Inhibitor of the bacterial type III secretion system

Abstract

The present invention is directed to novel organic compounds that exhibit the ability to inhibit effector toxin secretion or translocation mediated by the bacterial type III secretion system. The novel compounds contain zwitterionic moieties that provide polarity under suitable conditions for solubility, formulation, and delivery to patients, while allowing the compounds to be less polar under other conditions, such as binding to the T3SS needle target. Type III secretion system inhibitor compounds are useful for treating or preventing infections caused by bacteria, such as Pseudomonas species and Chlamydia species.

Description

Technical Field

[0001] Cross - Reference to Related Applications The subject matter of the present disclosure claims the benefit of U.S. Provisional Patent Application No. 63 / 349,173, filed on June 6, 2022, the disclosure of which is hereby incorporated by reference in its entirety. Statement Regarding Federally Sponsored Research The invention described herein was supported by National Institute of Allergy and Infectious Diseases (NIAID), DHHS / NIH grant numbers R43 AI068185, and R01 AI099269. Accordingly, the U.S. government has certain rights in this invention.

[0002] The present invention is in the field of treating bacterial infections and diseases. In particular, the present invention provides organic compounds that inhibit the type III secretion system of bacterial species.

Background Art

[0003] The bacterial type III secretion system (T3SS) is a complex multi-protein apparatus that facilitates the secretion and translocation of effector proteins directly from the bacterial cytoplasm into the mammalian host cell cytoplasm. This complex protein delivery apparatus is shared by more than 15 Gram-negative human pathogens, including Salmonella species, Shigella flexneri, Pseudomonas aeruginosa, Yersinia species, enteropathogenic and enteroinvasive Escherichia coli, and Chlamydia species (Non-Patent Document 1). In the opportunistic pathogen P. aeruginosa, the T3SS is a major virulence factor contributing to the establishment and dissemination of acute infections (Non-Patent Document 2). Four T3SS effectors have been identified in P. aeruginosa strains - ExoS, ExoT, ExoY, and ExoU. ExoS and ExoT are bifunctional proteins consisting of an N-terminal small G protein-activating protein (GAP) domain and a C-terminal ADP-ribosylation domain. ExoY is an adenylate cyclase. ExoU is a phospholipase (Non-Patent Document 3).

[0004] In studies using strains that produce each effector separately, ExoU and ExoS significantly contributed to persistence, dissemination, and death, while ExoT had a minor effect on virulence in a mouse lung infection model and ExoY did not appear to play a major role in P. aeruginosa pathogenesis (Non-Patent Document 4). Although not a prototypical effector toxin, flagellin (FliC) can also be injected from P. aeruginosa into the cytoplasm of host cells via the T3SS machinery, where it causes activation of the innate immune system via the nod-like receptor NLRC4 inflammasome (Non-Patent Document 5).

[0005] The presence of a functional T3SS is significantly associated with poor clinical outcomes and death in patients with lower respiratory tract infections and systemic infections caused by Pseudomonas aeruginosa (Non-Patent Document 6). Furthermore, the T3SS reduces survival in a Pseudomonas aeruginosa animal infection model (Schulert, et al., J. Infect. Dis., 188:1695-1706 (2003)) and is required for systemic dissemination of Pseudomonas aeruginosa in a murine acute pneumonia infection model (Non-Patent Document 7). The T3SS appears to contribute to the development of severe pneumonia by inhibiting the host's ability to contain and clear pulmonary bacterial infections. Secretion of T3SS toxins, particularly ExoU, blocks phagocyte-mediated clearance at the site of infection and promotes the establishment of infection (Non-Patent Document 8). As a result, essential components of the innate immune response are locally disrupted, creating an immunosuppressive environment in the lung. This not only allows Pseudomonas aeruginosa to persist in the lung but also promotes superinfection by other bacterial species.

[0006] Several antibacterial agents are effective against Pseudomonas aeruginosa, but even in patients with hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) who have been administered antibiotics active against the causative strain, high mortality rates in the range of 40% to a maximum of 69% (Non-Patent Document 9) and recurrence rates of over 30% (Non-Patent Document 10) associated with severe Pseudomonas aeruginosa infections reflect an increasing incidence of drug-resistant strains, highlighting the need for new therapeutic agents. (See Non-Patent Document 11) Conventional bacteriostatic and bactericidal antibiotics are insufficient to adequately address these infections, and new therapeutic approaches, such as inhibitors of Pseudomonas aeruginosa virulence determinants, may prove useful as adjunctive therapies (Non-Patent Document 12).

[0007] The potential of the type III secretion system as a therapeutic target has prompted several groups to screen for inhibitors of T3SS in various bacterial species, including Salmonella typhimurium, Yersinia pestis, Pseudotuberculosis, and Escherichia coli (Non-Patent Document 13). Since there is a high level of sequence conservation among various proteins including the T3SS apparatus, certain T3SS inhibitors may exhibit activity even in related species. The broad-spectrum activity of T3SS inhibitors identified in screening against Yersinia was demonstrated in Non-Patent Document 14.

[0008] Screening for Pseudomonas aeruginosa T3SS inhibitors has been reported (see, for example, Non-Patent Document 15), resulting in several selective inhibitors of P. aeruginosa T3SS-mediated secretion, one of which reproducibly inhibits both T3SS-mediated secretion and translocation (Non-Patent Document 16).

[0009] Nevertheless, there remains a medical need for optimized inhibitors of bacterial T3SS in P. aeruginosa and other bacterial species with drug-like properties. Specifically, there is a need for inhibitors that can be easily prepared in a clinically acceptable formulation, are safe and effective in animal models of infection, and exhibit appropriate pharmacokinetic parameters to ensure sufficient levels of inhibition in the appropriate tissues.

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Summary of the Invention

Problems to be Solved by the Invention

[0011] The present invention relates to the identification and characterization of novel antibacterial / antipathogenic agents that are active against current drug-resistant strains of Pseudomonas aeruginosa. The compounds of the present invention exhibit surprising and unexpected improved levels of efficacy, water solubility, in vivo pharmacokinetics, safety, and effectiveness compared to known T3SS inhibitor compounds, thereby providing a promising addition to the family of antibacterial / antipathogenic agents under development.

Means for Solving the Problems

[0012] The present invention provides novel bacterial type III secretion system (T3SS) inhibitor compounds. The T3SS inhibitor compounds described herein were identified by a program that performs structural modifications on a phenoxyacetamide (PhA) scaffold and subsequently tests novel analogs using cell-based secretion, translocation, and cytotoxicity assays. Advantageously, the present invention provides additional compounds having surprisingly improved T3SS inhibitory potency, solubility, in vivo tolerance, in vivo stability, lung, and other tissue levels, and in vivo efficacy in a mouse lung infection model, compared to previous phenoxyacetamides or other known small molecule T3SS inhibitors.

[0013] Without limitation, the surprisingly improved drug-like properties of the compounds disclosed herein may be related to their zwitterionic nature. As reported in Aiello et al., 2010, op. cit., further T3SS inhibitor analogs, including MBX-2359, were synthesized by structure / activity correlation (SAR) studies based on the compound named MBX-1641, i.e., having the structure:

Chemical formula

[0014] The present invention is the result of further advancing the SAR study of the phenoxyacetamide scaffold. The results provide novel analogs having zwitterionic moieties to provide polarity under suitable conditions for solubility, formulation, and delivery to patients or animal models, but to render the compounds less polar under other conditions such as binding to the T3SS needle target. Optimization of the zwitterionic component, its structure, and position in the analogs provided novel T3SS inhibitors having excellent potency as well as excellent drug-like properties for delivery and efficacy compared to known T3SS inhibitor compounds.

[0015] The novel analogs of the present invention provide excellent physical and ADMET properties relative to the prototype inhibitor scaffolds represented by MBX-1641 and MBX-2359.

[0016] For example, a zwitterionic substituent at the R5 position of the following formula has been discovered, which is the optimal substituent on this backbone and represents a structural difference from previously studied aryloxyacetamide inhibitor compounds.

Chemical formula

[0017] Successful drugs must meet a series of criteria that establish their safety, pharmacokinetics (PK), and efficacy. The chemical optimization of the phenoxyacetamide series demonstrates the complexity of this process, which simultaneously optimizes multiple properties. The optimization process is complex and often unpredictable, but the zwitterionic series of T3SS compounds described herein surprisingly meet all criteria. The challenges of minimizing non-specific interactions and toxicity, maximizing tissue levels, maintaining efficacy, and improving the solubility of inhibitory compounds to enable high concentrations in therapeutic formulations can be difficult to overcome. Surprisingly, according to the present invention, the addition of a zwitterionic moiety to the phenoxyacetamide scaffold has led to the unexpected identification of novel analogs that meet the desired drug-like properties required for developing these compounds into drugs for human or animal therapeutic treatment. Exemplary embodiments of these novel analogs include compounds MBX-5452A and MBX-6681B described below. Furthermore, the zwitterionic analogs MBX-5452A and MBX-6681B exhibit superior efficacy, selectivity, and drug-like properties compared to other known T3SS inhibitor analogs such as MBX-1641, which shows lower efficacy and an insufficient MLMt value of less than 1 minute, and MBX-2359, which shows insufficient water solubility and can be difficult to formulate at sufficiently high concentrations in clinically acceptable excipients. The T3SS inhibitor compounds of the present invention overcome these problems and thus provide a genus of novel T3SS inhibitor compounds suitable for safe and effective administration for the treatment of bacterial infections in mammalian subjects. 1 / 2 Values, and MBX-2359, which shows insufficient water solubility and can be difficult to formulate at sufficiently high concentrations in clinically acceptable excipients, exhibit superior efficacy, selectivity, and drug-like properties. The T3SS inhibitor compounds of the present invention overcome these problems and thus provide a genus of novel T3SS inhibitor compounds suitable for safe and effective administration for the treatment of bacterial infections in mammalian subjects.

[0018] To demonstrate, Table 1 below provides some examples of the properties of known T3SS inhibitor compounds (entries 1-7), and the subsequent discovery of novel compounds with clearly improved properties (entries 8-15), in particular, the unexpected superiority of the exemplary zwitterionic compounds MBX-5452 and MBX-6681B (entries 14 and 15, respectively).

[0019] [Table 1]

[0020] Thus, the T3SS inhibitor compounds described herein inhibit the T3SS-mediated secretion of bacterial exotoxins (effectors) from bacterial cells. More preferably, the T3SS inhibitor compounds described herein inhibit the T3SS-mediated secretion of effectors from bacterial cells and also inhibit the T3SS-mediated translocation of effectors from bacterial cells to host cells (e.g., human or other animal cells), particularly phagocytic cells of the innate immune response such as macrophages and neutrophils.

[0021] In one embodiment, the T3SS inhibitor compounds described herein inhibit the T3SS of Pseudomonas and the T3SS of bacteria of at least one other genus. Preferably, the Pseudomonas bacteria to be inhibited are Pseudomonas aeruginosa.

[0022] In another embodiment, the present invention relates to a composition for treating or preventing a bacterial infection, the composition comprising a novel bacterial T3SS inhibitor compound described herein. The compositions described herein are suitable for the treatment or prevention of bacterial infections, particularly Pseudomonas infections, more specifically Pseudomonas aeruginosa infections, in mammals, particularly humans.

[0023] In another embodiment, the present invention relates to a method for treating or preventing a bacterial infection, particularly a Pseudomonas infection, more particularly a Pseudomonas aeruginosa infection, in a mammal by administration of a novel bacterial T3SS inhibitor compound of the present invention. In a preferred embodiment, the mammal is a human.

[0024] The present invention also provides a pharmaceutical composition containing one or more of the T3SS inhibitor compounds disclosed herein and a pharmaceutically acceptable carrier or excipient. The use of one or more T3SS inhibitor compounds in the preparation of a medicament for combating bacterial infections is also disclosed.

[0025] Also disclosed is a pharmaceutical composition comprising a therapeutically effective amount of a novel T3SS inhibitor compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition is suitable for use in the disclosed methods for the treatment or prevention of bacterial infections, particularly Pseudomonas infections, more specifically Pseudomonas aeruginosa infections, in mammals. The pharmaceutical composition can be formulated for both oral administration and / or parenteral administration to a subject or patient in need thereof.

[0026] In another embodiment, the present invention relates to the use of a novel T3SS inhibitor compound described herein in a method of manufacturing a medicament for treating bacterial infections, particularly Pseudomonas infections, more specifically Pseudomonas aeruginosa infections, in mammals (e.g., humans), which comprises combining one or more of the disclosed T3SS inhibitor compounds, products, or compositions of the present invention with a pharmaceutically acceptable carrier or excipient. Thus, in one aspect, the present invention relates to a method for manufacturing a medicament comprising combining at least one disclosed T3SS inhibitor compound according to the present invention, or at least one disclosed product, with a pharmaceutically acceptable carrier or diluent.

[0027] In another embodiment, the present invention relates to the identification of bacterial T3SS inhibitors for the treatment and / or prevention of bacterial infections, particularly Pseudomonas infections. More specifically, the present invention relates to the identification and characterization of bacterial T3SS inhibitors suitable for the treatment and / or prevention of infections caused by Pseudomonas aeruginosa.

[0028] In another embodiment, the small molecule inhibitor of the present invention can optionally be administered to a subject in need thereof in combination with one or more known antibacterial or antiviral agents.

[0029] In another embodiment, the bacterial T3SS inhibitor of the present invention is formulated in a pharmaceutically acceptable carrier and is applied / administered to a subject in need thereof by injection including, but not limited to, intradermal, transdermal, intramuscular, intraperitoneal, and intravenous. According to another embodiment of the present invention, the administration is oral and the compound may be provided, for example, in the form of tablets or encapsulated in gelatin capsules or microcapsules, thereby facilitating oral application. The generation of these dosage forms is within the scope of the general knowledge of a person skilled in the art. Multiple routes of administration are envisioned for these drug-like molecules, and a highly cost-effective production strategy can be easily achieved.

[0030] In a preferred embodiment, the present invention provides a novel class of bacterial type III secretion system (T3SS) inhibitor compounds of formula I or a pharmaceutically acceptable salt thereof:

Chemical formula

Chem.

[0031] In another embodiment, the present invention provides a family of bacterial type III secretion system (T3SS) inhibitor compounds having the structure of formula I(a) or a pharmaceutically acceptable salt thereof:

Chem.

Chemical formula

[0032] In another embodiment, the present invention provides a bacterial type III secretion system (T3SS) inhibitor compound family having the structure of formula I(b) or a pharmaceutically acceptable salt thereof:

Chemical formula

[0033] In another embodiment, the present invention provides a family of bacterial type III secretion system (T3SS) inhibitor compounds having the structure of Formula I(c) or a pharmaceutically acceptable salt thereof: [ka] During the ceremony: A is CH or N; at least one X is Cl and the other X is hydrogen, Br, or Cl; W is a divalent group bridging Ar and U, selected from the group including -C(O)CH2-, -S(O)2-, -NHS(O)2-, -S(O)2NH-, -C(O)-, -CH2-, -CH(CH3)-, -NHC(O)-, -NHC(O)NH-, -NHC(NH)NH-, -NHC(NCH3)NH-, -NHS(O)2NH-, -N(CH3)C(O)-, -C(O)NH-, -C(O)N(CH3)-, -O(C(O)-, -(C(O)O-, -NH-, or -O-; U contains from 1 to 2 nitrogen atoms and 0 to 1 oxygen atom, and is part of a 4- to 7-membered non-aromatic heterocyclic ring having from 0 to 3 substituents selected from alkyl, cycloalkyl, aryl, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, or sulfinyl (in addition to the bonds between W and ring carbon / nitrogen and between U and ring carbon), and can be either nitrogen or carbon, or U is independently hydrogen or optionally an aliphatic group of less than nine carbon atoms which can be substituted with a group selected from haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, sulfinyl, carboxy, alkoxycarbonyl, or aminocarbonyl group, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, sulfonyl, or haloalkoxy, sulfonyl, or sulfinyl group, and is connected to a basic nitrogen in an inner ring or outer ring structure and is composed of a 3- to 7-membered atomic ring, and can be either nitrogen or saturated carbon, linked via a chain, carbocyclic ring, or heterocyclic ring of 1 to 6 carbon atoms in a monocyclic, bicyclic, or spiro ring system; R5 is a monovalent group selected from the group containing -CH2C(CH3)2C(O)OH, -CH2CH(OH)C(O)OH, -CH2C(O)OH, -CH2(oxetane)C(O)OH, -CH2C(O)OH, -CH2CH2CH2C(O)OH, -(cyclobutyl)C(O)OH, -CH2(cyclopropyl)C(O)OH, or CH2(cyclopropyl)C(O)OH; and its methylene or ethylene group is -C(O)NHOH, -C(O)N(CH3)OH, -C(O)NHOCH3, -NHP(O)(OH)NHCH3, -P(O)(OH)NHCH 3、 -P(O)(OH) 2、 -OP(O)(OH) 2、 -OP(O)(OH)NHCH 3、 -S(O)2Me, -S(O)2OH, -S(O)OH, -S(O)2CF3,

Chemical formula

[0034] The compounds according to the above formula were tested using an assay that shows specific inhibition of the T3SS of Pseudomonas aeruginosa.

