Isoquinoline alkaloid compounds and their preparation and use

Abstract

The present invention provides an isoquinoline alkaloid compound selected from the compounds represented by the following structural formulas, and a method for producing the compound and its application. The present invention relates to a compound that is capable of suppressing the production of isoquinoline alkaloids by suppressing the production of isoquinoline alkaloids in the nervous system, the cardiovascular system, and the pancreas. K We have demonstrated for the first time that Kv2.1 and TRSK channel inhibitors are inhibitors of potassium currents and background potassium currents. In particular, inhibitors of Kv2.1 and TRSK channels can be used to treat diseases such as stroke, arrhythmia, atrial fibrillation, diabetes, pain, respiratory depression, and depression. TIFF2024522236000010.tif31162

Description

[Technical field]

[0001] The present invention relates to the field of isolating effective ingredients from plants and the field of medical and health care, and more particularly to effective ingredients from Zanthoxylum chinense, their isolation and use, and more particularly to isoquinoline alkaloid compounds and their preparation and use. [Background technology]

[0002] Alkaloids are the main active substances in Zanthoxylum genus plants, and are considered to be the characteristic components that have analgesic and anti-inflammatory activities and bring about a unique tingling sensation. These alkaloids are classified into four types according to their mother nucleus: quinoline derivatives, isoquinoline derivatives, benzophenanthridine derivatives, and quinolone derivatives. Among them, isoquinoline alkaloids are the characteristic components due to their significant pharmacological activity.

[0003] Voltage-gated potassium channels (Kv) have been discovered so far, with 12 subfamilies, Kv1.x~Kv12.x, and approximately 80 family members. Among them, Kv5, Kv6, Kv8, and Kv9 subfamily members cannot form functional channels by themselves, and are called silent Kv subunits (KvS). KvS channels can form functional heteromeric channels with Kv2.1 channels, and can regulate the current width, activation and inactivation voltages, and kinetic characteristics of Kv2.1 channels. Current research has shown that Kv2.1 channels regulate delayed inward rectifier potassium currents (DIRs) in primary tissues and cells, such as dorsal ganglion neurons (DRGs), trigeminal ganglion neurons (TRGs), pancreatic islets, hippocampus, and cardiovascular system. KFor example, in cultured small diameter DRG neurons, Kv2.1 and Kv2.1 / KvS channels account for approximately 60% of the total, whereas Kv1.x and Kv3.x channels account for approximately 40% (Bocksteins, E. et al. Am J Physiol Cell Physiol.296(6): C1271-8, 2009).

[0004] I K Potassium currents and their major molecular basis, the Kv2.1 channel, play important roles in normal physiology and disease states in the human body. 2+ Among the pathways promoting neuronal death after stroke induced by I, overexpression of Kv2.1 on the neuronal membrane surface causes K+ extrusion, exacerbating neuronal death, and inhibition of Kv2.1 can exert neuroprotective and stroke therapeutic effects (Int J Mol Sci. 21(17):6107, 2020). In the circulatory system, I K Currents mediate the repolarization of cardiac action potentials and are involved in the regulation of heart rate and blood pressure (Madeja, M. et al. J Biol Chem. 285(44): 33898-905, 2010). In pancreatic islets, elevated blood glucose stimulates depolarization of the islet β-cell membrane potential, and the hyperpolarized membrane potential of activated Kv2.1 channels reduces neuronal excitability and decreases insulin secretion. Inhibition of Kv2.1 increases the duration and width of islet β-cell action potentials, further increasing insulin secretion and lowering blood glucose levels (Jacobson, DA et al. Cell Metab. 6(3):229-35, 2007). IB4-negative small-diameter DRG neurons are mainly I KIt is a current that hyperpolarizes the resting membrane potential, increases the width and duration of the after hyperpolarization potential (AHP), elevates the discharge threshold, and prolongs the action potential duration (Vydyanathan, A. et al. J Neurophysiol. 93(6): 3401-9, 2005). During repetitive stimulation discharges of hippocampal and trigeminal ganglion neurons, the regulatory effect of Kv2.1 on neuronal excitation depends on the stimulation schedule, inhibiting Kv2.1 channels to increase the firing frequency in the early phase and to suppress the firing frequency in the late phase (Liu PW et al. J Neurosci. 34(14): 4991-5002, 2014). Thus, it is important to consider whether Kv2.1 or I K Inhibiting the current induces a capsaicin-like nerve excitatory desensitization effect and exerts analgesic effects (Arora, V. et al. Pharmacol Ther. 220:107743, 2021). K By inhibiting the electrical current or the Kv2.1 channel, diseases such as stroke, arrhythmia, diabetes, and pain can be alleviated or treated.

