A seco - C19 diterpenoid alkaloid compound, its preparation method and application

Abstract

The present invention discloses a seco-C19 diterpenoid alkaloid compound, a preparation method thereof and an application thereof. The seco-C19 diterpenoid alkaloid compound provided by the present invention shows significant antidepressant activity in the mouse behavioral despair model experiment, which is significantly superior to the positive drug fluoxetine, and can be used for developing antidepressant drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and more specifically, relates to a seco-C19 diterpenoid alkaloid compound, a preparation method thereof, and an application thereof. Background Art

[0002] Nowadays, social competition is becoming increasingly fierce. The high-intensity and high-pressure life has a serious impact on people's physical and mental health. Depression is gradually increasing and seriously affects people's normal work and life. At present, the drugs for treating depression generally have relatively large side effects and have certain limitations in clinical applications.

[0003] The inventors prepared a novel class of C-15 / C-16 seco-C19 diterpenoid alkaloid compounds, Carmaloidline H and Carmaloidline I, from the traditional Chinese medicine Aconiti Lateralis Radix Praeparata, and found that they showed significant antidepressant activity in the mouse behavioral despair model experiment, superior to the positive drug fluoxetine, and can be used for developing antidepressant drugs. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defects existing in the prior art, and provide a class of seco-C19 diterpenoid alkaloid compounds, Carmaloidline H and Carmaloidline I, a preparation method thereof, a pharmaceutical composition, and an application thereof.

[0005] To achieve the above purpose, the technical solution of the present invention provides a seco-C19 diterpenoid alkaloid compound having a structure shown in formula (1) and / or formula (2):

[0006]

[0007] Further, the seco-C19 diterpenoid alkaloid compound is obtained by extraction and separation from Aconiti Lateralis Radix Praeparata.

[0008] The present invention further provides a preparation method of the above seco-C19 diterpenoid alkaloid compound, which includes the following preparation steps:

[0009] S1. Reflux and extract Aconiti Lateralis Radix Praeparata with a solvent, combine the extracts and concentrate to obtain an extract;

[0010] S2. Dissolve the extract obtained in step S1 in an acidic solution, suspend, filter to remove impurities to obtain a filtered acidic aqueous solution; the filtered acidic aqueous solution is extracted with dichloromethane to obtain an acidic aqueous solution A and an extract A;

[0011] Adjust the pH value of the acidic aqueous solution A to alkaline, and then extract with dichloromethane to obtain an extract B;

[0012] S3. The extract B is subjected to column chromatography separation and high performance liquid chromatography separation in sequence to obtain the seco-C19 diterpenoid alkaloid compounds;

[0013] Among them, the column chromatography separation includes silica gel column chromatography, ODS column chromatography, and gel column chromatography carried out in sequence.

[0014] Preferably, the gel column chromatography is Sephadex LH-20 gel column chromatography.

[0015] Furthermore, in step S1, the solvent is one or several of methanol, ethanol, acetone, chloroform, and petroleum ether.

[0016] Furthermore, in step S1, the solvent is an aqueous ethanol solution of 88-98 V%;

[0017] The added mass of the solvent is 8-10 times that of the aconite.

[0018] The number of reflux extractions is 2-4 times, and each extraction is for 1-3 h.

[0019] Furthermore, in step S2, the acidic solution is 0.1-1.0% dilute hydrochloric acid; the added amount of the acidic solution is 8-15 times the mass of the extract.

[0020] Furthermore, in step S2, the pH value of the acid aqueous solution A is adjusted to 8-11 by adding a base. Preferably, the pH value is adjusted to 9.

[0021] Furthermore, step S3 includes:

[0022] S31. The extract B is gradient eluted by a mobile phase through silica gel column chromatography to obtain fraction C; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C is 0.72-0.75;

[0023] S32. Fraction C is eluted by a mobile phase of methanol-water through ODS column chromatography to obtain fraction C3; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C3 is 0.50-0.60;

[0024] S33. Fraction C3 is subjected to gel column chromatography with a mobile phase of methanol-water to obtain fraction M1; the Rf value of fraction M1 is 0.30-0.39;

[0025] S34. Subject the fraction M1 to reverse-phase high performance liquid chromatography with acetonitrile-water as the mobile phase to obtain the seco-C19 diterpenoid alkaloid compound Carmaloidline H with the structural formula shown in formula (1), and the retention time of the compound Carmaloidline H is 10 - 12 min; and the seco-C19 diterpenoid alkaloid compound Carmaloidline I with the structural formula shown in formula (2), and the retention time of the compound Carmaloidline I is 13 - 15 min.

[0026] Further, based on a total volume of 100, in step S31, the silica gel column chromatography uses gradient elution, and the mobile phase uses a mixed solvent composed of solvent A and solvent B in a volume ratio of (0:100) - (100:0);

[0027] The solvent A is any one of dichloromethane, chloroform, and petroleum ether;

[0028] The solvent B is any one of chloroform, acetone, ethyl acetate, and methanol.

[0029] Further, in step S31, the mobile phase of the silica gel column chromatography is dichloromethane - methanol.

[0030] Further, in step S31, the mobile phase of the silica gel column chromatography is dichloromethane - methanol with a volume ratio of (93:7) - (91:9).

[0031] Preferably, in step S31, the mobile phase of the silica gel column chromatography is dichloromethane - methanol with a volume ratio of 92:8.

[0032] Further, in step S32, the volume ratio of methanol - water in the ODS column chromatography mobile phase is (35:65) - (45:55); the mobile phase also contains formic acid with a volume fraction of 0.01 - 0.1%.