[0035] In a particularly preferred embodiment of the present invention, the T3SS inhibitor compound blocks T3SS-mediated secretion from Pseudomonas aeruginosa cells and the translocation of one or more toxin effectors, is safe, and achieves a sufficiently high level and for a sufficient period in a suitable animal tissue that shows efficacy in animal models of infection such as the mouse lung infection model.

[0036] Even more preferably, the T3SS inhibitor compounds described herein have a translocation inhibition EC 50 <10 uM against 90% of the Pseudomonas aeruginosa clinical strains tested, are stable in human serum (>75% stability for more than 1 hour), are stable in human liver microsome preparations (t 1 / 2 >1 hour), are soluble at concentrations above 5 mg / mL in clinically acceptable formulations, and show in vivo efficacy in animal models of Pseudomonas aeruginosa infection.

[0037] The T3SS compounds described herein are useful as anti-pathogenic agents and can be used to treat bacterial infections. Thus, an infected or exposed individual with a bacterial infection, particularly a Pseudomonas infection or a Chlamydia infection, can be treated by administering an effective amount of a compound according to the present invention to the individual in need.

[0038] The use of one or more of the compounds disclosed herein, or combinations thereof, for treating infections caused by bacteria having a type III secretion system is contemplated herein. In particular, the use of one or more of the above compounds, or combinations thereof, for treating Pseudomonas or Chlamydia infections is contemplated herein. In particular, the use of one or more of the above compounds, or combinations thereof, for treating Pseudomonas aeruginosa or Chlamydia trachomatis infections is advantageously carried out according to the teachings herein.

[0039] The T3SS inhibitor compounds described herein, or combinations of T3SS inhibitor compounds, can be used as supportive or adjunctive therapy for the treatment of bacterial infections in an individual (human or other animal). In the case of an individual who is not severely immunocompromised, administration of the T3SS inhibitor compounds described herein to inhibit the T3SS of bacterial cells within or on the individual may be sufficient for the individual's own immune system to effectively remove or kill the infecting or contaminating bacteria from the individual's tissues. Alternatively, the T3SS inhibitor compounds described herein can be administered to an individual in combination with (i.e., in a mixture, sequentially, or simultaneously) antibacterial agents such as antibiotics, antibodies, or immunostimulants to provide both inhibition of the T3SS and inhibition of the growth of invading bacterial cells.

[0040] In yet another embodiment, a composition comprising a T3SS inhibitor, or combination of T3SS inhibitors, described herein can also include a second agent (second active ingredient, second active agent) having a desired therapeutic or prophylactic activity other than the activity of T3SS inhibition. Such second active agents include, but are not limited to, antibiotics, antibodies, antiviral agents, anti-cancer agents, analgesics (e.g., non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, opioids, COX-2 inhibitors), immunostimulants (e.g., cytokines), hormones (natural or synthetic), central nervous system (CNS) stimulants, anti-emetics, antihistamines, erythropoietin, complement stimulants, sedatives, muscle relaxants, anesthetics, anti-convulsants, anti-depressants, anti-psychotics, and combinations thereof.

[0041] The composition containing the T3SS inhibitor described in this specification can be formulated for administration to an individual (human or other animal) by any of various routes including, but not limited to, intravenous, intramuscular, subcutaneous, intra-arterial, parenteral, intraperitoneal, sublingual (under the tongue), buccal (cheek), oral (for swallowing), topical (epidermis), transdermal (absorbed into the underlying vascular structure through the skin and the lower dermal layer), intranasal (nasal mucosa), intralung (lung), intrauterine, intravaginal, endocervical, intrarectal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrarenal, nasojejunal, and intraduodenal.

Brief Description of the Drawings

[0042]

Figure 1

Figure 2

Figures 2A - C

Figures 2B - D

Figure 3

Figures 3A - C

Figures 3B - D

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Mode for Carrying Out the Invention

[0043] Definitions

[0044] For the present invention to be more clearly understood, the following abbreviations and terms are used as defined below.

[0045] Abbreviations for various substituents (side chain groups, radicals) of organic molecules are those commonly used in organic chemistry. Such abbreviations may include "abbreviated" forms of such substituents. For example, "Ac" is an abbreviation for an acetyl group, "Ar" is an abbreviation for an "aryl" group, "halo", or "halogen", indicates a halogen group (e.g., F, Cl, Br, I). "Me" and "Et" are abbreviations used to indicate a methyl (CH3-) and an ethyl (CH3CH2-) group, respectively. "OMe" (or "MeO") and "OEt" (or "EtO") indicate a methoxy (CH3O-) and an ethoxy (CH3CH2O-), respectively. Since the presence and position of hydrogen atoms in an organic molecular structure are understood and known by those skilled in the art, hydrogen atoms are not necessarily shown in an organic structure diagram (e.g., at the end of a drawn line representing a CH3 group), or may only be selectively shown in some structure diagrams. Similarly, carbon atoms are not necessarily specifically omitted as "C" since the presence and position of carbon atoms in a structure diagram are known and understood by those skilled in the art. Minutes are generally abbreviated as "min.", and time is generally abbreviated as "hr", or "h".

[0046] As used herein, a composition or method described as "comprising" one or more specified elements or steps is open-ended, meaning that the specified elements or steps are essential, but that other elements or steps can be added within the scope of the composition or method. To avoid redundancy, any composition or method described as "comprising" (or "including") one or more specified elements or steps also describes the corresponding more limited composition or method "consisting essentially of" (or "consists essentially of") the same specified elements or steps, meaning that the composition or method includes the specified essential elements or steps and may also include additional elements or steps that do not substantially affect the basic and novel characteristics (s) of the composition or method. Any composition or method described herein as "comprising" or "consisting essentially of" one or more specified elements or steps also describes the more limited, closed-ended composition or method "consisting of" (or "consists of") the specified elements or steps corresponding to the specified elements or steps, excluding any other unspecified elements or steps. In any composition or method disclosed herein, known or disclosed equivalents of any specified essential element or step may be substituted for that element or step. It is also understood that "selected from the group consisting of" an element or step refers to one or more of the following listed elements or steps, including any combination of two or more of the listed elements or steps.

[0047] As used herein, "halo" or "halogen" means fluorine, chlorine, bromine or iodine.

[0048] "Alkyl" means a linear or branched, monovalent or divalent group of saturated and / or unsaturated carbon atoms and hydrogen atoms, such as methyl (Me), ethyl (Et), propyl (Pr), isopropyl (iPr), butyl (Bu), isobutyl (iBu), sec-butyl (sBu), tert-butyl (tBu), etc., which may be unsubstituted or substituted with one or more suitable substituents found herein.

[0049] "Haloalkyl" means an alkyl moiety substituted with one or more identical or different halogen atoms, such as -CH2Cl, -CF3, -CH2CF3, -CH2CCl3, etc.

[0050] "Alkenyl" means a linear, branched or cyclic hydrocarbon group having 2 to 8 carbon atoms and at least one double bond, such as ethenyl, 3-buten-1-yl, 3-hexen-1-yl, cyclopenta-1-en-3-yl, etc., which may be unsubstituted or substituted with one or more suitable substituents found herein.

[0051] "Alkynyl" means a linear, branched or cyclic hydrocarbon group having 2 to 8 carbon atoms and at least one triple bond, such as ethynyl, 3-butyn-1-yl, 2-butyn-1-yl, 3-pentyn-1-yl, etc., which may be unsubstituted or substituted with one or more suitable substituents found herein.

[0052] As used herein, "cycloalkyl" means a non-aromatic monovalent or divalent, monocyclic or polycyclic group having 3 to 12 carbon atoms, each of which may be saturated or unsaturated, such as cyclopentyl, cyclohexyl, decalinyl, etc., may be unsubstituted or substituted with one or more suitable substituents found herein, and may be condensed with one or more aryl groups, heteroaryl groups, or heterocycloalkyl groups, which may themselves be unsubstituted or substituted with one or more suitable substituents found herein.

[0053] "Heterocycloalkyl" means a non-aromatic monovalent or divalent, monocyclic or polycyclic group having 2 to 12 carbon atoms and 1 to 5 heteroatoms selected from nitrogen, oxygen, and sulfur, each of which may be saturated or unsaturated, such as pyrrolidinyl, tetrahydropyranyl, morpholinyl, piperazinyl, oxiranyl, etc., and may be unsubstituted or substituted with one or more suitable substituents found herein, and may be condensed with one or more aryl groups, heteroaryl groups, or heterocycloalkyl groups, which may themselves be unsubstituted or substituted with one or more suitable substituents found herein.

[0054] "Aryl" means an aromatic monovalent or divalent, monocyclic or polycyclic group containing 6 to 18 carbon ring members, such as phenyl, biphenyl, naphthyl, phenanthryl, etc., and may be substituted with one or more suitable substituents found herein, and may be condensed with one or more heteroaryl groups or heterocycloalkyl groups, which may themselves be unsubstituted or substituted with one or more suitable substituents found herein.

[0055] "Heteroaryl" means an aromatic monovalent or divalent, monocyclic or polycyclic group containing 6 to 18 carbon ring members and at least one nitrogen heteroatom, such as pyridyl, pyrazyl, pyrimidyl, quinolyl, etc., and may be substituted with one or more suitable substituents found herein, and may be condensed with one or more aryl, heteroaryl groups, or heterocycloalkyl groups, which may themselves be unsubstituted or substituted with one or more suitable substituents found herein.

[0056] "Hydroxy" means the group -OH.

[0057] "Alkoxy" means the group -OR, where R is an alkyl or cycloalkyl group.

[0058] "Aryloxy" means the group -OAr, where Ar is an aryl group.

[0059] "Heteroaryloxy" means the group -O(HAr), where HAr is a heteroaryl group.

[0060] "Acyl" means the group -C(O)R, where R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl, for example, acetyl, benzoyl, etc.

[0061] "Carboxy" means the group -C(O)OH.

[0062] "Alkoxycarbonyl" means the group -C(O)OR, where R is alkyl, alkenyl, alkynyl, or cycloalkyl.

[0063] "Aryloxycarbonyl" means the group -C(O)OR, where R is aryl or heteroaryl.

[0064] "Amino" means the group -NH2.

[0065] "Alkylamino" means the group -NRR', where R and R' are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, or heterocycloalkyl.

[0066] "Acylamino" means the group -NHC(O)R, where R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl, for example, acetyl, benzoyl, etc., such as acetylamino, benzoylamino, etc.

[0067] "Carboxamide" means the group -C(O)NRR', where R and R' are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, or heterocycloalkyl.

[0068] "Sulfonylamide" means the group -NHSO2R, where R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl.

[0069] "Amidino" means the group -C(NR)NR´R", where R, R´, and R" are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, and R, R´, and R" may form a heterocycloalkyl ring, such as imidazolinyl, tetrahydropyrimidinyl.

[0070] "Guanidino" means the group -NHC(NR)NR´R", where R, R´, and R" are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, and R, R´, and R" may form a heterocycloalkyl ring.

[0071] "Alkylthio" means the group -SR, where R is an alkyl or cycloalkyl group.

[0072] "Arylthio" means the group -SAr, where Ar is an aryl group.

[0073] "Hydroxamide" means the group -C(O)NHOR, where R is an alkyl or cycloalkyl group.

[0074] "Thioacyl" means the group -C(S)R, where R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl.

[0075] "Alkylsulfonyl" means the group -SO2R, where R is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl.

[0076] "Aminosulfonyl" means the group -SO2NRR', where R and R' are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or heteroaryl, or heterocycloalkyl.

[0077] As used herein, the terms "bacterial type III secretion system inhibitor", "bacterial T3SS inhibitor", "bacterial T3SS inhibitor compound", and "T3SS inhibitor compound" are interchangeable and refer to a compound that, for example, specifically inhibits at least 15% of the bacterial type III secretion system at a concentration of 50 μM as measured by the T3SS effector transcriptional reporter assay, or that inhibits the bacterial T3SS, for example, as measured by the T3SS-mediated effector toxin secretion assay.

[0078] In connection with the therapeutic use of the T3SS inhibitor compounds described herein, the terms "treatment," "to treat," or "treating" refer to any use of a T3SS inhibitor compound that is calculated or intended to halt or inhibit the pathogenicity, or T3SS-mediated effector secretion, or translocation of bacteria having a type III secretion system. Thus, treatment of an individual can be carried out after any diagnosis indicative of a potential bacterial infection, i.e., whether an infection by a particular bacterium has been confirmed or whether the possibility of infection is only suspected, for example, after exposure to the bacterium or to another individual infected with the bacterium. Also, the inhibitors of the present invention affect the introduction of effector toxins into host cells and thus prevent or reduce the pathogenicity or toxicity resulting from the infection, it being recognized that the inhibitor compounds are not necessarily bactericidal and have no effect on inhibiting the growth or spread of bacterial cells. For this reason, it will be understood that the elimination of a bacterial infection is achieved by the host's own immune system, or by immune effector cells, or by the introduction of antibiotics. Thus, the compounds of the present invention are contemplated to be routinely combined with other active ingredients such as antibiotics, antibodies, antiviral agents, anticancer agents, analgesics (e.g., non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, opioids, COX-2 inhibitors), immunostimulants (e.g., cytokines, or synthetic immunostimulatory organic molecules), hormones (natural, synthetic, or semi-synthetic), central nervous system (CNS) stimulants, antiemetics, antihistamines, erythropoietin, agents that activate complement, sedatives, muscle relaxants, anesthetics, anticonvulsants, antidepressants, antipsychotics, and combinations thereof.

[0079] The meaning of other terms will be understood in the context understood by those skilled in the art, including the fields of organic chemistry, pharmacology, and microbiology.

[0080] Detailed Description of the Invention

[0081] The present invention provides novel organic compounds that inhibit the bacterial type III secretion system ("T3SS") that secretes and translocates bacterial-produced effectors (also referred to as effector toxins, exotoxins, cytotoxins, bacterial toxins) from bacterial cells into human or animal host cells. Effectors translocated into host cells can effectively inactivate the host immune response, such as by killing phagocytic cells (e.g., macrophages and neutrophils), thereby disabling the host innate immune response. Thus, T3SS is an important pathogenicity factor in establishing bacterial infections in an individual (human or other animal), and is particularly important for opportunistic Pseudomonas aeruginosa infections in human patients whose immune systems are compromised or who are otherwise predisposed to infection by bacteria such as P. aeruginosa.

[0082] The present invention provides certain novel organic compounds that inhibit the T3SS of Pseudomonas, particularly Pseudomonas aeruginosa. Structural analogs of previously studied T3SS inhibitors were evaluated for inhibition of T3SS-mediated secretion of an effector toxin-β-lactamase fusion protein (ExoS'-βLA) using the Pseudomonas strain MDM973 (PAK / pUCP24GW-lacI Q -lacPO-exoS::blaM, Table 1). Examples 1 and 2 below describe the screening and validation of initial T3SS inhibitors.

[0083] In a series of experiments to compare the effect of modifying a phenoxyacetamide scaffold, where compound MBX-1641 is a prototype example,

Chemical formula

[0084] [Chem.]

