Artificial water channels with amino acid modifications and methods of making and uses thereof

Abstract

The present invention relates to an amino acid-modified compound comprising a pillararene moiety, a linker having a divalent triazole ring, and an amino acid-modified group, a method for preparing the same, and a use thereof. The amino acid-modified compound of the present invention can be obtained in a simple and high-yield synthesis method, and the amino acid-modified compound is capable of spontaneously fusing into a cell membrane, exhibits high water transport efficiency and selectivity, is capable of significantly accelerating cell migration, and has excellent eardrum repair ability, and has low ototoxicity.

Description

Technical Field

[0001] This disclosure relates to an amino acid-modified compound comprising a columnar aromatic moiety, a linking group having a divalent triazole ring, and an amino acid-modifying group, a method for preparing the compound, and its uses. Background Technology

[0002] Pillar aromatics are a class of macrocyclic compounds, which are cyclic oligomers formed by the para-linking of hydroquinone or hydroquinone ethers through methylene bridges on the benzene ring. Since their discovery, pillar aromatics have been used in liquid crystals, nanoparticle synthesis, and sensing.

[0003] Aquaporins are a class of naturally occurring channel proteins responsible for the passive and selective transport of water across cell membranes. Phenotypic analysis has shown that aquaporins participate in a variety of important physiological and pathophysiological functions. The crucial functions of aquaporins in biological systems have inspired chemists to develop artificial analogues. Summary of the Invention

[0004] In view of the above-mentioned state of the prior art, the inventors of this invention have conducted extensive and in-depth research in the field of columnar aromatics in order to discover a columnar aromatic compound with excellent performance. The inventors have discovered an amino acid-modified compound comprising a columnar aromatic structural moiety, a linking group having a divalent triazole ring, and an amino acid-modifying group. The synthesis method of the amino acid-modified compound is simple and can be obtained in high yield, and the amino acid-modified compound exhibits excellent tympanic membrane repair ability.

[0005] One aspect of the present invention provides an amino acid-modified compound comprising a columnar aromatic moiety, a linking group having a divalent triazole ring, and an amino acid-modifying group.

[0006] In the columnar aromatic hydrocarbon structure, the oxygen atom directly attached to the benzene ring all carries an amino acid modifying group.

[0007] The amino acid modifying group is connected to the oxygen atom in the columnar aromatic hydrocarbon structural portion via a linking group having a divalent triazole ring.

[0008] The amino acid modifying group has only a single amino acid or is a short peptide comprising two or three amino acids, wherein the terminal amino acid in the amino acid modifying group, i.e., the single amino acid and the terminal amino acid of the short peptide, is a hydrophobic amino acid, and wherein the amino acid modifying group carries a positive charge.

[0009] The column aromatic structure portion is column (5) aromatic structure portion, column (6) aromatic structure portion or column (7) aromatic structure portion.

[0010] Another aspect of the present invention provides a method for preparing the amino acid-modified compound.

[0011] Another aspect of the invention provides the use of the amino acid-modified compound. Attached Figure Description

[0012] Figure 1 The changes in light scattering intensity over time after liposomes were exposed to a hypertonic solution of 100 mM KCl in the presence of different concentrations of compound channel 1 were shown.

[0013] Figure 2 The TAMRA images in the image represent confocal laser scanning microscope images of cells cultured in a medium containing the compound Channel 1 labeled with 5(6)-carboxy-tetramethylrhodamine. Figure 2 The DiO images in the image represent confocal laser scanning microscope images of cells cultured in a medium containing the fluorescent membrane tracer DiO. Figure 2 The BF picture in the image indicates that it is a public event; Figure 2 The Merge image in the image represents the superposition of images of channel 1, DiO, and BF of the 5(6)-carboxy-tetramethylrhodamine-labeled compound.

[0014] Figure 3 The effect of compound channel 1 on in vivo tympanic membrane (TM) perforation healing was demonstrated. Detailed Implementation

[0015] This article discloses specific values ​​of relevant features (including endpoint values ​​of the disclosed range), which can be combined to form new ranges.

[0016] One aspect of the present invention relates to an amino acid-modified compound comprising a columnar aromatic moiety, a linking group having a divalent triazole ring, and an amino acid-modifying group.

[0017] In the columnar aromatic hydrocarbon structure, the oxygen atom directly attached to the benzene ring all carries an amino acid modifying group.

[0018] The amino acid modifying group is connected to the oxygen atom in the columnar aromatic hydrocarbon structural portion via a linking group having a divalent triazole ring.

[0019] The amino acid modifying group has only a single amino acid or is a short peptide comprising two or three amino acids, wherein the terminal amino acid in the amino acid modifying group, i.e., the single amino acid and the terminal amino acid of the short peptide, is a hydrophobic amino acid, and wherein the amino acid modifying group carries a positive charge.

[0020] The column aromatic structure portion is column (5) aromatic structure portion, column (6) aromatic structure portion or column (7) aromatic structure portion.

[0021] In this paper, the columnar aromatic structure can have the following formula:

[0022]

[0023] The variables, such as n1 and R, are as follows: 5 And m are defined below.

[0024] In one embodiment, the linking group has 3-8 carbon atoms, 3-6 carbon atoms, or 3 or 4 carbon atoms in addition to the divalent triazole ring. In another embodiment, the linking group includes an additional N atom in addition to the carbon atoms and the triazole ring, preferably the additional N atom is located at the end of the linking group (i.e., the terminal N atom), and the amino acid modifying group is attached to the terminal N atom of the linking group.

[0025] In this paper, the head of the linking group is connected to the oxygen atom in the columnar aromatic structure, and the tail of the linking group is connected to the amino acid modification group.

[0026] In one embodiment, the linking group, in addition to the triazole ring and the terminal N atom, comprises one or more heteroatoms selected from N, O, and S. In another embodiment, the linking group, in addition to the triazole ring and the terminal N atom, does not contain heteroatoms.

[0027] In one embodiment, the linking groups on the same side of the column aromatic hydrocarbon are of the same length. Equal linking group length means that the number of atoms on the backbone chain of the linking group (excluding side groups) is the same, for example, -CH2-CH(CH3)-CH2- has 3 atoms on its backbone chain.

[0028] In one embodiment, the lengths (i.e., the number of atoms on the backbone chain) of the linking groups on the same side of the columnar aromatic hydrocarbon are the same, and the lengths of the linking groups on both sides of the columnar aromatic hydrocarbon differ by no more than 3, preferably no more than 2, more preferably no more than 1 atom, for example, they are equal.

[0029] In one embodiment, the linking group contains no heteroatoms except for the triazole ring and the terminal N atom, and the linking groups are of uniform length.

[0030] The detailed definition of the divalent triazole ring is as follows: [R...] 6 The group is described in detail.

[0031] In one embodiment, the linking groups in the amino acid-modified compound are identical.

[0032] In one embodiment, the amino acid modifying groups on the same side of the column aromatic hydrocarbon are identical.

[0033] In one embodiment, the amino acid modifying groups are the same.

[0034] In one embodiment, the amino acid modifying groups are the same and the linking groups are the same.

[0035] Further definitions of the amino acid modifying groups are given below.

[0036] According to the present invention, the amino acid-modified compound has counterions, the number of which is selected such that the entire molecule is uncharged. The counterions in the amino acid-modified compound are detailed below.

[0037] In one embodiment, the amino acid-modified compound has formula (I):

[0038]

[0039] in

[0040] n1 is 5, 6, or 7;

[0041] R 1 It is an alkylene group having 2, 3, 4 or 5 carbon atoms;

[0042] R 2 It is an alkylene group having 1, 2, or 3 carbon atoms;

[0043] R 3 It is an alkylene group having 2, 3, 4 or 5 carbon atoms;

[0044] R 4 It is an alkylene group having 1, 2, or 3 carbon atoms;

[0045] Where R 1 R 2 R 3 and R 4 The alkylene groups may optionally be separated by one or more non-adjacent heteroatoms selected from N, O, and S;

[0046] R 5 It is bromine or C1-C2 alkyl;

[0047] R 6 It is a divalent triazole ring;

[0048] m is an integer between 0 and 2;

[0049] z is an integer, which is chosen so that the entire molecule is uncharged;

[0050] Z is the counter ion; and

[0051] Each R can be the same or different and can be an amino acid modification group as defined above.

[0052] In one implementation, m is 0, 1, or 2. In one implementation, R 5 It is either bromine or methyl.

[0053] In one implementation, the variables in equation (I) are defined as follows:

[0054] n1 is 5, 6, or 7;

[0055] R 1 It is an alkylene group having 2, 3, 4 or 5 carbon atoms;

[0056] R 2 It is an alkylene group having 1, 2, or 3 carbon atoms;

[0057] R 3 It is an alkylene group having 2, 3, 4 or 5 carbon atoms;

[0058] R 4 It is an alkylene group having 1, 2, or 3 carbon atoms;

[0059] m is 0;

[0060] R 6 It is a divalent triazole ring;

[0061] z is an integer, which is chosen so that the entire molecule is uncharged;

[0062] Z is the counter ion; and

[0063] Each R is identical and is an amino acid modification group as defined above.

[0064] In one implementation, m is 0 and n1, R 1 R 2 R 3 and R 4 The following definition applies: n1 is 5 or 6, preferably 5;

[0065] R 1 It is an alkylene group having 2, 3 or 4, preferably 2 or 3, more preferably 2 carbon atoms;

[0066] R 2 It is an alkylene group having one or two, preferably one, carbon atom;

[0067] R 3 It is an alkylene group having 2, 3 or 4, preferably 2 or 3, more preferably 2 carbon atoms;

[0068] R 4 It is an alkylene group having one or two, preferably one, carbon atom.

[0069] In one implementation, R 1 R 2 R 3 and R 4 The alkylene group in R is linear. In a preferred embodiment, R 1 and R 3 Same as R 2 and R 4 same.

[0070] In one embodiment, R on the same side of the column aromatic hydrocarbon 1 The number of atoms on the backbone chain plus R 2 The sum (x) of the number of atoms on the backbone chain and the number of amino acid molecules in R is equal.

