Phenazine compounds for measuring bacterial excretion

Abstract

The present invention relates to a compound of the following formula (I), in which X is an anion, and R1 and R2 are both -CH3 or C1-C4 alkyl (wherein R1 is C n+i alkyl and R2 is C n When R is alkyl and vice versa, n is an integer ranging from 1 to 3 and i is an integer ranging from 1 to 3, where n+i ranges from 1 to 4), and R is -CH or C2-C8 alkyl, wherein the C2-C8 alkyl is optionally substituted with one or more heteroatoms. [Formula 1] JPEG2025504776000032.jpg47170

Description

[Technical field]

[0001] The present invention relates to cyclic compounds, in particular their use in measuring bacterial excretion.

[0002] In biology, efflux is a mechanism by which cells release compounds that are toxic to their metabolism (e.g., antibiotics, heavy metals, drugs, etc.) into the external environment. Efflux is an active, energy-dependent transport mechanism mediated by transmembrane proteins known as efflux pumps.

[0003] Although many efflux systems have been described in prokaryotes, particularly bacteria, these mechanisms can also be observed in eukaryotes.

[0004] Due to the elimination of compounds to the external environment, mechanisms involved in efflux contribute to the emergence of resistance to various pharmacological treatments, such as antibiotic resistance or resistance to cancer chemotherapy. The broad spectrum of certain efflux pumps observed in certain bacteria can lead to the emergence of multi-resistant phenotypes, making the infection extremely difficult to treat.

[0005] Therefore, there is a need to measure shedding to predict bacterial resistance.

[0006] One object of the present invention is to provide compounds and methods for measuring bacterial shedding.

[0007] Another object of the present invention is to provide a method for assessing antibiotic resistance in bacteria.

[0008] The present invention relates to compounds of formula I:

[0009] [ka] (In the formula, X - is a halogen atom or an acceptable counterion, in particular Cl; In the formula, R1 and R2 are -All of these are -CH 3 Or -C 4 H 9 or or C1-C4 alkyl (R1 is Cn+i alkyl and R2 is Cn alkyl, and vice versa, n is an integer ranging from 1 to 3 and i is an integer ranging from 1 to 3, where n+i ranges from 1 to 4); -Or R1 is -C 4 H 7 and R2 is C 8 H 17 and In the formula, R3 is -CH 3 or C2-C8 alkyl, which is optionally substituted with one or more heteroatoms. or a salt or solvate of this compound of formula I, or a protonated form thereof.

[0010] In other words, the present invention provides a compound of formula I as described above. where X- is a halogen atom or an acceptable counterion, in particular Cl; In the formula, R1 and R2 are -All of these are -CH 3 Or -C 4 H 9 or - or C1-C4 alkyl, wherein - when R1 is a Cn+i alkyl, R2 is a Cn alkyl; - when R1 is a Cn+i alkyl, R2 is a Cn alkyl; n is an integer ranging from 1 to 3, i is an integer ranging from 1 to 3, and n+i is in the range of 2 to 4; -Or R1 is C 4 H 9 and R2 is C 8 H 17 and wherein R3 is -CH3 or C2-C8 alkyl, the C2-C8 alkyl being optionally substituted with one or more heteroatoms. or a salt or solvate of this compound of formula I, or a protonated form thereof.

[0011] Advantageously, the present invention provides a compound of formula I:

[0012] [ka] (In the formula, X - is a halogen atom or an acceptable counterion, in particular Cl; In the formula, R1 and R2 are -All of these are -CH 3 or or C1-C4 alkyl (R1 is Cn+i alkyl and R2 is Cn alkyl, and vice versa, n is an integer ranging from 1 to 3, i is an integer ranging from 1 to 3, where n+i ranges from 1 to 4); and In the formula, R3 is -CH 3 or C2-C8 alkyl, which is optionally substituted with one or more heteroatoms. or a salt or solvate of this compound of formula I, or a protonated form thereof.

[0013] The present invention is based on the surprising observation by the inventors that the above-mentioned compounds of formula I are coloured fluorescent compounds that are efficiently excreted by bacteria via various efflux pumps, such that they make it possible to measure bacterial efflux and thus to measure the potential resistance of the bacteria.

[0014] The compounds of formula I are phenaziniums, i.e., cationic protonated forms of phenazines. Alkylated forms of the phenaziniums of the present invention are also contemplated.

[0015] In the compound of formula I, X -are acceptable, in particular pharma- ceutically acceptable, counter anions, i.e. counter anions whose use is compatible with the physiology of animals or bacteria. Advantageous counter anions are in particular those in which X is a halogen atom, such as F, Cl, Br or I, the preferred halogen being Cl. Other possible counter anions include chlorate, bromate, bicarbonate, acetate, butyrate, formate, tartrate, aluminate, salicylate, mesylate, boron anions, phosphorus anions, such as in particular hexafluorophosphate (PF 6 - ) or tetrafluoroborate (BF 4 - ) etc.

[0016] Thus, compounds according to formula I are phenaziniums, where the groups R1, R2, and R3 are defined as follows: Either R1 or R2 is -CH 3 Group or -C 4 H 9 Is it a base? - or R1 and R2 are C1-C4 alkyl, i.e. C1, C2, C3 or C4 alkyl, preferably linear, so that R1 and R2 always have different carbon atoms, which is defined by a phase that specifies that if R1 is a Cn alkyl, then R2 is a Cn+i alkyl, and if R1 is a Cn+i alkyl, then R2 is a Cn alkyl, where i is an integer ranging from 1 to 3 and n+i ranging from 2 to 4; - or R1 is -C 4 H 7 and R2 is C 8 H 17 It is.

[0017] In other words, R1 and R2 are -All of these are -CH 3 or When R1 and R2 are linear C1-C4 alkyl, -R1=-CH 3 and R2 = -C 2 H 5 Or R1 = -C2 H 5 and R2=-CH 3 or -R1=-C2H 5 and R2 = -C 3 H 7 Or R1 = -C 3 H 7 and R2 = -C 2 H 5 or -R1=-C 3 H 7 and R2 = -C 4 H 9 Or R1 = -C 4 H 9 and R2 = -C 3 H 7 It is.

[0018] Of course, when R1 or R3 is a C3 alkyl, they are 3 H 7 (i.e., -CH 2 -CH 2 -CH3) or CH(CH 3 ) 2 and when they are C4 alkyl, they are -C 4 H 9 (i.e., -CH 2 -CH 2 -CH 2 -CH 3 ), -CH(CH 3 )-CH 2 -CH 3 , or -CH 2 -CH(CH 3 ) 2 , or -C(CH 3 ) 3 It could be.

[0019] Regarding R3, this is methyl (-CH 3 ) or C2-C8 alkyl, i.e. -C1, i.e. -CH 3 or -C2, i.e. -C2H 5 (i.e., -CH 2 -CH3 ) or -C3, i.e. -C 3 H 7 (i.e., -CH 2 -CH 2 -CH3) or -CH(CH 3 ) 2 or -C4, i.e. -C 4 H 9 (i.e., -CH 2 -CH 2 -CH 2 -CH 3 ), -CH(CH 3 )-CH 2 -CH 3 , or -CH 2 -CH(CH 3 ) 2 , or -C(CH 3 ) 3 or -C5, i.e. -C5H11 (i.e. -CH 2 -CH 2 -CH 2 -CH 2 -CH 3 ), -CH(CH 3 )-CH 2 -CH 2 -CH 3 , -CH 2 -CH(CH 3 )-CH 2 -CH 3 , -CH 2 -CH 2 -CH(CH 3 ) 2 , -C(CH 3 ) 2 -CH 2 -CH 3 , -CH(CH 3 )-CH(CH 3 )-CH 3 , -CH 2 -C(CH 3 ) 3 , or -CH(CH 3 )-CH(CH 3 ) 2 or -C6, i.e. -C6H13 (i.e. -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 3 ), or in a branched form, or -C7, i.e. -C7H15 (i.e. -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 2 -CH 3 ), or in a branched form, or -C8, i.e., -C8H17 (i.e., -CH2-CH2--CH2CH2-CH2-CH2-CH2-CH3), or a branched form of alkyl.

[0020] When R3 is a C2-C8 alkyl, each of the carbon atoms can be replaced by a heteroatom, i.e., an oxygen (O), nitrogen (N) or sulfur (S) atom, or even phosphorus (P). For example, a C4 alkyl can have one of its carbons replaced by oxygen, giving the following R3 residue: - -O-CH 2 -CH 2 -CH 3 or - -CH 2 -O-CH 2 -CH 3 or - -CH 2 -CH 2 -O-CH 3 or - -CH 2 -CH 2 -NH-CH 3 or - -CH2-CH2-CH2-OH is obtained.

[0021] These examples are illustrative and cannot limit the scope of the invention.

[0022] The protonated forms of the compounds of formula I above are also encompassed by the present invention.

[0023] Advantageously, the invention relates to compounds as defined above, when R1 and R2 are each C2-C4 alkyl, for which: - when R1 is a Cn+i alkyl, R2 is a Cn alkyl; or - when R2 is a Cn+i alkyl, R1 is a Cn alkyl; n ranges from 1 to 3, i is an integer ranging from 1 to 3, n+i ranges from 1 to 4, and R3 is as defined below.

[0024] Advantageously, the invention relates to compounds as defined above, when R3 is a C2-C4 alkoxy group, in particular methoxyethyl, dimethylaminoethyl or methylaminexethyl.

[0025] Even more advantageously, the present invention relates to a compound as described above, which is selected from the group of compounds of formula I, wherein R1, R2, and R3 are all as indicated in each row of the following table:

[0026] [Table 1]

[0027] Note that compound #392 is also called #44.

[0028] Even more advantageously, the present invention relates to a compound as described above, which is selected from the compounds of the following formulae 47, 52, 53, 392 and 29:

[0029] [ka]

[0030] [ka]

[0031] These three compounds are the most advantageous of the present invention and their synthesis is described in detail in Example 1 below.

[0032] Advantageously, the present invention relates to a compound as defined above as a medicament or for its use as a medicament. It is also envisaged to use a compound as defined above for the manufacture of a medicament.

