CN117327084A - Berberine derivatives, preparation method thereof and application thereof in antiviral and leukopenia treatment - Google Patents

Berberine derivatives, preparation method thereof and application thereof in antiviral and leukopenia treatment Download PDF

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Publication number
CN117327084A
CN117327084A CN202311281101.6A CN202311281101A CN117327084A CN 117327084 A CN117327084 A CN 117327084A CN 202311281101 A CN202311281101 A CN 202311281101A CN 117327084 A CN117327084 A CN 117327084A
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formula
pharmaceutically acceptable
acid
acceptable salt
isomer
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吕凯
岑山
刘明亮
衣岽戎
汪阿鹏
李泉洁
王静
赵建元
姜斌
秦晓瑜
宋慧娟
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Institute of Medicinal Biotechnology of CAMS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/18—Bridged systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Abstract

The invention belongs to the technical field of biological medicine, and in particular relates to a berberine derivative, a preparation method thereof and application thereof in antiviral aspect, which has a structure shown as a formula I or a formula II, and is proved by experimentsThe serial derivatives provided by the invention have good antiviral effect and effect of treating or preventing leucopenia, and have more ideal pharmacokinetic performance compared with berbamine.

Description

Berberine derivatives, preparation method thereof and application thereof in antiviral and leukopenia treatment
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a berberine derivative, a preparation method thereof and application thereof in antivirus and leukopenia treatment.
Background
Berbamine (structure formula is shown below) is a dibenzyl isoquinoline alkaloid existing in berberis plant, and has various biological activities. The berbamine dihydrochloride has been marketed under the generic name berbamine hydrochloride tablet, and has the trade name of Bai An, and can be used for treating leukopenia caused by various reasons, and also can be used for preventing leukopenia after cancer radiotherapy and chemotherapy. We haveRecent researches of research teams find that the berbamine has definite Ebola virus (eboov) resisting activity, and the berbamine targets GPcl protein acting on Ebola virus, and 100% protection can be realized by administration of 100mg/kg of the berbamine 1 day before infection on a mouse model infected by the eboov (blank control group 0%); 83% protection (blank 0%) was achieved 1 day after 100mg/kg infection (Acta pharm. Sin. B.2022, https:// doi. Org/10.1016/j. Apsb.2022.05.023). Furthermore, it has been reported that berbamine can exert anti-novel coronavirus (SARS-Cov-2) activity by preventing S-protein mediated membrane fusion, in vitro against the EC of SARS-Cov-2 50 1.73-1.89. Mu.M (Plos Neglect Trop D.2022, https:// doi.org/10.1371/journ.pntd.0010363).
However, during the post-development against berbamine, we found that the pharmacokinetic properties of berbamine were slightly worse, ICR mice were orally administered 25mg/kg, C max AUC of only 704ng/mL 0-t 8552 ng/mL.h. The use of the berbamine hydrochloride tablet is 28 mg/tablet, and the dosage of the use is 3 times a day and 4 tablets at a time. The administration mode shows that the berberine possibly has poor pharmacokinetic properties and poor absorption, so that the administration dosage is higher, the single dosage reaches 112mg, and the daily dosage reaches 336mg. Accordingly, it is necessary to find derivatives or analogues with better pharmacokinetic properties.
Disclosure of Invention
It is an object of the present invention to provide a series of berbamine derivatives.
It is still another object of the present invention to provide a process for preparing berbamine derivatives.
It is a further object of the present invention to provide a composition containing a berbamine derivative.
It is a further object of the present invention to provide the use of berbamine derivatives in the treatment and prevention of antiviral drugs.
It is a further object of the present invention to provide the use of berbamine derivatives in medicaments for the treatment and prevention of leukopenia.
Definition of terms
The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The nomenclature used herein and the laboratory procedures in organic chemistry, pharmaceutical chemistry, biology described herein are those well known and commonly employed in the art. Unless otherwise defined, 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 belongs.
As used in the description of embodiments of the invention and the appended claims, the singular forms "a," "an," "the," and "the" are used to refer to the singular and the plural of the article unless the context clearly dictates otherwise. For example, a compound includes one or more than one compound.
As used herein, "and/or" refers to and includes any and all possible combinations of one or more of the associated listed items.
As used herein, the term "disease" or "patient" refers to any change in a physical state or some organ that interrupts or interferes with the performance of its function and/or causes symptoms.
