Molecular Design of Novel Antibiotics and Antibiotic Adjuvants against MCR Strains

Abstract

The present invention relates to compounds comprising a hydrophobic moiety, a linker and an N-containing moiety. The present invention also relates to methods for synthesizing the compounds and the use of the compounds as antibiotics or antibiotic adjuvants.

Description

Technical Field

[0001] The present invention relates to compounds comprising a hydrophobic moiety, a linker and an N-containing moiety. The present invention also relates to methods for synthesizing such compounds and the use of such compounds as antibiotics or antibiotic adjuvants. Background Art

[0002] The ongoing emergence of carbapenemase-producing Enterobacteriacae bacteria, particularly Klebsiella pneumoniae strains with the KPC-2 and NDM1 genes conferring resistance to carbapenem antibiotics, has further exacerbated the healthcare crisis aggravated by a growing number of reports of elevated levels of pathogen resistance to existing antibiotics. Treatment options for these patients are limited, and colistin has become an important antibiotic of last resort. Colistin is a key "last resort" antibiotic because it disrupts the molecular organization of the outer membrane, allowing it to diffuse towards the inner membrane where it disrupts the structure, killing the bacteria by allowing water to enter the bacteria and losing the trans-membrane potential. Due to this mode of action, colistin avoids most types of resistance caused by gene mutations. As Figure 1 shown, the outer membrane of Gram-negative bacteria is covered with lipopolysaccharide (LPS), which is anchored in lipid A that forms the base of the LPS structure and is crucial for stabilizing the outer membrane. Colistin is a peptide, also known as polymyxin E, and is a member of the cationic polymyxin family including polymyxin B. Molecules of this family kill Gram-negative bacteria by disrupting the lipid A portion of the outer membrane, subsequently disrupting the inner membrane, and then water enters the bacteria, thereby lysing the organism.

[0003] Colistin has broad activity against Gram-negative bacteria but is rarely used due to severe side effects, nephrotoxicity, and neurotoxicity. Because of these side effects, 2 μg / ml is usually regarded as the breakpoint for the emergence of resistance. In contrast, in the United States and China, it is widely used in chicken and beef cattle agriculture. However, colistin resistance has recently emerged in the form of a mobilizable plasmid associated with E. coli, which was found in pigs in northwestern China in 2015. The spread of mobile colistin resistance (MCR) in E. coli carrying mcr-1 has been documented, indicating that as of 2015, mcr-1 had emerged in 10 countries / regions, including the United States. MCR-1 resistance has now been found in many Gram-negative pathogens, including E. coli, Salmonella enterica, Klebsiella pneumoniae, Enterobacter aerogenes, and Acinetobacter baumannii, posing a huge challenge to the treatment of these infections, especially since this trait confers a high propensity for antibiotic resistance to current antimicrobials, particularly to carbapenems. In addition, MCR-1 resistance has been documented in more than 30 countries / regions.

[0004] In addition, due to the plasmid-mediated mechanism of transfer of the MCR gene, horizontal transmission is prone to occur between different bacterial strains, and the situation is expected to deteriorate further. Recently, three other mutations, namely MCR-2, MCR-3, and MCR-4, have also been identified. However, to date, the mechanism of action has been the modification of lipid A by attaching phosphoethanolamine to lipid A ( Figure 2 ). Therefore, there is an urgent need for strategies to address the resistance conferred by the MCR gene. Particularly affected are patients with carbapenemase-resistant bacteria that are insensitive to other antibiotics.

[0005] Therefore, new methods for solving mcr-1 resistance that can at least partially improve the above-mentioned drawbacks are needed. Summary of the Invention

[0007] New methods have been developed for designing new antibiotics or antibiotic adjuvants against MCR strains, including MCR-1, MCR-2, MCR-3, and MCR-4. A molecular library that disrupts the outer membrane hydrogen-bonding network of MCR modification has been designed. This method combines a new computer modeling method that can greatly accelerate the development time while reducing the cost of new antimicrobial therapeutics. This method compares computer design with laboratory validation and in vivo testing of the effectiveness of treating MCR infections.

[0008] The design method is based on a fragment-based drug strategy and includes four steps: (i) computer modeling for target identification, (ii) ligand design, (iii) synthesis, and (iv) biological validation. Through one or more rounds of optimization, one or more lead compounds are revealed. For MCR-positive strains of Gram-negative bacteria, two targets are identified: the active site of the MCR-1 protein and the hydrogen bonding network of the outer membrane. Based on detailed atomic analysis, a compound library that destabilizes the outer membrane of E. coli with an mcr-1 plasmid is designed, which overcomes drug resistance and kills bacteria. Several compounds are selected from the library, and their MICs and their synergistic activity with colistin are tested. One of the compounds, namely GLA-DPA, shows limited antimicrobial activity against a group of clinically isolated colistin-resistant bacteria. However, it has been demonstrated that GLA-DPA can restore the sensitivity of E. coli with mcr-1 to colistin when combined with colistin. Therefore, when combined with the disclosed compounds, the effective dose of colistin can be reduced to a level that avoids toxicity.

[0009] On the one hand, there is provided a compound having the following formula (I):

[0010] Z 1 -L 1 -A-L 2 -Z 2 Formula (I)

[0011] Wherein A is a hydrophobic moiety;

[0012] L 1 and L 2 are independently linkers; and

[0013] Z 1 and Z 2 are independently N-containing moieties.

[0014] Advantageously, the compound can have a suitable structure that interacts with the bacterial membrane and disrupts the bacterial membrane. In other words, the compound can have two head group regions that interact with the bacterial membrane and a hydrophobic moiety that interacts with the lipid components of the bacterial membrane. The compound can have a suitable size to enable it to cross the bacterial membrane lipid bilayer. Advantageously, the head group can contain N-atoms, thus having a high pKa value. More advantageously, the hydrophobic moiety can be large. More advantageously, the hydrophobic moiety can be planar. In summary, the composition of the compound can endow the compound with high membrane interaction and antimicrobial properties.

[0015] Advantageously, the nitrogen-containing moiety, guanidine, and zinc chelating complex all have the same characteristic, namely a high affinity for the phosphate groups in the bacterial membrane. The end groups (Z 1 or Z 2)The strong interaction between the phosphate group of the membrane results in large membrane disruption.

[0016] Advantageously, the N-containing moiety can disrupt the electrostatic interactions (e.g., hydrogen bonding network) that stabilize the bacterial membrane. Advantageously, this can lead to bacterial cell killing. Alternatively, disruption of the hydrogen bonding network can facilitate the effective action of other antibiotics such as colistin, thereby killing bacterial cells. Even if the bacteria are resistant to colistin, colistin can interact with lipid A, and a minor perturbation of the outer membrane can allow the compound of formula (I) to enter the structure of lipid A towards the inner membrane together with colistin, resulting in fatal disruption of the inner membrane and bacterial killing.

[0017] The compound of formula (I) can effectively kill Gram-positive bacteria when administered alone or in combination with colistin. The compound of formula (I) can effectively kill Gram-negative bacteria that are MCR-positive when administered alone or in combination with colistin. In this case, the N-containing moiety can chelate zinc, and zinc can thus bind to zinc-dependent MCR and disrupt its activity. When administered in combination with colistin, the compound of formula (I) can also effectively kill Gram-negative bacteria that are MCR-negative but still resistant to colistin. In addition, when administered in combination with colistin, the compound of formula (I) can effectively kill a variety of Gram-negative bacteria, including those resistant to carbapenems. Therefore, the compound of formula (I) can overcome bacterial resistance to colistin, restore the action of colistin, and actually extend the action of colistin to Gram-negative bacteria resistant to either carbapenems alone or colistin. Advantageously, the compound of formula (I) can also effectively kill a large number of bacteria without having to change the drug concentration according to the bacterial species. The only other known antimicrobial agent capable of achieving this is alcohol, but alcohol is known to be toxic.

[0018] On the other hand, there is provided a pharmaceutical composition comprising the compound as defined above, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

[0019] On the other hand, there is provided a method for preparing the compound as defined above, comprising the step of contacting the hydrophobic moiety with the N-containing moiety under reaction conditions.

[0020] Advantageously, the method for preparing the compound is easy to perform, uses mild reaction conditions, and is conducive to the low-cost and large-scale synthesis of the compound.

[0021] On the other hand, there is provided the use of the compound as defined above or the pharmaceutical composition as defined above as an antibiotic.

[0022] On the other hand, there is provided the use of the compound as defined above or the pharmaceutical composition as defined above for killing or inhibiting the growth of microorganisms in vitro.

[0023] On the other hand, there is provided a compound as defined above for use in therapy.

[0024] On the other hand, there is provided a method for treating a bacterial infection, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound as defined above.

[0025] On the other hand, there is provided a compound as defined above for use in treating a bacterial infection.

[0026] On the other hand, there is provided the use of a compound as defined above in the preparation of a medicament for treating a bacterial infection.

[0027] Advantageously, the compound can act as an antibiotic in its own right or as an adjuvant to other antibiotics such as colistin by promoting the disruption of the outer membranes of MCR mutant bacteria as well as other Gram-negative bacteria without MCR mutations, and restoring the sensitivity of these strains to colistin. Advantageously, the compound can reduce the MIC of colistin by at least two-fold.

[0028] Definitions

[0029] Unless otherwise specified, "alkyl", as a group or part of a group, refers to a straight-chain or branched-chain aliphatic hydrocarbon group, preferably C 1 –C 20 alkyl, C 1 –C 12 alkyl, more preferably C 1 -C 10 alkyl, most preferably C 1 -C 6 . Suitable examples of straight-chain and branched-chain C 1 -C 6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, hexyl, and the like. The group can be a terminal group or a bridging group.

[0030] "Alkenyl", as a group or part of a group, denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which can be straight-chain or branched-chain, preferably having 2-20 carbon atoms in the straight chain, preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, and most preferably 2-6 carbon atoms. The group can contain multiple double bonds in the straight chain, and the orientation of each double bond is independently E or Z. Exemplary alkenyls include, but are not limited to, vinyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, and nonenyl. The group can be a terminal group or a bridging group.

[0031] "Alkynyl", as a group or part of a group, refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond and can be a straight-chain or branched-chain, preferably having 2-20 carbon atoms in the straight chain, more preferably 2-12 carbon atoms, still more preferably 2-10 carbon atoms, and even more preferably 2-6 carbon atoms. Exemplary structures include, but are not limited to, ethynyl and propynyl. The group can be a terminal group or a bridging group.

[0032] "Amino" refers to a group of the form –NR a R b wherein R a and R b are each independently selected from the group consisting of, but not limited to, hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl.

[0033] "Aminoalkyl" refers to an NH 2 -alkyl- group, wherein alkyl is as defined herein. The group can be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the rest of the molecule through the alkyl group.

[0034] "Aryl", as a group or part of a group, means (i) an optionally substituted monocyclic or fused polycyclic aromatic carbocyclic ring (a ring structure in which all ring atoms are carbon), preferably having 5-12 atoms in each ring. Examples of aryl include phenyl, naphthyl, etc.; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety, wherein a phenyl group and a C 5-7 cycloalkyl or a C 5-7 cycloalkenyl are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl, or indanyl. The group can be a terminal group or a bridging group. Generally, aryl is a C 6 -C 18 aryl.

[0035] "Guanidylalkyl" refers to an alkyl-NC(NH 2 ) 2 - group, wherein alkyl is as defined herein. The group can be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the rest of the molecule through the alkyl group.

[0036] "Heteroaryl", alone or as part of a group, refers to a group containing an aromatic ring (preferably a 5- or 6-membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring, with the remaining ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen, and sulfur. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isoxazole, furazane, phenoxazine, 2-, 3-, or 4-pyridyl, 2-, 3-, 4-, 5-, or 8-quinolyl, 1-, 3-, 4-, or 5-isoquinolyl, 1-, 2-, or 3-indolyl, and 2- or 3-thienyl. Heteroaryl is typically C 1 -C 18 heteroaryl. The heteroaryl may contain 3-8 ring atoms. The heteroaryl may contain 1-3 heteroatoms independently selected from N, O, and S. The group may be a terminal or bridging group.

[0037] "Heteroarylalkyl" refers to a heteroaryl and an alkyl moiety as defined herein, heteroaryl-alkyl. Preferred heteroarylalkyls contain a lower alkyl moiety. Exemplary heteroarylalkyls include pyridylmethyl. The group may be a terminal or bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkyl.

[0038] "Heterocycle" refers to a saturated, partially unsaturated, or fully unsaturated monocyclic, bicyclic, or polycyclic ring system containing at least one heteroatom selected from nitrogen, sulfur, and oxygen as ring atoms. Examples of heterocyclic moieties include heterocycloalkyl, heterocycloalkenyl, and heteroaryl.

[0039] "Halogen" represents chlorine, fluorine, bromine, or iodine.

