Berbamine derivatives, processes for their preparation and their use in antiviral and leukopenia therapy

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a berbamine derivative, a preparation method thereof and application thereof in antiviral aspects, which has a structure as shown in formula I or formula II. It is verified through experiments that the series of derivatives provided by the application have good antiviral effects and effects of treating or preventing leukopenia, and have more ideal pharmacokinetic properties compared with berbamine.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, this invention relates to a berberine derivative, its preparation method, and its application in antiviral treatment and the treatment of leukopenia. Background Technology

[0002] Berberine (structural formula shown below) is a dibenzylisoquinoline alkaloid found in plants of the genus Berberis, possessing various biological activities. Berberine dihydrochloride is already marketed, with the generic name berberine hydrochloride tablets and the trade name Shengbaian, used to treat leukopenia caused by various reasons, and can also be used to prevent leukopenia after cancer radiotherapy and chemotherapy. Our research team recently discovered that berberine has a clear anti-Ebola virus (EBOV) activity, targeting the GPcl protein of Ebola virus. In a mouse model of EBOV infection, administration of 100 mg / kg one day before infection achieved 100% protection (0% in the blank control group); administration of 100 mg / kg one day after infection achieved 83% protection (0% in the blank control group) (ActaPharm. Sin. B. 2022, https: / / doi.org / 10.1016 / j.apsb.2022.05.023). In addition, it has been reported that berberine can exert anti-SARS-CoV-2 activity by inhibiting S-protein-mediated membrane fusion, and its in vitro anti-SARS-CoV-2 ECG... 50 The range was 1.73–1.89 μM (Plos Neglect Trop D. 2022, https: / / doi.org / 10.1371 / journal.pntd.0010363).

[0003]

[0004] However, during the later stages of development of berberine, we found that its pharmacokinetic properties were slightly poor; after oral administration of 25 mg / kg to ICR mice, C max Only 704 ng / mL, AUC 0-t The concentration was 8552 ng / mL·h. According to the instructions for use of berberine hydrochloride tablets, the dosage is 28 mg / tablet, and the administration is 4 tablets three times a day. This administration method suggests that berberine may have poor pharmacokinetic properties and poor absorption, resulting in a high dosage, with a single dose reaching 112 mg and a daily dose as high as 336 mg. Therefore, it is necessary to find derivatives or analogs with better pharmacokinetic properties. Summary of the Invention

[0005] One of the objectives of this invention is to provide a series of berberine derivatives.

[0006] Another object of the present invention is to provide a method for preparing berberine derivatives.

[0007] Another object of the present invention is to provide a composition containing a berberine derivative.

[0008] Another object of the present invention is to provide the use of berberine derivatives in therapeutic and preventive antiviral drugs.

[0009] Another object of the present invention is to provide the use of berberine derivatives in drugs for the treatment and prevention of leukopenia.

[0010] Terminology Definition

[0011] The terminology used in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The nomenclature used herein and the laboratory procedures described herein in organic chemistry, medicinal chemistry, and biology are well-known and commonly used in the art. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0012] As used in the description of embodiments of the invention and the appended claims, the singular forms of “a,” “an,” “the,” and “its” are used to refer to the singular and plural of the article, unless the context clearly indicates otherwise. For example, a compound comprises one or more compounds.

[0013] As used in this article, “and / or” means any and all possible combinations of one or more of the related listed items.

[0014] As used herein, the term “disease” or “patient” refers to any change in physical condition or organ that interrupts or interferes with the functioning of the organ and / or causes symptoms.

[0015] As used herein, the term "treatment" aims to alleviate or eliminate a targeted disease state or symptom. A subject is successfully "treated" if, in accordance with the methods described herein, a therapeutic amount of a compound or its pharmaceutically acceptable salt, isomer, or pharmaceutical composition thereof is received, and the subject exhibits an observable and / or detectable reduction or improvement in one or more indications and symptoms. It should also be understood that treatment of the disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

[0016] Technical Topic 1

[0017] This invention provides berberine derivatives having the structure shown in Formula I or Formula II, their isomers, or pharmaceutically acceptable salts thereof:

[0018]

[0019] R stands for:

[0020]

[0021] in:

[0022] R 1 and R 2 Each of these can be independently represented by H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and —(CH2). m OH, —(CH2) m OCH3;

[0023] R 3 Represents H, methyl, ethyl, n-propyl, isopropyl, isobutyl, —CF3, —CH2Cl, —CH2F, —CH2NR y R x —NR y R x ;

[0024] X represents —CH2—, —O—, —S—, —NR a —、—CHNR y R x —;

[0025] R y R x and R a Each of these can be independently represented by H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

[0026] n1 and n2 independently represent 0, 1, and 2;

[0027] m represents 1, 2, or 3.

[0028] In some preferred embodiments of the present invention, the R 1 and R 2 Each of these can be represented independently as H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.

[0029] In some preferred embodiments of the present invention, the R 3 Selected from H, methyl, ethyl, n-propyl, isopropyl, -NR y R x —CF3.

[0030] In some preferred embodiments of the present invention, the derivative comprises the following structure: , , , , , , , , , , , , , , , , , .

[0031] In some preferred embodiments of the present invention, the pharmaceutical salt is a salt formed by a derivative of Formula I or Formula II and an inorganic or organic acid.

