Preparation method of natural alkaloid (+)-rhynchine E compound

Abstract

The invention belongs to the technical field of organic synthesis, and particularly relates to a preparation method of a natural alkaloid (+)-rhynchine E compound. According to the compound preparation method provided by the invention, the simple and easily available commercial raw material (R)-N-Boc-pyroglutamic acid tert-butyl ester is used for carrying out multi-step chemical combination synthesis, and finally, the successful preparation of the natural alkaloid (+)-rhynchine E is realized. The preparation method provided by the invention is clear in synthesis raw materials and process and high in product yield, a new synthesis strategy can be provided for preparation of the (+)-rhynchine E compound, and the problems of low yield, low efficiency and the like when the compound is separated from natural plants are solved. Particularly, biological activity tests show that the compound prepared by the invention has a remarkable inhibition effect on hERG, and shows application potential in research and development of new drugs for resisting arrhythmia and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, and particularly relates to a method for preparing a natural alkaloid (+)-rhynchine E compound. Background Art

[0002] Uncaria rhynchophylla, a plant of the Rubiaceae family, is a widely used Chinese herbal medicine that has the effects of calming wind and relieving convulsions, clearing away heat and calming the liver. It is mainly used to treat hypertension, headaches, dizziness and febrile convulsions. To date, researchers have isolated nearly 130 indole alkaloids from Uncaria rhynchophylla, mainly oxidized indole and coryneform types, many of which have good antihypertensive, antiepileptic and antidepressant activities.

[0003] (+)-rhynchine E is an indole alkaloid with a novel skeleton isolated from Uncaria rhynchophylla. However, as a secondary metabolite in plants, its natural content is very low, and the yield of such compounds isolated from natural plants is low, which limits its development and application process. At the same time, due to its own toxicity, absorption or metabolism problems, it must undergo certain structural modifications or transformations before it can be developed into a drug. From a synthetic point of view, (+)-rhynchine E contains a complex and novel fused ring and spiro ring skeleton and densely arranged chiral centers, and the molecular structure contains units such as indole, azacycloheptane, cyclopentane, and furan rings, which presents great synthetic challenges.

[0004] Therefore, conducting total synthesis research on (+)-rhynchine E and exploring simple and efficient general synthetic strategies are of great significance for promoting the application of (+)-rhynchine E in drug research and development. Summary of the invention

[0005] In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a method for preparing a natural alkaloid (+)-rhynchine E compound, which starts from simple and easily available commercial raw materials and is used for the preparation of the alkaloid (+)-rhynchine E compound, thereby solving the problems of low yield and low efficiency in isolating such compounds from natural plants.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for preparing a natural alkaloid (+)-rhynchine E compound comprises the following steps:

[0008] (1) reacting the compound of formula 1 with lithium hexamethyldisilazide, triphenyl phosphate, and ethyl trifluoromethanesulfonate, and then purifying to obtain a compound of formula 2;

[0009] (2) reacting the compound of formula 2 with borane dimethyl sulfide, and then purifying to obtain a compound of formula 3;

[0010] (3) reacting the compound of formula 3 with trimethylsilyl trifluoromethanesulfonate, and then purifying to obtain a compound of formula 4;

[0011] (4) subjecting the compound of formula 4 to an oxidation reaction with N-chlorosuccinimide, then adding allyl chloroformate to carry out an isomerization reaction, and then purifying to obtain a compound of formula 5;

[0012] (5) subjecting the compound of formula 5 to Michael addition reaction with dimethyl malonate in the presence of sodium hydride, followed by purification to obtain a compound of formula 6;

[0013] (6) reacting the compound of formula 6 with phenylsilane and (triphenylphosphine) palladium to remove the Alloc protecting group, and then purifying to obtain a compound of formula 7;

[0014] (7) subjecting the compound of formula 7 to amidation reaction with the compound of formula 8, followed by acid hydrolysis and purification to obtain a compound of formula 9;

[0015] (8) subjecting the compound of formula 9 to a Friedel-Crafts acylation reaction with cyanuric chloride, pyridine and aluminum chloride, followed by purification to obtain a compound of formula 10;

[0016] (9) subjecting the compound of formula 10 to a selective hydrogenation reaction with tris(triphenylphosphine)carbonyl rhodium hydride and phenylsilane, followed by purification to obtain a compound of formula 11;

[0017] (10) subjecting the compound of formula 11 to a hydroxymethylation reaction in the presence of scandium trifluoromethanesulfonate and formaldehyde, followed by oxidative cyclization in air, and then purification to obtain a compound of formula 12;

[0018] (11) subjecting the compound of formula 12 to a Krapcho decarboxylation reaction with potassium cyanide, followed by purification to obtain the natural alkaloid (+)-rhynchine E compound;

[0019] Wherein, the structures of the compounds of Formula 1 to Formula 12 are:

[0020]

[0021]

[0022] The structural formula of the natural alkaloid (+)-rhynchine E compound is shown in Formula E:

[0023]

[0024] As a preferred embodiment, in step (1), the molar ratio of the compound of formula 1, lithium hexamethyldisilazide, triphenyl phosphate, and ethyl trifluoromethanesulfonate is 1:1.2:1:1.1; in step (2), the molar ratio of the compound of formula 2 to borane dimethyl sulfide is 1:2; in step (3), the molar ratio of the compound of formula 3 to trimethylsilane trifluoromethanesulfonate is 1:0.5; in step (4), the molar ratio of the compound of formula 4, N-chlorosuccinimide, and allyl chloroformate is 1:1.2:2.3; in step (5), the molar ratio of the compound of formula 5, dimethyl malonate, and sodium hydride is 1:1.5:1.5; in step (6 ), the molar ratio of the compound of formula 6, phenylsilane and (triphenylphosphine) palladium is 1:5:0.05; in step (7), the molar ratio of the compound of formula 7 and the compound of formula 8 is 1:1; in step (8), the molar ratio of the compound of formula 9, cyanuric chloride, pyridine and aluminum chloride is 1:1.6:1:1.3; in step (9), the molar ratio of the compound of formula 10, tri(triphenylphosphine)carbonyl rhodium hydride and phenylsilane is 1:0.15:5; in step (10), the molar ratio of the compound of formula 11, scandium trifluoromethanesulfonate and formaldehyde is 1:2:5; in step (11), the molar ratio of the compound of formula 12 and potassium cyanide is 1:5.