[0040] As used herein, the term "substituted" means that the group referred to by this term can be substituted by one or more groups independently selected from the following: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkylalkenyl, heterocycloalkyl, cycloalkylheteroalkyl, cycloalkoxy, cycloalkenyloxy, cycloamino, halo, carboxyl, haloalkyl, haloalkenyl, haloalkynyl, alkynyloxy, heteroalkyl, heteroalkoxy, hydroxy, hydroxyalkyl, alkoxy, alkenyloxy, nitro, amino, alkylamino, dialkylamino, alkenylamine, aminoalkyl, alkynylamino, acyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkoxycarbonyl, alkoxycycloalkyl, alkoxyheteroaryl, alkoxyheterocycloalkyl, acylamino, alkylsulfonyloxy, heterocycle, heterocycloalkenyl, heterocycloalkyl, heterocycloalkylalkyl, heterocycloalkylalkenyl, heterocycloalkylheteroalkyl, heterocycloalkoxy, heterocycloalkenyloxy, heterocycloamino, haloheterocycloalkyl, alkanesulfinyl, alkanesulfonyl, aminosulfonyl, sulfinyl, sulfinylamino, sulfonyl, sulfonylamino, aryl, heteroaryl, heteroarylalkyl, heteroarylalkenyl, heteroarylheteroalkyl, heteroarylamino, heteroaryloxy, arylalkenyl, arylalkyl, aryloxy, arylsulfonyl, cyano, cyanate, isocyanate, -C(O)NH(alkyl) and -C(O)N(alkyl) 2 。

[0041] The term "pharmaceutically acceptable salt" refers to salts that retain the desired biological activity of the above compounds, including pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of the compound of formula (I) can be prepared from inorganic acids or organic acids. Examples of such inorganic acids are hydrochloric acid, sulfuric acid and phosphoric acid. Suitable organic acids can be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic organic acids, and examples thereof are formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, fumaric acid, maleic acid, alkylsulfonic acid, arylsulfonic acid. In the case where the pharmaceutical is a solid, those skilled in the art should understand that the compounds, pharmaceuticals and salts of the present invention can exist in different crystalline or polymorphic forms, all of which are intended to be within the scope of this disclosure and the specified formula.

[0042] The term "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Unless any conventional media or agents are incompatible with the compound, their use in therapeutic compositions as well as in therapeutic and prophylactic methods is contemplated. Supplementary active compounds can also be incorporated into the compositions of the present invention. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. As used herein, "unit dosage form" refers to physically discrete units suitable as unit doses for the individual to be treated; each unit containing a predetermined quantity of the compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. For convenience and effective administration, an effective amount of the compound can be formulated with a suitable pharmaceutically acceptable carrier in an acceptable unit dosage. Where the composition contains supplementary active ingredients, the dosage is determined with reference to the usual dosage and mode of administration of said ingredients.

[0043] It should be understood that the disclosed family of compounds includes isomeric forms in the form of "E" or "Z" configurational isomers or mixtures of E and Z isomers, including diastereoisomers, enantiomers, tautomers, and geometric isomers. It should also be understood that those skilled in the art can separate some of the isomeric forms, such as diastereoisomers, enantiomers, and geometric isomers, by physical and / or chemical methods.

[0044] Some of the compounds of the disclosed embodiments can exist as a single stereoisomer, racemate, and / or a mixture of enantiomers and / or diastereoisomers. All such single stereoisomers, racemates, and their mixtures are intended to fall within the scope of the described and claimed subject matter.

[0045] In addition, where applicable, the disclosed compounds are intended to cover solvated and non-solvated forms of the compounds. Thus, each formula includes compounds having the indicated structure, including hydrated and non-hydrated forms.

[0046] The term "substantially" does not exclude "completely", e.g., a composition "substantially free" of Y can be completely free of Y. Where necessary, the term "substantially" can be omitted from the definitions of the present invention.

[0047] Unless otherwise specified, the terms "comprising" and "comprise" and their grammatical variants are intended to denote "open" or "inclusive" language, such that they include the recited elements but also permit the inclusion of additional, unrecited elements.

[0048] In the context of the concentration of a formulation ingredient, the term "about" as used herein generally refers to + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0049] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed range. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges within that range as well as individual numerical values. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as the individual numerical values within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0050] Detailed Description of Optional Embodiments

[0051] Abbreviations

[0052] ArgOMe: Methyl arginate

[0053] ArgOEt: Ethyl arginate

[0054] BPD: Bipyridine

[0055] DMF: N,N-Dimethylformamide

[0056] DIC: N,N′-Diisopropylcarbodiimide

[0057] DPA: Dipicolylamine

[0058] EDTA: Ethylenediaminetetraacetic acid

[0059] EtBr: Ethidium bromide

[0060] EtOH: Ethanol

[0061] HOBt: Hydroxybenzotriazole

[0062] HPLC: High Performance Liquid Chromatography

[0063] LMMD: Ligand Positioning Molecular Dynamics

[0064] LPS: Lipopolysaccharide

[0065] MCR: Mobile Colistin Resistance

[0066] MCR strain: Bacteria with mobile colistin resistance.

[0067] MCR protein: An enzyme that catalyzes the modification of the lipid A with a PE group in MCR-1, MCR-2, MCR-3, and MCR-4 strains. The structures of these four proteins are highly conserved and have similar active sites.

[0068] MD simulation: Molecular dynamics simulation

[0069] MeOH: Methanol

[0070] MIC: Minimum inhibitory concentration.

[0071] NDM1: New Delhi metallo-beta-lactamase 1

[0072] PE group: Phosphoethanolamine group

[0073] TEA: Triethylamine

[0074] THF: Tetrahydrofuran

[0075] TPD: Terpyridine

[0076] Overall strategy for the design of MCR inhibitors

[0077] The present disclosure aims to design antibiotic molecules that are active against a specific target (in this case, the mcr-1 protein) or against bacteria, or to restore the sensitivity of colistin to bacteria with MCR plasmids, as Figure 3 shown.

[0078] E. coli with mcr-1 may be resistant to colistin and other antibiotics, E. coli without mcr-1 may be resistant to colistin and other antibiotics, resistant Pseudomonas aeruginosa (P. aeruginosa) may be resistant to carbapenems, aminoglycosides, and other antibiotics, resistant Acinetobacter baumannii may be resistant to carbapenems, colistin, and other antibiotics, and resistant Klebsiella pneumoniae may be resistant to carbapenems, colistin, and other antibiotics.

[0079] Based on the fragment-based strategy, a multidisciplinary approach is disclosed, including: (1) computer analysis and target identification; (2) ligand design; (3) synthesis; and (4) biological verification. As Figure 3 shown, the method begins with a computer analysis of the conformation of the MCR protein and the outer membrane structure to identify possible targets in the lipid A modified by MCR. Based on the fragment-based drug design strategy (FBDD), a series of chemical fragments with high affinity for the identified targets are selected. These chemical fragments are assembled together or incorporated into other designed fragments to form multiple antimicrobial scaffolds to obtain new antibiotics or antibiotic adjuvants.

[0080] To effectively disrupt the bacterial membrane, the proposed model contains three types of fragments: two cationic fragments that interact with the two headgroup regions of the bacterial membrane, one hydrophobic fragment that interacts with the lipid tails of the bacterial membrane, and two linker groups that connect the cationic groups and the hydrophobic fragment. A series of chemical fragments are designed and assembled together to obtain new antibiotics or antibiotic adjuvants. The basic principles for selecting the fragment structures are based on the following criteria ( Figure 4 ):

[0081] ● pKa of the N-containing group: Using model compounds, it was found that the pKa value of the terminal polar group is important. The higher the pKa value, the higher the membrane activity. Preferably, the pKa should be greater than 8, greater than 8.5, greater than 9, greater than 11 or greater than 13. Preferably, the pKa should be less than 20, less than 17 or less than 15.

[0082] ● Metal chelating properties of the nitrogen-containing group: When chelated with divalent cations such as zinc or calcium, the positively charged complex has an affinity for both the phosphate groups in the outer membrane and the MCR-1 protein, which contains zinc at its catalytic site ( Figure 6 ).

[0083] ● Size of the hydrophobic scaffold: Since the central hydrophobic scaffold needs to have a certain size, compounds composed of two or more aromatic rings with one or more hydrocarbon groups (such as isoprenyl groups) were tested. This is to ensure that the hydrophobic fragment has a certain size that allows the resulting molecule to cross the bacterial membrane. It was found that generally the larger the scaffold, the better the activity.

[0084] ● Shape of the hydrophobic scaffold: Planar hydrophobic scaffolds are preferred because they can easily penetrate into the membrane.

[0085] ● Overall hydrophobicity of the scaffold: According to the pharmacophore model, the hydrophobic scaffold interacts with the lipid tails. Therefore, the higher the hydrophobicity or the more favorable the transfer energy, the higher the membrane activity. Preferably, the logP of the compound should be greater than 4, greater than 6 or greater than 8. Preferably, the logP of the compound should be less than 10 or less than 9. Preferably, the free energy barrier should be negative. The more negative the free energy barrier, the higher the affinity of the fragment for the membrane.

[0086] ● Role of the isoprenyl group: The isoprenyl group has a high membrane affinity and can be used as a membrane probe; when the hydrophobic scaffold is modified with one or more isoprenyl groups, the membrane activity becomes higher.

[0087] Next, the designed molecules are synthesized and their antimicrobial activities against bacteria carrying the mobile plasmid mcr-1 are tested. Computer and biological insights may be iteratively used in the next round of fragment design. These rounds of structural optimization may lead to the development of one or more lead compounds.

[0088] Computer analysis and target identification

[0089] When simulating the interaction of colistin with the lipid A component of the outer membrane of normal Gram-negative bacteria, the anionic phosphate groups in lipid A undergo primary electrostatic interactions with cationic colistin, which ultimately leads to the rupture of the outer membrane.

[0090] The Mcr-1 gene encodes an enzyme similar to phosphoethanolamine transferase, which can modify the lipid A portion of the outer membrane by adding phosphoethanolamine (PE) groups, resulting in electrostatic interactions such as hydrogen bonding between lipid molecules and reducing the penetration of these compounds into the bacterial outer membrane. By crystallography, MCR-1 is characterized as a zinc-dependent metalloprotein, which makes it a good target for designing ligands that inhibit its activity.

[0091] As Figure 5 shown, the transferred groups allow for additional hydrogen bonding that crosslinks and stabilizes the lipid A portion of the outer membrane. Although each hydrogen bond may be weak by itself, when present in large numbers, they result in a very strong supramolecular scaffold that can maintain the integrity of lipid A. For colistin or polymyxin to kill bacteria, this supramolecular organization of lipid A must be disrupted.

[0092] In addition, in MCR-1 strains, PE-modified lipid A forms a large number of hydrogen bonds that crosslink lipid A together, thus stabilizing the outer membrane. However, the hydrogen bond network between the modified lipid molecules can also serve as an additional target. Molecules that disrupt the hydrogen bonding network will destabilize the outer membrane of MCR strains and may restore the sensitivity of these strains to colistin.

[0093] Colistin resistance in MCR-positive bacterial strains stems from the modification of lipid A by the MCR protein and subsequent changes in the properties of the outer membrane. Therefore, two targets can be identified in MCR strains: (1) the MCR protein; (2) the modified outer membrane. For the first target, a structural alignment of the extracellular domain of MCR-1 with the extracellular domain of the full-length PE transferase reveals a high degree of structural similarity. Using the crystal structures of the MCR-1 protein and the extracellular domain of the PE transferase as templates, based on this data, the structure of the entire MCR-1 protein was constructed using homology modeling ( Figure 7 ). Similar to the PE transferase, the MCR enzyme is a metalloprotein with a zinc atom in the catalytic domain, which facilitates the binding of the negatively charged portion of POPE lipids and catalyzes the subsequent modification of lipid A. The modified lipid A exhibits reduced interaction with colistin, resulting in colistin resistance. Therefore, molecules that bind to the active site of the MCR-1 protein will inhibit its activity and restore the sensitivity of MCR strains to colistin.

[0094] For the second target, the attachment of the PE group to lipid A reduces its electrostatic interaction with colistin, resulting in a decreased affinity of colistin for the outer membrane. In addition, molecular dynamics simulations of the PE-modified lipid A membrane have for the first time revealed the formation of a large number of hydrogen bonds between the lipid A molecular head groups ( Figure 7 ). The hydrogen bond network crosslinks LPS molecules together and stabilizes the outer membrane of MCR strains, thus conferring resistance to colistin. Therefore, the hydrogen bonding network in the outer membrane of MCR-positive strains can serve as another target for designing anti-MCR therapeutic agents. Fragments that disrupt / disturb the outer membrane hydrogen bonding network can directly kill bacteria or restore bacterial sensitivity to colistin. In summary, the MCR protein and the hydrogen bonding network in the outer membrane have been identified as two targets, and ligand mapping simulation has been used to search for more hidden binding pockets for designing MCR inhibitors.

[0095] Ligand design

[0096] Based on the atomic details of the identified targets, a series of fragments have been designed that can bind to the MCR protein or disrupt the hydrogen bonding network in the outer membrane. In terms of the mode of action, these fragments can be classified into three categories:

[0097] (1) Fragments that bind to the active site of the MCR-1 protein;

[0098] (2) Fragments that bind to the hidden pockets of the MCR-1 protein;

[0099] (3) Fragments that disrupt / disturb the hydrogen bonding network in the outer membrane.

