Antibacterial peptide having broad-spectrum antibacterial activity and preparation method therefor

Abstract

Provided are an antibacterial peptide and the use thereof. The antibacterial peptide has a structure represented by formula (I), wherein Z1 is selected from tetrapeptides, the tetrapeptides being optionally modified by -OH, -CHO, -COOH, -NH2, -SO3H or -C=O; S1 and S2 are each independently selected from amino acids having the structure of (I); Q1 and Q2 are each independently selected from dipeptides or tripeptides; and n is independently selected from any integer between 1 and 6. (Q2)4-(S2-Q1)2-S1-Z1

Description

Antibacterial peptide with broad-spectrum antibacterial activity and preparation method thereof TECHNICAL FIELD

[0001] The present application belongs to the field of chemical biotechnology, and particularly relates to an antibacterial peptide with broad-spectrum antibacterial activity and a preparation method thereof. BACKGROUND

[0002] Antimicrobial peptides (AMPs) are a class of small molecule polypeptides widely existing in the natural immune system of organisms, have broad-spectrum antibacterial effect, and mostly exist in linear AMPs and cyclic AMPs. The antibacterial peptide kills bacteria by physically destroying the bacterial biofilm. Compared with traditional antibiotics, the antibacterial peptide is less likely to produce drug resistance. In recent years, researchers have found a new type of antibacterial peptide, namely dendritic antibacterial peptide, which can also be called antimicrobial peptide dendrimer (AMPD). It is defined as a branched compound with several copies of peptide monomers connected to a template or core matrix. This new type of antibacterial peptide has higher bactericidal activity, better biocompatibility and stability, and has good application prospect.

[0003] The research group of the University of Bern first screened a combinatorial library through a growth inhibition test of Bacillus subtilis ("Membrane disrupting antimicrobial peptide dendrimers with multiple amino termini." Med Chem Commun. 2012, 3, 86), and identified the first membrane-disrupting third-generation AMPD - bH1, which has only one residue on each branch, and the positive charge is provided by the amino terminus. The AMPD shows good antibacterial activity against Bacillus subtilis, but only slight activity against Pseudomonas aeruginosa. Therefore, a third-generation AMPD - G3KL ("Combining Topology and Sequence Design for the Discovery of Potent Antimicrobial Peptide Dendrimers against Multidrug-Resistant Pseudomonas aeruginosa." Angew Chem Int Ed. 2014, 53, 12827-12831) was obtained through multivalent and sequence design, which increases the number of positive charges and hydrophobic groups in the dendritic branches, and can produce antibacterial activity against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii.

[0004] Due to the relatively large size of the third generation AMPD molecules and the difficulty in synthesizing them in high yield, the research group at the University of Bern has disclosed a series of second generation AMPD in WO2015 / 144928A1 and the literature ("Lipidated Peptide Dendrimers Killing Multidrug Resistant Bacteria." J. Am. Chem. Soc. 2018, 140, 423-432) which can kill quality control bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii, and a few polypeptides only show antibacterial activity against drug-resistant Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA).

[0005] It can be seen that the current second generation AMPD has limited antibacterial species against drug-resistant bacteria, which cannot meet the clinical needs of intensive care units (ICU), and cannot be used in humans, so researchers still need to work on more broad-spectrum and efficient dendritic antibacterial polypeptides. SUMMARY

[0006] The present application aims to at least partially solve at least one of the technical problems existing in the prior art, and therefore provides a new type of second generation dendritic antibacterial polypeptide with stronger antibacterial properties, which can effectively inhibit a variety of common bacteria and exhibit excellent antibacterial activity against widely existing multi-drug resistant bacteria.

[0007] Therefore, in a first aspect of the present application, an antibacterial peptide is provided. According to embodiments of the present application, the antibacterial peptide has a structure represented by Formula (I) or a stereoisomer, tautomer or salt of the structure represented by Formula (I):

[0008] (Q 2 )4-(S 2 -Q 1 ) - S 1 -Z 1

[0009] (I)

[0010] wherein Z 1 is selected from a tetrapeptide optionally modified with -OH, -CHO, -COOH, -NH2, -SO3H or -C=O; S 1 , S 2 are each independently selected from an amino acid having the structure ; Q 1 , Q 2 are each independently selected from a dipeptide or a tripeptide; and n are each independently selected from any integer between 1 and 6.

[0011] The antibacterial peptide provided by the present application not only has excellent antibacterial activity, but also shows more broad-spectrum antibacterial ability. In addition to having antibacterial effect on common gram-negative bacteria and gram-positive bacteria, the antibacterial peptide can also effectively inhibit various drug-resistant gram-negative bacteria and gram-positive bacteria. This broad-spectrum antibacterial activity enables the antibacterial peptide to be widely used in the field of antibacterial and can effectively deal with the increasing problem of drug-resistant bacteria.

[0012] In a second aspect of the present application, an antibacterial peptide is provided. According to an embodiment of the present application, there is provided an antibacterial peptide having a structure represented by formula (II) or a stereoisomer, a tautomer or a salt of the structure represented by formula (II):

[0013] (Q 4 ) - (K-Q 3 )2-K-Z 2

[0014] (II)

[0015] wherein Z 2 is selected from a tetrapeptide optionally modified by -OH, -CHO, -COOH, -NH2, -SO3H or -C=O; Q 3 , Q 4 are each independently selected from a dipeptide or a tripeptide; and K is lysine.

[0016] The antibacterial peptide provided by the present application not only has excellent antibacterial activity, but also shows more broad-spectrum antibacterial ability. In addition to having antibacterial effect on common gram-negative bacteria and gram-positive bacteria, the antibacterial peptide can also effectively inhibit various drug-resistant gram-negative bacteria and gram-positive bacteria. This broad-spectrum antibacterial activity enables the antibacterial peptide to be widely used in the field of antibacterial and can effectively deal with the increasing problem of drug-resistant bacteria.

[0017] In a third aspect of the present application, an antibacterial peptide is provided. According to an embodiment of the present application, there is provided an antibacterial peptide having a structure represented by formula (III) or a stereoisomer, a tautomer or a salt of the structure represented by formula (III):

[0018] (Q 6 )4-(K-Q 5 ) 2 -K-Z 3

[0019] (III)

[0020] wherein Z 3 is selected from a tetrapeptide; Q 5 , Q 6 are each independently selected from a dipeptide or a tripeptide consisting of lysine, leucine, phenylalanine and / or ornithine, and Q5 or Q 6 does not comprise two consecutive leucines; K is lysine.

[0021] The antibacterial peptide has excellent antibacterial activity, can effectively inhibit various bacteria, especially common gram-negative bacteria and gram-positive bacteria, in addition, the inventors find through a large number of experiments that the antibacterial peptide also exhibits excellent antibacterial effect on various drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria, has broad-spectrum antibacterial activity. This makes it a promising antibacterial agent, which is expected to play an important role in bacterial infection treatment and control.

[0022] In a fourth aspect of the present application, an antibacterial peptide is provided. According to embodiments of the present application, there is provided a compound having a structure represented by formula (IV) or a stereoisomer, a tautomer or a salt of the structure represented by formula (IV):

[0023] (Q 8 )4-(K-Q 7 )2-K-Z 4

[0024] (IV)

[0025] wherein Z 4 is a tetrapeptide selected from the group consisting of leucine and / or phenylalanine; Q 7 , Q 8 are each independently selected from the group consisting of dipeptide or tripeptide of lysine and / or leucine; K is lysine.

[0026] The antibacterial peptide has excellent antibacterial activity, can effectively inhibit various bacteria, especially common gram-negative bacteria and gram-positive bacteria, in addition, the inventors find through a large number of experiments that the antibacterial peptide also exhibits excellent antibacterial effect on various drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria, has broad-spectrum antibacterial activity. This makes it a promising antibacterial agent, which is expected to play an important role in bacterial infection treatment and control.

[0027] In a fifth aspect of the present application, an antibacterial peptide is provided. According to embodiments of the present application, there is provided a compound having a structure represented by formula (V) or a stereoisomer, a tautomer or a salt of the structure represented by formula (V):

[0028] (Q 10 )4-(K-Q 9 )2-K-LLLL

[0029] (V)

[0030] wherein Q 9 , Q 10are independently selected from dipeptides or tripeptides consisting of lysine and / or leucine; K is lysine, and L is leucine.

[0031] The antibacterial peptide has excellent antibacterial activity and can effectively inhibit various bacteria.

[0032] In a sixth aspect, the present application provides an antibacterial peptide. According to an embodiment of the present application, the antibacterial peptide has a structure represented by formula (VI) or a stereoisomer, a tautomer or a salt of the structure represented by formula (VI):

[0033] (Q 12 )4-(K-Q 11 )2-K-LLLL

[0034] (VI)

[0035] wherein Q 11 are independently selected from dipeptides or tripeptides consisting of lysine and / or leucine; Q 12 are independently selected from dipeptides consisting of lysine and / or leucine; K is lysine, and L is leucine.

[0036] The antibacterial peptide has excellent antibacterial activity and can effectively inhibit various bacteria.

[0037] In a seventh aspect of the present application, the present application provides an antibacterial peptide. According to embodiments of the present application, the antibacterial peptide has one of the following structures: (kl)4(kkl)2klllf, (kl)4(kkl)2klflf, (kl)4(kkl)2kvlvl, (lk)4(kkl)2kllll, (kl)4(klk)2kllll, (lk)4(klk)2kllll, (kll)4(kkl)2kllll, (klk)4(kkl)2kllll, (kl)4(kkll)2kllll, (kl)4(kkkl)2kllll, (of)4(kkl)2kllll, (kf)4(kkl)2kllll, (kl)4(kkf)2kllll, (kf)4(kof)2kllll, or (kl)4(kkl)2kllll.

[0038] The antibacterial peptide provided by the present application not only has excellent antibacterial activity, but also shows more broad-spectrum antibacterial ability. In addition to having antibacterial effects on common gram-negative bacteria and gram-positive bacteria, the antibacterial peptide can also effectively inhibit various drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria. This broad-spectrum antibacterial activity enables the antibacterial peptide to be widely used in the field of antibacterial treatment and to effectively deal with the increasing problem of drug-resistant bacteria.

[0039] In an eighth aspect of the present application, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition comprises the antibacterial peptide of the first aspect, the second aspect, the third aspect, the fourth aspect, the fifth aspect, the sixth aspect, and / or the seventh aspect. As described above, the antibacterial peptide of the present application exhibits excellent antibacterial activity and broad-spectrum antibacterial ability. Therefore, based on these characteristics, the pharmaceutical composition provided also has excellent effects and can effectively inhibit various bacteria, even including drug-resistant bacteria, and can be applied to the field of antibacterial treatment.

