A novel alkaloid derivative, its synthesis method and application

Abstract

The present invention discloses a novel alkaloid derivative, its synthesis method and application. Based on the active pregnane-type alkaloid skeleton structure in the Miao medicine "Sanyangyin", using epiandrosterone as a raw material, a series of C20-oxime pregnane-type alkaloid derivatives are obtained through reactions such as Wittig reaction, reduction reaction, Corey oxidation, reductive amination, and N-alkylation. This method has a simple and efficient preparation process and mild reaction conditions. The synthesized C20-oxime pregnane-type alkaloid derivatives have excellent anti-tumor activity and anti-inflammatory properties and can be used in anti-tumor and anti-inflammatory drugs. It provides a scientific basis for the new drug development and application of novel anti-tumor and anti-inflammatory pregnane-type alkaloids.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical synthesis, and relates to a synthesis method and application of a novel alkaloid derivative. Background Art

[0002] The Miao medicine "Three Liang of Silver" (also known as "Three Liang of Gold", Miao medicine name bub jid ngongx) is the root of Sarcococca ruscifolia, a plant of the genus Sarcococca in the Buxaceae family, and its common substitute Sarcococca hookeriana. It is a commonly used medicine in the Miao ethnic areas in Hunan and Guizhou. Literature records show that it can treat various diseases such as stomachache, physical weakness, sore throat, and traumatic injury. Modern research shows that its chemical components are mainly pregnane-type steroidal alkaloid compounds, which have a wide range of biological activities in aspects such as cholinesterase inhibition, anti-cancer, anti-ulcer, anti-inflammatory, and spasmolysis. The research group found in the investigation of Miao medicine that the resource-rich Miao medicine "Three Liang of Silver" is widely used in the treatment of malignant tumors (referred to as "evil toxin disease" by Miao medicine) and Alzheimer's disease ("stupid disease of the elderly") in the Miao folk, with significant curative effects. The research group previously conducted a relatively systematic chemical component study on S. ruscifolia and its common substitute S. hookeriana of the Miao medicine "Three Liang of Silver", and carried out in vitro anti-tumor and anti-inflammatory activity tests on this series of compounds, showing good anti-tumor and anti-inflammatory activities. At the same time, through chemical, pharmacological and computer virtual screening, the pharmacophores of this type of alkaloid were preliminarily analyzed at positions C2, 3, 4 of ring A and C20 of ring D of the parent nucleus. However, previous studies found that the structural diversity of the derivatives naturally produced by this type of alkaloid is relatively low, which limits the in-depth study of finding highly effective and low-toxic derivatives and their structure-activity relationships.

[0003] Therefore, in order to find highly effective and low-toxic pregnane-type steroidal alkaloid derivatives, based on the pregnane-type alkaloid skeleton structure in the Miao medicine Three Liang of Silver, the present invention uses epiandrosterone as a raw material, and through Wittig reaction, reduction reaction, Corey oxidation, reductive amination, and nucleophilic addition with hydroxylamine hydrochloride to obtain a series of new C20-oxime pregnane-type alkaloid derivatives. Further, through anti-tumor and anti-inflammatory activity evaluation, it is found that the synthesis of this series of derivatives shows significant anti-tumor and anti-inflammatory activities. The invention provides a scientific basis for finding novel anti-tumor and anti-inflammatory lead compounds with high selectivity and safety and developing novel anti-tumor and anti-inflammatory drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a novel alkaloid derivative.

[0005] The purpose of the present invention is to provide a synthesis method of a novel alkaloid derivative.

[0006] Another object of the present invention is to provide the use of novel alkaloid derivatives in anti-hepatocellular carcinoma, colorectal cancer, and anti-inflammation.

[0007] In order to achieve the object of the present invention, the present invention adopts the following technical solutions and steps to achieve:

[0008] The novel alkaloid derivative described in the present invention has a general structural formula shown as (Ⅰ):

[0009]

[0010] Among them, R1 is selected from aniline, p-methylaniline, o-methylaniline, m-methylaniline, p-chloroaniline, p-methoxyaniline, o-methoxyaniline, m-methoxyaniline.

[0011] There are 8 kinds of the derivative compounds described in the present invention, which are sequentially denoted as 5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h, and the corresponding relationship of R1 is as follows:

[0012]

[0013] The synthesis method of the novel alkaloid derivative described in the present invention includes the following steps:

[0014] (1) Synthesis of compound 1: Add potassium tert-butoxide and ethyltriphenylphosphonium bromide to a flask, evacuate, add an appropriate amount of anhydrous tetrahydrofuran, stir at room temperature for 1 h, add a pre-prepared solution of episterone in tetrahydrofuran, stir at room temperature for 3 h, monitor the completion of the reaction by TLC, then add saturated ammonium chloride solution to the reaction solution to quench the reaction, extract with ethyl acetate, wash with saturated brine, dry the organic layer with anhydrous magnesium sulfate, filter, concentrate under reduced pressure to obtain a crude product, and purify by silica gel chromatography with a mobile phase of petroleum ether: ethyl acetate = 5:1 to obtain compound 1;

[0015] (2) Synthesis of compound 2: Add BH3-Me2S to the tetrahydrofuran solution of compound 1, stir at 25 °C for 2 h, then dropwise add ethanol, NaOH, and H2O2, stir at 25 °C for 2 h, quench the mixture with Na2S2O3, and extract with ethyl acetate. Wash the synthesized organic solution with saturated brine, dry with Na2SO4, filter and concentrate to obtain compound 2;

[0016] (3) Synthesis of compound 3: Add compound 2, PCC, and an appropriate amount of silica gel to DCM, stir the mixture at room temperature for 3 h, filter the mixture, wash the filter cake with DCM, recover the solvent under reduced pressure, and perform silica gel column chromatography to obtain compound 3;

[0017] (4) Synthesis of Compound 4a: Place Compound 3 in a flask, add acetic acid, inject aniline, stir overnight, slowly add sodium borohydride in batches, stir at room temperature for 2 h, remove acetic acid under reduced pressure, add water to the solid, adjust the pH of the organic layer to neutral or weakly alkaline with dilute sodium hydroxide solution, extract with ethyl acetate, concentrate under reduced pressure to obtain a crude product, and purify by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate to obtain Compound 4a;

[0018] Synthesis of Compounds 4b - 4h: Operate according to the synthesis procedure of Compound 4a above. When aniline is replaced with the following corresponding compounds, react with p - toluidine to obtain Compound 4b; react with p - anisidine to obtain Compound 4c; react with p - chloroaniline to obtain Compound 4d; react with o - toluidine to obtain Compound 4e; react with m - toluidine to obtain Compound 4f; react with m - anisidine to obtain Compound 4g; react with o - anisidine to obtain Compound 4h.

