CN101531637B - A novel compound capable of restraining and blocking M2 ion channel activity and application thereof - Google Patents

Abstract

The invention discloses a compound in formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, which has the activity of inhibiting and blocking M2 ion channel, and the definition of each group in the formula is detailed in the description. The compound or its pharmaceutically acceptable salt or stereoisomer may inhibit and block the activity of wild-type and mutant influenza and avian influenza M2 ion channels through a new mechanism of action, so as to treat wild-type and mutant influenza and The medicinal function of bird flu, and they may also be used to treat hepatitis B, hepatitis C and AIDS, so as to provide more drug options for clinical treatment.

Description

Compound with activity of inhibiting and blocking M2 ion channel and application thereof

technical field

The present invention relates to a new compound with the activity of inhibiting and blocking M2 ion channel and its application in pharmacy.

Background technique

The recent H5N1 Influenza A and Avian Influenza, which endangered human life, have attracted global attention. The World Health Organization and various countries have taken active measures against possible outbreaks, including the development of vaccines and drugs. In the process of influenza A and avian influenza infection, the M2 ion channel of influenza A and avian influenza plays an important role. It is a key protein for the fusion of the viral membrane and the endosome membrane to release the gene from the virus into the cell. The M2 ion channel has been proved to be an effective drug target by clinical tests, and amantadine and rimantadine have been used clinically for decades as effective M2 ion channel blockers. However, due to long-term clinical use or/and drug abuse, a large number of amantadine and rimantadine-resistant influenza virus strains have emerged clinically in recent years.

Based on amantadine and rimantadine, a series of compounds have been designed, synthesized and pharmacologically studied (J.Med.Chem.2004, 47, 4975-4978, J.Am.Chem.Soc.2000, 122, 466- 473, J.Med.Chem.2003, 46, 2823-2833, US3391142, Eur J Med Chem 1996, 31, 105-110, Nature Reviews Drug Discovery 2006, 5, 1015-1025), because these compounds are basically the same as diamond Alkylamine and rimantadine have a similar mechanism of action and mode of action, so their resistant influenza virus strains have cross resistance (such as influenza virus strains resistant to amantadine are also resistant to BL-1743, J.VIROLOGY 1996, 70, 4246-4252), so it is very important to find compounds that inhibit M2 ion channels with new mechanisms of action and new frameworks.

Contents of the invention

The technical problem to be solved in the present invention is to provide a new compound capable of inhibiting and blocking M2 ion channel activity. The technical scheme that solves the above-mentioned technical problem is as follows:

Compounds in formula (I) or pharmaceutically acceptable salts or stereoisomers thereof

in,

R _is H, an acyclic hydrocarbon group containing or not containing fluorine, nitrogen, oxygen, sulfur heteroatoms, or an optionally substituted monocyclic or polycyclic hydrocarbon group;

_R2 is H, alkyl, acyl, or a structure containing at least one ring, which can be substituted arbitrarily;

R ₃ is H, OR ₇ , SR ₈ , NR ₉ R ₁₀ , or an optionally substituted chain or cyclic hydrocarbon group;

R ₄ and R ₅ are respectively H, alkyl, halogen, or a chain with an amino terminal or a cyclic hydrocarbon with an amino terminal; or R ₄ and R ₅ together form a cyclic hydrocarbon with an amino terminal;

R ₇ , R ₈ , R ₉ , and R ₁₀ are H, alkyl, aryl, or heterocyclic aryl, respectively;

m and n are integers from 0 to 6 respectively;

X is zero, S, O, NH, or _CH2 ;

Y is N, or CH.

Preferably, the compound or its pharmaceutically acceptable salt or stereoisomer has the structure of formula (II) when Y is CH:

Wherein R ₁₁ and R ₁₂ are H, alkyl or acyl respectively; Z and T are CH or N respectively.

Or preferably, when Y is CH it has the structure of formula (III):

Wherein R ₁₃ and R ₁₄ are H, alkyl or acyl respectively; the definitions of R ₁ , R ₂ and R ₃ are the same as above.

Z and T are CH or N, respectively.

Or preferably, the compound or its pharmaceutically acceptable salt or stereoisomer, when Y is N, it has the structure of formula (IV):

Wherein R ₁₅ and R ₁₆ are H, alkyl or acyl respectively; the definitions of R ₁ , R ₂ and R ₃ are the same as above;

Z and T are CH or N, respectively.

Or preferably, the compound or its pharmaceutically acceptable salt or stereoisomer, when Y is N, has the structure of formula (V):

Wherein R ₁₇ and R ₁₈ are H, alkyl or acyl respectively; the definitions of R ₁ , R ₂ and R ₃ are the same as above;

Z and T are CH or N, respectively.

Or preferably, when Y is N, it has the structure of formula (VI):

Wherein R ₁₉ and R ₂₀ are H, alkyl or acyl respectively; Z and T are CH or N respectively.

