Molecular design of new antibiotics and antibiotic adjuvants for MCR strains

Abstract

The present invention relates to the molecular design of novel antibiotics and antibiotic adjuvants against mcr strains, said molecules being compounds comprising a hydrophobic moiety, a linker and an N-containing moiety. The invention also relates to a method for synthesizing the compounds and to the use of the compounds as antibiotics or antibiotic adjuvants.

Description

[0001] This application is a divisional application of PCT international patent application PCT / SG2020 / 050292 with an application date of May 18, 2020, which entered the Chinese national phase with Chinese patent application number 202080051205.6 and the invention name being “Molecular design of new antibiotics and antibiotic adjuvants for MCR strains”. Technical Field

[0002] The present invention relates to a compound comprising a hydrophobic portion, a linker and an N-containing portion. The present invention also relates to a method for synthesizing the compound and the use of the compound as an antibiotic or an antibiotic adjuvant. Background Art

[0003] The healthcare crisis exacerbated by increasing reports of rising levels of pathogen resistance to existing antibiotics is further exacerbated by the continued emergence of carbapenemase-producing Enterobacteriacae bacteria, particularly strains of K. pneumoniae that possess the KPC-2 and NDM1 genes that confer resistance to penicillins. 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 toward 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 genetic mutations. As Figure 1 As shown in the figure, the outer membrane of Gram-negative bacteria is covered with lipopolysaccharide (LPS), which is anchored in lipid A, which forms the structural base of LPS and is crucial for outer membrane stability. Colistin, a peptide also known as polymyxin E, is a member of the cationic polymyxin family that includes polymyxin B. Molecules in this family kill Gram-negative bacteria by disrupting the lipid A portion of the outer membrane, which then disrupts the inner membrane. Water then enters the bacteria, lysing the organism.

[0004] Colistin has broad activity against Gram-negative bacteria but is rarely used due to severe side effects, nephrotoxicity, and neurotoxicity. Due to these side effects, a dose of 2 μg / ml is generally considered the breakpoint for resistance emergence. In contrast, it is widely used in chicken and beef cattle agriculture in the United States and China. However, colistin resistance has recently emerged in the form of a mobile or mobile plasmid associated with E. coli, which was discovered in pigs in northwestern China in 2015. Documented spread of mobile colistin resistance (MCR) in E. coli carrying mcr-1 indicates that, by 2015, mcr-1 had emerged in 10 countries. MCR-1 resistance has now been identified in numerous Gram-negative pathogens, including E. coli, Salmonella enterica, Klebsiella pneumoniae, Enterobacter aerogenes, and Acinetobacter baumannii, posing a significant challenge to the treatment of these infections, particularly because this trait confers a high propensity for antibiotic resistance to current antimicrobial agents, particularly carbapenems. Furthermore, MCR-1 resistance has now been documented in more than 30 countries.

[0005] Furthermore, the situation is expected to worsen further due to the ease of lateral transmission between different bacterial strains due to the transfer of the MCR gene via a plasmid-mediated mechanism. Recently, three other mutations, MCR-2, MCR-3, and MCR-4, have been identified. However, the mechanism of action to date has been lipid A modification ( Figure 2 Therefore, strategies to address resistance conferred by MCR genes are urgently needed. Particularly affected are patients with carbapenemase-resistant bacteria that are not susceptible to other antibiotics.

[0006] Therefore, new approaches to address mcr-1 resistance that at least partially ameliorate the above-mentioned drawbacks are needed. Summary of the Invention

[0007] A new approach has been developed for designing new antibiotics or antibiotic adjuvants against MCR strains (including MCR-1, MCR-2, MCR-3, and MCR-4). A library of molecules that disrupt the hydrogen-bonding network of the MCR-modified outer membrane was designed. This approach was combined with a novel computer modeling method that could significantly accelerate the development time and reduce the cost of new antimicrobial therapeutics. The computer designs were compared with laboratory validation and in vivo testing of their effectiveness in treating MCR infections.

[0008] The design approach 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 were revealed. For MCR-positive strains of Gram-negative bacteria, two targets were identified: the active site of the MCR-1 protein and the hydrogen bonding network of the outer membrane. Based on detailed atomic analysis, a library of compounds was designed that destabilizes the outer membrane of E. coli harboring the mcr-1 plasmid, overcoming drug resistance and killing the bacteria. Several compounds were selected from the library and tested for their MICs and their synergistic activity with colistin. One of the compounds, GLA-DPA, showed limited antimicrobial activity against a panel of clinically isolated colistin-resistant bacteria. However, it was demonstrated that GLA-DPA, when combined with colistin, can restore the sensitivity of E. coli harboring mcr-1 to colistin. Therefore, when combined with the disclosed compounds, the effective dose of colistin can be reduced to a level that avoids toxicity.

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

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

[0011] Where A is a hydrophobic part;

[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 may have a suitable structure for interacting with and disrupting bacterial membranes. In other words, the compound may have two headgroup regions that interact with bacterial membranes and a hydrophobic portion that interacts with the bacterial membrane lipid components. The compound may have a size suitable for enabling it to span the bacterial membrane lipid bilayer. Advantageously, the headgroup may contain N-atoms, thereby having a high pKa value. Even more advantageously, the hydrophobic portion may be large. Even more advantageously, the hydrophobic portion may be planar. In short, the composition of the compound may impart to the compound high membrane interaction and antimicrobial properties.

[0015] Advantageously, the nitrogen-containing moiety, guanidine, and zinc chelate complex all have the same characteristic, namely, a high affinity for phosphate groups in bacterial membranes. 1 or Z 2) with the phosphate groups of the membrane leading to large membrane perturbations.

[0016] Advantageously, the N-containing moiety can disrupt the electrostatic interactions (e.g., hydrogen bonding networks) that stabilize bacterial membranes. Advantageously, this can lead to bacterial cell killing. Alternatively, the disruption of the hydrogen bonding network can promote the effective function of other antibiotics such as colistin, thereby killing bacterial cells. Even if bacteria are resistant to colistin, colistin can interact with lipid A, and slight perturbations of the outer membrane can cause the compound of formula (I) and colistin to enter the structure of lipid A toward the inner membrane, resulting in fatal destruction of the inner membrane and bacterial killing.

[0017] Compounds of formula (I) are effective against Gram-positive bacteria when administered alone or in combination with colistin. Compounds of formula (I) are effective against MCR-positive Gram-negative bacteria when administered alone or in combination with colistin. In this case, the N-containing moiety can chelate zinc, which can then bind to zinc-dependent MCRs and disrupt their activity. When administered in combination with colistin, compounds of formula (I) are also effective against Gram-negative bacteria that are MCR-negative but still resistant to colistin. Furthermore, when administered in combination with colistin, compounds of formula (I) are effective against a wide range of Gram-negative bacteria, including those resistant to carbapenems. Thus, compounds of formula (I) can overcome bacterial resistance to colistin, restoring the effects of colistin and, in effect, extending the effects of colistin to Gram-negative bacteria resistant to either carbapenems or colistin alone. Advantageously, compounds of formula (I) are also effective against a wide range of bacteria without having to adjust drug concentrations based on bacterial species. The only other known antimicrobial that can achieve this is alcohol, but alcohol is notoriously toxic.

[0018] In another aspect, there is provided a pharmaceutical composition comprising the compound defined above, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

[0019] In another aspect, there is provided a process for preparing a compound as defined above, comprising the step of contacting a hydrophobic moiety with a N-containing moiety under reaction conditions.

[0020] Advantageously, the method for preparing the compound is readily available, employing mild reaction conditions, facilitating low-cost and large-scale synthesis of the compound.

[0021] In another aspect, there is provided the use of a compound as defined above or a pharmaceutical composition as defined above as an antibiotic.

[0022] In another aspect, there is provided the use of a compound as defined above or a pharmaceutical composition as defined above for killing or inhibiting the growth of microorganisms in vitro.

[0023] In another aspect, there is provided a compound as defined above for use in therapy.

[0024] In another aspect, there is provided a method of 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] In another aspect, there is provided a compound as defined above for use in the treatment of a bacterial infection.

[0026] In another aspect, there is provided the use of a compound as defined above in the preparation of a medicament for the treatment of a bacterial infection.

[0027] Advantageously, the compound can act as an antibiotic itself or as an adjuvant to other antibiotics, such as colistin, by promoting the destruction of the outer membrane of MCR mutant bacteria and other Gram-negative bacteria without MCR mutations, and restore the sensitivity of these strains to colistin. Advantageously, the compound can reduce the MIC of colistin by at least two-fold. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] [ Figure 1 ] is a schematic diagram showing the pathway to the plasma membrane of Gram-negative bacteria and the types of molecular interactions at each step.

[0029] [ Figure 2 ] is a cartoon illustration of the chemical structure of the outer membrane of Gram-negative bacteria and the lipid A portion of the LPS molecule.

[0030] [ Figure 3 ] Schematic flow chart relating to the proposed method for designing molecules that can restore the sensitivity of MCR strains to colistin.

[0031] [ Figure 4 The schematic diagram shows 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) via a linker (4002). The N-containing group (4001) interacts with the lipid head group (4004), while the hydrophobic scaffold (4003) interacts with the lipid tail (4005).

[0032] [ Figure 5 ] Schematic diagram showing the chemical modification of lipid A in MCR strains. Lipid A is modified by MCR-1 through the transfer of a phosphoethanolamine (PPEA) group, resulting in the formation of hydrogen bonds between adjacent segments of lipid A, leading to the formation of PPEA-4'-lipid A or PPEA-1'-lipid A.

[0033] [ Figure 6 [Image of the MCR-1 protein structure constructed by homology modeling] shows an image of the MCR-1 protein structure. The structure of the extracellular domain is highly conserved and contains a zinc atom in the active site.

[0034] [ Figure 7 Figures and plots show molecular dynamics simulations of the outer membrane of MCR-modified membranes. (a) Snapshot of a normal lipid A membrane; (b) Snapshot of a PE-modified lipid A membrane. PE groups form hydrogen bonds (shown as dashed lines) with neighboring lipid A molecules, forming a network that stabilizes the outer membrane. (c) Plot showing the number of hydrogen bonds in a membrane sheet of 64 lipid A molecules. (d) Plot showing the radial distribution function (RDF) between different groups, showing phosphate-phosphate (701), amine-amine (702), and phosphate-amine (703).

[0035] [ Figure 8 ] Representative structures involving type 1-16 fragments.

[0036] [ Figure 9 ] relates to the structure of a hydrophobic scaffold.

[0037] [ Figure 10 ] relate to the structures of molecules synthesized in the DPA-Zn series.

[0038] [ Figure 11 ] concerns the structure of molecules synthesized in the amine series.

[0039] [ Figure 12 ] concerns the structures of molecules synthesized in the guanidine series.

[0040] [ Figure 13 ] Relates to graphs showing time kill studies of (a) colistin and (b) LC100 in combination with colistin against colistin-resistant mcr-1(+) strain 6083655967.

[0041] [ Figure 14 ] Relates to graphs showing time kill studies of (a) colistin and (b) LC100 in combination with colistin against colistin-resistant mcr-1(+) strain 6075066346.

[0042] [ Figure 15 ] Relates to graphs showing time kill studies of (a) colistin and (b) LC100 in combination with colistin against the colistin-resistant mcr-1(-) strain 7023446108.

[0043] [ Figure 16 ] The graphs involved show the time-killing effect of the combination of B2088 and LC100.

[0044] [ Figure 17] The graphs depict the effects of (a) LC100, (b) colistin, and (c) LC100 combined with colistin on mcr-1 bacterial membranes using the fluorescent probe ethidium bromide.

