EP4626445A1

EP4626445A1 - Gallium-salophen antimicrobial compounds and methods of use thereof

Info

Publication number: EP4626445A1
Application number: EP23899008.9A
Authority: EP
Inventors: Fengtian Xue; Angela Wilks
Original assignee: University of Maryland Baltimore; University of Maryland College Park
Current assignee: University of Maryland Baltimore; University of Maryland College Park
Priority date: 2022-12-02
Filing date: 2023-12-01
Publication date: 2025-10-08
Also published as: WO2024119099A1

Abstract

Gallium-salophen compounds, and methods of using the same for the treatment of disease are disclosed.

Description

GALLTUM-SALOPHEN ANTIMICROBIAL COMPOUNDS AND METHODS OF USE

THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63/385,919, filed December 2, 2022, which is incorporated by reference herein in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[001] This invention was made with government support under Grant Number AH34886 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

[002] The disclosure relates generally to gallium-salophen compounds and methods of treating or preventing conditions associated with inhibition of HasAp protein activity and/or the P. aeruginosa siderophore iron uptake system.

BACKGROUND

[003] Pseudomonas aeruginosa is an opportunistic bacterium that causes life-threatening infections in immunocompromised patients. Multidrug-resistant P. aeruginosa has been classified by the CDC as a “serious threat” to public health owing to its ability to overcome many current treatment strategies. It frequently causes infection in immunocompromised patients and is a leading cause of nosocomial infections. P. aeruginosa is the second leading cause of ventilator-associated pneumonias in intensive care facilities, and the primary cause of respiratory infection in cystic fibrosis (CF) patients where it persists for decades. It is particularly problematic for cystic fibrosis (CF) patients, who suffer from life-threatening chronic infection where even direct delivery of the antibiotic tobramycin to the lung is becoming ineffective. While new antibiotics are entering the pipeline, many candidates in Phase I-III trials have minimal activity against P. aeruginosa and new developments are typically improved versions of existing inhibitors rather than new classes of antibiotics. These targets typically include essential cellular pathways such as peptidoglycan or protein synthesis, which often see resistance emerge within a few years of introduction.

SUMMARY

[004] The disclosure provides in one aspect a compound of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: formula (I) wherein in formula (I): each R¹ is a substituent independently selected at each occurrence from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl; each R² and R³ is a substituent independently selected at each occurrence from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl;

R⁴ is H or an optionally substituted Ci-Ce alkyl; n is an integer from 0 to 4; p is an integer from 0 to 4; and q is an integer from 0 to 4; with the proviso that when R⁴ is H, then at least one of p or q is an integer from 1 to 4; and a) when p is 1 and R² is selected from Group A, i) q cannot be 0; and ii) when q is 1 , R³ cannot be identical to R²; and b) when q is 1 and R³ is selected from Group A, i) p cannot be 0; and ii) when p is 1, R² cannot be identical to R³:

Group A: independently at each occurrence selected from Br , Cl , and I .

[005] In some embodiments, n is 0. In some embodiments, each R² and R³ is independently - OR^b wherein each R^b is independently selected at each occurrence from the group consisting of optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylcycloalkyl, and optionally substituted alkylheteroaryl. In some embodiments, each R² and , wherein ring A is an optionally substituted heterocyclyl, ring B is an optionally substituted aryl, each R^e is independently at each occurrence H or alkyl, each X is independently at each occurrence selected from Br, Cl ", and I" , and each s and v is independently at each occurrence an integer from 1 to 4. In some embodiments, s is 2. In some embodiments, v is 1. In some embodiments, each occurrence of R^c is H or Ci-Csalkyl, optionally

optionally fluorine, and ring C is optionally substituted heterocyclyl, optionally morpholine. In some embodiments, each R² is independently selected at each occurrence from endently at each occurrence selected from Br , Cl , and I . In some embodiments, each R³ is independently selected at each occurrence from

Br , Cl , and I . In some embodiments, p is 1 or 2. In some embodiments, q is 1. [006] The disclosure provides in one aspect a compound of formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: wherein in formula (II):

R² and R³ are each a substituent independently selected from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl; and

R⁴ is H or an optionally substituted Ci-Ce alkyl; with the proviso that when R⁴ is H, then at least one of p or q is an integer from 1 to 4; and a) when p is 1 and R² is selected from Group A, i) q cannot be 0; and ii) when q is 1, R³ cannot be identical to R²; and b) when q is 1 and R³ is selected from Group A, i) p cannot be 0; and ii) when p is 1, R² cannot be identical to R³:

Group A: independently at each occurrence selected from Br , Cl", and I".

[007] The disclosure provides in one aspect a compound of formula (III), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: formula (III) wherein in formula (III):

R³ is a substituent selected from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl; and R⁴ is H or an optionally substituted Ci-Ce alkyl; with the proviso that when R⁴ is H, then at least one of p or q is an integer from 1 to 4; and a) when p is 1 and R² is selected from Group A, i) q cannot be 0; and ii) when q is 1, R³ cannot be identical to R²; and b) when q is 1 and R³ is selected from Group A, i) p cannot be 0; and ii) when p is 1, R² cannot be identical to R³:

Group A: independently at each occurrence selected from Br , Cl , and I .

[008] The disclosure provides in one aspect a compound of any one of formulas 1001 to 1339, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof wherein X is independently at each occurrence selected from Br , Cl", or I".

[009] The disclosure provides in one aspect a compound of any one of formulas 2001 to

2031, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof wherein X is independently at each occurrence selected from Br , Cl , or I .

[0010] The disclosure provides in one aspect a compound, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, selected from:

[0011] In some embodiments, the compound inhibits HasAp protein activity. In some embodiments, the compound inhibits HemO protein activity. In some embodiments, the compound inhibits both HasAp protein activity and HemO protein activity.

[0012] In one aspect, the disclosure provides a pharmaceutical composition for treating a condition alleviated by inhibiting HasAp protein activity, the pharmaceutical composition comprising one or more compounds of the disclosure, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

[0013] In one aspect, the disclosure provides a pharmaceutical composition for treating a condition alleviated by inhibiting HemO protein activity, the pharmaceutical composition comprising one or more compounds of the disclosure, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

[0014] In one aspect, the disclosure provides a pharmaceutical composition for treating a condition alleviated by dual inhibition of HasAp protein activity and the HemO protein activity, the pharmaceutical composition comprising one or more compounds of the disclosure, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

[0015] In some embodiments, the condition is selected from sepsis, meningitis, endocarditis, osteomyelitis, otitis media, sinusitis, pneumonia, chronic respiratory tract infection, catheter infection, surgical implant infection (e.g., cardiac valve replacement, pacemakerjoint replacement (e.g., hip or knee replacement) and the like), postoperative peritonitis, postoperative biliary tract, tricuspid valve endocarditis, ecthyma gangrenosum, eyelid abscess, lacrimal cystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, orbital infection, urinary tract infection, complicated urinary tract infection, catheter infection, perianal abscess, severe burns, airway bums, pressure ulcer infections, cystic fibrosis, and a bacterial infection.

[0016] In one aspect, the disclosure provides a pharmaceutical composition for treating or preventing a bacterial infection, the pharmaceutical composition comprising one or more compounds of the disclosure, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

[0017] In some embodiments, the bacterial infection is caused by a bacterium selected from P. aeruginosa, Serratia marcescens, Bordetella pertussis, Bordetella bronchiseptica, Bordetella avium, Yersinia pestis, Yersinia pseudotuberculosis, Acinetobacter baumannii, and pan-resistant pneumonia, optionally wherein the bacterium is an extensively drug-resistant (XDR) bacterium. [0018] In one aspect, the disclosure provides a method of treating a condition by inhibiting HasAp protein activity in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of the disclosure or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

[0019] In one aspect, the disclosure provides a method of treating a condition by inhibiting HemO protein activity in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of the disclosure or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

[0020] In one aspect, the disclosure provides a method of treating a condition by dual inhibition of the HasAp protein activity and HemO protein activity in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of the disclosure or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

[0021] In some embodiments, the condition is selected from sepsis, meningitis, endocarditis, osteomyelitis, otitis media, sinusitis, pneumonia, chronic respiratory tract infection, catheter infection, surgical implant infection (e.g., cardiac valve replacement, pacemakerjoint replacement (e.g., hip or knee replacement) and the like), postoperative peritonitis, postoperative biliary tract, tricuspid valve endocarditis, ecthyma gangrenosum, eyelid abscess, lacrimal cystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, orbital infection, urinary tract infection, complicated urinary tract infection, catheter infection, perianal abscess, severe burns, airway bums, pressure ulcer infections, cystic fibrosis, and a bacterial infection.

[0022] In one aspect, the disclosure provides a method for treating or preventing a bacterial infection in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of the disclosure or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

[0023] In some embodiments, the bacterial infection is caused by a bacterium selected from P. aeruginosa, Serratia marcescens, Bordetella pertussis, Bordetella bronchiseptica, Bordetella avium, Yersinia pestis, Yersinia pseudotuberculosis, Acinetobacter baumannii, and pan-resistant pneumonia, optionally wherein the bacterium is an extensively drug-resistant (XDR) bacterium.

BRIEF DESCRIPTION OF THE FIGURES

[0024] FIG. 1 illustrates an X-Ray crystal structure of GaSal-2.

[0025] FIGS. 2A- 2E illustrate the binding, growth inhibition and transcriptional activation with GaSal and GaSal-2. FIG. 2A shows experimental data demonstrating GaSal results from three separate experiments. FIG. 2B shows experimental data demonstrating GaSal-2 results from three separate experiments. FIG. 2C shows experimental data demonstrating fluorescence quenching data from HasAp binding studies. Data represent the average and standard deviation of five independent experiments ****, p<0.00005

[0026] FIG. 3 illustrates a STD-NMR of GaSal-2 with HasAp. The top shows difference spectrum after saturation of 10 pM HasAp with 1 mM GaSal-2 in D2O. The bottom shows the reference 1D-^XH spectrum of GaSal-2 in D2O including GaSal-2 structure and proton assignments. [0027] FIGS 4A-4B show experimental data demonstrating the results of ICP-MS uptake studies with GaSal and GaSal-2.

[0028] FIGS. 5A-5C are graphs illustrating how GaSal and GaSal-2 bind to HemO and inhibit heme degradation. FIG. 5A shows experimental data demonstrating GaSal/HemO fluorescence quenching from three experiments. FIG. 5B shows experimental data demonstrating GaSal- 2/HemO fluorescence quenching from three experiments. Data were fit to a one-site binding model in GraphPad Prism. FIG. 5C shows experimental data demonstrating LC-MS/MS BVIX levels from cultures treated with heme (1 pM) and GaSal or GaSal-2 (10 pM). Data represent the average of three independent experiments corrected for ODeoo as well as an average extraction efficiency of 27%. Statistical differences were determined by two-tailed t-tests. *, p < 0.05; **, p < 0.005.

[0029] FIGS. 6A- 6D are graphs illustrating the effect of GaSal-2 on PAO1 infection in mice. Bacterial load in the lungs (FIG. 6A) and nasal wash (FIG. 6A B), lung weight (FIG. 6A C) and body temperature (FIG. 6A D) from infected CD1 mice for each strain as shown. GaSal -2 treatment was via nebulization (dose: 1 mg/mL). a P values by two-tailed student t-test *, P < 0.05. ** < 0.001.

[0030] FIG. 7A illustrates experimental data demonstrating the binding mode of GaSal-2 (yellow) in HasAp (PDB 3w8m), with the highlights of the Solubility Site (pink), Heme Site (black), and Affinity Site (gold). FIG. 7B illustrates experimental data demonstrating the binding mode of GaSal-2 (yellow) in HemO (PDB lsk7). GaSal-2 was overlaid with the SILCS apolar (green), H-bond-donor (blue), H-bond-acceptor (red), negative (orange), and positive (cyan) FragMaps. FragMaps are shown at contour levels of -1.0 kcal/mol for the generic apolar Hbond-donor and Hbond-acceptor maps and at -1.5 kcal/mol for the generic apolar, negative, and positive maps.

[0031] FIG. 8A illustrates the KD, IC50, solubility, and stability of salophen complexes. FIG.

8B illustrates an X-ray crystal structure of GaSal-2.

[0032] FIG. 9 illustrates experimental data demonstrating transcriptional activation of the Has signaling cascade. 10 pM of GaSal or GaSal -2 were used in the presence of heme (1 pM). Data represent the average and SD of five independent experiments ****, p<0.00005.

[0033] FIGS. 10A- 10B are graphs illustrating experimental data demonstrating ICP-MS uptake of GaSal and GaSal -2. PAO1 WT and phu . strains are prepared for ICP-MS in 6% FINCH. Data represent the average and SD of 3 independent experiments, with levels of Ga (FIG. 10A) and Fe (FIG. 10B) normalized to dry pellet weight.

[0034] FIG. 11 is a graph of experimental data demonstrating GaSal-2 inhibits HemO and BVIX p/5 formation. LC-MS/MS BVIX levels from cultures treated with heme (1 pM) and GaSal or GaSal-2 (10 pM). Data represent the average of 3 independent experiments corrected for extraction efficiency and OD600 and statistical differences were determined by two-tailed t-tests. *, p<0.05; **, p<0.005.

[0035] FIG. 12A illustrates regions showing the most significant changes in deuterium uptake labeled (purple) and mapped onto the structure of holo-HasAp. Heme was shown in blue and GaSal was shown in red. FIG. 12B is a graph of experimental data demonstrating the deuteration of a peptide of residues 26-54. FIG. 12C is a graph of experimental data demonstrating the thermal denaturation profiles of apo-, holo-, and GaSal-HasAp.

[0036] FIG. 13A illustrates binding mode of GaSal-2 (yellow) in HasAp (PDB 3w8m), with the highlights of the Solubility Site (pink), Heme Site (black), and Affinity Site (gold). FIG. 13B illustrates binding mode of GaSal -2 (yellow) in HemO (PDB lsk7). GaSal -2 was overlaid with the SILCS apolar (green), H-bond-donor (blue), H-bond-acceptor (red), negative (orange), and positive (cyan) FragMaps. FragMaps are shown at contour levels of -1.0 kcal/mol for the generic apolar Hbond-donor and Hbond-acceptor maps and at -1.5 kcal/mol for the generic apolar, negative, and positive maps.

[0037] FIGS. 14A-14C illustrates a non-limiting example of a selection of target GaSal-2 analogs. FIG. 14A is a non-limiting example of a structure and selection criteria of 54 target compounds. FIG. 14B shows structures of the top solubility arms (SAs) and affinity arms (AAs) of compounds of the disclosure. FIG. 14C is a table showing LGFE differences (kcal/mol) versus GaSal-2.

[0038] FIG. 15 is a non-limiting example of a strategic outline for the design, synthesis, evaluation and selection criteria of compounds of the disclosure.

[0039] FIG. 16 is a non-limiting example of the synthesis of CADD SILCS-designed new GaSal-2 analogs 11-154.

[0040] FIG. 17 is Structure of II.

[0041] FIGS. 18A-18B show the binding mode of previously characterized HemO inhibitor acitretin. FIG. 18A is a graph showing ^XH,¹⁵N HSQC (heteronuclear single quantum coherence) spectrum of 250 pM HemO with 5% DMSO (red) is superimposed onto the spectrum of HemO with 500 pM acitretin (blue). Residues that experience the most significant chemical shift perturbation (>2 x sd) are labeled. FIG. 18B is a graphical representation of the HemO based on PDB lsk7 is shown. Acitretin is shown in cyan, and residues perturbed upon binding of acitretin in magenta.

[0042] FIG. 19 illustrates a MALDI-MSI of PAO1 infected (A) versus uninfected (B) murine lung. Left: phosphatidyl choline (m/z 756) in red. Center: BVIX (m/z 583) in green Right: Merged ion image. H&E far left.

[0043] FIG. 20 illustrates a non-limiting example of a synthetic route to prepare NCE GalSal-2. [0044] FIG. 21 illustrates the structure, Kd and IC50 values of FeSal, GalSal, and GalSal-2. [0045] FIG. 22 illustrates non-limiting examples of compounds o the disclosure.

DETAILED DESCRIPTION

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs. All patents and publications referred to herein are incorporated by reference in their entireties. Definitions

[0047] As used herein, the terms “administer,” “administration” or “administering” refer to (1) providing, giving, dosing, and/or prescribing by either a health practitioner or his authorized agent or under his or her direction according to the disclosure; and/or (2) putting into, taking or consuming by the mammal, according to the disclosure.

[0048] The terms “co-administration,” “co-administering,” “administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients to a subject so that both active pharmaceutical ingredients and/or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.

[0049] The terms “active pharmaceutical ingredient” and “drug” include, but are not limited to, the compounds described herein and, more specifically: compounds of formula (I), formula (II), formula (III); compounds of any one of formulas 1001-1339 and 2001-2031 ; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal- 15, and their features and limitations as described herein. The terms “active pharmaceutical ingredient” and “drug” may also include those compounds described herein that bind HasAp and/or HemO and/or the P. aeruginosa siderophore iron uptake system, and thereby modulate HasAp protein activity and/or HemO protein activity and/or/⁵, aeruginosa siderophore iron uptake system activity, selectively bind HasAp, selectively bind HemO, selectively bind the P. aeruginosa siderophore iron uptake system, or dually bind HasAp and HemO, and/or bind P. aeruginosa siderophore iron uptake system.

[0050] The term “/// vzvo” refers to an event that takes place in a subject’s body.

[0051] The term “in vitro" refers to an event that takes places outside of a subject’s body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.

[0052] The term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc. which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells (e.g., increased sensitivity to apoptosis). The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.

[0053] A “therapeutic effect” as that term is used herein, encompasses a therapeutic benefit and/or a prophylactic benefit. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0054] The terms “QD,” “qd,” or “q.d.” mean quaque die, once a day, or once daily. The terms “BID,” “bid,” or “b i d.” mean bis in die, twice a day, or twice daily. The terms “TID,” “tid,” or “t.i.d.” mean ter in die, three times a day, or three times daily. The terms “QID,” “qid,” or “q.i.d .” mean quater in die, four times a day, or four times daily.

[0055] The term “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Preferred inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid. Preferred organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese and aluminum. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts. The term “cocrystal” refers to a molecular complex derived from a number of cocrystal formers known in the art. Unlike a salt, a cocrystal typically does not involve hydrogen transfer between the cocrystal and the drug, and instead involves intermolecular interactions, such as hydrogen bonding, aromatic ring stacking, or dispersive forces, between the cocrystal former and the drug in the crystal structure.

[0056] “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the disclosure is contemplated. Additional active pharmaceutical ingredients, such as other drugs disclosed herein, can also be incorporated into the described compositions and methods.

[0057] As used herein, the terms “treat,” “treatment,” and/or “treating” may refer to the management of a disease, disorder, or pathological condition, or symptom thereof with the intent to cure, ameliorate, stabilize, and/or control the disease, disorder, pathological condition or symptom thereof. Regarding control of the disease, disorder, or pathological condition more specifically, “control” may include the absence of condition progression, as assessed by the response to the methods recited herein, where such response may be complete (e.g., placing the disease in remission) or partial (e.g., lessening or ameliorating any symptoms associated with the condition).

[0058] As used herein, the terms “modulate” and “modulation” refer to a change in biological activity for a biological molecule (e.g., a protein, gene, peptide, antibody, and the like), where such change may relate to an increase in biological activity (e.g., increased activity, agonism, activation, expression, upregulation, and/or increased expression) or decrease in biological activity (e.g., decreased activity, antagonism, suppression, deactivation, downregulation, and/or decreased expression) for the biological molecule. In some embodiments, the biological molecules modulated by the methods and compounds of the disclosure to effect treatment may include the HasAp protein and/or the HemO protein and/or the protein of the P. aeruginosa siderophore iron uptake system.

[0059] As used herein, the term “prodrug” refers to a derivative of a compound described herein, the pharmacologic action of which results from the conversion by chemical or metabolic processes in vivo to the active compound. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxyl or carboxylic acid group of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by one or three letter symbols but also include, for example, 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, 3- methylhistidine, beta-alanine, gamma-aminobutyric acid, citrulline, homocysteine, homoserine, ornithine and methionine sulfone. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters (e.g., methyl esters and acetoxy methyl esters). Prodrug esters as employed herein includes esters and carbonates formed by reacting one or more hydroxyls of compounds of the method of the disclosure with alkyl, alkoxy, or aryl substituted acylating agents employing procedures known to those skilled in the art to generate acetates, pivalates, methylcarbonates, benzoates and the like. As further examples, free hydroxyl groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxy carbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxyl and amino groups are also included, as are carbonate prodrugs, sulfonate prodrugs, sulfonate esters and sulfate esters of hydroxyl groups. Free amines can also be derivatized to amides, sulfonamides or phosphonamides. All of the stated prodrug moi eties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities. Moreover, any compound that can be converted in vivo to provide the bioactive agent (e.g., a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15) is a prodrug within the scope of the disclosure. Various forms of prodrugs are well known in the art. A comprehensive description of pro drugs and prodrug derivatives are described in: (a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al., (Academic Press, 1996); (b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); (c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds., (Harwood Academic Publishers, 1991). In general, prodrugs may be designed to improve the penetration of a drug across biological membranes in order to obtain improved drug absorption, to prolong duration of action of a drug (slow release of the parent drug from a prodrug, decreased first-pass metabolism of the drug), to target the drug action (e.g. organ or tumor-targeting, lymphocyte targeting), to modify or improve aqueous solubility of a drug (e.g., i.v. preparations and eyedrops), to improve topical drug delivery (e.g. dermal and ocular drug delivery), to improve the chemical/enzymatic stability of a drug, or to decrease off-target drug effects, and more generally in order to improve the therapeutic efficacy of the compounds utilized in the disclosure.

[0060] Unless otherwise stated, the chemical structures depicted herein are intended to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds where one or more hydrogen atoms is replaced by deuterium or tritium, or wherein one or more carbon atoms is replaced by ¹³C- or ¹⁴C-enriched carbons, are within the scope of this disclosure.

[0061] When ranges are used herein to describe, for example, physical or chemical properties such as molecular weight or chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. Use of the term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary. The variation is typically from 0% to 15%, preferably from 0% to 10%, more preferably from 0% to 5% of the stated number or numerical range. The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) includes those embodiments such as, for example, an embodiment of any composition of matter, method or process that “consist of’ or “consist essentially of’ the described features.