[0040] In a ninth aspect of the present application, the present application provides use of the antibacterial peptide of the first aspect, the second aspect, the third aspect, the fourth aspect, the fifth aspect, the sixth aspect, and / or the seventh aspect or the pharmaceutical composition of the eighth aspect in the preparation of a medicament for preventing or treating bacterial infection.

[0041] In a tenth aspect of the present application, the present application provides use of the antibacterial peptide of the first aspect, the second aspect, the third aspect, the fourth aspect, the fifth aspect, the sixth aspect, and / or the seventh aspect or the pharmaceutical composition of the eighth aspect in preventing or treating bacterial infection.

[0042] In an eleventh aspect of the present application, the present application provides use of the antibacterial peptide according to the first aspect, the second aspect, the third aspect, the fourth aspect, the fifth aspect, the sixth aspect and / or the seventh aspect in the preparation of an antibiotic, a disinfectant, a cleaner or a preservative for treating a bacterial infection.

[0043] In a twelfth aspect of the present application, the present application provides a method for preventing or treating a bacterial infection. According to an embodiment of the present application, the method comprises administering to a subject an effective amount of the antibacterial peptide according to the first aspect, the second aspect, the third aspect, the fourth aspect, the fifth aspect, the sixth aspect and / or the seventh aspect or the pharmaceutical composition according to the eighth aspect. As described previously, the antibacterial peptide and the pharmaceutical composition of the present application have excellent antibacterial activity and broad-spectrum antibacterial ability. Thus, by using the method of the present application, a bacterial infection can be effectively prevented or treated.

[0044] Additional aspects and advantages of the present application will be set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the following description, or can be learned by practice of the present application. DETAILED DESCRIPTION

[0045] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not to be construed as limiting the present application.

[0046] As used in the description of the application and the appended claims, the singular forms "a", "an" and "the" are intended to include plural forms as well, unless the context clearly indicates otherwise.

[0047] The terms "comprise", "comprising", and "comprises" or "comprising" as used herein specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0048] As used herein, the term "optionally", "optional" or "optional" generally means that the event or circumstance subsequently described can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0049] As used herein, the term "amino acid" relates to naturally occurring amino acids, in particular proteinogenic amino acids, as well as non-natural amino acids. Amino acids can be shown in the three-letter code (Stryer, Biochemistry, 3rdedition, page 21) or the one-letter code. Amino acids can be in the D- or L-configuration. Based on the chemical properties of their side chains, amino acids can be classified as hydrophobic amino acids and cationic amino acids.

[0050] As used herein, the term "hydrophobic amino acid" is any alpha-aminocarboxylic acid with a side chain that has no hydrogen bond donor or acceptor. Hydrophobic amino acids include, but are not limited to, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, proline, or tryptophan.

[0051] As used herein, the term "cationic amino acid" is an alpha-aminocarboxylic acid with a side chain comprising a chemical functional group that exhibits a cation at physiological pH. Cationic amino acids include, but are not limited to, lysine, arginine, histidine, ornithine, aspartic acid, or L-beta-leucine.

[0052] As used herein, the anti-bacterial peptide sequences given herein are given from N-terminus to C-terminus. The terminal carboxyl group of the anti-bacterial peptides referred to herein can be a carboxylic acid, a carboxylate salt (COO-), or an amide (CONH2) group.

[0053] As used herein, the term "quality control organism" is a microorganism strain that has been specifically screened and identified to be stable, reproducible, standardized, and representative.

[0054] As used herein, the term "drug-resistant Gram-negative bacteria" is a Gram-negative bacteria that has developed resistance to the action of an anti-bacterial drug, and the term "drug-resistant Gram-positive bacteria" is a Gram-positive bacteria that has developed resistance to the action of an anti-bacterial drug.

[0055] Anti-bacterial peptide

[0056] In a first aspect of the present application, the present application proposes an anti-bacterial peptide. According to embodiments of the present application, the anti-bacterial peptide has a structure shown in Formula (I) or a stereoisomer, a tautomer, or a salt of the structure shown in Formula (I):

[0057] (Q 2 )4-(S 2 -Q 1 )2 - S 1 -Z 1

[0058] (I)

[0059] wherein Z 1 is selected from a tetrapeptide, which is optionally modified by -OH, -CHO, -COOH, -NH2, -SO3H, or -C=O; S 1 , S 2 are each independently selected from an amino acid having structure; Q 1 , Q 2 are each independently selected from a dipeptide or a tripeptide; and n are each independently selected from any integer between 1 and 6.

[0060] The antibacterial peptide provided by the present application not only has excellent antibacterial activity, but also shows more broad-spectrum antibacterial ability. In addition to having antibacterial effect on common gram-negative bacteria and gram-positive bacteria, the antibacterial peptide can also effectively inhibit various drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria. This broad-spectrum antibacterial activity enables the antibacterial peptide to be widely used in the field of antibacterial and can effectively deal with the increasing problem of drug-resistant bacteria.

[0061] It should be noted that the "tetrapeptide" described in the present application refers to a straight-chain polypeptide composed of four amino acids.

[0062] In some embodiments of the present application, the antibacterial peptide represented by the structure of formula (I) can further include at least one of the following additional technical features:

[0063] In some embodiments of the present application, the tetrapeptide includes at least one amino acid containing a hydrophobic side chain. Therefore, the presence of these amino acids can enhance the effect of the antibacterial peptide on the bacterial membrane, causing the bacterial membrane to be destabilized, thereby increasing the probability of bacterial death.

[0064] In some embodiments of the present application, the tetrapeptide includes at least three amino acids containing a hydrophobic side chain. Therefore, the presence of these amino acids can enhance the effect of the antibacterial peptide on the bacterial membrane, causing the bacterial membrane to be destabilized, thereby increasing the probability of bacterial death.

[0065] In some embodiments of the present application, the tetrapeptide includes amino acids containing a hydrophobic side chain and amino acids containing a cationic side chain. Therefore, the presence of these amino acids can enhance the effect of the antibacterial peptide on the bacterial membrane, causing the bacterial membrane to be destabilized, thereby increasing the probability of bacterial death.

[0066] In some embodiments of the present application, the dipeptide includes amino acids containing a hydrophobic side chain and amino acids containing a cationic side chain. Therefore, the presence of these amino acids can enhance the effect of the antibacterial peptide on the bacterial membrane, causing the bacterial membrane to be destabilized, thereby increasing the probability of bacterial death.

[0067] In some embodiments of the present application, the tripeptide includes amino acids containing a hydrophobic side chain and amino acids containing a cationic side chain. Therefore, the presence of these amino acids can enhance the effect of the antibacterial peptide on the bacterial membrane, causing the bacterial membrane to be destabilized, thereby increasing the probability of bacterial death.

[0068] It should be noted that the "dipeptide or tripeptide" described in the present application refers to a straight-chain polypeptide composed of two amino acids or three amino acids.

[0069] In some embodiments of the application, the hydrophobic side chain containing amino acid is selected from glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), proline (Pro, P), or tryptophan (Trp, W).

[0070] In some embodiments of the application, the cationic side chain containing amino acid is selected from lysine (Lys, K), arginine (Arg, R), histidine (His, H), ornithine (Orn, O), 2,3-diaminopropionic acid (Dap, B), or 2,4-diaminobutyric acid (Dab).

[0071] In some embodiments of the application, the Z 1 is selected from lllf, lllv, llla, llvv, llff, lflf, lvlv, vlvl, flfl, lllll, flll, vlll, vvll, ffll, lklk, lkkk, llkk, lllk, kllk, klkl, kkkl, klll, or kkll. Thus, the presence of these amino acids can enhance the effect of the antibacterial peptide on the bacterial membrane, causing it to destabilize, thus increasing the probability of bacterial death.

[0072] In some embodiments of the application, the S 1 , S 2 are each independently an amino-functionalized alpha-amino acid.

[0073] In some embodiments of the application, the S 1 , S 2 are each independently selected from lysine, ornithine, 2,3-diaminopropionic acid, or 2,4-diaminobutyric acid.

[0074] In some embodiments of the application, the Q 1 , Q 2 are each independently selected from kl, lk, kll, lkl, llk, klk, kkl, of, kf, ol, bl, lb, oll, bll, fk, ffo, vo, vb, wk, fk, fb, bf, rl, or lo. Thus, the presence of these amino acids can enhance the effect of the antibacterial peptide on the bacterial membrane, causing it to destabilize, thus increasing the probability of bacterial death.

[0075] In some embodiments of the present application, the antibacterial peptide of the present application is synthesized by solid phase peptide synthesis, and the antibacterial peptide (or dendritic antibacterial polypeptide) is extended by coupling the active carboxyl group with the amino group of the dendritic polypeptide grown on the solid phase support. In some embodiments of the present application, Z 1 is connected to S 1 through an amide bond between the amino functional group on Z 1 and the carboxylic acid carbon on S 1 , and Q 1 , Q 2 are connected to their respective binding partners S 1 , S 2 through an amide bond between the amino nitrogen on S 1 , S 2 and the carboxylic acid carbon on Q 1 , Q 2 , respectively. Wherein, Z 1 is the central moiety, and in the solid phase chemical method for preparing the antibacterial peptide of the present application, Z 1 is the starting point of synthesis.

[0076] In a second aspect of the present application, an antibacterial peptide is provided. According to embodiments of the present application, the antibacterial peptide has a structure shown in formula (II) or a stereoisomer, a tautomer or a salt of the structure shown in formula (II):

[0077] (Q 4 ) - (K-Q 3 )2-K-Z 2

[0078] (II)

[0079] wherein Z 2 is selected from a tetrapeptide optionally modified with -OH, -CHO, -COOH, -NH2, -SO3H or -C=O; Q 3 , Q 4 are each independently selected from a dipeptide or a tripeptide; and K is lysine.

[0080] The antibacterial peptide provided by the present application not only has excellent antibacterial activity, but also shows more broad-spectrum antibacterial ability. In addition to having antibacterial effect on common gram-negative bacteria and gram-positive bacteria, the antibacterial peptide can also effectively inhibit various drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria. This broad-spectrum antibacterial activity enables the antibacterial peptide to be widely used in the field of antibacterial and can effectively deal with the increasing problem of drug-resistant bacteria.

[0081] In some embodiments of the present application, the antibacterial peptide of the structure shown in formula (II) can further comprise at least one of the following additional technical features:

[0082] In some embodiments of the present application, the tetrapeptide comprises at least 1 amino acid with a hydrophobic side chain. The presence of these amino acids can therefore enhance the effect of the antibacterial peptide on the bacterial membrane, causing it to destabilize, thus increasing the probability of bacterial death.