As used herein, the term "treatment" is intended to reduce or eliminate the disease state or condition for which it is intended. A subject is successfully "treated" if the subject has received a therapeutic amount of a compound, or a pharmaceutically acceptable salt, isomer, or pharmaceutical composition thereof, according to the methods described herein, and the subject exhibits an observable and/or detectable decrease or improvement in one or more indications and symptoms. It is also to be understood that the treatment of the disease state or condition described includes not only complete treatment, but also less than complete treatment, but achieves some biologically or medically relevant result.
Subject 1
The invention provides a berbamine derivative with a structure shown in a formula I or a formula II, an isomer or a medicinal salt thereof:
r represents:
wherein:
R 1 and R is 2 Independently of one another, represents H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, - (CH) 2 ) m OH、—(CH 2 ) m OCH 3 ；
R 3 Represents H, methyl, ethyl, n-propyl, isopropyl, isobutyl, -CF 3 、—CH 2 Cl、—CH 2 F、—CH 2 NR y R x 、—NR y R x ；
X represents-CH 2 —、—O—、—S—、—NR a —、—CHNR y R x —；
R y 、R x And R is a Each independently represents H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl;
n1 and n2 independently represent 0,1,2;
m represents 1,2, 3.
In some preferred embodiments of the invention, the R 1 And R is 2 And each independently represents H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl.
In some preferred embodiments of the invention, the R 3 Selected from H, methyl, ethyl, n-propyl, isopropyl, -NR y R x 、—CF 3 。
In some preferred embodiments of the invention, the derivative comprises the following structure:
in some preferred embodiments of the invention, the pharmaceutically acceptable salt is a salt of a derivative of formula I or formula II with an inorganic or organic acid.
The inorganic acid comprises hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid and the like; organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic 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, pamoic acid, pectate acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric 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, glucoheptonic acid, glycerophosphate, aspartic acid, sulfosalicylic acid, and the like.
In some preferred embodiments of the invention, the pharmaceutically acceptable salt is a hydrochloride or, phosphate, sulfate, trifluoroacetate, methanesulfonic acid, tartaric acid, maleic acid.
As used herein, "isomer" refers to the fact that where the berbamine derivative of formula I or II contains one or more asymmetric centers, the compounds of the present invention can exist as racemates, racemic mixtures, single enantiomers, diastereomeric mixtures, single diastereomers, geometric isomers, and the like. These compounds may be represented by the symbol "R" or "S", depending on the configuration of substituents around the stereogenic carbon atom.
Subject matter II
The invention also provides a synthesis method of the compound shown in the formula I or the formula II, which comprises the following steps:
or alternatively
Reaction conditions: the solvent is dichloromethane or N, N-dimethylformamide; the base is triethylamine or potassium carbonate; the catalyst is 4-dimethylaminopyridine.
Subject III
The invention provides a composition comprising a berbamine derivative, an isomer or a pharmaceutically acceptable salt thereof, as described in formula I or formula II.
Further, the "pharmaceutical composition" may also comprise one or more pharmaceutically acceptable carriers or excipients, and be prepared into the forms of tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, wines, ointments, lozenges, mixtures, suppositories, injections, inhalants or sprays and the like.
As used herein, "pharmaceutically acceptable carrier or excipient" includes: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, coloring agents, anti-caking agents, humectants, chelating agents, plasticizers, viscosity enhancing agents, antioxidants, preservatives, stabilizers, surfactants and buffers, it will be understood by those skilled in the art that certain pharmaceutically acceptable excipients may be used in more than one function and in alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.
For example: when used for oral administration, oral preparations such as tablets (including ordinary tablets, enteric tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric capsules), granules, pills and the like, containing fillers (e.g., saccharide 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 sodium, acacia, 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 and the like), binders (e.g., gelatin, polyvinylpyrrolidone and polyethylene glycol), disintegrants (e.g., cellulose derivatives such as carboxymethyl cellulose sodium, polyvinylpyrrolidone), lubricants (e.g., talc, calcium stearate, magnesium stearate, waxes, boric acid, sodium benzoate, leucine), stabilizers (methyl parahydroxybenzoate, propyl parahydroxybenzoate and the like), corrigents (e.g., sour taste corrigents, sweet taste and the like) may be prepared.