[0019] (5) Synthesis of Compound 5a: Place Compound 4a in a flask, add potassium hydroxide and hydroxylamine hydrochloride, add ethanol, stir at 15 °C for 16 h, monitor the completion of the reaction by TLC, add water, extract with ethyl acetate, combine the organic phases, wash with saturated brine, dry with anhydrous magnesium sulfate, filter, concentrate under reduced pressure to obtain a crude product, and purify by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate to obtain Compound 5a;

[0020] Synthesis of Compounds 5b - 5h: Operate according to the synthesis procedure of Compound 5a above. React with Compound 4b to obtain Compound 5b; react with Compound 4c to obtain Compound 5c; react with Compound 4d to obtain Compound 5d; react with Compound 4e to obtain Compound 5e; react with Compound 4f to obtain Compound 5f; react with Compound 4g to obtain Compound 5g; react with Compound 4h to obtain Compound 5h.

[0021] The synthesis method of the novel alkaloid derivative described in the present invention specifically includes the following steps:

[0022] (1) Synthesis of Compound 1: Weigh 2.34 g of potassium tert - butoxide and 7.67 g of ethyltriphenylphosphonium bromide respectively, add them to a flask, evacuate, add an appropriate amount of anhydrous tetrahydrofuran, stir at room temperature for 1 h, add a pre - prepared tetrahydrofuran solution of epicatechol (1 g, 3.5 mmol), stir at room temperature for 3 h, monitor the completion of the reaction by TLC, then quench the reaction by adding saturated ammonium chloride solution, extract with ethyl acetate 3 times, 100 ml each time, combine the extracts, wash with saturated brine, dry the organic layer with anhydrous magnesium sulfate, filter, concentrate under reduced pressure to obtain a crude product, and purify by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate = 5∶1 to obtain Compound 1;

[0023] (2) Synthesis of Compound 2: Add BH3-Me2S (7.5 mL, 10 M, 75.0 mmol) to a solution of Compound 1 (5 g, 15.0 mmol) in THF (100 mL). After stirring at 25 °C for 2 h, add 10 mL of ethanol, NaOH (30 mL, 5 M), and H2O2 (15 mL, 10 M). After stirring at 60 °C for 1 h, quench the mixture with 400 mL of 10% Na2S2O3 solution and extract twice with ethyl acetate, 300 mL each time. Wash the combined organic solutions with 300 mL of saturated brine, dry over Na2SO4, filter, and concentrate to obtain Compound 2;

[0024] (3) Synthesis of Compound 3: Weigh 1 g of Compound 2 and 2.76 g of PCC, add an appropriate amount of silica gel to DCM (100 mL). Stir the mixture at room temperature for 3 h, filter the mixture, wash the filter cake with DCM, recover the solvent under reduced pressure, and perform silica gel column chromatography to obtain Compound 3;

[0025] (4) Synthesis of Compound 4a: Weigh 100 mg of Compound 3 into a 25 mL flask, add 1 mL of acetic acid, inject 23.70 μL of aniline, stir overnight, slowly add 22.70 mg of sodium borohydride in portions, stir at room temperature for 2 h, remove acetic acid under reduced pressure, add water to the solid, adjust the pH of the organic layer to neutral or weakly basic with dilute sodium hydroxide solution, extract with ethyl acetate, and concentrate the organic layer under reduced pressure to obtain the crude product. Purify by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate to obtain Compound 4a;

[0026] Synthesis of Compounds 4b - 4h: Operate according to the synthesis procedure of Compound 4a above, react with p-toluidine to obtain Compound 4b; react with p-methoxyaniline to obtain Compound 4c; react with p-chloroaniline to obtain Compound 4d; react with o-toluidine to obtain Compound 4e; react with m-toluidine to obtain Compound 4f; react with m-methoxyaniline to obtain Compound 4g; react with o-methoxyaniline to obtain Compound 4h.

[0027] (5) Synthesis of Compounds 5a - 5h: Weigh 50 mg of Compound 4a into a 25 mL flask, add 21.38 mg of potassium hydroxide and 18.07 mg of hydroxylamine hydrochloride, add 3 mL of ethanol, stir at 15 °C for 16 h, monitor the reaction completion by TLC, add water, extract three times with ethyl acetate, 20 mL each time, combine the organic phases, wash with saturated brine, dry over anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain the crude product. Purify by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate to obtain Compound 5a;

[0028] Synthesis of Compounds 5b - 5h: Operate according to the synthesis procedure of Compound 5a above, react with Compound 4b to obtain Compound 5b; react with Compound 4c to obtain Compound 5c; react with Compound 4d to obtain Compound 5d; react with Compound 4e to obtain Compound 5e; react with Compound 4f to obtain Compound 5f; react with Compound 4g to obtain Compound 5g; react with Compound 4h to obtain Compound 5h.

[0029] In step (1) of the present invention, the molar ratio of potassium tert - butoxide, ethyltriphenylphosphonium bromide to epiandrosterone is 6∶6∶1.

[0030] In step (2) of the present invention, the molar ratio of BH3 - Me2S to Compound 1 is 5∶1.