[0083] In some embodiments of the present application, the tetrapeptide comprises at least 3 amino acids with a hydrophobic side chain. The presence of these amino acids can therefore enhance the effect of the antibacterial peptide on the bacterial membrane, causing it to destabilize, thus increasing the probability of bacterial death.

[0084] In some embodiments of the present application, the tetrapeptide comprises an amino acid with a hydrophobic side chain and an amino acid with a cationic side chain. The presence of these amino acids can therefore enhance the effect of the antibacterial peptide on the bacterial membrane, causing it to destabilize, thus increasing the probability of bacterial death.

[0085] In some embodiments of the present application, the dipeptide comprises an amino acid with a hydrophobic side chain and an amino acid with a cationic side chain. The presence of these amino acids can therefore enhance the effect of the antibacterial peptide on the bacterial membrane, causing it to destabilize, thus increasing the probability of bacterial death.

[0086] In some embodiments of the present application, the tripeptide comprises an amino acid with a hydrophobic side chain and an amino acid with a cationic side chain. The presence of these amino acids can therefore enhance the effect of the antibacterial peptide on the bacterial membrane, causing it to destabilize, thus increasing the probability of bacterial death.

[0087] In some embodiments of the present application, the amino acid with a hydrophobic side chain is selected from glycine (Gly, G), alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), proline (Pro, P) or tryptophan (Trp, W).

[0088] In some embodiments of the present application, the amino acid with a cationic side chain is selected from lysine (Lys, K), arginine (Arg, R), histidine (His, H), ornithine (Orn, O), 2,3-diaminopropionic acid (Dap, B) or 2,4-diaminobutyric acid (Dab).

[0089] In some embodiments of the present application, the Z 2selected from lllf, lflf, vlvl or lllll. Therefore, the antibacterial peptide is able to form isomeric changes with the bacterial membrane structure, thereby facilitating the antibacterial activity of the antibacterial peptide.

[0090] In some embodiments of the present application, the Q 3 , Q 4 are each independently selected from kl, lk, kll, klk, kkl, of or kf. Therefore, the presence of these amino acids can enhance the effect of the antibacterial peptide on the bacterial membrane, causing the bacterial membrane to be destabilized, thereby increasing the probability of bacterial death.

[0091] In a third aspect of the present application, the present application provides an antibacterial peptide. According to embodiments of the present application, the antibacterial peptide has a structure shown in formula (III) or a stereoisomer, a tautomer or a salt of the structure shown in formula (III):

[0092] (Q 6 )4-(K-Q 5 )2-K-Z 3

[0093] (III)

[0094] wherein Z 3 is selected from a tetrapeptide; Q 5 , Q 6 are each independently selected from a dipeptide or a tripeptide consisting of lysine, leucine, phenylalanine and / or ornithine, and Q 5 or Q 6 does not contain two consecutive leucines; K is lysine.

[0095] The antibacterial peptide of the present application has excellent antibacterial activity and can effectively inhibit various bacteria, especially common gram-negative bacteria and gram-positive bacteria. In addition, the inventors have found through a large number of experiments that the antibacterial peptide also exhibits excellent antibacterial effect on various drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria, and has broad-spectrum antibacterial activity.

[0096] In some embodiments of the present application, the antibacterial peptide of the structure shown in formula (III) can further include at least one of the following additional technical features:

[0097] In some embodiments of the present application, the tetrapeptide includes at least 3 amino acids containing hydrophobic side chains. Therefore, the antibacterial peptide is able to form isomeric changes with the bacterial membrane structure, thereby facilitating the antibacterial activity of the antibacterial peptide.

[0098] In some embodiments of the present application, the amino acid containing a hydrophobic side chain is selected from valine, leucine or phenylalanine.

[0099] In some embodiments of the present application, the Q6 Q is selected from kl, lk, kkl, klk, of or kf. Thus, the antibacterial peptide is able to further improve the antibacterial activity, increasing the probability of bacterial death. 5 Q is not a dipeptide having kl structure. Thus, the antibacterial peptide is able to further improve the antibacterial activity.

[0100] In some embodiments of the present application, the Z 3 Q is selected from kl, lk, kkl, klk, of or kf. Thus, the antibacterial peptide is able to further improve the antibacterial activity, increasing the probability of bacterial death.

[0101] In some embodiments of the present application, the Q 5 Q is selected from kl, lk, kkl, klk, of or kf. Thus, the antibacterial peptide is able to further improve the antibacterial activity, increasing the probability of bacterial death. 6 Q is selected from kl, lk, kkl, klk, of or kf. Thus, the antibacterial peptide is able to further improve the antibacterial activity, increasing the probability of bacterial death.

[0102] In some embodiments of the present application, the Q 5 Q is selected from a dipeptide or tripeptide consisting of lysine and / or leucine, or a dipeptide consisting of ornithine and / or phenylalanine. Thus, the antibacterial peptide has superior antibacterial activity, with smaller MIC (minimum inhibitory concentration) values, regardless of Gram-positive or Gram-negative bacteria.

[0103] As used herein, the term "minimum inhibitory concentration" refers to the lowest drug concentration capable of inhibiting bacterial growth under specific conditions.

[0104] In some embodiments of the present application, the Q 5 Q is selected from kl, lk, kkl or of. Thus, the antibacterial peptide has superior antibacterial activity.

[0105] In some embodiments of the present application, the Q 5 Q is selected from kl, lk or kkl. Thus, the antibacterial peptide has superior antibacterial activity.

[0106] In some embodiments of the present application, the Q 6 Q is selected from a dipeptide consisting of lysine, leucine and / or phenylalanine. Thus, the antibacterial peptide has superior antibacterial activity, with smaller MIC (minimum inhibitory concentration) values, regardless of Gram-positive or Gram-negative bacteria.

[0107] In some embodiments of the present application, the Q 6 Q is selected from kl, lk or kf. Thus, the antibacterial peptide has superior antibacterial activity.

[0108] In some embodiments of the present application, the Q 6are independently selected from dipeptide or tripeptide consisting of lysine and / or leucine. Therefore, the antibacterial peptide has superior antibacterial activity, regardless of gram-positive bacteria or gram-negative bacteria, and has a smaller MIC (minimum inhibitory concentration) value.

[0109] In some embodiments of the present application, the Q 6 are independently selected from kl, lk or klk. Therefore, the antibacterial peptide has superior antibacterial activity.

[0110] In some embodiments of the present application, the Z 3 is llll or lflf. Therefore, the antibacterial peptide has superior antibacterial activity.

[0111] In some embodiments of the present application, when the Z 3 is llll, Q 6 is kl, Q 5 is not a dipeptide having the structure of kl or lk.

[0112] In a fourth aspect of the present application, an antibacterial peptide is provided. According to embodiments of the present application, the antibacterial peptide has a structure shown in formula (IV) or a stereoisomer, a tautomer or a salt of the structure shown in formula (IV):

[0113] (Q 8 )4-(K-Q 7 )2-K-Z 4

[0114] (IV)

[0115] wherein Z 4 is a tetrapeptide consisting of leucine and / or phenylalanine; Q 7 , Q 8 are independently selected from dipeptide or tripeptide consisting of lysine and / or leucine; and K is lysine.

[0116] The antibacterial peptide of the present application has excellent antibacterial activity and can effectively inhibit various bacteria, especially common gram-negative bacteria and gram-positive bacteria. In addition, the inventors have found through a large number of experiments that the antibacterial peptide also exhibits excellent antibacterial effect on various drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria, and has broad-spectrum antibacterial activity.

[0117] In some embodiments of the present application, the antibacterial peptide of the structure shown in formula (IV) can further include at least one of the following additional technical features:

[0118] In some embodiments of the present application, the Z 4At least one lysine is included. Therefore, the effectiveness of the antibacterial peptide against drug-resistant bacteria can be improved, the risk of drug resistance of bacteria can be reduced, and a new option for antibacterial treatment is provided.

[0119] In some embodiments of the present application, the Q 7 , Q 8 At least one lysine is included. Therefore, the effectiveness of the antibacterial peptide against drug-resistant bacteria can be improved, the risk of drug resistance of bacteria can be reduced, and a new option for antibacterial treatment is provided.

[0120] In some embodiments of the present application, the Q 7 , Q 8 does not contain two consecutive leucines, or the Z 4 contains two phenylalanines, or when the Q 7 is a dipeptide of lk structure, the Q 8 is not a dipeptide of kl structure. Therefore, the effectiveness of the antibacterial peptide against drug-resistant bacteria can be improved, and it is expected to play an important role in the treatment and control of bacterial infections.

[0121] In some embodiments of the present application, the Z 4 is selected from lflf or llll. Therefore, the effectiveness of the antibacterial peptide against drug-resistant bacteria can be improved.

[0122] In some embodiments of the present application, the Q 7 , Q 8 are independently selected from kl, lk, klk or kkl, respectively. Therefore, the effectiveness of the antibacterial peptide against drug-resistant bacteria can be improved.

[0123] In a fifth aspect of the present application, an antibacterial peptide is provided. According to embodiments of the present application, the antibacterial peptide has a structure shown in formula (V) or a stereoisomer, a tautomer or a salt of the structure shown in formula (V):

[0124] (Q 10 )4-(K-Q 9 )2-K-LLLL

[0125] (V)

[0126] wherein Q 9 , Q 10 are independently selected from a dipeptide or tripeptide consisting of lysine and / or leucine; K is lysine, and L is leucine.

[0127] The antibacterial peptide has excellent antibacterial activity and can effectively inhibit various bacteria. The antibacterial peptide has antibacterial effect on common gram-negative bacteria and gram-positive bacteria. In addition, the inventors find through a large number of experiments that the antibacterial peptide can effectively inhibit the growth of various drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria, especially drug-resistant gram-negative bacteria. Therefore, the antibacterial peptide has excellent antibacterial activity and broad-spectrum antibacterial ability, so that it can have a wide application prospect in the antibacterial field and is expected to play an important role in the treatment and control of bacterial infection.

[0128] In some embodiments of the present application, the antibacterial peptide of the structure shown in formula (V) can further include at least one of the following additional technical features:

[0129] In some embodiments of the present application, the Q 9 , Q 10 are independently selected from kl, klk or kkl. Therefore, the antibacterial activity of the antibacterial peptide can be improved, especially the inhibitory activity on drug-resistant bacteria.

[0130] In some embodiments of the present application, the Q 9 and Q 10 are not lk.

[0131] In some embodiments of the present application, the Q 10 is not a dipeptide of lk structure. Therefore, the antibacterial activity of the antibacterial peptide can be improved, especially the inhibitory activity on drug-resistant bacteria.