[0062] “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to ten carbon atoms (e-g., (C i -io)alkyl or Ci-io alkyl). Whenever it appears herein, a numerical range such as “1 to 10” refers to each integer in the given range - e.g., “1 to 10 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the definition is also intended to cover the occurrence of the term “alkyl” where no numerical range is specifically designated. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, w-butyl, isobutyl, sec-butyl isobutyl, tertiary butyl, pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl and decyl. The alkyl moiety may be attached to the rest of the molecule by a single bond, such as for example, methyl (Me), ethyl (Et), //-propyl (Pr), 1 -methylethyl (isopropyl), //-butyl, //-pentyl, 1, 1 -dimethylethyl (/-butyl) and 3 -methylhexyl. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted by one or more of substituents which are independently heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SR^a, - OC(O)-R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, - N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)_tR^a (where t is 1 or 2), - S(O)tR^a (where t is 1 or 2), -S(O)tOR^a (where t is 1 or 2), -S(O)tN(R^a)2 (where t is 1 or 2), or PO3(R^a)2 where each R^a is independently hydrogen, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0063] “Alkylaryl” refers to an -(alkyl)aryl radical where aryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for aryl and alkyl respectively.

[0064] “Alkylhetaryl” refers to an -(alkyl)hetaryl radical where hetaryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for aryl and alkyl respectively.

[0065] “Alkylheterocycloalkyl” refers to an -(alkyl) heterocyclyl radical where alkyl and heterocycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heterocycloalkyl and alkyl respectively. [0066] An “alkene” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an “alkyne” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic.

[0067] “Alkenyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to ten carbon atoms (z. ., (C2-io)alkenyl or C2-10 alkenyl). Whenever it appears herein, a numerical range such as “2 to 10” refers to each integer in the given range - e.g., “2 to 10 carbon atoms” means that the alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. The alkenyl moiety may be attached to the rest of the molecule by a single bond, such as for example, ethenyl (i.e., vinyl), prop-l-enyl (i.e., allyl), but-l-enyl, pent-l-enyl and penta- 1,4-dienyl. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted by one or more substituents which are independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaiyl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, - OR^a, -SR^a, -OC(O)-R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, - N(R^a)C(O)OR^a, -N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)tR^a (where t is 1 or 2), -S(O)tR^a (where t is 1 or 2), -S(O)_tOR^a (where t is 1 or 2), -S(O)tN(R^a)2 (where t is 1 or 2), or PO3(R^a)2, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclyl alkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkyl alkyl, heteroaryl or heteroarylalkyl.

[0068] “Alkenyl-cycloalkyl” refers to an -(alkenyl)cycloalkyl radical where alkenyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for alkenyl and cycloalkyl respectively.

[0069] “Alkynyl” refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to ten carbon atoms (/.<?., (C2-io)alkynyl or C2-10 alkynyl). Whenever it appears herein, a numerical range such as “2 to 10” refers to each integer in the given range - e.g., “2 to 10 carbon atoms” means that the alkynyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. The alkynyl may be attached to the rest of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl and hexynyl. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroaiylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SR^a, -OC(O)-R^a, - N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, -

N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)_tR^a (where t is 1 or 2), - S(O)tR^a (where t is 1 or 2), -S(O)_tOR^a (where t is 1 or 2), -S(O)tN(R^a)2 (where t is 1 or 2), or POs(R^a)2, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0070] “Alkynyl-cycloalkyl” refers to an -(alkynyl)cycloalkyl radical where alkynyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for alkynyl and cycloalkyl respectively.

[0071] “Carboxaldehyde” refers to a -(C=O)H radical.

[0072] “Carboxyl” refers to a -(C=O)OH radical.

[0073] “Cyano” refers to a -CN radical.

[0074] “Cycloalkyl” refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and may be saturated, or partially unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms (i.e. (C3-io)cycloalkyl or C3-10 cycloalkyl). Whenever it appears herein, a numerical range such as “3 to 10” refers to each integer in the given range - e.g., “3 to 10 carbon atoms” means that the cycloalkyl group may consist of 3 carbon atoms, etc., up to and including 10 carbon atoms. Illustrative examples of cycloalkyl groups include, but are not limited to the following moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and the like. Unless stated otherwise specifically in the specification, a cycloalkyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SIU, -OC(O)-R^a, - N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, - N(R^a)C(O)R^a, -N(R^a)C(0)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)_tR^a (where t is 1 or 2), - S(O)tR^a (where t is 1 or 2), -S(O)tOR^a (where t is 1 or 2), -S(O)tN(R^a)2 (where t is 1 or 2), or PO3(R^a)2, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0075] “Cycloalkyl-alkenyl” refers to a -(cycloalkyl)alkenyl radical where cycloalkyl and alkenyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and alkenyl, respectively.

[0076] “Cycloalkyl-heterocycloalkyl” refers to a -(cycloalkyl)heterocycloalkyl radical where cycloalkyl and heterocycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and heterocycloalkyl, respectively.

[0077] “Cycloalkyl-heteroaryl” refers to a -(cycloalkyl)heteroaryl radical where cycloalkyl and heteroaryl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for cycloalkyl and heteroaryl, respectively.

[0078] The term “alkoxy” refers to the group -O-alkyl, including from 1 to 8 carbon atoms of a straight, branched, cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy and cyclohexyloxy. “Lower alkoxy” refers to alkoxy groups containing one to six carbons. [0079] The term “substituted alkoxy” refers to alkoxy wherein the alkyl constituent is substituted (i.e., -©-(substituted alkyl)). Unless stated otherwise specifically in the specification, the alkyl moiety of an alkoxy group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SR^a, -OC(O)-R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, - C(O)N(R^a)₂, -N(R^a)C(O)OR^a, -N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, - N(R^a)S(O)_tR^a (where t is 1 or 2), -S(O)_tR^a (where t is 1 or 2), -S(O)tOR^a (where t is 1 or 2), -S(O)tN(R^a)₂ (where t is 1 or 2), or PO₂(R^a)₂, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0080] The term “alkoxy carbonyl” refers to a group of the formula (alkoxy )(C=O)- attached through the carbonyl carbon wherein the alkoxy group has the indicated number of carbon atoms. Thus a (Ci-e)alkoxy carbonyl group is an alkoxy group having from 1 to 6 carbon atoms attached through its oxygen to a carbonyl linker. “Lower alkoxycarbonyl” refers to an alkoxycarbonyl group wherein the alkoxy group is a lower alkoxy group.

[0081] The term “substituted alkoxycarbonyl” refers to the group (substituted alkyl)-O-C(O)- wherein the group is attached to the parent structure through the carbonyl functionality. Unless stated otherwise specifically in the specification, the alkyl moiety of an alkoxycarbonyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SR^a, - OC(O)-R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, - N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)_tR^a (where t is 1 or 2), - S(O)tR^a (where t is 1 or 2), -S(O)_tOR^a (where t is 1 or 2), -S(O)tN(R^a)₂ (where t is 1 or 2), or POs(R^a)₂, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroaryl alkyl.

[0082] “Acyl” refers to the groups (alkyl)-C(O)-, (aryl)-C(O)-, (heteroaryl)-C(O)-, (heteroalkyl)-C(O)- and (heterocycloalkyl)-C(O)-, wherein the group is attached to the parent structure through the carbonyl functionality. If the R radical is heteroaryl or heterocycloalkyl, the hetero ring or chain atoms contribute to the total number of chain or ring atoms. Unless stated otherwise specifically in the specification, the alkyl, aryl or heteroaryl moiety of the acyl group is optionally substituted by one or more substituents which are independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SR^a, - OC(O)-R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, - N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)_tR^a (where t is 1 or 2), - S(O)tR^a (where t is 1 or 2), -S(O)_tOR^a (where t is 1 or 2), -S(O)tN(R^a)2 (where t is 1 or 2), or POs(R^a)2, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0083] “Acyloxy” refers to a R(C=O)O- radical wherein R is alkyl, aryl, heteroaryl, heteroalkyl or heterocycloalkyl, which are as described herein. If the R radical is heteroaryl or heterocycloalkyl, the hetero ring or chain atoms contribute to the total number of chain or ring atoms. Unless stated otherwise specifically in the specification, the R of an acyloxy group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SR^a, - OC(O)-R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, - N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)_tR^a (where t is 1 or 2), - S(O)tR^a (where t is 1 or 2), -S(O)tOR^a (where t is 1 or 2), -S(O)tN(R^a)2 (where t is 1 or 2), or PO3(R^a)2, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0084] “Acylsulfonamide” refers a -S(O)2-N(R^a)-C(=O)- radical, where R^a is hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroaryl alkyl. Unless stated otherwise specifically in the specification, an acylsulfonamide group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, - OR^a, -SR^a, -OC(O)-R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, - N(R^a)C(O)OR^a, -N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)tR^a (where t is 1 or 2), -S(O)tR^a (where t is 1 or 2), -S(O)tOR^a (where t is 1 or 2), -S(O)tN(R^a)₂ (where t is 1 or 2), or PC>3(R^a)₂, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl

[0085] “Amino” or “amine” refers to a -N(R^a)₂ radical group, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, unless stated otherwise specifically in the specification. When a -N(R^a)₂ group has two R^a substituents other than hydrogen, they can be combined with the nitrogen atom to form a 4-, 5-, 6- or 7-membered ring. For example, -N(R^a)₂ is intended to include, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. Unless stated otherwise specifically in the specification, an amino group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SR^a, -OC(O)-R^a, - N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, - N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)_tR^a (where t is 1 or 2), - S(O)tR^a (where t is 1 or 2), -S(O)tOR^a (where t is 1 or 2), -S(O)tN(R^a)₂ (where t is 1 or 2), or POs(R^a)₂, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0086] The term “substituted amino” also refers to N-oxides of the groups -NHR^a, and NR^aR^a each as described above. N-oxides can be prepared by treatment of the corresponding amino group with, for example, hydrogen peroxide or m-chloroperoxybenzoic acid.

[0087] “Amide” or “amido” refers to a chemical moiety with formula -C(O)N(R)₂ or -NHC(O)R, where R is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroali cyclic (bonded through a ring carbon), each of which moiety may itself be optionally substituted. The R₂ of -N(R)₂ of the amide may optionally be taken together with the nitrogen to which it is attached to form a 4-, 5-, 6- or 7- membered ring. Unless stated otherwise specifically in the specification, an amido group is optionally substituted independently by one or more of the substituents as described herein for alkyl, cycloalkyl, aryl, heteroaryl, or heterocycloalkyl. An amide may be an amino acid or a peptide molecule attached to a compound disclosed herein, thereby forming a prodrug. The procedures and specific groups to make such amides are known to those of skill in the art and can readily be found in seminal sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd Ed., John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein by reference in its entirety.

[0088] ‘ ‘Aromatic” or “aryl” or “Ar” refers to an aromatic radical with six to ten ring atoms (e.g., C₆-Cio aromatic or Ce-Cio aryl) which has at least one ring having a conjugated pi electron system which is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl). Bivalent radicals formed from substituted benzene derivatives and having the free valences at ring atoms are named as substituted phenylene radicals. Bivalent radicals derived from univalent polycyclic hydrocarbon radicals whose names end in “-yl” by removal of one hydrogen atom from the carbon atom with the free valence are named by adding “-idene” to the name of the corresponding univalent radical, e.g., a naphthyl group with two points of attachment is termed naphthylidene. Whenever it appears herein, a numerical range such as “6 to 10” refers to each integer in the given range; e.g., “6 to 10 ring atoms” means that the aryl group may consist of 6 ring atoms, 7 ring atoms, etc., up to and including 10 ring atoms. The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of ring atoms) groups. Unless stated otherwise specifically in the specification, an aryl moiety is optionally substituted by one or more substituents which are independently alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SR^a, -OC(O)-R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, - OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, -N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)_tR^a (where t is 1 or 2), -S(O)tR^a (where t is 1 or 2), -S(O)_tOR^a (where t is 1 or 2), -S(O)tN(R^a)₂ (where t is 1 or 2), or PO3(R^a)₂, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0089] The term “aryl oxy” refers to the group -O-ary 1.

[0090] The term “substituted aryloxy” refers to aryloxy wherein the aryl substituent is substituted (i.e., -O-(substituted aryl)). Unless stated otherwise specifically in the specification, the aryl moiety of an aryloxy group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SR^a, -OC(O)-R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, - C(O)N(R^a)₂, -N(R^a)C(O)OR^a, -N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, - N(R^a)S(O)tR^a (where t is 1 or 2), -S(O)tR^a (where t is 1 or 2), -S(O)tOR^a (where t is 1 or 2), -S(O)tN(R^a)₂ (where t is 1 or 2), or PO3(R^a)₂, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroaryl alkyl.

[0091] “Aralkyl” or “arylalkyl” refers to an (aryl)alkyl-radical where aryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for aryl and alkyl respectively.

[0092] ‘ ‘Ester” refers to a chemical radical of formula -COOR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). The procedures and specific groups to make esters are known to those of skill in the art and can readily be found in seminal sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd Ed., John Wiley & Sons, New York, N.Y., 1999, which is incorporated herein by reference in its entirety. Unless stated otherwise specifically in the specification, an ester group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroaiylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR^a, -SR^a, -OC(O)- R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, - N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)_tR^a (where t is 1 or 2), - S(O)tR^a (where t is 1 or 2), -S(O)tOR^a (where t is 1 or 2), -S(O)tN(R^a)₂ (where t is 1 or 2), or PO₂(R^a)₂, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0093] “Fluoroalkyl” refers to an alkyl radical, as defined above, that is substituted by one or more fluoro radicals, as defined above, for example, trifluoromethyl, difluoromethyl, 2,2,2- trifluoroethyl, l-fluoromethyl-2-fluoroethyl, and the like. The alkyl part of the fluoroalkyl radical may be optionally substituted as defined above for an alkyl group. [0094] ‘ ‘Halo,” “halide,” or, alternatively, “halogen” is intended to mean fluoro, chloro, bromo or iodo. The terms “haloalkyl,” “haloalkenyl,” “haloalkynyl,” and “haloalkoxy” include alkyl, alkenyl, alkynyl and alkoxy structures that are substituted with one or more halo groups or with combinations thereof. For example, the terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine.

[0095] “Heteroalkyl,” “heteroalkenyl,” and “heteroalkynyl” refer to optionally substituted alkyl, alkenyl and alkynyl radicals and which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof. A numerical range may be given - e.g., C1-C4 heteroalkyl which refers to the chain length in total, which in this example is 4 atoms long. A heteroalkyl group may be substituted with one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroaryl alkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, -OR^a, -SR^a, -OC(O)-R^a, -N(R^a)2, -C(O)R^a, -C(O)OR^a, - OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, -N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O>R^a (where t is 1 or 2), -S(O)tR^a (where t is 1 or 2), -S(O OR^a (where t is 1 or 2), -S(O)tN(R^a)2 (where t is 1 or 2), or POs(R^a)2, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[0096] “Heteroalkylaryl” refers to an -(heteroalkyl)aryl radical where heteroalkyl and aryl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heteroalkyl and aryl, respectively.

[0097] “Heteroalkylheteroaryl” refers to an -(heteroalkyl )heteroaryl radical where heteroalkyl and heteroaryl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heteroalkyl and heteroaryl, respectively.

[0098] “Heteroalkylheterocycloalkyl” refers to an -(heteroalkyl)heterocycloalkyl radical where heteroalkyl and heterocycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heteroalkyl and heterocycloalkyl, respectively.

[0099] “Heteroalkylcycloalkyl” refers to an -(heteroalkyl)cycloalkyl radical where heteroalkyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the substituents described as suitable substituents for heteroalkyl and cycloalkyl, respectively. [00100] “Heteroaryl” or “heteroaromatic” or “HetAr” or “Het” refers to a 5- to 18-membered aromatic radical ( .g., C5-C13 heteroaryl) that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur, and which may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system. Whenever it appears herein, a numerical range such as “5 to 18” refers to each integer in the given range - e.g., “5 to 18 ring atoms” means that the heteroaryl group may consist of 5 ring atoms, 6 ring atoms, etc., up to and including 18 ring atoms. Bivalent radicals derived from univalent heteroaryl radicals whose names end in “-yl” by removal of one hydrogen atom from the atom with the free valence are named by adding “-idene” to the name of the corresponding univalent radical - e.g., a pyridyl group with two points of attachment is a pyridylidene. A N-containing “heteroaromatic” or “heteroaryl” moiety refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom. The polycyclic heteroaryl group may be fused or non-fused. The heteroatom(s) in the heteroaryl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl may be attached to the rest of the molecule through any atom of the ring(s). Examples of heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo [tTjthiazolyl, benzothiadiazolyl, benzo[/>][l,4]dioxepinyl, benzo[/>][l,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzofurazanyl, benzothiazolyl, benzothienyl(benzothiophenyl), benzothieno[3,2-</]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[l,2-rz]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[ ]pyrimidinyl, 6,7-dihydro-5/7-cyclopenta[4,5]thieno[2,3- ]pyrimidinyl, 5,6-dihydrobenzo[A]quinazolinyl, 5,6-dihydrobenzo[A]cinnolinyl, 6,7-dihydro-577- benzo[6,7]cyclohepta[l,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[<7]pyrimidinyl, 5,6,7,8,9,10- hexahydrocycloocta[ ]pyridazinyl, 5, 6, 7, 8, 9, 10-hexahydrocycloocta[ ]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6- naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a- octahydrobenzo[/?]quinazolinyl, 1 -phenyl- 1 //-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4- d]pyrimidinyl, pyridinyl, pyrido[3,2-<7]pyrimidinyl, pyrido[3,4- ]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidinyl, 6,7,8,9-tetrahydro-5Z/-cyclohepta[4,5]thieno[2,3- ]pyrimidinyl, 5, 6,7,8- tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, thiapyranyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-<7]pyrimidinyl, thieno[3,2-6/]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl (z.e., thienyl). Unless stated otherwise specifically in the specification, a heteroaryl moiety is optionally substituted by one or more substituents which are independently: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroaryl alkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethyl si lanyl, -OR^a, -SIU, -OC(O)- R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, -N(R^a)C(O)OR^a, - N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)_tR^a (where t is 1 or 2), - S(O)tR^a (where t is 1 or 2), -S(O)tOR^a (where t is 1 or 2), -S(O)tN(R^a)₂ (where t is 1 or 2), or PO3(R^a)₂, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[00101] Substituted heteroaryl also includes ring systems substituted with one or more oxide (- O-) substituents, such as, for example, pyridinyl N-oxides.

[00102] “Heteroarylalkyl” refers to a moiety having an aryl moiety, as described herein, connected to an alkylene moiety, as described herein, wherein the connection to the remainder of the molecule is through the alkylene group.

[00103] “Heterocycloalkyl” or “heterocyclyl” refer to a stable 3- to 18-membered non-aromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Whenever it appears herein, a numerical range such as “3 to 18” refers to each integer in the given range - e.g., “3 to 18 ring atoms” means that the heterocycloalkyl group may consist of 3 ring atoms, 4 ring atoms, etc., up to and including 18 ring atoms. Unless stated otherwise specifically in the specification, the heterocycloalkyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems. The heteroatoms in the heterocycloalkyl radical may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quatemized. The heterocycloalkyl radical is partially or fully saturated. The heterocycloalkyl may be attached to the rest of the molecule through any atom of the ring(s). Examples of such heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl [1 ,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo- thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, a heterocycloalkyl moiety is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroaryl alkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, - OR^a, -SR , -OC(O)-R^a, -N(R^a)₂, -C(O)R^a, -C(O)OR^a, -OC(O)N(R^a)₂, -C(O)N(R^a)₂, - N(R^a)C(O)OR^a, -N(R^a)C(O)R^a, -N(R^a)C(O)N(R^a)₂, N(R^a)C(NR^a)N(R^a)₂, -N(R^a)S(O)tR^a (where t is 1 or 2), -S(O)tR^a (where t is 1 or 2), -S(O)tOR^a (where t is 1 or 2), -S(O)tN(R^a)₂ (where t is 1 or 2), or PO3(R^a)₂, where each R^a is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl or heteroarylalkyl.

[00104] “Heterocycloalkyl” also includes bicyclic ring systems wherein one non-aromatic ring, usually with 3 to 7 ring atoms, contains at least 2 carbon atoms in addition to 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen, as well as combinations comprising at least one of the foregoing heteroatoms; and the other ring, usually with 3 to 7 ring atoms, optionally contains 1-3 heteroatoms independently selected from oxygen, sulfur, and nitrogen and is not aromatic.

[00105] “Nitro” refers to the -NO₂ radical.

[00106] “ Oxa” refers to the -O- radical.

[00107] “ Oxo” refers to the =0 radical.

[00108] “Isomers” are different compounds that have the same molecular formula. “Stereoisomers” are isomers that differ only in the way the atoms are arranged in space - i.e., having a different stereochemical configuration. “Enantiomers” are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1 : 1 mixture of a pair of enantiomers is a “racemic” mixture. The term “(±)” is used to designate a racemic mixture where appropriate. “Diastereoisomers” are stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The absolute stereochemistry is specified according to the Cahn- Ingold-Prelog R-S system. When a compound is a pure enantiomer the stereochemistry at each chiral carbon can be specified by either (R) or ( ). Resolved compounds whose absolute configuration is unknown can be designated (+) or (-) depending on the direction (dextro- or levorotatory) which they rotate plane polarized light at the wavelength of the sodium D line. Certain of the compounds described herein contain one or more asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R) or (S). The present chemical entities, pharmaceutical compositions and methods are meant to include all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures. Optically active (R)- and fS')-i somers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

[00109] “Enantiomeric purity” as used herein refers to the relative amounts, expressed as a percentage, of the presence of a specific enantiomer relative to the other enantiomer. For example, if a compound, which may potentially have an (R)- or an (S)-isomeric configuration, is present as a racemic mixture, the enantiomeric purity is about 50% with respect to either the (R)- or (5)-isomer. If that compound has one isomeric form predominant over the other, for example, 80% (5)-isomer and 20% (J?)-isomer, the enantiomeric purity of the compound with respect to the (5)-i someric form is 80%. The enantiomeric purity of a compound can be determined in a number of ways known in the art, including but not limited to chromatography using a chiral support, polarimetric measurement of the rotation of polarized light, nuclear magnetic resonance spectroscopy using chiral shift reagents which include but are not limited to lanthanide containing chiral complexes or Pirkle’s reagents, or derivatization of a compounds using a chiral compound such as Mosher’s acid followed by chromatography or nuclear magnetic resonance spectroscopy.

[00110] In some embodiments, the enantiomerically enriched composition has a higher potency with respect to therapeutic utility per unit mass than does the racemic mixture of that composition. Enantiomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred enantiomers can be prepared by asymmetric syntheses. See, for example, Jacques, etal., Enantiomers, Racemates and Resolutions, Wiley Interscience, New York (1981); E. L. Eliel, Stereochemistry of Carbon Compounds, McGraw-Hill, New York (1962); and E. L. Eliel and S. H. Wilen, Stereochemistry of Organic Compounds, Wiley- Inter science, New York (1994).