Or preferably, when Y is N, it has the structure of formula (VII):

More preferably, when _R is H it has the structure of formula (VIII):

R₁、R₂、R₃、R₄的定义与上述定义相同。

The definitions of R ₁ , R ₂ , R ₃ and R ₄ are the same as those defined above.

Specifically, the compound or its pharmaceutically acceptable salt or stereoisomer is preferably 1-(N-(1-adamantyl)-aminomethyl)histamine, or 1-(N-( 1-adamantyl)-1,2,6-hexanetriamine, or 1-(N-(1-adamantyl)-5-guanidino-1,2-pentanediamine; or N-adamantane Methylhistidine and esters, N-adamantylmethylhistidine; N-cyclopropanemethylhistidine and esters, N-cyclopentylmethylhistidine; N-cyclohexylmethylhistidine And esters, N-cyclohexylmethyl histidine amide, N-cyclohexyl histidine and esters, N-cyclohexyl histidine amide; N-cyclopentylmethyl histidine and esters, N-cyclopentyl methyl Histidine amide, N-cyclopentyl histidine and esters, N-cyclopentyl histidine amide; N-benzyl histidine and esters, N-benzyl histidine amide.

Another technical problem to be solved by the present invention is to provide the application of the above compound or its pharmaceutically acceptable salt or stereoisomer in medicine.

The technical scheme that solves the above-mentioned technical problem is as follows:

Application of the above compound or its pharmaceutically acceptable salt or stereoisomer in the preparation of influenza and avian influenza medicines.

The above compounds or pharmaceutically acceptable salts or stereoisomers thereof are used in the preparation of drugs against hepatitis C, HIV infection or fungal infection.

Another technical problem to be solved by the present invention is to provide a pharmaceutical composition.

A pharmaceutical composition, which mainly consists of the above-mentioned compound or its pharmaceutically acceptable salt or stereoisomer as an active ingredient and at least one pharmaceutically acceptable carrier.

The present invention designs and prepares a series of compounds with multiple amino structural units or their pharmaceutically acceptable salts or stereoisomers, and these rationally distributed polyamino structural units constitute multiple binding functional groups and functional regions of these compounds . These compounds may inhibit and block the activity of wild-type and mutant influenza and avian influenza M2 ion channels (proved to be an effective influenza drug target by clinical tests) through a new mechanism of action, so as to treat wild-type and mutant influenza and avian influenza. The pharmacodynamic function of influenza, and they may also be used to treat hepatitis B, hepatitis C and AIDS, so as to provide more drug options for clinical treatment.

Description of drawings

Figure 1 is a schematic diagram of the results of patch clamp detection of amantadine inhibiting M2 ion channel activity;

Fig. 2 Schematic diagram of the results of patch clamp detection of compound 2 inhibiting M2 ion channel activity.

Detailed ways

"Pharmaceutically acceptable salts" of the compounds of the present invention include conventional non-toxic salts of the compounds of the present invention formed by reacting the compounds of the present invention with inorganic or organic acids. For example, salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc. are included, as well as salts derived from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, Tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethane Salts of disulfonic acid, oxalic acid, isethionic acid, trifluoroacetic acid, etc.

Where the compound of the present invention is acidic, appropriate "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases including salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. For example, arginine, betaine, caffeine, choline, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, aminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine , Theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

The compounds of the present invention may have asymmetric centers, chiral axes and chiral faces, and exist as racemates, racemic mixtures and single diastereomers and all possible isomers and mixtures thereof Both optical isomers are included in the present invention. In addition, the compounds disclosed herein may exist as tautomers, and both tautomeric forms are included within the scope of the present invention, even if only one of the tautomeric structures is depicted. For example, any claimed compound A below is understood to include tautomeric structure B, and vice versa, as well as mixtures thereof.

When any variable (such as R ₃ , R ₄ , R ₅ , R ₆ , R _{7 ,} etc.) occurs more than once in any component, its definition for each occurrence is independent of the definition for each other occurrence. Likewise, substitutions are allowed combinations of radicals and variables provided that such combinations render the compound stable.

The term "hydrocarbyl" used in the present invention means to include C ₁ -C ₈ alkyl, C ₂ -C ₈ alkenyl, C ₂ -C ₈ alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, Heterocyclyl, heterocycloalkyl. A line drawn from a substituent into a ring system indicates that the indicated bond may be attached to any substitutable ring atom. If the ring system is polycyclic it means that such bonds are only to any suitable carbon atoms of adjacent rings. It is understood that one of ordinary skill in the art can select substituents and substitution patterns on the compounds of the present invention to provide compounds that are chemically stable and can be readily synthesized from readily available starting materials by skill in the art and by methods set forth below. If a substituent is itself substituted with more than one group, it is understood that these groups may be on the same carbon atom or on different carbon atoms, so long as the structure is stabilized.