[0032] The inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, or nitric acid; organic acids include formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, nicotinic acid, bamoic acid, pectinic acid, 3-phenylpropionic acid, and picric acid. Pteropenic acid, 2-hydroxyethanesulfonic acid, itaconic acid, aminosulfonic acid, trifluoromethanesulfonic acid, dodecyl sulfuric acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheponic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, etc.

[0033] In some preferred embodiments of the present invention, the pharmaceutical salt is a hydrochloride, phosphate, sulfate, trifluoroacetate, methanesulfonic acid, tartaric acid, or maleic acid.

[0034] As used herein, "isomer" refers to the presence of one or more asymmetric centers in the berberine derivatives represented by Formula I or Formula II. The compounds of the present invention can exist as racemic derivatives, racemic mixtures, single enantiomers, diastereomer mixtures, single diastereomers, geometric isomers, etc. These compounds may be represented by the symbol "R" or "S," depending on the configuration of the substituents surrounding the stereocarbon atom.

[0035] Technical Theme Two

[0036] The present invention also provides a method for synthesizing the compound shown in Formula I or Formula II, comprising the following steps:

[0037] ;

[0038] or:

[0039] ;

[0040] Reaction conditions: solvent is dichloromethane or N,N-dimethylformamide; base is triethylamine or potassium carbonate; catalyst is 4-dimethylaminopyridine.

[0041] Technical Theme 3

[0042] The present invention provides a composition comprising a berberine derivative of formula I or formula II, an isomer thereof, or a pharmaceutical salt thereof.

[0043] Furthermore, the "pharmaceutical composition" may also contain one or more pharmaceutically acceptable carriers or excipients, and be prepared in the form of tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, tinctures, decoctions, lozenges, mixtures, suppositories, injections, inhalants, or sprays.

[0044] As used herein, “pharmaceuticalally acceptable carriers or excipients” include: diluents, fillers, binders, disintegrants, lubricants, flow aids, granulators, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, thickeners, antioxidants, preservatives, stabilizers, surfactants, and buffers. Those skilled in the art will understand that some pharmaceutically acceptable excipients may be used for more than one function and for alternative functions, depending on the amount of said excipient present in the formulation and what other ingredients are present in the formulation.

[0045] For example, when intended for oral administration, it can be formulated into oral preparations such as tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, and pills, containing fillers (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, calcium carboxymethyl cellulose, and sodium carboxymethyl cellulose); Arabica... Gum; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (e.g., gelatin, polyvinylpyrrolidone, and polyethylene glycol), disintegrants (e.g., cellulose derivatives such as sodium carboxymethyl cellulose and polyvinylpyrrolidone), lubricants (e.g., talc, calcium stearate, magnesium stearate, cetyl, boric acid, sodium benzoate, leucine), stabilizers (methylparaben, propylparaben, etc.), flavoring agents (e.g., commonly used sweeteners, acidulants, and flavorings, etc.).

[0046] When used parenterally, the drug can be formulated as an injectable preparation, including sterile powder for injection and solvent for injection. The carrier or excipients used may include sterile water, Ringer's solution, and isotonic sodium chloride solution. Appropriate excipients such as antioxidants, buffers, antibacterial agents, solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators may be added depending on the properties of the drug. Solubilizers or co-solvents may include poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters may include phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; and osmotic pressure regulators may include sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder for injection, mannitol, glucose, etc., may also be added as a support agent. When used rectally, the drug can be formulated as suppositories, etc.

[0047] When intended for pulmonary administration, the drug may be formulated as an inhaler or spray, etc. Numerous resources available to those skilled in the art describe pharmaceutically acceptable excipients and can be used to select appropriate pharmaceutically acceptable excipients, such as books like *Remington's Complete Pharmacy*, *Chinese Pharmaceutical Yearbook*, and *Pharmaceutics*.

[0048] The compounds of this invention and their pharmaceutically acceptable salts can be formulated into conventional formulations, as well as sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0049] Technical Theme 4

[0050] The present invention also provides the use of berberine derivatives of Formula I or Formula II, their isomers or pharmaceutical salts thereof in the preparation of antiviral drugs.

[0051] Furthermore, the antiviral agent is selected from anti-Ebola virus, anti-coronavirus, anti-dengue virus, anti-Zika virus, or anti-influenza virus.

[0052] Furthermore, the coronavirus is selected from SARS-CoV, SAR-CoV-2, MERS-CoV, and HcoV 229E.

[0053] Technology Theme 5

[0054] The present invention also provides the use of berberine derivatives of Formula I or Formula II, their isomers or pharmaceutical salts thereof in the preparation of medicaments for treating leukopenia.

[0055] Furthermore, the leukopenia refers to leukopenia following cancer radiotherapy or chemotherapy, or leukopenia caused by various reasons.

[0056] The beneficial effects of this invention are as follows:

[0057] This invention provides a class of berberine derivatives, which have been verified to have good antiviral and leukopenia treatment effects, and whose pharmacokinetics are more ideal than those of berberine. Attached Figure Description

[0058] Figure 1 Changes in body weight during mouse experiments;

[0059] Figure 2 The effect of the compound on peripheral blood counts in mice with leukopenia; In the figure: A is white blood cells (WBC), C is platelets, D is hemoglobin, E is red blood cells, F is lymphocytes, G is monocytes and H is neutrophils;

[0060] Figure 3 The results are statistical findings on the nucleated cell count in mouse bone marrow.