[0033] Pharmaceutical compositions comprising the compound as defined above as active substance together with a pharma- ceutically acceptable vehicle are also envisaged. Preferably, the compound of formula (I) as defined above or a pharma- ceutically acceptable salt thereof is administered or can be administered or packaged in a unit dose of about 50 mg to about 1500 mg, in particular about 150 to about 200 mg. Also preferably, the compound of formula (I) as defined above or a pharma- ceutically acceptable salt thereof is administered or can be administered in a dosage regimen of 50 mg / day to about 1500 mg / day, in particular about 150 mg / day to about 200 mg / day. Preferably, the compound of formula (I) as defined above or a pharmaceutical salt thereof is administered or can be administered in a form suitable for oral or injectable administration. More preferably, the compound of formula (I) as defined above or a pharma- ceutically acceptable salt thereof is administered or can be administered in the form of a powder, tablet, gelatin capsule or sachet.

[0034] Even more advantageously, said compounds or pharmaceutical compositions are used in or for the treatment of Gram-positive bacterial infections. The said compounds have antibacterial properties which may prove useful in the treatment of infections.

[0035] More advantageously, the compounds described above are used as part of an antibacterial therapy to treat conditions or infections associated with contamination with gram-positive bacteria that are unable to excrete the compounds or that express few or no pumps responsible for antibiotic resistance.

[0036] In another aspect, the aforementioned compounds possessing antibiotic or at least antibacterial properties can be used as part of the decontamination of an inert surface infected or contaminated with bacteria. The compound is then contacted with the contaminated surface to kill and / or inhibit the growth of the contaminating bacteria. The compound can be used in the form of a powder that is spread on the surface to be decontaminated, or in the form of a solution that can be poured or sprayed on the surface to be decontaminated.

[0037] It is also envisaged to use the above-mentioned compounds for staining or for carrying out staining or fluorescent marking of Gram-positive bacteria, more particularly Gram-positive bacteria that are unable to efflux compounds, i.e. bacteria that express few or no pumps responsible for multidrug resistance (efflux pumps).

[0038] The invention also relates to the use of a compound as defined above for assessing or measuring, ie for observing qualitatively or quantitatively, the excretion of said compound by bacteria.

[0039] As shown in the examples, the compounds of the invention can be excreted from the bacteria by an efflux mechanism, and because the compounds of the invention are dyeable and / or fluorescent, it is easy to follow their excretion from the inside to the outside of the bacteria.

[0040] The compounds of the invention can also be used to identify whether a bacterial isolate is overproducing its efflux pumps, and by measuring or assessing the efflux of the compounds of the invention, it is possible to determine whether the pump is overproduced or overexpressed, or conversely, underproduced or underexpressed.

[0041] It is therefore possible to measure characteristics such as the efflux rate, which may provide information about the quantity of efflux pumps present in the bacterial membrane, or to measure the variation in pump activity (efflux capacity) following mutagenesis (induced or natural).

[0042] If the compound is staining and / or fluorescent, it is possible to measure the staining / fluorescence at the start of the experiment when the compound is in contact with the bacteria, and after a given time, measure either the fluorescence or color in the bacteria or the external environment.

[0043] More advantageously, the present invention relates to the above-mentioned use for assessing or measuring excretion by Gram-positive bacteria.

[0044] Several genes encoding efflux systems have been described in Gram-positive bacteria whose products form protein transporters that reduce the accumulation of antibiotics in the cell.

[0045] For example, the msrA and msrB genes are responsible for the MS-type resistance phenotype, i.e., inducible resistance to macrolides with 14- and 15-carbon nuclei (C14 and C15) and streptogramin B, after induction with erythromycin. The mef gene gives rise to a resistance phenotype known as M, characterized by limited resistance to C14 and C15 macrolides.

[0046] The vgaA and vgaB genes code for the sole efflux protein of synergistin A. All of these genes are found in different species of Staphylococcus aureus (SA) and coagulase-negative staphylococci (CNS).

[0047] These are merely examples and should not be considered as limiting the scope of the invention in any way.

[0048] More advantageously, the invention relates to the above-mentioned use for assessing or measuring excretion by gram-negative bacteria.

[0049] Advantageously, the above mentioned use concerns a measurement or evaluation.

[0050] In another aspect, the present invention provides a kit for detecting or measuring bacterial shedding, comprising: a compound as defined above, - at least one control bacterium, Related to the kit, including

[0051] Thus, the kits of the invention contain a compound according to the invention capable of being excreted by bacteria, and additionally an experimental control, a control bacterium known to excrete the compound.

[0052] This control bacterium can express either a wild-type pump, the efflux of which is known in a particularly precise and reproducible way, or a mutant pump that no longer allows efflux due to lack of expression of the proteins forming the pump or to structural modifications that affect its function. Other control bacteria can of course also be used, and in an advantageous embodiment the kit can be supplied with both a positive control (bacteria with a functional efflux pump) and a negative control (bacteria with a non-functional or absent efflux pump).

[0053] To the extent that the compounds of the invention are stainable and / or fluorescent, a color palette or fluorescence detection means to define whether or not the bacteria are able to excrete the compound can also be included in the kits of the invention.

[0054] Advantageously, the invention relates to the use of a kit as defined above for measuring or quantifying the excretion of said compounds by bacteria, in particular gram-positive bacteria, in particular Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis) and S. pneumoniae (S. pneumoniae).

[0055] Advantageously, the invention relates to the use of a kit as defined above for measuring or quantifying the excretion of said compounds by Gram-negative bacteria, and in particular by Escherichia coli.

[0056] Even more advantageously, the invention relates to a kit as described above, which additionally comprises a protonophore, in particular chosen from 2,4-dinitrophenol, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), carbonyl cyanide-m-chlorophenylhydrazone (CCCP), C4R1, ellipticine, 10-[2-(3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]oxydecyl-triphenyl-phosphonium bromide and 2-(2-hydroxyaryl)hexylphosphonium bromide, in particular CCCP.

[0057] When measuring shedding from gram-negative bacteria, the use of protonophores such as CCCP is particularly appropriate. The protonophores in the kits mentioned above range from 0.25 to 15 μg.mL -1 This means that the protonophore is intended for use at any dose within this range, particularly at 0.25 μg.mL -1 , 0.5 μg.mL -1 , 0.75 μg.mL -1 , 1μg.mL -1 , 1.25 μg.mL -1 , 1.5 μg.mL -1 , 1.75 μg.mL -1 , 2 μg.mL -1 , 2.25 μg.mL -1 , 2.5 μg.mL -1 , 2.75 μg.mL -1 , 3 μg.mL -1 , 3.25 μg.mL -1 , 3.5 μg.mL -1 , 3.75 μg.mL -1 , 4 μg.mL -1 , 4.25 μg.mL -1 , 4.5 μg.mL -1 , 4.75 μg.mL -1 , 5 μg.mL -1 , 5.25 μg.mL -1 , 5.5 μg.mL -1 , 5.75 μg.mL -1 , 6 μg.mL -1 , 6.25 μg.mL -1 , 6.5 μg.mL -1, 6.75 μg.mL -1 , 7 μg.mL -1 , 7.25 μg.mL -1 , 7.5 μg.mL -1 , 7.75 μg.mL -1 , 8 μg.mL -1 , 8.25 μg.mL -1 , 8.5 μg.mL -1 , 8.75 μg.mL -1 , 9 μg.mL -1 , 9.25 μg.mL -1 , 9.5 μg.mL -1 , 9.75 μg.mL -1 , 10 μg.mL -1 , 10.25 μg.mL -1 , 10.5 μg.mL -1 , 10.75 μg.mL -1 , 11 μg.mL -1 , 11.25 μg.mL -1 , 11.5 μg.mL -1 , 11.75 μg.mL -1 , 12 μg.mL -1 , 12.25 μg.mL -1 , 12.5 μg.mL -1 , 12.75 μg.mL -1 , 13 μg.mL -1 , 13.25 μg.mL -1 , 13.5 μg.mL -1 , 13.75 μg.mL -1 , 14 μg.mL -1 , 14.25 μg.mL -1 , 14.5 μg.mL -1 , 14.75 μg.mL -1 , or 15 μg.mL -1 This means that the drug may be used at a dose of 5 μg.mL. Those skilled in the art can determine the appropriate dose depending on the protonophore used. For example, when using CCCP, 5 μg.mL -1 In yet another aspect, the present invention relates to the use of a compound as defined above for assessing the resistance of bacteria to bacteriostatic or bactericidal compounds.

[0058] Compounds according to the invention can be effluxed by bacterial pumps and therefore can logically be used to assess the efficiency of bacterial efflux.

[0059] If it is established that the compounds according to the invention are effluxed, this means that the efflux is functional and thus the bacteria are able to eliminate the antibiotic, therefore, without having to measure resistance with an antibiotic, we can conclude that the bacteria concerned are antibiotic resistant via the efflux phenomenon.

[0060] Conversely, if the compounds of the invention are not effluxed, this means that the efflux pump is not functional and therefore the antibiotic is not effluxed either, thus making it possible to easily determine, without the use of antibiotics, that a bacterium is susceptible to said antibiotic due to lack of efflux, similar to resistance.

[0061] In yet another aspect, the present invention provides a method for assessing bacterial resistance to a bacteriostatic or bactericidal compound, in particular resistance due to efflux mechanisms, comprising the steps of: - culturing the bacteria in a suitable culture medium to obtain a bacterial culture; - contacting said bacterial culture with a compound as defined above to obtain a treated bacterial culture; - washing the treated bacterial culture and precipitating the bacteria contained in the treated bacterial culture to obtain a bacterial pellet; - observing the staining of the bacterial pellet, - if the bacterial pellet has the color of a bacterial pellet from an untreated bacterial culture, the bacteria is resistant to the bacteriostatic or bactericidal compound; and observing that if the bacterial pellet has a color different from the color of the bacterial pellet from the untreated bacterial culture, the bacteria is resistant to the bacteriostatic or bactericidal compound; The present invention relates to a method comprising the steps of:

[0062] Alternatively, the evaluation method may include a filtration step instead of the centrifugation step. In this variant, the bacterial culture is passed through a filter, and the bacteria are precipitated on the filter. The coloration of the solid deposit of bacteria present on the filter is then evaluated to determine whether the bacteria are resistant to bacteriostatic or bactericidal compounds, in particular due to efflux mechanisms. Thus, according to this embodiment, the invention provides a method for evaluating the resistance of bacteria to bacteriostatic or bactericidal compounds, in particular due to efflux mechanisms, comprising: - culturing the bacteria in a suitable culture medium to obtain a bacterial culture; - contacting said bacterial culture with a compound as defined above to obtain a treated bacterial culture; - washing the treated bacterial culture and filtering the bacteria contained in the treated bacterial culture to obtain, on the one hand, a filtrate and, on the other hand, a bacterial deposit on the filter; - observing the staining of solid deposits on the filter, - if the solid deposit on the filter has the same color as the solid deposit from an untreated bacterial culture, the bacteria is resistant to the bacteriostatic or bactericidal compound; and observing that if the solid deposit has a color different from the color of the solid deposit from the untreated bacterial culture, the bacteria is resistant to the bacteriostatic or bactericidal compound; The present invention relates to a method comprising the steps of:

[0063] Advantageously, the present invention relates to a method for assessing resistance due to bacterial efflux mechanisms to a bacteriostatic or bactericidal compound, comprising the steps of: - culturing the bacteria in a suitable culture medium to obtain a bacterial culture; - contacting said bacterial culture with a compound as defined above to obtain a treated bacterial culture; - washing the treated bacterial culture and precipitating the bacteria contained in the treated bacterial culture to obtain a bacterial pellet; - observing the staining of the bacterial pellet, - if the bacterial pellet has the color of the bacterial pellet from the untreated bacterial culture, the bacteria is resistant to the bacteriostatic or bactericidal compound by excretion of the bacteriostatic or bactericidal compound; and observing that if the bacterial pellet has a color different from the color of the bacterial pellet from the untreated bacterial culture, the bacteria are resistant to the bacteriostatic or bactericidal compound by excretion of the bacteriostatic or bactericidal compound; The present invention relates to a method comprising the steps of:

[0064] Even more advantageously, the present invention relates to a method as described above, wherein said method further comprises a step of incubating the bacterial culture with a protonophore.

