A class of lycorine derivatives, pharmaceutical compositions thereof and uses thereof in the preparation of antiviral drugs

Abstract

The application belongs to the technical field of medicine, and discloses a type of lycorine derivatives, a pharmaceutical composition thereof and the use of the lycorine derivatives in preparation of antiviral drugs, and specifically relates to a type of lycorine derivatives as shown in a general formula (I) and pharmaceutically acceptable salts, pharmaceutical compositions and applications thereof. The lycorine derivatives have obvious antiviral activity and can be used for treating viral diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a class of lycorine derivatives, a preparation method thereof, a pharmaceutical composition containing the same, and an application thereof in the preparation of antiviral drugs. BACKGROUND

[0002] Lycorine (LY) was first isolated from Narcissus pseudonarcissus of the Amaryllidaceae family in 1877, and it is widely distributed in the Amaryllidaceae family. Studies have shown that lycorine (structural formula as follows) has strong broad-spectrum antiviral effects, including coronavirus (SARS-CoV), dengue virus (DENV), West Nile virus (WNV), retrovirus (HIV-1), herpes simplex virus (HSV-1), poliovirus (PV), enterovirus (EV71), influenza virus (H5N1), hepatitis C virus (HCV), yellow fever virus (YFV), Zika virus (ZIKV), etc.

[0003] Recent research reports that lycorine has in-vitro anti-SARS-CoV-2 activity, and the intensity of the effect is comparable to that of remdesivir. Since lycorine itself has strong hydrophilicity, poor metabolic stability, and toxic side effects, etc., the development of lycorine-based broad-spectrum antiviral drugs is urgent. The inventors have also previously disclosed a class of lycorine derivatives having anti-hand-foot-mouth disease virus activity (authorized publication number CN 110759927B, application date: July 27, 2018).

[0004]

[0005] The present application focuses on the oxidation of the 2-hydroxyl group of lycorine to a carbonyl group for in-depth structural optimization exploration, and a class of lycorine derivatives are obtained, which show good anti-SARS-CoV-2 activity.

[0006] REFERENCES:

[0007] Zeng, B., et al. Research Progress on Pharmacological Activity and Structure-activity Relationship of Lycorine [J]. Natural Products Research and Development, 2021, 33(02): 342-351. SUMMARY

[0008] The technical problem solved by the present application is to provide a class of lycorine derivatives and pharmaceutically acceptable salts thereof, and an application thereof in the preparation of antiviral drugs.

[0009] To solve the technical problem of the present application, the present application provides the following technical solutions:

[0010] The first aspect of the technical solution of the present application provides a type of lycorine derivatives represented by the following general formula (I) and pharmaceutically acceptable salts thereof:

[0011]

[0012] Among them:

[0013] R is hydrogen, C3-6cycloalkylcarbonyl, difluoro-substituted cyclobutanecarbonyl, phenoxyacetyl or halogen-substituted phenoxyacetyl, phenylmercaptoacetyl or halogen-substituted phenylmercaptoacetyl, 2-(halogen-substituted phenoxy)propionyl, 2-(halogen-substituted phenoxy)isobutyryl, N-phthalimideacetyl.

[0014] The substituents of the halogen-substituted phenoxy and halogen-substituted phenylmercapto on the phenyl group are para, meta or ortho, and the number of substituents is single, double or multiple substitution.

[0015] The halogen is F, Cl, Br or I.

[0016] The most preferred lycorine derivatives and pharmaceutically acceptable salts thereof are selected from the following compounds:

[0017]

[0018] The second aspect of the technical solution of the present application provides a pharmaceutical composition comprising the lycorine derivative of any one of claims 1-4 and pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier or excipient. The compounds of the present application can be administered orally, for example, in the form of capsules, tablets, powders, granules, syrups or the like, or non-gastrointestinally, for example, by injection, ointment, suppository or the like. These pharmaceutical preparations can be produced by ordinary methods using adjuvants well known in the art, such as binding agents, excipients, stabilizers, disintegrating agents, flavoring agents, lubricants and the like. Although the dosage varies with the symptoms and the age of the patient, the nature and severity of the disease or disorder, and the route and mode of administration, the normal dosage of the compounds of the present application for oral administration in adult patients is 1 to 200 mg, preferably 5 to 50 mg, per day as a total daily dose in single or divided doses; for example, twice or three times a day; and for intravenous injection, 0.1 to 100 mg, preferably 5 to 50 mg, per day in one to three divided doses.