The term "alkyl" used in the present invention is meant to include branched, linear or cyclic saturated aliphatic hydrocarbon groups having a specified number of carbon atoms. The definition of "C ₁ -C ₈ " in "C ₁ -C ₈ alkyl" includes groups having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms in a linear or branched arrangement . For example, "C ₁ -C ₈ alkyl" specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, Cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl and the like.

The term "alkenyl" refers to a linear, branched or cyclic non-aromatic hydrocarbon group containing 2-8 carbon atoms and at least one carbon-carbon double bond. Thus, " _C2 - _C8 alkenyl" refers to an alkenyl group having 2-8 carbon atoms. Alkenyl includes ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl. A linear, branched or cyclic portion of an alkenyl group may contain double bonds and such portion may be substituted if a substituted alkenyl group is indicated.

The term "alkynyl" refers to a straight-chain, branched or cyclic non-aromatic hydrocarbon group containing 2-8 carbon atoms and at least one carbon-carbon triple bond. Thus, " _C2 - _C8alkynyl " refers to an alkynyl group having 2-8 carbon atoms. Alkynyl includes ethynyl, propynyl, butynyl, 3-methylbutynyl, and the like. Straight chain, branched or cyclic moieties of an alkynyl may contain triple bonds and such moieties may be substituted if substituted alkynyl is indicated.

The term "aryl" then refers to any stable monocyclic or bicyclic carbocyclic ring of up to 7 atoms in each ring, at least one of which is aromatic. Examples of aryl groups include phenyl, naphthyl, anthracenyl and biphenyl.

The term "aralkyl" includes groups in which the above-mentioned "alkyl" is replaced by the above-mentioned "aryl". Examples of aralkyl groups include benzyl, phenethyl, naphthylmethylene, anthracenylmethylene.

The term "heteroaryl" denotes a stable monocyclic ring of up to 7 atoms in the ring or a bicyclic carbocycle of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains 1 to 4 atoms selected from the group consisting of O, N and S heteroatoms. Heteroaryl groups within the scope of this definition include, but are not limited to, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furyl, thienyl, benzene Thienyl, benzofuryl, quinolinyl, isoquinolyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl.

The term "heteroaralkyl" includes groups in which the above-mentioned "alkyl" is replaced by the above-mentioned "heteroaryl". Examples of heteroaryl groups include furyl methylene, pyridyl ethyl, pyrromethene.

The term "heterocyclic group" refers to a 5-10 membered aromatic or non-aromatic heterocyclic ring containing 1-4 heteroatoms selected from O, N and S, and includes bicyclic groups. "Heterocyclyl" thus includes the above-mentioned heteroaryl groups, as well as dihydrogenated and tetrahydrogenated analogs thereof. Further examples of "heterocyclyl" include, but are not limited to, benzimidazolyl, benzofuranyl, benzopyranyl, benzopyrazolyl, benzotriazolyl, benzothienyl, benzoxazolyl , carbazolyl, carbolinyl, cinnolinyl, furyl, imidazolyl, indolinyl, indolyl, indazolyl, isobenzofuryl, isoindolyl, isoquinolyl, iso Thiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl (oxetanyl), pyranyl, pyrazinyl, pyrazolyl, pyridazine Base, pyridopyridyl, pyridazinyl, pyridyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydropyranyl, tetrazolyl, tetrazolopyridyl, Thiadiazolyl, thiazolyl, thienyl, triazolyl, azetidinyl, 1,4-dioxanyl, azepanyl (hexallydroazepinyl), piperazinyl, piperidinyl, pyridine -2-keto, pyrrolidinyl, morpholinyl, thiomorpholinyl (thiomorpholinyl), dihydrobenzimidazolyl, dihydrobenzofuranyl, dihydrobenzothienyl, dihydrobenzoxazole Dihydrofuryl, dihydroimidazolyl, dihydroindolyl, dihydroisoxazolyl, dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl, dihydropyrazinyl, Dihydropyrazolyl, dihydropyridyl, dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl, dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl, dihydrothienyl, Dihydrotriazolyl, dihydroazetidinyl, methylenedioxybenzoyl, tetrahydrofuranyl and tetrahydrothiophenyl and their N-oxides. Attachment of heterocyclic substituents can be via carbon atoms or via heteroatoms.

The term "polycyclic" includes structures in which at least 2 rings are formed, among which at least two rings are spiro rings with 1 common atom between them or amalgamated (joined) rings or bridges with at least 2 common atoms ( across) ring structure. Typical structures such as the basic structures of adamantane, norbornene, norbornene, pinane, (iso) campane, carane, etc. These rings may or may not have substituent structures, and these polycyclic structures may be heteropolycyclic.

