A novel compound and methods and uses thereof

Abstract

The present invention relates to compounds of formula (I), to methods of their use for inhibiting the efflux action of efflux pumps of microorganisms, and to the use as efflux pump inhibitors, wherein the symbols in formula (I) are defined as described in the description,

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemical drugs, in particular, to a new kind of dipeptide-like compound, their use and purposes, especially as efflux pump inhibitors. BACKGROUND

[0002] The multi-drug resistance of pathogenic bacteria is a very thorny problem in current clinical infections, one of the common causes of which is the efflux of various drugs by the efflux pump system of bacteria. Multi-drug resistant efflux pumps exist widely in various pathogenic bacteria. For bacteria, efflux genes account for 6%-18% of all transport proteins. The overexpression of the efflux system dominates the occurrence of bacterial drug resistance, so it is necessary to develop a new compound which can inhibit the efflux of the efflux pump of microorganisms, especially bacteria, so as to reduce the efflux of the microorganisms to drugs. This has important significance for improving the antibacterial activity of antibacterial drugs and improving the current situation of clinical treatment of bacterial infectious diseases. SUMMARY

[0003] The present application provides a new compound (also known as dipeptide-like compound) as an efflux pump inhibitor (EPI), thereby reducing the efflux capacity of microorganisms and reducing the drug resistance of microorganisms.

[0004] The present application provides a compound of formula (I), or a stereoisomer, solvate or pharmaceutically acceptable salt thereof,

[0005]

[0006] wherein,

[0007] X is CH or N,

[0008] n, m are each independently an integer from 0 to 3,

[0009] Q is hydrogen, (C1-C4)-alkyl,

[0010] R1 is guanidino, amino, (C6-C 14 )-aryl, said (C6-C 14 )-aryl being optionally substituted by one or more substituents selected from (C1-C6)-alkyl, (C1-C6)-alkoxy, halo-(C1-C6)-alkyl, halogen, carboxyl, (C1-C6)-alkylacyloxy, (C1-C4)-alkyloxycarbonyl, (C6-C 14 )-aryl-(C1-C4)-alkoxy, cyano, nitro, or

[0011] R1, Q and -(CH2) mCH2CHN- moieties together form a (C6-C 14 )-heteroaryl, wherein R1 is -(C6-C 14 )-arylene, the heteroatoms comprising at least nitrogen atoms, Q is (C1-C4)-alkylene;

[0012] W is (C6-C 14 )-heteroaryl, (C1-C4)-alkyl-(C6-C 14 )-heteroaryl; or is selected from one of the following formulae:

[0013]

[0014] wherein Ar1, Ar2, Ar3 are each independently (C6-C 14 )-aryl, said (C6-C 14 )-aryl is optionally substituted by one or more substituents selected from (C1-C6)-alkyl, (C1-C6)-alkoxy, halo-(C1-C6)-alkyl, halogen, carboxyl, (C1-C6)-alkylacyloxy, (C1-C6)-alkyloxycarbonyl, (C6-C 14 )-aryl-(C1-C4)-alkoxy, cyano, nitro; and

[0015] In case W is of formula (I-3), n is an integer from 1 to 3;

[0016] The wavy line indicates the point of attachment to the remainder of formula (I).

[0017] Furthermore, the present application also provides a method for inhibiting the efflux action of an efflux pump of a microorganism, by using a compound of formula (I) according to the present application.

[0018] In addition, the present application also provides the use of a compound of formula (I) as an efflux pump inhibitor.

[0019] Surprisingly, it has been found that the present application, by inserting a carbon atom in the dipeptide backbone (i.e. the amino group and the carbonyl group are attached to different carbon atoms, in particular to two adjacent carbon atoms), and / or by introducing a nitrogen-containing heteroaryl group (e.g. tetrahydroisoquinolinyl, indolyl) in the dipeptide backbone, leads to a new compound of formula (I) which has the effect of reducing the efflux capacity of microorganisms and can be used as an efflux pump inhibitor. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Hoechst-33342 accumulation curves over time are shown in the presence of 64 μg / mL of the compound 4g according to the present application, PAβN (positive control), and in the absence of EPI (blank, i.e. without efflux pump inhibitor), respectively.

[0021] Figure 2 Hoechst-33342 accumulation curves over time in the presence of 64 μg / mL of the compound of the application 4h, PAβN (positive control) and no EPI (blank), respectively.

[0022] Figure 3 Hoechst-33342 accumulation curves over time in the presence of 32 μg / mL of the compound of the application 4h, PAβN (positive control) and no EPI (blank), respectively.

[0023] Figure 4 Hoechst-33342 accumulation curves over time in the presence of 16 μg / mL of the compound of the application 4h, PAβN (positive control) and no EPI (blank), respectively.

[0024] Figure 5 Hoechst-33342 accumulation curves over time in the presence of 64 μg / mL of the compound of the application 4p, PAβN (positive control) and no EPI (blank), respectively.

[0025] Figure 6 Hoechst-33342 accumulation curves over time in the presence of 32 μg / mL of the compound of the application 4p, PAβN (positive control) and no EPI (blank), respectively.

[0026] Figure 7 Hoechst-33342 accumulation curves over time in the presence of 16 μg / mL of the compound of the application 4p, PAβN (positive control) and no EPI (blank), respectively.

[0027] Figure 8 Hoechst-33342 accumulation curves over time in the presence of 64 μg / mL of the compound of the application 4r, PAβN (positive control) and no EPI (blank), respectively.

[0028] Figure 9 Hoechst-33342 accumulation curves over time in the presence of 64 μg / mL of the compound of the application 8b, PAβN (positive control) and no EPI (blank), respectively.

[0029] Figure 10 Hoechst-33342 accumulation curves over time in the presence of 64 μg / mL of the compound of the application 8c, PAβN (positive control) and no EPI (blank), respectively.

[0030] Figure 11 Hoechst-33342 accumulation curves over time in the presence of 64 μg / mL of the compound 8d of the present application, PAβN (positive control), and no EPI (blank), respectively.

[0031] Figure 12 Hoechst-33342 accumulation curves over time in the presence of 64 μg / mL of the compound 8e of the present application, PAβN (positive control), and no EPI (blank), respectively.

[0032] Figure 13 Hoechst-33342 accumulation curves over time in the presence of 32 μg / mL of the compound 8e of the present application, PAβN (positive control), and no EPI (blank), respectively.

[0033] Figure 14 Hoechst-33342 accumulation curves over time in the presence of 64 μg / mL of the compound 8g of the present application, PAβN (positive control), and no EPI (blank), respectively.

[0034] Figure 15 Hoechst-33342 accumulation curves over time in the presence of 32 μg / mL of the compound 8g of the present application, PAβN (positive control), and no EPI (blank), respectively.

[0035] Figure 16 Hoechst-33342 accumulation curves over time in the presence of 64 μg / mL of the compound 15c of the present application, PAβN (positive control), and no EPI (blank), respectively.

[0036] Figure 17 Hoechst-33342 accumulation curves over time in the presence of 32 μg / mL of the compound 15c of the present application, PAβN (positive control), and no EPI (blank), respectively. DETAILED DESCRIPTION

[0037] GENERAL DEFINITIONS

[0038] The compounds of formula (I) of the present application and their stereoisomers, solvates and pharmaceutically acceptable salts, as well as the subgroups of compounds: compounds of formulae (II-1) to (II-4), compounds of formulae (III-1) to (III-4), compounds of formulae (IV-1) to (IV-4), compounds of formulae (V-1) to (V-4), are sometimes also referred to as "compounds of the present application" or "dipeptide mimetic compounds of the present application".

[0039] The terms "optionally", "optional" or "optionally" as used herein mean that the event, circumstance or material that follows can or can not occur or be present, and such description includes situations where the event, circumstance or material occurs or is present and situations where the event, circumstance or material does not occur or is not present.

[0040] The terms "comprise", "comprising", and "comprises" and "comprising" as used herein are to be construed as "including, but not limited to", and are not intended to exclude, for example, additional, optional, components, integers or steps.

[0041] In the present application, "microorganism" has the meaning commonly known in the art and includes bacteria, viruses and fungi, in particular bacteria, such as Gram-positive bacteria, Gram-negative bacteria and the like.

[0042] In the context of the present application, the term "alkyl", alone or in combination with other terms, such as haloalkyl, alkylcarboxy, alkylheteroaryl, is to be understood as meaning a saturated aliphatic hydrocarbon group, which can be branched or unbranched. Examples of (Ci-C6)-alkyl are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, i-pentyl, neopentyl, t-pentyl, 1-methylbutyl, 2-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl and hexyl. Of these alkyl groups, (Ci-C4)-alkyl is particularly preferred.

[0043] In the present application, the term "alkoxy", alone or in combination with other terms, such as arylalkyloxy, is to be understood as meaning alkyl-O-, wherein the term "alkyl" is as defined above. In particular, the alkyl groups here can be straight-chain or branched. Examples of (Ci-C6)-alkyl are methoxy, ethoxy, propyloxy, 1-methylethoxy, butyloxy, 1-methylpropyloxy, 2-methylpropyloxy, 1,1-dimethylethoxy. Of these alkoxy groups, (Ci-C4)-alkoxy is particularly preferred.

[0044] In the present application, the term "aryl" is to be understood as meaning an aromatic group having 6 to 14 carbon atoms (also referred to as (C6-Ci4)-aryl), preferably phenyl, biphenyl, naphthyl, anthryl or phenanthryl, more preferably diphenyl, phenyl, naphthyl. 14 )-aryl), preferably phenyl, biphenyl, naphthyl, anthryl or phenanthryl, more preferably diphenyl, phenyl, naphthyl.

[0045] In the present application, the term "halo-(Ci-C6)-alkyl" is to be understood as meaning an alkyl group which is substituted by one or more halogens, wherein the halogen is selected from fluorine, chlorine, bromine, iodine, the number of halogen substituents being 1 to the maximum number of substituents possible; examples thereof are CH2F, CHF2, CF3, CH3CH2F, CH2FCH2F.

[0046] In the present application, the term "alkylcarbonyloxy" (alkyl-C(=0)-0-) is to be understood as an alkyl group which is bound to the skeleton via a -C(=0)-0- group, for example (C1-C6)-alkylcarbonyloxy, (C1-C4)-alkylcarbonyloxy. Herein, the number of carbon atoms relates to the alkyl group in the alkylcarbonyloxy group.

[0047] In the present application, the term "alkylcarbonyloxy" (alkyl-C(=0)-0-) is to be understood as an alkyl group which is bound to the skeleton via a -C(=0)-0- group, for example (C1-C6)-alkylcarbonyloxy, (C1-C4)-alkylcarbonyloxy. Herein, the number of carbon atoms relates to the alkyl group in the alkylcarbonyloxy group.

[0048] In the present application, the term "arylalkyloxy" is to be understood as an aryl group which is bound to the skeleton via an oxygen atom in the alkyloxy group, for example (C6-C 14 )-aryl-(C1-C4)-alkyloxy.

[0049] In the present application, the term "heterocyclyl" is to be understood as a saturated monocyclic ring having carbon atoms and at least one heteroatom in the ring. Preferably, the heterocyclyl contains 4, 5, 6 or 7 carbon atoms and 1 or 2 heteroatoms selected from oxygen and nitrogen. Examples of heterocyclyl are azetidinyl, azetidinyl, azetidinyl, oxetanyl, oxetanyl, oxetanyl, dioxanyl, and tetrahydrofuranyl. Among these heterocyclyls, (C5-C8)-heterocyclyl is preferred, particularly (C5-C6)-heterocyclyl is preferred.

[0050] In the present application, the term "heteroaryl" - by itself or in combination with other terms, for example alkylheteroaryl - denotes a monocyclic, bicyclic or tricyclic heterocyclic radical of carbon atoms and at least one heteroatom, wherein at least one ring is aromatic; examples thereof are tetrahydroisoquinolinyl, indolyl, tetrahydroquinolinyl, thienyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, tetrazolyl, benzofuranyl, benzisofuranyl, benzothienyl, benzoisothienyl, isoindolyl, indazolyl, benzothiazolyl, benzoisothiazolyl, benzoxazolyl, benzoisoxazolyl, benzoimidazolyl, 2,1,3-benzoxadiazole, quinolinyl, isoquinolinyl. Among these heteroaryls, (C6-C 14 )-heteroaryl is preferred, particularly (C6-C12 )-heteroaryl.

[0051] In the present application, the term "alkylheteroaryl" is to be understood as a heteroaryl group attached to the backbone via an alkyl group, for example (Ci-C4)-alkyl-(C6-Ci0)-heteroaryl. 14 )-heteroaryl.

[0052] When a structure "is substituted by one or more substituents", this includes in each case the simultaneous substitution by one or more identical and / or different groups.

[0053] The term "solvate" refers to a form of a compound or salt thereof associated with a solvent, usually formed by a solvolysis reaction. This physical association can include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether and the like. The compounds described herein can be prepared, for example, in crystalline form, and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. In some cases, the solvate will be capable of isolation, for example, where one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" includes both solution-phase and isolatable solvates. Representative solvates include hydrates, ethanolates and methanolates.

[0054] If a compound can exist in one of more tautomeric forms, the disclosure includes all tautomeric forms of the compounds of formula (I) unless otherwise indicated under the particular tautomer being considered. For example, many carbonyl compounds can exist in either the keto or enol form, and both the keto and enol forms are included in the definition of the compounds of formula (I).

[0055] Depending on the nature and the way they are attached, the compounds of formula (I) can exist as stereoisomers. The possible stereoisomers, such as enantiomers, diastereomers, Z and E isomers, defined by their particular three-dimensional shape, are included in formula (I). For example, if one or more alkenyl groups are present, diastereomers (Z and E isomers) can occur. For example, if one or more asymmetric carbon atoms are present, enantiomers and diastereomers can occur. The stereoisomers can be obtained from mixtures obtained in the preparation by conventional separation methods. Chromatographic separation can be performed on an analytical scale to find enantiomeric or diastereomeric excess, or on a preparative scale to produce test samples for biological testing. The stereoisomers can likewise be prepared selectively by using stereoselective reactions with the use of optically active starting materials and / or auxiliaries. The present application therefore also relates to all stereoisomers embraced by formula (I) or its intermediates but not shown in its particular stereoisomeric form, and mixtures thereof.

[0056] According to the present application, the term "efflux pump" refers to a protein transport system of bacteria that expels intracellular drugs or toxic substances out of the cell, which requires proton exchange or hydrolysis of ATP to provide energy in the process of efflux. The efflux pump includes the resistance nodulation division Family (RND).

[0057] In the present application, all operations are carried out at room temperature and normal pressure, unless otherwise specified.

[0058] Pseudo-dipeptide compound of the present application

[0059] The present application provides a compound of formula (I), or a stereoisomer, solvate or pharmaceutically acceptable salt thereof,

[0060]

[0061] wherein,

[0062] X is CH or N,

[0063] n, m are each independently an integer from 0 to 3,

[0064] Q is hydrogen, (C1-C4)-alkyl,

[0065] R1 is guanidino, amino, (C6-C 14 )-aryl, said (C6-C 14 )-aryl being optionally substituted by one or more substituents selected from (C1-C6)-alkyl, (C1-C6)-alkoxy, halo-(C1-C6)-alkyl, halogen, carboxyl, (C1-C6)-alkylacyloxy, (C1-C4)-alkyloxycarbonyl, (C6-C 14 )-aryl-(C1-C4)-alkoxy, cyano, nitro, or

[0066] R1, Q and the -(CH2) m CH2CHN- moiety together form a (C6-C 14 )-heteroaryl, wherein R1 is -(C6-C 14 )-arylene, the heteroatoms comprising at least a nitrogen atom, Q is (C1-C4)-alkylene;

[0067] W is (C6-C 14 )-heteroaryl, (C1-C4)-alkyl-(C6-C 14 )-heteroaryl; or is selected from one of the following formulae:

[0068]

[0069] wherein Ar1, Ar2, Ar3are each independently (C6-C 14 )-aryl, which (C6-C 14 )-aryl is optionally substituted by one or more substituents selected from (C1-C6)-alkyl, (C1-C6)-alkoxy, halo-(C1-C6)-alkyl, halogen, carboxyl, (C1-C6)-alkylcarbonyloxy, (C1-C6)-alkyloxycarbonyl, (C6-C 14 )-aryl-(C1-C4)-alkoxy, cyano, nitro; and

[0070] n is an integer from 1 to 3, in case W is of formula (I-3);

[0071] the wave symbol indicates the point of attachment to the remainder of formula (I).