[0019] The third aspect of the technical solution of the present application provides the use of the lycorine derivative of the first aspect and pharmaceutically acceptable salts thereof or the pharmaceutical composition of the second aspect in the preparation of an antiviral drug, wherein the virus is selected from SARS-CoV-2.

[0020] Beneficial technical effects

[0021] The application focuses on providing a lycorine derivative with a general formula (I), which is mainly obtained by oxidizing a 2-hydroxyl group of lycorine into a carbonyl group and introducing different acyl groups on a 1-hydroxyl group. Through anti-novel coronavirus activity screening, the derivative has significant anti-novel coronavirus activity, indicating that the derivative has good antiviral prospects. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Inhibition of GD108 strain of novel coronavirus by all 2-carbonyl lycorine derivatives (10 μmol / L)

[0023] Figure 2 EC of three effective 2-carbonyl lycorine derivatives (LY155, LY208 and LY211) on wild strain GD108 of novel coronavirus 50 Determination results

[0024] Figure 3 Determination results of median toxicity concentration (CC 50 ) of three 2-carbonyl lycorine derivatives (LY155, LY208 and LY211) DETAILED DESCRIPTION

[0025] English name and abbreviation:

[0026] aq.: aqueous solution

[0027] CC 50 : median toxicity concentration

[0028] DCM: dichloromethane

[0029] DMEM: Dulbecco's modified Eagle's medium

[0030] DMF: N,N-dimethylformamide

[0031] DMSO: dimethyl sulfoxide

[0032] DMAP: 4-dimethylaminopyridine

[0033] EA: ethyl acetate

[0034] EC 50 : 50% inhibition concentration

[0035] EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0036] EtOH: ethanol

[0037] FBS: fetal bovine serum

[0038] LY: lycorine

[0039] MOI: multiplicity of infection

[0040] OD: optical density

[0041] PE: petroleum ether (60-90°C)

[0042] Py: pyridine

[0043] SI: selectivity index (CC 50 / EC 50 )

[0044] r.t.: room temperature

[0045] μM: μmol / L

[0046] Disclosed are a class of lycorine derivatives, the use of their salts, solvates, prodrugs and pharmaceutical compositions. Those skilled in the art can refer to the content of the present text and appropriately improve the process parameters. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and applications of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0047] The present application is further described below in conjunction with examples:

[0048] General synthesis method of compounds:

[0049] The reaction formula is as follows:

[0050]

[0051] Reagents and conditions: (a) Dess-Martin reagent, DMF-Py, r.t.; (b) EDCI, DMAP, carboxylic acid, DMF.

[0052] General synthesis method:

[0053] Into a 200 mL single-neck flask was added lycorine 1.15 g (4 mmol), followed by 75 mL of DMF and 50 mL of pyridine, and stirred at room temperature for a few minutes to clarify, then Dess-Martin reagent was added, and stirred at room temperature until the raw material disappeared by TLC monitoring. The reaction solution was rotary evaporated to remove most of the solvent, extracted with ethyl acetate and water, then washed with saturated NaCl, dried over anhydrous sodium sulfate, filtered and rotary dried to obtain brown solid 2-oxo-lycorine (yield 45%), which was directly used in the next step reaction synthesis.

