CN106892920A

CN106892920A - Aloperine derivative, Preparation Method And The Use

Info

Publication number: CN106892920A
Application number: CN201510956338.9A
Authority: CN
Inventors: 宋丹青; 蒋建东; 彭宗根; 张欣; 唐胜; 李迎红; 李玉环
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2015-12-18
Filing date: 2015-12-18
Publication date: 2017-06-27
Anticipated expiration: 2035-12-18
Also published as: CN106892920B

Abstract

The present invention provides a kind of aloperine derivative, Preparation Method And The Use, specifically, the invention provides compound shown in a kind of Formulas I, its optical isomer, solvate or pharmaceutically acceptable salt, the compound has excellent antiviral activity, especially to hepatitis type B virus (HBV), HCV (HCV), Ebola virus (EBOV), Middle East breath syndrome virus (MERS viruses) and influenza virus (such as H7N9 types avian influenza virus) show inhibitory activity higher, and with good druggability, this provides more more options for the novel antiviral drugs of development structure.

Description

Aloperine derivative, preparation method and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a aloperine derivative, a preparation method and application thereof.

Background

Sophora alopecuroides belongs to the plants of Sophora of Leguminosae, and is perennial herb, namely Sophora alopecuroides root, Sophora alopecuroides L, Sophora flavescens and the like, and has the effects of clearing away heat and toxic materials, dispelling wind and eliminating dampness, relieving pain and killing insects, relieving asthma and relieving cough and the. Clinically, the medicine is mainly used for treating various diseases such as dysentery, sore and furuncle, traumatic suppuration, eczema, stubborn dermatitis, trichomonas, stomach diseases and the like, and the sophora alopecuroides mainly comprises 2 chemical components: alkaloids and flavones. The main active ingredients are alkaloids with extensive biological activity, and 20 kinds of alkaloids have been successfully separated from aloperine by various experimental techniques, wherein 8 kinds of alkaloids are studied more deeply, and are matrine (matrine), oxymatrine (oxymatrine), sophocarpine (ophocarpine), oxysophocarpine (oxysophocarpine), sophoramine (sophoramine), sophoridine (sophoridine), lehmannine (1ehmannine), and aloperine (allopurine). Wherein the aloperine has the following structural formula:

research shows that the aloperine has various physiological activities including arrhythmia resistance, negative muscle strength resistance, antibiosis, antivirus, anti-inflammation, smooth muscle relaxation, immunosuppression, anticancer, free radical elimination, toxic and side effects, insecticidal action and the like.

Chinese patent No. CN101209252A discloses the use of aloperine in the medicine for treating chronic hepatitis b, which is characterized in that in hepg2.2.15 cell experiment, aloperine has good anti-HBV effect in vitro, especially has good inhibitory effect on hepatitis b virus protein, and its inhibitory effect on hepatitis b virus protein is superior to that of the same kind of matrine and oxymatrine, and also superior to lamivudine. In addition, the aloperine is combined with a drug for inhibiting HBV DNA replication, so that the anti-HBV curative effect can be improved, and the drug for treating chronic hepatitis B can be further prepared.

It is still expected to find new and effective antiviral drugs for antiviral therapy.

Disclosure of Invention

The present inventors have completed this invention by modifying the structure of aloperine to obtain a novel class of compounds with excellent antiviral activity.

The invention provides a compound shown in formula I, an optical isomer, a solvate or a pharmaceutically acceptable salt thereof,

wherein,

the X group being-CH₂-, -C (O) -or-S (O)_a-, wherein a is 1 or 2;

the Y radical being C_1-10An alkyl group, or a 3-15 membered cycloalkyl, aryl or heterocyclyl group formed by atoms selected from C, N, O and S; optionally, wherein said C_1-10Alkyl, 3-15 membered cycloalkyl, aryl or heterocyclyl are each independently substituted by one or more substituents selected from halogen, amino, cyano, nitro,Carboxy, C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkoxycarbonyl, C_1-6Alkyl acyl radical, C_1-6Alkylamine acyl, C_1-6Alkylamido radical, C_1-6Alkylsulfonyl and 8-15 membered benzoheterocyclyl.

In one embodiment of the present invention, in the compound of formula I,

the X group being-CH₂-, -C (O) -or-S (O)_a-, wherein a is 1 or 2;

the Y radical being C_1-10An alkyl group, or a 3-15 membered cycloalkyl group formed by atoms selected from C, N, O and S, an aryl group (e.g., phenyl or fused ring aryl), an aliphatic heterocyclic group, or an aromatic heterocyclic group (e.g., a monoaromatic heterocyclic group or a fused aromatic heterocyclic group); optionally, wherein said C_1-10Each of the alkyl, 3-15 membered cycloalkyl, aryl (e.g., phenyl or fused ring aryl), aliphatic heterocyclic or aromatic heterocyclic (e.g., monoaromatic heterocyclic or fused aromatic heterocyclic) groups is independently substituted with one or more groups selected from halogen, amino, cyano, nitro, carboxy, C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkoxycarbonyl, C_1-6Alkyl acyl radical, C_1-6Alkylamine acyl, C_1-6Alkylamido radical, C_1-6Alkylsulfonyl and 8-15 membered benzoheterocyclyl.

In one embodiment of the present invention, in the compound of formula I,

the X group being-CH₂-, -C (O) -or-S (O)_a-, wherein a is 1 or 2;

the Y radical being C_1-6Alkyl, or a 3-8 membered cycloalkyl formed by an atom selected from C, N, O and S, phenyl, naphthyl, a 3-8 membered lipoheterocyclyl, a 3-8 membered monoaromatic heterocyclyl or an 8-12 membered fused heteroaromatic heterocyclyl, optionally wherein said C is_1-6Alkyl, 3-8 membered cycloalkyl, phenyl, naphthyl, 3-8 membered lipoheterocyclyl, 3-8 membered monoarylheterocyclyl or 8-12 memberedThe condensed aromatic heterocyclic groups are each independently substituted by one or more groups selected from halogen, amino, cyano, nitro, carboxyl, C_1-4Alkyl radical, C_1-4Alkoxy, halo C_1-4Alkyl, halo C_1-4Alkoxy radical, C_1-4Alkoxycarbonyl, C_1-4Alkyl acyl radical, C_1-4Alkylamine acyl, C_1-4Alkylamido radical, C_1-4Alkylsulfonyl or 8-12 membered benzoheterocyclyl.

In one embodiment of the present invention, in the compound of formula I,

the X group being-CH₂-, -C (O) -or-S (O)_a-, wherein a is 1 or 2;

the Y radical being C_1-6Alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, oxiranyl, oxocyclobutyl, tetrahydrofuryl, tetrahydropyranyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, furanyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyranyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, benzofuranyl, indolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzooxadiazolyl, benzotriazolyl, benzothiadiazolyl, purinyl, quinolinyl, benzotetrahydrofuranyl, benzotetrahydropyrrolyl, benzotetrahydropyranyl, benzopiperidinyl, benzodioxanyl or benzopiperazinyl, optionally substituted with one or more groups selected from halogen, amino, cyano, nitro, carboxyl, C_1-4Alkyl radical, C_1-4Alkoxy, halo C_1-4Alkyl, halo C_1-4Alkoxy radical, C_1-4Alkoxycarbonyl, C_1-4Alkyl acyl radical, C_1-4Alkylamine acyl, C_1-4Alkylamido radical, C_1-4Alkylsulfonyl and phthalimido groups.

In one embodiment of the invention, the compound of formula I is selected from the following compounds:

12-N- (2-cyanobenzyl) aloperine (N-KD-1);

12-N- (2-methylbenzyl) aloperine (N-KD-2);

12-N- (4-methoxybenzyl) aloperine (N-KD-3);

12-N-benzylaloperine (N-KD-4);

12-N- (4-nitrobenzyl) aloperine (N-KD-5);

12-N- (pyridin-4-methyl) aloperine (N-KD-13);

12-N- (4-trifluoromethoxybenzyl) aloperine (N-KD-14);

12-N- (3-methoxybenzyl) aloperine (N-KD-15);

12-N- (2-methoxybenzyl) aloperine (N-KD-16);

12-N- (4-fluorobenzyl) aloperine (N-KD-17);

12-N- (3, 4-dichlorobenzyl) aloperine (N-KD-18);

12-N- (4-methoxybenzenesulfonyl) aloperine (N-KD-19);

12-N- (4-methoxycarbonylbenzoyl) aloperine (N-KD-20);

12-N- (4-acetylbenzenesulfonyl) aloperine (N-KD-21);

12-N- (4-methoxybenzoyl) aloperine (N-KD-22);

12-N- (3-cyanophenylsulfonyl) aloperine (N-KD-23);

12-N- (pyridine-4-sulfonyl) aloperine (N-KD-24);

12-N- (2-thiophenesulfonyl) aloperine (N-KD-25);

12-N- (4-acetamidobenzenesulfonyl) aloperine (N-KD-26);

12-N- (3, 4-dimethoxybenzenesulfonyl) aloperine (N-KD-27);

12-N- (3,4, 5-trimethoxybenzoyl) aloperine (N-KD-29);

12-N- (4-isopropoxybenzenesulfonyl) aloperine (N-KD-30);

12-N- (3-methyl-4-methoxybenzenesulfonyl) aloperine (N-KD-31);

12-N- (2-phthalimidoethylsulfonyl) aloperine (N-KD-32);

12-N- (2-methoxy-5-chlorophenylsulfonyl) aloperine (N-KD-33);

12-N- (4-methylsulfonylphenylsulfonyl) aloperine (N-KD-34);

12-N- (8-quinolinesulfonyl) aloperine (N-KD-35);

12-N- (2, 3-dihydro-1, 4-benzodioxan-6-sulfonyl) aloperine (N-KD-36);

12-N- (4-aminobenzenesulfonyl) aloperine (N-KD-38);

12-N- (4-carboxybenzoyl) aloperine (N-KD-39);

12-N- (2-methoxycarbonylphenylsulfonyl) aloperine (N-KD-40);

12-N- (2-carboxyphenylsulfonyl) aloperine (N-KD-41);

12-N- (4-methoxycarbonylphenylsulfonyl) aloperine (N-KD-42);

12-N- (4-carboxyphenylsulfonyl) aloperine (N-KD-43);

12-N- (2-aminoethanesulfonyl) aloperine (N-KD-44);

12-N- (2-cyanophenylsulfonyl) aloperine (N-KD-45);

12-N- (2-acetylamino-4-methyl-5-thiazolesulfonyl) aloperine (N-KD-46);

12-N- (4-cyanophenylsulfonyl) aloperine (N-KD-47);

12-N- (2-amino-4-methyl-5-thiazolesulfonyl) aloperine (N-KD-48);

12-N- (1-methyl-1H-imidazole-4-sulfonyl) aloperine (N-KD-49);

12-N- (4-trifluoromethoxybenzenesulfonyl) aloperine (N-KD-50);

12-N- (2,1, 3-benzothiadiazole-4-sulfonyl) aloperine (N-KD-51);

12-N- (2, 3-dihydrobenzofuran-5-sulfonyl) aloperine (N-KD-52); and

12-N- (4-methyl-1-piperazinesulfonyl) aloperine (N-KD-53).

A second aspect of the present invention relates to a process for the preparation of a compound according to any one of the first aspect of the present invention, an optical isomer, solvate or pharmaceutically acceptable salt thereof, which comprises the steps of:

carrying out nucleophilic substitution reaction on aloperine (1) and L-X-Y in the presence of alkali to obtain a compound shown in a formula I;

wherein L represents a leaving group, such as halogen, -OTs or-OCOR;

the base being an organic or inorganic base, e.g. K₂CO₃、Na₂CO₃、Cs₂CO₃、Et₃N, DIPEA, DMAP or NaH;

the remaining groups are as defined in any one of the first aspect of the invention.

A third aspect of the present invention relates to a pharmaceutical composition comprising a compound according to any one of the first aspect of the present invention, an optical isomer, solvate or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

A fourth aspect of the present invention relates to the use of a compound according to any one of the first aspect of the present invention, an optical isomer, solvate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of the third aspect of the present invention for the manufacture of an antiviral medicament.

In one embodiment of the present invention, wherein the virus is selected from the group consisting of Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), ebola virus (EBOV), middle east respiratory syndrome coronavirus (MERS virus), and influenza virus (preferably influenza a virus, more preferably viruses such as H5N1, H5N2, H7N2, H7N3, H7N7, H7N9, H9N2, and H10N 7).

A fifth aspect of the invention relates to the use of a compound according to any one of the first aspect of the invention, an optical isomer, solvate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of the third aspect of the invention, in the manufacture of a medicament for the treatment of hepatitis b, hepatitis c, ebola hemorrhagic fever, middle east respiratory syndrome, or influenza (e.g. avian influenza, preferably H7N9 avian influenza).

Another aspect of the present invention relates to a compound according to any one of the first aspect of the present invention, an optical isomer, solvate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of the third aspect of the present invention, for use in an antiviral.

Another aspect of the present invention relates to a compound according to any one of the first aspect of the present invention, an optical isomer, solvate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of the third aspect of the present invention, for use in the treatment of hepatitis b, hepatitis c, ebola hemorrhagic fever, middle east respiratory syndrome or influenza (e.g. avian influenza, preferably H7N9 avian influenza).

Another aspect of the present invention relates to an antiviral method comprising administering to a subject in need thereof a compound of any one of the first aspect of the present invention, an optical isomer, solvate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of the third aspect of the present invention.

