the third dimension using C8CE (reverse phase chromatography column), the mobile phase A was 25% ammonia-methanol (volume concentration 0.1%), B was 25% ammonia-water (volume concentration 0.1%), the gradient elution conditions were 0-5 min: 5% A, 5-50 min: 5-95% A, 50-60 min: 95% A, and 12 sub-fractions F3-1 to F3-12 were obtained by collecting according to the chromatographic peaks.

[0037] (8) The sub-fraction F3-12 obtained in step (7) was separated and purified in the fourth dimension using FC8HL (reverse phase chromatography column), the mobile phase A was (20 mM) ammonium formate-95% methanol / water (volume concentration), B was (20 mM) ammonium formate-water, and the gradient elution conditions were 0-2.38 min: 50% A, 2.38-13.88 min: 50%-100% A, 13.88-32 min: 100% A, and 8 sub-fractions were collected according to the chromatographic peaks to obtain F3-12-1 to F3-12-8.

[0038] (9) The subfraction F3-12-5 obtained in step (8) was separated and purified in the fifth dimension by C18HCE (reverse phase chromatography column), the mobile phase A was formic acid-methanol (volume concentration 0.1%), B was formic acid-water (volume concentration 0.1%), the gradient elution condition was 0-15min: 29% A, and compound 5 (t R =8.5min), named CorybungineE.

[0039] (10) The sub-fraction F4 obtained in step (3) was separated and purified in the third dimension using C8CE (reverse phase chromatography column), the mobile phase A was 25% ammonia-methanol (volume concentration 0.1%), B was 25% ammonia-water (volume concentration 0.1%), the gradient elution conditions were 0-5 min: 5% A, 5-50 min, 5-95% A, 50-60 min: 95% A, and 15 sub-fractions F4-1 to F4-15 were collected according to the chromatographic peaks.

[0040] (11) The subfraction F4-13 obtained in step (10) was separated and purified in the fourth dimension using FC8HL (reverse phase chromatography column), the mobile phase A was (20 mM) ammonium formate-95% methanol / water (volume concentration), B was (20 mM) ammonium formate-water, and the gradient elution conditions were 0-2.38 min: 50% A, 2.38-13.88 min: 50%-100% A, 13.88-32 min: 100% A, and 10 subfractions were collected according to the chromatographic peaks, namely F4-13-1 to F4-13-10.

[0041] (12) The subfraction F4-13-4 obtained in step (11) was separated and purified in the fifth dimension by C18HCE (reverse phase chromatography column), the mobile phase A was formic acid-methanol (volume concentration 0.1%), B was formic acid-water (volume concentration 0.1%), the gradient elution conditions were 0-5 min: 25% A, 5-15 min: 25-45% A, 15-20 min: 45% A, and compound 3 (t R =10.0min)、7(t R =15.4min)、8(t R =13.6min)、9(t R =18.1min), named Corybungine C, G, H and I respectively.

[0042] (13) The subfraction F4-13-7 obtained in step (11) was separated and purified in the fifth dimension by C18HCE (reverse phase chromatography column), the mobile phase A was formic acid-methanol (volume concentration 0.1%), B was formic acid-water (volume concentration 0.1%), the gradient elution conditions were 0-12 min: 25% A, 12-15 min: 25-45% A, 15-20 min: 45% A, and the compound 1 (t R =18.1min) and 2(t R =18.9min), named Corybungine A and B respectively.

[0043] (14) The sub-fraction F6 obtained in step (3) was separated and purified in the third dimension using C8CE (reverse phase chromatography column), the mobile phase A was 25% ammonia-methanol (volume concentration 0.1%), B was 25% ammonia-water (volume concentration 0.1%), the gradient elution conditions were 0-5 min: 5% A, 5-50 min: 5-95% A, 50-60 min: 95% A, and 18 sub-fractions F6-1 to F6-18 were obtained by collecting according to the chromatographic peaks.

