Dopamine D2 receptor agonist in stephania epigaea and application and pharmaceutical composition thereof

Abstract

The invention relates to discovery and application of targeted active molecules in a natural product, in particular to discovery and application of a dopamine D2 receptor new agonist in the natural product radix stephaniae tetrandrae. The novel natural agonist and the partial agonist of the dopamine D2 receptor comprise aporphine compounds such as (-)-pasteine and liavine and pharmaceutically acceptable salifying compounds of the aporphine compounds. In-vitro cell experiments show that the compound provided by the invention has an excitation or partial excitation effect on a dopamine D2 receptor, and current researches show that the dopamine D2 receptor agonist has a good clinical curative effect on treating diseases such as Parkinson's disease, schizophrenia and hyperprolactinoma; therefore, the novel natural agonist of the dopamine D2 receptor provided by the invention provides a candidate compound for the development of medicines for treating related diseases.

Description

Technical Field

[0001] The present invention relates to the discovery and application of targeted active molecules in natural products, and specifically to the discovery and application of new agonists of dopamine D2 receptors in natural products. The new natural agonists of dopamine D2 include aporphenic compounds such as (-)-pavoline and nelumboline and their pharmaceutically acceptable salt compounds. The application includes the treatment of one or more of Parkinson's disease, schizophrenia, hyperprolactinoma and the like. Background Art

[0002] Stephania epigaea HSLo is a perennial herbaceous vine of the genus Stephania in the family Menispermaceae. Stephania epigaea HSLo is a commonly used Chinese medicine with bitter taste, cold nature, and slight toxicity. It has the traditional effects of clearing away heat and detoxifying, calming, regulating qi, and relieving pain. It can be used to relieve sores and treat malaria and abdominal pain caused by food accumulation. Stephania epigaea HSLo is rich in alkaloids. So far, more than 40 alkaloids have been identified from Stephania epigaea, mainly including protoberberine, aporphenes, protoaporphenes, morphines, nephrines, and bisbenzylisoquinoline alkaloids (Chem. Res. Appl, 2016, 28(12): 1730-1734). Among the main ingredients of Diburong, cephalothin has pharmacological activity of increasing leukocytes; epiganine B and dehydrobicuculline have acetylcholinesterase (AChE) inhibitory activity (Fitoterapia, 2015, 104: 102-107.); tetrahydropalmatine and corydaline monophenol alkaloids have dopamine D1 receptor antagonist activity (Chin. J. Nat. Med, 2007, 5 (2): 112-114). Although Diburong has the traditional effects of regulating qi, sedation and analgesia, its pharmacological activity of sedation and analgesia and the mechanism of action of its active ingredients have not been reported.

[0003] Dopamine exerts its effects through its corresponding membrane receptors, which are a family of G protein-coupled receptors consisting of seven transmembrane regions. Five dopamine receptors have been isolated, which can be divided into D1 and D2 receptors according to their biochemical and pharmacological properties. D1 receptors include D1 and D5 receptors, and D2 receptors include D2, D3 and D4 receptors. Dopamine D2 receptor (Dopamine receptor D2, referred to as D2R) is a dopamine receptor translated from the DRD2 gene. It was first discovered by Philip Seeman in 1975 and named "antipsychotic dopamine receptor". D2R is the target of all current antipsychotic drugs and drugs for the treatment of Parkinson's disease (Pharmacol. Rev, 2011. 63 (1): 182-217), such as anti-Parkinson's drugs bromocriptine, cabergoline and anti-depressant drug cariprazine. In addition, D2R agonists are also used clinically to treat hyperprolactinoma. Therefore, the discovery of new D2R agonists is of great significance for the development of drugs against Parkinson's disease, mental illness and the treatment of hyperprolactinoma.

[0004] Natural products have always been an important source of drug discovery. They contain structurally rich compounds and clinical effectiveness, providing a source for new drug discovery. According to statistics. Currently, about 30%-40% of drugs are directly or indirectly derived from natural products. Based on the traditional efficacy of the Chinese medicine dibuxian, this patent will look for new D2 receptor agonists from it, laying the foundation for the development of safe and efficient anti-Parkinson's disease and mental illness drugs. We used a high-performance liquid chromatography preparative chromatography system to prepare dibuxian, and finally obtained two aporphyrin-like components with agonist activity on dopamine D2 receptors. At present, the D2 receptor agonist activity of this type of component has not been reported. Summary of the invention