[0033] Preferably, in step S32, the volume ratio of methanol - water in the mobile phase is 40:60;

[0034] And / or, the volume fraction of formic acid in the mobile phase of the ODS column chromatography is 0.05%.

[0035] Further, in step S33, the volume ratio of methanol - water in the gel column chromatography mobile phase is (0:100) - (10:90).

[0036] Preferably, in step S33, the volume ratio of methanol - water in the mobile phase is 0:100.

[0037] Further, in step S34, the high performance liquid chromatography uses a C18 chromatographic column, the mobile phase uses acetonitrile-water with a volume ratio of (26:74)-(30:70), and the mobile phase further contains trifluoroacetic acid with a volume fraction of 0.01-0.5%.

[0038] Further, in step S34, the volume ratio of acetonitrile to water in the mobile phase of the high performance liquid chromatography is 28:72; the volume fraction of trifluoroacetic acid in the mobile phase is 0.1%.

[0039] In the above technical solution, according to the brick-red spots developed by potassium bismuth iodide in thin layer chromatography identification and the characteristic ultraviolet absorption (λ max = 235 nm) of diterpenoid alkaloids observed by HPLC analysis, fraction M1 is separated in the next step. When separating fraction M1 by RP-HPLC method, the volume ratio of acetonitrile to water containing 0.1% trifluoroacetic acid in the mobile phase is (26:74)-(30:70).

[0040] Preferably, the retention time of compound Carmaloidline H with the structural formula shown in formula (1) is 11 min, and the retention time of compound Carmaloidline I with the structural formula shown in formula (2) is 14 min.

[0041] The third aspect of the present invention discloses a pharmaceutical composition, which comprises the above-mentioned seco-C19 diterpenoid alkaloid compounds.

[0042] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0043] Further, the pharmaceutical composition further comprises a synergist;

[0044] The synergist is one or several of the following substances:

[0045] Fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, amitriptyline, clomipramine, doxepin, maprotiline, moclobemide, Shugan Jieyu Capsule, Hypericum perforatum extract, Flupentixol Melitracen.

[0046] Further, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquids, infusion, dripping pills or pellets.

[0047] The fourth aspect of the present application discloses the use of the above-mentioned seco-C19 diterpenoid alkaloid compounds and / or the pharmaceutical composition in the preparation of antidepressant drugs.

[0048] For the sake of simplicity of description, the compounds having the structures of formula (1) and formula (2) are named as compound Carmaloidline H and compound Carmaloidline I in this application respectively.

[0049] Among them, the tautomers of the seco-C19 diterpenoid alkaloid compounds or pharmaceutically acceptable salts thereof are also within the protection scope of the present invention.

[0050] The advantages and beneficial effects of the present invention are as follows:

[0051] (1) The present invention provides a class of seco-C19 diterpenoid alkaloid compounds Carmaloidline H and Carmaloidline I that have not been reported so far, and further provides a method for extracting the above compounds from Aconitum carmichaelii Debx. with simple operation, good reproducibility and high extraction purity;

[0052] (2) The diterpenoid alkaloid compounds Carmaloidline H and Carmaloidline I show significant antidepressant activity in the mouse behavioral despair model experiment, which is superior to the positive drug fluoxetine (a first-line clinical drug for treating depression), indicating that Carmaloidline H and Carmaloidline I can be used for developing antidepressant drugs. Description of the Drawings

[0053] Figure 1 1H NMR spectrum (400 MHz, CD3OD) of compound 1 prepared in Example 1 of the present invention; 1 H NMR spectrum (400 MHz, CD3OD) of compound 1 prepared in Example 1 of the present invention;

[0054] Figure 2 13C NMR spectrum (100 MHz, CD3OD) of compound 1 prepared in Example 1 of the present invention; 13 13C NMR spectrum (100 MHz, CD3OD) of compound 1 prepared in Example 1 of the present invention;

[0055] Figure 3 HMBC spectrum of compound 1 prepared in Example 1 of the present invention;

[0056] Figure 4 1H NMR spectrum (400 MHz, DMSO-d6) of compound 2 prepared in Example 1 of the present invention; 1 1H NMR spectrum (400 MHz, DMSO-d6) of compound 2 prepared in Example 1 of the present invention;

[0057] Figure 5 13C NMR spectrum (100 MHz, DMSO-d6) of compound 2 prepared in Example 1 of the present invention; 13 13C NMR spectrum (100 MHz, DMSO-d6) of compound 2 prepared in Example 1 of the present invention;

[0058] Figure 6 HMBC spectrum of compound 2 prepared in Example 1 of the present invention. Detailed implementation manners

[0059] The following combines the accompanying drawings and embodiments to further describe the detailed implementation manners of the present invention. The following embodiments are only used to illustrate the technical solutions of the present invention more clearly, and cannot be used to limit the protection scope of the present invention.

[0060] (1) Preparation of C-15 / C-16 seco-C19 diterpenoid alkaloid compounds

[0061] Example 1

[0062] A preparation method of a class of C-15 / C-16 seco-C19 diterpenoid alkaloid compounds includes the following steps:

[0063] S1. Take dry Aconiti Lateralis Radix Praeparata (about 200.5 kg), use an ethanol aqueous solution of 95% with a mass 10 times that of Aconiti Lateralis Radix Praeparata as a solvent for reflux extraction three times, extract for 2 hours each time, and concentrate the combined extraction solution to obtain an extract (about 6 kg);

[0064] S2. Add the extract to 0.5% dilute hydrochloric acid (60 L) with a mass 10 times that of the extract, fully dissolve and suspend it, filter to remove impurities, and extract the filtered acidic aqueous solution with dichloromethane with a volume 1.5 times that of the acidic aqueous solution for 3 times to obtain acidified dichloromethane extract A (about 2.5 kg, discarded) and acidic aqueous solution A (about 1.2 kg).