[0044] (15) The subfraction F6-8 obtained in step (14) was separated and purified in the fourth dimension using FC8HL (reverse phase chromatography column), the mobile phase A was (20 mM) ammonium formate-95% methanol / water (volume concentration), B was (20 mM) ammonium formate-water, and the gradient elution conditions were 0-2.38 min: 50% A, 2.38-13.88 min: 50%-100% A, 13.88-32 min: 100% A, and 15 subfractions were collected according to the chromatographic peaks, namely F6-8-1 to F6-8-15.

[0045] (16) The subfraction F6-8-11 obtained in step (15) was separated and purified in the fifth dimension by C18HCE (reverse phase chromatography column), the mobile phase A was formic acid-methanol (volume concentration 0.1%), B was formic acid-water (volume concentration 0.1%), the gradient elution condition was 0-15min: 24% A, and compound 11 (t R =12.8min), named Corybungine K.

[0046] (17) The sub-fraction F8 obtained in step (3) was separated and purified in the third dimension using C8CE (reverse phase chromatography column), the mobile phase A was 25% ammonia-methanol (volume concentration 0.1%), B was 25% ammonia-water (volume concentration 0.1%), the gradient elution conditions were 0-5 min: 5% A, 5-50 min: 5-95% A, 50-60 min: 95% A, and 14 sub-fractions F8-1 to F8-14 were obtained by collecting according to the chromatographic peaks.

[0047] (18) The subfraction F8-10 obtained in step (17) was separated and purified in the fourth dimension using FC8HL (reverse phase chromatography column), the mobile phase A was (20 mM) ammonium formate-95% methanol / water (volume concentration), B was (20 mM) ammonium formate-water, and the gradient elution conditions were 0-2.38 min: 50% A, 2.38-13.88 min: 50%-100% A, 13.88-32 min: 100% A. Seven subfractions were collected according to the chromatographic peaks, namely F8-10-1 to F8-10-7.

[0048] (19) The subfraction F8-10-1 obtained in step (18) was separated and purified in the fifth dimension by C18HCE (reverse phase chromatography column), the mobile phase A was formic acid-acetonitrile (volume concentration 0.1%), B was formic acid-water (volume concentration 0.1%), the gradient elution condition was 0-15min: 20% A, and compound 12 (t R =12.1min), named Corybungine L.

[0049] (20) The subfraction F8-11 obtained in step (17) was separated and purified in the fourth dimension using FC8HL (reverse phase chromatography column), the mobile phase A was (20 mM) ammonium formate-95% methanol / water (volume concentration), B was (20 mM) ammonium formate-water, and the gradient elution conditions were 0-2.38 min: 50% A, 2.38-13.88 min: 50%-100% A, 13.88-32 min: 100% A, and 8 subfractions were collected according to the chromatographic peaks to obtain F8-11-1 to F8-11-8.

[0050] (21) The subfraction F8-11-2 obtained in step (20) was separated and purified in the fifth dimension by C18HCE (reverse phase chromatography column), the mobile phase A was formic acid-acetonitrile (volume concentration 0.1%), B was formic acid-water (volume concentration 0.1%), the gradient elution condition was 0-10min: 45% A, and compound 10 (t R =6.4min), named Corybungine J.

[0051] (22) The subfraction F8-13 obtained in step (17) was subjected to fourth-dimensional separation and purification using FC8HL (reverse phase chromatography column), the mobile phase A was (20 mM) ammonium formate-95% methanol / water (volume concentration), B was (20 mM) ammonium formate-water, and the gradient elution conditions were 0-2.38 min: 50% A, 2.38-13.88 min: 50%-100% A, 13.88-32 min: 100% A, and 7 subfractions were collected according to the chromatographic peaks to obtain F8-13-1 to F8-13-7.

[0052] (23) The subfraction F8-13-2 obtained in step (22) was separated and purified in the fifth dimension by C18HCE (reverse phase chromatography column), the mobile phase A was formic acid-methanol (volume concentration 0.1%), B was formic acid-water (volume concentration 0.1%), the gradient elution condition was 0-10min: 30% A, and compound 6 (t R =4.6min), named Corybungine F.