[0054] The corresponding organic acid (1.2 mmol), EDCI (383 mg, 2 mmol), DMAP (24 mg, 0.2 mmol) were dissolved in 5 mL of dry DMF and stirred at room temperature for 10 min. To this 2-oxo-2-thioxoacetic acid (about 1 mmol) was added and stirred at room temperature till TLC showed complete consumption of starting material. Most of the solvent was removed by rotary evaporation and the residue was extracted with ethyl acetate and saturated NaHC03. The organic layer was separated, washed with water, dried over anhydrous Na2S04, filtered and concentrated. The crude product was purified by column chromatography (petroleum ether-ethyl acetate = 1:1) to get the desired compound.

[0055] Example 12-oxo-2-thioxoacetic acid (LY-11)

[0056]

[0057] Brown solid, yield 45%. 1 H NMR (400 MHz, DMSO-d6) δ 6.84 (s, 1H, H-11), 6.71 (s, 1H, H-8), 5.97-5.96 (m, 2H, H-12), 5.82-5.80 (m, 1H, H-3), 4.41 (d, J = 2.8 Hz, 1H), 4.07 (d, J = 14.2 Hz, 1H, H-7), 3.41 (d, J = 14.2 Hz, 1H, H-7), 3.31 (t, J = 8.6 Hz, 1H), 3.09 (d, J = 10.1 Hz, 1H), 2.90-2.67 (m, 3H), 2.38 (q, J = 8.3 Hz, 1H); ESI-MS: 286 [M+H] + .

[0058] Example 21-cyclohexanecarbonyl-2-oxo-2-thioxoacetic acid (LY-155)

[0059]

[0060] White solid, yield 45%. 1 H NMR (400 MHz, CDCl3) δ 6.70 (s, 1H), 6.56 (s, 1H), 5.98 (s, 2H), 5.90 (s, 2H), 4.17 (d, J = 14.1 Hz, 1H), 3.60 (d, J = 14.1 Hz, 1H), 3.54-3.39 (m, 1H), 3.27 (d, J = 10.2 Hz, 1H), 3.17 (d, J = 9.9 Hz, 1H), 2.86 (s, 2H), 2.53 (q, J = 8.8, 8.3 Hz, 1H), 2.23-2.11 (m, 1H), 1.78-1.43 (m, 7H), 1.38-1.20 (m, 3H). 13C NMR (101 MHz, CDC13) δ 193.25, 174.50, 168.93, 146.72, 128.89, 125.42, 120.60, 107.38, 105.82, 101.20, 68.58, 62.61, 56.44, 53.37, 45.66, 42.85, 30.12, 28.85, 28.69, 25.74, 25.24, 25.18. ESI-MS: 396 [M+H] + .

[0061] Example 31 - Cyclopentanecarbonyl-2-carbonyl ajoene (LY-185)

[0062]

[0063] White solid, yield 40%. 1 H NMR (400 MHz, CDC13) δ 6.70 (s, 1H), 6.56 (s, 1H), 5.98 (s, 2H), 5.90 (s, 2H), 4.16 (d, J = 14.1 Hz, 1H), 3.59 (d, J = 14.1 Hz, 1H), 3.45 (dt, J = 9.1, 4.5 Hz, 1H), 3.26 (d, J = 10.0 Hz, 1H), 3.16 (d, J = 9.7 Hz, 1H), 2.86 (s, 2H), 2.65 - 2.47 (m, 2H), 1.74 - 1.64 (m, 2H), 1.65 - 1.40 (m, 6H). 13 C NMR (101 MHz, CDC13) δ 193.25, 174.50, 168.93, 146.72, 128.89, 125.42, 120.60, 107.38, 105.82, 101.20, 68.58, 62.61, 56.44, 53.37, 45.66, 42.85, 30.12, 28.85, 28.69, 25.74, 25.24, 25.18. ESI-MS: 396 [M+H] + .