[0071] In one embodiment, R on the same side of the column aromatic hydrocarbon 3 The number of atoms on the backbone chain plus R 4 The sum (y) of the number of atoms on the backbone chain and the number of amino acid molecules in R is equal.

[0072] In one implementation, the absolute value of the difference between x and y does not exceed 3, preferably not more than 2, and more preferably not more than 1, for example, x equals y.

[0073] In this article, R 1 R 2 R 3 and R 4 The number of atoms on the backbone chain does not include any side groups (if any), for example, the group -CH2-CH(CH3)-CH2- has 3 atoms on the backbone chain.

[0074] In one implementation, R 6 It is a divalent 1H-1,2,3-triazole ring. In a preferred embodiment, the divalent 1H-1,2,3-triazole ring is linked to the remainder of an amino acid-modified compound via positions 1 and 4, such as R of compound (I). 1 and R 3 The compound of formula (I) is attached to the 1-position of a divalent 1H-1,2,3-triazole ring and R 2 and R 4 It is attached to the 4-position of the divalent 1H-1,2,3-triazole ring, or to the R of the compound of formula (I). 1 and R 3 The compound of formula (I) is attached to the 4-position of a divalent 1H-1,2,3-triazole ring at the same position as R. 2 and R 4 It is attached to the 1-position of the divalent 1H-1,2,3-triazole ring.

[0075] In a preferred embodiment, R6 It has the following structure (1H-1,2,3-triazole ring):

[0076]

[0077] In one implementation, m is 0 and n1, R 1 R 2 R 3 R 4 and R 6 The following definition applies: n1 is 5 or 6, preferably 5;

[0078] R 1 It is an alkylene group having 2, 3 or 4, preferably 2 or 3, more preferably 2 carbon atoms;

[0079] R 2 It is an alkylene group having one or two, preferably one, carbon atom;

[0080] R 3 It is an alkylene group having 2, 3 or 4, preferably 2 or 3, more preferably 2 carbon atoms;

[0081] R 4 It is an alkylene group having one or two, preferably one, carbon atom.

[0082] R 6 It has the following structure:

[0083] Especially the R of compound (I) 1 and R 3 The R of compound (I) is attached to the 1-position of the divalent 1H-1,2,3-triazole ring. 2 and R 4 It is attached to the 4-position of the divalent 1H-1,2,3-triazole ring.

[0084] In one embodiment, the terminal amino acid of the amino-modifying group is selected from tryptophan, phenylalanine, valine, leucine, isoleucine, alanine, proline, or methionine, preferably tryptophan or phenylalanine, and most preferably tryptophan.

[0085] In one embodiment, the terminal amino acid of the amino-modifying group carries a positive charge.

[0086] In one embodiment, the amino group of the amino acid modifying group carries a positive charge, or the amino acid modifying group carries a -NH3 group. + Group or -NH2 + - group, preferably the amino group is a terminal amino group of an amino group in which the amino group has a positive charge or is -NH3. + Group or -NH2+ - group.

[0087] In a preferred embodiment, the amino acid modifying group is a short peptide comprising two or three, preferably two amino acids.

[0088] In one embodiment, the amino acids other than the terminal amino acid in the short peptide are selected from hydrophobic amino acids and glycine, more preferably from tryptophan, phenylalanine, valine, leucine, isoleucine, alanine, proline, methionine and glycine, even more preferably from leucine, isoleucine and glycine, and most preferably leucine.

[0089] In a preferred embodiment of the invention, when the amino acid modifying group is a short peptide comprising two or three amino acids, the amino acids in the short peptide are linked alternately in D- and L-forms. For example, when the short peptide has two amino acids, the two amino acids are linked in DL or LD; when the short peptide has three amino acids, the three amino acids are linked in DLD or LDL. The terminal amino acid can be either D-form or L-form.

[0090] In a preferred embodiment, the amino acid modifying group is a short peptide comprising leucine and tryptophan:

[0091]

[0092] or

[0093] The following are short peptides containing leucine and phenylalanine:

[0094]

[0095] In a preferred embodiment of the present invention, the variables in equation (I) have the following meanings:

[0096] n1 is 5;

[0097] R 1 It is 1,2-ethylene;

[0098] R 2 It is methylene;

[0099] R 3 It is 1,2-ethylene;

[0100] R 4 It is methylene;

[0101] m is 0;

[0102] R 6 It has the following structure:

[0103] Especially the R of compound (I) 1 and R 3 The compound of formula (I) is attached to the 1-position of the divalent 1H-1,2,3-triazole ring at the R position. 2 and R 4 It is attached to the 4 position of the divalent 1H-1,2,3-triazole ring;

[0104] z is an integer, which is chosen so that the entire molecule is uncharged;

[0105] Z is the counter ion; and

[0106] Short peptides containing leucine and tryptophan, where all R values ​​are the same and they are of the following type:

[0107]

[0108] or

[0109] The variables in equation (I) have the following meanings:

[0110] n1 is 6;

[0111] R 1 It is 1,2-ethylene;

[0112] R 2 It is methylene;

[0113] R 3 It is 1,2-ethylene;

[0114] R 4 It is methylene;

[0115] m is 0;

[0116] R 6 It has the following structure:

[0117] Especially the R of compound (I) 1 and R 3 The compound of formula (I) is attached to the 1-position of the divalent 1H-1,2,3-triazole ring at the R position. 2 and R 4 It is attached to the 4th position of the divalent 1H-1,2,3-triazole ring, where z is an integer, and is selected so that the entire molecule is uncharged;

[0118] Z is the counter ion; and

[0119] Short peptides containing leucine and tryptophan, where all R values ​​are the same and they are of the following type:

[0120]

[0121] or

[0122] The variables in equation (I) have the following meanings:

[0123] n1 is 7;

[0124] R 1 It is 1,2-ethylene;

[0125] R 2 It is methylene;

[0126] R 3 It is 1,2-ethylene;

[0127] R 4 It is methylene;

[0128] m is 0;

[0129] R 6 It has the following structure:

[0130] Especially the R of compound (I) 1 and R 3 The compound of formula (I) is attached to the 1-position of the divalent 1H-1,2,3-triazole ring at the R position. 2 and R 4 It is attached to the 4th position of the divalent 1H-1,2,3-triazole ring, where z is an integer, and is selected so that the entire molecule is uncharged;

[0131] Z is the counter ion; and

[0132] Short peptides containing leucine and tryptophan, where all R values ​​are the same and they are of the following type:

[0133]

[0134] or

[0135] The variables in equation (I) have the following meanings:

[0136] n1 is 5;

[0137] R 1 It is 1,2-ethylene;

[0138] R 2 It is methylene;

[0139] R 3 It is 1,2-ethylene;

[0140] R 4 It is methylene;

[0141] m is 0;

[0142] R 6 It has the following structure:

[0143] Especially the R of compound (I) 1 and R 3 The compound of formula (I) is attached to the 1-position of the divalent 1H-1,2,3-triazole ring at the R position. 2 and R 4 It is attached to the 4th position of the divalent 1H-1,2,3-triazole ring, where z is an integer, and is selected so that the entire molecule is uncharged;

[0144] Z is the counter ion; and

[0145] Short peptides containing leucine and phenylalanine with all R's identical and belonging to the following formula:

[0146]

[0147] Any type of organic or inorganic anion Z commonly known in the field of amino acids can be used as a counterion. Specific examples of suitable counterions are selected from hydroxide, chloride, bromide, nitrate, sulfate, monomethyl sulfate, formate, acetate, propionate, and trifluoroacetate ions.

[0148] In one embodiment, the amino acid-modified compound, such as the compound of formula (I), has formula (I-1):

[0149]

[0150] Where m, n1, and R 1 R 2 R 3 R 4 R 5 R, z, and Z have the definitions given above, especially the preferred definitions.

[0151] In a preferred embodiment of the present invention, the variables in formula (I-1) have the following meanings:

[0152] n1 is 5;

[0153] R 1 It is 1,2-ethylene;

[0154] R 2 It is methylene;

[0155] R 3 It is 1,2-ethylene;

[0156] R 4 It is methylene;

[0157] m is 0;

[0158] z is an integer, which is chosen so that the entire molecule is uncharged;

[0159] Z is the counter ion; and

[0160] Short peptides containing leucine and tryptophan, where all R values ​​are the same and they are of the following type:

[0161]

[0162] Methods for preparing amino acid-modified compounds

[0163] One aspect of the present invention relates to a method for preparing the amino acid-modified compounds of the present invention, preferably compounds of formula (I), particularly compounds of formula (I-1), comprising:

[0164] 1) Reacting compound (II) with formaldehyde yields compound (III).

[0165]

[0166] Where X is chlorine, bromine, or iodine, preferably bromine; and

[0167] n1, m, R 1 R 3 and R 5 As defined above;

[0168] 2) Reacting compound (III) with an alkali metal azide yields compound (IV):

[0169]

[0170] Where n1, m, R 1 R 3 and R 5 As defined above;

[0171] 3) Compound (IV) is reacted with compounds (V-1) and (V-2) via click chemistry to yield compound (VI):

[0172]

[0173] in

[0174] R 2 and R 4 As defined above;

[0175] R' corresponds to R as defined above, but the reactive amino group in the amino acid molecule of R' is protected by Pro; and

[0176] Pro is the protecting group for the amino group in the amino acid molecule of R';

[0177]

[0178] Where n1, m, R 1 R 2 R 3 R 4 R 5 R' and Pro are as defined above;

[0179] 4) Remove the protecting group Pro from the amino group in compound (VI) and give the amino group a positive charge to obtain the amino acid-modified compound.

[0180] According to the present invention, the formaldehyde in step 1) can be monomeric formaldehyde or paraformaldehyde, preferably paraformaldehyde.

[0181] According to the present invention, the reaction in step 1) is carried out in the presence of a Lewis acid, preferably selected from FeCl3, AlCl3, SnCl4, BF3, BF3·O(C2H5)2 and organic sulfonic acids, more preferably BF3·O(C2H5)2. The molar ratio of the Lewis acid to the compound of formula (II) can be at least 1.2:1, at least 1.5:1, at least 1.8:1, at least 2:1, or can be 1.2:1-3.5:1, 1.5:1-3:1 or 1.8:1-2.5:1.