[0065] Advantageously, this is the case when the invention relates to a method for assessing resistance due to bacterial efflux mechanisms to a bacteriostatic or bactericidal compound, comprising the steps of: - culturing the bacteria in a suitable culture medium to obtain a bacterial culture; - contacting said bacterial culture with a protonophore, in particular with a protonophore as mentioned above, to obtain a treated bacterial culture; - contacting said pre-treated bacterial culture with a compound as defined above to obtain a treated bacterial culture; - washing the treated bacterial culture and precipitating the bacteria contained in the treated bacterial culture to obtain a bacterial pellet; - observing the staining of the bacterial pellet, - if the bacterial pellet has the color of a bacterial pellet from an untreated bacterial culture, the bacteria is resistant to the bacteriostatic or bactericidal compound by excretion of the bacteriostatic or bactericidal compound; and observing that if the bacterial pellet has a color different from the color of the bacterial pellet from the untreated bacterial culture, the bacteria are resistant to the bacteriostatic or bactericidal compound by excretion of the bacteriostatic or bactericidal compound; The present invention relates to a method, including the steps of:

[0066] This pretreatment method is particularly advantageous for measuring the excretion of Gram-negative bacteria.The above mentioned compounds are very simple and are based on the color properties of the compounds according to the invention.

[0067] By incubating bacteria with a compound according to the invention, the compound accumulates in the bacteria.

[0068] When the efflux pump is functional, the compound is excreted and released into the medium, the bacteria no longer have any of the compound, and the bacterial pellet obtained from the culture has the color of a pellet that has not been treated with the coloring compound.

[0069] Conversely, if the efflux pump does not function, the compound accumulates in the bacteria and the pellet then takes on a color that is the color of the pigmented compound (with some variation in intensity).

[0070] After washing to eliminate residual compounds that have been excreted or not taken up by the bacteria, a simple colorimetric test can be used to determine whether the bacteria are capable of excreting compounds and therefore whether the bacteria are resistant to antibiotics.

[0071] Colorimetric tests can be carried out using techniques known to those skilled in the art, particularly L * a * b * It can be implemented according to the CIE 1976 color space. This color space is particularly common for characterizing surface colors. Three quantities characterize a color: lightness L * comes from the surface brightness. Two parameters a * and b * represents the deviation of a color from the color of a gray surface of the same lightness. [Brief description of the drawings]

[0072] The invention will be better understood on reading the following description, given purely by way of example, and on referring to the accompanying drawings, in which: [Figure 1]Figure 1 is a representative photograph of a test for NorA efflux pump activity in Staphylococcus aureus strains using compound 053 at a concentration of 0.75 μg.mL-1. Pellet A represents the pellet of CIP 76.26, a reference strain with normal efflux activity, which is light pink in color. Pellet B represents the pellet of SA 1199 strain, a clinical strain with mild to moderate efflux activity, which is orange in color. Pellet C represents the pellet of SA 1199 B strain, a mutant strain with high efflux activity, which is white / off-white in color. Pellet D represents the pellet from SA K1758, a mutant strain lacking efflux pumps, which is bright dark pink in color. [Diagram 2] FIG. 2 is a graph showing the fluorescence intensity as a function of wavelength (nm): A: buffer, B: SA K1758 strain, C: CIP 76.26 strain, D: SA 1199 B strain, and E: SA K1758 strain. [Diagram 3] FIG. 3 is a histogram showing the maximum fluorescence emission values ​​at 642 nm (after excitation at a wavelength of 530 nm): A: buffer, B: SA K1758 strain, C: CIP 76.26 strain, D: SA 1199 B strain, and E: SA K1758 strain. [Figure 4] Figure 4 shows a representative photograph of a test with compound 053 at a concentration of 0.75 μg.mL-1 on a mutant of the Bacillus subtilis BmrA efflux pump. Pellet A represents a wild-type BmrA pellet, which is light pink in color. Pellet B represents a pellet from a strain overexpressing BmrA, which is white in color. Pellet C represents a bacterial pellet with a BmrA deletion (BmrAΔ), which is dark pink in color. [Diagram 5] FIG. 5 is a graph showing the fluorescence intensity as a function of wavelength (nm): A: buffer, B: BrmA-overexpressing B. subtilis strain, C: wild-type B. subtilis strain, and D: BrmAΔ strain. [Figure 6]FIG. 6 is a histogram showing maximum fluorescence emission values ​​at 642 nm (after excitation at a wavelength of 530 nm): A: buffer, B: BrmA-overexpressing B. subtilis strain, C: wild-type B. subtilis strain, and D: BrmAΔ strain. [Figure 7] Figure 7 is a histogram showing the fluorescence intensity measured by flow cytometry for different Staphylococcus aureus strains treated with compound 053. A: wild type strain corresponding to CIP 76.25, B: reference strain corresponding to SA, C: NorA overproducing strain corresponding to SA1199B, and D: NorA deletion strain corresponding to SA K1758. ns: non-specific. * indicates significant difference with p value less than 0.05; ****=significant difference with p value less than 0.001 (one-way ANOVA test with Tukey test). [Figure 8] 8 is a histogram showing the relative fluorescence intensity (arbitrary units) for each Staphylococcus aureus strain treated with compound 053 and fixed with PFA or ethanol: A: wild type strain corresponding to CIP 76.25 fixed with PFA, B: reference strain corresponding to SA fixed with PFA, C: NorA overproducing strain corresponding to SA1199B fixed with PFA, D: NorA deletion strain corresponding to SA K1758 fixed with PFA, E: wild type strain corresponding to CIP 76.25 fixed with ethanol, F: reference strain corresponding to SA fixed with ethanol, G: NorA overproducing strain corresponding to SA1199B fixed with ethanol, and H: NorA deletion strain corresponding to SA K1758 fixed with ethanol. [Figure 9] Figure 9 shows representative pictures of a test with compound 053 at a concentration of 1 μg.mL-1 against mutants of the PatA-PatB transporter of Streptococcus pneumoniae. Pellet "9A" represents a wild-type PatA-PatB pellet. Pellet "9B" represents a pellet from a strain overexpressing PatA / PatB. Pellet "9C" represents a pellet from a strain with a PatA / PatB deletion. [Figure 10]Figure 10 is a histogram showing the maximum fluorescence emission values ​​at 642 nm (after excitation at a wavelength of 530 nm) for different Streptococcus pneumoniae strains: A: wild-type PatA-PatB strain, B: strains overexpressing PatA / PatB, and C: strains with PatA / PatB deletions. [Figure 11] Figure 11 is a representative photograph of a test for AcrAB efflux pump activity in Escherichia coli strains with compound 392 at a concentration of 1 μg.mL-1. Pellet "A" represents the pellet of the ATCC11775 strain, a reference strain with normal efflux activity. Pellet "B" represents the pellet of a wild-type strain (AG100) with normal efflux activity. Pellet "C" represents the pellet of the AG100A strain, which corresponds to a mutant strain deleted from the AcrAB efflux pump. Pellet "C" represents the pellet from AG102, a mutant strain overexpressing AcrAB. [Figure 12] Figure 12 depicts a representative graph of a test for AcrAB efflux pump activity in Escherichia coli strains with compound 392 at a concentration of 1 μg.mL-1. The graph shows the fluorescence intensity as a function of wavelength (nm). "A" represents the ATCC11775 strain, a reference strain with normal efflux activity. "B" represents the wild-type strain (AG100) with normal efflux activity. "C" represents the strain AG100A, which corresponds to a mutant strain deleted from the AcrAB efflux pump. "D" represents the pellet from the AG102 strain, a mutant strain overexpressing AcrAB. [Figure 13]Figure 13 is a representative histogram of a test for AcrAB efflux pump activity in Escherichia coli strains with compound 392 at a concentration of 1 μg.mL-1. Figure 13 shows the maximum fluorescence emission values ​​at 642 nm (after excitation at a wavelength of 530 nm) for different strains of Escherichia coli as bars. "A" represents the ATCC11775 strain, a reference strain with normal efflux activity. "B" represents the wild-type strain (AG100) with normal efflux activity. "C" represents the strain AG100A, which corresponds to a mutant strain deleted from the AcrAB efflux pump. "D" represents the pellet from the AG102 strain, a mutant strain overexpressing AcrAB. [Figure 14] Figure 14 is a representative photograph of a test for AcrAB efflux pump activity in Escherichia coli strains with compound 029 at a concentration of 1 μg.mL-1. Pellet "A" represents the pellet of strain ATCC11775, a reference strain with normal efflux activity. Pellet "B" represents the pellet of a wild-type strain (AG100) with normal efflux activity. Pellet "C" represents the pellet of strain AG100A, which corresponds to a mutant strain deleted from the AcrAB efflux pump. Pellet "D" represents the pellet from strain AG102, a mutant strain overexpressing AcrAB. [Figure 15] Figure 15 shows a representative graph of the test for AcrAB efflux pump activity in Escherichia coli strains with compound 029 at a concentration of 1 μg.mL-1. The graph shows the fluorescence intensity as a function of wavelength (nm). "A" represents the ATCC11775 strain, a reference strain with normal efflux activity. "B" represents the wild-type strain (AG100) with normal efflux activity. "C" represents the strain AG100A, which corresponds to a mutant strain deleted from the AcrAB efflux pump. "D" represents the pellet from the AG102 strain, a mutant strain overexpressing AcrAB. [Figure 16]Figure 16 is a representative histogram of the test for AcrAB efflux pump activity in Escherichia coli strains with compound 029 at a concentration of 1 μg.mL-1. Figure 16 shows the maximum fluorescence emission values ​​at 642 nm (after excitation at a wavelength of 530 nm) for different strains of Escherichia coli as bars. "A" represents the ATCC11775 strain, a reference strain with normal efflux activity. "B" represents the wild-type strain (AG100) with normal efflux activity. "C" represents the strain AG100A, which corresponds to a mutant strain deleted from the AcrAB efflux pump. "D" represents the pellet from the AG102 strain, a mutant strain overexpressing AcrAB. [Figure 17] FIG. 17 is a photograph representative of a test for AcrAB efflux pump activity in Escherichia coli strains pretreated with Carbonyl Cyanide m-Chlorophenylhydrazone (CCCP) using compound 053 at a concentration of 1 μg.mL-1. Pellet "A" represents the pellet of the ATCC11775 strain, a reference strain with normal efflux activity. Pellet "B" represents the pellet of a wild-type strain (AG100) with normal efflux activity. Pellet "C" represents the pellet of the AG100A strain, which corresponds to a mutant strain deleted from the AcrAB efflux pump. Pellet "D" represents the pellet from the AG102 strain, a mutant strain overexpressing AcrAB. [Figure 18] Figure 18 is a graph representative of a test for AcrAB efflux pump activity in Escherichia coli strains pretreated with carbonyl cyanide m-chlorophenylhydrazone at a concentration of 1 μg.mL-1 using compound 053. The graph shows the fluorescence intensity as a function of wavelength (nm). "A" represents the ATCC11775 strain, a reference strain with normal efflux activity. "B" represents the wild-type strain (AG100) with normal efflux activity. "C" represents strain AG100A, which corresponds to a mutant strain deleted from the AcrAB efflux pump. "D" represents the pellet from strain AG102, a mutant strain overexpressing AcrAB. [Figure 19] Figure 19 is a histogram representative of a test for AcrAB efflux pump activity in Escherichia coli strains pretreated with carbonyl cyanide m-chlorophenylhydrazone at a concentration of 1 μg.mL-1 of compound 053. Figure 19 shows the maximum fluorescence emission values ​​at 642 nm (after excitation at a wavelength of 530 nm) for different strains of Escherichia coli as bars. "A" represents the ATCC11775 strain, a reference strain with normal efflux activity. "B" represents the wild-type strain (AG100) with normal efflux activity. "C" represents the strain AG100A, which corresponds to a mutant strain deleted from the AcrAB efflux pump. "D" represents the pellet from the AG102 strain, a mutant strain overexpressing AcrAB. EXAMPLES