[0005] Two-pore domain potassium channels (K2P) play an important role in maintaining the resting membrane potential of cells through background potassium ion leak currents and in generating and releasing the regulating action potential. Currently, 15 K2P channels have been cloned in mammals, and TWI K , TREK, TASK, TALK, THI K, and TRESK. Alpha-hydroxysanshool, isolated from Japanese pepper, inhibits the TRSK (KCNK18) and TASK1 / TASK3 (KCNK3 / KCNK9) channels in two-pore potassium channel DRG and TRG neurons, respectively, promoting the emission of small and large diameter neurons and causing numbness (Bautista DM, et al. Nat Neurosci. 11(7): 772-9, 2008). In addition, antagonists of TRSK and TASK1 / TASK3 channels are thought to have the effect of treating atrial fibrillation, respiratory depression and depression (Mathie, A. et al., Annu Rev Pharmacol Toxicol. 61: 401-420, 2021).

[0006] Although many active ingredients, including isoquinoline derivatives, have been isolated from Japanese pepper, the mechanism of action of these active ingredients is generally unknown. In this field, active molecules with novel structures, clear mechanisms, and specific types of applicable diseases are also needed. Summary of the Invention

[0007] The present inventors have discovered and disclosed three isoquinoline alkaloid compounds extracted from Japanese pepper that have not been reported in the literature. After in-depth research, it was found that these isoquinoline alkaloid compounds are involved in the regulation of the nervous system, cardiovascular system, and pancreas I K It was the first time that this isoquinoline alkaloid compound was demonstrated to be an inhibitor of potassium current, especially an inhibitor of the Kv2.1 channel. This isoquinoline alkaloid compound also has an inhibitory effect on the TRSK potassium channel, and is more active than the previously reported α-hydroxysanshool. Based on this, the present invention demonstrated that this isoquinoline derivative has analgesic activity in vivo using a typical inflammatory pain animal model.

[0008] Accordingly, it is an object of the present invention to provide isoquinoline alkaloid compounds.

[0009] Another object of the present invention is to provide a process for producing the above isoquinoline alkaloid compounds.

[0010] Another object of the present invention is to provide uses of the above isoquinoline alkaloid compounds.

[0011] In one aspect, the present invention provides an isoquinoline alkaloid compound selected from the group consisting of compounds represented by the following structural formulas: [ka]

[0012] In another aspect, the present invention provides a method for producing the isoquinoline alkaloid compound, comprising the steps of:

[0013] (1) crushing dried Japanese pepper, infusing it with a solvent, and concentrating the extract to obtain a Japanese pepper concentrated extract;

[0014] (2) dissolving the concentrated extract of Japanese pepper in water, and extracting it with petroleum ether, ethyl acetate, and n-butanol to obtain a petroleum ether phase, an ethyl acetate phase, an n-butanol phase, and an aqueous phase;

[0015] (3) The ethyl acetate phase was subjected to gradient elution on a 200-300 mesh silica gel column using a petroleum ether-acetone mixture as an eluent, with the volume ratios of petroleum ether:acetone being 50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 2:1, and 1:1, respectively, to obtain six fractions, Fr.1-Fr.6, of which Fr.4 was subjected to gradient elution on a reversed-phase silica gel C18 column using a water-methanol mixture as an eluent, with the volume ratios of water:methanol being 1:1, 1:2, and 1:4, to obtain eight fractions, Fr.4-1-Fr.4-8; fraction Fr.4-6 was subjected to Megrez Eluting the C18 column with a water-methanol mixture as an eluent, among which 90% volume fraction of methanol / water is used to obtain compound HJ-68; and 80% volume fraction of methanol / water is used to obtain HJ-69 and HJ-70, respectively; The present invention provides a method of manufacturing a semiconductor device, comprising:

[0016] In the above step (3), for example, six fractions Fr.1 to Fr.6 are produced by receiving the eluent in a 500 mL beaker, sequentially obtaining a series of eluents, evaporating 500 mL of the solution per beaker, and transferring the solution to a sample bottle after evaporation; when the elution of the ethyl acetate phase is completed, spotting the plate with thin layer chromatography, and merging samples of the same components into the same fraction, thereby sequentially obtaining the six fractions Fr.1 to Fr.6. However, the present invention is not limited to these.

[0017] In the above step (3), for example, the eight fractions Fr.4-1 to Fr.4-8 are produced by receiving the eluent in a 50 mL graduated test tube, sequentially obtaining a series of eluents, evaporating 50 mL of the solution per test tube, and transferring the solution to a sample bottle after evaporation; when the elution is completed, spotting the plate with a reversed-phase high-performance thin-layer chromatography plate, and merging samples of the same components into the same fraction, thereby sequentially obtaining the eight fractions Fr.4-1 to Fr.4-8. However, the present invention is not limited to these.