[0100] The first two types of fragments directly inhibit MCR-1 activity and restore the sensitivity of MCR-1 strains to colistin, while the third type of fragment disrupts / disturbs the PE-modified outer membrane, which can independently kill MCR bacteria or act synergistically with colistin. Based on these three modes of action, 6 types of fragments have been proposed, Figure 8 showing the structures of representative examples of each type.

[0101] i. PE group. The PE group and its analogs bind to the active site of the MCR-1 protein.

[0102] ii. Zinc chelating groups such as the DPA group, terpyridine group, 2,2'-bipyridine group, 1,10-phenanthroline group, porphyrin group, 8-hydroxyquinoline group, and carboxyl group. The zinc chelating group can inhibit the activity of the MCR-1 protein by binding to the zinc atom in the active site of the MCR-1 protein. In addition, when chelating with zinc or other divalent cations, the complex is positively charged and can form salt bridges with phosphate groups, thus disrupting the hydrogen bonding network in the mcr-1 outer membrane and leading to the instability of the PE-modified outer membrane.

[0103] iii. Cyclic polyamines. Cyclic polyamines have dual functions: (a) forming hydrogen bonds with the head groups of PE-modified lipids, resulting in the disruption of the hydrogen-bonding network in the outer membrane; (b) chelating zinc atoms to inhibit the MCR-1 protein and disrupt the mcr-1 outer membrane.

[0104] iv. Amines and linear or branched polyamines. Polyamines can form hydrogen bonds with the phosphate groups of lipids and can disrupt the hydrogen-bonding network of the outer membrane.

[0105] v. Guanidines and polyguanidine groups. Polyguanidine groups can form bidentate hydrogen bonds with phosphate groups and can disrupt the hydrogen-bonding network of the outer membrane.

[0106] vi. Basic amino acid groups such as arginine, polyarginine, lysine, polylysine, poly-ε-lysine, arginine derivatives, lysine derivatives. Groups containing basic amino acids can form hydrogen bonds with the phosphate groups of lipid molecules and can disrupt the hydrogen-bonding network of the outer membrane.

[0107] All of the above fragments have the same characteristic, that is, they have a high affinity for phosphate groups in the bacterial membrane. The strong interaction between the above fragments and the phosphate groups in the bacterial membrane results in large membrane disruption.

[0108] Using the above fragments, a compound library was designed as a possible antibiotic or antibiotic adjuvant against MCR bacteria that synergizes with colistin. Molecules were designed by assembling two or more fragments together or coupling the fragments with other scaffolds. The structures of the library molecules and their modes of action are described below.

[0109] X-Cn-Y-Cn-X, where X is an N-containing moiety, Y is a hydrophobic scaffold, and Cn is an alkyl chain having n carbon atoms. The N-containing moiety X can be Figure 8 a fragment in Figure 9 , and Y can be any hydrophobic scaffold in

[0110] There is provided a compound having the following formula (I):

[0111] Z 1 -L 1 -A-L 2 -Z 2 Formula (I)

[0112] wherein A is a hydrophobic moiety;

[0113] L 1 and L 2 are independently linkers; and

[0114] Z 1 and Z2 Independently, it is an N-containing moiety.

[0115] A may comprise at least one substituted or unsubstituted aryl.

[0116] A may further comprise at least one alkenyl.

[0117] A may be planar or substantially planar. That is, A may be within the plane or substantially within the plane.

[0118] A may further comprise at least one isoprenyl.

[0119] A may be selected from:

[0120] and any mixture thereof.

[0121] Where the broken bond indicates the position where the structure is connected to the remainder of formula (I).

[0122] A may be selected from:

[0123]

[0124] and any mixture thereof.

[0125] Z 1 and Z 2 The pKa values of may independently be greater than 8. The pKa value may be greater than 8.5, greater than 9, greater than 11 or greater than 13. The pKa value may be less than 20.

[0126] Z 1 and Z 2 may independently have the following structures:

[0127]

[0128] Where the broken bond indicates the position where the structure is connected to the remainder of formula (I);

[0129] R 1 and R 2 may independently be hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aminoalkyl, guanidine, substituted or unsubstituted guanidinoalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, amino acid or peptide, or R 1 and R 2 together form a saturated or unsaturated, substituted or unsubstituted heterocycle; and

[0130] R 3may be absent, or may be hydrogen or a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted alkynyl group, a substituted or unsubstituted aminoalkyl group, guanidine, a substituted or unsubstituted guanidylalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted heteroarylalkyl group, an amino acid or a peptide,

[0131] wherein when R 3 is present, the nitrogen atom may be a cationic tetravalent nitrogen.

[0132] Z 1 and Z 2 may independently comprise one selected from the group consisting of amine, guanidine, pyrrolidine, pyrrole, imidazolidine, pyrazolidine, imidazole, pyrazole, triazole, piperidine, pyridine, piperazine, diazine, hydroxamic acid, hydrazine, N-hydroxyurea, squaric acid, carbamolyphosphonate, oxazoline, pyrimidine trione, 1-hydroxy-2(1H)-pyridone (1,2-HOPO), and any combination thereof.

[0133] R 1 、R 2 and R 3 may independently be selected from methyl, ethyl, propyl, butyl, -(CH 2 ) x NR’R”, -(CH 2 ) x OH, -(CH 2 ) x PO 3 、-(CH 2 ) x CR’R”R”’, -(C(NH 2 )NHC(NH 2 ))x-NH 2 、guanidine, 2-methylpyridine, 1-methylimidazole, pyridine, bipyridine, terpyridine, phenanthroline, 3-methylpyrrole, cyclen (1,4,7,10-tetraazacyclododecane), cyclam (1,4,8,11-tetraazacyclotetradecane), 1,8-dimethyl-1,4,8,11-tetraazacyclotetradecane, 1,4,7-triazacyclononane, arginine, polyarginine, lysine, polylysine, poly-ε-lysine, and any mixture thereof, where x may be any integer from 1 to 10, and R’, R” and R”’ may independently be hydrogen or a substituted or unsubstituted alkyl group.

[0134] Z 1 and Z 2 may independently be selected from:

[0135] and any combination thereof.

[0136] L 1 and L 2 may independently be a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted alkynyl, an ester, an amide, an ether, -(O-CH2-CH2-O) n- 、L 3 or any combination thereof, wherein L 3 has the following structure:

[0137] wherein n can be an integer from 1 to 10 and R 4 may be an amino or a heteroalkyl and the broken bond indicates the position where this structure is attached to the remainder of formula (I).

[0138] L 1 and L 2 may independently be selected from -(CH 2 ) 4 -, an amide, and any combination thereof, or

[0139] The logP value of the compound of formula (I) may be greater than 4. The logP value may be greater than 6 or greater than 8.

[0140] The compound of formula (I) may have the following structure:

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] Each N-containing moiety may independently chelate with zinc.

[0150] Each N-containing moiety may independently have a positive charge.

[0151] There is provided a pharmaceutical composition comprising a compound as defined above, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

[0152] The pharmaceutically acceptable carrier may be saline.

[0153] There is provided a method for preparing a compound as defined above, comprising the step of contacting a hydrophobic moiety with an N-containing moiety under reaction conditions.

[0154] The method as defined above may further comprise the step of contacting the hydrophobic moiety with a linker before contacting with the N-containing moiety.

[0155] A covalent bond may be formed between the hydrophobic moiety and the N-containing moiety, or between the hydrophobic moiety and the linker, or between the linker and the N-containing moiety.

[0156] The hydrophobic moiety may be selected from:

[0157] and any mixture thereof.

[0158] The linker may be selected from 1,4-dibromobutane, 1,3-diiodobutane, ethyl iodoacetate, hydroxybenzotriazole, 1,2-dibromoethane, 1,3-dibromopropane, methyl iodoacetate, methyl bromoacetate, 1,4-diiodobutane, and any combination thereof.

[0159] The N-containing moiety may be selected from dipyridylmethylamine, cyclen, 1,8-dimethyl-1,1,4,8,11-pentaazacyclotetradecane, diethylamine, -NH[(CH 2 ) 3 N(CH 3 )] 2 , -NH 2 (CH 2 ) 3 N(CH 3 ) 2 , 5-bromopentyltrimethylammonium bromide, 3-bromopentyltrimethylammonium bromide, 4-bromobutyltriethylammonium bromide, 3-(4-bromobutyl)-1-methylimidazolium bromide, 1-(4-bromobutyl)pyridinium bromide, 1-(4-bromobutyl)pyridinium bromide, and any combination thereof.

[0160] The method as defined above may include the step of adding zinc after the N-containing moiety is covalently bonded to the linker or the hydrophobic moiety.

[0161] The contacting may be carried out in a solvent selected from acetone, methanol, ethanol, propanol, butanol, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, pyridine, water, and any mixture thereof.

[0162] The contacting step can be carried out at a temperature of from about 21°C to about 160°C, from about 21°C to about 60°C, from about 21°C to about 100°C, from about 60°C to about 100°C, from about 60°C to about 160°C or from about 100°C to about 160°C.

[0163] The contacting step can be carried out for from about 2 hours to about 36 hours, from about 2 hours to about 8 hours, from about 2 hours to about 12 hours, from about 2 hours to about 24 hours, from about 8 hours to about 12 hours, from about 8 hours to about 24 hours, from about 8 hours to about 36 hours, from about 12 hours to about 24 hours, from about 12 hours to about 36 hours or from about 24 hours to about 36 hours.

[0164] Use of a compound as defined above or a pharmaceutical composition as defined above as an antibiotic is provided.

[0165] Use of a compound as defined above or a pharmaceutical composition as defined above for killing or inhibiting the growth of microorganisms in vitro is provided.

[0166] The compound as defined above or the pharmaceutical composition as defined above can be used for killing or inhibiting the growth of microorganisms on a surface, including but not limited to topical application, in plasters, eye drops, nasal sprays, mouthwashes and hand sanitizers. The compound as defined above or the pharmaceutical composition as defined above can also be used as a food preservative, a disinfectant, a surface cleaner or a medical device.

[0167] The microorganism can be a bacterium, an archaeon, a fungus, a protist or any mixture thereof.

[0168] The microorganism can be a Gram-negative bacterium or a Gram-positive bacterium.

[0169] The bacterium can be MCR-positive or MCR-negative.

[0170] The bacterium can be resistant to carbapenems.

[0171] The bacterium can be selected from E. coli, E. cloacae, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumoniae, Enterobacter aerogenes and Acinetobacter baumannii.

[0172] The compound as defined above can be used in combination with colistin.

[0173] A compound as defined above for use in therapy is provided.

[0174] There is also provided a method for treating a bacterial infection, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound as defined above.

[0175] The method as defined above can further comprise the step of administering a therapeutic amount of colistin and a compound as defined above.

[0176] The above-defined compounds are provided for the treatment of bacterial infections.

[0177] The above-defined compounds can be administered in combination with colistin.

[0178] Use of the above-defined compounds in the preparation of a medicament for the treatment of bacterial infections is provided.

[0179] According to the present invention, when used for the treatment or prevention of microbial infections, the compounds of the present invention can be administered alone. Alternatively, the compound can be administered as a pharmaceutical, veterinary, agricultural or industrial preparation, which comprises at least one compound of the present invention. The compound can also exist as a suitable salt, including a pharmaceutically acceptable salt.

[0180] The above-defined use may also include the above-defined compounds administered in combination with colistin.

[0181] The above-defined method, the above-defined compound or the above-defined use is provided, wherein the above-defined compound can be present in the following amounts: about 2 μg / mL to about 75 μg / mL, about 2 μg / mL to about 12 μg / mL, about 2 μg / mL to about 25 μg / mL, about 2 μg / mL to about 50 μg / mL, about 12 μg / mL to about 25 μg / mL, about 12 μg / mL to about 50 μg / mL, about 12 μg / mL to about 75 μg / mL, about 25 μg / mL to about 50 μg / mL, about 25 μg / mL to about 75 μg / mL or about 50 μg / mL to about 75 μg / mL.

[0182] The above-defined method, the above-defined compound or the above-defined use, wherein the above-defined compound and colistin can be present in the same weight amount, or by weight, the above-defined compound can be about 1.5 to about 6 times, about 1.5 to about 3 times, about 1.5 to about 4.5 times, about 3 times to about 4.5 times, about 3 times to about 6 times or about 4.5 times to about 6 times in excess of colistin.

[0183] The method, compound or use as defined above, wherein colistin can be present in the following ranges: about 1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 2 mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg or about 5 mg / kg to about 10 mg / kg, and the above-defined compound can be present in the range of about 10 mg / kg to about 50 mg / kg, about 10 mg / kg to about 20 mg / kg or 20 mg / kg to about 50 mg / kg.

[0184] The method as defined above, the compound as defined above, or the use as defined above, wherein the compound as defined above can be administered intramuscularly, intraperitoneally, topically, subcutaneously or intravenously.

[0185] In one embodiment, the compound can be administered by injection. In the case of an injectable solution, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using surfactants. The action of microorganisms can be prevented by including various antibacterial and / or antifungal agents. Suitable agents are well known to those skilled in the art and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, etc. In many cases, it may be preferable to include isotonic agents, such as sugars, polyols such as mannitol, sorbitol, sodium chloride, in the composition. The absorption of the injectable composition can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0186] If desired, a sterile injectable solution can be prepared by incorporating the required amount of the analogue and a combination of one or more of the above ingredients into a suitable solvent and then filtering to sterilize. Generally, a dispersion is prepared by incorporating the analogue into a sterile vehicle containing a basic dispersion medium and the other ingredients required from those listed above.