The term "heterocyclic group" includes groups in which the above-mentioned "alkyl group" is substituted with the above-mentioned "heterocyclic group". Examples of heterocyclyl groups include imidazolidine, pyrrolidinylethyl, tetrazolymethylene.

The term "halogen" is meant to include fluorine, chlorine, bromine and iodine.

The term "pKa" means the negative logarithm of the equilibrium constant formed by the dissociation of a protonated nitrogen from a proton therein. Examples of "hydrocarbyl with pKa not less than 4" include, but are not limited to, substituted or unsubstituted amino, pyrrolidinyl, quinuclidinyl, piperidinyl; pyridyl, quinoline, isoquinoline, imidazolyl.

The target compound is prepared by ammoniation reduction of the corresponding aldehyde with a suitable ammonia (amine) or reduction after amide formation of carboxylic acid and carboxylic acid ester with ammonia (amine). Used raw material aldehyde, carboxylic acid or ester, ammonia (amine) etc. except that market buys, following is corresponding basic preparation method and principle, those skilled in the art can prepare described any kind of compound according to the present invention:

1) The preparation of aldehydes, carboxylic acids or esters can be synthesized according to the needs of the target compound, such as a series of conversions starting from amino acids to form corresponding aldehydes; alcohols can be oxidized into corresponding carboxylic acids and can be converted into corresponding esters; through carboxylic acids or Esters are partially reduced to aldehydes, or carboxylic acids or esters are completely reduced to alcohols and then oxidized to aldehydes.

2) The preparation of ammonia (amine) can be converted from corresponding alcohols, halides, aldehydes, carboxylic acids, amides, etc.

Example 1. N-adamantylmethylhistidine methyl ester (compounds 2 and 3)

Take 0.43g (11.3mmol) of lithium aluminum hydride and place it in a 50ml two-necked bottle, add 15ml of anhydrous diethyl ether, stir in an ice bath for 10min, slowly add a THF solution of methyl 1-adamantanecarboxylate (1.62g, 8.4mmol soluble In 3.5mlTHF), the temperature was raised to reflux for 5h after the drop. After the reaction was completed, put it in an ice bath, slowly add 0.5ml H2O, 0.5ml 15% NaOH solution, and _1.5mlH2O dropwise in sequence, continue stirring for 30min, return to room temperature, filter, wash the filter cake with ether, combine the organic phases, anhydrous Na2SO4 dried, filtered, concentrated and dried to obtain 1.3 g of 1-adamantanemethanol (yield 92.8%).

Take 1 g (6 mmol) of 1-adamantanemethanol, dissolve it in 50 ml CH ₂ Cl ₂ , add 2.6 g (12 mmol) of PCC under ice-cooling, stir for 10 min to return to room temperature, and continue the reaction for 1.5 h. After the reaction was completed, ethyl acetate was slowly added in an ice bath, the clear liquid was passed through a 60-100 thick silica gel column, the filtrate was collected, concentrated and dried to obtain 0.86 g of 1-adamantanecarbaldehyde (compound 1) (yield 87.6%).

Dissolve 0.09g (0.53mmol) of L-histidine methyl ester or D-histidine methyl ester in 5ml MeOH, add 0.106g (0.65mmol) of 1-adamantanecarbaldehyde, stir at room temperature for 1.5h, and Add 0.07 g (1.11 mmol) of NaBH ₃ CN, return to room temperature, and perform TLC until the reaction is completed. Slowly add saturated NaHCO ₃ solution under ice bath, extract three times with CH2Cl2, combine the organic phases, dry over anhydrous Na ₂ SO ₄ , filter and concentrate, and separate on a silica gel column (MeOH/CH ₂ Cl ₂ =2/98) to obtain N-adamantane Methyl histidine methyl ester (compound 2 or 3) 0.126 g (31% yield).

Example 2. N-myrtenene methyl histidine methyl ester (compound 4)

Take D-histidine methyl ester 0.15g (0.89mmol) dissolved in 7ml MeOH, add myrtenal ((-)-Myrtenal) 0.5mmol, stir at room temperature for 1.5h, add NaBH ₃ CN 0.25 g (3.98mmol), return to room temperature, TLC until the reaction is complete. Slowly add saturated NaHCO ₃ solution under ice bath, extract three times with CH ₂ Cl ₂ , combine the organic phases, dry over anhydrous Na ₂ SO ₄ , filter and concentrate, and separate on a silica gel column (MeOH/CH ₂ Cl ₂ =5/95) to obtain N - 50 mg of myrtenyl methyl histidine methyl ester (compound 4) (yield 40%).