In one embodiment of the invention, the subject is a mammal, e.g., a bovine, equine, ovine, porcine, canine, feline, rodent, primate; among these, particularly preferred subjects are humans.

Another aspect of the present invention relates to a method of treating hepatitis b, hepatitis c, ebola hemorrhagic fever, middle east respiratory syndrome, or influenza (e.g. avian influenza, preferably avian influenza H7N 9), comprising administering to a subject in need thereof a compound of any one of the first aspect of the present invention, an optical isomer, solvate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of the third aspect of the present invention.

Interpretation of terms

The term "alkyl" as used herein refers to a straight or branched chain saturated hydrocarbon radical, e.g. C_1-10Alkyl radical, C_1-6Alkyl or C_1-4Non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like. The above-mentionedThe alkyl group may be substituted or unsubstituted, and when substituted, the substituent is selected from the group consisting of halogen, amino, alkoxy, and the like, and non-limiting examples of substituted alkyl groups include trifluoromethyl, trifluoroethyl, hydroxymethyl, hydroxyethyl, aminomethyl, aminoethyl, and the like.

As used herein, the term "alkoxy" refers to a group having the structure "R-O-", wherein R is an alkyl group, e.g., C_1-10Alkoxy radical, C_1-6Alkoxy or C_1-4Alkoxy, non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy and the like. When the alkyl group in the alkoxy group is substituted with halogen, the "haloalkyl" as described herein is obtained, and non-limiting examples of the haloalkyl include trifluoromethoxy, trifluoroethoxy, and the like.

The term "alkoxycarbonyl" as used herein refers to a group having the structure "R-O-C (O) -", wherein R is alkyl, and the alkoxycarbonyl group can be C_1-6Alkoxycarbonyl, C_1-4Alkoxycarbonyl and the like, non-limiting examples of alkoxycarbonyl include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, tert-butoxycarbonyl, and the like.

The term "alkanoyl" as used herein refers to a group having the structure "R-C (O) -", wherein R is alkyl, and the alkanoyl may be C_1-6Alkyl acyl radical, C_1-4Alkanoyl and the like, non-limiting examples of alkanoyl include acetyl, propionyl, butyryl and the like.

The term "alkylaminoacyl" as used herein refers to a group having the structure "RR 'N-C (O) -", wherein R and R' are alkyl groups, and the alkylaminoacyl group can be C_1-6Alkylamine acyl, C_1-4Alkylaminoacyl groups and the like, non-limiting examples of alkylaminoacyl groups include methyl aminoacyl, ethyl aminoacyl, N-dimethyl aminoacyl, N-diethyl aminoacyl, and the like.

As used herein, the term "alkylamido" refers to a group having the structure "R-C (O) NH-", wherein R is alkyl, and the alkylamido can be C_1-6Alkylamido radical, C_1-4Alkylamido and the like, non-limiting examples of alkylamido include carboxamido, acetamido, and the like.

The term "alkylsulfonyl" as used herein means having the meaning of "R-S (O)₂- "where R is alkyl, the alkylsulfonyl group may be C_1-6Alkylsulfonyl radical, C_1-4Alkylsulfonyl and the like, non-limiting examples of alkylsulfonyl include methylsulfonyl, ethylsulfonyl and the like.

The term "cycloalkyl" as used herein refers to a saturated or partially saturated cyclic hydrocarbon group, and the number of carbon atoms constituting the cycloalkyl group may be 3 to 15, for example 3 to 8. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

The term "aryl" as used herein refers to any functional group or substituent derived from a benzene or fused aromatic ring, such as a 3-15 membered aryl, non-limiting examples of which include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, fluorenyl, and the like.

The term "heterocyclic group" as used herein means that the ring-constituting atoms contain at least one hetero atom selected from N, O or S in addition to carbon atoms, and is classified into an alicyclic heterocyclic group and an aromatic heterocyclic group, wherein the aromatic heterocyclic group includes a monoaromatic heterocyclic group and a fused aromatic heterocyclic group formed by fusing a benzene ring and one or more than two monoaromatic heterocyclic groups. Such as 3-15 membered heterocyclic groups (including 3-15 membered aliphatic heterocyclic groups and aromatic heterocyclic groups), such as 3-8 membered heterocyclic groups, such as 3-8 membered aliphatic heterocyclic groups, such as 3-8 membered monoaromatic heterocyclic groups, and also such as 8-12 membered fused aromatic heterocyclic groups. Non-limiting examples of heterocyclic groups include, but are not limited to, oxirane, oxocyclobutyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrrole, piperidinyl, piperazinyl, furyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyranyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, benzofuran, indole, benzothiophene, benzoxazole, benzimidazole, benzothiazole, benzoxadiazole, benzotriazole, benzothiadiazole, quinoline, benzotetrahydrofuran, benzotetrahydropyrrole, benzopyridine, benzodioxacyclo, phthalimido, benzopyrazine, or purine, and the like.

The term "isomer" as used herein includes all possible isomeric (e.g., enantiomeric, diastereomeric, geometric, conformational, epimeric, and rotational isomers, etc.) forms of the compounds of formula I of the present invention. For example, the respective R and S configurations of the asymmetric centers, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers are within the scope of the present invention.

The compounds of formula I of the present invention or pharmaceutically acceptable salts thereof may also form solvates, such as hydrates, alcoholates and the like. In general, the solvate forms with pharmaceutically acceptable solvents such as water, ethanol, and the like are comparable to the non-solvate forms.

The compounds of formula I according to the invention may also be prodrugs or may be in a form which releases the active ingredient after metabolic changes in the body. The selection and preparation of suitable prodrug derivatives is well known to those skilled in the art and is not intended to be limiting.

The compound of formula I or a pharmaceutically acceptable salt thereof according to the present invention may also exist in the form of crystals, which means that molecules, atoms or ions constituting the compound are arranged in a spatially repeated arrangement according to a regular cycle, and the arrangement has a three-dimensional spatial periodicity, and is repeated at a certain distance. The compounds can exist in two or more crystalline states, and molecules with the same structure are crystallized into different solid forms, which are called polymorph, i.e. polymorphic forms, polymorphic forms and the like. When referring to a particular crystalline form or polymorph, collectively referred to as "crystal form", the term "crystalline form" as used herein, encompasses any crystalline form of the compound of formula I or a pharmaceutically acceptable salt thereof.

The term "effective amount" as used herein refers to an amount sufficient to achieve a desired prophylactic and/or therapeutic effect, e.g., an amount that achieves prevention or alleviation of symptoms associated with the disease to be treated.

The term "treatment" as used herein refers to both therapeutic treatment and prophylactic measures, the purpose of which is to prevent or delay (lessen) the disease state or condition being addressed. A subject is successfully "treated" if the subject receives a therapeutic amount of a compound of formula I, an optical isomer, solvate, or pharmaceutically acceptable salt thereof, according to the methods described herein, and the subject exhibits an observable and/or detectable decrease or improvement in one or more of the indications and symptoms of the subject. It is also understood that the prevention or treatment of a disease state or condition as described includes not only complete prevention or treatment, but also less than complete prevention or treatment, but also achievement of some biologically or medically relevant result.

The term "pharmaceutical composition" as used herein means a composition containing one or more compounds of formula I, as described herein, an optical isomer, solvate or pharmaceutically acceptable salt thereof, and other ingredients such as a pharmaceutically acceptable carrier or excipient. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity. Vectors described herein include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin. The excipient refers to an additive in the medicinal preparation except the main medicament. The composition has stable properties, no incompatibility with main drug, no side effect, no influence on curative effect, no deformation at room temperature, no crack, mildew, moth-eaten feeling, no harm to human body, no physiological effect, no chemical or physical effect with main drug, no influence on content determination of main drug, etc. Such as binders, fillers, disintegrants, lubricants in tablets; wine, vinegar, medicinal juice, etc. in the Chinese medicinal pill; base portion in semisolid formulations ointments, creams; preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, tonicity adjusting agents, colorants and the like in liquid preparations can all be referred to as excipients.

The compound shown in the formula I, the optical isomer, the solvate or the pharmaceutically acceptable salt thereof can be administered by the following routes: parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intramuscular routes, or as inhalants. The compositions may optionally be administered in combination with other agents that have at least some effect in the treatment of various diseases.

The compound shown in the formula I, the optical isomer, the solvate or the pharmaceutically acceptable salt thereof can be prepared into various suitable dosage forms according to the administration route.

When administered orally, the compounds of the present invention may be formulated in any orally acceptable dosage form, including but not limited to tablets, capsules, aqueous solutions or suspensions. Among these, carriers for tablets generally include lactose and corn starch, and additionally, lubricating agents such as magnesium stearate may be added. Diluents used in capsule formulations generally include lactose and dried corn starch. Aqueous suspension formulations are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents. Optionally, some sweetener, aromatic or colorant may be added into the above oral preparation.

When applied topically to the skin, the compounds of the present invention may be formulated in the form of suitable ointment, lotion or cream formulations wherein the active ingredient is suspended or dissolved in one or more carriers. Carriers that may be used in ointment formulations include, but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; carriers that can be used in lotions or creams include, but are not limited to: mineral oil, sorbitan monostearate, tween 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The compounds of the present invention may also be administered in the form of sterile injectable preparations, including sterile injectable aqueous or oleaginous suspensions or solutions, and may also be in lyophilized form. Among the carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

The pharmaceutical preparation of the present invention includes any preparation which can be pharmaceutically practiced, for example, oral preparations, parenteral preparations and the like.

The pharmaceutical compositions and preparations of the invention may contain 0.01 to 2000mg of a compound of the invention, preferably 0.1 to 1000mg of a compound of the invention, preferably 1 to 800mg of a compound of the invention, more preferably 10 to 600mg of a compound of the invention, and particularly preferably 50 to 500mg of a compound of the invention.

In embodiments of the invention, suitable in vitro or in vivo assays are performed to determine the effectiveness of the compositions of the invention and whether administration is suitable for treating a disease or medical condition in an individual. Examples of such assays are described below in the non-limiting examples in connection with specific diseases or medical treatments. Generally, an effective amount of a composition of the invention sufficient to achieve a prophylactic or therapeutic effect is from about 0.001 mg/kg body weight/day to about 10,000 mg/kg body weight/day. Suitably, the dose is from about 0.01 mg/kg body weight/day to about 1000mg/kg body weight/day. The dosage range may be about 0.01 to 1000mg/kg of host body weight per day, every second day, or every third day, more usually 0.1 to 500mg/kg of host body weight. Exemplary treatment regimens are once every two days or once a week or once a month. The agent is typically administered multiple times, and the interval between single doses may be daily, weekly, monthly or yearly. Alternatively, the agent may be administered in a sustained release formulation, in which case less frequency of administration is required. The dose and frequency will vary depending on the half-life of the agent in the subject. It may also vary depending on whether prophylactic or therapeutic treatment is carried out. In prophylactic applications, relatively low doses are administered chronically at relatively infrequent intervals. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until the progression of the disease is delayed or halted, and preferably until the individual exhibits a partial or complete improvement in the symptoms of the disease, after which a prophylactic regimen can be administered to the patient.

Advantageous effects of the invention

According to the invention, a series of compounds with excellent antiviral activity are obtained by modifying the 12 th N of aloperine, and in the embodiment of the invention, the compounds have higher inhibitory activity on Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Ebola virus (EBOV), middle east respiratory syndrome virus (MERS virus) and influenza virus (such as H7N9 avian influenza virus), and have good drug forming property, so that more choices are provided for developing antiviral drugs with novel structures.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

General method for synthesizing 12-N substituted aloperine derivative 1

0.5g (0,002mol) of aloperine (1) was taken and added to 20ml of acetonitrile, and 1.8g (0.006mol) of anhydrous potassium carbonate and substituted bromobenzyl or chlorobenzyl (0.003mol) were sequentially added to the reaction mixture and stirred at room temperature. And (5) detecting by TLC until the reaction is complete. Concentrating the organic phase, evaporating to dryness, adding dichloromethane solvent for dissolving, and washing with water and saturated saline solution sequentially. Drying dichloromethane layer, evaporating solvent with rotary evaporator to obtain 12-N-benzylDissolving the crude aloperine derivative with 20ml dichloromethane solvent, mixing with silica gel, and separating by column chromatography to obtain pure 12-N-benzyl aloperine derivative (2). The resulting compound was dissolved in 5ml of diethyl ether and then 1N HCl/Et was added₂Adjusting pH to 6-7 by O, and filtering to obtain hydrochloride of the pure 12-N-benzyl aloperine derivative.

General procedure for the synthesis of 12-N substituted aloperine derivatives 2:

0.5g (0.002mol) of aloperine (1) is taken and added into 20ml of dichloromethane at the temperature of 0 ℃, 1.8g (0.006mol) of anhydrous potassium carbonate and substituted benzenesulfonyl chloride or benzoyl chloride (0.0018mol) are sequentially added into the reaction solution, and the mixture is stirred at room temperature. And (5) detecting by TLC until the reaction is complete. Concentrating the organic phase, evaporating to dryness, adding dichloromethane solvent for dissolving, and washing with water and saturated saline solution sequentially. Drying dichloromethane layer, evaporating solvent with rotary evaporator to obtain crude 12-N-acyl or sulfonyl aloperine derivative, dissolving in 20ml dichloromethane solvent, mixing with silica gel, and separating by column chromatography to obtain pure 12-N-acyl or sulfonyl aloperine 3 or 4.