[0187] Preferably, the pharmaceutical composition can also include a suitable buffer to minimize acid hydrolysis. Suitable buffers are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof.

[0188] Dispersions of the compounds of the present invention can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and in oils. Under ordinary storage and use conditions, the pharmaceutical formulations can contain preservatives to prevent the growth of microorganisms.

[0189] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the case of water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. It is desirable that the composition be stable under the conditions of manufacture and storage and can include preservatives to stabilize the composition against the contaminating action of microorganisms such as bacteria and fungi.

[0190] Sustained-release formulations are also included within the scope of the present invention.

[0191] The compounds of the present invention can also be administered in the form of "prodrugs". A prodrug is an inactive form of a compound that is converted into an active form in the body. Suitable prodrugs include esters, phosphonates, etc. of the active form compound.

[0192] Single or multiple administrations of the pharmaceutical compositions of the present invention can be carried out. Those skilled in the art should be able to determine, through routine experiments, the effective and non-toxic dosage levels of the compounds and / or compositions of the present invention and the mode of administration suitable for treating the diseases and / or infections for which they are applicable.

[0193] In addition, it is obvious to those of ordinary skill in the art that conventional course determination tests can be used to determine the optimal course, such as the number of doses of the compounds or compositions of the present invention administered daily over a defined number of days.

[0194] Brief Description of the Drawings

[0195] Figure 1 is a schematic diagram showing the pathway to the cytoplasmic membrane of Gram-negative bacteria and the types of molecular interactions at each step.

[0196] Figure 2 is a cartoon of the chemical structure of the outer membrane of Gram-negative bacteria and the lipid A part of the LPS molecule.

[0197] Figure 3 relates to a schematic flow chart of the proposed method for designing molecules that can restore the sensitivity of MCR strains to colistin.

[0198] Figure 4 relates to a schematic diagram showing how the components of the antibiotic of the present invention interact with the lipid bilayer of the bacterial membrane. The N-containing group (4001) is connected to the hydrophobic scaffold (4003) through a linker (4002). The N-containing group (4001) interacts with the head group (4004) of the lipid, while the hydrophobic scaffold (4003) interacts with the lipid tail (4005).

[0199] Figure 5 relates to a schematic diagram showing the chemical modification of lipid A in MCR strains. Lipid A is modified by MCR-1 through the transfer of phosphoethanolamine (PPEA) groups, resulting in the formation of hydrogen bonds between adjacent segments of lipid A, thus leading to the formation of PPEA-4'-lipid A or PPEA-1'-lipid A.

[0200] Figure 6 shows an image of the MCR-1 protein structure constructed by homology modeling. The structure of the extracellular domain is highly conserved and contains a zinc atom at the active site.

[0201] Figure 7 ​​​​​​​The images and plots involved show the molecular dynamics simulations of the outer membrane of the MCR-modified membrane. (a) Snapshot of the normal lipid A membrane; (b) Snapshot of the PE-modified lipid A membrane. The PE groups form hydrogen bonds (shown as dashed lines) with adjacent lipid A molecules, thus forming a network that stabilizes the outer membrane. (c) Plot showing the number of hydrogen bonds in a membrane patch of 64 lipid A molecules. (d) Plot showing the radial distribution function (RDF) between different groups, showing phospho-phospho (701), amine-amine (702), and phospho-amine (703).

[0202] Figure 8 Representative structures involving serotypes 1-16 fragments.

[0203] Figure 9 Structures involving hydrophobic scaffolds.

[0204] Figure 10 Structures of molecules synthesized in the DPA-Zn series.

[0205] Figure 11 Structures of molecules synthesized in the amine series.

[0206] Figure 12 Structures of molecules synthesized in the guanidine series.

[0207] Figure 13 The plots involved show time-kill studies for (a) colistin and (b) the combination of LC100 and colistin against the colistin-resistant mcr-1(+) strain 6083655967.

[0208] Figure 14 The plots involved show time-kill studies for (a) colistin and (b) the combination of LC100 and colistin against the colistin-resistant mcr-1(+) strain 6075066346.

[0209] Figure 15 The plots involved show time-kill studies for (a) colistin and (b) the combination of LC100 and colistin against the colistin-resistant mcr-1(-) strain 7023446108.

[0210] Figure 16 The plots involved show the time-kill effect of the combination of B2088 and LC100.

[0211] Figure 17 The plots involved show the effect of (a) LC100, (b) colistin, and (c) the combination of LC100 and colistin on the mcr-1 bacterial membrane using the fluorescent probe ethidium bromide.

[0212] ​​​​​​​​​​Figure 18 is a coordinate graph showing the interaction between LC100 and MCR1 proteins using fluorescence quenching.

[0213] Figure 19 The coordinate graph involved shows the hydrophobic surface area in the bacterial membrane in the presence and absence of polymyxin B.

[0214] Figure 20 The coordinate graph involved shows the calcein leakage of LC100 and LC101 using 100 nm large unilamellar vesicles. The lipid composition of the vesicles is DOPE / DOPG = 3 / 1, thus mimicking the bacterial inner membrane. As the concentration of LC100 or LC101 increases, significant fluorescence is detected, indicating that the vesicles are disrupted and the dye molecules are released. Due to the fluorescence quenching effect, a further increase in LC100 or LC101 results in a decrease in fluorescence.

[0215] Figure 21 The coordinate graph involved shows the efficacy test of the combination of LC100 and colistin in a neutropenic mouse thigh infection model infected with MCR-positive E. coli clinical isolate 6083655967. The starting inoculum was 1.87x10 6 , N = 3. "Combination" refers to colistin 10 mg / kg + LC100 50 mg / kg. Each treatment was performed intraperitoneally 1 hour after infection. 6 hours after treatment, the mouse thigh tissues were homogenized to obtain viable CFU. Abdominal distension was found in the mice in the LC100 group and the combination group. Compared with the LC100 group, the mice in the combination group seemed weaker, colder, and less active before being sacrificed.

[0216] Detailed description of the drawings

[0217] Figure 1 is a schematic diagram showing the pathway of Gram-negative bacteria to the cytoplasmic membrane and the types of molecular interactions at each step. 1001 refers to step 1, where t ~ ns, and the adsorption on the LPS surface occurs through electrostatic interactions. 1002 refers to step 2, where the outer membrane is permeabilized, and the electrostatic interactions, hydrogen bonds with PO4 3- and hydrophobic interactions with the lipid tails disrupt the PO 4 3- salt bridge between Ca 2+ / Mg 2+ . 1003 refers to step 3, where t ~ ns, and the adsorption on the cytoplasmic membrane occurs through electrostatic interactions with anionic lipids, hydrogen bonds with the head groups, and hydrophobic interactions with the lipid tails. 1004 refers to step 4, where t > μs, where the cytoplasmic membrane is disrupted.

[0218] Figure 3 ​​​​​​Schematic flowchart of the proposed method for designing molecules that can restore the sensitivity of MCR strains to colistin. Perform MD simulations of the outer membrane and structural analysis of the MCR enzyme (3001), identify the target (3002), generate fragments (3003), incorporate fragments (3004), synthesize new antibiotics (3005), verify the antibiotics (3006), then decipher the mechanism of action (3007), and feedback this information (3008) to fragment generation (3003).

[0219] Experiments and methods

[0220] Example 1: Materials

[0221] Strains with "ATCC" in their names were purchased from the American Type Culture Collection (ATCC), Virginia, USA. All other bacterial strains were purchased from Tan Tock Seng Hospital, Singapore, Singapore.

[0222] The hydrophobic scaffolds LC003, LC007, LC008, AM000 were purchased from Chengdu Biopurify Phytochemicals (Chengdu, China). The cationic moieties N,N,N-trimethyl-1,3-propanediamine and 3,3'-iminobis(N,N-dimethylpropylamine) were purchased from Bio-etc (Singapore, Singapore). The MCR-1 protein was purchased from the A*STAR Institute of BioProcessing Technology (Singapore, Singapore). Cyclen and 1,8-dimethyl-1,4,8,11-tetrazacyclotetradecane were purchased from TCI (Tokyo, Japan). The solvents ethyl acetate, hexane, acetone, butanol, methanol, DMF were purchased from Aik Moh (Singapore, Singapore). All other chemicals were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

[0223] Example 2: MIC study

[0224] According to the guidelines of the Clinical and Laboratory Standards Institute (CLSI), the MIC experiment of antimicrobial agents was determined using a 96-well culture plate by the modified broth microdilution method. Briefly, serial dilutions of all plates were prepared at different concentrations (μg). The inoculum suspension was prepared by adding 18 - 20-hour isolated colonies on TSA plates to phosphate buffer (0.31 mM, pH 7.2) or water for injection (BBraun) and adjusting the suspension to a turbidity equivalent to the 0.5 McFarland standard. The final bacterial concentration in each well was approximately 5×10 5 colony-forming units (CFU) / mL. The plates were covered and incubated at 35 °C for 24 hours. The MIC is the lowest concentration of antimicrobial agent that completely inhibits the growth of microorganisms detected by the naked eye in a test tube or microdilution well. For microdilution plates, the turbidity in the wells can be measured at OD 600 using a TECAN infinite M200Pro. The MIC was determined as the highest concentration that produced 99% inhibition.

[0225] Example 3: Synergy study

[0226] The MIC of individual drugs and combinations with the LC compound was determined using the broth microdilution technique recommended by CLSI, whereby two antimicrobial agents were added in equal amounts. (For example: 50 μL colistin: 50 μL LC compound). The diluted medium containing the compound and the antibiotic was serially diluted 2-fold, and the LC compound and the antibiotic alone or in combination were tested at fixed synergy concentrations. In all cases, the lowest concentration at which no visible growth occurred was recorded as the MIC value of the individual and combined antimicrobial agents. The results were obtained after 24 hours of incubation.

[0227] Example 4: Time-kill study

[0228] The time-kill assay was used to determine the rate at which antimicrobial agents kill bacterial isolates. The bacterial suspension was adjusted to 10 6 to 10 7CFU / mL for comparison in separate tubes incubated at 35°C. For time-kill in combination: Two different antimicrobial agents were added in equal amounts as antibiotic / LC compound to make a final volume of 1 mL (e.g., 500 μL antibiotic: 500 μL LC compound), and then 1 mL of the prepared bacterial suspension was added to the same tube. Aliquots of 100 μL samples were taken at different time intervals (0, 1, 2, 4, 8, 24 hours) and plated on Mueller Hinton Agar (MHA) after serial dilution. Subsequently, after 24 and 48 hours, bacterial growth on all plates was examined, and the colony-forming unit (CFU / mL) count of the colonies was calculated, and a time-kill curve was plotted with the logarithm of viable count against time. The results for various concentrations of the antibiotic and the control were plotted.

[0229] Example 5: In Vivo Mouse Thigh Infection Model

[0230] In a neutropenic mouse thigh infection model, the combination of colistin and antimicrobial peptide (LC100) was tested against the E. coli clinical isolate MCR-positive strain 6083655967. C57BL / 6 mice were rendered neutropenic by cyclophosphamide and dosed at 150 mg kg -1 and 100 mg kg -1 delivered on days -4 and -1 prior to infection. The bacteria were suspended in sterile saline and adjusted to a concentration of approximately 1.876×10 6 CFU per infection site (100 μL) and injected intramuscularly into the right thigh of 5 mice in each treatment group. One hour after infection, the mice received colistin (15 mg kg -1 , i.p. n = 5), LC100 (50 mg kg -1 , i.p. n = 5), untreated (n = 5) or combination (n = 5). Mice were euthanized 6 hours after infection; thigh tissues were collected under sterile conditions, homogenized, serially diluted in PBS, and plated on solid plates supplemented with TSA. The plates were incubated overnight at 37°C and the colonies were quantified to determine the bacterial load.

[0231] Example 6: Results of Molecular Dynamics Studies

[0232] Molecular dynamics simulations were performed to understand the molecular origin of colistin resistance. The structures and dynamics of mcr-1(+) and intact lipid A membranes were studied. For each membrane, a pre-assembled lipid A bilayer was first assembled by placing the required number of lipid A molecules (from mcr-1(+) or mcr-1(-) strains) on a grid in the xy dimension. Next, the pre-assembled bilayer was solvated with water molecules. Calcium ions were used to neutralize the system. MD simulations of 300 ns were performed to equilibrate each lipid A bilayer. At the end of the MD simulations, all calcium ions were found to be adsorbed on the bilayer surface. In the mcr-1(-) strain, calcium ions were found to form salt bridges between the phosphate groups, which counteracted the repulsion between the negatively charged lipid A molecules and stabilized the lipid A membrane. Disrupting the salt bridges with an antimicrobial peptide such as colistin destabilized the outer membrane. However, in the case of the mcr-1(+) strain, MD simulations revealed Figure 7 ). Since the phosphate and amine groups carry opposite charges, they interact with strong electrostatic attractive forces that further stabilize the lipid A membrane. As a result, short-range lipid-lipid hydrogen bonds and long-range electrostatic interactions crosslink the lipid A molecules of the mcr-1(+) strain, leading to resistance to the action of colistin. The results suggest that disruption of the hydrogen bonds or electrostatic interactions of the lipid A membrane can destabilize the outer membrane of the mcr-1(+) strain.