Example 3. N-cyclopropanemethyl histidine methyl ester (compound 5)

Dissolve 0.15g (0.89mmol) of histidine methyl ester in 7ml of MeOH, add 0.07ml (0.94mmol) of cyclopropaneformaldehyde, stir at room temperature for 1.5h, add 0.25g (3.98mmol) of NaBH3CN under ice bath, return to room temperature , TLC until the end of the reaction. Slowly add saturated NaHCO ₃ solution under ice bath, extract three times with CH ₂ Cl ₂ , combine the organic phases, dry over anhydrous Na ₂ SO ₄ , filter and concentrate, and separate on a silica gel column (MeOH/CH ₂ Cl ₂ =5/95) to obtain N - Cyclopropanemethylhistidine methyl ester (compound 5) 0.025 g (yield 12.6%).

Example 4.2-N-substituted histidine methyl ester compound (compound 6)

Dissolve 0.36mmmol of L-histidine methyl ester in 10ml methanol, add 0.43mmol (1.2eq) of aldehyde, add NaBH3CN27mg (0.43mmol, 1.0eq) after 2h, react at room temperature for 8h, concentrate, and purify by silica gel column chromatography ( _{CH2Cl 2} /CH ₃ OH) to obtain 50 mg of 2-N-substituted histidine methyl ester compound ie compound 6. Yield 32%.

Example 5. N-Adamantylmethylhistidine (Compounds 7 and 8)

Suspend 0.5mmol L-histidine or D-histidine hydrochloride in 5ml methanol, add 0.55mmol triethylamine and 0.6mmol adamantanal (2) successively, and stir the reaction system at room temperature for 5 hours, carefully After degassing, a catalytic amount of Pd/C is added, and then filled with hydrogen. The reductive amination reaction takes about 8 hours to complete, the catalyst is removed by filtration, and silica gel column chromatography (MeOH/DCM 5% to 15%) gives N-adamantylmethylhistidine (compounds 7 and 8) (53% yield) .

Example 6.1-(N-(1-adamantyl)-aminomethyl) histidine compounds

Take 2g (12.90mmol) of L-histidine or D-histidine and dissolve it in 10ml of saturated NaHCO ₃ , then add 30ml of dioxane, and slowly add 7.0g (32.25mmol) of Boc ₂ O in an ice-water bath. 10ml dioxane dissolved), reacted at room temperature for 24 hours. After the reaction, wash with 3×60ml petroleum ether, adjust the pH value of the aqueous phase to 3-4 with saturated NaHSO ₄ aqueous solution, extract with 3×60ml ethyl acetate, dry over anhydrous Na ₂ SO ₄ , filter and concentrate, and use a silica gel column ( Methanol/dichloromethane=1/20, 300 mL eluent plus 1 mL formic acid) isolated 4.27 g of product 9 or 10 (93% yield).

Take 1.59g (8.45mmol) of amantadine hydrochloride and put it into 100ml of dichloromethane, add 3ml of triethylamine, and after 30 minutes, put in 3g (8.45mmol) of product 9 or 10, 2.72g of TBTU (8.47mmol) and 1ml of N -Methylmorpholine, reacted at room temperature for 18 hours. After the reaction was completed, the mixture was concentrated through a column and separated by silica gel column chromatography (methanol/dichloromethane=1/30) to obtain 1.9 g of product 11 or 12 (yield 87%).

Take 0.9g of 11 or 12 (1.84mmol) and dissolve it in 20ml of dichloromethane, inject 1ml of TFA into the ice bath, after the addition, move the reaction to room temperature and continue to stir for 5 hours. After the reaction was completed, the mixture was concentrated and separated on a silica gel column (methanol/dichloromethane=1/15, 100 ml of eluent plus 1 ml of ammonia water) to obtain 0.51 g of product 13 or 14 (93% yield).

Take 0.1g (0.347mmol) of 13 or 14 and 0.35g (0.868mmol) of Lawesson reagent into 20ml of benzene, heat to reflux for 20h; evaporate the solvent and add 20ml of 80% tert-butanol (20% water content) and 1.0g of Raney Ni , After heating to reflux for 8h. After the reaction was completed, it was concentrated by filtration and separated on a silica gel column (methanol/dichloromethane=1/5) to obtain 0.072 g of the product 15 or 16 (the two-step yield was 76%).

Dissolve 0.5g of 15 or 16 (1.82mmol) in 20ml of water, inject 2ml of triethylamine, and slowly add 0.80g of Boc ₂ O (3.6mmol, dissolved in 3ml of dioxane) dropwise in an ice-water bath. Moved to room temperature to continue the reaction for 20h. After the reaction, wash with 3×50ml petroleum ether, extract the aqueous phase with 3×50ml ethyl acetate, dry over anhydrous Na ₂ SO ₄ , filter and concentrate, and separate on a silica gel column (methanol/dichloromethane=1/15) to obtain 0.76 g product 19 or 20 (88% yield).

Dissolve 0.45g of 19 or 20 (0.949mmol) in 20ml of methanol, add 0.2g of NaHCO ₃ (2.3mmol) and 0.674g of iodomethane (4.74mmol), and react at room temperature for 20h. After the reaction was completed, it was concentrated and separated on a silica gel column (methanol/dichloromethane=1/20) to obtain 0.41 g of product 21 or 22 (yield 89%).