Example 1Synthesis of 12-N- (2-cyanobenzyl) aloperine hydrochloride (N-KD-1):

referring to general procedure 1 of the above synthesis, 0.6g of o-cyanobenzyl was added to give a yellow solid (767mg, 85%), m.p.: decomposing at 198 deg.C.¹H NMR(400MHz,DMSO):11.56(s,1H),8.13(d,J＝7.8Hz,1H),8.09(s,1H),8.00(d,J＝7.6Hz,1H),7.85(t,J＝7.6Hz,1H),7.70(t,J＝7.6Hz,1H),5.88(d,J＝5.5Hz,1H),4.98(d,J＝13.6Hz,4H),4.55(s,1H),4.26(d,J＝12.3Hz,1H),3.87(t,J＝11.7Hz,1H),3.64(dd,J＝24.9,12.9Hz,2H),3.45(d,J＝13.0Hz,1H),3.28(d,J＝12.7Hz,1H),3.16(s,1H),3.06(s,1H),2.48(s,1H),2.41(s,1H),2.35–2.15(m,3H),1.83–1.76(m,1H),1.68(dd,J＝23.9,14.2Hz,4H),1.45(d,J＝12.6Hz,1H)；¹³C NMR(400MHz,DMSO):134.8,134.2,134.0,133.8,131.0,128.6,117.8,114.5,110.0,65.7,58.9,54.4,54.2,52.5,45.5,32.9,30.6,27.6,23.4,23.3,22.5,20.8,18.2； HRMS:calcd for C₂₃H₃₀N₃·2HCl[M-2HCl+H]⁺:348.2434，found:348.2436。

Example 2Synthesis of 12-N- (2-methylbenzyl) aloperine hydrochloride (N-KD-2):

referring to the above general procedure for synthesis 1, 0.6g o-methylbenzyl bromide was added to give a pale yellow solid (630mg, 87%), melting point: 130 ℃ and 132 ℃.¹H NMR(400MHz,DMSO):10.92(d,J＝7.2Hz,1H),8.32(s,1H),7.75(d,J＝7.5Hz,1H),7.38–7.25(m,3H),5.87(d,J＝6.0Hz,1H),4.69(dd,J＝12.2,10.4Hz,4H),4.63–4.53(m,1H),4.27(dd,J＝13.5,9.5Hz,1H),3.93–3.80(m,1H),3.62(d,J＝12.3Hz,1H),3.51(d,J＝13.6Hz,1H),3.31–3.20(m,1H),3.20–3.10(m,1H),3.05(s,2H),2.48(s,3H),2.41(s,1H),2.32–2.12(m,3H),1.85–1.59(m,6H),1.48(dd,J＝17.8,8.3Hz,1H)；¹³CNMR(400MHz,DMSO):139.1,135.3,132.9,131.4,130.0,128.8,128.1,126.7,66.0,59.0,54.5,53.4,51.5,45.6,32.9,30.7,27.5,23.5,23.4,22.4,20.6,20.2,18.3；HRMS:calcdfor C₂₃H₃₃N₂·2HCl[M-2HCl+H]⁺:337.2638,found:337.2633。

Example 3Synthesis of 12-N- (4-methoxybenzyl) aloperine hydrochloride (N-KD-3):

referring to general procedure 1 of the above synthesis, 0.6g of 4-methoxybenzyl bromide was added to give a white solid (804mg, 88%), melting point: 112 ℃ and 114 ℃.¹H NMR(400MHz,DMSO):11.15(d,J＝7.3Hz,1H),8.06(s,1H),7.55(d,J＝8.7Hz,2H),7.01(d,J＝8.7Hz,2H),5.85(d,J＝6.0Hz,1H),4.62(d,J＝12.0Hz,1H),4.27(s,4H),4.10(dd,J＝13.2,8.0Hz,1H),3.79(s,3H),3.65–3.55(m,2H),3.47(d,J＝13.1Hz,1H),3.30–3.22(m,1H),3.17(d,J＝11.9Hz,1H),2.98(dd,J＝12.3,9.6Hz,2H),2.48–2.38(m,2H),2.28–2.10(m,3H),1.83–1.62(m,5H),1.53–1.41(m,1H)；¹³C NMR(400MHz,DMSO):160.5,135.0,133.7,133.7,128.5,121.5,114.6,114.6,64.6,58.8,55.7,55.3,54.4,51.8,45.6,32.9,30.8,27.6,23.4,23.2,22.5,20.8,18.2.HRMS:calcd for C₂₃H₃₃N₂O·2HCl[M-2HCl+H]⁺:353.2587,found:353.2586。

Example 4Synthesis of 12-N-benzyl Sophora alopecuroide hydrochloride (N-KD-4):

referring to the above general procedure for synthesis 1, 0.5g of bromobenzyl was added to give a pale yellow solid (704mg, 83%), m.p.: 113 ℃ and 115 ℃.¹H NMR(400MHz,DMSO)；11.23(s,1H),8.09(s,1H),7.70–7.59(m,2H),7.50–7.41(m,3H),5.86(d,J＝5.8Hz,1H),5.24(s,2H),4.73(d,J＝12.6Hz,1H),4.38(s,1H),4.18(dd,J＝13.1,8.4Hz,1H),3.86(t,J＝11.2Hz,1H),3.60(d,J＝13.2Hz,1H),3.47(d,J＝13.1Hz,1H),3.25(t,J＝13.1Hz,1H),3.13(t,J＝11.5Hz,1H),3.05(d,J＝21.1Hz,1H),2.43(dd,J＝15.5,10.3Hz,3H),2.30–2.12(m,3H),1.80(d,J＝8.1Hz,2H),1.68(dd,J＝24.8,13.3Hz,4H),1.47(dd,J＝17.9,8.3Hz,1H)；¹³C NMR(400MHz,DMSO):135.0,132.1,132.1,130.0,129.9,129.2,129.2,128.5,65.0,58.8,55.9,54.4,52.0,45.6,32.9,30.7,27.6,23.4,23.2,22.5,20.7,18.2；HRMS:calcd for C₂₂H₃₃N₂·2HCl[M-2HCl+H]⁺:323.2482,found:323.2481。

Example 5Synthesis of 12-N- (4-nitrobenzyl) aloperine hydrochloride (N-KD-5):

referring to general procedure 1 of the above synthesis, 0.7g of 4-nitrobenzyl bromide was added to give a yellow solid (765mg, 80%), mp 154-.¹H NMR(400MHz,DMSO):11.53(s,1H),8.31(d,J＝8.6Hz,2H),7.98(d,J＝8.5Hz,2H),7.95–7.89(m,1H),5.87(d,J＝5.5Hz,1H),4.89(d,J＝12.8Hz,1H),4.43(s,2H),3.92–3.81(m,1H),3.60(d,J ＝12.4Hz,1H),3.49(d,J＝13.0Hz,1H),3.39(s,1H),3.27(t,J＝12.6Hz,1H),3.12(s,2H),3.01(s,1H),2.50–2.39(m,3H),2.21(dd,J＝31.3,13.3Hz,3H),1.83–1.62(m,6H),1.46(t,J＝12.8Hz,1H)；¹³C NMR(400MHz,DMSO):148.6,137.5,134.7,133.7,128.7,124.0,63.3,58.8,54.8,54.3,52.4,45.6,33.0,30.7,27.7,23.4,23.2,22.5,20.8,18.2；HRMS:calcd for C₂₂H₃₀N₃O₂·2HCl[M-2HCl+H]⁺:368.2333,found:368.2329。

Example 6Synthesis of 12-N- (pyridine-4-methyl) aloperine hydrochloride (N-KD-13):

referring to the general procedure for the synthesis of 12-position N-benzyl substituted aloperine, 0.4g of 4-chloromethylpyridine was added, along with a catalytic amount of potassium iodide, to give a pale yellow solid. (665mg, 78%), melting point: decomposing at 236 ℃.¹H NMR(400MHz,DMSO):11.79(s,1H),9.01(d,J＝6.2Hz,2H),8.31(d,J＝5.8Hz,2H),7.88(s,1H),5.88(d,J＝4.7Hz,1H),5.02(d,J＝11.3Hz,1H),4.53(d,J＝65.5Hz,2H),3.84(t,J＝11.1Hz,1H),3.76–3.52(m,2H),3.46(d,J＝12.9Hz,1H),3.33–3.14(m,3H),2.96(s,1H),2.48–2.39(m,2H),2.21(dd,J＝27.9,13.0Hz,3H),1.85–1.60(m,7H),1.46(t,J＝12.7Hz,1H)；¹³C NMR(500MHz,DMSO):148.9,142.9(2),134.5,129.9(2),129.0,65.6,58.7,54.2,54.2,53.0,45.6,33.0,30.6,27.7,23.2,23.2,22.5,21.0,18.1；HRMS:calcd for C₂₁H₃₀N₃·2HCl[M-2HCl+H]⁺:324.2434,found:324.2433。

Example 7Synthesis of 12-N- (4-trifluoromethoxybenzyl) aloperine hydrochloride (N-KD-14):

referring to the above general procedure 1 for synthesis, 0.8g of 4-trifluoromethoxybenzyl bromide was added to give a white solid (836mg, 81%), melting point: 138 ℃ and 140 ℃.¹H NMR(400MHz,DMSO):11.38(s,1H),7.98(s,1H),7.81(d,J＝8.1Hz,2H),7.47(d,J＝7.8Hz,2H),5.87(d,J＝ 5.2Hz,1H),4.76(d,J＝11.9Hz,1H),4.34(s,1H),4.24(s,1H),3.85(d,J＝11.7Hz,1H),3.68–3.55(m,2H),3.47(d,J＝13.0Hz,1H),3.26(t,J＝11.3Hz,1H),3.19–2.90(m,3H),2.47–2.35(m,3H),2.20(d,J＝21.7Hz,3H),1.84–1.61(m,6H),1.44(d,J＝11.1Hz,1H)；¹³C NMR(400MHz,DMSO):149.5(2),134.8,134.4(2),129.4,128.7,124.3,122.5,121.7,121.6,120.1,119.2,65.1,58.8,54.9,54.3,52.1,45.6,32.9,30.7,27.6,23.4,23.2,22.5,20.8,18.2；HRMS:calcd for C₂₃H₃₀F₃N₂O·2HCl[M-2HCl+H]⁺:407.2305,found:407.2302。

Example 8Synthesis of 12-N- (3-methoxybenzyl) aloperine hydrochloride (N-KD-15):

referring to general procedure 1 of the above synthesis, 0.6g of 3-methoxybenzyl bromide was added to give a white solid (695mg, 82%), melting point: 151 ℃ and 153 ℃.¹H NMR(400MHz,DMSO):11.21(s,1H),8.09(s,1H),7.36(dd,J＝16.2,8.3Hz,2H),7.15(d,J＝7.3Hz,1H),7.05–7.00(m,1H),5.86(d,J＝5.5Hz,1H),4.71(d,J＝12.6Hz,1H),4.33(s,1H),4.09(d,J＝8.6Hz,2H),3.81(s,3H),3.60(d,J＝12.9Hz,2H),3.46(d,J＝13.0Hz,1H),3.25(t,J＝12.7Hz,1H),3.14(d,J＝11.5Hz,1H),3.01(d,J＝19.3Hz,2H),2.49–2.36(m,3H),2.20(dd,J＝23.4,11.7Hz,3H),1.85–1.61(m,6H),1.44(d,J＝12.2Hz,1H)；¹³C NMR(400MHz,DMSO):159.8,134.9,131.5,130.4,128.5,124.0,117.1,115.7,65.2,58.8,56.1,55.8,54.4,52.2,45.5,32.9,30.7,27.6,23.4,23.2,22.5,20.8,18.2；HRMS:calcd for C₂₃H₃₃N₂O·2HCl[M-2HCl+H]⁺:353.2587,found:353.2584。

Example 9Synthesis of 12-N- (2-methoxybenzyl) aloperine hydrochloride (N-KD-16):

referring to general procedure 1 of the above synthesis, 0.5g of 2-methoxybenzyl chloride was added to give a white solid (700mg, 83%), melting point: 211 ℃ and 213 ℃.¹H NMR(400MHz,DMSO):10.45(d,J＝6.7Hz,1H),8.56(s,1H),7.53(dd,J＝7.4,1.4Hz,1H),7.51–7.45(m,1H),7.13(d,J＝8.1Hz,1H),7.03(t,J＝7.3Hz,1H),5.85(d,J＝5.9Hz,1H),4.63(d,J＝11.7Hz,3H),4.36(s,1H),4.06(dd,J＝12.9,8.9Hz,1H),3.86(d,J＝6.4Hz,3H),3.56(t,J＝14.4Hz,2H),3.26(dd,J＝31.3,17.5Hz,2H),3.09(s,2H),2.60(dd,J＝9.6,4.0Hz,1H),2.48–2.36(m,2H),2.31–2.10(m,3H),1.86–1.61(m,6H),1.46(d,J＝13.1Hz,1H)；¹³C NMR(400MHz,DMSO):158.4,135.3,134.2,132.2,128.2,120.9,117.6,111.8,65.3,58.8,56.0,54.7,52.4,51.7,44.8,32.9,30.8,27.6,23.6,23.2,22.5,20.6,18.3；HRMS:calcd for C₂₃H₃₃N₂O·2HCl[M-2HCl+H]⁺:353.2587,found:353.2586。