[00111] The terms “enantiomerically enriched” and “non-racemic,” as used herein, refer to compositions in which the percent by weight of one enantiomer is greater than the amount of that one enantiomer in a control mixture of the racemic composition (e.g. , greater than 1 : 1 by weight). For example, an enantiomerically enriched preparation of the (S)-enantiomer, means a preparation of the compound having greater than 50% by weight of the (5)-enantiomer relative to the (A)-enantiomer, such as at least 75% by weight, or such as at least 80% by weight. In some embodiments, the enrichment can be significantly greater than 80% by weight, providing a “substantially enantiomerically enriched” or a “substantially non-racemic” preparation, which refers to preparations of compositions which have at least 85% by weight of one enantiomer relative to other enantiomer, such as at least 90% by weight, or such as at least 95% by weight. The terms “enantiomerically pure” or “substantially enantiomerically pure” refers to a composition that comprises at least 98% of a single enantiomer and less than 2% of the opposite enantiomer.

[00112] “Moiety” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule. [00113] “ Tautomers” are structurally distinct isomers that interconvert by tautomerization. “Tautomerization” is a form of isomerization and includes prototropic or proton-shift tautomerization, which is considered a subset of acid-base chemistry. “Prototropic tautomerization” or “proton-shift tautomerization” involves the migration of a proton accompanied by changes in bond order, often the interchange of a single bond with an adjacent double bond. Where tautomerization is possible (e.g., in solution), a chemical equilibrium of tautomers can be reached. An example of tautomerization is keto-enol tautomerization. A specific example of keto-enol tautomerization is the interconversion of pentane-2, 4-dione and 4- hydroxypent-3-en-2-one tautomers. Another example of tautomerization is phenol-keto tautomerization. A specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridin-4(H7)-one tautomers. [00114] A “leaving group or atom” is any group or atom that will, under selected reaction conditions, cleave from the starting material, thus promoting reaction at a specified site. Examples of such groups, unless otherwise specified, include halogen atoms and mesyloxy, p- nitrobenzensulphonyloxy and tosyloxy groups.

[00115] “Protecting group” is intended to mean a group that selectively blocks one or more reactive sites in a multifunctional compound such that a chemical reaction can be carried out selectively on another unprotected reactive site and the group can then be readily removed or deprotected after the selective reaction is complete. A variety of protecting groups are disclosed, for example, in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, New York (1999).

[00116] “Solvate” refers to a compound in physical association with one or more molecules of a pharmaceutically acceptable solvent.

[00117] “ Substituted” means that the referenced group may have attached one or more additional groups, radicals or moi eties individually and independently selected from, for example, acyl, alkyl, alkylaryl, cycloalkyl, aralkyl, aryl, carbohydrate, carbonate, heteroaryl, heterocycloalkyl, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, ester, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, oxo, perhaloalkyl, perfluoroalkyl, phosphate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea, and amino, including mono- and di-substituted amino groups, and protected derivatives thereof. The substituents themselves may be substituted, for example, a cycloalkyl substituent may itself have a halide substituent at one or more of its ring carbons. The term “optionally substituted” means optional substitution with the specified groups, radicals or moieties.

[00118] “Sulfanyl” refers to groups that include -S-(optionally substituted alkyl), -S-(optionally substituted aryl), -S-(optionally substituted heteroaryl) and -S-(optionally substituted heterocycloalkyl).

[00119] “Sulfinyl” refers to groups that include -S(O)-H, -S(O)-(optionally substituted alkyl), -S(O)-(optionally substituted amino), -S(O)-(optionally substituted aryl), -S(O)- (optionally substituted heteroaryl) and -S(O)-(optionally substituted heterocycloalkyl).

[00120] “Sulfonyl” refers to groups that include -S(O2)-H, -S(O2)-(optionally substituted alkyl), -S(O2)-(optionally substituted amino), -S(C>2)-(optionally substituted aryl), -S(O2)- (optionally substituted heteroaryl), and -S(O2)-(optionally substituted heterocycloalkyl). [00121] “Sulfonamidyl” or “sulfonamido” refers to a -S(=O)2-NRR radical, where each R is selected independently from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). The R groups in -NRR of the -S(=O)2-NRR radical may be taken together with the nitrogen to which it is attached to form a 4-, 5-, 6- or 7-membered ring. A sulfonamido group is optionally substituted by one or more of the substituents described for alkyl, cycloalkyl, aryl, heteroaryl, respectively.

[00122] “Sulfoxyl” refers to a -S(=O)2OH radical.

[00123] “ Sulfonate” refers to a -S(=O)2-OR radical, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). A sulfonate group is optionally substituted on R by one or more of the substituents described for alkyl, cycloalkyl, aryl, heteroaryl, respectively. [00124] Compounds of the disclosure also include crystalline and amorphous forms of those compounds, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms of the compounds, as well as mixtures thereof. “Crystalline form” and “polymorph” are intended to include all crystalline and amorphous forms of the compound, including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, unsolvated polymorphs (including anhydrates), conformational polymorphs, and amorphous forms, as well as mixtures thereof, unless a particular crystalline or amorphous form is referred to.

[00125] For the avoidance of doubt, it is intended herein that particular features (for example integers, characteristics, values, uses, diseases, formulae, compounds or groups) described in conjunction with a particular aspect, embodiment or example of the disclosure are to be understood as applicable to any other aspect, embodiment or example described herein unless incompatible therewith. Thus such features may be used where appropriate in conjunction with any of the definition, claims or embodiments defined herein. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and/or steps are mutually exclusive. The disclosure is not restricted to any details of any disclosed embodiments. The disclosure extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[00126] Moreover, as used herein, the term “about” means that dimensions, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, a dimension, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is noted that embodiments of very different sizes, shapes and dimensions may employ the described arrangements.

[00127] Furthermore, the transitional terms “comprising”, “consisting essentially of’ and “consisting of’, when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of’ excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of’ limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. All embodiments of the disclosure can, in the alternative, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

[00128] Inhibitors of heme assimilation system (Has) and/or the the P. aeruginosa siderophore iron uptake system

[00129] The antibiotic development pipeline falls critically short of its need, with only two of 26 antibiotics in the clinical development pipeline target WHO-priority multi-drug resistant Gram-negative pathogens and nearly 80% of new antibiotics are developments of existing classes with well-known resistance mechanisms. As such, new antibiotic strategies are critically needed, with many approaches turning to nontraditional mechanisms such as antibodies, phagebased strategies or immunomodulators. Research targeting virulence traits is also of great interest, as such pathways are important for infection but not as critical for survival outside the hose and are therefore expected to exert less selective pressure and slow resistance development. The strategies aim to inhibit pathways such as quorum sensing (for bacterial communication and virulence regulation), virulence factor secretion and regulation, or biofilm formation.

[00130] Another virulence-based strategy is to target the high nutritional iron requirement in pathogenic bacteria, as several virulence pathways are linked to bacterial iron acquisition and metabolic pathways. To this extent, many strategies targeting iron sensing, uptake and trafficking have been reported. Further, bacteria such as Pseudomonas aeruginosa express systems for the uptake of ferric and ferrous iron as well as heme, which accounts for 75-90% of iron in a host. The ability to adapt to acquire iron in multiple forms is an important evolutionary trait for the bacteria but creates significant therapeutic challenges wherein iron acquisition pathways are abundant and adaptable. Approaches such as siderophore-drug conjugates have shown preliminary success and are well-reviewed, leading to the recent approval of Cefiderocol, a first- in-class iron-chelating cephalosporin antibiotic. These approaches, however, are limited if the pathogens counteract this mechanism by shifting to heme as an iron source and decrease reliance on siderophores, as P. aeruginosa does in chronic infection.

[00131] Pa is a WHO priority 1 “ESKAPE” pathogen that causes life-threatening infections. Multi drug resistant (MDR)P has been classified by the CDC as a “serious threat” to public health owing to its ability to overcome many current treatment strategies. Pa commonly causes infection in immune-compromised patients, and is a leading cause of nosocomial infections. It is the second leading cause of ventilator-associated pneumonias in intensive care units, and the primary cause of respiratory infection in cystic fibrosis patients.

[00132] Iron is essential for TA/ survival; numerous investigators have attempted to blunt Pa infection by chelating iron. However, these attempts have generally failed, because blood contains an abundance of iron, complexed in heme. The NCEs are the gallium salophen compounds GalSal and GalSal-2, which circumvent this issue by preventing Pa from using heme as an iron source. They accomplish this by inhibiting Pa’s ability to sense heme and by reducing the transport of heme into the bacterial cell. Because gallium is electrochemically similar to iron, it also competes with iron for uptake by Pa. Finally, gallium salophen compounds are toxic to Pa and result in the death of the bacteria. This Mil allowed us to extend the in vitro results into a relevant murine model of acute Pa lung infection that is highly clinically relevant. [00133] A potential strategy for targeting bacterial iron utilization that can be adapted to both heme and non-heme iron is the use of gallium as an iron mimic. Ga³⁺ mimics the charge and ionic radius of Fe³⁺ and adapts similar coordination environments but is critically redox-inert under physiological conditions and this inhibits important iron-dependent metabolic pathways. This has been shown through the use of gallium-siderophores as well as gallium heme mimics as well as recent clinical trial data repurposing the FDA-approved Ganite (Ga(NOs)3) as an antibacterial agent. In many cases, antibiotic resistance outpaces antibiotic development and the use of gallium is no exception. Reports of gallium-resistant/³, aeruginosa infections in cystic fibrosis patients suggest the potential for decreased gallium intake as well as the potential for gallium efflux when using gallium nitrate. In embodiments, effective gallium-based strategies account for this and successful formulations are likely to utilize more than one mechanism of action.

[00134] With this in mind, the development of gallium therapeutics must consider the important interplay between heme and non-heme iron-targeting pathways. The use of Gallium Salophen (GaSal) as a heme mimic targeting the extracellular hemophore, HasAp, in P. aeruginosa has previously been reported. This hemophore delivers heme to the outer-membrane receptor, HasR as part of the Has (Heme Assimilation System) system, which is primarily responsible for the sensing of exogenous heme and distinct from the higher-capacity Phu (Pseudomonas Heme Uptake) system. The ability to sense and respond to extracellular heme through the Has system is critical to survival and adaptation in the host, where both systems are highly upregulated in acute infection, and is therefore an attractive extracellular target of inhibition. In our previous work, we showed that GaSal binds HasAp and reduced the HasAp/HasR signaling cascade through increased conformational flexibility of the heme-coordinating H32 and Y75 loops. Results disclosed herein also showed that GaSal inhibits growth in a manner independent of HasR and PhuR, leading to a proposed dual mechanism wherein the GaSal-HasAp complex inhibits the heme-sensing signaling cascade and the uptake of GaSal through potential xenosiderophore receptors leads to intracellular gallium toxicity. In embodiments, this equilibrium carries several advantages in targeting both heme and siderophore-based iron acquisition in that the bacteria cannot simply switch between the systems as both mechanisms are important for iron acquisition and thus, the development of resistance will be more challenging. [00135] It has been shown that P. aeruginosa within the CF lung adapts to utilize heme and Fe²⁺, while decreasing its dependence on the major siderophore py overdine. Within the host, P. aeruginosa adapts to utilize heme through positive selection for mutations within the phuR promoter, leading to increased expression of heme transporter PhuR. Mutations in the phu promoter are concomitant with the loss of the pyoverdine biosynthesis genes. It was also confirmed that overtime, this adaption in longitudinal clinical isolates becomes more efficient at metabolizing heme, while decreasing pyoverdine production. Interestingly, studies have shown heme levels are unusually high in the lungs of CF patients because of spontaneous bleeding as a result of chronic airway inflammation. Lungs removed from CF patients who underwent transplantation contain large quantities of iron and heme, suggesting that bleeding events occur over the lifetime of the patient. Such micro-bleeds coupled with periodic pulmonary exacerbations promote P. aeruginosa infection. The ability to adapt and utilize heme as the primary iron source requires the ability to sense the extracellular environment via the has system. Indeed, the need to sense heme during in infection is supported in a murine acute lung infection model where dual RNA-seq showed upregulation of hasAp (~300-fold), hasR (~70-fold), and phuR (~30-fold) compared to P. aeruginosa in vitro. Deletion of the signaling receptor HasR (RhasR) showed significantly lower bacterial load in the murine acute lung infection compared to PAO1.

[00136] In one aspect of the disclosure, the compounds disclosed herein exhibit the dual activity of inhibiting the heme dependent CSS cascade while acting as a substrate for siderophore receptor uptake. In some embodiments, the compounds described herein target HasAp and block the ability of P. aeruginosa to sense its environment while also limiting iron availability. In some embodiments, compounds described herein are actively taken up as a xenosiderophore, and therefore the ability of P. aeruginosa to switch from heme to iron-uptake increases the potential for toxicity and dysregulation of iron homeostasis. In some embodiments, the compounds described herein target HemO. The heme sensing and uptake systems represent virulence mechanisms that can be targeted within the host, but are not essential for survival outside of the host, and therefore bacteria face less selective pressure to develop resistance. In another aspect of the disclosure, the compounds disclosed herein represent a novel formulation strategy for gallium, which was found to be an effective an iron-mimicking element. [00137] In some embodiments, the compounds described herein inhibit heme sensing and intracellular iron homeostasis, resulting in dysregulation of related metabolic and virulence pathways. In some embodiments, the compounds described herein target the extracellular hemophore HasAp and xenosiderophore receptor uptake. In some embodiments, the compounds described herein inhibit the extracellular hemophore HasAp and act as a substrate for the siderophore receptor uptake. In some embodiments, the compounds described herein inhibit both heme sensing/transport and iron uptake. In some embodiments, the compounds described herein inhibit the interaction between HasAp and HasR. In some embodiments, the compounds described herein target the extracellular hemophore HasAp and HemO.

[00138] In some embodiments, the compounds described herein inhibit HasAp protein activity. In some embodiments, the compounds described herein inhibit the P. aeruginosa siderophore iron uptake system. In some embodiments, the compounds described herein inhibit both HasAp protein activity and the /< aeruginosa siderophore iron uptake system. In some embodiments, the compounds described herein inhibit both HasAp protein activity and HemO protein activity.

[00139] The disclosure provides in one aspect a compound of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: formula (I) wherein in formula (I): each R¹ is a substituent independently selected at each occurrence from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl; each R² and R³ is a substituent independently selected at each occurrence from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl;

R⁴ is H or an optionally substituted Ci-Ce alkyl; n is an integer from 0 to 4; p is an integer from 0 to 4; and q is an integer from 0 to 4; with the proviso that when R⁴ is H, then at least one of p or q is an integer from 1 to 4; and a) when p is 1 and R² is selected from Group A, i) q cannot be 0; and ii) when q is 1, R³ cannot be identical to R²; and b) when q is 1 and R³ is selected from Group A, i) p cannot be 0; and ii) when p is 1, R² cannot be identical to R³:

Group A: independently at each occurrence selected from Br , Cl , and I .

[00140] In some embodiments, n is 0.

[00141] In some embodiments, each R² and R³ is independently -OR^b wherein each R^bis independently selected at each occurrence from the group consisting of optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylcycloalkyl, and optionally substituted alkylheteroaryl. [00142] In some embodiments, each R² and R³ is independently selected from-C₃-C6alkoxy, - ally substituted heterocyclyl, ring B is an optionally substituted aryl, each R^c is independently at each occurrence H or alkyl, each X is independently at each occurrence selected from Br , Cl , and I , and each s and v is independently at each occurrence an integer from 1 to 4.

[00143] In some embodiments, s is 1 to 3. In some embodiments, s is 2.

[00144] In some embodiments, v is 1 to 3. In some embodiments, v is 1.

[00145] In some embodiments, each occurrence of R^c is H or Ci-C₃alkyl. In some embodiments, R^c is methyl. [00147] In some embodiments, ring , wherein X is halogen and ring C is optionally substituted heterocyclyl. In some embodiments, X is Cl or F. In some embodiments, X is F. In some embodiments, ring C is morpholine.

[00148] In some embodiments, each R² is independently selected at each occurrence from independently at each occurrence selected from Br , Cl , and I .

[00149] In some embodiments, each R³ is independently selected at each occurrence from independently at each occurrence selected from Br , Cl , and I .

[00150] In some embodiments, p is 1 or 2. In some embodiments, p is 1. In some embodiments, p is 2.

[00151] In some embodiments, q is 1.

[00152] The disclosure provides in one aspect a compound of formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: wherein in formula (II):

R⁴ is H or an optionally substituted Ci-Ce alkyl; with the proviso that when R⁴ is H, then at least one of p or q is an integer from 1 to 4; and a) when p is 1 and R² is selected from Group A, i) q cannot be 0; and ii) when q is 1, R³ cannot be identical to R²; and b) when q is 1 and R³ is selected from Group A, i) p cannot be 0; and ii) when p is 1, R² cannot be identical to R³: Group A: independently at each occurrence selected from Br , Cl", and I".

[00153] The disclosure provides in one aspect a compound of formula (III), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: formula (III) wherein in formula (III):

R³ is a substituent selected from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl; and

Group A: independently at each occurrence selected from Br , Cl , and I .

[00154] In some embodiments, the compound is selected from Table A, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, wherein X is independently at each occurrence selected from Br, Cl , and I":

Table A

[00155] In some embodiments, the compound is selected from Table B, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, wherein X is independently at each occurrence selected from Br , CB,and I":

Table B

[00156] The disclosure provides in one aspect a compound, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, selected from:

[00157J In some embodiments, compounds of any one of formula (1), formula (II), formula (III), formulas 1001-1339 and 2001-2031, GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, and GaSal-15 form a salt with a counterion, as would be understood by one of ordinary skill in the art. Non-limiting examples of counterions include nitrate (NO?-).

Methods of Treatment

[00158] The compounds and compositions described herein can be used in methods for treating diseases and conditions. In some embodiments, the compounds and compositions described herein can be used in methods for treating a disease or a condition associated with inhibiting HasAp protein activity and/or inhibiting HemO protein activity and/or inhibiting the P. aeruginosa siderophore iron uptake system. In some embodiments, the compounds and compositions described herein are HasAp inhibitors. In some embodiments, the compounds and compositions described herein are /< aeruginosa siderophore iron uptake system inhibitors. In some embodiments, the compounds and compositions described herein are dual HasAp protein activity inhibitors and P. aeruginosa siderophore iron uptake system inhibitors. In some embodiments, the compounds and compositions described herein are dual HasAp protein activity inhibitors and HemO protein inhibititors. The compounds and compositions described herein may also be used in treating other diseases and conditions as described herein and in the following paragraphs.

[00159] In some embodiments, the compounds described herein are dual HasAp/P. aeruginosa siderophore iron uptake system inhibitors. In some embodiments, the compounds described herein are dual HasAp/HemO inhibitors. Compounds described herein include, but are not limited to: compounds of formula (I), formula (II), formula (III), and their features and limitations as described herein; compounds of formulas 1001-1339 and 2001-2031, and their features and limitations as described herein; compounds Gal Sal -2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, and their features and limitations as described herein.

[00160] In some embodiments, the condition is selected from sepsis, meningitis, endocarditis, osteomyelitis, otitis media, sinusitis, pneumonia, chronic respiratory tract infection, catheter infection, surgical implant infection (e.g., cardiac valve replacement, pacemaker, joint replacement (e.g., hip or knee replacement) and the like), postoperative peritonitis, postoperative biliary tract, tricuspid valve endocarditis, ecthyma gangrenosum, eyelid abscess, lacrimal cystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, orbital infection, urinary tract infection, complicated urinary tract infection, catheter infection, surgical implant infection (e.g., cardiac valve replacement, pacemakerjoint replacement (e.g., hip or knee replacement) and the like), perianal abscess, severe burns, airway burns, pressure ulcer infections, cystic fibrosis, and a bacterial infection.

[00161] In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a compound of formula (I), formula (II), formula (III), and their features and limitations as described herein; a compound of any one of formulas 1001-1339 and 2001-2031, and their features and limitations as described herein; GalSal-2, GaSal-2, GaSal- 4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, and their features and limitations as described herein.

[00162] In one aspect of the disclosure, the compounds and compositions described herein can be used for the treatment of bacterial infections. In one aspect, a method of treating or preventing a bacterial infection in a subject in need thereof is provided. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a compound of formula (I), formula (II), formula (III), and their features and limitations as described herein; a compound of any one of formulas 1001-1339 and 2001-2031, and their features and limitations as described herein; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, and their features and limitations as described herein. [00163] In one aspect, a method of reducing virulence of a bacteria in a subject is provided. In another aspect, a method of reducing pathogenicity and/or cytoxicity of a bacteria in a subject is provided. In one aspect, a method of reducing or preventing development of drug resistance in a bacteria is provided. In some embodiments, the subject is an animal. In some embodiments, the subject is a human.

[00164] In some embodiments, the bacteria is P. aeruginosa. As would be understood by one of ordinary skill in the art, any bacterial infection or condition caused by a bacterium having a similar sigma factor receptor to P. aeruginosa can also can also be treated or prevented using the compounds and compositions described herein. Non-limiting examples of bacteria having a similar sigma factor receptor to P. aeruginosa include Serratia marcescens, Bordetella pertussis, Bordetella bronchi septica, Bordetella avium, Yersinia pestis, Yersinia pseudotuberculosis, and Acinetobacter baumannii .

[00165] In another aspect of the disclosure, the compounds and compositions described herein can be used for the treatment of cancer. In one aspect, a method of treating or preventing cancer in a subject in need thereof is provided. In some embodiments, the method comprises administering to the patient a therapeutically effective amount of a compound of formula (I), formula (II), formula (III), and their features and limitations as described herein; a compound of any one of formulas 1001-1339 and 2001-2031, and their features and limitations as described herein; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, and their features and limitations as described herein.

[00166] In some embodiments, the cancer is selected from the group consisting of pancreatic cancer, breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms’ tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi’s sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin’s disease, non-Hodgkin’s lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma, and the like.

[00167] Efficacy of the methods, compounds, and combinations of compounds described herein in treating, preventing and/or managing the indicated diseases or disorders can be tested using various animal models known in the art.

Pharmaceutical Compositions

[00168] In an embodiment, the disclosure provides a pharmaceutical composition for use in the treatment of the diseases and conditions described herein.

[00169] The pharmaceutical compositions are typically formulated to provide a therapeutically effective amount of a compound of formula (I), formula (II), formula (III), and their features and limitations as described herein; a compound of any one of formulas 1001-1339 and 2001-2031, and their features and limitations as described herein; Gal Sal -2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, and their features and limitations as described herein, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, as the active ingredient. Typically, the pharmaceutical compositions also comprise one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fdlers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants.

[00170] The pharmaceutical compositions described above are preferably for use in the treatment of sepsis, meningitis, endocarditis, osteomyelitis, otitis media, sinusitis, pneumonia, chronic respiratory tract infection, catheter infection, surgical implant infection (e.g., cardiac valve replacement, pacemakerjoint replacement (e.g., hip or knee replacement) and the like), postoperative peritonitis, postoperative biliary tract, tricuspid valve endocarditis, ecthyma gangrenosum, eyelid abscess, lacrimal cystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, orbital infection, urinary tract infection, complicated urinary tract infection, catheter infection, perianal abscess, severe burns, airway bums, pressure ulcer infections, cystic fibrosis, a bacterial infection, and cancer.