Take 0.2g of 21 or 22 (0.41mmol) and dissolve it in 10ml of dichloromethane, inject 1ml of TFA in an ice bath, drop it and move it to room temperature to continue the reaction for 3h. After the reaction was completed, it was concentrated and separated on a silica gel column (methanol/dichloromethane=1/10, 100 ml of eluent plus 1 ml of ammonia water) to obtain 0.11 g of the final product 23 or 24 (94% yield).

Example 7.2-(N-adamantylmethyl)-arginine ester

Take 25 1g (1.54mmol) and dissolve it in 40ml of dichloromethane, add 2.3ml of piperidine, and stir at room temperature for 40min. After the reaction was completed, most of the solvent was evaporated, 40ml of water was added, extracted with 4×40ml petroleum ether, the aqueous layer was extracted with 4×40ml ethyl acetate, the ethyl acetate layer was collected, dried over anhydrous Na ₂ SO ₄ , concentrated, Silica gel column (methanol/dichloromethane=1/15) yielded 0.59 g of product 26 (yield 91%).

Dissolve 260.852g (2.0mmol) in 30ml methanol, add 0.361g AdCHO (2.2mmol) and 3g anhydrous Na ₂ SO ₄ , react at room temperature for 25h, then add 0.69g sodium cyanoborohydride (10mmol) in an ice-water bath, After 20 min, move to room temperature and stir overnight. After the reaction, silica gel column (methanol/dichloromethane=1/15) was used to obtain 1.02 g of product 27 (yield 89%).

Take 270.7 g (1.18 mmol) and dissolve it in 20 ml of methanol, add a catalytic amount of DMAP and 6 ml of thionyl chloride, and heat to reflux for 16 h after 5 min. After the reaction was completed, it was filtered, and 20 ml of ethyl acetate was added for recrystallization to obtain 0.267 g of product 28 (yield 65%).

Example 8.5-(N-adamantylamino)-2-aminovalerate compounds (compounds 31 and 32)

Dissolve 291.0g (2.9mmmol) in 50ml methanol, add 0.65g (3.47mmol, 1.2eq) of adamantane hydrochloride, add 2.0g (14.5mmol, 5eq) of ZnCl ₂ in methanol solution under ice-cooling, and move to room temperature for 2h Then add NaBH ₃ CN 220mg (3.47mmol, 1.0eq), add 10ml water after 4h, NaHCO ₃ saturated solution, ethyl acetate extraction, saturated brine wash, anhydrous Na ₂ SO4 dry, silica gel column chromatography (with 2% ammonia water 1.1 g of compound 30 was obtained by elution with saturated solution of CH ₂ Cl ₂ /CH ₃ OH. Yield 79%.

Compound 301.0g (2.1mmmol) was dissolved in 30ml of anhydrous CH ₂ Cl ₂ , protected by N2, TFA was added dropwise under ice-cooling, and the reaction was complete after moving to room temperature for 4 hours, concentrated to dryness, and purified by silica gel column chromatography (with 2% ammonia water) CH ₂ Cl ₂ /CH ₃ OH saturated liquid elution) to obtain compound 310.55g. Yield 94%.

100mg (0.36mmmol) of compound 31 was dissolved in 10ml of methanol, AdCHO 65mg (0.39mmol, 1.1eq) was added, and NaBH ₃ CN 23mg (0.36mmol, 1.0eq) was added after 2h, reacted at room temperature for 8h, concentrated, purified by silica gel column chromatography (CH ₂ Cl ₂ /CH ₃ OH) to obtain 65 mg of compound 32. Yield 42%.

Example 9.2-(N-adamantyl methyl) lysine ester (compound 35) and 2,6-two (N-adamantyl methyl) lysine ester (compound 36)

Fmoc-Lys(Boc)-OH, 1.00g was placed in a 100ml reaction flask, vacuumed and injected with nitrogen, then injected with 50ml redistilled CH ₂ Cl ₂ and 2ml piperidine, and stirred at room temperature. After two hours the reaction was complete. CH ₂ Cl ₂ was extracted twice, and the combined organic phases were washed twice with saturated NaCl solution. Dry over anhydrous NaSO ₄ , mix 3.0 g of crude silica gel (60-100 mesh) after desolventization, and purify by silica gel column chromatography (elution polarity: CH ₃ OH:CH ₂ Cl ₂ , 1:3) to obtain 480 mg of white crystals Namely compound 33, the yield is 91.4%.