[0064] Example 41 - Cyclobutanecarbonyl-2-carbonyl ajoene (LY-187)

[0065]

[0066] White solid, yield 41%. 1H NMR (400 MHz, CDC13) δ 6.72 (s, 1H), 6.56 (s, 1H), 5.98 (s, 2H), 5.90 (s, 2H), 4.16 (d, J = 14.1 Hz, 1H), 3.58 (d, J = 14.1 Hz, 1H), 3.45 (dt, J = 9.1, 4.5 Hz, 1H), 3.26 (d, J = 11.7 Hz, 1H), 3.14 (d, J = 9.9 Hz, 1H), 2.98 (p, J = 8.3 Hz, 1H), 2.86 (s, 2H), 2.52 (q, J = 8.7 Hz, 1H), 2.16 - 2.05 (m, 2H), 2.03 - 1.93 (m, 2H), 1.89 - 1.75 (m, 2H). 13 CNMR (101 MHz, CDC13) δ 193.16, 174.04, 168.98, 146.60, 128.84, 125.29, 120.44, 107.27, 105.56, 101.08, 68.65, 62.42, 56.33, 53.24, 45.56, 37.79, 30.00, 25.04, 18.41. ESI-MS: 368 [M+H] +

[0067] Example 51 - Cyclopropylcarbonyl-2-carbonyl-lycorine (LY-186)

[0068]

[0069] White solid, yield 43%. 1 H NMR (400 MHz, CDC13) δ 6.72 (s, 1H), 6.56 (s, 1H), 5.98 (s, 2H), 5.90 (s, 2H), 4.16 (d, J = 14.1 Hz, 1H), 3.58 (d, J = 14.1 Hz, 1H), 3.45 (dt, J = 9.1, 4.5 Hz, 1H), 3.26 (d, J = 11.7 Hz, 1H), 3.14 (d, J = 9.9 Hz, 1H), 2.98 (p, J = 8.3 Hz, 1H), 2.86 (s, 2H), 2.52 (q, J = 8.7 Hz, 1H), 2.16 - 2.05 (m, 2H), 2.03 - 1.93 (m, 2H), 1.89 - 1.75 (m, 2H). 13C NMR (101 MHz, CDC13) δ 193.09, 173.66, 168.95, 146.79, 128.84, 125.41, 120.59, 107.41, 105.60, 101.21, 68.96, 62.45, 56.38, 53.35, 45.62, 30.09, 29.81, 12.93, 9.10. ESI-MS: 354 [M+H] +

[0070] Example 61 -(3,3-difluorocyclobutyl)formyl-2-oxoaffinidine (LY-206)

[0071]

[0072] Pale yellow solid, yield 33%. 1 H NMR (400 MHz, CDC13) δ 6.67 (s, 1H), 6.57 (s, 1H), 6.00 (s, 2H), 5.91 (d, J = 3.7 Hz, 2H), 4.17 (d, J = 14.1 Hz, 1H), 3.59 (d, J = 14.1 Hz, 1H), 3.47 (dt, J = 9.1, 4.5 Hz, 1H), 3.30 (d, J = 9.7 Hz, 1H), 3.13 (d, J = 10.0 Hz, 1H), 2.95 - 2.40 (m, 8H). 13 CNMR (101 MHz, CDC13) δ 192.58, 172.08, 169.41, 146.90, 146.80, 129.10, 124.98, 120.49, 119.99 (dd, J = 269, 267 Hz), 107.54, 105.34, 101.30, 69.66, 62.47, 56.39, 53.33, 45.56, 38.63 (t, J = 24.6 Hz), 30.17, 26.43 (dd, J = 13.9, 6.3 Hz). ESI-MS: 404 [M+H] +

[0073] Example 71 -(N-phtalimido)acetyl-2-oxoaffinidine (LY-20)

[0074]

[0075] Off-white solid, yield 52%. 1H NMR (400 MHz, CDC13) δ 7.79-7.69 (m, 4H, Ar-H), 6.57 (s, 1H, H-11), 6.40 (s, 1H, H-8), 5.98-5.88 (m, 4H, H-1, 3, 12), 4.28 (s, 1H, N-CH2), 4.04 (d, J = 14.0 Hz, 1H, H-7), 3.40 (s, 1H), 3.30-3.24 (m, 2H), 2.99 (s, 1H), 2.82 (s, 2H), 2.39 (s, 1H). 13 CNMR (126 MHz, CDC13) δ 191.74, 167.07, 166.15, 147.20, 147.08, 134.35, 132.05, 128.56, 124.65, 123.74, 120.73, 107.49, 105.33, 101.46, 70.77, 62.47, 56.31, 53.47, 45.45, 39.06, 30.14. HRMS (ESI) m / z C 26 H 21 N2O7[M+H] + Calculated: 473.1343, Found: 473.1327.