[0031] In step (3) of the present invention, the molar ratio of Compound 2 to PCC is 1∶4.

[0032] In step (4) of the present invention, the molar ratio of Compound 3, aniline to sodium borohydride is 1.6∶1.3∶3.

[0033] In step (5) of the present invention, the molar ratio of Compound 4a, potassium hydroxide to hydroxylamine hydrochloride is 1∶3∶2.

[0034] Application of the novel alkaloid derivative of the present invention in the preparation of anti - liver cancer, anti - colorectal cancer and anti - inflammatory drugs.

[0035] The synthesis procedure of the present invention is as follows:

[0036]

[0037] Advantages of the present invention:

[0038] 1. For the synthesis method of the present invention, the raw materials used are cheap and easily available, the reaction conditions are mild, and the operation is simple.

[0039] 2. The obtained C20 - oxime - pregnane - type alkaloid derivative of the present invention is a new compound. Through the cytotoxic activity test on tumor cell lines and the in vitro anti - inflammatory activity test of the synthesized new C20 - oxime - pregnane - type alkaloid, it shows that this kind of compound has anti - tumor and anti - inflammatory effects; it provides a scientific basis for searching for novel anti - tumor and anti - inflammatory lead compounds with high selectivity and safety and developing novel anti - tumor and anti - inflammatory drugs. Brief Description of the Drawings

[0040] Figure 1a 1H - NMR spectrum of Compound 5a 1 1H - NMR spectrum

[0041] Figure 1b 13C - NMR spectrum of Compound 5a 13 13C - NMR spectrum

[0042] Figure 2a 1H-NMR spectrum of Compound 5b 1 1H-NMR spectrum

[0043] Figure 2b 13C-NMR spectrum of Compound 5b 13 13C-NMR spectrum

[0044] Figure 3a 1H-NMR spectrum of Compound 5c 1 1H-NMR spectrum

[0045] Figure 3b 13C-NMR spectrum of Compound 5c 13 13C-NMR spectrum

[0046] Figure 4a 1H-NMR spectrum of Compound 5d 1 1H-NMR spectrum

[0047] Figure 4b 13C-NMR spectrum of Compound 5d 13 13C-NMR spectrum

[0048] Figure 5a 1H-NMR spectrum of Compound 5e 1 1H-NMR spectrum

[0049] Figure 5b 13C-NMR spectrum of Compound 5e 13 13C-NMR spectrum

[0050] Figure 6a 1H-NMR spectrum of Compound 5f 1 1H-NMR spectrum

[0051] Figure 6b 13C-NMR spectrum of Compound 5f 13 13C-NMR spectrum

[0052] Figure 7a 1H-NMR spectrum of Compound 5g 1 1H-NMR spectrum

[0053] Figure 7b 13C-NMR spectrum of Compound 5g 13 13C-NMR spectrum

[0054] Figure 8a 1H-NMR spectrum of Compound 5h 1 1H-NMR spectrum

[0055] Figure 8b 13C-NMR spectrum of Compound 5h 13 13C-NMR spectrum

[0056] Figure 9 Cytotoxicity of the compounds against RAW264.7 cells

[0057] Figure 10Effect of the compound on NO in LPS-induced RAW264.7 cells Detailed implementation manners

[0058] Example 1: Synthesis method of a novel alkaloid derivative

[0059] 1. Synthesis of Compound 1

[0060] Potassium tert-butoxide (2.34 g, 21 mmol) and ethyltriphenylphosphonium bromide (7.67 g, 21 mmol) were added to a flask. After evacuation, an appropriate amount of anhydrous tetrahydrofuran was added, and the mixture was stirred at room temperature for 1 h. A pre-prepared solution of epiatriol (1 g, 3.5 mmol) in tetrahydrofuran was added, and the mixture was stirred at room temperature for 3 h. The reaction was monitored by TLC until completion. A saturated ammonium chloride (NH4Cl) solution was added to quench the reaction, and the mixture was extracted with ethyl acetate (3 × 100 mL), washed with saturated sodium chloride NaCl(aq), the organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel chromatography with a mobile phase of petroleum ether∶ethyl acetate = 5∶1 to obtain Compound 1.

[0061] 2. Synthesis of Compound 2

[0062] BH3-Me2S (7.5 mL, 10 M, 75.0 mmol) was added to a solution of Compound 1 (5 g, 15.0 mmol) in THF (100 mL). After stirring at 25 °C for 2 h, ethanol (10 mL), NaOH (30 mL, 5 M), and H2O2 (15 mL, 10 M) were added dropwise. After stirring at 60 °C for 1 h, the mixture was quenched with Na2S2O3 (400 mL, 10%), and extracted with ethyl acetate (2 × 300 mL). The synthesized organic solution was washed with saturated sodium chloride (300 mL), dried over Na2SO4, filtered and concentrated to obtain Compound 2.

[0063] 3. Synthesis of Compound 3

[0064] Compound 2 (1 g, 2 mmol) and PCC (2.76 g, 8 mmol), together with an appropriate amount of silica gel, were added to DCM (100 mL), and the mixture was stirred at room temperature for 3 h. The mixture was filtered, and the filter cake was washed with DCM. The solvent was recovered under reduced pressure, and silica gel column chromatography was performed to obtain Compound 3.

[0065] 4. Synthesis of Compound 4a

[0066] Compound 3 (100 mg, 0.32 mmol) was placed in a 25 mL flask, 1 mL of acetic acid was added, and aniline (23.70 μL, 0.26 mmol) was added by injection. Stirred overnight, sodium borohydride (22.70 mg, 0.6 mmol) was added slowly in portions. Stirred at room temperature for 2 h, acetic acid was removed under reduced pressure, water was added to the solid, the organic layer was adjusted to neutral or weakly alkaline with dilute sodium hydroxide solution, extracted with ethyl acetate, the organic layer was extracted, concentrated under reduced pressure to obtain a crude product, and purified by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate to obtain compound 4.