[0061] Figure 4 The effects of the compound on the thymus and spleen indices in a mouse model of leukopenia. Detailed Implementation

[0062] The present invention is illustrated below with reference to specific embodiments. These embodiments are not intended to limit the scope of the invention, but rather to provide guidance to those skilled in the art for the preparation and use of the compounds and compositions of the present invention. The chemical names of the compounds described in this application are generally derived from ChemDraw Ultra (Chambridge Soft) and are generated / or generally follow the principles of IUPAC nomenclature.

[0063] The synthetic routes of some of the compounds in this embodiment are as follows:

[0064] or

[0065] ;

[0066] Example 1

[0067] (Compound 1)

[0068] At room temperature, triethylamine (66 μL, 0.48 mmol), DMAP (6 mg, 0.05 mmol), and dimethylcarbamoyl chloride (30 μL, 0.32 mmol) were added sequentially to a solution of berberine hydrochloride (100 mg, 0.15 mmol) in dichloromethane (20 mL). The mixture was stirred overnight at the same temperature, quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated, and the residue was subjected to silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain the above compound as a white solid (69 mg, 68%). 1 H NMR (600 MHz, DMSO) δ 7.42 – 7.39 (m, 1H), 6.93 –6.90 (m, 2H), 6.89 – 6.86(m, 1H), 6.79 – 6.76 (m, 1H), 6.74 (s, 1H), 6.46 (d,J = 9.6 Hz, 2H), 6.26 (dd, J = 8.2, 2.6 Hz, 1H), 5.45 (d, J = 2.0 Hz, 1H), 4.03 – 4.01 (m, 1H), 3.73 (s, 3H), 3.60 (s, 3H), 3.53 (brs, 1H), 3.25 – 3.21(m, 1H), 3.13 – 3.00 (m, 9H), 2.94 – 2.85 (m, 6H), 2.75 – 2.69 (m, 2H), 2.65– 2.59 (m, 1H), 2.54 (s, 3H), 2.52 – 2.51 (m, 2H), 2.36 – 2.26 (m, 3H). ESI-MS: 680 [M+H] + .

[0069] Example 2

[0070] (Compound 2)

[0071] The preparation method was the same as in Example 1, but diethylcarbamoyl chloride was used instead of dimethylcarbamoyl chloride to obtain the above-mentioned white solid compound, with a yield of 75%. 1H NMR (500 MHz, DMSO) δ 7.40 (d, J = 8.4 Hz, 1H), 6.89 (dd,J = 24.5, 7.9 Hz, 3H), 6.76 (d, J = 8.4 Hz, 1H), 6.73 (s, 1H), 6.46 (s, 2H), 6.25 (dd, J = 8.2, 2.6 Hz, 1H), 5.44 (s, 1H), 4.05 (s, 1H), 3.72 (s, 3H), 3.59 (s, 3H), 3.55 (s, 1H), 3.24 (d, J = 15.0 Hz, 3H), 3.08 (s, 5H), 3.03 (s,1H), 2.94 – 2.83 (m, 4H), 2.78 – 2.70 (m, 3H), 2.62 (d, J = 17.2 Hz, 2H), 2.55 (s, 3H), 2.30 (s, 3H), 1.12 (d, J = 54.7 Hz, 7H); MS-ESI (m / z): 708.2 (M+H) + .

[0072] Example 3

[0073] (Compound 3)

[0074] The preparation method was the same as in Example 1, but diisopropylcarbamoyl chloride was used instead of dimethylcarbamoyl chloride to obtain the above-mentioned white solid compound, with a yield of 64%. 1 H NMR (500 MHz, DMSO) δ 7.40 (s, 1H), 6.87 (dd, J =28.3, 8.1 Hz, 3H), 6.80 – 6.70 (m, 2H), 6.46 (d, J = 5.3 Hz, 2H), 6.25 (t, J= 12.4 Hz, 1H), 5.44 (s, 1H), 3.99 (s, 4H), 3.72 (s, 3H), 3.59 (s, 3H), 3.31(s, 5H), 3.07 (s, 4H), 2.88 (t, J = 13.6 Hz, 3H), 2.78 – 2.70 (m, 2H), 2.55(s, 4H), 2.32 (s, 1H), 1.21 (s, 14H);MS-ESI (m / z): 736.3 (M+H) + .

[0075] Example 4

[0076] (Compound 4)

[0077] The preparation method was the same as in Example 1, but 1-piperidine carbonyl chloride was used instead of dimethylcarbamoyl chloride to obtain the above-mentioned white solid compound, with a yield of 71%. 1 H NMR (500 MHz, DMSO) δ 7.39 (d, J = 8.3 Hz, 1H), 6.88 (dd, J= 21.3, 8.2 Hz, 3H), 6.75 (d, J = 8.5 Hz, 1H), 6.72 (s, 1H), 6.45 (d, J = 5.5Hz, 2H), 6.25 (d, J = 7.6 Hz, 1H), 5.43 (s, 1H), 4.01 (s, 1H), 3.72 (s, 3H), 3.59 (s, 3H), 3.56 – 3.51 (m, 3H), 3.36 (s, 3H), 3.23 (d, J = 14.8 Hz, 2H),3.08 (s, MS-ESI (m / z): 720.3 (M+H) + .