[0076] Example 8 Phenoxyacetyl-2-carbonyl-lycorane (LY-18)

[0077]

[0078] Yellow solid, yield 86%. 1 H NMR (500 MHz, CDC13) δ 7.35-7.32 (m, 2H, Ar-H), 7.07-6.99 (m, 4H, Ar-H), 6.57 (s, 1H, H-8), 6.06 (s, 1H, H-3), 5.95 (s, 1H, H-12), 5.93 (s, 1H, H-12), 5.57 (d, J = 13.0 Hz, 1H, H-1), 4.93 (s, 1H, O-CH2), 4.14 (d, J = 14.6 Hz, 1H, H-7), 3.62 (d, J = 14.6 Hz, 1H, H-7), 3.48-3.43 (m, 2H), 3.06 (d, J = 10.5 Hz, 1H), 2.85 (s, 2H), 2.57-2.52 (m, 1H). 13C NMR (126 MHz, CDC13) δ 192.14, 168.43, 168.26, 157.79, 146.79, 146.69, 129.63, 128.73, 127.15, 121.77, 120.46, 114.69, 107.15, 107.10, 101.20, 78.05, 66.59, 65.41, 56.76, 53.67, 44.48, 29.89. HRMS (ESI) m / z r C 24 H 22 NO6[M+H] + Calcd: 420.1442, Found: 420.1429. Example 94-fluorophenoxyacetyl-2-oxo-lycorane (LY-188)

[0079]

[0080] Yellowish solid, yield 31%. 1 H NMR (400 MHz, CDC13) δ 6.79 (t, J = 8.5 Hz, 2H), 6.69 (s, 1H), 6.55 (d, J = 7.4 Hz, 3H), 6.09 - 5.88 (m, 4H), 4.51 (s, 2H), 4.11 (d, J = 14.0 Hz, 1H), 3.51 - 3.32 (m, 2H), 3.26 (d, J = 9.6 Hz, 1H), 2.99 - 2.70 (m, 3H), 2.43 (q, J = 8.8 Hz, 1H). 13 C NMR (101 MHz, CDC13) δ 192.05, 169.47, 167.89, 157.65 (d, J = 239.2 Hz), 153.74, 153.72, 146.96, 146.82, 129.06, 124.76, 120.45, 115.90 (d, J = 23.2 Hz), 115.44 (d, J = 8.1 Hz), 107.59, 105.45, 101.34, 69.96, 65.53, 62.26, 56.30, 53.30, 45.56, 30.10, 29.83. ESI-MS: 438 [M+H] +

[0081] Example 10 phenylmercaptoacetyl-2-oxo-lycorane (LY-16)

[0082]

[0083] Yellow oil, yield 46%. 1H NMR (400 MHz, CDC13) δ 7.17-7.10 (m, 5H, Ar-H), 6.69 (s, 1H, H-11), 6.52 (s, 1H, H-8), 5.97-5.94 (m, 2H, H-1, H-3), 5.89 (d, J = 1.5 Hz, 1H, H-12), 5.87 (d, J = 1.5 Hz, 1H, H-12), 4.11 (d, J = 14.1 Hz, 1H, H-7), 3.51-3.47 (m, 3H, H-7, S-CH2), 3.45-3.40 (m, 1H), 3.25-3.22 (m, 1H), 2.98 (dd, J = 10.0, 2.1 Hz, 1H), 2.84-2.80 (m, 2H), 2.59 (q, J = 8.4 Hz, 1H). 13 C NMR (126 MHz, CDC13) δ 192.19, 169.31, 168.31, 163.36, 160.90, 146.75, 146.63, 132.88, 32.80, 129.46, 129.43, 128.84, 124.82, 120.33, 116.15, 115.93, 107.34, 105.41, 101.15, 70.06, 62.16, 56.24, 53.18, 45.50, 37.12, 29.98. HRMS (ESI) m / z C 24 H 22 NO5S[M+H] + Calculated: 436.1213, Found: 436.1215.