[0045] [ Figure 18 ] is a coordinate diagram showing the interaction between LC100 and MCR1 protein using fluorescence quenching.

[0046] [ Figure 19 ] The graphs involved show the hydrophobic surface area in bacterial membranes in the presence and absence of polymyxin B.

[0047] [ Figure 20 The graphs depict calcein leakage from 100 nm large unilamellar vesicles (LUVs) containing LC100 and LC101. The vesicles have a lipid composition of DOPE / DOPG = 3 / 1, mimicking the bacterial inner membrane. Significant fluorescence was detected with increasing concentrations of LC100 or LC101, indicating vesicle disruption and dye release. Further additions of LC100 or LC101 resulted in decreased fluorescence due to fluorescence quenching.

[0048] [ Figure 21 The graph depicts the efficacy 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 1.87 x 10 6 N = 3. "Combination" refers to colistin 10 mg / kg + LC100 50 mg / kg. Each treatment was administered intraperitoneally 1 hour after infection. Six hours after treatment, thigh tissue was homogenized to obtain viable CFU. Abdominal distension was observed in both the LC100 and combination groups. Compared to the LC100 group, mice in the combination group appeared weaker, cooler, and less active before sacrifice.

[0049] Detailed description with accompanying drawings

[0050] [ 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 adsorption on the LPS surface occurs through electrostatic interactions. 1002 refers to step 2, where the outer membrane is permeabilized, electrostatic interactions, and PO4 3- The hydrogen bonds and hydrophobic interactions with the lipid tails destroy the PO4 3- With Ca 2+ / Mg 2+1003 refers to step 3, where t~ns, and 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, and the cytoplasmic membrane is disrupted.

[0051] [ Figure 3 Schematic flow chart of the proposed method for designing molecules that can restore the sensitivity of MCR strains to colistin. MD simulations of the outer membrane and structural analysis of the MCR enzyme are performed (3001), the target is determined (3002), fragments are generated (3003), the fragments are incorporated (3004), new antibiotics are synthesized (3005), the antibiotics are validated (3006), and the mechanism of action is then deciphered (3007), with this information fed back (3008) to the fragment generation (3003). DETAILED DESCRIPTION

[0052] definition

[0053] Unless otherwise specified, "alkyl" as a group or part of a group refers to a straight-chain or branched aliphatic hydrocarbon group, preferably C1-C 20 Alkyl, C1–C 12 Alkyl, more preferably C1-C 10 Alkyl, most preferably C1-C6. Examples of suitable straight chain and branched C1-C6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, hexyl, etc. The group can be a terminal group or a bridging group.

[0054] "Alkenyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and can be straight or branched, preferably having 2-20 carbon atoms in the normal 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 normal chain, and the orientation of each double bond is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, and nonenyl. The group can be a terminal group or a bridging group.

[0055] "Alkynyl" as a group or part of a group refers to an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched, preferably having 2 to 20 carbon atoms in the normal chain, preferably 2 to 12 carbon atoms, more preferably 2 to 10 carbon atoms, and more preferably 2 to 6 carbon atoms. Exemplary structures include, but are not limited to, ethynyl and propynyl. The group may be a terminal group or a bridging group.

[0056] "Amino" refers to -NR a R b A group of the form, where R a and Rb Individually selected from groups including, but not limited to, hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl.

[0057] "Aminoalkyl" refers to an NH2-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.

[0058] "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 the ring atoms are carbon), preferably having 5 to 12 atoms per ring. Examples of aryl include phenyl, naphthyl, etc.; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which the phenyl and C 5-7 Cycloalkyl or C 5-7 Cycloalkenyl groups are fused together to form a ring structure, such as tetrahydronaphthyl, indenyl or indanyl. This group can be a terminal group or a bridge group. Usually, the aryl group is C6-C 18 Aryl.

[0059] "Guandinoalkyl" refers to an alkyl-NC(NH2)2- group, where 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 remainder of the molecule through the alkyl group.

[0060] "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, the remaining ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulfur. Examples of heteroaryl groups 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-thiophene. Heteroaryl is usually C1-C 18 Heteroaryl. A heteroaryl group may contain 3-8 ring atoms. A heteroaryl group may contain 1-3 heteroatoms independently selected from N, O, and S. This group may be a terminal group or a bridging group.

[0061] "Heteroarylalkyl" refers to a heteroaryl-alkyl group with a heteroaryl and alkyl moieties as defined herein. Preferred heteroarylalkyl groups contain a lower alkyl moiety. Exemplary heteroarylalkyl groups include pyridylmethyl. The group can be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkyl group.

[0062] "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 a ring atom. Examples of heterocyclic moieties include heterocycloalkyl, heterocycloalkenyl and heteroaryl.

[0063] "Halogen" represents chlorine, fluorine, bromine or iodine.

[0064] As used herein, the term "substituted" means that the group to which the term refers may be substituted by one or more groups independently selected from the group consisting of 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, alkoxyheteroalkyl, alkyl, alkylthiophene, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl, alkylthiophene-based groups, such as aryl,

[0065] The term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the above-mentioned compound, including pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of compounds of formula (I) can be prepared from inorganic 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 acids, and sulfonic acid organic acids, examples of which 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 medicament is a solid, it will be understood by those skilled in the art that the compounds of the present invention, medicaments, and salts 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.

[0066] 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 pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the compound, their use in therapeutic compositions and methods of treatment and prevention is contemplated. Supplementary active compounds may 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 dosage unit form. As used herein, "dosage unit form" refers to a physically discrete unit suitable as a unit dose for an individual to be treated; each unit is calculated to contain a predetermined amount of the compound and the required pharmaceutical carrier to produce the desired therapeutic effect. For convenient and effective administration, an effective amount of the compound may be formulated in an acceptable dosage unit together with a suitable pharmaceutically acceptable carrier. In the case where the composition contains a supplementary active ingredient, the dosage is determined by reference to the conventional dose and mode of administration of the ingredient.

[0067] It is understood that the disclosed compound family includes isomeric forms in the form of "E" or "Z" configuration isomers or mixtures of E and Z isomers, including diastereomers, enantiomers, tautomers and geometric isomers. It is also understood that those skilled in the art can separate some isomeric forms, such as diastereomers, enantiomers and geometric isomers, by physical and / or chemical methods.

[0068] Some compounds of the disclosed embodiments may exist as single stereoisomers, racemates, and / or mixtures of enantiomers and / or diastereomers. All such single stereoisomers, racemates, and mixtures thereof are intended to fall within the scope of the described and claimed subject matter.

[0069] Additionally, where applicable, the disclosed compounds are intended to encompass both solvated and unsolvated forms of the compounds. Thus, each formula includes compounds having the indicated structure, including hydrated and unhydrated forms.

[0070] The word "substantially" does not exclude "completely", for example, a composition "substantially free of" Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

[0071] Unless otherwise stated, the terms "comprising" and "comprise" and grammatical variations thereof are intended to represent "open" or "inclusive" language such that they include the listed elements but also allow for additional, non-listed elements.

[0072] As used herein, the term "about" in the context of formulation ingredient concentrations typically 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.

[0073] Throughout this disclosure, certain embodiments may be disclosed in range format. It should be understood that the description of range format is merely for convenience and brevity and should not be construed as an inflexible limitation of the scope of the disclosed range. Therefore, it should be considered that the description of a range has specifically disclosed all possible subranges and each numerical value within the range. For example, a description of a range, such as from 1 to 6, should be considered to have specifically disclosed subranges, such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as single digits within the range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the scope.

[0074] Detailed description of alternative implementations

[0075] abbreviation

[0076] ArgOMe: Arginine methyl ester

[0077] ArgOEt: Arginine ethyl ester

[0078] BPD: Bipyridine

[0079] DMF: N,N-dimethylformamide

[0080] DIC: N,N′-diisopropylcarbodiimide

[0081] DPA: Dipicolylamine

[0082] EDTA: Ethylenediaminetetraacetic acid

[0083] EtBr: ethidium bromide

[0084] EtOH: ethanol

[0085] HOBt: Hydroxybenzotriazole

[0086] HPLC: High Performance Liquid Chromatography

[0087] LMMD: Ligand-localized molecular dynamics

[0088] LPS: Lipopolysaccharide

[0089] MCR: Mobile Colistin Resistance

[0090] MCR strains: bacteria with mobile colistin resistance.

[0091] MCR protein: An enzyme that catalyzes the modification of lipid A with a PE group in strains MCR-1, MCR-2, MCR-3, and MCR-4. The structures of these four proteins are highly conserved and have similar active sites.

[0092] MD simulation: molecular dynamics simulation

[0093] MeOH: methanol

[0094] MIC: minimum inhibitory concentration.

[0095] NDM1: New Delhi metallo-beta-lactamase 1

[0096] PE group: phosphoethanolamine group

[0097] TEA: triethylamine

[0098] THF: Tetrahydrofuran

[0099] TPD: Terpyridine

[0100] Overall strategy for designing MCR inhibitors

[0101] 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 colistin sensitivity to bacteria with MCR plasmids, e.g. Figure 3 shown.

[0102] 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.

[0103] Based on the fragment-based strategy, a multidisciplinary approach was disclosed, including: (1) computer analysis and target identification (2) ligand design; (3) synthesis and (4) biological validation. Figure 3 As shown, the approach begins with a computational analysis of the conformation of the MCR protein and its outer membrane structure to identify potential targets within MCR-modified lipid A. Based on a fragment-based drug design (FBDD) strategy, a series of chemical fragments with high affinity for the identified targets are selected. These chemical fragments are then assembled together or incorporated with other designed fragments to form multiple antimicrobial scaffolds to obtain new antibiotics or antibiotic adjuvants.

[0104] To effectively disrupt bacterial membranes, the proposed model contains three types of fragments: two cationic fragments that interact with the two headgroup regions of the bacterial membrane, a hydrophobic fragment that interacts with the lipid tail of the bacterial membrane, and two linker groups that connect the cationic groups and the hydrophobic fragments. A series of chemical fragments were designed and assembled together to obtain new antibiotics or antibiotic adjuvants. The rationale for selecting the fragment structure was based on the following criteria ( Figure 4 ):

[0105] pKa of the N-containing group: Using model compounds, the pKa value of the terminal polar group was found to be 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.

[0106] ● Metal chelating properties of nitrogen-containing groups: When chelated with divalent cations such as zinc or calcium, the positively charged complex has affinity for both the phosphate groups in the outer membrane and the MCR-1 protein, which contains zinc in its catalytic site ( Figure 6 ).

[0107] Hydrophobic Scaffold Size: Because the central hydrophobic scaffold must be of a certain size, compounds composed of two or more aromatic rings with one or more hydrocarbon groups (e.g., prenyl groups) were tested. This ensured that the hydrophobic segments were of a size that would allow the resulting molecule to cross bacterial membranes. It was found that, in general, larger scaffolds resulted in better activity.

[0108] • Shape of the hydrophobic scaffold: Flat hydrophobic scaffolds are preferred because they can be easily cut into the membrane.

[0109] Overall hydrophobicity of the scaffold: According to the pharmacophore model, a hydrophobic scaffold interacts with the lipid tails, so higher hydrophobicity, or favorable transfer energy, indicates higher membrane activity. Preferably, the compound's logP should be greater than 4, greater than 6, or greater than 8. Preferably, the compound's logP 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 membranes.

[0110] ●The role of isoprenyl: Isoprenylic groups have high membrane affinity and can be used as membrane probes; when the hydrophobic scaffold is modified with one or more isoprenyl groups, the membrane activity becomes higher.