[0067] Synthesis of compounds 4b - 4h: Operated according to the synthesis procedure of compound 4a above, reacted with p - toluidine to obtain compound 4b; reacted with p - anisidine to obtain compound 4c; reacted with p - chloroaniline to obtain compound 4d; reacted with o - toluidine to obtain compound 4e; reacted with m - toluidine to obtain compound 4f; reacted with m - anisidine to obtain compound 4g; reacted with o - anisidine to obtain compound 4h.

[0068] 5. Synthesis of compounds 5a - 5h

[0069] Compound 4a (50 mg, 0.13 mmol) was placed in a 25 mL flask, potassium hydroxide (21.38 mg, 0.39 mmol) and hydroxylamine hydrochloride (18.07 mg, 0.26 mmol) were added, and ethanol (3 mL) was added. Stirred at 15 °C for 16 h, the reaction was monitored by TLC until completion, water was added, extracted with ethyl acetate (3 × 20 mL), the combined organic phases were washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure to obtain a crude product, and purified by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate to obtain compound 5a.

[0070] Synthesis of compounds 5b - 5h: Operated according to the synthesis procedure of compound 5a above, reacted with compound 4b to obtain compound 5b; reacted with compound 4c to obtain compound 5c; reacted with compound 4d to obtain compound 5d; reacted with compound 4e to obtain compound 5e; reacted with compound 4f to obtain compound 5f; reacted with compound 4g to obtain compound 5g; reacted with compound 4h to obtain compound 5h.

[0071] Compound 5a: White powder, yield 20%. HR - ESI - MS m / z calcd for C 27 H 41 N2O([M + H] + ) 409.3213, found 409.3205; 1H-NMR (400 MHz, CDCl3) δ 7.19–7.13 (2H, m), 6.73–6.58 (3H, m), 3.25 (1H, dt, J = 11.2, 5.7 Hz), 2.22 (1H, t, J = 9.2 Hz), 2.07 (1H, q, J = 10.7, 10.0 Hz,), 1.96 (1H, d, J = 13.5 Hz), 1.88 (3H, s), 1.86 (1H, d, J = 2.6 Hz), 1.78–1.65 (5H, m), 1.62–1.47 (3H, m), 1.43–1.06 (11H, m), 0.97–0.86 (1H, m), 0.83 (3H, d, J = 6.6 Hz), 0.80–0.69 (1H, m), 0.62 (3H, s); 13 C-NMR (100 MHz, CDCl3) δ 159.2, 129.4, 113.1, 57.0, 56.1, 54.6, 45.8, 44.2, 40.5, 39.1, 37.8, 36.3, 36.0, 35.8, 33.1, 32.7, 32.1, 28.7, 25.9, 24.3, 23.2, 21.3, 20.9, 15.3, 13.5, 12.5, 11.7.

[0072] The structural formula of compound 5a is as follows:

[0073]

[0074] Compound 5b: Yellow needle-like crystals, yield 33%. HR-ESI-MS m / z calcd for C 28 H 43 N2O ([M + H] + ) 423.3370, found 423.3363; 1 H-NMR (400 MHz, CDCl3) δ 8.08 (1H, s), 7.00–6.94 (2H, m), 6.58–6.51 (2H, m), 3.22 (1H, tt, J = 10.8, 4.2 Hz), 2.23 (4H, s), 2.12–2.02 (1H, m), 1.99–1.92 (1H, m), 1.88 (4H, s), 1.78–1.49 (7H, m), 1.41–1.01 (12H, m), 0.92 (1H, qd, J = 11.8, 4.9 Hz), 0.81 (3H, s), 0.72 (1H, td, J = 12.1, 11.3, 3.9 Hz), 0.62 (3H, s); 1313C-NMR (100 MHz, CDCl3) δ 159.2, 129.9, 114.1, 57.0, 56.1, 54.7, 53.4, 45.8, 44.2, 39.1, 37.8, 36.0, 35.9, 35.8, 32.1, 29.5, 28.8, 24.3, 23.2, 21.3, 20.5, 15.3, 13.5, 12.5.

[0075] The structural formula of compound 5b is as follows:

[0076]

[0077] Compound 5c: Yellow powder, yield 41%. HR-ESI-MS m / z calcd for C 28 H 43 N2O2 ([M + H] + ) 439.3319, found 439.3317; 1 1H-NMR (400 MHz, CDCl3) δ 8.14 (1H, s), 6.80–6.74 (2H, m), 6.60 (2H, dd, J = 17.0, 8.7 Hz), 3.74 (3H, s), 3.59 (1H, p, J = 3.4 Hz), 2.22 (1H, t, J = 9.0 Hz), 2.06 (1H, q, J = 10.5, 9.6 Hz), 1.88 (3H, s), 1.87–1.84 (1H, m), 1.76–1.64 (5H, m), 1.61–1.48 (3H, m), 1.36 (2H, tt, J = 10.0, 4.5 Hz), 1.26 (4H, td, J = 7.1, 6.4, 3.5 Hz), 1.23–1.09 (5H, m), 0.99–0.87 (1H, m), 0.82 (3H, d, J = 6.5 Hz), 0.76–0.66 (1H, m), 0.62 (3H, s); 13 13C-NMR (100 MHz, CDCl3) δ 158.5, 151.6, 114.8, 114.7, 114.4, 56.4, 55.5, 55.4, 54.0, 45.2, 43.5, 39.8, 38.4, 37.2, 35.6, 35.4, 35.2, 32.7, 31.4, 28.0, 25.2, 23.6, 22.6, 20.3, 14.6, 12.9, 11.9, 11.0.