[0084] Example 11 4-Fluorobenzenethioacetyl-2-oxo-3-methylthiopine (LY-11)

[0085]

[0086] Pale yellow solid, yield 41%. 1 H NMR (400 MHz, CDC13) δ 7.15 (t, J = 8.5 Hz, 2H), 6.88 (t, 1H), 6.67 (s, J = 7.4 Hz, 3H), 6.54 (s, 4H), 5.96 (d, 2H), 5.90 (t, J = 14.0 Hz, 2H), 4.15 (d, 2H), 3.53 (d, 1H), 3.45 (s, 2H), 3.42 (d, 1H), 2.85 (d, 1H), 2.83 (m, 2H), 1.26 (q, J = 8.8 Hz, 1H). 13CNMR (101 MHz, CDC13) δ 192.05, 169.47, 167.89, 157.65 (d, J = 239.2 Hz), 153.74, 153.72, 146.96, 146.82, 129.06, 124.76, 120.45, 115.90 (d, J = 23.2 Hz), 115.44 (d, J = 8.1 Hz), 107.59, 105.45, 101.34, 69.96, 65.53, 62.26, 56.30, 53.30, 45.56, 30.10, 29.83. HRMS (ESI) m / z C 24 H 21 NFO5S[M+H] + Calcd: 454.1119, Found: 454.1113.

[0087] Example 124-chlorophenoxypropionyl-2-oxoaffinidine (LY-209)

[0088]

[0089] White solid, yield 31%. 1 H NMR (400 MHz, CDC13) δ 7.25 (d, J = 6.0 Hz, 1H), 7.07 (s, 1H), 6.94 - 6.79 (m, 2H), 6.74 - 6.63 (m, 1H), 6.55 (s, 1H), 6.01 - 5.80 (m, 4H), 4.13 (d, J = 14.0 Hz, 1H), 3.68 - 3.32 (m, 3H), 3.32 - 3.16 (m, 1H), 3.05 - 2.71 (m, 3H), 2.48 (d, J = 11.4 Hz, 1H), 1.28 (d, J = 7.7 Hz, 3H). 13 C NMR (101 MHz, CDC13) δ 192.50, 171.35, 162.71 (d, J = 248.2 Hz), 146.83, 146.75, 136.13 (d, J = 8.3 Hz), 135.44 (d, J = 8.4 Hz), 128.83, 125.01, 120.49, 115.95 (d, J = 21.8 Hz), 107.45, 105.76, 105.39, 101.22, 69.88, 62.29, 56.25, 53.25, 44.82, 30.05, 17.03, 16.84. ESI-MS: 468 [M+H] +

[0090] Example 134-fluorophenylmercaptopropionyl-2-oxoaffinidine (LY-177)

[0091]

[0092] White solid, yield 31%. 1 H NMR (400 MHz, CDC13) δ 7.25 (d, J = 6.0 Hz, 1H), 7.07 (s, 1H), 6.94-6.79 (m, 2H), 6.74-6.63 (m, 1H), 6.55 (s, 1H), 6.01-5.80 (m, 4H), 4.13 (d, J = 14.0 Hz, 1H), 3.68-3.32 (m, 3H), 3.32-3.16 (m, 1H), 3.05-2.71 (m, 3H), 2.48 (d, J = 11.4 Hz, 1H), 1.28 (d, J = 7.7 Hz, 3H). 13 C NMR (101 MHz, CDC13) δ 192.50, 171.35, 162.71 (d, J = 248.2 Hz), 146.83, 146.75, 136.13 (d, J = 8.3 Hz), 135.44 (d, J = 8.4 Hz), 128.83, 125.01, 120.49, 115.95 (d, J = 21.8 Hz), 107.45, 105.76, 105.39, 101.22, 69.88, 62.29, 56.25, 53.25, 44.82, 30.05, 17.03, 16.84. ESI-MS: 468 [M+H] +