[0132] In a sixth aspect of the present application, an antibacterial peptide is provided. According to embodiments of the present application, the antibacterial peptide has a structure shown in formula (VI) or a stereoisomer, a tautomer or a salt of the structure shown in formula (VI):

[0133] (Q 12 )4-(K-Q 11 )2-K-LLLL

[0134] (VI)

[0135] wherein Q 11 are independently selected from a dipeptide or a tripeptide consisting of lysine and / or leucine; Q 12 are independently selected from a dipeptide consisting of lysine and / or leucine; K is lysine and L is leucine.

[0136] The antibacterial peptide of the present application has excellent antibacterial activity and can effectively inhibit various bacteria, and has antibacterial effect on common gram-negative bacteria and gram-positive bacteria. In addition, the inventors have found through a large number of experiments that the antibacterial peptide can also effectively inhibit the growth of various drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria, especially drug-resistant gram-positive bacteria. Therefore, the antibacterial peptide has excellent antibacterial activity and broad-spectrum antibacterial ability, so that it can have a wide application prospect in the field of antibacterial, and is expected to play an important role in the treatment and control of bacterial infection.

[0137] In some embodiments of the present application, the antibacterial peptide of the structure shown in formula (VI) can further include at least one of the following additional technical features:

[0138] In some embodiments of the present application, the Q 11 is not a dipeptide of lk structure. Therefore, the antibacterial activity of the antibacterial peptide can be improved, especially the inhibitory activity on drug-resistant bacteria.

[0139] In some embodiments of the present application, the Q 11 are respectively and independently selected from kl or kkl. Therefore, the antibacterial activity of the antibacterial peptide can be improved, especially the inhibitory activity on drug-resistant bacteria.

[0140] In some embodiments of the present application, the Q 12 are respectively and independently selected from kl or lk. Therefore, the antibacterial activity of the antibacterial peptide can be improved, especially the inhibitory activity on drug-resistant bacteria.

[0141] In some embodiments of the present application, the Q 12 is not a dipeptide of lk structure. Therefore, the antibacterial peptide has excellent activity against drug-resistant bacteria, and has excellent inhibitory activity on drug-resistant gram-positive bacteria and drug-resistant gram-negative bacteria, and the MIC value is small.

[0142] In a seventh aspect, the present application provides an antibacterial peptide. According to embodiments of the present application, the antibacterial peptide has one of the following structures: (kl)4(kkl)2klllf, (kl)4(kkl)2klflf, (kl)4(kkl)2kvlvl, (lk)4(kkl)2kllll, (kl)4(klk)2kllll, (lk)4(klk)2kllll, (kll)4(kkl)2kllll, (klk)4(kkl)2kllll, (kl)4(kkll)2kllll, (kl)4(kkkl)2kllll, (of)4(kkl)2kllll, (kf)4(kkl)2kllll, (kl)4(kkf)2kllll, (kf)4(kof)2kllll, or (kl)4(kkl)2kllll. The antibacterial peptide provided by the present application not only has excellent antibacterial activity, but also exhibits a more broad-spectrum antibacterial ability. In addition to having antibacterial effects on common gram-negative bacteria and gram-positive bacteria, the antibacterial peptide can also effectively inhibit various drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria. This broad-spectrum antibacterial activity enables the antibacterial peptide to be widely used in the antibacterial field and to effectively address the increasing problem of drug-resistant bacteria.

[0143] In the present application, the antibacterial peptide has a branching moiety at the leftmost (N-terminal) amino acid within the first bracket from right to left. In the given examples, the single amino acid between the bracketed moiety and the Z moiety is the branching moiety. For example, in Formula (I), S 2 and S 1 are the branching moieties; in Formula (II), K is the branching moiety.

[0144] Pharmaceutical composition

[0145] In an eighth aspect, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition comprises the antibacterial peptide of any one of the first to seventh aspects. As described above, the antibacterial peptide of the present application exhibits excellent antibacterial activity and broad-spectrum antibacterial ability. Therefore, based on these characteristics, the provided pharmaceutical composition also has excellent effects and can effectively inhibit various bacteria, even including drug-resistant bacteria. The pharmaceutical composition can be applied in the field of antibacterial treatment.

[0146] In some embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier.

[0147] In some embodiments of the application, the pharmaceutical compositions of the application can be formulated in a conventional manner using one or more pharmaceutically acceptable excipients, including vehicles, adjuvants or diluents, which facilitate processing of compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. The pharmaceutical compositions of the application can be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, intravitreal, mucosally.

[0148] In some embodiments of the application, the pharmaceutical compositions are formulated into injectable compositions. The injectable compositions are prepared in any conventional form, such as a liquid solution, suspension, emulsion, or solid form suitable for producing a liquid solution, suspension or emulsion. The injectable formulations can include sterile and / or non-pyrogenic solutions ready for injection, sterile dry dissolvable products ready for mixing with a solvent prior to use, such as lyophilized powders, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready for combination with a carrier prior to use, and sterile and / or non-pyrogenic emulsions. The solutions can be aqueous or non-aqueous.

[0149] In some embodiments of the application, the injectable compositions are packaged in ampules, vials or syringes with needles. The injectable compositions should be sterile and non-pyrogenic, as is known and practiced in the art.

[0150] In some embodiments of the application, the pharmaceutical compositions are formulated into compositions that can be administered transmucosally, using penetrants appropriate to the barrier to be permeated. Such penetrants are well known in the art.

[0151] In some embodiments of the application, the pharmaceutical compositions of the application are administered to a patient (preferably a human patient) in need of medical intervention. The pharmaceutical compositions can be administered alone or in combination with other drugs / pharmaceutical compositions. These other drugs / pharmaceutical compositions can be administered simultaneously or non-simultaneously with the pharmaceutical compositions of the application.

[0152] In this context, "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and / or toxicologically, with the other ingredients comprising a formulation, and / or the mammal being treated with it. Preferably, "pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or state government or the United States Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0153] In this context, the term "pharmaceutically acceptable carrier" includes any and all solvents, pharmaceutical stabilizers, or combinations thereof, which are known to one of skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0154] In the present context, the term "pharmaceutically acceptable excipient" can include any solvent suitable for the particular dosage form of interest. Except insofar as any conventional excipient is incompatible with the antibacterial peptide of the present application, for example, by producing any adverse biological effect or by interacting in an deleterious manner with any other component(s) of the pharmaceutically acceptable composition, their use is contemplated to be within the scope of this disclosure.

[0155] Uses and methods

[0156] In a ninth aspect of the present application, the present application provides use of the antibacterial peptide according to any one of the first to seventh aspects or the pharmaceutical composition according to the eighth aspect in the manufacture of a medicament for preventing or treating a bacterial infection.

[0157] In a tenth aspect of the present application, the present application provides use of the antibacterial peptide according to any one of the first to seventh aspects or the pharmaceutical composition according to the eighth aspect in preventing or treating a bacterial infection.

[0158] In an eleventh aspect of the present application, the present application provides use of the antibacterial peptide according to any one of the first to seventh aspects in the manufacture of an antibiotic, a disinfectant, a cleaning agent or an antiseptic for treating a bacterial infection.

[0159] In some embodiments of the present application, the above-mentioned use can further include at least one of the following additional technical features:

[0160] In some embodiments of the present application, the bacteria are Gram-positive bacteria and / or Gram-negative bacteria and / or drug-resistant Gram-positive bacteria and / or drug-resistant Gram-negative bacteria.

[0161] In some embodiments of the present application, the Gram-negative bacteria include, but are not limited to, at least one of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii.

[0162] In some embodiments of the present application, the Gram-negative bacteria include Klebsiella pneumoniae.

[0163] In some embodiments of the present application, the Gram-positive bacteria include, but are not limited to, at least one of Staphylococcus aureus and Staphylococcus epidermidis.

[0164] In some embodiments of the present application, the Gram-positive bacteria include Staphylococcus epidermidis.

[0165] In some embodiments of the present application, the drug-resistant Gram-negative bacteria include, but are not limited to, at least one of carbapenem-resistant Acinetobacter baumannii, drug-resistant Pseudomonas aeruginosa, extended-spectrum beta-lactamase-producing drug-resistant Klebsiella pneumoniae, KPC-2 carbapenemase drug-resistant Klebsiella pneumoniae, extended-spectrum beta-lactamase-producing drug-resistant Escherichia coli, and New Delhi metallo-beta-lactamase-1 drug-resistant Escherichia coli.

[0166] In some embodiments of the present application, the drug-resistant Gram-negative bacteria include extended-spectrum beta-lactamase-producing drug-resistant Escherichia coli.

[0167] In some embodiments of the present application, the drug-resistant Gram-positive bacteria include, but are not limited to, at least one of methicillin-resistant Staphylococcus aureus, drug-resistant Enterococcus faecium, and drug-resistant Enterococcus faecalis.

[0168] In some embodiments of the present application, the drug-resistant Gram-positive bacteria include drug-resistant Enterococcus faecium.

[0169] In the present disclosure, the term "treatment" refers to intervention by the use of drugs to diseases and symptoms.

[0170] In the present disclosure, the term "prevention" refers to taking proactive measures to reduce or eliminate the probability of occurrence of potential risks, threats, or problems as much as possible.

[0171] In a twelfth aspect of the present application, a method for preventing or treating bacterial infection is provided. According to embodiments of the present application, the method includes administering to a subject an effective amount of the antibacterial peptide of any one of the first to seventh aspects or the pharmaceutical composition of the eighth aspect. As described above, the antibacterial peptide and the pharmaceutical composition of the present application have excellent antibacterial activity and broad-spectrum antibacterial ability. Thus, by using the method of the present application, bacterial infection can be effectively prevented or treated.

[0172] In some embodiments of the present application, the method can further include at least one of the following additional technical features:

[0173] In some embodiments of the present application, the bacteria are Gram-positive bacteria and / or Gram-negative bacteria and / or drug-resistant Gram-positive bacteria and / or drug-resistant Gram-negative bacteria.

[0174] In some embodiments of the present application, the Gram-negative bacteria include, but are not limited to, at least one of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii.

[0175] In some embodiments of the present application, the Gram-negative bacteria include Klebsiella pneumoniae.

[0176] In some embodiments of the application, the Gram-positive bacteria include, but are not limited to, at least one of Staphylococcus aureus and Staphylococcus epidermidis.

[0177] In some embodiments of the application, the Gram-positive bacteria include Staphylococcus epidermidis.

[0178] In some embodiments of the application, the drug-resistant Gram-negative bacteria include, but are not limited to, at least one of carbapenem-resistant Acinetobacter baumannii, drug-resistant Pseudomonas aeruginosa, extended-spectrum beta-lactamase-producing drug-resistant Klebsiella pneumoniae, KPC-2 carbapenemase drug-resistant Klebsiella pneumoniae, extended-spectrum beta-lactamase-producing drug-resistant Escherichia coli, and New Delhi metallo-beta-lactamase-1 drug-resistant Escherichia coli.