[0182] In step 1), the molar ratio of formaldehyde (based on monomeric formaldehyde) to the compound of formula (II) can be at least 1.1:1, at least 1.2:1, at least 1.3:1, at least 1.4:1 or at least 1.5:1, or can be 1.1:1-2.5:1, 1.2:1-2:1 or 1.3:1-1.8:1.

[0183] The reaction in step 1) can be carried out in the presence of a solvent. The solvent can be a haloalkane (such as a haloalkane) having 1-8, 1-6, 1-4, or 1-2 carbon atoms, preferably a chlorinated hydrocarbon (such as a chlorinated alkane), for example dichloromethane, 1,2-dichloroethane, or chloroform. 1,2-dichloroethane is preferred when n1 is 5, and chloroform is preferred when n1 is 6 or 7.

[0184] The reaction temperature in step 1) can be 15-35℃ or 18-30℃, for example, room temperature. Alternatively, the reactant compound (II) can be mixed with formaldehyde first, then the Lewis acid can be added under ice bath conditions, and then the reaction mixture can be warmed to the reaction temperature. The reaction time in step 1) can be 1-8 h, or 1.5-5 h, or 1.5-3 h.

[0185] In step 2), the compound of formula (III) is reacted with an alkali metal azide.

[0186] The molar ratio of the alkali metal azide to the X group (preferably bromine) in the compound of formula (III) can be at least 1.1:1, at least 1.2:1, at least 1.3:1, at least 1.4:1, at least 1.5:1, or can be 1.1:1-2.5:1, 1.2:1-2:1, or 1.3:1-1.8:1.

[0187] The alkali metal azide in step 2) is preferably potassium azide or sodium azide.

[0188] The reaction in step 2) is preferably carried out under an inert atmosphere, such as a nitrogen or argon atmosphere.

[0189] The reaction in step 2) can be carried out in a suitable solvent, such as an aprotic solvent (e.g., DMF).

[0190] The reaction temperature in step 2) can be 25-55℃ or 30-50℃. The reaction time in step 2) can be 3-20h, 4-15h, or 5-10h.

[0191] In step 3), compound (IV) is reacted with compounds (V-1) and (V-2) via a click chemistry reaction to obtain compound (VI).

[0192] The Pro group in compounds of formulas (V-1) and (V-2) can be a suitable amino protecting group, such as tert-butoxycarbonyl.

[0193] In this article, a reactive amino group refers to an amino group that can react with a carboxyl group to form an amide bond.

[0194] In compounds of formulas (V-1) and (V-2), R' corresponds to R as defined above, but the reactive amino group in the amino acid molecule of R' is protected by Pro. For example, when R is a short peptide comprising leucine and tryptophan:

[0195]

[0196] R' can be:

[0197]

[0198] R in compounds of formulas (V-1) and (V-2) 2 and R 4 As defined above. As stated above, R 2 and R 4 They can be the same or different. In a preferred embodiment, R 2 and R 4 They are the same.

[0199] The molar ratio of the total amount of compounds of formula (V-1) and (V-2) to the azide groups in compound of formula (IV) can be at least 1.1:1, at least 1.2:1, at least 1.3:1, at least 1.4:1, at least 1.5:1, or can be 1.1:1-2.5:1, 1.2:1-2:1, or 1.3:1-1.8:1.

[0200] According to the present invention, the click chemistry reaction in step 3) can be carried out in the presence of a metal catalyst. The metal may include Cu (e.g., Cu(I)), such as copper (e.g., Cu(I)) produced in situ from Cu(II) and ascorbic acid, ruthenium (e.g., Ru(II)), and Ag, Ni, Pt, Pd, Rh, and Ir, preferably Cu, such as copper (e.g., Cu(I)) produced in situ from Cu(II) and ascorbic acid. The amount of metal catalyst, based on the molar amount of the azide group in the compound of formula (IV), may be 15-50 mol% or 20-40 mol%.

[0201] The reaction in step 3) can be carried out in a suitable solvent, such as an aprotic solvent (DMF and / or THF).

[0202] The reaction temperature in step 3) can be 25-55℃ or 30-50℃. The reaction time in step 3) can be 3-20h, 4-16h, or 5-14h.

[0203] According to the invention, step 4) is carried out in the presence of a protic acid (such as trifluoroacetic acid). According to the invention, the amount of protic acid should be sufficient to eliminate the protecting group and cause the amino-modified group to carry a positive charge.

[0204] According to the present invention, the amino acid-modified compound, preferably the compound of formula (I), and especially the compound of formula (I-1), can also be prepared by a method comprising the following steps:

[0205] i) Reacting compound (II) with an alkali metal azide to obtain compound (A).

[0206]

[0207] Where X is chlorine, bromine, or iodine, preferably bromine; and

[0208] m、R 1 R 3 and R 5 As defined above;

[0209] ii) Reacting compound (A) with compounds (V-1) and (V-2) via click chemistry to obtain compound (B):

[0210]

[0211] in

[0212] R 2 and R 4 As defined above;

[0213] R' corresponds to R as defined above, but the reactive amino group in the amino acid molecule of R' is protected by Pro; and

[0214] Pro is the protecting group for the amino group in the amino acid molecule of R';

[0215]

[0216] Where m, R 1 R 2 R 3 R 4 R 5 R' and Pro are as defined above; iii) react the compound of formula (B) with formaldehyde to give the compound of formula (VI):

[0217]

[0218] Where n1 is defined as above;

[0219] m、R 1 R 2 R 3 R 4 and R 5 R' and Pro are as defined above;

[0220] iv) Remove the protecting group Pro from the amino group in the compound of formula (VI) and make the amino group positively charged to obtain the amino acid-modified compound.

[0221] In step i), the compound of formula (II) is reacted with an alkali metal azide.

[0222] The molar ratio of the alkali metal azide to the X group (preferably bromine) in the compound of formula (II) can be at least 1.1:1, at least 1.2:1, at least 1.3:1, at least 1.4:1, at least 1.5:1, or can be 1.1:1-2.5:1, 1.2:1-2:1, or 1.3:1-1.8:1.

[0223] The alkali metal azide in step i) is preferably potassium azide or sodium azide.

[0224] The reaction in step i) is preferably carried out under an inert atmosphere, such as a nitrogen or argon atmosphere.

[0225] The reaction in step i) can be carried out in a suitable solvent, such as an aprotic solvent (e.g., DMF).

[0226] The reaction temperature in step i) can be 25-55℃ or 30-50℃. The reaction time in step i) can be 3-20h, 4-15h, or 5-10h.

[0227] In step ii), compound (A) is reacted with compounds (V-1) and (V-2) via a click chemistry reaction to obtain compound (B).

[0228] The Pro group in compounds of formulas (V-1) and (V-2) can be a suitable amino protecting group, such as tert-butoxycarbonyl.

[0229] In compounds of formulas (V-1) and (V-2), R' corresponds to R as defined above, but the reactive amino group in the amino acid molecule of R' is protected by Pro. For example, when R is a short peptide comprising leucine and tryptophan:

[0230]

[0231] R' can be:

[0232]

[0233] R in compounds of formulas (V-1) and (V-2) 2 and R 4 As defined above. As stated above, R 2 and R 4 They can be the same or different. In a preferred embodiment, R 2 and R 4 They are the same.

[0234] The molar ratio of the total amount of compounds of formula (V-1) and (V-2) to the azide groups on compound of formula (A) can be at least 1.1:1, at least 1.2:1, at least 1.3:1, at least 1.4:1, at least 1.5:1, or can be 1.1:1-2.5:1, 1.2:1-2:1, or 1.3:1-1.8:1.

[0235] According to the present invention, the click chemistry reaction in step ii) can be carried out in the presence of a metal catalyst. The metal may include Cu (e.g., Cu(I)), such as copper (e.g., Cu(I)) produced in situ from Cu(II) and ascorbic acid, ruthenium (e.g., Ru(II)), and Ag, Ni, Pt, Pd, Rh, and Ir, preferably Cu, such as copper (e.g., Cu(I)) produced in situ from Cu(II) and ascorbic acid. The amount of metal catalyst, based on the molar amount of the azide group in the compound of formula (A), may be 15-50 mol% or 20-40 mol%.

[0236] The reaction in step ii) can be carried out in a suitable solvent, such as an aprotic solvent (DMF and / or THF).

[0237] The reaction temperature in step ii) can be 25-55℃ or 30-50℃. The reaction time in step ii) can be 3-20h, 4-16h, or 5-14h.

[0238] According to the present invention, the formaldehyde in step iii) can be monomeric formaldehyde or paraformaldehyde, preferably paraformaldehyde.

[0239] According to the present invention, the reaction in step iii) is carried out in the presence of a Lewis acid, preferably selected from FeCl3, AlCl3, SnCl4, BF3, BF3·O(C2H5)2 and organic sulfonic acids, more preferably BF3·O(C2H5)2. The molar ratio of the Lewis acid to the compound of formula (B) can be at least 1.2:1, at least 1.5:1, at least 1.8:1, at least 2:1, or can be 1.2:1-3.5:1, 1.5:1-3:1 or 1.8:1-2.5:1.

[0240] In step iii), the molar ratio of formaldehyde (based on monomeric formaldehyde) to the compound of formula (B) may be at least 1.1:1, at least 1.2:1, at least 1.3:1, at least 1.4:1 or at least 1.5:1, or may be 1.1:1-2.5:1, 1.2:1-2:1 or 1.3:1-1.8:1.

[0241] The reaction in step iii) can be carried out in the presence of a solvent. The solvent can be a haloalkane (such as a haloalkane) having 1-8, 1-6, 1-4, or 1-2 carbon atoms, preferably a chlorinated hydrocarbon (such as a chlorinated alkane), for example dichloromethane, 1,2-dichloroethane, or chloroform. 1,2-dichloroethane is preferred when n1 is 5, and chloroform is preferred when n1 is 6 or 7.