[0072] In a preferred embodiment, the compounds of the present application are compounds of formula (I), wherein

[0073] X is CH or N,

[0074] n is 0 or 1,

[0075] m is an integer from 0 to 2,

[0076] Q is hydrogen, methyl or ethyl,

[0077] R1is guanidino, amino, phenyl, naphthyl, which phenyl is optionally substituted by one or more substituents selected from tert-butyl, methoxy, trifluoromethyl, benzyloxy, cyano, methyl, fluorine, chlorine, bromine, iodine, nitro, carboxyl; or

[0078] R1, Q and the -(CH2) m CH2CHN- moiety together form a tetrahydroisoquinolinyl group, wherein the heteroatoms comprise at least a nitrogen atom, Q is methylene, preferably in case W is of formula (I-3);

[0079] W is tetrahydroisoquinolinyl, methylenetetrahydroisoquinolinyl, indolyl, methyleneindolyl, naphthyl, biphenyl, tetrahydroquinolinyl, thienyl; or is selected from one of the following formulae:

[0080]

[0081] wherein Ar1is phenyl, naphthyl, which phenyl, naphthyl is optionally substituted by one or more substituents selected from tert-butyl, benzyloxy, fluorine, chlorine, bromine, iodine, methoxy, trifluoromethyl, methyl, cyano, nitro;

[0082] Ar2, Ar3are each independently phenyl; and

[0083] n is an integer from 1 to 3, in the case where W is of formula (I-3);

[0084] the wave symbol indicates the point of attachment to the remainder of formula (I).

[0085] In one particular aspect, the compound of the application is selected from at least one compound of the following formulae (II-1) to (II-4):

[0086]

[0087]

[0088] wherein,

[0089] X is CH or N,

[0090] m is an integer from 0 to 2, preferably m is 2,

[0091] R1is guanidino, amino, (C6-C 12 )-aryl, said (C6-C 12 )-aryl being optionally substituted by one or more substituents selected from (C1-C4)-alkyl, (C1-C4)-alkoxy, halo-(C1-C4)-alkyl, halogen, carboxyl, (C1-C4)-alkylacyloxy, (C1-C4)-alkyloxycarbonyl, (C6-C 12 )-aryl-(C1-C4)-alkoxy, cyano, nitro; preferably R1is guanidino, amino,

[0092] R2, R3are each independently hydrogen, (C6-C 12 )-aryl, said (C6-C 12 )-aryl being optionally substituted by one or more substituents selected from (C1-C4)-alkyl, (C1-C4)-alkoxy, halo-(C1-C4)-alkyl, halogen, carboxyl, (C1-C4)-alkylacyloxy, (C1-C4)-alkyloxycarbonyl, (C6-C 14 )-aryl-(C1-C4)-alkoxy, cyano, nitro, and R2and R3cannot be hydrogen at the same time;

[0093] Preferably, R2is phenyl, naphthyl, quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl, indolyl, said phenyl being optionally substituted by one or more substituents selected from halogen, methoxy, tert-butyl, benzyloxy, cyano, methyl, fluoro, nitro, trifluoromethyl, carboxyl,

[0094] Preferably, R3is hydrogen or phenyl.

[0095] In another embodiment, the compounds of the present application are selected from at least one compound of the following formulae (III-1) to (III-4):

[0096]

[0097] wherein,

[0098] X is CH or N,

[0099] k is an integer from 0 to 2, preferably k is 2,

[0100] R4 is (C6-C 12 )-aryl, which (C6-C 12 )-aryl is optionally substituted by one or more substituents selected from (C1-C4)-alkyl, (C1-C4)-alkoxy, halo-(C1-C4)-alkyl, halogen, carboxyl, (C1-C4)-alkylcarbonyloxy, (C6-C 12 )-aryl-(C1-C4)-alkoxy, cyano, nitro; preferably phenyl, naphthyl, which phenyl is optionally substituted by one or more substituents selected from halogen, methoxy, tert-butyl, benzyloxy, cyano, methyl, fluorine, nitro, trifluoromethyl, carboxyl,

[0101] R5 is guanidino, amino, (C6-C 12 )-aryl, which (C6-C 12 )-aryl is optionally substituted by one or more substituents selected from (C1-C4)-alkyl, (C1-C4)-alkoxy, halo-(C1-C4)-alkyl, halogen, carboxyl, (C1-C4)-alkylcarbonyloxy, (C6-C 12 )-aryl-(C1-C4)-alkoxy, cyano, nitro; preferably guanidino, amino.

[0102] In the present application, the compounds of formulae (II-1) to (II-4) and (III-1) to (III-4) are present in the form of specific stereoisomers, which have a chiral carbon atom in a specific position, and are obtained by modifying the dipeptide backbone, in particular by inserting a carbon atom, especially one carbon atom, into the single peptide bond of the dipeptide backbone (amino and carbonyl are attached to the same carbon atom, e.g. -NH-CH2-CO-). These compounds have the effect of reducing the efflux capacity of microorganisms and can be used as efflux pump inhibitors.

[0103] In yet another embodiment, the compounds of the present application are selected from at least one compound of the following formulae (IV-1) to (IV-4):

[0104]

[0105] wherein,

[0106] X is CH or N,

[0107] q is an integer from 1 to 2,

[0108] E is (C6-C 12 )-heteroaryl, wherein the heteroatoms comprise at least N atoms; preferably tetrahydroquinolinyl, indolyl, tetrahydroisoquinolinyl,

[0109] R6 is guanidyl, amino, (C6-C 12 )-aryl, said (C6-C 12 )-aryl being optionally substituted by one or more substituents selected from (C1-C4)-alkyl, (C1-C4)-alkoxy, halo-(C1-C4)-alkyl, halogen, carboxyl, (C1-C4)-alkylacyloxy, (C1-C4)-alkyloxycarbonyl, (C6-C 12 )-aryl-(C1-C4)-alkoxy, cyano, nitro; preferably R1 is guanidyl, amino.

[0110] In the present application, the compounds of formulae (IV-1) to (IV-4) exist in the form of specific stereoisomers having a chiral carbon atom in a specific position and are obtained by modifying the dipeptide backbone, in particular by linking the dipeptide backbone to a nitrogen-containing heteroaryl group (e.g. tetrahydroquinolinyl, indolyl, tetrahydroisoquinolinyl). These compounds have the effect of reducing the efflux capacity of microorganisms and can be used as efflux pump inhibitors.

[0111] In yet another embodiment, the compounds of the present application are selected from at least one compound of (V-1) to (V-4)

[0112]

[0113]

[0114] wherein,

[0115] X is CH or N,

[0116] m is 0,

[0117] R1 is -(C6-C 14 )-arylene, preferably phenylene, naphthylene;

[0118] Q is methylene

[0119] Y is -(C1-C6)-alkyleneamino, preferably propyleneamino (-(CH2)3-NH2);

[0120] Preferably, R1, Q and the -(CH2) m CH2CHN- moiety together form a tetrahydroisoquinoline group.

[0121] In the present application, the compounds of formula (V-1) to (V-4) exist in the form of stereoisomers, which have chiral carbon atoms in specific positions, and are obtained by modifying the dipeptide backbone, specifically, by inserting a carbon atom (especially one carbon atom) and a nitrogen-containing heteroaryl group (such as tetrahydroquinoline, indole, tetrahydroisoquinoline) in a single peptide bond (such as -NH-CH2-CO-) of the dipeptide backbone. These compounds have the effect of reducing the efflux ability of microorganisms and can be used as efflux pump inhibitors.

[0122] In a more preferred embodiment, the compound of the present application is selected from at least one of the compounds in Table 1 below:

[0123] Table 1

[0124]

[0125]

[0126]

[0127]

[0128] It was unexpectedly found that the compounds of the present application, the compounds of formula (I) and its subgroups: the compounds of formula (II-1) to (II-4), the compounds of formula (III-1) to (III-4), the compounds of formula (IV-1) to (IV-4), the compounds of formula (V-1) to (V-4), are advantageous for the accumulation of drugs in cells, have excellent inhibitory effect on the efflux ability of the efflux pump of microorganisms, and compared with the efflux pump inhibitors known in the prior art (such as phenylalanine arginine-β-naphthylamide (PAβN)), the efflux inhibiting effect of the compounds of the present application is stronger, can improve the intracellular accumulation rate of drugs, and achieve satisfactory therapeutic effect. Therefore, the compounds of the present application can be used as efflux pump inhibitors to reduce the efflux of microorganisms, reduce the drug resistance of microorganisms, and improve the drug efficacy.

[0129] Preparation of the pseudo-dipeptide compounds of the present application

[0130] The present application also provides a method for preparing the compound of formula (I).

[0131] The compound of formula (I) is prepared by the following scheme:

[0132]

[0133] In the above formulae, PG represents an amino protecting group; X, R1, Q and W have the same meanings as above, and W1is a W group in which the amino group has a protecting group GP.

[0134] The amino protecting group PG is a commonly used amino protecting group in the art. As the "amino protecting group", if it is a protecting group having this function, it is not particularly limited, and examples include -Boc (tert-butyloxycarbonyl), -Cbz (benzyloxycarbonyl), -Teoc (trimethylsilylethoxycarbonyl), -Tos (p-toluenesulfonyl), -Trt (trityl), and -Bn (benzyl). The person skilled in the art can reasonably select them according to the actual needs. Preferably, PG is -Boc (tert-butyloxycarbonyl).

[0135] The above-mentioned step a (step a) and step c (step c) are amidation reactions, the synthesis methods of which are familiar to the person skilled in the art, such as the activated ester method, the carbodiimide condensing agent method or the onium salt condensing agent method. In the present application, the activated ester method and the carbodiimide condensing agent method are preferred.

[0136] The above-mentioned step b (step b) and step d (step d) are deprotection reactions of the amino protecting group PG, and the reaction conditions of the reaction are conventional deprotection conditions, which vary depending on PG, as described in A. G. Myers, J. Gleason, T. Yoon, D. W. Kung, J. Am. Chem. Soc, 1997, 119, 656; and M. Frankel, D. Ladkany, C. Gilon, Y. Wolman, Tetrahedron Lett., 1966, 7, 4765.

[0137] In the present application, in the above-mentioned step (a), an amidation reaction is carried out. Specifically, in an organic solvent, an acylation activator (such as 4-N,N-dimethylpyridine (DMAP), 1-hydroxybenzotriazole (HOBT)) and a carbodiimide condensing agent (such as dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU)) are used.

[0138] (DCC), diisopropylcarbodiimide (DIC), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide (EDCI) are added to the carboxylic compound 2 as acylating agent, the mixture is stirred at 60 to 70 °C for 25 to 40 min; then, the amine compound 1 is added to the mixture, and stirred until the reaction is complete. The reaction solution is extracted, the organic phases are combined, dried, concentrated to give a residue, and column chromatography is used to isolate the intermediate 3.

[0139] As the organic solvent used in the reaction of step a above, for example, dichloromethane, ethyl acetate, chloroform, toluene, tetrahydrofuran, N,N-dimethylformamide, acetonitrile, methanol, ethanol, isopropanol, and mixtures thereof can be exemplified. Preferably, the organic solvent is super dry dichloromethane and / or super dry ethyl acetate. These super dry organic solvents are prepared by a solvent super drying system or are commercially available.

[0140] In step b above, the reaction of deprotection of the amino protecting group is carried out. Specifically, the intermediate 3 and trifluoroacetic acid are added to an organic solvent, and stirred at room temperature until the reaction is complete; the reaction solution is extracted, the organic phases are combined, dried, concentrated to give a residue, and column chromatography is used to isolate the intermediate 4.

[0141] As the organic solvent used in the reaction of step b above, for example, dichloromethane can be exemplified. Preferably, the organic solvent is super dry ethyl acetate or super dry dichloromethane.

[0142] In step c above, the amidation reaction is carried out, including two methods c-1 and c-2. Method c-1 is similar to step a above, specifically, in an organic solvent, l-hydroxybenzotriazole (HOBT) and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) are added to the carboxylic compound 5 as acylating agent, the mixture is stirred at 60 to 70 °C for 25 to 40 min; then, the intermediate 4 is added to the mixture, and stirred until the reaction is complete. The reaction solution is extracted, the organic phases are combined, dried, concentrated to give a residue, and column chromatography is used to isolate the intermediate 6.

[0143] In the method c-2, in an organic solvent, l-hydroxybenzotriazole (HOBT) and a basic substance (such as triethylamine (TEA), diisopropylethylamine (DIEA), N-methylmorpholine (NMM)) are added to the carboxylic compound 5 as acylating agent, and the reaction mixture is stirred at room temperature for 25 to 40 min. Then, at 0 °C, the intermediate 4 and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) are added, and stirred until the reaction is complete, dried, concentrated to give a residue, and column chromatography is used to isolate the intermediate 6.

[0144] The organic solvent used in the reaction of the above-mentioned steps c-1 and c-2 can also use the organic solvent of the above-mentioned step a, preferably super-dry ethyl acetate or super-dry dichloromethane.

[0145] In the above-mentioned step d, the reaction of removing the deamination protecting group PG is carried out. Specifically, intermediate 6 and trifluoroacetic acid are stirred in an organic solvent at room temperature until the reaction is complete; the residue is concentrated and column chromatography is used to isolate the target product, i.e. the compound of formula (I). The amino protecting group PG in the W1 moiety of intermediate 6 is removed by this step.

[0146] The organic solvent used in the reaction of the above-mentioned step d can also use the organic solvent of the above-mentioned step b, preferably super-dry ethyl acetate or super-dry dichloromethane.

[0147] In one specific embodiment, the compounds of formula (II-1) to (II-4) of the present application are obtained by method c-2 in step c and step d, for example compounds 4a to 4u, and compounds 8a to 8f, and 8i; wherein intermediate 4 and compound 5 can be commercially available from Inokai Reagent, Bide Reagent and the like manufacturers;

[0148] Specifically, intermediate 4 is selected from at least one of the following:

[0149]

[0150] Compound 5 is of the following formula:

[0151]

[0152] wherein R1, X, m, R2and R3are as defined above in the compounds of formula (II-1) to (II-4), n is 1, and PG is as defined above for the amino protecting group.

[0153] In another specific embodiment, the compounds of formula (III-1) to (III-4) of the present application are obtained by steps a to d, wherein method c-1 is used in step c, for example compounds 15a to 15o, wherein compound 1, 2 and compound 5 are commercially available from Inokai Reagent, Bide Reagent and the like manufacturers;

[0154] Specifically, compound 2 is selected from at least one of the following:

[0155]

[0156] Compound 5 is selected from at least one of the following:

[0157]

[0158] wherein R4, R5and k are as defined above in the compounds of formula (III-1) to (III-4), and PG is as defined above for an amino protecting group.

[0159]

[0160] In yet another embodiment, the compounds of formula (IV-1) to (IV-4) of the present application are obtained by process c-2 in step c and step d, for example compounds 4a to 4u, and compounds 8g and 8h; wherein the intermediates 4 and compounds 5 are commercially available from vendors such as Inokai Reagent, Bide Reagent, etc.

[0161] wherein the intermediates 4 are selected from at least one of the following:

[0162]

[0163] the compounds 5 are selected from at least one of the following:

[0164]

[0165] wherein R6, E and q are as defined above in the compounds of formula (IV-1) to (IV-4), and PG is as defined above for an amino protecting group; more specifically, the compounds 5 are selected from at least one of the following:

[0166]

[0167] In yet another embodiment, the compounds of formula (V-1) to (V-4) of the present application are obtained by steps a to d, wherein process c-1 is used in step c, for example compound 15p, wherein the compounds 1, 2 and compounds 5 are commercially available from vendors such as Inokai Reagent, Bide Reagent, etc.

[0168] Specifically, the compounds 2 are selected from at least one of the following:

[0169]

[0170] the compounds 5 are selected from at least one of the following:

[0171]

[0172] wherein R1, Q, m and Y are as defined above in the compounds of formula (V-1) to (V-4), and PG is as defined above for an amino protecting group; more specifically, the compounds 2 are selected from at least one of the following:

[0173]

[0174] ​Although the preparation method of the compound of the present application is exemplarily described herein, it is obvious to those skilled in the art that the preparation method is provided only by way of example and in no way limits the present application.