[0065] Adjust the pH value of acidic aqueous solution A to 9 with sodium hydroxide, and then still extract with dichloromethane to obtain a dichloromethane extract, that is, extract B (about 500 g).

[0066] S3. The extract B is successively subjected to column chromatography separation and high-performance liquid chromatography separation to obtain seco-C19 diterpenoid alkaloid compounds.

[0067] Step S3 specifically includes the following steps:

[0068] S31. Use dichloromethane-methanol for gradient elution of the extract B through a silica gel column chromatography to obtain fraction C; when using silica gel thin-layer chromatography for detection, the Rf value corresponding to fraction C is 0.72 - 0.75. The mobile phase of the silica gel column chromatography is dichloromethane-methanol with a volume ratio of (93:7)-(91:9). Preferably, the volume ratio of the mobile phase dichloromethane-methanol is 92:8.

[0069] It should be noted that during gradient elution, dichloromethane-methanol with a volume ratio of (93:7)-(91:9) can all elute fraction C. When using silica gel thin-layer chromatography for detection, the fraction with an Rf value of 0.72 - 0.75 is fraction C. Among them, the elution efficiency of dichloromethane-methanol with a volume ratio of 92:8 is the highest. The principle of the "preferred volume ratio" in the following gradient elution process is the same.

[0070] Specifically, the extract B is subjected to silica gel column chromatography and eluted with dichloromethane-methanol gradients with volume ratios of 100:0, 95:5, 92:8, 90:10, 85:15, 75:25, 65:35, 50:50, 0:100 in sequence to obtain fractions 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90. After collecting 90 fractions in total, silica gel thin layer chromatography is used for identification. According to the brick-red spots shown by Dragendorff's reagent, the Rf values are observed to be 0.82 - 0.86 (fractions 1-10), 0.76 - 0.80 (fractions 11-20), 0.72 - 0.75 (fractions 21-30), 0.65 - 0.70 (fractions 31-40), 0.56 - 0.62 (fractions 41-50), 0.50 - 0.54 (fractions 51-60), 0.42 - 0.48 (fractions 61-70), 0.30 - 0.38 (fractions 71-80), 0.25 - 0.28 (fractions 81-90). Similar fractions are combined to obtain fractions A, B, C, D, E, F, G, H, and I in sequence; according to the brick-red spots shown by Dragendorff's reagent in thin layer chromatography identification and the characteristic ultraviolet absorption (λ max = 235 nm) of diterpenoid alkaloids observed by HPLC analysis, fraction C is selected for the next separation. Among them, fractions 21-30 are combined to obtain fraction C.

[0071] S32. Fraction C is eluted with the mobile phase methanol-water through ODS column chromatography to obtain fraction C3; when silica gel thin layer chromatography is used for identification, the Rf value corresponding to fraction C3 is 0.50 - 0.60. The volume ratio of the mobile phase methanol-water for ODS column chromatography is (35:65) - (45:55). Preferably, it is 40:60. The mobile phase also contains formic acid with a volume ratio of 0.01 - 0.1%, and preferably, the mobile phase includes formic acid with a volume ratio of 0.05%.

[0072] The specific operation is as follows: Fraction C is subjected to ODS column chromatography and eluted with methanol-water gradients with volume ratios of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, where the mobile phase methanol-water also contains formic acid with a volume ratio of 0.05%.

[0073] Then, fractions 1-5, 6-15, 16-25, 26-35, 36-45, 46-50, 51-55, 56-60, and 61-68 were obtained successively. A total of 68 fractions were collected and identified by silica gel thin-layer chromatography. According to the brick-red spots shown by Dragendorff's reagent, the Rf values were observed to be 0.35-0.44 (fractions 1-5), 0.45-0.49 (fractions 6-15), 0.50-0.60 (fractions 16-25), 0.61-0.69 (fractions 26-35), 0.70-0.74 (fractions 36-55), 0.75-0.79 (fractions 56-60), and 0.80-0.89 (fractions 61-68). Similar fractions were combined into 7 fractions C1-C7. After combination, fractions 16-25 gave fraction C3.

[0074] Based on the brick-red spots shown by Dragendorff's reagent in thin-layer chromatography identification and the characteristic ultraviolet absorption (λ max = 235 nm) of diterpenoid alkaloids observed by HPLC analysis, fraction C3 was selected for the next separation step.

[0075] S33. Gel column chromatography was performed on fraction C3 using the mobile phase methanol-water to obtain fraction M1; the Rf value of fraction M1 was 0.30-0.39; the volume ratio of methanol-water in the mobile phase for gel column chromatography was (0:100)-(10:90). Preferably, the volume ratio of methanol-water was 0:100.

[0076] The specific operation includes: subjecting fraction C3 to Sephadex LH-20 column chromatography and eluting with a methanol-water gradient of volume ratios 0:100, 30:70, 70:30, and 100:0. Fractions 1-6, 7-14, 15-20, and 21-29 were collected successively. After collecting a total of 29 fractions, silica gel thin-layer chromatography identification was carried out. According to the brick-red spots shown by Dragendorff's reagent, the Rf values were observed to be 0.30-0.39 (fractions 1-6), 0.40-0.54 (fractions 7-14), 0.55-0.61 (fractions 15-20), and 0.62-0.85 (fractions 21-29). Similar fractions were combined successively to obtain 4 fractions M1-M4. Among them, fractions 1-6 were combined to give fraction M1.