[0018] The three compounds isolated from Japanese pepper, HJ-68, HJ-69, and HJ-70, are all isoquinoalkaloids, and their structural formulas are as follows: [ka]

[0019] In step (1) of the production method of the present invention, the solvent may be one or a mixed solvent of two or more selected from water, methanol, ethanol, acetone, and dichloromethane, and examples thereof include, but are not limited to, alcohols, such as 70% aqueous ethanol solution, ethanol, 70% aqueous methanol solution, methanol, acetone, dichloromethane, or combinations thereof.

[0020] In step (1) of the production method of the present invention, the leaching may be room temperature immersion or heating reflux extraction. The leaching may be carried out one or more times. It is preferable to carry out immersion extraction with a solvent three times at room temperature for 5 to 7 days each time, or to carry out heating reflux extraction three times for 1 to 2 hours each time.

[0021] In step (3) of the production method of the present invention, the liquid phase separation conditions for compound HJ-68 are preferably as follows: methanol / water=90 / 10 (v / v) as the mobile phase, the flow rate is 4 mL / min; the column model is Megres C18 column, and the column length×diameter is 250 mm×20 mm.

[0022] In step (3) of the production method of the present invention, the liquid phase separation conditions for compound HJ-69 are preferably as follows: methanol / water=80 / 20 (v / v) as the mobile phase, the flow rate is 4 mL / min; the column model is Megres C18 column, and the column length×diameter is 250 mm×20 mm.

[0023] In step (3) of the production method of the present invention, the liquid phase separation conditions for compound HJ-70 are preferably as follows: methanol / water=80 / 20 (v / v) as the mobile phase, and the flow rate is 4 mL / min; the column model is Megres C18 column, and the column length×diameter is 250 mm×20 mm.

[0024] The present invention relates to a method for the treatment of DRG neurons in which the isoquinoline alkaloid compounds are effective in activating delayed rectifier potassium currents (I K ) and inhibited the sodium current and the instantaneous outward potassium current (I) of DRG neurons. A ) was shown for the first time to have no significant effect.

[0025] The present invention further relates to the isoquinoline alkaloid compound I K As an antagonist of the Kv2.1 potassium channel, which is the main molecular basis of the I K It was demonstrated that it has inhibitory activity equivalent to that of the current.

[0026] Thus, in yet another aspect, the present invention provides a method for measuring delayed rectifier potassium current (I K ), in particular the use of the isoquinoline compounds in the manufacture of antagonists of the Kv2.1 channel.

[0027] Specifically, the present invention relates to K The present invention provides the use of the above isoquino alkaloid compounds as antagonists of electrical currents, particularly the Kv2.1 channel, and thus the above isoquino alkaloid compounds are useful as drugs for alleviating or treating diseases such as stroke, arrhythmia, diabetes, and pain, particularly pain.

[0028] In addition, the present invention is the first to disclose the above-mentioned isoquinoalkaloid compounds as antagonists of K2P two-hole potassium channels, particularly TRSK channels, and the present invention has confirmed that the inhibitory activity of the above-mentioned isoquinoalkaloid compounds against K2P channels is stronger than that of the reported α-hydroxysanshool.

[0029] Thus, in a further aspect, the present invention provides the use of an isoquinoline compound as defined above in the manufacture of an antagonist of a K2P two-pore potassium channel, in particular a TRSK channel.

[0030] Specifically, the present invention provides the use of the above isoquinoalkaloid compounds as antagonists of K2P two-pore potassium channels, in particular TRSK channels, and thus the above isoquinoalkaloid compounds are useful as drugs for alleviating or treating diseases such as, for example, atrial fibrillation, respiratory depression and / or depression.

[0031] Thus, in a further aspect, the present invention provides the use of an isoquinoline compound as defined above in the manufacture of a medicament for the treatment of stroke, arrhythmia, diabetes, pain, atrial fibrillation, respiratory depression and / or depressive disorders. [Brief description of the drawings]

[0032] [Figure 1]The effect of HJ-70 on the frequency of spontaneous discharges in hippocampal neurons and DRG evoked discharges is shown. Here, A is the effect of 10uM HJ-70 on the frequency of spontaneous discharges in hippocampal neurons. B is a statistical diagram of the effect of 10uM HJ-70 on the frequency of spontaneous discharges in hippocampal neurons. C is the effect of 10uM HJ-70 on the frequency of evoked discharges in DRG neurons. D is a statistical diagram of the effect of 10uM HJ-70 on the frequency of evoked discharges in DRG neurons. ***P≦0.001.