[0179] In some embodiments of the application, the drug-resistant Gram-negative bacteria include extended-spectrum beta-lactamase-producing drug-resistant Escherichia coli.

[0180] In some embodiments of the application, the drug-resistant Gram-positive bacteria include, but are not limited to, at least one of methicillin-resistant Staphylococcus aureus, drug-resistant Enterococcus faecium, and drug-resistant Enterococcus faecalis.

[0181] In some embodiments of the application, the drug-resistant Gram-positive bacteria include drug-resistant Enterococcus faecium.

[0182] In this context, the term "effective amount" refers to an amount of a therapeutic agent, i.e., an antibacterial peptide, a pharmaceutical composition of the present application, that reduces or prevents bacterial growth and colonization, or that shows a detectable therapeutic or prophylactic effect. The effect can be detected by, for example, culturing a biopsy and assaying for bacterial activity, or by any other appropriate method of assessing the progression or severity of a bacterial infection. The exact effective amount for an individual depends on the individual's body weight and health status, the nature and extent of the disease state, and the therapeutic agent or combination of therapeutic agents selected for administration. In particular, the compositions of the present application can be used to reduce or prevent a bacterial infection and / or concomitant biological or physical manifestations, such as, for example, fever reduction. Methods for establishing an initial dosage by a clinician are known in the art. The dosage administered must be safe and effective.

[0183] In this context, the term "subject" is an animal, preferably a mammal, more preferably a human, and includes, but is not limited to, a consumer of a health product and a patient having a disease, disorder, and / or symptoms. The subject in the present application is preferably a mammal. The term "mammal" refers primarily to warm-blooded vertebrate mammals, including, but not limited to, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (e.g., rats, mice), pigs, cows, sheep, horses, humans, and the like, preferably primates, and more preferably humans.

[0184] Generally, the dendrimeric antimicrobial peptides of the present application can be prepared by the methods described herein, and the following reaction schemes and examples are provided to further illustrate the present application. It is to be understood that the examples and features of the examples in the present application can be arbitrarily combined with each other in the absence of conflict.

[0185] Those skilled in the art will recognize that the chemical reactions described herein can be used to practice the present application with a wide variety of dendrimeric antimicrobial peptides, and that the protection of the present application is not limited to the specific examples described herein. Other methods for preparing dendrimeric antimicrobial peptides of the present application are deemed to be within the scope of the present application.

[0186] In the examples described below, reagents were purchased from commercial suppliers such as the carrier resin, protected amino acids (Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, etc.), condensing agents, solvents, cleaving reagents, etc., and were used without further purification unless otherwise noted. General reagents were purchased from Xi'an Lanxiao Science and Technology, Chengdu Zhengyuan Biochemical, Suzhou Haofan, Guangdong Xilong Chemical, and National Pharmaceutical Reagent, etc.

[0187] The experimental conditions for high performance liquid chromatography (HPLC) data are shown in Table 1:

[0188] Table 1 HPLC experimental conditions

[0189] Note: Blank solution is 20% acetonitrile: 80% water

[0190] The conditions for mass spectrometry (MS) data are as follows: a sample solution with a concentration of 1 mg / mL is taken, a solvent (acetonitrile: water = 2:8) is used, and the filtered solution is directly injected into a Thermo LTQ-XL type LC-MS to obtain molecular ion peak data.

[0191] The following abbreviations are used throughout the present application:

[0192] Fmoc: 9-fluorenylmethyloxycarbonyl

[0193] Boc: tert-butyloxycarbonyl

[0194] DMF: N,N-dimethylformamide

[0195] DCM: dichloromethane

[0196] HoBT: 1-hydroxybenzotriazole

[0197] DIC: N,N'-diisopropylcarbodiimide

[0198] DIEA: N,N-diisopropylethylamine

[0199] TBTU: O-benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0200] MTT: thiazolyl blue

[0201] K(k): Lys, lysine

[0202] L(l): Leu, leucine

[0203] F(f): Phe, phenylalanine

[0204] V(v): Val, valine

[0205] O(o): Orn, ornithine

[0206] The synthesis method of the antibacterial peptide described in the present application is shown in the following figure:

[0207] wherein: each n is independently any integer from 1 to 6; each m is independently 0 or 1; each R is the same or different, and is independently an amino acid side chain group.

[0208] The specific steps are as follows:

[0209] 1. Synthesis of single-chain peptide: Compound 1 (resin with exposed amino group) is coupled with Fmoc-protected amino acid to obtain compound 2, and then the Fmoc group is removed to obtain compound 3. The coupling-deprotection reaction process is repeated three times to obtain compound 4, which is a resin-attached tetrapeptide. After coupling and deprotection of compound 4 and Fmoc-protected diaminomethyl carboxylic acid moiety, compound 5 is obtained, and the reaction of the single-chain peptide is completed, and the last amino acid is the amino acid at the branch point.

[0210] 2. Synthesis of double-chain peptide: Compound 5 is repeated for two or three times of coupling-deprotection reaction to obtain compound 6, which is a dipeptide or tripeptide on the double-chain. After coupling and deprotection of compound 6 and Fmoc-protected diaminomethyl carboxylic acid moiety, compound 7 is obtained, and the reaction of the double-chain peptide is completed, and the last amino acid is the amino acid at the branch point.

[0211] 3. Synthesis of four-chain peptide: Compound 7 is repeated for two or three times of coupling-deprotection reaction to obtain compound 8, which is a dipeptide or tripeptide on the four-chain. After cleavage and purification, the resin is filtered out to obtain the final product compound 9.

[0212] Example 1: Preparation of (kl)4(kkl)2klllf

[0213] Structure of (kl)4(kkl)2klllf

[0214] (1) Preparation of Fmoc-Phe-Rink Amide-AM resin

[0215] Prepare the deprotection solution: weigh 125 mL of piperidine, add DMF to make up to 500 mL, and shake well for use.

[0216] Weigh 1 g of Rink Amide-AM resin with a substitution degree of 0.50 mmol / g and place it in a glass chromatographic synthesis column. The synthesis scale is 0.5 mmol. Swell the resin in 10 mL of DCM for 15 min, and then dry the solvent. Add 10 mL of the deprotection solution to the swelled resin and react for 5-10 min, and then dry the solvent. Wash the resin with 10 mL of DMF for 3 times, and then wash it with 10 mL of DCM for 2 times, and then dry the solvent. Weigh 0.97 g of Fmoc-Phe-OH and 0.34 g of HoBt into a reagent bottle, dissolve them in 10 mL of DMF, and then add 0.39 mL of DIC. Shake well, and then add it to the synthesis column and react for 30-60 min. After the reaction is completed, dry the reaction solution. Wash the resin with 10 mL of DMF for 3 times, and then wash it with 10 mL of DCM for 2 times, and then dry the solvent to obtain Fmoc-Phe-Rink Amide-AM resin. Nitrogen with a purity of 99.99% is used to stir the reaction and washing processes in this step. The same stirring method is used in the following steps.

[0217] (2) Preparation of Fmoc-Leu-Phe-Rink Amide-AM resin

[0218] Add 10 mL of the deprotection solution to the resin prepared in step (1) and react for 5-10 min, and then dry the solvent. Wash the resin with 10 mL of DMF for 3 times, and then wash it with 10 mL of DCM for 2 times, and then dry the solvent. Weigh 0.88 g of Fmoc-Leu-OH and 0.34 g of HoBt into a reagent bottle, dissolve them in 10 mL of DMF, and then add 0.39 mL of DIC. Shake well, and then add it to the synthesis column and react for 30-60 min. After the reaction is completed, dry the reaction solution. Wash the resin with 10 mL of DMF for 3 times, and then wash it with 10 mL of DCM for 2 times, and then dry the solvent to obtain Fmoc-Leu-Phe-Rink Amide-AM resin.

[0219] (3) Preparation of Fmoc-Leu-Leu-Phe-Rink Amide-AM resin

[0220] The prepared resin in step (2) was added with 10 mL of deprotection solution for reaction for 5-10 min, and the solvent was extracted. The resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was extracted. 0.88 g of Fmoc-Leu-OH and 0.34 g of HoBt were weighed in a reagent bottle, and 10 mL of DMF was added to dissolve, and then 0.39 mL of DIC was added, and then the synthesis column was added for reaction for 30-60 min. After the reaction was completed, the reaction solution was extracted, the resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was extracted, to obtain Fmoc-Leu-Leu-Phe-Rink Amide-AM resin.

[0221] (4) Preparation of Fmoc-Leu-Leu-Leu-Phe-Rink Amide-AM resin

[0222] The prepared resin in step (3) was added with 10 mL of deprotection solution for reaction for 5-10 min, and the solvent was extracted. The resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was extracted. 0.88 g of Fmoc-Leu-OH and 0.34 g of HoBt were weighed in a reagent bottle, and 10 mL of DMF was added to dissolve, and then 0.39 mL of DIC was added, and then the synthesis column was added for reaction for 30-60 min. After the reaction was completed, the reaction solution was extracted, the resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was extracted, to obtain Fmoc-Leu-Leu-Leu-Phe-Rink Amide-AM resin.

[0223] (5) Preparation of Fmoc-Lys(Fmoc)-Leu-Leu-Leu-Phe-Rink Amide-AM resin

[0224] The prepared resin in step (4) was added with 10 mL of deprotection solution for reaction for 5-10 min, and the solvent was extracted. The resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was extracted. 1.48 g of Fmoc-Lys(Fmoc)-OH and 0.34 g of HoBt were weighed in a reagent bottle, and 10 mL of DMF was added to dissolve, and then 0.39 mL of DIC was added, and then the synthesis column was added for reaction for 30-60 min. After the reaction was completed, the reaction solution was extracted, the resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was extracted, to obtain Fmoc-Lys(Fmoc)-Leu-Leu-Leu-Phe-Rink Amide-AM resin.

[0225] (6) Preparation of (Fmoc-Leu)2-Lys-Leu-Leu-Leu-Phe-Rink Amide-AM resin

[0226] The prepared resin in step (5) was added with 10 mL of the deprotection solution and reacted for 5-10 min, repeated twice, and the solvent was drawn off. The resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was drawn off. 1.76 g of Fmoc-Leu-OH and 0.68 g of HoBt were weighed into a reagent bottle, dissolved in 10 mL of DMF, and then 0.78 mL of DIC was added, shaken, and then added to the synthesis column and reacted for 30-60 min. After the reaction was completed, the reaction solution was drawn off, the resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was drawn off, to obtain (Fmoc-Leu)2-Lys-Leu-Leu-Leu-Phe-Rink Amide-AM resin.