[0073] Example 1 - Synthesis 1. General scheme for the synthesis of compounds according to the invention Synthetic schemes for various compounds of the invention are provided below. Synthetic details, including intermediate steps, for three specific compounds are also provided below.

[0074] [ka]

[0075] 2-Synthesis of Compound 053 2a-Step 1

[0076] [ka]

[0077] To a stirred solution of 1,4-dinitrofluorobenzene (DFNB-2 mL, 2.9 g, 18.23 mmol, 1 equiv.) in dichloromethane (DCM-50 mL) at 0° C. was added propylamine (1.5 mL, 1.08 g, 18.23 mmol, 1 equiv.), followed by N,N-diisopropylethylamine (DIPEA-3.49 mL, 2.6 g, 20 mmol, 1.1 equiv.). The reaction mixture was allowed to warm to room temperature (over 30 min), after which the reaction was complete (monitored by thin-layer chromatography (TLC)). The solvent was evaporated in vacuo to give a yellow residue which was dissolved in 100 mL of SiO 2 Purification by gel column chromatography afforded the desired product FBP-002 as a yellow oil (2.74 g, 13.8 mmol, 76%).

[0078] 1 H-NMR (400 MHz; CDCl 3 ): δ8.16(m,2H),6.46(dd,J 1 =11.6Hz,J 2 =2.4Hz,1H),6.34(ddd,J 1 =9.6Hz,J 2 =4.8Hz,J 3 =2.4Hz,1H),3.21(td,J 1 =7Hz,J 2 =5.2Hz,2H),1.75(sext,J=7.3Hz,2H),1.05(t,J=7.4Hz,3H), 13 C-NMR (101 MHz; CDCl 3 ): δ167.7(d,J C-F =257Hz), 147.7(d,J C-F = 14Hz), 130.1(d,J C-F =13Hz), 128.8, 103.9(d,J C-F = 25Hz), 99.3(d,J C-F =27Hz), 45.1, 22.1, 11.6.

[0079] 2b-Step 2

[0080] [ka]

[0081] To a solution of FBP-002 (3 g, 15 mmol, 1.0 equiv) in tetrahydrofuran (THF-40 mL) was added butylamine (3.32 g, 4.48 mL, 45 mmol, 3 equiv) and DIPEA (3.3 g, 4.3 mL, 25.5 mmol, 1.7 equiv). The reaction mixture was stirred at 60° C. overnight. The solvent was then evaporated and the residue was taken up in ethyl acetate (EtOAc-150 mL) and washed with saturated aqueous ammonium chloride (2×150 mL) and brine (100 mL). The combined organic layers were then diluted with MgSO 4 The mixture was dried at 40° C., filtered and evaporated in vacuo. The resulting crude was purified by SiO2 gel column chromatography to give the desired product FBP-043 as an orange oil (1.2 g, 4.8 mmol, 32% yield).

[0082] 1 H-NMR (400 MHz; CDCl 3 ): δ8.16(m,2H),6.46(dd,J 1 =11.6Hz,J 2 =2.4Hz,1H),6.34(ddd,J 1 =9.6Hz,J 2 =4.8Hz,J 3 =2.4Hz,1H),3.21(td,J 1 =7Hz,J 2 =5.2Hz,2H),1.75(sext,J=7.3Hz,2H),1.05(t,J=7.4Hz,3H), 13 C-NMR (101 MHz; CDCl 3 ): δ167.7(d,J C-F =257Hz), 147.7(d,J C-F = 14Hz), 130.1(d,J C-F =13Hz), 128.8, 103.9(d,J C-F = 25Hz), 99.3(d,J C-F =27Hz), 45.1, 22.1, 11.6.

[0083] 2c-Step 3

[0084] [ka]

[0085] A solution of FBP-043 (1.174 g, 4.67 mmol, 1 equiv.) in THF (70 mL) was hydrogenated overnight (P=20 bar) in the presence of Pd / C (5%, 100 mg, 1.0 mol%). After depressurization, the solution was degassed by bubbling argon into the mixture. 1,5-Difluoro-2,4-dinitrobenzene (905 mg, 4.4 mmol, 0.95 equiv.) was added at 0° C. with stirring. This condition was maintained for 20 min, and the completion of the reaction was detected by TLC (DCM / CH, 70 / 30). Pd / Ca was then removed by filtration through a Celite® plug. The crude product was purified by silica gel column chromatography (DCM / EP, 50 / 50) to give the desired product FB-005 as a deep reddish solid (1.466 g, 3.62 mmol, 77% yield).

[0086] 1 H-NMR (400 MHz; CDCl 3 ):δ9.30(br,1H),9.14(d,J=8Hz,1H),6.82(d,J=8.4Hz,1H),6.53(d,J=13.6Hz,1H),5.99(dd,J 1 =8.4Hz,J 2 =2.4Hz,1H),5.95(d,J=2.4Hz,1H),3.75(br,1H),3.69(br,1H),3.13(t,J=6Hz,2H),3.06(q,J=6.4 Hz,1H),1.67-1.51(m,4H),1.46(sext,J=7.4Hz,2H),0.97(t,J=7.4Hz,3H),0.92(t,J=7.4Hz,3H), 13 CRMN (101MHz;CDCl 3 ): δ159.9(d,J C-F =271Hz), 150.5(d,J C-F=13Hz),150.3,145.2,128.7,127.8,126.9,110.8,103.9(d,J C-F =27Hz),101.7,95.2,45.3,43.6,43.2,31.7,22.6,20.4,14.0,11.7.

[0087] 1d-Step 4

[0088] [ka]

[0089] To a stirred solution of FB-045 (343 mg, 0.85 mmol, 1 equiv.) in acetonitrile (15 mL) was added methoxyethylamine (221 μL, 191 mg, 2.54 mmol, 3 equiv.). The mixture was stirred at room temperature for 2 h, at which time completion was monitored by TLC. After evaporation of the solvent, hot ethanol (10 mL) was added to the crude residue. The resulting asphalt-like material was collected by filtration, washed with hot water (30 mL), and then recovered by dissolving in dichloromethane and removing 10 mL of anhydrous MgSO. 4 After removing the solvent under reduced pressure and drying the solid in vacuum, the desired product FBP-051 was isolated as a dark asphalt-like solid (383 mg; 0.83 mmol; 98%).

[0090] 1 H-NMR (400 MHz; CDCl 3 ):δ9.26(s,1H),9.13(br,1H),8.36(t,J=4.6Hz,1H),6.86(d,J=8Hz,1H),5.99(dd,J 1 =8.4Hz,J 2=2.4Hz,1H),5.95(d,J=2.4Hz,1H),5.71(s,1H),3.79(br,1H),3.67(br,1H),3.54(t,J=5.4Hz,2H),3.35(s,3H),3.19-3. 10(m,4H),3.06(t,J=7Hz,2H),1.67-1.51(m,4H),1.45(sext,J=7.4Hz,2H),0.97(t,J=7.4Hz,3H),0.91(t,J=7.4Hz,3H), 13 CRMN (101MHz;CDCl 3 ):δ149.7,149.3,148.6,145.4,129.5,128.8,125.2,124.5,112.1,101. 6,95.4,93.2,69.6,59.1,45.4,43.8,42.9,31.8,22.7,20.4,14.0,11.7.