[0175] The present application also provides a method for inhibiting the efflux of the efflux pump of microorganisms, which is carried out by using the compound of the present application, and specifically comprises contacting the compound of the present application with microorganisms. Among them, the microorganisms are bacteria, such as gram-negative bacteria, including but not limited to Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa (also known as Pseudomonas aeruginosa; or P. aeruginosa), Acinetobacter baumannii, Enterobacter cloacae, Enterobacter aerogenes, Salmonella typhi, Serratia marcescens, Citrobacter freundii, Providencia rettgeri, Proteus vulgaris, Proteus mirabilis, Pseudomonas maltophilia, Shigella flexneri. In an embodiment, the bacteria are, for example, Escherichia coli, Pseudomonas aeruginosa. In a specific embodiment, the bacteria are, for example, Pseudomonas aeruginosa N150ABM (source: Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, preparation method according to patent CN1876832A). However, those skilled in the art will understand that these bacteria are only exemplary and in no way limit the present application.

[0176] The compound of the present application can inhibit the efflux of the efflux pump of microorganisms (particularly Pseudomonas aeruginosa) at a concentration of not less than 16 μg / mL, and the result is obtained by measuring the RFFI value of the efflux inhibition of Hoechst 33342, and measuring the intracellular accumulation rate of H33342, see the literature (Tambat R, Mahey N, Chandal N, et al.“A Microbe-Derived Efflux Pump Inhibitor of the Resistance-Nodulation-Cell Division Protein Restores Antibiotic Susceptibility in Escherichia coli and Pseudomonas aeruginosa”. ACS Infect Dis. 2022; 8(2): 255-270).

[0177] In one embodiment, compounds 4c, 4o, 8c, 8f, 15c, 4h and 4p are capable of inhibiting the efflux of efflux pumps of microorganisms (especially P. aeruginosa) at a concentration of no less than 16 μg / mL, for example, 16-64 μg / mL, and have improved inhibitory effect on the efflux pumps of microorganisms as compared with PAβN; in another embodiment, compounds 8d, 8e, 8g and 15p are capable of inhibiting the efflux of efflux pumps of microorganisms (especially P. aeruginosa) at a concentration of no less than 32 μg / mL, for example, 32-64 μg / mL; in yet another embodiment, compounds 4g, 4m, 4q, 4r, 8b, 8c and 8d of the present application are capable of inhibiting the efflux of efflux pumps of microorganisms (especially P. aeruginosa) at a concentration of 64 μg / mL.

[0178] The present application also provides the use of the compounds of the present application for inhibiting the efflux of efflux pumps of microorganisms, thereby improving the antibacterial activity of antibacterial drugs and improving the clinical treatment of bacterial infectious diseases.

[0179] The present application is described in more detail below by way of examples.

[0180] Examples

[0181] The solvents and raw materials used in the preparation methods are commercially available or self-made, and the solvent dichloromethane is obtained by a solvent super dry system (manufacturer JC-Meyer, USA, model Phoenix SDS) to obtain super dry dichloromethane; the super dry ethyl acetate is commercially available from a reagent manufacturer.

[0182] The rule for the intermediate numbers below is that the number of the final compound is connected with the number of the intermediate by a symbol "-", wherein the number of the final compound is consistent with the number described in the subheadings, and the number of the intermediate refers to the number consistent with the above synthesis scheme, wherein the group variable in the intermediate is the corresponding group in the raw material used. For example, in the preparation example 1, the intermediate number is 4a-6, wherein R1 is guanidino group, and W1 is (S)-2-(Boc-amino)-3-phenylpropane-1-yl. In the characterization data 1 HNMR and 13 The δ unit in C NMR is ppm.

[0183] Preparation Example

[0184] Synthesis of compound 4a: (S)-2-((S)-3-amino-4-phenylbutyramido)-5-guanidino-N-(naphthalen-2-yl)pentanamide

[0185]

[0186] Step 1 (step c): To a stirred solution of (S)-3-(Boc-amino)-4-phenylbutyric acid (137 mg, 0.5 mmol) in super dry dichloromethane (15 mL) was added 1-hydroxybenzotriazole (HoBt) (60 mg, 0.45 mmol) and N-methylmorpholine (NMM) (90 mg, 0.9 mmol) and the reaction mixture was stirred at room temperature for 30 min. To this mixture was then added L-arginine beta-naphthylamide hydrochloride (150 mg, 0.45 mmol) and EDCI (110 mg, 0.59 mmol) at 0 °C. The reaction was stirred overnight. TLC indicated the reaction was complete. The reaction was concentrated and the resulting residue was purified by silica gel flash column chromatography, dichloromethane / methanol (containing about 1.5 vol% ammonia water, i.e. methanol was added to 15 mL of ammonia water and made up to 1000 mL) gradient elution from 1:10 to 1:15 by volume to give the intermediate 4a-6 as a light yellow solid 130 mg, 52% yield;

[0187] Step 2 (step d): The intermediate (4a)-6 was added to a flask containing super dry dichloromethane (10 mL) and TFA (2 mL) was added and stirred at room temperature for 3 hours, TLC indicated the reaction was complete. The organic phase was concentrated. The residue was purified by silica gel flash column chromatography, dichloromethane / methanol (containing about 1.5 vol% ammonia water, i.e. methanol was added to 15 mL of ammonia water and made up to 1000 mL) gradient elution from 1:10 to 1:25 by volume to give the target compound 4a as a white solid 65 mg, 61% yield;

[0188] 1 H NMR (600 MHz, Methanol-d4) d (ppm) 8.24 (d, J = 2.1 Hz, 1H), 7.82 (dd, J = 10.3, 8.4 Hz, 2H), 7.78 (d, J = 8.2 Hz, 1H), 7.65 (dd, J = 8.8, 2.1 Hz, 1H), 7.46 (ddd, J = 8.2, 6.7, 1.3 Hz, 1H), 7.41 (ddd, J = 8.1, 6.8, 1.3 Hz, 1H), 7.38 - 7.35 (m, 2H), 7.31 - 7.28 (m, 3H), 4.57 (dd, J = 8.7, 5.4 Hz, 1H), 3.85 - 3.81 (m, 1H), 3.28 - 3.25 (m, 2H), 3.09 (dd, J = 13.7, 6.3 Hz, 1H), 2.96 (dd, J = 13.7, 6.3 Hz, 1H), 2.71 (dd, J = 16.3, 4.5 Hz, 1H), 2.66 (dd, J = 16.3, 8.1 Hz, 1H), 2.02 - 1.96 (m, 1H), 1.89 - 1.69 (m, 3H).

[0189] Synthesis of compound 4b: (S)-2-((S)-3-amino-4-(4-methoxyphenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0190]

[0191] Following the procedure for the synthesis of compound 4a, using (S)-3-(Boc-amino)-4- (4-methoxyphenyl)butanoic acid in place of (S)-3-(Boc-amino)-4-phenylbutanoic acid, the target compound 4b was obtained as a white solid in 35% yield over two steps;

[0192] Target compound 4b: 1 H NMR (500 MHz, Methanol-d4) d 8.25 (d, J = 2.3 Hz, 1H), 7.85 (t, J = 9.0 Hz, 2H), 7.80 (d, J = 8.2 Hz, 1H), 7.63 (dt, J = 8.8, 2.0 Hz, 1H), 7.53 - 7.41 (m, 2H), 7.24 - 7.22 (m, 2H), 6.95 - 6.92 (m, 2H), 4.56 (dd, J = 8.1, 5.8 Hz, 1H), 3.80 (d, J = 1.7 Hz, 3H), 3.31 - 3.21 (m, 3H), 3.01 (dd, J = 14.0, 6.1 Hz, 1H), 2.89 (dd, J = 13.9, 8.5 Hz, 1H), 2.71 (m, 1H), 2.65 - 2.56 (m, 1H), 1.98 (m, 1H), 1.92 - 1.67 (m, 3H).

[0193] 13 C NMR (151 MHz, Methanol-d4) d 171.2, 170.9, 159.2, 157.3, 135.5, 133.8, 130.8, 130.2 (2), 128.2, 127.2, 127.1, 127.06, 126.2, 124.8, 119.8, 116.7, 114.1 (2), 54.3, 54.0, 50.3, 40.6, 37.4, 35.3, 28.9, 25.1.

[0194] HRMS: calculated for C 27 H 34 N6O3 [M+H] 491.2765, found 491.2767.

[0195] Synthesis of compound 4c: (S)-2-((S)-3-amino-4-(4-trifluoromethylphenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0196] Synthesis of compound 4c: (S)-2-((S)-3-amino-4-(4-trifluoromethylphenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0197] Referring to the synthetic procedure for the preparation of compound 4a, (S)-3-(Boc-amino)-4-(4- trifluoromethylphenyl)butyric acid was used in place of (S)-3-(Boc-amino)-4-phenylbutyric acid to give the target compound 4c as a light yellow solid in 51% yield over two steps;

[0198] Target compound 4c: 1 H NMR (500 MHz, Methanol-d4) d 8.25 (s, 1H), 7.84 (t, J = 9.2 Hz, 2H), 7.79 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 7.9 Hz, 2H), 7.62 (dt, J = 8.8, 1.7 Hz, 1H), 7.54 (d, J = 7.9 Hz, 2H), 7.49 (t, J = 7.5 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H), 4.55 (t, J = 7.1 Hz, 1H), 3.97 - 3.82 (m, 1H), 3.45 - 3.31 (m, 5H), 3.27 (q, J = 7.6, 7.0 Hz, 2H), 3.17 (dd, J = 13.8, 6.3 Hz, 1H), 3.06 (m, 1H), 2.76 - 2.57 (m, 2H), 1.98 (m, 1H), 1.91 - 1.70 (m, 3H).

[0199] 13 C NMR (151 MHz, Methanol-d4) d 171.2, 170.7, 157.3, 140.0, 135.5, 133.8, 130.8, 129.9 (2), 128.3, 127.2, 127.1, 126.2, 125.5, 125.5, 124.8, 119.8, 117.8, 116.7, 115.8, 54.0, 49.8, 40.6, 37.8, 35.1, 28.8, 25.1.

[0200] HRMS: calculated for C 27 H 31 F3N6O2 [M+H] 529.2533, found 529.2539.

[0201] Synthesis of compound 4d: (S)-2-((S)-3-amino-4-(4-methylphenyl)butanamido)-5-guanidino-N- (naphthalen-2-yl)pentanamide

[0202]

[0203] The synthetic method for preparing compound 4a was referred to, and (S)-3-(Boc-amino)-4-(4- methylphenyl)butyric acid was used instead of (S)-3-(Boc-amino)-4-phenylbutyric acid to obtain the target compound 4d as a white solid with a two-step yield of 67%;

[0204] Target compound 4d: 1 H NMR (500 MHz, Methanol-d4) δ 8.24 (d, J = 2.2 Hz, 1H), 7.84 (t, J = 8.3 Hz, 2H), 7.79 (d, J = 8.3 Hz, 1H), 7.63 (dd, J = 8.9, 2.1 Hz, 1H), 7.46 (dt, J = 26.3, 7.2 Hz, 3H), 7.19 (s, 3H), 4.55 (dd, J = 8.6, 5.3 Hz, 1H), 3.83 - 3.78 (m, 1H), 3.27 (t, J = 7.1 Hz, 2H), 3.04 (dd, J = 13.7, 6.2 Hz, 1H), 2.96 - 2.85 (m, 1H), 2.66 (dd, J = 20.8, 6.3 Hz, 2H), 2.34 (s, 3H), 2.04 - 1.93 (m, 1H), 1.87 - 1.70 (m, 3H).

[0205] 13 C NMR (151 MHz, Methanol-d4) δ 171.2, 170.9, 157.3, 136.9, 135.5, 133.8, 132.2, 130.8, 129.3 (2), 128.9 (2), 128.2, 127.2, 127.1, 126.2, 124.8, 119.9, 116.7, 54.0, 50.2, 40.6, 37.8, 35.3, 28.8, 25.1, 19.7.

[0206] HRMS: calculated for C 27 H 34 N6O2[M+H] 475.2816, found 475.2807.

[0207] Synthesis of compound 4e: (S)-2-((S)-3-amino-4-cyanophenylbutanamido)-5-guanidino-N- (naphthalen-2-yl)pentanamide

[0208]

[0209] The synthetic method for preparing compound 4a was referred to, and (S)-3-(Boc-amino)-4-(4- cyanophenyl)butyric acid was used instead of (S)-3-(Boc-amino)-4-phenylbutyric acid to obtain the target compound 4e as a light yellow solid with a two-step yield of 19%.

[0210] Compound 4e: 1 H NMR (500 MHz, Methanol-d4) d 8.31 - 8.15 (m, 3H), 7.85 - 7.86 (m, 3H), 7.61 (dd, J = 17.7, 7.7 Hz, 3H), 7.52 - 7.35 (m, 2H), 4.58 - 4.48 (m, 1H), 3.99 - 3.85 (m, 1H), 3.31 - 3.17 (m, 3H), 3.12 (dd, J = 13.8, 8.4 Hz, 1H), 2.75 - 2.64 (m, 1H), 2.00 - 1.95 (m, 1H), 1.92 - 1.67 (m, 3H).

[0211] 13 C NMR (151 MHz, Methanol-d4) d 171.3, 170.7, 157.3, 141.3, 133.7, 135.5, 133.7, 132.4 (2), 130.8 (2), 130.3, 128.2, 127.2, 127.1, 126.2, 124.8, 119.9, 118.22, 117.8, 115.8, 110.9, 54.1, 49.7, 40.6, 38.1, 35.3, 28.8, 25.1.

[0212] HRMS: calculated for C 27 H 31 N7O2[M+H] 486.2612, found 486.2612.

[0213] Synthesis of compound 4f: (S)-2-((S)-3-amino-4-(4-nitrophenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0214]

[0215] Following the procedure for the synthesis of reference compound 4a, using (S)-3-(Boc- amino)-4-(4-nitrophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid, the target compound 4f was obtained as a light yellow solid with a two-step yield of 16%;

[0216] Compound 4f: 1H NMR (500 MHz, Methanol-d4) δ 8.31 - 8.15 (m, 3H), 7.81 (dt, J = 28.6, 9.9 Hz, 3H), 7.61 (dd, J = 17.7, 7.7 Hz, 3H), 7.52 - 7.35 (m, 2H), 4.58 - 4.48 (m, 1H), 3.99 - 3.85 (m, 1H), 3.31 - 3.17 (m, 3H), 3.12 (dd, J = 13.8, 8.4 Hz, 1H), 2.75 - 2.64 (m, 1H), 2.00 - 1.95 (m, 1H), 1.92 - 1.67 (m, 3H).

[0217] 13 C NMR (151 MHz, Methanol-d4) δ 171.2, 170.6, 157.3, 147.4, 143.2, 135.5, 133.8, 130.8, 130.4 (2), 128.2, 127.2, 127.1, 126.2, 124.8, 123.6 (2), 119.8, 116.67, 54.1, 49.7, 40.67, 37.7, 35.2, 28.8, 25.1.

[0218] HRMS: calculated for C 26 H 31 N7O4 [M+H] 506.2503, found 506.2510.

[0219] Synthesis of compound 4g: (S)-2-((S)-3-amino-4-(4-tert-butylphenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0220]

[0221] The synthesis of reference compound 4a was followed using (S)-3-(Boc-amino)-4-(4-tert- butylphenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 4g as a white solid in 48% yield over two steps;

[0222] Compound 4g: 1H NMR (500 MHz, Methanol-d4) δ 8.26 (d, J = 2.5 Hz, 1H), 7.88 - 7.81 (m, 2H), 7.79 (d, J = 8.2 Hz, 1H), 7.63 (dt, J = 8.9, 2.4 Hz, 1H), 7.52 - 7.46 (m, 1H), 7.46 - 7.40 (m, 3H), 7.27 - 7.21 (m, 2H), 4.59 - 4.55 (m, 1H), 3.85 - 3.76 (m, 1H), 3.38 (d, J = 2.6 Hz, 9H), 3.28 - 3.25 (m, 2H), 3.07 - 2.99 (m, 1H), 2.95 - 2.90 (m, 1H), 2.71 (dt, J = 16.6, 3.5 Hz, 1H), 2.64 - 2.59 (m, 1H), 2.02 - 1.94 (m, 1H), 1.90 - 1.72 (m, 3H).