[0093] Example 144-Fluorophenoxybutyryl-2-carbonyl-ajmaline (LY-153)

[0094]

[0095] White solid, yield 33%. 1 H NMR (400 MHz, CDC13) δ 6.92 (d, J = 8.4 Hz, 2H), 6.78 (s, 1H), 6.57 (s, 1H), 6.45 (d, J = 8.4 Hz, 2H), 5.99 (s, 2H), 5.94 (s, 2H), 4.07 (d, J = 13.9 Hz, 1H), 3.44-3.30 (m, 2H), 3.26 (d, J = 9.7 Hz, 1H), 2.92-2.75 (m, 3H), 2.39 (q, J = 8.5 Hz, 1H), 1.43 (s, 3H), 1.41 (s, 3H). 13C NMR (101MHz, CDCl3) δ192.19,172.76,169.55,153.86,146.88,146.73,129.00,126.63,124.91,120.35,119. 54,107.67,105.63,101.83,79.26,70.35,62.38,56.27,53.22,45.12,30.08,26.22,24.62.ESI-MS:482[M+H] +

[0096] Pharmacological experiments

[0097] Experimental Example 1: Preliminary Screening Results of the In Vitro Anti-SARS-CoV-2 Activity of Lycorine Derivatives (10 μmol / L) Figure 1 )

[0098] (1) Compound preparation and challenge experiment

[0099] With a density of 2×10 5 Vero cells were seeded at a density of 100 μL / mL in 96-well plates and cultured overnight. Appropriate amounts of lycorine and its derivative were weighed and prepared as a 10 mM stock solution using DMSO. Before use, the drug was prepared to 10 μM using DMEM or DMEM containing 2% FBS. Vero cells were treated with the drug diluted to 10 μM in DMEM for 1 hour (100 μL / well in a 96-well plate). Then, the cells were infected with virus (100 μL / well) for 1 hour (MOI = 0.05). The drug-virus mixture was discarded, and the drug diluted in DMEM with 2% FBS was added again (10 μM concentration). The cells were incubated at 37°C for 48 hours. Cytopathic effect (CPE) was observed after drug treatment. 100 μL of the cell culture supernatant was treated with 200 μL of triazole, and RNA was extracted using a nucleic acid extraction kit for subsequent experiments.

[0100] (2) Real-time fluorescence quantitative detection of viral RNA copy number

[0101] Primers for the SARS-CoV-2 N protein (Nucleocapsid protein) gene and a carboxyfluorescein (FAM) fluorescent probe (synthesized by Sangon Biotech) were diluted to 5 μM. The N plasmid (constructed in Peng Xiaozhong's lab) was used as a standard, and a ten-fold serial dilution was performed starting at 10 ng / μL, for a total of eight concentration gradients, to calculate the standard curve. The copy number of the viral N gene was calculated (absolute quantification) based on the standard curve. The following components were then added to 384-well white plates and centrifuged:

[0102]

[0103] Obtain a total volume of 10 μL of the reaction system, set the PCR instrument to run as follows:

[0104]

[0105] After the instrument runs, the sample N gene copy number will be calculated according to the standard curve, and the inhibition rate of the compound on the virus = (virus control group N gene copy number - drug group N gene copy number) / virus control group N gene copy number x 100% is calculated by the formula to calculate the inhibition rate of the compound on the virus replication.

[0106] From Figure 1 The anti-coronavirus GD108 strain primary screening results show that lycoramine, LY155, LY208, LY211 and LY14 have significant inhibitory effect on the coronavirus strain GD108, and show a certain inhibition rate at a drug concentration of 10 μM. Compound LY155 has a similar inhibition rate to lycoramine at 10 μM, suggesting that the three effective compounds LY155, LY208 and LY211 can be further determined for EC 50 values.