[0005] The present invention relates to the discovery and application of new dopamine D2 receptor agonists in natural products. One of the purposes is to provide compounds in natural products that can stimulate dopamine D2 receptors, including (-)-pavoline, nelumboline, and pharmaceutically acceptable carriers or excipients. The second purpose is to provide the clinical application scope of such compounds, including clinical treatment of one or more diseases among Parkinson's disease, schizophrenia, and hyperprolactinoma. The chemical structures of (-)-pavoline and nelumboline are as follows:

[0006]

[0007] Nelumbosine

[0008] In vitro cell experiments show that the natural compounds in the present invention have an agonist effect on dopamine D2 receptors. Current studies have shown that dopamine D2 receptor agonists have good clinical efficacy in treating Parkinson's disease, schizophrenia, hyperprolactinoma and other diseases. Based on this, the new natural agonists of dopamine D2 receptors mentioned in the present invention provide candidate compounds for the development of drugs for these related diseases.

[0009] Beneficial effects of the present invention

[0010] In vitro cell experiments show that the compounds of the present invention have agonist or partial agonist effects on dopamine D2 receptors.

[0011] The present invention discloses two natural compounds from diabetic rats that have agonistic effects on dopamine D2 receptors. Current studies have shown that dopamine D2 receptor agonists have good clinical efficacy in treating Parkinson's disease, schizophrenia, hyperprolactinoma and other diseases. Based on this, the new natural agonists of dopamine D2 receptors mentioned in the present invention provide candidate compounds for the development of drugs for these related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : One-dimensional preparative chromatogram of dichloromethane layer of Dibucon extract.

[0013] Figure 2 :Chromatograms of the multidimensional preparative purification process of (-)-pavoline (independent fraction number: DBR_F2-2-3-5_TY). A. 2D preparative chromatogram of the 1D component DBR_F2; B. 3D preparative chromatogram of the 2D fraction DBR_F2-2; C. 4D preparative chromatogram of the 3D fraction DBR_F2-2-3; D. Refined desalting spectrum of the 4D fraction DBR_F2-2-3-5. The fractions where (-)-pavoline is located are marked with asterisks. Figure 3 : Chromatograms of the multidimensional preparative purification process of nelumbinine (independent fraction number: DBR_F2-4-9-1-1). A. 2D preparative chromatogram of the 1D component DBR_F2; B. 3D preparative chromatogram of the 2D fraction DBR_F2-4; C. 4D preparative chromatogram of the 3D fraction DBR_F2-4-9; D. 5D preparative chromatogram of the 4D fraction DBR_F2-4-9-1. The fractions where nelumbinine is located are marked with asterisks.

[0014] Figure 4: A—Excitatory concentration-response DMR spectra of compounds (-)-papovaline and nephrine on CHO-D2 cells; B—Excitatory concentration-response curves of compounds (-)-papovaline and nephrine on cells (data points are marked with circles); Concentration-response curves of 5 nM dopamine on CHO-D2 cells after pretreatment with different concentrations of compounds (-)-papovaline and nephrine for 1 hour (data points are marked with squares); Concentration-response curves of compounds (-)-papovaline and nephrine on CHO-D2 cells after pretreatment with different concentrations of raclopride for 1 hour (data points are marked with triangles).

[0015] Table 1: High-resolution NMR analysis of compounds (-)-pavoline and nelumbinine. DETAILED DESCRIPTION

[0016] The present invention is further described with reference to examples, which are intended to illustrate the present invention but not to limit the present invention.

[0017] Example 1: Preparation method of total alkaloids of Dioscorea paniculate

[0018] 100 kg of ground herbs were crushed and thrown into a 2m 3 The extraction tank was filled with 70% ethanol at 60°C (3 extractions in total, each heating extraction time was 2 hours, and the amount of 70% ethanol per extraction was 1m 3 , after each extraction, the extract was collected, and 70% ethanol was added to the solid for the next extraction). The extracts were combined and concentrated in a concentrator to obtain an extract with a volume of about 100 liters and a mass of 93 kg. 10 grams of the extract was heated and dried, and the solid content was measured to be 15.7%. The extract was diluted with 4 times its pure water and acidified to pH = 2 with 2% sulfuric acid by mass concentration, and the neutral components were extracted with petroleum ether. The aqueous phase was adjusted to pH = 10 with 2M sodium hydroxide solution, and then extracted with dichloromethane. After the dichloromethane layer was evaporated under reduced pressure, about 4 kg of crude alkaloids were obtained.