[0078] The structural formula of compound 5c is as follows:

[0079]

[0080] Compound 5d: Yellow powder, yield 39%. HR-ESI-MS m / z calcd for C 27 H 40 N2OCl([M+H] + ) 443.2824, found 443.2819; 1 1H-NMR (400 MHz, CDCl3) δ 7.12–7.07 (2H, m), 6.53 (2H, dd, J = 10.8, 8.7 Hz), 3.26–3.15 (1H, m), 2.23 (1H, dd, J = 10.1, 8.2 Hz), 2.07 (1H, q, J = 10.6, 9.7 Hz), 1.93 (1H, d, J = 12.8 Hz), 1.88 (3H, s), 1.78–1.64 (5H, m), 1.57 (1H, t, J = 9.8 Hz), 1.39–1.23 (9H, m), 1.20–1.07 (5H, m), 0.92 (1H, ddd, J = 24.7, 12.4, 6.0 Hz), 0.82 (3H, d, J = 6.5 Hz), 0.62 (3H, s); 13 13C-NMR (100 MHz, CDCl3) δ 159.1, 145.6, 129.2, 114.9, 57.0, 56.0, 54.6, 45.7, 44.2, 39.0, 37.7, 36.3, 36.0, 35.8, 35.6, 32.1, 29.8, 29.2, 28.7, 24.3, 23.2, 21.3, 15.3, 13.5, 12.5.

[0081] The structural formula of Compound 5d is as follows:

[0082]

[0083] Compound 5e: Yellow powder, yield 60%. HR-ESI-MS m / z calcd for C 28 H 43 N2O([M+H] + ) 422.3910, found 422.3907; 11H-NMR (400 MHz, CDCl3) δ 8.07 (1H, s), 6.85 (1H, td, J = 7.7, 1.4 Hz), 6.77 (1H, dd, J = 7.9, 1.5 Hz), 6.72–6.56 (2H, m), 3.86 (3H, d, J = 13.9 Hz), 3.68–3.62 (1H, m), 2.22 (1H, td, J = 9.2, 8.4, 3.8 Hz), 2.12–2.02 (1H, m), 1.88 (3H, d, J = 0.9 Hz), 1.79–1.64 (5H, m), 1.62–1.49 (3H, m), 1.44–1.32 (3H, m), 1.31–1.05 (10H, m), 0.98–0.89 (1H, m), 0.84 (3H, d, J = 3.4 Hz), 0.62 (3H, s); 13 13C-NMR (100 MHz, CDCl3) δ 159.2, 146.9, 121.5, 109.7, 57.0, 56.1, 55.6, 54.5, 45.8, 44.2, 40.5, 39.1, 37.8, 36.3, 36.1, 35.8, 33.2, 32.8, 32.0, 28.6, 26.1, 24.2, 23.2, 21.3, 20.9, 15.3, 13.5, 12.6, 11.6.

[0084] The structural formula of compound 5e is as follows:

[0085]

[0086] Compound 5f: yellow powder, yield 44%. HR-ESI-MS m / z calcd for C 28 H 42 N2O ([M + H] + ) 423.3370, found 423.3359; 1 1H-NMR (400 MHz, CDCl3) δ 8.38 (1H, s), 7.08–7.02 (1H, m), 6.55–6.38 (3H, m), 3.68–3.64 (1H, m), 2.27 (3H, s), 2.22 (1H, dd, J = 8.4, 2.6 Hz), 2.12–2.01 (1H, m), 1.89 (4H, s), 1.77–1.65 (5H, m), 1.51 (1H, p, J = 4.0, 3.3 Hz), 1.36 (3H, d, J = 15.2 Hz), 1.30–1.24 (7H, m), 1.22–1.11 (5H, m), 0.83 (3H, d, J = 6.4 Hz), 0.63 (3H, s); 1313C-NMR (100 MHz, CDCl3) δ 159.1, 139.2, 139.2, 129.3, 57.0, 56.1, 54.7, 45.8, 44.2, 40.5, 39.1, 36.3, 36.0, 35.8, 33.1, 32.8, 32.0, 29.8, 28.6, 26.0, 24.2, 23.2, 21.8, 20.9, 15.3, 13.5, 12.5, 11.7.

[0087] The structural formula of compound 5f is as follows:

[0088]

[0089] Compound 5g: Yellow powder, yield 37%. HR-ESI-MS m / z calcd for C 28 H 43 N2O2 ([M + H] + ) 439.3319, found 439.3316; 1 1H-NMR (400 MHz, CDCl3) δ 7.07 (1H, t, J = 8.1 Hz), 6.23 (2H, ddd, J = 12.6, 6.1, 3.4 Hz), 6.16 (1H, t, J = 2.3 Hz), 3.77 (3H, d, J = 1.2 Hz), 3.65 (1H, p, J = 3.1 Hz), 2.23 (1H, td, J = 8.0, 2.9 Hz), 2.10–2.00 (2H, m), 1.90–1.84 (4H, m), 1.76–1.65 (5H, m), 1.55–1.49 (2H, m), 1.44–1.41 (2H, m), 1.28 (10H, dd, J = 12.7, 2.1 Hz), 1.20 (4H, dq, J = 7.0, 4.0, 2.7 Hz), 0.82 (3H, d, J = 6.8 Hz), 0.62 (3H, s); 13 13C-NMR (100 MHz, CDCl3) δ 161.0, 159.1, 130.1, 57.0, 56.1, 55.2, 54.6, 44.2, 40.5, 39.0, 36.2, 35.8, 33.1, 32.7, 32.1, 32.0, 31.8, 30.4, 29.8, 29.8, 28.6, 26.0, 24.2, 23.2, 20.9, 15.3, 13.5, 11.6.