[0233] Example 7: Chemical Synthesis

[0234] Selected synthetic schemes are as Figure 10 、 Figure 11 and Figure 12 shown.

[0235] Synthesis of LC004: 1,4-Dibromobutane (313.8 μL, 2.65 mmol) and K 2 CO 3 (111.7 mg, 0.808 mmol) were added to isobavachalcone LC003 (57.2 mg, 0.176 mmol) dissolved in 3 mL of acetone. The reaction was kept under reflux conditions and stirred overnight. The acetone solvent was removed in vacuo. The crude product was diluted with ethyl acetate and extracted with saturated sodium chloride solution. Pure LC004, 77.3 mg, was obtained by silica gel column chromatography using an elution gradient (hexane:EtOAc (v / v), 26:1). Yield: 73.8%.

[0236] Synthesis of LC003-I: 1,4-Diiodobutane (601 μL, 4.557 mmol) and K 2 CO 3(198 mg, 1.43 mmol) was added to LC003 (98.6 mg, 0.304 mmol) dissolved in acetone. The reaction workup was similar to the synthesis of LC004. Silica gel column chromatography of the crude product using a solvent ratio of hexane:EtOAc (v / v) 40:1 gave 113.5 mg of LC003-I as a yellow powder. Yield: 54%.

[0237] Synthesis of LC003-DPA: To a 2.5 mL acetone solution of LC003-I (25.5 mg, 0.037 mmol) was added K 2 CO 3 (26.2 mg, 0.190 mmol) and dipyridylmethylamine (99 μL, 0.550 mmol). The reaction mixture was stirred at 55 °C overnight. Acetone was removed in vacuo. The crude product was diluted with EtOAc and extracted 3 times with saturated NaCl solution. Purification by preparative HPLC gave 17.8 mg of LC003-DPA as a bright yellow gel. Yield: 58%.

[0238] Synthesis of LC003-DPA-Zn: A 1 mL methanol solution of 5.44 mg ZnCl 2 was mixed with 8.3 mg of LC003-DPA and stirred for 3 h. The solvent was removed in vacuo to give the LC003-DPA-Zn complex (LC003-DPA:Zn = 1:4).

[0239] Synthesis of LC008-I: 1,4-Diiodobutane (478 μL, 1.86 mmol) and K 2 CO 3 (180 mg, 1.302 mmol) were added to LC008 (61.8 mg, 0.241 mmol) dissolved in acetone. The reaction workup was similar to the synthesis of LC004. Silica gel column chromatography of the crude product using a solvent ratio of hexane:EtOAc (v / v) 25:1 gave 49.6 mg of LC008-I as a yellow powder. Yield: 33%.

[0240] Synthesis of LC008-DPA: To a 2 mL acetone solution of LC008-I (32.1 mg, 0.052 mmol) was added K 2 CO 3 (42.0 mg, 0.304 mmol) and dipyridylmethylamine (140 μL, 0.778 mmol). The reaction mixture was stirred at 55 °C overnight. Acetone was removed in vacuo. The crude product was diluted with EtOAc and extracted 3 times with saturated NaCl solution. Purification by preparative HPLC gave 16.7 mg of LC008-DPA as a bright yellow gel. Yield: 42%.

[0241] Synthesis of LC008-DPA-Zn: A 1 mL methanol solution of 5.44 mg ZnCl 2 was mixed with 7.62 mg LC003-DPA and stirred for 3 hours. The solvent was removed under vacuum to obtain the LC008-DPA-Zn complex (LC008-DPA:Zn = 1:4).

[0242] Synthesis of AM005: 1,4-Dibromobutane (434 μL, 3.66 mmol) and K 2 CO 3 (168 mg, 1.22 mmol) were added to AM000 (100 mg, 0.244 mmol) dissolved in 3 mL of acetone. The reaction was worked up similarly to the synthesis of LC004. Silica gel column chromatography of the crude product using a solvent ratio of hexane:EtOAc (v / v) 20:1 gave 98 mg of AM005 as a yellow powder. Yield: 59%.

[0243] Synthesis of AM000-DPA: To a 3 mL acetone solution of AM005 (68 mg, 0.1 mmol) was added K 2 CO 3 (84.0 mg, 0.6 mmol) and dipyridylmethylamine (269 μL, 1.5 mmol). The reaction mixture was stirred at 55 °C overnight. Acetone was removed under vacuum. The crude product was diluted with EtOAc and extracted 3 times with saturated NaCl solution. Purification by preparative HPLC gave 38.4 mg of AM000-DPA as a bright yellow gel. Yield: 47%.

[0244] Synthesis of LC104 (AM000-DPA-Zn): A 1 mL methanol solution of 5.44 mg ZnCl 2 was mixed with 9.16 mg AM000-DPA and stirred for 3 hours. The solvent was removed under vacuum to obtain the LC104 complex (AM000-DPA:Zn = 1:4).

[0245] Synthesis of Orc-I: 1,4-Diiodobutane (1 mL, 7.58 mmol) and K 2 CO 3 (546 mg, 3.95 mmol) were added to orcinol (62.9 mg, 0.506 mmol) dissolved in acetone. The reaction was worked up similarly to the synthesis of LC004. Silica gel column chromatography of the crude product using a solvent ratio of hexane:EtOAc (v / v) 100:1 gave 164.7 mg of LC008-I as a yellow powder. Yield: 67%.

[0246] Synthesis of Orc-DPA: To a 2 mL acetone solution of Orc-I (66 mg, 2.83 mmol) was added K2 CO 3 (122 mg, 0.883 mmol) and dipyridylmethylamine (364 μL, 2.022 mmol). The reaction mixture was stirred overnight at 55 °C. Acetone was removed in vacuo. The crude product was diluted with EtOAc and extracted three times with saturated NaCl solution. Purification by preparative HPLC gave 66.4 mg of bright yellow gel-like LC008-DPA. Yield: 85%.

[0247] Synthesis of Orc-DPA-Zn: A 1 mL methanol solution of 5.44 mg ZnCl 2 was mixed with 6.3 mg of Orc-DPA and stirred for 3 h. The solvent was removed in vacuo to give the Orc-DPA-Zn complex (Orc-DPA:Zn = 1:4).

[0248] Synthesis of LC007-I: 1,4-Diiodobutane (373 μL, 2.83 mmol) and K 2 CO 3 (147.3 mg, 1.07 mmol) were added to glabridin (62.2 mg, 0.192 mmol) dissolved in acetone. The reaction workup was similar to the synthesis of LC004. Silica gel column chromatography of the crude product using a solvent ratio of hexane:EtOAc (v / v) 30:1 gave 66.6 mg of yellow powder-like LC007-I. Yield: 50.0%.

[0249] Synthesis of GLA-DPA: K 2 CO 3 (37.5 mg, 0.271 mmol) and dipyridylmethylamine (123 μL, 0.683 mmol) were added to a 2 mL acetone solution of GLA-I (31.5 mg, 0.0456 mmol). The reaction mixture was stirred overnight at 55 °C. Acetone was removed in vacuo. The crude product was diluted with EtOAc and extracted three times with saturated NaCl solution. Purification by preparative HPLC gave 24.4 mg of yellow gel-like GLA-DPA. Yield: 65%.

[0250] Synthesis of GLA-DPA-Zn (LC007-DPA-Zn): A 1 mL methanol solution of 5.44 mg ZnCl 2 was mixed with 8.3 mg of GLA-DPA and stirred for 3 h. The solvent was removed in vacuo to give the GLA-DPA-Zn complex (GLA-DPA:Zn = 1:4).

[0251] Synthesis of LC300: Cyclen (68.8 mg, 0.4 mmol) was added to a 3 mL DMF solution of LC010 (34.85 mg, 0.05 mmol). The reaction mixture was stirred at room temperature for 4 hours. The crude product was dissolved in butanol and then washed three times with an aqueous K 2 CO 3 aqueous solution and saturated NaCl solution and dried in vacuo. The crude product was then redissolved in a 1 mmol ZnCl 2 / MeOH solution. Purification by preparative HPLC gave 20.2 mg of yellow-gel-like LC300 (GLA-Cyclen-Zn). Yield: 40.5%.

[0252] Synthesis of LC301: Cyclen (68.8 mg, 0.4 mmol) was added to a 3 mL DMF solution of LC003-I (34.85 mg, 0.05 mmol). The reaction mixture was stirred at room temperature for 4 hours. The crude product was dissolved in butanol and then washed three times with an aqueous K 2 CO 3 aqueous solution and saturated NaCl solution and dried in vacuo. The crude product was then redissolved in a 1 mmol ZnCl 2 / MeOH solution. Purification by preparative HPLC gave 18.4 mg of yellow-gel-like LC301 (LC003-Cyclen-Zn). Yield: 37%.

[0253] Synthesis of LC302: Cyclen (68.8 mg, 0.4 mmol) was added to a 3 mL DMF solution of AM005 (34.0 mg, 0.05 mmol). The reaction mixture was stirred at room temperature for 4 hours. The crude product was dissolved in butanol and then washed three times with an aqueous K 2 CO 3 aqueous solution and saturated NaCl solution and dried in vacuo. The crude product was then redissolved in a 1 mmol ZnCl 2 / MeOH solution. Purification by preparative HPLC gave 24.6 mg of yellow-gel-like LC302 (AM000-Cyclen-Zn). Yield: 45%.

[0254] Synthesis of LC304: Cyclen (68.8 mg, 0.4 mmol) was added to a 3 mL DMF solution of LC008-I (34.0 mg, 0.05 mmol). The reaction mixture was stirred at room temperature for 4 hours. The crude product was dissolved in butanol and then washed three times with an aqueous K 2 CO 3 aqueous solution and saturated NaCl solution and dried in vacuo. The crude product was redissolved in a 1 mmol ZnCl 2In a / MeOH solution. It was purified by preparative HPLC to obtain 25.3 mg of yellow gel-like LC302 (LC008 - cyclen - Zn). Yield: 54%.

[0255] Synthesis of LC014: LC004 (62.1 mg, 0.104 mmol) and 1,8 - dimethyl - 1,4,8,11 - tetracyclotetradecane (61.9 μL, 0.376 mmol) were dissolved in 4 mL of DMF and stirred overnight at room temperature. The crude product was diluted with butanol, extracted 3 times with saturated NaCl solution, and dried under vacuum. Then the crude product was redissolved in 1 mmol ZnCl 2 / MeOH solution. It was purified by preparative HPLC to obtain 10.9 mg of yellow gel-like LC014. Yield: 12%.

[0256] Synthesis of LC100: LC004 (89 mg, 0.150 mmol) was added to a mixture of 3 mL of diethylamine and 3 mL of DMSO and stirred at room temperature for 3 hours. The crude product was washed with K 2 CO 3 aqueous solution and saturated NaCl solution. The crude sample was purified by silica gel chromatography (EtOAc / MeOH / TEA (v / v / v), 150 / 1 / 1). 63.8 mg of product was obtained. Yield: 73.6%.

[0257] Synthesis of LC097: LC004 (59.4 mg, 0.10 mmol) was added to a mixture of 1 mL of 3,3'-iminobis(N,N - dimethylpropylamine) and 3 mL of DMSO and stirred at room temperature for 4 hours. The crude product was washed with K 2 CO 3 aqueous solution and saturated NaCl solution. It was purified by preparative HPLC to obtain 41.8 mg of product. Yield: 52%.

[0258] Synthesis of LC098: LC004 (59.4 mg, 0.10 mmol) was added to a mixture of 1 mL of N,N,N - trimethyl - 1,3 - propanediamine and 3 mL of DMSO and stirred at room temperature for 4 hours. The crude product was washed with K 2 CO 3 aqueous solution and saturated NaCl solution. It was purified by preparative HPLC to obtain 43.2 mg of product. Yield: 65%.

[0259] Synthesis of LC095: LC010 (29.7 mg, 0.05 mmol) was added to a mixture of 1 mL of 3,3'-iminobis(N,N - dimethylpropylamine) and 3 mL of DMSO and stirred at room temperature for 4 hours. The crude product was washed with K2 CO 3 Washed with aqueous CO solution and saturated NaCl solution. Purified by preparative HPLC to obtain 36 mg of the product. Yield: 44.8%.

[0260] Synthesis of LC096: Added LC010 (29.7 mg, 0.05 mmol) to a mixture of 0.6 mL of N,N,N-trimethyl-1,3-propanediamine and 3 mL of DMSO, and stirred at room temperature for 4 hours. The crude product was washed with K 2 CO 3 aqueous solution and saturated NaCl solution. Purified by preparative HPLC to obtain 46.5 mg of the product. Yield: 70%.