[0077] Based on the brick-red spots shown by Dragendorff's reagent and the characteristic ultraviolet absorption (λ max = 235 nm) of diterpenoid alkaloids observed by HPLC analysis, fraction M1 was selected for the next separation step.

[0078] S34. Subject the fraction M1 to reversed-phase high-performance liquid chromatography with a mobile phase of acetonitrile-water having a volume ratio of (26:74)-(30:70), and the mobile phase further contains trifluoroacetic acid with a volume ratio of 0.01-0.5%; obtain the seco-C19 diterpenoid alkaloid compound Carmaloidline H with the structural formula shown in formula (1), and the retention time of the compound Carmaloidline H is 10-12 min; and the seco-C19 diterpenoid alkaloid compound Carmaloidline I with the structural formula shown in formula (2), and the retention time of the compound Carmaloidline I is 13-15 min.

[0079] The specific operation is as follows: adopt the HPLC method, use acetonitrile-water with a volume ratio of 28:72 as the mobile phase, and the mobile phase further contains trifluoroacetic acid with a volume ratio of 0.1%. Use a C18 chromatographic column to prepare the seco-C19 diterpenoid alkaloid compound 1 (i.e., the compound Carmaloidline H) (t R = 11 min, purity 98%) and the compound 2 (i.e., the compound Carmaloidline I) (t R = 14 min, purity 99%) from the fraction M1.

[0080] The physical properties and detection data of the compound 1 prepared in Example 1 are as follows:

[0081] Physical properties: yellow powder, soluble in methanol.

[0082] High-resolution mass spectrometry (HR-ESI-MS) m / z 466.2432 ([M+H] + The calculated value is 466.2435), combined with 1 1H NMR ( Figure 1 ), 13 13C NMR spectra ( Figure 2 ), determine its molecular formula as C 24 H 36 NO8, and calculate its degree of unsaturation as 8.

[0083] 1 1H NMR (CD3OD, 400 MHz) spectra ( Figure 1 ) show 1 olefinic proton signal δ H 6.73 (1H, s); 18 proton signals on the carbon atoms connected to oxygen / nitrogen δ H4.80 (1H, s), 4.45 (1H, dd, J = 6.6, 1.9 Hz), 4.38 (1H, t, J = 3.2 Hz), 3.72 (3H, s), 3.55 (2H, abq), 3.44 (3H, s), 3.35 (1H, m), 3.33 (3H, s), 3.21 (1H, dd, J = 13.2, 6.9 Hz), 3.10 (1H, overlapped), 3.02 (1H, overlapped); hydrogens on the carbons adjacent to two double bonds, δ H 3.10 (1H, overlapped), 2.83 (2H, m) and at δ H one methyl, two methylene and three methine proton signals between 2.76 - 1.36. The hydrogen spectrum information suggests that compound 1 may be an amine alcohol - type C19 diterpenoid alkaloid with a double bond.

[0084] 13 13C NMR (CD3OD, 100 MHz) spectrum ( Figure 2 ) shows a total of 24 carbon signals. Combining with the HSQC spectrum, it is determined that the compound contains two carbonyl signals δ C 176.2 (C - 15), 166.6 (C - 16); a set of olefin signals δ C 142.6 (C - 13), 137.4 (C - 14); ten carbon signals connected to oxygen / nitrogen δ C 83.5 (C - 6), 81.5 (C - 8), 79.6 (C - 18), 71.1 (C - 1), 63.4 (C - 17), 59.5 (18 - OCH3), 58.8 (C - 19), 58.7 (6 - OCH3), 52.1 (16 - OCH3), 50.4 (C - 20); one methyl carbon signal δ C 10.4 (C - 21); three methylene carbon signals δ C 35.7 (C - 12), 29.5 (C - 2), 28.4 (C - 3); three methine carbon signals δ C 51.1 (C - 7), 50.9 (C - 10), 44.5 (C - 5) and two quaternary carbon signals δ C 50.2 (C - 11), 39.4 (C - 5).

[0085] Combining 1 the 1H NMR and 13 13C NMR spectra (Table 1), it is speculated that compound 1 also contains two carbonyl carbon signals, one carbon - carbon double bond, ten carbon signals connected to oxygen / nitrogen and one methyl; combining the above information, it is speculated that compound 1 may be a seco - C19 diterpenoid alkaloid.

[0086] In the HMBC spectrum ( Figure 3 ), H-14 (δ H 6.73) shows long-range correlations with C-16 (δ C 166.6), C-10 (δ C 50.9), and C-12 (δ C 35.7). Combining with the chemical shifts, it indicates that there is a carbon-carbon double bond at C-13 / C-14 and simultaneously determines the connection position of C-16; the long-range correlation between H-7 (δ H 2.49) and C-15 (δ C 176.2) determines the connection position of C-15. Combining with the chemical shifts and the molecular formula, it shows that the compound is a C19-type diterpenoid alkaloid with C-15 / C-16 oxidative cleavage, and C-15 is oxidized to carboxylic acid and C-16 is oxidized to methyl ester. Combining the HMBC spectrum and chemical shifts, the structure of compound 1 is confirmed.