[0223] 13 C NMR (151 MHz, Methanol-d4) δ 171.2, 170.9, 157.3, 150.2, 135.5, 133.8, 132.21, 130.9, 128.8, 128.3, 127.2, 127.1, 126.2, 125.7, 124.8, 119.8, 116.7, 53.9, 50.2, 48.5, 40.7, 37.7, 35.2, 33.9, 30.3, 28.9, 25.1.

[0224] HRMS: C 30 H 40 N6O2 calcd [M+H] 517.3278, found 517.3285.

[0225] Synthesis of compound 4h: (S)-2-((S)-3-amino-4-(4-benzyloxyphenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0226]

[0227] The target compound 4h was obtained as a white solid in 64% yield over two steps by following the procedure for the synthesis of reference compound 4a, using (S)-3-(Boc-amino)-4-(4-benzyloxyphenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid.

[0228] Compound 4h: 1H NMR (500 MHz, Methanol-d4) d 8.25 (s, 1H), 7.84 (dd, J = 12.5, 5.6 Hz, 2H), 7.79 (d, J = 7.8 Hz, 1H), 7.62 (dt, J = 10.2, 2.7 Hz, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.45 - 7.43 (m, 3H), 7.40 - 7.37 (m, 2H), 7.33 (d, J = 7.8 Hz, 1H), 7.25 - 7.18 (m, 2H), 7.03 - 6.97 (m, 2H), 5.08 (d, J = 3.0 Hz, 2H), 4.55 (q, J = 5.3, 3.2 Hz, 1H), 3.81 - 3.73 (m, 1H), 3.35 - 3.30 (m, 2H), 3.29 - 3.21 (m, 2H), 3.04 - 2.96 (m, 1H), 2.92 - 2.84 (m, 1H), 2.70 (dt, J = 16.1, 3.7 Hz, 1H), 2.63 - 2.58 (m, 1H), 2.01 - 1.92 (m, 1H), 1.89 - 1.68 (m, 3H).

[0229] 13 C NMR (151 MHz, Methanol-d4) d 171.2, 170.9, 161.9, 161.6, 158.3, 157.3, 137.3, 135.5, 133.8, 130.9, 130.2, 128.3, 128.1, 127.5, 127.4, 127.2, 127.1, 127.1, 126.2, 124.8, 119.9, 117.8, 116.7, 115.9, 115.1, 69.6, 53.9, 50.3, 40.6, 37.4, 35.3, 28.9, 25.1.

[0230] HRMS: C 33 H 38 N6O3 calcd [M+H] 567.3075, found 567.3078.

[0231] Synthesis of compound 4i: (S)-2-((S)-3-amino-4-(4-fluorophenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0232]

[0233] The target compound 4i was obtained as a white solid in 33% yield over two steps by following the procedure for the synthesis of reference compound 4a, using (S)-3-(BOC- amino)-4-(4-fluorophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid.

[0234] Compound 4i: 1 H NMR (500 MHz, Methanol-d4) d 8.24 (d, J = 3.6 Hz, 1H), 7.86-7.81 (m, 2H), 7.79 (dd, J = 8.6, 3.6 Hz, 1H), 7.62 (dt, J = 8.8, 2.8 Hz, 1H), 7.51-7.46 (m, 1H), 7.45-7.41 (m, 1H), 7.34 (m, 2H), 7.10 (m, 2H), 4.56-4.53 (m, 1H), 3.87-3.78 (m, 1H), 3.27 (dt, J = 10.3, 5.4 Hz, 2H), 3.09-3.04 (m, 1H), 2.98-2.93 (m, 1H), 2.75-2.57 (m, 2H), 2.03-1.93 (m, 1H), 1.91-1.67 (m, 3H).

[0235] HRMS: C 26 H 31 FN6O2 calculated [M+H] 479.2565, found 479.2577.

[0236] Compound 4j: (S)-2-((S)-3-amino-4-(2-fluorophenyl)butanamido)-5-guanidino-N- (naphthalen-2-yl)pentanamide

[0237]

[0238] The target compound 4j was obtained as a white solid in 47% yield over two steps by following the procedure for the synthesis of reference compound 4a, using (S)-3-(Boc-amino)-4-(2-fluorophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid.

[0239] Compound 4j: 1 H NMR (500 MHz, Methanol-d4) d 8.24 (s, 1H), 7.89-

[0240] 7.81 (m, 2H), 7.79 (d, J = 8.4 Hz, 1H), 7.62 (dt, J = 8.8, 2.4 Hz, 1H), 7.50-7.36 (m, 4H), 7.23-7.15 (m, 2H), 4.56-4.53 (m, 1H), 3.91-3.87 (m, 1H), 3.28-3.25 (m, 2H), 3.17-3.14 (m, 1H), 3.03 (dd, J = 13.4, 7.7 Hz, 1H), 2.74-2.62 (m, 2H), 2.02-1.93 (m, 1H), 1.91-1.64 (m, 3H).

[0241] 13 C NMR (151 MHz, Methanol-^) d 171.2, 170.6, 157.3, 135.5, 133.8, 131.7, 131.7, 130.85, 129.6, 128.3, 127.2, 127.1, 126.2, 124.8, 124.6, 124.6, 122.2, 119.8, 116.7, 53.9, 48.9, 40.6, 35.3, 31.8, 28.9, 251.

[0242] HRMS: C 26 H 31 FN6O2 calcd [M+H] 479.2565, found 479.2557.

[0243] Synthesis of compound 4k: (S)-2-((S)-3-amino-4-(2,4,5-fluorophenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0244]

[0245] The synthesis of reference compound 4a was followed using (S)-3-(BOC-amino-)--4-(2,4,5- fluorophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 4k as a light yellow solid in 26% yield over two steps;

[0246] Compound 4k: 1 H NMR (500 MHz, Methanol-^) d 8.25 (d, J = 2.2 Hz, 1H), 7.84 (t, J = 8.7 Hz, 2H), 7.79 (d, J = 8.2 Hz, 1H), 7.62 (dd, J = 8.9, 2.1 Hz, 1H), 7.50 - 7.47 (m, 1H), 7.45 - 7.40 (m, 2H), 7.24 (td, J = 10.0, 6.6 Hz, 1H), 4.54 (dd, J = 8.1, 5.8 Hz, 1H), 3.89 - 3.84 (m, 1H), 3.27 (t, J = 7.1 Hz, 2H), 3.11 (dd, J = 14.1, 6.4 Hz, 1H), 3.01 (dd, J = 14.2, 8.2 Hz, 1H), 2.73 (dd, J = 16.5, 4.5 Hz, 1H), 2.66 (dd, J = 16.5, 7.9 Hz, 1H), 2.01 - 1.96 (m, 1H), 1.90 - 1.69 (m, 3H).

[0247] 13C NMR (151 MHz, Methanol- de) δ 171.2, 170.5, 157.3, 135.5, 133.8, 130.8, 128.2 (2), 127.2, 127.1, 126.2 (2), 124.8, 119.8, 119.4, 119.3, 116.6, 105.6, 105.44, 54.1, 48.7, 48.2, 40.7, 35.3, 31.1, 28.9, 25.1.

[0248] HRMS: C 26 H 29 F3N6O2 Calculated [M+H] 515.2376, found 515.2369.

[0249] Synthesis of compound 4l: (S)-2-((S)-3-amino-4-(4-bromophenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0250]

[0251] The synthesis of reference compound 4a was followed using (S)-3-(Boc-amino)-4-(4- bromophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 4l as a white solid in 54% yield over two steps.

[0252] Target compound 4l: 1 H NMR (500 MHz, Methanol- de) δ 8.25 (d, J = 2.1 Hz, 1H), 7.85 (t, J = 9.0 Hz, 2H), 7.80 (d, J = 8.2 Hz, 1H), 7.62 (dd, J = 8.9, 2.2 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.49 (t, J = 7.4 Hz, 1H), 7.44 (t, J = 7.4 Hz, 1H), 7.27 (d, J = 8.1 Hz, 2H), 4.54 (dd, J = 8.1, 5.9 Hz, 1H), 3.86-

[0253] 3.81 (m, 1H), 3.27 (t, J = 7.0 Hz, 2H), 3.05 (dd, J = 13.8, 6.2 Hz, 1H), 2.93 (dd, J = 13.8, 8.6 Hz, 1H), 2.70 (dd, J = 16.5, 4.4 Hz, 1H), 2.61 (dd, J = 16.5, 7.8 Hz, 1H), 2.00 - 1.94 (m, 1H), 1.89 - 1.66 (m, 3H).

[0254] 13C NMR (151 MHz, Methanol- de) d 171.2, 170.7, 157.3, 135.5, 134.6, 133.8, 131.8 (2), 131.1 (2), 130.9, 128.3, 127.2, 127.1, 126.2, 124.8, 121.0, 119.8, 116.7, 54.1, 49.8, 40.7, 37.5, 35.1, 28.9, 25.1.

[0255] HRMS: C 26 H 31 BrN6O2 Calcd [M+H] 539.1764, found 539.1766, 541.1748.

[0256] Synthesis of compound 4m: (S)-2-((S)-3-amino-4-(3-bromophenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0257]

[0258] The synthesis of reference compound 4a was followed using (S)-3-(Boc-amino)-4-(3- bromophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 4m as a white solid in 63% yield over two steps.

[0259] Compound 4m: 1 H NMR (500 MHz, Methanol- de) d 8.26 (s, 1H), 7.87-7.84 (m, 2H), 7.80 (d, J = 8.4 Hz, 1H), 7.64-7.61 (m, 1H), 7.57 (s, 1H), 7.54-7.47 (m, 2H), 7.46-7.43 (m, 1H), 7.33 (d, J = 4.8 Hz, 2H), 4.55 (t, J = 7.1 Hz, 1H), 3.86 (d, J = 11.6 Hz, 1H), 3.27 (d, J = 7.2 Hz, 2H), 3.07 (dd, J = 14.0, 6.4 Hz, 1H), 2.96 (dd, J = 13.4, 8.0 Hz, 1H), 2.73-2.61 (m, 2H), 2.03-1.94 (m, 1H), 1.89-1.65 (m, 3H).

[0260] 13C NMR (151 MHz, Methanol-d4) δ 171.1, 170.6, 157.3, 137.9, 135.5, 133.8, 132.1, 130.8, 130.5, 130.4, 128.3, 128.0, 127.2, 127.1, 126.2, 124.8, 122.6, 119.8, 116.7, 54.0, 49.8, 40.7, 37.7, 35.2, 28.9, 25.1.

[0261] HRMS: C 26 H 31 BrN6O2Calcd [M+H] 539.1764, found 539.1763, 541.1746.

[0262] Synthesis of compound 4n: (S)-2-((S)-3-amino-4-(2-bromophenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0263]

[0264] The synthesis of reference compound 4a was followed using (S)-3-(Boc-amino)-4-(2- bromophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 4n as a white solid in 41% yield over two steps.

[0265] Compound 4n: 13 C NMR (151 MHz, Methanol-d4) δ 171.2, 170.7, 157.3, 135.5, 134.8, 133.8, 133.1, 131.8, 130.9, 129.3, 128.3, 127.9, 127.2, 127.1, 126.2, 124.1, 124.3, 119.8, 116.7, 54.0, 48.5, 40.6, 38.4, 35.1, 28.9, 25.1.

[0266] HRMS: C 26 H 31 BrN6O2Calcd [M+H] 539.1764, found 539.1765, 541.1748.

[0267] Synthesis of compound 4o: (S)-2-((S)-3-amino-4-(4-chlorophenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0268]

[0269] The synthesis of compound 4a was followed using (S)-3-(Boc-amino)-4-(4- chlorophenyl)butyric acid instead of (S)-3-(Boc-amino)-4-phenylbutyric acid to give the target compound 4o as a white solid in 63% yield over two steps.

[0270] Compound 4o: 1 H NMR (600 MHz, Methanol-d4) δ 8.23 (d, J = 2.2 Hz, 1H), 7.82 (dd, J = 10.9, 8.4 Hz, 2H), 7.78 (d, J = 8.2 Hz, 1H), 7.61 (dd, J = 8.8, 2.1 Hz, 1H), 7.47 (ddd, J = 8.1, 6.7, 1.3 Hz, 1H), 7.41 (ddd, J = 8.1, 6.8, 1.3 Hz, 1H), 7.38 - 7.35 (m, 2H), 7.31 - 7.30 (m, 2H), 4.53 (dd, J = 8.5, 5.6 Hz, 1H), 3.82 (m, 1H), 3.34 - 3.32 (m, 1H), 3.26 - 3.24 (m, 2H), 3.06 (dd, J = 13.8, 6.2 Hz, 1H), 2.94 (dd, J = 13.8, 8.6 Hz, 1H), 2.69 (dd, J = 16.3, 4.4 Hz, 1H), 2.62 (dd, J = 16.4, 7.9 Hz, 1H), 1.99 - 1.94 (m, 1H), 1.89 - 1.68 (m, 3H).

[0271] HRMS: C 26 H 31 ClN6O2 Calcd [M+H] 495.2269, found 495.2268.

[0272] Synthesis of compound 4p: (S)-2-((S)-3-amino-4-(4-iodophenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0273]

[0274] The synthesis of compound 4a was followed using (S)-3-(Boc-amino)-4-(4- chlorophenyl)butyric acid instead of (S)-3-(Boc-amino)-4-phenylbutyric acid to give the target compound 4o as a white solid in 63% yield over two steps.

[0275] Compound 4p: 1H NMR (600 MHz, Methanol-^) δ 8.24 (d, J = 2.2 Hz, 1H), 7.82 (dd, J = 11.2, 8.4 Hz, 2H), 7.78 (d, J = 8.2 Hz, 1H), 7.72 - 7.70 (m, 2H), 7.63 (dd, J = 8.8, 2.1 Hz, 1H), 7.48 - 7.45 (m, 1H), 7.11 (d, J = 8.3 Hz, 2H), 4.54 (dd, J = 8.5, 5.5 Hz, 1H), 3.84 - 3.79 (m, 1H), 3.37 - 3.32 (m, 1H), 3.26 (t, J = 7.0 Hz, 2H), 3.04 (dd, J = 13.8, 6.2 Hz, 1H), 2.91 (dd, J = 13.8, 8.6 Hz, 1H), 2.69 (dd, J = 16.4, 4.4 Hz, 1H), 2.62 (dd, J = 16.4, 7.9 Hz, 1H), 2.00 - 1.94 (m, 1H), 1.88 - 1.68 (m, 3H).

[0276] 13 C NMR (151 MHz, Methanol-^) δ 171.3, 170.8, 157.3, 137.9 (2), 135.6, 135.2, 133.8, 131.3 (2), 130.8, 128.2, 127.2, 127.1, 126.2, 124.79, 119.9, 116.7, 92.2, 54.0, 49.9, 40.6, 37.7, 35.3, 28.8, 25.1.

[0277] HRMS: C 26 H 31 IN6O2 calcd [M+H] 587.1626, found 587.1623.

[0278] Synthesis of compound 4q: (S)-2-((R)-3-amino-4-(4-bromophenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0279]

[0280] The target compound 4q was obtained as a white solid in 29% yield over two steps by following the procedure for the synthesis of reference compound 4a, using (R)-3-(Boc- amino)-4-(4-bromophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid;

[0281] Compound 4q: 1H NMR (600 MHz, Methanol-^) δ 8.21 (d, J = 2.1 Hz, 1H), 7.83 (t, J = 8.5 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.58 (dd, J = 8.8, 2.2 Hz, 1H), 7.54 - 7.52 (m, 2H), 7.49 - 7.46 (m, 1H), 7.44 - 7.41 (m, 1H), 7.24 - 7.22 (m, 2H), 4.53 (dd, J = 8.2, 5.9 Hz, 1H), 3.83 - 3.80 (m, 1H), 3.26 (t, J = 7.0 Hz, 2H), 3.03 (dd, J = 13.9, 6.4 Hz, 1H), 2.94 (dd, J = 13.9, 8.3 Hz, 1H), 2.73 (dd, J = 16.4, 4.5 Hz, 1H), 2.56 (dd, J = 16.4, 8.2 Hz, 1H), 2.00 - 1.94 (m, 1H), 1.88 - 1.68 (m, 3H).