[00171] In some embodiments, the concentration of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal- 15, or a pharmaceutically acceptable salt thereof, provided in the pharmaceutical compositions of the disclosure is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceutical composition.

[00172] In some embodiments, the concentration of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal- 15, or pharmaceutically acceptable salt thereof, provided in the pharmaceutical compositions of the disclosure is independently greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%,

19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%,

16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%,

13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%,

10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical composition.

[00173] In some embodiments, the concentration of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal- 15, or pharmaceutically acceptable salt thereof, provided in the pharmaceutical compositions of the disclosure is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w/w, w/v or v/v of the pharmaceutical composition.

[00174] In some embodiments, the concentration of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal- 15, or pharmaceutically acceptable salt thereof, provided in the pharmaceutical compositions of the disclosure is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v or v/v of the pharmaceutical composition.

[00175] In some embodiments, the amount of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal- 4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, provided in the pharmaceutical compositions of the disclosure is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007 g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g, 0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or 0.0001 g.

[00176] In some embodiments, the amount of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal- 4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, provided in the pharmaceutical compositions of the disclosure is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g,

0.07 g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25 g, 0.3 g, 0.35 g, 0.4 g,

0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g, 0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5 g,

3 g, 3.5, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or 10 g.

[00177] Each of the compounds provided according to the disclosure is effective over a wide dosage range. For example, in the treatment of adult humans, dosages independently ranging from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.

[00178] Described below are non-limiting pharmaceutical compositions and methods for preparing the same.

Pharmaceutical Compositions for Oral Administration

[00179] In some embodiments, the disclosure provides a pharmaceutical composition for oral administration containing: a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; Gal Sal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, and a pharmaceutical excipient suitable for administration.

[00180] In some embodiments, the disclosure provides a solid pharmaceutical composition for oral administration containing: (i) an effective amount of: a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal- 14, GaSal-15, or pharmaceutically acceptable salt thereof, and (ii) a pharmaceutical excipient suitable for administration. In some embodiments, the composition further contains (iii) an effective amount of an additional active pharmaceutical ingredient. For example, additional active pharmaceutical ingredients, as used herein, may include one or more compounds that induce cell cycle arrest and/or apoptosis in cells containing functional Mcl-1 and/or Bcl-2 proteins. Such additional active pharmaceutical ingredients may also include those compounds used for sensitizing cells to additional agent(s), such as inducers of apoptosis and/or cell cycle arrest, and chemoprotection of normal cells through the induction of cell cycle arrest prior to treatment with chemotherapeutic agents.

[00181] In some embodiments, the pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption.

[00182] Pharmaceutical compositions of the disclosure suitable for oral administration can be presented as discrete dosage forms, such as capsules, sachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, a water-in-oil liquid emulsion, powders for reconstitution, powders for oral consumptions, bottles (including powders or liquids in a bottle), orally dissolving films, lozenges, pastes, tubes, gums, and packs. Such dosage forms can be prepared by any of the methods of pharmacy, but all methods include the step of bringing the active ingredient(s) into association with the carrier, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient(s) with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. In some embodiments, the compounds of [[]] are administered as an aerosol spray. [00183] The disclosure further encompasses anhydrous pharmaceutical compositions and dosage forms since water can facilitate the degradation of some compounds. For example, water may be added (e.g., 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. Anhydrous pharmaceutical compositions and dosage forms of the disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms of the disclosure which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.

[00184] Active pharmaceutical ingredients can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.

[00185] Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, com starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.

[00186] Examples of suitable fillers for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate {e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. [00187] Disintegrants may be used in the compositions of the disclosure to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which disintegrate in the bottle. Too little may be insufficient for disintegration to occur, thus altering the rate and extent of release of the active ingredients from the dosage form. Thus, a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the compounds disclosed herein. The amount of disintegrant used may vary based upon the type of formulation and mode of administration, and may be readily discernible to those of ordinary skill in the art. About 0.5 to about 15 weight percent of disintegrant, or about 1 to about 5 weight percent of disintegrant, may be used in the pharmaceutical composition. Disintegrants that can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums or mixtures thereof.

[00188] Lubricants which can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, calcium stearate, magnesium stearate, sodium stearyl fumarate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, com oil, and soybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, or mixtures thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, silicified microcrystalline cellulose, or mixtures thereof. A lubricant can optionally be added in an amount of less than about 0.5% or less than about 1% (by weight) of the pharmaceutical composition.

[00189] When aqueous suspensions and/or elixirs are desired for oral administration, the active pharmaceutical ingredient(s) may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof. [00190] The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil. [00191] Surfactants which can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.

[00192] A suitable hydrophilic surfactant may generally have an HLB value of at least 10, while suitable lipophilic surfactants may generally have an HLB value of or less than about 10. An empirical parameter used to characterize the relative hydrophilicity and hydrophobicity of nonionic amphiphilic compounds is the hydrophilic-lipophilic balance (“HLB” value). Surfactants with lower HLB values are more lipophilic or hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have greater solubility in aqueous solutions. Hydrophilic surfactants are generally considered to be those compounds having an HLB value greater than about 10, as well as anionic, cationic, or zwitterionic compounds for which the HLB scale is not generally applicable. Similarly, lipophilic (z.e., hydrophobic) surfactants are compounds having an HLB value equal to or less than about 10. However, HLB value of a surfactant is merely a rough guide generally used to enable formulation of industrial, pharmaceutical and cosmetic emulsions.

[00193] Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acyllactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof. [00194] Within the aforementioned group, ionic surfactants include, by way of example: lecithins, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkyl sulfates; fatty acid salts; sodium docusate; acyllactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and diglycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.

[00195] Ionic surfactants may be the ionized forms of lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidyl serine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG- phosphatidylethanolamine, PVP -phosphatidylethanolamine, lactylic esters of fatty acids, stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides, mono/diacetylated tartaric acid esters of mono/diglycerides, citric acid esters of mono/diglycerides, cholyl sarcosine, caproate, caprylate, caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate, linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate, lauroyl carnitines, palmitoyl carnitines, myristoyl carnitines, and salts and mixtures thereof.

[00196] Hydrophilic non-ionic surfactants may include, but not limited to, alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, derivatives, and analogues thereof; polyoxyethylated vitamins and derivatives thereof; polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof; polyethylene glycol sorbitan fatty acid esters and hydrophilic transesterification products of a polyol with at least one member of the group consisting of triglycerides, vegetable oils, and hydrogenated vegetable oils. The polyol may be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, or a saccharide. [00197] Other hydrophilic-non-ionic surfactants include, without limitation, PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG- 15 stearate, PEG-32 distearate, PEG-40 stearate, PEG- 100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40 castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-60 com oil, PEG-6 caprate/caprylate glycerides, PEG-8 caprat e/capry late glycerides, polyglyceryl- 10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30 soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE-10 oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, tocopheryl PEG- 100 succinate, PEG-24 cholesterol, polyglyceryl- 10 oleate, Tween 40, Tween 60, sucrose monostearate, sucrose monolaurate, sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG 15-100 octyl phenol series, and poloxamers.

[00198] Suitable lipophilic surfactants include, by way of example only: fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acids esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and di-glycerides; hydrophobic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols; oilsoluble vitamins/vitamin derivatives; and mixtures thereof. Within this group, preferred lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters, and mixtures thereof, or are hydrophobic transesterification products of a polyol with at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils, and triglycerides. [00199] In an embodiment, the composition may include a solubilizer to ensure good solubilization and/or dissolution of the compound of the present disclosure and to minimize precipitation of the compound of the present disclosure. This can be especially important for compositions for non-oral use - e.g, compositions for injection. A solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

[00200] Examples of suitable solubilizers include, but are not limited to, the following: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediols and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycols having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (glycofurol) or methoxy PEG; amides and other nitrogen-containing compounds such as 2-pyrrolidone, 2-piperidone, E-caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone; esters such as ethyl propionate, tributyl citrate, acetyl triethylcitrate, acetyl tributyl citrate, tri ethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, . epsilon. - caprolactone and isomers thereof, 3-valerolactone and isomers thereof, P-butyrolactone and isomers thereof; and other solubilizers known in the art, such as dimethyl acetamide, dimethyl isosorbide, N-methyl pyrrolidones, monooctanoin, diethylene glycol monoethyl ether, and water. [00201] Mixtures of solubilizers may also be used. Examples include, but not limited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide. Particularly preferred solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol and propylene glycol.

[00202] The amount of solubilizer that can be included is not particularly limited. The amount of a given solubilizer may be limited to a bioacceptable amount, which may be readily determined by one of skill in the art. In some circumstances, it may be advantageous to include amounts of solubilizers far in excess of bioacceptable amounts, for example to maximize the concentration of the drug, with excess solubilizer removed prior to providing the composition to a patient using conventional techniques, such as distillation or evaporation. Thus, if present, the solubilizer can be in a weight ratio of 10%, 25%, 50%, 100%, or up to about 200% by weight, based on the combined weight of the drug, and other excipients. If desired, very small amounts of solubilizer may also be used, such as 5%, 2%, 1% or even less. Typically, the solubilizer may be present in an amount of about 1% to about 100%, more typically about 5% to about 25% by weight.

[00203] The composition can further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, antifoaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

[00204] In addition, an acid or a base may be incorporated into the composition to facilitate processing, to enhance stability, or for other reasons. Examples of pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, tri ethyl amine, triisopropanolamine, trimethylamine, tris(hydroxymethyl)aminomethane (TRIS) and the like. Also suitable are bases that are salts of a pharmaceutically acceptable acid, such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid, and the like. Salts of polyprotic acids, such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate can also be used. When the base is a salt, the cation can be any convenient and pharmaceutically acceptable cation, such as ammonium, alkali metals and alkaline earth metals. Example may include, but not limited to, sodium, potassium, lithium, magnesium, calcium and ammonium.

[00205] Suitable acids are pharmaceutically acceptable organic or inorganic acids. Examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, and the like. Examples of suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p- toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid and uric acid.

Pharmaceutical Compositions for Injection

[00206] In some embodiments, the disclosure provides a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal- 15, or pharmaceutically acceptable salt thereof, described herein, and a pharmaceutical excipient suitable for injection. Components and amounts of compounds in the compositions are as described herein.

[00207] The forms in which the compositions of the disclosure may be incorporated for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, com oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles.

[00208] Aqueous solutions in saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol and liquid polyethylene glycol (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, for the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.

[00209] Sterile injectable solutions are prepared by incorporating: a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031;

GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal- 12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, in the required amounts in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, certain desirable methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Pharmaceutical Compositions for Topical Delivery

[00210] In some embodiments, the disclosure provides a pharmaceutical composition for transdermal delivery containing: a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, and a pharmaceutical excipient suitable for transdermal delivery.

[00211] Compositions of the present disclosure can be formulated into preparations in solid, semi-solid, or liquid forms suitable for local or topical administration, such as gels, water soluble jellies, creams, lotions, suspensions, foams, powders, slurries, ointments, solutions, oils, pastes, suppositories, sprays, emulsions, saline solutions, dimethylsulfoxide (DMSO)-based solutions. In general, carriers with higher densities are capable of providing an area with a prolonged exposure to the active ingredients. In contrast, a solution formulation may provide more immediate exposure of the active ingredient to the chosen area.

[00212] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients, which are compounds that allow increased penetration of, or assist in the delivery of, therapeutic molecules across the stratum corneum permeability barrier of the skin. There are many of these penetration-enhancing molecules known to those trained in the art of topical formulation. Examples of such carriers and excipients include, but are not limited to, humectants (e.g., urea), glycols (e.g., propylene glycol), alcohols (e.g, ethanol), fatty acids (e.g., oleic acid), surfactants (e.g., isopropyl myristate and sodium lauryl sulfate), pyrrolidones, glycerol monolaurate, sulfoxides, terpenes (e.g, menthol), amines, amides, alkanes, alkanols, water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

[00213] Another exemplary formulation for use in the methods of the present disclosure employs transdermal delivery devices (“patches”). Such transdermal patches may be used to provide continuous or discontinuous infusion of: a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal- 15, or pharmaceutically acceptable salt thereof, described herein, in controlled amounts, either with or without another active pharmaceutical ingredient.

[00214] The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Patent Nos. 5,023,252; 4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

Pharmaceutical Compositions for Inhalation

[00215] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner. Dry powder inhalers may also be used to provide inhaled delivery of the compositions.

Other Pharmaceutical Compositions

[00216] Pharmaceutical compositions may also be prepared from compositions described herein and one or more pharmaceutically acceptable excipients suitable for sublingual, buccal, rectal, intraosseous, intraocular, intranasal, epidural, or intraspinal administration. Preparations for such pharmaceutical compositions are well-known in the art. See, e.g., Anderson, et al., eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; and Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, N.Y., 1990, each of which is incorporated by reference herein in its entirety.

[00217] Administration of: a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; Gal Sal-2, GaSal-2, GaSal-4, GaSal-6, GaSal- 7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, or a pharmaceutical composition of these compounds can be effected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, intraarterial, subcutaneous, intramuscular, intravascular, intraperitoneal or infusion), topical transdermal application), rectal administration, via local delivery by catheter or stent or through inhalation. The compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, can also be administered intraadiposally or intrathecally. In some embodiments, the compounds of [[]] are administered orally. In some embodiments, the compounds of [[]] are administered by intramuscular injection.

[00218] The compositions of the disclosure may also be delivered via an impregnated or coated device such as a stent, for example, or an artery -inserted cylindrical polymer. In some embodiments, the compounds and compositions of the disclosure can be used in conjunction with stents to treat or prevent infections and/or biofdms in the blood vessels or heart. A compound of the disclosure may be administered, for example, by local delivery from the struts of a stent, from a stent graft, from grafts, or from the cover or sheath of a stent. In some embodiments, a compound of the disclosure is admixed with a matrix. Such a matrix may be a polymeric matrix, and may serve to bond the compound to the stent. Polymeric matrices suitable for such use, include, for example, lactone-based polyesters or copolyesters such as polylactide, polycaprolactonglycolide, polyorthoesters, polyanhydrides, polyaminoacids, polysaccharides, polyphosphazenes, poly(ether-ester) copolymers (e.g., PEO-PLLA); poly dimethylsiloxane, poly(ethylene-vinylacetate), acrylate-based polymers or copolymers (e.g., polyhydroxy ethyl methylmethacrylate, polyvinyl pyrrolidinone), fluorinated polymers such as polytetrafluoroethylene and cellulose esters. Suitable matrices may be nondegrading or may degrade with time, releasing the compound or compounds. A compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal- 14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, may be applied to the surface of the stent by various methods such as dip/spin coating, spray coating, dip-coating, and/or brush-coating. The compounds may be applied in a solvent and the solvent may be allowed to evaporate, thus forming a layer of compound onto the stent. Alternatively, the compound may be located in the body of the stent or graft, for example in microchannels or micropores. When implanted, the compound diffuses out of the body of the stent to contact the arterial wall. Such stents may be prepared by dipping a stent manufactured to contain such micropores or microchannels into a solution of the compound of the disclosure in a suitable solvent, followed by evaporation of the solvent. Excess drug on the surface of the stent may be removed via an additional brief solvent wash. In yet other embodiments, compounds of the disclosure may be covalently linked to a stent or graft. A covalent linker may be used which degrades in vivo, leading to the release of the compound of the disclosure. Any bio-labile linkage may be used for such a purpose, such as ester, amide or anhydride linkages. A compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001- 2031; Gal Sal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, may additionally be administered intravascularly from a balloon used during angioplasty. Extravascular administration of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, via the pericard or via advential application of formulations of the disclosure may also be performed.

[00219] Exemplary parenteral administration forms include solutions or suspensions of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001 - 1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal- 10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.

[00220] The disclosure also provides kits. The kits a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal- 15, or pharmaceutically acceptable salt thereof, described herein, in suitable packaging, and written material that can include instructions for use, discussion of clinical studies and listing of side effects. Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. The kit may further contain another active pharmaceutical ingredient. In some embodiments, the compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, and another active pharmaceutical ingredient are provided as separate compositions in separate containers within the kit. In some embodiments, the compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal- 11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, and the agent are provided as a single composition within a container in the kit. Suitable packaging and additional articles for use (e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.

[00221] The kits described above are preferably for use in the treatment of the diseases and conditions described herein. In a preferred embodiment, the kits are for use in the treatment of conditions associated with inhibiting HasAp protein activity and/or inhibiting the P. aeruginosa siderophore iron uptake system.

[00222] In some embodiments, the kits described herein are for use in the treatment of sepsis, meningitis, endocarditis, osteomyelitis, otitis media, sinusitis, pneumonia, chronic respiratory tract infection, catheter infection, surgical implant infection (e.g., cardiac valve replacement, pacemaker, joint replacement (e.g., hip or knee replacement) and the like), postoperative peritonitis, postoperative biliary tract, tricuspid valve endocarditis, ecthyma gangrenosum, eyelid abscess, lacrimal cystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, orbital infection, urinary tract infection, complicated urinary tract infection, catheter infection, perianal abscess, severe burns, airway bums, pressure ulcer infections, cystic fibrosis, a bacterial infection, and cancer.

Dosages and Dosing Regimens

[00223] The amounts of: a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; Gal Sal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, administered will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the compounds and the discretion of the prescribing physician. However, an effective dosage of each is in the range of about 0.001 to about 100 mg per kg body weight per day, such as about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, such as about 0.05 to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect - e.g. by dividing such larger doses into several small doses for administration throughout the day. The dosage of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, may be provided in units of mg/kg of body mass or in mg/m² of body surface area.

[00224] In some embodiments, a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein is administered in multiple doses. In a preferred embodiment a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein is administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per day. Dosing may be once a month, once every two weeks, once a week, or once every other day. In other a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001- 1339 and 2001 -2031 ; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal- 10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, is administered about once per day to about 6 times per day. In some embodiments a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, is administered once daily, while in other embodiments, a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001 - 1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal- 10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein is administered twice daily, and in other a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-1 1, GaSal-12, GaSal- 14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, is administered three times daily.

[00225] Administration of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031 ; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal- 7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, may continue as long as necessary. In some embodiments, a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In some embodiments a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein is administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031 ; Gal Sal-2, GaSal-2, GaSal-4, GaSal-6, GaSal- 7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein is administered chronically on an ongoing basis - e.g., for the treatment of chronic effects. In another embodiment, the administration of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001- 2031; Gal Sal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, continues for less than about 7 days. In yet another embodiment, the administration continues for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous dosing is achieved and maintained as long as necessary.

[00226] In some embodiments, an effective dosage of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal- 15, or pharmaceutically acceptable salt thereof, described herein, is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 10 mg to about 200 mg, about 20 mg to about 150 mg, about 30 mg to about 120 mg, about 10 mg to about 90 mg, about 20 mg to about 80 mg, about 30 mg to about 70 mg, about 40 mg to about 60 mg, about 45 mg to about 55 mg, about 48 mg to about 52 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about 105 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 202 mg.

[00227] In some embodiments, an effective dosage of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal- 15, or pharmaceutically acceptable salt thereof, described herein, is in the range of about 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6 mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about 2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about 2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg to about 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kg to about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kg to about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kg to about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg to about 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kg mg to about 1.6 mg/kg, about 1 .35 mg/kg to about 1.5 mg/kg, about 2.15 mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4 mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7 mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85 mg/kg to about 2.95 mg/kg.

[00228] In some instances, dosage levels below the lower limit of the aforesaid ranges may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect - e.g., by dividing such larger doses into several small doses for administration throughout the day.

[00229] An effective amount of a compound of formula (I), formula (II), formula (III); a compound of any one of formulas 1001-1339 and 2001-2031; GalSal-2, GaSal-2, GaSal-4, GaSal-6, GaSal-7, GaSal-8, GaSal-9, GaSal-10, GaSal-11, GaSal-12, GaSal-14, GaSal-15, or pharmaceutically acceptable salt thereof, described herein, may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, or as an inhalant.

[00230] The disclosure will be further described in the following embodiments, which do not limit the scope of the disclosure described in the claims.

[00231] Embodiment 1. A compound of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: formula (I) wherein in formula (I): each R¹ is a substituent independently selected at each occurrence from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl; each R² and R³ is a substituent independently selected at each occurrence from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl; R⁴ is H or an optionally substituted Ci-Ce alkyl; n is an integer from 0 to 4; p is an integer from 0 to 4; and q is an integer from 0 to 4; with the proviso that when R⁴ is H, then at least one of p or q is an integer from 1 to 4; and (a) when p is 1 and R² is selected from Group A, (i) q cannot be 0; and (ii) when q is 1, R³ cannot be identical to R²; and (b) when q is 1 and R³ is selected from Group A, (i) p cannot be 0; and (ii) when p is 1, R² cannot be identical to R³; wherein Group A is: each occurrence selected from Br , Cl , and I .

[00232] Embodiment 2. The compound of Embodiment 1, wherein n is 0.

[00233] Embodiment 3. The compound of Embodiment 1 or 2, wherein each R² and R³ is independently -OR^b wherein each R^b is independently selected at each occurrence from the group consisting of optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylcycloalkyl, and optionally substituted alkylheteroaryl.

[00234] Embodiment 4. The compound of Embodiment 3, wherein each R² and R³ is independently selected from-Ca-Cealkoxy, -O(CH2)_sN(R^e)3⁺X , -O(CH2)_SN(R^C)2, -

, , optionally substituted aryl, each R^c is independently at each occurrence H or alkyl, each X is independently at each occurrence selected from Br , Cl", and I" , and each s and v is independently at each occurrence an integer from 1 to 4.

[00235] Embodiment 5. The compound of Embodiment 4, wherein s is 2.

[00236] Embodiment 6. The compound of Embodiment 4, wherein v is 1.

[00237] Embodiment 7. The compound of any one of Embodiments 3-6, wherein each occurrence of R^cis H or Ci-Csalkyl, optionally methyl.

[00238] Embodiment 8. The compound of any one of Embodiments 3-7, wherein ring A is

[00239] Embodiment 9. The compound of any one of Embodiments 3-7, wherein ring B is , wherein X is halogen, optionally fluorine, and ring C is optionally substituted heterocyclyl, optionally morpholine. [00240] Embodiment 10. The compound of any one of Embodiments 1-9, wherein each R² is independently selected at each occurrence from

P° , and . | «-o , wherein X is independently at each occurrence selected from

Br , Cl", and I"

[002411 Embodiment 11. The compound of any one of Embodiments 1-10, wherein each R³ is independently selected at each occurrence from P^ , wherein X is independently at each occurrence selected from

Br , Cl , and I .

[00242] Embodiment 12. The compound of any one of Embodiments 1-11, wherein p is 1 or 2. [00243] Embodiment 13. The compound any one of Embodiments 1 -12, wherein q is 1.