[0087] In summary, the structure of the new compound 1 (compound Carmaloidline H) is determined as follows:

[0088]

[0089] The physical properties and detection data of compound 2 prepared in Example 1 are as follows:

[0090] Physical properties: Yellow powder, soluble in methanol.

[0091] High-resolution mass spectrometry (HR-ESI-MS) m / z 466.2427 ([M+H] + calculated value is 466.2435). Combining with 1 1H NMR( Figure 4 ), 13 13C NMR spectrum( Figure 5 ), the molecular formula is determined to be C 24 H 35 NO8, and its degree of unsaturation is calculated to be 8.

[0092] 1 The 1H NMR (DMSO-d6, 400 MHz) spectrum( Figure 4 ) shows 20 proton signals on carbons connected to oxygen / nitrogen at δ H5.19 (1H, dd, J = 5.4, 4.0 Hz), 4.23 (1H, t, J = 3.9 Hz), 4.04 (1H, dd, J = 6.4, 1.7 Hz), 3.67 (3H, s), 3.48 (1H, abq, J = 8.3 Hz), 3.44 (1H, abq, J = 8.3 Hz), 3.34 (3H, s), 3.24 (3H, s), 3.14 (1H, overlapped), 3.14 (2H, overlapped), 3.14 (1H, overlapped), 3.08 (1H, overlapped), 3.04 (1H, overlapped) and at δ H between 2.79 - 1.17, one methyl, three methylene and four methine proton signals. The 1H NMR information suggests that compound 2 might be an amino alcohol type C19 diterpenoid alkaloid.

[0093] 13 13C NMR (DMSO-d6, 100 MHz) spectrum ( Figure 5 ) showed a total of 24 carbon signals. Combining with the HSQC spectrum, it was determined that the compound contained two carbonyl signals at δ C 176.5 (C-15), 170.2 (C-16); eleven oxygen / nitrogen-linked carbon signals at δ C 84.0 (C-14), 82.9 (C-6), 78.5 (C-8), 78.0 (C-18), 69.2 (C-1), 61.3 (C-17), 58.7 (18-OCH3), 57.9 (6-OCH3), 56.9 (C-19), 51.8 (16-OCH3), 49.2 (C-20); one methyl carbon signal at δ C 9.4 (C-21); three methylene carbon signals at δ C 28.2 (C-2), 27.9 (C-12), 27.1 (C-3); four methine carbon signals at δ C 47.6 (C-9), 47.0 (C-10), 44.7 (C-7), 44.5 (C-5) and two quaternary carbon signals at δ C 49.1 (C-11), 37.8 (C-15). Comparing the above data with the known compounds isolated in this project, it was found that the compound might be a seco-C19 diterpenoid alkaloid.

[0094] In the HMBC spectrum ( Figure 6 ), the long-range correlations of H-12b (δ H 1.88), H-13 (δ H 3.08) and C-16 (δ C 170.2) and 16-OCH3 (δH 3.67) and C-16 (δ C 170.2), C-13 (δ C 46.7), the long-range correlations determined the connection position of C-16; H-9 (δ H 2.79), H-14 (δ H 5.19) and C-15 (δ C 176.5), the long-range correlations combined with the molecular formula and chemical shifts determined that C-15 was connected to C-8 and linked to C-14 through a lactone ring. In summary, it was shown that compound 2 was a C19 diterpenoid alkaloid with C-15 / C-16 oxidative cleavage, and C-15 was oxidized to carboxylic acid and formed a lactone ring with the hydroxyl group of C-14, and C-16 was oxidized to methyl ester. Combining the HMBC spectrum and chemical shifts, the structure of compound 2 was confirmed.

[0095] In summary, the structure of the new compound 2 (compound Carmaloidline I) was determined as follows:

[0096]

[0097] 1 H NMR, 13 The signal assignments of 13C NMR are shown in Table 1.

[0098] Table 1 1 H NMR, 13 Signal assignments of 13C NMR δ (ppm)

[0099]

[0100]

[0101] Comparative Example 1

[0102] The preparation method was the same as that of Example 1, except that: in step S34, in the mobile phase used for separating fraction M1 by HPLC method, the aqueous solution for preparing the mobile phase did not contain trifluoroacetic acid.

[0103] The peak shape of the compound prepared in Comparative Example 1 was seriously tailing during the preparation process. After preparation, the analysis by liquid chromatography detected a large amount of impurities, and the purities of compounds 1 and 2 prepared were less than 40%.

[0104] Comparative Example 2

[0105] The preparation methods were the same as those of Example 1, except that: in step S32, when fraction C was passed through an ODS column chromatography, methanol-water with an initial volume ratio of 40:60 was used for elution, and the mobile phase did not contain formic acid.

[0106] Compound could not be detected during the preparation of subsequent fractions in Comparative Example 2:

[0107] The results showed that Compounds 1 and 2 could not be prepared.

[0108] (II) Verification experiment

[0109] Experimental Example 1: Mouse behavioral despair model experiment of Carmaloidline H and Carmaloidline I (the gold standard for screening antidepressant drugs)

[0110] 1. Experimental materials and instruments

[0111] Fluoxetine hydrochloride was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; demethylcoclaurine and methyllycaconitine were purchased from Shanghai Standard Technology Service Co., Ltd.; total alkaloid extract of Aconitum carmichaeli Debx., Carmaloidline H and Carmaloidline I were all prepared in the laboratory.