[0242] The reaction temperature in step iii) can be 15-35℃ or 18-30℃, for example, room temperature. Alternatively, the reactant compound (B) can be mixed with formaldehyde first, then the Lewis acid can be added under ice bath conditions, and the reaction mixture can be warmed to the reaction temperature. The reaction time in step iii) can be 1-8 h, 1.5-5 h, or 1.5-3 h.

[0243] According to the invention, step iv) is carried out in the presence of a protic acid (such as trifluoroacetic acid). According to the invention, the amount of protic acid should be sufficient to eliminate the protecting group and cause the amino-modified group to carry a positive charge.

[0244] According to the present invention, the alkyne derivatives of formula (V-1) and formula (V-2) can be obtained by reacting the corresponding amino acid with the corresponding amine containing an alkyne group.

[0245] Compounds of formula (VI) and compounds of formula (I')

[0246] One aspect of the present invention relates to compounds of formula (VI):

[0247]

[0248] in

[0249] n1, m, R 1 R 2 R 3 R 4 and R 5 As defined above;

[0250] R' corresponds to R as defined above, but the reactive amino group in the amino acid molecule of R' is protected by Pro; and

[0251] Pro is the protecting group for the amino group in the amino acid molecule of R'.

[0252] One aspect of the present invention relates to compounds of formula (I'):

[0253]

[0254] in

[0255] n1, m, R 1 R 2 R 3 R 4 R 5 and R 6 As defined above; R” corresponds to R as defined above, but R” does not carry a positive charge. For example, when R is a short peptide containing leucine and tryptophan:

[0256] R can be:

[0257]

[0258] In one embodiment, the compound of formula (I') has formula (I”):

[0259]

[0260] in

[0261] n1, m, R 1 R 2 R 3 R 4 and R 5 As defined above;

[0262] R” corresponds to R as defined above, but R” does not carry a positive charge.

[0263] According to the present invention, the compound of formula (I') can be obtained by reacting with a protic acid (such as trifluoroacetic acid) to give the compound of formula (I). Similarly, the compound of formula (I”) can be obtained by reacting with a protic acid (such as trifluoroacetic acid) to give the compound of formula (I-1). The compound of formula (I) can be obtained by reacting with a base (such as an alkali metal hydroxide or an alkaline earth metal hydroxide or an alkali metal bicarbonate, such as sodium bicarbonate) to give the compound of formula (I'), and similarly, the compound of formula (I”) can be obtained from the compound of formula (I-1). The compound of formula (I') or formula (I”) can also be obtained by appropriately controlling the pH in step 4) or iv).

[0264] Uses and Compositions

[0265] One aspect of the present invention relates to a pharmaceutical composition comprising an amino acid-modified compound according to the present invention.

[0266] One aspect of the invention relates to the use of the amino acid-modified compounds according to the invention as artificial aquaporins, or in the preparation of medicaments for wound repair, particularly for tympanic membrane repair.

[0267] The amino acid-modified compounds of the present invention can be obtained by a simple and high-yield synthetic method, and the amino acid-modified compounds can spontaneously integrate into the cell membrane and exhibit high water transport efficiency and selectivity, significantly accelerate cell migration, have excellent tympanic membrane repair capabilities, and have low ototoxicity.

[0268] Example

[0269] The technical solutions of this invention are further described below with reference to specific embodiments, but should not be construed as limiting the scope of protection of this invention. The embodiments described below are only some embodiments of this invention, not all embodiments. Other embodiments proposed by other personnel skilled in the art based on the embodiments listed in this invention without creative effort are all within the scope of protection of this invention. Unless otherwise stated, percentages in the embodiments are weight percentages, and parts in the embodiments are parts by weight.

[0270] Materials and methods

[0271] Paraformaldehyde was purchased from Sigma-Aldrich.

[0272] The chloroform solution of egg yolk L-α-phosphatidylcholine (EYPC) was obtained from Avanti Polar Lipids.

[0273] CountBright TM 5(6)-Carboxytetramethylrhodamine (TAMRA) was obtained from ThermoFisher Scientific.

[0274] The wound healing culture plugin is from ibidi.

[0275] All required amino acids were purchased from Jier Biochemical (Shanghai) Co., Ltd. The alkyne derivatives in the examples were obtained by reacting the corresponding amino acids with propargylamine.

[0276] Other chemicals were purchased directly from local chemical companies and used without further purification.

[0277] C57BL / 6J mice aged 4-5 weeks were obtained from the Department of Experimental Animal Science, Fudan University.

[0278] The mouse fibroblast cell line (L929) was obtained from the Chinese Academy of Sciences Cell Bank / Stem Cell Bank.

[0279] Recorded at 298K using a Mercury Plus 400 spectrometer. 1 H and 13 C10 NMR spectrum. Chemical shift reference residual solvent.

[0280] Mass spectra can be recorded using a Bruker MicroTOF II spectrometer in either positive or negative mode.

[0281] Stopped-flow experiments were conducted using an Applied Photophysics SX-20 instrument in both light scattering and fluorescence modes.

[0282] Fluorescence microscopy images were collected using a Leica DMi8 fluorescence microscope.

[0283] Images of a confocal laser scanning microscope were collected using a Zeiss confocal microscope (LSM 800).

[0284] Example 1 – Synthesis of amino acid-modified compounds

[0285] Example 1-A: Synthesis of Compound 1

[0286]

[0287] Boc = tert-Butyloxycarbonyl

[0288] Synthesis Route 1

[0289] Compound 3

[0290] The solution of 1,4-bis(2-bromoethoxy)benzene (29.6 g, 92.0 mmol) and paraformaldehyde (4.14 g, 138.0 mmol) in 1,2-dichloroethane (400 mL) was cooled in an ice bath. Boron trifluoride diethyl ether (26.1 g, 184.0 mmol) was added to the solution, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched by adding water. The organic phase was separated, washed with water (3 × 400 mL), and dried over Na₂SO₄. The solvent was evaporated to give the crude product, which was purified by column chromatography (PE:CH₂Cl₂ = 1:2) to give compound 3 as a white solid (8.60 g, 25% yield). 1 H NMR (400MHz, CHCl3) δ6.91 (s, 10H), 4.23 (t, J = 5.4Hz, 20H), 3.85 (s, 10H), 3.63 (t, J = 5.5Hz, 20H). 13 C10 NMR (101 MHz, CHCl3) δ 149.9, 129.2, 116.3, 69.2, 30.9, 29.6. HR-MS (ESI-TOF): Calculation of C10 NMR values. 55 H 60 O 10 Br 10 [M+NH4] + :1697.6256, Actual measurement:1697.6269.

[0291] Compound 4

[0292] Compound 3 (1.00 g, 0.60 mmol) was dissolved in anhydrous DMF (20 mL) under a nitrogen atmosphere. After adding NaN3 (0.58 g, 8.92 mmol), the reaction mixture was heated at 40 °C for 8 hours, and then poured into water (200 mL). The precipitate was collected by filtration and washed with H2O to give compound 4 (0.70 g, 91% yield) as a white solid. 1 H NMR (400MHz, CDCl3) δ6.84 (s, 10H), 4.01 (t, J = 4.8Hz, 20H), 3.84 (s, 10H), 3.55 (t, J = 4.8Hz, 20H). 13 C NMR (101MHz, CDCl3) δ 150.1, 129.1, 115.9, 67.7, 51.1, 29.8. HR-MS (ESI-TOF): Calculation of C 55 H 60 N 30 O 10 [M+Na] + :1323.5001, actual measurement 1323.5000.

[0293] Compound 5

[0294] An alkyne derivative (1.05 g, 2.31 mmol) (its structure is shown in the synthetic route above), sodium ascorbate (17.8 mg, 0.09 mmol), and CuSO4·5H2O (11.2 mg, 0.05 mmol) were added to a solution of compound 4 (0.20 g, 0.15 mmol) in DMF / THF (3 mL / 1 mL). The mixture was stirred at 40 °C for 12 hours and then poured into H2O (40 mL). The precipitate was collected by filtration and washed with H2O. The crude residue was purified by column chromatography (CH2Cl2:MeOH = 40:1-10:1) to give compound 5 (0.55 g, 61% yield) as a pale yellow solid. 1 H NMR (400MHz, DMSO-d6) δ10.79(s,10H),8.34(s,10H),8.12–7.96(m,20H),7.64–6.69(m,70H),4.81( s,20H),4.55–3.93(m,60H),3.24–2.69(m,30H),1.54–0.96(m,120H),0.66(dd,J=30.6,5.1Hz,60H). 13C NMR (101MHz, DMSO-d6) δ 172.1, 172.0, 155.5, 155.4, 148.7, 145.1, 145.0, 136.0, 128.3, 127.2, 123.7, 122.7, 120.8, 118.4, 118.1, 111.2, 109.7, 78.2, 67.2, 59.7, 55.6, 50.9, 49.6, 48.6, 40.4, 34.4, 28.0, 27.6, 27.3, 23.8, 23.0, 21.0, 20.7, 14.1. HR-MS (ESI-TOF): Calculation of C 305 H 400 N 70 O 50 [M+3H] 3+ :1949.7072, Actual measurement:1949.7085.

[0295] Compound 1

[0296] Trifluoroacetic acid (TFA) (2 mL) was added to a solution of compound 5 (0.40 g, 0.07 mmol) in CH2Cl2 (2 mL). The mixture was stirred at room temperature for 8 hours, and then concentrated under vacuum. The crude product was washed with water to give compound 1 (0.27 g, 82% yield) as a pale yellow solid. 1 H NMR(400MHz,DMSO-d6)δ10.83(s,10H),8.48(s,10H),8.03(s,20H),7.70–6.60(m,60H),4.80(s,20H) ),4.56-4.02(m,60H),3.81–3.00(m,20H),2.67(s,10H),1.82–1.02(m,30H),0.72(d,J=5.8Hz,60H). 13 C NMR (101MHz, DMSO-d6) δ 172.3, 171.9, 148.7, 145.0, 136.3, 128.3, 127.2, 124.1, 122.9, 120.9, 118.5, 118.2, 114.8, 111.3, 109.2, 67.2, 54.3, 50.9, 49.7, 41.1, 34.2, 31.9, 29.9, 29.2, 27.1, 24.0, 22.9, 21.3. HR-MS (ESI-TOF): Calculation of C 255 H 320 N 70 O 30 [M+4H] 4+ :1212.1504, Actual measurement:1212.1501.