[0078] Example 5

[0079] (Compound 5)

[0080] The preparation method was the same as in Example 1, but 4-morpholine carbonyl chloride was used instead of dimethylcarbamoyl chloride to obtain the above-mentioned white solid compound, with a yield of 55%. 1H NMR (500 MHz, DMSO) δ 7.40 (d, J = 8.2 Hz, 1H), 6.91 (dt, J = 28.1, 8.4 Hz, 3H), 6.78 (d, J = 7.9 Hz, 1H), 6.73 (s, 1H), 6.46 (d, J = 7.4Hz, 2H), 6.28 – 6.23 (m, 1H), 5.46 (s, 1H), 4.01 (t, J = 4.3 Hz, 1H), 3.73(s, 3H), 3.64 – 3.51 (m, 10H), 3.40 (s, 4H), 3.23 (d, J = 14.6 Hz, 2H), 3.14– 2.99 (m, 5H), 2.98 – 2.67 (m, 6H), 2.67 – 2.52 (m, 4H), 2.44 – 2.26 (m,2H); MS-ESI (m / z): 722.2 (M+H) + .

[0081] Example 6

[0082] (Compound 6)

[0083] The preparation method was the same as in Example 1, but 4-thiomorpholine carbonyl chloride was used instead of dimethylcarbamoyl chloride to obtain the above-mentioned white solid compound in 80% yield. 1 H NMR (500 MHz, DMSO) δ 7.44 (s, 1H), 6.93 (ddd, J =21.2, 17.8, 8.2 Hz, 3H), 6.80 (d, J = 8.0 Hz, 1H), 6.75 (s, 1H), 6.49 (s,2H), 6.29 (d, J = 8.1 Hz, 1H), 5.45 (s, 1H), 3.85 (s, 3H), 3.74 (s, 4H), 3.68(s, 3H), 3.61 (s, 4H), 3.10 (s, 5H), 3.04 (s, 1H), 2.99 – 2.84 (m, 4H), 2.63(d, J = 36.0 Hz, 12H), 2.31 (s, 1H);MS-ESI (m / z): 738.3 (M+H) + .

[0084] Example 7

[0085] (Compound 7)

[0086] The preparation method was the same as in Example 1, but 4-methylpiperazine-1-formyl chloride was used instead of dimethylcarbamoyl chloride to obtain the above-mentioned white solid compound, with a yield of 67%. 1 H NMR (500 MHz, DMSO) δ 7.38 (d, J = 8.5 Hz, 1H), 6.97– 6.84 (m, 3H), 6.76 (d, J = 8.2 Hz, 1H), 6.72 (s, 1H), 6.45 (d, J = 6.2 Hz, 2H), 6.23 (s, 1H), 5.43 (s, 1H), 4.00 (s, 1H), 3.71 (s, 3H), 3.59 (s, 4H), 3.52 (s, 1H), 3.39 (s, 2H), 3.22 (d, J = 14.8 Hz, 2H), 3.07 (s, 5H), 2.96 –2.79 (m, 3H), 2.76 – 2.68 (m, 2H), 2.53 (s, 4H), 2.49 (s, 3H), 2.30 (d, J =10.1 Hz, 7H), 2.15 (d, J = 11.8 Hz, 3H); MS-ESI (m / z): 735.3 (M+H) + .

[0087] Example 8

[0088] (Compound 8)

[0089] At 0°C, triphosgene (208 mg, 0.7 mmol) was added to a flask under argon protection. Anhydrous dichloromethane (10 mL) was added, followed by dropwise addition of pyridine (166 μL, 2.1 mmol). Finally, 3-(diethylamino)tetrahydropyrrole (100 mg, 0.7 mmol) was added, and the reaction was carried out at 0°C for 12 hours. The reaction solution was then directly concentrated and added to the next step.

[0090] At room temperature, triethylamine was added to a 20 mL solution of berberine hydrochloride (154 mg, 0.23 mmol) in dichloromethane to adjust the pH to 7. Then, triethylamine (96 μL, 0.69 mmol), DMAP (17 mg, 0.14 mmol), and 3-(diethylamino)tetrahydropyrrole-1-formyl chloride (100 mg, 0.52 mmol) were added sequentially. The mixture was stirred overnight at room temperature, quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated, and the residue was subjected to silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain the above compound as a white solid (128 mg, yield 79%). 1 H NMR (500MHz, DMSO) δ 7.40 (d, J = 8.4 Hz, 1H), 6.99 – 6.82 (m, 3H), 6.77 (d, J = 8.3Hz, 1H), 6.73 (s, 1H), 6.46 (s, 2H), 6.26 (d, J = 8.2 Hz, 1H), 5.44 (s, 1H), 4.02 (s, 1H), 3.72 (s, 3H), 3.60 (s, 3H), 3.50 (d, J = 40.1 Hz, 4H), 3.24 (d,J = 15.2 Hz, 4H), 3.15 – 2.97 (m, 6H), 2.94 – 2.80 (m, 4H), 0.94 (s, 6H);MS-ESI (m / z): 708.2 (M+H) + .