[0033] [Diagram 2] The effect of HJ-70 on the potassium ion channel current on DRG neurons. Here, A is the effect of the control external solution on the potassium ion channel in DRG neurons. B is the effect of 3uM HJ-70 on the potassium ion channel in DRG neurons. C is the effect of 10uM HJ-70 on the potassium ion channel in DRG neurons. D is the result of dissolving the drug into the potassium ion channel of DRG neurons. E is a statistical diagram of the inhibition of the potassium ion channel of DRG neurons by 3uM HJ-70. F is a statistical diagram of the inhibition of the potassium ion channel of DRG neurons by 10uM HJ-70. G is a statistical diagram of the effect of dissolving the drug into the potassium ion channel of DRG neurons. ** represents P≦0.01, *** represents P≦0.001.

[0034] [Diagram 3]The effect of HJ-70 on the potassium ion channel current on hippocampal neurons is shown. Here, A is the effect of 1uM HJ-70 on the potassium ion channel in hippocampal neurons. B is the effect of 3uM HJ-70 on the potassium ion channel in hippocampal neurons. C is the effect of 10uM HJ-70 on the potassium ion channel in hippocampal neurons. D is the effect of the positive drug 5mM TEA on the potassium ion channel in hippocampal neurons. E is the statistical diagram of the effect of 1uM HJ-70 on the potassium ion channel in hippocampal neurons. F is the statistical diagram of the effect of 3uM HJ-70 on the potassium ion channel in hippocampal neurons. G is the statistical diagram of the effect of 10uM HJ-70 on the potassium ion channel in hippocampal neurons. H is the statistical diagram of the effect of the positive drug 5mM TEA on the potassium ion channel in hippocampal neurons. ns represents no statistical difference, ** represents P ≤ 0.01, and *** represents P ≤ 0.001.

[0035] [Figure 4] The effect of HJ-70 on sodium ion channel current in hippocampal neurons is shown by voltage clamping at -90mV. Here, A is the effect of 10uM HJ-70 on sodium ion channels in hippocampal neurons. B is the statistical effect of 10uM HJ-70 on sodium ion channel inhibition in hippocampal neurons. *** indicates P≦0.001.

[0036] [Diagram 5] The effect of HJ-70 on the instantaneous voltage-gated potassium ion channel of CHO cells is shown. A is a graph showing the current effect of 10uM HJ-70 on the Kv2.1 ion channel. B is a statistical graph showing the effect of 10uM HJ-70 on the Kv2.1 ion channel. C is a graph showing the current effect of 10uM HJ-70 on the TRSK ion channel. D is a statistical graph showing the effect of 10uM HJ-70 on the TRSK ion channel. *** indicates P≦0.001.

[0037] [Figure 6] 1 shows the effect of HJ-70 on formalin-induced inflammatory pain behavior-dependent reduction. Here, A is the alleviating effect of 100 mg / kg HJ-70 on formalin pain behavior. B is the alleviating effect of 50 mg / kg HJ-70 on formalin model pain behavior. C is the alleviating effect of 30 mg / kg HJ-70 on formalin model pain behavior. D is a statistical diagram of inflammatory pain licking time and pain behavior score in the first phase (0-10 min) of the formalin model by HJ-70. E is a statistical diagram of inflammatory pain licking time and pain behavior score in the second phase (10-60 min) of the formalin model by HJ-70. * indicates P≦0.05, ** indicates P≦0.01, and *** indicates P≦0.001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The essence and effects of the present invention will be further explained below based on examples. However, these examples are for the purpose of explaining the present invention and are not intended to limit the present invention.

[0039] Working Example

[0040] The experimental methods used in the following examples are conventional unless otherwise noted.

[0041] Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

[0042] Extract sample: Japanese pepper.

[0043] Leaching, extraction solvents and solvents for separating purified compounds: ethanol, water, petroleum ether, ethyl acetate, n-butanol.

[0044] Equipment: Extraction tank (homemade), Tokyo Rika 20L rotary evaporator (Tokyo Rika, Japan), R-210 rotary evaporator (BUCHI, Switzerland), analytical high-performance liquid chromatograph with a maximum flow rate of 10mL (Jiangsu Hanbang Technology Co., Ltd.), Bruker Avance III-400 nuclear magnetic resonance spectrometer (Bruker, Germany), Bruker microTOF-Q II high-resolution mass spectrometer (Bruker, Germany), IFS120HR 670 FT-IR infrared spectrometer (Bruker, Germany), Lambda 35 UV-Vis spectrophotometer (Perkin Elmer, USA).

[0045] Example 1: Extraction method of active ingredients from Japanese pepper

[0046] 1.1 Dried Japanese pepper was crushed and extracted with 10 times its mass of 70% ethanol at room temperature for 3 times. The extracts were combined and concentrated until they no longer had an alcoholic taste, to obtain a concentrated Japanese pepper extract.

[0047] The concentrated extract of Japanese pepper was dissolved in water and extracted with petroleum ether, ethyl acetate, and n-butanol, respectively, to obtain a petroleum ether phase, an ethyl acetate phase, an n-butanol phase, and an aqueous phase.