[0091] 2e-Process 5

[0092] [ka]

[0093] A solution of compound FBP-051 (433 mg, 0.94 mmol, 1 equiv.) in methanol (20 mL) was hydrogenated (20 bar) in the presence of Pd / C (5 wt%, 36 mg) and HCl (12 M, 0.30 mL) for 6 h. The mixture was then stirred in air for 16 h. Pd / Ca was removed by filtration through a Celite® plug, which was rinsed several times with methanol and dichloromethane. After removing the solvent under reduced pressure, the resulting solid was taken up in dichloromethane (50 mL) and extracted with brine (2×50 mL), then the organic layer was dried over MgSO4, filtered, and evaporated in vacuum to obtain a crude black solid, which was then purified on an alumina gel chromatography column (DCM / MeOH) to obtain a deep purple solid of compound FBP-053 or compound 053 (177 mg, 0.44 mmol, 49% yield).

[0094] 1H-NMR (400MHz; CD 3 OD): δ7.68(d,J=9.2Hz,1H),7.17(dd,J 1 =9.2Hz,J 2 =1.6Hz,1H),6.94(d,J=1.6Hz,1H),6.78(s,1H),6.52(br,1H),4.52(t,J=8Hz ,2H),3.73(t,J=5.2Hz,2H),3.45(t,J=5.2Hz,2H),3.42(s,3H),3.32(t,J=7. 2Hz,2H),3.29(sext,J=1.6Hz,2H),1.96(sext,J=7.6Hz,2H),1.71(quint,J=7Hz,2H),1.51(sext,J=7Hz,2H),1.19(t,J=7.4Hz,3H),1.01(t,J=7.4Hz,3H), 13 C-NMR (101MHz; CD 3 OD): δ150.9,139.2,138.4,135.5,132.9,131.4,130.9,92.4,69.9, 57.8, 43.3, 42.7, 30.5, 20.1, 19.7, 12.9, 10.1; 5. The signal is repeated again. High resolution energy mass analysis method: C 22 H 32 N 5 O + Calculate value: 382.2601; get value: 382.2593

[0095] 3-Synthesis of compound 052 3a-Project 1

[0096]

change

[0097] To a stirred solution of DFNB (2 mL, 2.9 g, 18.23 mmol, 1 equiv) in DCM (50 mL) at 0° C., propylamine (1.5 mL, 1.08 g, 18.23 mmol, 1 equiv) was added, followed by DIPEA (3.49 mL, 2.6 g, 20 mmol, 1.1 equiv). The reaction mixture was allowed to warm to room temperature (over 30 min), after which the reaction was complete (monitored by TLC). The solvent was evaporated in vacuo to give a yellow residue, which was purified by SiO2 gel column chromatography to give the desired product FBP-002 as a yellow oil (2.74 g, 13.8 mmol, 76%).

[0098] 1 H-NMR (400 MHz; CDCl 3 ): δ8.16(m,2H),6.46(dd,J 1 =11.6Hz,J 2 =2.4Hz,1H),6.34(ddd,J 1 =9.6Hz,J 2 =4.8Hz,J 3 =2,4Hz,1H),3.21(td,J 1 =7Hz,J 2 =5.2Hz,2H),1.75(sext,J=7.3Hz,2H),1.05(t,J=7.4Hz,3H).

[0099] 13 C-NMR (101 MHz; CDCl 3 ): δ167.7(d,J C-F =257Hz), 147.7(d,J C-F = 14Hz), 130.1(d,J C-F =13Hz), 128.8, 103.9(d,J C-F = 25Hz), 99.3(d,J C-F =27Hz), 45.1, 22.1, 11.6.

[0100] 3b-Step 2

[0101] [ka]

[0102] To a solution of FBP-002 (3 g, 15 mmol, 1.0 equiv) in THF (40 mL) was added butylamine (3.32 g, 4.48 mL, 45 mmol, 3 equiv) and DIPEA (3.3 g, 4.3 mL, 25.5 mmol, 1.7 equiv). The reaction mixture was stirred at 60 °C overnight. The solvent was then evaporated and the residue was taken up in EtOAc (150 mL) and washed with saturated aqueous ammonium chloride (2 x 150 mL) and brine (100 mL). The combined organic layers were then diluted with MgSO 4 The mixture was dried at 40° C., filtered and evaporated in vacuo. The resulting crude was purified by SiO2 gel column chromatography to give the desired product FBP-043 as an orange oil (1.2 g, 4.8 mmol, 32% yield).

[0103] 1 H-NMR (400 MHz; CDCl 3 ):δ8.52(br,1H),7.99(d,J=9.2Hz,1H),5.89(d,J=9.6Hz,1H),5.61(s,1H),4.41(br,1H),3.18(m,4H),1.75(sext ,J=7.2Hz,2H),1.62(quint,J=7.2Hz,2H),1.43(sext,J=7.5Hz,2H),1.04(t,J=7.4Hz,3H),0.96(t,J=7.2Hz,3H).

[0104] 13 C-NMR (101 MHz; CDCl 3 ):δ154.5,148.7,129.3,123.7,104.8,90.0,44.8,43.0,31.3,22.2,20.3,13.9,11.8.

[0105] 3c-Process 3

[0106] [ka]

[0107] A solution of FBP-043 (1.174 g, 4.67 mmol, 1 equiv.) in THF (70 mL) was hydrogenated overnight (P=20 bar) in the presence of Pd / C (5%, 100 mg, 1.0 mol%). After depressurization, the solution was degassed by bubbling Ar into the mixture. 1,5-Difluoro-2,4-dinitrobenzene (905 mg, 4.4 mmol, 0.95 equiv.) was added at 0° C. with stirring. This condition was maintained for 20 min, and the completion of the reaction was detected by TLC (DCM / CH, 70 / 30). Pd / Ca was then removed by filtration through a Celite® plug. The crude product was purified by silica gel column chromatography (DCM / EP, 50 / 50) to give the desired product FB-045 as a deep reddish solid (1.466 g, 3.62 mmol, 77% yield).

[0108] 1 H-NMR (400 MHz; CDCl 3 ):δ9.30(br,1H),9.14(d,J=8Hz,1H),6.82(d,J=8.4Hz,1H),6.53(d,J=13.6Hz,1H),5.99(dd,J 1 =8.4Hz,J 2 =2.4Hz,1H),5.95(d,J=2.4Hz,1H),3.75(br,1H),3.69(br,1H),3.13(t,J=6Hz,2H),3.06(q,J=6.4 Hz,1H),1.67-1.51(m,4H),1.46(sext,J=7.4Hz,2H),0.97(t,J=7.4Hz,3H),0.92(t,J=7.4Hz,3H).

[0109] 13 C-NMR (101 MHz; CDCl 3 ): δ159.9(d,J C-F =271Hz), 150.5(d,J C-F =13Hz),150.3,145.2,128.7,127.8,126.9,110.8,103.9(d,J C-F =27Hz),101.7,95.2,45.3,43.6,43.2,31.7,22.6,20.4,14.0,11.7.

[0110] 3d-process 4

[0111] [ka]

[0112] To a stirred solution of FB-045 (327 mg, 0.81 mmol, 1 equiv.) in acetonitrile (15 mL) was added octylamine (398 μL, 311 mg, 2.41 mmol, 3 equiv.). The mixture was stirred at room temperature for 2 h, during which completion was monitored by TLC. After evaporation of the solvent, hot ethanol (10 mL) was added to the crude residue. The resulting asphalt-like material was collected by filtration, washed with hot water (30 mL), and then recovered by dissolving in dichloromethane, drying over anhydrous MgSO4, and filtering. After removing the solvent under reduced pressure and drying the solid in vacuum, the desired product FBP-050 was isolated as a dark solid (392 mg; 0.76 mmol; 94% yield).

[0113] 1 H-NMR (400 MHz; CDCl 3 ):δ9.26(s,1H),9.12(s,1H),8.20(t,J=5Hz,1H),6.86(d,J=8.4Hz,1H),5.99(dd,J 1 =8.2Hz,J 2 =2.4Hz,1H),5.95(d,J=2.4Hz,1H),5.69(s,1H),3.79(t,J=5Hz,1H),3.65(br,1H),3,13(t,J=7Hz,2H),3.06(q,J=6.3Hz,2H),2.99(q,J =6.3Hz,2H),1.97-1.50(m,8H),1.45(sext,J=8Hz,2H),1.30(m,8H),0.97(t,J=7.2Hz,3H),0.91(t,J=7.4Hz,3H),0.87(t,J=7.4Hz,3H).

[0114] 13 C-NMR (101 MHz; CDCl 3):δ149.7,149.2,148.6,129.6,128.8,125.0,124.5,112.1,101.6,95.3,93.1,45.4,4 3.9,43.2,31.9,31.8,29.3,29.2,28.4,27.0,22.7,22.6,20.4,14.2,14.0,13.9,11.7.

[0115] 3e-Process 5

[0116] [ka]

[0117] A solution of compound FBP-050 (316 mg, 0.61 mmol, 1 equiv.) in methanol (20 mL) was hydrogenated (20 bar) in the presence of Pd / C (5 wt%, 24 mg) and HCl (12 M, 0.25 mL) for 6 h. The mixture was then stirred in air for 16 h. Pd / Ca was removed by filtration through a Celite® plug, which was rinsed several times with methanol and dichloromethane. After removing the solvent under reduced pressure, the resulting solid was taken up in dichloromethane (50 mL) and extracted with brine (2×50 mL), then the organic layer was dried over MgSO4, filtered, and evaporated in vacuum to obtain a crude black solid, which was then purified on an alumina gel chromatography column (DCM / MeOH) to obtain a deep purple solid of compound FBP-052 or compound 052 (129 mg, 0.27 mmol, 45% yield).

[0118] 1 H-NMR (400 MHz; CD 3 OD):δ7.72(d,J=9.6Hz,1H),7.18(dd,J 1 =9.2Hz,J 2=2Hz,1H),6.96(s,1H),6.75(s,1H),6.56(br,1H),4.55(t,J=8.2Hz,2H),3.32(m,2H),3.28(quint,J=1.6Hz,2H),3.25(t,J=7.4Hz,2H),1.98(se xt,J=8Hz,2H),1.80-1.66(m,4H),1.49(quint,J=8Hz,4H),1.41-1.26(m,8H),1.20(t,J=7.4Hz,3H),1.01(t,J=7.4Hz,3H),0.88(t,J=7.4Hz,3H).

[0119] 13 C-NMR (101 MHz; CDCl 3 ): δ 153.3, 150.9, 139.2, 138.6, 135.4, 131.3, 130.9, 102.8, 92.4, 48.9, 43.7, 42.7, 31.7, 30.5, 29.3, 29.1, 28.1, 27.1, 22.4, 20.1, 19.7, 13.1, 12.9, 10.1. The three signals are masked or overlapping.