[0085] Minor modifications of the A aryl group can be tolerated without increasing the inhibitory concentration level (IC 50 ) beyond the minimum threshold (i.e., 200 μM). However, the introduction of a third substituent onto the A aryl group improved the IC 50 (>5-fold). Studying alternative linker moieties for the A aryl group and the B aryl group, their positions were found to have a significant impact on the overall properties of the resulting compounds. Similarly, changes to remove the methyl group at the chiral center (α-carbon) or increase the size of the substituents also had a significant effect on the T3SS inhibitory properties. A wide range of substitutions on the B aryl group have proven to be highly promising, having excellent in vitro potency and ADME properties. More specifically, zwitterionic groups represent privileged motifs required for both potency and microsomal stability. In general, the structure / activity relationships revealed experimentally were characteristic of discoveries regarding alternative compounds reacting with a single target binding site.

[0086] From programs of analog synthesis and comparative testing, a new family of compounds has emerged that rivals and in many cases exceeds the T3SS inhibitory properties of previously described phenoxyacetamide inhibitor compounds.

[0087] In one embodiment, a family of novel T3SS inhibitor compounds is defined by formula I or a pharmaceutically acceptable salt thereof: [Chem.] Wherein, A is independently selected from CH or N; X is independently selected from hydrogen, halogen, or hydroxyl; Z is O, S, NH, or NR', where R' is alkyl; R1, R2, and R3 are each independently selected from hydrogen, halogen, alkyl, hydroxy, alkoxy, alkylthio, or cyano, with no more than two of the preceding groups being hydrogen; In NR4, R4 is hydrogen, a straight-chain aliphatic group, a branched-chain aliphatic group, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy, alkylamino, alkylthio, sulfonyl, sulfinyl, carboxy, alkoxycarbonyl, or aryl; Y is a straight-chain or branched-chain alkyl, alkenyl, or alkynyl group having 1 to 6 carbon atoms, or a cyclic alkyl, alkenyl, or alkynyl group having 3 to 6 carbon atoms, which may contain one or more heteroatoms and which may be unsubstituted or substituted with up to 4 substituents selected from halo, cyano, hydroxy, amino, alkyl, cycloalkyl, alkylamino, carboxyl, alkoxycarbonyl, carboxamide, acylamino, amidino, sulfonamide, aminosulfonyl, alkylsulfonyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, oxygen, oxime, or NR", where R" is hydrogen, alkyl, cycloalkyl; hydrogen may be replaced by deuterium, Ar is an aryl or heteroaryl group forming a 5- or 6-membered ring that may be further fused with 1 to 3 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings, and the Ar group is unsubstituted or substituted with up to 4 substituents selected from halo, cyano, hydroxy, amino, alkyl, cycloalkyl, alkylamino, carboxyl, alkoxycarbonyl, carboxamide, acylamino, amidino, sulfonamide, aminosulfonyl, alkylsulfonyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, and any two substituents may together form an aromatic or non-aromatic ring structure fused to the aryl or heteroaryl group Ar, or the substituents on Ar may optionally covalently bond to either Y or R4, or both Y and R4, to form a heterocyclic or carbocyclic system, which may be aromatic, heteroaromatic, or partially aromatic (i.e., one or more rings are aromatic and one or more rings are non-aromatic (saturated)); W is a divalent group bridging Ar and U selected from the group consisting of -C(O)CH2-, -S(O)2-, -NHS(O)2-, -S(O)2NH-, -C(O)-, -CH2-, -CH(CH3)-, -NHC(O)-, -NHC(O)NH-, -NHC(NH)NH-, -NHC(NCH3)NH-, -NHS(O)2NH-, -N(CH3)C(O)-, -C(O)NH-, -C(O)N(CH3)-, -O(C(O)-, -(C(O)O-, -NH-, or -O-; U contains 1 to 2 nitrogen atoms and 0 to 1 oxygen atom, and has 0 to 3 substituents selected from alkyl, cycloalkyl, aryl, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, or sulfinyl (in addition to the bonds between W and the ring carbon / nitrogen and between U and the ring carbon), and is part of a 4- to 7-membered non-aromatic heterocycle, and can be either nitrogen or carbon, or U is independently hydrogen, or optionally, an aliphatic group having less than 9 carbon atoms and substituted with a group selected from haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, sulfinyl, carboxy, alkoxycarbonyl, or aminocarbonyl group, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, sulfonyl, or haloalkoxy, sulfonyl, or sulfinyl group, and is connected to a basic nitrogen in an inner ring or outer ring structure, and is composed of a 3- to 7-membered atomic ring, and can be either nitrogen or saturated carbon, and is linked via a chain, carbocycle, or heterocycle having 1 to 6 carbon atoms in a monocyclic, bicyclic, or spiro ring system; R5 is a monovalent group selected from the group consisting of -CH2C(CH3)2C(O)OH, -CH2CH(OH)C(O)OH, -CH2C(O)OH, -CH2(oxetane)C(O)OH, -CH2C(O)OH, -CH2CH2CH2C(O)OH, -(cyclobutyl)C(O)OH, -CH2(cyclopropyl)C(O)OH, or CH2(cyclopropyl)C(O)OH; and its methylene or ethylene group is -C(O)NHOH, -C(O)N(CH3)OH, -C(O)NHOCH3, -NHP(O)(OH)NHCH3, -P(O)(OH)NHCH 3、 -P(O)(OH) 2、 -OP(O)(OH) 2、 -OP(O)(OH)NHCH 3、 -S(O)2Me, -S(O)2OH, -S(O)OH, -S(O)2CF3,

Chemical formula

[0088] In another embodiment, the present invention provides a family of bacterial type III secretion system (T3SS) inhibitor compounds having the structure of formula I(a) or a pharmaceutically acceptable salt thereof:

Chemical formula

Chemical Structure

[0089] In another embodiment, the present invention provides a family of bacterial type III secretion system (T3SS) inhibitor compounds having the structure of formula I(b) or a pharmaceutically acceptable salt thereof:

Chemical formula

Chem.

[0090] In another embodiment, the present invention provides a family of bacterial type III secretion system (T3SS) inhibitor compounds having the structure of formula I(c) or a pharmaceutically acceptable salt thereof:

Chem.

Chemical Structure

[0091] Preferred embodiments of the present invention, which are examples of the above formula I, formula I(a), (b), or (c), include the following compounds or pharmaceutically acceptable salts thereof: [Table 2] TIFF2025524366000024.tif254169TIFF2025524366000025.tif254168TIFF2025524366000026.tif249170TIFF2025524366000027.tif254170TIFF2025524366000028.tif249170TIFF2025524366000029.tif236170TIFF2025524366000030.tif237170TIFF2025524366000031.tif238170TIFF2025524366000032.tif247170TIFF2025524366000033.tif247170TIFF2025524366000034.tif163170

[0092] The compounds of the present invention inhibit the T3SS-dependent translocation of exotoxins into phagocytic cells of the innate immune response (neutrophils and macrophages), effectively rescue these cells from destruction, and enhance the host innate immune system rather than directly killing the invading bacteria, thereby enabling the enhancement of the activity of existing antibacterial agents and being designed to function by a novel anti-pathogenic approach. Although not classical innate immune regulators, these anti-T3SS agents are thought to act indirectly on host targets by protecting phagocytic cells of the innate immune system from most of the acute cytotoxic effects of bacteria having a type III secretion system such as Pseudomonas aeruginosa. As a therapeutic agent, the compounds of the present invention may reduce the frequency of polymicrobial VAP infections, which are thought to be due to local innate immune suppression by Pseudomonas aeruginosa T3SS effector toxins. (Diaz et al., Infect. Immun., 76:4414-4421 (2008)). Furthermore, the compounds of the present invention are species-specific, as a result, they spare the normal flora and advantageously align with a new understanding of the protective role of the normal flora in infectious diseases and this therapeutic approach. Parillo and Dellinger, Critical Care Medicine: Principles of Diagnosis and Management in the Adult, 2 nd ed. (Moseby, New York 2007), pp.800-802. When applied in combination with antibacterial agents, the novel T3SS inhibitor compounds do not contribute to the elimination of the normal flora and may enable the use of low-dose co-administered antibiotics. Finally, these T3SS inhibitor compounds are equally potent against multiple Pseudomonas aeruginosa strains (including clinical isolates), are not affected by the Pseudomonas aeruginosa efflux mechanism, do not exert a selective pressure for the development of resistance in vitro, and are expected to exert only a relatively weak selective pressure during treatment. This combination of favorable features and a novel mechanism of action of the compounds provides a novel approach for improving the treatment and prevention of acute Pseudomonas aeruginosa infections such as VAP and bacteremia.

[0093] The T3SS inhibitor compounds described herein inhibit T3SS effector transcription by at least 15% at a concentration of 50 μM using a transcriptional reporter assay, or inhibit effector secretion by at least 50% at a concentration of 100 μM or less (IC 50 ≤ 100 μM) in an effector secretion assay. The above compounds show more than 15% T3SS-specific inhibition in Pseudomonas using an exoT-lux transcriptional reporter construct transferred into Pseudomonas aeruginosa PAO1 (reporter strain MDM852 described herein), and / or the effector toxin-β-lactamase reporter fusion protein assay described herein using Pseudomonas strain MDM973 (PAK / pUCP24GW-lacI Q -lacPO-exoS::blaM) shows an IC 50 of less than 100 μM for T3SS as measured in an assay of T3SS-mediated secretion. See Table 1 below. Compounds that inhibit effector transcription by less than 15% or have an IC 50 above 10 μM are generally not useful as T3SS inhibitors in the compositions and methods described herein.

[0094] In a particularly preferred embodiment, the T3SS inhibitor compounds useful in the compositions and methods described herein have an IC 50 value of less than 200 μM as measured in the T3SS-mediated effector toxin-β-lactamase reporter fusion protein secretion assay (or equivalent assay) described herein, and relatively low cytotoxicity to human cells, e.g., a CC 50 value of 200 μM or more (CC 50 ≥ 200 μM) when measured in the standard cytotoxicity assay described herein or when used in the pharmaceutical field of antibiotics. Such standard cytotoxicity assays can use any human cells typically used in cytotoxicity assays of antibiotics, including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, Hep-2 cells, human embryonic kidney (HEK) 293 cells, 293T cells, etc.

[0095] Even more preferably, the T3SS inhibitor compounds described herein have an IC 50 value of ≤ 50 μM when measured in the T3SS-mediated effector toxin-β-lactamase reporter fusion protein secretion assay described herein, or an equivalent assay.

[0096] In a particularly preferred embodiment of the invention, the T3SS inhibitor compound blocks T3SS-mediated secretion from the cells of Pseudomonas aeruginosa and the translocation of one or more toxin effectors, is safe, and achieves a sufficiently high level and for a sufficient period in a suitable animal tissue that shows efficacy in an animal model of infection such as a mouse lung infection model.

[0097] Even more preferably, for use as a drug, the T3SS inhibitor compounds described herein show a translocation inhibition EC 50 <10 μM against 90% of the tested Pseudomonas aeruginosa clinical strains, are stable in human serum (> 75% stability for over 1 hour), are stable in a human liver microsome preparation (t 1 / 2 > 1 hour), are soluble at a concentration of > 5 mg / mL in a clinically acceptable formulation, and show in vivo efficacy in an animal model of Pseudomonas aeruginosa infection.

[0098] In yet another embodiment, the T3SS inhibitor compounds described herein specifically inhibit T3SS and have a sufficiently high minimum inhibitory concentration (MIC) that shows no effect on the viability of bacterial cells in culture in the absence of phagocytes.

[0099] In a particularly preferred embodiment of the invention, the T3SS inhibitor compound blocks T3SS-mediated secretion from the cells of Pseudomonas aeruginosa and the translocation of one or more toxin effectors, is safe, and achieves a sufficiently high level and for a sufficient period in a suitable animal tissue that shows efficacy in an animal model of infection such as a mouse lung infection model.

[0100] Compositions and Methods

[0101] The T3SS inhibitor compounds described herein can also be synthesized using established chemistry.

[0102] The following schemes provide suitable synthetic schemes for preferred embodiments.

[0103] Scheme 1: Synthesis of common intermediate 4

Chem.

[0104] R = ethyl: (S)-methyl 2-hydroxybutanoate (2): (S)-2-hydroxybutanoic acid (1) (24.47 g, 235.1 mmol) was dissolved in MeOH (1 L), to which H2SO4 (about 0.5 mL) was added, and the mixture was heated to reflux vigorously for 18 h (the reaction can be confirmed by crude NMR). The mixture was then concentrated to an oil on a rotary evaporator. The oil was diluted with ethyl ether (300 mL), washed with saturated aqueous NaHCO3 (2×, about 300 mL in total), the aqueous phase was washed with ethyl ether (2×, about 300 mL in total), the combined organic phases were dried over MgSO4, filtered, and concentrated to a pale yellow oil, 18.86 g (69%). 1 1H NMR (CDCl3): 4.20 - 4.16 (m, 1H), 3.80 (s, 3H), 2.71 (d, 1H), 1.89 - 1.64 (m, 2H), 0.97 (t, 3H).

[0105] R = methyl can be prepared by starting from (S)-2-hydroxypropanoic acid following the same procedure.

[0106] (R)-Methyl 2-((3,5-dichloropyridin-2-yl)oxy)butanoate (3): (S)-Methyl 2-hydroxybutanoate (13.18 g, 111.6 mmol), 3,5-dichloro-2-hydroxypyridine (21.96 g, 133.9 mmol, 1.2 equiv), triphenylphosphine (35.12 g, 133.9 mmol, 1.2 equiv) were combined in a 350 mL flask, dissolved in dry DMF (100 mL), and cooled in an ice bath under a N2 atmosphere. DIAD (26.4 mL, 133.9 mmol, 1.2 equiv) diluted with DMF (25 mL) was added to the reaction mixture via a dropping funnel over 30 minutes. The resulting mixture was warmed to room temperature with stirring overnight. The mixture was diluted with EtOAc (200 mL), then washed with water (2 ×, total 300 mL), then washed once with brine (100 mL). The organics were then extracted from the combined aqueous portions with EtOAc (3 ×, total 300 mL), the combined organics were dried over MgSO4, filtered, and adsorbed onto silica gel (ca. 400 mL). The product was isolated by column chromatography; linear gradient of 0 to 15% acetone / hexane using 210 nm UV detection. Pale yellow oil; 16.51 g (56%), 1 1H NMR (CDCl3): 7.92 (d, 1H), 7.66 (d, 1H), 5.12 (t, 1H), 3.73 (s, 3H), 2.04 (quint, 2H), 1.11 (t 3H).

[0107] (R)-2-((3,5-Dichloropyridin-2-yl)oxy)butanoic acid (4): (R)-Methyl 2-((3,5-dichloropyridin-2-yl)oxy)butanoate (15.01 g, 56.83 mmol) was dissolved in MeOH (200 mL), diluted with THF (200 mL), and thereto, via an addition funnel, 1 M LiOH (300 mL, freshly prepared) was added over 2 h, then stirred overnight at room temperature. The mixture was concentrated on a rotavap until it became a residue / solid, then diluted with water (ca. 500 mL) and washed with ethyl ether (3×, total ca. 400 mL). The ether portion was then washed with 0.7 M NaOH solution (2×, ca. 200 mL), the combined aqueous portions were acidified to pH ca. 0 - 1 with concentrated HCl, then washed with DCM (4×, total ca. 600 mL), which was then dried over MgSO4 and the solvent removed. The residue was dissolved in ethyl ether, evaporated to a solid, and dried under high vacuum overnight, 13.81 g (97%) of an off-white or light brown solid, 1 1H NMR (CDCl3): 11.02 (br, 1H), 7.94 (d, 1H), 7.67 (d, 1H), 5.15 (t, 1H), 2.08 (quint, 2H), 1.13 (t, 3H).