[0048] The ethyl acetate phase was gradient eluted with a petroleum ether-acetone mixture as an eluent on a 200-300 mesh silica gel column (volume ratios of petroleum ether to acetone in the mixture were 50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 2:1, and 1:1, respectively), the eluent was received in a 500 mL beaker, and 500 mL of solution per beaker was evaporated and transferred to a sample bottle after evaporation. After the elution of the ethyl acetate phase was completed, the plate was spotted by thin layer chromatography, and samples of the same components were merged into the same fraction to obtain a total of six fractions, Fr.1 to Fr.6.

[0049] Among them, F.4 was subjected to gradient elution on a reversed-phase silica gel C18 column using a water-methanol mixture as the eluent, with the volume ratios of water:methanol being 1:1, 1:2, and 1:4, respectively, and finally the column was run with methanol, the eluent was received in a 50 mL graduated test tube, 50 mL of solution per test tube was evaporated, and after evaporation, it was transferred to a sample bottle. After the elution was completed, the plate was spotted on a reversed-phase high-performance thin-layer chromatography plate, and samples with the same components were merged into the same fraction, thereby obtaining a total of eight fractions, F.4-1 to F.4-8.

[0050] Fractions Fr.4-6 were separated using an analytical liquid phase with a maximum flow rate of 10 mL / min, a 10 mm x 250 mm column (model number Megres C18), and a mobile phase flow rate of 4 mL / min to obtain compounds HJ-68 (mobile phase: 90% methanol / water by volume, peak out time: 5.4 min); HJ-69 (mobile phase: 80% methanol / water by volume, peak out time: 10.1 min); and HJ-70 (mobile phase: 80% methanol / water by volume, peak out time: 12.0 min).

[0051] The experimental data of the compound N-13-isobutyl rutecarpine (HJ-68) are as follows: Yellow needle crystals; Infrared spectrum IR (film) ν max 2926, 255 2858, 1728, 1669, 1637, 1587, 1465, 1292, 1130 cm -1 ;Ultraviolet spectrum UV(CH 3 OH) λ max (log ε) 216 (3.06), 234 (2.99), 329 (2.99), 345 (3.05), 362 (2.95) nm; proton magnetic resonance 1 H and carbon magnetic resonance 13 C NMR shown in Table 1; High resolution mass spectrometry HRESIMS m / z 344.1764 [M+H] + (C 22 H 22 N 3 Calculated value for O, 344.1757).

[0052] The experimental data of the compound N-13-methylpropyl ether rutecarpine (HJ-69) are as follows: Yellow needle crystals; Infrared spectrum IR (film) ν max 2924, 2872, 1662, 1585, 1534, 1465, 1332, 1203, 1114 cm -1 ;Ultraviolet spectrum UV(CH 3 OH) λ max (log ε) 215 (3.34), 235 (3.28), 328 (3.28), 344 (3.44), 362 (3.24) nm; proton magnetic resonance 1 H and carbon magnetic resonance 13 C NMR shown in Table 1; High Resolution Mass Spectroscopy HRESIMS m / z 382.1530 [M + Na] + (C 22 H 21 N 3 O 2 Calculated value for Na, 382.1526).

[0053] The experimental data of the compound N-13-n-propanol rutecarpine (HJ-70) are as follows: Yellow needle crystals; Infrared spectrum IR (film) ν max 3430, 2925, 2857, 1729, 1665, 1586, 1466, 1291, 1048 cm -1 ;Ultraviolet spectrum UV(CH 3 OH) λ max (log ε) 216 (3.33), 235 (3.26), 329 (3.24), 344 (3.29), 361 (3.18) nm; proton magnetic resonance 1 H and carbon magnetic resonance 13 C NMR shown in Table 1; High resolution mass spectrometry HRESIMS m / z 346.1542 [M+H] + (C 21 H 20 N 3 O 2 Calculated value for, 346.1550). [Table 1]

[0054] Example 2: Effects of HJ-70 on dorsal root ganglion action potential and potassium ions

[0055] 2.1 Isolation and culture of dorsal root ganglion: 4-6 week old C57BL / 6 mice (Shanghai Slake Laboratory Animal Co., Ltd.) were collected for acute isolation of dorsal root ganglion neuron cells. First, the mice were killed by decapitation, the surface skin was disinfected with 75% alcohol, the neck skin incision was cut to the buttocks, the spinal column from the neck to the waist was cut, excess muscle and blood clots were removed, and the spinal column was placed in pre-cooled phosphate buffer PBS (Hyclone), the spinal column was cut vertically along the midline, the white spinal cord and blood clots in the middle were removed with ophthalmic tweezers, the intervertebral foramen was exposed, and the dorsal root nerve node in it was taken out with fine tweezers, and the fiber connection was peeled off to the large node in the waist. The ganglia were then microdissected and digested in a mixture of 1 ml of collagenase (1 mg / mL) and trypsin (0.25 mg / mL) (Sigma-Aldrich) for 15 min. Digestion was then stopped and diluted with DMEM / F12 medium (Gibco) containing 10% fetal bovine serum. The cells were repeatedly blown off and large tissue chunks were filtered through a 70 μM cell filter. The remaining liquid was inoculated onto a glass plate previously coated with polylysine (Sigma-Aldrich) and incubated at 4°C for 15 min in 5% CO. 2 The cells were cultured in an incubator at 37°C.