[0025] As a preferred embodiment, in step (1), the reaction is carried out at -80 to -70°C for 1 to 3 hours; in step (2), the reaction is carried out under heating reflux for 6 to 8 hours; in step (3), the reaction is carried out at -5 to 5°C for 4 to 10 minutes; in step (4), the oxidation reaction is carried out at -5 to 5°C for 1 to 2 hours, and the isomerization reaction is carried out under heating reflux for 1 to 3 hours; in step (5), the Michael addition reaction is carried out at 20 to 30°C for 6 to 8 hours; in step (6), the reaction is carried out at 20 to 30°C for 20 to 40 minutes; in step (7), the amidation reaction is carried out at 20 ~30℃ for 0.5~2h, and the acid hydrolysis is reacted at 20~30℃ for 4~6h; in step (8), the Friedel-Crafts acylation reaction is first reacted at 20~30℃ for 20~40min, and then reacted under heating reflux for 10~15h; in step (9), the selective hydrogenation reaction is reacted at 20~30℃ for 0.5~2h; in step (10), the hydroxymethylation reaction is reacted at 20~30℃ for 1~3h, and the oxidative cyclization is reacted at 20~30℃ for 10~15h; in step (11), the Krapcho decarboxylation reaction is reacted at 70~90℃ for 3~6h.

[0026] As a preferred embodiment, in step (1), the reaction is carried out in tetrahydrofuran; in step (2), the reaction is carried out in tetrahydrofuran; in step (3), the reaction is carried out in dichloromethane; in step (4), the oxidation reaction is carried out in dichloromethane; in step (5), the Michael addition reaction is carried out in tetrahydrofuran; in step (6), the reaction is carried out in dichloromethane; in step (7), the amidation reaction is carried out in dichloromethane; in step (8), the Friedel-Crafts acylation reaction is carried out in dichloromethane; in step (9), the selective hydrogenation reaction is carried out in tetrahydrofuran; in step (10), the hydroxymethylation reaction is carried out in tetrahydrofuran; in step (11), the Krapcho decarboxylation reaction is carried out in a dimethyl sulfoxide aqueous solution.

[0027] As a preferred embodiment, in step (7), the acid hydrolysis is carried out by adding formic acid or acetic acid.

[0028] As a preferred embodiment, in step (10), the oxidative cyclization is carried out after adding a saturated sodium bicarbonate aqueous solution.

[0029] As a preferred solution, the reactions in step (2), step (9) and step (10) are carried out in a protective atmosphere. As a further preferred solution, the protective atmosphere is argon.

[0030] As a preferred embodiment, in steps (1) to (11), the purification is performed by silica gel column chromatography.

[0031] The beneficial effects of the above technical solution of the present invention are:

[0032] The method for preparing the natural alkaloid (+)-rhynchine E compound provided by the present invention starts from the simple and easily available commercial raw material (R)-N-Boc-t-butyl pyroglutamate, and performs a multi-step chemical synthesis, and finally achieves the successful preparation of the natural alkaloid (+)-rhynchine E compound. The above-mentioned preparation method provided by the present invention has clear synthetic raw materials and processes, and a high product yield. It can provide a new synthetic strategy for the preparation of natural alkaloid (+)-rhynchine E compounds, and solve the problems of low yield and low efficiency in isolating such compounds from natural plants. In particular, the biological activity test shows that the (+)-rhynchine E compound prepared by the present invention has a significant inhibitory effect on hERG (IC 50 value 5.74μM), demonstrating its potential for application in the development of new drugs for diseases such as arrhythmias. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1is the concentration-response curve of the positive control terfenadine in the test example of the present invention;

[0034] Figure 2 The concentration-response curve of the natural alkaloid (+)-rhynchine E compound in the test example of the present invention. DETAILED DESCRIPTION

[0035] The technical scheme and technical effects of the present invention will be clearly and completely described below in conjunction with specific embodiments and test examples. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical scheme of the present invention and should not be regarded as limiting the scope of protection of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the raw materials used are commonly used in the art, available to the public or available through commercial channels unless otherwise specified. In the following examples, room temperature has the conventional meaning in the art, specifically 25±5°C.

[0036] The present invention provides a method for preparing a natural alkaloid (+)-rhynchine E compound, comprising the following steps:

[0037] (1) reacting the compound of formula 1 with lithium hexamethyldisilazide, triphenyl phosphate, and ethyl trifluoromethanesulfonate, and then purifying to obtain a compound of formula 2;

[0038] (2) reacting the compound of formula 2 with borane dimethyl sulfide, and then purifying to obtain a compound of formula 3;

[0039] (3) reacting the compound of formula 3 with trimethylsilyl trifluoromethanesulfonate, and then purifying to obtain a compound of formula 4;

[0040] (4) subjecting the compound of formula 4 to an oxidation reaction with N-chlorosuccinimide, then adding allyl chloroformate to carry out an isomerization reaction, and then purifying to obtain a compound of formula 5;

[0041] (5) subjecting the compound of formula 5 to Michael addition reaction with dimethyl malonate in the presence of sodium hydride, followed by purification to obtain a compound of formula 6;

[0042] (6) reacting the compound of formula 6 with phenylsilane and (triphenylphosphine) palladium to remove the Alloc protecting group, and then purifying to obtain a compound of formula 7;