[0282] 13 C NMR (151 MHz, Methanol-^) δ 171.1, 171.0, 158.7, 134.6, 133.8, 131.8 (2), 131.0 (2), 130.9, 128.2, 127.2, 127.1, 126.2, 124.8, 121.1, 119.8, 116.73, 53.9, 49.8, 40.7, 37.7, 35.1, 28.9, 25.1.

[0283] HRMS: C 26 H 31 BrN6O2 Calcd [M+H] 539.17646, Found 539.1770, 541.1751.

[0284] Synthesis of compound 4r: (S)-2-((R)-3-amino-4-(4-tert-butylphenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0285]

[0286] The target compound 4r was obtained as a light yellow solid in 36% yield over two steps by following the procedure for the synthesis of reference compound 4a, using (R)-3-(BOC-amino)-4-(4-tert-butylphenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid;

[0287] Compound 4r: 1H NMR (500 MHz, Methanol-d4) δ 8.21 (d, J = 2.3 Hz, 1H), 7.84 (t, J = 7.6 Hz, 2H), 7.78 (d, J = 8.1 Hz, 1H), 7.60 (dd, J = 8.8, 2.2 Hz, 1H), 7.50 - 7.47 (m, 1H), 7.45 - 7.42 (m, 3H), 7.24 - 7.20 (m, 2H), 4.56 (dd, J = 8.2, 5.6 Hz, 1H), 3.82 (t, J = 6.0 Hz, 1H), 3.28 (t, J = 7.1 Hz, 2H), 3.04 (dd, J = 13.8, 6.2 Hz, 1H), 2.93 (dd, J = 13.8, 8.2 Hz, 1H), 2.75 (dd, J = 16.4, 4.2 Hz, 1H), 2.58 (dd, J = 16.4, 8.5 Hz, 1H), 2.01 - 1.97 (m, 1H), 1.90 - 1.73 (m, 3H).

[0288] 13 C NMR (151 MHz, Methanol-d4) δ 171.2, 171.1, 157.3, 150.2, 135.5, 133.8, 132.2, 130.85, 128.8 (2), 128.2, 127.2, 127.1, 126.2, 125.6 (2), 124.8, 119.9, 116.8, 53.98, 50.240.7, 37.8, 35.1, 33.9, 30.3 (3), 28.8, 25.1.

[0289] Synthesis of compound 4s: (S)-2-((S)-3-amino-4-(4-benzyloxyphenyl)butanamido)-5- amino-N-(naphthalen-2-yl)pentanamide

[0290]

[0291] Following the procedure for the synthesis of reference compound 4a, replacing (S)-3- (Boc-amino)-4-phenylbutanoic acid with (S)-3-(Boc-amino)-4-(4-benzyloxyphenyl)butanoic acid, the target compound 4s was obtained as a white solid with a yield of 53% over two steps;

[0292] Compound 4s: 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.60 (d, J = 7.6 Hz, 1H), 8.29 (d, J = 2.0 Hz, 1H), 7.89-7.80 (m, 6H), 7.64 (dd, J = 8.9, 2.1 Hz, 1H), 7.49-7.46 (m, 1H), 7.45-7.42 (m, 2H), 7.41-7.39 (m, 3H), 7.35-7.33 (m, 1H), 7.19-7.18 (m, 2H), 7.00-6.98 (m, 2H), 4.54-4.48 (m, 1H), 3.62 (s, 1H), 3.35 (br s, 3H), 3.17 (s, 1H), 2.88 (dd, J = 13.7, 5.8 Hz, 1H), 2.81 (s, 2H), 2.72 (dd, J = 13.8, 8.3 Hz, 1H), 1.84-1.78 (m, 1H), 1.70-1.57 (m, 3H).

[0293] 13 C NMR (151 MHz, DMSO-d6) δ 170.9, 170.1, 157.9, 137.6, 136.9, 133.8, 130.9, 130.3, 128.9 (3), 128.9, 128.6, 128.3, 128.1 (3), 127.9, 127.7, 126.9, 125.2, 120.4, 115.9, 115.4, 69.7, 53.5, 49.9, 38.9, 37.7, 36.2, 29.4, 24.1.

[0294] HRMS: C 32 H 36 N4O3 calcd [M+H] 525.2860, found 525.2872.

[0295] Synthesis of compound 4t: (S)-2-((S)-3-amino-4-(4-tert-butylphenyl)butanamido)-5-amino-N-(naphthalen-2-yl)pentanamide

[0296]

[0297] The target compound 4t was obtained as a white solid in 66% yield over two steps by following the procedure for the synthesis of reference compound 4a, using (S)-3-(Boc-amino)-4-(4-tert-butylphenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid;

[0298] Compound 4t: 1H NMR (600 MHz, Methanol-d4) δ 8.26 (d, J = 2.1 Hz, 1H), 7.85 - 7.81 (m, 2H), 7.78 (d, J = 8.2 Hz, 1H), 7.62 (dd, J = 8.8, 2.1 Hz, 1H), 7.49 - 7.46 (m, 1H), 7.44 - 7.40 (m, 3H), 7.23 (d, J = 8.3 Hz, 2H), 4.59 (dd, J = 8.1, 5.6 Hz, 1H), 3.83 - 3.78 (m, 1H), 3.05 - 2.99 (m, 3H), 2.91 (dd, J = 13.8, 8.7 Hz, 1H), 2.69 (dd, J = 16.5, 4.0 Hz, 1H), 2.61 (dd, J = 16.5, 8.4 Hz, 1H), 2.03 - 1.97 (m, 1H), 1.90 - 1.78 (m, 3H), 1.31 (s, 9H).

[0299] 13.8,8.7Hz,1H),2.69(dd,J=16.5,4.0Hz,1H),2.61(dd,J=16.5,8.4Hz,1H),2.03-1.97(m,1H),1.90-1.78(m,3H),1.31(s,9H)。

[0300] 13 C NMR (151 MHz, Methanol-d4) δ 170.9, 170.9, 150.2, 135.5, 133.8, 132.2, 130.8, 128.8 (2), 128.2, 127.2, 127.1, 126.2, 125.6 (2), 124.8, 119.8, 116.7, 53.7, 50.2, 38.9, 37.7, 35.2, 33.9, 30.3 (3), 28.6, 23.7.

[0301] HRMS: C 29 H 38 N4O2 calcd [M+H] 475.3067, found 475.3073.

[0302] Synthesis of compound 4u: (S)-2-((S)-3-amino-4-(4-bromophenyl)butanamido)-5- amino-N-(naphthalen-2-yl)pentanamide

[0303]

[0304] The synthesis of reference compound 4a was followed using (S)-3-(Boc-amino)-4- (4-bromophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 4u as a white solid in 55% yield over two steps;

[0305] Compound 4u: 1H NMR (600 MHz, Methanol-d4) δ 8.21 (d, J = 2.2 Hz, 1H), 7.81 (t, J = 7.7 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.58 (dd, J = 8.8, 2.1 Hz, 1H), 7.46 - 7.44 (m, 3H), 7.42 - 7.39 (m, 1H), 7.17 (d, J = 8.2 Hz, 2H), 4.52 (dd, J = 9.0, 5.3 Hz, 1H), 3.44 - 3.33 (m, 1H), 2.78 - 2.68 (m, 4H), 2.40 (dd, J = 14.3, 4.4 Hz, 1H), 2.30 (dd, J = 14.3, 8.9 Hz, 1H), 2.00 - 1.94 (mz, 1H), 1.82 - 1.76 (m, 1H), 1.71 - 1.58 (m, 2H).

[0306] 13 C NMR (151 MHz, Methanol-d4) δ 173.2, 171.5, 137.7, 135.7, 133.8, 131.3 (2), 131.0 (2), 130.8, 128.2, 127.2, 127.1, 126.1, 124.7, 120.0, 119.9, 116.8, 53.9, 50.2, 42.7, 41.8, 40.3, 29.0, 27.9.

[0307] HRMS: C 25 H 29 BrN4O2 Calcd [M+H] 497.15467, Found 497.1551, 499.1528.

[0308] Synthesis of compound 8a: (S)-2-((S)-3-amino-4-(2-naphthalenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0309]

[0310] The synthesis of reference compound 4a was followed using (S)-3-(Boc-amino)-4-(2- naphthalenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 8a as a white solid in 53% yield over two steps;

[0311] Compound 8a: 1H NMR (600 MHz, Methanol-d4) δ 8.26 (d, J = 2.1 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.87 - 7.83 (m, 4H), 7.82 - 7.78 (m, 3H), 7.62 (dd, J = 8.8, 2.1 Hz, 1H), 7.51 - 7.47 (m, 3H), 7.45 - 7.40 (m, 2H), 4.51 (dd, J = 8.2, 5.9 Hz, 1H), 4.00 - 3.94 (m, 1H), 3.26 - 3.20 (m, 3H), 3.11 (dd, J = 13.7, 8.8 Hz, 1H), 2.74 (dd, J = 16.5, 4.3 Hz, 1H), 2.65 (dd, J = 16.5, 7.9 Hz, 1H), 1.95 - 1.89 (m, 1H), 1.80 - 1.63 (m, 3H).

[0312] 13 C NMR (151 MHz, Methanol-d4) δ 171.0, 170.1, 157.2, 136.9, 134.1, 133.8, 133.5, 132.6, 130.3, 128.9, 128.7, 128.6, 128.1, 128.0, 127.9 (2), 127.8, 127.7, 126.9, 126.7, 126.3, 125.2, 120.5, 115.9, 53.9, 49.6, 38.7, 36.3, 29.6, 27.3, 25.7.

[0313] HRMS: C 30 H 34 N6O2 calcd [M+H] 511.2816, found 511.2810.

[0314] Synthesis of compound 8b: (S)-2-((R)-3-amino-4-(2-naphthalenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0315]

[0316] Following the procedure for the synthesis of reference compound 4a, using (R)-3-(BOC- amino)-4-(2-naphthalenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid, the target compound 8b was obtained as a white solid in 55% yield over two steps;

[0317] Compound 8b: 1H NMR (500 MHz, Methanol-d4) δ 8.21 (d, J = 2.1 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.87-7.85 (m, 2H), 7.84-7.80 (m, 3H), 7.77 (d, J = 8.2 Hz, 1H), 7.60 (dd, J = 8.8, 2.1 Hz, 1H), 7.49 (td, J = 6.2, 3.0 Hz, 3H), 7.46-7.42 (m, 2H), 4.55 (dd, J = 8.4, 5.6 Hz, 1H), 4.00-3.95 (m, 1H), 3.34 (t, J = 1.7 Hz, 4H), 3.29-3.24 (m, 3H), 3.16 (dd, J = 13.8, 8.3 Hz, 1H), 2.81 (dd, J = 16.4, 4.5 Hz, 1H), 2.67 (dd, J = 16.4, 8.2 Hz, 1H), 2.02-1.94 (m, 1H), 1.89-1.70 (m, 3H).

[0318] 13 C NMR (151 MHz, Methanol-d4) δ 171.2, 171.0, 157.3, 135.5, 133.8, 133.7, 132.8, 132.8, 130.8, 128.5, 128.2, 128.0, 127.3 (2), 127.2, 127.1, 126.7, 126.1, 126.1, 125.7, 124.8, 119.9, 116.8, 53.9, 50.1, 40.6, 38.5, 35.4, 28.8, 25.1.

[0319] HRMS: C 30 H 34 N6O2 calcd [M+H] 511.2816, found 511.2807.

[0320] Synthesis of compound 8c: (S)-2-((S)-3-amino-4-(2-naphthalenyl)butanamido)-5- guanidino-N-(naphthalen-2-yl)pentanamide

[0321]

[0322] The synthesis of reference compound 4a was followed using (S)-3-(Boc-amino)-4-(2- naphthalenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 8c as a white solid with a two-step yield of 50%;

[0323] Compound 8c: 1H NMR (600 MHz, Methanol-d4) δ 8.22 (d, J = 2.1 Hz, 1H), 7.84 - 7.80 (m, 6H), 7.77 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 1.6 Hz, 1H), 7.59 (dd, J = 8.8, 2.2 Hz, 1H), 7.48 - 7.40 (m, 6H), 4.55 (dd, J = 8.7, 5.4 Hz, 1H), 3.63 - 3.58 (m, 1H), 2.97 (d, J = 6.9 Hz, 2H), 2.88 - 2.85 (m, 2H), 2.50 (dd, J = 14.6, 4.4 Hz, 1H), 2.42 (dd, J = 14.6, 8.9 Hz, 1H), 2.01 - 1.95 (m, 1H), 1.83 - 1.67 (m, 3H).

[0324] 13 C NMR (151 MHz, Methanol-d4) δ 173.1, 171.2, 135.6, 135.5, 133.8, 133.7, 132.5, 130.8, 128.2, 127.9, 127.6, 127.2, 127.2, 127.2 (2), 127.1, 126.1, 125.8, 125.2, 124.8, 120.0, 116.9, 53.7, 50.3, 42.9, 41.1, 39.5, 28.7, 25.6.

[0325] HRMS: C 29 H 32 N4O2 calcd [M+H] 469.2598, found 469.2598.

[0326] Synthesis of compound 8d: (S)-2-((S)-3-amino-4,4-diphenyl-butyrylamido)-5-guanidino-N- (naphthalen-2-yl)pentanamide

[0327]

[0328] The synthesis of reference compound 4a was followed using (S)-3-(Boc-amino)-4,4-diphenyl- butyric acid instead of (S)-3-(Boc-amino)-4-phenylbutyric acid to give the target compound 8d as a light yellow solid in 33% yield over two steps;

[0329] Compound 8d: 1H NMR (600 MHz, Methanol-d4) δ 8.24 (d, J = 2.1 Hz, 1H), 7.84 (dd, J = 12.5, 8.4 Hz, 2H), 7.79 (d, J = 8.1 Hz, 1H), 7.61 (dd, J = 8.8, 2.2 Hz, 1H), 7.49 - 7.48 (m, 3H), 7.44 - 7.37 (m, 6H), 7.34 - 7.32 (m, 2H), 7.30 - 7.27 (m, 1H), 7.26 - 7.23 (m, 1H), 4.53 - 4.47 (m, 2H), 4.12 (d, J = 11.4 Hz, 1H), 3.24 (t, J = 7.0 Hz, 2H), 2.65 (dd, J = 16.5, 3.2 Hz, 1H), 2.54 (dd, J = 16.5, 8.8 Hz, 1H), 1.97 - 1.91 (m, 1H), 1.82 - 1.66 (m, 4H).

[0330] 13 C NMR (151 MHz, Methanol-d4) δ 171.4, 171.2, 157.3, 140.7, 139.8, 135.5, 133.8, 130.9, 129.0 (2), 128.8 (2), 128.3, 127.8 (2), 127.8 (2), 127.4, 127.2, 127.1, 127.1, 126.2, 124.8, 119.9, 116.7, 55.8, 54.0, 51.4, 40.6, 36.3, 28.7, 25.0.

[0331] HRMS: C 32 H 36 N6O2 calcd [M+H] 537.2972, found 537.2974.

[0332] Synthesis of compound 8e: (S)-2-((S)-3-amino-4-(-phenyl)butanamido)-5-guanidine-N- (naphthalen-2-yl)pentanamide

[0333]

[0334] The synthesis of reference compound 4a was followed by replacing (S)-3-(Boc-amino)-4- phenylbutyric acid with Boc-(S)-l,2,3,4-tetrahydroisoquinoline-3-acetic acid to give the target compound 8e as a white solid with a two-step yield of 40%;

[0335] Compound 8e: 1H NMR (500 MHz, Methanol-d4) δ 8.24 (s, 1H), 7.85 (t, J = 8.4 Hz, 2H), 7.80 (d, J = 8.3 Hz, 1H), 7.63 (d, J = 9.1 Hz, 1H), 7.51 - 7.48 (m, 1H), 7.45 (t, J = 7.5 Hz, 1H), 7.37 - 7.24 (m, 4H), 4.66 - 4.59 (m, 1H), 4.45 (d, J = 8.6 Hz, 2H), 3.97 (d, J = 11.1 Hz, 1H), 3.31 (t, J = 7.1 Hz, 2H), 3.22 (dd, J = 17.6, 4.4 Hz, 1H), 3.08 (dd, J = 17.4, 10.6 Hz, 1H), 2.98 - 2.88 (m, 2H), 2.07 - 2.01 (m, 1H), 1.96 - 1.73 (m, 3H).