[0053] (24) The above compounds have the following physicochemical properties and spectroscopic characteristics:

[0054] Corybungine A: brown powder; UV(MeOH)λ max (logε)305(4.11),283(4.22),218(4.46),202(4.50)nm; ECD(MeOH)λ max (Δε)λ 307 (+0.83),λ 275 (+0.35),λ 239 (-3.79),λ 217 (+1.45); HRESIMS m / z 418.1652[M+H] + (calcd for C 25 H 24 NO 3 ,418.1654).

[0055] Corybungine B: brown powder; UV (in MeOH) λ max (log ε) 307 (4.05), 281 (4.08), 221 (4.39), 202 (4.40) nm; ECD (in MeOH) λ max (Δε) λ 309 (+0.30), λ 279 (+0.20), λ 238 (-1.39), λ 212 HRESIMS m / z 418.1663 [M + H] + (calcd for C 25 H 24 NO 3 , 418.1654).

[0056] Corybungine C: Brown powder; UV (in MeOH) λ max (log ε) 325 (3.42), 265 (3.92), 207 (4.29) nm; ECD (in MeOH) λ max (Δε) λ 272 (+0.78), λ 232 (-3.64), λ 212 HRESIMS m / z 458.1801 [M + H] + (calcd for C 24 H 28 NO 8 , 458.1815).

[0057] Corybungine D: Black powder; UV (in MeOH) λ max (log ε) 317 (4.26), 263 (4.64), 205 (4.80) nm; ECD (in MeOH) λ max λ 233 (-0.52); HRESIMS m / z 446.1817 [M + H] + (calcd for C 23 H 28 NO 8 , 446.1815).

[0058] Corybungine E: Black powder; UV (in MeOH) λ max(logε) 308 (3.39), 267 (3.76), 223 (4.28) nm; ECD (MeOH) λ max (Δε) λ 269 (-0.80), λ 235 (+2.21); HRESIMS m / z 376.1310 [M+H] + (calcd for C 20 H 22 35 ClNO 4 , 376.1316); m / z 378.1286 [M+H] + (calcd for C 20 H 22 37 ClNO 4 , 378.1286).

[0059] Corybungine F: Black powder; UV (MeOH) λ max (logε) 317 (3.18), 279 (3.58), 216 (3.85) nm; ECD (MeOH) λ max (Δε) λ 276 (+0.38), λ 236 (-2.17), λ 212 (+0.92); HRESIMS m / z 328.1543 [M+H] + (calcd for C 19 H 22 NO 4 , 328.1549).

[0060] Corybungine G: Brown powder; UV (MeOH) λ max (logε) 285 (3.74), 204 (4.68) nm; ECD (MeOH) λ max (Δε) λ 207 (-1.70); HRESIMS m / z 464.2054 [M+H] + (calcd for C 27 H 30 NO 6 , 464.2073).

[0061] Corybungine H: Brown powder; UV (MeOH) λ max(logε)283(3.71),255(4.03),204(4.49)nm; HRESIMS m / z 462.1905[M] + (calcd for C 27 H 28 NO 6 + ,462.1911).

[0062] Corybungine I: brown powder; UV(MeOH)λ max (logε)283(4.14),254(4.52),210(4.61)nm; HRESIMS m / z554.2168[M] + (calcd for C 33 H 32 NO 7 + ,554.2173).

[0063] Corybungine J: brown powder; UV(MeOH)λ max (logε)293(3.58),253(3.82),220(4.23),210(4.22)nm; HRESIMS m / z 402.1549[M+H] + (calcd for C 21 H 24 NO 7 ,402.1553); m / z424.1368[M+Na] + (calcd for C 21 H 23 NNaO 7 ,424.1372).

[0064] Corybungine K: yellow powder; UV(MeOH)λ max (logε)312(3.58),257(4.29),210(4.30)nm; HRESIMS m / z366.0970[M] + (calcd for C 20 H 16 NO 6 + ,366.0972).

[0065] Corybungine L: yellow powder; UV(MeOH)λ max(logε)289(3.15),235(3.34),207(3.64)nm; HRESIMS m / z 416.1341[M+H] + (calcd for C 21 H 22 NO 8 ,416.1345).