[00244] Embodiment 14. The compound of any one of Embodiments 1-13, wherein the compound of formula (I) is a compound of formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: formula (II) wherein in formula (II): R² and R³ are each a substituent independently selected from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl; and R⁴ is H or an optionally substituted Ci-Ce alkyl; with the proviso that when R⁴ is H, then at least one of p or q is an integer from 1 to 4; and (a) when p is 1 and R² is selected from Group A, (i) q cannot be 0; and (ii) when q is 1, R³ cannot be identical to R²; and (b) when q is 1 and R³ is selected from Group A, (i) p cannot be 0; and (ii) when p is 1, R² cannot be identical to R³:

OH , wherein X is independently at each occurrence selected from Br , Cl , and I .

[00245J Embodiment 15. The compound of any one of Embodiments 1-13, wherein the compound of formula (I) is a compound of formula (III), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: formula (III) wherein in formula (III): R³ is a substituent selected from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl; and R⁴ is H or an optionally substituted Ci-Ce alkyl; with the proviso that when R⁴ is H, then at least one of p or q is an integer from 1 to 4; and (a) when p is 1 and R² is selected from Group A, (i) q cannot be 0; and (ii) when q is 1, R³ cannot be identical to R²; and (b) when q is 1 and R³ is selected from Group A, (i) p cannot be 0; and (ii) when p is 1, R² cannot be identical to R³; wherein Group A is: each occurrence selected from Br , Cl ,

[00246] Embodiment 16. The compound of Embodiment 11, wherein the compound is of any one of formulas 1001 to 1339, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, wherein X is independently at each occurrence selected from Br, Cl", or I . [00247] Embodiment 17. The compound of Embodiment 1 or 15, wherein the compound is of any one of formulas 2001 to 2031, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, wherein X is independently at each occurrence selected from Br , Cl", or I .

[00248] Embodiment 18. The compound of Embodiment 1, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, wherein the compound is selected from:

[00249] Embodiment 19. The compound of any one of Embodiments 1-18, wherein the compound inhibits HasAp protein activity.

[00250] Embodiment 20. The compound of any one of Embodiments 1-19, wherein the compound inhibits HemO protein activity.

[00251] Embodiment 21. The compound of any one of Embodiments 1-20, wherein the compound inhibits both HasAp protein activity and HemO protein activity.

[00252] Embodiment 22. A pharmaceutical composition for treating a condition alleviated by inhibiting HasAp protein activity, the pharmaceutical composition comprising one or more compounds according to any of Embodiments 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

[00253] Embodiment 23. A pharmaceutical composition for treating a condition alleviated by inhibiting HemO protein activity, the pharmaceutical composition comprising one or more compounds according to any of Embodiments 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

[00254] Embodiment 24. A pharmaceutical composition for treating a condition alleviated by dual inhibition of HasAp protein activity and the HemO protein activity, the pharmaceutical composition comprising one or more compounds according to any of Embodiments 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

[002551 Embodiment 25. The pharmaceutical composition of any of Embodiments 22 to 24, wherein the condition is selected from sepsis, meningitis, endocarditis, osteomyelitis, otitis media, sinusitis, pneumonia, chronic respiratory tract infection, catheter infection, surgical implant infection (e.g., cardiac valve replacement, pacemaker, joint replacement (e.g., hip or knee replacement) and the like), postoperative peritonitis, postoperative biliary tract, tricuspid valve endocarditis, ecthyma gangrenosum, eyelid abscess, lacrimal cystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, orbital infection, urinary tract infection, complicated urinary tract infection, catheter infection, perianal abscess, severe bums, airway burns, pressure ulcer infections, cystic fibrosis, and a bacterial infection.

[00256] Embodiment 26. A pharmaceutical composition for treating or preventing a bacterial infection, the pharmaceutical composition comprising one or more compounds according to any of c Embodiments 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

[00257] Embodiment 27. The pharmaceutical composition of Embodiment 26, wherein the bacterial infection is caused by a bacterium selected from P. aeruginosa, Serratia marcescens, Bordetella pertussis, Bordetella bronchiseptica, Bordetella avium, Yersinia pestis, Yersinia pseudotuberculosis, Acinetobacter baumannii, and pan-resistant pneumonia, optionally wherein the bacterium is an extensively drug-resistant (XDR) bacterium.

[00258] Embodiment 28. A method of treating a condition by inhibiting HasAp protein activity in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of any of Embodiments 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

[00259] Embodiment 29. A method of treating a condition by inhibiting HemO protein activity in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of any of Embodiments 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

[00260] Embodiment 30. A method of treating a condition by dual inhibition of the HasAp protein activity and HemO protein activity in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of any of Embodiments 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

[002611 Embodiment 31. The method of any of Embodiments 28 to 30, wherein the condition is selected from sepsis, meningitis, endocarditis, osteomyelitis, otitis media, sinusitis, pneumonia, chronic respiratory tract infection, catheter infection, surgical implant infection (e.g., cardiac valve replacement, pacemaker, joint replacement (e.g., hip or knee replacement) and the like), postoperative peritonitis, postoperative biliary tract, tricuspid valve endocarditis, ecthyma gangrenosum, eyelid abscess, lacrimal cystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, orbital infection, urinary tract infection, complicated urinary tract infection, catheter infection, perianal abscess, severe burns, airway burns, pressure ulcer infections, cystic fibrosis, and a bacterial infection.

[00262] Embodiment 32. A method for treating or preventing a bacterial infection in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of any of Embodiments 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

[00263] Embodiment 33. The method of Embodiment 32, wherein the bacterial infection is caused by a bacterium selected from P. aeruginosa, Serratia marcescens, Bordetella pertussis, Bordetella bronchiseptica, Bordetella avium, Yersinia pestis, Yersinia pseudotuberculosis, Acinetobacter baumannii, and pan-resistant pneumonia, optionally wherein the bacterium is an extensively drug-resistant (XDR) bacterium.

[00264] Embodiment 34. A pharmaceutical composition for treating cancer, the pharmaceutical composition comprising one or more compounds according to any of Embodiments 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

[00265] Embodiment 35. A method for treating or preventing cancer in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of any of Embodiments 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof. EXAMPLES

[00266] The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.

Example 1: The water-soluble gallium salophen complex GaSal-2 acts as an antiPseudomonas agent by targeting bacterial heme sensing and utilization

[00267] Abstract

[00268] Pseudomonas aeruginosa is one of several opportunistic pathogens that causes lifethreatening infections in immunocompromised patients due it its hallmark resistance to antibiotics coupled with a stark lack of new options in clinical development. In infection, P. aeruginosa can utilize both labile iron stores from host iron-storage proteins as well as heme, and this dual uptake pathway further contribute to the difficulty of targeting iron and heme acquisition to therapeutic benefit. To this extent, the use of GaSal was previously reported, a gallium-based heme mimic with a mechanism that targets both extracellular heme sensing through the secreted hemophore HasAp as well as intracellular iron utilization through the gallium core. In this work, the development of GaSal is continued and report a water-soluble analog, GaSal-2 which includes an ethylene linker capped with a dimethylamino group to increase polarity and ionizability. GaSal-2 retains the activity against P. aeruginosa heme signaling and growth, with ICP-MS experiments confirming the uptake of the gallium center independent of the heme uptake pathway. Though the specific uptake receptors are yet undetermined, it was shown that intracellular GaSal-2 can target heme degradation by inhibiting HemO, leading to a decrease in the levels of the heme metabolites biliverdin IX-p/8. Further, GaSal-2 can be nebulized as an aerosol and delivered directly to the airway and lungs in mice, providing a significant protective effect in a murine acute infection model. It is envisioned that new generations of soluble GaSal analogs are useful in the treatment of P. aeruginosa and that the simultaneous targeting of both heme and iron uptake pathways lowers the chance of resistance development.

[00269] Introduction [00270] The potential for intracellular effects of GaSal or potential analogs have not been characterized. As disclosed herein, a water-soluble analog is reported, GaSal-2, and the mechanistic characterization expanded to include intracellular targets and anti-infective capability. In some embodiments, structural modifications to GaSal affect the ability of the complex to be transported into the cell or bind to HasAp and so GaSal-2 represents an investigation into such effects on signal inhibition and uptake, which are in a careful equilibrium and may have independent effects on activity. It was sought to further characterize the uptake of GaSal and GaSal-2 and whether GaSal or GaSal-2 inhibits heme oxygenase (HemO), which is critical for the breakdown of exogenous heme and use of iron, regulating heme flux into the cell and the generation of the heme metabolites biliverdin IX-p/8 (BVIX-p/8) which have their own regulatory effect on HasAp as well as potential links to virulence traits.

[00271] The development of GaSal-2 also significantly increased the solubility of such a complex, which is planar, aromatic, and hydrophobic by nature. Reports of a gallium porphyrin mimic (gallium phthalocyanine, GaPc) demonstrate the success of combining gallium and HasAp-targeting, yet such work commented on the expected insolubility of the Pc macrocycle and suggested that, since HasAp could solubilize such a ligand, the GaPc-HasAp complex could be lyophilized and used as a delivery vehicle. Instead, more soluble analogs of the hememimicking inhibitors that rely on the proposed mechanism were developed rather than hemophore-based delivery. The synthesis of GaSal-2 also includes a common intermediate that can be used to generate additional analogs which can provide an understanding of the structure/activity relationship as it pertains to HasAp binding and cellular uptake, solubility, and the potential for further functionalization. The ability to maintain affinity towards several points along the iron uptake and utilization pathways of P. aeruginosa decreases the potential for resistance as the design of such complexes is not necessarily tailored to each target but allows for activity at several targets in an intricately linked pathway. Continued characterization of these compounds is useful for the development of critically-needed therapeutic strategies and provides further insight into the importance and connections between iron/heme sensing, uptake, utilization, and trafficking pathways.

[00272] Results

[00273] Development and Synthesis of GaSal-2

[00274] Scheme 1. Synthesis ofGaSal-2

[00275] Based on the previous characterization of GaSal, analogs were developed and their mechanistic activity analyzed. As solubility is often a roadblock to medicinal chemistry campaigns, GaSal-2 was designed to include solubility-enhancing dimethylethylamine tails linked to the salicylaldehyde ring (Scheme 1). The presence of a basic tertiary nitrogen allows protonation under physiological conditions to increase polarity and solubility. The synthesis of GaSal-2 began with commercially available 2,4-dihydroxybenzaldehyde (1). Regioselective alkylation of compound 1 using recently developed conditions generated bromide 2 in good yields. Nucleophilic substitution of the bromide with dimethylamine gave tertiary amine 3 in high yields. Next, condensation of 1,2-diaminobenzene with 2 equivalents of aldehyde 3 gave ligand 4, which was allowed to react with Ga(NO3).3 to provide the final complex GaSal-2 in modest yields. GaSal -2 was readily soluble in water with a solubility > 30 mg/mL, which presents a significant advantage over the irst inhibitor which was soluble in DMSO and methanol but could be diluted into aqueous buffer.

[00276] The formation of GaSal-2 has been confirmed by small molecule X-ray crystallography (FIG. 1). The two N atoms (N15 and N22) and two O atoms (013 and 030) from the salophen ligand, along with one water O atom from both the bottom (037) and top (038) of the plane were arranged around the Ga³⁺ in an octahedral fashion.

[00277] Binding and Signal Inhibition [00278] To further validate GaSal-2 as an inhibitor with the same mechanism of action as GaSal, it was determined the HasAp binding affinity as well as the an -Pse domonas activity in shaking culture and in the use of the HasR transcriptional reporter assay previously employed to show that GaSal compounds inhibit that Has system (FIGS. 2A-2E). Through these experiments, it was found that GaSal-2 retained low micromolar binding affinity and reasonable growth inhibition while vastly improving aqueous solubility (>10 mg/mL, FIG. 2C). To confirm that the additional tails did not have a negative effect on the Has signaling cascade, a transcriptional reporter assay was employed at 3 hours based on prior understanding of this system. Consistent with GaSal, GaSal-2 also showed decreased signaling activity and confirmed the potential for later analogs based on the ethylene linker to retain the vital Has system inhibition.

[00279] Further development of such solubilizing tails is of interest, so the GaSal-2/HasAp complex by STD-NMR (FIG. 3) was tested. This technique allows regions of a ligand that interact with a protein to receiver saturation transfer from an irradiated protein to report on ligand binding epitopes. This has been used to successfully to show the binding of GaSal to HasAp in cellular supernatant and in this case, and was used to investigate the contributions of the tail to binding. The results show that the aromatic region of GaSal-2 binds to HasAp and receives saturation transfer. One of the two ethylene linker regions is weakly represented. Although not wishing to be bound by any particular theory, this result suggests that the linker does not interact strongly with HasAp or that only part of the linker interacts significantly with the protein. The terminal amine, however, appears very clearly in the STD spectrum which suggests that the end of the tails may regain some interactions with the H32 or Y75 loops that could be important to binding or, through some other mechanism. In embodiments, GaSal analogs can be designed through this strategy with a common linker intermediate and varied terminal functionalities for solubility, binding, or further conjugation to other substrates.

[00280] Non-limiting examples of compounds of the disclosure are found in FIG. 22.

[00281] Uptake Studies

[00282] Cultures were grown using PAO1 WT and mutant strains and prepared for ICP-MS in 6% nitric acid. Data represent the average and standard deviation of three independent experiments, with levels of gallium and iron normalized to dry pellet weight. A) Gallium levels. B) Iron levels. [00283] In the previous characterization of GaSal, it was reported that GaSal-HasAp is responsible for signal inhibition while GaSal is taken into the cell where it can lead to gallium toxicity. This showed that the cellular activity was independent of either heme receptor, HasR and PhuR and that pyochelin and py overdine were also likely not responsible for cellular uptake. Although not wishing to be bound by any particular theory, this result suggests that GaSal-2 is a substrate for the many xenosiderophore receptors encoded by P. aeruginosa, including those that transport siderophore-like compounds. To confirm the uptake of gallium, internal metal levels using ICP-MS were measured. The data show that in both WT and deletion strains lacking the primary heme uptake receptor PhuR that intracellular gallium is still detectable (FIG. 4A). Additionally, the structural differences in GaSal-2 do not appear to affect the gallium uptake relative to GaSal as both inhibitors show similar uptake patterns and levels. Interestingly, the use of GaSal or GaSal-2 does not appear to inhibit the acquisition of iron supplied as heme in the growth media.

[00284] GaSal Binds to HemO and Decreases Biliverdin Levels

[00285] With the confirmation that gallium is taken into the cell, it was then reasoned that GaSal-2 could bind the P. aeruginosa heme oxygenase, HemO, as the design is functionally a heme mimic. Through fluorescence quenching assays, the inhibitors show low-micromolar binding activity to HemO in vitro. Although not wishing to be bound by any particular theory, this result suggests that HemO is an intracellular target of such complexes (FIGS. 5A and 5B) and therefore a potential mechanism of intracellular toxicity. Given the experience with the development of HemO inhibitors, an LCMS-MS assay was used to detect the levels of the heme metabolite biliverdin IX [3/5 (BVIX- /5). To this effect, PAO1 was treated with heme (1 pM) and GaSal or GaSal -2 (10 pM) and analyzed the resulting BVIX levels (FIG. 5C). If the complexes bind and inhibit HemO in cells, the resultant metabolite levels should be reduced, whereas degraded complexes would not inhibit HemO. Using this assay, reduced total levels of BVIX were seen. Of note, BVIXa was below the limit of quantification and is not produced by HemO but is produced by a separate heme oxygenase, BphO that does not interact with exogenous heme. Though a reduction in average BVIX|3 levels was seen with GaSal-2, this did not achieve the typical cutoff for statistical significance but is important to note given the reductions in total levels and levels of BVIX5.

[00286] GaSal-2 Prevents Colonization in Acute Murine Infection [00287] Mice were nebulized with PBS or GaSal-2 and infected intranasally with 10⁷ CFU of WT PAO1. Bacterial burden was determined in the nares and lung 24 h post infection and data represented are the CFU detected in the total sample volume (1 mL PBS nasal wash or lung homogenate) and represent individual mice. Data were analyzed by two-tailed t-test using GraphPad Prism (*”, p<0.0005; ****, p<0.00005).

[00288] Mechanistic characterization is important for the understanding of therapeutic targets but can often be limited in the determination of efficacy when confined to simple shaking culture or in vitro experiments. The limited solubility of GaSal in aqueous buffer hindered further development in animal models. The development of GaSal-2 therefore proved a vital step in continued characterization of this class of compounds using a mouse infection model. In this experiment, mice were prophylactically dosed with nebulized with PBS or GaSal-2 to deliver the compound to the site of infection, similar to the inhalation of tobramycin as a therapeutic, rather than rely on a typical injection. To ensure consistent bacterial loads, an acute infection model was used, and mice were subjected to a dose of 2 x 10⁷ CFU one hour after nebulization. Mice were euthanized after 24 hours and bacterial burden was analyzed in both nasal washes and lung homogenate. As shown, GaSal-2 provides a clear protective effect, similar to the decreased colonization observed in a PAO1 HasR deletion strain previously studied (FIGS. 6A-6D).

[00289] Binding modes studies of GaSal-2 to HasAp and HemO

[00290] The CADD method SILCS was applied to both HasAp and HemO (FIG. 7A-7B), to direct new compound design and selection. Using SILCS, the 3D functionality probability distribution maps (termed FragMaps) are obtained, which identify surface regions where different types of functional groups have favorable interactions. SILCS allows for 1) qualitative analysis of the binding sites of a protein target to drive the design of synthetically accessible structure modifications and 2) quantitative predictions of changes in binding affinity using LGFE (ligand grid free energy). As shown in FIG. 7A, the binding mode of GaSal-2 in HasAp indicated that the gallium salophen core fit into the heme binding site. The gallium ion was sitting at the position of the heme iron forming favorable interactions with the key heme-binding residues His32 and Tyr75. The middle phenyl group of GaSal-2 fit snugly into the deep hydrophobic pocket formed by residues Phe46, Tyr56, Val85 and Vai 137. One of the flexible amino arms was mostly solvent exposed although it is in proximity to the acidic residue Asp39 for a potential charge-charge interaction. The other amino arm binds into a narrow channel that opens to the back site of the protein, with the phenolic oxygen atom close to the sidechain of Tyrl38 and the amino tail in the range of an H-bonding to the residue Argl29. On the other hand, the binding mode of GaSal-2 in HemO indicated that the gallium salophen core bind to the heme-binding active site of the enzyme (FIG. 7B), with the middle phenyl ring fit into the deep hydrophobic pocket formed by three phenylalanine residues F48, F55, and F189. The two arms extruded outside of the heme-binding pocket. One of the amino groups locates closely to residues N19 and W158. The phenolic oxygen atom of the other arm was in close proximity to residue Lysl32 that is used to form key ionic interaction with one of the propanoic tails of heme. At the same time, the amino group of this arm was in close proximity to Glu30. This binding mode of GaSal- 2 indicated that modification of its arms probably won’t impair the binding of new compounds to HemO.

[00291] Discussion

[00292] The previous characterization of GaSal demonstrated the potential for targeting several points along the iron and heme acquisition pathways of P. aeruginosa. In structural studies, it was determined that a more conformationally-flexible H32 and Y75 loop, as determined by HDX-MS, likely prevented ligand release from HasAp and subsequent activation of the signaling cascade. In the design of GaSal-2, the inclusion of solubilizing tails maintains the core salophen scaffold critical for binding in the heme site and provides handles for potential derivatization. This Examples shows that the structural modifications of GaSal to yield GaSal-2 have minimal impact on the reduction in HasAp signaling. Although not wishing to be bound by any particular theory, this result suggests that the tails do not interact with HasAp or HasR in such a way that restores the conformation needed to fully activate the HasR signaling cascade. Instead, it was observed that the linker region has minimal interaction with HasAp relative to the aromatic region (as expected based on the contribution of hydrophobic interactions to binding) even though the methyl groups appear strongly in the STD experiment. This result suggests that subsequent design can use the ethylene linker with varied end groups to probe structure-activity relationships as well as potential conjugation to other targeting moi eties or further functionalization. Wherein a dimethylamino group was used, the inclusion of a quaternary ammine could retain the solubility of such complexes by providing a permanent positive charge as well as act as a flexible tail with a mechanism of action similar to other quaternary ammonium antimicrobial agents that disrupt membranes. [00293] It was previously hypothesized that the effect of GaSal was independent of either HasR or PhuR and so an alternative mechanism was proposed for uptake wherein GaSal could be transported as a potential xenosiderophore. Though a specific receptor for this transport has yet to be identified, P. aeruginosa encodes a suite of several receptors used for siderophore piracy in co-infection. These receptors are not only able to transport xenosiderophores, but show some preferential uptake of siderophore-like catecholate compounds. In this study, it was shown that gallium is transported into the cell and that it is detectable with similar levels and patterns independent of the heme uptake receptor PhuR. As the proposed mechanism involves the equilibrium between GaSal-HasAp formation (responsible for signal inhibition) and uptake (intracellular toxicity), modifications to GaSal, such as those in GaSal-2, may shift the equilibrium if they favor HasAp binding or affect the ability to be transported as a siderophore substrate. It was observed that GaSal-2 does not have significantly different uptake results compared to GaSal, indicating that the equilibrium is preserved. Additionally, a decrease in intracellular iron was not seen when GaSal or GaSal-2 were added to cultures. The uptake of GaSal as a metal chelate is unlikely to inhibit heme acquisition through HasR and PhuR. These data suggest that GaSal is not necessarily a direct inhibitor of heme uptake and that intracellular mechanisms of toxicity lie with the disruption of iron utilization whereas the GaSal-HasAp complex inhibits extracellular heme sensing but not heme uptake. Taken together, these results suggest that GaSal and analogs dampen the ability of P. aeruginosa to sense and utilize extracellular heme but does not dampen acquisition. This may be beneficial, as the direct inhibition of iron uptake may cause an iron-starvation response which is typically linked to the upregulation of virulence factors. Instead, this approach likely dampens the infectious capabilities of P. aeruginosa, which is a highly desirable approach.

[00294] The use of ICP-MS also is limited to the detection of the metal itself and not metal complexes. Though formation of GaSal-HasAp complexes was shown previously, evidence had not yet been reported for the stability and uptake of GaSal or more recently, GaSal-2 as an in-tact complex. The use of the LCMS/MS assay therefore shows the effect of these complexes on heme degradation. GaSal or GaSal-2 can bind to HemO in vitro as they are similar to heme, and thus have the potential to inhibit HemO activity, which is another target under investigation in the lab. If the complex were degraded and only gallium enters, then no effect on HemO would be observed and similar levels of BVIX would be expected. It was seen that GaSal and GaSal -2 reduced metabolite levels by inhibiting HemO. Together, these results further demonstrate the dual mechanisms wherein HasAp is an extracellular target for the inhibition of heme sensing and HemO is an intracellular target for the inhibition of heme utilization. There is not yet a direct readout of HasAp-mediated toxicity caused by signal inhibition, the lack of the HasR receptor and subsequent reduction in acute infection colonization suggests its importance for the establishment of infection.