[0336] 13 C NMR (151 MHz, Methanol-d4) δ 171.2, 170.4, 157.3, 135.5, 133.8, 130.9, 130.8, 128.7, 128.3, 127.9, 127.5, 127.2, 127.1, 126.9, 126.3, 126.2, 124.8, 119.8, 116.7, 54.1, 51.2, 44.6, 40.7, 36.2, 30.6, 28.9, 25.1.

[0337] HRMS: C 27 H 32 N6O2 calcd [M+H] 473.2659, found 473.2656.

[0338] Synthesis of compound 8f: (S)-2-((R)-3-amino-4-(-phenyl)butanamido)-5-guanidine-N- (naphthalen-2-yl)pentanamide

[0339]

[0340] The synthesis of reference compound 4a was followed using Boc-(R)-1,2,3,4- tetrahydroisoquinoline-3-acetic acid instead of (S)-3-(Boc-amino)-4-phenylbutyric acid to give the target compound 8f as a white solid in 43% yield over two steps;

[0341] Compound 8f: 1H NMR (600 MHz, Methanol-d4) δ 8.24 (d, J = 2.1 Hz, 1H), 7.85 - 7.78 (m, 2H), 7.79 (d, J = 8.1 Hz, 1H), 7.62 (dd, J = 8.8, 2.2 Hz, 1H), 7.49 - 7.47 (m, 1H), 7.44 - 7.41 (m, 1H), 7.31 - 7.27 (m, 2H), 7.25 - 7.22 (m, 2H), 4.60 (dd, J = 8.3, 5.9 Hz, 1H), 4.47 - 4.40 (m, 2H), 3.96 - 3.92 (m, 1H), 3.37 (s, 1H), 3.28 (t, J = 7.0 Hz, 2H), 3.20 (dd, J = 17.2, 4.7 Hz, 1H), 3.06 (dd, J = 17.2, 10.8 Hz, 1H), 2.94 (dd, J = 16.2, 5.3 Hz, 1H), 2.85 (dd, J = 16.2, 7.7 Hz, 1H), 2.05 - 1.99 (m, 1H), 1.93 - 1.73 (m, 3H).

[0342] 13 C NMR (151 MHz, Methanol-d4) δ 171.1, 170.5, 157.3, 135.5, 133.8, 130.9, 130.7, 128.7, 128.3, 127.9, 127.5, 127.2, 127.1, 126.9, 126.3, 126.2, 124.8, 119.9, 116.7, 54.1, 51.3, 44.6, 40.7, 36.3, 30.6, 28.9, 25.1.

[0343] Synthesis of compound 8g: (S)-2-((S)-3-amino-4-(-phenyl)butanamido)-5-guanidine-N- (naphthalen-2-yl)pentanamide

[0344]

[0345] The synthesis of reference compound 4a was followed using (R)-N-tert-butoxycarbonyl (Boc)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid instead of (S)-3-(Boc-amino)-4- phenylbutyric acid to give the target compound 8g as a white solid in 55% yield over two steps;

[0346] Compound 8g: 1H NMR (600 MHz, Methanol-d4) δ 8.23 (d, J = 2.1 Hz, 1H), 7.84 - 7.81 (m, 2H), 7.78 (dd, J = 8.2, 1.2 Hz, 1H), 7.61 (dd, J = 8.8, 2.1 Hz, 1H), 7.49 - 7.47 (m, 1H), 7.44 - 7.41 (m, 1H), 7.28 - 7.23 (m, 3H), 7.19 (dd, J = 7.0, 2.0 Hz, 1H), 4.67 (dd, J = 8.5, 5.7 Hz, 1H), 4.32 (s, 2H), 4.13 (dd, J = 11.5, 4.7 Hz, 1H), 3.37 (s, 2H), 3.29 (td, J = 7.0, 2.3 Hz, 2H), 3.15 (dd, J = 16.6, 11.5 Hz, 1H), 2.09 - 2.03 (m, 1H), 1.95 - 1.89 (m, 1H), 1.85 - 1.71 (m, 2H).

[0347] 13 C NMR (151 MHz, Methanol-d4) δ 170.7, 170.4, 157.3, 135.4, 133.8, 131.3, 130.9, 129.9, 128.6, 128.3, 127.3, 127.2, 127.1, 126.8, 126.2, 126.1, 124.8, 119.9, 116.8, 55.5, 53.8, 44.9, 40.6, 30.2, 29.2, 25.1.

[0348] Synthesis of compound 8h: (S)-2-((R)-3-amino-4-(-phenyl)butanamido)-5-guanidine-N- (naphthalen-2-yl)pentanamide

[0349]

[0350] Following the procedure for the synthesis of reference compound 4a, using (S)-N-tert- butyloxycarbonyl-l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid instead of (S)-3-(Boc- amino)-4-phenylbutyric acid, the target compound 8h was obtained as a white solid in 47% yield over two steps;

[0351] Compound 8h: 1H NMR (600 MHz, Methanol-d4) δ 8.25 (d, J = 2.1 Hz, 1H), 7.84 - 7.81 (m, 2H), 7.79 (d, J = 8.2 Hz, 1H), 7.62 (dd, J = 8.8, 2.2 Hz, 1H), 7.49 - 7.46 (m, 1H), 7.43 - 7.41 (m, 1H), 7.31 - 7.26 (m, 3H), 7.22 - 7.20 (m, 1H), 4.70 (dd, J = 8.6, 5.4 Hz, 1H), 4.42 (s, 2H), 4.31 (dd, J = 12.0, 4.8 Hz, 1H), 3.48 (dd, J = 16.9, 4.8 Hz, 1H), 3.34 - 3.32 (m, 1H), 3.29 (td, J = 7.0, 1.8 Hz, 2H), 3.20 (dd, J = 16.9, 12.0 Hz, 1H), 2.09 - 2.03 (m, 1H), 1.95 - 1.91 (m, 1H), 1.89 - 1.73 (m, 2H).

[0352] 13 C NMR (151 MHz, Methanol-d4) δ 170.6, 168.9, 157.3, 135.5, 133.8, 130.8, 130.7, 128.6, 128.3, 128.0, 127.8, 127.2, 127.2, 127.1, 126.2, 126.2, 124.8, 119.9, 116.7, 55.1, 53.9, 44.3, 40.6, 29.7, 29.2, 25.0.

[0353] Synthesis of compound 8i: (S)-2-((S)-3-amino-4-(-phenyl)butanamido)-5-guanidine-N- (naphthalen-2-yl)pentanamide

[0354]

[0355] Following the procedure for the synthesis of reference compound 4a, using (S)-3-(Boc- amino)-4-(4-phenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid, the target compound 8i was obtained as a white solid in 15% yield over two steps;

[0356] 1H NMR (600 MHz, Methanol-d4) δ 8.23 (s, 1H), 7.83-7.79 (m, 2H), 7.77-7.76 (m, 1H), 7.63-7.60 (m, 2H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 2H), 7.25 (s, 1H), 7.16-7.13 (m, 1H), 7.07-7.05 (m, 1H), 4.55 (dd, J = 8.6, 5.6 Hz, 1H), 3.93-3.89 (m, 1H), 3.34-3.20 (m, J, 3H), 3.16-3.12 (m, 1H), 2.77 (dd, J = 16.3, 4.6 Hz, 1H), 2.68 (dd, J = 16.3, 8.2 Hz, 1H), 2.00-1.93 (m, 1H), 1.85-1.65 (m, 3H).

[0357] Compound 8i: HRMS: C 28 H 33 Calcd [M+H] 500.27685, Found 500.27643.

[0358] Synthesis of compound 15a: (S)-2,5-diamino-N-((S)-4-(naphthalen-2-ylamino)-4- oxo-1-phenylbutan-2-yl)pentanamide

[0359]

[0360] Step 1 (step a): 1-hydroxybenzotriazole (HOBT) (169 mg, 1.25 mmol) and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) (312 mg, 1.63 mmol) were added to a solution of (S)-3-(Boc-amino)-4-phenylbutanoic acid (385 mg, 1.37 mmol) in super dry ethyl acetate (15 mL) under stirring, the reaction mixture was stirred at 65 °C for 30 min. Then to the mixture was added 2-naphthylamine (179 mg, 1.25 mmol) and stirred for 8 h. TLC indicated the reaction was complete. The organic phase was concentrated. The residue was purified by flash column chromatography on silica gel, eluting with petroleum ether and ethyl acetate in a gradient from 1:10 to 1:20 by volume to give intermediate 15a-3 as a white solid 380 mg, 75% yield;

[0361] Step 2 (step b): solid intermediate 15a-3 was added to a flask containing super-dry dichloromethane, then trifluoroacetic acid TFA (20% vol, based on the volume of solvent dichloromethane) was added, stirring at room temperature for 3 hours, TLC detection of reaction completion. The organic phase was concentrated. The residue was purified by flash column chromatography on silica gel, gradient elution with dichloromethane / methanol (1.5 vol% ammonia, i.e. 15 mL of ammonia was added to methanol and made up to 1000 mL) in volume ratio from 1:10 to 1:20, to obtain intermediate 15a-4 as a light yellow solid, 198 mg, 77% yield;

[0362] Step 3 (step c): 1-hydroxybenzotriazole (HOBT) (87 mg, 0.64 mmol) and 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) (162 mg, 0.84 mmol) were added to a solution of (S)-2,4-bis((tert-butoxycarbonyl)amino)pentanoic acid (Boc-Orn(Boc)-OH, 0.71 mmol) in super-dry ethyl acetate (15 mL) under stirring, the reaction mixture was stirred at 65°C for 30 min, then intermediate 15a-4 (198 mg, 0.65 mmol) was added to the mixture, stirring for 8 h, TLC detection of reaction completion. The organic phase was concentrated, the residue was purified by flash column chromatography on silica gel, gradient elution with dichloromethane and methanol in volume ratio from 1:10 to 1:20, to obtain intermediate 15a-6 as a white solid, 350 mg, 87% yield;

[0363] Step 4 (step d): intermediate 15a-6 was added to a flask containing super-dry dichloromethane, TFA (20% vol, based on the volume of solvent dichloromethane) was added, stirring at room temperature for 3 hours, TLC detection of reaction completion. The organic phase was concentrated. The residue was purified by flash column chromatography on silica gel, gradient elution with dichloromethane / methanol (about 1.5 vol% ammonia, i.e. 15 mL of ammonia was added to methanol and made up to 1000 mL) in volume ratio from 1:10 to 1:20, to obtain the white target compound 15a, 86% yield;

[0364] Compound 15a: 1H NMR (500 MHz, Methanol-d4) δ 8.14 - 8.13 (m, 1H), 7.81 (t, J = 8.1 Hz, 2H), 7.77 (d, J = 8.1 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.48 - 7.44 (m, 1H), 7.42 - 7.39 (m, 1H), 7.34 - 7.30 (m, 4H), 7.25 - 7.22 (m, 1H), 4.70 - 4.64 (m, 1H), 3.79 (t, J = 6.1 Hz, 1H), 3.00 (dd, J = 13.7, 7.0 Hz, 1H), 2.92 (dd, J = 13.7, 7.1 Hz, 1H), 2.76 - 2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.88 - 1.73 (m, 2H), 1.69 - 1.60 (m, 2H).

[0365] 13 C NMR (151 MHz, Methanol-d4) δ 170.3, 168.1, 137.7, 135.7, 133.8, 130.8, 129.1 (2), 128.3, 128.2 (2), 127.2, 127.1, 126.4, 126.2, 124.8, 120.1, 116.8, 52.4, 48.9, 40.4, 40.2, 38.5, 28.4, 22.3.

[0366] Synthesis of compound 15b: (S)-2,5-diamino-N-((S)-1-(4-(tert-butyl)phenyl)-4-(naphthalen-2- ylamino)-4-oxobutan-2-yl)pentanamide

[0367]

[0368] Following the procedure for the synthesis of reference compound 15a, replacing (S)-3-(Boc-amino)-4- phenylbutanoic acid with N-tert-butoxycarbonyl-(S)-3-amino-4-(4-tert-butylphenyl)butanoic acid, the target compound 15b was obtained as a white solid in 12% yield over four steps;

[0369] Compound 15b: 1 H NMR (600 MHz, Methanol-d4) δ 8.18 (m, 1H), 7.80 - 7.75 (m, 3H), 7.55 (dd, J = 8.8, 2.1 Hz, 1H), 7.46 - 7.43 (m, 1H), 7.40 - 7.37 (m, 1H), 7.35 - 7.33 (m, 2H), 7.23 - 7.22 (m, 2H), 4.66 - 4.61 (m, 1H), 2.95-

[0370] 2.87 (m, 2H), 2.74-2.65 (m, 3H), 2.61 (dd, J = 14.5, 8.8 Hz, 1H), 1.69-1.48 (m, 5H), 1.28 (s, 9H).

[0371] 13 C NMR (151 MHz, Methanol-deuterium) δ 161.8, 161.6, 135.8, 134.8, 133.8, 128.9 (2), 128.1, 127.2, 127.1, 126.1, 125.0 (2), 124.7, 120.0, 117.8, 116.5, 115.9, 53.8, 48.5, 48.2, 40.8, 38.9, 33.8, 31.3, 30.4 (3), 23.6.

[0372] Synthesis of compound 15c: (S)-2,5-diamino-N-((S)-1-(4-(benzyloxy)phenyl)-4-(naphthalen-2-ylamino)-4-oxobutan-2-yl)pentanamide

[0373]

[0374] Following the procedure for the synthesis of reference compound 15a, replacing (S)-3-(Boc-amino)-4-phenylbutanoic acid with (3S)-4-[4-(benzyloxy)phenyl]-3-(tert- butoxycarbonylamino)butanoic acid, the target compound 15c was obtained as a white solid in 24% yield over four steps;

[0375] Compound 15c: 1 H NMR (600 MHz, Methanol-deuterium) δ 8.12 (m, 1H), 7.80 (t, J = 8.7 Hz, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.53-7.51 (m, 1H), 7.47-7.44 (m, 1H), 7.41-7.39 (m, 3H), 7.37-7.34 (m, 2H), 7.31-7.28 (m, 1H), 7.23-

[0376] 7.20 (m, 2H), 6.95-6.94 (m, 2H), 5.02 (s, 2H), 4.64-4.61 (m, 1H), 3.88-3.86 (m, 1H), 2.94 (dd, J = 13.8, 6.7 Hz, 1H), 2.85 (dd, J = 13.8, 7.2 Hz, 1H), 2.75-2.70 (m, 3H), 2.58-2.54 (m, 1H), 1.91-1.78 (m, 2H), 1.70-1.64 (m, 2H).

[0377] 13C NMR (151 MHz, Methanol-d4) δ 170.3, 167.5, 157.8, 137.4, 135.7, 133.8, 130.8, 130.1 (2), 129.8, 128.3, 128.1 (2), 127.5, 127.3, 127.1 (3), 126.2, 124.8, 120.1, 116.8, 114.7 (2), 69.6, 52.3, 49.1, 40.5, 39.5, 38.5, 28.1, 22.2.

[0378] Synthesis of compound 15d: (S)-2,5-diamino-N-((S)-1-(4-cyanophenyl)-4-(naphthalen-2- ylamino)-4-oxobutan-2-yl)pentanamide

[0379]

[0380] The target compound 15d was obtained as a white solid in 21% yield over four steps by following the synthetic procedure of reference compound 15a, using N-tert-butoxycarbonyl-(S)-3-amino-4-(4-cyanophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid;

[0381] Compound 15d: 1 H NMR (500 MHz, Methanol-d4) δ 8.14 - 8.13 (m, 1H), 7.81 (t, J = 8.8 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.69 - 7.68 (m, 2H), 7.53 - 7.49 (m, 3H), 7.48 - 7.45 m, 1H), 7.43 - 7.37 (m, 1H), 4.75 - 4,69 (m, 1H), 3.85 (t, J = 6.1 Hz, 1H), 3.05 (dd, J = 7.0, 3.1 Hz, 2H), 2.77 (dd, J = 14.7, 4.4 Hz, 1H), 2.74 - 2.69 (m, 2H), 2.59 (dd, J = 14.7, 9.4 Hz, 1H), 1.91 - 1.76 (m, 2H), 1.69 - 1.62 (m, 2H).

[0382] 13 C NMR (151 MHz, Methanol-d4) δ 169.9, 167.6, 143.7, 135.6, 133.8, 132.0 (2), 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 120.0, 118.3, 116.8, 110.3, 52.2, 40.6, 40.2, 38.5, 28.1, 27.6, 22.2.