[0091] Example 9

[0092] (Compound 9)

[0093] The preparation method was the same as in Example 8, but 1,2-dimethylpiperazine was used instead of 3-(diethylamino)tetrahydropyrrole to obtain the above-mentioned white solid compound in 62% yield. 1H NMR (500 MHz, DMSO) δ 7.41 (d, J = 8.4 Hz, 1H), 6.94 – 6.86 (m, 3H), 6.78 (d, J = 8.3 Hz, 1H), 6.74 (s, 1H), 6.47 (d, J = 4.3Hz, 2H), 6.26 (d, J = 8.2 Hz, 1H), 5.46 (s, 1H), 4.03 (s, 2H), 3.73 (s, 3H), 3.61 (s, 2H), 3.55 (s, 1H), 3.28 (d, J = 48.3 Hz, 8H), 3.19 – 3.01 (m, 5H),2.89 (dq, J = 27.1, 12.0, 11.2 Hz, 3H), 2.75 – 2.71 (m, 3H), 2.55 (s, 4H), 2.51 (s, 3H), 2.33 – 2.27 (m, 2H), 2.17 (s, 4H), 0.99 (s, 2H); (m / z):749.2 (M+H) + .

[0094] Example 10

[0095] (Compound 10)

[0096] The preparation method was the same as in Example 1, but 1-pyrrolidine carbonyl chloride was used instead of dimethylcarbamoyl chloride to obtain the above-mentioned white solid compound, with a yield of 27%. 1H NMR (500 MHz, DMSO) δ 7.38 (d, J = 7.4 Hz, 1H), 6.93 –6.83 (m, 3H), 6.76 – 6.71 (m, 2H), 6.44 (d, J = 10.3 Hz, 2H), 6.24 (dd, J =8.2, 2.6 Hz, 1H), 5.43 (s, 1H), 3.98 (d, J = 5.1 Hz, 1H), 3.71 (s, 4H), 3.59(s, 3H), 3.52 – 3.45 (m, 4H), 3.32 – 3.28 (m, 3H), 3.21 (d, J = 14.4 Hz, 1H),3.13 – 2.99 (m, 6H), 2.93 – 2.82 (m, 2H), 2.78 (dd, J = 12.3, 6.7 Hz, 1H), 2.65 (ddd, J = 65.0, 15.6, 5.0 Hz, 4H), 2.36 – 2.22 (m, 3H), 1.85 (dq, J =20.7, 6.8 Hz, 5H).

[0097] Example 11

[0098] (Compound 11)

[0099] The preparation method was the same as in Example 8, but N-ethylpiperazine was used instead of 3-(diethylamino)tetrahydropyrrole to obtain the above-mentioned yellow solid compound in 45% yield. 1H NMR (500 MHz, DMSO) δ 7.38 (d, J = 8.4 Hz, 1H), 6.94 –6.84 (m, 3H), 6.77 – 6.72 (m, 2H), 6.44 (d, J = 9.0 Hz, 2H), 6.24 (dd, J =8.2, 2.6 Hz, 1H), 5.43 (s, 1H), 3.98 (d, J = 5.0 Hz, 1H), 3.71 (s, 3H), 3.59(s, 5H), 3.51 (s, 1H), 3.38 (s, 4H), 3.30 (s, 2H), 3.21 (d, J = 14.5 Hz, 1H),3.12 – 2.99 (m, 5H), 2.89 (td, J = 16.0, 9.6 Hz, 2H), 2.82 – 2.56 (m, 5H), 2.30 (ddd, J = 19.7, 13.6, 8.3 Hz, 10H), 0.98 (t, J = 7.2 Hz, 3H).

[0100] Example 12

[0101] (Compound 12)

[0102] The preparation method was the same as in Example 8, but 1-isopropylpiperazine was used instead of 3-(diethylamino)tetrahydropyrrole to obtain the above-mentioned yellow oily compound, with a yield of 44%. 1 H NMR (500 MHz, DMSO) δ 7.38 (dd, J = 8.4, 2.2 Hz, 1H), 6.93 – 6.83 (m, 3H), 6.77 – 6.70 (m, 2H), 6.44 (d, J = 9.5 Hz, 2H), 6.24 (dd,J = 8.2, 2.6 Hz, 1H), 5.43 (s, 1H), 3.99 (s, 1H), 3.71 (s, 4H), 3.55 (d, J =40.0 Hz, 7H), 3.36 (s, 2H), 3.21 (d, J = 14.4 Hz, 1H), 3.13 – 2.98 (m, 6H),2.95 – 2.83 (m, 2H), 2.82 – 2.56 (m, 7H), 2.46 – 2.26 (m, 7H), 0.93 (d, J =6.5 Hz, 7H).

[0103] Example 13

[0104] (Compound 13)

[0105] The preparation method was the same as in Example 1, but methylcarbamoyl chloride was used instead of dimethylcarbamoyl chloride to obtain the above-mentioned yellow oily compound, with a yield of 50%. 1 H NMR (500 MHz, DMSO) δ 7.54 (q, J = 4.6 Hz, 1H), 7.37 (s,1H), 6.91 – 6.82 (m, 3H), 6.74 (d, J = 8.4 Hz, 2H), 6.45 (d, J = 12.0 Hz,2H), 6.22 (s, 1H), 5.42 (s, 1H), 3.99 (d, J = 5.1 Hz, 1H), 3.71 (s, 4H), 3.59(s, 3H), 3.50 (s, 1H), 3.29 (d, J = 11.2 Hz, 2H), 3.21 (d, J = 14.8 Hz, 1H),3.03 (d, J = 35.6 Hz, 6H), 2.89 (td, J = 16.2, 15.4, 4.9 Hz, 2H), 2.81 – 2.68(m, 4H), 2.66 – 2.55 (m, 5H), 2.40 – 2.24 (m, 3H).