[00295] Mechanistic analysis in laboratory culture, is often impacted by growth conditions and does not always reflect realistic biological results. Given understanding of the importance of heme sensing to infection and the increased knowledge of potential GaSal targets, an acute infection model is more representative than a growth-inhibition experiment. While the murine infection model still has limitations, and GaSal-2 was dosed prophylactically, the reduction in bacterial load observed. These results also highlight the ability of soluble GaSal analogs to be delivered through aerosols, which could be a useful method of direct delivery to respiratory infections in which P. aeruginosa is often found. In this model, it also suggests that with an abundance of GaSal at the site of the infection, the heme signaling cascade is significantly disrupted and the infection has less of a chance to take hold, which is harder to recapitulate in cell culture and further shows potential of such compounds especially at the early stages on infection. The use of GaSal-2 in these studies also highlights the importance of solubility, as initial tests with GaSal needed the aid of a DMSO/PEG300 vehicle rather than nebulization in PBS buffer.

[00296] The mechanisms of GaSal and GaSal-2 are expanded by these data, which previously reported the characterization of the GaSal-HasAp complex and its effect on heme signaling. Here, the analysis was continued on an intracellular level and propose additional points along the iron/heme uptake and utilization pathways in P. aeruginosa. The use of such a strategy with the potential to interfere with several important processes in an extended pathway is beneficial as a therapeutic strategy as it complicates the evolution of resistance in each individual target, as well as both iron and heme uptake pathways.

[00297] Conclusion

[00298] This characterization builds on the previous report that GaSal inhibits heme sensing and can also be taken into the cell to afford additional mechanisms of toxicity. This example demonstrates intracellular effects by showing the uptake of gallium and iron via ICP-MS as well as the inhibition of HemO. The design of GaSal analogs successfully produced a water-soluble compound, which showed preliminary success in acute murine infection. These results demonstrate the use of the disclosed metallotherepeautics for interfering with several levels of P. aeruginosa iron and heme trafficking. In embodiments, compounds of the disclosure can be used to probe binding affinity to HasAp and increase potency, although HasAp binding affinity may not be as critical a factor given the equilibrium between signaling inhibition and uptake. In a non-limiting example, the synthetic strategy can provide a common linker with varied ends, which also allows for expedient generation of analogs for various purposes such as conjugation with various targeting moieties, solubility enhancers, and asymmetrical salophen construction. Further characterization of uptake as well as complex stability and the fate of the gallium core is also of significant interest to thestudies and the elucidation of such pathways is under development.

[00299] Methods

[00300] Pseudomonas aeruginosa Strains and Growth Protocol

[00301] Pseudomonas aeruginosa (PAO 1 and mutants) strains were stored as glycerol stocks in LB at -80 °C and were freshly streaked on Pseudomonas isolation agar (BD Biosciences) before transferring to liquid culture medium. PAO1 (wild type) was used as reported The PAO1 lacZ fusion was constructed as described previously. The heme receptor deletion strains (PAO1 AhasR, PAO Ap/mR) were constructed as reported.

[00302] For subsequent experiments, strains were cultured overnight from a single colony in 5 mL LB broth with shaking with shaking at 37 °C. Cells were harvested by centrifugation and resuspended in M9 media and used to inoculate fresh M9 minimal medium at Aeoo = 0.05 and then cultured for 3 h to deplete bacterial iron stores. Supplements were added as described below for each experiment.

[00303] Expression and Purification of WT apo-HasAp

[00304] WT, full-length HasAp was prepared from freshly transformed E. coli BL21(DE3) competent cells as previously described in 4 L of M9 media. Cells pellets were resuspended and lysed with an LM-20 microfluidizer at 18,000 psi. Lysate was clarified by centrifugation and applied to a Q-Sepharose column. HasAp was eluted over a gradient from 20 to 600 mM NaCl in 20 mM Tris-HCl (pH 7.5) and desired fractions as determined by SDS-PAGE were pooled. Apo- HasAp was further on a Butyl Sepharose Fast Flow column equilibrated with 50 mM sodium phosphate buffer with 0.7 M ammonium sulfate (pH 7.0). Holo-HasAp and other proteins eluted within 2-3 bed volumes of 50 mM sodium phosphate containing 0.5 M ammonium sulfate. Apo- HasAp was then eluted with a linear gradient of sodium phosphate buffer (50 to 20 mM) containing ammonium sulfate (0.5 to 0 M). Fractions were once again analyzed by SDS-PAGE and the apo-HasAp was pooled, concentrated (Amicon stirred cell with Ultracel 10 kDa filter) and exchanged into 20 mM sodium phosphate buffer.

[00305] Expression and Purification of WT HemO

[00306] HemO was expressed and purified as previously described from freshly transformed cells in LB broth. Lysate containing HemO protein was purified by Q-Sepharose Fast Flow chromatography as described with HasAp. Protein was eluted with a 20 mM Tris (pH 8.0 at 4 °C), 100 to 500 mM NaCl gradient. Protein fractions were monitored by SDS-PAGE analysis, and the fractions were further pooled and purified using a Sephadex 200 column equilibrated and eluted with 20 mM Tris buffer (pH 8).

[00307] Chemical Synthesis

[00308] All the reagents and solvents were purchased from commercial sources and used as received, unless otherwise stated. The ¹ H and ¹³C NMR spectra were obtained in DMSC , on a 400 MHz spectrometer with chemical shifts referenced to tetramethyl silane (TMS). Purity (>95%, HPLC) was confirmed by HPLC as only singular peaks were detected.

[00309] Synthesis of GaSalophen Nitrate. GaSal was synthesized as previously described. To a solution of metal-free salophen (5.269 g, 16.8 mmol) in ethanol (50 mL) was added gallium (III) nitrate hydrate (4.73 g, 18.5 mmol). The reaction mixture was refluxed for 3 h and cooled to room temperature. The final product was collected by filtration and washed with ethanol and diethyl ether to afford yellow crystals (2.58 g, 33%). ¹ H NMR (400 MHz, DMSOt/e): 5 9.37 (s, 2 H), 8.16-8.13 (m, 2 H), 7.68-7.66 (dd, 2 H), 7.66-7.51 (m, 4 H), 7.02-6.99 (d, 2 H), 6.90-6.86 (t, 2 H). ¹³C NMR (400 MHz, DMS< ): 8 167.9, 162.9, 137, 136.6, 135.4, 129, 122.3, 118.3, 117.1, 117. HRMS (ESI) m/z: [M -NO₃]⁺ Calc’d for C2oHi₄N₂02Ga 383.0311; Found 383.0301 [00310] Synthesis of GaSal-2 Nitrate 4-(2-(dimethylamino)ethoxy)-2-hydroxybenzaldehyde (3). To a solution of compound 2 (732 mg, 3.0 mmol) in anhydrous THF (3.0 mL) was added dimethylamine (2 M solution in THF, 4.5 mL, 9.0 mmol). The reaction mixture was heated at 50 °C for 16 h, then cooled down to room temperature and concentrated by rotary evaporator. The crude product was purified using flash chromatography (5% MeOH in DCM) to give compound 3 as a white solid (500 mg, 2.5 mmol, 83%): 'H-NMR (400 MHz, CDC1₃) 8 11.48 (br s, 1H), 9.72 (s, 1H), 7.43-7.42 (d, J= 6.4 Hz, 1H), 6.60-6.50 (dd, J= 1.2, 6.4 Hz, 1H), 6.45-6.44 (d, J = 1.2 Hz, 1H), 4.13-4.10 (t, J= 4.0 Hz, 2H), 2.77-2.75 (t, J= 4.0 Hz, 2H), 2.34 (s, 6H); 'H-NMR (400 MHz, DMSOde) 8 11.00-10.00 (br s, 1H), 10.00 (s, 1H), 7.62-7.60 (d, J= 6.8 Hz, 1H), 6.60-6.50 (dd, .7= 1.2, 6.8 Hz, 1H), 6.49-6.48 (d, J= 1.2 Hz, 1H), 4.13-4.10 (t, .7= 4,0 Hz, 2H), 2.65-2.62 (t, J= 4.0 Hz, 2H), 2.22 (s, 6H); ¹³C-NMR (100 MHz, DMSCH) 8 191.1, 165.2, 163.1, 132.2, 116.2, 107.7, 101.3, 66.2, 57.3, 45.4.

[00311] GaSal-2. To a solution of compound 3 (105 mg, 0.5 mmol) in MeOH (10 mL) was added 1,2-diaminobenzene (54 mg, 0.5 mmol). The resulting solution was stirred at 80 °C for 1 h, then cooled down to room temperature. To the orange-colored solution (4) was added gallium (III) nitrate hydrate (200 mg, 0.5 mmol). The reaction mixture was heated at 60 °C for an additional 3 h. After cooled to the room temperature, The resulting yellowish red solution was added to cold ether (200 mL) with vigorous stirring. The yellow precipitate was collected by vacuum fdtration, washed with ether (30 mL), and dried under vacuum to give GaSal-2 as a yellow powder (334 mg, 54%): 'H-NMR (400 MHz, DMSC ) 8 9.80-9.50 (br s, 2H), 9.24 (s, 2H), 8.07-8.04 (dd, J= 3.2, 5.6 Hz, 2H), 7.60-7.50 (d, J= 8.8 Hz, 2H), 7.50-7.40 (dd, J= 3.2, 5.6 Hz, 2H), 6.65-6.55 (d, J= 8.8 Hz, 2H), 6.53 (s, 2H), 4.41 (br s, 4H), 3.54 (br s, 4H), 2.89 (s, 12H); ¹³C-NMR (100 MHz, DMS( ) 8 170.1, 164.9, 160.8, 138.1, 135.3, 128.1, 116.4, 113.3, 107.0, 105.0, 62.7, 55.5, 43.1.

[00312] Crystallography

[00313] A clear yellow needle crystal of GaSal-2 with dimensions 0.243 x 0.060 x 0.040 mm was mounted on a MiteGen MicroMesh using a small amount of Cargille Immersion Oil. Data were collected on a Bruker three-circle platform diffractometer equipped with a PHOTON II CP AD detector. The crystals were irradiated using a Ips microfocus CuKa source ( = 1.54178) with Montel optics. Data was collected at room temperature (20°C). Data collection and refinement parameters are listed in the Supplementary Information.

[00314] Preparation ofHasAp Complexes

[00315] Apo-HasAp was prepared at a desired concentration in 20 mM sodium phosphate buffer (pH 7.4, 25 °C). To this solution was added a 3-fold molar excess of ligand prepared in the same buffer solution. Excess unbound ligand was removed using 7K MWCO centrifugal desalting columns (Zeba™ Spin, Thermo Fisher Scientific) according to manufacturer’s recommendati ons .

[00316] Binding A ffinity Determination by Fluorescence Quenching

[00317] Binding affinities were determined on an ISS K2 multifrequency fluorometer in Informal using 1 cm quartz cuvettes. To a 1 pM solution of apo-HasAp or HemO in 20 mM sodium phosphate buffer (pH 7.4, 25 °C) was titrated ligand of interest. Emission spectra were recorded (300-500 nm) following excitation at 295 nm. The decrease in maximum emission was plotted against the ligand concentration (accounting for dilution) and fit to one-site binding using GraphPad Prism 9.

[00318] Saturation Transfer Difference NMR

[00319] STD experiments were performed as described previously, with a 600 final volume containing 1 mM GaSal-2 and 10 pM HasAp (as a negative control, a protein-free sample was used) in D2O. Experiments were performed at 25 °C on an Agilent DD2 500 MHz spectrometer. [00320] Growth Inhibition Assays

[00321] After culturing as described above, cells were plated in three wells per condition on 96- well plates (Costar clear, flat-bottom with lid, Corning Inc) at Aeoo ~ 0.05 to a final volume of 200 pL in M9 medium containing 1 pM heme. Inhibitors (2 pL) were added as 100* stock solutions. Growth curves were recorded at 600 nm with a Biotek Synergy™ HT plate reader over 16 h of growth with shaking at 37 °C and technical replicates were averaged per condition. IC50 values were determined by plotting the final ODeoo value as a function of inhibitor concentration and analyzing by nonlinear regression in GraphPad Prism 9. Data presented are the average of at least three independent experiments.

[00322] Transcriptional Reporter Assays

[00323] The chromosomal fusion hasR-lacZ was previously constructed and used to report on transcriptional activation of the Has signaling cascade. After the described growth protocol, cultures were supplemented with 1 pM of either holo-HasAp, GaSal-HasAp, or GaSal2-HasAp. Aliquots (1 mL) were collected at 3 hours post supplementation and assayed for />-gal activity as described previously. Data represent the average of three or more independent experiments.

[00324] ICP-MS Uptake Studies [00325] After iron starvation as described, heme and inhibitors were added at 1 and 10 pM, respectively, and the cultures were incubated for an additional 6 h. For each time point, the ODeoo was recorded and 2 mL of culture was collected for analysis.

[00326] Each sample was pelleted and washed with fresh M9 media. Dried pellet weights were recorded for normalization and the pellets were then dissolved in 200 pL of trace metal-free 67% nitric acid and incubated at room temperature for 2-3 days. Samples were added to 2 mL of ultrapure water to a final nitric acid concentration of 6.7% and analyzed by on an Agilent 7700 ICP-MS (Agilent Technologies). ICP-MS runs were calibrated with high-purity iron and gallium standard solutions (Sigma- Aldrich), and values were corrected for drift using values for scandium and germanium (CPI International) as internal standards and added to samples during processing. Corrected values were then normalized to cell pellet weights. Reported values represent the average of three independent experiments.

[00327] Biliverdin Extraction and Analysis by LC-MS/MS

[00328] Cultures were grown as described above. Heme and inhibitors were added at 1 and 10 pM, respectively, and the cultures were incubated for an additional 4 h. Cells were harvested by centrifugation in 50 mL conical tubes at 6000 rpm for 20 minutes at 4 °C. Supernatants were collected and fdtered through 0.22 pm syringe fdters and stored at -80 °C until extraction.

[00329] Biliverdin was extracted and analyzed as previously reported. Briefly, supernatants were supplemented with dimethyl ester BVIXa as an internal standard (10 ng/mL final concentration), acidified to pH 2.5 with 10% TFA, and loaded over a Cl 8 Sep-Pak column (Waters) equilibrated with 2 mL each of acetonitrile (ACN), H2O, 0.1% TFA in H2O, and methanol (MeOH) in 0.1% TFA (10:90). After sample application, the column was washed with 4 mL of 0.1% TFA, 4 mL of an ACN/0.1% TFA mixture (20:80), and 2 mL of a MeOH/0.1% TFA mixture (50:50) and eluted with 1 mL of MeOH. Purified BVIX samples were dried and stored up to 1 week at -80 °C prior to LC-MS/MS analysis.

[00330] For analysis, samples were resuspended in of DMSO (10 pL), diluted to 100 pL with a mobile phase of ACN and H2O [50:50 (v/v)], and centrifuged at 14000 rpm for 5 min at 4 °C. BVIX isomers (2 pL) were separated on an Ascentis RP-amide 2.7 mm C18 column (10 cm x 2.1 mm) at a flow rate of 0.4 mL/ min and analyzed by LC-MS/MS (Waters TQ-XS triple quadrupole mass spectrometer with an AQUITY H-Class UPLC instrument). The mobile phase consisted of solvent A (H2O and 0.1% formic acid) and solvent B (ACN and 0.1% formic acid). The initial gradient is 64% A and 36% B and then 5 min at 55% A and 45% B, 8 min at 40% A and 60% B, 8.5 min at 5% A and 95% B, and 10 min at 64% A and 36% B. Using multiplereaction monitoring (MRM), fragmentation patterns of the precursor ions were detected at 583.21 (BVIX). The source temperature was set to 150 °C, the capillary voltage to 3.60 kV, and the cone voltage to 43 V. The column was kept at 30 °C during separation. The precursor ions used in MRM for BVIXa,-P, and -5 isomers were 297.1, 343.1, and 402.2 with collision energies of 38, 36, and 30 V, respectively. Analysis was performed by converting the BVIX ion intensities to concentrations via a standard curve, normalizing to the ODeoo and correcting for BVIX extraction efficiency (average = 27%) using dimethyl ester BVIXa ion intensities. Data represent the average of three independent experiments.

[00331] Murine Challenge Model

[00332] PAOlwas grown overnight from frozen stock on Lysogeny Agar (LA; Miller formulation) at 37°C. A single colony was picked and used to start 3 mL cultures in Lysogeny Broth (LB; Miller formulation) that were incubated overnight at 37 °C under constant shaking. The culture was then diluted 1 : 100 in LB and incubated for 4-5 hours at 37 °C under constant shaking to reach exponential phase. Bacteria were then washed by centrifugation at 5000 x g for 3 min and resuspension in phosphate buffered saline (PBS). The infectious dose was adjusted to 2 xlO⁷ colony forming units (CFU)/20pL using optical density (ODeoo; SpectraMax i3 and Cerillo Stratus plate readers).

[00333] Five weeks-old outbred CD-I mice (Charles River) were placed in a mouse restrainer and submitted to nebulization using an Aeroneb delivery system (Kent Scientific). GaSal-2 was prepared at a concentration of lOmg/ml. A total of lOOyl of either GaSal-2 (approximate dose of 50mg/kg) or PBS were then nebulized and delivered over the course of 20s. One-hour post- nebulization, mice were anesthetized by intraperitoneal administration of ketamine (77 mg/kg) (Patterson Veterinary #07-803-6637) and xylazine (7.7 mg/kg) (Patterson Veterinary #07-808- 1939) in 0.9% saline, as described previously. Mice were then infected by intranasal administration of 20uL of bacterial suspension containing 2 xlO⁷ CFUs and allowed to recover from anesthesia. Fourteen hours post-challenge, mice were euthanized by intraperitoneal injection of Euthasol® (390 mg pentobarbital/kg; Patterson Veterinary #07-805-9296) in 0.9% NaCl. Immediately following euthanasia, body temperature was recorded using a rectal probe thermometer (Kent Scientific). Lung were aseptically removed, weighted, and homogenized using a Polytron PT 2500 E homogenizer (Kinematica) in 1 mL of sterile PBS. Tn addition, nasal wash was collected by flushing 1 mL of sterile PBS through the nasal cavity. Bacterial burden in the lung and the nares were quantified by serial dilutions and plating on Pseudomonas Isolation Agar.

[00334] Abbreviations Used: ACN, acetonitrile, BVIX, biliverdin IX; GaSal, gallium salophen; Has, heme assimilation system; LC-MS/MS, liquid chromatography with tandem mass spectrometry; MRM, multiple reaction monitoring; Phu, Pseudomonas heme uptake; PPIX, protoporphyrin IX; STD-NMR, saturation transfer difference NMR; TFA, trifluoroacetic acid

Example 2: Therapeutic strategies to target Gram-negative MDR Pa Infections

[00335] GaSal complexes simultaneously inhibit HasAp and HemO, while functioning as siderophore mimics. The gallium-salophen complex GaSal were characterized as antipseudomonal agents (FIG. 8A-8B). Similar to the iron analog FeSal, GaSal binds to HasAp with a KD value of 1.2 pM (KD of heme to HasAp was 0.4 pM). Interestingly, different from FeSal which supported Pa growth, GaSal showed antipseudomonal activity (IC50 = 9.7 pM). Further growth studies with a Pa hasR phuR strain using FeSal demonstrated that growth was independent of uptake by the heme receptors, suggesting FeSal is actively taken up as a xenosiderophore, by xenosiderophore receptors such as PfuA and PiuA. GaSal, upon uptake via a similar mechanism, is toxic to the cell. The recent results show GaSal also binds to HemO with a low micromolar affinity (FIG. 8A). Although GaSal provides an exciting multi-targeting scaffold for the Pa heme sensing, uptake, and iron acquisition system, its further in vivo evaluation was prohibited by its limited solubility (< 0.1 mg/mL in H2O).

[00336] GaSal-2 is an improved lead: GaSal-2 is a dimethylaminoethyl ether analog of GaSal (FIG..8B). It has not only maintained similar KD and IC50 values as those of the parent GaSal, but also indicated excellent aqueous solubility (> 50 mg/mL in H2O). The structure of GaSal-2 has been confirmed by small molecule X-ray crystallography (FIG. 8B). GaSal-2 was found stable in deuterated PBS buffer (pH values of 6, 7, and 8) for at least one month confirmed by 'H NMR.

[00337] GaSal-2 inhibits transcriptional activation of the has signaling cascade: It was previously reported that GaSal inhibited the transcriptional activation of the has operon relative to heme or GaPPIX in Pa WT, suggesting the scaffold is key for blocking the CSS cascade. To confirm that GaSal-2 could also inhibit the same CSS cascade, p-galactosidase transcriptional reporter assays were perfomed in the presence of compound GaSal-2 in comparison with the parent GaSal (FIG. 9). Consistent with GaSal, GaSal-2 also showed decreased activation of the signaling cascade. These results demonstrate that additional GaSal-2 analogs based on a similar chemical scaffold can retain inhibition of the signaling system.

[00338] GaSal-2 is actively taken up into the cell: To confirm the uptake of Ga, the intracellular metal levels were measured using ICP-MS (FIGS. 10A-10B). In both WT and the Pa AphuR strains, Ga was detectable (FIG. 10A). The structural differences in GaSal -2 and GaSal did not affect the uptake as both complexes showed similar Ga levels. Importantly, the use of GaSal-2 or GaSal does not inhibit iron acquisition from heme (FIG.10B). These results confirm that the GaSal complexes target heme and iron uptake through iron-dysregulation rather than starvation. Thus, the treatment of Pa with GaSal analogs would not activate iron-regulated virulence factors that lead to drug resistance.

[00339] GaSal-2 decreases BVIXf/5 formation by inhibiting HemO : A cellular LC-

MS/MS assay was reported to follow the inhibition of HemO by detecting the levels of the heme metabolite BVIX-p/8. As shown in Fig.11, the PAO1 strain was treated with heme (1 pM) with or without compounds GaSal (10 pM) or GaSal-2 (10 pM). Treatments of both GaSal and GaSal-2 led to decreased levels of BVIX0 and BVIX5, indicating that the complexes are taken up into the cell and inhibit the heme-degrading enzyme HemO. The BVIXa levels were below the quantification limit as they are produced by a second heme oxygenase BphO, that does not utilize exogenous heme.