[0383] Synthesis of compound 15e: (S)-2,5-diamino-N-((S)-4-(naphthalen-2-ylamino)-4- oxo-1-(p-tolyl)butan-2-yl)pentanamide

[0384]

[0385] The synthesis of reference compound 15a was followed using (S)-N-tert- butyloxycarbonyl-3-amino-4-(4-methylphenyl)butanoic acid instead of (S)-3-(Boc- amino)-4-phenylbutanoic acid to give the target compound 15e as a white solid in 34% yield over four steps;

[0386] Compound 15e: 1 H NMR (500 MHz, Methanol-d4) d 8.12 (m, 1H), 7.81 (t, J = 7.9 Hz, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.51 (dd, J = 8.8, 2.1 Hz, 1H), 7.47 - 7.44 (m, 1H), 7.42 - 7.39 (m, 1H), 7.19 - 7.12 (m, 4H), 4.67 - 4.61 (m, 1H), 3.85 (t, J = 6.1 Hz, 1H), 2.96 (dd, J = 13.7, 6.8 Hz, 1H), 2.87 (dd, J = 13.7, 7.2 Hz, 1H), 2.75 - 2.70 (m, 3H), 2.55 (dd, J = 14.7, 9.5 Hz, 1H), 2.29 (s, 3H), 1.91 - 1.77 (m, 2H), 1.69 - 1.63 (m, 2H).

[0387] 13 C NMR (151 MHz, Methanol-d4) d 170.3, 167.5, 136.1, 135.6, 134.5, 133.8, 130.8, 129.0 (2), 128.8 (2), 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 52.3, 49.0, 40.4, 39.9, 38.5, 28.1, 22.2, 19.7.

[0388] Synthesis of compound 15f: (S)-2,5-diamino-N-((S)-1-(4-fluorophenyl)-4-(naphthalen-2- ylamino)-4-oxobutan-2-yl)pentanamide

[0389]

[0390] The synthesis of compound 15a was followed using (S)-N-tert-butoxycarbonyl-3- amino-4-(4-fluorophenyl)butanoic acid instead of (S)-3-(Boc-amino)-4- phenylbutanoic acid to give the target compound 15f as a white solid in 21% yield over four steps;

[0391] Compound 15f: 1 H NMR (600 MHz, Methanol-d4) δ 8.13 (m, 1H), 7.82-7.79 (m, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.32-7.30 (m, 2H), 7.06-7.02 (m, 2H), 4.66-4.63 (m, 1H), 3.86 (t, J = 6.1 Hz, 1H), 2.95 (qd, J = 13.9, 7.0 Hz, 2H), 2.76-2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.89-1.79 (m, 2H), 1.69-1.64 (m, 2H).

[0392] Synthesis of compound 15g: (S)-2,5-diamino-N-((S)-4-(naphthalen-2-ylamino)-1-(4- nitrophenyl)-4-oxobutan-2-yl)pentanamide

[0393]

[0394] The synthesis of compound 15a was followed using Boc-(S)-3-amino-4-(4- nitrophenyl)-butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 15g as a white solid in 8% yield over four steps;

[0395] Compound 15g: 1 H NMR (600 MHz, Methanol-d4) δ 8.13 (m, 1H), 7.82-7.79 (m, 2H), 7.77 (d, J = 8.2 Hz, 1H), 7.52 (dd, J = 8.8, 2.1 Hz, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.32-7.30 (m, 2H), 7.06-7.02 (m, 2H), 4.66-4.63 (m, 1H), 3.86 (t, J = 6.1 Hz, 1H), 2.95 (qd, J = 13.9, 7.0 Hz, 2H), 2.76-2.69 (m, 3H), 2.57 (dd, J = 14.7, 9.4 Hz, 1H), 1.89-1.79 (m, 2H), 1.69-1.64 (m, 2H).

[0396] 3.85 (m, 1H), 3.14-3.06 (m, 2H), 2.82-2.72 (m, 3H), 2.64-2.60 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).

[0397] 13 C NMR (151 MHz, Methanol-d4) δ 169.9, 167.6, 147.0, 145.7, 135.6, 133.8, 130.8, 130.2 (2), 128.3, 127.3, 127.1, 126.3, 124.9, 123.2 (2), 120.0, 116.8, 52.2, 48.5, 40.7, 39.9, 38.5, 28.1, 22.2.

[0398] Synthesis of compound 15h: (S)-2,5-diamino-N-((S)-1-(4-methoxyphenyl)-4-(naphthalen-2- ylamino)-4-oxobutan-2-yl)pentanamide

[0399]

[0400] Following the procedure for the synthesis of reference compound 15a, replacing (S)-3-(Boc-amino)-4- phenylbutyric acid with Boc-S-3-amino-4-(4-methoxyphenyl)-butyric acid, the target compound 15h was obtained as a white solid in 11% yield over four steps;

[0401] Compound 15h: 1 H NMR (600 MHz, Methanol-d4) δ 8.13 (s, 1H), 7.82-7.79 (m, 2H), 7.76 (d, J = 8.2 Hz, 1H), 7.53-7.51 (m, 1H), 7.47-7.44 (m, 1H), 7.42-7.39 (m, 1H), 7.22-7.20 (m, 2H), 6.88-6.86 (m, 2H), 4.64-4.60 (m, 1H), 3.87-3.86 (m, 1H), 3.75 (m, 3H), 2.93 (dd, J = 13.9, 6.9 Hz, 1H), 2.87-2.83 (m, 1H), 2.75-2.70 (m, 3H), 2.58-2.53 (m, 1H), 1.91-1.77 (m, 2H), 1.69-1.63 (m, 2H).

[0402] 13 C NMR (151 MHz, Methanol-d4) δ 170.4, 167.5, 158.7, 135.7, 133.8, 130.8, 130.0 (2), 129.5, 128.3, 127.2, 127.1, 126.2, 124.8, 120.1, 116.8, 113.6 (2), 54.2, 52.3, 49.1, 40.4, 39.4, 38.5, 28.1, 22.2.

[0403] Synthesis of compound 15i: (S)-2,5-diamino-N-((S)-l-(naphthalen-2-yl)-4-(naphthalen-2- ylamino)-4-oxobutan-2-yl)pentanamide

[0404]

[0405] Following the procedure for the synthesis of compound 15a, replacing (S)-3-(Boc-amino)-4- phenylbutanoic acid with Boc-(S)-3-amino-4-(2-naphthyl)-butanoic acid, the target compound 15i was obtained as a white solid in 11% yield over four steps;

[0406] Compound 15i: ¾ NMR (500 MHz, Methanol-d4) d 8.11 (m, 1H), 7.85-7.79 (m, 5H), 7.77-7.75 (m, 2H), 7.51-7.43 (m, 5H), 7.42-7.39 (m, 1H), 4.89-4.76 (m, 1H), 3.84 (t, J = 6.1 Hz, 1H), 3.18 (dd, J = 13.7, 7.2 Hz, 1H), 3.11 (dd, J = 13.7, 7.0 Hz, 1H), 2.81 (dd, J = 14.7, 4.3 Hz, 1H), 2.69 (t, J = 7.4 Hz, 2H), 2.63 (dd, J = 14.7, 9.3 Hz, 1H), 1.89-1.76 (m, 2H), 1.68-1.61 (m, 2H).

[0407] 13 C NMR (151 MHz, Methanol-d4) d 170.3, 167.6, 135.6, 135.2, 133.8, 133.7, 132.6, 130.8, 128.3, 127.9, 127.6, 127.2 (4), 127.1, 126.2, 125.8, 125.3, 124.8, 120.1, 116.9, 52.3, 49.0, 40.6, 40.3, 38.5, 28.2, 22.2.

[0408] Synthesis of compound 15j: (S)-2,5-diamino-N-((S)-4-oxo-l-phenyl-4-(quinolin-3- ylamino)butan-2-yl)pentanamide

[0409]

[0410] Following the procedure for the synthesis of compound 15a, replacing 2-naphthylamine with 3- aminoquinoline, the target compound 15j was obtained as a white solid in 4% yield over four steps;

[0411] Compound 15j: 1H NMR (600 MHz, Methanol-d4) δ 8.97 - 8.96 (m, 1H), 8.63 - 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90 - 7.89 (m, 1H), 7.72 - 7.69 (m, 1H), 7.63 - 7.60 (m, 1H), 7.32 - 7.29 (m, 4H), 7.23 - 7.20 (m, 1H), 4.68 - 4.66 (m, 1H), 3.89 - 3.87 (m, 1H), 3.03 (dd, J = 13.8, 7.1 Hz, 1H), 2.94 (dd, J = 13.7, 7.1 Hz, 1H), 2.86 - 2.83 (m, 2H), 2.78 (dd, J = 14.9, 4.6 Hz, 1H), 2.68 - 2.64 (m, 1H), 1.95 - 1.90 (m, 1H), 1.86 - 1.80 (m, 1H), 1.76 - 1.71 (m, 2H).

[0412] 13 C NMR (151 MHz, Methanol-d4) δ 170.7, 167.8, 144.0, 143.8, 137.7, 132.5, 129.1 (2), 128.7, 128.4, 128.2 (2), 127.5, 127.4, 127.2, 126.4, 124.8, 52.3, 48.9, 40.5, 40.1, 38.6, 28.2, 22.4.

[0413] Synthesis of compound 15k: (S)-2,5-diamino-N-((S)-1-(naphthalen-2-yl)-4-oxo-4- (quinolin-3-ylamino)butan-2-yl)pentanamide

[0414]

[0415] The target compound 15k was obtained as a white solid in 11% yield over four steps by referring to the synthetic procedure of compound 15a, replacing 2-naphthylamine with 3- aminoquinoline and Boc-(S)-3-amino-4-(2-naphthyl)butanoic acid with (S)-3-(Boc-amino)-4- phenylbutanoic acid;

[0416] Compound 15k: 1H NMR (600 MHz, Methanol-d4) δ 8.88 - 8.87 (m, 1H), 8.52 - 8.51 (m, 1H), 7.97 - 7.95 (m, 1H), 7.85 - 7.77 (m, 5H), 7.70 - 7.67 (m, 1H), 7.61 - 7.57 (m, 1H), 7.49 - 7.47 (m, 1H), 7.45 - 7.39 (m, 2H), 4.81 - 4.76 (m, 1H), 3.87 - 3.85 (m, 1H), 3.22 - 3.19 (m, 1H), 3.13 - 3.09 (m, 1H), 2.86 - 2.79 (m, 3H), 2.71 - 2.66 (m, 1H), 1.92 - 1.79 (m, 2H), 1.73 - 1.68 (m, 2H).

[0417] 13 C NMR (151 MHz, Methanol-d4) δ 170.6, 167.8, 144.2, 135.2, 133.6, 132.5, 132.4, 128.5, 128.3, 127.9, 127.7, 127.4 (3), 127.3, 127.2 (3), 125.8, 125.3, 124.5, 52.3, 48.9, 40.7, 40.3, 38.5, 28.3, 22.4.

[0418] Synthesis of compound 15l: (S)-2,5-diamino-N-((S)-1-(4-nitrophenyl)-4-oxo-4-(quinolin-3- ylamino)butan-2-yl)pentanamide

[0419]

[0420] The synthesis of reference compound 15a was followed using 3-aminoquinoline instead of 2-naphthylamine and Boc-(S)-3-amino-4-(4-nitrophenyl)-butyric acid instead of (S)-3-(Boc-amino)-4- phenylbutyric acid to give the target compound 15l as a white solid in 33% yield over four steps;

[0421] Compound 15l: 1H NMR (600 MHz, Methanol-d4) δ 8.96 - 8.95 (m, 1H), 8.62 (s, 1H), 8.17 (m, 2H), 7.98 (d, J = 8.3 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.72 - 7.69 (m, 1H), 7.63 - 7.60 (m, 1H), 7.56 - 7.54 (m, 2H), 4.77 - 4.72 (m, 1H), 3.88 - 3.87 (m, 1H), 3.12 - 3.09 (m, 2H), 2.87 - 2.80 (m, 3H), 2.69 (dd, J = 14.5, 8.5 Hz, 1H), 1.91 (m, 1H), 1.85 - 1.79 (m, 1H), 1.74 - 1.71 (m, 2H).

[0422] 13 C NMR (151 MHz, Methanol-d4) δ 170.3, 167.8, 147.0, 145.7, 144.2 (2), 132.4, 130.2 (2), 128.5, 128.3, 127.5, 127.4 (2), 124.4, 123.2 (2), 52.2, 48.4, 40.7, 39.8, 38.5, 28.2, 22.4.

[0423] Synthesis of compound 15m: (S)-2,5-diamino-N-((S)-l-(4-chlorophenyl)-4-oxo-4(quinolin-3- ylamino)butan-2-yl)pentanamide

[0424]

[0425] The synthesis of reference compound 15a was followed using 3-aminoquinoline instead of 2-naphthylamine and Boc-(S)-3-amino-4-(4-chlorophenyl)-butyric acid instead of (S)-3-(Boc-amino)-4- phenylbutyric acid to give the target compound 15m as a white solid in 33% yield over four steps;

[0426] Compound 15m: 1 H NMR (600 MHz, Methanol-d4) δ 8.99 (s, 1H), 8.64 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.91 - 7.90 (m, 1H), 7.73 - 7.70 (m, 1H), 7.64 - 7.61 (m, 1H), 7.30 (m, 4H), 4.68 - 4.63 (m, 1H), 3.88 (s, 1H), 3.00 - 2.93 (m, 2H), 2.85 - 2.77 (m, 3H), 2.65 (dd, J = 14.3, 8.6 Hz, 1H), 1.92 (s, 1H), 1.84 - 1.73 (m, 3H).

[0427] 13 C NMR (151 MHz, Methanol-d4) δ 170.5, 167.8, 144.2, 144.1, 136.4, 132.5, 132.3, 130.7 (2), 128.5, 128.3 (3), 127.5 (2), 127.3, 124.5, 52.3, 48.7, 40.5, 39.3, 38.6, 28.2, 22.4.

[0428] Synthesis of compound 15n: (S)-2,5-diamino-N-((S)-1-(4-(benzyloxy)phenyl)-4-oxo-4- (quinolin-3-ylamino)butan-2-yl)pentanamide

[0429]

[0430] The synthesis of reference compound 15a was followed using 3-aminoquinoline instead of 2-naphthylamine and (3S)-4-[4-(benzyloxy)phenyl]-3-(tert-butoxycarbonylamino)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 15n as a white solid in 7% yield over four steps;

[0431] Compound 15n: 1 H NMR (600 MHz, Methanol-d4) δ 8.97-8.96 (m, 1H), 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.88 (m, 1H), 7.72-7.69 (m, 1H), 7.62-7.60 (m, 1H), 7.37-7.28 (m, 5H), 7.23-7.19 (m, 2H), 6.92 (d, J = 7.4 Hz, 2H), 4.98 (s, 2H), 4.65-4.60 (m, 1H), 3.90-3.88 (m, 1H), 2.96 (dd, J = 13.8, 6.8 Hz, 1H), 2.87-2.81 (m, 3H), 2.76 (dd, J = 14.5, 5.2 Hz, 1H), 2.62 (dd, J = 14.8, 8.7 Hz, 1H), 1.95-1.80 (m, 2H), 1.76-1.71 (m, 2H).

[0432] 13C NMR (151 MHz, Methanol-^) d 170.7, 167.7, 157.8, 144.2, 144.1, 137.3, 132.5, 130.1 (2), 129.8, 128.5, 128.3, 128.1 (2), 127.5 (3), 127.3, 127.1 (2), 124.5, 114.6 (2), 69.6, 52.3, 49.0, 40.4, 39.4, 38.6, 28.2, 22.4.