[0106] Example 14

[0107] (Compound 14)

[0108] The preparation method was the same as in Example 8, but 4-methylpiperidine was used instead of 3-(diethylamino)tetrahydropyrrole to obtain the above-mentioned yellow solid compound in 63% yield. 1H NMR (500 MHz, DMSO) δ 7.42 – 7.36 (m, 1H), 6.93 – 6.83(m, 3H), 6.74 (d, J = 14.7 Hz, 2H), 6.45 (d, J = 3.5 Hz, 2H), 6.25 (d, J =8.3 Hz, 1H), 5.42 (d, J = 2.0 Hz, 1H), 4.14 – 3.90 (m, 3H), 3.71 (s, 4H), 3.59 (s, 3H), 3.52 (s, 1H), 3.27 (d, J = 41.8 Hz, 2H), 3.13 – 2.67 (m, 13H), 2.54 (s, 3H), 2.30 (dt, J = 19.6, 10.2 Hz, 3H), 1.58 (dd, J = 22.1, 9.7 Hz, 3H), 1.15 – 0.96 (m, 2H), 0.86 (d, J = 6.2 Hz, 4H).

[0109] Example 15

[0110] (Compound 15)

[0111] The preparation method was the same as in Example 8, but 4-(trifluoromethyl)piperidine was used instead of 3-(diethylamino)tetrahydropyrrole to obtain the above-mentioned white solid compound in 60% yield; 1H NMR (500 MHz, DMSO) δ 7.37 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 6.87 (dd, J = 17.4, 9.0 Hz, 2H), 6.75 (d, J = 19.3Hz, 2H), 6.44 (d, J = 8.6 Hz, 2H), 6.23 (d, J = 5.8 Hz, 1H), 5.43 (s, 1H), 4.23 (s, 1H), 4.06 (s, 1H), 3.99 (d, J = 5.1 Hz, 1H), 3.71 (s, 4H), 3.59 (s,3H), 3.51 (s, 1H), 3.30 (s, 1H), 3.21 (d, J = 14.5 Hz, 1H), 3.13 – 2.98 (m,7H), 2.88 (q, J = 12.0 Hz, 4H), 2.81 – 2.56 (m, 7H), 2.37 – 2.24 (m, 3H), 1.82 (d, J = 12.3 Hz, 2H), 1.40 (d, J = 70.1 Hz, 2H).

[0112] Example 16

[0113] (Compound 16)

[0114] The preparation method was the same as in Example 1, except that tetrandrine was used instead of berberine hydrochloride to obtain the above-mentioned yellow solid compound in 80% yield. 1H NMR (500 MHz, DMSO) δ 7.49 (s, 1H), 7.09 (dd, J = 8.2, 2.6 Hz,1H), 6.94 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 10.1 Hz, 1H), 6.70 (d, J = 18.2Hz, 2H), 6.45 (s, 1H), 6.32 (s, 2H), 5.78 (s, 1H), 3.83 (s, 4H), 3.65 (s,3H), 3.55 – 3.45 (m, 2H), 3.35 (s, 2H), 3.30 (d, J = 10.0 Hz, 2H), 3.20 (dd,J = 12.6, 5.9 Hz, 1H), 2.86 (dq, J = 18.0, 9.1 Hz, 3H), 2.75 (dd, J = 11.8,6.9 Hz, 3H), 2.71 – 2.62 (m, 4H), 2.58 (brs, 4H), 2.45 – 2.37 (m, 1H), 2.20(s, 6H); ESI-MS: 680 [M+H] + .

[0115] Example 17

[0116] (Compound 17)

[0117] The preparation method was the same as in Example 4, except that tetrandrine was used instead of berberine hydrochloride to obtain the above-mentioned yellow solid compound, with a yield of 70%. 1H NMR (500 MHz, DMSO) δ 7.46 (d, J = 8.1 Hz, 1H), 7.07 (dd, J =8.2, 2.6 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.82 – 6.74 (m, 1H), 6.64 (s,2H), 6.44 (s, 1H), 6.31 (s, 2H), 5.77 (s, 1H), 3.86 (s, 1H), 3.81 (s, 3H), 3.63 (s, 3H), 3.52 – 3.41 (m, 2H), 3.30 (s, 5H), 3.18 (dd, J = 12.5, 6.0 Hz,2H), 2.99 (s, 1H), 2.93 – 2.61 (m, 8H), 2.57 (s, 3H), 2.41 (dd, J = 16.4, 4.9Hz, 1H), 2.27 (d, J = 13.6 Hz, 1H), 2.17 (s, 3H), 1.40 (s, 6H).

[0118] Example 18

[0119] (Compound 18)

[0120] The preparation method was the same as in Example 3, except that tetrandrine was used instead of berberine hydrochloride to obtain the above-mentioned yellowish-white solid compound, with a yield of 80%. 1 H NMR (500 MHz, DMSO) δ 7.44 (s, 1H), 7.05 (s, 1H), 6.92 (d, J =8.2 Hz, 1H), 6.76 (d, J = 10.1 Hz, 1H), 6.67 (d, J = 32.9 Hz, 2H), 6.43 (s,1H), 6.31 (d, J = 17.6 Hz, 2H), 5.76 (s, 1H), 3.81 (s, 4H), 3.62 (s, 4H), 3.47 (s, 3H), 3.30 (s, 3H), 3.17 (s, 2H), 2.92 – 2.60 (m, 7H), 2.53 (s, 2H), 2.41 (d, J = 16.1 Hz, 1H), 2.30 (d, J = 13.2 Hz, 1H), 2.16 (s, 3H), 1.06 (d,J = 6.9 Hz, 6H), 0.93 (brs, 4H), 0.74 (s, 3H).