When used parenterally, injections, including sterile powders for injection and solvents for injection, may be formulated with carriers or excipients including sterile water, ringer's solution and isotonic sodium chloride solution, and appropriate additives such as antioxidants, buffers, bacteriostats, solubilizers, co-solvents, pH adjusting agents, and osmotic pressure adjusting agents may be added depending on the nature of the drug. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol, glucose, etc. can be added as propping agent for preparing lyophilized powder for injection. When used for rectal administration, the medicament may be formulated as suppositories and the like.
For pulmonary administration, the medicament may be formulated as an inhalant or spray, or the like. There are many sources available to those skilled in the art which describe pharmaceutically acceptable excipients and which can be used to select suitable pharmaceutically acceptable excipients, for example books such as Leimden pharmaceutical university, chinese pharmaceutical annual de-gress, pharmaceutics, etc.
The compound and the pharmaceutically acceptable salt thereof can be prepared into common preparations, slow release preparations, controlled release preparations, targeted preparations and various microparticle administration systems.
Technical subject IV
The invention also provides application of the berbamine derivative shown in the formula I or the formula II, isomers or medicinal salts thereof in preparing antiviral medicaments.
Further, the antiviral is selected from the group consisting of anti-ebola virus, anti-coronavirus, anti-dengue virus, anti-zika virus, or anti-influenza virus.
Further, the coronavirus is selected from SARS-CoV, SAR-CoV-2, MERS-CoV, hcov 229E.
Subject five technology
The invention also provides application of the berbamine derivative shown in the formula I or the formula II, an isomer or a medicinal salt thereof in preparing medicaments for treating leucopenia.
Further, the leukopenia is leukopenia after cancer radiotherapy, chemotherapy or a leukopenia caused by various reasons.
The beneficial effects of the invention are as follows:
the invention provides a kind of berberine derivatives, which has good antiviral and leukopenia treatment effects and more ideal pharmacokinetics compared with berberine through verification.
Drawings
FIG. 1 is the body weight change during the mice test;
FIG. 2 is the effect of compounds on peripheral blood images of leukopenia mice; in the figure: a is White Blood Cell (WBC), C is platelet, D is hemoglobin, E is red blood cell, F is lymphocyte, G monocyte, and H neutrophil;
FIG. 3 is a statistical result of the bone marrow nucleated cell count of the mice;
FIG. 4 is the effect of compounds on thymus and spleen index in mice with leukopenia models.
Detailed Description
The present invention is illustrated below in conjunction with specific examples which are not intended to limit the scope of the invention, but rather to provide guidance to those skilled in the art in making and using the compounds, compositions of the present invention. Chemical names of the compounds described in this application are generally from ChemDraw Ultra (chambridge soft) and generated/or generally follow the principles of IUPAC nomenclature.
The synthetic routes for the compounds of this example section are as follows:
example 1
To a solution of berbamine dihydrochloride (100 mg,0.15 mmol) in dichloromethane (20 mL) was added triethylamine (66. Mu.L, 0.48 mmol), DMAP (6 mg,0.05 mmol) and dimethylcarbamoyl chloride (30. Mu.L, 0.32 mmol) in this order at room temperature, stirred overnight at room temperature, quenched with saturated aqueous sodium bicarbonate, extracted with dichloromethane, washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated, the residue was purified by silica gel column chromatography (dichloromethane: methanol=20:1) to give the above compound as a white solid (69 mg, 68%), 1 H NMR(600MHz,DMSO)δ7.42-7.39(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＝9.6Hz,2H),6.26(dd,J＝8.2,2.6Hz,1H),5.45(d,J＝2.0Hz,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] + .
example 2
The preparation method was the same as in example 1, using diethylcarbamoyl chloride instead of dimethylcarbamoyl chloride to prepare the above white solid compound in 75% yield, 1 H NMR(500MHz,DMSO)δ7.40(d,J＝8.4Hz,1H),6.89(dd,J＝24.5,7.9Hz,3H),6.76(d,J＝8.4Hz,1H),6.73(s,1H),6.46(s,2H),6.25(dd,J＝8.2,2.6Hz,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＝15.0Hz,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＝17.2Hz,2H),2.55(s,3H),2.30(s,3H),1.12(d,J＝54.7Hz,7H)；MS-ESI(m/z):708.2(M+H) + .