[0111] Next, the designed molecules will be synthesized and tested for antimicrobial activity against bacteria carrying the mobile plasmid mcr-1. Computational 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.

[0112] In silico analysis and target identification

[0113] When the mimetic colistin interacts 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 the cationic colistin, which ultimately leads to outer membrane rupture.

[0114] The Mcr-1 gene encodes an enzyme similar to phosphoethanolamine transferase, which modifies the lipid A portion of the outer membrane by adding phosphoethanolamine (PE) groups, leading to electrostatic interactions such as hydrogen bonds between lipid molecules and reducing the penetration of these compounds into the bacterial outer membrane. Through crystallography, MCR-1 has been characterized as a zinc-dependent metalloprotein, making it a good target for designing ligands to inhibit its activity.

[0115] like Figure 5 As shown, the transferred groups allow for additional hydrogen bonding that crosslinks and stabilizes the lipid A portion of the outer membrane. While each hydrogen bond may be weak on its own, when present in large numbers, they result in a very strong supramolecular scaffold that maintains the integrity of lipid A. For colistin or polymyxin to be effective in killing bacteria, this supramolecular organization of lipid A must be disrupted.

[0116] Furthermore, in the MCR-1 strain, PE-modified lipid A forms numerous hydrogen bonds that cross-link the lipid A molecules together, thereby stabilizing the outer membrane. However, the hydrogen-bonding network between the modified lipid molecules could also serve as an additional target. Molecules that disrupt this hydrogen-bonding network would destabilize the outer membrane of MCR strains and potentially restore their susceptibility to colistin.

[0117] Colistin resistance in MCR-positive bacterial strains stems from the modification of lipid A by MCR proteins and the subsequent changes in outer membrane properties. Therefore, two targets in MCR strains can be identified: (1) MCR proteins; and (2) modified outer membranes. For the first target, structural alignment of the MCR-1 ectodomain with the full-length PE transferase ectodomain revealed a high degree of structural similarity. Using the crystal structures of the MCR-1 protein and the PE transferase ectodomain as templates, the structure of the entire MCR-1 protein was constructed using homology modeling based on this data ( Figure 7 Like PE transferases, MCR enzymes are metalloproteins with a zinc atom in their catalytic domain, which facilitates the binding of the negatively charged portion of the POPE lipid and catalyzes the subsequent modification of lipid A. Modified lipid A exhibits reduced interaction with colistin, leading to colistin resistance. Therefore, molecules binding to the active site of the MCR-1 protein inhibit its activity and restore the sensitivity of MCR strains to colistin.

[0118] For the second target, the attachment of PE groups to lipid A reduced its electrostatic interaction with colistin, resulting in a decrease in the affinity of colistin to the outer membrane. In addition, molecular dynamics simulations of PE-modified lipid A membranes revealed for the first time the formation of a large number of hydrogen bonds between the head groups of lipid A molecules ( Figure 7 ). The hydrogen bonding network cross-links LPS molecules together and stabilizes the outer membrane of MCR strains, thereby producing 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 destroy / disrupt the outer membrane hydrogen bonding network can directly kill bacteria or restore bacterial sensitivity to colistin. In summary, the MCR protein and hydrogen bonding network of the outer membrane have been identified as two targets, and ligand mapping simulations have been used to find more hidden binding pockets for designing MCR inhibitors.

[0119] Ligand design

[0120] Based on the atomic details of the identified target, a series of fragments were designed that can bind to the MCR protein or disrupt the hydrogen bonding network of the outer membrane. In terms of the mode of action, these fragments can be divided into three categories:

[0121] (1) Fragments that bind to the active site of the MCR-1 protein;

[0122] (2) a fragment that binds to the hidden pocket of the MCR-1 protein;

[0123] (3) Fragments that disrupt / destroy the hydrogen bonding network of the outer membrane.

[0124] 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 fragments destroys / disrupts the PE-modified outer membrane, which can kill MCR bacteria independently or synergistically with colistin. Based on these three modes of action, six types of fragments were proposed. Figure 8 The structures of representative examples of each type are shown.

[0125] i. PE group: The PE group and its analogs bind to the active site of the MCR-1 protein.

[0126] ii. Zinc chelating groups, such as DPA, terpyridine, 2,2'-bipyridine, 1,10-phenanthroline, porphyrin, 8-hydroxyquinoline, and carboxyl groups. Zinc chelating groups can inhibit the activity of MCR-1 by binding to the zinc atom in the active site of the protein. Furthermore, when chelated with zinc or other divalent cations, the complex becomes cationic and can form salt bridges with phosphate groups, disrupting the hydrogen bonding network in the outer membrane of MCR-1 and leading to instability of the PE-modified outer membrane.

[0127] iii. Cyclic polyamines. Cyclic polyamines have dual functions: (a) forming hydrogen bonds with the head groups of PE-modified lipids, leading to disruption of the hydrogen bonding network in the outer membrane; and (b) chelating zinc atoms, thereby inhibiting the MCR-1 protein and disrupting the mcr-1 outer membrane.

[0128] 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.

[0129] v. Guanidine and polyguanidine groups. Polyguanidine groups can form bidentate hydrogen bonds with phosphate groups and can disrupt the hydrogen bonding network of the outer membrane.

[0130] vi. Basic amino acid groups, such as arginine, polyarginine, lysine, polylysine, poly-ε-lysine, arginine derivatives, and 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.

[0131] All of the fragments share the same characteristic: they bind with high affinity to phosphate groups in bacterial membranes. The strong interaction between these fragments and phosphate groups in bacterial membranes leads to large membrane perturbations.

[0132] Using these fragments, we designed a library of compounds as potential antibiotics or antibiotic adjuvants that synergize with colistin against MCR bacteria. Molecules were designed by assembling two or more fragments together or coupling the fragments to other scaffolds. The structures of the library molecules and their modes of action are described below.

[0133] X-Cn-Y-Cn-X, wherein X is a nitrogen-containing moiety, Y is a hydrophobic scaffold, and Cn is an alkyl chain having n carbon atoms. The nitrogen-containing moiety X can be Figure 8 The fragment in , and Y can be Figure 9 The molecule can act as (i) an MCR-1 protein inhibitor; (ii) a hydrogen bonding network disruptor.

[0134] Provided are compounds having the following formula (I):

[0135] Z 1 -L 1 -AL 2 -Z 2 Formula (I)

[0136] Where A is a hydrophobic part;

[0137] L 1 and L 2 are independently linkers; and

[0138] Z 1 and Z2 are independently N-containing moieties.

[0139] A may contain at least one substituted or unsubstituted aryl group.

[0140] A may also contain at least one alkenyl group.

[0141] A can be planar or substantially planar. That is, A can lie within a plane or lie substantially within a plane.

[0142] A may also contain at least one isoprenyl group.

[0143] A can be selected from:

[0144]

[0145] and any mixture thereof.

[0146] Wherein a bond break indicates the position at which the structure is attached to the remainder of formula (I).

[0147] A can be selected from:

[0148]

[0149] and any mixture thereof.

[0150] Z 1 and Z 2 The pKa value of each of the compounds can independently be greater than 8. The pKa value can be greater than 8.5, greater than 9, greater than 11, or greater than 13. The pKa value can be less than 20.

[0151] Z 1 and Z 2 Can independently have the following structures:

[0152]

[0153] wherein the broken bond indicates the position at which the structure is attached to the rest of formula (I);

[0154] R 1 and R 2 R 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 heterocyclic ring; and

[0155] R 3may be absent, or may be hydrogen or 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,

[0156] When R 3 When present, the nitrogen atom may be a cationic quaternary nitrogen.

[0157] Z 1 and Z 2 The compound may independently comprise an amine, guanidine, pyrrolidine, pyrrole, imidazolidine, pyrazolidine, imidazole, pyrazole, triazole, piperidine, pyridine, piperazine, diazine, hydroxamic acid, hydrazine, N-hydroxyurea, squaric acid, carbamolyphosphonate, oxazoline, pyrimidinetrione, 1-hydroxy-2(1H)-pyridinone (1,2-HOPO), and any combination thereof.

[0158] R 1 、R 2 and R 3 Can be independently selected from methyl, ethyl, propyl, butyl, -(CH2) x NR'R", -(CH2) x OH, -(CH2) x PO3, -(CH2) x CR'R"R"', -(C(NH2)NHC(NH2))x-NH2, guanidine, 2-methylpyridine, 1-methylimidazole, pyridine, bipyridine, terpyridine, phenanthroline, 3-methylpyrrole, cyclen (1,4,7,10-tetraazacyclododecane), cyclopentane (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, wherein x can be any integer from 1 to 10, and R', R" and R'" can independently be hydrogen or a substituted or unsubstituted alkyl group.

[0159] Z 1 and Z 2 Can be independently selected from:

[0160]

[0161]

[0162] and any combination thereof.

[0163] L 1and L 2 may independently be substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, ester, amide, ether, -(O-CH2-CH2-O) n- , L 3 or any combination thereof, wherein L 3 Has the following structure:

[0164] Where n can be an integer from 1 to 10 and R 4 It can be amino or heteroalkyl and the bond break indicates the position at which this structure is attached to the remainder of formula (I).

[0165] L 1 and L 2 Can be independently selected from -(CH2)4-, amide, and any combination thereof, or

[0166] 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.

[0167] The compound of formula (I) may have the following structure:

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] Each N-containing moiety can independently chelate zinc.

[0177] Each N-containing moiety may independently have a positive charge.

[0178] Provided is a pharmaceutical composition comprising the compound defined above, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

[0179] The pharmaceutically acceptable carrier may be saline.

[0180] There is provided a process for preparing a compound as defined above comprising the step of contacting a hydrophobic moiety with an N-containing moiety under reaction conditions.

[0181] The method defined above may further comprise the step of contacting the hydrophobic moiety with a linker prior to contacting with the N-containing moiety.

[0182] Covalent bonds can 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.

[0183] The hydrophobic portion may be selected from:

[0184]

[0185] and any mixtures thereof.

[0186] 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.

[0187] The N-containing portion can be selected from dispicolylamine, cyclopentane, 1,8-dimethyl-l,1,4,8,11-tetraazacyclotetradecane, diethylamine, -NH[(CH2)3N(CH3)]2, -NH2(CH2)3N(CH3)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.

[0188] The method defined above may comprise the step of adding zinc after the N-containing moiety has been covalently bonded to the linker or the hydrophobic moiety.

[0189] The contacting may be carried out in a solvent selected from the group consisting of acetone, methanol, ethanol, propanol, butanol, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, pyridine, water, and any mixture thereof.

[0190] The contacting step may be performed at a temperature of about 21°C to about 160°C, about 21°C to about 60°C, about 21°C to about 100°C, about 60°C to about 100°C, about 60°C to about 160°C, or about 100°C to about 160°C.

[0191] The contacting step can be carried out for about 2 hours to about 36 hours, about 2 hours to about 8 hours, about 2 hours to about 12 hours, about 2 hours to about 24 hours, about 8 hours to about 12 hours, about 8 hours to about 24 hours, about 8 hours to about 36 hours, about 12 hours to about 24 hours, about 12 hours to about 36 hours, or about 24 hours to about 36 hours.

[0192] There is provided a compound as defined above or a pharmaceutical composition as defined above for use as an antibiotic.

[0193] There is provided the use of a compound as defined above or a pharmaceutical composition as defined above for killing or inhibiting the growth of microorganisms in vitro.