[0019] The crude alkaloids were purified using a chromatographic column with a strong cation exchange filler HSCX (Zhejiang Huapu Xinchuang Technology Co., Ltd., 100×250 mm, 40 μm, filler batch number D2020113001). The sample load for each preparation was 70 g, the sample load was about 3%, the mobile phase A was 90% methanol / 0.1% FA (V / V), the mobile phase B was 70% methanol / 200 mM ammonium formate (V / V), and the gradients were 0-8 minutes: 100% A; 8-60 minutes: 100% B. The preparation chromatogram is shown in Figure 1. Figure 1 According to the peak time of the liquid phase spectrum, the fraction after 15 minutes was collected, which was the alkaloid segment. After spin drying, the total alkaloid component of dibronzi was obtained, which was independently numbered DBR_F2.

[0020] Example 2: Preparation and Identification of (-)-Papovaline

[0021] Total alkaloids of DBR-F2 (DBR-F2) were separated and purified using a C18HCE column (Zhejiang Huapu Xinchuang Technology Co., Ltd., 10 μm, 100×264 mm, filler batch number Z2019082901, 21041901G). The mobile phase was A: water / 0.1% formic acid (V / V); B: methanol / 0.1% formic acid (V / V), and the elution gradient (V / V) was: 0-7 min, 10% B; 7-10 min, 10-18% B; 10-20 min, 18% B; 20.1-30 min, 23% B; 30.1-40 min, 28% B; 40.1-50 min, 33% B; 50.1-60 min, 43% B; 60-62 min, 43-90% B; 62-80 min, 90% B. See the preparation chromatogram. Figure 2 A, 8 fractions were collected according to the chromatographic peaks, of which the second fraction was the fraction containing (-)-pavoline, and the main fraction was numbered DBR_F2-2, which was concentrated and then used for the next separation.

[0022] DBR_F2-2 was prepared by three-dimensional purification using a C8CE column (Zhejiang Huapu Xinchuang Technology Co., Ltd., 10 μm, 100×250 mm, filler batch number Z207101301). The mobile phase was A: 0.125% ammonia water (V / V); B: methanol, and the elution gradient was (V / V): 0-50 min, 45-70% B; 50-52 min, 70-90% B, 52-70 min, 90% B. See the chromatogram for details. Figure 2 B. A total of 9 fractions were collected according to the chromatographic peaks, among which the third fraction was the fraction containing (-)-pavoline, and the independent fraction was numbered DBR_F2-2-3. After concentration, the next step of separation and purification was carried out.

[0023] DBR_F2-2-3 was prepared by four-dimensional purification using HSCX column (Zhejiang Huapu Xinchuang Technology Co., Ltd., 10 μm, 300×250 mm, filler batch number Z2019091602). The mobile phase was A: final concentration 80 mM ammonium formate / 0.2% formic acid / water (V / V); B: final concentration 80 mM ammonium formate / 0.2% formic acid / methanol (V / V), and the elution gradient was: 0-50 min, 15-70% B; 50-52 min, 70-90% B; 52-60 min, 90% B. See the chromatogram. Figure 2 C. A total of 6 fractions were collected according to the chromatographic peaks, among which the fifth fraction was the fraction containing (-)-pavoline, and the independent fraction was numbered DBR_F2-2-3-5. After concentration, the next step of separation and purification was carried out.

[0024] DBR_F2-2-3-5 was desalted using a C18HCE column (Zhejiang Huapu Xinchuang Technology Co., Ltd., 7μm, 50×250mm, D2020122401). The mobile phase was A: 0.1% FA / water (V / V); B: methanol, and the elution gradient was (V / V): 0-15min, 5% B; 15-25min, 90% B. The main peak with a retention time of 14.5 minutes to 16.7 minutes was collected, and the fraction number was DBR_F2-2-3-5-TY. The collected fraction was concentrated by rotary evaporation and freeze-dried to obtain white needle-shaped crystals.

[0025] The NMR structure identification was completed on a Bruker 600M NMR spectrometer, and the hydrogen and carbon spectrum peaks and attribution information of the compound are shown in Table 1. It was identified that DBR_F2-2-3-5-TY is a known compound (-)-pavoline, CAS number: 6871-21-2.

[0026] Example 3: Preparation and identification of nelumbinine

[0027] Total alkaloids of DBR-F2 were separated and purified using a C18HCE column (Zhejiang Huapu Xinchuang Technology Co., Ltd., 10 μm, 100×264 mm, Z2019082901, 21041901G). The mobile phase was A: water / 0.1% formic acid; B: methanol / 0.1% formic acid, and the elution gradient was: 0-7 min, 10% B; 7-10 min, 10-18% B; 10-20 min, 18% B; 20.1-30 min, 23% B; 30.1-40 min, 28% B; 40.1-50 min, 33% B; 50.1-60 min, 43% B; 60-62 min, 43-90% B; 62-80 min, 90% B. See the preparation chromatogram. Figure 3 A, 8 fractions were collected according to the chromatographic peaks, among which the fourth fraction was the fraction containing nelumbinine, and the independent fraction was numbered as DBR_F2-4. After concentration, it was separated in the next step.