example 3
The preparation method was the same as in example 1, using diisopropylcarbamoyl chloride instead of dimethylcarbamoyl chloride to prepare the above white solid compound in 64% yield, 1 H NMR(500MHz,DMSO)δ7.40(s,1H),6.87(dd,J＝28.3,8.1Hz,3H),6.80-6.70(m,2H),6.46(d,J＝5.3Hz,2H),6.25(t,J＝12.4Hz,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＝13.6Hz,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) + 。
example 4
The preparation method was the same as in example 1, except that 1-piperidinecarbonyl chloride was used instead of dimethylcarbamoyl chloride to prepare the above white solid compound in 71% yield, 1 H NMR(500MHz,DMSO)δ7.39(d,J＝8.3Hz,1H),6.88(dd,J＝21.3,8.2Hz,3H),6.75(d,J＝8.5Hz,1H),6.72(s,1H),6.45(d,J＝5.5Hz,2H),6.25(d,J＝7.6Hz,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＝14.8Hz,2H),3.08(s,5H),2.88(q,J＝14.8,11.4Hz,3H),2.71(s,2H),2.53(s,5H),2.43(s,1H),2.29(s,2H),1.52(d,J＝33.4Hz,6H)；MS-ESI(m/z):720.3(M+H) + 。
example 5
The preparation method is the same as in example 1, 4-morpholinecarbonyl chloride is used for replacing dimethylcarbamoyl chloride to prepare the white solid compound, the yield is 55%, 1 H NMR(500MHz,DMSO)δ7.40(d,J＝8.2Hz,1H),6.91(dt,J＝28.1,8.4Hz,3H),6.78(d,J＝7.9Hz,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＝4.3Hz,1H),3.73(s,3H),3.64-3.51(m,10H),3.40(s,4H),3.23(d,J＝14.6Hz,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) + 。
example 6
The preparation method is the same as in example 1, and 4-thiomorpholinecarbonyl chloride is used for replacing dimethylcarbamoyl chloride to prepare the white solid compound, the yield is 80%, 1 H NMR(500MHz,DMSO)δ7.44(s,1H),6.93(ddd,J＝21.2,17.8,8.2Hz,3H),6.80(d,J＝8.0Hz,1H),6.75(s,1H),6.49(s,2H),6.29(d,J＝8.1Hz,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＝36.0Hz,12H),2.31(s,1H)；MS-ESI(m/z):738.3(M+H) + 。
example 7
The preparation was carried out in the same manner as in example 1 using 4-methylpiperazine-1-carbonyl chloride instead of dimethylcarbamoyl chloride to give the above white solid compound in 67% yield, 1 H NMR(500MHz,DMSO)δ7.38(d,J＝8.5Hz,1H),6.97-6.84(m,3H),6.76(d,J＝8.2Hz,1H),6.72(s,1H),6.45(d,J＝6.2Hz,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＝14.8Hz,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＝10.1Hz,7H),2.15(d,J＝11.8Hz,3H)；MS-ESI(m/z):735.3(M+H) + .
example 8
Triphosgene (208 mg,0.7 mmol) was added to the flask at 0deg.C, argon protected, anhydrous dichloromethane (10 mL) was added, pyridine (166 μL,2.1 mmol) was added dropwise, and finally 3- (diethylamino) tetrahydropyrrole (100 mg,0.7 mmol) was added and reacted at 0deg.C for 12 hours. The reaction solution was concentrated directly to the next step.
To a solution of berberine dihydrochloride (154 mg,0.23 mmol) in dichloromethane (20 mL) was added triethylamine to adjust ph=7, followed by triethylamine (96 μl,0.69 mmol), DMAP (17 mg,0.14 mmol) and 3- (diethylamino) tetrahydropyrrole-1-carbonyl chloride (100 mg,0.52 mmol) in sequence, stirred overnight at room temperature, quenched with saturated aqueous sodium bicarbonate, extracted with dichloromethane, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane: methanol=20:1) to give the above compound as a white solid (128 mg, yield 79%), 1 H NMR(500MHz,DMSO)δ7.40(d,J＝8.4Hz,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＝8.2Hz,1H),5.44(s,1H),4.02(s,1H),3.72(s,3H),3.60(s,3H),3.50(d,J＝40.1Hz,4H),3.24(d,J＝15.2Hz,4H),3.15-2.97(m,6H),2.94-2.80(m,4H),2.77-2.62(m,4H),2.54(s,6H),2.44(s,1H),2.36-2.26(m,2H),2.10-2.00(m,1H),1.74(dt,J＝29.0,10.7Hz,1H),0.94(s,6H)；MS-ESI(m/z):708.2(M+H) + .