[0194] The compounds defined above or the pharmaceutical compositions defined above may be used to kill or inhibit the growth of microorganisms on surfaces, including but not limited to topical applications, in ointments, eye drops, nasal sprays, mouthwashes and hand sanitizers. The compounds defined above or the pharmaceutical compositions defined above may also be used as food preservatives, disinfectants, surface cleaners or medical devices.

[0195] The microorganism may be a bacterium, an archaeon, a fungus, a protist, or any mixture thereof.

[0196] The microorganisms can be gram-negative bacteria or gram-positive bacteria.

[0197] Bacteria can be MCR-positive or MCR-negative.

[0198] Bacteria may be resistant to carbapenems.

[0199] The bacteria may be selected from the group consisting of E. coli, E. cloacae, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumoniae, Enterobacter aerogenes and Acinetobacter baumannii.

[0200] The compounds defined above may be used in combination with colistin.

[0201] There is provided a compound as defined above for use in therapy.

[0202] Also provided is a method of treating a bacterial infection comprising the step of administering to a patient in need thereof a therapeutically effective amount of a compound as defined above.

[0203] The method defined above may further comprise the step of administering a therapeutic amount of colistin and a compound defined above.

[0204] There is provided a compound as defined above for use in the treatment of a bacterial infection.

[0205] The compounds defined above may be administered in combination with colistin.

[0206] There is provided the use of a compound defined above in the preparation of a medicament for treating a bacterial infection.

[0207] According to the present invention, when used to treat or prevent microbial infections, the compounds of the present invention can be administered alone. Alternatively, the compounds can be administered as pharmaceutical, veterinary, agricultural or industrial formulations comprising at least one compound of the present invention. The compounds can also exist as suitable salts, including pharmaceutically acceptable salts.

[0208] The use defined above may also comprise the administration of a compound defined above in combination with colistin.

[0209] Provided are methods as defined above, compounds as defined above, or uses as defined above, wherein the compound as defined above is present in an amount of 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.

[0210] The method as defined above, the compound as defined above or the use as defined above, wherein the compound as defined above and colistin may be present in the same amount by weight, or the compound as defined above may be in an excess of 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 by weight of colistin.

[0211] The method, compound or use as defined above, wherein colistin may be present in the range of 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 compound as defined above may 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.

[0212] The method as defined above, the compound as defined above or the use as defined above, wherein the compound as defined above is administered intramuscularly, intraperitoneally, topically, subcutaneously or intravenously.

[0213] In one embodiment, the compound can be administered by injection. In the case of injectable solutions, 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.), a suitable mixture thereof, and vegetable oil. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size and by using a surfactant in the case of dispersion. The effect of microorganisms can be prevented by including various antibacterial and / or antifungal agents. Suitable medicaments 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, isotonic agents are included in the composition, such as sugar, polyols such as mannitol, sorbitol, sodium chloride, which may be preferred. The absorption of the injectable composition can be extended by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0214] As needed, sterile injectable solutions can be prepared by incorporating the desired amount of the analog and one or more of the above ingredients into an appropriate solvent, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the analog into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from those listed above.

[0215] Preferably, the pharmaceutical composition may further 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.

[0216] Dispersions of the compounds of the invention can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, the pharmaceutical preparation may contain a preservative to prevent the growth of microorganisms.

[0217] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Ideally, the compositions are stable under the conditions of manufacture and storage, and preservatives may be included to stabilize the compositions against the contaminating effects of microorganisms such as bacteria and fungi.

[0218] Delayed release formulations are also included within the scope of this invention.

[0219] 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 vivo. Suitable prodrugs include esters, phosphonates, and the like of the active form of the compound.

[0220] The pharmaceutical compositions of the present invention may be administered in single or multiple doses. One skilled in the art will be able to determine by routine experimentation the effective, non-toxic dosage levels of the compounds and / or compositions of the present invention and the modes of administration suitable for treating the diseases and / or infections for which they are intended.

[0221] Furthermore, it will be apparent to one of ordinary skill in the art that routine treatment course determination testing can be used to determine the optimal treatment course, such as the number of doses of a compound or composition of the invention to be administered per day over a defined number of days.

[0222] The present invention relates to the following specific embodiments.

[0223] Item 1. A compound having the following formula (I):

[0224] Z 1 -L 1 -AL 2 -Z 2 Formula (I)

[0225] Where A is a hydrophobic part;

[0226] L 1 and L 2 are independently linkers; and

[0227] Z 1 and Z 2 are independently N-containing moieties.

[0228] Item 2. The compound according to Item 1, wherein A comprises at least one substituted or unsubstituted aromatic group.

[0229] Item 3. The compound according to Item 2, wherein A further comprises at least one alkenyl group.

[0230] Item 4. A compound according to Item 3, wherein A is planar or substantially planar.

[0231] Item 5. The compound according to Item 4, wherein A further comprises at least one isoprenyl group.

[0232] Item 6. The compound according to any one of the preceding items, wherein A is selected from:

[0233]

[0234]

[0235] and any mixture thereof,

[0236] wherein the broken bond represents the position where the structure is connected to the rest of formula (I). Item 7. The compound according to Item 6, wherein A is selected from:

[0237]

[0238] and any mixtures thereof.

[0239] Item 8. The compound according to any one of the preceding items, wherein Z 1 and Z 2 The pKa values ​​of the compounds are independently greater than 9.

[0240] Item 9. The compound according to any one of the preceding items, wherein Z 1 and Z 2 Independently has the following structure:

[0241]

[0242] wherein the bond break represents the position at which the structure is attached to the remainder of formula (I);

[0243] R 1 and R 2 R is independently 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 heterocyclic ring; and

[0244] R 3 is absent or is hydrogen or 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,

[0245] When R 3 When present, the nitrogen atom is a cationic tetravalent nitrogen.

[0246] Item 10. The compound according to Item 9, wherein R 1 、R 2 and R 3Independently comprises a functional group 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, carbamoylphosphonate, oxazoline, pyrimidinetrione, 1-hydroxy-2(1H)-pyridinone (1,2-HOPO), and any combination thereof.

[0247] Item 11. The compound according to Item 9 or 10, wherein R 1 、R 2 and R 3 independently selected from methyl, ethyl, propyl, butyl, -(CH2) x NR'R", -(CH2) x OH, -(CH2) x PO3, -(CH2) x CR'R"R"', -(C(NH2)NHC(NH2))x-NH2, guanidine, 2-methylpyridine, 1-methylimidazole, pyridine, bipyridine, terpyridine, phenanthroline, 3-methylpyrrole, cyclamen (1,4,7,10-tetraazacyclododecane), cyclamen (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, wherein x can be any integer from 1 to 10, and R', R" and R"' are independently hydrogen or substituted or unsubstituted alkyl.

[0248] Item 12. The compound according to any one of Items 9-11, wherein Z 1 and Z 2 Independently selected from:

[0249]

[0250]

[0251] and any combination thereof.

[0252] Item 13. The compound according to any one of the preceding items, wherein L 1 and L 2 are independently substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, ester, amide, ether, -(O-CH2-CH2-O) n -、L 3 or any combination thereof, wherein L 3 Has the following structure:

[0253] Where n is an integer from 1 to 10, R4 is amino or heteroalkyl, and the bond break indicates the position at which the structure is attached to the remainder of Formula (I).

[0254] Item 14. The compound according to Item 13, wherein L 1 and L 2 Independently selected from -(CH2)4-, amide, and any combination thereof or

[0255] Item 15. The compound according to any one of the preceding items, wherein the logP value of the compound of formula (I) is greater than 4.

[0256] Item 16. The compound according to any one of the preceding items, wherein the compound of formula (I) has the following structure:

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265] Item 17. The compound according to any one of the preceding items, wherein each N-containing moiety independently chelates with zinc.

[0266] Item 18. The compound according to any one of the preceding items, wherein each N-containing moiety independently has a positive charge.

[0267] Item 19. A pharmaceutical composition comprising the compound according to any one of the preceding items or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

[0268] Item 20. The pharmaceutical composition according to Item 19, wherein the pharmaceutically acceptable carrier is saline.

[0269] Item 21. A method for preparing a compound according to any one of items 1-18, comprising the step of contacting a hydrophobic portion with an N-containing portion under reaction conditions.

[0270] Item 22. The method according to Item 21, further comprising the step of contacting the hydrophobic portion with a linker before contacting the N-containing portion.

[0271] Item 23. The method according to Item 21 or 22, wherein a covalent bond is formed between the hydrophobic portion and the N-containing portion, or between the hydrophobic portion and the linker, or between the linker and the N-containing portion.

[0272] Item 24. The method according to any one of items 21-23, wherein the hydrophobic portion is selected from:

[0273]

[0274] and any mixtures thereof.

[0275] Item 25. A method according to any one of Items 21-24, wherein the linker is 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.

[0276] Item 26. A method according to any one of Items 21-25, wherein the N-containing portion is selected from disicolamine, cyclopentane, 1,8-dimethyl1,4,8,11-tetracyclotetradecane, diethylamine, -NH[(CH2)3N(CH3)]2, -NH2(CH2)3N(CH3)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.

[0277] Item 27. The method according to any one of Items 21-26, comprising the step of adding zinc after the N-containing portion is covalently bonded to the linker or hydrophobic portion.

[0278] Item 28. A method according to any one of items 21-27, 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.

[0279] Item 29. The method of any one of items 21-28, wherein the contacting step is performed at a temperature of about 21°C to about 160°C.

[0280] Item 30. The method according to any one of items 21-29, wherein the contacting step is performed for a duration of about 2 hours to about 36 hours.

[0281] Item 31. Use of the compound according to any one of Items 1 to 16 or the pharmaceutical composition according to Item 19 or 20 for killing microorganisms or inhibiting the growth of microorganisms in vitro.

[0282] Item 32. The use according to Item 31, wherein the microorganism is a bacterium, an archaea, a fungus, a protist or any mixture thereof.

[0283] Item 33. The use according to Item 32, wherein the microorganism is a Gram-negative bacterium or a Gram-positive bacterium.

[0284] Item 34. The use according to Item 32 or 33, wherein the bacterium is MCR-positive or MCR-negative.

[0285] Item 35. The use according to any one of Items 32 to 34, wherein the bacterium is carbapenem-resistant.

[0286] Item 36. The use according to any one of Items 31 to 35, wherein the compound according to any one of Items 1 to 16 is used in combination with colistin.

[0287] Item 37. A compound according to any one of Items 1-16 for use in therapy.

[0288] Item 38. A method for treating bacterial infection, comprising the step of administering a therapeutically effective amount of a compound according to any one of Items 1-16 to a patient in need thereof.

[0289] Item 39. The method according to Item 38, wherein the method further comprises the step of administering a therapeutic amount of colistin in combination with a compound according to any one of Items 1-16.

[0290] Item 40. A compound according to any one of Items 1-16, for use in treating bacterial infection.

[0291] Item 41. The compound according to Item 40, wherein the compound according to any one of Items 1 to 16 is administered in combination with colistin.

[0292] Item 42. Use of the compound according to any one of Items 1 to 16 in the preparation of a medicament for treating bacterial infection.

[0293] Item 43. The use according to Item 42, wherein the compound according to any one of Items 1 to 16 is administered in combination with colistin.

[0294] Item 44. The method according to items 38 and 39, the compound according to items 40 and 41, or the use according to items 42 and 43, wherein the compound according to any one of items 1-16 is present in an amount of about 2 μg / mL to about 75 μg / mL.

[0295] Item 45. The method according to Item 39, the compound according to Item 41, or the use according to Item 43, wherein the compound according to any one of Items 1-16 and colistin are present in equal amounts by weight, or the compound according to Items 1-16 is present in an excess of about 1.5 to about 6 times by weight of colistin.