[0108] Scheme 2: Synthesis of N-Boc protected urea intermediate

[0109] (A) Linear synthesis

Chemical formula

[0110] (R)-N-(3-Aminobenzyl)-2-((3,5-dichloropyridin-2-yl)oxy)butanamide (6): (R)-2-((3,5-Dichloropyridin-2-yl)oxy)butanoic acid 4 (1.00 g, 4.00 mmol), HBTU (1.82 g, 4.88 mmol, 1.2 equiv) were dissolved in DMF (dry, 10 mL), diisopropylethylamine (0.91 mL, 5.20 mmol, 1.3 equiv) was added, and tert-butyl 3-aminobenzylcarbamate 5 (1.07 g, 4.88 mmol, 1.2 equiv) was added to the reaction mixture in portions of approximately 4 equiv over 5 minutes. The reaction was stirred at room temperature for 18 h. The reaction mixture was diluted with water (ca. 120 mL), the precipitate was filtered off and dried overnight under vacuum, 1.935 g (>99%) of an off-white solid, 1 H NMR (CDCl3): 7.99 (d, 1H), 7.66 (d, 1H), 7.28 - 7.26 (m, 1H + CHCl3), 7.21 (d, 2H), 6.88 (br, 1H), 6.65 (br, 1H), 5.44 (t, 1H), 4.44 (d, 2H), 2.68 (m, 2H), 1.51 (s, 9H), 1.02 (t, 3H).

[0111] (R)-tert-Butyl (3-((2-((3,5-dichloropyridin-2-yl)oxy)butanamido)methyl)phenyl)carbamate (1.817 g, 4.00 mmol) was dissolved in DCM (70 mL), cooled to 0 °C in an ice bath, and trifluoroacetic acid (20 mL) was added. The reaction was complete by LC / MS after 3 h. The mixture was concentrated to a solid residue on a rotary evaporator and dried overnight under high vacuum. The residue was then dissolved in DCM (ca. 100 mL), washed with saturated aqueous NaHCO3 (2 × 50 mL), the organics were back-extracted from the aqueous portion with two DCM washes (each ca. 50 mL), the combined organics were dried over MgSO4, filtered, concentrated to a solid on a rotary evaporator, and then dried overnight under high vacuum, 1.103 g (78%) of a light brown solid, 11H NMR (CDCl3): δ 8.00 (d, 1H), 7.67 (d, 1H), 7.08 (t, 1H), 6.60 - 6.53 (m, 4H), 5.46 (t, 1H), 4.39 (d, 2H), 2.91 (br, 2H), 2.08 (m, 2H), 1.02 (t, 3H).

[0112] General manufacturing procedure of urea:

[0113] Method A: (CDI): To (R)-N-(3-aminobenzyl)-2-((3,5-dichloropyridin-2-yl)oxy)butanamide (as salt or free base) dissolved in N-methylimidazole (about 0.15 mM), 1 pellet / 0.5 mmol of 4 Å molecular sieves was added, cooled to 0 °C in an ice bath, then 1,1'-carbonyldiimidazole (1.2 equivalents) was added, stirred in the ice bath for 1 hour, then diamine (1.5 equivalents) was added, the mixture was warmed to room temperature, diluted with water (≥ 10-fold volume of NMI), the product precipitated, which was filtered and dried under vacuum overnight {Example of compound NMR}.

[0114] Method B: To a solution of (trichloromethyl) chloroformate (0.167 g, 0.565 mmol) in DCM (0.3 mL) was added dropwise at 0 °C to a solution of (R)-N-(3-aminobenzyl)-2-((3,5-dichloropyridin-2-yl)oxy)butanamide (6) (0.200 g, 0.568 mmol), triethylamine (0.94 mL, 6.77 mmol), and DCM (11 mL). The resulting mixture was stirred at room temperature for 30 minutes. A solution of tert-butyl 3-(1-aminocyclopropyl)azetidine-1-carboxylate (0.143, 0.678 mmol), triethylamine (0.55 mL) in DCM (3.6 mL) was added dropwise to the reaction mixture, and the resulting reaction mixture was stirred at room temperature for 2 hours. After completion of 2 hours by LC / MS, the mixture was quenched with MeOH (5 mL) and concentrated under reduced pressure. The crude compound was purified by Combiflash using a 20 g silica gel column eluting with a 0 to 10% MeOH / DCM gradient to give 269 mg (80%) of the product as a pale yellow solid, 1H NMR (CDCl3): 7.98 (s, 1H), 7.66 (s, 1H), 7.38 - 7.12 (m, 4H), 6.85 (d, 1H), 6.75 (m, 1H), 5.88 (bs, 1H), 5.42 - 5.39 (t, 1H), 4.42 (bs, 2H), 3.97 - 3.91 (t, 2H), 3.68 (bs, 2H), 2.85 - 2.78 (m, 1H), 2.06 (m, 2H), 1.42 (s, 9H), 1.01 (t, 3H), 0.95 - 0.70 (m, 4H).

[0115] (B) Convergent synthesis

Chemical formula

[0116] General synthesis of urea: 13: 3-Cyanophenyl isocyanate (1.0 equiv) was dissolved in THF (0.5 M), trimethylamine (1.1 equiv) was added, followed by addition of a monoprotected diamine (11, 1.0 equiv), and the reaction mixture was stirred at room temperature for 18 h. The material was then adsorbed onto celite and purified by silica gel column (0 to 100% EtOAc / hexane), white foam / solid. Example: tert-Butyl 6-(3-(3-cyanophenyl)ureido)-2-azaspiro[3.3]heptane-2-carboxylate, 1 H NMR (CDCl3): 7.87 (s, 1H), 7.67 (s, 1H), 7.60 (d, 1H), 7.36 - 7.22 (m, 2H + CHCl3), 5.74 (br, 1H), 4.16 (m, 1H + EtOAc), 3.94 (s, 2H), 3.84 (s, 2H), 2.59 (m, 2H), 2.00 (m, 2H + EtOAc), .....

[0117] Nitrile reduction (14): Nitrile-urea (13, 1.0 equiv, ~20 - 25 mmol) was dissolved in MeOH (ca. 50 mL) in a Parr shaker vessel, and to this mixture was added Raney Ni slurry (2800 mesh, 5 mL), and concentrated ammonium hydroxide (5 mL). The vessel was placed in a Parr shaker, charged with H2 gas at 50 psi, and shaken at room temperature while maintaining a pressure of H2 gas at 40 - 50 psi for 16 h. The reaction mixture was carefully filtered through a celite filter cake, and the filtrate was concentrated to give a yellow-colored residue / foamy solid. Example: tert-Butyl 6-(3-(3-aminomethyl)phenyl)ureido)-2-azaspiro[3.3]heptane-2-carboxylate, 1 H NMR (CDCl3): 7.51 (br, 1H), 7.28 (m, 1H + CHCl3), 7.19 (m, 2H), 6.90 (d, 1H), 5.72 (br, 1H), 4.10 (q, 1H), 3.86 (m, 2H), 3.76 - 3.62 (m, 4H), 2.51 (br t, 2H), 2.26 (br, 2H + H2O), 1.92 (br t, 2H), 1.43 (s, 9H).

[0118] Procedure for the amide coupling of 4 + 14 = urea (7): To a mixture of (R)-2-((3,5-dichloropyridin-2-yl)oxy)butanoic acid 4 (0.200 g, 0.803 mmol), HBTU (0.364 g, 0.964 mmol, 1.2 equiv.) dissolved in DMF (dry, 2 mL) at 0 °C was added diisopropylethylamine (0.44 mL, 2.55 mmol, 2.5 equiv.). After 5 min, a solution of tert-butyl 6-(3-(3-(aminomethyl)phenyl)ureido)-2-azaspiro[3.3]heptane-2-carboxylate 14 (0.304 g, 0.843 mmol, 1.05 equiv.) in DMF (2 mL) was added, and the mixture was allowed to warm to room temperature overnight. LC / MS showed the reaction was complete, as well as significant guanidine by-product formation. The reaction mixture was diluted dropwise into water (100 mL) and the solid precipitate was collected by vacuum filtration. The product was purified by silica chromatography (20 g packed column, 0 to 5% MeOH linear gradient in DCM), 0.312 g (66%) white solid, 1 H NMR(MeOD)7.41(s,1H),7.27(s,1H),7.19-7.12(m,3H),6.89-6.81(m,2H),4.59-4.52(m,1H),4.34(d,2H),4.11 -4.05(t,1H),3.96(s,2H),3.84(s,2H),2.57(t,2H),2.09(t,2H),2.01-1.94(m,2H),1.40(s,9H),1.07(t,3H).

[0119] Scheme 3: Synthesis of N-Boc protected urea intermediate [ka]

[0120] Removal of Boc protection (8) of the di - amine moiety: N - Boc urea (7, 1.0 equiv) was dissolved in DCM (0.1 - 0.25 mM) cooled to 0 °C in an ice bath, trifluoroacetic acid (equivalent amount to DCM) was added, and after 3 hours, the reaction was completed as determined by LC / MS and warmed to room temperature. The mixture was concentrated to a solid residue on a rotary evaporator and dried under high vacuum overnight. The residue was then dissolved in DCM (about 100 mL), washed with saturated aqueous NaHCO3 (2×50 mL), and the organic matter was back - extracted from the aqueous portion with two DCM washes (each about 50 mL). The combined organic matter was dried over MgSO4, filtered, and concentrated to a solid on a rotary evaporator. Evaporation from MeOH (2×), DCM (2×), and then Et2O (2×) was carried out, and it was dried under high vacuum overnight. Example: (R)-N-(3-((2-((3,5 - dichloropyridin - 2 - yl)oxy)butanamido)methyl)phenyl)piperazine - 1 - carboxamide, TFA salt, 1 1H NMR (MeOD - d4) δ 7.99 (s, 1H), 7.91 (s, 1H), 7.31 (s, 1H), 7.24 (m, 2H), 6.97 (m, 1H), 5.11 (1H), 4.38 (s, 2H), 3.80 (m, 4H), 3.60 - 3.20 (m, 4H), 2.10 - 1.95 (m, 2H), 1.78 (t, 3H).

[0121] Scheme 4: General scheme for the synthesis of zwitterions

Chemical formula

[0122] Step 1: Addition of the carboxylic acid moiety (9):

[0123] (A) (Michael Addition): To a solution of (R)—N-(3-(3-(1-(azetidin-3-yl)cyclopropyl)ureido)benzyl)-2-((3,5-dichloropyridin-2-yl)oxy)butanamide, TFA salt (0.05 g, 0.082 mmol), DIPEA (0.09 mL, 6.3 mmol) in MeOH (1.5 mL) was added methyl acrylate (0.015 mL, 2.12 mmol) and the reaction mixture was stirred at room temperature for 2 hours. After 2 hours, the reaction was complete by LC / MS. The reaction was concentrated and the crude compound was purified by Combiflash using 0 to 10% MeOH / DCM to give 27 mg (57%) as a colorless solid. 1 H NMR(300MHz,MeOD)7.96(s,1H),7.89(m,1H),7.32-7.17(m,3H),6.88(d,1H),5.08(t,1H),4.64(s,1H),4.33(s,2H), 4.03(m,2H),3.89(m,2H),3.76-3.69(m,3H),2.91(m,1H),2.63(t,2H),2.02-1.98(m,2H),1.08(t,3H),0.84(d,4H).

[0124] (B) (Reductive amination): The deprotected amine (8, 1.0 equiv.) was dissolved in a mixture of MeOH:AcOH (10:1, 0.5 mM) to which was added a carbonyl (aldehyde or ketone, 2.0 equiv.). The mixture was stirred at room temperature for 1 h. Borane-2-methylpyridine complex (2.0 equiv.) was then added, and the reaction mixture was stirred at room temperature for 2–72 h. The reaction was concentrated under reduced pressure to a residue, which was subsequently dissolved in DCM (approximately 10 reaction volumes) and washed with saturated aqueous NaHCO3 (approximately 20 reaction volumes). The organics were then extracted from the aqueous portion with two DCM washes (10 reaction volumes each). The combined organic layers were dried over MgSO4, filtered, and the solvent removed. The product was purified on a silica gel column (0–100% MeOH / DCM linear gradient elution) to give a white solid product. Example: ethyl (R)-2-(4-((3-((2-((3,5-dichloropyridin-2-yl)oxy)butanamido)methyl)phenyl)carbamoyl)piperazin-1-yl)acetate,1 1H NMR (MeOD-d4) δ 8.00 (s, 1H), 7.91 (s, 1H), 7.30 - 7.15 (m, 3H), 6.94 (d, 1H), 5.11 (t, 1H), 4.37 (s, 2H), 4.25 - 4.18 (q, 2H), 3.60 (m, 4H), 3.37 (s, 2H), 2.66 (m, 4H), 2.03 (m, 2H), 1.30 (t, 3H), 1.11 (t, 3H).

[0125] (C)(S N (2): The amine (1.0 equivalent) was dissolved in DCM (0.3 mM), DiPEA (3.5 equivalents) was added, and then the alkyl halide (1.05 equivalents) was added. The mixture was stirred at 22 °C, and after 6 hours, it was found that the reaction was complete by LC / MS. The product was purified by preparative HPLC. Example: (R)-methyl 4-(6-(3-((2-((3,5-dichloropyridin-2-yl)oxy)butanamido)methyl)benzamido)-2-azaspiro[3.3]heptan-2-yl)butanoate, 0.023 g (38%), white solid, 1 1H NMR (CDCl3) δ 12.35 (br, 1H), 7.96 (d, 1H), 7.66 - 7.62 (m, 3H), 7.30 - 7.26 (m, 2H + CHCl3), 7.17 (d, 1H), 6.94 (br, 1H), 5.39 (t, 1H), 4.46 - 4.36 (m, 5H), 3.82 - 3.68 (m, 5H), 3.11 (br, 2H), 2.76 (m, 1H), 2.65 (m, 1H), 2.47 - 2.30 (m, 4H), 2.08 - 2.01 (m, 2H), 1.88 - 1.80 (m, 2H), 1.01 (t, 3H).

[0126] Step 2: Hydrolysis of the ester (10):

[0127] The carboxylic acid ester (9, 1.0 equivalent) was dissolved in THF (0.1 M), and 1 M aqueous LiOH solution (3.0 equivalents) was added thereto, followed by stirring at room temperature for 4 to 18 hours. The product was adsorbed onto celite and purified by a silica gel column (linear gradient eluent from 0 to 100% MeOH / DCM) to obtain a white solid product. Example: Lithium (R)-2-(6-(3-(3-((2-((3,5-dichloropyridin-2-yl)oxy)butanamido)methyl)phenyl)ureido)-2-azaspiro[3.3]heptan-2-yl)acetate, 1 1H NMR (DMSO): 9.31 (s, 1H), 8.49 (t, 1H), 8.15 (m, 2H), 7.48 (d, 1H), 7.32 (s, 1H), 7.26 (d, 1H), 7.11 (t, 1H), 6.72 (d, 1H), 5.08 (dd, 1H), 4.20 (m, 2H), 4.01 (dt, 1H), 3.89 (s, 2H), 3.96 (s, 2H), 2.56 (m, 2H), 2.10 (m, 2H), 1.91 (m, 2H), 0.99 (t, 3H).

[0128] Scheme 5: Synthesis of Amides Containing Zwitterions [Chemical formula]

[0129] (R)-Methyl 3-((2-((3,5-dichloropyridin-2-yl)oxy)butanamido)methyl)benzoate (15): (R)-2-((3,5-Dichloropyridin-2-yl)oxy)butanoic acid (15.00 g, 59.98 mmol), HBTU (27.30 g, 71.98 mmol, 1.2 equiv) were dissolved in DMF (dry, 300 mL), cooled in an ice bath, and a solution of methyl 3-(aminomethyl)benzoate hydrochloride (14.51 g, 71.98 mmol, 1.2 equiv), diisopropylethylamine (26.1 mL, 150 mmol, 2.5 equiv) was added via an addition funnel over 2 h to the solution of HBTU and (R)-2-((3,5-dichloropyridin-2-yl)oxy)butanoic acid, then stirred at room temperature for 16 - 18 h. The reaction mixture was diluted with water (about 4 L), the precipitate was filtered off and dried under vacuum overnight, 26.36 g (>99%), light brown solid, 1 H NMR (CDCl3): 7.95 - 7.90 (m, 3H), 7.67 (d, 1H), 7.45 - 7.36 (m, 2H), 6.70 (br, 1H), 5.45 (t, 1H), 4.53 (d, 2H), 3.91 (s, 3H), 2.14 - 2.04 (m, 2H), 1.03 (t, 3H).