[0120] 4-Synthesis of Compound 047 4a-Step 1

[0121] [ka]

[0122] To a stirred solution of 1,4-dinitrofluorobenzene (DFNB-2 mL, 2.9 g, 18.23 mmol, 1 equiv.) in dichloromethane (DCM-50 mL) at 0° C. was added propylamine (1.5 mL, 1.08 g, 18.23 mmol, 1 equiv.), followed by N,N-diisopropylethylamine (DIPEA-3.49 mL, 2.6 g, 20 mmol, 1.1 equiv.). The reaction mixture was allowed to warm to room temperature (over 30 min), after which the reaction was complete (monitored by thin-layer chromatography (TLC)). The solvent was evaporated in vacuo to give a yellow residue which was dissolved in 100 mL of SiO 2Purification by gel column chromatography afforded the desired product FBP-002 as a yellow oil (2.74 g, 13.8 mmol, 76%).

[0123] 1 H-NMR (400 MHz; CDCl 3 ): δ8.16(m,2H),6.46(dd,J 1 =11.6Hz,J 2 =2.4Hz,1H),6.34(ddd,J 1 =9.6Hz,J 2 =4.8Hz,J 3 =2.4Hz,1H),3.21(td,J 1 =7Hz,J 2 =5.2Hz,2H),1.75(sext,J=7.3Hz,2H),1.05(t,J=7.4Hz,3H), 13 C-NMR (101 MHz; CDCl 3 ): δ167.7(d,J C-F =257Hz), 147.7(d,J C-F = 14Hz), 130.1(d,J C-F =13Hz), 128.8, 103.9(d,J C-F = 25Hz), 99.3(d,J C-F =27Hz), 45.1, 22.1, 11.6.

[0124] 4b-Step 2

[0125] [ka]

[0126] To a solution of FBP-002 (3 g, 15 mmol, 1.0 equiv) in tetrahydrofuran (THF-40 mL) was added butylamine (3.32 g, 4.48 mL, 45 mmol, 3 equiv) and DIPEA (3.3 g, 4.3 mL, 25.5 mmol, 1.7 equiv). The reaction mixture was stirred at 60° C. overnight. The solvent was then evaporated and the residue was taken up in ethyl acetate (EtOAc-150 mL) and washed with saturated aqueous ammonium chloride (2×150 mL) and brine (100 mL). The combined organic layers were then diluted with MgSO 4 The mixture was dried at 40° C., filtered and evaporated in vacuum. The resulting crude was purified by SiO2 gel column chromatography to give the desired product FBP-043 as an orange oil (1.2 g, 4.8 mmol, 32% yield).

[0127] 1 H-NMR (400 MHz; CDCl 3 ): δ8.16(m,2H),6.46(dd,J 1 =11.6Hz,J 2 =2.4Hz,1H),6.34(ddd,J 1 =9.6Hz,J 2 =4.8Hz,J 3 =2.4Hz,1H),3.21(td,J 1 =7Hz,J 2 =5.2Hz,2H),1.75(sext,J=7.3Hz,2H),1.05(t,J=7.4Hz,3H), 13 C-NMR (101 MHz; CDCl 3 ): δ167.7(d,J C-F =257Hz), 147.7(d,J C-F = 14Hz), 130.1(d,J C-F =13Hz), 128.8, 103.9(d,J C-F = 25Hz), 99.3(d,J C-F =27Hz), 45.1, 22.1, 11.6.

[0128] 4c-Step 3

[0129] [ka]

[0130] A solution of FBP-043 (1.174 g, 4.67 mmol, 1 equiv.) in THF (70 mL) was hydrogenated overnight (P=20 bar) in the presence of Pd / C (5%, 100 mg, 1.0 mol%). After depressurization, the solution was degassed by bubbling argon into the mixture. 1,5-Difluoro-2,4-dinitrobenzene (905 mg, 4.4 mmol, 0.95 equiv.) was added at 0° C. with stirring. This condition was maintained for 20 min, and the completion of the reaction was detected by TLC (DCM / CH, 70 / 30). Pd / Ca was then removed by filtration through a Celite® plug. The crude product was purified by silica gel column chromatography (DCM / EP, 50 / 50) to give the desired product FB-005 as a deep reddish solid (1.466 g, 3.62 mmol, 77% yield).

[0131] 1 H-NMR (400 MHz; CDCl 3 ):δ9.30(br,1H),9.14(d,J=8Hz,1H),6.82(d,J=8.4Hz,1H),6.53(d,J=13.6Hz,1H),5.99(dd,J 1 =8.4Hz,J 2 =2.4Hz,1H),5.95(d,J=2.4Hz,1H),3.75(br,1H),3,69(br,1H),3.13(t,J=6Hz,2H),3.06(q,J=6.4 Hz,1H),1.67-1.51(m,4H),1.46(sext,J=7.4Hz,2H),0.97(t,J=7.4Hz,3H),0.92(t,J=7.4Hz,3H), 13 CRMN (101MHz;CDCl 3 ): δ159.9(d,J C-F =271Hz), 150.5(d,J C-F =13Hz),150.3,145.2,128.7,127.8,126.9,110.8,103.9(d,J C-F=27Hz),101.7,95.2,45.3,43.6,43.2,31.7,22.6,20.4,14.0,11.7.

[0132] 4d-process 4

[0133] [ka]

[0134] To a stirred solution of FB-045 (341 mg, 0.7 mmol, 0.84 mmol, 1 equiv) in acetonitrile (15 mL) was added butylamine (249 μL, 186 mg, 2.52 mmol, 3 equiv). The mixture was stirred at room temperature for 2 h, during which completion was monitored by TLC. After evaporation of the solvent, hot ethanol (10 mL) was added to the crude residue. The resulting asphalt-like material was collected by filtration, washed with hot water (30 mL), and then recovered by dissolving in dichloromethane, drying over anhydrous MgSO4, and filtering. After removing the solvent under reduced pressure and drying the solid in vacuum, the desired product was isolated as a dark solid (354 mg; 0.67 mmol; 96%).

[0135] 1 H-NMR (400 MHz; CDCl 3 ):δ9.26(s,1H),9.11(s,1H),8.20(t,J=5Hz,1H),6.86(d,J=8.4Hz,1H),5.98(dd,J 1 =8.4Hz,J 2 =2.4Hz,1H),5.96(d,J=2.4Hz,1H),5.69(s,1H),3.80(t,J=5.2Hz,1H),3.67( br,1H),3.13(t,J=7Hz,2H),3.06(q,J=6.7Hz,2H),3.01(q,J=6.7Hz,2H),3.0 6(t,J=7Hz,2H),1.67-1.51(m,4H),1.45(sext,J=7.4Hz,2H),1.33(sext,J=7 .4Hz,2H),0.97(t,J=7.4Hz,3H),0.91(t,J=7.4Hz,3H),0.88(t,J=7.4Hz,3H).

[0136] 13 C-NMR (101 MHz; CDCl 3 ):δ149.7,149.2,148.6,145.4,129.6,128.8,125.0,124.5,112.1,101.6,9 5.3,93.1,45.4,43.9,42.9,31.8,30.4,22.7,20.4,20.2,14.0,13.8,11.7.

[0137] 4e-Process 5

[0138] [ka]

[0139] A solution of compound FBP-046 (340 mg, 0.74 mmol, 1 equiv.) in methanol (20 mL) was hydrogenated (20 bar) in the presence of Pd / C (5 wt%, 29 mg) and HCl (12 M, 0.3 mL) for 6 h. The mixture was then stirred in air for 16 h. The Pd / Ca was removed by filtration through a Celite® plug, which was rinsed several times with methanol and dichloromethane. After removing the solvent under reduced pressure, the resulting solid was taken up in dichloromethane (50 mL) and extracted with brine (2×50 mL), and the organic layer was then washed with MgSO 4 The mixture was dried at 40° C., filtered and evaporated in vacuo to give a crude black / purple solid, which was then purified on an alumina gel chromatography column (DCM / MeOH) to give compound FBP-047 or compound 047 as a deep purple solid (130 mg, 0.31 mmol, 42% yield).

[0140] 1 H-NMR (400 MHz; CD 3 OD):δ7.72(d,J=9.2Hz,1H),7.19(dd,J 1 =9.2Hz,J 2=2Hz,1H),6.97(s,1H),6.76(s,1H),6.5(br,1H),4.55(t,J=8Hz,2H),3.35-3.24(m,8H),1.98(sext,J=7.6Hz,2H) ,1.80-1.64(m,4H),1.51(quint,J=7.2Hz,4H),1.20(t,J=7.4Hz,3H),1.02(t,J=7.4Hz,3H),1.01(t,J=7.4Hz,3H).

[0141] 13 C-NMR (101 MHz; CD 3 OD): δ 153.3, 150.8, 139.2, 138.5, 135.3, 132.6, 131.3, 130.8, 119.7, 102.7, 92.4, 89.3, 48.9, 43.4, 42.8, 30.5, 30.2, 20.3, 20.1, 19.7, 13.0, 10.1. One signal is masked or overlapped.

[0142] 5-Synthesis of Compound 029

[0143] [ka]

[0144] A solution of compound FBP-025 (356 mg, 0.78 mmol, 1 equiv.) in methanol (20 mL) was hydrogenated (20 bar) in the presence of Pd / C (5 wt%, 30 mg) and HCl (12 M, 0.3 mL) for 6 h. The mixture was then stirred overnight in air. The Pd / Ca was removed by filtration through a Celite® plug, which was rinsed several times with methanol and dichloromethane. After removing the solvent under reduced pressure, the resulting solid was taken up in dichloromethane (50 mL) and extracted with brine (2×50 mL), and the organic layer was then washed with MgSO 4 The mixture was dried at 40° C., filtered and evaporated in vacuo to give a crude black solid which was then purified on an alumina gel chromatography column (Brockmann activity I) (DCM / MeOH) to give a deep purple solid (160 mg, 39 mmol; 50%).

[0145] 1 H-NMR (400 MHz; CD 3 OD):δ7.68(d,J=9.2Hz,1H),7.16(dd,J 1 =9.2Hz,J 2 =2Hz,1H),6.93(s,1H),6.70(s,1H),6.5(br,1H),4.54(t,J=8Hz,2H),3.32-3.19(m,8H),1.89(m,2H),1.79(quint,J=7.4 Hz,2H),1.73-1.58(m,4H),1.49(quint,J=7.5Hz,2H),1.1(t,J=7.2Hz,3H),1.08(t,J=7.4Hz,3H),1.01(t,J=7.4Hz,3H).