[0090] The structural formula of compound 5g is as follows:

[0091]

[0092] Compound 5h: Yellow powder, yield 52%. HR-ESI-MS m / z calcd for C 28 H 43 N2O2([M+H] + ) 439.3319, found 439.3316; 1 1H-NMR (400 MHz, CDCl3) δ 7.83 (1H, s), 6.85 (1H, td, J = 7.6, 1.4 Hz), 6.77 (1H, dd, J = 8.0, 1.4 Hz), 6.72–6.57 (2H, m), 3.86 (3H, d, J = 13.2 Hz), 3.69–3.64 (1H, m), 2.21 (1H, qd, J = 9.3, 7.5, 3.1 Hz), 2.12–2.03 (1H, m), 1.88 (4H, s), 1.79–1.64 (5H, m), 1.62–1.49 (3H, m), 1.44–1.07 (12H, m), 0.93 (1H, ddd, J = 17.7, 15.4, 9.3 Hz,), 0.84 (3H, d, J = 3.3 Hz), 0.62 (3H, s); 13 13C-NMR (100 MHz, CDCl3) δ 159.3, 121.5, 109.6, 57.0, 56.1, 56.1, 55.6, 54.5, 44.2, 40.5, 39.1, 36.3, 36.1, 35.8, 33.2, 32.0, 28.6, 26.0, 24.2, 23.2, 20.9, 15.2, 13.5, 11.6.

[0093] The structural formula of Compound 5h is as follows:

[0094]

[0095] Experimental Example 1: The present invention screened the cytotoxic activities of the synthesized novel C20-oxime pregnane alkaloids against human liver cancer cells (HepG2, SK-Hep1) and human colorectal cancer cells (HCT116, SW480, DLD-1), and further illustrated the present invention through the following embodiments.

[0096] I. Study on the cytotoxic activity of the compounds of the present invention:

[0097] 1. Experimental principle: The full English name of CCK-8 is Cell Counting Kit-8. The main chemical drug is WST-8, whose chemical name is 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt. Its core group is the same as that of MTT, and it is an upgraded version of MTT. In the presence of an electron-coupling reagent (that is, when the cells are alive, breathing, and have energy metabolism), it can be oxidized and reduced by NAD+ to form a water-soluble yellow formazan product. The more living cells there are, the more formazan is produced and the darker the color will be.

[0098] 2. Experimental steps: Take cells in the logarithmic growth phase. After digesting with 0.25% EDTA (1×) trypsin until the cells are in a suspended state, add a medium containing serum to dilute the trypsin concentration and terminate the digestion. Prepare a single-cell suspension with a density of 2×10 4 cells / mL and inoculate 100 μL / well into a 96-well plate. Add 100 μL of PBS buffer to the outer cell culture wells to reduce the influence of edge effects on the experimental results. Incubate statically in a 37 °C, 5% CO2 incubator for 24 h. After observing cell attachment, add 100 μL of the culture medium containing the alkaloid derivative to be tested. The concentration range of the alkaloid derivative test solution is set as: 20, 10, 5, 2.5, 1.25, 0.625 μM, and 3 replicate wells are set for each concentration. Leave three wells with only the cell culture medium as the blank control, and use the well with the culture medium containing only DMSO without adding the drug as the negative control. After culturing for 72 h, discard the supernatant, add 100 μL of the CCK-8 working solution of 10% CCK-8, continue to incubate in the incubator for 2 h, then measure the absorbance value at a wavelength of 450 nm with an enzyme-linked immunosorbent detector. After subtracting the blank control value from all absorbance values first, the relative cell inhibition rate of the experimental group drug is obtained by comparing with the control group value. The drug cell inhibition rate = [OD control - OD experiment] / [OD control - OD negative] × 100%. The concentration at which the growth inhibition rate of the drug on tumor cells is 50% can be obtained from the growth inhibition rates of different concentrations of the same drug, that is, the half-maximal inhibitory concentration IC 50 . Since each alkaloid derivative at the same concentration occupies 3 wells in the 96-well plate, that is, the cells are averaged three times at the same concentration and the average value is taken, and each experiment is repeated three times. The obtained data are expressed as "mean + standard error" (mean + SEM), and a Student's t-test is used for significance analysis. A significant difference is considered when P < 0.05.

[0099] 3. Experimental results:

[0100] Table 1 IC of the compounds against HepG2, SK-Hep-1, HCT-116, SW480, DLD-1 50 (Unit: μmol / mL)

[0101]

[0102] Note: * represents the IC 50 superior to the positive control

[0103] The test results of the target derivatives against hepatocellular carcinoma cells (HepG2, SK-Hep1) showed (Table 1): The IC 50 values of compounds 5b, 5c, 5e, 5f, 5g, 5h against hepatocellular carcinoma cell HepG2 were all superior to the positive control sorafenib; the IC 50 values of compounds 5a, 5b, 5d, 5e, 5f, 5h against hepatocellular carcinoma cell SK-Hep1 were all superior to the positive control; the test results of the target derivatives against colorectal cancer cells (HCT116, SW480, DLD-1) showed (Table 1): The IC 50 value of compound 5f against HCT116 was 0.05 μmol / mL, which was superior to the positive control.

[0104] II. The present invention screened the in vitro anti-inflammatory activity of the newly synthesized C20 oxime-based pregnane alkaloids, tested the activity of the synthesized derivatives on RAW264.7 cells, and then starting from the perspective of inflammation theory, an inflammatory cell model was established by inducing mouse mononuclear macrophage cell line (RAW264.7) with lipopolysaccharide. Subsequently, whether the synthesized compounds had anti-inflammatory effects was judged by detecting the nitric oxide release amount of the cells. And the following experiments were carried out to further illustrate the present invention.

[0105] 1. Experimental principle: The basic principle of the CCK-8 method is the same as above. NO is easily oxidized to NO 2- in vivo or in aqueous solution and other environments. However, under acidic conditions, NO can undergo a series of reactions to form a pink special substance, which has the maximum absorption peak at 540 nm, and the NO concentration can be calculated by the OD value.

[0106] 2. Experimental steps:

[0107] (1) The experimental steps of the cytotoxicity of pregnane alkaloid derivatives against RAW264.7 cells are the same as above.