Take 900mg of compound 33 and 500mg of AdCHO in a 100ml one-necked flask, inject 30ml of CH ₃ OH, and stir overnight at room temperature. On the next day, 194 mg of NaBH ₃ CN was weighed and dissolved in 5 ml of CH ₃ OH, and then slowly injected into the reaction system. After two hours, it was checked that the reaction was complete. The system was diluted with water and extracted three times with CH ₂ Cl ₂ to combine the organic phases. Dry over anhydrous NaSO ₄ . After desolventization, mix the sample with crude silica gel and purify by silica gel column chromatography (elution polarity: CH ₃ OH:CH ₂ Cl ₂ , 1:6). 730 mg of white crystal 34 was obtained with a yield of 50.6%.

Weigh 100 mg of compound 34 into a 25 ml two-neck flask, inject 10 ml CH ₃ OH, slowly inject 1.5 ml SOCl ₂ , and heat to reflux for 12 hours to stop the reaction. The system was directly spin-dried to remove residual SOCl ₂ , and purified by silica gel column chromatography (elution polarity: CH ₃ OH:CH ₂ Cl ₂ , 1:6). 60 mg of light yellow crystals, that is, compound 35, was obtained with a yield of 77%.

The preparation of compound 36 was similar to the preparation of 34.

Example 10.2-(N-adamantylmethyl) glutamate diester (compound 37)

Dissolve 100mg (0.57mmol) of dimethyl glutamate in 10ml of methanol, add 103mg of AdCHO (0.63mmol, 1.1eq), add NaBH ₃ CN 36mg (0.57mmol, 1.0eq) after 2h, react at room temperature for 6h, and then concentrate on silica gel Purification by column chromatography (eluted with CH ₂ Cl ₂ /CH ₃ OH) yielded 83 mg of compound 37 with a yield of 45%.

Example 11. 5-(N-adamantylamino)-2-(N-myrtenylamino)valeric acid ester compounds (compound 38)

Dissolve 100mg (0.36mmmol) of compound 31 in 10ml methanol, add (-)-Myrtenal ((-)-Myrtenal) 64mg (0.43mmol, 1.2eq), add NaBH ₃ CN 27mg (0.43mmol, 1.0eq) after 2h, After reacting at room temperature for 8 h, it was concentrated and purified by silica gel column chromatography (eluted with CH ₂ Cl ₂ /CH ₃ OH) to obtain 51 mg of compound 38 with a yield of 35%.

Example 12. 5-(N-adamantylamino)-2-(N-cycloalkyl)valeric acid ester compounds (compound 39)

Dissolve 100mg (0.36mmmol) of compound 31 in 10ml methanol, add 0.43mmol (1.2eq) of cyclohexanone, add NaBH ₃ CN 27mg (0.43mmol, 1.0eq) after 2h, react at room temperature for 8h, concentrate, and purify by silica gel column chromatography ( CH ₂ Cl ₂ /CH 3 OH (eluted with CH 2 Cl 2 /CH ₃ OH) afforded 45 mg of compound 39, yield 34%.

Example 13.5-(N-adamantylamino)-2-(N-cycloalkyl)valeric acid ester compound (compound 40)

Dissolve 100mg (0.36mmmol) of compound 31 in 10ml of methanol, add 0.43mmol (1.2eq) of cyclopentanone, add NaBH ₃ CN 27mg (0.43mmol, 1.0eq) after 2h, react at room temperature for 8h, concentrate, and purify by silica gel column chromatography ( CH ₂ Cl ₂ /CH 3 OH (eluted with CH 2 Cl 2 /CH ₃ OH) afforded 40 mg of compound 40 with a yield of 33%.

Example 14. 5-(N-adamantylamino)-2-(N-cycloalkyl)valeric acid ester compounds (compound 41)

Dissolve 100mg (0.36mmmol) of compound 31 in 10ml of methanol, add 0.43mmol (1.2eq) of aldehyde, add NaBH ₃ CN 27mg (0.43mmol, 1.0eq) after 2h, react at room temperature for 8h, concentrate, and purify by silica gel column chromatography (CH ₂ Cl ₂ /CH ₃ OH (elution) yielded 50 mg of compound 41 with a yield of 32%.

Example 15. Inhibitory activity of compounds on wild-type and mutant H5N1-M2 ion channels

1. Construction of cell lines stably expressing wild-type and mutant H5N1-M2 ion channels, see literature (Sun, J.; Li, C.; Xu, W.; Li, Z.; Liu, J.; Chen, L. Chin J Biotech 2008, 24, 1902).

2. Patch clamp detection of the ability of some compounds to block H5N1-M2 ion channels

After 293T-rex cells expressing H5N1-M2 ion channels were induced by 1 μg/ml tetracycline for 24-48 hours, the current changes were recorded using patch clamp (Axopatch 200B) at room temperature (refer to Li, Z. et al. J Neuroscience 2004, 24, 7378-7386). Dosing and pH adjustment use an automatic dosing system.