Example 10Synthesis of 12-N- (4-fluorobenzyl) aloperine hydrochloride (N-KD-17):

referring to general procedure 1 of the above synthesis, 0.5g of 4-fluorobenzyl bromide was added to give a yellow solid (660mg, 80%), melting point: 198-200 ℃.¹H NMR(400MHz,DMSO):11.30(s,1H),8.00(s,1H),7.71(dd,J＝8.5,5.5Hz,2H),7.30(dd,J＝15.6,6.8Hz,2H),5.86(d,J＝5.9Hz,1H),4.71(d,J＝12.8Hz,1H),4.35(s,1H),4.21(dd,J＝13.0,8.2Hz,1H),3.84(t,J＝11.5Hz,1H),3.61(t,J＝13.3Hz,2H),3.47(d,J＝13.0Hz,1H),3.26(dd,J＝17.9,8.1Hz,1H),3.18–2.96(m,3H),2.41(dd,J＝26.3,13.5Hz,3H),2.19(dd,J＝31.3,13.1Hz,3H),1.87–1.59(m,6H),1.51–1.37(m,1H)；¹³C NMR(500MHz,DMSO):150.8,134.9,134.6,134.6,128.6,126.3,116.2,116.0,64.9,58.8,54.96,54.3,51.9,45.6,32.9,30.7,27.6,23.4,23.2,22.5,20.8,18.2；HRMS:calcdfor C₂₃H₃₀FN₂·2HCl[M-2HCl+H]⁺:341.2388,found:341.2388。

Example 11Synthesis of 12-N- (3, 4-dichlorobenzyl) aloperine hydrochloride (N-KD-18):

referring to the above general procedure 1 for synthesis, 0.7g of 3, 4-dichlorobenzyl bromide was added to give a yellow solid (750mg, 82%), melting point: 181 ℃ and 183 ℃.¹H NMR(500MHz,DMSO):11.46(s,1H),8.03(s,1H),7.89(s,1H),7.76(d,J＝8.0Hz,1H),7.65(d,J＝7.9Hz,1H),5.87(d,J＝5.1Hz,1H),4.75(d,J＝11.1Hz,1H),4.28(d,J＝45.5Hz,2H),3.82(s,1H),3.68(d,J＝12.4Hz,1H),3.60(d,J＝12.0Hz,1H),3.49(d,J＝12.8Hz,1H),3.27(t,J＝12.8Hz,1H),3.16(d,J＝6.5Hz,1H),3.09(s,1H),2.97(s,1H),2.50–2.32(m,3H),2.22(t,J＝18.0Hz,3H),1.87–1.61(m,6H),1.45(d,J＝12.1Hz,1H)；¹³C NMR(500MHz,DMSO)134.7,134.2,132.9,132.6,131.8,131.3,131.0,128.8,65.2,58.8,54.5,54.3,52.2,45.6,33.0,30.7,27.7,23.4,23.2,22.5,20.9,18.2；HRMS:calcdfor C₂₃H₂₉Cl₂N₂·2HCl[M-2HCl+H]⁺:391.1702,found:391.1710。

Example 12Synthesis of 12-N- (4-methoxybenzenesulfonyl) aloperine (N-KD-19):

referring to general procedure 2 of the above synthesis, 0.4g of 4-methoxybenzenesulfonyl chloride was added to give a yellow solid (623mg, 80%), melting point: 139 ℃ and 141 ℃.¹H NMR(500MHz,DMSO):7.84(d,J＝8.7Hz,2H),7.12(d,J＝8.7Hz,2H),5.53(d,J＝5.2Hz,1H),4.19(d,J＝5.2Hz,1H),3.86(s,3H),3.57(dd,J＝14.2,7.3Hz,1H),3.45–3.36(m,1H),3.04(dd,J＝11.4,3.5Hz,1H),2.74(d,J＝12.8Hz,1H),2.62(dd,J＝25.0,12.1Hz,2H),2.45(d,J＝11.6Hz,1H),2.08(s,1H),1.97–1.88(m,2H),1.86–1.74(m,3H),1.65(d,J＝13.0Hz,1H),1.59–1.46(m,2H),1.43–1.30(m,2H),1.26–1.17(m,1H),1.06(dd,J＝27.5,13.9Hz,2H)；¹³C NMR(500MHz,DMSO):162.8,135.1,132.7,129.5,129.5,128.2,114.8,114.8,60.2,59.3,56.1,54.3,46.7,41.6,35.3,34.7,27.5,26.1,24.9,24.1,23.8,19.4；HRMS:calcd for C₂₂H₃₁N₂O₃S[M+H]⁺:403.2050,found:403.2058。

Example 1312-N- (4-methoxycarbonylbenzoyl)Group) synthesis of aloperine (N-KD-20):

referring to general procedure 2 of the above synthesis, 0.4g of methyl p-chlorocarbonyl benzoate was added to give a yellow solid (553mg, 78%), melting point: 76-78 ℃.¹H NMR(500MHz,DMSO):8.03(d,J＝8.1Hz,2H),7.53(d,J＝8.1Hz,2H),5.64(s,1H),4.71(s,1H),3.89(d,J＝7.0Hz,3H),3.60–3.47(m,1H),3.20–3.03(m,2H),2.79(d,J＝12.9Hz,1H),2.67(t,J＝12.3Hz,1H),2.56(d,J＝11.7Hz,1H),2.47(d,J＝16.7Hz,1H),2.28–2.13(m,2H),1.99(d,J＝12.4Hz,1H),1.94–1.72(m,4H),1.65(dd,J＝25.9,12.5Hz,2H),1.56–1.33(m,2H),1.00–1.25(m,3H)；¹³C NMR(500MHz,DMSO):169.2,166.2,142.1,135.8,130.4,129.9,129.9,128.1,127.1,127.1,59.2,58.3,54.2,52.8,46.5,43.4,34.8,31.2,27.8,26.2,24.9,24.7,23.8,19.2；HRMS:calcd for C₂₄H₃₁N₂O₃[M+H]⁺:395.2329,found:395.2335。

Example 14Synthesis of 12-N- (4-acetylbenzenesulfonyl) aloperine (N-KD-21):

referring to general procedure 2 of the above synthesis, 0.4g of 4-acetylbenzenesulfonyl chloride was added to give a pale yellow solid (596mg, 80%), melting point: 126 ℃ and 128 ℃.¹H NMR(500MHz,DMSO):8.16–8.13(m,2H),8.08–8.04(m,2H),5.54(d,J＝5.5Hz,1H),4.28(d,J＝5.6Hz,1H),3.65(dd,J＝14.2,7.5Hz,1H),3.53–3.42(m,1H),3.05(dd,J＝11.5,3.5Hz,1H),2.74(d,J＝13.2Hz,1H),2.66(s,3H),2.64(d,J＝2.6Hz,1H),2.59(dd,J＝18.9,7.0Hz,1H),2.46(d,J＝11.7Hz,1H),2.11(s,1H),1.97–1.90(m,2H),1.87–1.74(m,3H),1.70–1.56(m,2H),1.55–1.44(m,1H),1.39(dd,J＝9.4,4.0Hz,2H),1.23–1.12(m,1H),1.05(dd,J＝25.1,13.0Hz,2H)；¹³C NMR(500MHz,DMSO)197.8,144.6,140.1,134.8,129.6(2),128.5,127.7(2),60.4,59.1,54.2,46.5,41.8,35.3,34.7,27.5,27.4,26.1,24.8,24.0,23.7,19.3；HRMS:calcd for C₂₃H₃₁N₂O₃S[M+H]⁺:415.2050,found:415.2054。

Example 15Synthesis of 12-N- (4-methoxybenzoyl) aloperine (N-KD-22):

referring to general procedure 2 of the above synthesis, 0.3g of 4-methoxybenzoyl chloride was added to give a pale yellow solid (520mg, 79%), melting point: 141 ℃ and 143 ℃.¹H NMR(400MHz,DMSO):7.33(d,J＝8.6Hz,2H),6.98(d,J＝8.6Hz,2H),5.62(d,J＝4.8Hz,1H),4.65(s,1H),3.80(d,J＝9.6Hz,3H),3.50(s,1H),3.40(s,1H),3.06(dd,J＝11.4,2.9Hz,1H),2.78(d,J＝12.8Hz,1H),2.67(t,J＝12.1Hz,1H),2.55(d,J＝11.7Hz,1H),2.47(d,J＝11.5Hz,2H),2.26–2.08(m,2H),1.95(s,1H),1.82(dd,J＝22.5,11.6Hz,4H),1.66(d,J＝13.0Hz,1H),1.56(s,2H),1.40(dd,J＝26.3,13.2Hz,1H),1.06(t,J＝10.1Hz,2H)；¹³C NMR(400MHz,DMSO):170.4,160.2,136.1,129.7,128.6(2),127.8,114.1(2),59.3,58.3,55.6(2),54.2,46.6,34.8,31.3,27.9,26.2,24.9,24.8,23.8,19.2；HRMS:calcd for C₂₃H₃₁N₂O₂[M+H]⁺:367.2380,found:367.2375。

Example 16Synthesis of 12-N- (3-cyanophenylsulfonyl) aloperine (N-KD-23):

general Synthesis methods as described aboveMethod 2, addition of 0.4g of 3-cyanobenzenesulfonyl chloride, gave a white solid (571mg, 80%), melting point: 177 and 179 ℃.¹H NMR(400MHz,DMSO-D6):8.44(d,J＝1.6Hz,1H),8.26–8.23(m,1H),8.18–8.14(m,1H),7.81(dd,J＝11.6,4.2Hz,1H),5.53(d,J＝5.3Hz,1H),4.34(d,J＝5.4Hz,1H),3.70–3.61(m,1H),3.50–3.40(m,1H),3.04(dd,J＝11.5,3.5Hz,1H),2.72(d,J＝12.4Hz,1H), 2.65(dd,J＝13.0,2.4Hz,1H),2.54(d,J＝11.4Hz,1H),2.46(d,J＝11.8Hz,1H),2.08(s,1H),1.98–1.89(m,2H),1.86–1.73(m,3H),1.68–1.56(m,2H),1.45–1.29(m,3H),1.16–0.97(m,3H)；¹³C NMR(400MHz,DMSO-D6):142.4,136.9,134.9,131.8,131.3,131.1,128.5,118.1,113.1,60.5,59.1,54.3,46.5,41.8,35.4,34.7,27.4,26.2,24.8,24.0,23.6,19.3；HRMS:calcd for C₂₂H₂₈N₃O₂S[M+H]⁺:398.1897，found:398.1914。

Example 17Synthesis of 12-N- (pyridine-4-sulfonyl) aloperine (N-KD-24):

referring to general method 2 of the above synthesis, 0.3g of 4-pyridinesulfonyl chloride was added to give a white solid (543mg, 81%), melting point: 100-102 ℃.¹H NMR(400MHz,DMSO-D6):9.07(d,J＝2.1Hz,1H),8.85(dd,J＝4.8,1.5Hz,1H),8.38–8.31(m,1H),7.70–7.58(m,1H),5.53(d,J＝5.4Hz,1H),4.29(d,J＝5.4Hz,1H),3.71–3.59(m,1H),3.52–3.39(m,1H),3.03(dd,J＝11.5,3.6Hz,1H),2.72(d,J＝13.4Hz,1H),2.67–2.58(m,1H),2.53(d,J＝11.4Hz,1H),2.49–2.42(m,1H),2.07(s,1H),1.98–1.88(m,2H),1.80(t,J＝13.4Hz,3H),1.66–1.55(m,2H),1.48–1.33(m,3H),1.20–0.96(m,3H)；¹³C NMR(101MHz,DMSO-D6):153.9,147.8,137.5,135.4,134.7,128.6,124.9,60.4,59.1,54.3,46.5,41.7,35.3,34.7,27.4,26.2,24.8,24.0,23.7,19.3；HRMS:calcdfor C₂₀H₂₈N₃O₂S[M+H]⁺:374.1897，found:374.1908。

Example 18Synthesis of 12-N- (2-thiophenesulfonyl) aloperine (N-KD-25):

referring to general procedure 2 of the above synthesis, 0.3g of 2-thiophenesulfonyl chloride was added to give a light red solid (544mg, 80%), melting point: 133 ℃ and 135 ℃.¹H NMR(400MHz,DMSO-D6):7.98(dd, J＝5.0,1.3Hz,1H),7.76(dd,J＝3.7,1.3Hz,1H),7.22(dd,J＝5.0,3.7Hz,1H),5.55(d,J＝5.1Hz,1H),4.24(d,J＝5.7Hz,1H),3.62(dd,J＝14.2,7.5Hz,1H),3.50–3.41(m,1H),3.05(dd,J＝11.5,3.5Hz,1H),2.73(d,J＝12.8Hz,1H),2.67–2.55(m,2H),2.46(d,J＝11.6Hz,1H),2.11(s,1H),2.02–1.89(m,2H),1.88–1.73(m,3H),1.68–1.52(m,3H),1.46–1.24(m,3H),1.05(t,J＝15.0Hz,2H)；¹³C NMR(400MHz,DMSO-D6):141.2,134.8,133.3,132.6,128.6,128.6,60.5,59.2,54.3,46.7,42.1,35.2,34.7,27.6,26.2,24.9,24.1,23.6,19.4；HRMS:calcd forC₁₉H₂₇N₂O₂S₂[M+H]⁺:379.1509,found:379.1540。