[0227] (7) Preparation of [Fmoc-Lys(Boc)-Leu]2-Lys-Leu-Leu-Leu-Phe-Rink Amide-AM resin

[0228] The prepared resin in step (6) was added with 10 mL of the deprotection solution and reacted for 5-10 min, repeated twice, and the solvent was drawn off. The resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was drawn off. 2.34 g of Fmoc-Lys(Boc)-OH and 0.68 g of HoBt were weighed into a reagent bottle, dissolved in 10 mL of DMF, and then 0.78 mL of DIC was added, shaken, and then added to the synthesis column and reacted for 30-60 min. After the reaction was completed, the reaction solution was drawn off, the resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was drawn off, to obtain [Fmoc-Lys(Boc)-Leu]2-Lys-Leu-Leu-Leu-Phe-Rink Amide-AM resin.

[0229] (8) Preparation of [Fmoc-Lys(Fmoc)-Lys(Boc)-Leu]2-Lys-Leu-Leu-Leu-Phe-Rink Amide-AM resin

[0230] Step (7) 10 mL of deprotection solution was added to the prepared resin and reacted for 5-10 min, repeated 2 times, and the solvent was removed by suction. The resin was washed 3 times with 10 mL of DMF and 2 times with 10 mL of DCM, and the solvent was removed by suction. 2.96 g of Fmoc-Lys(Fmoc)-OH and 0.68 g of HoBt were weighed into a reagent bottle, dissolved in 10 mL of DMF, and then 0.78 mL of DIC was added, shaken, and then added to the synthesis column and reacted for 30-60 min. After the reaction was completed, the reaction solution was removed by suction, the resin was washed 3 times with 10 mL of DMF and 2 times with 10 mL of DCM, and the solvent was removed by suction to obtain [Fmoc-Lys(Fmoc)-Lys(Boc)-Leu]2-Lys-Leu-Leu-Leu-Phe-Rink Amide-AM resin.

[0231] (9) Preparation of [Fmoc-Leu]4-[Lys-Lys(Boc)-Leu]2-Lys-Leu-Leu-Leu-Phe-Rink Amide-AM resin

[0232] Step (8) 10 mL of deprotection solution was added to the prepared resin and reacted for 5-10 min, repeated 4 times, and the solvent was removed by suction. The resin was washed 3 times with 10 mL of DMF and 2 times with 10 mL of DCM, and the solvent was removed by suction. 1.76 g of Fmoc-Leu-OH and 0.68 g of HoBt were weighed into a reagent bottle, dissolved in 10 mL of DMF, and then 0.78 mL of DIC was added, shaken, and then added to the synthesis column and reacted for 30-60 min. After the reaction was completed, the reaction solution was removed by suction, the resin was washed 3 times with 10 mL of DMF and 2 times with 10 mL of DCM, and the solvent was removed by suction. 1.76 g of Fmoc-Leu-OH and 1.61 g of TBTU were weighed into a reagent bottle, dissolved in 10 mL of DMF, and then 0.83 mL of DIEA was added, shaken, and then added to the synthesis column and reacted for 30-60 min. After the reaction was completed, the reaction solution was removed by suction, the resin was washed 3 times with 10 mL of DMF and 2 times with 10 mL of DCM, and the solvent was removed by suction to obtain [Fmoc-Leu]4-[Lys-Lys(Boc)-Leu]2-Lys-Leu-Leu-Leu-Phe-Rink Amide-AM resin.

[0233] (10) Preparation of [Boc-Lys(Boc)-Leu]4-[Lys-Lys(Boc)-Leu]2-Lys-Leu-Leu-Leu-Phe-Rink Amide-AM resin

[0234] Step (9) The prepared resin was added with 10 mL of deprotection solution for reaction for 5-10 min, repeated for 4 times, and the solvent was drawn dry. The resin was washed with 10 mL of DMF for 3 times and 10 mL of DCM for 2 times, and the solvent was drawn dry. 1.73 g of Boc-Lys(Boc)-OH and 0.68 g of HoBt were weighed into a reagent bottle, dissolved in 10 mL of DMF, and then 0.78 mL of DIC was added, stirred, and then added to the synthesis column for reaction for 30-60 min. After the reaction was completed, the reaction solution was drawn dry, the resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was drawn dry. 1.73 g of Boc-Lys(Boc)-OH and 1.61 g of TBTU were weighed into a reagent bottle, dissolved in 10 mL of DMF, and then 0.83 mL of DIEA was added, stirred, and then added to the synthesis column for reaction for 30-60 min. After the reaction was completed, the reaction solution was drawn dry, the resin was washed with 10 mL of DMF for 3 times, and 10 mL of DCM for 2 times, and the solvent was drawn dry. [Boc-Lys(Boc)-Leu]4-[Lys-Lys(Boc)-Leu]2-Lys-Leu-Leu-Leu-Phe-Rink Amide-AM resin was obtained, 10 mL of methanol was added for stirring to shrink the resin for 3 times, the resin was transferred to a vacuum drying oven for drying to constant weight, and 2.67 g of peptide resin was obtained.

[0235] (11) Cleavage, purification

[0236] A 30 mL cleavage solution was prepared, and the proportion of the cleavage solution was trifluoroacetic acid:water = 98:2. The peptide resin obtained in step (10) was added to the cleavage solution for stirring reaction for 120 min, the resin was filtered out, the filtrate was precipitated by adding 150 mL of ice methyl tert-butyl ether (≤0°C), resuspended and washed for 2 times, and dried at room temperature under reduced pressure to obtain 1.62 g of a crude product (containing trifluoroacetate salt). The crude product was dissolved in 30 mL of purified water and filtered (0.22 μm), and purified by semi-preparative HPLC. The sample with a purity of ≥95% was collected and freeze-dried.

[0237] (kl)4(kkl)2klllf was obtained as a white powder solid (0.72 g, yield 61.55%, purity 99.4%) after semi-preparative HPLC purification.

[0238] MS (ESI, pos.ion) m / z: 2337.24 [M+1];

[0239] The HPLC peak time was 14.505 min.

[0240] Example 2: Preparation of (kl)4(kkl)2klflf

[0241] (kl)4(kkl)2klflf structure

[0242] Prepared according to a procedure similar to that described in Example 1.

[0243] (kl)4(kkl)2klflf was obtained as a white powder-like solid (0.75 g, yield 62.86%, purity 99.6%) after purification by semi-preparative HPLC.

[0244] MS (ESI, pos.ion) m / z: 2371.23 [M+1];

[0245] HPLC retention time was 14.621 min.

[0246] Example 3: Preparation of (kl)4(kkl)2kvlvl

[0247] (kl)4(kkl)2kvlvl structure

[0248] Prepared according to a procedure similar to that described in Example 1.

[0249] (kl)4(kkl)2kvlvl was obtained as a white powder-like solid (0.68 g, yield 59.8%, purity 97.8%) after purification by semi-preparative HPLC.

[0250] MS (ESI, pos.ion) m / z: 2275.22 [M+1];

[0251] HPLC retention time was 12.603 min.

[0252] Example 4: Preparation of (lk)4(kkl)2kllll

[0253] (lk)4(kkl)2kllll structure

[0254] Prepared according to a procedure similar to that described in Example 1.

[0255] (lk)4(kkl)2kllll was obtained as a white powder-like solid (0.68 g, yield 58.64%, purity 98.4%) after purification by semi-preparative HPLC.

[0256] MS (ESI, pos.ion) m / z: 2303.28 [M+1];

[0257] HPLC retention time was 12.109 min.

[0258] Example 5: Preparation of (kl)4(klk)2kllll

[0259] (kl)4(klk)2kllll structure

[0260] Prepared according to the procedure described in Example 1.

[0261] (kl)4(klk)2kllll was obtained as a white powdery solid (0.46 g, yield 39.53%, purity 97.3%) after purification by semi-preparative HPLC.

[0262] MS (ESI, pos.ion) m / z: 2303.04 [M+1];

[0263] HPLC retention time was 13.809 min.

[0264] Example 6: Preparation of (lk)4(klk)2kllll

[0265] (lk)4(klk)2kllll structure

[0266] Prepared according to the procedure described in Example 1.

[0267] (lk)4(klk)2kllll was obtained as a white powdery solid (0.45 g, yield 38.92%, purity 98.5%) after purification by semi-preparative HPLC.

[0268] MS (ESI, pos.ion) m / z: 2303.06 [M+1];

[0269] HPLC retention time was 12.23 min.

[0270] Example 7: Preparation of (klk)4(kkl)2kllll

[0271] (klk)4(kkl)2kllll structure

[0272] Prepared according to the procedure described in Example 1.

[0273] (klk)4(kkl)2kllll was obtained as a white powdery solid (0.17 g, yield 11.94%, purity 91.2%) after purification by semi-preparative HPLC.

[0274] MS (ESI, pos.ion) m / z: 2815.57 [M+1];

[0275] HPLC retention time was 12.361 min.

[0276] Example 8: Preparation of (kl)4(kkkl)2kllll

[0277] Structure of (kl)4(kkkl)2kllll

[0278] Prepared according to the procedure described in Example 1.

[0279] Obtained (kl)4(kkkl)2kllll as white powder solid (0.84 g, yield 65.94%, purity 96.5%) after purification by semi-preparative HPLC.

[0280] MS (ESI, pos.ion) m / z: 2559.47 [M+1];

[0281] HPLC retention time 12.929 min.

[0282] Example 9: Preparation of (of)4(kkl)2kllll

[0283] Structure of (of)4(kkl)2kllll

[0284] Prepared according to the procedure described in Example 1.

[0285] Obtained (of)4(kkl)2kllll as white powder solid (0.63 g, yield 52.61%, purity 98.4%) after purification by semi-preparative HPLC.

[0286] MS (ESI, pos.ion) m / z: 2383.2 [M+1];

[0287] HPLC retention time 14.809 min.

[0288] Example 10: Preparation of (kf)4(kkl)2kllll

[0289] Structure of (kf)4(kkl)2kllll

[0290] Prepared according to the procedure described in Example 1.

[0291] Obtained (kf)4(kkl)2kllll as white powder solid (0.67 g, yield 54.68%, purity 97.1%) after purification by semi-preparative HPLC.

[0292] MS (ESI, pos.ion) m / z: 2438.95 [M+1];

[0293] HPLC retention time of 14.767 min.

[0294] Example 11: Preparation of (kl)4(kkf)2kllll

[0295] Structure of (kl)4(kkf)2kllll

[0296] Prepared according to a procedure similar to that described in Example 1.

[0297] (kl)4(kkf)2kllll was obtained as a white powdery solid (0.55 g, yield 46.07%, purity 94.9%) after purification by semi-preparative HPLC.

[0298] MS (ESI, pos.ion) m / z: 2371.17 [M+1];

[0299] HPLC retention time of 14.371 min.