Please refer to Table 1. The results of the patch clamp experiment show that the half inhibitory activity of amantadine, the positive drug for influenza wild-type H5N1-M2 ion channel, has an IC ₅₀ of 4.2 μM, but amantadine has no inhibitory effect on influenza variant M2 ion channel ; The new compound 2 has a good inhibitory activity against the wild type and the mutant type of influenza, and the activity is equivalent, and the half inhibitory activity IC ₅₀ is 5-10 μ M; The new compound 5 has certain inhibitory activity against the wild type and the mutant type of influenza, and the activity Correspondingly, the IC ₅₀ of the half inhibitory activity is 0.3-0.4mM; the new compound 13 has a certain inhibitory activity on the wild type.

Table 1. Some compounds inhibit the activity of wild-type and mutant H5N1-M2 ion channels

NA means no inhibitory activity; ND means not detected

Figure 1 and Figure 2 are the results of patch clamp detection of compound inhibition of H5N1-M2 ion channel activity, where (A) is to maintain the potential difference between the inside and outside of the cell membrane at 40mV, change the extracellular pH from 6.8 to 5.5, express wild-type, amantadine Membrane current changes in 293T-rex cells resistant to M2 ion channels of H5N1-S31N and H5N1-L26I/S31N. The blue line is the control, and the red line is the current after using the drug; the amount of drug used has been marked. (B) is the relationship between the dosage of the compound and the inhibition rate, and the number of repetitions of the data is 4-6.

Example 16. Compound anti-M2 ion channel wild-type and variant influenza virus H1N1 activity

Cells: MDCK dog kidney cells; Culture medium: DMEM+10%FBS; Maintenance solution: DMEM+2%FBS; Viruses: H1N1 influenza virus A/PR/8/34 (M2 wild type) and A/WS/33 ( M2S31N variant).

1. Determination of virus virulence (TCID50 of virus virulence is ^10-5..5 )

MDCK cells were inoculated in a 96-well plate at 5×10 ⁴ /well. After the cells grew into a single layer, 100 μl of 10-fold serially diluted virus solution of 6 concentrations (the original virus solution was derived from chicken embryo proliferation and diluted in DMEM) was added to absorb 1 After -2 hours, pour out the virus, wash it twice with PBS, add maintenance solution and culture, observe the cytopathic changes (CPE) every day with an inverted microscope, measure the hemagglutination titer and observe the cell viability and virus amplification by MTT staining method, The median infectious dose (TCID50) of the virus was calculated by the Reed-Muench method, and 8 replicate wells were set up for each concentration, and the experiment was repeated to determine the virulence.

2. Antiviral experiment

After obtaining the TCID50 of the virus and the CC0 of the drug, the antiviral activity was screened on MDCK cells, and the cells were seeded in a 96-well plate at 5×10 ⁴ /well. After the cells grew into a monolayer, the experiment was carried out in the following two ways:

(1) In the drug-adding group (referred to as group A) after attacking the virus first, the drug (2-fold dilution, generally starting from the maximum non-toxic concentration) and the virus (100TCID50) were added to the cells at the same time to start the infection. After 2 hours of adsorption, the virus was poured out. Wash twice with PBS, and then add 200 μl of maintenance solution containing the same drug.

(2). In the poison and drug simultaneous action group (referred to as group B), add 100 μl (100 TCID50) virus infection to each well, and after 2 hours of adsorption, pour out the virus, wash twice with PBS, and then add 200 μl 2-fold gradient dilution to be prepared. Test drugs.

A virus control, a cell control and a positive drug control ("Rabavirin", Amantadine, Tamiflu) were set up in the test. The test was terminated when more than 75% of the lesions appeared in the virus control wells. The pathogenic effect (CPE) on the cells was observed with an inverted microscope (24, 48, 72). At the same time, the supernatant of the culture medium was taken at 24 hours and 48 hours to measure the hemagglutination titer, and the MTT test was carried out 72 hours later. The half effective concentration (EC50) was calculated by Reed-Muench method.

The research results of inhibiting H1N1 virus activity showed that: 1) The inhibitory activity of compounds on H1N1 was related to the order of compound addition, and compound 35 was less affected by the time of adding drugs; 2) Compounds 32, 35, 38, and 41 had better inhibition of M2 wild-type and mutant H1N1 influenza virus activity.

Table 2. Anti-M2 ion channel wild-type and mutant influenza virus H1N1 activities of some compounds

H1N1-M2 wild-type virus: A/PR/8/34; H1N1-M2 variant virus: A/WS/33.

Claims (3)

1. A compound having the structural formula or a pharmaceutically acceptable salt or stereoisomer thereof,

2. the compound of claim 1 or a pharmaceutically acceptable salt or a stereoisomer thereof, as a substance with the activity of inhibiting and blocking M2 ion channel, and the application thereof in preparing influenza and avian influenza drugs.

3. A pharmaceutical composition consisting essentially of a compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof as an active ingredient in combination with at least one pharmaceutically acceptable carrier.

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