[00340] GaSal-2 shows in vivo efficacy in an acute murine lung infection: As GaSal is relatively insoluble, the a series of GaSal analogs with increased solubility were synthesized. Specifically, the analog GaSal-2 was shown to have significantly increased solubility with a similar binding affinity and cellular activity as the parent GaSal (FIGS. 6A-6D and 8A-11C). The antipseudomonal efficacy was further tested of GaSal-2 in an acute murine lung infection model (FIGS. 6A-6D). The drug was delivered to the lungs of the animals via nebulization. It was successfully demonstrated that the treatment of infected animals with GaSal-2 can effectively decrease the bacterial burden in both the lungs (FIG. 6A) and nasal wash (FIG. 6B). Moreover, following treatment, the animals did not show any significant drop in their lung weight (FIG. 6C) and maintained normal body temperature (FIG. 6D). Furthermore, GaSal-2 is well-tolerated in the mice with a dose as high as 500 mg/kg. In sum, GaSal-2 represents a compound useful for further design and development of new classes of inhibitors.

[00341] The GaSal-HasAp binds to HasR with a similar affinity as holo-HasAp and inhibits heme sensing/uptake. The formation of the GaSal-HasAp complex was confirmed by STD NMR. It was also confirmed that the GaSal-HasAp and holo-HasAp bind to HasR with similar affinities using surface plasmon resonance (SPR). Utilizing the cellular assays outlined in FIGS. 11 A-l IB, it was also shown that that GaSal-HasAp inhibits activation of the HasAp-HasR CSS signaling cascade and decreases the production of BVIX0. To determine if the decreased transcriptional activation is the result of conformational differences between the GaSal- and holo-HasAp, the apo-, holo- and GaSal-HasAp complexes were analyzed by HDX-MS (FIGS. 12A-12C). Both heme and GaSal binding to HasAp resulted in significant protection from deuteration in the H32 and Y75 loops as well as the heme-binding site ([3-sheets 1, 2, 3, and 5), consistent with loop closure and decreased solvent exposure upon ligand binding (FIG. 12A). However, the H32 and Y75 loops showed significant differences between the two ligand-bound complexes. GaSal binding increased protection from deuteration on the H32 loop relative to the apo form, but not as strongly as heme binding most likely due to the decreased interaction of the smaller scaffold with the H32 loop backbone (FIG. 12B). To assess if this observation carried a thermodynamic consequence, the melting temperatures of the four different HasAp complexes were measured by circular dichroism (FIG. 12C). All HasAp complexes showed similar CD spectra at 25°C, indicating that the overall secondary structure is not significantly different upon ligand binding. However, the thermal stability of apo-HasAp (58°C) was increased significantly on heme binding (>90°C). In contrast, while the FeSal- (69°C) and GaSal-HasAp (66°C) showed an increase in thermal stability compared to apo-HasAp, this was far less than that on heme binding. Taken together the HDX-MS and CD results indicate significant differences between the holo- and Fe/GaSal-HasAp complexes that were not reflected in the X-ray crystal structure. The differences in flexibility of GaSal-HasAp may have consequences beyond the inability to trigger heme signaling and uptake, where the decreased stability of GaSal-HasAp may increase susceptibility to proteolysis within the host.

[00342] New GaSal-2 analogs have been designed and prioritized by a novel CADD method SILCS. CADD SILCS characterization of HasAp and HemO: The CADD method was applied SILCS to both HasAp and HemO (FIG. 13A-13B), to direct new compound design and selection. Using SILCS, the 3D functionality probability distribution maps (termed FragMaps) are obtained, which identify surface regions where different types of functional groups have favorable interactions. SILCS allows for 1) qualitative analysis of the binding sites of a protein target to drive the design of synthetically accessible structure modifications and 2) quantitative predictions of changes in binding affinity using LGFE (ligand grid free energy). As shown in FIG. 13B, the binding mode of GaSal-2 in HasAp indicated that the gallium sal ophen core fit into the Heme Site (black). One of the flexible amino arms was solvent exposed (pink) while the other amino arm binds into a narrow channel that opens to the Affinity Site (gold), indicating the opportunity for enhanced affinity and specificity of compounds by extending the chemical structure into this pocket. On the other hand, the binding mode of GaSal-2 in HemO (FIG. 13B) indicated that the gallium salophen core bind to the heme-binding active site of the enzyme, with the two arms extruded outside of the heme-binding pocket. This binding mode of GaSal-2 indicated that modification of its arms probably won’t impair the binding of new compounds to HemO. In the past, the Xue Lab has employed these CADD tools to design new ligands in efforts targeting transcriptional factor BCL6, GPCR mGluR5, and nuclear receptor hCAR.

[00343] Selection and prioritization of target GaSal-2 analogs. Guided by the SILCS

FragMaps of HasAp (FIG. 13A), GaSal-2 analogs shown in Fig.l4A were designed. The new compounds were based on a gallium salophen core equipped with a solubility arm (SA) to occupy the Solubility Site, and an affinity arm (AA) to extend into the Affinity Site. An initial collection of 10 SAs and 257 AAs were screened against the SILCS FragMaps of HasAp. 7 SAs 15 AAs with an LGFE-HasAp value < -11.0 kcal/mol were selected for a second SILCS analysis using the HemO FragMaps (FIG. 13B). Then 6 SAs & 17 AAs showing an LGFE-HemO value < -9.5 kcal/mol were considered as dual inhibitors for HasAp and HemO. Considering the synthesis accessibility, a final number of 54 (6SAs & 9AAs, FIG. 14B) GaSal-2 analogs are selected for synthesis. As shown in FIG. 14C, 54 target compounds are prioritized according to the LGFE differences (kcal/mol) of compounds vs GaSal-2 (LGFE-HasAp -9.97 kcal/mol). Priority is given to the ones shown in red followed by those in yellow.

[00344] The array of CADD, Medchem, analytical, and biophysical tools combined with the suite of in vitro and in vivo assays provides a strong foundation for the development of first- in-class multi-targeting GaSal complexes directed toward several pathways essential for MDR Pa infections.

[003451 DISCUSSION

[00346] Disclosed herein is the development of a first -in-class poly-pharmacological drug targeting the heme-dependent CSS cascade and HemO activity while taking advantage of active xenosiderophore uptake. Simultaneously targeting HasAp and HemO with GaSal-2 blocks the ability of Pa to sense heme while also disrupting homeostasis without inducing iron starvation and increased virulence. The overall effect of targeting both proteins limits iron availability and reduces the levels of BVIX0 which has been shown to have a positive effect on the ability of P. aeruginosa to adapt to a sessile lifestyle.

[00347] Targeting multiple steps in the same pathway can reduce selective pressure for resistance development. This novel multi-targeting strategy disrupting heme utilization, iron homeostasis, and virulence, increases the barrier to resistance.

[00348] GaSal-2 and its analogs described herein represent a novel formulation method and multi-targeting strategy for gallium, an iron-mimicking element that has been successfully used as an antimicrobial agent in the clinic.

[00349] Exploiting the social behavior of a bacterial population by targeting beneficial virulence mechanisms may render the inhibitor susceptible population phenotypic “cheaters” that take advantage of the public goods (i.e. quorum sensing molecules such as the recently identified BVIXP) produced by resistant strains. Therefore as the “cheater” population increases and dominates they are selective by “starving” the resistant community.

[00350] This Example includes an exemplary example of small -molecule therapeutic development using an established compound evaluation platform in the development of GaSal-2 analogs that simultaneously target the Pa heme sensing, uptake, and iron acquisition system (FIG. 15). A collection of 54 novel GaSal-2 analogs designed and prioritized by the CADD methodology are synthesized SILCS in order to simultaneously target the characterized binding sites of both HasAp and HemO. Then, the binding affinities (XD values) of synthesized GaSal analogs to both HasAp and HemO is determined by dose responses using the HTS FQ assays. For the compounds indicating dual inhibitory activity against HasAp and HemO (XD <500 nM for both proteins), their inhibition for heme signaling and uptake are tested using the transcriptional gene reporter assay and ¹³C-heme LC-MS/MS assay, respectively. The uptake of new GaSal-2 analogs by the siderophore receptors are quantified by measuring the intracellular Ga levels using ICP-MS. These efforts generate 6-8 selected candidates prioritized for further evaluation. IC50 values are determined, resistance analysis is performed, and biofilm inhibition is studied of the 6-8 selected compounds on a panel of Pa strains. The HasAp and HemO binding epitope for the top three compounds is then determined by NMR, HDX-MS, and X-ray crystallography. Three compounds with desirable properties are then advanced to preliminary pharmacokinetic (PK) and toxicity studies. For the best two, their in vivo antipseudomonal efficacy is evaluated in a murine acute lung infection model. The distribution of the best two compounds is studied in the lungs using MALDI mass spectrometry imaging (MALDI-MSI). The multitargeting small molecule approach permits the identification, validatation, and characterization of novel GaSal-based dual inhibitors against HasAp and HemO. In aspects, these studies provides tractable therapeutic candidate useful for blocking heme sensing and utilization while reducing virulence by modulating the activity of both the extracellular hemophore HasAp and the heme-degrading enzyme HemO to provide a first-in-class antipseudomonal targeting heme and iron uptake systems.

[00351] Synthesize GaSal-2 analogs that target HasAp, HemO, and xenosiderophore receptor uptake. 54 new GaSal-2 analogs are synthesized. After determining the j>s, those with KD <500 nM for both HasAp and HemO is tested for inhibition of heme signaling and uptake using transcriptional reporter assays and ¹³C-heme LC-MS/MS assay, respectively. Compound uptake by the siderophore receptors is quantified by measuring the intracellular Ga levels using ICP-MS. Additional rounds of refinement are expected to be required following biological and biophysical studies (FIG. 15).

[00352] Synthesizing new gallium-salophen complexes. It has been shown that GaSal-2 and its parent GaSal bind to both HasAp and HemO, and inhibits Pa heme sensing, uptake, and iron utilization while simultaneously acting as a substrate for xenosiderophore receptors (FIGS. 8A-8B). SILCS CADD are also used to design compounds that bind to the heme site and the HasR docking site of HasAp (FIGS. 13A-13B). A new compound GaSal-2 was synthesized by including a hydrophobic tail designed to fit into the HasR docking site of HasAp. Compared to parent GaSal, the ether fragment of compound GaSal-2 can occupy the Affinity Site of HasAp (FIG. 13 A). To further optimize the potency of inhibitors, specific modifications have been undertaken using CADD (FIG. 16) and 54 new GaSal-2 analogs identified for synthesis (FIGS. 14A-14B). The synthesized new compounds are evaluated for their biological efficacy in Tasks 2-11. Synthesis of 11-154 begins with aldehyde 1. Using a previousy-reported method, regioselective alkylation of aldehyde 1 using alkyl bromide (2) in the presence ofCsHCCh provides ether 3. Condensation of 3 with 1,2-diaminobenzene 4 yields the half ligand 5, which further reacts with aldehyde 6 to give ligand 7. Treatment of ligand 7 with Ga(NO3)3 yields the final compounds 11-154.

[00353] HasAp and HemO affinities: determine binding affinities (KD by FQ. The KD values of GaSal-2 were determined for both HasAp and HemO (FIG. 8A) using FQ assays previously discussed. Binding affinities of synthesized compounds is determined in a final volume of 200 pL in black flat-bottom 96-well plates using a microplate reader. HasAp or HemO concentration is kept at 1.0 pM in Tris-HCl buffer (20 mM, pH 8.0). Inhibitors solvated in DMSO are added across a concentration range of 0.01 to 100 pM. The excitation (295 nm) and emission (332 nm) spectrum is recorded. The KD values are calculated from plots of total binding vs ligand concentration by fitting the data to a sigmoidal dose-response equation in GraphPad Prism, where total binding corresponds to the fraction of binding sites represented by the decrease in fluorescence at the maximum emission (332 nm). All experiments are performed in triplicate. The assay was optimized for temperature, pH value, and concentration of HasAp or HemO in the 96-well format; several independent test runs were performed to assess the assay parameters.

[00354] Testing 12 selected dual inhibitors for inhibition of heme sensing by transcriptional reporter assays. GaSal-2 and its parent inhibited transcriptional activation of the has cascade using transcriptional gene reporter assays (FIG. 9). The effects of HasAp and HemO inhibition are tested on the cell surface signaling cascade using the same assay. Assays are performed by growing the PAO1 VhasR-lacZ transcriptional reporter strain under iron limiting conditions in M9 media supplemented with 1.0 pM heme in the presence or absence of selected inhibitors. Samples are removed at various times (0.5-5 h) and assayed for /3-Gal activity and corrected for differences in ODeoo. Besides, as deletion of the OM signaling receptor HasR attenuates virulence, it is believed that inhibition of the signaling pathway combined with decreased heme uptake is a novel synergistic multi-targeting strategy. Moreover, a VhasR- mScarlet transcriptional reporter was constructed that allows for the measurement of inhibition of HasAp-dependent transcriptional activation in real-time as a function of fluorescence. [00355] Testing the cellular HemO inhibition of 12 selected dual inhibitors by ¹³C-heme isotopic labeling. A quantitative isotopic ¹³C-heme labeling LC-MS/MS method was developed to distinguish BVIX metabolites derived from extracellular uptake (¹³C-heme) versus those derived from intracellular biosynthesis (¹²C-heme). This assay allows for direct readout of ¹³C- BVIXP and 1X5 isomer levels as a measure of inhibition of heme uptake and utilization by HemO inhibition. Decreased ¹³C-BVIXP levels as a consequence of the inhibition of HemO also leads to decreased transcriptional activation of hasAp which is assessed. GaSal-2 decreased BVIXp/5 formation by inhibiting HemO (FIG. 11). This assay was also utilized in studies of HasAp mutants and with inhibitors of HemO. Briefly, BVIX extracted from cultures at 2, 5 and 7 h (spiked with internal standard) is re-suspended in DMSO (10 pL) and diluted to 40 pL with the mobile phase and filtered through a 0.45 pm PTFE syringe. The BVIX isomers are separated and analyzed by multiple reaction monitoring (MRM) on a Waters TQD triple quadrupole mass spectrometer with an AQUITY H-Class UPLC fitted with Ascends RP -amide 2.7 pm C18 column (10 cm * 2.1 mm) at a flow rate of 0.4 mL/min with a mobile phase of A: H2O:0.1% formic acid and B: ACN:0.1% formic acid and a step gradient: 64% A:36% B; 5 min 55%A:45%B; 8 min 40%A:60%B; 8.5 min 5%A:95%B and 10 min 64%A:36%B.

[00356] Testing the uptake of GaSal analogs by ICP-MS. GaSal-2 and its parent yielded active uptake of Gallium (FIG. 10A-10B). The same method is used to study new GaSal-2 analogs. Briefly, aliquots (2 mL) are removed from cultures grown as described for the heme uptake studies, pelleted, and washed in M9 media. Pellets are dissolved in trace-metal-free ultrapure 20% HNO3 and boiled overnight at 100 °C. Samples are diluted with ultrapure H2O to a final concentration of 2% HNO3 and subjected to ICP-MS (Agilent 7700 ICP-MS). ICP-MS runs are calibrated with high purity iron standard solution, and raw ICP-MS data (ppb) are corrected for drift using values for scandium and germanium added as internal standards. Corrected values are normalized to culture density as determined by the ODeoo values for a total of six biological replicates. qPCR analysis of the xenosiderophore receptors piuA,pfuA, and femA is performed to determine if any are upregulated in response to GaSal treatment. Given a recent proteomics report that the addition of exogenous siderophores repressed pyoverdine production, the pvd biosynthesis genes pvdE,pvdF, and pvdG are also studied.

[00357] The characterization of GaSal-2 as a dual mechanism inhibitor of heme signaling and iron uptake provides a platform for the development of more potent complexes. In embodiments, the FQ assay can generate false positives, however, the robust suite of assays including transcriptional reporters, inhibition of heme uptake, Ga-uptake, and cellular HemO inhibition assays further validate compounds with K values (< 500 nM) (FIG. 15). In embodiments, initial efforts result in a collection (15-20) of inhibitors with varying degrees of potencies. The selection of compounds (12) are based on those that further disrupt heme signaling, while not significantly decreasing siderophore uptake. ICP-MS measures Ga uptake and not the intact salophen, however, the stability of GaSal complexes in the secreted media supports transport via a siderophore receptor. The uptake of GaSal analogs by performing ¹³C- salophen uptake studies similar to those described for heme. If there is limited success enhancing affinity by modification of the salophen scaffold I, the focus switches to the metasubstituted ligand (II) that indicated favorable LGFE values to the Affinity Site (FIG. 17). [00358] The assays have been used successfully to assess Pa mutant strains and HasAp- HemO dual inhibitors. In some embodiments, it is possible to inhibit one of the two protein targets, either HasAp or HemO, while having little effect on the other. These inhibitors have the potential to be optimized to increase potency in both systems. The transcriptional reporter assays are a valuable tool to assess the effect of inhibitors on heme signaling, however, as the cells require lysing to monitor -Gal activity this assay is not adaptable to HTS. A transcriptional reporter assay was developed using mScarlet that allowed for direct monitoring of fluorescence in real-time and is adaptable to HTS.

[00359] Test the effects of multi-targeting of HasAp, HemO, and xenosiderophore receptor uptake. The efficacy of 6-8 compounds are tested by IC50 screening, resistance analysis, and biofilm inhibition assays. Next, the top two candidates are evaluated in the murine acute lung infection model, and confirm the binding epitope by NMR, HDX-MS, and X-ray crystallography.

[00360] Determine IC50S of 6-8 selected dual inhibitors in defined media. Pa growth inhibition is performed on a Bioscreen C automated growth curve 100x100 sample well system. Briefly, the high throughput liquid handling system is used to seed the plates with Pa at an ODeoo of 0.05, following which the inhibitor or the solvent (0.1% DMSO or MeOH) is added to a final volume of 200 pF. Growth at 600 nm is recorded every 30 min over a period of 12 h. Growth inhibition studies are performed in minimal media (M9) supplemented with or without heme, and in SSM which mimics the growth environment of the CF lung. Using a similar protocol, the killing effects of selected hits on 15 clinical isolates obtained from CF lung, eye infections, and skin wounds are screened.

[003611 Resistance analysis using subinhibitory concentration of 6-8 selected hits. Serial dilution analysis of selected dual inhibitors from the growth inhibition screen above using the Pa lab strain PAO1 are performed. Briefly, cultures from a single susceptible colony (IxlO⁴ CFUs) are grown at subinhibitory concentrations that give rise to 25%, 50%, and 75% CFUs. Samples are removed from the culture and serially passaged at 1 : 1000 dilution every 24 h for 14 days in fresh culture. The percentage of resistant cells in each culture is monitored by plating ~10⁵ cells from cultures at 14 days onto LB agar containing the lead hit at the concentration that inhibits 90% growth. Any colonies evident on the plates are re-streaked on the same inhibitor concentration to confirm resistance. The selected colonies are further analyzed by sequencing hemO to determine if the resistance is due to decreased binding to HemO versus reduced heme uptake. Decreased heme uptake is determined by ¹³C-heme uptake studies as described in Task 4. Colonies exhibiting reduced heme uptake are analyzed for decreased expression of the heme receptors (qPCR) or mutation in hasR and/or phuR by DNA sequencing.

[00362] Assaying biofilm inhibition of 6-8 selected hits. Minimal biofilm eradication concentrations (MB EC) of the 6-8 selected compounds are measured in 96-well MBEC plates. Plates containing M9 are inoculated with Pa (1 * 10⁸ CFU/mL) and incubated at 37 °C for 48 h, allowing biofilms to form. After incubation, the peg lid is washed with 0.9% saline to remove non-adherent bacteria, then transferred to a fresh 96-well challenge plate containing M9 supplemented with selected inhibitors (9-300 pg/mL) and either 10 pM heme (pH 7.5) or 10 pM FeCE. Biofilms are incubated at 37 °C for 48 h, at which point the pegs are washed with 0.9% saline, stained with 0.1% crystal violet solution for 10 min, and rinsed with water. After drying the peg lid is destained in 200 pL of acetic acid (30%), diluted 1 in 10 and the OD595 measured. MBEC values represent the minimum inhibitor concentrations where at least 3 of 4 biological replicates have OD595 < 0.1 the breakpoint for 99.9% biofilm eradication.

[00363] Confirm binding of 3-4 selected dual inhibitors by STD-NMR. The binding of

GaSal-2 and its parent to HasAp were confirmed by STD-NMR. STD-NMR is performed for HemO (5-10 pM) in potassium phosphate (50 mM, pH 7.4, D2O). To this solution, the testing compound is added (50 pL, 500 pM) in DMSO-t/s. After a 4 h incubation period, compound 4,4-dimethyl-4-silapentane-l-sulfonic acid (DSS, 3 pL in D2O) is added as an internal standard and NMR spectra are recorded as previously described. NMR. These techniques have been used to determine the binding epitope of Pa HemO inhibitors, while HSQC-NMR studies of HasAp have been reported, HSQC-NMR is performed using 512 (tl) x 4096 (t2) complex points with a width of 1800 and 9000 Hz, respectively. Data are collected using an interscan delay of 1 s and 16 transients per fid, and processed with NMR- Pipe. Combined chemical shift differences between the apo and each of the titration points are calculated using eq 1. The nitrogen shift changes are multiplied by 0.2 to compensate for the difference in the chemical shift range between proton and nitrogen. Samples contain HasAp (125 pM), compound (250 pM) phosphate (50 mM), KC1 (100 mM) in H2O:DMSO- e (95:5, v:v). As shown in FIG. 18A, HSQC-NMR were used to determine the binding site of a HemO inhibitor acitretin.

[00365] Analyze solution structures of HasAp and HemO in the presence of dual inhibitors by HDX-MS. HDX-MS was used to study the solution structures of both HasAp (FIGS. 12A- 12C) and HemOin the presence of small molecule inhibitors. Experiments are performed with 100 pM protein in 20 mM HEPES (pH 7.4) diluted 50-fold with 20 mM HEPES buffer in D2O. Aliquots (100 pmol) are removed at 0, 10, and 30 min, and the deuteration reaction quenched by lowering the pH to 2.5 with HC1. For reactions of inhibitor-bound protein, the inhibitor required for 95% binding is estimated from the Ko, and a 10 to 20-fold excess is incubated with the protein. A 2-fold excess of inhibitor is added to the deuteration buffer to account for exchange during incubation. Quenched samples are analyzed on a Waters nanoACQUITY UPLC system. The protein is digested online at 10°C by rapid passage over an Enzymate BEH Pepsin Column. The digest is trapped and desalted online on an ACQUITY Vanguard BEH C 18 pre-column at 0°C for 4 min at a flow rate of 125 pL/min in 0.1% formic acid. Peptides are separated on an ACQUITY UPLC BEH C18 column at 0°C over a 15 min acetonitrile gradient (5-50%) with 0.1 % formic acid at a flow rate of 40 pL/min. The eluent is directed into the ion source of a coupled SYNAPT G2 HDMS mass spectrometer. Mass spectra are acquired in MSE mode (m/z range 50-2000). Non-deuterated peptides generated on digestion of the protein and identified using Waters ProteinLynx Global Server software is used to generate a peptide list for import into Waters DynamX software for identification of deuterated peptides, and to calculate their relative deuterium incorporation using the 0 s sample as a reference.