[0297] Example 1-B: Synthesis of compound 1'

[0298] Compound 1'

[0299]

[0300] Compound 1 was washed with a saturated sodium bicarbonate aqueous solution to deprotonate the protonated amino group, yielding a pale yellow solid compound 1'. 1 H NMR(400MHz,DMSO-d6)δ10.81(s,10H),8.47(s,10H),8.10–8.01(m,20H),7.65–6.59(m,60H), 4.80(s,20H),4.62–3.96(m,60H),3.65–2.60(m,30H),1.66–0.93(m,30H),0.88-0.54(m,60H). 13 C NMR (101MHz, DMSO-d6) δ 174.5, 172.1, 148.7, 145.0, 136.3, 128.3, 127.2, 123.7, 122.9, 120.8, 118.5, 118.2, 114.8, 111.3, 110.6, 67.2, 55.2, 50.8, 49.7, 41.3, 34.3, 31.9, 29.8, 29.2, 27.1, 24.0, 23.0, 21.4. HR-MS (ESI-TOF): Calculation of C 255 H 320 N 70 O 30 [M+2H] 2+ :2423.2939, Actual measurement:2423.2960.

[0301] Example 1-C: Synthesis of compounds 11 and 13

[0302]

[0303] Synthesis Route 2

[0304] Compounds 6 and 7

[0305] The solution of 1,4-bis(2-bromoethoxy)benzene (29.6 g, 92.0 mmol) and paraformaldehyde (4.14 g, 138.0 mmol) in chloroform (400 mL) was cooled in an ice bath. Boron trifluoride diethyl ether (26.1 g, 184.0 mmol) was added to the solution, and the mixture was stirred at room temperature for 2 hours. The reaction was quenched by adding water. The organic phase was separated, washed with water (3 × 400 mL), and dried over Na₂SO₄. The solvent was evaporated to give the crude product, which was purified by column chromatography (PE:CH₂Cl₂ = 1:2–1:6) to give compound 6 (8.11 g, 22% yield) and compound 7 (0.43 g, 1% yield) as white solids.

[0306] Compound 6: 1 H NMR (400MHz, CDCl3) δ6.78 (s, 12H), 4.17 (t, J = 5.9 Hz, 24H), 3.87 (s, 12H), 3.55 (t, J = 5.9 Hz, 24H). 13 C NMR (101MHz, CDCl3) δ 150.4, 128.7, 116.0, 69.2, 30.80, 30.4. HR-MS (ESI-TOF): Calculation of C 66 H 72 O 12 Br 12 [M+NH4] + :2035.5450, actual measurement 2035.5422.

[0307] Compound 7: 1 H NMR (400MHz, CDCl3) δ6.70 (s, 14H), 4.14 (t, J = 5.9 Hz, 28H), 3.90 (s, 14H), 3.51 (t, J = 5.9 Hz, 28H). 13 C10 NMR (101 MHz, CDCl3) δ 150.6, 128.6, 115.9, 69.3, 31.1, 30.3. HR-MS (ESI-TOF): Calculation of C10 NMR values. 77 H 84 O 14 Br 14 [M+Na] + :2372.4200, actual measurement 2372.4242.

[0308] Compound 8

[0309] Compound 6 (1.00 g, 0.50 mmol) was dissolved in anhydrous DMF (20 mL) under a nitrogen atmosphere. After adding NaN3 (0.58 g, 8.90 mmol), the reaction mixture was heated at 40 °C for 8 hours, and then poured into water (200 mL). The precipitate was collected by filtration and washed with H2O to give compound 8 (0.70 g, 90% yield) as a white solid. Compound 8: 1 H NMR (400MHz, CDCl3) δ6.75 (s, 12H), 3.98 (t, J = 4.9 Hz, 24H), 3.88 (s, 12H), 3.47 (t, J = 4.9 Hz, 24H). 13 C10 NMR (101MHz, CDCl3) δ 150.5, 128.5, 115.5, 67.7, 50.8, 30.7. HR-MS (ESI-TOF): Calculation of C10 NMR values. 66 H 72 N 36 O 12 [M+Na] + :1583.6023, actual measurement 1583.6019.

[0310] Compound 9

[0311] Compound 7 (0.40 g, 0.17 mmol) was dissolved in anhydrous DMF (20 mL) under a nitrogen atmosphere. After adding NaN3 (0.22 g, 3.42 mmol), the reaction mixture was heated at 40 °C for 8 hours, and then poured into water (200 mL). The precipitate was collected by filtration and washed with H2O to give compound 9 (0.27 g, 88% yield) as a white solid. Compound 9: 1 H NMR (400MHz, CDCl3) δ6.63 (s, 14H), 4.00–3.88 (m, 42H), 3.42 (t, J = 4.8Hz, 28H). 13 CNMR (101MHz, CDCl3) δ 150.7, 128.4, 115.2, 67.92, 50.7, 30.7. HR-MS (ESI-TOF): Calculation of C 77 H 84 N 42 O 14 [M+Na] + :1843.7044, actual measurement 1843.7113.

[0312]

[0313] Boc = tert-Butyloxycarbonyl

[0314] Synthesis Route 3

[0315] Compound 10

[0316] An alkyne derivative (1.06 g, 2.31 mmol) (its structure is shown in the synthetic route above), sodium ascorbate (15.2 mg, 0.07 mmol), and CuSO4·5H2O (10.0 mg, 0.04 mmol) were added to a solution of compound 8 (0.20 g, 0.13 mmol) in DMF / THF (3 mL / 1 mL). The mixture was stirred at 40 °C for 12 hours and then poured into H2O (40 mL). The precipitate was collected by filtration and washed with H2O. The crude residue was purified by column chromatography (CH2Cl2:MeOH = 40:1-10:1) to give compound 10 (0.54 g, 60% yield) as a pale yellow solid. 1 H NMR(400MHz,DMSO-d6)δ10.79(s,12H),8.32(s,12H),8.19–7.85(m,24H),7.63–6.65(m,84H),4 .71(s,24H),4.42–3.95(m,72H),3.10–2.83(m,36H),1.50–1.03(m,144H),0.85–0.49(m,72H). 13 C NMR (101MHz, DMSO-d6) δ 172.4, 155.9, 150.1, 145.4, 136.5, 127.7, 124.2, 123.2, 121.2, 118.8, 118.6, 115.4, 111.6, 110.14, 78.7, 67.3, 56.0, 51.3, 49.8, 40.8, 34.8, 28.5, 27.8, 24.3, 23.5, 21.9, 21.4. HR-MS (ESI-TOF): Calculation of C 366 H 480 N 84 O 60 [M+2H] 2+ :3508.8684, Actual measurement:3508.8694.

[0317] Compound 11

[0318] Trifluoroacetic acid (TFA) (2 mL) was added to a solution of compound 10 (0.40 g, 0.06 mmol) in CH2Cl2 (2 mL). The mixture was stirred at room temperature for 8 hours, and then concentrated under vacuum. The crude product was washed with water to give compound 11 (0.26 g, 80% yield) as a pale yellow solid. 1H NMR(400MHz,DMSO-d6)δ10.89(s,12H),8.53(s,12H),8.27(s,12H),7.99(s,12H),7.60–6.55(m,72H), 4.69(s,24H),4.27(s,72H),3.85–2.97(m,24H),2.81(s,12H),1.52–1.12(m,36H),0.9–0.49(m,72H). 13 C NMR (101MHz, DMSO-d6) δ 174.9, 172.6, 150.1, 145.4, 136.7, 127.8, 127.2, 124.2, 123.8, 121.3, 118.9, 118.7, 115.5, 111.7, 111.1, 67.3, 55.7, 51.2, 49.9, 41.7, 34.7, 32.4, 31.6, 29.8, 24.5, 23.4, 21.9. HR-MS (ESI-TOF): Calculation of C 306 H 384 N 84 O 36 [M+3H] 3+ :1939.0368, Actual measurement:1939.0376.

[0319]

[0320] Boc = tert-Butyloxycarbonyl

[0321] Synthesis Route 4

[0322] Compound 12

[0323] An alkyne derivative (1.00 g, 2.20 mmol) (its structure is shown in the synthetic route above), sodium ascorbate (15.2 mg, 0.07 mmol), and CuSO4·5H2O (10.0 mg, 0.04 mmol) were added to a solution of compound 9 (0.20 g, 0.11 mmol) in DMF / THF (3 mL / 1 mL). The mixture was stirred at 40 °C for 12 hours and then poured into H2O (40 mL). The precipitate was collected by filtration and washed with H2O. The crude residue was purified by column chromatography (CH2Cl2:MeOH = 40:1-10:1) to give compound 12 (0.49 g, 55% yield) as a pale yellow solid. 1H NMR(400MHz,DMSO-d6)δ10.78(s,14H),8.31(s,14H),8.16–7.82(m,28H),7.63–6.62(m,98H),4 .67(s,28H),4.50–3.97(m,84H),3.18–2.76(m,42H),1.56–0.97(m,168H),0.75–0.51(m,84H). 13 C NMR (101MHz, DMSO-d6) δ 172.0, 155.5, 150.1, 145.0, 136.0, 127.3, 126.6, 123.7, 122.7, 120.8, 118.4, 118.1, 111.2, 109.7, 78.2, 67.1, 55.6, 54.9, 50.9, 49.3, 40.4, 34.3, 28.0, 27.6, 27.4, 23.8, 23.0, 21.0. HR-MS (ESI-TOF): Calculation of C 427 H 560 N 98 O 70 [M+3H] 3+ :2729.4552, Actual measurement: 2729.4548.