[0028] DBR_F2-4 was prepared by three-dimensional purification using a C8CE column (Zhejiang Huapu Xinchuang Technology Co., Ltd., 10 μm, 100×250 mm, filler batch number Z207101301). The mobile phase was A: 0.125% ammonia water; B: methanol, and the elution gradient was: 0-3 min, 25% B; 3-5 min, 25-55% B; 5-45 min, 55-70% B; 45-47 min, 70-90% B; 47-60 min, 90% B. See the chromatogram for details. Figure 3 B. 10 fractions were collected according to the chromatographic peaks, among which the ninth fraction was the fraction containing nelumbinine, and the independent fraction was numbered DBR_F2-4-9. After concentration, the next step of separation and purification was carried out.

[0029] DBR_F2-4-9 was prepared by four-dimensional purification using a C18HCE column (Zhejiang Huapu Xinchuang Technology Co., Ltd., 7 μm, 30×250 mm, D2020122401). The mobile phase was A: 0.1% FA / water; B: methanol, and the elution gradient was: 0-40 min, 20-30% B; 40-42 min, 30-90% B; 42-55 min, 90% B. See the chromatogram for details. Figure 3 C, the main peak with a retention time of 20-25 minutes was taken, and the fraction number was DBR_F2-4-9-1. After concentration, the next step of separation and purification was carried out.

[0030] Considering the orthogonality of separation, DBR_F2-4-9-1 was prepared by five-dimensional purification using supercritical fluid chromatography. The chromatographic column was 2-PIC (Zhejiang Huapu Xinchuang Technology Co., Ltd., 4.6×250 mm, 5 μm), the mobile phase was A: supercritical CO2; B: methanol / 0.1% triethylamine, and the elution gradient was 0-8min: 95-85% A; 5-15% B; 8-12min: 85-45% A; 15-55% B. See the chromatogram for details. Figure 3 D, the peak with a retention time of 4-4.8 minutes was taken, and the fraction number was DBR_F2-4-9-1-1. White needle-shaped crystals were obtained after rotary evaporation concentration and freeze-drying.

[0031] The NMR structure identification was completed on a Bruker 600M NMR spectrometer, and the hydrogen and carbon spectrum peaks and attribution information of the compound are shown in Table 1. It was identified that DBR_F2-2-3-5-TY is a known compound, nelumbinine, CAS number: 548-08-3.

[0032] Example 4: Target specificity verification of active compounds

[0033] The stable cell line was constructed as a target model of dopamine D2 receptor. The specific steps are as follows. Chinese hamster ovary cells (CHO-K1) were cultured with 10% fetal bovine serum (Gibco TM , Thermo Fisher Scientific) Ham's F-12 medium (Gibco TM ) were cultured at 37°C in air / 5% CO2. 24 μL of Lipofectamine 2000 reagent (Invitrogen TM, Thermo Fisher Scientific) and 8 μg D2 plasmid were transfected into CHO-K1 cells. 24 hours after transfection, the cells were cultured in 600 μg / mL bleomycin (Shanghai Biyuntian Biotechnology Co., Ltd.) for 3 weeks to obtain the CHO-K1-D2 stable cell line. CHO-K1-D2 cells were cultured in F-12 medium containing 10% fetal bovine serum and 200 μg / mL bleomycin at 37°C, air / 5% CO2.