[0043] (7) subjecting the compound of formula 7 to amidation reaction with the compound of formula 8, followed by acid hydrolysis and purification to obtain a compound of formula 9;

[0044] (8) subjecting the compound of formula 9 to a Friedel-Crafts acylation reaction with cyanuric chloride, pyridine and aluminum chloride, followed by purification to obtain a compound of formula 10;

[0045] (9) subjecting the compound of formula 10 to a selective hydrogenation reaction with tris(triphenylphosphine)carbonyl rhodium hydride and phenylsilane, followed by purification to obtain a compound of formula 11;

[0046] (10) subjecting the compound of formula 11 to a hydroxymethylation reaction in the presence of scandium trifluoromethanesulfonate and formaldehyde, followed by oxidative cyclization in air, and then purification to obtain a compound of formula 12;

[0047] (11) subjecting the compound of formula 12 to a Krapcho decarboxylation reaction with potassium cyanide, followed by purification to obtain the natural alkaloid (+)-rhynchine E compound;

[0048] Wherein, the structures of the compounds of Formula 1 to Formula 12 are:

[0049]

[0050] The structural formula of the natural alkaloid (+)-rhynchine E compound is shown in Formula E:

[0051]

[0052] Among them, in the following examples, the compounds of formula 1 to formula 12 involved are respectively recorded as compounds 1 to 12. Compound 1 is (R)-N-Boc-pyroglutamic acid tert-butyl ester, which is derived from conventional commercial channels (Anhui Zesheng Technology Co., Ltd.). Compound 3 is a pyrrole derivative; Compound 4 is tert-butyl proline; Compound 5 is a Michael addition precursor; Compound 6 is a diastereoisomer; Compound 7 is a chiral proline fragment; Compound 8 is indoleacetic acid, which is derived from conventional commercial channels; Compound 10 is a 6 / 5 / 7 / 5 tetracyclic intermediate; Compound 11 is a tertiary amine.

[0053] Example 1

[0054] This embodiment provides a method for preparing a natural alkaloid (+)-rhynchine E compound, comprising the following steps:

[0055] (1) Preparation of compound 2, the technical route is shown below:

[0056]

[0057] To a tetrahydrofuran solution (200 mL) of compound 1 (20.00 g, 70.16 mmol, 1.0 equiv) was added a lithium hexamethyldisilazide solution (abbreviated as LiHMDS, 84 mL, concentration 1.00 M in 200 mL) at -78 °C. THF, 1.2 equiv); 1 hour later, triphenyl phosphate (22.80 g, 70.16 mmol, 1.0 equiv) was added to the system, followed by ethyl trifluoromethanesulfonate (9.4 mL, 77.18 mmol, 1.1 equiv), stirred at -78 ° C for 2 h, quenched the reaction mixture with saturated ammonium chloride solution, and extracted the aqueous layer with ethyl acetate 3 times; then washed the combined organic layer with saturated brine, dried with anhydrous sodium sulfate, filtered, and finally evaporated to dryness; separated and purified the product by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate = 15:1), and 10.33 g of compound 2 and 5.27 g of compound 2' were obtained, with yields of 47% and 24%, respectively. The structural characterization results of compound 2 are as follows: 1 H NMR(600MHz,Chloroform-d)δ4.37(dd,J=8.8,6.1Hz,1H),2.60-2.28(m,2H),2.02-1.76 (m,1H),1.65(dd,J=12.5,6.4Hz,1H),1.50(s,9H),1.47(s,10H),0.96(t,J=7.4Hz,3H); 13 C NMR (151MHz, Chloroform-d) δ175.4,170.7,149.7,83.4,82.2,58.2,44.2,28.0(6C),27.2,24.4,11.6ppm.

[0058] Compound 2' is further transformed into compound 2, and the technical route is shown as follows:

[0059]

[0060] Under argon atmosphere, to a stirred solution of 2' (3.00 g, 9.59 mmol, 1.0 equiv) in tetrahydrofuran (40 mL) at room temperature was added tetrabutylammonium fluoride (10.16 mL, 1.0 M in THF, 1.06 equiv); the resulting mixture was stirred under reflux for 1.5 h, then cooled to room temperature and quenched by the addition of saturated ammonium chloride solution, the aqueous layer was extracted three times with ethyl acetate, and the combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate and concentrated in vacuo; the product was separated and purified by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate = 15:1) to obtain 1.80 g of compound 2 with a yield of 30%, and 2.70 g of compound 2' with a yield of 45%.

[0061] (2) Preparation of compound 3, the technical route is shown below:

[0062]

[0063] Under argon atmosphere, borane dimethyl sulfide (BH3·SMe2, 30.35mL, 60.70mmol, 2.0equiv) was added dropwise to a stirred solution of compound 2 (9.50g, 30.35mmol, 1.0equiv) in tetrahydrofuran (100mL); the resulting solution was heated and stirred under reflux for 7 hours; after cooling to room temperature, the reaction mixture was quenched with cold water and extracted with ethyl acetate three times; the combined organic layer was dried over anhydrous sodium sulfate, filtered, and finally evaporated to dryness. The product was separated and purified by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate = 17:1) to obtain 7.08g of compound 3 with a yield of 78%. The structural characterization results of compound 3 are as follows: 1 H NMR(600MHz,Chloroform-d)δ4.09(dt,J=29.3,8.3Hz,1H),3.76(dd,J=10.5,7.2Hz,1H),2.94(td,J=10.1,6.7Hz,1H), 2.44-2.32(m,1H),2.01(ttd,J=17.0,14.1,10.7,8.5,2.9Hz,1H),1.51-1.33(m,21H),0.90ppm(td,J=7.5,3.4Hz,3H); 13 C NMR (151MHz, Chloroform-d) δ172.2,153.8,80.6,79.4,59.8,52.0,39.9,36.4,28.2(6C),25.8,12.5ppm.