[0112] ZIL-2 type mouse spontaneous activity box (Shanghai Xinman Science and Education Equipment Co., Ltd.); YLS-18A type mouse tail suspension apparatus (Anhui Zhenghua Biological Instrument Equipment Co., Ltd.); electronic balance (Sartorius Scientific Instruments Co., Ltd., Beijing).

[0113] 2. Experimental animals

[0114] ICR mice, male, weighing 18 - 22 g, SPF grade, provided by Vital River Laboratories Inc., animal license number: SYXK(Beijing)2023 - 0001. The animals were housed in an environment with a temperature of 23 ± 2 °C and a humidity of 50% ± 10%, and the lighting time was 12 h per day (7:00 - 19:00 lighting). After the animals were housed for 3 days, various experiments were carried out. The animals were fasted for 6 h before the start of the behavioral experiments and had free access to water.

[0115] 3. Experimental methods

[0116] ICR male mice were arranged in a snake-like pattern and divided into 9 groups in descending order of the number of spontaneous activities during animal screening. They were the blank group (normal saline of the same volume), the positive drug fluoxetine hydrochloride group (10 mg / kg), the demethylcoclaurine group (10 mg / kg), the methyllycaconitine group (10 mg / kg), the total alkaloids of aconite group (10 mg / kg), the low-dose group of Carmaloidline H (0.03 mg / kg), the high-dose group of Carmaloidline H (0.3 mg / kg), the low-dose group of Carmaloidline I (0.03 mg / kg), and the high-dose group of Carmaloidline I (0.3 mg / kg). Each group had 10 mice, which were numbered separately. The drugs were all made into suspension with normal saline. Each group was given the drug by gavage once a day, and the administration volume was 0.1 ml / 10 g body weight. After continuous administration for 3 days, the spontaneous activity experiment was carried out on the 4th day, and the tail suspension experiment was carried out on the 5th day.

[0117] 4. Experimental results

[0118] As shown in Table 2, compared with the blank group, the low- and high-dose groups of Carmaloidline H and Carmaloidline I (0.03, 0.3 mg / kg) could significantly shorten the immobility time of mice in the tail suspension experiment, showing significantly better antidepressant effects than the positive control drugs fluoxetine (10 mg / kg, the first-line chemical drug for clinical treatment of depression), demethylcoclaurine (10 mg / kg), methyllycaconitine (10 mg / kg), and the total alkaloids of aconite (10 mg / kg).

[0119] Table 2 Effects of each group on the immobility time of mice in the tail suspension experiment

[0120]

[0121] (Compared with the blank control group, *** P < 0.001)

[0122] As shown in Table 3, in the open field experiment, compared with the blank group, each group had no significant effect on the spontaneous activity of mice, indicating that the effect of each group on the immobility time of mice in the tail suspension experiment was not related to nerve excitability.

[0123] Table 3 Effects of each group on the number of horizontal grid crossings of mice in the open field experiment

[0124]

[0125]

[0126] In summary, the two novel C-15 / C-16 ring-split C19 diterpenoid alkaloid compounds, Carmaloidline H and Carmaloidline I, of the present invention have significant antidepressant activity, which is significantly superior to the positive drug fluoxetine, and can be used as drug precursors for the treatment of depression.

[0127] Application Example 1

[0128] The application example of the present invention discloses a capsule using Carmaloidline H as the raw material drug, and its components are as follows:

[0129]

[0130] The specific preparation process is as follows:

[0131] Take Carmaloidline H, starch, and sodium metabisulfite, mix them evenly, add absolute ethanol to make a soft material, pass through a 24-mesh sieve, make granules, dry them, add magnesium stearate, mix well, and fill into capsules.

[0132] Application Example 2

[0133] The application example of the present invention discloses a granule using the compound Carmaloidline H as the raw material drug, and its components are as follows:

[0134] Carmaloidline H 4.0mg

[0135] Starch 6.0g

[0136] Sodium bisulfite 0.2g

[0137] Magnesium stearate 0.2g

[0138] Absolute ethanol Appropriate amount

[0139] Make 100 bags.

[0140] The specific preparation process is as follows:

[0141] Take Carmaloidline H, mix it with starch and sodium bisulfite, add absolute ethanol to make a soft material, pass through a 24-mesh sieve, make granules, dry them, add magnesium stearate, mix well, and bag them.

[0142] Application Example 3

[0143] The application example of the present invention discloses an oral liquid using the compound Carmaloidline H as the raw material drug, and its components are as follows:

[0144]

[0145] The specific preparation process is as follows:

[0146] After mixing the above components, use the conventional preparation method for oral liquid and then carry out sub-packaging.

[0147] Application Example 4

[0148] The application example of the present invention discloses an injection with compound Carmaloidline H as the raw material medicine, and its components are as follows:

[0149] Carmaloidline H 4.0mg

[0150] Vitamin C 0.2g

[0151] Sodium chloride 6.0g

[0152] Sodium bicarbonate 0.5g

[0153] Water for injection 1000.0mL;

[0154] Manufacture 100 vials.

[0155] The specific preparation process is as follows:

[0156] After mixing the above components, use the conventional preparation method for injection, and then 100 vials can be obtained.

[0157] Application Example 5

[0158] The application example of the present invention discloses a tablet with compound Carmaloidline H and fluoxetine as the raw material medicines, and its components are as follows:

[0159] Carmaloidline H 3.5mg

[0160] Fluoxetine 20.0mg

[0161] Hydroxypropyl methylcellulose 18.0g

[0162] Talcum powder 0.4g

[0163] Lactose 0.2g

[0164] Magnesium stearate 0.2g

[0165] Anhydrous ethanol appropriate amount

[0166] Manufacture 100 tablets.