[0296] Item 46. The method, compound or use according to Item 45, wherein colistin is present in the range of about 1 mg / kg to about 10 mg / kg, and the compound according to any one of Items 1-16 is present in the range of 10 mg / kg to about 50 mg / kg.

[0297] Item 47. The method according to items 38-39, the compound according to items 42 and 42, or the use according to item 43, the use according to item 40 or 41, or the use according to items 42 and 43, wherein the compound according to items 1-16 is or is to be administered intramuscularly, intraperitoneally, topically, subcutaneously or intravenously.

[0298] Experiments and methods

[0299] Example 1: Materials

[0300] Strains with “ATCC” in their names were purchased from the American Type Culture Collection (ATCC), Virginia, U.S.A. All other bacterial strains were purchased from Tan Tock Seng Hospital, Singapore.

[0301] The hydrophobic scaffolds LC003, LC007, LC008, and AM000 were purchased from Chengdu Biopurify Phytochemicals (Chengdu, China). The cationic moieties N,N,N-trimethyl-1,3-propanediamine and 3,3'-3,3'-iminobis(Iminobis)(N,N-dimethylpropylamine) were purchased from Bio-etc (Singapore, Singapore). MCR-1 protein was purchased from A*STAR Bioprocess Technology Institute (Singapore, Singapore). Cyclotrimethylenetetradecane and 1,8-dimethyl-1,4,8,11-tetracyclotetradecane were purchased from TCI (Tokyo, Japan). The solvents ethyl acetate, hexane, acetone, butanol, methanol, and DMF were purchased from Aik Moh (Singapore, Singapore). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0302] Example 2: MIC studies

[0303] According to the Clinical and Laboratory Standards Institute (CLSI) guidelines, 96-well culture plates were used to determine the MIC of antimicrobial agents by a modified broth microdilution method. Briefly, serial dilutions of all plates were prepared at different concentrations (μg). Inoculum suspensions were prepared by adding isolated colonies from 18-20 hours 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 a 0.5 McFarland standard. The final bacterial concentration contained in each well was approximately 5 × 10 5 The plate was 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 in a test tube or microdilution well as detected by the naked eye. For microdilution plates, the TECANinfinite M200Pro can be used to measure the OD 600 The turbidity in the wells was measured at 4 °C. The MIC was determined as the highest concentration that produced 99% inhibition.

[0304] Example 3: Synergistic Effect Study

[0305] MICs for individual drugs and combinations with LC compounds were determined using the CLSI-recommended broth microdilution technique, whereby both antimicrobials were added in equal amounts (e.g., 50 μL colistin: 50 μL LC compound). The dilution medium containing the compound and antibiotic was serially diluted two-fold, and the LC compound and antibiotic were tested alone or in combination at fixed synergistic concentrations. In all cases, the lowest concentration at which no visible growth occurred was recorded as the MIC value for both the individual and combined antimicrobials. Results were obtained after a 24-hour incubation period.

[0306] Example 4: Time Kill Study

[0307] The time kill assay is used to determine the rate at which an antimicrobial agent kills a bacterial isolate. The bacterial suspension is adjusted to 10 6 to 10 7 CFU / mL for comparison in separate test tubes incubated at 35°C. For synergistic time kill: two different antimicrobial agents, antibiotics / LC compounds, were added in equal amounts to a final volume of 1 mL (e.g., 500 μL antibiotic: 500 μL LC compound), followed by addition of 1 mL of the prepared bacterial suspension to the same tube. Aliquots of 100 μL of sample 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, all plates were checked for bacterial growth, and the colony forming unit (CFU / mL) counts of the colonies were calculated, and a time kill curve was plotted using the viable counts versus the logarithm of time. The results for various concentrations of antibiotics and controls were plotted.

[0308] Example 5: In vivo mouse thigh infection model

[0309] In a neutropenic mouse thigh infection model, a combination of colistin and an antimicrobial peptide (LC100) was tested against the E. coli clinical isolate MCR-positive strain 6083655967. C57BL / 6 mice were rendered neutropenic with cyclophosphamide and 150 mg kg was delivered on days -4 and -1 prior to infection. -1 and 100 mg kg -1 Dosage administration was performed. Bacteria were suspended in sterile saline and adjusted to approximately 1.876×10 per infection site (100 μL). 6 The concentration of CFU was determined and injected intramuscularly into the right thigh of 5 mice in each treatment group. One hour after infection, mice received colistin (15 mg kg -1 , ipn = 5), LC100 (50 mg kg-1 , ip n = 5), untreated (n = 5), or the combination (n = 5). Mice were euthanized 6 hours after infection; thigh tissue was collected under sterile conditions, homogenized, serially diluted in PBS, and plated on solid plates supplemented with TSA. Plates were incubated overnight at 37°C, and colonies were quantified to determine bacterial load.

[0310] Example 6: Molecular dynamics study results

[0311] Molecular dynamics simulations were performed to understand the molecular origins of colistin resistance. The structure and dynamics of mcr-1(+) and intact lipid A membranes were studied. For each membrane, a preassembled lipid A bilayer was first assembled by placing the required number of lipid A molecules (from either the mcr-1(+) or mcr-1(-) strain) on a grid in the xy dimension. Next, the preassembled bilayer was solvated with water molecules. Calcium ions were used to neutralize the system. MD simulations for 300ns 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. Disruption of the salt bridges with antimicrobial peptides such as colistin would destabilize the outer membrane. However, in the case of the mcr-1(+) strain, MD simulations revealed the presence of a large number of intermolecular hydrogen bonds ( ) between the phosphate groups of the mcr-1 membrane and the mcr-1 membrane. Figure 7 Because the phosphate and amine groups carry opposite charges, they interact with strong electrostatic forces that further stabilize the lipid A membrane. As a result, short-range inter-lipid hydrogen bonds and long-range electrostatic interactions cross-link the lipid A molecules of the mcr-1(+) strain, leading to resistance to colistin. The results suggest that disruption of hydrogen bonds or electrostatic interactions within the lipid A membrane can destabilize the outer membrane of the mcr-1(+) strain.

[0312] Example 7: Chemical synthesis

[0313] The selected synthetic scheme is as follows Figure 10 、 Figure 11 and Figure 12 shown.

[0314] Synthesis of LC004: 1,4-dibromobutane (313.8 μL, 2.65 mmol) and K2CO3 (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 maintained under reflux 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%.

[0315] Synthesis of LC003-I: 1,4-Diiodobutane (601 μL, 4.557 mmol) and K2CO3 (198 mg, 1.43 mmol) were added to LC003 (98.6 mg, 0.304 mmol) dissolved in acetone. The reaction was processed similarly to the synthesis of LC004. Silica gel column chromatography of the crude product using a solvent ratio of hexane:EtOAc (v / v) 40:1 afforded 113.5 mg of LC003-I as a yellow powder. Yield: 54%.

[0316] Synthesis of LC003-DPA: To a 2.5 mL acetone solution of LC003-I (25.5 mg, 0.037 mmol) was added KCO (26.2 mg, 0.190 mmol) and dipicolylamine (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 three times with a saturated NaCl solution. Purification was performed using preparative HPLC to give 17.8 mg of LC003-DPA as a bright yellow gel. Yield: 58%.

[0317] Synthesis of LC003-DPA-Zn: A solution of 5.44 mg of ZnCl2 in 1 mL of methanol was mixed with 8.3 mg of LC003-DPA and stirred for 3 hours. The solvent was removed in vacuo to obtain the LC003-DPA-Zn complex (LC003-DPA:Zn=1:4).

[0318] Synthesis of LC008-I: 1,4-Diiodobutane (478 μL, 1.86 mmol) and KCO (180 mg, 1.302 mmol) were added to LC008 (61.8 mg, 0.241 mmol) dissolved in acetone. The reaction was processed similarly to the synthesis of LC004. The crude product was purified by silica gel column chromatography using a solvent ratio of hexane:EtOAc (v / v) 25:1 to obtain 49.6 mg of LC008-I as a yellow powder. Yield: 33%.

[0319] Synthesis of LC008-DPA: To a solution of LC008-I (32.1 mg, 0.052 mmol) in 2 mL of acetone was added KCO (42.0 mg, 0.304 mmol) and dipicolylamine (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 three times with saturated NaCl solution. Purification by preparative HPLC gave 16.7 mg of LC008-DPA as a bright yellow gel. Yield: 42%.

[0320] Synthesis of LC008-DPA-Zn: A 1 mL methanol solution of 5.44 mg ZnCl2 was mixed with 7.62 mg LC003-DPA and stirred for 3 hours. The solvent was removed in vacuo to obtain the LC008-DPA-Zn complex (LC008-DPA:Zn=1:4).

[0321] Synthesis of AM005: 1,4-Dibromobutane (434 μL, 3.66 mmol) and KCO (168 mg, 1.22 mmol) were added to AM000 (100 mg, 0.244 mmol) dissolved in 3 mL of acetone. The reaction was processed similarly to the synthesis of LC004. The crude product was chromatographed on a silica gel column using a solvent ratio of hexane:EtOAc (v / v) 20:1 to obtain 98 mg of AM005 as a yellow powder. Yield: 59%.

[0322] Synthesis of AM000-DPA: To a solution of AM005 (68 mg, 0.1 mmol) in 3 mL of acetone was added KCO (84.0 mg, 0.6 mmol) and dipicolylamine (269 μL, 1.5 mmol). The reaction mixture was stirred at 55°C overnight. Acetone was removed in vacuo. The crude product was diluted with EtOAc and extracted three times with saturated NaCl solution. Purification by preparative HPLC afforded 38.4 mg of AM000-DPA as a bright yellow gel. Yield: 47%.

[0323] Synthesis of LC104 (AM000-DPA-Zn): A solution of 5.44 mg of ZnCl2 in 1 mL of methanol was mixed with 9.16 mg of AM000-DPA and stirred for 3 hours. The solvent was removed in vacuo to obtain the LC104 complex (AM000-DPA:Zn=1:4).

[0324] Synthesis of Orc-1: 1,4-Diiodobutane (1 mL, 7.58 mmol) and KCO (546 mg, 3.95 mmol) were added to orcinol (62.9 mg, 0.506 mmol) dissolved in acetone. The reaction was processed similarly to the synthesis of LC004. The crude product was purified by silica gel column chromatography using a solvent ratio of hexane:EtOAc (v / v) of 100:1 to obtain 164.7 mg of LC008-1 as a yellow powder. Yield: 67%.

[0325] Synthesis of Orc-DPA: To a solution of Orc-I (66 mg, 2.83 mmol) in 2 mL of acetone were added KCO (122 mg, 0.883 mmol) and dipicolylamine (364 μL, 2.022 mmol). The reaction mixture was stirred at 55°C overnight. The acetone was removed in vacuo. The crude product was diluted with EtOAc and extracted three times with saturated NaCl solution. Purification by preparative HPLC afforded 66.4 mg of LC008-DPA as a bright yellow gel. Yield: 85%.

[0326] Synthesis of Orc-DPA-Zn: A solution of 5.44 mg of ZnCl2 in 1 mL of methanol was mixed with 6.3 mg of Orc-DPA and stirred for 3 hours. The solvent was removed in vacuo to obtain the Orc-DPA-Zn complex (Orc-DPA:Zn=1:4).