[00366] Obtain X-ray crystal structures of the three selected dual inhibitors in complex with HemO. The crystal structures of the Pa and N. meningitidis HemO enzymes were solved. Structures of the three optimal dual inhibitors are obtained.

[00367] Obtain X-ray crystal structures of the three selected dual inhibitors in complex with HasAp. Co-crystal structures of HasAp with small -molecule metal-complex ligands have been reported. A large quantity of purified HasAp protein and structures of the three optimal dual inhibitors are obtained.

[00368] Results. The growth inhibition assays were successfully utilized to screen for selective HasAp and HemO inhibitors. In some embodiments, other experimental approaches (e.g., resistance analysis, biofilm inhibition, and binding mode studies) are used. HasAp and HemO were crystalized. The screening assays described herein can be used to provide 2-4 optimal dual inhibitors for in vivo efficacy studies in Aim 3.

[00369] Evaluate the in vivo efficacy of the top compounds. The in vivo efficacy of dual inhibitors with desirable properties is assessed. The PK and Tox profiles of three selected multi-targeting compounds are screened and the best two for antipseudomonal efficacy is determined using an acute murine lung infection model. In addition, MALDI mass spectrometry imaging (MALDI-MSI) is performed to image the drug (top two GaSal analogs) localization in the lung.

[00370] PK study. Initial delivery of the GaSal analogs are via nebulization dosing used successfully for the delivery of tobramycin in CF lung infection. The drug clearance rate, volume of distribution, C_max, and in vivo half-life of the three top compounds are determined. These studies allow us to estimate the time needed to reach steady-state plasma concentration. Heart, liver, kidney, lung, and brain are collected for future determination of compound bioavailability in each organ. The dose and time points are altered for the two follow-up studies. For clearance kinetics, a tm are 3-4 h or longer, indicating a multiple dosing protocol could achieve the desired steady-state plasma concentration within 14 h. Once the range of clinically relevant plasma concentrations is established, the first estimate of bioavailability is made for oral dosing.

Completion isdentifies a lead candidate(s) with plasma clearance (CL) of less than 30% of blood flow, a half - life ( 1/2) of more than 3.5 h, and a distribution volume of more than 0.75 L/kg. If these are not obtained, studies are conducted on the backup compounds.

[00371] Establish maximum tolerated doses (MTDs). This study of the three top compounds provides the estimate of the MTDs and therapeutic window. CD-I mice are treated with a single nebulized dose of the testing compounds. 20 mice form four groups (5 mice/group; 3 dose levels of the testing compound and one vehicle control). Compound dosages are set by and half-log intervals employed. Mice are weighed and observed for two weeks, then euthanized (earlier if they show 20% weight loss or signs of distress). Metabolic monitoring using a comprehensive lab animal monitoring system (CLAMS) is performed to assess basal metabolism, cage movement, and food intake. Upon sacrifice, liver, heart, kidney, lung, and brain tissue is formalin-fixed. Tissues are embedded, sectioned, and hematoxylin & eosin stained then evaluated by a trained observer for tissue damage. Completion yields leads one or more candidate with the MTDs > 500 mg/kg.

[00372] Efficacy of two top compounds in a murine acute lung infection model. The therapeutic effect of GaSal-2 in mice (FIGS. 6A-6D) was studied and the two top compounds are tested. 30 CD-I mice form 5 groups (6 mice/group; 2 dose levels of testing compounds from Tasks 9 & JO, 1 dose of tobramycin (0.1 pg/mL), 1 dose of testing compounds plus tobramycin, and one vehicle control group. Dosages of testing compounds are determined. Mice are treated with a single nebulized dose of testing compounds (or multiple doses if PK is less favorable). When the compound reaches steady-state (Task 9,' estimate at ~14 h) mice are challenged with 5xl0⁷ colony- forming units of Pa strain PAO1. 16 h post-infection, established proxies are measured for the severity of infection: body temperature, wet lung weight, and bacterial burden in the nares and in the lung. Completion provides the lowest dose with therapeutic effect.

[00373] MALDI-MSI of GaSal analogs in murine acute lung infection. MALDI-MSI

(spatial distribution and relative abundance) and LC-MS/MS (absolute quantification) is used to characterize the top two top GaSal candidates’ distribution in the lung. MALD-MSI provides critical information on the spatial distribution of the GaSal drugs as it relates to colonization and morphology (e.g., biofilm), and inflammation. Briefly, frozen lung sections (10 pm) are thawmounted, dried, and processed as in earlier studies. Imaging is carried out on a Bruker Solarix Fourier transform-ion cyclotron resonance (FT-ICR) or a Bruker UltrafleXtreme matrix-assisted laser desorption/ionization-time of flight/time of flight (MALDI-TOF/TOF) mass spectrometer. Following MALDI-MSI, the MALDI matrix is washed from the lung sections prior to staining with hemotoxylin and eosin (H&E). Stained sections are microscopically scanned and coregistered with the MALDI dataset for analysis. LC-MS/MS is used to quantify and validate Ga- Sal analogs identified by MALDI-MSI. Compounds of interest are identified directly from tissue sections using the accurate mass measurements of the parent and fragment ions for each molecule. FIG. 19 shows BVIX is only detected in the infected lung and appears to be more prominent in the bronchi consistent with the site of PAO1 colonization.

[00374] Data Interpretation and Statistical Analysis: MALDI-MSI datasets are processed and co-registered with their representative histology image using the Fleximaging software.

Statistical analysis is carried out with SCiLs Lab software, designed to process MALDI imaging datasets. Supervised analysis is carried out based on known histological regions and spectra compared using univariate analysis methods. Unsupervised multivariate analysis of the data implements automated spatial segmentation using either hierarchical clustering, bisecting k- means or k-means clustering methods. Spatial segmentation uses clustering methods to group spectra based on their similarity using a ‘distance’ metric to quantify the similarity and place similar spectra into one cluster. All spectra of a given cluster are assigned a selected color and displayed in a manner by which all pixels are color-coded according to their cluster assignment. Spatial segmentation enables the automated identification of different anatomical features based on their molecular profiles. Univariate statistical hypothesis tests are applied to determine whether an m/z value can be used to confidently discriminate between anatomical regions of interest within the groups. For all m/z values identified the significance level is calculated using ANOVA. LC-MS/MS data is analyzed in Xcalibur and statistical tests are performed for in vivo data where p < 0.05 vs. control.

[00375] Results. Optimal dual inhibitors for HasAp and HemO are obtained based on the GaSal-2 scaffold, which indicates favorable PK and Tox profiles, and desirable efficacy in the murine acute lung infection models. Additional spatial imaging of drug distribution related to colonization and infection provides a qualitative and quantitative assessment of drug delivery to the site of action. As GaSal-2 and its analogs are highly fluorescent, live fluorescence imaging is used, as a complementary method to MALDI-MSI, to study the distribution of selected compounds. Lead compounds for the development of a first-in-class antipseudomonal drug synergistically targeting multiple pathways are developed. [00376] This Example provides studies for advancing the understanding of extracellular heme utilization and regulation, and its role in bacterial pathogenesis. As both HasAp and HemO play essential roles in iron acquisition, regulation of the extracellular heme sensing system, and the activation of critical virulence factors, the development inhibitors that modulate the flux of heme through these protein targets offer several advantages for antibacterial drug development. First, while both HasAp and HemO are required for colonization and infection within the host it is not essential for survival outside of the host, secondly, modulating the flux of heme and BVIX levels targets several metabolic and virulence pathways. These factors along with the potential to develop synergistic inhibitors targeting both heme and iron uptake, offer a powerful therapeutic approach with a reduced propensity for drug resistance development. The completion of the proposed aims provide a platform for the development of a first-in-class multitargeting antipseudomonal therapeutic candidate.

[00377] Bacterial strains are checked by PCR to ensure the absence or complementation of a gene or cross-contamination. All plasmid assays are checked by PCR following the assay. Plasmids for over-expression of protein are routinely checked by resequencing. All qRT-PCR and reporter assays are performed in triplicate on a minimum of 3 biological replicates. Purified proteins are analyzed by SDS-PAGE and checked by MALDI-TOF mass spectrometry to ensure homogeneity or removal of affinity tags. For metabolite analysis, all experiments are performed on a minimum of 5 biological replicates and 3 technical replicates per sample. Metabolite extraction efficiency is determined by the addition of internal standard (BVIXy) and BVIX concentrations from calibration curves generated for each isomer. Verification of instrumentation sensitivity is determined with internal standards to assess day-to-day variation of instrumentation. All SMPs and inhibitors are verified by NMR and high accuracy mass spectrometry prior to use.

Example 3 : New Chemical Entities (NCEs)

[00378] Overview

[00379] A family of New Chemical Entities (NCEs) were developed with the potential to serve as antibiotics that specifically target Pseudomonas aeruginosa (Pa) by a novel and unique mechanism of action. [00380] Traditional antibiotics that target Pa interfere with cell wall synthesis or with protein translation. Unfortunately, resistance arises quickly, and agents are toxic to nerves and kidneys. In June 2020, the Gallium Salophen (GalSal, PMID 32551497; Fig. 20) was reported. It inhibits Pa growth (IC50 = 7 pM) by blocking the import of heme-bound iron, and by allowing the import of cytotoxic Ga³⁺ into the bacterium to interfere with Fe³⁺. It attaches to Pa" s heme-binding extracellular hemophore protein HasAp. By doing so, GalSal keeps heme from reaching HasR, the transmembrane “heme importing” protein. At the same time, Pa uses its siderophore receptors to import GalSal. Both heme and sal ophen can be complexed with Fe³⁺, or with the similar metal ion Ga³⁺. Either’s binding to HasAp is almost unaffected by the choice of trivalent cation. The salophen KD’S are only ~3-fold higher than those for heme. Despite the similarity of sal ophen to heme and this relatively tight binding, Pa does not import sal ophen-bound Fe³⁺ (or Ga³⁺) by the heme pathway. While both heme-Fe and FeSal support the growth of Pa in iron-deficient M9 medium, a different entry mechanism was discovered. Heme-Fe (and heme-Ga) binds to HasAp and stimulates transcription of the HasR operon; FeSal and GalSal fail to do so. Subsequent studies with the hasRAphiiR strain showed that FeSal uptake is independent of the heme receptor system. FeSal and GalSal are instead taken up via a siderophore receptor.

[00381] This Example describes antibiotic development, including early animal studies. 5 g of GalSal with >95% purity was synthesized, purified, and characterized. Because Gal Sal’s aqueous solubility was unexpectedly low, a backup lead compound GalSal-2 was created. GalSal- 2 was designed using the computer-aided drug design method SILCS. This highly soluble GalSal analog displays potent inhibitory activity for HasAp and kills Pa strains. Additionally, GalSal-2 inhibits HemO with low micromolar binding affinity. Moreover, GalSal-2 demonstrated excellent efficacy in alleviating Pa bacterial burden in an acute murine lung infection model. With these data in hand, Mil development efforts were switched over to this compound.

[00382] Results

[00383] The synthesis of GalSal was completed, but in the Pharmokinetics phase, it was discovered that its solubility in water was disappointingly low - 0.1 mg/mL. Therefore, related compound Gal Sal-2 was developed.

[00384] Synthesis of GalSal and GalSal-2 [00385] Early on, 5 grams of Gal Sal were synthesized, then used that pathway as the basis for creating Gal Sal -2 (FIG. 20). Key starting compounds were diaminobenzene, 2,4- dihydroxybenzyladehyde, and 1,2-dibromoethane. Each of the four steps of the procedure was optimized for synthesis of GalSal-2 at the 1-gram (or larger) scale. Final products and intermediates were characterized by 'H-N R, ¹³C-NMR, and high-resolution mass spectrometry (HRMS). Purity was >95%. This alleviates concerns about synthesis efficiency for large-scale production.

[00386] PK Study

[00387] The LCMS method for the quantification of GalSal-based complexes has been established. In the initial PK studies, GalSal was given at a single dose of 40 pg/g body weight using a vehicle containing 25% PEG300/5% DMSO/70% PBS. Higher doses could not be achieved due to the limited solubility of GalSal. Blood was collected at 0.08, 0.25, 0.5, 1, 2, 4, 6, 12 and 24 h after compound GalSal administration. Samples were analyzed using an LC/MS/MS method. However, it was not sensitive enough to quantitate plasma GalSal in individual mice. After finding no evidence of in vivo efficacy at the dose of 40 pg/g body weight, the focus was shifted to the identification of soluble analogs of GalSal. Five GalSal analogs were then designed and synthesized (FIG. 21).

[00388] The structures of the final complexes have been confirmed by 'H/^C NMR and high-resolution mass spectrometry. HasAp binding KD values and Pa growth inhibition IC50 have been determined for the new compound GalSal-2, which is highly soluble in water (>50 mg/mL water), maintains potent inhibitory activity for HasAp, and kills Pa strains. Additionally, GaSal-2 inhibits the intracellular heme degrading enzyme heme oxygenase (HemO) with low micromolar binding affinity.

[00389] Toxicology study

[00390] Acute toxicology studies were conducted. In the acute studies, mice were given GalSal (vehicle, 10 pg/g and 40 pg/g; n=5 mice/group) or Gal Sal-2 (vehicle, 40 pg/g, 200 pg/g, and 500 pg/g; n=5 mice/group) by a single IP injection. The mice of all groups showed no other signs of toxicity. Body weight monitor evaluation indicated no signs of toxicity by GalSal at 40 p.g/g for 7 days. GalSal-2 at 500 p.g/g for 7 days was similarly negative. Overall, GalSal-2 was well tolerated in mice at all doses used. A dose-escalation strategy is employed to determine if an MTD > 500 mg/kg can be achieved. Additional compounds from the pipeline which passed the in vitro cell toxicity evaluation are assessed in vivo.

[00391] GalSal-2 efficacy in a murine model of Pa Lung Infection

[00392] The antipseudomonal efficacy of GaSal-2 in an acute murine lung infection model was tested (FIGS. 6A-6D). The drug was delivered to the lungs of the animals via nebulization. It was demonstrated that the treatment of infected animals with GaSal-2 can decrease the bacterial burden in lungs (FIG. 6A) and in nasal wash (FIG. 6B). Moreover, following treatment, the animals did not show a significant drop in their lung weight (FIG. 6C) and maintained normal body temperature (FIG. 6D).

[00393] The efficacy of the drug and the fact that high doses (500 mg/kg) are well-tolerated in the mice are promising for further optimization of this new class of inhibitors. In the future, the anti-pseudomonal efficacy of GalSal-2 and additional related compounds are evaluated with this and other lung infection models (including chronic lung infection models).

[00394] Impact of Results on Commercialization Plans

[00395] GalSal-2 is a useful for the treatment of Pa infections, including hospital- and community-based pneumonia. Acquired antibiotic resistance in Pa is mediated by multiple mechanisms, including enzyme expression, reductions to membrane permeability, active efflux, and acquisition of exogenous resistance plasmids. For infections resistant to first-line agents (piperacillin, cephalosporins, and carbapenems), the Infectious Disease Society of America recommends ceftolozane-tazobactam or ceftazidime-avibactam, with laboratory confirmation of susceptibility. Cefiderocol and imipenem-cilastatin-relebactam are second-line agents, while polymyxins are typically deployed only when no other options are available.

[00396] Persistent and inappropriate use of antibiotics over decades has resulted in the emergence of MDR and extensively drug resistant (XDR) bacteria. GalSal-2 targets two steps in an essential metabolic process, heme uptake. The development of novel therapeutics to treat M/XDR infections is now critical to combat growing antibiotic resistance worldwide. [00397] In a non-limiting example, GalSal-2 can be useful in IND-directed preclinical studies as an aerosol adjunctive therapy for the treatment of XDR or Pan-resistant pneumonias. In a non-limiting example, Gal Sal -2 can be delivered orally or by intramuscular injection rather than intravenously.

References

[00398] A number of patent and non-patent publications are cited herein in order to describe the state of the art to which this disclosure pertains. The entire disclosure of each of these publications is incorporated by reference herein.

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[00399] While certain embodiments of the present disclosure have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. The present disclosure is, therefore, not limited to the particular embodiments described and/or exemplified, but is capable of considerable variation and modification without departure from the scope and spirit of the appended claims.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: formula (I) wherein in formula (I): each R¹ is a substituent independently selected at each occurrence from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl; each R² and R³ is a substituent independently selected at each occurrence from halogen, optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylheteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted haloalkyl, optionally substituted alkoxy, and optionally substituted heteroaryl;

Group A: independently at each occurrence selected from Br , Cl , and I .

2. The compound of claim 1, wherein n is 0.

3. The compound of claim 1 or 2, wherein each R² and R³ is independently -OR^b wherein each R^b is independently selected at each occurrence from the group consisting of optionally substituted alkyl, optionally substituted alkylaryl, optionally substituted alkylcycloalkyl, and optionally substituted alkylheteroaryl.

4. The compound of claim 3, wherein each R² and R³ is independently selected from-Cs- ring A is an optionally substituted heterocyclyl, ring B is an optionally substituted aryl, each R^c is independently at each occurrence H or alkyl, each X is independently at each occurrence selected from Br , Cl , and I , and each s and v is independently at each occurrence an integer from 1 to 4.

5. The compound of claim 4, wherein s is 2.

6. The compound of claim 4, wherein v is 1.

7. The compound of any one of claims 3-6, wherein each occurrence of R^c is H or Ci- C alkyl, optionally methyl.

9. The compound of any one of claims 3-7, wherein ring , wherein X is halogen, optionally fluorine, and ring C is optionally substituted heterocyclyl, optionally morpholine. he compound of any one of claims 1 -9, wherein each R² is independently selected at , wherein X is independently at each occurrence selected from Br , Cl , and I . he compound of any one of claims 1-10, wherein each R³ is independently selected , wherein X is independently at each occurrence selected from

Br , Cl , and I ..

12. The compound of any one of claims 1-11, wherein p is 1 or 2.

13. The compound any one of claims 1-12, wherein q is 1.

14. The compound of any one of claims 1-13, wherein the compound of formula (I) is a compound of formula (II), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: wherein in formula (II):

R⁴ is H or an optionally substituted Ci-Ce alkyl; with the proviso that when R⁴ is H, then at least one of p or q is an integer from 1 to 4; and a) when p is 1 and R² is selected from Group A, i) q cannot be 0; and ii) when q is 1, R³ cannot be identical to R²; and b) when q is 1 and R³ is selected from Group A, i) p cannot be 0; and ii) when p is 1, R² cannot be identical to R³: Group A: independently at each occurrence selected from Br , Cl , and I .

15. The compound of any one of claims 1-13, wherein the compound of formula (I) is a compound of formula (III), or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof: formula (III) wherein in formula (III):

Group A: independently at each occurrence selected from Br , Cl , and I

16. The compound of claim 11, wherein the compound is of any one of formulas 1001 to 1339, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, wherein X is independently at each occurrence selected from Br, Cl", or I":

17. The compound of claim 1 or 15, wherein the compound is of any one of formulas 2001 to 2031, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, wherein X is independently at each occurrence selected from Br , Cl", or I":

18. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, wherein the compound is selected from:

19. The compound of any one of claims 1 -18, wherein the compound inhibits HasAp protein activity.

20. The compound of any one of claims 1-1 , wherein the compound inhibits HemO protein activity.

21. The compound of any one of claims 1-20, wherein the compound inhibits both HasAp protein activity and HemO protein activity.

22. A pharmaceutical composition for treating a condition alleviated by inhibiting HasAp protein activity, the pharmaceutical composition comprising one or more compounds according to any of claims 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

23. A pharmaceutical composition for treating a condition alleviated by inhibiting HemO protein activity, the pharmaceutical composition comprising one or more compounds according to any of claims 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

24. A pharmaceutical composition for treating a condition alleviated by dual inhibition of HasAp protein activity and the HemO protein activity, the pharmaceutical composition comprising one or more compounds according to any of claims 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of any of claims 22 to 24, wherein the condition is selected from sepsis, meningitis, endocarditis, osteomyelitis, otitis media, sinusitis, pneumonia, chronic respiratory tract infection, catheter infection, surgical implant infection (e.g., cardiac valve replacement, pacemaker, joint replacement (e.g., hip or knee replacement) and the like), postoperative peritonitis, postoperative biliary tract, tricuspid valve endocarditis, ecthyma gangrenosum, eyelid abscess, lacrimal cystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, orbital infection, urinary tract infection, complicated urinary tract infection, catheter infection, perianal abscess, severe burns, airway burns, pressure ulcer infections, cystic fibrosis, and a bacterial infection.

26. A pharmaceutical composition for treating or preventing a bacterial infection, the pharmaceutical composition comprising one or more compounds according to any of claims 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26, wherein the bacterial infection is caused by a bacterium selected from P. aeruginosa, Serratia marcescens, Bordetella pertussis, Bordetella bronchiseptica, Bordetella avium, Yersinia pestis, Yersinia pseudotuberculosis, Acinetobacter baumannii, and pan-resistant pneumonia, optionally wherein the bacterium is an extensively drug-resistant (XDR) bacterium.

28. A method of treating a condition by inhibiting HasAp protein activity in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of any of claims 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

29. A method of treating a condition by inhibiting HemO protein activity in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of any of claims 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

30. A method of treating a condition by dual inhibition of the HasAp protein activity and HemO protein activity in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of any of claims 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

31. The method of any of claims 28 to 30, wherein the condition is selected from sepsis, meningitis, endocarditis, osteomyelitis, otitis media, sinusitis, pneumonia, chronic respiratory tract infection, catheter infection, surgical implant infection (e g., cardiac valve replacement, pacemaker, joint replacement (e.g., hip or knee replacement) and the like), postoperative peritonitis, postoperative biliary tract, tricuspid valve endocarditis, ecthyma gangrenosum, eyelid abscess, lacrimal cystitis, conjunctivitis, corneal ulcer, corneal abscess, panophthalmitis, orbital infection, urinary tract infection, complicated urinary tract infection, catheter infection, perianal abscess, severe burns, airway bums, pressure ulcer infections, cystic fibrosis, and a bacterial infection.

32. A method for treating or preventing a bacterial infection in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of any of claims 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.

33. The method of claim 32, wherein the bacterial infection is caused by a bacterium selected from P. aeruginosa, Serratia marcescens, Bordetella pertussis, Bordetella bronchiseptica, Bordetella avium, Yersinia pestis, Yersinia pseudotuberculosis, Acinetobacter baumannii, and pan-resistant pneumonia, optionally wherein the bacterium is an extensively drug-resistant (XDR) bacterium.

34. A pharmaceutical composition for treating cancer, the pharmaceutical composition comprising one or more compounds according to any of claims 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof, and a pharmaceutically acceptable carrier.

35. A method for treating or preventing cancer in a patient in need of said treatment, the method comprising administering to the patient a therapeutically effective amount of a compound of any of claims 1 to 21, or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.