Example 19Synthesis of 12-N- (4-acetamidobenzenesulfonyl) aloperine (N-KD-26):

referring to general procedure 2 of the above synthesis, 0.4g of 4-acetamidophenylsulfonyl chloride was added to give a white solid (633mg, 82%), melting point: 120-122 ℃.¹H NMR(500MHz,CDCl₃):8.13(s,1H),7.82(d,J＝8.7Hz,2H),7.71(d,J＝8.6Hz,2H),5.54(d,J＝4.9Hz,1H),4.24(d,J＝4.7Hz,1H),3.65(dd,J＝14.2,7.4Hz,1H),3.62–3.48(m,1H),3.08(dd,J＝11.4,2.9Hz,1H),2.80(d,J＝12.6Hz,1H),2.72(d,J＝10.0Hz,1H),2.50(d,J＝7.5Hz,1H),2.23(s,4H),2.01–1.78(m,6H),1.73–1.57(m,3H),1.52–1.36(m,3H),1.12(dd,J＝34.7,11.3Hz,2H)；¹³C NMR(500MHz,CDCl₃)169.1,142.0,135.4,134.9,128.2(3),119.2(2),60.4,59.5,54.3,46.7,42.0,35.1,34.8,27.8,26.0,24.9,24.7,24.4,23.8,19.5；HRMS:calcd for C₂₃H₃₂N₃O₃S[M+H]⁺:430.2159,found:430.2178。

Example 20Synthesis of 12-N- (3, 4-dimethoxybenzenesulfonyl) aloperine (N-KD-27):

referring to general procedure 2 of the above synthesis, 0.4g of 3, 4-dimethoxybenzenesulfonyl chloride was added to give a white solid (583mg, 75%), m.p.: 197 ℃ and 199 ℃.¹H NMR(400MHz,CDCl₃):7.54(d,J＝8.4Hz,1H),7.39(s,1H),6.96(d,J＝8.5Hz,1H),5.57(s,1H),4.29(s,1H),3.97(d,J＝6.7Hz,6H),3.77–3.50(m,2H),3.10(d,J＝9.3Hz,1H),2.83(d,J＝11.3Hz,1H),2.72(dd,J＝30.9,12.2Hz,2H),2.51(d,J＝11.0Hz,1H),2.27(s,1H),2.04–1.81(m,5H),1.68(dd,J＝28.8,11.1Hz,3H),1.46(s,4H),1.13(dd,J＝26.5,9.4Hz,1H)；¹³C NMR(500MHz,CDCl₃)152.8,149.2,137.2,131.2,126.0,121.2,110.6,109.7,59.0,57.8,56.4(2),56.2(2),54.6,45.4,43.8,33.8,27.6,23.8,22.4,21.2,18.7；HRMS:calcd for C₂₃H₃₃N₂O₄S[M+H]⁺:433.2156,found:433.2157。

Example 21Synthesis of 12-N- (3,4, 5-trimethoxybenzoyl) aloperine (N-KD-29):

referring to general method 2 of the above synthesis, 0.4g of 3,4, 5-trimethoxybenzoyl chloride was added to give a white solid (654mg, 79%), melting point: 75-77 ℃.¹H NMR(500MHz,CDCl₃):6.63(s,2H),5.65(s,1H),4.84(s,1H),3.91–3.81(m,9H),3.70–3.37(m,2H),3.13(d,J＝11.2Hz,1H),2.76(d,J＝7.4Hz,2H),2.61(d,J＝11.6Hz,2H),2.51(d,J＝10.8Hz,1H),2.28(dd,J＝23.9,12.9Hz,2H),1.99(s,1H),1.88(d,J＝10.6Hz,4H),1.77–1.60(m,3H),1.50–1.38(m,1H),1.17–1.03(m,2H)；¹³CNMR(500MHz,CDCl₃):170.3,153.2(2),138.7,135.8,132.8,128.0,103.7(2),60.9,59.4,58.6,56.2(2),54.4,46.9,43.6,35.0,31.1,28.0,26.2,25.2,24.9,24.0,19.2；HRMS:calcd for C₂₅H₃₅N₂O₄[M+H]⁺:427.2591，found:427.2601。

Example 22Synthesis of 12-N- (4-isopropoxybenzenesulfonyl) aloperine (N-KD-30):

referring to general procedure 2 of the above synthesis, 0.4g of 4-isopropoxybenzenesulfonyl chloride was added to give a yellow solid (642mg, 83%), melting point: 59-61 ℃.¹H NMR(500MHz,CDCl₃):7.81(d,J＝8.8Hz,2H),6.94(d,J＝8.7Hz,2H),5.53(s,1H),4.64(m,J＝12.1,6.0Hz,1H),4.26(s,1H),3.72–3.63(m,1H),3.55(d,J＝8.4Hz,1H),3.06(d,J＝9.8Hz,1H),2.80(d,J＝12.0Hz,1H),2.75–2.59(m,2H),2.48(d,J＝11.4Hz,1H),2.25(s,1H),2.02–1.77(m,5H),1.74–1.57(m,3H),1.44(s,3H),1.38(d,J＝6.1Hz,6H),1.10(dd,J＝34.7,11.4Hz,2H)；¹³C NMR(500MHz,CDCl₃):161.0,135.2,132.5,129.2,129.2,128.1,115.5,115.5,70.3,60.3,59.5,54.3,46.7,41.7,35.1,34.9,27.8,26.1,25.0,24.4,23.9,21.9,21.8,19.5；HRMS:calcd for C₂₄H₃₅N₂O₃S[M+H]⁺:431.2363，found:431.2369。

Example 23Synthesis of 12-N- (3-methyl-4-methoxybenzenesulfonyl) aloperine (N-KD-31):

referring to general procedure 2 of the above synthesis, 0.4g of 3-methyl-4-methoxybenzenesulfonyl chloride was added to give a white solid (614mg, 82%), melting point: 138 ℃ and 140 ℃.¹H NMR(500MHz,CDCl₃):7.72(dd,J＝8.6,2.3Hz,1H),7.66(s,1H),6.88(d,J＝8.6Hz,1H),5.54(s,1H),4.25(s,1H),3.90(s,3H),3.66(s,1H),3.54(s,1H),3.07(d,J＝7.2Hz,1H),2.86–2.60(m,3H),2.48(d,J＝10.5Hz,1H),2.27(d,J＝12.7Hz,4H),2.04–1.77(m,5H),1.66(dd,J＝38.7,10.3Hz,3H),1.43(s,3H),1.21–1.01(m,2H)；¹³C NMR(500MHz,CDCl₃):160.8,135.3,132.3,129.3,128.1,127.5,126.9,109.4,60.3,59.6,55.6,54.3,46.8,41.7,35.1,34.9,27.8,26.1,24.9,24.5,23.9,19.5,16.3；HRMS:calcd for C₂₃H₃₃N₂O₃S[M+H]⁺:417.22064,found:417.22100。

Example 24Synthesis of 12-N- (2-phthalimidoethylsulfonyl) aloperine (N-KD-32):

referring to general procedure 2 of the above synthesis, 0.5g of 2-phthalimidoethanesulfonyl chloride was added to give a yellow solid (709mg, 84%), melting point: 89-91 ℃.¹H NMR(500MHz,CDCl₃):7.88(dd,J＝5.4,3.0Hz,2H),7.76–7.72(m,2H),5.63(d,J＝4.6Hz,1H),4.37(d,J＝5.2Hz,1H),4.26–4.13(m,2H),3.65–3.55(m,2H),3.47–3.38(m,1H),3.36–3.28(m,1H),3.12(dd,J＝11.7,3.3Hz,1H),2.82(d,J＝13.2Hz,1H),2.74–2.70(m,1H),2.54(d,J＝11.9Hz,1H),2.31–2.24(m,1H),2.23–2.15(m,2H),2.11–2.01(m,1H),1.95(s,1H),1.86(t,J＝11.3Hz,2H),1.66(dd,J＝28.6,13.1Hz,3H),1.44(d,J＝6.6Hz,3H),1.10(dd,J＝29.4,12.9Hz,2H)；¹³C NMR(500MHz,CDCl₃):167.7(2),134.9,134.2(2),132.0,128.6,123.5(2),60.5,59.3,54.2,49.7,46.6,41.8,34.9,34.9,32.8,28.0,26.9(2),26.1,24.9,24.2,19.3；HRMS:calcd forC₂₅H₃₂N₃O₄S[M+H]⁺:470.2108，found:470.2114。

Example 25Synthesis of 12-N- (2-methoxy-5-chlorobenzenesulfonyl) aloperine (N-KD-33):

referring to general procedure 2 of the above synthesis, 0.4g of 2-methoxy-5-chlorobenzenesulfonyl chloride was added to give a white solid (637mg, 81%). Melting point: 158 ℃ and 160 ℃.¹H NMR(500MHz,CDCl₃):7.97(d,J＝2.7Hz,1H),7.46(dd,J＝8.8,2.5Hz,1H),6.95(d,J＝8.8Hz,1H),5.57(s,1H),4.61(s,1H),3.95(s,3H),3.68(dd,J＝13.8,7.5Hz,1H),3.57(dd,J＝11.6,4.6Hz,1H),2.95(d,J＝9.4Hz,1H),2.77(d,J＝12.1Hz,1H),2.72–2.55(m,2H),2.47(d,J＝11.4Hz,1H),2.12(d,J＝12.5Hz,2H),1.96–1.74(m,5H),1.64(dd,J＝26.2,12.4Hz,3H),1.45(dd,J＝34.7,21.9Hz,2H),1.11(dd,J＝31.6,11.8Hz,2H)；¹³C NMR(500MHz,CDCl₃)155.2,135.3,133.6, 131.1,130.9,128.1,125.4,113.3,60.6,59.6,56.2,54.4,46.7,42.0,34.9,34.8,28.0,26.1,24.8,24.6,24.5,19.6；HRMS:calcd for C₂₂H₃₀N₂O₃S[M+H]⁺:437.1660，found:437.1650。

Example 26Synthesis of 12-N- (4-methylsulfonylphenylsulfonyl) aloperine (N-KD-34):

referring to general procedure 2 of the above synthesis, 0.5g of 4-methylsulfonylbenzenesulfonyl chloride was added to give a white solid (640mg, 79%), melting point: 221-223 ℃.¹H NMR(500MHz,CDCl₃):8.11(s,3H),5.56(d,J＝4.2Hz,1H),4.29(d,J＝4.6Hz,1H),3.72(dd,J＝14.0,7.6Hz,1H),3.64(dd,J＝12.2,6.1Hz,1H),3.12(s,3H),3.10–3.03(m,1H),2.79(d,J＝12.9Hz,1H),2.68(dd,J＝17.9,12.5Hz,2H),2.51(d,J＝11.9Hz,1H),2.24(s,1H),2.05–1.98(m,1H),1.94(s,1H),1.91–1.80(m,3H),1.73–1.55(m,4H),1.53–1.39(m,2H),1.32(d,J＝10.9Hz,1H),1.10(dd,J＝33.6,12.7Hz,2H)；¹³C NMR(500MHz,CDCl₃)146.5,143.7,134.2,128.9,128.3,128.3,128.1,128.1,61.0,59.2,54.2,46.5,44.4,42.0,35.2,34.8,27.7,26.1,24.9,24.1,23.8,19.3；HRMS:calcd for C₂₂H₃₁N₂O₃S₂[M+H]⁺:451.1720，found:451.1713。

Example 27Synthesis of 12-N- (8-quinolinesulfonyl) aloperine (N-KD-35):

referring to general procedure 2 of the above synthesis, 0.4g of 8-quinolinesulfonyl chloride was added to give a white solid (640mg, 79%), melting point: 194 ℃ and 196 ℃.¹H NMR(500MHz,CDCl₃):9.12(dd,J＝4.2,1.7Hz,1H),8.56(dd,J＝7.3,1.2Hz,1H),8.25(dd,J＝8.3,1.6Hz,1H),8.06–8.00(m,1H),7.62(t,J＝7.8Hz,1H),7.53(dd,J＝8.3,4.2Hz,1H),5.50 (d,J＝4.4Hz,1H),5.24(d,J＝4.4Hz,1H),3.90(dd,J＝14.1,7.2Hz,1H),3.67–3.55(m,1H),2.86(dd,J＝11.7,3.8Hz,1H),2.70(d,J＝12.9Hz,1H),2.61(dd,J＝15.8,5.9Hz,2H),2.43(d,J＝11.9Hz,1H),2.34(d,J＝15.2Hz,1H),1.95–1.80(m,4H),1.79–1.72(m,1H),1.68(d,J＝12.9Hz,1H),1.65–1.57(m,1H),1.52(dd,J＝20.5,10.4Hz,1H),1.44–1.32(m,2H),1.28(dd,J＝20.4,10.6Hz,1H),1.13–1.02(m,2H)；¹³CNMR(500MHz,CDCl₃):151.1,144.2,139.6,136.5,135.9,133.0,132.7,129.0,127.8,125.6,121.9,60.6,59.8,54.3,47.0,42.1,35.1,35.0,28.0,26.1,24.9,24.8,24.3,19.7；HRMS:calcd for C₂₄H₃₀N₃O₂S[M+H]⁺:424.2053，found:424.2043。