[0056] 2.2 Recording of action potential and potassium current of dorsal root neurons: Whole-cell patch clamp recording was performed using a digital-to-analog converter Digidata 1440A Axon CNS and a patch clamp amplifier axonpatch700b (Molecular Devices). The borosilicate glass electrodes used in the manual patch clamp experiments were manufactured using a Sutter-P1000 electrode manufacturing system (Sutter). The electrode infusion resistance was about 3 MΩ, and the perfusion rate was about 2 mL / min. For the action potential recording, the cells were clamped at 0 pA, and action potential-evoked discharge recording was performed with a current stimulus of 200 pA injection intensity and 500 ms duration. Electrophysiological experiment recording of potassium current of neurons: The cells were clamped at a voltage of -50 mV, hyperpolarized to -110 mV for 600 ms, and then directly depolarized to 40 mV to record whole-cell potassium current (I Total ) was recorded; when the cells were first depolarized to -50 mV, maintained for 50 ms, and then depolarized to 40 mV, a delayed rectifier potassium current (I K The efficacy of HJ-70 was detected by perfusion of 10 μM, and 5 mM TEA was administered as a positive control. [Table 2] [Table 3]

[0057] 2.3 Acute isolation of hippocampal neurons: 9 mg of protease (type XXIII, Sigma), 5 mg of bovine serum albumin (BSA, Shanghai Biotechnology Co., Ltd.) and trypsin inhibitor (Sigma-Aldrich Co., Ltd.) were taken in advance, and 3 mg / mL and 5 mg / mL of enzyme and stop solution were prepared in the digestion solution, respectively, and oxygenated in a water bath at 32 °C. Two 1-7 day old SD rat mice (Shanghai Slake Laboratory Animal Co., Ltd.) were taken, decapitated, the cortex and skull were cut, the complete brain was removed, and the brain was divided into two parts along the center line. The hippocampus is located in the temporal lobe of the cerebral cortex, the temporal lobe cortex was scraped aside, the hippocampal gyrus was exposed, and dissection was performed. The hippocampal tissue was then slightly moistened with the digestion solution, thinly sliced ​​horizontally with a blade, the slices were transferred to the digestion solution and digested for 8 min, the digestion solution was discarded, the stop solution was added, oxygen was passed, and the tissue was left for 1-2 hours. Three or four hippocampal-shaped slices were placed in a digestion solution and exposed to the air to form a suspension. Slightly larger pieces of tissue were allowed to settle and the suspension was dropped onto the prepared dish. After leaving the tissue to stand for a few minutes, half of the digestion solution was replaced with extracellular fluid, which was then promptly administered to perform patch clamp experiments on potassium and sodium channels on neurons.

[0058] 2.4 Primary culture of hippocampal neurons: The same concentration of enzyme and stop solution were prepared in PBS, and the medium of 90% DMEM / F12 + 10% FBS, neuron culture medium (2% B-27 + 1% mixture of penicillin and streptomycin + 0.5 mM 1% GlutaMAX Neurobasal-A), enzyme and stop solution (all purchased from Gibco) were preheated. Cover glass was precoated with 20 ug / mL polylysine in a 24-well plate. All operations were performed in a biosafety cabinet. Three 1-3 day old SD rat newborn mice were taken, and the hippocampus was removed by the above method, cut into pieces with a microdissection cutter, put into the enzyme and digested for 8 min, and gently shaken every 2-3 min to make the digestion uniform, then sucked out the enzyme solution, added the stop solution to terminate, gently sprayed until it became a suspension, filtered through a 70uM filter, and the filtrate was centrifuged at 1100 rpm for 5 min. Discard the supernatant, add 5 mL of medium, resuspend the cells, count them using a cell counter, and adjust the final concentration to 10 5The concentration of cells was adjusted to 1 / mL. The cells were cultured in a carbon dioxide incubator, and after 6 hours, the entire medium was replaced with neuronal culture medium. After that, half of the medium was replaced every 3 days. After 14 hours of culture, electrophysiological recording of spontaneous neuronal discharge was performed by perfusion administration. [Table 4] [Table 5]

[0059] 2.5 Analysis of experimental results: Data were processed with Clampfit 10.2 and imported into an Excel table for statistical analysis, and then the data was organized with GraphPad Prim5 software and subjected to significant difference analysis. Instantaneous outward potassium current was obtained by subtracting delayed rectifier potassium current from whole-cell potassium current. All electrophysiological experimental data were expressed as mean ± standard error (mean ± SEM), and significant difference comparison for the data was performed by paired t-test (Student's paired). * P ≤ 0.05; ** P ≤ 0.01; *** P≦0.001 indicated significance between the two groups and statistical significance.