[0167] The specific preparation process is as follows:

[0168] Mix Carmaloidline H, fluoxetine, hydroxypropyl methylcellulose, talc, lactose, and magnesium stearate evenly, add absolute ethanol to make soft materials, pass through a 24-mesh sieve to make granules, dry, add magnesium stearate, mix evenly, and press into tablets.

[0169] Application Example 6

[0170] The application example of the present invention discloses a capsule using compound Carmaloidline H and St. John's wort as raw materials, and its components are as follows:

[0171] Carmaloidline H 3.5mg

[0172] St. John's wort 300.0mg

[0173] Starch 6.0g

[0174] Sodium metabisulfite 0.2g

[0175] Magnesium stearate 0.2g

[0176] Absolute ethanol appropriate amount

[0177] Make 100 capsules.

[0178] The specific preparation process is as follows:

[0179] Take Carmaloidline H, St. John's wort, starch, and sodium metabisulfite, mix evenly, add absolute ethanol to make soft materials, pass through a 24-mesh sieve to make granules, dry, add magnesium stearate, mix evenly, and fill into capsules.

[0180] Application Example 7

[0181] The application example of the present invention discloses an injection using compound Carmaloidline H and paroxetine as raw materials, and its components are as follows:

[0182] Carmaloidline H 5.0mg

[0183] Paroxetine 20.0mg

[0184] Vitamin C 0.2g

[0185] Sodium chloride 6.0g

[0186] Sodium bicarbonate 0.5g

[0187] Water for injection 1000.0mL;

[0188] Make 100 vials.

[0189] The specific preparation process is as follows:

[0190] After mixing the above components, 100 injections can be obtained by using the conventional preparation method for injections.

[0191] Application Example 8

[0192] The application example of the present invention discloses a capsule using Carmaloidline I as the raw material drug, and its components are as follows:

[0193]

[0194]

[0195] The specific preparation process is as follows:

[0196] Take Carmaloidline I, starch, and sodium metabisulfite, mix them evenly, add anhydrous ethanol to make a soft material, pass through a 24-mesh sieve, make granules, dry, add magnesium stearate, mix evenly, and fill into capsules.

[0197] Application Example 9

[0198] The application example of the present invention discloses a granule using compound Carmaloidline I as the raw material drug, and its components are as follows:

[0199] Carmaloidline I 4.0mg

[0200] Starch 6.0g

[0201] Sodium bisulfite 0.2g

[0202] Magnesium stearate 0.2g

[0203] Anhydrous ethanol appropriate amount

[0204] Make 100 bags.

[0205] The specific preparation process is as follows:

[0206] Take Carmaloidline I, mix it with starch and sodium bisulfite, add anhydrous ethanol to make a soft material, pass through a 24-mesh sieve, make granules, dry, add magnesium stearate, mix evenly, and bag.

[0207] Application Example 10

[0208] The application example of the present invention discloses an oral liquid using compound Carmaloidline I as the raw material drug, and its components are as follows:

[0209]

[0210]

[0211] The specific preparation process is as follows:

[0212] After mixing the above components, use the conventional preparation method for oral liquid and then carry out sub-packaging.

[0213] Application Example 11

[0214] The application example of the present invention discloses an injection with compound Carmaloidline I as the raw material drug, and its components are as follows:

[0215] Carmaloidline I 4.0mg

[0216] Vitamin C 0.2g

[0217] Sodium chloride 6.0g

[0218] Sodium bicarbonate 0.1mL

[0219] Water for injection 1000.0mL;

[0220] Make 100 vials.

[0221] The specific preparation process is as follows:

[0222] After mixing the above components, use the conventional preparation method for injection, and then 100 vials can be obtained.

[0223] Application Example 12

[0224] The application example of the present invention discloses a tablet with compound Carmaloidline I and fluoxetine as the raw material drugs, and its components are as follows:

[0225]

[0226]

[0227] The specific preparation process is as follows:

[0228] Take Carmaloidline I, fluoxetine, hydroxypropyl methylcellulose, talc, lactose, and magnesium stearate, mix them evenly, add anhydrous ethanol to make soft materials, pass through a 24-mesh sieve to make granules, dry them, add magnesium stearate, mix them evenly, and then press tablets.

[0229] Application Example 13

[0230] The application example of the present invention discloses a capsule with compound Carmaloidline I and St. John's wort as the raw material drugs, and its components are as follows:

[0231] Carmaloidline I 3.5mg

[0232] St. John's wort 300.0mg

[0233] Starch 6.0 g

[0234] Sodium metabisulfite 0.2 g

[0235] Magnesium stearate 0.2 g

[0236] Absolute ethanol q.s.

[0237] Manufacture 100 capsules

[0238] The specific preparation process is as follows:

[0239] Take Carmaloidline I, Seroxat and starch, sodium metabisulfite, mix them evenly, add absolute ethanol to make soft materials, pass through a 24-mesh sieve, make granules, dry them, add magnesium stearate, mix them evenly, and fill into capsules.

[0240] Application Example 14

[0241] The application example of the present invention discloses an injection with compound Carmaloidline I and paroxetine as raw materials, and its components are as follows:

[0242] Carmaloidline I 5.0 mg

[0243] Paroxetine 20.0 mg

[0244] Vitamin C 0.2 g

[0245] Sodium chloride 6.0 g

[0246] Sodium bicarbonate 0.5 g

[0247] Water for injection 1000.0 mL

[0248] Manufacture 100 injections

[0249] The specific preparation process is as follows:

[0250] After mixing the above components, using the conventional preparation method for injections, 100 injections can be obtained.