Example 28Synthesis of 12-N- (2, 3-dihydro-1, 4-benzodioxan-6-sulfonyl) aloperine (N-KD-36):

referring to general procedure 2 of the above synthesis, 0.4g of 2, 3-dihydro-1, 4-benzodioxole-6-sulfonyl chloride was added to give a white solid (619mg, 80%), m.p.: 205 ℃ and 207 ℃.¹H NMR(500MHz,CDCl₃):7.43(d,J＝1.2Hz,1H),7.39(dd,J＝8.5,1.9Hz,1H),6.96(d,J＝8.4Hz,1H),5.55(s,1H),4.33(dd,J＝12.5,4.9Hz,4H),4.25(s,1H),3.60(d,J＝52.2Hz,2H),3.07(s,1H),2.84–2.65(m,2H),2.49(d,J＝7.6Hz,1H),2.34–2.18(m,1H),2.01(s,1H),1.96–1.80(m,4H),1.66(d,J＝29.5Hz,4H),1.46(s,3H),1.27(s,1H),1.11(d,J＝29.5Hz,1H)；¹³C NMR(500MHz,CDCl₃):147.1,143.4,135.2,133.8,128.2,120.8,117.7,116.8,64.5,64.2,60.4,59.5,54.3,46.6,41.8,35.0,27.8,26.0,24.9,24.4,23.9,22.4,19.4；HRMS:calcd for C₂₃H₃₁N₂O₄S[M+H]⁺:431.1999，found:431.1995。

Example 29Synthesis of 12-N- (4-aminobenzenesulfonyl) aloperine (N-KD-38):

referring to general synthetic method 2 above, 0.4g of 4-acetamidobenzenesulfonyl chloride was added to give N-KD-26 as a white solid, which was hydrolyzed under heating in 20% aqueous NaOH to give a pale yellow solid (253mg, 80%), melting point: decomposing at 220 ℃.¹H NMR(500MHz,DMSO):7.49(d,J＝8.7Hz,2H),6.61(d,J＝8.7Hz,2H),5.99(s,2H),5.50(d,J＝5.4Hz,1H),4.10(d,J＝5.3Hz,1H),3.49(dd,J＝14.2,7.4Hz,1H),3.40–3.29(m,1H),3.01(dd,J＝11.4,3.7Hz,1H),2.73(d,J＝13.2Hz,1H),2.62(dd,J＝21.1,7.6Hz,2H),2.43(d,J＝11.6Hz,1H),2.03(s,1H),1.97–1.86(m,2H),1.85–1.72(m,3H),1.70–1.50(m,3H),1.43–1.24(m,3H),1.05(dd,J＝27.2,12.1Hz,2H)；¹³C NMR(500MHz,DMSO):153.2,135.5,129.2(2),127.8,125.9,113.2(2),59.8,59.3,54.3,46.7,41.5,35.2,34.8,27.6,26.2,24.9,24.2,23.8,19.5；HRMS:calcd for C₂₁H₃₀N₃O₂S[M+H]⁺:388.2053，found:388.2049。

Example 30Synthesis of 12-N- (4-carboxybenzoyl) aloperine (N-KD-39):

referring to general synthetic method 2 above, 0.4g of methyl p-chlorocarbonyl benzoate was added to give N-KD-20 as a yellow solid, which was hydrolyzed in 20% aqueous NaOH to give a white solid (237mg, 62%), melting point: 206 and 208 ℃.¹HNMR(500MHz,DMSO):8.00(d,J＝8.2Hz,2H),7.57(d,J＝7.9Hz,2H),7.41(s,1H),5.65(s,1H),4.79(s,1H),3.53(d,J＝20.1Hz,2H),3.34–3.26(m,1H),3.22(s,1H),3.11(s,3H),2.72(s,1H),2.38–2.20(m,3H),2.13(s,1H),2.00(d,J＝9.5Hz,1H),1.85–1.61(m,5H),1.55(d,J＝10.8Hz,2H),1.49–1.37(m,1H)；¹³C NMR(500MHz,DMSO)170.1,167.2(2),141.1,132.0,129.8(3),127.3(2),63.3,57.3,54.1,49.0,45.8(2),44.6,33.6,29.6,27.4,23.4,22.5,19.0；HRMS:calcd for C₂₃H₂₉N₂O₃[M+H]⁺: 381.21727，found:381.21685

Example 31Synthesis of 12-N- (2-methoxycarbonylphenylsulfonyl) aloperine (N-KD-40):

referring to general procedure 2 of the above synthesis, 0.4g methyl 2-chlorosulfonylbenzoate was added to give a pale yellow solid (657mg, 85%), m.p.: 155 ℃ and 157 ℃.¹H NMR(500MHz,CDCl₃):7.98–7.93(m,1H),7.60–7.53(m,2H),7.44(dd,J＝7.2,1.7Hz,1H),5.54(s,1H),4.53(s,1H),3.93(d,J＝4.6Hz,3H),3.76(dd,J＝14.0,7.2Hz,1H),3.60(d,J＝20.9Hz,1H),3.02(d,J＝9.5Hz,1H),2.74(dd,J＝50.2,10.1Hz,3H),2.50(d,J＝10.0Hz,1H),2.14(s,1H),2.05–1.96(m,1H),1.84(dd,J＝38.6,28.1Hz,4H),1.66(dd,J＝25.5,12.2Hz,4H),1.43(dd,J＝25.1,10.8Hz,2H),1.14–0.99(m,2H)；¹³C NMR(500MHz,CDCl₃)168.8,138.8,135.2,133.1,132.0,130.1,128.6,127.98(2),60.5,59.3,54.2,53.1,46.4,41.7,35.0,34.9,27.7,26.1,25.0,24.2,24.0,19.3；HRMS:calcd for C₂₃H₃₁N₂O₄S[M+H]⁺:431.1999，found:431.1994。

Example 32Synthesis of 12-N- (2-carboxyphenylsulfonyl) aloperine (N-KD-41):

referring to general synthetic method 2 above, 0.4g of methyl 2-chlorosulfonylbenzoate was added to give a pale yellow solid, N-KD-40, which was hydrolyzed in 20% aqueous NaOH to give a white solid (300mg,72%), melting point: 226-228 ℃.¹H NMR(500MHz,CDCl₃):8.90(s,1H),7.97(d,J＝5.5Hz,1H),7.57(dd,J＝30.1,20.1Hz,3H),5.73(s,1H),4.60(s,1H),3.73(d,J＝14.5Hz,4H),3.47(s,2H),3.34(t,J＝14.2Hz,1H),2.68(s,1H),2.46–2.25(m,2H),2.16–1.96(m,3H),1.92–1.62(m,7H),1.54(s,2H)；¹³C NMR(500MHz,CDCl₃):169.0,137.8,136.2,134.0,133.1,130.0, 128.7,128.4,125.7,59.4,58.0,54.48,45.6,45.2,33.6,33.2,28.4,23.7,22.6,22.4,21.6,18.6；HRMS:calcd for C₂₂H₂₉N₂O₄S[M+H]⁺:417.1843，found:417.1839。

Example 33Synthesis of 12-N- (4-methoxycarbonylphenylsulfonyl) aloperine (N-KD-42):

referring to general procedure 2 of the above synthesis, 0.4g 4-chlorosulfonylbenzoate was added to give a pale yellow solid (642mg, 83%), m.p.: 67-69 ℃.¹H NMR(500MHz,CDCl₃):8.16(d,J＝8.4Hz,2H),7.99–7.95(m,2H),5.53(d,J＝4.8Hz,1H),4.28(d,J＝5.2Hz,1H),3.96(s,3H),3.71(dd,J＝14.3,7.6Hz,1H),3.62–3.54(m,1H),3.05(dd,J＝11.7,3.4Hz,1H),2.78(d,J＝13.4Hz,1H),2.68(d,J＝12.7Hz,2H),2.49(d,J＝11.9Hz,1H),2.23(s,1H),2.00–1.75(m,5H),1.71–1.53(m,3H),1.49–1.37(m,2H),1.35–1.26(m,1H),1.08(dd,J＝33.9,12.9Hz,2H)；¹³C NMR(500MHz,CDCl₃)165.78,145.16,134.62,133.39,130.22,128.49,127.05,60.73,59.28,54.21,52.63,46.53,41.83,35.17,34.84,27.65,26.10,24.93,24.15,23.82,19.31；HRMS:calcd for C₂₃H₃₁N₂O₄S[M+H]⁺:431.1999，found:431.1995。

Example 34Synthesis of 12-N- (4-carboxyphenylsulfonyl) aloperine (N-KD-43)The composition is as follows:

referring to general synthetic procedure 2 above, 0.4g methyl 2-chlorosulfonylbenzoate was added to give N-KD-42 as a pale yellow solid, which was hydrolyzed in 20% aqueous NaOH to give a pale yellow solid (293mg, 71%), melting point: 245 ℃ and 247 ℃.¹H NMR(500MHz,DMSO):8.12 (d,J＝8.3Hz,2H),8.00(d,J＝8.3Hz,2H),5.54(d,J＝4.4Hz,1H),4.26(d,J＝3.7Hz,1H),3.62–3.58(m,1H),3.49–3.41(m,2H),3.19(d,J＝3.4Hz,1H),2.86(s,1H),2.82–2.70(m,2H),2.64(s,1H),2.21(s,1H),2.02–1.89(m,3H),1.81(dd,J＝22.8,12.8Hz,2H),1.65(dd,J＝43.4,12.8Hz,2H),1.39(d,J＝12.6Hz,3H),1.20(dd,J＝21.5,12.6Hz,3H)；¹³C NMR(500MHz,DMSO)172.2,148.0,139.8,135.3(3),132.8,132.2(2),64.8,64.0,58.9,53.8,51.1,47.0,39.5,39.0,32.2,29.8,28.8,27.8,24.0；HRMS:calcd for C₂₂H₂₉N₂O₄S[M+H]⁺:417.1843,found:417.1840。

Example 35Synthesis of 12-N- (2-aminoethanesulfonyl) aloperine (N-KD-44):

referring to general procedure 2 of the above synthesis, 0.5g of 2-phthalimidoethanesulfonyl chloride was added to give N-KD-32, 3 drops of hydrazine hydrate were added and column chromatography was performed to give a white solid (390mg, 64%). Melting point: 117 ℃ and 119 ℃.¹H NMR(500MHz,CDCl₃)；5.61–5.54(m,1H),4.27(d,J＝5.6Hz,1H),3.55–3.47(m,2H),3.22–3.13(m,3H),3.09–2.96(m,2H),2.79–2.74(m,1H),2.65(dd,J＝18.6,7.7Hz,2H),2.49(d,J＝12.0Hz,1H),2.27–2.18(m,1H),2.13–2.01(m,3H),2.00–1.94(m,1H),1.90(s,1H),1.86–1.77(m,2H),1.69–1.53(m,3H),1.43–1.33(m,1H),1.06(dd,J＝30.8,12.8Hz,2H)；¹³C NMR(500MHz,CDCl₃)134.8,128.6,60.2,59.3,55.3,54.2,46.6,41.6,37.1,35.0,34.8,28.0,26.0,24.8,24.7,24.2,19.3；HRMS:HRMS:calcd for C₁₇H₃₀N₃O₂S[M+H]⁺:340.2053,found:340.2051。

Example 36Synthesis of 12-N- (2-cyanophenylsulfonyl) aloperine (N-KD-45):

referring to general procedure 2 of the above synthesis, 0.4g of 2-cyanobenzenesulfonyl chloride was added to give a white solid (605 mg, 85%), melting point: 171 ℃ and 173 ℃.¹H NMR(500MHz,CDCl₃):8.14(d,J＝7.8Hz,1H),7.88(d,J＝7.5Hz,1H),7.75(t,J＝7.5Hz,1H),7.68(t,J＝7.3Hz,1H),5.58(s,1H),4.52(s,1H),3.82(s,1H),3.69(d,J＝8.2Hz,1H),2.99(d,J＝9.8Hz,1H),2.80(d,J＝11.0Hz,1H),2.67(d,J＝10.4Hz,2H),2.50(d,J＝10.2Hz,1H),2.10(s,2H),1.93(s,1H),1.87–1.55(m,8H),1.42(d,J＝12.1Hz,1H),1.09(dd,J＝34.0,9.5Hz,2H)；¹³C NMR(600MHz,CDCl₃)144.0,135.5,134.5,133.0,132.4,129.8,128.6,116.7,110.2,65.8,61.0,59.2,54.2,46.5,42.3,34.8,27.8,26.2,24.9,24.4,24.2,19.3；HRMS:calcd for C₂₂H₂₈N₃O₂S[M+H]⁺:398.1897,found:398.1893。

Example 37Synthesis of 12-N- (2-acetamido-4-methyl-5-thiazolesulfonyl) aloperine (N-KD-46):

with reference to general Synthesis procedure 2 described above, 0.5g of 2-acetamido was addedYl-4-methyl-5-thiazolesulfonyl chloride to give a light yellow solid (631mg, 78%), melting point: 132 ℃ and 134 ℃.¹H NMR(500MHz,CDCl₃):5.59(s,1H),4.38(s,1H),3.66(s,2H),3.09(s,1H),2.71(s,3H),2.58(s,3H),2.49(s,1H),2.33(s,3H),2.27–2.20(m,1H),2.13–2.03(m,1H),1.98–1.83(m,4H),1.76(dd,J＝47.4,30.3Hz,5H),1.59–1.38(m,2H),1.10(dd,J＝15.0,7.6Hz,2H)；¹³C NMR(151MHz,cd₃od)170.1,159.5,151.3,134.5,128.3,123.2,60.6,59.8,54.2,46.7,41.9,35.0,34.8,27.8,25.6,24.5,24.4,23.7,21.5,19.4,15.7；HRMS:calcd for C₂₁H₃₁N₄O₃S₂[M+H]⁺:451.1832,found:451.1828。