[0060] As a result, we found that 10uM HJ-70 could significantly suppress the spontaneous discharge of cultured hippocampal neurons (Fig. 1A and B). In addition, 10uM HJ-70 could significantly suppress the frequency of discharge induced by action potentials of dorsal root neurons (DRG) (Fig. C and D). Therefore, HJ-70 can suppress the excitability of central and peripheral neurons.

[0061] Considering that sodium and potassium currents are typical molecular basis mediating neuronal discharge, and HJ-70 inhibits neuronal electrical excitability, the inventors further detected the effects of HJ-70 on potassium and sodium currents in DRG and hippocampal neurons. As shown in Figures 2B and 2C, HJ-70 inhibited the I KThe current was inhibited in a dose-dependent manner, but I A There was no significant effect on the current. K The inhibitory effect of HJ-70 on the current was completely eliminated. K The inhibition of DRG neuron activity in the peripheral nervous system by HJ-70 was also reversible, and it dose-dependently suppressed the I current in hippocampal neurons. K Suppresses the current and I A There was no effect on the current (Figure 3).

[0062] Furthermore, we detected the effect of HJ-70 on sodium currents in hippocampal neurons, and found that 10 μM HJ-70 had no effect on sodium currents in hippocampal neurons (Fig. 3A), whereas 1 μM of a sodium channel-selective inhibitor could completely suppress sodium currents (Fig. 3B).

[0063] In summary, HJ-70 dose-dependently regulates I expression in neurons. K Suppresses the current and I A It had no effect on potassium and sodium currents and reduced the electrical excitability of hippocampal and DRG neurons.

[0064] Example 3: Inhibition of Kv2.1 and TRSK channel currents by HJ-70

[0065] 3.1 Cell electrophysiology experiment: First, TRSK plasmid (gift from Professor Limin Hui, Johns Hopkins University) and Kv2.1 plasmid (gift from Professor Limin Hui, Johns Hopkins University) were co-transfected with green fluorescent protein EGFP plasmid (gift from Researcher Li Jia, Shanghai Institute of Materia Medica) at a ratio of 9:1 into CHO-K1 cells (ATCC). Cells with green fluorescence were selected under a fluorescent microscope for the experiment. The cells were clamped at -70mV, hyperpolarized to -130mV, and then subjected to a ramp depolarization stimulus to 20mV, maintained for 100ms, and then hyperpolarized to 0mV to record the potassium channel current. 10uM HJ-70 was perfused to detect its efficacy.

[0066] 3.2 Analysis of experimental results: The current study shows that Kv2.1 channels are I K This is the main molecular basis for the current (see Background section for details). K In view of the inhibition of current, the inventors further detected the effect on Kv2.1 current. As shown in Figure 5A and Figure 5B, 10uM HJ-70 completely inhibited the current mediated by Kv2.1 channel, which was consistent with the activity in neurons. In addition, α-hydroxysanshool, another active ingredient in Japanese pepper, inhibited TRESK and TASK1 / TASK3 channels in neurons, with a median effective dose of about 50uM. Therefore, the effect of HJ-70 on TRESK channel was evaluated, and it was found that 10uM HJ-70 almost completely inhibited TRSK channel current (Figure 5C and D), suggesting that HJ-70 has stronger inhibitory activity against TRESK than α-hydroxysanshool. These results indicate that HJ-70 is an inhibitor of Kv2.1 and TRESK potassium channels.

[0067] Example 4: Significant relief of inflammatory pain caused by formaldehyde with HJ-70

[0068] 4.1 Pain model experiment: 10% formaldehyde solution was diluted 10 times to prepare 1% formaldehyde solution, and 100mg / kg and 30mg / kg HJ-70 compound was prepared with 5% DMSO, 5% Tween-80 and 90% saline (0.9% NaCl solution). 30min before the experiment, the mice were administered with a dose of 0.2mL / 10g per mouse, and then 20μL of 1% formaldehyde solution was injected from the sole of the microinjection needle to create a formalin model. The number of times of licking the rear nail, shaking the rear nail, and lifting the nail, as well as the time of licking the nail and lifting the nail for a long time were recorded within 60min. Nail licking was scored 3 points, nail shaking was scored 2 points, and nail lifting was scored 1 point. The pain behavioral score and time were tallied to determine the analgesic activity of HJ-70.