[0324] Compound 13

[0325] Trifluoroacetic acid (TFA) (2 mL) was added to a solution of compound 12 (0.40 g, 0.05 mmol) in CH2Cl2 (2 mL). The mixture was stirred at room temperature for 8 hours, and then concentrated under vacuum. The crude product was washed with water to give compound 13 (0.27 g, 80% yield) as a pale yellow solid. 1 H NMR(400MHz,DMSO-d6)δ10.81(s,14H),8.45(s,14H),8.16–7.80(m,28H),7.73–6.60(m,84H),4.66 (s,28H),4.29(s,84H),3.62–2.90(m,28H),2.68(s,14H),1.57–1.04(m,42H),0.88–0.34(m,84H). 13C NMR (101MHz, DMSO-d6) δ 174.5, 172.1, 150.1, 144.9, 136.2, 127.3, 126.6, 123.7, 122.9, 120.8, 118.5, 118.2, 111.3, 110.6, 67.1, 55.2, 50.7, 49.4, 41.2, 34.1, 31.9, 31.1, 29.3, 24.0, 23.0, 21.4. HR-MS (ESI-TOF): Calculation of C 357 H 448 N 98 O 42 [M+3H] 3+ :2261.8746, Actual measurement:2261.8912.

[0326] Example 1-D: Synthesis of Compound 15

[0327]

[0328] Boc = tert-Butyloxycarbonyl

[0329] Synthesis Route 5

[0330] Compound 14

[0331] The alkyne derivative (0.96, 2.31 mmol) (its structure is shown in the synthetic route above), sodium ascorbate (17.8 mg, 0.09 mmol), and CuSO4·5H2O (11.2 mg, 0.05 mmol) were added to a solution of compound 4 (0.20 g, 0.15 mmol) in DMF / THF (3 mL / 1 mL). The mixture was stirred at 40 °C for 12 hours and then poured into H2O (40 mL). The precipitate was collected by filtration and washed with H2O. The crude residue was purified by column chromatography (CH2Cl2:MeOH = 40:1-10:1) to give compound 14 (0.47 g, 56% yield) as a pale yellow solid. 1 H NMR(400MHz, DMSO-d6)δ8.36(s,10H),8.07(d,J=8.2Hz,10H),8.00(s,10H),7.30–7.14(m,50H),7.03(d,J=7.3Hz,10H),6. 75(s,10H),4.80(s,20H),4.58–3.94(m,60H),3.16(s,10H),2.96–2.67(m,20H),1.42–1.06(m,120H),0.80–0.56(m,60H). 13C NMR (101MHz, DMSO-d6) δ 171.9, 171.4, 155.4, 148.7, 145.0, 137.6, 129.2, 128.3, 127.9, 126.1, 122.8, 114.8, 78.2, 67.2, 56.0, 50.8, 49.6, 40.6, 37.4, 34.4, 28.0, 23.7, 21.2. HR-MS (ESI-TOF): Calculation of C 285 H 390 N 60 O 50 [M+2Na] 2+ :2750.4832, Actual measurement:2750.4811.

[0332] Compound 15

[0333] Trifluoroacetic acid (TFA) (2 mL) was added to a solution of compound 14 (0.40 g, 0.07 mmol) in CH2Cl2 (2 mL). The mixture was stirred at room temperature for 8 hours, and then concentrated under vacuum. The crude product was washed with water to give compound 15 (0.27 g, 83% yield) as a pale yellow solid. 1 H NMR(400MHz,DMSO-d6)δ8.46(s,10H),8.02(s,10H),7.95(s,10H),7.28–7.07(m,50H),6.74(s,10H),4.80(s,20H) ,4.54–3.99(m,50H),3.5–3.05(m,20H),2.80–2.78(m,10H),2.68–2.54(m,10H),1.61–1.14(m,30H),0.70(m,60H). 13 CNMR (101MHz, DMSO-d6) δ 174.3, 172.1, 148.7, 145.1, 138.7, 129.2, 128.3, 128.0, 126.0, 122.9, 114.8, 67.3, 56.2, 50.7, 49.7, 41.2, 41.2, 34.3, 23.9, 23.0, 21.5. HR-MS (ESI-TOF): Calculate C 235 H 310 N 60 O 30 [M+3H] 3+ :1486.8290, Actual measurement:1485.8317.

[0334] Example 2 - Performance of compounds 1, 11, and 13 (hereinafter also referred to as compounds 1, 11, and 13) as artificial channels in liposome bilayer membranes

[0335] (a) Preparation of homogeneous monolayer liposomes: Egg yolk L-α phosphatidylcholine (EYPC) (25 mg / mL, 0.8 mL) was diluted in CHCl3 (10 mL). The solution was evaporated under reduced pressure to form a lipid film, which was then further dried under high vacuum for 3 hours. The lipid film was hydrated by vortexing with HEPES buffer (2 mL, 10 mM HEPES, pH 7.4) for 5 minutes to obtain a milky white suspension. The resulting suspension was subjected to six freeze-thaw cycles using liquid nitrogen freezing and a 50°C warm water bath thawing. The suspension was extruded nine times through a polyethersulfone membrane (Collins, 0.22 μm) and then diluted to 40 mL with the same buffer. The final lipid concentration was 0.65 mM. The diameter of the liposomes was measured to be 200 nm using ZetaView (Z-NTA, Particle Metrix), and the number of particles (N) was determined. v The value is 2.4 × 10 12 per ml.

[0336] (b) Membrane binding efficiency (E1) (Ref. [S1]): The DMSO solution of compound channel 1 was mixed with the prepared liposome suspension (2 mL). The resulting suspension was then loaded into a dialysis membrane tube and dialyzed overnight with HEPES buffer (10 mM HEPES, pH = 7.4) to remove free compound channel 1. The UV-Vis absorbance (A) of the suspension at 282 nm before and after dialysis was recorded. Equation A was used. 后 / A 前 Calculate the membrane binding efficiency (E1) of compound channel 1 at a given concentration using equation N × 100%. c / v =N c ×E1 / N v Calculate the number of channels (N) on each liposome. c / v ), N c This indicates the number of channels at a given concentration.

[0337] Table 1 - Absorbance before and after dialysis

[0338]

[0339]

[0340] mRL%: Molar ratio of compound channel 1 relative to lipids (phospholipids, 25 mg / mL, 0.8 mL).

[0341] Table 2 - Combination Efficiency

[0342] Compound channel 1 concentration (mRL%) <![CDATA[Number N of experimental channels c / v > <![CDATA[Combination efficiency % E1]]> 0.10 81 48.7 0.15 103 40.9 0.30 182 36.2 0.45 262 34.8 0.60 324 32.3

[0343] (c) Water permeability: The water permeability of compound channel 1 was measured in light scattering mode using a stop-flow experiment (refer to [S2]). Generally, a dimethyl sulfoxide solution of compound channel 1 (20 μL of desired concentration) was added to a liposome suspension (2.0 mL) to embed the compound channel 1 molecules into the lipid bilayer. The liposome solution and a hypertonic solution (2.0 mL, 200 mM KCl, 10 mM HEPES, pH 7.4) were then placed in two separate syringes. These two solutions were rapidly mixed in a stop-flow chamber, abruptly exposing the liposomes to the hypertonic solution, creating an osmotic pressure across the lipid bilayer. Driven by this osmotic pressure, water flowed from the inside of the liposomes to the outside, causing the liposomes to contract and thus increasing their scattering intensity. The size change of the liposomes was measured by monitoring the change in light scattering intensity at a wavelength of 594 nm and an observation angle of 90°. Data acquisition intervals were 12.5 μs and dead time was 1.2 ms. For each measurement, the data were the average of five individual runs. Figure 1 The changes in light scattering intensity over time after liposomes were exposed to a hypertonic solution of 100 mM KCl in the presence of compound channel 1 are shown. Figure 1 In the middle, the mRL (%) from top to bottom are 0.60%, 0.45%, 0.30%, 0.15%, 0.10%, and 0%. Figure 1 The results showed that, compared with the blank liposomes, the liposomes in the presence of compound channel 1 exhibited a more significant increase in scattering intensity.

[0344] The water permeability (P) of the liposome membrane bound to compound channel 1 f Calculated using the following formula:

[0345]

[0346] in,

[0347] k refers to the kinetic coefficient of liposome size change, which is obtained by fitting the scattering curve with a single exponential function (see [S3]);

[0348] S0 and V0 refer to the initial surface area and volume of the liposome, respectively, which can be calculated based on the diameter of the liposome (200 nm).

[0349] V w It is the molar volume of water;

[0350] Δosm is the osmotic pressure difference across the lipid bilayer, which can be accurately measured using a freezing point osmoremeter (Osmomat 3000basic, Gonote).

[0351] Table 3

[0352] Compound channel 1 concentration (mRL%) <![CDATA[P f,ch (μm / s)]]> 0.1 13.354±2.351 0.15 17.598±3.944 0.30 35.593±7.213 0.45 63.269±9.646 0.60 103.461±11.327

[0353] Depend on Figure 1 As shown in the table above, the water permeability (P) of the lipid bilayer is... f The concentration of compound channel 1 (expressed as the molar ratio of compound channel 1 to lipid (mRL)) increased with increasing concentration. This indicates that compound channel 1 can spontaneously insert into the lipid bilayer to form transmembrane channels to mediate water transport.

[0354] Water permeability of compound channel 1 (P) f,ch The water permeability (P) of the phospholipid membrane embedded with channel 1 f,cm Subtract the water permeability of the blank phospholipid membrane (P) f,m The value was obtained as 23.467 ± 0.835. The single-channel water permeability (P1) of compound channel 1 was further calculated using the following formula. sc ):

[0355]

[0356] in

[0357] S in This is the initial internal surface area of ​​the liposome;

[0358] P f,ch Water permeability of compound channel 1; and

[0359] N c / v This is the number of channels per liposome, calculated in section (b) above.

[0360] Table 4

[0361] Compound channel 1 concentration (mRL%) <![CDATA[P sc (H2O·s -1 ·aisle -1 )]]> 0.1 <![CDATA[6.22±1.05×10 8 ]]> 0.15 <![CDATA[6.51±1.40×10 8 ]]> 0.30 <![CDATA[7.41±1.44×10 8 ]]> 0.45 <![CDATA[9.16±1.34×10 8 ]]> 0.60 <![CDATA[1.21±0.13×10 9 ]]>

[0362] The data in the table above shows that P sc The value remained essentially constant with changes in the concentration of compound channel 1, indicating that compound 1 forms a monomolecular channel in the lipid bilayer.