[0327] Synthesis of LC007-I: 1,4-Diiodobutane (373 μL, 2.83 mmol) and KCO (147.3 mg, 1.07 mmol) were added to glabridin (62.2 mg, 0.192 mmol) dissolved in acetone. The reaction was processed similarly to the synthesis of LC004. The crude product was purified by silica gel column chromatography using a solvent ratio of hexane:EtOAc (v / v) of 30:1 to obtain 66.6 mg of LC007-I as a yellow powder. Yield: 50.0%.

[0328] Synthesis of GLA-DPA: To a solution of GLA-I (31.5 mg, 0.0456 mmol) in 2 mL of acetone was added KCO (37.5 mg, 0.271 mmol) and dipicolylamine (123 μL, 0.683 mmol). The reaction mixture was stirred at 55°C overnight. 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 GLA-DPA as a yellow gel. Yield: 65%.

[0329] Synthesis of GLA-DPA-Zn (LC007-DPA-Zn): A solution of 5.44 mg of ZnCl2 in 1 mL of methanol was mixed with 8.3 mg of GLA-DPA and stirred for 3 hours. The solvent was removed in vacuo to obtain a GLA-DPA-Zn complex (GLA-DPA:Zn=1:4).

[0330] Synthesis of LC300: To a solution of LC010 (34.85 mg, 0.05 mmol) in 3 mL of DMF was added cyclopentane (68.8 mg, 0.4 mmol). The reaction mixture was stirred at room temperature for 4 hours. The crude product was dissolved in butanol, washed three times with aqueous KCO and saturated NaCl solution, and dried under vacuum. The crude product was then redissolved in 1 mmol of ZnCl / MeOH solution. Purification by preparative HPLC yielded 20.2 mg of LC300 (GLA-cyclopentane-Zn) as a yellow gel. Yield: 40.5%.

[0331] Synthesis of LC301: Cyclodextrin (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, subsequently washed three times with a K CO aqueous solution and a saturated NaCl solution and dried in vacuo. The crude product was then redissolved in a 1 mmol ZnCl / MeOH solution. Purification was performed using preparative HPLC to obtain 18.4 mg of a yellow gel-like LC301 (LC003-Cyclodextrin-Zn). Yield: 37%.

[0332] Synthesis of LC302: To a 3 mL DMF solution of AM005 (34.0 mg, 0.05 mmol) was added cyclopentane (68.8 mg, 0.4 mmol). The reaction mixture was stirred at room temperature for 4 hours. The crude product was dissolved in butanol, subsequently washed three times with aqueous KCO and saturated NaCl solution and dried under vacuum. The crude product was then redissolved in a 1 mmol ZnCl / MeOH solution. Purification was performed using preparative HPLC to obtain 24.6 mg of LC302 (AM000-cyclopentane-Zn) as a yellow gel. Yield: 45%.

[0333] Synthesis of LC304: To a 3 mL DMF solution of LC008-1 (34.0 mg, 0.05 mmol) was added cyclopentane (68.8 mg, 0.4 mmol). The reaction mixture was stirred at room temperature for 4 hours. The crude product was dissolved in butanol, subsequently washed three times with a K2CO3 aqueous solution and a saturated NaCl solution and dried in vacuo. The crude product was redissolved in a 1 mmol ZnCl2 / MeOH solution. Purification was performed using preparative HPLC to obtain 25.3 mg of a yellow gel-like LC302 (LC008-cyclopentane-Zn). Yield: 54%.

[0334] 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 at room temperature overnight. The crude product was diluted with butanol, extracted three times with saturated NaCl solution, and dried under vacuum. The crude product was then redissolved in 1 mmol of ZnCl2 / MeOH solution. Purification by preparative HPLC gave 10.9 mg of LC014 as a yellow gel. Yield: 12%.

[0335] 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 aqueous KCO 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%.

[0336] 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 aqueous KCO and saturated NaCl solution. Purification by preparative HPLC afforded 41.8 mg of product. Yield: 52%.

[0337] 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 aqueous KCO and saturated NaCl. Purification by preparative HPLC afforded 43.2 mg of product. Yield: 65%.

[0338] 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 aqueous KCO and saturated NaCl. Purification by preparative HPLC afforded 36 mg of product. Yield: 44.8%.

[0339] Synthesis of LC096: LC010 (29.7 mg, 0.05 mmol) was added 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 aqueous KCO and saturated NaCl. Purification by preparative HPLC afforded 46.5 mg of product. Yield: 70%.

[0340] Synthesis of LC149: 1,2-Dibromoethane (259 μL, 3 mmol) and K2CO3 (138 mg, 0.1 mmol) were added to isopsoralea corylifolia chalcone LC003 (64.8 mg, 0.2 mmol) dissolved in 3 mL of acetone. The reaction was maintained under reflux 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 product, was obtained by silica gel column chromatography using an elution gradient (hexane:EtOAc (v / v), 15:1). Yield: 59.1%.

[0341] Synthesis of LC143: LC149 (26.9 mg, 0.05 mmol) was added 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 aqueous KCO and saturated NaCl. Purification by preparative HPLC afforded 7.3 mg of LC143. Yield: 29.4%.

[0342] Synthesis of LC150: LC149 (53.8 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. The crude product was washed with aqueous KCO and saturated NaCl. Purification by preparative HPLC afforded 35.5 mg of LC150. Yield: 68%.

[0343] Synthesis of LC011: 1,4-dibromobutane (99.0 μL, 0.829 mmol) and K2CO3 (83.3 mg, 0.603 mmol) were 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 in vacuo. 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%.

[0344] 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%.

[0345] Synthesis of LC010: 1,4-Dibromobutane (548.5 μL, 4.62 mmol) and K2CO3 (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 in vacuo. 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%.

[0346] 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%.

[0347] Synthesis of LC310: Orcinol (62 mg, 0.5 mmol), 5-bromopentyltrimethylammonium bromide (418 mg, 2 mmol), and KCO (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 three times with saturated NaCl solution. The butanol was removed in vacuo. Purification by preparative HPLC yielded 32 mg of LC310. Yield: 16.8%.

[0348] Synthesis of LC311: LC003 (97 mg, 0.3 mmol), 5-bromopentyltrimethylammonium bromide (289 mg, 1 mmol), and KCO (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 three times with saturated NaCl solution. The butanol was removed in vacuo. Purification by preparative HPLC afforded 44.2 mg of LC311. Yield: 28%.

[0349] Synthesis of LC312: LC003 (97 mg, 0.3 mmol), 3-bromopropyltrimethylammonium bromide (265 mg, 1 mmol), and KCO (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 three times with saturated NaCl solution. The butanol was removed in vacuo. Purification by preparative HPLC yielded 48.5 mg of LC312. Yield: 31%.

[0350] Synthesis of LC315: LC003 (97 mg, 0.3 mmol), 4-bromobutyltriethylammonium bromide (317 mg, 1 mmol), and KCO (272 mg, 2 mmol) were dissolved in 4 mL of DMF. The mixture was stirred at 60°C for 6 hours. After the reaction, diethyl ether was added, and the precipitate was dissolved in methanol and purified by preparative HPLC to obtain 59.1 mg of LC315. Yield: 31%.

[0351] Synthesis of LC316: LC007 (97 mg, 0.3 mmol), 5-bromopentyltrimethylammonium bromide (289 mg, 1 mmol), and KCO (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 three times with saturated NaCl solution. The butanol was removed in vacuo. Purification by preparative HPLC yielded 55.7 mg of LC316. Yield: 35%.

[0352] Synthesis of LC317: LC007 (97 mg, 0.3 mmol), 4-bromobutyltriethylammonium bromide (317 mg, 1 mmol), and KCO (272 mg, 2 mmol) were dissolved in 4 mL of DMF. The mixture was stirred at 60°C for 6 hours. After the reaction, diethyl ether was added, and the precipitate was dissolved in methanol and purified by preparative HPLC to obtain 63 mg of LC317. Yield: 33%.

[0353] Synthesis of LC350: LC003 (97 mg, 0.3 mmol), 3-(4-bromobutyl)-1-methylimidazolium bromide (298 mg, 1 mmol), and KCO (272 mg, 2 mmol) were dissolved in 4 mL of DMF. The mixture was stirred at 50°C for 5 hours. After the reaction, diethyl ether was added, and the precipitate was dissolved in methanol and purified by preparative HPLC to obtain 46.5 mg of LC350. Yield: 25%.

[0354] Synthesis of LC365: LC003 (97 mg, 0.3 mmol), 1-(4-bromobutyl)pyridinium bromide (295 mg, 1 mmol), and KCO (272 mg, 2 mmol) were dissolved in 4 mL of DMF. The mixture was stirred at 50°C for 5 hours. After the reaction, diethyl ether was added, and the precipitate was dissolved in methanol and purified by preparative HPLC to obtain 49.7 mg of LC365. Yield: 28%.

[0355] Synthesis of LC370: LC003 (97 mg, 0.3 mmol), 3-(4-bromobutyl)-1-methylimidazolium bromide (298 mg, 1 mmol), and KCO (272 mg, 2 mmol) were dissolved in 4 mL of DMF. The mixture was stirred at 50°C for 5 hours. After the reaction, diethyl ether was added, and the precipitate was dissolved in methanol and purified by preparative HPLC to obtain 56.1 mg of LC370. Yield: 30%.

[0356] Synthesis of LC375: LC003 (97 mg, 0.3 mmol), 1-(4-bromobutyl)pyridinium bromide (295 mg, 1 mmol), and KCO (272 mg, 2 mmol) were dissolved in 4 mL of DMF. The mixture was stirred at 50°C for 5 hours. After the reaction, diethyl ether was added, and the precipitate was dissolved in methanol and purified by preparative HPLC to obtain 65.1 mg of LC375. Yield: 37%.

[0357] Synthesis of LC005: Ethyl iodoacetate (25.3 μL, 0.213 mmol) and KCO (82.9 mg, 0.60 mmol) were added to LC003 (31.6 mg, 0.097 mmol) in 3 mL of acetone. The reaction was processed similarly to the synthesis of LC004. The pure acetate analog was obtained by silica gel chromatography using an elution gradient (hexane:EtOAc (v / v), 12.5:1) to obtain 42.8 mg. Yield: 88.5%.

[0358] 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 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 Na2SO4 overnight. The solvent was removed in vacuo, and the product was used in subsequent reactions 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. DIC (106 μL, 0.696 mmol) was then added. The mixture was mixed at room temperature overnight. Sample preparation was performed using diethyl ether precipitation before injection. Purification by preparative HPLC yielded 18.3 mg of LC101. Yield: 16.8%.

[0359] 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 hours and then cooled to room temperature. The solvent was removed in vacuo and the product was used in subsequent reactions without further purification. HOBt (35.0 mg, 0.23 mmol) and arginine amide dihydrochloride (61.5 mg, 0.25 mmol) were introduced into the previously dried product dissolved in DMF. DIC (130 μL, 0.85 mmol) and N,N-diisopropylethylamine (71.8 μL, 0.51 mmol) were then added. The mixture was mixed at room temperature overnight. Sample preparation before injection was performed using diethyl ether precipitation. Purification was performed using preparative HPLC to obtain 17.4 mg of LC127. Yield: 42%.

[0360] 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 subsequent reactions without further purification. HOBt (35.0 mg, 0.23 mmol) and arginine amide dihydrochloride (61.5 mg, 0.25 mmol) were introduced into the previously dried product dissolved in DMF. DIC (130 μL, 0.85 mmol) and N,N-diisopropylethylamine (71.8 μL, 0.51 mmol) were then added. The mixture was mixed at room temperature overnight. Sample preparation before injection was performed using diethyl ether precipitation. Purification was performed using preparative HPLC to obtain 13.3 mg of LC131. Yield: 32%.