[0069] 4.2 Analysis of experimental results: Data were organized and statistically analyzed in the same manner as electrophysiological data processing. Then, one-way ANOVA was performed using GraphPad Prism5 software. *P<0.05, ** P<0.01, *** P<0.001 indicates a significant difference between the two groups and is statistically significant.

[0070] The inventor is I K Considering the important roles of potassium currents, especially Kv2.1 and TRESK channels in stroke, arrhythmia, diabetes, pain, atrial fibrillation, respiratory depression and / or depressive diseases (see background section for details), we evaluated the effect of HJ-70 on pain. The formalin-induced mouse pain model is a typical inflammatory pain animal model and is widely used in analgesic drug discovery and research. Consistent with the results of in vitro experiments, the inventors found that intraperitoneal administration of HJ-70 dose-dependently alleviated the pain behavior response induced by formalin injection into the plantar of mice (Figure 5D, E). This result indicated that HJ-70 has clear in vivo activity.

Claims

1. An isoquinoline alkaloid compound selected from compounds represented by the following structural formula:

2. A method for producing the isoquinoline alkaloid compound according to claim 1, comprising the steps of: (1) crushing dried Japanese pepper, infusing it with a solvent, and concentrating the extract to obtain a Japanese pepper concentrated extract; (2) dissolving the concentrated extract of Japanese pepper in water, and extracting it with petroleum ether, ethyl acetate, and n-butanol to obtain a petroleum ether phase, an ethyl acetate phase, an n-butanol phase, and an aqueous phase; (3) The ethyl acetate phase was subjected to gradient elution on a 200-300 mesh silica gel column using a petroleum ether-acetone mixture as an eluent, with the volume ratios of petroleum ether:acetone being 50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 2:1, and 1:1, respectively, to obtain six fractions, Fr. 1 to Fr. 6, of which Fr. 4 was subjected to gradient elution on a reversed-phase silica gel C18 column using a water-methanol mixture as an eluent, with the volume ratios of water:methanol being 1:1, 1:2, and 1:4, respectively, to obtain eight fractions, Fr. 4-1 to Fr. 4-8; eluting 4-6 into a C18 column using a water-methanol mixture as an eluent, among which elution with 90% volume fraction of methanol / water to obtain compound HJ-68; elution with 80% volume fraction of methanol / water to obtain HJ-69 and HJ-70, respectively; A manufacturing method comprising:

3. In step (1), the solvent is one or a mixed solvent of two or more selected from water, methanol, ethanol, acetone, and dichloromethane; The method according to claim 2, wherein in step (1), the leaching is room temperature soaking or heating reflux extraction.

4. In step (1), the solvent is an aqueous ethanol solution having a volume fraction of 70%, ethanol, an aqueous methanol solution having a volume fraction of 70%, methanol, acetone, dichloromethane, or a combination thereof; The process of claim 2, wherein the leaching is carried out one or more times.

5. The method of claim 2, wherein in step (1), the leaching is performed by immersion extraction with a solvent at room temperature three times, each time for 5 to 7 days, or by heating and reflux extraction three times, each time for 1 to 2 hours.

6. The method according to claim 2, wherein in step (3), the liquid phase separation conditions for compound HJ-68 are methanol / water=90 / 10 (v / v) as the mobile phase and a flow rate of 4 mL / min; the column model is a C18 column, and the column length×diameter is 250 mm×20 mm.

7. The method according to claim 2, wherein in step (3), the liquid phase separation conditions for compound HJ-69 are methanol / water = 80 / 20 (v / v) as the mobile phase and a flow rate of 4 mL / min; the column model is a C18 column, and the column length x diameter is 250 mm x 20 mm.

8. The method according to claim 2, wherein in step (3), the liquid phase separation conditions for compound HJ-70 are methanol / water = 80 / 20 (v / v) as the mobile phase and a flow rate of 4 mL / min; the column model is a C18 column, and the column length x diameter is 250 mm x 20 mm.

9. Delayed rectifier potassium current (I Ｋ 2. Use of the isoquinoline alkaloid compound of claim 1 in the manufacture of an antagonist of .

10. Use of an isoquinoline alkaloid compound described in claim 1 in the manufacture of an antagonist of Kv2.1 channel.

11. 2. Use of the isoquinoline alkaloid compound of claim 1 in the manufacture of an antagonist of K2P two-pore potassium channel.

12. Use of the isoquinoline alkaloid compound described in claim 1 in the manufacture of an antagonist of a TRSK channel.

13. 13. Use of the isoquinoline alkaloid compound of claim 1 in the manufacture of a medicament for the treatment of stroke, arrhythmia, diabetes, pain, atrial fibrillation, respiratory depression and / or depression disorders.

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