[0107] Experimental Example 2 Determination of EC 50 of effective compounds in vitro

[0108] (1) Compound preparation and challenge experiment

[0109] Vero cells with a density of 2 x 10 5 / mL were plated in a 96-well plate, 100 μL per well, and incubated overnight. An appropriate amount of lycoramine and lycoramine derivatives were weighed and prepared into 10 mM stock solutions with DMSO. The drugs were diluted 2-fold by volume (starting from 10 μM) with DMEM or DMEM containing 2% FBS, for a total of 7 concentration gradients. After treating Vero cells with the test drugs diluted with DMEM for 1 hour (96-well plate, 100 μL / well), adding virus (100 μL / well) to infect Vero cells for 1 hour (MOI = 0.05), discarding the drug-virus mixture, and re-adding 2% FBS DMEM diluted drugs, incubating in a 37°C incubator for 48 hours. Observe the cytopathic effect (CPE) after drug treatment, take 100 μL of the culture supernatant, add 200 μL triazole, and use the nucleic acid extraction kit to extract RNA for subsequent experiments.

[0110] (2) Real-time fluorescent quantitative detection of viral RNA copy number

[0111] The detection method is the same as in Experimental Example 1. After calculating the inhibition rate of the compound at different gradient concentrations on virus replication, the statistical software GraphPad Prism 8 is used to plot and calculate the half maximal inhibitory concentration (EC 50, 50% inhibitory concentration).

[0112] By further testing of LY155, LY208 and LY211 on wild-type GD108 strain of new coronavirus, the derivatives LY155 and LY208 have relatively small EC 50 values Figure 2 , and the compound LY211 has a larger EC 50 value.

[0113] The CC 50 of the most effective compound was determined (CCK8 method) 50

[0114] The CC 50 determination method is as follows:

[0115] Vero cells with a density of 2×10 5 / mL were added to 96-well plates at 100 μL per well and cultured overnight. Gradient dilution was performed to 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 μM with DMEM (Dulbecco's modified Eagle's medium) or DMEM medium containing 2% FBS. The culture medium in the 96-well plate was discarded, 200 μL of DMEM-diluted compound was added to each well, and incubation was performed in a 37°C incubator for 48 hours. Discard the culture medium, add 100 μL of CCK-8-containing DMEM medium, and incubate at 37°C for 2 hours. Set up negative control wells (add 0.1% DMSO) and blank control wells (no cells, add DMEM). The absorbance value was measured at 450 nm wavelength using a microplate reader, and the cell inhibition rate of the compound was calculated by the formula: inhibition rate = (negative control group - drug group) / (negative control group - blank group) × 100%, wherein the negative is the average absorbance (OD) of the negative control group, the drug group is the average OD of the compound group, and the blank is the average OD of the blank group, and the CC 50 was calculated using statistical software GraphPad prism 8.

[0116] As can be seen from Figure 3 , the experimentally determined half toxicity concentrations (CC 50 ) of LY155, LY208 and LY211 on Vero cells are 164.6, 281.8 and 158.8 μM, respectively, showing that their toxicity is better than that of lycorine (CC 50 53.31 μM).

[0117] ​The SI values of the three compounds are 96.88, 92.48 and 12.46 respectively, and the SI value of lycorine is 80.17. The results show that the compounds LY155 and LY208 have significant anti-SARS-CoV-2 effect and are worth further development.

Claims

1. A lycorine derivative or a pharmaceutically acceptable salt thereof for treating SARS-CoV-2 infection, characterized in that, Selected from the following: 。 2. A pharmaceutical composition for treating COVID-19 infection, characterized in that, The pharmaceutical composition comprises the lycorine derivative of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical compositions include tablets, capsules, pills, injections, eye drops, sprays, and ointments.

4. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition is selected from controlled-release dosage forms, sustained-release dosage forms, and various microparticle delivery systems.

5. The use of the lycorine derivative or a pharmaceutically acceptable salt thereof as described in claim 1 in the preparation of an anti-COVID-19 drug.

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