example 9
The preparation method is implemented in the same wayExample 8 the above white solid compound was prepared using 1, 2-dimethylpiperazine instead of 3- (diethylamino) tetrahydropyrrole in 62% yield, 1 H NMR(500MHz,DMSO)δ7.41(d,J＝8.4Hz,1H),6.94-6.86(m,3H),6.78(d,J＝8.3Hz,1H),6.74(s,1H),6.47(d,J＝4.3Hz,2H),6.26(d,J＝8.2Hz,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＝48.3Hz,8H),3.19-3.01(m,5H),2.89(dq,J＝27.1,12.0,11.2Hz,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)；MS-ESI(m/z):749.2(M+H) + .
example 10
The preparation method was the same as in example 1, except that 1-pyrrolidone carbonyl chloride was used instead of dimethylformamide to prepare the above white solid compound in 27% yield, 1 H NMR(500MHz,DMSO)δ7.38(d,J＝7.4Hz,1H),6.93-6.83(m,3H),6.76-6.71(m,2H),6.44(d,J＝10.3Hz,2H),6.24(dd,J＝8.2,2.6Hz,1H),5.43(s,1H),3.98(d,J＝5.1Hz,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＝14.4Hz,1H),3.13-2.99(m,6H),2.93-2.82(m,2H),2.78(dd,J＝12.3,6.7Hz,1H),2.65(ddd,J＝65.0,15.6,5.0Hz,4H),2.36-2.22(m,3H),1.85(dq,J＝20.7,6.8Hz,5H).
example 11
The preparation method is the same as in example 8, and N-ethylpiperazine is used for replacing 3- (diethylamino) tetrahydropyrrole to prepare the yellow solid compound with the yield of 45%, 1 H NMR(500MHz,DMSO)δ7.38(d,J＝8.4Hz,1H),6.94-6.84(m,3H),6.77-6.72(m,2H),6.44(d,J＝9.0Hz,2H),6.24(dd,J＝8.2,2.6Hz,1H),5.43(s,1H),3.98(d,J＝5.0Hz,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＝14.5Hz,1H),3.12-2.99(m,5H),2.89(td,J＝16.0,9.6Hz,2H),2.82-2.56(m,5H),2.30(ddd,J＝19.7,13.6,8.3Hz,10H),0.98(t,J＝7.2Hz,3H)。
example 12
The preparation method is the same as in example 8, and 1-isopropyl piperazine is used for replacing 3- (diethylamino) tetrahydropyrrole to prepare the yellow oily compound with the yield of 44%, 1 H NMR(500MHz,DMSO)δ7.38(dd,J＝8.4,2.2Hz,1H),6.93-6.83(m,3H),6.77-6.70(m,2H),6.44(d,J＝9.5Hz,2H),6.24(dd,J＝8.2,2.6Hz,1H),5.43(s,1H),3.99(s,1H),3.71(s,4H),3.55(d,J＝40.0Hz,7H),3.36(s,2H),3.21(d,J＝14.4Hz,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＝6.5Hz,7H).
example 13
The preparation method is the same as in example 1, and methylamino formyl chloride is used for replacing dimethylcarbamoyl chloride to prepare the yellow oily compound with the yield of 50 percent, 1 H NMR(500MHz,DMSO)δ7.54(q,J＝4.6Hz,1H),7.37(s,1H),6.91-6.82(m,3H),6.74(d,J＝8.4Hz,2H),6.45(d,J＝12.0Hz,2H),6.22(s,1H),5.42(s,1H),3.99(d,J＝5.1Hz,1H),3.71(s,4H),3.59(s,3H),3.50(s,1H),3.29(d,J＝11.2Hz,2H),3.21(d,J＝14.8Hz,1H),3.03(d,J＝35.6Hz,6H),2.89(td,J＝16.2,15.4,4.9Hz,2H),2.81-2.68(m,4H),2.66-2.55(m,5H),2.40-2.24(m,3H).
example 14
The preparation method is the same as in example 8, and 4-methylpiperidine is used for replacing 3- (diethylamino) tetrahydropyrrole to prepare the yellow solid compound, the yield is 63%, 1 H NMR(500MHz,DMSO)δ7.42-7.36(m,1H),6.93-6.83(m,3H),6.74(d,J＝14.7Hz,2H),6.45(d,J＝3.5Hz,2H),6.25(d,J＝8.3Hz,1H),5.42(d,J＝2.0Hz,1H),4.14-3.90(m,3H),3.71(s,4H),3.59(s,3H),3.52(s,1H),3.27(d,J＝41.8Hz,2H),3.13-2.67(m,13H),2.54(s,3H),2.30(dt,J＝19.6,10.2Hz,3H),1.58(dd,J＝22.1,9.7Hz,3H),1.15-0.96(m,2H),0.86(d,J＝6.2Hz,4H).