Example 38Synthesis of 12-N- (4-cyanophenylsulfonyl) aloperine (N-KD-47):

referring to general procedure 2 of the above synthesis, 0.4g of 4-cyanobenzenesulfonyl chloride was added to give a white solid (614mg, 86%), melting point: 155 ℃ and 157 ℃.¹H NMR(500MHz,CDCl₃):8.02(d,J＝8.4Hz,2H),7.82(d,J＝8.4Hz,2H),5.55(d,J＝4.9Hz,1H),4.27(d,J＝5.3Hz,1H),3.70(dd,J＝14.3,7.6Hz,1H),3.66–3.55(m,1H),3.06(dd,J＝11.8,3.4Hz,1H),2.77(d,J＝13.3Hz,1H),2.72–2.58(m,2H),2.50(d,J＝12.0Hz,1H),2.22(s,1H),2.06–1.75(m,5H),1.71–1.55(m,3H),1.51–1.37(m,2H),1.35–1.24(m,1H),1.08(dd,J＝33.6,13.0Hz,2H)；¹³C NMR(600MHz,CDCl₃):145.5,134.2,132.8(2),128.8,127.6(2),117.5,115.9,60.9,59.2,54.2,46.5,41.9,35.1,34.8,27.6,26.1,24.9,24.1,23.9,19.3；HRMS:calcd for C₂₂H₂₈N₃O₂S[M+H]⁺:398.1897,found:398.1892。

Example 3912-N- (2-amino-4-methyl-5-thiazolesulfonic acidAcyl) synthesis of aloperine (N-KD-48):

referring to general synthetic procedure 2 above, 0.5g of 2-acetamido-4-methyl-5-thiazolesulfonyl chloride was added to give N-KD-46 as a pale yellow solid, which was then hydrolyzed in 20% aqueous NaOH to give a pale yellow solid (473mg, 58%), melting point: 186 ℃ and 188 ℃.¹H NMR(500MHz,CDCl₃):5.87(s,2H),5.62(s,1H),4.32(s,1H),3.60(s,2H),3.12(s,1H),2.78(d,J＝61.0Hz,3H),2.47(s,4H),2.17(s,1H),1.89(s,7H),1.74–1.40(m,5H),1.19–1.06(m,1H).HRMS:calcd for C₁₉H₂₉N₄O₂S₂[M+H]⁺:409.1726,found:409.1722。

Example 40Synthesis of 12-N- (1-methyl-1H-imidazole-4-sulfonyl) aloperine (N-KD-49):

referring to general procedure 2 of the above synthesis, 0.3g of 1-methyl-1H-imidazole-4-sulfonyl chloride was added to give a white solid (540mg, 80%), melting point: 152 ℃ and 154 ℃.¹H NMR(500MHz,CDCl₃):7.49–7.45(m,1H),7.41(d,J＝4.0Hz,1H),5.53(s,1H),4.32(s,1H),3.80–3.66(m,4H),3.54(s,1H),3.04(s,1H),2.72(d,J＝65.2Hz,3H),2.46(s,1H),2.27(s,1H),2.00(s,1H),1.84(t,J＝26.0Hz,5H),1.72–1.53(m,3H),1.41(dd,J＝12.6,9.3Hz,2H),1.08(d,J＝26.3Hz,2H)；¹³C NMR(500MHz,CDCl₃)140.7,138.9,135.7,128.0,123.5,77.3,60.3,59.6,54.3,46.8,42.3,34.9,34.0,27.8,26.1,24.9,24.4,23.8,19.5.HRMS:calcd for C₁₉H₂₉N₄O₂S[M+H]⁺:377.2006,found:377.2002。

EXAMPLE 41Synthesis of 12-N- (4-trifluoromethoxybenzenesulfonyl) aloperine (N-KD-50):

referring to general procedure 2 of the above synthesis, 0.5g of 4-trifluoromethoxybenzenesulfonyl chloride was added to give a white solid (697mg, 85%), melting point: 93-95 ℃.¹H NMR(500MHz,CDCl₃):7.96(d,J＝8.7Hz,2H),7.34(d,J＝8.2Hz,2H),5.55(s,1H),4.27(s,1H),3.78–3.54(m,2H),3.06(d,J＝10.1Hz,1H),2.79(d,J＝12.1Hz,1H),2.68(s,2H),2.50(d,J＝11.4Hz,1H),2.24(s,1H),2.03–1.78(m,5H),1.65(dd,J＝30.3,12.2Hz,3H),1.53–1.31(m,3H),1.09(dd,J＝33.3,11.7Hz,2H)；¹³C NMR(600MHz,CDCl₃)151.8,139.7,134.6,129.1(2),128.5,121.1,120.8(2),60.6,59.3,54.2,46.6,41.8,35.0,34.8,27.6,26.1,24.9,24.2,23.9,19.3；HRMS:calcd forC₂₂H₂₈F₃N₂O₃S[M+H]⁺:457.1767,found:457.1760。

Example 42Synthesis of 12-N- (2,1, 3-benzothiadiazole-4-sulfonyl) aloperine (N-KD-51):

referring to general procedure 2 of the above synthesis, 0.4g of 2,1, 3-benzothiadiazole-4-sulfonyl chloride was added to give a bright yellow solid (640mg, 83%), m.p.: 198-200 ℃.¹H NMR(500MHz,CDCl₃):8.30(dd,J＝7.0,0.8Hz,1H),8.21(dd,J＝8.8,0.8Hz,1H),7.70(dd,J＝8.8,7.1Hz,1H),5.50(d,J＝5.3Hz,1H),4.94(d,J＝5.5Hz,1H),3.87(dd,J＝14.2,6.6Hz,1H),3.68–3.56(m,1H),2.94(dd,J＝11.7,3.8Hz,1H),2.76–2.55(m,3H),2.45(d,J＝12.0Hz,1H),2.31(s,1H),1.95–1.73(m,5H),1.72–1.56(m,2H),1.46–1.32(m,2H),1.32–1.23(m,2H),1.06(dd,J＝24.9,13.6Hz,2H)；¹³C NMR(126MHz,CDCl₃):155.6,149.6,135.0,133.8,131.4,128.4,128.3,125.9,60.8,59.4,54.2,46.7,41.9,35.2,34.9,27.8,26.1,24.9,24.3,24.2,19.4；HRMS:calcd forC₂₁H₂₇N₄O₂S₂[M+H]⁺:431.1570,found:431.1563。

Example 43Synthesis of 12-N- (2, 3-dihydrobenzofuran-5-sulfonyl) aloperine (N-KD-52):

referring to general procedure 2 of the above synthesis, 0.4g of 2, 3-dihydrobenzofuran-5-sulfonyl chloride was added to give a pale yellow solid (610mg, 82%), melting point: 171 ℃ and 173 ℃.¹H NMR(500MHz,CDCl₃):7.70(s,1H),7.66(dd,J＝8.5,1.5Hz,1H),6.82(d,J＝8.4Hz,1H),5.53(s,1H),4.67(t,J＝8.8Hz,2H),4.22(s,1H),3.60(dd,J＝34.5,27.9Hz,2H),3.26(t,J＝8.8Hz,2H),3.04(s,1H),2.85–2.60(m,3H),2.46(d,J＝9.2Hz,1H),2.23(s,1H),2.04–1.75(m,5H),1.63(t,J＝25.5Hz,3H),1.50–1.34(m,3H),1.18–1.00(m,2H)；¹³C NMR(151MHz,CDCl₃)163.4,135.2,132.9,128.4,128.0,124.3,109.3(2),72.2,60.3,59.5,54.3,46.7,41.7,35.0,34.9,29.1,27.9, 26.0,24.9,24.4,23.9,19.5；HRMS:calcd for C₂₃H₃₁N₂O₃S[M+H]⁺:415.2050,found:415.2047。

Example 44Synthesis of 12-N- (4-methyl-1-piperazinesulfonyl) aloperine (N-KD-53):

referring to general procedure 2 of the above synthesis, 0.4g of 4-methyl-1-piperazinesulfonyl chloride was added to give the compound as a yellow oil (565mg, 80%).¹H NMR(500MHz,CDCl₃):5.46(d,J＝4.9Hz,1H),4.20(d,J＝5.0Hz,1H),3.34(m,J＝21.3,13.8,6.6Hz,2H),3.12(d,J＝5.1Hz,4H),2.98(dd,J＝11.6,3.8Hz,1H),2.68(d,J＝13.3Hz,1H),2.58(t,J＝12.1Hz,2H),2.42–2.30(m,5H),2.19(s,3H),2.10(dd,J＝25.0,14.9Hz,3H),2.01–1.91(m,1H),1.82(s,1H),1.74(d,J＝11.7Hz,2H),1.69–1.62(m,1H),1.61–1.44(m,3H),1.37–1.24(m,1H),0.99(dd,J＝28.8,13.1Hz,2H)；¹³C NMR(500MHz,CDCl₃):135.5,127.6,65.7,60.8,59.4,54.4,54.2,46.8,46.0,45.8,42.4,34.8,34.1,27.8,26.0,24.8,24.6,24.3,19.4,15.2；HRMS:calcd for C₂₀H₃₅N₄O₂S[M+H]⁺:395.2475，found:395.2492。

Biological activity

Experimental example 1Anti-hepatitis C Virus Activity of Compounds of the invention

The test principle is as follows: the huh7.5 cells were used as hepatitis C virus vectors, and the samples were assayed for inhibition of DNA replication by hepatitis C virus.

Test materials and methods:

1. cell lines: huh7.5 cells; stored in the room.

2. Sample treatment: the compounds of the examples of the present invention were prepared in 50mM, 20mM stock solutions with a maximum concentration of 500. mu.M (200. mu.M) in DMSO, and then diluted 3-fold with culture medium at 8 dilutions each.

3. Positive control drug: aloperine, highest concentration 200. mu.M, was diluted 5-fold with culture medium, 8 dilutions each. Storing at 4 ℃.

4. The main reagents are as follows: the DNA extraction kit is a product of Beijing all-purpose gold biotechnology limited company.

5. The test method comprises the following steps:

MTT: huh7.5 cell seed 96-well culture plate and seedAdding sample and positive control drug according to the dilution after 48 hr, setting cell control hole, 50 μ l liquid medicine +50 μ l culture medium, adding 10 μ l MTT into each hole 3 days after adding drug, standing at 37 deg.C for 3-4h, sucking off liquid, adding 100 μ l DMSO into each hole, oscillating for 10min, measuring OD value with ultraviolet analyzer, performing data analysis, calculating CC₅₀。

The drug effect is as follows: the huh7.5 cell culture plate with 96 wells was seeded 48 hours later, the sample and the positive control drug were added according to the above dilution, and at the same time, virus control wells, 50. mu.l of the drug solution + 50. mu.l of the virus solution were set, and after 3 days, RNA was extracted. Detecting HCV DNA in cells by adopting Real time PCR method, and respectively calculating EC₅₀And SI.

The data show that the compound has good inhibitory activity and lower cytotoxicity on Hepatitis C Virus (HCV), particularly the compounds N-KD-1-5, 14, 18, 23, 25, 30, 33, 44, 50, 51, 53, 45 and 47, and the HCV inhibitory activity of the compound is obviously superior to that of the precursor compound aloperine.

Experimental example 2The compounds of the invention are active against hepatitis B virus

The test principle is as follows: 2.2.15 cells are taken as hepatitis B virus vectors, and the sample is determined to inhibit the hepatitis B virus from DNA replication.

Test materials and methods:

1. cell lines: 2.2.15 cells; stored in the room.

2. Sample treatment: the sample DMSO was prepared as 20mg/ml stock solution, diluted 3-fold with culture medium just before use, 8 dilutions each.

3. Positive control drug: lamivudine (3TC), produced by Beijing NodeHengxin chemical technology, Inc., under the batch number of NDS0111122, stored at 4 ℃;

aloperine.

5. The test method comprises the following steps:

the drug effect is as follows: 2.2.15 cell culture plate with 96 wells, adding sample and positive control drug according to above dilution after 48 hr, setting cell control wells, changing culture solution containing samples with different dilution concentrations after 3 days, detecting HBV DNA in cells 2.2.15 after 6 days, calculating IC, respectively₅₀And SI.

MTT 2.2.15 cells in exponential growth phase were seeded in 96-well culture plates, 2 × 10⁵Adding culture solution containing PFA diluted at different concentration multiple ratio into each cell/well, repeating for 3 wells for each dilution, and placing at 37 deg.C in CO₂Incubating for 48 hours; discarding the supernatant, adding 100. mu.l MTT (0.5mg/ml) prepared from the culture solution, and continuing to culture at 37 ℃ for 4 hours; adding 100 μ l of 50% DMF-20% SDS decolorized solution into each well, and standing overnight at 37 deg.C; the absorbance (OD) at a wavelength of 570nm was measured on a microplate reader₅₇₀)。

Each experiment was performed in 3 wells for cell control and blank control, and the results were expressed as the following formula: (cell control OD₅₇₀-medicated cell OD₅₇₀) Cell control OD₅₇₀Calculating the cell death rate (%), and calculating the half toxic concentration CC by a Reed-Muench method₅₀。

The results show that the activity of the compound of the invention against Hepatitis B Virus (HBV) is equivalent to that of the positive drug 3 TC.