[0066] Compounds 1-12 1 H-NMR and 13 The C-NMR data are shown in Tables 1, 2, 3, and 4. 1 H-NMR and 13 The C-NMR spectrum is as follows Figure 1 As shown, the key 2D NMR information of the compound is as follows Figure 2 shown.

[0067] Table 1 Compounds 1-6 1 H-NMR data (δ H , J in Hz, MeOD)

[0068]

[0069] Table 2 Compounds 1-6 13 C-NMR data (δ C , MeOD)

[0070] No. 1 2 3 4 5 6 1 144.0,C 144.2,C 145.0,C 147.2,C 144.6,C 143.4,C 1a 117.1,C 117.4,C 117.4,C 128.4,C 126.1,C 121.3,C 1b 123.0,C 123.8,C 124.7,C 129.2,C 128.2,C 122.2,C 2 149.5,C 149.5,C 149.6,C 152.0,C 153.4,C 147.0,C 3 107.8,CH 108.1,CH 108.6,CH 116.5,CH 113.1,CH 113.6,CH 3a 126.1,C 126.4,C 126.0,C 129.4,C 130.3,C 122.2,C 4 <![CDATA[27.7,CH 2 ]]> <![CDATA[28.0,CH 2 ]]> <![CDATA[27.7,CH 2 ]]> <![CDATA[30.1,CH 2 ]]> <![CDATA[29.0,CH 2 ]]> <![CDATA[27.1,CH 2 ]]> 5 <![CDATA[54.0,CH 2 ]]> <![CDATA[54.1,CH 2 ]]> <![CDATA[54.0,CH 2 ]]> <![CDATA[56.6,CH 2 ]]> <![CDATA[53.6,CH 2 ]]> <![CDATA[54.1,CH 2 ]]> 6a 63.4,CH 63.5,CH 63.1,CH 66.6,CH 63.6,CH 63.7,CH 7 <![CDATA[33.1,CH 2 ]]> <![CDATA[32.8,CH 2 ]]> <![CDATA[26.0,CH 2 ]]> <![CDATA[35.5,CH 2 ]]> <![CDATA[32.2,CH 2 ]]> <![CDATA[26.3,CH 2 ]]> 7a 130.8,C 126.4,C 123.9,C 137.1,C 122.5,C 128.3,C 8 114.0,CH 119.6,C 155.8,C 129.4,CH 144.0,C 146.5,C 9 152.3,C 148.1,C 118.0,CH 128.8,CH 150.8,C 152.9,C 10 146.5,C 151.1,C 129.1,CH 128.0,CH 113.7,CH 111.8,C 11 120.3,CH 113.2 123.1,CH 129.2,CH 127.3,C 126.3,C 11a 124.5,C 126.9,C 132.7,C 133.2,C 124.2,C 127.0,C <![CDATA[-OCH 2 The-]]> <![CDATA[102.6,CH 2 ]]> <![CDATA[102.7,CH 2 ]]> <![CDATA[102.7,CH 2 ]]> <![CDATA[OCH 3 -1]]> <![CDATA[61.2,CH 3 ]]> <![CDATA[62.4,CH 3 ]]> <![CDATA[OCH 3 -2]]> <![CDATA[56.5,CH 3 ]]> <![CDATA[OCH 3 -8]]> <![CDATA[61.2,CH 3 ]]> <![CDATA[OCH 3 -9]]> <![CDATA[56.5,CH 3 ]]> <![CDATA[56.9,CH 3 ]]> <![CDATA[56.2,CH 3 ]]> <![CDATA[OCH 3 -10]]> <![CDATA[56.7,CH 3 ]]> <![CDATA[N-CH 3 ]]> <![CDATA[42.0,CH 3 ]]> <![CDATA[42.3,CH 3 ]]> <![CDATA[42.1,CH 3 ]]> <![CDATA[43.4,CH 3 ]]> <![CDATA[41.8,CH 3 ]]> 1′ 152.1,C 151.2,C 103.7,CH 102.7,CH 2′ 119.3,CH 120.7,CH 74.9,CH 75.0,CH 3′ 116.9,CH 117.1,CH 78.1,CH 78.3,CH 4′ 153.9,C 154.7,C 71.4,CH 71.5,CH 5′ 116.9,CH 117.1,CH 78.3,CH 78.4,CH 6′ 119.3,CH 120.7,CH <![CDATA[62.5,CH 2 ]]> <![CDATA[62.6,CH 2 ]]>

[0071] Table 3 Compounds 7-12 1 H-NMR data (δ H , J in Hz, MeOD)

[0072]

[0073]

[0074] Note: * indicates the signal is obscured by the solvent.