[0363] Similarly, P was obtained from compound channels 11 and 13. f,ch (μm / s) and P sc (H2O / s / channel) is shown in the table below:

[0364] Table 5

[0365] Compound channel (mRL = 0.6%) <![CDATA[P f,ch (μm / s)]]> <![CDATA[P sc (H2O / s / channel)]]> 1 103.461±11.327 <![CDATA[1.21±0.13×10 9 ]]> 11 71.365±7.411 <![CDATA[6.77±0.70×10 8 ]]> 13 21.362±3.150 <![CDATA[1.70±0.25×10 8 ]]>

[0366] (d)H + Transport activity: H was investigated using a stop-flow fluorescence experiment. +Transport activity. Liposomes containing HPTS were prepared using a phosphate buffer (10 mM potassium phosphate, 100 mM KCl, pH 7.2) containing HPTS (0.1 mM), following a similar procedure to that described in Section (a) above. HPTS is an H+ phosphate buffer solution. + Sensitive dyes. H is driven by exposing liposomes to a buffer solution at pH 6.0. + The liposomes were pretreated with valinomycin (10 μM) before being mixed with an acidic solution. H was assessed by monitoring fluorescence intensity at 515 nm. + Transport activity (excited at 460 nm).

[0367] Table 6

[0368]

[0369]

[0370] The results in the table above indicate that compound channel 1 can effectively exclude H + Transport. However, the presence of the natural ion channel gramicidin A (gA) leads to a significant decrease in fluorescence intensity.

[0371] Example 3: Performance of Compound Channel 1 in Living Cell Membranes

[0372] (a) Cell culture: Mouse fibroblasts (L929) were cultured in standard Dulbecco modified Eagle medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% penicillin / streptomycin under a humid atmosphere of 5% CO2 / 95% air at 37°C.

[0373] (b) Cell membrane binding efficiency of compound channel 1: The feasibility of compound channel 1 embedding into the cell membrane was first investigated using confocal laser scanning microscopy. In the experiment, L929 cells were first cultured for 10 minutes in a medium containing the fluorescent membrane tracer DiO (2.0 μM), and then washed three times with PBS buffer to remove free DiO. The cells were then cultured for 30 minutes in a medium containing 5(6)-carboxy-tetramethylrhodamine (TAMRA)-labeled compound channel 1 (1.0 μM). After washing three times with PBS buffer to remove free TAMRA-labeled compound channel 1, the cells were subsequently imaged. The fluorescence of DiO was excited by a 488 nm laser, and the emission light was collected at 500–550 nm. The fluorescence of TAMRA-labeled compound channel 1 was excited by a 561 nm laser, and the emission light was collected at 570–620 nm. Representative images of the cells are shown below. Figure 2 As shown.

[0374] Figure 2 The TAMRA images in the image represent confocal laser scanning microscope images of cells cultured in a medium containing the compound Channel 1 labeled with 5(6)-carboxy-tetramethylrhodamine. Figure 2 The DiO images in the image represent confocal laser scanning microscope images of cells cultured in a medium containing the fluorescent membrane tracer DiO. Figure 2 The BF picture in the image indicates that it is a public event; Figure 2 The Merge image in the image represents the superposition of images of channel 1, DiO, and BF of the 5(6)-carboxy-tetramethylrhodamine-labeled compound.

[0375] Figure 2 The TAMRA image shows cells in red, the DiO image shows cells in green, and the Merge image shows cells in yellow, which clearly indicates that the channel 1 molecule of compound is embedded in the cell membrane.

[0376] Cell membrane binding efficiency was further quantified using fluorescence measurements. Cells were cultured for 3 h in a medium containing compound channel 1 (1.0 μM). The fluorescence intensity (F) of the medium at 490 nm (excitation at 245 nm) was measured before and after incubation. The equation (F) was used to... 后 -F 前 ) / F 前 The cell membrane binding efficiency (E2) of the channel at a given channel concentration was calculated by multiplying by 100%; the result was 26.3%.

[0377] (c) Number of channels per cell (N) c / c ): CountBright TM Absolute counting beads were mixed with cell samples and analyzed by flow cytometry. The absolute number of cells (N) in the sample could be calculated by comparing the bead-to-cell ratio. 细胞 The number of channels (N) per cell is calculated using the following formula: N = N c ×E2 / N 细胞 , where N c N is the number of channels in channel 1 (1.0 μM) of a given concentration of compound. 细胞 The cell number is N, and E2 is the cell membrane binding efficiency of compound channel 1. The calculated N = 1.7 × 10⁻⁶. 7 Channels / cells.

[0378] (d) D2O transport study: L929 cells were cultured in medium containing fluorescent yellow glucan (LYD) (25 μM) and then washed with LYD-free medium to obtain cells loaded with the D2O-sensitive fluorescent probe, fluorescent yellow glucan (LYD) (reference [S4]). These cells were incubated for 3 hours in medium containing compound channel 1 (1.0 μM). Before imaging, LYD-loaded L929 cells were treated for 30 minutes with 170 mOsm Tris-HCl buffer (H2O-TBS, 5.7 mM NaCl, 1.79 mM KCl, 0.42 mM CaCl2, 0.27 mM MgCl2, 1.87 mM glucose, 20 mM Tris-HCl, and 4 mM NaHCO3, pH 7.20). H2O-TBS was then completely removed. Subsequently, D2O-TBS was added to the resulting cells, and the fluorescence intensity of the cells was measured (excitation: 450-490 nm, emission: LP 515 nm). The results are shown in the table below.

[0379] Table 7

[0380] LYD fluorescence intensity (F, %) Cells treated with compound channel 1 37.312±4.516 Cells lacking compound channel 1 (null) 11.239±1.342

[0381] (e) Measurement of P in cell membranes sc The permeability of compound channel 1 in the cell membrane was determined using a stop-flow apparatus in fluorescence mode. The cell suspension was placed in one syringe of the instrument. D2O-TBS was placed in another syringe. The LYD fluorescence intensity (λ) was recorded as the cell solution in the instrument was rapidly mixed with the D2O-TBS. 激发 =430nm,λ 发射 =525nm) as a function of time. Five parallel tests were performed, and the curves are the average results. Intracellular [D2O] was calculated using the standard curve of LYD fluorescence intensity versus [D2O] (refer to [S5]). 内 ).

[0382] Table 8

[0383]

[0384]

[0385] The single-channel water permeability of compound channel 1 is calculated using the following formula:

[0386] P sc =v0N A V0 / N

[0387] Where v0 is the initial velocity determined by the slope of the curve at t = 0 s. N AV is Avogadro's constant; V0 is the cell volume, roughly calculated based on the cell diameter in the suspension (26.4 μm); N is the number of channels per cell, calculated in section c of Example 3; P on the cell membrane was calculated. sc =4.6×10 8 Water / second / channel.

[0388] (f) Cell depolarization assay (refer to [S6]): L929 cells were obtained following the same procedure as described in Section 3(a) of Example 3. After trypsin digestion, a cell suspension was obtained and diluted with PBS buffer to a final concentration of 2.5 × 10⁻⁶. 5 Cells / mL. Add cell suspension (2 mL) and saffron O (3.0 μM) to a test tube and stir gently. Continuously monitor fluorescence intensity at 581 nm (excitation at 522 nm) until the fluorescence intensity reaches a stable value (1 min). Then, add DMSO solution of compound channel 1 with stirring to reach a final concentration of 1.0 μM, and record fluorescence intensity for another 2 min.

[0389] Table 9

[0390] 0min 1min 2min 3min Fluorescence intensity of cells treated with compound channel 1 95.121 93.647 95.186 93.951 Fluorescence intensity of cells without compound channel 1 94.995 95.923 95.648 95.129 fluorescence intensity of cells treated with gA 94.808 94.369 82.445 81.008

[0391] Due to the difference in ion concentrations inside and outside the cell, the active cell membrane maintains a resting potential. This potential can be monitored using the fluorescent probe saffron O. As shown in the table above, the binding of compound channel 1 to the cell membrane resulted in a negligible change in the fluorescence intensity of saffron O. However, the presence of the natural ion channel bacitracin A (gA) led to a significant decrease in fluorescence intensity. These observations indicate that compound channel 1 also effectively excludes ions within the cell membrane. Despite the complex environment of the cell membrane, embedding compound channel 1 into the cell membrane did not result in a significant reduction in permeability and selectivity. The high water permeability and high selectivity exhibited by compound channel 1 in living cell membranes clearly demonstrate its potential as an artificial aquaporin in biological systems.

[0392] Example 4 – Effect of Compound Channel 1 on In Vivo Tympanic Membrane Perforation Healing

[0393] (a) Induction and treatment of tympanic membrane perforation: Mice were anesthetized by intraperitoneal injection of a combination of tiletamine (15 mg / kg), zolazepam (15 mg / kg / kg), and dexmedetomidine (5 mg / kg). Electrootoscopy ruled out middle ear effusion. Subsequently, acute perforations were created in the pars tensa of both tympanic membranes using custom-made needles (reference [S7]). The needles had an outer diameter of 0.45 mm and a black tip, and were held by a manual micromanipulator (Marzhauser Wetzlar, Germany). To ensure that the tympanic membrane perforations were of equal size, the needles were inserted into the tympanic membranes until their black tips were no longer visible. One day after the tympanic membrane perforation, a piece of gelatinous sponge larger than the perforation was soaked in Compound Channel 1 (1.0 μM saline) and placed on the residual tympanic membrane of the right ear. Each animal served as a self-control, with the right ear as the treatment side of Compound Channel 1 and the left ear as the control side of saline. Animals were euthanized at the following time points: 0 days (4 mice), 2 days (3 mice), 4 days (3 mice), or 6 days (14 mice) after tympanic membrane perforation.