[0130] (R)-3-((2-((3,5-dichloropyridin-2-yl)oxy)butanamido)methyl)benzoic acid (16): (R)-Methyl 3-((2-((3,5-dichloropyridin-2-yl)oxy)butanamido)methyl)benzoate (26.36 g, 66.36 mmol) was dissolved in THF (200 mL). To this was added 2 M aqueous NaOH (200 mL) and additional THF (100 mL), and the mixture was stirred at room temperature for 16-18 hours. The mixture was concentrated on a rotovap, and the residue was dissolved in water (approximately 200 mL) and washed three times with DCM (total volume approximately 500 mL), and the aqueous portion was acidified to pH 1 with concentrated HCl. The precipitate was filtered and dried in a vacuum oven overnight at 65 °C in the presence of PO, 23.00 g (>99%), off-white solid, NMR (DMSO): 12.93 (br, 1H), 8.62 (t, 1H), 8.15 (m, 2H), 7.85-7.80 (m, 2H), 7.45-7.40 (m, 2H), 5.08 (t, 1H), 4.34 (qd, 2H), 1.90 (m, 2H), 1.00 (t, 3H).

[0131] Amide formation (17): (R)-3-((2-((3,5-dichloropyridin-2-yl)oxy)butanamido)methyl)benzoic acid (1.0 equiv.) and HBTU (1.2 equiv.) were dissolved in DMF (0.25 mM), then diisopropylethylamine (1.3 equiv.) and mono-Boc diamine (1.1 equiv.) were added, and the mixture was stirred at room temperature for 16-18 h. The reaction was diluted dropwise with water (>10 reaction volumes), and the precipitated product was filtered and dried in a vacuum oven at 70 °C overnight in the presence of PO as a white / off-white solid. Example: (R)-tert-butyl 6-(3-((2-((3,5-dichloropyridin-2-yl)oxy)butanamido)methyl)benzamido)-2-azaspiro[3.3]heptane-2-carboxylate. 11H NMR (DMSO): δ 8.63 - 8.57 (m, 2H), 8.17 (m, 2H), 7.72 - 7.66 (m, 2H), 7.39 - 7.36 (m, 2H), 5.06 (t, 1H), 4.36 - 4.25 (m, 3H), 3.91 (s, 2H), 3.79 (s, 2H), 2.23 (t, 2H), 1.89 (m, 2H), 1.37 (s, 9H), 1.00 (t, 3H).

[0132] The steps of preparing Compounds 18, 19, and 20 are the same as those of Compounds 8, 9, and 10.

[0133] Scheme 6: Synthesis of Benzyl Zwitterion [Chemical formula]

[0134] (R)-2-((3,5-Dichloropyridin-2-yl)oxy)-N-(3-formylbenzyl)butanamide (22): (R)-2-((3,5-Dichloropyridin-2-yl)oxy)butanoic acid (1.0 equiv), HBTU (1.2 equiv) were dissolved in DMF (0.1 mM), diisopropylethylamine (1.3 equiv) was added, and the mixture was stirred at room temperature for 30 min. Then, (3-(1,3-dioxolan-2-yl)phenyl)methanamine (1.1 equiv) dissolved in DMF (3 mM) was added dropwise at a rate of 2 mL / min, and the mixture was stirred at room temperature for 16 - 18 h. The product was precipitated by diluting the reaction mixture by dropping it into water (>10 reaction volumes), the precipitate was filtered, and dried under vacuum. The obtained solid of (R)-N-(3-(1,3-dioxolan-2-yl)benzyl)-2-((3,5-dichloropyridin-2-yl)oxy)butanamide (21) was suspended in DCM (0.5 mM), trifluoroacetic acid (20 equiv) was added thereto over 10 min, and the mixture was stirred at room temperature for 2 h. The reaction was quenched by shaking in a separatory funnel with water (2 reaction volumes), then the aqueous portion was washed with EtOAc (3 times), the combined organic layers were washed with brine, and then concentrated to a solid. Passing the material through a silica gel column using a 0 to 50% EtOAc linear gradient in hexane gave an oily residue, which was dissolved in DCM, washed with saturated aqueous NaHCO3, the product was extracted from the aqueous portion with EtOAc (2 times), the combined organic layers were dried over MgSO4, filtered, and concentrated to obtain a solid product, a white solid, 1 1H NMR (CDCl3): 9.98 (s, 1H), 8.01 (d, 1H), 7.79 - 7.73 (m, 2H), 7.68 (d, 1H), 7.53 - 7.45 (m, 2H), 6.77 (br, 1H), 5.46 (t, 1H), 4.66 - 4.50 (m, 2H), 2.14 - 2.04 (m, 2H), 1.03 (t, 3H).

[0135] (R)-N-(3-(Aminomethyl)benzyl)-2-((3,5-dichloropyridin-2-yl)oxy)butanamide (25): (R)-2-((3,5-Dichloropyridin-2-yl)oxy)butane (0.500 g, 1.999 mmol), HBTU (0.910 g, 2.399 mmol, 1.2 equiv) were dissolved in DMF (dry, 4 mL), and diisopropylethylamine (0.45 mL, 2.599 mmol, 1.3 equiv) was added. tert-Butyl 3-(aminomethyl)benzylcarbamate (0.567 g, 2.399 mmol, 1.2 equiv) was dissolved in DMF (dry, 1 mL) and added dropwise to the reaction mixture via syringe. The reaction was stirred at room temperature for 18 h. The reaction mixture was diluted with water (ca. 70 mL), the precipitate was filtered off and dried under vacuum overnight. The product was isolated by column chromatography (0 to 75% EtOAc / hexane linear gradient), 0.807 g (86%), white solid, 1 1H NMR (CDCl3): δ 8.00 (d, 1H), 7.68 (d, 1H), 7.30 - 7.11 (m, 4H + CHCl3), 6.67 (br t, 1H), 5.47 (t, 1H), 4.83 (br, 1H), 4.47 (d, 2H), 4.28 (d, 2H), 2.11 - 2.06 (m, 2H), 1.46 (s, 9H), 1.02 (t, 3H). (R)-tert-Butyl 3-((2-((3,5-dichloropyridin-2-yl)oxy)butanamido)methyl)benzylcarbamate (5.619 g, 12.00 mmol) was dissolved in DCM (50 mL), cooled in an ice bath, and 4.0 M HCl in dioxane (20 mL) was added via dropping funnel. The reaction was complete after 4 h, the mixture was diluted with ethyl ether (500 mL), the solid precipitate was filtered off and dried under vacuum. The solid was then dissolved in MeOH (100 mL) and evaporated twice. The resulting solid was triturated with ethyl ether, filtered off and dried under high vacuum. >5 g was recovered (yield >100%, the product still holds some dioxane which can be removed by lyophilization), light brown solid, 11H NMR (DMSO): δ 8.72 (t, 1H), 8.59 (br, 3H), 8.21 (d, 1H), 8.15 (d, 1H), 7.42 - 7.22 (m, 4H), 5.11 (t, 1H), 4.28 (d, 2H), 3.95 (d, 2H), 1.95 - 1.90 (m, 2H), 1.00 (t, 3H). The HCl salt and excess dioxane were removed by partitioning between DCM and saturated aqueous Na₂CO₃, then the aqueous portion was washed twice more with DCM, the combined organic layers were dried over MgSO₄, filtered, the solvent removed, and dried under vacuum.

[0136] Reductive amination (23):

[0137] (A) Benzaldehyde: (R)-2-((3,5-Dichloropyridin-2-yl)oxy)-N-(3-formylbenzyl)butanamide (1.0 equiv), and amino acid (1.1 equiv) were dissolved in MeOH (0.1 - 0.2 mM), a catalytic amount of acetic acid (ca. 1% of the reaction volume), and NaHB(OAc)₃ (1.2 equiv) were added, and the mixture was stirred at room temperature and monitored by LC / MS. When the reaction was shown to be complete, the product was purified by preparative HPLC and lyophilized.

[0138] (B) Benzylamine: (R)-N-(3-(Aminomethyl)benzyl)-2-((3,5-dichloropyridin-2-yl)oxy)butanamide (1.0 equiv), and aldehyde, or ketone (1.1 equiv) were dissolved in DCM:MeOH (1:1 volume, 0.1 - 0.2 mM), a catalytic amount of acetic acid (ca. 1% of the reaction volume), and NaHB(OAc)₃ (1.2 equiv) were added, and the mixture was stirred at room temperature and monitored by LC / MS. When the reaction was shown to be complete, the product was purified by preparative HPLC and lyophilized.

[0139] Ester removal (24): Prepared as for compound (10).

[0140] [Table 3] TIFF2025524366000043.tif254170TIFF2025524366000044.tif254170TIFF2025524366000045.tif254170TIFF2025524366000046.tif251170TIFF2025524366000047.tif254170TIFF2025524366000048.tif251170TIFF2025524366000049.tif254170TIFF2025524366000050.tif122170

[0141] Unless otherwise indicated, descriptions of the use of T3SS inhibitor compounds in a composition or method also include embodiments in which combinations of two or more T3SS inhibitor compounds are used as a source of T3SS inhibitory activity in the compositions or methods of the present invention.

[0142] The pharmaceutical compositions according to the present invention comprise a T3SS inhibitor compound as described herein, or a pharmaceutically acceptable salt thereof, the "active ingredient", and a pharmaceutically acceptable carrier, the "vehicle", which can be a liquid, solid, or semi-solid compound. A "pharmaceutically acceptable" compound or composition is one in which the compound or composition is not biologically, chemically, or in any other way incompatible with the chemistry and metabolism of the body and does not adversely affect the activity of any other ingredient that may be present in the composition in such a way as to impair the desired therapeutic and / or prophylactic benefit to the patient. Pharmaceutically acceptable carriers useful in the present invention include those known in the art of preparing pharmaceutical compositions and include, but are not limited to, water, physiological pH buffers, physiologically compatible salt solutions (e.g., phosphate buffered saline), and isotonic solutions. The pharmaceutical compositions of the present invention may also include one or more excipients, i.e., compounds or compositions other than the active ingredient that contribute to or enhance the desired properties of the composition.

[0143] Various aspects of the formulation of pharmaceutical compositions, including examples of excipients, dosages, dosage forms, modes of administration, etc., are known to those skilled in the art of pharmaceutical compositions and are also available in standard pharmaceutical texts such as Remington´s Pharmaceutical Sciences, 18th edition, Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, PA 1990), Remington: The Science and Practice of Pharmacy, Volumes 1&2, 19th edition, Alfonso R. Gennaro, ed., (Mack Publishing Co., Easton, PA 1995), or other standard texts regarding the preparation of pharmaceutical compositions.

[0144] The pharmaceutical composition can be in any of various dosage forms, particularly suitable for the intended mode of administration. Such dosage forms include, but are not limited to, aqueous solutions, suspensions, syrups, elixirs, tablets, lozenges, pills, capsules, powders, films, suppositories, and powders for inhalable formulations. Preferably, the pharmaceutical composition is in a unit dosage form suitable for single administration of an exact dosage and can be a part or multiple of a dosage calculated to provide effective inhibition of the T3SS.

[0145] Compositions comprising the T3SS inhibitors (or combinations of T3SS inhibitors) described herein may also optionally contain a second active ingredient (also referred to as the "second agent", "second active agent") having one or more desired therapeutic or prophylactic activities other than T3SS inhibitory activity. Such second agents useful in the compositions of the present invention include, but are not limited to, antibiotics, antibodies, antiviral agents, anti-cancer agents, analgesics (e.g., non-steroidal anti-inflammatory drugs (NSAIDs), acetaminophen, opioids, COX-2 inhibitors), immunostimulants (e.g., cytokines), hormones (natural, synthetic, or semi-synthetic), central nervous system (CNS) stimulants, antiemetics, antihistamines, erythropoietin, complement stimulants, sedatives, muscle relaxants, anesthetics, anticonvulsants, antidepressants, antipsychotics, and combinations thereof.

[0146] The pharmaceutical compositions described herein can be administered to humans and other animals in the same manner as those used in other known therapeutic or prophylactic agents, particularly those used in therapeutic aromatic or polycyclic antibiotics. The dosage and mode of administration to an individual depend on various factors including age, weight, gender, the condition of the patient, and genetic factors, and will ultimately be determined by a qualified healthcare provider responsible therefor.

[0147] Pharmaceutically acceptable salts of the T3SS inhibitor compounds described herein include salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, malic acid, palmitic acid, pamoic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, nicotinic acid, acetic acid, citric acid, methanesulfonic acid, ascorbic acid, aspartic acid, salicylic acid, glutamic acid, oxalic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, tannic acid, carboxymethylcellulose, polylactic acid, polyglycolic acid, gentisic acid, fumaric acid, adipic acid, camphoric acid, capric acid, caprylic acid, carbonic acid, cinnamic acid, ethanesulfonic acid, lauric acid, mandelic acid, cyclamic acid, gluconic acid, glucuronic acid, glutaric acid, glycerophosphoric acid, hypophosphoric acid, isobutyric acid, oleic acid, propionic acid, stearic acid, thiocyanic acid, pyroglutamic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, or benzenesulfonic acid, or an alkyl-quaternary ammonium salt formed from a positively charged N-alkyl group of methyl, ethyl, propyl, isopropyl, cyclopropyl, or cyclobutyl and a negatively charged counterion of chloride, bromide, iodide, methyl sulfoxide, trifluoromethyl sulfoxide, or p-toluenesulfoxide.

[0148] Examples of suitable bases include, but are not limited to, ammonia, lithium, sodium, potassium, calcium, magnesium, and iron.

[0149] The present invention also contemplates "quaternization" of any basic nitrogen-containing group of the compounds described herein, provided that such quaternization does not destroy the ability of the compound to inhibit the T3SS. Such quaternization may be particularly desirable for enhancing solubility. Any basic nitrogen may be quaternized with any of a variety of compounds including, but not limited to, lower (e.g., C1-C4) alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate), long-chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), and aralkyl halides (e.g., benzyl bromide and phenethyl bromide).

[0150] In the case of solid compositions, conventional non-toxic solid carriers may be used including, but not limited to, mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate.

[0151] Pharmaceutical compositions can be formulated for administration to a patient by any of a variety of oral, and parenteral routes or modes. Such routes include, but are not limited to, intravenous, intramuscular, intra-articular, intraperitoneal, intracranial, paravertebral, perijoint, periosteal, subcutaneous, intradermal, intrasynovial, intrasternal, intramedullary, intralesional, intratracheal, sublingual, pulmonary, topical, rectal, nasal, buccal, intravaginal, or via an implanted reservoir. The implanted reservoir can function by mechanical means, osmotic means, or other means. In general, and particularly when administration is via the intravenous, arterial, or intramuscular route, the pharmaceutical composition can be administered as a bolus, as two or more doses separated in time, or as a constant or non-linear infusion.

[0152] The pharmaceutical composition can be in the form of a sterile injectable preparation, for example, a sterile aqueous injection solution or an oily suspension. Such preparations can be formulated according to techniques known in the art using suitable dispersing agents or wetting agents (e.g., polyoxyethylene 20 sorbitan monooleate (also referred to as "polysorbate 80"), TWEEN® 80, ICI Americas, Inc., Bridgewater, New Jersey), and suspending agents. Acceptable vehicles and solvents that can be used in injectable preparations include mannitol, water, Ringer's solution, isotonic sodium chloride solution, and 1,3 - butanediol solution. Furthermore, sterile fixed oils can conventionally be used as solvents or suspending media. For this purpose, non - irritating fixed oils containing synthetic monoglycerides or diglycerides can be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils including olive oil or castor oil, particularly their polyoxyethylated versions.

[0153] The T3SS inhibitors described herein can be formulated in any of a variety of orally administrable dosage forms including, but not limited to, capsules, tablets, caplets, pills, films, aqueous solutions, oily suspensions, syrups, or elixirs. In the case of oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. Capsules, tablets, pills, films, lozenges, and caplets can be formulated for delayed release or sustained release.