[0146] 13 C-NMR (101 MHz; CD 3 OD):δ153.3,150.8,139.2,138.5,135.3,132.6,131.3,130.8,119.7,102.7, 92.4,89.3,48.9,43.4,43.3,42.6,30.5,30.2,20.3,20.2,19.7,13.0,10.2.

[0147] 6-Synthesis of Compound 392 or Compound 44 A solution of compound TM390 (3.08 g, 5.8 mmol, 1 eq) in methanol (60 mL) was hydrogenated (40 bar) in the presence of Pd / C (5 wt%, 617 mg) and HCl (12 M, 1 mL) for 16 h. The mixture was then stirred in air for 16 h. Pd / Ca was removed by filtration through a Celite® plug, which was rinsed several times with methanol and dichloromethane. After removing the solvent under reduced pressure, the resulting solid was purified on an alumina gel chromatography column (DCM / MeOH 95:5 to 90:10) to give TM 392 as a deep purple solid (3% yield).

[0148] 1 H-NMR (400 MHz; CD 3 OD): δ7.56(d,3 J=8.97Hz,1H),7.11(dd, 3 J=9.25Hz, 4 J=1.87Hz,1H),6.84(s,1H),6.60(s,1H),6.34(br,1H),4.44(t, 3 J=7.99Hz,2H),3.76(t, 3 J=5.29Hz,2H),3.44(s,3H),3.41(t, 3 J=5.41Hz,2H),3.27(t, 3 J=7.09Hz,2H),1.85-1.32(mm,16H),1.04(t, 3 J=7.2Hz,3H),0.92(t, 3 J=7.08Hz,3H).

[0149] [ka]

[0150] Example 2 - Identification of antibiotic excretion and quantification of its efficacy Antibiotic shedding is a phenomenon that is recognized as very serious by health authorities around the world. It concerns all bacteria, but is clearly of greatest concern in hospital settings such as intensive care, orthopedic and thoracic surgery, and in diabetes-related infections.

[0151] Due to their multispecificity, antibiotic efflux has long been identified as complementary to more conventional resistance mechanisms (targeted mutations, enzymes such as beta-lactamases, etc.), but more recently as intervening upstream of other mechanisms to drive their selection.

[0152] Although methods exist for identifying the primary resistance mechanisms in biology laboratories, shedding is not identified or identifiable by any standardized method, except by estimation, which is not always very reliable.

[0153] Therefore, there is a need to develop methods to identify antibiotic efflux and quantify its efficacy. The staining and fluorescent properties of compound 053 make it an excellent tool for this type of project.

[0154] Example 2-1: Staphylococcus aureus Materials and Methods: Bacterial strains: We used the following Staphylococcus aureus strains: - CIP 76.25, a reference strain that produces basal levels of NorA; - SA1199, a reference strain that produces basal levels of NorA; -SA 1199B, an overproducer of the NorA pump, and -SA K1758, in which the NorA gene has been deleted (Kaatz et al., 2003, Antimicrob Agents Chemother. February 2003; 47(2):719-26).

[0155] Compound: Compound 053 (see synthesis in Example 1) is prepared in pure DMSO (Sigma) at a concentration of 20 mg / mL. It is then diluted with milliQ water to obtain a stock solution of 1000 μg / mL.

[0156] Bacterial staining: Exponentially growing bacteria are incubated for 15 minutes in the presence of compound 053. The bacteria are then washed twice with phosphate buffer, centrifuged, and the pellet is visually observed.

[0157] Fluorescence measurements: Exponentially growing bacteria are incubated for 15 minutes in the presence of compound 053. The optical density at 600 nm is then adjusted to 0.5 and 50 μL is transferred to a black microtiter plate. Fluorescence is read on a Tecan M200 microplate reader.

[0158] The following protocol was followed: 1. Grow bacteria on freshly prepared Mueller-Hinton II (MH-II) agar plates. Incubate the Petri dishes overnight at 37 °C (Note: incubation temperature and growth time depend on the bacteria used in the experiment). 2. The next morning, pick one or two colonies from the overnight plate and inoculate them into an Erlenmeyer flask containing 20 mL of MH-II broth. 3. Incubate the Erlenmeyer at 37 °C and 160 rpm on an orbital shaker for 3-4 hours. Note: This step makes it possible to obtain cells in the logarithmic phase, so it is necessary to take into account the doubling time of the bacteria used. 4. After incubation, transfer the cells to a centrifuge tube and centrifuge at 4500 rpm for 10 minutes. Discard the supernatant. Add fresh MH-II broth to the centrifuge tube and adjust the optical density of the cells to 0.5. 5. Transfer an aliquot (5 mL) of cells with adjusted optical density to a new centrifuge tube (50 mL). 3. Add 75 μL of compound 053 stock solution (1000 μg / mL) to this tube. Cap the tube and shake thoroughly to mix the compound. Then keep the centrifuge tube on the table at room temperature for 15 minutes. Note: It is preferable to have a larger surface area to facilitate stirring. The final concentration of 053 used in the experiment is 0.75 μg / mL. Do not incubate for more than 15 minutes. 6. After 15 minutes of contact time, centrifuge the tube at 4500 rpm for 10-15 minutes to obtain a cell pellet and discard the supernatant. Wash the cell pellet thoroughly with 5 mL of phosphate or potassium phosphate buffered saline (PBS, pH 7) and repeat this operation twice. Note: Pipette several times with a 5 mL or 1 mL pipette during washing. Make sure there are no clumps while pipetting. The purpose of the wash is to remove excess dye from the cell surface. 7. A color change in the cells is visible to the naked eye on the cell pellet after two washes. a. Cells that overexpress efflux pumps (i.e., resistant cells) are white or off-white (default cell color). b. Cells with normal or moderate efflux activity are slightly pink in color. c. Cells without efflux pumps or with dysfunctional efflux pumps stain dark pink. d. Occasionally, the cell pellet is orange, indicating moderate efflux activity, mainly due to the inherent yellow color of the cells.

[0159] result: Figures 1-3 show the color (1A) and spectrofluorimetric (1B) differences observed in different reference strains (strain A, CIP 76.25), clinical strains (strain B, SA1199), and laboratory constructed NorA efflux pump mutants of strain B: NorA overexpression (strain C, SA 1199B) and NorA deletion (strain D, SA K1758). Since strain C has an overexpressed efflux pump, 053 efflux is stronger and the cell pellet appears white (053 is completely eliminated by efflux). However, since strain D does not have an efflux pump, the cell pellet appears dark pink. Intermediate colors can be observed for the reference strain (strain A) and clinical strain (strain B), suggesting mild to moderate activity. This is confirmed by fluorescence spectroscopy (Figures 2 and 3). Figure 2 shows the fluorescence emission spectrum of compound 053 in bacterial cells after excitation at 530 nm. FIG. 3 shows a histogram obtained by plotting the fluorescence intensity values ​​at 642 nm obtained after excitation at 530 nm.

[0160] The results obtained using the colorimetric analysis (Figures 1 to 3) clearly show that compound 053 makes it possible to visually distinguish three levels of strain coloration: 1. Dark pink color corresponding to a strain lacking the NorA efflux pump (SAK 1758). 2. No staining for the strain overproducing the NorA pump (SA 1199B). 3. Medium pale pink coloration for strains producing basal pump levels (SA 1199 and CIP 76.25).

[0161] It is therefore possible to identify which isolates are overproducing the NorA efflux pump and therefore have a very high potential for antibiotic resistance, and to distinguish them from those that produce basal levels.

[0162] Spectrofluorimetric results (Figure 2) show that compound 053 fluoresces when accumulated in bacteria, and that, like coloration, fluorescence is dependent on the level of NorA pump production. 1. The fluorescence of the overproducing strain (SA 1199B) is almost zero (Figures 2 and 3, green curves and histograms). 2. The NorA pump-free mutant (SAK 1758) has high fluorescence at 670 nm (FIGS. 2 and 3, black curve and histogram). 3. Two strains (CIP 76.25, SA 1199) that produce low levels of pump show intermediate levels of fluorescence (Figures 2 and 3, red and blue curves and histograms).

[0163] Unlike colorimetric tests, which do not require special reading equipment, fluorescence requires an appropriate spectrofluorometer, whereas fluorescence measurements are quantitative and allow accurate determination of the level of efflux activity of the isolate.

[0164] We also carried out fluorescence readings by flow cytometry to verify the results previously obtained by spectrofluorimetry. Advantageously, flow cytometry only takes into account the intracellular fluorescence and not the surrounding fluorescence, thus allowing a direct assessment of the concentration of compound 053 within the cells without the need for rinsing.

[0165] The results are consistent with those obtained previously. Figure 7 shows the fluorescence intensity measured by flow cytometry for the wild type strain (WT-A) corresponding to CIP 76.25, the reference strain (Ref-B) corresponding to SA, the NorA overproducing strain (NorA overproducing-C) corresponding to SA1199B, and the NorA deletion strain (NorA deletion-D) corresponding to SA K1758. The cytometry results showed that compound 53 moderately accumulated in the wild type and reference strains, very little accumulated in the NorA overexpressing strain, and accumulated heavily in the NorA deletion strain. We also tested the effect that fixatives such as 1% w / v paraformaldehyde (PFA) or 70% cold ethanol may have on cells treated with compound 053. Figure 8 shows the relative fluorescence intensity for each strain treated with compound 053 and PFA (A-D) or ethanol (E-H). Whatever the binding agent used, the emission differences between the different strains remained visible, demonstrating the robustness of the method regardless of the binding environment.

[0166] Example 2-2: Bacillus subtilis Materials and Methods: Bacterial strains: We used - Reference strain Bacillus subtilis 168 (DSM 402) which produces basal levels of BmrA - Bacillus subtilis BmrA+++, which overproduces the BmrA pump, and - Bacillus subtilis BmrAΔ, in which the BmrA gene has been deleted (see Steinfels E et al., Biochemistry 43:7491-7502 and Krugel H et al., FEMS Microbiol Lett. 2010 December;313(2):155-63).

[0167] The protocol used was the same as that used in Example 2-1.