[0361] 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 subsequent reactions 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. DIC (130 μL, 0.85 mmol) and N,N-diisopropylethylamine (156.8 μL, 0.9 mmol) were then added. The mixture was mixed at room temperature overnight. Sample preparation before injection was performed using diethyl ether precipitation. Purification was performed using preparative HPLC to obtain 46.3 mg of LC137. Yield: 60.8%.

[0362] 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 Na2SO4 overnight. The solvent was removed in vacuo, and the product was used in subsequent reactions without further purification.

[0363] 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. DIC (164 μL, 1.089 mmol) was then added. The mixture was mixed at room temperature overnight. Sample preparation was performed using diethyl ether precipitation before injection. Purification was performed using preparative HPLC to obtain 34.9 mg of LC106. Yield: 20.6%.

[0364] Synthesis of LC012: Ethyl iodoacetate (175 μL, 1.48 mmol) and KCO (208.9 mg, 1.51 mmol) were added to a solution of LC007 (95.8 mg, 0.295 mmol) in 4 mL of acetone. The reaction was processed similarly to the synthesis of LC010. The pure acetate analog LC012 was obtained by silica gel chromatography using an elution gradient (hexane:EtOAc (v / v), 20:1). Yield: 73.5%.

[0365] 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 aqueous KCO and saturated NaCl. Purification by preparative HPLC afforded 22.5 mg of LC140. Yield: 37%.

[0366] Synthesis of LC148: 1,3-Dibromopropane (305 μL, 3 mmol) and K2CO3 (138 mg, 0.1 mmol) were added to isopsoralea corylifolia chalcone LC003 (64.8 mg, 0.2 mmol) dissolved in 3 mL of acetone. The reaction was maintained under reflux 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%.

[0367] 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. The crude product was washed with aqueous KCO and saturated NaCl. Purification by preparative HPLC afforded 34.6 mg of LC204. Yield: 63.1%.

[0368] Synthesis of LC206: To a solution of LC148 (56.6 mg, 0.1 mmol) in 3 mL of DMF was added cyclopentane (86 mg, 0.5 mmol). The reaction mixture was stirred at room temperature for 5 hours. The crude product was dissolved in butanol, subsequently washed three times with aqueous KCO and saturated NaCl solution and dried under vacuum. Purification by preparative HPLC yielded 26.2 mg of LC2O6 as a yellow gel. Yield: 35%.

[0369] Synthesis of LC013: Ethyl iodoacetate (341.4 μL, 2.88 mmol) and KCO (252.3 mg, 1.83 mmol) were added to a solution of LC008 (92.4 mg, 0.361 mmol) in 4 mL of acetone. The reaction was processed similarly to the synthesis of LC011. The pure acetate analog LC013 (112.7 mg) was obtained by silica gel chromatography using a gradient of Hex:EtOAc (v / v), 10:1. Yield: 73.1%.

[0370] 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 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 Na2SO4 overnight. The solvent was removed in vacuo, and the product was used in subsequent reactions 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 at room temperature overnight. Sample preparation was performed using diethyl ether precipitation before injection. Purification was performed using preparative HPLC to obtain 8.4 mg of LC108. Yield: 18.7%.

[0371] Example 8: Biological Studies

[0372] in vitro

[0373] In Gram-negative bacteria, both the outer and inner membranes are lipid bilayers. Each bilayer consists of a hydrophobic region of the lipid tail and two head groups facing the aqueous solution, forming a sandwich structure. In order to disrupt the bacterial outer and inner membranes, many bola-type amphiphilic compounds composed of two cationic end groups and a hydrophobic scaffold were synthesized. The cationic end groups and the hydrophobic scaffold can interact with the two anionic head groups and the lipid tail region respectively ( Figure 4 The hydrophobic scaffold was 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 amines ( Figure 8 ). As a result, a library of compounds was synthesized by assembling a hydrophobic core and a cationic group.

[0374] The antimicrobial activity and synergistic effects of various 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 for colistin was 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.

[0375] Table 1a. MIC (μg / ml) of DPA-Zn compounds against a panel of colistin-resistant strains.

[0376]

[0377] Table 1b. MIC (μg / ml) of amine compounds against a panel of colistin-resistant strains.

[0378]

[0379] Table 1c. MIC (μg / ml) of amine compounds against a panel of colistin-resistant strains.

[0380]

[0381] Table 1d. MIC (μg / ml) of amine compounds against a panel of colistin-resistant strains.

[0382]

[0383] Table 1e. MICs (μg / ml) of LC137, LC140, LC143, LC150, LC204, and LC206 against a panel of colistin-resistant strains.

[0384]

[0385] In addition, due to some other mechanisms of sensitivity to colistin combinations, other E. coli are also resistant to colistin, but do not have the mcr-1 mutation, referred to as mcr-1(-). Most of the compounds of the present invention showed antimicrobial activity against these mcr(-) resistant strains or acted synergistically with the colistin combination (Tables 2a-2e). This indicates that the combination of the present invention works by similar mechanisms against mcr-1(-) and mcr-1(+) bacteria.

[0386] Table 2a. Colistin MIC (μg / ml) against a panel of colistin-resistant strains in the presence of DPA-Zn compounds.

[0387]

[0388] Table 2b. Colistin MICs (μg / ml) against a panel of colistin-resistant strains in the presence of amine analogs.

[0389]

[0390] Table 2c. Colistin MIC (μg / ml) against a panel of colistin-resistant strains in the presence of amine analogs.

[0391]

[0392] Table 2d. Colistin MICs (μg / ml) against a panel of colistin-resistant strains in the presence of amine analogs.

[0393]

[0394] Table 2e. Colistin MIC (μg / ml) against a panel of colistin-resistant strains in the presence of LC137, LC140, LC143, LC150, LC204, LC206.

[0395]

[0396] Four classes of compounds were developed. The first class of compounds were DPA derivatives in which two DPA-Zn moieties were linked to a selected hydrophobic scaffold via two linker groups. The MICs and synergistic activities are shown in Tables 1a and 2a. The first proof-of-concept compound was Orc-DPA, which showed synergy with colistin against mcr-1 bacteria at 50 μg / ml. In contrast, EDTA only worked at concentrations above 250 μg / ml. The hydrophobic scaffold was then optimized and a number of DPA derivatives were synthesized. One compound, GLA-DPA, synergized with colistin at 5 μg / ml.

[0397] The second class of compounds is 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, and LC105. Most of these compounds showed synergy with colistin against mcr-1 bacteria, with the results summarized in Tables 1b-1e and 2b-2e. One of these compounds, LC100, showed no activity when used alone but exhibited excellent synergy with 2 μg / ml colistin. LC100 has been selected as a lead compound for in vivo and biophysical studies, which are discussed in further detail below.

[0398] The third class of compounds is quaternary amine analogs, including LC311, LC312, LC315, LC316, LC317, LC350, LC365, LC370, and LC375. The corresponding MIC and synergistic data are shown in Tables 1b-1d and 2b-2d. Compared with amine and polyamine analogs, quaternary amine analogs not only act synergistically with colistin but also exhibit antimicrobial activity when used alone.

[0399] The fourth class of compounds is guanidine analogs, including LC101, LC137, LC131, LC106, LC127, and LC108. Two compounds, LC101 and LC137, developed using isopsoralea corylifolia chalcone (LC003) as a hydrophobic core, showed excellent synergistic effects with colistin. Furthermore, LC101 and LC137 also exhibited moderate antimicrobial activity (MIC range of 12.5-25 μg / ml) when used alone.

[0400] Example 9: LC100

[0401] To further test the antimicrobial activity and synergistic activity of the compounds of the present invention with colistin, LC100 was selected as a model compound to further examine the antimicrobial spectrum and understand the mechanism of action. This is because LC100 is inactive alone but exhibits excellent synergistic activity with colistin. Synergy was first tested using varying concentrations of LC100. Table 3 shows that synergy becomes stronger with increasing LC100 concentrations. LC100 concentrations greater than 5 μg / ml can reduce the colistin MIC to as low as 0.0975 μg / ml, a concentration at which colistin is significantly less toxic. Next, LC100 was examined for synergistic activity with polymyxin B (PMB), a peptide that functions in a manner similar to colistin but differs from it by one residue. The data in Table 4 demonstrate that this combination is equally effective against polymyxin B. Table 5 extends the efficacy of this combination to three multidrug-resistant forms of Gram-negative bacteria: Acinetobacter baumannii (ACBA), Klebsiella pneumoniae (KLPN), and Escherichia coli cloacae. It should be noted that the KLPN strain was identified as a carbapenem-resistant Enterobacteriaceae (CRE) by genetic analysis. The results indicate that the combination is effective against these carbapenem-resistant and multidrug-resistant strains.

[0402] It should also be noted that the combination is effective against a wide range of bacteria without having to vary the drug concentration depending on the bacterial species. The only other way to achieve this is with disinfectants, such as alcohol, which are known to be toxic.

[0403] Table 3. Colistin MIC (μg / ml) against a panel of colistin-resistant strains in the presence of different concentrations of LC100

[0404]

[0405] Table 4. Synergistic effect of LC100 and colistin against colistin-sensitive strains.

[0406]

[0407] Table 5. Synergistic effect of LC100 and colistin against multidrug-resistant strains

[0408]

[0409] The combination of LC100 and colistin was found to be able to kill bacteria such as E. coli with mcr-1 that is resistant to colistin and other antibiotics, E. coli without mcr-1 that is resistant to colistin and other antibiotics, Pseudomonas aeruginosa that is resistant to carbapenems, aminoglycosides, and other antibiotics, Acinetobacter baumannii that is resistant to carbapenems, colistin, and other antibiotics, and Klebsiella pneumoniae that is resistant to carbapenems, colistin, and other antibiotics. Table 3 shows that the amount of colistin required to kill bacteria changes at different concentrations of LC100, 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 the fact that this phenomenon is true across a wide range of bacteria.

[0410] To understand the effects 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 resulted in a 3-log reduction within 2 hours. In the presence of 10 μg / ml LC100, colistin at 1 / 8 the MIC achieved the same killing effect (in terms of a 3-log reduction) within 2 hours. The rapid killing kinetics suggest that the combination of LC100 and colistin acts on the bacterial membrane.

[0411] 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. B2088 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 1 μg / ml level in killing E. coli with mcr-1 mutations, thereby increasing the activity by more than 10-fold. Figure 16As shown in Table 6. The combination of LC100 and colistin was further tested for activity against a wide range of bacterial strains (Table 7). Table 7 covers a wide range of bacteria, many of which are highly resistant to antibiotics. It appears that the combination of 1 μg / ml colistin and 5 μg / ml LC100 is effective against all pathogens except Klebsiella pneumoniae (KLPN). This combination also appears to be safe for use.

[0412] The antimicrobial activity of the combination of the present invention was also tested against Gram-positive bacteria, including multidrug-resistant strains such as MRSA. Table 8 shows that LC100 or LC101 alone exhibited good activity against Gram-positive bacterial strains, indicating that LC100 or LC101 alone has a strong effect on the bacterial inner membrane, as Gram-positive bacteria lack an outer membrane. This means that the combination of LC100 / LC101 and colistin has a broad antimicrobial spectrum, active against both Gram-positive and Gram-negative bacteria.

[0413] Table 6. Synergistic study results of LC100 or LC101 and B2088

[0414]

[0415] Table 7. Pathogen sensitivity of the compounds of the present invention as colistin adjuvants in various pathogens

[0416]

[0417] Table 7. Continued

[0418]

[0419] Table 7. Continued

[0420]

[0421] Table 8. Antimicrobial effects of LC100 and LC101 against Gram-positive bacteria.