[0300] Example 12: Preparation of (kf)4(kof)2kllll

[0301] Structure of (kf)4(kof)2kllll

[0302] Prepared according to a procedure similar to that described in Example 1.

[0303] (kf)4(kof)2kllll was obtained as a white powdery solid (0.52 g, yield 41.97%, purity 97.8%) after purification by semi-preparative HPLC.

[0304] MS (ESI, pos.ion) m / z: 2479.27 [M+1];

[0305] HPLC retention time of 15.102 min.

[0306] Example 13: Preparation of (kl)4(kkl)2kllll

[0307] Structure of (kl)4(kkl)2kllll

[0308] Prepared according to a procedure similar to that described in Example 1.

[0309] (kl)4(kkl)2kllll was obtained as a white powdery solid (1.61 g, yield 34.95%, purity 99.5%) after semi-preparative HPLC.

[0310] MS (ESI, pos. ion) m / z: 2303.6 [M+1];

[0311] HPLC retention time was 15.565 min.

[0312] Example 14: Preparation of (kll)4(kkl)2kllll

[0313] Structure of (kll)4(kkl)2kllll

[0314] Prepared according to a procedure similar to that described in Example 1.

[0315] (kll)4(kkl)2kllll was obtained as a white powdery solid (0.48 g, yield 34.78%, purity 94.5%) after purification by semi-preparative HPLC.

[0316] MS (ESI, pos. ion) m / z: 2755.74 [M+1];

[0317] HPLC retention time was 16.922 min.

[0318] Example 15: Preparation of (kl)4(kkll)2kllll

[0319] Structure of (kl)4(kkll)2kllll

[0320] Prepared according to a procedure similar to that described in Example 1.

[0321] (kl)4(kkll)2kllll was obtained as a white powdery solid (0.50 g, yield 39.26%, purity 94.2%) after purification by semi-preparative HPLC.

[0322] MS (ESI, pos. ion) m / z: 2528.9 [M+1];

[0323] HPLC retention time was 17.657 min.

[0324] Comparative Example 1

[0325] Compounds MSt-146 ((kl)4(kkl)2(kkl)) and mis-03 ((kkl)4(kkkl)2(kkkl)) were synthesized according to a procedure described in CN106132979B.

[0326] Test Example 1: Test of minimum inhibitory concentration (MIC) of antibacterial peptides of the present application on different quality control bacteria

[0327] In order to study whether the antibacterial peptide of the present application can have inhibitory effect on gram-negative bacteria and gram-positive bacteria, the inventors carried out antibacterial test on different quality control bacteria using the antibacterial peptide, and obtained the minimum inhibitory concentration, and the specific steps are as follows:

[0328] (1) Preparation of bacterial strains:

[0329] The following strains: Escherichia coli (8099), Staphylococcus aureus (CMCC(B)26003), Staphylococcus epidermidis (CMCC(B)26069), Pseudomonas aeruginosa (ATCC27853, CMCC(B)10104), Klebsiella pneumoniae (CICC10870), Acinetobacter baumannii (CICC10980) were subjected to bacterial amplification to obtain working bacterial liquid. The inoculation culture medium of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis was Luria-Bertani (LB) broth medium, and the culture conditions were 150 r / min, 32℃, 16-18h. The inoculation culture medium of Klebsiella pneumoniae and Acinetobacter baumannii was nutrient broth medium (Beijing Luqiao, #220317), and the culture conditions were 150 r / min, 37℃, 16-18h. Recovery was carried out by coating the inoculation loop on Tryptone Soy Agar (TSA) plate culture medium, and the culture was incubated in a bacterial incubator at 37℃ for 18-24h.

[0330] A certain volume of the original bacterial liquid was diluted with physiological saline to an appropriate concentration, and observed and counted under a microscope and a hemocytometer. The number of bacteria in the bacterial culture stock sample was calculated, and then physiological saline was used to prepare a bacterial liquid with a concentration of 1×10 8 CFU / mL for standby. Then Mueller-Hinton (MH) broth medium was used to dilute 200 times to prepare a bacterial liquid with a concentration of 5×10 5 CFU / mL for standby.

[0331] (2) Preparation of compounds:

[0332] Antibacterial peptide 1mg was weighed and added to physiological saline (Shandong Qidu, #R15B23010902) to prepare a stock solution with a concentration of 1mg / mL, mixed well, and filtered through a 0.22μm sterile filter membrane. The antibacterial peptide was diluted to 256μg / mL with MH broth (Beijing Luqiao, #220104), mixed well, and used for standby.

[0333] In the 96-well plate, 100 μL of MH broth was added to the wells except the starting concentration column, and 3 parallel tests were set for each group. A negative control group and a positive control group were set. 200 μL of the prepared antibacterial peptide solution was added to the starting concentration column (the first column), and the dilution was carried out by hole by hole ratio from the second column. The starting concentration of all test antibacterial peptides was 256 μg / mL, and the concentration was diluted to 0.125 μg / mL in the 12th column. After the antibacterial peptide was diluted to the final concentration, the excess antibacterial peptide was discarded.

[0334] (3) Detection:

[0335] The prepared 5×10 5 CFU / mL of bacteria solution was added to each well except the negative control, and 100 μL was added to each well (at this time, the working concentration of each compound was 1 / 2 of the original concentration per well), and the amount of bacteria added to each well was 5×10 4 CFU. At this time, the concentration of the test antibacterial peptide in the well plate was 128 μg / ml-0.0625 μg / ml. After the sample was added to the well plate, the sealing film was sealed, and incubation was carried out in a constant temperature shaker (RH-Q, Changzhou Guanjun). The incubation conditions were 35℃, 150r / min, and 16-18h.

[0336] The MIC results on the 96-well plate were read and photographed after MTT staining. When observing with the naked eye, the lowest drug concentration contained in the concave hole without bacterial growth was the MIC. Note: When the micro-broth dilution method shows a single jump hole, the highest antibacterial peptide concentration inhibiting bacterial growth should be recorded. If multiple jump holes appear, the results should not be reported, and the test should be repeated.

[0337] Table 2 is the MIC of the antibacterial peptide of the application against various quality control bacteria. The experimental results show that the antibacterial peptide prepared by the application has broad-spectrum antibacterial ability and can simultaneously inhibit various gram-negative bacteria and gram-positive bacteria. Compared with MSt-146 and MIS-03, the antibacterial peptide prepared by the application can achieve better antibacterial effect.

[0338] The MIC value of the antibacterial peptide prepared by the application for the above-mentioned various gram-negative quality control bacteria is in the range of 1-32 μg / mL, and the MIC value for the above-mentioned various gram-positive quality control bacteria is not more than 16 μg / mL, achieving a strong antibacterial effect. Among them, the antibacterial activity of the antibacterial peptides prepared in Example 6 and Example 8 is particularly outstanding.

[0339] Table 2 measures the MIC value of different antibacterial peptides on quality control bacteria

[0340] Test Example 2: Test of MIC of the antibacterial peptide of the application on drug-resistant bacteria

[0341] Based on the results in Test Example 1, i.e. the antibacterial peptide has good inhibitory effect on both gram-negative quality control bacteria and gram-positive quality control bacteria, the inventors further studied whether the antibacterial peptide can have good inhibitory effect on drug-resistant gram-negative bacteria and drug-resistant gram-positive bacteria, and the specific steps are as follows:

[0342] (1) Strain preparation:

[0343] The carbapenem-resistant Acinetobacter baumannii, drug-resistant Pseudomonas aeruginosa, super broad-spectrum beta-lactamase-producing drug-resistant Klebsiella pneumoniae, KPC-2 carbapenemase-resistant Klebsiella pneumoniae, super broad-spectrum beta-lactamase-producing drug-resistant Escherichia coli, and New Delhi metallo-beta-lactamase-1 drug-resistant Escherichia coli were picked up with a sterile inoculation ring from the well-grown colonies on the agar medium, stirred and mixed in physiological saline to serve as the working original bacterial liquid. The operation method for subsequent strain preparation refers to Test Example 1.

[0344] The methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant Enterococcus faecium, and drug-resistant Enterococcus faecalis were picked up with a sterile inoculation ring from the well-grown colonies on the agar medium, stirred and mixed in physiological saline to serve as the working original bacterial liquid. The operation method for subsequent strain preparation refers to Test Example 1.

[0345] The above-mentioned clinical strains are from the Clinical Microbiology Room of the Siming Branch of the First Affiliated Hospital of Xiamen University.

[0346] (2) Compound preparation:

[0347] The antibacterial peptide was weighed and added to physiological saline (Shandong Qidu, #R15B23010902) to prepare a mother liquor with a concentration of 1 mg / mL, and mixed well. The 15 antibacterial peptides were diluted to 512 μg / ml with MH broth (Beijing Luqiao, #220104) respectively, and mixed well for standby use.

[0348] The subsequent operation method refers to Test Example 1.

[0349] (3) Detection:

[0350] The prepared 5×10 5 CFU / mL bacterial liquid was added to each well at an amount of 100 μL, and the bacterial addition amount in each well was 5×10 4 CFU. At this time, the concentration of the tested antibacterial peptide in the well plate was 256 μg / ml-0.125 μg / ml. After the well plate was completely added, the well plate was sealed with a sealing film and incubated in a constant temperature shaker or incubator (RH-Q Changzhou Guanjun). Incubation conditions: 37°C, 150 r / min, 16-20 h.

[0351] The MIC results on the 96-well plate were read and photographed after MTT staining.

[0352] Table 3 is the minimum inhibitory concentration of the antibacterial peptide of the present application to clinically isolated drug-resistant gram-negative bacteria. The experimental results show that the antibacterial peptide prepared in the present application can produce antibacterial effect on various drug-resistant gram-negative bacteria, and has broad-spectrum antibacterial activity.

[0353] Table 4 is the minimum inhibitory concentration of the antibacterial peptide of the present application to clinically isolated drug-resistant gram-positive bacteria. The experimental results show that the antibacterial peptide prepared in the present application can produce antibacterial effect on various drug-resistant gram-positive bacteria, and has broad-spectrum antibacterial activity.

[0354] Table 3 measures the MIC value of different antibacterial peptides to drug-resistant gram-negative bacteria

[0355] * indicates that the drug resistance of Pseudomonas aeruginosa is unknown

[0356] Table 4 measures the MIC value of different antibacterial peptides to drug-resistant gram-positive bacteria

[0357] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0358] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and cannot be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. An antibacterial peptide, characterized in that, having the structure of Formula (I) or a stereoisomer, tautomer, or salt of the structure of Formula (I): (Q 2 )4-(S 2 -Q 1 )2-S 1 -Z 1 (I) wherein, Z 1 a tetrapeptide optionally modified with -OH, -CHO, -COOH, -NH2, -SO3H, or -C=O; S 1 , S 2 are each independently selected from the group consisting of the amino acid of the structure; Q 1 , Q 2 are each independently selected from a di- or tripeptide; n are each independently selected from any integer between 1 and 6.