[0168] result: Figures 4-6 show the color (Figure 4) and spectrofluorimetric (Figures 5 and 6) differences observed for different Bacillus subtilis strains. Strain A is a wild-type strain of B. subtilis 168 (DSM 402), strain B, BmrA+++, is a mutant overexpressing BmrA, and strain C is a BmrA deletion mutant (BmrAΔ). Since strain B shows overexpression of the BmrA efflux pump, compound 053 is completely effluxed from the cells and the cell pellet appears white. However, for strain C, which does not have an efflux pump, the cell pellet appears dark pink. Strain A is a normal wild-type strain expressing normal efflux activity; therefore, compound 053 is partially effluxed and the cell pellet appears pale pink, suggesting mild to moderate activity. Similar activity is confirmed and quantified using fluorescence spectroscopy (Figures 5 and 6).

[0169] As observed in FIG. 4 in the colorimetric analysis, it is clear that compound 053 visually distinguishes the BmrA mutants at three coloration levels: 1. Dark pink coloration, corresponding to a strain lacking the BmrA efflux pump (BmrAΔ); 2. There is no coloration for strains overproducing the BmrA pump (BmrA+++). 3. Medium pale pink for strains producing basal (normal, wild-type) levels of pump.

[0170] It is therefore possible to identify among clinical isolates those that overproduce the BmrA efflux pump, a medium-sized ABC (ATP-binding cassette) transporter involved in multidrug resistance.

[0171] The spectrofluorimetric results (Figures 5 and 6) are consistent and quantitatively validate the results: Compound 053 becomes fluorescent when accumulated in bacteria, and like coloration, fluorescence is dependent on the level of BmrA pump production.

[0172] 1. The fluorescence of the overproducing strain (BmrA+++) is almost zero (Figures 5 and 6, curves and histograms). 2. The pump-free mutant (BmrAΔ) is very high, with a maximum at 642 nm (FIGS. 5 and 6, curves and histograms). 3. The wild type strain (DSM 402), which produces low levels of pump, shows intermediate levels of fluorescence (Figures 5 and 6, curves and histograms).

[0173] Unlike colorimetric tests, which do not require special reading equipment, fluorescence requires an appropriate spectrofluorometer, whereas fluorescence measurements are quantitative and allow accurate determination of the level of efflux activity of the isolate.

[0174] Example 2-3: Streptococcus pneumoniae The inventors compared the staining induced by compound 053 for different strains of Streptococcus pneumoniae bacteria: a wild-type strain ("WT"), a strain overexpressing PatA-PatB, a Streptococcus pneumoniae ABC transporter ("OVP") for transporting molecules involved in the resistance of this pathogen to fluoroquinolone antibiotics out of the cell, and a strain deleted for PatA-PatB ("DEL").

[0175] Materials and Methods: Bacterial strains: We used -WT: Streptococcus pneumoniae reference strain producing basal levels of PatA-PatB -OVP: PatA-PatB overproducing strain -DEL: strain with deleted PatA-PatB coding genes The protocol used was the same as that used in Example 2-1.

[0176] result: Figures 9 and 10 show the colour (Figure 9) and spectrofluorimetric (Figure 10) differences observed with different strains of Streptococcus pneumoniae.

[0177] Since the OVP strain shows overexpression of the PatA-PatB transporter, compound 053 is strongly exported out of the cells and the cell pellet appears white. In contrast, in the WT and DEL strains lacking the PatA-PatB transporter, the cell pellet appears dark pink. Similar activity is confirmed and quantified using fluorescence spectroscopy (Figure 10).

[0178] Therefore, compound 053 can be used to qualitatively and quantitatively distinguish strains that overexpress the ABC PatA-PatB transporter from strains that do not overexpress or do not express this transporter at all.

[0179] Example 2-4: Escherichia coli (a) Compound 392 and Compound 29.

[0180] We tested the use of compound 392 and compound 29 on different E. coli strains with varying expression levels of the AcrAB efflux pump.

[0181] [ka]

[0182] Materials and Methods: The experimental protocol is the same as that described in the previous example, except for the bacterial strain used. Briefly, compound 29 and compound 392 (1 μg / mL) were added to 5 mL of cell suspension of different E. coli strains (OD=1.5), mixed by tilting, and left on the table at room temperature for 15 minutes. The cells were then centrifuged at 3500 rpm for 10 minutes and rinsed with potassium phosphate buffer (PBP). The cell pellet was washed only once.

[0183] [Table 2]

[0184] result: Figures 11-16 show the color (Figures 11 and 14) and spectrofluorimetric (Figures 12, 13, 15, and 16) differences observed in different Escherichia coli strains. Strain EC (Reference) is a reference strain (ATCC11775) that shows normal efflux, strain AG100 is a wild type strain of E. coli (Wild) that also shows normal efflux, strain AG102 is an AcrAB overexpression mutant (acrAB+++) that shows strong efflux, and strain AG100A (ΔacrAB) is an acrAB deletion mutant that shows weak efflux.

[0185] As can be seen in Figures 11 and 14, bacteria with normal efflux (EC and AG100) are characterized by a very light pink color, while a strain in which the gene encoding the AcrAB pump has been deleted (AG100A) shows a dark pink staining. In contrast, a strain with AcrAB overexpression (AG102) shows no staining. The same differences in efflux activity between the different strains are confirmed by fluorescence spectroscopy (Figures 12, 13, 15, and 16), and are even more remarkable.

[0186] (b) Compound 053 We also tested compound 053 (1 μg / mL) on the same Escherichia coli strains with various levels of AcrAB efflux pump expression. We slightly modified the protocol to introduce a pretreatment step with carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP is a chemical inhibitor of oxidative phosphorylation. CCCP interferes with the transmembrane electrochemical gradient and the proton motive force, thereby reducing ATP production. The effect of CCCP is to reduce the efficiency of the efflux pump, making it easier to perceive the staining differences between different strains.

[0187] [ka]

[0188] Chemical structure of CCCP Materials and Methods: The experimental protocol was the same as that described in the Examples, except that the cells were treated with CCCP (5 μg / mL) in phosphate buffered saline (PPB) for 1 h. After CCP treatment, all experiments were carried out in PPB instead of MH-II broth from step 4 onwards.

[0189] result: As can be seen in Figure 17, it is possible to visually distinguish strains with normal efflux (EC, AG100) or no efflux (AG100A) from the strain overexpressing AcrAB (AG102). Fluorescence spectroscopy (Figures 18 and 19) confirms this observation.

Claims

1. A compound of formula I: 【Chemistry 1】 (In the formula, X － is a halogen atom or an acceptable counterion, in particular Cl; In the formula, R1 and R2 are - In both cases, -CH ３ Or -C ４ H ９ or - or C １ ~C ４ Alkyl (R1 is C ｎ＋ｉ alkyl and R2 is C ｎ alkyl, and vice versa, n is an integer ranging from 1 to 3 and i is an integer ranging from 1 to 3, where n+i ranges from 1 to 4; or - or R1 is -C ４ H ７ and R2 is C ８ H １７ and and, In the formula, R3 is -CH ３ Or C ２ ~C ８ alkyl, ２ ~C ８ The alkyl is optionally substituted with one or more heteroatoms. or a salt or solvate of this compound of formula I, or a protonated form thereof.

2. The compound of claim 1: (Wherein, R1 and R2 are - In both cases, -CH ３ or - or C １ ~C ４ Alkyl (R1 is C ｎ＋ｉ alkyl and R2 is C ｎ alkyl, and vice versa, n is an integer ranging from 1 to 3 and i is an integer ranging from 1 to 3, where n+i ranges from 1 to 4; or and, In the formula, R3 is -CH ３ Or C ２ ~C ８ alkyl, ２ ~C ８ The alkyl is optionally substituted with one or more heteroatoms. or a salt or solvate of said compound of formula I, or a protonated form thereof.

3. R1 and R2 are each C ２ ~C ４ is an alkyl group, and -R1 is C ｎ＋ｉ When R is alkyl, R is C ｎ is alkyl, or -R2 is C ｎ＋ｉ When R is alkyl, R is C ｎ is alkyl, 3. The compound of claim 1 or claim 2, wherein n ranges from 1 to 3, i is an integer ranging from 1 to 3, and n+i ranges from 1 to 4.

4. The compound according to any one of claims 1 to 3, wherein R3 is C2-C4 alkoxy, dimethylaminoethyl, or methylaminexethyl.

5. The compound is selected from the group of compounds of formula I, wherein R1, R2, and R3 are all selected from the following table: 【Table 1】 The compound according to any one of claims 1 to 4, as indicated in each row of:

6. The compound according to any one of claims 1 to 5, wherein the compound is selected from the compounds of the following formulas 47, 52, 53, 392, and 29: 【Chemistry 2】 【Chemistry 3】

7. Use of a compound according to any one of claims 1 to 6 for assessing or measuring the excretion of said compound by bacteria.

8. 8. Use according to claim 7 for assessing or measuring excretion by Gram-positive bacteria.

9. 8. Use according to claim 7 for assessing or measuring excretion by gram-negative bacteria.

10. A bacterial shedding detection or measurement kit, comprising: - a compound according to any one of claims 1 to 6, - at least one control bacterium, A bacterial shedding detection or measurement kit comprising:

11. 11. The detection kit according to claim 10, additionally comprising a protonophore, in particular selected from 2,4-dinitrophenol, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), carbonyl cyanide-m-chlorophenylhydrazone (CCCP), C4R1, ellipticine, 10-[2-(3-hydroxy-6-oxo-xanthen-9-yl)benzoyl]oxydecyl-triphenyl-phosphonium bromide, and 2-(2-hydroxyaryl)hexylphosphonium bromide.

12. Use of a compound according to any one of claims 1 to 6 for assessing bacterial resistance to bacteriostatic or bactericidal compounds.

13. 1. A method for assessing bacterial efflux resistance to a bacteriostatic or bactericidal compound, comprising: - culturing the bacteria in a suitable culture medium to obtain a bacterial culture; - contacting said bacterial culture with a compound according to any one of claims 1 to 6 to obtain a treated bacterial culture; - washing the treated bacterial culture and precipitating the bacteria contained in the treated bacterial culture to obtain a bacterial pellet; - observing the staining of the bacterial pellet, - if the bacterial pellet has the color of the bacterial pellet from the untreated bacterial culture, the bacteria is resistant to the bacteriostatic or bactericidal compound by excretion of the bacteriostatic or bactericidal compound; and observing that if the bacterial pellet has a color different from the color of the bacterial pellet from the untreated bacterial culture, the bacteria are resistant to the bacteriostatic or bactericidal compound by excretion of the bacteriostatic or bactericidal compound; A method comprising:

14. 14. The method of claim 13, wherein the method further comprises incubating the bacterial culture with a protonophore.

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