[0154] Tablets, and other solid, or semi-solid formulations that rapidly disintegrate, or dissolve, in the mouth of an individual can be prepared. Such rapidly disintegrating, or rapidly dissolving formulations can eliminate, or significantly reduce, the use of exogenous water as a swallowing aid. Further, rapidly disintegrating, or rapidly dissolving formulations are also particularly useful for the treatment of individuals having difficulty swallowing. In the case of such formulations, a small amount of saliva is usually sufficient to effect tablet disintegration in the mouth. The active ingredient (the T3SS inhibitor described herein) can then be partially, or completely, absorbed into the circulation from blood vessels underlying the oral mucosa (e.g., sublingual, and / or buccal mucosa), or swallowed as a solution that is absorbed from the gastrointestinal tract.

[0155] When an aqueous suspension is administered orally, whether for absorption by the oral mucosa, or for absorption via the intestine (stomach, and intestine), the composition containing the T3SS inhibitor can be advantageously combined with an emulsifier, and / or a suspending agent. Such compositions can be in the form of a liquid, a soluble film, a soluble solid (e.g., a lozenge), or a semi-solid (chewable, and digestible). If desired, such orally administrable compositions can also contain one or more other excipients such as sweeteners, flavoring agents, taste modifiers, coloring agents, and combinations thereof.

[0156] The pharmaceutical composition containing the T3SS inhibitor described herein can also be formulated as a suppository for vaginal, or rectal administration. Such compositions are prepared by mixing the T3SS inhibitor compound described herein with a suitable non-irritating excipient that is solid at room temperature but liquid at body temperature, and thus melts in the appropriate body space to release the T3SS inhibitor, and any other desired components of the composition. Particularly useful excipients for such compositions include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0157] Local administration of a T3SS inhibitor may be useful when the desired treatment involves areas accessible by topical application, such as the epidermis, superficial wounds, or areas accessible during surgery, or organs that can be reached by topical application. Carriers for the local administration of the T3SS inhibitors described herein include, but are not limited to, mineral oil, liquid petroleum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsifying wax, and water. Alternatively, a topical composition containing the T3SS inhibitor described herein can be formulated with a suitable lotion or cream containing the inhibitor suspended or dissolved in a suitable carrier to promote absorption of the inhibitor by the upper dermis without significant penetration into the lower dermis and underlying vascular structures. Carriers particularly suitable for topical administration include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. The T3SS inhibitor can also be formulated for topical application as a jelly, gel, or emollient. Local administration can also be achieved via a skin patch.

[0158] Those skilled in the art of topical and transdermal formulations recognize that the selection of various components, such as absorption enhancers, emollients, and other agents, and the formulation can provide compositions particularly suitable for topical administration (i.e., remaining primarily on the surface or upper dermis with minimal or no absorption by the lower dermis and underlying vascular structures) or transdermal administration (absorption through the upper dermis and into the lower dermis and underlying vascular structures).

[0159] The pharmaceutical composition containing the T3SS inhibitor described in this specification can be formulated for nasal administration, in which case absorption can occur via the nasal cavity or the mucosa of the lungs. Such a mode of administration typically requires that the composition be provided in the form of a powder, solution, or liquid suspension, which is then mixed with a gas (e.g., air, oxygen, nitrogen, or a combination thereof) to produce an aerosol, or a suspension of droplets or particles. The inhalable powder composition preferably uses a low-irritant or non-irritant powder carrier, such as mannitol (mericitol). Such compositions are prepared according to techniques well known in the field of pharmaceutical formulations and can be prepared as a solution in physiological saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. The pharmaceutical composition containing the T3SS inhibitor described in this specification for administration via the nasal cavity or the lungs can be particularly effective in the treatment of lung infections such as hospital-acquired pneumonia (HAP).

[0160] The pharmaceutical compositions described herein can be packaged in a variety of ways suitable for the dosage form and mode of administration. These include, but are not limited to, vials, bottles, cans, packets, ampoules, cartons, flexible containers, inhalers, and nebulizers. Such compositions can be packaged for single or multiple administrations from the same container. A kit can include a composition containing the T3SS inhibitor and preferably a suitable diluent, which can preferably be provided in a dry powder or lyophilized form, which is combined with the dry or lyophilized composition immediately prior to administration as described in the accompanying instructions for use. The pharmaceutical composition can also be packaged in a single-use prefilled syringe, or an autoinjector, and a cartridge for a needleless jet injector. For multi-use packaging, it may be necessary to add antibacterial agents, such as phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, benzalkonium chloride, and benzethonium chloride, at concentrations that prevent the growth of bacteria, fungi, etc. and are non-toxic when administered to the patient.

[0161] All components that come into contact with, or are included in, the pharmaceutical composition, in accordance with good manufacturing practices currently used in the pharmaceutical industry and well known to those skilled in the art, are sterilized and must be periodically tested for sterility according to industry standards. Methods for sterilization include ultrafiltration, autoclaving, dry and wet heat, exposure to gases such as ethylene oxide, exposure to liquids such as oxidizing agents including sodium hypochlorite (bleach), and exposure to high-energy electromagnetic radiation (e.g., ultraviolet light, X-rays, gamma rays, ionizing radiation). The choice of sterilization method is made by those skilled in the art for the purpose of achieving the most efficient sterilization that does not significantly alter the desired biological function of the T3SS inhibitor or other components of the composition.

[0162] Further embodiments and features of the present invention will become apparent from the following non-limiting examples.

Examples

[0163] Example 1. Materials and methods for characterizing the T3SS inhibitor.

[0164] Strains, plasmids, and growth media.

[0165] The bacterial strains and plasmids used in the assay are described in Table 4 below. All Pseudomonas aeruginosa strains were derivatives of PAO1 (Holloway et al., Microbiol. Rev., 43:73-102 (1979)), PAK (Bradley, D.E., Virology, 58:149-163 (1974)), or PA99 (Feltman et al., Microbiology, 147:2659-2669 (2004)). E. coli TOP10 (Invitrogen), E. coli DB3.1 (GATEWAY® host, Invitrogen), E. coli SM10 (V. de Lorenzo and K. N. Timmis, Methods Enzymol., 235:386-405 (1994)), and E. coli S17-1 (ATCC 47055) were used as hosts for molecular cloning. Luria-Bertani (LB) medium (liquid and agar) was purchased from Difco. LB was supplemented with 30 μg / mL gentamicin (LBG) with or without 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 5 mM EGTA (LBGI and LBGIE, respectively).

[0166] [Table 4]

[0167] (1) Aiello et al., Antimicrob. Agents Chemother., 54:1988-1999 (2010). (2) Bowlin et al., Antimicrob. Agents Chemother., 58:2211-2220 (2014). (3) Shaver and Hauser, Infect Immun., 72:6969-6977 (2004).

[0168] Plasmid pGSV3-Lux was kindly supplied by Dr. Donald Woods (U. Calgary) (4) Moore et al., Infect Immun,;72:4172-4187(2004).

[0169] Luciferase transcriptional reporter assay of T3SS-mediated secretion by luminescence signal.

[0170] A transcriptional fusion of the Photorhabdus luminescens lux operon (luxCDAB) to the Pseudomonas aeruginosa PAO1 T3SS effector gene exoT (PA0044) was constructed and used to construct the MDM852 strain as previously described (Aiello et al., Antimicrob Agents Chemother, 54:1988-1999(2010)). Induction of the T3SS operon by depletion of Ca ++ through EGTA addition results in the secretion of the negative regulator of T3SS, ExsE, and the production of high levels of T3SS components including the luxCDABE gene products, resulting in luminescence that is easily detected by a microplate reader. Inhibition of the T3SS-mediated secretion of the negative regulator ExsE by a small molecule inhibitor causes a decrease in the production of the luxCDABE gene products and a decrease in luminescence. Inhibitor IC 50 For the test, the compounds were added to 96-well microplates in a concentration dilution series. The reporter strain MDM852 was grown in LBG at 37 °C to an OD 600Grow to between 0.025 - 0.05, transfer to a microplate (50 μL / well) containing the test compound and EGTA (5 μL of 0.1 M stock solution), and cover this with a semi-transparent gas-permeable seal (Abgene, Inc., catalog number AB-0718). Control wells contained cells with fully induced T3SS (EGTA and test compound diluent DMSO, columns 1 and 2), and uninduced T3SS (DMSO only, columns 11 and 12). The plates were incubated at room temperature for 300 minutes. Luminescence was then measured with an Envision multi-label microplate reader (PerkinElmer). The Z'-factor (see Zhang, et al., J. Biomol. Screen., 4:67 - 73 (1999)), a screening window coefficient defined as the ratio of the positive control separation band and the negative control separation band to the signal dynamic range of the assay, was on average 0.7 for the assay. IC 50 (The concentration of inhibitor that causes 50% inhibition of T3SS-mediated secretion) was calculated for each test article and used to rank the inhibitors by potency.

[0171] Effector-β-lactamase (βLA) reporter assay of T3SS-mediated secretion with an absorption signal.

[0172] The gene encoding the exoS promoter / regulatory region and the ExoS-β-lactamase (βLA) fusion protein (composed of the full-length Pseudomonas aeruginosa T3SS effector ExoS fused in-frame to the TEM-1 β-lactamase gene lacking the secretion signal codon) was constructed by overlapping extension PCR (splicing according to (K. Choi and H. Schweizer, BMC Microbiol., 5:30 (2005)), the sequence was confirmed, cloned into miniCTX, and introduced into the Pseudomonas aeruginosa strain PAO1 to generate strains MDM1746 and MDM1838 as previously described for the MDM1710 strain (Bowlin et al., Antimicrob Agents Chemother., 58:2211-2220 (2014)). Secretion of the ExoS-β-lactamase fusion protein after addition of EGTA to induce the T3SS operon was detected by measuring the hydrolysis of the chromogenic β-lactamase substrate nitrocefin in clear 96-well microplates in a modification of the previously described assay (Lee, et al., Infect. Immun., 75:1089-1098 (2007)). Cells of the MDM1746 strain, or the negative control MDM1838 (which did not perform T3SS secretion due to ΔpscF), were subcultured mid-morning from an overnight growth in 0.1 mL of LBGE in LBG with or without the test compound and grown for 150 minutes. Nitrocefin (final 100 ug / mL) was added and 3 measurements were taken every minute for 15 minutes with a Victor 490 V 1420 Multilabel HTS Counter (PerkinElmer). The gradient was calculated as a relative measure of the amount of secreted effector-βLA fusion protein and was absolutely dependent on induction by EGTA and the presence of a functional pscF gene in Pseudomonas aeruginosa cells. A typical signal:background ratio was 6-10. IC 50 (the concentration of inhibitor that causes 50% inhibition of T3SS-mediated secretion) was calculated for each test article and used to rank the inhibitors by potency.

[0173] Assay for ExoU-dependent CHO cell killing and T3SS-mediated translocation by Pseudomonas aeruginosa:

[0174] The rescue of CHO cells from T3SS-mediated cytotoxicity by translocated effector protein ExoU by Pseudomonas aeruginosa strain MDM1561 (PA99U) was measured using lactate dehydrogenase (LDH) as previously reported, except that the infection with Pseudomonas aeruginosa was performed for 2 hours in the absence of gentamicin ((Lee, et al., Infect. Immun., 73:1695-1705 (2005)). The percent cytotoxicity (% LDH release) was calculated relative to the percent cytotoxicity (% LDH release) of non-infected control cells set at 0% LDH release and the percent cytotoxicity (100% LDH release) of cells infected with Pseudomonas aeruginosa strain MDM1561 (PA99U) in the absence of the test inhibitory compound. The LDH released from uninhibited infected cells reached at least 80% of the value obtained from complete lysis with 1% Triton X-100 within the 2-hour time frame of this experiment. Pseudolipasin, which acts by direct inhibition of the ExoU phospholipase, was used as a control inhibitor in some experiments ((Lee et al, Infect. Immun., 75:1089-1098 (2007)). EC 50 (The concentration of inhibitor that causes a 50% inhibition of T3SS-mediated translocation) was calculated for each test article and used to rank the inhibitors by potency.

[0175] Minimum inhibitory concentration (MIC) assay

[0176] MIC determinations were performed by the microbroth dilution method described in the CLSI (formerly NCCLS) guidelines, IC 50 、and CC 50It was expressed in μM to facilitate comparison with values. Refer to NCCLS, Approved standard M7-A4: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. National Committee for Clinical Laboratory Standards, Wayne, PA (1997). T3SS inhibitors did not affect the growth of Pseudomonas aeruginosa during in vitro culture, and thus, compounds with detectable MIC values were discarded. th Refer to th . T3SS inhibitors did not affect the growth of Pseudomonas aeruginosa during in vitro culture, and thus, compounds with detectable MIC values were discarded.

[0177] Determination of mammalian cell cytotoxicity

[0178] The cytotoxic concentration (CC 50 ) of the compound against cultured mammalian cells (HeLa, ATCC CCL-2; American Type Culture Collection, Manassas, VA) was determined as the concentration of the compound that inhibits 50% of the conversion of MTS to formazan (Marshall et al., Growth Regul., 5:69 - 84 (1995)). Briefly, HeLa cells were seeded at a density of 4×10 3 / well in serum-free VP-SFM medium (Frazzati-Gallina et al., J. Biotechnol., 92:67 - 72 (2001)) in the presence or absence of serial dilutions of the compound dissolved in DMSO in 96-well plates. After incubation at 37°C for 3 days in VP-SFM, cell viability was measured using the vital tetrazolium salt stain 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide according to the manufacturer's instructions (Promega, Madison, Wisconsin). Values were determined in triplicate using dilutions of inhibitory compounds from 100 μM to 0.2 μM.

[0179] Liver microsomal stability To investigate the potential for first-pass metabolism of the analogs in the liver, the stability of the analogs was measured in the presence of mouse and human liver microsomal preparations (XenoTech). The stability of the compounds in the presence of NADPH was measured using the method of Kuhnz (Kuhnz, W. and Gieschen, H., Drug Metab. Dispos., 26:1120 - 1127 (1998)). Analogs that were stable in the presence of mouse and human microsomal preparations (t 1 / 2 > 1 hour) were prioritized.

[0180] Cytochrome P450 inhibition

[0181] Solubility The maximum aqueous solubility of the analogs was determined using nephelometry as described by Bevan, C. D. and Lloyd, R. S., Anal. Chem., 72:1781 - 1787 (2000). Particularly promising compounds were confirmed using LC / MS as described (Lee et al.. Antimicrob Agents Chemother., 35:2505 - 2508 (1991)).

[0182] Protein binding Plasma protein binding was determined for mouse, rat, dog, and human using equilibrium dialysis (Banker et al., J. Pharm. Sci., 92:967 - 974 (2003)).

[0183] Evaluation of off-target effects of the prioritized compounds To evaluate potential off-target effects, the inventors evaluated the prioritized hits, which were validated by in vivo testing, in the Eurofins Panlabs HitProfiling Screen + CYP450 assay panel, which was performed by Eurofins as a service. The assay panel consists of assays for 30 receptors and transporters + 5 major CYP450 enzymes (1A2, 2C19, 2C9, 2D6, 3A4). These assays were used to verify that potential preclinical candidates do not exhibit potential off-target toxicity.

[0184] For in vivo formulation studies, MBX-5452 was formulated in 0.9% saline (physiological saline) or a solution of 0.45% sodium bicarbonate and 1.5% polysorbate 80 (Tween 80). MBX-6681 was formulated in either a solution of 0.45% sodium bicarbonate and 5% Cremophor EL.

[0185] Single-dose tolerance determination in mice CD-1 female mice weighing 18 - 22 g were administered a single dose of the test agent at a predetermined concentration via up to three predetermined routes of administration (IP, SC, or IV). If the first animal appeared healthy, two additional animals were dosed at the same concentration and observed for up to 48 hours. For dose concentrations without adverse events, the concentration was increased and administered to up to three naive mice. This study scheme continued with escalating doses until the animals showed adverse effects to the dose administration or until the defined upper limit dose concentration was reached. If the animals showed adverse effects to the dose administration, the compound concentration was halved (1 / 2-X) and administered to up to three animals and observed. The MTD was defined as the highest dose concentration that did not show adverse effects 48 hours after administration. The animals were observed continuously for 1 hour after dose administration and at least five more times during the 48-hour period (two more times during the first 24 hours and three observation periods between 24 - 48 hours). All animals that received a dose of the test agent were euthanized at the end of the test (48 hours). If any animals showed adverse effects at any time after dose administration, they were removed from the test and euthanized. In these series of tests, acute tolerance was not expected to differ between male and female mice. To increase efficiency and reduce the number of animals used, the inventors utilized only female mice for single-dose tolerance.