example 15
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The preparation was carried out in the same manner as in example 8 using 4- (trifluoromethyl) piperidine instead of 3- (diethylamino) tetrahydropyrrole to obtain the above white solid compound in 60% yield; 1 H NMR(500MHz,DMSO)δ7.37(d,J＝8.4Hz,1H),6.93(d,J＝8.1Hz,1H),6.87(dd,J＝17.4,9.0Hz,2H),6.75(d,J＝19.3Hz,2H),6.44(d,J＝8.6Hz,2H),6.23(d,J＝5.8Hz,1H),5.43(s,1H),4.23(s,1H),4.06(s,1H),3.99(d,J＝5.1Hz,1H),3.71(s,4H),3.59(s,3H),3.51(s,1H),3.30(s,1H),3.21(d,J＝14.5Hz,1H),3.13-2.98(m,7H),2.88(q,J＝12.0Hz,4H),2.81-2.56(m,7H),2.37-2.24(m,3H),1.82(d,J＝12.3Hz,2H),1.40(d,J＝70.1Hz,2H).
example 16
The preparation method is the same as in example 1, and tetrandrine is used for replacing berberine to prepare the yellow solid compound with the yield of 80 percent, 1 H NMR(500MHz,DMSO)δ7.49(s,1H),7.09(dd,J＝8.2,2.6Hz,1H),6.94(d,J＝8.2Hz,1H),6.80(d,J＝10.1Hz,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＝10.0Hz,2H),3.20(dd,J＝12.6,5.9Hz,1H),2.86(dq,J＝18.0,9.1Hz,3H),2.75(dd,J＝11.8,6.9Hz,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] + .
example 17
The preparation method is the same asExample 4, the yellow solid compound was prepared using tetrandrine instead of berbamine, in 70% yield, 1 H NMR(500MHz,DMSO)δ7.46(d,J＝8.1Hz,1H),7.07(dd,J＝8.2,2.6Hz,1H),6.92(d,J＝8.2Hz,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＝12.5,6.0Hz,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＝13.6Hz,1H),2.17(s,3H),1.40(s,6H).
example 18
The preparation method is the same as in example 3, and tetrandrine is used for replacing berberine to prepare the yellow-white solid compound with the yield of 80 percent, 1 H NMR(500MHz,DMSO)δ7.44(s,1H),7.05(s,1H),6.92(d,J＝8.2Hz,1H),6.76(d,J＝10.1Hz,1H),6.67(d,J＝32.9Hz,2H),6.43(s,1H),6.31(d,J＝17.6Hz,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＝16.1Hz,1H),2.30(d,J＝13.2Hz,1H),2.16(s,3H),1.06(d,J＝6.9Hz,6H),0.93(brs,4H),0.74(s,3H).
example 19 antiviral Activity test and results
1. Test method
Method 1:
in order to examine the broad-spectrum antiviral activity of the berberine hydrochloride derivative, the inhibition of pseudoviruses containing target virus envelope proteins to the entering process is examined first. The pseudovirus containing the reporter gene is constructed by utilizing ebola virus envelope (EBOV-Zair-GP) or coronavirus envelope (SARS-CoV-S, SARS-CoV2-S, MERS-CoV-S) combined with HIV virus core protein (HIV-NL 4-3-Luc). After the berbamine hydrochloride derivative is dissolved in DMSO, drug concentration gradients of 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10 and 20 mu M are set, and the mixture is mixed with pseudovirus (MOI=0.1) and then added into HEK293T (or ACE2 over-expressed Hela (applicable to coronavirus)) cells for incubation for 4 hours, cell culture supernatant is discarded, and new culture medium and small molecular compound with equal concentration are added. After further culturing for 48 hours, cells were collected, lysed using cell lysates, and the antiviral activity of the berbamine hydrochloride derivatives was evaluated by measuring intracellular luciferase activity. Solvent DMSO is used as a negative control group, and the luciferase activity of the experimental group and the luciferase activity of the DMSO group are compared to calculate the virus inhibition rate.