[0064] (3) Preparation of compound 4, the technical route is shown below:

[0065]

[0066] Trimethylsilyl trifluoromethanesulfonate (2.00 mL, 11.03 mmol, 0.5 equiv) was added to a dichloromethane (60 mL) solution of compound 3 (6.60 g, 22.07 mmol, 1.0 equiv), and the reaction mixture was quenched with a saturated sodium bicarbonate solution (1 mL / mmol) and stirred for 15 minutes; the aqueous layer was then extracted 3 times with dichloromethane, the combined organic layer was dried with anhydrous sodium sulfate, filtered, and finally evaporated to dryness; the product was separated and purified by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate = 1:1) to obtain 3.73 g of compound 4 with a yield of 85%. The structural characterization results of compound 4 are as follows:1 HNMR(600MHz,Chloroform-d)δ3.65(t,J=8.0Hz,1H),3.05(dd,J=9.8,7.0Hz,1H),2.64(t,J=9.1Hz,1H),2.29(dd,J=12 .8,7.7Hz,1H),2.25(d,J=7.2Hz,1H),2.01(p,J=7.6Hz,1H),1.45(s,9H),1.39-1.31(m,3H),0.89ppm(t,J=7.4Hz,3H); 13 C NMR (151MHz, Chloroform-d) δ175.0,81.1,60.6,52.8,41.9,37.0,28.2(3C),26.8,13.0ppm.

[0067] (4) Preparation of compound 5, the technical route is shown below:

[0068]

[0069] To a solution of compound 4 (3.53 g, 17.74 mmol, 1.0 equiv) in dichloromethane (40 mL) at 0°C, triethylamine (6.15 mL, 44.35 mmol, 2.5 equiv) and N-chlorosuccinimide (2.83 g, 21.29 mmol, 1.2 equiv) were added in sequence; after stirring for 1.5 hours, pyridine (3.28 mL, 40.80 mmol, 2.3 equiv) and allyl chloroformate (4.32 mL, 40.80 mmol, 2.3 equiv) were added at -20°C; the resulting solution was stirred and heated to reflux for 2 hours; the reaction mixture was quenched with saturated ammonium chloride solution; the aqueous layer was then extracted with dichloromethane three times, the combined organic layers were dried over anhydrous sodium sulfate, filtered, and finally evaporated to dryness; the product was separated and purified by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate = 15:1) to obtain 4.34 g of compound 5 with a yield of 87%. The structural characterization results of compound 5 are: 1 H NMR(600MHz,Chloroform-d)δ6.09-5.83(m,1H),5.71(d,J=2.8Hz,1H),5.46-5.23(m,1H),5.20(dq,J=10.5,1.3Hz,1H),4.78-4.40(m,2 H),4.04(dd,J=11.3,9.8Hz,1H),3.57(dd,J=11.3,6.7Hz,1H),2.98-2.67(m,1H),1.50(s,9H),1.49-1.39(m,2H),0.91(t,J=7.4Hz,3H);13 C NMR (151MHz, Chloroform-d) δ161.2,153.5,137.3,132.7,122.9,118.0,82.1,66.5,54.5,43.1,28.1,27.3(3C),11.5ppm.

[0070] (5) Preparation of compound 6, the technical route is shown below:

[0071]

[0072] At 0°C, sodium hydride (800 mg, 20.00 mmol, 1.5 equiv) and dimethyl malonate (2.30 mL, 20.00 mmol, 1.5 equiv) were added to anhydrous tetrahydrofuran (40 mL); after 30 minutes, compound 5 (4.04 g, 13.33 mmol, 1.0 equiv) was added; the reaction mixture was stirred at room temperature for 7 hours; the reaction was quenched with saturated ammonium chloride solution and extracted with ethyl acetate three times; the combined organic layer was dried over anhydrous sodium sulfate, filtered, and finally evaporated to dryness. The product was separated and purified by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate = 5:1) to obtain 4.88 g of compound 6 with a yield of 89%. The structural characterization results of compound 6 are as follows: 1 H NMR(600MHz,Chloroform-d)δ5.89(dddt,J=35.7,16.3,10.7,5.5Hz,1H),5.28(ddd,J=19.0,17.2,1.8Hz, 1H),5.21-5.10(m,1H),4.62-4.49(m,2H),4.19(dd,J=12.4,4.6Hz,1H),3.84-3.71(m,7H),3.57(dd,J=7.1 ,3.2Hz,1H),3.19(dt,J=11.2,6.1Hz,1H),2.71(dt,J=6.5,5.0Hz,1H),2.00(ddt,J=34.4,15.2,6.4Hz,1H ),1.58-1.49(m,1H),1.44(d,J=13.5Hz,9H),1.33(ddd,J=13.7,9.3,7.1Hz,1H),0.88ppm(q,J=7.0Hz,3H); 13 C NMR (151MHz, Chloroform-d) δ170.9,168.2(2C),154.4,132.9,117.5,81.9,66.1,63.0,54.0,52.8(2C),51.2,48.0,43.0,28.0(3C),26.5,12.4ppm.

[0073] (6) Preparation of compound 7, the technical route is shown below:

[0074]

[0075] At room temperature, phenylsilane (abbreviated as PhSiH3, 6.71 mL, 54.48 mmol, 5.0 equiv) and (triphenylphosphine) palladium (abbreviated as Pd(PPh3)4, 630 mg, 0.54 mmol, 0.05 equiv) were added to a dichloromethane (40 mL) solution of compound 6 (4.50 g, 10.90 mmol, 1.0 equiv) in sequence. After 30 minutes, the reaction was quenched with water and extracted with dichloromethane three times; the combined organic layer was dried over anhydrous sodium sulfate, filtered, and finally evaporated to dryness; the product was separated and purified by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate = 1:2) to obtain 3.17 g of compound 7 with a yield of 88%. The structural characterization results of compound 7 are as follows: 1 H NMR(600MHz,Chloroform-d)δ3.74-3.69(m,6H),3.60-3.53(m,2H),3.04(dd,J=10.8,7.0Hz,1H),2.72(dd,J=10.8,6.0Hz,1H),2.51(d t,J=7.6,5.0Hz,1H),2.26(s,1H),1.86-1.77(m,1H),1.51-1.41(m,10H),1.24(dtd,J=13.5,7.1,2.0Hz,1H),0.84ppm(t,J=7.4Hz,3H); 13 CNMR(151MHz,Chloroform-d)δ173.4,168.9,168.8,81.5,64.5,54.8,52.6,52.5,51.9,486,45.5,28.1(3C),27.0,12.5ppm.