[0075] Table 4 Compounds 7-12 13 C-NMR data (δ C , MeOD)

[0076]

[0077]

[0078] Note: * indicates the signal is obscured by the background. a Indicates that the signal is read out from the HMBC correlation.

[0079] Activity test example:

[0080] Cell culture and transfection experiments:

[0081] Chinese hamster ovary (CHO) cells were obtained from the National Collection of Authenticated Cell Cultures. CHO cells were cultured in F-12K medium supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO. 2 Cultivate under air conditions. The generation of CHO cells stably expressing dopamine D2 receptors was achieved using the standard lipofectamine method. The transfection of human D2 receptors into CHO cells was performed as follows: CHO cells were transfected with 8 μg of D2 plasmid mixed with 24 μL of lipofectamine2000 reagent (Invitrogen). 24 hours after transfection, the cells were cultured in 600 μg / mL zeocin for two weeks. Untransfected cells died during these periods and cell cloning was performed using complete medium containing 600 μg / mL zeocin. Stable clones (CHO-D2 cells) were cultured in modified F-12K medium supplemented with 10% fetal bovine serum (FBS) and 300 μg / mL Zoecin at 37°C and 5% CO 2 The cells were cultured under air conditions for another two weeks.

[0082] Dynamic mass redistribution assay (DMR):

[0083] The Epic system (Corning Inc.) is a wavelength interrogation readout system tailored for resonant waveguide grating (RWG) biosensors in microtiter plates for DMR assays. Whole-cell DMR experiments were performed by seeding CHO-D2 cells directly in Epic 384-well biosensor plates at 15,000 cells per well and incubating overnight to form a confluent monolayer in cell culture medium. The culture medium in the 384-well biosensor plate was then replaced with 30 μL Hank's balanced salt solution (1× HBSS) and then measured after further incubation in the system for 1 hour. The compounds were dissolved in dimethyl sulfoxide at a concentration of 100 mM, diluted to different concentrations with HBSS buffer, and then automatically added to the wells. At the same time, 6.25 nM dopamine was prepared as a D2 receptor agonist probe.

[0084] For the dopamine D2 dose activity assay of compounds 1, 3, and 8, a 2-minute baseline was first established. In the first step, the compounds were added in gradient dilutions, with the highest final concentration being 100 μM, 4-fold gradient dilutions, and 7 concentration points (100 μM, 25 μM, 6.25 μM, 1.563 μM, 0.391 μM, 0.098 μM, and 0.024 μM), and the DMR response triggered by the compounds within 1 hour was recorded. The second step of drug addition: re-establish the baseline, add a D2 agonist dopamine at a final concentration of 6.25 nM to each well, and then record the DMR response triggered by dopamine and the test compound within 1 hour. The results are shown in Figure 2. Figure 3 All ICs described in this invention 50 The values ​​were calculated based on the maximum DMR signal within 1 hour after compound stimulation. The DMR response induced by the assay buffer containing 0.1% dimethyl sulfoxide was used as a negative control. All DMR responses were background corrected.

[0085] Table 5 Dopamine D2 receptor antagonist activity data of compounds

[0086]

[0087] The dopamine D2 receptor antagonist activity data of corybungine A, C, and H are shown in Table 5. The test results show that the three compounds all caused dopamine desensitization, indicating that they have relatively obvious D2 receptor antagonist activity. Therefore, the compounds of the present invention can be used to develop lead compounds for treating pain, schizophrenia, Lesch-Nyhan syndrome, and other nervous system diseases.