[0034] First, the compounds (-)-pavoline and nephrine were respectively detected by excitation analysis to see whether they produce concentration-dependent and saturated receptor ligand properties. The specific steps are as follows. CHO-K1-D2 cells were inoculated into a 384-well label-free detection plate (Corning) at a density of 10k / well and cultured at 37°C, air / 5% CO2 for 24 hours to make the cell confluence reach 95%. The compounds (-)-pavoline and nephrine were acted on CHO-K1-D2 cells. The final concentration gradient of each compound in the well was 50μM, 25μM, 12.5μM, 6.25μM, 3.12μM, 1.56μM, 0.78μM, 0.39μM, 0.195μM, 97.7nM, 48.8nM, 24.4nM, 12.2nM and 6.1nM. The BT label-free detection system (Corning) continuously recorded the DMR signal for 60 min. Figure 4 A is the DMR signal spectrum caused by different concentrations of the two compounds. It can be seen that the amplitude of the DMR signal increases with the increase of concentration. The compound (-)-pavoline can cause the maximum signal of about 300pm, while the maximum response signal caused by the compound nelumbinine is relatively small. The concentration-response concentration curve of the agonist effect is shown in Figure 4 B (data points are marked with dots), the concentration response curves of (-)-pavoline and nelumbinine are both single-phase "S" type. The saturation response signal of (-)-pavoline is equivalent to that of dopamine, and the corresponding EC 50 The response signal of compound nelumbosityl at the highest concentration was weaker than the maximum DMR signal of dopamine, indicating that it is a partial agonist. The corresponding EC 50 It is 0.82±0.22μM.

[0035] Then, the desensitization analysis method was used to verify the specificity of the compounds (-)-pavoline and nelumboline on the dopamine D2 receptor. The cell inoculation and culture methods were the same as above. CHO-K1-D2 cells were pretreated for 60 minutes with (-)-pavoline and nelumboline at final concentrations of 50μM, 25μM, 12.5μM, 6.25μM, 3.12μM, 1.56μM, 0.78μM, 0.39μM, 0.195μM, 97.7nM, 48.8nM, 24.4nM, 12.2nM and 6.1nM, respectively. Then, 5nM dopamine was added and the DMR signal was monitored for another 60 minutes. The concentration-response curve of the desensitization effect is shown in FIG. Figure 4 B (data points are marked with squares). As shown in the figure, (-)-pavoline and nelumboline both showed concentration-dependent desensitization of the receptor, and the corresponding half-maximal inhibitory concentration (IC 50 ) were 0.29±0.01μM and 1.66±0.20μM, respectively.

[0036] Finally, the antagonistic analysis method was used to verify the specificity of the compounds (-)-pavoline and nephrine on the D2 receptor. The cell inoculation and culture methods were the same as above. CHO-K1-D2 cells were pretreated with the dopamine D2 receptor selective antagonist raclopride (Shanghai Haoyuan Pharmaceutical) at final concentrations of 125nM, 62.5nM, 31.25nM, 15.62nM, 7.81nM, 3.91nM, 1.95nM, 0.98nM, 0.49nM, 0.24nM, 0.12nM, 0.061nM, 0.031nM and 0.015nM for 1 hour, and then (-)-pavoline and nephrine were added at a concentration of 12.5μM. The concentration-response curve of the antagonistic effect is shown in Figure 4 B. As shown in the figure, high concentrations of raclopride can completely inhibit the response of compounds (-)-pavoline and nephrine, with IC 50 The values ​​are 8.19±1.32nM and 1.32±0.24nM respectively, which shows that the compounds (-)-pavoline and nephrine do have a selective agonist effect on dopamine D2 receptors. In summary, (-)-pavoline and nephrine are agonists of dopamine D2 receptors.

[0037]

[0038] Table 1

Claims

1. An agonist of dopamine D2 receptor in diabetic rats, characterized in that: The invention uses one or more of (-)-pavoline and nelumboline compounds and pharmaceutically acceptable salts of the above compounds as active ingredients.

2. The agonist according to claim 1, characterized in that: The agonist also includes a pharmaceutically acceptable carrier or excipient.

3. The agonist according to claim 1, characterized in that: The half-maximal effect concentration (EC) of the compounds (-)-pavoline and nelumboline on the agonist activity of dopamine D2 receptors at the cellular level 50 ) were 0.35±0.04μM and 0.82±0.22μM, respectively.

4. Use of the dopamine D2 receptor agonist in Dibuconazole as claimed in any one of claims 1 to 3 in the preparation of drugs for treating one or more of Parkinson's disease, schizophrenia and prolactinoma.

5. Use of one or more of (-)-pavoline and nelumboline compounds and pharmaceutically acceptable salts of the above compounds in the preparation of dopamine D2 receptor agonists, or in the preparation of drugs for the treatment of one or more of Parkinson's disease, schizophrenia and prolactinoma.

6. A pharmaceutical composition, characterized in that: The invention uses one or more of (-)-pavoline and nelumboline compounds and pharmaceutically acceptable salts of the above compounds as active ingredients.

7. The pharmaceutical composition according to claim 6, characterized in that: The agonist also includes a pharmaceutically acceptable carrier or excipient, which is a drug for treating one or more of Parkinson's disease, schizophrenia and high prolactinoma.

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