Experimental example 3anti-H7N 9 Activity of Compounds of the invention

The test principle is as follows: chemical luminescence method

The test method comprises the following steps: the compound is prepared from 1: 40 to 1: a five-fold dilution gradient is prepared in the 25000 range, and the prepared concentration depends on the degree of efficacy to be evaluated. To each EP tube, 550. mu.L of the above dilution and an equal amount of the virus-carrying solution were added and mixed. At 37 ℃ 5% CO₂Was incubated for one hour, 500. mu.L of the mixture was then added to Vero cell-plated plates, the various gradients were repeated, and then at 37 ℃ with 5% CO₂Shake-culturing for 2 hours under the conditions of (1), and adding 3ml of a covering medium containing 0.5% agarose thereto. On the fourth day, the virus plaques were stained with 0.1% crystal violet on PBS (containing 0.2% formaldehyde) and visualized and counted. NT₅₀Is defined as the reciprocal of the compound concentration at 50% reduction of virus on viral plaques. IC (integrated circuit)₅₀Means NT₅₀The reciprocal of (c).

The results show that the compound has certain anti-influenza activity after structural modification.

Experimental example 4anti-EBOV Activity of Compounds of the invention

The test principle is as follows: a chemiluminescence method;

positive control drug: aloperine;

the test method comprises the following steps: the compound is prepared from 1: 40 to 1: a five-fold dilution gradient is prepared in the 25000 range, and the prepared concentration depends on the degree of efficacy to be evaluated. To each EP tube, 550. mu.L of the above dilution and an equal amount of the virus-carrying solution were added and mixed. At 37 ℃ 5% CO₂Under the conditions of (1) cultivating a smallIn this case, 500. mu.L of the suspension was then applied to Vero cell-plated plates, and the gradients were repeated, followed by 5% CO at 37 ℃₂Shake-culturing for 2 hours under the conditions of (1), and adding 3ml of a covering medium containing 0.5% agarose thereto. On the fourth day, the virus plaques were stained with 0.1% crystal violet on PBS (containing 0.2% formaldehyde) and visualized and counted. NT₅₀Defined as the reciprocal of the compound dilution requirement at 50% reduction of virus on viral plaques.

The 12-N-substituted aloperine derivative has medium activity on EBOV, wherein N-KD-16, N-KD-17 and N-KD-18 have strong inhibitory activity on EBOV virus.

Experimental example 5anti-MERS Activity of Compounds of the invention

The test principle is as follows: chemical luminescence method

The test method comprises the following steps: the compound is prepared from 1: 40 to 1: a five-fold dilution gradient is prepared in the 25000 range, and the prepared concentration depends on the degree of efficacy to be evaluated. To each EP tube, 550. mu.L of the above dilution and an equal amount of the virus-carrying solution were added and mixed. At 37 ℃ 5% CO₂Was incubated for one hour, 500. mu.L of the mixture was then added to Vero cell-plated plates, the various gradients were repeated, and then at 37 ℃ with 5% CO₂Shake-culturing for 2 hours under the conditions of (1), and adding 3ml of a covering medium containing 0.5% agarose thereto. On the fourth day, the virus plaques were stained with 0.1% crystal violet on PBS (containing 0.2% formaldehyde) and visualized and counted. NT₅₀Defined as the reciprocal of the compound dilution requirement at 50% reduction of virus on viral plaques.

After activity evaluation, the compound has certain MERS resistance activity.

Experimental example 6Acute toxicity of the Compounds of the invention

Kunming mice (purchased from animal research institute of Chinese academy of medical sciences) 18-20g, weighing and randomly grouping, each group having 10 mice each with half male and female, recording body weight, and fasting for 5 hours before administration. 250mg/kg, 500mg/kg and 1000mg/kg are respectively administered by one-time intragastric administration, and the blank control group is treated with physiological saline with the same amount, closely observed for 7 days, and the weight and death condition are recorded. LD50 values were calculated from the cumulative mortality of each dose using SPSS software.

The results show that the compound is safe within 500mg/kg after single administration, wherein the safe range of the single administration of the N-KD-23, the N-KD-49 and the N-KD-53 can reach 1000 mg/kg.

Experimental example 7Pharmacokinetic Studies of Compounds of the invention

Healthy male SD rats were taken 3 per group (male, body weight 200-250g) and orally administered 25mg/kg, respectively, in a volume of 10 ml/kg. The dosing solution was formulated one hour prior to dosing. After administration, about 0.3ml of whole blood is taken through a jugular vein cannula according to a specified time point (0.25h, 0.5h, 1.5h, 2h, 4h, 6h and 24h), the whole blood is placed into a centrifuge tube containing K3EDTA as an anticoagulant, and a whole blood sample is collected and then subjected to centrifugal separation of plasma. And (3) treating the plasma sample by using an organic solvent, and determining by using a liquid chromatography-tandem mass spectrometry method. And calculating pharmacokinetic parameters during sample analysis, and storing the collected plasma sample in a refrigerator at-20 ℃.

The results show that the compound has excellent pharmacokinetic properties, particularly the N-KD-44, the N-KD-50 and the N-KD-53 are more prominent.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A compound shown in formula I, an optical isomer, a solvate or a pharmaceutically acceptable salt thereof,

wherein,

the X group being-CH₂-, -C (O) -or-S (O)_a-, wherein a is 1 or 2;

the Y radical being C_1-10Alkyl, or 3-15-membered cycloalkyl, aryl or heterocyclyl; optionally, wherein said C_1-10The alkyl, 3-15 membered cycloalkyl, aryl or heterocyclyl is each independently substituted by one or more groups selected from halogen, amino, cyano, nitro, carboxy, C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkoxycarbonyl, C_1-6Alkyl acyl radical, C_1-6Alkylamine acyl, C_1-6Alkylamido radical, C_1-6Alkylsulfonyl and 8-15 membered benzoheterocyclyl.

2. A compound of formula I according to claim 1, an optical isomer, solvate or pharmaceutically acceptable salt thereof,

wherein,

the X group being-CH₂-, -C (O) -or-S (O)_a-, wherein a is 1 or 2;

3. A compound of formula I according to claim 2, an optical isomer, solvate or pharmaceutically acceptable salt thereof,

wherein,

the X group being-CH₂-, -C (O) -or-S (O)_a-, wherein a is 1 or 2;

the Y radical being C_1-6Alkyl, or a 3-8 membered cycloalkyl formed by an atom selected from C, N, O and S, phenyl, naphthyl, a 3-8 membered lipoheterocyclyl, a 3-8 membered monoaromatic heterocyclyl or an 8-12 membered fused heteroaromatic heterocyclyl, optionally wherein said C is_1-6Alkyl, 3-8 membered cycloalkyl, phenyl, naphthyl, 3-8 membered lipoheterocyclyl, 3-8 membered monoaromatic heterocyclyl or 8-12 membered fused heteroaromatic heterocyclyl are each independently substituted by one or more groups selected from halogen, amino, cyano, nitro, carboxy, C_1-4Alkyl radical, C_1-4Alkoxy, halo C_1-4Alkyl, halo C_1-4Alkoxy radical, C_1-4Alkoxycarbonyl, C_1-4Alkyl acyl radical, C_1-4Alkylamine acyl, C_1-4Alkylamido radical, C_1-4Alkylsulfonyl or 8-12 membered benzoheterocyclyl.

4. The compound of claim 3, an optical isomer, solvate or pharmaceutically acceptable salt thereof,

wherein,

the X group being-CH₂-, -C (O) -or-S (O)_a-, wherein a is 1 or 2;

the Y radical being C_1-4Alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, naphthyl, oxiranyl, oxocyclobutyl, tetrahydrofuryl, tetrahydropyranyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, furanyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyranyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, benzofuranyl, indolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzooxadiazolyl, benzotriazolyl, benzothiadiazolyl, purinyl, quinolinyl, benzotetrahydrofuranyl, benzotetrahydropyrrolyl, benzotetrahydropyranyl, benzopiperidinyl, benzodioxanyl or benzopiperazinyl, optionally substituted with one or more groups selected from halogen, amino, cyano, nitro, carboxyl, C_1-4Alkyl radical, C_1-4Alkoxy, halo C_1-4Alkyl, halo C_1-4Alkoxy radical, C_1-4Alkoxycarbonyl, C_1-4Alkyl acylBase, C_1-4Alkylamine acyl, C_1-4Alkylamido radical, C_1-4Alkylsulfonyl and phthalimido groups.

5. The compound of claim 1, an optical isomer, solvate or pharmaceutically acceptable salt thereof, selected from the group consisting of:

12-N- (2-cyanobenzyl) aloperine (N-KD-1);

12-N- (2-methylbenzyl) aloperine (N-KD-2);

12-N- (4-methoxybenzyl) aloperine (N-KD-3);

12-N-benzylaloperine (N-KD-4);

12-N- (4-nitrobenzyl) aloperine (N-KD-5);

12-N- (pyridin-4-methyl) aloperine (N-KD-13);

12-N- (4-trifluoromethoxybenzyl) aloperine (N-KD-14);

12-N- (3-methoxybenzyl) aloperine (N-KD-15);

12-N- (2-methoxybenzyl) aloperine (N-KD-16);

12-N- (4-fluorobenzyl) aloperine (N-KD-17);

12-N- (3, 4-dichlorobenzyl) aloperine (N-KD-18);

12-N- (4-methoxybenzenesulfonyl) aloperine (N-KD-19);

12-N- (4-methoxycarbonylbenzoyl) aloperine (N-KD-20);

12-N- (4-acetylbenzenesulfonyl) aloperine (N-KD-21);

12-N- (4-methoxybenzoyl) aloperine (N-KD-22);

12-N- (3-cyanophenylsulfonyl) aloperine (N-KD-23);

12-N- (pyridine-4-sulfonyl) aloperine (N-KD-24);

12-N- (2-thiophenesulfonyl) aloperine (N-KD-25);

12-N- (4-acetamidobenzenesulfonyl) aloperine (N-KD-26);

12-N- (3, 4-dimethoxybenzenesulfonyl) aloperine (N-KD-27);

12-N- (3,4, 5-trimethoxybenzoyl) aloperine (N-KD-29);

12-N- (4-isopropoxybenzenesulfonyl) aloperine (N-KD-30);

12-N- (3-methyl-4-methoxybenzenesulfonyl) aloperine (N-KD-31);

12-N- (2-phthalimidoethylsulfonyl) aloperine (N-KD-32);

12-N- (2-methoxy-5-chlorophenylsulfonyl) aloperine (N-KD-33);

12-N- (4-methylsulfonylphenylsulfonyl) aloperine (N-KD-34);

12-N- (8-quinolinesulfonyl) aloperine (N-KD-35);

12-N- (2, 3-dihydro-1, 4-benzodioxan-6-sulfonyl) aloperine (N-KD-36);

12-N- (4-aminobenzenesulfonyl) aloperine (N-KD-38);

12-N- (4-carboxybenzoyl) aloperine (N-KD-39);

12-N- (2-methoxycarbonylphenylsulfonyl) aloperine (N-KD-40);

12-N- (2-carboxyphenylsulfonyl) aloperine (N-KD-41);

12-N- (4-methoxycarbonylphenylsulfonyl) aloperine (N-KD-42);

12-N- (4-carboxyphenylsulfonyl) aloperine (N-KD-43);

12-N- (2-aminoethanesulfonyl) aloperine (N-KD-44);

12-N- (2-cyanophenylsulfonyl) aloperine (N-KD-45);

12-N- (2-acetylamino-4-methyl-5-thiazolesulfonyl) aloperine (N-KD-46);

12-N- (4-cyanophenylsulfonyl) aloperine (N-KD-47);

12-N- (2-amino-4-methyl-5-thiazolesulfonyl) aloperine (N-KD-48);

12-N- (1-methyl-1H-imidazole-4-sulfonyl) aloperine (N-KD-49);

12-N- (4-trifluoromethoxybenzenesulfonyl) aloperine (N-KD-50);

12-N- (2,1, 3-benzothiadiazole-4-sulfonyl) aloperine (N-KD-51);

12-N- (2, 3-dihydrobenzofuran-5-sulfonyl) aloperine (N-KD-52); and

12-N- (4-methyl-1-piperazinesulfonyl) aloperine (N-KD-53).

6. A process for the preparation of a compound according to any one of claims 1 to 5, an optical isomer, solvate or pharmaceutically acceptable salt thereof, which comprises the steps of:

wherein L represents a leaving group, such as halogen, -OTs or-OCOR;

the remaining groups are as defined in claim 1.

7. A pharmaceutical composition comprising a compound of any one of claims 1-5, an optical isomer, solvate or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

8. Use of a compound as claimed in any one of claims 1 to 5, an optical isomer, solvate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as claimed in claim 7 for the manufacture of an antiviral medicament.

9. Use according to claim 8, wherein the virus is selected from the group consisting of Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Ebola virus (EBOV), middle east respiratory syndrome virus (MERS virus) and influenza virus (preferably influenza A virus, more preferably viruses such as H5N1, H5N2, H7N2, H7N3, H7N7, H7N9, H9N2 and H10N 7).

10. Use of a compound according to any one of claims 1 to 5, an optical isomer, solvate or pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 7, for the manufacture of a medicament for the treatment of hepatitis b, hepatitis c, ebola hemorrhagic fever, middle east respiratory syndrome or influenza (e.g. avian influenza, preferably avian influenza H7N 9).