[0261] Synthesis of LC149: Added 1,2-dibromoethane (259 μL, 3 mmol) and K 2 CO 3 (138 mg, 0.1 mmol) to isopsoralen chalcone LC003 (64.8 mg, 0.2 mmol) dissolved in 3 mL of acetone. The reaction was maintained under reflux conditions and stirred for two days. The acetone solvent was removed in vacuo. The crude product was diluted with ethyl acetate and extracted with saturated sodium chloride solution. Pure LC149, 63.4 mg of the product, was obtained by silica gel column chromatography using an elution gradient (hexane:EtOAc (v / v), 15:1). Yield: 59.1%.

[0262] Synthesis of LC143: Added LC149 (26.9 mg, 0.05 mmol) to a mixture of 1 mL of ethylenediamine and 3 mL of DMSO, and stirred at room temperature for 5 hours. The crude product was washed with K 2 CO 3 aqueous solution and saturated NaCl solution. Purified by preparative HPLC to obtain 7.3 mg of LC143. Yield: 29.4%.

[0263] Synthesis of LC150: Added LC149 (53.8 mg, 0.1 mmol) to a mixture of 2 mL of diethylamine and 3 mL of DMSO, and stirred at room temperature for 5 hours. The crude product was washed with K 2 CO 3 aqueous solution and saturated NaCl solution. Purified by preparative HPLC to obtain 35.5 mg of LC150. Yield: 68%.

[0264] Synthesis of LC011: Added 1,4-dibromobutane (99.0 μL, 0.829 mmol) and K 2 CO 3(83.3 mg, 0.603 mmol) was added to isoliquiritigenin LC008 (30.8 mg, 0.120 mmol) dissolved in 4 mL of acetone. The reaction was heated at 62 °C and stirred overnight. The acetone solvent was removed under vacuum. The crude product was diluted with ethyl acetate and extracted with saturated sodium chloride solution. Pure LC011, 37.7 mg, was obtained by silica gel column chromatography using an elution gradient (PE:EtOAc (v / v), 50:1). Yield: 45.1%.

[0265] Synthesis of LC107: LC011 (23.1 mg, 0.044 mmol) was added to a mixture of 2 mL of diethylamine and 3 mL of DMSO and stirred at room temperature for 3 hours. The crude product was washed with saturated NaCl solution. The crude sample was purified by silica gel chromatography (EtOAc / MeOH / TEA (v / v / v), 100 / 1 / 1). 20.3 mg of LC107 was obtained. Yield: 90.6%.

[0266] Synthesis of LC010: 1,4-Dibromobutane (548.5 μL, 4.62 mmol) and K 2 CO 3 (213 mg, 1.54 mmol) were added to glabridin LC007 (101.6 mg, 0.313 mmol) dissolved in 4 mL of acetone. The reaction was heated at 60 °C and stirred overnight. The acetone solvent was removed under vacuum. The crude product was diluted with ethyl acetate and extracted with saturated sodium chloride solution. Pure LC010, 145 mg, was obtained by silica gel column chromatography using an elution gradient (PE:EtOAc (v / v), 30:1). Yield: 79.5%.

[0267] Synthesis of LC105: LC010 (69.2 mg, 0.116 mmol) was added to a mixture of 2 mL of diethylamine and 3 mL of DMSO and stirred at room temperature for 3 hours. The crude product was washed with saturated NaCl solution. The crude sample was purified by silica gel chromatography (EtOAc / MeOH / TEA (v / v / v), 200 / 1 / 1). 35.5 mg of LC105 was obtained. Yield: 52.7%.

[0268] Synthesis of LC310: Oroxylin A (62 mg, 0.5 mmol), 5-bromopentyltrimethylammonium bromide (418 mg, 2 mmol) and K 2 CO 3 (272 mg, 2 mmol) were dissolved in 3.5 mL of DMF. The mixture was stirred at 80 °C for 24 hours. After the reaction, the mixture was dissolved in butanol and washed 3 times with saturated NaCl solution. The butanol was removed under vacuum. Purification was carried out by preparative HPLC to obtain 32 mg of LC310. Yield: 16.8%.

[0269] Synthesis of LC311: LC003 (97 mg, 0.3 mmol), 5-bromopentyltrimethylammonium bromide (289 mg, 1 mmol) and K 2 CO 3 (272 mg, 2 mmol) were dissolved in 4 mL of DMF. The mixture was stirred at 60 °C for 6 hours. After the reaction, the mixture was dissolved in butanol and washed 3 times with saturated NaCl solution. Butanol was removed in vacuo. Purification by preparative HPLC gave 44.2 mg of LC311. Yield: 28%.

[0270] Synthesis of LC312: LC003 (97 mg, 0.3 mmol), 3-bromopropyltrimethylammonium bromide (265 mg, 1 mmol) and K 2 CO 3 (272 mg, 2 mmol) were dissolved in 4 mL of DMF. The mixture was stirred at 60 °C for 6 hours. After the reaction, the mixture was dissolved in butanol and washed 3 times with saturated NaCl solution. Butanol was removed in vacuo. Purification by preparative HPLC gave 48.5 mg of LC312. Yield: 31%.

[0271] Synthesis of LC315: LC003 (97 mg, 0.3 mmol), 4-bromobutyltriethylammonium bromide (317 mg, 1 mmol) and K 2 CO 3 (272 mg, 2 mmol) were dissolved in 4 mL of DMF. The mixture was stirred at 60 °C for 6 hours. After the reaction, ether was added, the precipitate was dissolved in methanol, and purified by preparative HPLC to obtain 59.1 mg of LC315. Yield: 31%.

[0272] Synthesis of LC316: LC007 (97 mg, 0.3 mmol), 5-bromopentyltrimethylammonium bromide (289 mg, 1 mmol) and K 2 CO 3 (272 mg, 2 mmol) were dissolved in 4 mL of DMF. The mixture was stirred at 60 °C for 6 hours. After the reaction, the mixture was dissolved in butanol and washed 3 times with saturated NaCl solution. Butanol was removed in vacuo. Purification by preparative HPLC gave 55.7 mg of LC316. Yield: 35%.

[0273] Synthesis of LC317: LC007 (97 mg, 0.3 mmol), 4-bromobutyltriethylammonium bromide (317 mg, 1 mmol) and K 2 CO 3(272 mg, 2 mmol) was dissolved in 4 mL of DMF. The mixture was stirred at 60 °C for 6 hours. After the reaction, ether was added, the precipitate was dissolved in methanol, and purified by preparative HPLC to obtain 63 mg of LC317. Yield: 33%.

[0274] Synthesis of LC350: LC003 (97 mg, 0.3 mmol), 3-(4-bromobutyl)-1-methylimidazolium bromide (298 mg, 1 mmol) and K 2 CO 3 (272 mg, 2 mmol) was dissolved in 4 mL of DMF. The mixture was stirred at 50 °C for 5 hours. After the reaction, ether was added, the precipitate was dissolved in methanol, and purified by preparative HPLC to obtain 46.5 mg of LC350. Yield: 25%.

[0275] Synthesis of LC365: LC003 (97 mg, 0.3 mmol), 1-(4-bromobutyl)pyridinium bromide (295 mg, 1 mmol) and K 2 CO 3 (272 mg, 2 mmol) was dissolved in 4 mL of DMF. The mixture was stirred at 50 °C for 5 hours. After the reaction, ether was added, the precipitate was dissolved in methanol, and purified by preparative HPLC to obtain 49.7 mg of LC365. Yield: 28%.

[0276] Synthesis of LC370: LC003 (97 mg, 0.3 mmol), 3-(4-bromobutyl)-1-methylimidazolium bromide (298 mg, 1 mmol) and K 2 CO 3 (272 mg, 2 mmol) was dissolved in 4 mL of DMF. The mixture was stirred at 50 °C for 5 hours. After the reaction, ether was added, the precipitate was dissolved in methanol, and purified by preparative HPLC to obtain 56.1 mg of LC370. Yield: 30%.

[0277] Synthesis of LC375: LC003 (97 mg, 0.3 mmol), 1-(4-bromobutyl)pyridinium bromide (295 mg, 1 mmol) and K 2 CO 3 (272 mg, 2 mmol) was dissolved in 4 mL of DMF. The mixture was stirred at 50 °C for 5 hours. After the reaction, ether was added, the precipitate was dissolved in methanol, and purified by preparative HPLC to obtain 65.1 mg of LC375. Yield: 37%.

[0278] Synthesis of LC005: Ethyl iodoacetate (25.3 μL, 0.213 mmol) and K 2 CO3 (82.9 mg, 0.60 mmol) was added to LC003 (31.6 mg, 0.097 mmol) in 3 mL of acetone. The workup of the reaction was similar to the synthesis of LC004. Pure acetate analogue, 42.8 mg, was obtained by silica gel chromatography using an elution gradient (hexane:EtOAc (v / v), 12.5:1). Yield: 88.5%.

[0279] Synthesis of LC101: LC005 (69.1 mg, 0.139 mmol) in 2 mL of THF was added to LiOH (31.6 mg, 1.32 mmol) dissolved in 1 mL of water. The solution was stirred at room temperature for 2 h and then cooled to room temperature. Acetic acid was added to the mixture and stirred for 5 min. After dilution with butanol, the solution was washed with saturated NaCl solution and dried over Na 2 SO 4 overnight. The solvent was removed in vacuo and the product was used in the subsequent reaction without further purification. HOBt (55.0 mg, 0.360 mmol) and ArgOMe (94.4 mg, 0.361 mmol) were introduced into the previously dried product dissolved in DMF. Subsequently, DIC (106 μL, 0.696 mmol) was added. The mixture was stirred overnight at room temperature. Sample preparation before injection was carried out by precipitation with diethyl ether. Purification was performed by preparative HPLC to obtain 18.3 mg of LC101. Yield: 16.8%.

[0280] Synthesis of LC127: LC012 (24.8 mg, 0.05 mmol) in 2 mL of THF was added to LiOH (31.6 mg, 1.32 mmol) dissolved in 1 mL of water. The solution was stirred at room temperature for 2 h and then cooled to room temperature. The solvent was removed in vacuo and the product was used in the subsequent reaction without further purification. HOBt (35.0 mg, 0.23 mmol) and arginamide dihydrochloride (61.5 mg, 0.25 mmol) were introduced into the previously dried product dissolved in DMF. Subsequently, DIC (130 μL, 0.85 mmol) and N,N-diisopropylethylamine (71.8 μL, 0.51 mmol) were added. The mixture was stirred overnight at room temperature. Sample preparation before injection was carried out by precipitation with diethyl ether. Purification was performed by preparative HPLC to obtain 17.4 mg of LC127. Yield: 42%.

[0281] Synthesis of LC131: LC005 (24.8 mg, 0.05 mmol) in 2 mL of THF was added to LiOH (31.6 mg, 1.32 mmol) dissolved in 1 mL of water. The solution was stirred at room temperature for 2 hours and then cooled to room temperature. The solvent was removed in vacuo and the product was used in the subsequent reaction without further purification. HOBt (35.0 mg, 0.23 mmol) and arginamide dihydrochloride (61.5 mg, 0.25 mmol) were introduced into the previously dried product dissolved in DMF. Subsequently, DIC (130 μL, 0.85 mmol) and N,N-diisopropylethylamine (71.8 μL, 0.51 mmol) were added. The mixture was stirred overnight at room temperature. Sample preparation before injection was carried out using diethyl ether precipitation. Purification was performed by preparative HPLC to obtain 13.3 mg of LC131. Yield: 32%.

[0282] Synthesis of LC137: LC005 (42.6 mg, 0.086 mmol) in 2 mL of THF was added to LiOH (31.6 mg, 1.32 mmol) dissolved in 1 mL of water. The solution was stirred at room temperature for 2 hours and then cooled to room temperature. The solvent was removed in vacuo and the product was used in the subsequent reaction without further purification. HOBt (63.0 mg, 0.410 mmol) and ArgOEt (118 mg, 0.43 mmol) were introduced into the previously dried product dissolved in DMF. Subsequently, DIC (130 μL, 0.85 mmol) and N,N-diisopropylethylamine (156.8 μL, 0.9 mmol) were added. The mixture was stirred overnight at room temperature. Sample preparation before injection was carried out using diethyl ether precipitation. Purification was performed by preparative HPLC to obtain 46.3 mg of LC137. Yield: 60.8%.

[0283] Synthesis of LC106: LC012 (107.7 mg, 0.217 mmol) in 2 ml of THF was added to LiOH (43.6 mg, 1.820 mmol) dissolved in 1 mL of water. The solution was stirred at room temperature for 2 hours and then cooled to room temperature. Acetic acid was added to the mixture and stirred for 5 minutes. After dilution with butanol, the solution was washed with saturated NaCl solution and dried over Na 2 SO 4 overnight. The solvent was removed in vacuo and the product was used in the subsequent reaction without further purification.

[0284] HOBt (85.4 mg, 0.558 mmol) and ArgOMe (153.8 mg, 0.589 mmol) were introduced into the previously dried product dissolved in DMF. Subsequently, DIC (164 μL, 1.089 mmol) was added. The mixture was stirred overnight at room temperature. Sample preparation before injection was carried out by precipitation with diethyl ether. Purification was performed by preparative HPLC to obtain 34.9 mg of LC106. Yield: 20.6%.