[0076] (7) Preparation of compound 9, the technical route is shown below:

[0077]

[0078] Indoleacetic acid (compound 8, 1.17 g, 6.68 mmol, 1.0 equiv) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.91 g, 10.03 mmol, 1.5 equiv) were added to a stirred solution of compound 7 (2.20 g, 6.68 mmol, 1.0 equiv) in dichloromethane (45 mL) at room temperature; the resulting mixture was stirred at room temperature for 1 hour, and then formic acid (15 mL) was added; the resulting mixture was stirred at room temperature for 5 h, and then concentrated in vacuo; 2.38 g of compound 9 was obtained by silica gel column chromatography (silica gel, dichloromethane / methanol = 20:1), with a yield of 83%. The structural characterization results of compound 9 are as follows: 1 HNMR(600MHz,Chloroform-d)δ8.52(s,1H),7.53(d,J=7.9Hz,1H),7.29(d,J=8.1Hz,1H) ,7.19-7.13(m,1H),7.12-7.08(m,2H),4.75(d,J=5.9Hz,1H),3.90-3.75(m,3H),3.69(s ,6H),3.65-3.59(m,1H),3.17(dd,J=10.7,7.9Hz,1H),2.90(t,J=6.5Hz,1H),2.14-2.04 (m,1H),1.53(ddd,J=13.4,7.5,5.4Hz,1H),1.25-1.21(m,1H),0.80ppm(t,J=7.3Hz,3H); 13 C NMR(151MHz,Chloroform-d)δ172.8,171.3,167.4,167.3,135.1,126.2,122.5,120.8,11 8.3,117.1,110.5,106.2,62.0,51.7,51.7,51.6,51.4,44.9,42.3,30.7,24.4,11.1ppm.

[0079] (8) The preparation of compound 10, the technical route is shown as follows:

[0080]

[0081] Cyanuric chloride (883 mg, 4.80 mmol, 1.6 equiv) and pyridine (0.24 mL, 3.0 mmol, 1.0 equiv) were added to a stirred solution of compound 9 (1.29 g, 3.0 mmol, 1.0 equiv) in dichloromethane (100 mL) at room temperature; the resulting mixture was stirred at room temperature for 30 minutes, and then aluminum chloride (519 mg, 3.9 mmol, 1.3 equiv) was added; the reactants were stirred at reflux for 12 h, then cooled to room temperature and water (40 mL) was added; the layers were separated, and the aqueous layer was extracted 3 times with dichloromethane; the combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate and concentrated in vacuo; the product was separated and purified by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate = 2:1) to obtain 840 mg of compound 10 with a yield of 68%. The structural characterization results of compound 10 are as follows: 1 H NMR(600MHz,Chloroform-d)δ9.38(s,1H),7.59(d,J=8.2Hz,1H),7.45(dt,J=8.3,0.9Hz,1H),7.37(ddd,J=8.2,6.9,1.1Hz,1H ),7.15(ddd,J=8.0,6.9,1.0Hz,1H),5.19(d,J=5.3Hz,1H),4.14(dd,J=11.9,7.5Hz,1H),3.86(d,J=16.0Hz,1H),3.83(s,2.75 H),3.76(s,0.25H),3.81-3.80(m,1H),3.79(s,2.75H),3.75(s,0.25H),3.60(d,J=16.0Hz,1H),3.47(dt,J=7.8,5.3Hz,1H),2 .80(dd,J=12.0,8.0Hz,1H),2.03(dtd,J=9.4,5.0,1.7Hz,1H),1.72-1.54(m,1H),1.38-1.26(m,2H),0.94ppm(t,J=7.4Hz,3H); 13 CNMR(151MHz,Chloroform-d)δ185.7,169.9,169.4,168.5,137.1,130.1,127.5,126.7,1 21.1,120.9,120.3,112.5,65.8,53.2,52.9,51.8,50.5,43.0,42.3,34.1,25.8,12.5ppm.

[0082] (9) The preparation of compound 11, the technical route is shown as follows:

[0083]

[0084] Under argon atmosphere, tris(triphenylphosphine)carbonyl rhodium hydride (abbreviated as [Rh(H)(CO)](PPh3)3], 140 mg, 0.15 mmol, 0.1 equiv) and PhSiH3 (0.94 mL, 7.65 mmol, 5.0 equiv) were added to a stirred solution of compound 10 (630 mg, 1.53 mmol, 1.0 equiv) in tetrahydrofuran (60 mL) in sequence; the resulting mixture was stirred at room temperature for 1 hour and then quenched by adding saturated aqueous ammonium chloride solution. The aqueous layer was extracted 3 times with ethyl acetate; the combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate and concentrated in vacuo; the product was separated and purified by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate 8:1) to obtain 383 mg of compound 11 with a yield of 63%. The structural characterization results of compound 11 are as follows: 1 H NMR(600MHz,Chloroform-d)δ9.12(s,1H),7.63(dd,J=8.1,1.1Hz,1H),7.39(dt,J=8.3,1.0Hz,1H),7.34(ddd, J=8.2,6.9,1.1Hz,1H),7.12(ddd,J=8.0,6.8,1.0Hz,1H),3.90(d,J=6.8Hz,1H),3.78(s,3H),3.75(s,3H),3.6 5(d,J=5.3Hz,1H),3.43-3.22(m,2H),3.17-3.04(m,2H),3.04-2.95(m,2H),2.83(dd,J=8.8,2.8Hz,1H),2.04- 1.94(m,1H),1.49(ddd,J=13.1,7.5,5.5Hz,1H),1.37(ddd,J=13.7,9.4,7.2Hz,1H),0.86ppm(t,J=7.4Hz,3H); 13 CNMR(151MHz,Chloroform-d)δ194.7,169.4,169.3,136.9,131.9,127.9,126.8,123.9,1 21.4,120.2,112.2,74.8,58.5,55.4,52.6,52.4,52.0,47.6,42.3,28.4,26.3,12.7ppm.