2. The antimicrobial peptide according to claim 1, characterized in that, the tetrapeptide comprises at least 1 amino acid comprising a hydrophobic side chain; and / or, the tetrapeptide comprises at least 3 amino acids comprising a hydrophobic side chain; and / or, the tetrapeptide comprises an amino acid comprising a hydrophobic side chain and an amino acid comprising a cationic side chain; and / or, the dipeptide comprises an amino acid comprising a hydrophobic side chain and an amino acid comprising a cationic side chain; and / or, the tripeptide comprises an amino acid comprising a hydrophobic side chain and an amino acid comprising a cationic side chain.

3. An antibacterial peptide, characterized in that, having the structure of Formula (II) or a stereoisomer, tautomer, or salt of the structure of Formula (II): (Q 4 )4-(K-Q 3 )2-K-Z 2 (II) wherein, Z 2 a tetrapeptide optionally modified with -OH, -CHO, -COOH, -NH2, -SO3H, or -C=O; Q 3 , Q 4 are each independently selected from a dipeptide or tripeptide; K is lysine.

4. The antimicrobial peptide according to claim 3, characterized in that, the tetrapeptide comprises at least 1 amino acid comprising a hydrophobic side chain; and / or, the tetrapeptide comprises at least 3 amino acids comprising a hydrophobic side chain; and / or, the tetrapeptide comprises an amino acid comprising a hydrophobic side chain and an amino acid comprising a cationic side chain; and / or, the dipeptide comprises an amino acid comprising a hydrophobic side chain and an amino acid comprising a cationic side chain; and / or, the tripeptide comprises an amino acid comprising a hydrophobic side chain and an amino acid comprising a cationic side chain.

5. An antibacterial peptide, characterized in that, having the structure of Formula (III) or a stereoisomer, tautomer, or salt of the structure of Formula (III): (Q 6 )4-(K-Q 5 )2-K-Z 3 (III) wherein, Z 3 selected from tetrapeptides; Q 5 , Q 6 are each independently selected from a di- or tripeptide consisting of lysine, leucine, phenylalanine and / or ornithine, and Q 5 or Q 6 does not comprise two consecutive leucines; K is lysine.

6. The antimicrobial peptide according to claim 5, wherein, The Q 6 When phenylalanine is included, the Q 5 Is not a dipeptide having the structure of kl.

7. The antimicrobial peptide of claim 5, wherein said Q 6 and Q 5 are each independently selected from a di- or tripeptide consisting of lysine and / or leucine; The Z 3 For llll or lflf; and when Z is H, Q is not a dipeptide having the structure kl or lk. 3 when Z is H, Q is not a dipeptide having the structure kl or lk. 6 when Z is H, Q is not a dipeptide having the structure kl or lk. 5 when Z is H, Q is not a dipeptide having 8. The antimicrobial peptide of claim 5, wherein, the tetrapeptide comprises at least 3 amino acids comprising a hydrophobic side chain; and / or, the amino acid comprising a hydrophobic side chain is selected from valine, leucine or phenylalanine.

9. An antibacterial peptide, characterized in that, having the structure of Formula (IV) or a stereoisomer, tautomer, or salt of the structure of Formula (IV): (Q 8 )4-(K-Q 7 )2-K-Z 4 (IV) wherein, Z 4 a tetrapeptide selected from the group consisting of leucine and / or phenylalanine; Q 7 , Q 8 are each independently selected from a di- or tripeptide consisting of lysine and / or leucine; K is lysine; and / or, the Z 4 at least two leucines; and / or, the Q 7 , Q 8 at least one lysine; and / or, the Q 7 , Q 8 does not comprise two consecutive leucines, or, the Z 4 comprises two phenylalanines, or, when the Q 7 is a dipeptide of the structure lk, the Q 8 is not a dipeptide of the structure kl.

10. An antibacterial peptide, characterized in that, having the structure of Formula (V) or a stereoisomer, tautomer, or salt of the structure of Formula (V): 10 )4-(K-Q 9 )2-K-LLLL (V) wherein, Q 9 , Q 10 are each independently selected from a di- or tripeptide consisting of lysine and / or leucine; K is lysine and L is leucine; and / or, the Q 10 with Q 9 are not identical to lk.

11. The antimicrobial peptide according to claim 2 or 4, characterized in that, the amino acid comprising a hydrophobic side chain is selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, proline or tryptophan; and / or, the amino acid comprising a cationic side chain is selected from lysine, arginine, histidine, ornithine, 2,3-diaminopropionic acid or 2,4-diaminobutyric acid.

12. The antimicrobial peptide of claim 1, wherein, The Z 1 is selected from lllf, lllv, lll a, lllvv, lllff, lflf, lvlv, vlvl, flfl, llll, flll, vlll, vvll, ffll, lklk, lkkk, llkk, lllk, kllk, klkl, kkkl, klll or kkll; and / or, the S 1 , S 2 are each independently selected from lysine, ornithine, 2,3-diaminopropionic acid or 2,4-diaminobutyric acid; and / or, the Q 1 , Q 2 are each independently selected from kl, lk, kll, lkl, llk, klk, kkl, of, kf, ol, bl, lb, oll, bll, fk, ffo, vo, vb, wk, fk, fb, bf, rl, or lo.

13. The antimicrobial peptide of claim 3, wherein The Z 2 is selected from lllf, If If, vlvl, or llll; and / or, the Q 3 , Q 4 are each independently selected from kl, lk, kll, klk, kkl, of or kf.

14. The antimicrobial peptide of claim 5, wherein The Z 3 is selected from lllf, If If, vlvl, or llll; and / or, the Q 5 , Q 6 are each independently selected from kl, lk, kkl, klk, of or kf.

15. The antimicrobial peptide of claim 9, wherein, The Z 4 is selected from lflf or llll; and / or, the Q 7 , Q 8 are each independently selected from kl, lk, klk or kkl.

16. The antimicrobial peptide of claim 10, wherein, said Q 9 , Q 10 are each independently selected from lk, kl, klk or kkl.

17. An antibacterial peptide, characterized in that, having one of the following structures: (kl)4(kkl)2klllf, (kl)4(kkl)2klflf, (kl)4(kkl)2kvlvl, (lk)4(kkl)2kllll, (kl)4(klk)2kllll, (lk)4(klk)2kllll, (kll)4(kkl)2kllll, (klk)4(kkl)2kllll, (kl)4(kkll)2kllll, (kl)4(kkkl)2kllll, (of)4(kkl)2kllll, (kf)4(kkl)2kllll, (kl)4(kkf)2kllll, (kf)4(kof)2kllll or (kl)4(kkl)2kllll.

18. A pharmaceutical composition, characterized by, comprising the antibacterial peptide of any one of claims 1 to 17.

19. The pharmaceutical composition of claim 18, wherein, further comprising a pharmaceutically acceptable excipient or carrier.

20. Use of the antibacterial peptide of any one of claims 1 to 17 or the pharmaceutical composition of any one of claims 18 to 19 in the manufacture of a medicament for the prevention or treatment of a bacterial infection.

21. Use of the antibacterial peptide of any one of claims 1 to 17 or the pharmaceutical composition of any one of claims 18 to 19 in the prevention or treatment of a bacterial infection.

22. Use of the antibacterial peptide of any one of claims 1 to 17 in the manufacture of an antibiotic, disinfectant, cleaner or antiseptic for the treatment of a bacterial infection.

23. Use according to any one of claims 20 to 22, characterized in that, The bacteria are gram-positive bacteria and / or gram-negative bacteria and / or drug-resistant gram-positive bacteria and / or drug-resistant gram-negative bacteria; and / or, the gram-negative bacteria comprise at least one of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii; and / or, the gram-negative bacteria comprise Klebsiella pneumoniae; and / or, the gram-positive bacteria comprise at least one of Staphylococcus aureus and Staphylococcus epidermidis; and / or, the gram-positive bacteria comprise Staphylococcus epidermidis; and / or, the drug-resistant gram-negative bacteria comprise at least one of carbapenem-resistant Acinetobacter baumannii, drug-resistant Pseudomonas aeruginosa, extended-spectrum β-lactamase-producing drug-resistant Klebsiella pneumoniae, KPC-2-type carbapenemase drug-resistant Klebsiella pneumoniae, extended-spectrum β-lactamase-producing drug-resistant Escherichia coli and New Delhi metallo-β-lactamase-1 drug-resistant Escherichia coli; and / or, the drug-resistant gram-negative bacteria comprise extended-spectrum β-lactamase-producing drug-resistant Escherichia coli; and / or, the drug-resistant gram-positive bacteria comprise at least one of methicillin-resistant Staphylococcus aureus, drug-resistant Enterococcus faecium and drug-resistant Enterococcus faecalis; and / or, the drug-resistant gram-positive bacteria comprise drug-resistant Enterococcus faecium.

24. A method of preventing or treating a bacterial infection, comprising administering to a subject in need thereof an effective amount of a compound of any one of claims 1-23, or a pharmaceutically acceptable salt thereof. Comprise: administering to the subject an effective amount of the antibacterial peptide of any one of claims 1-17 or the pharmaceutical composition of any one of claims 18-19.

25. The method of claim 24, wherein, The bacteria are gram-positive bacteria and / or gram-negative bacteria and / or drug-resistant gram-positive bacteria and / or drug-resistant gram-negative bacteria; and / or, the gram-negative bacteria comprise at least one of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii; and / or, the gram-negative bacteria comprise Klebsiella pneumoniae; and / or, the gram-positive bacteria comprise at least one of Staphylococcus aureus and Staphylococcus epidermidis; and / or, the gram-positive bacteria comprise Staphylococcus epidermidis; and / or, the drug-resistant gram-negative bacteria comprise at least one of carbapenem-resistant Acinetobacter baumannii, drug-resistant Pseudomonas aeruginosa, extended-spectrum β-lactamase-producing drug-resistant Klebsiella pneumoniae, KPC-2-type carbapenemase drug-resistant Klebsiella pneumoniae, extended-spectrum β-lactamase-producing drug-resistant Escherichia coli and New Delhi metallo-β-lactamase-1 drug-resistant Escherichia coli; and / or, the drug-resistant gram-negative bacteria comprise extended-spectrum β-lactamase-producing drug-resistant Escherichia coli; and / or, the drug-resistant gram-positive bacteria comprise at least one of methicillin-resistant Staphylococcus aureus, drug-resistant Enterococcus faecium and drug-resistant Enterococcus faecalis; and / or, the drug-resistant gram-positive bacteria comprise drug-resistant Enterococcus faecium.

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