Method 2:
to examine the effect of drugs on virulence, we used laboratory-stored coronaviruses (HCoV-OC 43, HCoV-229E), dengue viruses, zika viruses, influenza a viruses, etc., dissolved the drugs with DMSO, set drug concentration gradients of 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40 μm, mixed with viruses (moi=0.1) and added to cell culture media (Huh 7 cells (HCoV-OC 43, HCoV-229E), vero cells (dengue viruses, zika viruses), MDCK cells (influenza a viruses)), incubated at 37 ℃ for 4 hours, while the medium was replaced with the corresponding concentration of drugs, after further culturing for 48 hours, the cells were collected, total cellular RNAs were extracted, and intracellular viral RNA levels were detected using RT-qPCR techniques. In addition, supernatants were collected and virus titers were calculated by serial gradient dilution combined with Cytopathic (CPE) microscopy. The antiviral activity of the drug was analyzed by combining the changes in intracellular viral nucleic acid and the supernatant virus titer.
2. Results
2.1 results of in vitro anti-ebola virus activity of the compounds of the invention as measured by method 1 are shown in table 1:
table 1: results of in vitro anti-ebola virus Activity of example Compounds
2.2 results of in vitro anti-coronavirus activity of the compounds of the invention as measured by method 2 are shown in Table 2:
TABLE 2 results of in vitro anti-coronavirus Activity of example Compounds
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2.3 results of in vitro anti-influenza virus activity of the compounds of the invention as measured by method 2 are shown in Table 3:
TABLE 3 results of in vitro anti-influenza Virus Activity of example Compounds
EXAMPLE 20 in vivo pharmacokinetic Properties
After single-dose oral administration of the target in ICR female mice, blood samples are collected at different time points, the concentration of the compound in the plasma of the rats is measured by LC-MS/MS, relevant drug generation parameters are calculated, and the exposure condition of the compound in the mice is examined. ICR 6 mice, supplied by Suzhou Zhaoyan laboratory animal Limited, were subjected to the experiment in accordance with Table 4.
TABLE 4 pharmacokinetic dosing regimen
Each mouse took 0.030mL of blood per orbital, EDTAK2 was anticoagulated, and the collection time point was po. groups: 15min,30min,1h,2h,4h,6h,8h,24h after administration of the test substance. Blood samples were collected and stored on ice and plasma was centrifuged (centrifugation conditions: 5000 rpm, 10 min, 4 ℃) for 30 minutes. The sample was stored at-80℃before analysis.
The data acquisition and control system software is Analyst1.5.1 software (Applied Biosystem). The peak integration mode of the map sample is automatic integration; regression was performed using the ratio of the peak area of the sample to the peak area of the internal standard as an index, and the concentration of the sample. Regression mode: linear regression, weight coefficient 1/X 2 . Pharmacokinetic parameters were determined using WinNonlin Professional v 6.3.6.3 (Pharsight, USA) using non-atrioventricular cellsAnd (5) model analysis and treatment. C (C) max The area under the blood concentration-time curve AUC is the measured maximum blood concentration (0→t) Calculated by a trapezoid method, T max Peak time is reached for the blood concentration after administration. Experimental data are expressed as "Mean ± standard deviation" (Mean ± ICR, n.
TABLE 5 pharmacokinetic Properties of Compound 1
a ICR mice (n=3) oral compound 1, 27.92mg/kg (equimolar concentration), testing plasma berberine and compound 1 for blood concentration; b ICR mice (n=3) were orally administered berbamine, 25mg/kg, and plasma berbamine plasma concentrations were tested.
Example 21 detection and results of Compounds for treatment of leukopenia in mice
1. Detection method
Establishing a leukopenia mouse model: in addition to the normal control group, BALB/C mice were intraperitoneally injected with 60mg/kg cyclophosphamide once a day for three consecutive days, and normal group mice were intraperitoneally injected with an equal volume of physiological saline. From the beginning of the molding, compound 1 or compound 16 was suspended in a 0.5% CMC-Na solution, and the experimental animals were administered by gastric lavage (30 mg/kg) once daily for 7 consecutive days. 24 hours after the last administration, the eyeball was bled, 200 μl was collected with an anticoagulant tube added with heparin sodium for routine blood detection; and then the isoflurane anesthesia broken ends are killed, thymus and spleen are weighed and used for calculating thymus indexes and spleen indexes, and femur is used for bone marrow nucleated cell technology. The thymus and spleen taken out are weighed after the liquid on the surface of the organ is sucked by the absorbent paper, and the ratio of the lifting capacity to the body weight is the spleen index and the thymus index. The muscle and connective tissue on the removed femur was removed, PBS was extracted by injection, bone marrow was flushed from the bone marrow cavity, and after centrifugation, 10 μl of PBS was resuspended and removed for cell counting.