[0186] Determination of Repeated-Dose Tolerance in Mice Based on the results of the single-dose tolerance test, a high-tolerance dose concentration and an administration route were selected. CD-1 female and male mice (n ≤ 10) were administered once or twice a day (BID or TID) for up to 3 days. The body weight before administration and the daily body weight were recorded during the last dose administration and 3 days later. The animals were observed up to a maximum of 2 times per dose event and up to a maximum of 2 times per day. The observations were made for up to a total of 6 days. Any animal showing adverse effects at any point during the study was removed from the study and immediately euthanized. All remaining animals were sacrificed at the end of the test.

[0187] Determination of Single-Dose Pharmacokinetics (PK) in Mice CD-1 female mice weighing 18 - 22 g were administered a single dose of the test article by up to 3 routes (IV, SC, or IP) based on the results of the single-dose and repeated-dose tolerance tests. At the selected time points, 3 mice were euthanized, and then, for analysis, blood, lung, spleen, and / or liver samples were collected post-euthanasia. Typically, each test article was evaluated at 7 time points (e.g., 0.083, 0.25, 1, 4, 6, 8, and 12 hours). Using the plasma concentrations from the biological analysis, T max , C max , half-life, volume of distribution, and AUC were calculated. Additionally, T max , C max , and AUC values were often determined from the epithelial lining fluid (ELF) levels from BAL (bronchoalveolar lavage) samples and calculated from lung, spleen, and liver levels.

[0188] In this series of studies, the pharmacokinetic profile was expected to be the same in male and female mice. Therefore, to increase efficiency and reduce the number of animals used, the inventors utilized only female mice for the single-dose PK test.

[0189] Repeated-dose PK determination in mice. CD-1 male and female mice weighing 18 - 22 g were administered the test article once or twice daily (BID or TID, respectively) for up to 3 days. Based on single-dose and repeated-dose tolerance studies, the test article was administered via one route (IV, SC, or IP) at the dose concentrations. At selected time points, 5 mice were euthanized and then, for analysis, blood and / or lung, BAL (bronchoalveolar lavage), liver, and spleen samples were collected post-euthanasia. For each test article, samples were collected at up to 24 time points for analysis over up to 2 days of test article administration and up to 3 days of further observation.

[0190] Using plasma concentrations from the biological analysis, T max , C max , half-life, volume of distribution, and AUC were calculated. Additionally, T max , C max , and AUC values were often calculated from epithelial lining fluid (ELF) levels determined from BAL samples and levels from the lung, spleen, and liver.

[0191] Single-Dose Pharmacokinetics (PK) Determination in Mice Male Sprague Dawley rats from Charles River Laboratories were acclimated for 5 days prior to the start of the study. During acclimation, animals were housed three per cage with free access to food and water. Prior to weighing and dosing, animals were individually identified by tail marking. All procedures were conducted in accordance with the NeoSome IACUC policy and guidelines, as well as OLAW regulations. The test article MBX-5452A was formulated in 0.9% saline, 5% PS80 at 25 mg / mL. The test article was administered by intraperitoneal or subcutaneous injection at a volume of 10 mL / kg. Body weight, dose volume, and dose administration time were recorded. At each time point after dose administration, blood was collected through the saphenous vein into K2EDTA collection tubes and inverted several times to ensure complete mixing. Blood was kept on wet ice until centrifugation (5 minutes, 4°C, 8,000 RPM). Plasma was decanted into labeled tubes and stored at -80°C until processed for bioanalysis. Using plasma concentrations from bioanalysis, T max 、C max 、half-life, volume of distribution, and AUC were calculated.

[0192] Pseudomonas aeruginosa Infection - Neutrophil-Nonreduced Mouse Pneumonia Model with Bacterial Load Immunocompetent (neutrophil-nonreduced) BALB / c mice (male and female) were utilized in these studies. On Day 0, at 0 hours, each mouse was administered intranasally as follows: 5×10 6 ~1×10 7CFU bacteria (e.g., a T3SS-producing Pseudomonas aeruginosa strain such as PA99 grown in LB broth) were challenged. Mice were anesthetized with isoflurane, put to sleep once, the mice were held in a vertical position, and 50 μL of the prepared bacterial inoculum was dropped into the nostrils. Once the entire volume of the inoculum was delivered, the mice were returned to their home cages and allowed to recover from anesthesia. The number of CFU in the inoculum was determined by the pathogenicity of the bacterial isolate through previously conducted bacterial titration tests. The bacterial challenge of the mice was performed in a dedicated BSL-2 suite designed for the containment of infectious agents. The test compound was administered starting 1 hour after infection via the specified route (IV, SC, or IP) in the above 5 - 20 mg / mL formulation, and further doses were administered at intervals of 6 hours (QID), 8 hours (TID), or 12 hours (BID) by the same route based on pharmacokinetic data to achieve compound levels in the appropriate tissues that exceeded EC 50 during the time between doses.

[0193] The animals were observed frequently for signs of pain and distress for the next 24 hours. Animals that appeared moribund, assumed a hunched posture, were inactive, or had cold contacts were removed from the study and immediately euthanized. 24 hours after the start of treatment (25 hours after infection), the mice were euthanized by CO2 inhalation, and appropriate tissues (lungs, spleen, and / or liver) were aseptically removed, weighed, homogenized, and serially diluted. The diluted samples were plated on bacterial growth agar for CFU counting after an overnight incubation. For each group of animals, the mean log 10 CFU and standard deviation were determined. Preferably, 8 - 10 mice per group were used to allow for appropriate statistical analysis between the treatment and non-treatment groups and the inherent variability of this model, with the standard deviation being 0.5 log for any test group 10It was made possible to be less than the CFU variation. A Mann-Whitney statistical test was performed between the treatment group and the infection control group (using GraphPad Prism software, GraphPad Software, LLC, San Diego, CA), and a P-value of less than 0.05 was considered to be significantly different. The test design included, for up to 8 groups, each with N = 10 (e.g., up to 80 mice per study), one infection control group (in 1 hour), one or more experimental compound groups with up to 5 dose concentrations, and up to 3 dose concentrations of a positive control agent (e.g., ciprofloxacin, or meropenem) at a low level of effectiveness (to show a significant decrease in CFU or to show a synergistic or additive effect with the test compound).

[0194] Example 2. Improvement of in vitro properties of zwitterionic T3SS inhibitors

[0195] The results of these studies highlighted the unexpected effect of the zwitterionic moiety on the phenoxyacetamide T3SS inhibitor scaffold. As shown in Table 5, some zwitterionic PhA T3SS inhibitors (see Table 1 for structures) showed improved potency in the in vitro T3SS activity assay, particularly in the T3SS translocation assay that measures the translocation of the pseudomonas exotoxins ExoS or ExoU into mammalian cells, compared to the previous PhA compounds MBX-1641 and MBX-2359. For example, the lead compounds of this series, MBX-5452A and MBX-6681B (highlighted in Table 5), showed increased potency in the in vitro T3SS translocation assay (see Figure 1) compared to the early-generation inhibitors exemplified by MBX-2416 and MBX-2359. As shown in Table 6, the EC 50 of MBX-5452A and MBX-6681B was that of MBX-1641 and MBX-2359's EC 502 to 4 times lower than. Furthermore, MBX-5452A and MBX-6681B exhibit potent T3SS inhibitory activity against a diverse panel of clinical isolates of Pseudomonas aeruginosa that express exotoxin ExoS or ExoU and various antibiotic resistance genes (see Tables 7 and 8). Some isolates of the strain panel are multi-drug resistant (MDR). As shown in Table 7, MBX-5452A exhibits potent T3SS inhibitory activity against all strains of the clinical isolate panel, and the EC 50 of MBX-5452A against 90% of the 26 clinical strains tested was 1.9 μg / mL. Similarly, MBX-6681B exhibits potent T3SS inhibitory activity against all strains of the clinical isolate panel, and the EC 50 of MBX-6681B against 90% of the 22 clinical strains tested was 1.4 μg / mL. Clearly, the zwitterionic moiety on the PhA scaffold provides a surprising improvement in the inhibitory activity against the T3SS of Pseudomonas aeruginosa.

[0196] The zwitterionic moiety provides improved in vitro potency against the T3SS activity assay, but the most unexpected effect of these modifications to the PhA scaffold was the improvement in the predicted drug-like properties of these compounds, as highlighted by the results of in vitro ADME experiments. Table 9 shows the results of a panel of in vitro ADME experiments for the selected zwitterions shown in Table 2. Some zwitterions have surprisingly high levels of water solubility above 200 μM, undetectable mammalian cell cytotoxicity (CC 50 ≥ 200 μM), favorable levels of serum protein binding (≤ 95% binding), mouse liver microsomes (T 1 / 2 ≥ 139 minutes), and hepatocyte cultures (T 1 / 2showed stability in the presence of ≧200 points). Table 10 shows a comparison of the drug-like properties of two initial PhA compounds (MBX-1641 and MBX-2359), as well as the lead compounds MBX-5452A and MBX-6681B. Although the data available for MBX-1641 and MBX-2359 are limited, it is clear that zwitterionic compounds show a 4- to 8-fold increase in solubility and a >100-fold increase in mouse liver microsomal stability. Finally, as demonstrated by the results of in vitro safety assays against a panel of human cytochrome P450, receptors, and ion channels performed by PanLabs / Eurofins (Taipei City, Taiwan), zwitterionic substitution resulted in an unexpected improvement in the predicted safety of these compounds during use in humans. The results of these assays for MBX-5452A (assayed at 100 μM), MBX-6681 (assayed at 10 μM), and the initial PhA compound MBX-4532 (assayed at 10 μM) are shown in Table 11. MBX-5452A did not show >50% inhibition in any of the safety assays when tested at a concentration of 100 μM, while MBX-4532 showed >50% inhibition against five safety targets (highlighted in bold font) when tested at a concentration of 10 μM. MBX-6681 did not show >50% inhibition in any of the safety assays when tested at a concentration of 10 μM. Clearly, zwitterionic substitution provides a surprising improvement in drug-likeness and safety in in vitro assays compared to the initial PhA T3SS inhibitors.

[0197] Example 3. Improvement of the in vitro properties of zwitterionic T3SS inhibitors

[0198] The surprising improvements in drug-likeness and safety provided by zwitterionic PhA substitution lead to improvements in pharmacokinetics, tolerance, and efficacy in an immunocompetent mouse model of acute pneumonia.

[0199] As shown in Figures 2A and B, the pharmacokinetics (PK) of MBX-5452A in mice after a single dose of 250 mg / kg via the subcutaneous (SC) or intraperitoneal (IP) administration route showed excellent exposure to plasma (AUClast 272,338 hr*ng / mL) and related tissues at 5 - 6 hours and 3 - 4 hours after SC and IP administration, respectively. The concentration of MBX-5452A in plasma exceeded the EC 50 for 90% (1900 ng / mL) of the clinical isolates tested over 5 - 6 hours. The levels of the compound in the spleen, ELF, lung, and liver indicate that the compound distributes rapidly into tissues. Similarly, Figure 3 shows the favorable pharmacokinetics (PK) of MBX-6681B in mice after a single dose administration of 50 mg / kg via intravenous (IV) administration and 100 mg / kg via subcutaneous (SC) administration. The data demonstrate that MBX-6681 distributes rapidly into tissues after a 100 mg / kg SC dose and provides overall good exposure (AUClast 105,719 hr*ng / mL), which is approximately equivalent to the AUClast of MBX-5452A normalized by the dose level. The concentration of MBX-6681B in plasma exceeded the EC 50Exceeds. As shown in Figure 4, the PK analysis of repeated SC doses of 100 mg / kg TID (q8h) for 40 hours of MBX-5452A (Panel A) and MBX-6681B (Panel B) did not result in significant accumulation of the compound in plasma, ELF, or lung after repeated dosing, and the peak plasma levels were on average about 100 μg / mL immediately after each dose. Finally, MBX-5452A showed favorable pharmacokinetics in rats after a single IP or SC dose of 250 mg / kg (Panel A), and the calculated PK parameters are shown in Panel B. The plasma concentration curves from mice and the PK parameters after a single IP or SC dose of 250 mg / kg are included in Panels A and B, respectively, for comparison. The data show that the half-life and MRTINF_obs are significantly higher in rats, and the plasma concentration of MBX-5452A after SC administration is higher than the T3SS translocation EC 50 for more than 16 hours, indicating that the PK parameters of rats and mice are similar, except that. In summary, zwitterionic PhA shows favorable PK characteristics consistent with its efficacy in animal models of infection.

[0200] To determine whether multiple doses of MBX-5452A are tolerated by mice, a repeated-dose tolerance study was conducted in which repeated SC or IP doses of 750 mg / kg / day (250 mg / kg, TID of MBX-5452A) were administered to mice and body weight and clinical signs were monitored for 4 days. As shown in Figure 6, repeated dosing of MBX-5452A had no significant effect on the body weight (Panel A) and body weight change rate (Panel B) of CD-1 mice over 4 days. Furthermore, no adverse events were reported during the course of the experiment, indicating that repeated dosing of MBX-5452A was well tolerated by mice, which is consistent with the results of in vitro safety experiments.

[0201] To determine whether zwitterionic PhA MBX-5452A and MBX-6681B are effective in relevant infected animal models, these compounds were used to treat experimental infections in an immunocompetent mouse model of acute pneumonia after challenging Pseudomonas aeruginosa PA99 as a single agent or in combination with a sub-effective dose of the anti-Pseudomonas aeruginosa antibiotic meropenem (MEM). In this model, CD-1 mice were infected with 1×10 7 bacteria via the intranasal route, treatment was initiated 1 hour after infection, and the mice were sacrificed 24 hours after infection to determine the bacterial load in the lungs. As shown in Figure 7, MBX-5452A (panel A) and MBX-6681B (panel B), when administered at doses of 125 and 250 mg / kg TID, significantly reduced the bacterial load (CFU / g lung tissue) in the lungs by 1-2 Log 10 (P<0.0001) as a single agent. Importantly, the data in Figure 8 show that MBX-5452A (panel A) and MBX-6681 (panel B) significantly increased the effectiveness of a sub-effective amount of meropenem (MEM, 2 mg / kg TID) in reducing the bacterial load in the infected lungs to levels similar to those of the positive control (MEM, 10 mg / kg TID). Since the Log10 reduction in the combination of CFUs was significantly greater than the sum of the Log10 reductions of the individual treatments, both compounds showed a synergistic effect when combined with 2 mg / kg of MEM. These results provide proof of concept for the use of these compounds as adjuvants to anti-Pseudomonas aeruginosa antibiotics for the treatment of nosocomial infections and ventilator-associated pneumonia caused by Pseudomonas aeruginosa.

[0202]

Table 5

[0203]

Table 6

[0204]

Table 7

[0205]

Table 8

[0206]

Table 9

[0207] Footnotes: a, Dynamic solubility in water (turbidimetric assay); b, Cytotoxicity against HeLa cells; c, Serum binding test using mouse (M) serum; d, Serum binding test using human (H) serum; e, Stability in human liver microsomes (HLM); f, Stability in mouse liver microsomes (MLM); g, Stability in the presence of mouse hepatocytes.