[0085] (10) The preparation of compound 12, the technical route is shown as follows:

[0086]

[0087] Under argon atmosphere, scandium trifluoromethanesulfonate (abbreviated as Sc(OTf)3, 640 mg, 1.30 mmol, 2.0 equiv) and aqueous formaldehyde solution (264 mg, 3.25 mmol, 5.0 equiv, 37% w / w) were added to a stirred solution of compound 11 (260 mg, 0.65 mmol, 1.0 equiv) in tetrahydrofuran (7 mL) in sequence; the resulting mixture was stirred at room temperature for 2 hours, and then a saturated aqueous sodium bicarbonate solution was added, and the resulting mixture was stirred at room temperature for 12 hours, and then water (20 mL) was added; the layers were separated, and the aqueous layer was extracted 3 times with ethyl acetate; the combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate and concentrated in vacuo; the product was separated and purified by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate 5:1) to obtain 230 mg of compound 12 with a yield of 83%. The structural characterization results of compound 12 are as follows: 1 H NMR(600MHz,Chloroform-d)δ8.84(s,1H),7.83-7.47(m,1H),7.42-7.27(m,2H),7.12(ddd,J=8.0,6.4,1.4Hz, 1H),4.54(d,J=9.4Hz,1H),4.44(d,J=9.4Hz,1H),3.88(d,J=4.7Hz,1H),3.81(s,3H),3.77(s,3H),3.60(ddd,J=

[0088] 13.0,8.6,4.2Hz,1H),3.33(dd,J=8.7,6.8Hz,1H),3.27-3.15(m,2H),3.01(ddd,J=13.2,5.7,4.2Hz,1H),2.77(dd,J =8.8,5.1Hz,1H),1.99-1.71(m,1H),1.54(ddd,J=13.5,7.5,5.9Hz,1H),1.46-1.31(m,1H),0.89ppm(t,J=7.4Hz,3H); 13 C NMR(151MHz,Chloroform-d)δ187.8,169.9,169.0,137.1,131.0,127.6,126.9,125.4,121. 4,120.4,112.1,107.3,72.6,65.6,58.4,57.0,53.3,52.7,46.5,40.0,27.7,25.2,12.4ppm.

[0089] (11) The preparation of (+)-rhynchine E is as follows:

[0090]

[0091] To a stirred solution of compound 12 (1.20 g, 2.82 mmol, 1.0 equiv) in DMSO / H2O (36 mL / 1.8 mL) at room temperature was added KCN (potassium cyanide, 917 mg, 14.10 mol, 5.0 equiv); the mixed solution was heated to 80°C and stirred for 4 hours, then cooled to room temperature and added water (10 mL); the layers were separated, and the aqueous layer was extracted 3 times with ethyl acetate; the combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate and concentrated in vacuo; the product was isolated and purified by silica gel column chromatography (silica gel, petroleum ether / ethyl acetate 12:1) to obtain 35 mg of a light yellow solid (+)-rhynchine E compound with a yield of 51%. The structural characterization results of the (+)-rhynchine E compound are as follows: 1 H NMR(600MHz,Chloroform-d)δ8.83(s,1H),7.63(d,J=8.1Hz,1H),7.36-7.31(m,2H),7.11(ddd ,J=8.0,6.4,1.4Hz,1H),4.21(dd,J=9.0,7.1Hz,1H),4.15(dd,J=9.0,7.6Hz,1H),3.78(s,3H) ,3.62(ddd,J=13.7,10.0,4.0Hz,1H),3.40-3.34(m,2H),3.21-3.15(m,2H),3.04(dt,J=13.4, 4.7Hz,1H),2.96-2.92(m,2H),1.86-1.83(m,1H),1.47-1.41(m,2H),0.92ppm(t,J=7.4Hz,3H). 13 C NMR(151MHz,Chloroform-d)δ188.5,172.5,137.1,130.8,127.9,126.8,124.4,121.5 ,120.3,112.0,105.9,69.7,58.0,55.4,52.3,51.2,45.0,44.1,26.9,26.1,12.4ppm.

[0092] Test example: Inhibitory effects of (+)-rhynchine E and terfenadine on hERG current

[0093] (1) Cell preparation: After HEK-293-hERG cells are subcultured to an appropriate state, the cells are rinsed with PBS (or DPBS), digested and separated with Tryple solution, and the cells are resuspended with culture medium and stored in a centrifuge tube. After centrifugation, the supernatant is discarded, and the cells are resuspended with extracellular fluid for later use and stored at 2-8°C. Before patch clamp recording, the cells are added dropwise to the culture dish to ensure that the cells have a certain density and are in a single isolated state.

[0094] (2) Electrophysiological test: The hERG current was recorded using the whole-cell patch clamp technique. The cell suspension was added to a small culture dish and placed on an inverted microscope stage. After the cells adhered to the wall, they were perfused with extracellular fluid. The recommended flow rate was 1–2 mL / min. The glass microelectrode was pulled in two steps by a microelectrode pulling instrument. After the electrode was filled with internal fluid, its water resistance was 2-5 MΩ.