[0422] MCR1 strain LC100 LC101 SA ATCC 29213 3.125 3.125 SA ATCC 6538 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

[0423] Example 10: Mechanism of Action of LC100 and LC101 (with mcr-1)

[0424] The compounds of the present invention are believed to be adjuvant compounds that enhance the effect of colistin against Gram-negative bacteria, particularly E. coli with the mcr-1 mutation, because these compounds act synergistically, 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 over 6 μg / ml to less than 0.5 μg / ml. At 6 μg / ml, colistin is considered unsafe for use due to concentration-dependent side effects. Synergistic effects with these two compounds have also been observed against other forms of resistant Gram-negative bacteria, such as E. coli, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0425] Although mcr-1 bacteria are resistant to colistin, reports indicate that colistin can still disrupt the outer membrane of mcr-1 bacteria. It is important to note that disruption of the outer membrane alone does not directly lead to cell death; the inner membrane must be affected, causing a loss of transmembrane potential and allowing water to enter the bacteria. Therefore, it is hypothesized that in the case of a combination of LC100 and colistin, colistin and LC100 work together to disrupt the outer membrane, allowing LC100 to reach the inner membrane. Because LC100 has a strong effect on the inner membrane, bacterial cell death occurs due to inner membrane rupture.

[0426] To test this hypothesis, we performed fluorescence experiments using the dye molecule EtBr, which fluoresces strongly when bound to DNA. However, EtBr is membrane-impermeable, meaning it can only enter bacterial cells and bind to DNA, and it can only fluoresce when both the outer and inner bacterial membranes are disturbed. Figure 17 The results showed that LC100 and colistin induced only mild fluorescence when used alone, indicating that neither compound disrupted the bacterial outer membrane. However, in the presence of LC100 and colistin combined, strong fluorescence was observed, indicating that both the outer and inner membranes of mcr-1 bacteria were disrupted, confirming the hypothesis that the combined action of LC100 and colistin is on the bacterial membrane. LC100 may also have 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 various concentrations of LC100. The MCR-1 protein contains several tryptophan residues and emits autofluorescence at 323 nm. Figure 18 The fluorescence quenching effect showed that the fluorescence decreased with the increase of LC100 concentration, indicating that there was a strong interaction between MCR-1 protein and LC100.

[0427] Colistin and polymyxin B can interact with the phosphate groups and disrupt the head group, thereby creating a cavity that exposes the lipid tail of the membrane to the aqueous phase. For example, in the presence of cationic polymyxin B, a colistin analog, the hydrophobic surface area of ​​the bacterial membrane increases ( Figure 19), thereby allowing hydrophobic association between the lipid tail of polymyxin B and the lipid tail of the bacterial outer membrane, leading to resistance to colistin.

[0428] It is hypothesized that the efficacy of the combination of LC100 and colistin arises from the ability of the cationic groups 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 appears to be sufficient for the LC100 molecule to "drill" through the outer membrane, reach the inner membrane, and kill the bacteria. Microbiological data suggest that the concentration of colistin required for this synergistic effect can be reduced by 4-100 times compared to the amount required for colistin to be effective on its own. The observation that LC100 disrupts the lipid bodies that mimic 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 form cavities that facilitate the diffusion of LC100 into the inner membrane.

[0429] Example 11: Mechanism of Action of LC100 and LC101 (without mcr-1)

[0430] E. coli is a favorite host of mcr-1 and is often resistant to colistin in the absence of mcr-1 mutations. It has been proposed that this may be due to the presence of arabinose. The sugar molecule has several hydroxyl groups that can also participate in hydrogen-bonding networks to stabilize bacterial membranes, leading to colistin resistance. As mentioned above, when the combination of LC100 and colistin was tested against several resistant bacteria without mcr-1, E. coli was found to be very sensitive to low concentrations of colistin in the presence of LC100, down to concentrations of <0.5 μg / ml. The mechanism of action may be similar to that proposed above for bacteria with mcr-1, where cationic colistin may temporarily disrupt the intramembrane hydrogen-bonding network, allowing the transient appearance of a hydrophobic cavity in the membrane. This, in turn, facilitates the entry of LC100, with its strongly hydrophobic core, into the fatty acid layer of lipid A and subsequently downward to disrupt the bacterial inner membrane.

[0431] Carbapenem-resistant bacteria have also been found to be susceptible to the combination of LC100 and colistin. Carbapenem resistance is caused by mutations in bacterial cytoplasmic biosynthetic enzymes. These bacteria have variable sensitivity to colistin. It is likely that in the presence of the combination of LC100 and colistin, the interaction of colistin with the outer membrane facilitates the entry of LC100 into the inner membrane, resulting in LC100's ability to kill the bacteria.

[0432] Example 12: In vivo studies of LC100

[0433] To evaluate the in vivo efficacy of the LC100 and colistin combination, a neutropenic mouse thigh infection model was used. Thirty mice were divided into five groups: control, 10 mg / kg colistin, 50 mg / kg LC100, 100 mg / kg meropenem, and a combination of 50 mg / kg LC100 and 10 mg / kg colistin. Each treatment was administered intraperitoneally 1 hour after infection. The initial inoculum was 1.87 x 10 6 , 6 h after treatment, mouse thigh tissues were homogenized to obtain viable CFU. Figure 21 LC100 or colistin alone were unable to reduce bacterial load, while the combination resulted in a 1.2-log reduction within 6 hours. Because 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 mice's immune system. As a control, meropenem showed more significant activity (a 2-log reduction) due to the high concentration and penem-susceptibility of the bacterial strain.

[0434] Industrial Applicability

[0435] The disclosed compounds can be used to interact with and disrupt bacterial membranes. The disclosed compounds can effectively kill Gram-positive bacteria when administered alone or in combination with colistin. The disclosed compounds can effectively kill MCR-positive Gram-negative bacteria when administered alone or in combination with colistin. The disclosed compounds can also effectively kill Gram-negative bacteria that are MCR-negative but still resistant to colistin when administered in combination with colistin. In addition, the disclosed compounds can effectively kill a wide range of Gram-negative bacteria, including bacteria resistant to carbapenems, when administered in combination with colistin.

[0436] 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.

[0437] The disclosed compounds can be used to kill or inhibit the growth of microorganisms on surfaces, including but not limited to topical application, in ointments, eye drops, nasal sprays, mouthwashes and hand sanitizers. The disclosed compounds can also be used as food preservatives, disinfectants, surface cleaners or medical devices.

[0438] The disclosed methods for preparing the disclosed compounds can be facile, employ mild reaction conditions, and facilitate low-cost and large-scale synthesis of the compounds.

[0439] 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 invention, and it is therefore intended that all such modifications and adaptations fall within the scope of the appended claims.

Claims

1. A compound having the following formula (I): Z 1 -L 1 -A-L 2 -Z 2 Formula (I) Where A is selected from and any mixture thereof, wherein the broken bond represents the position at which the structure is attached to the remainder of formula (I); L 1 and L 2 are independently linkers; and Z 1 and Z 2 are independently N-containing moieties.

2. The compound according to claim 1, wherein Z 1 and Z 2 The pKa values ​​of the compounds are independently greater than 9.

3. The compound according to claim 1 or 2, wherein Z 1 and Z 2 Independently has the following structure: wherein the bond break represents the position at which the structure is attached to the remainder of formula (I); R 1 and R 2 R is independently 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 heterocyclic ring; and R 3 is absent or is hydrogen or 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, When R 3 When present, the nitrogen atom is a cationic tetravalent nitrogen.

4. The compound according to claim 3, wherein R 1 、R 2 and R 3 independently comprising a functional group 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, carbamoylphosphonate, oxazoline, pyrimidinetrione, 1-hydroxy-2(1H)-pyridinone (1,2-HOPO), and any combination thereof, and / or where R 1 、R 2 and R 3 independently selected from methyl, ethyl, propyl, butyl, -(CH2) x NR'R", -(CH2) x OH, -(CH2) x PO3, -(CH2) x CR'R"R"', -(C(NH2)NHC(NH2))x-NH2, guanidine, 2-methylpyridine, 1-methylimidazole, pyridine, bipyridine, terpyridine, phenanthroline, 3-methylpyrrole, cyclamen (1,4,7,10-tetraazacyclododecane), cyclamen (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, wherein x may be any integer from 1 to 10, and R', R" and R'" are independently hydrogen or substituted or unsubstituted alkyl, and / or where Z 1 and Z 2 Independently selected from: and any combination thereof.

5. A compound according to any one of the preceding claims, wherein L 1 and L 2 are independently substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, ester, amide, ether, -(O-CH2-CH2-O) n -、L 3 or any combination thereof, wherein L 3 Has the following structure: Where n is an integer from 1 to 10, R 4 is amino or heteroalkyl, and the bond break indicates the position at which the structure is attached to the remainder of Formula (I); Preferably, L 1 and L 2 Independently selected from -(CH2)4-, amide, and any combination thereof, or 6. A compound according to any one of the preceding claims, wherein the compound of formula (I) has a logP value greater than 4; and / or The compound of formula (I) has the following structure: and / or wherein each N-containing moiety independently chelates zinc, and / or wherein each N-containing moiety independently has a positive charge.

7. A pharmaceutical composition comprising the compound according to any one of the preceding claims or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

8. A method for preparing the compound according to any one of claims 1 to 6, comprising the following steps: 1) contacting a hydrophobic moiety with a linker under reaction conditions, wherein the hydrophobic moiety is selected from: 2) contacting the linker with the N-containing moiety under reaction conditions.

9. The method of claim 8, wherein a covalent bond is formed between the hydrophobic portion and the linker, or between the linker and the N-containing portion; and / or wherein the linker is 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, and / or wherein the N-containing moiety is selected from the group consisting of dipyridylamine, cyclopentane, 1,8-dimethyl-1,4,8,11-tetracyclotetradecane, diethylamine, -NH[(CH2)3N(CH3)]2, -NH2(CH2)3N(CH3)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; and / or wherein the method comprises the step of adding zinc after the N-containing moiety is covalently bonded to the linker or hydrophobic moiety; and / or wherein said contacting is carried out in a solvent selected from the group consisting of acetone, methanol, ethanol, propanol, butanol, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, pyridine, water and any mixture thereof; and / or wherein the contacting step is performed at a temperature of about 21°C to about 160°C; and / or wherein the contacting step is performed for a duration of about 2 hours to about 36 hours.

10. Use of the compound according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 for killing microorganisms or inhibiting microbial growth in vitro, Preferably, the compound according to any one of claims 1 to 6 is used in combination with colistin.

11. A compound according to any one of claims 1 to 6 for use in therapy.

12. Use of a compound according to any one of claims 1 to 6 in the preparation of a medicament for treating bacterial infection, Preferably, the compound according to any one of claims 1 to 6 is administered in combination with colistin.

13. The use according to claim 12, wherein the compound according to any one of claims 1 to 6 is present in an amount of about 2 μg / mL to about 75 μg / mL, and / or wherein the compound according to any one of claims 1 to 6 and colistin are present in equal amounts by weight, or the compound according to claims 1 to 6 is present in an excess of about 1.5 to about 6 times by weight of colistin, Preferably wherein colistin is present in the range of about 1 mg / kg to about 10 mg / kg and the compound according to any one of claims 1 to 6 is present in the range of 10 mg / kg to about 50 mg / kg.

14. Use according to claims 12 and 13, wherein the compound according to claims 1 to 6 is or is to be administered intramuscularly, intraperitoneally, topically, subcutaneously or intravenously.

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