2. Results
2.1 Effect of Compound 1 and Compound 16 on body weight of experimental mice
As shown in fig. 1, cyclophosphamide mice significantly lost weight compared to the normal group. The body weight of the mice in the berberine, compound 1 and compound 16 treatment groups was recovered compared with cyclophosphamide group, and the body weight of the mice in the compound 16 treatment group was not significantly different from that of the normal group.
2.2 Effect of Compounds 1 and 16 on peripheral blood image of leukopenia mice
Compared with the normal group, the contents of leucocytes, lymphocytes and neutrophils in the blood of the cyclophosphamide group mice are obviously reduced, and other parameters such as erythrocytes, hemoglobin and the like have no obvious change. The treatment group of berbamine, compound 16 and compound 1 was significantly more effective at increasing the number of white blood cells and the percentage of lymphocytes than the cyclophosphamide group.
2.3 Effect of Compounds 1 and 16 on bone marrow nucleated cells
The number of bone marrow nucleated cells was significantly reduced in cyclophosphamide mice compared to normal (PBS). Whereas the number of bone marrow nucleated cells of the mice in the compound 16 treated group was significantly recovered.
2.4 Effect of Compound 1 and Compound 16 on the thymus and spleen index in mice
Spleen index and thymus index reflect the strength of the immune function of the body to a certain extent. As shown in fig. 4, the thymus and spleen of cyclophosphamide treated group had atrophy and the spleen and thymus index was significantly decreased compared to the normal group. The spleen and thymus index was significantly elevated in the berbamine and compound 16 mice compared to the model group.

Claims (10)

1. A berbamine derivative, an isomer thereof, or a pharmaceutically acceptable salt thereof, characterized by having a structure as shown in formula I or formula II:
r represents:
wherein:
R 1 and R is 2 Independently of one another, represents H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, - (CH) 2 ) m OH、—(CH 2 ) m OCH 3 ；
R 3 Represents H, methyl, ethyl, n-propyl, isopropyl, isobutyl, -CF 3 、—CH 2 Cl、—CH 2 F、—CH 2 NR y R x 、—NR y R x ；
X represents-CH 2 —、—O—、—S—、—NR a —、—CHNR y R x —；
R y 、R x And R is a Each independently represents H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl;
n1 and n2 independently represent 0,1,2;
m represents 1,2, 3.
2. The berbamine derivative, isomer or pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 And R is 2 And each independently represents H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl.
3. The berbamine derivative, isomer thereof, or pharmaceutically acceptable salt thereof according to claim 1, comprising the structure:
4. the berberine derivative, isomer or pharmaceutically acceptable salt thereof according to claim 1, wherein the pharmaceutically acceptable salt is a salt of the structure shown in formula I or formula II with an inorganic or organic acid.
5. A method of synthesizing a compound of formula I or formula II as claimed in any one of claims 1 to 4 comprising the steps of:
or alternatively
Reaction conditions: the solvent is dichloromethane or N, N-dimethylformamide; the base is triethylamine or potassium carbonate; the catalyst is 4-dimethylaminopyridine.
6. A pharmaceutical composition comprising a berbamine derivative of formula I or formula II, an isomer thereof, or a pharmaceutically acceptable salt thereof.
7. The pharmaceutical composition of claim 6, further comprising one or more pharmaceutically acceptable carriers or excipients.
8. Use of a berbamine derivative, an isomer thereof, or a pharmaceutically acceptable salt thereof according to any one of claims 1-4 in the manufacture of an antiviral medicament.
9. Use according to claim 8, wherein said antiviral is selected from the group consisting of anti-ebola virus, anti-coronavirus, anti-dengue virus, anti-zika virus or anti-influenza virus, preferably said coronavirus is selected from the group consisting of SARS-CoV, SAR-CoV-2, MERS-CoV, hcoV 229E.
10. Use of a berbamine derivative, an isomer thereof, or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment or prevention of leukopenia, preferably leukopenia following cancer radiotherapy, chemotherapy, or leukopenia due to various causes.
CN202311281101.6A 2022-10-08 2023-10-07 Berberine derivatives, preparation method thereof and application thereof in antiviral and leukopenia treatment Pending CN117327084A (en)

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