[0394] (b) Histological examination of tympanic membrane perforation repair: After collecting the audible bullae, the samples were fixed overnight in 4% paraformaldehyde at 4°C. The next day, the fixed tympanic membrane was washed in 1×PBS and then decalcified in 120mM EDTA. For tympanic membrane spread staining, the entire tympanic membrane was dissected and used for staining; for tympanic membrane section staining, the decalcified tympanic membrane was dehydrated in 30% sucrose, embedded in OCT, and then cut into 14μm thick sections perpendicular to the malleus handle using a cryostat. For immunofluorescence staining, the tympanic membrane spreads or sections were blocked with 1% Triton X-100 and 10% donkey serum at room temperature, and then the samples were incubated overnight at 4°C with mouse anti-Vimentin (1:200) primary antibody. The next day, after washing three times in PBS, the samples were incubated with secondary antibody at room temperature for 2 hours, and then incubated with DAPI for 5 minutes for nuclear staining. The tympanic membrane was covered with a coverslip and allowed to dry, then scanned using a Zeiss confocal microscope (LSM 800) under 10X or 40X objectives. Images were edited using Adobe Photoshop and ImageJ software. Tools in ImageJ were used to determine the scale bar and perforation area. Figure 3 The morphology of the tympanic membrane after treatment with compound channel 1 or saline is shown. Immunofluorescence staining of vimentin and DAPI in the tympanic membrane indicates perforation healing. The perforation edges of the tympanic membrane healed with compound channel 1 (experimental group) were relatively smooth, indicating that embedding compound channel 1 into the cell membrane can effectively promote the healing of tympanic membrane perforations. Compared with the control group, the perforation area in the experimental group was smaller at 4 and 6 days after perforation formation.

[0395] Table 11 - Perforation area after treatment with compound channel 1

[0396]

[0397]

[0398] (c) Hearing Tests: The ototoxicity of compound channel 1 was further evaluated in mice with healed perforations via auditory brainstem response (ABR). Hearing thresholds were measured using an RZ6 acoustic system (Tucker-Davis Technologies, Alachua, FL, USA) before and 6 days after tympanic membrane perforation via ABR. Mice were anesthetized and placed on a heating pad to maintain a body temperature of 37°C. Gelatin sponges (if available) were gently removed from the external auditory canal under a microscope using forceps. During signal acquisition, an earpiece was placed inside the external auditory canal. Recording, reference, and grounding electrodes were placed subcutaneously at the midline of the mouse skull, the mastoid process of the recording ear, and the contralateral mastoid process, respectively. Hearing thresholds were assessed at five frequencies (4, 8, 16, 24, and 32 kHz). Stimulus levels started at 90 dB and decreased in 5 dB increments to 20 dB. Hearing thresholds were defined as the lowest sound stimulus capable of evoking a reproducible ABR waveform. The detailed protocol for measurement is described in (Ref. [S8]).

[0399] Table 12 - Hearing thresholds at different frequencies before tympanic membrane perforation (data in the table are average values ​​for 14 mice)

[0400]

[0401] Table 13 - Hearing thresholds at different frequencies after tympanic membrane perforation (data in the table are average values ​​for 13 mice).

[0402]

[0403] The hearing thresholds at different frequencies before and 6 days after tympanic membrane perforation are shown in the table above. The hearing thresholds of mice treated with compound channel 1 were very similar to those of untreated mice, indicating that in vivo administration of compound channel 1 to the tympanic membrane does not cause ototoxicity.

[0404] References:

[0405] [S1]Xiao,Q.;Haoyang,W.-W.;Lin,T.;Li,Z.-T.;Zhang D.-W.;Hou,J.-L.,Unimolecular artificial transmembrane channels showing reversible ligand-gating behavior.Chem.Commun.,2021,57,863-866.

[0406] [S2]Yan,Z.-J.;Wang,D.;Ye,Z.;Fan,T.;Wu,G.;Deng,L.;Yang,L.;Li,B.;Liu,J.;Ma,T.;Dong,C.Li,Z.-T.;Xiao,L.;Wang,Y.;Wang,W.;Hou,J.-L.,Artificialaquaporin that restores wound healing of impaired cells.J.Am.Chem.Soc.,2020,142,15638-15643.

[0407] [S3]Li,Q.;Li,X.;Ning,L.;Tan,C.H.;Mu,Y.;Wang,R.,Hyperfast watertransport through biomimetic nanochannels from peptide-attached(pR)-pillar[5]arene.Small,2019,15,1804678.

[0408] [S4]Li,P.;Hua,M.,Wang,C.;Feng,X.;Zhao,Z.;Yang,Y.;Sahoo,N.;Gu,M.;Yang,Y.;Xiao,S.;Sah,R.;.Cover,T.L.;Chou,J.;Geha,R.;Benavides,F.;Hume,R.I.;Xu,H.,LRRC8 family proteins within lysosomes regulate cellular osmoregulation andenhance cell survival to multiple physiological stresses.Proc.Natl.Acad.Sci.U.S.A.,2020,117,29155-29165.

[0409] [S5]Licsandru,E.;Kocsis,I.;Shen,Y.X.;Murail,S.;Legrand,Y.-M.;van derLee,A.;Tsai,D.;Baaden,M.;Kumar,M.;Barboiu,M.,J.Am.Chem.Soc.,2016,138,5403-5409.

[0410] [S6]Zhang,M.;Zhu,P.-P.;Xin,P.;Si,W.;Li,Z.-T.;Hou,J.-L.,Syntheticchannel specifically inserts into the lipid bilayer of gram-positive bacteriabut not that of mammalian erythrocytes.Angew.Chem.Int.Ed.,2017,56,2999-3003.

[0411] [S7](a)Chari,D.A.;Frumm,S.M.;Akil,O.;Tward,A.D.,Cellular dynamics inearly healing of mouse tympanic membranes.Otol.Neurotol.,2019,40,e160-e166.(b)Goncalves,S.;Bas,E.;Goldstein,B.J.;Angeli,S.,Effects of cell-based therapyfor treating tympanic membrane perforations in mice.Otolaryngol.Head NeckSurg.,2016,154,1106-1114.

[0412] [S8]He, Y.; Li, W.; Zheng, Z.; Zhao, L.; Li, W.; Wang, Y.; Li, H., Inhibition ofProtein arginine methyltransferase 6reduces reactive oxygen species production and attenuates aminoglycoside-and cisplatin-induced hair celldeath. Theranostics, 2020, 10, 133-150.

[0413] The above description is merely a preferred embodiment of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made within the scope of the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. An amino acid-modified compound having the formula (I-1): (I-1) in n1 is 5; R 1 It is 1,2-ethylene; R 2 It is methylene; R 3 It is 1,2-ethylene; R 4 It is methylene; z is an integer, which is chosen so that the entire molecule is uncharged; Z is the counter ion; and Short peptides containing leucine and tryptophan, where all R values ​​are the same and they are of the following type: 。 2. A method for preparing an amino acid-modified compound as defined in claim 1, comprising: 1) Reacting compound (II) with formaldehyde yields compound (III). (II) (III) Where X is chlorine, bromine, or iodine; and n1, R 1 and R 3 As defined in claim 1; 2) Reacting compound (III) with an alkali metal azide yields compound (IV): (IV) Where n1, R 1 and R 3 As defined above; 3) Compound (IV) is reacted with compounds (V-1) and (V-2) via a click chemistry reaction to yield compound (VI): (V-1) (V-2) in R 2 and R 4 As defined in claim 1; R' corresponds to R as defined in claim 1, but the reactive amino group in the amino acid molecule of R' is protected by Pro; and Pro is the protecting group for the amino group in the amino acid molecule of R'; (WE) Where n1, R 1 R 2 R 3 R 4 R' and Pro are as defined above; 4) Remove the protecting group Pro from the amino group in compound (VI) and make the amino group positively charged to obtain the amino acid-modified compound.

3. The method according to claim 2, wherein X is bromine.

4. A method for preparing an amino acid-modified compound as defined in claim 1, comprising: i) Reacting compound (II) with an alkali metal azide to give compound (A) (II) (A) Where X is chlorine, bromine, or iodine; and R 1 and R 3 As defined in claim 1; ii) Reacting compound (A) with compounds (V-1) and (V-2) via click chemistry to obtain compound (B): (V-1) (V-2) in R 2 and R 4 As defined in claim 1; R' corresponds to R as defined in claim 1, but the reactive amino group in the amino acid molecule of R' is protected by Pro; and Pro is the protecting group for the amino group in the amino acid molecule of R'; (B) Where R 1 R 2 R 3 R 4 R' and Pro are as defined above; iii) Reacting compound (B) with formaldehyde to yield compound (VI): (WE) Wherein n1 is defined as in claim 1; R 1 R 2 R 3 R 4 R' and Pro are as defined above; iv) Remove the protecting group Pro from the amino group in the compound of formula (VI) and make the amino group positively charged to obtain the amino acid-modified compound.

5. The method according to claim 4, wherein X is bromine.

6. The method according to claim 2 or 4, wherein the reactions in steps 1) and iii) are carried out in the presence of a Lewis acid.

7. The method according to claim 6, wherein the Lewis acid is selected from FeCl3, AlCl3, SnCl4, BF3, BF3·O(C2H5)2 and organic sulfonic acids.

8. The method according to claim 2 or 4, wherein steps 4) and iv) are carried out in the presence of a protic acid.

9. Compounds of formula (VI): (WE) in n1, R 1 R 2 R 3 and R 4 As defined in claim 1; R' corresponds to R as defined in claim 1, but the reactive amino group in the amino acid molecule of R' is protected by Pro; and Pro is the protecting group for the amino group in the amino acid molecule of R'.

10. Compounds of formula (I''): (I’’) in n1, R 1 R 2 R 3 and R 4 As defined in claim 1; R” corresponds to R as defined in claim 1, but R” does not carry a positive charge.

11. A pharmaceutical composition comprising an amino acid-modified compound as defined in claim 1.

12. Use of the amino acid-modified compound as defined in claim 1 in the preparation of a medicament for repairing the tympanic membrane.

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