[0208]

Table 10

[0209]

Table 11

Claims

1. A bacterial type III secretion system (T3SS) inhibitor compound of Formula I or a pharmaceutically acceptable salt thereof: 【Chemical 1】 During the ceremony, A is independently selected from CH or N; X is independently selected from hydrogen, halogen, or hydroxyl; Z is O, S, NH, or NR ´ and R ´ is alkyl; R １ , R ２ , and R ３ are independently selected from hydrogen, halogen, alkyl, hydroxy, alkoxy, alkylthio, or cyano, and no more than two of the preceding groups are hydrogen, NR ４ and R ４ is hydrogen, a straight chain aliphatic group, a branched chain aliphatic group, cycloalkyl, haloalkyl, hydroxyalkyl, alkoxy, alkylamino, alkylthio, sulfonyl, sulfinyl, carboxy, alkoxycarbonyl, or aryl; Y is a straight or branched chain alkyl, alkenyl, or alkynyl group of 1 to 6 carbon atoms, or a cyclic alkyl, alkenyl, or alkynyl group of 3 to 6 carbon atoms, which may contain one or more heteroatoms, and which may be unsubstituted or substituted with up to four substituents selected from halo, cyano, hydroxy, amino, alkyl, cycloalkyl, alkylamino, carboxyl, alkoxycarbonyl, carboxamido, acylamino, amidino, sulfonamido, aminosulfonyl, alkylsulfonyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, oxygen, oxime, or NR", where R" is hydrogen, alkyl, cycloalkyl; Ar is an aryl or heteroaryl divalent group forming a 5- or 6-membered ring which may be further fused with 1 to 3 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings, the Ar group can be unsubstituted or substituted with up to 4 substituents selected from halo, cyano, hydroxy, amino, alkyl, cycloalkyl, alkylamino, carboxyl, alkoxycarbonyl, carboxamido, acylamino, amidino, sulfonamido, aminosulfonyl, alkylsulfonyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, any two substituents can together form an aromatic or non-aromatic ring structure fused with said aryl or heteroaryl group Ar, or the substituents on Ar can optionally be selected from Y, R ４ or Y and R ４ may be covalently linked to both to form a heterocyclic or carbocyclic ring system, which ring system may be aromatic, heteroaromatic, or partially aromatic, i.e., one or more rings are aromatic and one or more rings are non-aromatic; W is —C(O)CH ２ -, -S(O) ２ -, -NHS(O) ２ -, -S(O) ２ NH-, -C(O)-, -CH ２ -, -CH(CH ３ )-, -NHC(O)-, -NHC(O)NH-, -NHC(NH)NH-, -NHC(NCH ３ )NH-, -NHS(O) ２ NH-, -N(CH ３ )C(O)-, -C(O)NH-, -C(O)N(CH ３ a divalent group bridging Ar and U, selected from the group consisting of —O(C(O)—, —(C(O)O—, —NH—, or —O—; U can be either nitrogen, or carbon that is part of a 4-7 membered non-aromatic heterocycle containing 1-2 nitrogen atoms, 0-1 oxygen atoms, and having 0-3 substituents (in addition to bonds between W and the ring carbon / nitrogen, and U and the ring carbon) selected from alkyl, cycloalkyl, aryl, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, or sulfinyl; or U can be independently hydrogen or, optionally, haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, or sulfinyl. a monocyclic, bicyclic, or spirocyclic system consisting of a 3-7 membered ring connected to a basic nitrogen in an endocyclic or exocyclic configuration, which may be either nitrogen or saturated carbon, linked via a chain, carbocyclic, or heterocyclic ring, of 1-6 carbon atoms, with an aliphatic group of less than 9 carbon atoms, which may be substituted with a group selected from: sulfonyl, sulfinyl, carboxy, alkoxycarbonyl, or aminocarbonyl group, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, sulfonyl, or haloalkoxy, sulfonyl, or sulfinyl group; R ５ But -CH ２ C(CH ３ ) ２ C(O)OH, -CH ２ CH(OH)C(O)OH, -CH ２ C(O)OH, -CH ２ (oxetane)C(O)OH, —CH ２ C(O)OH, -CH ２ CH ２ CH ２ C(O)OH, -(cyclobutyl)C(O)OH, -CH ２ (cyclopropyl)C(O)OH, or CH ２ (cyclopropyl)C(O)OH; the methylene or ethylene group is selected from the group consisting of -C(O)NHOH, -C(O)N(CH ３ )OH, -C(O)NHOCH ３ , -NHP(O)(OH)NHCH ３ , -P(O)(OH)NHCH ３、 -P(O)(OH) ２、 -OP (O) (OH) ２、 -OP(O)(OH)NHCH ３、 -S(O) ２ Me, -S(O) ２ OH, -S(O)OH, -S(O) ２ CF ３ , 【Chemistry 2】 is connected to an acid or acid isostere group, including

2. A bacterial type III secretion system (T3SS) inhibitor compound of formula I(a) or a pharmaceutically acceptable salt thereof: 【Chemistry 3】 During the ceremony: A is independently selected from CH or N; X is independently selected from hydrogen or halogen; Z is O, S, NH, or NR ´ and R ´ is alkyl; R ４ is hydrogen or methyl; Y is a straight or branched chain alkyl, alkenyl, or alkynyl group of 1 to 6 carbon atoms, or a cyclic alkyl, alkenyl, or alkynyl group of 3 to 6 carbon atoms, which may contain one or more heteroatoms, and which may be unsubstituted or substituted with up to four substituents selected from halo, cyano, hydroxy, amino, alkyl, cycloalkyl, alkylamino, carboxyl, alkoxycarbonyl, carboxamido, acylamino, amidino, sulfonamido, aminosulfonyl, alkylsulfonyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, oxygen, oxime, or NR", where R" is hydrogen, alkyl, cycloalkyl; Ar is an aryl or heteroaryl divalent group forming a 5- or 6-membered ring which may be further fused with 1 to 3 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings, the Ar group can be unsubstituted or substituted with up to 4 substituents selected from halo, cyano, hydroxy, amino, alkyl, cycloalkyl, alkylamino, carboxyl, alkoxycarbonyl, carboxamido, acylamino, amidino, sulfonamido, aminosulfonyl, alkylsulfonyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, any two substituents can together form an aromatic or non-aromatic ring structure fused with said aryl or heteroaryl group Ar, or the substituents on Ar can optionally be selected from Y, R ４ or Y and R ４ may be covalently linked to both to form a heterocyclic or carbocyclic ring system, which ring system may be aromatic, heteroaromatic, or partially aromatic, i.e., one or more rings are aromatic and one or more rings are non-aromatic; W is —C(O)CH ２ -, -S(O) ２ -, -NHS(O) ２ -, -S(O) ２ NH-, -C(O)-, -CH ２ -, -CH(CH ３ )-, -NHC(O)-, -NHC(O)NH-, -NHC(NH)NH-, -NHC(NCH ３ )NH-, -NHS(O) ２ NH-, -N(CH ３ )C(O)-, -C(O)NH-, -C(O)N(CH ３ a divalent group bridging Ar and U, selected from the group consisting of —O(C(O)—, —(C(O)O—, —NH—, or —O—; U can be either nitrogen, or carbon that is part of a 4-7 membered non-aromatic heterocycle containing 1-2 nitrogen atoms, 0-1 oxygen atoms, and having 0-3 substituents (in addition to bonds between W and the ring carbon / nitrogen, and U and the ring carbon) selected from alkyl, cycloalkyl, aryl, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, or sulfinyl; or U can be independently hydrogen or, optionally, haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, or sulfinyl. a monocyclic, bicyclic, or spirocyclic system consisting of a 3-7 membered ring connected to a basic nitrogen in an endocyclic or exocyclic configuration, which may be either nitrogen or saturated carbon, linked via a chain, carbocyclic, or heterocyclic ring, of 1-6 carbon atoms, with an aliphatic group of less than 9 carbon atoms, which may be substituted with a group selected from: sulfonyl, sulfinyl, carboxy, alkoxycarbonyl, or aminocarbonyl group, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, sulfonyl, or haloalkoxy, sulfonyl, or sulfinyl group; R ５ But -CH ２ C(CH ３ ) ２ C(O)OH, -CH ２ CH(OH)C(O)OH, -CH ２ C(O)OH, -CH ２ (oxetane)C(O)OH, —CH ２ C(O)OH, -CH ２ CH ２ CH ２ C(O)OH, -(cyclobutyl)C(O)OH, -CH ２ (cyclopropyl)C(O)OH, or CH ２ (cyclopropyl)C(O)OH; the methylene or ethylene group is selected from the group consisting of -C(O)NHOH, -C(O)N(CH ３ )OH, -C(O)NHOCH ３ , -NHP(O)(OH)NHCH ３ , -P(O)(OH)NHCH ３、 -P(O)(OH) ２、 -OP (O) (OH) ２、 -OP(O)(OH)NHCH ３、 -S(O) ２ Me, -S(O) ２ OH, -S(O)OH, -S(O) ２ CF ３ , 【Chemistry 4】 is connected to an acid or acid isostere group, including

3. A bacterial type III secretion system (T3SS) inhibitor compound of formula I(b) or a pharmaceutically acceptable salt thereof: 【Chemistry 5】 During the ceremony: A is CH or N; X is independently selected from hydrogen or halogen; Y is -CH ２ -, -CH(CH ３ ) - or -C(CH ３ ) ２ - and; Ar is an aryl or heteroaryl divalent group forming a 5- or 6-membered ring which may be further fused with 1 to 3 aryl, heteroaryl, cycloalkyl, or heterocycloalkyl rings, the Ar group being unsubstituted or substituted with up to 4 substituents selected from halo, cyano, hydroxy, amino, alkyl, cycloalkyl, alkylamino, carboxyl, alkoxycarbonyl, carboxamido, acylamino, amidino, sulfonamido, aminosulfonyl, alkylsulfonyl, aryl, heteroaryl, alkoxy, alkylthio, aryloxy, and heteroaryloxy, any two substituents may together form an aromatic or non-aromatic ring structure fused with said aryl or heteroaryl group Ar, or substituents on Ar may, optionally, be covalently linked to Y to form a heterocyclic or carbocyclic ring system which may be aromatic, heteroaromatic, or partially aromatic, i.e., one or more rings are aromatic and one or more rings are non-aromatic; W is —C(O)CH ２ -, -S(O) ２ -, -NHS(O) ２ -, -S(O) ２ NH-, -C(O)-, -CH ２ -, -CH(CH ３ )-, -NHC(O)-, -NHC(O)NH-, -NHC(NH)NH-, -NHC(NCH ３ )NH-, -NHS(O) ２ NH-, -N(CH ３ )C(O)-, -C(O)NH-, -C(O)N(CH ３ a divalent group bridging Ar and U, selected from the group consisting of —O(C(O)—, —(C(O)O—, —NH—, or —O—; U can be either nitrogen, or carbon that is part of a 4-7 membered non-aromatic heterocycle containing 1-2 nitrogen atoms, 0-1 oxygen atoms, and having 0-3 substituents (in addition to bonds between W and the ring carbon / nitrogen, and U and the ring carbon) selected from alkyl, cycloalkyl, aryl, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, or sulfinyl; or U can be independently hydrogen or, optionally, haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, or sulfinyl. a monocyclic, bicyclic, or spirocyclic system consisting of a 3-7 membered ring connected to a basic nitrogen in an endocyclic or exocyclic configuration, which may be either nitrogen or saturated carbon, linked via a chain, carbocyclic, or heterocyclic ring, of 1-6 carbon atoms, with an aliphatic group of less than 9 carbon atoms, which may be substituted with a group selected from: sulfonyl, sulfinyl, carboxy, alkoxycarbonyl, or aminocarbonyl group, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, sulfonyl, or haloalkoxy, sulfonyl, or sulfinyl group; R ５ But -CH ２ C(CH ３ ) ２ C(O)OH, -CH ２ CH(OH)C(O)OH, -CH ２ C(O)OH, -CH ２ (oxetane)C(O)OH, —CH ２ C(O)OH, -CH ２ CH ２ CH ２ C(O)OH, -(cyclobutyl)C(O)OH, -CH ２ (cyclopropyl)C(O)OH, or CH ２ (cyclopropyl)C(O)OH; the methylene or ethylene group is selected from the group consisting of -C(O)NHOH, -C(O)N(CH ３ )OH, -C(O)NHOCH ３ , -NHP(O)(OH)NHCH ３ , -P(O)(OH)NHCH ３、 -P(O)(OH) ２、 -OP (O) (OH) ２、 -OP(O)(OH)NHCH ３、 -S(O) ２ Me, -S(O) ２ OH, -S(O)OH, -S(O) ２ CF ３ , 【Chemistry 6】 is connected to an acid or acid isostere group, including

4. A bacterial type III secretion system (T3SS) inhibitor compound of formula I(c) or a pharmaceutically acceptable salt thereof: 【Chemistry 7】 During the ceremony: A is CH or N; at least one X is Cl and the other X is hydrogen, Br, or Cl; W is —C(O)CH ２ -, -S(O) ２ -, -NHS(O) ２ -, -S(O) ２ NH-, -C(O)-, -CH ２ -, -CH(CH ３ )-, -NHC(O)-, -NHC(O)NH-, -NHC(NH)NH-, -NHC(NCH ３ )NH-, -NHS(O) ２ NH-, -N(CH ３ )C(O)-, -C(O)NH-, -C(O)N(CH ３ a divalent group bridging Ar and U, selected from the group consisting of —O(C(O)—, —(C(O)O—, —NH—, or —O—; U can be either nitrogen, or carbon that is part of a 4-7 membered non-aromatic heterocycle containing 1-2 nitrogen atoms, 0-1 oxygen atoms, and having 0-3 substituents (in addition to bonds between W and the ring carbon / nitrogen, and U and the ring carbon) selected from alkyl, cycloalkyl, aryl, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, or sulfinyl; or U can be independently hydrogen or, optionally, haloalkyl, nitro, halogen, alkoxy, alkylthio, haloalkoxy, sulfonyl, or sulfinyl. a monocyclic, bicyclic, or spirocyclic system consisting of a 3-7 membered ring connected to a basic nitrogen in an endocyclic or exocyclic configuration, which may be either nitrogen or saturated carbon, linked via a chain, carbocyclic, or heterocyclic ring, of 1-6 carbon atoms, with an aliphatic group of less than 9 carbon atoms, which may be substituted with a group selected from: sulfonyl, sulfinyl, carboxy, alkoxycarbonyl, or aminocarbonyl group, heteroaryl, haloalkyl, nitro, halogen, alkoxy, alkylthio, sulfonyl, or haloalkoxy, sulfonyl, or sulfinyl group; R ５ But -CH ２ C(CH ３ ) ２ C(O)OH, -CH ２ CH(OH)C(O)OH, -CH ２ C(O)OH, -CH ２ (oxetane)C(O)OH, —CH ２ C(O)OH, -CH ２ CH ２ CH ２ C(O)OH, -(cyclobutyl)C(O)OH, -CH ２ (cyclopropyl)C(O)OH, or CH ２ (cyclopropyl)C(O)OH; the methylene or ethylene group is selected from the group consisting of -C(O)NHOH, -C(O)N(CH ３ )OH, -C(O)NHOCH ３ , -NHP(O)(OH)NHCH ３ , -P(O)(OH)NHCH ３、 -P(O)(OH) ２、 -OP (O) (OH) ２、 -OP(O)(OH)NHCH ３、 -S(O) ２ Me, -S(O) ２ OH, -S(O)OH, -S(O) ２ CF ３ , 【Chemistry 8】 is connected to an acid or acid isostere group, including

5. The compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: 【Chemistry 9】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】 【change】

6. A pharmaceutical composition comprising one or more bacterial T3SS inhibitor compounds according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier or excipient.

7. A method of treating or preventing a bacterial infection in a mammalian subject comprising administering a compound of any one of claims 1 to 6.

8. 8. The method of claim 7, wherein the bacterial infection is caused by Pseudomonas or Chlamydia.

9. 9. The method of claim 8, wherein the bacterial infection is caused by Pseudomonas aeruginosa.

10. The method of claim 7, wherein the mammal is a human.

11. Use of a composition according to any one of claims 6 to 10 for the treatment of bacterial infections.

12. Use of a composition according to any one of claims 6 to 10 for the manufacture of a medicament for the treatment of a bacterial infection.

13. 13. Use of the composition according to claim 11 or 12, wherein the bacterial infection is caused by Pseudomonas or Chlamydia.

14. 14. The use of the composition of claim 13, wherein the Pseudomonas is Pseudomonas aeruginosa.

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