[0121] Example 19 Antiviral activity test and results

[0122] 1. Testing Method

[0123] Method 1:

[0124] To investigate the broad-spectrum antiviral activity of berberine hydrochloride derivatives, their inhibitory effect on the entry process of pseudoviruses containing target viral envelope proteins was first examined. Pseudoviruses containing reporter genes were constructed using Ebola virus envelope (EBOV-Zaire-GP) or coronavirus envelope (SARS-CoV-S, SARS-CoV2-S, MERS-CoV-S) combined with HIV core protein (HIV-NL4-3-Luc). Berberine hydrochloride derivatives were dissolved in DMSO, and drug concentration gradients of 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, and 20 μM were set. These were mixed with pseudoviruses (MOI=0.1) and added to HEK293T cells (or ACE2-overexpressing HeLa cells (suitable for coronaviruses)) for 4 hours. The cell culture supernatant was discarded, and fresh culture medium and an equal concentration of the small molecule compound were added. After culturing for another 48 hours, cells were collected and lysed using cell lysis buffer. The antiviral activity of the berberine hydrochloride derivative was assessed by measuring intracellular luciferase activity. DMSO was used as a negative control. The luciferase activity of the experimental group and the DMSO group was compared to calculate the virus inhibition rate.

[0125] Method 2:

[0126] To investigate the effect of drugs on the infectivity of true viruses, we used laboratory-preserved coronaviruses (HCoV-OC43, HCoV-229E), dengue virus, Zika virus, and influenza A virus. After dissolving the drugs in DMSO, we set up drug concentration gradients of 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM, mixed with the virus (MOI=0.1), and added to cell culture media (Huh7 cells (HCoV-OC43, HCoV-229E), Vero cells (dengue virus, Zika virus), MDCK cells (influenza A virus)). After incubation at 37°C for 4 hours, the culture medium was replaced, and the corresponding drug concentration was added. After further culturing for 48 hours, cells were collected, total cellular RNA was extracted, and intracellular viral RNA levels were detected using RT-qPCR. In addition, the supernatant was collected, and viral titers were calculated by serial dilution combined with cytopathic effect (CPE) microscopic examination. The antiviral activity of the drug was analyzed by combining changes in intracellular viral nucleic acid and viral titer in the supernatant.

[0127] 2. Results

[0128] 2.1 The in vitro anti-Ebola virus activity of the compounds of the present invention, as determined by Method 1, is shown in Table 1:

[0129] Table 1: Results of in vitro anti-Ebola virus activity of compounds from the examples

[0130]

[0131] 2.2 The in vitro anticoronavirus activity results of the compounds of the present invention, obtained by method 2, are shown in Table 2:

[0132] Table 2. Results of in vitro anticoronavirus activity of the compounds in the examples.

[0133]

[0134] 2.3 The in vitro anti-influenza virus activity results of the compounds of the present invention, obtained by method 2, are shown in Table 3:

[0135] Table 3. Results of in vitro anti-influenza virus activity of the compounds in the examples.

[0136]

[0137] Example 20 In vivo pharmacokinetic properties

[0138] Following a single oral administration of the target compound to female ICR mice, blood samples were collected at different time points. The concentration of the compound in rat plasma was determined by LC-MS / MS, and relevant pharmacokinetic parameters were calculated to investigate the compound's exposure in mice. Six ICR mice, provided by Suzhou Zhaoyan Experimental Animal Co., Ltd., were used in the experiment according to Table 4.

[0139] Table 4. Pharmacokinetic Dosing Regimen

[0140]

[0141] 0.030 mL of blood was collected from each mouse via the orbital cavity each time, anticoagulated with EDTAK2. Collection time points were: po. Group: 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after administration of the test substance. Blood samples were stored on ice after collection and centrifuged within 30 minutes to separate plasma (centrifugation conditions: 5000 rpm, 10 min, 4°C). The plasma was stored at –80°C before analysis.

[0142] The data acquisition and control system software was Analyst 1.5.1 (Applied Biosystems). The peak integration method for the spectral data was automatic integration; the ratio of the sample peak area to the internal standard peak area was used as an indicator, and regression was performed with the sample concentration. The regression method was linear regression, with a weighting coefficient of 1 / X.2 Pharmacokinetic parameters were analyzed using a non-compartmental model with WinNonlin Professional v6.3 (Pharsight, USA). max The measured maximum plasma concentration is represented by the area under the plasma concentration-time curve (AUC). (0→t) T is calculated using the trapezoidal method. max The time to peak plasma concentration is given. Experimental data are expressed as mean ± standard deviation (Mean ± ICR, n ≥ 3) or mean (Mean, n = 2), and the results are shown in Table 5.