[0433] Synthesis of compound 15o: (S)-2,5-diamino-N-((S)-l-(4-(tert-butyl)phenyl)-4-oxo-4- (quinolin-3-ylamino)butan-2-yl)pentanamide

[0434]

[0435] The synthesis of reference compound 15a was followed using 3-aminoquinoline instead of 2-naphthylamine and N-tert-butoxycarbonyl-(S)-3-amino-4-(4-tert-butylphenyl)butanoic acid instead of (S)-3-(Boc-amino)-4-phenylbutanoic acid to give the target compound 15o as a white solid in 33% yield over four steps;

[0436] Compound 15o: 1 H NMR (600 MHz, Methanol-^) d 8.97 (m, 1H), 8.62 (m, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.90-7.88 (m, 1H), 7.72-7.70 (m, 1H), 7.63-7.60 (m, 1H), 7.34 (d, J = 7.6 Hz, 2H), 7.24-7.22 (m, 2H), 4.68-4.63 (m, 1H), 3.91-3.89 (m, 1H), 3.00 (dd, J = 13.7, 6.5 Hz, 1H), 2.90-2.84 (m, 3H), 2.77 (dd, J = 14.9, 4.8 Hz, 1H), 2.62 (dd, J = 14.8, 8.8 Hz, 1H), 1.96-1.84 (m, 2H), 1.77-1.73 (m, 2H), 1.26 (s, 9H).

[0437] 13C NMR (151 MHz, Methanol-deuterium) d 170.7, 167.7, 149.4, 144.2, 144.1, 134.4, 132.5, 128.8 (2), 128.5, 128.3, 127.5, 127.4, 127.3, 125.1 (2), 124.4, 52.3, 48.8, 40.3, 39.6, 38.6, 33.8, 30.4 (3), 28.2, 22.4.

[0438] Synthesis of compound 15p: 2-((S)-2-((S))-2,5-diaminopentanoyl)-1,2,3,4- tetrahydroisoquinolin-3-yl)-N-(naphthalen-2-yl)acetamide

[0439]

[0440] Referring to the synthesis method of target compound 15a, N-tert-butoxycarbonyl- (S)-1,2,3,4-tetrahydroisoquinoline-3-acetic acid is used instead of (S)-3-(Boc-amino)-4- phenylbutyric acid to obtain the target compound 15p as a white solid with a four-step yield of 4%;

[0441] HRMS: C 26 H 30 N4O2 calculated value [M+H] 431.2472, found value 431.2455.

[0442] Effect example

[0443] Effect example 1: determination of relative final fluorescence index (RFFI)

[0444] The ability of the dipeptide mimetic compounds of the present application to inhibit the efflux of Hoechst 33342 in Pseudomonas aeruginosa N150ABM was evaluated using the relative final fluorescence index.

[0445] Sterile LB Broth (containing 200 μg / mL of carbenicillin corresponding to plasmid resistance, PM0011L LB Broth, Lennox / ReadyLB Broth (Lennox) purchased from Beijing Coolab Technology Co., Ltd.):

[0446] Test samples: compounds synthesized in the above preparation examples and PAβN (purchased from MedChemExpress);

[0447] Test strain: Pseudomonas aeruginosa N150ABM (also known as P. aeruginosa 26) (transformed with plasmid pAK1900-mexAB-oprM to knock out the major efflux pump of P. aeruginosa strain N150 and make MexAB-OprM specific overexpression, the preparation method is described in patent CN1876832A, Example 2), which is from the Institute of Medical Biotechnology, Chinese Academy of Medical Sciences, and N150 was donated by Nobuhisa Masuda, Biological Research Laboratories, Sankyo Co., Ltd., Shinagawa-ku, Tokyo 140-8710, reference: Masuda N, Sakagawa E, Ohya S, Gotoh N, Tsujimoto H, Nishino T. Substrate specificities of MexAB-OprM, MexCD-OprJ, and MexXY-oprM efflux pumps in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2000 Dec;44(12):3322-3327.

[0448] Test method: The test was performed according to the method described in the literature (Tambat R, Mahey N, Chandal N, et al. A Microbe-Derived Efflux Pump Inhibitor of the Resistance-Nodulation-Cell Division Protein Restores Antibiotic Susceptibility in Escherichia coli and Pseudomonas aeruginosa. ACS Infect Dis. 2022;8(2):255-270). The specific steps are as follows:

[0449] (1) Preparation of bacterial suspension: Under sterile conditions, a colony of P. aeruginosa N150ABM from the LB agar medium containing the corresponding carbenicillin 200 μg / mL was inoculated into 2 mL sterile LB broth in a sterile 20 mL universal tube, and the test was inoculated in LB broth medium for overnight culture. Then incubate in a 37°C incubator with shaking (200 rpm) overnight.

[0450] The overnight culture was subcultured into 40 mL of pre-warmed (37°C) LB broth at 1% volume ratio and incubated at 37°C with shaking (200 rpm) under sterile conditions until the OD600 nm reached 1.0.

[0451] The bacteria were collected by centrifugation at 3220 x g for 10 min at 4°C from the centrifuge tube containing 20 mL of bacterial solution. The supernatant was discarded and the bacterial pellet was resuspended in 10 mL of phosphate buffer solution (PBS, 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 were dissolved in 800 mL of distilled water. The pH was adjusted to 7.4 with HC1, and then distilled water was added to make up to 1000 mL. The solution was divided into aliquots, and autoclaved at 20 min at 15 psi (1.05 kg / cm2) of high-pressure steam, and stored at room temperature). The precipitated bacteria were collected again by centrifugation at 3220 x g for 10 min at 4°C, and the bacterial pellet was resuspended in 0.1 mL of detection buffer (110 mM of NaCl, 7 mM of KCl, 50 mM of NH4Cl, 0.4 mM of Na2HPO4, 52 mM of Tris base, pH 7.5), and then diluted with PBS to an OD600 nm of 0.6, which was the bacterial suspension. 2 ) high-pressure steam for 20 min at 15 psi (1.05 kg / cm2), and stored at room temperature). The precipitated bacteria were collected again by centrifugation at 3220 x g for 10 min at 4°C, and the bacterial pellet was resuspended in 0.1 mL of detection buffer (110 mM of NaCl, 7 mM of KCl, 50 mM of NH4Cl, 0.4 mM of Na2HPO4, 52 mM of Tris base, pH 7.5), and then diluted with PBS to an OD600 nm of 0.6, which was the bacterial suspension.

[0452] (2) Addition of efflux pump inhibitor: The bacterial suspension prepared above was diluted 2-fold with the efflux pump inhibitor to a final concentration of 64, 32, 16, 0 μg / mL per 48 μl. 2 μl of 62.5 μM Hoechst 33342 was added to each to a final concentration of 2.5 μM.

[0453] (3) Incubation: After incubation at room temperature for 30 min, the Hoechst 33342-pretreated cells obtained in step (2) above were centrifuged at 15,000 x g for 3 min at 4°C, the supernatant was discarded, and resuspended in 50 μl of PBS, and then transferred to a black 96-well half-plate. After incubation at room temperature for 45 min, the fluorescence intensity was measured in a microplate reader (Model: EnSpire™ 2300 Multilabel Reader, Manufacturer: PerkinElmer) at excitation and emission wavelengths of 350 nm and 460 nm, respectively.

[0454] (4) Calculation: RFFI is calculated according to the formula: RFFI = (45 min treated RFF - 45 min untreated RFF) / 45 min untreated RFF, wherein 45 min treated RFF is the fluorescence intensity of the treated group determined; 45 min untreated RFF is the fluorescence intensity of the untreated group determined; the group with a final concentration of 0 μg / mL of efflux pump inhibitor is the untreated group (blank group, i.e. without efflux pump inhibitor), and the group with a final concentration of 64, 32, 16 μg / mL of efflux pump inhibitor is the treated group.

[0455] The RFFI value is greater than zero, i.e. the fluorescence intensity of the treated group is higher than that of the untreated group, indicating that the accumulation of Hoechst 33342 in the treated group of cells is enhanced compared with the untreated group of cells, and the efflux pump inhibitor of the treated group has the ability to reduce the efflux of bacteria (Pseudomonas aeruginosa N150ABM); the greater the RFFI value, the higher the degree of accumulation of Hoechst 33342 in the cells, and the stronger the effect of the efflux pump inhibitor on reducing the efflux ability of bacteria (Pseudomonas aeruginosa N150ABM). Generally, when the RFFI value is greater than 1, it is considered that the efflux pump inhibitor significantly enhances the accumulation of Hoechst 33342 in the cells, and the inhibitory effect on the efflux ability of bacteria is obviously improved, and the efflux pump inhibitor has excellent inhibitory effect on the efflux ability of bacteria.

[0456] To ensure the reliability of the data, three parallel tests were performed for each sample, and the test data were the average values of the three parallel tests.

[0457] According to the above-described determination method, the pseudo-dipeptide compound of the application and PaβN as the efflux pump inhibitor were determined respectively, and the test results are shown in Tables 2 to 4.

[0458] Table 2: RFFI value of efflux pump inhibitor at a concentration of 16 μg / mL for the efflux inhibition of Hoechst 33342 in Pseudomonas aeruginosa

[0459]

[0460] Table 3: RFFI value of efflux pump inhibitor at a concentration of 32 μg / mL for the efflux inhibition of Hoechst 33342 in Pseudomonas aeruginosa

[0461]

[0462]

[0463] Table 4: RFFI value of efflux pump inhibitor at a concentration of 64 μg / mL for the efflux inhibition of Hoechst 33342 in Pseudomonas aeruginosa

[0464]

[0465] Table 2 to Table 4 show that the RFFI values of the compounds of the present application at 16, 32 and 64 pg / mL are significantly improved in P. aeruginosa; and, under the condition of using the same efflux pump inhibitor, the RFFI values increase as the concentration of the efflux pump inhibitor increases; thus indicating that the compounds of the present application exhibit a dose-dependent effect of Hoechst 33342 accumulation and inhibition of bacterial efflux ability as an efflux pump inhibitor, and the compounds of the present application are significantly higher than the control PAβN in terms of such an effect of inhibiting bacterial efflux ability.

[0466] Effect Example 2: Hoechst 33342 intracellular accumulation curve

[0467] By testing the intracellular accumulation rate of Hoechst 33342 in bacteria by the pseudo-dipeptide compounds of the present application, it is further proved that the pseudo-dipeptide compounds of the present application can reduce the efflux of H33342 by the efflux pump, and can be used as an efflux pump inhibitor.

[0468] The sterile LB broth, test sample and test strain are as described in Effect Example 1.

[0469] Test method: The test was performed according to the method for determining the accumulation rate of Hoechst 33342 as described in the literature (Tambat R, Mahey N, Chandal N, et al. “A Microbe-Derived Efflux Pump Inhibitor of the Resistance-Nodulation-Cell Division Protein Restores Antibiotic Susceptibility in Escherichia coli and Pseudomonas aeruginosa”. ACS Infect Dis. 2022; 8(2): 255-270). The specific experimental operation is as follows:

[0470] (1) Preparation of bacterial suspension: Under sterile conditions, Pseudomonas aeruginosa N150ABM colonies from LB agar medium (containing 200 μg / mL of carbenicillin corresponding to plasmid resistance) overnight culture were placed in 2 mL of sterile LB broth in a sterile 20 mL universal tube, and inoculated in LB broth medium for overnight culture of the test. Then, the overnight culture was incubated in a 37°C incubator with shaking (200 rpm) overnight. The above overnight culture was transferred at a volume ratio of 1% into 40 mL of preheated (37°C) LB broth, and incubated at 37°C with shaking (200 rpm) under sterile conditions until the OD600nm reached 1.0.

[0471] 4°C, 3220 x g for 10 min, and the bacteria were collected from the centrifuge tube containing 20 mL of bacterial solution. The supernatant was discarded, and the bacterial pellet was resuspended in 10 mL of detection buffer (110 mM NaCl, 7 mM KCl, 50 mM NH4Cl, 0.4 mM Na2HPO4, 52 mM Tris base, pH 7.5), and the precipitated bacteria were collected by centrifugation at 3220 x g for 10 min at 4°C. The bacterial pellet was resuspended in a small amount of detection buffer, and diluted with detection buffer to an OD600nm of 0.3 to obtain the bacterial suspension.

[0472] (2) Addition of efflux pump inhibitors: The bacterial suspension prepared above was diluted 2-fold to obtain a final concentration of 64, 32, and 16 μg / mL of efflux pump inhibitors in the detection system, and added to a black 96-well half-plate at 48 μL per well. The untreated group was not added with efflux pump inhibitors, and was also referred to as the blank group. 2 μL of 62.5 μM Hoechst 33342 was added to each well to obtain a final concentration of 2.5 μM. The efflux pump inhibitors were the compounds synthesized in the above preparation examples, and were used as the test group. The efflux pump inhibitors were the control PAβN, and were used as the positive control group.

[0473] (3) Testing: The fluorescence intensity was measured for 45 min at an interval of 5 min each time in a microplate detector with an excitation wavelength of 355 nm and an emission wavelength of 465 nm, and was the fluorescence intensity of Hoechst-33342 (a commonly used RND efflux pump substrate that can bind to DNA). To ensure reliable data, each sample was tested in triplicate, and the test data were the average values of the triplicate tests.

[0474] The determination method described above was used to determine the peptidomimetic compounds of the present application and PAβN as the efflux pump inhibitors. In order to clearly characterize the relationship between the determined fluorescence intensity and time, the curve of the fluorescence intensity versus time was plotted, as shown in Figures 1 to 16 .

[0475] The results of the determination of the peptidomimetic compounds of the present application and PAβN as the efflux pump inhibitors are shown in Table 1.Figures 1-17 As can be seen, the fluorescence intensity of the test group containing the efflux pump inhibitor of the present application is significantly higher than that of the blank group (untreated group) compared to the positive control group PAβN and the blank group (untreated group without efflux pump inhibitor), and the fluorescence intensity of the test group is significantly higher than that of the positive control group PAβN under the condition of containing the efflux pump inhibitor with the same concentration. Thus, it is indicated that the efflux pump inhibitor of the present application has a higher intracellular accumulation rate of Hoechst 33342, indicating that it can effectively inhibit the efflux of the substrate Hoechst 33342 by the efflux pump, and the efflux capacity of the efflux pump of the microorganism is significantly inhibited.

Claims

1. A compound of formula (I), or a stereoisomer thereof, and a pharmaceutically acceptable salt, in, X is CH. n and m are each an independent integer from 0 to 3. Q is hydrogen. R1 is guanidino, amino, or phenyl, wherein the phenyl group is substituted with a benzyloxy group; W is tetrahydroisoquinolinyl, methylenetetrahydroisoquinolinyl; or selected from one of the following formulas: Wherein, Ar1 is phenyl or naphthyl, and the phenyl is optionally substituted by one or more substituents selected from the following: (C1-C6)-alkyl, benzyloxy, halogen, or halo-(C1-C6)-alkyl. Ar2 and Ar3 are each independently phenyl; and Under the condition that W is the same as equation (I-3), n is an integer from 1 to 3; The tilde indicates the connection point with the rest of equation (I).

2. The compound according to claim 1, wherein... n is 0 or 1, m is an integer from 0 to 2. Ar1 is phenyl or naphthyl, wherein the phenyl group is optionally substituted by one or more substituents selected from the following: tert-butyl, benzyloxy, fluorine, chlorine, bromine, iodine, trifluoromethyl, methyl; Under the condition that W is equation (I-3), n is 1.

3. The compound according to claim 1, wherein the compound is selected from at least one compound of formula (II-1) to (II-4): in, X is CH. m is 2. R1 is a guanidinyl or amino group. R2 is phenyl or naphthyl, wherein the phenyl group is optionally substituted with one or more substituents selected from the following: halogen, tert-butyl, benzyloxy, methyl, trifluoromethyl. R3 is hydrogen; or R2 and R3 are each independently phenyl.

4. The compound according to claim 1, wherein the compound is selected from at least one compound of formula (III-1) to (III-4): in, X is CH. k is 2, R4 is a phenyl group, wherein the phenyl group is substituted with a benzyloxy group. R5 is guanidino or amino.

5. The compound according to claim 1, wherein the compound is selected from at least one compound of formula (IV-1) to (IV-4): in, X is CH. q is an integer between 1 and 2. E stands for tetrahydroisoquinolinyl. R6 is a guanidino or amino group.

6. The compound according to claim 1, wherein the compound is selected from at least one of the following compounds:

7. A method for inhibiting the efflux of microbial efflux pumps for purposes other than disease diagnosis and treatment, wherein the method is carried out by using one or more compounds according to any one of claims 1-6.

8. The method of claim 7, further comprising contacting one or more compounds of any one of claims 1-6 with microorganisms; The microorganisms mentioned are Escherichia coli and Pseudomonas aeruginosa.

9. Use of the compound of any one of claims 1-6 for the preparation of an efflux pump inhibitor.

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