[0285] Synthesis of LC012: Ethyl iodoacetate (175 μL, 1.48 mmol) and K 2 CO 3 (208.9 mg, 1.51 mmol) were added to a 4 mL acetone solution of LC007 (95.8 mg, 0.295 mmol). The reaction was worked up similarly to the synthesis of LC010. Pure acetate analogue LC012, 107.7 mg, was obtained by silica gel chromatography using an elution gradient (hexane:EtOAc (v / v), 20:1). Yield: 73.5%.

[0286] Synthesis of LC140: LC149 (53.8 mg, 0.1 mmol) was added to a mixture of 2 mL of N,N,N-trimethyl-1,3-propanediamine and 3 mL of DMSO and stirred at room temperature for 3 hours. The crude product was washed with an aqueous K 2 CO 3 solution and saturated NaCl solution. Purification was performed by preparative HPLC to obtain 22.5 mg of LC140. Yield: 37%.

[0287] Synthesis of LC148: 1,3-Dibromopropane (305 μL, 3 mmol) and K 2 CO 3 (138 mg, 0.1 mmol) were added to isopsoralen chalcone LC003 (64.8 mg, 0.2 mmol) dissolved in 3 mL of acetone. The reaction was maintained under reflux conditions and stirred for two days. The acetone solvent was removed in vacuo. The crude product was diluted with ethyl acetate and extracted with saturated sodium chloride solution. Pure LC148, 58.1 mg of product, was obtained by silica gel column chromatography using an elution gradient (hexane:EtOAc (v / v), 20:1). Yield: 52%.

[0288] Synthesis of LC204: LC148 (56.6 mg, 0.1 mmol) was added to a mixture of 2 mL of diethylamine and 3 mL of DMSO and stirred at room temperature for 5 hours. With K 2 CO 3The crude product was washed with aqueous solution and saturated NaCl solution. Purification was carried out by preparative HPLC to obtain 34.6 mg of LC204. Yield: 63.1%.

[0289] Synthesis of LC206: Cyclen (86 mg, 0.5 mmol) was added to a 3 mL DMF solution of LC148 (56.6 mg, 0.1 mmol). The reaction mixture was stirred at room temperature for 5 h. The crude product was dissolved in butanol and then washed three times with K 2 CO 3 aqueous solution and saturated NaCl solution and dried in vacuo. Purification was carried out by preparative HPLC to obtain 26.2 mg of yellow gel-like LC2O6. Yield: 35%.

[0290] Synthesis of LC013: Ethyl iodoacetate (341.4 μL, 2.88 mmol) and K 2 CO 3 (252.3 mg, 1.83 mmol) were added to a 4 ml acetone solution of LC008 (92.4 mg, 0.361 mmol). The reaction work-up was similar to that of the synthesis of LC011. Pure acetate analogue LC013, 112.7 mg, was obtained by silica gel chromatography with an elution gradient (Hex:EtOAc (v / v), 10:1). Yield: 73.1%.

[0291] Synthesis of LC108: LC013 (27 mg, 0.063 mmol) in 2 mL of THF was added to LiOH (14.5 mg, 0.605 mmol) dissolved in 1 mL of water. The solution was stirred at room temperature for 2 h and then cooled to room temperature. Acetic acid was added to the mixture and stirred for 5 min. After dilution with butanol, the solution was washed with saturated NaCl solution and dried with Na 2 SO 4 overnight. The solvent was removed in vacuo and the product was used in the subsequent reaction without further purification. HOBt (24.2 mg, 0.158 mmol) and ArgOMe (42.7 mg, 0.164 mmol) were introduced into the previously dried product dissolved in DMF. Subsequently, (48 μL, 0.315 mmol) was added. The mixture was mixed overnight at room temperature. Sample preparation before injection was carried out by precipitation with diethyl ether. Purification was carried out by preparative HPLC to obtain 8.4 mg of LC108. Yield: 18.7%.

[0292] Example 8: Biological studies

[0293] In vitro

[0294] In Gram-negative bacteria, the outer membrane and the inner membrane are both lipid bilayers. Each bilayer consists of a hydrophobic region of lipid tails and two head-group regions facing the aqueous solution, forming a sandwich structure. To disrupt the bacterial outer and inner membranes, many bola-shaped amphiphilic compounds composed of two cationic end-groups and a hydrophobic scaffold have been synthesized, where the cationic end-groups and the hydrophobic scaffold can interact with the two anionic head-group regions and the lipid tail regions, respectively ( Figure 4 ). The hydrophobic scaffold is selected from a series of natural products ( Figure 9 ). Many cationic groups have been selected, such as DPA-Zn, polyamines, tertiary amines, quaternary amines, guanidines, and basic amino acids ( Figure 8 ). As a result, a library of compounds has been synthesized by assembling the hydrophobic core and the cationic groups.

[0295] The antimicrobial activities and synergisms of a variety of compounds were tested against a panel of colistin-resistant strains, including 7 mcr-1(+) strains and 4 mcr-1(-) strains. Tables 1a - 1e show the minimum inhibitory concentrations (MICs) of the synthesized compounds. Colistin showed an MIC of 6.25 μg / ml; however, due to its toxicity, the breakpoint of colistin is 2 - 4 μg / ml, indicating that E. coli strains carrying mcr-1 are resistant to colistin. It also shows that mcr-1-negative E. coli strains are also resistant.

[0296] Table 1a. MIC (μg / ml) of DPA-Zn compounds against a panel of colistin-resistant strains.

[0297]

[0298] *E.Coll

[0299] Songqiu Enterobacter cloacae

[0300] Table 1b. MIC (μg / ml) of amine compounds against a panel of colistin-resistant strains.

[0301]

[0302] *E.Coli

[0303] Lailv* Enterobacter cloacae

[0304] Table 1c. MIC (μg / ml) of amine compounds against a panel of colistin-resistant strains.

[0305]

[0306] *E.Coli

[0307] **Enterobacter cloacae

[0308] Table 1d. MIC (μg / ml) of amine compounds against a panel of colistin-resistant strains.

[0309]

[0310] *E.Coli

[0311] **Escherichia cloacae

[0312] Table 1e. MIC (μg / ml) of LC137, LC140, LC143, LC150, LC204 and LC206 against a panel of colistin-resistant strains.

[0313]

[0314] In addition, due to some other mechanisms sensitive to colistin combinations, other E. coli are also resistant to colistin but do not have the mcr-1 mutation, called mcr-1(-). Most of the compounds of the present invention show antimicrobial activity or synergistic action in combination with colistin against these mcr(-) resistant strains (Tables 2a-2e). This indicates a similar working mechanism of the combinations of the present invention against mcr-1(-) and mcr-1(+) bacteria.

[0315] Table 2a. Colistin MIC (μg / ml) against a panel of colistin-resistant strains in the presence of DPA-Zn compounds.

[0316]

[0317] *E.Coli

[0318] **Escherichia cloacae

[0319] Table 2b. Colistin MIC (μg / ml) against a panel of colistin-resistant strains in the presence of amine analogs.

[0320]

[0321] Table 2c. Colistin MIC (μg / ml) against a panel of colistin-resistant strains in the presence of amine analogs.

[0322]

[0323] *E.Coli

[0324] Escherichia cloacae

[0325] Table 2d. Colistin MIC (μg / ml) against a panel of colistin-resistant strains in the presence of amine analogs.

[0326]

[0327] *E.Coli

[0328] Klebsiella oxytoca

[0329] Table 2e. Colistin MIC (μg / ml) against a panel of colistin-resistant strains in the presence of LC137, LC140, LC143, LC150, LC204, LC206.

[0330]

[0331] Four classes of compounds were developed. The first class of compounds are DPA derivatives in which two DPA-Zn moieties are linked to a selected hydrophobic scaffold via two linker groups. The MIC and synergy activities are shown in Table 1a and Table 2a. The first proof-of-concept compound is Orc-DPA, which shows synergy with colistin against mcr-1 bacteria at 50 μg / ml. In contrast, EDTA only works at concentrations above 250 μg / ml. Then the hydrophobic scaffold was optimized and many DPA derivatives were synthesized. One compound, GLA-DPA, synergizes with 5 μg / ml of colistin.

[0332] The second class of compounds are amine and polyamine derivatives. Using different amine or polyamine groups, a series of analogs were developed, such as LC100, LC098, LC097, LC096, LC095, LC300, LC301, LC302, LC304, LC140, LC204, LC206, LC100, LC107, LC143, LC014, LC105. Against mcr-1 bacteria, most of these compounds show synergy with colistin and the results are summarized in Tables 1b-1e and 2b-2e. One of the compounds, LC100, shows no activity when used alone but shows excellent synergy with 2 μg / ml of colistin. LC100 has been selected as the lead compound for in vivo and biophysical studies and will be discussed in further detail below.

[0333] The third class of compounds are quaternary amine analogs, including LC311, LC312, LC315, LC316, LC317, LC350, LC365, LC370 and LC375. The corresponding MIC and synergy data are shown in Tables 1b-1d and 2b-2d. Compared with the amine and polyamine analogs, the quaternary amine analogs not only synergize with colistin but also show antimicrobial activity when used alone.

[0334] The fourth class of compounds are guanidine analogs, including LC101, LC137, LC131, LC106, LC127, LC108. Two compounds, LC101 and LC137, developed using isobavachalcone (LC003) as the hydrophobic core showed excellent synergy with colistin. In addition, LC101 and LC137 also showed moderate antimicrobial activity when used alone (MIC range of 12.5 - 25 μg / ml).

[0335] Example 9: LC100

[0336] To further test the antimicrobial and synergistic activities of the compounds of the present invention with colistin, LC100 was selected as a model compound to further test the antimicrobial spectrum and understand the mechanism of action, because LC100 has no activity alone but shows excellent synergistic activity with colistin. First, the synergy was tested using different concentrations of L100. Table 3 shows that as the concentration of LC100 increased, the synergy became stronger. LC100 at concentrations greater than 5 μg / ml could reduce the MIC of colistin to as low as 0.0975 μg / ml, at which concentration the toxicity of colistin is much lower. Next, it was examined whether LC100 was synergistic with polymyxin B (PMB), a peptide that functions in a similar manner to colistin but has one residue difference from colistin. The data in Table 4 show that the combination is equally effective for polymyxin B. Table 5 extends the action of this combination to three multi-drug resistant forms of Gram-negative bacteria: Acinetobacter baumannii (ACBA), Klebsiella pneumoniae (KLPN), and Escherichia cloacae. It should be noted that the KLPN strain was genetically analyzed as carbapenem-resistant Enterobacteriaceae (CRE). The results show that the combination is effective against these carbapenem-resistant and multi-drug resistant strains.

[0337] It should also be noted that the combination can effectively kill a large number of bacteria without changing the drug concentration according to the bacterial species. The only other way to achieve this is to use disinfectants such as alcohol, but disinfectants are known to be toxic.

[0338] Table 3. Colistin MIC (μg / ml) against a panel of colistin-resistant strains in the presence of different concentrations of LC100

[0339]

[0340] *E.Coli

[0341] **Escherichia cloacae

[0342] Table 4. Synergistic effect of LC100 and colistin against colistin-sensitive strains.

[0343]

[0344] Table 5. Synergistic effect of LC100 and colistin against multidrug-resistant strains

[0345]

[0346] It was found that the combination of LC100 and colistin was able to kill bacteria such as E. coli with mcr-1 that was resistant to colistin and other antibiotics, E. coli without mcr-1 that was resistant to colistin and other antibiotics, Pseudomonas aeruginosa that was resistant to carbapenems, aminoglycosides, and other antibiotics, Acinetobacter baumannii that was resistant to carbapenems, colistin, and other antibiotics, and Klebsiella pneumoniae that was resistant to carbapenems, colistin, and other antibiotics. Table 3 shows that at different concentrations of LC100, the amount of colistin required to kill the bacteria changed, confirming that LC100 actually acts as an adjuvant. This was also found in E. coli with mcr-1 and other bacteria such as Enterobacter cloacae, supporting that this phenomenon is true across a wide range of bacteria.

[0347] To understand the effect of LC100 in combination with colistin, a time-kill experiment was performed using the combination of the present invention in three colistin-resistant strains including two mcr-1(+) and one mcr-1(-) strains. Figure 13 , Figure 14 and Figure 15 Colistin concentrations equal to or above the MIC were shown to cause a 3 log reduction in 2 hours. In the presence of 10 μg / ml LC100, colistin at 1 / 8 the MIC achieved the same killing effect (in terms of 3 log reduction) in 2 hours. The rapid killing kinetics indicate that the combination of LC100 and colistin acts on the bacterial membrane.

[0348] LC100 and LC101 can not only synergistically enhance the activity of colistin or polymyxin B, but also synergize with other cationic peptides. This was tested by replacing colistin with another compound, B2088, which has a completely different chemical nature and is a positively charged peptide that also has physicochemical interactions with the outer membrane. In synergy studies, it was found that LC100 or LC101 synergized with B2088 at the 1μg / ml level in killing E. coli with mcr-1 mutations, thereby increasing the activity by more than 10 times, as shown in Figure 2. Figure 16As shown in Table 6. Using the combination of LC100 and colistin, the activity of a wide range of strains was further tested (Table 7). Table 7 covers a wide range of bacteria, many of which are highly resistant to antibiotics. It seems that the combination of 1 μg / ml of colistin and 5 μg / ml of LC100 is effective against all pathogens except Klebsiella pneumoniae (KLPN). This combination also seems to be safe to use.