[0143] Table 5. Pharmacokinetic properties of compound 1

[0144]

[0145] a ICR mice (n = 3) were orally administered compound 1 at 27.92 mg / kg (equimolar concentration), and the plasma concentrations of berberine and compound 1 were tested. b ICR mice (n = 3) were orally administered berberine at 25 mg / kg, and the plasma concentration of berberine was tested.

[0146] Example 21: Detection and Results of Compound Treatment for Leukopenia in Mice

[0147] 1. Detection Method

[0148] A mouse model of leukopenia was established: Except for the normal control group, BALB / C mice were intraperitoneally injected with 60 mg / kg cyclophosphamide once daily for three consecutive days. Mice in the normal control group were intraperitoneally injected with an equal volume of physiological saline. From the start of modeling, compound 1 or compound 16 was suspended in 0.5% CMC-Na solution and administered to the experimental animals by gavage (30 mg / kg) once daily for 7 consecutive days. Twenty-four hours after the last administration, blood was collected from the eyeballs, and 200 μl was collected in an anticoagulant tube containing heparin sodium for complete blood count (CBC). The animals were then euthanized by decapitation under isoflurane anesthesia. The thymus and spleen were harvested and weighed to calculate the thymus index and spleen index. The femur was harvested for bone marrow cytology. The removed thymus and spleen were blotted dry with absorbent paper and weighed; the ratio of the weight to the body weight was the spleen index and thymus index, respectively. Remove the muscles and connective tissue from the femur, inject PBS to flush the bone marrow from the medullary cavity, collect it by centrifugation, resuspend it in 200 μl of PBS, and take 10 μl for cell counting.

[0149] 2. Results

[0150] 2.1 Effects of Compound 1 and Compound 16 on the body weight of experimental mice

[0151] like Figure 1 As shown, the mice in the cyclophosphamide group had a significantly reduced body weight compared to the normal group. Compared to the cyclophosphamide group, the mice in the berberine, compound 1, and compound 16 treatment groups all recovered their body weight, while the body weight of the mice in the compound 16 treatment group was not significantly different from that of the normal group.

[0152] 2.2 Effects of Compound 1 and Compound 16 on Peripheral Blood Counts in Leukopenic Mice

[0153] Compared with the normal group, the cyclophosphamide group showed a significant decrease in the levels of white blood cells, lymphocytes, and neutrophils in the blood, while other parameters such as red blood cells and hemoglobin showed no significant changes. Compared with the cyclophosphamide group, the treatment groups of berberine, compound 16, and compound 1 showed significant efficacy in increasing the number of white blood cells and the percentage of lymphocytes.

[0154] 2.3 Effects of Compound 1 and Compound 16 on Bone Marrow Nucleated Cells

[0155] Compared with the normal group (PBS), the number of nucleated cells in the bone marrow of mice in the cyclophosphamide group was significantly reduced. However, the number of nucleated cells in the bone marrow of mice treated with compound 16 was significantly restored.

[0156] 2.4 Effects of Compound 1 and Compound 16 on the thymus and spleen indices in mice

[0157] The spleen index and thymus index reflect the strength of the body's immune function to some extent. For example... Figure 4 As shown, compared with the normal group, the thymus and spleen of the cyclophosphamide-treated group atrophied, and the spleen and thymus indices decreased significantly. Compared with the model group, the spleen and thymus indices of mice in the berberine and compound 16 groups were significantly increased.

Claims

1. A berberine derivative or its pharmaceutical salt, characterized in that, Its structure is one of the following: (1) Its structure is as shown in Formula I. ； Where R represents: ; R 3 Represents H, methyl, ethyl, n-propyl, isopropyl, isobutyl, -CF3, -NR y R x ; X represents —CH2—, —O—, —S—, —NR a —、—CHNR y R x —; R y R x and R a Each of these can be independently represented as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl. n1 and n2 independently represent 0, 1, and 2; (2) Its structure is selected from one of the following: , , or .

2. The berberine derivative or its pharmaceutical salt according to claim 1, characterized in that, Includes the following structure: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. The berberine derivative or its pharmaceutical salt according to claim 1, characterized in that, The medicinal salt is a salt formed by the compound of claim 1 and an inorganic or organic acid.

4. A method for synthesizing the compound of formula I as described in claim 1, characterized in that, Includes the following steps: Reaction conditions: solvent is dichloromethane or N,N-dimethylformamide; base is triethylamine or potassium carbonate; catalyst is 4-dimethylaminopyridine.

5. A pharmaceutical composition, characterized in that, Includes the berberine derivative or its pharmaceutical salt as described in any one of claims 1-3.

6. The pharmaceutical composition according to claim 5, characterized in that, It also includes one or more pharmaceutically acceptable carriers or excipients.

7. The use of the berberine derivative or its pharmaceutical salt as described in any one of claims 1-3 in the preparation of antiviral drugs.

8. The application according to claim 7, characterized in that, The antiviral agents are selected from those against Ebola virus, coronavirus, dengue virus, Zika virus, or influenza virus.

9. The application according to claim 8, characterized in that, The coronaviruses mentioned are selected from SARS-CoV, SAR-CoV-2, MERS-CoV, and HcoV 229E.

10. The use of the berberine derivative or its pharmaceutical salt as described in any one of claims 1-3 in the preparation of a medicament for treating or preventing leukopenia.

11. The application according to claim 10, characterized in that, The leukopenia mentioned refers to leukopenia following cancer radiotherapy or chemotherapy, or leukopenia caused by various reasons.

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