Claims

1. A method for preparing an isoquinoline alkaloid compound, Features: Compounds 1 and 3 have the following structures: ； ； The following steps are involved: (1) Extraction of medicinal materials: Take the dried whole herb with roots of Corydalis eryngii, crush it to obtain medicinal material powder; use 50% to 90% ethanol by volume to heat and extract at 60 to 70°C for 6 to 24 hours, filter the extract with a 70-80 mesh filter, and then remove the solvent by rotary evaporation to obtain an extract; dissolve the extract in 0.01 to 0.2 mol / L sulfuric acid water, adjust the pH to 2 to 3, add petroleum ether in a volume ratio of 1:1 to 1:2 to extract, and take the water layer; adjust the pH of the obtained water layer to 9 to 10 with 0.01 to 2 mol / L sodium hydroxide, add dichloromethane in a volume ratio of 1:1 to 1:2 to extract, and the obtained dichloromethane layer is the crude alkali; (2) The crude alkali obtained in step (1) is separated and purified in the first dimension using a C18HCE reverse phase chromatography column, wherein the mobile phase A is formic acid-methanol with a volume concentration of 0.1% to 2%, and the mobile phase B is formic acid-water with a volume concentration of 0.1% to 2%. The gradient elution conditions are: 0 to 8 min: 25% A, 8 to 23 min: 40% A, 23 to 38 min: 80% A, 38 to 55 min: 100% A, and the sample is taken from 4.2 to 26 min, and concentrated as a sample of total alkaloids of berberine; (3) The total alkaloids of bitter herb obtained in step (2) were separated and purified in the second dimension using a C18HCE reverse phase chromatography column, wherein the mobile phase A was formic acid-methanol with a volume concentration of 0.1% to 2%, and the mobile phase B was formic acid-water with a volume concentration of 0.1% to 2%. The gradient elution conditions were: 0 to 5 min: 20% A, 5 to 50 min: 20 to 45% A, 50 to 60 min: 95% A, and 11 sub-fractions F1 to F11 were collected according to the chromatographic peaks; (4) The sub-fraction F4 obtained in step (3) was subjected to third-dimensional separation and purification using a C8CE reverse phase chromatographic column, wherein the mobile phase A was 0.1% to 2% ammonia-methanol, wherein the mass concentration of ammonia was 20-25%; and the mobile phase B was 0.1% to 2% ammonia-water, wherein the mass concentration of ammonia was 20-25%. The gradient elution conditions were 0 to 5 min: 5% A, 5 to 50 min: 5 to 95% A, and 50 to 60 min: 95% A. Fifteen sub-fractions F4-1 to F4-15 were obtained by collecting the chromatographic peaks. (5) The sub-fraction F4-13 obtained in step (4) was separated and purified in the fourth dimension using an FC8HL reverse phase chromatography column, wherein the mobile phase A was 5-50 mM ammonium formate-95% volume concentration methanol / water, and the mobile phase B was 5-50 mM ammonium formate-water. The gradient elution conditions were 0-2.38 min: 50% A, 2.38-13.88 min: 50%-100% A, and 13.88-32 min: 100% A. Ten sub-fractions were collected according to the chromatographic peaks, namely F4-13-1 to F4-13-10. (6) The subfraction F4-13-4 obtained in step (5) was separated and purified in the fifth dimension by a C18HCE reverse phase column, wherein the mobile phase A was formic acid-methanol with a volume concentration of 0.1% to 2%, and the mobile phase B was formic acid-water with a volume concentration of 0.1% to 2%. The gradient elution conditions were 0 to 5 min: 25% A, 5 to 15 min: 25 to 45% A, and 15 to 20 min: 45% A. Compound 3 was obtained by collecting the chromatographic peaks. t R =9.40~10.56min, named Corybungine C; (7) The subfraction F4-13-7 obtained in step (6) was separated and purified in the fifth dimension by a C18HCE reverse phase column, wherein the mobile phase A was formic acid-methanol with a volume concentration of 0.1% to 2%, and the mobile phase B was formic acid-water with a volume concentration of 0.1% to 2%. The gradient elution conditions were 0 to 12 min: 25% A, 12 to 15 min: 25 to 45% A, and 15 to 20 min: 45% A. Compound 1 was obtained by collecting the chromatographic peaks. t R =17.74~18.54min, named Corybungine A.

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