[0108] (2) Determination of the NO secretion amount in RAW264.7 cells by the Griess method

[0109] Take cells in the logarithmic growth phase and prepare a density of 1×10 5A single-cell suspension of cells / mL was inoculated into a 96-well plate at 100 μL / well. 100 μL of PBS buffer was added to the outer cell culture wells to reduce the influence of edge effects on the experimental results. It was statically cultured in an incubator at 37 °C and 5% CO2 for 20 h. After observing cell adhesion, 100 μL of the culture medium containing the alkaloid derivative to be tested was added. The concentration range of the alkaloid derivative test solution was set as: 200, 100, 50, 25, 12.5, 6.25 μM, and 3 replicate wells were set for each concentration. Three wells were reserved with only the cell culture medium as the normal group, and the culture medium well with only DMSO added without drug was used as the blank group. After statically culturing in an incubator at 37 °C and 5% CO2 for 1 h, LPS with a concentration of 1 μg / mL was added to stimulate the production of inflammation, and finally it was cultured in an incubator at 37 °C and 5% CO2 for 20 h.

[0110] Take out Griess Reagent I and II, let them return to room temperature, make a standard curve according to the reagent instruction manual. Add the standard product and samples to a new 96-well plate at 50 μL / well for the cell supernatant of the cultured cells. Add Griess Reagent I to each well at 50 μl / well first, then Griess Reagent II, and finally measure its absorbance at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the concentration of nitric oxide in the sample according to the standard product curve.

[0111] 3. Experimental results

[0112] The survival rate of C20 oxime-based pregnane alkaloids on RAW264.7 cells was determined by the CCK8 experiment at 6 different concentrations. As Figure 9 shown: For compounds 5a - 5h, when the dosing concentration of compound 5a was below 200, there was no significant difference compared with the normal group and no cytotoxicity. Compounds 5c and 5g would not affect macrophages RAW264.7 at a dose below 25 μM; the safe concentration ranges of the target compounds varied greatly.

[0113] Nitric oxide (NO) is a signal molecule that plays a key role in the inflammatory pathogenesis mechanism. It is a pro-inflammatory mediator that is over-secreted under abnormal conditions and then causes inflammation. When RAW264.7 cells are stimulated with LPS, a large amount of NO is released from the cells into the culture medium and exists in the form of nitrite, and the content of nitrite can be measured with Griess reagent. Due to the different survival rates of different compounds, we set different concentration gradients within their safe concentration ranges to explore the effect of the compounds on the NO expression level in the RAW264.7 cell inflammation model stimulated by LPS (1 μg / mL) after 24 h. The experimental results showed that all compounds could reduce the secretion of NO to a certain extent. As Figure 10Shown as follows: all the administered concentrations of compound 5b and 5h had extremely significant differences compared with the model group (P≤0.01), and could well inhibit the secretion of NO in cells; for compound 5a, there was no significant difference in other administered concentrations compared with the model group except 6.25 μM.

[0114] Although the present invention has been described in detail in the above text with general descriptions, specific embodiments and experiments, some modifications or improvements can be made on the basis of the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. An alkaloid derivative, characterized in that, There are 8 kinds of the alkaloid derivative compounds, which are sequentially denoted as 5a, 5b, 5c, 5d, 5e, 5f, 5g, and 5h, and the corresponding relationships of R1 are as follows:

2. A method for synthesizing the alkaloid derivative according to claim 1, characterized in that, The steps include the following: (1) Synthesis of compound 1: Potassium tert-butoxide and ethyltriphenylphosphonium bromide are added to a flask, evacuated, an appropriate amount of anhydrous tetrahydrofuran is added, stirred at room temperature for 1 h, a pre-prepared tetrahydrofuran solution of epiatestosterone is added, stirred at room temperature for 3 h, the reaction is monitored by TLC until completion, then a saturated ammonium chloride solution is added to the reaction solution to quench the reaction, extracted with ethyl acetate, washed with saturated brine, the organic layer is dried with anhydrous magnesium sulfate, filtered, concentrated under reduced pressure to obtain a crude product, and purified by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate = 5∶1 to obtain compound 1; (2) Synthesis of compound 2: BH3-Me2S is added to the tetrahydrofuran solution of compound 1, stirred at 25 °C for 2 h, then ethanol, NaOH, and H2O2 are added dropwise, stirred at 25 °C for 2 h, the mixture is quenched with Na2S2O3, and extracted with ethyl acetate. The synthesized organic solution is washed with saturated brine, dried with Na2SO4, filtered and concentrated to obtain compound 2; (3) Synthesis of compound 3: Compound 2, PCC, and an appropriate amount of silica gel are added to DCM, the mixture is stirred at room temperature for 3 h, the mixture is filtered, the filter cake is washed with DCM, the solvent is recovered under reduced pressure, and silica gel column chromatography is carried out with the mobile phase of petroleum ether∶ethyl acetate to obtain compound 3; (4) Synthesis of compound 4a: Compound 3 is placed in a flask, acetic acid is added, aniline is added by injection, stirred for 12 h, sodium borohydride is added slowly in small portions in multiple batches, stirred at room temperature for 2 h, acetic acid is removed under reduced pressure, water is added to the solid, and the pH is adjusted to neutral or weakly alkaline with dilute sodium hydroxide solution, extracted with ethyl acetate, dried with Na2SO4, concentrated under reduced pressure to obtain a crude product, and purified by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate for gradient elution to obtain compound 4a; Synthesis of compounds 4b - 4h: Operate according to the synthesis steps of compound 4a above, react with p-toluidine to obtain compound 4b; react with p-methoxyaniline to obtain compound 4c; react with p-chloroaniline to obtain compound 4d; react with o-toluidine to obtain compound 4e; react with m-toluidine to obtain compound 4f; react with m-methoxyaniline to obtain compound 4g; react with o-methoxyaniline to obtain compound 4h; (5) Synthesis of compound 5a: Compound 4a is placed in a flask, potassium hydroxide and hydroxylamine hydrochloride are added, ethanol is added, stirred at 15 °C for 16 h, the reaction is monitored by TLC until completion, water is added, extracted with ethyl acetate, the organic phases are combined, washed with saturated brine, dried with anhydrous magnesium sulfate, filtered, concentrated under reduced pressure to obtain a crude product, and purified by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate for gradient elution to obtain compound 5a; Synthesis of Compounds 5b - 5h: Operate according to the synthesis procedure of Compound 5a described above, react with Compound 4b to obtain Compound 5b; react with Compound 4c to obtain Compound 5c; react with Compound 4d to obtain Compound 5d; react with Compound 4e to obtain Compound 5e; react with Compound 4f to obtain Compound 5f; react with Compound 4g to obtain Compound 5g; react with Compound 4h to obtain Compound 5h; The synthesis procedure is as follows:

3. A method for synthesizing the alkaloid derivative according to claim 1, characterized in that, The specific synthesis method includes the following steps: (1) Synthesis of Compound 1: Weigh 2.34 g of potassium tert - butoxide and 7.67 g of ethyltriphenylphosphonium bromide respectively, add them to a flask, evacuate, add 15 mL of anhydrous tetrahydrofuran, stir at room temperature for 1 h, add a pre - prepared tetrahydrofuran solution of 1 g (3.5 mmol) of epiandrosterone, stir at room temperature for 3 h, monitor the completion of the reaction by TLC, then add saturated ammonium chloride solution to quench the reaction, extract with ethyl acetate 3 times, 100 mL each time, combine the extracts, wash with saturated brine 2 times, 100 mL each time, dry the organic layer with anhydrous magnesium sulfate, filter, concentrate under reduced pressure to obtain a crude product, and purify it by silica gel chromatography with the mobile phase of petroleum ether: ethyl acetate = 5:1 to obtain Compound 1; (2) Synthesis of Compound 2: Add 10 M BH3 - Me2S to a 100 mL THF solution of 5 g (15.0 mmol) of Compound 1, stir at 25 °C for 2 h, then add 10 mL of ethanol, 30 mL of 5 M NaOH and 15 mL of 10 M H2O2 dropwise, stir at 60 °C for 1 h, quench the mixture with 400 mL of 10% Na2S2O3 solution, and extract with ethyl acetate 2 times, 300 mL each time. Wash the synthesized organic solution with 300 mL of saturated brine, dry with Na2SO4, filter and concentrate to obtain Compound 2; (3) Synthesis of Compound 3: Weigh 1 g of Compound 2 and 2.76 g of PCC, add an appropriate amount of silica gel to 100 mL of DCM, stir the mixture at room temperature for 3 h, filter the mixture, wash the filter cake with DCM 2 times, 100 mL each time, recover the solvent under reduced pressure, and perform silica gel column chromatography to obtain Compound 3; (4) Synthesis of Compound 4: Weigh 100 mg of Compound 3 and place it in a 25 mL flask, add 1 mL of acetic acid, inject 23.70 μL of aniline, stir overnight, slowly add 22.70 mg of sodium borohydride in portions, stir at room temperature for 2 h, remove acetic acid under reduced pressure, add water to the solid, adjust the pH to neutral or weakly alkaline with dilute sodium hydroxide solution, extract with ethyl acetate, concentrate under reduced pressure to obtain a crude product, and purify it by silica gel chromatography with the mobile phase of petroleum ether: ethyl acetate to obtain Compound 4; Synthesis of compounds 4b - 4h: Operate according to the synthesis procedure of compound 4a above, react with p - toluidine to obtain compound 4b; react with p - anisidine to obtain compound 4c; react with p - chloroaniline to obtain compound 4d; react with o - toluidine to obtain compound 4e; react with m - toluidine to obtain compound 4f; react with m - anisidine to obtain compound 4g; react with o - anisidine to obtain compound 4h; (5) Synthesis of compounds 5a - 5h: Weigh 50 mg of compound 4a and place it in a 25 - mL flask, add 21.38 mg of potassium tert - butoxide and 18.07 mg of hydroxylamine hydrochloride, add 3 mL of ethanol, stir at 15 °C for 16 h, monitor the completion of the reaction by TLC, add water, extract with ethyl acetate three times, 20 mL each time, combine the organic phases, wash with saturated brine, dry over anhydrous magnesium sulfate, filter, concentrate under reduced pressure to obtain the crude product, and purify by silica gel chromatography with the mobile phase of petroleum ether∶ethyl acetate to obtain compound 5a; Synthesis of compounds 5b - 5h: Operate according to the synthesis procedure of compound 5a above, react with compound 4b to obtain compound 5b; react with compound 4c to obtain compound 5c; react with compound 4d to obtain compound 5d; react with compound 4e to obtain compound 5e; react with compound 4f to obtain compound 5f; react with compound 4g to obtain compound 5g; react with compound 4h to obtain compound 5h.

4. The synthesis method of the alkaloid derivative according to claim 2, wherein, In the said step (1), the molar ratio of potassium tert - butoxide, ethyltriphenylphosphonium bromide to epiandrosterone is 6∶6∶1.

5. The synthesis method of the alkaloid derivative according to claim 2, characterized in that, In the said step (2), the molar ratio of BH3 - Me2S to compound 1 is 5∶1.

6. The synthesis method of the alkaloid derivative according to claim 2, wherein, In the said step (3), the molar ratio of compound 2 to PCC is 1∶4.

7. The synthesis method of the alkaloid derivative according to claim 2, wherein, In the said step (4), the molar ratio of compound 3, aniline to sodium borohydride is 1.6∶1.3∶3.

8. The synthesis method of the alkaloid derivative according to claim 2, characterized in that, In the said step (5), the molar ratio of compound 4a, potassium hydroxide to hydroxylamine hydrochloride is 1∶3∶2.

9. Use of the alkaloid derivative as claimed in claim 1 in the preparation of anti - liver cancer, anti - colorectal cancer and anti - inflammatory drugs.

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