[0251] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the technical principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a seco - C19 diterpenoid alkaloid compound, characterized in that, The seco-C19 diterpenoid alkaloid compounds have the structures shown in formula (1) and / or formula (2): The seco-C19 diterpenoid alkaloid compounds are extracted and separated from Aconitum carmichaeli Debx.; The preparation method comprises the following preparation steps: S1. Aconitum carmichaeli Debx. is refluxed and extracted with a solvent, and after the extraction solutions are combined and concentrated, an extract is obtained; the solvent is an ethanol aqueous solution with 88-98V%. S2. The extract obtained in step S1 is dissolved in an acidic solution, and after being suspended and filtered to remove impurities, a filtered acidic aqueous solution is obtained; the filtered acidic aqueous solution is extracted with dichloromethane to obtain an acidic aqueous solution A and an extract A; the acidic solution is 0.1-1.0% dilute hydrochloric acid; The pH value of the acidic aqueous solution A is adjusted to be alkaline, and then it is extracted with dichloromethane to obtain an extract B; S3. The extract B is subjected to column chromatography separation and high performance liquid chromatography separation in sequence to obtain the seco-C19 diterpenoid alkaloid compounds; Among them, the column chromatography separation includes silica gel column chromatography, ODS column chromatography, and gel column chromatography carried out in sequence; Step S3 includes: S31. The extract B is subjected to gradient elution with a mobile phase through silica gel column chromatography to obtain a fraction C; when detected by silica gel thin layer chromatography, the Rf value corresponding to the fraction C is 0.72-0.75; the mobile phase for silica gel column chromatography is dichloromethane-methanol with a volume ratio of (93:7)-(91:9); S32. The fraction C is eluted with a mobile phase of methanol-water through ODS column chromatography to obtain a fraction C3; when detected by silica gel thin layer chromatography, the Rf value corresponding to the fraction C3 is 0.50-0.60; the volume ratio of the mobile phase methanol-water for ODS column chromatography is (35:65)-(45:55); and the mobile phase also contains formic acid with a volume fraction of 0.01-0.1%; S33. The fraction C3 is subjected to gel column chromatography with a mobile phase of methanol-water to obtain a fraction M1; the Rf value of the fraction M1 is 0.30-0.39; the volume ratio of the mobile phase methanol-water for gel column chromatography is (0:100)-(10:90); S34. The fraction M1 is subjected to reversed-phase high performance liquid chromatography with a mobile phase of acetonitrile-water; the seco-C19 diterpenoid alkaloid compound Carmaloidline H with the structure shown in formula (1) is obtained, and the retention time of the compound Carmaloidline H is 10-12 min; and the seco-C19 diterpenoid alkaloid compound Carmaloidline I with the structure shown in formula (2) is obtained, and the retention time of the compound Carmaloidline I is 13-15 min; In step S34, the high performance liquid chromatography uses a C18 chromatographic column, and the mobile phase uses acetonitrile-water with a volume ratio of (26:74)-(30:70), and the mobile phase also contains trifluoroacetic acid with a volume fraction of 0.01-0.5%.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass of the solvent added is 8-10 times that of Aconitum carmichaeli Debx.; The number of times of reflux extraction is 2-4 times, and each extraction is for 1-3 h.

3. The preparation method according to claim 1, wherein, In step S2, the addition amount of the acidic solution is 8-15 times the mass of the extract.

4. The preparation method according to claim 1, characterized in that, In step S2, the pH value of the acidic aqueous solution A is adjusted to 8-11 by adding a base thereto.

5. The preparation method according to claim 1, characterized in that, In step S31, the mobile phase for silica gel column chromatography is dichloromethane-methanol with a volume ratio of 92:

8.

6. The preparation method according to claim 1, characterized in that, In step S32, the volume ratio of the mobile phase methanol-water is 40:60; and, the mobile phase includes formic acid with a volume proportion of 0.05%.

7. The preparation method according to claim 1, characterized in that, In step S34, the volume ratio of acetonitrile-water in the mobile phase of the high performance liquid chromatography is 28:72; the volume proportion of trifluoroacetic acid in the mobile phase is 0.1%.

8. A pharmaceutical composition, characterized in that, It includes seco-C19 diterpenoid alkaloid compounds having the structure shown in formula (1); 9. The pharmaceutical composition according to claim 8, wherein It further includes a pharmaceutically acceptable carrier or excipient.

10. The pharmaceutical composition according to claim 8, wherein It further includes a synergist; The synergist is one or several of the following substances: Fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, amitriptyline, clomipramine, doxepin, maprotiline, moclobemide, Shuganjieyu Capsule, Hypericum perforatum extract, Flupentixol Melitracen.

11. The pharmaceutical composition according to claim 8, characterized in that, The dosage form of the pharmaceutical composition is tablet, capsule, granule, oral liquid, infusion, dropping pill or pellet.

12. Use of the composition according to any one of claims 8-11 in the preparation of an antidepressant drug.

13. A seco - C19 diterpenoid alkaloid compound, characterized in that, The seco-C19 diterpenoid alkaloid compound has the structure shown in formula (1):

14. Use of seco - C19 diterpenoid alkaloid compounds in the preparation of antidepressant drugs; characterized in that, The seco-C19 diterpenoid alkaloid compound has the structure shown in formula (1) and / or formula (2):

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