[0349] The antimicrobial activity of the combination of the present invention was also tested against Gram-positive bacteria, including multi-drug resistant strains such as MRSA. Table 8 shows that LC100 or LC101 alone exhibits good activity against Gram-positive bacterial strains, indicating that LC100 or LC101 alone has a strong effect on the bacterial inner membrane because Gram-positive bacteria do not have an outer membrane. This means that the combination of LC100 / LC101 and colistin has a broad antimicrobial spectrum and is active against both Gram-positive and Gram-negative bacteria.

[0350] Table 6. Results of the synergistic study of LC100 or LC101 with B2088

[0351]

[0352] Table 7. Pathogen susceptibility of the compounds of the present invention as colistin adjuvants in various pathogens

[0353]

[0354] Table 7. Continued

[0355]

[0356] Table 7. Continued

[0357]

[0358] Table 8. Antimicrobial effect of LC100 and LC101 against Gram-positive bacteria.

[0359] MCR 1 strain LC 100 LC 101 SA ATCC 29213 3.125 3.125 SA ATCC6538 3.125 3.125 SA DM 4400R 3.125 3.125 MRSA ATCC 43300 3.125 6.25 MRSA ATCC 700699 3.125 6.25 MRSA DM 21455 3.125 3.125

[0360] Example 10: Mechanism of action of LC100 and LC101 (with mcr-1)

[0361] The compounds of the present invention are considered to be adjuvant compounds that enhance the action of colistin against Gram-negative bacteria, especially E. coli with the mcr-1 mutation, because these compounds act in a synergistic manner, thereby reducing the effective dose of colistin by 10-fold or more. When used in combination with LC100 or LC101, the MIC of colistin changes from more than 6 μg / ml to less than 0.5 μg / ml. At 6 μg / ml, colistin is considered unsafe to use due to concentration-dependent side effects. Synergistic effects with these two compounds have also been observed on other forms of resistant Gram-negative bacteria, such as E. coli, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0362] Although mcr-1 bacteria are resistant to colistin, it has been reported that colistin can still disrupt the outer membrane of mcr-1 bacteria. It should be noted that simply disrupting the outer membrane does not directly lead to cell death; the inner membrane must be affected, resulting in the loss of transmembrane potential and the entry of water into the bacteria. Therefore, it is hypothesized that in the case of the combination of LC100 and colistin, colistin and LC100 act together to disrupt the outer membrane, thereby allowing LC100 to reach the inner membrane. Since LC100 has a strong effect on the inner membrane, the bacterial cells die due to rupture of the inner membrane.

[0363] To verify this hypothesis, we conducted fluorescence experiments using the dye molecule EtBr, which emits strong fluorescence when bound to DNA. However, EtBr is membrane-impermeable, which means it can only enter bacterial cells and bind to DNA, and can only fluoresce when both the outer and inner membranes of the bacteria are disrupted. Figure 17 Results showed that LC100 and colistin alone induced only slight fluorescence, indicating that neither of these two compounds can disrupt the bacterial outer membrane. However, in the presence of the combination of LC100 and colistin, there was strong fluorescence, indicating that both the outer and inner membranes of mcr-1 bacteria were disrupted, confirming the hypothesis that the combination of LC100 and colistin acts on the bacterial membrane. It is also possible that LC100 has other targets. One possible target is the MCR-1 protein. To understand the interaction between LC100 and the MCR-1 protein, the fluorescence of the MCR-1 protein was measured in the presence of different concentrations of LC100. The MCR-1 protein contains several tryptophan residues that emit autofluorescence at 323 nm. Figure 18 Results showed that as the concentration of LC100 increased, the fluorescence decreased. The fluorescence quenching effect indicates a strong interaction between the MCR-1 protein and LC100.

[0364] Colistin and polymyxin B can interact with phosphate groups and disrupt the head groups, thereby creating cavities that expose the lipid tails of the membrane to the aqueous phase. For example, in the presence of the cationic polymyxin B, which is a colistin analogue, the hydrophobic surface area of the bacterial membrane increases ( Figure 19), thereby allowing for hydrophobic association between the lipid tail of colistin B and the lipid tail of the bacterial outer membrane, resulting in resistance to colistin.

[0365] It is hypothesized that the efficacy of the combination of LC100 and colistin stems from the ability of the cationic group of colistin to transiently disrupt the hydrogen-bonding network within the membrane, thereby exposing the hydrophobic lipid groups in the bacterial membrane. This transient exposure seems sufficient for LC100 molecules to "drill into" and cross the outer membrane, reach the inner membrane, and kill the bacteria. Microbiological data indicate that the concentration of colistin required for this synergistic effect can be reduced 4 - 100-fold compared to the amount needed for colistin to act alone. The observation that LC100 disrupts liposomes mimicking the inner membrane strengthens this hypothesis ( Figure 20 ). Another proposed mode of action is that colistin molecules form channel-like oligomers in the membrane, which may create cavities that facilitate the diffusion of LC100 into the inner membrane.

[0366] Example 11: Mechanism of action of LC100 and LC101 (without mcr-1)

[0367] E. coli is the favorite host of mcr-1 and is also often resistant to colistin without the mcr-1 mutation. It has been proposed that this may be due to the presence of arabinose. Sugar molecules have several hydroxyl groups, which can also participate in the hydrogen-bonding network to stabilize the bacterial membrane, resulting in resistance to colistin. As described above, when testing the effect of the combination of LC100 and colistin on several resistant bacteria without mcr-1, it was found that in the presence of LC100, E. coli was very sensitive to low concentrations of colistin, down to levels as low as <0.5 μg / ml. The mechanism of action may be similar to the mechanism proposed above for bacteria with mcr-1, where cationic colistin may temporarily disrupt the hydrogen-bond network within the membrane, causing a transient appearance of hydrophobic cavities in the membrane, which in turn facilitates the entry of LC100, which has a strong hydrophobic core, into the fatty acid layer of lipid A, and then down to disrupt the inner membrane of the bacteria.

[0368] It was also found that carbapenem-resistant bacteria are sensitive to the combination of LC100 and colistin. Carbapenem resistance is caused by mutations in the biosynthetic enzymes of the bacterial cytoplasm. These bacteria have variable sensitivity to colistin. Most likely, in the presence of the combination of LC100 and colistin, the interaction of colistin with the outer membrane promotes the entry of LC100 into the inner membrane, resulting in LC100 being able to kill the bacteria.

[0369] Example 12: In vivo study of LC100

[0370] To evaluate the in vivo efficacy of the combination of LC100 and colistin, a neutropenic mouse thigh infection model was used. Thirty mice were divided into 5 groups: control, 10 mg / kg colistin, 50 mg / kg LC100, 100 mg / kg meropenem, and the combination of 50 mg / kg LC100 and 10 mg / kg colistin. Each treatment was administered intraperitoneally 1 hour after infection. The initial inoculum was 1.87x10 6 , and 6 hours after treatment, the mouse thigh tissues were homogenized to obtain viable CFUs. Figure 21 It was shown that neither LC100 alone nor colistin could reduce the bacterial load, while the combination resulted in a 1.2 log reduction within 6 hours. Since neutropenic mice were used in the study, the 1.2 log reduction in bacterial load was due to the combination of LC100 and colistin rather than the mouse immune system. As a control, meropenem showed more significant activity (a 2 log reduction) because of the high concentration and the susceptibility of the bacterial strain to penem.

[0371] Industrial Applicability

[0372] The disclosed compounds can be used to interact with bacterial membranes and disrupt them. The disclosed compounds can effectively kill Gram-positive bacteria when administered alone or co-administered with colistin. The disclosed compounds can effectively kill mcr-positive Gram-negative bacteria when administered alone or co-administered with colistin. The disclosed compounds can also effectively kill MCR-negative but colistin-resistant Gram-negative bacteria when co-administered with colistin. In addition, the disclosed compounds can effectively kill a broad range of Gram-negative bacteria, including those resistant to carbapenems, when co-administered with colistin.

[0373] The disclosed compounds can be used as antibiotics to kill or inhibit the growth of microorganisms in therapy, and / or for the treatment of bacterial infections.

[0374] The disclosed compounds can be used to kill or inhibit the growth of surface microorganisms, including but not limited to topical applications, in plasters, eye drops, nasal sprays, mouthwashes, and hand sanitizers. The disclosed compounds can also be used as food preservatives, disinfectants, surface cleaners, or medical devices.

[0375] The method for preparing the disclosed compounds can be simple, using mild reaction conditions, which is beneficial for the low-cost and large-scale synthesis of the compounds.

[0376] Obviously, after reading the above disclosure, various other modifications and adaptations of the present invention will be obvious to those skilled in the art without departing from the spirit and scope of the present invention. Therefore, it is stipulated that all such modifications and adaptations fall within the scope of the appended claims.

Claims

1. A compound of formula (I): Z 1 -L 1 -A-L 2 -Z 2 Formula (I) wherein A is a hydrophobic moiety selected from the group consisting of: L 1 and L 2 are connectors, where the connectors are independently C 2 -C 6 alkyl; and Z 1 and Z 2 each independently has the following structure: where the broken bond indicates the position where the structure is attached to the remainder of formula (I).

2. The compound according to claim 1, wherein the logP value of the compound of formula (I) is greater than 4.

3. The compound according to claim 1, wherein the compound of formula (I) has the following structure:

4. A compound of formula (I): Z 1 -L 1 -A-L 2 -Z 2 Formula (I) wherein A is a hydrophobic moiety selected from the group consisting of: L 1 and L 2 are joints, wherein said joints are independently C 2 -C 6 alkyl; and Z 1 and Z 2 each independently has the following structure: where the broken bond indicates the position where the structure is attached to the remainder of formula (I).

5. The compound according to claim 4, wherein the compound of formula (I) has the following structure:

6. A pharmaceutical composition comprising the compound according to claim 4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable carrier is saline.

8. A method for preparing a compound, wherein the compound has formula (I): Z 1 -L 1 -A-L 2 -Z 2 Formula (I) wherein A is a hydrophobic moiety selected from the group consisting of: L 1 and L 2 are connectors, where the connectors are independently C 2 -C 6 alkyl; and Z 1 and Z 2 independently has the following structure: where the broken bond indicates the position where the structure is attached to the remainder of formula (I), and wherein the method comprises the following steps: 1) contacting the hydrophobic moiety with a linker under reaction conditions; and 2) contacting the linker with an N-containing moiety under reaction conditions.

9. A method for preparing a compound, wherein the compound has formula (I): Z 1 -L 1 -A-L 2 -Z 2 Formula (I) wherein A is a hydrophobic moiety selected from the group consisting of: L 1 and L 2 are connectors, wherein said connectors are independently C 2 -C 6 alkyl; and Z 1 and Z 2 independently has the following structure: where the broken bond indicates the position where the structure is attached to the remainder of formula (I), and wherein the method comprises the following steps: 1) contacting the hydrophobic moiety with a linker under reaction conditions; 2) contacting the linker with an N-containing moiety under reaction conditions; and 3) adding zinc after the N-containing moiety is covalently bonded to the linker.

10. The method according to claim 8 or 9, wherein a covalent bond is formed between the hydrophobic moiety and the linker or between the linker and the N-containing moiety.

11. The method according to claim 8 or 9, wherein the linker is selected from 1,4-dibromobutane, 1,3-diiodobutane, 1,2-dibromoethane, 1,3-dibromopropane, 1,4-diiodobutane, and any combination thereof.

12. The method according to claim 8 or 9, wherein the contacting is carried out in a solvent selected from acetone, methanol, ethanol, propanol, butanol, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, pyridine, water, and any mixture thereof.

13. The method according to claim 8 or 9, wherein the contacting step is carried out at a temperature of 21 °C to 160 °C.

14. The method according to claim 8 or 9, wherein the contacting step is carried out for a duration of 2 hours to 36 hours.

15. Use of the compound according to claim 4 or the pharmaceutical composition according to claim 6 for killing bacteria in vitro, wherein the compound according to claim 4 is used in combination with colistin.

16. The use according to claim 15, wherein the bacteria are Gram-negative bacteria or Gram-positive bacteria.

17. Use according to claim 15, wherein the bacterium is MCR positive or MCR negative.

18. Use according to claim 15, wherein the bacterium is carbapenem-resistant.

19. Use of the compound according to claim 4 in the preparation of a medicament for treating a bacterial infection, wherein the compound according to claim 4 is administered in combination with colistin.

20. Use according to claim 19, wherein the compound according to claim 4 is present in an amount of 2 μg / mL to 75 μg / mL.

21. Use according to claim 19, wherein the compound according to claim 4 and colistin are present in equal amounts by weight, or the compound according to claim 4 is present in an excess of 1.5 to 6 times that of colistin by weight.

22. Use according to claim 21, wherein colistin is present in the range of 1 mg / kg to 10 mg / kg, and the compound according to claim 4 is present in the range of 10 mg / kg to 50 mg / kg.

23. Use according to claim 19, wherein the compound according to claim 4 will be administered topically.

24. Use according to claim 19, wherein the compound according to claim 4 will be administered intramuscularly, intraperitoneally, subcutaneously or intravenously.

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