[0095] After the whole-cell recording mode was established, the stimulation parameters for hERG current recording were as follows: first, the clamp potential was maintained at -80 mV. A depolarizing voltage was applied to +60 mV for 850 ms, and then repolarized to -50 mV for 1275 ms to elicit hERG tail current. This set of pulse procedures was repeated every 15 seconds throughout the experiment.

[0096] After the current stabilizes, the drug is administered by continuous extracellular perfusion from low concentration to high concentration. Starting from a low concentration, perfusion is continued until the drug effect is stable, and then the next concentration is perfused. This experiment will test the blocking effect of each concentration of the test product and the positive control (terfenadine) on the hERG tail current (N≥2); the specific actual concentration can be adjusted according to the actual solubility and effect, and it is not considered a deviation from the protocol.

[0097] The definition of stable drug efficacy is that the current value change of the last 5 stimulation strips in each concentration administration stage is less than 10% of the mean (when the current is greater than or equal to 200pA) or less than 30% of the mean (when the current is less than 200pA). If it is unstable, the data of this concentration will not be taken.

[0098] (3) Data acquisition and analysis: PatchMaster software was used for stimulation and signal acquisition; the patch clamp amplifier amplified the signal. FitMaster, EXCEL, Graphpad Prism or SPSS21.0 were used for further data analysis and curve fitting. The data were expressed as mean and standard deviation. In data processing, when judging the blocking effect on hERG, the peak value of the tail current and its baseline were corrected. The inhibition rate (IR) of the tail current was used to represent the effect of each compound at different concentrations. The SD of %IR of all cells at each concentration was ≤15, which was accepted as the standard (except for abnormal data).

[0099] IR=100%×(peak value of tail current before administration-peak value of tail current after administration) / peak value of tail current before administration.

[0100] IC 50 The value is obtained by fitting the Hill equation. If the maximum inhibition rate of all concentrations of the test substance is less than 50%, the IC is not calculated. 50 Numeric value.

[0101] Among them, y: I / I control ; max: 100%; min: 0%; [drug]: test sample concentration; n H : Hill slope; IC 50 : The half maximum inhibitory concentration of the test substance.

[0102] ① Test of the inhibitory effect of terfenadine on hERG current: In order to prove the reliability of the test results, the present invention first uses terfenadine as a positive control to measure its inhibitory effect on hERG current. The final concentration of terfenadine is 0.001, 0.01, 0.1, and 1 μM, and the final concentration of DMSO in the extracellular fluid is 0.3%. The inhibitory effect of terfenadine on hERG current is shown in Table 1, and its concentration-response curve is shown in Figure 1 shown.

[0103] Table 1. Inhibitory effect of terfenadine on hERG current

[0104]

[0105] From Table 1 and Figure 1 The results show that the inhibitory effect of terfenadine on hERG potassium channels is concentration-dependent. The IC of terfenadine on hERG current inhibition was obtained by fitting the Hill equation. 50 The value is 0.040 μM (N=2). This result is consistent with the historical results of the test of the present invention and is also consistent with the results reported in the literature, which shows that the test results of the present invention are stable and reliable.

[0106] ② Test of the inhibitory effect of (+)-rhynchine E on hERG current: The (+)-rhynchine E compound prepared in Example 1 of the present invention was dissolved in DMSO, and the final concentration of DMSO in the extracellular fluid was 0.3%, and the final concentrations of the (+)-rhynchine E compound were 0.1, 0.3, 1, 3 and 10 μM. The inhibitory effect of the (+)-rhynchine E compound on hERG current is shown in Table 2, and the concentration-response curve is shown in Figure 2 shown.

[0107] Table 2. Inhibitory effect of (+)-rhynchine E on hERG current

[0108]

[0109]

[0110] From Table 2 and Figure 2 The results show that the effect of (+)-rhynchine E on hERG potassium channel current is concentration-dependent. The IC of (+)-rhynchine E on hERG current inhibition was obtained by fitting the Hill equation. 50 The value was 5.74 μM (N=2).

[0111] In summary, the present invention uses manual patch clamp to test the inhibitory effect of (+)-rhynchine E on hERG current in HEK293 cells stably transfected with hERG potassium channel at room temperature. The results show that the effect of (+)-rhynchine E on hERG potassium channel current is concentration-dependent. The IC value of (+)-rhynchine E on hERG current inhibition is obtained by fitting the Hill equation. 50 The value was 5.74 μM (N=2). Rapid activation of human delayed rectifier outward potassium current (I Kr ) is mainly mediated by hERG ion channels and participates in the repolarization of human myocardial cells. Drugs blocking this current will lead to the clinical appearance of QT interval prolongation syndrome, which is easy to induce acute arrhythmias and even sudden death. The present invention uses manual patch clamp to detect the effect of compound E on hERG potassium current in stable cell lines transfected with hERG potassium channels, thereby demonstrating the application potential of (+)-rhynchine E in the development of antiarrhythmic drugs.

[0112] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a natural alkaloid (+)-rhynchine E compound, characterized in that: The following steps are involved: (1) reacting the compound of formula 1 with lithium hexamethyldisilazide, triphenyl phosphate, and ethyl trifluoromethanesulfonate, and then purifying to obtain a compound of formula 2; (2) reacting the compound of formula 2 with borane dimethyl sulfide, and then purifying to obtain a compound of formula 3; (3) reacting the compound of formula 3 with trimethylsilyl trifluoromethanesulfonate, and then purifying to obtain a compound of formula 4; (4) subjecting the compound of formula 4 to an oxidation reaction with N-chlorosuccinimide, then adding allyl chloroformate to carry out an isomerization reaction, and then purifying to obtain a compound of formula 5; (5) subjecting the compound of formula 5 to Michael addition reaction with dimethyl malonate in the presence of sodium hydride, followed by purification to obtain a compound of formula 6; (6) reacting the compound of formula 6 with phenylsilane and (triphenylphosphine) palladium to remove the Alloc protecting group, and then purifying to obtain a compound of formula 7; (7) subjecting the compound of formula 7 to amidation reaction with the compound of formula 8, followed by acid hydrolysis and purification to obtain a compound of formula 9; (8) subjecting the compound of formula 9 to a Friedel-Crafts acylation reaction with cyanuric chloride, pyridine and aluminum chloride, followed by purification to obtain a compound of formula 10; (9) subjecting the compound of formula 10 to a selective hydrogenation reaction with tris(triphenylphosphine)carbonyl rhodium hydride and phenylsilane, followed by purification to obtain a compound of formula 11; (10) subjecting the compound of formula 11 to a hydroxymethylation reaction in the presence of scandium trifluoromethanesulfonate and formaldehyde, followed by oxidative cyclization in air, and then purification to obtain a compound of formula 12; (11) subjecting the compound of formula 12 to a Krapcho decarboxylation reaction with potassium cyanide, followed by purification to obtain the natural alkaloid (+)-rhynchine E compound; Wherein, the structures of the compounds of Formula 1 to Formula 12 are: The structural formula of the natural alkaloid (+)-rhynchine E compound is shown in Formula E:

2. The method for preparing the natural alkaloid (+)-rhynchine E compound according to claim 1, characterized in that: In step (1), the molar ratio of the compound of formula 1, lithium hexamethyldisilazide, triphenyl phosphate, and ethyl trifluoromethanesulfonate is 1:1.2:1:1.1; in step (2), the molar ratio of the compound of formula 2 to borane dimethyl sulfide is 1:2; in step (3), the molar ratio of the compound of formula 3 to trimethylsilane trifluoromethanesulfonate is 1:0.5; in step (4), the molar ratio of the compound of formula 4, N-chlorosuccinimide, and allyl chloroformate is 1:1.2:2.3; in step (5), the molar ratio of the compound of formula 5, dimethyl malonate, and sodium hydride is 1:1.5:1.5; in step (6), the compound of formula The molar ratio of the compound of formula 6, phenylsilane and (triphenylphosphine) palladium is 1:5:0.05; in step (7), the molar ratio of the compound of formula 7 and the compound of formula 8 is 1:1; in step (8), the molar ratio of the compound of formula 9, cyanuric chloride, pyridine and aluminum chloride is 1:1.6:1:1.3; in step (9), the molar ratio of the compound of formula 10, tri(triphenylphosphine)carbonyl rhodium hydride and phenylsilane is 1:0.15:5; in step (10), the molar ratio of the compound of formula 11, scandium trifluoromethanesulfonate and formaldehyde is 1:2:5; in step (11), the molar ratio of the compound of formula 12 and potassium cyanide is 1:

5.

3. The method for preparing the natural alkaloid (+)-rhynchine E compound according to claim 1, characterized in that: In step (1), the reaction is carried out at -80 to -70°C for 1 to 3 hours; in step (2), the reaction is carried out under heating reflux for 6 to 8 hours; in step (3), the reaction is carried out at -5 to 5°C for 4 to 10 minutes; in step (4), the oxidation reaction is carried out at -5 to 5°C for 1 to 2 hours, and the isomerization reaction is carried out under heating reflux for 1 to 3 hours; in step (5), the Michael addition reaction is carried out at 20 to 30°C for 6 to 8 hours; in step (6), the reaction is carried out at 20 to 30°C for 20 to 40 minutes; in step (7), the amidation reaction is carried out at 20 to 30°C. The step (8) comprises the following steps: the Friedel-Crafts acylation reaction is first carried out at 20 to 30°C for 20 to 40 minutes and then under heating reflux for 10 to 15 hours; the selective hydrogenation reaction is carried out at 20 to 30°C for 0.5 to 2 hours; the hydroxymethylation reaction is carried out at 20 to 30°C for 1 to 3 hours and the oxidative cyclization reaction is carried out at 20 to 30°C for 10 to 15 hours; and the Krapcho decarboxylation reaction is carried out at 70 to 90°C for 3 to 6 hours.

4. The method for preparing the natural alkaloid (+)-rhynchine E compound according to claim 1, characterized in that: In step (1), the reaction is carried out in tetrahydrofuran; in step (2), the reaction is carried out in tetrahydrofuran; in step (3), the reaction is carried out in dichloromethane; in step (4), the oxidation reaction is carried out in dichloromethane; in step (5), the Michael addition reaction is carried out in tetrahydrofuran; in step (6), the reaction is carried out in dichloromethane; in step (7), the amidation reaction is carried out in dichloromethane; in step (8), the Friedel-Crafts acylation reaction is carried out in dichloromethane; in step (9), the selective hydrogenation reaction is carried out in tetrahydrofuran; in step (10), the hydroxymethylation reaction is carried out in tetrahydrofuran; in step (11), the Krapcho decarboxylation reaction is carried out in a dimethyl sulfoxide aqueous solution.

5. The method for preparing the natural alkaloid (+)-rhynchine E compound according to any one of claims 1 to 4, characterized in that: In step (7), the acid hydrolysis is carried out by adding formic acid or acetic acid.

6. The method for preparing the natural alkaloid (+)-rhynchine E compound according to any one of claims 1 to 4, characterized in that: In step (10), the oxidative cyclization is carried out after adding a saturated sodium bicarbonate aqueous solution.

7. The method for preparing the natural alkaloid (+)-rhynchine E compound according to any one of claims 1 to 4, characterized in that: The reactions in step (2), step (9) and step (10) are carried out in a protective atmosphere.

8. The method for preparing the natural alkaloid (+)-rhynchine E compound according to any one of claims 1 to 4, characterized in that: In steps (1) to (11), the purification is performed by silica gel column chromatography.

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